Enzyme Linked Immunosorbent Assay

BACKGROUND

In this article we’ll discuss about Enzyme linked immunosorbent assay (ELISA). Enzyme immuno assay (EIA) was introduced in 1972 by Engvall and Perlmann, for applications where RIA would also be an option. This general class of assay can involve the detection of antibody or antigen of inters.

Enzyme linked immunosorbent assay

Enzyme linked immunosorbent assay (ELISA) typically involves a two-stage incubated immune reaction.

First the target Ag to Ab a solid phase. Non-bound materials are washed away and an enzyme labelled antibody, called a conjugate, binds to form a ‘sandwich’ complin.

Finally, the antigen-antibody is introduced to a substrate where a chromogen is used to give a colour change.

Enzyme linked immunosorbent assay are designed for detecting a quantitating substance such as peptides, proteins, antibodies and hormones other names such as EIA, are also used to describe the same process, most commonly, ELISAs are performed in 96 well polystyrene plates, which will passively bind antibodies and proteins.

It is this binding and immobilization of reagents that makes ELISAs so easy to design and perform, having the reactants of the ELISA immobilized to the microplate surface make it easy to separate bound from unbound material during the assay.

This ability to wash away non specifically bound materials makes Enzyme linked immunosorbent assay a powerful tool for measuring specific analytes with in crude proportion.

REQUIREMENTS

Microtiter plate

Coating antibody

Coating buffer

Blocking buffer

Standard antigen

Test sample

Antibody HRP conjugate

Aluminium foil

Blotting sheet/ filter paper

Substrate

Dilution of TMB/H2O2: 0.1 ml of TMB 20X concentration + 1.9 ml distilled water (dilute just before use)

Assay buffer (PBS-T)

100 ml 1X assay buffer, 10 ml of 10X assay buffer, 90 ml of distilled water

Stop solution (5X)

30ml 1X stop solution, 5ml of 5X stop solution, 25 ml of distilled water

PROCEDURE

1. Coating of the antibody ^[1]
   1. Prepare 10 µg/ml of antibody for coating buffer, i.e., 20 µl of coating antibody in 1980 µl of coating buffer.
   2. Pipette out 200 µl of the diluted antibody to each well of the microtiter plate
   3. Microtiter plate covered with aluminium foil and incubate at 37˚ C for 1 hour or overnight at 4˚ C.
2. Blocking
   1. Remove the coating solution from microtiter plate
   2. Wash three times with 1X assay buffer immediately
   3. Add 200 µl of the blocking buffer and incubate for 1 hour at room temperature.
3. Antigen capturing ^[2]
   1. Discard the blocking buffer from the microtiter plate and wash with 1X assay buffer three times for two minutes each
   2. Discard the content completely and tap the wells on a blotting sheet to completely remove the liquid.
   3. Prepare the standard antigen solution with different concentration
   4. Add 200 µl of different diluted antigen to each well and 200 µl of 1X assay buffer to the control well
   5. Incubate the microtiter plate at room temperature for 45 min.
   6. Discard the content of the well and wash with 1X assay buffer for 3 times, tap the microtiter plate on a blotting sheet to completely remove the liquid.
4. Addition of Antibody-HRP conjugate
   1. The antibody HRP conjugate is diluted to 1:2000 times i.e., 2 µl of Ab-HRP in 4 ml of 1X assay buffer and 200 µl of diluted conjugate is added to all the wells including the control.
5. Addition of the substrate ^[1]
   1. Discard the microtiter plate contents and wash with 1X assay buffer for 3 times
   2. Dispense 200 µl diluted substrate into each well.
   3. After 5-10 min. add 10 µl of the 1X stop solution to each well
   4. Transfer the contents to tubes containing 1.7 ml of 1X stop solution
   5. Read the absorbance at 450 nm
   6. Plot the graph with concentration of antigen on X-axis and O.D. at 450 nm on Y-axis.

CONCLUSION

By following above procedure of sandwich Enzyme linked immunosorbent assay (ELISA), we observe the Ag-Ab reaction by using microtiter plate and taking its absorbance at 450 nm.

REFERENCES

1. Peter Hornbeck, Sara Fuller, Scott Winston, “Enzyme-linked immunosorbent assays (ELISA)”, Current protocols in molecular biology, June 2001.
2. PATRICK JACQUIER, BRUNO GOTTSTEIN, YVONNE STINGELIN, AND JOHANNES ECKERT, “Immunodiagnosis of Toxocarosis in Humans: Evaluation of a New Enzyme-Linked Immunosorbent Assay Kit”, JOURNAL OF CLINICAL MICROBIOLOGY, Vol. 29 (9), September 1991.
3. Stephanie D. Gan and Kruti R. Patel, “Enzyme Immunoassay and Enzyme-Linked Immunosorbent Assay”, The Society for Investigative Dermatology, September 2013.
4. Mandy Alhajj, Aisha Farhana, “Enzyme Linked Immunosorbent Assay”, StatPearls, January 2022.

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