scholarly journals False Positivity in a Cyto-ELISA for Anti-Endothelial Cell Antibodies Caused by Heterophile Antibodies to Bovine Serum Proteins

2000 ◽  
Vol 46 (2) ◽  
pp. 273-278 ◽  
Author(s):  
Ronan Revelen ◽  
Anne Bordron ◽  
Maryvonne Dueymes ◽  
Pierre Youinou ◽  
Josiane Arvieux
Keyword(s):  
Endothelial Cell ◽  
Bovine Serum ◽  
Serum Proteins ◽  
Solid Phase ◽  
Calf Serum ◽  
Blocking Agent ◽  
Screening Tests ◽  
Tween 20 ◽  
Serum Samples ◽  
Antibody Absorption

Abstract Background: ELISAs with fixed endothelial cells or cell lines are widely used screening tests for anti-endothelial cell antibodies (AECAs), but spurious increases occur. We examined interferences by heteroantibodies and means to eliminate them. Methods: AECAs were measured by ELISA on fixed layers of the human endothelial cell line, EA.hy 926, in a panel of 60 patient serum samples diluted in bovine serum albumin. Heteroantibodies against fetal calf serum (FCS) proteins were demonstrated and characterized in an ELISA—the interference assay—that used FCS-coated plates and Tween 20-containing buffer as blocking agent and sample diluent, as well as by immunoblotting. Results: In 12 of 60 patient serum samples, spurious increases of AECA titers were produced by endogenous antibodies reacting with FCS proteins from culture medium that were coated onto the solid-phase at the time of cell plating. This mechanism of interference was supported experimentally by exposing extracellular matrix, varying cell density, and incubating wells with FCS alone. The heterophile antibodies were mainly IgG and IgA, and in inhibition experiments, they recognized serum proteins from goat, sheep, and horse. Washing cells free of FCS before plating, or adding FCS (100 mL/L) to the patient sample diluent eliminated spurious signals from all 30 tested sera, but the latter method had practical advantages. Conclusions: Antibodies against animal serum proteins are a frequent cause of erroneous results in cyto-ELISAs. The interference can be eliminated by simple antibody absorption in FCS-containing dilution buffer.

Detection, Quantitation, and Identification of Residual Aminopenicillins by High-Performance Liquid Chromatography after Fluorescamine Derivation

2000 ◽  
Vol 63 (10) ◽  
pp. 1421-1425 ◽  
Author(s):  
CHIH-CHUN HONG ◽  
FUSAO KONDO
Keyword(s):  
Bovine Serum ◽  
Mobile Phase ◽  
High Performance ◽  
Serum Proteins ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Excitation Wavelength ◽  
Column Temperature ◽  
Solid Phase Extraction Cartridge ◽  
Mobile Phases

A high-performance liquid chromatographic (HPLC) method with fluorescence detection after precolumn fluorescamine derivation was developed to detect residues of two aminopenicillins, amoxicillin (AMPC) and ampicillin (ABPC), in bovine serum. Proteins in serum samples spiked with each of these penicillins were precipitated with sodium tungstate and sulfuric acid, centrifuged, and removed by passage through a C18 solid-phase extraction cartridge. After precolumn treatment of the extraction products of AMPC and ABPC with fluorescamine solution, HPLC analysis with fluorescence spectrophotometric detection at an excitation wavelength of 390 nm and an emission wavelength of 485 nm was performed to identify these products. Two mobile phases were used for residual analysis by the isocratic HPLC system. An ODP column (polyvinyl alcohol bonded with an octadecyl functional group) that can be used with strongly alkaline mobile phases (pH 2.0 to 13) was selected, and the column temperature was set at 40°C. A mobile phase comprising 100-mM K2HPO4 solution and acetonitrile (72:28, vol/vol), which yielded AMPC and ABPC retention times of 4.1 and 7.9 min, respectively, was suitable for detection of residual ABPC but not for residual AMPC because interference was caused by peaks of other extracted substances. When a mobile phase comprising a different ratio of 100-mM K2HPO4 solution and acetonitrile (78:22, vol/vol) was used, the retention times of AMPC and ABPC were 7.3 and 26.3 min, respectively, and both penicillins could be analyzed using this system. The calculated standard curves of the reaction products with both mobile phases were linear, and the correlation coefficients were greater than 0.999. The lower limit of detection was 10 ng/ml for both penicillins. Analysis of extracts from bovine serum spiked with AMPC and ABPC at a concentration of 1 μg/ml yielded recovery rates of 102.2 ± 5.5% and 79.0 ± 5.2%, respectively. This detection method may be useful for routine laboratory testing of AMPC and ABPC.

scholarly journals Validation of a Dissociation Enhanced Lanthanide Fluorescence Immunoassay for the Screening of 17 -Estradiol in Bovine Serum According to European Union Decision 2002/657/EC

2007 ◽  
Vol 90 (5) ◽  
pp. 1427-1431 ◽  
Author(s):  
Daniela Scalas ◽  
Stefania Squadrone ◽  
Marilena Gili ◽  
Daniela Marchis ◽  
Marino Prearo ◽  
...  
Keyword(s):  
European Union ◽  
Bovine Serum ◽  
False Negative ◽  
Screening Tests ◽  
Rapid Screening ◽  
Fluorescence Immunoassay ◽  
Serum Samples ◽  
Related Substances ◽  
The Matrix ◽  
Negative Effect

Abstract A validation study was carried out in order to evaluate the performances of a dissociation enhanced lanthanide fluorescence immunoassay (DELFIA) for rapid screening of 17 -Estradiol in bovine serum. This validation was performed according to European Union (EU) Decision 2002/657/EC, which establishes criteria and procedures for determination of detection capability (CC), selectivity/specificity, and applicability/ruggedness/stability for qualitative screening tests. To determine these performance characteristics, 20 blank serum samples of cattle were collected and spiked with 17 -Estradiol at 40 pg/mL, corresponding to the maximum residue limit permitted by Italian legislation. According to the EU Decision CC criterion, spiked samples must have <5 probability to be classified as a false negative. 17 -Estradiol was detected in each spiked sample, and the CC results were <40 pg/mL. There was also no observed interference effect due to chemically related substances or from the matrix. Moreover, slight variations of some critical factors in the DELFIA procedure, deliberately introduced for ruggedness evaluation, did not result in any negative effect on the 17 -Estradiol detection. The proposed method is suitable for qualitative screening analysis of 17 -Estradiol according to EU performance requirements.

scholarly journals Determination of Haptoglobin in Bovine Serum using Polyclonal and Monoclonal Anti-human Haptoglobin Antibodies

2010 ◽  
Vol 79 (1) ◽  
pp. 105-112 ◽  
Author(s):  
Paulina Jawor ◽  
Tadeusz Stefaniak ◽  
Iwona Kątnik-Prastowska
Keyword(s):  
Monoclonal Antibody ◽  
Detection Limit ◽  
Bovine Serum ◽  
Solid Phase ◽  
Low Cost ◽  
Reference Method ◽  
Serum Samples ◽  
Incubation Periods ◽  
Short Time

Two ELISA procedures to determine haptoglobin (Hp) in bovine serum were developed. Equine haemoglobin was used as the solid phase. Self-developed goat polyclonal antibody (variant I) and monoclonal antibody (variant II) raised against human Hp were used. The results were compared with the guaiacol method. High correlation was found (r = 0.96 and r = 0.90, respectively) based on the results of 548 bovine serum samples, of which 357 were from clinically healthy cows and 191 from cows and calves monitored during treatment for the most common diseases. The Hp detection limit of ELISA using polyclonal Ab was 0.1 mg/l and using MoAb 0.21 mg/l. The addition of 2% PEG 6000 at the antibody-binding steps enabled major shortening of the incubation periods. The relatively short time, low cost of reagents, and high correlation with the reference method support the use of these ELISA variants in bovine diagnostics.

Solid-Phase Radioimmunoassay of Thyroxine in Untreated Serum

1975 ◽  
Vol 21 (10) ◽  
pp. 1406-1413 ◽  
Author(s):  
John Seth ◽  
Frederick J Rutherford ◽  
Ian McKenzie
Keyword(s):  
Serum Proteins ◽  
Clinical Laboratory ◽  
Solid Phase ◽  
Gel Filtration ◽  
Serum Samples ◽  
Total Thyroxine ◽  
Solid Phase Radioimmunoassay ◽  
Technical Simplicity ◽  
Routine Clinical Laboratory ◽  
Antigen Antibody

Abstract We describe a simple, convenient solid-phase radioimmunoassay of total thyroxine in unextracted serum. Serum samples are added directly to the assay incubation mixture, interference in the antigen/antibody reaction by the thyroxine-binding serum proteins being almost completely eliminated by the addition of 8-anilino-1-naphthalene sulfonic acid and incubation at pH 10.5. Residual interference is compensated for by including thyroxine-free serum in the standards. Use of thyroxine antibodies that are coupled to a solid support permits separation of free and antibody-bound hormone by a single washing step, followed by centrifugation. The method is specific, accurate, and reasonably precise. The results obtained compare well with those for radioimmunoassay of thyroxine in serum freed of protein by gel filtration, and with results of a competitive protein-binding method. The technical simplicity of the procedure should readily permit automation. These features suggest that the technique should be well suited for routine clinical laboratory use.

scholarly journals OC 8435 MULTI-BIOMARKER TEST STRIP FOR POINT-OF-CARE SCREENING FOR ACTIVE TUBERCULOSIS: A FIVE-COUNTRY MULTI-CENTRE TEST EVALUATION

2019 ◽  
Vol 4 (Suppl 3) ◽  
pp. A6.2-A6
Author(s):  
Paul Corstjens ◽  
Anouk Van Hooij ◽  
Elisa Tjon Kon Fat ◽  
Shannon Herdigein ◽  
Anna Ritah Namuganga ◽  
...  
Keyword(s):  
Respiratory Diseases ◽  
Serum Proteins ◽  
Point Of Care ◽  
Low Cost ◽  
Test Strip ◽  
Screening Tests ◽  
Rapid Screening ◽  
Serum Samples ◽  
Multiple Test ◽  
Biomarker Selection

BackgroundInexpensive rapid screening tests that can be used at the point-of-care (POC) are vital to combat tuberculosis. Particularly, less invasive non-sputum-based biomarker tests for all TB forms can help controlling transmission. Availability of such tests would significantly accelerate and streamline diagnostic approaches, improve cost-efficiency and decrease unnecessary costly GeneXpert referrals.MethodsMulti-biomarker test (MBT) devices measuring levels of selections of up to six serum proteins simultaneously on a single lateral flow (LF) strip were produced. The strip contains individual capture lines for a biomarker selection allowing discrimination of TB-patients from other respiratory diseases (ORD). Only biomarkers successfully evaluated with singleplex strips (single biomarker tests) were applied to the MBT device. Quantitative signals are recorded with a low-cost handheld reader compatible with the applied luminescent up-converting particle (UCP) label. Biomarker selection and algorithms used to distinguish potential-TB and ORD are flexible.ResultsResults obtained with MBT strips containing multiple test lines correlate well with singleplex LF strips. Using LF tests for 5 selected biomarkers a sensitivity of 94% and specificity of 96% could be achieved with a confirmed South African selection of 20 TB and 31 non-TB samples. Patients were designated TB positive when scoring a value above the cut-off threshold for at least 3 out of 5 biomarkers. Serum samples of potential TB patients collected at five medical research institutes (Ethiopia, Namibia, South Africa, The Gambia, Uganda) were tested locally with MBT strips comprised of CRP, SAA, IP-10, Ferritin, ApoA-I and IL-6 and results analysed to obtain an overall pan-Africa applicable signature.ConclusionEvaluated POC applicable UCP-LF devices detecting serum biomarker signatures can help to distinguish active TB from other respiratory diseases and as such can prioritise highest-risk patients for further care. Ongoing prospective studies evaluate the MBT strip with fingerstick blood and do not require a laboratory or trained phlebotomist anymore.

scholarly journals Computer-supported Detection of M-Components and Evaluation of Immunoglobulins after Capillary Electrophoresis

2001 ◽  
Vol 47 (1) ◽  
pp. 110-117 ◽  
Author(s):  
Magnus Jonsson ◽  
Joyce Carlson ◽  
Jan-Olof Jeppsson ◽  
Per Simonsson
Keyword(s):  
Capillary Electrophoresis ◽  
Decision Support ◽  
Protein Separation ◽  
Serum Proteins ◽  
Cost Effective ◽  
Clinical Samples ◽  
Serum Samples ◽  
Computerized Decision Support ◽  
Cost Effective Method ◽  
Mathematical Algorithms

Abstract Background: Electrophoresis of serum samples allows detection of monoclonal gammopathies indicative of multiple myeloma, Waldenström macroglobulinemia, monoclonal gammopathy of undetermined significance, and amyloidosis. Present methods of high-resolution agarose gel electrophoresis (HRAGE) and immunofixation electrophoresis (IFE) are manual and labor-intensive. Capillary zone electrophoresis (CZE) allows rapid automated protein separation and produces digital absorbance data, appropriate as input for a computerized decision support system. Methods: Using the Beckman Paragon CZE 2000 instrument, we analyzed 711 routine clinical samples, including 95 monoclonal components (MCs) and 9 cases of Bence Jones myeloma, in both the CZE and HRAGE systems. Mathematical algorithms developed for the detection of monoclonal immunoglobulins (MCs) in the γ- and β-regions of the electropherogram were tested on the entire material. Additional algorithms evaluating oligoclonality and polyclonal concentrations of immunoglobulins were also tested. Results: CZE electropherograms corresponded well with HRAGE. Only one IgG MC of 1 g/L, visible on HRAGE, was not visible after CZE. Algorithms detected 94 of 95 MCs (98.9%) and 100% of those visible after CZE. Of 607 samples lacking an MC on HRAGE, only 3 were identified by the algorithms (specificity, 99%). Algorithms evaluating total gammaglobulinemia and oligoclonality also identified several cases of Bence Jones myeloma. Conclusions: The use of capillary electrophoresis provides a modern, rapid, and cost-effective method of analyzing serum proteins. The additional option of computerized decision support, which provides rapid and standardized interpretations, should increase the clinical availability and usefulness of protein analyses in the future.

scholarly journals UHPLC-ESI-MS/MS Quantification of Relevant Substrates and Metabolites of the Kynurenine Pathway Present in Serum and Peritoneal Fluid from Gastric Cancer Patients—Method Development and Validation

2021 ◽  
Vol 22 (13) ◽  
pp. 6972
Author(s):  
Ilona Sadok ◽  
Katarzyna Jędruchniewicz ◽  
Karol Rawicz-Pruszyński ◽  
Magdalena Staniszewska
Keyword(s):  
Gastric Cancer ◽  
Cancer Patients ◽  
Peritoneal Fluid ◽  
Method Development ◽  
Solid Phase ◽  
Kynurenine Pathway ◽  
Serum Samples ◽  
Esi Ms ◽  
Gastric Cancer Patients ◽  
Development And Validation

Metabolites and enzymes involved in the kynurenine pathway (KP) are highly promising targets for cancer treatment, including gastrointestinal tract diseases. Thus, accurate quantification of these compounds in body fluids becomes increasingly important. The aim of this study was the development and validation of the UHPLC-ESI-MS/MS methods for targeted quantification of biologically important KP substrates (tryptophan and nicotinamide) and metabolites(kynurenines) in samples of serum and peritoneal fluid from gastric cancer patients. The serum samples were simply pretreated with trichloroacetic acid to precipitate proteins. The peritoneal fluid was purified by solid-phase extraction before analysis. Validation was carried out for both matrices independently. Analysis of the samples from gastric cancer patients showed different accumulations of tryptophan and its metabolites in different biofluids of the same patient. The protocols will be used for the evaluation of tryptophan and kynurenines in blood and peritoneal fluid to determine correlation with the clinicopathological status of gastric cancer or the disease’s prognosis.

scholarly journals Associations between the thyroid panel and serum protein concentrations across pregnancy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Barbara Lisowska-Myjak ◽  
Agnieszka Strawa ◽  
Hanna Zborowska ◽  
Artur Jakimiuk ◽  
Ewa Skarżyńska
Keyword(s):  
Serum Proteins ◽  
Thyroid Stimulating Hormone ◽  
Second Trimester ◽  
Serum Concentrations ◽  
Metabolic Processes ◽  
Serum Samples ◽  
Serum Parameters ◽  
Active Regulation ◽  
Potential Use

AbstractEstablishing any characteristic associations between the serum parameters of thyroid function and serum proteins in pregnancy may aid in elucidating the role of the thyroid gland in the regulation of pregnancy-specific metabolic processes and in selecting candidate biomarkers for use in their clinical assessment. Concentrations of thyroid stimulating hormone (TSH), free tri-iodothyronine (fT3) and free thyroxine (fT4), six electrophoretically separated protein fractions (albumin, alpha-1-, alpha2-, beta-1-, beta-2- and gamma-globulins), representative proteins—albumin (ALB), transferrin (TRF), alpha-2-macroglobulin (AMG) and ceruloplasmin (CER) were measured in 136 serum samples from 65 women in their consecutive trimesters of pregnancy. The concentrations of TSH, fT4 and fT3 were significantly correlated (p < 0.05) with the concentrations of the albumin, alpha-2- and beta-1 globulin fractions. Significant correlations (p < 0.05) which were positive between fT4 and ALB and negative between fT4 and TRF were established throughout pregnancy. Significant negative correlations (p < 0.05) were demonstrated for fT3 with alpha-2-globulin, AMG and CER. Changes in the serum concentrations of thyroid hormones seen between the trimesters were found to correlate with the concentrations of high-abundance serum proteins. Opposite directions of correlations between fT4 and ALB and fT4 and TRF observed throughout pregnancy may indicate the shared biological role of these parameters in maintaining maternal homeostasis and they suggest their potential use in the clinic as a simple biomarker panel. A negative correlation of fT3 with CER in the second trimester possibly reflects their involvement in the active regulation of metabolic processes.

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