THE DEVELOPMENT OF A RADIOIMMUNOASSAY FOR ARGININE-VASOPRESSIN: PRODUCTION OF ANTISERA AND LABELLED HORMONE; SEPARATION TECHNIQUES; SPECIFICITY AND SENSITIVITY OF THE ASSAY IN AQUEOUS SOLUTION

1972 ◽  
Vol 52 (2) ◽  
pp. 279-288 ◽  
Author(s):  
C. R. W. EDWARDS ◽  
T. CHARD ◽  
MURIEL J. KITAU ◽  
MARY L. FORSLING ◽  
J. LANDON
Keyword(s):  
Arginine Vasopressin ◽  
Specific Activity ◽  
Limit Of Detection ◽  
High Specific Activity ◽  
Ion Exchange Resin ◽  
Cross Reactivity ◽  
Amino Acid Sequences ◽  
Simple Method ◽  
Specificity And Sensitivity ◽  
Chloramine T

SUMMARY A radioimmunoassay for vasopressin was developed using antibodies produced against conjugated and non-conjugated arginine vasopressin. Despite the fact that the vasopressin molecule has only eight amino acids, cross reactivity studies showed that these antibodies were specific for different amino acid sequences. Labelled hormone of high specific activity (350–800 μCi/μg) was produced by a modification of the chloramine-T method. Unreacted iodide was removed by the batchwise addition of an ion-exchange resin. Other techniques of purification produced no advantage over this simple method. Several methods of separating antibody-bound and free hormone were studied. All except chromatoelectrophoresis proved satisfactory. Ammonium sulphate or ethanol precipitation of bound hormone was chosen because of simplicity, speed and reproducibility. The lower limit of detection of the assay was 80 pg arginine vasopressin/ml diluent buffer. Therefore an extraction and concentration procedure is necessary for the measurement of basal circulating levels of the hormone.

RADIOIMMUNOASSAY OF [8-ARGININE]-VASOPRESSIN

1975 ◽  
Vol 80 (3) ◽  
pp. 444-452 ◽  
Author(s):  
P. Czernichow ◽  
U. Merkelbach ◽  
M. B. Vallotton
Keyword(s):  
Antidiuretic Hormone ◽  
Arginine Vasopressin ◽  
Association Constant ◽  
Specific Activity ◽  
Limit Of Detection ◽  
High Specific Activity ◽  
High Association ◽  
Theoretical Maximum

ABSTRACT A radioimmunological method for the determination of the human antidiuretic hormone, [8-arginine]-vasopressin (AVP), is described in detail. The antiserum has been raised in rabbits injected with AVP adsorbed onto charcoal particles and is used at a final dilution of 1:200 000. It contains antibodies directed specifically against the C-terminal tripeptide and possesses a high association constant of 8.2× 1012 1/mole. AVP is labelled in monoiodinated form (as proven after pronase digestion) at a high specific activity close to the theoretical maximum after purification from unlabelled hormone on Sephadex gel. The standard curves are characterized by a Bo of 55 %, a limit of detection ascertained at least at 3 pg (10 % displacement) and a 50 % displacement achieved with 35.5 pg. The index of precision λ ranges from 0.033 to 0.042. The conditions of the assay (buffer's composition and pH, timing) were systematically varied and tested. In addition the result of immunization of chicken with AVP is reported. The assay is adequate for the measurement of AVP in urine and plasma and will be described in forthcoming papers.

Synthesis and N.M.R. Spectra of Substituted Aminoiodoacridines

1979 ◽  
Vol 32 (12) ◽  
pp. 2637 ◽  
Author(s):  
RF Martin ◽  
DP Kelly
Keyword(s):  
Electron Density ◽  
Electrophilic Substitution ◽  
Specific Activity ◽  
Diazonium Salt ◽  
High Specific Activity ◽  
High Yield ◽  
Model Compounds ◽  
Iodide Ion ◽  
Ion Substitution ◽  
Chloramine T

3-Amino-6-iodoacridine (10), 3,6-diiodoacridine (11) and 9-amino-2-ethoxy-6-iodoacridine (14) were prepared by iodide ion substitution of the corresponding diazonium salt whereas 3,6-diamino-4,5-diiodoacridine (12) and 6,9-diamino-2-ethoxy-5-iodoacridine (13) were prepared by direct iodination with iodide ion in the presence of chloramine-T. The latter reaction proceeded in relatively high yield and has been used for the synthesis of high specific activity 125I-labelled compounds (12), (13). The 1H and 13C N.M.R. spectra of (10)-(14) and model compounds indicate higher electron density at C4(C5) than at C2(C7) in 3(6)-amino-substituted acridines in agreement with the observed pattern of electrophilic substitution.

scholarly journals Selection and Identification of Novel Aptamers Specific for Clenbuterol Based on ssDNA Library Immobilized SELEX and Gold Nanoparticles Biosensor

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2337 ◽  
Author(s):  
Xixia Liu ◽  
Qi Lu ◽  
Sirui Chen ◽  
Fang Wang ◽  
Jianjun Hou ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Retention Rate ◽  
Pcr Amplification ◽  
Cross Reactivity ◽  
Enriched Library ◽  
Amplification Curve ◽  
Specificity And Sensitivity

We describe a multiple combined strategy to discover novel aptamers specific for clenbuterol (CBL). An immobilized ssDNA library was used for the selection of specific aptamers using the systematic evolution of ligands by exponential enrichment (SELEX). Progress was monitored using real-time quantitative PCR (Q-PCR), and the enriched library was sequenced by high-throughput sequencing. Candidate aptamers were picked and preliminarily identified using a gold nanoparticles (AuNPs) biosensor. Bioactive aptamers were characterized for affinity, circular dichroism (CD), specificity and sensitivity. The Q-PCR amplification curve increased and the retention rate was about 1% at the eighth round. Use of the AuNPs biosensor and CD analyses determined that six aptamers had binding activity. Affinity analysis showed that aptamer 47 had the highest affinity (Kd = 42.17 ± 8.98 nM) with no cross reactivity to CBL analogs. Indirect competitive enzyme linked aptamer assay (IC-ELAA) based on a 5′-biotin aptamer 47 indicated the limit of detection (LOD) was 0.18 ± 0.02 ng/L (n = 3), and it was used to detect pork samples with a mean recovery of 83.33–97.03%. This is the first report of a universal strategy including library fixation, Q-PCR monitoring, high-throughput sequencing, and AuNPs biosensor identification to select aptamers specific for small molecules.

125I-labeled gonadoliberin and high specific activity and immunoreactivity: method of iodination and rapid separation.

1980 ◽  
Vol 26 (5) ◽  
pp. 573-578
Author(s):  
A K Sarda ◽  
M A Barnes ◽  
R M Nair
Keyword(s):  
Exchange Resin ◽  
Specific Activity ◽  
High Specific Activity ◽  
Ion Exchange Resin ◽  
Free Form ◽  
Paper Strip ◽  
Peptide Fragments ◽  
Separation And Purification ◽  
Rapid Separation ◽  
Assay Procedure

Abstract We describe optimum conditions for iodinating gonadoliberin with use of relatively large proportions of Na 125I. Products of the iodination are separated on an anion-exchange resin (Amberlite IRA-400). The 125I-labeled gonadoliberin thus obtained has a high specific activity (1400 to 1590 Ci/g); because of the conditions of iodination, we believe that the predominant species of the labeled decapeptide is the mono-iodinated one. Our separation and purification of the labeled substance on ion-exchange resin is rapid, economical, and less cumbersome than the use of a Biogel P-2 column. There is no adsorption of the labeled hormone onto the resin, as evidenced by analytical recovery studies with tritium-labeled gonadoliberin. Paper-strip chromatoelectrophoresis showed no free Na 125I or radiolabeled damaged peptide fragments after purification on the resin. When antiserum was used at a concentration 32-fold that used in the regular assay procedure, only 4% of the radioactivity remained in the free form, indicating the high immunoreactivity of the labeled hormone.

Development and evaluation of a radioimmunoassay for Arg8-vasopressin, after extraction with Sep-Pak C18.

1985 ◽  
Vol 31 (6) ◽  
pp. 861-863 ◽  
Author(s):  
K A Ysewijn-Van Brussel ◽  
A P De Leenheer
Keyword(s):  
Human Plasma ◽  
Arginine Vasopressin ◽  
Specific Activity ◽  
Cross Reaction ◽  
High Avidity ◽  
Plasma Samples ◽  
Displacement Analysis ◽  
Double Antibody ◽  
Chloramine T ◽  
Dilution Curves

Abstract We describe a specific double-antibody radioimmunoassay for measuring arginine vasopressin (AVP) in human plasma. Antisera of high avidity were obtained from rabbits that had been injected with AVP coupled to bovine thyroglobulin. The antibody reacts with both the tripeptide tail and the pentapeptide ring of the molecule, thereby eliminating cross reaction with oxytocin. Synthetic AVP was labeled with 125I by a modification of the Chloramine-T technique. The specific activity of the labeled hormone was 29 MBq/micrograms of AVP, as estimated by self-displacement analysis. The assay involves Sep-Pak C18 extraction of acidified (pH 4) plasma. Recovery of [3H]AVP added to plasma averaged 86.6 (SD 6.1)% (n = 14). Dilution curves for plasma showed linearity of response with concentration. The overall sensitivity was 0.3 ng/L when 2-mL plasma samples were extracted. The intra-assay CV was 7.8% at 4.8 ng/L (n = 12) and the interassay CV was 12.3% (n = 16) and 6.3% (n = 14) at 2.7 and 4.1 ng/L concentrations, respectively.

Fragment D Immunoreacivity: The Influence of Iodination and Calcium Environment

1979 ◽  
Author(s):  
B. Kudryk ◽  
M. Blombäck
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Specific Activity ◽  
High Specific Activity ◽  
Antigenic Determinants ◽  
Affinity Binding ◽  
Compact Structure ◽  
High Affinity Binding ◽  
High Affinity ◽  
Chloramine T

Human fragment D (Fg-Ds) has heen iodinated using both the Chloramine-T and lactoperoildaae methods. The specific activity was similar regardless of the method used. However, binding to a specific antibody was different for each preparation. The antigen labeled by the Chloramine-T method bound to a maximum of 40% the other labeled product bound up to 85%. A correlation between the decree of immunoreactivity and avidity for a fihrinmcnomer conjugate vas found also. Fibrinmonomer bound about twice the ajnount of lactoperoxidase iodinated Fg-Da ae it did the Chloramine-T product. The use of these conjugates in the purification of immunoreactive Fg-Ds of high specific activity will be discussed. High affinity binding sites for calcium have recently been demonstrated in fibrinogen. Tha presence of bound calcium is also believed to protect Fg-Ds f m further digestion by plasmin. This is probably due to the formation of a more compact structure. However, conformational changes for calcium bound fibrinogen or Fg-Ds have not been observed. We tested the immunoreactivity of the lactoperoxidase iodinated Fg-Ds in presence and absence of calcium. Differences were found and this data suggests that soma modification of antigenic determinants takes place as a consequence of calcium in the environment.

scholarly journals Purification and characterization of casein kinase I from broccoli

1993 ◽  
Vol 293 (1) ◽  
pp. 283-288 ◽  
Author(s):  
L J Klimczak ◽  
A R Cashmore
Keyword(s):  
Molecular Mass ◽  
Specific Activity ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Casein Kinase ◽  
Casein Kinase I ◽  
Native Molecular Mass ◽  
Specificity And Sensitivity ◽  
Sds Page

Casein kinase I from broccoli was purified approximately 65,000-fold by chromatography on phosphocellulose, phenyl-Sepharose, CM-Sephacel, and affinity chromatography on N-(2-aminoethyl)-5-chloroisoquinolone-8-sulphonamide (CKI-7)-Sepharose. The catalytic subunit of casein kinase I was identified as a 36-38 kDa polypeptide doublet by using the technique of activity gel assay after SDS/PAGE with casein as a gel-incorporated substrate. A silver-stained polypeptide doublet of the same molecular mass constituted at least 95% of the protein in the final preparation, corresponding to a specific activity of approximately 1800 nmol/min per mg of protein. The enzyme was found to be a monomer by gel filtration and glycerol gradient sedimentation; the native molecular mass was calculated to be 34.2 kDa. These characteristics, as well as other essential features of plant casein kinase I activity, such as substrate specificity and sensitivity to inhibitors, were found to be similar to those established for animal casein kinase I. Broccoli casein kinase I showed weak immunological cross-reactivity with antibodies raised against bovine casein kinase I.

scholarly journals Development and validation of an enzyme immunoassay for detection and quantification of SARS-CoV-2 salivary IgA and IgG

2021 ◽  
Author(s):  
Veronica Costantini ◽  
Kenny Nguyen ◽  
Zoe Lyski ◽  
Shannon Novosad ◽  
Ana C Bardossy ◽  
...  
Keyword(s):  
Immune Response ◽  
Enzyme Immunoassay ◽  
Limit Of Detection ◽  
Natural Infection ◽  
High Specificity ◽  
Cross Reactivity ◽  
Igg Antibodies ◽  
Antibody Isotype ◽  
Salivary Iga ◽  
Specificity And Sensitivity

Oral fluids offer a non-invasive sampling method for the detection of antibodies. Quantification of IgA and IgG antibodies in saliva allows studies of the mucosal and systemic immune response after natural infection or vaccination. We developed and validated an enzyme immunoassay (EIA) to detect and quantify salivary IgA and IgG antibodies against the prefusion-stabilized form of the SARS-CoV-2 spike protein. Normalization against total antibody isotype was performed to account for specimen differences, such as collection time and sample volume. Saliva samples collected from 187 SARS-CoV-2 confirmed cases enrolled in 2 cohorts and 373 pre-pandemic saliva samples were tested. The sensitivity of both EIAs was high (IgA: 95.5%; IgG: 89.7%) without compromising specificity (IgA: 99%; IgG: 97%). No cross reactivity with seasonal coronaviruses was observed. The limit of detection for SARS-CoV-2 salivary IgA and IgG assays were 1.98 ng/mL and 0.30 ng/mL, respectively. Salivary IgA and IgG antibodies were detected earlier in patients with mild COVID-19 symptoms than in severe cases. However, severe cases showed higher salivary antibody titers than those with a mild infection. Salivary IgA titers quickly decreased after 6 weeks in mild cases but remained detectable until at least week 10 in severe cases. Salivary IgG titers remained high for all patients, regardless of disease severity. In conclusion, EIAs for both IgA and IgG had high specificity and sensitivity for the confirmation of current or recent SARS-CoV-2 infections and evaluation of the IgA and IgG immune response.

MRP4/ABCC4 As a New Therapeutic Target: Meta-Analysis to Determine cAMP Binding Sites as a Tool for Drug Design

2019 ◽  
Vol 26 (7) ◽  
pp. 1270-1307 ◽  
Author(s):  
Agustín Yaneff ◽  
Ana Sahores ◽  
Natalia Gómez ◽  
Alejandro Carozzo ◽  
Carina Shayo ◽  
...  
Keyword(s):  
Therapeutic Target ◽  
Binding Sites ◽  
Rational Design ◽  
Specific Activity ◽  
Meta Analysis ◽  
High Specific Activity ◽  
Amino Acid Sequences ◽  
Pharmacological Agents ◽  
Attractive Therapeutic Target ◽  
Analysis Platform

MRP4 transports multiple endogenous and exogenous substances and is critical not only for detoxification but also in the homeostasis of several signaling molecules. Its dysregulation has been reported in numerous pathological disorders, thus MRP4 appears as an attractive therapeutic target. However, the efficacy of MRP4 inhibitors is still controversial. The design of specific pharmacological agents with the ability to selectively modulate the activity of this transporter or modify its affinity to certain substrates represents a challenge in current medicine and chemical biology. The first step in the long process of drug rational design is to identify the therapeutic target and characterize the mechanism by which it affects the given pathology. In order to develop a pharmacological agent with high specific activity, the second step is to systematically study the structure of the target and identify all the possible binding sites. Using available homology models and mutagenesis assays, in this review we recapitulate the up-to-date knowledge about MRP structure and aligned amino acid sequences to identify the candidate MRP4 residues where cyclic nucleotides bind. We have also listed the most relevant MRP inhibitors studied to date, considering drug safety and specificity for MRP4 in particular. This meta-analysis platform may serve as a basis for the future development of inhibitors of MRP4 cAMP specific transport.

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