Human MICB ELISA Kit (ab100593)

Thank you for contacting Abcam.

After reviewing your data, it looks as though you are experiencing very high background (>0.8 OD). In general, the background response should be ≤0.2 OD so as you can see, the background that the customer measured are extremely high. Please could you answer the following question

· What were the OD values for the MICB kit?
· What temperature was the Assay Diluent B stored at?
· How long was the Assay Diluent B store for?
· What dilution fold was used to prepare the HRP-streptavidin?
The good news is that high background issues are easily correctable. Below are the most common causes of high background and how to minimize it:

Common Causes of High Background

Too much HRP-Streptavidin may cause background. Briefly centrifuge the vial of HRP-streptavidin concentrate (Item G) and pipette up and down to mix gently before using, since precipitation may form during storage.
Too much detection antibody may also contribute to high background. Make sure of correct dilution fold of both the biotinylated antibody (Item F) and HRP-Streptavidin. Both should be diluted with 1x Assay Diluent B.
Long incubation times in some steps (i.e. overnight sample/standard incubation) can cause high background.
And finally, the wash steps may introduce background: if the plate is insufficiently washed, or if the wash buffer itself was contaminated. Wash buffer must be removed completely from the wells after each wash. We recommend using an automated plate washer or multi-channel pipettor. Squirt bottles are not advised.


If the high background persists after checking all these steps, try reducing the amount of HRP-streptavidin (by increasing the dilution by 1.5 or 2-fold).

Please let me know if you have any questions or comments.

Thank you for taking the time to complete our questionnaire. Reviewing this case, I would like to offer some suggestions to help optimise the results from this kit:

1) Too much HRP-streptavidin may cause a high signal. Double check the preparation of the HRP Streptavidin; it is important to mix the liquid thoroughly by pipetting up and down, as precipitate may form during storage. You could even try increasing the dilution of HRP-streptavidin to 30,000x or 50,000x.

2) Long incubation times may cause high signal. Try incubating the sample at 2.5 hours at room temperature rather than overnight.

3) Be sure the incubation temperature is 25 degrees C or less; higher temperatures may cause a higher signal.

4) Vigorous shaking may cause high signal. We use a Rota-Shake Genie rocker set to 2 cycles per second.

5) If necessary, you may try decreasing the concentration of the standards to a range that is more workable for you. For instance, you could make their highest point 5000 pg/ml or so.

If you wish you can also send us the data from the experiment so we can find out what may be causing the saturated results.

In any case, do not hesitate to contact us again if after reviewing these steps the results are not still satisfactory, and I will be more than happy to help you further.

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.


I look forward to receiving your reply.