Thyroxine T4 ELISA 试剂盒
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Quick Overview for Thyroxine T4 ELISA 试剂盒 (ABIN649045)
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See all Thyroxine T4 (T4) ELISA试剂盒适用
检测方法
实验类型
应用范围
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原理
- The essential reagents required for a solid phase sequential enzyme immunoassay include immobilized antibody, enzyme-antigen conjugate and native antigen. Upon mixing immobilized antibody, and a whole blood sample containing the native antigen, a binding reaction results between the native antigen for a limited number of insolubulized binding sites. After removing any unreacted native antigen by a wash step, the enzyme-conjugated antigen is introduced. The conjugate reacts with sites of the antibody unoccupied by the native antigen. After a short second incubation, the antibody-bound fraction is separated from unbound antigen by decantation or aspiration. The enzyme activity in the antibody-bound fraction is inversely proportional to the native antigen concentration. By utilizing several different calibrators 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. Neo-T4 Calibrator (Dried Blood Spots, Two rows by six dots levels, 2 x 6). Six levels of T4 Antigen in dried blood spots at approximate concentrations of 0(A), 1. 5 (B), 3. 5(C), 7(D), 14(E) and 25(F) µg/dl placed on S&S type 903 filter paper. Store at 2-8°C. A preservative has been added. Note 1: The exact values are printed on the outside of the aluminum pouch. Note 2: The Lot Specific calibrators, whole human blood based, were calibrated using analytically pure T4 (greater than 99% by weight). This material exceeds the specifications set by USP. B. Whole Blood Contrls (I, II & III). Three controls for thyroxine at varying concentrations (batch specific) made in whole blood spotted on S&S filter paper supplied in a zip-lock foil bag with a desiccant. Please see the bag label for ranges for different controls. Store at 2-8°C. A preservative has been added. C. Neo-T4 Elution Reagent: One vial containing 13 ml of buffer with binding protein inhibitors, surfactants and preservatives. Store at 2-8°C. D. Neo-T4 Conjugate Buffer (13 ml). One vial containing 13 ml of buffer, red dye, surfactants and preservatives. Store at 2-8°C. E. NT4 Enzyme Reagent (1. 5ml/via). One vial of thyroxine-horseradish peroxidase (HRP) conjugate in a protein-stabilizing matrix. A preservative has been added. Store at 2-8°C. F. fT4 Antibody Coated Plate (96 wells). One 96-well microplate coated with purified Mouse anti-Thyroxine IgG and packaged in an aluminum bag with a drying agent. Store at 2-8°C. G. Wash Solution Concentrate (20ml). One vial containing surfactant, buffer and saline. Store at 2-30°C. H. Substrate Solution (12ml/vial). One bottle containing tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) in buffer. Store at 2-8°C. I. Stop Solution (8ml/vial). One bottle containing a strong acid (1N H2SO4). Store at 2-30°C. J. 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: Do not use reagents that look cloudy or turbid. They may be contaminated. Note 4: Do not exchange reagents between different batches.
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试剂未包括
- 1. Laboratory Shaker capable of 150rpm rotation. 2. Dispenser(s) for repetitive deliveries of, 0. 050ml, 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 dilutions. 4. 1/8th inch hole punch. 5. Microplate washer or a squeeze bottle (optional). 6. Microplate Reader capable of absorbance readings at 450nm and 620nm. 7. Test tubes for making working enzyme conjugate. 8. Absorbent Paper for blotting the microplate well. 9. Plastic wrap and microplate cover for incubation step. 10. Vacuum aspirator (optional) for wash step. 11. Time. 12. External quality control materials.
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Thyroxine T4 (T4) ELISA Kit
T4 适用: Various Species Colorimetric Competition ELISA 3.7 ng/mL - 300 ng/mL Plasma, Serum
Thyroxine T4 (T4) ELISA KitT4 适用: Various Species, 人 Colorimetric Sandwich ELISA Cell Culture Supernatant, Fecal, Plasma (EDTA), Plasma (heparin), Serum, Urine
Thyroxine T4 (T4) ELISA KitT4 适用: 大鼠 Colorimetric Competition ELISA 0.703 ng/mL - 45 ng/mL Plasma, Serum, Tissue Homogenate
Thyroxine T4 (T4) ELISA KitT4 适用: Various Species Colorimetric Competition ELISA 0.703 ng/mL - 45 ng/mL Plasma, Serum, Tissue Homogenate
Thyroxine T4 (T4) ELISA KitT4 适用: Pig Colorimetric Competition ELISA 0.703 ng/mL - 45 ng/mL Plasma, Serum, Tissue Homogenate
Thyroxine T4 (T4) ELISA KitT4 适用: Cow Colorimetric Competition ELISA 0.703 ng/mL - 45 ng/mL Plasma, Serum, Tissue Homogenate
Thyroxine T4 (T4) ELISA KitSmall Sample T4 适用: Various Species Colorimetric Competition ELISA 3.7 ng/mL - 300 ng/mL Plasma, Serum
Thyroxine T4 (T4) ELISA KitT4 适用: Various Species Colorimetric Sandwich ELISA 1.56-100 nM/L Cell Culture Supernatant, Plasma, Serum, Tissue Homogenate
Thyroxine T4 (T4) ELISA KitT4 适用: 鱼 Colorimetric Competition ELISA 20-320 ng/mL Plasma, Serum, Tissue Homogenate
Thyroxine T4 (T4) ELISA KitT4 适用: 大鼠 Colorimetric Competition ELISA 20-320 ng/mL Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate
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应用备注
- Precautions: All products that contain human blood 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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- Working T4-Enzyme Reagent Solution: Dilute the T4-enzyme reagent 1: 11 with Neo T4 Enzyme Conjugate Buffer in a suitable, clean 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 two-three hours for maximum performance of the assay. General Formula: Amount of Buffer required equal Number of wells 0. 1 Quantity of NT4 Enzyme necessary equal Number of wells ( 0. 01 i. e. equal 16 x 0. 1 equal 1. 6ml for NT4 Conjugate Buffer16 x 0. 01 equal 0. 16ml (160µl) for NT4 enzyme reagent2. Wash Buffer: Dilute contents of wash concentrate to 1000ml with distilled or deionized water in a suitable storage container. Store at 2-30°C.
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样品收集
- The sampling from neonates is performed by lancing the heels of the infants and then spotting enough whole blood on S&S filter paper card to fill the marked circle. Allow the filter paper to dry at room temperature overnight away from heat and moisture. Enclose the dry blood specimen (DBS) in a moisture barrier plastic bag with desiccant and send to the laboratory. The specimen should be collected 3-7 days post partum, Physical data including age and weight of the infant, whether a multiple birth, or a premature birth etc should accompany the sample. It is important for the clinician to know these facts in order to properly assess the thyroid status of the infant. The dried blood samples are stable at 2-8°C for 2-3 weeks if stored in zip-lock, moisture resistant bags with desiccants.
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结果分析
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A dose response curve is used to ascertain the concentration of Neo-natal T4 in unknown specimens. 1. Record the absorbance obtained from the printout of the microplate readr. 2. Plot the absorbance for each duplicate serum reference versus the corresponding NNT4 concentration in µg/dl 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 NNT4 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 µg/dl) from the horizontal axis of the graph (the duplicates of the unknown may be averaged as indicated).
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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 and patient samples to room temperature (20 - 27°C). 1. Assemble the required number of microwells for each calibrator, control and patient sample to be assayed in duplicate. Replace any unused microwell strips back into the aluminum bag, seal and store at 2-8°C. Punch out 1/8” blood dot out of each calibrator, control and specimens into the assigned wells. (NOTE: Do not punch blood dots from areas that are printed or that are near the edge of the blood spot). 3. Add 0. 100 ml (100µl) of Neo-T4 Elution Reagent to all the wells. 4. Shake the microplate gently for 20-30 seconds to mix. (NOTE: Make sure that all blood dots are fully submerged in the liquid and not stuck to the walls of the microwells). 5. Cover with a microplate cover and rotate for 90 minutes at ambient temperature using a laboratory rotator set @ 150rpm. (Note: see alternative overnight incubation). 6. Discard the contents of the microplate by decantation or aspiration. If decanting, blot the plate dry with absorbent paper. NOTE: Make sure all the blood dots are removed at this point. There should be no dots left in the microwells. 7. Add 350µl of wash buffer (see Reagent Preparation Section), decant (tap and blot) or aspirate. 8. Repeat four additional times for a total of five washes. An automatic or manual plate washer can be used. Follow the manufacturer’s instruction for proper usage. 9. 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 four additional times. 10. Add 100 µl of working Neo-T4 Enzyme Reagent to each well. Cover the microplate and rotate for 45 minutes at ambient temperature using a laboratory rotator set @ 150rpm. (Note: see alternative overnight incubation). Repeat wash step Number7. 11. Add 0. 100 ml (100µl) of substrate solution to each well. 12. Cover the microplate and incubate for 15 minutes at ambient temperature. No rotation is required for this step. 13. Add 0. 050ml (50µl) of stop solution to each well and gently mix for 15-20 seconds. NOTE: Always add reagents in the same order to minimize reaction time differences between wells. 14 . 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 15 minutes of adding the stop solution. Alternative overnight procedure: 1. Substitute overnight incubation (12-16hrs) for the 90 minutes with rotation (Step 5). No rotator is required. Seal the plate(s) with plastic wrap. 2. Substitute 1 hr incubation for the 45 minute incubation with rotation (Step 8). No rotator isrequired. 3. All other steps remain the same.
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储存条件
- 4 °C/-20 °C
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- Thyroxine T4 (T4)
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别名
- Thyroxine
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物质类
- Amino Acid
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背景
- Summary and Explanation of the Test: Determination of hypothyroidism within the first few days of birth has been recognized as the single most important diagnostic test in neonates by the American Thyroid Association. The need for its early detection and treatment has resulted in the establishment of screening centers by federal and state health departments. A program of early screening of neonates for congenital hypothyroidism was started in Quebec, Canada in the early seventies. They used dry blood spots on filter paper as the sampling device. Very soon the program was followed by other major public health institutions in Canada and the US. By 1978, almost one million infants had been screened and an incidence rate of congenital hypothyroidism was established to be approximately 1 in 7000 births. Congenital hypothyroidism is probably the single most common preventable cause of mental retardation. Diagnosis and treatment of congenital hypothyroidism within the first 1-2 months after birth appears to be necessary in order to prevent severe mental retardation This microplate enzyme immunoassay methodology provides the technician with optimum sensitivity while requiring few technical manipulations. In this method, calibrators, patient specimen, or controls, all made and dried in whole blood are first added to a microplate well. A buffer containing essential ingredients to isolate T4 from blood proteins is added. The blood from the filter paper dots is allowed to elute in the buffer. In the process T4 (Thyroxine) dissociates from the serum (blood) proteins and binds to the antibody that is immobilized on the inside of the microwells. Excess blood is removed using a wash step. Enzyme-T4 conjugate is added. The enzyme labeled T4 binds to the sites on the antibody left available by the native T4 that came from the sample. After the completion of the required incubation period, excess enzyme conjugate is removed using 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 references, made in whole blood, of known thyroxine concentration permits construction of a dose response curve (DRC-graph) of activity and oncentration. Intended Use: The Quantitative Determination of Total Thyroxine Concentration in Human (Neonates) whole blood by a Microwell Enzyme Immunoassay. Q. C. Paramters: In order for the assay results to be considered valid the following criteria should be met. The absorbance (OD) of Calibrator 0 µg/dl should be greater than 1.3. 2. Four out of six quality control pools should be within the established ranges.
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