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     

Immunohistochemical study of angiotensin-converting enzyme in human tissues using monoclonal antibodies

The localization of angiotensin-converting enzyme (ACE) in human tissues has been studied by the PAP-method with the use of monoclonal antibody 9 B9 against human lung ACE. The enzyme was detected on the surface of endothelial cells in lung, myocardium, liver, intestine and testis as well as in the epithelial cells of the kidney proximal tubules and intestine. The monoclonal antibody 9 B9 did not react with ACE in the epithelial cells of the testis seminiferous tubules. These data suggest that the antibody 9 B9 recognizes epitope which is shared by the ACE molecule of endothelial cells and renal and intestinal epithelial cells but is not present in testicular ACE, or is not accessible there to the antibody.     

Production and characterization of monoclonal antibodies to rat liver arginase

Rat liver arginase was purified and five monoclonal antibodies were produced by fusion of spleen cells from a Balb/c mouse and the myeloma cell line P3-X36-Ag-U1. One, R2D19, of five antibodies belonged to the IgG2a subclass, the other four, R1D81, R1G11, R2E10, and R2G51, were of the IgG1 type. The R1D81 cross-reacted with human liver arginase. This antibody inhibited the arginase activity, competing with arginine. These results suggest that R1D81 binds to the catalytic site of arginase. The R2D19 also inhibited the enzyme activity but acted as a noncompetitive inhibitor. With the use of R1D81 and a polyclonal anti-human liver arginase antibody conjugated with alkaline phosphatase, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of human arginase. Specificity of monoclonal antibodies for rat liver arginase was examined by means of the sandwich ELISA. Eight pairs of monoclonal antibodies could form a sandwich with the arginase. Only the R2E10 could be used for both the first and the second antibody in the sandwich system. In other cases, monoclonal antibodies could not be interchanged between solid and liquid phase.     

Proteomics-based generation and characterization of monoclonal antibodies against human liver mitochondrial proteins

Monoclonal antibodies (mAbs) have the potential to be a very powerful tool in proteomics research to determine protein expression, quantification, localization and modification, as well as protein-protein interactions, especially when combined with microarray technology. Thus, a large amount of well-characterized and highly qualified antibodies are needed in proteomics. Purified antigen, which is not always available, has proven to be one of the rate-limiting steps in mAb large-scale generation. Here we describe our strategies to establish a murine hybridoma cell bank for human liver mitochondria using unknown native proteins as the immunogens. The antibody-recognized mitochondrial proteins were identified by MS following immunoprecipitation (IP), and by screening of human liver cDNA expression library. We found that the established antibodies reacted specifically with a number of important enzymes in mitochondria. The subcellular localization of these antigens in mitochondria was further confirmed by immunohistocytochemistry. A panel of antibodies was also tested for their ability to capture and deplete the targeting proteins and complexes from the total mitochondrial proteins. We believe these well-characterized antibodies would be useful in various applications for Human Liver Proteome Project (HLPP) when the scale of this hybridoma cell bank is enlarged significantly in the near future.     

Immunofluorescent localisation of cytokeratin antigens in mitotic HeLa cells using monoclonal antibodies

The organisation of cytokeratin filaments in mitotic HeLa cells has been analysed by immunofluorescence microscopy using a monoclonal antibody which recognises proteins with apparent subunit molecular weights of 52 kDa and 57 kDa and which binds exclusively to cytokeratin-type filaments. Mitotic cells were prepared for microscopic analysis by hypotonic swelling, centrifugation onto glass slides, brief pre-extraction with 0.1% Triton X-100 and fixation in 80% ethanol. This procedure gave particularly good resolution of intermediate filaments and preservation of chromosome morphology. In prometaphase-metaphase cells the antigen was present in an anastomosing filament network which completely or partially enclosed the chromosomes, in filament fragments and in cytoplasmic aggregates. The epichromosomal filament network was absent from cells in anaphase or later stages of mitosis. In these cells non-filamentous antigen was often located in a narrow band defining the periphery of individual chromosomes and in variable numbers of cytoplasmic filaments or fragments. The results suggest that extensive disaggregation and reformation of cytokeratin filaments occurs during mitosis and that disaggregated cytokeratin proteins are frequently located adjacent to mitotic chromosomes.     

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.     

Inhibitory monoclonal antibodies against rat liver alcohol dehydrogenase

Eleven hybridoma clones which secrete monoclonal antibodies against purified rat liver alcohol dehydrogenase (EC 1.1.1.1) were isolated. Antibodies (R-1-R-11) were identified by their ability to bind to immobilized pure alcohol dehydrogenase in an enzyme-linked immunoadsorbent assay, in which antibody R-9 showed the highest binding capacity. Except for R-1 and R-7, all antibodies inhibited catalytic activity of the enzyme isolated from inbred (Fischer-344) or outbred (Sprague-Dawley) strains (R-11 greater than R-9 greater than R-4 greater than R-6 greater than R-10 greater than R-8 greater than R-2 = R-3 = R-5). The inhibition of enzyme activity by antibodies was noncompetitive for ethanol and NAD+, and was dependent on antibody concentration and incubation time. Antibodies R-4, R-9, and R-11 were most effective when enzyme activity was assayed below pH 7.7-7.8, a condition thought to protonate the enzyme's active center. These three antibodies did not inhibit horse liver alcohol dehydrogenase activity, indicating their species specificity. Such antibodies will be useful to delineate structural and functional roles of rat liver alcohol dehydrogenase.     

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 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.     

Immunohistochemical localisation of cyclooxygenase in the human uterus

The distribution in the human uterus of the enzyme cyclooxygenase, involved in prostaglandin synthesis, was studied using the peroxidase-antiperoxidase immunohistochemical technique. Specific staining was observed only during the luteal phase of the menstrual cycle. It was found that cyclooxygenase was localised in the surface and glandular epithelium of the endometrium but could not be demonstrated in the endometrial stroma, myometrium or blood vessels. The location of specific staining for cyclooxygenase and its variation during the menstrual cycle was the same regardless of the amount of menstrual blood loss. Tissue samples obtained from early pregnant and post-menopausal patients failed to exhibit any specific staining.     

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 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.     

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.     

Production and characterization of monoclonal antibodies against human ceruloplasmin

Ceruloplasmin (CP) is the major plasma antioxidant and copper transport protein. Monoclonal antibodies (mAbs) against human CP were produced and characterized. A total of five hybridoma cell lines were established (CP2, CP10, CP20, CP25, CP30). From the epitope mapping analysis, two subgroups of mAbs recognize different peptide fragments were identified. When the purified CP was incubated with the mAbs, the ferroxidase activity of CP was inhibited up to a maximum 57 %. Immunoblotting with various tissue homogenates indicated that all the mAbs specifically recognize a single protein band of 130 kDa. They also appear to be extensively cross-reactive among different mammalian including human and avian sources. These results demonstrated that only one type of immunologically similar CP is present in all of the mammalian tissues including human. The CP mAbs could be of great benefit to design the diagnostic kit for CP-related diseases such as Wilson's disease.     

Preparation and characterization of monoclonal antibodies against human myeloperoxidase

We established 11 hybridomas producing monoclonal antibodies (MoAbs), designated AM, against human myeloperoxidase (MPO), by immunizing mice with the three forms of MPO (I, II, and III) purified from healthy human polymorphonuclear leukocytes (PMN) and characterized the specificity of the AM MoAbs. Ten of the AM MoAbs reacted similarly to each of the three forms using an enzyme-linked immunosorbent assay. When a cetyltrimethylammonium bromide (CETAB) extract of human PMN was electrophoresed in a CETAB polyacrylamide gel and transferred to a nitrocellulose filter, IgG1 class AM MoAbs immunostained only the MPO band of the proteins of the extract. In addition, the AM MoAbs reacted to two radioactive bands of 94 and 92 kDa in a HL-60 cell lysate labeled with [35S]methionine for 1 h. After a chase period of 24 h, these bands were replaced by four radioactive bands of 64.5, 43, 16.7, and 13.4 kDa, demonstrating that the MoAbs recognize not only mature MPO but also the MPO precursors of 94 and 92 kDa. The data also indicated that the two major bands of 64.5 and 13.4 kDa corresponded to heavy and light chains of mature MPO, respectively, and the additive intermediate bands of 43 and 16.7 kDa were MPO-related proteins. Moreover, AM MoAbs reacted to a similar extent to the deglycosylated form of MPO III with endo-beta-N-acetylglucosaminidase H (Endo-H). Thus, IgG1 class AM MoAbs recognized MPO with high specificity and reacted to the structure which is commonly conserved in the three mature forms of MPO (I, II, and III), MPO precursors, and deglycosylated MPO with Endo-H. AM MoAbs also specifically reacted to PMN and/or monocytes but did not react to lymphocytes when the cell staining method was used.     

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.     

Characterization of monoclonal antibodies against human parvovirus B19

Eleven hybridoma cell lines producing mouse monoclonal antibodies (mAbs) against human parvovirus B19 were established. Their specificity was as follows. Approximately 5% of fetal erythroid cells inoculated with B19 reacted with all the mAbs and with anti-B19 positive human serum, but not with negative serum by indirect double immunofluorescence staining. All the mAbs recognized both VP-1 (84 kDa) and VP-2 (58 kDa) capsid proteins of B19 virions propagated in vitro and in vivo by Western blotting, and immunoprecipitated B19 virions.     

Characterization of monoclonal antibodies against human leukocyte 5-lipoxygenase

Four mouse monoclonal IgG1 antibody-producing cell lines (5LO-1, 5LO-2, 5LO-3, 5LO-4), produced against highly purified human leukocyte 5-lipoxygenase have been characterized. The monoclonal antibodies produced by these cell lines exhibited differential reactivity against 5-lipoxygenase as determined by ELISA and immunoprecipitation analyses. Monoclonal antibodies 5LO-2 and 5LO-3 inhibited the activity of recombinant human leukocyte 5-lipoxygenase in a dose-dependent manner. This inhibition was selective for 5-lipoxygenase activity since these monoclonal antibodies did not inhibit human leukocyte 15-lipoxygenase or porcine leukocyte 12-lipoxygenase.     

Immunohistochemical analysis of human lung cancers with anti-ras p21 monoclonal antibodies

The expression of ras oncogene product p21 in human malignant pleurisy and primary lung cancer was studied immunocyto-histochemically with monoclonal antibodies (MoAbs) rp-28 and rp-35 against ras p21. In pleural effusion cells, cancer cells revealed more intensively positive reaction with MoAb rp-35 than with MoAb rp-28, especially in the plasma membrane, and no positive reaction was obtained in any kind of inflammation cells with the exception of faintly positive reaction in the cytoplasm of macrophages. In primary lung cancers, well or moderately differentiated adenocarcinoma tissues showed higher reactivity with MoAb rp-28 than those of poorly differentiated adenocarcinoma or any other histological subtype of lung cancer. With MoAb rp-35, intensively positive reaction was obtained in most of cases with all different histological subtypes of lung cancer. The staining in cancer cells was usually localized intensively to the plasma membrane and weakly to the cytoplasm with both MoAbs. Normal bronchial epithelial and glandular tissues showed only cytoplasmic staining. These two MoAbs, especially MoAb rp-35, may be useful in clinicopathological applications for the diagnosis of malignant pleurisy and primary lung cancer.