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Panose (O-α-D-glucosyl-(16)-O-α-D-glucosyl-(14)-α-D-glucose) and maltose (O-α-D-glucosyl-(14)-α-D-glucose) coupled to bovine serum albumin (BSA) by means of an azophenyl linkage provided the synthetic antigens panosyl-BSA and maltosyl-BSA, used to examine the anti-hapten specificity of rabbit sera prepared against panosyl-BSA. The combining site specificity of cross-reactive antibodies in carrier-absorbed anti-panoside sera was investigated by quantitative hapten inhibition of maltosyl BSA-anti-panoside cross-precipitation and compared with data obtained by inhibition of homologous precipitation. Immunochemical data obtained demonstrate a heterogeneity in the combining site specificity of anti-hapten produced in response to panosyl-BSA and establish the occurrence of at least two distinct kinds of antibody directed against the α-(16)-α-(14) sequence of glucose units. One type of anti-panoside, inhibited more effectively by isomaltose than by maltose, shows isomaltose end group immunodominance. A second type of anti-panoside, cross-precipitated by maltosyl-BSA and more effectively inhibited by maltose than by isomaltose, shows maltose immunodominance.

Panosyl-BSA, a synthetic sugar-azoprotein conjugate consisting of the trisaccharide panose (glucosyl α-(1,6)-glucosyl-α(1,4) glucose) coupled to bovine serum albumin by an azphenyl linkage, cross precipitates antibody from types II and XX antipneumococcal horse sera. Hapten inhibition indicates that these cross reactions of synthetic antigen are mediated by the α-isomaltosyl moiety of the introduced panosyl haptenic grouping. While interaction of conjugate with horse anti-XX involves a fraction of antibody directed against an α-isomaltosyl end group, interaction with horse anti-II involves a fraction of antibody directed against the α-isomaltosyl end group. Purified fractions of antibody, cross reactive with panosly-BSA were specifically from antipneumococcal horse sera and their behavior with homologous antigen examined and compared with unfractionated serum.

Plasma glutathione peroxidase (GSHPx) (glutathione: H2O2 oxidoreductase) is a unique selenoglycoprotein. Treatment of this enzyme with glycopeptidase F partially deglycosylates it and establishes the presence of N-linked sugar moieties. Antibodies raised in a rabbit against the purified enzyme from plasma were found to be specific, noninhibitory, and capable of precipitating the enzymatic activity. The antibodies precipitated greater than 90% of the GSHPx activity of normal plasma, thus indicating that the selenoenzyme is the main if not the sole GSHPx activity of plasma. The antibodies did not precipitate RBC GSHPx. A slight cross-reactivity of the antibodies was found with rat plasma GSHPx. A GSHPx activity precipitation assay of normal plasma in the presence of selenium (Se)-deficient plasma indicates that no cross-reactive protein in the Se-deficient plasma interferes with the precipitation of the GSHPx activity from normal plasma. Thus, GSHPx protein as well as activity is deficient in plasma in the absence of Se. Antibodies against GSHPx either from RBCs or from plasma were used to specifically immunoprecipitate most of the GSHPx activity from RBCs or plasma, respectively, in healthy individuals to determine the amount of Se associated with the protein. GSHPx accounts for approximately 15% of the Se in RBCs and 12% of the Se in plasma. Thus, in normal individuals, these proteins account for only a fraction of plasma and RBC Se.

Conjugates consisting of methyl 2( p -azobenzamido)2-deoxy-α- d -glucopyranoside and methyl 2( p -azobenzamido)-2-β- d -glucopyranoside coupled to bovine serum albumin were used as synthetic antigens. Rabbits immunized with artificial antigen provided specific anti-methyl α-glucosaminide or anti-methyl β-glucosaminide anti-hapten sera. Quantitative hapten inhibition was employed to elucidate the specificity of anti-hapten combining sites and to evaluate some structural and configurational features contributing to the immunochemical interaction with the antibody. It is shown that the configuration at the anomeric carbon atom makes a major contribution to the immunochemical specificity of the anti-hapten antibodies against α- and β-methyl glycosides.

Following mild acid treatment (pH 1.1 for 1 h at 100°C), the extracellular polysaccharide of Xanthomonas campestris (NRRL B-1459) acquires a weak cross-reactivity with horse antipneumococcal type VIII (anti-Pn VIII) serum. The interaction of mild acid-treated xanthomonal polysaccharide with anti-Pn VIII was examined by quantitative precipitation and hapten inhibition and compared with data similarly obtained for oat glucan. Immunoglobulins isolated by a cellobiose-strong salt extraction of specific precipitates formed by anti-Pn VIII with xanthomonal antigen, oat glucan, lichenin, and types III and VIII Pn capsular polysaccharides were identified by the use of monospecific anti-equine heavy chain reagents. Various oligosaccharides, including di-, tri-, tetra-, and pentasaccharides of β -(1 →4)-linked glucose as well as G β (1→3) G β (1→4)G and G β (1→3) G β (1→4) G β (1→4)G, were assayed for their ability to inhibit anti-Pn VIII precipitation by xanthomonal antigen. The inhibition data obtained show that combining sites of antibody cross-precipitated by xanthomonal antigen optimally accommodate a cellodextrin structure consisting of a terminal or internal sequence of only two β -(1→4)-linked glucose units. Inhibition data showing an ordering (cellopentaose = cellotetraose > cellotriose > cellobiose) in the relative ability of cellodextrins to inhibit oat glucan precipitation establish that a sequence for four consecutive glucose units in β -(1→4) linkage provides the optimal cellodextrin ligand structure accommodated by combining sites of the anti-Pn VIII fraction interacting with oat glucan.

The isolation and properties of a protein obtained in crystalline form from bovine liver, kidney, and spleen are described. The protein, which appears not to have been previously described, was originally named football protein by Sumner and Dounce. Football (FTBL) protein is a tetramer having a molecular weight of about 264,000 A monomer molecular weight of about 66,000 was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A s 20 =11.54, a rough diffusion constant of 3.68×10 −7 cm 2 /s, av=0.739, and an amino acid composition were obtained for the protein. The protein was also prepared from lamb liver in a concentrated but noncrystalline form. Anti-FTBL protein, prepared by immunizing rabbits with 2x crystallized beef liver protein, was used as a reagent to determine the tissue distribution of FTBL protein. The protein is present in beef liver, spleen, kidney, and white blood cells, but absent from beef red blood cells. An immunochemically related but not identical protein is present in rat liver, spleen, and kidney, but is not detected in rat thymus, pancreas, or heart muscle. As determined by immunodiffusion analysis, beef liver, kidney, and spleen protein and the lamb liver protein are immunologically indistinguishable from one another. Although tested on various substrates, no enzymatic activity has been found yet which can be attributed to crystalline beef FTBL protein. A list of the enzymes tested is given.

Human plasma glutathione peroxidase (GSHPx) has been shown to be a glycosylated selenoprotein distinct enzymatically, structurally, and antigenically from known cellular glutathione peroxidases. The extracellular location of the enzyme and the fact that it is glycosylated suggested that it is a secreted protein. Utilizing mutually non-cross-reactive antibodies to human cellular and plasma GSHPx, we conducted a search to determine the tissue of origin for plasma GSHPx. The cells screened were endothelial cells because they are the main source of extracellular superoxide dismutase, HL-60 cells (myeloid cell line) because they are the main source of extracellular H2O2, and Hep G2 cells (hepatic cell line) because they are the source of many plasma proteins. Human umbilical vein endothelial cells were metabolically labeled with either [35S]methionine or [75Se]selenious acid, and HL-60 cells and Hep G2 cells were metabolically labeled with [75Se]selenious acid. Proteins were immunopurified from the labeled cells and their media with either anti-red blood cell (RBC) GSHPx IgG or with anti-plasma GSHPx IgG. Utilizing anti-RBC GSHPx IgG, only the cellular form of the enzyme was precipitated from all the cells tested but not from their media. When anti-plasma GSHPx IgG was applied to the cells and their media, a selenoprotein was precipitated only from the media of Hep G2 cells. When Hep G2 cells were incubated in the presence of the carboxylic ionophore monensin, an intracellular selenoprotein could be detected using anti-plasma GSHPx IgG. The precipitation of the cellular form from all three cell types was partially inhibited by preincubation of the anti-RBC GSHPx IgG with purified RBC GSHPx while the precipitation of the selenoprotein from the medium of Hep G2 cells by anti-plasma GSHPx IgG was prevented by preincubation of the antibody with purified plasma GSHPx. We suggest that plasma GSHPx is synthesized by and secreted from hepatic cells. This is, to the best of our knowledge, the only known selenoprotein with a defined function that has been shown to be synthesized for secretion by mammalian cells.

Salmonella telaviv, Salmonella tranoroa, and Salmonella illinois were examined for their ability to interact with 15 purified lectins of known sugar specificity. The only interaction observed was between the lectin of Maclura pomifera and S. telaviv. M. pomifera lectin specifically agglutinated suspensions of S. telaviv and precipitated with its purified lipopolysaccharide and isolated lipid A free O polysaccharide. Quantitative inhibition assays showing methyl-alpha-D-galactopyranoside and N-acetyl-D-galactosamine to be potent inhibitors of Maclura lectin precipitation by S. telaviv O polysaccharide suggest that the interaction is mediated by D-galactose or N-acetyl-D-galactosamine units of bacterial polysaccharide structure, or both.

Interactions of polymeric McPC 870 and MOPC 384 mouse IgA myeloma proteins with a streptococcal cell wall tetraheteroglycan (T4-antigen), Salmonella tranoroa lipopolysaccharide (LPS), S. telaviv LPS and its lipid A free polysaccharide were examined by the quantitative precipitin method. Hapten inhibition of myeloma protein precipitation by T4-antigen or S. telaviv LPS indicates a complex anticarbohydrate specificity for 870 and 384 protein combining sites involving several sugars. While the combining site of 870 protein appears to bind alpha-D-mannopyranosyl, alpha-D-galactopyranosyl and alpha-D-glucopyranosyl residues, the combining site of 384 protein can accommodate both the alpha (1----2)-linked glucobiose (kojibiose) and alpha-D-galactopyranosyl but not the alpha-D-mannopyranosyl structure.

The crystalline beef liver protein of Sumner and Dounce (A. L. Dounce, P. Z. Allen, and G. A. Mourtzikos (1978) Arch. Biochem. Biophys. 188 , 251–265 (1)) termed FTBL (football) protein because of the shape of its crystals, has been identified as a crystalline leucine aminopeptidase (LAP), on the basis of its high specific LAP activity and coincidence of its N terminal amino acid sequence (30 amino acids) with that of beef eye lens LAP. Amino acid analyses of the two proteins are also in reasonable agreement when based on the exact monomer molecular weight of beef eye lens protein obtained by the van Loon group ((1982) J. Biol. Chem. 257 , 7077–7081) . Our previously published (1) monomer molecular weight of the FTBL protein was 25% too high, leading to the erroneous conclusion that the beef liver FTBL-LAP protein was a tetramer rather than a hexamer, as found by the van Loon group for beef lens LAP. The present report, taken together with our first paper on the FTBL protein establishes that the FTBL-LAP protein has been isolated from beef kidney and beef spleen as well as from beef liver. We now find that the properties of FTBL-LAP protein indicate that it is the same protein as beef eye lens LAP. The cellular and intracellular distributions of the FTBL-LAP protein have been considered in our first publication on the FTBL protein (1).

Pneumococcal type 37 capsular polysaccharide was obtained free of contaminants by affinity chromatography on Con-A, wheat germ agglutinin, Maclura pomifera lectin and HOPC-8 mouse myeloma protein affinity columns. The immunochemical reactivity of native and periodate oxidized borohydride reduced type 37 polysaccharide antigen with polyclonal rabbit and monoclonal mouse anti-Pn37 hybridoma antibodies was studied by quantitative precipitation. Quantitative hapten inhibition studies, employing the isomeric series of alpha- and beta-(1----2), (1----3), (1----4) and (1----6)-linked glucobioses as competitive inhibitors of antibody precipitation establish a specificity for anti-Pn37 antibody directed at least in part, against the Glc beta(1----2) Glc (sophorosyl) unit. A high mol. wt, D-glucose containing polysaccharide antigen, cross-reactive with rabbit anti-Pn37 is reported which was found to occur in the culture medium of 7 of 19 strains of Actinomyces examined.

1. 1. Antigenic analysis employing rabbit antisera specific for horse immunoglobulins established the occurrence of IgG, IgG(T), IgM and γ 1 -component in the normal serum of the donkey, mule, hinny and zebra. 2. 2. With the rabbit reagents employed, the corresponding immunoglobulins of each equine specie and the interspecific hybrids were immunologically indistinguishable. 3. 3. Antigenic analysis of sera obtained from horse umbilical cord blood showed only IgM to be present; IgG(T), γ 1 -component and IgG were not detected. 4. 4. IgG-globulins of the donkey, mule, hinny and zebra were isolated from normal serum by chromatography on columns of DEAE-cellulose and their extinction and sedimentation coefficients determined.

Structural features of antigenic groupings of oat glucan involved in the cross reaction with Type VIII horese antipneumococcal serum were examined by quantitative hapten inhibition. Oligosaccharides of the cellodextrin series up to cellotetraose were examined and their molar inhibitory potency found to increase with the number of β(1→4) linked glucose units present. Employing mixed oligosaccharides as inhibitors possessing either terminal or subterminal β(1→4) linked glucose units, the cross reactive portion of anti-VIII could be shown to interact as well as terminal non-reducing cellodextrinyl groups. The pentameric structural unit of oat glucan possessing a sequence of four glucosyl residues joined through three consecutive β(1→4) linkages probably provides the internal groupings largely responsible for cross reactivity with anti-VIII.

Antiserum to crude lactoperoxidase was employed in agar diffusion and immunoelectrophoretic analyses of fractions obtained in the isolation and purification of lactoperoxidase from cow's milk by ion-exchange chromatography and gel filtration. The presence of seven antigenic components could be detected in a crude enzyme preparation by immunological analysis and their fate monitored during the purification procedure. Purified lactoperoxidase, prepared by ion-exchange chromatography and gel filtration, was found by immunological analysis to consist of a single antigenic component.

The production of specific anti-enzyme in response to immunization with purified lactoperoxidase could be demonstrated by assays of peroxidase activity on the supernatant fluids and precipitates obtained from quantitative precipitin determinations. Spectrophotometric analysis of specific precipitates formed in antibody excess for a distinctive antigen marker showed all the hemoprotein added as antigen to be quantitatively precipitated by antibody. Lactoperoxidase isolated from the milk of ruminants such as the goat and sheep were examined for their cross reactivity with anti-bovine lactoperoxidase and found to be immunochemically indistinguishable from cow's milk lactoperoxidase in their immunodiffusion and quantitative precipitin behavior. Bovine lactoperoxidase is immunologically distinct from bovine transferrin, ceruloplasmin, red protein, and lactollin as well as dog myeloperoxidase. Exposure of lactoperoxidase to proteolytic enzymes or 8 M urea does not result in any change detectable by immunodiffusion. The diffusion coefficient of bovine milk lactoperoxidase was estimated by immunodiffusion to be 5.2 × 10 −7 cm sec − .

1. 1. Antisera prepared against equine IgG globulin, its Fab, Fc fragments and L chains were employed in immunodiffusion analysis to examine their cross reactivity with canine IgGa,b globulins and a canine IgGd paraprotein. 2. 2. Interspecies antigenic similarities were found and cross reactive groupings assigned to various parts of the canine γG-globulin molecule. 3. 3. Immunodiffusion and cross absorption studies with anti-equine IgG, showed canine IgGa,b and IgGd to share some antigenic determinants in common with equine IgG and IgB absent from equine IgG(T).

Bovine tissues were analyzed for the presence of lactoperoxidase by immunodiffusion analysis. A protein antigenically identical to lactoperoxidase was shown to be present in the sublingual, submaxillary, and parotid glands. An antigenically related but not identical protein was present in pig salivary glands. As the enzyme could not be detected in any of the other tissues employed, it could be concluded that if present, it represented less than 0.04% of the extracted protein. Most notable among the tissues which did not contain the enzyme, are the thyroid, uterus, and lymph node. A method is presented for the isolation of lactoperoxidase from the submaxillary gland. The enzyme is a major protein of this tissue, comprising between 1 and 2% of the wet weight of the tissue. The relationship of lactoperoxidase to peroxidase activity reported in other studies of the salivary gland is discussed. It is clear that the “salivary peroxidase” and “iodide peroxidase” can be attributed to lactoperoxidase.

Rabbit antiserum to a BSA-cellobiose conjugate was examined by immunodiffusion and quantitative hapten inhibition employing the synthetic polyvalent hapten phloroglucinol triazophenyl-s- d -cellobioside as antigen. The isomeric series of s-linked glucobioses and mixed oligosaccharides possessing either terminal nonreducing or internal (subterminal) cellobiose were assayed for their ability to inhibit precipitation. Anticonjugate was more effectively inhibited by oligosaccharides possessing a terminal non-reducing cellobiose unit than by compounds with subterminal cellobiose groups. antibody could readily distinguish the s-(1,4) from the s-(1,3) and s-(1,6) glucosidic linkages. The portion of horse anti-VIII, precipitable by BSA-Cello was examined by hapten inhibition and p- nitrophenyl -s- d -cellobioside , cellotriose and cellotetraose found to be equipotent inhibitors of precipitation suggesting a specificity directed against the s- d -cellobiosyl or cellotriose unit.

The immunochemical behavior and enzymic activity of α-amylase from Aspergillus oryzae were examined before and after treatment of enzyme with N -bromosuccinimide or H 2 O 2 in the presence of Cu ++ . Under conditions resulting in the loss of 60–90% of the enzymic activity, preparations still reactive with antibody were obtained which showed a decrease in capacity per unit weight in precipitating antibody. Under certain conditions, 60% of the amylase activity could be destroyed by N -bromosuccinimide without any detectable change in immunochemical behavior. Immunochemical reactivity and enzymic activity thus show a difference in susceptibility toward these oxidizing reagents. Amylase-antiamylase specific precipitates formed with 7S antibody or bivalent 5S antibody fragments and soluble complex formed with univalent 3.8S antibody fragments showed only a small fraction of the activity of uncombined enzyme when starch was used as substrate. Similar findings were obtained when maltotriose or maltotetraose were substituted for starch in the assay of residual activity. These findings suggest that decreased activity shown by insoluble complexes is not due to aggregate formation but rather to the location of antibody attachment with respect to active sites. A Lineweaver-Burk treatment of kinetic data with antibody complexes is consistent with this interpretation.

The immunochemical behavior of native and partially dexylosylated glucuronic acid-containing polysaccharides of Cryptococcus laurentii (NRRL Y-1401) and of haploid cells of Tremella mesenterica (Y-6151) was examined with Type II anti-pneumococcal horse serum. Cryptococcal polysaccharide and its derivatives cross reacted while the corresponding Tremella preparations did not. The specificity of cross reactive antibodies precipitating with C. laurentii polysaccharide and oxidized cellulose (Oxycel) was examined by quantitative hapten inhibition and compared with the homologous S II-anti-S II system. Differences were found in the relative ability of sodium glucuronate and p-nitrophenyl-β- d -glucuronide to inhibit precipitation of anti-S II antibodies of C. laurentii, Oxycel and Type II pneumococcal polysaccharide.

The trisaccharide panose, 6- O - α -d-glucopyranosyl-4- O - α -d-glucopyranosyl-d-glucose, coupled to bovine serum albumin (BSA) via an azophenyl linkage was used as an artificial antigen to immunize rabbits. Antisera rendered specific for the introduced haptenic grouping by absorption with whole calf serum were studied by immunodiffusion, quantitative precipitation and hapten inhibition. Anti-hapten showed a specificity directed against the introduced p -phenylazo- s -panoside group. Hapten inhibition assays employing various di- and triglucosides, including two structural isomers of panose, established the relative importance of an α-configuration and a nonreducing terminal (1,6)-(1,4) ordered sequence of glucoside bonds for optimal interaction with antibody. A heterogeneity in the specificity of antibodies formed in response to s-panosyl-BSA was evidenced from cross reactions obtained between anti-panoside and various sugar azoprotein conjugates.

Sirishinha, Stitaya (University of Rochester School of Medicine and Dentistry, Rochester, N.Y.), and Peter Z. Allen . Immunochemical studies on α-amylase. III. Immunochemical relationships among amylases from various microorganisms. J. Bacteriol. 90: 1120–1128. 1965.—Immunochemical relationships among amylases obtained from a selected group of microorganisms were examined, and a cross-reaction was detected between the α-amylases of Bacillus stearothermophilus and B. subtilis . Immunodiffusion and quantitative precipitin studies, as well as cross-neutralization tests, indicate that B. stearothermophilus α-amylase reacts with a portion of antibody present in antisera to crystalline B. subtilis α-amylase. Amylases from these two species thus have some aspects of structure in common. Limited data obtained by immunodiffusion suggest that groupings which confer cross-reactivity to the B. stearothermophilus enzyme are lost after exposure to mercaptoethanol in the presence of ethylenediamine-tetraacetate, followed by treatment with iodoacetamide. With the antisera employed and within the concentration range examined, no immunochemical cross-reaction was observed among amylases from Aspergillus oryzae, B. subtilis, B. polymyxa, B. macerans, Pseudomonas saccharophila , and Euglena sanguinis . Immunoelectrophoresis of partially purified B. stearothermophilus α-amylase by use of antiserum to the crude enzyme, together with localization of amylase activity in immunoelectrophoretic plates, suggests that B. stearothermophilus α-amylase is antigenic in the rabbit.

Investigation of bovine lacrimal and harderian glands revealed the presence of the enzyme lactoperoxidase, which was isolated and purified. A nonheme, iron-containing protein was identified at the same time. Both proteins are present in milk, mammary glands, and salivary glands. Their roles are discussed: The lactoperoxidase may be important in controlling bacterial flora.

Selenium deficiency can develop in patients receiving intravenous hyperalimentation. This depletion can result in a decrease in both cellular and plasma glutathione peroxidase. Previous investigations have revealed that with repletion of selenium, there is a rapid increase in the recovery of plasma glutathione peroxidase and a more slow recovery in red blood cell glutathione peroxidase. This suggests that the plasma enzyme is not present as a result of hemolysis of red blood cells.

The chemical and immunochemical properties of lipopolysaccharides (LPS) isolated from pyocin 103-sensitive and -resistant Neisseria gonorrheae were investigated. Marked differences were found in immunochemical behavior of LPS from pyocin-sensitive gonococcal strain JW31 and its isogenic pyocin-resistant variant JW31R. JW31 LPS readily precipitated wheat-germ agglutinin, soybean lectin, and rabbit anti-Streptococcus faecalis or horse anti-type 14 pneumococcal antibody. In contrast, JW31R LPS precipitated only soybean lectin. The combining-site specificity of anti-S. faecalis cross-precipitated by JW31 LPS, or type 14 pneumococcal capsular polysaccharide, was examined by hapten inhibition, and lactose found to be the most potent inhibitor. Horse anti-pneumococcal type 14 antibodies, cross-precipitated by JW31 LPS and streptococcal lactose polymer, exhibited heterogeneity with respect to combining site specificity. Gel filtration of LPS-derived core oligosaccharide showed both strain JW31 and JW31 R to possess R-type lipopolysaccharide with cores having a Mr approximately 1800. JW31R LPS contains more galactose but less hexosamine than JW31 LPS. Both JW31 and JW31R core oligosaccharides possess D-glucosamine and D-galactosamine, probably N-acetylated, as the only nonreducing end-groups, and (1 leads to 4)-linked D-glucose residues. Chemical data support immunochemical findings which indicate that lactose units occur as a structural feature of JW31 gonococcal LPS.

Immunodiffusion analysis of papain digestion products, heavy and light chains of horse IgG-globulins with several rabbit and anti-horse IgG sera, have permitted the demonstration of five antigenic specificities (Fc 1 , Fc 2 , L, L sp and Fab sp ) associated with these equine antigens. Reactivity with anti-Fc 1 is shown by both F′c and Fc fragments, while anti-Fc 2 reactivity is shown only by Fc fragment. Absorption of anti-Fab serum with L chain Fc fragment provides a reagent (anti-Fab sp ) which precipitates only with Fab fragment, IgG-globulin or reduced and alkylated IgG. Upon exposure to detergent, reduced and alkylated, horse IgG loses its precipitability by anti-Fab sp , while reactivity with anti-L or anti-Fc reagents remains unaffected; reactivity is recoverable, however, following removal of detergent. Antisera to horse L chain show two kinds of specificity, one type (anti-L) directed against L chain determinants of IgG and Fab fragment, while another fraction of antibodies (anti-L sp ) is directed against determinants present on purified, reduced and alkylated horse L chains. Anti-L sp reagent does not precipitate with native IgG or Fab fragment, but does not react with free L chains and urea or detergent or heat-treated IgG-globulins.

Purified levan is antigenic in man and subcutaneous injection of 1 mg. leads to the production of precipitins and skin sensitivity. Apple amylopectin, maize glycogen, and synthetic polyglucose did not stimulate antibody production in small numbers of individuals injected. Quantitative precipitin studies carried out with several levan preparations employing human antilevan show variations in immunochemical behavior indicating structural differences among levans. Evidence that precipitins formed in response to levan injection are antilevan antibodies was obtained by analysis of specific precipitates formed in the region of antibody excess for ketosugar. Injection of levan giving rise to production of antilevan failed in one individual to give a non-specific anamnestic rise in either antidextran or antiblood group A precipitins. Laminarin, a neutral polysaccharide, was found to give precipitation with normal human sera.

Laminaribiose ( 3-o-β- d -glucopyranosyl- d -glucose ) was coupled to bovine serum albumin (BSA) by an azophenyl linkage to provide the synthetic antigen BSA-p-phenylazo-β-laminaribioside. The behavior of antisera prepared in rabbits immunized with the β-laminaribiosyl conjugate was examined by immunodiffusion, quantitative precipitation and hapten inhibition. Anticonjugate absorbed with carrier protein showed the greatest reactivity with the homologous β-laminaribiosyl-BSA antigen, but also showed some cross precipitation with β-cellobiosyl, β-sophorosyl and β-gentiobiosyl-BSA conjugates. Glucobioses linked through the β-(1 → 2), β-(1 → 3), β-(1 → 4) and β-(1 → 6) positions, as well as laminaridextrins and tri and tetrasaccharides of β-linked glucose possessing a laminaribiose moiety either at a nonreducing end location or at a subterminal location, were assayed for their ability to inhibit antilaminaribioside precipitation. Hapten inhibition data showed anticonjugate a possess a high degree of specificity directed against the terminal nonreducing β-laminaribiosyl end group.