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I have ran the ELISA using your last kit ((ab100747). As requested I am sending the absorbance values we got on the Standard. From my previous experience I noticed rather lighter colour reaction for the Standardcompared to the previous Standards. You can give comments on these values, may be we can improve the assay procedure.
Another thing I was thinking about whether your precoated plate needs to be blocked, although blocking is not a critical factor as it is in immunohistochemistry.
I also would like to know what is chemistry for adding STOP solution ? Will it have any differential effect on lipopolysaccharide(LPS) in terms of hydrolysis?
Asked on Sep 20 2012
Thank you very much for your email and for sending your absorbance values.
I've reviewed this data with our lab, and we do agree that these standard absorbances are weaker than expected. Did yourun any samples as well?This would help narrow down whether the issue is with just the standard protein only or with a common detection reagent.
All of the kits that you've used are from the same production batch, so we wouldn't expect much inherent differences between the kits, but if you think there is something wrong with an individual component we can certainly replace the kit again for you.
I’ve attached some tips from the lab regarding how to get strong signal from the standards, and also forimproving inter-assay variability which might be helpful. Some of the more useful points:
Once reconstituted (50ng/ml), the standard should be stored at ≤-20 for no more than 1-2 weeks. An extra vial of Item C is included in every kit to limit storage issues.
All other reagents once thawed need to be stored at 4°C for no more than 1 month. Do not store any reagent in their diluted form, they are not as stable diluted.
Do not vortex the standard to mix. Rather gently mix by pipetting the reconstituted standard solution up and down several times with a pipette.
Briefly centrifuge reagent vials prior to opening to ensure complete recovery of the lyophilized powder or liquid concentrate as power/liquid can adhere to the inside walls or cap during transit.
It isn't necessary to block the pre-coated plate, it is ready to use. Generally these prepared plates are already blocked afterthe capture antibody is coatedto the wells. The stop solution is sulfuric acid, which is commonly used to stop the reaction that is occuring between the HRP andTMB. Before sulfuric acid is added, the reaction mixture is producing a cation free radical that is blue, but adding the acid results in a diimine terminal product (yellow) that is more stable than the blue radical.
Iapologize if I'mmisunderstanding the question about LPS hydrolysis, but based on a search of the literature it does look like sulfuric acid itself can cause LPS hydrolysis-
I hope that this information will be useful, and I look forward to hearing from you. Please let me know if you have any further questions or if there is anything else that we can do for you.
Answered on Sep 20 2012