Shared antigens of human prostate cancer cell lines as defined by monoclonal antibodies

Eight monoclonal antibodies (MAbs) raised against human prostate cancer cell lines are described. One MAb was derived from the fusion of mouse myeloma P3x63Ag8-653 cells with spleen cells of mice immunized with DU145 prostate cancer cells. The other seven were from the fusion of myeloma lines P3x63Ag8-653 or SP2/0 with spleen cells of mice immunized with PC3, DU145 and 1013L prostate cancer cells. All of the antibodies also reacted with cell lines of other human cancer types, especially carcinomas. Immunoperoxidase staining on fixed tissue revealed strong reactivity only with antibody PrN10. Seven other antibodies seemed to bind to cell surface-associated (glyco)proteins. Antibodies PrL22 and PrO11 showed similar reactivity in radioimmunoassay, and immunoprecipitated a 160 kD molecular weight polypeptide from [125I]lactoperoxidase-labeled cells. Antibodies PrHk an PrQ12 bound to molecules with apparent MW of 115 kD and 100 kD, respectively; antibodies PrM24 and PrP14 revealed a more complex picture in immunoprecipitation of surface-labeled cells.     

Polyreactivity of human monoclonal antibodies: Human IgM anti-rhesus monoclonal antibodies are frequently polyreactive

The aim of this study was to characterize human anti-Rhesus monoclonal antibodies cross-reacting with tissue antigens. Of the 155 monoclonal alloantibodies tested, 49 also reacted with intracellular antigens, as demonstrated by immunofluorescence assay on cryostat sections of animal and human tissues. This cross-reactivity was mainly a property of monoclonal alloantibodies belonging to the IgM isotype (among the 49 cross-reacting Mabs, 37 were IgM). The results confirm that during an immune response against a foreign antigen (alloantigen), B cells that produce polyreactive antibodies are not excluded from the pool of responding cells.     

Cell surface antigens of L-1210 leukemia recognized by monoclonal antibodies

Conventional antisera to L-1210 leukemia were being prepared in our laboratory for nearly a decade and consistently the only specificity detectable was anti Mammary Leukemia antigen (ML). Serological analysis of five monoclonal antibodies obtained following the same immunization schedule showed more diverse pattern of reactivity. Two antigens detected belong to oncofetal category. The third one is differentiation antigen Ly-6 and the nature of two others, expressed on leukemic cells only, remains at present unclear. Thus none of the clones analysed produces antibodies to ML antigen. Our previous analysis of cell surface antigens of L-1210 leukemia with the use of conventional antisera has already been described. This paper presents the results of applying monoclonal antibodies in a comparable studies.     

Schistosoma mansoni: Analysis of monoclonal antibodies reactive with gut-associated antigens

The analysis of a series of monoclonal antibodies (mAbs) developed in our laboratory against gutassociated antigens ofSchistosoma mansoni is described. It was found that mAbs that recognized epitopes of antigens in the gut and on the eggshell were mainly of the IgM isotype; these epitopes are likely to be carbohydrate in composition. Of a number of mAbs that were reactive with antigens important to the human humoral immune response, 75% appeared to be reactive with the circulating cathodic antigen.     

Production of human monoclonal antibodies against Epstein-Barr virus-specific antigens by the virus-immortalized lymphoblastoid cell lines

The possible production of human monoclonal antibodies against Epstein-Barr virus (EBV) was assessed through the EBV immortalization technique. When individual lymphocyte samples from 50 clinical patients and healthy donors were immortalized by EBV, 4 lymphoblastoid lines yielded antibodies to EBV antigens. These positive lines were cloned and each line yielded cultures that secreted monoclonal antibodies against either viral capsid antigen (VCA) or membrane antigen (MA) component. Above all, a clonal line TAKA-SP-8 produced 5 micrograms MA antibody/10(6) cells/ml for more than 12 months. The culture fluid specifically immunoprecipitated a single polypeptide with a size of 93K from both P3HR-1 and B95-8 cell extracts. FUKA-SP-3, on the other hand, secreted 5 micrograms VCA antibody/10(6) cells/ml for at least 8 months. This antibody recognized two polypeptides with sizes of 123K and 120K, from P3HR-1 and B95-8 cell extracts, respectively. When B95-8 and P3HR-1 EBV were treated with the human MA monoclonal, both nuclear antigen (EBNA) synthesis and early antigen (EA) induction were strongly inhibited. All EBV antibody-producing cultures were exclusively achieved from splenic lymphocytes of patients with autoimmune diseases, but not from other donors.     

Murine monoclonal antibodies reactive with human heart and group A streptococcal membrane antigens.

Ten selected murine hybridoma cell lines that produce monoclonal antibodies against M type 5 Streptococcus pyogenes and human heart antigen were isolated. All of the monoclonal antibodies studied were determined to be the immunoglobulin M isotype. The antibodies were characterized on the basis of their reactions with Triton X-100-extracted whole human heart antigens, sodium dodecyl sulfate-extracted sarcolemmal antigens, and whole streptococci or their membranes. Enzyme-linked immunosorbent assays and Western immunoblotting techniques were used to compare the reactivity of the monoclonal antibodies. All 10 of the antibodies were first selected for their reactivity with Triton X-100-extracted heart antigens and whole group A, M type 5 streptococci. These antibodies were then divided into two categories: strong reactors or weak reactors with human sarcolemmal and streptococcal membranes. Among the strong reactors, two different types of monoclonal antibodies were observed on the basis of their immunobanding patterns with sarcolemmal and streptococcal membranes on Western blots. Monoclonal antibodies that were strong reactors with sarcolemmal and group A streptococcal membrane antigen were directed against a determinant on a family of proteins. The major reactants of sarcolemmal extracts were high-molecular-weight proteins near 200,000. Some monoclonal antibodies demonstrated more specificity for the heart than did others when reacted with separated Triton X-100-extracted tissue antigens from the heart, kidney, and skeletal muscle. One of the monoclonal antibodies that reacted with group A streptococci reacted with a Triton X-100-extracted heart antigen ca. 40,000 daltons in size. None of these monoclonal antibodies opsonized type 5 Streptococcus pyogenes, and in enzyme-linked immunosorbent assays most of the antibodies were found to react to a lesser degree with other groups of streptococci. Monoclonal antibody was used to probe normal and rheumatic sarcolemma for differences in reactivity. Although the rheumatic heart reacted more intensely, no major differences between the immunobanding patterns of normal and rheumatic hearts were observed.     

Immunohistochemical analysis of human lymphomas with monoclonal antibodies to B cell and Ia antigens reactive in paraffin sections

Monoclonal antibodies (MoAbs) to B cell- and T cell-specific antigens uniformly have been restricted in their use to cell suspension or frozen section techniques because the antigens that they identify are either masked or lost in the fixation or paraffin-embedding processes. Antiimmunoglobulin antisera, although readily identifying cytoplasmic immunoglobulin in paraffin sections, has not been as useful as originally hoped since the majority of B cell lymphomas express surface immunoglobulins, which requires cell suspensions or frozen sections for detection. Because cell suspension procedures disrupt tissue architecture and frozen section techniques grossly distort morphology, neither method allows the combination of optimal morphologic and immunologic classification of lymphomas. We recently reported two MoAbs, LN-1 and LN-2, that react with B cells in paraffin sections. LN-1 reacts with the surface membrane and cytoplasm of germinal center B cells. LN-2 reacts uniquely with the nuclear membrane and cytoplasm of mantle zone and germinal center B cells and interdigitating histiocytes. We have also identified a new MoAb, LN-3, that reacts with the HLA-DR antigen in paraffin sections. We now report the use of LN-1, LN-2, and LN-3 in the analysis of paraffin sections from 58 non-Hodgkin's lymphomas and 15 cases of Hodgkin's disease. The types of cells reactive with these MoAbs in neoplastic lymphoid proliferations largely recapitulate their benign morphologic and immunologic counterparts. As a panel, LN-1, LN-2, and LN-3 were reactive with 98% of B cell lymphomas, and LN-1 and LN-2 were negative on all T cell lymphomas. In addition to identifying the cell of origin of these malignant proliferations, these MoAbs were also useful for identifying architectural features in neoplastic lymph nodes. Thus, these reagents provide the ability to assess the immunologic phenotype of neoplastic lymphocytes in conjunction with the critical morphologic criteria requiring paraffin embedding.     

Human leukocyte antigens (HLA) class I (Bg) on red cells studied with monoclonal antibodies

Monoclonal antibodies, capable of detecting monomorphic epitopes on HLA class I polypeptides and beta-microglobulin (Beta2-M), have been used by a variety of techniques to ascertain the type of structure detected on red blood cells (RBCs). Hemgglutination with class I monoclonal antibodies confirmed the reported relationship between Bg blood groups and HLA. It also established that the expression of HLA on RBCs which do not have nuclei is not normally strong, but may be enhanced in patients, notably those with systemic lupus erythematosus (SLE). Estimates of the number of class I molecules on mature RBCs by a radioligand-binding assay have confirmed that all HLA-B7 (Bga) individuals have higher numbers but that SLE patients usually have the most (124/RBC). Class I polypeptides were not elevated in the plasma of SLE patients and all RBCs lost molecules on aging in the circulation. These two facts suggest that HLA on RBCs is not acquired from plasma. when RBCs from SLE patients were immunoblotted with monoclonal antibodies, a complete 45 kDa intrinsic transmembrane heavy chain of HLA class I and a light chain of 11 kDa (Beta2-M) were detected. Chloroquine treatment and acid elution of RBCs did not remove HLA class I but only Beta2-M, As most antibodies recognize epitopes that depend on close association of class I with Beta2-M, the lost reactivity of treated RBCs may be understood.     

Complement-dependent cytotoxicity of antibodies reactive with HIV-induced cell surface antigens in HIV-carrying haemophiliacs

Sera obtained from HIV-infected as well as uninfected haemophiliacs and from healthy subjects were investigated for the presence of lymphocytotoxic antibodies. Using the 51Cr-release test, HIV-infected haemophiliacs were found to produce serum antibodies exerting complement-dependent cytotoxic effect on HIV-infected T4 cells. The antibodies were reactive mainly when HIV-infected target cells were stimulated with concanavalin-A. Results of complement-dependent antibody cytotoxicity and indirect membrane immunofluorescence tests suggest that envelope antigen(s) of HIV may be the target(s) for cytotoxic antibodies.     

Moloney leukemia virus-induced cell surface antigen mimicry by monoclonal antibodies

We have investigated antigen-independent modulation of immune responses by monoclonal antibodies directed against both viral and nonviral antigens. BALB/c mice were immunized with monoclonal IgM (i.e. Ab1) specific for either Moloney murine leukemia virus-induced cell surface antigen (MCSA) or the hapten 2,4-dinitrophenyl (DNP). Injection with either Ab1 activated a functional idiotypic (Id) network as evidenced by production of both anti-Id (Ab2) antibodies and anti-anti-Id (Ab3) antibodies. A subset of induced Ab3 (designated Ab1), exhibited specificity for antigen (virus or DNP). In mice immunized with anti-Id antibodies (Ab2), production of Ab3 and Ab1′ was also observed. In the MCSA system, antibody-induced Ab1′ responses were effective in protecting mice from tumor development upon subsequent challenge with live virus. Furthermore, antigen-independent modulation of immunity to both viral and nonviral antigens was found to be thymus-dependent. Similar findings in other viral systems suggest that antibody-induced activation of Id networks may prove a viable alternative vaccine strategy that can elicit antigen-specific responses, and in some cases protection, in the apparent absence of exposure to antigen.     

Characterization of cell surface antigens of human mammary epithelial cells with monoclonal antibodies prepared against human milk fat globule

Hybridomas have been prepared that secrete monoclonal antibodies against three different surface antigens of normal human mammary epithelial cells by fusion of mouse myeloma cells with spleen cells from mice and rats immunized with delipidated human milk fat globules. Using a novel method for molecular weight determination, the three different monoclonal antibodies, BLMRL-HMFG-Mc3, BLMRL-HMFG-McR2, and BLMRLHMFG-Mc5, were found to identify molecules with apparent molecular weights of 46,000, 70,000, and 400,000 daltons, respectively. The latter is a mucin-like glycoprotein with a high sugar content and has not previously been described as a component of the human milk fat globule or of human mammary epithelial cell membranes. Single-cell quantitation of binding of monoclonal BLMRL-HMFG-Mc5 to three breast tumor cell lines using a Microscope Spectrum Analyzer and indirect immunofluorescence revealed a heterogeneous expression. Further, using a competitive radioimmunoassay, it was found that breast tumor cell lines differed by at least 10-fold in the 400,000-molecular-weight antigen content. None of the three antigens are detectable on several nonbreast cell lines, including normal breast fibroblasts.     

Equilibrium binding characteristics of monoclonal antibodies recognizing melanoma cell surface antigens

The equilibrium binding characteristics of a panel of six monoclonal antibodies (MAb) recognizing melanoma cell surface antigens (125 kdal cell surface melanoma associated glycoprotein antigen, 125kD-MAA; high molecular weight melanoma associated antigen, HMW-MAA; and a non-protein melanoma associated antigen, NP-MAA) were investigated using the cell lines SK-MEL-2, SK-MEL-5, and M21. The MAbs displayed equilibrium association constant (K) values ranging from 10(7) M-1 to 10(10) M-1 and maximum MAb binding values (Qmax) from 2 x 10(4) to 2 x 10(6) MAb molecules bound per cell. High trypsin concentrations were shown to have deleterious effects on Qmax values obtained for antibodies recognizing the 125kD-MAA, and even low trypsin concentrations affected Qmax values obtained for MAbs recognizing the HMW-MAA (although a complete linear recovery of HMW-MAA antigen was observed in 20-25 hours). Significant changes in Qmax were also noted for different cell passages. Except for MAb 43.2, little variation in K was observed when different cell lines were used. Linear Scatchard plots were obtained for all MAbs except 43.2 in which case concave down behavior was observed suggesting the existence of positive cooperativity between the binding sites of this MAb.     

Human myeloid differentiation antigens identified by monoclonal antibodies to the myelomonocytic leukemia cell line RC-2A

Murine monoclonal antibodies to human myeloid cell surface differentiation antigens were prepared using the myelomonocytic leukemia cell line RC-2A as immunogen. Using a highly sensitive colorimetric assay, antibodies were selected as myeloid-associated based on their binding to RC-2A cells, but not to cells of the autologous EBV-transformed* B cell line Cess-B. Antibodies to five distinct cell surface antigens were extensively characterized for their binding to normal and leukemic hemopoietic cells, and to tissue sections. Three antibodies may identify antigens previously described in the International Leucocyte Typing Workshops (CD14, CD11b and CD31). The other two antigens appear to be expressed at low levels on the surface of RC-2A cells, and do not correspond to existing CD groups. One of these is also present on monocytes and neutrophils. Both were present on myeloid progenitor cells, as judged by depletion experiments with antibody and complement, although neither bound appreciably to myeloid leukemic cells as judged by indirect immunofluorescence. The other three antibodies bound preferentially to leukemic specimens displaying monocytic differentiation. Four of the antibodies could be demonstrated to bind to cells in frozen sections of tonsil and small intestine and all gave distinct patterns of reactivity. In particular, these antibodies differed markedly in their binding to endothelium, follicular dendritic cells and various types of tissue macrophages. These antibodies may be useful in the study of the differentiation of myeloid cells and in studies of immunologically mediated disease such as allograft rejection.     

Internalization and processing of antibodies to surface antigens on human B cells. Monoclonal anti-IgM antibodies are processed differently than monoclonal antibodies towards non-Ig surface receptors

The internalization and intracellular processing of monoclonal antibody to immunoglobulin mu heavy chain (Mamu) have been investigated in two human Burkitt lymphoma cell lines (Ramos and Raji), in a human B cell lymphoma and in normal human peripheral blood B cells. In addition to the degradation of 125I-labeled Mamu to trichloroacetic acid (TCA) soluble material, a distinct pattern of larger 125I-Mamu fragments was detected in all sources of B cells tested. The particular fragmentation pattern, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis, involved the cleavage of both peptide bonds and disulfide bridges. This type of antibody fragmentation appeared to be a selective mechanism associated with sIgM, as no other degradation than that leading to TCA-soluble material could be detected after the internalization and degradation of radiolabeled monoclonal antibodies towards a variety of non-Ig B cell surface receptors. Three fragments of 125I-Mamu degradation were also detected in the supernatant of Ramos cells, implying that the recycling and exocytosis of certain 125I-Mamu fragments also took place.     

Acquired heterophile antigens on the surface of human cell lines.

Chicken IgM antibodies raised against sheep erythrocytes (SE) reacted with a heterophile carbohydrate antigen designated as "HC". This antigen was visualized by SE rosette formation with antibody-sensitized and formaldehyde-fixed target cells. Cells from eight lymphoblastoid lines and ten lines of diverse histogenetic origin all expressed HC on their surface to a greater or lesser degree when grown in fetal calf serum (FCS). Lymphoblastoid cell lines, HSB2 and Namalva, lost most of their HC antigen after three cell divisions when grown in medium which contained normal human serum (NHS) instead of FCS. The acquired origin of HC was demonstrated by the conversion of HSB2-HC- into HSB2-HC+ cells after incubation in the presence of FCS or fetuin for 18 h at 37 degrees C. The acquisition of HC antigen depended on the concentration of fetuin as well as the time and temperature of incubation. The presence of NHS in the culture medium reduced the degree of HC incorporation. The level of HC expressed on the cell surface was reduced by treating HSB2 cells with sodium periodate but not by proteolytic enzymes. Competitive inhibition with hog blood group substance and sheep IgG suggested similar specificity of HC on target cells which had been sensitized with either fetuin or sheep IgM (Eur. J. Immunol. 1977.7:204). The avidity of chicken antibodies to HC in either of these systems was several orders of magnitude lower than the binding to SE.     

A neomycin-resistant cell line for improved production of monoclonal antibodies to cell surface antigens

Overgrowth of hybridomas by proliferating spleen T cells often hinders the production of monoclonal antibodies to certain antigens. To overcome this problem we derived neomycin-resistant fusion partner SP2 neoR.1 by transfection of SP2/0-Ag14 cells with plasmid pSVTK neo beta. Hybridomas obtained with SP2 neoR.1 grew optimally in the presence of the neomycin analog G418 at concentrations which blocked the proliferative response of T cells to mitogenic stimuli. The advantage of using SP2 neoR.1 and G418 under conditions where spleen cell proliferation occurs after fusion was demonstrated with hybridomas derived from a rat immunized with mouse helper T cell lines.     

Immunodiagnosis of human cysticercosis (Taenia solium) with antigens purified by monoclonal antibodies.

Monoclonal antibodies were generated from mice immunized with scolex protein antigen of Cysticercus cellulosae. Three monoclonal antibodies specific for cysticercal antigens, which did not show any cross-reactivity with Taenia solium or Taenia saginata antigens, were selected. Each monoclonal antibody coupled to Sepharose could purify one antigen, which appeared as a single band on polyacrylamide gel electrophoresis. When antigens purified by monoclonal antibodies were used to detect antibody in serum samples taken from patients with cysticercosis, taeniasis, and other parasitic infections in an enzyme-linked immunosorbent assay, cross-reactivity was observed until a serum dilution of 1:128 was reached. Since serum samples from unexposed subjects showed positive reactions until a dilution of 1:64 was reached, we chose a discriminative dilution (1:128) above which no cross-reaction was observed. The percent positive serum samples from cysticercosis patients was 100% by the enzyme-linked immunosorbent assay with any of the antigens purified by monoclonal antibodies.     

Generation of monoclonal antibodies to human lymphocyte cell surface antigens using insect cells expressing recombinant proteins.

We have expressed human CD40 and human B7 in insect cells using the baculovirus expression system and have used these insect cells to immunize mice for the generation of monoclonal antibodies. We demonstrate here that specific monoclonal antibodies to human CD40 and human B7 were obtained using this approach. One significant advantage of this method is that immunizing mice with insect cells did not evoke an immune response to human cells and, therefore, EBV-transformed human B cells could be used to screen for specific antibody production by the hybridoma clones.     

分泌人源性单克隆抗体永生化淋巴细胞系的构建

目的;构建分泌人源性单允隆抗体（ＭｃＡｂ）的永生化Ｂ淋巴细胞系。方法：用正丁醇法提取的人卵巢癌细胞系ａｏｌ１０／１７抗原在含免疫活化剂的无血清培基中免疫人淋巴细胞,将制备ａｏ １０／１７细胞ＤＮＡ转染经体外免疫的淋巴细胞。采用ＥＬＩＳＡ筛选和有限稀释法克隆化。