DOCSLIB.ORG

Ultrasensitive Enzymatic Radioimmunoassay: Application to Detection of Cholera Toxin and Rotavirus

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Ultrasensitive Enzymatic Radioimmunoassay: Application to Detection of Cholera Toxin and Rotavirus Proc. Natl. Acad. Sci. USA Vol. 76, No. 10, pp. 5336-5339, October 1979 Medical Sciences Ultrasensitive enzymatic radioimmunoassay: Application to detection of cholera toxin and rotavirus (enzyme-linked immunosorbent assay/enteritis/adenosine 5'-monophosphate) CURTIS C. HARRIS*, ROBERT H. YOLKENt, HANS KROKAN1, AND IH CHANG HSu* *Human Tissue Studies Section, Laboratory of Experimental Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205; tDepartment.of Pediatrics, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205; and *University of Tromso, Institute of Medical Biology, Tromso, Norway Communicated by Gerald N. Wogan, July 30, 1979 ABSTRACT Rotavirus and enterotoxin-producing bacteria MATERIALS AND METHODS are major causes of diarrheal disease in humans. A method of rapid diagnosis, ultrasensitive enzymatic radioimmunoassay, Reagents. [3H]AMP (generally 3H-labeled, 15 Ci/mmol, has been developed to quantitatively detect cholera toxin and New England Nuclear; 1 Ci = 3.7 X 10O° becquerels) was pu- rotavirus. The method uses features of both enzyme-linked rified by column chromatography with DEAE-Sephadex (A-25; immunosorbent assay and radioimmunoassay; however, the Pharmacia) in a stepwise manner as follows: (i) 2 mCi of sensitivity of the assay is 100- to 1000-fold more sensitive than [3H]AMP in 0.2 ml of distilled water was applied to a disposable the two parent assays. Ultrasensitive enzymatic radioimmu- column (Isolab, Akron, OH) containing 2 ml of DEAE-Sepha- noassay should also be useful in measuring other biologically dex; (ii) the contaminating [3H]adenosine was eluted with important agents such as drugs and hormones. distilled water and discarded; (iii) [3H]AMP was eluted with 0.2 M ammonium carbonate; and (iv) after lyophilization to In recent years, the capability to detect small quantities of bi- remove the ammonium carbonate, the [3H]AMP was dissolved ologically important molecules has been greatly expanded by in 50% (vol/vol) ethanol. The column-purified [3H]AMP con- the development of radioimmunoassays (RIA) (1, 2). These tained less than 0.2% [3H]adenosine and was stable at -20°C assays have been especially useful for the detection of circu- for at least 3 months. lating substances, such as hormones, drugs, and infectious Alkaline phosphatase-conjugated goat IgG was prepared as agents. However, RIA have certain practical limitations. The described by Engvall and Perlman (8). Burro anti-cholera toxin short half-life of the isotopes used limits the shelf life of the was obtained from John Robbins (Bureau of Biologics, Bethesda, reagents. In addition, the need for y-emitting isotopes subjects MD). Guinea pig anti-cholera toxin was prepared as reported the users of RIA to a radiation hazard. Enzyme immunoassays (6). (also known as enzyme-linked immunosorbent assays or ELISA) The antisera to rotavirus (9) were obtained from Anthony have been developed in an attempt to overcome those problems Kalica and Albert Kapikian (Laboratory of Infectious Diseases, (3-5). ELISA is similar in design to solid-phase RIA except that National Institute of Allergy and Infectious Diseases, Bethesda, an enzyme is used as the immunoglobulin marker instead of a MD). Cholera toxin was obtained from Schwarz/Mann. The 'y-emitting isotope. This enzyme-antibody conjugate is bound rotavirus specimen tested was a 2% filtrate prepared from the to the solid phase by a series of antibody-antigen reactions and stool of a genobiotic calf experimentally infected with human essentially converts the substrate to products with a visible rotavirus (10). Adenosine and p-nitrophenylphosphate were yellow color, which can be measured spectrophotometrically. purchased from Sigma. The fact that a single molecule of enzyme is capable of reacting USERIA. The optimal dilutions of reagents were determined with a large number of substrate molecules provides for am- by checkerboard titration (5). The method for measuring plification and, thus, a high degree of sensitivity (3, 6). Because cholera toxin is, briefly, as follows. (i) Polyvinyl, round-bot- stable enzymes such as alkaline phosphatase [orthophospho- tomed microtiter plates (Cooke, Alexandria, VA) were coated ric-monoester phosphohydrolase (alkaline optimum), EC with a 0. 1-mIn aliquot of burro anti-cholera toxin diluted 3.1.3.1] can be used, ELISA also has the advantage of using 1:10,000 in 60 mM carbonate buffer (pH 9.8) for 14 hr at 4°C. reagents with a long shelf life. (ii) The wells of the plates were washed five times in phos- In practice, however, the sensitivity of ELISA systems has phate-buffered saline (pH 7.4) (Pi/NaCl) containing 0.5 ml of not significantly exceeded that of RIA (7). In order to combine Tween 20 per liter (Pi/NaCl/Tween). (iii) A 0.1-ml aliquot of the advantages of both RIA and ELISA, we developed a prac- either diluted cholera toxin or solvent (negative control) was tical ultrasensitive enzymatic radioimmunoassay (USERIA) for added in duplicate to the wells. The negative controls consisted the detection of antigens such as human rotavinus (an important of five filtrates from strains of Escherichia coli that do not cause of infantile gastroenteritis) and cholera toxin. These assay produce any detectable toxin. (iv) The plates were incubated systems are a 1000-fold more sensitive than both RIA and for 14 hr at 40C. (v) After another washing procedure as in step ELISA for the detection of these agents. ii, a 0.l-ml aliquot of guinea pig anti-cholera toxin diluted 1: 4000 in Pi/NaCl/Tween containing 1% normal horse serum The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviations: Pi/NaCl, phosphate-buffered saline; RIA, radioim- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate munoassays; ELISA, enzyme-linked immunosorbent assay(s); USERIA, this fact. ultrasensitive enzymatic radioimmunoassay. 5336 Downloaded by guest on September 24, 2021 Medical Sciences: Harris et al. Proc. Natl. Acad. Sci. USA 76 (1979) 5337 Q, c c -o0) 'D(D o x 0) CD 4CC.t 0 am C, CU W 4-cm 0n am .CCU 7 E 7) I/ 10-21 1o-20 10-'9 10o-l 10-17 Alkaline phosphatase, mol FIG. 2. Determination of the sensitivity of the assay. Various dilutions of alkaline phosphatase (10-17-10-21 mole) were included xI in 0.1-ml reaction volumes of 10 mM diethanolamine buffer (pH 9) containing 100 pmol of [3H]AMP as substrate. The products were isolated by DEAE-Sephadex column chromatography. The activity is expressed as adenosine (dpm) released in 90 min. - 1/K, sine formed in the absence of the enzyme. The enzyme-specific 10 activity was determined by subtracting this value from that 1 /substrate, mM-' obtained from the test specimen. In addition, five filtrates from FIG. 1. (A) Percent of AMP converted by alkaline phospha- non-cholera toxin-producing organisms were run in each test. tase-IgG conjugate to adenosine. Alkaline phosphatase conjugate (2 A test well was considered positive for cholera toxin if it yielded X 10-8 was to mg/mI) added 10 mM diethanolamine buffer (pH 9.8) an enzyme that was 2 standard containing various concentrations of AMP. The reaction mixtures specific activity deviations greater mean value. The assays were were incubated at 370C for 30 min. The [3Hladenosine released was than the toxin-free control determined by DEAE-Sephadex column chromatography. (B) Dou- run in duplicate; the variation between duplicates was less than ble-reciprocal plot of the Michalis-Menten equation of the alkaline 5%. phosphatase assay in A. The USERIA for rotavirus was performed in a manner sim- ilar to that described above except that goat anti-rotavirus serum (GIBCO) was added and the plates were incubated for 1 hr at was substituted for burro anti-cholera toxin, guinea pig anti- 370C. [Normal horse serum was added to the diluent both to saturate unbound sites in the well and to minimize the cross- reactivity with the burro serum bound to the well (6).] (vi) After another washing procedure as in step ii, 0.1-ml aliquots of al- 6 - 5 kaline phosphatase-conjugated goat anti-guinea pig IgG were x added to each well. (vii) After another washing procedure (five loo00-- / times with Pi/NaCI/Tween and twice with 100 mM dietha- .> 4 nolamine buffer, pH 9.8), 0.1-ml aliquots of [3H]AMP (1 X 106 CU 00 cpm; 0.1 nmol) diluted in 0.01 M diethanolamine (pH 9.0) were Xo 3( added to each well. 0. After 10, 100, or 1000 min, the [3H]adenosine produced by the reaction of the alkaline phosphatase-IgG conjugate with Do was :La, the [3H]AMP substrate separated by applying a 0.05-ml - aliquot of the reaction mixture to a column packed with 1.5 ml of DEAE-Sephadex A-25. The [3H]adenosine was then eluted, with a total of 4.5 ml of 10 mM diethanolamine buffer (pH 9.8) Ir 6 12 1T8 24 30 36 42 48 containing 0.1 mM adenosine, in two fractions. Twenty milli- Time, hr liters of Aquasol (New England Nuclear) was added to each fraction, and the [3H]adenosine was measured in a liquid FIG. 3. Time course of alkaline phosphatase-IgG conjugate re- scintillation counter. As an alkaline phosphatase negative action. Triplicates of the assay mixture containing 100 pmol of control, 0.1-ml aliquots of [3H]AMP were incubated at 370C [3HJAMP and 10-8 mg of enzyme conjugate in 0.1 ml of 10 mM buffer (pH 9.0) were incubated at 370C for 0-48 hr. Aliquots of 100 Ml of the without the enzyme same as conjugate for the length of time reaction mixtures were withdrawn for determination of enzyme ac- the test specimens, and 0.05 ml was passed through the DEAE tivity at 0.5, 1.5, 4.5, 24, and 48 hr.
Load more

Recommended publications
  • 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 .
    [Show full text]
  • 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).
    [Show full text]
  • 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.
    [Show full text]
  • 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).
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • I M M U N O L O G Y Core Notes
    II MM MM UU NN OO LL OO GG YY CCOORREE NNOOTTEESS MEDICAL IMMUNOLOGY 544 FALL 2011 Dr. George A. Gutman SCHOOL OF MEDICINE UNIVERSITY OF CALIFORNIA, IRVINE (Copyright) 2011 Regents of the University of California TABLE OF CONTENTS CHAPTER 1 INTRODUCTION...................................................................................... 3 CHAPTER 2 ANTIGEN/ANTIBODY INTERACTIONS ..............................................9 CHAPTER 3 ANTIBODY STRUCTURE I..................................................................17 CHAPTER 4 ANTIBODY STRUCTURE II.................................................................23 CHAPTER 5 COMPLEMENT...................................................................................... 33 CHAPTER 6 ANTIBODY GENETICS, ISOTYPES, ALLOTYPES, IDIOTYPES.....45 CHAPTER 7 CELLULAR BASIS OF ANTIBODY DIVERSITY: CLONAL SELECTION..................................................................53 CHAPTER 8 GENETIC BASIS OF ANTIBODY DIVERSITY...................................61 CHAPTER 9 IMMUNOGLOBULIN BIOSYNTHESIS ...............................................69 CHAPTER 10 BLOOD GROUPS: ABO AND Rh .........................................................77 CHAPTER 11 CELL-MEDIATED IMMUNITY AND MHC ........................................83 CHAPTER 12 CELL INTERACTIONS IN CELL MEDIATED IMMUNITY ..............91 CHAPTER 13 T-CELL/B-CELL COOPERATION IN HUMORAL IMMUNITY......105 CHAPTER 14 CELL SURFACE MARKERS OF T-CELLS, B-CELLS AND MACROPHAGES...............................................................111
    [Show full text]
  • Immunological Relationships Among Hexosaminidases of Different Species
    Immunochemistry, 1976, Vol. 13, pp. 485490. Pergamon Press. Printed in Great Britam IMMUNOLOGICAL RELATIONSHIPS AMONG HEXOSAMINIDASES OF DIFFERENT SPECIES BENJAMIN GEIGER, YOAV BEN-YOSEPH and RUTH ARNON Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot, Israel (First received 10 November 1975; in reaised.form 8 December 1975) Abstract-The immunological inter-relationships between hexosaminidases* of several mammals were established. Qualitative immunochemical analysis indicated similarity between the enzymes derived from man, Rhesus monkey, Iris maccaque, bovine, pig, goat and guinea-pig. Quantitation of the extent of antigenic similarity among the various species, using antigen binding assay and radioimmunoassay, correlated well with the phylogenetic relationships between the species investigated. The evolutionary distance between the species used as sources of enzyme and the immunized animal affects the capacity to distinguish between closely related enzymes. Thus, the use of closer animals for immunization enables the detection of subtle differences between the tested enzymes, whereas a phylogenetically remote animal recognizes better their similarity. INTRODUCTION cleaves the terminal P-linked N-acetyl glucosamine or In the course of evolutionary process, many changes N-acetyl galactosamine. Purification and characteri- and variations occur in the primary sequence of both zation of hexosaminidases from different animals, in- structural and biologically active proteins, which are cluding rat (Sellinger et aI., 1973; Robinson et nl., manifested in variations among isofunctional homolo- 1967) bovine (Robinson et a1.,1972; Frohwein & Gatt, gous proteins. Several approaches have been pro- 1967; Hayase et al., 1973), sheep (Winchester, 1971), posed for the investigation of such phylogenetic inter- pig (Findlay & Levvy, 1960), mouse (Hunter & Mil- relationships, including the application of immuno- son, 1966) and man (Robinson & Stirling, 1968; Sand- chemical methods.
    [Show full text]
  • Comparison of Immunofluorescence and Radioimmunoassay for Detecting Igm Antibody in Infants with the Congenital Rubella Syndrome
    J. Hyg., Camb. (1979), 83, 413 413 Printed in Great Britain Comparison of immunofluorescence and radioimmunoassay for detecting IgM antibody in infants with the congenital rubella syndrome BY J. E. CRADOCK-WATSON, MARGARET K. S. RIDEHALGH Public Health Laboratory, Withington Hospital, Manchester M20 SLR J. R. PATTISON, MARY J. ANDERSON Department of Medical Microbiology, King's College Hospital Medical School, London SE5 8RX AND H. O. KANGRO Department of Virology, The London Hospital Medical College, Turner Street, London El 2AD {Received 5 April 1979) SUMMARY Immunofluorescence (IF) and radioimmunoassay (RIA) have been compared as methods for detecting IgM antibody in 124 infants with confirmed or suspected congenital rubella. IF was used to test sucrose density gradient fractions and RIA to test fractions and whole serum. When fractions were tested IF and RIA were equally specific and distinguished clearly between IgM and IgG, but RIA was the more sensitive method. The RIA titre in whole serum was always greater than in the peak IgM fraction and there was no evidence that testing the serum, rather than the fraction, could result in failure to detect IgM. With some sera RIA gave low titres which became negative after absorption with IgG-coated latex beads. The mechanism of this 'false positive' effect, which may have been due to IgM with anti-IgG activity, was not investigated, but if it can be removed by absorption it need not reduce the specificity of the test. During the first 6 months of life IgM antibody was detected by RIA in 30 out of 32 unfractionated sera and by IF in fractions from 28 of these.
    [Show full text]
  • Radioimmunoassay Quality Control and Troubleshooting
    Radioimmunoassay Quality Control and Troubleshooting Helen H. Drew 111e Johns Hopkins Hospital, Baltimore, Maryland This is the third continuing education article in a four-part series tal errors define the overall random error associated with a on radioimmunoassay. After reading and studying this article, the single measurement. Precision studies assess random errors reader should be able to: I) describe the procedures used for immu­ and tell us how closely the same measurement can be repeated noassay quality control; 2) state acceptable limits of results; and within the same assay (intra-assay precision) or between assays 3) discuss troubleshooting approaches. at different times (interassay precision). Therefore, the goal of a quality control program defines acceptable error limits Quality control (QC) is a vital function of every laboratory without compromising the medical significance ofthe results. performing immunoassays and, when applied in a knowledge­ able and sensible fashion, it can be a laboratory's most valuable QUALITY CONTROL STATISTICS tool. A properly designed QC program ensures that results are within acceptable limits of accuracy and precision. In A QC program uses the fact that repeated measurements simpler terms, a quality control program helps you answer of a single sample fluctuate over time because of random er­ the following important questions about your assay: Are to­ rors or imprecision. It must be determined whether these day's results reliable? May the same assay be performed tomor­ fluctuations are within acceptable limits or reflect an analytical row using the same reagents with the same reliable results? error requiring troubleshooting action. The distribution of In addition to answering these simple questions, quality con­ these errors is assumed to be Gaussian, a curve of normal trol will help you unravel the mystery of why when your assay distribution.
    [Show full text]
  • Immunosorbent Assay, Solid-Phase Radioimmunoassay, and Indirect Immunofluorescence for Detection of Human Rotavirus Antigen in Faeces
    J Clin Pathol: first published as 10.1136/jcp.32.7.700 on 1 July 1979. Downloaded from Journal of Clinical Pathology, 1979, 32, 700-705 Comparison of electron microscopy, enzyme-linked immunosorbent assay, solid-phase radioimmunoassay, and indirect immunofluorescence for detection of human rotavirus antigen in faeces CHRISTOPHER J. BIRCH, NOREEN I. LEHMANN, ALISON J. HAWKER, JOHN A. MARSHALL, AND IAN D. GUST From the Virology Department, Fairfield Hospital for Communicable Diseases, Yarra Bend Road, Fairfield 3078, Victoria, Australia SUMMARY Four techniques were compared for their practicability, speed, and sensitivity for the detection of human rotavirus. Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were found to be the most sensitive means of identifying rotavirus, and, once processed, up to 40 specimens could be examined daily. Electron microscopy, although less sensitive than these techniques, had the advantage of being able to detect other viral agents present in faecal extracts. Indirect immunofluorescence failed to detect rotavirus as often as the other three methods. In laboratories where routine examination of faecal specimens from patients with gastroenteritis is required, ELISA and RIA are useful alternatives to electron microscopy. Rotaviruses are recognised as important causative and Johnsson, 1976; Birch et al., 1977), indirect http://jcp.bmj.com/ agents of acute gastroenteritis in children and the immunofluorescence (11F) (Bryden et al., 1976), free young of various other species (Flewett and Woode, viral immunofluorescence (Yolken et al., 1977b), a 1978), and there is a special need for a sensitive and viral RNA detection technique (Espejo et al., 1977), rapid method for their identification in faeces.
    [Show full text]