Differential cross-reactivity of monoclonal antibody OPD4 (anti-CD45RO) in macaques

Immunologic research in nonhuman primates is occasionally limited by the availability of reagents that cross-react in nonhuman primates. One major limitation has been the lack of a monoclonal antibody to CD45RO. Although the monoclonal antibody UCHL-1 is used to detect CD45RO isoforms in humans, it does not react with nonhuman primates, mandating the use of alternative strategies to define "memory" T cell responses in nonhuman primates. The current study examined the reactivity and specificity of another antibody against CD45RO, clone OPD4, in macaques. Here we demonstrate that OPD4 specifically labels memory CD4+ T cells in approximately 44% of rhesus macaques (Macaca mulatta) of Indian but not Chinese origin. In contrast, tissues from pigtail macaques (Macaca nemestrina) react with this clone, indicating that OPD4 may be useful for examining memory CD4+ T cells in certain macaques, but its utility may be limited in other species or even among individual macaques.     

Immunohistochemical characterization of Ki-M6 monoclonal antibody in Bouin-fixed,paraffin-embedded sections of normal and neoplastic human tissues

Summary A monoclonal antibody (Ki-M6) against the CD 68 antigen, which labels cells of the monocyte/macrophage system, was tested on Bouin-fixed, paraffin-embedded samples of normal, reactive and neoplastic tissues by an avidin-biotin-peroxidase complex method, with the aim of establishing its value in diagnostic pathology. In normal human tissues, Ki-M6 reactivity was confined to the so-called resident macrophages populating normal organs under physiological conditions. Moreover, restricted reactivity against cells of macrophage lineage was observed in reactive and inflammatory lesions. Granulocytes, monocyte/macrophage-related immune accessory cells, and other analysed normal tissue structures did not reveal any reactivity. Ki-M6 was strongly reactive with the cases of benign (4/4) and malignant (15/15) fibrous histiocytomas, in addition to the true histiocytic lymphomas (3/3). Cases of granular cell tumour (2/3) showed strong reactivity with Ki-M6, whereas only few immunoreactive cells, with weak staining, were seen in the other Ki-M6-positive neoplasms [neurofibroma (3/3), benign schwannoma (1/2), ganglioneuroma (1/1), malignant schwannoma (5/9), melanoma (9/28), dermatofibrosarcoma protuberans (1/1), myelomonocytic leukaemia (3/3)]. Among the epithelial malignancies tested (47 cases), Ki-M6 was positive only in renal cell carcinoma (11/14). Malignant lymphomas of the Hodgkin (56 cases) and non-Hodgkin type (67 cases) were uniformly non-reactive. From these data, Ki-M6 appears to be an excellent marker of monocyte/macrophage-related cells and appears to be a reliable indicator for fibrous histiocytomas and true histiocytic malignancies. The availability of this additional antibody capable of staining routinely processed tissue is of practical interest.     

Immunohistochemical characterization of osteoclasts and osteoclast-like cells with monoclonal antibody MB1 on paraffin-embedded tissues

In this study we provide evidence that MB1, a newly developed monoclonal antibody which reacts with B lymphocytes and a proportion of T cells and monocytes, can be successfully used for the direct immunohistochemical identification of osteoclasts on paraffin-embedded surgical specimens. The antigen(s) recognized by MB1 is present at high density in the cytoplasm of osteoclasts of fetal bone and in the multinucleated cells of human giant cell tumour of bone (osteoclastoma), but is weakly expressed or absent in the giant cells of granulomas. MB1 is thus proposed as a new immunohistochemical marker for osteoclasts on paraffin-embedded material.     

False-positive immunostaining of normal epithelia and carcinomas with ascites fluid preparations of antimelanoma monoclonal antibody HMB45

HMB45 is a melanoma-specific monoclonal antibody that has found widespread use in diagnostic pathology. Recent reports, however, have suggested that this antibody may cross-react with a small number of carcinomas and other epithelial cells. The authors tested the hypothesis that these latter reports represent examples of false-positive immunostaining by comparing the immunostaining on breast, salivary gland, and lung tumors with the following: (1) a commercial ascites preparation of this monoclonal antibody; (2) a protein A-purified antibody preparation derived from ascites fluid; and (3) supernatant fluid obtained from the hybridoma cell line. The authors found that all examples of nonmelanoma immunostaining in the carcinomas tested were eliminated with the nonascites fluid preparations, whereas strong immunostaining of melanomas was retained. The authors conclude that contaminated commercial ascites fluid preparations of HMB45 may account for most, if not all, of the reports of nonmelanoma immunostaining with HMB45.     

A monoclonal antibody to human leukocyte common antigen, SHL-1, and its use for formalin-fixed, paraffin-embedded tissues

The authors describe a newly characterized murine monoclonal antibody to the human leukocyte surface antigen, SHL-1. The antigen belongs to the leukocyte common antigen (LCA) family, and its molecular weight is about 180,000 daltons, which is similar to that of some previously characterized LCAs. The SHL-1 antigen is resistant to conventional tissue-fixation and embedding procedures. This antibody can therefore be used in the immunohistochemical staining of paraffin-embedded tissue sections. Wide screening with a sufficient number of both fresh and routinely processed paraffin-embedded tissues was done with indirect immunoperoxidase technique. With this procedure, SHL-1 labeled the majority of normal leukocytes and hematopoietic malignancies. Some B-cell malignancies were not stained with this antibody. The non-hematologic malignancies posing diagnostic problems of differentiation from lymphomas or leukemias were completely negative to SHL-1. The immunoreactivity to SHL-1 of samples from 24 leukemic patients and 15 human tumor cell lines was determined by the immunofluorescence method. Of 24 leukemic preparations, 23 were strongly reactive to this antibody. One case of B-cell leukemia did not react with SHL-1. No immunoreactivity was demonstrated in non-hematopoietic tumor cell lines. The overall reaction pattern of SHL-1 proved its usefulness in both diagnostic and research practice in hematological disorders. This antibody detected cell surface antigens of the T cell series more effectively than those of the B-cell series in terms of the positive number of cells and mean fluorescence intensity.     

Leu-M1--a marker for Reed-Sternberg cells in Hodgkin''s disease. An immunoperoxidase study of paraffin-embedded tissues.

Monoclonal antibody to Leu-M1, a granulocyte-related differentiation antigen, represents a highly effective reagent for detection of diagnostic Reed-Sternberg (R-S) cells and variants in paraffin-embedded tissues of Hodgkin's disease. In 69 of 73 cases of Hodgkin's disease (41 nodular sclerosis, 25 mixed cellularity, 4 lymphocyte predominance, and 3 lymphocyte depletion types), R-S cells were strongly immunoreactive for Leu-M1. Four cases of lymphocyte predominance Hodgkin's disease (nodular) were uniformly nonreactive for Leu-M1. In most of the positive cases (57/69, 83%), the majority (60-90%) of R-S cells and variants exhibited immunoreactivity for Leu-M1. A characteristic staining pattern included granular and/or vesicular cytoplasmic immunoreactivity, often with a prominent globular paranuclear reaction product, and membrane staining with highly irregular cytoplasmic borders. Evaluation of B-cell (37 specimens), T-cell (20 specimens), and true histiocytic (3 specimens) neoplasms and a case of mastocytosis revealed immunoreactivity for Leu-M1 only in 1 B-cell and 4 T-cell malignancies. The staining patterns in these cases, however, clearly differed from that observed for R-S cells. Studies of nonneoplastic lymphoid tissues (38 total) demonstrated that lymphoid cells were typically nonreactive; histiocytes revealed variable reactivity for Leu-M1. Occasional histiocytes of the sinusoidal network of lymph nodes, particularly in toxoplasmic lymphadenitis, exhibited a staining pattern (membranous/cytoplasmic/paranuclear) similar to that observed for R-S cells. Leu-M1 represents a potentially helpful diagnostic discriminant in the assessment of Hodgkin's disease and its distinction from non-Hodgkin's lymphomas and other lymphoid proliferations.     

A novel monoclonal antibody (OPD4) recognizing a helper/inducer T cell subset. Its application to paraffin-embedded tissues.

A novel monoclonal antibody (MAb), OPD4, reactive with a helper/inducer (H/I) subset of T cells in formalin-fixed, paraffin-embedded tissue sections, has been identified through immunization with an activated H/I T cell line, namely DL40. The antibody is an IgG1 antibody and it recognizes an antigen with a molecular weight of 200 kd, corresponding to that of leukocyte common antigen. OPD4+/CD4+ T cells provided better help for pokeweed mitogen-stimulated polyclonal IgG production than OPD4-/CD4+ T cells. OPD4 recognized the H/I T cell subset even in paraffin-embedded tissue sections, but did not recognize nonhematopoietic cells, suppressor/cytotoxic T cells, B cells, monocytes in the peripheral blood, or other normal hematopoietic cells as examined by the flow cytometric and immunoperoxidase methods. Besides the lymphoid cells, OPD4 reacted with a number of histiocytes (epithelioid cells) in tissues from sarcoidosis and tuberculosis. For the neoplastic lesions, OPD4 reacted with approximately half of the cases of T cell lymphomas. Consequently, OPD4 may be useful for the diagnosis and study of malignant lymphomas and other related lesions.     

TDM35--a new monoclonal antibody to the XH1 cervical carcinoma cell line. Characterization and immunoperoxidase localization in benign and malignant tissues.

The murine monoclonal IgG1 kappa antibody TDM35 was raised against the cervical carcinoma cell line XH1. The antibody recognizes 18.5-66 kDa NCA-like glycoproteins and immunostains a variety of formalin-fixed, paraffin-embedded normal, benign, and malignant tissues. It is of value in the diagnosis of carcinoma of the exocrine pancreas and it identifies foci of squamous and glandular differentiation in other tumours. TDM35 should form a useful addition to a panel of antibodies for the evaluation of epithelial lesions.     

A new murine monoclonal antibody for the diagnosis of erythroleukemia.

The authors have developed a murine monoclonal antibody, RC-82.4, against an antigen expressed by a human erythroleukemia cell line OCI-MIR. The antibody reacts with an antigen expressed by proerythroblasts, normoblasts, and some reticulocytes but not expressed in erythrocytes, granulocytes, monocytes, megakaryocytes, plasma cells, or lymphocytes. The authors have established an immunocytochemical method for studying bone marrow smears with RC-82.4. By studying bone marrow smears from 11 patients with M-6 erythroleukemia and 104 patients with various other hematologic and nonhematologic malignancies, the authors have found that RC-82.4 has great sensitivity and specificity in recognizing erythroid differentiation in blasts. The authors have used RC-82.4 and antihemoglobin antibodies to identify erythroblasts in acute and secondary acquired cases of erythroleukemia that would have been unclassifiable by morphologic and all other conventional cytochemical and immunocytochemical criteria.     

A role for anti-CD45RB monoclonal antibody treatment upon dendritic cells

Selective interference with CD45RB isoform by monoclonal antibody (anti-CD45RBmAb) reliably induces donor-specific tolerance. Dendritic cells (DCs) are the most potent antigen-presenting cells that are capable of activating na?ve T cells. The purposes of the present study were to investigate the roles of anti-CD45RBmAb on the phenotypes and functioning of DCs and to further illustrate the mechanism of anti-CD45RBmAb-inducing immunologic tolerance. DCs from C57BL/6 mice were cultured and treated with various doses of anti-CD45RB monoclonal antibody. Cell phenotype, cycle and phagocytic ability were detected by flow cytometry. The production of IL-10 and IL-12 in the supernatants of mature DCs was measured with ELISA. Exosomes (Dex) were recovered from the supernatant of DCs cultured for 6 days in depleted medium, and effects of DCs and Dex on the ability of T-cell proliferation were detected by mixed lymphocyte culture. Anti-CD45RBmAb could inhibit DCs maturation in a dose-dependent manner, and the effects of exosomes (Dex) on DCs enhance or inhibition proliferation of T cells were also in a dose-dependent manner. Anti-CD45RBmAb could profoundly inhibit the maturation and functioning of DCs and generate tolerogenic dendritic cells (tDCs) as well as Dex, suggesting mechanistic contributions to tolerance development from the DCs through interactions with T cells.     

A new murine monoclonal antibody against human hepatoma.

A murine monoclonal antibody (MAb 336) reactive with human hepatocellular carcinoma has been raised after immunizing BALB/c mice with whole HepG2 cells. MAb 336 (IgG1) was reactive with HepG2 (whole cells and membrane fractions), but not normal liver or peripheral blood cells. Immunohistological studies indicated that 12/16 hepatocellular carcinoma and 6/11 cirrhotic livers expressed MAb 336-associated antigen, and most normal human tissues and tissues derived from other cancers were unstained. Direct and competitive binding assays ruled out the possibility that this MAb reacts with alpha-fetoprotein, carcinoembryonic antigen, or ferritin. Western blot analysis indicated that MAb 336 reacts with an antigen of approximately 30,000 daltons. This MAb may be potentially useful for studying antigenic expression in hepatocellular carcinoma and as a targeting agent for radioimmunodetection and immunoconjugate therapy.     

Development of new rabbit monoclonal antibody to estrogen receptor: immunohistochemical assessment on formalin-fixed, paraffin-embedded tissue sections.

Evaluation of estrogen and progesterone receptors in breast cancer is widely used for the prediction of the response to endocrine therapy and as a biologic parameter closely related to disease prognosis. Immunohistochemistry is considered a specific, sensitive, and economic method for the determination of estrogen receptor/progesterone receptor status. The authors developed the first rabbit antiestrogen receptor monoclonal antibody (clone SP1) used in immunohistochemistry on formalin-fixed, paraffin-embedded tissue sections especially from breast carcinomas. This new antibody, compared with currently available antiestrogen receptor antibodies, has important advantages, including its reactivity even without heat-based antigen retrieval of fixed, embedded tissue sections in immunohistochemistry, and the predominance of nuclear immunostaining with only a very low cytoplasmic signal. A comparative study of immunohistochemistry on 61 histologic specimens from breast cancer cases showed that SP1 yields the same results as the well-known, standardized mouse monoclonal antibody to estrogen receptor (clone 1D5). Antibody affinity of SP1 is 8 times higher than that of 1D5. Thus, SP1 may prove of great value in the assessment of estrogen receptor status in human breast cancer.     

A method for the use of immunofluorescence on paraffin-embedded tissues.

Immunofluorescence is increasingly used for the visualization of antigens and antibodies in various tissues. The commonly used frozen sections present several disadvantages, which can be avoided by the present adaptation of paraffin-embedded sections for immunofluorescence. In this method, Bouin's solution achieves fast, deep fixation which preserves well the capacity of antigens to react with specific FITC-labeled antibodies. The method gives superior resolution of morphology, lower nonspecific fluorescent background, and permanent sections suitable for indefinite storage and retrospective studies. It has been successfully applied thus far to the study of immunofluorescence of lymphoid, thyroid, renal, pulmonary, and intestinal tissues.     

New immunohistochemical "sandwich" staining method for mdr1 P-glycoprotein detection with JSB-1 monoclonal antibody in formalin-fixed,paraffin-embedded human tissues.

We have developed a new immunoperoxidase "sandwich" staining method for amplified detection of P-glycoprotein (Pgp) that is suitable for use on formalin-fixed, paraffin-embedded (conventional) tissue sections. This was accomplished by substantially changing the procedure described by Chan (1988) so as to increase specific staining intensity and to decrease nonspecific background staining. To determine the most appropriate primary antibody for the assay, we compared the immunoreactivity of JSB-1, C494, and C219 monoclonal antibodies recognizing internal epitopes of Pgp, and MRK16 and 4E3 monoclonal antibodies recognizing external epitopes of Pgp. Paraffin sections of Pgp-positive normal human tissues (adrenal, liver, kidney, and brain), of renal tumors, and of cell pellets of sensitive and multidrug resistant human tumor cell lines (MCF-7, KB) were used for comparisons. Immunostaining was excellent with JSB-1, moderate with C494, and very weak with C219. MRK16 and 4E3 showed no reaction. Nonspecific background staining was reduced by 1) omitting immunoglobulin G from secondary antibodies; 2) decreasing the concentration of peroxidase-antiperoxidase complex; and 3) utilizing casein solution for blocking and washing. Pretreatment of sections before immunostaining was also simplified. Using JSB-1, the threshold for detection of elevated Pgp corresponded to less than two-fold relative resistance to doxorubicin. Applying this method, we found two of 26 non-small cell lung cancers were positive for Pgp, consistent with previous results of others using frozen sections. This new immunoperoxidase sandwich staining method using JSB-1 now allows reliable Pgp detection in sections of formalin-fixed, paraffin-embedded (archived) surgical specimens and small biopsy materials commonly used for diagnostic purposes.     

Immunocytochemical characterization of a monoclonal antibody directed against mitochondria reactive in paraffin-embedded sections.

The monoclonal antibody mES 13 was previously produced against bacterially expressed BALB ras p21 and was reported to have both membrane and cytoplasmic reactivity in formalin-fixed, paraffin-embedded tissue sections. In the current study, the cytoplasmic reactivity of mES 13 is investigated and demonstrated to be mitochondrial. Immunoelectron microscopic studies showed specific labeling of mitochondria without labeling of other organelles. In normal tissues, the antibody strongly labeled tissues known to have large amounts of mitochondria such as renal tubules, hepatocytes, and myocardium. The pattern of reactivity of tumors generally mimicked that of normal tissues, with carcinomas and melanomas usually showing stronger staining than sarcomas and lymphomas. Two granular cell tumors were negative. Among renal neoplasms, mES 13 strongly labeled renal oncocytomas and granular cell renal cell carcinomas and showed weaker staining of clear cell and chromophobe cell tumors. The mES 13 antibody should be useful in the characterization and diagnosis of tumors in which oncocytoma is in the differential diagnosis, especially when only paraffin-embedded tissue is available for study.     

Detection of a monocyte/macrophage differentiation antigen in routinely processed paraffin-embedded tissues by monoclonal antibody Ki-M1P

A new monoclonal antibody Ki-M1P that is raised against supernatants of detergent solubilized human lymph node tissue is described. Ki-M1P recognizes in particular monocytes and their macrophage derivatives as tested by light- and electron-microscopic immunohistochemistry. Granulocytes, dendritic cells as the accessory cells of humoral and cellular immune response, and epithelial, endothelial, neural, and mesenchymal cells do not react with Ki-M1P. In extensive application Ki-M1P has proven to be a useful marker for distinguishing monocytic leukemias within FAB groups M4 and M5. The recognized antigen is composed of five proteins with molecular masses of about 60, 92, 98, 124, and 150 kDa in blood monocytes, whereas tissue macrophages tested so far expressed only the 60-kDa protein. Because the Ki-M1P antigen is not destroyed or masked during routine fixation and paraffin embedding of biopsy tissue samples, Ki-M1P represents a useful diagnostic reagent for the identification of physiological functional and pathologic reaction forms as well as neoplastic variants of the human monocyte/macrophage system even in retrospective studies.     

A monoclonal antibody (OPT1) to T cells which is available for paraffin-embedded materials

A monoclonal antibody (MAb), OPT1, reactive with T cells in formalin-fixed, paraffin-embedded tissue sections, has been identified through immunization with activated T cells from peripheral blood lymphocytes (PBL). The antibody is an IgG1 antibody as demonstrated by the Ouchterlony technique. By cytofluorometric analysis, almost all CD3+ lymphocytes and only a few CD20+ lymphocytes of peripheral blood expressed the OPT1 antigen. Nonhematolymphoid cell lines were negative for OPT1 by the immunoperoxidase staining using acetone-fixed cell lines. On the contrary, peripheral T cells, cells of two T cell lines out of four and a part of the cells of one B cell line out of two were positive for OPT1. The immunoperoxidase staining of paraffin-embedded tissue sections revealed that most of lymphocytes in T cell areas of lymph nodes expressed OPT1 antigen. Some lymphocytes in both cortex and medulla of the thymus and erythroid precursors of the bone marrow were OPT1+. In the malignant lymphoma series, approximately 90% of T cell lymphomas and 6% of B cell lymphomas reacted with OPT1. None of the Reed-Sternberg cells nor Hodgkin cells in Hodgkin's disease were positive. Consequently, OPT1 may be useful for the diagnosis and study of malignant lymphomas and other related lesions.     

Diagnostic usefulness of dipeptidyl aminopeptidase IV monoclonal antibody in paraffin-embedded thyroid follicular tumours.

Monoclonal antibodies to dipeptidyl aminopeptidase IV (DAP IV, EC 3.4.14.5) were raised and selectively applied to paraffin-embedded sections of thyroid carcinoma. Five monoclonal antibodies were found to stain paraffin sections of thyroid carcinomas. Using one of these antibodies (44-4), we studied retrospectively aberrant expression of DAP IV in thyroid carcinoma to determine whether immunohistochemical staining with DAP IV antibody is useful in pathological diagnosis. In almost all cases of thyroid follicular and papillary carcinoma, tumour cells were positive (99.0 per cent) with DAP IV, whereas the cases of follicular adenoma showed a low incidence (27.1 per cent) of positive staining. Follicular adenoma with incomplete capsular invasion had a higher positive incidence (50 per cent) than follicular adenoma without incomplete capsular invasion (9.6 per cent). In positive staining cases previously diagnosed as benign tumours, 11 benign cases reacting positively with DAP IV were rediagnosed as carcinoma after re-examination of more thyroid paraffin block sections or serial sections. These findings suggest that DAP IV monoclonal antibody is very useful in distinguishing thyroid follicular carcinoma from follicular adenoma.     

Detection of HLA-DR antigens in paraffin-embedded thyroid epithelial cells with a monoclonal antibody.

The human Class II major histocompatibility (MHC) antigens, or Ia antigens, which are thought to regulate immune cell interaction, can be detected in paraffin-embedded tissues by immunoperoxidase staining with a recently developed monoclonal antibody (LK8D3). HLA-DR antigens were observed in lymphoid tissues, Langerhans cells of the skin, some epithelial cells, and pulmonary alveolar macrophages. The expression of HLA-DR antigens was analyzed in formalin-paraffin sections by immunoperoxidase in 86 normal and abnormal thyroid epithelial tissues. All patients with Hashimoto's disease (8/8) and most patients with Graves' disease (6/8) expressed HLA/DR antigens in the thyroid epithelial cells and in adjacent inflammatory cells. Most papillary carcinomas (12/18), including 3 of 5 follicular variant of papillary thyroid carcinomas, had HLA-DR antigens detected in epithelial cells; whereas medullary thyroid carcinomas (0/5), follicular carcinomas (0/5), and multinodular goiters (0/4) did not have detectable HLA-DR immunoreactivity. A few other thyroid lesions had HLA-DR antigens detected in epithelial cells, including anaplastic carcinomas (2/5), Hurthle-cell tumors (1/16), and thyroid lymphomas (2/2). Monoclonal antibody LK8D3 and two other commercially available monoclonal antibodies against HLA-DR-stained tissues equally well in cryostat sections, but only antibody LK8D3 was effective in formalin-fixed paraffin-embedded tissue sections. These results indicate that epithelial cells from thyroids of patients with autoimmune diseases commonly express HLA-DR antigens. The presence of HLA-DR antigens in most papillary thyroid carcinomas may be helpful diagnostically in cases of follicular variants of papillary carcinomas. The role of HLA-DR expression in autoimmune thyroid disease and in papillary thyroid carcinoma remains to be determined.     

Anti-IL-6 receptor monoclonal antibody as a new treatment of endometriosis

Immunologic Research - Many pro-inflammatory cytokines especially tumor necrotic factor alpha (TNFα), interleukin (IL)-1β, and IL-6 have crucial role in the pathogenesis of endometriosis....