4.4. Preparation of Polyclonal Antibody against Ustiloxins

In the initial immunization, 2 mg USTG-BSA conjugate was dissolved in a sterilized 0.5 mL NaCl solution (0.9%) and then emulsified with an equal volume of FCA. Three 6-week-old female BALB/c mice were immunized by multiple-point subcutaneous injection with the final water-in-oil emulsion described above. Booster injections were performed with an equal volume of the FIA-emulsified antigen three times at 3-week intervals. To investigate the immune response to immunogen, the antisera were collected from the tails of the four mice at day 9 post immunization and assayed with ustiloxin G-OVA by indirect noncompetitive ELISA.

To examine the sensitivity and specificity of antibodies, the inhibition experiments of mycotoxin AFB1; DON; ZEA; OTA; and UST-A, B, and G were conducted using a traditional indirect competitive ELISA, and their 50% inhibition concentrations (IC₅₀) were calculated by the classic four-parameter equation [27]. The specificity was usually expressed as cross-reactivity, which was calculated according to the following formula.

Briefly, the procedures of the traditional indirect competitive ELISA were as follows: (1) The plates were coated with 100 mL/well USTG-OVA at an appropriate concentration in 0.05 M PBS (pH 7.4) and stood for 2 h at 37 °C (2). After washing three times with 300 mL 0.05% PBST, 200 mL of 5% OVA in the PBST solution was added to each well and incubated for 1 h at 37 °C. (3) After another three washes, the 100 mL/well generic polyclonal antibody against ustiloxins at an appropriate solution was added into each well of the plates; (4) after 1-h incubation at 37 °C, the plates were rewashed, IgG-HRP (diluted at 1/8000 in the PBST, 100 mL/well) was added, and then the plates were incubated for 45 min at 37 °C; (5). After six washes, the color was developed by adding 100 mL freshly prepared substrate solution (containing 9.5 mL phosphate-citrate buffer (pH 5.0) and 0.5 mL TMB (2 mg/mL) dissolved in ethanol), and 32 mL urea-hydrogen peroxide (3%, w/v)), and the mixture was incubated for 15 min at 37 °C in the dark; (6) the 50 mL of the stopping coloring solution (H₂SO₄) was added to each well, and the absorbance at 450 nm was measured with a microplate reader.