Estrogen (El/E2/E3) ELISA KIT | Creative Diagnostics DEIA6828-U product information

product summary

company name :

Creative Diagnostics

product type :

ELISA/assay

product name :

Estrogen (El/E2/E3) ELISA KIT

catalog :

DEIA6828-U

quantity :

96T

more info or order :

Creative Diagnostics product webpage

product information

Catalog :

DEIA6828-U

proudctName :

Estrogen (El/E2/E3) ELISA KIT

common :

Estrogen

Short Name :

Estrogen (El/E2/E3) ELISA KIT

Detection Method :

cELISA

Common name :

Estrogen

Species :

Human

Application :

Quantitative

Sample :

Serum

Size :

96T

Storage :

The kit should be stored at 2-8°C upon receipt.

Species Reactivity :

Human

Intended Use :

The Estrogen ELISA Kit is an immnoassay for detection of Estrogen in human serum.

General Description :

Estrogens are a group of compounds named for their importance in the estrous cycle of humans and other animals. They are the primary female sex hormones. Natural estrogens are steroid hormones, while some synthetic ones are non-steroidal. The name comes from the Greek, literally meaning "gadfly" but figuratively sexual passion or desire, and the suffix -gen, meaning "producer of". Estrogens are synthesized in all vertebrates as well as some insects. Their presence in both vertebrates and insects suggests that estrogenic sex hormones have an ancient evolutionary history.

Principle Of The Test :

The total estrogens ELISA is a competitive immunoassay. Competition occurs between total estrogens (estrone, estradiol, and estriol) present in standards, controls and samples and an enzyme-labelled antigen (conjugate) for a limiting number of anti-estrogen antibody binding sites on the microplate wells. After a washing step that removes unbound materials the enzyme substrate is added and approximately 15–20 minutes later the enzymatic reaction is terminated by addition of stopping solution. The resulting optical density (OD), measured with a microplate reader, is inversely proportional to the concentration of total estrogens in the sample. A standard curve is plotted with a provided set of standards to calculate directly the concentration of total estrogens in samples and controls.

Reagents And Materials Provided :

1. Rabbit Anti-Estrogens Antibody-Coated Break-Apart Well Microplate — Ready To Use. One 96-well (12×8) polyclonal antibody-coated microplate in a resealable pouch with desiccant. Unopened: Stable until the expiry date printed on the label. After Opening: Stable for three weeks. 2. Estrogen-HRP Conjugate — Ready to Use. Estrogen-HRP conjugate in a protein-based buffer with a nonmercury preservative. 20 mL/bottle. Stability: Unopened: Stable until the expiry date printed on the label. After Opening: Stable for three weeks. 3. Calibrators — Ready To Use. Seven vials containing estrogen in a protein-based buffer with a non-mercury preservative. Prepared by spiking buffer with a defined quantity of estrogens. * Listed below are approximate concentrations, please refer to vial labels for exact concentrations. 4. Controls — Ready to Use. Two vials containing estrogen in a protein-based buffer with a non-mercury preservative. Prepared by spiking serum with defined quantities of total estrogens. Refer to vial labels for the acceptable range. 1.0 mL/vial. Unopened: Stable until the expiry date printed on the label. After Opening: Stable for three weeks. 5. Wash Buffer Concentrate — Requires Preparation ×10 One bottle containing buffer with a non-ionic detergent and a non-mercury preservative. 50 mL/bottle. 12 months or as indicated on label. Dilute 1:10 in distilled or deionized water before use. If the whole microplate is to be used dilute 50 mL of the wash buffer concentrate in 450 mL of distilled or deionized water. Stable until the expiry date printed on the label. After Opening: Stable for three weeks. Working wash buffer stability: Following preparation, the working wash buffer is stable for up to 2 weeks when stored at 2-8°C. 6. TMB Substrate — Ready To Use. One bottle containing tetramethylbenzidine and hydrogen peroxide in a non-DMF or DMSO containing buffer. 16 mL/bottle. Unopened: Stable until the expiry date printed on the label. After Opening: Stable for three weeks. 7. Stopping Solution — Ready To Use. One bottle containing 1M sulfuric acid. 6 mL/bottle.

Materials Required But Not Supplied :

1. Single-channel pipette to dispense 50 μL 2. Multi-channel pipettes to dispense 50 μL and 150 μL 3. Multi-channel pipettes to dispense 350 μL (if washing manually) 4. Disposable pipette tips 5. Distilled or deionized water 6. Microplate reader with a filter set at 450 nm and an upper OD limit of 3.0 or greater 7. Microplate washer

Specimen Collection And Preparation :

Serum Approximately 0.1 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 a later date. Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling. No specimen pretreatment is necessary.

Assay Procedure :

Bring reagents, samples, and the microplate to room temperature before use. Test the calibrators, controls and specimen samples in duplicate. Once the procedure has started, complete all steps without interruption. 1. Prepare the working solution of wash buffer. 2. Remove the required number of well strips from the microplate. 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. Incubate the microplate on a microplate shaker** for 30 minutes at room temperature. 5. Pipette 150 μL of the Estrogen-HRP conjugate into each well. (We recommend using a multichannel pipette.) 6. Incubate for 2 hours at room temperature (no shaking). Cover the plate to avoid any contamination. 7. Wash the wells 3 times with 350 μL of diluted wash buffer per well. Tap the plate firmly against absorbent paper to ensure that no droplets remain in the wells. The use of a microplate washer is recommended. If a washer is not available, ensure the wash buffer reaches the top edge of the wells and no liquid remains in the plate after the final washing.) 8. Pipette 150 μL of the TMB substrate into each well at timed intervals. 9. Incubate for 30 minutes at room temperature (or until calibrator A attains dark blue color). 10. Pipette 50 μL of stopping solution into each well at the same timed intervals as in step 8. Gently tap the side of the microplate to mix the contents of the wells. 11. Read the plate on a microplate reader at 450 nm within 20 minutes after addition of the stopping solution.

Quality Control :

When assessing the validity of the test results, the following criteria should be evaluated: 1. The calibrator A mean optical density meets the acceptable range as stated in the QC Certificate. 2. The calibrator with the highest concentration meets the % binding acceptable ranges as stated in the QC Certificate. % Binding = (OD of calibrator / OD of calibrator A) x 100. 3. The values obtained for the kit controls are withing the acceptable ranges as stated in the QC certificate. 4. The results of any external controls that were used meet the acceptable ranges.

Calculation :

1. Calculate the mean optical density of each calibrator duplicate. 2. Use a 4-parameter curve fit with immunoassay software to generate concentration results. 3. If no software is available 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. 4. Read the values of the unknowns directly off the calibrator curve. 5. If a sample reads more than 10,000 pg/mL then dilute it with calibrator A at a dilution of no more than 1:10. The result obtained should be multiplied by the dilution factor.

Typical Standard Curve :

Sample data only. Do not use to calculate results.

Interferences :

Hemoglobin up to 2 g/L, Bilirubin conjugated and unconjugated up to 10 mg/dL, Triglycerides up to 5 mg/mL, Biotin up to 2.4 μg/mL, Daidzein and Resveratrol up to 200 ng/mL, Genistein up to 100 ng/mL, HAMAS up to 1.2 μg/mL, and Rheumatoid Factor up to 1.2 IU/mL did not interfere with the assay.

Precautions :

1. This kit is intended for research use only. 2. Practice good laboratory practices when handling kit reagents and specimens. This includes: • Do not smoke, drink, or eat in areas where specimens or kit reagents are handled. • Wear protective clothing and disposable gloves. • Wash hands thoroughly after performing the test. • Avoid contact with the eyes, flush eyes with water immediately and contact a doctor. 3. 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. 4. Control materials should be included in every run at a high and low level for assessing the reliability of results 5. Use deionized or distilled water to dilute wash buffer concentrate. 6. In order to reduce exposure to potentially harmful substances, gloves should be worn when handling kit reagents and human specimens. 7. 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. 8. A calibrator curve must be established for every run. 9. The controls should be included in every run and fall within established confidence limits. 10. 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. 11. When reading the microplate, the presence of bubbles in the wells will affect the optical densities (ODs). Carefully remove any bubbles before performing the reading step. 12. 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. 13. When dispensing the substrate and stopping solution, do not use pipettes in which these liquids will come into contact with any metal parts. 14. To prevent contamination of reagents, use a new disposable pipette tip for dispensing each reagent, sample, standard and control. 15. 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. 16. 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 total estrogens in human serum. 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 will lead to false results. 5. This kit is intended for research use only and should not be used as a diagnostic tool.

applicatons :

The Estrogen ELISA Kit is an immnoassay for detection of Estrogen in human serum.

Contents of Kit :

Standard Curve :

Sample data only. Do not use to calculate results.

Tetramethylbenzidine :