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C-Peptide ELISA 试剂盒

适用: 人 Colorimetric Sandwich ELISA
产品编号 ABIN649046
发货至: 中国
  • 抗原 See all C-Peptide ELISA试剂盒
    C-Peptide
    适用
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    检测方法
    Colorimetric
    实验类型
    Sandwich ELISA
    应用范围
    ELISA
    原理
    Immunoenzymometric assay (TYPE 3): The essential reagents required for an immunoenzymometric assay include high affinity and specificity antibodies (Ab). (enzyme conjugated and immobilized), with different and distinct epitope recognition, in excess, and native antigen (Ag). In this procedure, the immobilization takes place during the assay at the surface of a microplate well through the interaction of streptavidin coated on the well and exogenously added biotinylated monoclonal C-peptide antibody. Upon mixing monoclonal biotinylated antibody, the enzyme-labeled antibody and a serum containing the native antigen, reaction results between the native antigen and the antibodies, without competition or steric hindrance, to form a soluble sandwich complex. After equilibrium is attained, the antibodybound fraction is separated from unbound antigen by decantation or aspiration. The enzyme activity in the antibodybound fraction is directly proportional to the native antigen concentration. By utilizing several different serum references of known antigen values, a dose response curve can be generated from which the antigen concentration of an unknown can be ascertained.
    Analytical Method
    Quantitative
    组件
    A. C-Peptide Calibrators (2. 0 ml/vial, Dried). Six vials of references for C-Peptide antigen at levels of 0 (A), 0. 2 (B), 1. 0 (C), 2. 0 (D), 5. 0 (E), and 10. 0 (F) ng/ml. Reconstitute each vial with 2. 0ml of distilled or deionized water. The reconstituted calibrators are stable for 7 days at 2-8°C. In order to store for a longer period of time aliquot the reconstituted calibrators in cryo vials and store at -20 °C. DO NOT FREEZE THAW MORE THAN ONCE. A preservative has been added. Note: The calibrators, human serum based, were calibrated using a reference preparation, which was assayed against the WHO 1st IRR 84/510. B. C-Peptide Enzyme Reagent (13ml/vial): One vial containing enzyme labeled affinity purified monoclonal mouse antibody, biotinylated monoclonal mouse IgG in buffer, dye, and preservative. Store at 2-8°C. C. Streptavidin Plate (96 wells). One 96-well microplate coated with streptavidin and packaged in an aluminum bag with a drying agent. Store at 2-8°C. D. Wash Solution Concentrate (20 ml). One vial containing a surfactant in buffered saline. A preservative has been added. Store at 2-30°C. E. Substrate A (7. 0ml/vial). One bottle containing tetramethylbenzidine (TMB) in buffer. Store at 2-8°C. F. Substrate B (7. 0ml/vial). One bottle containing hydrogen peroxide (H2O2) in buffer. Store at 2-8°C. G. Stop Solution (8. 0ml/vial). One bottle containing a strong acid (1. 0 N HCl). Store at 2-30°C. H. Product Instructions.
    试剂未包括
    1. Pipette (s) 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% (optional). 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. Storage container for storage of wash buffer. 10. Distilled or deionized water. 11. Quality Control Materials.
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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
    样本量
    50 μL
    板类型
    Pre-coated
    试剂准备
    1. Wash Buffer: Dilute contents of wash solution 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.
    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. For accurate comparison to established normal values, a fasting morning serum sample should be obtained. The blood should be collected in a plain red-top venipuncture tube without additives. Allow the blood to clot. Centrifuge the specimen to separate the serum from the cells. Samples may be refrigerated at 2-8°C for a maximum period of two (2) 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.
    结果分析

    A dose response curve is used to ascertain the concentration of C-Peptide 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 C-Peptide concentration in ng/ml on linear graph paper (do not average the duplicates of the serum references before plotting). Draw the best-fit curve through the plotted points. 4. To determine the concentration of C-Peptide 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/ml) from the horizontal axis of the graph (the duplicates of the unknown may be averaged as indicated). Note: Computer data reduction software designed for IEMA (ELISA) assays may also be used for the data reduction.

    限制
    仅限研究用
  • 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 microplates' wells for calibrator, 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 calibrators, controls and samples into the assigned wells. 3. Add 0. 100 ml (100µl) of the C-Peptide Enzyme Reagent to each well. It is very important to dispense all reagents close to the bottom of the microwell. 4. Swirl the microplate gently for 20-30 seconds to mix. Cover with a plastic wrap. 5. Incubate for 120 minutes at room temperature (20-25°C). 6. Discard the contents of the microplate by decantation or aspiration. If decanting, tap and 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 used, fill each well to the top by squeezing the container. Avoiding air bubbles. 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). 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 mix gently for 15-20 seconds. 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: Always add reagents in the same order to minimize reaction time differences between wells.
    储存条件
    4 °C/-20 °C
  • 抗原 See all C-Peptide ELISA试剂盒
    C-Peptide
    Abstract
    C-Peptide 产品
    别名
    insulin 2 ELISA Kit, Ins2 ELISA Kit
    背景
    Summary and Explanation of the test: Diabetes is one of the leading causes of disability and death in the U. S. It affects an estimated 16 million Americans, about one third of them do not even know they have the disease. The causes of diabetes are not precisely known, but both genetic and environmental factors play a significant role. The disease is marked by deficiencies in the body's ability to produce and properly use insulin. The most common forms of diabetes are type 1, in which the body's ability to produce insulin is destroyed, and type 2, in which the body is resistant to insulin even though some amount of insulin may be produced. In-vitro determination of insulin and C-Peptide levels help in the differential diagnosis of liver disease, acromegaly, Cushing's syndrome, familial glucose intolerance, insulinoma, renal failure, ingestion of accidental oral hypoglycemic drugs or insulin induced factitious hypoglycemia. Both insulin and C-Peptide are produced by enzymatic cleavage of proinsulin. Proinsulin is stored in the secretory granules of pancreatic alpha cells and is split into a 31 amino acid connecting peptide (C-Peptide, MW 3600) and insulin (MW 6000). C-Peptide is devoid of any biological activity but appears to be necessary to maintain the structural integrity of insulin. Although insulin and C-Peptide are secreted into portal circulation in equimolar concentrations, fasting levels of C-Peptide are 5-10 fold higher than those of insulin owing to the longer half-life of C-Peptide. The liver does not extract C-Peptide however, it is removed from the circulation by degradation in the kidneys with a fraction passing out unchanged in urine. Hence urine C-Peptide levels correlate well with fasting C-Peptide levels in serum. The glucagon stimulated C-Peptide determination is often used for differential diagnosis of insulin-dependent from non-insulin-dependent diabetic patients. Intended Use: The Quantitative Determination of Circulating C-Peptide Concentrations in Human Serum by a Microplate Immunoenzymometric assay.
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