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20.3: Agglutination Assays - Biology

20.3: Agglutination Assays - Biology


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Learning Objectives

  • Compare direct and indirect agglutination
  • Identify various uses of hemagglutination in the diagnosis of disease
  • Explain how blood types are determined
  • Explain the steps used to cross-match blood to be used in a transfusion

In addition to causing precipitation of soluble molecules and flocculation of molecules in suspension, antibodies can also clump together cells or particles (e.g., antigen-coated latex beads) in a process called agglutination ([link]). Agglutination can be used as an indicator of the presence of antibodies against bacteria or red blood cells. Agglutination assays are usually quick and easy to perform on a glass slide or microtiter plate (Figure (PageIndex{1})). Microtiter plates have an array of wells to hold small volumes of reagents and to observe reactions (e.g., agglutination) either visually or using a specially designed spectrophotometer. The wells come in many different sizes for assays involving different volumes of reagents.

Agglutination of Bacteria and Viruses

The use of agglutination tests to identify streptococcal bacteria was developed in the 1920s by Rebecca Lancefieldworking with her colleagues A.R. Dochez and Oswald Avery.1 She used antibodies to identify M protein, a virulence factor on streptococci that is necessary for the bacteria’s ability to cause strep throat. Production of antibodies against M protein is crucial in mounting a protective response against the bacteria.

Lancefield used antisera to show that different strains of the same species of streptococci express different versions of M protein, which explains why children can come down with strep throat repeatedly. Lancefield classified beta-hemolytic streptococci into many groups based on antigenic differences in group-specific polysaccharides located in the bacterial cell wall. The strains are called serovars because they are differentiated using antisera. Identifying the serovars present in a disease outbreak is important because some serovars may cause more severe disease than others.

The method developed by Lancefield is a direct agglutination assay, since the bacterial cells themselves agglutinate. A similar strategy is more commonly used today when identifying serovars of bacteria and viruses; however, to improve visualization of the agglutination, the antibodies may be attached to inert latex beads. This technique is called an indirect agglutination assay (or latex fixation assay), because the agglutination of the beads is a marker for antibody binding to some other antigen (Figure (PageIndex{2})). Indirect assays can be used to detect the presence of either antibodies or specific antigens.

To identify antibodies in a patient’s serum, the antigen of interest is attached to latex beads. When mixed with patient serum, the antibodies will bind the antigen, cross-linking the latex beads and causing the beads to agglutinate indirectly; this indicates the presence of the antibody (Figure (PageIndex{3})). This technique is most often used when looking for IgM antibodies, because their structure provides maximum cross-linking. One widely used example of this assay is a test for rheumatoid factor (RF) to confirm a diagnosis of rheumatoid arthritis. RF is, in fact, the presence of IgM antibodies that bind to the patient’s own IgG. RF will agglutinate IgG-coated latex beads.

In the reverse test, soluble antigens can be detected in a patient’s serum by attaching specific antibodies (commonly mAbs) to the latex beads and mixing this complex with the serum (Figure (PageIndex{3})).

Agglutination tests are widely used in underdeveloped countries that may lack appropriate facilities for culturing bacteria. For example, the Widal test, used for the diagnosis of typhoid fever, looks for agglutination of Salmonella enterica subspecies typhi in patient sera. The Widal test is rapid, inexpensive, and useful for monitoring the extent of an outbreak; however, it is not as accurate as tests that involve culturing of the bacteria. The Widal test frequently produces false positives in patients with previous infections with other subspecies of Salmonella, as well as false negatives in patients with hyperproteinemia or immune deficiencies.

In addition, agglutination tests are limited by the fact that patients generally do not produce detectable levels of antibody during the first week (or longer) of an infection. A patient is said to have undergone seroconversion when antibody levels reach the threshold for detection. Typically, seroconversion coincides with the onset of signs and symptoms of disease. However, in an HIV infection, for example, it generally takes 3 weeks for seroconversion to take place, and in some instances, it may take much longer.

Similar to techniques for the precipitin ring test and plaque assays, it is routine to prepare serial two-fold dilutions of the patient’s serum and determine the titer of agglutinating antibody present. Since antibody levels change over time in both primary and secondary immune responses, by checking samples over time, changes in antibody titer can be detected. For example, a comparison of the titer during the acute phase of an infection versus the titer from the convalescent phase will distinguish whether an infection is current or has occurred in the past. It is also possible to monitor how well the patient’s immune system is responding to the pathogen.

Watch this video that demonstrates agglutination reactions with latex beads.

Exercise (PageIndex{1})

  1. How is agglutination used to distinguish serovars from each other?
  2. In a latex bead assay to test for antibodies in a patient's serum, with what are the beads coated?
  3. What has happened when a patient has undergone seroconversion?

Hemagglutination

Agglutination of red blood cells is called hemagglutination. One common assay that uses hemagglutination is the direct Coombs’ test, also called the direct antihuman globulin test (DAT), which generally looks for nonagglutinating antibodies. The test can also detect complement attached to red blood cells.

The Coombs’ test is often employed when a newborn has jaundice, yellowing of the skin caused by high blood concentrations of bilirubin, a product of the breakdown of hemoglobin in the blood. The Coombs’ test is used to determine whether the child’s red blood cells have been bound by the mother’s antibodies. These antibodies would activate complement, leading to red blood cell lysis and the subsequent jaundice. Other conditions that can cause positive direct Coombs’ tests include hemolytic transfusion reactions, autoimmune hemolytic anemia, infectious mononucleosis (caused by Epstein-Barr virus), syphilis, and Mycoplasma pneumonia. A positive direct Coombs’ test may also be seen in some cancers and as an allergic reaction to some drugs (e.g., penicillin).

The antibodies bound to red blood cells in these conditions are most often IgG, and because of the orientation of the antigen-binding sites on IgG and the comparatively large size of a red blood cell, it is unlikely that any visible agglutination will occur. However, the presence of IgG bound to red blood cells can be detected by adding Coombs’ reagent, an antiserum containing antihuman IgG antibodies (that may be combined with anti-complement) (Figure (PageIndex{4})). The Coombs’ reagent links the IgG attached to neighboring red blood cells and thus promotes agglutination.

There is also an indirect Coombs’ test known as the indirect antiglobulin test (IAT). This screens an individual for antibodies against red blood cell antigens (other than the A and B antigens) that are unbound in a patient’s serum (Figure (PageIndex{4})). IAT can be used to screen pregnant women for antibodies that may cause hemolytic disease of the newborn. It can also be used prior to giving blood transfusions. More detail on how the IAT is performed is discussed below.

Antibodies that bind to red blood cells are not the only cause of hemagglutination. Some viruses also bind to red blood cells, and this binding can cause agglutination when the viruses cross-link the red blood cells. For example, influenza viruses have two different types of viral spikes called neuraminidase (N) and hemagglutinin (H), the latter named for its ability to agglutinate red blood cells (see Viruses). Thus, we can use red blood cells to detect the presence of influenza virus by direct hemagglutination assays (HA), in which the virus causes visible agglutination of red blood cells. The mumps and rubella viruses can also be detected using HA.

Most frequently, a serial dilution viral agglutination assay is used to measure the titer or estimate the amount of virus produced in cell culture or for vaccine production. A viral titer can be determined using a direct HA by making a serial dilution of the sample containing the virus, starting with a high concentration of sample that is then diluted in a series of wells. The highest dilution producing visible agglutination is the titer. The assay is carried out in a microtiter plate with V- or round-bottomed wells. In the presence of agglutinating viruses, the red blood cells and virus clump together and produce a diffuse mat over the bottom of the well. In the absence of virus, the red blood cells roll or sediment to the bottom of the well and form a dense pellet, which is why flat-bottomed wells cannot be used (Figure (PageIndex{5})).

A modification of the HA assay can be used to determine the titer of antiviral antibodies. The presence of these antibodies in a patient’s serum or in a lab-produced antiserum will neutralize the virus and block it from agglutinating the red cells, making this a viral hemagglutination inhibition assay (HIA). In this assay, patient serum is mixed with a standardized amount of virus. After a short incubation, a standardized amount of red blood cells is added and hemagglutination is observed. The titer of the patient’s serum is the highest dilution that blocks agglutination (Figure (PageIndex{6})).

Exercise (PageIndex{2})

  1. What is the mechanism by which viruses are detected in a hemagglutination assay?
  2. Which hemagglutination result tells us the titer of virus in a sample?

ANIMALS IN THE LABORATORY

Much of what we know today about the human immune system has been learned through research conducted using animals—primarily, mammals—as models. Besides research, mammals are also used for the production of most of the antibodies and other immune system components needed for immunodiagnostics. Vaccines, diagnostics, therapies, and translational medicine in general have all been developed through research with animal models.

Consider some of the common uses of laboratory animals for producing immune system components. Guinea pigs are used as a source of complement, and mice are the primary source of cells for making mAbs. These mAbs can be used in research and for therapeutic purposes. Antisera are raised in a variety of species, including horses, sheep, goats, and rabbits. When producing an antiserum, the animal will usually be injected at least twice, and adjuvants may be used to boost the antibody response. The larger animals used for making antisera will have blood harvested repeatedly over long periods of time, with little harm to the animals, but that is not usually the case for rabbits. Although we can obtain a few milliliters of blood from the ear veins of rabbits, we usually need larger volumes, which results in the deaths of the animals.

We also use animals for the study of disease. The only way to grow Treponema pallidum for the study of syphilis is in living animals. Many viruses can be grown in cell culture, but growth in cell culture tells us very little about how the immune system will respond to the virus. When working on a newly discovered disease, we still employ Koch’s postulates, which require causing disease in lab animals using pathogens from pure culture as a crucial step in proving that a particular microorganism is the cause of a disease. Studying the proliferation of bacteria and viruses in animal hosts, and how the host immune system responds, has been central to microbiological research for well over 100 years.

While the practice of using laboratory animals is essential to scientific research and medical diagnostics, many people strongly object to the exploitation of animals for human benefit. This ethical argument is not a new one—indeed, one of Charles Darwin's daughters was an active antivivisectionist (vivisection is the practice of cutting or dissecting a live animal to study it). Most scientists acknowledge that there should be limits on the extent to which animals can be exploited for research purposes. Ethical considerations have led the National Institutes of Health (NIH) to develop strict regulations on the types of research that may be performed. These regulations also include guidelines for the humane treatment of lab animals, setting standards for their housing, care, and euthanization. The NIH document “Guide for the Care and Use of Laboratory Animals” makes it clear that the use of animals in research is a privilege granted by society to researchers.

The NIH guidelines are based on the principle of the three R’s: replace, refine, and reduce. Researchers should strive to replace animal models with nonliving models, replace vertebrates with invertebrates whenever possible, or use computer-models when applicable. They should refine husbandry and experimental procedures to reduce pain and suffering, and use experimental designs and procedures that reduce the number of animals needed to obtain the desired information. To obtain funding, researchers must satisfy NIH reviewers that the research justifies the use of animals and that their use is in accordance with the guidelines.

At the local level, any facility that uses animals and receives federal funding must have an Institutional Animal Care and Use Committee (IACUC) that ensures that the NIH guidelines are being followed. The IACUC must include researchers, administrators, a veterinarian, and at least one person with no ties to the institution, that is, a concerned citizen. This committee also performs inspections of laboratories and protocols. For research involving human subjects, an Institutional Review Board (IRB) ensures that proper guidelines are followed.

Visit this site to view the NIH Guide for the Care and Use of Laboratory Animals.

Blood Typing and Cross-Matching

In addition to antibodies against bacteria and viruses to which they have previously been exposed, most individuals also carry antibodies against blood types other than their own. There are presently 33 immunologically important blood-type systems, many of which are restricted within various ethnic groups or rarely result in the production of antibodies. The most important and perhaps best known are the ABO and Rh blood groups (see [link]).

When units of blood are being considered for transfusion, pretransfusion blood testing must be performed. For the blood unit, commercially prepared antibodies against the A, B, and Rh antigens are mixed with red blood cells from the units to initially confirm that the blood type on the unit is accurate. Once a unit of blood has been requested for transfusion, it is vitally important to make sure the donor (unit of blood) and recipient (patient) are compatible for these crucial antigens. In addition to confirming the blood type of the unit, the patient’s blood type is also confirmed using the same commercially prepared antibodies to A, B, and Rh. For example, as shown in Figure (PageIndex{7}), if the donor blood is A-positive, it will agglutinate with the anti-A antiserum and with the anti-Rh antiserum. If no agglutination is observed with any of the sera, then the blood type would be O-negative.

Following determination of the blood type, immediately prior to releasing the blood for transfusion, a cross-match is performed in which a small aliquot of the donor red blood cells are mixed with serum from the patient awaiting transfusion. If the patient does have antibodies against the donor red blood cells, hemagglutination will occur. To confirm any negative test results and check for sensitized red blood cells, Coombs’ reagent may be added to the mix to facilitate visualization of the antibody-red blood cell interaction.

Under some circumstances, a minor cross-match may be performed as well. In this assay, a small aliquot of donor serum is mixed with patient red blood cells. This allows the detection of agglutinizing antibodies in the donor serum. This test is rarely necessary because transfusions generally use packed red blood cells with most of the plasma removed by centrifugation.

Red blood cells have many other antigens in addition to ABO and Rh. While most people are unlikely to have antibodies against these antigens, women who have had multiple pregnancies or patients who have had multiple transfusions may have them because of repeated exposure. For this reason, an antibody screen test is used to determine if such antibodies are present. Patient serum is checked against commercially prepared, pooled, type O red blood cells that express these antigens. If agglutination occurs, the antigen to which the patient is responding must be identified and determined not to be present in the donor unit.

Exercise (PageIndex{3})

  1. If a patient's blood agglutinates with anti-B serum, what is the patient’s blood type?
  2. What is a cross-match assay, and why is it performed?

Table (PageIndex{1}) summarizes the various kinds of agglutination assays discussed in this section.

Table (PageIndex{1}): Mechanisms of Select Antibody-Antigen Assays
Type of AssayMechanismExample
AgglutinationDirect: Antibody is used to clump bacterial cells or other large structuresSerotyping bacteria
Indirect: Latex beads are coupled with antigen or antibody to look for antibody or antigen, respectively, in patient serumConfirming the presence of rheumatoid factor (IgM-binding Ig) in patient serum
HemagglutinationDirect: Some bacteria and viruses cross-link red blood cells and clump them togetherDiagnosing influenza, mumps, and measles
Direct Coombs’ test (DAT): Detects nonagglutinating antibodies or complement proteins on red blood cells in vivoChecking for maternal antibodies binding to neonatal red blood cells
Indirect Coombs’ test (IAT): Screens an individual for antibodies against red blood cell antigens (other than the A and B antigens) that are unbound in a patient’s serum in vitroPerforming pretransfusion blood testing
Viral hemagglutination inhibition: Uses antibodies from a patient to inhibit viral agglutinationDiagnosing various viral diseases by the presence of patient antibodies against the virus
Blood typing and cross-matching: Detects ABO, Rh, and minor antigens in the bloodMatches donor blood to recipient immune requirements

Key Concepts and Summary

  • Antibodies can agglutinate cells or large particles into a visible matrix. Agglutination tests are often done on cards or in microtiter plates that allow multiple reactions to take place side by side using small volumes of reagents.
  • Using antisera against certain proteins allows identification of serovars within species of bacteria.
  • Detecting antibodies against a pathogen can be a powerful tool for diagnosing disease, but there is a period of time before patients go through seroconversion and the level of antibodies becomes detectable.
  • Agglutination of latex beads in indirect agglutination assays can be used to detect the presence of specific antigens or specific antibodies in patient serum.
  • The presence of some antibacterial and antiviral antibodies can be confirmed by the use of the direct Coombs’ test, which uses Coombs’ reagent to cross-link antibodies bound to red blood cells and facilitate hemagglutination.
  • Some viruses and bacteria will bind and agglutinate red blood cells; this interaction is the basis of the direct hemagglutination assay, most often used to determine the titer of virus in solution.
  • Neutralization assays quantify the level of virus-specific antibody by measuring the decrease in hemagglutination observed after mixing patient serum with a standardized amount of virus.
  • Hemagglutination assays are also used to screen and cross-match donor and recipient blood to ensure that the transfusion recipient does not have antibodies to antigens in the donated blood.

Multiple Choice

We use antisera to distinguish between various ________ within a species of bacteria.

A. isotypes
B. serovars
C. subspecies
D. lines

B

When using antisera to characterize bacteria, we will often link the antibodies to ________ to better visualize the agglutination.

A. latex beads
B. red blood cells
C. other bacteria
D. white blood cells

A

The antibody screening test that is done along with pretransfusion blood typing is used to ensure that the recipient

A. does not have a previously undetected bacterial or viral infection.
B. is not immunocompromised.
C. actually does have the blood type stated in the online chart.
D. is not making antibodies against antigens outside the ABO or Rh systems.

D

The direct Coombs’ test is designed to detect when people have a disease that causes them to

A. have an excessively high fever.
B. quit making antibodies.
C. make too many red blood cells.
D. produce antibodies that bind to their own red blood cells.

D

Viral hemagglutination assays only work with certain types of viruses because

A. the virus must be able to cross-link red blood cells directly.
B. the virus must be able to lyse red blood cells.
C. the virus must not be able to lyse red blood cells.
D. other viruses are too dangerous to work with in a clinical lab setting.

A

Fill in the Blank

In the major cross-match, we mix ________ with the donor red blood cells and look for agglutination.

patient serum

Coombs’ reagent is an antiserum with antibodies that bind to human ________.

immunoglobulins/antibodies and/or complement

Short Answer

Explain why the titer of a direct hemagglutination assay is the highest dilution that still causes hemagglutination, whereas in the viral hemagglutination inhibition assay, the titer is the highest dilution at which hemagglutination is not observed.

Why would a doctor order a direct Coombs’ test when a baby is born with jaundice?

Critical Thinking

When shortages of donated blood occur, O-negative blood may be given to patients, even if they have a different blood type. Why is this the case? If O-negative blood supplies were depleted, what would be the next-best choice for a patient with a different blood type in critical need of a transfusion? Explain your answers.

Footnotes

  1. 1 Lancefield, Rebecca C., “The Antigenic Complex of Streptococcus haemoliticus. I. Demonstration of a Type-Specific Substance in Extracts of Streptococcus haemolyticus,” The Journal of Experimental Medicine 47, no. 1 (1928): 91-103.

Learning Objectives

In addition to causing precipitation of soluble molecules and flocculation of molecules in suspension, antibodies can also clump together cells or particles (e.g., antigen-coated latex beads) in a process called agglutination (See Figure 19.9). Agglutination can be used as an indicator of the presence of antibodies against bacteria or red blood cells. Agglutination assays are usually quick and easy to perform on a glass slide or microtitre plate (Figure 21.15). Microtitre plates have an array of wells to hold small volumes of reagents and to observe reactions (e.g., agglutination) either visually or using a specially designed spectrophotometer. The wells come in many different sizes for assays involving different volumes of reagents.

Figure 21.15. Microtitre plates are used for conducting numerous reactions simultaneously in an array of wells. [Credit: modification of work by “Microrao”/Wikimedia]


What Is Agglutination? (with pictures)

In biology, agglutination refers to the bunching together of particles. This process is especially important as part of the immune system, the response process that an organism uses to fight disease. Hemagglutination, the clumping of red blood cells, has special applications in medicine, where it is used to determine blood type and find the concentration of infecting viruses or bacteria in the bloodstream.

Foreign bacteria or viruses entering the body contain specific components called antigens, which trigger an immune response in the host. White blood cells in the body produce proteins known as antibodies in response to the presence of antigens. The antibodies bind with the antigens through a structural mechanism similar to a key and a lock, and can either neutralize the antigen directly or mark it for destruction by the immune system.

Agglutination is one way in which antibodies mark antigens for destruction. Antibodies have at least two sites where an antigen can bind, so they are capable of binding with more than one bacterium or virus. When this happens, the invading particles begin to agglutinate, or form clumps, through a network of antibodies. The clumps eventually become too large to remain in solution in the bloodstream, and precipitate out of solution.

Once the clumps of particles are large enough, they become easy prey for phagocytes — a type of white blood cell that ingests foreign material. Phagocytes ingest and break down the clump, neutralizing the threat of disease. In this way, agglutination enables the body to disarm and remove harmful invading particles.

Hemagglutination, by contrast, is not a naturally occurring process in the body but is instead used to perform assays and testing procedures in molecular biology. Blood type is determined through this process. In blood typing, specific antibodies are added that bind to specific types of red blood cells. If the antibody binds to the red blood cells in a sample, agglutination occurs, and blood type can be confirmed based on which antibody was used.

The concentration of bacteria or viruses in a sample can sometimes be determined using a test called a hemagglutination assay. Certain bacteria and viruses contain compounds that allow them to bind to red blood cells, creating a clump network. In the assay, a diluted sample of virus is added to a diluted blood cell sample, and agglutination is allowed to occur for about 30 minutes. The concentration of the virus can be determined by counting the number of clumps or lattices formed in the mixed sample.


20.3: Agglutination Assays - Biology

Sometimes when the blood of 2 people is mixed together, it clump or forms visible island in the liquid plasma the red cell becomes attached to one another. this agglutination when different types blood are mixed within the body the reaction can be bursting of the red cells as well as agglutination different types of blood are recognized on the molecular level and sometimes rejected ,by being destroyed and ultimately filtred out by kidney in order to expel them from the body along with urine in this case of transfusion mistake .there can be so much of the wrong types of blood in the system that its can results in kidney failure and death this is due to fact that, when the kidney try to filter the blood they essentially become clogged as they are overwhelmed and causes being effective filters. the compositional different between clogged as they are overwhelmed so that they are relatively large protein elect that provides the biological signature of an individual blood types within blood there-there are substances called antibodies which distinguish particular antigens from other causing bursting 01 agglutination of the red cells when alien antigens are found the antibodies binds to antigens In case of angulation the antibodies "glue" together the antigens from different red cells there by sticking the red cell together.

the specific types of antigens on our red blood cell determine our blood types there are 27 known human blood system or group for which each of us can be typed as a result, there are one or more antigens for each of these blood groups since many of these blood systems also are found in apes and monkey. it is likely that they evolved prior to to the time that we become a separate species.

long before the phenomenon of blood antigen antibodies interaction was discovered surgeons experimented with human transfusion .in an attempt to save the lives of patients who were dying from severe blood loss and the resulting shocks the 1st attempt may have been an English physician during severe blood loss and the resulting shock during 19 centuries Europeans and American doctors , used transfusions in a last ditch attempts to save soldiers and other patient vice versa a rubber tube with hypodermic needles at each ends this occasionally resulted in success but more often than not killed the recipient the results seemed to be random Doctors in the "19 the century" also experimented with a variety of blood substitutes including milk water and even oil it was discovery of the ABO blood types in 1900 that finally led us to understand how to consistently use transfusions to save liver even with this knowledge however life threatening reaction still occurs in about 80000 transfusion development nation.

Blood types

A
AB
B
O
blood group A means the presence of Aantigen but the absence of B antigens with the presence of receive gene.
blood group B means the presence of B antigen but an absence of A antigens with the presence of receive gene.
blood groupAB means presence of B antigen and of A antigens with the presence of receive gene.
blood group O means the absence of B antigen and of A antigens with the presence of receive gene.

source:en.wikipedia.org

blood is categorized as one of 4 types O,A,B,AB and another identifier of blood types is the RH factors indicated as either positive or negative. the RH factors are the team for the substance found on the surface of the red blood cell and is named for the rheus monkey where it was originated found RH factors are generally only an interset in obstetrics.

Genetic inheritance of blood Groups

" Scientists Bernstein's in 1924 AD was the first to find out multiple alleles in human blood group ABO which based on 3 allell".

RH Factors

the RH factors genetics information is also inherited from our parents but it is inherited independently of ABO blood type allele. there are 2 different alleles for the RH factors knowns as RH + and RH -
Someone who is Rh positive has least one RH+ allele but could have two their genotypes could be either RH+ or RH - .some one RH - has a like ABO alleles each biological arents donates one their .this couple couldA father who is RH+ could pass either an RH + or RH allel to his son or daughter this couple would have RH + children or RH - children.

Rh blood types were discovered is 1940 by Kal land Steiner and alexander wiener this was 40 years after land Steiner had discovered the ABO blood group over ,the last half century we have learned far more about the processes responsible for RH types this blood group ,may be the most complex genetically of all blood types system ,since for RH types this blood group may be most complex genetically of all blood types systems since it involves 45 different antigens on the surface of red cell that are controlled by 2 closed linked genes chromosomes 1 the RH system was named after rhesus monkey since they were initially used in the research to make the antiserum for typing blood sample.

clinically the RH factors like ABO factors can lead to serious medical complication the greatest problem with RH group is not so many incompatibilities following transfusion .as those between a mother and her developing fetus mother-fetus incompletely occurs as those between a mother nad fetus the risk increase with each pregnancy Europeans are like to have this problem 13 % of new born babies are at risk .actually these babies of European birth have complication with prevention treatment this number can be cut down even further less than 1 % of those treated have trouble, however, Rh blood types incomplatiblitely is still the leading cause of potentially fatal blood related problems of the newborn.

source:www.keyword-suggestions.com
figure Rh factors


Agglutination of bacteria and viruses

The use of agglutination tests to identify streptococcal bacteria was developed in the 1920s by Rebecca Lancefield working with her colleagues A.R. Dochez and Oswald Avery . Lancefield, Rebecca C., &ldquoThe Antigenic Complex of Streptococcus haemoliticus . I. Demonstration of a Type-Specific Substance in Extracts of Streptococcus haemolyticus ,&rdquo The Journal of Experimental Medicine 47, no. 1 (1928): 91-103. She used antibodies to identify M protein , a virulence factor on streptococci that is necessary for the bacteria&rsquos ability to cause strep throat. Production of antibodies against M protein is crucial in mounting a protective response against the bacteria.

Lancefield used antisera to show that different strains of the same species of streptococci express different versions of M protein, which explains why children can come down with strep throat repeatedly. Lancefield classified beta-hemolytic streptococci into many groups based on antigenic differences in group-specific polysaccharides located in the bacterial cell wall. The strains are called serovars because they are differentiated using antisera. Identifying the serovars present in a disease outbreak is important because some serovars may cause more severe disease than others.

The method developed by Lancefield is a direct agglutination assay , since the bacterial cells themselves agglutinate. A similar strategy is more commonly used today when identifying serovars of bacteria and viruses however, to improve visualization of the agglutination, the antibodies may be attached to inert latex beads . This technique is called an indirect agglutination assay (or latex fixation assay ), because the agglutination of the beads is a marker for antibody binding to some other antigen ( [link] ). Indirect assays can be used to detect the presence of either antibodies or specific antigens.

Antibodies against six different serovars of Group A strep were attached to latex beads. Each of the six antibody preparations was mixed with bacteria isolated from a patient. The tiny clumps seen in well 4 are indicative of agglutination, which is absent from all other wells. This indicates that the serovar associated with well 4 is present in the patient sample. (credit: modification of work by American Society for Microbiology)


20.3: Agglutination Assays - Biology

Lab 6. Agglutination

When antibodies are mixed with their corresponding antigens on the surface of large, easily sedimented particles such as animal cells, erythrocytes, or bacteria, the antibodies cross-link the particles, forming visible clumps. This reaction is termed agglutination. Agglutination is a serological reaction and is very similar to the precipitation reaction we learnt last week. Both reactions are highly specific because they depend on the specific antibody and antigen pair. The main difference between these two reactions is the size of antigens. For precipitation, antigens are soluble molecules, and for agglutination, antigens are large, easily sedimented particles. As you will see from this lab exercise, agglutination is more sensitive than precipitation reaction because it takes a lot of more soluble antigens and antibody molecules to form a visible precipitation. To make the detection of soluble antigen and antibody reaction more sensitive, a precipitation reaction can be transformed into an agglutination reaction by attaching soluble antigens to large, inert carriers, such as erythrocytes or latex beads.

Agglutination reactions have many applications in clinical medicine. Agglutination reactions can be used to type blood cells for transfusion, to identify bacterial cultures, and to detect the presence and relative amount of specific antibody in a patient’s serum. Agglutination has been commonly used to determine whether a patient had or has a bacterial infection. For example, if a patient is suspected of having typhoid fever, the patient’s serum is mixed with a culture of Salmonella typhi. If an agglutination reaction occurs, shown as clumping of the bacteria, the patient either had or has an S. typhi infection. Since certain antibodies can persist in a patient’s blood for years after the patent has recovered from the infection, a positive reaction does not mean that the patient currently has the infection. To determine whether a patient is currently suffering from typhoid fever, the amount or titer of the antibody will be determined at the onset of illness and two weeks later. If the titer of antibody in the patient’s serum has increased at least four-fold between the two tests, the patient is currently fighting off the infection, and the pathogen causing the illness is confirmed.

In this lab exercise, you will learn two different methods of employing agglutination reactions, rapid slide agglutination and microtiter test. These two tests are valuable methods commonly used in clinical laboratories. Applications of agglutination include A-B-O blood typing tests and rapid bacterial identification. The microtiter test is used to quantify the amount of antibody in patient’s blood.


Part 1. Rapid Slide Agglutination


SAFETY NOTE: This lab uses your own blood for ABO blood typing. After obtaining your drops of blood, put on gloves. Dispose of waste in the biohazard container.

Procedure

1. Label three slides A, B and A+B.

2. Spray your left “ring “ finger with 70% ethanol or wipe it with an alcohol wiper and let it air dry.

3. Take a sterile lancet and puncture your fingertip. If you have calluses, aim a little to the side. DO NOT LANCET ANYONE OTHER THAN YOURSELF. When finished with your lances, place them in the BIOHAZARD CONTAINER .

4. Place a small drop of blood on each of three microscope slides. At this point, put on GLOVES. DO NOT TOUCH ANYONE ELSE’S BLOOD.

5. On the A slide, place a 20 m l of anti-A antiserum. Place anti-B on the B slide and anti-A + anti-B on the A+B slide. Mix the antisera in with the blood using a separate toothpick for each slide. Place toothpicks in the biohazard waste.

6. After several minutes, observe agglutination and determine your blood type.

Part 2. Microtiter Test

In this lab exercise, you will learn how to use the microtiter test to determine the amount of anti-sheep red blood cell (RBC) antibody.

Procedure

1. In a round-bottom microtiter plate, add 50 m l of PBS to columns 2-9 in rows A-C. Leave column 1 empty.

2. Add 100 m l of rabbit anti-sheep RBC to column 1 in rows A-C.

3. In each row, transfer 50 m l from column 1 to column 2 and mix well. Now take 50 m l from column 2 and transfer to column 3. Repeat this process across the columns. Discard the final 50 ml taken from row 9.

4. Add 50 m l of a 2% suspension of sheep RBC to all wells (columns 1-9, rows A-C). Make sure the RBCs stay adequately resuspended as you are using them. Periodically invert the capped tube to keep them resuspended evenly.

5. Incubate the plate for 24 hours at room temperature.

6. Come back the next day and observe agglutination. Positive wells will exhibit a diffuse and confluent settling of the RBC and Ab, while in negative wells, all cells will roll down to the bottom and it will look more like a dot.

Reading
Immunobiology A-7

Study Questions

1. What is the difference between precipitation and agglutination? Which one of these two methods is more sensitive and why?

2. What type of structure on your red blood cells decides your blood type?

3. You used the slide agglutination test to determine your blood type. You got the following result:
Anti-serum A B AB
Agglutination - + +

What is your blood type?

If you receive type AB blood for blood transfusion, what is going to happen and why?

4. Describe how to determine whether a patient has ever had a Salmonella typhi infection, the name of method, reagents used, possible results, conclusions deduced from results and why.

5. In preparing her immunology lab, an instructor purified IgG antibodies that are specific to sheep red blood cells (SRBCs) and digested some of the antibodies into Fab, Fc, and F(ab’)2 fragments. She placed each preparation in a separate tube, labeled the tubes with a water-soluble marker, and left them in an ice bucket. When the instructor returned, she discovered that the labels had smeared and were unreadable. Determined to salvage the antibodies, she relabeled the tubes 1, 2, 3, and 4 and proceeded. Based on the test results described below, indicate which preparation was contained in each tube and explain why you so identified the contents.

a. The preparation in tube 1 agglutinated SRBCs but did not lyse them in the presence of complement.

b. The preparation in tube 2 did not agglutinate SRBCs or lyse them in the presence of complement. However, when this preparation was added to SRBCs before the addition of whole anti-SRBC, it prevented agglutination of the cells by the whole anti-SRBC antiserum.

c. The preparation in tube 3 agglutinated SRBCs and also lysed the cells in the presence of complement.

d. The preparation in tube 4 did not agglutinate or lyse SRBCs and did not inhibit agglutination of SRBCs by whole anti-SRBC antiserum.


  1. Obtain a preparation of virus (e.g. influenza viruses) with known HA titer or determine its HA titer
  2. Prepare two-fold dilutions of patient/test serum to be tested e.g. from 1:4 to 1:1024.
  3. Add a fixed amount of virus to every well of a 96-well plate, equivalent to 4 HA units (varies according to the virus), except for the serum control wells.
  4. Allow the plate to stand at room temperature for 60 minutes (time varies according to specific requirements).
  5. Add red blood cells (RBC) and incubate at 4°C for 30 minutes.
  6. Read the wells.

The highest dilution of serum (Ab) that prevents hemagglutination is called the HAI titer of the serum. A smooth or jagged shield of cells or an irregular button indicates agglutination. Observation of movement of the button of red cells when the plate is tilted may help to clarify the endpoint.

This virus sample has an HAI titer of 1280, which means that the greatest dilution of antibody that still blocked hemagglutination from occurring was at 1280 dilution. At this dilution, the antibodies were still capable of recognizing and binding to the antigens on the virus.


Immunodiagnostic Assays for the Investigation of Fungal Outbreaks

Fungal pathogens can affect humans, animals, and plants, and they can be found in the environment or as part of the host microbiome. Fungal diseases present a broad clinical spectrum, ranging from superficial to invasive infections, and can cause outbreaks. During an outbreak investigation, the laboratory plays an essential role in verifying the diagnosis and helping to confirm the source of the outbreak. Immunodiagnostic assays are important tools and often relied upon for the diagnosis of fungal infections, since the gold standard assays of culture and histopathology are time-consuming and often require invasive procedures. Immunodiagnostic assays range from complement fixation and immunodiffusion to enzyme immunoassays and, most recently, to point-of-care lateral flow devices. In general, these assays provide results faster and offer good analytical performance. These characteristics make immunodiagnostic assays good laboratory tools for outbreak investigations. The aim of this review is to describe the principles, advantages, limitations, and availability of immunodiagnostics assays in outbreak investigations, based on the experience of a reference laboratory.

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Antibody Functions

Antibodies, part of the humoral immune response, are involved in pathogen detection and neutralization.

Learning Objectives

Differentiate among affinity, avidity, and cross-reactivity in antibodies

Key Takeaways

Key Points

  • Antibodies are produced by plasma cells, but, once secreted, can act independently against extracellular pathogen and toxins.
  • Antibodies bind to specific antigens on pathogens this binding can inhibit pathogen infectivity by blocking key extracellular sites, such as receptors involved in host cell entry.
  • Antibodies can also induce the innate immune response to destroy a pathogen, by activating phagocytes such as macrophages or neutrophils, which are attracted to antibody-bound cells.
  • Affinity describes how strongly a single antibody binds a given antigen, while avidity describes the binding of a multimeric antibody to multiple antigens.
  • A multimeric antibody may have individual arms with low affinity, but have high overall avidity due to synergistic effects between binding sites.
  • Cross reactivity occurs when an antibody binds to a different-but-similar antigen than the one for which it was raised this can increase pathogen resistance or result in an autoimmune reaction.

Key Terms

  • avidity: the measure of the synergism of the strength individual interactions between proteins
  • affinity: the attraction between an antibody and an antigen

Antibody Functions

Differentiated plasma cells are crucial players in the humoral immunity response. The antibodies they secrete are particularly significant against extracellular pathogens and toxins. Once secreted, antibodies circulate freely and act independently of plasma cells. Sometimes, antibodies can be transferred from one individual to another. For instance, a person who has recently produced a successful immune response against a particular disease agent can donate blood to a non-immune recipient, confering temporary immunity through antibodies in the donor’s blood serum. This phenomenon, called passive immunity, also occurs naturally during breastfeeding, which makes breastfed infants highly resistant to infections during the first few months of life.

Antibodies coat extracellular pathogens and neutralize them by blocking key sites on the pathogen that enhance their infectivity, such as receptors that “dock” pathogens on host cells. Antibody neutralization can prevent pathogens from entering and infecting host cells, as opposed to the cytotoxic T-cell-mediated approach of killing cells that are already infected to prevent progression of an established infection. The neutralized antibody-coated pathogens can then be filtered by the spleen and eliminated in urine or feces.

Mechanisms of antibody action: Antibodies may inhibit infection by (a) preventing the antigen from binding to its target, (b) tagging a pathogen for destruction by macrophages or neutrophils, or (c) activating the complement cascade.

Antibodies also mark pathogens for destruction by phagocytic cells, such as macrophages or neutrophils, because they are highly attracted to macromolecules complexed with antibodies. Phagocytic enhancement by antibodies is called opsonization. In another process, complement fixation, IgM and IgG in serum bind to antigens, providing docking sites onto which sequential complement proteins can bind. The combination of antibodies and complement enhances opsonization even further, promoting rapid clearing of pathogens.

Affinity, avidity, and cross reactivity

Not all antibodies bind with the same strength, specificity, and stability. In fact, antibodies exhibit different affinities (attraction) depending on the molecular complementarity between antigen and antibody molecules. An antibody with a higher affinity for a particular antigen would bind more strongly and stably. It would be expected to present a more challenging defense against the pathogen corresponding to the specific antigen.

Antibody affinity, avidity, and cross reactivity: (a) Affinity refers to the strength of single interactions between antigen and antibody, while avidity refers to the strength of all interactions combined. (b) An antibody may cross-react with different epitopes.

The term avidity describes binding by antibody classes that are secreted as joined, multivalent structures (such as IgM and IgA). Although avidity measures the strength of binding, just as affinity does, the avidity is not simply the sum of the affinities of the antibodies in a multimeric structure. The avidity depends on the number of identical binding sites on the antigen being detected, as well as other physical and chemical factors. Typically, multimeric antibodies, such as pentameric IgM, are classified as having lower affinity than monomeric antibodies, but high avidity. Essentially, the fact that multimeric antibodies can bind many antigens simultaneously balances their slightly-lower-binding strength for each antibody/antigen interaction.

Antibodies secreted after binding to one epitope on an antigen may exhibit cross reactivity for the same or similar epitopes on different antigens. Cross reactivity occurs when an antibody binds not to the antigen that elicited its synthesis and secretion, but to a different antigen. Because an epitope corresponds to such a small region (the surface area of about four to six amino acids), it is possible for different macromolecules to exhibit the same molecular identities and orientations over short regions.

Cross reactivity can be beneficial if an individual develops immunity to several related pathogens despite having been exposed to or vaccinated against only one of them. For instance, antibody cross reactivity may occur against the similar surface structures of various Gram-negative bacteria. Conversely, antibodies raised against pathogenic molecular components that resemble self molecules may incorrectly mark host cells for destruction, causing autoimmune damage. Patients who develop systemic lupus erythematosus (SLE) commonly exhibit antibodies that react with their own DNA. These antibodies may have been initially raised against the nucleic acid of microorganisms, but later cross-reacted with self-antigens. This phenomenon is also called molecular mimicry.


Molecular and Immunological Diagnostic Techniques of Medical Viruses

Viral infections are causing serious problems in human population worldwide. The recent outbreak of coronavirus disease 2019 caused by SARS-CoV-2 is a perfect example how viral infection could pose a great threat to global public health and economic sectors. Therefore, the first step in combating viral pathogens is to get a timely and accurate diagnosis. Early and accurate detection of the viral presence in patient sample is crucial for appropriate treatment, control, and prevention of epidemics. Here, we summarize some of the molecular and immunological diagnostic approaches available for the detection of viral infections of humans. Molecular diagnostic techniques provide rapid viral detection in patient sample. They are also relatively inexpensive and highly sensitive and specific diagnostic methods. Immunological-based techniques have been extensively utilized for the detection and epidemiological studies of human viral infections. They can detect antiviral antibodies or viral antigens in clinical samples. There are several commercially available molecular and immunological diagnostic kits that facilitate the use of these methods in the majority of clinical laboratories worldwide. In developing countries including Ethiopia where most of viral infections are endemic, exposure to improved or new methods is highly limited as these methods are very costly to use and also require technical skills. Since researchers and clinicians in all corners of the globe are working hard, it is hoped that in the near future, they will develop good quality tests that can be accessible in low-income countries.

1. Introduction

Viruses are small segments of nucleic acid, deoxyribonucleic acid (DNA), or ribonucleic acid (RNA) within a protein coat or lipoprotein coat (envelope). Viruses require host resources for their replication because they are obligate intracellular parasites. Once viruses enter the host cells, they take over or hijack the cells’ biosynthetic machineries for the replication of their genomes and other components [1, 2].

Viral infections are the most common cause of human diseases. Millions of people are still dying because of human immunodeficiency virus (HIV) and hepatitis viruses worldwide. The emerging viruses are also causing serious problems in human population. For example, avian influenza A (H5N1) in 1997, the severe acute respiratory syndrome-coronavirus (SARS-CoV) in 2002–2003, pandemic swine influenza A (H1N1) virus in 2009, Ebola virus in 2014, Zika virus (ZIKV) in 2015, and pandemic SARS-CoV-2 recently, among others, have caused several outbreaks in different countries [3–9].

The morbidity and mortality rates of human viral infections are significantly high [10]. For example, the pandemic swine influenza A (H1N1) infection in 2009 occurred in 214 countries with more than 18,036 deaths [5]. In 2010 alone, the number of human deaths due to rabies globally was estimated to be 61,000, with 84% of the deaths occurred in rural areas [11]. In 2013, approximately, 35,000,000 people were infected with HIV worldwide [10]. The World Health Organization (WHO) reported 1.34 million deaths of viral hepatitis in 2015 [12]. As on 6 th January 2015, H5N1 viruses have killed 402 out of 694 laboratory-confirmed human infections in 16 countries [13], with a mortality rate of around 58%. Recently, the world is challenged by the novel coronavirus disease 2019 (COVID-19). The disease is caused by the novel coronavirus (SARS-CoV-2). The pathogen first emerged in Wuhan city, Hubei province, China, which has now quickly gained worldwide spread [9, 14]. On 11 th March 2020, the WHO declared the COVID-19 outbreak a global pandemic. According to the WHO, 9, 129, 146 confirmed cases of COVID-19 have been reported worldwide, including 473, 797 deaths since 31 st December 2019 and as of 24 th June 2020 [15]. Therefore, good diagnostic techniques are required to detect these viral infections early and accurately. Early and accurate detection of viral diseases plays a significant role in selecting appropriate therapy timely, minimizing therapy costs, minimizing unnecessary loss of human lives, and controlling the disease. It also helps to develop appropriate disease prevention and treatment strategies, like development of antiviral vaccines and new therapeutic agents [14, 16, 17].

Traditionally, laboratory diagnoses of medical viruses are carried out by isolating viruses in embryonated chicken eggs, in tissue culture, or in laboratory animals and visual examination of viral particles in sample using electron microscopy among others [16]. Many conventional diagnostic tools tend to be cumbersome, time-consuming, expensive, and poorly reproducible [18, 19]. In contrast, molecular techniques have revolutionized diagnostic virology by detecting the presence or absence of viral nucleic acids in a patient’s sample [18]. Immuno-based techniques still play a great role for the detection and serosurveillance of human viral infections despite the fact that many of the traditional methods are replaced by nucleic acid-based techniques [20]. Immunological methods detect viral infections by identifying antiviral antibodies or viral antigens in clinical samples [21, 22]. Here, we describe some of the molecular and immunological diagnostic approaches for the detection of medical viruses.

2. Molecular Diagnostic Techniques of Medical Viruses

Nucleic acid-based molecular detection techniques have revolutionized diagnostic virology with their faster, highly sensitive, and highly specific diagnosis [14, 23, 24]. Since these methods detect specific nucleic acid sequences, nucleic acid-based diagnostic tests can be applied for the detection of virtually any virus that affects humans [1].

2.1. Nucleic Acid-Based Amplification Techniques

Molecular techniques that involve the amplification of viral genomic material are extremely sensitive and specific, provide rapid diagnosis, and allow the detection of several viruses at same time [16]. Nucleic acid amplification techniques are very useful for the detection of viruses that are uncultivable or difficult and harmful to culture, slow growing viruses in culture, and viruses that display antigenic variations [1, 25]. The nucleic acid amplification tests are very popular in the diagnosis of viral infections caused by several viruses, including hepatitis C virus (HCV), human immunodeficiency virus (HIV), dengue virus, Epstein–Barr virus (EBV), influenza viruses, Zika virus (ZIKV), Ebola virus, and coronavirus [26–32]. Several nucleic acid amplification methods are currently available for the laboratory diagnosis of viral infections worldwide, and their advantages and limitations will be summarized in Table 1.

2.1.1. Polymerase Chain Reaction (PCR)

PCR is a typical example of nucleic acid amplification assay. It has revolutionized the field of molecular diagnosis since developed by Mullis and Faloona [50]. PCR is based on extraction and purification of DNA molecule and exponential amplification of the target sequence, using a thermostable DNA polymerase and two specific oligonucleotide primers. After the PCR reaction, the amplified product can be detected by several techniques, including gel electrophoresis, colorimetric methods, and sequencing [10, 51, 52]. Since its inception, PCR has been used for the detection of human viral infections with overall clinical sensitivity ranging from 77.8% to 100% and clinical specificity ranging from 89% to 100% [28, 53–55]. These reports suggest that PCR can be employed for the detection of medical viruses in a variety of specimen types. Conventional PCR is still in use by some clinical laboratories worldwide, but it is rapidly replaced by more advanced variants of the technique.

PCR is a highly versatile technique. A number of variants of the conventional PCR have been developed, but the most important variants are reverse transcription-PCR and real-time PCR [1, 10]. The first method was devised to amplify ribonucleic acid (RNA) targets [1] the second technique was introduced to quantify deoxyribonucleic acid (DNA) in real time throughout the PCR reactions [56].

2.1.2. Reverse Transcription-PCR (RT-PCR)

RT-PCR was designed to amplify RNA targets. In this technique, reverse transcriptase (RT) is used to convert viral RNA targets into complementary DNA (cDNA), and then the resulting cDNA is amplified by conventional PCR. Since its development, RT-PCR has been used for the diagnosis of human infection by RNA viruses. Conventional RT-PCR demonstrated overall sensitivity ranging from 73% to 100% and specificity ranging from 99% to 100% in the detection of viral infection [29, 57, 58]. These data indicate that RT-PCR is an excellent technique for the diagnosis of human infection by RNA viruses. Nowadays, however, the method is not used commonly in clinical specimens owing to its high cost and time-consuming process [14].

2.1.3. Real-Time PCR

In real-time PCR system, viral nucleic acid amplification and detection steps are carried out at the same time. The detection of the amplification product is relied on the amount of fluorescence emission from the specimen. The fluorescence emission from the specimen is monitored by special thermal cycler. The computer, with appropriate software connected to the thermal cycler, records the data and produces an amplification plot at every reaction cycle [51, 59]. The detection and quantification of amplification products can be done by using SYBR green, the TaqMan, and molecular beacon chemistries. The SYBR green dye binds to the minor groove of double-stranded DNA (dsDNA) product and upon excitation by appropriate light, it exhibits improved fluorescence, which is directly proportional to the accumulated dsDNA product. The TaqMan probe is a DNA oligonucleotide with a fluorescent dye termed reporter attached to one end (5′ base) and quencher on the other (3′ base) (Figure 1). TaqMan probes are designed to hybridize to an internal region of a PCR product. During the annealing stage of the PCR, both the primer and the TaqMan probe bind to the template strand. When the Taq DNA polymerase extends the primer, the polymerase cleaves the probe by its the 5′-3′ exonuclease activity. Cleavage of the probe leads to the release of the fluorescent dye (Figure 1), resulting in fluorescence emission. The amount of fluorescence is directly proportional to the PCR product. Molecular beacon is a small DNA molecule with a fluorescent dye at the 5′ end and a quencher at the 3′ end. The sequences at the very 3′ and 5′ ends are complementary to each other. The internal part of the molecule is designed to be complementary to the target sequence of a PCR product. When molecular beacon is free in solution, it will adopt a hairpin structure. This brings the fluorophore and quencher in close proximity, leading to absorption of emitted light of the florescent dye by the quencher and hence fluorescence is not detected (Figure 2 A). However, when a molecular beacon hybridizes to the target sequence, the fluorophore and quencher are separated, leading to the emission of fluorescence (Figure 2 B). The amount of fluorescence is directly proportional to the PCR product [16, 42, 51, 60].

Owing to high sensitivity and specificity, short turnaround time for results, and ease of performance [33, 61], most laboratories across the globe employ real-time PCR for the detection and quantification of medical DNA and RNA viruses in clinical specimens. For example, Boppana et al. [39] used real-time PCR for the detection of cytomegalovirus (CMV) in liquid saliva with overall sensitivity of 100% and specificity of 99.9%, compared with standard rapid culture. The method was also employed for the diagnosis of primary of EBV infection with overall sensitivity of 95.7% and specificity of 100%, compared to serologic assays [62]. Real-time PCR was also served to determine viral load in herpes simplex encephalitis patients [40]. The determination of viral loads in patient specimens is crucial as it provides prognostic and predictive information. In this study, patients with higher viral loads in their cerebrospinal fluid (CSF) found to require acyclovir therapy for a longer duration and had a poorer clinical outcome than the patients with lower viral loads in their CSF [40]. The assay can also be used for the multiplex identification of different viruses. Both TaqMan probe and molecular beacon play crucial roles for multiplex identification of different viruses in a single PCR reaction. In multiplexing assays, different probes/beacons are labeled with different fluorescent dyes [41]. In multiplex assay, sensitivity of 100% and specificity of 99.6% were reported, compared to immunofluorescence assay, for real-time PCR in the detection of human adenovirus B, C, and E in the throat swab samples [63]. Ramamurthy and his colleagues [33] compared multiplex real-time PCR with multiplex conventional PCR for the detection of neurotropic viruses (CMV, EBV, herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) Japanese encephalitis virus (JEV), and varicella-zoster virus (VZV)) in CSF. Out of 147 CSF samples collected from patients with neurological disorders, real-time PCR detected viral pathogens in 88 samples while conventional PCR could only detect the viruses in six samples, suggesting that real-time PCR has higher sensitivity than conventional PCR. Qiu et al. [64] developed a triplex quantitative real-time PCR assay for rapid and differential detection of human adenovirus (hAdV) serotypes 2, 3, and 7 for potential clinical use. The analytical sensitivity (limit of detection LoD) of this assay was 10 2 DNA copies/reaction for each of serotypes and no cross-reactions with other respiratory pathogens. The authors concluded that the assay is sensitive and specific and has the potential for clinical use in the rapid and differential detection and quantitation of hAdV serotypes 2, 3, and 7 in human specimens.

By the incorporation of reverse transcription step, real-time PCR can be combined with the conventional reverse transcription PCR (RT-PCR) to form reverse transcription quantitative real-time PCR (RT-qPCR). RT-qPCR has a number of advantages over the conventional RT-PCR technique, including reduction of contamination, possibility of quantifying the amplicons, and quick assay time since there are no post-PCR processing activities [14, 51]. As a result, RT-qPCR is widely deployed for the detection and quantification of several RNA viruses in clinical specimens, including ZIKV, Ebola virus, coronavirus, HCV, respiratory syncytial virus (RSV), dengue virus, HIV-1, and influenza A virus [30–32, 65–69]. Recently, Corman et al. [32] developed RT-qPCR for the detection of SARS-CoV-2. The assay targeted envelope protein (E) gene and RNA-dependent RNA polymerase (RdRp) gene of SARS-CoV-2. High LoDs of 5.2 copies/reaction for E-gene and 3.8 copies/reaction for RdRp gene were demonstrated and no cross-reaction with other coronaviruses, suggesting the usefulness of the method for sensitive and specific diagnosis of COVID-19. The RT-qPCR assay (Quanty ZEBOV FAST assay) was evaluated for the detection of Ebola virus in clinical samples. CLONIT Srl (Italy) developed the assay during outbreak of Ebola in Sierra Leone, and it had overall sensitivity of 100% and specificity of 98.63%, compared to RealStar® Filovirus Screen RT-qPCR Kit 1.0 (Altona Diagnostics) [31]. Gueudin et al. [68] used RT-qPCR for diagnosis and monitoring of HIV-1 group O infection with LoD of 40 copies/ml and specificity of 100%. The method was applied for ZIKV detection in human serum and urine, and it had LoDs of 2.5 PFU/ml and 250 PFU/ml in urine and serum, respectively [30]. Júnior et al. [70] used RT-qPCR for the detection of respiratory viruses in outpatients with acute respiratory infection. They also compared the performance of RT-qPCR with indirect immunofluorescence assay (IFA). Accordingly, RT-qPCR managed to detect viral pathogens in 88 (88/162) nasopharyngeal aspirates, but IFA detected viral pathogens in only 33 (33/162) specimens. The data indicated that the use of RT-qPCR increased the viral detection by 33.9%. Today, several real-time RT-PCR kits are available commercially. For example, real-time-qPCR test developed by Cepheid AB (Sunnyvale, CA, USA) is commercially available for the qualitative detection of Ebola virus in EDTA venous whole blood or buccal swabs. The assay targets viral nucleoprotein and glycoprotein genes of Ebola Zaire virus. The assay has a LoD of 82 RNA copies/reaction with turnaround time of 98 minutes. Simplexa™ Dengue RT-PCR assay developed by Focus Diagnostics (Cypress, CA, USA) is a commercial kit for detection and typing of dengue virus serotypes 1, 2, 3, and 4 in human serum. The assay targets four serotype specific regions, namely, dengue 1 (nonstructural (NS)5 gene ), dengue 2 (NS3 gene), dengue 3 (NS5 gene), and dengue 4 (capsid gene). The LoDs of the assay are 0.16 PFU/ml, 2.0 PFU/ml, 0.2 PFU/ml, and 0.2 PFU/ml for dengue 1, dengue 2, dengue 3, and dengue 4, respectively. Real-Star Zika Virus RT-PCR kit 1.0 is available, developed by Altona Diagnostics (Hamburg, Germany), for qualitative detection of ZIKV specific RNA in human serum or urine. The LoD of the assay is 0.61 RNA copies/μl. Abbott RealTime HCV quantitative assay developed by Abbott Laboratories (Rungis, France) is commercially available for HCV RNA quantitation in human serum and plasma. The target sequence for the assay is in the highly conserved 5′untranslated region (UTR) of the HCV genome. The LoD of the assay is 12 IU/ml when testing human plasma or serum. COBAS TaqMan HIV-1 test developed by Roche Diagnostics (Branchburg, USA) is commercially available for the quantitation of HIV-1 in human plasma. The real-time RT-PCR targets two highly conserved regions of the HIV-1 genome, namely, gag and long terminal repeat (LTR). The assay has LoD of 20 HIV-1 RNA copies/ml. Recently, several developers of diagnostic tests have developed real-time RT-PCR kits for COVID-19, and they are now seeking marking and emergency use authorization (EUA) from regulatory agencies. For example, Co-Diagnostics (Salt Lake City, USA) has developed real-time RT-PCR kit (Logix Smart COVID-19 test) for qualitative detection of nucleic acid from the SARS-CoV-2 in lower respiratory samples (e.g., bronchoalveolar lavage, sputum, and tracheal aspirate) and upper respiratory specimens (e.g., oropharyngeal swabs, nasal swabs, and nasopharyngeal swabs). The kit has received EUA from United States Food and Drug Administration (US FDA) and CE-IVD marking approval. The assay targets RdRp gene of SARS-CoV-2. The LoD of the assay is 9.35 × 10 3 RNA copies/ml with thermocycler run time of 63–90 minutes, depending on PCR equipment. US Centers for Disease Control and Prevention (CDC) has developed three real-time RT-PCR assays for the detection of SARS-CoV-2 genetic material in upper and lower respiratory specimens, and this panel has been granted EUA by the US FDA. CDC real-time RT-PCR panel targets 3 candidate regions of nucleocapsid (N) gene of SARS-CoV-2. The LoD of all assays are 5 RNA copies/reaction. The Agency for Science, Technology and Research (A∗STAR) and Tan Tock Seng Hospital (TTSH) (Singapore) have developed real-time RT-PCR test (Fortitude Kit 2.0) for qualitative detection of SARS-CoV-2 genetic material in oropharyngeal swabs. The kit has received Singapore Health Sciences Authority’s (HAS) provisional authorization for clinical use. The US FDA has not yet approved the kit for clinical use. The assay has LoD of 1000 RNA copies/ml in in oropharyngeal swabs. The kit developers have not yet disclosed the target gene for this assay. BGI Group (Beijing, China) has developed real-time fluorescent RT-PCR kit for the qualitative detection of nucleic acid from the SARS-CoV-2 in throat (oropharyngeal) swabs, nasal swabs, nasopharyngeal swabs, and other respiratory specimens. The company has received an EUA form the China’s National Medical Product Administration and the US FDA for its test kit. The assay targets open reading frame 1a and b (ORF1ab) genes of SARS-CoV-2. It has LoD of 150 copies/ml in throat samples with turnaround time of 4 hours. RADI COVID-19 Real-Time PCR kit developed by KH Medical (Korea) has CE-IVD marking and is used for qualitative detection of SARS-CoV-2 in human nasal swab or sputum sample. The assay targets two genes, namely, spike protein (S) gene and RdRP gene of SARS-CoV-2. It has LoD of 0.66 copies/μl with turnaround time of 80 minutes.

2.1.4. Transcription-Based Amplification Methods

Transcription-based amplification method includes nucleic acid sequence-based amplification (NASBA) and transcription-mediated amplification (TMA). NASBA and TMA are similar to each other. They are isothermal amplification methods. The entire amplification process is carried out at the temperature of 41°C. In both cases, the viral RNA target is first converted into cDNA with RT and then RNA polymerase synthesizes multiple copies of viral RNA product. The only difference between TMA and NASBA in the amplification process is two enzymes (RT and RNA polymerase) are utilized in case of TMA while NASBA utilizes three enzymes (avian myeloblastosis virus reverse transcriptase (AMV-RT), RNase H, and T7 RNA polymerase) [42, 51].

As depicted in Figure 3, in the NASBA process, three enzymes and two primers work together to exponentially amplify a target viral RNA. Primer 1 (P1) carries at its 5′ end T7 RNA polymerase promotor region and at its 3′ end, P1 carries sequence that is complementary to a target viral RNA sequence. Primer 2 (P2) carries a sequence complementary to cDNA strand. The amplification reaction begins with the production of cDNA copy of the viral RNA by RT using P1. RNase H degrades the viral RNA from RNA-DNA hybrid molecules. Then, RT synthesizes dsDNA molecules using P2 and the released DNA strand. Finally, T7 RNA polymerase uses dsDNA molecules as templates to transcribe many viral RNA copies. The above cycle is repeated several times, resulting in the accumulation of many viral RNA copies and ds DNA molecules. The amplified product can either be detected by gel electrophoresis at the end of the assay or in real time using molecular beacon [16, 42, 43, 71]. Transcription-based amplification methods have several advantages, for example, they do not require a thermal cycler, so developing countries and budget-restricted laboratories can afford to perform the assays, they have rapid kinetics (requires fewer cycles), and they produce a single-stranded RNA product that is suitable for detection by various techniques [42, 51, 71, 72]. Transcription-based amplification methods are suitable for the diagnosis of human viral infections caused by RNA viruses. They can amplify viral genomic RNA, messenger RNA, or ribosomal RNA [51, 71, 73]. Ayele et al. [44] developed NASBA assay that uses gag-based molecular beacons to distinguish between HIV-1 subtype C (C and C′) circulating in Ethiopia. The assay demonstrated high levels of sensitivity and specificity for both beacons (90.5% sensitivity and 100% specificity for the C beacon and 100% sensitivity and 95.2% specificity for the C′ beacon) by considering sequencing as gold standard for genotyping. Moore et al. [74] also used NSABA for the detection of influenza A H5N1 virus in clinical specimens with a LoD of 10 RNA copies/μl along with the same sensitivity as RT-PCR and average turnaround time of 4 hours. The NASBA assay was also used for the detection of dengue viral RNA with LoD of 1 PFU/ml for all of 4 dengue virus serotypes, no cross-reaction with JEV, and turnaround time of 3 hours [27]. Ender et al. [26] used TMA for screening of blood donations for HIV-1 and HCV RNA. The TMA assay had LoDs of 16.2 IU/ml for HIV-1 and 3.5 IU/ml for HCV. A multiplex NASBA assay was used for simultaneous detection of HIV-1 and HCV in plasma samples. The LoD of the assay for both HIV-1 and HCV was determined to be 1000 copies/ml and no cross-reactions with other selected viruses [45]. Swenson and his colleagues [75] used real-time TMA for the detection of HSV-1 and HSV-2 in lesion swab specimens with overall sensitivities of 98.2% and 99.4%, respectively, and specificity of 97.8% and 94.5%, respectively, compared to culture. In one study, real-time NASBA assay was more sensitive than the conventional RT-PCR in the detection of norovirus. In this study, RT-PCR detected 10 pg of standard viral RNA, while the real-time NASBA assay could detect 100 fg of standard viral RNA [76]. These data indicate that the assays are sensitive, specific, and cost-effective for the detection of human infection by RNA viruses.

TMA-based assays for the detection of HCV and HIV-1 are commercially available, developed by Hologic (San Diego, CA, USA). The Aptima HCV RNA qualitative assay is used for the detection of HCV RNA in human plasma or serum. The assay utilizes TMA to amplify conserved regions within the 5′-UTR of the HCV genome. The assay has LoD of 7.5 IU/ml with a specificity of 99.6%. NASBA-based kits for detection of HIV-1, CMV, enterovirus, and RSV are also commercially available, developed by bioMérieux Clinical Diagnostics. The NucliSens Easy Q RSV A and B assay is developed by bioMérieux (Marcy l’Etoile, France), and it is used for qualitative detection of RSV in respiratory samples of different types. The assay is based on real-time NASBA, and it targets F gene of RSV. Moore et al. [77] evaluated the performance of the commercial test kit using 508 respiratory specimens that were tested by direct immunofluorescence and culture. The assay was found to be more sensitive than culture and immunofluorescence assay. The sensitivity and specificity of the assay were determined to be 99% and 87%, respectively, compared to immunofluorescence assay with turnaround time of <4 hours.

2.1.5. Loop-Mediated Isothermal Amplification (LAMP)

LAMP is another isothermal nucleic acid amplification method that is extensively utilized for sensitive, specific, rapid, and cost-effective detection of both DNA and RNA viruses in human specimens. The method was first developed by Notomi et al. [78] and rapidly gained popularity in diagnostic virology. The method employs four to six unique primers and DNA polymerase with strand-displacement activity to amplify target DNA [78, 79]. The addition of RT in LAMP reaction (RT-LAMP) permits the amplification of RNA target [80]. Primer sets for LAMP initially reported by Notomi et al. [78] include the forward inner primer (FIP), backward inner primer (BIP), forward outer primer (F3), and backward outer primer (B3). The primers are specifically designed to recognize six precise regions from a targeted nucleic acid sequence. Nagamine et al. [79] later added two loop primers, namely, forward loop primer (LF) and backward loop primer (LB), to accelerate LAMP assay. Owing to the use of four to six specific primers, LAMP assay has outstanding sensitivity and specificity in the detection of target nucleic acids [79, 81]. A detailed description of the LAMP reaction mechanism is available in reviews by Becherer et al. [81], Tomita et al. [82], and Silva et al. [83], which use illustrations to explain the mechanism. The LAMP reaction is performed in constant temperature between 60–65°C, alleviating the need for expensive specialized equipment. The method requires only inexpensive heating block or water bath, making it very useful under poor laboratory settings [84]. The LAMP reaction takes turnaround time of <1 hour and the amplified product can be detected by several methods, including the real-time measurement of the turbidity caused by precipitated magnesium pyrophosphate using a turbidimeter, visual detection of magnesium pyrophosphate precipitation following completion of the reaction, detection of fluorescence under ultraviolet light or natural light by adding an intercalating fluorescent dye to the final reaction mixture, and visualization of the bands with various sizes using agarose gel electrophoresis [84–87].

LAMP assay has been successfully utilized to the rapid detection of a number of DNA viruses in human specimens, such as HSV-1 with LoD of 10 copies of HSV-1 DNA/μl and no cross-reactions with other selected viruses [85], hAdV40 and hAdV41 with LoD of between 50 and 100 copies of DNA/reaction, no cross-reactions with other selected viruses, and turnaround time of 60 minutes [88], EBV with sensitivity of 86.4%, specificity of 100%, compared to serological assay, and only 45 minutes of amplification of the target sequences [89], and CMV with LoD of 10 DNA copies/μl, no cross-reactivity with other viruses, and turnaround time of 1 hour after RNA extraction [90].

The utility of LAMP is expanded by merging it with reverse transcription (RT) into RT-LAMP to allow the rapid detection of RNA viruses in clinical specimens. Recently, for instance, Huang et al. [91] developed a rapid RT-LAMP assay for diagnosis of SARS-CoV-2 with LoD of 80 copies of viral RNA/ml in a sample within a 30 minutes reaction. This assay was validated by using 16 clinical samples (8 positives and 8 negatives) that were also tested by RT-qPCR. The testing results of the assay were consistent with RT-qPCR method, suggesting RT-LAMP assay can be used for rapid, simple, cost-effective, and sensitive detection of SARS-CoV-2 in respiratory samples. Similarly, Lu et al. [92] developed the RT-LAMP method for rapid detection of SARS-CoV-2 with LoD of 30 RNA copies/reaction and turnaround time of 40 minutes . Further, Baek et al. [93] developed a rapid RT-LAMP assay for early detection of SARS-CoV-2. The assay has LoD of 100 RNA copies/reaction, which is close to that of RT-qPCR with a rapid detection span of 30 minutes . RT-LAMP assay has also been developed to detect Middle East respiratory syndrome coronavirus (MERS-CoV) with LoD of 3.4 copies of MERS-CoV RNA/reaction along with the same sensitivity as MERS-CoV RT-qPCR, no cross-reaction to other respiratory viruses, and results available in <1 hour [87]. Kurosaki et al. [94] detected acute Ebola virus infection by RT-LAMP coupled with a portable device. The sensitivity and specificity of the assay was 100% each, compared to RT-qPCR and turnaround time of 35 minutes . In one study, RT-LAMP was more sensitive than conventional RT-PCR and NASBA [95]. The assay has been also developed for rapid detection of dengue virus [84], influenza A (H1N1) pdm09 virus [96], H5N1 avian influenza virus [97], HCV [98], HIV-1 [99], RSV [46], and ZIKV [100] in clinical samples. RT-LAMP-based commercial test kits are available for the detection of SARS-CoV-2 in respiratory specimens. The assay is developed by Color Genomics (USA), and it uses three SARS-CoV-2 specific primer sets targeting N gene , E gene , and ORF1a region, respectively, and a fourth control primer set targeting human ribonuclease P (RNaseP). It has LoD of 0.75 copies of viral RNA/μl with 70 minutes reaction. The assay received EUA from the US FDA in respiratory specimens. Abbott Diagnostic Scarborough, Inc. (USA) has also developed RT-LAMP-based test (ID NOW™ COVID-19 assay) for direct detection of SARS-CoV-2 in nasal, nasopharyngeal, or throat swabs. The kit has received EUA from US FDA. The assay targets RdRp gene of SARS-CoV-2. The LoD of the test is 125 genome equivalents (GE)/ml with positive results in <5 minutes and negative results in 13 minutes . LAMP primer sets such as the Loopamp primer set for avian flu H5 and H7 and FluA influenza are commercially available from Eiken Chemical Co., Ltd. (Japan).

2.2. DNA Microarrays

DNA microarray technologies have the capacity to identify medical viruses [101]. In DNA microarray diagnosis, fluorescently labeled viral nucleic acids in a test sample are used to screen an array of oligonucleotide probes immobilized on a solid surface (e.g., glass slide). The oligonucleotide probes used here are specific for the genome of the target virus. The results of hybridization between immobilized probes and target sequences labeled with fluorescent dyes are detected and quantified by fluorescence-based detection [16, 51, 72].

Extensive literature exists demonstrating the utility of DNA microarray for the detection of medical viruses in human specimens. Chiu et al. [102] used DNA microarray for high-throughput multiplex detection of viruses in nasopharyngeal aspirate samples originated from children infected with respiratory viruses. The assay demonstrated overall sensitivity of 87% to 90% and specificity of ≥99% in the detection of RSV, influenza A virus, and rhinovirus/enterovirus compared to RT-PCR. In one study, DNA microarray was utilized for simultaneous detection of HSV-1/2, VZV, EBV, CMV, human herpesvirus-6 types A and B (HHV-6 A/B), and adenovirus in clinical samples with LoD of 10 GE/reaction for each virus without cross-reactivity [103]. DNA microarray was also used to identify viral causes of meningitis and encephalitis with overall sensitivity of 93% and specificity of 100%, compared to single-virus PCR [104]. DNA microarray was also utilized for high-throughput multiplex detection of gastrointestinal viruses [105], viruses transmitted by small mammals and arthropods [106], herpesviruses, enteroviruses and flaviviruses [107], HIV-1, HIV-2, and hepatitis viruses [108] and dual infection with two dengue virus serotypes [109] in human specimens. DNA microarray was used to identify and genotype drug-resistant mutations of HIV [110, 111] to detect and genotype drug-resistant hepatitis B virus (HBV) mutations [112], to detect and genotype SARS coronavirus [113], and to detect and determine lineage of influenza B viruses [114]. During an outbreak of SARS in China in 2002, DNA microarray also served for the discovery of a new member of the coronavirus family [115].

DNA microarray technology is a high-throughput tool as it allows multiplex detection of a large number of potential viral pathogens in clinical specimens [102–105]. The technique does have a number of limitations nevertheless, including being too expensive to be used for routine clinical diagnosis, labor-intensive, and time-consuming (the hybridization process may take hours to days to complete). Nonspecific hybridization between test materials and immobilized probes can affect the sensitivity of the assay. In addition, designing of specific probes requires almost complete information of the genetic makeup the virus of interest. The assay detects only those viral pathogens that have target probes on the array [72, 116, 117].

2.3. Next-Generation Sequencing (NGS)

NGS finds itself very useful in diagnostic virology as it can directly analyze viral nucleic acid fragments extracted from clinical specimens [118, 119]. Generally, NGS involves preparation of test sample, sequencing of the target nucleic acid fragments using one of the available NGS platforms, and analysis of the sequence data using suitable bioinformatic tools [10, 120]. Several companies produce different NGS machines that use different methods of sequencing, reagents, and data analysis tools [119]. For example, pyrosequencing (Roche 454) detects release of pyrophosphate following incorporation of nucleotides in a DNA polymerization process. Illumina’s NGS platforms detect release of fluorescent labels from incorporated nucleotides in a DNA polymerization process. The emerging technologies like Oxford nanopore (MinIon) platform sequences the target nucleic acid by sensing the ionic current of DNA/RNA molecules that pass through the nanopores [10, 119, 120]. Despite its high sequence error rate (up to 38.2%) [121], MinION nanopore sequencer has merits over other NGS platforms. Firstly, it can generate longer read lengths (up to 882 kb) in real time [122], making it suitable for whole genome sequencing of viral pathogens with short turnaround time [123, 124]. Secondly, it is portable and no Internet is required for analysis, making it deployable in the field during outbreaks of viral infections [125]. Thirdly, it has low capital cost, making it affordable in low-income countries and budget-restricted laboratories across the globe [121].

NGS has been used in diagnostic virology for several applications. Recently, Kustin et al. [126] used NGS for rapid and robust identification of respiratory viruses in clinical samples. It was applied to track influenza A (H1N1) pdm09 virus [127]. Dessilly et al. [128] used NGS for the detection of HIV-1 drug resistance mutations. NGS was also conducted to discover a new Ebola virus [129].

Unlike PCR and DNA microarray methods, NGS does not require prior knowledge of genomic sequences of the viral pathogens. It does not also require target specific PCR primers and oligonucleotide probes [126, 130]. However, the use of NGS in clinical laboratories is limited because of the following reasons: the turnaround time, the number of samples per run, cost of sequencers, and requirement of skills in bioinformatics [10, 128, 131].

3. Immunological Diagnostic Techniques of Medical Viruses

The humoral branch of the immune system makes antibodies in response to viral infections. This natural response of the human body against viral infection is utilized for the development of immunological diagnostic methods. Several immunological diagnostic techniques are available for the detection human viral infections in clinical samples, including enzyme-linked immunosorbent assay, western blotting, immunofluorescence assay, and hemagglutination inhibition assay. The principles of these assays rely on the formation of antigen-antibody complex and consist of clinical specimens, whole virus or viral antigen, and an indicator [10, 19, 20].

3.1. Enzyme-Linked Immunosorbent Assay (ELISA)

In ELISA, enzyme conjugated antibody is utilized to detect the presence of specific antiviral antibody or viral antigen in human specimens. In positive sample, the reaction between an enzyme conjugated with an antibody and colorless chromogenic substrate leads to the formation of a colorful product. In the absence of antigen/antibody in the clinical specimen, no color is produced. The intensity of the color is directly proportional to the amount of antigen-antibody complex formed. The color change can be observed by the naked eye or read by a spectrophotometer, which can measure the absorbance. Several enzymes, including alkaline phosphatase, horseradish peroxidase (HRP), and β-galactosidase, have been used for ELISA. There are several variants of ELISA, but the two main types are antigen-capture ELISA (also called sandwich ELISA) and antibody-capture ELISA (also called indirect ELISA) [19, 132]. As illustrated in Figure 4, the first method detects viral antigen by immobilizing antibody specific for the viral protein of interest on a microtiter well [22] the second technique detects antiviral antibody in a patient sample by coating whole virus or viral protein on a microtiter well [133].

ELISA is sensitive and specific, easy to perform, and has a short turnaround time for results. Consequently, the assay has been developed and extensively utilized for the detection and serosurveillance of human viral pathogens. Recently, Adams et al. [134] developed antibody-capture ELISA for the detection of SARS-CoV-2 IgM or IgG in human plasma samples. The assay was tested by using 40 plasma samples from RT-PCR-confirmed SARS-CoV-2 infected patients and 50 plasma samples from healthy control. It demonstrated overall sensitivity of 85%, compared to RT-PCR and specificity of 100% in the detection of anti-SARS-CoV-2 IgG or IgM in plasma samples. This assay detected IgG levels in all of RT-PCR positive individuals from ≥10 days following symptoms onset with a sensitivity of 100%. Similarly, Colavita et al. [135] developed antibody-capture ELISA for the detection of anti-SARS-CoV-2 IgG, IgM, and IgA in serum samples. The assay was validated using 553 serum samples collected from suspected and confirmed SARS-CoV-2 infection cases, healthy donors, and patients positive for other infections or autoimmune conditions. The assay showed overall sensitivity of 91.7% and 97.9% for the detection of IgG and IgA in serum sample, respectively, and specificity of >96% for all antibody types, compared to IFA reference test. Chen et al. [22] also developed antigen-capture ELISA for the detection of MERS-CoV in clinical specimens. The assay demonstrated a LoD of <1 ng of MERS-CoV-recombinant nucleocapsid protein/ml and specificity of 100%. Antigen-capture ELISA was developed for rapid detection of dengue virus NS1 and differentiation of DENV serotypes in human specimens. The overall sensitivity and specificity of the test were 84.85% and 100%, respectively, compared to RT-qPCR, and the sensitivity rates for serotyping were 88.2%, 94.7%, 75%, and 66.6% for DENV serotype 1 (DENV1), DENV2, DENV3, and DENV4, respectively, with no cross-reactivity among serotypes [136]. ELISA was also employed for the detection of several other medical viruses, including Ebola virus [133], HSV-2 [137], SARS-CoV [138], hepatitis viruses [139], H5N1 influenza virus [140], and ZIKV [141].

Commercial antibody-capture ELISA-based test kit (Anti-ZIKV IgA, IgG or IgM ELISA) is available, developed by Euroimmun AG (Germany), for serodiagnosis of acute and past ZIKV infections. The assay uses ZIKV-specific NS1 recombinant antigen. The overall sensitivity and specificity of the assay are 100% and 94%, respectively, compared to RT-PCR. Creative Diagnostics (USA) also developed sandwich ELISA-based commercial kit (HIV 1 and 2 Ag/Ab ELISA kit) for qualitative determination of antigens or antibodies to HIV-1 and HIV-2 in human serum or plasma samples. The assay uses recombinant HIV antigens (HIV-1 glycoprotein (gp)41, gp120, and HIV-2 gp36) and anti-HIV viral gag protein p24 antibodies. The LoD of the assay for the detection of HIV p24 antigen is about 1pg/ml. Moreover, Bio-Rad (France) developed NS1 Ag capture ELISA-based commercial kit (Platelia Dengue NS1 Ag) for the qualitative or semiquantitative detection of dengue virus NS1 antigen in human serum or plasma samples. The assay employs anti-NS1 monoclonal antibody (Mab) as capture antibody and anti-NS1 Mab-HRP conjugate as detection antibody. The sensitivity rates of the assay related to virus serotype are 88.9%, 87.1%, 100%, and 93.3% for DENV1, DENV2, DENV3, and DENV4, respectively, compared to RT-PCR, and specificity of the assay is 100% for all serotypes. Recently, Euroimmun AG (Germany) has developed antibody-capture ELISA-based kit (Anti-SARS-Cov-2 ELISA IgG) for qualitative detection of IgG to SARS-CoV-2 in human serum or plasma samples. The assay uses recombinant S1 protein of SARS-CoV-2 as capture antigen. The assay has received EUA from US FDA for use in authorized laboratories. The estimated sensitivity and specificity of the assay are 90% and 100%, respectively, compared to nucleic acid amplification test. Epitope Diagnostics, Inc. (USA) has also developed two types of ELISA kits (COVID-19 IgG ELISA and COVID-19 IgM ELISA Kits) for the detection of anti-SARS-CoV-2 IgG and IgM in human serum samples, respectively. COVID-19 IgG ELISA kit uses SARS-CoV-2 recombinant antigen and HRP labeled anti-human IgG antibody. COVID-19 IgM ELISA employs anti-human IgM antibody and HRP labeled SARS-CoV-2 recombinant antigen. The assays have a LoD of 5IU/ml. The kits are approved by FDA for clinical and research use.

3.2. Western Blotting Analysis

Western blotting (also known as immunoblotting) assay detects viral proteins or antiviral antibodies. For detection of viral proteins, denatured whole viral proteins are first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Viral proteins are then electrotransferred onto nitrocellulose membrane. The membrane is then incubated with enzyme conjugated antibodies specific for the viral proteins. If the viral proteins are bound by enzyme labeled antibody, addition of a chromogenic substrate leads to the formation of colored bands at the sites of the viral antigens (Figure 5) [19, 132]. For detection of antiviral antibodies, viral specific denatured proteins are electrophoretically blotted onto nitrocellulose membrane after subjected to SDS-PAGE. The membrane is then incubated with patient serum. If the patient serum contains antibodies against the viral proteins, they will bind to their specific viral proteins. The addition of enzyme conjugated secondary anti-human antibody and a chromogenic substrate results in the production of colored bands at the locations of the viral proteins [142].

Immunoblotting has been used in clinical diagnosis for serosurveillance and as confirmatory tests for human viral infection. He et al. [143] developed western blot assay for detection of antibodies against SARS-CoV in human serum samples. The assay demonstrated a sensitivity of 98.3% and specificity of 90.9%, compared to IFA. Western blotting assay was also used for the detection of anti-Chikungunya virus antibody in human serum. Sensitivity of 83.3% and specificity of 96.7% were demonstrated by the assay using 30 sera from confirmed Chikungunya virus infected patient and 30 normal sera [144]. In one study, western blotting was a promising method for surveillance of HIV-1 infection in resource-limited regions [145]. The assay was also used for the detection and confirmation of HCV and HIV infections [146–148]. Western blotting assay is also commercially available. For example, J. Mitra and Co. Pvt. Ltd (Mumbai, India) developed commercial kit (HIV 1 and HIV 2 western blot) for the detection of antibodies to HIV-1 and HIV-2 in human serum or plasma samples. The assay uses preblotted nitrocellulose membrane strips with resolved HIV-1 viral lysate and HIV-2 antigen (gp36). The assay has 100% sensitivity and 100% specificity when compared with licensed western blot test. GS HIV-1 Western Blot kit for the detection of antibodies to HIV-1 in human serum, plasma, or dried blood spots is also available developed by Bio-Rad Laboratories (Redmond, USA). The assay uses preblotted nitrocellulose membrane strips with resolved HIV-1 viral proteins. The assay has 100% sensitivity and 87.2% specificity, compared to licensed HIV-1 western blot test.

3.3. Immunofluorescence Assay

Immunofluorescence assay is commonly conducted for the detection of viral antigens or antiviral antibodies in clinical samples. The assay is conducted in two formats: direct immunofluorescence assay (DFA) that detects viral antigens in patient sample [149] and indirect immunofluorescence assay (IFA) that detects antiviral antibody [150] or viral antigen [151] in clinical specimen. In the DFA, antibody that recognizes viral antigen is directly conjugated to fluorescent dye. In the IFA, viral antigen specific antibody is unlabeled and is detected with a second fluorescently labeled anti-human antibody (Figure 6). IFA is more sensitive than DFA because several fluorescently labeled anti-immunoglobulin antibodies bind to each antiviral antibody, increasing the intensity of fluorescence at the site of each antiviral antibody. The most widely used fluorescent dye in diagnostic virology is fluorescein isothiocyanate (FITC), which emits an intense yellow-green fluorescence, but rhodamine, which emits a deep red fluorescence, is also available. After staining, the specimen is examined under fluorescence microscope with a source of incident UV light [1, 16, 132].

IFA was used for the diagnosis of SARS. The assay showed 100% sensitivity and 100% specificity in the detection of anti-SARS-CoV IgG in human serum samples when compared to RT-PCR [150]. Madhusudana et al. [152] developed IFA for the detection of anti-rabies virus antibodies in human serum and CSF. When compared to the mouse neutralization test, the assay demonstrated a sensitivity of 97.2% and a specificity of 97.9%. IFA was also used for direct detection of HSV antigen in clinical specimens with sensitivity of 84.6% and specificity of 95.7%, compared to the tissue culture method [151]. Moreover, IFA was applied for subtyping of influenza A virus with 100% agreement to RT-PCR [153]. IFA was also used for the detection of EBV [21] and as a confirmatory test for HIV-1 [154]. Concerning DFA, in one study, it showed 60% sensitivity and 96% specificity in the detection of pandemic influenza A (H1N1) pdm09 in children when compared to RT-qPCR [149]. In another study, DFA showed high specificity (99–100%) in comparison to RT-qPCR for the detection of RSV in children [155]. IFA-based commercial test kit (Anti- ZIKV IIFT) is available, developed by Euroimmun AG (Germany), for the detection of ZIKV infection. The assay uses the complete ZIKV particles as antigen. Consequently, cross-reactivities with antibodies against viruses of the flavivirus family can occur. De Ory et al. [156] evaluated the performance of the assay using 126 positive and 102 negative samples. The assay showed 96.8% sensitivity and 72.5% specificity. OXOID Limited (UK) developed DFA-based kit (IMAGEN influenza virus A and B test) for the detection and differentiation of influenza A virus and influenza B virus in human specimens. The assay uses FITC labeled anti-influenza A virus or influenza B virus monoclonal antibodies. The assay has 100% sensitivity and 100% specificity, compared to the cell culture method.

3.4. Hemagglutination Inhibition (HI) Assay

Some viruses such as dengue virus, adenovirus, rubella virus, measles virus, and influenza virus have hemagglutinin antigen on their surfaces that binds and agglutinates RBCs termed hemagglutination (HA). The inhibition of the ability of the viruses to agglutinate RBCs is utilized for the development of HI assay. In the HI assay, serial dilutions of serum sample are prepared in a microtiter plate. Then, a specified amount of viral hemagglutinin is added. Finally, appropriate RBCs are added. The absence of HA indicates a positive reaction. This is judged by tilting the microtiter plate, which allows free RBCs to stream (Figure 7). The dilution rate where complete inhibition of agglutination of RBCs occurred is recorded. The HI titer, therefore, is the reciprocal of the last serum dilution which completely inhibits HA [10, 132, 157]. HI was utilized for a number of applications in diagnostic virology. The assay was used for serosurveillance of influenza A (H1N1) pdm09 virus [158] and measles virus [159]. In one study, HI assay was applied to assess the efficacy of pandemic influenza vaccine [160]. In a validation study using sera from 79 RT-qPCR-confirmed cases and 176 sera from a nonexposed population, HI assay showed high sensitivity (92%) and specificity (91%) for the detection of human infection with 2009 pandemic H1N1 virus [161].

Immunological diagnostic methods are widely employed in routine clinical diagnosis of human viral infections worldwide. The methods have several advantages, such as high sensitivity and specificity, relatively simple to conduct, rapidity, and possibility of testing several specimens simultaneously [10, 138]. However, immunological-based assays do have several limitations. The assays are subject to interferences. Interferences in immunoassays may result from the presence of (a) cross-reactive agents in the sample that carry similar or the same epitopes as the viral antigen of interest, leading to false-positive result [10, 162] (b) endogenous antibodies, like autoantibodies, heterophilic antibodies, or human anti-animal antibodies in the specimen. Despite the fact that viral antigen is not present in the sample, endogenous antibodies may interact with antiviral antibodies or detection antibodies, leading to false-positive result [162, 163]. The specificity of immunoassay may be affected when they are used in malaria-endemic areas. As it is known, Plasmodium induces nonspecific polyclonal B-cell activation that leads to generation of nonspecific antibodies [164]. These broad specific antibodies may react with a variety of antigens, leading to false-positive test. In one study, of 34 samples from PCR confirmed malaria patients, 14 samples were positive or borderline for anti-ZIKV antibodies in commercially available ZIKV ELISA test kit. When these 14 samples were tested using virus neutralization assay, ZIKV infection was not demonstrated in the 11 samples [165]. HI assay is laborious and time-consuming. The interpretation of the assay results between laboratories may be different as no standard reagents are available for the assay [153, 157, 166]. In case of IF assay, prolonged exposure of specimen to UV light leads to fading of fluorescence that could result in false-negative test [167]. Reagents and equipment that are used in some of the immunoassays are expensive [10, 11].

4. Status of Diagnostic Methods of Medical Viruses in Ethiopia

Most of viral diseases are endemic to Ethiopia [168]. Serosurveys have demonstrated the high prevalence rate of HBV [169, 170], HCV [170], HIV [171], and HSV-2 [137]. The population is vulnerable to rabies [172] and influenza [173]. Rotaviral diarrhea is the leading cause of morbidity and mortality in children [174]. Recently, like other nations in the globe, public health and economic sectors of Ethiopia are heavily challenged by COVID-19 pandemic.

Immunological methods, mostly commercial ELISA test kits [137, 169] and immunochromatographic test kits [170, 175], are used for the detection of viral infections in most clinical laboratories in the country. IFA technology is available only in Ethiopian Public Health Institute for the detection of rabies virus infection in suspected dogs that bit humans [176]. Conventional RT-PCR is used for the detection of influenza virus in human specimens in National Influenza Laboratory [173]. Recently, the RT-qPCR technique is widely used in several research institutes, universities, and clinical laboratories for the detection of SARS-CoV-2 in clinical samples. In general, few molecular techniques such as conventional PCR and RT-PCR are utilized in research institutes and universities for research purposes. Since most laboratories are budget-restricted and do not have trained laboratory personnel, molecular methods are not used for routine clinical diagnosis of human viral infections in the country. Nationwide use of RT-qPCR technologies for the diagnosis of COVID-19 and the experiences obtained will open the door to introduce molecular techniques for routine laboratory testing of other human viral infections.

5. Conclusion

The introduction of nucleic acid-based diagnostic tests into diagnostic virology has made tremendous improvement in the detection of human viral infections. Since nucleic acid-based diagnostic tests are highly sensitive and specific, they play a crucial role in the diagnosis and control of medical viruses. Molecular diagnostic methods diagnose viral infections by detecting viral RNA or DNA. Therefore, these techniques can pick infected individuals before antibody response is mounted against the virus in question. This is especially important in young, elderly, and immunosuppressed patients. However, they are beyond the reach of resource-limited nations due to their high cost, instrumentation complexity, and requirement for technical expertise. Immunoassays also play a significant role in the diagnosis and serosurveillance of viral infections worldwide. Although immunotechniques are easy to perform and inexpensive compared to molecular methods, they are not widely available in low-income countries. Consequently, scientists are working hard to develop inexpensive good quality tests in low-income nations. Moreover, most countries in the developing world are training their citizens abroad and inland at postgraduate level by opening relevant departments and institutes.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

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Copyright © 2020 Daniel Hussien Reta et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Comments:

  1. Donavan

    A completely coincidental coincidence

  2. Duzuru

    I do not know, I do not know

  3. Danaus

    I am ready to help you, ask questions. Together we can find a solution.



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