Fisher Terrence L, Nocera MaryAnn, Willis Richard A, Turner Michael J, Abdul Alim C Siddiq, Brown Deborah M, Bourne Patricia A, di Sant'Agnese P Anthony, Messing Edward M, Lord Edith M, Frelinger John G
Department of Microbiology and Immunology, School of Medicine and Dentistry, University of Rochester Cancer Center, 575 Elmwood Avenue, Rochester, NY 14642, USA.
Prostate. 2002 May 15;51(3):153-65. doi: 10.1002/pros.10071.
Human kallikrein 2 (hK2) and prostate-specific antigen (PSA) are serine proteases in the human kallikrein gene family that are 80% identical at the protein level. Like PSA, hK2 is expressed primarily in the prostate, making it an attractive bio-marker for prostate cancer development. In addition, its potent enzymatic activity may functionally affect the biology of prostate cancer. In order to further elucidate the possible roles of hK2 in prostate cancer, we have generated a panel of hK2-specific, non-PSA cross-reactive monoclonal antibodies.
A novel tumor-immunization strategy was used to produce monoclonal antibodies. Human hK2 cDNA was transfected into a BALB/c tumor cell line and used to immunize both BALB/c and PSA-expressing BALB/c.PSA transgenic mice. Because the BALB/c.PSA transgenic mouse showed a biased response towards hK2, a B cell fusion was performed using spleen cells from a transgenic mouse immunized in this fashion.
A panel of monoclonal antibodies was produced and shown to be hK2-specific using newly developed hK2-specific sandwich ELISA and ELIspot assays. One of the monoclonal antibodies (6B7) was used to detect hK2 in human prostate by immunohistochemistry. Interestingly, two of the antibodies affected the function of hK2. The 1F8 antibody enhanced the enzymatic activity of hK2 whereas the 3C7 antibody inhibited its function.
These hK2-specific antibodies illustrate a novel approach for constructing B-cell hybridomas and provide useful reagents to examine the role of hK2 in the biology and detection of prostate cancer.
人激肽释放酶2（hK2）和前列腺特异性抗原（PSA）是人类激肽释放酶基因家族中的丝氨酸蛋白酶，在蛋白质水平上具有80%的同一性。与PSA一样，hK2主要在前列腺中表达，使其成为前列腺癌发展的一个有吸引力的生物标志物。此外，其强大的酶活性可能在功能上影响前列腺癌的生物学特性。为了进一步阐明hK2在前列腺癌中的可能作用，我们制备了一组hK2特异性、非PSA交叉反应的单克隆抗体。
采用一种新型的肿瘤免疫策略来制备单克隆抗体。将人hK2 cDNA转染到BALB/c肿瘤细胞系中，并用于免疫BALB/c小鼠和表达PSA的BALB/c.PSA转基因小鼠。由于BALB/c.PSA转基因小鼠对hK2表现出偏向性反应，因此使用以此方式免疫的转基因小鼠的脾细胞进行B细胞融合。
制备了一组单克隆抗体，并使用新开发的hK2特异性夹心ELISA和ELIspot分析证明其为hK2特异性。其中一种单克隆抗体（6B7）用于通过免疫组织化学检测人前列腺中的hK2。有趣的是，其中两种抗体影响hK2的功能。1F8抗体增强了hK2的酶活性，而3C7抗体则抑制其功能。
这些hK2特异性抗体展示了一种构建B细胞杂交瘤的新方法，并为研究hK2在前列腺癌生物学和检测中的作用提供了有用的试剂。
