Mosquito ELISA difficulties - (Nov/16/2013 )

I began running an ELISA on malaria sporozoites of mosquitoes last week, but have since run across a little doubt concerning my results.  I'm not using a kit, my supplies: PBS, mAb capture and conjugated peroxidase, substrates, etc.  Most of these supplies came from sigma.  My problem:

The mosquitoes are being ground with Igepal and run on a high-affinity costar plate.  The steps include 30 min. capture Ab coating, blocking, loading of samples, wash 2x, peroxidase Ab, wash 3x, and substrates.  The analysis is preliminary, and only requires visual inspection, which will ultimately be ran with a plate reader in the future.  Following an hour of incubation with the substrates, I am getting fairly robust signals from my positive controls but virtually no background.  While I have recorded a series of positives in the past week, they appear to be relatively weak in strength - slightly fluoresced.  I'm unsure if this is something that I should be concerned about.  Are these valid results? I expected a stronger signal from the positives and a higher background, but I have only seen relatively weak signals.  Does a signal from my positives validate that the antibodies are viable? I have already tested the conjugated Ab's with my substrates, and they appear to be fine.

Any advice or insight would be greatly appreciated!

You can add an excess of unconjugated Ab during your peroxidase Ab step and see if the signal dissappears. This will somewhat validate the peroxidase-Ab.

Repeat this process with your coating Ab, i.e. add excess coating Ab during the sample addition step.

Hope this helps.

You also should establish something like a threshold from what you consider a signal as positive and negative. There are some recommendations but you should follow the ones that are used in your field of work.