I'm reposting my data combined with clarification from you reply, many points you made were very helpful and sensible. However, I compared my first post with your reply and got confused again. Data
Primary Antibody (B1-E12): Endogenous mouse anti-RSA polyclonal serum
Secondary Antibody (A1-E12): Goat-anti-mouse IgG-HRP conjugate
Substrate (A1-E12): OPD
2 wells: +Control: Mouse-anti-RSA polyclonal serum AU= 0.923, 0.922
2 wells: -Control: Preimmune mouse serum AU= 0.404, 0.410
2 wells: RSA,coating buffer, blocking buffer, Goat anti-mouse IgG-HRP conjugate AU= 0.428, 0.404
Test sample (Primary antibody) Rows:
B- (saline injections) 0 Ring ID mouse serum in blocking buffer
C- (RSA immunized) 1 Ring ID mouse serum in blocking buffer
D- (RSA immunized) 2 Ring mouse serum in blocking buffer
E- (RSA immunized) 3 Ring mouse ID serum in blocking buffer
Last 3 rows F,G,and H: RSA, coating buffer, blocking buffer only Questions:
From the above data:
1. What is considered the non-specific binding control? What defines
2. What is the blank? (everything but the anti-RSA polyclonal serum right? Meaning Goat-anti-mouse IgG-HRP conjugate + OPD is included?) I know blanks are everything except for what is being assayed.
I'm sorry for re-asking the same question again, I realized that I left out and mixed up the given data previously. When you replied back (so amazingly), I couldn't decipher between the above two questions.
By the way, thank you so much for your reply; it's cleared up so much of the confusion!!!!!!!!!!!!!!!!!!!!!!!!
PS: If I can get my scanner working, I'll send a file your way as a direct message, in case my question is unclear.