3 3 5 5 tetramethylbenzidine (tmb)

See Key Resources Table for information on the sources of key reagents used in this study.
p-nitrophenyl phosphate (pNPP) and goat anti-human IgG–HRP antibody were obtained from Sigma-Aldrich, Dorset, UK. The semisynthetic macromolecular conjugate of unfractionated heparin and a human serum albumin, APAC, was from Aplagon Oy, Helsinki, Finland. Blocking medium (2.5% horse serum) and 3,3'-diaminobenzidine tetrahydrochloride (DAB) peroxidase substrate for immunohistochemistry were purchased from Vector Laboratories, Peterborough, UK and 3,3,5,5 tetramethylbenzidine (TMB) was from BD Biosciences, Wokingham, UK. Polyclonal phospho-specific G6b-B antibodies were generated by Biogenes, Berlin, Germany. Phalloidin-Alexa 488 was from Invitrogen Life Technologies, Paisley, UK. All other antibodies and chemicals were either purchased or generated as previously described (Mazharian et al., 2012 (link)).

Ag85A (catalog number NR-14871; BEI Resources), Ag85B (our purified recombinant), or Ag85C (catalog number NR-14858; BEI Resources) was used to coat wells at 0.5 µg/well in phosphate-buffered saline (PBS) and incubated overnight at 4°C. The plates were washed 3 times with PBS (pH 7.4) with 0.05% Tween 20 and blocked with PBS containing 1.0% BSA for 1 h. A starting concentration of 5 µg/ml of mAb 710, 711, or 712 was serially diluted, and 200 µl/well was incubated at room temperature for 2 h. Plates were washed 5 times and incubated with goat anti-mouse IgG-HRP (MP Biomedicals) for 1 h at room temperature. Plates were washed 7 times and developed with the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate according to the manufacturer’s instructions (BD). The reaction was stopped with 2 M sulfuric acid, and the absorbance was read at 450 nm with a Synergy H1 microplate reader (BioTek).

Nunc MaxiSorp™ 96-well microplates were coated with 100 ng/well of recombinant Receptor Binding Domain of the spike1 protein (RBD/S1 protein) (IP Tunis, Tunis, Tunisia) produced in SF9 cells [18 (link)] and incubated at 4 °C overnight. Plates were washed three times with PBS 1% Tween-20, then blocked with 5% skim milk in PBS-T for 1 h at 37 °C. After discarding the blocking buffer, the plates were washed three times with PBS, then 50 µL of each serum sample diluted at 1:400 in PBS-T was added in the wells and incubated for 2 h at 37 °C. After six washings with PBS-T, plates were incubated with 100 µL of 1:8000 diluted peroxidase-conjugated anti-human goat IgG for 1 h at 37 °C. After six washes, plates were revealed by adding 50 µL of horseradish peroxidase (HRP) chromogenic substrate [3,3′,5,5′-tetramethylbenzidine (TMB) (BD Biosciences, Franklin Lakes, NJ, USA, 555214)]. After 30 min, the reaction was stopped by adding 50 µL of sulfuric acid (2 N). The absorbance was monitored at 450/630 nm using a Multiskan™ FC Microplate Photometer. The cutoff value was estimated at 0.34 OD630, as the average OD of 50 pre-COVID human sera samples provided by the Institut Pasteur de Dakar plus 3 standard deviations (SDs). For each ELISA plate, positive and negative controls were used to validate the test. All serum samples were tested at least twice.

One milliliter of heparinized whole blood was transferred to a “Nil” tube (containing saline) and a “TB Antigen” tube (coated with peptides simulating ESAT-6, CFP-10, and TB 7.7) of the QFT system (Qiagen, Hilden, Germany). In addition, as a measure of cell viability, 1 ml blood was incubated with phytohemagglutinin (PHA) (Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS) at a final concentration of 10 µg/ml. The tubes were shaken according to the manufacturer’s instructions and incubated for 20–24 h at 37°C. Hereafter, the tubes were centrifuged at 1,600 × g for 10 min and plasma was harvested and stored at −80°C. Plasma samples were assayed in duplicate using a commercial bovine IFN-γ ELISA cross-reactive with IFN-γ of sheep and horses (Kit 3115-1H-20; Mabtech AB, Nacka Strand, Sweden) that has previously been shown to detect recombinant rhinoceros IFN-γ (data not shown). Reactions were visualized using 3,3′,5,5′-tetramethylbenzidine (TMB) (BD Biosciences, NJ, USA) as a color substrate. The IFN-γ concentration in each sample was measured as the OD of each well, at a wavelength of 450 nm, using a Labtech LT-4000 microplate reader (Lasec, Cape Town, South Africa). The M. bovis-specific release of IFN-γ was calculated as the mean OD derived for plasma harvested from the TB Ag tube minus the mean OD derived for plasma harvested from the Nil tube.

An indirect ELISA was used to detect GAA specific antibodies. Plates were coated with rGAA (Myozyme, Genzyme Corporation) overnight, rinsed, and blocked with 5% milk in PBS. Samples in serial dilutions, controls, and blanks were added and incubated for 2 h at 37 °C. HRP conjugated donkey anti-rat IgG (1:5000, r712–035-150; Jackson Immuno Research) followed by Streptavidin/HRP (1:1000, P 0397; DakoCytomation) was added and 3,3′,5,5′- Tetramethylbenzidine (TMB, BD Biosciences) was used as substrate. Reactions were stopped with 2 N H2SO4 and reading was determined at 450 nm. Positivity was determined with control sera collected prior to the injections using a cut-off value of 0.200 OD.

Microtiter 96-well plates (Nunc, Weisbaden, Germany) were coated overnight at 4°C with 100 μl of different concentrations (0 to 4 μg/well) of recombinant human LDH-A, recombinant human LDH-B, or recombinant Spn PspA in PBS. Plates were washed, blocked with 200 μl 5% bovine serum albumin (BSA) in PBS for 2 h, washed three times by T-PBS (PBS with 0.5% Tween 20), and then incubated with 2 μg/well of PspA_WU2 or A549 lysate at 37°C for 1 h. The plate was washed three times with T-PBS. Finally, the plate was incubated at room temperature for 1 h with mouse monoclonal anti-PspA (1b2.21) (60 (link)) or rabbit monoclonal anti-LDH (ab130923) antibody (1:1,000) in PBS with 5% BSA, washed three times with T-PBS, incubated for 1 h with 1:10,000 alkaline phosphatase-conjugated donkey anti-rabbit immunoglobulin antiserum IgG(H+L) (Southern Biotechnology Associates, Birmingham, AL), washed, and developed with p-nitrophenyl phosphate (Sigma) or 3,3′,5,5′ tetramethylbenzidine (TMB; BD Biosciences); the absorbance was read at 405 nm or 450 nm.

MaxiSorp plates were coated with Hcp and blocked with 5% skim milk in PBS-T. Patients' and controls' sera were added at a dilution of 1:100. Wells were incubated with goat anti-human antibody conjugated with HRP at a 1:5000 dilution and developed with 3,3′,5,5′ tetramethylbenzidine (TMB) (BD Biosciences). The reaction was stopped with H₂SO₄ and plates were read at 450 nm. A standard curve was generated by coating human IgG or IgM in a 10-fold dilution series (10 pg/mL – 100 μg/mL). Patients' sera were from culture-confirmed melioidosis patients presenting at Sappasithiprasong Hospital47 (link). The 20 patients were randomly selected from the patients recruited into the study. Control sera were from healthy consenting donors (age range 25–50).