Human albumin ELISA - all positive results (most of the time !) - (Jun/09/2006 )

hello.

I am using a humal albumin ELISA and after a short development time i have been getting good reproducable results. My background reading is a little high (0.45) but the ELISA was functioning well.

I then started to hit problems - i started to get all positive results (across the entire plate including standards). I put this down to contamination, and re ran the ELISA with disposable pots for each of the stages (antibody addition, wash buffer, substrate etc etc). This resulted in a success.

however on repeating this method (again using disposable pots) i keep getting all positive results. Yesterday i ran a plate that resulted in the samples looking about right but all the standards were saturated all giving high abs (even the zero protein one). I suspected this was again due to contamination so re made all solutions in new glasswear and ran again. unfortunatly i obtained another all positive plate.

At some point i started using a new batch of substrate (TMB) and a new elisa kit, but the new kit has worked on occasion.


if the kit or substrate wasnt working i would not expect the occasional success, or is this normal ?

has anyone got any ideas as this is driving me mad and i am running out of time (i am doing a phd)

i you need any more info please ask.

thanks so much for helping.

Iain

Do you block your plate with BSA? Does the ELISA cross react with this?

We had a problem once where we re-used plastic following it being washed. Unfortunately either the washer hadn't been turned on or there was alot of HRP conjugated abs from Western Blotting going into the washer. As the HRP contaminated our ELISA troughs (for use with multichannel pipettes) which reacted with the TMB regardless of what was on the ELISA plate.

Could your ELISA troughs not be clean? We tend to use a fresh trough each time or you can wash it out throughly in between steps? Or is your plate not being washed well enough.

All the best,
Ceri

many many thanks for getting back to me !


ok, i use 1 % BSA as a blocking agent in a TRIS buffer which does not contain any tween 20. This is as per the manufactureres guidelines.

The first time i had the problems i was re-using my troughs (washing them in between obviously). As i susspected contamination, i re-ran the elisa using disposable troughts for eah step and it worked ! However, i have continued to do this and still had problems.

We do not have a plate washer here, so i have to wash the plate by hand. i do this using a multichannel pippete - i aspirate the samples / standards with a new pippette tip (disposable) and then wash 3 times after capture ab, block and 5 times after sample addition and HRP addition.

I have tried increasing the wash step to 6 times but there wasnt any difference.

as it used to work with out problems i am suggesting that it is the detection antibody hrp or the substrates that seem to be the problem.


If it is blocking that is the issue, why did it work the previous 7 or so times ?

i have remade all the buffers and diluents in new glasswear to make sure no contamination is coming in from there.

The kit instructions state there is zero cross reactivity with BSA.

hope this info helps !!

thanks again for your input.

Iain

Sounds like some kind of contamination problem somewhere if all the wells are coming up blue.

hi iain2276 ! i read your posts but you didnot mention your wach buffer composition and concentration of T-20 you are using. however i liek to suggest you few things.
(a) spare 4 wells for controls
1.all components (blocking agent, primary antibody, secondary antibody and substrate( but with out Antigen
2.all components except BSA
3.all components except primary antibody
4. all components except secondary antibody.

you should not get any color in well no.1. if you are getiing any colour then your blocking might be insufficient in that case you should also get color in well no.3 as insufficient blocking leaves spaces for the secondary Ab to bind but in this case you shlould get more colour in the 2 well.. it also implies that your washings are not proper. in that case you should get colour in well not.

you should get color in well 2. because it leaves primary to bind and therefore secondary Ab. usually the color is equal or more than the color in the maximum standard. more color indicates improper blocking.

you should not get any color in well no. 3 if you are getting any background colour then you blocking might be insufficient (color in well 2 is very high)or your washings are not proper.

you should not get any colour in well no. 4 .if you are getting then it implies that this colour is due to background and one of your solutions are not proper.

( you might increase the concentration of BSA to 2%

© you might also increase the concentration of T-20 to 0.1%. i have seen people using T-20 up to 0.3%.

(d) i strongly doubt your washing step. you might be washing your wells by pipetting in and out. i this case you always face the problem of repeatability. use a rapid-shaker for washing.
i hope these suggestions are helpful to you
all the best

since you are using there might be cross contamination. you might use other blocking agents like gelatin.