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L S Rodkey

Publications and source records attributed to L S Rodkey.

At least 19 recordsLinked to original sources
PubMed1992

Direct detection of carrier ampholytes in immobilized pH gradients using picric acid precipitation.

A protocol is described for monitoring the heterogeneity of end products of organic syntheses yielding amphoteric molecules containing two or more amino groups. This protocol was found to be a valuable aid in synthesis of carrier ampholytes for specific isoelectric focusing applications. This method does not depend on the ampholytes themselves to dictate the conditions under which they are analyzed. Carrier ampholytes have been found previously to be insoluble in picric acid and the insolubility property was not dependent upon the pI of individual ampholyte species. This insolubility property was exploited in the protocol. Immobilized pH gradients were used to focus the carrier ampholytes. Ampholytes were then visualized in situ by picric acid precipitation. The data shows that the protocol is useful for analyzing the results of chemical manipulations for enhancing the resolution of carrier ampholytes. A direct relationship was shown between carrier ampholyte heterogeneity as demonstrated by this protocol and the resolution of complex protein mixtures in isoelectric focusing gels. Picric acid formed visible precipitates with a variety of organic compounds which contained more than one amino group.

Acrylamides↗
PubMed1992

Microheterogeneity of S-glycoprotein of mouse hepatitis virus temperature-sensitive mutants.

Mouse hepatitis virus (MHV) strain JHM (MHV-JHM) is a neurotropic coronavirus that causes acute fatal encephalomyelitis in 75-99% of infected mice. The surviving animals may subsequently develop demyelinating disease. We compared the S peplomer protein of the wild type (wt) and five temperature-sensitive (ts) mutants of MHV-JHM. In contrast with the wt, none of these five cause fatal disease (mortality less than 10%). Three of these ts mutants did not induce any demyelinating disease, a fourth caused demyelinating disease in 5% of the animals and a fifth, designated ts8, exhibited strong demyelinating properties and caused demyelination in 99% of the animals. SDS-PAGE analysis revealed no differences in the molecular weight of S peplomer protein of wt or ts MHV-JHM mutants. However, isoelectric focusing of the S protein of these five ts mutants and the wt MHV-JHM, followed by transfer to nitrocellulose sheets and immunoblotting with anti-S specific antibody revealed significant differences in the microheterogeneity of the S protein.

Animals↗
PubMed1992

Ultrasound-guided blood sampling of rabbit fetuses.

A rabbit animal model for hemolytic disease of the newborn has been previously described. However, evaluating the effects of this disease was limited to histologic and hematologic examinations of liveborn kitlings. To assess the feasibility of in utero blood sampling, we performed ultrasound-guided cardiac sampling of 50 fetuses in 16 New Zealand White does on days 26 and 27 of gestation. The overall rate of successful sampling was 80%. The procedure-related mortality declined to 35% by the third phase of the study. The mean (+/- SD) hematocrit (%) and reticulocyte values (#/100 RBCs) on day 26 were 26.3 +/- 3.3 and 35.6 +/- 5.1, respectively; values on day 27 were 31.3 +/- 4.9 and 27.5 +/- 7.6. The results of this study suggest that hematologic data can be obtained from rabbit fetuses in the majority of cases with only moderate fetal loss.

Animals↗
PubMed1990

Comparative studies of recycling isoelectric focusing and continuous flow electrophoresis: separation of proteins with minor charge differences.

Continuous flow zone electrophoresis (CFE) and recycling isoelectric focusing (RIEF) are two of the alternative formats for fluid phase preparative isolation of biological products in liquid separation media. The McDonnell Douglas CFE system has been used for both ground-based and microgravity separations. The ground-based McDonnell Douglas CFE and RIEF were compared for the ability to resolve mixtures of proteins with known charge differences. Mixtures of 1) cytochrome c, myoglobin, and ovalbumin or 2) beta-lactoglobulin and ovalbumin were used to evaluate the resolving capabilities of CFE and RIEF. Following separation, fractions were analyzed by determining absorbance at 280 nm and by analytical isoelectric focusing (IEF) using Coomassie Brilliant Blue or silver staining to detect focused proteins. Both CFE and RIEF apparently separated the components of both mixtures into individual peaks, separated by fractions which contained little or no detectable protein. Coomassie-stained analytical IEF gels supported this finding. However, when separated proteins were analyzed by silver staining of the analytical gels, the separation of ovalbumin from beta-lactoglobulin by CFE was not complete. Ovalbumin was free of beta-lactoglobulin but beta-lactoglobulin was contaminated by trace amounts of ovalbumin. RIEF clearly separated each protein with no detectable contamination. These data demonstrate the superiority of RIEF over CFE for resolution of protein mixtures having only minor charge differences. RIEF may be more efficient due to the documented electrodissociation of noncovalent protein:protein complexes which occurs during RIEF separations.

Cytochrome c Group↗
PubMed1990

Induction and immunochemical properties of a novel anti-antibody.

This paper describes results which characterize an induced antibody in normal outbred rabbits which we have, for convenience, called parareactant (PR). PR resulting from autoimmunization of rabbits with either keyhole limpet hemocyanin-anti-tetanus toxoid F(ab')2 or with tetanus toxoid-anti-tetanus toxoid F(ab')2 complexes was studied. PR activity was directed solely to autologous, homologous or heterologous F(ab')2 fragments regardless of their specificity. PR failed to react with intact antibodies or with antigen-antibody complexes consisting of homologous antibody bound to specific antigen. Radioimmunoassay and ELISA inhibition assays showed that reactivity between PR and autologous anti-tetanus toxoid F(ab')2 or homologous anti-bovine serum albumin F(ab')2 fragments was specifically inhibited with antigen. Anti-allotypic antibodies specific for a2 and b6 markers strongly inhibited binding of 125I-anti-micrococcal carbohydrate F(ab')2 (a2, b6) with PR (a3, b4, b5). PR specificity thus appears to be directed against non-idiotypic determinants present in Fv regions. Affinity immunoblotting was used to analyze clonality of PR in the sera collected from individual rabbits during the course of an active immune response. PR-positive sera displayed clonally restricted spectrotype patterns. PR molecules were predominantly IgG with isoelectric points of 5.9-6.8. These results strongly suggest that these PR molecules are coded by a small number of V region genes.

Animals↗
PubMed1990

Free flow cell electrophoresis using zwitterionic buffer.

Studies of a zwitterionic buffer formulated for cell electrophoresis were done using the McDonnell-Douglas Continuous Flow Electrophoresis System. Standard buffers were analyzed for their stability in the electrical field and the results showed that both buffers tested were inherently unstable. Further, titration studies showed that the standards buffers buffered poorly at the pH employed for electrophoresis. The zwitterionic buffer buffered well at its nominal pH and was shown to be stable in the electrical field. Comparative studies of the buffer with standard cell separation buffers using formalin fixed rabbit and goose red blood cells showed that the zwitterionic buffer gave better resolution of the fixed cells. Studies with viable hybridoma cells showed that buffer Q supported cell viability equal to Hank's Balanced Salt Solution and that hybridoma cells in different stages of the growth cycle demonstrated reproducible differences in electrophoretic mobility.

Animals↗
PubMed1989

Adoptive immunity transferred by naive donor cells immunized in vitro.

Speedy restoration of immune responsiveness in bone marrow recipients has been the objective of studies in which the donor was immunized so that specific immunologic memory could be transferred adoptively and selectively. Using unrelated rabbits, matched for major histocompatibility antigens but mismatched for their immunoglobulin allotypes, it could be shown that recipients of lymphoid cells from naive donors became B cell chimeras but did not use donor-derived B cells for their antibody responses to test antigens. In contrast, cells from donors primed for such antigens dominated antibody production in recipients in response to specific challenge. Clonal restriction in such adoptive responses was demonstrated. We now show that the induction of effective memory in cells from naive donors can be achieved in vitro during the preparation of donor cells for transfer to the recipient. Early challenge of the recipient enhances expression of the transferred immune response quantitatively and also results in the establishment or preservation of a larger diversity of clones from the donor.

Animals↗
PubMed1988

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.

Cytochrome c Group↗
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↗
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↗