A sensitive enzyme‐linked immunosorbent assay for determination of bovine β‐lactoglobulin in infant feeding formulas and in human milk

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Abstract
We developed a sensitive sandwich‐type ELISA for measuring low levels of cow's milk (CM) β‐lactoglobulin. Purified anti‐β‐lactoglobulin was used as coating antibody and also as second antibody conjugated with alkaline phosphatase. Polyethylene glycol 6000 was added to the incubation buffers to improve sensitivity. The detection limit of the assay was 0.002 μg/l, which is much better than sensitivities reported for other β‐lactoglobulin assays. The sensitivity was not impaired by the presence of other CM proteins. The recovery from breast milk was 93% and from the diluting buffer 127%. The coefficient of variation within day was 5–15% and between days 10%. One hour after oral intake of milk, P‐lactoglobulin could be detected in the breast milk of three mothers at concentrations of about 1–2 μg/l. Widely different concentrations of β‐lactoglobulin were measured in two protein hydrolysates based on CM whey and casein proteins; the observed concentrations were 200 and 0.0056 μg P‐lactoglobulinμ/g dry weight, respectively.