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A series of 14 new mouse monoclonal antibodies (MAbs) to keratins is described and the data suggesting their potential value in the differential diagnosis of human tumours are reported. The specificities of individual MAbs of the 'C-series' presented here range from monospecificity for keratin No. 7 (MAbs C-18, C-35, C-62, and C-68), keratin No. 8 (MAbs C-15, C-43, and C-15), and keratin No. 18 (MAbs C-04 and C-08) up to the broadly reacting 'pan-keratin' MAb C-11, with the target epitopes of the remaining four MAbs being shared by different pairs of keratin polypeptides. The results of the biochemical characterization of the MAbs, together with their immunohistochemical staining patterns on frozen as well as on paraffin sections of normal human tissues, suggest that they represent a significant contribution to the growing list of anti-keratin MAbs applicable in both research and routine diagnostic pathology. The immunohistochemical examination of a wide range of human neoplasms with the new MAbs not only confirmed their value in making distinctions between carcinomas, on the one hand, and lymphomas, and gliomas, on the other, but also verified the possibility of more subtle subdivisions within the group of adenocarcinomas and their metastases. Furthermore, the identification of small subsets of breast carcinomas with decreased levels or apparent loss of the keratin No. 7 polypeptide and some cases of stomach carcinoma with apparently induced expression of this keratin suggests that such 'exceptions' must be considered when using keratin spectra as one of the criteria in differential diagnosis.

In an attempt to raise a set of monoclonal antibody (MAb) probes for the study of biology and diagnostic value of human keratin No. 8, a series of hybridomas was prepared and their characteristics investigated by immunohistochemistry and immunoblotting. The polypeptide specificities of individual MAbs ranged from monospecificity for keratin No. 8 (MAbs C-15, C-23, C-36, C-43, C-47, C-51, and C-61) up to the "pan-keratin" MAbs C-11 and C-66 recognizing a broad range of keratins. The target epitopes of the remaining 4 MAbs (C-10, C-22, C-50, C-69) appear to be shared by various pairs of keratin polypeptides. The immunohistochemical investigation of tissues and/or cultured cells from 10 animal species revealed considerable variability of the interspecies crossreactivity of individual MAbs. The target epitopes of 5 MAbs appear to be phyllogenetically conserved from man to Xenopus, 6 of 12 MAbs tested show reactivity with several mammalian species (but not chicken or Xenopus) and the MAb C-15 reacts with human and sheep keratin only. The data obtained demonstrate that at least 9, and possibly as many as 12, nonidentical epitopes of the human keratin 8 polypeptide can be distinguished by this set of antibodies. The results of the present study suggest that our MAbs represent a significant contribution to the list of reagents applicable in both research of intermediate filament (IF) biology and diagnostic histopathology.

A comparative study with 21 recently raised monoclonal antibodies (3 of which are reported here for the first time) to human keratin polypeptides was performed on a wide range of paraffin-embedded tissues and tumors, aimed at the examination of effects of four different fixatives and protease pretreatment on the immunohistochemical detection of keratins. Our data demonstrated that: (a) formaldehyde-based fixatives modified by acidification and/or addition of methanol gave results superior to those achieved by routinely used formol saline; (b) relatively rare antibodies (4 out of 21) could be identified which gave reliable immunostaining patterns even on routine formalin-fixed material; (c) a proteolytic digestion step preceding the immunostaining was beneficial for the performance of the majority of antibodies in our panel. Additional options which could potentially lead to further improvement of keratin immunohistochemistry in paraffin embedded specimens are also suggested. This work provides the necessary basis for wider application of the anti-keratin antibodies of the C-series in both routine oncopathology and research-oriented retrospective studies.

Reactivity patterns of seven mouse monoclonal antibodies to human keratin 7 were compared using immunoblotting and immunohistochemistry on cultured cells and normal human and animal tissues. Differences in keratin specificities as determined by two-dimensional immunoblots and interspecies cross-reactivity data on 8 mammalian species suggest that at least six nonidentical epitopes of the keratin 7 molecule are recognized by this panel of reagents. Immunohistochemical examination of a panel of various human neoplasms with monoclonal antibodies monospecific for keratins 7, 18 and 19 revealed potential value of keratin subtyping in differential diagnosis of tumors in general and in subclassification of carcinomas in particular.

Establishment of a mouse-mouse hybridoma and partial characterization of IgM monoclonal antibody (M-04) identifying cytoplasmic filamentous structures is described. Immunofluorescence performed on a panel of various cultured human cell types as well as on frozen sections of normal and tumour tissues revealed specificity of M-04 antibody for cells of epithelial origin. Using MCF-7 cell line as a model, staining patterns of microtubules, microfilaments and M-04-target filaments in untreated cells were compared with those pretreated with Colcemid and Cytochalasin B. From both differential staining of various cell types and the results of drug studies it is concluded that monoclonal antibody M-04 binds to intermediate filaments of cytokeratin type. Furthermore, restricted expression of M-04 target determinant among epithelial tissues is suggested from the lack of reaction in stratified skin epithelium.

The monoclonal antibodies BA16 and BA17, reacting specifically with human keratin 19 (40 kD) have been tested by immunohistochemical staining methods for their reaction with a wide range of human tumours and cultured cells. Primary adenocarcinomas and their metastases showed a homogeneously positive reaction with greater than 95% of the tumour cells staining. Non-epithelial tumours, basaliomas and squamous cell carcinomas were unstained, while benign breast lesions and a thyroid adenoma show a mosaic pattern of stained and unstained (5-40%) cells. These three staining patterns were also seen in cultured cells. Positive homogeneous staining was seen in all breast cancer cell lines examined with the exception of PMC42, which exhibits stem cell characteristics, and which showed the heterogeneous pattern of staining seen in milk cell cultures. Non-epithelial lines and strains, two cell lines from cervical carcinomas and three SV40 transformed breast epithelial lines were unstained. The antibodies BA16 and 17 are potentially useful reagents for distinguishing adenocarcinomas (and their metastases) from non-epithelial tumours and from squamous carcinomas. They may also discriminate between benign and malignant breast lesions, and identify a specific differentiation phenotype in the secretory cell lineage.

A panel of 17 monoclonal antibodies (MAbs) recognizing various keratin polypeptides has been used to define their binding on non-epithelial elements in 28 bone-marrow samples and 14 lymph nodes, in order to establish their limitations for use as a possible tool for immunodiagnosis of carcinoma spread. Immunocytochemical studies have shown that only 8 antibodies consistently exhibited no false-positive staining of marrow cells. All the remaining MAbs labelled (mostly in a non-specific manner) a few cells of marrow samples derived from patients with either haematological disorders or malignant lymphomas. Fine granules and droplet-like cytoplasmic inclusions were predominant patterns of positive reactions. Homogeneous cytoplasmic staining reminiscent of specific keratin immunolabelling was occasionally seen as well. The positive cells could be also identified in some lymph nodes free of tumour infiltration. All antibodies visualized cytoplasmic droplets in scattered cells of lymph nodes taken from a patient with non-Hodgkin lymphoma. This type of positivity was mostly associated with positive histochemical reactions for iron. Quite significant was the detection of fibrillar positivity in the extrafollicular reticular cells in all nodes examined. Such a specific type of staining was exclusively induced by antibodies directed against epitopes of keratin 8 and 18, whereas those MAbs recognizing keratin 7 and 19 always gave negative results. Our data indicate that caution is required when such MAbs, considered as markers of specific cell types, are being used as an immunodiagnostic tool to identify single carcinoma cells. A series of criteria, including morphological ones, must be utilized in order to obtain meaningful results.

Three monospecific monoclonal antibodies (BA16, BA17 and A53-B/A2) recognizing different epitopes of the human keratin 19 were used to determine tissue distribution of this 40 kDa keratin polypeptide. Immunohistochemical methods revealed four different staining patterns among normal human epithelial tissues: firstly, complete negativity of the epidermis, sebaceous glands, hepatocytes and other tissues; secondly, homogeneous positivity as seen for example in the gall bladder and urinary bladder epithelium, endometrium and many other epithelia; thirdly, a mosaic of positive and negative cells among mammary gland luminal cells, prostate epithelia and some other epithelia and fourthly, a more complex heterogeneous pattern found in non-keratinizing squamous epithelia and hair follicles with generally the basal layer being the most strongly or sometimes exclusively stained. The pattern seen in non-keratinizing squamous epithelia varied considerably according to the fixation method and the antibody used as well as among different donors and in different areas of the same organ. The other three staining patterns were on the other hand nearly identical with all three antibodies on both frozen sections and sections of methacarn-fixed paraffin-embedded tissues. Our results provide evidence for differential expression of the human keratin 19 at the single cell level, an observation which could be exploited in the study of epithelial differentiation and pathology.

A comparative study of six mouse monoclonal antibodies against human 45 kDa keratin polypeptide (keratin No. 18) was undertaken using three experimental approaches: immunohistochemistry on normal human tissues, examination of interspecies cross-reactivity and identification of the target polypeptides in 1-D and 2-D immunoblots. The data suggest that at least five different antigenic sites of keratin 18 are recognized by this panel of reagents. The C-04 epitope is keratin 18-specific and widely conserved among mammalian species, while the antibodies DA7 and DC10 also react specifically with the 45 kDa keratin but stain simple epithelia of human origin only. Two antibodies, C-11 and C-66, decorate simple as well as stratified epithelia in human and in all seven animal species tested, but their respective target epitopes are shared by different groups of keratin polypeptides, which indicates their non-identity. In contrast to keratin specificity of the five above mentioned antibodies, the C-08 antibody cross-reacts with a 70 kDa nuclear lamina protein found in human and bovine tissues. The results of the present study provide the necessary basis for future applications of these antibodies in both routine immunodiagnostic work and as probes to study the biology of epithelial cells in general and the significance of keratin intermediate filaments in particular.