Competitive immunochromatographic assay for the detection of thiodiglycol sulfoxide, a degradation product of sulfur mustard.

An immunochromatographic assay (ICA) based on the competitive antigen-coated format using colloidal gold as the label was developed for the detection of thiodiglycol sulfoxide (TDGO), an important metabolite and degradation compound of sulphur mustard (SM). The ICA test strip consisted of a membrane with a detection zone, a sample pad and an absorbent pad. The membrane was separately coated with hapten-OVA conjugate (test line) and anti-rabbit mouse IgG (control line). The visual detection limit for TDGO by ICA detection was found to be 10 µg mL(-1). For validation, the ICA results obtained for spiked water samples were in good agreement with those obtained by indirect competitive inhibition enzyme-linked immunosorbent assay (ELISA) for TDGO. The assay time for detection was less than 10 min. The developed ICA has the potential to be a useful on-site screening tool for the retrospective detection of SM in environmental samples.

Fermentation and downstream process for high yield production of Plasmodium falciparum recombinant HRP II protein and its application in diagnosis.

Malaria represents the world's greatest public health problem in terms of number of people affected, levels of morbidity and mortality in tropical and subtropical countries. Malaria parasites are members of the Apicomplexa, family of Plasmodiidae. Histidine-rich protein-II secreted by Plasmodium falciparum is known to be a compelling marker in malaria diagnosis and follow-up. In our present study, we have optimized the batch fermentation and downstream process for large scale production of recombinant P. falciparum HRP-II 62 kDa protein for diagnostic application. The culture broth was effectively induced with IPTG twice at different time intervals to sustain induction for a long period. Batch fermentation resulted in a wet weight of 61.34 g/L and dry cell biomass 12.81 g/L. With the improved downstream process, purified recombinant protein had a yield of 304.60 mg/L. The authenticity of the purified recombinant protein was confirmed via western blotting using indigenously developed HRP-II specific monoclonal antibodies and known positive human clinical sera samples. Further, the reactivity of recombinant HRP-II protein was validated using commercially available immuno chromatographic strips. Indirect ELISA using recombinant purified protein recognized the P. falciparum specific antibodies in suspected human sera samples. Our results clearly suggest that the recombinant HRP-II protein produced via batch fermentation has immense potential for routine diagnostic application.

Direct hapten-linked competitive inhibition enzyme-linked immunosorbent assay (CIELISA) for the detection of O-pinacolyl methylphosphonic acid.

Immunoassay detection of O-pinacolyl methylphosphonic acid (PMPA) employing direct coating of N-2-aminoethyl-O-pinacolyl methylphosphonate (hapten B) on microtiter plates is reported. Coating was achieved by covalently linking hapten B to a glutaraldehyde (GA) polymer network directly bound to the polystyrene (PS) surface of a standard 96-well microtiter plate. 4-(2-(O-Pinacolylmethylphosphoryl amino)ethyl amino)-4-oxobutanoic acid (hapten A)-ovalbumin (OVA) conjugate served as the coating antigen for comparison with direct hapten B-coated plates in the CIELISA format. The developed assay employing direct hapten B coated plates demonstrated enhanced sensitivity with the IC(50) value for PMPA being 0.027 µg mL(-1). The assay could detect PMPA even at the concentration of 0.006 µg mL(-1). The mean recovery of standard PMPA (spiked in water) was found to be 83.7%.

Amperometric immunosensor based on gold nanoparticles/alumina sol-gel modified screen-printed electrodes for antibodies to Plasmodium falciparum histidine rich protein-2.

We report herein the amperometric immunosensor for antibodies to Plasmodium falciparum histidine rich protein-2 (PfHRP-2). Screen-printed electrodes (SPEs) were modified with alumina sol-gel (Al(2)O(3) sol-gel) derived film and gold nanoparticles i.e. AuNPs/Al(2)O(3)sol-gel/SPE. A thin film was formed by dripping Al(2)O(3) sol on SPE followed by electrochemical deposition of gold nanoparticles (AuNPs). The modified SPEs were characterized by scanning electron microscopy/energy dispersive X-ray analysis (SEM-EDAX), Raman spectra and voltammetric experiments. Antibodies in rabbit serum sample were allowed to react with the PfHRP-2 protein that was immobilized on the modified SPE to form antigen-antibody immune complex (PfHRP-2/anti-PfHRP-2). The bound antibodies were quantified by alkaline phosphatase (AP) enzyme labeled secondary antibodies (anti-rabbit immunoglobulins-AP conjugate). Enzymatic substrate, 1-naphthyl phosphate was converted to 1-naphthol by AP and an electroactive product was quantified using amperometry. The performances of the developed immunosensor and Dot-ELISA were tested against different dilutions of hyper immune serum (rabbit anti-PfHRP-2). Dot ELISA and the developed immunosensor (AuNPs/Al(2)O(3)sol-gel/SPE) results for the hyper immune serum containing anti-PfHRP-2 were distinctly positive when diluted upto 8 times (1 : 12800 dilution) and 11 times (1 : 102400 dilution), respectively. The developed immunosensor was applied for antibodies to PfHRP-2 in human clinical samples.

Evaluation of PCR, DNA hybridization and immunomagnetic separation - PCR for detection of Burkholderia mallei in artificially inoculated environmental samples.

Glanders is highly contagious disease of equines, caused by Burkholderia mallei. The disease though rare, can be transmitted to humans. Here, we report a strategy for rapid detection of B. mallei from environmental samples. Different bacteriological media were evaluated and brain heart infusion broth medium with selective supplements (BHIB-SS) of penicillin (200 U/ml) and crystal violet (1:10,00000) was found to support the maximum growth of B. mallei even in the presence of other bacteria like Escherichia coli and Staphylococcus aureus. A polymerase chain reaction (PCR) and a DNA hybridization method was standardized for 823 bp specific dNA sequence of B. mallei. To enable the quicker and direct enrichment of B. mallei bacteria from environmental samples, an immunomagnetic separation (IMS) method was also standardized. Water, husk, grass and gram samples were artificially contaminated by B. mallei bacteria and after enrichment of B. mallei in BHIB-SS, detection was carried out by PCR and DNA hybridization. PCR was found to be a better method of the two with a detection limit of 10(4)-10(6) CFU/ml (6 h enrichment in BHIB-SS) in water and other particulate matrices. Detection by PCR in the above samples without enrichment in BHIBSS was carried out following IMS where the detection limit was about 1-2 log higher than PCR following enrichment in BHIB-SS. We recommend PCR for 823 bp for detection of B. mallei from environmental samples either following enrichment in BHIB-SS or IMS. IMS-PCR method may be preferred in situations where numbers of B. mallei bacteria are expected to be high and results are required in short time.

Th1-type immune response to infection by pYV-cured phoP-phoQ null mutant of Yersinia pseudotuberculosis is defective in mouse model.

The PhoP-PhoQ two-component system of Yersinia pseudotuberculosis, a Gram-negative enteric pathogen which causes a variety of gastrointestinal and extraintestinal infections in humans, has been shown to be necessary for virulence. A phoP-phoQ null mutant of a strain of Y. pseudotuberculosis cured of its native plasmid pYV was obtained and studied for generation of immune response in mouse model following intravenous inoculation. The phoP-phoQ null mutant elicited much weaker IgG antibody response to whole cell sonicated (WCS) antigen, in particular that of IgG2a isotype. Interferon-gamma levels were also significantly reduced in cultured splenocytes of mice immunized with phoP-phoQ null mutant. The null mutant was found to be about 72-fold less virulent than the parent isogenic strain of Y. pseudotuberculosis. Average counts in spleen of mice inoculated with the null mutant were observed to reduce by at least four logs when compared with the counts in the spleen of mice inoculated with parent isogenic strain. We can thus suggest that the Th1-type immune response of the phoP-phoQ null mutant of Y. pseudotuberculosis is diminished in mice.

Antimicrobial properties of CuO nanorods and multi-armed nanoparticles against B. anthracis vegetative cells and endospores.

Two different kinds of CuO nanoparticles (NPs) namely CuO nanorods (PS2) and multi-armed nanoparticles (P5) were synthesized by wet and electrochemical routes, respectively. Their structure, morphology, size and compositions were characterized by SEM, EDX and XRD. The NPs demonstrated strong bactericidal potential against Bacillus anthracis cells and endospores. PS2 killed 92.17% of 4.5 × 10(4) CFU/mL B. anthracis cells within 1 h at a dose of 1 mg/mL. Whereas P5 showed a higher efficacy by killing 99.92% of 7 × 10(5) CFU/mL B. anthracis cells within 30 min at a dose of 0.5 mg/mL and 99.6% of 1.25 × 10(4) CFU/mL B. anthracis cells within 5 min at a dose of 2 mg/mL. More than 99% of spores were killed within 8 h with 2 mg/mL PS2 in LB media.

Real-time polymerase chain reaction assay for rapid and sensitive detection of anthrax spores in spiked soil and talcum powder.

Real-time polymerase chain reaction (real-time PCR) is a laboratory technique based on PCR. This technique is able to detect sequence-specific PCR products as they accumulate in "real time" during the PCR amplification, and also to quantify the number of substrates present in the initial PCR mixture before amplification begins. In the present study, real-time PCR assay was employed for rapid and real-time detection of Bacillus anthracis spores spiked in 0.1 g of soil and talcum powder ranging from 5 to 10(7) spores. DNA was isolated from spiked soil and talcum powder, using PBS containing 1 % Triton-X-100, followed by heat treatment. The isolated DNA was used as template for real-time PCR and PCR. Real-time PCR amplification was obtained in 60 min under the annealing condition at 60°C by employing primers targeting the pag gene of B. anthracis. In the present study, the detection limit of real-time PCR assay in soil was 10(3) spores and 10(2) spores in talcum powder, respectively, whereas PCR could detect 10(4) spores in soil and 10(3) spores in talcum powder, respectively.

Surface plasmon resonance immunosensor for the detection of Salmonella typhi antibodies in buffer and patient serum.

Surface plasmon resonance (SPR) immunosensor using 4-mercaptobenzoic acid (4-MBA) modified gold SPR chip was developed first time for the detection of flagellin specific antibodies of Salmonella typhi (S. typhi). Flagellin protein of S. typhi was prepared by recombinant DNA technology. The modification of gold chip with 4-MBA was in-situ characterized by SPR and electrochemical impedance spectroscopy. By using kinetic evaluation software, K(D) and B(max) values were calculated and found to be 26.3 fM and 62.04 m°, respectively, for the immobilized monoclonal antibody (Moab) of recombinant flagellin (r-fla) protein of S. typhi (r-fla S. typhi). In addition, thermodynamic parameters such as ΔG, ΔH and ΔS were determined first time for r-fla S. typhi and Moab of r-fla S. typhi interactions and the values revealed the interaction between r-fla S. typhi and Moab of r-fla S. typhi as spontaneous, endothermic and entropy driven one. Moreover, healthy human serum samples and patient sera (Widal positive and Widal negative) were subjected to SPR analysis. The present SPR based approach provides an alternative way for S. typhi detection in less than 10 min.

Design and synthesis of immunoconjugates and development of competition inhibition enzyme-linked immunosorbent assay (CIEIA) for the detection of O-isopropyl methylphosphonofluoridate (sarin): an organophosphorous toxicant.

Three haptens of the organophosphorus (OP) toxicant 'sarin' having different spacer arm were designed and synthesized. Haptens were conjugated with BSA (bovine serum albumin) and ovalbumin (OVA) for raising antibody and coating antigen. High antibody titer with higher specificity was obtained from 4-(4-(isopropoxy(methyl)phosphoryloxy)phenylamino)-4-oxobutanoic acid (hapten B) having reasonable long spacer arm. For the standard curve, an IC(50) (inhibitory concentration) of free antigen was found to be 0.415 µg mL(-1) on the basis of indirect competitive ELISA. The study revealed that heterology in competition inhibition enzyme immunoassay (CIEIA) produced remarkable improvement in the sensitivity and specificity of the assay. Under the optimized conditions, the quantitative working range was found to be 0.19-1.56 µg mL(-1) with a limit of detection (LOD) of 0.05 µg mL(-1). The antibodies showed negligible cross reactivity (CR) with other OP toxicants and pesticides, which makes the assay suitable for the selective detection of sarin.

A novel piezoelectric immunosensor for the detection of malarial Plasmodium falciparum histidine rich protein-2 antigen.

A novel piezoelectric (PZ) immunosensor for the direct detection of malarial Plasmodium falciparum histidine rich protein-2 (PfHRP-2) antigen was developed. The mixed self-assembled monolayers (SAMs) of thioctic acid and 1-dodecanethiol were formed on gold surface of quartz crystal. Cyclic voltammetry, electrochemical impedance spectroscopy and surface Raman spectroscopy techniques were used to characterize the mixed SAMs. The rabbit anti-PfHRP-2 antibodies were coupled on mixed SAM modified gold surface of quartz crystal via NHS/EDC activation method. The PZ immunosensor was applied to detect PfHRP-2 in the linear range of 15-60 ng/ml with a detection limit of 12 ng/ml. It was also found that even after 14 days of storage, 50% of the activity still remained. Clinical human serum samples were tested with this method, and the results were in agreement with those obtained from commercially available ICT kit (NOW(®) Malaria).

Real-time loop-mediated isothermal amplification assay for rapid and sensitive detection of anthrax spores in spiked soil and talcum powder.

Loop-mediated isothermal amplification (LAMP) assay is a powerful and innovative gene amplification technique that specifically amplifies the target gene under isothermal conditions with a high degree of sensitivity, rapidity and specificity. The major advantage of the LAMP assay is monitoring of amplified products without the requirement of any sophisticated equipment. In the present study a real time LAMP assay was employed for rapid and real time detection of Bacillus anthracis spores spiked in 0.1 g of soil and talcum powder ranging from 2 to 10(7) spores. DNA was isolated from spiked soil and talcum powder using PBS containing 1% Triton X-100, and heat treatment. Isolated DNA was used as template for LAMP and PCR. LAMP amplification was obtained in 60 min under isothermal condition at 63°C by employing a set of six primers targeting the pag gene of B. anthracis. The detection limit of LAMP assay in soil and talcum powder was found to be as low as 5 spores, compared to 10(3) spores and 10(4) spores by PCR in talcum powder and soil, respectively. The findings suggest that LAMP is a more rapid and sensitive assay than PCR for detecting anthrax spores, additionally the methodology to prepare DNA from spiked samples is simple, rapid and cost effective.

Photocatalytic inactivation of spores of Bacillus anthracis using titania nanomaterials.

Studies on photocatalytic inactivation of spores of Bacillus anthracis have been carried out using nanosized titania materials and UVA light or sun light. Results demonstrated pseudo first order behaviour of spore inactivation kinetics. The value of kinetic rate constant increased from 0.4h(-1) to 1.4h(-1) indicating photocatalysis facilitated by addition of nanosized titania. Nanosized titania exhibited superior inactivation kinetics on par with large sized titania. The value of kinetic rate constant increased from 0.02 h(-1) to 0.26 h(-1) on reduction of size from 1000 nm to 16 nm depicting the enhanced rate of inactivation of Bacillus anthracis Sterne spores on the decrease of particle size.