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The instrument used is a Beckman Coulter DXI 800. The Access Vitamin B12 assay is a competitive-binding immunoenzymatic assay. A sample is added to a reaction vessel along with alkaline potassium cyanide and dithiothreitol. This treatment denatures B12 binding proteins and converts all forms of vitamin B12 to the cyanocobalamin form. After neutralization, intrinsic factor-alkaline phosphatase conjugate and paramagnetic particles coated with goat antimouse IgG:mouse monoclonal anti-intrinsic factor are added to the sample. Vitamin B12 in the sample binds to the intrinsic factor conjugate, preventing the conjugate from binding to the solid phase anti-intrinsic factor. After incubation in a reaction vessel, materials bound to the solid phase are held in a magnetic field, while unbound materials are washed away. The chemiluminescent substrate Lumi-Phos 530 is added to the vessel and light generated by the reaction is measured with a luminometer. The photon production is inversely proportional to the concentration of vitamin B12 in the sample. The amount of analyte in the sample is determined by means of a stored, multipoint calibration curve.(Instruction manual: Beckman Coulter Assay 2009, Beckman Coulter Inc, Fullerton, CA)
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