Determination of folate-binding proteins from milk by optical biosensor analysis

Milk from various mammalian species contains folate-binding proteins (FBP) to ensure an adequate supply of folate via milk to neonates. FBP has gained a renewed interest because of the capability to enhance bioavailability of folate in the gut. In the present publication we describe a rapid and sensitive determination of FBP from milk based on an optical biosensor, i.e. a Biacore(R) instrument, and the specific interaction between folic acid (pteroyl-L-glutamic acid, PGA) and FBP. It was demonstrated that the amine group in the pteridine moiety of PGA was essential for this interaction. The optical biosensor assay showed good compliance with an existing ELISA method. The repeatability (CV) of the biosensor assay was high, i.e. at an FBP concentration of 0.38 mug mL(-1) it was 1.2-3.6% within-assay on 3 different days and 6.4% between days. The developed biosensor method offers several advantages over conventional routine methods, e.g. ELISA techniques, both in speed and by means of automation. Since the biosensor method is based on the specific interaction between FBP and PGA, unless a sample pre-treatment to remove folate is applied, it will only determine free FBP. The difference between free FBP and FBP associated to folate can be of interest and exploited in certain situations. In the described biosensor assay, removal of folate prior to analysis is achieved by a combination of low pH and molecular weight cut-off filtration. (C) 2003 Elsevier Science Ltd. All rights reserved.