Sandwich ELISA Protocols
Comparison with other commercially-available anti-PEG antibodies
1. Comparison with Vendor B
Monoclonal anti-PEG antibodies from Vendor B (Mab 139 and 141) or Mab AGP4 were coated in microtiter plates at 5 µg/ml.
Graded concentrations of PEGylated compounds were added to the plate
Detection was performed with mAb 3.3-biotin and streptavidin-HRP
(A detailed protocol is available here)
Anti-PEG antibody AGP4 capture - 3.3-biotin detection
Vendor B mAb 141 captured PEGylated Q-dots poorly
Vendor B mAb 139 produced high background readings
Vendor B mAb 141 captured PEGylated Q-dots well
Vendor B mAb 139 produced high background readings
Vendor B mAb 141 captured PEGylated Q-dots poorly
Vendor B mAb 139 produced high background readings
2. Comparison with Vendor E
Rabbit monoclonal anti-PEG antibodies from Vendor E or Mab AGP4 were coated in microtiter plates at 5 µg/ml.
Graded concentrations of PEGylated compounds were added to the plate
Detection was performed with 5 µg/ml Vendor E rabbit monoclonal anti-PEG-biotin, 5 µg/ml mAb 3.3-biotin, 5 µg/ml AGP3-biotin or 1 or 5 µg/ml AGP4-biotin followed by streptavidin-HRP
Rabbit anti-PEG mAb worked well for detection of Lipodox
Similar detection limits were found for all antibodies
AGP4 capture with AGP4-biotin or 3.3-biotin detected PEGasys with about 10-fold more sensitivity as compared to Vendor E antibdoies
Vendor E antibodies detected Intron poorly
Vendor E antibodies detected Q-dots very poorly
Conclusions
The combination of AGP4 for capture and 3.3-biotin for detection allows quantization of all PEGylated compounds tested. In contrast to other commercial anti-PEG antibodies, AGP4 and 3.3 appear to be universally applicable.
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Mei Lee