Chapter-Xii Immunochemical Techniques
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An Enzyme-Linked Immunosorbent Assay (ELISA) for Pantothenate
Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 5-1981 An Enzyme-Linked Immunosorbent Assay (ELISA) for Pantothenate Allen H. Smith Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Biochemistry Commons Recommended Citation Smith, Allen H., "An Enzyme-Linked Immunosorbent Assay (ELISA) for Pantothenate" (1981). All Graduate Theses and Dissertations. 5295. https://digitalcommons.usu.edu/etd/5295 This Thesis is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. AN ENZYME- LINKED ThltviUNOSORBE!'IT ASSAY (ELISA) FOR PANTOTHENATE by Allen H. Smith A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Biochemistry UTAH STATE UNIVERSITY Logan, Utah 1981 ii ACKNOWLEDGEMENTS To Dr. R. G. Hansen, to Dr. B. W. Wyse, to Carl Wittwer, to Jack Brown, to Jan Pearson, to Nedra Christensen, to all those who have made this experience one of tremendous growth, I express my thanks. I express appreciation to the United States Department of Agriculture, under Grant #5901-0410-9-0288-0 with Utah State University, for financial support. Finally, I express thanks to my parents, who have come to realize that graduate school is also a part of life. Allen H. Smith "iii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ii LIST OF TABLES . vi LIST OF FIGURES. vii · ABSTRACT .. ix INTRODUCTION 1 REVIEW OF LITERATURE 4 Pantothenate Assays . -
Test Directory
800.541.7891 509.755.8600 Test Directory Fax 509.921.7107 Billing Code Test Code [sunquest] (1,3)-BETA-D-GLUCAN (FUNGITELL) 13BGA 13BGA Synonyms Fungitell; Glucan Container Type Red top tube (plain) Store and Transport Refrigerated Specimen Type Serum Preferred Volume 2 mL Minimum Volume 0.5 mL Specimen Processing Separate serum from cells within 2 hours of collection and transfer to a standard PAML aliquot tube Room Temp Unacceptable Refrigerated 2 weeks Frozen (-20°C) 2 weeks Unacceptable Condition Hemolyzed, lipemic and icteric samples Reference Laboratory ARUP Reference Lab Test Code 2002434 CPT Codes 87449 Test Schedule Mon-Fri Turnaround Time 2-4 days Method Semi-Quantitative Colorimetry Test Includes (1,3-beta-D-glucan, pg/mL; (1,3-beta D-glucan Interpretation. Notes Reference ranges for pediatric patients (less than 18 years old) have not been established. Assay ranges were validated in adult subjects. Supply Item Number 1372 Billing Code Test Code [sunquest] 1, 5 ANHYDROGLUCITOL (GLYCOMARK) GLYMAR GLYMAR Synonyms Anhydroglucitol, 1,5 AD; GlycoMark® Container Type Serum separator tube (gold, brick, SST, or corvac) Store and Transport Refrigerated Specimen Type Serum Preferred Volume 1 mL Minimum Volume 0.2 mL Specimen Processing Allow serum specimen to clot completely at room temperature. Separate serum or plasma from cells and transfer to a standard PAML aliquot tube Room Temp 1 week Refrigerated 1 week Frozen (-20°C) 1 month Alternate Specimens Lavender (EDTA) Reference Laboratory ARUP Reference Lab Test Code 0081335 CPT Codes 84378 Test Schedule Mon-Fri Turnaround Time 2-4 days Method Quantitative Enzymatic Test Includes GlycoMark, ug/mL. -
Detection of Virus-Specific Immunoglobulins Using a Doubly Labeled Fluorescein- 125I Antibody A
JOURNAL OF CLINICAL MICROBIOLOGY, June 1976, p. 637-639 Vol. 3, No. 6 Copyright © 1976 American Society for Microbiology Printed in U.S.A. Detection of Virus-Specific Immunoglobulins Using a Doubly Labeled Fluorescein- 125I Antibody A. J. PARKINSON* AND J. KALMAKOFF Department ofMicrobiology, University of Otago, Dunedin, New Zealand Received for publication 17 February 1976 Commercially prepared fluorescein-labeled antihuman antibodies were la- beled with 125I and used to compare specific herpes simplex virus antibody titers as determined by indirect fluorescent antibody and radioimmunoassay tech- niques. Total virus-specific immunoglobulin and virus-specific immunoglobulin G titers did not vary by more than one twofold dilution when compared by the two methods. Efforts are being made to develop a reliable calf serum, penicillin (100 U/ml) streptomycin radioimmunoassay (RIA) for the detection of (100 ,ug/ml), and 0.1% bicarbonate, were in- virus-specific immunoglobulins, acceptable for fected with the isolated virus. Uninoculated use in diagnostic serology (1, 2, 5-8). The estab- monolayers were maintained as controls. When lishment of a satisfactory RIA depends on the infected monolayers showed 75% cytopathic use of antibody with both a high avidity and effect, both inoculated and uninoculated cells selectivity for the material to be assayed (4). were dispersed, using 0.015% ethylenediamine- Consequently, a major obstacle to using RIA tetraacetic acid. Both cellular suspensions were routinely is the necessity of preparing specific standardized to contain 2.5 x 105 cells/ml in high-titer antibody against human immuno- phosphate-buffered saline. Using 0.025-ml vol- globulins (IgG, IgM, and IgA). -
Anti-Insulin Antibodies in Von Freien Und Totalen Anti-Insulin Dans Le Sérum Humain Human Serum Antikörpern in Humanserum
Février 2019 – Modèle 014 Cisbio Bioassays Parc Marcel Boiteux – BP 84175 – 30200 Codolet / France - Tél. 33 (0)4.66.79.67.00 ANTI-INSULIN AAI ANTIBODIES Trousse pour le dosage radioimmunologique Kit for radioimmunoassay for determination Kit zur radioimmunologischen Bestimmung des anticorps anti-insuline libres et totaux of free and total anti-insulin antibodies in von freien und totalen Anti-Insulin dans le sérum humain human serum Antikörpern in Humanserum La trousse contient : Kit content : Inhalt des Kits : Traceur ≤ 52 kBq 2 x 5 mL Tracer ≤ 52 kBq 2 x 5 mL Tracer ≤ 52 kBq 2 x 5 mL Solution précipitante 1 x 100 mL Precipitating solution 1 x 100 mL Präzipitationslösung 1 x 100 mL Réactif d’extraction 1 x 10 mL Extraction reagent 1 x 10 mL Extraktionsreagenz 1 x 10 mL Tampon de neutralisation 1 x 10 mL Neutralization buffer 1 x 10 mL Neutralisationspuffer 1 x 10 mL Contrôle négatif (C1) 1 x qsp 1 mL Negative control 1 x qs 1 mL Negativkontrolle 1 x qs 1 mL Contrôle positif (C2) 1 x qsp 1 mL Positive control 1 x qs 1 mL Positivkontrolle 1 x qs 1 mL Mode d’emploi 1 Instruction for use 1 Gebrauchsinformation 1 Attention : Certains réactifs contiennent de l’azoture de sodium Warning : Some reagents contain sodium azide Achtung : Einige Reagenzien enthalten Natriumazid Kit per il dosaggio radioimmunologico degli Equipo radioinmunológica para la Δοκιμασία για τον ραδιοανοσολογικό anticorpi anti-insulina liberi e totali nel siero determinación de los anticuerpos anti- προσδιορισμό των ελεύθερων και συνολικών umano insulina libres y totales en suero humano αντισωμάτων έναντι της ινσουλίνης στον ανθρώπινο ορό . -
Making Antibodies Work
MILESTONES conjugated to the enzyme alkaline MILESTONE 4 phosphatase. They named their assay the ‘enzyme-linked immunosorbent assay’, which resulted in the catchy Making antibodies acronym ‘ELISA’. In addition to its application in the detection and work quantification of serum components, ELISAs are routinely used to detect viral infections, such as infection with human immunodeficiency virus, and the technique remains a mainstay of laboratories around the world. In addition to detecting the tagging of antibodies to molecules or cells of interest, it was clearly desira- ble to be able to separate the tagged components. This was achieved in Crossed immunoelectropho- 1979 by David Parks, Virginia Bryan, resis—just one analytical application of antibodies— Vernon Oi and Leonard Herzenberg, can simultaneously identify who used the newly invented dozens of serum proteins. Courtesy T.C.Bøg-Hansen. fluorescence-activated cell sorter. The light-scattering and fluorescent prop- erties of the cells enabled cells bound with antigen-coupled microspheres to be distinguished and directed Because antibodies are able to between the binding of antibodies into alternative collection pots, thus specifically bind target molecules, to endogenous insulin versus their facilitating phenotypic separation, the possibility of their having an binding to radioactive insulin. This monoclonal description and categorization. analytical application was recognized radioimmunoassay was used to antibodies… Since the pioneering work of early on. Robin Coombs, Arthur measure insulin present in the blood César Milstein and Georges J. F. Mourant and Robert Race, working and provided greater sensitivity than have had Köhler (MILESTONE 9) there have for the UK’s Medical Research that of previous approaches. -
BLOOD TYPE) Methodology: Tube Agglutination BBK Set Up: Daily, As Ordered ABORH BLOOD TYPE 6.0 Ml Whole Blood (Pink) ABORH Report Available: Same Day
LAB OE TEST REFERENCE SPECIMEN ORDER ORDER PROCEDURE RANGE REQUIREMENTS MNEMONIC NAME ABORH GROUP (BLOOD TYPE) Methodology: Tube agglutination BBK Set up: Daily, as ordered ABORH BLOOD TYPE 6.0 mL whole blood (Pink) ABORH Report available: Same day CPT Code: 86900, 86901 ACA or ACLA - See Anti-Cardiolipin Antibodies ACE - see Angiotensin-1 Converting Enzyme ACETAMINOPHEN, SERUM Methodology: Immunoassay 1 mL blood (Gn -Li (PST)) Set up: Daily, as ordered or LAB ACETAMINOPHEN Accompanies report Report available: Same day 1 mL serum (SS) ACET Minimum: 0.5 mL CPT Code: 80329 ACETYLCHOLINE RECEPTOR 1.0 mL serum (SS) BINDING ANTIBODIES (QUEST 206) Minimum: 0.5 mL ACETYLCHOLINE BINDING Methodology: RIA LAB Accompanies report RECEP Set up: Tues-Sat Allow serum to clot at room ACETYL BIND Report available: 1-2 days temperature. Serum should be separated from cells within 1 CPT Code: 83519 hour of collection. ACETYLCHOLINE RECEPTOR BLOCKING ANTIBODIES (QUEST 34459) 1.0 mL serum (SS) centrifuge ACETYLCHOLINE Methodology: RIA with 1 hr of collection LAB Accompanies report BLOCKING RECEP Set up:Mon, Wed, Fri ACETYL BLO Report available: Next day Minimum:0.5 mL CPT Code: 83519 ACETYLCHOLINE RECEPTORMODULATING ANTIBODY (QUEST 26474) 1 mL serum (SS) ACETYLCHOL LAB Methodology: RIA Accompanies report MODULATING RECEP ACETYL MOD Set up: Tue,Thur,Sun Minimum: 0.5 mL Report available: 5 days CPT Code: 83519 ACETYLCHOLINESTERASE, QUALITATIVE, GEL ELECTROPHORESIS (QUEST 185314) This test is automatically performed on all 1.5 mL Amniotic fluid, ROOM Alpha-Fetroprotein -
Plasma and Cellular Retinoid-Binding Proteins and Transthyretin
Proc. Nati. Acad. Sci. USA Vol. 82, pp. 2488-2492, April 1985 Medical Sciences Plasma and cellular retinoid-binding proteins and transthyretin (prealbumin) are all localized in the islets of Langerhans in the rat (retlnoids/vitamin A/immunohistochemistry/radioimmunoassay) MICHIMASA KATO*t, KUNIYO KATO*t, WILLIAM S. BLANER*, BRUCE S. CHERTOWt, AND DEWITT S. GOODMAN*§ *Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, NY 10032; and tDepartment of Medicine, Marshall University School of Medicine and Veterans Administration Medical Center, Huntington, WV 25701 Communicated by Seymour Lieperman, December 6, 1984 ABSTRACT The immunohistochemical localization of roles that CRBP and CRABP play within cells have not been plasmia retinol-binding protein' (RBP), cellular retinol-binding established, it has been suggested that these binding proteins protein (CRBP), and transtiyretin (TTR) was studied in rat may be involved in the biological expression of retinoid pancreas. The studies employed antibodies purified by im- activity within cells (see ref. 6 for a recent review). munosorbent affinity chromatography, permitting the specific We have' recently reported studies on the immunohis- staining and localization of each antigen by the, unlabeled tochemical localization of RBP, TTR, and CRBP in rat liver peroxidase-antiperoxidase method. Specific immunostaining and kidney (7). Each' of these proteins was found to be for each of these three proteins was found localized to the islets localized in specific cells within each organ. Highly specific ofLangerhans. Both RBPand CRBP were localized in cells that localization of CRBP has also been observed in the testis and were, peripherally distributed within the islets,'with an ana- epididymis (8, 9). -
Radioimmunoassay for Human Alphar-Fetoprotein (Cancer/Diagnosis/Hepatoma/Hepatitis/Embryonie) HULBERT K
Proc. Nat. Acad. Sci. USA Vol. 70, No. 2, pp. 526-530, February 1973 Radioimmunoassay for Human Alphar-Fetoprotein (cancer/diagnosis/hepatoma/hepatitis/embryonie) HULBERT K. B. SILVER, PHIL GOLD, STEPHEN FEDER, SAMUEL 0. FREEDMAN, AND JOSEPH SHUSTER The Division of Clinical Immunology and Allergy and the McGill University Medical Clinic of the Montreal General Hospital, Montreal, 109, Quebec, Canada Communicated by Wilder Penfield, November 21, 1972 ABSTRACT A method of radioimmunoassay has been tion of goats with human fetal serum emulsified in Freund's developed for the quantitation of alpha,-fetoprotein in complete adjuvant. In order to render the antiserum mono- human serum. The assay requires 20 ;d of serum, can be completed in 8 hr, and can reproducibly detect concen- specific for AFP, it was absorbed with serum, from a normal trations of 20 ng of alpha1-fetoprotein per ml of serum. adult male, that had been lyophilized and dialyzed against Hence, the method is about 500-fold more sensitive for water, at a concentration of 200 mg of lyophilized serum the detection of alpha1-fetoprotein than the Ouchterlony per ml of antiserum. After absorption, the antiserum was technique currently in general use. The procedure is of potential clinical value as an aid in the diagnosis of can- centrifuged at 40,000 X g for 20 min. This preparation was cer and a number of noncancerous hepatic diseases. denoted as anti-AFP antiserum. In 1963, Abelev reported that some chemically-induced mouse Purification of Human AFP was accomplished by the tech- hepatomfts synthesized an alphal-globulin that was absent nique of Nishi (10), as modified by Silver et al. -
KPNW Specimen Requirements ***Please Hit 'Ctrl'+'F' to Open the 'Find on This Page' Funtion
Page 1 of 368 KPNW Specimen Requirements ***Please hit 'Ctrl'+'F' to open the 'Find on this page' funtion. Panel Name Panel Description Preferred Collection/Volume: RED 10 No Gel Barrier Tubes Volume: 1.0 mL Serum Alternative Collection: GRN Li Hep or LAV EDTA Volume: 1.0 mL Plasma TAT: Report available: 5 Days Test Schedule: Saturday Morning Method: Liquid Chromatography/Tandem Mass Spectrometry Test Facility: Quest Diagnostics Nichols Inst San Juan Capistrano 33608 Ortega Highway San Juan Capistrano, CA 92690-6130 Clinical Data: 11-Deoxycortisol (Compound S) is useful in diagnosing patients with 11-beta- hydroxylase deficiency (second leading cause of congenital adrenal hyperplasia) and 11-Deoxycortisol primary (adrenal failure) or secondary (hypothalmic-pituitary ACTH deficiency) adrenal insufficiency. Patient Preparation: An early morning specimen is preferred Specimen Stability: Room Temperature: 4 days Refrigerated: 4 days Frozen: 4 weeks Ship serum frozen or refrigerated Test Code: CHANTILLY T.C.30543 TO SJC TC 30543X Processing: Centrifuge and aliquot into Referred Tests aliquot tube. Note if serum or plasma on specimen. Freeze solid. Transport: Transport frozen on ice. Preferred Collection/Volume: Preserve 24-hour urine with 25 mL of 50% Acetic Acid (preferred) or 30 mL 6N HCl or 1 gram Boric Acid/100 mL (acceptable) during collection. Refrigerate during collection. TAT: Next Day Test Schedule: Monday-Friday Night Method: Colorimetric Test Facility: Quest Diagnostics Nichols Institute 14225 Newbrook Drive Chantilly, VA 20153 17 Ketosteroids Total Pediatric 24 Hr Urine Labeling: Please specify on the request form and on the urine container the patient's age, sex, and (Includes Creatinine the total 24-hour urine volume Ratio) Specimen Stability: Room Temperature: 8 hours Refrigerated: 7 days Frozen: 30 days (-20c) Please specify on the request form and on the urine container the patient's age, sex, and the total 24-hour urine volume. -
Immunological Techniques Radioimmunoassay (RIA)
Regular Office Hours: Tuesdays 11-12 Extra office hours: Wed, Feb 7 12-1pm Thurs, Feb 8 11am-12 Midterm Fri, Feb 9 2-4pm I WILL NOT BE HOLDING OFFICE HOURS ON TUESDAY Feb 13!! Extra Office Hours Dina, Tim, and I encourage all confused students to come to our office hours and discussion sections so we can try to help un-confuse you. Take No class on Tuesday Feb 13. First midterm: Thurs Feb 15 at 6pm in 155 Dwinelle (not 2050 VLSB as listed in the original schedule). Midterm will focus on material covered in lectures and will be designed to be taken in 90 min. (We have the room till 8pm.) The GSIs will conduct a review session in our regular class period on Thursday Feb 15. The extraordinary specificity of Immunological Techniques antibodies for their antigens, and the ability to generate polyclonal and monoclonal antibodies to virtually anything, makes them fantastically Monoclonal Antibodies useful reagents for detecting and quantitating substances. Radioimmune Assay (RIA) A large number of different assays have been developed to detect antigens based Enyzme Linked Immune Sorbant Assay (ELISA) on antibody binding that can be used in in fluids, tissues, or cells. Western blot Immunoprecipitation Immunofluorescence Flow cytometry Expression cloning Radioimmunoassay (RIA) •1960 Yalow and Berson (Nobel Prize) •Very sensitive: can detect material present at concentrations of <0.001 micrograms/ml. •Takes advantage of protein binding to Cpm plastic of tissue culture dish. •Generate standard curve with known RIA to detect amounts of unlabeled antigen •Measure unknown using standard curve. hepatitis Labeled antigen detects hepatitis virus in 1 microliter Amount unlabeled Antigen Unlabeled of blood. -
1 Detection of Antibodies Directed to the N-Terminal Region of GAD Is
Page 1 of 30 Diabetes Detection of antibodies directed to the N-terminal region of GAD is dependent on assay format and contributes to differences in the specificity of GAD autoantibody assays for type 1 diabetes Short running title: Improved specificity of autoantibodies to N-terminally truncated GAD Alistair JK Williams1, Vito Lampasona2, Michael Schlosser3, Patricia W Mueller4, David L Pittman5, William E Winter5, Beena Akolkar6, Rebecca Wyatt1, Cristina Brigatti2, Stephanie Krause7, Peter Achenbach7 and Participating Laboratories 1. School of Clinical Sciences, University of Bristol, Bristol, UK 2. Division of Genetics and Cell Biology & Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, Milan, Italy 3. Department of Medical Biochemistry and Molecular Biology and Institute of Pathophysiology, University Medical Center of Greifswald, Karlsburg, Germany 4. Molecular Risk Assessment Laboratory, Centers for Disease Control and Prevention, Atlanta, GA, USA 5. Department of Pathology, University of Florida, Gainesville, FL, USA 6. Division of Diabetes, Endocrinology, and Metabolic Diseases, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, USA 7. Institute of Diabetes Research, Helmholtz Zentrum München, and Forschergruppe Diabetes, Klinikum rechts der Isar, Technische Universität München, Neuherberg, Germany Correspondence to – Alistair Williams Diabetes and Metabolism, School of Clinical Sciences Learning and Research Building, Southmead Hospital Bristol BS10 5NB Fax: +44 117 323 5336 Tel: +44 117 414 7900 [email protected] Word count: 2836 Figures: 4 Supplemental figures: 3 Supplemental table: 1 Supplemental appendix: 1 1 Diabetes Publish Ahead of Print, published online May 13, 2015 Diabetes Page 2 of 30 ABSTRACT Autoantibodies to glutamate decarboxylase (GADA) are sensitive markers of islet autoimmunity and type 1 diabetes. -
Radioimmunoassay in Developing Countries: General Principles
XA9847613 Chapter 16 RADIOIMMUNOASSAY IN DEVELOPING COUNTRIES (General principles) R.D. Piyasena Radioimmunoassay (RIA) is probably the most commonly performed nuclear medicine technique. It is an in vitro procedure, where no radioactivity is administered to the patient. But this alone is not the reason for its widespread use. It provides the basis for extremely sensitive and specific diagnostic tests, and its use in present day medicine has brought a virtual information explosion in terms of understanding the pathophysiology of many diseases. The fact that the technology involved is within the technical and economic capabilities of the developing world is evident from the increasing demand for its introduction or expansion of existing services. RIA facilities need not be restricted to urban hospitals, as in the case of in vivo nuclear medicine techniques, but may be extended to smaller district hospitals and other laboratories in peripheral areas. It is also possible to send blood samples to a central laboratory so that a single centre can serve a wide geographical area. There are many laboratories in the industrialized world that receive a major proportion of samples for assay by mail. In recent years, substantial RIA services have been established in many of the developing countries in Asia and Latin America. The International Atomic Energy Agency (IAEA) and World Health Organisations (WHO) have made vital contributions to these activities and have played a catalytic role in assisting member states to achieve realistic goals. In the past five years, more than 250 individual RIA laboratories in developing member states have been beneficiaries of IAEA projects.