Cortisol Saliva ELISA

Transcription

1 Li StarFish S.r.l. Via Cavour, Cernusco S/N (MI), Italy Tel Fax Instructions for use Cortisol Saliva ELISA LDNSA E6000

2 Cortisol Saliva ELISA 1. Intended use For the quantitative determination of Cortisol by enzyme immunoassay in human saliva. For in vitro diagnostic use only. 2. Principle of the test The principle of the following enzyme immunoassay test follows the typical competitive binding scenario. Competition occurs between an unlabeled antigen (present in standards, control and patient samples) and an enzymelabelled antigen (conjugate) for a limited number of antibody binding sites on the microwell plate. The washing and decanting procedures remove unbound materials. After the washing step, the enzyme substrate is added. The enzymatic reaction is terminated by addition of the stopping solution. The absorbance is measured on a microtiter plate reader. The intensity of the colour formed is inversely proportional to the concentration of cortisol in the sample. A set of standards is used to plot a standard curve from which the amount of cortisol in patient samples and controls can be directly read. 3. Advice on handling the test 3.1 Reliability of the test results In order to assure a reliable evaluation of the test results it must be conducted according to the instructions included and in accordance with current rules and guidelines (GLP, RILIBÄK, etc.). Special attention must be paid to control checks for precision and correctness during the test; the results of these control checks have to be within the norm range. In case of significant discrepancies between the preset assay characteristics of this test and the actual results please contact the manufacturer of the test kit for further instructions. It is recommended that each laboratory establishes its own reference intervals. The values reported in this test instruction are only indicative. The results obtained with this test kit should not be taken as the sole reason for any therapeutic consequence but have to be correlated to other diagnostic tests and clinical observations. 3.2 Complaints In case of complaints please submit to the manufacturer a written report containing all data as to how the test was conducted, the results received and a copy of the original test printout. Please contact the manufacturer to obtain a reclamation form and return it completely filled in to the manufacturer. 3.3 Warranty This test kit was produced according to the latest developments in technology and subjected to stringent internal and external quality control checks. Any alteration of the test kit or the test procedure as well as the usage of reagents from different charges may have a negative influence on the test results and are therefore not covered by warranty. The manufacturer is not liable for damages incurred in transit. 3.4 Disposal Residual substances and/or all remaining chemicals, reagents and ready for use solutions, are special refuse. The disposal is subject to the laws and regulations of the federation and the countries. About the removal of special refuse the responsible authorities or refuse disposal enterprises inform. The disposal of the kit must be made according to the national official regulations. Legal basis for the disposal of special refuse is the cycle economic and waste law. The appropriate safety data sheets of the individual products are available on the homepage. The safety data sheets correspond to the standard: ISO Interference Do not mix reagents and solutions from different lots. Consider different transport and storage conditions. Inappropriate handling of test samples or deviations from the test regulation can the results affect. Use no kit components beyond the expiration date. Avoid microbiological contamination of the reagents and the washing water. Consider incubation periods and wash references. 3.6 Precautions Observe the incubation periods and washing instructions. Never pipette by mouth and avoid contact of reagents and specimens with skin. No smoking, eating or drinking in areas where samples or kit test tubes are handled. When working with kit components or samples, always wear protective gloves and wash your hand thoroughly as soon as you have finished the work. Avoid spraying of any kind. Avoid any skin contact with reagents. Use protective clothing and disposable gloves. All steps have to be performed according to the protocol. Optimal test results are only obtained when using calibrated pipettes. Sodium azide could react with lead and copper tubes and may form highly explosive metal azide. When clearing up, rinse thoroughly with large volumes of water to prevent such formation. All reagents of this testkit which contain human or animal serum or plasma have been tested and confirmed negative for HIV I/II, HbsAg and HCV by FDA approved procedures. All reagents, however, should be treated as potential biohazards in use and for disposal. Revision date: 03Aug2009 2/7

3 4. SPECIMEN COLLECTION AND STORAGE Approximately 1 ml of saliva is required per duplicate determination. Collect 45 ml of saliva into a clean glass tube (Salivette by Sarstedt may be used) without force or inducement and before eating, drinking or brushing the teeth. Simply rinse the mouth with water before collection. Do not use bloodcontaminated specimens. Store samples at 4 o C for up to 24 hours or at 10 o C or lower if the analyses are to be done at a later date. Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling. 5. SPECIMEN PRETREATMENT Specimen tubes are to be placed into a freezer and allowed to freeze. When ready to use, the specimens are to be thawed and centrifuged. The supernatants are to be collected and poured into freshly labelled tubes. 6. REAGENTS AND EQUIPMENT NEEDED BUT NOT PROVIDED 1. Precision pipettes to dispense 50, 100, 150 and 300 µl 2. Disposable pipette tips 3. Distilled or deionized water 4. Plate shaker 5. Benchtop centrifuge 6. Microwell plate reader with a filter set at 450nm and an upper OD limit of 3.0 or greater* (see assay procedure step 10). 7. REAGENTS PROVIDED SA E6031 Rabbit AntiCortisol Antibody Coated Microwell PlateBreak Apart Wells Ready To Use. Contents: One 96 well (12x8) polyclonal antibodycoated microwell plate in a resealable pouch with desiccant. Storage: Refrigerate at 28 o C AA E1740 CortisolHorseradish Peroxidase (HRP) Conjugate Concentrate 50x Contents: CortisolHRP conjugate in a proteinbased buffer with a nonmercury preservative. Volume: 300 µl/vial Storage: Refrigerate at 28 o C Preparation: Dilute 1:50 in assay buffer before use (eg. 40 µl of HRP in 2 ml of assay buffer). If the whole plate is to be used dilute 240 µl of HRP in 12ml of assay buffer. Discard any that is left over. Cortisol Saliva Standards Ready To Use. Contents: Six vials containing cortisol in a proteinbased buffer with a nonmercury preservative. Prepared by spiking buffer with a defined quantity of cortisol. *Listed below are approximate concentrations, please refer to vial labels for exact concentrations: Catalogue no. Standard Concentration Volume/Vial SA E6001 Standard A 0 ng/ml 2.0 ml SA E6002 Standard B 1 ng/ml 0.6 ml SA E6003 Standard C 3 ng/ml 0.6 ml SA E6004 Standard D 10 ng/ml 0.6 ml SA E6005 Standard E 30 ng/ml 0.6 ml SA E6005 Standard F 0 ng/ml 2.0 ml Storage: Refrigerate at 28 o C Stability: 12 months in unopened vials or as indicated on label. Once opened, the standards should be used within 14 days or aliquoted and stored frozen. Avoid multiple freezing and thawing cycles. AA E1751 Control Ready To Use Contents: One vial containing cortisol in a proteinbased buffer with a nonmercury preservative. Prepared by spiking buffer with a defined quantity of cortisol. Refer to vial label for expected value and acceptable range. Volume: 0.6 ml/vial Storage: Refrigerate at 28 o C Stability: 12 months in unopened vial or as indicated on label. Once opened, the control should be used within 14 days or aliquoted and stored frozen. Avoid multiple freezing and thawing cycles. Revision date: 03Aug2009 3/7

4 AA E0030 Wash Buffer Concentrate X10 Contents: One bottle containing buffer with a nonionic detergent and a nonmercury preservative. Volume: 50 ml/bottle Storage: Refrigerate at 28 o C Preparation: Dilute 1:10 in distilled or deionized water before use. If the whole plate is to be used dilute 50 ml of the wash buffer concentrate in 450 ml of water. SA E6013 Assay Buffer Ready To Use Contents: One vial containing a proteinbased buffer with a nonmercury preservative. Volume: 15 ml/vial Storage: Refrigerate at 28 o C AA E0055 TMB Substrate Ready To Use Contents: One bottle containing tetramethylbenzidine and hydrogen peroxide in a nondmf or DMSO containing buffer. Volume: 16 ml/bottle Storage: Refrigerate at 28 o C AA E0080 Stopping Solution Ready To Use Contents: One vial containing 1M sulfuric acid. Volume: 6 ml/vial Storage: Refrigerate at 28 o C 8. Test procedure All reagents must reach room temperature before use. Calibrators, controls and specimen samples should be assayed in duplicate. Once the procedure has been started, all steps should be completed without interruption. 1. Prepare working solutions of the cortisolhrp conjugate and wash buffer. 2. Remove the required number of microwell strips. Reseal the bag and return any unused strips to the refrigerator. 3. Pipette 50 µl of each calibrator, control and specimen sample into correspondingly labelled wells in duplicate. 4. Pipette 100 µl of the conjugate working solution into each well (We recommend using a multichannel pipette). 5. Incubate on a plate shaker (approximately 200 rpm) for 45 minutes at room temperature. 6. Wash the wells 3 times with 300 µl of diluted wash buffer per well and tap the plate firmly against absorbent paper to ensure that it is dry (the use of a washer is recommended). 7. Pipette 150 µl of TMB substrate into each well at timed intervals. 8. Incubate on a plate shaker for 1520 minutes at room temperature (or until calibrator A attains dark blue colour for desired OD). 9. Pipette 50 µl of stopping solution into each well at the same timed intervals as in step Read the plate on a microwell plate reader at 450nm within 20 minutes after addition of the stopping solution. * If the OD exceeds the upper limit of detection or if a 450nm filter is unavailable, a 405 or 415nm filter may be substituted. The optical densities will be lower, however, this will not affect the results of patient/control samples. Revision date: 03Aug2009 4/7

5 9. CALCULATIONS 1. Calculate the mean optical density of each calibrator duplicate. 2. Draw a calibrator curve on semilog paper with the mean optical densities on the Yaxis and the calibrator concentrations on the Xaxis. If immunoassay software is being used, a 4parameter curve is recommended. 3. Calculate the mean optical density of each unknown duplicate. 4. Read the values of the unknowns directly off the calibrator curve. 5. If a sample reads more than 100 ng/ml then dilute it with calibrator A at a dilution of no more than 1:8. The result obtained should be multiplied by the dilution factor. TYPICAL TABULATED DATA Calibrator OD 1 OD 2 Mean OD Value (ng/ml) A B C D E F Unknown TYPICAL CALIBRATOR CURVE Sample curve only. Do not use to calculate results OD 450nm Cortisol (ng/ml) 10. ASSAY CHARACTERISTICS SENSITIVITY The lower detection limit is calculated from the standard curve by determining the resulting concentration of the mean OD of Calibrator A (based on 10 replicate analyses) minus 2 SD. Therefore, the sensitivity of the Cortisol Saliva ELISA kit is 1.0 ng/ml. SPECIFICITY (CROSS REACTIVITY) The following compounds were tested for crossreactivity with the Direct Cortisol Saliva ELISA kit with cortisol crossreacting at 100%. Steroid %Cross Reactivity Cortisol 100 Prednisolone 13.6 Corticosterone 7.6 Deoxycorticosterone 7.2 Progesterone 7.2 Cortisone 6.2 Deoxycortisol 5.6 Pednisone 5.6 Dexamethasone 1.6 No cross reaction was detected with DHEAS and Tetrahydrocortisone. Please note that there is an observed crossreactivity of 13.6% with prednisolone. Since prednisone is converted to prednisolone in vivo, caution must be exercised when assaying the cortisol levels of patients undergoing either therapy. Revision date: 03Aug2009 5/7

6 INTRAASSAY PRECISION Three samples were assayed ten times each on the same calibrator curve. The results (in ng/ml) are tabulated below: Sample Mean SD CV% INTERASSAY PRECISION Three samples were assayed ten times over a period of four weeks. The results (in ng/ml) are tabulated below: Sample Mean SD CV% RECOVERY Spiked samples were prepared by adding defined amounts of cortisol to three patient saliva samples (1:1). The results (in ng/ml) are tabulated below: Sample Obs.Result Exp.Result Recovery% Unspiked Unspiked Unspiked LINEARITY Three patient saliva samples were diluted with calibrator A. The results (in ng/ml) are tabulated below: Sample Obs.Result Exp.Result Recovery% 1 1:2 1:4 1: :2 1:4 1:8 3 1:2 1:4 1: EXPECTED NORMAL VALUES As for all clinical assays each laboratory should collect data and establish their own range of expected normal values. Random male and female samples were taken in the early morning and had an absolute range of: ng/ml Revision date: 03Aug2009 6/7

7 For actual literature, information about clinical significance or any other information please contact your local supplier. Symbols: Storage temperature Expiry date Consult instructions for use Caution Manufacturer Batch code Content Catalogue number Contains sufficient for <n> tests For invitro diagnostic use only! CE labelled For research use only! Revision date: 03Aug2009 7/7