Immune Adherence Assay Research Articles

A human monoclonal antibody that detects HLA-A1, A23 and A24 antigens.

We report the production and characterization of a human monoclonal IgM (mu, kappa) antibody recognizing the HLA A1, A23 and A24 antigens. B lymphocytes obtained from a multiparous Japanese woman were transformed in vitro by Epstein-Barr virus, screened with an immune adherence assay, and fused with a murine myeloma cell line, P3-X63-Ag8.653. After subcloning by limiting dilution three times, a stable antibody-secreting hybridoma cell line, 4-35-7, was identified. The culture supernant had a titer of 1:32-64 against each of A1-, A23- and A24-positive lymphocyte panels, and showed complete correlation (r = 1.00) with the A1, A23 and A24 antigens on a lymphocyte panel of 287 unrelated, class I HLA-typed donors by the NIH cytotoxicity assay. Monoclonality of the antibody was ensured by Southern blot analysis of the human immunoglobulin heavy chain gene of 4-35-7. In view of the published data on HLA class I nucleotide sequences, the antibody may recognize an antigeneic determinant including two amino acid residues, Asp-166 and Gly-167, in the alpha 2 helix of the class I molecule that are specific for A1, A23 and A24 so far analyzed.

Antibody responses to ganglio-series gangliosides in different strains of inbred mice

We studied antibody responses after immunization with ganglio-series gangliosides against 10 strains of inbred mice, including Balb/c, C57BL/6, A/J, C3H/HeN, C3H/HeJ, CBA/N, AKR/N, NZB/N, DBA/2 and nu/nu Balb/c. Twelve gangliosides having NeuAc as their sialic acid moiety (GM4, GM3, GM2, GM1, GD3, O-Ac-GD3, GD2, GD1a, GD1b, GT1a, GT1b and GQ1b), four gangliosides having NeuGc (GM3, GM2, GM1 and GD3) and four asialo-gangliosides (GA4, GA3, GA2 and GA1) were injected intravenously adsorbed to Salmonella minnesota. The antibody titers of the mice sera were determined by an enzyme-linked immunosorbent assay and an immune adherence assay. Antibody responses were found to depend not only on the ganglioside used as an immunogen but also on the mouse strain. Gangliosides having a trisaccharide sequence (NeuAcα2 → 8NeuAcα2 → 3Gal-) such as GD3, GD2, GDlb, GT1a and GQ1b, in particular O-Ac-GD3, induced high-titer antibody responses, whereas those having a disaccharide sequence (NeuAcα2 → 3Gal-) such as GM4, GM3, GM2, GM1, GDla and GTl1 induced low-liter antibody responses. On the other hand, gangliosides with NeuGc developed minimum titers. In contrast, asialo-gangliosides induced much higher responses than the corresponding gangliosides. The differences in ceramide portions of these gangliosides did not appear to be involved in inducing antibody responses. Mice could be divided into three groups according to the magnitude of their antibody responses: Group 1, those that produce the highest antibody responses (C3H/HeN and A/J); Group 2, those that demonstrate moderate antibody titers (Balb/c, C57BL/6, DBA/2 and nu/nu Balb/c); and Group 3, those that make minimum responses (AKR/N, C3H/HeJ, CBA/N and NZB/N). The pattern of reactivity to the various gangliosides was similar in all the strains tested.

Anti-ganglioside antibodies in myasthenia gravis (MG)

We studied antibody responses after immunization with ganglio-series gangliosides against 10 strains of inbred mice, including Balb/c, C57BL/6, A/J, C3H/HeN, C3H/HeJ, CBA/N, AKR/N, NZB/N, DBA/2 and nu/nu Balb/c. Twelve gangliosides having NeuAc as their sialic acid moiety (GM4, GM3, GM2, GM1, GD3, O-Ac-GD3, GD2, GD1a, GD1b, GT1a, GT1b and GQ1b), four gangliosides having NeuGc (GM3, GM2, GM1 and GD3) and four asialo-gangliosides (GA4, GA3, GA2 and GA1) were injected intravenously adsorbed to Salmonella minnesota. The antibody titers of the mice sera were determined by an enzyme-linked immunosorbent assay and an immune adherence assay. Antibody responses were found to depend not only on the ganglioside used as an immunogen but also on the mouse strain. Gangliosides having a trisaccharide sequence (NeuAcα2 → 8NeuAcα2 → 3Gal-) such as GD3, GD2, GDlb, GT1a and GQ1b, in particular O-Ac-GD3, induced high-titer antibody responses, whereas those having a disaccharide sequence (NeuAcα2 → 3Gal-) such as GM4, GM3, GM2, GM1, GDla and GTl1 induced low-liter antibody responses. On the other hand, gangliosides with NeuGc developed minimum titers. In contrast, asialo-gangliosides induced much higher responses than the corresponding gangliosides. The differences in ceramide portions of these gangliosides did not appear to be involved in inducing antibody responses. Mice could be divided into three groups according to the magnitude of their antibody responses: Group 1, those that produce the highest antibody responses (C3H/HeN and A/J); Group 2, those that demonstrate moderate antibody titers (Balb/c, C57BL/6, DBA/2 and nu/nu Balb/c); and Group 3, those that make minimum responses (AKR/N, C3H/HeJ, CBA/N and NZB/N). The pattern of reactivity to the various gangliosides was similar in all the strains tested.

Serological analysis of cell surface antigens in choriocarcinoma cell lines.

Major histocompatibility complex (MHC) antigens as well as blood group related antigens were investigated in four newly established gestational choriocarcinoma cell lines (NaUCC-1, -2, -3, and -4) using protein A and immune adherence assays. Antibodies to both monomorphic determinants of HLA class I antigens and beta 2 microglobulin reacted with all of the choriocarcinoma cell lines at levels equal to or much greater than SCH, which was teratoid choriocarcinoma cell line. Antibodies to polymorphic determinants not only of the patient's HLA type, but also of the husband's type, reacted to NaUCC-1 and -2. These results indicated that all four newly established choriocarcinoma cell lines express class I HLA antigens. However, we could not demonstrate expression of class II antigens. No expression of blood group A and B antigens could be established, and binding of antibody to Rho(D) antigens was only positive in the NaUCC-1 cell line. These results suggest that some choriocarcinoma cell lines actually express alloantigens and that choriocarcinomas have the character of transplanted tumors that have activities to induce transplantation immunity of the patients.

Autologous antibodies to human bladder cancer.

The autologous serologic reactivity of 13 patients with bladder cancer was evaluated using cell lines derived from each individual's own tumor as targets. Protein A and immune adherence assays were employed to determine antibody binding to the tumor targets at varying passage numbers. Autologous reactivity was found in 6 of the 13 cell lines tested. However, the titer was usually low regardless of the passage number. Seven autologous serum/cell line combinations were tested using both low and high passage cells as targets. In six of these combinations, the degree of antibody binding was similar with both low and high passage target cells. The incidence of autologous reactivity in the 12 patients with urothelial tumors was 50%.

Human melanoma antigen O-acetylated ganglioside GD3 is recognized by Cancer antennarius lectin.

A lectin that can specifically bind to O-acetylsialic acids, found in glycoproteins and gangliosides, was purified to homogeneity from a crab Cancer antennarius (crab lectin) (Ravindranath, M. H., Higa, H. H., Cooper, E. L., and Paulson, J. C. (1985) J. Biol. Chem. 260, 8850-8856; Correction (1986) J. Biol. Chem. 261, 1983; Ravindranath, M. H., and Paulson, J. C. (1987) Methods Enzymol. 138, 520-527). We tested lectin binding to human melanoma cell lines to identify O-acetylsialylated gangliosides on the melanoma cell surface. The highest degree of binding of the crab lectin was demonstrated on a melanoma cell line, UCLASO-M25. To confirm that the binding was due to O-acetylsialic acid in the alkali-labile gangliosides, the gangliosides were isolated and purified from M25 cells and individually coated onto sheep asialoerythrocytes, which served as targets in an agglutination assay using the lectin. The crab lectin agglutinated the asialo-sheep erythrocytes coated with alkali-labile gangliosides, but the lectin failed to agglutinate the asialoerythrocytes coated with GM3, GD3, and base-treated gangliosides. Subsequently, the purified alkali-labile M25 ganglioside was base-treated and applied to TLC, and we found that it was converted to a slower migrating species identical to the disialolactosylceramide (GD3). These results indicate that O-acetyl GD3 expressed on the melanoma cell surface is recognized by the lectin. Because O-acetyl GD3 is not expressed on human normal tissues, we examined the capability of O-acetyl GD3 to induce immune responses in man. Sera from patients with melanoma were tested against M25 cells in an immuneadherence assay, and those positive to the M25 cell line were further tested for specificity to O-acetyl gangliosides. The presence of autoantibodies to O-acetyl-GD3 in melanoma sera was confirmed by blocking of the antigen sites on M25 cells by the lectin or preabsorption of the sera with erythrocytes bearing O-acetyl gangliosides. The data provide evidence that O-acetyl-GD3 may represent an important tumor marker for detection and treatment of human melanoma.

Expression of the Leukocyte Functional Molecule (LFA-1) on Mouse Platelets

Abstract Platelet involvement in adhesion, hemostasis, and immune adherence is mediated by functionally associated cell surface molecules. Platelets are also involved in cytolytic reactions, but little is known about the mechanisms or biologic significance of these processes. To further investigate cell surface molecules concerned with platelet function, antisera against mouse platelets, thymocytes, and macrophages and monoclonal antibodies against Mac-1 (complement receptor type 3) and leukocyte function-associated glycoprotein type 1 (LFA-1) were used to demonstrate LFA-1--like molecules on mouse platelets. The alpha subunits of platelet and thymocyte LFA-1 showed identical electrophoretic mobility but differed significantly from the alpha subunit of macrophage Mac-1. Peptide mapping demonstrated the identity of the beta subunits of these three molecules but showed that the alpha subunit of Mac-1 was distinct from the alpha subunits of platelet and thymocyte LFA-1. Platelet LFA-1, as demonstrated by surface iodination with lactoperoxidase and by labeling sialic acid residues with sodium borohydride, was not a major component of the platelet membrane. The functional significance of LFA-1 on mouse platelets has yet to be demonstrated, monoclonal antibodies against LFA-1 having little effect on adenosine diphosphate-induced platelet aggregation and immune adherence. In contrast, although Mac-1 could not be demonstrated on mouse platelets in immunoprecipitation studies, its presence was clearly demonstrated by low levels of antibody binding in enzyme-linked immunosorbent assays and by the ability of M1/70 monoclonal antibody to inhibit platelet immune adherence. Human platelets, which are inactive in immune adherence assays, are shown to lack LFA-1 and Mac-1.

Expression of the leukocyte functional molecule (LFA-1) on mouse platelets

Platelet involvement in adhesion, hemostasis, and immune adherence is mediated by functionally associated cell surface molecules. Platelets are also involved in cytolytic reactions, but little is known about the mechanisms or biologic significance of these processes. To further investigate cell surface molecules concerned with platelet function, antisera against mouse platelets, thymocytes, and macrophages and monoclonal antibodies against Mac-1 (complement receptor type 3) and leukocyte function-associated glycoprotein type 1 (LFA-1) were used to demonstrate LFA-1--like molecules on mouse platelets. The alpha subunits of platelet and thymocyte LFA-1 showed identical electrophoretic mobility but differed significantly from the alpha subunit of macrophage Mac-1. Peptide mapping demonstrated the identity of the beta subunits of these three molecules but showed that the alpha subunit of Mac-1 was distinct from the alpha subunits of platelet and thymocyte LFA-1. Platelet LFA-1, as demonstrated by surface iodination with lactoperoxidase and by labeling sialic acid residues with sodium borohydride, was not a major component of the platelet membrane. The functional significance of LFA-1 on mouse platelets has yet to be demonstrated, monoclonal antibodies against LFA-1 having little effect on adenosine diphosphate-induced platelet aggregation and immune adherence. In contrast, although Mac-1 could not be demonstrated on mouse platelets in immunoprecipitation studies, its presence was clearly demonstrated by low levels of antibody binding in enzyme-linked immunosorbent assays and by the ability of M1/70 monoclonal antibody to inhibit platelet immune adherence. Human platelets, which are inactive in immune adherence assays, are shown to lack LFA-1 and Mac-1.

The serologic response of patients with stage II melanoma to allogeneic melanoma cell vaccines

Seventeen patients with Stage II malignant melanoma were treated with vaccines prepared from three allogeneic melanoma cell lines in an attempt to induce a humoral immune response against melanoma cell surface antigens. The patients were free of detectable melanoma at the time of vaccination. Vaccines were prepared from three melanoma cell lines that expressed highly restricted melanocyte differentiation antigens. One of these cell lines also expressed an antigen found only on this particular line. The antigens were initially identified by antibodies in autologous serum; they were thus known to be recognized by the human immune system. In addition, two of the cell lines expressed HLA-A, -B, -C, and -DR antigens; no HLA antigens were detectable on the third line. The vaccines were administered sequentially by subcutaneous injection, mixed with bacillus Calmette-Guerin (BCG) or Corynebacterium parvum. The patients' sera were tested for antibodies against cell surface antigens of the vaccine cells in protein A assays and immune adherence assays, and the specificity of observed reactions was defined by absorption tests. Antibodies against alloantigens of the vaccine cells developed in 16 patients and in 15 patients, against antigens related to fetal calf serum in the culture medium. The magnitude of the antibody response to alloantigens varied considerably, with no difference between patients who received BCG or C. parvum with their vaccines. Antibodies against the restricted melanocyte differentiation antigens or the unique melanoma antigen expressed by the vaccine cells were not detected.

Human Renal Carcinoma: Characterization of Five New Cell Lines

Abstract Five human renal carcinoma cell lines have been established in long-term tissue culture. Two of the cell lines, UM-RC-2 and UM-RC-3, produced clear cell tumors in athymic nude mice. The cell lines have been characterized by staining with oil red O, doubling time in vitro, and number of chromosomes. Although protein A assay reactivity of autologous combinations of patient's sera and tumor cells were seen with all five cell lines, similar binding was also found with autologous normal kidney cultures. However, the immune adherence assay demonstrated low titer autologous reactivity with two renal carcinoma cell lines but not with the corresponding normal kidney cultures. This strongly suggest host recognition of tumor-associated antigens. Characterization of cell surface antigens with murine monoclonal antibodies demonstrated shared reactivity between normal kidney tubular cells and renal carcinoma cells. Antibody A68.11 reacted strongly with all five cell lines. Antibody A80 bound to only UM-RC-3 and UM-RC-6.

Human renal carcinoma: characterization of five new cell lines.

Five human renal carcinoma cell lines have been established in long-term tissue culture. Two of the cell lines, UM-RC-2 and UM-RC-3, produced clear cell tumors in athymic nude mice. The cell lines have been characterized by staining with oil red O, doubling time in vitro, and number of chromosomes. Although protein A assay reactivity of autologous combinations of patient's sera and tumor cells were seen with all five cell lines, similar binding was also found with autologous normal kidney cultures. However, the immune adherence assay demonstrated low titer autologous reactivity with two renal carcinoma cell lines but not with the corresponding normal kidney cultures. This strongly suggest host recognition of tumor-associated antigens. Characterization of cell surface antigens with murine monoclonal antibodies demonstrated shared reactivity between normal kidney tubular cells and renal carcinoma cells. Antibody A68.11 reacted strongly with all five cell lines. Antibody A80 bound to only UM-RC-3 and UM-RC-6.

Production and analysis of HH 10 monoclonal antibodies reactive to immature hematopoietic cells and their use for monitoring acute leukemia cells.

A clone of murine monoclonal antibody HH 10 (IgM) was raised by fusing NS-1 myeloma cells and spleen cells of a mouse hyperimmunized with acute promyelocytic leukemia cells. Serological analysis by means of immune adherence assays showed that HH 10 reacts with immature hematopoietic cells including thymocytes and myeloid precursor cells (defined as colony-forming units in culture assays). The antibodies were not reactive to either peripheral blood cells or bone marrow cells obtained from normal individuals. Lymphoid blasts induced with phytohemagglutinin, pokeweed mitogen, or concanavalin A were also non-reactive, and all non-hematopoietic cultured cells examined were also negative. The antibodies were, however, reactive to leukemia cells in 67 cases out of 91 cases (74%) of acute leukemia. In patients with acute lymphoblastic leukemia, 43 out of 52 cases (83%) were positive and, in particular, all of 12 T-cell-type acute leukemias were reactive to HH 10. Comparison of percent HH 10 positive cells in the bone marrow of patients with acute leukemia with the results of cytological studies showed a good correlation. Analysis of sequentially collected bone marrow cells of acute leukemia patients using HH 10 revealed its usefulness for monitoring leukemia cells.

Effect of neuraminidase treatment on serum reactivity to autologous leukemic blast cells.

The sera of 35 patients with acute lymphoblastic leukemia (ALL) and acute non-lymphoblastic leukemia (ANLL) were tested for reactivity against cell surface antigens of autologous leukemic blast cells by protein A assay (PA), immune adherence assay (IA), and anti-C3 mixed hemadsorption assay (C3-MHA). Autologous serum reactivity was detectable by PA in four cases and by IA and C3-MHA in about half the patients. Autologous serum reactivity occurred more often in ALL than in ANLL. Absorption studies revealed that in one patient only the autologous reactivity was directed against a restricted antigen, which could be detected only on the individual T-ALL blast cells. All other autologous antibodies detected unspecific antigens. Neuraminidase treatment had two effects: first, it increased antibody attachment to antigens which are also present on untreated cells; secondly, after neuraminidase treatment an antigen was detectable on the cell surface which could also be demonstrated on neuraminidase-treated non-leukemic cells (e.g., erythrocytes). Neither of these two effects of neuraminidase treatment seems to be tumor-specific. Possible therapeutic effects of neuraminidase are probably caused by unspecific adjuvant effects of the enzyme.

Serological analysis of cell surface antigens of HL-60 cells before and after treatment with a phorbol ester tumor promoter

The human HL-60 cell line derived from acute promyelocytic leukemia, consisting of promyelocytic type of cells, was able to differentiate into adherent cells with monocyte-macrophage features by the treatment with 12- O-tetradecanoyl phorbol-13-acetate (TPA). Cell surface antigens of HL-60 cells before and after TPA treatment were studied with monoclonal antibodies and four hybridoma clones producing IgM antibodies were established. Two antibodies (HL-21 and HL-47) reacted only with the immunizing TPA-treated HL-60 cells, and HL-1 antibody produced against untreated cells was reactive with both TPA-treated and untreated cells, but HL-5 antibody reacted predominantly with the immunizing untreated cells. Serological reactivity against various types of normal hematopoietic cells and acute leukemias (diagnosed by the French-American-British classification) was studied by immune adherence assay and immuno-electron microscopy. HL-21 antibody was reactive with monocytes and most cases of M4 and M5 types of acute non-lymphocytic leukemia cells. HL-47 antibody did not react with the cells of myelocyte-monocyte lineage or mature lymphocytes, but it did react with one-third of acute lymphocytic leukemia (L1 and L2) cases. Since all HL-47 + cases were included in the group of common ALL antigen positive cases, it was estimated that HL-47 is a differentiation antigen present on lymphocyte precursors, from which null-cell type acute lymphocytic leukemia cells generally originate. HL-1 antibody reacted with the cells of myelocyte-monocyte lineage as well as those of most acute non-lymphocytic leukemias. HL-5 antibody reacted with granulocytes and M2 type of acute myelocytic leukemia cases, and also with M5 type of acute monocytic leukemia cases. Serological studies of these antibodies revealed that TPA can induce to differentiate HL-60 cells not only into HL-21 + macrophage-like cells, but also into HL-47 + lymphoid stem cells. In addition, these antibodies were demonstrated to be very valuable for differential diagnosis of acute leukemias.

Human antibody to OFA-I, a tumor antigen, produced in vitro by Epstein-Barr virus-transformed human B-lymphoid cell lines.

We established two long-term human B-lymphoblastoid cell lines (L55 and L72) transformed by Epstein-Barr virus that produced IgM kappa antibodies to the human tumor antigen, OFA-I. Periphral blood lymphocytes obtained from melanoma patients were used as the source of the B lymphocytes. Antibody specificity was determined by the immune adherence assay using various human cancer and noncancer tissues as targets. L55 antibody (designated anti-OFA-I-1) reacted with a variety of human tumor types whereas L72 antibody (designated anti-OFA-I-2) reacted only with tumor cells of neuroectodermal origin (melanoma, glioma, and neuroblastoma). The levels of IgM detected in the spent medium of 1 X 10(6) L55 and L72 cells were 4 and 9 micrograms/ml, respectively, by radioimmunoassay.

Natural antibody to a human bladder carcinoma cell line.

Sera from 144 healthy blood donors were screened for the presence of antibody against the bladder carcinoma cell line RT4 by means of an immune adherence assay. Only one serum (9241) demonstrated high activity with a titer of 1/124. Qualitative and quantitative absorption analyses were carried out with a wide variety of cells to characterize the specificity of the reaction. Cells used for absorption were both cultured and non-cultured and normal and malignant. The reactivity was absorbed by the bladder cancer cell lines RT4 and 5637 and the lung cancer cell line SK-LC-LL. In addition, surgically obtained urothelium from a patient with transitional cell carcinoma abrogated reactivity. No other absorbing cells removed the reactivity of serum 9241 for RT4. The antigen defined by this serologic reaction is heat-stable and trypsin-resistant, as demonstrated by using heated or trypsinized RT4 cells for absorption. This study shows that naturally occurring antibody directed toward cell surface antigens of tumor cells is present in some normal individuals. This finding is important in the evaluation of a possible causal relationship between the presence of antibody specific for tumor cell antigens and the development of neoplasia.

Natural antibodies to cell‐surface antigens of human astrocytoma

Sera of 200 non-transfused healthy male blood donors were tested for antibody reactivity to cell-surface antigens of cultured astrocytoma cells. Positive reactions were observed only rarely by protein-A assay (PA), in about half the cases by immune adherence assay (IA) and in nearly all cases by anti-C3 mixed hemadsorption assay (C3-MHA). In general, titers were low and only seven sera showed reactivity at 1:1,000. Serum 537 showed the strongest reaction. The anti-astrocytoma reactivity in this serum was due to an IgG antibody. Extensive absorption analysis with a panel of cell lines and fresh cells of both benign and malignant origin, as well al fetal cells, revealed that this serum detected an antigen that was present on most neural-crest-derived tumors (astrocytomas, melanomas and neuroblastomas), on very few other malignant tumors and on fetal brain. The antigen detected by serum 537 shows close relationship to the astrocytoma antigen AJ which had been defined by the serum of a patient with astrocytoma. Both antigens appear to be differentiation antigens present predominantly on non-epithelial neoplasms. The antigen detected by serum 537 is heat-stable and pronase-resistant. The sera of two other healthy donors apparently had a similar specificity, whereas the four other high-titered sera and all other sera detected class-III antigens which were non-specific and not tumor-restricted.

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Inbred CDF rats (cesarean-delivered F344 rats) were immunized with either a nitrosourea-induced glioma EA-285 or its subline EA-285A and both with and without Corynebacterium parvum. A humoral immune response in syngeneic rats to the immunizing tumor was demonstrated with micromodifications of different rosette assays (immune adherence assay, protein-A assay, and anti-C3-mixed hemadsorption assay). Extensive absorption studies of two immune sera with the highest reactivity revealed two glioma-specific antigens (or two components of one antigen) on the cell surface of the cloned tumor subline. One antigen was present only on the cell surface of the subline of the glioma used for both immunization and absorption analyses. The other antigen appeared on the cell surface of both the cloned subline EA-285A and the parental cell line EA-285. These preliminary studies showed that CDF rats immunized against a syngeneic glioma mounted a humoral immune response that appeared to be individually tumor-specific.

Serologic analysis of human tumor antigens

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.

Complement fixation by IgM and IgG autoantibodies on cultured human glial cells.

The humoral immune response to autologous gliomas has been investigated in 16 patients using immune adherence and anti-C3 immunofluorescence assays of serum samples. Significant antiglioma antibody activity was detected in 56% of these sera. Fractionation experiments demonstrated that IgM class antibodies were more effective that IgG in their ability to fix complement on cultured glioma cell membranes. In addition, autologous IgG was shown to inhibit IgM complement-fixing activity in some experiments. "Capping" of immune complexes on the glioma cell surface is also described.