T3 EIA Kit – 96 Test:

ENZYME IMMUNOASSAY FOR THE QUANTITATIVE
DETERMINATION OF TRIIODOTHYRONINE (T3)
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.