Masaharu Naiki

Antigen of “serum sickness” type of heterophile antibodies in human sera: Identification as gangliosides with N-glycolylneuraminic acid

Abstract Antigen of “serum-sickness” type of heterophile antibodies in pathologic human sera was purified from equine and bovine erythrocyte stroma. The chemical nature of this antigen was glycosphingolipids with N-glycolylneuraminic acid. The antigen of equine erythrocytes was identified as hematoside with N-glycolylneuraminic acid, GlNeu(α, 2–3)Gal(β, 1–4)Glc(β,1-1) ceramide and the antigen of bovine erythrocytes was N-glycolylneuraminyl-paragloboside, GlNeu (α,2–3)Gal(β,1–4)GlcNAc(β,1–3)Gal(β,1–4)Glc(β,1-1) ceramide. The results indicate that “serum-sickness” antibodies react with a common disaccharide moiety of non-reducing end of the both glycosphingolipids.

Changes in serum C-reactive protein levels in dogs with various disorders and surgical traumas.

The serum levels of C-reactive protein (CRP) produced as an inflammatory response in dogs with various disorders and surgical traumas were measured by enzyme-linked immunoabsorbent assay and slide reversed passive latex agglutination test (RPLA). The CRP levels were greatly increased 1–2 days after surgery in most of the dogs (n=29) subjected to surgery. These levels had markedly decreased by the time the sutures were removed. In dogs with various disorders (n=58), the serum CRP levels at first diagnosis were high in infectious diseases. In dogs from which paired serum samples were examined, the serum CRP usually showed a decrease with improvement in the condition (n=11) or a terminal increase (n=4) but, conversely, some showed an increase with improvement in the condition (n=3).

A monoclonal antibody reacting with the zona pellucida of the oviductal egg but not with that of the ovarian egg of the golden hamster

A monoclonal antibody (mAb) designated AZPO-8 was produced by hybridizing a mouse myeloma with spleen cells from BALB/c mice immunized with materials obtained from the hamster oviduct. With an immunofluorescence test, AZPO-8 reacted with the zona pellucida (ZP) of ovulated eggs in the oviduct (ZP-OVI) but not with the zona pellucida of eggs in the ovary (ZP-OVA). Using indirect enzyme immunostaining, this mAb reacted with epithelial cells of the oviduct, the uterus (especially the cervical epithelium) and the gastric mucosa, but not with other hamster tissues examined. The reactivity of antigen-positive tissues was abrogated by pretreatment of the tissues with periodic acid. Western blotting analysis revealed that AZPO-8 reacted with substances of broad molecular weight range, and the strongest reactivity was detected at a molecular weight of approximately 200,000 in both cases when extract of ZP-OVI or the hamster oviduct was applied on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. AZPO-8 showed strong hemagglutination activity only to group A human red blood cells. These results indicated that (1) ZP-OVI had an antigen that was not detected on ZP-OVA, (2) ZP-OVI and the oviduct shared the same antigenicity, and (3) the antigenic determinant reactive with the mAb might be carbohydrate in nature. A possible role of this antigen in fertilization was discussed.

Isolation of canine C-reactive protein and characterization of its properties.

C-reactive protein (CRP) was isolated from the acute phase serum of dogs subjected to surgical stimulation. Its properties were characterized. Canine CRP was isolated by ion-exchange chromatography using DEAE-Sephacel and DEAE-Sephadex A-50 and affinity chromatography using protein A-Sepharose CL 4B in combination with agar-block electrophoresis. In immunoelectrophoresis, canine CRP had the same gamma-mobility as human gamma-type CRP. The molecular weight of purifined canine CRP was estimated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis to be approximately 157,000 and 155,000 respectively. This CRP was a thermolabile protein which completely lost its antigenicity by heating at 70 degrees C for 15 min. The serum concentration of CRP in normal beagle dogs ranged from 0.198 to 0.826 micrograms ml-1 (0.486 +/- 0.170 micrograms ml-1). The concentration was acutely increased by surgery as it was in man and was rapidly decreased with convalescence. Dogs can be a useful animal model for investigation of the mechanism of CRP production and the function of CRP.

Specificities of human heterophilic Hanganutziu and Deicher (H-D) antibodies and avian antisera against H-D antigen-active glycosphingolipids.

The precise specificities H-D antibodies and newly obtained avian antisera to the H-D antigen-active glycosphingolipids. N-glycolylneuraminyl (alpha 2-3) lactosyl ceramide (hematoside) and N-glycolylneuraminyl (alpha 2-3) lactoneotetraosyl ceramide (sialosylparagloboside) were compared by gel precipitation and hemagglutination inhibition using other gangliosides with a related structure and chemically modified hematoside derivatives and the oligosaccharides from both the antigenic glycosphingolipids. Human H-D antibodies were found to have the same affinity for both antigenic glycosphingolipids; however, the avian anti-hematoside or anti-sialosylparaglobosides showed a higher affinity for the homologous antigen. Human H-D antibodies were directed more exactly toward the terminal sialic acid residue and more roughly to the hydrophobic portion of both compounds; however, the two avian antibodies were directed more specificity to the whole structure of the homologous antigen glycosphingolipid.

Isolation and characterization of rainbow trout C-reactive protein

An acute phase serum component, C-reactive protein (CRP), was isolated from the sera of rainbow trout (Salmo gairdneri). The isolation was based on its calcium-dependent binding affinity for pneumococcal C-polysaccharide (CPS) according to the isolation procedure of human C-reactive protein. In SDS-PAGE, the nonreduced CRP showed two subunits with molecular weights of 43,700 and 26,600, respectively, at a molar ratio of 1:1. The reduced CRP showed a single subunit of 26,600. The molecular weight of the native protein was estimated as 66,000 by native gradient PAGE and 81,400 by sedimentation equilibrium analysis using ultracentrifugation. The antigenic determinant on CPS-reactive site was destroyed by periodate oxidation, indicating that rainbow trout CRP is a glycoprotein. CRP levels in rainbow trout serum measured by the CPS-ELISA procedure showed that the rainbow trout CRP could behave as an acute phase reactant, following experimental infection with the fish pathogen Vibrio anguillarum.

Bovine haptoglobin: Single radial immunodiffusion assay of its polymeric forms and dramatic rise in acute-phase sera

Using purified bovine haptoglobin (Hp) and specific antisera, a single radial immunodiffusion (SRID) assay method has been developed to measure the serum Hp level in cattle. Bovine Hp is a highly polymerized protein showing heterogeneous molecular forms in serum. After treatment with cysteine or glutathione, Hp was partially reduced to a homogeneous form, suitable for SRID assay. This method gives values comparable to those obtained by hemoglobin-binding capacity assay, and has the advantage of being simple and convenient. Although serum Hp was not detectable in healthy cattle, it was found more than 50-fold after invasive surgery, indicating that Hp is a characteristic acute-phase protein in cattle.

Isolation and characterization of conglutinin as an influenza A virus inhibitor.

Normal horse and guinea pig sera contain alpha 2-macroglobulin which inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H2 and H3 subtypes. On the other hand, normal bovine serum contains a component termed beta inhibitor that inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H1 and H3 subtypes. To investigate the nature of the beta inhibitor of influenza A virus, we purified the conglutinin and examined its characteristics. First, we found a high correlation between the hemagglutination inhibition(HI) titer and conglutinin titer in several bovine sera (r = 0.906, p less than 0.005). The HI of bovine serum was mainly dependent on conglutinin because the HI activity was abrogated by N-acetylglucosamine but not by D-mannose. The conglutinin, purified from bovine serum, had neutralizing-activity as well as HI activity on influenza A viruses of the H1 and H3 subtypes. The HI activity of conglutinin was heat stable (56 degrees C, 30 min), Ca(++)-dependent, and resistant to both neuraminidase and periodate treatments. The HI activity of purified conglutinin was blocked by N-acetylglucosamine but not by D-mannose. The conglutinin was bound to hemagglutinin which had high mannose and complex sugar chains and its binding was inhibited by N-acetylglucosamine and dependent on divalent cations. These data indicate that the beta-like inhibitor activity of bovine serum is mainly dependent on conglutinin which inhibits hemagglutination and neutralizes the virus infectivity by its binding to a carbohydrate site at the HA.

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 [GM3(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, GM3(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 GM3(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.

Immunological Identification of Blood Group Pk Antigen on Normal Human Erythrocytes and Isolation of Anti-Pk with Different Affinity

Abstract. The P1 and Pk blood group glycolipid antigens have the common terminal disaccharide, Gal(α, 1–4)Gal, but previous studies indicated that anti‐P1 from P2 individuals does not cross‐react with Pk antigen. In this paper, the specificities of anti‐P1 and anti‐Pkwere analyzed carefully by complement fixation and hemagglutination techniques and the following results were obtained: (1) Anti‐P1 from P2 serum was not absorbed with the Pk glycolipid (CTH), but this antigen absorbed all anti‐P1 and anti‐Pk (anti‐P1Pk) antibodies from the sera of four p individuals. Most of the anti‐P1Pk antibodies were IgG, but the anti‐P1 from the P2 individual was IgM. (2) The Pk antigen on normal P2 erythrocytes was not ‘cryptic’. It was reactive with p serum from which the anti‐P antibodies were removed by absorption with the P glycolipid (globoside). This was not appreciated previously because, in order to make anti‐Pk reagents, p sera (anti‐P1PPk) were absorbed with P1 cells which contain CTH. (3) The anti‐P1Pk antibodies in p sera were separated by partial absorption with P1 erythrocytes and elution from the absorbing cells, into two fractions that differ markedly in their affinity for α‐methyl‐D‐galactoside and the oligosaccharides prepared from CTH.