I am standardizing indirect dot ELISA for detecting anti-epsilon antibodies in Enterotoxaemia vaccinated goats. I have coated the combs with toxin antigen, where titrations of increasing/decreasing antigens yielded ''dots'' as positive signals, but i am getting positive ''dots'' in PBS coated combs as antigen controls! How to address this problem?
Hello Gururaj,
do you use any blocking agent like BSA to prevent unspecific binding ?
I would suggest to use 5% milk as the blocking agent and use this blocking soluction also to dilute antibodies. Casein has an excellent blocking effect.
I am using 3% non-fat dry milk. But i am diluting serum in PBS. I have tried with BSA too, but still there was background signal!
It s important to dilute primary ad secondary antibodies in blocking solution.
If you are using blocking protein at each and every level than you titrate antibody conjugated with enzyme. You might be using low dilution of enzyme conjugated antibody. You use high dilution of enzyme conjugated antibody. This may resolve problem of getting positive in PBS control.
With all the best.
I find that Sigma sodium casein at about 0.2% works better than milk. Milk tends to reduce signal especially with antibodies that have weak affinity. It takes a while to get it to dissolve so give it time. If you add some healthy sample to the buffer at the rate of 1/1000 in the IgG and enzyme conjugate steps this can also help? This especially helps if you were going to be testing plant samples.
https://www.sigmaaldrich.com/catalog/product/sigma/c8654?lang=en®ion=US
I found that most sodium caseinates do not perform as well as the Sigma material so it is best to not take a shortcut there. My experience: I have 25 years experience developing ELISA tests for mostly plant samples but have experience with animal and human samples. I developed an ELISA protocol for a company which calls it Compound Direct ELISA. I also developed buffers that work well with plant samples.
Fish protein, cold water fish protein are especially stable, egg albumin and Bovine Serum Albumin (BSA) are also commonly used as blockers. Commercially available Prionex® has been found useful.
Yes the buffers I used generally started with PBS Tween or PBST. We also wash the plates with PBST in between steps.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies