scholarly journals Evaluating Sandwich Immunoassays in Microarray Format in Terms of the Ambient Analyte Regime

2004 ◽  
Vol 50 (10) ◽  
pp. 1907-1920 ◽  
Author(s):  
Petri Saviranta ◽  
Ryan Okon ◽  
Achim Brinker ◽  
Masaki Warashina ◽  
Joerg Eppinger ◽  
...  
Keyword(s):  
Detection Limit ◽  
Serum Proteins ◽  
Resonance Light Scattering ◽  
Mass Action ◽  
Mouse Serum ◽  
Experimental Setup ◽  
Assay Sensitivity ◽  
Antibody Microarrays ◽  
Titration Curves ◽  
Assay Volume

Abstract Background: Conceptionally, antibody microarrays are simply multiplexed sandwich immunoassays in a miniaturized format. However, from the amounts of capture antibodies used, it is not apparent whether such assays are ambient analyte (Ekins. Clin Chem 1998;44:2015–30) or mass-sensing devices (Silzel et al. Clin Chem 1998;44:2036–43). We evaluated multiplexed microarray sandwich assays for 24 mouse serum proteins in these terms within the boundaries of our experimental setup and based on theoretical considerations of the law of mass action. Methods: Capture antibodies for 24 mouse serum proteins were printed on planar microarray substrates. After incubation with mixtures of purified antigens for 1 or 18 h, mixtures of biotinylated detection antibodies were used. High assay sensitivity was achieved by use of resonance-light-scattering particles for signal generation. Titration curves were generated for assay volumes of 20, 40, and 80 μL, and detection limits were calculated and compared. The assays were modeled theoretically based on the amounts of capture antibodies and the assay volumes used. Results: As predicted, experimental variations of the assay volume by up to fourfold did not appreciably affect detection. Even for the most sensitive assay, <2% of the analyte molecules present in the sample were captured and generated signal at the detection limit. However, increasing the sample incubation time from 1 to 18 h on average lowered the detection limit threefold. Conclusions: In our experimental setup, all 24 sandwich microarray assays fulfill the criteria of the “ambient analyte” regime because depletion of analyte molecules from the assay volume is insignificant.

scholarly journals Development of a Chemiluminescence Competitive PCR for the Detection and Quantification of Parvovirus B19 DNA Using a Microplate Luminometer

1999 ◽  
Vol 45 (9) ◽  
pp. 1391-1396 ◽  
Author(s):  
Fabiana Fini ◽  
Giorgio Gallinella ◽  
Stefano Girotti ◽  
Marialuisa Zerbini ◽  
Monica Musiani
Keyword(s):  
Detection Limit ◽  
Parvovirus B19 ◽  
Quantitative Detection ◽  
Specific Method ◽  
Competitive Pcr ◽  
Serum Samples ◽  
Titration Curves ◽  
Viral Dnas ◽  
Assessment And Monitoring ◽  
Detection And Quantification

Abstract Background: Quantitative PCR of viral nucleic acids can be useful clinically in diagnosis, risk assessment, and monitoring of antiviral therapy. We wished to develop a chemiluminescence competitive PCR (cPCR) for parvovirus B19. Methods: Parvovirus DNA target sequences and competitor sequences were coamplified and directly labeled. Amplified products were then separately hybridized by specific biotin-labeled probes, captured onto streptavidin-coated ELISA microplates, and detected immunoenzymatically using chemiluminescent substrates of peroxidase. Chemiluminescent signals were quantitatively analyzed by a microplate luminometer and were correlated to the amounts of amplified products. Results: Luminol-based systems displayed constant emission but had a higher detection limit (100–1000 genome copies) than the acridan-based system (20 genome copies). The detection limit of chemiluminescent substrates was lower (20 genome copies) than colorimetric substrates (50 genome copies). In chemiluminescence cPCR, the titration curves showed linear correlation above 100 target genome copies. Chemiluminescence cPCR was positive in six serum samples from patients with parvovirus infections and negative in six control sera. Conclusions: The chemiluminescence cPCR appears to be a sensitive and specific method for the quantitative detection of viral DNAs.

Nonimmunological Assay of Urinary Albumin Based on Laser-Induced Fluorescence

1992 ◽  
Vol 38 (10) ◽  
pp. 2089-2092 ◽  
Author(s):  
M A Kessler ◽  
M R Hubmann ◽  
B A Dremel ◽  
O S Wolfbeis
Keyword(s):  
Detection Limit ◽  
Flow Injection ◽  
Flow Injection Analysis ◽  
Serum Proteins ◽  
Early Stage ◽  
Laser Induced Fluorescence ◽  
Urinary Albumin ◽  
Coefficients Of Variation ◽  
Flow Injection Analysis System ◽  
Analysis System

Abstract We describe the first nonimmunological assay of albumin in urine with a detection limit of 1 mg/L. The method is simple, rapid, and accurate. It is based on the probe Albumin Blue 670, which becomes highly fluorescent on binding to albumin. An inexpensive diode laser was used as the light source for measurement of laser-induced fluorescence. The assay was coupled to a flow-injection analysis system capable of running 20 samples per hour. The working range was 1-100 mg/L, which covered albumin concentrations found in nonpathological urine and in urine with slightly increased albumin. This range makes prediction of nephropathy possible at an early stage. Other serum proteins and hemoglobin do not interfere. The coefficients of variation were < 4% and < 7% within one day and from day to day, respectively. A correlation coefficient of 0.990 (n = 100) was obtained for comparison with the Behring nephelometric assay.

Radioimmunoassay for Serum Triiodothyronine: Evaluation of Simple Techniques to Control Interference from Binding Proteins

1974 ◽  
Vol 20 (9) ◽  
pp. 1150-1154 ◽  
Author(s):  
Victor S Fang ◽  
Samuel Refetoff
Keyword(s):  
Binding Sites ◽  
Binding Proteins ◽  
Serum Proteins ◽  
Cross Reactivity ◽  
Heat Inactivation ◽  
Nonspecific Binding ◽  
High Concentration ◽  
Assay Sensitivity ◽  
Thyroxine Binding Globulin ◽  
Fixed Concentration

Abstract Simple techniques for controlling interference from binding proteins in serum, such as thyroxine-binding globulin, in radioimmunoassay for triiodothyronine (T3) have been evaluated for their efficacy, and their effect on assay sensitivity and on recovery of added T3. Ethanol precipitation of serum proteins decreased the assay sensitivity, nonspecific binding was increased, and recoveries of added T3 were inconsistent. Heat-inactivation of thyroxine-binding globulin or use of 8-anilino-1naphthalene sulfonic acid (ANS) to displace T3 from thyroxine-binding globulin produced comparable recovery rates. The heat-inactivation method slightly decreased the sensitivity of the assay and prolonged the procedure, whereas use of ANS is simple, and the assay sensitivity is maintained. When sera contain a high concentration of thyroxine-binding globulin, a fixed concentration of ANS (175 µg/100 µl of serum) might be too low to displace T3 from all its binding sites, but a concentration of ANS greater than 200 µg/100 µl of serum interferes with T3 quantitation by competitively binding to the antibodies. The cross-reactivity of thyroxine to T3-antibodies varies with the antiserum. Thyroxine-binding globulin appears to be the only protein in serum that competes with the antibody for T3 binding.

Electrostatic field induced changes in mouse serum proteins

1974 ◽  
Vol 30 (11) ◽  
pp. 1274-1275 ◽  
Author(s):  
A. A. Marino ◽  
T. J. Berger ◽  
R. O. Becker ◽  
F. X. Hart
Keyword(s):  
Electrostatic Field ◽  
Serum Proteins ◽  
Mouse Serum ◽  
Induced Changes

Determination of Luteolin by Fluorescence Spectrometry

2014 ◽  
Vol 941-944 ◽  
pp. 994-997
Author(s):  
Wei Huang ◽  
Feng Wang ◽  
Hong Long Zhu
Keyword(s):  
Light Scattering ◽  
Detection Limit ◽  
Absorption Spectra ◽  
Resonance Light Scattering ◽  
Linear Range ◽  
Average Content ◽  
Buffer Solution ◽  
Average Recovery ◽  
Higher Sensitivity

It is showed that in the buffer solution of Borax-HCl (pH=6.00), the presence of LU could quench the fluorescence of OFLO, and the quenched level is linear with the concentration of luteolin in a certain range. The detection limit of luteolin is 1.27×10-9g·mL-1.The linear range of luteolin is 2.00×10-7- 8.00×10-5mol·L-1with R =0.9955. The average recovery is 99.2%, RSD = 0.75% . The average content of luteolin in honeysuckle is 212.0μg·g-1. At the same time, its mechanism is discussed by resonance light scattering and absorption spectra. Compared with other analysis methods for Luteolin which have been reported,this method has a relatively wider linear range and higher sensitivity.

Pharmacokinetics, Serum Stability and Immunogenicity of A Polypeptide Inhibitor of B2GPI/Antibody Complexes Tested in Mice.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2205-2205
Author(s):  
Andrew Porter ◽  
Natalia Beglova
Keyword(s):  
Human Serum ◽  
Serum Proteins ◽  
Conflicts Of Interest ◽  
Intraperitoneal Administration ◽  
Gel Filtration ◽  
Reversed Phase ◽  
Mouse Serum ◽  
Carrier Protein ◽  
Serum Stability

Abstract Abstract 2205 Background: Antiphospholipid syndrome (APS) is an autoimmune disease with clinical features of thrombosis and pregnancy loss. Beta2-glycoprotein I (B2GPI) is the major antigen for APS-related antibodies. We engineered a polypeptide consisting of two ligand-binding A1 modules from the ApoE receptor 2. Previously, we demonstrated that this polypeptide, A1-A1, preferentially binds B2GPI/antibody complexes compared to B2GPI alone and efficiently inhibits the binding of B2GPI/antibody complexes to negatively charged phospholipids. Therefore, A1-A1 effectively interferes with two pathological mechanisms of B2GPI/antibody complexes: the binding to anionic phospholipids and ApoER2. In order to use A1-A1 to study pathological mechanisms of B2GPI/antibody complexes in vivo, we tested its pharmacokinetic, serum stability and immunogenicity in mice. To visualize A1-A1, we labeled it with a fluorescent probe Atto-488 attached to the N-terminus. Results: We monitored clearance of A1-A1 from the circulation after intraperitoneal and intravenous administration. After intraperitoneal administration, the concentration of A1-A1 in the blood reached its maximum at 30 min after injection and cleared from the blood in 6–8 hours. When A1-A1 was injected intravenously, 14% of A1-A1 remained in the blood 1 hour after administration and decreased to 4% in 3 hours. We assessed the binding of A1-A1 to serum proteins in both mouse and human serum by gel-filtration chromatography. Chromatograms of A1-A1 in both mouse and human serum collected just after mixing of A1-A1 with serum were almost identical to those collected after 2 hours of incubation at 37° C. About 90% of A1-A1 stays free from serum proteins. Previously, we demonstrated that A1-A1 has a favorable stability in human serum. More than 35% of A1-A1 remained in human serum after 15 days of incubation at 37° C. Here, we determined whether A1-A1 is cleaved by proteases in mouse serum. Degradation of A1-A1 was monitored by the reversed-phase HPLC by comparing the peak corresponding to intact A1-A1 and A1-A1 incubated with serum for 2 hours at 37° C. After incubation with mouse serum, A1-A1 eluted at the same time as intact A1-A1 and the intensity of the elution peak did not decrease, indicating that A1-A1 remains intact in mouse serum. To evaluate immunogenecity of A1-A1, we immunized mice with A1-A1, A1-A1 in the presence of adjuvant and A1-A1 conjugated to a carrier protein. A1-A1 did not induce detectable anti-A1-A1 IgG production even in the presence of adjuvant or carrier protein. Conclusions: A1-A1 has favorable properties for use in vivo. It stays in the circulation for more than one hour following intravenous injection. After intraperitoneal administration, A1-A1 is rapidly absorbed into blood and cleared in about 6 hours. A1-A1 is resistant to both human and mouse proteases, has low immunogenicity and its amount in the blood is not depleted by binding to serum proteins. Disclosures: No relevant conflicts of interest to declare.

Stabilization of turbidimetric immunoassay by covalent coupling of antibody to latex particles

1991 ◽  
Vol 37 (7) ◽  
pp. 1248-1251 ◽  
Author(s):  
H Thakkar ◽  
C L Davey ◽  
E A Medcalf ◽  
L Skingle ◽  
A R Craig ◽  
...  
Keyword(s):  
Latex Particle ◽  
Serum Proteins ◽  
Assay Sensitivity ◽  
Latex Particles ◽  
Covalent Coupling ◽  
Signal Change ◽  
Analytical Recovery ◽  
Turbidimetric Assay ◽  
High Concentrations ◽  
Turbidimetric Immunoassay

Abstract Turbidimetric immunoassay is commonly used to quantify serum proteins. Latex-particle enhancement of this type of assay has been primarily associated with increasing assay sensitivity. However, covalent coupling of an antibody to a latex particle can offer other advantages that are also pertinent in measurement of high concentrations of analytes. By using a common antibody with IgG as a model analyte, we describe the development of a nonenhanced and a latex-particle-enhanced turbidimetric assay for measuring serum IgG. Both assays show adequate analytical recovery and parallelism, and results compare well with those by rate nephelometry. The latex-enhanced assay has equivalent sensitivity, working range, and interassay precision, but much greater signal change and calibration stability than the nonenhanced assay. In addition, with latex particles, less antiserum is needed. Coupling antibodies to latex particles offers considerable advantages, even when an improved assay detection limit is not required.

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