Sheep Estrogen ELISA Kit

1.

When diluting samples and reagents, they must be mixed completely and evenly. Before adding TMB into wells, equilibrate TMB Substrate for 30 minutes at 37°C. It is recommended to plot a standard curve for each test.

2.

Set standard, test samples, control (blank) wells on the pre-coated plate respectively, and then, records their positions.
It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (blank) wells!

3.

Add 50ul of Standard, Blank, or Sample per well. The blank well is added with Sample/Standard Dilution Buffer. Immediately add 50ul Biotin-labeled Antibody Working Solution into each well. Cover with the Plate sealer we provided. Gently tap the plate to ensure thorough mixing. Incubate for 45 minutes at 37°C.
(Solutions are added to the bottom of microplate well, avoiding inside wall touching and foaming as much as you can.)

4.

Remove the cover, and wash plate 3 times with Wash Buffer, and let the wash buffer stay in the wells for 1 minute each time. After the last wash, remove any remaining Wash Buffer by aspirating or decanting

5.

Add 100ul SABC Working Solution into each well. Cover it with a new Plate sealer.
Incubate for 30 minutes at 37°C.

6.

Remove the cover and wash plate 5 times with Wash Buffer, and let the wash buffer stay in the wells for 1-2 minutes each time.

7.

Add 90ul TMB Substrate into each well, cover the plate and incubate at 37°C in dark within 10-20
minutes. (Note: The reaction time can be shortened or extended according to the actual color change, but not more than 30 minutes. You can terminate the reaction when apparent gradient appeared in standard wells.)

8.

Add 50ul Stop Solution into each well. The color will turn yellow immediately. The adding order of Stop Solution should be as the same as the TMB Substrate Solution

9.

Read the O.D. absorbance at 450nm in Microplate Reader immediately after adding the stop solution.

ELISA Microplate(Dismountable)

8x12

2-8°C/-20°C

Lyophilized Standard

2 vial

2-8°C/-20°C

Sample Dilution Buffer

20ml

2-8°C

Biotin-labeled Antibody(Concentrated)

60ul

2-8°C(Avoid Direct Light)

Antibody Dilution Buffer

10ml

2-8°C

HRP-Streptavidin Conjugate(SABC)

120ul

2-8°C(Avoid Direct Light)

SABC Dilution Buffer

10ml

2-8°C

TMB Substrate

10ml

2-8°C(Avoid Direct Light)

Text

10ml

2-8°C

Wash Buffer(25X)

30ml

2-8°C

Plate Sealers

5

Serum

Place whole blood sample at room temperature for 2 hours or put it at 2-8°C overnight and centrifugation for 20 minutes at approximately 1000×g, Collect the supernatant and carry out the assay immediately. Blood collection tubes should be disposable, non-pyrogenic, and non-endotoxin.