Immunoprecipitation of Triton X-100-solubilized Mycoplasma mycoides proteins.

Mycoplasma mycoides subsp. mycoides (PG1 and strain Y) proteins were solubilized in Triton X-100, and the antigenic proteins were precipitated from this complex mixture by addition of antiserum and then separated by two-dimensional gel electrophoresis. Of the 300 proteins solubilized, about 10 were precipitated. Proteins of PG1, a slow-growing, small colony (SC) strain, were precipitated by antiserum to PG1 and by antiserum to strain Y, a fast-growing, large colony (LC) strain. Similarly, strain Y proteins were precipitated by antiserum to PG1 and by antiserum to strain Y. The few proteins precipitated in this way gave similar patterns after two-dimensional gel electrophoresis indicating that many of the dominant protein antigens of PG1 and strain Y are shared. Antiserum to Mycoplasma mycoides subsp. capri (PG3) also precipitated some proteins of strain Y. Antiserum to Mycoplasma gallisepticum gave no reaction with any M. mycoides antigens. It was concluded that, in addition to the polysaccharide antigens, there are proteins in M. mycoides that are antigenic and that some of these are found in both the SC and LC strains of subsp. mycoides and also in subsp. capri.     

The 1,629-nucleotide open reading frame located downstream of the Autographa californica nuclear polyhedrosis virus polyhedrin gene encodes a nucleocapsid-associated phosphoprotein.

A 78-kDa protein was produced in bacteria from a clone of the 1,629-nucleotide open reading frame located immediately downstream from the polyhedrin gene of Autographa californica nuclear polyhedrosis virus. The identity of this protein was confirmed by its reactivity with peptide antiserum and amino terminal peptide sequencing after purification from transformed bacteria. The polypeptide was used to produce polyclonal antisera in rabbits. Immunoblot analysis of insect cells infected with the baculovirus indicated that two related proteins with molecular masses of 78 and 83 kDa were synthesized late in infection. Biochemical fractionation studies indicated that both of these proteins were present in purified nucleocapsids from budded and occluded virus preparations. Immunoprecipitation of 32P-labeled proteins and treatment of purified nucleocapsids with alkaline phosphatase demonstrated that the 83-kDa protein was a phosphorylated derivative of the 78-kDa protein. Furthermore, immunoelectron microscopy revealed that the proteins were localized to regions of nucleocapsid assembly within the infected cell and appeared to be associated with the end structures of mature nucleocapsids.     

Soluble Antigens of Vaccinia-infected Mammalian Cells II. Time Course of Synthesis of Soluble Antigens and Virus Structural Proteins

Virus-induced soluble antigens produced in mammalian cells after infection with vaccinia virus can be divided into two classes on the basis of molecular weight. Synthesis of the low molecular weight antigens begins early in the course of infection (1 to 2 hr), and is switched-off rather abruptly 4 to 5 hr after infection in a manner similar to that reported for the early enzymes characteristic of this same system. It was demonstrated, however, that these antigens do not include virus-induced thymidine kinase, a major virus-induced enzyme, nor is it likely that the low molecular weight antigens described here share identity with any of the virus-induced enzymes. A portion of the low molecular weight antigens appear to be incorporated into the structure of newly synthesized virus, probably as internal proteins. In contrast, synthesis of the high molecular weight antigen class is initiated later in the course of infection (4 to 5 hr), just prior to the appearance of newly synthesized virus. Antiserum directed specifically against virus structural proteins forms precipitin bands with all of the high molecular weight antigens recognizable by immunoelectrophoresis. This evidence, coupled with the observation that the high molecular weight antigen fraction elicits production of specific virus-neutralizing antibody, strongly suggests that this antigen class represents virus structural subunits produced in excess.     

Immunochemical characterization of proteins from mouse liver plasma membranes

1. Antiserum was prepared in rabbits against a purified mouse liver plasma-membrane fraction. 2. The antiserum was made to react with an ¹²⁵I-labelled alkaline-EDTA extract of the plasma membranes, and the immunoprecipitate analysed by polyacrylamide-gel electrophoresis. Seven proteins were immunoprecipitated and a single glycoprotein present in the alkaline-EDTA-soluble fraction was found to be a major component. 3. The alkaline-EDTA-soluble fraction was analysed by two-dimensional immunoelectrophoresis and this procedure indicated the presence of six antigenic components. 4. The plasma membranes were also extracted with 1% deoxycholate–1% Triton X-100; 50% of the protein, 80% of the alkaline phosphodiesterase activity and 30% of the 5′-nucleotidase activity were solubilized. 5. Two-dimensional immunoelectrophoresis of the deoxycholate–Triton X-100 extract indicated the presence of six antigens. 6. The relative distribution of the six antigens among the fractions obtained during the extraction procedure was examined immunoelectrophoretically to provide information on their disposition within the membrane.     

Isolation and characterization of a folate receptor from human placenta

While folate binding proteins have been described in serum and a variety of tissues, the function of these proteins is unknown. A particulate folate binding protein from human placenta has been isolated and characterized following solubilization with Triton X-100. The protein was purified 61,000-fold using affinity chromatography on pteroylglutamic acid-Sepharose as the major purification step. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified protein gave a single band with a Mr = 38,500. Stoichiometry of binding indicated that 1 mol of folate was bound per mol of protein. The protein was a glycoprotein that contained 12% carbohydrate. Antiserum was raised in a rabbit, and on immunodiffusion, gave a single precipitin line with the purified placental folate binding protein. Immunoprecipitation studies using this antiserum indicated that the purified placental folate binding protein shared antigenic determinants with both the large Mr and small Mr folate binding proteins from human milk. Immunofluorescent studies with this antiserum and human erythrocytes revealed the presence of an immunologically similar protein on the plasma membrane of these cells suggesting that this protein may function as a folate receptor.     

Immunochemical characterization of the outer membrane complex of Serratia marcescens and identification of the antigens accessible to antibodies on the cell surface

Serratia marcescens New CDC O14:H12 contains major outer membrane proteins of 43.5 kDal, 42 kDal (the porins) and 38 kDal (the OmpA protein) which can be separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Immunoblotting of whole cell or outer membrane preparations using antiserum raised against the whole cells revealed similar complex patterns of antigens. The OmpA protein was the major immunogen, although six other outer membrane proteins were also detected; the porins reacted only weakly with antibodies in this system. Immunoabsorption of antisera with whole cells showed that only the O antigenic chains of lipopolysaccharide and the H (flagella) antigens were accessible to antibody on the cell surface. Failure to detect the OmpA protein and other envelope antigens in this way suggests that their antigenic sites are not able to react with antibodies and are possibly masked by the O antigen.     

A highly antigenic proteoglycan-like component of cholinergic synaptic vesicles

A monoclonal antibody, tor70, recognizes an antigenic determinant on the inside surface of synaptic vesicles, purified from the electric organ of Narcine brasiliensis. The antigenic determinant appears to be unique to vesicles since it co-purifies with vesicle content and is blocked by an antiserum specific for synaptic vesicle antigens. Immunoblotting of vesicle proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the antigen has a low heterogeneous electrophoretic mobility and corresponds to a major protein component of pure synaptic vesicles. Synaptic vesicles contain a proteoglycan-like material since proteolytic digestion yields a ruthenium red-binding material that migrates during electrophoresis with a mammalian heparin standard. The only major vesicle component with which the proteoglycan-like material co-elutes during chromatography on Sepharose 6B is the material recognized by tor70. The antigen adsorbs specifically to beads coated with the lectin wheat germ agglutinin. Isolation of the tor70 antigen by velocity sedimentation in sodium dodecyl sulfate-sucrose gradients shows it to contain glucosamine (0.75 nmol/microgram of protein) and uronic acid but no galactosamine. Earlier work has shown that specific antiserum to pure synaptic vesicles could be used to identify nerve terminals, quantitate vesicle components, purify membranes, and monitor exocytosis. We now know that one of the components recognized by the antiserum is a molecule with properties of a proteoglycan, attached to the inside surface of vesicle membranes.     

Structural Proteins of Mammalian Oncogenic RNA Viruses: Multiple Antigenic Determinants of the Major Internal Protein and Envelope Glycoprotein

The antigenic determinants of two purified protein constituents of mammalian C-type RNA viruses, the major structural protein of about 30,000 daltons, and the membrane glycopeptides of about 70,000 daltons were examined by competition radioimmunoassay. By the appropriate choice of antiserum and competing proteins, it was possible to distinguish type-specific, group-specific, and interspecies determinants. Both of the viral constituents were found to contain each of these three classes of antigens. The results suggested that the majority of the determinants of the major structural protein were group specific, 5% to 30% were interspecies, and a small fraction were type specific. In the case of the envelope glycopeptides, the chief determinants were type and group specific, and a small fraction were interspecies.     

Structural proteins of mammalian RNA tumor viruses: relatedness of the interspecies antigenic determinants of the major internal protein.

The relatedness of antigenic determinants of purified major core proteins of the murine, feline, RD 114/baboon, and woolly monkey/gibbon ape groups of RNA tumor viruses was examined by competition radioimmunoassay. In assay systems of a homologous antigen and antiserum, high affinity competition for binding to all of the antibodies was observed only with the homologous unlabeled protein; the core proteins of other groups of viruses showed only low affinity binding of a small fraction of antibodies, presumably those reactive with the interspecies determinants, at concentrations of competing protein 10- to 100-fold greater than that of the labeled antigen. The cross-reactive (interspecies) antigens of every two viruses were selectively examined by precipitating the purified 125-I-labeled protein with antiserum against each of the other proteins. The extent to which these shared determinants were common to the other viruses was then tested by the effectiveness of the proteins of each virus to compete for antibody binding. Several classes of interspecies determinants were distinguished: those common to two of the groups of viruses, others to three, and some to all four. Moreover, an even greater variety of interspecies determinants was indicated by differences in the affinity of the individual proteins for antibody binding, supporting the hypothesis that there are at least several, if not many, different interspecies determinants with a broad spectrum of antigenic cross-reactivity. These studies suggest that the murine and feline viruses are closely related as they contain cross-reactive antigenic determinants not shared with the other viruses, that the feline virus is more closely related to the woolly monkey virus than to RD 114, and that the RD 114 and woolly monkey viruses retain interspecies determinants shared relatively equally with each of the other viruses.     

Isolation of a human B lymphocyte membrane protein with Ia-like properties.

A rapid method for isolation of a major surface membrane glycoprotein from whole, unfractionated cultured human B lymphoblasts is described. After detergent solubilization the method uses gel filtration followed by affinity chromatography on Sepharose Con A and then alkaline acrylamide gel electrophoresis. Specific high-titre, rabbit antisera to the isolated protein reacted with cultured and normal peripheral blood B lymphocytes, as well as peritoneal macrophages from a renal dialysis patient. The antisera selectively inhibited the mixed lymphocyte reaction at high dilution. The protein reacted with a heterologous antiserum to HL-B antigens and contained subunits of MW 33 000 and 27 000. Resolution of the subunits, however, required a discontinuous SDS gel system. These properties indicate its similarity to murine Ia antigens. The protein was not associated with beta 2 microglobulin and showed no structural or antigenic similarity to the major erythocyte glycoprotein, glycophorin. Antisera to the protein failed to precipitate surface-radiodinated components from similarily treated extracts of cultured human T lymphoblasts. This method now makes available a reference membrane glycoprotein from a differentiated, nucleated human cell in sufficient purity and quantity for kinetic and biosynthetic studies.     

Preparation of variant-specific anti-human placental phosphatase antisera by immunoabsorption.

Antiserum raised in rabbits sensitized with purified human placental alkaline phosphatase of the rare FD phenotype was absorbed on purified FF phenotype enzyme conjugated to Sepharose. The absorbed antiserum was not able to bind to the F-variant, but was still capable of binding to the D-variant enzyme, determined by electrophoretic retardation and gel filtration. It therefore appears that some allelic variants of placental phosphatase differ in their antigenic structure.     

Identification, purification, and characterization of major antigenic proteins of Campylobacter jejuni

Evidence from developing countries and volunteer studies indicates that immunity to Campylobacter jejuni and Campylobacter coli may be acquired, but the antigenic basis for this protection is poorly defined. We have purified to homogeneity four proteins with molecular weights of 28,000 (PEB1), 29,000 (PEB2), 30,000 (PEB3), and 31,000 (PEB4) from epidemic C. jejuni strain 81-176 using acid extraction and sequential ion-exchange, hydrophobic interaction, and gel filtration chromatography. The relative amino acid compositions of these four proteins are similar. NH2-terminal sequence analysis indicates that all four proteins are different, although the first 35 amino acids of PEB2 and PEB3 are 51.4% homologous. Isoelectric focusing showed that all four are basic proteins with pI of 8.5 for PEB1 protein and greater than 9.3 for the others. Use of the purified proteins as antigens in an IgG enzyme-linked immunosorbent assay (ELISA) found that seroconversion to the PEB1 or PEB3 proteins occurred in 15 of 19 patients with sporadic C. jejuni or C. coli infection. In comparison, only two, six, and 14 of these patients seroconverted to PEB2, PEB4, or the acid extract antigen. In an ELISA with whole bacterial cells as antigens, antiserum to the acid-extracted antigens showed broad recognition of C. jejuni, C. coli, C. fetus, C. lari, and Helicobacter pylori. Antiserum to PEB1 recognized all 35 C. jejuni and all 15 C. coli strains but none of the isolates of the other three bacterial species. The PEB1 and PEB3 proteins appear to be candidate antigens for both a Campylobacter vaccine and for serological assays for the pathogen.     

Localization of phosphoprotein C23 in nucleoli by immunological methods

Antiserum to a major phosphorylated nucleolar protein. C23 (MW 103000, pI 5.2) from Novikoff hepatoma was produced in rabbits. By immunodiffusion analysis, the antiserum produced precipitin bands and with various crude extracts of nucleoli, but not with extranucleolar or cytosol fractions. The specificity of the antibody was assessed using acid-urea polyacrylamide gel electropherograms of acid-soluble nucleolar proteins in which the separated proteins were transferred to nitrocellulose sheets. The purified antibody reacted predominantly with protein C23 as visualized by the immunoperoxidase procedure. By the indirect immunofluorescence technique, protein C23 was localized predominantly to nucleoli of Novikoff hepatoma or normal rat liver cells. In Novikoff hepatoma cells, traces of fluorescence were seen near the inner layer of the nuclear envelope. Additional narrow regions of fluorescence extended from the nucleoli into the extranucleolar areas of some Novikoff cells. The nucleolar areas of fluorescence were smaller but brighter in the normal liver than in Novikoff hepatoma, consistent with the small size of rat liver nucleoli. These data indicate that the major location of protein C23 is the nucleolus.     

Simultaneous Purification of Murine Mammary Tumor Virus Structural Proteins: Analysis of Antigenic Reactivities of Native gp34 by Radioimmunocompetition Assays

All the structural proteins (gp47, gp34, p27, p23, p16, and p12) of the murine mammary tumor virus (MuMTV) were simultaneously purified utilizing alkylagarose chromatography as the initial fractionation step. Least-hydrophobic MuMTV polypeptides (p23, p16) and the slightly hydrophobic p27 were separated from moderately hydrophobic proteins gp47 and p12 by passage through octylimino (C(8))-agarose; the gp47 and p12 could be removed from the matrix by elution with ethylene glycol, whereas the most hydrophobic MuMTV protein, gp34, was eluted using nonionic detergent together with ethylene glycol. Subsequent purification steps involved ion-exchange or gel filtration chromatography. The resulting protein preparations appeared near-homogeneous on analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Recoveries of MuMTV proteins, based on their approximate individual contribution to total virus protein, ranged from about 20% for gp47 to greater than 100% for the minor structural component p23, the major phosphoprotein of MuMTV. Antiserum against purified C3H MuMTV gp34, together with purified, radioiodinated gp34, was used to develop a radioimmunoassay which showed that from 13 to 14% of total MuMTV protein by weight is gp34. Using this assay system, the group-specific antigenic reactivity of gp34 was also demonstrated. When solubilized preparations of C3H, RIII, and GR MuMTV's were used as competing antigens in gp34 radioimmunoassays with anti-C3H MuMTV serum, both group- and type-specific differences in antigenic reactivity were found.     

Photosynthetic apparatus in chilling-sensitive plants

Proteins of fresh, cold and dark-stored and illuminated tomato leaves were fractionated by SDS electrophoresis. The total soluble proteins extracted from fresh leaves were separated into 5 main fractions with MWs of 54,000, 45,000, 32,000, 23,000 and 14,000. The cold and dark storage of the leaves causes a marked reduction mainly in the fraction with MW of 45,000 which increased with the illumination of the cold and dark-storaged leaves. The polypeptides with MWs of 54,000 and 14,000 (probably large and small subunits of ribulose, bisphosphate carboxylase) were stable under these conditions. In contrast, the polypeptides with MWs of 54,000 and 14,000 are decreased following the storage of tomato leaves in the dark at room temperature. Chloroplast soluble proteins were seperated by SDS electrophoresis into fractions with MWs of 64,000, 54,000, 20,000 and 14,000. The same fractions in similar proportions were observed in soluble-chloroplast proteins from fresh as well as coold and dark-stored and illuminated leaves. No drastic changes in structural polypeptides were observed following cold and dark-storage and illumination of the leaves. The results indicated that the main protein fraction, which degradated following cold and dark storage of tomato leaves and synthetized during illumination, is the fraction of cytoplasmic protein which in SDS electrophoresis gives polypeptides of about 45,000 MW. The fractions of chloroplast proteins were stable under such conditions.Abbreviations DCIP 2,6-dichlorophenolindophenol - FFA free fatty acid - MW molecular weight - RuBP carboxylase ribulose 1,5-bisphosphate carboxylase - SDS sodium dodecyl sulphate - TCA trichloroacetic acid     

Production of polyclonal antiserum specific to the 27.5 kDa envelope protein of white spot syndrome virus

A truncated version of the white spot syndrome virus (WSSV) 27.5 kDa envelope protein was expressed as a histidine tag fusion protein in Escherichia coli. The bacterial expression system allowed the production of up to 10 mg of purified recombinant protein per liter of bacterial culture. Antiserum from a rabbit immunized with the recombinant protein recognized the 27.5 kDa viral envelope protein of WSSV isolated from different geographic regions. The antiserum did not recognize any of the other known WSSV structural proteins. A sensitive immunodot assay for WSSV was developed using the specific rabbit polyclonal antiserum.     

Localization of a 56-kDa antigen that is present in multiple developmental stages of Neospora caninum

The purpose of the present study was to characterize the intracellular distribution of a native Neospora caninum 56-kDa protein that is recognized by sera from N. caninum-infected dairy cattle. The complementary DNA coding for this protein was expressed in Escherichia coli as a polyHis fusion protein to which antiserum was prepared and used to localize the antigen in N. caninum tachyzoites and bradyzoites. By sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting, antirecombinant Nc56 serum recognized a major 56-kDa protein and 2 minor (43 and 39 kDa) proteins of N. caninum tachyzoites. Antiserum to recombinant 56-kDa protein showed this antigen to be present in both N. caninum tachyzoites and bradyzoites/cysts as detected by immunofluorescence staining. Immunoelectron microscopy revealed the 56-kDa antigen to be present in the apical end of both tachyzoites and bradyzoites and possibly extracellularly secreted by tachyzoites.     

Antigenic identification of virion structural proteins from infectious spleen and kidney necrosis virus

Infectious spleen and kidney necrosis virus (ISKNV), belonging to the genus Megalocytivirus in the family Iridoviridae, is one of the major agents causing mortality and economic losses to the freshwater fish culture industry in Asian countries. Currently, little information regarding the antigenic properties of Megalocytivirus (especially ISKNV) is available. Our previous study using four different workflows with systematic and comprehensive proteomic approaches led to the identification of 38 ISKNV virion-associated proteins (J. Virol. 2869-2877, 2011). Thus, in this report, the antigenicity of 31 structural proteins from ISKNV virion was investigated. A one-dimensional gel electrophoresis immunoblot profile coupled with MALDI-TOF-TOF MS/MS was applied to identify six immunogenic viral proteins, namely, ORFs major capsid protein (006L), 054L, 055L, 101L, 117L, and 125L. Then, the antigenicity of 31 structural proteins was characterized by Western blot by using pooled sera from mandarin fish that survived ISKNV infection. Of the 31 viral proteins, 22 were recognized by the fish ISKNV antiserum. Furthermore, this antiserum neutralizes MFF-1 cells ISKNV infection. To our knowledge, this study is the first report on the immunogenicity of viral proteins and characterization of the proteome of megalocytivirus infective agents. Our findings are expected to promote the development of effective vaccine candidates.     

Heat-stable proteases from psychrotrophic pseudomonads: comparison of immunological properties.

A heat-stable extracellular protease from Pseudomonas fluorescens was purified by chromatography on a DEAE-cellulose column and gel filtration on a Sephadex G100 column. The homogeneous enzyme preparation was used to prepare antiserum in rabbits. The rabbit antiserum was used to study the antigenic relatedness of proteases from 19 psychrotrophic pseudomonads isolated from raw milk. The inhibition of the proteases by the antiserum and the gel precipitin reactions revealed similar antigenic determinants in proteases from different isolates. Rabbit antiserum to the purified protease gave precipitin bands with antigens (proteases) from 10 different isolates. However, the same antiserum did not inhibit the protease activity in cell extracts of isolates T10, T13, and T24. By determining serological cross-reactions, proteases from psychrotrophic pseudomonads were shown to be different from one another.