Biotinylated chain specific anti human immunoglobulins

Portions of Sprague–Dawley rat sural nerves or NXS2 tumors grown in A/J mice were embedded in Tissue Tek-II O.C.T. (Miles, Naperville, IL), snap frozen in liquid nitrogen, and stored at −80°C. Ten micrometer-sections were cut, fixed in acetone, and stained with chimeric mAbs c.8B6 or ch14.18 for 1 hour. After washing with PBS, mAb binding was detected by stepwise incubation with biotinylated chain specific anti-human immunoglobulins (1∶500, diluted) (Southern Biotech, Birmingham, AL, USA) for 1 hour at room temperature, followed by streptavidin-horseradish peroxidase complex (1∶1000, diluted) (Southern Biotech, Birmingham, AL, USA) for 1 hour at room temperature. After rinsing, the bound antibody was detected with DAB chromogen substrate solution (Dako, Glostrup, Denmark), which was used to produce a brown deposit. Rituximab was used as a negative control. A mAb specific to mouse neural cell adhesion molecule (CD56) purchased from Abbiotec (San Diego, CA, USA) was used as positive control. The concentration 5 µg/mL was selected for the study because it would result in minimal background and maximal detection signal. Slides were counter-stained with hematoxylin before immunocytological evaluation. Staining was graded as positive or negative according to the presence or absence of immunoreactivity, respectively.

Detection of GD2 and OAcGD2 was performed by TLC-immunostaining (I-TLC) with mAb ch14.18 and mAb c.8B6 respectively as described by Cerato et al.[11] (link). Briefly, the developed TLC plates were fixed by 0.05% polyisobutylmethacrylate in hexane and treated with PBS-1% BSA for 1 h at 25°C. They were then overlaid with either antibody ch14.18 or c.8B6 at 10 µg/mL in PBS-0.1% BSA overnight at 4°C. After three washings with PBS, mAb binding was detected by stepwise incubation with biotinylated chain specific anti-human immunoglobulins (1∶2000, diluted) (Southern Biotech, Birmingham, AL, USA) for 1 h at room temperature, followed by streptavidin-horseradish peroxidase complex (1∶1000, diluted) (Southern Biotech, Birmingham, AL, USA) for 1 h at room temperature. After extensive washings with PBS, the bound peroxydases were visualized with 4-chloro-1-naphtol solution (Sigma Aldrich, St. Louis, MO, USA).