FOR FLUID MILK SAMPLES With 2% FAT
Enzyme immunoassay kit for the quantitative determination of Vitamin-A in fluid milk containing 2% milk fat.
For in vitro quantification use only.
I. PROPRIETARY NAME
The VitaKit A contains sufficient material to assay 96 tests.
II. APPLICATION AND INTENDED USE
Dairy milk is fortified with vitamins A & D, as milk has become the major source of these vitamins for human beings. Regulatory agencies have set standards specifying the amount of vitamins A and D to be added to milk products. The methodology has been designed to extract Vitamin A (all trans retinol) from milk fat, and to directly quantify the Vitamin A content using an ELISA based assay. Other methods that can detect vitamins in dairy milk are time consuming and require specialized laboratory equipment and trained personnel.
The VitaKit ATM provides materials for the quantitative measurement of vitamin-A in fluid milk containing 2% milk fat for routine quality control purpose. This assay is intended for in vitro quantification only.
III. PRINCIPLE OF THE METHOD
The vitamin A ELISA test is based on the principle of a sandwich enzyme-linked immunosorbent assay. The assay system utilises immobilization of a monoclonal antibody directed against a distinct antigenic determinant on the vitamin-A molecule (retinol) on a solid phase. A second Anti-vitamin-A monoclonal antibody conjugated to horseradish peroxidase (HRP) is used as the detecting antibody in the assay. The test sample is allowed to react sequentially with the two antibodies, resulting in the vitamin-A molecules being sandwiched between the solid phase and enzyme-linked antibody. After the incubation, the wells are washed with distilled water to remove all unbound labelled antibodies. TMB solution is added as a substrate, resulting in the development of a blue color. The reaction is stopped with the addition of 0.2M H2SO4, changing the color to yellow. The concentration of vitamin A is directly proportional to the color intensity of the test sample. Absorbence is measured spectrophotometrically using a plate reader at 450 nm.
PERFORMANCE CHARACTERISTICS
Sensitivity: The range for this assay under the specified conditions is from 0.055 I.U./mL to 0.45 I.U./mL.
Limit of Detection: The minimum detection limit for this assay is 33 IU/100 mL or 0.33 IU/mL.
Precision & reproducibility: The relative standard deviation for interassay and intrassay was determined to be 8.6 % and 1.9% respectively.
Cross Reactivity and Specificity: The kit did not exihibit any significant cross reactivity with cholesterol and vitamin D3 and is specific for Vitamin A.
Standard curve Linearity Linearity was determined to be 0.996
(Average of six independent assays) with %RSD of 1.5%.
