Membrane associated antigens of human malignant melanoma V: Serological typing of cell lines using antisera from nonhuman primates

Summary Antisera against various melanoma cell lines were raised in nonhuman primates (Cercopithecus aethiop.). After exhaustive absorption with AB Rh + red blood cells and pooled platelets from about 200 donors the sera were still reactive to various degrees in the microimmune adherence test with other melanoma lines, with embryonic fibroblasts, and with non-melanoma lines. As proven by absorption experiments, the main-specificity of the antisera was not directed against components of the fetal calf serum used for cell culture or against mycoplasma grown from commercial fetal calf serum. In addition, no cross-reactivity was observed with Bacillus Calmette-Gérin, and in blocking experiments no reactivity against extracts of common bacterial antigens or mixed molds was detected. Absorption with embryonic fibroblasts or embryonic tissue showed that the reactivity of most antisera was directed against melanoma-associated antigens expressed also on fetal tissue. It was not possible to determine whether the remaining reactivity on some cell lines was melanoma-specific or directed against fetal antigens not contained in the fetal material used for absorption. Cross-absorption of antisera with other melanoma cells revealed that various cell lines express different patterns of tumor-associated antigens with no, or only partial, overlap. The cross-absorption experiments made it possible to type the cell lines according to their surface antigens and arrange the cell lines in order according to the degree of mutual antigenic relationship.     

Serologic analysis of human tumor antigens

Summary The immune adherence (IA) assay was used to measure serum reactivity of patients with melanoma and osteosarcoma against paired cell lines (tumor cells and normal skin fibroblasts obtained from the same individuals) grown in tissue culture. Sera from 224 patients with various stages of melanoma were compared with sera from 100 normal age- and sex-matched donors. None of the 18 stage I sera (0%), 23 of 166 (14%) stage II sera, 3 of 40 (7%) stage III sera, and 3 of 100 (3%) normal sera were highly reactive to a standard allogeneic melanoma-fibroblast pair. None of the sera exhibited unique activity against melanoma. There was no correlation between stage of melanoma and high serum reactivity, nor was this reactivity predictive of recurrence. Sera from 39 tumor-bearing osteosarcoma patients prior to amputation were compared with sera from 50 normal age-and sex-matched donors. Eight of 39 (21%) patient sera and 1 of 50 (2%) normal sera were highly reactive to an osteosarcoma-fibroblast pair. No sera had reactivity uniquely directed against osteosarcoma. Eight osteosarcoma and two melanoma patients were tested simultaneously against their autologous cultured tumor and skin cells. Only one of these patients exhibited high reactivity towards autologous cells, and this reactivity was equal against both osteosarcoma and normal cells. None of seven highly reactive osteosarcoma or six highly reactive melanoma sera had residual tumor-specific reactivity against allogeneic osteosarcoma or melanoma after absorption with cultured fibroblasts, cultured fetal fibroblasts, or fetal calf serum.     

Evaluation of antibody inhibition assays of melanoma antigens in sera to monitor tumor growth in melanoma patients

Summary It was reported previously that melanoma leukocyte-dependent antibody (LDA) in the sera of melanoma patients was inhibited by small-molecular-weight (small-mol.-wt.) glycoproteins which were similar to cell surface antigens identified in cell membrane extracts of melanoma cells. The present study was to determine whether measurement of the levels of these factors in sera may be a useful monitor of tumor growth in melanoma patients. Small-mol.-wt. fractions were obtained by gel filtration or membrane chromatography of acidified sera and tested for their ability to inhibit LDA in ⁵¹Cr release cytotoxic assays. A panel of LDA was used, consisting of three antisera from melanoma patients, which appeared relatively specific for melanoma, and three non-melanoma antisera against carcinoembryonic antigen, ₂ microglobulin, and fetal antigens. The results showed that in patients with melanoma, approximately 70% had melanoma LDA-inhibitory activity detected in the small-mol.-wt. fractions of their sera when these were tested against the panel of melanoma LDA. The specificity of the inhibitory activity for melanoma LDA was shown by failure of the serum fractions to inhibit non-melanoma LDA and by absence of inhibitory activity in equivalent serum fractions from non-melanoma carcinoma patients for melanoma LDA. The levels of melanoma LDA-inhibitory activity in the serum fractions appeared to correlate with tumor growth, as shown by clearance of the inhibitory activity after surgical removal of melanoma and reappearance in the serum of patients who subsequently developed recurrent melanoma. The 30% false-negative rate indicated that the assays could not be used to reliably exclude melanoma, but the close correlation with tumor growth and the low number of false-positive results suggested that in 70% of patients detection of these small-mol.-wt. antigens would be of value to detect recurrence from melanoma and to monitor the effectiveness of therapy.     

Heterogeneity of primary and metastatic human malignant melanoma as detected with monoclonal mntibodies in cryostat sections of biopsies

Summary Monoclonal antibodies were generated against established melanoma cell lines and characterized by their reactivity with various sublines. The antibodies selected for their reaction with melanoma-associated antigens were tested on cryostat sections of melanoma tissue from various stages and on other tumors. The reactivity with normal tissues was also determined. Of 30 antibodies reacting with melanoma cell lines 11 did not react with melanoma biopsies. Of the remaining 19 antibodies nine displayed broad cross-reactivity with normal cells and structures and other benign or malignant tumor cells. Among the remaining antibodies five types were defined that detected antigens (nevocellular I, nevocellular II, neural, endothelial, basal cell) found on certain normal tissues and structures and on certain tumor phenotypes. Even though there seems to be a tendency for some antigens to be preferentially associated with certain stages of melanoma, it has not yet been possible to establish any clear-cut correlation between the expression of one of the differentiation antigens and a particular stage or malignancy potential of melanoma.     

Characterization of human malignant melanoma cell lines. III. Membrane immunofluorescence reactivity with sera from patients with melanoma

Summary Seven well-characterized human malignant melanoma cell lines have been evaluated in terms of their reactivity in membrane immunofluorescence tests with sera from 48 patients with melanoma, 23 patients with other forms of cancer and 28 normal controls. There was a significantly greater degree of reactivity of melanoma sera (33.7%) than of sera of normal controls (22.2%) or of sera from patients with other forms of cancer (24.2%). The incidence of strong reactors among the melanoma patients was found to be inversely proportional to the extent of disease in the melanoma patients: Stage I, 54.5%, Stage III, 36.8% and Stage IV, 29.4%. Reactivity against non-melanoma cell lines was similar in the three subject groups and was unaffected by stage of disease in the melanoma patients. No single cell line showed preferential reactivity with melanoma sera. There was an increased overall incidence of reactivity of all three subject groups against non-pigmented cell lines.A-B-0 antigens and heterophile antigens were excluded as a cause of seropositivity. The antigen(s) was trypsin-sensitive and neuraminidase-resistant.These data suggest that long term cultures of human melanoma may contain melanoma-associated antigens which may be useful in the further study and search for melanoma-specific antigens.     

Delayed-type cutaneous hypersensitivity to melanoma antigens and its implication in active specific immunotherapy in cancer

Summary In this study, we explored whether soluble tumor-cell surface-associated antigens (TAA) might be derived from autochthonous as well as allogeneic sources as immunogens for active specific immunotherapy. Using two popular cell membrane-bound antigen extraction techniques (3 M KCl and isotonic-hypotonic NaCl), we examined the immunogenic potential of such TAA and the specificity of immunologic host reactivity through a delayed-type cutaneous hypersensitivity reaction (DTH) as a guideline for their immunogenic potential in a human malignant melanoma model system. We found that either extraction technique could provide soluble TAA from both autochthonous and allogeneic sources capable of eliciting DTH. While evidence of positive DTH with autochthonous TAA reaffirms the immunogenicity of such TAA, the specificity of host reactivity against TAA derived from allogeneic sources is extremely difficult to establish, even with TAA partially purified by column chromatography in Sephadex G-200. Patients exhibited reactivity to other TAA derived from tumors of different histologies and often to more than one component isolated by column chromatography. Furthermore, when a group of melanoma patients was tested against a panel of melanoma antigens in any random combination, DTH to allogeneic TAA was seen in an unpredictable order and with inconsistent frequency. We conclude, therefore, that while autochthonous antigen immunizations may be justified, more careful studies will be necessary to define the antigenic profile of a given tumor (individual specificity vs shared specificity), establish specificity of alloantigens, and devise suitable methods for testing immunologic specificity for alloantigens, before rational immunotherapy with allogeneic tumor antigens will be feasible.     

Tumour-associated antigens in culture medium of malignant melanoma cell strains

Summary The presence of melanoma-associated antigens naturally shed from cultured melanoma cells in spent culture medium was investigated by means of a leukocyte migration test and culture medium from four melanoma and two control cell strains.Leukocytes from 29/64 melanoma patients showed a positive reaction with spent culture medium from at least one melanoma cell strain, whereas leukocytes from only 4/25 patients with other cancers and 1/30 normal donors reacted. On the other hand, leukocytes from only 8/51 melanoma patients reacted with control culture medium. Only melanoma patients' leukocytes reacted with two or more of the melanoma cell strains used. Culture media from two melanoma cell strains were more reactive (25.3% and 29.4% positive tests with melanoma patients' leukocytes) than others (12.5% and 17.2% positive tests); this may represent either a qualitative difference (i.e., different antigens) or a quantitative one (i.e., different levels of antigen expression according to tissue culture conditions). Both inhibition and stimulation of migration were observed, but with one exception, on a given occasion, leukocytes from the same donor always reacted in the same way (i.e., either inhibition or stimulation). Migration stimulation was observed mainly with melanoma patients' leukocytes, and more especially when leukocytes were sampled from patients within a few weeks from tumour removal; migration stimulation may thus reflect a particular state of sensitization in patients.From the evidence obtained in these studies, it is concluded that spent culture medium from melanoma cell strains contains melanoma-associated antigen (s) that is (are) reactive in the leukocyte migration test and that this may contribute to the study of specific antitumour reactivity in patients and to the study and purification of tumour-associated antigens by providing an homogeneous source of antigens spontaneously released from tumour cells in conditions close to natural ones.     

Effect of glutaraldehyde treatment of human melanoma cells on the expression of melanoma-associated antigens

Summary Human melanoma cells were treated with different concentrations of glutaraldehyde, and retention of serological reactivity with antisera against melanoma-associated antigens, HLA antigen, and 2-microglobulin was assessed by quantitative absorption analysis in mixed hemadsorption microassays. Glutaraldehyde concentrations of 0.025% or greater significantly impaired binding to melanoma cells of antibody against melanoma-associated antigens. At a concentration of 0.0025% antibody binding was not decreased although plating efficiency was reduced to less than 1%. Glutaraldehyde concentrations of 0.25% or greater significantly reduced binding to the same melanoma cells of antisera against HLA antigen and 2-microglobulin. Glutaraldehyde treatment (up to 2.5%) of HT-29 colon carcinoma cells failed to reduce reactivity of antisera against CEA and blood group A isoantigen, which are present on these cells. These studies indicate that the effect of glutaraldehyde treatment of cells on retention of surface antigens is critically dependent on the concentration of glutaraldehyde used and the type of antigens involved. Abbreviations used in this paper: MAA, melanoma-associated antigens; GA, glutaraldehyde; FCS, fetal calf serum; RPMI, Roswell Park Memorial Institute; 2M, 2-microglobulin; CEA, carcinoembryonic antigen; PBS, phosphate-buffered saline; NGP, normal glycoprotein cross-reacting with CEA; SRBC, sheep red blood cells     

In vitro sensitization of human lymphoid cells to antigens on cultured melanoma cells: II. Sensitization against melanoma-associated antigens

Peripheral blood lymphoid cells from patients with malignant melanoma can be sensitized on allogeneic or autochthonous melanoma monolayers. Peak cytotoxicity occurred after 5 days of sensitization. Sensitization appeared to be directed against melanoma-associated antigens, as judged by the pattern of cytotoxic reactivity. Sensitized cells were cytotoxic against autochthonous or allogeneic melanoma cells, but not against autochthonous fibroblasts or allogeneic tumor cells of different histologic types. Sensitization of responder lymphoid cells from melanoma patients on allogeneic melanoma cells usually resulted in more pronounced cytotoxicity against autochthonous melanoma target cells than did sensitization on autochthonous melanoma monolayers. These results indicate that cell cultures of human malignant melanoma contain tumor-associated antigens which can sensitize human peripheral blood lymphoid cells in vitro. These results also support the concept that there are cross-reactive tumor-associated antigens in human malignant melanomas.     

Analysis of sera from breast cancer patients by radioimmunoassays

Summary The antibody reactivity of human breast cancer sera was evaluated by means of radioimmunoassays and established breast cancer cell lines. When tested against the MDA-MB 231 cell line, 30 of 324 sera had detectable antibody reactivity. All the positive sera, however, reacted with other cell lines as well, generally including cultures initiated from sites other than breast cancers, and often including animal cell cultures. In competition radioimmunoassays the positive sera fell into various groups, indicating that a diversity of antigens was being detected. Two patients' sera identified antigens that were expressed on breast cancer cells but that were not expressed on an assortment of other cell types. Sera like these two, which identify potentially important tumor markers, could serve as valuable reagents for the analysis of the tumor-assiciated antigens of human breast cancer cells.     

Serial studies of autologous antibody reactivity to squamous cell carcinoma of the head and neck

Summary In previous studies we evaluated the incidence and specificity of autologous antibody reactivity against squamous cell carcinoma of the head and neck (SCCHN). We were able to demonstrate that autologous antibody reactivity is present in native sera but was usually of too low a titer to allow further analysis. Dissociation of immune complexes by acidification and ultrafiltration of serum augmented autologous antibody reactivity in nine out of nine autologous systems tested. Native antibody and antibody derived from immune complexes produced by the host and reactive with autologous tumor cells may be directed against physiologically relevant antigens. Therefore, correlations of antibody titers with clinical course may provide insight into the nature of the host response to cancer. In the present analysis, serological studies of six patients with SCCHN were performed with serum samples obtained over many months. Results of serial serological assays were correlated to tumor progression and clinical course. Fluctuations in autologous antibody reactivity were noted over time. In four cases, rises in autologous antibody titers preceded the clinical diagnosis of recurrence by several months. Drops in autologous antibody reactivity were noted in two cases following surgery or radiation therapy. In two cases of long-term survivors, no correlation between antibody reactivity and clinical course was noted. Specificity analysis of the six autologous systems demonstrated reactivity against autologous and allogeneic SCCHN as well as melanoma cell lines. These sera did not react with glioma, neuroblastoma, renal cell, breast, bladder and colon carcinoma cell lines nor with fetal calf serum, pooled lymphocytes, red blood cells and platelets. Autologous serial serological studies may provide a means by which to evaluate the host/tumor relationship in patients with SCCHN.     

Bowes human melanoma cell line. An immunocytochemical study

Bowes human melanoma cell line was investigated immunocytochemically to localize S-100 protein, HMB-45 and intermediate filament proteins. The majority of the cells showed strong positive staining with antibodies directed against S-100 protein, HMB-45 and vimentin filaments. Antibodies directed against the other cytoskeletal proteins failed to show any reactivity. These findings suggest that this transformed cell line does not dedifferentiate in culture and continues to express the specific antigens of human melanoma cells. Thus, Bowes cell line provides a useful model for studying the cellular biology and pathology of malignant melanoma.     

Expression of fetal antigens by normal human skin cells grown in tissue culture.

Normal human sera are capable of causing complement-mediated lysis of normal human skin cells grown in tissue culture. This lytic reactivity can be completely removed by absorption with first trimester fetal tissue. Absorption with a variety of normal adult human tissues including lymphocytes, decidua, skin, and muscle are incapable of absorbing reactivity. Absorption of reactivity by fetal tissue is specific and not due to the introduction of anti-complementary or other nonspecific factors, as evidenced by the inability of simultaneous fetal absorption to remove reactivity from antisera with specificity for HLA antigens. Similarly, absorption of lytic sera with fetal calf serum proteins was incapable of removing reactivity against normal cells in tissue culture. It thus appears that normal human cells in tissue culture express antigens shared by the first trimester human fetus, but not present on a variety of adult human tissues. This "neoantigen" present on normal human cells when grown in tissue culture is a potential source of confusion and must be accounted for in searching for human tumor-specific antigens utilizing tissue culture cells.     

Demonstration of the specificity of a monkey antiserum against human melanoma

Summary The reactivity spectrum of a monkey antiserum raised against in vitro-cultured human melanoma cells was compared by means of three different assays: complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and mixed hemadsorption (MHA). After absorption with a pool of cells from T- and B-lymphoid cell lines the antiserum was specifically cytotoxic in CDC for cultured melanoma cells. The melanoma specificity of the antiserum was confirmed by quantitative absorption experiments with cultured melanoma and nonmelanoma cells. When the lymphoid cell-absorbed antiserum was assayed by MHA, however, a high reactivity against both melanoma and nonmelanoma cells was observed. Further absorption with nonmelanoma tumor cells removed the reactivity of the antiserum with different nonmelanoma cell lines, but did not abolish its reactivity with melanoma cells. After this second absorption, the antiserum remained cytolytic against melanoma cells in CDC. In ADCC experiments this antiserum was not able to induce any cytotoxicity even before absorption.Analysis of Sephadex G 200 fractions of the antiserum revealed that the melanoma-specific antibodies cytotoxic in CDC were localized exclusively in the IgM peak. This finding was confirmed in similar studies with four other nonhuman primate melanoma antisera. Abbreviations used in this paper: CDC, complement dependent cytotoxicity: ADCC, antibody dependent cell mediated cytotoxicity; MHA, mixed hemadsorption; FCS, fetal calf serum.     

Trypanosoma cruzi: identification of specific epimastigote antigens by human immune sera

Soluble antigens from epimastigotes of Trypanosoma cruzi were analyzed by western blot in terms of their reactivity with sera from patients with Chagas' disease. In addition, sera from patients with visceral (AVL) and tegumental leishmaniasis (ATL) were also tested in order to identify cross-reactivities with Trypanosoma cruzi antigens. Twenty eight polypeptides with molecular weights ranging from 14 kDa to 113 kDa were identified with sera from Chagas' disease patients. An extensive cross-reactivity was observed when sera from human visceral leishmaniasis were used, while only a slight cross-reaction was observed with sera from tegumental leishmaniasis. On the other hand, 10 polypeptides specifically reacting with sera from Chagas' disease patients were identified. Among them, the antigens with molecular weights of 46 kDa and 25 kDa reacted with all sera tested and may be good candidates for specific immunodiagnosis of Chagas' disease.     

Shared antigens between human malignant melanoma cells and Mycobacterium bovis (BCG).

This study was undertaken to investigate the antigenic relationships between human malignant melanoma cells and Mycobacterium bovis (BCG). Rabbits were immunized with sonicates of BCG or with malignant melanoma cells from different patients and the resulting antisera were tested for their capacity to bind radiolabeled soluble extracts prepared from BCG and melanoma cells. The binding of antibodies to radiolabeled antigens was studied by precipitation of radiolabeled antigen-antibody complexes by anti-rabbit immunoglobulin. Antibodies in sera from rabbits immunized with either BCG (anti-BCG) or melanoma cells (anti-melanoma) bound both the labeled BCG and melanoma antigens. Control antisera, from rabbits immunized with human acute or chronic lymphatic leukemia cells or with normal human spleen cells, did not bind significant amounts of radiolabeled BCG. Antibodies in sera from rabbits immunized with normal spleen cells bound small but significant amounts of radiolabeled melanoma antigens. Binding by anti-BCG and anti-melanoma to the radiolabeled antigens was studied before and after absorption of antisera with cells from human melanoma, leukemia, guinea pig hepatoma, and normal human spleen cells. Inhibition studies using unlabeled BCG extracts also were carried out. The absorption and inhibition studies confirmed that the binding reactions were specific and that antigens from five melanoma patients shared antigenic determinants with BCG.     

Prognostic significance of expression of antigens on melanoma cells

Summary The expression of antigens on 33 human melanoma cells obtained directly from surgically excised tumours was investigated by means of antibody-dependent cell-mediated cytotoxic assays. Antisera used in the study were two antisera from human melanoma patients against different tumour-associated antigens on melanoma cells and antisera against carcinoembryonic antigen (CEA) and ₂ microglobulin (₂M). Considerable heterogeneity was observed in the expression of both melanoma-associated and non-melanoma antigens on melanoma cells from 33 different patients.Patients whose tumours were reactive with the melanoma-associated antiserum (CHI) had a significantly longer remission period to stage 2 melanoma. The period to development of stage 3 melanoma also appeared longer, but this was not statistically significant with the number of patients available for study. The expression of CEA, ₂M, and the tumour-associated antigen TIN was not significantly related to the recurrence-free interval. There appeared to be a reciprocal expression of the two melanoma-associated antigens, and patients with tumours expressing CHI but not TIN had a significantly longer recurrence-free interval than patients whose tumours had the opposite antigenic pattern.In the limited number of patients available for study the expression of the antigen CHI did not appear related to thickness of the primary tumour or to immune response of the patients to melanoma cells in leucocyte-dependent antibody and natural killer cell assays. Although the nature of the association between expression of this antigen and longer remission-free period is unknown these results suggest that the expression of certain melanoma antigens on the cell surface may be an important additional variable which has prognostic and therapeutic importance.     

A 125I-protein A-binding assay detecting antibodies to cell surface antigens

A 125I-protein A-binding assay detecting antibodies to cell surface antigens on human blood cells was developed and evaluated using sera from multitransfused nonleukemic patients sensitized against HLA antigens. The binding assay was found to be reproducible and more sensitive than conventional HLA testing. Seven patients with acute myelogenous leukemia and two patients with acute lymphoblastic leukemia successfully treated by chemotherapy were then investigated. Sera from seven of the patients studied in partial or complete remission demonstrated significant binding to autochthonous leukemic cells obtained from bone marrow or peripheral blood. In two cases sera taken during the leukemic stage demonstrated the most pronounced binding to the patients' own leukemic cells. Sera from four patients with demonstrable significant binding to autochthonous leukemic cells failed to bind to autochthonous remission cells when both types of target cells were tested in parallel. Differences in serum concentrations of IgG, IgA, and IgM were not the cause of the demonstrated increased binding of leukemic sera to autochthonous target cells. We propose that the 125I-protein A-binding assay presented in this paper detects antibodies reacting selectively with acute leukemia cells.     

Serological response of non-human primates to human melanoma disialoganglioside GD3

Summary The immunogenicity of the disialoganglioside, GD3, a melanoma-tumor-associated antigen, has been evaluated in non-human primates. Sera from four chimpanzees and two monkeys were evaluated for anti-GD3 antibody activity by solid-phase radioimmunoassay using GD3 and control gangliosides as targets. Serum from one monkey, immunized with cells from a melanoma cell line, was strongly reactive with GD3, having a titer of >2500. In contrast, serum from this animal was non-reactive with several other gangliosides including the structurally similar GM3. Anti-GD3 reactivity was also demonstrable, albeit in low titer, in the sera of an additional monkey and a chimpanzee. Each of these animals had likewise been immunized using cells from melanoma cell lines. On the basis of these observations, suggestive of a primate anti-GD3 antibody response, we initiated a series of immunizations of chimpanzee using purified GD3 bound to Salmonella minnesota, R595. IgG reactive with melanoma cells in the cell-binding assay was first detected in sera collected after 4 immunizations and increased in titer against each reactive melanoma cell line during the immunizations. Reactivity of this serum with melanoma cell lines demonstrated a direct correlation with the expression of GD3 by the respective cell line. Anti-GD3 reactivity was evident in solid-phase radioimmunoassay against purified GD3 beginning with serum collected after 11 immunizations. By comparison with its binding to the control ganglioside panel, this serum demonstrated strong specificity for GD3 (titer=640) while having only marginal reactivity with GM3 (titer=40). Immune serum from this animal was also able specifically to block subsequent binding of a murine IgM anti-GD3 antibody (DMab7) to target GD3 in solid-phase radioimmunoassay. Together, these observations suggest that GD3, in the form of a purified molecule bound to a bacterial matrix or as part of the intact melanoma cell membrane, can be immunogenic in non-human primates, and is able to elicit an antibody response of appropriate specificity.Supported in part by grant CA32672 from the National Cancer Institute, Veterans Administration Program 821 and by the Yerkes Regional Primate Center, Atlanta, Georgia. The Yerkes Center is fully accredited by the American Association for Accreditation of Laboratory Animal Care     

Detection of phenotypic differences on human malignant melanoma lines and their variant sublines with monoclonal antibodies

The hybridoma technique was used to generate monoclonal antibodies against a wide spectrum of melanoma-associated surface antigens. Mice were immunized against the human melanoma lines Mel A-375, SK Mel-25, and Mel S-5 (subclone of SK Mel-25), which differ with respect to a number of biological and biochemical properties. Spleen cells were fused with P3 X 63-AG8.653 myeloma cells. Twenty hybridomas producing antibodies that were negative on platelets, leukocytes, and monocytes but positive on melanoma cells were isolated and recloned. The specificity of antibodies was investigated on 30 human melanoma and nonmelanoma lines. Five groups of antibodies could be distinguished by their reactivity (1) with few melanoma lines and embryonic fibroblasts; (2) with melanoma, neuroblastoma, and teratoma; (3) with melanoma, neuroblastoma, glioblastoma, teratoma, and carcinoma; (4) with melanoma, teratoma, and carcinoma; and (5) with melanoma, neuroblastoma, teratoma, glioblastoma, carcinoma, embryonic fibroblasts, and B-lymphoblastoid cells. The antigen expression was qualitatively and quantitatively different from cell line to cell line. No evidence for melanoma-specific antigens was found. Eight antibodies were isolated detecting phenotypic differences on sublines of SK Mel-25.