Hrp conjugated anti m13 major coat protein antibody

Deparaffinized the biliary system of mouse infected with O. viverrini section were treated with 0.3% H₂O₂ for 30 minutes to digest endogenous peroxidase. After blocking nonspecific staining with 10% skim milk for 30 min. These sections were incubated overnight at 4°C with 10¹² PFU/ml of phage display KKU505 Fab and KKU505 monoclonal antibody (1:500). After washing, the sections were sequentially incubated for 90 minutes with HRP-conjugated anti-M13 major coat protein antibody (1:1000, Santa Cruz Biotechnology, sc-53004 HRP) for phage system and HRP-anti-mouse antibody (Envision) for monoclonal system and then developed in diaminobenzidine tetrahydrochloride (DAB). After that, hematoxylin was used to counterstaining the section. These sections were dehydrated, cleared and mounted. KKU505 mAb was used for positive pattern with mouse Envision Systems. The localization of O. viverrini antigens was performed using the Immunohistochemistry with HRP-conjugated anti-M13 major coat protein antibody for phage system and diaminobenzidine tetrahydrochloride (DAB) for monoclonal system.

Microtiter plates were coated with 1 μg/well of crude O. vivirrini in carbonate/bicarbonate buffer pH 8.6 overnight at 4°C. The plates were then blocked with 300 μl of 5% bovine serum albumin (BSA) in PBS for 2 hours at 37°C. The wells were washed with 0.02% Tween-20 in TBS and 100 μl of 10¹² PFU/ml of phage in PBS were added and incubated for 2 hours 37°C. The unbound phage was washed out by with 0.01% Tween-20 in TBS for 5 times. HRP-conjugated anti-M13 major coat protein antibody (1:800, Santa Cruz Biotechnology, sc-53004 HRP) was added and incubated for 2 hours at 37°C. Bound phage was detected by adding 100 μL/well of 3,3_,5,5_-tetramethylbenzidine (TMB) substrate (KPL, Gaithersburg, MD), which containing with substrate A and B at 1:1 (v/v) ratio, after that it was incubated in the dark at room temperature for 30 minutes. After the blue color was developed, in each well was added 50 μL of the stop reaction solution (2N H2PO4). The blue color changed to yellow color and was measured the absorbance (optical density; O.D.) at 450 nm by Sunrise ELISA plate reader (Tecan, Austria) with Magellen7 software (Tecan, Austria).