TRUSTwell Vitamin-D (Total) ELISA

TEST PRINCIPLE

The TRUSTwell Vitamin-D (Total) ELISA is a solid-phase enzyme-linked immunosorbent assay for the quantitative determination of Vitamin-D level in human serum. 

The TRUSTwell Vitamin-D (Total) ELISA is comprised of five key components: 

1) Solid microwells pre-coated with anti-Vit-D antibody 
2) Vit D Calibrators 
3) Vit D Assay Controls comprised of human serum with the Vit D concentration on the label. Contains preservative. Store at 2-8°C. 
4) Vit D Releasing Agent comprised of Vitamin D binding protein releasing agents, 
5) Vit D Enzyme Reagent comprised of 25-OH Vitamin D3 analog-HRP conjugate in a protein stabilizing matrix (Vit-D-HRP). 
 

During the assay, the calibrator, control, or test specimen and Vit D Enzyme Concentrate mixture in the releasing agent are added to the microwell coated with Vit D antibody. During the 45-minute incubation, the Vit D in the patient specimen competes with the HRP-Vit-D to bind to the plate coated antibody. Excess conjugate and serum protein are then removed by washing. In the next step, the TMB single substrate is added. The presence of the HRP-Vit-D is shown by the development of a blue color. The reaction is then terminated with the Stop Solution and absorbance is determined using a spectrophotometer at 450/620-690 nm. The color development is indirectly proportional to the concentration of Vit D in the calibrators, controls, or specimens. 

A Dose Response Curve (DRC) can be generated to determine the concentration of unknown by plotting the calibrator Vit D concentrations on the x-axis and the OD values on the y-axis. By interpolating the absorbance, the concentrations for unknown specimens can be determined.

STORAGE AND STABILITY

All reagents except the concentrated wash buffer are ready to use as supplied. Store all components at 2-8°C. Do not freeze. Avoid strong light. Ensure that the reagents are brought to room temperature before opening. Reseal the microwells after removing the desired number of wells. Place unused wells in the resealable plastic bag provided and return to 2-8°C. All reagents are stable through the expiration date printed on the label if not opened. Once opened, the kit is stable for 60 days at 2-8°C, or until the labeled expiration date, whichever is earlier.