Question:

I have been using your antigen capture kit (cat. # IAV-142-5) with reproduceable results and only recently have learned of a potential problem with the wash step---namely, that if the wash step is not properly executed the potential for having the negative controls (provided in your kit) and the postitive controls (also provided in your kit) display undiscernable optical densities (the negative and positive control OD are approximately equal, but well above the background/blank control).

Is it reasonable to suspect that a clumsy incomplete wash may explain why the negative and positive controls provided in the kit would dispaly comparable values, versus the blank/background control?

Resolution:

You are correct in saying that the wash is critical. An incomplete or clumsy wash will most definitely affect the results in the way you describe.

Incubation at higher temperatures than room temperature can also approximate these results.

You are adding 100 µl sample + 100 µl conjugate to the well for a total of 200 µl volume. If this is not washed very well, addition of the TMB will result in non-specific color generation.

Please wash well (gently - not vigorously) with a full well of wash buffer 6 times as specified in the package insert.