Cell Envelope Antigen Research Articles

Development and validation of immunoassay for whole cell detection of Brucella abortus and Brucella melitensis

Brucella is alpha-2 Proteobacteria mainly responsible for multi-factorial bacterial zoonotic disease brucellosis with low concentration (10–100 CFU) required to establish the infection. In this study, we developed sandwich ELISA with detection range of 102 to 108 cells mL−1 and limit of detection at 103 cells mL−1 by employing polyclonal rabbit IgG (capture antibody, 10 µg mL−1) and mice IgG (detection antibody, 50 µg mL−1) antibody for its detection. Surface Plasmon Resonance evaluated the interaction of detection antibody with whole cell spiked serum samples at LOD of 102 cells mL−1 along with non co-operative interaction of protein albumin. Further, kinetic evaluation study using detection antibody against cell envelope antigen was performed whereby, Equilibrium Dissociation Constant (KD) and Maximum Binding Capacity (Bmax) were found to be 16.48 pM and 81.67 m° for Brucella abortus S99 and 0.42 pM and 54.50 m° for Brucella melitensis 16 M, respectively. During interference study, sandwich ELISA assay cross-reacted with either of the polyclonal antibody of above Brucella species. Upon validation, no cross-reactivity observed with bacteria-closely related to Brucella. In conclusion, developed semi-quantitative sandwich immunoassay is sensitively rapid in whole cell detection of Brucella and will be useful in development of detection assays from environmental and clinical matrices.

Evaluation of Diagnostic Potential of Recombinant Outer Membrane Protein (rOmp28) of Brucella Melitensis for Serodiagnosis of Ovine and Caprine Brucellosis

Brucella melitensis is the main causative agent of brucellosis in small ruminants. The diagnosis of brucellosis is mostly done by isolation of bacteria from aborted material, udder secretions or from tissues of infected animals. The presumptive diagnosis of Brucellosis is attempted by elucidating the serological responses to Brucella antigens. The present study was designed to evaluate the diagnostic potential of rOmp28 antigen of Brucella melitensis for ovine and caprine brucellosis. A total of 163 clinical samples (n=79 samples of ovine and n=84 samples of caprine) were tested in an indirect plate-ELISA format using rOmp28 antigen. Results of rOmp28 antigen based indirect ELISA were also compared with the native antigens [cell envelope antigen (CE) and whole cell sonicated antigen (SA)] based ELISA and with conventional Standard Tube Agglutination Test (STAT). Recombinant Omp28 antigen showed high sensitivity and specificity i.e., 71.4%, 97.7% for ovine samples and 74%, 87.8% for caprine samples as compared with CE antigen (40%, 75%) and (44%, 67.6%) and SA antigen (37.1%, 84%) and (42%, 70.5%) for ovine and caprine samples respectively. This study demonstrated that rOmp28 can be a good candidate antigen in the serodiagnosis of ovine and caprine brucellosis in India and also further in the development of rapid field-adaptable diagnostic assay for screening of ovine and caprine brucellosis.

Comparative Evaluation of Native Antigens for the Development of Brucellosis Antibody Detection System

Brucellosis is a highly infectious zoonotic disease and an economically important infection of humans and livestock with a worldwide distribution. The main mode of transmission of this disease to humans is through the consumption of infected milk, milk products, and uncooked or raw meat. The present study was designed to prepare few native antigens, that is, sonicated antigen (SA), cell envelope (CE) antigen, and freeze and thaw (FT) antigen fromBrucella abortusS99 culture and to test them in a highly sensitive and specific indirect enzyme-linked immunosorbent assay (I-ELISA) in both a microtiter plate and a dot-blot format for the development of field-based diagnosis. All 50 suspected bovine samples were tested by plate as well as in dot ELISA formats for all the three antigens prepared. The CE antigen was found to be more suitable as it had the maximum agreement with the Rose Bengal plate agglutination test results followed by the SA and the least agreement was found with that of the FT antigen. This detection system in microtiter plates and a dot-blot format will be useful for the rapid screening of samples for the disease surveillance and routine diagnosis.

Comparative Evaluation of Native Antigens for the Development of Brucellosis Antibody Detection System

Brucellosis is a highly infectious zoonotic disease and an economically important infection of humans and livestock with a worldwide distribution. The main mode of transmission of this disease to humans is through the consumption of infected milk, milk products, and uncooked or raw meat. The present study was designed to prepare few native antigens, that is, sonicated antigen (SA), cell envelope (CE) antigen, and freeze and thaw (FT) antigen from Brucella abortus S99 culture and to test them in a highly sensitive and specific indirect enzyme-linked immunosorbent assay (I-ELISA) in both a microtiter plate and a dot-blot format for the development of field-based diagnosis. All 50 suspected bovine samples were tested by plate as well as in dot ELISA formats for all the three antigens prepared. The CE antigen was found to be more suitable as it had the maximum agreement with the Rose Bengal plate agglutination test results followed by the SA and the least agreement was found with that of the FT antigen. This detection system in microtiter plates and a dot-blot format will be useful for the rapid screening of samples for the disease surveillance and routine diagnosis.

Acute poststaphylococcal glomerulonephritis superimposed on diabetic glomerulosclerosis

Acute poststaphylococcal glomerulonephritis superimposed on diabetic glomerulosclerosis

Staphylococcus aureus cell envelope antigen is a new candidate for the induction of IgA nephropathy

IgA nephropathy is the most common form of glomerulonephritis worldwide. We previously reported a novel form of glomerulonephritis with glomerular IgA deposits following methicillin-resistant Staphylococcus aureus (S. aureus) infection. We investigated the role of S. aureus related antigens in the immunopathogenesis of IgA nephropathy by producing several monoclonal antibodies against S. aureus surface antigens and determining the epitopes of deposited antigens in patients with IgA nephropathy. Cell membrane proteins were isolated from cultured S. aureus. Mouse monoclonal antibodies against these proteins were generated, and their target epitopes were determined by antibody affinity chromatography and amino acid sequence analysis, and by monoclonal antibody screening of Escherichia coli clones transfected with plasmids from the Lambda S. aureus Genomic Library. Renal biopsy specimens from 116 patients with IgA nephropathy and 122 patients with other forms of renal disease were examined for glomerular antigen depositions by immunofluorescence microscopy. . The major antigen recognized by monoclonal antibodies against S. aureus cell membrane was identified as the S. aureus cell envelope antigen designated 'probable adhesin' (ACCESSION AP003131-77, Protein ID; BAB41819.1). In 68.1% (79/116) of renal biopsy specimens from patients with IgA nephropathy, S. aureus cell envelope antigen was localized in the glomeruli, and the data confirmed that S. aureus cell envelope antigen was co-localized with IgA antibody in the glomeruli. No deposition of this antigen was detected in the glomeruli of patients with non-immune complex deposit forms of glomerulonephritis. S. aureus cell envelope antigen is a new candidate for the induction of IgA nephropathy.

Immunoglobulin G subclass response of localized juvenile periodontitis patients to Actinobacillus actinomycetemcomitans Y4 lipopolysaccharide.

Sera from patients with localized juvenile periodontitis (LJP) often contain markedly elevated immunoglobulin G (IgG) antibody titers to serospecific determinants of the lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans. The objective of the present study was to define the subclass distribution of the IgG antibody response of LJP patients to this key cell envelope antigen. IgG subclass antibody responses to A. actinomycetemcomitans LPS were quantified in an enzyme-linked immunosorbent assay with human IgG subclass-restricted monoclonal antibodies. Serum antibody concentrations were calculated by heterologous interpolation of a dose-response curve constructed by using human-mouse chimeric antibodies. Sixteen of 17 LJP serum samples tested contained significantly greater concentrations of IgG2 than IgG1 antibodies reactive toward A. actinomycetemcomitans LPS. Geometric mean antibody concentrations of IgG1 and IgG2 were 7.8 and 136.5 micrograms/ml, respectively, among LJP patients with elevated IgG titers to LPS (94% of whom were black). However, both IgG1 and IgG2 antibody concentrations were significantly greater than the corresponding values obtained from sera from LJP patients with low IgG titers to LPS. Among LJP patients with elevated IgG titers to A. actinomycetemcomitans LPS, serum IgG2 concentration and total IgG concentration were also significantly elevated compared with both low-titered LJP sera and sera from periodontally healthy race-matched controls. The results of this study indicate that the humoral response of a predominantly black population of LJP patients to A. actinomycetemcomitans includes the production of LPS-reactive IgG antibodies which are primarily of the IgG2 subclass.

Expression of virulence factors by classical and El TorVibrio choleraein vivo and in vitro

We examined the production of virulence factors of Vibrio cholerae O1 bacteria, and especially compared expression in vitro under near optimal growth conditions with that in vivo during experimental cholera infection. The results show that the in vitro formation of cholera toxin (CT), soluble hemagglutinin (SHA), colonizing pilus TCP, and the biotype associated hemagglutinins FSHA and MSHA, as well as of various cell envelope antigens often rather poorly reflected expression in vivo. For instance, production of CT by vibrios of classical biotype and of TCP by the El Tor biotype were enhanced in vivo, while production of SHA was instead suppressed. Likewise significant differences in cell envelope antigen composition were found between bacteria grown in vivo and in vitro. A more precise definition of the role of different postulated virulence factors in the processes of infection and immunity should include in vivo studies as outlined by this study.

Expression of virulence factors by classical and El Tor Vibrio cholerae in vivo and in vitro

We examined the production of virulence factors of Vibrio cholerae O1 bacteria, and especially compared expression in vitro under near optimal growth conditions with that in vivo during experimental cholera infection. The results show that the in vitro formation of cholera toxin (CT), soluble hemagglutinin (SHA), colonizing pilus TCP, and the biotype associated hemagglutinins FSHA and MSHA, as well as of various cell envelope antigens often rather poorly reflected expression in vivo. For instance, production of CT by vibrios of classical biotype and of TCP by the El Tor biotype were enhanced in vivo, while production of SHA was instead suppressed. Likewise significant differences in cell envelope antigen composition were found between bacteria grown in vivo and in vitro. A more precise definition of the role of different postulated virulence factors in the processes of infection and immunity should include in vivo studies as outlined by this study.

Immunoelectrophoretic comparison of cell envelope antigens in pathogenic and non-pathogenic Agrobacterium tumefaciens

The cell envelope antigens of a pathogenic strain (57P) of Agrobacterium tumefaciens were compared with those of a non-pathogenic strain (57sr) by means of quantitative Immunoelectrophoresis. Strain 57P was derived from 57sr by conjugative transfer of the tumour inducing (Ti) plasmid from strain B6Sm, and should differ from 57sr only with respect to characters encoded by the Ti plasmid. Both strains, when grown on glucose and (NH 4 ) 2 SO 4 , contained one cell envelope antigen (A) which was absent from 57P when grown on octopine as the sole carbon and nitrogen source. Another antigen (D) was present only in the pathogenic strain, when grown on octopine. Two more antigens (B and C) were restricted to the pathogen but were produced on both media. The possible identity of these antigens and their role in tumorigenesis is discussed.

Antigenic analysis of Pasteurella multocida (serotype 1) by crossed immunoelectrophoresis: characterization of cytoplasmic and cell envelope associated antigens

The application of crossed immunoelectrophoresis to the analysis of a reference cytoplasm and cell envelope preparation from Pasteurella multocida serotype 1 revealed antigenic complexity not previously found. At least 55 cytoplasmic and 19 cell envelope antigens were clearly distinguished. Variation of anticytoplasm immunoglobulin concentration was a major determining factor in resolving the maximum array of cytoplasmic antigens. The use of intermediate gel modification of crossed immunoelectrophoresis permitted the recognition of antibodies in the preimmune rabbit serum against a number of cytoplasmic antigens and a single cell envelope antigen. This technique also demonstrated that reference cytoplasm obtained by 105,000 x g centrifugation of sonically disrupted pasteurellae and repeatedly washed reference cell envelope preparation contained antigens of either origin in amounts sufficient to elicit antibody responses in the host. Antisera to whole cells in the intermediate gel indicated that formalin killed P. multocida were capable of eliciting immune responses to both reference systems.

Detection of Neisseria meningitidis cell envelope antigen by enzyme-linked immunosorbent assay in patients with meningococcal disease

Sera from various populations and cerebrospinal fluid (CSF) from meningitis patients were tested for Neisseria meningitidis cell envelope antigen by an enzyme-linked immunosorbent assay (ELISA) sandwich system. The minimum optical density (OD) for antigen detection in CSF was defined as the mean value obtained with specimens from a group of tuberculous meningitis patients plus two standard deviations. By this criterion, antigen was detected by ELISA in four of five CSF specimens from group A meningococcal meningitis patients. Very high ELISA values were obtained with sera from fulminating cases but control sera and sera obtained from meningococcal meningitis nonfulminating cases could not be clearly differentiated by this technique. The possible reasons for this are discussed. The results of this study show that as little as 15 ng/ml (protein) of cell envelope antigen can be detected by the ELISA sandwich test and suggest that the technique may be of value in clinical diagnosis of meningococcal disease.

Identification of Bacteroides fragilis by indirect immunofluorescence.

Rabbit antiserum against live or heat-killed Bacteroides fragilis NCTC 9343 bacteria was titrated against hot phenol water-extracted polysaccharide antigens from five different species of the 'B. fragilis group' of bacteria using an enzyme immunoassay and shown to be specific for the B. fragilis NCTC 9343 polysaccharide. When the antiserum was used in indirect immunofluorescence, 97.1% of 244 B. fragilis strains were correctly identified. Only 8 of the other 312 Bacteroides strains were stained by the anti-B fragilis serum. The 'cross-reactive' Bacteriodes strains all belonged to the "B. fragilis group" of bacteria (i.e., B; distasonis, B. ovatus, B. thetaiotaomicron, B. uniformis, and B. vulgatus). None of the 425 aerobic enteric bacteria representing Salmonella, E. coli, Proteus, Yersinia, Shigella, Klebsiella, Enterobacter, Citrobacter, and Pseudomonas were positive using the anti-B. fragilis serum. Likewise, all the 59 gram-positive strains representing Streptococcus, Bacillus, Peptostreptococcus, Peptococcus, Propionibacterium, Lactobacillus, Eubacterium, and Clostridium did not stain. Our data shows in accordance with other findings [11], that B. fragilis strains possess a species-specific cell envelope antigen(s) which promises to be important for production of antisera, making a rapid identification of the species possible.