Immunology Biochemistry - epgp.inflibnet.ac.in

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Biochemistry Immunology

Immunotechnology-I

Paper : 16 Immunology Module : 11 Immunotechnology-I

Principal Investigator

Dr. Sunil Kumar Khare,Professor

Dept. of Chemistry,

I.I.T. Delhi

Content Reviewer:

Dr. M.N.Gupta, Emeritus Professor

Dept. of Biochemical Engg. and

Biotechnology, I.I.T. Delhi

Paper Coordinator

and

Content Writer

Dr. Prashant Mishra, Professor

Dept. of Biochemical Engg. and

Biotechnology, I.I.T. Delhi

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Immunotechnology-I

Description of Module

Subject Name Biochemstry

Paper Name 16 Immunology

Module Name/Title 11 Immunotechnology-I

Dr. Vijaya Khader Dr. MC Varadaraj

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Immunotechnology-I

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

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Immunotechnology-I

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.

Figure 1

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Immunotechnology-I

Figure 2

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.

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Immunotechnology-I

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.

Table 1: List of some commonly used adjuvants and the way they help in the increase

in antigenicity

Adjuvants Mechanism

Compounds of aluminium and calcium

beryllium sulphate

Freund’s adjuvants

Creates antigen defects

Creates and stimulates T-cells

Creates and enhances via other

mechanism as indicated in the text

Mycobacteria Facilitates antigen processing and T-

cell response

Bordetella pertulis Increases response in terms of B and T-

cells, macrophages

Bacterial lipopolysaccharides Increases B-cell activity

Saponin, lysolecithin analogs, Vitamin A and

E, Polyanions and liposomes

Facilitates antigen processing

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

Figure 3

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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.

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Figure 4

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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.

Figure 5

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.

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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”.

Figure 6

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Figure 7

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.

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Figure 8

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

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Immunotechnology-I

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.

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Figure 11

A slight modification of rocket electrophoresis: 2 D method

Figure 12

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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.

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Figure 13

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.

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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

Immunology Biochemistry - epgp.inflibnet.ac.in

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