Immunochemical Characterization of Cell Wall Protein Antigen Purified from the Cell Wall Autolysate of Clostridium botulinum Type A

The cell wall protein antigen was solubilized from the isolated cell walls of Clostridium botulinum type A by autolysis and purified by diethylaminoethyl-cellulose column chromatography followed by gel filtration on Sephadex G-150. The two fractions showed a high degree of the serological activity and produced a main fused precipitin line in immunodiffusion tests against the homologous antiserum. The fact that antigenic fractions contained various kinds of amino acids but no detectable amounts of amino sugars or carbohydrates suggests that the antigens were principally composed of proteins. The protein antigen possessed multiple antigenic components on immunoelectrophoresis. As serological activity, the antigen was heat-stable and resistant to tryptic digestion but sensitive to the actions of pronase, nagarse or pepsin. The protein antigen appeared to be responsible for the common antigenicity among the proteolytic strains of C. botulinum.     

Demonstration and characterization of the cell wall carbohydrate and protein antigens from Clostridium botulinum type E Saroma

Two different cell wall antigens, carbohydrate (CHO) and protein (P), from Clostridium botulinum type E Saroma were extracted with sodium dodecyl sulfate (SDS) and purified by chromatography on DEAE-Sepharose CL-6B and Sephadex G-75 or G-100. The CHO antigen was composed of glucose, galactose, glucosamine, galactosamine, alanine and phosphorus with a molar ratio of 1.5:1.5:0.25:0.25:1:1. The P antigen was an acidic protein with a molecular weight of 60 kDa, in which the major amino acids were aspartate, glutamate and serine, while the minor ones were cysteine and methionine. Thin sections of the intact or SDS-extracted cells of the organism demonstrated that the cell wall was composed of a two-layered structure, an inner layer about 20 nm thick and an outer layer about 10 nm, and by the extraction with SDS, the outer layer disappeared from the cell surface, leaving the inner layer. Immunogel diffusion tests demonstrated that either CHO antigen or P antigen was common among the nonproteolytic strains of C. botulinum.     

Chemical composition and serological analysis of the cell wall of Peptostreptococcus

Bahn, Arthur N. (Northwestern University, Chicago, Ill.), Patrick C. Y. Kung, and James A. Hayashi. Chemical composition and serological analysis of the cell wall of Peptostreptococcus. J. Bacteriol. 91:1672-1676. 1966.-Chemical and serological analyses were made of the cell wall of Peptostreptococcus to characterize taxonomically this genus of anaerobic streptococci. Cell wall hydrolysates of P. putridus strains 06 and 85, P. intermedius strains 11 and 87, and P. elsdenii strain B-159 were prepared, and the cell wall sugars were measured quantitatively by paper chromatography. Strain 85 contained only glucose, whereas strain 06 contained 93% glucose and 7% mannose. Strain 87 contained only rhamnose, and strain 11 contained approximately equal amounts of glucose and rhamnose. Strain B-159 differed from all the other strains in having a low (3.1%) content of total carbohydrate, consisting of rhamnose, galactose, and glucose. Quantitative amino acid analyses showed that the major amino compounds present in the cell wall were glutamic and aspartic acids, alanine, lysine, muramic acid, glucosamine, and galactosamine. Strains 06 and 85 possessed this complement of amino compounds, but strains 11 and 87 had relatively little aspartic acid. Strain B-159 was markedly different in having a high content of glycine and diaminopimelic acid, with only traces of lysine; it was the only strain in which teichoic acid was found. Serological analyses were made with the use of cell wall extracts as antigenic material and with homologous antisera, as well as streptococcal group antisera for groups A through S. The only strong agglutination was obtained between strain 87 antigen and group C antisera; weak agglutination was obtained with 87 against N, O, and K, and between strain 11 and groups E and F. All other antisera gave negative reactions. It is concluded that strain B-159 does not belong to the genus Peptostreptococcus, that strains 06 and 85 are members of P. putridus, and that strains 11 and 87 may be members of two different genera.     

N-terminal amino acid sequences of the polyoma middle-size T antigen are important for protein kinase activity and cell transformation.

We constructed deletion mutations which removed N-terminal coding sequences of various lengths from a cloned polyoma middle-size T antigen (MT antigen) gene. We introduced the MT antigen genes into a simian virus 40 expression vector so that they were expressed at high levels under the control of the simian virus 40 late promoter in COS-1 cells. The deletion mutant genes synthesized truncated MT antigens whose size was consistent with the deletion of either 70 or 106 amino acids from N termini, owing to initiation of translation at internal methionine codons in the MT antigen-coding region. The truncated MT antigens were found in cell membrane fractions but failed to show MT antigen-associated protein kinase activity. The cloned deletion mutant DNAs failed to transform rat F2408 or mouse NIH 3T3 cells. Therefore, N-terminal amino acid sequences of the polyoma MT antigen, as well as C-terminal sequences, are important for protein kinase activity and cell transformation.     

Immunologic properties and role of the low-molecular component in a preparation of S. typhi Vi antigen]

The author compared the serological, immunogenic and protective activity of the Vi-antigen and its high- and low-molecular fractions; an interaction between these fractions in administration of their mixture to the animals was studied. The low-molecular antigen (the 2nd fraction), contained in the preparation of the Vi-polysaccharide differed considerably (by properties) from the high-molecular antigen. The 2nd fraction, whose antigenic substance possessed the least immunogenic and protective capacity, failed to induce or to resolve the immunological memory, and also prevented the manifestations of the high immunogenicity of the 1st fraction. Therefore the nonfractional preparation of the Vi-antigen, consisting of 80% of a high-molecular substance of the 1st fraction and having the same serological activity as the 1st fraction, possessed a lesser immunogenic and protective activity.     

The preparation and chemical composition of fractions from Aspergillus fumigatus wall and protoplasts possessing antigenic activity.

A detergent-soluble fraction was prepared from the fragmented wall of Aspergillus fumigatus mycelium using the non-ionic detergent Triton X-100, and a wall-free extract was prepared from the same source in the form of protoplasts, released by a lytic enzyme system from Trichoderma harzianum. These extracts were examined by polyacrylamide gel electrophoresis and their detailed chemical composition was established. They were compared with the water-soluble fraction prepared from total mycelium, which is used routinely in this laboratory for serological tests. All fractions had immunological reactivity towards an antiserum prepared in rabbits against this water-soluble fraction of the mycelium, as shown by double diffusion. Both protein and carbohydrate moieties appear to be involved in the antigenic sites, with carbohydrate reactivity predominantly associated with the protoplast fraction. The fact that all preparations contained at least one common antigenic determinant, as judged by lines of identity to a single antiserum, is discussed in relation to antigen location.     

Inability of EDTA to prevent damage mediated by cytolytic T-lymphocytes.

To analyze the nature of the target cell determinants recognized and bound by killer lymphocytes during lymphocyte-mediated cytolysis (LMC), the specific binding of serologically active tumor cell membrane fractions to cytotoxic T lymphocytes has been investigated. Particulate membrane fractions and soluble antigen preparations (extracted by papain or 3 M KCl) from tumor target cells were tested for their ability to inhibit the destruction of intact ⁵¹Cr-labeled target cells by killer lymphocytes in vitro. The effect of papain-solubilized tumor cell antigen on the binding of killer lymphocytes to tumor cell monolayers was also evaluated. Direct assays to determine the extent of binding of unlabeled or radioiodinated soluble antigen (extracted by papain or deoxycholate) to cytotoxic lymphocytes were carried out. In marked contrast to their serological activity, all of these particulate and soluble preparations failed to inhibit LMC or bind to killer lymphocytes in an immunologically specific way. It is suggested that killer lymphocytes recognize and bind to an antigenic complex whose organization is dependent upon the integrity of the target cell membrane.     

Evaluation of diagnostic efficiency of the recombinant protein modeling immunodominant epitope V3 of envelope gp120 for immunoenzyme detection for HIV-1 infection antibodies

The third hypervariable domain V3 of the human immunodeficiency virus type 1 gpl20 envelope glycoprotein contains neutralizing epitopes and plays an important role in the diagnosis of HIV infection . Neutralizing antibodies bind to conserved epitope with sequence GPG of V3 loop. The effect of sequence variation on the antigenic properties of the V3 epitope gp120 was studied using five synthetic peptides. The amino acid sequence of the peptide corresponding to the V3 region gp120 of HIV-1 subtype C showed the highest immunoreactivity. The DNA fragment encoding V3-C region gp120 was synthesized by polymerase chain reaction and cloned into pET41b vector. The recombinant plasmid was expressed in the E. coli cells, and recombinant protein was purified using glutathione-S sepharose affinity chromatography. The serological activity of the recombinant protein was tested using ELISA and compared to activity of similar synthetic peptide. The results of this study showed that most immunoreactive agent was the amino acid sequence of V3 region gp120 of HIV-1 subtype C. The recombinant antigen comprising this sequence was more antigenic than synthetic peptide with the same sequence. The evaluation of this antigen shows that this protein is a good candidate for the immunoassay development.     

Immunochemical study of the antigenic structure of bacteria of genus Bordetella. IV. Fractionation of B. pertussis extracts and study of the immunochemical and biological properties of the isolated fractions]

Fractionation of an extract of pertussis microbes was carried out with the aid of gel-filtration on Sephadex G-100, ion-exchange chromatography on DEAE-cellulose, and preparative electrophoresis. Fractions differing in serological, immunogenic activity and the content of antigenic components were isolated. In using the method of gel-filtration of sefadex G-100 the greatest serological, immunogenic and histamine-sensitizing activity was possessed by the high-molecular fraction containing 8 of 11 antigenic components detected in the initial extract. The antigenic components were distributed into 5 fractions by the ion exchange chromatography on DEAE-cellulose. The greatest serological activity was possessed by fractions exiting from the column at the 0.01--0.04M interval of the phosphate buffer concentration. A method of preparative electrophoresis from the pertussis microbes extract was applied and two fractions were isolated from the anode and the cathode zones, each containing 4 antigenic components only, but possessing serological and immunogenic activity and having no histamine-sensitizing properties.     

Immunochemical study of Clostridium botulinum type B toxin

Fractionation of type B. Cl. botulinum toxin, a protein complex, was carried out; as a result, 3 fractions, alpha, beta, and gamma, were isolated in a pure form, alpha-fraction, or neurotoxin, is highly toxic (5-10.10(7) LDm per 1 mg of protein), beta-fraction showed hemagglutinating activity (64-128 HAU per 1 mg of protein), gamma-fraction was not biologically active. The molecular weight of alpha and gamma-fractions was 150,000. All these fractions had antigenic properties. alpha-fraction was serologically specific. beta- and gamma-fractions showed incomplete serologic identity.     

Activity of the glycoprotein antigenic fractions of Mycoplasma pneumoniae in immunological reactions

Glycoprotein fractions exhibiting pronounced antigenic properties in immunological in vitro reactions have been isolated from sonicated M. pneumoniae cells by extraction with glacial acetic acid and ethanol. Glycoprotein fraction 3 of M. pneumoniae antigen shows the highest blood-sensitive and precipitating activity and may be recommended as a diagnosticum for serological tests.     

Immunological Analysis of Mycoplasma Membranes

The antigens responsible for the production of antibodies to Mycoplasma laidlawii and M. gallisepticum causing growth and metabolic inhibition of these organisms were localized in the cell membrane. Various membrane fractions were tested for serological activity. Membrane lipids were completely or almost completely inactive, whereas several preparations of defatted membrane proteins retained some serological activity, shown by their ability to stimulate metabolic inhibition antibody in rabbits and to adsorb metabolic inhibition antibody and form precipitation lines with an antiserum to the membrane. When the membranes were heated to 65 C for 1 hr, they virtually lost their ability to adsorb metabolic inhibition antibody, which suggests that the antigenic determinants are proteins. Serological activity was retained in reaggregated membranes obtained by dialysis against Mg²⁺ of membranes solubilized in sodium dodecyl sulfate. The amount of solubilized membrane protein and lipid incorporated into the reaggregated membranes could be regulated by varying the Mg²⁺ concentration. As the serological tests indicated that the various membrane antigens were selectively incorporated into the different reaggregated membranes, the use of controlled reaggregation of solubilized membranes is suggested as a new tool for the fractionation and antigenic analysis of membrane proteins.     

B细胞表位作图研究进展

抗原-抗体的特异性结合是由抗体表面的抗原决定簇与抗原表面的表位基序间的特异性互补识别决定的。B细胞表位作图既包括B细胞抗原表位基序的鉴定(即确定抗原分子上被B细胞表面受体或抗体特异性识别并结合的氨基酸基序),也包括绘制抗原蛋白的全部或接近全部的B细胞表位基序在其一级或高级结构上的分布图谱的过程。B细胞表位作图是研发表位疫苗、治疗性表位抗体药物和建立疾病免疫诊断方法的重要前提。目前,已经建立了多种B细胞表位鉴定或绘制抗原蛋白B细胞表位图谱的实验方法。基于抗原-单抗复合物晶体结构的X-射线晶体学分析的B细胞表位作图和基于抗原蛋白或抗原片段的突变体库筛选技术的B细胞表位作图可以在氨基酸水平,甚至原子水平上揭示抗原分子上与单抗特异性结合的关键基序;其它B细胞表位作图方法(如基于ELISA的肽库筛选技术)常常只能获得包含B细胞表位的抗原性肽段,因而,很少用于最小表位基序的鉴定;而改良的生物合成肽法多用于B细胞表位的最小基序鉴定和精细作图。鉴于每种B细胞作图方法都存在各自的优势与不足,B细胞表位作图往往需要多种作图方法的有机结合。本文对目前常用的B细胞表位作图的实验方法及其在动物疫病防控中的应用进行综述,以期为研究者设计最佳的表位作图方案提供参考。     

The cell wall of Micromonospora purpurea contains a high conductance channel

In this communication it is demonstrated that the cell wall of the gram-positive bacterium Micromonospora purpurea contains a cell wall channel for the passage of hydrophilic solutes. The channel-forming protein was identified in sucrose step-density-gradient fractions of the cell envelope and in whole cell extracts using either organic solvent or detergent and the lipid bilayer technique. The fractions of the sucrose step-density centrifugation were assayed for NADH-oxidase activity and for the formation of ion-permeable channels in lipid bilayers. The highest NADH-oxidase activity and the highest channel-forming ability were found in different fractions. The cell wall fraction was identified by the presence of meso-diaminopimelic acid and contained an ion-permeable channel with the extremely high single-channel conductance of about 14 nS in 1 M KCl. The channel-forming unit was purified to homogeneity by FPLC on a HiTrap-Q column. It was identified as a heat- and SDS-resistant 200-kDa band on SDS-PAGE and formed the same general diffusion pores in lipid bilayer membranes as those formed by detergent extracts of the cell wall fraction of the sucrose step-density centrifugation. The channels were slightly selective for potassium ions over chloride, possibly caused by an excess of negative charges in or near the channel.     

A T helper cell hybridoma produces an antigen-specific regulatory activity. Relationship to the T cell receptor by serology and antigenic fine specificity.

We have previously shown that a T cell hybridoma, A1.1, constitutively produces an Ag-specific regulatory factor with specificity for poly-18, a synthetic polypeptide. This cell also responds to poly-18 plus I-Ad by producing lymphokines. The antigenic specificity of the factor and the T cell appeared to be the same. This suggested the possibility that some part of the TCR, responsible for antigenic specificity of the cell, also imparts specificity to the A1.1-derived factor. This was supported by the observation that the factor was bound and eluted from a monospecific anti-TCR antiserum. Further, we demonstrated that antisense oligodeoxynucleotides corresponding to the TCR V alpha of A1.1 (but not TCR V beta) block production of the Ag-specific factor. Herein, we report recent findings that strengthen the proposed relationship between the TCR and the A1.1-derived factor. The factor was bound and eluted from a monoclonal anti-TCR C alpha antibody, but not from anti-TCR beta, anti-V beta 6, nor anti-CD3 epsilon. The anti-TCR C alpha antibody bound a Mr 46-kDa protein from A1.1 supernatants, which is the same apparent size at which activity could be eluted from an SDS-PAGE gel separation of concentrated factor. Antigenic fine-specificity analysis revealed that two amino acids in poly-18 are critical for the recognition of the antigen by the Ag-specific factor. These two amino acids appear to be those recognized by the TCR. The factor that was bound and eluted from the monoclonal anti-TCR C alpha showed this fine-specificity as well. This, combined with our earlier studies, supports the view that the A1.1-derived factor is encoded, at least in part, by TCR-alpha.     

Structural studies of the antigen III cell wall polysaccharide of Trichosporon domesticum

Cell wall and soluble polysaccharides that reacted with Trichosporon domesticum factor III serum were isolated from the type strain of T. domesticum. The fractions contained O-acetyl groups, which contributed to the serological reactivity. The antigenic structure was characterized by chromatographic and spectroscopic methods. The polysaccharide has an alpha-(1-->3)-D-mannan backbone with hetero-oligosaccharide side chains consisting of a 2-O-substituted beta-D-glucuronic acid residue bound to O-2 of the mannose residue, beta-D-xylopyranosyl residues located in the middle of the side chain, and a nonreducing terminal alpha-L-arabinopyranosyl residue bound to 0-4 of xylose. The mannan backbone is O-acetylated at O-6 of the mannose residues.     

Antigenic and Immunogenic Characteristics of Nuclear and Membrane-Associated Simian Virus 40 Tumor Antigen

Antisera were prepared in syngeneic hosts against subcellular fractions of simian virus 40 (SV40)-transformed cells (MoalphaPM, MoalphaNuc), glutaraldehydefixed SV40-transformed cells (HaalphaH-50-G, MoalphaVLM-G), and electrophoretically purified denatured SV40 tumor antigen (T-ag) (RaalphaT). Immune sera were also collected from animals bearing tumors induced by SV40-transformed cells (HaalphaT, MoalphaT, HAF) and from SV40-immunized animals that had rejected a transplant of SV40-transformed cells (HaalphaS, MoalphaS). Immunological reagents prepared against cell surface (MoalphaPM, HaalphaS, MoalphaS, HaalphaH-50-G, MoalphaVLM-G) reacted exclusively with the surface of SV40-transformed cells by indirect immunofluorescence or protein A surface antigen radioimmunoassay. Immunological reagents prepared against the nuclear fraction (MoalphaNuc) or whole-cell determinants (HaalphaT, MoalphaT, HAF, RaalphaT) reacted with both the nuclei and surface of SV40-transformed or -infected cells. All reagents were capable of immunoprecipitating 96,000-molecular weight large T-ag from solubilized whole cell extracts of SV40-transformed cells. The exclusive surface reactivity of HaalphaS exhibited in immunofluorescence tests was abolished by solubilization of subcellular fractions, which then allowed immunoprecipitation of T-ag by HaalphaS from both nuclear and plasma membrane preparations. Specificity was established by the fact that all T-reactive reagents failed to react in serological tests against chemically transformed mouse cells, and sera from mice bearing transplants chemically transformed mouse cells (MoalphaDMBA-2) failed to react with SV40-transformed mouse or hamster cells. Reagents demonstrating positive surface immunofluorescence and protein A radioimmunoassay reactions against SV40-transformed cells were capable of blocking the surface binding of RaalphaT to SV40-transformed cells in a double-antibody surface antigen radioimmunoassay. This blocking ability demonstrated directly that a component specificity of each surface-reactive reagent is directed against SV40 T-ag. A model is presented which postulates that the differential detection of T-ag by the various serological reagents is a reflection of immunogenic and antigenic differences between T-ag polypeptides localized in nuclei and plasma membranes.     

Characteristics of the low molecular antigens of pertussis bacteria]

The authors elaborated a method of obtaining pertussis soluble antigenic complex by dialysis through the cellophane membrane against the physiological saline at a temperature of 4 degrees C. An antigen which was active in the passive hemagglutination and neutralization of antibodies tests was revealed in the dialyzate. The amount of this antigen in the dialyzate increased gradually up to the 7th day and then became stabilized. The serological activity of the antigen after evaportation increased 4-16 times. The results of the antibody neutralization test pointed to the presence in the dialysate of substances common to those contained in the 1a and 1Da fractions isolated from the pertussis bacteria with the aid of ammounium sulfate.     

Study of the heterogenetic antigens in vaccinal preparations of V. cholerae]

There proved to be a serological similarity between the antigens of the human small intestine, the stomach and the liver, and the antigens of various cholera vibrio fractions. No antigenic similarity was revealed in examination of the heart and kidney. Heterogenous antigen was found not only in the somatic V. cholerae antigen, strain 569 (B), but also in the cholerogen-toxoid obtained from it. At present it is the most widespread prophylactic preparation.     

Deglycosylation effect of the mammalian sperm maturation antigen (SMA2) on serological reaction and acrosome reaction

Spermatozoal membrane proteins are considered to possess several immunological unique characteristics as the cell is formed behind the blood-testes barriers. Major goat sperm maturation antigen (SMA2) contains one hexosamine along with mannose, galactose and glucose. In the present study, effects of deglycosylation of SMA2 antigen on immuno-reactivity and the serological activity was investigated. SMA2 glycoantigen showed positive immunoreactivity after treatment with sodium borohydride (NaBH(4)) and moreover this generated a 44kDa protein band which was negative for periodic acid Schiff reagent. Trifluoromethanesulfonic acid (TFMS) caused aggregation and restricted the free mobility of the treated antigen on SDS-PAGE and the protein band generated by TFMS treatment also showed positive immuno-reactivity. The results supported the views that the protein portion retains its immuno-reactivity even after oxidation of the vicinal hydroxyl group of saccharide component of SMA2 antigen. These data suggest that immunodominent epitopes exist on the core protein by which the SMA2 antigen retains its immuno-reactivity even after disruption of the saccharide portion. Additional experiments demonstrate that protein epitopes have a role in capacitation and the acrosome reaction (AR) in presence of antibody which is raised against this protein part of SMA2 using the negative staining of FITC-PSA (fluorescein isothiocyanate-labeled Pisum sativum agglutinin) probe. Altogether these findings indicate that the protein portion of SMA2 might fulfill the serological activity of the antigen as well as the protein epitope affects the acrosome reaction. In view of this property, we propose that the protein portion of SMA2 antigen might be considered as a potential antigenic target for an immune response.