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[Development and preliminary application of a double mAb sandwich ELISA for detecting soluble CD226]
Shuang-Fei Ning1, Wei Jia, Xing-Hai Zhang
1Department of Immunology, Fourth Military Medical University, Xi'an 710032, China.
Aim:
To develop a double mAb sandwich ELISA for detecting sCD226 and apply it to clinical specimens.
Methods:
The optimal concentrations of coating mAb and HRP-mAb, and the optimal diluents were determined. A double mAb sandwich ELISA then was established. The specificity, sentivity and stability of the method were identified. And clinical specimens were detected by this method.
Results:
The optimal concentrations of coating mAb LeoA1 and HRP-mAb FMU3 were 2.5 mg/L and 1:400, respectively, and the optimal diluents for standard antigen and HRP-mAb were 1 g/L BSA 1 mL/L Tween20-PBS and 30 g/L PEG-PBS.The ELISA achieved satisfactory results in respect to specificity (no cross-reaction to human IgG, in block test blocking effect was does-dependent), sensitivity (the detected threshold was 110 ng/L) and stability (CV<10.2%). Statistical analysis showed that the serum sCD226 level in patients with rheumatoid arthritis (RA) was significantly higher than that in healthy adults.
Conclusion:
A double mAb sandwich ELISA with high specifity, and relative sensitivity for detection of sCD226 has been developed. It is shown to be an applicable method of detecting sCD226 in clinical specimens.
