[34] Competitive enzyme-linked immunosorbent assay for biotin

Shiuan, David; Wu, Chwen-Huey; Chang, Yuo-Sheng; Chang, Re-Jiin

doi:10.1016/s0076-6879(97)79036-0

Cited by 10 publications

(4 citation statements)

References 6 publications

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“…Sensitivity of the assay was in£uenced by the [ 125 I]iodostreptavidin dilution. 15 As shown in Fig. 1, a tracer amount of 0¢4 ng/tube is more e¡ective for determination of low biotin concentrations, whereas a tracer amount of 3 ¢ 5 ng/tube is better for high biotin concentrations.…”

Section: Resultsmentioning

confidence: 90%

A sensitive and practical competitive radioassay for plasma biotin

Harthé

Bruno

2003

Ann Clin Biochem

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Section: Resultsmentioning

confidence: 90%

A sensitive and practical competitive radioassay for plasma biotin

Harthé

Bruno

2003

Ann Clin Biochem

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“…[6][7][8][9] We used the following procedure with a small amount of biotinylated BSA, which was different from the procedure reported previously.…”

Section: Quantitation Of Biotin Using Biotinylated Bsa-coated Multiwementioning

confidence: 99%

“…[6][7][8][9] We used the following procedure with a small amount of biotinylated BSA, which was different from the procedure reported previously. 6,7) Multiwell microplates (96 wells) were coated with biotinylated BSA by adding 50 ml of 0.1 mg ml Ϫ1 biotinylated BSA in PBS and 0.02% NaN 3 (PBS-N) to each well, then incubating overnight at 6°C. After the wells were washed five times with PBS-N, 300 ml of 1% BSA in PBS-N was added to each well.…”

mentioning

confidence: 99%

Biotin-Protein Ratios and Stability of Biotinylated Immunoglobulins as Standards for the Quantitation of Biotin-Binding Immunoglobulins

Yazawa

Fukuoka

Honda

et al. 2006

Biological & Pharmaceutical Bulletin

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Immunoglobulins to which biotin was covalently linked were found in human sera for the first time in 1993.1) The prevalence of biotin-binding immunoglobulin was higher in patients with atopic dermatitis, other dermatitis, allergic disorders, and autoimmune disease. [1][2][3] Recently, biotin-binding IgG was detected in a multiwell microplate format for the first time. 4) We developed a quantitative assay for biotinbinding IgG without any purification of samples using F(ab') 2 anti-human IgG-coated multiwell microplates. 5) We showed that the ratio of biotin molecules to protein molecules (biotin-protein ratio) in biotinylated IgG in human sera affected the detection limit in this assay.5) The slope of doseresponse curve of the assay decreased as the biotin-protein ratio of standards decreased and the observed values of biotinylated IgG in human sera depended on the biotin-protein ratio of standards. Thus it was very important to measure the biotin-protein ratio of biotinylatd immunoglobulins which were used as standards in the assay.In this study, we investigated a synthesis method for biotinylated human immunoglobulins, how to determine the biotin-protein ratio of the biotinylated proteins, and their stability. MATERIALS AND METHODS MaterialsThe following materials were obtained from the sources as indicated: flat-bottomed multiwell microplates (Immulon I) from Dynatech Laboratories, Inc., U.S.A.; N-hydroxysuccinimidobiotin (NHS-biotin) and BCA Protein Assay Reagent kit from Pierce Chemical Co., Rockford, U.S.A.; biotin, horseradish peroxidase (HRP)-labeled streptavidin, dimethyl sulfoxide (DMSO), human IgG, human IgA, human IgM, and BSA from Sigma Chemical Co., St. Louis, U.S.A.; ELISA Color Reagent kit (Type O) from Sumitomo Bakelite Co., Ltd., Tokyo, Japan; Biotin Assay Medium ® from Nissui Pharmaceutical Co., Ltd., Tokyo, Japan; Microcon YM-3 from Millipore Co., Beldford, U.S.A. All other chemicals were of reagent grade or better. The water used was 17-Mohm grade.Biotinylation of IgA, IgM, IgG, and BSA Biotinylation of BSA was also examined, since biotinylated BSA was used for the quantitation of biotin in this study. Twenty-five microliters of NHS-biotin DMSO solution at various concentrations was added to 1 ml of 2 mg ml Ϫ1 IgA, IgM, IgG, or BSA in 50 mmol l Ϫ1 sodium bicarbonate/HCl buffer (pH 8.6). The mixture was gently stirred at room temperature for a fixed time. The reaction solution was dialyzed against 0.15 mol l Ϫ1NaCl and finally against Dulbecco's phosphate buffered saline without Ca 2ϩ and Mg 2ϩ (PBS). The amounts of biotinylated IgA, IgM, IgG, and BSA were expressed as the amounts of IgA, IgM, IgG, and BSA, respectively, which were determined using a BCA Protein Assay kit with human IgG and BSA as standards for biotinylated IgA, IgM, and IgG and for BSA, respectively. Quantitation of Biotin Using Biotinylated BSA-Coated Multiwell MicroplateThe principle of biotin quantitation was the same as has been previously reported. [6][7][8][9] We used the following procedure with a small amount of biot...

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“…The amount of DAP produced in this period of time is measured by a spectrometer, which detects the absorbance at 490nm of the DAP. In a competitive ELISA scheme, a lower signal indicates that higher amounts of free GGT were present in the serum to compete with the primary antibody, giving an indication of serum GGT levels when compared to other samples of known GGT concentration (Kumar K, 2014;Shiuan et al, 1997).…”

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confidence: 99%

Biomarker identification and development of aptamers for the diagnosis of sub-clinical facial eczema

Livingston¹

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Facial eczema is a disease caused by the ingestion of fungal spores on pasture grass, causing irreversible liver damage. The disease causes significant decreases in production (i.e. meat, milk and reproduction) for affected animals and primarily affects ruminants. As ruminants contribute enormously to New Zealand’s primary industries, facial eczema is an economically relevant disease. Anthropogenic changes in New Zealand’s climate are likely to increase the fungi’s prevalence in our pastures, driving a need for innovations in treatment and prevention of facial eczema. A key area for improvement is the current facial eczema test, which measures the liver enzyme gamma-glutamyltransferase (GGT) to assess the degree of liver damage. This is normally employed in a small selection of the wider herd due to logistical constraints, as blood samples must be taken from each animal and returned to a veterinary laboratory. This greatly limits the number of animals in a herd that can be tested, forcing treatment to be non-specific and herd-wide. If facial eczema is detected, then herd-wide treatment measures are implemented at significant cost and with substantial side effects for the animals being treated. An alternative method could utilise aptamers in a diagnostic test that could be performed farmside at a reduced cost, and studying this possibility in greater detail formed the core of this research project. Aptamers are single-stranded oligonucleotide sequences developed using systematic evolution of ligands via exponential enrichment (SELEX), an iterative process of selection acting upon an oligonucleotide library against a target of choice. Their properties enable them to form unique secondary structure conformations that bind the specific target molecule they are generated to. As the GGT family has four major isozymes with significant structural variation, it was necessary to develop an enzyme-linked immunosorbent assay (ELISA) using GGT isozyme-specific antibodies to identify the isozyme that is elevated during facial eczema infection. In this study, these ELISAs were developed and used to test a wide range of serum samples collected from sheep that were known to be positive or negative for facial eczema. It was determined that GGT1 was the best candidate isozyme for a facial eczema biomarker. Due to time constraints, the target molecule used for SELEX was a combination of a crude GGT preparation, and serum that was positive for facial eczema. A counter-SELEX step using serum that was negative for facial eczema was successful in removing substantial oligonucleotide sequences that exhibited non-specific binding. The enriched oligonucleotide library was then sequenced producing 21 unique aptamer candidates. In summary, this study identified the GGT isozyme most likely to be an accurate biomarker of facial eczema, and generated 21 potential aptamer candidates that may be used in future studies to develop a novel, cheap and easy on-farm detection test for facial eczema.

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[34] Competitive enzyme-linked immunosorbent assay for biotin

Cited by 10 publications

References 6 publications

A sensitive and practical competitive radioassay for plasma biotin

A sensitive and practical competitive radioassay for plasma biotin

Biotin-Protein Ratios and Stability of Biotinylated Immunoglobulins as Standards for the Quantitation of Biotin-Binding Immunoglobulins

Biomarker identification and development of aptamers for the diagnosis of sub-clinical facial eczema

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