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The serum anti-kisspeptin antibodies were determined using an indirect enzyme-linked immunosorbent assay (ELISA) with kisspeptin peptide as a coating antigen as previously described22. The HPR-goat anti-rat antibody IgG (Bioss, Beijing, China) in 1:5000 dilutions was used as a secondary antibody. The antibody endpoint titers were determined using the formula N > μ + 2 SD as previously described25. Where: N is the absorbance of the highest serum dilution; μ and SD are the mean and standard deviation of negative control samples at the same dilution.
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