Immunohistochemical analysis of human peripheral blood and lymphoid tissues using monoclonal antibodies immunoreactive with non-lymphoid cells

Immunoperoxidase methods were used to study human peripheral blood and lymphoid tissues using a panel of monoclonal antibodies to non-lymphoid cells. The majority of peripheral blood monocytes were immunoreactive for LeuM1, LeuM2, LeuM3 and LeuM5. Rare peripheral blood monocytes were immunoreactive for R4/23. The different antibodies showed characteristic patterns of immunoreactivity in peripheral lymphoid tissues. LeuM1 was immunoreactive with scattered cells in the paracortex of lymph node and tonsil and with many cells in the marginal zone of the spleen. LeuM2 was immunoreactive with endothelial cells in lymph node and tonsil. A few cells in the red pulp of the spleen were immunoreactive for LeuM2. LeuM3 and R4/23 showed distinctive immunoreactivity in germinal centers of secondary follicles, giving a "lacy" pattern. LeuM3 was also immunoreactive with endothelium in lymph node and tonsil and with sinus lining cells in lymph node. LeuM5 was immunoreactive with macrophages in the germinal center, fibroblastic reticulum cells in the mantle zone and interdigitating reticulum cells in the paracortex of lymph node and tonsil.     

Immunohistochemical analysis of human peripheral blood and lymphoid tissues using monoclonal antibodies immunoreactive with non-lymphoid cells

Summary Immunoperoxidase methods were used to study human peripheral blood and lymphoid tissues using a panel of monoclonal antibodies to non-lymphoid cells. The majority of peripheral blood monocytes were immunoreactive for LeuM1, LeuM2, LeuM3 and LeuM5. Rare peripheral blood monocytes were immunoreactive for R4/23. The different antibodies showed characteristic patterns of immunoreactivity in peripheral lymphoid tissues. LeuM1 was immunoreactive with scattered cells in the paracortex of lymph node and tonsil and with many cells in the marginal zone of the spleen. LeuM2 was immunoreactive with endothelial cells in lymph node and tonsil. A few cells in the red pulp of the spleen were immunoreactive for LeuM2. LeuM3 and R4/23 showed distinctive immunoreactivity in germinal centers of secondary follicles, giving a lacy pattern. LeuM3 was also immunoreactive with endothelium in lymph node and tonsil and with sinus lining cells in lymph node. LeuM5 was immunoreactive with macropages in the germinal center, fibroblastic reticulum cells in the mantle zone and interdigitating reticulum cells in the paracortex of lymph node and tonsil.     

Immunohistochemical localisation of arginase in human liver using monoclonal antibodies against human liver arginase

Monoclonal antibodies against human liver arginase were raised in order to determine the exact distribution of arginase in human liver using a modified indirect unlabelled immunoperoxidase method. In normal human liver specific immunohistochemical staining was found in the cytoplasm of hepatocytes. Portal components (bile ducts and veins) and fibrous tissue were non-reactive, while erythrocytes were slightly positive. The specificity of the immunological reaction was confirmed by control tests. Spectrophotometry was used to quantitate the immunohistochemical reaction product, and the results indicated that arginase is homogeneously distributed in the liver lobule.     

Immunohistochemical localisation of arginase in human liver using monoclonal antibodies against human liver arginase

Summary Monoclonal antibodies against human liver arginase were raised in order to determine the exact distribution of arginase in human liver using a modified indirect unlabelled immunoperoxidase method. In normal human liver specific immunohistochemical staining was found in the cytoplasm of hepatocytes. Portal components (bile ducts and veins) and fibrous tissue were non-reactive, while erythrocytes were slightly positive. The specificity of the immunological reaction was confirmed by control tests. Spectrophotometry was used to quantitate the immunohistochemical reaction product, and the results indicated that arginase is homogeneously distributed in the liver lobule.Present address: Biologisches Institut der Universität Stuttgart, Ulmerstrasse 227, D-7000 Stuttgart 60, Federal Republic of Germany     

Solid-phase enzyme immunoassay of urokinase using monoclonal antibodies

Using two monoclonal antibodies directed against urokinase, we have developed a micro enzyme-linked immunosorbant assay (ELISA) to detect and measure urokinase in biological fluids. The system presents the following characteristics: simple and rapid procedure, reproducibility, sensitivity (urokinase levels down to 1 ng/ml) and evaluation of the enzyme in biological fluids such as urine, pleural effusions, and ascitic fluids without preliminary purification.     

Immunohistochemical heterogeneity of alpha-tubulin in human epithelia revealed with monoclonal antibodies

Summary Four monoclonal antibodies raised to alpha subunit of pig brain tubulin (TU-01, TU-02, TU-03, TU-04) were used to study immunohistochemical heterogeneity of alpha tubulin in human epithelia. Selective reactivity was detected in the skin and trachea/bronchi, whereas all other epithelia investigated reacted uniformly with all four monoclonal antibodies. In the skin TU-01 reacted very strongly with all layers except the basal layer; TU-02 reacted strongly with granular layer and was unreactive or only weakly reactive with others; TU-03 reacted very strongly with basal layer and weakly to moderately with superficial layers; TU-04 reacted strongly with the granular layer of epidermis and was unreactive with other layers. In the trachea and major bronchi TU-01 reacted with the entire epithelial layer; TU-02 reacted only with superficial layer; TU-03 reacted with superficial and basal layer; TU-04 reacted only with superficial layer. Different staining patterns obtained with these four monoclonal antibodies indicate that there is immunohistochemical heterogeneity of alpha tubulin in some but not all normal human epithelia.     

Monoclonal antibodies to the angiotensin-converting enzyme from the human lung

The hybridoma producing monoclonal antibody (IgG1) to human angiotensin-converting enzyme (ACE) has been prepared by fusion of murine myeloma P3O1 with spleen cells of BALB/c mice immunised with a purified human lung ACE preparation. A high specificity of monoclonal antibody (MAb) binding to immobilized ACE has been demonstrated by ELISA; that of soluble ACE--by immunoadsorption test. The latter technique permits the use of impure ACE preparations for the screening procedure. This MAb did not effect ACE activity. This antibody is believed useful not only for immunoassay and immunopurification of ACE, but also as a tool for investigation of enzyme distribution in tissue as well as for studying the structure and mechanism of ACE action.     

Immunohistochemical heterogeneity of alpha-tubulin in human epithelia revealed with monoclonal antibodies

Four monoclonal antibodies raised to alpha subunit of pig brain tubulin (TU-01, TU-02, TU-03, TU-04) were used to study immunohistochemical heterogeneity of alpha tubulin in human epithelia. Selective reactivity was detected in the skin and trachea/bronchi, whereas all other epithelia investigated reacted uniformly with all four monoclonal antibodies. In the skin TU-01 reacted very strongly with all layers except the basal layer; TU-02 reacted strongly with granular layer and was unreactive or only weakly reactive with others; TU-03 reacted very strongly with basal layer and weakly to moderately with superficial layers; TU-04 reacted strongly with the granular layer of epidermis and was unreactive with other layers. In the trachea and major bronchi TU-01 reacted with the entire epithelial layer; TU-02 reacted only with superficial layer; TU-03 reacted with superficial and basal layer; TU-04 reacted only with superficial layer. Different staining patterns obtained with these four monoclonal antibodies indicate that there is immunohistochemical heterogeneity of alpha tubulin in some but not all normal human epithelia.     

Immunohistochemical demonstration of estrophilin in mouse tissues using a biotinylated monoclonal antibody

We have developed a direct avidin-biotin-peroxidase complex (ABC) immunohistochemical method for localization of estrophilin in mouse tissues. The method has been found especially useful for microscopic demonstration of the receptor in mouse liver, since the indirect alternative, autoradiography after injection of radiolabeled estrogens, is of no value in this organ. The ABC technique employs a biotinylated monoclonal antibody to human estrophilin (Abbot H222) which was previously shown to crossreact with the murine receptor. Cryostat-cut tissue sections which were briefly fixed were incubated with the modified antibody, and the estrophilin was revealed by subsequent exposure to ABC followed by H2O2/diaminobenzidine.     

Immunohistochemical detection of human gastrointestinal glutathione peroxidase in normal tissues and cultured cells with novel mouse monoclonal antibodies.

This is the first report to describe the successful detection of human gastrointestinal glutathione peroxidase in normal tissues by Western blotting and immunohistochemical staining techniques. Four hybridoma clones producing monoclonal antibodies (MAbs) against the human gastrointestinal glutathione peroxidase were established from mice immunized with a gastrointestinal glutathione peroxidase-derived peptide. The MAbs did not crossreact with other members of the glutathione peroxidase family, be it cellular glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, or extracellular glutathione peroxidase. Although the MAbs were found to react with a 24-kD protein in a Western blotting assay using gastric carcinoma cell extracts as antigen, they did not react with a B-lymphoblastoid cell extract. Immunohistochemical staining showed gastrointestinal glutathione peroxidase localized in the cytoplasm and in the nucleus of gastric carcinoma cells. Moreover, gastrointestinal glutathione peroxidase was detected in tissue extracts of human stomach, small intestine, large intestine, liver, and gallbladder by Western blotting, and its localization was immunohistochemically confirmed in the mucosal epithelia of the basal area of gastric pits and intestinal crypts.     

Immunohistochemical characteristics of chicken spleen ellipsoids using newly established monoclonal antibodies

Ellipsoids, the extra-vasculature sites surrounding penicilliary capillaries of the chicken spleen, play critical roles in the immune response and also in the clearance of pathogens or other particles. The meshwork of ellipsoids is formed by fibroblastic reticular cells. To characterize ellipsoidal reticular cells, a series of monoclonal antibodies against the chicken spleen have been developed. Of these antibodies, CSA-1 antibody reacts with fibroblastic reticular cells in ellipsoids and with endothelial cells. The reticular nature of positive cells in ellipsoids is indicated by immuno-electron microscopy, and by double-staining with anti-heat-shock protein 47 kDa (hsp47) antibody. The reaction of CSA-1 with reticular cells is limited in ellipsoids; CSA-1 does not react with reticular cells in other lymphoid organs. These findings indicate that ellipsoidal reticular cells share the antigen with endothelial cells. Ontogenic studies reveal that, on embryonic day 18, the development of ellipsoids is completed, penicilliary capillaries become fenestrated, and CSA-1 expression in ellipsoids begins. These findings suggest that CSA-1 is expressed on the cell surface of ellipsoidal reticular cells once they are exposed to blood flow.     

Localization of alpha 1-microglobulin (HC protein) in normal human tissues: an immunohistochemical study using monoclonal antibodies

Summary Alpha-1-microglobulin is a low molecular weight (approximately 30 000 d) glycoprotein present in biological fluids. It is heterogeneous in charge. A monoclonal antibody was used to investigate the tissue distribution of the protein in normal human tissues and cell lines by indirect immunofluorescence and immunoperoxidase techniques. The protein was demonstrable in cells of the monocyte-macrophage lineage, in thymus and T cell dependent areas of spleen, lymph node and tonsils. It was detected in several lymphoid or nonlymphoid cell lines but not in peripheral blood lymphocytes. The microglobulin was also detectable in the cytoplasm of hepatocytes. Finally, it was observed in glandular secretions (sudoral glands and mucosal glands of the digestive tract) where it may be associated with IgA. Possible explanations for the highly divergent results previously reported with polyclonal antisera to ₁ microglobulin are discussed.     

Immunohistochemical characterization of B cells and T cells in musk shrew (Suncus murinus) lymphoid tissues using monoclonal antibodies

We have developed and characterized three monoclonal antibodies (mAbs), which recognize the surface antigens of musk shrew B cells and T cells. About 30% of all lymph node cells reacted with the mAb ST1. Staining of frozen sections showed that ST1-positive cells were located in the primary lymphoid follicles of lymph nodes, and were absent in the thymus. mAbs ST4 and ST2 were also surface-reactive. The ST1-negative (about 60% of the total) lymph node cells reacted positively with ST4, and about 30% of these ST4-positive cells were recognized by ST2. The distribution of ST4-positive cells was shown by staining of lymph node sections to be identical to that of T cells reported in other species. Western blot analysis showed that the apparent molecular weights of the antigens recognized by ST1 and ST4 were 70000 and 74000, and 60000 and 64000, respectively. These findings suggest that the antigens detected by ST1, ST4, and ST2 are the musk shrew homologues of pan-B cells, pan-T cells, and T cell subsets, respectively. The three mABs may facilitate immunohistochemical analysis of the cellular immune response in this species.     

Immunohistochemical investigations of the human palatine tonsil from surgical specimens using polyclonal and monoclonal antikeratin antibodies

The keratinization process of tonsillary epithelium in patients with different age, who underwent surgery because of chronic inflammatory processes, has been studied by employing low (40 KD), medium (52-56-58 KD), high (56-64 and 68 KD) as well as broad spectrum antikeratin antibodies. The findings thus obtained outline the various keratin patterns of the tonsil in the outer covering and, even more, in the crypt labyrinth wall. These findings have been compared with those obtained by histochemical reactions for acid phosphatase and non-specific esterase activities (Favrz et al. 1986 II). In crypt epithelium keratinization, 56-64 KD keratins are involved in cells where also high enzymic activities are taking place, namely 1. in those elements delimiting the lumen as well as in those belonging to the reticulum mesh which are nearest to it and host lymphoid elements active in immunity reactions; 2. in elements at the bottom of small and large crypts, where excavation continues into the lymphoid tissue and interfollicular spaces. 68 KD keratin, although present in lower amounts, is synthesized almost everywhere, but only irregularly. It marks surface epithelium surrounding some dermal papillae, some meshes of the crypt reticulum, the "gallery" bottom as well as elements of interfollicular proliferation developing in the lymphoid tissue like arborescence. The path of congested blood vessels in the crypt wall towards the surface is marked by keratinized epitheliocytes. 56-64 KD keratin is present in the most superficial elements and is generally accompanied by the wall growing thinner, its blistering and breaking. The most frequent pathologic alterations of the epithelium (like cellular hypertrophy) involve variations in cytoplasm keratin pattern, in still un-flattened polyhedral elements of the intermediate layers.     

Antigenic study of the differentiation of the human kidney using monoclonal antibodies

The production of monoclonal antibodies against human embryonic renal cells allowed to display on the adult human kidney some antigens typical of certain structures or tissues: the proximal convoluted tubule for EG 9-11 and EG 19-6 monoclonal antibodies, the glomerular basement membrane for EG 14-1, the urothelium for EE 24-6, the connective tissue for EK 8-1 and EK 17-1 and probably the capsular and tubular basement membranes for EK 8-1. Simultaneously, we could follow the spatial and temporal repartition of the antigens during the renal development. One of them (EI 16-1) seemed to disappear in the adult and might correspond to a foetal type-antigen.     

Immunohistochemical phenotyping of human solid tumors with monoclonal antibodies in devising biotherapeutic strategies

Summary A panel of 14 monoclonal antibodies (MoAbs) (4 raised against breast cancer, 6 against colon cancer and 4 against melanoma) were used to phenotype frozen sections of tumor biopsies obtained from 110 patients, by avidin-biotin-peroxidase complex techniques. We observed heterogeneity of antigen expression among the multiple metastatic lesions of single patients, as well as among tumor lesions from different patients with similar tumor histotypes. A wide range of cross-reactivity of anti-(breast-carcinoma) and anti-(colon-carcinoma) MoAbs with other carcinoma histotypes and limited reactivity with melanoma and sarcoma was detected. Some of our anti-melanoma MoAbs were also found to cross-react with selected carcinomas. Nine of the 14 MoAbs most reactive with carcinomas of diverse histotypes have been identified. A mixture or cocktail of different MoAbs could be selected for each individual patient in order to achieve binding of MoAbs with most, if not 100% of tumor cells. This study illustrates the approach that we have taken to individualize the cocktail of MoAbs for the development of patient-specific therapeutic immunoconjugates.     

Immunohistochemical localization of aldehyde and xanthine oxidase in rat tissues using polyclonal antibodies

Tissues from male Wistar rats, fixed with 4% paraformaldehyde and embedded in paraffin, were studied with immunoperoxidase techniques using polyclonal antibodies raised against aldehyde oxidase or xanthine oxidase purified from rat liver. Immunohistochemical studies demonstrated that aldehyde oxidase-bearing cells were strongly stained in renal tubules, esophageal, gastric, intestinal and bronchial epithelium as well as liver cytoplasm. Weak but positive immunoreactivity was observed on the pulmonary alveolar epithelial cells, gastric glands and intestinal goblet cells. In contrast, it was demonstrated that cells with xanthine oxidase were strongly stained in renal tubules, esophageal, gastric, and small and large intestinal and bronchial epithelia etc. Positive immunostaining was also found in adrenal gland, skeletal muscle, spleen and cerebral hippocampus. Immunoreactivity againt aldehyde oxidase was not found in adrenal gland, spleen, mesentery or aorta, while immunoreactivity against xanthine oxidase was not found in mesentery or aorta. Although the significance of this ubiquitous and similar localization of aldehyde and xanthine oxidase seems unclear at present, these results may provide a clue as to the full understanding of the pathophysiological role of these oxidases in tissues.     

Detection of P-glycoprotein in human leukemias using monoclonal antibodies

Overexpression of a Mr 170,000 membrane glycoprotein (P-glycoprotein) is consistently associated with multidrug resistance in cell lines. Two monoclonal antibodies (Mab) against P-glycoprotein (265/F4 and C 219) were used to examine tumour samples from patients with leukemias for evidence of P-glycoprotein overexpression. High levels of P-glycoprotein (greater than 5% positive cells) were detected with both antibodies in samples from 3 out of 18 patients suggesting that a multidrug resistant phenotype may also occur in human leukemias.