identical results in ELISA assay - I must be doing somthing wrong, but what? (Sep/20/2005 )

I'm running a normal sandwich ELISA assay in the sequence; Primary ab, 2x wash, block, 2x wash, antigen, 4x wash, secondary ab, 4x wash, detect.

When I detect I'm getting near identical results for every well, including the serial dilutions of my pure protein standards and the alledged blanks (Primary ab and secondary ab but no antigen). However my wells with either no primary ab or no secondary ab added show a blank result.

Any ideas why?

At the moment I'm using a 1xPBS-T wash solution with a PBS wash for the final secondary ab wash (ie. 3x wash plate with secondary ab in PBS-T + 1x wash plate with PBS), a primary ab concentration of 1:2500 and a secondary ab concentration of 1:1500 (ab datasheet for secondary says 1:1000 - 1:2000), and a 3% BSA + sodium azide Blocking buffer (my lable is alkaline phosphatase so that shouldn't be a problem)

Could I be using too much primary ab? Or could it be another problem?

Thanks
Rosie

Hi Rosie,
It sounds like you're saying you have high ODs in all your wells other than when you leave out the capture (primary) or detection (secondary) antibodies. So it sounds like your antibodies are able to interact independently of their antigen (??).

How do you coat your plate with the primary/capture ab ?

Is the secondary/detection ab directly conjugated to alkaline phosphatase or do you add something like a biotinylated ab plus streptavidin-AP mix/ an ab specific to your protein plus an anti-mouse or rabbit Ig-AP conjugate?

Also what % Tween-20 do you use in the PBS wash buffer and do you use a plate washer?

I've mainly used commercially available ELISA kits but I set my own up with some R&D abs which hadn't at the time been validated for ELISA using an ELISA development guide they produce. This might be quite useful for you as they suggest concentrations of abs and coating conditions. It's available from their website as a downloadable pdf (?). The address is http://www.rndsystems.com/additional_literature.aspx

Hope this is of some help.

Ceri

The R&D guide troubleshooting guide suggest possible causes as insufficient washing (increase number of washes), too much HRP/AP enzyme conjugate (use more dilute) or insufficient blocking.

We always use 3x 300µl washes on a plate washer with PBS-0.05% Tween 20 for each wash step and block in 5% BSA/PBS which is almost the same as your protocol.

The capture/primary ab concentrations given by R&D are 0.5-8µg/ml for a monoclonal ab or 0.2-0.8ng/ml for a polyclonal ab.

The detection/secondary ab concentrations given by R&D are 0.25-2µg/ml for a monoclonal ab or 50-400ng/ml for a polyclonal ab.

All the best,
Ceri

Hi Ceri,
I coat my plate with the primary by adding 50µl of 1:2500 diluted ab to the plate, covering with saren wrap, and leaving overnight at 4 degrees.

My wash is 1ml of Tween-20 in 1L PBS (0.1% I guess) I don't use a plate washer though

My secondary is directly conjugated with AP

I'm increasing the washing steps to 6 each time and I've tried changing the temperature.

Thanks
Rosie

Hi Rosie,
Increasing your wash steps seems like a good idea. The research assistant in my previous lab did ELISAs without a plate washer. I remember that he was very careful to remove each wash from the wells by hitting (tapping doesn't really describe it) the plate against a paper towel laid out on the bench.

I would have thought that insufficient washing would have also given you postitive wells in your secondary ab only control though. That's why I asked about the secondary ab being an mouse anti-your protein plus an anti-mouse AP conjugate for example. I thought if the coating ab was also a mouse ab, the anti-mouse AP would bind and it might explain why both abs are needed for the high background.

Since both your abs are specific and one is directly conjugated they should only bind to your protein, not in the absence of it.

How do the dilutions you're using relate to concentrations of ab?

Hope the increased washing works.

All the best,
Ceri

Hi Guys,

I just had a quick look at this and one thing that stands out is that you are using alkaline phosphatase as your detection method.

Inorganic phosphate is a strong competitive inhibitor of alkaline phosphatase so you should use something other than PBS to wash your plates as this can potentially be a big problem. I would use TBS for this instead....

Hi Rosie
Might I ask what species yr antibodies are ie is there a cross reraction between the 3 antibody species, also from your format would it be true to say that you don't control for the secondary antibody coating itself to the plate regardless of antigen being present. What rationale did you use for the block step. Has it been validated elsewhere or did you pick it as it worked in another assay. Could be its not working. Plenty of assays can require up to 10% of a protein to solution to block, some don't need any blocking at all.
I would block as heavy as possible, run a chessboard of antigen versus the secondary ab right out till you get no signal. Get everthing optimised and then gradually reduce the blocking step to ensure sensitivity. If you are getting Ods in the absence of the antigen then you might suspect either the 2nday ab coating itself to the plate, cross reactvity to the primary ab or the alk-phos conjugate. Excess washing will interfere with your assay I think, 4 washes per step should be fine, but yes remember to tap all the froth out at the end if you are doing it by hand (pipette).
S

Thanks guys I'll give your suggestions a go!

I've been told not to give up on my Western blotting either so I'm now running two experiments in tandem!