Production and characterisation of an antimelanoma monoclonal antibody KBA.62 using a new melanoma cell line reactive on paraffin wax embedded sections.

AIMS--To generate new monoclonal antibodies directed against melanoma associated antigens using a new melanoma cell line, KAL. METHODS--The melanoma cell line was established in culture from a lymph node metastasis of malignant melanoma. Normal Balb/c mice were immunised with KAL cells. Splenocytes were used for fusion experiments using standard techniques. Hybridoma supernatants were tested for antibody binding activity using an indirect immunoperoxidase method on frozen sections from KAL tumour cells xenografted onto nude mice and human tonsils. KBA.62 was selected because of its reactivity with melanocytic proliferations on both frozen and paraffin wax sections. RESULTS--On immunoblotting, KBA.62 reacted with three bands of 140, 135 and 128 kD and two weak bands of 88 and 73 kD. In normal human tissues basal melanocytes in the epidermis did not react with this antibody and only occasional labelling of endothelial cells was noted. Of the human tumours, KBA.62 reacted strongly and uniformly with the majority of benign (21/21) and malignant (75/86) melanocytic proliferations. Staining was localised predominantly to the cell membrane with little or no cytoplasmic reactivity. Negative staining was observed in the majority of human non-melanocytic neoplasms, the exceptions being some carcinomas (11/89), particularly the well differentiated squamous cell type. This, however, was not thought to present a diagnostic problem. CONCLUSIONS--KBA.62 appears to be potentially useful in ascertaining the immunomorphological diagnosis of malignant melanoma in routinely processed paraffin wax sections.     

T311--an anti-tyrosinase monoclonal antibody for the detection of melanocytic lesions in paraffin embedded tissues

Tyrosinase is a key enzyme in melanin biosynthesis and represents a marker of melanocytic differentiation. We previously generated T311, a murine monoclonal antibody to the tyrosinase recombinant protein. This study was performed to evaluate T311 as a diagnostic immunohistochemical reagent for use on formalin-fixed paraffin-embedded pathological material. We analyzed the specificity of the antibody on a panel of normal and neoplastic tissues, and we assessed its sensitivity in a large number of metastatic and primary malignant melanomas, nevi, three angiomyolipomas, and two vitiligo specimens. T311 revealed intense reactivity on paraffin-embedded material. Immunoreactivity was limited to cells of melanocytic differentiation and no immunostaining was present in unrelated normal tissues and tumors. Eighty-four percent of metastatic malignant melanomas were immunoreactive with T311 and showed predominantly a homogeneous expression pattern. However, in primary melanomas of the desmoplastic/spindle cell type, T311 revealed a poor immunoreactivity. Nevi showed intense staining at the junctional zone, while the dermal component revealed decreasing reactivity towards deeper areas. Only one angiomyolipoma was focally immunoreactive with T311. Vitiligo specimens were immunonegative. We conclude that T311 is a specific and sensitive marker for the detection of melanocytic lesions in formalin-fixed paraffin-embedded tissues and a useful serological reagent for diagnostic pathology.     

Use of monoclonal anti-actin as a megakaryocyte marker in paraffin wax embedded bone marrow biopsy specimens.

Monoclonal anti-actin was used as a marker of megakaryocytes in Zenker's fixed, paraffin wax embedded bone marrow tissue, using an immunoperoxidase staining method. Twenty bone marrow samples were studied, including controls, and different myeloproliferative and myelodysplastic syndromes. The results were compared with those obtained using factor VIII related antigen (F VIII RAg) immunolabelling. Anti-actin is as good a marker for megakaryocytes as anti-FVIIIRAg and is potentially clinically useful when morphological identification is difficult, when bone marrow aspiration is unsuccessful, or when quantitative evaluation of tissue sections is required.     

Production of monoclonal antibody against electrophoretically purified RNA polymerase II subunits using in vitro immunization

A procedure has been developed for the production of MAb against weakly immunogenic subunits of a multisubunit enzyme. This procedure takes into account the problems of insufficient antigen, single epitope immunodominance and the difficulty of mapping non-sequential determinants. Small quantities of mammalian RNA polymerase II subunits were purified by SDS-polyacrylamide gel electrophoresis and were used to immunize splenocytes in vitro. After fusion with plasmacytoma cells, the hybrid cells were cloned and screened by ELISA utilizing native RNA polymerase II. This procedure is biased towards the production of MAb directed against sequential epitopes accessible on the native enzyme. Monoclonal antibodies, produced by in vitro immunization, were shown to be useful in protein transblot analyses, to inhibit enzyme activity in vitro and to have binding affinities comparable with MAbs produced by in vivo immunization.     

Observations favouring Pneumocystis carinii pneumonia as a primary infection: a monoclonal antibody study on paraffin sections

Pneumocystis carinii pneumonia is characteristic of immunodeficiency and the organism is probably acquired during early childhood. Since infection is only manifest in the lungs, it has been presumed that the organism lies dormant in these tissues following the primary infection. Conventional staining procedures have, however, failed in the absence of pneumonia to demonstrate consistently any forms of Pneumocystis carinii. To study this problem further, lung sections and hilar lymph nodes from immunodepressed adults with and without Pneumocystis carinii pneumonia as well as lung sections from presumed immunocompetent patients were examined for the cyst and trophozoite forms of Pneumocystis carinii using a monoclonal antibody. The organism was only identified in areas of pneumonia, and the source of the organism in these patients may therefore be a new infection with a different human subtype and not, as previously thought, reactivation of a primary infection.     

Use of imprints for monoclonal antibody studies: suitability of air-dried preparations from lymphoid tissues with an immunohistochemical method

Air-dried imprint preparations are conveniently produced from human lymphoid samples without the special methods required for snap-freezing tissues or rendering them into suspensions. T-cells, T-cell subsets (helper and suppressor), and HLA-DR-positive cells (B-lymphocytes, monocytic-histiocytic cells) can be identified in such imprints by the use of commercially obtained mouse hybridoma antibodies with a simple two-step immunoperoxidase method. Direct nuclear morphologic correlation with surface determinants is achieved by this method. Immunoreactivity is retained only eight to 10 days in such air-dried preparations, and attempts to prolong reactivity have been unsuccessful so far.     

Monoclonal antibody SM047 as an immunohistochemical marker of ovarian adenocarcinoma

AIMS: This study describes the generation of a monoclonal antibody designated SM047 which binds to an epitope that is displayed by a multivalent antigen associated with the glycocalyx of ovarian adenocarcinoma cells. The study also investigates SM047 staining in adenocarcinomas of diverse sites in order to determine whether the antibody is specific for ovarian adenocarcinoma and of value in the confirmation of an ovarian origin when the site of primary tumour is unknown. METHODS AND RESULTS: SM047, an IgM monoclonal antibody, was the product of hybridoma cells derived from fusion of SP2 myeloma cells with splenocytes of a mouse that had been immunized with a membrane preparation of tumour (ovarian serous cystadenocarcinoma) and boosted with cells from a cell line established from a similar tumour in a different patient. Sixty-two primary ovarian adenocarcinomas (28 serous, 23 mucinous, five endometrioid and six clear cell), 69 adenocarcinomas arising primary at other sites and 10 mesotheliomas were stained with SM047. There was positive membrane staining, which was usually strong and widespread, in 27 of 28 ovarian serous carcinomas and in all ovarian endometrioid and clear cell carcinomas. Most ovarian mucinous tumours were negative or exhibited weak cytoplasmic staining. Staining was variable in the other tumours but there was positive staining of most endometrial, endocervical and pancreatic adenocarcinomas. Most colonic adenocarcinomas were negative or exhibited weak cytoplasmic staining. CONCLUSIONS: SM047 is strongly expressed in most ovarian serous adenocarcinomas and in other female genital tract adenocarcinomas, with the exception of ovarian mucinous tumours. The antibody may be useful in confirming the ovarian origin of an adenocarcinoma when used as part of a larger panel. This is especially so in the distinction between a non-mucinous ovarian adenocarcinoma, which usually exhibits strong membranous staining, and a colonic adenocarcinoma which is usually negative or exhibits weak cytoplasmic staining. These findings need to be confirmed by further study of larger numbers of cases.     

A method of processing small biopsy specimens embedded in paraffin using xylene

The method proposed for the processing of gastrobiopsies is as follows: 30-40 min in solution I (seventy per cent 96% ethyl alcohol and 30% of turpentine), then 30-40 min in solution II (100 parts of turpentine, 5 parts of balzam and 8 parts of castor oil), 4 portions of melted paraffin for 7.5-10 min in each portion. Small biopsies (up to 5 mm in diameter) are treated in the same way but till 3 hrs in solution I, 6-8 hrs in solution II and 30 min in each portion of paraffin. For an instant processing of biopsies of operation and autopsy material the basic solution is prepared: 1000 ml of xylene, 100-120 g of balzam, 150 ml of ol. ricini. The material is treated in two portions of ethyl alcohol (30-150 min in each portion); two portions of mixture A; 80% of acetone and 20% of the basic solution), 3 hrs in each portion; two portions of mixture B (20% of acetone and 80% of basic solution), 2 hrs in each portion; and in 6 portions of melted paraffin, 30 min in each portion. The technique provides good results for the processing of small biopsies from other organs.     

Two new ELISAs using monoclonal H-Y antibody

Biotin-conjugated monoclonal H-Y antibody was used to detect soluble H-Y antigen, directly, in two novel enzyme-based systems: a sandwich or 'capture' ELISA and an inhibition ELISA. In each case, soluble H-Y was readily detected in samples known to contain the antigen, provided that positive and negative samples were compared on an equivalent protein basis. By adding increasing concentrations of inhibitor (antigen) we define the useful range of the assays to be between 0.6 and 20.0 micrograms of added protein. Both sandwich and inhibition ELISAs gave similar results with antigens tested. The methods can accordingly be applied as direct diagnostics in the study of sex determination and in the assignment of gender.     

The detection of mast cell subpopulations in formalin-fixed human tissues using a new monoclonal antibody specific for chymase

Chymase is an important marker for human mast cells as well as a mediator of inflammation and matrix remodelling, but research into chymase-containing mast cell subpopulations has been hampered by the lack of reagents suitable for use with formalin-fixed tissue. A monoclonal antibody to chymase (designated CC1) was prepared by immunizing a mouse with chymase purified from human skin, fusing the splenocytes with NS-1 myeloma cells, and screening the hybridoma supernatants by ELISA with recombinant human prochymase isolated from a baculovirus expression system. This antibody bound to chymase in western blots and bound selectively to cells with the morphology and distribution of mast cells in paraffin wax sections of skin, synovium, lung, and heart. In sequential sections and with double-labelling experiments, chymase was localized to cells which contained mast cell tryptase; in contrast to previous reports, no evidence was found for its presence in endothelial cells or any other cell type. The antibody permitted chymase-containing mast cells to be detected in formalin-fixed tissues, and the numbers identified were similar to those in tissues fixed with Carnoy's or ethanol fixatives. Immunocytochemistry with antibody CC1 provides for the first time a sensitive and specific means for the detection of chymase in routinely fixed tissues and should prove valuable in studying mast cell subsets in disease. Copyright © 1999 John Wiley & Sons, Ltd.     

Production of a monoclonal antibody against SARS-CoV spike protein with single intrasplenic immunization of plasmid DNA

Severe acute respiratory syndrome (SARS) is a highly infectious disease caused by a novel coronavirus (SARS-CoV). Specific monoclonal antibodies (mAbs) against the SARS-CoV are vital for early diagnosis and pathological studies of SARS. Direct intrasplenic inoculation of plasmid DNA encoding antigen is an effective and fast approach to generate specific mAb when the protein antigen is difficult to prepare or dangerous in use. In this study, we selected one fragment of SARS-CoV spike protein (S1-₃) as antigenic determinant by immunoinformatics. Single intrasplenic immunization of plasmid DNA encoding S1-₃ induced anti-spike protein antibodies. We established one hybridoma cell line secreting specific mAb and evaluated this mAb with murine leukemia virus pseudotyped with SARS-CoV spike protein (MLV/SARS-CoV). The mAb could recognize the spike protein on the MLV/SARS-CoV-infected Vero E6 cells albeit with no neutralizing effect on the infectivity of the pseudotype virus. Our results show that a single-shot intrasplenic DNA immunization is efficient for the production of specific mAb against SARS spike protein, and a linear epitope of the spike protein is recognized in this study.     

Production and characterization of a new monoclonal antibody effective in recognizing the CD3 T-cell associated antigen in formalin-fixed embedded tissue

Phenotypic analysis of lymphoproliferative disorders is now considered mandatory for accurate classification which is the basis for optimum patient management. This is presently carried out in most cases using a range of antibodies recognizing B and T-cell antigens effective in paraffin sections, and an antibody to CD3 is currently a key member of such panels, indicating T-cell phenotype. Current antibodies to CD3 are polyclonal with the inherent disadvantages of this type of reagent compared to monoclonal antibodies. In this study, we have used a recombinant fusion protein representing part of the epsilon subunit of the CD3 molecule to generate a novel monoclonal antibody (NCL-CD3-PS1) effective in paraffin sections. The antibody has been characterized biochemically and by immunohistochemistry using a wide range of normal and pathological tissues. Lineage and phenotype specificity have been supported in our study and results from other laboratories are awaited with interest.     

Production and characterization of a new monoclonal antibody effective in recognizing the CD3 T-cell associated antigen in formalin-fixed embedded tissue

Phenotypic analysis of lymphoproliferative disorders is now considered mandatory for accurate classification which is the basis for optimum patient management. This is presently carried out in most cases using a range of antibodies recognizing B and T-cell antigens effective in paraffin sections, and an antibody to CD3 is currently a key member of such panels, indicating T-cell phenotype. Current antibodies to CD3 are polyclonal with the inherent disadvantages of this type of reagent compared to monoclonal antibodies. In this study, we have used a recombinant fusion protein representing part of the epsilon subunit of the CD3 molecule to generate a novel monoclonal antibody (NCL-CD3-PS1) effective in paraffin sections. The antibody has been characterized biochemically and by immunohistochemistry using a wide range of normal and pathological tissues. Lineage and phenotype specificity have been supported in our study and results from other laboratories are awaited with interest.     

A new approach to monoclonal antibody production. In vitro immunization with antigens on nitrocellulose using Drosophila myosin heavy chain as an example

A methodology of broad applicability is described for the production of monoclonal antibodies to antigens with interesting developmental properties using Drosophila myosin heavy chain to exemplify the procedure. The technique consists of two parts. (1) Identification of the antigen with any tool which allows its detection on a Western blot. In the present case, this was achieved by monospecific polyclonal antibodies, prepared as described by Smith and Fisher (J. Cell Biol. (1984) 99, 20), which usefully define the characteristic properties of the antigen. (2) In vitro immunization of splenocytes with antigen on nitrocellulose whose position on Western blots was detected using monospecific polyclonal antibodies, followed by generation of monoclonal antibodies. A total of 19 hybridomas were selected by immunohistochemistry screening and at least six of them were directed against antigens possessing the specific characteristic properties of myosin heavy chain.     

A simplified new method for HLA-DR typing using the TM1 monoclonal antibody

A new modification of an HLA-DR typing technique is described which makes DR typing as rapid and simple as routine HLA-A,B,C typing. In this new method, designated the TM1 technique, carboxyfluoresceindiacetate labeled peripheral blood lymphocytes are added directly to DR typing trays. The T cells are then lysed by addition of TM1, a pan-T cytotoxic IgM monoclonal antibody, and residual B-cell reactivity with cytotoxic DR alloantibodies is read as in routine fluorochromasia microlymphocytotoxicity. HLA-DR typing by the TM1 technique compares favorably to typing by methods using B cells enriched by sheep red blood cell rosetting or by Degalan bead columns. The TM1 technique also works well with cells that have been cryopreserved as well as with cells that have been separated from whole blood drawn as much as 3 days earlier. Finally, because TM1 is so effective in lysing normal T lymphocytes, this antibody may prove useful in functional in vitro and in vivo studies requiring T-cell depletion.     

Murine monoclonal anti-idiotype antibody (alpha) as a probe to detect human monoclonal antibody bound to human tumor tissues

A new immunohistochemical assay was developed for the detection of human monoclonal antibody (HuMAb) bound to human biopsied tumor tissues. A murine anti-idiotype monoclonal antibody, alpha type, 18C6 (IgGl), was raised against an IgM HuMAb, L612, defining a tumor-associated ganglioside antigen (GM3) and used as a probe in a three step cell-binding assay (HuMAb + anti-id + biotinylated anti-mouse Ig). Anti-id 18C6 has an exclusive binding specificity for HuMAb L612, but does not interfere with the binding of L612 to antigen positive melanoma cell lines or to a purified antigen, GM3. The applicability of 18C6 in the three step cell-binding assay was tested first using a melanoma cell line, UCLASO-M12. L612 bound to M12 cells was specifically detected by 18C6 without any background reactivity in ELISA. When this assay was compared with the standard two-step cell-binding assay (HuMAb + peroxidase-conjugated anti-human IgM) using various cultured tumor cell lines, parallel reactivity was observed. The three-step cell-binding assay was then applied to various fresh-frozen human tumor sections. Positive reactivity was demonstrated on various histologic types of human tumor tissues: primary melanoma (10/10), metastatic melanoma (4/4), nevus (10/10), lung cancer (3/6), breast cancer (2/6), and colon cancer (1/1). Adjacent normal tissues were unstained. Control experiments included the cell-binding assay with L612 alone, 18C6 alone. L612 + unrelated mouse IgG, and unrelated IgM HuMAb (L72) + 18C6; but biotinylated anti-mouse IgG did not react with these control preparations. The results indicate that anti-id 18C6 is a highly specific probe to assess the expression of the ganglioside antigenic epitope recognized by the L612 HuMAb on biopsied human tumor tissues.