Detection of specific proteins the immunoassay

The most commonly used technique for quantification of allergenic or antigenic substances is the enzyme-linked immunosorbent assay (ELISA). ELISA has the advantage over radioimmunoassay (RIA) of being more cost-effective and, with modern techniques, not compromising sensitivity. The specificity of all immunoassays is in part dependent on the efficiency of the capture and detector antibodies. Once optimised and standardised the ELISA is relatively economical, and large numbers of samples can be analysed on each test run. The assay is carried out in standard plastic 96 well plates designed for use in ELISA. The wide use of such plates has led to a variety of plate washing and reading systems being available. The sensitivity of the antibodies in forming a complex with the protein is paramount for the sensitivity and specificity of the assay. The sensitivity of the basic assay may be further increased by using indirect labelling or amplification techniques. In non-competitive assays all the constituents are in excess, apart from the protein to be detected. The optimum quantities of each constituent are determined by preliminary experiments. An alternative is the inhibition ELISA, also highly sensitive, but this technique is susceptible to nonspecific interactions.

Sandwich enzyme-linked immunosorbent assay

The two-site or sandwich ELISA is ideal for detecting proteins in complex mixtures (Fig. 7.1). The antibodies may be from monoclonal or polyclonal origin. Monoclonal antibodies are most often used for capture, since polyclonal antibodies with wider specificity may theoretically mask the binding site for the monoclonal antibody. It is important that the capture antibody does not interact directly with any of the subsequent assay stages, or vice versa, as this leads to abnormally high background values that reduce sensitivity. In the majority of assays antibodies from different animal species are used to avoid this. An assay may utilise polyclonal antibodies both capture and detector stages. Monoclonal antibodies may be used for both capture and detector stages. In this situation the assay designer usually ensures that the capture and detector antibodies are directed against different parts of the molecule (so-called two-site assays) to avoid competition or interference between the antibodies. The example given in Fig. 7.1 is a direct assay where the enzyme label is directly conjugated to the antibody. Amplification steps are described below.

(a) Protein, in this case 'capture' antibody, is incubated in the well, usually of alkaline pH, and small quantities become absorbed onto the plastic surface.

(b) The protein solution to be tested is added to the plate at the appropriate dilution in duplicate or triplicate.

(c) The capture antibody binds the specific protein, anchoring it to the plate surface. Any unbound proteins will be washed off the plate and discarded.

(d) A 'detector' antibody specific for the protein is then used to link an enzyme label to the protein.

(e) The enzyme then acts on a colourless substrate to form a coloured product that is quantitated using a specialised optical density plate reader. The generation of standard curves using known antigen concentrations allows for the accurate estimation in weight/

volume.

Fig. 7.1 The enzyme-linked immunosorbent assay (ELISA) - antigen detection by direct sandwich.

Choice of antibody

The assay specificity and to some extent its sensitivity are primarily linked to the efficiency of the antibodies used. Polyclonal antibodies are cheaper to prepare than monoclonal; both types of antibody should be purified to reduce nonspecific interactions, and in the case of polyclonal antibodies they may also need to be affinity purified. Polyclonal antibody preparations contain a heterogeneous mix of antibodies directed against any number of epitopes on the protein surface. This gives the assay an advantage if food processing results in some epitopes being denatured or masked, but increases the likelihood of non-specific interactions between the antibodies and unrelated proteins. Monoclonal antibody preparations have a specificity directed against one epitope; this may increase specificity greatly if the epitope is not present on unrelated proteins. The assay may prove to be less versatile if the epitope is more susceptible to denaturing than the allergenic epitopes.

Amplification systems

The detector antibody may be conjugated directly to the enzyme label - direct assay. Or various amplification steps may be used:

• Conjugation of the detector antibody to biotin, a compound that binds to avidin with a great affinity and specificity, thus amplifying the signal -indirect biotinylated assay

• The detector antibody, for example IgG from rabbit, may be unlabelled, and subsequently detected using a third enzyme-labelled antibody specific for the detector antibody - indirect assay.

Substrates

Substrates of choice in traditional assays gave a coloured product or chromogen, but more recently fluorescent or luminescent substrates are being used. The

(a) In the direct ELISA the primary antibody is conjugated to the label.

the strong specific interaction between biotin and streptavidin. The primary antibody is conjugated to biotin, and the avidin molecules are conjugated to the enzyme label.

(b) In the indirect ELISA the primary antibody is unlabelled and a secondary antibody carries the label. There must be no interaction between the capture and secondary antibodies.

in which the enzyme is conjugated with biotin and avidin forms a bridge between the enzyme and the antibody.

(c) In the biotinylated assay use is made of (d) An adaptation of the biotinylated assay the strong specific interaction between biotin and streptavidin. The primary antibody is conjugated to biotin, and the avidin molecules are conjugated to the enzyme label.

in which the enzyme is conjugated with biotin and avidin forms a bridge between the enzyme and the antibody.

Fig. 7.2 Amplification of the ELISA.

coloured product had the advantage of being cheap and requiring fairly inexpensive optical density plate readers. Fluorescent or luminescent assays have the advantage of increased sensitivity, but the disadvantage of higher backgrounds, and expensive costs for the small laboratory or institution.

Standardisation

Reagents and procedures must be standardised in order to produce reliable results. Calibration and the appropriate quality controls ensure that the results can be compared between assay runs and between laboratories. Use of a single standard or reference preparation worldwide (such as that of the National Institute for Biological Standards and Control) allows universal comparison of results. However, at the time of this book going to press, none are available for food allergens, so appropriately stored in-house or secondary standards must be used. Assays must be validated for the types of samples to be processed. It is not sufficient to determine by experiment that an assay has a sensitivity of so many Mg/ml for peanut in flour and then assume that it would have the same sensitivity for peanut contamination of chocolate. The sample matrix or composition may affect the assay, giving artificially high or low readout values. Spiking a pure preparation of known allergen with the analyte-free food will reveal what these effects are.

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