Generation of rabbit monoclonal antibodies.

Rabbit hybridomas are gaining wide acceptance as serologic reagents to identify a variety of antigens, including proteins, small peptides, phosphorylated proteins, and polysaccharides. Rabbits make high-affinity IgG antibodies, all of which bind with high affinity to Protein A from Staphylococcus aureus and Protein G from Group G Streptococcus. Consequently, rabbit monoclonal antibodies of desired specificity can be rapidly detected using Protein A/G as secondary reagents. Here we describe the method for generating rabbit monoclonal antibodies using the rabbit hybridoma fusion partner 240E-1. The method begins with the immunization of rabbits and ends with the cloning the antigen-specific hybridomas.

Suppression of B lymphopoiesis at a lymphoid progenitor stage in adult rabbits.

B lymphopoiesis in rabbits is robust early in ontogeny, but is arrested by 16 weeks of age at which time no proB or preB cells are found in bone marrow (BM). To determine if BM cells from adults retain B lymphopoietic potential, we transferred BM from adult green fluorescent protein (gfp) transgenic rabbits into young rabbits. We found gfp+ preB cells arising in the young recipients, indicating that BM cells from adults can differentiate into B cell precursors. We identified a population of MHCII-IL-7-binding BM cells in adults that collectively expresses Tdt, EBF and Pax5-genes known to be expressed in murine lymphoid progenitors. Upon co-culture with OP9 or OP9 delta-like 1+ stromal cells, we found that these cells both expanded in number and differentiated into B and T cell precursors, respectively, showing that early lymphoid progenitors, designated rLP for rabbit lymphoid progenitors, are present within the MHCII-IL-7-binding BM cell population. Further, IL-7 was required for rLPs to expand and differentiate into B cell precursors in vitro. The arrest of B lymphopoiesis in adults, however, is not likely due to the absence of IL-7, because the level of IL-7 transcripts was higher in BM from adults than in young rabbits. B lymphopoiesis was re-initiated in adults after sub-lethal irradiation as shown by the reappearance of B cell precursors and the presence of B cell receptor excision circles in BM. We conclude that B lymphopoiesis in adults is suppressed at a lymphoid progenitor stage (MHCII-IL-7 binding) of development.

B lymphocyte deficiency in IgH-transgenic rabbits.

We developed IgH-transgenic rabbits carrying a productive VDJ-Cmu Tg and found the rabbits were B cell-deficient, with a 50-100% reduction in serum IgM and IgG levels. The bone marrow of newborn Tg rabbits contained severely reduced levels of preB cells and almost no B cells. The few preB cells present in the bone marrow were large, cycling cells that expressed the VDJ-Cmu Tg, indicating that the block in B cell development likely occurred at or before the transition from large (early) preB to small (late) preB cells. By immunoprecipitation, the Tg mu-chain paired with VpreB and lambda5, suggesting that the B cell deficiency is not due to an inability to form a preB cell receptor. Despite the block in B cell development, a few B cells, expressing predominantly endogenous mu-chains, began the second stage of development in GALT. B cells were localized in and beneath the follicle-associated epithelium of GALT prior to B cell follicle formation, suggesting to us that B cell follicle formation is initiated near the follicle-associated epithelium, possibly through contact with intestinal microbiota. These IgH-Tg rabbits should provide a useful model for studies of B cell development both in bone marrow and in GALT.

A negative regulatory element in the rabbit 3'IgH chromosomal region.

Mouse and human IgH loci contain several 3'IgH enhancers. In rabbit, a single hs1,2 enhancer is located 3' of the distal germ line Calpha gene, Calpha13. We searched for additional regulatory elements in this region by using a luciferase reporter assay and nucleotide sequence analysis. Within 8 kb 3' of Calpha13, we identified a 1-kb fragment that negatively regulated the hs1,2 enhancement of the Ialpha promoter. This negative regulatory element, Calpha-NRE, contains a conserved 300-bp region that is associated with 8 of the 13 germ line Calpha genes. This conserved region contains an E box that, by electrophoretic mobility shift assay, binds an E47-like protein. At the 5' end, Calpha-NRE also includes a 270-bp region with 20-bp repeats nearly identical to those 3' of mouse and human Calpha genes, and these repeats bind unidentified nuclear protein(s). Calpha-NRE appears to be a novel regulatory element that may contribute to the regulation of IgH gene expression.

Functional analysis of I alpha promoter regions of multiple IgA heavy chain genes.

The 13 nonallelic IgA H chain genes of rabbit are differentially expressed in vivo. They can be grouped into those expressed at high levels (Calpha4, Calpha5, Calpha6, Calpha9, Calpha10, Calpha12, and Calpha13), those expressed at low levels (Calpha1, Calpha2, Calpha7, and Calpha11), and those that are not expressed (Calpha3 and Calpha8). We tested whether the differential in vivo expression is due to differential responses of the Ialpha promoters to TGF-beta stimulation. We stimulated the rabbit B cell line 55D1 with TGF-beta and, using single-cell RT-PCR, found that expression of germline (GL) transcripts of alpha3 and alpha8 could not be induced. By luciferase reporter gene assay and EMSA we found that the promoters of the unexpressed isotypes Calpha3 and Calpha8 are defective, thereby explaining the absence of IgA3 and IgA8 in vivo. When comparing the promoter activities of the other isotypes we found that the activities did not reflect the degree of in vivo expression. Instead, the promoters of the isotypes expressed at high or low levels promoted expression of the luciferase gene to a similar degree, except for the Ialpha4 promoter, which had much higher activity. Also the degree to which TGF-beta induced GL expression of the various isotypes in 55D1 B cells did not reflect in vivo expression. However, most of the TGF-beta-stimulated cells expressed GL mRNA of multiple isotypes; no isotype was expressed preferentially. These results suggest that the final switch to a single isotype is regulated in a step subsequent to GL transcription, rather than by induction of GL transcripts by the Ialpha promoter.