Development and Validation of an ELISA Immunoassay for Clinical Pharmacokinetic Study

    ELISA Immunoassay

    The recent advances made to the field of biotechnology has introduced an increasing number of protein-based drugs across several different clinical settings. In fact, this has turned out to be the first choice of treatment for several medical conditions. Protein-dependent drugs are known to possess certain characteristics such as strong physiological activities, high specificities, and low dosage requirements. Furthermore, their absorption, distribution, metabolism, and excretion have also been established to be different from the routine small molecule analysis. This makes their pharmacokinetic evaluation to stand different from the routinely employed pharmacokinetic technique for the small molecule bioanalysis.  Also, the low concentrations and interferences caused by endogenous proteins makes it difficult to analyse the pharmacokinetic of the protein drugs being administered to the patients.

    The pharmacokinetics of such protein-dependent drugs are routinely carried out through both radioactive tracer techniques as well as ELISA or enzyme-linked immunosorbent assay methods. However, both the techniques results in different pharmacokinetic testing results.

    An ELISA development strategy consists of the following 4 technical elements ?

    • Plate coating strategy
    • Antibody pairs and antigen resources
    • Conjugating or labelling strategy
    • Enzyme and chromogen

    Plate coating strategy ?

    While developing an ELISA technique for a specific antigen, the first and the foremost step is determining and finalizing the immobilizing strategy as well as optimizing the plate-coating conditions both for the antigen or the capture antibody. Usually, polyvinyl or polystyrene 96 or 384 well plates are employed for this purpose.

    Antigen or antibody ?

    Both antigen and antibody are two key factors responsible for the determination of sensitivity as well as the specificity of the assay under analysis. Both the purity as well as the stability of the antigen are key considerations affecting the ELISA performance. If the antigen is highly pure, it can result in subsequently enhancing the capability of the capture antibody thereby resulting in an increase in the assay sensitivity.

    Conjugate strategy ?

    Enzyme labelled antibody plays a key role for generating the ELISA output. Enzyme conjugation to antibodies results in the formation of a stable and covalent linkage between the antigen specific antibody (either monoclonal or polyclonal) and the enzyme. Various reporter enzymes such as alkaline phosphatases or horseradish peroxidases can be employed for attaching the antibodies to the proteins of interest. This is usually accomplished through differential coupling chemistries that ensures maximised retention activities of both the proteins as well the enzymes.

    Enzymes and Chromogen ?

    The last and the final stage in ELISA technique is dictation wherein the signal intensity produced by the substrate is directly proportional to the antigen quantity captured on to the plate and bound to the detection reagents.

    Validation of ELISA ?

    The developed ELISA assay kits are then validated for testing both their safety and efficacy for determining their applicability as immunoassay techniques. There are 10 key immunoassay validation parameters ? robustness, precision, trueness, uncertainty, quantitation limits, dilutional linearity, parallelism, recovery, selectivity, and sample stability.

    Validation of the immunoassays in critical before its practical application to the fields of pharmacokinetics clinical trials to ensure the generation of reliable and interpretative test results.

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