抗猪、兔卵透明带单克隆抗体的分析

Characterization of monoclonal antibodies against both pig zona pellucida and rabbit zona pellucida

以热溶解的猪卵透明带和热溶解的兔卵透明带免疫BALB/c小鼠建立了三株与兔卵透明带有较强交叉反应的抗猪透明带单克隆抗体。以“EIA—AB”ABC试剂盒进行单克隆抗体的分析,其中二株,IA_1,AC_4是IgG_1,一株,KA_3是IgM,全部为k轻链。以ELISA测定腹水中单克隆抗体的滴度,对猪卵透明带,AC_4为1∶400,000,IA_1为1∶16,000,KA_3为1∶100,对兔卵透明带,AC_4为1∶800,000,IA_1为5.120,KA_3为1∶100。在SDS—双向丙烯酰胺电泳中显示三株单克隆抗体具有相同分子量的轻链。猪卵透明带双向电泳后以^(125)Ⅰ标记的蛋白A作免疫印迹,IA_1主要结合第三组分的酸性端的抗原。二株单克隆抗体,IA_1和KA_3,可抑制体外猪和兔的精神结合(p<0.001和p<0.01),另一株,AC_4则在体外对两种动物的精卵结合无抑制。

Three monoclonal antibodies (MABS) were raised against both pig zona pellucida and rabbit zona pellucida with a dual immunization protocol employing heat solution pIg zona (HSPZ ) and heat Solution Rabbit zona (HSRZ).Of 140 wells screened, 12 wells we(?)e positive to both HSPZ and HSRZ and 27 wells were positive to HSPZ only and Non-well was positive to HSRZ only by solid-phase, emzyme-like immunoassay (ELISA ) using ABC kit. Determination of class and subclass of MAbs was performed by means of ELISA assay w with the 'EIA-Ab'ELISA kit . Two MAbs (IA_1,AC_4 ) were IgG_1 and one MAb(KA_3) was IgM and all light chains of MAbs were kappa. The titers of these MAbs in ascites fluid were determined by ELISA and they were. 1: 400000, 1:16000, 1:100 to HSPZ and 1:800000, 1:5120 and 1:100 to HSRZ individually. The light chains of MAbs were one band on the SDS-PAEG and on the SDS-PAGE autoradiography with^(35) S methionine. These MAbs had the same molecular weight of each MAb light chain on the 2DPAGE. One MAb, IA_1, recognized the major antigen of acid end of I band on 2D-1 PAGE by western immunoblot using ^(125)I-protein A autoradiography. Two MAbs, IA, and KA_3, inhibited both boar sperm binding to pig zona pellucida (P<0.001) and rabbit sp erm finding to rabbit zona pellucida (P<0.01), but one MAb, AC_4, did not to both (P>0.05).