Use of monoclonal antibody XCI for studying cell proliferation PREPARATION OF RECOMBINANT PARTS OF THE K 167 ANTIGEN

Aim: To characterise a newly developed mouse monoclonal antibody JC1 which recognises a nuclear antigen present in proliferating cells in normal tissues and neoplastic lesions, and which is absent in resting cells. Methods: The methodology was established using a representative range of frozen sections from normal tissues and from certain tumours which were immunostained with antibodies Ki67 and JC1. The molecular weight of the antigen recognised by JC1 was obtained by western blot analysis and this was compared with that of Ki67. IM-9 cell lysates containing Ki67 derived plasmids were also tested with JC1 antibody. Results: Biochemical investigation indicated that the antigen recognised by JCl gives two molecular weight bands of 212 and 123 kilodaltons, which is distinct from the well characterised anti-proliferation monoclonal antibody Ki67 (395-345 kilodaltons). In addition recombinant Ki67 protein is not recognised by JC1. Immunohistological reactivity was seen in areas known to contain numerous proliferating cells such as lymphoid germinal centres, splenic white matter, cortical thymocytes and undifferentiated spermatogonia. In tumours many cells from adenocarcinomas, oat cell carcinomas, squamous cell carcinomas of lung, and seminomas were labelled by JC1 with a distribution and proportion similar to that seen with Ki67. In normal tissues the only apparent difference was in testis where JC1 stained a considerably greater number of cells than Ki67. In all cases studied the new antibody showed nuclear reactivity only. JC1 did not show any cytoplasmic crossreactivity with squamous cells as is frequently seen with Ki67. Conclusion: Antibody JCI, which recognises a nuclear antigen present in proliferating cells, should provide a useful adjunct to Ki67 as a marker of proliferation especially in those cases such as squamous cell carcinomas where a Ki67 index cannot be determined. ( Clin Pathol 1992;45:860-865) The rate at which a tumour proliferates has long been considered to indicate its clinical course.1̀ Histopathologists have therefore sought means of determining this as an adjunct to diagnosis. The development of monoclonal antibodies51 recognising antigens present in cells during proliferation has brought this possibility within the realm of a general diagnostic laboratory. The monoclonal antibody Ki67 is perhaps the best known of such reagents and has been widely used to detect proliferating cells.'0 There are now several studies which indicate a correlation between tumour proliferation rate as assessed by Ki67 immunoreactivity and clinical outcome. 11-14 However, Ki67 has a well reported crossreaction with some epithelial cells, particularly of squamous type. This can lead to strong cytoplasmic reactivity making the determination of proliferation related staining impossible to define. This has led to the elimination of cases from experimental study, and, more importantly, will not allow some individual cases to be assessed in diagnostic practice. `-16 Here we report the production of a new mouse monoclonal antibody (designated JC 1) that is similar to Ki67 in that it seems to recognise selectively nuclei in proliferating cells. However, it differs from the latter antibody in showing little or no cytoplasmic labelling in squamous or other cell types. In addition, it does not react with the same antigen as Ki67, thus providing an independent assessment of tumour proliferation which might be valuable in clinical practice when individual cases are being evaluated.