A monoclonal antibody (MAb) was generated against a synthetic peptide corresponding to amino acids 148 to 163 of the rfp protein with zinc finger domains. The MAb, designated RFP-1 (IgM), which was positive with the immunizing peptide in enzyme-linked immunosorbent assay, was reactive in immunoblotting with an in vitro translated rfp product as well as with native proteins in cell extracts made from mouse testis and HL-60 human leukemia cell line, both of which were previously shown to express high levels of rfp mRNA. When HL-60 cells were fractionated into nuclear and cytoplasmic components, the protein reactive with RFP-1 MAb was detectable only in the nuclear fraction. By the avidin-biotin complex immunoperoxidase method, this MAb strongly stained over 90% of the nuclei of human and mouse spermatogenic cells, except mature spermatozoon, and of human testicular tumor cells. In other human adult tissues, up to 60% of positive cells were observed. These antibody activities were clearly absorbed by pre-incubation of RFP-1 MAb with the immunizing peptide. These results thus indicated that RFP-1 MAb recognizes a nuclear protein which is expressed at high levels in male germ cells.
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