抗前列腺特异性膜抗原膜外区多肽单克隆抗体的研制及初步应用

目的建立前列腺特异性膜抗原（PSMA）膜外区多肽杂交瘤细胞株，并对其分泌的PSMA单克隆抗体进行初步鉴定，为PSMA的功能研究和人源化抗体的制备奠定基础，以求进一步用于前列腺癌的诊断和治疗。方法使用人工合成多肽免疫BABL／c小鼠，采用PEG融合技术建立杂交瘤细胞株，制备单克隆抗体。通过免疫荧光法、酶联免疫吸附法及斑点金标法确定单克隆抗体的交叉反应性、亲和力及免疫球蛋白的类型和亚类。结果获得两株可稳定分泌PSMA单克隆抗体的杂交瘤细胞，4F4为IsG1类，1F1为IgC3类。两株单抗均能识别LNCap细胞表达的PSMA蛋白，与不表达PSMA的PC-3、SP2／0等细胞无交叉反应。杂交瘤细胞株1F1培养上清效价为1：40。腹水效价为1：6400；而杂交瘤细胞株4F4培养上清效价为1：80。腹水效价为1：8000。结论成功地制备出两株抗PSMA单克隆抗体，均具有良好的特异性和亲和力，为进一步建立免疫分析方法。进行PSMA相关研究奠定了基础。

Preparation and Identification of Monoclonal Antibodies against PSMA

Objective To generate and characterize monoclonal antibody against artificial synthetiCal poptides derived from prostate specific membrane antigen （PSMA） extracellular domain by traditional method of preparing mAb as the foundation of diagnosis and therapy of prostate cancer. Methods Amino acid sequences of peptides with predicted antigenic determinant derived from PSMA were designed and synthesized by the method of solid-phase. The synthetic peptide was conjugated with carrier proteins such as KLH and BSA via maleimide linkage. BALB/c mice were immunized with purified of KLH conjugated peptide and antibody level was determined by inununofluorescence staining and ELISA. The fused cells that secreted specific monoclonal antibody were cloned continually by the limited dilution method, and then selected and analyzed further by the immunofluorescence staining. The affinity and specificity of mAb were measured by ELISA and Immunofluorescence staining. Results The hybridoma cell lines that could serect mAb steadily were identified by screening with indirect ELISA and 4 rounds of cloning. The isotype of the mAb 1F1 and 4F4 were IgG3 and IgG1, respectively. Hybridoma cell culture supematants titer of 1F1 was 1 ： 40 and that of 4F4 was 1 ： 80. The ascites titers of 1F1 and 4F4 were 1 ： 6400 and 1 ： 8000, respectively. Specificity analysis proved that all these mAbs reacted only with the LNCap cells which expressed PSMA and had no cross-reaction to PC-3 or SP2/0 cells which do not express PSMA. Relative affinity of monoclonal antibody of 4F4 was greater then that of 1F1. Conclusion It is the first report of preparation of mAbs specific to PSMA in China and the mAbs have high specificity and affinity which can be used for the further research on the PSMA function and the treatment of prostate cancer.