Application of orthogonal design in the development of Echinococcosis ELISA kit and its evaluation

Abstract

Objective To select the optimum conditions of Echinococcosis ELISA kit and kit preparation by using orthogonal experiments, then to test their detection performance. Methods Taking the absorbance of the samples as the indicator， orthogonal experiments of three factors four levels were taken to optimize the conditions of hydatid disease detection ELISA kit. Through L16（43）orthogonal experiments， the concentration of the antigen(A), the dilution multiple of the sample (B) and the dilution multiple of the enzyme labeled antigen (C) were studied. The specimen were provided by The First Affiliated Hospital of Xinjiang Medical University specimen library collected between March 2011 and June 2013, including 36 sera confirmed alveolar Echinococcus by the gold standard, 56 sera confirmed Echinococcus granulosus disease by the gold standard and 72 sera as the control group(including healthy people, cirrhosis, hepatitis, etc). Detecting sensitivity and specificity were compared using F test for statistical analysis. Results Orthogonal design showed the size proportion of three factors of echinococcosis ELISA kit: antibody dilution>sample dilution>antigen coating concentration. The optimal preparation conditions were A1B2C4, that was, the concentration of antigen was 1mg/L ,sample dilution of 1∶100, dilution of secondary antibody of 1∶160 000.Through the 3-factor and 4-level orthogonal design using F-test analysis, the fact of the concentration of the kit antigen(A), F=1.181, P=0.393; the fact of the dilution multiple of the sample (B), F=2.544, P=0.152, the fact of enzyme labeled antigen dilution (C) F=2.544, P=0.039. The sensitivity of the kit to Echinococcus granulosus disease and alveolar Echinococcus were 83%(30/36), and 91%(51/56), respectively; the specificity was 91%(29/32) and 83%(33/40); the misdiagnosis rate of 17% (6/36) and 9% (5/56); the misdiagnosis rate 9%(3/32) and 18%(7/40); positive likelihood ratio 8.86%(83/9) and 5.20%(91/18); negative likelihood ratio of 0.18%(17/91) and 0.11%(9/83). Conclusion Orthogonal design is a good method that can find out optimal conditions of preparation for Echinococcosis ELISA kit and improve the test performance. （Chin J Lab Med,2014,37:544-547） Key words: Echinococcosis; Enzyme-linked immunosorbent assay; Reagent kits,diagnostic; Sensitivity and specificity