Development of a monoclonal antibody-based immunoassay for specific quantification of bovine milk alkaline phosphatase

The detection of alkaline phosphatase (ALP) activity is used as a legal test to determine whether milk has been adequately pasteurized or recontaminated with raw milk. However, a wide variety of microorganisms produce both heat labile and heat stable ALPs which cannot be differentiated from the milk ALP by current enzymatic methods. Monoclonal antibodies specific of the bovine milk ALP were obtained in mice from a raw bovine milk ALP preparation. Coated in microtitre plates, these antibodies specifically capture the bovine milk ALP from dairy products. After washing, the enzymatic activity of the captured ALP is revealed by adding p-nitrophenyl-phosphate as a substrate. This simple immunoassay does not react with ALPs of intestinal or bacterial origin and, once optimized, was found to be the first immunoassay suitable to detect raw milk in boiled milk down to a 0.02% dilution. Moreover, in contrast with competitive indirect ELISA formats, the capture immunoassay does not require purified ALP.     

Application of a commercial immunoassay to the direct determination of insecticide imidacloprid in fruit juices

An ELISA was used to directly determine residual imidacloprid in fruit juices. Imidacloprid could be determined by only diluting samples without any pre-treatments such as filtration, centrifugation, and clean-up procedures. The ELISA enabled imidacloprid to accurately determine down to about 5 μg/L in apple and grape juice samples and down to about 20 μg/L in orange juice sample. Recovery and precision of the ELISA were evaluated by spiking fruit juice samples with imidacloprid in the 10–400 μg/L ranges. Coefficients of variation were lower than 20% in all cases, and average recoveries were 94.2%, 113.2%, and 104.2% for apple, grape, and orange juice samples, respectively. No false positive results were found. The results obtained with the proposed ELISA well correlated with the reference HPLC for each fruit juice sample (r > 0.99).     

Simultaneous determination of sulphamerazine,sulphamethazine and sulphadiazine in honey and chicken muscle by a new monoclonal antibody-based fluorescence polarisation immunoassay

A new monoclonal antibody (Mab) against sulphamerazine (SMR) was produced and a fluorescence polarisation immunoassay (FPIA) based on the Mab was developed and optimized for the simultaneous qualitative screening of SMR, sulphamethazine (SMZ) and sulphadiazine (SDZ). The Mab, raised from mice immunized with SMR, was bound to bovine serum albumin (BSA) using glutaraldehyde as the coupling reagent. Fluorescein-labelled SMR and SMZ (tracer) were synthesized and purified by thin layer chromatography (TLC). Cross-reactivities below 3.6% were displayed in the optimized FPIA for another 14 sulphonamides when both tracers were employed. The limits of detection (LOD) were 0.9 ng g^?1 for SMR, 2 ng g^?1 for SMZ and 3.1 ng g^?1 for SDZ. Analysis of SMR, SMZ and SDZ fortified chicken muscle and honey samples by the FPIA showed average recoveries of 86–131% with a standard deviation (SD) of 4.6–32. Comparative analyses of a SMZ-treated chicken muscle sample by both FPIA and high performance liquid chromatograph (HPLC) showed a good correlation (r?=?0.9991). The study demonstrates the practical application of FPIA in screening chicken muscle and honey samples for sulphonamides residues.     

Simultaneous determination of sulphamerazine, sulphamethazine and sulphadiazine in honey and chicken muscle by a new monoclonal antibody-based fluorescence polarisation immunoassay

A new monoclonal antibody (Mab) against sulphamerazine (SMR) was produced and a fluorescence polarisation immunoassay (FPIA) based on the Mab was developed and optimized for the simultaneous qualitative screening of SMR, sulphamethazine (SMZ) and sulphadiazine (SDZ). The Mab, raised from mice immunized with SMR, was bound to bovine serum albumin (BSA) using glutaraldehyde as the coupling reagent. Fluorescein-labelled SMR and SMZ (tracer) were synthesized and purified by thin layer chromatography (TLC). Cross-reactivities below 3.6% were displayed in the optimized FPIA for another 14 sulphonamides when both tracers were employed. The limits of detection (LOD) were 0.9 ng g(-1) for SMR, 2 ng g(-1) for SMZ and 3.1 ng g(-1) for SDZ. Analysis of SMR, SMZ and SDZ fortified chicken muscle and honey samples by the FPIA showed average recoveries of 86-131% with a standard deviation (SD) of 4.6-32. Comparative analyses of a SMZ-treated chicken muscle sample by both FPIA and high performance liquid chromatograph (HPLC) showed a good correlation (r = 0.9991). The study demonstrates the practical application of FPIA in screening chicken muscle and honey samples for sulphonamides residues.     

Inactivation-denaturation kinetics of bovine milk alkaline phosphatase during mild heating as determined by using a monoclonal antibody-based immunoassay

A monoclonal antibody based capture immunoassay has been recently developed for the specific quantitation of bovine milk alkaline phosphatase (ALP) without interference by contaminating microbial or fungal ALPs (Geneix et al. 2007). This immunoassay was used to study the kinetics of ALP heat denaturation in bovine milk over a range 50-60 degrees C for 5 to 60 min using a colorimetric quantification of the enzyme activity as a reference test. A denaturation midpoint was obtained at 56 degrees C for a 30 min heating. Thermal inactivation was found to follow first order kinetics and is characterized by z value of 6.7 deg C (D60 degrees C=24.6 min) and 6.8 (D60 degrees C=23.0 min) for respectively immunoassay and colorimetric assay. The high values of enthalpy of activation and the positive values of the entropy of activation and free energy of activation indicate that during denaturation ALP underwent a large change in conformation. The results of the immunoassay were highly correlated (r=0.994) with those obtained by the colorimetric assay. A similar high correlation (r=0.998) was obtained when industrially thermized milks (62-67 degrees C for 20-90 s) were analysed by both techniques. These results indicated that 1) thermally induced epitopic structural changes recognized by the capture monoclonal antibody are concomitant with or occur after the loss of enzymatic activity and 2) quantification of ALP by the specific immunoassay is appropriate for determining mild time/temperature treatment of milk and for the control of milk pasteurization.     

Simultaneous determination of sulphamerazine, sulphamethazine and sulphadiazine in honey and chicken muscle by a new monoclonal antibody-based fluorescence polarisation immunoassay.

A new monoclonal antibody (Mab) against sulphamerazine (SMR) was produced and a fluorescence polarisation immunoassay (FPIA) based on the Mab was developed and optimized for the simultaneous qualitative screening of SMR, sulphamethazine (SMZ) and sulphadiazine (SDZ). The Mab, raised from mice immunized with SMR, was bound to bovine serum albumin (BSA) using glutaraldehyde as the coupling reagent. Fluorescein-labelled SMR and SMZ (tracer) were synthesized and purified by thin layer chromatography (TLC). Cross-reactivities below 3.6% were displayed in the optimized FPIA for another 14 sulphonamides when both tracers were employed. The limits of detection (LOD) were 0.9 ng g(-1) for SMR, 2 ng g(-1) for SMZ and 3.1 ng g(-1) for SDZ. Analysis of SMR, SMZ and SDZ fortified chicken muscle and honey samples by the FPIA showed average recoveries of 86-131% with a standard deviation (SD) of 4.6-32. Comparative analyses of a SMZ-treated chicken muscle sample by both FPIA and high performance liquid chromatograph (HPLC) showed a good correlation (r = 0.9991). The study demonstrates the practical application of FPIA in screening chicken muscle and honey samples for sulphonamides residues.     

A monoclonal antibody-based sensitive enzyme-linked immunosorbent assay (ELISA) for the analysis of the organophosphorous pesticides chlorpyrifos-methyl in real samples

Chlorpyrifos-methyl hapten, O-methyl-O-(3,5,6-trichloro-2-pyridinyl)-N-(2-carboxyethyl)-phosphoramidothionte (H1), was synthesized and conjugated with bovine serum albumin (BSA) and ovalbumin (OVA) by the active ester method. Then H1–OVA conjugate was used as coating antigen, while H1–BSA conjugate was used as immunogen for producing monoclonal antibody. After optimisation, a monoclonal antibody-based effective competitive indirect enzyme-linked immunsorbent assay (ELISA) was developed and applied for determination of chlorpyrifos-methyl with a novel combination of antibody/antigen, I₅₀ of which was 75.22 ng/ml, limit detection (LD) was 0.32 ng/ml, and there was relative high cross-reactivity (CR) only with chlorpyrifos (1.4%), and CRs with other tested pesticides were all below 1% and regarded as negligible. The recoveries obtained by standard chlorpyrifos-methyl addition to real samples, including grape, Chinese cabbages, water and soil were all from 82.4% to 110.2%. Therefore, the optimised ELISA might become a convenient and satisfied analytical tool for monitoring chlorpyrifos-methyl residues in agriculture ecosystem.     

Detection of the sour-rot pathogen Geotrichum candidum in tomato fruit and juice by using a highly specific monoclonal antibody-based ELISA

Geotrichum candidum is a common soil-borne fungus that causes sour-rot of tomatoes, citrus fruits and vegetables, and is a major contaminant on tomato processing equipment. The aim of this work was to produce a monoclonal antibody and diagnostic assay for its detection in tomato fruit and juice. Using hybridoma technology, a cell line (FE10) was generated that produced a monoclonal antibody belonging to the immunoglobulin class M (IgM) that was specific to G. candidum and the closely related teleomorphic species Galactomyces geotrichum and anamorphic species Geotrichum europaeum and Geotrichum pseudocandidum in the G. geotrichum/G. candidum complex. The MAb did not cross-react with a wide range of unrelated fungi, including some likely to be encountered during crop production and processing. The MAb binds to an immunodominant high molecular mass (> 200 kDa) extracellular polysaccharide antigen that is present on the surface of arthroconidia and hyphae of G. candidum. The MAb was used in a highly specific enzyme-linked immunosorbent assay (ELISA) to accurately detect the fungus in infected tomato fruit and juice. Specificity of the ELISA was confirmed by sequencing of the internally transcribed spacer (ITS) 1-5.8S-ITS2 rRNA-encoding regions of fungi isolated from naturally-infected tomatoes.     

Development of a class-specific monoclonal antibody-based ELISA for aflatoxins in peanut

Three class-specific monoclonal antibodies against aflatoxins were screened by a designed strategy in which aflatoxin G₂ was used as competitor in the screening ELISA system. With a high cross-reactivity (65%) to aflatoxin G₂, antibody 10C9 had the most similar sensitivity for five aflatoxins (AFB₁, AFB₂, AFG₁, AFG₂ and AFM₁), whose I₅₀ values were in a range of 2.1–3.2 ng ml⁻¹. So, antibody 10C9 was selected to develop an ELISA for determination of aflatoxin B₁, B₂, G₁, G₂ and total of them in peanut samples. And spiked recoveries were from 87.5% to 102.0%. The results indicate that the ELISA developed can accurately determine total aflatoxins in samples of peanuts after the simple and rapid extraction procedure.     

An ultra-sensitive monoclonal antibody-based competitive enzyme immunoassay for aflatoxin M1 in milk and infant milk products

A sensitive and specific monoclonal antibody (Mab) against aflatoxin M₁ (AFM₁), named as 2C9, was selected by semi-solid HAT medium. It exhibited high affinity for AFM₁ of 1.74 × 10⁹ L/mol and no cross-reactivity to aflatoxin B₁, B₂, G₁ and G₂. Based on the antibody, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for AFM₁ in milk and infant milk products. Assays were performed in the AFM₁-BSA coated (0.0625 μg/mL) ELISA format in which the antibody was diluted 1:10,000. Several physicochemical factors (pH, ionic strength and blocking solution) that influence assay performance were optimised. Finally, the limits of detection were 3 ng/L for milk and 6 ng/L for milk-based cereal weaning food, inter-assay and intra-assay variations were less than 10%, and the recovery ranged from 91% to 110%. Thirty samples were analysed, and concordant results were obtained when the data were compared with a reference high-performance liquid chromatography method.     

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of the furaltadone metabolite,AMOZ, in fortified shrimp samples

3-Amino-5-morpholinomethyl-2-oxazolidone (AMOZ) is a tissue bound metabolite of furaltadone, a synthetic nitrofuran antibiotic widely used in veterinary practices both to treat infectious diseases and as a growth promoter. Because AMOZ is carcinogenic and poses a potential health hazard for human consumers, its parent compounds, the nitrofurans, are banned from being used in animals destined for human food consumption in various countries. To enforce this, foods are routinely monitored for nitrofuran metabolite residues including AMOZ. Thus, reliable high throughput analytical methods to detect AMOZ have been developed but these are mostly based on chemical analysis. In contrast, sensitive and specific detection methods based on immunoassays for AMOZ using monoclonal antibodies have yet to be established. In this study, we report the generation of two monoclonal antibodies with high specificity for AMOZ and the development of a monoclonal antibody-based ELISA to detect AMOZ in shrimp samples using one of these clones (clone 2E5.1). Clone 2E5.1 yielded the highest sensitivity and specificity and cross reacted solely to furaltadone and AMOZ and not to other antibiotics. Competitive ELISA with 2E5.1 gave IC₅₀ values for AMOZ, CPAMOZ and furaltadone of 5.33, 0.023 and 1.330 ng/ml, respectively. When applied in ELISA to detect AMOZ in fortified shrimp samples, the detection capability and limit of detection were 0.3 and 0.16 μg/kg, respectively. Taken together, a sensitive and specific monoclonal antibody-based ELISA for AMOZ detection has been developed that could facilitate the economic and reliable high throughput monitoring of AMOZ in shrimps and potentially other food.     

Improvement of the ammonia measurement using a clarifying reagent and application to evaluate heat damage in commercial milk samples

The enzymatic Boehringer procedure for ammonia measurement in milk was improved to be rapid and simple using a clarifying reagent without any pretreatment of the sample. A good correlation (r=0.99, n=36) existed between the proposed enzymatic method (EM) and the specific electrode method (SEM) when applied on raw and commercial milk samples. The repeatability of the EM applied to UHT milk was satisfactory with a relative standard deviation of 5.4% (n=26). The EM was used to evaluate heat damage in commercial half-skimmed pasteurised, direct or indirect or unlabelled UHT and in-bottle sterilised milk. The EM was well correlated with absorbance at 340 nm (A₃₄₀) of transparent modified milk (r=0.97, n=42), with lactulose determined by HPLC and by capillary electrophoresis (r=0.95, n=17 and r=0.95, n=26, respectively). All parameters differentiated significantly (P<0.001) between pasteurised, UHT- and in-bottle sterilised milk, and also direct-UHT and indirect-UHT samples. More intensive heat treatment produced higher contents of ammonia, lactulose and A₃₄₀ values.     

Evaluation of a novel malathion immunoassay for groundwater and surface water analysis

Malathion is an organophosphorus insecticide commonly used in crops and indoor applications. Negative effects of malathion on human health and ecosystems are of growing concern. In this work, novel malathion haptens are synthesized to develop an ELISA screening method. The immunoassay is based on a conjugate-coated format and shows a limit of detection of 0.11 ng/mL, an IC50 of 1.58 ng/mL, a dynamic range between 0.23 and 10.94 ng/ mL, and a cross-reactivity of <2% with structurally related compounds. The developed ELISA has been used to quantify malathion in groundwater and surface water samples. The good recoveries achieved and the agreement with those given by the GC-MS reference method indicate the potential of the assay for environmental monitoring of malathion in natural waters without purification or preconcentration steps. The effect of dissolved organic matter (humic acids) on the ELISA was evaluated, resulting in the conclusion that the immunoassay can be successfully performed in surface water samples with a humic acid content up to 10 mg/L, without sample pretreatment. Samples with a high humic acid content can be analyzed through ELISA after solid-phase extraction, eluting with acetone or acetonitrile.     

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the analysis of the new fungicide 2-allylphenol in strawberry fruits

2-Allylphenol (2-AP) is a recently registered fungicide in China to control fungal diseases on tomato, strawberry and apple. Four haptens were designed to produce 2-AP specific antibodies in the present work. A sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed with mAb225^# for the analysis of 2-AP after it is derivatized with 4-aminobenzoic acid. The icELISA showed a concentration of 4-carboxy-3′-allyl-4′-hydroxyazobenzene (CAH) as 2-AP equivalent producing 50% inhibition (IC₅₀) and a calibration range of 0.29 and 0.09–1.09 ng/mL, respectively. No competitive inhibition was observed up to 10,000 ng/mL of 4-aminobenzoic acid, 2(2-hydroxypropyl) phenol, salicylic acid, and 4-butanoylphenol. Average recoveries of 2-AP from strawberry samples spiked at concentrations from 156 to 5000 ng/g ranged from 83% to 95%. Comparable concentrations (R², 0.996) of 2-AP in strawberry samples determined with the icELISA and high performance liquid chromatography suggest that the icELISA is suitable for analyses of 2-AP residues in strawberry.     

Simultaneous separate and group determination of tylosin and tilmicosin in foodstuffs using single antibody-based immunoassay

An approach to modify the specificity of competitive indirect ELISA for determination of veterinary macrolides tylosin (TYL) and tilmicosin (TMN) using different structural design of coating antigen is described. The homologous and heterologous assay formats using rabbit antiserum against BSA-TYL conjugate were developed. The first format allowed selective determination of TYL (cross-reactivity for TMN was negligible). Heterologous hapten desmycosin (DMN) based conjugate being immobilised on the plates changed the specificity to group recognition (cross-reactivity for TMN was 103.4%). Using two coating antigens but single antibody and the same TYL standard solutions the described tandem test was capable for simultaneous determination of the respective analytes with their possible differentiation. The detection limit of assay that was far below the current MRLs and achieved 0.07 ng mL⁻¹ for TYL and 0.14 ng mL⁻¹ for TMN allowed to minimise matrix effect by sample dilution. The ELISAs of antibiotics in milk, eggs, honey and chicken muscle were optimised and showed acceptable recovery rate. A range of foodstuff samples was analysed using the developed tandem test.     

Identification of chemical detected in kiwi fruit during analysis for residual pesticide

An unknown peak was detected in a GC chromatogram of many kiwi fruit extracts during analysis for pesticide residues. It was identified by GC/MS as diphenyl 2-ethylhexyl phosphate (DPEHP), used as a plasticizer and flame retardant. The concentration of DPEHP was investigated in 15 samples of kiwi fruit, and it was detected at between 0.02 and 0.14 microgram/g in 10 of the samples. It might be due to migration of DPEHP into the fruit from the printed portion of the polyethylene terephthalate (PET) package.     

Detection of sulphathiazole in honey samples using a lateral flow immunoassay

A lateral flow immunoassay (LFIA) was developed in the competitive reaction format and applied to test sulphathiazole (STZ) residues in honey samples. To prepare the assay test, a hapten conjugate and goat antirabbit antiserum as capture and control reagent, respectively, were dispensed on nitrocellulose membrane. Polyclonal antiserum against sulphathiazole was conjugated to colloidal gold nanoparticles and used as the detection reagent. The visual limit of detection (cut-off value) of the sulphathiazole LFIA was 15 ng/g, reaching qualitative results within 10 min. The assay was evaluated with STZ spiked honey samples from different geographical origins (n = 25). The results were in good agreement with those obtained from liquid chromatography separation and mass spectroscopy detection (LC–MS), indicating that the LFIA test might be used as a qualitative method for the determination of sulphathiazole residues without expensive equipment. The test was also highly specific, showing no cross-reactivity to other chemically similar antibiotics. To our knowledge, this is the only work where a development of LFIA tests for the detection of sulphathiazole residues is performed.     

Evaluation and application of a simple and rapid method for the analysis of aflatoxins in commercial foods from Malaysia and the Philippines.

For application to the analysis of aflatoxins (AF) in commercial peanut and corn products, the ISOLUTE multimode column (IMC, solid phase multifunctional column) method was validated by comparing with the modified Florisil column (MFC) method. Twenty-two peanut and eight corn products from Malaysia and the Philippines were analysed for AFB1, AFB2, AFG1 and AFG2 firstly by the MFC method and then by the IMC method. For peanut products, 14 out of 22 samples were positive by the two methods in the range of 1-378 micrograms/kg of AF, and correlation coefficients (r) for AFB1 and AFB2 were 0.987 and 0.997, respectively. For corn and corn products, all the samples were positive in the range of 1-130 micrograms/kg, and r values were 0.992 and 0.805 for AFB1 and AFB2 respectively. Thus, the results were significantly (p < 0.01) in close agreement, particularly for lower range of 1-50 micrograms/kg of AF concentrations in all the samples. For the occurrence of AF, 11 (65%) of peanut products from Malaysia were contaminated with AF at a mean level of 50 micrograms/kg (maximum 180 micrograms/kg) and two (40% products from the Philippines were contaminated with as high as 375 micrograms/kg and 177 micrograms/kg of AF, respectively. All the corn products from the Philippines were contaminated with AF at a mean level of 44 micrograms/kg (maximum 130 micrograms/kg). Contamination of commercial foods with high levels of AF is a very important issue to both the countries since these foods are very popular among children.     

新设备在果蔬组织结构分析中的应用

尽管对产品的色、香、味和外观的研究方面是经过长期研究并早已确立的领域,但是在对产品组织结构(Texture)的研究迄今为止还是不够成熟和不充分的领域.一个时期以来,水果和蔬菜加工业者与对于成品质量相关联的咬嚼度(Crunchiness)--俗称咬头、坚硬度(Firmness)和咀嚼度(Chewiness)等几个特性给予定义,同时也进行了若干研究和调查.     

Development of a sensitive ELISA for the analysis of the organophosphorous insecticide fenthion in fruit samples

Two fenthion haptens, 4-(4-(dimethoxyphosphorothioyloxy)-2-methylphenylamino)-4-oxobutanoic acid (H1) and 6-(methoxy(4-(methylthio)phenoxy)phosphorothioylamino)hexanoic acid (H2), were synthesized. H1 was conjugated with bovine serum albumin (BSA) and H2 with ovalbumin (OVA) by the active ester method. Then H2-OVA conjugate was used as coating antigen, while H2-BSA conjugate was used to produce polyclonal antibodies. After optimization, an effective competitive indirect enzyme-linked immunosorbent assay (ELISA) for determination of fenthion was established with the new combination of antibody/antigen, I₅₀ of which was 0.01 ng/ml, and there was only cross reactivity (CR) with fenitrothion (4.5%), and CRs with other tested pesticides were all below 0.1%. The recoveries obtained by standard fenthion addition to the different fruit samples such as grape, peach, pear and tomato were all from 79.8% to 106.0%. Therefore, the optimized ELISA may become a new convenient and satisfied analytical tool for monitoring fenthion residues in agricultural samples.