Immunological detection of pyrazine-2-carboxylic acid for the detection of pyrazinamide resistance in Mycobacterium tuberculosis

Abstract

Pyrazinamide (PZA) susceptibility testing in Mycobacterium tuberculosis (Mtb) is a current area of development and PZA-resistant strains are increasingly prevalent. Previous studies have demonstrated that the detection of pyrazinoic acid (POA), the metabolite produced by the deamidation of PZA, is a good predictor for PZA resistance since a resistant strain would not convert PZA into POA at a critical required rate, whereas a susceptible strain will do, expelling POA to the extracellular environment at a certain rate, and allowing for quantification of this accumulated analyte. In order to quantify POA, an indirect competitive ELISA (icELISA) test using hyperimmune polyclonal rabbit serum against POA was developed: for this purpose, pure POA was first covalently linked to the highly immunogenic Keyhole Limpet Hemocyanine, and inoculated in rabbits. A construct made of bovine serum albumin (BSA) linked to pure POA and fixed at the bottom of wells was used as a competitor against spiked samples and liquid Mtb culture supernatants. When spiked samples (commercial POA alone) were analyzed, the half maximal inhibitory concentration (IC50) was 1.16 mg/mL, the limit of detection 200 μg/mL and the assay was specific (it did not detect PZA, IC50 > 20 mg/mL). However, culture supernatants (7H9-OADC-PANTA medium) disrupted the competition and a proper icELISA curve was not obtainable. We consider that, although we have shown that it is feasible to induce antibodies against POA, matrix effects could damage its analytical usefulness; multiple, upcoming ways to solve this obstacle are suggested.

Fig 1. Structural formulas.

Constructs (not to scale) used in the present study. Green dotted lines separate the original components. Acronyms are explained in Table 1. mcKLH being a massive molecule with an estimated weight of 8 million Daltons displays great immunogenicity in an mammal subject (rabbit). In comparison, molecular weight of the rest of reactant molecules is indicated.

Fig 2. Indirect competitive ELISA.

The principle of this assay can be explained as follows: In a positive sample, the POA analyte (purple) competes for rabbit polyclonal antibodies (orange) against synthetic analyte which is attached to BSA (blue, in this study denominated “PLB”). After several washes, a secondary anti-rabbit antibody (dark blue) labeled with horseradish peroxidase (HRP) is added to develop a colorimetric reaction that is inversely proportional to the amount of analyte: Positive samples will show absence of color and negative samples will show full intensity of color.

Fig 3. Optimized icELISA inhibition standard curve.

The Y axis represents the ratio (%) of inhibition, inversely proportional to analyte concentration, where signal is maximal when no analyte is present or bound (B0) and reduced when increasing concentrations of analyte are bound to the BSA linked to POA (PLB) at the bottom of the well (B). Assays performed in triplicate. Data are presented as mean absorbances, and error bars represent SD. A 5 parameter logistic asymmetrical sigmoid curve was fitted with R2 = 0.9995. The X axis is displayed as log2 transformed.

Fig 4. Reproducibility.

Multiple inhibition curves showed inter-assay and inter-aliquot consistency when POA alone in liquid phase (no culture medium) was used. Inset: Representative bar chart showing that post immunization serum was highly reactive versus pre immunization against PLB, in a consistent manner, indicating a strong response (ratio = 1:49).

Fig 5. icELISA of POA in culture medium.

7H9-OADC-PANTA medium was used as reaction medium, where the orange line depicts raw ODs and the blue line represents B/B0 ratios (similar to Fig 4). The dotted blue line marks the B/B0 ratio of 1.