Development of an immunochromatographic method to determine β-lactoglobulin at trace levels

Abstract

β-Lactoglobulin (βLG) is one of the main components of whey proteins. Among other reasons, its allergenic character makes necessary its determination in hypoallergenic foods and biopharmaceutical products at very low concentration making use of very sensitive methods. In this paper, the development of an enzyme linked immunoaffinity chromatographic (ELIAC) method in sandwich format is described. The optimized method includes the use of a minicolumn (4.9 mm × 1 mm) packed with Nucleosil ® 4000-7OH derivatized with high density of antibody coverage, the employment of rabbit serum and Zwittergent ® 3–12 as mobile phase additives, and the incubation of the sample containing βLG with anti βLG labeled with horseradish peroxidase prior to their injection into the immunochromatographic system. The method allows determination of βLG with a practical detection limit of 20.7 pM and a sensitivity of 1.05 × 10 9 AU mol −1 L. The application of this ELIAC method to determine βLG in a commercial hypoallergenic formula based on caseins is shown. The amount of allergen, 0.32 pmol βLG per gram of sample, is about 5 million times lower than that naturally occurring in cow's milk.