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Antibodies, part of the humoral immune response, are involved in pathogen detection and neutralization.
- Differentiate among affinity, avidity, and cross-reactivity in antibodies
- 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.
- avidity: the measure of the synergism of the strength individual interactions between proteins
- affinity: the attraction between an antibody and an antigen
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.
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.
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.
Functions of Antibodies
In the setting of infectious diseases, antibody function refers to the biological effect that antibody has on a pathogen or its toxin. Thus, assays that measure antibody function are differentiated from those that strictly measure the ability of an antibody to bind to its cognate antigen. Examples of antibody functions include neutralization of infectivity, phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), and complement-mediated lysis of pathogens or of infected cells.
Antibodies can impact pathogens in the presence or in the absence of effector cells or effector molecules such as complement, and experiments can often sort out with precision the mechanisms by which an antibody inhibits a pathogenin vitro. In addition, in vivo models, particularly those engineered to knock in or knock out effector cells or effector molecules are excellent tools for understanding antibody functions. However, it is highly likely that multiple antibody functions occur simultaneously or sequentially in the presence of an infecting organism in vivo.
The most critical incentive for measuring antibody functions is to provide a basis for vaccine development and for the development of therapeutic antibodies. In this respect, some functions, such as virus neutralization, serve to inhibit the acquisition of a pathogen or limit its pathogenesis. However, antibody can also enhance replication or contribute to pathogenesis. This chapter will emphasize those functions of antibody that are potentially beneficial to the host a separate chapter is devoted to a discussion of antibody-dependent enhancement of infection. In addition, this chapter will focus on the effects of antibodies on organisms themselves, rather than on the toxins the organisms may produce. Finally, the role of antibody in modulating T cell immunity is not discussed in detail.
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, as illustrated in Figure 2. An antibody with a higher affinity for a particular antigen would bind more strongly and stably, and thus would be expected to present a more challenging defense against the pathogen corresponding to the specific antigen.
Figure 2. (a) Affinity refers to the strength of single interaction 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. 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 describes when an antibody binds not to the antigen that elicited its synthesis and secretion, but to a different antigen.
Cross reactivity can be beneficial if an individual develops immunity to several related pathogens despite having only been exposed to or vaccinated against 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 and cause 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.
The biological function of antibodies
Antibody is an immunoglobulin produced by the body’s immune system and stimulated by antigen to proliferate and differentiate from B lymphocytes or memory cells and specifically bind to the corresponding antigen. So what are the major biological functions of antibodies?
1. Specific binding of the corresponding antigen
Antibody hypervariable region and antigenic determinants of the three-dimensional structure must be consistent in order to bind the antibody and the antigen binding is highly specific. Antibody molecules that specifically bind antigen can mediate a variety of physiological and pathological effects in vivo.
Antibody and antigen binding by non-covalent bond is reversible, and electrolyte concentration, PH, temperature and the integrity of the antibody structure can affect the ability of antibodies and antigen binding. The binding valence of IgG is bivalent the binding valence of IgM is theoretically deca-valent but is practically pentavalent due to steric hindrance and the dimeric IgA is tetravalent.
2. Activation of complement
When the IgG1, IgG2, IgG3 and IgM antibody molecules specifically bind to the corresponding antigen, their conformation changes. The complement of the complement binding site, CH2 of IgM or CH2 of IgG is bound to Clq and the complement system is activated by the traditional pathway. For IgG, at least two closely adjacent IgG molecules are needed to activate complement when they are bound to the corresponding antigen. Aggregates of other Ig molecules, such as IgG4 and IgA, activate complement by alternative pathways. Human natural anti-A and anti-B blood group antibody is IgM, and when blood group does not meet the blood transfusion, the antigen-antibody reaction activates complement hemolysis, causing rapid and serious transfusion reactions.
3. Binding Fc receptors
After binding the corresponding antigen through the V region, Ig can bind trough Fc segment to a variety of cell surface Fc receptors, and stimulate different effector functions.
3.1 Opsonization promotes phagocytosis
IgG molecules binds to bacteria and other particulate antigen, then pass through the Fc segment and mononuclear phagocytes and neutrophils corresponding receptors (FcγR), and thus promotes its phagocytosis called opsonization. Complement and antibody play the role of conditioning phagocytosis, known as the joint conditioning effect. Neutrophils, monocytes and macrophages have high affinity or low affinity for FcγRI (CD64) and FcrRII (CD32), and IgG, particularly human IgGl and IgG3 subclasses, plays major roles in opsonophagocytosis. Eosinophils have affinity FcyRII, and IgE and the corresponding antigen can promote phagocytosis of eosinophils.
3.2 Mediated allergic reactions
Fc fragments of IgE, upon binding to the corresponding receptors on the surface of mast cells and basophils (FcεR), sensitize these cells and under the action of allergens, degranulate these cells to release bioactive substances such as Histamine, bradykinin, causing local telangiectasia, increased permeability, stimulate type I hypersensitivity.
3.3 Antibody-dependent cellular cytotoxicity, ADCC effect
IgG binds to corresponding target cells, such as virus-infected cells and tumor cells, and exerts an ADCC effect by binding its Fc fragment to the corresponding receptor (FcγR) on NK cells. Mononuclear phagocytes and neutrophils, which have IgG Fc receptors on the surface, also produce ADCC effects on target cells that bind to IgG as described above.
4. Through the placenta
Among the five types of Ig, IgG is the only Ig that can be transferred from the mother to the fetus through the placenta, and the immunity obtained by the fetus in this manner is called natural passive immunity. Studies have shown that maternal IgG may be transported to the fetus by binding to the corresponding receptor on the surface of the placental trophoblast—FcγR.
5. Immune regulation
Antibodies have a positive and a negative regulatory effect on immune response, and through the unique and anti-unique type of network involve in the body’s immune regulation. The above briefly described the five biological functions of antibodies, which are a specific function with the antigen, activation of complement, binding of Fc receptors and transplacental and immunoregulation. Resulting from a single B cell clone, monoclonal antibody is highly uniform and only binds to specific antigenic epitopes and polyclonal antibodies are hybrid antibodies that stimulate various types of monoclonal antibodies produced by various epitopes. All of these antibodies have the basic biological function of antibodies and are widely used in many types of research and diagnosis.
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
Michelle M. Chang, Leonid Gaidukov, Wen Allen Tseng, Sepideh Dolatshahi, Douglas A. Lauffenburger, Timothy K. Lu & Ron Weiss
Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA, USA
Michelle M. Chang, Leonid Gaidukov, Giyoung Jung, Wen Allen Tseng, Jonathan L. Lyles, Sepideh Dolatshahi, Nevin M. Summers, Timothy K. Lu & Ron Weiss
Department of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA
Cell Line Development, Biotherapeutics Pharmaceutical Sciences, Pfizer Inc., Andover, MA, USA
Analytical Research and Development, Biotherapeutics Pharmaceutical Sciences, Pfizer Inc., Andover, MA, USA
Richard Cornell, Jeffrey K. Marshall, Paul Sakorafas, An-Hsiang Adam Chu, Kaffa Cote & Boriana Tzvetkova
Culture Process Development, Biotherapeutics Pharmaceutical Sciences, Pfizer Inc., Andover, MA, USA
The field of therapeutic antibodies has undergone rapid growth in recent years, becoming a dominant force in the therapeutics market. However, there is still significant growth potential for the therapeutic antibody field. Traditionally, antibodies have been used for the treatment of cancer, autoimmune diseases, and infectious diseases. If the molecular mechanisms of a specific disease can be clearly elucidated and the specific proteins or molecules involved in pathogenesis can be identified, antibodies may provide an effective therapeutic option. For example, anti-CGRP receptor antibodies (erenumab, galcanezumab, or fremanezumab) have been developed for the prevention of migraine. Anti-proprotein convertase subtilisin/kexin type 9 (PCSK9) antibodies (evolocumab or alirocumab) are used for the treatment of hypercholesterolemia. Anti-fibroblast growth factor 23 (FGF23) antibody (burosumab) is used to treat X-linked hypophosphatemia. Anti-IL6R antibody (sarilumab and tocilizumab) can be used for the treatment of rheumatoid arthritis. Anti-Factor IXa/Xa antibody (emicizumab) is a valuable treatment for hemophilia A. Anti-von Willebrand factor antibody (caplacizumab) is approved for the treatment of thrombotic thrombocytopenic purpura, and other antibodies will be approved for new indications in the near future.
Therapeutic antibodies can roughly be separated into two broad categories (Fig. 5). In the first category, the naked antibody is directly used for disease therapy. Cancer treatments from this category may act through several different mechanisms, including mediated pathways (e.g., ADCC/CDC), direct targeting of cancer cells to induce apoptosis, targeting the tumor microenvironment, or targeting immune checkpoints. In mediated pathways, the antibody kills cancer cells by recruiting natural killer cells or other immune cells. Recently, new technological developments have been made to enhance the therapeutic effects of ADCC or CDC, such as antibody Fc point mutations [251,252,253] or modification of glycosylation [254,255,256,257,258] to improve cancer cell killing capabilities. The direct induction apoptosis in cancer cells has traditionally been the preferred mechanism for therapeutic antibodies. With regard to targeting the tumor microenvironment, antibodies can inhibit tumorigenesis by targeting factors involved in cancer cell growth. For example, Avastin targets vascular endothelial growth factor (VEGF) to inhibit blood vessel growth around the tumor, shutting down the supply of nutrients required for the cancer cell growth. Immune checkpoints have proven to be valuable targets for cancer treatment. In the future, studies evaluating synergistic effects of antibodies and chemotherapeutic drugs, radiotherapy or other biologic agents will greatly benefit the further development of antibody therapeutics. Furthermore, the identification of novel biomarkers may improve the efficacy and specificity of antibody-based therapy for human diseases.
Schematic overview showing the development of antibody-based therapeutics for the treatment of cancer. Therapeutic antibodies can be roughly separated into two broad categories. The first category involves the direct use of the naked antibody for disease therapy. Antibodies in this category are used for cancer treatment and elicit cell death by different mechanisms, including ADCC/CDC, direct targeting of cancer cells to induce apoptosis, targeting the tumor microenvironment, or targeting immune checkpoints. For antibodies in the second category, additional engineering is performed to enhance their therapeutic efficacy. Some general approaches for the use of these antibodies include immunocytokine, antibody-drug conjugate (ADC), antibody-radionuclide conjugate (ARC), bispecific antibody, immunoliposome, and CAR-T
In the second category of antibody drugs, additional modifications are made to the antibody in order to enhance its therapeutic value. Some general approaches include immunocytokines, antibody-drug conjugate, antibody-radionuclide conjugates, bispecific antibodies, immunoliposomes, and chimeric antigen receptor T cell (CAR-T) therapy. To create an immunocytokine, a selected cytokine is fused to an antibody to enhance delivery specificity . Antibody drug conjugates consist of an antibody that targets a cancer-specific marker conjugated to the small molecule drug the antibody enhances delivery to the tumor site, increasing the efficacy of the small molecule while reducing side effects by reducing non-specific toxicity to non-target tissues . The antibody may also be conjugated to a radionuclide, in order to direct radiotherapy more specifically to the tumor site . For bispecific antibodies, antibodies targeting two receptors are engineered to further enhance therapeutic effects . Antibody-engaged effector cell functions may enhance the therapeutic efficacy of bispecific antibodies. With regard to immunoliposomes, the binding site of the antibody (scFv or Fab) is cleaved from the constant region and subsequently conjugated to different nano-drug delivery systems, such as liposomal drugs, to provide more specific targeting [263, 264]. Lastly, CAR-T involves inserting the gene for a chimeric T cell receptor-antibody targeting a specific cancer marker into T cells, such that the engineered cells target and kill cancer cells [265, 266]. In recent years, this approach has garnered major attention from the scientific and medical community due to its significant clinical benefits to cancer patients. In many cases, patients have experienced complete remission or even been completely cured of cancer [267,268,269,270,271].
Although new methods have been well-established for generating fully human antibodies, such as human antibody transgenic mice and human single B cell antibody techniques, phage display still has advantages as an antibody drug discovery platform, based on its efficient and economical in vitro selection methodology. Recently, some advanced techniques have been applied in antibody discovery, including high-throughput robotic screening , next generation sequencing  and single cell sequencing [274, 275]. These techniques are expected to greatly accelerate the identification of specific phage binders, facilitating mAb development for use in research, clinical diagnostics, and pharmaceuticals for the treatment of human disease.
By reviewing currently approved mAbs, one may easily see how sophisticated formats were developed in response to challenges posed by therapeutic indications. These mAb engineering solutions are highlighted by antibody-drug conjugates, glycoengineered mAbs, immunomodulators, bispecific mAbs, and CAR-T cells.
The function of immunoglobulin A in immunity
The vast surfaces of the gastrointestinal, respiratory, and genitourinary tracts represent major sites of potential attack by invading micro-organisms. Immunoglobulin A (IgA), as the principal antibody class in the secretions that bathe these mucosal surfaces, acts as an important first line of defence. IgA, also an important serum immunoglobulin, mediates a variety of protective functions through interaction with specific receptors and immune mediators. The importance of such protection is underlined by the fact that certain pathogens have evolved mechanisms to compromise IgA-mediated defence, providing an opportunity for more effective invasion. IgA function may also be perturbed in certain disease states, some of which are characterized by deposition of IgA in specific tissues. This review details current understanding of the roles played by IgA in both health and disease.
Copyright 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Rare Detection of Antiviral Functions of Polyclonal IgA Isolated from Plasma and Breast Milk Compartments in Women Chronically Infected with HIV-1
The humoral response to invading mucosal pathogens comprises multiple antibody isotypes derived from systemic and mucosal compartments. To understand the contribution of each antibody isotype/source to the mucosal humoral response, parallel investigation of the specificities and functions of antibodies within and across isotypes and compartments is required. The role of IgA against HIV-1 is complex, with studies supporting a protective role as well as a role for serum IgA in blocking effector functions. Thus, we explored the fine specificity and function of IgA in both plasma and mucosal secretions important to infant HIV-1 infection, i.e., breast milk. IgA and IgG were isolated from milk and plasma from 20 HIV-1-infected lactating Malawian women. HIV-1 binding specificities, neutralization potency, inhibition of virus-epithelial cell binding, and antibody-mediated phagocytosis were measured. Fine-specificity mapping showed IgA and IgG responses to multiple HIV-1 Env epitopes, including conformational V1/V2 and linear V2, V3, and constant region 5 (C5). Env IgA was heterogeneous between the milk and systemic compartments (Env IgA, τ = 0.00 to 0.63, P = 0.0046 to 1.00). Furthermore, IgA and IgG appeared compartmentalized as there was a lack of correlation between the specificities of Env-specific IgA and IgG (in milk, τ = -0.07 to 0.26, P = 0.35 to 0.83). IgA and IgG also differed in functions: while neutralization and phagocytosis were consistently mediated by milk and plasma IgG, they were rarely detected in IgA from both milk and plasma. Understanding the ontogeny of the divergent IgG and IgA antigen specificity repertoires and their effects on antibody function will inform vaccination approaches targeted toward mucosal pathogens.IMPORTANCE Antibodies within the mucosa are part of the first line of defense against mucosal pathogens. Evaluating mucosal antibody isotypes, specificities, and antiviral functions in relationship to the systemic antibody profile can provide insights into whether the antibody response is coordinated in response to mucosal pathogens. In a natural immunity cohort of HIV-infected lactating women, we mapped the fine specificity and function of IgA in breast milk and plasma and compared these with the autologous IgG responses. Antigen specificities and functions differed between IgG and IgA, with antiviral functions (neutralization and phagocytosis) predominantly mediated by the IgG fraction in both milk and plasma. Furthermore, the specificity of milk IgA differed from that of systemic IgA. Our data suggest that milk IgA and systemic IgA should be separately examined as potential correlates of risk. Preventive vaccines may need to employ different strategies to elicit functional antiviral immunity by both antibody isotypes in the mucosa.
Keywords: HIV-1 IgA effector functions mucosal immunity.
Copyright © 2019 Tay et al.
Env antigen specificities of milk…
Env antigen specificities of milk IgG and IgA and plasma and milk IgAs…
Heterogeneity of the Env antigen…
Heterogeneity of the Env antigen specificities of breast milk (BM) and plasma (PLA)…
Env antigen-specific IgA and IgG…
Env antigen-specific IgA and IgG responses in milk and plasma seldom correlate with…
Milk and plasma IgG, but not IgA, mediate tier 1 HIV MW965 neutralization.…
IgA-mediated tier 1 HIV-1 neutralization…
IgA-mediated tier 1 HIV-1 neutralization is not enhanced in FcαRI-expressing cells or peripheral…
Breast milk and plasma IgAs…
Breast milk and plasma IgAs may inhibit C.1086 HIV-1 virion binding to epithelial…
Breast milk and plasma IgGs,…
Breast milk and plasma IgGs, but not IgAs, mediate phagocytosis of HIV-1 virions…
Lack of detectable milk IgA-mediated…
Lack of detectable milk IgA-mediated phagocytosis despite increasing IgA concentration. To determine if…
Functional IgG activity in milk…
Functional IgG activity in milk and plasma are correlated and linked to antibodies…
Correlations between antibody functions and…
Correlations between antibody functions and epitope specificity in plasma IgG. (A) To determine…
Virion phagocytosis is not dependent…
Virion phagocytosis is not dependent on spinoculation and requires specific Fc-FcR interaction. (A)…
Immunocytochemistry differs from immunohistochemistry  in that the former is performed on samples of intact cells that have had most, if not all, of their surrounding extracellular matrix removed. [ citation needed ] This includes individual cells that have been isolated from a block of solid tissue, cells grown within a culture, cells deposited from suspension, or cells taken from a smear. In contrast, immunohistochemical samples are sections of biological tissue, where each cell is surrounded by tissue architecture and other cells normally found in the intact tissue. Immunocytochemistry is a technique used to assess the presence of a specific protein or antigen in cells (cultured cells, cell suspensions) by use of a specific antibody, which binds to it, thereby allowing visualization and examination under a microscope. It is a valuable tool for the determination of cellular contents from individual cells. Samples that can be analyzed include blood smears, aspirates, swabs, cultured cells, and cell suspensions.
There are many ways to prepare cell samples for immunocytochemical analysis. Each method has its own strengths and unique characteristics so the right method can be chosen for the desired sample and outcome.
Cells to be stained can be attached to a solid support to allow easy handling in subsequent procedures. This can be achieved by several methods: adherent cells may be grown on microscope slides, coverslips, or an optically suitable plastic support. Suspension cells can be centrifuged onto glass slides (cytospin), bound to solid support using chemical linkers, or in some cases handled in suspension.
Concentrated cellular suspensions that exist in a low-viscosity medium make good candidates for smear preparations. Dilute cell suspensions existing in a dilute medium are best suited for the preparation of cytospins through cytocentrifugation. Cell suspensions that exist in a high-viscosity medium, are best suited to be tested as swab preparations. The constant among these preparations is that the whole cell is present on the slide surface. For any intercellular reaction to take place, immunoglobulin must first traverse the cell membrane that is intact in these preparations. Reactions taking place in the nucleus can be more difficult, and the extracellular fluids can create unique obstacles in the performance of immunocytochemistry. In this situation, permeabilizing cells using detergent (Triton X-100 or Tween-20) or choosing organic fixatives (acetone, methanol, or ethanol) becomes necessary.
Antibodies are an important tool for demonstrating both the presence and the subcellular localization of an antigen. Cell staining is a very versatile technique and, if the antigen is highly localized, can detect as few as a thousand antigen molecules in a cell. In some circumstances, cell staining may also be used to determine the approximate concentration of an antigen, especially by an image analyzer.
There are many methods to obtain immunological detection on tissues, including those tied directly to primary antibodies or antisera. A direct method involves the use of a detectable tag (e.g., fluorescent molecule, gold particles, etc., ) directly to the antibody  that is then allowed to bind to the antigen (e.g., protein) in a cell.
Alternatively, there are many indirect methods. In one such method, the antigen is bound by a primary antibody which is then amplified by use of a secondary antibody which binds to the primary antibody. Next, a tertiary reagent containing an enzymatic moiety is applied and binds to the secondary antibody. When the quaternary reagent, or substrate, is applied, the enzymatic end of the tertiary reagent converts the substrate into a pigment reaction product, which produces a color (many colors are possible brown, black, red, etc.,) in the same location that the original primary antibody recognized that antigen of interest.
Some examples of substrates used (also known as chromogens) are AEC (3-Amino-9-EthylCarbazole), or DAB (3,3'-Diaminobenzidine). Use of one of these reagents after exposure to the necessary enzyme (e.g., horseradish peroxidase conjugated to an antibody reagent) produces a positive immunoreaction product. Immunocytochemical visualization of specific antigens of interest can be used when a less specific stain like H&E (Hematoxylin and Eosin) cannot be used for a diagnosis to be made or to provide additional predictive information regarding treatment (in some cancers, for example).
Alternatively the secondary antibody may be covalently linked to a fluorophore (FITC and Rhodamine are the most common) which is detected in a fluorescence or confocal microscope. The location of fluorescence will vary according to the target molecule, external for membrane proteins, and internal for cytoplasmic proteins. In this way immunofluorescence is a powerful technique when combined with confocal microscopy for studying the location of proteins and dynamic processes (exocytosis, endocytosis, etc.).
In addition to antibody isotypes and subtypes, allelic variation is found among the antibody subtypes. These polymorphic epitopes of immunoglobulins that can differ between individuals and ethnic groups are known as allotypes. Exposure of an individual to a non-self allotype can induce an anti-allotype response (1, 2). However, not all variations are immunogenic because this sequence may be found in other isotypes or subtypes and so these are known as isoallotypic variants. In fact, a recent study suggests that allotypic differences in human IgG1 do not represent a significant risk for induction of immunogenicity (3) and to date little evidence has been found for significant anti-allotype responses to therapeutic antibodies, e.g. adalimumab (4) or infliximab (5).
Allotpyes have been identified on the g1, g3 and a2 heavy chains (designated G1m, G3m and A2m allotypes respectively) and on the kappa light chain (Km allotypes). Although variants of g2 and g4 exist these are isoallotypic as the amino acids present are also found in other subclasses.
The remainder of this page will focus on G1m allotypes. See IMGT for further information on G3m, A2M or Km allotypes.
G1m17 and G1m3
G1m17, also known as G1m(z), corresponds to Lys (K) at position 214 in the CH1 domain (EU numbering).
G1m3, also known as G1m(f), corresponds to Arg (R) at position 214 in the CH1 domain.
G1m1 and nG1m1
G1m1, also known as G1m(a), corresponds to Asp (D) and Leu (L) at positions 356 and 358 in the CH3 domain (EU numbering).
nG1m1, also known as nG1m(a) corresponds to Glu (E) and Met (M) at positions 356 and 358.
G1m2 and nG1m2
G1m2, also known as G1m(x), corresponds to Gly (G) at position 431 in the CH3 domain (EU numbering).
nG1m2, also known as nG1m(x), corresponds to Ala (A) at position 431 in the CH3 domain.
Figure. Sequence alignment of human G1m allotypes.
The main allelic forms for IgG1 are G1m (z,a), G1m (f), and G1m (f,a) (6,7). The G1m (f) allele is only found in Caucasians, whereas the G1m (f,a) allele is common in Orientals, but other variants, G1m (z,a,x) and G1m (z,a,v), have also been described (8,9).