LH-ELISA - DIAsource Immunoassays
Contents
1. Ab Ag Ab Ag CONJ CHROM CHROM SUB STOP INC BUF Ab CART SAV PIPETTE WASH SOLN Simbolos utilisados Consultar las instrucciones de uso Limitacion de temperatura Fecha de caducidad Codigo de lote Numero de catalogo Control Producto sanitario para 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 Tamp n de incubaci n Acetonitrilo Suero Diluyente de Muestra 7 Tamp n de diluci n Antisuero i Inmunoadsorbente Diluyente de calibrador 4 Soluci n de Reconstituci n Glicol Polietileno 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 Placa de microvaloraci n HRP Conjugado HRP Conjugado HRP Conjugado concentrada HRP Conjugado concentrada Tamp n 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 AP Conjugado Sustrato PNPP Concentrado de2. Ed W B Saunders Co 1981 10 Marshall J C Clinics in Endocrinal Metab 4 545 567 1975 11 Acosta A A M D and Wright G L Journal of Clinical Immunoassays 6 41 1983 12 Brown J Wood eds Churchill Livingston Co New York 7 42 1980 13 Yen S S C Vela P and Rankin J J Clin Endocrinol Metab 30 434 442 1970 14 Cohen K L Metabolism 26 1165 1177 1977 15 Engvall E Methods in Endocrinology Volume 70 Van Vunakis H and Langone J J eds Academic Press New York 419 492 1980 16 Uotila M Ruoslahti E and Engvall E J Immunol Methods 42 11 15 1981 ae O ON Revision date 2014 01 27 Catalogue Nr KAPD1289 P I Number 1701238 en Revision Nr 140127 1 LH ELISA KAPD1289 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 LH ELISA proporciona los materiales necesarios para la determinaci n cuantitativa del Hormona Luteinizante LH en suero Este ensayo est dise ado solo para diagn stico in vitro 2 FUNDAMENTO DEL ENSAYO El Kit DIAsource LH 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 policlonal dirigido contra un nico foci antig nico en u
3. I Number 1701238 en Revision Nr 140127 1 10 3 High Dose Hook Effect No hook effect was observed in this test up to 10 000 mIU mL of LH 11 LEGAL ASPECTS 111 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 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 DIAsource 112 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 comp
4. conjugado de biotina 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 l Estreptavidina HRP Pipeta Tamp n de lavado Revision nr 130513
5. 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 calibrador 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 KAPD1289 P I Number 1701238 es Revision Nr 140127 1 Calibrador Unidades Opticas 450 nm 7 VALORES ESPERADOS Se recomienda encarecidamente que cada laboratorio determine sus valores normales e inusuales En un estudio con adultos aparentemente sanos utilizando el DIAsource LH ELISA se observaron los siguientes valores Mujeres Fase folicular y luteal lt 20 Pico de LH 20 200 Mujeres posmenopausia 20 100 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 estan 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 refiere
6. DA Ge CE LH ELISA KAPD1289 DIAsource ImmunoAssays S A Rue du Bosquet 2 B 1348 Louvain la Neuve Belgium LOT 140127 1 LH ELISA KAPD1289 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 LH ELISA Kit is an enzyme immunoassay for the quantitative in vitro diagnostic measurement of the Luteinizing Hormone LH in serum 1 2 Summary and Explanation Luteinizing hormone LH is produced in both men and women from the anterior pituitary gland in response to luteinizing hormone releasing hormone LH RH or Gn RH which is released by the hypothalamus 1 3 LH also called interstitial cell stimulating hormone ICSH in men is a glycoprotein with a molecular weight of approximately 30 000 daltons 4 It is composed of two non covalently associated dissimilar amino acid chains alpha and beta 5 The alpha chain is similar to that found in human thyroid stimulating hormone TSH follicle stimulating hormone FSH and human chorionic gonadotropin hCG The difference between these hormones lie in the amino acid composition of their beta subunits which account for their immunological differentiation 6 8 The basal secretion of LH in men is episodic and has the primary function of stimulating the interstitial cells Leydig cells to produce testosterone The variation in LH conce
7. U 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 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 Catalogue Nr KAPD1289 P I Number 1701238 es Revision Nr 140127 1 10 2 Interferencias con drogas Hasta ahora no se han encontrado sustancias drogas conocidas por nosotros que tengan influencia en la medida de Hormona Luteinizante en una muestra 10 3 Efecto Gancho Dosis Elevada No se ha observado efecto gancho en este ensayo hasta 10 000 mlU mL de Hormona Luteinizante 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
8. ay The within assay variability is shown below Sampe 1 2 3 Mean mIU mL 15 72 28 33 SD mUmL 6 9 4 2 Inter Assay The between assay variability is shown below Mean mIU mL 15 36 28 96 SD mIU mL cv 11 02 ne gt fo Catalogue Nr KAPD1289 P I Number 1701238 en Revision Nr 140127 1 9 5 Recovery Samples have been spiked by adding LH 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 Endogenous Measured Conc Expected Recovery LH LH LH mIU mL mIU mL mIU mL Endogenous LH 2 added LH because of a 1 1 dilution of serum with spike material 9 6 Linearity Measured Conc mIU mL Expected Conc Recovery mIU mL 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 LH in a sample Catalogue Nr KAPD1289 P
9. be treated as potential biohazards in use and for disposal 3 Before starting the assay read the instructions completely and carefully Use the valid version of the package insert provided with the kit Be sure that everything is understood 4 The microplate contains snap off strips Unused wells must be stored at 2 C to 8 C in the sealed foil pouch and used in the frame provided 5 Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for each step Na Catalogue Nr KAPD1289 P I Number 1701238 en Revision Nr 140127 1 6 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 7 Mixthe contents of the microplate wells thoroughly to ensure good test results Do not reuse microwells 8 Do not let wells dry during assay add reagents immediately after completing the rinsing steps 9 Allow the reagents to reach room temperature 21 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 10 Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes 11 Do not smoke eat drink or apply cosmetics in areas where spec
10. clar 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 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 Asegurar el n mero deseado de pocillos en el recipiente 2 Dispensar 25 uL de cada Calibrador Control y muestras con puntas nuevas en los pocillos adecuados Dispensar 100 pL de Enzyme Conjugate a cada pocillo Mezclar totalmente durante 10 segundos Es importante
11. conception the developing embryo produces hCG which causes the corpus luteum to continue producing progesterone and estradiol The corpus luteum regresses if pregnancy does not occur and the corresponding drop in progesterone and estradiol levels results in menstruation The hypothalamus initiates the menstrual cycle again as a result of these low hormone levels 12 Patients suffering from hypogonadism show increased concentrations of serum LH A decrease in steroid hormone production in females is a result of immature ovaries primary ovarian failure polycystic ovary disease or menopause in these cases LH secretion is not regulated 10 13 A similar loss of regulatory hormones occurs in males when the testes develop abnormally or anorchia exists High concentrations of LH may also be found in primary testicular failure and Klinefelter syndrome although LH levels will not necessarily be elevated if the secretion of androgens continues Increased concentrations of LH are also present during renal failure cirrhosis hyperthyroidism and severe starvation 10 14 A lack of secretion by the anterior pituitary may cause lower LH levels As may be expected low levels may result in infertility in both males and females Low levels of LH may also be due to the decreased secretion of GnRH by the hypothalamus although the same effect may be seen by a failure of the anterior pituitary to respond to GnRH stimulation Low LH values may therefore indicate some d
12. de almacenarse hasta que se encuentre una soluci n Despu s de esto deben ser eliminados de acuerdo con las leyes oficiales 5 MUESTRAS En este ensayo debe usarse s lo suero 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 02 1388 001 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 48 horas a 2 C 8 C antes del ensayo Las muestras almacenadas por un per odo de tiempo mas largo 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 KAPD1289 P I Number 1701238 es Revision Nr 140127 1 5 3 Dilucion 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 O mez
13. do al kit durante su transporte no est sujeto a la responsabilidad del fabricante 12 REFERENCIAS BIBLIOGRAFIA Consultar el manual de usuario en ingl s Fecha de revisi n 2014 01 27 Catalogue Nr KAPD1289 P I Number 1701238 es Revision Nr 140127 1 P I Number 1701000 CAL CAL CONTROL Ag Ab Ag Ab T INC BUF ACETONITRILE SERUM DIL SPE DIL BUF ANTISERUM IMMUNOADSORBENT DIL CAL REC SOLN PEG EXTR ELU GEL PRE NEUTR TRACEUR Ab Ag Ab Ag CONJ CHROM CHROM 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 Tubes Manufacturer Contains sufficient for lt n gt tests Wash solution concentrated Zero calibrator Calibrator Control Tracer Tracer Tracer concentrated Tracer concentrated Incubation bu
14. ffer Acetonitrile Serum Specimen diluent Dilution buffer Antiserum Immunoadsorbent Calibrator diluent Reconstitution solution Polyethylene glycol Extraction solution Elution solution i Bond Elut Silica cartridges Pre treatment solution Neutralization solution Tracer buffer Microtiterplate HRP Conjugate HRP Conjugate HRP Conjugate concentrate HRP Conjugate concentrate Conjugate buffer Chromogenic TMB concentrate Chromogenic TMB solution Substrate buffer Stop solution Incubation serum i Buffer i AP Conjugate Substrate PNPP Biotin conjugate concentrate Assay buffer Avidine HRP concentrate Biotin conjugate Specific Antibody Streptavidin HRP concentrate Non specific binding 2nd Antibody Acidification Buffer Distributor Incubation trays PMSF solution Protect from light Dot Strip Substrate Extraction Buffer Concentrate l Cartridge Streptavidin HRP Pipette Wash buffer Revision nr 130513 P I Number 1701000 WASH CAL CAL CONTROL Ag Ab Ag Ab T INC BUF ACETONITRILE SERUM DIL SPE DIL BUF ANTISERUM IMMUNOADSORBENT DIL CAL REC PEG EXTR ELU GEL PRE NEUTR TRACEUR
15. he 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 4 Absorbance is a function of the incubation time and temperature Before starting the assay it is recommended that all reagents are ready caps removed all needed wells secured in holder etc This will ensure equal elapsed time for each pipetting step without interruption 5 As a general rule the enzymatic reaction is linearly proportional to time and temperature 6 Pipetting of all calibrators samples and controls should be completed within 6 minutes note this especially for manual pipetting 6 2 Test Procedure Each run must include a calibration curve Secure the desired number of Microtiterwells in the holder 2 Dispense 25 uL of each Calibrator controls and samples with new disposable tips into appropriate wells 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 Catalogue Nr KAPD1289 P I Number 1701238 en Revision Nr 140127 1 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 co
16. imens or kit reagents are handled 12 Wear disposable latex gloves when handling specimens and reagents Microbial contamination of reagents or specimens may give false results 13 Handling should be done in accordance with the procedures defined by an appropriate national biohazard safety guideline or regulation 14 Do not use reagents beyond expiry date as shown on the kit labels 15 All indicated volumes have to be performed according to the protocol Optimal test results are only obtained when using calibrated pipettes and microtiterplate readers 16 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 17 Avoid contact with Stop Solution containing 0 5 M H2SO4 It may cause skin irritation and burns 18 Some reagents contain Proclin 300 BND and or MIT as preservatives In case of contact with eyes or skin flush immediately with water 19 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 20 Chemicals and prepared or used reagents have to be treated as hazardous waste according to the national biohazard
17. 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 en el ensayo 11 3 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 provoca
18. 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 uL 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 Fe Or OO O 6 3 Calculo de los Resultados Calcular los valores de absorbancia media para cada conjunto de calibradores controles y muestras de pacientes Construir una curva calibrador 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 IFU se han calculado autom ticamente usando una curva de regresi n 4 PL 4 Par metros Log sticos Otras
19. mmended 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 this 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 Catalogue Nr KAPD1289 P I Number 1701238 en Revision Nr 140127 1 9 PERFORMANCE CHARACTERISTICS 9 1 Assay Dynamic Range The range of the assay is between 1 27 200 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 LH in Serum mIU mL NOTE Pregnancy results in elevated levels of hCG the use of the LH enzyme immunoassay test is not recommended during pregnancy or immediately post partum 9 3 Sensitivity The analytical sensitivity of the DIAsource ELISA was calculated by adding 2 standard deviations to the mean of 20 replicate analyses of the Zero Calibrator SO and was found to be 1 27 mIU mL 9 4 Reproducibility 9 4 1 Intra Ass
20. n Nr 140127 1 4 COMPONENTES DEL KIT 4 1 Componentes del Kit Microtiterwells Placas multipocillo 12x8 tiras separables 96 pocillos Pocillos recubiertos con anticuerpo anti Hormona Luteinizante monoclonal Calibrador Calibrador 0 5 6 viales liofilizados 1 mL Concentraciones 0 10 20 40 100 200 mIU mL Los calibradores est n calibrados seg n WHO 2nd International Calibrador parar LH IRP 80 552 Ver Preparaci n de los Reactivos Contiene 0 03 Proclin 300 0 015 BND y 0 010 MIT como conservante Enzyme Conjugate Conjugado enzim tico 1 vial 11 mL listo para usar Anticuerpo anti Hormona Luteinizante conjugado con la Peroxidasa de r bano Contiene 0 03 Proclin 300 0 015 BND y 0 010 MIT como conservante CHROM TMB Substrate Solution Soluci n de sustrato 1 vial 14 mL listo para usar Tetrametilbencidina TMB Stop Solution Soluci n de parada 1 vial 14 mL listo para usar SOLN contiene 0 5M H2SO Evitar el contacto con la Soluci n de parada Puede causar irritaci n y quemaduras en al piel AL 7 HE i gt STOP Y 4 E BND 5 bromo 5 nitro 1 3 dioxane Bromonitrodioxano MIT 2 methyl 2H isothiazol 3 one Metilisotiazolona 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 p
21. n 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 reactivos 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 1 27 200 mIU mL 9 2 Especificidad de los Anticuerpos Reactividad Cruzada Consultar el manual de usuario en ingl s NOTA El embarazo produce niveles elevados de hCG El uso del inmunoensayo del enzima LH no se recomienda durante el embarazo o inmediatamente tras el parto 9 3 Sensibilidad Anal tica La sensibilidad anal tica se calcul a partir de la media mas dos desviaciones calibrador de veinte 20 r plicas del Calibrador O y result ser 1 27 ml
22. na mol cula de Hormona Luteinizante Se incuba una al cuota de una muestra perteneciente a un paciente que contiene Hormona Luteinizante end gena en los pocillos recubiertos con el enzima conjugado que es un anticuerpo anti Hormona Luteinizante 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 Hormona Luteinizante 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 Hormona Luteinizante en la muestra del paciente 3 PRECAUCIONES e Este kit es solamente para diagn stico in vitro 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 1 11 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 H2SO 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 mues
23. ntrations in women is subject to the complex ovulatory cycle of healthy menstruating women and depends upon a sequence of hormonal events along the gonado hypothalamic pituitary axis The decrease in progesterone and estradiol levels from the preceeding ovulation initiates each menstrual cycle 9 10 As a result of the decrease in hormone levels the hypothalamus increases the secretion of gonadotropin releasing factors GnRF which in turn stimulates the pituitary to increase FSH production and secretion 4 The rising FSH levels stimulate several follicles during the follicular phase one of these will mature to contain the egg As the follicle develops estradiol is secreted slowly at first but by day 12 or 13 of a normal cycle increasing rapidly LH is released as a result of this rapid estradiol rise because of direct stimulation of the pituitary and increasing GnRF and FSH levels These events constitute the pre ovulatory phase 11 Ovulation occurs approximately 12 to 18 hours after the LH reaches a maximum level After the egg is released corpus luteum is formed which secretes progesterone and estrogen two feedback regulators of LH 3 10 The luteal phase rapidly follows this ovulatory phase and is characterized by high progesterone levels a second estradiol increase and low LH and FSH levels 12 Low LH and FSH levels are the result of the negative feedback effects of estradiol and progesterone on the hypotalamic pituitary axis After
24. 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 i 7 EXPECTED NORMAL VALUES It is strongly recommended that each laboratory should determine its own normal and abnormal values In a study conducted with apparently normal healthy adults using the DIAsource LH ELISA the following values are observed Population LH mIU mL Adult Female Follicular and luteal phase LH Surge 20 200 Female post menopausal 20 100 The results alone should not be the only reason for any therapeutic consequences The results should be correlated to other clinical observations and diagnostic tests 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 It is also reco
25. onents 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 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 12 REFERENCES 1 Harris G W and Naftolinf The Hypothalamus and Control of Ovulation Brit Med Bullet 26 1 9 1970 2 Knobil E The Neuroendocrine Control of the Menstrual Cycle Rec Prog Horm Res 36 52 88 1980 3 Jeffcoate S L Clinics in Endocrinol Metab 4 521 543 1975 Whitely R J Keutmann H T and Ryan R J Endocrinology 102 1874 1978 Pierce J G and Parsons T F Glycoprotein hormones Structure and Function Annual Rev Biochem 50 465 495 1981 Bardin C W and Paulsen C A The Testes in Textbook of Endocrinology ed R H Williams M D W B Saunders Co 1981 Shome B and Parlow A F J Clin Endocrinol Metabl 39 199 202 1974 Shome B and Parlow A F J Clin Endocrinol Metabl 39 203 205 1974 Ross G T VandeWeile R L and Frantz A G Chapter 7 in The Ovaries and the Breasts in Textbook of Endocrinology R H Williams
26. recisi 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 6 semanas si se almacenan como se ha descrito arriba 4 4 Preparaci n de los Reactivos Dejar que todos los reactivos y el numero 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 calibradores reconstituidos son estables durante 2 meses a 2 C 8 C Para periodos mas largos congelar a 20 C 4 5 Eliminacion 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 DlAsource 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
27. rrect 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 uL of Stop Solution to each well Determine the absorbance OD of each well at 450 10 nm with a microtiter plate reader It is recommended that the wells be read within 10 minutes after adding the Stop Solution 0100 AD 6 3 Calculation of Results 1 Calculate the average absorbance values for each set of calibrators controls and patient samples 2 Using semi logarithmic graph paper 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 ifu have been calculated automatically using a 4 pl 4 parameter logistics curve fit 4 parameter logistics is the preferred method 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 or reported as gt 200 miu ml For the calculation of the concentrations this dilution factor has to be taken into account 6 3 1 Example
28. s 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 02 1388 001 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 48 hours at 2 C to 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 Calibrator O 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 O mix thoroughly b dilution 1 100 10 uL dilution a 1 10 90 uL Calibrator O mix thoroughly 6 ASSAY PROCEDURE 6 1 General Remarks 1 All reagents and specimens must be allowed to come to room temperature before use All reagents must be mixed without foaming 2 Once t
29. safety guideline or regulation 21 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 DlAsource 4 REAGENTS 4 1 Reagents provided Microtiterwells 12 x 8 break apart strips 96 wells Wells coated with anti LH monoclonal antibody CAI LH Calibrators N 0 to 5 6 vials lyophilized 1 mL Concentration 0 10 20 40 100 200 mIU mL The calibrators are calibrated against WHO 2 International Calibrator for LH IRP 80 552 See Preparation of Reagents Contain 0 03 Proclin 300 0 015 BND and 0 010 MIT as preservative y 8 E 3 Al Enzyme Conjugate 1 vial 11 mL Ready for use Anti LH antibody conjugated to horseradish peroxidase Contains 0 03 Proclin 300 0 015 BND and 0 010 Mit as preservative gt ICHROM TMB Substrate Solution 1 vial 14 mL Ready for use Tetramethylbenzidine TMB 5 SOUN Stop Solution 1 vial 14 mL Ready for use Contains 0 5 M H SO4 Avoid contact with the stop solution It may cause skin irritations and burns BND 5 bromo 5 nitro 1 3 dioxane MIT 2 methyl 2H isothiazol 3 one Note Additional Calibrator 0 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 Di
30. stilled or deionized water Timer Semi logarithmic graph paper or software for data reduction 4 3 Storage Conditions When stored at 2 C to 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 to8 C Microtiter wells must be stored at 2 C to 8 C Once the foil bag has been opened care should be taken to close it tightly again Opened kits retain activity for six weeks if stored as described above Catalogue Nr KAPD1289 P I Number 1701238 en Revision Nr 140127 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 C to 8 C For longer storage freeze at 20 C 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 4 6 Damaged Test Kits In case of any severe damage to the test kit or components DIAsource 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 regulation
31. tras 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 resultados 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 Las hojas de los datos de seguridad se ajustan a las demandas de las gu a europea EU Guideline 91 155 EC Catalogue Nr KAPD1289 P I Number 1701238 es Revisio
32. ysfunction of the pituitary or hypothalamus but the actual source of the problem must be confirmed by other tests 10 In the differential diagnosis of hypothalamic pituitary or gonadal dysfunction assays of LH concentration are routinely performed in conjunction with FSH assays since their roles are closely interrelated Furthermore the hormone levels are used to determine menopause pinpoint ovulation and monitor endocrine therapy 2 PRINCIPLE OF TEST The DlAsource LH ELISA Kit is a solid phase enzyme linked immunosorbent assay ELISA based on the sandwich principle The microtiter wells are coated with a monoclonal mouse antibody directed towards a unique antigenic site on a LH molecule An aliquot of patient sample containing endogenous LH is incubated in the coated well with enzyme conjugate which is an anti LH 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 LH in the sample Having added the substrate solution the intensity of colour developed is proportional to the concentration of LH 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 HBsAg and HCV by FDA approved procedures All reagents however should
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