Bioindex T4 EIA Hormone Test Kit – 96 Test:

Enzyme Immunoassay for the Quantitative
Determination of Total Thyroxine (T4) in
Human Serum

REAGENTS
Materials provided with the kit:

 Goat Anti-Mouse IgG Coated Microtiter Wells, 96 wells
 Enzyme Conjugate Concentrate (11x), 1.3 ml.
 Enzyme Conjugate Diluent, 13 ml.
 T3 Reference Standards, 0, 0.75, 1.5, 3.0, 6.0 and 10.0 ng/ml, 1
set, 1.0 ml each, ready to use.
 Antibody Reagent, 7 ml.
 TMB Reagnet (One-Step), 11 ml.
 Stop Solution (1N HCl), 11 ml

Materials required but not provided:

 Precision pipettes: 50 l, 100 l and 1.0 ml.
 Disposable pipette tips.
 Distilled water.
 Vortex mixer or equivalent.
 Absorbent paper or paper towel.
 Graph paper.
 Microtiter plate reader.

ASSAY PROCEDURE (how to use)
1. Secure the desired number of coated well in the holder. Make
data sheet with sample identification.
2. Pipette 50 l of standard, samples, and controls into
appropriate wells.
3. Dispense 50 l of the Antibody Reagent into each well. Mix
thoroughly for 30 seconds.
4. Add 100 l of Working Conjugate Reagent into each well.
Mix thoroughly for 30 seconds. It is important to have a
complete mixing in this step.
5. Incubate at room temperature for 60 minutes.
6. Remove the incubation mixture by flicking plate contents into a
waste container.
7. Rinse and flick the microtiter wells 5 times with distilled or
deionized water. (Please do not use tap water.)
8. Strike the wells sharply onto absorbent paper to remove
residual water droplets.
9. Dispense 100 l TMB Reagent into each well. Gently mix for
10 seconds.
10. Incubate at room temperature in the dark for 20 minutes
without shaking.
11. Stop the reaction by adding 100 l of Stop Solution to each
well.
12. Gently mix for 30 seconds.
 It is important to make sure that the blue color changes to
yellow color completely.
13. Read OD at 450 nm with a microtiter reader within 15
minutes.

STANDARD CURVE
Concentration
(ug/dL) O.D 450 nm
0 2.669
2 1.687
5 1.188
10 0.832
15 0.576
25 0.307
This curve was obtained on the date of performance testing of the components. It is for the only
purpose of illustration. Each laboratory should establish its own standard curve