capture elisa - (Feb/22/2006 )

last 20 days iam doing captureelisa only.i purified 8 antibodies of p 24 .i used those as a capture antibody and some 4 antibodies i have biotinylated those iam using as a detector antibody.iam checking the antigen sensitivity.in 1 well iam not adding the antigen but iam getting od near to positive.iam not understanding this.buffers everything iam using fresh.blocking with sheep serum(10%).dilutions iam done with 1%this sheep serum.washes with tbs+0.1%tweene.what is the problem can u people fiind this problem as soon as and tell me iam waiting for that

Are you using purifed protein as your standard?
Are you using Streptavidin-HRP in combination with your detection?
How many washes are you using?

Is it possible that you're using an anti-species ab in the detection step which is binding to you capture antibody?

Have you tried increasing the wash steps or titrating your ab dilutions?

R&D systems (Ab manufacturer) produce a manual for setting up an capture ELISA with suggested starting concentrations and a matrix grid to try different concentrations of antigen, capture and detection abs on the same plate.

All the best,
Ceri

iam using as a capture ab is whatever i purified which has recognized n term and detector ab whatever i have biotinylatedwhich has recognized c term.in no antigen control also iam getting near to pur positive control.washes with tbs+tweene and blocking with 10%nss

OK, so I'm guessing you use a streptavidin-HRP conjugate to bind your secondary antibody and after washing a TMB substrate of some kind then add sulphuric acid to stop the reaction.

How many washes in TBS-Tween do you use between each step?

What concentration are your capture and detection abs in µg/ml or ng/ml?

Have you tried a range of dilutions of both capture and detection abs ?

All the best,

Ceri

hello praveen,
did you check the specificty of your antibodies? what are the dilutions they are working?
because if your capture and detector antibodies working at low dilutions then there may be other possibility of heterophilicity of your p24 antibodies.
given case if your are working at appropriate concentrations of antibodies as CERI commented, then other probability could be that without p24 protein still capture and detector antibodies can form a bridge and finally give high OD.
probable solution for this problem may be that you can preincubate your detector antibody with an isotype control(given case that your p24 is recombinant rather from plasma), then use that sample for detection step and see what results you get.

gud luck