Myelin Basic Protein ELISA. MyBioSource.com. For the determination of Myelin Basic Protein (MBP) in human cerebrospinal fluid (CSF).

Transcription

1 Product information Userś Manual Myelin Basic Protein ELISA For the determination of Myelin Basic Protein (MBP) in human cerebrospinal fluid (CSF). 96 Storage: 2-8 C RUO For research use only, not for use in diagnostic procedures. 1

2 Contents 1 INTRODUCTION PRINCIPLE OF THE TEST WARNINGS AND PRECAUTIONS REAGENTS COLLECTION AND HANDLING OF UNKNOWNS ASSAY PROCEDURE QUALITY CONTROL PERFORMANCE CHARACTERISTICS LIMITATIONS OF USE LEGAL ASPECTS REFERENCES / LITERATURE... 7 SYMBOLS USED WITH ELISAS... 8 Updated

3 1 INTRODUCTION 1.1 Intended Use For the determination of Myelin Basic Protein (MBP) in human cerebrospinal fluid (CSF). For research use only, not for use in diagnostic procedures. 1.2 Summary and Explanation MBP constitutes about one-third of the proteins in myelin sheath and appears to be specific for myelin (1). It consists of a single chain of 170 amino acid residues (molecular weight:18,400 Dalton) whose sequence is very similar in different species (1, 2). Elevated concentrations of MBP in CSF have been found in subjects with active multiple sclerosis (3, 4), following neurosurgery (5), head trauma (6,7) and with acute stroke (8). It has been claimed that MBP determination in CSF is valuable in following the activity of multiple sclerosis in its acute phase (9, 10) and also as a marker for the severity of brain damage in head trauma (7) and acute stroke (8). It has also been shown that determination of MBP in CSF may be of significance in the study of necrotizing leukoencephalopathy in children of young adults with acute lymphoblasic leukemia (11), or it may serve as an index of severity of disease in subjects with neuro-becher s disease (12) and in subjects with encephalitis of various origins (13-18). 2 PRINCIPLE OF THE TEST The MBP test kit employs a solid phase enzyme linked immunosorbant assay procedure. Unknowns are added to MBP specific antibody coated microplate wells and incubated. During the incubation period endogenous MBP binds to the antibody. The microplate is then washed to remove unreacted materials and incubated with horseradish peroxidase labeled antibody specific to MBP. The microplate is again washed 3,3,5,5 tetramethylbenzidine, a chromogen and an enzyme substrate, peroxide are then added and incubated. The enzyme reaction is terminated by the addition of stop solution and the color developed is read in an EIA colorimetric analyzer. The measured absorbance is directly related to the concentration of MBP bound to the microplate wells. The concentration of MBP in CSF is determined from a calibration curve. 3 WARNINGS AND PRECAUTIONS 1. This kit is for research use only, not for use in diagnostic procedures. 2. All materials are for in-vitro diagnostic use only and not for internal or external use in animals as humans. 3. Some of the reagents in this kit contain merthiolate. Merthiolate may be toxic if ingested. Care should be taken to avoid ingestion. 4. Some of the reagents in this kit contain sodium azide. Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. On disposal, flush with a large volume of water to prevent azide accumulation. Sodium azide is also toxic. Care should be taken to avoid ingestion. 5. Handle all compounds and all unknowns as if capable of transmitting hepatitis and the acquired immunodeficiency syndrome. This kit contains components of human origin. When tested by approved methods, the components were found negative for Hepatitis B surface antigen and HIV antibody. However, no known tests can guarantee that such material does not contain the causative agent of viral hepatitis of acquired immunodeficiency syndrome. 6. This kit is designed to measure MBP in CSF unknowns. Any CSF contaminated with blood should be either spun down or not be assayed. Updated

4 4 REAGENTS 4.1 Reagents provided Calibrator A - 0 ng/ml, liquid Description Calibrator B - 4 ng/ml, freeze dried Control Low - Concentration is indicated on the certificate of analysis. Control High - Concentration is indicated on the certificate of analysis. Antibody enzyme conjugate reagent - Buffered reagent containing MBP specific antibody horseradish peroxidase enzyme conjugate. Merthiolate (0.01%) added as preservative. MBP antibody coated microplate - 96 well microplate containing MBP specific antibody adsorbed, packaged in a zip-lock bag. The plate consists of twelve polystyrene removable strips mounted in a frame. Each strip includes eight wells. Chromogen and substrate solution - Buffered reagent containing 3.3:5.5 tetramethylbenzidine and peroxidase. Merthiolate (0.01%) added as a preservative. Stop solution - One vial containing stop solution. Quantity 10 ml 2 vials 2 vials 2 vials 5 ml 1 plate 5 ml 10 ml Buffer solution - One bottle containing concentrated wash buffer solution. 0.01% merthiolate added as a preservative. 60 ml * All calibrators are buffered reagent containing sodium azide (0.01%) as a preservative. 4.2 Materials required but not provided EIA microplate analyzer capable of reading at 450 nm. Refrigerator (2-8 C). Distilled or deionated water. 50 and 100 μl precision pipets. Volumetric pipet, 1-10 ml. Container, 1, 100, 500 ml. Pasteur Pipets and a bulb. 4.3 Storage Conditions / Expiration Unopened test kit is stable until the expiration date shown on the kit label. Store the unopened test kit in refrigerator (2-8 C). Discard the calibrators and controls after each use. Store all other reagents in refrigerator (2-8 C) Updated

5 4.4 Reagent Preparation All components must be at ambient temperature before use. A. Prepare a 1:10 dilution of desired volume of 10x concentrated wash buffer with distilled water. B. Reconstitute Calibrator B with 1.0 ml of Calibrator A. C. Dilute 1:2 an aliquot of Calibrator B with Calibrator A (e.g 0.5 ml B ml A) and label it as Calibrator C, 2.0 ng/ml. D. Dilute 1:2 an aliquot of Calibrator C with Calibrator A (e.g 0.5 ml C ml A) and label it as calibrator D, 1.0 ng/ml. E. Dilute 1:2 an aliquot of Calibrator D with Calibrator A (e.g 0.5 ml D ml A) and label it as Calibrator E, 0.5 ng/ml. F. Reconstitute low and high controls with 1.0 ml of Calibrator A. 4.5 Disposal of the Kit Note: A. All dilutions and reconstitutions must be done immediately prior to assay. B. All reconstituted and diluted calibrators and controls must be discarded after one use. The disposal of the kit must be made according to the national regulations. Special information for this product is given in the Material Safety Data Sheet. 4.6 Damaged Test Kits In case of any severe damage to the test kit or components, has to be informed in writing, at the latest, one week after receiving the kit. Severely damaged single components should not be used for a test run. They have to be stored until a final solution has been found. After this, they should be disposed according to the official regulations. 5 COLLECTION AND HANDLING OF UNKNOWNS 5.1 Preparation and Storage of Unknowns Collect 1-2 ml CSF in a plastic tube and remove any insoluble material by centrifuging in cold at 2000 RPM for five minutes. The procedure calls for 50 μl of CSF per assay. The unknowns may be stored under refrigeration (2-8 C) for 24 hours or frozen (-20 C or lower) if kept for a longer period of time. Aliquot the unknowns before freezing to avoid repeated freeze-thaw cycles. Any CSF contaminated with blood should be spun down or not be used. 6 ASSAY PROCEDURE 6.1 Test Procedure 1. Arrange wells in duplicate. 2. Pipet 50 μl of calibrators, controls, and unknowns to the appropriate wells. 3. Incubate at room temperature for 2 hours. 4. After the incubation, decant the content from each well, wash the wells five times with prepared wash buffer. Tap the wells gently and blot the rims to remove all residual droplets. 5. Add 50 μl enzyme antibody conjugate to each well. 6. Incubate at room temperature for 1 hour. 7. After the incubation, decant the content from each well, wash the wells five times with buffer. Tap the wells gently and blot the rims to remove all residual droplets. 8. Add 50 μl chromogen substrate reagent to each well. 9. Incubate at room temperature for 15 minutes. 10. Add 100 μl stop solution to each well to stop the color development. Mix well by a slow shaking of the wells for a few seconds. 11. Determine the absorbance of all the wells in an EIA colorimetric analyzer at 450 nm against air blank (use reference wavelength 630 nm) within 5 minutes. Updated

6 6.2 Results 1. Subtract calibrator 1 ( 0 ng/ml) absorbancy (wells no. 1 and 2) from absorbancy of the other wells. 2. Using a linear-linear graph, plot absorbancy on the vertical axis (y-axis), against concentration (ng/ml) on the horizontal axis (x-axis) for each of the calibrators. MBP concentrations for the controls and unknowns may then be estimated from the graph by interpolation. 3. Unknowns with values greater than the highest calibrator should be diluted with calibrator 1 (0 ng/ml) diluent and reassayed. 7 QUALITY CONTROL The reliability of test results should be monitored by the routine use of control reagents of known MBP concentrations. Two quality control pools are supplied with the kit and should be analyzed with each assay. The results should be charted from assay to assay and the overall performance checked periodically. 8 PERFORMANCE CHARACTERISTICS 8.1 Precision a. Intra-assay Within assay variation was determined from 9-10 single determination of MBP in three separate pools. The CV varied from 4-9%. b. Inter-assay Between assay variation was determined from three separate runs in duplicate of three pools of MBP. The CV varied from 2-4%. 8.2 Sensitivity Ten 0 ng/ml calibrator wells were processed along with a calibration curve. Mean and standard deviation were calculated for the absorbance of the ten 0 ng/ml calibrator wells. Analytical sensitivity was then calculated from 3 standard deviation and was found to be 0.5 ng/ml. 8.3 Recovery Recovery studies were done by spiking two samples with different concentration of MBP. The recovery varied from %. 8.4 Parallelism Three samples were serially diluted with the kit s 0 ng/ml calibrator. The recovery varied from %. 9 LIMITATIONS OF USE Please Note: For Research Use Only, Not for Use In Diagnostic Procedures. Reliable and reproducible results will be obtained when the assay procedure is performed with a complete understanding of the package insert instruction and with adherence to good laboratory practice. Any improper handling of samples or modification of this test might influence the results. 10 LEGAL ASPECTS 10.1 Reliability of Results The test must be performed exactly as per the manufacturer s instructions for use. Moreover the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable national standards and/or laws. This is especially relevant for the use of control reagents. It is important to always include, within the test procedure, a sufficient number of controls for validating the accuracy and precision of the test. The test results are valid only if all controls are within the specified ranges and if all other test parameters are also within the given assay specifications. In case of any doubt or concern please contact. Updated

7 10.2 Liability Any modification of the test kit and/or exchange or mixture of any components of different lots from one test kit to another could negatively affect the intended results and validity of the overall test. Such modification and/or exchanges invalidate any claim for replacement. Claims submitted due to customer misinterpretation of laboratory results subject to point are also invalid. Regardless, in the event of any claim, the manufacturer s liability is not to exceed the value of the test kit. Any damage caused to the test kit during transportation is not subject to the liability of the manufacturer. 11 REFERENCES / LITERATURE 1. Norton, W.T., and Poduslo, S.E. J. Neurochem 21, 749, Rumsby, M.G. and Crang, A.J., In The synthesis, assembly and turnover of cell surface components (Eds. Poste, G., and Nicholson, G.L.) Elsevier, Amsterdam, P257, Cohen, S.R., Brune, M.J., Herndon, R.M., and McKhann, G.M. Adv. Exp. Med. Biol. 100,513, Whitaker, J.N. Neurology 27, 911, Alling, C., Karlsson, B., and Vallfors, B.J., Neurol. 223, Palfreyman, J.W., Johnston, R.V., Ratcliff, J.G., Thomas, D.G.T., and Forbes, C.D. Clin Chim Acta 92, 403, Mukherjee, A., Vogt, R.F., and Linthicum, D.S., Clin. Biochem. 18, 304, Strand, T., Alling, C., Karlsson, B., Karlsson, I., and Winblad, B., Stroke 15, 138, Cohen, S.R., Brooks, B.R., Jubelt, B., Herndon, R.M., and McKhann, G.M. In Neurobiology of cerebrospinal fluid (Ed. Wood, J.H.) Plenum Press, New York, p 487, Cohen, S.R., Brooks, B.R., Jubelt, H.B., and McKhann, G.M., Ann Neurol. 8, 25, Gangji, D., Reaman, G.H., Cohen, S.R., Bleyer, W.A., and Poplack, D.G., N.Engl. J. Med. 303, 19, Ohta, M., Nishirani, H., Matsubara, F., and Inaba, G.N. Engl. J. Med. 302, 1093, Jacque, C., Delassalle, A., Rancurel, G., Raoul, M., Lesourd, B., and Legrand, J.C., Arch. Neurol. 39, 557, Carson, J.H., Barbarese, E., Braun, P.E., and McPherson, T.A., Proc. Natl. Acad. Sci. (USA) 75, 1976, Bashir, R.M., and Whitaker, J.N. Ann. Neurol. 7, 50, 1980 Updated

8 SYMBOLS USED WITH MyBioSource ELISAS Symbol English Deutsch Français Español Italiano European Conformity Consult instructions for use CE-Konfirmitätskennzeichnung Gebrauchsanweisung beachten Conforme aux normes européennes Consulter les instructions d utilisation Conformidad europea Conformità europea Consultare le istruzioni per l uso RUO In vitro diagnostic device For research use only In-vitro-Diagnostikum Nur für Forschungszwecke Usage Diagnostic in vitro Seulement dans le cadre de recherches Para uso Diagnóstico in vitro Sólo para uso en investigación Per uso Diagnostica in vitro Solo a scopo di ricerca Catalogue number Katalog-Nr. Référence Número de catálogo No. di Cat. Lot. No. / Batch code Chargen-Nr. No. de lot Número de lote Lotto no Contains sufficient for <n> tests/ Ausreichend für n Ansätze Contenu suffisant pour n tests Contenido suficiente para <n> ensayos Contenuto sufficiente per n saggi Storage Temperature Expiration Date Lagerungstemperatur Consulte las Instrucciones Mindesthaltbarkeitsdatum Température de conservation Temperatura de conservacion Temperatura di conservazione Date limite d utilisation Fecha de caducidad Data di scadenza Legal Manufacturer Hersteller Fabricant Fabricante Fabbricante Distributed by Distributor Vertreiber Distributeur Distribuidor Distributore Content Content Inhalt Contenu Contenido Contenuto Volume/No. Volume / No. Volumen/Anzahl Volume/Numéro Volumen/Número Volume/Quantità Microtiterwells Microtiterwells Mikrotiterwells Plaques de microtitration Placas multipocillo Micropozzetti Antiserum Antiserum Antiserum Antisérum Antisuero Antisiero Enzyme Conjugate Enzyme Completique Complexe enzyma- Enzyme Complex Enzymkomplex Complex enzimático Substrate Solution Stop Solution Stop Solution Stopplösung Solution d arrêt Solución de parada Soluzione d arresto Enzyme Conjugate Enzymkonjugat Conjugué enzymatique Conjugado enzimático Tracciante enzimatico Complesso enzimatico Substrate Solution Substratlösung Solution substrat Solución de sustrato Soluzione di substrato Zero Standard Zero Standard Nullstandard Zero Standard Estándar cero Standard zero Standard Standard Standard Standard Estándar Standard Control Control Kontrolle Contrôle Control Controllo Assay Buffer Assay Buffer Assaypuffer Tampon d essai Tampón de ensayo Tampone del test Wash Solution Wash Solution Waschlösung Solution de lavage Solución de lavado Soluzione di lavaggio 1N NaOH 1N NaOH 1N NaOH 1N NaOH 1N NaOH 1 N HCl 1 N HCl 1 N HCl 1N HCl 1 N HCl Sample Diluent Conjugate Diluent Sample Diluent Conjugate Diluent Probenverdünnungsmedium Konjugatverdünnungsmedium Solution pour dilution de l échantillon Solution pour dilution du conjugué Solución para dilución de la muestra Solución para dilución del conjugado 1N NaOH (idrossido di sodio 1N) Diluente dei campioni Diluente del tracciante Updated