Elisa Kit Vitamin B12 PRINCIPLE OF THE ASSAY This assay employs the direct competitive inhibition enzyme immunoassay technique. Antibody specific for VB12 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells with biotin-conjugated VB12. A competitive inhibition reaction is launched between VB12 (Standards or samples) and biotin-conjugated VB12 with the pre-coated VB12 antibody. After washing, avidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound reagent, a substrate solution is added to the wells and color develops in opposite to the amount of VB12 bound in the initial step. The color development is stopped and the intensity of the color is measured. DETECTION RANGE 1.56 pg/ml-90 pg/ml. SENSITIVITY The minimum detectable dose of human VB12 is typically less than 1.498 pg/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest human VB12 concentration that could be differentiated from zero. It was determined the mean O.D value of 20 replicates of the zero standard added by their three standard deviations. SPECIFICITY This assay has high sensitivity and excellent specificity for detection of human VB12. No significant cross-reactivity or interference between human VB12 and analogues was observed. Note: Limited by current skills and knowledge, it is impossible for us to complete the cross-reactivity detection between human VB12 and all the analogues, therefore, cross reaction may still exist. OTHER SUPPLIES REQUIRED  Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm.  An incubator which can provide stable incubation conditions up to 37°C±0.5°C.  Squirt bottle, manifold dispenser, or automated microplate washer.  Absorbent paper for blotting the microtiter plate.  100ml and 500ml graduated cylinders.  Deionized or distilled water.  Pipettes and pipette tips.  Test tubes for dilution. PRECAUTIONS The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material. SAMPLE COLLECTION AND STORAGE  Serum Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 15 minutes at 1000 ×g. Remove serum and assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles.  Plasma Collect plasma using EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 ×g at 2-8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles.