Development of a Quantitative Sandwich Enzyme-Linked Immunosorbent Assay for Detecting the MPT64 Antigen ofMycobacterium tuberculosis

Abstract

PurposeTuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed.Materials and MethodsThe MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains.ResultsThe sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7×104 CFU/mL and 2.0×106 CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%).ConclusionThe sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis.

Validation of nanodiamond-extracted CFP-10 antigen as a biomarker in clinical isolates of Mycobacterium tuberculosis complex in broth culture media

“…Moreover, the sensitivity and specificity of the ELISA-based assay to detect MPT64 were both reported as 100%. The limit of the ELISA-based assay can reach 2.1 ng/mL of MPT64 [30]. We can conclude that by detecting MPT64, ICAs and ELISAs can differentiate MTC from NTM with high sensitivity and specificity.…”

Aluminum Foil vs. Gold Film: Cost-Effective Substrate in Sandwich SERS Immunoassays of Biomarkers Reveals Potential for Selectivity Improvement

“…The slopes on calibration plots were higher on gold than on Al foil, but blank Raman intensities and standard deviations of the blank were significantly lower on Al foil; therefore, on average, the ratio of the standard deviation of the blank to the slope of the calibration plot was slightly lower on Al foil in comparison to gold film. Overall, the results demonstrated a slightly lower LOD for the SERS sandwich immunoassay of MPT64 using Al foil (1.8 or 1.9 ng/mL or just 54 or 57 pg of the biomarker) with commercial antibodies, which were produced several months before the assay date, in comparison to the LOD reported for sandwich ELISA using custom-made “fresh” antibodies of 2.0 ng/mL [ 38 ]. Since we used non-lyophilized antibodies and antigens particularly sensitive to storage conditions (−20 °C or less recommended), with a process of delivery to NU in Kazakhstan a few weeks long, including customs clearance, this relatively high assay sensitivity showed the significant robustness of the SERS sandwich immunoassay method using both gold and Al foil substrates.…”

“…In this paper, for the first time, we report the SERS immunoassay detection of tuberculosis biomarker protein MPT64 using aluminum foil, and we compare its performance to gold film. The detection of MPT64 using a sandwich ELISA with freshly made in-house antibodies, is reported [ 38 ]. We demonstrate that the reported sensitivity of SERS can be matched or improved by the application of a low-cost /high-availability substrate, Al foil, even when commercial antibodies are used.…”

Ultrasensitive enzyme-linked immunosorbent assay for the detection of MPT64 secretory antigen to evaluate Mycobacterium tuberculosis viability in sputum

“…Although these studies measured MPT64 directly mixed with other TB-specific antigens in sputum, the sensitivity was lower than the current study for diagnosing active PTB cases, which was probably due to the low sensitivity of conventional ELISA. Ji et al established a quantitative ELISA method to detect MPT64 in cultured MTB samples and determined that 1.7 Â 10 4 CFU/mL MTB was the lower limit of MTB detection with the conventional sandwich ELISA method (Ji et al, 2014). This finding suggests that the threshold level for detecting viable MTB with the conventional ELISA method is similar to the threshold level of the direct sputum smear (Rouillon et al, 1976;Smithwick, 1979).…”