Immunocytochemical staining of cells in pleural and peritoneal effusions with a panel of monoclonal antibodies.

A panel of seven monoclonal antibodies was applied to smears of cell deposit from 70 pleural and peritoneal fluids, using an immunoalkaline phosphatase (IAP) procedure. The cases were chosen to show typical cytological patterns, both benign and malignant, and in this way the diagnostic value of the method could be assessed. The antibodies used were 2D1 (anti-leucocyte), Ca 1, HMFG-2 (anti-milk fat globule membrane), LE61 and M73 (both anti-intermediate filament antibodies), anti-CEA, and K92 (anti-keratin). The anti-leucocyte antibody was found useful for distinguishing lymphoma from carcinoma. Anti-CEA gave positive reactions in 80% of carcinoma cases and was not seen to react with any other cell types. Ca 1 was positive with some cells in 95% of carcinoma cases, but mesothelial cells reacted with it in two cases. A strong reaction with the anti-milk fat globule membrane antibody was very constant in carcinoma but was also seen in mesothelial cells in 30% of benign effusions. The anti-keratin reacted with malignant cells in only a small proportion of cases. The antibodies against epithelial intermediate filaments reacted equally strongly with benign mesothelial cells and carcinoma cells, but gave negative reactions with lymphoma cells. It is concluded that a suitably chosen panel of monoclonal antibodies can be of great value in identifying neoplastic cells in serous effusions.     

Bovine rotavirus type detection by neutralizing monoclonal antibodies

Summary A series of monoclonal antibodies were developed against bovine rotavirus Q₁₇. Among the five high affinity antibodies characterized, two (RQ 31 and RQ 4) were able to neutralize type G 6 viruses and may be specific for PB 1 type virus. Seventy seven feces from diarrheic calves were tested by double sandwich ELISA using four monoclonal and one polyclonal anti-rotavirus antibodies. The combination of mono- and polyclonal antibodies thus appears to be a more efficient strategy for detection and typing of bovine rotaviruses.     

Immunocytochemical reaction of Ca1 and HMFG2 monoclonal antibodies with cells from serous effusions.

The Ca1 antibody was used in an alkaline phosphatase immunocytochemical method on cells obtained from 150 specimens of pleural and ascitic fluids. The results were compared with the routine cytology report based on the light microscopical appearances. The Ca1 antibody identified tumour cells in 51 of 57 specimens with malignant cells. The exceptions were four small cell carcinomas, one malignant lymphoma, and one adenocarcinoma. A further seven specimens reported as containing atypical cells but without conclusive evidence of malignancy were Ca1 positive. The Ca1 antibody did not give a positive reaction with benign mesothelial cells. Similar results were obtained with the HMFG2 antibody and malignant cells, but in eight of 18 benign effusions it reacted with mesothelial cells.     

Sensitivity of immunofluorescence with monoclonal antibodies for detection of Chlamydia trachomatis inclusions in cell culture.

Monoclonal antibodies which recognize the species-specific major outer membrane protein antigen of Chlamydia trachomatis were used for immunofluorescence staining of chlamydial inclusions in cell culture. A total of 115 clinical specimens were inoculated onto replicate HeLa 229 cell monolayers and assayed for chlamydial inclusions by immunofluorescence staining and Giemsa staining. Of the isolates, 38 were detected by immunofluorescence staining on passage 1 and 1 was detected on passage 2; 23 isolates on passage 1 and 13 isolates on passage 2 were detected by Giemsa staining. Immunofluorescence staining was significantly more sensitive than Giemsa staining for detecting chlamydial inclusions, particularly from specimens containing low titers of Chlamydia.     

Dendritic cells of the mouse recognized by two monoclonal antibodies

A new monoclonal antibody, MIDC-8, is described which shows a comparable tissue distribution as the recently described NLDC-145 antibody. It recognizes interdigitating cells, veiled cells and Langerhans cells in lymphoid organs and the skin of the mouse. In contrast to NLDC-145 it recognizes a cytoplasmic component of these cell types. Its distribution is more restricted to the T cell-dependent areas than NLDC-145. Isolation of dendritic cells from lymph nodes, spleen and thymus revealed that both antibodies react with the in vitro isolated dendritic cells. The results show that these antibodies can be used to study dendritic cells in vitro and emphasize the relationship between the in vivo interdigitating cells of the T cell areas and the in vitro isolated dendritic cells.     

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.     

Xenopus NK cells identified by novel monoclonal antibodies

Early-thymectomized (Tx) Xenopus frogs, which are permanently deficient in T cells, are used as a model sytem for the characterization of novel monoclonal antibodies (mAb) which identify candidate NK cells at the amphibian level of evolution. Hybridomas, generated from mice immunized with splenocytes from Tx Xenopus following B cell and thrombocyte depletion, were screened by flow cytometry. Three mAb (1F8, 4D4 and 1G5) were identified that stained increased proportions of splenocytes from Tx compared with control frogs. These mAb identified lymphoid populations from Xenopus spleen, liver and gut which, after 48 h culture in growth factor-rich medium, exhibited spontanous killing of MHC-deficient allotumor targets. mAb-defined splenocytes also rapidly induced apoptosis of such tumor targets. Dual color analysis confirmed that NK cells are neither T nor B cells. Cytospins of splenocytes isolated with anti-NK mAb revealed large lymphoid cells with distinct pseudopodia. Immunohistology indicated each anti-NK mAb routinely labeled cells within the gut epithelium but NK cells were difficult to visualize in spleen sections. Western blotting of spleen, liver and intestinal lysates subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that 1G5 reacted strongly with protein bands of approximately 70 - 85 kDa, whereas mAb 1F8 and 4D4 stained less intensely, but identified similar protein bands.     

Cytogenetic study of cancer cells in effusions

A cytogenetic study of 71 malignant effusions caused by different histopathologic types of cancer was performed by a direct technique. Abnormal metaphases were present in 50 effusions (70.4%); a detailed analysis of banded karyotypes was possible in 20 of these. Trisomies and monosomies were present in 18 of 20 cases. The most common trisomies were +19, +3, +5, +8, +9, +10, +16 and +22; the most frequent monosomies were -X, -2, -5, -7, and -21. Clonal structural abnormalities were identified in 18 of 20 cases. In most instances, they were extensive, and some chromosomes were involved in a nonrandom fashion. Chromosomes #1, #13, and #6 were the most frequently involved in these rearrangements. A common marker chromosome, t(7;9)(p11;q12), was observed in two patients with cancer of the endometrium. Double minutes (DMs) were present in two patients, and there was a homogeneously stained region (HSR) in one.     

Antigenic phenotype of the myeloid leukaemic cells defined by monoclonal antibodies

The antigenic characteristics of the leukaemic cell population in 31 patients with acute myeloid leukaemia (AML) and 5 patients with acute undifferentiated leukaemia (AUL) was investigated using a panel of 15 monoclonal antibodies (Mc Abs). We chose 14 Mc Abs that react with lineage--and stage related myeloid antigens and L243 Mc Ab that reacts with HLA-DR antigen. In AML cases we correlated the antigenic phenotype with morphological FAB classification. The study indicates a substantial antigenic heterogeneity of the surface antigen expression on leukaemia cells particularly in M1, M2 and M4 AML cases. The morphological subtype of these leukaemias tended to correlate with the immunologic phenotype, particularly in more differentiated AML cases such as M3 or M5. The most immature cell phenotype characterised "undifferentiated" AML, which was expressed by the reactivity or L243, BI3C5, MY9, VIM-2 and S3-13 Mc Abs with the majority of the patients. The analysis shows that although there is a tendency for the morphology to correlate with the surface antigen phenotype each morphological group contains patients having different surface antigen phenotype.     

Long-term passive enhancement of allogeneic skin grafts with monoclonal antibodies

Monoclonal antibodies (mAb) to graft-specific class I or class II major histocompatibility antigens were tested for their ability to enhance the survival of allogeneic skin transplants. Mutant mouse strains were grafted with wild type tissue to restrict the antigenic differences being recognized. For allogeneic recognition of the class I antigen Ld, mutant BALB/c-H-2dm2 (dm2) mice were grafted with wild type BALB/cKh skin, and two dm2 anti-BALB/cKh mAb, 23-10-1 and 30-5-7, were tested for their ability to enhance. The anti-Ld antibody 23-10-1 (IgM) was found not to enhance the survival of BALB/c skin on dm2 mice. 30-5-7, however, an IgG2a antibody of indistinguishable specificity from 23-10-1, prolonged graft survival for approximately 5 days. For recognition of selected Iab determinants, mutant B6.C-H-2bm12 (bm12) mice were grafted with wild type B6/Kh skin, and mAb specific for the serological change(s) in bm12 were tested for their ability to enhance. The anti-Iab antibody 25-9-17 (IgG2a) was found not to enhance B6 grafts on bm12 mice. However, the enhancement seen with 25-9-17 using (C3H X bm12)F1 recipients was extraordinary, such that treated mice had a mean survival time three times that of the controls. Since 25-9-17 is of C3H origin, these results suggest that allotype (or possibly idiotype) compatibility is important in antibody enhancement. Another anti-Iab antibody 28-16-8 (IgM), also of C3H origin, failed to enhance a B6 graft on (C3H X bm 12)F1 mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro generated mast cells investigated by monoclonal antibodies

In the present study we have investigated the development of mast cells under in vitro conditions after depletion of mature mast cells of male Wistar rats by intraperitoneal application of distilled water. The studies were carried out both by monoclonal antibodies (MAb) against rat peritoneal mast cells and by cytochemical techniques. During cultivation the amount of MAb-positive cells increased from less than 5% to 64% after 3 weeks, which was accompanied by increased histamine levels and cell size. After 2 weeks the first Alcian blue stained mast cells were detectable, while safranin-positive granules appeared only after 3 weeks. These findings suggest that there are mast cell precursors in the peritoneum which may differentiate into mature mast cells. The antigen on the cell surface recognized by the MAb seems to be expressed during mast cell maturation.     

Subsets of normal mononuclear cells in the peripheral blocd defined by monoclonal antibodies

We studied the peripheral blood of 20 healthy adult individuals for the distribution of subsets within the mononuclear cell population using indirect immunofluorescence staining. Normal ranges are given for several cell populations reacting with a panel of monoclonal antibodies. The analyzed immunological reagents included the monoclonal antibodies Leu-1 (Pan-T), Leu-2a (Suppressor-T), Leu-3a (Helper-T), Leu-7 (Natural Killer-T), MCS-2 (Pan-myeloid), NU-B1 (Pan-B), FMC-7 (B-subset), Tac (IL-2-receptor) and sheep erythrocyte rosetting (E). Cells were examined for positivity under an epi-immunofluorescence microscope and by flow cytometry. The data obtained by these two different approaches were in close agreement. The majority of the cells from the total mononuclear cell fraction in the peripheral blood were Leu-l+ (mean value of 64% by microscope/59% by flow cytometry) and E+ (61%). The T-populations could be broken down into Leu-3a+ (48%/43%), Leu-2a+ (31%/31%) and Leu-7+ (13%/ 14%). Between 2 and 5% of the cells showed double expression of antigens detected by Leu-2a and Leu-3a. The pan-myeloid reagent MCS-2 was positive on 25%/18% of the total cell population; the 8-cells accounted for 5%/ 8%. Tac stained 7%/6% of the cells.     

Distinction between cells in serous effusions using a panel of antibodies

Summary In serous effusions the distinction between reactive mesothelial cells and malignant cells (especially adenocarcinoma cells and malignant mesothelial cells) is frequently a cause of diagnostic difficulty. The present paper describes the immunocytochemical staining of cells in 76 effusions from 71 patients with different malignancies. In 91% of the effusions obtained from patients with adenocarcinomas, the cells stained positive for anti-EMA, 94% for anti-CEA, 64% for anti-LeuM1-antigen and 75% for anti-keratins. In more than 90% of the cases the reactive mesothelial cells stained positive for anti-keratins, but not for anti-EMA, anti-CEA or anti-LeuM 1-antigen. It is concluded that a panel of the antibodies against EMA, CEA, LeuM 1-antigen and keratins is valuable in the distinction between adenocarcinoma cells, malignant mesothelial cells and reactive mesothelial cells in serous effusions.     

Characterization of immunoregulatory T cells during pregnancy by monoclonal antibodies

T Lymphocyte subpopulations were characterized by means of monoclonal antibodies in 44 pregnant women at different stages of gestation, and in 45 healthy controls. In the pregnant subjects, there is a small but significant reduction of the whole circulating T Lymphocytes and of their T helper subset identified respectively by OKT3 and OKT4 monoclonal antibodies. There is no influence of pregnancy on the proportions of T cytotoxic-suppressor cells (OKT8 positive) nor of lymphocytes bearing Ia antigen. Low counts of circulating cells with a thymocyte phenotype (OKT6 positive) are found in the two groups of subjects. It is concluded that pregnancy has a marginal effect on maternal immunocompetence.     

Phenotype of the hairy cells of leukemic reticuloendotheliosis defined by monoclonal antibodies

Hairy cell populations of greater than 90 purity were prepared from six samples obtained from four patients with leukemic reticuloendotheliosis (LRE). These then were analyzed for surface immunoglobulin (SIg) and antigens specified by a panel of monoclonal antibodies. The hairy cells from all patients displayed SIg and antigens reactive with OKIa-1 and OKM-1 antibodies; these markers often were expressed simultaneously. T-cell-specific antigens were not displayed on the surface of hairy cells. The simultaneous expression of SIg and OKM-1 which ordinarily are unique for B cells or myeloid cells, respectively, suggests that hairy cells may represent an aberrant form of either cell type with defective regulation of antigen expression. It alternatively suggests the possibility that B cells and myeloid elements develop along a common pathway and that the hairy cell is a component of such a pathway.     

Role of monoclonal antibodies in the study of breast cancer

Breast cancer is the most frequent cancer among women in western countries, where it represents about one third of tumors. Many laboratories utilized monoclonal antibodies technology for studying this pathology and for clinical applications. Different immunisation strategies have been utilized in the aim to produce monoclonal antibodies specific of mammary epithelial cells, of cancer cells or of cell secretions products. Despite efforts none of these are entirely specific, either for the normal tissue or for malignant tumors derived from it. However several of them had applications in fundamental study of oncogenesis and/or in tumor diagnostic, and in therapeutic assays. In this review we analysed described monoclonal antibodies, their reactive antigens and their applications.     

Characterization of rabbit cells by monoclonal and polyclonal antibodies.

Reagents for the identification of rabbit cell markers have been developed at a relatively slow rate. In this paper, rabbit cells are being characterized by polyclonal antibodies against a T-cell antigen (RTLA), a B-cell antigen (RABELA) and an analogue of murine Ia antigen. A number of monoclonal antibodies, specific for lymphocytes and/or bone marrow and/or polymorphonuclear leucocytes, have been used for the analysis of cells with identifiable membrane antigens. Populations that have cells with two of the above antigens in the membranes were identified. To these ends, complement-mediated cell kill by antisera alone and in mixtures was employed.     

Improved detection ofPneumocystis carinii by an immunofluorescence technique using monoclonal antibodies

To assess whether a recently developed indirect immunofluorescent stain using monoclonal antibodies was more sensitive in detectingPneumocystis carinii than the combination of Giemsa and methenamine silver nitrate stains which has routinely been used in the laboratory, 88 lavage fluid specimens and 34 induced sputum specimens were examined. All specimens were stained by five techniques: immunofluorescence using a combination of three monoclonal antibodies (from the National Institutes of Health, USA), immunofluorescence using a single monoclonal antibody (from Dakopatts), Giemsa, methenamine silver nitrate and toluidine blue O. Immunofluorescence using the monoclonal antibodies from the NIH was significantly more sensitive than any other single staining method and than the combination of Giemsa and methenamine silver nitrate staining. The study also showed that the cytospin centrifuge was very suitable for the preparation of slides with lavage fluid and processed induced sputum. Finally, the sensitivity of examination of induced sputum to detectPneumocystis carinii was found to be 50 % when compared with bronchoalveolar lavage fluid. However, this sensitivity may increase through practice.