Serum antibodies to human leucocyte antigen (HLA)-E, HLA-F and HLA-G in patients with systemic lupus erythematosus (SLE) during disease flares: Clinical relevance of HLA-F autoantibodies.

T lymphocyte hyperactivity and progressive inflammation in systemic lupus erythematosus (SLE) patients results in over-expression of human leucocyte antigen (HLA)-Ib on the surface of lymphocytes. These are shed into the circulation upon inflammation, and may augment production of antibodies promoting pathogenicity of the disease. The objective was to evaluate the association of HLA-Ib (HLA-E, HLA-F and HLA-G) antibodies to the disease activity of SLE. The immunoglobulin (Ig)G/IgM reactivity to HLA-Ib and ß2m in the sera of 69 German, 29 Mexican female SLE patients and 17 German female controls was measured by multiplex Luminex(®)-based flow cytometry. The values were expressed as mean flourescence intensity (MFI). Only the German SLE cohort was analysed in relation to the clinical disease activity. In the controls, anti-HLA-G IgG predominated over other HLA-Ib antibodies, whereas SLE patients had a preponderance of anti-HLA-F IgG over the other HLA-Ib antibodies. The disease activity index, Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)-2000, was reflected only in the levels of anti-HLA-F IgG. Anti-HLA-F IgG with MFI level of 500-1999 was associated with active SLE, whereas inactive SLE revealed higher MFI (>2000). When anti-HLA-F IgG were cross-reactive with other HLA-Ib alleles, their reactivity was reflected in the levels of anti-HLA-E and -G IgG. The prevalence of HLA-F-monospecific antibodies in SLE patients was also associated with the clinical disease activity. Anti-HLA-F IgG is possibly involved in the clearance of HLA-F shed from lymphocytes and inflamed tissues to lessen the disease's severity, and thus emerges as a beneficial immune biomarker. Therefore, anti-HLA-Ib IgG should be considered as a biomarker in standard SLE diagnostics.

Immunoglobulin (Ig)G purified from human sera mirrors intravenous Ig human leucocyte antigen (HLA) reactivity and recognizes one's own HLA types, but may be masked by Fab complementarity-determining region peptide in the native sera.

Intravenous immunoglobulin (IVIg) reacted with a wide array of human leucocyte antigen (HLA) alleles, in contrast to normal sera, due possibly to the purification of IgG from the pooled plasma. The reactivity of IgG purified from normal sera was compared with that of native sera to determine whether any serum factors mask the HLA reactivity of anti-HLA IgG and whether IgG purified from sera can recognize the HLA types of the corresponding donors. The purified IgG, unlike native sera, mirrored IVIg reactivity to a wide array of HLA-I/-II alleles, indicating that anti-HLA IgG may be masked in normal sera - either by peptides derived from soluble HLA or by those from antibodies. A < 3 kDa peptide from the complementarity-determining region (CDR) of the Fab region of IgG (but not the HLA peptides) masked HLA recognition by the purified IgG. Most importantly, some of the anti-HLA IgG purified from normal sera - and serum IgG from a few donors - indeed recognized the HLA types of the corresponding donors, confirming the presence of auto-HLA antibodies. Comparison of HLA types with the profile of HLA antibodies showed auto-HLA IgG to the donors' HLA antigens in this order of frequency: DPA (80%), DQA (71%), DRB345 (67%), DQB (57%), Cw (50%), DBP (43%), DRB1 (21%), A (14%) and B (7%). The auto-HLA antibodies, when unmasked in vivo, may perform immunoregulatory functions similar to those of therapeutic preparations of IVIg.

Suppression of blastogenesis and proliferation of activated CD4(+) T cells: intravenous immunoglobulin (IVIg) versus novel anti-human leucocyte antigen (HLA)-E monoclonal antibodies mimicking HLA-I reactivity of IVIg.

Activated CD4(+) T cells undergo blastogenesis and proliferation and they express several surface receptors, including ß2-microglobulin-free human leucocyte antigen (HLA) heavy chains (open conformers). Intravenous immunoglobulin (IVIg) suppresses activated T cells, but the mechanism is unclear. IVIg reacts with HLA-Ia/Ib antigens but its reactivity is lost when the anti-HLA-E Ab is adsorbed out. Anti-HLA-E antibodies may bind to the peptides shared by HLA-E and the HLA-I alleles. These shared peptides are cryptic in intact HLA, but exposed in open conformers. The hypothesis that anti-HLA-E monoclonal antibodies (mAbs) that mimic HLA-I reactivity of IVIg may suppress activated T cells by binding to the shared peptides of the open conformers on the T cell surface was tested by examining the relative binding affinity of those mAbs for open conformers coated on regular beads and for intact HLA coated on iBeads, and by comparing the effects on the suppression of phytohaemagglutinin (PHA)-activated T cells of three entities: IVIg, anti-HLA-E mAbs that mimic IVIg [Terasaki Foundation Laboratory (TFL)-006 and (TFL)-007]; and anti-HLA-E antibodies that do not mimic IVIg (TFL-033 and TFL-037). Suppression of blastogenesis and proliferation of those T cells by both IVIg and the anti-HLA-E mAbs was dose-dependent, the dose required with mAbs 50-150-fold lower than with IVIg. TFL-006 and TFL-007 significantly suppressed blastogenesis and proliferation of activated CD4(+) T cells, but neither the non-IVIg-mimicking mAbs nor control antibodies did so. The suppression may be mediated by Fab-binding of TFL-006/TFL-007 to the exposed shared peptides. The mAb binding to the open conformer may signal T cell deactivation because the open conformers have an elongated cytoplasmic tail with phosphorylation sites (tryosine(320)/serine(335)).

Suppression of allo-human leucocyte antigen (HLA) antibodies secreted by B memory cells in vitro: intravenous immunoglobulin (IVIg)†versus a monoclonal anti-HLA-E IgG that mimics HLA-I reactivities of IVIg.

B memory cells remain in circulation and secrete alloantibodies without antigen exposure > 20 years after alloimmunization postpartum or by transplantation. These long-lived B cells are resistant to cytostatic drugs. Therapeutically, intravenous immunoglobulin (IVIg) is administered to reduce allo-human leucocyte antigen (HLA) antibodies pre- and post-transplantation, but the mechanism of reduction remains unclear. Recently, we reported that IVIg reacts with several HLA-I alleles and the HLA reactivity of IVIg is lost after its HLA-E reactivity is adsorbed out. Therefore, we have generated an anti-HLA-E monoclonal antibody that mimics the HLA-reactivity of IVIg to investigate whether this antibody suppresses IgG secretion, as does IVIg. B cells were purified from the blood of a woman in whose blood the B memory cells remained without antigen exposure > 20 years after postpartum alloimmunization. The B cells were stimulated with cytokines using a well-defined culture system. The anti-HLA-E monoclonal antibody (mAb) significantly suppressed the allo-HLA class-II IgG produced by the B cells, and that this suppression was far superior to that by IVIg. These findings were confirmed with HLA-I antibody secreted by the immortalized B cell line, developed from the blood of another alloimmunized woman. The binding affinity of the anti-HLA-E mAb for peptide sequences shared (i.e. shared epitopes) between HLA-E and other ß2-microglobulin-free HLA heavy chains (open conformers) on the cell surface of B cells may act as a ligand and signal suppression of IgG production of activated B memory cells. We propose that anti-HLA-E monoclonal antibody may also be useful to suppress allo-HLA IgG production in vivo.

Interleukin-2 binds to ganglioside GD(1b).

We have developed a solid matrix immunoassay to determine the binding of interleukin-2 (IL-2) to specific gangliosides. The assay establishes that recombinant human IL-2 binds to ganglioside GD(1b) but not to any other gangliosides (GM(1), GM(2), GM(3), GD(1a), GD(2), GD(3), and GT(1b)). The binding varies with the ratio of GD1b and IL-2. This assay enables distinguishing the nature of the sugar moiety of the ganglioside recognized by IL-2 and establishes the dosimetry of the ganglioside-IL-2 interaction. Since rIL-2 is administered systematically into stage IV melanoma patients, we have examined 45 tumor biopsies for GD(1b) content. The incidence of GD(1b) in tumor biopsies is 51%. We postulate that GD(1b) associated on the tumor or in the circulation of cancer patients may bind to rIL-2 and prevent the availability of rIL-2 to augment antitumor-immune response.

Does human melanoma express carcinoembryonic antigen?

BACKGROUND: Several investigators have proposed that carcinoembryonic antigen (CEA), an immunogenic antigen expressed by colon carcinoma, may also be expressed by human melanoma. Because sialyl Lewisx (sLex), the carbohydrate moiety of CEA, has been identified in melanoma, we compared CEA and sLex levels in colon carcinoma cells and melanoma cells. METHODS: CEA levels were assessed for expression on the cell surface and in cell lysates of cutaneous melanoma cell lines by two different kinds of ELISA, and by Western blot analysis of immunoprecipitated CEA using monoclonal antibodies (Mabs) T84-66 and COL-1, which have defined specificities for CEA. Colon carcinoma cells and purified CEA were positive controls. RESULTS: Both Mabs reacted strongly with cell surface and cell lysates of colon cancer. Mab T84-66 reacted well with cell surface but not cell lysates of melanoma. COL-1 reacted poorly with cell surface but its binding increased with the density of melanoma cell lysates. Both Mabs intensely stained the blots of purified CEA and colon carcinoma lysates immunoprecipitated with the respective Mabs, but failed to stain the immunoprecipitates of melanoma cell lysates. Both Mabs bound to lysates immunoprecipitated with anti-sLex Mab in colon carcinoma, but not in melanoma. Cell-surface expression of CEA and sLex was significantly correlated (r2: 0.88) in colon cancer cells but not in melanoma. CONCLUSION: Our results confirm the presence of CEA in colon carcinoma but not in human cutaneous melanoma cell lines.

Gangliosides as targets for immunotherapy for pancreatic adenocarcinoma.

BACKGROUND: Pancreatic adenocarcinoma cells express gangliosides and sialyl Lewis (sLe) antigens. It is not known whether these carbohydrate antigens can be targeted by immunotherapy. The authors measured the expression of GM(2) and sLe antigens on the surface of pancreatic carcinoma cells and the serum levels of total gangliosides, GM(2), and antiganglioside antibodies in patients with pancreatic carcinoma. METHODS: Cell surface GM(2) and sLe antigens were measured by cell suspension enzyme linked immunoadsorbent assay (ELISA) in four pancreatic carcinoma cell lines. Sera from 20 pancreatic carcinoma patients and 20 age- and gender-matched healthy volunteers were analyzed for antiganglioside and anti-sLe immunoglobulin (Ig) M titers by ELISA. Serum levels of total gangliosides and GM(2) also were measured. RESULTS: All cell lines expressed GM(2) and sLe antigens. When compared with age- and gender-matched volunteers, patients had significantly higher serum levels of total gangliosides (25.6 +/- 9.0 mg/dL vs. 15.6 +/- 2.7 mg/dL; P < 0.001), GM(2) (0.278 +/- 0.415 mg/dL vs. 0.013 +/- 0.018 mg/dL; P = 0.02), ELISA units of anti-GM(2) IgM antibody (368 +/- 95 vs. 155 +/- 25; P = 0.04) and anti-GD(1b) IgM antibody (351 +/- 91 vs. 138 +/- 26; P = 0.03), but not anti-sLe(x) IgM (1389 +/- 345 vs. 1081 +/- 224; P = 0.46) or anti-sLe(a) IgM antibody (1097 +/- 253 vs. 1200 +/- 315; P = 0.80). Patients with unresectable tumors had higher serum levels of total gangliosides compared with patients with resectable tumors, and a serum level > 25 mg/dL was found to correlate significantly with poor overall survival (P < 0.02). CONCLUSIONS: Increased serum levels of total gangliosides and GM(2) may reflect shedding or release of gangliosides from the surface of tumor cells. Production of IgM antibody against GM(2) and GD(1b) indicates that these gangliosides are immunogenic antigens that may be potential targets for effective active immunotherapy.

Immunology of gangliosides.

This review discusses the immunology of gangliosides from the perspective of tumor, neuronal and general immunology. Antiganglioside antibodies in human sera are invariably IgM and are found in healthy individuals. Their titers decline with age. Persistent high titer of IgM is associated with several diseases, particularly neuropathies. Membrane-bound gangliosides are important tumor-associated antigens and targets for immune attack. Cells enriched with gangliosides can be used as cancer vaccines. Efficacy of these vaccines depends on the viability of whole cells, integrity of the cell membranes, adjuvants and topography of the tumor-associated antigens. The role of antiganglioside IgM is to eliminate the immunosuppressive gangliosides shed from tissues during ageing, degeneration of neural and extraneural tissues, and tumor growth and necrosis. In addition, in vitro observations with human and murine monoclonal antibodies suggest that they are capable of complement dependent cytotoxicity and apoptosis.

Ratio of IgG:IgM antibodies to sialyl Lewis(x) and GM3 correlates with tumor growth after immunization with melanoma-cell vaccine with different adjuvants in mice.

Human melanoma cells (from biopsies and culture) express sialyl-Lewis(x) and sialyl Lewis(a), the ligands for ECAM. These ligands may facilitate tumor progression and metastasis in human cancers. To test whether the antibodies to these ligands inhibit tumor progression, IgG and IgM responses to sLe(x) and sLe(a) were induced in C57BL/6j mice (n = 76) by immunization with human melanoma cells, with or without adjuvants (BCG, MPL, KLH). Control mice were treated with saline or BCG. Tumor growth and antigen expression were monitored after challenge with B16 mouse melanoma cells expressing sLe(x), sLe(a) and the ganglioside GM3. Tumor growth was reduced in mice immunized with BCG alone or cells with BCG or MPL, while tumors in mice receiving cells without adjuvants grew larger than in the control. Augmentation of IgM titers to sLe(x) and GM3 after immunization with BCG, or with cells with BCG or MPL correlated with retarded tumor growth, while increased IgG titers to sLe(x) significantly correlated with aggressive tumor growth in mice immunized with cells without adjuvants. SLe(x), sLe(a) and GM3 were expressed in tumors from mice treated with saline or BCG. SLe(x) expression, in particular, was lost in tumors growing in mice immunized with cells with or without adjuvants. Anti-sLe(x) antibodies may promote or prevent tumor growth by antigenic modulation or by cytotoxic killing of tumor cells. Since early anti-sLe(x) IgM correlated with tumor regression, in contrast to anti-sLe(x) IgG, it may serve as a potential early endpoint for the effectiveness of melanoma vaccines expressing the antigens.

Augmentation of natural antiganglioside IgM antibodies in lower motor neuron disease (LMND) and role of CD5+ B cells.

IgM antibodies directed against neuronal gangliosides GM1, GM2, GD1a, GD1b and GT1b occur in normal individuals and their level significantly decreases with age. Patients with lower motor neuron disease (LMND) produce high levels of these autoantibodies. AntiGM1 IgM is selectively augmented. In these patients, the CD5+ (B1) and CD5- (B2) subsets of B cells are not distinct entities but range from those without detectable CD5 marker to those with high CD5+ expression. B1 B cells were sorted to homogeneity, but B2 B cell cannot be isolated to homogeneity because of the presence of B1 cells with low CD5 expression. In short term cultures both the subsets produced IgM antibodies, but the antibodies reacted better with desialylated GM1 than with GM1. Cycloheximide (Cx) (0.35 mM) largely blocked IgM synthesis of the B1 B cells but inhibition of the B2 B cells was incomplete, possibly due to shedding of cytophilic antibodies as well as to the presence of B1 phenotype with loss of CD5 expression. CD5+ B cells may be involved in the production of antiglycolipid IgM.

Endothelial-selectin ligands sialyl Lewis(x) and sialyl Lewis(a) are differentiation antigens immunogenic in human melanoma.

BACKGROUND: Sialyl Lewis(x) (sLe(x)) and sialyl Lewis(a) (sLe(a)), the endothelial-selectin ligands involved in extravasation of neutrophils and carcinomas, have been identified in human melanoma. This study explored the following issue: If these ligands are immunogenic tumor-differentiation antigens, they would be potential targets for immunotherapy because of their putative roles in extravasation and metastasis. METHODS: Using a cell-suspension enzyme-linked immunosorbent assay (ELISA), the expression of sLe(x) and sLe(a) on the surface of normal melanocytes, melanoma cells from biopsies, and cell lines (M10-v, M24, and M101) constituting melanoma cell vaccine (MCV) were quantitated. Melanoma patients were immunized with the MCV expressing these antigens. Sera of normal individuals, sera of patients, and sera that adsorbed to sLe(x) and sLe(a) were titrated for anti-sLe antibodies by ELISA to verify the immunogenicity of the ligands. RESULTS: The normal melanocytes did not express sLe(x) and poorly expressed sLe(a). Melanoma cells from tumor biopsies and MCV lines expressed both sLe(x) and sLe(a). Sialyl Le(x) was associated with glycoprotein(s) in M10-v, and sLe(a) occurred as a glycolipid moiety in M24. MCV recipients developed high titers for immunoglobulin (Ig)M but not IgG to both ligands. IgM titers to these ligands were low in normal subjects. In some of the preimmune sera of patients, the titers were threefold above normal. Six of 13 MCV recipients developed at least a twofold increase in anti-sLe titers above preimmune level after the second or third immunization. Adsorption studies suggested that both ligands were immunogenic. CONCLUSIONS: The melanoma-associated sLe(x) and sLe(a) are immunogenic neoplasm-differentiation antigens and are therefore potential targets for passive and active specific immunotherapy in the treatment of melanoma.

Cellular cancer vaccine induces delayed-type hypersensitivity reaction and augments antibody response to tumor-associated carbohydrate antigens (sialyl Le(a), sialyl Le(x), GD3 and GM2) better than soluble lysate cancer vaccine.

Allogenic whole cell and lysate cancer vaccines are associated with very different clinical outcome, which could be due to different immune responses to critical tumor-associated antigens. We used a guinea pig model to evaluate the immune responses to melanoma-associated carbohydrate antigens administered in whole cell and soluble lysate vaccines produced from the same cell lines and administered with or without Bacille Calmette-Guerin (BCG). Animals immunized with whole cell vaccine developed a significantly higher delayed-type hypersensitivity (DTH) reaction. The IgG response to all tumor-associated carbohydrate antigens except GD2 was significantly higher in animals immunized with whole cell vaccine than lysate vaccine. This study indicates that whole cell vaccine is superior to soluble or lysate vaccine because it induces a better immune response against cell-surface antigens. The addition of BCG significantly increased the antibody response, suggesting that an exogenous adjuvant may immunopotentiate antigens better in the presence of an intact cell membrane.

Development of a human monoclonal antibody to ganglioside G(M2) with potential for cancer treatment.

A human B-lymphoblastoid cell clone, L55-81, that produces human monoclonal antibody (MAb) to ganglioside G(M2) was established from peripheral blood B lymphocytes of a melanoma patient. L55-81 secretes IgMkappa light chain antibody in a serum-free medium. G(M2) specificity of the antibody was tested by immune adherence assay, TLC immunostaining, and ELISA. Anti-G(M2) antibody was shown to have the ability to kill the G(M2)-rich human melanoma cell line M14 in the presence of human or rabbit complement. A purified L55-81 MAb (>99.5% purity in protein concentration) was biotinylated and tested for its reactivity to various histological-type biopsied tumor and normal tissues in an avidin-biotin detection system. L55-81 MAb (20 microg/ml) reacted with several types of tumor tissues such as melanoma (7 of 10), colon carcinoma (4 of 5), ovary carcinoma (4 of 5), breast carcinoma (1 of 5), kidney carcinoma (1 of 5), and prostate carcinoma (1 of 5). None of the normal tissues derived from 24 different organs and adjacent normal tissues surrounding the cancerous tissues were stained. Production of the antibody in a serum-free medium, the cytotoxic potential with human complement, the inability to react to normal tissues, and the ability to target antigen-specific target cells make L55-81 a potential therapeutic agent for the treatment of cancers expressing ganglioside G(M2).

Quantitation of the density of cell surface carbohydrate antigens on cancer cells with a sensitive cell-suspension ELISA.

The density of carbohydrate epitopes on the surface of tumor cells is a governing factor for immune recognition and antibody-mediated targeting of tumor-associated carbohydrate antigens in cancer immunotherapy. A sensitive cell-suspension ELISA (cs-ELISA) is developed for quantitation of the functionally exposed carbohydrate epitopes on the cell surface. The factors affecting the measurement of tumor-cell surface glycoconjugates are evaluated using three human melanoma cell lines before and after exposure to various cell preservation treatments. The results of cs-ELISA are compared with the quantitative profile obtained by biochemical and flow cytometry assays. Cs-ELISA measures the density of the functionally exposed specific sugar epitopes on the surface of tumor cells, even in the presence of other similar carbohydrate antigens, provided that the monoclonal antibodies to carbohydrate epitopes are monospecific and sensitive, and that the cells are viable and present in optimal density. Of the three melanoma cell lines, M10-v and M101 expressed disialolactosyl residues of GD3 at concentrations of 5-6 pmol/10(6) cells and 2-3 pmol/10(6) cells, respectively. In both cell lines, the cell-surface GD2 was less than 1.0 pmol/10(6) cells. M24 melanoma cells expressed trace quantities (< 0.1 pmol/10(6) cells) of GD3 and GD2. Trypsinization of M10-v and M101 cells significantly reduced the cell-surface expression of GD3, suggesting GD3 loss, but increased the expression of GD2, suggesting crypticity of membrane-bound GD2. Cs-ELISA results showed that cryopreservation with 10% DMSO and irradiation at 15 krad decreased melanoma cell viability and ganglioside expression for M10-v but not M101 and M24. Formalinization did not affect cs-ELISA measurement of cell-surface carbohydrates. Cs-ELISA was used to monitor the quantity of incorporation of exogenous GD3 onto the surface of GD3-deficient M24 cells. Cs-ELISA for assessment of density of cell surface carbohydrate epitopes may be useful to characterize different types of tumors, to develop carbohydrate-based whole cell vaccines from tumor biopsies, to monitor the effects of cell preservation treatments commonly used in a whole cell vaccine preparation, and to evaluate the incorporation of a particular glycolipid (antigen or adjuvant) into glycolipid-deficient cells that are useful for carbohydrate-based active specific immunotherapy.

Attachment of monophosphoryl lipid A (MPL) to cells and liposomes augments antibody response to membrane-bound gangliosides.

Antibodies to gangliosides are found in low levels in normal individuals, and attempts to augment their production have had limited success. Murine studies suggest that the antibody response to membrane-bound cryptic antigens, such as phospholipids and gangliosides, can be induced and augmented by attaching lipid A to membranes. Therefore, we assessed the ability of monophosphoryl lipid A, a non-toxic derivative of lipid A, to augment antibody response against membrane-associated gangliosides. Anti-ganglioside antibodies were IgM after the first and second immunizations; in contrast, anti-phospholipid antibodies were IgM after the first immunization and IgG after the second immunization. Mice (BALB/c) immunized with MPL-attached human cells as well as mice (C57BL/6J) immunized with MPL-attached syngeneic tumor cells (B16 melanoma) produced a significant IgM response. Mice (C57BL/6J) immunized with MPL-attached liposomes containing GM3 developed significantly higher IgM responses than those immunized with purified gangliosides, MPL or MPL-free B16 cells. However, the antibody response after immunization with MPL-GM3-liposomes is similar to that after immunization with MPL-attached tumor cells, even though the MPL-liposomes contained a 27-fold higher level of gangliosides than the tumor cells. Our results emphasize that co-expression of MPL with membrane-bound gangliosides is necessary to augment the anti-ganglioside antibody response. These findings may shed light on the elevated titers of anti-ganglioside IgM antibodies found in patients with motor neuron diseases, various neuropathies and classical ALS, and are relevant to clearance of circulating immunosuppressive gangliosides in cancer patients.

Efficacy of tumor cell vaccine after incorporating monophosphoryl lipid A (MPL) in tumor cell membranes containing tumor-associated ganglioside.

Murine B16 melanoma expresses the ganglioside GM3. GM3 shed from tumor cells is immunosuppressive and promotes tumor growth. Reduction or elimination of the shed GM3 could be therapeutic, and the anti-GM3 antibodies may reduce and clear the shed ganglioside. To test this hypothesis, mice were challenged with tumor cells, with or without inducing anti-GM3 antibody response. Since gangliosides are poor immunogens and T-cell independent antigens, an adjuvant (monophosphoryl lipid A (MPL), a non-toxic lipid A of Salmonella), directed against B-cells, was employed. MPL was incorporated onto liposomes and into the surface membrane of B16 mouse melanoma cells; both are rich in GM3. C57BL/6J mice immunized with MPL-liposomes or MPL-B16 cells responded with elevated levels of anti-GM3 IgM. Non-immunized mice or mice immunized with B16 cells alone or ganglioside GM3 alone (without MPL) elicited poor anti-GM3 IgM response, confirming the GM3's immunologic crypticity and MPL's immunopotentiating effect. MPL's immunopotentiating effect was improved by coupling it to melanoma cell membranes. C57BL/6J mice were immunized with irradiated B16 alone or MPL alone or MPL-conjugated irradiated B16. After three weekly immunizations, each mouse received a challenge dose of viable syngeneic B16. Neither MPL alone nor B16 alone had a significant effect on tumor growth or host survival; however, administration of MPL-conjugated B16 cells significantly prevented tumor growth and prolonged survival. Our results indicate that MPL-incorporated B16 cells augment the anti-GM3 IgM response, which may reverse GM3-induced immunosuppression by eliminating tumor-derived GM3, and restore immunocompetence.

An improved method for the measurement of total lipid-bound sialic acids after cleavage of alpha 2,8 sialic acid linkage with Vibrio cholerae sialidase in the presence of cholic acid, SDS and Ca2+.

In the measurement of total lipid-bound sialic acids involving periodic acid oxidation, as in the periodate-resorcinol assay, the inner sialic acids of disialoglycolipids (such as GD3 and GD2) are not involved because their alpha 2,8 ketosidic linkages are resistant to periodic acid oxidation, even after acid/enzyme hydrolysis or alkali pretreatment. However, the sialic acids from these glycolipids can be recovered completely after cleavage of alpha 2,8 linkages by V. cholerae sialidase in the presence of cholic acid, sodium dodecyl sulphate and calcium. Interestingly, removal of calcium or detergent(s) or both significantly minimizes the sialidase action on the disialyl residues of these gangliosides. Therefore, we recommend sialidase (Vibrio cholerae) pretreatment of the glycolipids in the presence of cholic acid, SDS and Ca2+ for complete recovery of sialic acids from di- and polysialogangliosides and for accurate measurement of total lipid-bound sialic acids by periodate-resorcinol assay.

Factors affecting the fine specificity and sensitivity of serum antiganglioside antibodies in ELISA.

The major problem associated with ELISA of serum antiganglioside antibodies is the high background values (absorbancy of sera added to wells without ganglioside), which interfere with the accurate assessment of the fine specificity and sensitivity of these antibodies. This investigation identifies factors elevating the background values and/or decreasing the fine specificity, and describes strategies to minimize their influence. Using sera of neuropathy and melanoma patients, we found that highest background values were observed with the polystyrene 'tissue culture' microtiter plates; of the various 'non-tissue culture' microtiter plates tested, the lowest background values (> 0.060) were observed with Costar-3590 (H), Immunolon-3, Immunolon-1, Falcon-3915 (in increasing order). Background artifact of polystyrene microtest plates was significantly reduced by gamma irradiation (at 40 kRad) and/or use of detergent Tween-20 (0.1%) in the washing step. Even after controlling the background values, the fine specificity, namely, the ability of the antibody to distinguish between the target epitope of an antigen and epitopes of related antigens (when moles of antigen/well is constant) varied with different microtiter plates. Using sera with high affinity and specificity for GM2, GD3 or GM3, we observed that Immunolon-1, Immunolon-3 and particularly Falcon-3915 were superior for assessing the abilities of the antibodies to distinguish closely related epitopes found on other gangliosides. The reactivity of antiganglioside antibodies was more consistent after detergent treatment. The reactivity of antibodies to GD3 is significantly enhanced after treatment with Tween-20, but that of antibodies reacting to GM1 and GM2 is reduced. Fine specificity of the antiglycolipid antibodies was resolved better by coating glycolipids in mol/well rather than by weight/well. Based on these results, a protocol for a sensitive and reproducible ELISA for serum antiganglioside antibodies is recommended. The protocol takes into consideration the suitability of polystyrene plates, coating based on the number of molecules, pertinency of the solvent for coating, use of human serum albumin for blocking, dilution and washing steps and use of 0.1% Tween-20 to further minimize the background absorbancy.