Immunoassay specificity


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Specificity is an important characteristic of any laboratory test which describes its ability to distinguish between true (or specific) and non-specific results. With immunoassay methods, interferences which affect specificity can be categorized into two major classes: 1) those which affect the binding event between the antibody and an antigen in a general way, such as pH or ionic strength; or 2) those substances which affect binding of antigen by competing for the specific binding site on the antibody. These 'specific' interferences are often referred to as 'cross-reactants'. In the analysis of pesticide residues, it is often desirable to have high levels of cross-reactivity with related compounds and metabolites of the parent compounds, so that broad screen can be performed.

The specificity of an immunoassay may be characterized by adding increasing amounts of a potential cross reacting substance to a sample and measuring the response in the immunoassay. The results of this experiment can be reported several ways.

One method of representing the comparative reactivity of these compounds is to determine the concentration of each compound required to displace the same amount of labeled antigen from antibody. For example, one commonly calculates cross-reactivity using the concentrations required to displace 50% of the label or 50% B/B0. The concentration is called the ED50 (A) or estimated dose at 50% B/Bo. A ratio of the resulting concentrations can be referred to as the 'percent cross-reactivity at the ED50 (D). Cross-reactivity can also be calculated at other levels of displacement such as 20% or the ED20 (B,E). Depending on the slope and shape of the response curve the % cross-reactivity may be different at different displacement levels (compare D and E).

Another method to report cross-reactivity may be to simply report the concentration of cross-reactant required to displace a given amount of labeled antigen. For example, one might report the concentration of cross-reactant required to displace 50% of the label i.e. the ED50 (A). Again different displacement levels can be used but the absolute result and possibly the relative results will change (compare A and B). If one chooses the lowest level of displacement which can be reliably distinguished from zero displacement than the resulting concentrations could be represented as a least detectable dose (LDD) (C) for each cross-reactant.

Translating this information into expected results in naturally-occurring samples with mixtures of compounds present can be very complex. However, it is important to recognized that one cannot simply add together the expected results.

In summary, there are many ways in which the same cross-reactivity data can be represented. The choice of one method over another is largely dependent on how the information is being used. However, it is always important when reviewing information characterizing an immunoassay to understand how cross-reactivity is calculated so that the data is interpreted appropriately.

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