Binding Proteins As Reagents In Enzyme-linked Competitive Binding Assays (immunoassay, Folate).

Research concerning the feasibility of replacing antibodies with binding proteins as the specific binders in enzyme-linked competitive binding assays is described. The relative merits of these techniques compared to traditional enzyme immunoassays are presented for both heterogeneous and homogeneous arrangements. The folate binding protein (FBP)-folate system is used as a model to demonstrate the analytical properties of such systems. A theoretical model for heterogeneous enzyme-linked assays is detailed. This model compares the use of antibodies and binding proteins as binders in these assays and demonstrates that by using such binding reagents the overall dose-response characteristics of the assays could be improved, particularly when immunological bridging group recognition diminishes the sensitivity of conventional antibody-based methods. A heterogeneous assay for the determination of folate is described. This assay uses an enzyme-folate conjugate and immobilized FBP. Dose-response curves demonstrate the sensitivity and selectivity of the assay. Assays with detection limits sufficient to quantitate folate in serum are presented. Unique biphasic dose-response curves are evident when low enzyme-folate conjugate concentrations are used. This observation suggests a positive cooperativity binding mechanism between the immobilized FBP and the enzyme-folate conjugate. The accuracy of the method was tested by assaying folate in infant formula samples. The kinetic and thermodynamic properties of the FBP-folate are discussed. The employment of enzyme- rather than radio-labeled folate provides further insight into the complex binding mechanism between folate and FBP and indicates that polymerization of the binding protein is not a prerequisite for the cooperative behavior. The effect of surfactants on this system is also demonstrated. A homogeneous competitive binding assay for folate is also described. This method utilizes a highly substituted glucose-6-phosphate dehydrogenase-folate conjugate in conjunction with FBP. The binding protein, when bound to the enzyme-folate conjugate, inhibits the enzymatic activity of the conjugate. The inhibition is reversed in an amount proportional to the quantity of unlabeled folate in the sample. Extremely steep dose-response curves are obtained which are used for the selective determination of folate in multivitamin preparations.Ph.D.Analytical chemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/127840/2/8612467.pd