Evaluation of variability and quality control procedures for a receptor-binding assay for paralytic shellfish poisoning toxins

Abstract

The receptor-binding assay (RBA) method for determining saxatoxin (STX) and its numerous analogues, which cause paralytic shellfish poisoning (PSP) in humans, was evaluated in a single laboratory study. Each step of the assay preparation procedure including the performance of the multi-detector TopCount® instrument was evaluated for its contribution to method variability. The overall inherent RBA variability was determined to be 17%. Variability within the 12 detectors was observed; however, there was no reproducible pattern in detector performance. This observed variability among detectors could be attributed to other factors, such as pipetting errors. In an attempt to reduce the number of plates rejected due to excessive variability in the method's quality control parameters, a statistical approach was evaluated using either Grubbs’ test or the Student's t-test for rejecting outliers in the measurement of triplicate wells. This approach improved the ratio of accepted versus rejected plates, saving cost and time for rerunning the assay. However, the potential reduction in accuracy and the lack of improvement in precision suggests caution when using this approach. The current study has recommended an alternate quality control procedure for accepting or rejecting plates in place of the criteria currently used in the published assay, or the alternative of outlier testing. The recommended procedure involves the development of control charts to monitor the critical parameters identified in the published method (QC sample, EC₅₀, slope of calibration curve), with the addition of a fourth critical parameter which is the top value (100% binding) of the calibration curve.

Keywords:

• receptor-binding assay
• paralytic shellfish poisoning
• saxitoxins
• variability

Acknowledgements

This study was supported under NOAA grant NA04NOS4780239 from the Monitoring and Event Response for Harmful Algal Bloom (MERHAB) programme. This is MERHAB Publication Number 152. The authors would like to thank Roger Ho for helping with the Graphpad Prism data analysis; Clive Kittredge and Vanessa Zubkousky for the preparation and analysis of plates; and American Radiolabeled Chemicals Inc. for providing ³H-STX reagents. Special thanks to Sherwood Hall of the US Food and Drug Administration's Office of Regulatory Science for FDA STX reference standards and helpful discussions on this study. The authors would also like to thank Fran van Dolah and Greg Doucette for their guidance in the RBA method.