Brucella IgG/IgM immunoassay was performed using a home-made kit according Sippel J.E., El-Marry N.A., Farid Z. (1982) Diagnosis of human brucellosis with ELISA. Lancet, 3, 19, modified by myself. In brief, dispensed 200 µl of suspension Brucella antigen (Sclavo) diluted 1:10 in PBS (dissolve in dH2O, 8.0 g/L NaCl, 0.2 g/L KCl, 1.15 g/L Na2HPO4, 0.2 g/L KH2PO4 obtaining a solution having pH 7.4) in wells of HB-MTP (BioHit) and incubate at 4 °C for 48 hours. After one wash with washing solution (PBS+0.05% Tween-20), dispense 200 µl of blocking solution (2.5% BSA in washing solution or blocked for 90 min with PBS containing 0.1% [w/v] casein, 0.5 M NaCI and 0.1% Tween 20) and incubate for 24 hours at 4 °C. After twice wash as above, the plate is dried ad store at 4 °C in sealer plastic bag. At the time of the assay, take the number strips useful for the duplicate samples/controls assay and blank and curry up to room temperature. Dispense 100 µl serum samples and controls (positive and negative sera), diluted 1:100 in diluent assay (1% BSA in washing solution) in respectively wells, sealer the wells whit adhesive plastic to avoid evaporation, and incubate for 1 hour at room temperature. Wash thrice the IgG wells with physiologic saline, do not to use washing solution, and for IgM wells, once with RF neutralization buffer (citrate buffer 0.1 M at pH 4.8) and twice with physiologic saline. Dispense 100 µl of stable solution of goat anti-human IgG or IgM phosphatase alkaline conjugate (dilute 1:100 goat anti-human IgG or IgM alkaline phosphatase conjugate in 1% BSA in PBS containing 1% NaN3. Store in transparent glass bottle at 4 °C, resulting stable 8 months) diluted, just before use, 1:5 in 1% BSA in PBS and incubate for 1 hour at room temperature or for 30 minutes at 37 °C in a moist chamber. Repeat step wash with only physiologic saline and add 100 µl of pNPP substrate 10 mg in 10 ml DEA buffer (dilute in 300 ml dH2O, 2.9 ml [30 mM] of DEA, adjust at pH 9.8 with 1 M HCl at 25 °C. Dissolve 0.514 g [5.4 mM] MgCl2 anhydrous and 17 mg (0.124 mM) ZnCl2 and mix for 15 min. Check again pH, and add 0.22 ml of Tween-20, continue to mix avoid foam, not less than 30 mins. And add 15.2 ml (0.053%) Proclin 300 and fill to 1 liter with dH2O, mixing for further 30 mins. Aliquote in opaque plastic bottle and store at 4 °C resulting stable for several months) and incubate for 30 min. at room temperature protect from light. Stop reaction dispensing 50 µl of 3 N NaOH. Mix and read the Abs at 405 nm. Under the described conditions, is considered positive values when Abs is > to 0.500 for IgG and > 0.200 for IgM
