Validation of an ELISA-type immune-enzymatic test to quantify levels of diphtheria antitoxin in human serum

Abstract

Introduction: Diphtheria still persists in many countries. In Cuba, studies conducted in different age groups have demonstrated that there are non-protective levels of diphtheria antitoxin in the population, so it is necessary to have methods that allow the serologic survey of population immunity. The quantification of antibodies against vaccine antigens such as diphtheria toxin is also a useful, rapid and economic method to evaluate the immune response.Objective: To validate an ELISA-type immune-enzymatic test to quantify the levels of diphtheria antitoxin in human serum.Material and Method: An experimental study of technological development was carried out in the Immunology Laboratory of the National Medical Genetics Center, Havana, Cuba. The optimal values of the variables that influence on the result of the indirect heterogeneous immune-enzymatic test for the quantification of diphtheria antitoxin were determined. The calibration curve obtained was evaluated against the WHO standard (Diphtheria Antitoxin Human Serum 00/496). The analytical validation of the standardized method was performed.Results: The intra-assay and inter-assay coefficients of variation were less than 10% and 20%, respectively. Recovery values between 90 and 110% were found in accuracy and selectivity. The parallelism between the standard curve and the samples studied showed a coefficient of variation lower or equal to 10%. The limit of quantification was 0,015 IU/mL and the one of detection was 0,0039 IU/mL. Conclusions: The result obtained in the precision, accuracy and selectivity of the ELISA-type immune-enzymatic test developed and validated in the National Medical Genetics Center demonstrated that it can be used in the clinical practice to quantify the values of diphtheria antitoxin in human serum.Keywords: diphtheria antitoxin, immune-enzymatic test, ELISA, validation.