A monoclonal antibody, NCRC-11, raised to human breast carcinoma. 1. Production and immunohistological characterization

The production of a mouse monoclonal IgM antibody, NCRC-11, raised against human breast carcinoma is described. It has been characterized immunohistologically. The antigen recognised has a wide but highly specific distribution in normal tissues, being virtually confined to the surface of certain epithelial cell types. It is found in some forms of epithelial metaplasia and most epithelial malignancies, particularly adenocarcinomas. The heterogeneity of staining in mammary carcinomas is outlined and is of particular interest. The immunohistological staining distribution of NCRC-11 is similar to other antibodies, including anti-epithelial membrane antigen, which were raised against human milk fat globule membrane. A competition experiment with some of these antibodies, using a flow cytofluorimeter, showed competition with one antibody, LICR LON/M8.     

Cell lineage specificity of newly raised monoclonal antibodies against gastric mucins in normal, metaplastic, and neoplastic human tissues and their application to pathology diagnosis

The specificity of monoclonal antibodies against gastric mucins (designated as HIK1083, PGM 36, and PGM 37) was studied immunohistochemically in normal, metaplastic, and neoplastic human tissues. These antibodies labeled class III mucin-producing cells identified by paradoxical concanavalin A staining in normal stomach, duodenum (Brunner gland), biliary tract, and main pancreatic duct; in mucinous metaplasia of pancreas and gallbladder; and in adenocarcinomas of stomach (90%), bile duct (80%), gallbladder (100%), pancreas (80%), lung (100% of goblet cell type adenocarcinomas), ovary (67% of mucinous carcinomas), and uterine cervix (100% of adenoma malignum tumors). Normal and neoplastic cells of esophagus, colon, salivary gland, kidney, endometrium, breast, prostate, and liver, as well as normal small intestine, lung, and uterine cervix, were all negative. The antibodies used should be valuable for the detection of class III mucin and class III mucin-producing cells in normal, metaplastic, and neoplastic tissues.     

Monoclonal antibody EBM/11: high cellular specificity for human macrophages.

A monoclonal antibody, EBM/11, was raised against isolated human lung macrophages. Immunohistochemically this antibody reacted with freshly isolated lung macrophages and blood monocytes, mononuclear cells (presumptive macrophages) in sections of lung, skin, stomach, small and large bowel, pancreas, spleen, tonsil, placenta, liver, gall bladder, heart, thyroid, pituitary, brain, and peritubular and mesangial cell in kidney. Microglial cells and osteoclasts also labelled with EBM/11. The antibody reacted with cytoplasmic structures rather than with cell membranes. The epitope recognised by EBM/11 was present on four polypeptides (of 120, 70, 64 and 22 kilodaltons). It did not react with any other cell type in the tissues screened except the epithelium of renal proximal tubules. This antibody may be useful in identifying and elucidating the function of macrophages in pathological processes.     

Widespread distribution in human tissues of an antigenic determinant of granulocytes.

The monoclonal antibodies AGF4 .48 and AGF4 .36 have previously been shown to distinguish human granulocyte lineage cells from other peripheral blood and bone marrow cells. The AGF4 .48 antigen, which is carbohydrate in nature, together with similar antigens described by numerous investigators have been considered specific differentiation antigens of myeloid cells. In immunohistological studies of a wide range of normal tissues, the AGF4 .48 antibody selectively stained cells in several apparently unrelated tissues. These included proximal tubules and descending thin limbs in the kidney, parietal cells in the stomach, a variety of other epithelial cells, astrocytes in the brain, and cells in the anterior pituitary containing adrenocorticotrophic hormone. The AGF4 .36 antibody gave similar results on kidney, stomach and pituitary. These findings emphasise the importance of assessing the binding of monoclonal antibodies, which appear unique in their reactivity with blood cells, to non-haemopoietic tissues before assigning specificity to reagents. The distribution of cells expressing the AGF4 .48 and AGF4 .36 antigen correlates with the occurrence of the 3-fucosyl-N-acetyllactosamine carbohydrate structure in various secreted glycoproteins.     

A murine monoclonal antibody raised against human non-small cell carcinoma of the lung

Murine monoclonal antibodies (MAbs) reactive with human non-small cell carcinoma of the lung (NSCCL) were produced following immunization with a membrane preparation of Calu-3, a human adenocarcinoma of the lung cell line grown in nude mice. Positive hybrids unreactive with normal liver membranes and human peripheral white blood cells were selected for further testing. One MAb (RS5-4H6) recognized an antigen expressed in a variety of lung and other carcinoma cell lines as detected by flow cytometry. Immunohistology showed a selectivity for normal and neoplastic lung epithelium, as well as other cancers. The antigen was detected by immunohistology in 87% of tumor specimens derived from the lung, breast, colon, kidney, and ovary. Most other normal human tissues were not stained but occasional cells of the stomach, salivary and other glands, as well as kidney tubules were reactive. This MAb is an IgG1. Western blot analysis indicated that the antibody reacts selectively with an antigen greater than 300 kD. The antigen is resistant to neuraminidase treatment and periodate oxidation, but sensitive to pronase treatment, suggesting that the epitope is peptide in nature. This antibody may be potentially useful as a targeting agent for radioimmunodetection and immunoconjugate therapy.     

Identity of the neoplastic alkaline phosphatase as revealed with monoclonal antibodies to the placental form of the enzyme

A panel of six monoclonal antibodies and a conventional polyclonal antibody raised against human placental alkaline phosphatase (ALP) were used to characterize the alkaline phosphatase detected by means of histochemistry on tumors of breast, ovary, lung, gastrointestinal tract, and kidney. Complete antigenic identity between the tumor ALP and the placental ALP was found only in one lung tumor. However, ten tumors reacted with the polyclonal antibody and some monoclonal antibodies, thus exhibiting partial identity with the placental ALP.     

Production of monoclonal antibody SP-21 to colon-ovarian tumor antigen, COTA

An IgG monoclonal antibody, SP-21, directed against colon-ovarian tumor antigen, COTA, is reported. The antibody had no reactivity with CEA, normal colonic mucin, CSAp, ABO blood group antigens, or with normal human lung, liver, spleen, kidney, plasma and saliva in studies using the enzyme-linked immunoassay method (ELISA). Immunoperoxidase staining of colon, lung, kidney, and prostate cancer tissues and benign and inflammatory colon disease tissues revealed a specificity identical to that of the polyclonal (goat) anti-COTA antibodies.     

Extraglomerular distribution of immunoreactive Goodpasture antigen

The distribution of Goodpasture antigen (GA) was studied in a range of human tissues using indirect immunofluorescence and immunoperoxidase techniques. Frozen sections were stained using (1) a mouse monoclonal antibody (P1) raised against the autoantigenic component of human glomerular basement membrane, (2) autoantibodies eluted from the kidneys of patients with Goodpasture's syndrome, (3) antibodies eluted from the kidneys of a sheep with Steblay nephritis, and (4) mouse monoclonal and guinea pig polyclonal antibodies to human type IV collagen. The same pattern of staining was demonstrated using the eluted antibodies and monoclonal antibody P1. The presence of GA was confirmed in the lung and choroid plexus. GA was also detected in basement membranes at a number of previously unreported sites in the eye, thyroid, pituitary, adrenal, breast, and liver. GA was absent from other sites at which type IV collagen could be demonstrated. Direct immunofluorescence studies of tissue from a patient with Goodpasture's syndrome revealed deposition of IgG in the choroid plexus and eye, as well as in the kidney and lung.     

Characterization of a panel of novel anti-p21Waf1/Cip1 monoclonal antibodies and immunochemical analysis of p21Waf1/Cip1 expression in normal human tissues.

As a universal inhibitor of cyclin-dependent kinases and one of the target genes of the tumor suppresser p53, p21Waf1/Cip1 can act as a tumor suppresser through its ability to control cell cycle progression. To study the function of p21Waf1/Cip1 protein and to investigate its tissue distribution, a panel of anti-p21Waf1/Cip1 monoclonal antibodies was generated. These anti-p21Waf1/Cip1 monoclonal antibodies were initially raised against a GST-p21Waf1/Cip1 fusion protein produced in bacteria. Detailed characterization of the antibodies showed that they can specifically detect p21Waf1/Cip1 by immunoblotting, immunoprecipitation, and immunostaining. The specific induction of p21Waf1/Cip1 expression in response to gamma-radiation in cells containing p53 was also detected by these antibodies. The ability to detect p21Waf1/Cip1 expression in conventionally fixed tissue sections allowed us to investigate the distribution of p21Waf1/Cip1 in 23 different types of normal human tissues, and p21Waf1/Cip1 expression was found in most tissues. A close inverse relationship between p21Waf1/Cip1 expression and proliferation was seen in some tissues, including gastrointestinal tract. However, such association is not universal. In tissues such as lung, kidney, thyroid, pancreatic ducts and acini, and liver, despite the fact that most of the cells are quiescent, expression of p21Waf1/Cip1 was detected only in occasional epithelial cells. All these suggest that the expression of p21Waf1/Cip1 varies among different human tissues. Finally, epitope mapping of the anti-p21Waf1/Cip1 antibodies using a peptide library covering the entire p21Waf1/Cip1 protein sequence indicates that two of the antibodies recognize a region of p21Waf1/Cip1 close to that bound by proliferating cell nuclear antigen. These two monoclonal antibodies will therefore be additionally useful in further understanding the functions of p21Waf1/Cip1 both in vitro and in vivo.     

Monoclonal antibodies specific for human monocytes, granulocytes and endothelium

Four monoclonal antibodies against antigens of human myeloid cells have been produced and thoroughly characterized in terms of their reactions with peripheral blood cells, cell lines, nine lymphoid and non-lymphoid tissues and the polypeptides with which they react. UCHM1 and SmO identify antigens present on the majority of blood monocytes and a variable, but lower, proportion of tissue macrophages. From their morphology and location in tissues, these cells appear to be recirculating monocytes. SMO antigen is also present on platelets. In addition, both antibodies stained endothelial cells, SMO in all tissues examined and UCHM1 variably. Biochemical investigation indicated that the UCHM1 antigen is a protein of 52,000 MW while the SMO antigen could not be indentified. The antibodies TG1 and 28 identify antigens mainly present on granulocytes. While mAb 28 reacted with neutrophils, TG1 also stained eosinophils and stained strongly a proportion of monocytes. TG1 also reacted variably with some non-haemopoietic cell lines. Both antibodies reacted predominantly with granulocytes in tissue sections. MAb TG1 precipitated a single polypeptide of 156,000 MW from monocytes and granulocytes, while mAb 28 precipitated non-convalently associated polypeptides of 83,000 and 155,000 MW from granulocytes but only a single molecule from monocytes, corresponding to the lower MW chain of 83,000. The epitope with which mAb 28 reacts appears not to be exposed on the surface of intact monocytes. This suggests that a similar or identical 83,000 MW molecule is made by both neutrophils and monocytes, but that its expression differs according to cell type.     

Expression of a colorectal antigen defined by a new monoclonal antibody, CO-TL1

A murine monoclonal antibody (MoAb CO-TL1, IgG1) has been raised by differential screening of hybridoma supernatants on sections of human large and small intestines, followed by screening on colon adenomas as well as on colorectal carcinomas. In both paraffin sections and cryostat sections, the antibody stained strongly all cell types in adult, neonatal and fetal human colorectal epithelium, that is, the goblet cells, the columnar cells and the endocrine cells. No staining was observed in the remaining parts of the normal gastrointestinal tract and other tissues. As revealed by immuno electron microscopy the epitope was present in the apical and basolateral cell membranes, the Golgi complex, secretory vesicles of goblet and columnar cells, and also in granules of the endocrine cells. The epitope in colorectal tissue sections was resistant to the deglycosylation enzymes neuramidase, diastase and hyaluronidase indicating its proteinaceous nature. This colorectal antigen remained expressed in 100% of colorectal adenomas (n = 39) and 86% (n = 29) of colorectal carcinomas. The expression was reduced in undifferentiated carcinomas. The CO-TL1 antibody detected also most other gastrointestinal adenocarcinomas and a few carcinomas of the ovary, uterus, breast, gallbladder and pancreas. However, it never detected carcinomas derived from the thyroid, lung, liver, bladder, kidney, prostate, testis, serous membranes of body cavities and skin. A wild-type variant protein of > 300 kDa of the colorectal antigen was identified in normal colorectal epithelium. In colorectal tumours, however, two tumour variant forms were found of 160-200 and 115-140 kDa, respectively. Our data indicate that this new MoAb CO-TL1 can be considered as a useful marker, which identifies normal colorectal epithelium and gastrointestinal tumours and especially colorectal tumours with high accuracy and excludes tumours originated from thyroid, lung, liver, bladder, kidney, prostate, testis, mesothelium and skin.     

Immunohistochemical reactivity of a monoclonal antibody prepared against human breast carcinoma

Summary The reactivity profile of an IgM monoclonal antibody, MBR1, raised against the human breast cancer cell line MCF7, was studied in a variety of human tumours and non-neoplastic tissues by light microscopic immunohistochemistry. The range of reactivity included specific types of non-neoplastic epithelial cells and a number of epithelial tumours. Most mammary carcinomas reacted with MBR1, but adenocarcinomas and squamous carcinomas from different sites were also strongly positive. Different patterns of immunoreactivity were apparent in microscopically normal tissues, in tissues with inflammatory changes and in carcinomas. Heterogeneous staining, despite morphological similarities, was documented in neoplastic and non-neoplastic epithelial cells. The reactivity of MBR1 was different from that reported for other monoclonal antibodies, but revealed similarities to that of monoclonal antibodies and polyclonal sera against human milk fat globule membrane.     

Reactivity of human trophoblast monoclonal antibodies with marmoset monkey trophoblast cultures

The aim of this study was to determine whether cultured trophoblast tissues, derived from the trophectoderm of marmoset monkey blastocysts, contain homologues of human trophoblast antigens. This is an essential prerequisite to determine whether the marmoset may be a suitable model for preclinical testing of a human antitrophoblast antigen for fertility regulation. Previously evaluated monoclonal antibodies from the Flinders University laboratory, which reacted with human trophoblast with a high degree of specificity, were tested for immunohistochemical reactivity using an immunoperoxidase detection method on both frozen and paraformaldehyde-fixed sections of the cultured marmoset monkey trophoblast. All monoclonal antibodies raised against human placenta reacted positively, when compared to controls, suggesting that human and marmoset trophoblast cells share common epitopes. The specificity of the monoclonal antibodies was investigated by determining whether there was cross-reactivity with other marmoset monkey tissues, including adrenal, spleen, kidney, liver, muscle, ovary and testis. The specificities of the monoclonal antibodies on these marmoset tissues were similar to those previously found on the corresponding human tissues. We have concluded that marmoset monkey trophoblast exhibits homologues of human trophoblast antigens. The findings also suggest that marmoset monkeys should be evaluated further as a primate model to test suitable target antigens for antitrophoblast vaccines that may be useful contragestation agents in humans.      

Glycoprotein PAS-0 from the milk fat globule membrane carries antigenic determinants for epithelial membrane antigen

Epithelial membrane antigen (EMA) can be found in human tissues using antisera raised against defatted human cream. PAS-0 is a glycoprotein which has been extracted from human milk fat globule membranes. Using a polyclonal antiserum and a series of monoclonal antibodies, we have shown that the major antigenic determinant for EMA is carried by PAS-0. A more detailed comparison of the two glycoproteins has been made by establishing a set of radioimmunoassays using the different antibodies.     

Human mast cells detected by monoclonal antibodies.

We report the establishment of seven mouse-mouse hybridoma cell lines secreting monoclonal antibodies with specificity for granule components of all human mast cells. Reactivity is directed against a molecule which is also found intracytoplasmically in human mature small intestinal enterocytes, liver parenchymal cells, and kidney proximal tubule epithelial cells. No reactivity of these antibodies was found with any other human or animal cell type examined. In particular, the antibodies did not react with basophils or other haemopoietic cell types. This study shows the potential of specific monoclonal antibodies as a tool for identifying and enumerating infiltrating mast cells in tissues. Such antibodies should be of value in investigations into the role of the mast cell in immunological reactions and hypersensitivity diseases.     

Cellular Location of ''Liver-Specific'' Antigen F

Three mouse monoclonal antibodies against Type 1 murine 'liver-specific' antigen F and one rat monoclonal antibody against human antigen F were produced. These monoclonals recognize antigenic determinants common to Type 1 and Type 2 mouse and human antigen F. By immunolabelling with beta-galactosidase, antigen F could be demonstrated in the cytoplasm of hepatocytes, tubules of the kidney, and perikarya of neurons of the central nervous system. F antigens isolated from liver, kidney, and brain all have molecular weights of around 40,000.     

Characterisation of renal antigens on distinct parts of the human nephron by monoclonal antibodies

Summary Ten monoclonal antibodies (TN 1-TN 10) directed against different renal antigens of distinct sites of the human nephron were derived from a fusion between P3-NS1/1-Ag4-1 mouse myeloma and spleen cells of a mouse hyperimmunized against isolated human kidney cells. Two of these reagents (TN 1, TN 10) were shown by immunoperoxidase labelling on frozen sections of five normal kidneys and of other selected human organs, as well as by immunofluorescence studies on normal peripheral blood cells and selected lymphohematopoietic cell lines, to detect antigens exclusively expressed on visceral glomerular or proximal tubular epithelial cells. The other eight antibodies were found to react with different determinants shared between renal structures, muscle cells, different epithelia, B-lymphocytes and granulocytes. In heterogeneous cultures of isolated kidney cells these monoclonal reagents could be used to identify distinct cell types of tubular origin. Thus such hybridoma-derived antibodies provide new tools to correlate structural characteristics of various renal epithelial cells with their functional properties and will contribute to the study of their influence on immunologically mediated kidney injuries in different forms of glomerulonephritis in man.This study was supported by two grants from the Deutsche Forschungsgemeinschaft, DFG, Mu 523/3-1 and SFB 120, Leukämieforschung und Immungenetik, Project A2 and A3     

The differential diagnosis of hairy cell leukemia with a panel of monoclonal antibodies

A panel of monoclonal antibodies reactive with normal human lymphoid cells and with hairy cells has been applied to the immunocytochemical analysis of hairy cell leukemia. Staining was performed by immunoenzymatic methods on frozen sections of bone marrow trephines and extramedullary tissues and on cell smears. Hairy cells reacted with antibodies against HLA-DR, leukocyte common antigen, B-cell antigens (antibodies To15 and B1) and with three anti-hairy cell monoclonal antibodies (S-HCL3, HC1, and HC2). Neoplastic cells in other B-cell lymphoproliferative disorders also expressed HLA-DR, leukocyte common, and B-cell antigens but were consistently negative for the antigen detected by monoclonal antibody S-HCL3. Furthermore, hairy cells differed from other neoplastic B-cells in that they were unreactive with monoclonal antibodies against C3b receptors, anti-Leu-1, Tü1, Tü33, and lacked a meshwork of dendritic reticulum cells. These findings establish a distinctive antigenic phenotype for hairy cell leukemia and indicate that it may be diagnosed reliably by immunoenzymatic labeling of tissue sections or cell smears.     

Background staining of visualization systems in immunohistochemistry: comparison of the Avidin-Biotin Complex system and the EnVision+ system.

The aim of this study was to evaluate specific immunostaining and background staining in formalin-fixed, paraffin-embedded human tissues with the 2 most frequently used immunohistochemical detection systems, Avidin-Biotin-Peroxidase (ABC) and EnVision+. A series of fixed tissues, including breast, colon, kidney, larynx, liver, lung, ovary, pancreas, prostate, stomach, and tonsil, was used in the study. Three monoclonal antibodies, 1 against a nuclear antigen (Ki-67), 1 against a cytoplasmic antigen (cytokeratin), and 1 against a cytoplasmic and membrane-associated antigen and a polyclonal antibody against a nuclear and cytoplasmic antigen (S-100) were selected for these studies. When the ABC system was applied, immunostaining was performed with and without blocking of endogenous avidin-binding activity. The intensity of specific immunostaining and the percentage of stained cells were comparable for the 2 detection systems. The use of ABC caused widespread cytoplasmic and rare nuclear background staining in a variety of normal and tumor cells. A very strong background staining was observed in colon, gastric mucosa, liver, and kidney. Blocking avidin-binding capacity reduced background staining, but complete blocking was difficult to attain. With the EnVision+ system no background staining occurred. Given the efficiency of the detection, equal for both systems or higher with EnVision+, and the significant background problem with ABC, we advocate the routine use of the EnVision+ system.