Characterization of pandemic influenza A (H1N1) virus hemagglutinin specific polyclonal antibodies for biosensor applications.

In this study, recombinant hemagglutinin protein (rH1N1HA) of Pandemic influenza virus and polyclonal antibodies against it for biosensor applications have been characterized. For rapid and high sensitive detection of H1N1 virus or its antibodies, PCR-free and label free detection method based on a surface plasmon resonance technique has been proposed. The glycosylated H1N1HA protein was expressed in yeast and the authenticity of the expressed protein was confirmed by Western blotting. Rabbit polyclonal antibodies developed against rH1N1HA protein were evaluated for their ability to neutralize H1N1 virus through plaque reduction neutralization test and indirect ELISA. Affinity purified anti-H1N1HA IgG were characterized further for their specificity, affinity of interaction, the association and dissociation rates at which they interact through surface plasmon resonance technique. The equilibrium constant and maximum binding capacity of analyte was found to be 49.7 nM and 47.28m°, respectively. The assay could detect a lowest IgG of 0.5 ng on a rH1N1HA coated chip. Combined with the high sensitivity of surface plasmon resonance technique and specificity of the reagents, it is possible to develop a rapid detection assay for monitoring influenza infections.

Surface Plasmon Resonance Biosensor for Detection of Bacillus anthracis, the Causative Agent of Anthrax from Soil Samples Targeting Protective Antigen.

Bacillus anthracis, the causative agent of anthrax is one of the most important biological warfare agents. In this study, surface plasmon resonance (SPR) technology was used for indirect detection of B. anthracis by detecting protective antigen (PA), a common toxin produced by all live B. anthracis bacteria. For development of biosensor, a monoclonal antibody raised against B. anthracis PA was immobilized on carboxymethyldextran modified gold chip and its interaction with PA was characterized in situ by SPR and electrochemical impedance spectroscopy. By using kinetic evaluation software, KD (equilibrium constant) and Bmax (maximum binding capacity of analyte) were found to be 20 fM and 18.74, respectively. The change in Gibb's free energy (∆G = -78.04 kJ/mol) confirmed the spontaneous interaction between antigen and antibody. The assay could detect 12 fM purified PA. When anthrax spores spiked soil samples were enriched, PA produced in the sample containing even a single spore of B. anthracis could be detected by SPR. PA being produced only by the vegetative cells of B. anthracis, confirms indirectly the presence of B. anthracis in the samples. The proposed method can be a very useful tool for screening and confirmation of anthrax suspected environmental samples during a bio-warfare like situation.

Fabrication and Dosimetric Characteristics of Silicon Elastomer-Based Bolus Using External Beam Radiotherapy.

OBJECTIVE: A study on dosimetric characteristics of silicon elastomer-based bolus was carried out using a Linear accelerator (Varian - Unique Performance). The study is performed to know if the silicone elastomer based bolus can be used in the radiotherapy. A bolus is a tissue equivalent material used to provide uniform dose to the uneven surface contours. It is exposed during the radiation therapy and also provides maximum dose (dmax) to treat surface tumors in case of high energy photons like megavoltage therapy photons. It is used in the case of external beam radiation therapy. METHODS: In this study, the bolus was fabricated using PDMS substrate with a curing agent by the ratio of 10:1. The bolus was fabricated in two thicknesses 0.5cm and 1cm. The dosimetric characteristics like transmission factor, mass attenuation coefficient, durability, homogeneity, density test of the fabricated bolus were studied. RESULTS: The dosimetric characteristics of the silicone elastomer based bolus were studied over a period of one month by exposing it in a 6MV photon. The result of the study shows that the silicone elastomer based bolus fabricated, satisfies the dosimetric characteristics needed for a tissue equivalent bolus to be used in the radiation therapy. CONCLUSIONS: The fabricated bolus could increase the percentage surface dose, reduce skin-sparing effect, and protect OAR. The aim of this is to provide an adjustable, transparent, and easily fabricated, less expensive, nontoxic bolus which can be used in the radiotherapy.

Construction of a single-chain variable-fragment antibody against the superantigen Staphylococcal enterotoxin B.

Staphylococcal food poisoning (SFP) is one of the most prevalent causes of food-borne illness throughout the world. SFP is caused by 21 different types of staphylococcal enterotoxins produced by Staphylococcus aureus. Among these, staphylococcal enterotoxin B (SEB) is the most potent toxin and is a listed biological warfare (BW) agent. Therefore, development of immunological reagents for detection of SEB is of the utmost importance. High-affinity and specific monoclonal antibodies are being used for detection of SEB, but hybridoma clones tend to lose their antibody-secreting ability over time. This problem can be overcome by the use of recombinant antibodies produced in a bacterial system. In the present investigation, genes from a hybridoma clone encoding monoclonal antibody against SEB were immortalized using antibody phage display technology. A murine phage display library containing single-chain variable-fragment (ScFv) antibody genes was constructed in a pCANTAB 5E phagemid vector. Phage particles displaying ScFv were rescued by reinfection of helper phage followed by four rounds of biopanning for selection of SEB binding ScFv antibody fragments by using phage enzyme-linked immunosorbent assay (ELISA). Soluble SEB-ScFv antibodies were characterized from one of the clones showing high affinity for SEB. The anti-SEB ScFv antibody was highly specific, and its affinity constant was 3.16 nM as determined by surface plasmon resonance (SPR). These results demonstrate that the recombinant antibody constructed by immortalizing the antibody genes from a hybridoma clone is useful for immunodetection of SEB.

Synthesis and characterization of metal ion imprinted nano-porous polymer for the selective recognition of copper.

Selective recognition of metal ions utilizing metal ion-imprinted polymers (MIIPs) received much importance in diverse fields owing to their high selectivity for the target metal ions. In the present study, a copper ion imprinted polymer was synthesized without an additional complexing ligand or complex with a broad aim to avoid the conventional extra metal ion complexing ligand during the synthesis of MIIP. The complete removal of the copper metal ion from the MIIP was confirmed by AAS and SEM-EDX. SEM image of the MIIP exhibited nano-patterns and it was also found to be entirely different from that of non-imprinted polymer and polymer with copper metal ions. BET surface area analysis revealed more surface area (47.96 m(2)/g) for the Cu(II)-MIIP than non-imprinted control polymer (41.43 m(2)/g). TGA result of polymer with copper metal ion indicated more char yield (18.41%) when compared to non-imprinted control polymer (8.3%) and Cu(II)-MIIP (less than 1%). FTIR study confirmed the complexation between Cu(II)-MIIP and Cu(II) metal ion through carbonyl oxygen of acryl amide. The Cu(II)-MIIP exhibited an imprinting efficiency of 2.0 and it was showing 8% interference from a mixture of Zn, Ni and Co ions. A potentiometric ion selective electrode devised with Cu(II)-MIIP showed more potential response for Cu(II) ion than that was fabricated from non-imprinted polymer.

Plastic antibody for the recognition of chemical warfare agent sulphur mustard.

Molecularly imprinted polymers (MIPs) known as plastic antibodies (PAs) represent a new class of materials possessing high selectivity and affinity for the target molecule. Since their discovery, PAs have attracted considerable interest from bio- and chemical laboratories to pharmaceutical institutes. PAs are becoming an important class of synthetic materials mimicking molecular recognition by natural receptors. In addition, they have been utilized as catalysts, sorbents for solid-phase extraction, stationary phase for liquid chromatography and mimics of enzymes. In this paper, first time we report the preparation and characterization of a PA for the recognition of blistering chemical warfare agent sulphur mustard (SM). The SM imprinted PA exhibited more surface area when compared to the control non-imprinted polymer (NIP). In addition, SEM image showed an ordered nano-pattern for the PA of SM that is entirely different from the image of NIP. The imprinting also enhanced SM rebinding ability to the PA when compared to the NIP with an imprinting efficiency (alpha) of 1.3.

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.

Supersensitive detection of T-2 toxin by the in situ synthesized π-conjugated molecularly imprinted nanopatterns. An in situ investigation by surface plasmon resonance combined with electrochemistry.

A π-conjugated molecularly imprinted polymer (MIP) with nanopatterns for T-2 toxin (T-2) was prepared on SPR chip by in situ electropolymerization of 3-aminophenylboronicacid (3-APBA) with T-2. The complete removal of T-2 from polymer was confirmed in situ by SPR and EIS and also ex situ by SEM, EDAX, fluorescence microscopy and Raman spectroscopy. SEM image of T-2 MIP exhibited nanopatterns due to imprinting of T-2. The MIP of T-2 showed a linear response for T-2 from 2.1 fM to 33.6 fM with a detection limit of 0.1 fM (0.05 pg/mL). In this study, thermodynamic parameters such as change in Gibb's free energy (ΔG), change in enthalpy (ΔH) and change in entropy (ΔS) were determined and the values revealed that the interaction between T-2 and T-2 MIP as spontaneous, endothermic and entropy driven one. Moreover, interactions of very high concentration of interferents with T-2 MIP showed very less response due to the presence of nanopatterns of T-2 in the T-2 MIP. Equilibrium constant (12.7 fM) obtained in this study indicates the super binding affinity of T-2 with T-2 MIP. Moreover, the present methodology provides an outline to develop field-detection equipment capable of detecting T-2 toxin at or well below the guideline concentrations recommended by American subcommittee on military field drinking water.

Molecularly imprinted nanopatterns for the recognition of biological warfare agent ricin.

Molecularly imprinted polymer (MIP) for biological warfare agent (BWA) ricin was synthesized using silanes in order to avoid harsh environments during the synthesis of MIP. The synthesized MIP was utilized for the recognition of ricin. The complete removal of ricin from polymer was confirmed by fluorescence spectrometer and SEM-EDAX. SEM and EDAX studies confirmed the attachment of silane polymer on the surface of silica gel matrix. SEM image of Ricin-MIP exhibited nanopatterns and it was found to be entirely different from the SEM image of non-imprinted polymer (NIP). BET surface area analysis revealed more surface area (227 m(2)/g) for Ricin-MIP than that of NIP (143 m(2)/g). In addition, surface area study also showed more pore volume (0.5010 cm(3)/g) for Ricin-MIP than that of NIP (0.2828 cm(3)/g) at 12 nm pore diameter confirming the presence of imprinted sites for ricin as the reported diameter of ricin is 12 nm. The recognition and rebinding ability of the Ricin-MIP was tested in aqueous solution. Ricin-MIP rebound more ricin when compared to the NIP. Chromatogram obtained with Ricin-MIP exhibited two peaks due to imprinting, however, chromatogram of NIP exhibited only one peak for free ricin. SDS-PAGE result confirmed the second peak observed in chromatogram of Ricin-MIP as ricin peak. Ricin-MIP exhibited an imprinting efficiency of 1.76 and it also showed 10% interference from the structurally similar protein abrin.