An immunoassay to quantify αS1-casein (αS1-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-αS1-CN antibodies directed against each extremity of the molecule. This strategy permits only intact αS1-CN to be quantified and not its degradation products. The calibration curve was obtained using a reference milk powder with a known αS1-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 μg/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 αS1-CN, with no pre-treatment of the sample. An initial analysis of 48 milk samples permitted αS1-CN concentrations ranging from 8·8 to 12·06 mg/ml to be obtained.
beta-Casein was quantified in milk and cheese, using an optical immunosensor, based on surface plasmon resonance (SPR) measurement. The assay consists of a two-step sandwich strategy, with two anti-beta-casein antibodies directed against each extremity of the casein. This strategy permits only native beta-casein to be quantified and not its degradation products. The calibration curve was obtained with a reference milk powder of known beta-casein concentration. The analysis time per sample was less than 10 minutes. The antibody-coated surface could be used for more than 250 determinations. The detection limit was established at 85 ng x mL(-)(1) and the intra- and inter-assay variation coefficients were 2.6 and 6.2% respectively. The method was applied to raw milk to quantify intact beta-casein, with no pretreatment of the sample. A second application was realized with cheese, to follow the proteolysis of beta-casein during ripening.
Dairy industries are interested in knowing the heat treatment undergone by milk so as to control the quality of drinking milks or to control their heating systems. Among the different techniques available to characterize the heat treatment of milk, estimation of the denaturation of proteins has been widely used. However, because the concentration of the proteins in raw milk can fluctuate significantly, determining only the concentration of a native protein without knowing its concentration in the raw milk before undergoing heat treatment can lead to significant imprecision. The objective of this study was to develop, on Biacore 3000, a biosensor assay for determining the denaturation index of alpha-lactalbumin by quantifying separately the native and "heat-denatured" forms of alpha-lactalbumin with specific monoclonal antibodies. alpha-Lactalbumin denaturation index is independent of the concentration of alpha-lactalbumin in the original raw milk. The technique developed is discriminating, fast, repeatable, fully automated, and requires no pretreatment of the milk sample.
Two recent techniques using optical immunosensor technology were developed for the quantification of milk proteins in dairy products. The first application is the simultaneous quantification of the 3 major caseins (αS1, β, and κ). This assay consists of a 2-step sandwich strategy, with 2 monoclonal antibodies directed against the N- and C-terminal extremities of each of the caseins, respectively. This strategy permits only intact caseins to be quantified, and not their degradation products. The technique is fast (10 min), sensitive (detection limit about 0.87 μg/mL), and has been applied successfully to raw and drinking milks. In the second application, the severity of the heat treatment sustained by a milk of unknown origin is determined by quantifying separately the native and heat-denatured forms of α-lactalbumin with specific monoclonal antibodies. The technique allows discrimination of the different heat treatments studied (pasteurization, direct and indirect ultra-high temperature, sterilization), is fast (4 min), repeatable, fully automated, and requires no pretreatment of the milk sample.
k-casein (k-CN) plays an important role in the stability and coagulation properties of milk. An immunoassay to quantify k-casein 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-k-casein antibodies directed against each extremity of the casein to quantify only native k-casein. The analysis time per sample was less than ten minutes. The antibody-coated surface could be used for more than 150 determinations. The detection limit was established at 0.45 mg.ml (1 and the intra-and inter-assay variation coefficients were 4.28% and 6.8%, respectively. The method was applied to raw milk in order to quantify intact k-casein, with no pretreatment of the sample. It also allowed the monitoring of k-CN concentration during milk coagulation after addition of rennet.
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