Immobilization of invertase on sepharose-linked enzyme glycosyl recognizing polyclonal antibodies

Polyclonal antibodies directed against the yeast invertase glycosyls were raised by immunizing rabbits with neoglycoprotein-I and neoglycoprotein-II. The neoglycoproteins were prepared by separately coupling the N-linked large and small molecular weight yeast invertase oligosaccharides respectively to bovine serum albumin with the help of glutaraldehyde. Antibodies specifically recognizing the invertase oligosaccharides were purified from the sera of rabbits immunized with either neoglycoprotein using an affinity column of sepharose 4B-linked yeast invertase. Specific immunoaffinity supports for the immobilization of invertase were constructed by coupling the affinity-purified antineoglycoprotein-I or antineoglycoprotein-II antibodies to cyanogen bromide activated sepharose-4B. Both the affinity adsorbants were effective in binding and improving the thermal stability of invertase. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 605-609, 1997.     

黄鳝IgM的分离纯化及兔抗黄鳝IgM抗血清的制备

目的 分离纯化黄鳝血清免疫球蛋白,制备其兔抗血清,并检测抗血清的特异性。方法 用Protein A亲和层析的方法纯化黄鳝血清免疫球蛋白,通过SDS-聚丙烯酰胺凝胶电泳检测蛋白的纯度,免疫大耳白兔制备抗血清,利用免疫双扩散检测抗血清的效价,通过western blotting检测抗血清的特异性。结果 纯化了黄鳝血清免疫球蛋白,免疫双扩散法测定兔抗黄鳝免疫球蛋白血清效价为1∶32,western blotting结果显示抗血清具有很好的特异性。结论 成功纯化了黄鳝免疫球蛋白,制备了兔抗黄鳝IgM抗血清,为建立黄鳝的血清学检测系统奠定了基础。     

Invertase beta-galactosidase hybrid proteins fail to be transported from the endoplasmic reticulum in Saccharomyces cerevisiae.

The yeast SUC2 gene codes for the secreted enzyme invertase. A series of 16 different-sized gene fusions have been constructed between this yeast gene and the Escherichia coli lacZ gene, which codes for the cytoplasmic enzyme beta-galactosidase. Various amounts of SUC2 NH2-terminal coding sequence have been fused in frame to a constant COOH-terminal coding segment of the lacZ gene, resulting in the synthesis of hybrid invertase-beta-galactosidase proteins in Saccharomyces cerevisiae. The hybrid proteins exhibit beta-galactosidase activity, and they are recognized specifically by antisera directed against either invertase or beta-galactosidase. Expression of beta-galactosidase activity is regulated in a manner similar to that observed for invertase activity expressed from a wild-type SUC2 gene: repressed in high-glucose medium and derepressed in low-glucose medium. Unlike wild-type invertase, however, the invertase-beta-galactosidase hybrid proteins are not secreted. Rather, they appear to remain trapped at a very early stage of secretory protein transit: insertion into the endoplasmic reticulum (ER). The hybrid proteins appear only to have undergone core glycosylation, an ER process, and do not receive the additional glycosyl modifications that take place in the Golgi complex. Even those hybrid proteins containing only a short segment of invertase sequences at the NH2 terminus are glycosylated, suggesting that no extensive folding of the invertase polypeptide is required before initiation of transmembrane transfer. beta-Galactosidase activity expressed by the SUC2-lacZ gene fusions cofractionates on Percoll density gradients with ER marker enzymes and not with other organelles. In addition, the hybrid proteins are not accessible to cell-surface labeling by 125I. Accumulation of the invertase-beta-galactosidase hybrid proteins within the ER does not appear to confer a growth-defective phenotype to yeast cells. In this location, however, the hybrid proteins and the beta-galactosidase activity they exhibit could provide a useful biochemical tag for yeast ER membranes.     

Immunodiffusion Analysis of Yaba Poxvirus Structural and Associated Antigens

Yaba poxvirus virions were extracted and purified from Rhesus monkey tumors. A saline-soluble virion fraction (Y-xp), obtained by mechanical fractionation of purified virions with an X-press, contained seven components in acrylamide gel electrophoresis; five of these components were reactive in immunodiffusion with whole virion and Y-xp antisera produced in rabbits and monkeys. The saline-insoluble residue remaining after X-press treatment was hydrolyzed with sodium dodecyl sulfate, urea, and 2-mercaptoethanol (SUM). This fraction, Y-sum, contained five components, four of which were demonstrable by immunodiffusion. There was no evidence of antigenic relationships between Y-xp and Y-sum antigens in immunodiffusion. In acrylamide gel electrophoresis, one Y-xp and one Y-sum component had similar mobilities. Y-xp but not Y-sum antisera contained viral-neutralizing antibodies. Virus-free saline extracts of Yaba tumor prepared with Genetron (YS) were essentially devoid of virion structural antigens. They failed to induce precipitating antibodies for virion antigens, were nonreactive in immunodiffusion with virion antisera, and gave low complement-fixation titers with virion antisera. Yaba virion antigens were recovered from the Genetron tumor sediment by SUM and alkaline hydrolysis. Antisera prepared to YS extracts gave a maximum of 17 precipitin lines in immunodiffusion with YS extracts; none was identified as a virion structural antigen. Saline extracts of tumor prepared without Genetron contained immunogenic amounts of 5 virion antigens and 12 to 14 associated antigens. Animals immunized with infected cell culture extracts (virus-free) formed antibodies to six to seven virion antigens. The implications of using extracts of Yaba poxvirus-infected tissues in complement-fixation tests to measure virion antibodies were discussed.     

Cross-reactions among carbonic anhydrases I,II, and III studied by binding tests and with monoclonal antibodies

Cross-reactions among carbonic anhydrases (CAs) I, II, and III were studied using a variety of antisera: (1) a rabbit antiserum to bovine CA III, (2) mouse antisera to human CA I, CA II, and CA III; and (3) five monoclonal antibodies prepared by the hybridoma technique using splenocytes from a mouse immunized with human CAs I and II and bovine CA III. Cross-reactions between CAs were readily found by binding assays using these antisera. Human CA I, but not human CA II, inhibited the reaction of the rabbit anti-CA III with its homologous antigen. Mouse antisera to CA I or CA II bound the homologous I or II with nearly as great efficiency as the autologous isozyme and sometimes weakly bound CA III. Mouse antisera to CA III frequently bound CA I or II. These cross-reactions were confirmed by the first use of hybridoma-prepared, monoclonal antibodies to CAs. The mouse monoclonal antibodies to CA isozymes varied in the amount of cross-reactivity among I, II, and III: at one extreme, one monoclonal was highly specific for the autologous CA III; at the other extreme, one monoclonal weakly reacted with some examples of CAs I, II, and III.This work was supported by NIH Grant GM-24681 and a grant from the National Foundation-March of Dimes.     

Coincident localization of secretory and plasma membrane proteins in organelles of the yeast secretory pathway.

Immunoelectron microscopy of Saccharomyces cerevisiae cells embedded in Lowicryl K4M has been used to localize invertase and plasma membrane (PM) ATPase in secretory organelles. sec mutant cells incubated at 37 degrees C were prepared for electron microscopy, and thin sections were incubated with polyclonal antibodies, followed by decoration with protein A-gold. Specific labeling of invertase was seen in the lumen of the endoplasmic reticulum, Golgi apparatus, and secretory vesicles in mutant cells that exaggerate these organelles. PM ATPase accumulated within the same organelles. Double-immune labeling revealed that invertase and PM ATPase colocalized in secretory vesicles. These results strengthen the view that secretion and plasma membrane assembly are biosynthetically coupled in yeast.     

Specific subtyping of influenza A virus using a recombinant hemagglutinin protein expressed in baculovirus

Influenza A viruses are subtyped according to antigen characterization of hemagglutinin (HA) and neuraminidase surface glycoproteins. The hemagglutination inhibition (HI) assay using reference antiserum is currently applied to serologic screening of subtype-specific antibodies in sera. The reference antiserum is made by injecting chickens with live or inactivated whole virus preparations. Nonspecific inhibitors of antisera prepared by the conventional method may affect the specificity of HI assay. In this study, highly pure recombinant proteins generated using baculovirus expression vector system based on full-length of HA (HAF) and antigenic region of HA₁ genes of H9 subtype, and also inactivated whole virus were used to immunization of chickens. Measurable antibody titers were present for treated birds after 3 weeks and generally increased after each boost. The performance of the prepared antisera was evaluated by testing a panel of known standard strains of influenza virus representing five HA subtypes. Relative to the conventional method using whole virus immunization and recombinant HAF protein, the antiserum prepared by recombinant HA₁ had a specificity of 100% for all tested subtypes. The antiserum prepared by expression of HA1 protein in baculovirus has the potential for rapid and specific HA subtyping of influenza viruses without producing antibodies specific to other viral proteins.     

伤寒沙门菌与甲、乙、丙副伤寒沙门菌质控血清的制备

应用免疫学原理,将伤寒沙门菌O901、H901和甲、乙、丙型副伤寒沙门菌分别制成全菌体抗原,免疫实验兔获取免疫血清。依据伤寒沙门菌和副伤寒沙门菌的抗原成分的异同性,选择适当的吸收菌除去免疫血清中的交叉反应抗体和类属凝集素,而保留其特异性的抗体。通过对诊断菌液的验证试验,证实吸收充分的免疫血清具有质控血清的特性。具备可靠性能的质控血清,适用于伤寒沙门菌与副伤寒沙门菌的菌种检定及其效价检测;亦有利于肥达氏诊断菌液的质量控制。     

Binding of antibodies to acetylcholine receptors in Electrophorus and Torpedo electroplax membranes

Antisera against purified acetylcholine receptors from the electric tissues of Torpedo californica and of Electrophorus electricus were raised in rabbits. The antisera contain antibodies which bind to both autologous and heterologous receptors in solution as shown by an immunoprecipitation assay. Antibodies in both types of antisera bind specifically to the postjunctional membrane on the innervated surface of the intact electroplax from Electrophorus electric tissue as demonstrated by an indirect immunohistochemical procedure using horseradish peroxidase conjugated to anti-rabbit IgG. Only anti-Electrophorus receptor antisera, however, cause inhibition of the receptor-mediated depolarization of the intact Electrophorus electroplax. The lack of inhibition by anti-Torpedo receptor antibodies, which do bind, suggests that the receptor does not undergo extensive movement during activity. The binding of anti-Torpedo antibodies to receptor-rich vesicles prepared by subcellular fractionation of Torpedo electric tissue was demonstrated by both direct and indirect immunohistochemical methods using ferritin conjugates. These vesicles can be conveniently collected and prepared for electron microscopy on Millipore filters, a procedure requiring only 25 micrograms of membrane protein per filter. In addition, it was possible to visualize the binding of anti-Torpedo receptor antibodies directly, without ferritin. These anti-Torpedo receptor antibodies, however, do not inhibit the binding of acetylcholine or of alpha-neurotoxin to receptor in Torpedo microsacs but do inhibit binding of alpha-neurotoxin to Torpedo receptor in Triton X-100 solution. It is likely that the principal antigenic determinants on receptor are at sites other than the acetylcholine-binding sites and that inhibition of receptor function, when it occurs, may be due to a stabilization by antibody binding of an inactive conformational state.     

Antibodies specific to two deoxyribotrinucleotide sequences.

Antibodies to the deoxyribotrinucleotides dpApTpA and dpApApT were prepared by injecting the bovine serum albumin conjugates of the respective haptens in rabbits. The specificities of the antibodies were determined by estimating the inhibition of the binding of the tritiated haptens to the immunoglobulins by various nonradioactive mono- and oligonucleotides, using nitrocellulose membrane binding assay. Anti-dpApTpA and anti-dpApApT antisera were found to contain antibodies which were highly specific to the respective hapten sequence.     

腮腺炎病毒抗血清的制备

以制备适用于疫苗生产检定中病毒鉴别试验和外源因子检查的高效价腮腺炎病毒抗血清为目的。用腮腺炎病毒接种SPF鸡胚尿囊腔,培养收取病毒尿液免疫SPF鸡,采集抗血清。腮腺炎病毒接种Vero细胞,培养病毒抗原经PEG沉淀,超速离心法纯化后免疫家兔采集抗血清。比较两种免疫方法所得病毒抗血清效价。结果显示SPF鸡抗腮腺炎病毒血清中和抗体GMT为1:1716,兔抗腮腺炎病毒血清中和抗体GMT为1:732。两种动物抗血清均适用于疫苗生产相关检定。免疫SPF鸡制备的病毒抗血清无特定病原及抗体污染,是毒种外源因子检测和疫苗鉴别试验的理想试剂。免疫SPF鸡制备病毒抗血清的程序简单,结果易于验证,有利于生物试剂标准化。     

Immunological comparison of desmosomal components from several bovine tissues

A panel of monoclonal antibodies and conventional antisera directed against desmosomal proteins from bovine muzzle epidermis was used to identify immunologically related proteins from two other bovine stratified squamous epithelia, cornea and esophagus. Desmosome-enriched tissue fractions were prepared from epidermis, cornea, and esophagus. These tissue extracts were electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels, blotted onto nitrocellulose paper, and labeled using an indirect immunoperoxidase technique. Labeling with the conventional antisera demonstrates that each of the previously characterized epidermal desmosomal proteins or protein families has an immunologically cross-reacting counterpart in cornea and esophagus. However, chemical differences between homologous desmosomal proteins in these three tissues have also been detected. The corresponding proteins in the different tissues have similar but not always identical apparent molecular weights. Moreover, tissue-restricted antigenic determinants were detected in two of the desmosomal proteins families using four monoclonal antibodies, each of which recognizes a distinct antigenic determinant.     

X-ray diffraction structure of a plant glycosyl hydrolase family 32 protein: fructan 1-exohydrolase IIa of Cichorium intybus

Fructan 1-exohydrolase, an enzyme involved in fructan degradation, belongs to the glycosyl hydrolase family 32. The structure of isoenzyme 1-FEH IIa from Cichorium intybus is described at a resolution of 2.35 A. The structure consists of an N-terminal fivefold beta-propeller domain connected to two C-terminal beta-sheets. The putative active site is located entirely in the beta-propeller domain and is formed by amino acids which are highly conserved within glycosyl hydrolase family 32. The fructan-binding site is thought to be in the cleft formed between the two domains. The 1-FEH IIa structure is compared with the structures of two homologous but functionally different enzymes: a levansucrase from Bacillus subtilis (glycosyl hydrolase family 68) and an invertase from Thermotoga maritima (glycosyl hydrolase family 32).     

Characterization and immunogenicity of Vibrio cholerae ghosts expressing toxin-coregulated pili

Bacterial ghosts are attractive for use as non-living vaccines and as carriers of heterologous antigens of vaccine relevance. Ghosts were prepared from Vibrio cholerae strains of O1 or O139 serogroup after growth under culture conditions, which favor or repress the production of toxin-coregulated pili (TCP). Immunoblotting confirmed the TCP status of these V. cholerae ghosts (VCG), which retained the cellular morphology and envelope sub-component profile of viable bacteria. Rabbits were immunized with VCGs prepared from O139 bacteria with TCP-positive or TCP-negative phenotypes and the resulting sera assayed for antibodies to lipopolysaccharide (LPS) and to TCP. Regardless of the TCP status of the VCG preparations used for immunization, all animals produced antibodies to LPS as demonstrated in bactericidal assays. These antibodies were probably responsible for the capacity of the antisera to confer passive immunity to challenge with the homologous O139 strain in the infant mouse cholera model (IMCM). Only following immunization with TCP-positive VCG, however, were antibodies to TCP generated, as judged by the potential of antisera to mediate protection against a challenge strain of heterologous serogroup.     

Separation of a Soluble Antigen and Infectious Particles of Bovine Viral Diarrhea Viruses and their Relationship to Hog Cholera

A soluble antigen present in infectious tissue culture fluids was separated from the infective virus particle by ultracentrifugation of two serologically related strains of bovine viral diarrhea viruses, NADL-MD and Oregon C24V. Neutralizing antibodies against the two viruses were absent in four hog cholera antisera, but present in significant titer in the commercially prepared antiserum. Precipitin tests utilizing the agar double diffusion technique formed a single line of identity between the concentrated soluble antigen of both viruses and NADL-MD and hog cholera antisera. No lines were observed using concentrated virus pellet and noninfected BEK cell antigens or control SPF calf and swine sera.     

Immunochemical analysis of cartilage proteoglycans. Antigenic determinants of substructures.

Antibodies were raised in rabbits by injection of cartilage proteoglycan monomers, isolated hyaluronic acid-binding region, polysaccharide-peptides prepared by trypsin digestion of proteoglycans and link-protein. The rabbits injected with the proteoglycan monomers made antibodies reacting with the intact proteoglycan. The antiserum contained antibodies specific for, and also reacting with, the isolated hyaluronic acid-binding region and the keratan sulphate-rich region. In addition there were probably antibodies reacting with other structures of the proteoglycan monomer. When isolated hyaluronic acid-binding region was used for immunization the antibodies obtained reacted specifically with the hyaluronic acid-binding region. The antibodies obtained from rabbits immunized with the polysaccharide-peptides reacted with the proteoglycan monomers and showed a reaction identical with that of the chondroitin sulphate-peptides isolated after trypsin digestion of proteoglycans. The antibodies prepared with the link-protein as the antigen reacted only with the link-protein and not with any preparation from the proteoglycan monomer. Neither did any of the antisera raised against the proteoglycan monomer or its substructures react with the link-protein. Separately it was shown that the peptide 'maps' prepared from trypsin digests of the link-protein and the hyaluronic acid-binding region were different. Therefore it appears that the link-protein is not structurally related to the proteoglycan or the hyaluronic acid-binding region. Digestion of proteoglycan monomers or isolated hyaluronic acid-binding region with trypsin did not destroy the antigenic sites of the hyaluronic acid-binding region. In contrast trypsin digests of previously reduced and alkylated preparations did not react with the anti-(hyaluronic acid-binding region). The trypsin digests, however, reacted with both the antibodies directed against the chondroitin sulphate-peptides and those against the keratan sulphate-peptides. Trypsin digestion of the link-proteins destroyed the antigenic site and the reactivity with the antibodies. By combining immunoassay of proteoglycan preparations before and after trypsin digestion it is feasible to quantitatively determine its substructures by using the antisera described above.     

Biotin sulfone tagged oligomannosides as immunogens for eliciting antibodies against specific mannan epitopes

Biotinylated tri and tetrasaccharide: α Man (1→3) α Man (1→2) α Man; α Man (1→3) α Man (1→2) α Man (1→2) α Man were prepared using methyl tertbutyl phenyl thioglycosides glycosyl donors (MBP) and biotin sulfone strategy. Three key mannosyl thioglycosidic donors have been prepared: one for 1→2 linkage and two for the 1→3 linkage (protected with a 4,6-O-benzylidene or a 4,6-di-O-benzyl). The benzyliden protected one was not found reactive enough, and the benzylated donor was preferred. These biotinylated oligomanosides were evaluated as antigen in Crohn disease diagnosis and used coupled to streptavidin as hapten for eliciting polyclonal antibodies in mice.     

Antibodies against p-aminophenyl-beta-D-galactopyranoside-containing proteins

The antibodies were prepared from antisera of rabbits immunized with bovine serum albumin containing covalently bound p-aminophenyl-beta-D-galactopyranoside (APG) and purified by affinity chromatography on APG-containing ovalbumin immobilized by BrCN-activated Sepharose 4B. The antibodies possessed a selective specificity for APG and interacted with different APG-containing proteins, including APG-containing lysosomal alpha-glucosidase. The purified antibodies are immunoglobulins of G type as was determined from the molecular weights of native and dissociated antibodies and from the immunochemical assays with antibodies against rabbit IgG.     

Antibodies specific to isoniazid and isonicotinic acid.

Rabbit antisera to isoniazid (INH) and its major metabolite, isonicotinic acid (INA), were prepared by immunization with conjugates of these compounds with human serum albumin. The antisera were rendered hapten-specific by exhaustive absorption with the immunizing carrier. Purified anti-hapten antibodies were also isolated with appropriate immunosorbents. As demonstrated by inhibition of the quantitative precipitin curves and of precipitating immune complexes in immunodiffusion tests, the antibodies to the two haptens reacted with either INH or INA, and also with isonicotinamide (INC); these three related molecules share the isonicotinyl group. The relative effectiveness of inhibition by free hapten of precipitating immune complexes consisting of either anti-INH or anti-INA antibodies and the related hapten-protein conjugates was INH greater than INC greater than INA.     

Idiotypic analysis of anti-GL phi antibodies. I. Identification and strain distribution of GL-1 idiotypes

We utilized both the inhibition of antigen binding and direct idiotype binding methods to identify a new set of common idiotype determinants on anti-GL antibodies of various mouse strains. Three anti-idiotypic antisera, each prepared against individually purified B10.WB anti-GL phi antibodies, were able to detect antibody-combining site-associated common idiotypic determinants, designated GL-1 idiotype(s), in antisera with GLT-binding activity obtained from all mouse strains except strains bearing Igh-1e allotype. Anti-GL phi antisera obtained from rabbits, guinea pigs, and rats did not express detectable levels of GL-1 idiotypes. Nonresponder mice to GL phi, upon immunization with GL-F gamma G or GL phi-F gamma G produced anti-GL antibodies expressing GL-1 idiotypes. Although the magnitude of the immune response to various GL-containing polymers is controlled by distinct Ir genes, the common GL-related antigenic determinants on these polymers are able to induce anti-GL antibodies with GL-1 idiotypic specificities.