ANP RIA. Radioimmunoassay for the quantitative determination of Human Atrial Natriuretic Peptide in plasma.

Transcription

1 Instructions for Use ANP RIA Radioimmunoassay for the quantitative determination of Human Atrial Natriuretic Peptide in plasma. ED For illustrative purposes only. To perform the assay the instructions for use provided with the kit have to be used. Distributed by: I B L I N T E R N A T I O N A L G M B H Flughafenstrasse 52a Phone: +49 (0) IBL@IBLInternational.com D22335 Hamburg, Germany Fax: +49 (0)

2 TABLE OF CONTENTS PAGE: 1. Intended use Clinical application Principle of the test Precautions Specimen collection Materials and equipment required Quality Control Shelflife and Storage Contents of the kit Preparation of reagents Performance... 7 Extraction procedure... 7 Assay procedure Calculation of results Example of sample data Assay characteristics Literature

3 1. INTENDED USE The EuroDiagnostica ANP kit contains reagents and instructions for the quantitative measurement in plasma samples. After extraction with Seppak C18, ANP concentrations are measured by radioimmunoassay. 2. CLINICAL APPLICATION Mammalian atria contain specific granules, which resemble secretory granules in endocrine cells (1,2). DeBold et al. (3) showed that the number of these atrial granules was inversely correlated to the volume of the extracellular fluid. From atrial extracts, peptides have been isolated, sequenced, and synthesized (410). In human atria, three peptides seem to be important: a 28amino acids component (alfa ANP), and a biosynthetic precursor (gammaanp, 126 amino acids). Biological activity was highest for alfaanp (11). In vitro and animal experiments have shown that alfaanp may possesse important biological actions: it has diuretic and natriuretic activities, relaxes smooth muscle, its secretion is stimulated by volume loading, and it opposes the aldosterone secretion (reviewed in 1217). These effects are thought to be mediated by alfaanp circulating in peripheral blood. Measurements of this peptide in human plasma (1830) could be of value in determining the role of ANP in the endocrine system and in various pathophysiological states in man (3134). This kit decribes a simple, sensitive and reliable radioimmunoassay for ANP in human plasma. 3. PRINCIPLE OF THE TEST The method is based on a radioimmunoassay that is specific for hanp. Quantification of ANP is achieved using a nonequilibrium radioimmunoassay with delayed addition of the iodinated tracer. The bound and free hormone are separated by the second antibody/polyethylene glycol system. Centrifugation separates the bound and free hormone fractions. The precipitate, which contains the bound fraction, is counted. The final concentration of ANP is calculated and expressed in picograms/ml plasma. (pg/ml). 3

4 4. PRECAUTIONS 1. Materials derived from human blood and used in the preparation of this kit were tested and found negative for hepatitis B surface antigen (HBsAg) and for HIV. However, handle all components as a possible source of infection. 2. The radioactive material included may be received, acquired, possessed and used only by physicians, clinical laboratories or hospitals for invitro clinical or laboratory tests not involving internal or external administration of the material, or the radiation therefrom, to human beings or animals. Its receipt, acquisition, possession, use and transfer are subject to the regulation of each country. Adherence to the basic rules of radiation safety should provide adequate protection. Do not eat, drink, smoke or apply cosmetics where radioactive materials are used. Do not pipette radioactive solutions by mouth. Avoid direct contact with all radioactive materials by using protective articles such as lab coats and disposable gloves. All radiological work should be done in a designated area. Radioactive materials should be stored in original containers in a designated area. Laboratory equipment and glassware which are subject to contamination should be segregated to prevent crosscontamination of different radioisotopes. Any radioactive spills should be taken care of immediately in accordance with established procedures. All radioactive materials must be disposed of in accordance with the prevailing regulations and guidelines of the agencies jurisdiction over the laboratory. 5. SPECIMEN COLLECTION EDTAplasma is recommended to perform the human ANP radioimmunoassay. Collect blood by venipuncture on crushed ice. EDTA is used as anticoagulant. Centrifuge within one hour at 4 C for 15 min. at 1500 g. Add 200 KIU of aprotinin/ml (Trasylol, Bayer) plasma and store in polystyrene tubes at 20 C or lower. 6. MATERIALS AND EQUIPMENT REQUIRED Disposable polystyrene tubes; 12 x 75 mm. Gammacounter. Vortex mixer. Pipetting devices to deliver 100, 200 and 1000 µl. Centrifuge 1500 g Seppak C18 cartridge Acetic Acid 96% to be diluted to 4% Ethanol 86%, ethanol 100% Methanol 100% Device for use in the extraction/purfication procedure to provide vacuum. Glass container of 100 ml. 4

5 7. QUALITY CONTROL Controls should be carried out in each assay run. A reference control is included in the kit, for score, AFTER EXTRACTION, refer to the QC sheet. Use controls as recommended by the control plasma manufacturer and in accordance with reference laboratories' practice to monitor the accuracy and precision of reagents and techniques. 8. SHELFLIFE AND STORAGE This kit is stable until the stated expiry date if stored as specified. Upon receipt of the kit, all reagents should be stored at 28 C. 9. CONTENTS OF THE KIT Item Nr. of Vials Containing Assay buffer M Borate + 0.2% BSA, ph 8.4 Antihuman ANP serum (sheep) HumanANP 125 I tracer 1 lyophilized antiserum in 0.2 M Borate + 0.2% BSA 1 lyophilized 125 IANP in 0.2 M Borate + 0.2% BSA Precipitating reagent 1 lyophilized horse antisheep antiserum Reference control 1 lyophilized ANP reference control Human ANP stock standard 1 lyophilized ANP human standard 5

6 10. PREPARATION OF REAGENTS The kit contains reagents sufficient for 100 tubes. NOTE: prepare reagents after extraction procedure has been completed. (reconstitute 30 minutes before use) 1. Assay buffer (zero standard) Reconstitute the buffer with 30 ml distilled or deionized water. Allow the buffer to stand for 20 min. on ice for complete reconstitution. The buffer is used for preparing the standard curve (one to one serial dilution), zero standard and nonspecific binding. After extraction and evaporation the samples are dissolved in this buffer. After use, store the assay buffer at 20 C. 2. Antihuman ANP antiserum Reconstitute the content of the vial with 2 distilled water at 4 C. Allow the antibody to stand for 20 min. for complete reconstitution. Mix gently, do not shake. After use, store at 20 C or lower. The antibody is raised in sheep against human ANP. 3. Human ANP 125 I tracer Reconstitute with 2 distilled water at 4 C. Allow the tracer to stand for 20 minutes for complete reconstitution. Mix gently, do not shake. After use, store at 20 C. The tracer contains 37 kbq of radioactivity. 4. Precipitating reagent Reconstitute the horse antisheep reagent with 40 ml distilled water at room temperature. Allow to stand for 20 minutes. Transfer this reconstituted reagent in a 150 ml container and add an additional 65 ml distilled water. Mix gently. After use, store at 20 C. 5. Reference control Reconstitute with 1.2 ml distilled water. For exact values, refer to quality control sheet. 6. Human ANP stock standard Reconstitute the standard with 1.0 ml distilled water. Allow to stand at 4 C for 20 minutes for complete reconstitution. This stock standard contains 100 pg/ (A). Make serial dilutions in polystyrene tubes on ice. A scheme for preparation of standard solution is shown next. 6

7 11. PERFORMANCE Do not extract standards! A. Extraction procedure for samples and ref. control 1. Extraction on Seppak C18 cartridge. Subsequently pretreat with: 5 ml 4% acetic acid (96%) in 86% ethanol 5 ml methanol 5 ml distilled water 5 ml 4% acetic acid (96%) 2. Acidify 0.5 ml plasma with 1.5 ml 4% acetic acid and apply to the cartridge. 3. Wash twice with 3 ml distilled water. 4. Elute with 3 ml acetic acid in 86% ethanol. 5. Evaporate under a stream of nitrogen during 1 hour at 37 C. Add of ethanol 100%, mix and evaporate to dryness. 6. Dissolve residue in 250 µl assay buffer. 7. Introduce samples into the radioimmunoassay procedure. Each laboratory should establish the extraction recovery under its own experimental conditions. B. Preparation of standard solutions Dilution Conc. ANP Stock Standard A 100 pg/ 250 µl stock solution A µl assay buffer 250 µl stock solution B µl assay buffer 250 µl stock solution C µl assay buffer 250 µl stock solution D µl assay buffer 250 µl stock solution E µl assay buffer 250 µl stock solution F µl assay buffer 250 µl stock solution G µl assay buffer Standard B 50 pg/ Standard C 25 pg/ Standard D 12.5 pg/ Standard E 6.25 pg/ Standard F 3.12 pg/ Standard G 1.56 pg/ Standard H 0.78 pg/ After use, store the stock standard A at 20 C 7

8 C. Assay procedure 1. Pipette of ANP standard solutions and of plasma extracts and ref. control extracts into labelled test tubes. 2. Add sheep anti ANP antibody (except NSB and TCtubes). Correct NSB volume with assay buffer. 3. Vortex and incubate all tubes at 4 C for 18 hours. 4. Add of ANP [ 125 I] Tracer to all tubes. 5. Vortex all tubes and incubate overnight at 4 C. 6. Add precipitating reagent to all tubes, except TC tubes. 7. Vortex, and incubate for 30 min. at roomtemperature. 8. Centrifuge 30 min at 2000 g, at roomtemperature. 9. Decant (or aspirate) supernatant immediately. 10. Count residue for 1 min. 8

9 Flow Chart Concentration in pg/ Assay buffer Standard or Sample Antiserum Tracer Prec. reagent Total counts NSB Standard 0 Standard 0.78 Standard 1.56 Standard 3.12 Standard 6.25 Standard 12.5 Standard 25 Standard 50 Standard µl Vortex and incubate 18 hrs at 4 C Vortex and incubate 18 hrs at 4 C Incubate 30 min. at R.T. Centrifuge 30 min. at R.T. at 2000 g. Aspirate or decant supernatant Unknown 9

10 12. CALCULATION OF RESULTS Substract the average count rate (cpm) of the NSB from the average countrate (cpm) of the replicates of standards, ref. controls and patient samples. A standardcurve can be generated by plotting cpm or % B/Bo of preciptated bound fraction against the concentration of the ANP calibrators. To obtain the ANP concentration in patient samples and ref. controls, their cpm or % B/Bo of precipitated bound fractions are interpolated now from the generated standardcurve. The calibration curve can also be constructed by computer methods. For automated data reduction, both logit/log or Spline methods can be used. Note: ANP concentration in samples read from standard curve have to be multiplied by 5 due to the dilution factor with the extraction procedure. Correct all patient sample results by % recovery: ANP pg/ x 5 x 100 = pg/ml % recovery 13. EXAMPLE OF SAMPLE DATA cpm correcte d cpm % B/Bo results pg/100 µl Total counts NSB 435 Standard 0 Standard 0.78 Standard 1.56 Standard 3.12 Standard 6.25 Standard 12.5 Standard 25 Standard 50 Standard 100 pg/ pg/ pg/ pg/ pg/ pg/ pg/ pg/ pg/ Unknown

11 14. ASSAY CHARACTERISTICS Specificity The following fragments were checked for crossreactivity: Fragment human αanp [128] human αanp [528] human αanp [728] human αanp [1328] human αanp [1828] human αanp [111] rat αanp [128] αmsh ArgVasopressin ACTH [139) % cross reactivity <0.2 <0.2 < <0.2 <0.2 <0.2 Recovery Mean recovery after addition of different amounts of ANP to of plasma is 90%. Linearity Different samples were diluted with zero calibrator Sample no. Undiluted (pg/ml) 1/2 (pg/ml) 1/4 (pg/ml)

12 Precision Withinrun variation The withinrun variation was calculated from 7 replicate determinations. n mean pg/ml SD % c.v. Betweenrun variation The betweenrun variation was calculated from 6 independent assay runs. n mean pg/ml SD % c.v. Sample Samp le Sensitivity The sensitivity of the assay, judged as 3 Sd change from zero calibrator is 3.5 pg/ml. Expected values Each laboratory should establish its own normal range. The normal range as determined at the department of Experimental and Chemical Endocrinology of the University of Nijmegen, The Netherlands, is 968 pg/ml, with a mean value of 26.0 ± 15.5 pg/ml (n = 25). 12

13 15. LITERATURE 1. Kirsch B Exp. Med. Surg. 14: Jamieson JD; Palade GE, J.Cell. Biol. 23: DeBold AJ Proc. Soc. Exp.B iol. Med.1 61: Flynn TG; debold ML; debold AJ Biochem. Biophys. Res. Comm. 117:3: Seidah NG; Lazure C; Chretien M. et al Proc. Natl. Acad. Sci. USA. 81:9: Misono KS; Fukumi H; Grammar RT; Inagami T Biochem. Biophys. Res. Comm. 119:2: Currie MG; Geller DM; Cole BR. et al Science 223: DM; Currie MG; Wakitani K; et al Biochem.Biophys. Res. Comm. 120:2: Misono KS; Grammar RT; Fukumi H; Inigami T Biochem. Biophys. Res. Comm. 123:2: Kangawa K; Matsuo H Biochem. Biophys. Res. Comm. 118:1: Kangawa K; Fukuda A; Matsuo H nature. 313: Cantin M; Genest J Endocr. Rev. 6:2: Seymour AA Clin. Exper. Theory. Pract. A7: Ballerman BJ; Brenner BM J. Clin. Invest. 76:6: Ackerman U Clin. Chem. 32:2: Ballermann BJ; Brenner BM Circul. Res. 58:5: Atlas S; Laragh H Ann. Rev. Med. 37: Larose P; Meloche S; du Souich P; de Laen A; Ong H Biochem. Biophys. Res. Comm. 130:2: Sugawara A; Nakao K; Morii N. et al Biochem. Biophys. Res. Comm. 129:2: Gutkowska J; Bourassa M; Roy D. et al Biochem. Biophys. Res. Comm. 128:3: Hartter E; Woloszczuk W; Stummvoll H Clin. Chem. 32:3: Yamaji T; Ishibashi M; Takaku F; J. Clin. Invest. 76:4: Arendt RM; Stangl E; Zahringer J; Liebisch DC; Herz A FEBS. 198:1: Naruse M; Naruse K; Obana K. et al Peptides. 7:1: Marumo F; Sakamoto H; Ando K; Ishigami T; Kawakami M Biochem. Biophys. Res. Comm. 137:1: Bated ER; Shenker Y; Grekin RJ Circul. 73:6: Sugawara A; Nakao K; Morii N. et al Hypertension. 8:4:2: Weidmann P; Hellmueller B; UUehlinger D, et al J. Clin. Endocr. Metab. 62:5: Tan ACITL; Rosmalen FMA; Theelen BGA; Kloppenborg PWC; Benraad HB; Benraad TJ Annals of Clin. Biochem. in press. 30. Rosmalen FMA. et al Clin. Chem. Acta. in press. 31. Tan ACITL; Rosmalen FMA; Hofman JA; Kloppenborg PWC; Benraad TJ World Contress Biol. Act. Atrial Pept. New York. 32. Rosmalen FMA et al XIII Int. Congr. of Clin. Chem. VII European Congr. of Clin. Chem. The Hague. 33. Steegers EAP. et al World Congress Biol. Act. Atrial Pept. New York. 34. Visser W; vd Dorpel MA; Derkx FHM; Wallenburg HCS; Schalenkamp ADH Third European Meeting on Hypertensbion. Milan July 1995/L1

14 Symbols / Symbole / Symbôles / Símbolos / Símbolos / Σύµβολα REF LOT CONC LYO IVD Cat.No.: / Kat.Nr.: / No. Cat.: / Cat.No.: / N.º Cat.: / N. Cat.: / ΑριθµόςΚατ.: LotNo.: / ChargenBez.: / No. Lot: / LotNo.: / Lote N.º: / Lotto n.: / Αριθµός Παραγωγή: Use by: / Verwendbar bis: / Utiliser à: / Usado por: / Usar até: / Da utilizzare entro: / Χρησιµοποιείται από: No. of Tests: / Kitgröße: / Nb. de Tests: / No. de Determ.: / N.º de Testes: / Quantità dei tests: / Αριθµός εξετάσεων: Concentrate / Konzentrat / Concentré / Concentrar / Concentrado / Concentrato / Συµπύκνωµα Lyophilized / Lyophilisat / Lyophilisé / Liofilizado / Liofilizado / Liofilizzato / Λυοφιλιασµένο In Vitro Diagnostic Medical Device. / InvitroDiagnostikum. / Appareil Médical pour Diagnostics In Vitro. / Dispositivo Médico para Diagnóstico In Vitro. / Equipamento Médico de Diagnóstico In Vitro. / Dispositivo Medico Diagnostico In vitro. / Ιατρική συσκευή για InVitro ιάγνωση. Evaluation kit. / Nur für Leistungsbewertungszwecke. / Kit pour évaluation. / Juego de Reactivos para Evaluació. / Kit de avaliação. / Kit di evaluazione. / Κιτ Αξιολόγησης. Read instructions before use. / Arbeitsanleitung lesen. / Lire la fiche technique avant emploi. / Lea las instrucciones antes de usar. / Ler as instruções antes de usar. / Leggere le istruzioni prima dell uso. / ιαβάστε τις οδηγίες πριν την χρήση. Keep away from heat or direct sun light. / Vor Hitze und direkter Sonneneinstrahlung schützen. / Garder à l abri de la chaleur et de toute exposition lumineuse. / Manténgase alejado del calor o la luz solar directa. / Manter longe do calor ou luz solar directa. / Non esporre ai raggi solari. / Να φυλάσσεται µακριά από θερµότητα και άµεση επαφή µε το φως του ηλίου. Store at: / Lagern bei: / Stocker à: / Almacene a: / Armazenar a: / Conservare a: / Αποθήκευση στους: Manufacturer: / Hersteller: / Fabricant: / Productor: / Fabricante: / Fabbricante: / Παραγωγός: Caution! / Vorsicht! / Attention! / Precaución! / Cuidado! / Attenzione! / Προσοχή! Symbols of the kit components see MATERIALS SUPPLIED. Die Symbole der Komponenten sind im Kapitel KOMPONENTEN DES KITS beschrieben. Voir MATERIEL FOURNI pour les symbôles des composants du kit. Símbolos de los componentes del juego de reactivos, vea MATERIALES SUMINISTRADOS. Para símbolos dos componentes do kit ver MATERIAIS FORNECIDOS. Per i simboli dei componenti del kit si veda COMPONENTI DEL KIT. Για τα σύµβολα των συστατικών του κιτ συµβουλευτείτε το ΠΑΡΕΧΟΜΕΝΑ ΥΛΙΚΑ. IBL AFFILIATES WORLDWIDE IBL International GmbH Flughafenstr. 52A, D22335 Hamburg, Germany IBL Deventer B.V. Zutphenseweg 55, NL7418 AH Deventer, The Netherlands IBL Transatlantic Corp. 288 Wildcat Road, Toronto, Ontario M3J 2N5 Tel.: + 49 (0) Fax: 11 IBL@IBLInternational.com WEB: Tel.: Fax: IBL@IBLInternational.com WEB: Toll free: +1 (866) Tel.: +1 (416) Fax: IBL@IBLTransatlantic.com WEB: LIABILITY: Complaints will only be accepted in written and if all details of the test performance and results are included (complaint form available from IBL or supplier). Any modification of the test procedure or exchange or mixing of components of different lots could negatively affect the results. These cases invalidate any claim for replacement. Regardless, in the event of any claim, the manufacturer s liability is not to exceed the value of the test kit. Any damage caused to the kit during transportation is not subject to the liability of the manufacturer. Symbols Version 3.5 /