Protocol: PK Antigen Capture Format ELISA for Use with Anti-Ramucirumab Antibodies
Protocol: PK Antigen Capture Format ELISA for Use with Anti-Ramucirumab Antibodies
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Pharmacokinetic (PK) Bridging ELISA: For use with Anti-Ramucirumab Monoclonal Antibody catalog TZA053 / TZA053P
This method provides a procedure for carrying out a PK ELISA Antigen Capture Format with HRP-conjugated Anti-Ramucirumab (Drug/Target Complex) Antibody TZA053 / TZA053P (detection antibody), using ramucirumab for the standard curve. Anti-ramucirumab drug/target complex antibody recognizes ramucirumab only when bound to its target human VEGFR2. It does not recognize the free drug or unbound human VEGFR2. The method should always be used in conjunction with product and batch specific information provided with each vial (see product datasheets). This protocol will need to be adjusted for use with different detection methods and immunoassay technology platforms.
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Reagents
- BSA (Sigma-Aldrich, A7906)
HISPEC Assay Diluent (BUF049)
Human Serum (Sigma-Aldrich, H4522)
- PBS
- 136 mM NaCl
2.68 mM KCl
8.1 mM Na2HPO4
1.46 mM KH2PO4
- PBST
- PBS with 0.05% Tween 20 (Merck Millipore, 817072)
- SpyCatcher Reagents e.g. BiSpyCatcher2:HRP (TZC002P). Contact us to discuss alternative SpyCatcher options
- QuantaBlu Fluorogenic Peroxidase Substrate (Thermo Fisher Scientific, 15169)
Materials
- 384-well microtiter plate, black, square flat-bottom wells, for example, Black 384-Well Immuno Plates (Thermo Fisher Scientific, 460518)
- Fluorescence plate reader
- 96-well plates can be used instead of 384-well plates, (black, flat-bottom wells) for example, Black 96-Well Immuno Plates (Thermo Fisher Scientific, #437111). For the 96-well format, use 100 μl (instead of 20 μl) of antigen, antibodies, or substrate and 300 μl for the blocking step.
Method
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Prepare human VEGFR2-His (capture antigen) at 1 µg/ml in PBS. Coat the required number of wells of a 384-well microtiter plate with 20 µl per well of the prepared capture antigen, and incubate overnight at 4°C.
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If using TZA053P (AbD53034pap) for detection proceed to step 3. If using TZA053 for detection, prepare the detection Anti-Ramucirumab (Drug/Target Complex) Antibody: Couple TZA053 (AbD53034ad) to a suitable SpyCatcher Reagent (e.g.TZC002P) using the appropriate coupling protocol.
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Wash the microtiter plate five times (5x) with PBST.
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Block the microtiter plate by adding 100 µl 5% BSA in PBST to each well, and then incubate for 1 hr at RT.
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Wash the microtiter plate 5x with PBST.
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For the standard curve, prepare a dilution series of ramucirumab in 10% human serum in PBST in triplicate. Final concentration of ramucirumab should cover the range from 0.001 ng/ml to 1,000 ng/ml. Include a zero ramucirumab concentration as the background value.
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Add 20 µl of ramucirumab dilution per well (in triplicate for each standard recommended). Add 20 µl of each test sample to the other wells (in triplicate for each sample recommended). Incubate for 1 hr at RT.
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Wash the microtiter plate 5x with PBST.
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To each well, add 20 µl of either (Bi)SpyCartcher:HRP-coupled detection Anti-Ramucirumab (Drug/Target Complex) Antibody, TZA053 (AbD53034ad), or HRP-conjugated detection Anti-Ramucirumab (Drug/Target Complex) Antibody, TZA053P (AbD53034pap) at 0.5 µg/ml in HISPEC assay diluent. Incubate for 1 hr at RT.
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Wash the microtiter plate 10x with PBST.
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Add 20 µl QuantaBlu Fluorogenic Peroxidase Substrate to each well and measure the fluorescence after 30 min.