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Previously great ELISAs have stopped working


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5 replies to this topic

#1 lab ratty

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Posted 16 March 2010 - 08:17 AM

Hey,
Our lab is having a never-ending ELISA issue, our assays which were all previously working beautifully and giving consistent results have stopped working right after we returned from holiday shut-down, as in no color change or very little color will develop along with a loss of proportionality. Here's our protocol:

Incubate overnight @ 4C in 0.2M sodium carbonate, 0.35M sodium bicarbonate, 0.03M sodium azide pH 9.6

Block with 1% BSA in PBS with 0.02% azide for 1 hr at RT while rocking

Incubate overnight @ 4C with primary in diluent (PBS, 0.05% Tween, 0.1% BSA, 0.02% azide)

Wash 5x in wash buffer (PBS, 0.05% Tween, 0.02% azide) and blot plate after each wash

Incubate 1 hr @ RT in secondary (alkaline phosphatase conjugated)

Wash 5x, then develop in KPL BluePhos substrate (BCIP based)

All assays have stopped working regardless of which primary antibody was used, and here's the list of everything we have done to try and fix the problem which we now believe may have to do with our water source (river water + spring runoff = bad???):

1) tried different lots of the binding plate (Corning high-binding)
2) had our water purification system serviced - new reverse osmosis membrane and thorough cleaning of the system and reservoir tank
3) purchased new coating buffer chemicals
4) purchased pre-made coating buffer capsules from Sigma, 0.05M carbonate and bicarbonate
5) purchased distilled water from the local CVS
6) tested all protein lysates and antibodies in westerns and dot blots
7) tried binding in PBS

Nothing has worked completely, some of our primaries will give a bit of decent color (~60% of former OD values) but we just can't find that magical combination that gets us back to where we were with all of our primaries.

Our coating buffer used to come out right at pH 9.6 without any adjustment, but now after our water service visit the pH starts around 10.1 to 10.2. We have brought aliquots of coating buffer back down to 9.6 with HCl, but it seems like we lose some carbon as CO2 gas during the process. If anyone has experienced something similar or has any suggestions please help, we're really at our wit's end and we're trying to graduate two students who need ELISA data for their dissertation projects. Thanks in advance for reading this and for any suggestions!

Lab ratty

#2 Gerard

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Posted 16 March 2010 - 11:44 PM

First question, did you test the secondary with the substrate as you haven't it in your list and is it working?
Ockham's razor
Pluralitas non est ponenda sine necessitate
-- "You must assume no plural without necessity".

#3 sgt4boston

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Posted 17 March 2010 - 02:23 AM

You said "all previously working" there has to be one common reagent between them...the conjugate is a very good place to begin looking...if it is common to all the assays you describe.

#4 lab ratty

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Posted 18 March 2010 - 06:33 PM

Hi Gerard and sgt4boston, thanks for your suggestions. We have tested our conjugate, our AP-conjugated secondary is the same detection method we use for our Westerns, all of which are consistently nice so it seems like we're ok in that respect. A few thoughts we have had recently is that we don't have direct evidence of a lack of binding to the plates, it's more of an assumption, so we'll do a quick Sypro stain and see if anything sticks (and whether there is proportionality), and we had also considered using TBS-based buffers in place of the PBS since our Western protocol is so very similar to the ELISA and the Westerns are all in good shape. Not sure what a reviewer would say about TBS, but we're willing to try just about anything at this point!
Lab ratty

#5 Gerard

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Posted 19 March 2010 - 01:27 AM

Did you make fresh coating buffer, the pH change by different water isn't logical it's a buffer and no pH change is to be expected.
Did you check the pH of the substrate? And a question as I don't know the KPL BluePhos substrate, can the azide be a problem? Wrong made, 0.2% azide in the wash buffer.
Ockham's razor
Pluralitas non est ponenda sine necessitate
-- "You must assume no plural without necessity".

#6 lab ratty

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Posted 21 March 2010 - 04:14 PM

Yes, we have remade coating buffer numerous times. The BluePhos is something we have used for years and it has never been impaired by the presence of azide in our other buffers, plus the ELISAs stopped working halfway through the batch of the BluePhos we were using at the time so that probably isn't it (didn't check pH but since it is a commercial product the pH is something that would be checked by Qual Control before it leaves the supplier, so I'm not sure we could get much info out of the pH, plus we don't know what the pH was before since we've never measured it). After the problems started we remade every single solution and double-checked our measurements and dilutions of chemicals including the azide solution, and it made no difference to our results. Buying pre-coated plates just isn't worth the money, so at the moment we're pretty well stuck. We did recently have an idea regarding how we dilute samples prior to coating the plate; we usually make all of our dilutions in the coating buffer directly, but we saw a product from a company to be used for diluting samples prior to coating which is supposed to enhance adsorption of antigen to the plate and stabilize the tertiary structure (ImmunoChemisrty Technologies 5x antigen coating buffer). Anyone have any experience with commercial diluting buffers?




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