Human ferritin,FE ELISA Kit​

Materials provided with the kit

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 24ng/ml,16 ng/ml,8 ng/ml,4ng/ml and 2ng/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 Human Ferritin (FE) were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Human Ferritin (FE) were tested on 3 different plates, 8 replicates in each plate.

CV(%) = SD/meanX100

Intra-Assay: CV<10%

Inter-Assay: CV<12%

Assay range

0.4ng/ml-25ng/ml