Meng

Preparation of Monoclonal Antibody for Melamine and Development of an Indirect Competitive ELISA for Melamine Detection in Raw Milk, Milk Powder, and Animal Feeds

Melamine (MEL) has been involved in several food recalls after the discovery of severe kidney damages in children and pets poisoned by melamine-adulterated food. To detect MEL residue in foods and animal feeds, an indirect competitive ELISA (cELISA) method was developed in this study based on preparation of monoclonal antibodies (MAbs) to MEL. The immunogen was prepared by linking MEL hapten with carrier protein via carbodiimide method. The method is applicable in the range of 5.0-135.0 microg L(-1) MEL in buffer solution, with an IC(50) value of 22.6 +/- 1.9 microg L(-1). The MAbs showed high specificity with low cross-reactivity (< or =1%) toward cyanurate, ammelide, and ammeline. The method was utilized in the detection of MEL in raw milk, milk powder, and animal feeds, with detection limits of 0.1 mg L(-1) for milk, 0.2 mg kg(-1) for milk powder, and 0.5 mg L(-1) for feeds. The recovery ratio was 79-110% for all matrices. The intra-assay and interassay coefficients of variation were <12.0 and <13.0%, respectively. Finally, the application of the cELISA in quantity evaluation of MEL in various feeds from local markets was evaluated and discussed.

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.

Chemiluminescence enzyme immunoassay using magnetic nanoparticles for detection of neuron specific enolase in human serum

To detect a biomarker for small cell lung carcinoma, neuron specific enolase (NSE), a sensitive and specific chemiluminescence enzyme immunoassay was developed. Fluorescein isothiocyanate (FITC) labeled NSE capture antibody connected with NSE and alkaline phosphatase (ALP) labeled NSE detection antibody in a sandwich-type detection manner. This immune complex was further reacted with anti-FITC coated magnetic beads. In a magnetic field, the complex was enriched, and the sensitivity was thus enhanced. The limit of detection (LOD) of this method was <0.2 ng mL(-1). The proposed immunoassay was highly selective, and not interfered by hook effect. The recovery was >83.0% and the coefficient of variation was <10.0%. Human sera from 120 patients were tested with the presented and traditional chemiluminescence enzyme immunoassay. An excellent linear relationship was obtained between two techniques. Overall, this immunoassay offers a promising alternative for NSE detection than traditional clinical examinations.

Preparation of anti-Sudan red monoclonal antibody and development of an indirect competitive enzyme-linked immunosorbent assay for detection of Sudan red in chilli jam and chilli oil

Sudan dyes are banned to be used in food additives because of the carcinogenicity of their metabolites. A rapid and sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect the residues of Sudan dyes. Novel immunogen and coating antigen were synthesized via glutaraldehyde linking. The hapten-bovine serum albumin (BSA) was applied as immunogen and the hapten-ovalbumin (OVA) was served as coating antigen. The monoclonal antibody obtained showed high sensitivity to Sudan I with an IC(50) value of 1.7 μg L(-1) in buffer and was suitable to detect the residues of Sudan red in food products. The specificity of the assay was studied by measuring cross-reactivity of the antibody with the structurally related compounds of Sudan II (<1%), Sudan IV (<1%) and para red (120%). Chilli jam and chilli oil samples spiked with Sudan dyes were analyzed by the method. The detection limit (LOD) of the ELISA method applied in chilli jam and chilli oil was 9.0 μg L(-1) and 19.6 μg L(-1), respectively. The recovery rates of Sudan-I in chilli oil and chilli jam were in the range of 80%-110% with coefficients of variation <25%. The intra-assay variation and inter-assay variation in buffer were both <9%.

Determination of sodium benzoate in food products by fluorescence polarization immunoassay.

A rapid and sensitive fluorescence polarization immunoassay (FPIA), based on a polyclonal antibody, has been developed for the detection of sodium benzoate in spiked samples. The immunogen and fluorescein-labeled analyte conjugate were successfully synthesized, and the tracer was purified by TLC. Under the optimal assay conditions, the FPIA shows a detection range of 0.3-20.0 μg mL(-1) for sodium benzoate with a detection limit of 0.26 μg mL(-1) in the borate buffer. In addition, the IC₅₀ value was 2.48 μg mL(-1), and the cross-reactivity of the antibodies with ten structurally and functionally related analogs were detected respectively. Four kinds of food samples (energy drink, candy, ice sucker, RIO(TM) cocktail) were selected to evaluate the application of FPIA in real systems. The recoveries were 96.68-106.55% in energy drink; 95.78-100.80% in candy, 86.97-102.70% in ice sucker, and 103.58-109.87% in benzoate contained sample RIO(TM) cocktail, and coefficients of variation of this method were all lower than 11.25%. Comparing with the detection results of HPLC, the developed FPIA has comparative performance in the real sample determination. The results suggest that the FPIA developed in this study is a rapid, convenient and simple method, which is suitable to be used as a screening tool for homogeneous detection of sodium benzoate in food products.

Preparation of a monoclonal antibody and development of an indirect competitive ELISA for the detection of chlorpromazine residue in chicken and swine liver

BACKGROUND Chlorpromazine is a typical antipsychotic drug used to make food-producing animals calm and promote growth as feed additives. Accumulation of chlorpromazine in animal bodies would cause side effects in the circulatory and nervous systems, and have adverse effects on blood cells, the skin and the eye. To detect the chlorpromazine residue in food producing animals, an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed based on preparation of an anti-chlorpromazine monoclonal antibody. RESULTS The antibody generated from immunogen of cationic bovine serum albumin (cBSA) coupled with chlorpromazine showed high sensitivity toward chlorpromazine with an IC(50) value of 0.73 ppb. The ELISA method was applied to detect swine liver and chicken samples spiked by chlorpromazine and satisfactory results were obtained. The recovery rates in chicken and swine liver were in the range of 88-95% and 86-95%, respectively; the intra-assay coefficients of variation were both < 15.3% and < 13.5%, respectively. CONCLUSION An indirect competitive ELISA method based on a monoclonal antibody towards chlorpromazine with excellent sensitivity and specificity has been successfully developed. The immunoassay provided in this study was a hopeful alternative to chromatography spectrometry for regulatory analysis of chlorpromazine residue in food-producing animals.

Indirect Competitive Immunoassay for Detection of Vitamin B2 in Foods and Pharmaceuticals

An indirect immunoassay for the determination of vitamin B2 in food samples and vitamin tablets was developed. A carbodiimide-modified active ester method was used to synthesize the immunogen for vitamin B2. The coupling ratio of vitamin B2 to carrier protein in immunogen was 19.98:1. The titer of the polyclonal antibody was 1:64000, and the antibody showed high specificity in the presence of vitamin B2 photolytic products and other B group vitamins. The immunoassay showed detection limits (LODs) of 1.07 ng/mL in PBS, 24.6 ng/g in vitamin drink, and 0.50 mg/kg in milk powder. Recovery was 99.58-110.91% in milk powder and 70.20-100.5% in vitamin drink. Vitamin B2 samples were analyzed by high-pressure liquid chromatography (HPLC) and the immunoassay, and results showed good agreement. Finally, this method was applied to detect vitamin B2 in commercial milk powder and vitamin tablets, and the detected amount correlated well with the labeled amount.

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.

Preparation of anti-melamine antibody and development of an indirect chemiluminescent competitive ELISA for melamine detection in milk

An indirect chemiluminescent competitive ELISA (CL-ELISA) method was developed to detect residues of melamine in milk. A high-quality polyclonal antibody towards melamine cyanurate (MC) was prepared based on synthesis of a novel immunogen. The method is applicable over the range of 0.5–7.0 µg.mL−1 of MC, with an IC50 value of 1.7 µg.mL−1. The developed method was used in the detection of melamine residue in milk with the detection limit of 1 ng.mL−1. There was no cross-reactivity with commonly used veterinary drugs. The CL-ELISA method developed provides an alternative to chromatography spectrometry for regulatory analysis of melamine in milk and could be promisingly used to improve the sensitivity of the available ELISA test kits.