13 Immunotechnology-II

Dr. M. N. Gupta

  1. Objectives

To understand:

  •  In many cases concentration of the antigen or antibody is too small to be measured by their precipitation or by following an agglutination
  •  The higher sensitivity in measuring immunocomplexes is achieved by labelling antigens/antibodies
  •  These labels can be a radioactive one, an enzyme or simply an affinity ligand
  1. Concept Map

 

  1. Description Primary Binding Tests

In the earlier module, we looked at some methods which allow us to detect/measure antigen or antibody in a given sample. These methods were based upon some phenomena which are a consequence of antigen-antibody interaction: precipitin formation and agglutination. There are another set of methods which are called primary binding tests. These tests/methods are based upon the formation of immune complex under equilibrium conditions. The first common key strategy element in these methods is to separate the immune complex. The second common key strategy is to use some “label” which allows us to measure the amount of immune complex. For any given pair of antigen and antibody (we have already learnt), there is an equilibrium constant known which is governed by avidity. So, if one of the pair (say antibody) is kept constant, the amount of the other member of the pair (antigen) in the immune complex will depend upon the amount of this antigen present in the immune complex. If the antibody is “labelled”, the amount of label ending in the complex will depend upon the antigen concentration!

 

The two labels which are discussed in this module are : a.) a radionuclide. Generally Iodine radio isotopes are used. The amount of radionuclide in the immune complex can be directly measured in a gamma counter. b.) an enzyme. The amount of enzyme is measured in terms of units of activity. Hence the enzyme (in the bound form) is assayed under the standard assay conditions using a well known substrate. In principle, all the numerous conditions used for enzyme assays can be used. In practice, enzymes generating coloured products are used. This enables such assays to be conducted in multititre plates.

Primary binding tests in general are more sensitive than secondary binding tests which we discussed in the last module.

 

Radioimmuno Assays (RIA)

 

S.A. Berson and Rosalyn Yalow developed RIA in 1960 for quantifying the amount of insulin present in plasma. Rosalyn Yalow was awarded the Nobel Prize in 1977 for the development of RIA.

 

RIA can measure an antigen at pictogram level (10-12 g). The wide range of applications of RIA include:

  • Hormones
  • Steroids
  • Drugs
  • Prostaglandins
  • Viruses
  • Surface antigens

 

Farr Method

 

This method is generally used for the estimation of an antibody against an antigen in the given antisera. Hence, Antigen is labelled and added in a reasonable excess to the antiserum. The amount of label ending in the antigen antibody complex is proportional to the antibody present in the antiserum.

 

Farr method relies upon the precipitation of immunoglobulins in the presence of 40-50% ammonium sulphate. It is assumed that the antigen is not precipitated under these conditions, although precipitation of the uncomplexed antibody expectedly does take place and does not matter. The method in its unmodified form cannot be used with those antigens which also precipitate under these conditions. Subsequently, 2% polyethylene glycol (PEG) has also been used in place of ammonium sulphate.

 

 

Figure 3: Determination of an antigen combining capacity of an antiserum using antigen coupled to 125I or some other label. The radioactivity of the precipitate provides a measure of the antigen-combining capacity

 

The Farr method or Farr technique can be used with a small amount of antiserum and is more precise than agglutination method described in the last module.

 

Anti-immunoglobulin Procedure

 

In a variation of the Farr technique, salt or PEG can be replaced by anti immunoglobulin for precipitation of the complex and free antibody. This makes it possible to work with those antigens which are precipitated with 40-50% ammonium sulphate or even 2% PEG.

In all variants of Farr technique, adequate amount of the non specific immunoglobulins are incorporated to facilitate complete precipitation.

 

The dissolution of the complex, for example in SDS enables radioautography of the SDS-PAGE. Anti immunoglobulin procedure also has one additional versatility. The distribution of antibody among the various classes of immunoglobulin can be estimated. For example, if anti-IgA is used as the precipitating immunoglobulin, only free IgA and IgA-antigen complex from the population of Ig in antibody will be precipitated and be estimated.

 

RIA for antigens

 

Quite often, it is the antigen which needs to be assayed in biological fluids such as blood sera or in similar complex mixtures. In such cases, the antigen can be added to a microtitre plate. The binding to the plate material is through non covalent bonds. The unbound antigen is washed away. The rest of the plate surface is blocked by a protein such as BSA to prevent non-specific binding during the subsequent steps. The test antibody is added. Unbound antibody is washed away. Now, a labelled ligand is added which is often anti-immunoglobulin to the known test antibody or even IgA. The bound radioactivity can be counted. A standard curve between the radioactivity vs amount of test antibody added can be generated. A short linear range (over and above the background count) before the plateau region is obtained. The plateau binding should be about 20-100 times the background in a well run assay.

 

The competition format makes sure that the result is indeed due to specific binding between the suspected antigen and test antibody. Also, it allows a greater linear range. In this format, rather than addig just the test antibody, increasing amount of free suspected antigen is added along with a fixed amount of test antibody. Higher this amount of right free antigen, lower is the subsequent count. The method is based upon the fact that antigen –antibody complex doest not bind to the antigen bound to the plate.

 

The Radioallergosorbent Test (RAST)

 

RAST is essentially a RIA adopted to estimate IgE specific to a suspected allergen. In both classical RIA and RAST, the antigen (allergen in RAST) can also be covalently bound to the plate. In this case the radiolabelled ligand is the immunoglobulin against human IgE.

 

Radioimmunosorbent Test (RIST)

 

RIST is a RIA conducted in the competition format for measuring the total serum IgE. In RIST, the surface of the well or even a cellulose disc has the anti-IgE immunoglobulin bound to it. Now increasing amount of labelled IgE (acting as antigen in this case!) is added to determine 80% of the maximum IgE which can bind. In the second stage, this amount of labelled IgE (antigen) is mixed with the test serum (in which IgE is being estimated) and added to the well containing the anti-IgE. Both labelled IgE and unlabeled IgE in the test serum competes with the bound anti-IgE (antibody). As before, a standard curve can be generated.

Sometimes, the non-competitive RIST is also used. In this case, the patient antibody is added to the solid phase anti-IgE. It binds specifically. As a second step, the enzyme labelled anti-immunoglobulin is added and it binds to the patient antibody. The excess enzyme is washed away. The color development is proportional to the patient antibody present.

Radiolabelling

 

Two γ-emitting radioisotopes are available: 125I and 131I. The advantage of γ-emitter isotopes is that the label can be estimated directly in a γ-counter and the count rate is much higher than β-emitting radionucleotides. Of the two radio isotopes, 125I with a higher half life of 60 days is preferred.

 

The Chloramine T method

 

This is the most widely used method for radioiodination. This is also most extensively used for labelling antibodies. Radioactive I is oxidized by Chloramine T. However, this involves strong oxidizing conditions and it is not recommended for proteins which are likely to denature under such conditions.

The Lactoperoxidase Method

 

The enzyme lactoperoxidase oxidizes 125I- to 125I2 or 125I+. These radioactive specie iodate mostly the tyrosine residues. The oxidation can be stopped by dilution. In this case the presence of traces of H2O2 is necessary. While it is a milder method as compared to Chloramine T, but the optimum reaction conditions vary greatly from system to system.

The Bolten-Hunter Method

 

This is an aylation method. The acylating agent is N-(succinimidyl-3-[4-hydroxyphenyl]propionate).

 

A water soluble Bolten Hunter reagent is also available.

 

This acylating agent reacts with Lys side chains. This is valuable for proteins which lack Tyr or His residues or are essential residues for activity. Also, addition of a hydroxyphenyl group makes it possible to further iodinate the protein.

 

Enzyme Linked Immunosorbent Assay (ELISA)

 

While about 20 different enzymes have been used in enzyme immunoassays, three are most commonly used:

Alkaline phosphatase, beta-galactosidase and Peroxidase

 

The main considerations are that these enzymes are inexpensive, ready to assay, stable and have good turnover number. If required appropriate substrates which generate fluorescent products enhances the sensitivity of the method are also used.

 

In this system, the ligand is a molecule that can detect the antibody and is covalently coupled to an enzyme such as peroxidase. This binds the test antibody, and after free ligand is washed away (6) the bound ligand is visualized by the addition of chromogen (7) – a colorless substrate that is acted on by the enzyme portion of the ligand to produce a colored end-product. A developed plate (8) is shown in the lower panel. The amount of test antibody is measured by assessing the amount of colored end product by optical density scanning of the plate

Sandwich ELISA

Difference between ELISA and Sandwich ELISA

 

These assays are called direct assays as these are not dependant upon a consequence of antigen-antibody interaction , e.g.: precipitin reaction or agglutination. The separation of the bound label (actually labelled reagent) from unbound label is a common strategy. The bound label is quantified. When a solid support is used, such as in RIA or ELISA, non specific binding id avoided by prior coating of the surface with various materials such as BSA.

 

RIA or ELISA, both can be used to quantify antigen or antibody. However, in each one, one has to be clear about the strategy. Neither methods allow direct measurement of either antigen or antibody when it is present in a sample of unknown composition. However, the format of the competitive inhibition assay allows it as the antigen in the unknown sample competes for pure labelled antigen. A standard curve is generated with unlabelled antigen and the labelled antigen is used to quantify the antigen in the given sample of unknown composition.

 

Summary

 

In this lecture, we learnt:

  •  Antigen and antibody can be estimated by mixing them. One of these is labelled. Which one is labelled depends on what is being estimated and what is the design of the immunoassay.
  •  In RIA and its variants like RAST and RIST, the label id generally 125 Some methods which are generally used for labelling antigen or antibody were also outlined.
  •  In ELISA, the label is an enzyme which generates a coloured product.