Tumor-associated antigens in effusions of malignant and benign origin

Summary We determined the concentration and effusion/serum ratio of mucin-like carcinoma-associated antigen (MCA) in comparison to carcinoembryonic antigen, carbohydrate antigen 19-9, cancer antigen 125, and cancer antigen 15-3 in 80 sera and 99 effusions from 64 patients with histologically confirmed malignancies (4 patients out of this group showed various effusions simultaneously, which were analyzed separately) and 31 patients with various nonneoplastic diseases. Tumor cells were detected by cytological examination in 41 effusions (60.3%) from patients with neoplastic diseases, while in another 27 cases this method failed to demonstrate the malignant origin of the effusion. Of the cytological positive malignant effusions 90% were also correctly identified by an elevated MCA concentration at a cutoff level of 10 U/ml, whereas only one effusion of benign origin (3%) showed a slightly elevated MCA concentration of 10.5 U/ml. In 33% of cytologically negative effusions of patients with neoplastic diseases, the MCA concentration was also elevated, with a maximum of 453 U/ml. Increased MCA levels in cytologically confirmed malignant effusions were not restricted to metastatic breast cancer. All 17 cytologically positive non-breast cancer effusions were correctly identified by their MCA concentrations. None of the other tumor markers reached this high sensitivity at the same level of specificity. The ratio of effusion/serum concentration of all tumor markers as well as the concentration of cancer antigen 125 in effusions was of little diagnostic value. Our results indicate that the MCA concentration in an effusion correlates very closely with its malignant origin and is superior to all the other antigens tested. Accordingly, the concurrent MCA determination could improve the diagnostic accuracy of the cytological examination of effusions.Abbreviations MCA mucin-like carcinoma-associated antigen - CEA carcinoembryonic antigen - CA 15-3 cancer antigen 15-3 - CA 125 cancer antigen 125 - CA 19-9 carbohydrate antigen 19-9     

Analysis of the repertoire of anti-HLA antibodies with anti-idiotypes to a murine anti-HLA-A2,A28 monoclonal antibody

Xenoantibodies to idiotypes of the anti-HLA-A2,A29 MoAb CR11–351 were isolated from an antiserum raised in rabbit #81 by immunizations with purified MoAb CR11–351. The purification procedure involved absorption with insolubilized mouse immunoglobulis and monoclonal antibodies and affinity chromatography on insolubilized MoAb CR11–351. Two antibody populations were identified in the xenoantibody preparation #81: one recognizes a recurrent idiotope expressed by the MoAb CR10–215, CR10–402, Q1/28, Q6/64, and 6/31 to monomorphic determinants of HLA-A,B antigens and by the MoAB CR10–343, CR11–462, and Q516 to human la antigens. The other antibody population recognizes a private idiotope. Neither idiotope was detected on the anti-HLA-A2, A28 variant (A28¹) MoAbs BB7.2, MA2.2, and PA2.1, on the anti-HLA-A2,B17 MoAb MA2.1 and on antibody populations in conventional anti-HLA-A2,A28 antisera. The idiotopes were also not detected on the anti-HLA-A2,A28 MoAb A2,A28 M1 which recognizes a determinant spatially close to that identified by the MoAb CR11–351. The idiotope(s) recognized by the xenoantibodies #81 may be located in the combining site of the MoAb CR11–351, since its incubation with the anti-idiotype antibodies specifically blocks the reactivity with lymphoid cells with the appropriate HLA phenotype.     

Utility of cytokeratin 20 in identifying the origin of metastatic carcinomas in effusions

Using a commercially available monoclonal antibody (K_s20.1) and the avidin-biotin peroxidase method on cytospins and cell blocks, we analyzed cytokeratin (CK) 20 expression in 169 serous effusions. Cytoplasmic staining was observed in 44/151 malignant fluids. Colon, gastric, and pancreatic adenocarcinomas and mucinous ovarian tumors were most frequently positive. Single cases of transitional-cell and squamous cell carcinomas were reactive as well. Lung and breast cancers were mostly negative. Nonmucinous ovarian tumors were invariably unlabeled as were mesotheliomas and normal mesothelial cells. the study shows that CK 20 is valuable in distinguishing tumor cell origin in effusions. in particular, it identifies a set of carcinomas with the majority arising from the gastrointestinal tract, and represents a highly characteristic marker for colorectal cancer. © 1995 Wiley- Liss, Inc.     

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.     

Development of a rapid microELISA assay for screening hybridoma supernatants for murine monoclonal antibodies

A micro enzyme-linked immunosorbent assay (ELISA) utilizing a filtration method has been developed which allows the rapid, simple, and sensitive detection of monoclonal antibodies that recognize either soluble or cell surface antigens. This assay involves the immobilization of target cells (or soluble antigen) onto glass fiber filter discs followed by an incubation with the test hybridoma supernatant and subsequent analysis by ELISA. A specially designed 96-well filtration device is employed which serves both as an incubation chamber and as a filtration manifold. This microELISA requires small volumes of antiserum, few target cells, and can be completed in less than 2 h. This assay is well suited for the rapid screening of murine hybridoma supernatants and can be adapted to detect monoclonal antibodies from other species.     

Detection of bone marrow metastases in neuroblastoma using a short term tissue culture technique.

AIMS: To improve detection of neuroblastoma metastases in the bone marrow: morphological evaluation of bone marrow is a routine and important component of the clinical staging of neuroblastoma and it depends on the successful identification of tumour cells which may only be present at extremely low levels. METHODS: Bone marrow mononuclear cells from patients with neuroblastoma were incubated in a simple suspension culture and examined regularly using an inverted objective microscope. In some cases cytospins of the cultured cells were examined further using morphological and immunocytological techniques. RESULTS: In some cultures spheroidal clumps of cells could be seen growing after only a few days. If the marrow was cultured for a longer period these spheroids continued to increase in size, became adherent to the stromal cell layer on the culture flask floor, and put out long characteristic processes (neurites). Morphological and immunocytological examination of cytospins from these cultures confirmed these cells as neuroblasts. CONCLUSIONS: This method has provided the sole evidence of marrow metastases in several newly diagnosed cases of neuroblastoma in which the bone marrow had shown no evidence of tumour using standard morphological and fluorescent immunocytological techniques. Although negative cultures do not preclude the presence of neuroblastoma, this method is a useful adjunct to the standard techniques.     

Routine large-scale production of monoclonal antibodies in a protein-free culture medium

A defined culture medium and cultivation technique are described which allow the long-term production on a large scale of monoclonal antibodies and myeloma proteins without the use of serum, serum proteins, or protein-containing liposomes. The high initial purity of the culture supernatants obtained with protein-free medium facilitates the purification of the monoclonal antibodies, the immunoglobulin (Ig) purity of which is limited only by the genetic homogeneity of the cells. Successful growth of 8 hybridoma and 2 myeloma lines has been achieved. SDS-PAGE analysis revealed that the levels of Ig production in the protein-free medium were comparable to those achieved with serum-supplemented media and with media supplemented with purified serum proteins. Stability of Ig production during continuous cultivation in the protein-free medium over an extended period was studied for a myeloma and a hybridoma line, both of which produced high levels (50–200 μg/ml) of Ig for periods of 11 and 22 weeks, respectively.     

Detection of cytomegalovirus by a rapid culture system: a comparison of monoclonal antibodies in a clinical setting.

A monoclonal antibody cocktail, directed against immediate early antigens of cytomegalovirus, was assessed in a rapid culture system for detection of CMV (DEAFF) in clinical samples. The antibody cocktail was shown to be equivalent in sensitivity and specificity to our currently used monoclonal antibody. A comparison of DEAFF (using either monoclonal preparation) and conventional cell culture shows that DEAFF detects a smaller proportion of CMV positive samples from those receiving antiviral chemotherapy. The reasons for the discrepancies observed between these techniques are discussed.     

The origin of cells in the glomerular crescent investigated by the use of monoclonal antibodies

A study was made of the cells forming the crescents in human crescentic glomerulonephritis. The investigation was performed using a panel of antibodies with immunoperoxidase techniques in formalin fixed, paraffin embedded renal biopsy material. Some of the cells of glomerular crescents were found to contain cytokeratin intermediate filaments, as did some of the cells of the normal parietal epithelium of Bowman's capsule. Leucocytes were also found in crescents, often in the outer part, and their presence was associated with a mantle of inflammatory cells around the glomerulus. The use of paraffin embedded rather than frozen tissue allowed better histological assessment than has been possible in previous studies. The glomerular crescents appeared to be primarily epithelial in origin, with leucocytes contributing to the overall inflammatory response.     

A Quantitative Nitrocellulose Enzyme Immunoassay and its Application to the Screening of Hybrbidomas for the Detection of Uncharacterized Tumor-Associated Antigens in Sera of Cancer Patients

Abstract

An enzyme-linked immunosorbent assay on nitrocellulose based microtiter plates for the detection of uncharacterized tumor associated antigens in squamous cell carcinoma and adenocarcinoma cancer patients' sera is described. Nitrocellulose microtiter plates are more sensitive than the plastic plates of polystyrene and polyvinyl chloride for the detection of antigens in serum. Monoclonal antibodies were selected for their net reactivities toward cancer patients' sera as compared to nomal sera. Sera from benign liver and kidney disease patients and activated human peripheral blood leukocyte supernatant were used to reduce potential false positives toward inflammatory and benign diseases. Using this system, fourteen antibodies were selected out of over eight hundred antibodies for their potential serodiagnostic application.     

Expression in normal adult, fetal, and neoplastic tissues of a carbohydrate differentiation antigen recognised by antigranulocyte mouse monoclonal antibodies.

The distribution in paraffin fixed human tissues of a carbohydrate antigen defined by two monoclonal antibodies raised against human granulocytes has been studied by means of an immunoperoxidase technique. In addition to granulocytes, the antigen has been detected in adult tissues on identifiable cell types of the stomach, kidney, adrenal medulla, and brain and on the mucins of the gastrointestinal tract and other secretions. In fetal tissue, epithelial cells of the alimentary tract, lung, brain, and kidney express the antigen. Adenocarcinoma of the colon, stomach, breast, and lung are stained strongly, as are other types of lung cancer. The monoclonal antibodies give a staining pattern similar but not identical to other monoclonal antibodies raised against granulocytes or neoplastic cell lines which recognise the antigen 3-fucosyl N-acetyllactosamine. The use of antibodies against this oncofetal antigen in the study of differentiation and as tumour markers is discussed.     

Cell mediated cytotoxicity against HLA-D region products expressed in monocytes and B lymphocytes. III. Inhibition by monoclonal antibodies and study of an HLA-B/DR recombinant

Attempts to further define the antigens recognized by HLA-D region specific cytotoxic lymphocytes were undertaken using monocytes and transformed B cell lines as target cells. Monoclonal antibody against framework determinants of HLA-DR antigens partially blocked cell mediated lysis, suggesting that at least a portion of the D-region specific cytotoxic cells recognized the HLA-DR determinants. The study of a family with an HLA-B/DR recombinant showed that the determinants recognized by allogeneic anti-HLA-D-region cytotoxic lymphocytes are encoded outside of HLA-B. In addition, cytotoxic lymphocytes specific for the HLA-D region could be generated with cells identical throughout the interval from HLA-A to B and disparate only to the left of HLA-B.     

Detection of blood group A and H-related antigens in normal and neoplastic bladder epithelium: a comparative study using monoclonal antibodies with defined fine specificities.

Three monoclonal anti-blood group H and four monoclonal anti-blood group A antibodies directed at the blood group antigens on Type 1 or Type 2 backbone structures were evaluated as immunohistochemical reagents by indirect immunofluorescence of normal and neoplastic bladder epithelia. The results were compared with fluorescence using polyclonal rabbit anti-A or H sera or Ulex europaeus lectin. The monoclonal antibodies gave less intense or more restricted immunofluorescence than the conventional reagents but showed considerable variation in the extent of their reactivities with urothelial samples from different individuals. In some cases they failed to give immunofluorescence with tissue samples known to contain the immunodominant blood group structures they recognize. In addition, hitherto unsuspected heterogeneity was revealed in the expression of the Type 2-based blood group H and A-structures in the endothelia of neighbouring small blood vessels.     

Epitope mapping and the detection of transmissible gastroenteritis viral proteins in cell culture using biotinylated monoclonal antibodies in a fixed-cell ELISA

Summary A fixed-cell ELISA was developed using swine testicle (ST) cells infected with the virulent Miller strain of transmissible gastroenteritis virus (TGEV) and purified biotinylated monoclonal antibodies (b-MAbs). Five of the b-MAbs were specific for the peplomer (E2), five reacted to the nucleocapsid (N), and one reacted to the E1 protein of the Miller strain of TGEV. Protein A-Sepharose purification of MAbs yielded protein concentrations ranging from 0.40 to 3 mg per ml of ascites. Separate pools of N-MAbs and E2-MAbs, and the E1-MAb were used to monitor synthesis of TGE viral antigen in ST cells from 0 to 16 h post-infection at various multiplicities of infection (MOI). Epitopes of N proteins appeared sooner and at a lower MOI than those for the E1 and E2 proteins. The fixed-cell ELISA was also used to examine relative binding affinities of TGEV MAbs. Concentrations of b-MAbs producing a half-maximal signal ranged from 0.11 to 3.8 µg/ml for E2-MAbs, from 0.05 to 0.82 µg/ml for N-MAbs, and 6 µg/ml for the E1-MAb. The assay was used to determine the 50% neutralization concentrations for four neutralizing E2-MAbs (0.1 µg/ml to 6.9 µg/ml) and one E1-MAb (1.2 µg/ml). Competition assays between b-MAbs and unlabeled competitors indicated that at least two major antigenic sites exist on the E2-protein and 2 to 3 antigenic sites are present on the N-protein of Miller TGEV.     

Gamma heavy chain disease in man: synthesis of a deleted gamma3 immunoglobulin by lymphoid cells in short and long term tissue culture.

Bone marrow cells were obtained from a patient with gamma heavy chain disease (HCD) whose serum contained a deleted immunoglobulin heavy chain. Incubation of the marrow cells with radioactive amino acids in short term tissue culture resulted in the synthesis of the labelled HCD protein. A permanent cell line was established from the peripheral blood of the patient. Similar labelling studies with the cell line and its cloned progeny demonstrated the synthesis of a protein identical in size and antigenicity to that synthesized by the marrow cells and found in the patient's serum. These experiments clearly demonstrated that, in this case of heavy chain disease, the deleted protein was the synthetic product of a clone of malignant lymphoid cells.     

A porcine cell surface receptor identified by monoclonal antibodies to SWC3 is a member of the signal regulatory protein family and associates with protein-tyrosine phosphatase SHP-1

SWC3 was defined at the First International Swine CD Workshop as a specific myelomonocytic antigen of 230 kDa with mAbs 74-22-15, 6F3 and DH59B. In this report, we describe two new mAbs (BL1H7 and BA1C11) that react selectively with granulocytes, monocytes and macrophages. These monoclonal antibodies (mAbs) recognize a molecule in the range of 90-115 kDa in immunoprecipitation and/or Western blotting analyses. Two-colour FACS analyses showed that the distribution of BL1H7 and BA1C11 antigens was identical to that of SWC3. Moreover, in this assay, mAb 74-22-15 appeared to partially block the binding of mAbs BL1H7 and BA1C11, suggesting that all these mAbs reacted with the same or spatially close epitopes. Cross-blocking analyses indicated that it was the case with mAbs 74-22-15 and BL1H7. Immunoprecipitation experiments with mAbs 74-22-15, BL1H7 and BA1C11, followed by immunoblotting with mAb BL1H7 confirmed that all three mAbs recognize the same molecule. Analysis of the N-terminal sequence carried out on the affinity purified protein revealed homology with members of signal regulatory protein (SIRP) family. Like other members of this family, after treatment with sodium pervanadate, SWC3 became phosphorylated in tyrosines, and associated with the protein-tyrosine phosphatase SHP-1.     

Use of monoclonal antibodies in a study of the development of T lymphocytes in the human fetus.

A panel of monoclonal antibodies (OKT3, 4, 6, 8, 10, 11) was used for the identification of T lymphocyte subpopulations in cell suspensions of human fetal liver, thymus, bone marrow and spleen. In liver suspensions of 8-16 week old fetuses and in bone marrow suspensions (12-20 weeks) less than 5% of lymphocytes reacted with either OKT3, 11, 4, 8 or 6, whereas the OKT10 antibody bound to, respectively, 35 and 86% of lymphocytes in these tissues. In liver suspensions of 17-20 week old fetuses, about 20% of lymphocytes carried either the T3, 11, 4 or 8 antigen and more than 60% of lymphocytes were OKT10+. The maturation stages in fetal thymus (11-20 weeks) are comparable to those in the post-natal thymus, with the exception that a substantial proportion of fetal thymocytes expresses the T3 and T6 antigen simultaneously. In the fetal spleen (12-20 weeks), 40% of lymphocytes reacts with OKT3. These OKT3+ spleen cells may be divided into two subsets expressing either the T4 antigen or the T8 antigen. These OKT3+/OKT4+ and OKT3+/OKT8+ lymphoid cells of the fetal spleen can be further subdivided into a T10+ and T10- subpopulation. These data suggest that T lymphoid precursor cells, reacting with either none of the monoclonal antibodies or only with OKT10, are generated in fetal liver (up till 16 weeks gestational age) and bone marrow. Further maturation takes place in the fetal thymus, but also to a certain extent in peripheral lymphoid organs such as the fetal spleen, as evidenced by the coexistence of a T3+/T10+ and T3+/T10- subpopulation in this organ.     

Cancer diagnostic test of Field and Caspary: Part I. Spontaneous aggregation of lymphocytes in saline suspension is blocked by albumin and histone in normals but not in various disease states: the lymphocyte aggregation blocking test

A blood test is described which is often positive in diverse disease states. In this test washed patient lymphocytes are incubated with histone or albumin. Early aggregation indicates that the patient might have inflammatory, malignant, or other disease, while absence of aggregation is the pattern found in normals. The test is called the lymphocyte aggregation blocking (LAB) test. Positive results when testing cancer patients were 86%, normals 14%, and non-malignant hospitalized patients were 50%. Lymphocyte aggregation was not an active process mediated by added protein. All washed lymphocyte suspensions will aggregate in the test conditions, whether, from normals, cancer or non-cancer patients if no protein is added. Normals' lymphocyte suspensions are prevented from aggregating by the added histone or albumin.     

Pseudotumoral amyloidosis of beta 2-microglobulin origin in the buttock of a patient receiving long term haemodialysis.

A 52 year old man who had been receiving haemodialysis for 13 years, with a history of renal tuberculosis, right ischial tuberculous osteomyelitis, and dialysis arthropathy, developed a soft tissue tumour in his left buttock. Histological analysis, immunohistological staining, and electron microscopic examination of the surgically removed tumour showed massive deposits of beta 2-microglobulin (beta 2-M) amyloid. This case shows the expanding clinical spectrum of this type of amyloidosis, and it is suggested that amyloid infiltration should be considered in the differential diagnosis of gluteal tumours in these patients.