A statistical model to optimize enzyme-linked immunosorbent assay parameters for detection of Mycoplasma gallisepticum and M. synoviae antibodies in egg yolk.

An enzyme-linked immunosorbent assay (ELISA) was developed to quantify Mycoplasma gallisepticum (MG) and M. synoviae (MS) antibodies in egg-yolk extract. Various parameters of ELISA were evaluated and optimized. A statistical model was developed to study the relationship between ELISA absorbance (A) and antigen concentration, antibody concentration, and time of reading of the test. These factors explained 62% of the variability in A for the MG antigen and 74% of the variability in A for the MS antigen. The optimum concentration of MG antigen was 4 micrograms protein/ml, and that for MS antigen was 3 micrograms protein/ml. A similar model was developed to study the relation between A and conjugate concentration, antibody concentration, and time of reading of the test. The optimum concentration of the conjugate was found to be 1:4000. The within-run and between-run coefficients of variation for MG were 8% and 9%, respectively, and those for MS were 9% and 7%, respectively, indicating a high degree of reproducibility of these tests. There was a high correlation between ELISA and hemagglutination-inhibition test results.