Heart cross-reactive antigens of mutans streptococci share epitopes with group A streptococci and myosin

Monoclonal antibodies (mAb) generated by immunization of mice with membranes of Streptococcus pyogenes Manfredo (M type 5) were tested for their reactivity with sodium dodecyl sulfate extracts of Streptococcus mutans GS5, Streptococcus rattus BHT (formerly S. mutans, serotype b), and S. mutans Ingbritt 175 in the Western blot. The reaction of the sodium dodecyl sulfate-extracted proteins of whole S. mutans, serotype b), and S. rattus with the mAb revealed that the shared cross-reactive antigens were near m.w. 62,000 to 67,000. Several higher m.w. proteins in S. mutans Ingbritt 175 reacted with the mAb but were not observed in the other two strains of streptococci. Cytoplasmic membranes of S. rattus BHT reacted with these mAb in the enzyme-linked immunosorbent assay and Western blot. The most reactive BHT membrane component detected by these mAb was a 62-kDa polypeptide that was similar in m.w. to the membrane component recognized by these antibodies in purified S. pyogenes membranes. The reactivity of the mAb with BHT membranes in the enzyme-linked immunosorbent assay was absorbed by rabbit skeletal muscle myosin. The patterns of reactivity observed in the BHT membrane immunoblots in this study closely resemble those previously obtained using polyclonal rabbit anti-human heart sera.     

Characterization of monoclonal antibodies raised toStreptococcus mutans

Monoclonal antibodies (MAb) to whole cells ofStreptococcus mutants P-4 (serotype e) were generated. Of the four MAb used in this investigation, three were found to cross-react with mouse heart tissue (MAb-HT) in the solid-phase ELISA, while the remaining MAb was found to be specific forS. mutants only (MAb-SM). Serotypic characterization has shown that MAb-SM as well as MAb-HT recognize strains ofS. mutans representing all of the eight currently known serological groups. In addition, MAb-HT showed reactivity withStreptococcus pyogenes, a Lancefield group-A streptococcus frequently implicated in rheumatic fever. Western blot analysis has shown that MAb-SM recognizes a 14,000-dalton component present in a partially purified membrane fraction recovered following glass-bead breakage ofS. mutans P-4 cells. Two of the MAb-HT were found to react with a 35,000-dalton component from this same preparation.     

Comparison of extracellular protein profiles of seven serotypes of mutans streptococci grown under controlled conditions

Extracellular proteins produced by the four human commensal species of mutans streptococci were analysed. The organisms used were Streptococcus mutans, serotypes c, e and f, Streptococcus cricetus, serotype a, Streptococcus rattus, serotype b, and Streptococcus sobrinus, serotypes d and g. They were grown in continuous culture at different generation times and pH values in media containing either glucose or fructose to determine the extent of variation in extracellular protein production that could occur for an individual strain. The results for different organisms grown under the same conditions were then compared. The total amount of protein of molecular mass greater than or equal to 60 kDa varied considerably with the growth conditions and with the strain. Generally more protein was present at a higher pH, conditions under which the organisms also form more lipoteichoic acid. With respect to individual protein components SDS-PAGE proved better than isoelectric focusing for detecting phenotypic responses by a particular strain to environmental changes and differences between the different strains. Differences in the molecular masses of protein components were particularly pronounced in the regions designated P1 (185-200 kDa), P2 (130-155 kDa) and P3 (60-95 kDa). Every strain produced at least one component in the P1 region that cross-reacted with antiserum to the purified protein from S. mutans serotype c, a protein which is indistinguishable from antigens B and I/II. Two components in the P2 region were dominant in the case of S. cricetus and S. sobrinus strains and showed glucosyltransferase (GTF) activity. GTF activity was also detected in the P3 region, particularly with S. mutans strains.     

Heart-reactive antibodies in rabbit anti-Streptococcus mutans sera fail to cross-react with Streptococcus mutans

Immunization of rabbits with Streptococcus mutans antigens results in the production of serum antibodies that bind in vitro to human, rabbit, and monkey cardiac muscle. Antibodies to heart, however, have also been reported to occur at lower titers in the sera of unimmunized rabbits. In this study, the specificities of heart-reactive antibodies (HRA) in sera of unimmunized and S. mutans-immunized rabbits were compared using indirect immunofluorescence, Western blot, and Bio-Dot immunoassays. Both groups of sera gave striational indirect immunofluorescence-staining patterns on thin sections of native human and monkey cardiac muscle. Western blot analyses revealed that antibodies in normal sera bound 9 to 20 components of human, rabbit, and monkey heart. The major bands had Mr of 205,000, 160,000, 135,000, and 70,000. Several of the normal sera did not have antibody activity to S. mutans antigens, indicating that these HRA do not cross-react with these bacteria. Although immunization of rabbits with S. mutans caused increased titers of HRA (two to three doubling dilutions), Western blot assays using anti-S. mutans sera showed banding patterns qualitatively similar to those of normal sera on heart extracts. Antibodies to skeletal muscle myosin were detected in both serum groups. Of eighteen normal rabbit sera sixteen had antimyosin titers of 10 to 40, whereas all eighteen anti-S. mutans sera had titers of 10 to 160. Affinity-purified antimyosin antibodies isolated from anti-S. mutans serum did not bind to S. mutans components. Conversely, affinity-purified antibodies to S. mutans antigens did not bind to myosin or to other cardiac muscle components. Among these were antibodies to the 185-kDa cell wall protein (also known as B, I/II, IF, Spa A, and P1) previously believed to possess antigenic mimicry. HRA were removed from anti-S. mutans sera by absorption with S. mutans but this effect was not specific, because a non-cross-reactive internal standard antibody was also absorbed to the same extent. Because previous evidence for antigenic mimicry between S. mutans and cardiac muscle was based on serum cross-absorption experiments, this immunologic relationship is not substantiated. These results indicated that naturally occurring antibodies to cardiac muscle components are present in the sera of unimmunized rabbits and that immunization with S. mutans does not stimulate production of new heart-reactive antibody, but rather serves to boost antibody production by preexisting clones of self-reactive B-lymphocytes.     

Chemical composition of Streptococcus mutans cell walls and their susceptibility to Flavobacterium L-11 enzyme

The susceptibility to a cell wall lytic L-11 enzyme from Flavobacterium sp. and the quantitative and/or qualitative composition of the cell walls of some strains of cariogenic Streptococcus mutans and a non-cariogenic strain of Streptococcus mitis were determined. The purified cell walls of S. mutans strains HS-1 (serotype a), BHT (b), NCTC10449 (c), C67-1 (c), C67-25 (c), OMZ 176 (d), MT703 (e), MT557 (f), OMZ65 (g), and AHT (g), and S. mitis CHT contained glutamic acid, alanine, and lysine as well as muramic acid and glucosamine as a peptidoglycan component. Besides these amino acids, significant amounts of threonine were detected in strains HS-1, OMZ65, and AHT cell walls, and considerable amounts of aspartic acid and/or threonine as well as several other amino acids in OMZ176, OMZ65, and CHT cell walls. Rhamnose was a common special component of the cell walls of S. mutans strains BHT, NCTC10449, MT703, B2 (e), MT557, and AHT, and S. mitis CHT. An additional sugar component, glucose, was detected in the cell walls of all of these strains except BHT, and galactose was found in BHT, AHT, and CHT cell walls. Galactosamine was present in S. mitis CHT cell walls. Varying amounts of phosphorus were detected in the cell walls of all the strains examined. The cell walls of all these streptococcal strains except MT703, 6715, and AHT were susceptible to the lytic action of the L-11 enzyme to various extents. No consistent relationship was observed between the amino acid and sugar composition of these cell walls and their susceptibility to the L-11 enzyme. The chemical composition of these cell walls is discussed in terms of the serological classification of S. mutans.     

A comparative study on cell wall antigens and cell surface hydrophobicity in clinical isolates ofCandida albicans

Characterization of common cell surface-bound antigens inCandida albicans strains, particularly those expressed in the walls of mycelial cells might be useful in the diagnosis of systemic candidiasis. Hence, antigenic similarities among wall proteins and mannoproteins fromC. albicans clinical serotype A and B isolates, were studied using polyclonal (mPAbs) and monoclonal (MAb 4C12) antibodies raised against wall antigens from the mycelial form of a commonC. albicans serotype A laboratory strain (ATCC 26555). Zymolyase digestion of walls isolated from cells of the different strains studied grown at 37°C (germination conditions), released, in all cases, numerous protein and mannoprotein components larger than 100 kDa, along with a 33–34 kDa species. The pattern of major antigens exhibiting reactivity towards the mPAbs preparation was basically similar for all the serotype A and B isolates, though minor strain-specific bands were also observed. The immunodeterminant recognized by MAb 4C12 was found to be absent or present in very low amounts inC. albicans isolates other than the ATCC 26555 strain, yet high molecular weight species similar in size (e.g., 260 kDa) to the wall antigen against which MAb 4C12 was raised, were observed, particularly in wall digests from serotype A strains. Cell surface hydrophobicity, an apparently important virulence factor inC. albicans, of the cell population of each serotype B strain was lower than that of the corresponding serotype A counterparts, which is possibly due to the fact that the former strains exhibited a reduced ability to form mycelial filaments under the experimental conditions used.Abbreviations CSH cell surface hydrophobicity - IIF indirect immunofluorescence     

Cell wall glycoproteins of Candida albicans as released by different methods

Different methods of extraction frequently used in other studies were used to release glycoproteins from both intact cells and isolated cell walls of yeast and hyphal forms of Candida albicans. Extracts were obtained from whole cells by treatment (i) with 2-mercaptoethanol (beta ME) at pH 8.6 and 37 C degrees and (ii) with zymolyase after treatment with beta ME. Extracts were obtained from isolated and washed cell walls (i) by boiling with beta ME and sodium dodecyl sulphate (SDS), (ii) by boiling with SDS and (iii) by treatment with zymolyase after SDS. The extracts were separated by SDS-polyacrylamide gel electrophoresis and analysed by Western blotting with four reagents. Analysis with concanavalin A (ConA) revealed different glycoprotein populations depending on the treatment. Three possible germ-tube-specific constituents were observed; and 80 kDa component released by beta ME from both intact cells and cell walls, and 47 kDa and 43 kDa moieties released by zymolyase only from intact cells. MAb 4C12, specific for the protein portion of a large germ tube constituent, recognized polydisperse material which just entered the gel in beta ME extracts and in the region extending up from 200 kDa to near the top of the gel in zymolyase extracts. MAb 24.17, specific for a carbohydrate determinant of yeast phase cells, reacted with disperse material in the region from the top of the gel to one-third to two-thirds the distance to the 220 kDa mass marker. Antiserum specific for the serotype A determinant of mannan reacted with large disperse component(s) migrating in the region from the top of the gel to about two-thirds the distance to the 220 kDa mass marker and with a 180 kDa component. The components recognized by MAb 4C12, but not those recognised by MAb 24.17 and serotype A antiserum, were effected by treatment with endo-beta-N-acetylglucosamidase H. The various analyses revealed that the method of extraction affected the composition and size of the constituents recognized by the reagents.     

Monoclonal antibodies to Streptococcus mutans serotype f polysaccharide cross-react with the 'mutans group' of oral streptococci

Abstract Eleven stable clones producing monoclonal antibodies (MAbs) against serotype polysaccharide f were generated from rat IR 983F myeloma cells and splenocytes from rats immunized with formalin-killed whole Streptococcus mutants OMZ 175. These MAbs were IgG 2b, they had different affinities towards polysaccharide f and all but three were able to inhibit saliva binding to S. mutants OMZ 175 cells. Two specificities were recognized on S. mutans serotype f polysaccharide. One MAb (K24) recognized an epitope on the poly-rhamnose core, as determined by competitive ELISA using different sugars. The ten other MAbs recognized an epitope which seems principally formed of glucose subunits. Purified polysaccharides from other 'mutans streptococci' were able, to different extents, to inhibit the binding of the MAbs to polysaccharide f, confirming the presence of common epitopes in the different strains.     

Immunochemical characteristics of Streptococcus mutans serotype h carbohydrate antigen

Serotype h carbohydrate antigen was prepared from cell walls of Streptococcus mutans strain MFe28 of monkey origin. The h antigen was extracted from the cell walls with 5% trichloracetic acid at 4 C, and purified by DEAE-Sephadex A-25 ion exchange chromatography followed by Sephacryl S-300 gel filtration. The purified antigen was composed of galactose (75%), glucose (16%), and rhamnose (3%). Although the antiserum against whole cells of S. mutans MFe28 gave a strong cross reaction with serotype d S. mutans, serotype h-specific antiserum could be obtained by adequate adsorption. The precipitin reactions and hapten inhibition test using serotype h-specific antiserum showed that galactose, glucose, and their derivative sugars were markedly potent inhibitors. It was concluded that the serotype h antigen is immunologically distinguishable from the known serotypes of S. mutans, although it is closely related to serotype d antigen of S. mutans.     

Wall-associated protein antigens of Streptococcus mutans.

When heat-killed whole organisms of Streptococcus mutans strain Ingbritt (serotype c) were injected into rabbits, antibodies to at least 12 antigens were detectable by crossed immunoelectrophoresis. In contrast, when rabbits were immunized with organisms which had been subjected to extraction with the detergent sodium dodecyl sulphate (SDS), antibodies to only two protein antigens were found. These two proteins (A and B), while existing in a form apparently closely associated with peptidoglycan, could also be recovered from homogenates of whole organisms after sonication and from culture filtrates. Antigenic material was excreted throughout growth. SDS-polyacrylamide gradient gel electrophoresis showed A to have a molecular weight of 29 000, while B had a molecular weight of 190 000. Antigen B was purified to apparent homogeneity as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing. All of six strains of serotype c examined produced antigen B. Strains of serotypes e and f also produce antigenically identical proteins and strains of serotypes d and g produce proteins which cross-reacted with antigen B. Antigen B was specifically precipitated by rabbit antiserum to human heart tissue.     

Genetic heterogeneity in Streptococcus mutans

The genetic homogeneity among eight cariogenic strains of Streptococcus mutans was assessed by deoxyribonucleic acid (DNA)-DNA reassociation experiments. DNA species were extracted from strains GS5, Ingbritt, 10449, FAl, BHT, E49, SLl, and KlR. Labeled DNA ((14)C-DNA) was extracted from strains 10449, FAl, and SLl. Denatured (14)C-DNA fragments were allowed to reassociate, i.e., form hybrid duplexes, with denatured DNA immobilized on membrane filters incubated in 0.45 m NaCl-0.045 m sodium citrate at 67 or 75 C. At 67 C, 10449 (14)C-DNA reassociated extensively only with GS5 and Ingbritt DNA. FAl (14)C-DNA hybridized extensively only with BHT DNA, and SLl (14)C-DNA reassociated with KlR and E49 DNA. DNA which hybridized extensively at 67 C also reassociated to a high degree at 75 C. Thermal elution of (14)C-FAl-BHT duplexes showed that the hybrid duplexes were thermostable. The results indicate that S. mutans is a genetically heterogeneous species. The strains studied can be divided into three (possibly four) genetic groups, and these groups closely parallel antigenic groups.     

Human IgG and Streptococcus mutans SR protein contain cross-reactive epitopes

Antigen B, a glycoprotein present on the cell surface of "mutans streptococci," mediates bacterial adherence to teeth surfaces and has been implicated in cross-reactivity with human heart components. Elevated levels of anti-IgG antibodies were generally found in sera of rabbits immunized with protein SR, a B-related protein from Streptococcus mutans serogroup f, or recombinant protein SR (rSR). These anti-IgG antibodies could be involved in the previously mentioned heart cross-reactivities. Results from immunoblots and ELISA analyses demonstrate that these anti-IgG antibodies recognize common epitopes on SR, rSR, and human IgG2 and IgG4 probably located on the Fab region. Furthermore, control experiments with biotinylated human IgG show that the cross-reactions between IgG and SR were not mediated by an FcR mechanism. Direct competition between rSR and human IgG in binding to anti-IgG or anti-SR antibodies confirm that S. mutans SR protein possesses Ag mimicry with human IgG. Our studies provide some evidence that S. mutans SR protein and human IgG H chains share autoimmune epitopes which could play a role in the induction of anti-IgG antibodies and therefore could explain the enhancement of anti-IgG antibody levels observed in rabbits immunized with either S. mutans whole cells or purified B-related Ag.     

Purification and immunochemical properties of a protein antigen from serotype g Streptococcus mutans

A proteinaceous antigen (PAg) was purified from the culture supernatant of Streptococcus mutans 6715 (serotype g) by ultrafiltration, ammonium sulfate precipitation, DEAE-Sephacel ion-exchange chromatography, Phenyl-Sepharose CL-4B hydrophobic chromatography, and subsequent Sephacryl S-300 gel filtration. A yield of 0.1 mg of PAg was obtained from a liter of culture supernatant. The isoelectric point and molecular weight of PAg were pH 4.6 and 210,000, respectively. It contained 35% sugar, which was identified as glucose by gas-liquid chromatography. Amino acid analysis revealed that PAg contains 28% acidic and 11% basic amino acid residues. PAg retained its antigenicity after heating at 80 C for 10 min in deionized water, or after treatment with 0.1 M HC1 or 0.1 M NaOH at 37 C for 1 hr. Immunodiffusion and immunoelectrophoresis analyses revealed that PAg is serologically distinct from other cell-surface antigens such as serotype-specific polysaccharide and lipoteichoic acid. A cross-reaction between PAg and a protein antigen similarly prepared from serotype c S. mutans was observed in immunodiffusion tests.     

Subcellular localization of the Streptococcus mutans P1 protein C terminus.

To determine the subcellular location of the Streptococcus mutans P1 protein C-terminal anchor, cell envelope fractionation experiments were conducted in combination with Western immunoblotting, using monoclonal antibody MAb 6-8C specific for an epitope that maps near the C terminus of P1 protein and also a polyclonal antibody preparation directed against the P1 C-terminal 144 amino acids (P1COOH). P1 protein was detected in cell walls but not the membrane purified from S. mutans cells by the monoclonal antibody. In contrast, P1 protein was not detected in the same cell wall preparation using the anti-P1COOH polyclonal antibody. However, proteins released from the cell walls by treatment with mutanolysin contained antigen that was recognized by the anti-P1COOH antibody, suggesting that the epitopes recognized by the antibody were masked by peptidoglycan in the cell wall preparations. When cell walls were treated with boiling trichloroacetic acid to solubilize cell-wall-associated carbohydrate, P1 antigen could not be detected in either the solubilized carbohydrate, or in the remaining peptidoglycan, regardless of whether polyclonal or monoclonal antibody was used. However, when the peptidoglycan was treated with mutanolysin, P1 antigen could be detected in the mutanolysin solubilized fraction by MAb 6-8C. Collectively, these data suggest that the C-terminal 144 amino acids of the P1 protein are embedded within the cell wall, and associated exclusively with the peptidoglycan. Furthermore, the ability of the anti-P1COOH antibody to recognize P1 antigen only after mutanolysin treatment of cell walls suggests these C-terminal 144 amino acids are tightly intercalated within the peptidoglycan strands.     

Complete Genome Sequence of Streptococcus mutans GS-5, a Serotype c Strain

Streptococcus mutans, a principal causative agent of dental caries, is considered to be the most cariogenic among all oral streptococci. Of the four S. mutans serotypes (c, e, f, and k), serotype c strains predominate in the oral cavity. Here, we present the complete genome sequence of S. mutans GS-5, a serotype c strain originally isolated from human carious lesions, which is extensively used as a laboratory strain worldwide.     

A comparative study of the extracellular glucosyl- and fructosyltransferases from cariogenic and non-cariogenic Streptococcus mutans strains of two different serotypes

Extracellular proteins from continuous cultures of serotype c and g Streptococcus mutans strains were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Gels stained with raffinose after electrophoresis revealed that although serotype c strains secrete two fructosyltransferases of molecular mass 68 kDa and 79 kDa, no fructosyltransferase was secreted by the serotype g strain K1. A sucrose activity stain was used to detect two glucosyltransferases (GTF) of molecular mass 162 kDa (bifunctional 1,6-alpha-D-glucan 3-alpha- and 6-alpha GTF or 'dextransucrase') and 153 kDa (a 1,3-alpha-D-glucan 3-alpha-GTF) in samples from cariogenic serotype c strains. Neither the 153 kDa protein nor the corresponding GTF activity was secreted by the non-cariogenic mutant C 67-25. The molecular masses of the corresponding 1,3-alpha and 1,6-alpha-GTF proteins from the serotype g strain K1 were 164 kDa and 158 kDa, respectively. All of the GTF proteins were degraded to discrete bands of lower molecular mass on storage at 4 degrees C even after extensive purification. The results provide an explanation for several outstanding controversies in the GTF literature.     

DnaK expression in response to heat shock of Streptococcus mutans

Abstract The oral pathogen, Streptococcus mutans , persistently colonizes human hosts and initiates oral disease despite extreme variations in environmental conditions. To begin to investigate the role of the stress protein, DnaK (Hsp70), in environmental stress responses by S. mutans , pulse—chase experiments were initially used to establish that a functional heat shock response existed in this organism. A C-terminal fragment of the S. mutans dnaK gene was cloned and engineered to be expressed with a histidine tag. Using the recombinant DnaK protein that had been purified by nickel affinity chromatography, an antibody specific for the S. mutans DnaK protein was generated to analyse DnaK expression under homeostatic and heat shock conditions. Western blot analysis indicated that the anti-recombinant DnaK antibody specifically recognized a protein (molecular mass approx. 68 kDa) which was induced in response to thermal stress. Elucidating the role of DnaK in responses by S. mutans to various environmental Stressors will provide a better understanding of how DnaK is involved in survival of extreme environments and the contribution of the DnaK protein to the virulence of S. mutans .     

Production and characterization of monoclonal antibodies against vegetative cells of Bacillus cereus

Two monoclonal antibodies (MAbs) against Bacillus cereus were produced. The MAbs (8D3 and 9B7) were selected by enzyme-linked immunosorbent assay for their reactivity with B. cereus vegetative cells. They reacted with B. cereus vegetative cells while failing to recognize B. cereus spores. Immunoblotting revealed that MAb 8D3 recognized a 22-kDa antigen, while MAb 9B7 recognized two antigens with molecular masses of approximately 58 and 62 kDa. The use of MAbs 8D3 and 9B7 in combination to develop an immunological method for the detection of B. cereus vegetative cells in foods was investigated.     

Characteristics of a high molecular weight extracellular protein of Streptococcus mutans

A high molecular weight protein antigen, designated P1, has been isolated from the culture fluid of chemostat-grown Streptococcus mutans strain Ingbritt and shown to be free of other antigens including glucosyltransferase. Antiserum against the protein was used in rocket immunoelectrophoresis to confirm and extend the previous observation that there were major differences in the amount of the protein produced under different growth conditions. Physico-chemical and serological studies indicated that protein P1 was indistinguishable from antigens B, I/II and IF isolated in other laboratories. Mammalian tissue cross-reactivity of protein P1 was demonstrated by binding of antiserum to P1 to sections of normal rabbit tissues, particularly heart. There was also a statistically significant increase in the number of mononuclear leucocytes in heart tissue of rabbits which had been injected with protein P1, when compared with the levels in control uninjected rabbits; injection with whole cells of S. mutans Ingbritt did not produce this effect.     

Immunochemical characterization of the human blood cell membrane glycoprotein recognized by the monoclonal antibody 12E7.

The 12E7 murine monoclonal antibody recognizes a protease-sensitive component of human red cells, platelets and lymphocytes which could not be detected on granulocytes. Scatchard analyses indicated that the 125I-labelled antibody binds to 1000, 4000 and 27,000 antigen sites on each red cell, platelet and lymphocyte respectively, with a binding constant ranging from 4 x 10(7) to 9 x 10(7) M-1. The membrane components recognized by the monoclonal antibody were characterized by immunostaining on nitrocellulose sheets. A 28 kDa sialoglycoprotein was visualized following electrophoretic transfer of the red cell and lymphocyte membrane proteins separated by SDS/polyacrylamide-gel electrophoresis. Another component of 25 kDa was also clearly identified in the lymphocyte and platelet lysates, but was barely detectable in the red cell membrane preparations. Enzyme treatment of intact platelets, as well as analysis of the membrane and cytosolic preparations from these cells, have shown that the 25 kDa component was of cytoplasmic origin. The mobility of the 28 kDa membrane component is decreased following neuraminidase treatment of intact blood cells, but these cells still react normally with the monoclonal antibody, indicating that sialic acids are not required for binding. The 28 kDa component is present on red cell membranes prepared from S-s-U-, En(a-) and Gerbich(-) individuals, demonstrating that it is a new sialoglycoprotein not derived from glycophorins A, B, C or D. The 28 kDa component was totally solubilized with 0.1% Triton X-100 from red cell membranes and behaves like the other red cell membrane sialoglycoproteins since it was extracted in the aqueous phase following chloroform/methanol/water or butanol/water partitionings. The 28 kDa component could be partially purified by h.p.l.c. gel permeation chromatography and preparative SDS/polyacrylamide-gel electrophoresis. The material finally obtained strongly inhibits the 12E7 monoclonal as well as human anti-Xga antibodies, suggesting either that the 28 kDa glycoprotein carries both antigens or that the 12E7 and Xga-active molecules copurified.