Tmb hrp substrate

The blood were taken from retro-orbital plexus on days 7, 28, 42, and 65 for therapeutic treatment group and days 7, 27, 51, and 65 for preventive treatment group, centrifuged at 3,500 g for 10 min at 4°C, and the serum was collected and frozen at −80°C. Levels of serum Tm-specific IgE, IgG2a, and IgG1 were measured by ELISA as previously described with some modifications (31 (link)). In brief, serum samples were 1/20 diluted, and the secondary antibodies used in the ELISA tests were HRP-conjugated rat anti-mouse IgE, IgG2a, or IgG1 (Southern Biotechnology Associates, Birmingham, AL, USA). All the secondary antibodies were 1/6,000 diluted. After the HRP substrate TMB (eBioscience, San Diego, CA, USA) added, the absorbance was determined at 450 nm. Results were expressed as optical densities at 490 nm (OD490). Serum histamine was measured in 1/100 diluted serum samples with a commercial kit (Baomanbio, Shanghai, China) according to the manufacturer’s instructions, with a detection limit of 0.1 ng/ml.

L. donovani promastigote lysate was prepared by lysing cells in 1% NP-40 in PBS (Phosphate-buffered saline) with proteinase inhibitors at the concentration of 4 × 10⁸ cells/ml. After centrifugation at 4 °C at high speed for 15 min, the supernatant was stored at -20 °C until use.
The ELISA plate (Immulon cat#: 62402–972 from VWR) was coated with 50 μl (per well) cell lysate diluted in carbonate/bicarbonate buffer (1.89 g NaHCO₃ and 0.954 g Na₂CO₃ in 500 ml H₂O, pH 9.6) and incubated at 4 °C in a moist chamber overnight. The plate was washed 2 times with 200 μl wash buffer (0.05% Tween 20 in PBS) then blocked with 200 μl/well blocking buffer (5% nonfat dry milk in 0.1% PBS/T) for 1 h at 37 °C (covered with an adhesive plastic). The plate was washed 2 times with wash buffer, and 100 μl rabbit antiserum in 1:2000 dilution in blocking buffer was added to each well for 1.5 h at 37 °C or overnight at 4 °C. The plate was washed 3 times with wash buffer and 100 μl of horseradish peroxidase (HRP)-linked anti-rabbit antibody (ECL NA934V 1:5000 in blocking buffer) was added into each well for 1 h at 37 °C. The plate was washed 4 times with wash buffer followed by adding 50 μl/well of the HRP substrate TMB (eBioscience). After 10 min incubation at room temperature, the reactions were stopped with 25 μl of 1 N H₂SO₄. The color change in wells was read at 450 nm absorbance.