L. Scott Rodkey

Affinity immunoblotting. High resolution isoelectric focusing analysis of antibody clonotype distribution.

A sensitive and specific method has been developed for analyzing specific antibody clonotype changes during an immune response or comparing multiple sera for antibody clonotype similarities. Polyclonal serum antibodies were separated by flatbed acrylamide isoelectric focusing (IEF) and analyzed by an affinity immunoblotting method using antigen-coated nitrocellulose membranes. Antibodies present on the surface of the acrylamide gels after IEF bound the antigen on the nitrocellulose when the coated nitrocellulose was laid over the gels. Non-specific protein binding was inhibited with Tween 20. Bound IgG antibody clonotypes were detected using peroxidase-conjugated anti-IgG. This method has been used for the analysis of Ig clonotypes specific for five protein antigens and two carbohydrate antigens. Optimal antigen concentration for coating the nitrocellulose membranes ranged from 10 to 100 micrograms/ml. The reactions could be inhibited by saturating the nitrocellulose with soluble protein antigen or free hapten prior to immunoblotting. Antibodies of alternative allotypic forms were detected by probing the immunoblot with biotinylated anti-allotype antibodies instead of anti-IgG. The simultaneous analysis of alternative allelic forms of antibodies of defined specificity is not possible with methods which use labelled antigen for clonotype detection.

An animal model for hemolytic disease of the fetus and newborn. II. Fetal effects in New Zealand rabbits

OBJECTIVE The addition of ultrasonography and ultrasonographically directed fetal blood sampling was attempted in an effort to study the fetal effects of red blood cell alloimmunization in a rabbit model. STUDY DESIGN Nineteen New Zealand does were alloimmunized to incompatible red blood cells. Sensitized does were bred twice, once with a homozygous buck of incompatible blood type and once with a homozygous buck of compatible blood type. Ultrasonographic examinations were performed on days 20 and 27 of gestation (term 28 to 31 days). Fetal blood sampling was undertaken on day 27 of gestation, and hematologic data were compared between compatible and incompatible litters. RESULTS A total of 41 pregnancies occurred in 19 does. Fetal hemoglobin was higher in the compatible litters (9.7 gm/dl vs 5.8 gm/dl, p < 0.001), whereas no difference could be detected between the respective reticulocyte counts (31.9 vs 36.0/100 red blood cells, p = 0.2). Hydrops fetalis was noted in none of 18 compatible litters versus 12 of 19 incompatible litters (p < 0.01). CONCLUSION A disease analogous to human hemolytic disease of the newborn can be induced in the rabbit fetus.

Comparison of the immune response to Ars-BGG in germfree or conventional piglets.

Neonatal germfree (GF) colostrum-deprived and conventional (CV) colostrum-fed piglets were immunized IP with p-azo-phenyl-arsonate-bovine gamma globulin (Ars-BGG) in Freunds adjuvant to study the development of the immune response in the absence or presence of maternal antibodies and environmental antigens. Overall, the immune response varied greatly within each group but did not differ in GF from CV piglets statistically. Affinity immunoblot analysis suggested that anti-Ars antibody was more restricted in GF than CV piglets and clonotype shifts occurred more in GF than CV piglets after each antigenic stimulation. In contrast, the clonotype pattern of the anti-BGG antibody was similarly heterogeneous in the two groups. Based on the affinity immunoblot data the antibodies generated to the Ars-haptenic group in CV piglets are more heterogeneous than GF piglets and suggest that clonotype generation is influenced by maternal antibodies and environmental antigens.

Natural Regulation of Antibody Clones by Auto‐Anti‐Idiotypic Antibodies in Rabbitsa

The purpose of this paper is to review and summarize data accumulated in our laboratories that relate to the ability of normal outbred rabbits to produce autoanti-idiotypic (AAI) antibodies specific for their own previously synthesized antibody idiotopes. Data collected during the last seven to eight years by numerous laboratories suggest that the immune system can recognize and respond to both foreign and self epitopes. Recent data suggests that a variety of strategies may be employed by the immune system to regulate different elements and compartments within the immune system using the idiotope as the element of recognition. The outbred rabbit has been used in these studies as a model more similar to the human than the inbred strains of mice, and the rationale behind many of the experimental approaches is to determine what strategies of specific immunoregulation predominate under specified conditions in an outbred species. Further, the wellcharacterized immunoglobulin (Ig) allotypic markers in rabbits provide convenient markers for controlling purity of reagents and for use as antigens. The a locus markers a l , a2, and a3 are particularly useful because they are known to be V-region markers and are heterogenous, with each set possessing several subsets of molecules that share some epitopes and also express unique epitopes.

Ionic binding properties of carrier ampholytes

Radioactive ampholytes were synthesized with specific activity of 638 microCi/g. These were used in studies of ampholyte binding to target proteins under non-ionic conditions. These radioactive ampholytes bound to target proteins but were dissociable in sodium chloride solutions with dissociation occurring in a concentration dependent way. The ampholytes could be dissociated from target molecules using excess unlabelled ampholytes synthesized in the laboratory as well as commercial ampholytes. Radioactive ampholytes were bound to target proteins with different isoelectric points and the bound ampholytes were eluted and analyzed by recycling isoelectric focusing. The results showed that acidic proteins bound basic ampholytes and basic proteins bound acidic ampholytes. Acidic radioactive ampholytes were selectively bound by Sephacryl S-200 and ampholyte exchange from protein to Sephacryl S-200 was shown.

Selective survival and expression of B-lymphocyte memory cells during long-term serial transplantation

The expression of individual clonal products during long-term in vivo culture was investigated using a rabbit model system of bone marrow transplantation. RLA (MHC) matched rabbits were deliberately mismatched for kappa light chain immunoglobulin allotype to facilitate identification of antibodies as being of donor or recipient origin. Recipients of cells from antigen-primed donors responded to antigen stimulation with antibody of donor origin, showing that cells were effectively triggered for antibody production in the recipient. Isoelectric focusing followed by affinity immunoblotting of the expressed antibodies showed that the responding B-cell clonotype repertoire remained virtually unchanged throughout the extensive cell transfer protocol used. These results suggest that B-memory-cell stimulation, rather than stem cell differentiation, was responsible for the observed response patterns. There was no detectable increase in the heterogeneity of the donor-derived antibody response with time and no new clonotypes appeared which were not present in the cell donor. Unlike previous studies, early stimulation with antigen was not required for successful engraftment and memory cell establishment. However, our data suggest that the timing of antigenic challenge may determine which of the donor-derived clones will dominate a response after antigen challenge of the recipient.