Evaluation of several parameters influencing ELISA

In general, in ELISA, nonspecific adsorption can be reduced by including in the medium, during the incubation and washing steps, a nonionic detergent, such as Tween-20, either alone or supplemented with a protein, e.g. bovine serum albumin or gelatin. How ever, it must be realized that even under the best defined conditions, there will always be nonspecific adsorption of the enzyme conjugate by the coated solid phase.

The choice of the enzyme label depends essentially on how quickly the results are needed as well as on the number of samples to be examined. When a large number of samples has to be tested simultaneously, it is preferable to use enzymes for which stable substrates are available and therefore allowing reactions of long duration. The substrates of alkaline phosphatase and p-galactosidase are more stable than those used to measure peroxidase. Consequently, peroxidase is not well suited to ELISA requiring reactions of long duration but it is particularly effective in rapid heterogeneous enzyme immunoassays. Comparative assays performed using chromogenic sub strates and conjugates of peroxidase, glucose oxidase, alkaline phosphatase and p-galactosidase have shown that, after establishing the optimal conditions, these conjugates had virtually identical sensitivities and were capable of measuring equally small quantities.

Enzymes used as labels can be measured using several highly sensitive procedures. Thus with light emission techniques, peroxidase can be detected at the femtogram level. Using 4-methyIumbelliferyl-(3-D-galactoside, the fluorogenic substrate of (3-galacto-sidase, 5000 molecules of this enzyme in solution are easily measured. Similarly, alkaline phosphatase can be measured in minute amounts using either fluorogenic substrates or amplifying enzymatic cyclic systems. One would expect therefore that, by using such sensitive procedures for the measurement of enzymes, a considerable increase in sensitivity of ELISA would occur. However, when ELISA using either conventional chromogenic substrates or highly sensitive procedures of detection are first optimized and then compared in a given antigen-antibody system, usually no increase in sensitivity is noted or, sometimes, a maximum 5- to 10-fold increase is achieved. For example, when estradiol is measured by a competitive assay using fi-galactosidase-labeled estradiol and either the chromogenic (o-nitrophenvl-P-D-galactoside) or the fluorogenic (4-methylumbel-liferyl-(3-D-galactoside) substrate of this enzyme, the detection limit is the same (100 pgml"1). However, in order to achieve this result, 18 h of incubation are needed with the chromogenic substrate, while 2 h are sufficient with the fluorogenic one. Thus although the intensity and the rapidity of the enzymatic response is improved by using highly sensitive measurement procedures, the detection limit, i.e. the lowest concentration of antigen (or antibody) that can be measured, remains essentially unchanged.

Therefore, it appears that in ELISA, and probably in all the immunological procedures based on the use of labeled substances, the limiting factor is not the detection or the measurement of the enzyme level. The limiting factor is the binding affinity (avidity) of the antibody which will determine the ultimate sensitivity of the heterogeneous enzyme immunoassays.

See also: Affinity; Autoantibodies, tests for; Enzyme labeling of antibodies and antigens; Enzyme-linked immunosorbent assay (ELISA); Immunoassays.

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