High-Resolution Description of Antibody Heavy-Chain Repertoires in Humans

Antibodies' protective, pathological, and therapeutic properties result from their considerable diversity. This diversity is almost limitless in potential, but actual diversity is still poorly understood. Here we use deep sequencing to characterize the diversity of the heavy-chain CDR3 region, the most important contributor to antibody binding specificity, and the constituent V, D, and J segments that comprise it. We find that, during the stepwise D-J and then V-DJ recombination events, the choice of D and J segments exert some bias on each other; however, we find the choice of the V segment is essentially independent of both. V, D, and J segments are utilized with different frequencies, resulting in a highly skewed representation of VDJ combinations in the repertoire. Nevertheless, the pattern of segment usage was almost identical between two different individuals. The pattern of V, D, and J segment usage and recombination was insufficient to explain overlap that was observed between the two individuals' CDR3 repertoires. Finally, we find that while there are a near-infinite number of heavy-chain CDR3s in principle, there are about 3–9 million in the blood of an adult human being

Generation of murine monoclonal antibodies to Haemophilus influenzae type b capsular polysaccharide by in vivo immunization

Haemophilus influenzae type b (Hib) is a pathogenic gram-negative bacterium associated with human disease, especially in young children. Protective immune response to Hib results from antibodies developed against the polyribosylribitol phosphate (PRP) capsular polysaccharide of the bacterium. Several investigators in the study of immune response to Hib have produced human monoclonal antibodies to PRP. Only two previous groups have reported the generation of murine anti-PRP monoclonal antibodies using either immunizing mice with inactivated Hib bacteria or a combination of in vivo and in vitro immunization of mice with PRP conjugate antigen (PRP-D). In this present study, we generated murine anti-PRP monoclonal antibody secreting hybridomas for the first time by simple in vivo immunization with PRP conjugate antigen (PRP-T). The anti-PRP antibodies from one hybridoma clone (B10) are further characterized and potential applications are discussed