抗多环芳烃单克隆抗体的制备及初步鉴定

Development and identification of monoclonal antibodies against polycyclic aromatic hydrocarbons

目的本研究制备了2株抗多环芳烃单克隆抗体,并对其免疫学特性进行了初步鉴定。方法以芘丁酸(PBA)为半抗原,采用活性酯法分别与BSA和OVA偶联合成免疫抗原和包被抗原,4次免疫Balb/c小鼠后,选择抗体效价1∶25 600的小鼠脾细胞与Sp2/0细胞采用PEG进行细胞融合。ELISA法筛选阳性细胞株,有限稀释法进行亚克隆,体内诱生腹水法制备单克隆抗体。抗体经Protein G亲和纯化后,SDS-PAGE检测抗体纯度,测定抗体水平和亚类。间接ELISA法测定抗体效价、亲和力常数;竞争ELISA法测定2株抗体特异性和交叉反应率。结果 PBA-BSA免疫成功,共获得2株可稳定分泌抗多环芳烃单克隆抗体的杂交瘤细胞株,命名为#1、#6-B7。2株单克隆抗体纯化后经鉴定为Ig G1亚类,质量浓度分别为4.1和3.6 mg/ml,效价分别为1∶25 600和1∶51 200,亲和常数分别为7.52×108和1.25×109。结论 2株抗体均对高环PAHs具有较高的交叉反应率,而无法识别低环的PAHs。

This study aimed to develop two monoclonal antibodies（m Abs） against polycyclic aromatic hydrocarbons and identify their immunological characteristics. By activated ester method, hapten 1-hyrenylbutyric acid（PBA） was covalently linked to the carrier proteins BSA and OVA for the synthesis of immunogen and coated antigen. The splenocytes of two mice with the titer of 25 600 were fused with Sp2/0 cells using PEG method after four times immunization. The hybridoma cell lines were screened by ELISA based on the coated antigen PBA-OVA,and then the positive clones were subcloned with limiting dilution method. At last, monoclonal antibodies were prepared with in vivo ascites induction method. The antibody purity was tested by SDS-PAGE after purification by protein G affinity column, while the concentrations and the subtypes were measured by using the reagent kits; the titers and affinity constants were tested by indirect ELISA and the cross-reaction with 16 polycyclic aromatic hydrocarbons were tested by competitive ELISA. The results showed that PBA-BSA artificial antigen was synthesized successfully and two hybridoma cell lines（#1 and #6-B7） secreting anti-PAHs m Abs were established.The two m Abs were purified and identified as Ig G1 subtype, with the concentration of 4.1 mg/ml and 3. 6 mg/ml, the titter of 1 ∶25 600 and 1 ∶51 200, the affinity constant of 7.52 ×108and 1.25 ×109, respectively. Furthermore, two antibodies are highly cross-reactive to high ring PAHs, but do not recognize the low ring PAHs.