Objective To prepare anti-human B7 homolog 4(B7-H4)egg yolk immunoglobulins(IgY)polyclonal antibody and establish a double-antibody sandwich ELISA for the detection of soluble B7-H4(sB7-H4)protein in human serum.Methods Bioinformatics was used to screen specific B cell epitope peptides of human sB7-H4.New Hyland Grey laying hens were immunized with these peptides,and the eggs from the immunized hens were collected to purify chicken anti-human B7-H4 IgY antibody.The purity,concentration and titer of the antibody were detected,and its specificity and function of the antibodies were verified by using ELISA,Western blot analysis and flow cytometry,respectively.A double-antibody sandwich ELISA was established to detect sB7-H4 in clinical samples by using the IgY antibody.Comparative detection was performed using a commercialized ELISA kit on the same set of clinical samples.Results The chicken anti-human B7-H4 IgY antibodies were successfully prepared and proven to be highly specific for the human B7-H4 protein.The ELISA established with the IgY polyclonal antibody detected significantly higher levels of soluble B7-H4 in the serum of patients with ovarian cancer and benign ovarian tumors compared to healthy controls.These results were consistent with the detection results obtained using a commercialized ELISA kit.However,the ELISA with IgY antibody exhibited higher sensitivity than the commercialized kit.Conclusion The chicken polyclonal antibody against human B7-H4 IgY is successfully prepared,and a double-antibody sandwich ELISA suitable for detecting sB7-H4 protein in human serum is established.
维普
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