12 Immunotechnology-I

Dr. M. N. Gupta

1. Objectives

To understand:

a.) What experimental techniques are used to understand the functioning of the immune system

b.) What techniques are based upon cells/molecules related to the immune system

c.) How some of these techniques are now the basis of invaluable diagnostic tests/design of biosensors for analytes/infectious agents.

2. Concept Map

3. Description

Immunization

Immunology started with efforts to understand how our bodies respond to infectious agents and what we can do to avoid/minimize consequences of this invasion. However, right in the early years, immunologists developed protocols to raise antibodies in experimental animals against known (well defined or ill defined) antigen preparation.

Choice of the experimental animals

1. The animal should be healthy and as far removed as possible on the evolutionary tree from the source of the antigen. As we learnt in the module on antigens and epitopes, “foreignness” is one essential criterion for antigenicity

2. Depending upon the amount of anti-serum required, the animal with appropriate size has to be chosen.

Adjuvants

Adjuvants are substances which when mixed with an immunogen or antigen (before injection), results in a better immune response from the host. In practical terms, higher amount of antibody are obtained in the antiserum.

In cases, the idea is to study the primary response and the secondary response separately; the adjuvant should not be used during immunization. This is because the adjuvants function by avoiding dispersal of the antigen rapidly from the injection site and helping the antigen persist for a larger time in the tissues.

Jules Freund described water-in-mineral oil emulsions. Herbert in 1968 confirmed that antigens injected along with such an emulsion persisted upto 544 days. On the other hand oil-in-water emulsions were nowhere good. Such emulsions are called Freund’s incomplete adjuvant. There is a complete Freund’s adjuvant also described which additionally has dried heat killed bacteria such as Mycobacterium tuberculosis. The presence of glycolipid in this also enhances cell mediated immunity and activity of the other associated immune system members such as macrophages.

The mode of action of some adjuvants is further illustrated in the following figure:

Freund‟s complete adjuvant is not used in humans as it produces chronic inflammation around emulsion depots.

Many adjuvants are specifically used for stimulation of pathological conditions in animals. For example, Complete Freund‟s adjuvant can induce autoimmunity.

Finally, many immunoenhancing drugs can also be used as adjuvants. For example, levamisole, Isoprinosine and Avridine.

The Precipitin Reactions

The antigen molecules combine with the antibody molecules in a serum within few minutes. When univalent haptens are used as antigens, these antigen-antibody complexes remain in soluble form. Same is the case if monovalent antibody fragments Fab are used in place of antibody molecules.

If multivalent antigens and atleast a bivalent antibody molecule is used, after 12-24 h, a precipitate becomes visible. This precipitate results from the formation of a lattice like structure. This is called precipitin reaction.

At the equivalence zone, the precipitate is maximum. In this equivalence zone, most of the antigen and antibody molecules are part of this precipitin complex and do not exist free.

The precipitation was studied by Kraus in 1897. Kraus also established that this test works only with homologous antiserum.

This technique can measure the potency of different antisera against a given antigen. It can also establish relative heterogeneity of the given antigen-antibody pairs.

The precipitate can be weighed after centrifugation and thus the method adapts itself to quantification.

The mechanism of precipitate formation was studied by Marrack, Heidelberg and Kendall.

The interlocking three dimensional lattice after a critical size cannot remain in solution.

Sedimentation rate of the lattice α V x (ρ – ρ0) x g

Where:

V= volume of the complex

ρ = density of the particle/complex ρ0 = density of the solvent medium

Protein antigens in the molecular weight range of 40 kDa-160 kDa tend to result in precipitin curve with a sharp peak. Denatured proteins, some viruses, polysaccharides on the other hand tend to result in precipitin curve with a broad maxima.

Amino acid composition at the antigen binding site of the antibody plays an important role in forming this lattice. Similarly, highly charged antigen molecules disrupt this lattice formation due to electrostatic repulsion and do not form well defined precipitin curve.

Precipitin Reactions in Gels

Oudin in 1946 demonstrated that precipitin reactions can be carried out in agar gels. Antigen and antibody molecules can diffuse through the semi-solid agar-gel and give precipitin reaction at a point where their relative concentrations fall in the “equivalence zone”.

Agar gels are poured into the petriplates and allowed to set. Generally 1-2% agar in buffer at pH 7-8.5 is used. Wells are then punched into the gel and the test solutions of antigen (Ag) and antibody (Ab, generally in the centre) are added in different wells. The solutions diffuse out and where Ag and Ab meet they bind to each other, crosslink and precipitate leaving a line of precipitation. Depending upon the different types of antigens which react with the antibody, different lines of precipitation emerge.

The double diffusion method developed by Ouchterlony is very useful in establishing the identity and non-identity in antigen and antibody samples.

The double diffusion technique may be used to determine the relationship between antigens and a particular test antibody. Three basic patterns appear. In the case (A) (that is reaction of identity) the precipitin arcs formed between the antibody and the two test antigens fuse indicating that the antibody is precipitating identical epitopes in each preparation. In the case (B) (that the case of non –identity), the antibody recognizes the two different antigens and develops two different precipitation lines which intersect. In case (C) (that is the case of partial identity) the antibody recognizes that the two antigens share the same epitope but the second antibody also has an additional epitope. Thus a spur appears.

These reactions can also be done in agar plates as seen earlier. The precipitin bands can be better visualized by washing the gel to remove the soluble proteins and then staining the precipitin arcs with a protein stain such as Coomassie Blue.

Immunoelectrophoresis

This essentially has two steps:

1.) Separation of various antigens in a sample by electrophoresis in agar gel

2.) Double diffusion analysis with the corresponding sera containing antibody for various antigens

Immunoelectrophoresis was developed by Grabar and Williams in the 1950s. It is very useful clinically. The antibodyconcentration in the range of 3-20 µg/ mL of the antiserum can be detected qualitatively.

Rocket Electrophoresis

This is a variation of Immunoelectrophoresis and is also called Laurell technique. It is a single step method as the antibody containing the anti-serum is incorporated in the agar gel itself. The precipitin arcs have the shape of rockets. The pH is chosen so that the antibodies contained in the gel are immobile and the antigens are negatively charged. The precipitin arcs have the shape of rockets. The height of the rocket is proportional to the antigen concentration. The sensitivity of this method is 10-20 fold more than the double diffusion method.

Agglutination Reactions

If the antigens are in the form of cells (such as RBCs) or particles (antigens immobilized on latex beads), the reaction between these multivalent „antigens‟ and bivalent or multivalent antibody forms clumps. The clump formation is maximum in the equivalence zone. However, in the quantitative version, generally serial dilution technique is used to determine maximum dilution of the antiserum at which clump formation can be detected.

The clump formation is the result of agglutination of the antigen via crosslinking with the antibody molecules.

The highest dilution at which agglutination/clump formation is observed is called the titre value.

The agglutination tests are semi-quantitative but are sensitive enough to detect antibody concentration of 0.001 µg/ mL of the serum.

The method provides a simple, rapid and sensitive way of identifying type of RBCs, bacteria, fungi and other cells.

Summary

In this lecture we learnt about:

a.) Role of adjuvant in immunization

b.) The precipitin reactions in solution

c.) The precipitin reactions in agar gels

d.) Immunoelectrophoretic techniques

e.) Agglutination reactions