Indirect Rosette Microassay to Characterize Human Melanoma-associated Antigens Recognized by Operationally Specific Xenoantisera Kohzoh Imai and Soldano Ferrone Cancer Res 1980;40:2252-2256. Published online July 1, 1980.
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[CANCER RESEARCH 40. 2252-2256, 0008-5472/80/0040-OOOOS02.00
July 1980]
Indirect Rosette Microassay to Characterize Human Melanoma-associated Antigens Recognized by Operationally Specific Xenoantisera1 Kohzoh Imai2 and Soldano Ferrone3 Department of Molecular Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037
ABSTRACT
MATERIALS
The indirect rosette assay which detects the interaction of antibodies with target cells by their ability to rosette with sheep red blood cells chemically coated with purified anti-immuno-
Cultured Cells and Murine Fetal Cells. Human melanoma (BW5, M10, M14, M21, Colo 38, M51 ), carcinoma (D98, HT29, MANO), sarcoma (S1), fetal fibroblast(IMR90), lymphoblastoid cell lines (Molt 4, 1301, Raji, RPMI 1788, RPMI 4098, RPMI 6410, RPMI 8866, Victor, WI-L2), and murine melanoma cell line S91 were grown in Roswell Park Memorial Institute Medium 1640 with 10% fetal calf serum. These cell lines have been described elsewhere (12). The identities of human cell lines were periodically confirmed by HLA typing. Polyclonal Xenoantisera to MAA and Cell Surface Markers and Monoclonal Antibodies to HLA Antigens. Four types of xenoantisera to MAA were used in this study. One (No. 6522) was from a rabbit immunized with cultured melanoma cells mixed with a xenoantiserum to cultured human lymphoid cells. After extensive absorption with cultured human lymphoid cells, the xenoantiserum reacted with melanoma cells and with other types of tumor of nonlymphoid origin (12). Characterization of the xenoantiserum by indirect immunoprecipitation showed that it contained one antibody population reacting with an antigenic structure (M.W. 240,000) which has been detected thus far only on melanoma cells and also one antibody popu lation reacting with an antigenic structure (M.W. 94,000) ex pressed both on melanoma cells and on other human tumors of nonlymphoid origin (3). Xenoantiserum 8986 was from a rabbit immunized with hybrids derived from the fusion of human melanoma cells with murine fibroblasts; following absorption with cultured lymphoid cells, the xenoantiserum contained a major antibody population which immunochemical studies showed to react with the above-mentioned M.W. 94,000 com ponent (3). Xenoantiserum 9456 came from a rabbit immunized with MAA solubilized from cultured melanoma cells with 3 M KCI and partially purified by affinity chromatography on lentil lectin immunoadsorbents. The major antibody population in this xenoantiserum reacted with the above mentioned M.W. 240,000 antigenic structure (3). Xenoantiserum 9446 came from a rabbit immunized with MAA isolated from spent medium of cultured melanoma cells by affinity chromatography on ricin immunoadsorbents. The major antibody population in this xenoantiserum reacted with a M.W. 94,000 antigenic structure (3). The xenoantisera to MAA used in this study had been absorbed previously with pooled cultured B-lymphoid cell lines to remove antibodies to HLA antigens and to human speciesspecific antigens. A specific xenoantiserum to human ß2-fiwas produced in a cow immunized with purified urinary ß2-fi(16). Rabbit antisera to common determinants of HLA-A.B and HLA-DR antigens have been prepared and characterized as described (20, 22). The monoclonal antibodies 28 to framework determinants of HLA-A.B antigens and 513 to framework determinants of HLADR antigens were prepared using the hybridoma technique (9)
globulin antibodies or with protein A from Staphylococcus aureus has been used to analyze xenoantisera to human mel anoma-associated antigens. The test has been developed as a microassay and performed in microtiter plates, thus facilitating the screening of large numbers of samples. When modified as an inhibition assay, the assay has been successfully used (a) to compare the specificities of xenoantisera elicited with cul tured melanoma cells, hybrids derived from the fusion of cul tured human melanoma cells, and murine fibroblasts and mel anoma-associated antigens purified by biochemical proce dures and (b) to investigate the relationship of melanomaassociated antigens with /?2-microglobulin and HLA antigens on the membrane of melanoma cells.
INTRODUCTION Human melanoma cells express various types of membranebound antigens (5, 6), which have been characterized with autologous (17), allogeneic (4, 10, 15), and xenogeneic (12, 18) antisera using a variety of assays (5). In our studies of MAA4, we have elected to use serological assays since they are simple and rapid and they allow the screening of a large number of samples in a limited time. Binding assays have been preferred to lytic assays to avoid the interference of changes in susceptibility to lysis of target cells on the outcome of the reaction. The aim of this study is to describe an indirect rosette microassay to characterize xenoantisera to MAA and to inves tigate their spatial relationship with other surface markers on cell membrane. The test, which detects binding of antibodies to melanoma cells by their ability to rosette with sheep erythrocytes chemically coated with anti-immunoglobulin antibodies or with protein A from Staphylococcus aureus, is performed in microtiter plates, thus facilitating the handling of large numbers of samples.
' This work was supported by USPHS grants. This is Publication
1989 from
Scripps Clinic and Research Foundation. 2 Visiting investigator from Sapporo Medical College (Japan) and recipient of a USPHS International Research Fellowship (1 F05 TW02817-01). To whom requests for reprints should be addressed. 1Recipient of an American Heart Association Established Investigatorship Award. AThe abbreviations used are: MAA. melanoma-associated antigens; /32-/i. ßimicroglobulin; SPA. protein A from Staphylococcus aureus; SPA-E, sheep erythrocytes coated with protein A from S. aureus; Ab-E, sheep erythrocytes coated with anti-immunoglobulin antibody; MEM, minimum essential medium. Received December 3. 1979; accepted April 10. 1980.
AND METHODS
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RESEARCH
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Rosette Microassay and MAA and characterized as described elsewhere (13, 21). Coating of Sheep Erythrocytes with Protein A from SPA and with Purified Antibodies to Immunoglobulin. SPA was purchased from Pharmacia Fine Chemicals, Piscataway, N. J. Antibodies to rabbit immunoglobulin and to mouse immunoglobulin were purified from goat anti-rabbit immunoglobulin antisera and rabbit anti-mouse immunoglobulin antisera by affinity chromatography on immunoadsorbent columns. SPA and purified antibodies to immunoglobulin were coupled to sheep erythrocytes by using the chromium chloride method (14). Briefly, 1 volume of packed washed erythrocytes was incubated with 1 volume of chromium chloride solution [0.1% (w/v) in 0.15 M NaCI] and 1 volume of SPA (1 mg/ml) solution or purified antibodies (1 mg/ml) for 5 min at 23°.SPA-E or AbE then were washed 3 times with 0.15 M NaCI and stored for up to 7 days at 4°. Indirect Rosette Microassay. Twenty /il of target cell sus pensions (5 x 106/ml) were incubated with 20 fi\ of serial
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Dilution Chart
1. Comparison
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of the efficiency
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binding of rabbit MAA antibodies to cultured melanoma cells. Cultured melanoma cells M21 and BW5 were incubated with anti-MAA Xenoantiserum 8986 (O. •) or with normal rabbit serum (D, •)and then rosetted with SPA-E (O. D) and AbE (••)
10CK
dilutions of antisera in microtiter plates (Microtiter U plates; Cooke Laboratory Products, Alexandria, Va.) at 4°for 60 min.
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Then, the sensitized cells were washed 6 times with MEM and pelleted by centrifugation at 200 x g at 4°for 5 min. Twentyfive /il of a 2% suspension of Ab-E or SPA-E then were added, and the plates were centrifuged at 200 x g for 4 min at 4°. The pellets were resuspended by vigorous pipetting and stained with 25 /il of toluidine blue solution (0.1% in 0.15 M NaCI). One drop of the suspension on a slide was examined under a light microscope at 800 x g for resetted and unrosetted cells. A cell was considered rosetted when firmly sur rounded by at least 5 Ab-E or SPA-E. Two hundred consecutive cells were scored, and the percentage of rosettes was deter mined. In the rosette inhibition assay, 0.2 x 106 target cells were incubated with 20 /il of an antiserum for 60 min at room temperature. Where indicated, the cells were washed 3 times with MEM and incubated with 20/il of a "sandwich" antiserum (goat anti-rabbit immunoglobulin antiserum) for 30 min at 37° to induce capping of antigens. After 3 washings with MEM, the cells were incubated with an anti-MAA antiserum, and the rosette assay was continued as described above. Absorption. Xenoantisera were incubated with absorbing cells at room temperature for 60 min.
RESULTS Indirect Rosette Microassay. Preliminary studies compared the efficiency of SPA-E for detecting the binding of xenoantibodies to melanoma cells with that of Ab-E. No significant difference was found between the titration curves of an antiMAA xenoantiserum performed with the 2 types of indicator cells, although at each dilution of the antiserum the percentage of rosettes obtained with SPA-E is slightly greater than with AbE (Chart 1). With both types of indicator cells, the reproducibility of the indirect rosette microassay is high, as indicated by the fact that titration curves of anti-MAA xenoantisera per formed on different days are almost superimposable (Chart 2). However, there is a certain degree of variation in the percent age of rosettes detected with Ab-E when the value is lower than 50%. The rosettes formed between antibody-sensitized melanoma cells and SPA-E or Ab-E are stable up to 4 days of
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Dilution (Reciprocal) Chart 2. Reproducibility of the indirect rosette microassay performed with SPA-E and Ab-E. Titration curves of Xenoantiserum 8986 with cultured melanoma M21 cells were performed on 5 days, using SPA-E (left) and Ab-E (right) as indicator cells. •. reactivity of melanoma cells with normal rabbit serum
storage at 4°,which eliminates the need to read the reactions at the end of the centrifugation. Reactivity of Xenoantisera to MAA with a Panel of Tumor Cell Lines in the Indirect Rosette Microassay. The xenoanti sera to MAA were tested with a panel of tumor cell lines which included human melanoma cell lines, human B- and T-lymphoid cell lines, human carcinoma cell lines, human sarcoma cell lines, human fetal fibroblasts, and the murine melanoma cell line S91. All the anti-MAA xenoantisera reacted with all of the human melanoma cell lines tested with a titer of at least 1:80; none of the xenoantisera reacted with the human lymphoid cell lines and with the murine melanoma cell line S91. Furthermore, Xenoantisera 6522, 8986, and 9446 reacted with carcinoma and sarcoma cell lines, while Xenoantiserum 9456 did not react with these types of tumor cell lines. Representative results of the patterns of reactions obtained are shown in Table 1. Two lines of evidence indicate that the reaction detected with the indirect rosette microassay is specific. Target cells incubated with normal rabbit serum did not rosette with Ab-E and SPA-E (Chart 1). Furthermore, melanoma cells did not rosette with SPA-E or Ab-E following incubation with anti-MAA xenoantisera from which antibodies had been absorbed with melanoma cells. Representative results are shown in Chart 3. Rosette Inhibition Assay to Compare the Specificity of
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K. Imai and S. Ferrane Anti-MAA Xenoantisera and to Investigate the Relationship of MAA with Other Surface Markers. The rosette inhibition assay was used to compare the specificity of anti-MAA xenoantisera produced with cultured melanoma cells, hybrids be tween human melanoma cells and murine fibroblasts, and purified MAA. In this test, cultured melanoma cells were first sensitized with a given anti-MAA xenoantiserum; following re distribution of antigens by incubation with a goat anti-rabbit immunoglobulin antiserum, the cells were tested with another anti-MAA xenoantiserum in the indirect rosette microassay. SPA-E were used as indicators since SPA does not bind to goat immunoglobulin (8). Representative results are shown in Table 2. Melanoma M21 cells treated with Xenoantisera 6522 and 9446, both of which contain antibodies to a M.W. 94,000 structure expressed on tumor cells of nonlymphoid origin, showed a significantly reduced reactivity with Xenoantiserum 8986 which contains antibodies to the M.W. 94,000 compo nent; treatment of melanoma M21 cells with Xenoantiserum 9456, which contains antibodies to a M.W. 240,000 compo nent detected only on melanoma cells did not affect the reac tivity of the melanoma cells with Xenoantiserum 8986. Con versely, melanoma M21 cells treated with Xenoantiserum 8986 under capping conditions lost their reactivity with Xenoanti serum 9446 but did not have altered reactivity with Xenoanti sera 6522 and 9456. These results indicate that Xenoantiserum 8986 reacts with the same antigenic structures recognized by Xenoantiserum 9446 and by one of the 2 antibody populations present in Xenoantiserum 6522, but not with those recognized by Xenoantiserum 9456. The rosette inhibition assay can also be performed by taking advantage of the specificity of the anti-immunoglobulin anti bodies bound to sheep RBC, if the antisera being used have been elicited in different animal species. This approach was used to investigate the spatial relationship of MAA with /?2-ju and HLA-A.B and HLA-DR antigens, using rabbit anti-MAA antisera, cow anti-human ß2-nantisera, and murine monoclonal antibodies to framework determinants of HLA-A.B and HLA-DR
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Chart 3. Reactivity of Xenoantiserum 8986 with the cultured melanoma cells BW5. M10, M14, and M21 after absorption with melanoma cells Colo 38 (•), M10 (•)and M21 (A). O, reactivity of the unabsorbed xenoantiserum with the melanoma cell lines.
Table 2 Comparison of the specificity of anti-MAA Xenoantiserum Xenoantisera 6522, 9446. and 9456 withcSensitization Resetted
8986 with that of
of ro settes"1009278999682936 with3MEMMEMMEMMEM652294469456898689868986Capped serum8986652294469456898689868986652294469456Tite withGtaRblg"GtaRblgGtaRblgGtaRblgGtaRblgGtaRblgGtaRblgGtaRblgGtaRblgG
Table 1 Reactivity of an xenoantiserum to MAA solubilized from human cultured melanoma cells Xenoantiserum 9456 was from a rabbit immunized with MAA solubilized from cultured melanoma cells by 3 M KCI and purified by affinity chromatography on lentil lectin. ofTumor typeMelanomaCultured
% of rosettes at titer lineM21M51RajiRPMI
1:16072 4352 30
