We have been working on the ELISA Methods to detect CTB011 and CTB012 in human serum. We have made progress with both ELISAs. The CTB012 ELISA, project code REOF, is in the last stage of development should go into validation soon. The CTB011 ELISA, project code REOA, is still in development. We will need to send you a contract modification for the CTB011.
Our initial protocol(s) closely followed the method found in Synermore Report Titled Validation of the ELISA method for the quantitation of CTB011 and CTB012 in beagle dog serum”. There were some alternations to the protocol from the outset.
1) We set the calibrators up in 100% Matrix or Assay Diluent. We would dilute to the calibrators to the assays MRD (1:50) in Assay Diluent.…show more content… We tested two Jackson Immunoresearch Antibodies, a HRP conjugated Goat anti-Human IgG Antibody with minimal cross-reaction to bovine, horse, and mouse serum proteins and a HRP conjugated Mouse anti-Human IgG Antibody. We will be using the Jackson Immunoresearch HRP conjugated Goat anti-Human IgG Antibody for the CTB012 ELISA and the Jackson Immunoresearch HRP conjugated Goat anti-Human IgG Antibody for the CTB011 ELISA. Any potential HAMA reaction in the CTB011 ELISA will be blocked by adding Mouse IgG at 0.7 ug/mL to the Assay Diluent.
We have also changed the assay diluent from PBS to a high salt PBS (500 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1.47 mM KH2PO4). The matrix background noise in both ELISAs was shown to be reduced with the high salt PBS being used in the Assay Diluent (1% BSA in PBS with 0.05% Tween 20). We may change the plate block solution to blocker casein in PBS (Thermo Scientific) for the CTB011 ELISA. When the blocker Casein in PBS was used to block the plate in the CTB012 ELISA the matrix background noise was
