Triiodothyronine T3 ELISA Kit

Produktnummer ABIN649027

Human Triiodothyronine T3 ELISA Kit, Colorimetric Assay für die Quantifizierung von Human Triiodothyronine T3.

Kurzübersicht für Triiodothyronine T3 ELISA Kit (ABIN649027)

Target: Triiodothyronine T3 (T3)

Reaktivität: Human

Nachweismethode: Colorimetric

Methodentyp: Competition ELISA

Applikation: ELISA

Produktdetails Triiodothyronine T3 ELISA Kit

Verwendungszweck: Competitive Enzyme Immunoassay (TYPE 5): The essential reagents required for a solid phase enzyme immunoassay include immobilized antibody, enzyme-antigen conjugate and native antigen. Upon mixing immobilized antibody, enzyme-antigen conjugate and a serum containing the native antigen, a competition reaction results between the native antigen and the enzyme-antigen conjugate for a limited number of insolubulized binding sites. After equilibrium is attained, the antibodybound fraction is separated from unbound antigen by decantation or aspiration. The enzyme activity in the antibodybound fraction is inversely proportional to the native antigen concentration. By utilizing several different serum references of known antigen concentration, a dose response curve can be generated from which the antigen concentration of an unknown can be ascertained.

Analytische Methode: Quantitative

Bestandteile: A. Human Serum References (1ml/vial). Six vials of serum reference for triiodothyronine at concentrations of 0 (A), 0. 5 (B), 1. 0 (C), 2. 5 (D), 5. 0 (E) and 7. 5 (F) ng/ml. Store at 2-8°C. A preservative has been added. For SI units: ng/ml x 1. 536 equal nmol/L. B. T3 Enzyme Reagent (1. 5ml/vial). One vial of T3-horseradish peroxidase (HRP) conjugate in an albumin-stabilizing matrix. A preservative has been added. Store at 2-8°C. C. T3/T4 Conjugate Buffer (13ml). One bottle reagent containing buffer, red dye, preservative, and binding protein inhibitors. Store at 2-8°C. D. T3 Antibody Coated Plate (96 wells). One 96-well microplate coated with Sheep anti-T3 serum and packaged in an aluminum bag with a drying agent. Store at 2-8°C. E. Wash Solution Concentrate (20ml). One vial containing a surfactant in buffered saline. A preservative has been added. Store at 2-30°C. F. Substrate A (7 ml/vial). One bottle containing tetramethylbenzidine (TMB) in buffer. Store at 2-8°C. G. Substrate B (7 ml/vial). One bottle containing hydrogen peroxide (H2O2) in buffer. Store at 2-8°C. H. Stop Solution (8ml/vial). One bottle of stop solution containing a strong acid (1N HCL). Store at 2-30°C. I. Product Instructions: Note 1: Do not use reagents beyond the kit expiration date. Note 2: Opened reagents are stable for 60 days when stored at 2-8°C. Note 3: See end of this product insert for various configurations of reagents by kit size.

Benötigtes Material: 1. Pipettes capable of delivering 50µl volumes with a precision of better than 1. 5%. 2. Dispenser(s) for repetitive deliveries of 0. 100ml and 0. 350ml volumes with a precision of better than 1. 5%. 3. Adjustable volume (20-200µl) and (200-1000µl) dispenser(s) for conjugate and substrate dilutions. 4. Microplate washers or a squeeze bottle (optional). 5. Microplate Reader with 450nm and 620nm wavelength absorbance capability. 6. Test tubes for dilution of enzyme conjugate and substrate A and B. 7. Absorbent Paper for blotting the microplate wells. 8. Plastic wrap or microplate cover for incubation steps. 9. Vacuum aspirator (optional) for wash steps. 10. Timer. 11. Quality control materials.

Anwendungsinformationen

Applikationshinweise: Precautions: All products that contain human serum have been found to be non-reactive for Hepatitis B Surface Antigen, HIV 1&2 and HCV Antibodies by FDA required tests. Since no known test can offer complete assurance that infectious agents are absent, all human serum products should be handled as potentially hazardous and capable of transmitting disease. Good laboratory procedures for handling blood products can be found in the Center for Disease Control / National Institute of Health, Biosafety in Microbiological and Biomedical Laboratories, 2nd Edition, 1988, HHS Publication No. (CDC) 88-8395.

Plattentyp: Pre-coated

Aufbereitung der Reagenzien:

1. Working Reagent A - T3-enzyme Conjugate Solution: Dilute the T3-enzyme conjugate 1:11 with Total T3/T4 conjugate buffer in a suitable container. For example, dilute 160µl of conjugate with 1. 6ml of buffer for 16 wells (A slight excess of solution is made). This reagent should be used within twenty-four hours for maximum performance of the assay. Store at 2-8°C. General Formula: Amount of Buffer required equal Number of wells 0. 1, Quantity of T3-Enzyme necessary equal Number of wells 0. 01, i. E. equal 16 x 0. 1 equal 1. 6ml for Total T3/T4 Conjugate Buffer 16 x 0. 01 equal 0. 16ml (160µl) for T3 enzyme conjugate. 2. Wash Buffer: Dilute contents of wash concentrate to 1000ml with distilled or deionized water in a suitable storage container. Store at room temperature 20-27 °C for up to 60 days. 3. Working Substrate Solution: Pour the contents of the amber vial labeled Solution A into the clear vial labeled Solution B. Place the yellow cap on the clear vial for easy identification. Mix and label accordingly. Store at 2 - 8 °C. Note: Do not use the working substrate if it looks blue.

Probennahme: The specimens shall be blood, serum or plasma in type and the usual precautions in the collection of venipuncture samples should be observed. For accurate comparison to established normal values, a fasting morning serum sample should be obtained. The blood should be collected in a plain redtop venipuncture tube without additives or anti-coagulants (for serum) or evacuated tube(s) containing EDTA or heparin. Allow the blood to clot for serum samples. Centrifuge the specimen to separate the serum or plasma from the cells. Samples may be refrigerated at 2-8°C for a maximum period of five days. If the specimen(s) cannot be assayed within this time, the sample(s) may be stored at temperatures of -20 °C for up to 30 days. Avoid repetitive freezing and thawing. When assayed in duplicate, 0. 100ml of the specimen is required.

Ergebnisberechnung:

A dose response curve is used to ascertain the concentration of triiodothyronine in unknown specimens. 1. Record the absorbance obtained from the printout of the microplate reader. Plot the absorbance for each duplicate serum reference versus the corresponding T3 concentration in ng/ml on linear graph paper (do not average the duplicates of the serum references before plotting). 3. Draw the best-fit curve through the plotted points. 4. To determine the concentration of T3 for an unknown, locate the average absorbance of the duplicates for each unknown on the vertical axis (y-axis) of the graph, find the intersecting point on the curve, and read the concentration (in ng/ml) from the horizontal axis (X-axis) of the graph (the duplicates of the unknown may be averaged as indicated).

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Handhabung: Before proceeding with the assay, bring all reagents, serum references and controls to room temperature (20-27°C). 1. Format the microplates' wells for each serum reference, control and patient specimen to be assayed in duplicate. Replace any unused microwell strips back into the aluminum bag, seal and store at 2-8°C. 2. Pipette 0. 050 ml (50µl) of the appropriate serum reference, control or specimen into the assigned well. 3. Add 0. 100 ml (100µl) of Working Reagent A, T3 Enzyme Reagent to all wells (see Reagent Preparation Section). 4. Swirl the microplate gently for 20-30 seconds to mix and cover. 5. Incubate 60 minutes at room temperature. 6. Discard the contents of the microplate by decantation or aspiration. If decanting, blot the plate dry with absorbent paper. 7. Add 350µl of wash buffer (see Reagent Preparation Section), decant (tap and blot) or aspirate. Repeat two additional times for a total of three washes. An automatic or manual plate washer can be used. Follow the manufacturer's instruction for proper usage. If a squeeze bottle is employed, fill each well by depressing the container (avoiding air bubbles) to dispense the wash. Decant the wash and Repeat two additional times. 8. Add 0. 100 ml (100µl) of working substrate solution to all wells (see Reagent Preparation Section). Always add reagents in the same order to minimize reaction time differences between wells. DO NOT SHAKE THE PLATE AFTER SUBSTRATE ADDITION. 9. Incubate at room temperature for 15 minutes. 10. Add 0. 050ml (50µl) of stop solution to each well and gently mix for 15-20 seconds. Always add reagents in the same order to minimize reaction time differences between wells. 11. Read the absorbance in each well at 450nm (using a reference wavelength of 620-630nm to minimize well imperfections) in a microplate reader The results should be read within 30 minutes of adding the stop solution. Note: For re-assaying specimens with concentrations greater than 7. 5ng/ml, pipette 25µl of the specimen and 25µl of the 0 serum reference into the sample well (this maintains a uniform protein concentration). Multiply the readout value by 2 to obtain the triiodothyronine concentration.

Lagerung: 4 °C/-20 °C

Details zu Triiodothyronine T3

Target: Triiodothyronine T3 (T3)

Andere Bezeichnung: Triiodothyronine

Substanzklasse: Amino Acid

Hintergrund: Summary and Explanation of the test: Measurement of serum triiodothyronine concentration is generally regarded as a valuable tool in the diagnosis of thyroid dysfunction. This importance has provided the impetus for the significant improvement in assay methodology that has occurred in the last two decades. The advent of monospecific antiserum and the discovery of blocking agents to the T3 binding serum proteins have enabled the development of procedurally simple radioimmunoassays (1,2). This microplate enzyme immunoassay methodology provides the technician with optimum sensitivity while requiring few technical manipulations. In this method, serum reference, patient specimen, or control is first added to a microplate well. Enzyme-T3 conjugate is added, and then the reactants are mixed. A competition reaction results between the enzyme conjugate and the native triiodothyronine for a limited number of antibody combining sites immobilized on the well. After the completion of the required incubation period, the antibody bound T3-enzyme conjugate is separated from the unbound T3-enzyme conjugate by aspiration or decantation. The activity of the enzyme present on the surface of the well is quantitated by reaction with a suitable substrate to produce color. The employment of several serum references of known triiodothyronine concentration permits construction of a graph of activity and concentration. From comparison to the dose response curve, an unknown specimen's activity can be correlated with T3 concentration. Intended Use: The Quantitative Determination of Total Triiodothyronine Concentration in Human Serum or Plasma by a Microplate Enzyme Immunoassay. Q. C. Parameters: In order for the assay results to be considered valid the following criteria should be met: 1. The absorbance (OD) of calibrator 0 ng/ml should be greater than 1.3. 2. Four out of six quality control pools should be within the established ranges.