Hi Saeed,

To check whether your blocking agent gives a signal, you need to include some wells in your experiment where you do not coat the wells with sample, but just block. These act as your negative controls (you should also do a control where you do not add primary antibody to wells coated with your sample to rule out signal generated by direct binding of the secondary antibody). If you see a significant signal with a blocking agent in the absence of sample, this means either the primary or secondary antibody is binding to the blocking agent. I have usually used BSA at 5%; this is better for ELISA as it gives a clear solution, but casein & fat-free milk powder can also be used. To reduce background, I also add some BSA to washing solutions (0.1%), and always dilute my antibodies in blocking buffer.

Edit: I also recommend blocking and washing with a larger volume than you use for coating/antibody incubations. If you coat with e.g. 100 ul, you should block your wells with 150 ul.

Hi Saeed,

To check whether your blocking agent gives a signal, you need to include some wells in your experiment where you do not coat the wells with sample, but just block. These act as your negative controls (you should also do a control where you do not add primary antibody to wells coated with your sample to rule out signal generated by direct binding of the secondary antibody). If you see a significant signal with a blocking agent in the absence of sample, this means either the primary or secondary antibody is binding to the blocking agent. I have usually used BSA at 5%; this is better for ELISA as it gives a clear solution, but casein & fat-free milk powder can also be used. To reduce background, I also add some BSA to washing solutions (0.1%), and always dilute my antibodies in blocking buffer.

Edit: I also recommend blocking and washing with a larger volume than you use for coating/antibody incubations. If you coat with e.g. 100 ul, you should block your wells with 150 ul.

Have you tried to reduce the concentration of your first or second antibody ? You may also try to purify your first antibody: run a gel with a single well containing your antigen, then transfer to a nitrocellulose or PVDF membrane and stain a strip on the side to locate the band of interest. Cut the strip corresponding to the antigen band and incubate it with your antiserum. Wash with blocking buffer, and elute the antibodies that remain bound to the strip with a pH 2 buffer. Then neutralize the preparation with Tris buffer.

Saeed , also recommend Dr. Jack C Leo answer

I have usually used BSA at 5% , the incubation for blocking is 2 hours.

I also dilute secondary antibodies and conjugates with PBS that has 1% BSA added,

Regards

@jack thank you so much for your suggestion, last time i used BSA upto 2% so now i want to increase it till 5%. i used some of the well without my antigen (purified protein and still it gives me a reading. but i think your suggestion will help me out of this problem.

@pierre thanks for your suggestion, actually my antigen is bacterial protein which is purified by NI-NTA column of bio rad, and my elisa is just like direct competitive ELISA. and antibody is the conjugate of my drug with HRP.

@Nora thanks for your time, i will try to follow your suggestion

Add 0.05-0.1% tween80 in all buffer. I do not understand your ELISA. If you need a blocking solution for an antigen-antibody reaction BSA is ok, but if your reaction is a protein-drug-HRP reaction probably BSA is not a good choice.

thanks @Paola Di Bonito, i want to explain my Direct ELISA like method, i coat my bacterial protein on plate for overnight, then after washing with pbst block with (BSA, OVA, GELATIN and also tried casein) then after washing i add Hrp-drug and after washing add substrate TMB.