Free T4 ELISA Kit

Free T4 ELISA kit description:

The Diagnostic Automation Free T4 ELISA is intended for the quantitative determination of the Free Thyroxine (T4) concentration in human serum.

Materials provided with Free T4 ELISA Test Kit:

1. Anti-T4 Antibody-coated ELISA Microplate (96 wells)

2. enzyme conjugate reagent: 1 Vials contains T4-HRP with preservative buffer, ready to use.(1 Vials, 12 ml)

3. standard set, contains 0, 0.5, 1, 2.5, 5, 10 ng/dL,ready for use. (6 vials. 0.5 ml)

4. TMB Substrate: Hydrogen peroxide and tetra-methyl benzidine in preservative buffer, ready to use. (1 vial of 11ml)

5. Stop Solution: a normal hydrochloric acid (1 vial, 6 ml).

6. Concentrated washing buffer: PBS-Tween (1 vial 30ml) (20X)

7. Control serum: Free T4 in serum with preservatives(1Vial, 0.5 ml)

Materials Required, not Provided:

1. Precision pipettes(20,50,100, µl)

2. Distilled or deionized water

3. EIA kit Microplate Washer

4. EIA kit Microplate Reader with a 450 nm, If possible, 630 nm as reference.

5. Absorbent paper

Introduction

Thyroxine is the main hormone of the thyroid. Most thyroxine in blood circulation is predominantly bound to the thyroxine binding globulin (TBG) and to a lesser extent to the thyroxine binding prealbumin and albumin. A very small percentage of thyroxine remains unbound in blood as free. These unbound fractions of the thyroid hormone are thought to be responsible for the biologic activity. The metabolic processes are regulated entirely by the concentration of the free thyroid hormones. As the concentrations of the carrier proteins alter, the total thyroxine level changes so that the free thyroxine concentration remains constant. Thus, measurement of free thyroxine concentrations correlates better with clinical status than total thyroxine levels. The changes in total thyroxine concentrations associated with pregnancy, Liver failure and kidney failure occasionally result in total T4 levels over the limits of normal while the free thyroxine concentration remains within the normal reference range. The total T4 can be elevated or lowered. The free T4 concentration shows the real state of thyroid function and measurement of this free T4 is very important.

Principle of the assay

This ELISA test is based on the use of specific monoclonal antibody against a distinct antigenic determinant on the intact T4 molecule. Anti- T4 monoclonal antibody with a certain concentration is used for coating the wells. A certain amount of serum and enzyme conjugate containing the bounded T4 to HRP-T4 enzyme. In the incubation period, the present free T4 and T4-HRP in serum, compete for binding to the coated Anti-T4 monoclonal antibody in wells; After 45-minute incubation at room temperature, the well is washed with washing solution. Then substrate solution and chromogen solution are added and incubated for 15 minutes, resulting in the development of blue color. The color development is stopped with the addition of stop solution, and the resulting yellow color is measured spectrophotometrically at 450 nm. The concentration of Free T4 is inversely proportional to the color intensity of the test sample.