Development of real-time immuno-PCR for the quantitative detection of mycobacterial PstS1 in tuberculosis patients

Sharma, Sunil; Raj, Ankush; Singh, Netrapal; Dahiya, Bhawna; Sheoran, Anita; Gupta, K. B.; Mehta, Promod K

doi:10.1016/j.mimet.2016.12.006

Cited by 14 publications

(11 citation statements)

References 16 publications

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“…During the last two decades, an unprecedented interest has been emerged for designing new TB diagnostic tools but still a reliable and accurate test for rapid diagnosis for all forms of TB, ie, pulmonary, extrapulmonary, TB-HIV coinfection and latent TB is lacking. We developed immuno-PCR (I-PCR) assay based on the detection of array of Mycobacterium tuberculosis antigens, ie, Ag85B (Antigen 85B, Rv1886c), ESAT-6 (early secreted antigenic target-6), cord factor (trehalose 6,6′-dimycolate) and PstS1 (phosphate-specific transporter lipoprotein, Rv0934), [2][3][4] which certainly revealed better sensitivity than ELISA. ESAT-6 encoded by region of difference (RD1) is M. tuberculosis complex specific and is considered as a potential biomarker for the diagnosis of TB by ELISA and I-PCR, either alone or in combination with other RD1 and RD2 antigens such as CFP-10 (culture filtrate protein-10, Rv3874) and MPT64 (mycobacterial protein from species tuberculosis-64, Rv1980c).…”

Section: Introductionmentioning

confidence: 99%

Detection of <em>Mycobacterium tuberculosis</em> purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay

Singh¹,

Dahiya²,

Radhakrishnan³

et al. 2018

IJN

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PurposeImmuno-PCR (I-PCR), an ultrasensitive method, combines the versatility of ELISA with the exponential amplification capacity of PCR. Coupling of detection antibodies with the reporter DNA is a critical step of I-PCR. Gold nanoparticles (GNPs) and magnetic beads (MBs) are relatively easy to attach with the antibodies and DNA. Therefore, we designed MB-coupled GNP-based I-PCR (MB-GNP-I-PCR) assay for the detection of Mycobacterium tuberculosis antigen.MethodsGNPs were synthesized by chemical reduction and seed-mediated synthesis. Functionalized GNPs were prepared by coupling GNPs with the detection antibodies and reporter DNA and were characterized. Detection limit of M. tuberculosis-specific purified early secreted antigenic target-6 (ESAT-6) (Rv3875) was determined by MB-GNP-I-PCR.ResultsTransmission electron microscopy revealed spherical and slightly polydispersed GNPs of ~20 and ~60 nm size. Coupling of antibodies to GNPs was indicated by a shift in absorption maxima from 524 to 534 nm, which was confirmed by transmission electron microscopy. A color reaction with ELISA and the presence of 76 bp product by PCR further validated the coupling of detection antibodies and signal DNA to the functionalized GNPs. Also, attachment of capture antibodies with MBs was confirmed by magneto-ELISA. Detection limit of purified ESAT-6 by MB-GNP-I-PCR was determined to be 10 fg/mL, 105-fold lower than analogous ELISA. Notably, no sample matrix effect was observed in the saliva samples of healthy individuals spiked with the purified ESAT-6.ConclusionUnlike conventional I-PCR (solid format), MB-GNP-I-PCR (liquid format) is relatively simple with the reduced background signals, which can be further exploited for the clinical diagnosis of tuberculosis.

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Section: Introductionmentioning

confidence: 99%

Detection of <em>Mycobacterium tuberculosis</em> purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay

Singh¹,

Dahiya²,

Radhakrishnan³

et al. 2018

IJN

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“…PstS1 is a 38-kDa phosphate-binding periplasmatic protein, one of three subunits of the Mtb phosphate-specific transporter (Pst) complex 12 . PstS1 is an immunodominant marker for ATB [28][29][30] . PstS1 is also necessary for Mtb virulence; PstS1-deletion mutants were attenuated in a mouse-infection model 13 .…”

Section: Discussionmentioning

confidence: 99%

Human antibodies targeting a Mycobacterium transporter protein mediate protection against tuberculosis

Watson

et al. 2021

Nat Commun

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Mycobacterium tuberculosis (Mtb) exposure drives antibody responses, but whether patients with active tuberculosis elicit protective antibodies, and against which antigens, is still unclear. Here we generate monoclonal antibodies from memory B cells of one patient to investigate the B cell responses during active infection. The antibodies, members of four distinct B cell clones, are directed against the Mtb phosphate transporter subunit PstS1. Antibodies p4-36 and p4-163 reduce Mycobacterium bovis-BCG and Mtb levels in an ex vivo human whole blood growth inhibition assay in an FcR-dependent manner; meanwhile, germline versions of p4-36 and p4-163 do not bind Mtb. Crystal structures of p4-36 and p4-170, complexed to PstS1, are determined at 2.1 Å and 2.4 Å resolution, respectively, to reveal two distinctive PstS1 epitopes. Lastly, a prophylactic p4-36 and p4-163 treatment in Mtb-infected Balb/c mice reduces bacterial lung burden by 50%. Our study shows that inhibitory anti-PstS1 B cell responses arise during active tuberculosis.

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“…PstS1 is a 38-kDa phosphate-binding periplasmatic protein, one of three subunits of the Mtb phosphate-specific transporter (Pst) complex 12 . PstS1 is as an immunodominant marker for ATB 26–28 . PstS1 is also necessary for Mtb virulence; PstS1-deletion mutants were attenuated in a mouse-infection model 13 .…”

Section: Discussionmentioning

confidence: 99%

Human Antibodies Targeting a Transporter Mediate Protection Against Tuberculosis

Watson¹,

Li²,

Ma³

et al. 2020

Preprint

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Evidence has emerged that some healthy individuals with a history of exposure to Mycobacterium tuberculosis (Mtb) can develop protective antibody responses. However, it is not known whether patients with active tuberculosis elicit protective antibodies, and if they do, which bacterial antigens are targeted. To investigate the B cell responses during active infection, we generated a panel of monoclonal antibodies isolated from memory B cells of one patient. The antibodies, members of four distinct B cell clones, were directed against the Mtb phosphate transporter subunit PstS1. Antibodies p4-36 and p4-163 from two different B cell clones showed protective efficacy against Mtb and Mycobacterium bovis-BCG in an ex vivo human whole blood growth inhibition assay. Germline versions of p4-36 and p4-163 could no longer bind Mtb, implying that affinity maturation was vital for their activity. Crystal structures of p4-36 and a closely related clonal variant of p4-163, p4-170, complexed to PstS1 were determined at a resolution of 2.1A and 2.4A and revealed that the two antibodies recognize two distinctive epitopes on PstS1. As a proof of principle, p4-36 and p4-163 were used in a passive vaccination setting in aerosol Mtb-infected Balb/c mice, where both antibodies reduced bacterial lung burden by 50% after a single injection prior to Mtb infection. Our study shows that inhibitory B cell responses arise during active tuberculosis and identifies PstS1 as a target for elicitation of anti-Mtb antibodies.

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Development of real-time immuno-PCR for the quantitative detection of mycobacterial PstS1 in tuberculosis patients

Cited by 14 publications

References 16 publications

Detection of <em>Mycobacterium tuberculosis</em> purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay

Detection of <em>Mycobacterium tuberculosis</em> purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay

Human antibodies targeting a Mycobacterium transporter protein mediate protection against tuberculosis

Human Antibodies Targeting a Transporter Mediate Protection Against Tuberculosis

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