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R A Reisfeld

Publications and source records attributed to R A Reisfeld.

At least 181 records · Page 10Linked to original sources
PubMed1981

Production and characterization of monoclonal antibody to a melanoma specific glycoprotein.

An immunogen consisting of a 4M urea extract derived from human melanoma cells (M14), that was devoid of HLA-A,B,C, HLA-DR antigens and fibronectin was adsorbed to lens culinaris lectin-Sepharose 4B and used to immunize mice for production of monoclonal antibody to a melanoma-specific glycoprotein. Screening for hybridomas secreting antibodies to melanoma associated antigens was facilitated by use of a solid phase target antigen of chemically defined medium of melanoma cells (CDM). Use of these procedures allowed us to select 40 hybridomas secreting antibody which recognized determinants on melanoma cells not found on lymphoid cells. Further characterization of one of these hybridomas, 9.2.27, indicated that the antibody it secreted recognized a 240K dalton glycoprotein found on all melanoma cell lines tested but not on carcinoma, lymphoid, or fibroblastoid cultures. These results demonstrate the utility of soluble antigen preparations devoid of strongly immunogenic non tumor-specific molecules in the elicitation of tumor specific antibody. Preliminary results suggest that immunogens of this kind are superior to intact melanoma cells for production of tumor specific hybridomas.

Animals↗
PubMed1981

Cellular and molecular heterogeneity of H-2Kk antigens.

Up to 70% of mouse spleen cells expressing H-2Kk alloantigens reacted with the monoclonal anti-H-2Kk antibody 11-4.1 in an indirect rosetting assay. When the cells that rosetted with the monoclonal antibody 11-4.1 were removed by density centrifugation, the residual unreactive cells reacted with the polyclonal anti-H-2Kk alloantiserum D23 but not with the monoclonal antibody 11-4.1 indicating the existence of two cel populations. In sequential immunoprecipitation experiments, the glycoprotein fraction of NP40 extracts from H-2Kk cells, after removal of antigens reacting with the monoclonal antibody 11-4.1 still contained structures reactive with the alloantiserum. Therefore, there are at least two antigenically distinct H-2Kk molecules which are differentially expressed on two subpopulations of spleen cells.

Animals↗
PubMed1981

Human melanoma-associated antigens: role of carbohydrate in shedding and cell surface expression.

The shedding of 2 tumor-specific glycoprotein antigens from human melanoma cells into spent culture medium was selectively inhibited by nontoxic doses (0.5 microgram/ml) of tunicamycin, an inhibitor of N-asparagine-linked glycosylation. The inhibition of shedding of these 2 antigens with m.w. of 240K and 94K is complete within 24 hr after addition of tunicamycin. During this time interval, our data suggest that these glycosylated cell surface antigens are replaced by their nonglycosylated forms. Removal of tunicamycin or addition of N-acetyl glucosamine restores shedding of these melanoma-associated antigens with initially reduced glycosylation. This same selective inhibition of shedding was observed with cultures adapted to grow in high doses (2.5 microgram/ml) of tunicamycin that otherwise killed > 98% of the cells upon first exposure. In contrast to other glycoproteins found in spent culture medium of melanoma cells, the shedding of melanoma-associated antigens is strictly dependent on glycosylation.

Animals↗
PubMed1981

Tumor-associated antigens in spent medium of human melanoma cells: immunochemical characterization with xenoantisera.

Xenoantisera to human melanoma cells and to partially purified melanoma-associated antigens were coupled to protein A-bearing Staphylococcus Aureus or protein A-Sepharose and used as immunoadsorbents for the indirect immunoprecipitation of intrinsically radiolabeled proteins released into culture medium from various cultured human tumor and nontumor cell lines. These radiolabeled immunoprecipitates when analyzed by SDS-PAGE revealed highly reproducible molecular profiles of proteins and glycoproteins released by various cultured tumor lines and control cells into their spent culture media. A comparison of molecular profiles together with data indicating the binding specificity of known xenoantisera produced against human melanoma cells or their extracts led to the discovery of 2 macromolecules that are associated with human melanoma cells; a glycoprotein with a subunit m.w. of 240,000 (240K) and a single-chain glycoprotein of 94,000 (94K) m.w. also found in association with human carcinoma cells are described in this report.

Animals↗
PubMed1981

Molecular profiles of human melanoma-associated antigens.

Melanoma-associated antigens (MAA) shed into spent culture medium of intrinsically radiolabeled melanoma cells react specifically with monoclonal and polyclonal anti-melanoma xenoantiserums and are represented by two glycoproteins with molecular weights of 240,000 (240K) and 94,000 (94K): 240K is present only on melanoma cells whereas 94k is also found on carcinoma cells and on fetal melanocytes. Both 240K and 94K have been obtained radiochemically pure by utilizing cellulose ion-exchange and antibody affinity chromatography. The two antigens have different charge properties, as 240K binds to CM-cellulose while 94K does not. A difference in carbohydrate moieties is also indicated since 240K binds selectively to lentil lectin and 94K to ricin lectin. Two-dimensional gel electrophoresis an tryptic peptide maps of the two antigens reveal distinct and characteristics profiles. Subunit structure determination of both antigens suggests 94K to be a single chain whereas 240K appears to be a subunit of a larger molecular structure.

Antibodies, Neoplasm↗
PubMed1980

Human melanoma associated antigens: a solid-phase assay for detection of specific antibody.

A solid-phase radioimmunometric binding assay is described utilizing 125I-labeled protein A for the detection of antibody to human melanoma associated antigens. The novel aspect of this assay is the use of chemically defined spent culture medium of melanoma cells at target antigens previously depleted of fibronectin by affinity chromatography. This makes it possible to screen for antibody in unabsorbed antiserum. Sensitivity, reproducibility and ease of performance of the assay are optimized by conjugating target antigens to a background of bovine serum albumin dried onto polyvinyl 96-well microtiter plates and cross-linked with glutaraldehyde. The use of an immobilized soluble antigen target derived from a large pool of spent culture medium facilitates direct interassay comparisons and permits extensive absorption analysis of antisera. The assay has considerable potential in screening for alloantibody and both poly- and monoclonal xeonoantibody to human melanoma associated antigens.

Animals↗
PubMed1980

Lack of association of serologically detectable human melanoma-associated antigens with beta 2 microglobulin: serologic and immunochemical evidence.

Serologic and immunochemical assays showed that human melanoma-associated antigens (MAA) identified with operationally specific xenoantisera were neither spatially nor structurally associated with beta 2-microglobulin (beta 2-mu), the light chain of the HLA-A,B antigen molecular complex; i.e., cultured melanoma cells coated with a specific anti-beta 2-mu xenoantiserum maintained their reactivity with anti-MAA xenoantisera. Furthermore, soluble MAA were not bound by a beta 2-mu immunoadsorbent. Finally, MAA were shed into the culture medium of melanoma cells and then were immunoprecipitated with specific anti-MAA xenoantisera, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, they appeared as two distinct structures with molecular weights of 240,000 and 94,000 but comprised no structure with the characteristic 12,000 molecular weight of beta 2-mu. Conversely immunoprecipitates obtained by the reaction of spent culture medium of [3H]valine-labeled melanoma cells with anti-beta 2-mu xenoantiserum had the 12,000-molecular-weight component but no structures with the molecular weights established for MAA. Thus the data refute the contention that serologically detectable MAA have a molecular structure similar to that of HLA antigens.

Antibodies, Neoplasm↗
PubMed1979

Antibody-coated protein A-bearing Staphylococcus aureus: a versatile and stable immune reagent.

The human beta2-microglobulin antigen-antibody system was used as a model to illustrate the versatility of a microradioimmunoassay technique using protein A-bearing Staphylococcus aureus Cowan I strain (SACI) bacteria as a non-specific immunoadsorbent in place of a second antibody. Experimental conditions are described for a sensitive microassay which makes it possible to process large numbers of samples more rapidly and with minimum handling. Furthermore, SACI coated with specific antibodies by mixing with unfractionated antisera are a versatile reagent not only for radioimmunoassays but also for use as molecular probes to characterize cell surface antigens. Antibody-coated SACI could be lyophilized and proved extremely stable in storage thus providing a unique advantage for use in binding inhibition assays and as versatile reagent for clinical and investigative immunology.

Animals↗
PubMed1979

A radioimmunometric antibody-binding assay for evaluation of xenoantisera to melanoma-associated antigens.

A radioimmunometric antibody-binding assay was developed with the use of 125I-labeled protein A of Staphylococcus aureus (SpA) for the evaluation of xenoantisera to human melanoma-associated antigens. Antisera were produced in New Zealand male albino rabbits by the injection of cultured human melanoma cells or soluble, partially purified melanoma-associated antigens isolated from these cells. Xenoantisera were rendered operationally specific for melanoma-associated antigens by absorption with human red cells and cultured lymphoblasts. The methodologic parameters and the quantitative relationships among xenoantisera, cultured melanoma target cells, and 125I-labeled SpA and their effect on the measurement of xenoantibody binding were critically evaluated. Data indicated the usefulness of the radioimmunometric assay in monitoring the efficacy of absorption and in characterizing the specificity of xenoantisera to melanoma-associated antigens. The radioimmunometric binding assay when modified and used as a binding inhibition assay was effective in the assessment of the serologic activity of soluble melanoma-associated antigens and thus may be used to monitor the progress of antigen purification.

Animals↗
PubMed1979

Expression of a sheep red blood cell receptor by a murine lymphoma.

Murine 6C3HED lymphoma cells were found to rosette with sheep red blood cells (SRBC). Normal C3H lymphocytes did not exhibit this property. This rosetting capacity of 6C3HED cells was found to be an accurate and reproducible means for discriminating between normal and tumor cells. The SRBC receptor on these lymphoma cells appeared to be serologically distinct from that expressed by normal human T lymphocytes since reciprocal blocking experiments demonstrated that inhibitory antisera were not cross-reactive. The expression of the SRBC receptor by the 6C3HED cells appeared to correlate with the expression of a tumor-associated antigen and was spatially related to an antigen expressed by 6C3HED and normal neonatal but not adult mouse cells.

Animals↗
PubMed1979

Isolation and immunochemical characterization of antibodies from the sera of cancer patients which are reactive against human melanoma cell membranes by affinity chromatography.

Immunoglobulins were isolated by affinity chromatography from sera of two patients with melanoma, one with sarcoma, and one with carcinoma. The affinity columns were prepared by covalently linking the membrane-rich fraction of biopsied melanoma cells to cyanogen bromide-activated agarose beads. The membrane-rich fractions were prepared by two methods: (a) hypotonic cell lysis, and (b) homogenization and differential centrifugation. Melanoma sera were autologous to melanoma membrane preparations. The isolated immunoglobulins showed immunoreactivity against antigens prepared from melanoma, sarcoma, and carcinoma cells by complement fixation but not against antigens prepared from normal human liver and lung tissues. Absorption of the isolated immunoglobulins with rabbit anti-human immunoglobulin immunobeads resulted in complete elimination of the complement-fixing antibody titer in one instance, whereas reduction occurred in other samples. Similar absorption with rabbit anti-human immunoglobulin M immunobeads resulted in reduction, but not complete elimination, of the antibody titers against target tumor cell preparations. These results suggest the presence of immunoreactive immunoglobulin G in all immunoglobulins and immunoglobulin M in some. Absorption of the isolated immunoglobulins with cultured sarcoma cells reduced but did not completely abolish antibody activity against autologous or allogeneic melanoma target antigen, whereas it did completely abolish activity against sarcoma target antigen. However, absorption with cultured allogeneic melanoma cells abolished the antibody activity against melanoma as well as sarcoma target antigens. The antibody titers of the isolated immunoglobulins were not affected by absorption with cultured lymphoblastoid cells. Since cultured melanoma and sarcoma cells were known to contain oncofetal antigen(s), these results suggest that the isolated immunoglobulins from cancer sera by melanoma membrane affinity chromatography were of at least two specificities: (a) antioncofetal; and (b) antitumor associated. The former group may be comprised of antibody to cross-reactive antigens associated with different histological types of tumors. However, it was apparent that a portion of the antibody activity was against common tumor-associated antigen(s). These results provide further evidence for the presence of common antigen(s) associated with biopsy specimens of human malignant melanoma.

Adult↗
PubMed1979

Physical association of histocompatibility antigens and tumor-associated antigens on the surface of murine lymphoma cells.

Serologic and immunochemical techniques were used to characterize plasma membrane antigens of the murine lymphoma 6C3HED. Syngeneic C3H/HeJ anti-6C3HED antisera were prepared and were shown by cytotoxicity, absorption, and immunoprecipitation tests to be specifically reactive with the lymphoma cells. Alloantisera to H-2.23 were found to be unreactive with intact lymphoma cells by cytotoxicity and absorption assays. However, H-2.23 antigens were detectable in tumor cell lysates by inhibition of cytotoxicity and immunoprecipitation. SDS-PAGE electropherograms of immunoprecipitates of radiolabeled tumor cell extracts obtained with anti-6C3HED and anti-H2.23 antisera were essentially identical; proteins with Mr of approximately 70,000, 45,000, and 12,000 were apparent in both. An immuno-adsorbent of insolubilized anti-6C3HED removed H-2.23 antigens detectable in lymphoma extracts, but had no effect on H-2.23 antigens in normal splenocyte extracts. These data indicate that H-2.23 and TAA are physically associated on 6C3HED cells.

Animals↗
PubMed1978

Murine Ia and human DR antigens: homology of amino-terminal sequences.

Murine Ia and human DR antigens were isolated and purified by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis with allo- and xenoantisera, respectively. The I-A subregion antigen consists of two chains, designated Aalpha and Abeta, with molecular weights of 35,000 and 26,000, respectively. The I-C subregion antigen likewise consists of two chains, designated Calpha and Cbeta, with molecular weights of 32,000 and 29,000, respectively. Under nonreducing conditions, the Cbeta chain migrates appreciably more rapidly on sodium dodecyl sulfate/polyacrylamide gels than the reduced Cbeta chain, reflecting the presence of an intrachain disulfide bond. The human DR antigen is also a two-chain unit and contains DRalpha and DRbeta components with molecular weights of 34,000 and 28,000, respectively. The DRbeta chain migrates more rapidly before reduction than afterward, like the murine Cbeta chain. The DRbeta and Cbeta chains are also strikingly homologous if a single amino acid shift is imposed on one of those chains. Thus, human DR antigens strongly resemble the murine I-C subregion antigens.

Amino Acid Sequence↗
PubMed1978

Continuous production of carcinoembryonic antigen in hollow fiber cell culture units: brief communication.

A line of cultured cells derived from a primary human adenocarcinoma of the colon was grown in the extracapillary spaces of hollow fiber tissue culture units in the absence of serum components of high molecular weight (greater than 10,000). The cells were capable of producing more than 30 microgram of carcinoembryonic antigen (CEA) per day, provided that the extracapillary fluid was changed frequently. The concanavalin-A binding and molecular sieve chromatography properties of the tissue culture-derived CEA were similar to those of CEA isolated from metastatic colon cancer tissue.

Adenocarcinoma↗
PubMed1978

Purification and immunologic evaluation of human melnoma-associated antigens.

Melanoma-associated antigens (MAA) were isolated and their functional immunologic properties were evaluated. Spent fetal calf serum-free culture media and 3-m KCI extracts of cultured human melanoma cells grown in this medium were used as antigen sources. Ultracentrifugal flotation on KBr was used to separate MAA and HLA antigens present in the extracts or spent culture media; thus interference by histocompatibility antigens was prevented in subsequent tests of tumor antigenic activity. MAA purified in this manner retained their immunologic functions as evidenced by their ability to produce delayed cutaneous hypersensitivity reactions in patients with melanoma, specifically combine with antimelanoma xenoantibody, and elicit production of functionally specific xenoantibody. Possible structural differences between HLA antigens and MAA were considered in evaluation of the data.

Animals↗