Vasopressin ELISA Kit (Arginine Vasopressin)

Details for Product AVP ELISA Kit No. ABIN2815106, Supplier: Log in to see
Antigen
  • adh
  • arvp
  • avrp
  • avp-npii
  • copeptin
  • vasotocin
  • ADH
  • ALCOHOL DEHYDROGENASE
  • ARABIDOPSIS THALIANA ALCOHOL DEHYDROGENASE
  • ATADH
  • ATADH1
  • F22K20.19
  • F22K20_19
  • alcohol dehydrogenase 1
  • Adh
  • Adh1a
  • Adh1c
  • ARVP
  • AVP-NPII
  • AVRP
  • VP
  • DI
  • Vas
  • Vp
  • Vsp
  • arginine vasopressin
  • alcohol dehydrogenase 1
  • alcohol dehydrogenase 1 (class I)
  • AVP
  • avp
  • ADH1
  • Adh1
  • Avp
Alternatives
Mouse (Murine) Vasopressin ELISA Kit
Reactivity
All Species, Human
Alternatives
Kits with alternative reactivity to:
25
25
18
5
5
4
2
2
1
1
1
1
1
1
Method Type
Sandwich ELISA
Application
ELISA
Options
Supplier
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Purpose The DetectX® Arg8-Vasopressin (AVP) Immunoassay kit is designed to quantitatively measure AVP present in serum, plasma and tissue culture media samples.
Brand DetectX®
Sample Type Serum, Plasma, Tissue Culture Medium
Analytical Method Quantitative
Detection Method Chemiluminescent
Cross-Reactivity (Details) The following cross reactants were tested in the assay and calculated at the 50 % binding point. Steroid Cross Reactivity: Arg8-Vasopressin 100 %, Arg8-Vasotocin 26.2 %, Oxytocin 0.06 %, Isotocin < 0.01 %, Lys8-Vasopressin < 0.01 %
Components Coated White 96 Well Plates White plastic microtiter plate(s) with break-apart strips coated with goat anti-rabbit IgG. 1 Or 5 Each
Arg-Vasopressin Standard AVP at 100,000 pg/mL in a special stabilizing solution. 25 μL Or 125 μL
DetectX® Arg-Vasopressin Antibody A rabbit polyclonal antibody specific for AVP in a special stabilizing solution. 3 mL Or 13 mL
DetectX® Arg-Vasopressin Conjugate AVP-peroxidase conjugate in a special stabilizing solution. 3 mL Or 13 mL
Assay Buffer Concentrate Assay Buffer, 5X concentrate that should be diluted with deionized or distilled water. 28 mL Or 55 mL
Extraction Solution A special extraction solution for treatment of serum and plasma samples to extract AVP. 50 mL Or 250 mL
Wash Buffer Concentrate A 20X concentrate that should be diluted with deionized or distilled water. 30 mL Or 125 mL
Substrate Solution A 6 mL Or 28 mL
Substrate Solution B 6 mL Or 28 mL
Plate Sealer 1 Or 5 Each
Material not included Distilled or deionized water.
A Speedvac or other centrifugal vacuum concentrator or a manifold and inert gas supply, such as nitrogen or helium, to evaporate extracted samples.
Repeater pipet, such as an Eppendorf repeater, with disposable tips to accurately dispense 25, 50 and 100 μL.
A microplate shaker. 96 well microplate reader capable of reading glow chemiluminescence.
A list of some models of suitable readers can be found on our website at www.ArborAssays.com/documents/.
All lu- minometers read Relative Light Units (RLU).
These RLU readings will vary with make or model of plate reader.
The number of RLUs obtained is dependant on the sensitivity and gain of the reader used.
If you are unsure of how to properly configure your reader contact your plate reader manufacturer or carry out the following protocol: Dilute 5 μL of the AVP Conjugate Concentrate into 495 μL of deionized water.
Pipet 5 μL of this dilution into an uncoated white well and add 100 μL of prepared CLIA substrate (see page 8 for details).
This well will give you an intensity 0.7-0.8 times the maximum binding for the assay.
Ad- just the gain or sensitivity so that your reader is giving close to the readers maximum signal.
To properly analyze the data, software will be required for converting raw RLU readings from the plate reader and carrying out four parameter logistic curve (4PLC) fitting.
Alternative Name -Vasopressin (AVP ELISA Kit Abstract)
Background The neuropeptides, oxytocin and vasopressin, were isolated and synthesized by Vincent du Vigneaud at Cornel Medical College in 1953, work for which he received the Nobel Prize in Chemistry in 19551. The neuro- hypophysial hormone arginine vasopressin (AVP), which is also known as an antidiuretic hormone, is involved in a wide range of physiological regulatory processes, including renal water reabsorption, cardiovascular ho- meostasis, hormone secretion from the anterior pituitary, and modulation of social behavior and emotional status2. AVP and the structurally related posterior pituitary hormone, oxytocin (OT), are synthesized in the paraventricular nucleus and the supraoptic nucleus of the hypothalamus3. AVP is a 9 amino acid peptide with a 6-member disulfide ring. It is structurally related to oxytocin differing by 2 amino acids. Arg8-Vasopressin AVP is released in response to sexual stimulation, uterine dilatation, stress, and dehydration. AVP V2 recep- tors in the kidney are antidiuretic, whereas the receptors V1a and V1b receptors in the vascular tree, adrenal gland, uterus, and other tissues mediate the diverse peripheral effects of this peptide4. AVP acts principally on renal collecting tubules to increase water reabsorption. Diabetes insipidus (DI) is characterized by the in- ability to appropriately concentrate urine in response to volume and osmol stimuli. The main causes for DI are decreased AVP production (central DI) or decreased renal response to AVP (nephrogenic DI). AVP can also be secreted inappropriately in certain situations, particularly in elderly patients, leading to water retention and dilutional hyponatremia. Inappropriate AVP secretion might be observed with central nervous system pathol- ogy, such as head injury, stroke, or cerebral tumor, or as a side effect of central acting drugs that interfere with the hypothalamic regulation or AVP. Noncentral causes of inappropriate AVP secretion include peripheral stimuli that mimic central vascular hypovolemia, in particular severe low-output cardiac failure, and ectopic AVP secretion (usually by a bronchogenic carcinoma)
Pathways cAMP Metabolic Process
Application Notes This assay has been validated for serum, EDTA and heparin plasma, and tissue culture samples.
Samples containing visible particulate matter should be centrifuged before use.
Platelet poor EDTA plasma is recommended for AVP measurements and should be collected according to the protocol from WHO International Agency for Research on Cancer working group report, page 24 at: http://www.iarc.fr/en/publications/pdfs-online/wrk/wrk2/standardsBRC-8.pdf AVP is identical across almost all species and we expect this kit may measure AVP from a wide variety of sources other than human.
Pigs have Lys8-vasopressin (LVP) instead of AVP and as this assay has essentially zero reactivity to LVP porcine samples should not be used.
This assay has 26.2 % cross reactivity for Arg8-vasotocin, the reptile and avian analogue of Arg8-vasopressin.
It will therefore be useful in measuring samples from most species including mammals, reptiles, fish and birds.
The end user should evaluate recoveries of AVP in other samples being tested.
Plate Pre-coated
Protocol An AVP standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve.
Standards or diluted samples are pipetted into a white microti- ter plate coated with an antibody to capture rabbit antibodies.
An AVP-peroxidase conjugate is added to the standards and samples in the wells.
The binding reaction is initiated by the addition of a polyclonal antibody to AVP to each well.
After an overnight incubation at 4 °C the plate is washed and supplied substrate is added.
The substrate reacts with the bound AVP-peroxidase conjugate.
The intensity of the generated chemiluminescent signal is detected in a microtiter plate reader capable of measuring luminescence.
The concentration of the AVP in the sample is calculated, after making suitable correction for the dilution of the sample, using software available with most plate readers
Reagent Preparation

Allow the kit reagents to come to room temperature for 30 minutes.
We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine AVP concentrations.
Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit.
Assay Buffer Dilute Assay Buffer Concentrate 1:5 by adding one part of the concentrate to four parts of deion- ized water.
Once diluted this is stable at 4 °C for 3 months.
Wash Buffer Dilute Wash Buffer Concentrate 1:20 by adding one part of the concentrate to nineteen parts of deionized water.
Once diluted this is stable at 4 °C for 3 months.
Standard Preparation Label test tubes as #1 through #8.
Pipet 990 μL of Assay Buffer into tube #1 and 300 μL into the remaining tubes.
The Arg-Vasopressin stock solution contains an organic solvent.
Prerinse the pipet tip several times to ensure accurate delivery.
Carefully add 10 μL of the AVP stock solution to tube #1 and vortex completely.
Take 200 μL of the AVP solution in tube #1 and add it to tube #2 and vortex completely.
Repeat the serial dilutions for tubes #3 through #8.
The concentration of AVP will be 1,000, 400, 160, 64, 25.6, 10.24, 4.096 and 1.638 pg/mL.
Use all Standards within 2 hours of preparation.

Sample Preparation

Serum and Plasma Samples Serum and plasma samples should be extracted with the provided Extraction Solution, or with a solid phase C18 column extraction protocol prior to running in the kit. Protocol Using Extraction Solution: Mix 1 part sample with 1.5 parts of Extraction Solution. Vortex and then nutate at room temperature for 90 minutes. Centrifuge for 20 minutes at 4 °C at 1660 x g. Speedvac supernatant to dryness at 37 °C. Reconstitute sample with 250 µL of Assay Buffer. Use all samples within 2 hour of preparation.

Assay Procedure
  1. Use the plate layout sheet on the back page to aid in proper sample and standard identification. Determine the number of wells to be used and return unused wells to the foil pouch with desiccant. Seal the ziploc plate bag and store at 4ºC.
    2. Pipet 100 μL of samples or standards into wells in the plate.
    3. Pipet 100 μL of Assay Buffer into wells to act as maximum binding wells (Bo or 0 pg/mL).
    4. Pipet 125 μL of Assay Buffer into the non-specific binding (NSB) wells.
    5. Add 25 μL of the DetectX® AVP Conjugate to each well using a repeater pipet.
    6. Add 25 μL of the DetectX® AVP Antibody to each well, except the NSB wells, using a repeater pipet.
    7. Shake the plate in a plate shaker at room temperature for 15 minutes to ensure adequate mixing of the reagents. Cover the plate with the plate sealer and store at 4 °C for 16-18 hours.
    8. The following day remove the Chemiluminescent Substrate from the refrigerator and allow to come to room temperature for at least 30 minutes. Addition of cold Substrate will cause depressed signal.
    9. Aspirate the plate and wash each well 4 times with 300 μL wash buffer. Tap the plate dry on clean absorbent towels. 10. Add 100 μL of the mixed Chemiluminescent Substrate to each well, using a repeater pipet. 11. Incubate the plate at room temperature for 5 minutes without shaking. 12. Read the luminescence generated from each well in a mutimode or chemiluminescent plate reader using a 0.1 second read time per well. The chemiluminescent signal will decrease about 40 % over 60 minutes. 13. Use the plate reader's built-in 4PLC software capabilities to calculate AVP concentration for each sample.
Calculation of Results

All luminometers read Relative Light Units (RLU).
These RLU readings will vary with make or model of plate reader.
Average the duplicate RLU readings for each standard and sample.
Cre- ate a standard curve by reducing the data using the 4PLC fitting routine on the plate reader, after subtracting the mean RLU's for the NSB.
The sample concentrations obtained, calculated from the %B/B0 curve, should be multiplied by the dilution factor to obtain neat sample values.
Or use the online tool from http://www.myassays.com/arbor-assays-arg8-vasopressin-clia-kit.assay to calculate the data. *The MyAssays logo is a registered trademark of MyAssays Ltd. typical data Sample Mean RLU NetRLU % B/B0 AVP Conc. (pg/mL) NSB 10,445 0 - - Standard 1 48,115 37,670 22.88 1,000 Standard 2 56,605 46,160 28.03 400 Standard 3 65,020 54,575 33.14 160 Standard 4 79,215 68,770 41.76 64 Standard 5 102,215 91,770 55.73 25.6 Standard 6 125,960 115,515 70.15 10.24 Standard 7 146,890 136,445 82.86 4.096 Standard 8 157,205 146,760 89.13 1.638 B0 175,105 164,660 100.0 0 Sample 1 90,840 80,395 48.82 40.30 Sample 2 120,995 110,550 67.14 12.62 Always run your own standard curve for calculation of results.
Do not use this data. ® Conversion Factor: 1 ng/mL of AVP is equivalent to 0.922 nM. www.ArborAssays.com 10 Typical Standard Curves 100 160,000 90 140,000 80 120,000 70 100,000 60 50 %B/B0 80,000 %B/B0 Net RLU 40 60,000 30 40,000 20 20,000 10 0 0 1 10 100 1,000 Arg8-Vasopressin Conc. (pg/mL) Always run your own standard curves for calculation of results.
Do not use this data.
ValidatiOn data Sensitivity and Limit of Detection Sensitivity was calculated by comparing the RLUs for twenty wells run for each of the RLUs and standard #8.
The detection limit was determined at two (2) standard deviations from the RLUs along the standard curve.
Sensitivity was determined as 0.88 pg/mL.
The Limit of Detection for the assay was determined in a similar manner by comparing the RLUs for twenty runs for each of the zero standard and a low concentration sample.
Limit of Detection was determined as 1.16 pg/mL

Assay Precision Two samples were diluted with Assay Buffer and run in replicates of 20 in an assay.
Inter Assay Precision:
Two samples were diluted with Assay Buffer and run in duplicates in 19 assays run over multiple days by four operators.
Restrictions For Research Use only
Preservative Sodium azide
Precaution of Use As with all such products, this kit should only be used by qualified personnel who have had labo- ratory safety instruction.
The complete insert should be read and understood before attempting to use the product.
The antibody coated plate needs to be stored desiccated.
The silica gel pack included in the foil ziploc bag will keep the plate dry.
The silica gel pack will turn from blue to pink if the ziploc has not been closed properly.
This kit utilizes a peroxidase-based readout system.
Buffers, including other manufacturers Wash Buffers, containing sodium azide will inhibit color production from the enzyme.
Make sure all buffers used for samples are azide free.
Ensure that any plate washing system is rinsed well with deionized water prior to using the supplied Wash Buffer as prepared on Page 8.
Storage 4 °C
Storage Comment This kit should be stored at 4°C until the expiration date of the kit.
Supplier Images
 image for Arginine Vasopressin (AVP) ELISA Kit (ABIN2815106) Arginine Vasopressin (AVP) ELISA Kit
Product cited in: Whiting, Ogier, Forbush, Bucko, Gopalan, Seternes, Langeberg, Scott: "AKAP220 manages apical actin networks that coordinate aquaporin-2 location and renal water reabsorption." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 113, Issue 30, pp. E4328-37, 2016 (PubMed).

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