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L S Rodkey

Publications and source records attributed to L S Rodkey.

At least 37 records · Page 2Linked to original sources
PubMed1988

Isoelectric focusing analysis of antibody clonotype changes occurring during immune responses using immobilized pH gradients.

Serum was collected from rabbits at 2-day intervals following a single injection with tetanus toxoid or at weekly intervals following multiple injections with Micrococcus lysodeikticus cell walls. These sera were analyzed for the presence of individual clonotypes of specific anti-tetanus or anti-micrococcal antibodies by isoelectric focusing in immobilized pH gradients with added carrier ampholytes followed by affinity immunoblotting. The affinity immunoblots obtained clearly defined both the rapid disappearance and late appearance of distinct subsets of antibody clonotypes during the response. These data demonstrate the application of affinity immunoblotting combined with immobilized pH gradients for detecting the subtle changes in specific antibody clonotype patterns which occur during an immune response.

Animals↗
PubMed1987

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.

Animals↗
PubMed1987

Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis.

Quality control of murine hybridoma secretory products was performed using two variations of the isoelectric focusing affinity immunoblot analysis. The first approach employed antigen-coated nitrocellulose placed on top of an acrylamide gel containing isoelectrically focused ascites to bind antigen specific monoclonal antibody (MoAb). Murine antibody bound to the insolubilized antigen was then detected with enzyme-conjugated anti-mouse IgG. In a second variation, focused ascites proteins were passively blotted onto nitro-cellulose and specific monoclonal antibody was detected with enzyme-conjugated antigen. Several batches of ascites containing anti-human IgG antibodies that were produced by 6 hybridomas over a 1-3 year period were assessed by IEF-affinity immunoblot analysis. Both immunoblot approaches permitted effective monitoring of immunoreactive antibody for pI microheterogeneity. IEF-affinity immunoblot patterns of unprocessed ascites displayed specific MoAb banding patterns with narrow pI ranges (less than or equal to 0.6 pH units), in contrast to the reported 5.5-8.0 pI range of polyclonal mouse IgG. Banding patterns obtained in the IEF affinity immunoblot typically displayed 3-5 major dense bands flanked by 2-4 minor fainter bands. Batches of ascites obtained years apart produced similar immunoblot patterns, indicating constant antibody production and confirming the stability of these hybridoma clones. Minor bands appeared in 2 earlier lots of ascites, suggesting possible modification of antibody during storage. IEF affinity immunoblot analysis is a useful tool for monitoring MoAb pI microheterogeneity as an indicator of antibody quality without the need for isolation of monoclonal antibody from culture medium or ascites.

Animals↗
PubMed1986

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.

Antibodies↗
PubMed1983

Regulation of natural antiallotype antibody responses by idiotype network-induced auto-antiidiotypic antibodies.

This study was designed to determine whether natural immune responses could elicit immunoregulatory auto-antiidiotypic antibodies. Female rabbits heterozygous at the a and b Ig loci were bred to homozygous males. Offspring of one such breeding were studied for natural production of antibodies specific for the noninherited allotypes and for the production of immunoregulatory auto-antiidiotypic antibodies. All offspring mounted natural antiallotype responses. The anti-a1 responses cycled as a function of time whereas the anti-b5 responses were invariant. Anti-a1 responses from two offspring were shown to change specificity for different a1 subsets as they cycled. Anti-a1 was purified from the first cycle and was used to assay for auto-antiidiotypic responses. Auto-antiidiotypic antibodies were detected and were found to cycle in an inverse way with the anti-a1 cycles. The idiotopes detected using the natural auto-antiidiotypic antisera were strongly cross-reactive. Subsequent deliberate immunization showed that antibodies specific for all a1 subsets could be elicited after auto-antiidiotypic regulation had functioned. The data support the interpretation that idiotype network interactions indeed function in naturally occurring immunologic situations and are not merely laboratory curiosities or artifacts.

Aging↗
PubMed1983

Properties of narrow-range ampholytes isolated from wide-range ampholyte preparations.

A simple procedure for obtaining useful narrow-pH-range ampholytes from inexpensive laboratory-synthesized ampholytes by preparative isoelectric focusing in Pevikon is described. The narrow-range ampholytes prepared in this way are comparable to commercial ampholyte preparations as judged by conductivity, buffer capacity, pH gradient formation, and resolving power. These inexpensive narrow-range ampholytes are particularly well suited to preparative isoelectric focusing applications requiring large quantities of ampholytes.

Ampholyte Mixtures↗
PubMed1983

Destruction of antibody idiotopes with ultra-low concentrations of reducing agents.

The nature of the idiotopes present on F(ab')2 fragments prepared from rabbit anti-micrococcal carbohydrate antibodies and the loss of idiotypic reactivity of these F(ab')2 fragments upon iodination were examined. Rabbit anti-micrococcal idiotopes were shown to be exquisitely sensitive to treatment with very low concentrations of sodium metabisulfite or 2-mercaptoethanol. The treatment destroyed anti-micrococcal idiotopes, as shown by the loss of idiotopes on F(ab')2 fragments after reduction; the allotype epitopes and the antigen binding capacity of the F(ab')2 fragments were unaffected. The destruction of the idiotopes by very low concentrations of reducing agents indicated that an extremely labile disulfide bond is involved in the structure of the idiotope or in the maintenance of the conformation of the anti-micrococcal idiotopes. Identical reduction-sensitive anti-micrococcal idiotopes have been demonstrated in a number of related outbred rabbits, and in each case they induced a natural auto-anti-idiotype (AAI) antibody response. Recognition of the existence of these reduction-sensitive idiotopes and their properties could provide a basis for further study of these idiotopes and may lead to a better understanding of the idiotope network.

Animals↗
PubMed1982

Naturally induced auto-anit-idiotypic antibodies. Induction by identical idiotopes in some members of an outbred rabbit family.

Naturally induced auto-anti-idiotypic (AAI) antibody responses specific for antimicrococcal antibody idiotypes were detected in 42% of the rabbits in a family immunized with Micrococcus lysodeikticus. The natural AAI response of each rabbit recognized only a portion (11-41%) of that individual's total antimicrococcal antibody population. Cross-reactions of idiotypes were observed within the group of rabbits exhibiting natural AAI responses. Examination of the basis for the cross-reactions showed that the natural AAI antisera recognized identical idiotopes on the antimicrococcal F(ab')2 fragments from each rabbit that made an AAI response. The cross-reactive idiotopes were shown to be of paternal origin and were found in the antimicrococcal antibodies of each offspring. The data strongly support the idiotypic network concept that naturally induced AAI responses may occur routinely in outbred normal individuals as a result of antigenic stimulation. Further, the data suggest that the induction of regulatory AAI antibody responses in outbred rabbits may depend on the expression of particular germ line idiotopes.

Animals↗
PubMed1979

Autoregulation of an antibody response via network-induced auto-anti-idiotype.

The antibody response of a single outbred rabbit was studied throughout three rounds of injections with Micrococcus lysodeikticus vaccine over a 31-mo period. The first-round response was characterized by a vigorous anti- micrococcus response and a strong anti-IgG rheumatoid factor response. The second-round response consisted of a triad of interacting molecules: anti- micrococcal antibodies, autoanti-idiotypic antibodies specific for distinct clonotypes of the first-round anti-micrococcal antibodies, and Fc-specific anti-IgG rheumatoid factor. The interacting triple complex was detected because of the formation of an immune complex that became insoluble upon dilution of the serum. Complex formation was inhibited in the presence of saccharide compounds known to be major immunodominant determinants of the micrococcal cell-wall carbohydrate polymer. The same saccharides did not affect the reaction of rheumatoid factor with IgG. Direct-binding radioimmunoassays ruled out mediation of the dilution-precipitation reaction by soluble micrococcal antigens. Specific absorption of rheumatoid factor inhibited the dilution-precipitation reaction. Auto-anti-idiotypic antibodies were specifically purified from second-round sera, directly confirming the presence of these antibodies. Suppressive effects of auto-anti-idiotypic antibodies on distinct antibody clonotypes were shown by gel isoelectric focusing of first-, second-, and third-round sera. Clonotypes expressed in the first round of immunizations were reduced in quantity or absent when auto-anti-idiotypic antibodies were detectable. Greatly enhanced levels or initial synthesis of new clonotypes of anti-micrococcal antibodies were detected during the period of auto-anti-idiotype synthesis. The third-round sera, devoid of detectable auto-anti-idiotype, contained clonotypes characteristic of both first- and second-round antisera. Thus, auto-anti- idiotypic-mediated suppression appeared to be reversible. The data are interpreted as lending strong support for concepts of autoregulation of immune processes in normal outbred animals via an idiotypic network.

Animals↗
PubMed1979

Distinct functions of monoclonal IgG antibody depend on antigen-site specificities.

Intraveneous hyperimmunization of selectivity bred rabbits with streptococcal group A-variant vaccines elicits antibody responses of restricted heterogeneity at high antibody levels. All antisera contain two functionally distinct antibody populations, which can be isolated in single-band purity upon analytical isoelectric focusing. Typical examples of these two kinds of single-band antibodies were investigated in great detail for several parameters by a variety of methods. 85--99% of the streptococcal group A-variant polysaccharide (Av-CHO)-specific antibody in the antisera does not precipitate the isolated 5,000 daltons poly-L-rhamnose antigen, neither agglutinates nor lyses in the presence of complement Av-CHO-coated sheep erythrocytes (SRBC), binds the radio-labeled Av-CHO with an association constant in the ragne of 10(5)--10(6) M-1, and is of terminal specificity (nonreducing end) for the linear Av-CHO. In contrast, the minor fraction of Av-CHO-specific antibody (1--15%) does precipitate the linear Av-CHO, both agglutinates and lyses Av-CHO-coated SRBC in the presence of complement, has an affinity range of 10(8)--10(9) M-1, and is of internal specificity for the Av-CHO. The antigenic determinants of the Av-CHO for the antibodies are nonoverlapping, only one Fab of the low affinity antibody can be bound whereas four Fab of the high affinity antibody are accommodated. Hence, the determinant specificity explains the functional differences observed, for there is no indication of subclass differences. A mechanistic model of the A-variant carbohydrate presentation on the vaccine appears to account best for the unbalanced levels of low and high affinity antibody.

Animals↗
PubMed1979

Clonal dominance of low-affinity antibodies in rabbit hyperimmune anti-streptococcal group A-variant polysaccharide antisera.

Intraveneous hyperimmunization of selectively bred rabbits with streptococcal group A-variant vaccines elicits antibody responses of restricted heterogeneity at high antibody levels. In these antisera, IgG with dissociation constants Kd = 10(-6) M constitutes 90% and IgG with Kp = 10(-9) M accounts for only 10% of the group A-variant polysaccharide-specific antibodies. The low affinity antibody fraction represents the dominant clonotypes. Preparative isoelectric focusing in granulated (Ultrodex G-75) gels was used to successfully purify single-band material belonging to dominant spectrotypes. Affinity studies with these antibody fractions with the highest reported degree of purity yielded Kd = 10(-6) M values, thus confirming that clonal dominance is exclusively associated with low-affinity antibodies. Since it is known from previous work (M. Cramer and D. G. Braun, Scand. J. Immunol. 1975. 4:63) and from the rabbit antisera used here that clonal dominance of this sort is long-lived, this work fails to support the argument of immune maturation. The data more logically relate to antibodies that emerge with different subspecificities -- recognized in the antigen as a function of time in immunization procedures -- rather than to an inherent property of the immune system.

Animals↗