Antigen-Antibody Reaction: Definition, Stages, Properties, and Types

What is Antigen-Antibody reaction?

“During an immune response, the antigens and antibodies interact with each other, called antigen-antibody reaction.” It is also known as Ag-Ab reaction. This reaction occurs under highly specific conditions. An antigen reacts only with antibodies made by itself or similar antigens.

This reaction is essential for humoral or antibody-mediated immunity. It helps the body detect infectious agents and certain non-specific antigens, such as enzymes.

Stages of Antigen-Antibody reaction

The antigen and antibody reaction mostly happens in vitro (outside the body), called serological reaction. This reaction involves three different stages:

1. The first stage includes binding antigens and antibodies to form an Ag-Ab complex.
2. The second stage displays the visible results, such as precipitation or agglutination.
3. The third stage involves the destruction or neutralization of antigens to prevent harm.

Salient Features of Antigen-Antibody Reaction

Here are some key features of antigen-antibody reaction:

1. Immune Complex

When antigen and antibody come close, they bind to each other, and a complex molecule is formed called an immune complex or antigen-antibody complex. These binding triggers complement activation (marking targets for removal) and cellular activation (phagocytosis, cell killing). This process helps the body fight infections but can damage tissues if overly activated.

2. Specificity

The antigen-antibody reaction is highly specific, meaning an antibody binds only with the antigen, which causes its production. The antibody’s binding site, like a lock and key, is designed for that specific antigen. For example, the antibody produced against kidney antigen will only bind with kidney-antigen.

3. Binding Site of Antigen-Antibody Reaction

The specific site of antigen, where the antibody attaches, is called epitope (also known as antigenic determinant). The epitope is the site of antigen that is recognized by antibodies. The part of the antibody that recognizes and binds to the epitope is called the paratope.

4. The binding force of Antigen-antibody reaction

The binding between an antigen and antibody depends on Closeness, non-covalent bonds, and antibody affinity (strength of the bond).

Properties of Antigen-Antibody reaction

• It is a visible reaction, meaning it can be observed directly.
• Occurs under specific conditions
• The antigen-antibody binding includes intermolecular forces, i.e. ionic bonds, hydrogen bonds, hydrophobic interactions, and Van der Waals forces.
• The antibody and antigen are not denatured. They maintain their original shape and function during and after the reaction.
• It is a reversible reaction.
• Affinity: It shows the strength of the bond between the antigen’s epitope and the antibody’s paratope, and is measured by affinity constant K.
• Avidity: It shows the total binding strength of the Ag-Ab complex. It involves affinity and the number of binding sites (valencies) on both antigen and antibody.
  • Avidity measures how well an antibody binds to a multivalent antigen.
  • A multivalent antigen has many epitopes (parts that trigger immune responses).
  • Each epitope stimulates the production of a specific antibody.
  • Different antibodies bind to the same antigen’s various parts (epitopes/determinants).
• Cross-Reactivity: Sometimes, an antibody binds to similar sites on different antigens. The antibody that performs this reaction is known as cross-reactive Ab.
  • The bonds involved in cross-reactions are weak.

Types of Antigen-Antibody Reaction

There are five types of antibody-antigen reactions, which are described below:

1. Precipitation Reaction
2. Agglutination Reaction
3. Complement Fixation
4. Immunofluorescence
5. ELISA – Enzyme-Linked ImmunoSorbent Assay

1. Precipitation Reaction

When antibodies react with a soluble antigen in an electrolyte (salt like NaCl) at a specific pH and temperature, they form an insoluble complex called a ‘precipitate’. The antibody that causes this precipitate is known as precipitin, and the process is called the precipitation reaction.

Precipitation takes place in two media:

a) Liquid Precipitation

An antigen-antibody reaction is performed by adding a constant amount of antibody to many tubes, and then an increased amount of antigen is added to the tubes. The antibodies and antigens attach to each other and form a precipitate. We can get a precipitation curve by plotting the amount of precipitate against increasing antigen amount.

The precipitation curve displays three zones:

• Antibody (Ab) axis zone
• Equivalence zone
• Antigen (Ag) axis zone

b) Gel Precipitation

In this method, plates or petri dishes are used with gels. Both the antibody and antigen spread (diffusion) freely through the gel in all directions. Based on the diffusion rate and number of reactants, they form a visible precipitate at a specific point, known as a zone of equivalence.

Multiple bands can form if the antigen and antibody preparations are complex. It involves two types: single diffusion and double diffusion.

2. Agglutination Reaction

The reaction in which an antibody binds with a particular (insoluble) antigen results in clumping, is known as agglutination. It occurs in the presence of electrolytes, suitable pH, and temperature. The antibodies that cause this reaction are called agglutinins, and the antigens that form these clumps are Agglutinogens.

Agglutination reactions involve the following types:

a) Slide Agglutination

This method is used to check the quantity of agglutinating antibodies. A uniform antigen sample is added to a drop of saline and antiserum on a ‘slide’. Then, the slide is mixed gently. If it shows the clumps, the test result is positive. It takes a minute and is visible to the naked eye or can be checked under a microscope (if needed).

b) Tube Agglutination

In this method, the serum is diluted with saline in ‘test tubes’, adding a specific amount of antigen. A control tube without serum is also placed. The tubes are incubated, and the visible clumps appear. The tube with the highest agglutination level is known as ‘titre’. This test is used to diagnose several diseases. 

c) Passive agglutination

This test is like the haemagglutination test, but the physical nature of the reaction is changed. Antigens are attached to particles like RBCs, latex beads, or bentonite. This way, a precipitation reaction is changed into an agglutination reaction. Sometimes, RBCs with polystyrene coating (tanned RBCs) are used. When these particles are added to the patient’s serum, clumping (agglutination) occurs. This test is used to diagnose rheumatoid arthritis.

3. Complement Fixation

In this test, a complement system includes a group of 11 proteins in serum to check for the antigen-antibody reaction.  In the first step, the serum is heated to 56°C (to inactivate its natural complement). Then, the test antigen (Ag) and serum are mixed with a known amount of complement and are incubated for 18 hours at 4°C. If the antibody (Ab) specific to the antigen (Ag) is present in the serum, an antigen-antibody complex is formed, which binds the complement,

4. Immunofluorescence

Fluorescence is the ability to absorb light with one particular wavelength and emit light with a different wavelength. Fluorescent dyes glow brightly when exposed to UV radiation as they convert UV light into visible light.

In 1942, Coons et al. described that fluorescent dyes, along with antibodies, can be used to detect antigens. For example:

• Fluorescein is a commonly used dye, which absorbs blue light of 490 nm and emits a bright yellow-green light of 517 nm.
• Phycoerythrin absorbs light effectively and is 30 times more efficient than fluorescein. It emits a bright red fluorescence. That’s why it is used widely as a popular label for immunofluorescence techniques.

5. ELISA – Enzyme-Linked Immunosorbent Assay

In 1971, some sensitive tools, like enzyme-labelled antigens and antibodies, were developed to detect antigens and antibodies. These tests are more safe and affordable than radioimmunoassay (RIA). In this process, a ligand molecule attached to the antibody is combined with an enzyme like peroxidase or alkaline phosphatase.

ELISA involves three types:

a) Indirect ELISA

This test is used to detect HIV. The proteins that are developed by recombinant technology are coated on a plate. Then, the patient’s serum is mixed with it to check the presence of antibodies.

b) Sandwich ELISA

This method is used to detect antigens in a sample. An antigen-specific antibody is coated on a plate, and the sample is added. The labelled antibody detects the antigen, and the colour change can indicate its amount. The more the intensity of colour, the more antigen is present.  After the reaction, the unbound antigen is washed out.

c) Competitive ELISA

This is another method to measure the antigen levels in the sample. Firstly, the sample containing the antigen is mixed with the antibody. Then, it is added to an antigen-coated plate to check the antigen in the sample. If the amount of antigen is high, fewer antibodies will bind to the plate (as they are already attached to the antigen in the sample). That is how the enzyme-linked antibody helps detect the binding level. Then, a secondary antibody (linked with an enzyme) can be used to detect the quantity of antibodies that are attached to the plate.

Applications of Antigen-Antibody reaction

Here are some main applications of antigen and antibody reactions:

• It helps identify blood groups for safe blood transfusions.
• Diagnosis of infectious diseases.
• Detecting certain proteins in blood
• Measurement of substances in the body through immunoassays.
• Detection of specific immune system disorders.