LAMP2单克隆抗体的制备及初步鉴定

Preparation and Characterization of a Specific Monoclonal Antibody against LAMP 2 protein

目的:制备抗-lamp2单克隆抗体,并对其生物学特性进行初步鉴定。方法:根据已发表的文献通过人工合成多肽,并将此多肽分别连接KLH和BSA,以peptide-KLH作为抗原免疫BALB/c小鼠,取血清效价大于1:10000的小鼠的脾细胞与骨髓瘤细胞SP2/0进行融合,以peptide-BSA包被,通过酶联免疫吸附实验(ELISA)检测杂交瘤细胞的上清进行筛选;得到能稳定分泌单克隆抗体的杂交瘤细胞株;通过鼠单克隆抗体亚类分型试剂盒鉴定单克隆抗体的亚类,采用免疫组化方法证明新制备的单克隆抗体能识别目的蛋白。结果:得到一株能稳定分泌抗-LAMP2单克隆抗体的杂交瘤细胞株,压用免疫组化方法证明新制备的抗-LAMP2单克隆抗体能识别天然的LAMP2;此单克隆抗体的亚类为。结论:杂交瘤细胞株能稳定分泌目的单克隆抗体;免疫组化方法证明新制备的单克隆抗体能识别自己天然的目的蛋白,为更好地研究LAMP2的生物学功能奠定了基础。

Objective：To develop and identify monoclonal antibody（MAb） against LAMP2 based on synthesized peptide.Method：According to the publication,peptides were synthesized and coupled to keyhole limpet hemocyanin（peptide - KLH） or to bovine serum albumin （peptide - BSA）.BALB/c mice were intraperitoneally immunized with peptide - KLH.Hybridomas were produced by fu.sin.g SP2/0 myeloma cells with spleen cells from the immunized BALB/c mouse whose serum titer of the anti - peptide - BSA greater than 1：10000 determined by ELISA.Superna.tan.t of the growing hybridoma was used to screen for the presence of anti - peptide - BSA antibody with ELISA.BALB/c mice were boosted with the hybridoma to produce a deal of MAb.A commercially available mouse MAb isotyping kit was used to identify the isotype of this MAb.Immunohistochemistry proved that the new prepared MAb could bind both native and denatured purpose protein.Result：One hybridoma.sec.reting MAb against LAMP2 was produced.Immunogolobulin subclass determination identified that this MAb was of the IgG2a/κtype.Immunohistochemistry showed that the new prepared MAb against LAMP2 could specifically recognize LAMP2 in its denatured and native form.Conclusion：This cell lines could stably.sec.ret their own purpose MAb.The new prepared MAb could respectively recognize their own purpose protein in experiments of Immunohistochemistry,suggesting that this MAb could bind both native and denatured LAMP2,which is very impor.tan.t in the bio.log.ical function study of LAMP2.