- Antigen–antibody interactions are referred to as antigen–antibody responses.
- The responses are extremely specific, and an antigen will only react with antibodies made from its own antigen or antigens that are closely related.
- Since these reactions are extremely specific, they have been utilised in numerous in vitro diagnostic procedures for the detection of antigen or antibody.
- Antigen and antibody responses are also the cornerstone of in vivo immunity against microbial infections. It may induce tissue damage in the host during hypersensitivity reactions and autoimmune disorders.
- It is the fundamental biological mechanism by which the body defends itself against invading particles such as viruses and their harmful compounds.
- Antibodies create an antigen-antibody complex in the blood when they strongly and precisely bind to antigens.
- Subsequently, the immune complex is transmitted to cellular systems where it can be removed or inactivated.
What is Antigen (Ag)?
(Anti = opposite; gen = anything that causes)
- Immunogens are any foreign chemicals that, once entering the human body, typically trigger a cascade of immunological responses.
- While others, known as haptens, require the aid of additional molecules (carrier proteins) to elicit an immune response.
- All immunogens and haptens are classified as antigens.
- It is possible that they be polysaccharides, lipids, proteins, or peptides.
- An epitope is a site where antibodies bind.
What is Antibody (Ab)?
Antibodies are immune system components produced in response to antigens. Consequently, antigens induce the formation of antibodies. Together, they produce an immune reaction. The following are the general properties of an antibody:
- Antibody is also referred to as immunoglobulin (Ig).
- They are formed like an inverted Y.
- Generated by B-cells in plasma.
- The name of the antigen binding site is paratope.
- There are five types of immunoglobulins: IgG, IgA, IgM, IgE, and IgD.
Features and Strength of Antigen–Antibody Interaction/Reaction
Similar to how proteins bind to their biological receptors or enzymes bind to their substrates, antigen and antibody bind through noncovalent linkages. In contrast, antigen–antibody reactions do not result in irreversible chemical changes in either participant, i.e. the antigen or the antibody. The interaction between antigen and antibody is reversible and can be blocked or disrupted by a strong ionic force or an extreme pH. The following are some general characteristics of these interactions:
- Antigen–antibody interactions involve electrostatic connections, hydrogen bonds, van der Waals bonds, and hydrophobic interactions as intermolecular forces.
- All of these intermolecular pressures are dependent upon the close proximity of antigen and antibody molecules. Determining the stability of the antigen–antibody reaction, therefore, is the “excellent match” between an antigenic determinant and an antibody-combining site.
- Multiple interactions between the antigen and antibody guarantee that the antigen is securely bound to the antibodies.
- Affinity denotes the intensity of antigen and antibody attraction.
- Low-affinity antibodies bind antigen poorly and tend to dissociate quickly, whereas high-affinity antibodies bind antigen more strongly and remain bound for a longer period of time.
- It is hypothesised that a highly close match between the antigen-binding sites and the corresponding antigenic determinants favours the formation of strong noncovalent interactions between antigen and antibody, resulting in high-affinity binding.
Affinity quantifies the strength of contact between an epitope and the antigen-binding site of an antibody. It is defined by the same fundamental thermodynamic rules that govern any biomolecular interaction that is reversible:
- KA = affinity constant
- [Ab] = molar concentration of unoccupied binding sites on the antibody
- [Ag] = molar concentration of unoccupied binding sites on the antigen
- [Ab-Ag] = molar concentration of the antibody-antigen complex.
In other words, KA specifies the amount of antibody-antigen complex that occurs at equilibrium. The time required for this to occur is proportional to the diffusion rate and is the same for every antibody. However, high-affinity antibodies bind more antigen in less time than low-affinity antibodies. Therefore, KA can range from below 105 mol-1 to above 1012 mol-1 for antibodies, and can be affected by variables such as pH, temperature, and buffer composition.
- The overall strength of non-covalent interactions between a single Ag- binding site on an antibody and a single epitope is the affinity of the antibody for that epitope.
- Low affinity Ab: poorly bind Ag and rapidly dissociate.
- High affinity Ab: Bind Ag tightly and remain bound for an extended period of time.
- Avidity is a measure of the overall binding strength of multiple antigenic determinants and multivalent antibodies to an antigen.
- In actual biological systems, avidity is a better predictor of the strength of interactions than affinity.
- Consequently, the avidity of an antigen–antibody response depends on the valencies of both antigens and antibodies and is greater than the sum of the individual affinities.
- Avidity is influenced by three variables;
- The binding affinity: The binding affinity is the strength of the connection between two molecules at a single binding site.
- The valency: The total number of involved binding sites.
- The structural arrangement: the antigen and antibody structures involved.
- All antibodies are multivalent e.g. IgMs are decavalent while IgGs are bivalent. The greater the valency (number of antigen binding sites) of an immunoglobulin, the more antigen it can bind. Antigens can also exhibit multivalency since they can bind to several antibodies. The stability of an antibody and antigen is facilitated by their multimeric interactions.
- Additionally, a favourable structural arrangement between the antibody and antigen can result in a more stable antibody-antigen complex.
- Avidity is the strength of numerous interactions between multivalent Ab and Ag. Avidity is a more accurate indicator of the binding potential of an antibody than affinity. A high avidity might make up for a poor affinity.
- Specificity refers to the ability of an antibody-combining site to react with a single antigenic determinant or the ability of a population of antibody molecules to respond with a single antigen.
- Typically, antigen–antibody responses have a high degree of specificity.
- Despite this, cross-reactions between antigens and antibodies sometimes occur and are occasionally responsible for producing diseases in hosts and generating erroneous diagnostic test findings.
- Antibodies can recognise specific distinctions in:
- primary structure of an antigen.
- isomeric forms of an antigen.
- secondary and tertiary structure of an antigen.
- Although antigen–antibody responses are very specific, in some instances an antibody produced by an unrelated antigen might cross-react.
- This occurs when two distinct antigens share an identical or highly similar epitope.
- In the latter instance, the affinity of the antibody for the cross-reacting epitope is typically lower than its affinity for the original epitope.
- Antisera containing polyclonal antibodies frequently cross-react with immunogens somewhat related to those used for immunisation, as a result of the existence of common epitopes or epitopes with comparable conformations.
Properties of Antigen-Antibody Reaction in Brief
- Exceptionally particular reaction.
- Manifests in a notable manner.
- Non-covalent processes (Ionic bonds, Van der Waals forces, Hydrophobic interactions, Hydrogen bonds).
- Neither antibodies nor antigens are denatured.
- This refers to the degree to which an antigen binds to a certain antigen-binding site on an antibody.
- The concept of avidity is more comprehensive than that of affinity. It represents the entire strength of the Ag-Ab combination. It depends on: the affinity of the antibody, Antibody and antigen valencies (the number of binding sites), How epitopes and paratopes are ordered structurally.
- This word refers to the ability of an antibody to bind to comparable epitopes on other antigens.
Antigen-Antibody Interaction/Reaction Stages
There are two stages to the antigen–antibody reaction: primary and secondary.
1. Primary Stage
- The initial contact between antigen and antibody is known as the primary stage. It is quick and reversible, but its consequences are invisible.
- The ionic bonds, hydrogen bonds, van der Waals forces, and hydrophobic interactions are the weaker intermolecular forces that bind antigen and antibodies together in this primary stage.
- However, covalent binding, a stronger intermolecular force between antigen and antibody, does not occur at this stage.
2. Secondary Stage
- The secondary stage involves an irreversible interaction between antigen and antibody, accompanied by observable results such as agglutination, precipitation, neutralisation, complement fixation, and immobilisation of motile organisms.
- Covalent binding occurs between the antigen and antibody during this step.
- A single antibody is capable of producing a variety of antigen–antibody interactions, and a single antigen is capable of stimulating the creation of immunoglobulin classes with distinct biological features.
- Generally, titers are used to express the outcomes of agglutination, precipitation, neutralisation, and other tests.
- Titer is defined as the highest serum dilution that produces a positive test result. Higher titer indicates a higher concentration of antibodies in serum. A serum with a titer of 1/128 has more antibodies than one with a titer of 1/8, for example.
Factors Affecting Antigen-Antibody Interaction/Reaction
Numerous variables can affect the antigen-antibody reaction. Some of the most prevalent factors include:
- The optimal temperature for antigen-antibody reactions will rely on the chemical nature of the epitope and paratope, as well as the types of bonds involved in their interaction. For instance, hydrogen bond creation is typically exothermic.
- These linkages are more stable at lower temperatures and may be of more importance when dealing with antigens containing carbohydrates.
- The pH range between 6.5 and 8.4 influences the equilibrium constant of the antigen-antibody complex. Below pH 6.5 and above pH 8.4, the antigen-antibody interaction is hindered to a great extent.
- At pH 5.0 or 9.5, the equilibrium constant is one hundred times smaller than between pH 6.5 and pH 7.
- Under conditions of severe pH, antibodies may undergo conformational changes that degrade their compatibility with antigen.
3. Ionic Strength
- In blood group serology, the effect of ionic strength on antigen-antibody reactivity is very significant. Here, sodium and chloride ions greatly influence the process. In normal saline solution, for instance, Na+ and Cl cluster around the complex and partially neutralise charges, potentially interfering with the binding of antibody to antigen.
- This could be troublesome when using antibodies with low affinity.
- It is widely known that when -globulins are subjected to extremely low ionic strengths, they aggregate and form reversible complexes with red blood cell lipoproteins, resulting in their sedimentation.
4. Enzyme treatment
- Numerous proteolytic enzymes are known to promote the antigen-antibody interaction, although papain, ficin, and bromelin are the most common.
- Enzymatic pretreatment nearly increases the quantity of anti-D attached to red cells that are positive for D.
5. Polymers and other potentiators
- The majority of chemical compounds used to enhance the detection of red cell antibodies do not affect the first stage of agglutination, the antigen-antibody response, but rather the second, agglutination proper.
- Examples of typical polycations are polybrene, protamine, and methylcellulose. The exceptions are albumin and polyethylene glycol (PEG).
6. Concentrations of antigen and antibody
- Antigen and antibody concentrations that are increased boost the reaction.
7. Serum/cell ratio
- The serum/cell ratio is increased in order to improve the sensitivity of the antiglobulin test.
8. No. of antigen-binding sites
- More antigen-binding sites on the antibody increases the likelihood of contact.
- IgM, which is a pentamer and hence has 10 binding sites, will bind to antigens more efficiently than IgG, which is a monomer and therefore has just 2 binding sites.
9. Duration of incubation
- Allow the antigen-antibody response to reach equilibrium for optimal sensitivity. At 37 °C20 and normal ionic strength, this process can take up to four hours.
10. Structural arrangement
- If the structure of the epitope and paratope is such that they might function as a lock and key, then the interaction between the antigen and antibody is enhanced.
Chemical Bonds Responsible for the Interaction/Reaction
Similar to how proteins bind to their cellular receptors or how enzymes bind to their substrates, the interaction between the Ab-binding site and the epitope consists only of noncovalent bonds. High ionic strength or excessive pH can prevent or disrupt the binding. Following intermolecular forces contribute to Ag–Ab binding:
- Electrostatic bonds: These are the result of the attraction between the oppositely charged ionic groups of two protein side chains, such as an ionised amino group (NH4 +) on a lysine residue in the Ab and an ionised carboxyl group (COO_) on an aspartate residue in the Ag.
- Hydrogen bonding: When Ag and Ab are in close proximity, weak hydrogen bonds can form between hydrophilic groups (such as OH and C=O, NH and C=O, and NH and OH).
- Hydrophobic interactions: Hydrophobic groups, such as the side chains of valine, leucine, and phenylalanine, tend to combine and agglomerate in an aqueous environment, preventing water molecules from their surroundings due to Van der Waals interactions. As a result, the space between them reduces, heightening the attraction forces at play. It is estimated that this type of interaction contributes up to fifty percent of the total binding strength between Ag and Ab.
- Van der Waals bonds: These forces depend on interactions between the “electron clouds” that surround the Ag and Ab molecules. The contact has been compared to that which might exist between alternating dipoles in two molecules, alternating in such a way that, at any given time, oppositely orientated dipoles are present in closely adjacent regions of the Ag and Ab molecules.
Each of these non-covalent contacts operates over a very short distance (usually less than 1 Å), hence Ag-Ab interactions rely on an extremely close match between antigen and antibody.
Types of Antigen-Antibody Interaction/Reaction
Serological tests are commonly used to identify serum antibodies or antigens in order to diagnose a vast array of infectious disorders. These serological tests are also utilised for the identification of autoimmune illnesses as well as the typing of blood and tissues prior to transplantation. Here are some instances of antigen–antibody reactions:
- complement-dependent serological tests
- neutralization test
- enzyme immunoassay
- Western blotting
- chemiluminescence assay
- immunoelectronmicroscopic tests.
1. Precipitation Reaction
- It is a type of antigen–antibody response wherein the antigen is soluble.
- It is a test in which antibody reacts with soluble antigen in the presence of electrolyte at a predetermined pH and temperature to form a precipitate. Antigens and antibodies form a lattice; in certain instances, it is apparent as an insoluble precipitate.
- Precipitins are antibodies that aggregate soluble antigens. When precipitate remains suspended as floccules rather than settling, the process is known as flocculation.
- Formation of an antigen–antibody lattice is dependent on both the valency of the antigen and the antibody.
- The antibody must be bivalent; monovalent Fab fragments will not create a precipitate.
- The antigen must be either bivalent or polyvalent, with at least two copies of the same epitope or various epitopes that respond with different antibodies present in polyclonal antisera.
Types of precipitation reactions
Precipitation reactions often fall into three categories:
- Precipitation in solution: Precipitation in solution is illustrated by the ring and flocculation tests.
- Agar precipitation: The agar gel precipitation test is known as the immunodiffusion test. In this test, reactants are introduced to the gel, and the antigen–antibody combination takes place through diffusion. The rate of diffusion is influenced by the particle size, temperature, gel viscosity, quantity of hydration, and matrix-reactant interactions. Immunodiffusion reactions are characterised according to (a) the number of diffusing reactants and (b) the direction of diffusion:
- Single diffusion in one dimension
- Single diffusion in two dimensions
- Double diffusion in two dimension
- Double diffusion in one dimension
- Precipitation in agar with an electric field: Examples of Precipitation in agar using an electric field include Immunoelectrophoresis, Counter-current immunoelectrophoresis, Rocket electrophoresis, Two-dimensional immunoelectrophoresis, Turbidimetry, and nephelometry.
Application of Precipitation reaction
- Detection of an unknown antibody to diagnose an infection, for instance. The VDRL test detects syphilis.
- Toxins and antitoxins should be standardised.
- Identifying Bacteria, such as E. coli Clusters of streptococci that have been calcified
- Identification of bacterial component, such as the thermoprecipitin test for Bacillus anthracis developed by Ascoli.
2. Agglutination Reaction
- The interaction between an antibody and a particle antigen results in a phenomenon known as agglutination.
- Antibodies that induce such responses are known as agglutinins. IgM antibody agglutination is superior to IgG antibody agglutination.
- Antibody excess also reduces the agglutination response; this inhibition is known as the prozone phenomenon.
- Antibody detection is more sensitive via agglutination than precipitation.
- Optimal agglutination occurs when antigens and antibodies react in quantities that are equal.
- When an antibody or antigen is overproduced, the prozone phenomenon may be observed. Although incomplete or monovalent antibodies bind with the antigen, they do not generate agglutination.
- They may function as blocking antibodies, inhibiting agglutination by the subsequent addition of the full antibody.
Types of agglutination
Agglutination is divided into 3 groups such as;
- Direct agglutination: Direct agglutination can be of the following types: (a) slide agglutination, (b) tube agglutination, (c) heterophile agglutination, and (d) antiglobulin (Coombs’) test.
- Passive agglutination: Passive agglutination utilises antigen-coated carrier particles. This is typically done to transform precipitation reactions into agglutination reactions, as the latter are simpler to run and understand and more sensitive for detecting antibodies than precipitation reactions.
- Reverse passive agglutination: Reverse passive agglutination refers to the detection of antigens by adsorption of the antibody instead of antigens on the carrier particle.
Application of Agglutination reaction
- Cross-matching and blood grouping
- Characterization of Bacteria Serotyping of Vibrio cholera, Salmonella Typhi, and Paratyphi, for example.
- Diagnosis of numerous diseases using serology. Example: Rapid plasma regains (RPR) and Antistreptolysin O (ASO) tests for syphilis and rheumatic fever, respectively.
- Detection of an unidentified antigen in clinical specimens. Detection of the Vi antigen of Salmonella Typhi in the urine, for instance.
3. Complement-Dependent Serological Tests
- Normal serum contains a set of serum proteins known as the complement system. Twenty or more serum proteins interact with one another and the cell membrane to form the system.
- It is a metabolic cascade that helps rid the body of infections.
- It assists antibodies in lysing germs, stimulating phagocytosis, and increasing immunological adhesion.
Types of Complement-Dependent Serological Tests
The following types of complement-dependent serological assays are possible: 1. Complement fixation test 2. Immune adherence test 3. Immobilization test 4. Cytolytic or cell-killing reactions
a. Complement fixation test
- The complement fixation test is based on the premise that when antigen and IgM or IgG antibodies are combined, complement is “fixed” to the antigen–antibody combination.
- If this occurs on the surface of RBCs, the complement cascade will be activated, resulting in hemolysis.
- Two antigen–antibody complement systems comprise the complement fixation test: (a) an indicator system and (b) a test system.
b. Immune adherence test
- Certain infections (e.g., Vibrio cholerae, Treponema pallidum, etc.) respond with certain antibodies in the presence of complement and adhere to erythrocytes or platelets during an immune adherence test.
- Immune adherence is the adhesion of cells to bacteria, which aids the phagocytosis of germs.
c. Immobilization test
- Certain live bacteria, such as T. pallidum, are immobilised when mixed with a patient’s serum in the presence of complement in an immobilisation test dependent on complement.
- This is the basis for the immobilisation test for T. pallidum. A positive result indicates that the serum contains treponemal antibodies.
d. Cytolytic or cytocidal reactions
- When a live bacteria such as V. cholerae is combined with its particular antibody in the presence of complement, it is destroyed and lysed.
- This is the basis of the test used to assess serum anti-cholera antibodies.
Application of Complement-Dependent Serological Tests
Historically, complement fixation reactions were used to diagnose numerous illnesses, including:
- The Wassermann test is used to detect syphilis.
- Antibody screenings against M. smegmatis pneumoniae, Bordetella pertussis, numerous other viruses, and to fungus (such as Cryptococcus spp., Histoplasma, and Coccidioides immitis). This test is no longer administered since it is technically very complicated and frequently difficult.
4. Neutralization Tests
- Neutralization is an antigen–antibody response in which homologous antibodies known as neutralising antibodies negate the biological effects of viruses and poisons.
Types of Neutralization Tests
Generally speaking, these tests fall into two categories: (a) viral neutralisation tests and (b) toxin neutralisation testing.
a. Virus neutralization tests
- Testing for the neutralisation of viruses by their specific antibodies is known as virus neutralisation. Inoculation of viruses into cell cultures, eggs, and animals leads to viral replication and proliferation.
- Injecting virus-specific neutralising antibodies into these systems inhibits viral replication and proliferation.
- This is the foundation for the virus neutralisation test. The viral hemagglutination inhibition test is an example of a virus neutralisation test commonly used to diagnose viral diseases like influenza, measles, and mumps.
- If the patient’s serum includes antibodies against particular viruses that have the property of agglutinating red blood cells, these antibodies will bind to the viruses and prevent agglutination of the red blood cells.
b. Toxin neutralization tests
Toxin neutralisation tests are based on the premise that the biological effect of a toxin can be negated by specialised neutralising antibodies known as antitoxins. The following are examples of neutralisation tests:
- In vivo—(a) Schick test to demonstrate immunity against diphtheria and (b) Clostridium welchii toxin neutralization test in guinea pig or mice.
- In vitro—(a) antistreptolysin O test and (b) Nagler reaction used for rapid detection of C. welchii.
- By combining with opsonin, a particulate antigen becomes more vulnerable to phagocytosis through the process of opsonization.
- The opsonin is a heat-labile substance found in normal fresh serum.
- Unlike opsonin, bacteriotropin is a heat-stable serum constituent with similar functions to opsonin.
- The definition of the word “opsonic index” is the ratio of the phagocytic activity of a patient’s blood for a certain bacterium to the phagocytic activity of blood from a healthy individual.
- It is used to examine the progression of resistance during the disease’s progression. It is determined by incubating fresh citrated blood with a bacterial suspension at 37 °C for 15 minutes and calculating the average number of phagocytic bacteria from stained blood films.
- Fluorescence is the property of some dyes to absorb light rays at a specific wavelength (ultraviolet light) and emit them at an other wavelength (visible light).
- Antibody molecules can be attached to fluorescent dyes such fluorescein isothiocyanate and lissamine rhodamine.
- Under ultraviolet (UV) rays, they emit blue-green and orange-red fluorescence, respectively, in the fluorescence microscope.
- This is the basis for the immunological examination. Immunofluorescence tests have numerous diagnostic and research applications.
Types of Immunofluorescence
These examinations often fall into two categories: 1. Test of direct immunofluorescence 2. Test by indirect immunofluorescence
a. Direct immunofluorescence test
- Utilizing a tagged antibody that interacts directly with the unknown antigen, a direct immunofluorescence test is used to identify unknown antigen in a cell or tissue.
- If antigen is present, it reacts with labelled antibody, and the antibody-coated antigen can be detected under UV fluorescent light.
- The requirement to prepare a distinct labelled antibody for each pathogen is the most significant drawback of the direct immunofluorescence test.
b. Indirect immunofluorescence test
- The indirect immunofluorescence test is utilised for the serodiagnosis of numerous infectious disorders to identify particular antibodies in serum and other body fluids. Immunofluorescence indirecte is a two-step method.
- In the initial step, a known antigen is attached to a slide.
- The testing patient’s serum is then put to the slide, followed by a thorough washing. If the patient’s serum contains antigen-specific antibodies, they will bind to the antigen on the slide.
- In the second step, the combination of antibody and antigen can be identified by adding a fluorescent dye-labeled antibody to human IgG, which is then analysed using a fluorescence microscope.
- In the first step of the indirect immunofluorescence test, a fixed antigen (e.g., in a cell or tissue) is incubated with an unlabeled antibody, which binds to the antigen.
- After a thorough washing, a fluorescent antibody (e.g., anti-IgG with a fluorescent label) is added to the smear. This second antibody will bind to the first, allowing visualisation of the antigen–antibody combination under a fluorescent microscope.
- The indirect method has the advantage that a single labelled antiglobulin (antibody to IgG) can be used as a “universal reagent” to detect a variety of unique antigen–antibody responses.
- Typically, the test is more sensitive than direct immunofluorescence.
- Immunofluorescence is limited in that it requires (a) a costly fluorescence microscope and chemicals, (b) trained workers, and (c) a subjective aspect that may lead to inaccurate results.
Application of Immunofluorescence
- Direct immunofluorescence test for antemortem diagnosis of rabies: This test is used to detect rabies virus antigen in the skin smear taken from the nape of the neck of humans and the saliva of dogs.
- Also employed for N detection. gonorrhoeae, C. T. diphtheriae pallidum, etc. directly in relevant clinical specimens.
- Detect particular antibodies for the serodiagnosis of infectious disorders such as syphilis, leptospirosis, amoebiasis, and toxoplasmosis.
- Utilize fluorescent antibodies specific for various immunoglobulin isotypes to determine the class of a given antibody.
- Utilizing monoclonal antibodies and cytofluorographs, identify and quantify lymphocyte subpopulations.
- In autoimmune illnesses, detect autoantibodies, such as antinuclear antibodies.
7. Enzyme Immunoassays
- Enzyme immunoassays (EIAs) can be used to detect antigens or antibodies in the patient’s serum and other body fluids.
- Enzyme-labeled antigen or antibody is utilised in EIA methods. EIA tests utilise alkaline phosphatase, horseradish peroxidase, and galactosidase as enzymes.
- EIAs are routinely employed enzyme-linked immunosorbent tests (ELISAs). Peter Perlmann and Eva Engvall of Stockholm University, Sweden, first conceptualised and developed the ELISA technique.
- In these tests, an immunosorbent specific to the antigen or antibody is utilised.
- After the antigen-antibody reaction, an enzyme-specific chromogenic substrate (o-phenyldiamine dihydrochloride for peroxidase, p-nitrophenyl phosphate for alkaline phosphatase, etc.) is added.
- To detect the reaction, the optical density is measured. Typically, the unknown concentrations of antigen or antibody are determined using a standard curve based on known concentrations of antigen or antibody.
Types of Enzyme Immunoassays
There are a variety of ELISAs for detecting and quantifying antigens or antibodies in serum and other body fluids.These include: (a) indirect ELISA, (b) sandwich ELISA, (c) competitive ELISA, and (d) ELISPOT assay.
- Indirect ELISA: The indirect ELISA is used to quantify antibody concentrations in serum and other bodily fluids.
- Sandwich ELISA: Sandwich ELISA is utilised for antigen detection. This test involves coating and immobilising the known antibody on the wells of microtiter plates. The antigen-containing test sample is added to the wells and allowed to react with the antibodies in the wells.
- Competitive ELISA: Competitive ELISA is another method for determining the amount of antibodies in a sample, such as serum. The test employs two specific antibodies, one coupled with an enzyme and the other present in the test serum (if the serum is antibody-positive). There is competition between the two antibodies for the identical antigen.
- ELISPOT Assay: The ELISPOT assay is a variation of the ELISA assay. It enables the quantitative assessment of the number of cells in a population that produce antibodies specific for a certain antigen or an antigen for which a specific antibody exists. These tests have found widespread application in cytokine quantification.
Application of Enzyme Immunoassays
- Sandwich ELISA is utilised to detect rotavirus and Escherichia coli enterotoxin in faeces.
- Competitive ELISA is the most popular method for detecting HIV antibodies in the serum of HIV-positive patients.
- The test is widely utilised to determine blood antibodies for the diagnosis of human immunodeficiency virus (HIV) infection, Japanese encephalitis, dengue, and numerous other viral infections.
- When radioisotopes rather than enzymes are employed to mark antigens or antibodies, the technique for detecting the antigen–antibody combination is known as radioimmunoassay (RIA).
- Solomon Berson and Rosalyn Yalow of the New York Veterans Administration Hospital were the first to describe RIA in 1960 for measuring endogenous plasma insulin.
- Yalow was given the 1977 Nobel Prize in Medicine for the discovery of the RIA for peptide hormones, but Berson was unable to share the award due to his tragic death in 1972.
- The classical RIA methods are founded on the competitive binding principle. In this technique, unlabeled antigen competes with radiolabeled antigen for binding to the particular antibody.
- When mixtures of radiolabeled and unlabeled antigen are incubated with the matching antibody, the amount of unbound radiolabeled antigen is directly proportional to the amount of unlabeled antigen in the mixture.
- In the first step of the test, mixtures of known variable amounts of cold antigen and fixed amounts of labelled antigen are generated, as well as mixtures of samples with unknown concentrations of antigen and identical amounts of labelled antigen. A comparable amount of antibody is added to each mixture.
- Antigen–antibody complexes are precipitated by either crosslinking with a second antibody or by adding chemicals that facilitate antigen–antibody complex precipitation.
- By counting the radioactivity in the precipitates, the amount of radiolabeled antigen precipitated with the antibody can be determined.
- Antigen concentrations in patient samples are inferred using a standard curve created by graphing the fraction of antibody-bound radiolabeled antigen against known quantities of a standardised unlabeled antigen.
- RIA’s primary benefit is its extraordinarily high sensitivity.
- Antigens and antibodies in the serum can be determined down to the nanogram level using this test.
- This test is utilised to determine the concentration of hormones, medications, HBsAg, and other viral antigens.
- The principal disadvantages of the RIA include:
- the price of equipment and chemicals.
- The limited shelf life of radiolabeled substances.
- the issues linked with radioactive waste disposal.
9. Western Blotting
- Western blotting is so named because it is comparable to Southern blotting, which Edwin Southern invented for the detection of DNA.
- Western blotting is used to detect proteins, whereas Southern blotting is used to detect DNA. Western blotting is often performed on homogenates or extracts of tissue.
- It employs SDS-PAGE (sodium dodecyl sulphatepolyacrylamide gel electrophoresis), a form of gel electrophoresis, to initially separate proteins in a mixture based on their shape and size using gel electrophoresis.
- The resulting protein bands are transferred to a nitrocellulose or nylon membrane, where they are “probed” with antibodies specific to the target protein.
- The antigen–antibody complexes that form on the band containing the antibody-recognized protein can be seen in a number of different ways.
- If the target protein was captured by a radioactive antibody, its position on the blot can be identified by exposing the membrane to a sheet of X-ray film, a technique known as autoradiography.
- However, the most common detection methods involve enzyme-linked antibodies to the protein.
- After the enzyme–antibody combination has bound to the target antigen, the addition of a chromogenic substrate that yields a highly coloured and insoluble product promotes the formation of a colourful band at the site of the antigen.
- If a chemiluminescent chemical and suitable boosting agents are used to produce light at the antigen site, the site of the target protein can be determined with far greater sensitivity.
Application of Western Bloting
- It is used to identify a specific protein within a complicated protein mixture. Antigens of well-defined molecular weight are separated by SDS-PAGE and blotted onto nitrocellulose in this procedure. The separated bands of known antigens are next examined with a sample thought to have antibodies specific to one or more of these antigens. Using either a radiolabeled or enzyme-linked secondary antibody that is specific for the species of the antibodies in the test sample, the reaction of an antibody with a band is identified.
- It is also used to estimate the protein’s size and the amount of protein in a combination.
- The Western blot test is most commonly employed as a confirming test for the HIV diagnosis, where it is utilised to evaluate whether or not the patient has antibodies that respond with one or more viral proteins.
- For the diagnosis of neurocysticercosis and tuberculous meningitis, Western blotting is also performed to demonstrate the presence of particular antibodies in the blood.
10. Immunoelectronmicroscopic Tests
These are the types of antigen–antibody interactions that can be directly observed using an electron microscope. These fall into the following categories:
- This test is used to directly detect rotavirus and hepatitis A virus in faeces.
- In this test, viral particles are combined with specific antisera and observed under an electron microscope as clusters of virion particles.
b. Immunoenzyme test
- This test is used to directly detect antigen in tissue samples by treating tissue sections with peroxidase-labeled antisera to detect corresponding antigen.
- Under an electron microscope, the peroxidase-antigen complex is visualised.
c. Immunoferritin test
- Electron-dense compounds, such as ferritin, are conjugated with antibody, allowing the electron microscope to visualise labelled antibodies reacting with antigen.
11. Chemiluminescence Assay
- During antigen–antibody reactions, the chemiluminescence assay employs chemiluminescent substances that emit energy in the form of light.
- The emitted light is measured, and the concentration of the substance being analysed is computed.
- The assay is a completely automated approach often used to evaluate Mycobacterium TB for drug sensitivity.
Alternatives to Antigen-Antibody Reactions
- Some bacteria have evolved the ability to produce proteins that bind to the Fc region of IgG molecules with high affinity (Ka ~ 108) as a protection against host antibodies.
- Protein A is present in the cell walls of some strains of Staphylococcus aureus, while protein G is present in the cell walls of group C and G Streptococcus.
- By cloning the genes for protein A and protein G and making a hybrid of both, it is possible to create protein A/G, a recombinant protein that combines some of the best characteristics of each.
- These molecules are advantageous because they can bind IgG from numerous species. Consequently, they can be labelled with flourochromes, radioactivity, or biotin to detect IgG molecules in antigen-antibody complexes generated during ELISA, RIA, or fluorescence-based tests such as flow cytometry or fluorescence microscopy.
- These bacterial IgG-binding proteins can also be employed to create IgG-isolating affinity columns. Egg whites include avidin, a protein that binds biotin, a necessary nutrient for fat formation.
- Avidin is thought to have evolved as a protection against nest-robbing rats that consume the stolen eggs. The binding between avidin and biotin is extraordinarily selective and has a significantly greater affinity (Ka ~ 1015) than any known antigen-antibody response.
- Streptavidin, a bacterial protein produced by streptomyces avidinii, possesses comparable affinity and specificity. The high affinity and perfect specificity of these proteins’ interactions with biotin are routinely utilised in numerous immunological processes.
- After labelling the main or secondary antibody with biotin and allowing it to react with the target antigen, the unbound antibody is removed by washing.
- The bound antibody is then detected using streptavidin or avidin coupled with an enzyme, fluorochrome, or radioactive marker.
Antigen-Antibody Interaction Limitations
- RDTs are incapable of consistently detecting low parasitemia densities (200 parasites/L).
- Require specialised knowledge and equipment that are typically unavailable in developing or impoverished nations. Therefore, these procedures are not commonly employed in such locations.
- In certain instances of antibody detection, it is difficult to distinguish between early and late infections since antibodies stay in our blood for a long time after the illness has been cured.
Antigen-Antibody Interaction Applications
- The most prevalent application is blood group determination, or blood typing.
- This is the basis for rapid diagnostic test kits used for detecting pregnancy as well as a variety of diseases such as malaria, dengue, etc. They demand less time for the examinations.
- Determination of exposure to infectious pathogens by serology.
- Quantification of medicines, hormones, and viral antigens, among other substances.
- Detection of protein presence or absence in serum.
- To investigate the characteristics of various immunodeficiency disorders.
- To conduct confirmation tests for infections, such as the Western Blot for HIV.
- Reverberi R, Reverberi L. Factors affecting the antigen-antibody reaction. Blood Transfus. 2007 Nov;5(4):227-40. doi: 10.2450/2007.0047-07. PMID: 19204779; PMCID: PMC2581910.
- MILLER, J. J., & LEVINSON, S. S. (1996). INTERFERENCES IN IMMUNOASSAYS. Immunoassay, 165–190. doi:10.1016/b978-012214730-2/50008-x