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目的:观察抗原中残留的洗涤剂成分在间接ELISA筛选阳性杂交瘤克隆时,对抗原包被效率的影响以及相应的对策。方法:以卵白蛋白(OVA)、卵运铁蛋白(OT)和本室制备的相应的单克隆抗体(mAb)作为实验模型,在抗原中加入不同浓度梯度的Triton X-100包被ELISA板,用相应的mAb细胞株培养上清做间接ELISA,确定Triton X-100对包被效率的影响。结果:当包被抗原中Triton X-100的浓度为3×10-6(v/v)时,可以明显抑制包被效率,当TritonX-100的浓度为1.6×10-4(v/v)时几乎可以完全抑制抗原的包被;在含有一定浓度Triton X-100的条件下,提高抗原的浓度,或适当增大包被过程中的稀释倍数,可以明显提高包被效率。结论:洗涤剂对抗原包被有较强的影响,在抗体制备过程中,应尽量提高抗原的初始浓度,降低洗涤剂的浓度,设计合理的针对含有洗涤剂抗原的ELISA筛选方案,是mAb制备成功的关键之一。
OBJECTIVE: To observe the effect of detergent composition remaining in antigen on the efficiency of antigen coating when indirect ELISA screening of positive hybridoma clones and the corresponding countermeasures. Methods: OVA, OT and the corresponding monoclonal antibody (mAb) prepared in our laboratory were used as experimental models. Triton X-100 with different concentration gradient was coated on the ELISA plate, Using the corresponding mAb cell line culture supernatant indirect ELISA to determine the Triton X-100 on the coating efficiency. Results: When the concentration of Triton X-100 in coating antigen was 3 × 10-6 (v / v), the coating efficiency could be obviously inhibited. When the concentration of TritonX-100 was 1.6 × 10-4 (v / v) Almost completely inhibited the antigen coating; in a certain concentration of Triton X-100 conditions, increasing the concentration of antigen, or appropriate to increase the dilution of the coating process, can significantly improve the coating efficiency. Conclusion: The detergent has strong influence on antigen coating. In the process of antibody preparation, the initial concentration of antigen should be raised and the concentration of detergent should be reduced. The rational design of ELISA screening program for detergent antigen is to prepare mAb One of the keys to success.