Poor blocking in ELISA - any suggestions? - (Jan/20/2014 )

HI all,

I'm working on an ELISA for HPV-E7 protein (human). I'm using a polyclonal capture and a monoclonal biotinylated detection antibody format. The following is my protocol, I am getting a high background and also high signal for my negative control. I am using HeLa cell lysate to optimize the assay as it contains HPV18 E7. I'm using C33A cells as the negative control, as they're an HPV-free cancer strand. However, the C33A cells still show a relatively high signal (compared to 1% BSA only). Any thoughts on what is wrong with my protocol, or do you think it's the antibodies? Thanks

Ben

Hi,

I'm using the 37515 pierce superblock and I'm getting quite a high background. I am using a sandwich ELISA assay with a NUNC maxisorp plate.

1. polyclonal capture antibody (8 ug/mL) in coating buffer placed incubated overnight at 4 C.

2. Washed 3x with 300 uL 1% BSA in TBST (.05% tween 20).

3. Blocked 4x with 300 uL 37515, no incubation time

4. Washed 3x with 300 uL 1% BSA in TBST (.05% tween 20)

5. Monoclonal biotinylated detection antibody applied (0.5 ug/mL) diluted in 1% TBST-BSA for 1 hour at room temperature with gentle agitation

6. Washed 3X with 300uL 1%BSA-TBST

7. 100 uL poly-HRP (100 ng/mL)applied for 1 hour at room temperature

8. Washed 6x with 300 uL 1%BSA-TBST

9. 100 uL ultra-TMB applied for 30 minutes

10. 100 uL 2M H2S04 Applied

Do you have any suggestions to better this protocol? Thank you

-bdg25-

This may not be your problem but I think your blocking is insufficient. I've never encountered an ELISA protocol that does not contain a blocking incubation step. Perhaps you should try blocking for 30min at 37C or 1hr at RT. Also, 8ug/ml is likely quite a bit of excess so if reagent supply is an issue you could probably scale back to 2ug/ml.

What is you 'poly-HRP'? A biotinylated monoclonal would normaly be probed with a streptavidin HRP...

-Missle-

Missle on Mon Jan 20 19:28:31 2014 said:

This may not be your problem but I think your blocking is insufficient. I've never encountered an ELISA protocol that does not contain a blocking incubation step. Perhaps you should try blocking for 30min at 37C or 1hr at RT. Also, 8ug/ml is likely quite a bit of excess so if reagent supply is an issue you could probably scale back to 2ug/ml.

What is you 'poly-HRP'? A biotinylated monoclonal would normaly be probed with a streptavidin HRP...

Thanks, I think I'll try blocking at room temp for an hour with nonfat dry milk or BSA instead of this prioprietary solution. I agree instanteous blocking sounds suspect.

sorry poly-HRP is http://www.piercenet.com/product/streptavidin-poly-hrp-conjugate . It's a streptavidin with polymers of HRP attached to increase sensitivity. Might produce high background though so I could also possibly switch to a different streptavidin-HRP, I'll order some now.

Thanks for your advice on the capture, I'll try reducing to somewhere around 2 ug/mL. I attached the actual results - I'm looking for E7 which should be present in HeLa cells only (not C33A). It worries me that capture antibody + TBST-BSA alone + detection antibody yields a high background, and it's confusing to me that TBST-BSA alone + detection antibody does not. Could avidin have an affinity for my capture antibody? I don't understand why else capture antibody + negative control is so much higher than no capture + negative control. Thanks again for the help

Ben

-bdg25-

Hi.

I agree with Missie that you should block the plate with incubation time. I always block for 1hr, other than that - our protocol is very similar.

The danger with using milk as a blocking buffer is that milk can contain free Biotin - this might lead to a higher background if you have HRP present.

I would try using bovine gelatin, 0,1% in PBS for blocking buffer, that always works for me.

Good luck!

-swebus-

Thanks for sending the data. You have many good controls so it helps a lot! I think the reason you have the high background is not the avidin reacting with your capture antibody (although you could confirm this by adding a control that has your capture - TBST-BSA- NO biotin Ab - then avidin-HRP) but rather an interaction between your capture antibody and your biotinylated monoclonal antibody. I've seen this several times before. If you include the extra control that I just mentioned, it would confirm. If it is the poly capture interacting with your mono-tracer, then there isn't a lot you can do about that other than to change one of the antibodies in your pair.

-Missle-

How long do you incubate your lisates for? and do you add them neat or diluted?

Also, if I remember correclty, you need to wash without detergent before adding TMB!

-almost a doctor-

Thank you Missle, Swebus and almost a doctor for all of your help.

First of all, the protocol I listed never talks about when I added lysates so that doesn't help. I added lysate the day after incubating the capture antibody, after washing and blocking. The lysate was in a concentration of 1.8 million cells/mL. I diluted to 250 K cell/mL in TBST and added 100 uL.

Swebus - good point about the milk; that isn't a good idea with Biotin. I'll try the bovine gelatin.

Missle - I'll try with that new control that's a good idea. The only reason I think blocking is definitely a big culprit is in the control that I had no capture antibody at all, then blocked, the added lysate, I still had quite a strong signal. I would think if I had effective blocking that shouldn't be the case. However, binding of the detection and capture is certainly another likely candidate so I need to run that control.

Almostadoctor - Incubated lysates for 1 hour. They were diluted in TBST. I did not know that about TMB - I will change that last wash step

thank you

Ben

-bdg25-

almost a doctor on Tue Jan 21 12:57:23 2014 said:

Also, if I remember correclty, you need to wash without detergent before adding TMB!

Just one note about this, contacted Pierce thermo just to make sure, and although some detergents contain ingredients that will interfere with TMB, tween-20 is fine to wash with prior to TMB

-bdg25-