Asymmetric contribution to Ig repertoire diversity by V kappa exons: differences in the utilization of V kappa 10 exons.

The mouse has approximately 140 germline V kappa genes, and functional V kappa exons are expressed at roughly equivalent levels in the preimmune repertoire. We have examined the expression of individual members of the V kappa 10 family. V kappa 10A and V kappa 10B genes have been utilized in numerous hybridomas and myelomas, while V kappa 10C has not. In this study, we have cloned the V kappa 10C gene and shown that it is structurally functional, has the expected promoter elements and recombination signal sequences, and that it is capable of recombination. V kappa 10C mRNA, however, is present at levels at least 1000-fold lower than V kappa 10A and V kappa 10B in adult spleens. While there are no sequence differences in the octamer or TATA box between V kappa 10C and V kappa 10A, there are three nucleotide changes in the promoter region. These promoters equally drive the expression of a reporter gene in B cells or plasma cells, but the V kappa 10A promoter is able to drive expression in pre-B cell lines significantly better than the V kappa 10C promoter (p < 0.05). V kappa 10C rearrangements can be detected in bone marrow and splenic DNA. Therefore, the lack of V kappa 10C expression may reflect the inability of V kappa 10C-rearranged cells to undergo positive or negative selection. Our results suggest that the available Ab repertoire is shaped not only by the number of structurally functional genes, but also by the ability of assembled genes to be expressed at critical points during B cell maturation.

Histamine measurement in whole blood and cell supernatants. A comparative study of two different radioimmunoassays.

Histamine as an important mediator of the allergic reaction has been measured by a number of methods both in whole blood and cell supernatants after in vitro release. We compared the fluorometric histamine assay with two radioimmunoassays and studied detection limit, recovery, cross-reactivity and accuracy in buffer-based standards as well as the sensitivity after IgE-mediated in vitro release from washed cells and whole blood. Our data indicate that the double-antibody RIA (Pharmacia) can be used for studies of basophil histamine release in both whole blood and cell supernatants. Due to significant cross-reactivity to N-methyl-histamine it cannot be used for histamine determinations in plasma. The second radioimmunoassay using succinyl-glycine as an acylating reagent and monoclonal antibodies, which have been raised to acylated histamine, is the most sensitive assay without any cross-reactivity with methylhistamine.