Rat Estradiol,E2 ELISA Kit
Notes:
- Sample extraction and ELISA assay should be performed as soon as
possible after sample collection. The samples should be extracted
according to the relevant literature. If ELISA assay can not be
performed immediately, samples can be stored at -20℃.Repeated
freeze-thaw cycles should be avoided.
- Our kits can not be used for samples with NaN3 which can inhibit
the activity of HRP.
Materials provided with the kit
| Materials provided with the kit | 48 determinations | 96 determinations | Storage |
| 1 | User manual | 1 | 1 | R.T. |
| 2 | Closure plate membrane | 2 | 2 | R.T. |
| 3 | Sealed bags | 1 | 1 | R.T. |
| 4 | Microelisa stripplate | 1 | 1 | 2-8℃ |
| 5 | Standard:180 pg/ml | 0.5ml×1 bottle | 0.5ml×1 bottle | 2-8℃ |
| 6 | Standard diluent | 1.5ml×1 bottle | 1.5ml×1 bottle | 2-8℃ |
| 7 | HRP-Conjugate reagent | 3ml×1 bottle | 6ml×1 bottle | 2-8℃ |
| 8 | Sample diluent | 3ml×1 bottle | 6ml×1 bottle | 2-8℃ |
| 9 | Chromogen Solution A | 3ml×1 bottle | 6ml×1 bottle | 2-8℃ |
| 10 | Chromogen Solution B | 3ml×1 bottle | 6ml×1 bottle | 2-8℃ |
| 11 | Stop Solution | 3ml×1 bottle | 6ml×1 bottle | 2-8℃ |
| 12 | Wash solution | 20ml (20X)×1bottle | 20ml (30X)×1bottle | 2-8℃ |
Procedure
Dilution of Standards
1.Ten wells are set for standards in a Microelisa stripplate. In
Well 1 and Well 2, 100μl Standard solution and 50μl Standard
Dilution buffer are added and mixed well. In Well 3 and Well 4,
100μl solution from Well 1 and Well 2 are added respectively. Then
50μl Standard Dilution buffer are added and mixed well. 50μl
solution is discarded from Well 3 and Well 4. In Well 5 and Well 6,
50μl solution from Well 3 and Well 4 are added respectively. Then
50μl Standard Dilution buffer are added and mixed well. In Well 7
and Well 8, 50μl solution from Well 5 and Well 6 are added
respectively. Then 50μl Standard Dilution buffer are added and
mixed well. In Well 9 and Well 10, 50μl solution from Well 7 and
Well 8 are added respectively. Then 50μl Standard Dilution buffer
are added and mixed well. 50μl solution is discarded from Well 9
and Well 10. After dilution, the total volume in all the wells are
50μl and the concentrations are 120 pg/ml, 80 pg/ml, 40 pg/ml, 20
pg/ml and 10pg/ml, respectively
2. In the Microelisa stripplate, leave a well empty as blank
control. In sample wells, 40μl Sample dilution buffer and 10μl
sample are added (dilution factor is 5). Samples should be loaded
onto the bottom without touching the well wall. Mix well with
gentle shaking.
3. Incubation: incubate 30 min at 37℃ after sealed with Closure
plate membrane.
4. Dilution: dilute the concentrated washing buffer with distilled
water (30 times for 96T and 20 times for 48T).
5. Washing: carefully peel off Closure plate membrane, aspirate and
refill with the wash solution. Discard the wash solution after
resting for 30 seconds. Repeat the washing procedure for 5 times.
6. Add 50 μl HRP-Conjugate reagent to each well except the blank
control well.
7. Incubation as described in Step 3.
8. Washing as described in Step 5.
9. Coloring: Add 50 μl Chromogen Solution A and 50 μl Chromogen
Solution B to each well, mix with gently shaking and incubate at
37℃ for 15 minutes. Please avoid light during coloring.
10. Termination: add 50 μl stop solution to each well to terminate
the reaction. The color in the well should change from blue to
yellow.
11. Read absorbance O.D. at 450nm using a Microtiter Plate Reader.
The OD value of the blank control well is set as zero. Assay should
be carried out within 15 minutes after adding stop solution.
Precision
Intra-assay Precision (Precision within an assay): 3 samples with
low, middle and high level E2 were tested 20 times on one plate,
respectively.
Inter-assay Precision (Precision between assays): 3 samples with
low, middle and high level E2 were tested on 3 different plates, 8
replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%
Assay range
1.8 pg/ml -150 pg/ml
