Coated antigen conc. used 50ng/100ul
antigen1 (is an antibody) conc. 100ng/100ul + purified Mab conc. 1000ng/100ul
antigen1 is an recombinant Mab with human Fc region against peptide conjugated to BSA (peptide conjugated with BSA is coating antigen). antigen1 is immunized in mouse to raise antibodies against the whole antigen1. Purified anti antigen1 MAbs have mouse Fc. Therefore secondary Ab Goat anti-Human only binds to antigen1.
With purified MAbs an OD of 0.04 was observed meaning both Fab and Fc of antigen1 were blocked by the anti antigen1 MAbs.
In case of supernatants an OD 0.4 is observed which is contributed thru the Fc region of antigen1? Does this mean FBS proteins are occluding the interaction of anti antigen1 MAbs? (antigen1 conc. used was 100ng/100ul) Excluding FBS gives us an OD of 0.04? or is it due to insufficient concentration of anti antigen1 MAbs.
Its difficult to follow the expwrimental design. Aclearer explanation giving the usual names for antibodies ad antigens would probably help.
Are you coating with an antibody or not? In which order are the reagents added? what are the controls?
purified Mab + antigen is the positive control OD should be equivalent to blank, antigen alone is another positive control an OD of 2 is expected, Supernatants are the test samples.
1 Coated antigen conc. used 50ng/100ul (peptide conjugated with BSA ).
2 primary antibody is a mixture of antigen + antibody in this case antigen (which is human antibody (Fab specific to the peptide coated) + idiotype antibodies raised against the whole human antibody (Fab + Fc) in mice)
3 secondary antibody is goat anti-Human
plate layout:
A1 antigen+purified antibody mixture as primary, positive control
A2 antigen alone as primary, positive control
A3 supernatants (monoclones) that secrete Mabs against the antigen (antigen+supernatant as primary).
A4 Blank
Secondary is anti-Human antibody which is specific to the antigen (Human Fc) but not to the Mabs raised against it cuz they were raised in the mice.
Hello,
It is quite hard to understand the naming for the variables and control. I think you need to fix the naming of the reagents. I have a question. Are you investigating affinity of your sample (MAbs from mice) with the human MAbs?
Thank you.
Sincerely yours,
Indra
Hello,
You are trying to test the affinity of your MAbs produced in the mouse toward human Fc region. Firstly, Human Fc was immobilized in the wells and you add the primary antibody (purified MAbs as the positive control (low OD) and your sample ). Then, you add secondary antibody (goat MAbs anti-human Fc). Negative control is without primary antibody (high OD). Blank was also used indicating low OD. Your hypothesis was your MAbs from mouse will bind to human Fc and shows low OD as secondary antibody cannot bind to the human Fc. The problem you are facing now is the wells containing primary antibody which is your mouse MAbs shows high OD. Did i understand your situation correctly?
Thank you.
Sincerely yours,
Indra
Nope
I coated the plates with peptide conjugated with BSA
Primary antibody Gh76 Fab is specific to that peptide.
Now the mice was immunized with this Gh76 so they are MAbs against this Mab.
Now if we mix both Gh76 + MAbs raised against it. Gh76 Fab and Fc are blocked right. So the secondary antibody anti-human doesn't bind to this Gh76.
Hello,
Increasing the concentration of the anti-Gh76 in the supernatant should make some improvement. What blocking agent for the plate did you use? FBS or BSA?
If the problem is not solved, i think you should cconsider lowering the antigen1 concentration. For the secondary antibody, how many dillution did you do?
thank you.
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