Development and assessment of a novel magnetic nanoparticle antibody-conjugate and aptamer-based assay (MNp-Ab-Ap assay) for the rapid diagnosis of pleural tuberculosis

Abstract

Background: Pleural tuberculosis (pTB) is a diagnostic challenge because of its non-specific clinical features, lack of accurate diagnostic tools and paucibacillary nature of the disease. Methods: We, here describe the development of a novel magnetic nanoparticle antibody-conjugate and aptamer-based assay (MNp-Ab-Ap assay) targeting 4 different Mycobacterium tuberculosis (M. tb.) antigens (GlcB, MPT51, MPT64 and CFP-10) for pTB diagnosis. The MNp-Ab-Ap assay was developed by conjugating polyclonal antibodies on the surface of magnetic nanoparticles (MNPs) by using EDC-NHS chemistry. These conjugated MNPs were used to capture M. tb. antigens present in the pleural fluid samples. The resulting antigen-antibody complex was detected by antigen-specific 5'-biotinylated aptamers. All assays were standardized using samples of the 'Development set' (n=17) and evaluated in the 'Validation set' (n=114) in a blinded manner. Patient categorization was done using a 'Composite Reference Standard'. Assay cut-offs were determined from the 'Development set' (n=17; 'Definite & Probable' pTB; n=9 and 'Non-TB'; n=8) by calculating mean+3SD of OD₄₅₀ values of the 'Non-TB' group and applied to 'Validation set' (n=114; 'Definite' pTB; n=8, 'Probable' pTB; n=34, 'Possible' pTB; n=28 and 'Non-TB'; n=44). Results: Out of the 4 assays, MPT51-based MNp-Ab-Ap assay performed the best with 66.6% (95%CI;50.4-80.4) sensitivity and 95.4% (95%CI;85.1-99.4) specificity in the combined 'Definite and Probable' pTB group. Xpert MTB/RIF assay detected only six samples in the 'Validation set'. Binary logistic regression analysis indicated that MPT51-based MNp-Ab-Ap assay provided an incremental advantage over the existing diagnostic algorithm for pTB. Conclusions: We conclude that MPT51-based MNp-Ab-Ap assay is a novel technique that can pave the way towards rapid and accurate diagnosis of pTB.

Figure 1

Flowchart of the study. MNpAb-Ap assay: Magnetic nanoparticle antibody-conjugate and aptamer-based assay.

Figure 2

Scatterplots for OD₄₅₀ values of MNp-Ab-Ap assay in pleural fluid samples of 'Validation set' (n=114) of the study. a) GlcB, b) MPT51, c) MPT64, and d) CFP-10. Horizontal line across the graph represents the cut-off generated from mean+3SD of the OD₄₅₀ values of Non-TB samples of the 'Development set' (n=17) of the study.

Figure 3

Scatterplots for OD₄₅₀ values of ELISA in pleural fluid samples of 'Validation set' (n=114) of the study. a) GlcB, b) MPT51, c) MPT64, and d) CFP-10. Horizontal line across the graph indicates the cut-off calculated as mean+3SD of the OD₄₅₀ values of Non-TB samples included in the 'Development set' (n=17) of the study.

Figure 4

Binary logistic regression analysis for the 'clinical and biochemical' predictors along with MPT51-based MNp-Ab-Ap assay as a 'single test' in the 'Definite and Probable' pTB group. Receiver operating characteristic curves for MPT51-based MNp-Ab-Ap assay generated for 'Definite and Probable' pTB group vs. 'Non-TB' group. AUC: area under the curves.

Figure 5

Development of MNp-Ab-Ap assay. The steps involved in the development includes a) the conjugation of polyclonal antibodies on the surface of the magnetic nanoparticles (conjugated beads), followed by b) incubation of appropriate dilution of pleural fluid with conjugated beads, c) washing of the beads by using an in-house magnetic apparatus and blocking of beads, d) incubation of beads with antigen-specific DNA aptamers, e) washing of beads and incubation with streptavidin-HRP polymer, f) development of assay by using 3,3',5,5'-tetramethylbenzidine (TMB) substrate, g) stopping the reaction using 50 μl of 2 N H₂SO₄ and h) absorbance measurement at 450 nm.