Peroxidase conjugated anti human igg antibody

Specific anti-saliva IgG antibodies were measured by ELISA. The wells (NUNC, Maxisorp Roskilde, Denmark) were coated with SGE (0.5 glands/well) in 0.1 M carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C. After three washes with PBS-0.05% Tween, free binding sites in the plates were blocked for 1 hour at 37°C with PBS containing 0.1% Tween 20 and 0.5% gelatine (PBS-T-G). Human sera were diluted (1:200) in PBS-T-G and incubated for 2 hours at 37°C. After washing, wells were incubated with peroxidase-conjugated anti-human IgG antibodies (Sigma) at a 1:5000 dilution in PBS-T-G, for 1 hour at 37°C. Tetramethylbenzidine (TMB) substrate (Sigma) was added for 10 min at room temperature to visualize antibody-antigen complexes. The absorbance was measured using an automated ELISA reader (Thermo Labsystems Multiskan Ex) at 450 nm to 620 nm wavelengths. All sera were tested in duplicate and the mean value was recorded. Results were expressed as relative OD (ROD) defined as the ratio of sample OD/mean OD of sera from 20 negative controls (+ two standard deviations). ROD superior or equal to 2 was considered positive. Negative sera were obtained from healthy controls living outside Tunisia in sand fly-free regions.

Human IgG antibodies against Lsa46 and Lsa77 were evaluated by ELISA. Serum samples of negative and positive MAT from confirmed leptospirosis patients and of febrile unrelated diseases, were diluted 1:100 and evaluated for total IgG using peroxidase-conjugated anti-human IgG antibodies (1:3,000, Sigma, USA). Commercial healthy human sera were used as control, and cutoff values were set at three standard deviations above the mean OD₄₉₂ of sera from control (healthy human sera).

ELISA plates (ThermoFisher Scientific) were coated with SGH (28 ng of proteins per well; corresponding to 0.2 gland per well) or recombinant proteins (exact amounts specified in S1 Table) diluted in 20mM carbonate-bicarbonate buffer (pH 9.5) at 4°C overnight. After washing with phosphate-buffered saline with 0.05% Tween 20 (PBS-Tw), plates were blocked with 6% non-fat dried milk (Bio-Rad) diluted in PBS-Tween and incubated for one hour at 37°C. After another washing step, plates were incubated with sera diluted in 2% non-fat dried milk for 1.5 hour at 37°C (sera dilutions specified in S1 Table). Plates were washed and incubated for 45 minutes at 37°C with peroxidase-conjugated Anti-Human IgG antibody (Sigma-Aldrich) diluted 1:1000 in PBS-Tween. The chromogenic reaction was developed for six minutes in McIlwain phosphate-citrate buffer (pH 5.5) with OPD (orthophenylendiamine, Sigma-Aldrich) and hydrogen peroxide in dark. The reaction was stopped by adding 10% sulfuric acid and measured at 492 nm using a Tecan Infinite M200 microplate reader (Schoeller). In each step, 100 μl of solution was used and each sample was tested in replicates.