Application of a heterogeneous immunoassay for the quality control testing of release-active forms of diclofenac

- We first developed potency assay for drug containing release-active form of substance.
- The assay focused on the ability of the drugs to modify antigen-antibody interaction.
- Importance of the source of diclofenac was studied.
- Antigen concentration is a critical parameter for the proposed assay.

We report on a specially designed diclofenac-ELISA for the determination of diclofenac in the presence of release-active forms of diclofenac in lactose dissolved in water solutions according to a predefined schedule in single-blind experiments. In accordance with the objective of this project, a number of experiments were conducted to determine the optimal ELISA conditions for detecting potential modulatory effects of release-active forms of diclofenac depending on their ability to affect the binding of diclofenac to anti-diclofenac antibodies. As a feature, the diclofenac antibodies were previously incubated with manufactured pharmaceutical samples containing release-active forms of diclofenac or placebo. For comparison of the sample types, measured in ELISA optical densities were chosen. For statistic analysis, Student's two-sample t-test and single-factor ANOVA were applied. The extremely low concentrations of diclofenac of 0.01, 0.05 and 0.1 ng mL− 1 seem most appropriate for routine assay performance. The source of diclofenac used for standard solution preparation is not important but it could be important as the source of diclofenac for release active form of diclofenac preparation. As an outcome, the ELISA appeared to be suitable for the detection of the modifying effects of release-active forms of diclofenac toward the pharmaceutical substance in vitro.