V E T E R I N A R Y M E D I C A L S E R V I C E S i n V e t e r i n a r y M e d i c i n e Corticosteroids Help Ease the Itch of Dermatitis by Keith Hnilica, DVM, MS, Dipl. ACVD C R E D E N T I A L S . Keith Hnilica, DVM, MS, Dipl. ACVD, teaches and consults in dermatology at the University of Tennessee. His clinical and research interests are focused on clinical veterinary d
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Microsoft word - fr e-2000 en.docInstructions for use
For the direct quantitative determination of Estradiol by enzyme immunoassay in human serum.
For in vitro diagnostic use only.
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 patient 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 stopping solution. The absorbance is measured
on a microtiter plate reader. The intensity of the colour 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 patient samples and controls can be directly read.
Estradiol is one of the main components of naturally occurring estrogens and is 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
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's 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. When the use of water is specified for dilution or reconstitution, use deionized or distilled water. In order to reduce exposure to potentially harmful substances, gloves should be worn when handling kit reagents and human specimens. 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. A calibrator curve must be established for every run. The controls (included in kit) should be included in every run and fall within established confidence limits. 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. 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. The substrate solution (TMB) is sensitive to light and should remain colourless if properly stored. Instability or contamination may be indicated by the development of a blue colour, in which case it should not be used. When dispensing the substrate and stopping solution, do not use pipettes in which these liquids will come into contact with any metal parts. To prevent contamination of reagents, use a new disposable pipette tip for dispensing each reagent, sample, standard and control. 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. Kit reagents must be regarded as hazardous waste and disposed of according to national regulations.
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 saliva, plasma or other specimens of human or animal origin. Do not use grossly hemolyzed, grossly lipemic, icteric or improperly stored serum. Any samples or control sera containing azide or thimerosal are not compatible with this kit, as they may lead to false results. Only calibrator A may be used to dilute any high serum samples. The use of any other reagent may lead to false results. The results obtained with this kit should never be used as the sole basis for a clinical 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 patient’s 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. The reagents
should be considered a potential biohazard and handled with the same precautions as applied to any blood
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 4oC
for up to 24 hours or at -10oC 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.
This assay is a direct system; no specimen pretreatment is necessary.
REAGENTS AND EQUIPMENT NEEDED BUT NOT PROVIDED
Precision pipettes to dispense 50, 100, 150 and 300 µl Microwell plate reader with a filter set at 450nm and an upper OD limit of 3.0 or greater* REAGENTS PROVIDED
Wash Buffer Concentrate – X10
One bottle containing buffer with a non-ionic detergent and a non-mercury preservative. 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.
TMB Substrate - Ready To Use.
One bottle containing tetramethylbenzidine and hydrogen peroxide in a non-DMF or DMSO containing buffer.
Stopping Solution - Ready To Use.
Calibrators and Controls- Ready To Use.
Listed below are approximate concentrations, please refer to vial labels for exact concentrations: Calibrator
Estradiol in a protein-based buffer with a non-mercury preservative. Prepared by spiking buffer with a defined quantity of estradiol. 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.
Assay Buffer - Ready To Use.
One vial containing a protein-based buffer with a non-mercury preservative.
Rabbit Anti-Estradiol Antibody Coated Microwell Plate-Break Apart
Wells - Ready To Use.
One 96 well (12x8) polyclonal antibody-coated microwell plate in a resealable pouch with desiccant.
Estradiol-Biotin : Avidin-Horseradish Peroxidase (HRP) Conjugate
Estradiol-Biotin and Avidin-HRP conjugate in a protein-based buffer with a non-mercury preservative. 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 12 mL of assay buffer. Discard any that is left over. ASSAY 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.
Prepare working solutions of the estradiol-biotin: avidin-HRP conjugate and wash buffer.
Remove the required number of microwell strips. Reseal the bag and return any unused strips to the refrigerator. Pipette 50 µl of each calibrator, control and specimen sample into correspondingly labelled wells in
Pipette 100 µL of the conjugate working solution into each well.
(We recommend using a multichannel pipette). Incubate on a plate shaker (approximately 200 rpm) for 1 hour at room temperature.
Wash the wells 3 times with prepared wash buffer (300 µL/well for each wash) and tap the plate firmly
against absorbent paper to ensure that it is dry (The use of a washer is recommended).
Pipette 150 µL of TMB substrate into each well at timed intervals.
Incubate the plate on a plate shaker at room temperature for 10-15 minutes.
(or until Calibrator A attains dark blue colour for desired OD). Pipette 50 µl of stopping solution into each well at the same timed intervals as in step 7.
10. Read the plate on a microwell plate reader at 450 nm within 20 minutes after addition of the stopping
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 CALCULATIONS
• Calculate the mean optical density of each calibrator duplicate.
• Draw a calibrator curve on semi-log paper with the mean optical densities on the Y-axis and the calibrator
concentrations on the X-axis. If immunoassay software is being used, a 4-parameter or 5-parameter curve is recommended. • Calculate the mean optical density of each unknown duplicate. • Read the values of the unknowns directly off the calibrator curve. • 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:
OD 1 OD 2 Mean OD
TYPICAL CALIBRATOR CURVE
Sample curve only. Do not use to calculate results.
The detection limit is defined as the concentration of estradiol needed to give a B/B0 values equivalent to the
point where B is equal to B0 minus 2X the SD of B0. Based on 20 replicate analyses of standard A, the
sensitivity is 10 pg/ml.
SPECIFICITY (CROSS REACTIVITY)
The following compounds were tested for cross-reactivity with the Direct Estradiol ELISA kit with estradiol
cross-reacting at 100%.
Three samples were assayed ten times each on the same calibrator curve. The results (in pg/ml) are tabulated
Three samples were assayed ten times. The results (in pg/ml) are tabulated below:
Three human serum samples were spiked with defined amounts of estradiol. The recovery results (in pg/ml)
are tabulated below:
Obs.Result Exp.Result Recovery%
Three human serum samples were diluted with calibrator A. The linearity results (in pg/ml) are tabulated
Sample Obs.Result Exp.Result Recovery%
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):
Baird, D.T. & 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. 6. 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. Linder, M.R., et al., Steroids, 19:357, 1972. Marcus, G.L., et al., J. Steroid Biochem. 29:207, 1988. McConway, M.G., et al., Clin. Chem. Acta. 158:59, 1986. Midgley, A.R., et al., recent Progr. Horm. Res. 27:235, 1971. Sankolli, G.M., et al., Ann. Clin. Biochem. 25:288, 1988. Thomas, K., et al., Int. J. Fertil. 18:65, 1973. Tiefenauer, L., et al., J. Steroid Biochem. 485:133, 1986. Tiefenauer, L., et al., J. Immunol. Methods 74:293, 1984. Tulchinsky, D., et al., Am. J. Obstet. Gynecol., 117:884, 1973. Weinstein, A., et al., Steroids, 20:789, 1972. 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.
Suri Karthikeyan, M.D. Gastroentorology & Hepatology Suri Karthikeyan, M.D., M.R.C.P., F.R.C.P. (c) INSTRUCTIONS FOR COLONOSCOPIC PREP WITH MIRALAX AND CLEAR LIQUIDS READ CAREFULLY–DO NOT EXCEED RECOMMENDED DOSAGE AS SERIOUS SIDE EFFECTS MAY OCCUR. Name: _____________________________ You have been scheduled for the above examination. Please report to: Lakeside Memori