Good work

you should dilute your sample . Along with serum sample you are using should be kept in -80 degree celsius

Good work

when you first set up an Elisa it is important to carry out as many controls as you can. Do you carry out the following controls with no IGF

1. No antibodies

2. Secondary Ab only

3. Primary Ab only

4. Primary/Secondary Ab

This should tell you where the problem is. I would also dilute your secondary antibody further and re-test.

Set up a grid plate for testing different Ab dilutions using only 2-3 IGF concentrations (10 fold difference in concn= High, Middle and low) with all controls. You need to get a feel for the range of the assay then you can tweek things after this.

Try not to wash after blocking, incubation of sample too long 1h at room temp or 37 , it enough.

Dilute sample with 1% BSA in PBST, how much coating you use?

Use high bond plates.

Try using as blocker 10% animal serum

Good luck

Oded Babai Thank you, I am using between 20

ng/ml - 2 pg/ml IGF-1 for coating, Is that low or ok?

@ Ian R Wilkinson,

Thank you for your reply. I am using the concentration of IGF-1 between 20 ng/ml - 2pg/ml. Also, I am using no antibodies and secondary antibody alone control. Shall try doing it as you said.

Dear Raji

From my experience it rather low I usually start at 0.1ug -0.5ug/well (100ul).you sould try high concentration just to get positive , responed.

Try other antibody against IGF-1, maybe rabbit or mouse. Goat some time have cross with the secondary.

I agree with Dr.Raji

If you are getting negative absorbance values, you need to dilute your standard according to dilutiom required

For getting value in a line in graph you must change the shape of trendline in graph and increase its width..

If it is still not are coming in a line then try to plot graph between concentration vs 1/absorbance. It may help

Good work