Antigen-Antibody Interaction Reaction – Definition, Types, Examples, Properties

Antigen-antibody interaction reaction is a specific binding reaction between an antigen and its corresponding antibody. In this reaction the antibody recognizes the specific part of antigen, which is called epitope. The binding site of antibody is present in the hypervariable region and it is complementary to the epitope.

This reaction is very important in humoral immunity. It helps the body to identify and remove foreign antigen. Same reaction is also used in many diagnostic tests, because antigen and antibody bind with high specificity.

The reaction is not a permanent covalent reaction. It is a reversible reaction. The binding is mainly due to weak non-covalent forces such as electrostatic interaction, hydrogen bond, van der Waals force and hydrophobic interaction.

During this interaction, antigen and antibody first come near each other by attraction. Then water molecules present between binding surfaces are removed. After this, short range weak bonds are formed and the antigen-antibody complex becomes stable.

The stability of this reaction depends on proper fitting between antigen and antibody. If the shape of epitope and antibody binding site are complementary, many weak bonds are formed together. This gives more strength to the antigen-antibody complex.

The strength of single binding site is called affinity. It means the force of attraction between one antibody binding site and one antigenic epitope. High affinity antibody binds strongly with antigen.

The total binding strength between whole antibody and antigen is called avidity. It depends on number of binding sites and total strength of all bonds. So antigen-antibody interaction is mainly controlled by specificity, affinity and avidity.

Antigen-antibody interaction reaction is a specific binding reaction between antigen and its corresponding antibody. In this reaction epitope of antigen combines with paratope of antibody by weak non-covalent forces. This forms antigen-antibody complex.

Basic Concept of Antigen and Antibody

Antigen (Ag) is a foreign substance which enters into the body and is recognized by immune system. It may be bacteria, virus, toxin or any other foreign molecule. It can stimulate immune response and can bind with specific antibody or immune receptor.

The active part of antigen is called epitope or antigenic determinant. It is the small region of antigen where antibody binds. The whole antigen is not always involved in binding, only the specific epitope region takes part in reaction.

Epitopes may be of two types. Linear epitope is formed by continuous sequence of amino acids in a protein chain. Conformational epitope is formed when distant amino acids come close due to folding of protein in three dimensional structure.

Antigens may be small or large in size. Small antigens include haptens and short peptides. Large antigens are generally complex proteins and polysaccharides. They may be soluble in body fluid or may be particulate like whole bacterial cells, red blood cells or latex particles.

Antibody (Ab) is a special protein produced by plasma cells against antigen. It is also called immunoglobulin. It recognizes the specific antigen and helps in neutralization or removal of that antigen.

Antibody is generally Y-shaped protein. It is made up of two identical heavy chains and two identical light chains. These chains are joined together by disulfide bonds.

The antigen binding site of antibody is present at the tips of the Y-shaped arms. This site is called paratope. It contains variable region and complementarity determining regions (CDRs) which fit with the epitope of antigen.

The lower constant part of antibody is called Fc region. After antigen binding, this region remains exposed. It helps in activation of complement system and also helps phagocytic cells like macrophages to remove antigen.

There are five main classes of antibody. These are IgG, IgM, IgA, IgE and IgD. IgG is common in blood and can cross placenta. IgM appears first in immune response. IgA protects mucosal surface. IgE takes part in allergy and parasitic infection. IgD is mostly present on B-cell surface.

The binding between antigen and antibody depends on affinity and avidity. Affinity is the strength between one epitope and one antibody binding site. Avidity is the total strength of binding between whole antigen and antibody.

Principle of Antigen–Antibody Interaction

Antigen-antibody interaction is based on the specific union of antigen and antibody. The combining site of antigen is called epitope and the combining site of antibody is called paratope. These two parts come close and fit with each other due to complementary structure.

This reaction is not a permanent chemical reaction. No covalent bond is formed in it. The antigen and antibody are held together by weak forces like hydrogen bond, electrostatic force, van der Waals force and hydrophobic interaction.

The weak forces act only when antigen and antibody are very close. So the shape of epitope and paratope is important. If their shape is not suitable, proper binding does not occur and stable complex is not formed.

The binding is reversible. The antigen-antibody complex may dissociate again because the bonds are weak. But when many weak bonds are formed at the same time, the complex becomes more stable.

The strength of single binding between one epitope and one antibody binding site is called affinity. High affinity means antibody binds antigen strongly. Low affinity means the binding is weak.

The total strength of binding between antigen and antibody is called avidity. It depends on number of binding sites and total weak bonds present in the complex. Multivalent antigen and antibody show more avidity.

Structure Involved in Binding of Antigen–Antibody

1. Paratope – The antigen binding site of antibody is called paratope. It is present at the tip of antibody molecule. This site binds with the specific part of antigen.
2. Variable region – The binding site is formed by variable regions of heavy and light chains. These regions are called VH and VL regions. They are present at amino terminal end of antibody.
3. CDRs – The most important part of variable region is Complementarity Determining Regions (CDRs). These are hypervariable loops present in antibody. They directly take part in antigen binding.
4. CDR loops – Each variable domain has CDR1, CDR2 and CDR3. Three CDRs are present on heavy chain and three are present on light chain. These six loops together form the actual antigen binding surface.
5. CDR3 – CDR3 is the most variable region among the CDRs. It gives more specificity to the antibody. It mainly helps in proper fitting with antigenic determinant.
6. Epitope – The binding site present on antigen is called epitope. It is also called antigenic determinant. This is the part of antigen which is recognized by antibody.
7. Linear epitope – Linear epitope is made by continuous amino acid sequence of a polypeptide chain. The amino acids are present one after another. This type of epitope may remain recognized even after partial unfolding.
8. Conformational epitope – Conformational epitope is formed by folding of protein. In this, distant amino acids come close due to three dimensional structure. If the protein is denatured, this epitope may be lost.
9. Pocket or groove – Some antibodies have deep binding pocket or groove between heavy and light chain variable regions. These pockets are mainly used for binding small antigens like haptens, drugs and short peptides.
10. Extended surface – Large antigens bind with broad and flat or slightly curved antibody surface. This type of surface involves many CDR loops. It is used for binding large molecules like viral envelope proteins and cell surface receptors.
11. Binding surface – The binding surface of antigen and antibody should be complementary in shape. The close fitting of epitope and paratope allows weak forces to act between them. This forms the antigen-antibody complex.

Forces Involved in Antigen–Antibody Binding

1. Electrostatic force – This force is formed between oppositely charged groups of antigen and antibody. The positive charged amino group may attract negative charged carboxyl group. Sometimes these attractions form salt bridge like structure.
2. Hydrogen bond – It is formed when hydrogen atom is shared between two electronegative atoms. These atoms are mainly oxygen and nitrogen. This bond helps in holding the epitope and paratope together.
3. Van der Waals force – These are very weak short range forces. It acts only when antigen and antibody surfaces come very close to each other. So proper fitting of both surfaces is necessary for this force.
4. Hydrophobic interaction – This force occurs between non-polar amino acid side chains. These groups do not like water, so they come together and water is removed from the binding site. This helps the antigen-antibody complex to become more stable.

All these forces are weak when present separately. But many bonds are formed at the same time between antigen and antibody. Due to this, the binding becomes stable but still reversible.

Characteristics of Antigen–Antibody Reaction

• Specificity – Antigen-antibody reaction is specific type reaction. The antibody combines only with the particular antigen. The paratope of antibody fits with the epitope of antigen, and this gives the specific nature of reaction.
• Reversibility – This reaction is reversible. The antigen and antibody may combine and may also separate again. It occurs because no strong permanent bond is present between them.
• Weak bond formation – In this reaction, covalent bond is not formed. The binding is due to weak forces like hydrogen bond, electrostatic force, van der Waals force and hydrophobic interaction. These forces act only when antigen and antibody come very close.
• Affinity – Affinity means the strength of union between one combining site of antibody and one antigenic determinant. When the fitting is close, the affinity is more. When the fitting is loose, the affinity becomes less.
• Avidity – Avidity is the total strength of binding between whole antigen and antibody. It is more when many binding sites are involved in the reaction. So multivalent antigen and antibody gives stronger reaction.
• Cross reaction – Sometimes one antibody may react with another antigen also. This occurs when the second antigen has same or nearly same epitope. This type of reaction is called cross reaction.
• Visible reaction – The reaction may become visible when antigen and antibody are present in proper amount. They form large network or lattice. This is used in many diagnostic tests.
• Precipitation – When soluble antigen reacts with antibody, insoluble complex may be formed. This visible deposit is called precipitation. It is one type of antigen-antibody reaction.
• Agglutination – When particulate antigen like cell, bacteria or latex particle reacts with antibody, clumping occurs. This clumping is called agglutination. It is used in blood grouping and other serological tests.

Stages of Antigen–Antibody Reaction

1. Long range attraction – In the first stage, antigen and antibody come near to each other. They are attracted by long range forces. Mainly electrostatic force and hydrophobic force take part in this stage.
2. Desolvation – In this stage, water molecules present between antigen and antibody surface are removed. The attractive forces overcome the hydration layer. So the binding surface of epitope and paratope comes close.
3. Short range stabilization – After removal of water, antigen and antibody come in close contact. Then short range forces like hydrogen bond and van der Waals force are formed. These forces make the antigen-antibody complex stable.
4. Primary phase – This is also called sensitization phase. In this phase antibody binds with the specific epitope present on antigen surface. This reaction occurs rapidly but it is not visible by naked eye.
5. Secondary phase – In this phase many antibodies bind with many antigen particles. The antibodies form bridge between adjacent antigen particles. This produces large network or lattice formation.
6. Visible reaction – After lattice formation, visible clumping or deposit is formed. In case of particulate antigen, agglutination occurs. In case of soluble antigen, precipitation occurs.

Factors Affecting Antigen–Antibody Interaction

• Temperature – Temperature affects the reaction between antigen and antibody. At suitable temperature, movement of molecules increases and they collide more easily. Too much heat may denature the protein and the complex may break.
• pH – The reaction occurs properly only at suitable pH. Very high or very low pH changes the charge of amino acid groups. Due to this electrostatic force and hydrogen bond may be disturbed.
• Ionic strength – Salt concentration of the medium affects antigen-antibody binding. High salt concentration may interfere with charged groups. It reduces salt bridge formation and weakens the reaction.
• Antigen-antibody ratio – Proper amount of antigen and antibody is needed for visible reaction. In zone of equivalence, both are present in suitable amount and lattice is formed. In prozone, antibody is excess and in postzone, antigen is excess, so visible reaction may not occur.
• Affinity – Affinity is the strength of binding between one epitope and one antibody binding site. Higher affinity gives stronger binding. Low affinity gives weak and unstable binding.
• Avidity – Avidity is the total strength of binding between antigen and antibody. It depends on number of combining sites and total bonds formed. More avidity makes the complex more stable.
• Antibody class – Type of antibody also affects the reaction. IgM is pentameric and has more binding sites, so it forms lattice more effectively. IgG is monomeric and has less cross-linking ability than IgM.
• Physical motion – Shaking, stirring or centrifugation may increase contact between antigen and antibody. This increases collision between them and reaction may occur faster.
• Solvents and detergents – Organic solvents, polar solvents and detergents may disturb the binding surface. They affect hydrophobic interaction and hydrogen bond. So antigen-antibody complex may become weak.
• Molecular crowding – In body fluid, many molecules are present together. This crowding affects movement of antigen and antibody. It may change diffusion and rate of antigen-antibody reaction.

Specificity of Antigen–Antibody Reaction

Specificity of antigen-antibody reaction means the antibody combines with only particular antigen. This depends on the fitting of epitope of antigen and paratope of antibody. If the antigenic determinant is different, the antibody usually does not combine with it.

1. First the antibody comes near the antigen. The antibody has binding site at its variable region. This binding site is made by CDRs or Complementarity Determining Regions.
2. The CDRs form a special surface on antibody. This surface is called paratope. It has particular shape and chemical nature.
3. The antigen has a small combining part called epitope. It may be present on the surface of antigen. This epitope is the part which is recognized by antibody.
4. The paratope of antibody and epitope of antigen must be complementary. The shape, charge and chemical groups should fit with each other. Otherwise the reaction does not occur properly.
5. When both are suitable, long range attraction starts between them. Electrostatic force and hydrophobic force help to bring antigen and antibody close.
6. After coming close, water molecules between the two surfaces are removed. This is called desolvation. It helps the two binding surfaces to come in direct contact.
7. Then short range forces are formed. Hydrogen bond and van der Waals force act only when the surfaces are very close. So small mismatch also decreases the binding.
8. If the fitting is proper, many weak bonds are formed together. This makes the antigen-antibody complex stable. If fitting is poor, the complex is not stable and separates easily.
9. The strength of this specific union is called affinity. High affinity means strong binding between one epitope and one antibody binding site.
10. In this way, antibody selects only its matching antigen. This is why antigen-antibody reaction is called specific reaction.

Types of Antigen–Antibody Reactions

1. Precipitation reaction – This reaction occurs between soluble antigen and soluble antibody. When they are present in proper proportion, insoluble antigen-antibody complex is formed. This visible complex is called precipitin.
2. Precipitation tests – Some important precipitation tests are precipitin ring test, Ouchterlony double immunodiffusion, radial immunodiffusion (RID) and flocculation test. VDRL test is an example of flocculation reaction.
3. Agglutination reaction – This reaction occurs when particulate antigen reacts with specific antibody. The antigen may be whole bacterial cell, red blood cell or latex particle. Antibody cross links the particles and visible clumping is formed.
4. Agglutination types – Agglutination may be direct agglutination, passive agglutination and hemagglutination. In direct agglutination, natural particulate antigen is used. In passive agglutination, antigen is coated on carrier particle. In hemagglutination, red blood cells are involved.
5. Neutralization reaction – In this reaction antibody binds with active site of virus or bacterial toxin. After binding, virus or toxin cannot attach with host cell. So its infective or toxic action is blocked.
6. Neutralization test – Plaque Reduction Neutralization Test (PRNT) is an example of neutralization assay. It is used to measure the neutralizing antibody against virus. Less plaque formation shows more neutralizing activity.
7. Complement fixation reaction – This reaction is based on use of complement by antigen-antibody complex. If specific antibody is present in patient serum, antigen-antibody complex fixes the complement. Then complement is not available for lysis of indicator red blood cells.
8. ELISA – Enzyme-Linked Immunosorbent Assay (ELISA) is a sensitive antigen-antibody reaction. In this method antigen or antibody is fixed on solid surface like microtiter plate. Enzyme labelled antibody and substrate produce colour or fluorescence.
9. Types of ELISA – ELISA may be direct, indirect, sandwich and competitive type. These are used for detection of antigen or antibody in clinical sample.
10. Western blotting – Western blotting is also called immunoblotting. In this method proteins are first separated by gel electrophoresis according to molecular weight. Then proteins are transferred to membrane and detected by specific antibody.
11. Immunofluorescence – In this reaction antibody is labelled with fluorescent dye. The labelled antibody binds with specific antigen and fluorescence is observed under fluorescent microscope.
12. Radioimmunoassay – Radioimmunoassay (RIA) uses radio labelled antigen or antibody. It is very sensitive method. It is used for detection of very small amount of antigen or hormone.
13. Immunochromatography – This is a strip based antigen-antibody reaction. The sample moves on strip by capillary action. If antigen or antibody is present, coloured line is formed on the test region.

Formation of Immune Complex

1. Antigen and antibody first come near to each other. The epitope of antigen and paratope of antibody are brought close within very small distance. In this stage weak attraction starts between them.
2. The first attraction is due to long range forces. Mainly electrostatic force and hydrophobic force take part here. These forces help antigen and antibody to come more close.
3. After this, water molecules present between antigen and antibody surface are removed. This process is called desolvation. Due to removal of water, the binding surfaces can touch more properly.
4. When the two surfaces come very close, short range forces start to act. Hydrogen bonds and van der Waals forces are formed between antigen and antibody. These forces help to stabilize the complex.
5. The antigen-antibody complex is then formed. It is not formed by covalent bond. It is formed by many weak bonds which act together between epitope and paratope.
6. In the primary phase, antibody combines with single antigenic determinant present on antigen surface. This step is also called sensitization. This reaction occurs fast but it cannot be seen by naked eye.
7. In the secondary phase, more antibodies bind with more antigenic sites. One antibody may join two antigen particles and form bridge between them. This starts the cross-linking process.
8. After cross-linking, many antigen and antibody molecules join together. They form a large network like structure. This is called lattice formation.
9. The lattice becomes stable when antigen and antibody are present in proper proportion. This condition is called zone of equivalence. In this condition immune complex becomes more visible.
10. When antigen is soluble, the immune complex may appear as precipitation. When antigen is particulate, such as cell or latex particle, clumping occurs and it is called agglutination.

Biological Significance of Antigen–Antibody Interaction

• Neutralization – In this reaction antibody binds with the active site of virus, bacteria or toxin. After binding, the pathogen cannot attach with host cell receptor. So entry of pathogen and toxic action is blocked.
• Opsonization – Antibody coats the surface of foreign antigen. This coating acts as tag for phagocytic cells. Then macrophages and neutrophils recognize the antigen easily and engulf it.
• Complement activation – When IgM or IgG binds with antigen, it activates the classical complement pathway. This reaction forms many complement products. These products help in killing and removal of pathogen.
  • Membrane attack complex – During complement activation, MAC is formed on the surface of target cell. It makes pore in the cell membrane. Due to this the pathogen cell is lysed and destroyed.
  • C3b formation – Complement system also produces C3b. It gets deposited on the pathogen surface. This works as additional opsonin and increases phagocytosis.
  • Inflammation – Some small complement peptides are released during this reaction. They attract immune cells towards the site of infection. This produces local inflammation and helps in removal of antigen.
• ADCC – Antibody-dependent cellular cytotoxicity (ADCC) occurs when antibody binds with antigen on virus infected cell or cancer cell. The Fc region of antibody is recognized by Natural Killer (NK) cells. Then NK cells release perforin and granzymes and the target cell undergoes apoptosis.
• Clearance of dead cells – Antibody also helps in removal of dead or apoptotic cells. The cell fragments are marked by antibody and then removed by phagocytic cells. This keeps the tissue clean from cellular debris.
• Maintenance of self tolerance – Proper clearance of cell debris is important for preventing immune reaction against own tissue. If dead cell materials are not removed properly, they may act as autoantigens. So antigen-antibody interaction also helps to reduce autoimmune reaction.

Laboratory Methods Based on Antigen–Antibody Reactions

• Precipitation assay – This method is based on reaction between soluble antigen and soluble antibody. When both are present in proper amount, insoluble antigen-antibody complex is formed and it is called precipitin.
  • Precipitin ring test – Antigen and antibody solutions are layered in a tube. A visible ring is formed at the junction where both meet in equivalent amount.
  • Ouchterlony double immunodiffusion – Antigen and antibody are placed in separate wells of agar gel. They diffuse and form precipitin line, which shows whether antigens are same, partly same or different.
  • Radial immunodiffusion (RID) – Antigen diffuses into agar containing antibody and forms circular precipitin ring. The diameter of ring depends on antigen concentration.
  • Flocculation test – Fine insoluble antigen particles form small floccules with specific antibody. VDRL test for syphilis is an example.
• Agglutination assay – This reaction occurs between antibody and particulate antigen like bacteria, red blood cell or latex bead. Antibody cross links the particles and visible clumping is formed.
  • Direct agglutination – Antigen is naturally present on particle surface and antibody directly produces clumping. ABO blood grouping and Widal test are examples.
  • Passive agglutination – Soluble antigen is coated on carrier particle like latex bead or gelatin particle. It is used to detect antibody in patient serum.
  • Reverse passive agglutination – Antibody is coated on carrier particle and it is used to detect soluble antigen in sample.
  • Coombs test – It is a hemagglutination test using red blood cells and anti-human globulin. Direct Coombs test detects antibody on red blood cells and Indirect Coombs test detects free antibody in serum.
• Neutralization assay – This test is based on blocking of virus or toxin by specific antibody. The antibody binds with active site and prevents infection or toxic effect.
  • Plaque reduction neutralization test (PRNT) – Neutralizing antibody prevents virus infection in cell culture. So plaque formation is reduced.
  • Hemagglutination inhibition assay (HIA) – Specific antibody blocks virus from agglutinating red blood cells. So hemagglutination does not occur.
• Complement fixation test (CFT) – This test is based on fixation of complement by antigen-antibody complex. If specific antibody is present, complement is used up and added sheep red blood cells are not lysed.
• Labeled immunoassays – These tests use labelled antigen or antibody for detection of antigen-antibody reaction. The label may be enzyme, radioisotope or fluorescent dye.
  • ELISA – Enzyme-Linked Immunosorbent Assay is done on microtiter plate. Antigen or antibody is fixed on plate and enzyme linked antibody with substrate gives colour reaction.
  • Types of ELISA – Direct, indirect, sandwich and competitive ELISA are used for detection of antigen or antibody in clinical sample.
  • Radioimmunoassay (RIA) – It uses radio labelled antigen or antibody. It is very sensitive and used for very small amount of antigen, antibody or hormone.
  • Immunofluorescence – Antibody is labelled with fluorescent dye. It binds with antigen and fluorescence is seen under fluorescent microscope.
• Western blotting – It is also called immunoblotting. Proteins are first separated by gel electrophoresis and then transferred to membrane. Specific labelled antibody is used to detect the target protein antigen.
• Immunoelectrophoresis (IEP) – This method combines electrophoresis and precipitation reaction. Proteins are first separated in gel by electric current and then antibody forms precipitin arcs. It is used for abnormal immunoglobulin detection, such as in multiple myeloma.

Clinical Applications of Antigen–Antibody Reactions

• Blood grouping – Antigen-antibody reaction is used for ABO and Rh blood group detection. The red cell antigen reacts with specific antisera and clumping is formed. By this blood group is identified.
• Cross matching – Before blood transfusion, donor blood and recipient serum are tested. If antigen and antibody are incompatible, agglutination or hemolysis may occur. So this test helps to prevent transfusion reaction.
• Rh incompatibility – It is used for detection of hemolytic disease of newborn. Maternal antibody may react with fetal red cells. This causes destruction of fetal red blood cells.
• Infectious diseases – Many infection are diagnosed by antigen-antibody reaction. The test may detect microbial antigen or antibody in serum. It is used for bacterial, viral and fungal diseases.
  • VDRL, RPR and Wasserman test are used in syphilis.
  • Widal test is used in typhoid fever.
  • Serological tests are used in brucellosis and leptospirosis.
  • HIV, hepatitis A, hepatitis B, hepatitis C, COVID-19, dengue, Zika and influenza are detected by antigen or antibody test.
• Infectious mononucleosis – Epstein-Barr virus infection is detected by antigen-antibody based test. It helps in diagnosis of infectious mononucleosis.
• Autoimmune disease – In autoimmune disease, antibody is formed against own body antigen. Rheumatoid factor is detected in rheumatoid arthritis. Antinuclear antibody (ANA) is detected in systemic lupus erythematosus.
• Autoimmune hemolytic anemia – Direct Coombs test is used here. It detects antibody attached on red blood cells. Positive test indicates immune destruction of red cells.
• Pregnancy test – Pregnancy test is based on detection of hCG in urine. The hCG reacts with specific antibody on the strip. Colour line is formed when the test is positive.
• Hormone detection – Many hormones are measured by antigen-antibody reaction. Testosterone, prolactin, LH and FSH are detected by immunoassay. These tests are useful in endocrine disorders.
• Tumor marker detection – Cancer markers are detected by antigen-antibody reaction. PSA and CEA are common examples. Their level is used in diagnosis and follow up of cancer.
• Multiple myeloma – Abnormal monoclonal protein is detected by immunological methods. These proteins are produced by abnormal plasma cells. It helps in diagnosis of multiple myeloma.
• Vaccine evaluation – Antigen-antibody reaction is used to measure neutralizing antibody after vaccination. PRNT is used for this purpose. It shows the protective antibody level against virus.
• Allergy test – Allergy test detects specific IgE antibody against allergen. Food allergens and environmental allergens are tested by this method. It helps to identify the cause of allergy.
• Drug and toxin detection – Small drugs, toxins and chemicals are detected by immunoassay. Competitive ELISA is commonly used for such small molecules. It is used in toxicology and drug screening.

Limitations of Antigen–Antibody Reactions

• Prozone effect – When antibody is present in very excess amount, proper lattice is not formed. The antigen sites are separately coated by antibodies. So visible reaction may not occur and false negative result is obtained.
• Postzone effect – When antigen is present in very excess amount, the antibodies cannot cross link the antigen properly. Due to this large immune complex is not formed. This also gives false negative reaction.
• Cross reaction – Sometimes antibody reacts with another antigen having same or similar epitope. This gives non-specific reaction. So false positive result may occur in some test.
• False positive reaction – Some unrelated infections, high serum protein and autoimmune condition may also give positive reaction. In such condition the test result does not always indicate the actual disease.
• Low antigen level – If antigen or antibody is present in very small amount, the test may not detect it. Negative result does not always mean absence of disease. The amount may be below detection limit of the test.
• Early infection – In early stage of infection, antibody may not be formed in detectable amount. This period is before seroconversion. If test is done at this time, false negative result may be found.
• Effect of pH and temperature – The reaction depends on weak non-covalent forces. So change in pH, temperature and salt concentration can disturb the binding. Improper condition may reduce or stop the reaction.
• Interpretation problem – Some tests depend on visual reading. Weak clumping, faint precipitin line or small colour change may be difficult to judge. So human error may occur.
• Time consuming method – Some old antigen-antibody tests are slow and laborious. Complement fixation test and some precipitation methods need careful steps and proper control. So they are less convenient than automated methods.

Prozone Phenomenon

Prozone phenomenon is also called zone of antibody excess. It occurs when antibody is present in very high amount than antigen. Due to this the antigen-antibody reaction does not show visible clumping or precipitation.

In this condition, excess antibodies bind separately with all available epitopes of antigen. Each antigenic site becomes covered by antibody. But one antibody cannot make proper bridge between two antigen particles.

For visible reaction, antigen and antibody should form a large network. This network is called lattice formation. In prozone, lattice is not formed because antibodies are too much and they block cross-linking between antigen particles.

As a result no visible agglutination or precipitation is seen. The test appears negative, although antigen or antibody is actually present in the sample. This is called false-negative result.

Sometimes blocking antibodies or non-specific inhibitors present in serum may also produce prozone like effect. They interfere with normal antigen-antibody union and reduce visible reaction.

To avoid this error, serum is diluted serially. Dilution decreases antibody concentration. After proper dilution, antigen and antibody reach zone of equivalence and visible reaction is formed.

Comparison of Advantages and Limitations of Various Agglutination Tests

Difference between Prozone and Postzone Phenomena

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