Help! Why did IL-1b monoclonal antibody work in vitro very well, but not in vivo?
We infused IL-1b mAb (10mg/kg) to the sheep fetus, and took blood samples at 1, 2, 4, 24 hr after infusion. Then, we tried to measure the IL-1b cytokine level in the plasma using Sandwich ELISA to prove if IL-1b was neutralized by IL-1b mAb.
Before starting the experiments, we optimized the experiments protocols e.g. blocking agents, dilution factors, incubation time... We detected nice IL-1b signals from sham and baseline (before infusion) samples, however we found also same strong signal from mAb-treated samples?!
We did also in vitro experiments to mimic the in vivo situation. We found the IL-1b mAb neutralized the IL-1 very well!
But why we still have signals from plasma samples? What should I do for the next step? Is the antigen-mAb complex broken by the Frozon-Difrozen process?
By the way, our mAb is mouse IgG1 against sheep.
Thank you very much for help in advance!
Re: Help! Why did IL-1b monoclonal antibody work in vitro very well, but not in vivo?
Anti-IL-1β neutralizing monoclonal antibody (Mab), binding with precise affinity IL-1β epitope/s, IL-1β in vivo and in vitro, with scope to neutralize activity of IL-1β, inhibit the expression in culture (fibroblast/lymphocytes) of IL-6/IL-8. If antibody (poly- or monoclonal) functions in vitro it doesn't mean that it will work well in vivo, that linked to both environmental conditions and epitopes accessibility. Insofar, effecacies of antibodies neutralization in vitro can't be correlated in vivo always. From this it follows that motive so Mab different types search, from different clones, that same effecacie neutralizing in vivo/vitro show, particularly when these are employed for "therapeutic use". Is shown that Mabs that don't neutralize in vitro are instead able to neutralize in alive. Besides, a different pharmacology occur when Mab is intravenously or peritoneal injected, this is linked to different and complex molecular interactions that can prevent the action or not detect it. Therefore, a different behavior, vivo/vitro, between antigen vs Mab is possible linked to : a) power of interaction; b) affinity; c) Mab isotype; d) epitope accessibility. From this it is convenient to use Mab from other clones, primarily assaying in vitro culture.
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