Preparation of Artificial Antigen and Development of Indirect Competitive ELISA Based on Chicken IgY for the Detection of Acid Orange II in Food Samples

To evaluate the feasibility of specific egg yolk antibodies (IgY) technology for the detection of hazardous chemical residues in foodstuffs, IgY were generated to detect acid orange II (AO2) residues. Bovine serum albumin (BSA) was made to bind with AO2 by succinic anhydride (SA), and the conjugate was used to immunize the laying chickens. PEG-6000 precipitation was used to extract IgY antibodies. The titer of anti-AO2 IgY attained the peak (1:12,800) after fifth booster immunization. Checkerboard titration showed that 1:640 dilution of anti-AO2 IgY could approximately give an optical density (OD) 1.0 at 5 mu g/mL AO2-OVA coating concentration. The anti-AO2 IgY based indirect competitive ELISA (ic-ELISA) showed that the IC50 value of anti-AO2 IgY was 10.72 mu g/mL and regression curve equation was y =-24.41 x +75.11 (R (2) = 0.946). The ic-ELISA showed less cross-reactivity in range between 0.09 and 21.07 %. Recoveries from dried bean curd, sausage, and chilli powder samples were from 78.80 to 152.90 %, with relative standard deviation lower than 19.13 %. The study suggests that generated anti-AO2 IgY antibodies could be used in routine screening analysis of AO2 residues in food samples.