Immunofluorescence Assay, Radioimmunoassay and Enzyme Immuno Assay

1. Immunofluorescence assay (IFA)

Fluorescence is the phenomenon exhibited by certain molecules or compounds they absorb light energy of one wavelength and emit light energy of higher wavelength. If a fluorescent compound receives the invisible UV light, it will emit a light within the visible spectrum.

For an example of fluorescent compounds used in this technique is fluorescein, rhodamine B etc.

Fluorescence is commonly used as an isothiocyanate (i.e. fluorescence isothiocyanate) which makes its subsequent attachment to antigen or antibody easier. The phenomenon of fluorescence, when used in an antigen-antibody reaction, is known as immunofluorescence.

The immunofluorescence technique can be of 3 types:

1. Direct immunofluorescence assay
2. Indirect immunofluorescence assay
3. Sandwich immunofluorescence assay

Direct immunofluorescence assay (Direct IFA)

Direct IFA can be used to identify an unknown antigen which can be attached to a solid phase solid as a microscopic slide. The clinical specimen which may contain a specific antigen is smeared on a glass slide. The air dried smear is fixed with acetone for 1-2 minutes. This fixed antigen is then reacted with a known fluorescent labeled antibody. Evidence of the fluorescence in the smear under fluorescence microscope indicates a positive test for the antigen in the specimen.

Indirect immunofluorescence assay

In indirect IFA, the reactive antigen and antibody are both unlabelled. The antigen-antibody complex then defeated by an antihuman immunoglobulin labeled with a fluorescent compound.

Sandwich IFA

Unknown antigen in the specimen can also be detected by the sandwich techniques known unlabelled antibody specific for the antigen is attached to the solid surface. The specimen which is being tested for the antigen is then added. After incubation and washing, fluorescence is detached it is an indication of the presence of antigen in the specimen. Here, the antigen is sandwiched between unlabeled and labeled antibody.

B. Radioimmunoassay (RIA)

Radioimmunoassay (RIA) is another level technique used for detection of an antibody or more commonly antigen. The isotope I¹²⁵ is the most commonly used detection system. In most cases, a direct RIA is employed. The direct assay is a two step procedure.

Steps:

1. Binding antigen to well of a microtitre plate.
2. Add excess antibody that is labeled with I ¹²⁵; wash to remove unbound antibody.
3. Count antibody in gamma- radiation counter. Labeled in 1, 2, 3, and 4 established a standard curve with known amount of antigen. The radioactivity in the last well indicates, by comparison to the standard curve, how much antigen is present in a known amount of specimen.

Procedure description

• First, radioactive antigen-specific antibodies are added to a series of microtiter wells containing known concentration of a pure antigen which is first bound to the wells. The radioactivity in each of this standard curve for antigen concentration.
• Next, the antigen sample for a patient is allowed to bind to another well and radioactive antibodies are needed and radioactivity is measured as before. The amount of radioactivity found in the case of patients sample is then compared to a standard plot generated from the data obtained using the pure antigen and the concentration of the antigen in a patient's sample is interpolated from the standard plot.

C .Enzyme inhibition test

Enzymes are the protein which acts on their specific substrate and brings about chemical changes. Due to their chemical nature, enzymes can also be antigenic. Enzyme inhibition test makes use of this phenomena by reacting a specific antibody with its enzyme antigen which inhibits the enzymes ability to react with its substrate.

The best example of this phenomenon is the detection of antistreptolysin o which is an antibody to the hemolytic enzymes streptolysin 0. Patients serum which may contain the antibody is serially diluted and mixed with a constant amount of streptolysin o, followed by addition of a suspension of RBCs. Neutralization of streptolysin o by ASO (antistreptolysin o) will result in inhibition of hemolysis of the added red cells.

Labeling techniques

There are four types of techniques which involve labeling and tagging either antigen or antibody in order to detect the presence of one of the reactant. They are:

1. Enzyme immune assay (EIA)
2. Immunofluorescent assay
3. Radioimmunoassay (RIA)
4. Western blot (Immunoblot)

Enzyme Immuno Assay (EIA)

Enzymes are molecules which function as a catalyst for certain biochemical reactions. A small amount of enzyme can react with the larger amount of substrate to produce detectable levels of breakdown products in a given time. Enzymes which bring about chromogenic changes in their substrates are selected for enzyme Immunoassay.

EIA can be carried out entirely in solution. This is called homogenous enzymes linked assay. When some reactants are adsorbed on a solid phase, it is called heterogeneous enzymes linked assay or enzyme-linked Immunosorbent assay (ELISA)

In this technique, antibodies are conjugated or labeled with an enzyme in such a way that the requesting conjugates retain both immunological and enzyme activity. They can be assayed by their ability to break down a suitable substrate by into colored product. The most commonly used enzymes are alkaline phosphatase, peroxidase, beta-galactosidase are etc. The color change in the substrate due to enzyme activity is assayed visually or in a simple spectrophotometer. ELISA technique can be used to detect either antigen or antibody qualitative or quantitatively. The two main technique of ELISA are:

• Direct ELISA
• Indirect ELISA

Direct ELISA or Sandwich ELISA or Double antibody ELISA or for detection of antigen

This technique is used for the detection of antigen in the sample. A known antibody is adsorbed on the solid phase to which the test sample which may contain antigen is added. During incubation, the antibody binds the antigen to the solid phase. After washing, enzyme labeled specific antibody is added. At this stage, the antigen is sandwiched between the two antibody molecules, one on the solid phase and other with the enzyme labeled. On addition of the substrate, a color change indicates that the enzymes labeled antibody is present on the solid surface and this is due to the presence of antigen in the test sample.

Procedure

1. Antibody to microbial antigen is bound to wells of a microtitre plate.
2. Add patient’s samples suspected of containing microbial antigens and wash with buffer.
3. Add specific antibodies conjugated with an enzyme.
4. Wash with buffer
5. Add substrate for enzyme and measure the amount of colored product

Indirect ELISA

In this technique, a known antigen is attached to a solid phase such as the walls of the microtitre plate well. It is reacted with a patient serum which may contain antibody. The test is incubated and followed by the wash with buffer. Subsequently, an enzyme labeled anti-human globulin is reacted with the antibody in the test sample that has attached to the antigen on the solid phase. After incubation, the uncombined, the uncombined enzyme labeled anti-human globulin is washed off. Retention of the enzyme on the surface is detected by the addition of appropriate substrate which changes color after incubation. The concentration of antibody in the test sample sound bound by the antigen on a solid phase is proportional to the intensity of the color produced.

Procedure

1. Antigen is coated on the surface of microtitre well
2. Suspected patients serum is added to microtitre well
3. Wash with buffer
4. Add secondary anti-human antibody coated with specific enzyme and wash with buffer.
5. Add specific substrate and measure the color produced

Competitive ELISA

In this competitive ELISA which is an inhibitory type of assay, the concentration of antigen is inversely proportional to the antigen produced.

It is a variation for measuring amounts of antigens. In this technique, an antibody is first incubated in a solution with a sample containing antigen. The antigen-antibody mixture is then added to an antigen coated microtitre well. The more antigen present in a sample, the less free antibody will be available to bind the antigen coated on the surface of well. The addition of enzyme labeled secondary antibody specific for primary antibody can be used to determine the amount of primary. Antibody bound to the well as in the case of indirect ELISA, however, the higher the concentration of antigen in the original sample, the lower the adsorption.

REFERENCE

Cheesbrough, M. Medical Laboratory Manual for Tropical Countries. Vol. Vol 2. ELBS London, 2007.

Tille, P. Diagnostic Microbiology. 13th. Elsevier, 2014.

D, Grenwood, Slack RCB and Peutherer J. Medical Microbiology. Dunclude Livingstone: ELBS, 2001.