ANTIGEN – ANTIBODY INTERACTIONS

BY: RIA FAZULBHOY (MSIWM 031)

Antigen- antibody interactions are used to detect a number of immune diseases, check for humoral immunity and identify biological molecules. There is noncovalent interaction between the epitopes/antigenic determinants of antigens and the variable region (Vh & Vl) domain of antibodies. Noncovalent bonds include ionic bonds, hydrophobic bonds, hydrogen bonds and van der Waals forces.

Noncovalent bonds between antigen and antibody

Different Antigen – Antibody interactions:

  1. PRECIPITATION REACTIONS:

Precipitation reactions occur between antibodies (Ab) and soluble antigens (Ag) present in aqueous solution. They bind by noncovalent bonds to give Ag-Ab complexes known as lattices, which are in turn seen as precipitate. The term precipitins is given to antibodies which aggregate with soluble antigens.

The formation of Ag-Ab lettuces depends on the valencies of both antigens and antibodies:

  • Antibodies must be bivalent.
  • Antigens must be bivalent or polyvalent.

Precipitation reactions in fluids form a precipitation curve:

  1. Constant amount of antibodies is taken in a series of test tubes.
  2. Soluble antigens are added in an increasing amount to each test tube.
  3. Precipitates are formed in each test tube, and this precipitate is then centrifuged in order to form a pellet. Amount of precipitate is measured by the pellet.
  4. By plotting the graph of the amount of precipitate against increasing antigen concentration, we get a precipitin curve.
  5. Maximum precipitation occurs in the zone of equivalence where ration of antigen : antibody is optimum.
  6. If there is an excess of antigens or antibodies, such extensive lattices are not formed and precipitation is not seen.

                                                                       Excess Antibodies   Equivalence   Excess Antigens

    2) AGGLUTINATION REACTIONS

Definition of agglutination states that it is the interaction between antibodies and particulate antigens which results in visible clumping known as agglutination. Antibodies participating in such reactions are called agglutinins. Agglutination reactions gave a principle similar to precipitation reactions (based on cross-linking of polyvalent antigens). Excess of antibodies inhibits agglutination and this effect is known as the prozone effect.

There are 2 types of agglutination reactions:

  1.  Active (natural) agglutination
  2. Epitopes of the antigen are naturally found on the test particle.
  3. Eg. Antigens found on RBCs, bacteria, and fungal cells
  • Passive (chemically fixed) agglutination
  • Epitopes and soluble antigens do not occur naturally on the surface of the cells or particles
  • They need to be chemically fixed onto either RBCs (with the help of tannins/ chromium chloride) or synthetic materials like latex beads and polystyrene.
  • The synthetic materials offer more stability, uniformity and consistency.
  • Eg: soluble antigens, viral diseases.

            Examples of agglutination reactions

  1. Hemagglutination in blood typing

This is done to detect the blood group of patients and carry out proper blood transfusion. In typing for ABO antigens, red blood cells are mixed on a slide with antisera to the A or B blood group antigens. If antigen is present on the cells, they visibly clump due to agglutination taking place.

  1. Agglutination inhibition

Agglutination inhibition is used to detect use of illicit drugs and also used in pregnancy tests. It is also used to detect viral infections in patients.

3) RADIOIMMUNOASSAY (RIA)

Radioimmunoassay is a technique used to detect the binding of antigen and antibodies in the given sample. It is based on the principle that there is a competition for binding between radio-labelled antigens and unlabelled antigens when they are in the same vicinity as high affinity antibodies. The antibody does not distinguish between labelled and unlabelled antigens, thus there is competitive binding between the two.

>The radio-labelled antigen is generally labelled with:

  • Gamma emitting isotope like I125
  • Beta emitting isotope like 3H (tritium)

>The test sample which contains unlabelled antigens is a complex mixture like serum or other body fluids.

How does Radioimmunoassay take place?

  1. First the radio-labelled antigen (Ag*) is mixed with the antibodies at a concentration such that the antigens saturate the antigen binding site of the antibody. This concentration of antibodies which should bind to labelled antigens should be anywhere between 50-70%.
  2. An increasing amount of unknown test sample of non labelled antigens is added to the mixture.
  3. As the amount of non labelled antigens increase and bind to the antibody, the number of radio labelled antigens which bind to the antibody decreases. They compete to bind to the samples.
  4. To determine the amount of labelled and non labelled antigens bound to the antibody, the Ag-Ab complex is precipitated to separate from unbound, free antigen.
  5. Unbound antigens are separated by various methods like use of formalin killed S.aureus, other antibodies which react with free antigens, use of solid-phase RIAs, etc.
  6. The precipitated Ag-Ab complex’s radioactivity is measured with the help of a radiation counter.
  7. A standard curve can be obtained in order to plot and determine the amount of antigen present in the test sample.

Radioimmunoassay (RIA)

BY- SAI MANOGNA (MSIWM014)

Radioimmunoassay (RIA):

Radioimmunoassay (RIA) is one of the most responsive antigen or antibody detection techniques. In 1960, this procedure was first developed by two endocrinologists A. Berson and Rosalyn Yalow, to evaluate levels in diabetics of insulin-anti-insulin complexes. While their technique addressed some skepticism, at concentrations of 0.001 micrograms per millilitre or less, it soon proved its usefulness for testing hormones, serum proteins, medicines, and vitamins. The importance of the technique was recognised in 1977, some years after Berson ‘s death, by the granting of the Nobel Prize to Yalow.

Principle : RIA’s technique involves the competitive binding to a high-affinity antibody of radiolabeled antigen and unlabeled antigen. The labeled antigen is mixed with an antibody at a concentration that saturates the antigen binding sites of the antibody.. Then, in increasingly greater quantities, test samples of unlabeled antigen of unknown concentration are added. The antibody does not differentiate between labelled and unlabeled antigen, so the two kinds of antigen compete against the antibody for available binding sites. If the concentration of unlabeled antigen increases, it will displace more labelled antigen from the binding sites. In order to assess the amount of antigen present in the test sample, the decrease in the quantity of radiolabeled antigen bound to a particular antibody in the presence of the test sample is measured.

Procedure :

A gamma-emitting isotope such as 125I is commonly labelled with the antigen, but beta-emitting isotopes such as tritium (3H) are often used as labels. The radiolabeled antigen is part of the assay mixture; a complex mixture, such as serum or other body fluids, containing the unlabeled antigen (test sample).

1. The first step in setting up an RIA is to decide the amount of antibody required in the assay mixture to bind 50 percent to 70 percent of a fixed amount of radioactive antigen.

2. Unlabeled antigen applied to the sample mixture would also compete for the limited supply of antibodies with radiolabeled antigen. (Even a small amount of unlabeled antigen added to the labelled antigen and antibody assay mixture will cause the amount of radioactive antigen bound to decrease, and this decrease will be proportional to the amount of unlabeled antigen added).

3. The Ag-Ab complex is precipitated to distinguish it from free antigen (antigen not bound to Ab) to assess the quantity of labelled antigen bound, and the radioactivity in the precipitate is calculated.

4. Using unlabeled antigen samples of a known concentration (in place of the test sample), a standard curve can be produced and the amount of antigen in the test mixture can be accurately calculated from this map.

For the separation of the bound antigen from the free antigen in RIA, several methods have been established. One strategy involves precipitating the complex of Ag-Ab with a secondary antiserum anti-isotype.

For example, if rabbit IgG antibodies are included in the Ag-Ab complex, then goat anti-rabbit IgG will bind to rabbit IgG and precipitate the complex.

Another technique makes use of the fact that Staphylococcus aureus protein A has a high IgG affinity. If the Ag-Ab complex produces an IgG antibody, it can be combined with formalin-killed S to precipitate the complex. The amount of free labeled antigen remaining in the supernatant can be determined in a radiation counter after removal of the complex by one of these methods; subtracting this value from the total amount of labelled antigen added yields the amount of labeled antigen bound.

Advantages:

  1. Used to detect very small amounts of serum antigens and antibodies.
  2. Used to quantify hormones, pharmaceutical products, HBsAg and other viral antigens.
  3. Analysis of nanomolar and picomolar concentrations in biological fluids.

Disadvantages:

  1. Cost effective
  2. Radio-labeled compounds have short shelf life
  3. The concerns surrounding the handling of radioactive (nuclear) wastes.