The principles of ELISA and its application - Essay Example

The sensitivity of simple enzyme assays together with the specificity of antibodies/antigens aid in qualitative and quantitative determination of antigen or antibody concentration in samples. The various biological samples, like plasma, serum, urine, and cell extracts containing unknown antigen/antibody concentration can be analysed in ELISA. This biochemical technique employs polyclonal or monoclonal antibodies depending upon the specificity requirement for the sample to be assayed. The assays involving monoclonal antibodies increases specificity; and therefore give reproducible and accurate results.

The enzymes employed in ELISA should be simple, easily analysed and should have a high turnover number. The substrates used for the enzyme assay should be stable, safe, inexpensive, and should generate soluble end products. The chromogenic colourless substrates are utilized which give colored reaction products after the reaction. This visible color reaction is quantified spectrophotometrically. Other options for detection include analysis using fluorescent or radioactive probes.The most commonly used enzymes in ELISA are horse raddish peroxidase, alkaline phosphatase, urease, β-galactosidase etc.

(Axel 1999). When alkaline phosphatase is used for labeling the antibody, p-nitrophenylphosphate (pNPP) is used as substrate which forms yellow color product p-nitrophenol after the reaction. The chromogneic substrates employed with peroxidase are 2,2’-azo-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD) and 3,3’5,5’- tetramethylbenzidine base (TMB), which gives end products with green, orange and blue colors, respectively. A 96-well microtiter plate is used to execute the ELISA constituted of polystyrene or PVC.

Direct ELISA: In this technique, a sample containing the target antigen is adsorbed in a well of the microtiter plate. An enzyme labeled primary antibody reacts directly with the antigen. Direct ELISA excludes the use of