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AGT ELISA Kit

AGT Reactivity: Human, Mouse, Rat Colorimetric Competition ELISA 0.1-1.000 pg/mL Serum
Catalog No. ABIN1979314
  • Target See all AGT ELISA Kits
    AGT (Angiotensinogen (serpin Peptidase Inhibitor, Clade A, Member 8) (AGT))
    Reactivity
    • 4
    • 3
    • 3
    • 2
    • 2
    • 2
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    Human, Mouse, Rat
    Detection Method
    Colorimetric
    Method Type
    Competition ELISA
    Detection Range
    0.1-1.000 pg/mL
    Minimum Detection Limit
    0.1 pg/mL
    Application
    ELISA
    Purpose
    Human/Mouse/Rat Angiotensin II EIA Kit optimized for serum. Competition-based ELISA on a 96-well strip plate.
    Sample Type
    Serum
    Analytical Method
    Quantitative
    Specificity
    This kit can theoretically detect all active angiotensins, including ANGI, ANGII, ANGIII and ANGIV. However, it does not detect inactive angiotensinogen.

    Cross Reactivity: This EIA kit shows no cross-reactivity with any of the cytokines tested: Ghrelin, Nesfatin, NPY and APC.
    Cross-Reactivity (Details)
    This ELISA kit shows no cross-reactivity with any of the cytokines tested: Ghrelin, Nesfatin, NPY and APC. However, it does not detect inactive angiotensinogen.
    Sensitivity
    2.62 pg/mL
    Characteristics
    • Strip plates and additional reagents allow for use in multiple experiments
    • Quantitative protein detection
    • Establishes normal range
    • The best products for confirmation of antibody array data
    Components
    • Pre-Coated 96-well Strip Microplate
    • Wash Buffer
    • Standard Peptide
    • Assay Diluent(s)
    • Biotinylated Peptide
    • HRP-Streptavidin
    • TMB One-Step Substrate
    • Stop Solution
    • Assay Diagram
    • Positive Control Sample
    • Capture Antibody
    • User Manual
    Material not included
    • Distilled or deionized water
    • Precision pipettes to deliver 2 μL to 1 mL volumes
    • Adjustable 1-25 mL pipettes for reagent preparation
    • 100 mL and 1 liter graduated cylinders
    • Tubes to prepare standard and sample dilutions
    • Orbital shaker
    • Aluminum foil
    • Saran Wrap
    • Absorbent paper
    • Microplate reader capable of measuring absorbance at 450nm
    • SigmaPlot software (or other software that can perform four-parameter logistic regression models)
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  • Application Notes
    Recommended Dilution for serum and plasma samplesHuman: 2X / Mouse: 2X / Rat: 2X
    Sample Volume
    100 μL
    Assay Time
    5 h
    Plate
    Pre-coated
    Protocol
    1. Prepare all reagents, samples and standards as instructed.
    2. Add 100 μL detection antibody to each well.
    3. Incubate 1.5 h at RT or O/N at 4 °C.
    4. Add 100 μL standard or sample to each well.
    5. Incubate 2.5 h at RT.
    6. Add 100 μL prepared streptavidin solution.
    7. Incubate 45 min at RT.
    8. Add 100 μL TMB One-Step Substrate Reagent to each well.
    9. Incubate 30 min at RT.
    10. Add 50 μL Stop Solution to each well.
    11. Read plate at 450 nm immediately.
    Reagent Preparation
    1. Keep kit reagents on ice during steps. Equilibrate plate to room temperature before opening the sealed pouch.
      2. Briefly centrifuge the Anti-Angiotensin II Antibody vial (Item N) before use. Add 50 µL of 1x Assay Diluent E into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently.
      3. The antibody concentrate should then be diluted 100-fold with 1x Assay Diluent E. This is your anti-Angiotensin II antibody working solution, which will be used in step 2 of the Assay Procedure. NOTE: the following steps may be done during the antibody incubation procedure (step 2 of Assay Procedure).
      4. Briefly centrifuge the vial of biotinylated Angiotensin II peptide (Item F) before use. Add 10 µL of Item F to 5 mL of the 1X Assay Diluent E. Pipette up and down to mix gently. The final concentration of biotinylated Angiotensin II will be 200 pg/mL. This solution will only be used as the diluent in step 5 of Reagent Preparation.
      5. Preparation of Standards: Label 6 microtubes with the following concentrations: 10000 pg/mL, 1000 pg/mL, 100 pg/mL, 10 pg/mL, 1 pg/mL and 0 pg/mL. Pipette 450 mL of biotinylated Angiotensin II solution into each tube, except for the 10000 pg/mL (leave this one empty). It is very important to make sure the concentration of biotinylated Angiotensin II is 200 pg/mL in all standards. a. Briefly centrifuge the vial of standard Angiotensin II peptide (Item C). In the tube labeled 10000 pg/mL, pipette 8 µL of Item C and 792 µL of 200 pg/mL biotinylated Angiotensin II solution (prepared in step 4 above). This is your Angiotensin II stock solution (10000 pg/mL Angiotensin II, 200 pg/mL biotinylated Angiotensin II). Mix thoroughly. This solution serves as the first standard. b. To make the 1000 pg/mL standard, pipette 50 µL of Angiotensin II stock solution the tube labeled 1000 pg/mL. Mix thoroughly. c. Repeat this step with each successive concentration, preparing a dilution series as shown in the illustration below. Each time, use 450 mL of biotinylated Angiotensin II and 50 mL of the prior concentration until 1 pg/mL is reached. Mix each tube thoroughly before the next transfer. d. The final tube (0 pg/mL Angiotensin II, 200 pg/mL biotinylated Angiotensin II) serves as the zero standard (or total binding).
      6. Prepare a 10-fold dilution of Item F. To do this, add 2 mL of Item F to 18 mL of the 1XAssay Diluent E. This solution will be used in steps 7 and
      9.
      7. Positive Control Preparation: Briefly centrifuge the positive control vial (Item M). To the tube of Item M, add 101 µL 1x Assay Diluent E. Also add 4 µL of 10-fold diluted Item F (prepared in step 6) to the tube. This is a 2-fold dilution of the positive control. Mix thoroughly. The positive control is a cell culture medium sample that is meant to be a system control (to verify that the detection & kit components are working). It may be diluted further if desired, but be sure the final concentration of biotinylated Angiotensin II is 200 pg/mL.
      8. If Item B (20X Wash Concentrate) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 mL of Wash Buffer Concentrate into deionized or distilled water to yield 400 mL of 1X Wash Buffer. 10000 1000 100 10 1 0 pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL 50 mL 50 mL 50 mL 50 mL
      9. Sample Preparation: Use 1X Assay Diluent E + biotinylated ANGII to dilute samples, including serum/plasma, cell culture medium and other sample types. It is very important to make sure the final concentration of the biotinylated Angiotensin II is 200 pg/mL in every sample.
      Example: to make a 4-fold dilution of sample, mix together 5 µL of 10-fold diluted Item F (prepared in step 6), 182.5 mL of 1X Assay Diluent E, and 62.5 µL of your sample, mix gently. The total volume is 250 µl, enough for duplicate wells on the microplate. Do not use Item F diluent from Step 5 for sample preparation. If you plan to use undiluted samples, you must still add biotinylated Angiotensin II to a final concentration of 200 pg/mL.
      Example: Add 5 mL of 10-fold diluted Item F to 245 mL of sample.
      10. Briefly centrifuge the HRP-Streptavidin vial (Item G) before use. The HRP-Streptavidin concentrate should be diluted 200- fold with 1X Assay Diluent E.
    Sample Preparation

    Use 1X Assay Diluent E + biotinylated ANGII to dilute samples, including serum/plasma, cell culture medium and other sample types. It is very important to make sure the final concentration of the biotinylated Angiotensin II is 200 pg/mL in every sample. EXAMPLE: to make a 4-fold dilution of sample, mix together 5 µL of 10-fold diluted Item F (prepared in step 6), 182.5 mL of 1X Assay Diluent E, and 62.5 µL of your sample, mix gently. The total volume is 250 µl, enough for duplicate wells on the microplate. Do not use Item F diluent from Step 5 for sample preparation. If you plan to use undiluted samples, you must still add biotinylated Angiotensin II to a final concentration of 200 pg/mL. EXAMPLE: Add 5 mL of 10-fold diluted Item F to 245 mL of sample.

    Assay Procedure
    1. Keep kit reagents on ice during reagent preparation steps. It is recommended that all standards and samples be run at least in duplicate.
      2. Add 100 µL anti-Angiotensin II antibody (see Reagent Preparation step 3) to each well. Incubate for 1.5 hours at room temperature with gentle shaking (1-2 cycles/sec). You may also incubate overnight at 4 degrees C. 0
      3. Discard the solution and wash wells 4 times with 1x Wash Buffer (200-300 µL each). Washing may be done with a multichannel pipette or an automated plate washer. Complete removal of liquid at each step is essential to good assay performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
      4. Add 100 µL of each standard (see Reagent Preparation step 5), positive control (see Reagent Preparation step 7) and sample (see Reagent Preparation step 9) into appropriate wells. Be sure to include a blank well (Assay Diluent only). Cover wells and incubate for 2.5 hours at room temperature with gentle shaking (1-2 cycles/sec) or overnight at 4 °C.
      5. Discard the solution and wash 4 times as directed in Step
      3.
      6. Add 100 µL of prepared HRP-Streptavidin solution (see Reagent Preparation step 10) to each well. Incubate for 45 minutes with gentle shaking at room temperature. It is recommended that the incubation time should not be shorter or longer than 45 minutes.
      7. Discard the solution and wash 4 times as directed in Step
      3.
      8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking (1-2 cycles/sec).
      9. Add 50 µL of Stop Solution (Item I) to each well. Read absorbances at 450 nm immediately.
    Calculation of Results

    Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the blank optical density. Plot the standard curve using SigmaPlot software (or other software which can perform four-parameter logistic regression models), with standard concentration on the x-axis and percentage of absorbance on the y-axis. Draw the best-fit curve through the standard points.

    Assay Precision
    Intra-Assay: CV < 10 %
    Inter-Assay: CV < 15 %
    Restrictions
    For Research Use only
  • Handling Advice
    Avoid repeated freeze/thaw cycles.
    Storage
    -20 °C
    Storage Comment
    Standard, Biotinylated Angiotensin II peptide, and Positive Control should be stored at -20°C after arrival. Avoid multiple freeze-thaws. The remaining kit components may be stored at 4°C. Opened Microplate Wells and antibody (Item N) may be stored for up to 1 month at 2° to 8°C. Return unused wells to the pouch containing desiccant pack and reseal along entire edge.
    Expiry Date
    6 months
  • Shigemura, Iwata, Yasumatsu, Ohkuri, Horio, Sanematsu, Yoshida, Margolskee, Ninomiya: "Angiotensin II modulates salty and sweet taste sensitivities." in: The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 33, Issue 15, pp. 6267-77, (2013) (PubMed).

    Sun, Chang, Wu: "Uremic toxins induce kidney fibrosis by activating intrarenal renin-angiotensin-aldosterone system associated epithelial-to-mesenchymal transition." in: PLoS ONE, Vol. 7, Issue 3, pp. e34026, (2012) (PubMed).

  • Target See all AGT ELISA Kits
    AGT (Angiotensinogen (serpin Peptidase Inhibitor, Clade A, Member 8) (AGT))
    Alternative Name
    Angiotensin II, Angiotensinogen (AGT Products)
    Synonyms
    ANHU ELISA Kit, SERPINA8 ELISA Kit, AI265500 ELISA Kit, AngI ELISA Kit, AngII ELISA Kit, Aogen ELISA Kit, Serpina8 ELISA Kit, ANRT ELISA Kit, Ang ELISA Kit, PAT ELISA Kit, wu:fb62f06 ELISA Kit, wu:fj87b02 ELISA Kit, zgc:111892 ELISA Kit, AGT ELISA Kit, angt ELISA Kit, ANGT ELISA Kit, angiotensinogen ELISA Kit, angiotensinogen (serpin peptidase inhibitor, clade A, member 8) ELISA Kit, AGT ELISA Kit, Agt ELISA Kit, agt ELISA Kit
    Background
    Angiotensin II has been associated with a number of important physiological processes in heart, brain, adrenal gland and kidney. For cardiovascular effect, Angiotensin II is a potent direct vasoconstrictor, constricting arteries and veins and increasing blood pressure. It is also the most important Gq stimulator of the heart during hypertrophy. For neural effects, Angiotensin II increases thirst sensation (dipsogen) through the subfornical organ (SFO) of the brain, decreases the response of the baroreceptor reflex, and increases the desire for salt. It increases secretion of ADH in the posterior pituitary and secretion of ACTH in the anterior pituitary. For adrenal effects, Angiotensin II acts on the adrenal cortex, causing it to release aldosterone. For renal effects, Angiotensin II has a direct effect on the proximal tubules to increase Na + absorption. Angiotensin, a key player in the renin-angiotensin system, is a peptide hormone that causes vasoconstriction, increased blood pressure, and release of aldosterone from the adrenal cortex. It is derived from the precursor molecule angiotensinogen produced in the liver. Angiotensin II is formed from Angiotensin I, which is removed of two terminal residues by the enzyme Angiotensin-converting enzyme (ACE). Angiotensin II acts as an endocrine, autocrine/ paracrine, and intracrine hormone. Angiotensin II is degraded to angiotensin III by angiotensinases that are located in red blood cells and the vascular beds of most tissues. It has a half-life in circulation of around 30 seconds, while in tissue, it may be as long as 15-30 minutes. The effect of obesity on Angiotensin II has recently been reported.
    UniProt
    P11859
    Pathways
    JAK-STAT Signaling, ACE Inhibitor Pathway, EGFR Signaling Pathway, Peptide Hormone Metabolism, Regulation of Systemic Arterial Blood Pressure by Hormones, Regulation of Lipid Metabolism by PPARalpha, Protein targeting to Nucleus, Feeding Behaviour, Monocarboxylic Acid Catabolic Process, Dicarboxylic Acid Transport, Positive Regulation of Response to DNA Damage Stimulus, Regulation of long-term Neuronal Synaptic Plasticity
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