三唑磷酶联免疫吸附测定法中包被抗体稳定剂的研究

选择了甘油、山梨醇、聚乙二醇(PEG)、卵清蛋白(OVA)、多肽、糖、氨基酸等试剂,通过经验法和正交法相结合的手段配制了一系列稳定剂,对吸附在酶标板上的三唑磷多克隆抗体进行处理(37℃,1 h)后,再在37℃下连续贮存 7 d,利用直接竞争ELISA法对不同稳定剂处理的包被抗体免疫活性、亲合性及检测灵敏度进行检测,并与未经稳定剂处理的对照进行比较,筛选得到效果较好的稳定剂 1 (质量分数:甘油2.5%,氨基酸1.5%,蛋白胨3.0%,离子螯合剂0.1%,防腐剂0.01%)。用稳定剂 1 处理包被抗体后,4~6℃下保存半年及37℃下保存14 d的试验结果表明,抗体的活性相对保持率分别为97.8%和94.2%;其免疫活性、亲合性(I50分别为68.43和54.38 ng/mL)及灵敏度(I10分别为3.72和 3.22 ng/mL)与常规方法包被的抗体(包被好后不贮存,直接检测,I50为60.73 ng/mL,I10为 3.11 ng/mL)无明显差异;冻融试验表明,经稳定剂 1 处理的三唑磷抗体在反复冻-融次数不超过8次时其活性也是稳定的。说明筛选出的稳定剂可以显著提高三唑磷多克隆包被抗体的稳定性,可用于三唑磷ELISA试剂盒的生产。

Study on the Stabilizers for Coated Antibodies in ELISA of Triazophos

Mixtures of glycerol,sorbitol,amino acid,peptide,peptone,protein,chelator,antiseptic and so on as stabilizers were designed and prepared by means of experimental and orthogonal method.The anti-triazophos polyclonal antibodies (PAbs) coated on 96 microplates were treated with these stabilizers individually (37℃,1 h),then stored at 37℃ for 7 days.The immunoreactivities of the PAbs were determined by direct competitive ELISAs.PAbs were found to be stabilized under 3 stabilizers.After the further investigation of comparing the immunoreactivity,the affinity and the detection limit of the PAbs which treated with the three stabilizers individually,an optimized stabilizer was then selected.That was stabilizer 1 (m/m;glycerol 2.5%,amino acid 1.5%,peptone 3.0%,chelator 0.1%,antiseptic agent 0.01%).When the incubation of coated PAbs were in the presence of the stabilizer 1 ,with storage at 4~6℃ for half a year or 37℃ for 14 days,the relative immunoreactivity was 97.8% and 94.2%,respectively.And the affinity (I50 68.43 and 54.38 ng/mL,respectively) and the detection limit (I10,3.72 and 3.22 ng/mL,respectively) remained almost the same as those of the PAbs which were just coated on the plate without storage(I50 60.73 ng/mL,I10 3.11 ng/mL).Freeze-thaw test also demonstrated that the optimized stabilizer 1 was fit for the PAbs when the freeze-thaw was not more than 8 times.It indicated that the optimized stabilizer could improve the coated PAbs stability evidently,and could be applied to the production of the triazophos ELISA kit.