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Immunoprobes for thermally-induced alterations in whey protein structure and their application to the analysis of thermally-treated milks

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Polyclonal antisera have been raised to the whey proteins α-lactalbumin [α-La] and -lactoglobulin [-Lg], variants A and B. These antibody preparations have been used to develop enzyme-linked immunosorbent assays (ELISAs) for each of these proteins, which had limits of detection of 13 ng/ml [α-La], 27 ng/ml [-Lg, variant A], and 20 ng/ml [-Lg, variant B]. The α-La ELISA did not show any cross-reaction with -Lg, and neither of the -Lg ELISAs showed a cross-reactivity with α-La. However, despite the almost identical sequences of variants A and B of -Lg, the variant A ELISA had a cross-reactivity of 66% with variant B, whilst the variant B ELISA had a cross-reactivity of more than 200% with variant A. The effect of thermal treatment on the immunoreactivity of purified whey proteins was studied by ELISA and related to changes in secondary and tertiary structure determined using CD and fluorescence spectroscopy. The immunoreactivity of α-La determined by ELISA decreased on heating above 90°C, these changes coinciding with the protein denaturation as indicated by a loss of secondary structure. In contrast, the ELISA immunoreactivity of both -Lg variants increased after heating, a change that also coincided with changes in -Lg secondary and tertiary structure as determined by intrinsic fluorescence of the protein. Similar thermally-induced changes in whey protein immunoreactivity were observed following heat-treatment of raw milk, the immunoreactivity of α-La being reduced whilst that of the -Lg variants increased. When used in combination these ELISAs were able to discriminate between milks which had been pasteurized or subjected to more severe heat-treatments such as sterilization and ultra heat treatments (UHT). These data demonstrate that such immunoassays have the potential to be used as quality control methods for determining the thermal history of milks.
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Keywords: heat-treatment; immunoassay; secondary structure; α-lactalbumin; -lactoglobulin

Document Type: Research Article

Publication date: June 1, 2003

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