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

Abstract

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.

Ultra-sensitive AAV capsid detection by immunocapture-based qPCR following factor VIII gene transfer

“…Similar to the iqPCR method reported herein, previous immuno-PCR methods also demonstrated better sensitivity than corresponding traditional immunoassays [ 23 , 24 , 32 – 34 ]. The use of beads, rather than plates for antibody-mediated capsid capture in iqPCR also created an opportunity to perform an elution step.…”

“…We, therefore, sought to develop a novel AAV capsid detection method that would have comparable sensitivity to conventional qPCR methods used for measuring total vector DNA in clinical shedding studies [ 1 , 2 ]. To this end, we utilized the format of immunocapture-based qPCR (iqPCR), described previously to detect intact adenovirus particles, microbial antigens, and cytokines [ 20 – 24 ]. The method described herein capitalizes on the high target specificity of monoclonal antibodies directed against the AAV5 capsid and the exceptional sensitivity offered by qPCR detection of encapsidated vector DNA.…”

Tuberculosis Presenting as Isolated Wrist Swelling: A Case Report and Review of Literature

“…Immuno-PCR is a technique which combines amplification of DNA by PCR and coupling with ELISA technique. This was refined by the use of TB-specific Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay which has higher sensitivity compared with conventional immuno-PCR [23]. For the fast and efficient detection of TB, the use of mycolic acids of tuberculin bacilli was also described.…”

Broad-Spectrum Theranostics and Biomedical Application of Functionalized Nanomaterials

“…To detect mycobacterium tuberculosis MPT64 and CFP-10 proteins in the bodily fluids of TB patients, an MB-AuNP-I-PCR assay was designed using the functionalized gold nanoparticles (AuNPs) coupled with detection antibodies, oligonucleotides and magnetic beads (MBs), and found that sensitivity was much higher than magneto-ELISA and GeneXpert assays. This, thus, suggeststhat it leads to the development of a diagnostic kit [ 207 ]. In a study, a gold nanoparticle probe-based assay (GNPA) was developed for the detection of hepatitis C virus’ (HCV) core antigen.…”