Solid-phase enzyme-receptor assay (SPERA): a competitive-binding assay for glycopeptide antibiotics of the vancomycin class.

1985 ◽  
Vol 31 (10) ◽  
pp. 1606-1610 ◽  
Author(s):  
A Corti ◽  
C Rurali ◽  
A Borghi ◽  
G Cassani
Keyword(s):  
Human Serum ◽  
Binding Assay ◽  
Solid Phase ◽  
Quantitative Detection ◽  
Synthetic Analog ◽  
Bacterial Cells ◽  
Glycopeptide Antibiotics ◽  
Competitive Binding Assay ◽  
Analytical Recovery ◽  
Receptor Assay

Abstract A solid-phase enzyme-receptor assay (SPERA) has been developed for glycopeptide antibiotics of the vancomycin class such as teicoplanin, vancomycin, ristocetin, avoparcin, actaplanin, A-47934, A-41030, and A-35512-B. The assay exploits the mechanism of most action of these antibiotics, which is based on their interaction with acyl-D-alanyl-D-alanine, a constituent of the walls of most growing bacterial cells. The antibiotics and enzyme-labeled teicoplanin compete for a synthetic analog of the biological receptor, albumin-epsilon-aminocaproyl-D-alanyl-D-alanine. The various antibiotics produced different competition curves, 50% displacement being obtained with antibiotic concentrations ranging from 0.04 to 4 mg/L, vancomycin and actaplanin being the weakest and strongest competitors, respectively. For teicoplanin in human serum the intra-assay CV was 7.2%, the interassay CV was 11.2%, and the analytical recovery 94%. Teicoplanin concentrations obtained by SPERA (chi) correlated well with those obtained by microbiological assay (y): y = 1.03 chi + 0.053 (r = 0.943; n = 60). We conclude that SPERA is a powerful tool for identification and quantitative detection of glycopeptide antibiotics, even in complex media.

Solid-phase enzymoimmunoassay for osteocalcin in human serum or plasma, with use of a monoclonal antibody.

1989 ◽  
Vol 35 (10) ◽  
pp. 2087-2092 ◽  
Author(s):  
M J Power ◽  
P F Fottrell
Keyword(s):  
Monoclonal Antibody ◽  
Detection Limit ◽  
Horseradish Peroxidase ◽  
Human Serum ◽  
Solid Phase ◽  
Sample Volume ◽  
Microtiter Plates ◽  
Analytical Recovery ◽  
Peroxidase Conjugate

Abstract In this solid-phase enzymoimmunoassay on microtiter plates for osteocalcin in serum or plasma, we use an osteocalcin-horseradish-peroxidase conjugate and a monoclonal antibody raised against bovine osteocalcin. We thoroughly standardized the assay for measurement of osteocalcin in both serum and plasma, demonstrating independence of sample volume, and determining the analytical recovery and within-and between-assay CVs. The detection limit was between 0.6 and 1.1 micrograms/L and the ED50 was 16 micrograms/L for a 5-microL sample volume. The intra-assay CV over the range 3 to 74 micrograms/L was less than or equal to 15%. The interassay CV over the range 3.6 to 46 micrograms/L was less than or equal to 16%. Results by this assay and by an in-house radioimmunoassay in which the same monoclonal antibody was used correlated well (r2 = 0.948). Osteocalcin concentrations in serum and plasma as measured with the present assay agreed well with published values.

Improved radioassay for human antithyroglobulin

1982 ◽  
Vol 101 (4) ◽  
pp. 555-561 ◽  
Author(s):  
Gildon N. Beall ◽  
Margaret E. Parslow ◽  
Sally R. Kruger ◽  
Azra Zaidi
Keyword(s):  
Binding Assay ◽  
Solid Phase ◽  
Specific Binding ◽  
Serum Thyroglobulin ◽  
Competitive Binding Assay ◽  
Thyroglobulin Antibodies ◽  
High Concentrations ◽  
Standard Serum ◽  
Positive Results ◽  
Very High

Abstract. Human antithyroglobulin (anti-HTg) in serum and tissue cultures can be assayed by coprecipitation with 125I-labelled human thyroglobulin (HTg). Coprecipitation of the antibody from different sera gives roughly parallel curves, so that a standard serum can be used for quantitiation of other sera. By assessing the binding of 125I-labelled HTg in antigen excess we estimate the antibody content of the standard serum to be 0.14 ng anti-HTg per ml. Shortened incubation minimizes non-specific binding of HTg to serum globulins and obviates pre-assay absorption of HTg. Radioassay for thyroglobulin antibodies in serum correlated with values obtained by solid-phase competitive-binding assay (SPCB). One unit in our assay corresponds to 0.014 unit by the latter assay and to 0.12 units of MRC Research Standard A. We confirm that serum thyroglobulin seriously interferes with SPCB assay, giving what appears to be positive results for anti-HTg where none is detected with our assay. Values for anti-HTg are depressed by serum HTg but only in the presence of very high concentrations in serum.

Chemiluminescence receptor assay for measuring vitamin B12 in serum evaluated

1994 ◽  
Vol 40 (4) ◽  
pp. 537-540 ◽  
Author(s):  
S Wentworth ◽  
J A McBride ◽  
W H Walker
Keyword(s):  
Vitamin B12 ◽  
Magnetic Particle ◽  
Reference Range ◽  
Binding Assay ◽  
Radioligand Binding ◽  
Reference Ranges ◽  
Radioligand Binding Assay ◽  
Analytical Recovery ◽  
Receptor Assay ◽  
Local Reference

Abstract We evaluated a chemiluminescence receptor assay for vitamin B12 in serum (Magic Lite; Ciba Corning Diagnostics), in which an acridinium ester label is used with magnetic particle separation. Within- and between-batch precisions were generally acceptable, except at low analyte concentrations. The reference range determined from 104 elective preoperative patients was 120-610 pmol/L, compared with 150-590 pmol/L for our in-house radioligand-binding assay. Magic Lite discriminated between normal and abnormal results as effectively as the in-house method when local reference ranges were applied. Magic Lite demonstrated a negative bias at low analyte concentrations and was unable to detect any vitamin B12 in two B12-deficient patients. Assay accuracy--judged from analytical recovery and comparisons with the in-house method and two other radioassay kits (Quantaphase, Bio-Rad Labs., and Immophase, Ciba Corning Diagnostics)--was poor at low B12 concentrations when the manufacturer's recommended two-point calibration was used. This problem was partially corrected by using a full set of calibrators.

A Sensitive Heparin-Protamine Competitive Binding Assay

1981 ◽  
Author(s):  
J Dawes ◽  
D S Pepper
Keyword(s):  
Binding Assay ◽  
Antithrombin Iii ◽  
Solid Phase ◽  
Biological Fluids ◽  
Heparan Sulphate ◽  
Competitive Binding ◽  
Platelet Factor ◽  
Competitive Binding Assay ◽  
Agarose Beads ◽  
Potential Binding

Using protamine-agarose beads, with 125I- heparin as a tracer, we have developed a sensitive competitive binding assay with very wide applications. We are able to measure the concentrations of heparin and heparan sulphate in protease-treated biological fluids down to 40 ng. ml-1. Chondroitin and dermatan sulphates do not interfere with the assay, which can also be used to:(1) measure other acidic polysaccharides e.g. dextran sulphate, semisynthetic heparin analogues, and carrageenans.(2) assay proteins which bind to heparin, such as platelet factor 4, antithrombin III and protamine.(3) screen polymers and solid phases as potential binding materials for these molecules.(4) compare a series of polymers e.g. assess the degree of sulphation of heparins, or the affinity of basic polymers for heparin.Solid phase polyelectrolytes such as Dowex are potentially more robust substitutes for protamine- agarose, being resistant to proteases.

scholarly journals Solid-phase competitive-binding radioimmunoassay for detecting antibody to the M antigen of histoplasmin

1977 ◽  
Vol 6 (6) ◽  
pp. 598-604
Author(s):  
E Reiss ◽  
H Hutchinson ◽  
L Pine ◽  
D W Ziegler ◽  
L Kaufman
Keyword(s):  
Complement Fixation Test ◽  
Histoplasma Capsulatum ◽  
Binding Assay ◽  
Complement Fixation ◽  
Solid Phase ◽  
Competitive Binding ◽  
Fixation Test ◽  
Competitive Binding Assay ◽  
Mycotic Infections ◽  
Specific Rabbit

A radioimmunoassay (RIA) was designed and compared with complement fixation and immunodiffusion tests for their relative ability to detect antibodies in sera of histoplasmosis patients. M antigen, purified from histoplasmin, was fixed to microtiter wells as the solid phase, and specific rabbit 125I-labeled anti-M globulin was the source of indicator antibodies. The optimal concentrations for the competitive-binding assay were 1.6 ng per well for M antigen and 650 ng per well for the 125I-labeled anti-M globulin. A panel of sera from 29 histoplasmosis patients and from patients with other mycoses was screened for RIA activity and in complement fixation and immunodiffusion tests that used histoplasmin and Histoplasma capsulatum yeast-form antigens. The sera of 22 histoplasmosis patients reacted in the RIA, 21 in the complement fixation, and 16 in the immunodiffusion tests. Sera of patients with other mycotic infections did not react in the RIA, with the exception of those of one blastomycosis patient and one candidiasis patient. The RIA could be modified to quantitate M antigen; as little as 125 pg could be detected. The evaluation of this panel of histoplasmosis patients' sera showed that the RIA was about equivalent in sensitivity to the complement fixation test. Some advantages of the RIA over the complement fixation test were that RIA was less prone to cross-reactions and gave better quantitation of low-titered sera. The RIA was a 1-day test, was not hindered by the anti-complementary activity of some sera, and could be modified to quantitate minute amounts of M antigen.

Determination of cyclosporine by a competitive binding assay with cyclophilin

1991 ◽  
Vol 37 (3) ◽  
pp. 403-410 ◽  
Author(s):  
G Steiner ◽  
B Küllinger ◽  
G Mayer ◽  
Y Jie ◽  
H Leibl ◽  
...  
Keyword(s):  
Binding Assay ◽  
Biological Fluids ◽  
Bioactive Metabolites ◽  
Binding Affinities ◽  
Competitive Binding Assay ◽  
Protein Binding Assay ◽  
Analytical Recovery ◽  
Competitive Protein Binding Assay ◽  
Competitive Protein Binding

Abstract A competitive protein-binding assay for cyclosporine based on use of the intracellular cyclosporine-binding protein cyclophilin (CYP) was used to measure cyclosporin A (CsA) and its bioactive metabolites in whole blood. CYP from cytoplasmic extracts or erythrocyte lysates was applied in the binding assay with use of [3H]CsA as tracer and charcoal adsorption for separating bound from free tracer. Binding affinities of various CsA analogs and metabolites correlated well with their reported in vitro immunosuppressive activities. The assay detected as little CsA as 50 micrograms/L (1 g = 0.832 mmol of CsA), analytical recovery was greater than 80%, and CVs were less than 8% for intra-assay and less than 11% for interassay precision in the range of 150-1000 micrograms/L. We used this assay to measure CsA concentrations in blood and compared the results with those measured by HPLC or by CsA-specific (monoclonal) and CsA-nonspecific (polyclonal) radioimmunoassays. Binding assay results were, in nearly all cases, less than those measured by the nonspecific RIA and frequently greater than 20% above the values determined by the CsA-specific assays. Individual patients had pronounced differences in the relative proportions of CsA, CYP-binding (bioactive) metabolites, and cross-reacting CsA metabolites. Because the presence of bioactive metabolites may considerably contribute to the immunosuppressive activity of CsA, we consider the binding assay clinically useful for measuring CsA in biological fluids.

New enzyme-linked immunosorbent assay for glycocalicin in plasma

1991 ◽  
Vol 37 (2) ◽  
pp. 169-172 ◽  
Author(s):  
ShinjI Kunishima ◽  
Klyotaka Hayashi ◽  
Sentaro Kobayashi ◽  
Tomokl Naoe ◽  
Ryuzo Ohno
Keyword(s):  
Immunosorbent Assay ◽  
Binding Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Glycoprotein Ib ◽  
Proteolytic Fragment ◽  
Competitive Binding Assay ◽  
Analytical Recovery ◽  
Sandwich Type ◽  
Assay Time ◽  
The Mean

Abstract A new sandwich-type enzyme-linked immunosorbent assay for quantifying glycocalicin, a proteolytic fragment of platelet membrane glycoprotein Ib, is described. The assay is based on the use of two monoclonal antibodies raised against glycoprotein Ib and involves the avidin-biotin technique. The detection limit is 7 micrograms/L and the range of glycocalicin determined in plasma is 0.01 to 1 mg/L. Assay time is 2 h. The intra-assay CV ranged from 3.6% to 5.2%, the interassay CV from 5.4% to 8.0%. Analytical recovery of purified glycocalicin added to a plasma pool averaged 96%. In 36 healthy subjects, the mean glycocalicin concentration in plasma was 0.36 (SD 0.07) mg/L (2.7 nmol/L). We conclude that this assay is suitable for measuring glycocalicin in plasma and is also more sensitive and precise than the previously published immunoassays based on competitive binding assay.

scholarly journals Use of DNA-aptamers for enrichment of low abundant proteins in cellular extracts for quntitative detection by selected reaction monitoring

2018 ◽  
Vol 64 (1) ◽  
pp. 5-9
Author(s):  
K.G. Ptitsyn ◽  
S.E. Novikova ◽  
Y.Y. Kiseleva ◽  
A.A. Moysa ◽  
L.K. Kurbatov ◽  
...  
Keyword(s):  
Solid Phase ◽  
Fold Increase ◽  
Target Protein ◽  
Selected Reaction Monitoring ◽  
Quantitative Detection ◽  
Reaction Monitoring ◽  
Bacterial Cells ◽  
Affinity Enrichment ◽  
Human Thrombin ◽  
The Relationship

The relationship between the amount of a target protein in a complex biological sample and its amount measured by selected reaction monitoring (SRM) mass spectrometry upon the affinity enrichment of target protein with aptamers immobilized on a solid phase was studied. Human thrombin added in known concentrations to cellular extracts derived from bacterial cells was used as model target protein. It has been demonstrated that the affinity enrichment of thrombin in cellular extracts by means of the thrombin-binding aptamer immobilized on the surface of magnetic microbeads results in an approximately 10-fold increase of the concentration of target protein and a 100-fold decrease of the low limit of a target protein concentration range where its quantitative detection by SRM is possible without an interference from other peptides present in a tryptic digest.

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