有机膦半抗原诱导的抗神经性毒剂单克隆抗体的制备及鉴定

目的 :制备新化合物 1 羟基 1 对胺基苯基甲磷酸单频哪基醇酯 (P4 )的单克隆抗体 (mAb)。方法 :以P4为半抗原 ,与血蓝蛋白 (LPH)化学偶联制备人工抗原 (P4 LPH) ,并以其免疫BALB/c小鼠 ,制备抗P4的mAb。用竞争抑制酶免疫分析法 ,测定mAb对梭曼等 7种配体的结合活性 ,结果以mAb与P4 BSA的结合被抑制 5 0 %时的配体浓度 (IC50 ) ,表示mAb的交叉反应性。结果 :建立了 9株对P4 BSA阳性反应的mAb ,它们分属 3种Ig亚类 ;6株 (2B2、3A10、3D5、4F1、6C9和 6H4 )为IgG1,1株 (3B9)为IgG2b ,2株 (1B3和 6H5 )为IgM。 9株mAb中 ,8株具有与半抗原P4的结合的特异性。4株 (1B3、2B2、3D5和 4F1)具有梭曼结合活性 ,其IC50 值分别为 10 -3 、10 -3 、10 -5和 10 -6mol/L ;1株 (4F1)具有与对氧磷结合的特异性 ,其IC50 值为 10 -5mol/L。 4株腹水的mAb鼠滴度高 ,分别达 10 -6(1B3和 2B2 )和 10 -8(3D5和 4F1)。结论 :用新的半抗原P4成功地制得抗梭曼和抗对氧磷的mAb ,可用于梭曼抗体酶、梭曼的免疫抗毒和免疫检测研究

Preparation of monoclonal antibodies against neurotoxic agent raised from an organophosphorus hapten and its characterization

AIM: To develop monoclonal antibodies(mAbs)against a novel hapten, pinacolyl 1 p aminophenyl 1 hydroxymethylphosphonate(P4). METHODS: P4 was coupled chemically with Limulus polyphemus hemocyanin (LPH) to form a coupling agent P4 LPH, and then BALB/c mice were immunized with the P4 LPH. MAbs were prepared by hybridoma technique. Cross reactivities of mAbs to 7 ligands were detected by competitive inhibition enzyme immunoassay. The cross reactiveities were expressed by the concentration of ligand which inhibited 50% reaction of mAbs(IC 50 ). RESULTS: Nine mAbs were developed. Eight of them were specific for P4, four (1B3, 2B2, 3D5 and 4F1) could react to soman, their IC 50 were 10 -3 , 10 -3 , 10 -5 and 10 -6 mol/L respectively, and one (4F1) could recognize paraoxon, IC 50 being 10 -5 mol/L. All of which were effective with a titer as high as 10 6 to 10 8 in dilution. CONCLUSION: mAbs specific for soman and paraoxon are developed successfully, which may be used for research on soman abzyme, immuno detoxification of soman poisoning and immunoassay for soman or/and paraoxon.