FSH-ELISA - DIAsource Immunoassays
Contents
1. Como regla general la reacci n enzim tica es linealmente proporcional al tiempo y a la temperatura El pipeteo de todos los calibradores muestras y controles debe estar completado en 6 minutos Tenerlo en cuenta especialmente para el pipeteo manual 6 2 Procedimiento de ensayo Cada uno debe incluir una curva de calibradores 1 Asegurar el n mero deseado de pocillos en el recipiente 2 Dispensar 25 pL de cada Calibrador Control y muestras con puntas nuevas en los pocillos adecuados 3 Dispensar 100 pL de Enzyme Conjugate a cada pocillo Mezclar totalmente durante 10 segundos Es importante mezclar completamente en este paso 4 Incubar durante 30 minutes a temperatura ambiente 5 Sacudir en rgicamente el contenido de los pocillos Lavar los pocillos 5 veces con agua destilada 400 uL por pocillo Realizar un golpe seco de los pocillos contra el papel absorbente para eliminar las gotas residuales Nota importante La sensibilidad y la precisi n de este ensayo se ve marcadamente influenciada por la realizaci n correcta del proceso de lavado Adicionar 100 pL de Substrate Solution a cada pocillo Incubar durante 10 minutes a temperatura ambiente Parar la reacci n enzim tica mediante la adici n de 50 uL de Stop Solution a cada pocillo Leer la OD a 450410 nm con un lector de microplacas dentro de los 10 minutos despu s de la adici n de la Stop Solution OND 6 3 C lculo de los Resultados 1 Calcular l2. Substrate Extraction Buffer Concentrate Cartridge Streptavidin HRP E Pipette Wash buffer Revision nr 140723 P I Number 1701000 ACETONITRILE SERUM SPE BUF ANTISERUM IMMUNOADSORBENT DIL CAL REC SOLN PEG EXTR ELU GEL PRE NEUTR TRACEUR Ab Ag Ab Ag CONJ CHROM CHROM AGENT HRP BUF ACID DIST TRAY PMSF A STRIP SUB EXTR CART SAV PIPETTE WASH Simbolos utilisados Consultar las instrucciones de uso Limitaci n de temperatura l Fecha de caducidad E Codigo de lote g l N mero de cat logo Control Producto sanitario jara diagn stico in vitro Fabricante Contenido suficiente para lt n gt ensayos Soluci n de lavado concentrada Calibrador cero Calibrador Control Trazador Trazador Trazador concentrada Trazador concentrada Tubos Tampon de incubacion Acetonitrilo Suero Diluyente de Muestra l Tamp n de diluci n Antisuero Inmunoadsorbente iluyente
3. 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 2 REFERENCES LITERATURE Marshall J C Clinic in Endocrinol Metab 4 545 1975 Jeffcoate S L Clinic in Endocrinol Metab 4 521 1975 Cohen K L Metabolism 26 1165 1977 Shome B and Parlow A F J Clin Endocrinol Metab 39 199 1974 Lundy L E Lee S G Levy W et al Obstet Gynecol 44 14 1974 Ross F T Vande Wiele R L and Franty A G Text of Endocrinol Chapter 7 Ed R H Williams W B Saunders Philadelphia 1981 7 Speroff L Clinic Gynecol Endocrinol and Infert Chapter 3 Ed L Speroff R H Glass and M G Kase Williams amp Wilkins Baltimore 1978 8 Rebar R W Erickson G F and Yen S S C Fertil Steril 37 35 1982 9 Catt K J and Pierce J G Reprod Endocrinol Chapter 2 Ed S S C Yen and R B Jaffe Philadelphia 1978 10 Leonard J M Leach R B Couture M and Paulsen C A J Clinic Endocrinol 34 209 1972 11 Reiter E O and Lulin H E J Clinic Endocrinol 33 957 1971 12 Abraham G E Ed Radioassay Systems in Clinic Endocrinol Marcel Dekker Inc New York 1981 13 Engvall E Methods in Enzymology Volume 70 VanVunakis H and Langone J J eds Academic Press New York 419 1980 14 Uotila M Ruoslah
4. with the kit Be sure that everything is understood The microplate contains snap off strips Unused wells must be stored at 2 c to 8 in the sealed foil pouch and used in the frame provided Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for each step Use reservoirs only for single reagents This especially applies to the substrate reservoirs Using a reservoir for dispensing a substrate solution that had previously been used for the conjugate solution may turn solution colored Do not pour reagents back into vials as reagent contamination may occur Mix the contents of the microplate wells thoroughly to ensure good test results Do not reuse microwells Do not let wells dry during assay add reagents immediately after completing the rinsing steps Allow the reagents to reach room temperature 21 C to 26 C before starting the test Temperature will affect the absorbance readings of the assay However values for the patient samples will not be affected Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes 2 Do not smoke eat drink or apply cosmetics in areas where specimens or kit reagents are handled Catalogue Nr KAPD1288 P I Number 1701236 en Revision Nr 150622 1 Wear disposable latex gloves when handling specimens and reagents Microbial contamination of reagents or specimens may give false results Handling shoul
5. FSH conjugado con la Peroxidasa de r bano oo 4 mme Substrate Solution Soluci n de sustrato 1 vial 14 mL listo para usar HRON TME Tetrametilbencidina TMB Stop Solution Soluci n de parada 1 vial 14 mL listo para usar STOP contiene 0 5M H2SO4 Evitar el contacto con la Soluci n de parada Puede causar irritaci n y quemaduras en al piel BBH Nota Se puede solicitar el Calibrador 0 para la diluci n de la muestra 4 2 Equipamiento y material requerido pero no provisto Lector de microplacas calibrado 450 10 nm ej Microtiter Plate Reader Micropipetas de precisi n variable calibradas Papel absorbente Agua destilada 4 3 Almacenamiento y estabilidad del kit Cuando se almacena a 2 C 8 C los reactivos sin abrir mantienen su reactividad hasta la fecha de caducidad No utilizar los reactivos mas alla de esta fecha Los reactivos abiertos han de almacenarse a 2 C 8 C Las placas multipocillo han de almacenarse a 2 C 8 C Una vez se ha abierto la bolsa hay que tener cuidado y cerrarla de nuevo Los kits abiertos conservan su actividad durante dos meses si se almacenan como se ha descrito arriba 4 4 Preparaci n de los Reactivos Dejar que todos los reactivos y el n mero requerido de tiras alcancen la temperatura ambiente antes de usarse Calibradores Reconstituir los contenidos liofilizados de los viales de los calibradores con 1 mL de agua destilada Nota Los calibrador
6. CID DIST TRAY PMSF STRIP SUB EXTR CART SAV PIPETTE WASH Used symbols Consult instructions for use Storage temperature Use by Batch code Catalogue number Control In vitro diagnostic medical device Manufacturer Contains sufficient for lt n gt tests Wash solution concentrated Zero calibrator Calibrator Control Tracer Tracer Tracer concentrated Tracer concentrated Tubes Incubation buffer Acetonitrile Serum j Specimen diluent Dilution buffer Antiserum Tamunoadsorbent Calibrator diluent Reconstitution solution Polyethylene glycol Extraction solution Elution solution Bond Elut Silica cartridges Pre treatment solution l Neutralization solution E Tracer buffer Microtiterplate i HRP Conjugate HRP Conjugate HRP Conjugate concentrate HRP Conjugate concentrate Conjugate buffer Chromogenic TMB concentrate Chromogenic TMB solution Substrate buffer Stop solution ncubation serum Buffer AP Conjugate Substrate PNPP Biotin conjugate concentrate Precipitating Agent Avidine HRP concentrate Assay buffer Biotin conjugate f Specific Antibody Streptavidin HRP concentrate Non specific binding 2nd Antibody Acidification Buffer Distributor ncubation trays PMSF solution Protect from light Dot Strip
7. CIONES e Este kit es solamente para diagn stico in vitro e Por favor se usa solo la versi n valida de la met dico t cnico incluido aqu en el lit e Para obtener informaci n de las sustancias peligrosas incluidas en el kit por favor mirar las hojas de los datos de seguridad del material e Todos los reactivos en este kit de ensayo que contienen suero o plasma humano se han ensayado y confirmado ser negativos para HIV I II HBsAg y HCV mediante procedimientos aprobados por la FDA Sin embargo todos los reactivos deben ser tratados tanto en su uso como dispensaci n como potencialmente biopeligrosos Evitar contacto con Stop Solution que contiene H2SO4 0 5 M Puede provocar irritaci n y quemaduras en la piel Nunca pipetear con la boca y evitar el contacto de los reactivos y las muestras con la piel y con membranas mucosas No fumar comer beber o usar cosm ticos en reas donde las muestras o los reactivos del kit est n siendo usados Usar guantes de l tex cuando se utilicen las muestras y los reactivos La contaminaci n microbiana de los reactivos o las muestras puede dar resultados err neos e El manejo debe realizarse de acuerdo a los procedimientos definidos por las gu as o regulaci n nacionales de seguridad de sustancias biopeligrosas e No utilizar los reactivos despu s de su fecha de caducidad que aparece en las etiquetas del kit e Todos los vol menes indicados han de ser realizados de acuerdo con el protocolo Los resu
8. DA Ge CE FSH ELISA KAPDI288 DIAsource ImmunoAssays S A Rue du Bosquet 2 B 1348 Louvain la Neuve Belgium LOT 150622 1 FSH ELISA KAPD1288 IN VITRO DIAGNOSTIC USE DlAsource ImmunoAssays SA Rue du Bosquet 2 B 1348 Louvain la Neuve Belgium Tel 32 10 84 99 11 Fax 32 10 84 99 90 1 INTRODUCTION 1 1 Intended Use The DiAsource FSH ELISA is an enzyme immunoassay for the quantitative in vitro diagnostic measurement of Follicle Stimulating Hormone FSH in serum 1 2 Summary and Explanation Follicle Stimulating Hormone FSH and Luteinizing Hormone LH are intimately involved in the control of the growth and reproductive activities of the gonadal tissues which synthesize and secrete male and female sex hormones through a negative feedback relationship 1 2 FSH is a glycoprotein secreted by the basophil cells of the anterior pituitary Gonadotropin releasing hormone GnRH produced in the hypothalamus controls the release of FSH from the anterior pituitary Like other glycoproteins such as LH TSH and HCG FSH consists of subunits designated as alpha and beta Hormones of this type have alpha subunits that are very similar structurally therefore the biological and immunological properties of each are dependent on the unique beta subunit 3 4 5 In the female FSH stimulates the growth and maturation of ovarian follicles by acting directly on the receptors located on the granulosa cells follicular steroido
9. actice or other applicable national calibrators 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 DlAsource 11 2 Therapeutic Consequences Therapeutic consequences should never be based on laboratory results alone even if all test results are in agreement with the items as stated under point 11 1 Any laboratory result is only a part of the total clinical picture of a patient Only in cases where the laboratory results are in acceptable agreement with the overall clinical picture of the patient should therapeutic consequences be derived The test result itself should never be the sole determinant for deriving any therapeutic consequences 11 3 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 11 2 are also invalid Regardless in the event of any claim
10. d be done in accordance with the procedures defined by an appropriate national biohazard safety guideline or regulation Do not use reagents beyond expiry date as shown on the kit labels All indicated volumes have to be performed according to the protocol Optimal test results are only obtained when using calibrated pipettes and microtiterplate readers Do not mix or use components from kits with different lot numbers It is advised not to exchange wells of different plates even of the same lot The kits may have been shipped or stored under different conditions and the binding characteristics of the plates may result slightly different Avoid contact with stop solution containing 0 5 m h2soz lt may cause skin irritation and burns Some reagents contain proclin bnd and mit as preservatives In case of contact with eyes or skin flush immediately with water Tmb substrate has an irritant effect on skin and mucosa In case of possible contact wash eyes with an abundant volume of water and skin with soap and abundant water Wash contaminated objects before reusing them If inhaled take the person to open air Chemicals and prepared or used reagents have to be treated as hazardous waste according to the national biohazard safety guideline or regulation For information on hazardous substances included in the kit please refer to Material Safety Data Sheets Material Safety Data Sheets for this product are available upon request directly from D
11. de calibrador luci n de Reconstitucion Glicol Polietileno l Soluci n de extracci n Soluci n de eluci n Cartuchos Bond Elut Silica Soluci n de Pre tratamiento Soluci n de Neutralizaci n Tamp n de trazador 7 Placa de microvaloraci n HRP Conjugado HRP Conjugado HRP Conjugado concentrada HRP Conjugado concentrada Tampon de Conjugado Crom gena TMB concentrada Soluci n Crom gena TMB Tamp n de sustrato Soluci n de Parada Suero de Incubaci n Tamp n i AP Conjugado Sustrato PNPP i Concentrado de conjugado de biotina Agente de precipitaci n Concentrado avidina HRP Tamp n de ensayo Conjugado de biotina Anticuerpo espec fico Estreptavidina HRP Concentrado Uni n no espec fica Segundo anticuerpo Tamp n de Acidificaci n Distribuidor Bandejas de incubaci n Soluci n de PMSF Proteger de la luz Tries Dot Sustrato Concentrado de tamp n de extracci n Cartucho Estreptavidina HRP l Pipeta 7 Tampon de lavado Revision nr 140723
12. e its own normal and abnormal values In a study conducted with apparently normal healthy adults using the DIAsource FSH ELISA the following values are observed Population 5 95 Percentile mIU mL Follicular Phase 2 0 10 0 Mid cycle 7 0 20 0 Luteal Phase 2 0 10 0 Post Menopausal 20 0 100 0 8 QUALITY CONTROL Good laboratory practice requires that controls be run with each calibration curve A statistically significant number of controls should be assayed to establish mean values and acceptable ranges to assure proper performance It is recommended to use control samples according to state and federal regulations The use of control samples is advised to assure the day to day validity of results Use controls at both normal and pathological levels The controls and the corresponding results of the QC Laboratory are stated in the QC certificate added to the kit The values and ranges stated on the QC sheet always refer to the current kit lot and should be used for direct comparison of the results Catalogue Nr KAPD1288 P I Number 1701236 en Revision Nr 150622 1 It is also recommended to make use of national or international Quality Assessment programs in order to ensure the accuracy of the results Employ appropriate statistical methods for analysing control values and trends If the results of the assay do not fit to the established acceptable ranges of control materials patient results should be considered invalid In t
13. eactivos condiciones de almacenamiento e incubaci n m todos de aspiraci n y lavado Despu s de comprobar los asuntos mencionados arriba sin encontrar ning n error contactar con su distribuidor o con DlAsource ImmunoAssays S A directamente 9 CARACTER STICAS DEL ENSAYO 9 1 Rango din mico del ensayo El rango del ensayo se encuentra entre 0 86 100 mIU mL 9 2 Especificidad de los Anticuerpos Reactividad Cruzada Consultar el manual de usuario en ingl s 9 3 Sensibilidad Anal tica La sensibilidad anal tica se calcul a partir de la media mas dos desviaciones est ndar de veinte 20 r plicas del Calibrador 0 y result ser 0 856 mIU mL 9 4 Precisi n Consultar el manual de usuario en ingl s 9 5 Recuperaci n Consultar el manual de usuario en ingl s 9 6 Linealidad Consultar el manual de usuario en ingl s Catalogue Nr KAPD1288 P I Number 1701236 es Revision Nr 150622 1 10 LIMITACIONES DE USO Cualquier manipulaci n inadecuada de las muestras o modificaciones del ensayo pueden influenciar los resultados 10 1 Sustancias que pueden interferir Hemoglobina hasta 4 mg mL Bilirrubina hasta 0 5 mg mL y Triglic ridos hasta 30 mg mL no influencian los resultados del ensayo 10 2 Interferencias con drogas Hasta ahora no se han encontrado sustancias drogas conocidas por nosotros que tengan influencia en la medida de FSH en una muestra 10 3 Efecto Gancho Dosis Elevada No se ha observado efec
14. en el ensayo 113 Responsabilidad Cualquier modificaci n del kit y o cambio o mezcla de cualquier componente procedentes de kits de lotes diferentes puede afectar negativamente a los resultados esperados y en la validez de todo el test Esas modificaciones y o cambios invalidad cualquier reclamaci n de reposici n Las reclamaciones emitidas debidas a una mala interpretaci n de los resultados de laboratorio por parte del comprador referidos al punto 11 2 son tambi n inv lidas A pesar de todo en el caso de cualquier reclamaci n la responsabilidad del fabricante no excede el valor del kit Cualquier da o provocado al kit durante su transporte no est sujeto a la responsabilidad del fabricante 12 REFERENCIAS BBLIOGRAF A Consultar el manual de usuario en ingl s Fecha de revisi n 2015 06 22 Catalogue Nr KAPD1288 P I Number 1701236 es Revision Nr 150622 1 P I Number 1701000 ACETONITRILE SERUM ANTISERUM IMMUNOADSORBENT DIL CAL REC SOLN PEG EXTR ELU GEL PRE NEUTR TRACEUR Ab Ag Ab Ag CONJ CHROM CHROM AGENT HRP BUF A
15. es reconstituidos son estables durante 2 meses a 2 8 Para per odos m s largos congelar a 20 C 4 5 Eliminaci n del Kit La eliminaci n del kit debe realizarse de acuerdo con las leyes nacionales En las hojas de datos de seguridad se proporciona informaci n especial de este producto 4 6 Kits de ensayo da ados En caso de que exista cualquier da o severo del kit de ensayo o de sus componentes ha de informarse por escrito a DIAsource ImmunoAssays S A no mas tarde de una semana despu s de recibir el kit No deben utilizarse componentes da ados para llevar a cabo un ensayo Han de almacenarse hasta que se encuentre una soluci n Despu s de esto deben ser eliminados de acuerdo con las leyes oficiales 5 MUESTRAS S lo debe utilizarse suero en este ensayo No usar muestras hemol ticas ict ricas o lip micas Tener en cuenta No deben usarse muestras que contengan acida s dica 5 1 Toma de muestras Suero Recoger la sangre por punci n en la vena ej Sarstedt Monovette para el suero permitir coagulaci n y separar el suero por centrifugaci n a temperatura ambiente No centrifugar antes de la coagulaci n completa Las muestras de pacientes que reciben terapia anticoagulante requieren m s tiempo para coagular 5 2 Almacenamiento de las muestras Las muestras deben ser tapadas y pueden ser almacenadas hasta 5 d as a 2 C 8 C antes del ensayo Las muestras almacenadas por un per odo de tiempo mas larg
16. genesis is promoted and LH production is stimulated The LH produced then binds to the theca cells and stimulates steroidogenesis Increased intraovarian estradiol production occurs as follicular maturation advances thereupon stimulating increased FSH receptor activity and FSH follicular binding FSH LH and estradiol are therefore intimately related in supporting ovarian recruitment and maturation in women 6 7 9 FSH levels are elevated after menopause castration and in premature ovarian failure The levels of FSH may be normalized through the administration of estrogens which demonstrate a negative feedback mechanism Abnormal relationships between FSH and LH between FSH and estrogen have been linked to anorexia nervosa and polycystic ovarian disease Although there are significant exceptions ovarian failure is indicated when random FSH concentrations exceed 40 mIU mL 8 The growth of the seminiferous tubules and maintenance of spermatogenesis in men are regulated by FSH However androgens unlike estrogens do not lower FSH levels therefore demonstrating a feedback relationship only with serum LH 10 11 12 For reasons not fully understood azospermic and oligospermic males usually have elevated FSH levels Tumors of the testes generally depress serum FSH concentrations but levels of LH are elevated as determined by radioimmunoassay lt has been postulated that the apparent LH increase may be caused by cross reactivity with hCG like substances
17. his case please check the following technical areas Pipetting and timing devices photometer expiration dates of reagents storage and incubation conditions aspiration and washing methods After checking the above mentioned items without finding any error contact your distributor or DIAsource directly 9 PERFORMANCE CHARACTERISTICS 9 1 Assay Dynamic Range The range of the assay is between 0 86 100 mIU mL 9 2 Specificity of Antibodies Cross Reactivity The following substances were tested for cross reactivity of the assay Produced Color Intensity Hormone Tested Concentration Equivalent to FSH in serum mIU mL 10 000 mIU mL 50 000 mIU mL 100 000 mIU mL 50 plU mL TSH WHO 2 IRP hCG WHO 1 IRP75 537 100 mIU mL LH WHO 1 IRP 68 40 250 mIU mL 500 mIU mL Prolactin WHO 1 IRP 100 ng mL 75 504 200 ng mL st 100 ng mL hGH WHO 1 IRP 66 217 Soo Halt 9 3 Sensitivity The analytical sensitivity was calculated from the mean plus two standard deviations of twenty 20 replicate analyses of Calibrator 0 and was found to be 0 856 mIU mL 9 4 Reproducibility 9 4 1 Intra Assay The within assay variability is shown below Sample 1 2 s fas Low 9 4 2 Inter Assay The between assay variability is shown below Sampe 1 2 3 SD mimo ov 6 gt ET Catalogue Nr KAPD1288 P I Number 1701236 en Revision Nr 150622 1 9 5 Recovery Samples have been spiked by adding FSH
18. ine sus valores normales e inusuales En un estudio con adultos aparentemente sanos utilizando el DIAsource FSH ELISA se observaron los siguientes valores oe Percentil 5 95 Hombres 0 89 11 72 Mujeres Fase Folicular 2 0 10 0 Ciclo menstrual 7 0 20 0 Fase Luteal 2 0 10 0 Posmenopausia 20 0 100 0 8 CONTROL DE CALIDAD Se recomienda usar muestras control de acuerdo con las leyes estatales y federales El uso de muestras control ser recomienda para asegurar la validez diaria de los resultados Usar controles tanto a niveles normal como patol gico Los controles y los correspondientes resultados del Laboratorio de control de calidad est n fijados en el certificado de control de calidad que acompa an al kit Los valores y los rangos fijados en la hoja del control de calidad se refieren siempre al kit actual y deben usarse para la comparaci n directa de los resultados Es recomendable tambi n hacer uso de programas de Aseguramiento de la Calidad nacionales o internacionales para asegurar la exactitud de los resultados Utilizar m todos estad sticos apropiados para el an lisis de los valores y tendencia de los controles Si los resultados del ensayo no se ajustan a los rangos aceptables establecidos en los controles los resultados obtenidos de los pacientes han de considerarse inv lidos En este caso por favor comprobar las siguientes reas t cnicas Pipeteo y tiempo empleado fot metro fecha de caducidad de los r
19. lAsource 4 REAGENTS 4 1 Reagents provided 1 Microtiterwells 12 x 8 break apart strips 96 wells Wells coated with anti FSH monoclonal antibody 2 CAL FSH Calibrators N 0 to 5 6 vials lyophilized 1 mL EEN Concentration 0 5 10 20 50 100 mIU mL Conversion 6 mlU mL The calibrators are calibrated against 1 International Calibrator for Follicle Stimulation Hormone FSH human recombinant for immunoassay NIBSC code 92 510 See Preparation of Reagents Contains non mercury preservative 3 Enzyme Conjugate 1 vial 11 mL Ready for use Anti FSH antibody conjugated to horseradish peroxidase Contains non mercury preservative 4 Substrate Solution 1 vial 14 mL Ready for use Tetramethylbenzidine TMB 5 Stop Solution 1 vial 14 mL Ready for use Contains 0 5 M H2SO4 Avoid contact with the stop solution It may cause skin irritations and burns Note Additional Calibrator O for sample dilution is available on request 4 2 Material required but not provided A microtiter plate calibrated reader 450 10 nm Calibrated variable precision micropipettes Absorbent paper Distilled or deionized water Timer Linear graph paper of software for data reduction 4 3 Storage Conditions When stored at 2 C 8 C unopened reagents will retain reactivity until expiration date Do not use reagents beyond this date Opened reagents must be stored at 2 C 8 C Microtiter wells mu
20. ltados ptimos del ensayo se obtienen solo cuando se utilizan pipetas y lectores de microplacas calibrados e No mezclar o usar componentes de kits con distinto n mero de lote Se recomienda no intercambiar pocillos de distintas placas incluso si son del mismo lote Los kits pueden haber sido enviados o almacenados bajo diferentes condiciones y las caracter sticas de uni n de las placas pueden resultar diferentes e Los compuestos qu micos y los reactivos preparados o utilizados han de tratarse como residuos peligrosos de acuerdo con las gu as o regulaci n nacionales de seguridad de sustancias biopeligrosas e Las hojas de los datos de seguridad de este producto est n disponibles bajo pedido directamente a DIAsource ImmunoAssays S A Catalogue Nr KAPD1288 P I Number 1701236 es Revision Nr 150622 1 4 COMPONENTES DEL KIT 4 1 Componentes del Kit 1 Microtiterwells Placas multipocillo 12 x 8 tiras separables 96 pocillos Pocillos recubiertos con anticuerpo anti FSH monoclonal Calibrador Calibrador 0 5 6 viales liofilizados 1 mL N Concentraciones 0 5 10 20 50 100 mIU mL Conversi n 6 mIU mL 1 ng mL Los calibradores est n calibrados seg n WHO 1 International Calibrador parar FSH NIBSC c digo 92 510 1 Intern Calibrador for Follicle Stimulation Hormone FSH human recombinant for immunoassay CAL E Enzyme Conjugate Conjugado enzim tico 1 vial 11 mL listo para usar Anticuerpo anti
21. mmended that the wells be read within 10 minutes after adding the Stop Solution OND 6 3 Calculation of Results 1 Calculate the average absorbance values for each set of calibrators controls and patient samples 2 Construct a calibration curve by plotting the mean absorbance obtained from each calibrator against its concentration with absorbance value on the vertical Y axis and concentration on the horizontal X axis 3 Using the mean absorbance value for each sample determine the corresponding concentration from the calibration curve 4 Automated method The results in the Instructions For Use have been calculated automatically using a 4 Parameter curve fit 4 parameter Rodbard or 4 Parameter Marquardt are the preferred methods Other data reduction functions may give slightly different results 5 The concentration of the samples can be read directly from this calibration curve Samples with concentrations higher than that of the highest calibrator have to be further diluted For the calculation of the concentrations this dilution factor has to be taken into account 6 3 1 Example of Typical Calibration Curve The following data is for demonstration only and cannot be used in place of data generations at the time of assay Calibrator Optical Units 450 nm Calibrator 0 0 mIU mL 0 07 Calibrator 1 5 mIU mL Calibrator 5 100 mIU mL 7 EXPECTED NORMAL VALUES It is strongly recommended that each laboratory should determin
22. o han de congelarse s lo una vez a 20 C antes del ensayo Las muestras descongeladas deben invertirse varias veces antes del ensayo Catalogue Nr KAPD1288 P I Number 1701236 es Revision Nr 150622 1 5 3 Diluci n de las muestras Si en un ensayo inicial se encuentra una muestra que presenta valores mayores que el calibrador mas concentrado ha de diluirse con Calibrador 0 y volver a ensayarse como se describe en el Procedimiento de Ensayo Para el c lculo de las concentraciones habr que tener en cuenta el factor de diluci n Ejemplo a diluci n 1 10 10 uL Suero 90 uL Calibrador 0 mezclar totalmente b diluci n 1 100 10 uL diluci n a 1 10 90 uL Calibrador O mezclar totalmente 6 PROCEDIMIENTO DE ENSAYO 6 1 Consideraciones generales Todos los reactivos y muestras han de estar a temperatura ambiente antes de su uso Todos los reactivos deben mezclarse sin formar espuma Una vez se ha comenzado el ensayo deben completarse todos los pasos sin interrupci n Utilizar puntas de pipeta de pl stico nuevas para cada calibrador control o muestra para evitar combinaciones cruzadas La absorbancia es funci n del tiempo de incubaci n y la temperatura Antes de comenzar el ensayo se recomienda que todos los reactivos est n preparados tapas removidas todos los pocillos que se necesiten asegurados en recipiente etc Esto asegurar un tiempo similar para cada paso de pipeteo sin que haya interrupciones
23. os valores de absorbancia media para cada conjunto de calibradores controles y muestras de pacientes 2 Construir una curva est ndar mediante la representaci n de la absorbancia media obtenida para cada calibrador frente a su concentraci n con el valor de absorbancia en el eje vertical Y y la concentraci n en el eje horizontal X 3 Usando el valor de absorbancia media de cada muestra determinar la concentraci n correspondiente a partir de la curva calibrador 4 M todo automatizado Los resultados en la Instrucciones de uso se han calculado autom ticamente usando una curva de regresi n 4 Par metros 4 Par metros Rodbard o 4 Par metros Marquardt son los m todos preferidos Otras funciones de regresi n dar n lugar a resultados sensiblemente diferentes 5 La concentraci n de las muestra puede leerse directamente de la curva de calibradores Las muestras con concentraciones superiores al mayor est ndar han de diluirse Para el c lculo de las concentraciones hay que tener en cuenta el factor de diluci n 6 3 1 Ejemplo de una Curva Calibrador T pica Los siguientes datos son solamente para la explicaci n y no pueden ser utilizados en lugar de los datos generados en el momento del ensayo Catalogue Nr KAPD1288 P I Number 1701236 es Revision Nr 150622 1 Calibrador Unidades pticas 450 nm Calibrador 1 5 miU Calibrador 5 100 mIU mL 7 VALORES ESPERADOS Se recomienda encarecidamente que cada laboratorio determ
24. rum allow to clot and separate serum by centrifugation at room temperature Do not centrifuge before complete clotting has occurred Patients receiving anticoagulant therapy may require increased clotting time 5 2 Specimen Storage and Preparation Specimens should be capped and may be stored for up to 5 days at 2 C 8 C prior to assaying Specimens held for a longer time should be frozen only once at 20 C prior to assay Thawed samples should be inverted several times prior to testing 5 3 Specimen Dilution If in an initial assay a specimen is found to contain more than the highest calibrator the specimens can be diluted with Ctandard 0 and reassayed as described in Assay Procedure For the calculation of the concentrations this dilution factor has to be taken into account Example a dilution 1 10 10 uL Serum 90 uL Calibrator 0 mix thoroughly b dilution 1 100 10 uL dilution a 1 10 90 uL Calibrator 0 mix thoroughly 6 ASSAY PROCEDURE 6 1 General Remarks All reagents and specimens must be allowed to come to room temperature before use All reagents must be mixed without foaming Once the test has been started all steps should be completed without interruption Use new disposal plastic pipette tips for each calibrator control or sample in order to avoid cross contamination Absorbance is a function of the incubation time and temperature Before starting the assay it is recommended that all reagent
25. s are ready caps removed all needed wells secured in holder etc This will ensure equal elapsed time for each pipetting step without interruption As a general rule the enzymatic reaction is linearly proportional to time and temperature Pipetting of all calibrators samples and controls should be completed within 6 minutes Note this especially for manual pipetting Catalogue Nr KAPD1288 P I Number 1701236 en Revision Nr 150622 1 6 2 Test Procedure Each run must include a calibration curve 1 Secure the desired number of Microtiterwells in the holder Dispense 25 HL of each Calibrator controls and samples with new disposable tips into appropriate wells 3 Dispense 100 pL Enzyme Conjugate into each well Thoroughly mix for 10 seconds It is important to have a complete mixing in this step 4 Incubate for 30 minutes at room temperature 5 Briskly shake out the contents of the wells Rinse the wells 5 times with aqua dest 400 uL per well Strike the wells sharply on absorbent paper to remove residual droplets Important note The sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure Add 100 uL of Substrate Solution to each well Incubate for 10 minutes at room temperature Stop the enzymatic reaction by adding 50 pL of Stop Solution to each well Determine the absorbance OD of each well at 450 10 nm with a microtiter plate reader It is reco
26. secreted by tumors of the testes 11 12 High levels of FSH in men may be found in primary testicular failure and Klinefelter syndrome Elevated concentrations are also present in cases of starvation renal failure hyperthyroidism and cirrhosis 1 3 2 PRINCIPLE OF TEST The DiAsource FSH ELISA Kit is a solid phase enzyme linked immunosorbent assay ELISA based on the sandwich principle The microtiter wells are coated with a monoclonal antibody directed towards a unique antigenic site on a FSH molecule An aliquot of patient sample containing endogenous FSH is incubated in the coated well with enzyme conjugate which is an anti FSH monoclonal antibody conjugated with horseradish peroxidase After incubation the unbound conjugate is washed off The amount of bound peroxidase is proportional to the concentration of FSH in the sample Having added the substrate solution the intensity of colour developed is proportional to the concentration of FSH in the patient sample 3 WARNINGS AND PRECAUTIONS This kit is for in vitro diagnostic use only For professional use only All reagents of this test kit which contain human serum or plasma have been tested and confirmed negative for hiv i ii hosag and hcv by fda approved procedures All reagents however should be treated as potential biohazards in use and for disposal Before starting the assay read the instructions completely and carefully Use the valid version of the package insert provided
27. solutions with known concentrations in a 1 1 ratio The Recovery has been calculated by multiplication of the ratio of the measurements and the expected values with 100 Measured Expected Conc k FSH FSH mIU mL mIU mL Endogenous Added FSH FSH mIU mL mIU mL Recovery Endogenous FSH 2 added FSH because of a 1 1 dilution of serum with spike material 9 6 Linearity Measured Expected Conc Conc mIU mL mIU mL Recovery 10 LIMITATIONS OF USE 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 1 Interfering Substances Haemoglobin up to 4 mg mL Bilirubin up to 0 5 mg mL and Triglyceride up to 30 mg mL have no influence on the assay results 10 2 Drug interferences Until today no substances drugs are known to us which have an influence to the measurement of FSH in a sample Catalogue Nr KAPD1288 P I Number 1701236 en Revision Nr 150622 1 10 3 High Dose Hook Effect No hook effect was observed in this test up to 1600 mIU mL of FSH 11 LEGAL ASPECTS 11 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 Pr
28. st be stored at 2 C 8 C Once the foil bag has been opened care should be taken to close it tightly again Opened kits retain activity for two months if stored as described above Catalogue Nr KAPD1288 P I Number 1701236 en Revision Nr 150622 1 4 4 Reagents Preparation Bring all reagents and required number of strips to room temperature prior to use Calibrators Reconstitute the lyophilized contents of the calibrator vial with 1 mL Aqua dest Note The reconstituted calibrators are stable for 2 months at 2 8 For longer storage freeze at 20 4 5 Disposal of the Kit 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 Sheets see chapter 13 4 6 Damaged Test Kits In case of any severe damage to the test kit or components DlAsource 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 SPECIMEN COLLECTION AND PREPARATION Only serum should be used in this assay Do not use haemolytic icteric or lipaemic specimens Please note Samples containing sodium azide should not be used in the assay 5 1 Specimen Collection Serum Collect blood by venipuncture e g Sarstedt Monovette for se
29. ti E and Engvall E J Immunol Methods 42 11 1981 Ds QUE ON Revision date 2015 06 22 Catalogue Nr KAPD1288 P I Number 1701236 en Revision Nr 150622 1 FSH ELISA KAPD1288 IN VITRO DIAGNOSTIC USE DlAsource ImmunoAssays SA Rue du Bosquet 2 B 1348 Louvain la Neuve Belgium Tel 32 10 84 99 11 Fax 32 10 84 99 90 1 INTRODUCCI N El Kit de inmunoensayo enzim tico DiAsource FSH ELISA proporciona los materiales necesarios para la determinaci n cuantitativa del Hormona estimulante folicular FSH en suero Este ensayo est dise ado solo para diagn stico in vitro 2 FUNDAMENTO DEL ENSAYO El Kit DIAsource FSH ELISA es un ensayo en fase s lida de inmunoadsorci n unido a enzimas ELISA basado en el principio del s ndwich Los pocillos de las placas est n recubiertos con un anticuerpo monoclonal dirigido contra un nico foci antig nico en una mol cula de FSH Se incuba una al cuota de una muestra perteneciente a un paciente que contiene FSH end gena en los pocillos recubiertos con el enzima conjugado que es un anticuerpo anti FSH conjugado con la peroxidasa end gena Despu s de la incubaci n se lava el conjugado que no se ha unido La cantidad de peroxidasa unida es proporcional a la concentraci n de FSH en la muestra Cuando se a ade la soluci n del sustrato de la peroxidasa la intensidad del color desarrollado es proporcional a la concentraci n de FSH en la muestra del paciente 3 PRECAU
30. to gancho en este ensayo hasta 1600 mIU mL de FSH 11 ASPECTOS LEGALES 11 1 Fiabilidad de los Resultados El ensayo debe realizarse exactamente de acuerdo a las instrucciones del fabricante Mas a n el usuario debe ajustarse estrictamente a las reglas BPL Buenas Pr cticas de Laboratorio o a otros calibradores y o leyes nacionales aplicables Esto es especialmente relevante para el uso de reactivos control Es importante incluir siempre dentro del procedimiento de ensayo un n mero suficiente de controles para validar la exactitud y la precisi n del ensayo Los resultados del ensayo son v lidos s lo si todos los controles se encuentran dentro de los rangos especificados y si todos los otros par metros del ensayo se encuentran dentro de las especificaciones dadas para el ensayo En cado de alguna duda o inquietud por favor contactar con DIAsource ImmunoAssays S A 11 2 Consecuencias Terap uticas Las consecuencias terap uticas nunca deben basarse s lo en los resultados de laboratorio incluso si todos los resultados del ensayo est n de acuerdo con los asuntos fijados en el punto 11 1 Cualquier resultado de laboratorio es solamente una parte del cuadro cl nico de un paciente Solamente en los casos donde los resultados de laboratorio est n en acuerdo con todo el cuadro cl nico de un paciente se pueden derivar consecuencias terap uticas Nunca deben derivarse consecuencias terap uticas a partir de solamente el resultado obtenido
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