A Simple and Efficient Genetic Immunization Protocol for the Production of Highly Specific Polyclonal and Monoclonal Antibodies against the Native Form of Mammalian Proteins.

We have generated polyclonal and monoclonal antibodies by genetic immunization over the last two decades. In this paper, we present our most successful methodology acquired over these years and present the animals in which we obtained the highest rates of success. The technique presented is convenient, easy, affordable, and generates antibodies against mammalian proteins in their native form. This protocol requires neither expensive equipment, such as a gene gun, nor sophisticated techniques such as the conjugation of gold microspheres, electroporation, or surgery to inject in lymph nodes. The protocol presented uses simply the purified plasmid expressing the protein of interest under a strong promoter, which is injected at intramuscular and intradermal sites. This technique was tested in five species. Guinea pigs were the animals of choice for the production of polyclonal antibodies. Monoclonal antibodies could be generated in mice by giving, as a last injection, a suspension of transfected cells. The antibodies detected their antigens in their native forms. They were highly specific with very low non-specific background levels, as assessed by immune-blots, immunocytochemistry, immunohistochemistry and flow cytometry. We present herein a detailed and simple procedure to successfully raise specific antibodies against native proteins.

Generation and characterization of polyclonal and monoclonal antibodies to human NTPDase2 including a blocking antibody.

Nucleoside triphosphate diphosphohydrolase-2 (NTPDase2) is an ectonucleotidase that modulates P2 receptor activation by hydrolyzing ATP to ADP. In rodents, NTPDase2 is expressed by several specialized cell types such as vascular adventitial cells, neuroglial cells, hepatic portal fibroblasts, gustatory type I cells, and cells within the connective tissues of reproductive and gastrointestinal organs. Much less is known regarding the expression and function of NTPDase2 in humans. Here, we developed specific research tools to study human NTPDase2. We generated mouse monoclonal antibodies and rabbit polyclonal antibodies specific to human NTPDase2 and validated their specificity by western blot, immunocytochemistry, immunohistochemistry, and flow cytometry. In addition, one monoclonal antibody named hN2-D5 s specifically inhibits human NTPDase2 enzymatic activity but not mouse nor rat NTPDase2. Using these antibodies, NTPDase2 immunoreactivity was detected on glial cells of the human enteric nervous system suggesting a function of the enzyme in intestinal motility. In conclusion, the new antibodies described in our work are novel tools that will enhance future studies of NTPDase2 expression and function in humans.