Sandwich operation video of ELISA Kit
example for Human Interleukin 6,IL-6 ELISA KIT
Procedure
1. Dilution of Standards
Dilute the standard by small tubes first, then pipette the volume of 50ul from each tube to microplate well,each tube use two wells, total ten wells.
60 ng/L
Standard No.1
300μl Original Standard + 150μl Standard diluents
40 ng/L
Standard No.2
300μl Standard No.1 + 150μl Standard diluents
20 ng/L
Standard No.3
150μl Standard No.2 + 150μl Standard diluent
10 ng/L
Standard No.4
150μl Standard No.3 + 150μl Standard diluent
5 ng/L
Standard No.5
150μl Standard No.4 + 150μl Standard diluent
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.
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