Free Triiodothyronine T3 ELISA 试剂盒
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Quick Overview for Free Triiodothyronine T3 ELISA 试剂盒 (ABIN649029)
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检测方法
实验类型
应用范围
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原理
- Competitive Enzyme Immunoassay TYPE 5(Analog Method for Free T3) The essential reagents required for a solid phase enzyme immunoassay include immobilized T3 antibody, enzyme-T3 conjugate and native free T3 antigen. The enzyme-T3 conjugate should have no measurable binding to serum proteins especially TBG and albumin. The method achieves this goal. Upon mixing immobilized antibody, enzyme-T3 conjugate and a serum containing the native free T3 antigen, a competition reaction results between the native free T3 and the enzyme-T3 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.
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Analytical Method
- Quantitative
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组件
- A. Human Serum References (1ml/vial). Six vials of serum reference for free triiodothyronine at approximate concentrations of 0 (A), 1. 0 (B), 3. 0 (C), 5. 0 (D), 8. 0 (E) and 16. 0 (F) pg/ml. 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: 1pg/ml x 1. 536 equal pmol/L). B. fT3- Enzyme Reagent (13ml/vial): One vial of triiodothyronine -horseradish peroxidase (HRP) conjugate in a bovine albumin-stabilizing matrix. A preservative has been added. Store at 2-8°C. C. T3 Antibody Coated Plate (96 wells). One 96-well microplate coated with sheep anti-triiodothyronine 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 (7ml/vial). One bottle containing tetramethylbenzidine (TMB) in buffer. Store at 2-8°C. F. Substrate B (7ml/vial). One bottle containing hydrogen peroxide (H2O2) in buffer. Store at 2-8°C. G. Stop Solution (8ml/vial). One bottle containing a strong acid (1N HCl). Store at 2-30°C. H. Product Instructions.
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试剂未包括
- 1. Pipette 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. Microplate washer 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. 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.
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Free Triiodothyronine T3 (fT3) ELISA Kit
fT3 适用: 小鼠 Colorimetric Competition ELISA 0.156 pg/mL - 50 pg/mL Plasma, Serum, Tissue Homogenate
Free Triiodothyronine T3 (fT3) ELISA KitfT3 适用: Various Species Colorimetric Competition ELISA 0.156 pg/mL - 50 pg/mL Plasma, Serum, Tissue Homogenate
Free Triiodothyronine T3 (fT3) ELISA KitfT3 适用: 人 Colorimetric Competition ELISA 0.156 pg/mL - 50 pg/mL Plasma, Serum, Tissue Homogenate
Free Triiodothyronine T3 (fT3) ELISA KitfT3 适用: 大鼠 Colorimetric Competition ELISA 0.156 pg/mL - 50 pg/mL Plasma, Serum, Tissue Homogenate
Free Triiodothyronine T3 (fT3) ELISA KitfT3 适用: 小鸡 Colorimetric Competition ELISA 0.156 pg/mL - 50 pg/mL Plasma, Serum, Tissue Homogenate
Free Triiodothyronine T3 (fT3) ELISA KitfT3 适用: Various Species Colorimetric Competition ELISA 740.7 fg/mL - 60000 fg/mL Plasma, Serum
Free Triiodothyronine T3 (fT3) ELISA KitfT3 适用: 大鼠 free Colorimetric Competition ELISA 2-32 pM/L Cell Lysate, Plasma, Serum, Tissue Homogenate
Free Triiodothyronine T3 (fT3) ELISA KitfT3 适用: 小鼠 free Colorimetric Competition ELISA 2-32 pM/L Cell Culture Supernatant, Plasma, Serum, Tissue Homogenate
Free Triiodothyronine T3 (fT3) ELISA KitfT3 适用: Pig Colorimetric Competition ELISA 2-32 pM/L
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应用备注
- 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.
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板类型
- Pre-coated
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试剂准备
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- 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.
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样品收集
- 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.
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结果分析
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A dose response curve is used to ascertain the concentration of free triiodothyronine 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 fT3 concentration in pg/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 fT3 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 pg/ml) from the horizontal axis of the graph (the duplicates of the unknown may be averaged as indicated). In the following example, the average absorbance (1. 855) (intersects the standard curve at (2. 1pg/ml) fT3 concentration.
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限制
- 仅限研究用
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注意事项
- 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 fT3-Enzyme Reagent solution 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 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.
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储存条件
- 4 °C/-20 °C
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- Free Triiodothyronine T3 (fT3)
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别名
- Triiodothyronine (free)
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物质类
- Hormone
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背景
- Summary and Explanation of the test: Triiodothyronine, a thyroid hormone, circulates in blood bound to carrier proteins (1,2). The main transport protein is thyroxine-binding globulin (TBG). However, only the free (unbound) portion of triiodothyronine is believed to be 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 triiodothyronine level changes so that the free triiodothyronine concentration remains constant. Thus, measurements of free triiodothyronine concentrations correlate more reliably with clinical status than total triiodothyronine levels. For example, the increase in total triiodothyronine levels associated with pregnancy, oral contraceptives and estrogen therapy result in higher total T3 levels while the free T3 concentration remains basically unchanged. This microplate enzyme immunoassay methodology provides the technician with optimum sensitivity while requiring few technical manipulations in a direct determination of free T3. In this method, serum reference, patient specimen, or control is first added to a microplate well. Enzyme-T3 conjugate (analog method) is added, and then the reactants are mixed. A competition reaction results between the enzyme conjugate and the free triiodothyronine for a limited number of antibody combining sites immobilized on the well. After the completion of the required incubation period, the antibody bound enzyme-triiodothyronine conjugate is separated from the unbound enzyme-triiodothyronine 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 free 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 free triiodothyronine concentration. Intended Use: The Quantitative Determination of Free Triiodothyronine Concentration in Human Serum by a Microplate Enzyme Immunoassay. Levels of fT3 are thought to reflect the amount of T3 available to the cells and may therefore determine the clinical metabolic status of T3. 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 A should be greater than 1.3. 2. Four out of six quality control pools should be within the established range.
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