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Results for “antibody characterization”

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At least 55 records · Page 3Linked to original sources
PubMed1979

Gamma-aminobutyric acid pathways in the cerebellum studied by retrograde and anterograde transport of glutamic acid decarboxylase antibody after in vivo injections.

Injections of characterized antibody against glutamic acid decarboxylase (GAD), the enzyme responsible for the synthesis of gamma-aminobutyric acid (GABA), were made into the cerebellum. Small cortical injections of anti-GAD antibody produced labeled stellate, basket, Purkinje, and Golgi cells and their processes at the injection site. Anterograde transport of GAD antigen-antibody complexes in Purkinje cell axons caused intense labelling of terminals in deep cerebellar and several vestibular nuclei. Small groups of mossy fiber rosettes labeled and produced retrograde labeling and GAD immunoreactivity in a small number of pleomorphic neurons in the deep cerebellar nuclei. Injections into the dentate nucleus produced retrograde labeling in Purkinje cell bodies and anterograde label in a small number of mossy fiber rosettes. All projections conformed to previously reported topographic distributions of corticonuclear and nucleocortical cerebellar pathways. These findings confirm the GABA content of most Purkinje cell-deep nuclei connections and provide new evidence for a GABA component in part of the nucleocortical pathway in the cerebellum. Immunocytochemical controls for specificity were conducted by injections of preimmune rabbit serum as a substitute for GAD antibody. Only nonspecific labeling was obtained in these cases. Colchicine caused a cumulative enhancement of GAD immunoreactivity in all cases. The present studies indicate that the method of in vivo antibody injections can be utilized to study chemically specific connections in nervous tissue.

Animals↗
PubMed1977

Immunological studies on beef-heart ubiquinol--cytochrome c reductase (complex III)

Antibodies against isolated beef-heart ubiquinol--cytochrome c reductase (complex III) have been characterized. Antibodies to complex III react strongly with isolated beef heart complex III and intact beef heart mitochondria, as shown by immunodiffusion and rocket electrophoresis experiments. The complex III content of intact mitochondria can be quantitated with rocket electrophoresis using isolated complex III as a standard. Antibodies to complex III also react with beef liver mitochondria and with both heart and liver mitochondria from rats. The latter are very weak antigens compared to beef heart material. Antibodies to complex III do not react with respiratory chain complexes I and IV, or F1-ATPase from beef heart mitochondria, but gives a slight, but variable, reaction with complex II and the membrane fraction isolated from complex V (oligomycin-sensitive ATPase). Antigenic sites are located on at least five of the seven peptides of complex III. These peptides are presumably lacking in respiratory chain complexes which do not react with antibodies to complex III, and are assumed to be uniquely located in complex III. Antiserum against complex III inhibitis duroquinol--cytochrome c reductase activity in isolated complex III and in complex III incorporated into phospholipid vesicles. Oxidation of NADH and succinate is not affected in submitochondrial particles treated with 6-times more antibody than required for complete inhibition of enzyme activity in free complex III or in complex III-phospholipid vesicles.

Animals↗
PubMed1976

Structural and serological relationships among different antibodies from the same rabbit antiserum. I. Isolation, chemical and allotypic characterization of ten antibody components from one anti-streptococcal serum.

The relationships among antibodies produced by an individual rabbit were examined using multiple antibodies elicited by hyperimmunization with Group C streptococcal vaccine. Immunoadsorbent chromatography followed by agarose block electrophoresis yielded nine different antibody fractions, all directed against Group C carbohydrate, from the immune plasma of rabbit 4295 (allotype a1/b5). Electrophoresis of the antibody light chains gave single bands for most of the antibodies. Amino acid sequence analyses carried out on several of the antibody L-chains revealed that five of these had aspartic acid at the amino terminus. By quantitative allotypic analyses all antibodies were allotype a1/b5. One antibody fraction, homogeneous by all other criteria, contained antibodies with two distinct group a allotypic subspecificities. The two antibodies in this fraction could be separated on an anti-a1 column. The ten antibodies were placed into a minimum of three groups based on differences in a1 allotype subspecificity.

Amino Acid Sequence↗
PubMed1977

Immunological analysis of plasma membranes of a T-strain of mycoplasma (Ureaplasma urealyticum).

The cell membranes of a T-strain of mycoplasma, obtained by ultrasonic disruption, were as effective as whole organisms in eliciting metabolism-inhibiting and complement-fixing antibodies. The soluble fraction separated from cell membranes by centrifugation at 35,000 X g showed a minor ability to elicit an antibody response as measured by metabolism inhibition and complement fixation tests. After a further centrifugation at 100,000 X g, the immunogenic activity of the soluble fraction was completely lost. Immunogenic determinants in mycoplasma membranes could also be demonstrated by adsorption tests; cell membranes were more effective than soluble fractions in adsorbing antibody capacity from the immune sera against whole cells. It has been shown by further experiments that cell membranes have at least two major antigenic determinants, which differ either in chemical nature or in capacity to adsorb and evoke antibodies, characterized by different serological behaviors.

Adsorption↗
PubMed1978

Role of nonagglutinating antibody in the protracted immunity of vaccinated mice to Pseudomonas aeruginosa infection.

Effective immunization against infection with Pseudomonas aeruginosa is difficult to evaluate because agglutinin levels decline rapidly. Because fractionation of hyperimmune sera often yields more specific antibody than can be accounted for by direct agglutination tests, an immunoglobulin-specific assay based on antiglobulin augmentation was used to characterize antibody responses of C3H/HeJ mice vaccinated with P. aeruginosa type 2 lipopolysaccharide. Nonagglutinating antibodies, initially detected at 2 weeks post-primary vaccination, were predominantly immunoglobulin G after 5 weeks, and they remained elevated at levels usually 32-fold higher than the direct titer throughout the 4-month study period. The sequential production of immunoglobulin M, then immunoglobulin G, followed that found in orthodox immunological responses. Sera that contained nonagglutinating antibodies but not direct agglutinins (14 to 16 weeks) enhanced phagocytosis of P. aeruginosa type 2 by macrophages from unimmunized mice and passively immunized mice against lethal challenge doses; bactericidal activity of these sera was not demonstrated in the presence or absence of complement. When challenged with 1, 10, and 100 50% lethal doses at 16 weeks, survival rates of actively immunized mice were significantly higher than those of unvaccinated mice (P < 0.001). Thus, at a time when no direct agglutinins were detectable, the augmented system detected nonagglutinating antibodies that could confer protracted resistance in vaccinated mice to pseudomonas infection.

Agglutinins↗
PubMed1977

Structural studies on induced antibodies with defined idiotypic specificities. V. The complete amino acid sequence of the light chain variable regions of anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype.

The complete amino acid sequence of the variable regions of light chains derived from anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype is reported. At least two and probably more than three distinct light chains are associated with this idiotypically characterized antibody. The antibodies have several differences in their "framework" structures but evidence is presented indicating that all three light chain hypervariable regions have a homogeneous sequence. The data are discussed in relation to the various theories of antibody diversity. In addition, the findings support the view that hypervariable regions, idiotypic determinants, and the antibody-combining site involve, to a large extent, the same molecular structures.

Amino Acid Sequence↗
PubMed1976

Affinity-immunoadsorbent fractionation of rat anti-streptococcal A carbohydrate antibodies of restricted heterogeneity.

Antisera obtained from selectively bred Sprague-Dawley rats after a primary series of immunization with group A streptococcal vaccine exhibit specific anti-carbohydrate antibodies of restricted heterogeneity. Separation of anti-carbohydrate antibodies was achieved on the basis of differences in the relative binding affinities of the antibodies for an insoluble, hapten (N-acetyl-D-glucosamine) immunoadsorbent. Evaluation of several eluting reagents including hapten, thiocyanate, urea and acid-salt demonstrated the chaotropic ion thiocyanate to be most effective in separating anti-hapten populations of individual affinity characteristics. In general, there was no consistent relationship between the net electrical charge of an antibody and its relative binding affinity for the immunoadsorbent. Furthermore, non-precipitating rat anti-streptococcal A carbohydrate antibodies were characterized as antibodies of relatively low binding affinity for the hapten immunoadsorbent. From a practical point of view, thiocyanate is recommended for the routine elution of anti-carbohydrate antibodies from innumoadsorbents.

Animals↗
PubMed1979

Cilia regeneration in starved tetrahymena: an inducible system for studying gene expression and organelle biogenesis.

Deciliated starved Tetrahymena recover motility with kinetics similar to those of growing cells and, like growing cells, require RNA and protein synthesis for regeneration. Comparisons of polysome profiles and electrophoretic analyses of newly synthesized proteins indicate, however, that the basal level of protein synthesis in starved cells is markedly lower than that in growing cells. This difference allows demonstration of changes in protein synthesis following deciliation of starved cells which cannot be detected (if they occur at all) in growing cells. Deciliation of starved cells induces a specific and orderly program of protein synthesis. The synthesis of an 80,000 dalton protein (deciliation-induced protein, DIP) begins shortly after deciliation, comprises 15% of the protein synthesized from 20-60 min, and declines around 60 min after deciliation, shortly after most cells have begun to regenerate cilia. The synthesis of a 55,000 dalton protein is also induced during regeneration and has been identified as tubulin using a well characterized antibody made to ciliary tubulin. Tubulin synthesis is undetectable during the first hour after deciliation even though 60-80% of the cells regain mobility and regenerate short but clearly visible cilia. Tubulin synthesis begins 60 min after deciliation and continues for 2 hr. At its peak, tubulin comprises 7-8% of the protein synthesized. The results of actinomycin D addition at different times after deciliation suggest that RNA required for DIP synthesis is synthesized early (0-30 min), while RNA required for tubulin is synthesized later and over a longer period (30-90 min). Thus deciliation of starved cells, an event occurring at the cell periphery, initiates a well defined and reproducible series of events culminating in cilia formation. This system should be useful in elucidating the molecular mechanisms regulating gene expression and organelle biogenesis in Tetrahymena.

Animals↗
PubMed1975

A biologically active hormonal fragment isolated from bovine parathyroid glands (BPTH 1-65).

Fresh frozen bovine parathyroid glands were defatted in acetone, when extracted with phenol. Following trichloroacetic acid precipitation, the resultant peptides were chromatographed on Sephadex G-100. Parathyroid hormone (BPTH) characteristically elutes in the fourth peak. However, we also observed significant hormonal activity, both biological and immunological, in the fifth elution peak. The peak V material had potent hypercalcemic activity in the rat and chick, and stimulated adenylate cyclase activity in the rat renal cortex bioassay. This material was further purified by ion exchange chromatography on carboxymethylcellulose in 8 M urea. The biological activity of the purified peptide (3700 MRC units/mg) was equivalent to that of the native hormone on a molar basis. Amino acid analysis, carboxypeptidase digestion, and partial Edman sequence analysis identified this material as BPTH 1-65, a hormonal fragment lacking the C-terminal 19 residues of the 84 residue hormone molecule. Several immunoassays using different anti-PTH antisera had variable reactivity toward the BPTH 1-65 fragment, showing that it may be useful for further characterizing antibody recognition sites. The presence of a lysine residue at position 65 suggests a tryptic-like cleavage may be responsible for the genesis of this hormonal fragment. Further investigation will be necessary to determine if this peptide has physiological significance.

Amino Acid Sequence↗
PubMed1978

alpha-Melanocyte stimulating hormone: immunohistochemical identification and mapping in neurons of rat brain.

alpha-Melanocyte stimulating hormone (alpha-melanotropin) immunofluorescence was observed in rat brain by means of a highly specific and well-characterized antibody. The hormone was contained in arcuate nucleus cell bodies and in varicose fibers. Dense populations of hormone-containing fibers were present in the septum, the nucleus interstitialis stria terminalis, and the medial preoptic, anterior hypothalamic, dorsomedial, and periventricular nuclei. Moderate numbers of fibers were seen in the paraventricular and arcuate nuclei, the amygdala, the region of the tractus diagonalis, the mammillary body, the central gray, the cuneiform nucleus, and the nucleus of the solitary tract. There is an interesting correlation of alpha-melanocyte stimulating hormone fibers with regions of noradrenergic axonal projections and terminal fields.

Adrenergic Fibers↗
PubMed1979

Investigation of a corticosteroid-binding globulin-like molecule in human placenta.

The existence of an endogenous corticosteroid-binding globulin (CBG)-like molecule in human placenta has been reported in studies which depended largely on immunological techniques. The experiments reported herein do not support that contention. Using a well characterized antibody to purified human CBG, we were unable to demonstrate CBG in placental cytosol. Although the immunological methods were too insensitive to detect CBG, the presence of small amounts of it were detected in binding experiments with [3H]cortisol. Simultaneous measurement of serum albumin indicated that the presence of CBG in the placental cytosol could be accounted for by contamination of the preparation with plasma.

Cytosol↗
PubMed1975

Adaptation of a T3-uptake test and of radioimmunoassays for serum digoxin, thyroxine, and triiodothyronine to an automated radioimmunoassay system--"Centria".

We report the adaptation of four radioassays to the prototype of an automated radioimmunoassay system ("Centria," Union Carbide). The system consists of three integrated modules: (a) an automated pipettor, which dispenses samples and reagents; (b) the key module, an incubator/separator, in which centrifugal force is used to initiate and terminate multiple radioassay incubations and separations simultaneously; and (c) a gamma-counter/computer, which counts three tubes simultaneously and converts counts into concentration units. Radioimmunoassays for thyroxine, triiodothyronine, and digoxin were developed with use of well-characterized antibodies and of prepackaged Sephadex-containing columns to separate bound and free radioactive ligand. A triiodothyronine-uptake test in which the same kind of columns were used was also adapted to the instrument. Results for clinical samples compared favorably with those obtained by manual procedures. We report data on correlation between different methods and preliminary data on precision of the prototype system.

Animals↗
PubMed2026

LCM-Enriched Proteomic Characterization of Antibody-Mediated Glomerular Damage and Complement Activation in Pre-Clinical Models.

Biologics, lipid nanoparticles, and other therapeutic modalities can result in adverse events, often detected as lesions during preclinical pathology assessments. Characterization of these lesions provides valuable information during drug development to contextualize mechanisms of injury and assess species translatability. Here, we investigated the utility of a laser capture microdissection (LCM)-enriched mass spectrometry proteomics approach to analyze two well-characterized preclinical models of regional (glomerular) injury: Passive Heyman Nephritis in rats and bovine gamma globulin-induced glomerular injury in nonhuman primates (NHPs). Using LCM-enriched proteomics, glomeruli were isolated from formalin-fixed paraffin-embedded kidney tissue in the rat model, enabling identification of 4,661 proteins and quantification of 3,410. Proteinuria measurements were compared with digital pathology metrics of glomerular morphology and proteomics results, with all modalities yielding concordant evidence of glomerular injury and proteomics confirming the role of complement activation. The same LCM- enriched proteomics workflow was applied to an NHP model of induced glomerular damage, identifying 4,623 proteins, quantifying 3,000, and confirming qualitative concordance with established features of complement-mediated glomerular injury. Together, these findings illustrate the applicability of LCM-enriched proteomics for region-specific characterization of antibody-mediated tissue injury and support its use as a hypothesis-generating platform in translational toxicologic pathology.

Animals↗
PubMed1975

Incomplete antibodies and immunoglobulin characterization in adult urodeles, Pleurodeles waltlii Michah. and Triturus alpestris Laur.

Humoral immunoglobulin synthesis has been studied in two adult urodeles, Pleurodeles waltlii Michah. and Triturus alpestris Laur. following SRBC immunization. The specific antibody response is detected after a long period of immunization and is due exclusively to 'incomplete' antibodies which are unable to induce agglutination. The antibody titre is essentially dependent on the number of stimulations rather than on the dose or nature of the antigen (papainized or normal erythrocytes). Antibodies are detected in only 50 per cent of the immunized animals, 50 per cent never respond. This suggests that the latter group does not possess the genetic equipment (Ir genes) to recognize the antigenic determinants and to synthesize the corresponding antibodies. The sedimentation coefficient of the synthesized immunoglobulins was investigated by sucrose density gradient centrifugation and their characterization was carried out by starch and polyacrylamide gel electrophoresis. With this peculiar antigen even after a booster injection, only one class of immunoglobulin, an 18-2S IgM could be detected.

Animals↗
PubMed1978

Isolation and partial characterization of an antibody enhancing factor from leukaemic owl monkey cell cultures.

Herpesvirus saimiri inoculated into owl monkeys (Aotus trivirgatus) causes leukaemia and lymphoma. Peripheral lymphocytes from leukaemic monkeys grown in culture are predominantly T lymphocytes. The supernatants from these cultures contain a factor which enhances the antibody response of murine B cells to sheep red blood cells (SRBC). The factor has been partially characterized by ammonium sulphate precipitation, DEAE-chromatography, gel filtration over Sephadex G-150 and disc-gel electrophoresis. The enhancing factor is a protein with a molecular weight of approximately 40,000.

Animals↗
PubMed1978

Acetylcholine receptor antibody in myasthenia gravis: purification and characterization.

Acetylcholine receptor antibody was measured in serum and IgG fractions from patients with myasthenia gravis using different analyses for antibody interfering with the toxin binding site and for antibody directed against other sites. No correlation was found between the concentration of receptor antibody as measured with the two assays. The different antibody activities also showed different isoelectric spectra. Receptor antibody was purified about 1000 times from IgG by affinity chromatography on a partially purified human skeletal muscle receptor preparation. The purified antibody was homogeneous in SDS-gel electrophroesis and showed a polyclonal pattern in agarose gel electrophoresis and isoelectric focusing. Both kinds of light chains were demonstrated. The results imply that the acetylcholine receptor antibody found in myasthenia gravis is heterogeneous and of multiclonal origin.

Acetylcholine↗