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.     

Development and application of one-step ELISA for the detection of neomycin in milk

A sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of neomycin (NEO) in milk was developed. Two conjugates were synthesized as the immunogen and coating antigen. Four BALB/c female mice were immunised and the antisera were screened by indirect competitive ELISA (icELISA). The icELISA was further optimised using different coating methods, pH values, ionic strengths of assay buffer and reaction times. After optimised, the indirect competitive ELISA (icELISA) could be finished in 85min with an IC₅₀ value of 0.74±0.05 ng/mL. One-step indirect competitive ELISA (icELISA) exhibited similar sensitivity with an IC₅₀ value of 0.73±0.03 ng/mL, which was sensitive enough for NEO detection and could be finished in 55 min. No cross-reactivity of the antibody was observed with other aminoglycosides based on icELISA, indicating that the antibody is highly specific for neomycin. Milk samples were further analyzed with recovery ranging from 85 to 110% at spiked level of 0.25–10 ng/mL, and the detection limit was 0.08 ng/mL.     

Development of an indirect competitive immunoassay for determination of L-hydroxyproline in milk

L-hydroxyproline can be used as a marker for hydrolysed leather protein. This is the first study reporting an indirect competitive enzyme-linked immunosorbent assay (ELISA) for determination of L-hydroxyproline in milk. A hapten for L-hydroxyproline was synthesised and used to produce a monoclonal antibody. The antibody was specific to the hapten and showed low crossreactivity to parent L-hydroxyproline. After evaluation of different coating antigen/antibody combinations, a heterologous ELISA was developed to determine L-hydroxyproline in milk. Milk samples were hydrolysed with concentrated sulfuric acid in microwave oven to release free L-hydroxyproline that was derivatised with p-hydroxybenzaldehyde. The p-hydroxyphenyl L-hydroxyproline was determined by the ELISA, and the limit of detection calculated as L-hydroxyproline was 0.04 µg/mL. The recoveries of L-hydroxyproline from fortified milk ranged from 88.6% to 102.5%, with coefficients of variation ranging from 4.1% to 9.7%. Therefore, ELISA could be used as a rapid method to monitor the presence of L-hydroxyproline in milk.     

Development of a monoclonal antibody-based sandwich ELISA for the detection of ovalbumin in foods

Hen egg is one of the most frequent causes of food allergy in infants and adults. Ovalbumin (OVA) has been identified as a major egg allergen. In order to detect OVA in foods, a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) based on two monoclonal antibodies (mAbs) was established. The 2 mAbs were selected out of 17 murine hybridomas secreting OVA-specific antibody. Using mAb17 as the capture antibody and mAb15 as the detection antibody, the detection limit of the ELISA method was 0.51 ng/mL, and the linear dynamic range was between 1.95 and 500 ng/mL. The recovery ranged from 85.6 to 115.2%, whereas the intra- and inter-assay coefficients of variation were less than 8.6 and 13.9%, respectively. Sample analysis verified that the produced anti-OVA mAb and the developed ELISA may provide a valuable tool for the sensitive determination of OVA in processed foods and for future studies on the mechanism of how OVA functions in anaphylaxis.     

Development of an indirect competitive ELISA for the detection of acenaphthene and pyrene

Polycyclic aromatic hydrocarbons (PAHs) have mutagenic and carcinogenic properties. Acenaphthene and pyrene were members of 16 PAHs which were listed as the priority pollutants in water environment by US Environmental Protection Agency. The reported instrumental methods and immunoassays did not meet the need for simple and sensitive detection of acenaphthene and pyrene. In this study, a monoclonal antiobody having high affinities with acenaphthene and pyrene was produced and an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed for sensitive detection of acenaphthene and pyrene in water sample. The linear range of the assay was between 3.53 and 41.98?ng?mL^?1. The sensitivity was 12.17?ng?mL^?1 which was more sensitive than those of reported ELISAs. The average recovery of acenaphthene and pyrene from three kinds of water samples was 99.08% and 98.45, respectively. The developed ELISA could be used for sensitive detection of acenaphthene and pyrene in water samples.     

Sandwich ELISA for detection of goats’ milk in ewes’ milk and cheese

A sandwich ELISA has been developed for the detection of defined amounts of goats’ milk (1–100%) in ewes’ milk and cheese. Polyclonal antibodies were raised in rabbits against goat caseins (GC). The resultant antibodies were affinity purified by immunoadsorption of the crude antiserum on to columns containing immobilized cow, ewe and goat caseins, followed by elution of the goats’ milk‐specific antibodies (anti‐GC) from the column containing the goat caseins. The anti‐GC antibodies bound to the wells of a microtiter plate were used to capture the goats’ caseins from milk or cheese mixtures. Further immunorecognition of the captured proteins was achieved with the same antibodies conjugated to biotin. ExtrAvidin‐peroxidase was used to detect biotinylated antibodies bound to their specific antigens. Subsequent enzymic conversion of the substrate gave clear absorbance differences when assaying mixtures of ewes’ milk and cheese containing variable amounts of goats’ milk.     

Heterologous immunoassay for screening macrolide antibiotics residues in milk based on the monoclonal antibody of tylosin

In this study, tylosin (TYL) was derivatized with 4-aminophenylacetic acid to synthesise a new hapten and the hapten was used to produce the monoclonal antibody. The obtained antibody simultaneously recognised TYL, tilmicosin, acetylisovaleryltylosin and the metabolite of TYL (desmycosin) with cross-reactivities of 100%, 62%, 97% and 93%, respectively. After evaluation of two coating antigens, a heterologous competitive indirect enzyme linked immunosorbent assay was developed to determine the four analytes in milk simultaneously. The limits of detection for the four analytes were in the range of 1.5–3.1 ng mL^?1. The recoveries from fortified milk were in the range of 76.3–97.4% with coefficients of variation of 5.3–15.7%.     

Development of an ELISA for detection of Sudan I in food samples using monoclonal antibody

A highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been developed using a new monoclonal antibody for detecting the food colourant Sudan I. The half-maximum inhibition concentrations (IC₅₀) and the limit of detection (calculated as IC₂₀) of ELISA for Sudan I were 2 and 0.01 ng mL^?1, respectively. The study showed little cross-activity with Sudan I structural analogues (below 0.01%). The average recoveries in intra- and interassays for Sudan I from fortified fresh tomato and chilli samples by ELISA were in ranges of 82–94% and 79–91%, respectively. The coefficients of variation of intra- and interassays were 6–8% and 6–10%, respectively. The IC₅₀ was approximately 2.0 ng mL^?1 after the Sudan I-Kit had been kept for 180 days.     

Detection of Penicillinase in Milk by Sandwich ELISA Based Polyclonal and Monoclonal Antibody

A sandwich ELISA has been developed using polyclonal and monoclonal antibody for the determination of penicillinase in milk. For this purpose, specific polyclonal and monoclonal antibodies against penicillinase were generated and characterized. Using penicillinase standards prepared from 1–128 ng/mL, the method indicated that the detection limit of the sandwich ELISA, as measured in an ELISA plate reader, was as low as 0.86 ng/mL of penicillinase. For determine the accuracy, raw milk containing 2, 8, 32, and 64 ng/mL of penicillinase were tested by sandwich ELISA. Recoveries were from 93–97.5%, and the coefficient of variation [CV (%)] were from 5.55–8.38%. For interassay reproducibility, recoveries were from 89.5–95.1%, the coefficient of variation [CV (%)] were from 5.26–9.58%. This sandwich ELISA provides a useful screening method for quantitative detection of penicillinase in milk.     

Use of monoclonal antibody in an ELISA test for the detection of antibodies to bovine leukaemia virus

A variant of the ELISA technique, involving a monoclonal anti-gp51 antibody yields a highly sensitive method for the detection of bovine leukaemia virus (BLV) antibodies. The gp51 antigen-coated microtitre plates are obtained by incubation of plastic-adsorbed monoclonal antibodies with a non-purified mixture of BLV antigens. Sera to be tested are incubated in the wells of the gp51-coated plates and bound antibodies are revealed by an enzyme-linked antibovine immunoglobulin reagent. This test is as sensitive as liquid phase radioimmunoassay using the same gp51 antigen and thus appears as a highly sensitive, practical, rapid and cheap method for the detection of BLV antibodies.     

Development of IC-ELISA and immunochromatographic strip assay for the detection of flunixin meglumine in milk

Flunixin meglumine (FM) is a novel nonsteroidal anti-inflammatory drug for animals, which has antipyretic, analgesic, and anti-inflammatory effects. The drug, which was originally used to relieve inflammation in horses, musculoskeletal disorders, and pain, has been approved for use in endotoxemia, infectious diseases in swine, etc. A sensitive anti-FM monoclonal antibody 2H4 was prepared and used to develop an indirect competitive enzyme-linked immunosorbent assay and immunochromatographic strip assay for the detection of FM in milk. The complete antigen and coating antigen were conjugated with bovine serum albumin and ovalbumin, respectively. The monoclonal antibody 2H4, with a half inhibition concentration of 0.29?ng/mL, had a limit of detection of 0.432?ng/mL and a linear range of detection of 0.08664–0.97226?ng/mL. A sensitive and convenient immunochromatographic strip assay was developed with an FM cutoff value of 0.29?ng/mL. The developed methods were suitable for the detection of FM in milk.     

A novel monoclonal antibodies-based sandwich ELISA for detection of serotype 4 fowl adenovirus

As a major causative agent for hepatitis-hydropericardium syndrome (HPS) in chickens, serotype 4 fowl adenovirus (FAdV-4) has caused huge economic losses in the poultry industry globally. However, there is no commercial diagnostic test for FAdV-4 antigens. To generate a rapid approach for specific detection of FAdV-4, a monoclonal antibodies (mAbs)-based sandwich ELISA was developed. In this ELISA, a purified mAb 4A3 and a HRP-labelled mAb 3C2 specific to the fiber-2 of FAdV-4 were used as the capture antibody and detection antibody respectively. Specificity assay revealed the ELISA only reacted with FAdV-4, not with other avian viruses tested. Sensitivity assay showed the limit of detection of the ELISA was 1000 TCID₅₀/ml and 12.5?ng/ml for the FAdV-4 and the purified GST-Fiber2 protein respectively. Moreover, the ELISA could be efficiently applied in detecting the FAdV-4 in tissue samples from a clinically-diseased chicken flock. All these data demonstrated that the ELISA developed here provides a promising tool for rapid and efficient diagnosis of clinical infection with FAdV-4.     

An improved indirect competitive Elisa for aflatoxin m1 in milk powders using novel monoclonal antibodies

Among three newly prepared monoclonal anti‐aflatoxin M₁ antibodies, named AM.1, AM.2 and AM.3, both AM.1 and AM.3 possessed high specificity and sensitivity towards aflatoxin M₁, while AM.2 exhibited lower specificity. Employing AM.3 and horseradish peroxidase‐labelled second antibody, an improved ELISA was developed with a detection limit of 1.0 pg ml^‐1 of aflatoxin M₁ in solution. The contents of aflatoxin M₁ in powdered milks suspended in water were assayed by the indirect ELISA. The detection limit was 5 pg g^‐1dry weight, and no clean‐up procedures were required. The reliability of the present ELISA with monoclonal antibody AM.3 was confirmed with reference powdered milks for aflatoxin M₁. A limited ELISA survey showed the presence of aflatoxin M₁ in commercial milk powders sampled in France, the US and Thailand at levels of 30–418 pg g^‐1, and it was confirmed by an improved HPLC analysis.     

一种简便快速筛选双位点单克隆抗体方法的建立

本文建立了一种无需对单克隆抗体(McAb)进行纯化和酶标记,利用市售酶标记抗体既可筛选双位点McAb的简易方法。其基本原理是,首先将待筛选McAb与酶标抗体制成McAb-酶标抗体复合物,再以此复合物作示踪剂进行夹心ELISA。若夹心孔OD492值高于第一、二阴性对照孔之和者, 提示所筛选McAb可能为双位点抗体。文中还对应用该方法时的注意事项进行了讨论。     

Development of an ELISA for nitrazepam based on a monoclonal antibody

We report the production of a specific monoclonal antibody against Nitrazepam (NZP). First, the hapten 7-aminonitrazepam (7-ANZP) was synthesized, then the hapten was coupled with bovine serum albumin and ovalbumin by the diazotization method, and used as immunogen and coating antigen. Factors affecting binding, ionic strength, pH, and methanol concentration in phosphate-buffered saline, were optimized. The monoclonal antibody had a satisfactory IC₅₀ of 0.2 ng/mL for NZP. The cross-reactivities with other analogs were all lower than 0.1% except for 23% with 7-NZP, which suggested that the enzyme-linked immunosorbent assay method possessed high specificity. The recoveries were in the range of 84–95%, indicating that the method could be applied for the detection of NZP in urine.     

Competitive direct ELISA based on a monoclonal antibody for detection of Ochratoxin A in dried fig samples

A monoclonal antibody (MAb) generated against Ochratoxin A (OTA) has been used in a competitive direct enzyme linked immunosorbent assay (cdELISA) for the detection of OTA in dried figs obtained from the Spanish retail market. Fifty per cent inhibition of the maximum binding was obtained with an OTA concentration of 2 ng/mL, and the detection limit for OTA in solution was 0.18 ng/mL, corresponding to 3.15 ng OTA per gram of sample. OTA was detected in 19 (54.3%) out of 35 samples of dried figs analysed, with concentrations that ranged from 3.15 to 277 ng/g. Five samples contained OTA concentrations above the tolerable level set by EC regulations for dried vine fruits (10 ng/g). The MAb-based cdELISA assay developed in this work could be effectively applied for OTA screening in dried figs.     

Inter-laboratory studies for the evaluation of ELISA kits for the detection of chloramphenicol residues in milk and muscle

The use of chloramphenicol in veterinary medicine was banned in the EU in 1994. As the Community Reference Laboratory for antibiotic residues in food of animal origin, one of our functions is to organize inter-laboratory studies. A first inter-laboratory study for the analysis of chloramphenicol (CAP) in milk by ELISA kits was organized in 2001 and a second one for the detection of CAP in pig muscle by ELISA kits in 2002. These studies were intended to allow participants to control their CAP ELISA methods when used routinely and also to compare the performances of various ELISA kits for the detection of chloramphenicol in milk (commercial or in-house kits). In 2001, 15 participants received ten randomly coded frozen milk samples (four blank samples and six spiked milk samples from 0.5 to 5.0 μg/l). In 2002, 20 participants received eight randomly coded frozen muscle samples (two blank samples and six incurred muscle samples from 2.1 to 6.5 μg/kg). They were asked to analyse each sample in triplicate with the ELISA kit of their choice. Different kits from different suppliers were used and compared in the two studies. The results of the two inter-laboratory studies on ELISA kits were satisfactory regarding qualitative results. The global rates of false compliant results of 2.2% for milk and 0.0% for pig muscle samples were lower than 5% whichever kit was used. The global rates of false non-compliant results (16.7% and 10% for milk and muscle respectively) were also satisfactory. The distribution of false non-compliant results depends on the kind of kit used and on the detection limit for milk as well as for muscle. Moreover the sample preparation was very important to avoid false non-compliant results. Finally, this study demonstrates that ELISA kits for chloramphenicol in milk and muscle globally show good repeatability and accuracy. So these kits could be considered as suitable tests for screening purposes.     

Inter-laboratory studies for the evaluation of ELISA kits for the detection of chloramphenicol residues in milk and muscle

The use of chloramphenicol in veterinary medicine was banned in the EU in 1994. As the Community Reference Laboratory for antibiotic residues in food of animal origin, one of our functions is to organize inter-laboratory studies. A first inter-laboratory study for the analysis of chloramphenicol (CAP) in milk by ELISA kits was organized in 2001 and a second one for the detection of CAP in pig muscle by ELISA kits in 2002. These studies were intended to allow participants to control their CAP ELISA methods when used routinely and also to compare the performances of various ELISA kits for the detection of chloramphenicol in milk (commercial or in-house kits). In 2001, 15 participants received ten randomly coded frozen milk samples (four blank samples and six spiked milk samples from 0.5 to 5.0 μg/l). In 2002, 20 participants received eight randomly coded frozen muscle samples (two blank samples and six incurred muscle samples from 2.1 to 6.5 μg/kg). They were asked to analyse each sample in triplicate with the ELISA kit of their choice. Different kits from different suppliers were used and compared in the two studies. The results of the two inter-laboratory studies on ELISA kits were satisfactory regarding qualitative results. The global rates of false compliant results of 2.2% for milk and 0.0% for pig muscle samples were lower than 5% whichever kit was used. The global rates of false non-compliant results (16.7% and 10% for milk and muscle respectively) were also satisfactory. The distribution of false non-compliant results depends on the kind of kit used and on the detection limit for milk as well as for muscle. Moreover the sample preparation was very important to avoid false non-compliant results. Finally, this study demonstrates that ELISA kits for chloramphenicol in milk and muscle globally show good repeatability and accuracy. So these kits could be considered as suitable tests for screening purposes.     

Development of an enzyme-linked immunosorbent assay for the detection of florfenicol in fish feed

A sensitive enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody was developed for routine screening of florfenicol (FF) in fish feed. FF succinate was synthesised and conjugated to human serum albumin by N-hydroxysuccinimide-activated ester method as immunogen, while it was conjugated to ovalbumin by mixed anhydride reaction as coating antigen. The ELISA for FF showed an IC₅₀ value of 2.5 ng/mL, and negligible cross-reactivities with other amphenicol family of antibiotics. The inter-assay recoveries of FF from fortified fish feed at levels of 2–20 mg/kg ranged from 98.8 to 117.6% with coefficients of variation (CV) of 9.3–14.2%, intra-assay recoveries ranged from 102.5 to 121.2% with CV of 10.9–13.8%. The developed ELISA were then validated by liquid chromatography method and liquid chromatography–tandem mass spectrometry method, the results indicated this ELISA could be used as convenient method for detection of FF in fish feed.     

Sensitive double antibody sandwich ELISA for the quantification of phosvitin

An effective double antibody sandwich ELISA (DAS-ELISA) method based on monoclonal (mAb) and chicken egg yolk IgY antibodies was developed to determine phosvitin (PV) content in therapeutic and functional products. Leghorn laying hens were immunized with purified PV to produce anti-PV IgY antibody in the egg yolk. High anti-PV IgY titer obtained from the egg yolks collected during 4–10 weeks of the immunization period contained approximately 6.2% of specific anti-PV IgY in total IgY. The PV detection range of the DAS-ELISA and biotinylated DAS-ELISA was 16.8–90 and 7.5–40?ng/mL, respectively. However, biotinylated DAS-ELISA was the better method for PV quantification in terms of accuracy and sensitivity. This highly efficient PV detection method may recuperate the performance of the existing protein assay methods as well as facilitate future research on PV bioactivities and applications.