Binding assays biotin

The basic methods to quantify biotin in food are bioassays, avidin-binding assays or fluorescent derivative assays. Although the avidin-binding assay of biotin and its metabolites after the separation by HPLC is considered as one of the best cnrrently available methods [587], it is still not largely diffnsed. 

Table 23 HPLC Methods for Quantitating Biotin in Foods (C18 Columns Detection by Fluorophore-Linked Protein Binding Assay)... 

In a protein-binding assay with fluorescence detection a microarray of biotin, HPYPP-peptide and WSHHPQFEK-peptide was screened against streptavidin-Cy3 and avidin-Cy5. By following the same principle an anti-human insulin monoclonal antibody was also screened against a set of different peptides. 

A protein-binding assay (BA) coupled with hplc provided a highly sensitive post-column reaction detection system for the biologically important molecule biotin and its derivative biocytin, biotin ethylenediamine, 6-(biotinoylamino) caproic acid, and 6-(biotinoylamino)caproic acid hydrazide (71). This detection system is selective for the biotin moiety and responds only to the class of compounds that contain biotin in their molecules. In this assay a conjugate of streptavidin with fluorescamine isothiocyanate (streptavidin—FITC) was employed. Upon binding of the analyte (biotin or biotin derivative) to streptavidin—FITC, an enhancement in fluorescence intensity results. This enhancement in fluorescence intensity can be directly related to the concentration of the analyte and thus serves as the analytical signal. The hplc/BA system is more sensitive and selective than either the BA or hplc alone. With the described system, the detection limits for biotin and biocytin were found to be 97 and 149 pg, respectively. 

The concept of MW enhancers was developed to increase polarization value changes caused by large size differences between substrate and product when small peptide substrates were used. The most frequently used approach is based on the attachment of a biotin molecule to the terminus of the peptide on the opposite site of the scissile bond to the fluorophore. After the incubation of the substrate with the protease, streptavidin is added to the assay. Streptavidin binds to biotin and enhances the masses of the substrate and the unlabeled product, thus causing an increase of the polarization... 

Consistency of ambient temperature was important for reproducible results. The signal of the TRF assay seemed to be very sensitive to temperature. At 25°C, the assay signal was only half the signal at 20°C, possibly due to the affinity of the Hsp90 for biotin-radicicol and/or the affinity of the Eu-anti-His antibody. For any binding assay that requires washing and subsequent incubations,... 

Takeuchi T, Dobashi A, Kimura K (2000) Molecular imprinting of biotin derivatives and its application to competitive binding assay using nonisotopic labeled ligands. Anal Chem 72 2418... 

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