Immunochimie et immunopathologie

Uncertainty of Measurement for ELISA in a Serological Testing Laboratory

Abstract

Subrata Biswas and Malay Kumar Saha

Background: Analytical results estimating Uncertainty of Measurement (MU) signifies the confidence level of the concerned assay. Enzyme-linked immunosorbent assay (ELISA) is an extensively used important tool for infectious disease sero-diagnosis, nevertheless, MU for ELISA result is not reported since procedure of MU estimation is hardly available. International Organization for Standardization (ISO) provides the Guide to the Expression of Uncertainty in Measurement (GUM) describing set of guidelines, however no detailed procedures or instructions for evaluating specific measurement processes is described. This article aims step by step procedure for MU estimation in a serology laboratory by describing the potential sources of uncertainty during each step of ELISA.

Methods: HIV sample was tested by commercial ELISA following routine procedure. Uncertainty was estimated by specification of measurand, identification of uncertainty sources, quantification of values attributed to the sources of uncertainty and calculation of the combined standard uncertainty following GUM by converting all the standard uncertainties of ELISA into dimensionless relative standard uncertainties. The combined standard uncertainty was calculated using the propagation principle

Results: Uncertainty arising from systematic error (RSD 0.10966) and routine analytical imprecision (RSD 0.04938) were considered as the sources of uncertainty contributing to the total MU of this routine qualitative diagnostic method. Analysis of the data revealed that results obtained for the relative expanded uncertainty U_rel was 24% with 95% confidence level (k=2).

Conclusion: Quantification of uncertainty by combining systematic error and analytical imprecision seems a feasible method of MU for ELISA. The estimated MU reflects the extent of variation for the cut-off absorbance for equivocal outcome of the qualitative interpretation.