sorry for bothering you on holiday.. i have some questions related to product with catalogue number : SL0960Hu IFN-Gamma

Human Interferon γ ,IFN-γ ELISA Kit

we got this for the product processing procedure

2. In the Microelisa strip plate, leave a well empty as a blank control. In sample wells, 40μl of the sample dilution buffer and 10μl of the sample are added (the dilution factor is 5). The samples should be loaded onto the bottom without touching the well wall. Mix well with gentle shaking.

3. Incubation: incubate for 30 min at 37℃ after sealing with Closure plate membrane.

4. Dilution: Dilute the concentrated washing buffer with distilled water (30 times for 96 T and 20 times for 48 T).

5. Washing: Carefully peel off the Closure plate membrane, aspirate, and refill with the wash solution. After resting for 30 seconds, discard the wash solution. Repeat the washing procedure five times.

6. Add 50 μl HRP-conjugate reagent to each 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 gentle 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 well's color should change from blue to yellow.

11. Use a microtiter plate reader to read absorbance O.D. at 450 nm. The OD value of the blank control well is set to zero. The assay should be carried out within 15 minutes after adding the stop solution.

this is my question:

1. for procedure no. 4, the question is how many times is the wash buffer diluted? Is it once? The stock is 30 times.

Dilute 20ml wash solution to 600ml with distilled water.

2. for procedure no. 5, the question is how many microliters is the washing volume? is it 300 or more?

450-500ul or fill each hole to the top.

3. for procedure no. 9, the question is, is it possible to mixed chomogenic solutions A and B and then put into the well?
It is ok.