Part of my project is to compare the total number of influenza (B/Phuket/3073/2013) binding antibodies in human serum and mucus supernatant. Unfortunately, only mouse monoclonal antibodies for B/Phuket/3073/2013 are available create the a standard.
Can the mouse antibodies be used as the standard? Is there a way to design the ELISA to use the mouse antibodies?
Thank you!
What's on your solid phase? Antigen or Antibody? Guessing the former if you wanted to use mouse Antibodies. Also are you looking at a Qualitative or Quantitative assay? Again I'm guessing latter if you want standards.
Technically, there's nothing to stop you from doing this, however there are considerations:
- The antibodies may recognise different epitopes on the antigen - not a like for like comparison.
- Affinity of antibodies to antigen may differ due to different species
- Are you looking at IgM or IgG or both? The mouse mAb will prob be IgG?
- You'd have to optimise two lots of conjugated antibodies (mouse and human).
- You'd basically have to dilute out your mouse mAb to known concentrations (ug/ul) and find the best dilution range to give you a good standard curve. Then calculate your human samples based on this.
One of my colleagues suggested trying to find a human sample that gives you a high read out and assign Arbitrary Units to it? Take sample and run neat and then do a rough half-log dilutions (3-fold) and then assign Neat = 1000 AUs, 1/3 = 333 AU, 1/10 = 100 AU, 1/33 - 33 AU, 1/100 = 10 AU, 1/333 = 3.3 AU, 1/1000 = 1 AU? This way you could use this to roughly quantify in relation to other samples?
Hope my rambling makes some kind of sense?
John
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