RESUMEN
AIM: To evaluate the potential of an intergeneric multidomain recombinant chimeric protein for the simultaneous detection of Bacillus anthracis, Yersinia pestis and Staphylococcal enterotoxin B. METHODS AND RESULTS: Truncated portions of protective antigen (pag) of B. anthracis, fraction 1 capsular antigen (F1) of Y. pestis and enterotoxin B (entB) of Staphylococcus aureus were PCR amplified and linked each other using ligation-dependent cloning. The fusion gene was codon-optimized for expression in Escherichia coli and encoded a 55 kDa recombinant PFE protein (rPFE). Hyperimmune antiserum raised against rPFE specifically reacted individually with the native PA of B. anthracis, F1 antigen of Y. pestis and SEB of S. aureus on Western blot analysis as well as in enzyme-linked immunosorbent assay (ELISA). For simultaneous detection of these three antigens from culture supernatants, common media consisting of BHI broth supplemented with 0·2% xylose were used. To assess the detection capability, a known number of these organisms (10(8) -10(2) CFU ml(-1)) were experimentally spiked on to the meat and blood samples, the polyclonal antibodies were again clearly able to identify all three target proteins up to a dilution of 10(5) CFU ml(-1). CONCLUSIONS: This recombinant chimeric protein-based immunodetection approach may eventually provide advantages over PCR formats during onsite investigations of biological emergencies or even during routine testing by laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: The trivalent recombinant PFE protein could be a novel intervention for possible diagnosis/detection of potential biological agents simultaneously in environmental and clinical samples to reduce the responding time and minimize the impact of the bioattack.
Asunto(s)
Antígenos Bacterianos/aislamiento & purificación , Proteínas Bacterianas/aislamiento & purificación , Toxinas Bacterianas/aislamiento & purificación , Enterotoxinas/aislamiento & purificación , Sueros Inmunes/química , Animales , Bacillus anthracis , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Femenino , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Staphylococcus aureus , Yersinia pestisRESUMEN
Monoclonal antibodies were generated against whole cell lysate of Burkholderia pseudomallei. Two out of 6 monoclonal antibodies were found specific and exhibited high affinity against B. pseudomallei, one of which, was utilized to develop sandwich ELISA for detection of specific B. pseudomallei antigen. Immunoassays were found to be specific as no reaction was observed with closely related Burkholderia and Pseudomonas species. Blood samples from experimentally infected mice were found positive for isolation till 4 days post infection (DPI) and ELISA till 10 DPI. One out of 40 sick animal serum samples tested in Thailand was found positive by sandwich ELISA that was earlier confirmed by isolation of B. pseudomallei. The results indicate the potentiality of the assay for its applicability in specific diagnosis of septicaemic melioidosis.
Asunto(s)
Infecciones por Burkholderia/diagnóstico , Burkholderia pseudomallei/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Burkholderia pseudomallei/patogenicidad , Ensayo de Inmunoadsorción Enzimática , Femenino , Ratones , Ratones Endogámicos BALB CRESUMEN
Microorganisms found in industrial effluents and near the sites of the contamination can be used to indicate pollution and detoxify the contaminated water resources. Emergence of xenobiotic resistant bacteria among them might be potential application in bioremediation. The objective of this study was to isolate and characterize fluoride resistant bacteria from soil and water samples of different regions of India. Five isolates were recovered from different samples which were found to be fluoride resistant. Two of them effectively reduced the fluoride from their media. Through the current study it can be predicted that fluoride pollution results in selective pressure that leads to the development of fluoride resistant among bacterial populations, probably through the mechanism which involved high affinity anion binding compounds called ionophores. Resistant microbes may play a bioremediative role by transforming and concentrating these anions so that they are less available and less dangerous.
Asunto(s)
Aeromonas hydrophila/metabolismo , Escherichia coli/metabolismo , Fluoruros/metabolismo , Micrococcus/metabolismo , Pseudomonas aeruginosa/metabolismo , ARN Ribosómico 16S/análisis , Aeromonas hydrophila/aislamiento & purificación , Proteínas Bacterianas/biosíntesis , Biodegradación Ambiental , Biotransformación , Electroforesis en Gel de Poliacrilamida , Escherichia coli/aislamiento & purificación , India , Ionóforos/metabolismo , Micrococcus/aislamiento & purificación , Pseudomonas aeruginosa/aislamiento & purificación , Contaminantes del Suelo/metabolismo , Contaminantes Químicos del Agua/metabolismoRESUMEN
The genotype and antibiotic resistance pattern of the toxigenic Vibrio cholerae strains associated with cholera outbreaks vary frequently. Fifty-one V. cholerae strains isolated from cholera outbreaks in Chennai (2002-2005) were screened for the presence of virulence and regulatory genes by multiplex polymerase chain reaction (PCR) assay. Genotyping of the isolates was done by VC1 primers derived from enterobacterial repetitive intergenic consensus (ERIC)-related sequence in V. cholerae. All the isolates possessed toxigenic genes, such as ctxA, ctxB, tcpA, ace, ompU, toxR and zot. Two different El Tor genotypes and one O139 genotype could be delineated by VC1-PCR. One of the El Tor genotypes was similar to the El Tor strains isolated from Bhind district and Delhi during 2004. Antibiotic susceptibility testing revealed greater variability among the isolates tested. All the isolates were found to be susceptible to norfloxacin, ciprofloxacin and tetracycline. Thiry-three per cent of the isolates were found to be resistant to more than 4 antibiotics and could be termed as multiple antibiotic resistant. Coexistence of O139 serogroup along with the El Tor biotype could be identified among the strains recovered during the period 2002-2004. The O139 isolates were found to be more susceptible to the antibiotics tested when compared to the El Tor isolates.
Asunto(s)
Antígenos Bacterianos/sangre , Leptospira/aislamiento & purificación , Leptospirosis/diagnóstico , Enfermedad Aguda , Adolescente , Adulto , Anciano , ADN Bacteriano/sangre , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina M/sangre , Leptospira/genética , Leptospira/inmunología , Leptospirosis/microbiología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa/métodos , LluviaAsunto(s)
Anticuerpos Antibacterianos/sangre , Leptospira/inmunología , Leptospirosis/epidemiología , Adolescente , Adulto , Agricultura , Niño , Femenino , Humanos , India/epidemiología , Leptospira/clasificación , Leptospirosis/microbiología , Masculino , Persona de Mediana Edad , Exposición Profesional , Ocupaciones/estadística & datos numéricos , Personal de Hospital , Prevalencia , Factores de Riesgo , Estudios Seroepidemiológicos , Serotipificación , VeterinariosRESUMEN
Monoclonal antibodies (MAbs) were generated against the recombinant plasminogen activator (Pla) protein of Yersinia pestis. These MAbs detected Pla in all the 18 isolates of Y. pestis obtained from the sputum of pneumonic plague patients and from the liver and spleen of rodents from plague-affected areas of India during 1994-1995 as well as in seven of the eight isolates obtained from rodents in the surveillance regions of Hosur and Palmner in India during 1998 by simple dot-ELISA. In immunoblotting, the MAbs reacted with the Pla antigen only in Y. pestis isolates at 37 and 35kDa region. These monoclonal antibodies, being strictly specific, can be used for detecting Y. pestis isolates that are Fraction 1 antigen-negative. Also, the radiolabelled pla fragment hybridized specifically to the representative DNA samples of Y. pestis isolates.
Asunto(s)
Anticuerpos Monoclonales , Peste/microbiología , Activadores Plasminogénicos/inmunología , Yersinia pestis/inmunología , Animales , Western Blotting , Clonación Molecular , ADN Bacteriano/química , ADN Bacteriano/genética , Brotes de Enfermedades , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Humanos , India , Ratones , Ratones Endogámicos BALB C , Hibridación de Ácido Nucleico , Peste/diagnóstico , Peste/inmunología , Plásmidos , Activadores Plasminogénicos/química , Activadores Plasminogénicos/genética , Reacción en Cadena de la Polimerasa , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Yersinia pestis/genética , Yersinia pestis/aislamiento & purificaciónRESUMEN
BACKGROUND & OBJECTIVE: Anthrax has been reported from almost every country and India is endemic for this disease. There is considerable under reporting of the disease because of lack of microbiological facilities and diagnostic reagents. In India only conventional methods which have limitations, are being used to diagnose the disease. Hence the aim of this study was to isolate and purify protective antigen (PA) using different protocols and to use this PA for detection of anti-PA antibodies from sera samples. METHODS: Protective antigen was isolated and purified from the Sterne strain of Bacillus anthracis. B. anthracis lacking pXO1 and pXO2 transformed with pYS5 (B. anthracis pYS5) and recombinant Escherichia coli transformed with pQE30 containing PA gene using hydroxyapatite (HA), Q-sepharose fast protein liquid chromatography (FPLC) and nickel-nitrilotriacetic acid (Ni-NTA) chromatographic methods, respectively. A mixture of PA and edema factor (EF) was injected subcutaneously into rabbits to test the biological activity of PA. The immunogenicity of PA was tested by inoculating the protein into rabbits along with adjuvant. Using this PA, 20 bovine sera samples (pre- and post-vaccinated) were tested by Western blotting (WB) for the presence of anti-PA antibodies. RESULTS: The 83 kDa PA protein was obtained from all the bacteria with the yields of 13, 50 and 9.0 mg/l from Sterne B. anthracis, B. anthracis pYS5 and recombinant Esch. coli, respectively. Formation of edematous ulcers at the site of PA+EF injection clearly confirmed the retention of biological activity of the proteins. Of the 10 post-vaccination sera tested, 9 showed clear positive by WB whereas none of the pre-vaccination sera showed the reaction. INTERPRETATION & CONCLUSION: The purified PA preparations obtained in the present study may possibly be utilized for detection of anti-PA antibodies in the sera of anthrax patients for timely diagnosis of the disease and, might also be tested for their efficacy and use as human anthrax vaccine.
Asunto(s)
Antígenos Bacterianos , Bacillus anthracis/metabolismo , Toxinas Bacterianas/química , Escherichia coli/metabolismo , Animales , Vacunas contra el Carbunco/inmunología , Toxinas Bacterianas/sangre , Materiales Biocompatibles/farmacología , Western Blotting , Bovinos , Cromatografía Liquida , Durapatita/farmacología , Electroforesis en Gel de Poliacrilamida , Venenos de Víboras/metabolismoRESUMEN
Monoclonal antibodies (MoAbs) were generated following immunization of BALB/c mice with protective antigen (PA) of B. anthracis. Five clones reactive to this protein were stabilized and preserved. These MoAbs could detect nanogram levels of PA when tested in ELISA. In Western blotting, they reacted with all PA preparations tested and no cross-reactivity was observed with lethal factor, edema factor of B. anthracis and with other organisms. These MoAbs could detect PA from 22 confirmed clinical isolates of B. anthracis on Western blotting and hold promise for direct detection of PA in clinical samples for diagnosing anthrax.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Antígenos Bacterianos/inmunología , Bacillus anthracis/inmunología , Toxinas Bacterianas , Animales , Carbunco/inmunología , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Western Blotting , Proteínas Portadoras/inmunología , Reacciones Cruzadas/inmunología , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones Endogámicos BALB C , Sensibilidad y Especificidad , Venenos de Víboras/inmunologíaRESUMEN
A simple protective antigen (PA)-reactive mAb dot-ELISA was standardized for confirmation of toxin-producing strains of Bacillus anthracis. Twenty-seven clinical isolates were collected from patients clinically suspected of having anthrax. PA was elaborated from these isolates using Casamino acids medium and the culture medium was boiled to kill the cells. PA in boiled culture supernatants was detected using a dot-ELISA. Of the 27 clinical isolates tested, PA was detected in 24 isolates. This was further confirmed by amplifying the PA gene by PCR. This testing procedure is simple to perform, specific and safer than existing procedures, which are added advantages over existing methods of identification of B. anthracis. This test system could be a valuable tool in confirming clinical and environmental isolates of B. anthracis.
Asunto(s)
Carbunco/diagnóstico , Carbunco/microbiología , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/análisis , Antígenos Bacterianos/inmunología , Bacillus anthracis/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/métodos , Aminoácidos , Bacillus anthracis/inmunología , Medios de Cultivo , Humanos , Sensibilidad y EspecificidadRESUMEN
A saprophytic Leptospira isolate recovered from tap water was utilized for serological testing. One hundred-twenty Serum samples comprising 55 cases from PUO/febrile jaundice and 65 samples from apparently healthy individuals were tested by MAT and HA using this environmental saprophytic strain and the results compared with that of Leptospira biflexa semaranga patoc, the standard saprophytic strain commonly employed for sero-diagnosis of leptospirosis. The MAT data showed 96.4 per cent correlation between the two strains. Similarly, the HA results were matching to the extent of 94.5 per cent. Results, therefore, suggest that local saprophytic Leptospira strain may serve as a substitute to serovar patoc for serodiagnosis of leptospirosis.
Asunto(s)
Antígenos Bacterianos/inmunología , Leptospira/inmunología , Leptospirosis/microbiología , Anticuerpos Antibacterianos/sangre , Humanos , Leptospirosis/diagnóstico , Pruebas SerológicasRESUMEN
Monoclonal antibodies (MoAbs) were generated following immunization of Balb/C mice with adenovirus type 5 grown in Hep2 cell line. Six clones reactive to hexon antigen of the virus were stabilized, of which 4 had mu-heavy chain specificity and 2 were of gamma-heavy chain type. Three of the clones (ADV-1, ADV-3 and ADV-5) had a high ELISA reactivity to the hexon antigen but exhibited differential specificity to the adenovirus types tested. In Western blotting, ADV-1 and ADV-3 reacted with all the adenovirus types tested (types 3,4,5,7 and 8) with reactions at 116 kDa region (hexon antigen), in addition, ADV-3 also had reactivity at 80 kDa region, the penton antigen. Reactivity to these adenoviral types by the 2 MoAbs was demonstrable by dot ELISA. ADV-5 had a type specific reaction only to adenovirus type 5 in dot ELISA with specificity in the hexon region in Western blotting. The reactivity of these 3 clones was not observed to the normal Hep2 tissue culture antigens and to the 3 enteroviruses tested (polio, coxsackie A9 and echo 4).
Asunto(s)
Adenovirus Humanos/inmunología , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Antivirales/biosíntesis , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Antivirales/aislamiento & purificación , Especificidad de Anticuerpos , Línea Celular , Humanos , Ratones , Ratones Endogámicos BALB CRESUMEN
Enterovirus specific IgM responses in 51 children aged 0-7 years with acute, clinically diagnosed paralytic syndrome and 8 children with chronic paralysis were detected by IgM antibody capture enzyme immunoassay. Twenty-nine out of 51 (56.86%) acute phase sera were positive for enterovirus (Polio, CVB3 and CVA7) IgM antibodies, in 21 of whom poliovirus antibodies were found in close association with CVB3 and CVA7. On the other hand, preponderance of CVA7 specific IgM was detected in 6 out of 8 sera samples of chronically paralytic children.
Asunto(s)
Infecciones por Coxsackievirus/inmunología , Enterovirus/inmunología , Inmunoglobulina M/biosíntesis , Parálisis/inmunología , Poliomielitis/inmunología , Enfermedad Aguda , Anticuerpos Antivirales/biosíntesis , Niño , Preescolar , Enfermedad Crónica , Enterovirus Humano B/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Lactante , Poliovirus/inmunología , SíndromeRESUMEN
Non specific binding (NSB) is an important factor affecting sensitivity and specificity in dot immunobinding assay (DIA). Several blocking agents e.g. egg albumin, casein, gelatin, milk powder and goat serum were evaluated for their relative efficacy vis-a-vis bovine serum albumin (BSA) for DIA system purported for detection of group B coxsackieviruses (CVB). The results suggest that egg albumin (5%) which is economical and readily available may act as an effective blocking agent in DIA system.
Asunto(s)
Enterovirus Humano B/aislamiento & purificación , Immunoblotting/métodos , OvalbúminaRESUMEN
Formalinized goose erythrocytes were used in haemagglutination inhibition (HI) and indirect fluorescent-antibody (IFA) tests to detect antibodies to Japanese encephalitis (JE) and West Nile (WN) viruses in equines. Paired serum samples from 31 cases having clinical symptoms of flaviviral infections (JE and WN viruses) and 45 controls were examined. For HI test, formalinized goose erythrocytes were used as such, whereas in IFA test, formalinized goose erythrocytes were first coated with respective viral antigens separately and later used to detect antibodies. By employing HI and IFA tests, paired samples having a titre same or less than two fold rise over the control sera were considered normal for both the viruses. IFA test was found to be a method of choice, due to its sensitivity over HI test.
Asunto(s)
Anticuerpos Antivirales/sangre , Técnica del Anticuerpo Fluorescente , Pruebas de Inhibición de Hemaglutinación , Enfermedades de los Caballos/diagnóstico , Infecciones por Togaviridae/veterinaria , Animales , Encefalomielitis Equina/diagnóstico , Caballos , Perisodáctilos , Infecciones por Togaviridae/diagnóstico , Fiebre del Nilo Occidental/diagnóstico , Fiebre del Nilo Occidental/veterinariaRESUMEN
Indirect enzyme linked immunosorbent assay (ELISA) was applied for the direct detection of coxsackieviruses in clinical samples viz. rectal swabs (RS) and throat swabs (TS) collected from patients admitted to various nursing homes and local hospitals. Results indicate the presence of different CVA types in 65 (62.5%) out of 104 RS, and 18 (52.9%) out of 34 TS samples. Dot-immunobinding assay was also standardized for the identification of CVA types employing 52 RS samples and the results compared with indirect ELISA. Dot-immunobinding detected more CVA types in a relatively larger number of specimens than indirect ELISA.