Has anyone conducted an ELISA for testing the presence of an active drug in plasma of mice? I have a PEGylated molecular which I am injected into mice and wanted to test the PK of this drug. What is the best approach to this?
Do you mean PK or TK (Toxicokinetics)? Generally, pre-clinical studies involve TK analysis in animals. You can try designing a sandwich ELISA with the anti-drug antibody as capture and an HRP-tagged anti-drug antibody as detection. The ideal approach for a Pegylated molecule is to generate antibodies against it in-house and then have it tagged with HRP. If this activity can be performed in two different animal species, the better. Then you would get the anti-Peg Drug antibody from species 1 as your capture. You can do HRP-tagging for the antibody from species 2 and use it as detection antibody. If, however this is not possible, you can still design an in-house ELISA with the non-pegylated molecule itself (The pegylation only ensures late clearance of your active API by increasing the drug size). Just ensure that your standard curve is using the Pegylated material that has been injected in your study. If you are successful in developing a functional ELISA, then, you shall observe a shift in the time-point of your peak plasma drug concentration. Also the plasma levels of drug stay readable and analysable for longer in plasma. But the flip side is that such an ELISA shall need rigorous method development and validation. All the best.
You need a kind of combination ELISA. If have no idea about your enzyme. I hope that a colour reaction is possible.
Than you can bind
1. an antibody against your enzyme at the solid phase (microtiter plate) (0.1 mol/l carbonate pH 9.2, antibody 2 ... 5 µg/ml)
2. incubate overnight at 4 °C
3. wash
4. incubate with your antigen/enzyme
5. wash
6. carry out your enzyme-substrate reaction
7. measure at a microplate reader at the required absorption of your enzyme substrate
8. wash
9. incubate with your second labeled antibody (e.i. HRP labeled)
10. wash
11. carry out your HRP-substrate reaction (e.i. TMB + H2O2)
12. stop with 1 mol/l H2SO4
13. measure at 492 nm.
so you will receive both: via the enzyme substrate reaction --> the activity of your enzyme
but: this will vary due to differences in the concentration,
therefore you need the second part of the ELISA, the detection of the antigen/enzyme concentration.
If you have just now both, you can calculate the "specific activity". This parameter is always the best to characterize an enzyme and to know how much is active and how much is inactive ...
Dear friend if ur interested in performing the PK of any drug this is never been conducted through ELISA. Some how this is always done through HPLC or LCMS. So please first try to under stand ur problem and than contact what actually ur interested to do.
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