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Antibody dilution curves

المؤلف:  Sue Jickells , Adam Negrusz (Editors)

المصدر:  Clarkes Analytical Forensic Toxicology

الجزء والصفحة:  p379-381

2026-11-04

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Antibody dilution curves

Once an immunisation is underway, the quality of the antiserum is assessed by means of an antiserum dilution curve. This demonstrates the binding of the antibody to the target drug and is an indication of the antibody’s affinity for the antigen. Using a heterogeneous enzyme immunoassay (EIA) as an example, a test bleed of rabbit anti-cotinine was coated onto a microplate at different dilutions in bicarbonate buffer (pH 9) using incubation overnight at room temperature. The resultant binding to different titres of horseradish peroxidase-labelled cotinine is shown in Figure 14.4. Dilutions of the enzyme–drug conjugate for this type of experi ment can be made in simple phosphate buffer (pH 7.4) with a small amount of protein (e.g. BSA at 0.05% w/v) to prevent nonspecific binding (NSB) of materials to the assay tube. The hook (or apparent peak) seen in Fig. 1 at high anti serum concentrations is caused by a combination of steric hindrance and saturation of coating antibody to the microplate.

Once the concentration of antibody (titre) that produces the desired response is selected, it is necessary to check that the antibody also responds as required with the target drug (i.e. that the target drug successfully competes with the labelled drug for binding to the antibody). The improved performance with subsequent test bleeds for samples taken from a rabbit immunised with a buprenorphine–protein immunogen is shown in Figure 2. It is possible to gain useful information by combining both the experiments described above and analysing both a positive and negative sample at each antibody dilution. In this way, binding and displacement can be seen at each antibody titre. Careful titring of the labelled drug derivative and antibody dilution can improve the assay characteristics, and the assay can be optimised further by the addition of other proteins, surfactants and stabilisers to the assay buffer. An immunisation programme usually involves the injection of between three and six animals with the same antigen. If suitable antibodies are not produced after several immunisations, it may be necessary to start the programme again with different animals and possibly a different immunogen.

Figure 1 Titration curve for rabbit anti-cotinine anti serum with cotinine (HRP, horseradish peroxidase).

Figure 2 Comparison of early, mid and final test bleeds for anti-buprenorphine antisera.