NT-proCNP 2nd generation
(EN) ENZYME IMMUNOASSAY FOR THE QUANTITATIVE DETERMINATION OF NT-proCNP IN SERUM, EDTA PLASMA, CITRATE PLASMA OR HEPARIN PLASMA CAT. NO. BI-20812 . 12 X 8 TESTS (DE)
ENZYM IMMUNOASSAY ZUR QUANTITATIVEN BESTIMMUNG VON NT-proCNP IN HUMAN SERUM, EDTA PLASMA, CITRAT PLASMA ODER HEPARIN PLASMA KAT. NR. BI-20812 . 12 X 8 TESTS
FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC PROCEDURES
For the measurement of NT-proCNP in human urine, cell culture supernatants, and non-human samples please visit our homepage www.bmgrp.com (see Validation Data).
rev.no. 171213 This kit was developed and manufactured by: Biomedica Medizinprodukte GmbH & Co KG, A-1210 Wien, Divischgasse 4 Tel. +43/1/291 07 45, Fax +43/1/291 07 85, E-mail
[email protected] 1/16
CONTENT / INHALT
ENGLISH DEUTSCH
Page 3 Seite 7
Detailed information on the assay characteristics including the validation data can be found on our website. Detaillierte Informationen zu den Testmerkmalen einschließlich der Validierungsdaten finden Sie auf unserer Website.
www.bmgrp.com
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1) INTRODUCTION
C–type natriuretic peptide (CNP) is a paracrine growth factor widely expressed in tissues, including the vascular
endothelium, where it is considered to provide vasoprotective functions. In endothelial cells and macrophages it is secreted in response to several stimuli, including inflammatory mediators. CNP is rapidly degraded in tissues and negligible quantities enter the circulation. However, the N-terminal portion of the pro-hormone is not degraded at source and circulates in significantly higher concentrations than CNP. Therefore NT-proCNP is a valuable biomarker to determine CNP synthesis in tissues. CNP plays a critical role in linear growth. It is produced in the growth plate and signals through a paracrine mechanism. Recent studies have shown that the plasma concentrations of NTproCNP correlate with linear growth velocity in all phases of skeletal growth and increase during rhGH therapy (1). Furthermore, serum NT-proCNP levels increased after initiation of GH treatment in patients with achondroplasia/ hypochondroplasia (2). Women with pregnancy complications, such as diminished fetal growth and pre-eclampsia show significantly increased NT-proCNP levels early in gestation (3, 4). NT-proCNP concentration at hospital admission has sufficient sensitivity and specificity to differentiate naturally occurring sepsis from non-septic systemic inflammatory response syndrome (SIRS) (5, 6). Recently, Prickett and colleages demonstrated in a cohort of over 2000 individuals, that in contrast to the close association of NT-proBNP with cardiac function, and predictive value for outcome after myocardial infarction, plasma NT-proCNP is highly correlated with renal function and is an independent predictor of mortality and cardiac readmission in individuals with unstable angina (7).
Areas of Interest Vascular diseasse Growth Skeletal development
Angiogenesis Sepsis
2) CONTENT OF THE KIT CONT PLATE WASHBUF ASYBUF STD CTRL CONJ SUB STOP
KIT COMPONENTS Polyclonal sheep anti NT-proCNP antibody precoated microtiter strips in stripholder packed in aluminium bag with desiccant Wash buffer concentrate 20x, natural cap Assay buffer, red cap, ready to use Standards, (0; 4; 8; 16; 32; 64; 128 pmol/l), white caps, lyophilised Controls A + B, yellow caps, lyophilised, exact concentrations see labels Conjugate (sheep anti NT-proCNP-HRPO), amber cap, ready to use Substrate (TMB solution), blue cap, ready to use STOP solution, white cap, ready to use
QUANTITY 12 x 8 tests 1 x 50 ml 1 x 8 ml 7 vials 2 vials 1 x 7 ml 1 x 13 ml 1 x 7 ml
3) ADDITIONAL MATERIAL ADDED TO THE KIT 1 self-adhesive plastic film QC protocol Protocol sheet Instruction manual 4) EQUIPMENT REQUIRED BUT NOT SUPPLIED Precision pipettes calibrated to deliver 20 µl, 50 µl, 100 µl, and 300 µl and disposable tips ELISA reader for absorbance at 450 nm (reference 630 nm) Graph paper or software for calculation of results Distilled or deionised water 5) REAGENTS AND SAMPLE PREPARATION
A ll reagents of the kit are stable at +4°C (2-8°C) until expiry date stated on the label of each reagent.
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Sample preparation: Collect venous blood samples by using standardized blood collection tubes. Perform serum/plasma separation by centrifugation according to supplier’s instructions of the blood collection devices as soon as possible. The acquired plasma or serum samples should be measured as soon as possible. For longer storage aliquot samples and store at -25°C or lower. All samples should undergo only 4 freeze-thaw cycles. Lipemic or haemolysed samples may give erroneous results. Samples should be mixed well before assaying. We recommend duplicates for all values. Samples with values above highest STD can be diluted 1+1 or 1+3 with ASYBUF (Assay buffer). For further information on sample stability please visit our website www.bmgrp.com (see Validation Data) or contact our customer service by e-mail
[email protected] or by phone +43/ 1/ 29107-45. Reconstitute as follows: WASHBUF (Wash buffer): Dilute the concentrate 1:20 (1+19), e.g. 50 ml WASHBUF + 950 ml distilled water. Crystals in the buffer concentrate will dissolve at room temperature. Undiluted WASHBUF is stable at +4°C (2-8°C) until expiry date stated on label. Diluted WASHBUF is stable at +4°C (2-8°C) for one month. Use only diluted WASHBUF to perform the assay. STD (Standards) + CTRL (Controls): Pipette 300 µl of distilled or deionised water into each vial. Leave at room temperature (18-26°C) for 15 min. Vortex gently. The exact concentration is printed on the label. Reconstituted STDs and CTRLs are stable at -25°C or lower until expiry date stated on the label. STDs and CTRLs are stable for 3 freeze-thaw cycles. 6) PRINCIPLE OF THE ASSAY
T his kit is a sandwich enzyme immunoassay for the determination of NT-proCNP in human samples. In a first step, assay buffer and sample are pipetted into the wells of the microtiter strips, which are pre-coated with polyclonal sheep anti NT-proCNP antibody, for a short incubation. Without the need of a washing step, conjugate (sheep anti human NT-proCNP-HRPO) is added into the wells. NT-proCNP present in the sample binds to the precoated antibody in the well and forms a sandwich with the detection antibody. In the washing step all non-specific unbound material is removed. After washing the substrate (TMB Tetramethylbenzidine) is pipetted into the wells. The enzyme catalysed colour change of the substrate is directly proportional to the amount of NT-proCNP present in the sample. This colour change is detectable with a standard microtiter plate ELISA reader.
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7) ASSAY PROTOCOL All reagents and samples have to be brought to room temperature (18-26°C) before they can be used in the assay. Mark position for STD/SAMPLE/CTRL (Standard/Sample/Control) on the protocol sheet. Take microtiter strips out of the aluminium bag. Unused strips can be stored with desiccant in the aluminium bag at +4°C (2-8°C) until the expiry date. 1. 2. 3. 4. 5. 6.
Pipette 50 µl ASYBUF (Assay buffer, red cap) into each well. Add 20 µl STD/CTRL/SAMPLE (Standard/Control/Sample) in duplicate into respective wells, swirl gently. Cover tightly and incubate for 20 minutes at room temperature (18-26°C). Add 50 µl CONJ (Conjugate, amber cap) into each well, swirl gently. Cover tightly and incubate for 3 hours at room temperature (18-26°C) in the dark. Aspirate and wash wells 5x with 300 µl diluted WASHBUF (Wash buffer), remove remaining WASHBUF by hitting plate against paper towel after the final washing step. 7. Add 100 µl SUB (Substrate, blue cap) into each well, swirl gently. 8. Incubate for 30 min at room temperature (18-26°C) in the dark. 9. Add 50 µl STOP (Stop solution, white cap) into each well, swirl gently. 10. Measure absorbance immediately at 450 nm with reference 630 nm, if available. For the measurement of NT-proCNP in human urine, cell culture supernatants and non-human samples please visit our website www.bmgrp.com (see Validation Data). 8) CALCULATION OF RESULTS
R ead the optical density (OD) of all wells on a plate reader using 450 nm wavelength (correction wavelength 630 nm). Construct the standard curve from the OD values of the STD. Use commercially available software or graph paper. Obtain sample concentration from this standard curve. The assay was evaluated with 4PL algorithm. Different curve fitting methods need to be evaluated by the user. Respective dilution factors have to be considered. Typical STD-curve:
The quality control (QC) protocol supplied with the kit shows the results of the final release QC for each kit at production date. Data for OD obtained by customers may differ due to various influences and/or due to the normal decrease of signal intensity during shelf life. However, this does not affect validity of results as long as an OD of 1.50 or more is obtained for the STD with the highest concentration and the value of the CTRL is in range (target range see label).
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9) ASSAY CHARACTERISTICS Method:
Sandwich ELISA, HRP/TMB, 12x8-well strips
Sample volume:
Serum, EDTA plasma, heparin plasma, and citrate plasma Protocols available for urine, cell culture supernatant and non-human species 0 to 128 pmol/l (7 standards and 2 controls in a human serum matrix) Standard points: 0 / 4 / 8 / 16 / 32 / 64 / 128 pmol/l 1 pg/ml = 0.201 pmol/l (MW: 4.985 kD) 1 pmol/l = 4.985 pg/ml 20 µl / well
Incubation time, temperature:
20 min / 3 h / 30 min, room temperature
Sensitivity: Specificity: Precision:
LOD: (0 pmol/l + 3 SD): 0.7 pmol/l; LLOQ: 0.5 pmol/l This assay recognizes endogenous and synthetic human NT-proCNP. Intra-assay (n=5) ≤ 6%, Inter-assay (n=8) ≤ 7%
Spike/Recovery (average recovery spiked with 64 pmol/l synthetic NT-proCNP):
Serum (n=6): 101%
Heparin plasma (n=2): 93%
EDTA plasma (n=6): 99%
Citrate plasma (n=2): 100%
Sample type: Standard range: Conversion factor:
Average % expected of dilution: Serum (n=6) Dilution linearity of EDTA plasma (n=6) endogenous NT-proCNP: Heparin plasma (n=2) Citrate plasma (n=2) Median serum (n=32) = 14.5 pmol/l Median EDTA plasma (n=33) = 15 pmol/l Values from apparently healthy Median heparin plasma (n=18) = 13.5 pmol/l Median citrate plasma (n=18) = 12 pmol/l individuals:
1+1 99 103 100 96
1+3 98 98 100 92
Each laboratory should establish its own reference range for the samples under investigation. Do not change sample type during the study. For further information on assay characteristics and antibody specificity please visit our website www.bmgrp.com (see Validation Data) or contact our customer service by e-mail
[email protected] or by phone +43/ 1/ 29107-45. 10) PRECISION Intra-assay: 2 samples were tested 5 times within 1 assay lot by 1 operator. Inter-assay: 2 samples were tested 8 times in 2 different kit lots by 2 different operators. Intra-assay (n=5) Mean (pmol/l) SD (pmol/l) CV (%)
Sample 1 7.9 0.47 6
Sample 2 65.3 1.25 2
Inter-assay (n=8) Mean (pmol/l) SD (pmol/l) CV (%)
Sample 1 8.2 0.54 7
Sample 2 64.1 1.42 2
11) TECHNICAL HINTS Do not mix or substitute reagents with those from other lots or sources. Do not mix stoppers and caps from different reagents or use reagents between lots. Do not use reagents beyond expiration date. Protect reagents from direct sunlight. Substrate solution should remain colorless until added to the plate. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Avoid foaming when mixing reagents.
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12) PRECAUTIONS
All test components of human source were tested against HIV-Ab, HCV-Ab and HBsAg; and were found negative. Nevertheless, they should be handled and disposed as if they were infectious. Liquid reagents contain ≤0.1% Proclin 300 as preservative. Proclin 300 is not toxic in concentrations used in this kit. It may cause allergic skin reactions – avoid contact with skin or eyes. Do not pipette by mouth. Do not eat, drink, smoke or apply cosmetics where reagents are used. Avoid all contact with the reagents by using gloves. The stop solution contains sulfuric acid, contact can lead to irritations of eyes and skin. Flush with water after contact! 13) LITERATURE 1. 2. 3. 4. 5. 6. 7. 8.
Dynamic response of C-type natriuretic peptide and its aminoterminal propeptide (NTproCNP) to growth hormone treatment in children with short stature. Olney RC et al., Clin Endocrinol, 2016; 85(4):561-568. Serum NT-proCNP levels increased after initiation of GH treatment in patients with achondroplasia/hypochondroplasia. Kubota T et al., Clin Endocrinol (Oxf), 2016; 84(6):845-850. C-type natriuretic peptide in complicated pregnancy: increased secretion precedes adverse events. Reid RA et al., J Clin Endocrinol Metab, 2014; 99(4):1470-1478. Effects of pre-eclampsia and fetal growth restriction on C-type natriuretic peptide. Espiner, E A et al., BJOG, 2015; 122:1236-1243. Prognostic value of circulating amino-terminal pro-C-type natriuretic peptide in critically ill patients. Koch et al., Critical Care, 2011; 15:R45. The prognostic value of concomitant assessment of NT-proCNP, C-reactive protein, procalcitonin and inflammatory cytokines in septic patients. Tomasiuk R et al., Crit Care, 2014; 25;18(3):440. C-Type Natriuretic Peptides in Coronary Disease. Prickett TCR et al., Clin Chem, 2017; 63(1):316-324. The natriuretic peptides system in the pathophysiology of heart failure: from molecular basis to treatment. Volpe M et al., Clinical Science, 2016; 130:57-77.
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1) EINLEITUNG
C-Typ-natriuretisches Peptid (CNP) ist ein parakriner Wachstumsfaktor, der in zahlreichen Geweben exprimiert
wird, einschließlich des vaskulären Endothels, wo es vasoprotektiv wirken soll. In endothelialen Zellen und Makrophagen wird es als Reaktion auf verschiedene Stimuli, einschließlich entzündlicher Mediatoren, sezerniert. CNP wird in Geweben schnell abgebaut und nur geringe Mengen gelangen in die Zirkulation. Dagegen wird der N-terminale Teil des Pro-Hormons nicht so rasch abgebaut und zirkuliert in deutlich höheren Konzentrationen als CNP. Deshalb ist NT-proCNP ein wertvoller Biomarker, um die CNP-Synthese im Gewebe zu bestimmen. CNP spielt eine maßgebliche Rolle beim Längenwachstum. Es wird in der Wachstumsplatte produziert und die Signale erfolgen durch einen parakrinen Mechanismus. Jüngste Studien haben gezeigt, dass die Plasmakonzentrationen von NT-proCNP mit der linearen Wachstumsgeschwindigkeit in allen Phasen des Skelettwachstums korrelieren und während der rhGH-Therapie zunehmen (1). Weiters sind Serum NT-proCNP Werte nach beginnender Behandlung mit Wachstumshormonen in Patienten mit Achondroplasie/ Hypochondroplasie erhöht (2). Frauen mit Schwangerschaftskomplikationen, wie vermindertem fötalen Wachstum und Präeklampsie, zeigen schon früh in der Schwangerschaft signifikant erhöhte NT-proCNP-Werte (3,4). Die NT-proCNP-Konzentration zum Zeitpunkt der Aufnahme hat eine ausreichende Sensitivität und Spezifität, um die natürlich vorkommende Sepsis vom nichtseptischen systemischen Entzündungsreaktionssyndrom (SIRS) (5, 6) zu unterscheiden. Vor kurzem demonstrierten Prickett und Kollegen in einer Kohorte von über 2000 Personen, dass im Gegensatz zu der engen Assoziation von NT-proBNP mit der Herzfunktion sowie als Prädiktor nach einem Myokardinfarkt, Plasma NTProCNP stark mit der Nierenfunktion korreliert. NT-proCNP ist daher ein unabhängiger Prädiktor für Mortalität und einer kardialen Wiederaufnahme bei Personen mit instabiler Angina. (7).
Interessensgebiete: Vaskuläre Erkrankungen Wachstum Knochenentwicklung
Angiognese Sepsis
2) INHALT DES KITS CONT PLATE WASHBUF ASYBUF STD CTRL CONJ SUB STOP
KIT KOMPONENTEN Polyklonaler Schaf anti NT-proCNP Antikörper, beschichtet auf Mikrotiterplattenstreifen im Streifenhalter, verpackt in einem Aluminiumbeutel mit Trockenmittel Waschpuffer, 20fach Konzentrat, durchsichtige Kappe Assay Puffer, rote Kappe, gebrauchsfertig Standards (0; 4; 8; 16; 32; 64; 128 pmol/l), weiße Kappen, lyophilisiert Kontrollen A+B, gelbe Kappen, lyophilisiert, exakte Konzentration siehe Etiketten Konjugat (Schaf anti NT-proCNP-HRPO), braune Kappe, gebrauchsfertig Substrat (TMB Lösung), blaue Kappe, gebrauchsfertig Stopplösung, weiße Kappe, gebrauchsfertig
MENGE 12 x 8 Teste 1 x 50 ml 1 x 8 ml 7 Fläschchen 2 Fläschchen 1 x 7 ml 1 x 13 ml 1 x 7 ml
3) ZUSÄTZLICHES MATERIAL IM KIT 1 selbstklebende Abdeckfolie QC Protokoll Protokollblatt Arbeitsanleitung (Beipacktext) 4) ZUSÄTZLICH BENÖTIGTES MATERIAL UND GERÄTE Kalibrierte Präzisionspipetten für 20 µl, 50 µl und 100 µl und, 300 µl, inkl. Pipettenspitzen Mikrotiterplattenphotometer mit 450 nm Filter (630 nm Referenz) Millimeterpapier oder Software Destilliertes oder deionisiertes Wasser
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5) REAGENZIEN UND PROBENVORBEREITUNG
A lle Reagenzien des Kits sind bei +4°C (2-8°C) bis zum Ablaufdatum (siehe Etikett des Reagenz) stabil. Probenvorbereitung: Abnahme von venösem Blut mittels standardisierter Blutabnahmeröhrchen zur Gewinnung von Serum oder Plasma. Durchführung der Zentrifugation des Blutes laut Herstellerangaben der Probenentnahmebehälter, so schnell wie möglich. Das gewonnene Serum oder Plasma soll so schnell wie möglich gemessen werden. Für Langzeit Lagerung sollen die Proben aliquotiert und bei -25°C oder tiefer gelagert werden. Bis zu 4 Frier/Tau-Zyklen verändern die Messwerte nicht. Aufgetaute Proben so bald wie möglich abarbeiten. Lipämische und hämolytische Proben können falsche Ergebnisse liefern. Proben vor Verwendung gut mischen. Wir empfehlen Doppelbestimmung für alle Werte. Proben mit Ergebnissen über STD7 können mit ASYBUF (Assay Puffer) verdünnt und erneut getestet werden. Weitere Informationen zur Probenstabilität finden Sie auf unserer Webseite www.bmgrp.com (s. Validation Data) oder kontaktieren Sie unseren Kundenservice, Email unter
[email protected], Tel. unter +43/ 1/ 29107- 45. Reagenzienvorbereitung: WASHBUF (Waschpuffer): Das mitgelieferte Konzentrat wird 1:20 verdünnt (zB. 50 ml WASHBUF + 950 ml destilliertes Wasser). Kristalle im Pufferkonzentrat lösen sich bei Raumtemperatur auf. Das Konzentrat ist bei 4°C (2-8°C) bis zum Ablaufdatum haltbar. Der verdünnte Puffer ist bei 4°C (2-8°C) bis zu einem Monat haltbar. Im Testsystem darf nur verdünnter WASHBUF (Waschpuffer) verwendet werden. STD (Standards) und CTRL (Kontrollen): Lösen Sie das Lyophilisat in jeweils 300 µl destilliertem oder deionisiertem Wasser bei Raumtemperatur (18-26°C) für 15 min. Gut mischen. Die genaue Konzentrationen sind auf den Etiketten vermerkt. Rekonstituierte STD und CTRL sind bei -25°C oder tiefer bis zum Ablaufdatum haltbar. STD und CTRL sind für 3 Frier/Tau-Zyklen stabil. 6) TESTPRINZIP
Siehe Kapitel 6) PRINCIPLE OF THE ASSAY im englischen Teil des Beipacktextes 7) TESTPROTOKOLL Im Test dürfen nur Reagenzien und Proben verwendet werden, welche Raumtemperatur (18-26°C) aufweisen. Markieren Sie die Positionen für STD/PROBE/CTRL (Standard/Probe/Kontrolle) am Protokollblatt. Nehmen Sie die benötigten Mikrotiterstreifen aus dem Aluminiumbeutel. Nicht verwendete Mikrotiterstreifen können mit Trockenmittel im Aluminiumbeutel bis zum angegebenen Ablaufdatum gelagert werden. 1. Pipettieren Sie 50 µl ASYBUF (Assay Puffer, rote Kappe) in alle Wells. 2. Pipettieren Sie 20 µl STD/PROBE/CTRL (Standard/Probe/Kontrolle) in Doppelbestimmung in die Mikrotiterstreifen, gut mischen. 3. Streifen abdecken und für 20 min bei Raumtemperatur (18-26°C) inkubieren. 4. Pipettieren Sie 50 µl CONJ (Konjugat, braune Kappe) in alle Wells, gut mischen. 5. Streifen abdecken und 3 Stunden bei Raumtemperatur (18-26°C) im Dunkeln inkubieren. 6. Inhalt der Wells verwerfen und 5x mit 300 µl verdünntem WASHBUF (Waschpuffer) waschen. Nach dem letzten Waschschritt Reste von Waschpuffer durch Ausklopfen auf saugfähigem Papier entfernen. 7. Pipettieren Sie 100 µl SUB (Substrat, blaue Kappe) in alle Wells, gut mischen. 8. 30 Minuten bei Raumtemperatur (18-26°C) im Dunkeln inkubieren. 9. Pipettieren Sie 50 µl STOP (Stopplösung, weisse Kappe) in alle Wells, gut mischen. 10. Sofort die Extinktion bei 450 nm messen, falls möglich 630 nm als Referenz verwenden. Protokolle für Urinproben, ZK Überstände und non-human Spezies finden Sie auf unserer Webseite www.bmgrp.com (s. Validation Data) oder kontaktieren Sie unseren Kundenservice per e-mail
[email protected] oder Telefon +43-1-29107-45
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8) BERECHNUNG DER ERGEBNISSE
M essen Sie die optische Dichte (OD) von allen Wells mit einem Mikrotiterplattenphotometer mit einem 450 nm Filter (Referenz 630 nm). Konstruieren Sie eine Standardkurve aus den OD Werten der STD unter Verwendung von kommerziell erhältlichem Millimeterpapier oder einer geeigneten Software. Das Testsystem wurde mit einem 4 Parameter Algorithmus evaluiert. Andere Auswerte Algorithmen müssen vom Verwender evaluiert werden. Die Konzentration der Proben wird aus der Standardkurve abgelesen. Eventuelle weitere Verdünnungen müssen berücksichtigt werden. Typische STD-Kurve: Siehe Kapitel 8) CALCULATION OF RESULTS im englischen Teil des Beipacktextes Auf dem beigepackten QC Protokoll sind die Resultate bei der QC Freigabe des Kits vermerkt. Vom Verwender erhaltene Daten der OD können abweichend sein, bedingt durch verschiedene Einflüsse und/oder dem normalen Signalverlust des Kits während der Laufzeit. Dieser mögliche Signalverlust hat keinen Einfluss auf die Gültigkeit der Resultate, so lange die OD des höchsten Standards den Wert 1,50 oder höher erreicht und die Werte der CTRL im gültigen Bereich sind (Bereiche siehe Etiketten). 9) TESTMERKMALE Methode:
Sandwich ELISA, HRP/TMB, 12x8-well strips
Probenvolumen:
Serum, EDTA Plasma, Heparin Plasma und Citrat Plasma Protokolle für Urin, ZK Überstände und nicht humane Proben verfügbar 0 to 128 pmol/l (7 Standards und 2 Kontrollen in humaner Serum Matrix) Standards: 0/4/8/16/32/64/128 pmol/l 1 pg/ml = 0,201 pmol/l (MW: 4,985 kDa) 1 pmol/l = 4,985 pg/ml 20 µl / Vertiefung
Inkubationszeiten:
20 min / 3 h / 30 min
Sensitivität:
LOD: (0 pmol/l + 3 SD): 0,7 pmol/l; LLOQ: 0,5 pmol/l
Spezifität:
Dieser Assay erkennt endogenes und synthetisches, humanes NT-proCNP.
Präzision: Wiederfindung (durchschnittiche Wiederfindung nach spike mit 64 pmol/l synth. NT-proCNP):
Intra-assay (n=5) ≤ 6%, Inter-assay (n=8) ≤ 7%
Probentyp: Standardbereich: Umrechnungsfaktor pg/ml zu pmol/l:
Verdünnungslinearität (endogene NT-proCNP Werte nach VD mit ASYBUF):
Werte von anscheinend gesunden Spendern:
Serum (n=6) = 101%
Heparin plasma (n=2) = 93%
EDTA plasma (n=6) = 99% Citrat plasma (n=2) = 100% Durchschnittle Wiederfindung (%) 1+1 1+3 nach Verdünnung Serum (n=6) 99 98 EDTA plasma (n=6) 103 98 Heparin plasma (n=2) 100 100 Citrat plasma (n=2) 96 92 Median Serum (n=32) = 14,5 pmol/l Median EDTA plasma (n=33) = 15 pmol/l Median heparin plasma (n=18) = 13,5 pmol/l Median citrate plasma (n=18) = 12 pmol/l Jedes Labor sollte den Normalbereich für seine Proben evaluieren. Wechseln Sie nicht die Probenmatrix innerhalb einer Studie.
Nähere Informationen zu den Testmerkmalen und der Antikörperspezifität finden Sie auf unserer Website unter www.bmgrp.com (s. Validation Data) oder kontaktieren Sie unseren Kundenservice per Email an
[email protected] oder telefonisch unter +43/ 1/ 29107-45. 10/16
10) PRÄZISION Intra-assay: 2 Proben wurden 5 mal in einem Test von 1 Operator getestet. Inter-assay: 2 Proben wurden 8 mal in 2 verschiedenen Lots von 2 verschiedenen Operatoren getestet Intra-assay (n=5) Durchschnitt (pmol/l) SD (pmol/l) VK (%)
Sample 1 7.9 0.47 6
Sample 2 65.3 1.25 2
Inter-assay (n=8) Durchschnitt (pmol/l) SD (pmol/l) VK (%)
Sample 1 8,2 0,54 7
Sample 2 64,1 1,42 2
11) TECHNISCHE MERKMALE Reagenzien von verschiedenen Lots oder Testen dürfen nicht gemischt werden. Stöpsel oder Verschlüsse von verschiedenen Reagenzien dürfen nicht vertauscht werden. Abgelaufene Reagenzien dürfen nicht verwendet werden. Reagenzien sind vor direktem Sonnenlicht zu schützen. Substratlösung muss vor Verwendung farblos sein. Mikrotiterstreifen müssen bei den Inkubationen mit Abdeckfolie abgedeckt sein. Vermeiden Sie Schaumbildung beim Mischen der Reagenzien. 12) VORSICHTSMASSNAHMEN
Alle Bestandteile humanen Ursprunges wurden auf HIV-Ak, HCV-Ak und HBsAg getestet und negativ gefunden.
Trotzdem sollten die Reagenzien als potentiell infektiös behandelt werden. Die flüssigen Reagenzien enthalten ≤ 0,1% Proclin 300 als Konservierungsmittel. Proclin 300 ist in der verwendeten Konzentration nicht toxisch. Vermeiden Sie Kontakt mit Augen, Haut und Schleimhaut. Allergische Reaktionen sind möglich. Nicht mit dem Mund pipettieren. Nicht Rauchen, Essen, Trinken oder Kosmetika benutzen während der Verwendung der Testreagenzien. Verwenden Sie Handschuhe zur Vermeidung jedes Kontaktes mit Reagenzien. Die Stopplösung enthält Schwefelsäure, welche bei Kontakt die Augen und Haut reizen kann. Bei Berührung gründlich mit Wasser spülen. 13) LITERATUR
Siehe Kapitel 13) LTERATURE im englischen Teil des Beipacktextes.
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Notes:
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Notes:
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Notes:
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SYMBOLS Expiry date / Verfallsdatum / Date de péremption / Data di scadenza /Fecha de caducidad / Data de validade / Uiterste gebruiksdatum / Udløbsdato / Utgångsdatum / Termin Ważności / Lejárati idő / Doba exspirácie / Doba exspirace Consider instructions for use / Bitte Gebrauchsanweisung beachten / Consultez la notice d'utilisation / Consultare le istruzioni per l'uso / Consulte las instrucciones de utilización / Consulte as instruções de utilização / Raadpleeg de gebruiksaanwijzing / Se brugsanvisningen / Läs anvisningarna före användning / Proszę przeczytać instrukcję wykonania / Vegyük figyelembe a használati utasításban foglaltakat / Postupujte podľa pokynov na použitie / Postupujte dle návodu k použití In vitro Diagnostic Medical Device (for in Vitro Diagnostic Use)/ In vitro Diagnostikum (zur Invitro-Diagnostik) / Dispositif médical de diagnostic in vitro (Pour usage diagnostique in vitro) / Dispositivo medico per diagnostica in vitro (per uso diagnostico in vitro) / Dispositivo médico de diagnóstico in vitro (para uso diagnóstico in vitro) / Dispositivo médico para diagnóstico in vitro (Para utilização de diagnóstico "in vitro") / Medisch hulpmiddel voor diagnostiek in vitro (Voor diagnostisch gebruik in vitro) / Medicinsk udstyr til in vitro-diagnostik (Udelukkende til in vitro diagnostisk anvendelse) / Medicinteknisk produkt avsedd för in vitro-diagnostik (För in vitro-diagnostiskt bruk) / Wyrób medyczny do Diagnostyki In Vitro / In vitro orvosdiagnosztikai termék / In vitro diagnostický zdravotnícky materiál (určené pre diagnostiku „in vitro“) / In vitro diagnostický zdravotnicky materiál (určeno pro diagnostiku „in vitro“) Lot-Batch Number / Charge-Chargennummer / Lot-Code du lot / Lotto-Numero di lotto / LoteCódigo de lote / Lote-Código do lote / Lot-Partijnummer / Lot-Batchkode / Lot-Satskod / Numer serii / Lot-Batch szám / Číslo šarže / Číslo šarže Manufactured by / Hergestellt von / Fabriqué par / Prodotto da / Fabricado por / Fabricado por / Vervaardigd door / Fabrikation af / Tillverkad av / Wyprodukowane pr / Gyártotta / Vyrobené / Vyrobeno Catalogue Number / Bestellnummer / Numéro de référence / Numero di riferimento / Número de referencia / Número de referência / Referentienummer / Referencenummer / Katalognummer / Numer katalogowy / Katalógusszám / Katalógové číslo / Katalogové číslo Store at between / Lagerung bei zwischen / Conserver à entre / Conservare a tra / Conservar a temp. entre / Armazene a entre / Bewaar bij tussen / Opbevares mellem / Förvaras vid / Przechowywać w / Tároljuk …… között / Skladujte v rozsahu / Skladujte v rozmezí Contains sufficient for x tests / Inhalt ausreichend für x Tests / Contient suffisant pour x tests / Contenuto sufficiente per x test / Contiene suficiente para x pruebas / Contém suficiente para x testes / Bevat voldoende voor x bepalingen / Indeholder tilstrækkeligt til x prøver / Innehållet räcker till x analyser / Zawartość na x testów / Tartalma X teszt elvégzésére elegendő / Obsahuje materiál pre x testov / Obsahuje materiál pro x testů
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BI-20812 NT-proCNP ASSAY PROTOCOL AND CHECKLIST PREPARATION OF REAGENTS:
Bring all reagents to room temperature (18-26°C).
Prepare reagents and samples as instructed.
Bring unused and prepared components to the storage temperature mentioned in the package insert.
Take microtiter strips out of the aluminium bag and mark positions on the protocol sheet.
TEST PROCEDURE:
1) Pipette 50 µl ASYBUF (Assay Buffer) into each well.
2) Add 20 µl STD/ SAMPLE/ CTRL (standard/ sample/ control) in duplicates into respective wells, swirl gently.
3) Cover tightly and incubate for 20 min at room temperature (18-26°C).
4) Add 50 µl CONJ (Conjugate, amber cap) into each well, swirl gently.
5) Cover tightly and incubate for 3 hours at room temperature (18-26°C) in the dark.
6) Aspirate and wash wells five times with 300 µl diluted WASHBUF (Wash buffer). Remove remaining buffer by hitting plate against paper towel.
7) Add 100 µl SUB (Substrate) into each well, swirl gently.
8) Incubate for 30 minutes at room temperature (18-26°C), in the dark.
9) Add 50 µl STOP (Stop solution) into each well, swirl gently.
10) Read optical density at 450 nm with reference 630 nm, if available.
For the measurement of NT-proCNP in human urine, cell culture supernatants and non-human samples please visit our website www.bmgrp.com (see Validation Data).
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NT-proCNP ELISA, BI-20812 - Validation Data File developed and manufactured by Biomedica
NT-proCNP ELISA (2 n d generation) for the quantitative determination of human NT-proCNP in serum, EDTA plasma, citrate plasma and heparin plasma Cat. No. BI-20812 . 12 x 8 tests
Content ASSAY CHARACTERISTICS Summary .............................................................................. 2 TYPICAL STANDARD CURVE ........................................................................................ 3 PRINCIPLE OF THE ASSAY .......................................................................................... 3 SAMPLE VALUES .......................................................................................................... 4 MATRIX COMPARISON .................................................................................................. 6 ASSAY PERFORMANCE CHARACTERISTICS ...................................................................... 7 RECOVERY................................................................................................................ 7 LINEARITY ................................................................................................................ 8 PRECISION ............................................................................................................... 9 SENSITIVITY .......................................................................................................... 10 SAMPLE STABILITY .................................................................................................. 10 SPECIFICITY ........................................................................................................... 11 CALIBRATION ......................................................................................................... 11 COMPARISON of NT-proCNP ELISA # BI-20812 (2nd generation) vs # BI-20872 .............. 11 MEASUREMENT OF NT-proCNP in human URINE, CELL CULTURE SUPERNATANTS and in NON-HUMAN SAMPLES ...................................................................................... 12 1. MEASUREMENT of NT-proCNP in HUMAN URINE SAMPLES......................................... 12 2. MEASUREMENT of NT-proCNP in CELL CULTURE SUPERNATANTS ............................... 13 3. MEASUREMENT OF NT-proCNP in NON-HUMAN SAMPLES .......................................... 15
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NT-proCNP ELISA, BI-20812 - Validation Data File developed and manufactured by Biomedica
ASSAY CHARACTERISTICS Summary
Method
Sandwich ELISA, HRP/TMB, 12x8-well strips
Sample type
Serum, EDTA plasma, citrate plasma, and heparin plasma Protocol for urine, cell culture supernatants and non-human species available
Standard range
0 to 128 pmol/l (7 standards and 2 controls in a human serum matrix. Standards: 0/4/8/16/32/64/128 pmol/l)
Conversion factor
1 pg/ml = 0.201 pmol/l (MW: 4.985 kDa) 1 pmol/l = 4.985 pg/ml
Sample volume
20 µl / well
Incubation time, temp.
20 min / 3 h / 30 min, room temperature
Sensitivity
LOD: (0 pmol/l + 3 SD): 0.7 pmol/l; LLOQ: 0.5 pmol/l
Specificity
This assay recognizes endogenous and synthetic human NT-proCNP
Precision
Intra-assay (n=5) ≤ 6% Inter-assay (n=8) ≤ 7%
Spike/Recovery
Average % recovery spiked with 64 pmol/l
Serum (n=6): 101 EDTA plasma (n=6): 99 Citrate plasma (n=2): 100 Heparin plasma (n=2): 93
Average % of expected of dilution: Dilution linearity of endogenous NT-proCNP
Values of apparently healthy individuals
1+1
1+3
99
98
EDTA plasma (n=6):
103
98
Heparin plasma (n=2):
100
100
Citrate plasma (n=2):
96
92
Serum (n=6):
Median Median Median Median
serum (n=32) = 14.5 pmol/l EDTA plasma (n=33) = 15 pmol/l heparin plasma (n=18) = 13.5 pmol/l citrate plasma (n=18) = 12 pmol/l
Each laboratory should establish its own reference range for the samples under investigation. Do not change sample type during the study.
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NT-proCNP ELISA, BI-20812 - Validation Data File developed and manufactured by Biomedica
TYPICAL STANDARD CURVE
PRINCIPLE OF THE ASSAY
CAB Coating Antibody: polyclonal goat antibody (AA 53-73 of Uniprot ID # P23582) DAB Detection Antibody: polyclonal goat antibody (AA 24-42 of Uniprot ID # P23582) AG Antigen: synthetic human propeptide of CNP (AA 24-73 of Uniprot ID # P23582) INFORMATION on the ANALYTE The 103–amino acid propeptide is cleaved either between residues 50 and 51 or 81 and 82 to produce one of two biologically active peptides, carboxy-terminal proCNP (51–103) or carboxy-terminal proCNP (82–103), respectively, and an amino-terminal congener, N-terminal proCNP (1).
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NT-proCNP ELISA, BI-20812 - Validation Data File developed and manufactured by Biomedica
From: Natriuretic Peptides, Their Receptors, and Cyclic Guanosine Monophosphate-Dependent Signaling Functions. Potter LR et al., 2006; Endocrine Reviews 27(1):47–72. doi: 10.1210/er.2005-0014
SAMPLE VALUES NT-proCNP levels in an apparently healthy cohort
Mean Median
Serum (n=32) 15.78
NT-proCNP [pmol/l] EDTA plasma Heparin plasma Citrate plasma (n=33) (n=18) (n=18) 16.18 15.83 14.06
14.5
15
13.5
12
34
28.3
27
25
5.65
9.4
8
8
Min
5
8
8
8
Max
34
29
27
25
Percentile 95% Percentile 5%
It is recommended to establish the normal range for each laboratory.
NT-proCNP levels in unselected hospital panels 4/17
NT-proCNP ELISA, BI-20812 - Validation Data File developed and manufactured by Biomedica
NT-proCNP [pmol/l]
Mean Median Percentile 95% Percentile 5% Min Max
EDTA plasma Mean Median Percentile 95% Percentile 5% Min Max
Heparin plasma Mean Median Percentile 95% Percentile 5% Min Max
Citrate plasma (n=8) 13.38
EDTA plasma (n=8) 19.38
Heparin plasma (n=8) 17
14
19
16
17 9 9 17
26 15 15 26
28 11 11 28
NT-proCNP [pmol/l] App. healthy Hospital cohort (n=33) (n=8, unselected) 16.18 19.38 15
19
28.3 9.4 8 29
26 15 15 26
NT-proCNP [pmol/l] App. healthy Hospital cohort (n=18) (n=8, unselected) 15.83 17 13.5
16
27 8 8 27
28 11 11 28
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NT-proCNP ELISA, BI-20812 - Validation Data File developed and manufactured by Biomedica
MATRIX COMPARISON Comparison of NT-proCNP serum and plasma sample values from apparently healthy individuals Comparison of human NT-proCNP sample concentrations between serum, EDTA plasma, and citrate plasma from an apparently healthy cohort (n=16). Human NT-proCNP was measured in four matrices from sixteen individual donors. NT-proCNP [pmol/l] Donor ID #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 #13 #14 #15 #16
EDTA plasma 17 17 13 14 16 22 15 14 15 11 24 14 13 10 28 29
Citrate plasma 14 16 11 12 17 19 13 12 11 9 20 11 10 8 23 25
Heparin plasma 16 18 12 12 18 21 14 13 12 9 22 12 10 8 26 27
Serum* 20 21 16 15 22 24 19 16 16 14 28 15 14 11 34 34
*Measured values of human NT-proCNP in serum are higher compared to plasma in an apparently healthy cohort (n=16). It has been shown that sample values for serum and plasma can differ for the measurement of chemistry analytes (2). Graph showing NT-proCNP levels dependence of sample concentrations on sample matrix
Sample Matrix Comparison [app.healthy pop.] 35,0
NT-proCNP [pmol/l]
30,0 25,0 20,0
EDTA plasma
15,0
Citrate plasma Serum
10,0
Heparin plasma 5,0 0,0 #1
#2
#3
#4
#5
#6
#7
#8
#9
#10 #11 #12 #13 #14 #15 #16
Donor ID
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NT-proCNP ELISA, BI-20812 - Validation Data File developed and manufactured by Biomedica
ASSAY PERFORMANCE CHARACTERISTICS RECOVERY Summary of data showing mean recovery of NT-proCNP: Matrix Serum (n=6) EDTA plasma (n=6) Citrate plasma (n=2) Heparin plasma (n=2)
+12.8 pmol/l Mean Range 102% 91-115% 99% 80-116% 100% 94-106% 92% 90-94%
Mean 101% 99% 100% 93%
+64 pmol/l Range 96-105% 94-101% 97-103% 92-95%
Experiments: Recovery of spiked samples was tested by adding 2 concentrations synthetic NT-proCNP (12.8 + 64 pmol/l) to different human sample matrices. Data showing spike/recovery of human serum samples: Sample ID #S1 #S2 #S3 #S4 #S5 #S6
Spike NT-proCNP [pmol/l] 0 12.8 64 32.4 42.3 83.6 34.4 42.6 81.1 20.5 30.7 74.8 24.0 34.4 73.8 28.5 39.6 79.9 25.5 37.7 78.6 Mean R [%]
S/R [%] 12.8 64 103 105 91 100 95 101 100 96 109 103 115 103 102 101
Data showing spike/recovery of human EDTA plasma samples: Sample ID #E1 #E2 #E3 #E4 #E5 #E6
Spike NT-proCNP [pmol/l] 0 12.8 64 29.0 31.5 25.3 28.4 27.7 21.3
36.3 43.1 35.5 38.7 38.0 30.9
78.7 78.9 77.2 78.2 73.9 74.4 Mean R [%]
S/R [%] 12.8 64 80 116 99 103 102 92 99
100 99 101 100 94 100 99
Data showing spike/recovery of human heparin plasma samples: Sample ID #H1 #H2
Spike NT-proCNP [pmol/l] 0 12.8 64 28 37 75 30 39 74 Mean R [%]
S/R [%] 12.8 64 90 95 94 92 92 93
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NT-proCNP ELISA, BI-20812 - Validation Data File developed and manufactured by Biomedica
Data showing spike/recovery of human citrate plasma samples: Sample ID #C1 #C2
Spike NT-proCNP [pmol/l] 0 12.8 64 24 35 78 29 38 77 Mean R [%]
S/R [%] 12.8 64 106 103 94 97 100 100
LINEARITY Dilution linearity of samples containing endogenous NT-proCNP Matrix Serum (n=6) EDTA plasma (n=6) Heparin plasma (n=2) Citrate plasma (n=2)
Mean 99 103 100 96
Recovery of dilution steps [%] 1+1 1+3 Range Mean 96-102 98 98-110 98 98-103 100 94-98 92
Range 93-119 92-104 97-102 86-98
Dilution linearity of samples containing synthetic NT-proCNP Matrix Serum (n=6) EDTA plasma (n=6) Heparin plasma (n=2) Citrate plasma (n=2)
1+1 Mean Range 93 89-97 99 95-102 102 94-111 95 94-97
Recovery of dilution steps [%] 1+3 1+7 Mean Range Mean Range 89 78-97 86 78-100 95 89-102 93 84-101 100 94-105 92 84-99 93 89-96 87 84-90
1+15 Mean Range 99 89-118 97 85-111 93 90-96 99 98-100
Experiment: Dilution linearity was assessed by serially diluting samples containing endogenous NT-proCNP with ASYBUF (assay buffer supplied in the kit). Data showing the dilution of endogenous NT-proCNP in serum samples: Sample ID #S1 #S2 #S3 #S4 #S5 #S6
NT-proCNP [pmol/l] ref 1+1 1+3 32.4 34.4 20.5 24.0 28.5 25.5
15.6 17.2 10.1 11.5 14.1 13.0
7.6 8.0 4.9 5.6 6.8 7.6 Mean R [%]
R [%] 1+1 1+3 96 100 99 96 99 102 99
94 93 94 94 96 119 98
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NT-proCNP ELISA, BI-20812 - Validation Data File developed and manufactured by Biomedica
Data showing the dilution of endogenous NT-proCNP in EDTA plasma samples: Sample ID #E1 #E2 #E3 #E4 #E5 #E6
NT-proCNP [pmol/l] 1+1 1+3
ref 29.0 31.5 25.3 28.4 27.7 21.3
14.3 17.4 12.8 13.9 15.2 10.7
R [%]
6.9 8.2 6.1 6.5 7.1 5.2 Mean R [%]
1+1
1+3
99 110 101 98 110 101 103
95 104 97 92 102 98 98
Data showing the dilution of endogenous NT-proCNP in citrate plasma samples: Sample ID #C1 #C2
NT-proCNP [pmol/l] 1+1 1+3
ref 24.1 28.8
11.4 14.1
R [%]
6.9 5.2 Mean R [%]
1+1
1+3
94 98 96
86 98 92
Data showing the dilution of endogenous NT-proCNP in heparin plasma samples: Sample ID #H1 #H2
NT-proCNP [pmol/l] ref 1+1 1+3 28.1 29.7
13.7 15.,3
R [%]
6.8 7.6 Mean R [%]
1+1
1+3
98 103 100
97 102 100
Recommendations for sample dilution High measuring samples outside of the calibration range of the curve should be diluted with ASYBUF (assay buffer, supplied in the kit). PRECISION Intra-assay precision & Inter-assay precision Intra-assay (n=5) ≤ 6%, Inter-assay (n=8) ≤ 7% Intra-assay: 2 samples of known concentrations were tested 5 times within 1 kit lot by 1 operator. Inter-assay: 2 samples of known concentrations were tested 8 times within 2 different kit lots by 2 different operators. Intra-assay (n=5) Mean (pmol/l) SD (pmol/l) CV (%)
Sample 1
Sample 2
7.9 0.47 6
65.3 1.25 2
Inter-assay (n=8) Mean (pmol/l) SD (pmol/l) CV (%)
Sample 1
Sample 2
8.2 0.54 7
64.1 1.42 2
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NT-proCNP ELISA, BI-20812 - Validation Data File developed and manufactured by Biomedica
SENSITIVITY Limit of detection (LOD) The LOD is defined as the mean value of the back calculated concentration plus three times the standard deviation. The LOD of the NT-proCNP ELISA is 0.7 pmol/l. Lower limit of quantification (LLOQ) The lower limit of quantification is defined as the lowest concentration where the following two criteria are met: 1) back fit of the calibration standards shall be within 75 – 125% and 2) precision shall be ≤ 25% (acc. to ICH [Ref. 1]). The LLOQ for the NT-proCNP ELISA is 0.5 pmol/l. SAMPLE STABILITY Sample preparation Collect venous blood samples by using standardized blood collection tubes for serum or plasma. Perform serum and plasma separation by centrifugation according to supplier’s instructions of the blood collection devices as soon as possible. The acquired serum or plasma samples should be measured as soon as possible. For longer storage aliquot samples and store at -25°C or lower. Freeze-thaw of serum samples containing endogenous NT-proCNP A set of samples (3 sera, 3 EDTA plasma, 2 citrate plasma) was aliquoted and freeze-thaw stressed. The reference samples are freeze thawed once. Samples can undergo 4 freeze-thaw cycles. The mean recovery of sample concentrations stressed by 4 F/T cycles is 96%. According to our data, samples can undergo at least 4 freeze-thaw cycles. NT-proCNP concentrations of samples after freeze-thaw cycles: Sample ID #S1 #S2 #S3 #E1 #E2 #E3 #C1 #C2
reference 16.0 55.1 21.0 14.2 10.7 15.1 14.6 16.2
NT-proCNP 2x 15.9 52.3 20.5 14.6 10.8 17.4 13.0 15.3
[pmol/l] 3x 16.1 52.2 18.9 14.3 10.4 15.6 13.1 15.6
4x 15.6 51.3 20.8 13.3 9.6 16.8 13.4 15.4 Mean R [%]
R [%] 4 F/T vs ref 98 93 99 94 90 112 92 95 96
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NT-proCNP ELISA, BI-20812 - Validation Data File developed and manufactured by Biomedica
F/T of samples NT-proCNP [pmol/l]
60,0 50,0 reference
40,0
1x
30,0
2x
20,0
3x
10,0
4x
0,0 #S1
#S2
#S3
#E1
#E2
#E3
#C1
#C2
Sample ID
SPECIFICITY This assay recognizes endogenous (natural) and synthetic human NT-proCNP. CALIBRATION This immunoassay is calibrated against synthetic human propeptide of CNP (AA 24-73 of Uniprot ID# P23582) (= NT-proCNP 1-50). COMPARISON of NT-proCNP ELISA # BI-20812 (2nd generation) vs # BI-20872 The two NT-proCNP assays show a poor correlation due to the use of different reagents (e.g standard material, highly purified antibodies, and buffer solutions). VALIDATION GUIDELINES The assay is fully validated for human serum and plasma samples according to ICH Q2 (R1) (3). LITERATURE 1) Amino-Terminal Pro–C-Type Natriuretic Peptide in Heart Failure. Wright SP et al., Hypertension, 2004; 43:94-100. 2) Comparison of Serum and Heparinized Plasma Samples for Measurement of Chemistry Analytes. Miles RR et al., Clin Chem, 2004; 50, 9: 1704-1705. 3) CPMP/ICH/381/95 ICH Topic Q2 (R1) „Validation of Analytical Procedures: Text and Methodology” including: ICH Q2A “Text on Validation of Analytical Procedures” ICH Q2B “Validation of Analytical Procedures: Methodology” Available on our Website www.bmgrp.com Instructions for Use (package insert) Protocols for urine, cell culture supernatants and non-human samples Material Safety Data Sheet
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NT-proCNP ELISA, BI-20812 - Validation Data File developed and manufactured by Biomedica
MEASUREMENT OF NT-proCNP in human URINE, CELL CULTURE SUPERNATANTS and in NON-HUMAN SAMPLES The following experiments have been performed to test the use of the NT-proCNP assay (cat. no. BI-20812) in human urine, human cell culture supernatants, and in rat, mouse, and pig samples. Note: the experiments performed for these samples did not undergo a full validation and are therefore merely a performance check. 1. MEASUREMENT of NT-proCNP in HUMAN URINE SAMPLES Undiluted human urine samples from apparently healthy subjects and from patients with kidney disease were tested. Summary: Urine samples (n=48) were assayed with the Biomedica NT-proCNP ELISA (BI-20812) following the standard protocol using undiluted urine. -
Endogenous NT-proCNP was detectable in samples from kidney patients.
-
Urine samples can be spiked – the average recovery of 8 human urine samples is 109%.
-
If required, dilute urine samples 1+1 with assay buffer. Dilution linearity of samples containing high values of endogenous NT-proCNP (n=2) with assay buffer is 103%.
-
Specificity was assessed by adding the coating antibody utilized in the NT-proCNP ELISA assay to urine samples containing elevated endogenous NT-proCNP levels (n=2). The samples showed a competition of 93%.
RECOVERY Recovery was assessed by adding STD7 (final concentration 64 pmol/l) synthetic human NT-proCNP directly to eight different human urine samples (ratio 1+1). Data showing spike/recovery of human urine samples: NT-proCNP c [pmol/l] Sample ID
Reference
+ 64 pmol/l
S/R [%]
#U1 #U2
1
68
106
59
111
128
#U3
1
67
105
#U4
1
67
104
#U5
0
63
98
#U6
80
116
118
#U7
0
64
100
#U8
1
70
109
Mean R [%]
109
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NT-proCNP ELISA, BI-20812 - Validation Data File developed and manufactured by Biomedica
LINEARITY Dilution linearity was assessed by diluting urine samples containing endogenous NT-proCNP with ASYBUF (assay buffer supplied in the kit). Data showing the dilution of endogenous NT-proCNP in urine samples: Sample ID
NT-proCNP [pmol/l]
Dil R [%]
Reference
Dil 1+1
#U1
69
33
95
#U2
92
51
111
Mean R [%]
103
COMPETITION Specificity was assessed by adding the coating antibody utilized in the NT-proCNP ELISA assay to urine samples containing elevated endogenous NT-proCNP levels. Data showing the competition of the signal: NT-proCNP [pmol/l] Sample ID
Reference
Competition
Comp R [%]
#U1
69
6
91
#U2
92
4
96
Mean R [%]
93
Comparison of panels from various donors NT-proCNP [pmol/l] App. healthy (n=4)
Kidney cohort I (n=24)
Kidney cohort II (n=20)
1.25 1
9.5 1
0.35 0
Percentile 95%
2
86.25
1.95
Percentile 5%
1
0
0
Min
1
0
0
Max
2
92
2
Mean Median
Suggested protocol for the measurement of human NT-proCNP in urine samples Follow standard protocol as indicated in the package insert: Pipette 20 µl of undiluted urine sample directly into the well of the microtiter plate. If required, dilute samples 1+1 with assay buffer (provided in the kit). 13/17
NT-proCNP ELISA, BI-20812 - Validation Data File developed and manufactured by Biomedica
2. MEASUREMENT of NT-proCNP in CELL CULTURE SUPERNATANTS Note: the experiments performed to measure NT-proCNP in cell culture supernatants are not a full validation but are merely a performance check. Cell culture medium (ccm: RPMI1640 containing 10% fetal calf serum) was tested undiluted and spiked with a final concentration of 64 pmol/l synthetic human NT-proCNP. The spiked solution was diluted 1+1, 1+3, 1+7 with the cell culture medium. As a comparison, the spike recovery and dilution linearity of the standard matrix (=STD1) and the dilutions with assay buffer is shown. OD values of spiked and diluted cell culture medium sample and standard matrix (STD1) OD Dil medium Sample ID ccm ASYBUF
Reference
+ 64 pmol/l
1+1
1+3
1+7
1+15
0.028 0.031
1.355 1.271
0.627 0.601
0.290 0.318
0.125 0.156
0.072 0.090
ccm STD1
Graph showing dilution of cell culture medium (ccm) and a comparison to the standard (STD1), both spiked with spiked with the same amount of synthetic NT-proCNP (64 pmol/l).
1,600 1,400 1,200
OD
1,000 0,800
ccm
0,600
STD1
0,400 0,200 0,000 0
10
20
30
40
50
60
70
c [pmol/l]
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NT-proCNP ELISA, BI-20812 - Validation Data File developed and manufactured by Biomedica
Suggested protocol for the measurement of human NT-proCNP in cell culture supernatants Preparation of a cell culture medium based standard curve: Reconstitute STD7 in 300 μl deionized water. Leave at room temperature (18-26°C) for 15 min and mix well prior to making dilutions. Use polypropylene tubes. For the preparation of the cell culture based standards always use the identical cell culture medium in which the samples are based on. - Mark tubes e.g. CC STD6, CC STD 5 … CC STD1. - Prepare a two-fold serial dilution to obtain STD6 to STD2. e.g.: Dispense 100 µl cell culture medium into vials labelled with CC STD6 to CC STD1. Pipette 100 µl of STD 7 into tube marked as CC STD6. Mix thoroughly. Transfer 100 µl of CC STD6 into vial marked as CC STD5. Mix thoroughly. Continue in the same fashion to obtain CC STD4 to CC STD2. - Cell Culture Medium serves as the zero standard (=CC STD1, 0 pmol/l). Attention: Concentrations defined for CTRL A and B are only valid for measuring NTproCNP in human serum or plasma. The controls cannot be used for cell culture measurements.
3. MEASUREMENT OF NT-proCNP in NON-HUMAN SAMPLES The sequence homology of NT-proCNP is very conserved among different species. We therefore assessed if the assay which is validated for human samples can be used in rat, mouse and pig samples. Other species types that share a high homology to human NT-proCNP can likely be measured with this assay (e.g. monkey, cats, dogs). Note: the experiments performed for non-human species are not a full validation but are merely a performance check. uniprot ID P23582 H2QJL6 H2P8X1 M3WH43 E2R4X2 P18104 P55207 Q9D288 Q544K5
species C-type natriuretic peptide Homo sapiens (Human) PANTR - Uncharacterized protein Pan troglodytes (Chimpanzee) PONAB - Uncharacterized protein - Pongo abelii (Sumatran Orangutan) FELCA - Uncharacterized protein - Felis catus (Cat) CANLF - Uncharacterized protein - Canis lupus family PIG - C-type natriuretic peptide Sus scrofa (Pig) RAT - C-type natriuretic peptide - Rattus norvegicus (Rat) MOUSE - Putative uncharacterized protein Mus musculus (Mouse) MOUSE - C-type natriuretic peptide (Mus musculus)
sequence homology [%] 100 98 98 96 96 94 92 86 86
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NT-proCNP ELISA, BI-20812 - Validation Data File developed and manufactured by Biomedica MOUSE - C-type natriuretic peptide Mus musculus (Mouse) RAT - Natriuretic peptide C - Rattus norvegicu
Q61839 A0A1L1WKH8
86 93
For more information on the sequence homology between various species for the natriutetic peptides, NT-proCNP, NT-proANP, and NT-proBNP please visit our website www.bmgrp.com, Sequence homology xNPs. Suggested protocol for the measurement of NT-proCNP in non-human samples (e.g. rat or pig samples) Follow standard protocol as indicated in the package insert: Pipette 20 µl of undiluted sample directly into the well of the microtiter plate. If required, dilute samples 1+1 with assay buffer (provided in the kit).
A. Measurement of NT-proCNP in rat samples - performance check Rat NT-proCNP shares a 92% homology to human NT-proCNP. According to our data, rat serum samples (n=8) showed a recovery of 97% and a linearity of 87%. The samples tested contained endogenous NT-proCNP concentrations between 9-38 pmol/ml. Competition of endogenous NT-proCNP concentrations from rat samples is 100%. RECOVERY and LINEARITY Eight undiluted rat serum samples were tested in the NT-proCNP ELISA (note: standards contain synthetic human NT-proCNP spiked in a human serum matrix). For Linearity experiments: rat samples were diluted 1+1 with ASYBUF (supplied in the kit). For Recovery experiments: STD7 was added to the rat serum samples in a ratio 1+1 (final concentration 64 pmol/l). Calculation of rat sample concentrations, and spike recovery NT-proCNP [pmol/l]
NT-proCNP
Sample ID
Reference
dilution 1+1
dil lin R [%]
+ 64 pmol/l
S/R [%]
#R1 #R2 #R3 #R4 #R5 #R6 #R7 #R8
16 32 31 10 26 38 23 9
8 13 11 5 11 15 10 4 Mean R [%]
95 83 72 101 84 79 85 93 87
68 76 76 68 78 81 74 66 Mean S/R [%]
94 95 95 99 101 97 97 97 97
COMPETITION Specificity was assessed by adding the coating antibody utilized in the human NT-proCNP ELISA assay to the rat serum samples. 16/17
NT-proCNP ELISA, BI-20812 - Validation Data File developed and manufactured by Biomedica
Data showing the competition of the signal: NT-proCNP [pmol/l] Sample ID #R1 #R2 #R3 #R4 #R5 #R6 #R7 #R8
Reference 15 28 29 10 27 34 22 9
+ coating AB 0 0 0 0 0 0 0 0 Mean R [%]
R [%] comp. 100 100 100 100 100 100 100 100 100
B. Measurement of NT-proCNP in mouse samples - performance check Mouse NT-proCNP shares an 86% homology to human NT-proCNP. According to our data, mouse serum samples (n=8) showed an average recovery of 45% when spiked with STD7 in a ratio 1+1 (final concentration 64 pmol/l). The mouse samples tested did not contain endogenous NT-proCNP concentrations. Linearity was not tested. C. Measurement of NT-proCNP in pig samples - performance check Pig NT-proCNP shares a 94% homology to human NT-proCNP. According to our data, pig serum samples (n=8) showed a recovery of 83%. The pig samples tested did not contain endogenous NT-proCNP concentrations. Linearity was not tested. RECOVERY Eight undiluted pig serum samples were tested in the NT-proCNP ELISA (note: standards contain synthetic human NT-proCNP spiked in a human serum matrix). For Recovery experiments: STD7 was added to the pig serum samples in a ratio 1+1 (final concentration 64 pmol/l). Calculation of pig sample concentrations, and spike recovery: Sample ID #P1 #P2 #P3 #P4 #P5 #P6 #P7 #P8
NT-proCNP [pmol/l] Reference + 64 pmol/l 0 58 0 57 0 55 55 0 0 52 0 51 0 48 0 50 Mean R [%]
S/R [%] 91 89 85 86 81 80 75 78 83
Date: December 2017
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