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首页> 外文期刊>Molecular biotechnology >Purification and characterization of monoclonal antibodies against the free alpha-subunit of human chorionic gonadotrophin.
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Purification and characterization of monoclonal antibodies against the free alpha-subunit of human chorionic gonadotrophin.

机译：抗人绒毛膜促性腺激素游离α亚基的单克隆抗体的纯化和表征。

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摘要

Monoclonal antibodies (Mabs) against human chorionic gonadotropin hormone (hCG) were raised by hybridoma technology using Sp2/0 myeloma cells as fusion partner. Sixty-five percent of the total culture wells exhibited hybrid growth and 8% of the total wells (13 culture wells) contained anti-hCG secreting hybrids. A positive hybrid cell line secreting antibodies against the free alpha-subunit of hCG was cloned twice by limiting dilution method and eighty four clones were obtained that secreted monoclonal antibodies anti-alpha hCG. One of these hybridoma clones (1C4) secreting monoclonal antibodies against the free alpha-subunit of hCG was selected for purification and characterization purposes. This hybridoma cell line secreted monoclonal antibodies of IgG1 subclass, which were purified by affinity chromatography on Protein A Sepharose CL-4B column with a final relative recovery of antibody activity of 75% and a purification factor of about 12. The purified preparation was analyzed by SDS-PAGE, native PAGE, and IEF. Specificity studies of this Mab revealed that it recognized specifically an epitope on the free alpha-subunits of hCG, FSH, LH, and TSH as determined by enzyme immunoassays. On the other hand, this Mab exhibited crossreactivity with other pituitary hormones either as free subunits or intact molecules as follows: alpha hCG 100%; intact hCG 1.8%; beta hCG 0.14%; alpha FSH 24.5%; intact FSH 0.8%; beta FSH 0.09%; alpha LH 20.5%; intact LH 0.9%; beta LH 0.08%; alpha TSH 50.5%; intact TSH 3.7%; beta TSH 0.07%; The affinity constant (K) of this Mab with respect to free alpha-subunit of hCG was found to be 1.5 x 10(7) I/mol as determined by the simple antibody dilution analysis method.
机译：利用Sp2 / 0骨髓瘤细胞作为融合伴侣，通过杂交瘤技术产生了抗人绒毛膜促性腺激素（hCG）的单克隆抗体（Mabs）。总培养孔中有65％表现出杂种生长，而总培养孔（13个培养孔）中有8％含有抗hCG分泌杂种。通过有限稀释法将分泌抗hCG的游离α-亚基的抗体的阳性杂交细胞系克隆两次，得到84个分泌抗αhCG的单克隆抗体的克隆。为了纯化和鉴定目的，选择分泌针对hCG的游离α-亚基的单克隆抗体的这些杂交瘤克隆（1C4）之一。该杂交瘤细胞系分泌的IgG1亚类的单克隆抗体，通过亲和层析在蛋白A Sepharose CL-4B柱上进行纯化，最终抗体活性相对回收率为75％，纯化系数约为12。 SDS-PAGE，本机PAGE和IEF。对该Mab的特异性研究表明，通过酶免疫法测定，它可以特异性识别hCG，FSH，LH和TSH的游离α亚基上的表位。另一方面，该单克隆抗体表现出与其他垂体激素作为游离亚基或完整分子的交叉反应性，如下所示：αhCG 100％;完整的hCG 1.8％; βhCG 0.14％; αFSH 24.5％;完整FSH 0.8％; βFSH 0.09％;甲LH 20.5％;完整LH 0.9％; beta LH 0.08％; αTSH 50.5％;完整的TSH 3.7％; βTSH 0.07％;通过简单的抗体稀释分析方法测定，发现该Mab相对于hCG的游离α-亚基的亲和常数（K）为1.5×10（7）I / mol。

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