Estradiol ELISA
Contents
1. SPECIFICITY CROSS REACTIVITY The following compounds were tested for cross reactivity with the Estradiol ELISA kit with estradiol cross reacting at 10096 Steroid 96 Cross Reactivity Estradiol 100 Estriol 1 6 Estrone 1 3 Progesterone 0 1 Cortisol 0 1 INTRA ASSAY PRECISION Three samples were assayed ten times each on the same calibrator curve The results in pg ml are tabulated below Sample Mean SD CV96 1 85 624 7 946 9 3 2 355 735 32 372 9 1 3 1104 385 51 243 4 6 INTER ASSAY PRECISION Three samples were assayed ten times The results in pg ml are tabulated below Sample Mean SD CV96 1 82 044 8 286 10 1 2 324 623 31 813 9 8 3 1153 301 71 505 6 2 Page 7 of 9 RECOVERY Three human serum samples were spiked with defined amounts of estradiol The recovery results in pg ml are tabulated below LINEARITY Sample Obs Result Exp Result Recovery 1 Unspiked 43 312 800 20 196 874 169 427 116 2 3200 10 360 670 330 284 109 2 3200 20 638 328 569 427 112 1 1 Unspiked 125 661 800 20 275 461 238 051 98 2 3200 10 415 680 405 146 102 6 3200 20 576 160 638 051 90 3 2 Unspiked 336 297 800 20 474 791 413 581 114 8 3200 10 600 214 596 634 100 6 3200 20 758 257 813 581 93 2 Three human serum samples were diluted with calibrator A tabulated belo2. HAZARDS Avoid contact with reagents containing TMB hydrogen peroxide and sulfuric acid If contacted with any of these reagents wash with plenty of water TMB is a suspected carcinogen SPECIMEN COLLECTION AND STORAGE Approximately 0 2 ml of serum is required per duplicate determination Collect 4 5 ml of blood into an appropriately labelled tube and allow it to clot Centrifuge and carefully remove the serum layer Store at 4 C for up to 24 hours or at 10 C or lower if the analyses are to be done at alater date Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling SPECIMEN PRETREATMENT This assay is a direct system no specimen pretreatment is necessary REAGENTS AND EQUIPMENT NEEDED BUT NOT PROVIDED 1 Precision pipettes to dispense 50 100 150 and 300 ul 2 Disposable pipette tips 3 Distilled or deionized water 4 Plate shaker 5 Microwell plate reader with a filter set at 450nm and an upper OD limit of 3 0 or greater see assay procedure step 10 Page 3 of 9 REAGENTS PROVIDED 1 Rabbit Anti Estradiol Antibody Coated Microwell Plate Break Apart Wells Ready To Use Contents One 96 well 12x8 polyclonal antibody coated microwell plate in a resealable pouch with desiccant Storage Refrigerate at 2 8 C Stability 12 months or as indicated on label 2 Estradiol Biotin Avidin Horseradish Peroxidase HRP Conjugate Concentrate Requires Preparation Co
3. established for every run 7 The controls included in kit should be i ncluded in every run and fall within established confidence limits 8 Improper procedural techniques imprecise pipetting incomplete washing as well as improper reagent storage may be indicated when assay values for the controls do not reflect established ranges 9 When reading the microplate the presence of bubbles in the microwells will affect the optical densities ODs Carefully remove any bubbles before performing the reading step 10 The substrate solution TMB is sensitive to light and should remain colorless if properly stored Instability or contamination may be indicated by the development of a blue color in which case it should not be used 11 When dispensing the substrate and stop solution do not use pipettes in which these liquids will come into contact with any metal parts 12 To prevent contamination of reagents use a new disposable pipette tip for dispensing each reagent sample standard and control Page 2 of 9 13 Do not mix various lot numbers of kit components within a test and do not use any component beyond the expiration date printed on the label 14 Kit reagents must be regarded as hazardous waste and disposed of according to national regulations LIMITATIONS 1 All the reagents within the kit are calibrated for the direct determination of estradiol in human serum The kit is not calibrated for the determination of estradiol in sa
4. FAL Estradiol ELISA For the quantitative determination of Estradiol in human serum For In Vitro Diagnostic use within the United States of America This product is for Research Use Only outside of the United States of America Catalog Number 11 ESTHU E01 Size 96 wells Version 6 2 August 09 2013 ALPCO September 19 2013 26 G Keewaydin Drive Salem NH 03079 P 800 592 5726 F 603 898 6854 ts alpco com www alpco com age 10 INTENDED USE For the direct quantitative determination of Estradiol by enzyme immunoassay in human serum For in vitro diagnostic use PRINCIPLE OF THE TEST The principle of the following enzyme immunoassay test follows the typical competitive binding scenario Competition occurs between an unlabeled estradiol present in standards controls and samples and an enzyme labelled estradiol 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 stop solution The absorbance is measured on a microtiter plate reader The intensity of the color formed is inversely proportional to the concentration of unlabeled estradiol in the sample A set of standards is used to plot a standard curve from which the amount of estradiol in samples and controls can be directly read PRINCIPLE OF TEST Estradiol is one of th
5. L et al J Steroid Biochem 29 207 1988 12 McConway M G et al Clin Chem Acta 158 59 1986 13 Midgley A R et al recent Progr Horm Res 27 235 1971 14 Sankolli G M et al Ann Clin Biochem 25 288 1988 15 Thomas K et al Int J Fertil 18 65 1973 16 Tiefenauer L et al J Steroid Biochem 485 133 1986 17 Tiefenauer L et al J Immunol Methods 74 293 1984 18 Tulchinsky D et al Am J Obstet Gynecol 117 884 1973 19 Weinstein A et al Steroids 20 789 1972 20 Check J H et al Falsely elevated steroidal assay levels related to heterophile antibodies against various animal species Gynecol Obstet Invest 40 139 140 1995 QD Oo 40 014 C5 n2 Page 9 of 9
6. affect the results of the patient control samples CALCULATIONS 1 Calculate the mean optical density of each calibrator duplicate 2 Draw a calibrator curve on semi log paper with the mean optical densities on the Y axis and the calibrator concentrations on t he X axis If immunoassay software is being used a 4 parameter or 5 parameter 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 3200 pg 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 pg ml 2 001 1 952 1 976 0 1 716 1 775 1 746 20 1 397 1 356 1 377 100 0 902 0 883 0 893 300 0 612 0 702 0 657 800 nmol S gt 0 365 0 368 0 367 3200 Unknown 1 428 1 451 1 440 7TT 417 Page 6 of 9 TYPICAL CALIBRATOR CURVE Sample curve only Do not use to calculate results 2 5 OD 450nm 20 100 300 800 3200 Estradiol pg ml PERFORMANCE CHARACTERISTICS SENSITIVITY The detection limit is defined as the concentration of estradiol needed to give a B BO values equivalent to the point where B is equal to BO minus 2X the SD of BO Based on 20 replicate analyses of standard A the sensitivity is 10 pg ml
7. cimen 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 estradiol biotin avidin HRP 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 ul of each calibrator control and specimen sample into correspondingly labelled wells in duplicate 4 Pipette 100 ul of the conjugate working solution into each well The use of a multichannel pipette is recommended 5 Incubate on a plate shaker approximately 200 rpm for 1 hour at room temperature Page 5 of 9 6 Wash the wells three times with 300 ul 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 ul of TMB substrate into each well at timed intervals 8 Incubate on a plate shaker for 10 15 minutes at room temperature or until calibrator A attains dark blue color for desired OD 9 Pipette 50 ul of stop solution into each well at the same timed intervals as in step 7 10 Read the plate on a microwell plate reader at 450nm within 20 minutes after addition of the stop 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
8. e 0 5 ml vial Storage Refrigerate at 2 8 C Stability 12 months in unopened vial or as indicated on label Once opened the controls should be used within 14 days or aliquoted and stored frozen Avoid multiple freezing and thawing cycles Page 4 of 9 5 Assay Buffer Ready To Use Contents One vial buffer containing a dissociate agent with a non mercury preservative Volume 15 ml vial Storage Refrigerate at 2 8 C Stability 12 months or as indicated on label 6 Wash Buffer Concentrate Requires Preparation Contents One bottle containing buffer with a non ionic detergent and a non mercury preservative Volume 50 ml bottle Storage Refrigerate at 2 8 C Stability 12 months or as indicated on label 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 7 TMB Substrate Ready To Use Contents One bottle containing tetramethylbenzidine and hydrogen peroxide in a non DMF or DMSO containing buffer Volume 16 ml bottle Storage Refrigerate at 2 8 C Stability 12 months or as indicated on label 8 Stop Solution Ready To Use Contents One vial containing 1M sulfuric acid Volume 6 ml vial Storage Refrigerate at 2 8 C Stability 12 months or as indicated on label ASSAY PROCEDURE Specimen Pretreatment None All reagents must reach room temperature before use Calibrators controls and spe
9. e main components of naturally occurring estrogens andis the major estrogen secreted during the menstrual cycle The serum levels of estradiol are low during the follicular phase rising gradually until about one day before ovulation when a marked rise in the estradiol level occurs Ovulatory Peak The estradiol level falls rapidly at or right after ovulation and is again within the levels of the follicular phase There is a second rise of estradiol around day 21 of the cycle Luteal Peak The levels then decline gradually to the lowest level at the onset of the next menstrual cycle PROCEDURAL CAUTIONS AND WARNINGS 1 Users should have a thorough understanding of this protocol for the successful use of this kit Reliable performance will only be attained by strict and careful adherence to the instructions provided 2 It is recommended to all customers to prepare their own control materials or serum pools that should be included in every run at a high and low level for assessing the reliability of results 3 When the use of water is specified for dilution or reconstitution use deionized or distilled water 4 In order to reduce exposure to potentially harmful substances gloves should be worn when handling kit reagents and human specimens 5 All kit reagents and specimens should be brought to room temperature and mixed gently but thoroughly before use Avoid repeated freezing and thawing of reagents and specimens 6 A calibrator curve must be
10. liva plasma or other specimens of human or animal origin 2 Do not use grossly hemolyzed grossly lipemic icteric or improperly stored serum 3 Any samples or control sera containing azide or thimerosal are not compatible with this kit as they may lead to false results 4 Only calibrator A may be used to dilute any high serum samples The use of any other reagent may lead to false results 5 The results obtained with this kit should never be us ed as the sole basis for a c linical diagnosis For example the occurrence of heterophilic antibodies in patients regularly exposed to animals or animal products has the potential of causing interferences in immunological tests Consequently the clinical diagnosis should include all aspects of a patients background including the frequency of exposure to animals products if false results are suspected SAFETY CAUTIONS AND WARNINGS POTENTIAL BIOHAZARDOUS MATERIAL Human serum that may be used in the preparation of the standards and controls has been tested and found to be non reactive for Hepatitis B surface antigen and has also been tested for the presence of antibodies to HCV and Human Immunodeficiency Virus HIV and found to be negative However no test method can offer complete assurance that HIV HCV and Hepatitis B virus or any infectious agents are absent T he reagents should be considered a pot ential biohazard and handled with the same precautions as applied to any blood specimen CHEMICAL
11. ntents Estradiol Biotin and Avidin HRP conjugate in a protein based buffer with a non mercury preservative Volume 300 ul vial Storage Refrigerate at 2 8 C Stability 12 months or as indicated on label Preparation Dilute 1 50 in assay buffer before use eg 40 ul of conjugate in 2 ml of assay buffer If the whole plate is to be used dilute 240 ul of HRP in 12 ml of assay buffer Discard any that is left over 3 Estradiol Calibrators Ready To Use Contents Six vials containing estradiol in a protein based buffer with a non mercury preservative Prepared by spiking buffer with a defined quantity of estradiol Listed below are approximate concentrations please refer to vial labels for exact concentrations Calibrator Concentration Volume Vial Calibrator A 0 pg ml 2 0 ml Calibrator B 20 pg ml 0 5 ml Calibrator C 100 pg ml 0 5 ml Calibrator D 300 pg ml 0 5 ml Calibrator E 800 pg ml 0 5 ml Calibrator F 3200 pg ml 0 5 ml Storage Refrigerate at 2 8 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 4 Controls Ready To Use Contents Two vials containing estradiol in a protein based buffer with a non mercury preservative Prepared by spiking serum with defined quantities of estradiol Refer to vial labels for the acceptable range Volum
12. w The linearity results in pg ml are Sample Obs Result Exp Result Recovery 1 638 328 1 2 272 247 319 164 85 3 1 4 140 592 159 582 88 1 1 8 74 844 79 791 93 8 2 576 160 1 2 324 092 288 080 112 5 1 4 168 957 144 040 117 3 1 8 73 646 72 020 102 3 3 758 257 1 2 335 908 379 129 88 6 1 4 186 152 189 564 98 2 1 8 78 103 94 782 82 4 EXPECTED NORMAL VALUES As for all clinical assays each laboratory should collect data and establish their own range of expected normal values The results of an expected range study with apparently normal healthy subjects yielded the following results all values are reported in pg ml Group n Mean Central 9596 Males 40 22 100 Follicular Phase 10 41 15 120 Ovulation 3 289 200 400 Lutueal Phase 10 193 175 325 Postmenopausal 30 28 90 Page 8 of 9 REFERENCES 1 Baird D T amp Guevara A J J Clin Endo 29 149 1969 Cameron E H D et al Steroids 20 737 1972 Corrie J et al Clin Chem 27 594 1981 Dean R D et al Steroids 18 593 1971 De Boever J et al Clin Chem 32 1895 1986 De Hertogh R et al J Clin Endocrinol Metab 40 93 1975 Flickinger G L et al Acta Endocrinol Kbh 54 30 1967 Huber P R et al Clin Chem 26 960 1980 Kuss E et al Steroids 19 509 1972 10 Linder M R et al Steroids 19 357 1972 11 Marcus G
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