ELISA ( Enzyme linked Immunosorbent): Principle,Procedure,Types and MCQ for NET JRF, GPAT

1. ELISA :-

Enzyme linked Immunosorbent assay is commonly known as ELISA. where Ag-Ab interaction is monitored by enzyme measurement.

It is similar in principle to RIA but depends on an enzyme rather than a radioactive labels.

An enzyme conjugated with anti body reacts with a colourless substrate is called a chromogenic substrate.

This technique was developed by in 1971 by peter peter Perlman and eva engvall at Stockholm University in Sweden.

PRINCIPLE :- 

Principal is based on the formation of Ag-Ab complex which is detected by chromogenic detection using enzyme conjugated secondary antibodies.

The conjugated enzyme act on a specific substrate called chromogenic substrate and generate a color reaction product.

This product is qualitative or quantitative read using an ELISA Plate reader.

TYPES OF ELISA :-

1. DIRECT ELISA :- It is used in the detection of antigen in the given biological sample. Microtiter wells are initially coated with antigen to e detected which is followed by an antibody linked to an enzyme conjugate. This follow the addition of substrate which produces colour detected using ELISA detector.
2. INDIRECT ELISA :- It is used for detection of an antibody in the given sample. Microtiter wells are initially coated with antigen specific for anti body to be detected followed by the addition of sample. Enzyme conjugated secondary antibody is added followed by the substrate which forms a colour reaction product.
3. SANDWICH ELISA :- It Is used for detecting an antigen in the given sample. Microtiter wells are initially coated with monoclonal antibodies (called captureantibody)  raised against antigen to be detected followed by addition of sample. Any trace of antigen is detected by adding primary antibody followed by enzyme conjugated secondary Ab & a chromogenic substrate or by directly adding an enzyme conjugate primary Ab.
4. COMPETITIVE ELISA :-  the variation of ELISA is used to quantitatively estimate the amount of antigen in the given sample. Ag & Ab are initially incibated so that they from Ag-Ab complex.this mixture is then added to microtiter wells coated with synthetic analogue of antigen to be detected any free antibody binds to these antigen. This complex is estimated by enzyme conjugated secondary antibody by chromogenic detection. More the amount of antigen in the sample lesser is the antibody available to bind to microtiter wells.

APPLICATION OF ELISA :-

1. Detection of mycobacterium antibodies in tuberculosis.
2. Detection of rota virus in feces.
3. Detection of hepatitis B markers im serum
4. Detection of HIV antibodies in blood sample.
5. Measuring the hormone levels,

• HCG (as a test for pregnancy)
• LH (Determining the time of ovulation)
• TSH, T3 & T4 (for thyroid function)

MCQ

1.which is working principle of ELISA ?

A.Ag-Ab neturalization

B.Ag-Ab complex

C. A and B

D.None of the above

2. which is not application of ELISA ?

A.Detection of hepatitis B markers im serum.

B.Percentage of Hb in blood.

C.Detection of HIV antibodies in blood sample.

D.Detection of mycobacterium antibodies in tuberculosis.

3.Indirect ELISA which is detected in sample ?

A.Antigen

B.Anti body

C.A and B

D.None of the above

4.Indirect ELISA which is detected in sample ?

A.Antigen

B.Anti body

C.A and B

D.None of the above

5.sandwich ELISA which is detected in sample?

A.Antigen

B.Anti body

C. A and B

D.None of the above

6.COMPETITIVE ELISA which is detected in sample ?

A.antigen

B.anti body

C.a and b

D.none of the above

ANSWER KEY :-

1.B

2.B

3.A

4.B

5.A

6.B

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REFERENCE:-

—Textbook of microbiology by Prescott, Harley.

—Practical analytical chemistry by BECKEET.