Background: Vitamin B12 is very important for the human body. Early diagnosis of vitamin B12 deficiency is essential because it is associated with many problems such as fatigue, lethargy, depression, poor memory, breathlessness, headaches, pale skin, mania, psychosis, etc. Most of the vitamin B12 molecules in serum bind to specific proteins as a complex; so, although researchers have developed some sensitive methods to quantify vitamin B12, few can be used for human serum detection of vitamin B12 because of disturbed releasing process. In this study, we are aiming to develop a rapid and accurate chemiluminescence immunoassay (CLIA) for human serum testing.
Methods: In this study, we studied and optimized labelling of biotin molecules to vitamin B12 binding protein which had been extracted from hog gastric mucus, labelling of vitamin B12 molecules to horse radish peroxidase (HRP), releasing of vitamin B12 from its bound state in serum, concentrations of reagents, and incubation times.
Results: The developed method shows good performance and thermo-stability. The limit of detection (LOD) is 41 pg/mL, the recovery rate is 90.7% - 107.4%. The intra-assay CV is 4.4% - 8.0% and the inter-assay CV is 4.5% - 12.1%. Cross reactivity (CR) values are 49.4% for hydroxocobalamin and lower than 0.1% for vitamin B1, B2, B3, B9, and C. The method comparison results show that the correlation coefficient (R2) is 0.91, with a slight negative bias of -4.1% [95% limits of agreement (± 1.96 SD); -39.6% to 31.6%].
Conclusions: The developed method shows high sensitivity and specificity and good correlation using a Beckman Coulter instrument while the whole test needs only 40 minutes. It can fully satisfy the very busy clinical labs.