Vitamin B12, serum or plasma

Equipment : Roche Cobas Pro e801

 

Method : Competition principle. Total duration of assay: 27 minutes.
▪ 1st incubation: By incubating the sample (9 µL) with the vitamin B12 pretreatment 1 and pretreatment 2, bound vitamin B12 is released. 

▪ 2nd incubation: By incubating the pretreated sample with the ruthenium‑labeled intrinsic factor, a vitamin B12‑binding protein complex is formed, the amount of which is dependent upon the analyte concentration in the sample.
▪ 3rd incubation: After addition of streptavidin-coated microparticles and vitamin B12 labeled with biotin, the still-vacant sites of the ruthenium‑labeled intrinsic factor become occupied, with formation of a ruthenium‑labeled intrinsic factor vitamin B12 biotin complex. The entire complex becomes bound to the solid phase via interaction of biotin and streptavidin. 
▪ The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed with ProCell II M. Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier.
▪ Results are determined via a calibration curve which is instrument specifically generated by 2‑point calibration and a master curve provided via the cobas link.