Glycosphingolipid Antigens Research Articles

Specificities of human IgM and IgG anticarbohydrate xenoantibodies found in the sera of diabetic patients grafted with fetal pig islets

Abstract: Serum samples were collected from four diabetic patients who had received intraportal injections of pig fetal islet‐like cell clusters (ICC). The binding of IgM and IgG antibodies to glycosphingolipid antigens prepared from different pig organs and separated on thin layer plates was investigated in pre‐ and posttransplant serum samples. Both IgM and IgG antibodies in the pretransplant serum samples bind to several glycolipid fractions from the tri‐, tetra‐ and pentasaccharide regions but also to compounds with longer carbohydrate chains. In the posttransplant serum samples stronger binding, compared to the pretransplant samples, was noted for both Ig‐classes. Strong binding was seen in the pentasaccharide region known to contain the “linear blood group B” structure Galαl‐3Galβl‐4GlcNAcβl‐3Galβl‐4Glcβl‐lCer. There was no convincing evidence of the I recognition of new specificities. Adsorption of a patient serum to a Synsorbcolumn with Galαl‐3Gal specificity did not grossly change the binding pattern of the IgG antibodies in the effluate. However, the eluate from the column showed strong binding to the “linear B” compound but also to glycolipids with longer carbohydrate chains presumably with the same terminal epitope. Identification of these are in progress.The human natural anti‐pig antibodies against carbohydrate antigens expressed on pig cells seem to detect a rather limitted number of epitopes. The “linear B” structure appears to be one major target. However, other structures may also be targets for xenoreactive antibodies. Immunisation with pig cells does not seem to initiate antibody production against new carbohydrate epitopes but leads to an increased production against the existing ones.

Glycosphingolipid antigens in cultured bovine brain microvascular endothelial cells: sulfoglucuronosyl paragloboside as a target of monoclonal IgM in demyelinative neuropathy [corrected] [published erratum appears in J Cell Biol 1994 Oct;127(1):265

Since a number of anti-glycosphingolipid (GSL) antibody activities have been demonstrated in patients with various neurological disorders, the presence of common antigens between brain microvascular endothelial cells (BMECs) and the nervous tissues presents a potential mechanism for the penetration of macromolecules from the circulation to the nervous system parenchyma. We first investigated GSL composition of cultured bovine BMECs. Bovine BMECs express GM3(NeuAc) and GM3(NeuGc) as the major gangliosides, and GM1, GD1a, GD1b, GT1b, as well as sialyl paragloboside and sialyl lactosaminylparagloboside as the minor species. Sulfoglucuronosyl paragloboside was also found to be a component of the BMEC acidic GSL fraction, but its concentration was lower in older cultures. On the other hand, the amounts of neutral GSLs were extremely low, consisting primarily of glucosylceramide. In addition, we analyzed the effect of anti-SGPG IgM antibody obtained from a patient of demyelinative polyneuropathy with macroglobulinemia against cultured BMECs. Permeability studies utilizing cocultured BMEC monolayers and rat astrocytes revealed that the antibody facilitated the leakage of [carboxy-14C]-inulin and 125I-labeled human IgM through BMEC monolayers. A direct cytotoxicity of this antibody against BMECs was also shown by a leakage study using [51Cr]-incorporated BMECs. This cytotoxicity depended on the concentration of the IgM antibody, and was almost completely blocked by preincubation with the pure antigen, sulfoglucuronosyl paragloboside. Our present study strongly supports the concept that immunological insults against BMECs induce the destruction or malfunction of the blood-nerve barrier, resulting in the penetration of the immunoglobulin molecule to attach peripheral nerve parenchyma.

Human cervical epidermal carcinoma-associated intracellular localization of glycosphingolipid with blood group A type 3 chain.

A monoclonal antibody, MRG‐1, was produced by immunizing a mouse with a human ovarian mucinous cyst adenocarcinoma‐derived cell line, RMUG‐L. By immunohistochemical staining, the antigen was found to be exclusively localized in the intracellular structures of the cells used as the antigen and of the epithelial cells in normal human cervical glands. However, although the antigen was predominantly detected in the plasma membrane and the intercellular structure of the middle layer of normal human cervical squamous epithelium (92%), it was also contained in the intracellular structure of cervical epidermal carcinoma at a high frequency (80%). The striking difference in the distribution of the MRG‐1 antigen between normal and cancerous tissues was found to be a cervical carcinoma‐associated phenomenon and a useful tumor marker for immunohistochemical examination. Since the antigen was found to be of a blood group A‐related nature by immunohistochemical staining of the tissues and to be a glycosphingolipid, it was purified from human erythrocytes of blood group A, and the structure was concluded to be GalNAcα1–3Gal(2–1αFuc)jβ1–3GalNAcα1–3Gal(2–1αFuc)‐ β1–4GlcNAcβ1–3Galβl‐4Glcβ1–1′Cer, blood group A type 3 chain‐containing glycosphingolipid, by NMR, negative ion FABMS and permethylation analysis. In the subcellular localization analysis of the antigen, type 3‐A glycosphingolipid antigen was detected in the Golgi body and the microsomes of RMUG‐L cells, and the distribution coincided with the finding by immunohistochemical staining. In addition, in cervical epidermal carcinoma, although the blood group A, mainly type 2‐A chain, was localized in the plasma membrane and the intercellular structure, the blood group A type 3 chain was selectively found in the perinuclear structure. Also, the blood group A type 3 chain in cervical dysplasia as well as that in normal cervix was predominant in the plasma membrane. Thus, the selective intracellular localization of blood group A type 3 chain was a phenomenon characteristic of cervical epidermal carcinoma and the carcinoma in situ.

Surface expression of forssman glycosphingolipid antigen on murine bone marrow-derived macrophages is subject to both temporal and population-specific regulation and is modulated by IL-4 and IL-6

The Forssman glycolipid antigen (Fo) has been shown to be a differentiation marker for mouse macrophages both in vivo and in vitro.In order to determine whether or not there is a relationship between stage of differentiation and Fo expression, we have analyzed the kinetics of Fo expression during the growth of cultured mouse bone marrow-derived macrophages (BMDM).BMDM were grown in serum free medium to avoid the possible influence of undefined serum factors.In this medium they could be maintained over a period of up to 20 days with cell yields comparable to those obtained with serum-supplemented media.Fo antigen was assayed with a specific antibody using both a whole cell ELISA and immunocytochemical staining of cells grown on slides.With increasing age in culture, BMDM showed a gradual quantitative increase in Fo expression and parallel increase in the Fo+ BMDM fraction from about 10% Fo+ cells on the 10th day of culture to a maximum of 50%–60% Fo+ cells between the 17th and 19th days.The temporal control over the development of the Fo+ cell fraction was intrinsic to BMDM maturation but was specific for Fo.During the same time period expression of MHC class II (Ia) remained consistently low, whereas expression of both Mac-1 (C3bR) and the macrophage-specific marker ER-BMDM-1 was always high.The interleukins IL-4 and especially IL-6 induced a premature expression of Fo at earlier stages of BMDM culture, but neither could promote further Fo expression once the intrinsically occurring maximum had been reached.No evidence in support of an autocrine regulation of Fo expression by IL-6 could be obtained, nor could a connection between cell cycle status and Fo expression be established.These data provide further evidence that Fo is a temporally regulated differentiation marker for a mouse macrophage subpopulation and for modulation of its expression by lymphokines.

Glycosphingolipid antigens of Leishmania (Leishmania) amazonensis amastigotes identified by use of a monoclonal antibody

Monoclonal antibodies directed against Leishmania (Leishmania) amazonensis amastigotes were produced. One monoclonal antibody (1C3) selected by indirect immunofluorescence reacted with both amastigotes and promastigotes of L. (L.) amazonensis. Glycolipid extraction from L. (L.) amazonensis amastigotes and separation by high-performance thin-layer chromatography followed by immunoblotting demonstrated that 1C3 reacts with two glycosphingolipids which migrate chromatographically similarly to ceramide-N-acetylneuraminic acid (GM1) and ceramide-N-tetrose-di-acetylneuraminic acid (GD1a). The antibody did not react with glycosphingolipids from L. (L.) amazonensis promastigotes. Immunoprecipitation of 125I- and 35S-methionine-labeled promastigotes demonstrated that 1C3 recognizes gp63 from L. (L.) amazonensis promastigotes. Biosynthetic incorporation of labeled lipids by L. (L.) amazonensis amastigotes indicated that the glycosphingolipids reactive with 1C3 contain oleic acid in their structures. Surface labeling with galactose oxidase and sodium boro[3H]hydride indicated that galactose is present in 1C3-reactive antigens, strongly suggesting that these glycosphingolipids are localized on the surface of L. (L.) amazonensis amastigotes. Inhibition experiments of macrophage infection implicated the 1C3-reactive glycosphingolipids from L. (L.) amazonensis amastigotes in Leishmania invasion. The role of gp63 in promastigote-macrophage attachment was also demonstrated by inhibition experiments performed with 1C3, consistent with data from the literature.

Serological and immunochemical characterization of anti-PP 1 P k (anti-Tj a) antibodies in blood group little p individuals. Blood group a type 4 recognition due to internal binding

Serum samples from 13 blood group little p individuals were tested by radioimmunoassay for their IgG antibody subclass distribution against the P, p 1 and P k antigens. There was no uniform subclass distribution pattern, although all but one had IgG3 antibodies against all the P system antigens tested. Studies were performed adsorbing anti-Tj a serum sequentially to columns with synthetic carbohydrate antigenic determinants within the P system coupled to silica beads (Synsorbs R). The effect on agglutinin and indirect antiglobulin titers was determined after adsorption to Synsorbs R with different P-system antigens (p 1 , P k, P). Adsorption to all the three Synsorbs R was needed to eliminate or strongly reduce antibody titers. The effect on IgM, IgG, IgA as well as IgG subclass antibody binding to P, P 1 and P k antigens was also determined by radioimmunoassay and chromatogram binding assay. Anti-PP 1 P k antibodies from a little p woman with repeated abortions were shown to bind to glycosphingolipid antigens prepared from one of the aborted placentae using a chromatogram binding assay. This binding was eliminated by serum adsorption to Synsorbs R with P 1, P k and P carbohydrates, Anti-PP 1P k antibodies were also shown to bind to extended structures in the globoseries, i.e. globopentaosylceramide, globohexaosylceramide (globo-H) and globoheptaosylceramide (globo-A). This binding is most probably due to antibodies recognizing internal sequences in the carbohydrate chain. Attempts were made to visualize the binding epitope of the antibodies by computer molecular modelling.

Characterisation of the anti-A antibody response following an ABO incompatible (A 2 to O) kidney transplantation

Anti-A,B antibodies produced in a blood group OLe(a—b—) recipient receiving a kidney graft from a blood group A 2 Le(a—b + ) donor have been analysed for their ability to bind to different glycosphingolipid antigens. Solid-phase RIA using pure glycosphingolipid antigens and a chromatogram binding assay using total nonacid glycosphingolipid fractions from erythrocytes of different human blood group phenotypes together with pure glycolipid antigens were used as assay systems. Serum antibodies were shown to bind equally well to A (types 1, 2, 3 and 4) and B (types 1 and 2) antigenic structures but no binding to H antigens (types 1, 2 and 4) was detected. After adsorption of serum antibodies on A 1 Le(a—b + ) erythrocytes there was a residual anti-A antibody activity which could not be adsorbed by synthetic A-trisaccharides coupled to crystalline silica (Synsorb®-A). These residual antibodies, which are not present in a pretransplant serum sample, had a specificity for the A antigen with type 1 core saccharide chain and the binding epitope obviously included both the N-acetylgalactosamine and the N-acetylglucosamine. The fucose residue was apparently not obligate for binding. The conformation of the sugar units involved in the binding epitope was determined.

In vitro immunization of rat spleen lymphocytes with Forssman glycosphingolipid antigen and the generation of a monoclonal antibody

A procedure for in vitro immunization of splenic lymphocytes with a glycolipid antigen is described. Culture medium supernatant of ConA- and PHA-stimulated spleen cells and that of Con A-stimulated human Jurkat T cell line (IL-2-rich medium) were used as sources of cytokines to support T and B cell stimulation, and anti-mu was used to support B cell differentiation. Unprimed rat spleen cells (2 x 10(6)/ml) were stimulated with 2 micrograms/ml Forssman glycolipid antigen coupled to Sepharose for 4 days. The cells were fused with a mouse myeloma cell line P3-X63-Ag8-U1. At initial screening, 12% of the colony forming wells were secreting specific antibody. After cloning, a stable hybridoma cell line (designated 4C3) was established which secreted a monoclonal IgM antibody directed against the carbohydrate moiety of Forssman glycosphingolipid (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-ceramide).

Biosynthesis and turnover of O-acetyl and N-acetyl groups in the gangliosides of human melanoma cells.

We and others previously described the melanoma-associated oncofetal glycosphingolipid antigen 9-O-acetyl-GD3, a disialoganglioside O-acetylated at the 9-position of the outer sialic acid residue. We have now developed methods to examine the biosynthesis and turnover of disialogangliosides in cultured melanoma cells and in Golgi-enriched vesicles from these cells. O-Acetylation was selectively expressed on di- and trisialogangliosides, but not on monosialogangliosides, nor on glycoprotein-bound sialic acids. Double-labeling of cells with [3H]acetate and [14C]glucosamine introduced easily detectable labels into each of the components of the ganglioside molecules. Pulse-chase studies of such doubly labeled molecules indicated that the O-acetyl groups turn over faster than the parent molecule. When Golgi-enriched vesicles from these cells were incubated with [acetyl-3H]acetyl-coenzyme A, the major labeled products were disialogangliosides. [Acetyl-3H]O-acetyl groups were found at both the 7- and the 9-positions, indicating that both 7-O-acetyl GD3 and 9-O-acetyl GD3 were synthesized by the action of O-acetyltransferase(s) on endogenous GD3. Analysis of the metabolically labeled molecules confirmed the existence of both 7- and 9-O-acetylated GD3 in the intact cells. Surprisingly, the major 3H-labeled product of the in vitro labeling reaction was not O-acetyl-GD3, but GD3, with the label exclusively in the sialic acid residues. Fragmentation of the labeled sialic acids by enzymatic and chemical methods showed that the 3H-label was exclusively in [3H]N-acetyl groups. Analyses of the double-labeled sialic acids from intact cells also showed that the 3H-label from [3H]acetate was exclusively in the form of [3H]N-acetyl groups, whereas the 14C-label was at the 4-position. Pulse-chase analysis of the 3H/14C ratio showed that the N-acetyl groups of both GD3 and of the monosialoganglioside GM3 were turning over faster than the parent molecules. Selective periodate oxidation showed that both the inner and outer sialic acid residues of GD3 incorporated 3H-label in the in vitro reaction, and showed similar turnover of N-acetylation in the pulse-chase study. Taken together, these results indicate that both the O- and N-acetyl groups of the sialic acid residues of gangliosides turn over faster than the parent molecules. They also demonstrate a novel re-N-acetylation reaction that predicts the existence of de-N-acetyl gangliosides in melanoma cells.

Heterogeneity of Hanganutziu-Deicher antigen glycoproteins in different species animal sera.

A heterophilic Hanganutziu-Deicher (HD) antigen is present in many animal sera except human and chicken sera. To visualize the antigenic molecules, nine species animal and human sera were analyzed by SDS-PAGE, followed by Western blotting and immunostaining with avain anti-N-glycolylneuraminyl-lactosyl ceramide antibody which recognizes the terminal N-glycolylneuraminic acid moiety of glycoconjugates as an epitope of the HD antigen. Several HD antigen-active glycoprotein bands were detected in the sera of fetal calf, calf, horse, goat, monkey, rabbit, guinea pig, rat and mouse, except for human serum. The HD antigenic proteins showed heterogeneities in their molecular weights and were not identical with any major band visualized with silver-staining, indicating that they are minor components of serum proteins in each animal. Neuraminidase treatment destroyed the antigenicity of all proteins, confirming that N-glycolylneuraminic acid (NeuGc) at the non-reducing terminal of carbohydrate chains is the antigenic epitope in serum glycoprotein molecules as already confirmed in glycosphingolipid (GSL) antigens. The finding of HD-antigenic glycoproteins in animal sera suggests that they also stimulate HD antibody production in patients who received animal antiserum for therapeutic aim.

The glycosphingolipid composition of the human hepatoma cell line, Hep-G2

The origin of plasma glycosphingolipids in normal individuals and the mechanisms by which tumor-associated glycosphingolipid antigens enter the plasma in patients with cancer are largely unknown. The Hep-G2 human hepatoma cell line retains many of the characteristics of differentiated hepatocytes including the ability to synthesize and secrete lipoproteins. Preliminary results indicated that newly synthesized Hep-G2 cell glycosphingolipids are coupled to the secreted lipoproteins. This suggests that this cell line may offer an interesting model for studying glycosphingolipid secretion, transfer, and shedding. We now report on the chemical and immunological characterization of Hep-G2 cell glycosphingolipids. Five major glycosphingolipids were purified and biochemically characterized: glycosylceramide, lactosyl ceramide, ceramide trihexoside, ganglioside GM 3, and lactosyl sulfatide. Four additional minor components (3-fucosyllactosamine containing glycolipids, asialo GM 2, galactosylgloboside, and ganglioside GM 1) were identified using a combination of exoglycosidase digestion and immunostaining of thin-layer chromatography plates with specific carbohydrate binding proteins. This demonstrates that although this cell line synthesizes a limited number of major glycosphingolipids, it retains the ability to produce at least small amounts of structures in the lactoneo, globo, and ganglio series of glycosphingolipids. These studies show that it will be possible to investigate the mechanisms of secretion by Hep-G2 cells of different classes of these molecules such as neutral glycosphingolipids, gangliosides, and sulfatides.

Monoclonal antibody-defined antigen of human uterine endometrial carcinomas is Leb.

A monoclonal antibody, MSN-1, generated by immunizing a mouse with a human uterine endometrial adenocarcinoma cell line, SNG-II, was strongly and specifically reactive with neutral glycosphingolipids from cancer tissues of patients with uterine endometrial adenocarcinomas. The glycosphingolipid antigen was purified from pooled human meconia, which contained the antigen at the concentration of 1.95 mumol/g dry weight. Its structure was determined by NMR, negative ion FABMS, permethylation analysis, and TLC-immunostaining with monoclonal anti-Lc4Cer antibody, and was concluded to be the III4IV2Fuc2Lc4Cer,Leb antigen of the human Lewis blood system. On ELISA, the monoclonal antibody was found to be strongly reactive with Leb, slightly with Lea and not at all with A, B, H, or IV2FucGg4Cer. The amount of Leb in cancerous regions in the patients with the Lea-b+ blood group was significantly increased compared to that in normal regions in the same patients, and it was a newly appearing antigen in the cancerous tissue of a patient with the Lea+b- blood group.

Difference in glycosphingolipid compositions of avian Marek's disease lymphoma-derived cell lines and lymphoid leukosis lymphoma-derived cell lines.

We previously reported that Marek's disease (MD) lymphoma-derived cells, transplantable MD tumor-derived cells and lymphoid leukosis (LL) lymphoma-derived cells, each express different glycosphingolipid (GSL) antigens. The GSL composition of each cell line was compared by thin-layer chromatography and immunological analysis. Six different cell lines derived from MD lymphoma showed a typically identical GSL composition, in which a unique neutral GSL with a carbohydrate chain consisting of more than five sugars was detected as a major component. However, transplantable MD tumor-derived cell lines and LL lymphoma cell lines showed a similar composition in which Forssman GSL, asialo GM1 ganglioside and other several components were detected. The specific presence of Forssman GSL and asialo GM1 in these two cell lines was confirmed by immunological reaction of GSLs from each cell line with antisera to each authentic GSL and by the specific antibody elevation in rabbits immunized with each cell line.

Tissue localization and migration of murine spleen macrophages carrying the Forssman glycosphingolipid antigen.

Glycosphingolipids (GSLs) are receptors for virus (1), bacteria (2) and toxins (3). Their functions, if any, for the cells on whose surface they are expressed are still unclear. Recent data suggest the involvement of a sulfated GSL (4) in the cell-cell interactions of developing nervous tissue (5).

Use of the enzyme-linked immunoadsorbent assay to monitor the purification of glycosphingolipid antigens by high-performance liquid chromatography

An enzyme-linked immunoadsorbent assay (ELISA) technique has been applied to the analysis of glycosphingolipid fractions separated by high-performance liquid chromatography. Nanogram amounts of selected fractions were placed in microtiter wells and analyzed for glycosphingolipids carrying carbohydrate epitopes recognized by monoclonal antibodies using an avidin-biotin enzyme system (ABC reagents). A large number of fractions (more than 100) can be conveniently evaluated for the presence of glycosphingolipids recognized by one or more monoclonal antibodies in a single analysis. This method is a rapid and sensitive procedure for monitoring the purification of glycosphingolipid antigens and can be used in conjunction with immunostaining of glycosphingolipids separated by thin-layer chromatography.

Predefined gene transfer for expression of a glycosphingolipid antigen by transfection with a cosmid genomic library prepared from a cell line in which the specific glycosphingolipid is highly expressed

The deliberate transfer of globotriaosylceramide (Gb 3) expression in mouse lymphoma L5178 cells was achieved by transfection with a cosmid DNA library prepared from human Burkitt lymphoma Ramos cells in which Gb 3 was highly expressed. The recipient mouse lymphoma cells did not contain Gb 3 but did contain its direct precursor, lactosylceramide. The transfected cells expressed Gb 3, detected both chemically and immunologically, and contained human DNA detected by an Alu sequence probe. This model demonstrates a general method for studying glycosyltransferase genes and other factors necessary for the expression of glycosphingolipid antigens.

Glycosphingolipid immunostaining: Detection of antibody binding with an avidin-biotin enzyme system

Glycosphingolipids carrying carbohydrate sequences recognized by antibodies and lectins can be detected on thin layer chromatograms using an avidin-biotin enzyme system (ABC reagents). This same method can be used to detect glycosphingolipids blot-transferred from thin layer chromatograms to nitrocellulose. This method has certain advantages over the original radioimmunoassay method, including development of positive bands in minutes after incubation with the substrate, avoidance of handling hazardous radioactive materials and stability of reagents. We have demonstrated the usefulness of this method for immunostaining glycosphingolipids with both monoclonal and polyclonal anti-carbohydrate antibodies. These reagents have previously been used to detect carbohydrate antigens in tissues and isolated cells and now it is possible to use the same reagents for the detection of glycosphingolipid antigens on chromatograms.

Detection of 4- O-acetyl- N-glycolylneuraminyl lactosylceramide as one of tumor-associated antigens in human colon cancer tissues by specific antibody

Antibody to 4- O-acetyl- N-glycolylneuraminyl lactosylceramide [GM 3(Neu4AcGc)] was prepared by immunizing chicken with the glycosphingolipid antigen. The specific antibody was purified by affinity chromatography columns of Octyl-Sepharose linked to the homologous immunogen and its deacetylated analogue [ N-glycolylneuraminyl lactosylceramide, GM 3(NeuGc)], respectively. The specificity of the purified antibody was confirmed by enzyme-linked immunosorbent assay (ELISA) and inhibition of equine erythrocyte hemagglutination using authentic glycosphingolipids as antigens. The results indicated that the antibody recognized both 4- O-acetyl and N-glycolyl groups of terminal sialic acid residue as the immunodeterminants. The purified specific antibody was applied in the confirmation of the presence of GM 3(Neu4AcGc) in ganglioside fractions of human colon cancer tissues, which were suspected to have this antigen by studies of alkaline, periodate or neuraminidase treatment [Higashi et al. (1985) Cancer Res. 45, 3796–3802.], by thin-layer chromatography (TLC)-immunostaining technique.

An improved semi-quantitative enzyme immunostaining procedure for glycosphingolipid antigens on high performance thin layer chromatograms

An immunoassay is described which allows the detection of glycosphingolipid (GSL) antigens on high performance thin layer chromatograms (HPTLC). The method involves: (1) the separation of GSL on HPTLCs; (2) incubation with specific antibodies against carbohydrate structures of GSL, and (3) the detection of specifically bound antibodies with alkaline phosphatase-conjugated second antibodies and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) as substrate. Using a monoclonal rat IgG2c antibody against Forssman GSL, a BALB/c monoclonal antibody against asialo GM 2, and polyclonal rabbit antibodies against asialo GM 1, it was shown that as little as 3 ng GSL antigen could be detected in a procedure taking only 4 h to perform. The assay should be useful for screening mono- and polyclonal antibodies with potential specificity for GSL antigens, for the detection and quantification of GSL-antigens in tissue extracts, and for defining the specificity of anti-GSL antibodies.

Specificities of human heterophile Hanganutziu and Deicher (HD) antibodies to glycosphingolipids and a glycoprotein.

The specificities of five heterophile Hanganutziu and Deicher (HD) antibody-containing sera from four different cancer patients and one other diseased patients were compared. Three glycosphingolipids and one glycoprotein antigens and their chemically modified derivatives were used. The antibodies of all whole sera showed similar specificities. IgG and IgM antibody fractions of each serum were separated. Although antibodies of the same class showed similar specificities, differences were detected between the specificities of IgG and IgM. IgG antibody specificities were dependent on the hydrophobic (ceramide) group while IgM antibodies were directed more to the terminal sialic acid moiety of the glycosphingolipid antigens. The results suggested that a similar population of IgG-producing lymphocytes is stimulated in patients. Due to the similarities in specificities of HD antibodies, the results of this study will facilitate the future isolation of either IgG or IgM antibody-producing lymphocyte(s) from a patient with HD antibodies and the establishment of a monoclonal antibody through hybridization with a human myeloma cell line.