[Designing of a mixed ELISA method for detection of IgM and IgA rheumatoid factors]

ABSTRACT

Different isotypes of antibody can be produced by immune system after antigen contact. Detection and measurement of different classes of antibody against the antigen is very important in some cases. The aim of this study is designing of an ELISA method on the basis of inhibition of enzyme activity by using a non-competitive inhibitor. Therefore in this study rheumatoid factor is used as a model for the detection of different other classes of antibodies against the antigen.In this cross sectional analytical study, we measured IgM and IgA rheumatoid factors in sera of 10 patients with rheumatoid arthritis and positive latex test, by mixed and routine ELISA. In mixed ELISA the activity of the first conjugated enzyme was blocked by a non-competitive inhibitor after adding the substrate, then the next conjugated antibody, which was specific for another isotype, was added. By optical density, results was comparisoned with routine ELISA.The obtained results showed that the average optical density is lower when compared with routine ELISA, but the difference is not statistically significant. However these two methods did not show any significant difference in quantifying antibody isotypes. Also there is a positive association between mixed and routine ELISA [r =0.9, p=0.001].Lower optical density in mixed ELISA is probably because of stick hindrance by the first conjugate. So, because there is no significant difference between the results of these two types of ELISA, and also no need to repeat the test for each isotype in this method, it is recommended to use the new method instead of the routine one to save time and reagents