Active-B12 (Holotranscobalamin) ELISA

Sample

serum

Species Reactivity

Human

Detection Method

sELISA

Intended Use

The Active-B12 (Holotranscobalamin) ELISA is an enzyme-immunoassay (ELISA) for the quantitative determination of holotranscobalamin (HoloTC) in human serum. HoloTC (vitamin B12 bound to transcobalamin) is used as an aid in the diagnosis and treatment of vitamin B12 deficiency.

Storage

Opened (In-Use) Kit Stability:
A kit was opened, and reused on three occasions over a three month period with no adverse effect on kit performance. Following use, components must be returned to storage at 2-8°C.
Unopened kit stability:
At 2-8°C unopened components are stable until as directed on the labels.

Performance Characteristics

Unit of Measure
The unit of measurement for the Active-B12 (Holotranscobalamin) assay is pmol/L.
Measuring Interval (Reportable Range)
The measureable range of the assay is 10 pmol/L to 128 pmol/L.
Accuracy
A correlation study was performed with serum specimens from apparently healthy adults. All specimens were analysed using the Active-B12 (Holotranscobalamin) ELISA and another commercially available Holotranscobalamin assay according to the CLSI document EP9-A211. Specimen concentrations ranged from 13.8 to 112.8 pmol/L in the assay. The data obtained gave the following statistical values:

Limit of Blank
In a representative study, Limit of Blank determinations were performed using two low-level holotranscobalamin samples and two reagent lots (120 replicates per reagent lot). The limit of blank of the Active-B12 (Holotranscobalamin) ELISA was found to be 4.9 pmol/L (rounded to 1 decimal place).
High Dose Hook
High dose hook is a phenomenon whereby very high level specimens may read within the dynamic range of the assay. For the Active-B12 (Holotranscobalamin) ELISA. No high dose hook effect was detected with two samples with a concentration of approximately 419 and 2236 pmol/L.

Detection Limit

Limit of Detection
In a representative study, Limit of Detection determinations were performed using five low-level holotranscobalamin samples and two reagent lots (120 replicates per reagent lot). The limit of blank of the Active-B12 (Holotranscobalamin) ELISA was found to be 8.1 pmol/L (rounded up to 1 decimal place).
Limit of Quantitation
Limit of quantitation determinations were performed using five low-level holotranscobalamin samples and two reagent lots (120 replicates per reagent lot). The limit of quantification of the Active-B12 (Holotranscobalamin) ELISA was found to be 8.3 pmol/L (rounded to 1 decimal place).

General Description

Three binding proteins are involved in the transport of vitamin B12 around the body - Intrinsic Factor (IF), transcobalamin (TC) and haptocorrin (HC). These binding proteins ensure the efficient uptake of the very small amounts of vitamin B12 available from the diet. When TC and HC bind vitamin B12 the resulting complexes are known as holotranscobalamin (HoloTC) and holohaptocorrin (HoloHC) to distinguish them from the proteins carrying no vitamin. The major fraction in the circulation, HoloHC, represents 70-90% of vitamin B12 in the blood but is biologically inert. HoloTC represents only 10-30% of vitamin B12 circulating in the blood but is the only form of vitamin B12 that can be taken up by cells in the body. The TC protein alone transports vitamin B12 from its site of absorption in the ileum to tissues and cells. The vitamin is then internalised as the HoloTC (vitamin B12 bound to transcobalamin) complex via a specific receptor-mediated uptake. This process delivers vitamin B12 into the cells of the body and provides the vitamin as a co-enzyme for essential cellular functions such as DNA synthesis. As HoloTC has a shorter circulating half-life compared to HoloHC the earliest change that occurs on entering negative vitamin B12 balance is very likely to be a decrease in serum HoloTC concentration. The measurement of Total Serum B12 suffers from some limitations; in particular, most of the measured cobalamin is bound to biologically inert HC. Several studies have been published which conclude that HoloTC would be a better indicator of vitamin B12 status than Total Serum B12. As expected, HoloTC levels are low in patients with biochemical signs of vitamin B12 deficiency. Low values have been reported in vegetarians, vegans, and in populations with a low intake of vitamin B1. Notably, low levels of HoloTC but not Total B12 in serum were reported in patients with Alzheimer's disease compared to levels in a healthy control group. HoloTC levels reflect vitamin B12 status, independent of recent absorption of the vitamin.

Standard Curve