The avidin-biotin complex in bioanalytical applications

Dendritic cells (DCs; 1) have been purified from mouse spleen in good yield. Spleen cell suspensions were floated on dense bovine plasma albumin (BPA) columns, and the low density fraction was adhered to glass (2). The adherent cells consisted of DCs and immature macrophages most of which eluted in a viable state from the culture dish after overnight incubation. The macrophages were then removed by selective rosetting with opsonized erythrocytes and recentrifugation on dense BPA. This protocol resulted in a purified DC fraction, containing 1--3 X 10(5) DCs/spleen, which was homogeneous and distinctive in its properties. All cells exhibited the phase contrast and transmission electron microscopy (EM) cytologic features that were previously described for freshly isolated adherent DCs. By scanning EM, most purified DCs exhibited a remarkable array of bulbous protrusions of varying length and shape, unlike any other lymphoid cell. All DCs expressed surface Ia and other major histocompatibility complex (MHC)- linked alloantigens. DCs, however, lacked surface Ig and T-cell antigens, and did not bind or interiorize opsonized erythrocytes. Purified DCs have been maintined in vitro for 3 days. Recovery of cultured purified cells was 70% or more of starting cell numbers. When [3H]uridine-tagged DCs were mixed with nonlabeled heterogeneous spleen cells, 70--80% of the labeled DCs were recovered as viable cells 2--3 days later. Purified DCs did not readhere to tissue culture surfaces and did not proliferate, even when cultured with mitogenic doses of concanavalin A and lipopolysaccharide. Finally, DCs did not change their cytologic or surface properties after 3 days of culture. These observations extend the evidence that DCs are a novel cell type and provide useful properties and techniques for their further study.

Lymphoid organs of mice. V. Purification of spleen dendritic cells, new surface markers, and maintenance in vitro.

Functional and phenotypic properties of porcine peripheral blood CD4/CD8 double-positive (DP) lymphocytes were examined. In cross-sectional and longitudinal studies involving a total of 103 pigs, this lymphocyte population was found to increase gradually in proportion with age, comprising < 2% of the total peripheral blood lymphocyte pool in 1-week-old swine and reaching 30-55% by 3 years of age. CD4/CD8 DP lymphocytes were able to proliferate in response to stimulation with recall viral antigen. Furthermore, these cells mostly expressed high levels of the surface antigen recognized by monoclonal antibody (mAb) 4B4 (4B4hi), which is specific for the human beta 1 integrin. The CD4+4B4hi lymphocytes from pseudorabies virus-immune swine, proliferated in response to stimulation with the homologous virus, while CD4+4B4lo lymphocytes did not. Stimulation of CD4 single-positive (SP) cells with recall viral antigen, but not with mitogen, resulted in the generation of lymphoblasts which were predominantly of CD4/CD8 DP phenotype, suggesting a role for recall antigen in the generation of this lymphocyte subset. More than half of the CD4+ lymphocytes from palatine tonsils of 6-month-old swine were CD4/CD8 DP, while in the lymph nodes CD4/CD8 DP cells accounted for only one-third or less of CD4+ cells. In contrast, CD4/CD8 DP lymphocytes were absent from the palatine tonsils of 3-day-old swine, which only contained CD4 SP cells. Together, these results indicate that porcine CD4/CD8 DP lymphocytes, exhibit properties of mature antigen-experienced cells, and are inducible by stimulation with recall antigen. These data are consistent with the hypothesis that this population in swine includes memory/effector T cells.

Functional and phenotypic analysis of porcine peripheral blood CD4/CD8 double-positive T cells.

CD40 signaling through tumor necrosis factor receptor-associated factors (TRAFs). Binding site specificity and activation of downstream pathways by distinct TRAFs.

Publisher Summary This chapter describes the results of a literature survey showing applications of avidin–biotin technology in tabular forms. One of the tables presents the experimental details from investigations that have used avidin columns for the isolation of target material. Native biotin-containing systems can be isolated directly on such columns. The isolation of other materials, such as membrane proteins and glycol-conjugates, is dependent on the mediation of a biotinylated binder. One of the problems in the use of avidin–biotin technology for isolation purposes is the difficulty encountered in eluting the bound material from the column. Other tables provide information relating to cytochemical localization investigations. These types of investigations usually involve the microscopic visualization of membrane-based sites mediated through the avidin–biotin interaction. In some cases, target sites have been directly biotinylated using group-specific biotinylating reagents. In others, biotinylated lectins, hormones, antibodies, and other binders were employed to mediate between the target molecule and the avidin-probe conjugate or complex.

Applications of avidin-biotin technology: literature survey.

Microdetermination of rabbit immunoglobulin allotypes by ELISA using specific antibodies conjugated with peroxidase or with biotin

During the preparation of rabbit-mouse hybridomas, an unusual degree of cellular proliferation and a high level of rabbit immunoglobulin secretion in culture was noted. The proliferation seemed to be dependent on the use of relatively high levels (10%) of fetal bovine serum because it was not seen when spleen cells were cultured using either horse serum or autologous serum in a variety of media. The peak of the proliferative response occurs at 7 to 10 d in culture, after which the lymphoblast population dies rapidly, leaving a large, granular, nonadherent cell as the predominant component in the culture. These cells did not seem to divide further but persisted in very gradually diminishing numbers for many months. This long lived cell has been characterized as a nonadherent macrophage by its morphology, intense esterase staining, expression of an Fc receptor for rabbit IgG, phagocytosis of latex particles and opsonized sheep erythrocytes, and mediation of antibody dependent cell mediated cytotoxicity on chicken erythrocytes.

Long lived nonadherent rabbit macrophages obtained from spleen cell cultures

The expression of latent allotypes is a well documented phenomenon in rabbits. Speculation about their molecular genetic basis and the mechanisms that control expression of these unexpected markers lead inevitably to questions about the Ig gene complement of the rabbit. This central question is under current study using probes derived originally from the mRNA of rabbit-mouse hybridomas secreting rabbit Ig chains. Some of the basic features of rabbit Ig genes are already clear from these studies, and DNA fragments that might encode latent allotypes have been identified with the first set of probes. Further gene closing sequencing should shortly provide a definitive answer to the question of latent allotypy and will also provide a detailed understanding of nominal Ig expression in rabbits.

The role of rabbit immunoglobulin allotypes in an immune network.

Publisher Summary This chapter provides an overview of antiallotypic antibodies. Allotypes are defined as intraspecies antigenic markers of immunoglobulins. The allotypic markers are distinguished from isotypes by the fact that allotypes are genetically determined whereas isotypes are present in every individual within a given species. The idiotype which is a marker for the variable portion of the immunoglobulin molecule was first defined by antisera prepared by injection of antibodies into allotypically identical donors. Although allotypes are defined as antigenic markers the specificities recognized directly relate to changes in the primary structure of the immunoglobulin chains. Allotype markers may be located in any portion of the immunoglobulin molecule and may correspond to amino acid differences at a single position or to differences at multiple positions. The chapter further explains preparation of antiallotypic antibodies.

[9] Antiallotypic antibodies

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Microdetermination of rabbit immunoglobulin allotypes by ELISA using specific antibodies conjugated with peroxidase or with biotin

Long lived nonadherent rabbit macrophages obtained from spleen cell cultures

The role of rabbit immunoglobulin allotypes in an immune network.

[9] Antiallotypic antibodies