The monoclonal antibody Ki-67 reacts with a human nuclear cell proliferation-associated antigen that is expressed in all active parts of the cell cycle. Recently we have raised monoclonal antibodies, MIB 1-3, against recombinant parts of the Ki-67 antigen. These antibodies are true Ki-67 equivalents, as demonstrated by immunostaining of fresh specimens, biochemistry, and molecular biological techniques. Formalin-fixed, paraffin-embedded sections routinely processed for immunohistochemistry failed to stain for Ki-67 and MIB 2. Antibodies MIB 1 and MIB 3 labelled mitotic figures, while non-mitotic proliferating cells were negative under these conditions. However, when dewaxed microwave oven-processed paraffin sections of formalin-fixed tissues were used, MIB 1 and MIB 3 gave strong nuclear staining of those cells presumed to proliferate under a variety of normal and neoplastic conditions. Moreover, routine decalcification or depigmentation techniques did not alter the immunoreactivity of MIB 1 and MIB 3 with microwave-processed paraffin sections. This method is highly reproducible, easy to perform at low cost, and no additional technical skill is needed because after microwave treatment just routine immunohistochemical methods are used. Since we have successfully applied this new method to sections obtained from paraffin blocks stored for a long time (in one case more than 60 years), the assessment of cell kinetics through the detection of Ki-67 antigen is now possible on archival material collected in histopathology departments all over the world.

Abstract. The antigen defined by mAb Ki-67 is a human nuclear protein the expression of which is strictly associated with cell proliferation and which is widely used in routine pathology as a "proliferation marker" to measure the growth fraction of cells in human tumors. Ki-67 detects a double band with apparent molecular weights of 395 and 345 kD in immunoblots of proteins from proliferating cells. We cloned and sequenced the full length eDNA, identified two differentially spliced isoforms of mRNA with open reading frames of 9,768 and 8,688 bp encoding for this cell proliferation-associated protein with calculated molecular weights of 358,761 D and 319,508 D, respectively. New mAbs against a bacterially expressed part and a synthetic polypeptide deduced from the isolated eDNA react with the native Ki-67 antigen, thus providing a circle of evidence that we have cloned the authentic Ki-67 antigen eDNA. The central part of the Ki-67 antigen eDNA contains a large 6,845-bp exon with 16 tandemly repeated 366-bp elements, the "Ki-67 repeats", each including a highly conserved new motif of 66 bp, the "Ki-67 motif", which encodes for the epitope detected by Ki-67. Computer analysis of the nucleic acid and the deduced amino acid sequence of the Ki-67 antigen confirmed that the cDNA encodes for a nuclear and short-lived protein without any significant homology to known sequences. Ki-67 antigen-specific antisense oligonucleotides inhibit the proliferation of IM-9 cell line cells, indicating that the Ki-67 antigen may be an absolute requirement for maintaining cell proliferation. We conclude that the Ki-67 antigen defines a new category of cell cycleassociated nuclear nonhistone proteins.

Enzymatic and non-enzymatic treatments for antigen unmasking on formalin-fixed, paraffin-embedded, dewaxed sections were optimized and compared by the use of a panel of antibodies of diagnostic relevance (anti-cytokeratins, vimentin, S-100, T- and B-cell receptors, Ki-67/MIB 1, muscle actin). Non-enzymatic unmasking was obtained by boiling the slides in a microwave oven in 0.01 M salt solution (pH 6) or in 6 M urea. Trypsin or pronase digestion was used for comparison and found to be necessary for some of the reagents. The investigation was then extended to 256 antibodies; the epitopic amino acid sequence was known for 48 of them. We found that enzymatic and non-enzymatic antigen unmasking are not dependent on the epitope sequence, but some antigens benefit selectively from one treatment but not from the other. Denaturation of proteins is the likely mechanism which leads to immunodetection on microwave oven-boiled slides; this suggestion is supported by the use of denaturating solutions and by the observation that endogenous enzymes were inactivated and a few antigens were no longer immunodetectable after boiling. Non-enzymatic methods for antigen unmasking are a powerful new tool for broadening the use of antibodies for immunostaining formalin-fixed, paraffin-embedded sections and should be used in parallel with the traditional enzymatic methods.

Ains-To prepare a rabbit antiserum equivalent to MIB 1 to permit the simultaneous assessment of cell proliferation and other markers of interest using double labelling studies. Methods-Rabbits were immunised with a synthetic peptide deduced from the cDNA sequence coding for the antigen. Serum samples were tested for immunoreactivity using different immunobiochemical methods.

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.