Immunochromatographic determination of beta-lactoglobulin and its antigenic peptides in hypoallergenic formulas

Abstract

The use of hydrolysed formulas for feeding babies is efficient for preventing allergic diseases. Several hypoallergenic formulas (HF) based on hydrolysed cows’ milk proteins have been commercialised. However, some children fed such formulas suffer allergic reactions. b-lactoglobulin (bLG) is the main allergen present in cows’ milk. In addition to the whole protein, bLG fragments have also shown allergenic character. Detection of bLG and its peptides can be a useful marker of the presence of milk in food. Enzyme linked immunoassays (ELISAs) are general methods used to analyse allergens in foods. These techniques are sensitive and selective but they have several drawbacks. Immunoassay methods performed in chromatographic systems keep the advantages of classic ELISA while avoiding the disadvantages. A sandwich enzyme linked immunoaffinity chromatography (ELIAC) method was applied to determine bLG and its peptides in several commercial HF based on different milk proteins. Samples were also fractionated by employing membranes with different molecular weight cut-offs, and the antigenicity of the peptide fractions was determined. Applying the ELIAC method, for which one analysis takes 30 min, bLG was determined at the pM level in formulas based only on caseins, and at the nM to mM level in formulas based on hydrolysed whey proteins. Peptides of molecular weight less than 3000 derived from bLG showed positive results in ELIAC assays, indicating antigenic character.