Bioanalysis

Conducting ELISAs: are duplicates always necessary or do singlets suffice?

Recently, the QPS Netherlands large molecule ligand-binding assay (LBA) group conducted a research project to compare ELISA performance based on duplicates and singlets.

Usually, in current regulated bioanalytical practice, LBA methods call for duplicate sample analysis in order to improve reliability of results. In the 2013 Crystal City V meeting the necessity and rationale for duplicate sample analysis in LBA was discussed, along with criteria according to which singlet analyses could be justified. This discussion was fed by the introduction of new technologies and platforms and by advances in LBA that can allow for singlet analysis.

The consensus reached at the Crystal City V meeting was an “Agreement on the concept of running singlet analysis for LBAs if the method allows this and the number of replicates can be driven by the data during validation. This can be based on inter-plate precision and scientific rationale.” An additional statement was agreed upon during the meeting, in reference to run design: “Two sets of QCs run as singlets in addition to a calibrator curve at the beginning of the run.”

QPS conducted a study by applying this agreement. A clinical study with a monoclonal antibody was conducted conventionally (in duplicate) and the results were used for regulatory purposes. A priori, to simultaneously evaluate singlet analysis, certain duplicates were designated for singlet evaluation as well. In addition, the method validation results were revisited and recalculated using singlets.

The results of the comparison were presented at the EBF workshop on tiered approach, in Brussels, June 2014. In summary, for this application, we showed that there were no significant differences between pre-study validation, in-study validation including ISR, and individual sample results, when carried out with duplicates or with singlets.

For more information on this subject, contact Jaap Wieling, Senior Vice-President, Bioanalysis & Technology R&D: jaap.wieling@qps.com

Conducting ELISA

ELISA Concentration

ELISA Concentration

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