Effectiveness of antibody specific for heat-processed milk proteins and incurred calibrants for ELISA-based quantification of milk in dark chocolate matrices

Although undeclared milk is prevalent in chocolate products, processing-induced alterations of milk proteins and matrix composition prevent reliable quantification of this allergen by current enzyme-linked immunosorbent assays (ELISA). High affinity antibodies (Abs) are required to detect milk protein epitopes that may be modified by manufacturing procedures. We therefore developed an Ab specific for heat-processed milk proteins (HPMP), which might allow enhanced milk quantification in processed complex matrices, including chocolate. When characterizing HPMP-Ab by immunoblot analysis, greater signal intensities were observed with heat modified proteins. Using a sandwich (s)-ELISA, HPMP-Ab reacted with high affinity to purified major milk proteins, proteins extracted from milk-derived ingredients and reference materials subjected to several thermal processing procedures. Milk-incurred cookies (0–17.7 ppm) and tempered dark chocolate samples (62% cacao: 0–134 ppm; 100% cacao: 0–75 ppm) were assessed using HPMP-Ab in a revised s-ELISA. To optimize protein extraction, samples were defatted with hexane; cookies were then extracted using a high salt buffer (HSB)/1% Tween (T)-20 while chocolate samples were extracted using HSB/1%T-20/5% polyvinylpyrrolidone (PVP)/1% fish gelatin. Despite reliable detection of milk proteins using incurred cookies and tempered 62% dark chocolate samples, as shown by acceptable dynamic ranges of the HPMP-Ab binding curves, high baseline absorbance/background persisted using 100% dark chocolate samples. The HPMP-Ab ELISA was highly specific; no cross reactivity was observed when testing a broad range of food commodities using incurred cookies as calibrants. The HPMP-Ab demonstrated the capacity to detect declared milk proteins in products containing a variety of milk-derived ingredients and undeclared milk proteins in products with precautionary labeling when used to analyze chocolate market samples (up to 85% cacao). Milk proteins were also detected in cross-contact samples produced on equipment used to manufacture milk chocolate, utilizing tempered 62% dark chocolate samples as calibrants. Our results establish the benefits of using HPMP-Ab and matrix-specific incurred calibrants for milk quantification in chocolate matrices by ELISA.