Enhanced production and purification of recombinant surface array protein (Sap) for use in detection of Bacillus anthracis

Puranik, Nidhi; Tripathi, Nagesh K.; Pal, Vijai; Goel, Ajay

doi:10.1007/s13205-018-1269-0

Cited by 5 publications

(11 citation statements)

References 18 publications

(15 reference statements)

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“…An amplicon of 2355 bp of sap gene was generated by PCR employing the primers, sap-F (5′-CATGGATCCGCAGGTAAAACATTCCCAG-3′) and sap-R (5′-ATACTCGAGTTTTGTTGCAGGTTTTGCTTCT-3′) as described earlier 19 . After purification, the PCR product was restricted with BamH1 and Xho1 restriction enzymes, cloned in the pET32a+ expression vector (Novagen, uSA) and transformed into E. coli BL21(DE3).…”

Section: Production Of Recombinant Sap (Rsap)mentioning

confidence: 99%

“…After purification, the PCR product was restricted with BamH1 and Xho1 restriction enzymes, cloned in the pET32a+ expression vector (Novagen, uSA) and transformed into E. coli BL21(DE3). From the clone, Sap protein was produced using shake flask culture as well as bioreactor, and purified by affinity chromatography and diafiltration as described elsewhere 19 .…”

Section: Production Of Recombinant Sap (Rsap)mentioning

confidence: 99%

“…Antibodies Anti-Sap polyclonal antibodies were produced in rabbit (New Zealand white) and mice (BALB/c) as described elsewhere 19 . Briefly, each rabbit (2 Nos) and each mouse (5 Nos) were immunised with 200 µg (subcutaneous) and 25 µg (intramuscular) of rSap, respectively in Freund complete adjuvant.…”

Section: Production and Purification Of Polyclonalmentioning

confidence: 99%

“…At an interval of two weeks, 3 successive booster doses were administered with Freund incomplete adjuvant. The blood was collected post one week of last booster and the serum titer was estimated employing plate ELISA 19 . The IgG was purified by affinity chromatography using protein A sepharose (Millipore, uSA).…”

Section: Production and Purification Of Polyclonalmentioning

confidence: 99%

“…Previously, we have shown that Sap may be utilised as a biomarker for the detection of B. anthracis 19 . In this report, we have shown it experimentally by developing a flow through sandwich based membrane ELISA for rapid detection of Sap, a biomarker of B. anthracis using anti-Sap polyclonal antibodies.…”

Section: Introductionmentioning

confidence: 99%

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A Rapid Flow through Membrane Enzyme Linked Immunosorbent Assay for Bacillus anthracis using Surface Array Protein as a Biomarker

et al. 2019

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Anthrax, caused by Bacillus anthracis is an important disease of biowarfare and public health importance. It is imperative to develop a simple system which can detect and differentiate B. anthracis from other closely related species. The surface array protein (Sap), which is secreted during the early growth phase of bacteria can be an important biomarker for detection of B. anthracis. In the present study, we have developed a rapid flow through membrane ELISA for detection of B. anthracis. Polyclonal antibodies were used to develop a sandwich plate ELISA, which could detect 3.9 ng/ml of recombinant Sap. B. anthracis bacteria grown in culture broth could be detected after 5 h of growth. Finally, a rapid flow through membrane ELISA was developed which can be accomplished just within 2 minutes, instead of 3-4 h as required in sandwich plate ELISA. The results established that the developed flow through membrane ELISA may be used for detection of B. anthracis. The proposed method is rapid, safe and user friendly for detection of B. anthracis culture. 2. Walsh, J.J.; Pesik, N.; Quinn, C.P.; urdaneta, V.;Dykewicz, C.A.; Boyer, A.E., et al. A case of naturally acquired inhalation anthrax: clinical care and analyses of anti-protective antigen immunoglobulin G and lethal factor. clin. Infect. Dis., 2007, 44(7), 968-971. 7. Jimenez, G.; urdiain, M.; Cifuentes, A.; Lopez-Lopez, A.; Blanch, A.R.; Tamames, J., et al. Description of Bacillus toyonensis sp. nov., a novel species of the Bacillus cereus group, and pairwise genome comparisons of the species of the group by means of ANI calculations. syst. appl.

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Section: Production Of Recombinant Sap (Rsap)mentioning

confidence: 99%

Section: Production Of Recombinant Sap (Rsap)mentioning

confidence: 99%

Section: Production and Purification Of Polyclonalmentioning

confidence: 99%

Section: Production and Purification Of Polyclonalmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A Rapid Flow through Membrane Enzyme Linked Immunosorbent Assay for Bacillus anthracis using Surface Array Protein as a Biomarker

et al. 2019

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The electrochemical detection of bioterrorism agents: a review of the detection, diagnostics, and implementation of sensors in biosafety programs for Class A bioweapons

2021

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During the past year, disease has shown us the iron grip it can hold over a population of people. Health systems can be overwhelmed, economies can be brought into recession, and many people can be harmed or killed. When weaponized, diseases can be manipulated to create a detriment to health while becoming an economic burden on any society. It is consequently prudent that easy detection of bioweapons is available to governments for protecting their people. Electrochemical sensing displays many distinct advantages, such as its low limit of detection, low cost to run, rapid generation of results, and in many instances portability. We therefore present a wide array of electrochemical sensing platforms currently being fabricated, a brief summary of Class A bioweapons, and the potential future of bioweapon detection and biosafety.

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Enhanced Production of Recombinant Extractable Antigen (EA1) an Extracellular Protein and its use in Detection of Spores of Bacillus anthracis the Causative Agent of Anthrax

et al. 2020

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Detection of spores of Bacillus anthracis, the causative agent of anthrax in human and animals in environment is cumbersome due to the presence of spores of other closely related Bacillus species. The Extractable Antigen 1 (EA1), an extracellular protein is considered as a biomarker for detection of B. anthracis spores. In the present work, we have cloned and expressed the recombinant EA1 protein in soluble form in Escherichia coli. Optimisation of culture conditions and cultivation media was carried out to achieve enhanced soluble expression of recombinant EA1 protein. Further, the batch fermentation process was also developed using optimised conditions for scale up production of recombinant EA1 protein. The final yield of protein purified employing affinity chromatography was 42.64 mg/l of culture during batch fermentation process. The polyclonal antibodies were raised against recombinant EA in rabbit and mice and used to develop an ELISA for detection of B. anthracis spores. The specificity of the developed assay was ascertained with spores of other Bacillus species. The results corroborated that the EA1 could be a suitable biomarker for detection of B. anthracis spores.

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Enhanced production and purification of recombinant surface array protein (Sap) for use in detection of Bacillus anthracis

Cited by 5 publications

References 18 publications

A Rapid Flow through Membrane Enzyme Linked Immunosorbent Assay for Bacillus anthracis using Surface Array Protein as a Biomarker

A Rapid Flow through Membrane Enzyme Linked Immunosorbent Assay for Bacillus anthracis using Surface Array Protein as a Biomarker

The electrochemical detection of bioterrorism agents: a review of the detection, diagnostics, and implementation of sensors in biosafety programs for Class A bioweapons

Enhanced Production of Recombinant Extractable Antigen (EA1) an Extracellular Protein and its use in Detection of Spores of Bacillus anthracis the Causative Agent of Anthrax

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