Novel sensitive monoclonal antibody based competitive enzyme-linked immunosorbent assay for the detection of raw and processed bovine beta-casein

Abstract

Cow milk protein allergy (CMPA) is the most common childhood food allergy, which can sometimes persist or can newly develop in adulthood with severe symptoms. CMPA's treatment is complete dietary avoidance of milk proteins. To achieve this task, patients have to be aware of milk proteins found as "hidden allergens" in food commodities. In regard to milk proteins, it has been reported that allergenicity of caseins remains unaffected upon heat treatment. For these reasons, we aimed to obtain monoclonal antibodies (mAbs) against native and denatured β-casein, one of the most abundant and antigenic caseins, in order to develop an indirect competitive ELISA (icELISA) to detect and quantify traces of this milk allergen in raw and processed foodstuffs. We developed two specific hybridoma clones, 1H3 and 6A12, which recognized β-casein in its denatured and native conformations by indirect ELISA (iELISA). Cross-reaction analysis by Western blot and iELISA indicated that these mAbs specifically recognized β-casein from bovine and goat milk extracts, while they did not cross-react with proteins present in other food matrixes. These highly specific mAbs enabled the development of sensitive, reliable and reproducible icELISAs to detect and quantify this milk protein allergen in food commodities. The extraction of β-casein from foodstuff was efficiently carried out at 60°C for 15 minutes, using an extraction buffer containing 1% SDS. The present study establishes a valid 1H3 based-icELISA, which allows the detection and quantification -0.29 ppm and 0.80 ppm, respectively- of small amounts of β-casein in raw and processed foods. Furthermore, we were able to detect milk contamination in incurred food samples with the same sensitivity as a commercial sandwich ELISA thus showing that this icELISA constitutes a reliable analytical method for control strategies in food industry and allergy prevention.

Sağlıklı ve Mastitisli Keçi Sütünde αs1-kazeini Belirleyen Monoklonal Antikor Geliştirilmesi

“…Casein expression level is an important index when evaluating the lactation function of the mammary gland. While mAbs against β-casein [10] and κ-casein [11] have been developed, no studies have reported the successful development of mAbs against αs1-casein, the major part (38%) of total caseins in milk [12] . In addition, considering that αs1-casein is the most important protein causing milk allergy [13] , mAbs with high specificity to goat αs1-casein are therefore of critical importance for people to avoid milk allergy.…”

Detection of Allergenic Proteins in Foodstuffs: Advantages of the Innovative Multiplex Allergen Microarray-Based Immunoassay Compared to Conventional Methods

“…For instance, ELISA commercial kits have been developed for the detection of individual allergens in foods, such as peanut Ara h 1, milk caseins, crustacean tropomyosin, etc. [ 83 , 84 , 85 ]. Commercial multiplex kits, providing the same high specificity associated with sandwich ELISA and allowing the detection of more than one allergen, are also available and exploit IgG antibodies specific for some foods, such as nuts, egg, milk and gluten [ 80 ].…”

Nano-Biosensing Platforms for Detection of Cow’s Milk Allergens: An Overview

“…Besides these advancements in immunosensors for faster allergen detection, all techniques based on antibodies (whether ELISA or immunosensors) suffer from difficulties in the analysis of processed food. In the case of processed food, the treatment and the processing of food matrix led to denaturation, hydrolysis, and conformational changes in proteins, due to which they cannot be recognized by antibodies properly [112]. Moreover, the quality of antibodies is also crucial for development of ultra-sensitive immunosensors.…”