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目的:建立可用于不同剂型重组人促卵泡激素(rhFSH)产品质量控制的特异性SDS-PAGE/Western blot鉴别方法。方法:用SDS-PAGE电泳分离由不同生产企业生产的不同剂型rhFSH产品,通过半干电泳将凝胶上的待测蛋白转移至硝酸纤维素膜(NC膜),采用特异性抗体在NC膜上进行免疫杂交。通过检测灵敏度、线性和专属性试验,进行方法学考察。结果:对rhFSH特异性检测灵敏度可达5ng;对3个厂家生产的3种注射液、1种注射用冻干制剂检测,均可得到阳性结果,且试验结果不受不同剂型中的防腐剂、稳定剂和其他辅料干扰;用本法对等量的其他重组糖蛋白激素如重组人促黄体激素(rhLH)、重组人绒促性素(rhCG)测定结果均为阴性。结论:本方法灵敏度高、专属性强,可作为rhFSH的特异性鉴别方法,用于不同剂型rhFSH产品的质量控制。
OBJECTIVE: To establish a specific SDS-PAGE / Western blot method for the quality control of recombinant human follicle-stimulating hormone (rhFSH) in different dosage forms. METHODS: Different dosage forms of rhFSH products produced by different manufacturers were separated by SDS-PAGE electrophoresis and the proteins to be tested were transferred to nitrocellulose membrane (NC membrane) by semi-dry electrophoresis. The specific antibodies were detected on NC membrane Immuno hybridization. Methodological studies were performed by testing for sensitivity, linearity and specificity. Results: The specificity of rhFSH was 5ng. The positive results were obtained on the three kinds of injection produced by three manufacturers and one kind of lyophilized preparation for injection, and the results were not affected by the preservatives, Stabilizers and other excipients interfere with this method for the equivalent amount of other recombinant glycoprotein hormones such as recombinant human luteinizing hormone (rhLH), recombinant human rumin (rhCG) determination results were negative. Conclusion: The method has high sensitivity and specificity, and can be used as a specific identification method of rhFSH for the quality control of rhFSH products with different dosage forms.