Free Thyroxine ELISA Kit

Catalog No. ABIN996944

Product Details Free Thyroxine ELISA Kit

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

Reactivity: Chemical

Detection Method: Colorimetric

Method Type: Competition ELISA

Detection Range: 0-7.40 ng/mL

Minimum Detection Limit: 0 ng/mL

Application: ELISA

Purpose: The Quantitative Determination of Free Thyroxine Concentration in Human Serum by a Microplate Enzyme Immunoassay.
The assay is based on a competitive enzyme immunoassay.

Analytical Method: Quantitative

Sensitivity: 0.314 ng/dL

Components:

1. Human Serum References -- 1 ml/vial - Icons A-F
   Six (6) 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 = pmol/L
2. fT4- Enzyme Reagent - 13 ml/vial
   One (1) vial of thyroxine-horseradish peroxidase (HRP) conjugate in a protein-stabilized matrix. A preservative has been added. Store at 2-8°C.
3. Antibody Coated Microplate -- 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.
4. Wash Solution Concentrate -- 20ml
   One (1) vial containing a surfactant in buffered saline. A preservative has been added. Store at 2-8°C.
5. Substrate A -7 ml/vial
   One (1) bottle containing tetramethylbenzidine (TMB) in acetate buffer. Store at 2-8°C.
6. Substrate B - 7 ml/vial
   One (1) bottle containing hydrogen peroxide (H2O2) in acetate buffer. Store at 2-8°C.
7. Stop Solution - 8 ml/vial
   One (1) bottle containing a strong acid (1N HCl). Store at 2-30 °C.
8. Product Instructions

Material not included:

1. Pipette capable of delivering 50 μL & 100 μL volumes with a precision of better than 1.5 % .
2. Dispenser(s) for repetitive deliveries of 0.100 mL and 0.350 mL 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.

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

1. It is important that the time of reaction in e ach well is held constant for reproducible results
2. Pipetting of samples should not extend beyond ten (10) minutes to avoid assay drift.
3. Highly lipemic, hemolyzed or grossly contamina ted specimen(s) should not be used.
4. If more than one (1) plate is used, it is reco mmended to repeat the dose response curve.
5. The additional of substrate solution initiates a kinetic reaction, which terminated by the addition of the stop soluti on. Therefore, the substrate and stop solution should be added in the same seq uence to eliminate any time- deviation during reaction.
6. Plate readers measure vertically. Do not touch the bottom of the wells.
7. Failure to remove adhering solution adequately in the aspiration or decantation wash steps(s) may result in poor replication and spurious results.
8. Use components from the same lot. No intermixi ng of reagents from different batches.
9. Accurate and precise pipetting, as well as fol lowing the exact time and temperature requirements prescribed are essen tial. Any deviation from DAI IFU may yield inaccurate results.
10. All applicable national standards, regulations and laws, including, but not limited to, good laboratory procedures, must be strictly followed to ensure compliance and proper device usage.
11. It is important to calibrate all the equipment e.g. Pipettes, Readers, Washers and/or the automated instruments used with th is device, and to perform routine preventative maintenance.

Sample Volume: 50 μL

Assay Time: 1 - 2 h

Plate: Pre-coated

Protocol: 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 insolubilized binding sites.

Reagent Preparation:

1. Wash Buffer:
   Dilute contents of Wash solution to 1000 mL with distilled or deionized water in a suitable storage container. Dilute buffer can be store at 2-30 °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.

Sample Collection: The specimens shall be blood, serum in type and the usual precautions in the collection of venipuncture samples should be observ ed. For accurate comparison to established normal values, a fasting morning serum sample should be obtained. The blood should be collected i n a plain redtop venipuncture tube without additives or anti-coagula nts. Allow the blood to clot. Centrifuge the specimen to separate the serum from the cell

Sample Preparation:

Specimen(s) may be refrigerated at 2-8 ° C for a maximum period of (5) 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 the use of contaminated devices. Avoid repetitive freezing and thawing. When assayed in duplicate, 0.100 mL of the specimen is required.

Assay Procedure:

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 ref erence, 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 (50μ l) of the appropriate ser um reference, control or specimen into the assigned well.
3. Add 0.100 mL (100μ l) 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 deca ntation or aspiration. If decanting, blot the plate dry with absorbent paper.
7. Add 350μ l of wash buffer (see Reagent Preparat ion Section), decant (tap and blot) or aspirate. Repeat two (2) additio nal times for a total of three (3) washes. An automatic or manual plate washer can be used. Fo llow the manufacturer's instruction for proper usage. If a squeeze bottle is employed, fill each well by depressing the co ntainer (avoiding air bubbles) to dispense the wash. Decant the was h and repeat two (2) additional times.
8. Add 0.100 mL (100μ l) of working substrate solu tion to all wells (see Reagent Preparation Section). Always add reagents in the same order to minimize reaction time differences between we lls. DO NOT SHAKE THE PLATE AFTER SUBSTRATE ADDITI ON Incubate at room temperature for fifteen (15) m inutes.
9. Add 0.050 mL (50μ l) of stop solution to each well and gently mix for 15-20 seconds. Always add reagents in the same order to minimize r eaction time differences between wells
10. Read the absorbance in each well at 450nm (usin g a reference wavelength of 620-630nm to minimize well imperfections) in a microplate reader. The results should be read within thirty (30) min utes of adding the stop solution.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Expiry Date: 12 months

Target Details for Free Thyroxine

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

Abstract: fT4 Products

Background: 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.