Vernekar Vandana J, Bandivdekar A H, Raghavan Vijaya P, Kamada M, Koide S S
National Institute for Research, in Reproductive Health, J. M. Street, Parel, Mumbai, India.
Am J Reprod Immunol. 2004 Feb;51(2):106-11. doi: 10.1111/j.1600-0897.2004.00109.x.
The 80 kDa human sperm antigen (HSA) is a sperm-specific and conserved antigen, capable of inducing immunological infertility. Partial N-terminal amino acid sequences of 80 kDa HSA (Peptide NT) and its peptides obtained by digestion with endoproteinase Lys-C (peptides 1-4) and endoproteinase Glu-C (peptides 5-6) did not show any sequence homology with reported known proteins deposited in the Gen-Bank. These sequenced peptides were synthesized and conjugated to key hole limpet haemocyanin (KLH) and evaluated for its antifertility effects. The present communication describes the characterization of these peptides and their antibodies.
Peptides NT, 1, 2, 3 and 4 were synthesized and conjugated to KLH. Antibodies to KLH conjugated peptides were raised in rabbits by active immunization and the antibody titer was determined by enzyme-linked immunosorbent assay (ELISA) using sperm extract coated wells. The binding specificity of the synthetic peptides or purified 80 kDa HSA to their antibodies was assessed in the presence of various doses of respective synthetic peptides or 80 kDa HSA. The binding specificity was further confirmed by Western blot analysis. Antipeptide antibodies were also checked for sperm agglutinating activity, in-vitro.
Active immunization of rabbits elicited significant antibody titers against the synthetic peptides, except for peptide 3. Antipeptide antibodies specifically recognized the native protein in an ELISA and induced in-vitro agglutination of human, rat and monkey sperm. In addition, Western blot analysis showed that these antipeptide antibodies specifically bind to the 80 kDa HSA band of the sperm extract.
Synthetic peptides of 80 kDa HSA are immunogenic and antibodies raised against these peptides recognize the native protein detected by ELISA, Western blot analysis. In addition, they possess sperm agglutinating activity. These findings suggest that they are promising candidates in the development of immunocontraceptives.
80 kDa人精子抗原（HSA）是一种精子特异性且保守的抗原，能够诱发免疫性不育。80 kDa HSA的部分N端氨基酸序列（肽段NT）以及通过内肽酶Lys-C消化获得的其肽段（肽段1 - 4）和内肽酶Glu-C消化获得的肽段（肽段5 - 6）与基因库中已报道的已知蛋白质均无序列同源性。合成这些测序的肽段并将其与钥孔血蓝蛋白（KLH）偶联，评估其抗生育作用。本通讯描述了这些肽段及其抗体的特性。
合成肽段NT、1、2、3和4并与KLH偶联。通过主动免疫在兔体内产生针对KLH偶联肽段的抗体，使用包被有精子提取物的微孔板通过酶联免疫吸附测定（ELISA）测定抗体效价。在存在不同剂量的相应合成肽段或80 kDa HSA的情况下，评估合成肽段或纯化的80 kDa HSA与其抗体的结合特异性。通过蛋白质印迹分析进一步确认结合特异性。还在体外检查抗肽抗体的精子凝集活性。
除肽段3外，兔的主动免疫产生了针对合成肽段的显著抗体效价。抗肽抗体在ELISA中特异性识别天然蛋白，并诱导人、大鼠和猴精子的体外凝集。此外，蛋白质印迹分析表明这些抗肽抗体特异性结合精子提取物的80 kDa HSA条带。
80 kDa HSA的合成肽段具有免疫原性，针对这些肽段产生的抗体可通过ELISA、蛋白质印迹分析识别天然蛋白。此外，它们具有精子凝集活性。这些发现表明它们是免疫避孕药物开发中有前景的候选物。
