Optimization of enzyme-linked immunosorbent assay (ELISA) for detection of anti GM-CSF autoantibodies in serum

Abstract

Patients with anti-GM-CSF autoantibodies (GMAb) are susceptible to opportunistic fungal infections such as Cryptococcosis. However, the detection of GMAb is not routinely available in the service laboratory. The purpose of this study is to optimize the condition of enzyme-linked immunosorbent assay (ELISA) for detection of GMAb in serum. To optimize the ELISA conditions, we varied the concentration of coating antigen, the types of blocking buffer, the incubation time, the reagent volume and the dilution of sera. For ELISA, plates were coated with recombinant GM-CSF and blocked. Then patients’ sera were added using primary antibody and labeled secondary antibody to detect the antigen. Finally, the colored product was developed by substrate and the absorbance values were read at 405 nm. Nineteen sera from patients were screened for anti-GM-CSF antibodies using an ELISA (indirect format) which was optimized proper circumstances. We coated the plates overnight with GM-CSF at 1.5 µg/ml and blocked the non-specific binding using 3% BSA in PBS overnight. All of the reagent volumes were reduced from 100 to 50 µl to decrease non-specific backgrounds. Serum dilution of 1:20 was used for screening of GMAb by ELISA. This condition was further applied for indirect ELISA for GMAb detection in clinical specimens. Out of 19 specimens, 11 positive and 8 negative samples were clearly identified. The results are similar to previous results performed by another laboratory using a different protocol. This study showed that our improved method has potential in quantifying anti-GM-CSF autoantibodies with low background. This can be applied for laboratory diagnosis in patients with disseminated fungal infections