Peroxidase labeling kit nh2

Bacterial cells were inoculated into 40 ml of lysogeny broth and grown to mid-log phase at 37 °C with shaking. MMC was then added to the culture at a final concentration of 500 ng/ml. At 5 h after MMC addition, 100 µl of the culture was collected and immediately subjected to sonication using a Bioruptor (Cosmo Bio, Tokyo, Japan). The soluble fractions of each cell lysate were obtained via centrifugation at 14,000 × g at 4 °C for 10 min. The Stx2 concentration in each cell lysate was determined using a previously described sandwich ELISA [69 (link)] using RIDASCREEN Verotoxin microtiter plates (R-Biopharm GmbH, Germany) coated with capture antibodies that recognize Stx2 and monoclonal antibodies against Stx2 (LSBio, WA, USA) conjugated with horseradish peroxidase using a Peroxidase Labeling Kit–NH2 (Dojindo, Kumamoto, Japan) as detection antibodies. Note that both Stx2a and Stx2g could be quantified at a certain level of accuracy using the detection antibodies [70 (link)].

AGE-BSA was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and labeled with horseradish peroxidase using a kit (Peroxidase Labeling Kit-NH₂, Dojindo Molecular Technologies, Inc., Tokyo, Japan). The interaction between AGE and RAGE was assayed using a protocol reported earlier [30 (link),31 (link),32 (link)]. Briefly, AGE-BSA and the test compounds were added to RAGE-coated, 96-well plates. The binding of the AGE-BSA to RAGE was determined using a 3,3′,5,5′-tetramethylbenzidine enzyme-linked immunosorbent assays (TMB-ELISA) substrate solution (Sigma). The inhibition of the AGE/RAGE interaction is expressed as the percentage of the binding of the AGE-BSA with RAGE in the presence of the test compounds.

A double-antibody sandwich ELISA assay was used for pairing experiments. Unlabeled mAbs were used as capture antibodies, HRP-labeled mAbs as detection antibodies, and TMB substrate was used for coloration. The OD_{450 nm} value was used to indirectly measure the antigen content. HRP was used to label 8 high effective mouse anti-HPV16 E7-HIS oncoprotein mAbs according to the instructions of the Peroxidase Labeling Kit-NH₂ (Dojindo, Sahnghai, China). HRP-labeled mAbs were used as detection antibodies, and mAb pairing experiments were performed with unlabeled mAbs as capture antibodies. A total of 56 pairs were paired. The pair of mAbs with the strongest pairing signal was subjected to conventional double-antibody sandwich ELISA to quantitatively detect the lowest limitation of the HPV16 E7-HIS oncoprotein.