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A homogeneous assay for biotin based on chemiluminescence energy transfer
Authors:E J Williams  A K Campbell
Affiliation:1. Department of Medical Biochemistry, University Hospital of Wales, Heath Park, Cardiff, CF4 4XW, United Kingdom;1. University of Wales College of Medicine, Heath Park, Cardiff, CF4 4XW, United Kingdom;1. Lipoprotein Metabolism Section, National Heart, Lung and Blood Institute, Bethesda, MD, USA;2. Section of Inflammation and Cardiometabolic Diseases, National Heart, Lung and Blood Institute, Bethesda, MD, USA;3. Bioinformatics and Computational Biology Core Facility, National Heart, Lung and Blood Institute, Bethesda, MD, USA;4. Advanced Cardiovascular Imaging Laboratory, National Heart, Lung and Blood Institute, Bethesda, MD, USA;1. Department of Cardiology, The Second Affiliated Hospital of Harbin Medical University, The Key Laboratory of Myocardial Ischemia, Chinese Ministry of Education, Harbin, China;2. Department of Cardiology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China;1. Diagnostics Development Unit, Department of Haematology, University of Cambridge, Science Village, Chesterford Research Park, Little Chesterford CB10 1XL, UK;2. National Microbiology Reference Laboratory, Southerton, Harare ST749, Zimbabwe;3. Kenya Medical Research Institute/US CDC Research and Public Health Collaboration (KEMRI-CDC), Kisumu, Kenya;4. Kiev Regional AIDS Center, Kyiv, Ukraine;5. Diagnostics for the Real World Ltd., 840 Del Rey Avenue, Sunnyvale 94085, CA, USA;6. Department of Genitourinary Medicine and HIV, Guy''s and St Thomas’ NHS Foundation Trust, London SE1 9RT, UK;7. Diagnostics for the Real World (Europe) Ltd., Science Village, Chesterford Research Park, Little Chesterford CB10 1XL, UK;8. Division of AIDS, Henry M. Jackson Foundation for Advancement of Military Medicine, National Institutes of Health, Fishers Lane, Rockville 5601, MD, USA;1. Department of Clinical Laboratory Medicine, Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo, Japan;2. Research Fellow of the Japan Society for the Promotion of Science, Tokyo, Japan;3. Pathology Laboratory, Shinraku-en Hospital, Niigata, Niigata, Japan;4. Center for Genomic and Regenerative Medicine, Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo, Japan;5. Research Institute for Diseases of Old Age, Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo, Japan;6. The Sulphuric Acid Association of Japan, Minato-ku, Tokyo, Japan;1. Department of Cardiology, The Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou, People''s Republic of China;2. Department of Cardiology, The First Affiliated Hospital of Anhui Medical University, Hefei, People''s Republic of China;3. Department of Biochemistry and Physiology, Faculty of Health and Medical Sciences, School of Biosciences and Medicine, University of Surrey, Guildford, UK
Abstract:Chemiluminescence energy transfer between aminobutylethylisoluminol (ABEI)-biotin and fluorescein-avidin was investigated in order to establish a homogeneous assay for serum biotin in the physiological range. ABEI chemiluminescence was measured at pH 7.4 using microperoxidase-hydrogen peroxide and the chemiluminescence at two wavelengths (460 and 525 nm) measured simultaneously to quantify chemiluminescence energy transfer. ABEI-biotin was synthesized by a mixed anhydride reaction and purified by TLC and HPLC. Binding of ABEI-biotin to fluorescein-avidin resulted in a quenching of the chemiluminescence. Chemiluminescence energy transfer was demonstrated by a 2.5-fold decrease in the ratio of blue (460 nm) to green (525 nm) light emission compared with unbound ABEI-biotin. This energy transfer was used to establish an assay for biotin in the range 1 to 10 nM by relating the concentration of biotin to the ratio of chemiluminescence monitored at 460 and 525 nm simultaneously. The assay was capable of detecting biotin in reference sera and in patients with malabsorption syndromes and chronic alcoholism. The reference range in normal subjects was 1.2 to 4.3 nmol/liter mean +/- SD = 2.41 +/- 0.91 nmol/liter (n = 20). The quenching of the chemiluminescence of ABEI-biotin when bound to fluorescein-avidin appeared to be the result of a direct interaction between the excited state product of ABEI and fluorescein.
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