Advice on setting up a new ELISA protocol - (Jan/26/2011 )
I've done a few elisas before but I've always used a protocol that was set up by someone else. Now I have my own samples and I have to see what will work for me.
My mice were orally gavaged with tobacco tissue containing an antigen and I want to see if they have had an immune response. I have three groups of mice: a) a positive control
1. I will be coating my plates with purified antigen in coating buffer (carbonate/bicarbonate buffer). Is there a guideline as to how much antigen there should be per well?
2. How do I know what to use as my block? I usually use Marvel, but I've heard other people say that BSA is better. How would I check which is more appropriate for me? Is two hours block at rt enough?
3. My usual washes are 3x200ul PBST/well. As far as I can tell, this seems pretty standard. Can I leave this as it is?
4. What sort of dilutions of my sample should I be using? Should I start at 1:50 and just keep going to see what sort of absorbance values I get to start off? Should I leave my serum on the plate overnight or will a few hours at rt do? I don't mind it taking the extra day if longer is better, I really just want to get this optimised as quickly as possible.
5. My antibodies (anti-mouse IgG1 and anti-mouse IgG2a) are recommended to use anything between 1/1000 to 1/10,000. What is the best way to determine the best concentration? Would it be enough to coat a plate with normal mouse serum and then try different concentrations of the antibodies to see if they plateau off?
Thanks for any help you can give. This is my last big experiment before starting the big bad thesis so I appreciate anything you can suggest to get me there a little quicker.
1. 100 ug/ml; 100 ul/well. this is high concentration you can use less if the ag is too expensive.
2. There are many blockers: BSA, milk, commercial ones as well from Surmodics, East Coast Biologicals, Candor etc. BSA is very common and a good starting point. Best blocker is determined through trial and error. 2 hrs blocking is fine...fill wells completely.
3. Washes as indicated are fine.
4. Samples serially dilute 1:3 and cover wide range (several logs). Run in addition to ag coated the non-ag coated; blocked wells as blank. Shake 15-60 seconds with 1-2 hr incubation fine. (make sure plate is sealed during mixing incubation using plate sealer.
5. Your conjugates are subclass specific. Would anti mouse IgG (all subclasses) be less expensive? With BOTH anti G1 and anti-G2 (two conjugates?) you are developing 2 assays...twice the work. Don't coat mouse sera for conugate dilution determination. You can start with 1:5000 and titer once you start getting preliminary results. Note: the signals may come up quite fast so be prepared to read your plate several times or stop the reaction before signals become too intense.
Thanks a lot for the advice. You've been really helpful.
My PI wants both anti-G1 and anti-G2a so I can't really get around that. I'll focus on getting one done and then the other. Hoping it won't be too much work.
Since you have to develop both...it would be wise to select the one subclass that would most likely respond ie become elevated. I did some checking and found this reply to questions comparing human and mouse subclasses and responsiveness of the subclasses: