INNOTEST * ß-AMYLOID (1-42)

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1 INNOTEST * ß-AMYLOID (1-42) Manufactured by: INNOGENETICS N.V. Technologiepark Ghent Belgium Innogenetics 2004 * INNOTEST is a Trademark of Innogenetics N.V v

2 INNOGENETICS * 2 TABLE OF CONTENTS Symbols used English Intended use Test Principle Reagents Description, preparation for use and recommended storage conditions Preparation of reagents Materials required but not provided Safety and environment Specimens Remarks and precautions Washing procedures Manipulations procedures Directions for sample dispensing on the uncoated polypropylene plate Test procedure Results Validation Calculation of the results Limitations of the procedure Test performance Clinical results Assay range Analytical specificity Detection limit Precision Example of a standard curve Français But recherché Principe du test Réactifs Description, préparation et recommandations de conservation..21 Préparation des réactifs Matériel nécessaire mais non fourni Règles de sécurité et protection de l'environnement Echantillons Remarques et précautions Procédures de lavage Procédures de manipulation *INNOGENETICS is a Registered Trademark of Innogenetics N.V.

3 3 INNOTEST ß-AMYLOID (1-42) Directives de distribution des échantillons sur la microplaque en polypropylène non sensibilisée Protocole du test Résultats Validation du test Calcul des résultats Limites du test Caractéristiques fonctionnelles Résultats cliniques Plages de concentration Spécificité analytique Limite de détection Précision Exemple de courbe standard Deutsch Beabsichtigter Verwendungszweck Funktionsweise des Tests Reagenzien Beschreibung, Vorbereitung und empfohlene Lagerung und Haltbarkeit Vorbereitung der Reagenzien Zusätzlich erforderliche Materialien Sicherheits- und Umwelthinweise Herstellung der Proben Hinweise und Vorsichtsmaßnahmen Waschanleitung Handhabung Anweisung für die Probenverteilung auf die unbeschichtete Polypropylenplatte Arbeitsablauf Ergebnisse Validierung Berechnung der Resultate Einschränkungen der Methode Leistungsfähigkeit des Tests Klinische Ergebnisse Testumfang Analytische Spezifität Bestimmungsgrenze Präzision Beispiel einer Standardkurve Italiano Uso Principio del test

4 INNOGENETICS 4 Reagenti Descrizione, preparazione per l'uso e condizioni di conservazione raccomandate Preparazione dei reagenti Materiali richiesti ma non forniti Sicurezza e ambiente Campioni Note e precauzioni Procedure di lavaggio Procedure di manipolazione Istruzioni per la dispensazione del campione sulla piastra di polipropilene non rivestita Procedura del test Risultati Validazione Calcolo dei risultati Limiti della procedura Performance del Test Risultati clinici Range del saggio Specificità analitica Limite di rilevazione Precisione Esempio di una curva standard Español Indicaciones de uso Principio del ensayo Reactivos Descripción, preparación para el uso y condiciones de almacenamiento recomendadas Preparación de los reactivos Materiales necesarios pero no suministrados Seguridad y medio ambiente Muestras Observaciones y precauciones Procedimientos de lavado Procedimientos de manipulación Instrucciones para preparar muestras en la placa de polipropileno sin recubrir Procedimiento del ensayo Resultados Validación Cálculo de los resultados Limitaciones del procedimiento

5 5 INNOTEST ß-AMYLOID (1-42) Realización del test Resultados clínicos Rango de ensayos Especificidad analítica Límite de detección Precisión Ejemplo de curva estándar Português Utilização Princípio do teste Reagentes Descrição, preparação para utilização e condições recomendadas de conservação Preparação de reagentes Material necessário não fornecido Segurança e ambiente Amostras Limites e precauções Procedimentos de lavagem Procedimentos de manipulação Indicações para a distribuição de amostras em placa de polipropileno não impregnada Procedimento Resultados Validação Cálculo dos resultados Limites do procedimento Desempenho do teste Resultados clínicos Intervalo do ensaio Especificidade analítica Limite de detecção Precisão Exemplo de uma curva padrão

6 INNOGENETICS 6 Symbols used Manufactured by Fabriqué par Hersteller Prodotto da Fabricado por In vitro diagnostic medical device Produit pour diagnostic in vitro Hilfsmittel für medizinische in-vitro-diagnostik Dispositivo medico - diagnostico in vitro Producto sanitario para diagnóstico in vitro Dispositivo médico de diagnóstico in vitro Lot number Numéro du lot Chargennummer Numero di lotto Número de lote Catalog number Référence catalogue Katalognummer Codice Número de catálogo Use by Utiliser avant Verwendbar bis Uso Para ser usado por Utilizado por Consult instructions for use Consulter les instructions d'emploi Für Anwendung siehe Anleitung Consultare le istruzioni per l'uso Consulte las instrucciones de uso Consultar instruções de utilização Temperature limits Limites de température Temperaturgrenzen Limiti di temperatura Límites de temperatura Limites de temperatura

7 7 INNOTEST ß-AMYLOID (1-42) Contains sufficient for < X > tests Conditionnement suffisant pour < X > tests Inhalt ausreichend für < X > Tests Contenuto sufficiente per < X > test Contenido suficiente para < X > ensayos Contém material suficiente para < X > testes Coated plate Microplaque sensibilisée Beschichtete Platte Piastra immobilizzata Placa recubierta Placa impregnada Standard (packed in separate box because of special storage conditions: REF 80325) Standard (conditionné dans une boîte séparée en raison des conditions de stockage spéciales: REF 80325) Standard (in separater Verpackung wegen besonderer Lagerbedingungen) Standard (confezionato in scatola separata a causa delle speciali condizioni di conservazione: REF 80325) Estándar (empaquetado en una caja independiente porque requiere condiciones de almacenamiento especiales: REF 80325) Padrão (embalado em caixa separada devido a condições de conservação especiais) Sample Diluent Diluant Échantillon Probenverdünnungspuffer Diluente del Campione Diluyente de muestra Diluente da amostra Conjugate 1 100x Conjugué 1 100x Konjugat 1, 100x konzentriert Coniugato 1 100x Conjugado 1 100x Conjugate 2 100x Conjugué 2 100x Konjugat 2, 100x konzentriert Coniugato 2 100x Conjugado 2 100x

8 INNOGENETICS 8 Conjugate Diluent 1 Diluant Conjugué 1 Konjugatverdünner 1 Diluente del Coniugato 1 Diluyente de Conjugado 1 Diluente de conjugado 1 Conjugate Diluent 2 Diluant Conjugué 2 Konjugatverdünner 2 Diluente del Coniugato 2 Diluyente de Conjugado 2 Diluente de conjugado 2 Substrate 100x Substrat 100x Substrat 100x Substrato 100x Sustrato 100x Substrate Buffer Tampon Substrat Substratpuffer Tampone Substrato Tampón sustrato Tampão de substrato Stop Solution Solution d'arrêt Stopplösung Soluzione di Stop Solución de parada Solução de paragem Wash Solution 25x Solution de Lavage 25x 25x konzentrierte Waschlösung Soluzione di Lavaggio 25x Solución de lavado 25x Solução de lavagem 25x

9 9 INNOTEST ß-AMYLOID (1-42) English Intended use The INNOTEST ß-AMYLOID (1-42) is a solid-phase enzyme immunoassay for the quantitative determination of ß-amyloid (1-42) in human cerebrospinal fluid (CSF). The combined use of CSF-ß-amyloid (1-42) and CSF-tau marker concentrations allows differentiation between Alzheimer's disease (AD) and normal aging or other neurological diseases such as depression (Hulstaert F et al. Neurology 1999;52:1555), provided that each laboratory validates the discrimination line described in the test performance section. Test Principle The INNOTEST ß-AMYLOID (1-42) is a solid-phase enzyme immunoassay in which the amyloid peptide is first captured by a monoclonal antibody (21F12) bound on the solid phase. CSF samples are added in 25-µl volumes and subsequently incubated with a biotinylated antibody (3D6). This antibody is then detected by a peroxidase-labeled streptavidin. After addition of substrate solution, positive samples will develop a blue color. The reaction is stopped by the addition of sulfuric acid which produces a yellow color. The absorbance is then measured at 450 nm. Reagents Description, preparation for use and recommended storage conditions - If kept at 2-8 C, and stored in the original vials, the reagents, opened or unopened, are stable until the expiry date of the kit. The concentrated standard has to be stored at -20 C or lower upon arrival. This standard is packed separately. Do not use the reagents beyond the expiry date. - All reagents and the aluminum foil bag containing the strips should be brought to room temperature (18-30 C) approximately 30 minutes before use and should be returned to the refrigerator immediately after use. To avoid water condensation into the wells, the aluminum foil bag must be kept closed until the strips are stabilized at room temperature. Reagents supplied: Component Quantity Ref. Description Coated Plate 1 x sealed bag containing a strip holder with 12 x 8 coated test wells and a silicagel bag as desiccant.

10 INNOGENETICS 10 Sample 1 x 30 ml Phosphate buffer with stabilizing Diluent proteins and 0.01% MIT/0.1% CAA as preservative, used to dilute the standard and samples with high concentrations of ß-amyloid (1-42). Conjugate 1 1 x 0.2 ml Mouse anti-ß-amyloid (1-42) IgG labeled with biotin in phosphate buffer with stabilizing proteins and 0.04% MIT/0.1% CAA as preservative. Dilute 100x with Conjugate Diluent 1 before use (see preparation of reagents). Conjugate working solution 1 is stable for 24 hours if kept at room temperature. Conjugate 2 1 x 0.3 ml Peroxidase-labeled streptavidin containing 0.02% MIT and 0.02% bromonitrodioxane as preservative. Dilute 100x with Conjugate Diluent 2 before use (see preparation of reagents). Conjugate working solution 2 must be prepared freshly for each test. Conjugate 1 x 20 ml Phosphate buffer with stabilizing proteins Diluent 1 and 0.01% MIT/0.1% CAA as preservative, used to dilute Conjugate 1. Conjugate 1 x 30 ml Phosphate buffer containing 0.05% Diluent 2 Proclin 300 as preservative, bovine casein as stabilizer and bovine aprotinin as protease inhibitor, used to dilute the Conjugate 2. Substrate 1 x 0.3 ml Tetramethyl benzidine (TMB) dissolved TMB in dimethyl sulfoxide (DMSO). Dilute 100x in Substrate Buffer before use (see preparation of reagents). Substrate working solution must be prepared freshly for each test. Substrate 1 x 30 ml Phosphate-citrate buffer containing Buffer 0.02% hydrogen peroxide, used to dilute the Substrate TMB. Stop Solution 1 x 30 ml N sulfuric acid Wash 1 x 60 ml Phosphate buffer containing 0.01% Solution MIT/0.09% CAA, to be diluted 25x with distilled or deionized water before use. Prepare at least 40 ml of diluted wash

11 11 INNOTEST ß-AMYLOID (1-42) solution for each test well. Salt crystals may be formed in the concentrated wash solution after storage at 2-8 C. These crystals must be completely redissolved. Diluted wash solution is stable for 24 hours if kept at room temperature. Standard 3 x 0.2 ml Allow the standard to thaw on the bench Variable and vortex before use. Prepare ± 1500 µl concentration of the highest standard (2000 pg/ml) in ( Sample Diluent, based on the pg/ml) concentration of the frozen standard to be used with (concentration on label). Use the the according 2000 pg/ml for the preparation of the kit lot number other standards (see table). Once standard stock has been thawed, it cannot be stored for further use. Dilutions must also be discarded after use. Use only polypropylene tubes for preparation of all standards. Uncoated 1 - For dispensing of the samples before polypropylene transferring to the coated plate to plate prevent reactivity shift. Plate sealers Minigrip bag 1 - For storage of unused strips. Preparation of reagents Preparation of 1500 µl of the 2000 pg/ml ß-amyloid (1-42) standard Y = concentration of the concentrated Standard delivered with the kit X = volume of Y to be used for preparation of 2000 pg/ml solution X = 1500 (= volume to be prepared) x 2000 (= concentration highest standard) µl Y Conc. (pg/ml) Standard Sample Diluent 2000 => A X µl X µl 1500 => B 150 µl of A 50 µl 1000 => C 200 µl of A 200 µl 500 => D 200 µl of C 200 µl 250 => E 200 µl of D 200 µl 125 => F 200 µl of E 200 µl

12 INNOGENETICS 12 NOTE: - Use a new tip for each standard level and mix each standard vial thoroughly before using it for further dilution. Preparation of conjugate working solutions 1 and 2, and substrate working solution 8 wells 16 wells 32 wells 64 wells 96 wells CONJ 1 in µl CONJ DIL 1 in ml wells 16 wells 32 wells 64 wells 96 wells CONJ 2 in µl CONJ DIL 2 in ml wells 16 wells 32 wells 64 wells 96 wells SUBS in µl SUBS BUF in ml Preparation of diluted wash solution 8 wells 16 wells 32 wells 64 wells 96 wells WASH SOLN 25x in ml H 2 O in ml Materials required but not provided - Distilled or deionized water. - Calibrated precision pipettes with disposable tip to deliver volumes in the ranges of µl. A calibrated multi-channel pipette to deliver 25 µl, 50 µl, 75 µl, 100 µl, 200 µl is recommended for addition of samples, conjugate working solutions, substrate working solution and Stop Solution. - Vortex mixer or equivalent. - Polypropylene tubes to dilute the samples and to make the standards. - Microplate washer; alternatively, washing can be performed by using a repeat pipette delivering 0.4 ml volumes and an aspirating device. - Timer. - Absorbent tissues. - Microplate reader with 450 ± 5 nm filter, optionally, 620 nm or 690 nm filter for dual wavelength analysis, and with a linear absorbency range of 0 to or higher. - Disposable vials for preparation of working solutions. - Appropriate biohazard waste containers for potentially contaminated materials. - Microplate shaker (1000 rpm); alternatively, mixing can be performed by tapping the side of the plate.

13 13 INNOTEST ß-AMYLOID (1-42) Safety and environment - Please refer to the manufacturer's safety data sheet and product labeling for information on potentially hazardous components. R43, S24-37 Irritant! (Xi) Avoid contact with skin. May cause sensitization by skin contact. Wear suitable gloves. Contains 2-Chloroacetamide: CONJ 1, CONJ DIL 1, SAMP DIL, ß-amyloid (1-42) STAND. R36/37/38, S Irritant! (Xi) Irritating to eyes, respiratory system and skin. Do not breathe vapour. Avoid contact with skin. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. Contains DMSO: SUBS TMB 100x. - Specimens should always be handled as potentially infectious. All biological materials should be considered as being potentially infectious and should be handled as such. Only adequately trained personnel should be permitted to perform the test procedure. All biological materials should be disposed of in accordance with established safety procedures. Autoclave for at least 15 minutes at 121 C. Incinerate disposable material. Mix liquid waste with sodium hypochlorite so that the final concentration is ± 1% sodium hypochlorite. Allow to stand overnight before disposal. CAUTION: Neutralize liquid waste that contains acid before adding sodium hypochlorite. REMARK: Special precautions for Transmissible Spongiform Encephalopathy (TSE) / Prion contaminated materials: Inactivation of samples Clinical samples, e.g. CSF, should be autoclaved or immersed in a solution of sodium hypochlorite resulting in 20,000 ppm free chlorine for 1 hour before final disposal by incineration. Waste disposal All material classified as clinical waste should be disposed of by incineration at an authorized incineration site. For the safe handling of clinical waste, use secure leak-proof containers, e.g. double bagging, where appropriate. Avoid external contamination of the container.

14 INNOGENETICS 14 Reference: - Advisory Committee on Dangerous Pathogens (UK) - Spongiform Encephalopathy Advisory Committee - Transmissible Spongiform Encephalopathy Agents: Safe Working and the Prevention of Infection - World Health Organization (WHO): WHO Infection Control Guidelines for Transmissible Spongiform Encephalopathies. - STOP SOLN contains 0.9N sulfuric acid, CONJ 2 contains MIT/Bromonitrodioxane as preservative and CONJ DIL 2 contains Proclin 300 as preservative. - Use of personal protective equipment is necessary: gloves and safety spectacles when manipulating dangerous or infectious agents. - Waste should be handled according to the institution's waste disposal guidelines. All federal, state, and local environmental regulations should also be observed. Specimens - Human cerebrospinal fluid stored at -20 C (preferably -80 C), collected in polypropylene tubes. The specimens must be free of cells and show no hemolysis. - The assay is not suitable for testing serum or cell culture supernatants. - It is recommended to dispense the samples in small aliquots in order to avoid repeated freeze/thaw cycles. Repeated freeze/thaw cycles will result in incorrect concentrations. - CSF samples with an OD 450 nm value above the highest standard can be diluted in Sample Diluent. - Insoluble material should be removed from all samples by centrifugation (4000 g for 10 minutes) before testing. - Glass or polystyrene can absorb ß-amyloid (1-42). The use of polypropylene vials is strongly recommended for making dilutions of the CSF or standard. Remarks and precautions - Do not mix components from kits of different lot numbers. - All vessels used to prepare conjugate and substrate working solutions must be thoroughly clean to avoid contamination. - Hold the ELISA plates by the sides to avoid contamination of the wells. - Avoid microbial contamination of reagents. - Ensure that samples and diluted standard solutions are homogeneous before use. - Use a new pipette tip for each specimen. - Ensure that specimen is added to the microwell. - To avoid contamination, do not touch the edge of the wells with the pipette tips when adding sample or conjugate working solution.

15 15 INNOTEST ß-AMYLOID (1-42) - Remove any air bubbles present by tapping the microtiter plate gently or by mixing on plate shaker for 1 minute at 1000 rpm. - Do not expose substrate working solution to strong light during incubation or storage. Place the plate in the dark during the incubation of the substrate. Substrate working solution must be colorless when used. If the solution is blue it must be replaced. - Stop Solution, substrate working solution or conjugate working solutions should not come into contact with metals or metal ions to avoid unwanted color formation. - If the wells cannot be filled with conjugate or substrate immediately after washing, place the strips upside down on an absorbent tissue soaked in Wash Solution for no longer than 15 minutes. Washing procedures In house washing is performed with an automatic washer: Columbus M8/2ch (Tecan). The protocol is available upon request. For other washers or protocols, carry out automatic washing as follows: - Pre-rinse the washer with wash solution. - Perform 5 wash cycles ensuring that: the fill volume is 400 µl/well the dispensing height is set to completely fill the well the time taken to complete one aspiration/wash/soak cycle is approximately 30 seconds. Perform manual wash as follows: - Aspirate the liquid completely from all wells by inserting an aspiration tip (aspiration device) gently to the bottom of each well. - Take care not to scratch the inside of the well. - After aspiration, fill the wells with 400 µl of diluted wash solution. - Remove the liquid from the wells. - Perform these steps five times. - After the washing procedure, invert the plate and tap dry on absorbent tissue. Incomplete washing will adversely affect the test outcome. Microbial contamination of wash solution and/or washer can cause extensive problems. If problems occur, disinfect the wash bottles and washer overnight with an appropriate disinfectant solution and rinse with water. Manipulations procedures Directions for sample dispensing on the uncoated polypropylene plate Since sample incubation is performed at room temperature (18-30 C) for 60 minutes, the dispensing time should be reduced to a minimum. In order to

16 INNOGENETICS 16 reduce a reactivity shift, an uncoated polypropylene microplate has been added to the kit as a "waiting station". According to the experience of the technician, we advise the use of this preparation plate when more than 3 strips are to be used. - dispense 60 µl of standard / sample diluent / sample into the wells of the preparation plate. With the use of a multi-channel pipet, 25 µl of the specimen can be transferred from the uncoated polypropylene microplate to the according coated test wells (see Test procedure). Test procedure Please read Remarks and precautions before performing the test. NOTE: - Allow all specimens and test reagents to reach room temperature (18-30 C) before use. - Have all reagents and specimens ready before starting the assay. Once the test has started, it must be performed without any interruption in order to achieve the most reliable and consistent results. 1. Place the required number of strips into the strip-holder: for each test run duplicate wells of the 6 standards and one blank should be prepared. The strips can be marked on one edge for identification. In case more than three strips are needed, it is necessary to use the polypropylene plate provided in the kit to predispense the samples. Place any unused strip in the plastic minigrip bag with the silicagel desiccant. 2. Prepare conjugate working solution 1 and add 75 µl to each well of the antibody-coated plate. 3.a. In case a limited number of samples has to be tested: Add 25 µl of each standard (including the blank or 25 µl Sample Diluent) and the samples to duplicate wells of the antibody-coated plate. CSF samples should be vortexed before testing. 3.b. In case a larger number of samples has to be tested (more than 3 strips): Using a multi-channel pipette, transfer 25 µl from each well of the polypropylene plate to the corresponding well on the antibody-coated plate (see Manipulations procedures, Uncoated polypropylene plate). 4. Make sure that standards and CSF samples are adequately mixed by carefully tapping the stripholder or by shaking 1 minute at 1000 rpm. Cover the strips with an adhesive sealer. Incubate for one hour at room temperature (18-30 C). 5. Prepare conjugate working solution 2 just before the end of step Wash each well 5 times (see Washing procedures). 7. Add 100 µl conjugate working solution 2 to each well. Cover the strips with a new adhesive sealer and incubate for 30 minutes at room temperature (18-30 C).

17 17 INNOTEST ß-AMYLOID (1-42) 8. Prepare substrate working solution just before the end of step Wash each well 5 times (see Directions for washing). 10. Add 100 µl substrate working solution to each well. Incubate for 30 minutes at C in the dark. 11. To stop the reaction, add 50 µl Stop Solution to each well in the same sequence and at the same time intervals as the substrate solution. Tap the stripholder carefully to ensure optimal mixing. 12. Read (within 15 minutes after step 11) the absorbance at 450 nm (single wavelength). For dual wavelength analysis, 690 nm or 620 nm can be used as the reference wavelength. Results Validation - The absorbance at 450 nm of the individual blanks should be lower than The absorbance at 450 nm of the highest standard should be higher than 1.5. NOTE: - Absorbance values for dual wavelength (450 nm, 620 nm) analysis differ approx. 50 mod from the single wavelength values. This does not affect the final outcome of the test. Calculation of the results Calculate the mean absorbance for the standard solutions and the unknown samples. Repeat the test if individual values differ by more than 20%. Construct the standard curve by plotting the mean absorbance values obtained for each of the ß-amyloid (1-42) standard solutions on the vertical (Y) axis versus the corresponding ß-amyloid (1-42) concentrations on the horizontal (X) axis. Draw the best fitting curve through these points. NOTE: - A sigmoidal curve fitting is recommended (sigmoidal dose-response with variable slope, four parameter logistic equation or Logit-Log). Using the mean absorbance value of each unknown CSF sample, determine the corresponding concentration of ß-amyloid (1-42) in pg/ml from the standard curve. The concentration of samples can only be determined if the absorbance is within the range of the standard curve. Extrapolation of results from OD values which lie above the highest standard point or below the lowest point of the standard curve can lead to incorrect results.

18 INNOGENETICS 18 Limitations of the procedure The INNOTEST ß-AMYLOID (1-42) assay procedure was designed to quantify ß-amyloid (1-42) in human cerebrospinal fluid. Insufficient data are available to interpret tests performed on other body fluids or brain tissue samples. Therefor, testing of such specimens with this test protocol is not recommended. Test performance Clinical results An external validation study was conducted in eight European and two US university centers involved in CSF research (Hulstaert F et al. Neurology 1999;52:1555). CSF levels of ß-amyloid (1-42) and tau protein were determined in residual CSF stored for research purposes of 150 AD patients (AD), 79 patients with non-ad types of dementia (NAD), 84 patients with other neurological disorders (ND), and 100 healthy volunteers or patients with disorders not associated with pathological conditions of the brain (CON). Quantification of ß-amyloid (1-42) was done using INNOTEST ß-AMYLOID (1-42), tau protein concentrations were measured using INNOTEST htau Ag. Median levels of ß-amyloid (1-42) were significantly lower in AD (487 pg/ml) than in CON (849 pg/ml; p=0.001), ND (643 pg/ml; p=0.001), and NAD (603 pg/ml; p=0.001). Discrimination of AD from CON and ND was significantly improved by the combined assessment of ß-amyloid (1-42) and tau protein (discrimination line Aß 42 = ,18 tau). At 85% sensitivity, specificity of the combined test was 86% (95% CI [81%, 91%]), compared with 55% (95% CI [47%, 62%]) for ß-amyloid (1-42) alone and 65% (95% CI [58%, 72%]) for tau protein alone. The combined test at 85% sensitivity was 58% (95% CI [47%, 69%]) specific for NAD. In conclusion, the combined measure of ß-amyloid (1-42) and tau protein concentrations meets the requirements set by the Consensus guidelines for discriminating AD from normal aging and specific neurological disorders such as depression: a useful biomarker should have a sensitivity and a specificity of more than 80% (Consensus report [no authors listed] Neurobiol Aging 1998;19:109). The combination of CSF-ß-amyloid (1-42) and CSF-tau marker concentrations may also be of use in the early diagnosis of AD (Andreasen N et al. Neurosci Lett 1999;273:5) or in other specific clinical settings (Andreasen N et al. Arch Neurol 2001;58:373). The discrimination of AD from non-ad types of dementia such as dementia with Lewy Bodies may further be improved using the quantification of CSF-phospho-

19 19 INNOTEST ß-AMYLOID (1-42) tau 181 (De Vreese K et al. [poster136] Abstract Book:18th International Conference of Alzheimer's Disease;2002 October 23-26;Barcelona, Spain). Assay range ß-amyloid (1-42) standard levels range between 125 and 2000 pg/ml (see standard curve in Fig. 1). Analytical specificity The INNOTEST ß-AMYLOID (1-42) is highly specific for amyloid peptides starting at amino acid 1 and ending at the carboxy-terminus at amino acid 42 or 43. The highest sensitivity was obtained with the (1-42) peptide, as confirmed by ELISA (Fig. 2) and BIAcore. Detection limit The lowest detection limit is ± 50 pg/ml and is calculated as the mean of 8 determinations of the sample diluent + 5 SD. Precision Intra-assay Standards The intra-assay variation was determined for several standard concentrations (2000, 1500, 1000, 750, 500, 250, 125 pg/ml), all tested six-fold. The coefficient of variation (%) for every standard was 2.5, 6.0, 7.3, 4.6, 7.6, 7.3, respectively. Samples The median CV (%) for 6 different 'quality control' samples (including two CSF), tested in six-fold, and with calculated values higher than 400 pg/ml, was 5.6. Inter-assay Samples The inter-assay variation was determined using 6 different 'quality control' samples (including two CSF), tested in triplicate, on six different test runs. The calculated median CV for all samples was 7.7%. Example of a standard curve A typical standard curve is shown below. This curve is for example only and a new standard curve must be generated for each assay.

20 INNOGENETICS 20 Table 1: Characteristic absorbance values for the standards Standard (pg/ml) Mean Figure 1: Example of a standard curve OD PEPTIDE Amyloid peptides (nm) Figure 2: Specificity of the INNOTEST ß-amyloid (1-42) More information can be found on

21 21 INNOTEST ß-AMYLOID (1-42) Français But recherché INNOTEST ß-AMYLOID (1-42) est un test immunoenzymatique destiné au dosage des protéines ß-amyloïde (1-42) dans le liquide céphalo-rachidien (LCR) humain. L'utilisation combinée de concentrations de protéine ß-amyloïde (1-42) et de marqueur tau dans le LCR permet d'établir une différentiation entre la maladie d'alzheimer (MA) et les troubles de sénescence normaux ou autres troubles neurologiques tels que la dépression (Hulstaert F et al. Neurology 1999;52:1555), pourvu que chaque laboratoire valide la ligne de discrimination décrite dans la section relative aux performances. Principe du test INNOTEST ß-AMYLOID (1-42) est un test immunoenzymatique en phase solide au cours duquel le peptide amyloïde est d'abord capturé par un anticorps monoclonal (21F12) lié sur la phase solide. Les échantillons de CSF sont ajoutés en volumes de 25 µl puis incubés avec un anticorps biotinylé (3D6). Cet anticorps est ensuite détecté à l'aide d'un conjugué streptavidine-peroxydase. Après addition d'une solution de substrat, les échantillons positifs vont développer une coloration (bleue). La réaction est stoppée par l'addition d'acide sulfurique qui produit une couleur jaune. La densité optique est alors mesurée à 450 nm. Réactifs Description, préparation et recommandations de conservation - Tous les réactifs, ouverts ou fermés, sont stables jusqu'à la date de péremption indiquée sur l'emballage s'ils sont conservés à 2-8 C dans leurs flacons d'origine. Le standard concentré doit être conservé à -20 C ou moins à son arrivée. Ce standard est conditionné séparément. Ne pas utiliser les réactifs après la date de validité. - Tous les réactifs, ainsi que le sachet aluminium contenant les bandelettes, doivent être ramenés à température ambiante (18-30 C) environ 30 minutes avant utilisation et remis immédiatement au réfrigérateur après usage. Pour éviter la condensation à l'intérieur des puits, le sachet en aluminium doit être maintenu fermé jusqu'à stabilisation des bandelettes à température ambiante.

22 INNOGENETICS 22 Réactifs fournis: Composant Quantité Réf. Description Microplaque 1 x sachet en aluminium contenant un sensibilisée support de microplaque avec 12 bandelettes de 8 puits sensibilisés et un sachet de silicagel comme dessiccant. Diluant 1 x 30 ml Tampon phosphate avec protéines Échantillon stabilisatrices et 0,01% MIT/0,1% CAA comme conservateur, utilisé pour diluer le standard et les échantillons à haute concentration de ß-amyloïde (1-42). Conjugué 1 1 x 0,2 ml IgG de souris anti-ß-amyloïde (1-42) marquées à la biotine, dans un tampon phosphate avec protéines stabilisatrices et 0,01% MIT/0,1% CAA comme conservateur. Diluer 100x avec du Diluant Conjugué 1 avant utilisation (voir préparation des réactifs). La solution de travail de Conjugué 1 est stable 24 heures conservée à température ambiante. Conjugué 2 1 x 0,3 ml Complexe streptavidine-peroxydase contenant 0,02% MIT et 0,02% bromonitrodioxane comme conservateurs. Diluer 100x avec du Diluant Conjugué 2 avant utilisation (voir préparation des réactifs). La solution de travail de Conjugué 2 doit obligatoirement être préparée extemporanément pour chaque test. Diluant 1 x 20 ml Tampon phosphate avec protéines Conjugué 1 stabilisatrices et 0,01% MIT/0,1% CAA comme conservateur, utilisé pour diluer le Conjugué 1. Diluant 1 x 30 ml Tampon phosphate contenant 0,15% Conjugué 2 Proclin 300, de la caséine bovine comme stabilisateur et de l'aprotinine bovine comme inhibiteur de protéase, utilisé pour diluer le Conjugué 1. Substrat 1 x 0,3 ml Tétraméthylbenzidine (TMB) dissous TMB dans du diméthylsulfoxide (DMSO). Diluer 100x dans du Tampon Substrat avant utilisation (Voir préparation des

23 23 INNOTEST ß-AMYLOID (1-42) réactifs). La solution de travail de Substrat doit être préparée extemporanément pour chaque test. Tampon 1 x 30 ml Tampon de citrate de phosphate Substrat contenant 0,02% de peroxyde d'hydrogène, utilisé pour diluer le TMB Substrat. Solution 1 x 30 ml Acide sulfurique 0,9 N d'arrêt Solution de 1 x 60 ml Tampon phosphate contenant 0,01% Lavage MIT/0,09% CAA, à diluer 25x dans de l'eau distillée ou déminéralisée avant utilisation. Préparer un minimum de 40 ml de solution de lavage diluée par puits. Des cristaux de sel peuvent s'être formés dans la solution de lavage concentrée après stockage à 2-8 C. Ces cristaux doivent être de nouveau entièrement dissous. La solution de lavage diluée est stable 24 heures conservée à température ambiante. Standard 3 x 0,2 ml Laisser le Standard décongeler sur la Concentration paillasse et vortexer avant utilisation. variable ( Préparer ± 1500 µl du standard le plus pg/ml) élevé (2000 pg/ml) dans du Diluant à utiliser avec Echantillon, en fonction de la le numéro de concentration du Standard gelé lot de kit (concentration sur l'étiquette). Utiliser la correspondant concentration 2000 pg/ml pour la préparation des autres standards (cf. tableau). Une fois décongelé, le Standard ne peut plus être conservé pour une utilisation ultérieure. De même, on éliminera les dilutions sériées après usage. Utiliser exclusivement des tubes en polypropylène pour la préparation de tous les standards. Plaque en 1 - Pour distribution des échantillons avant polypropylène non transfert sur la microplaque sensibilisée sensibilisée, afin de prévenir tout glissement de réactivité.

24 INNOGENETICS 24 Feuilles adhésives Sachet 1 - Pour la conservation des bandelettes plastique inutilisées. minigrip Préparation des réactifs Préparation de 1500 µl du standard ß-amyloïde (1-42) 2000 pg/ml Y = concentration du Standard concentré fourni avec le kit X = volume de Y à utiliser pour la préparation d'une solution 2000 pg/ml X = 1500 (= volume à préparer) x 2000 (= concentration du standard le plus élevé) µl Y Conc. (pg/ml) Standard Diluant Échantillon 2000 => A X µl X µl 1500 => B 150 µl de A 50 µl 1000 => C 200 µl de A 200 µl 500 => D 200 µl de C 200 µl 250 => E 200 µl de D 200 µl 125 => F 200 µl de E 200 µl REMARQUE: - Changer de cône pour chaque niveau de standard et mélanger soigneusement chaque flacon de standard avant de l'utiliser pour les dilutions sériées. Préparation de solutions de travail de Conjugué 1 et 2, et de solution de travail du substrat 8 puits 16 puits 32 puits 64 puits 96 puits CONJ 1 en µl CONJ DIL 1 en ml puits 16 puits 32 puits 64 puits 96 puits CONJ 2 en µl CONJ DIL 2 en ml puits 16 puits 32 puits 64 puits 96 puits SUBS en µl SUBS BUF en ml Préparation de la solution de lavage diluée 8 puits 16 puits 32 puits 64 puits 96 puits SOL LAV 25x en ml H 2 O en ml

25 25 INNOTEST ß-AMYLOID (1-42) Matériel nécessaire mais non fourni - Eau distillée ou déminéralisée. - Pipettes de précision avec des cônes à usage unique pour délivrer des volumes de µl. Une pipette multicanaux pour délivrer 25, 50, 75, 100 et 200 µl est recommandée pour l'addition des échantillons, des solutions de travail de Conjugué, de la solution de travail du Substrat et de la Solution d'arrêt. - Agitateur Vortex ou appareil équivalent. - Tubes en polypropylène pour la dilution des échantillons et la préparation des Standards. - Laveur de microplaques. En alternative, le lavage peut être réalisé à l'aide d'une pipette à répétition délivrant des volumes de 0.4 ml et d'un système d'aspiration. - Chronomètre. - Papier absorbant. - Lecteur de microplaques avec un filtre à 450 ± 5 nm et éventuellement 620 ou 690 nm pour des lectures en double longueur d'onde, dont la linéarité est comprise entre 0 et 3000 ou plus. - Flacons jetables pour la préparation des solutions de travail. - Récipient d'évacuation des matériels potentiellement contaminés. - Agitateur de microplaques (1000 tr/min). En alternative, le mélange peut être réalisé en tapotant le côté de la microplaque. Règles de sécurité et protection de l'environnement - Se référer à la fiche sécurité du fabricant et à l'étiquetage pour toute information sur les produits potentiellement dangereux. R43, S24-37 Irritant! (Xi) Eviter tout contact avec la peau. Peut entraîner des irritations par contact avec la peau. Porter des gants de protection. Contient du chloro-2 acétamide: CONJ 1, CONJ DIL 1, SAMP DIL, STAND ß-amyloïde (1-42). R36/37/38, S Irritant! (Xi) Irritant pour les yeux, la peau et le système respiratoire. Ne pas inhaler. Eviter tout contact avec la peau. En cas de contact avec les yeux, rincer immédiatement à grande eau et consulter de suite un médecin. Contient DMSO: SUBS TMB 100x.

26 INNOGENETICS 26 - Les échantillons doivent toujours être manipulés comme potentiellement infectieux. Tous les produits biologiques doivent être considérés comme potentiellement infectieux et seront manipulés avec les précautions d'usage. Seul un personnel qualifié doit être autorisé à réaliser ce test. L'élimination des produits biologiques doit être effectuée en respectant les règles appliquées en laboratoire. Autoclaver 15 minutes minimum à 121 C. Incinérer le matériel jetable. Mélanger les déchets liquides avec de l'hypochlorite de sodium de sorte que la concentration finale en hypochlorite de sodium soit de ± 1%. Laisser en contact une nuit avant élimination. ATTENTION: Les liquides contenant de l'acide doivent être neutralisés avant ajout d'hypochlorite de sodium. REMARQUE: Précautions spéciales pour le matériel contaminé par l'encéphalopathie spongiforme transmissible (ESP)/le prion: Inactivation des échantillons Les échantillons cliniques, par exemple le LCR, doivent être autoclavés ou immergés dans une solution d'hypochlorite de sodium donnant ppm de chlore libre pendant 1 heure, avant élimination finale par incinération. Elimination des déchets Tout le matériel classifié comme déchet clinique doit être éliminé par incinération dans un site d'incinération agréé. Pour la manipulation des déchets en toute sécurité, utiliser des conteneurs hermétiques, par exemple sacs doubles, en cas de nécessité. Eviter toute contamination externe du conteneur. Références: - Advisory Committee on Dangerous Pathogens (UK) - Spongiform Encephalopathy Advisory Committee - Transmissible Spongiform Encephalopathy Agents: Safe Working and the Prevention of Infection - Organisation Mondiale de la Santé (OMS): WHO Infection Control Guidelines for Transmissible Spongiform Encephalopathies. - STOP SOLN contient de l'acide sulfurique 0,9 N, CONJ 2 contient du MIT/Bromonitrodioxane comme conservateur et CONJ DIL 2 contient du Proclin 300 comme conservateur. - L'utilisation d'un équipement de protection individuelle est nécessaire: gants et lunettes de protection doivent être portés lors de la manipulation de matériel dangereux ou infectieux. - Les déchets doivent être traités en accord avec les règles d'élimination des déchets en vigueur dans l'institution. Toutes les réglementations fédérales, nationales et locales doivent également être observées.

27 27 INNOTEST ß-AMYLOID (1-42) Echantillons - Liquide céphalo-rachidien conservé à -20 C (de préférence à -80 C), recueilli dans des tubes polypropylène. Les échantillons doivent être dépourvus de cellules et non hémolysés. - Ce test n'est pas conçu pour tester des sérums ou des surnageants de cultures. - Il est recommandé de distribuer les échantillons en petites quantités aliquotées pour éviter des cycles de congélation/décongélation répétés. Des cycles de congélation/décongélation répétés produiront des concentrations incorrectes. - Les échantillons de LCR présentant une valeur de OD 450 nm supérieure à la valeur du standard le plus élevé peuvent être dilués dans du Diluant Echantillon. - Les échantillons présentant des agrégats insolubles doivent être centrifugés (4000g pendant 10 minutes) avant de procéder au test. - Le verre ou le polystyrène peuvent absorber la ß-amyloïde (1-42). L'utilisation de flacons en polypropylène est fortement recommandée pour la préparation des dilutions de LCR ou de standard. Remarques et précautions - Ne pas mélanger les composants provenant de coffrets portant un numéro de lot différent. - Tous les récipients utilisés pour la préparation des solutions de travail de Conjugué et de Substrat doivent être parfaitement propres pour éviter la contamination de ces solutions. - Tenir les microplaques ELISA par les côtés pour éviter de contaminer les puits. - Eviter toute contamination microbienne des réactifs. - S'assurer de l'homogénéité des échantillons et des Standards dilués avant utilisation. - Changer de cône pour chaque aliquote d'échantillon. - S'assurer du dépôt effectif de l'échantillon dans les micropuits. - Pour éviter toute contamination, ne pas toucher le haut des puits avec les cônes lors de l'ajout des échantillons ou de la solution de travail de Conjugué. - Eliminer toute bulle d'air présente en tapotant doucement la microplaque ou en mélangeant dans un agitateur pour microplaques pendant 1 minute à 1000 tr/min. - Ne pas exposer la solution de travail de Substrat à une lumière vive pendant l'incubation ou la conservation. Placer la plaque à l'obscurité pendant l'incubation du substrat. La solution de travail de substrat doit être incolore au moment de son utilisation. Si elle est bleue, elle doit être remplacée.

28 INNOGENETICS 28 - La Solution d'arrêt, la solution de travail de Substrat ou les solutions de travail de Conjugués ne doivent pas être mises en contact avec du métal ou des ions métalliques pour éviter la formation d'une coloration indésirable. - Si le Conjugué ou le Substrat ne peut être distribué immédiatement après le lavage, retourner les microplaques sur un papier absorbant imprégné de Solution de Lavage pour un temps n'excédant pas 15 minutes. Procédures de lavage La procédure de lavage en interne est établie avec un laveur automatique: Columbus M8/2ch (Tecan). Le protocole est disponible sur demande. Pour les autres laveurs ou protocoles, réaliser un lavage automatique comme suit: - Pré-rincer le laveur avec de la solution de lavage. - Réaliser 5 cycles de lavage respectant: un volume de remplissage de 400 µl/puits une hauteur de distribution permettant un remplissage complet du puits un temps nécessaire à un cycle aspiration/lavage/trempage égal à 30 secondes environ. Réaliser un lavage manuel de la manière suivante: - Aspirer complètement le liquide de toutes les puits en insérant délicatement un embout d'aspiration (dispositif d'aspiration) sur le fond de chaque puits. - Attention à ne pas griffer l'intérieur de la puits. - Après aspiration, remplir les puits à l'aide de 400 µl de solution de lavage. - Eliminer le liquide des puits. - Répéter ces manipulations cinq fois. - Après le lavage, retourner la microplaque et la tapoter sur un papier absorbant. Un lavage incomplet peut affecter notablement les résultats. La contamination microbienne de la Solution de lavage et/ou du dispositif de lavage peut entraîner des problèmes majeurs. En cas de problème, décontaminer les bouteilles de lavage et le dispositif de lavage durant la nuit avec une solution désinfectante appropriée. Rincer à l'eau distillée après décontamination. Procédures de manipulation Directives de distribution des échantillons sur la microplaque en polypropylène non sensibilisée L'incubation des échantillons s'effectuant à température ambiante (18-30 C) pendant 60 minutes, le temps de distribution doit être réduit au strict minimum. Afin de réduire le glissement de réactivité, une microplaque en polypropylène

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