Objective: To prepare the monoclonal antibody (mAb) against human myoglobin (MYO) of high titer and specificity and develop double-antibody sandwich ELISA for detecting MYO in human serum samples.
Methods: The BALB/c mice were immunized with natural human MYO, and the hybridoma cell lines secreting anti-MYO mAb were established using cell fusion and hybridoma screening techniques. The characteristics of the mAb were identified after affinity purification from ascites. Then the best antibody pair was selected from mAb to establish a one-step sandwich ELISA method. Sixty human serum samples were detected by the homemade ELISA kit and the imported one, respectively.
Results: Nine strains of hybridoma cell lines stably secreted anti-MYO mAb. Four strains named 2M1, 3M4, 5M7 and 10M4 could secrete high-quality mAb and the titers of them were in the range of 1.0×10(6) to 2.6×10(6) (A450 value was about 1.0). Three antibody pairs (2M1/HRP-3M4, 5M7/HRP-3M4, 10M4/HRP-5M7) were selected by double-antibody sandwich ELISA. Among them, the 5M7/HRP-3M4 had higher sensitivity and larger linear range. The homemade ELISA kit had a larger linear range (25-1000 ng/mL) than the imported one (25-500 ng/mL) and showed high accuracies in detecting human serum samples, being 95% (19/20) in positive samples and 100% (40/40) in negative samples.
Conclusion: With the anti-human MYO mAbs of high specificity and affinity, a one-step sandwich ELISA for detecting human MYO has been established successfully, which provides a basis for the development of domestic ELISA kit.
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