Contribution of murine IgG Fc regions to antibody binding to the capsule of Burkholderia pseudomallei

Immunoglobulin G3 (IgG3) is the predominant IgG subclass elicited in response to polysaccharide antigens in mice. This specific subclass has been shown to crosslink its fragment crystallizable (Fc) regions following binding to multivalent polysaccharides. Crosslinking leads to increased affinity through avidity, which theoretically should lead to more effective protection against bacteria and yeast displaying capsular polysaccharides on their surface. To investigate this further we have analyzed the binding characteristics of 2 IgG monoclonal antibody (mAb) subclass families that bind to the capsular polysaccharide (CPS) of Burkholderia pseudomallei. The first subclass family originated from an IgG3 hybridoma cell line (3C5); the second family was generated from an IgG1 cell line (2A5). When the Fc region of the 3C5 IgG3 is removed by proteolytic cleavage, the resulting F(ab')(2) fragments exhibit decreased affinity compared to the full-length mAb. Similarly, when the parent IgG3 mAb is subclass-switched to IgG1, IgG2b, and IgG2a, all of these subclasses exhibit decreased affinity. This decrease in affinity is not seen when the 2A5 IgG1 mAb is switched to an IgG2b or IgG2a, strongly suggesting the drop in affinity is related to the IgG3 Fc region

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