Microsoft word - estradiolmpb.doc

Intended Use
Add reagents directly to the bottom of the tube while avoiding
contact between the reagents and the pipet tip. This will
For the ultra-sensitive detection and quantitation of 17β- help assure consistent quantities of reagent in the test mixture. Estradiol in water (groundwater, surface water, well water). For Polystyrene tubes (33) are packaged in a bag. other applications contact the company for application bulletins Avoid cross-contaminations and carryover of reagents by using and/or specific matrix validation guidelines. clean pipets for each sample addition and by avoiding contact
Reagent Storage and Stability
between reagent droplets on the tubes and pipet tips. • Principle
Store all reagents at 2-8°C. Do not freeze. Reagents may be used until the expiration date on the box. The test tubes and The magnetic separation system consists of two parts: an The Abraxis 17β-Estradiol Kit applies the principles of enzyme Washing Solution require no special storage condition and may upper rack which will securely hold the test tubes and a lower linked immunosorbent assay (ELISA) to the determination of be stored separately from the reagents to conserve refrigerator 17β-Estradiol. The sample to be tested is added to a disposable space. separator which contains the magnets used to attract the test tube, and pre-incubated for 30 minutes with paramagnetic antibody coupled paramagnetic particles. During incubations the particles attached with antibodies specific to 17β-Estradiol. Consult state, local and federal regulations for proper disposal of upper rack is removed from the lower separator so that the Followed by the addition of an enzyme labeled estradiol paramagnetic particles remain suspended during the incubation. conjugate. At this point a competitive reaction occurs between For separation steps, the rack and the separator are
the estradiol which may be be in the sample and the enzyme • Materials Required but Not Provided
combined to pull the paramagnetic particles to the sides of
labeled estradiol for the antibody binding sites on the magnetic the tubes.
particles. The reaction is allowed to continue for ninety (90) In addition to the reagents provided, the following items are minutes. At the end of the incubation period, a magnetic field is essential for the performance of the test: To obtain optimum assay precision, it is important to perform applied to hold in the test tube the para-magnetic particles (with the separation steps carefully and consistently. Decant the rack Estradiol and labeled Estradiol bound to the antibodies on the by slowly inverting away from the operator using a smooth particles, in proportion to their original concentration), and allow turning action so the liquid flows consistently along only one the unbound reagents to be decanted. After decanting, the side of the test tube. While still inverted, place the rack on an particles are washed with Washing Solution. absorbent pad and allow to drain. Lifting the rack and replacing The presence of 17β-Estradiol is detected by adding the “Color gently onto the pad several times will ensure complete removal Solution”, which contains the enzyme substrate (hydrogen of the liquid from the rim of the tube. Do not bang the rack. peroxide) and the chromogen (3,3',5,5'-tetramethylbenzidine). The enzyme-labeled Estradiol bound to the Estradiol antibody Mix the antibody coupled paramagnetic particles just prior to catalyzes the conversion of the substrate/ chromogen mixture to Photometer* capable of readings at 450 nm a colored product. After an incubation period, the reaction is Do not use any reagents beyond their stated shelf life. stopped and stabilized by the addition of a diluted acid (Stopping * Please contact Abraxis for supplier information. Solution). Since the labeled Estradiol (conjugate) was in Avoid contact of Stopping Solution (diluted sulfuric acid) with competition with the unlabeled Estradiol (sample) for the • Sample Information
skin and mucous membranes. If this reagent comes in contact antibody sites, the color developed is inversely proportional
to the concentration of Estradiol in the sample.
This procedure is recommended fo use with water samples. Other samples may require modifications to the procedure and • Reagents
Samples containing gross particulate matter should be filtered The Abraxis Estradiol Kit contains the following items:
(e.g. 0.2 um Anotop™ 25 Plus, Whatman, Inc.) to remove The Abraxis 17β-Estradiol Assay will detect 17β-Estradiol 1. Estradiol Antibody Coupled Paramagnetic Particles Samples which have been preserved with monochloroacetic acid specifically. Very little cross-reactivity has been observed with Estradiol antibody (rabbit anti-17β-Estradiol) covalently bound to or other acids, should be neutralized with strong base e.g. 6N paramagnetic particles suspended in a buffered soution with other hormones tested. Refer to specificity table for data on several other steroid hormones. The Abraxis Estradiol Assay kit If the Estradiol concentration of a sample exceeds 25 ppt, the provides screening results. As with any analytical technique sample is subject to repeat testing using a diluted sample. A (GC, HPLC, etc.) positive results requiring some action should Horseradish peroxidase (HRP) labeled Estradiol analog diluted in ten-fold or greater dilution of the sample is recommended with a buffered solution with preservative and stabilizers. an appropriate amount of Diluent/Zero Standard or Sample Diluent. For example, in a separate test tube make a ten-fold The total time required for pipetting the magnetic particles 3. 17β- Estradiol Standards dilution by adding 100 uL of the sample to 900 uL of Three concentrations (2.5, 7.5, 25.0 pg/mL or ppt) of 17β- Diluent/Zero Standard. Mix thoroughly before assaying. should be kept to two (2) minutes or less, therefore the total Estradiol standards in distilled water with preservative and Perform the assay according to the Assay Procedure and obtain number of tubes that can be assayed in a run should be adjusted final results by multiplying the value obtain by the dilution factor accordingly. A concentration (approximately 10 ppt) of Estradiol in distilled water with preservative and stabilizers. A 2.0 mL volume is • Reagent Preparation
Quality Control
All reagents must be allowed to come to room temperature. The Distilled water with preservative and stabilizers without any antibody coupled paramagnetic particles should be mixed A control solution at approximately 10 ppt of 17β-Estradiol is provided with the Abraxis Estradiol Assay kit. It is recommended that it be included in every run and treated in the A solution of hydrogen peroxide and 3,3',5,5'- • Procedural Notes and Precautions
tetramethylbenzidine in an organic base. same manner as unknown samples. Acceptable limits should be As with all immunoassays, a consistent technique is the key to optimal performance. To obtain the greatest precision, be sure to treat each tube in an identical manner. • Assay Procedure
Read Reagent Preparation, Procedural Notes and Precautions 1. Label test tubes for standards, control, and samples. 2. Add 250 uL of the appropriate standard, control, or 3. Mix the Estradiol Antibody Coupled Paramagnetic Particles Testing For Potential Water Interferences
Various compounds and ions spiked into water samples were • Expected Results
tested to see if they interfered with the 17β-Estradiol ELISA. 4. Vortex for 1 to 2 seconds minimizing foaming. The presence of the following substances up to 20,000 PPM 5. Incubate for 30 minutes at room temperature. In a study with water samples from locations across the U.S., were found to have no significant effect in this assay: nitrate, 6. Add 250 uL of Estradiol Enzyme Conjugate to each tube. the Abraxis Estradiol Assay was shown to correlate well with a magnesium, calcium, sulfate, phosphate. Copper and fluoride up 7. Vortex for 1 to 2 seconds minimizing foam. commercial clinical Estradiol immunoassay (r = 0.971). to 2,000 PPM. Sodium Chloride up to 1M. Humic acid up to 5 8. Incubate for 90 minutes at room temperature. • Performance Data
9. Separate in the Magnetic Separation System for two (2)
Ordering informattion
10. Decant and gently blot all tubes briefly in a consistent
Abraxis 17β-Estradiol Assay Kit 100T PN 580002 _____________________________ Standard Set 11. Add 1 mL of Washing Solution to each tube and allow them to remain in the magnetic separation unit for two (2)
12. Decant and gently blot all tubes briefly in a consistent
For ordering or technical assistance contact: 13. Repeat Steps 11 and 12 an additional time. 14. Remove the rack from the separator and add 500 uL of Sensitivity
15. Vortex for 1 to 2 seconds minimizing foaming. The Abraxis Estradiol Assay has an estimated minimum 16. Incubate for 20 minutes at room temperature. detectable concentration of 1.5 ppt, it was calculated by 17. Add 500 uL of Stopping Solution to each tube. substracting 3 SD from the mean of fifty determinations of a sample known to be free of 17β-estradiol. Phone: (215) 357-3911 * Fax: (215) 357-5232 18. Add 1 mL Washing Solution to a clean test tube. Use as Email:*WEB: Recovery
19. Read results at 450 nm within 15 minutes after adding the Five (5) groundwater samples, were spiked with various levels of • General Limited Warranty
17β-Estradiol and then assayed using the Abraxis Estradiol Assay. The following results were obtained: Abraxis LLC warrants the products manufactured by the • Results
------------------ Recovery ---------------- Company, against defects and workmanship when used in accordance with the applicable instructions for a period not to extend beyond the product’s printed expiration date. Abraxis
Manual Calculations
makes no other warranty, expressed or implied. There is
1. Calculate the mean absorbance value for each of the no warranty of merchantability or fitness for a particular
2.06 0.39 82.4 purpose.
2. Calculate the %B/Bo for each standard by dividing the mean absorbance value for the standard by the mean absorbance value 20.0 3. Construct a standard curve by plotting the %B/Bo for each standard on vertical logit (Y) axis versus the corresponding 17β- Specificity
Estradiol concentration on horizontal logarithmic (X) axis on the The cross-reactivity of the Abraxis Estradiol Assay for various steroid hormones can be expressed as the dose required for 50% absorbance inhibition (50% B/Bo) divided by the 50% B/Bo 4. %B/Bo for controls and samples will then yield levels in ppt of concentration given by 17β-estradiol standards X 100. 17β-Estradiol by interpolation using the standard curve. (Contact Abraxis for detailed application information on specific photometers.) Photometric Analyzer
Some instrument manufacturers make available photometers allowing for calibration curves to be automatically calculated and stored. Refer to instrument operating manual for detailed instructions. To obtain results for the Abraxis Estradiol Assay on instruments allowing data tranformation the following


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