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.     

Detection of alkaline phosphatase by competitive indirect ELISA using immunoglobulin in yolk (IgY) specific against bovine milk alkaline phosphatase

Immunoglobulin in yolk (IgY) (with a titer of 1.3 × 10⁶) specific against bovine milk (BM) alkaline phosphatase (ALP) was obtained by intramuscularly immunizing hens on the thigh and was used as the primary antibody to conduct competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) to determine BMALP in ALPs from BM and Escherichia coli sources. A relationship between the ELISA value and the BMALP level (0.01–10 μg/mL) in whole milk (R² = 0.9019) or in skimmed milk (R² = 0.9402) was observed. The maximal inhibition (%) of BMALP on the microtiter plate by free BMALP at 10 μg/mL whole milk (3.89 mU/μg BMALP) was about 50%, while no inhibition (%) of BMALP by free E. coli ALP at concentrations between 0.01 to 10 μg/mL (60 mU/μg E. coli ALP) was determined. At BMALP levels higher than 0.1 μg/mL, CI-ELISA was proved to be effective in differentiating between BMALP and E. coli ALP and quantifying BMALP in whole milk or skimmed milk in the presence of E. coli ALP with an activity of 0.6 U/mL. Higher inhibition (about 70%) of BMALP on the microtiter plate by free BMALP in diluted (10¹–10⁴ fold) milk samples was observed. The optimal conditions for CI-ELISA in determining BMALP (0.1–10 μg/mL) from ALPs in milk samples were using 10³-fold diluted crude IgY specific against BMALP as primary antibody and 10³-fold diluted goat anti-chicken IgG–ALP conjugate as the secondary antibody.     

Dephosphorylation of bovine casein by milk alkaline phosphatase.

The pH of optimum activity of alkaline phosphatase from cow's milk depended on the substrate, being 10-1 for rho-nitrophenylphosphate, 8-6 for phosphoserine, 8-0 for phosvitin and 6-8 for casein. Individual casein components were dephosphorylated more rapidly than mixtures of alphas- and beta-caseins or of alphas-, beta-and kappa-caseins and micellar casein. Mixtures of 2 components involving kappa-casein were more readily dephosphorylated than alphas- and beta-casein mixtures. At pH 6-8, lactose, whey proteins and phosphate ions had an inhibitory effect. beta-Lactoglobulin had an inhibitory effect only when the pH of the reaction was lower than the optimum pH value of the enzyme. Mg2+ and Zn2+ were not inhibitory. The optimum conditions for dephosphorylation of casein are described.     

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 biosensor immunoassay for the quantification of alphas1-casein in milk

An immunoassay to quantify alphas1-casein (alphas1-CN) in milk using an optical biosensor, based on surface plasmon resonance (SPR) measurement, has been developed. The assay consists of a two-step sandwich strategy, with two anti-alphas1-CN antibodies directed against each extremity of the molecule. This strategy permits only intact alphas1-CN to be quantified and not its degradation products. The calibration curve was obtained using a reference milk powder with a known alphas1-CN concentration. Analysis time per sample was less than ten minutes. The antibody-coated surface could be used for more than 150 determinations. Detection limit was established at 0.87 microg/ml and the intra- and inter-assay variation coefficients were 2.86 and 5.31%, respectively. The method was applied to raw milk to quantify intact alphas1-CN, with no pretreatment of the sample. An initial analysis of 48 milk samples permitted alphas1-CN concentrations ranging from 8.8 to 12.06 mg/ml to be obtained.     

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.     

Kinetics of thermal inactivation of alkaline phosphatase in bovine and caprine milk and buffer

The thermal inactivation of alkaline phosphatase (ALP) in raw bovine and caprine milk was investigated in the temperature range 54 to 69 °C. To assess the stabilizing effect of milk compounds on ALP, inactivation experiments were also carried out in 0.1 m potassium phosphate buffer, pH 6.6. Each set of inactivation experiments was fitted simultaneously using kinetic models that were based on either one-step or two-step mechanisms. The parameters of the Arrhenius equation showed that the stabilization effect of milk compounds on ALP had an entropic character. They also indicated a different structure of bovine and caprine milk ALPs, which was reflected by a higher stability of the bovine milk enzyme.     

Development and application of an optical biosensor immunoassay for α-lactalbumin in bovine milk

An automated, label-free biosensor-based immunoassay for α-lactalbumin in bovine milk utilising surface plasmon resonance (SPR) detection is described. α-Lactalbumin content was estimated from the specific interaction with an anti-bovine α-lactalbumin antibody immobilised on the sensor surface in a direct-binding assay format, although an alternative inhibition assay format is also described. Samples were prepared for analysis by direct dilution into buffer. Ligand selection and analysis conditions are defined, and non-specific binding considerations evaluated. Performance parameters include a working range of 10–1000 ng mL^?1, a method detection limit of 0.12 mg mL^?1 in milk, an overall instrumental reproducibility relative standard deviation (RSD_R) of 5.71%, a mean inter-assay RSD_R of 7.61% for an infant formula control sample and a surface stability of approximately 500 cycles. Accuracy was confirmed by comparison against an independent liquid chromatographic method. The technique was applied to the measurement of the α-lactalbumin content of consumer milk, colostrum, whey protein concentrates and infant formulae, the temporal change during early bovine lactation and a preliminary study of thermal denaturation.     

The β-lactoglobulin content of bovine milk: Development and application of a biosensor immunoassay

An optical biosensor immunoassay exploiting surface plasmon resonance is described for the quantification of β-lactoglobulin in milk. Samples were diluted with buffer, and the protein estimated from binding with a polyclonal antibody immobilised on the sensor surface. Analytical method performance characteristics including range, detection limit, precision and accuracy were determined and reported. The temporal variability in the β-lactoglobulin content of milk from pasture-fed cows during early lactation and across a production season was investigated. The content of β-lactoglobulin decreased from >10 mg mL⁻¹ in early colostrum to <5 mg mL⁻¹ in mature milk, and the β-lactoglobulin content of skim milk powder trended from 25 to 60 mg g⁻¹ across a season. In view of its allergenicity, these data will improve understanding of the expression of innate β-lactoglobulin in the milk of pasture-grazed dairy herds, thereby providing information that is applicable to the formulation of bovine milk-based products.     

Modified immunoassay for the detection of soy milk in pasteurized skimmed bovine milk

Utilization of soy milk and soy protein as a food supplement or as a replacement for milk and milk products is increasing. This form of food modification presents several different problems: quality assurance cannot always be given, people with soy allergy may be unknowingly exposed and regulatory bodies have difficulty in detecting the cases of deliberate adulteration. Therefore, a reliable means of detecting and quantifying soy protein in milk is needed. In the present study we have modified an ELISA technique to overcome the problem of quantitation of soy protein in milk. The results are reproducible, show low cross-reactivity with milk protein and sensitivity is good (assay range 3.5–70 μg soy protein/cm³). The assay measures soy protein and the correlation with soy milk added to bovine milk is linear.     

Enzymatic-fluorometric quantification of cholesterol in bovine milk

The present paper describes an enzymatic-fluorometric method for the determination of cholesterol in milk and other opaque matrices. The initial step of the method is to liberate chemically and physically bound cholesterol from the milk fat globule membrane by enzymatic action. The method is able to discriminate between esterified and free cholesterol in milk. The analysis is cost effective and is developed to work directly on whole, fresh milk thereby eliminating time consuming and tedious pre-treatment procedures of the sample. More than 1000 milk samples were analysed on the day of sampling. The total concentration of milk cholesterol ranged from 80 to 756μM (n=1068; mean 351μM). Milk cholesterol was significantly correlated to milk fat concentration as analysed by mid-infra red spectrometry (r=0.630; n=853) and by an enzymatic-fluorometric method (triacylglycerol) (r=0.611; n=842).     

乳制品中牛IgG含量间接竞争性ELISA检测方法的研究

建立了牛初乳制品和添加了牛初乳成分的乳制品中牛IgG含量间接竞争性酶联免疫吸附测定方法,主要研究步骤包括:制备牛IgG保守区(Fc段),以Fc段为免疫原免疫Balb/C小鼠,取其脾细胞进行细胞融合,制备特异性单克隆抗体及合成适合实际测定用途的ELISA试剂盒。应用本研究建立的方法对标准品和实际样品进行牛IgG含量检测的结果表明,该方法回收率在78.9%～117.5%,批内变异系数小于10%,批间变异系数小于15%,检测结果稳定可靠,可满足目前国内牛初乳制品和添加了牛初乳成分的乳制品品质监控的需要。     

Effect of high pressure carbon dioxide on alkaline phosphatase activity and quality characteristics of raw bovine milk

Novel food processing techniques would always be pursuits of researchers and food industry to avoid unfavorable thermal effects, especially in dairy and milk processing. In this study, effects of high pressure carbon dioxide (HPCD) on the activity of alkaline phosphatase (ALP) and main quality indices of raw bovine milk at 20 MPa using a batch system were investigated. A complete inactivation of ALP activity as exposure to HPCD treatment at 50 °C and 20 MPa for 50 min was observed. The protein and lactose content of HPCD-treated bovine milk hold steady, while pH value and total solids content decreased, turbidity and average particle size increased significantly (p < 0.05). Although a significant decrease of viscosity (p < 0.05) was observed, the Newtonian flow behavior of raw bovine milk did not alter. More obvious change of quality characteristics of raw bovine milk were observed as subjected to HPCD treatment at higher temperature or treated for longer period. Therefore, a compromise between controlling endogenous enzymes (and/or spoilage and pathogenic microorganisms) and retention of original/fresh like quality of foods should be introduced due to the nature of HPCD processing. It's suggested to keep raw bovine milk with low ALP activity and great quality treated with a batch HPCD apparatus at 20 MPa and 50 °C for 20 min.     

Thermal inactivation kinetics of alkaline phosphatase in equine milk

Alkaline phosphatase (ALP) is widely used as an indicator of proper pasteurization in bovine milk. Due to interest in the use of equine ALP as a time/temperature integrator (TTI) for evaluation of the efficacy of thermal processing of equine milk, its inactivation kinetics were evaluated in whole and skimmed equine milk. Experimentally determined decimal reduction times showed that equine ALP is more readily inactivated in equine milk than its bovine counterpart. Thus, considering the required 6 D reduction of pathogens and the rather low enzyme level present in equine milk, equine ALP will not be suitable as indicator for correct pasteurization of equine milk under the conditions currently used in the reference method for the determination of ALP in milk-based products.     

Development of a highly sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay for determination of doxycycline in chicken muscle,liver and egg

A modified indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method was developed using a highly sensitive and specific monoclonal antibody (McAb) to determine doxycycline (DC) residues in chicken tissues and egg. The McAb against DC was produced by hybridoma technique and a modified ic-ELISA was characterised in terms of sensitivity, specificity, precision and accuracy. At optimal experimental conditions, the standard curve was constructed at concentrations ranged from 0.01 to 100 ng/ml. The IC₅₀ value was 1.32 ± 0.18 ng/ml. The limit of detection was 0.14 ± 0.02 ng/g. The recoveries of DC from spiked chicken liver, muscle, and egg at levels of 50–600 ng/g were 84.6–85.5%, 88.2–89.1%, and 84.4–89.3%, respectively. The coefficient variations (CVs) were 5.1–9.3%, 3.7–11.3%, and 4.7–9.8%, respectively. Linear regression analysis showed good correlation, with r² values 0.9909 for chicken liver and 0.9916 for chicken muscle.     

Evaluation of performance of a commercial monoclonal antibody-based fenitrothion immunoassay and application to residual analysis in fruit samples

The performance of a commercially available enzyme-linked immunosorbent assay (ELISA) kit containing highly sensitive monoclonal antibodies against fenitrothion was assessed. The experimentally estimated dynamic range (0.087 to 2 ng/g) agreed with that established by the kit manufacturer (0.075 to 1 ng/g). The linearity of the standard curve produced for the kit-assembled standard solutions (slope = -0.3829) agreed with that of the curve produced for the self-made standard solutions (slope = -0.3619). The sensitivity (I50 value) and the limit of detection for the kit were 0.23 and 0.033 ng/g, respectively. Selectivity of the ELISA indicates that the monoclonal antibody can readily distinguish the target pesticide from other structurally related analogs and some metabolites (oxon forms), with the exception of ethyl O-(p-nitrophenyl) phenyl phosphonothionate (EPN), parathion-methyl, and parathion. Methanol was the best organic solvent for the kit, with optimal sensitivity observed at a final concentration in the well of 10% (vol/vol) or less. Matrix interferences were minimized by direct dilution with water (60-fold) of the methanolic extracts fromapple and peach samples. To extract fenitrothion from these two agricultural products as simply and rapidly as possible, three extraction methods were used. The extraction method that involved shaking by hand for 3 min was the best among the three methods. High recovery percentages (116.6% for apple and 110.8% for peach) were obtained. Validation of the ELISA method was carried out using the gas chromatography-mass spectrometry method, resulting in high recovery and close correlation of results (r > 0.95). These findings strongly suggest that the ELISA kit may be routinely employed for on-site fenitrothion screening of fruit samples.     

Development and validation of a nephelometric immunoassay for IgG1 in milk

A nephelometric immunoassay, with a detection range of 0.3 to 5 g IgG1/l, was (leveloped for the determination of immunoglobulin in bovine milk. The assay exhibited no significant cross-reactivity with alphas1-casein, alphas2-casein, beta-casein, K-casein or beta-lactoglobulin and 39% cross-reactivity with IgG2. The nephelometric assay was compared with ELISA and RID (24 h and 48 h incubations) assays using 105 duplicate milk samples covering IgG1 values ranging from 0.45 to 1.8 g. The results obtained from all assays showed good agreement with the exception of those obtained by the RID assay (24 h incubation) which gave lower results in samples containing more than 1.2 g IgG1/l. It was concluded that the nephelometric assay is a reliable, rapid and convenient method suitable for the quantification of IgG1 in milk. The assay can be configured for routine high-throughput milk quality assurance for IgG1 in dairy laboratories.     

An optical biosensor-based immunoassay for the determination of bovine serum albumin in milk and milk products

An automated biosensor immunoassay, exploiting surface plasmon resonance detection, is described for the quantitation of bovine serum albumin (BSA) in milk products. Antibody selection, assay conditions and potential non-specific binding interferences were defined. Analytical performance includes a working range of 10–1000 ng mL⁻¹, a method detection limit of 0.02 mg g⁻¹ in milk, an instrument intermediate precision relative standard deviation (RSD_iR) of 3.7%, an intermediate precision RSD_iR of 8.9% for whey protein concentrate, and a single flow cell stable for at least 400 cycles. Accuracy was demonstrated by recovery, compliance with a certified reference material and comparison with a high performance liquid chromatographic method. The technique was applied to the estimation of BSA content of bovine milk, colostrum, whey protein fractions and infant formulae. The change in BSA expression during early bovine lactation and across a production season, and the thermal denaturation of BSA were also investigated.     

乳中碱性磷酸酶活性测定方法研究进展

碱性磷酸酶(ALP,EC 3.1.3.1)是乳中天然存在的一种酶,该酶作为一种热处理强度指指标被广泛应用于评价牛奶巴氏杀菌是否彻底或巴氏杀菌后是否污染有生鲜乳.关于乳中ALP活性的测定不断有新的方法提出,本文对乳中ALP活性的测定方法进行了综述.     

Establishment and optimization of monoclonal antibody-based heterologous dcELISA for 19-nortestosterone residue in bovine edible tissue

Abstract: This paper presents the generation of monoclonal antibodies (mAbs) with high specificity against 19‐nortestosterone (NT) through cell fusion procedures, and the development of mAb‐based heterologous direct competitive enzyme‐linked immunoabsorbent assay (dcELISA) methods to detect NT residue using one of these hybridomas (clone 3B8‐E6). Under optimal experimental conditions, this assay exhibited a working range of 0.004 to 19 ng/mL with IC₅₀ and limit of detection values of 0.28 and 0.002 ng/mL, respectively, when it was run in 0.01M phosphate‐buffered saline (pH 7.4). Except for minor cross‐reactivity with β‐boldenone (6.9%) and trenbolone (1.2%), other interference to the assay was negligible (<0.05%). No significant differences (P > 0.05) were found for IC₅₀ values when the pH of the assay buffer ranged from 6 to 8 and phosphate ion concentration was less than 20 mM. The dcELISA can tolerate higher concentrations of methanol than other organic solvents tested. When applied to bovine sample, the correlation coefficients (R) of the dcELISA and GC‐MS data were 0.9918 in muscle, 0.9834 in liver, and 0.9976 in kidney. Therefore, this assay has the potential to be incorporated into a quantitative monitoring program for the rapid screening of NT residue in food.