In fact, an increasing inhibition of IgGMS-M1 binding from 40% up to 80% on coated GlnBP-AFM1 inside the wells was obtained (Figure ?Figure33). Open in a separate window Figure 3 Indirect competitive ELISA in the presence of an increasing concentration of AFM1 from 0.125 Imexon ppt up to 8 ppt, during the preincubation time of IgGMS-M1 purified on EAH-AFM1 resin. 3.3. 50 mM Tris/HCl buffer, pH 7.4. The Imexon sample was recovered and stored at 4 C. Different dilutions 1:100 (2 L of conjugate and 198 L of sample for each assay) were used for each experiment, and the experiments were performed in triplicate. 2.9. Production of the Aflatoxin Strip The purified conjugate IgGMS-M1-INV was tested on nitrocellulose strips previously incubated with different protein solutions. The strips (1.0 x 3.0 cm2) were placed in an Eppendorf tube and incubated with 1.0 mL of different protein solutions. For this purpose, 150 g of OVA and 150 g of GlnBP were used as negative controls. This experiment was performed to verify the ability of the conjugates to detect a commercial protein conjugated to various aflatoxins used in the competitive ELISA kit. A solution of peroxidase-conjugated aflatoxin (50 L, aflatoxin-HRP, 6 mg/mL) was diluted in 1.0 mL of TBS (20 mM Tris/HCl, 130 mM NaCl) before incubating with the nitrocellulose strip for 1 h at room temperature under mild stirring. Subsequently, the nitrocellulose strips were incubated with 1 mL of 0.5% OVA TBS-T (0.05% Tween-20) for 1 h at room temperature under mild stirring. Three washing steps were performed in 1.0 mL of TBS-T, and the strips were incubated overnight at 4 C with a dilution of IgGMS-M1-INV. The IgGMS-M1-INV conjugates were used at a dilution of (1:100 of 0.5 % OVA in TBS-T). Three washing steps were performed (10 min with TBS-T). The strips were incubated at room temperature with 500 L of a buffer solution containing an excess of substrate (2:3 of 100 mM acetate buffer, pH 4.6, and 1:3 of a solution at 10% w/v sucrose) to verify the invertase activity. After performing various incubations at varying durations, withdrawals of 33 L and final 100 L were tested with the d-glucose kit (Megazyme) for the detection of glucose produced by the immunoconjugate IgGMS-M1-INV. To perform a test in real matrices, the conjugate (IgGMS-M1-INV) was added to a solution of milk and methanol for extraction/dilution of AFM1. The assay was performed by diluting the IgGMS-M1-INV conjugate in a solution of whole milk at 33% in 0.25% OVA TBS-T; in all samples, 10% Argireline Acetate of methanol with or without AFM1 dilution was added. All experiments were performed three times. 3.?Results and Discussion 3.1. Synthesis of the Aflatoxin M1 Derivative and Aflatoxin M1-GlnBP Conjugate AFM1 (Figure ?Figure11A) lacks anchoring sites for conjugation with biomolecules. Consequently, a short linker arm was introduced by condensation on the carbonyl to form an oxime.11 The obtained AFM1-oxime (Figure ?Figure11B) derivative was conjugated through the well-known EDC conjugation procedure at two different protein carriers. BSA-AFM1 conjugate was used to produce Imexon polyclonal antibodies against AFM1.12,13 The total IgGs obtained were purified (Figure ?Figure22A) and tested for their ability to bind with AFM1. For this purpose, to avoid carrier interference, the AFM1 derivative was conjugated to GlnBP isolated from em Escherichia coli /em .9 Open in a separate window Figure 1 Aflatoxin M1 structure and synthesis of the AFM1 derivate used in the conjugation reaction with GlnBP. Open in a separate window Figure 2 New SDS-PAGE of anti-AFM1 purified antibodies (IgGMS-M1) on EAH-AFM1 resin (A). ELISA test performed against different antigens (BSA/GlnBP/GlnBP-AFM1) with different collected samples during immunoaffinity purification (B) and western blotting (C). In Figure ?Figure22B, the results of the ELISA tests are reported. The purified monospecific IgG anti-AFM1 (IgGMS-M1) are able to recognize GlnBP-AFM1 and BSA (immunization carrier), but they.