Free Thyroxine ELISA Kit

Catalog No. ABIN649025

Product Details Free Thyroxine ELISA Kit

Target: Free Thyroxine (fT4) (Free Thyroxine T4 (fT4))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Competition ELISA

Application: ELISA

Purpose: Competitive enzyme immunoassay Analog method for free T4 (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 free antigen, a competition reaction results between the native free 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 free 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.

Analytical Method: Quantitative

Components: A. Human Serum References (1 ml/vial). Six vials of human serum based reference calibrators for free thyroxine at approximate concentrations of 0 (A), 0. 40 (B), 1. 25 (C), 2. 10 (D), 5. 00 (E) and 7. 40 (F) ng/dl. Store at 2-8°C. A preservative has been added. ( Exact levels are given on the labels on a lot specific basis. For SI units: ng/dL x 12. 9 equal pmol/L). B. fT4- Enzyme Reagent (13 ml/vial). One vial of thyroxine-horseradish peroxidase (HRP) conjugate in a protein-stabilized matrix. A preservative has been added. Store at 2-8°C. C. fT4 Antibody Coated Plate (96 wells). One 96-well microplate coated with anti-thyroxine serum and packaged in an aluminum bag with a drying agent. Store at 2-8°C. D. Wash Solution Concentrate (20ml). One vial containing a surfactant in buffered saline. A preservative has been added. Store at 2-30°C. E. Substrate A (7 ml/vial). One bottle containing tetramethylbenzidine (TMB) in acetate buffer. Store at 2-8°C. F. Substrate B (7 ml/vial). One bottle containing hydrogen peroxide (H2O2) in acetate buffer. Store at 2-8°C. G. Stop Solution (8 ml/vial). One bottle containing a strong acid (1N HCl). Store at 2-30°C. H. 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.

Material not included: 1. Pipette capable of delivering 50µl & 100µl 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. Microplate washers or a squeeze bottle (optional). 4. Microplate Reader with 450nm and 620nm wavelength absorbance capability. 5. Absorbent Paper for blotting the microplate wells. 6. Plastic wrap or microplate cover for incubation steps. 7. Vacuum aspirator (optional) for wash steps. 8. Timer. 9. Quality control materials.

Application Details

Application Notes: 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 licensed reagents. 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.

Plate: Pre-coated

Reagent Preparation:

1. 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. 2. 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.

Sample Collection: The specimens shall be blood serum in type and the usual precautions in the collection of venipuncture samples should be observed. The blood should be collected in a plain redtop venipuncture tube without additives or gel barrier. Allow the blood to clot. Centrifuge the specimen to separate the serum from the cells. Specimen(s) may be refrigerated at 2-8°C for a maximum period of 48 hours. If the specimen(s) cannot be assayed within 48 hours, 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. 100 ml of the specimen is required

Calculation of Results:

A dose response curve is used to ascertain the concentration of free T4 in unknown specimens. 1. Record the absorbance obtained from the printout of the microplate reader. 2. Plot the absorbance for each duplicate serum reference versus the corresponding fT4 concentration in ng/dl on linear graph paper (do not average the duplicates of the serum references before plotting). 3. Connect the points with a best-fit curve. 4. To determine the concentration of fT4 for an unknown, locate the average absorbance of the duplicates for each unknown on the vertical axis of the graph, find the intersecting point on the curve, and read the concentration (in ng/dl) from the horizontal axis of the graph (the duplicates of the unknown may be averaged as indicated). In the following example, the average absorbance (0. 964) intersects the dose response curve at (1. 65ng/dl) free T4 concentration. Note 1: Computer data reduction software designed for (ELISA) assays may also be used for the data reduction. Duplicates of the unknown may be averaged as indicated.

Restrictions: For Research Use only

Handling

Handling Advice: Before proceeding with the assay, bring all reagents, serum references and controls to room temperature (20-27°C). 1. Format the microplate 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 fT4 Enzyme Reagent to all wells. 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.

Storage: 4 °C/-20 °C

Target Details for Free Thyroxine

Target: Free Thyroxine (fT4) (Free Thyroxine T4 (fT4))

Abstract: fT4 Products

Background: Summary and Explanation of the test: Thyroxine, the principal thyroid hormone, circulates in blood almost completely bound to carrier proteins. The main carrier is thyroxine-binding globulin (TBG). However, only the free (unbound) portion of thyroxine is responsible for the biological action. Further, the concentrations of the carrier proteins are altered in many clinical conditions, such as pregnancy. In normal thyroid function as the concentrations of the carrier proteins alters, the total thyroxine level changes so that the free thyroxine concentration remains constant. Thus, measurements of free thyroxine concentrations correlate better with clinical status than total thyroxine levels. The increase in total thyroxine associated with pregnancy, oral contraceptives and estrogen therapy occasionally result in total T4 levels over the limits of normal while the free thyroxine concentration remains in the normal reference range. Masking of abnormal thyroid function can also occur in both hyper and hypothyroid conditions by alterations in the TBG concentration. The total T4 can be elevated or lowered by TBG changes such that the normal reference levels result. The free thyroxine concentration can help in uncovering the patient's actual clinical status. In this method, serum reference, patient specimen, or control is first added to a microplate well. Enzyme-T4 conjugate (analog method) is added and the reactants are mixed. A competition reaction results between the enzyme conjugate and the free thyroxine for a limited number of antibody combining sites immobilized on the well. After the completion of the required incubation period, the antibody bound enzyme-thyroxine conjugate is separated from the unbound enzyme-thyroxine conjugate via a wash step. 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 free thyroxine 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 free thyroxine concentration. Intended Use: The Quantitative Determination of Free Thyroxine Concentration in Human Serum 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/dl should be greater than 1.3. 2. Four out of six quality control pools should be within the established ranges.