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Abstract
A new, very simple method for increasing the sensitivity and recovery rate of enzyme‐linked immunosorbent assay (ELISA) for the precise quantification of antigen in human serum is described. The assay design uses CATNF6A4c IgG2a monoclonal antibody and biotinylated anti‐human tumor necrosis factor‐α (hTNF‐α) polyclonal mouse IgG as the capture and tracer antibodies, respectively. The assay is completed within 4 hours at room temperature and is capable of detecting both recombinant and native human TNF‐α.
The assay incorporates the use of saturated ammonium sulfate (SAS) as a component of the dilution buffer to amplify the resultant signal from antigen containing human serum and eliminating the endogenous interference of native human serum. SAS worked optimally at the final concentrations, ranging from 1.2% to 11%. The addition of SAS to the dilution buffer resulted in a dramatic increase in both sensitivity and recovery rate of the ELISA.
The results demonstrated that 50 µL of dilution buffer, containing SAS, enabled the precise quantification of human TNF‐α in 100 µL of human serum samples and eliminated the interference of native serum, which seemed to be related to complement proteins. Therefore, dilution buffer containing SAS, at a defined concentration, seemed to be a potential candidate for resolving sensitivity and recovery problems usually encountered in immunoassays when measurement was performed with native serum samples. The proposed technique provides an easy, practical, and consistent method for ELISA when using human native serum.
Acknowledgment
This study was partially supported by a grant from TUBITAK (SBAG‐AYD 427).