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 870U/ 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.
benzylpenicillin;benzylpenicilloyl;mAb;double?antibody Sandwich ELISA
目的: 製備抗青黴素的單株抗體(mAb)並建立雙抗體夾心ELISA檢測方法, 對臨床上引起青黴素過敏反應的過敏原青黴噻唑蛋白進行研究。方法: 將半抗原青黴素和載體蛋白偶聯後免疫BALB/c小鼠, 應用雜交瘤技術建立穩定分泌抗青黴素mAb的雜交瘤細胞株。常規製備腹水, 用辛酸?硫酸銨法純化, 並對純化的mAb進行特異性鑑定。通過對不同抗體組合的分析和條件的優化, 建立檢測過敏原的雙抗體夾心ELISA方法。結果: 經細胞融合、 篩選及克隆化, 共獲得9株穩定分泌抗青黴素mAb的雜交瘤細胞株, 其中5株親和力較高。建立了雙抗體夾心ELISA相對定量檢測方法, 該方法靈敏度達到870U/L, 平均回收率為107.81%, 批內變異係數平均為6.7%, 批間變異係數平均為9.3%, 可用於A群鏈球菌製劑中青黴噻唑蛋白的檢測。結論: 成功地製備了抗青黴素的mAb, 並建立了相對定量檢測青黴噻唑蛋白的.雙抗體夾心ELISA法。
青黴素; 青黴噻唑基; 單株抗體; 雙抗體夾心ELISA
青黴素因其高效、 低毒的特點在臨床上被廣泛應用, 但其結構中的β?內醯胺環很不穩定, 在溶液狀態尤其是鹼性條件下易開環與蛋白、 多肽的氨基發生親核反應生成穩定的青黴噻唑蛋白, 形成臨床主要的過敏原。青黴素類抗生素的過敏反應發生率居各種藥物之首, 青黴素不純物0.01 μg即可使青黴素高度敏感者產生嚴重的過敏反應。生理條件下青黴素開環後大約95%與蛋白質共價結合形成青黴噻唑基（benzyl penicilloyl, BPO）主要抗原決定簇, 而其他一些共扼物如penicillanyl, penicillenate、 青黴烯酸、 penamaldate, penaldate, D?青黴胺和penicoyl等則為次要抗原決定簇, 後者僅佔5%。理論上連線有兩個青黴噻唑基的蛋白、 多肽就可能與IgE分子形成橋式結構從而引發I型超敏反應。青黴素的這種不穩定性使得在製備過程中新增有青黴素的生物製品以及食品中都可能殘留有青黴噻唑蛋白, 而目前的檢測方法通常是針對遊離的有抗菌活性的青黴素, 尚無專門針對具有免疫原性且有潛在致敏危險的青黴噻唑蛋白的檢測方法。本研究中, 採用雜交瘤技術製備了穩定分泌抗青黴素mAb的雜交瘤細胞, 並建立了相對定量檢測青黴噻唑蛋白的雙抗體夾心ELISA法, 該方法特異性強、 靈敏度高, 有望對藥品、 食品中可能殘留的青黴噻唑蛋白進行痕量檢測。