Purification, characterization and serological properties of a glycolipid antigen reactive with a serovar-specific monoclonal antibody against Leptospira interrogans serovar canicola

A glycolipid antigen possessing a serovar-specific antigenic determinant of Leptospira interrogans serovar canicola was purified from a chloroform/methanol extract of the organism. The purification procedures included silicic acid column chromatography and preparative thin-layer chromatography (TLC). Antigenic activity was detected by a TLC-enzyme immunostaining technique using monoclonal antibody CT3, which specifically agglutinates serovar canicola and only weakly serovar sumneri but no other serovars of Leptospira. The purified glycolipid reacted with CT3 antibody, indicating that the glycolipid possessed a serovar-specific antigenic determinant. Infrared spectrum and proton nuclear magnetic resonance analyses showed that the glycolipid contained sugar and lipid moieties, which possessed amide linkages and an acetyl group. Gas-liquid chromatography-mass spectrometry analysis showed that the glycolipid contained two unknown sugars, one of which (unknown sugar II) appeared to be associated with the antigenic determinant specific for canicola. The serovar-specific antigenic determinant was destroyed by mild alkali treatment of the glycolipid. These findings suggested that the antigenic determinant was an alkali-labile moiety which may be related to the unknown sugar II.     

Nature of antigenic determinant of serovar-specific antigen of Leptospira interrogans serovar hebdomadis.

The nondialyzable delipidized serovar-specific main antigen (NDTM antigen) of Leptospira interrogans serovar hebdomadis strain Hebdomadis (a variant which can grow in a synthetic medium) showed a strong inhibition of the complement fixation between the serovar-specific main (TM) antigen of this strain and the homologous antiserum. The inhibitory effect of the NDTM antigen was completely lost by treating the antigen with proteolytic enzymes, and the fractions of TM antigen containing amino sugar, neutral sugars, and lipids did not show any inhibition of complement fixation, indicating that the antigenic determinant of this strain is related to proteins. NDTM antigen contained more hydrophilic amino acids than hydrophobic amino acids, whereas TM antigen contained more hydrophobic amino acids than hydrophilic amino acids. The amino acid compositions of NDTM antigens of hebdomadis strain Hebdomadis (variant) and kremastos strain Kyoto, which belonged to the same serogroup, were considerably similar. Difference was found in the amounts of methionine, arginine, lysine and glutamine acid.     

Immunochemical Characteristics and Localization on Cells of Protective Antigen (PAg) Prepared from Leptospira interrogans Serovar lai

Immuno-electron microscopic methods revealed that the protective antigen (PAg) of Leptospira interrogans serovar lai exists on the outer envelope sheathing the leptospiral cell body. PAg lost its protective activity after treatment by hydrolysis with 2 M formic acid at 100 C for 2 hr, or oxidation with periodate at 4 C for 40 hr. The antigenic oligosaccharide fraction was further purified from the hydrolyzed PAg by immunoaffinity column coupled with protective monoclonal antibody, LW2, and by gel filtration of HPLC. The antigenic oligosaccharide fraction contained two unknown sugars and 4-O-methylmannose (molar ratio 3:5:1). These findings suggested that these sugars are components of an antigenic determinant contributing to the protective immunity against serovar lai infection.     

Isolation of an antigenic oligosaccharide fraction from Leptospira interrogans serovar canicola with a monoclonal antibody

An oligosaccharide fraction containing the antigenic determinant of lipopolysaccharide antigen (TM antigen) from Leptospira interrogans serovar canicola, recognized by a monoclonal antibody (CT3) which agglutinates serovars canicola and broomi, was isolated by formic acid and successive sulphuric acid hydrolyses. Separation of the antigenic compounds was done by Bio-Gel P-2 and Sephadex G-25 gel filtration, and high-performance liquid chromatography with two different columns. The fraction finally obtained was a mixture of two oligosaccharides, both of which migrated as a single spot having a slightly higher mobility than an authentic tetrasaccharide (stachyose) on thin layer chromatography. The fraction contained rhamnose, arabinose and two major and two minor unknown sugars which were shown to be N- or O-acetylated and/or O-methylated sugars by nuclear magnetic resonance. The fraction inhibited the binding of CT3 antibody with TM antigen in enzyme-linked immunosorbent assay and microscopic agglutination of serovar canicola with the antibody. The inhibitory activity was destroyed by periodate oxidation or mild alkaline treatment, but was resistant to sodium borohydride reduction.     

Characterization of the Antigenic Subunits of the Envelope Protein of Yersinia pestis

Chemical, physical, and immunological properties of the envelope antigen of Yersinia pestis strains have been investigated. The antigen consists of two components with isoelectric points (pI) of 4.6 and 4.8. One component (pI 4.6) is a protein bound to a small carbohydrate moiety identified as an oligomeric galactan; the other component (pI 4.8) is a simple protein. These two components are antigenically identical. In buffered solution, the antigen exists as aggregates of molecular weights larger than 300,000. The aggregates dissociate into a variety of smaller molecular weight forms depending on the nature of the treatment for dissociation. Each aggregate can be further dissociated into a single antigenic subunit fraction containing protein and glycoprotein species with molecular weights in the range from 15,000 to 17,000. The subunits can be obtained by a dissociation treatment with 0.1% mercaptoethanol in 0.25% sodium dodecyl sulfate at 95 C for 5 min. The subunits will readily reaggregate into a variety of larger molecular weight forms on the removal of dodecyl sulfate.     

Identification of Surface Antigens of Sarcocystis muris (Coccidia)

Zoites of Sarcocystis muris were recovered from the skeletal muscles of infected mice by trypsin digestion. Extracts of zoites prepared by freeze-thaw, Triton X-100 (0.1%), or a combination of the two treatments contained antigenic components. Testing of these antigens by agar gel diffusion and immunoelectrophoresis against sera from infected mice showed one major precipitin band. SDS-polyacrylamide-gel electrophoresis (SDS-PAGE) of the extracts revealed at least eight detectable polypeptides ranging in molecular weight from 10,000 to 220,000. The antigenic components of the extract were identified by labeling the parasite surface with [¹²⁵I] and precipitation of the [¹²⁵I]-labeled antigens with immune sera. Analysis of the immunoprecipitates by SDS-PAGE and autoradiography revealed three antigens with molecular weights of 27,500, 43,000 and 90,000. The smallest of these was the predominant antigen as suggested by labeling intensity.     

Agglutination,Toxigenicity and Sorbitol Fermentation of Clostridium difficile

A total of 79 Clostridium difficile strains from different sources (50 strains from the fecal specimens of healthy adults, 13 from patients receiving antibiotics without gastrointestinal complications, 13 from antibiotic-associated pseudomembranous colitis (PMC) or diarrhea patients, and three strains from ATCC) were investigated for agglutinability, using formol-treated cells as antigen, in relation to toxigenicity. C. difficile strains tested were divided into four serovars, I, II, III, and IV, by the cross-agglutination test. The agglutinin absorption test revealed that strains of serovar I, agglutinable with high titers (5,120–10,240) to antiserum prepared against a highly toxigenic C. difficile strain, ATCC 17859, possessed the serovar-specific antigen. All of the strains of serovar I were highly toxigenic and all 13 strains isolated from the fecal specimens of antibiotic-associated PMC or diarrhea patients belonged to this serovar, whereas 19 (38%) out of 50 strains from healthy adults and four (30.8%) out of 13 strains from patients receiving antibiotics without gastrointestinal complications possessed this antigen. None of the strains of other clostridial species than C. difficile were agglutinated by the three reference antisera used. Further study on the sugar fermentation test disclosed that the sorbitol-fermenting property of C. difficile is very closely related to the toxigenicity and agglutinability.     

Characterization of the antigen recognized by monoclonal antibody 1D3

Human ovarian mucinous cystadenocarcinoma-associated antigen recognized by murine monoclonal antibody 1D3 (Bhattacharya et al., 1982) was characterized. Gel filtration and sodium dodecylsulfate polyacrylamide gel electrophoresis, followed by Western-blot analysis showed that 1D3 is a high molecular weight glycoprotein. Isoelectric focusing of 1D3 antigen showed 2 overlapping antigenic components with PI 2.5 and 2.6. 1D3 antigen was extremely stable (10 min at 100 degrees C) to heating. The antigenic activity was slightly stimulated by treatment with galactosidases, but neuraminidase treatment enhanced the antigenic activity about 3-fold. Antigen activity was completely stable to periodate oxidation. Pronase and trypsin treatment completely destroyed the antigenic activity. Properties of 1D3 antigen suggest that this is a high molecular weight (approximately 5-20 x 10(6) Dalton), sialomucin. Monoclonal antibody 1D3 recognizes only the protein part of this molecule.     

Detection and isolation of an Ia glycoprotein antigen from mouse serum

Ia antigenic activity is present in mouse serum and exists predominantly (>90 percent) as a dialyzable oligosaccharide which can be purified readily. This paper describes experiments which indicate, however, that a small proportion (<10 percent) of the Ia antigenic activity in serum, particularly from mitogen-injected mice, exists in a high molecular-weight form. Furthermore, this high molecular-weight Ia antigen, like the Ia oligosaccharide, appears to be secreted by T lymphocytes, particularly by T cells which have been activated recently by mitogens. The high molecular-weight Ia antigen was isolated from mouse serum by a procedure which utilized salt fractionation, gel filtration, density separation, and sucrose gradient sedimentation. This procedure gave a 190–260-fold increase in specific Ia antigenic activity, and up to 80 or 90 percent of high molecular-weight Ia antigenic activity detected in the original serum was recovered in the purified fraction. On the basis of sucrose gradient sedimentation studies, the high molecular-weight Ia antigen was estimated to have a molecular weight of approximately 500,000 daltons, if it is assumed that it exists as a globular protein. In addition, the protein has an ₂-macroglobulin-like electrophoretic mobility, contains little or no lipid, and, based on its ability to bind to Concanavalin A columns, is a glycoprotein. The relationship between this Ia glycoprotein antigen and other molecular forms of Ia antigen is discussed.     

Skin test-active substance prepared from culture filtrate of Fonsecaea pedrosoi

Ethanol-precipitated substance (EP) was prepared from culture filtrate of Fonsecaea pedrosoi. EP was separated into two components by passing through a Sephadex G-50 column; the faster passing component was referred to as EP-1, the slower as EP-2. EP-1 and EP-2 were evaluated as an antigen for detecting cutaneous delayed hypersensitivity in patients with chromomycosis. EP-1 elicited positive delayed skin reactions in all of 8 patients with chromomycosis, of which 7 caused by F. pedrosoi and one by Exophiala jeanselmei. Healthy subjects, patients with sporotrichosis and patients with tinea barbae failed to react to EP-1. These results indicate that EP-1 is a useful tool for detecting cutaneous delayed hypersensitivity in patients with chromomycosis caused by F. pedrosoi. It was found that precipitin test using EP-1 as an antigen had little diagnostic value in chromomycosis. EP-2 did not show antigenic activity in both skin and precipitin reactions.     

Isolation of the Sweet Potato Weevil Factor Causing Terpene Induction in Sweet Potato Roots

The larval homogenate of sweet potato weevil, Cylas formicarius, induced terpene formation in sweet potato root tissue. We indicated that the terpene-inducing factor of the larvae consisted of high molecular weight component(s) and low molecular weight component(s). The high molecular weight component(s) was heat-unstable and partially inactivated by pronase, indicating proteinacious properties. The low molecular weight components) was heat-stable.     

Erratum to: Evaluation of the Use of Selective PCR Amplification of LPS Biosynthesis Genes for Molecular Typing of <Emphasis Type="Italic">Leptospira</Emphasis> at the Serovar Level

Leptospirosis is an important epidemic zoonosis worldwide. Currently, there are more than 250 Leptospira pathogenic serovars known that can potentially infect humans. Conventional classification of leptospires with the serovar as the basic taxon, based on serological recognition of lipopolysaccharide (LPS) composition does not correlate well with species determination, based on general genomic features. Here, we investigate the selective amplification of polymorphic regions from the LPS biosynthesis loci (rfb) as a potential tool for serovar typing of Leptospira interrogans species. Eight pairs of primers were designed to target six ORFs from the rfb operon with varying levels of sequence polymorphism. They were tested both separately and multiplexed. Half of these primer pairs produced serovar-specific amplicons, allowing the identification of some specific serovars and also groups of serovars. It was shown that the serovar classification of Leptospira can be accessed by selective amplification of rfb operons in some cases, which may permit a parallel between the serological and the genomic classifications of Leptospira. As a conclusion, the selective amplification of rfb generated promising and already useful results, but it appears necessary to characterize a larger variety of Leptospira genomes or rfb operons to fully develop this method.     

Biochemical characterization of H9/25, an allospecificity encoded by the Ly-6 region

H9/25, an allospecificity encoded by the Ly-6 region, was biochemically characterized. It was sensitive to pepsin and heat treatment, but was resistant to periodate oxidation. Its apparent molecular weight was approximately 12 000 daltons by gel filtration. The antigenic molecule was partially purified by gel filtration and antibody affinity chromatography. The partially purified antigen molecule was radioiodinated, immunoprecipitated with monoclonal antibody H9/25, and analyzed by SDS-polyacrylamide gel electrophoresis. The autoradiograph showed the molecular weight of H9/25 to be approximately 15000 daltons under reducing conditions. These results indicate that H9/25 is a protein with a single polypeptide chain of 12000–15000 daltons molecular weight, and the antigenic specificity is carried by a peptide but not a carbohydrate moiety.     

Characterization of Monoclonal Antibodies against Etiological Agents of Weil's Disease

Monoclonal antibodies against etiological agents of Weil's disease were produced by cell fusion technology. Twenty hybridomas were produced through the fusion of P3×63Ag8.653 cells with spleen cells from BALB/c mice immunized against Leptospira interrogans serovar icterohaemorrhagiae RGA strain and serovar copenhageni Shiromizu and M20 strains. Reactivities of the antibodies produced by the hybridomas were determined by the microscopic agglutination test. Among the five hybridoma antibodies to the RGA strain, two reacted specifically to serovar icterohaemorrhagiae, two reacted to serovar icterohaemorrhagiae at a high titer and serovar copenhageni at a low titer, and one reacted to serovars icterohaemorrhagiae, copenhageni, pyrogenes, and canicola. Of the ten hybridoma antibodies to the Shiromizu strain, one reacted specifically to serovar copenhageni, seven reacted to both serovars copenhageni and icterohaemorrhagiae at almost the same titer, and two exhibited intermediate properties. Of the five hybridoma antibodies to the M20 strain, three reacted to both serovars copenhageni and icterohaemorrhagiae at almost the same titer, one reacted to serovar copenhageni at a low titer and serovar icterohaemorrhagiae at a high titer, and one reacted to serovars copenhageni, icterohaemorrhagiae, and pyrogenes. The results revealed that each serovar has its own antigen(s) and their common antigens. In addition, 20 strains of leptospires were recently isolated and tested with three monoclonal antibodies characterized by different reactivities. Twenty strains were clearly identified by their antibodies, i.e., 16 strains were identified as serovar icterohaemorrhagiae and three strains were identified as serovar copenhageni. The remaining strain, which was not agglutinated by three antibodies, was identified as serovar autumnalis by an agglutination test with immune rabbit sera.     

Identification of two types of immature antigenic molecules on chicken red cells

Surface-labelled antigen analysis of circulating embryonic erythrocytes shows that the population of 48 000 D antigenic molecules involves an agerelated antigenic complex referred to as E₁ 48 K component and, in addition, an antigenic molecule also expressed on immature red cells from anemic adult chickens referred to as Im 48 K component. A second immature antigenic molecular species of 140 000 D was detected on red cells from anemic birds and is referred to as Im 140 K component. The Im 48 K and Im 140 K components have no common determinants, although they are both found on immature red cells of anemic animals. This work suggests that these two immature antigens could be involved in the differentiation process, possibly at different levels of erythroid progenitors.     

Spontaneous and transient defence against bacteriophage by phase‐variable glucosylation of O‐antigen in Salmonella enterica serovar Typhimurium

As natural killers of bacteria, bacteriophages have forced bacteria to develop a variety of defence mechanisms. The alteration of host receptors is one of the most common bacterial defence strategies against phage infection, which completely blocks phage attachment but comes at a potential fitness cost to the bacteria. Here, we report the cost‐free, transient emergence of phage resistance in Salmonella enterica subspecies enterica serovar Typhimurium through a phase‐variable modification of the O‐antigen. Phage SPC35 typically requires BtuB as a host receptor but also uses the Salmonella O12‐antigen as an adsorption‐assisting apparatus for the successful infection of S. Typhimurium. The α‐1,4‐glucosylation of galactose residues in the O12‐antigen by phase variably expressed O‐antigen glucosylating genes, designated the ^{LT 2} gtrABC1 cluster, blocks the adsorption‐assisting function of the O12‐antigen. Consequently, it confers transient SPC35 resistance to Salmonella without any mutations to the btuB gene. This temporal switch‐off of phage adsorption through phase‐variable antigenic modification may be widespread among Gram‐negative bacteria‐phage systems.     

Studies on the functions of the RNA polymerase components by means of mutations

Summary It had been shown earlier, that RNA polymerase 13 S particles contain the large components with a molecular weight of about 3–10⁵ and small subunits with a molecular weight of 4·10⁴-1·10⁵. These polymerase components easily dissociate and reassociate with restoration of the enzyme activity.Both temperature-sensitive (tsX) and rifamycin-resistant (rif-r-I) mutations proved to affect the large polymerase component without changing the small subunits. These mutations were mapped at different, though closely linked, loci of metB-thi region of E. coli K12 chromosome. These results as well as certain literature data allow to conclude that the large RNA polymerase component consists of at least two polypeptides, one being altered by ts mutation, and the other—by rif-r mutation.The large polymerase component when separated from the small subunits retain the ability to bind to T2 phage DNA while the separate small subunits lack this property. Rifamycin does not affect RNA polymerase-T2 DNA binding while ts mutation leads to inability of the enzyme to form stable complexes with DNA. Therefore, it is likely that the polypeptide affected by ts mutation is responsible for the attachment of RNA polymerase to specific sites of DNA template. On the other hand, the small subunits as well as polypeptide of the large component, which determines RNA polymerase sensitivity to rifamycin, seem not to participate in the enzyme binding to DNA template. It is suggested, that the catalytic site of RNA polymerase is located in the large component and formed by rifamycin-binding polypeptide. The small subunits are supposed to have regulatory function and activate the large components.     

Phenotypic and genotypic features of new autoagglutinating Bacillus thuringiensis strains

A total of 28 autoagglutinating strains of Bacillus thuringiensis were isolated from different ecologic niches and distinct sites. Twenty-six strains demonstrated toxicity to mosquito larvae of Aedes aegypti and Culex quinquefasciatus. The electrophoretic protein profiles of the crystal components were studied. Twenty-three out of the 28 strains showed the same larvicidal activity and the same protein profiles as B. thuringiensis serovar israelensis. Using isoenzyme analysis (MLEE), it was observed the presence of three electrophoretic types (ETs). The mosquitocidal strains grouped into one ET. The random amplified polymorphic DNA analysis (RAPD) was evaluated using six primers, which demonstrated three different patterns for the 28 autoagglutinating strains, allowing correlation of the profiles obtained with the toxicity observed in the bioassays. The RAPD patterns for mosquitocidal strains were identical to the one of serovar israelensis. However, to strains of low toxicity, each primer generated distinctive RAPD patterns, which demonstrated that these strains belong to different serovars. Although the antigenic classification the 26 autoagglutinating strains of B. thuringiensis could not be determined by classical flagellar serotyping, MLEE and RAPD profiles proved these strains to be compatible with B. thuringiensis serovar israelensis.     

The stereospecific d-glucose transport protein in cholate extracts of human erythrocyte membranes Molecular sieve chromatography and estimation of molecular weight

Cholate extracts of human erythrocyte membranes (Lundahl, P., Acevedo, F., Fröman, G. and Phutrakul, S. (1981) Biochim. Biophys. Acta 644, 101–107) were fractionated by molecular sieve chromatography on Sepharose 6B, and the size and molecular weight of the active d-glucose transporter were estimated. The eluent contained 10 or 12.5 mM cholate, since higher concentrations inactivated the glucose transporter, and lower concentrations resulted in aggregation. The chromatographic distribution of the transport activity was reproducible, but was broader than one would expect for a homogeneous component. In the presence of 20 mM EDTA and 5 mM dithioerythritol, a combination which affords a highly stable transport activity, a molecular weight of $\text{400 000 ± 20 000}$ (Stokes' radius 5.9 nm) was estimated for the smallest active component. This value represents an upper limit, since the molecular weight of a non-spherical component would have been overestimated, and since bound cholate was calculated to represent about 12% of the molecular weight. The activity was completely recovered upon rechromatography. In 10 mM EDTA and 10 mM 2-mercaptoethanol, the estimated molecular weight of the smallest active component was $\text{210 000 ± 15 000}$, and this component was not stable upon rechromatography in 10 mM EDTA and 10 mM 2-mercaptoethanol. In the absence of chelating and reducing agents, cholate extracts from membranes which had been kept for 5 days at 4°C showed three additional active components smaller than 200 000 in molecular weight. Most of the phospholipids eluted later than the active components of molecular weight 400 000 or 210 000, in all experiments. Electrophoretic analysis in dodecyl sulfate of the chromatographic eluents indicates that at least one of the band 3-polypeptides (nomenclature according to Steck, T.L. (1974) J. Cell Biol. 62, 1–19) is a component of the active transporter. This band 3-polypeptide, which we denote 3.3, has an apparent molecular weight of 88 000. The stable transporter of molecular weight 400 000 might be a tetramer of the 3.3-polypeptide. Alternatively, a dimer of the 3.3-polypeptide in complex with lipids might account for this molecular weight. If the 3.3-polypeptide is the transporter subunit and if it binds cytochalasin B with high affinity ($\text{1.8 · 10}{}^5$ sites/cell) the recovered activity per 3.3-polypeptide is around 40% A degradation product of the 3.3-component (possibly a 4.5-component) might account for the unstable active transporter of molecular weight 210 000.     

Far‐eastern strains of bean common mosaic virus and methods of their immunodiagnostics

The paper presents data of investigation on the physico‐chemical and antigenic properties of capsid proteins of the Bean common mosaic virus isolated from Phaseolus plants in the Russian Far East (BCMV‐R) and from China (BCMV‐C). A method for isolation of the virus preparation was selected. The purified preparations of two isolates BCMV have been obtained. The presence of one polypeptide in structural proteins of virions was established and their molecular masses determined (BCMV‐R ‐ 31,6 kD; BCMV‐C ‐ 32,1 kD). Polyclonal antiserum was obtained with titre 1:12800 and the indirect and “sandwich"‐variants of ELISA were developed to detect this virus. The allied relationships were established with the bean yellow mosaic virus and with the type representative of the genus Potyvirus ‐ PVY. Based on the data of physico‐chemical and antigenic properties it was concluded that isolates BCMV‐R and BCMV‐C are two independent strains of this virus. The presence of strain‐, virus‐ and genusspecific epitopes of capsid proteine was revealed as a result of comparison of antigenic characteristics of the Russian Far Eastern and Chinese strains of BCMV. A high antigenic activity of capsid protein of the Russian Far Eastern strain was observed.