Purification, biotinylation, and testing of a monoclonal antibody to identify B cells in Trachemys scripta, the red-eared slider turtle

Abstract

Abstract There is a shortage of research in reptile immunity which is further hampered by lack of reptile-specific reagents. Evidence suggests there are important differences between reptile and mammalian immune strategies and our laboratory is interested in reptile B cell development and function. Our undergraduate research project involved the preparation of a previously developed monoclonal antibody (HL673) that recognizes turtle light chain protein. To begin, culture supernatant from the HL673 mAb murine cell line was received and was applied to a protein A affinity column. Unbound proteins were then washed away, and the bound proteins were removed from the column using low pH glycine-HCl buffer. The purified antibody proteins were collected in fractions, OD at 280nm measured, then positive fractions were pooled and dialyzed. The concentration of the purified antibodies was determined, and reactivity tested using an ELISA plate coated with dilute turtle serum. Dilutions of the purified HL673 were detected by anti-mouse IgG-horseradish peroxidase (HRP). Furthermore, some of the antibody preparation was conjugated to biotin. After dialysis, the HL673-biotin was tested by ELISA with dilute turtle serum and detected by streptavidin-HRP. The newly biotinylated antibody was incubated with blood and spleen samples from both adult and hatchling red-eared slider turtles. Bound antibodies were detected using streptavidin-fluorochrome and B cell populations identified using flow cytometry. Our results showed successful detection of turtle B cells using the labeled mAb in both hatchling and adult turtle cell samples. Future studies will use this reagent to investigate the distribution and function of B cells in reptile gut immunity.