Huyin Xue

Establishment of magnetic beads-based enzyme immunoassay for detection of chloramphenicol in milk

In this research, magnetic beads-based enzyme immunoassays were investigated for rapid analysis of chloramphenicol (CAP) in milk. To improve sensitivity of CAP determination, two kinds of immunomagnetic separation methods were designed and compared. Magnetic polystyrene microspheres were conjugated with anti-CAP antibody (Method I) or goat-anti-mouse IgG (Method II). The whole determination could be finished in 1.25 h. Both methods showed high sensitivity to CAP in buffer, and obtained an IC(50) value of 0.05 ng mL(-1) for Method I and 0.4 ng mL(-1) for Method II. The methods showed high specificity, only showing a little cross-reaction towards CAP succinate. The two methods were applied to detect CAP in milk. The recovery rates were 80-106% and the coefficients of variation (CVs) were 4.7-15%. The immunomagnetic assay showed promising potential in rapid screening field for CAP analysis. Between the two methods, Method I is more sensitive, and Method II is more suitable for producing a general assay by changing a primary antibody for another analyte.

Development of a polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of Sunset Yellow FCF in food samples.

Sunset Yellow FCF is widely used as food additives to make foods more attractive. Due to its abuse and potential risk to human health, Sunset Yellow FCF is precisely limited to use in food. To monitor the illegal use of Sunset Yellow FCF, a polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) with satisfactory sensitivity and specificity was developed. A carboxyl group was introduced to Sunset Yellow FCF, then the modified hapten was coupled with carrier proteins to synthesize the immunogen and coating antigen. The IC(50) value of 0.52 ng mL(-1) and detection limit of 25 pg mL(-1) (in buffer) were achieved by this method. The cross-reactivity values of the antibodies with six structurally related colorants were less than 1.5%, indicating the high selectivity. Three kinds of food samples (beverage, dried beancurd, braised pork) and serum were chosen to evaluate the application of the immunoassay in real systems. The limits of detection (LOD) in the above three food samples were 0.12, 0.04 and 1.11, respectively (mean+3SD). The recovery (94%-106%), intra-assay (<5%) and inter-assay (<12%) coefficients of variation in foods and serum samples were also acceptable. The results suggest that this ELISA method is a specific, sensitive and simple method for the determination of Sunset Yellow FCF additives.

Determination of folic acid in milk, milk powder and energy drink by an indirect immunoassay

BACKGROUND Folic acid (FA) is essential for healthy people (reference daily intake 400 µg day⁻¹) and pregnant women (600 µg day⁻¹). Insufficient intake of FA will increase the risk of neural tube defects in newborns. In this study an indirect enzyme-linked immunosorbent assay was developed for rapid and convenient detection of FA in vitamin-fortified foods. RESULTS A carbodiimide-modified active ester method was used to synthesise the immunogen (FA-bovine serum albumin (BSA) conjugate) to raise polyclonal antibodies for FA. The coupling ratio of FA with BSA was determined to be 14:1 (molar ratio). The detection limit of the immunoassay was 3.0 ng mL⁻¹ in buffer, 3.52 ng mL⁻¹ in energy drink, 11.91 ng mL⁻¹ in milk and 16.50 ng mL⁻¹ in milk powder. Intra- and inter-assay variability ranged from 6.6 to 15.1%. Analytical recoveries of FA-spiked samples were 88.3-108.9%. CONCLUSION The immunoassay developed in this study can be used as a simple, rapid and accurate method for fast semi-quantitative and quantitative on-site analysis of FA in food products.

Application of an enzyme immunoassay for the quantitative determination of azo dye (Orange II) in food products

This paper reports the preparation of polyclonal antibodies against a synthetic azo dye, Orange II, and the development of an indirect ELISA to detect Orange II in foods. The sulfonic group of Orange II was modified and linked with carrier protein to synthesise an artificial antigen. Based on the checkerboard titration, the method showed excellent sensitivity (IC50 = 0.61  ng g−1) to Orange II in the linear range of 0.05–10 ng g−1. The antibody had little cross-reactivity with Chromotrope FB, Gardenia Yellow, Ponceau 4R, Sunset Yellow and Sudan dyes. The ELISA had limits of detection (LOD) of 0.22, 0.97 and 0.74 ng g−1 in chilli powder, chilli oil and braised pork, respectively. The limits of quantification (LOQ) of the assay were 0.91 ng g−1 in chilli powder, 1.48 ng g−1 in chilli oil and 1.10 ng g−1 in braised pork. For food products fortified with 1–10 ng g−1 Orange II, the inter- and intra-assay variations were all less than 24.0% and 18.0%, respectively. Therefore, the proposed test could be used as a rapid screening method for Orange II detection in food samples.

Development of a Fluorescence Polarization Immunoassay for Rapid Determination of Sarafloxacin in Milk and Pig Urine: Development of a Fluorescence Polarization Immunoassay for Rapid Determination of Sarafloxacin in Milk and Pig Urine

To develop a rapid and sensitive fluorescence polarization immunoassay(FPIA) for the determination of sarafloxacin(SAR),fluorescein-labelled tracer(SAR-FITC) was synthesized and purified by TLC.The reaction time,tracer and polyclonal antibody concentration were optimized,and the FPIA method showed a dynamic range from 5.7 to 327.6 μg/L with IC50 value of 43.23 μg/L to SAR in buffer.The specificity of the FPIA for SAR was investigated using other 4 quinolones and the cross-reactivity for ciprofloxacin,enrofloxacin,gatifloxacin,ofloxacin were 3.3%,1.8%,1.7%,0.7%,respectively.The recoveries in milk samples ranged from 71%-94%,and those of pig urine samples were in the range of 74%-102%.The FPIA developed in this study is a rapid and convenient method,which is suitable to be used as a screening method to detect residues of sarafloxacin.