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Insulin ELISA Kit

INS Reactivity: Human Colorimetric Sandwich ELISA
Catalog No. ABIN649043
  • Target See all Insulin (INS) ELISA Kits
    Insulin (INS)
    Reactivity
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    Human
    Detection Method
    Colorimetric
    Method Type
    Sandwich ELISA
    Application
    ELISA
    Purpose
    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 Insulin 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
    Components
    A. Insulin Calibrators (2. 0 ml/vial, Dried). Six vials of references for Insulin antigen at levels of 0 (A), 5 (B), 25 (C), 50 (D), 100 (E), and 300 (F) µIU/ml. Reconstitute each vial with 2ml of distilled or deionized water. The reconstituted calibrators are stable for 60 days at 2-8°C. A preservative has been added. Note: The calibrators, human serum based, were calibrated using a reference preparation, which was assayed against the WHO 1st IRP 66/304. B. Insulin Enzyme Reagent (13ml/vial): One vial containing enzyme labeled affinity purified monoclonal mouse x-insulin IgG, biotinylated monoclonal mouse x-insulin IgG in buffer, dye, and preservative. Store at 2-8°C. C. Streptavidin Coated 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 (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: Above reagents are for a single 96-well microplate.
    Material not included
    1. Pipette (s) capable of delivering 50µl and 100µl volumes with a precision of better than 1. 5%. 2. Dispenser(s) for repetitive deliveries of 0. 100ml and 0. 3500ml 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 (The 620nm filter is optional). 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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  • 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 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
    Sample Volume
    50 μL
    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 or plasma 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 redtop venipuncture tube without additives or anti-coagulants (for serum) or evacuated tube(s) containing EDTA or heparin. Allow the blood to clot for serum samples. Centrifuge the specimen to separate the serum or plasma from the cells. Samples may be refrigerated at 2-8°C for a maximum period of five 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.
    Calculation of Results

    A dose response curve is used to ascertain the concentration of Insulin 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 Insulin concentration in µIU/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 Insulin 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 µIU/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.

    Restrictions
    For Research Use only
  • Handling Advice
    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 Insulin 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 (20-27°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. 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
  • Wu, Chen, Chen, Wang, Wang: "Anti-hyperlipidemic and anti-oxidative effects of gelsemine in high-fat-diet-fed rabbits." in: Cell biochemistry and biophysics, Vol. 71, Issue 1, pp. 337-44, (2015) (PubMed).

    Aghadavod, Zarghami, Farzadi, Zare, Barzegari, Movassaghpour, Nouri: "Evaluation of relationship between serum levels of anti-müllerian hormone, androgen, and insulin resistant with retrieval oocytes in overweight patients with polycystic ovary syndrome." in: Advanced biomedical research, Vol. 4, pp. 76, (2015) (PubMed).

    Dey, Saha, Chowdhuri, Sen, Sarkar, Haldar, Chaudhuri: "Assessment of anti-diabetic activity of an ethnopharmacological plant Nerium oleander through alloxan induced diabetes in mice." in: Journal of ethnopharmacology, Vol. 161, pp. 128-37, (2015) (PubMed).

    González-Ortiz, Martínez-Abundis, Mercado-Sesma, Álvarez-Carrillo: "Effect of pantoprazole on insulin secretion in drug-naïve patients with type 2 diabetes." in: Diabetes research and clinical practice, Vol. 108, Issue 1, pp. e11-3, (2015) (PubMed).

    Shabani, Naeimi Khaledi, Beigy, Emamgholipour, Parvaz, Poustchi, Doosti: "Circulating level of CTRP1 in patients with nonalcoholic fatty liver disease (NAFLD): is it through insulin resistance?" in: PLoS ONE, Vol. 10, Issue 3, pp. e0118650, (2015) (PubMed).

    Mazloom, Alizadeh, Pasalar, Esfahani, Meshkani: "Downregulated microRNA-155 expression in peripheral blood mononuclear cells of type 2 diabetic patients is not correlated with increased inflammatory cytokine production." in: Cytokine, (2015) (PubMed).

    Mansour, Mohajeri-Tehrani, Qorbani, Heshmat, Larijani, Hosseini: "Effect of glutamine supplementation on cardiovascular risk factors in patients with type 2 diabetes." in: Nutrition (Burbank, Los Angeles County, Calif.), Vol. 31, Issue 1, pp. 119-26, (2014) (PubMed).

  • Target See all Insulin (INS) ELISA Kits
    Insulin (INS)
    Alternative Name
    Insulin (INS Products)
    Synonyms
    IDDM2 ELISA Kit, ILPR ELISA Kit, IRDN ELISA Kit, MODY10 ELISA Kit, ins1 ELISA Kit, xins ELISA Kit, ins1-a ELISA Kit, Insulin ELISA Kit, AA986540 ELISA Kit, Ins-2 ELISA Kit, InsII ELISA Kit, Mody ELISA Kit, Mody4 ELISA Kit, proinsulin ELISA Kit, zgc:109842 ELISA Kit, igf2-A ELISA Kit, ins ELISA Kit, ins-a ELISA Kit, ins-b ELISA Kit, insulin ELISA Kit, insulin precursor ELISA Kit, insulin II ELISA Kit, preproinsulin ELISA Kit, insulin L homeolog ELISA Kit, insulin S homeolog ELISA Kit, INS ELISA Kit, INS-IGF2 ELISA Kit, ins ELISA Kit, Ins ELISA Kit, PIN ELISA Kit, Ins2 ELISA Kit, ins.L ELISA Kit, ins.S ELISA Kit
    Background
    Summary and Explanation of the test: Human insulin is a peptide produced in the beta cells of the pancreas and is responsible for the metabolism and storage of carbohydrates. As a result of biofeedback the insulin levels increase with intake of sugars and decline when sugar content is low for absorption. In the diabetic population the mechanism of insulin production is impaired because of genetic predispositions (Type I) or because of lifestyle and/or hereditary factors (Type II). In such cases either the insulin production has to be boosted by medication or it has to be supplemented by oral or intravenous methods. The quantitative determination of insulin can help in dose selection the patient has to be subjected to. On the other hand the circulatory insulin can be found at much higher levels in patients with pancreatic tumors. These tumors secrete abnormally high levels of insulin and thus cause hypoglycemia. Accordingly, fasting hypoglycemia associated with inappropriately high concentrations of insulin strongly suggests an islet-cell tumor (insulinoma). To distinguish insulinomas from factitious hypoglycemia due to insulin administration, serum C-peptide values are recommended. These insulinomas can be localized by provocative intravenous doses of tolbutamide and calcium. Intended Use: The Insulin Microplate Elisa test is intended to be used for the quantitative determination of insulin levels in human serum.
    Pathways
    NF-kappaB Signaling, RTK Signaling, Positive Regulation of Peptide Hormone Secretion, Peptide Hormone Metabolism, Hormone Activity, Carbohydrate Homeostasis, ER-Nucleus Signaling, Regulation of Carbohydrate Metabolic Process, Feeding Behaviour, Autophagy, Negative Regulation of intrinsic apoptotic Signaling, Brown Fat Cell Differentiation, Positive Regulation of fat Cell Differentiation
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