Enzyme Immuno Assay and Radioimmunoassay

In this article we will discuss about the Enzyme Immuno Assay and Radioimmunoassay.

Enzyme Immuno Assay (EIA):

Hormones, Proteins, etc. present in blood in minute concentration can be assayed by the recent ad­vanced technique of “Enzyme Immuno Assay” without involving any disadvantage. The basic reaction is the interaction between an antibody and an antigen.

Enzyme immuno assay (EIA) is classified into two ways:

1. Homogeneous enzyme immuno-assay (EHIA).

2. Heterogeneous enzyme immuno-assay (HTEIA) or Enzyme-linked immuno­sorbent assay (ELISA).

1. HEIA:

(a) It has got only liquid phase.

(b) Haptens like hormones, drugs, etc. are la­belled with enzyme like lysozyme, malate dehydrogenase, etc. with no loss of enzyme activity. They will be made to react with a limited number of antibody molecules.

(c) There is competition between the enzyme-labelled hapten and the free hapten for the limited number of antibody molecules.

(d) No enzyme activity will be available if only the enzyme-labelled haptens are present since all of them unite with the antibody.

(e) No accommodation will be attained for some enzyme-labelled haptens depending on the concentration of free haptens in a mixture of free haptens and enzyme-la­belled haptens. A colour will be produced after the reaction of this with the substrate.

(f) The intensity of colour is measured in a photometer.

(g) It is called homogeneous EIA since the reaction takes place in liquid phase only and is applicable for T₃, T₄, estriol, Corti­sol, etc.

(h) This technique couples the three aspects-immunology, enzymology, and photom­etry.

2. Heterogeneous EIA:

(a) The antibody is fixed to the wall of a small test tube.

(b) The test sample contains the antigen to be estimated.

(c) Only the antigen is taken up by the anti­body when the test sample is added. Any­thing else contained in the medium is re­moved by aspiration and rinsing. This is so-called bound-free separation.

(d) The competition principle is applicable in this type also just like homogeneous EIA.

(e) A competition is held for the antibody sites between the un-labelled antigen and labelled antigen.

(f) Some antigens which are bound may be labelled while some others may be un-la­belled.

(g) The extent of the labelled antigen bind­ing to the antibodies is governed by the concentration of un-labelled antigen in the specimen.

(h) The specimen liquid is then aspirated out and the tube is rinsed with a suitable liq­uid.

(i) Then the test tube is treated with H₂O₂ and a chromogen.

(j) The peroxidase attached to the antigen and fixed to the wall via the antibody then reacts with the substrate to form a colour.

(k) The absorbance of the colour is measured in a photometer.

(l) The concentration of the enzyme-labelled bound antigen is decided by that of the un-labelled antigen.

(m) The latter can be assayed using a standard curve with known amounts of un-labelled antigen.

In some cases, instead of development of col­our, turbidity may be produced. The increase in turbidity due to the antigen-antibody reaction is measured in a cuvette.

But in the titration principle, insulin labelled with enzyme is made to bind, to antibodies in com­petition with un-labelled insulin. The sites are filled with enzyme-labelled insulin in titrable quanti­ties.

The antigen can bind to more than one site of the antibody when applied to a-fetoprotein on the basis of Sandwich principle.

This states as follows:

1. It is involved in filling the antibody sites fixed to the wall with a-fetoproteins in the serum of a pregnant woman.

2. The antigen has multiple sites for antibody binding.

3. The antibody labelled with the enzyme is now added. This labelled antibody at­taches to the site still free on the antigen.

4. The contents are aspirated.

5. The greater the amount of antigen, the greater the amounts of labelled antibody remaining on the wall.

6. Colour reaction is performed for estimat­ing the enzyme.

Important points to be noted:

1. Except glass tube, polystyrene microtitre plate, cellulose, isothiocyanate and polyacrylamide can be used for fixing the antibodies.

2. The enzyme label for the antibody may be alkaline phosphatase, glucose oxidase, β-Galactosidase, glucose-6-phosphate de­hydrogenase, and penicillinase.

3. Except hormones, immunoglobulin’s, α- fetoproteins, etc. the antigens can be purified virus and virus components, bacterial antigens, tetanus or diphtheria toxoids, fungal antigens etc.

Radioimmunoassay (RIA):

1. It has significant role in the diagnosis of diseases.

2. The gravimetric and the colorimetric methods fail when the substances to be analysed is in very low concentrations.

3. Even the nanogram amounts of radio-labelled substance give suitable counts de­tectable by electronic counters. These counts are related to the concentration of the substance assayed.

4. Radioimmunoassay is employed for the estimation of Vitamins like B₂, and folic acid ; hormones like insulin, thyroxine (T₄), triiodothyronine (T₃), Cortisol, testo­sterone, dihydrotestosterone, estrogens; trophic hormones like ACTH, FSH, LH; drugs like digoxin, digi-toxin; antigens like the Australia antigen.

The Principle of the method:

1. A radio-labelled antigen (e.g., insulin la­belled with l¹²⁵) is made to compete with an unstable antigen, e.g., insulin to be esti­mated in the serum of a patient) for a lim­ited number of binding sites of a specific antibody raised against insulin.

2. The antigen binds to the antibody.

3. Owing to inadequate binding sites, some of the antigens will be free and will in­clude radio-labelled antigen also.

4. After equilibrium, the antigen-antibody complex is precipitated by using suitable reagents.

5. The supernatant is separated from the pre­cipitate by centrifugation.

6. Both the precipitate (the bound antigen, B-form) and the supernatant (the free anti­gen, F) will have radioactivity since they have I¹²⁵ – insulin.

7. Dextran – coated charcoal if is used, it re­moves the free antigen. The supernatant will have the bound form.

8. The extent of radioactivity of the two forms is measured in gamma ray well type scin­tillation counter.

9. The magnitude of radioactivity of the free form may be related to the concentration of the un-labelled antigen.

10. Alternately, the radioactivity of the sound form or the ratio of B/F is also related to the concentration of the un-labelled anti­gen.

11. Different concentrations of the un-labelled insulin standard are used separately with the same concentration of the labelled in­sulin.

12. A graph is prepared with the radioactivity of the free form and the concentrations of the standard insulin. The serum whose in­sulin content is to be determined is treated in the same way.

13. From the radioactivity of the free form and extrapolation, the concentration of the in­sulin in serum is determined.

14. The assay is very sensitive since the la­bels used for R1A have high specific ac­tivity.

15. Normally, an antibody is raised for any antigen to be estimated. The technique is said to be radio immuno-assay since it couples radio activity and immune func­tion (antigen binding to antibody).

Importance of RIA:

1. The receptor sites can even be assayed in addition to the estimation of hormones proper.

2. The patient does not require to take the radioisotope as the label is used only in vitro.

3. RIA has significance in diagnostic bio­chemistry. RIA comes in handy for the es­timation of T₃ and T₄ separately.

4. In some thyroid disorders, T₄ may be nor­mal but T₃ is elevated as in subclinical hyperthyroidism, ophthalmic Graves dis­ease, autonomous thyroid nodules, T₃ toxicosis, etc.

5. An apparent increase of the thyroid hor­mones is observed in pregnancy or in per­sons taking oral contraceptives due to the increased level of the serum thyroxine-binding globulin.

6. RIA can help to differentiate the basic bio­chemical lesion in endocrinology whether the increased level of a hormone is due to the production of the hormone as such or the tropic hormone.

7. This technique offers safety to the patient in the use of drugs if there is only a narrow margin between the therapeutic and toxic dosage. This is applied during digitalisation in the management of congestive heart failure.

8. Low levels of serum digoxin mean under digitalisation whereas high levels are toxic. The patient is given the optimum digitalisation by RIA of digoxin in serum.

9. This technique is also useful in diagnos­ing insulinomas, sex hormone sensitive tumors, etc. and this facilitates proper treatment of the diseases.