共查询到20条相似文献,搜索用时 15 毫秒
1.
C Jin 《Nihon saikingaku zasshi. Japanese journal of bacteriology》1992,47(2):373-385
The beta-lactam resistance of genus Streptococcus has been explained by the low binding affinity of penicillin-binding proteins (PBPs) to the drug. This study was carried out to resolve the mechanisms of resistance to beta-lactam antibiotics in the species of genus Enterococcus by means of binding affinities of PBPs. Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium and Enterococcus avium were employed as assay microbes. Cefepime (CFPM) and ampicillin (ABPC) were used as representatives of cephems and penicillins, respectively. All the PBP fractions of S. pyogenes manifested high binding affinities to CFPM and ABPC, whereas PBPs 1 and 4 of E. faecalis showed high binding affinities to ABPC but not to CFPM. In E. faecium, PBPs of an exceptionally penicillin-susceptible strain manifested a high binding affinity to ABPC, but PBPs 5 and 6 showed low affinities to CFPM. beta-lactam resistant strains of E. faecium possessed PBPs 5 and 6 with low binding affinities to CFPM and ABPC. All the fractions of PBPs but PBP 1 in E. avium showed low binding affinities to CFPM. Although all the PBP fractions but PBPs 3 and 6 manifested high binding affinities to ABPC, PBPs 3 and 6 showed low binding affinities to ABPC. A strain of E. avium, which is susceptible to ABPC but moderately resistant to CFPM, lacked PBP 6. In conclusion, the resistance of E. avium to CFPM is based upon low binding affinities of the many fractions to this drug, and ABPC resistance is based upon PBPs 3 and 6 with low binding affinities to ABPC. 相似文献
2.
Monoclonal Antibodies to Benzodiazepines 总被引:1,自引:0,他引:1
Angel L. De Blas Lakshmi Sangameswaran Steven A. Haney Dongeun Park Carl J. Abraham Jr. Carroll A. Rayner 《Journal of neurochemistry》1985,45(6):1748-1753
Four hybridoma lines secreting monoclonal antibodies to benzodiazepines were produced after BALB/c mice were immunized with a benzodiazepine-bovine serum albumin conjugate. The monoclonal antibodies were purified from ascites fluids, and their binding affinities for benzodiazepines and other benzodiazepine receptor ligands were determined. These antibodies have very high binding affinities for diazepam, flunitrazepam, Ro5-4864, Ro5-3453, Ro11-6896, and Ro5-3438 (the KD values are in the 10(-9) M range). However, these antibodies have low affinities for the benzodiazepine receptor inverse agonists (beta-carbolines) and antagonists (Ro15-1788 and CGS-8216). 相似文献
3.
Rathanaswami P Roalstad S Roskos L Su QJ Lackie S Babcook J 《Biochemical and biophysical research communications》2005,334(4):1004-1013
The high specificity and affinity of monoclonal antibodies make them attractive as therapeutic agents. In general, the affinities of antibodies reported to be high affinity are in the high picomolar to low nanomolar range and have been affinity matured in vitro. It has been proposed that there is an in vivo affinity ceiling at 100 pM and that B cells producing antibodies with affinities for antigen above the estimated ceiling would have no selective advantage in antigen-induced affinity maturation during normal immune responses. Using a transgenic mouse producing fully human antibodies, we have routinely generated antibodies with sub-nanomolar affinities, have frequently rescued antibodies with less than 10 pM affinity, and now describe the existence of an in vivo generated anti-hIL-8 antibody with a sub-picomolar equilibrium dissociation constant. This confirms the prediction that antibodies with affinities beyond the proposed affinity ceiling can be generated in vivo. We also describe the technical challenges of determining such high affinities. To further understand the importance of affinity for therapy, we have constructed a mathematical model to predict the relationship between the affinity of an antibody and its in vivo potency using IL-8 as a model antigen. 相似文献
4.
E A Dranovskaya K D Duisenov 《Journal of hygiene, epidemiology, microbiology, and immunology》1976,20(4):457-463
The composition of immunoglobulins in patients with brucellosis was studied. The method of ion-exchange chromatography on DEAE-cellulose columns was used to define more precisely the physico-chemical character of cysteine-resistant antibodies. The study of IgM, IgA and IgG fractions obtained from the patients sera showed the IgG fraction to possess the greatest serological activity in the agglutination reaction, in the passive haemagglutination reaction and in Coomb's test. Specific antibodies in the remaining 2 fractions (IgA and IgM) were found only in single patients in low titres, mainly in Coomb's test (incomplete antibodies). The study of IgM, IgA and IgG serum fractions before and after cysteine treatment revealed cysteine-resistant antibodies to be usually IgG globulins. The presence of specific IgG antibodies in the sera of patients was found to correlate with active clinical manifestations of brucellosis. 相似文献
5.
Repeated intravenous injections of maximally coupled dinitrophenylated bovine gamma-globulin elicited both 7S and 17S anti-dinitrophenyl antibodies in chickens. Only antibodies of low affinity were produced regardless of the priming dose, the interval between injections, and the number of injections. The 7S and 17S antibodies isolated from invididual animals had identical affinities and heterogeneity indices. The concentrations of antibodies formed were uniformly low despite many injections over a prolonged period. These studies indicate that stimulation by antigen alone may not be sufficient for the induction of predominant 7S antibody formation and for the synthesis of high affinity antibody. 相似文献
6.
Four hybridoma lines secreting monoclonal antibodies to benzodiazepines were produced after BALB/c mice were immunized with a benzodiazepine-bovine serum albumin conjugate. The monoclonal antibodies were purified from ascites fluids, and their binding affinities for benzodiazepines and other benzodiazepine receptor ligands were determined. These antibodies have very high binding affinities for diazepam, flunitrazepam, Ro5-4864, Ro5-3453, Ro11-6896, and Ro5-3438 (the Kd values are in the 10(-9) M range). However, these antibodies have very low affinities for the benzodiazepine receptor inverse agonists (beta-carbolines) and antagonists (Ro15-1788 and CGS-8216). One of the monoclonal antibodies (21-7F9) has been used to demonstrate the existence of benzodiazepine-like molecules in the brain and for the purification of these molecules. Immunocytochemical experiments show that these molecules are neuronal and not glial and that they are ubiquitously distributed throughout the brain. Immunoblots indicate the presence of benzodiazepine-like epitopes in several brain peptides. An endogenous substance that binds to the central-type benzodiazepine receptor with agonist properties has been purified to homogeneity from the bovine brain. The purification consisted on immunoaffinity chromatography on immobilized monoclonal anti-benzodiazepine antibody followed by gel filtration on Sephadex G-25 and two reverse phase HPLCs. The purified substance has a small molecular weight and its activity is protease resistant. The endogenous substance blocks the binding of agonists, inverse agonists and antagonists to the central-type benzodiazepine receptor but it does not inhibit the binding of Ro5-4864 to the peripheral-type benzodiazepine receptor. The neurotransmitter gamma-aminobutyric acid increases the affinity of the benzodiazepine receptor for the purified substance. Preliminary evidence indicates that the purified substance is a benzodiazepine with a molecular structure that is identical or very close to N-desmethyldiazepam. 相似文献
7.
Rabbit antisera to bovine serum albumin (BSA) conjugates of 3-(O-carboxymethyl)oximino-, 7-(O-carboxymethyl)oximino- and 7β-hemi-succinamido derivatives of 5α-dihydrotestosterone (DHT) were applied to four affinity columns bearing respectively these three antigens and a fourth 3β-hemisuccinamido-5α-androstan-17β-ol-BSA antigen as ligands.The antibodies retained on the columns were totally desorbed by an excess of DHT, but in DHT-bound form, whereas 1M mh4oh and electrophoretic elution allowed a recovery of 60% of the retained antibodies in unbound form. The antibody fractions (40%) remaining on the columns after NH4OH or electrophoretic elution were totally recovered by addition of DHT following the electrophoretic elution only. All the DHT-bound fractions were dissociated by dialysis but with a 70% loss of binding activity.The association constants for DHT of most of the antibody fractions were similar to those of the crude antisera (Ka ~ 1010M?1), with the exception of the antibodies recovered from the antibody fractions resistant to electrophoretic elution which had higher affinities (Ka ~ 2.0 to 30 × 1010M?1).The specificity charts of the antisera were in some cases considerably modified after fractionation, according to the choice of the ligand employed in the affinity columns as well as of the elution methods. The lowest cross-reactions with testosterone were observed after elution with 1M NH4OH (17–20%) or electrophoresis (23–25%) of the anti-7-(O-carboxymethyl)oximino-DHT antisera fractions retained on 3β-hemisuccinamido-5α-androstan-17β-ol-BSA-Sepharose columns. 相似文献
8.
The affinities of rabbit antibodies directed against the spin-label nitroxide group have been found to be of the order of 106 1/mole for a number of low molecular weight water soluble haptens. It is shown that the same antibodies have almost equal binding affinities to corresponding hydroxylamines. 相似文献
9.
The authors describe a novel method for the quantitation of differential levels of biomolecules using unlabeled samples and protein-binding arrays for assessing differential expression. Traditional affinity arrays, whether in microplates or protein microarrays, suffer from a few common problems-a shortage of characterized antibodies and highly variable affinities for those available. Also, the assayed proteins could be present in a wide range of concentrations and physicochemical properties, so that it becomes an onerous task to optimize assay conditions for each antibody-antigen pair. Currently, this restricts parallel affinity assays to a low number of carefully selected antibodies and restricts the development of highly multiplexed parallel affinity assays. A displacement strategy allows the use of a much wider range of antibodies, reducing the requirement for matched affinities. The competitive assays described here also show a much higher tolerance for nonspecific background noise. The range of assayed protein concentrations is only limited by the sensitivity of the detection system used. 相似文献
10.
Sergei A. Bobrovnik Miroslava Demchenko Sergei Komisarenko Fred Stevens 《Journal of molecular recognition : JMR》2010,23(5):448-456
Traditionally used methods of antibody affinity determination either by ELISA or by the surface plasmon resonance technique do not allow detection of the presence of low‐affinity antibodies in samples of high‐affinity antibodies. In this paper we demonstrate the possibility to reveal their presence and to determine the affinities of both categories of antibodies as well as the ratio of their concentrations. This is especially important since by using traditional methods for antibody affinity evaluation the admixture of low‐affinity antibodies in a sample diminishes the accuracy in determination of specific antibody affinity. In addition, the presence of an admixture of low‐affinity antibodies may be an important biological characteristic of the system under study; their revelation and the evaluation of their binding parameters may be valuable in many cases for obtaining a more complete characterization of the binding properties of the multiple antibodies generated in an immune response. Copyright © 2009 John Wiley & Sons, Ltd. 相似文献
11.
Monoclonal antibodies to subunits of bovine heart cytochrome c oxidase were prepared by immunizing mice with the isolated enzyme. The majority of antibody-producing cell lines were found to react with two different subunits of similar molecular mass, as shown by Western blotting and ELISA titrations with the HPLC-purified subunits. The affinities of the monoclonal antibodies to the subunits were determined by ELISA titrations with increasing concentrations of NH4SCN. Two monoclonal antibodies with a low affinity to subunit VIa had a high affinity to subunit VIc, whereas two other antibodies showed the same affinity to subunits VIIa and VIIb. The same affinity of monoclonal antibodies suggested an evolutionary relationship of subunits VIIa and VIIb, which was further supported by reactivity of these antibodies to subunits VIIa and VIIb of cytochrome c oxidase from different species and tissues. Also the evolutionary relationship between subunit VIa and VIc was shown by hybridization at low stringency of cDNAs for rat cytochrome c oxidase subunits VIc and VIa-h (heart-type), after amplification by the polymerase chain reaction, with a probe of VIa-l (liver-type). 相似文献
12.
Pereira Crott Luciana Simon Lucisano-Valim Yara Maria Silva Celio Lopes Barbosa José Elpidio 《Mycopathologia》1997,140(1):19-28
The aim of our study was to investigate differences that might exist in the activation of the human complement system by F1
fractions from four different isolates of P. brasiliensis. Isolates HC and 18 (virulent), 265 (low virulence), and 9 (intermediate
virulence, attenuated) were used; before the experiments, the virulence of isolates HC and 18 was recovered by in vivo passage
in guinea pigs. The four isolates of the fungus were processed for purification of F1 fractions and the activation of the
human complement system was studied by a kinetic method of hemolytic activity measurement. The incubation of F1 fractions
in normal human serum resulted in different degrees of inhibition of the classical and alternative pathways. The F1 fraction
from the low virulence isolate was more efficient than the F1 fraction from the virulent isolates (HC and 18). Previous absorption
of sera with F1 fractions completely abolished classical pathway activation. Using zymosan, instead of F1, in the absorption
process caused the same phenomenon, suggesting that natural or nonspecific antibodies are responsible for the classical pathway
activation. The alternative pathway activation did not depend on these antibodies, but was enhanced by their presence. On
the other hand, F1 fractions from virulent isolates were more active in the stimulation of neutrophil chemiluminescence compared
with the F1 fraction from the low virulence isolate. Whole P. brasiliensis yeast cells (WYC) from two distinct strains, 18
and 265, showed the same patterns of response of those observed with the F1 fractions in the functions tested. These differences
in the behavior of the F1 fractions as well as WYC in relation to human complement activation and consequently to neutrophil
stimulation may correlate with the virulence of individual isolates and may contribute to the understanding of the inflammatory
response generation and maintenance processes in paracoccidioidomycosis.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
13.
Hybridoma lines secreting monoclonal antibodies against a phospholipase-inactive derivative of the presynaptic neurotoxin, beta-bungarotoxin, have been established. These antibodies, either of the IgG1 or IgG2b isotype with affinities in the range 1-2 X 10(8) 1/mol, recognized a single immunodominant region of native beta-bungarotoxin, most probably located on the A (phospholipase homologue) chain of the toxin. Using plate-adsorbed radioimmunoassay procedures, antibodies reacted with native beta-bungarotoxin and other beta-bungarotoxin isotoxins as well as with the non-toxic phospholipase A also present in Bungarus multicinctus venom. Other phospholipase A enzymes and presynaptic phospholipase neurotoxins did not show any competition with beta-bungarotoxin in the radioimmunoassay. Globulin fractions of monoclonal antibodies partially inhibited the phospholipase activity of beta-bungarotoxin. 相似文献
14.
A mimotope from a solid-phase peptide library induces a measles virus-neutralizing and protective antibody response. 总被引:2,自引:1,他引:1
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A solid-phase 8-mer random combinatorial peptide library was used to generate a panel of mimotopes of an epitope recognized by a monoclonal antibody to the F protein of measles virus (MV). An inhibition immunoassay was used to show that these peptides were bound by the monoclonal antibody with different affinities. BALB/c mice were coimmunized with the individual mimotopes and a T-helper epitope peptide (from MV fusion protein), and the reactivity of the induced anti-mimotope antibodies with the corresponding peptides and with MV was determined. The affinities of the antibodies with the homologous peptides ranged from 8.9 x 10(5) to 4.4 x 10(7) liters/mol. However, only one of the anti-mimotope antibodies cross-reacted with MV in an enzyme-linked immunosorbent assay and inhibited MV plaque formation. Coimmunization of mice with this mimotope and the T-helper epitope peptide induced an antibody response which conferred protection against fatal encephalitis induced following challenge with MV and with the structurally related canine distemper virus. These results indicate that peptide libraries can be used to identify mimotopes of conformational epitopes and that appropriate immunization with these mimotopes can induce protective antibody responses. 相似文献
15.
M K Homonylo S J Wilmot J S Lam L A MacDonald C Whitfield 《Canadian journal of microbiology》1988,34(10):1159-1165
Monoclonal antibodies were produced against the capsular antigen of Escherichia coli serotype K(A)30, using a mouse hybridoma system. The antibodies also recognised the chemically identical capsular polysaccharide produced by Klebsiella K20. Chemical modification of the K30 polysaccharide indicated that the glucuronic acid residues found in the E. coli K30 capsular antigen were important in the epitope recognised by these antibodies. Use of the antibodies as molecular probes revealed the presence of two discrete forms of the K30 antigen. One form was comprised of high molecular weight polysaccharide, present as a surface capsular layer. The second form of the antigen was of low molecular weight and was associated with lipopolysaccharide fractions from cell surface polysaccharide extracts. Separation of lipopolysaccharide fractions using gel chromatography in the presence of detergent showed that the low molecular weight K-antigenic fraction comigrated with a lipopolysaccharide lipid A core fraction present in encapsulated E. coli K30 bacteria but absent in acapsular mutants. 相似文献
16.
Generation of polyclonal antibodies against nisin: immunization strategies and immunoassay development. 总被引:7,自引:4,他引:3
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Murine polyclonal antibodies reactive to the lantibiotic bacteriocin nisin A (nisA) have been produced by immunization with nisA-cholera toxin and nisA-keyhole limpet hemocyanin (nisA-KLH) conjugates. Mice immunized with nisA-cholera toxin developed nisA-specific antibodies with low relative affinities and poor sensitivities, while the immunization of mice with nisA-KLH conjugates resulted in the production of nisA-specific antibodies with high relative affinities and much-increased sensitivities. nisA antibodies could also be readily mass produced in less than 8 weeks in ascites fluid by using the nisA-KLH conjugate. A competitive direct enzyme-linked immunosorbent assay (ELISA) whereby nisA-horseradish peroxidase and free nisA competed for antibody binding was devised. The detection limit for nisA in the competitive direct ELISA with the nisA-KLH-generated antibodies was from 5 to 100 ng/ml, while the amount of free nisA required for 50% antibody binding inhibition ranged from 0.3 to 5 micrograms /ml. Both antisera and ascites polyclonal antibodies cross-reacted with nisZ either in the supernatant of a producer strain or with the pure lantibiotic but did not cross-react at all with non-lantibiotic-type bacteriocins. These polyclonal antibodies should find a wide usage from nisA ELISA analysis in foods and other matrices. 相似文献
17.
Specific DNA binding of the two chicken Deformed family homeodomain proteins, Chox-1.4 and Chox-a. 总被引:4,自引:2,他引:4
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The cDNA clones encoding two chicken Deformed (Dfd) family homeobox containing genes Chox-1.4 and Chox-a were isolated. Comparison of their amino acid sequences with another chicken Dfd family homeodomain protein and with those of mouse homologues revealed that strong homologies are located in the amino terminal regions and around the homeodomains. Although homologies in other regions were relatively low, some short conserved sequences were also identified. E. coli-made full length proteins were purified and used for the production of specific antibodies and for DNA binding studies. The binding profiles of these proteins to the 5'-leader and 5'-upstream sequences of Chox-1.4 and Chox-a coding regions were analyzed by immunoprecipitation and DNase I footprint assays. These two Chox proteins bound to the same sites in the 5'-flanking sequences of their coding regions with various affinities and their binding affinities to each site were nearly the same. The consensus sequences of the high and low affinity binding sites were TAATGA(C/G) and CTAATTTT, respectively. A clustered binding site was identified in the 5'-upstream of the Chox-a gene, suggesting that this clustered binding site works as a cis-regulatory element for auto- and/or cross-regulation of Chox-a gene expression. 相似文献
18.
非洲爪蟾的孵化液对卵黄膜和二甲基酷蛋白具有降妥活性。用非洲爪蟾孵化酶的特异性抗GST-UV.2抗体进行Western杂交的结果表明,孵化液中出现一种分子量为60kD的大组分,有时也会出现一种分子量为40kD的小组分。 相似文献
19.
Different binding properties and affinities for ATP and ADP among sulfonylurea receptor subtypes, SUR1, SUR2A, and SUR2B 总被引:12,自引:0,他引:12
Matsuo M Tanabe K Kioka N Amachi T Ueda K 《The Journal of biological chemistry》2000,275(37):28757-28763
ATP-sensitive potassium (K(ATP)) channels, composed of sulfonylurea receptor (SURx) and Kir6.x, play important roles by linking cellular metabolic state to membrane potential in various tissues. Pancreatic, cardiac, and vascular smooth muscle K(ATP) channels, which consist of different subtypes of SURx, differ in their responses to cellular metabolic state. To explore the possibility that different interactions of SURx with nucleotides cause differential regulation of K(ATP) channels, we analyzed the properties of nucleotide-binding folds (NBFs) of SUR1, SUR2A, and SUR2B. SURx in crude membrane fractions was incubated with 8-azido-[alpha-(32)P]ATP or 8-azido-[gamma-(32)P]ATP under various conditions and was photoaffinity-labeled. Then, SURx was digested mildly with trypsin, and partial tryptic fragments were immunoprecipitated with antibodies against NBF1 and NBF2. Some nucleotide-binding properties were different among SUR subtypes as follows. 1) Mg(2+) dependence of nucleotide binding of NBF2 of SUR1 was high, whereas those of SUR2A and SUR2B were low. 2) The affinities of NBF1 of SUR1 for ATP and ADP, especially for ATP, were significantly higher than those of SUR2A and SUR2B. 3) The affinities of NBF2 of SUR2B for ATP and ADP were significantly higher than those of SUR2A. This is the first biochemical study to analyze and compare the nucleotide-binding properties of NBFs of three SUR subtypes, and our results suggest that their different properties may explain, in part, the differential regulation of K(ATP) channel subtypes. The high nucleotide-binding affinities of SUR1 may explain the high ability of SUR1 to stimulate pancreatic K(ATP) channels. It is also suggested that the C-terminal 42 amino acids affect the physiological roles of SUR2A and SUR2B by changing the nucleotide-binding properties of their NBFs. 相似文献
20.
The thermodynamic quantities of the anti-dinitrophenyl antibody-hapten interaction are reported for rabbit, goat, and guinea pig antibodies. Rabbit and goat antibodies had similar exothermic enthalpy changes for their reaction with 2,4-dinitrophenyl-L-lysine (-13.9 and -14.8 kcal/mol, respectively). The enthalpy change with guinea pig antibody was much less exothermic (-8.7 kcal/mol), and this value was the same for two guinea pig antibody preparations that differed in affinity by almost two orders of magnitude. A heterogeneous goat anti-dinitrophenyl antibody preparation was fractionated on a molecular sieve column in the presence of a bivalent ligand, a procedure that has been reported to separate antibodies according to differences in the depth of interaction with the ligand. The relationship of these differences in apparent site depth to changes in interactions with the hapten tail was examined by comparing the affinities of various fractions for two haptens. The results show that the presumed deeper sites have stronger interactions with the hapten tail. These studies suggest that the heterogeneity of anti-dinitrophenyl antibodies with respect to affinity results from differences in entropy driven lysyl side-chain interactions which arise from a heterogeneity in antigen binding site depth. 相似文献