抗青霉素单克隆抗体的制备及初步应用

目的:制备抗青霉素的单克隆抗体(mAb)并建立双抗体夹心ELISA检测方法,对临床上引起青霉素过敏反应的过敏原青霉噻唑蛋白进行研究。方法:将半抗原青霉素和载体蛋白偶联后免疫BALB/c小鼠,应用杂交瘤技术建立稳定分泌抗青霉素mAb的杂交瘤细胞株。常规制备腹水,用辛酸-硫酸铵法纯化,并对纯化的mAb进行特异性鉴定。通过对不同抗体组合的分析和条件的优化,建立检测过敏原的双抗体夹心ELISA方法。结果:经细胞融合、筛选及克隆化,共获得9株稳定分泌抗青霉素mAb的杂交瘤细胞株,其中5株亲和力较高。建立了双抗体夹心ELISA相对定量检测方法,该方法灵敏度达到870 U/L,平均回收率为107.81%,批内变异系数平均为6.7%,批间变异系数平均为9.3%,可用于A群链球菌制剂中青霉噻唑蛋白的检测。结论:成功地制备了抗青霉素的mAb,并建立了相对定量检测青霉噻唑蛋白的双抗体夹心ELISA法。

Preparation and primary application of monoclonal antibody against benzylpenicillin

AIM:To develop monoclonal antibody(mAb) against benzylpenicillin and to establish a Sandwich ELISA method for relative quantitative analysis of the allergen which induced penicillin allergy commonly in clinic.METHODS: Penicillin,as a kind of hapten,was conjugated with carrier protein to form complete antigen and then was used to immunize BALB/c mice.Hybridoma cells secrecting mAb against penicillin were developed.Ascites from the immunized mice were purified by caprylic acid-ammonium sulfate precipitation.The specificity of the purified antibodies was detected and the suitable antibodies were used for establishing Sandwich ELISA.RESULTS: Nine hybridoma cells stably secrecting mAb were prepared through cell fusion,screening and cloning,and five of these purified mAb had relative high affinity.The double-antibody Sandwich ELISA for relative quantitative analysis of benzylpenicilloyl protein was established with a sensitivity of 870 U/L.The average recovery rate was 107.81% and the average intra-and inter-coefficient of varitation(CV) was 6.7% and 9.3% respectively.The method was applied for relative quantitative analysis of benzylpenicilloyl protein from Group A Streptococcus Preparation.CONCLUSION: Nine hybridoma cell strains stably secreting mAb against benzylpenicillin were obtained.The double-antibody Sandwich ELISA for relative quantitative analysis of benzylpenicilloyl protein was established.