C/N:EE 2131

1. BACKGROUND

Vitamin B12, also known as cobalamin, is a vitamin containing metallic elements. In nature, vitamin B12 is synthesized exclusively by microorganisms, and higher plants and animals cannot produce it. Vitamin B12 is the only vitamin that requires assistance from an intestinal secretion (intrinsic factor) for absorption. Individuals with gastrointestinal abnormalities that result in a deficiency of this intrinsic factor may develop pernicious anemia, even with adequate dietary intake. Vitamin B12 is virtually absent in plant-based foods. It remains in the intestines for an extended period, taking approximately three hours to be absorbed (compared to most water-soluble vitamins, which are absorbed within seconds). The primary physiological functions of vitamin B12 include participating in the production of bone marrow red blood cells to prevent pernicious anemia and protecting brain nerves from damage.

2. Principle

This kit’s assay is based on the principle of specific antigen-antibody interactions in immunology. The microplate is coated with a VB12 antigen. VB12 antibody and VB12 standards or sample extracts are added. The VB12 extracted from the standards or samples competes with the VB12 coating antigen on the plate to bind a fixed quantity of antibody. Unbound antibodies, VB12 from standards, or samples are removed during the washing step. An enzyme-labeled secondary antibody is then added to react with the VB12 antibodies in the microplate. Unbound enzyme-labeled secondary antibodies are removed by washing. A substrate solution is added and incubated, during which the enzyme conjugate converts the colorless chromogen into a blue product. After adding a stop solution, the color changes from blue to yellow, and the optical density (OD) of each well is measured at 450 nm. The OD value is inversely proportional to the vitamin B12 concentration in the sample.

3. Kit KIT CONTENTS

Antigen-coated 96-well microplate ‌: 1 plate (96 wells, 12 strips × 8 wells)

Vitamin B12 standards ‌: 0 ng/mL, 0.5 ng/mL, 4.0 ng/mL, 10 ng/mL, 50 ng/mL (1 bottle × 1.0 mL)

‌ VB12 antibody working solution ‌: 1 bottle × 10 mL

Enzyme-labeled secondary antibody ‌: 1 bottle × 15 mL

‌10 × concentrated washing buffer ‌: 1 bottle × 50 mL

Dilution buffer ‌: 1 bottle × 50 mL

‌ Chromogenic substrate ‌: 1 bottle × 13 mL

‌ Stop solution ‌: 1 bottle × 7 mL

‌ Kit instructions ‌: 1 copy

COA report