Extravidin alp

Microtiter plates were coated overnight at room temperature with a 1:1 mix of ESAT-6/CFP-10 (10 μg/ml each) prepared from M.tb H37Rv (BEI Resources). Native human IgG1 (2 μg/ml; AbD Serotec, UK) and PBS were included as positive and negative controls (respectively). After washing with PBS containing 0.05% Tween 20 (PBS/T) and blocking with casein, serum samples were tested in duplicate at 1:10 (diluted in casein). Casein was used for control wells. After incubation at room temperature, biotinylated secondary antibody (anti-IgG1; Life Technologies) at 1:1,000 dilution was added, followed by washing and the addition of Extravidin-ALP (1:5,000; Sigma Aldrich). pNPP substrate (Sigma Aldrich) was added and plates were read at 405 nm every 10 min using an ELx800 Microplate Reader with Gen5 software until the IgG1 isotype control reached an OD of 0.6 (previously determined from the gradient of a standard curve of native human IgG1 ranging from 0.1 to 100 μg/ml to avoid saturation). The mean OD of negative controls was subtracted from the mean OD of samples and readings with an OD > negative control plus three standard deviations (SD) were considered positive.

T cell memory responses from immunized splenocytes were quantified using enzyme-linked immunospot (ELISpot) assay. Multiscreen-IP filter plates (MSIPS4510, Merck) were coated with anti-mouse IFNγ antibody (5 μg/mL, 100 μL/well, clone AN18, MABTech, Nacka Strand, Sweden) or anti-mouse IL4 antibody (5 μg/mL, 100 μL/well, BVD4-1D11, BD Biosciences) overnight at 4 °C, washed 5 times with 200 μL/well sterile PBS and blocked with complete RPMI (150 μL/well) for a minimum of 1 h at 37 °C. Then, 15 μg/mL of MSP4/5 was used for recall with 5 × 10⁵ splenocytes. Medium alone was used as the negative control and 1 μg/mL Concavalin A as a positive control. IFNγ was labelled with biotin anti-mouse IFNγ antibody (1 μg/mL, 100 μL/well, R4-6A2-Biotin, MABTech) or biotin anti-mouse IL4 (1 μg/mL, 100 μL/well, BVD6-24G2, BD Biosciences) for 2 h at room temperature. This was then labelled with streptavidin-alkaline phosphatase (ALP) (1:1000, 100 μL/well, 3310-10, MABTech) or ExtrAvidin-ALP (1:3000, 100 μL/well, Sigma-Aldrich) for 1.5 h at room temperature. Spots were developed with an ALP conjugate substrate kit (Bio-Rad, Hercules, CA, USA) and imaged and counted with an ELISpot reader and software (AID, Strasburg, Germany).