Competitive Elisa

Antibody-based enzyme-linked immunosorbent asays (ELISAs) offer robustness and high sensitivity, making them the assay of choice for many routine diagnostic assays for detecting antigen within a biological fluid. Although the dual-antibody sandwich (DAS) ELISA typically provides the highest level for specificity and sensitivity, this method requires antibody "pairs," which may not be available. In such a situation, a number of alternative ELISA techniques can be used, including the direct ELISA and the competitive

From: Methods in Molecular Biology, vol. 295: Immunochemical Protocols, Third Edition. Edited by: R. Burns © Humana Press Inc., Totowa, NJ

Competitive Elisa Principle
Fig. 1. Principles of a typical competitive ELISA.

ELISA. The focus of this chapter, the competitive ELISA, usually is the preferred choice when a DAS ELISA is not available because it provides greater specificity than the direct ELISA, making it more reliable for the diagnostic procedures it is being used for (1,2).

There are many variations and adaptations of the competitive ELISA, although the general principle for all of these remains the same. A typical representation of this technique is portrayed in Fig. 1. The initial stage of this technique involves coating a high capacity protein-binding microtiter plate (ELISA plate) with an antibody that is specific for the antigen to be measured. This allows sample to bind to the plate with a high affinity. The basis of the technique lies in a competition between a preprepared enzyme-conjugated form of the antigen of known concentration with the sample, for binding sites on the ELISA plate. The conjugated form can then be detected using a suitable substrate to develop color in the

Estimating Antigen Concentration

increasing concentration of'competing' unconjugated standards

Fig. 2. Estimating antigen concentration using a typical competitive ELISA standard curve.

increasing concentration of'competing' unconjugated standards

Fig. 2. Estimating antigen concentration using a typical competitive ELISA standard curve.

presence of the enzyme. A reading (OD) can then be gained using a spectrophotometer. When the sample and the conjugated antigen are mixed prior to incubation on the ELISA plate, competition between the (unconjugated) antigen within the sample interferes with the ability of the enzyme-conjugated antigen to bind the capture antibody. Thus, unlike a standard sandwich ELISA, the readout is inversely associated with the amount of antigen (see Fig. 2).

It is also worth noting the "blocking ELISA" at this stage. In this variation of the competitive ELISA the sample to be measured is not mixed with the enzyme-conjugated antigen but is preincubated onto the coated plate prior to washing and addition of the conjugated antigen. Thus, the sample "blocks" rather than "competes" for the sites on the plate. This can result in a greater degree of sensitivity, although it is more time consuming as it relies on an additional step. The principle of the assay, however, remains the same as the competitive ELISA. Another common variation of the competitive ELISA is often used to measure levels of antibody in solution. For example in measuring the antibody response in serum to a pathogen in order to diagnose infection. In this technique, the antigen itself is often coated onto the ELISA plate and an enzyme-conjugated "detection antibody" is used to generate the OD reading. As with the previous example, the mixture of sample (in this case serum) with the detection antibody competes for the antigen coated onto the plates resulting in a reading that can be cross-referenced with a standard curve to gain a quantitative estimate of antibody in the sample.

Irrespective of the variation of the competitive ELISA being used, careful optimization of the technique is essential. In particular, the competitive ELISA relies on careful optimization of the standard curve. During this process a known concentration of conjugated standard antigen is used to give an OD reading that is then com-peted-out by the antigen within the sample. The amount of conjugated antigen used has a major impact on the sensitivity of the assay. For example, if too much conjugated antigen is used then it takes a larger amount of non-conjugated antigen in the sample to result in a measurable reduction on the OD that can be used to quantify that sample, thus resulting in a low sensitivity of the assay. Using too little of the conjugated antigen results in a low initial OD reading, reducing the potential range of the ELISA. A titration of the conjugated antigen must therefore be performed as the first step in setting up a competitive ELISA. In general, a concentration giving an OD just below the maximal OD obtained is required (approx 90% is usually appropriate). In practice, the amount of conjugated antigen used depends on the sensitivity that is required from the assay.

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Responses

  • scarlett
    Is das elisa same as competitive?
    6 years ago
  • simon
    How to develop competitive ELISA?
    5 years ago

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