抗青霉素单克隆抗体的制备及初步应用
Preparation and primary application of monoclonal antibody against benzylpenicillin
[Abstract] 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 870U/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.
[Keywords]benzylpenicillin;benzylpenicilloyl;mAb;double?antibody Sandwich ELISA
[摘 要] 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870U/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。
[关键词]青霉素; 青霉噻唑基; 单克隆抗体; 双抗体夹心ELISA
青霉素因其高效、 低毒的特点在临床上被广泛应用, 但其结构中的β?内酰胺环很不稳定, 在溶液状态尤其是碱性条件下易开环与蛋白、 多肽的氨基发生亲核反应生成稳定的青霉噻唑蛋白, 形成临床主要的过敏原[1]。青霉素类抗生素的过敏反应发生率居各种药物之首[2], 青霉素不纯物0.01 μg即可使青霉素高度敏感者产生严重的过敏反应[3]。生理条件下青霉素开环后大约95%与蛋白质共价结合形成青霉噻唑基（benzyl penicilloyl, BPO）主要抗原决定簇, 而其他一些共扼物如penicillanyl, penicillenate、 青霉烯酸、 penamaldate, penaldate, D?青霉胺和penicoyl等则为次要抗原决定簇, 后者仅占5%[4]。理论上连接有两个青霉噻唑基的蛋白、 多肽就可能与IgE分子形成桥式结构从而引发I型超敏反应。青霉素的这种不稳定性使得在制备过程中添加有青霉素的生物制品以及食品中都可能残留有青霉噻唑蛋白, 而目前的检测方法通常是针对游离的有抗菌活性的青霉素, 尚无专门针对具有免疫原性且有潜在致敏危险的青霉噻唑蛋白的检测方法。本研究中, 采用杂交瘤技术制备了稳定分泌抗青霉素mAb的杂交瘤细胞, 并建立了相对定量检测青霉噻唑蛋白的双抗体夹心ELISA法, 该方法特异性强、 灵敏度高, 有望对药品、 食品中可能残留的青霉噻唑蛋白进行痕量检测。
1 材料和方法
1.1 材料 青霉素钾标准品, 为本所制备,相对分子质量（Mr）为372.49, 纯度为99.9%; 牛血清白蛋白（BSA）、 卵清蛋白（OA）、 多聚赖氨酸（Poly?L?Lysine, PLL）为Sigma公司产品; DMEM培养液为GibcoBRL公司产品, 新生牛血清购自杭州四季清公司; HAT、 HT、 弗氏完全佐剂和弗氏不完全佐剂、 Ig亚类试剂盒均购自Sigma公司, PEG4000购自Fluke公司, BCA蛋白浓度测定试剂盒购自Pierce公司, 所用二抗均为Jakson公司产品, 其余均为国产分析纯试剂。Sp2/0细胞由本所细胞室提供, BALB/c 雌鼠（6~8周龄）由本所实验动物中心提供。
1.2 方法
1.2.1 完全抗原的制备 ①免疫原的制备:参照Haan的方法[5], 将25 mg青霉素钾溶于pH9.6 Na2CO3缓冲液中, 充分混匀后加入5 mg牛血清白蛋白BSA（或卵清蛋白OA）, 调节pH值至11, 37℃搅拌过夜, 在0.01 mol/L PBS中充分透析至透析液中不再检出有抑菌活性。将透析物分装于-20℃保存。②筛选用包被抗原的制备: 用直链结构的多聚赖氨酸（Poly?L?Lysine, PLL）与青霉素偶联成BPO?PLL, 方法同①。
1.2.2 BALB/c小鼠的免疫 分别用两种载体偶联的免疫原免疫小鼠, 每只20μg, 皮下多点及四脚掌初次免疫; 14 d后相同剂量腹腔加强免疫, 每2周加强1次, 加强后每隔7 d眼眶采血, 分别测定针对半抗原和载体蛋白的抗体效价, 于细胞融合前3 d尾静脉加强免疫。
