Vasopressin ELISA Kit Wako
- for Immunochemistry
- Manufacturer :
- FUJIFILM Wako Pure Chemical Corporation
- Storage Condition :
- Keep at 2-10 degrees C.
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96Tests
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In stock in Japan |
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Kit component
96 tests
| Antibody-coated Plate | 1 plate |
|---|---|
| Vasopressin Standard | 2 vials |
| Buffer | 60 mL |
| Biotin-conjugated Vasopressin | 2 vials |
| Peroxidase-conjugated Streptavidin Solution | 100 μL |
| Luminescent Reagent 1 | 6 mL |
| Luminescent Reagent 2 | 6 mL |
| Wash Solution (10x) | 100 mL |
| Sample Buffer 1 | 10 mL |
| Sample Buffer 2 | 5 mL |
| Sample Buffer 3 | 15 mL |
| Plate Seal | 4 sheets |
Product Overview
Vasopressin is a 9-amino acid peptide hormone produced in the hypothalamus and released into the bloodstream from the posterior pituitary gland. It is well known that vasopressin is referred to as an antidiuretic hormone (ADH). Recently, it was found that vasopressin and its receptors (V1a/V1b) is also widely distributed in the brain, and its function as neurotransmitter in the central nervous system has been attracting attention. Vasopressin has been reported to be involved in stress, social behavior, and learning. Furthermore, it has been linked to various psychiatric disorders such as depression, schizophrenia, eating disorders, etc. Conventionally, radioimmunoassay has been widely used to measure vasopressin. However, since it requires radioisotopes (RIs), special equipment is necessary for such measurements. ELISA kits are commercially available, but there are challenges such as the need for a large volume of sample and complicated pretreatment is required due to the abnormally high values obtained when chyle samples are measured.
Features
- Simple pretreatment
Pretreatment can be performed simply by adding the pretreatment solution provided with the kit, followed by stirring and centrifuging. - Measurable with small sample volume
Minimum required sample volume: 50 μL (n=2) - High sensitivity measurement
Lower limit of calibration curve: 1.00 pg/mL - Less susceptible to inaccuracies caused by chyle
Even chyle samples can be measured without abnormally high values being obtained. - Radioisotope (RI)-free
No need for special equipment for RI experiment
[Note]
A plate shaker and a plate reader for luminescence measurement are required since this product uses a highly sensitive competitive method.
Kit Performance
| Calibration curve range | 1.00-2,000 pg/mL |
|---|---|
| Assay target | (Arg8)-vasopressin |
| Analysis sample | Human saliva/urine/serum/plasma (EDTA) Mouse serum/plasma (EDTA) Rat serum/plasma (EDTA) |
| Sample volume | 50 μL (n=2） |
| Measurement duration | Approx. 2.5 hours |
| Detection method | Luminescent detection (a plate reader for luminescence detection is required) |
Example of Calibration Curve
Assay Principle
Since this product uses the principle of competitive method, as the amount of vasopressin derived from the sample becomes greater, the luminescence intensity becomes less.
- In advance, anti-vasopressin, rabbit polyclonal antibody, and anti-rabbit secondary antibody are solid phased in each well of the assay plate.
- Add vasopressin-biotin and the standard solution or the pretreated sample to wells of the assay plate. Allow them to react competitively.
- Allow biotin and HRP-conjugated streptavidin to react.
- Add a luminescent substrate for HRP.
- Assay the vasopressin concentration in the sample by measuring HRP activity (luminescence intensity) in the wells.
Data
Correlation with Conventional Product
Vasopressin in human saliva, urine, and serum was measured using this product and conventional product to determine the correlation between the measured values.
[Result]
There was a high correlation between this product and conventional product.
Effects of Chyle
Vasopressin in human serum samples was measured using this product and conventional product to determine the correlation between them. Conventional product resulted in abnormally high values being obtained for chyle samples. These values fell outside the calibration curve range. Therefore, measurement was performed again using the pretreatment method recommended by the manufacturer. The correlation of measured values was determined using all samples including (pretreated) chyle samples, or after excluding chyle samples only.
| Sample No. |
Measured value (pg/mL) | Description | |
|---|---|---|---|
| This product | Conventional product |
||
| 1 | 51 | 292 | |
| 2 | 114 | 494 | |
| 3 | 158 | 1,086 | |
| 4 | 170 | 1,065 | |
| 5 | 0.245* | 208 | |
| 6 | 48 | 3,874 | Chyle samples ++ (pretreated) |
| 7 | 156 | 5,722 | Chyle samples +++ (pretreated) |
| 8 | 204 | 4,177 | Chyle samples +++ (pretreated) |
| 9 | 66 | 3,132 | Chyle samples ++ (pretreated) |
| 10 | 67 | 3,280 | Chyle samples ++ (pretreated) |
[Result]
For chyle samples, conventional product resulted in abnormally high values being obtained, with measured values tending to increase even with pretreatment. On the other hand, when this product was used, chyle had no effect on the measured values. When chyle samples were excluded, there was a high correlation between the values obtained using this product and those obtained with conventional product.
Spiked Recovery Test
Human
| Amount spiked (pg/mL) |
Measured value (pg/mL) |
Recovery volume (pg/mL) |
Recovery rate (%) |
|
|---|---|---|---|---|
| Saliva | 0 | 18.4 | - | - |
| 10 | 28.9 | 10.5 | 105 | |
| 50 | 64.8 | 46.4 | 92.8 | |
| 100 | 110 | 91.6 | 91.6 | |
| 500 | 530 | 512 | 102 | |
| Average | 97.9 | |||
| Urine | 0 | 4.00 | - | - |
| 10 | 13.4 | 9.40 | 94.0 | |
| 50 | 48.8 | 44.8 | 89.6 | |
| 100 | 94.4 | 90.4 | 90.4 | |
| 500 | 526 | 522 | 104 | |
| Average | 94.5 | |||
| Serum | 0 | 43.0 | - | - |
| 10 | 52.1 | 9.10 | 91.0 | |
| 50 | 88.6 | 45.6 | 91.2 | |
| 100 | 136 | 93.0 | 93.0 | |
| 500 | 558 | 515 | 103 | |
| Average | 94.6 | |||
| Plasma (EDTA) |
0 | 34.2 | - | - |
| 10 | 44.3 | 10.1 | 101 | |
| 50 | 84.0 | 49.8 | 99.6 | |
| 100 | 133 | 98.8 | 98.8 | |
| 500 | 588 | 554 | 111 | |
| Average | 103 | |||
Mouse
| Amount spiked (pg/mL) |
Measured value (pg/mL) |
Recovery volume (pg/mL) |
Recovery rate (%) |
|
|---|---|---|---|---|
| Serum | 0 | 43.3 | - | - |
| 10 | 53.1 | 9.80 | 98.0 | |
| 50 | 87.9 | 44.6 | 89.2 | |
| 100 | 137 | 93.7 | 93.7 | |
| 500 | 601 | 558 | 112 | |
| Average | 98.2 | |||
| Plasma (EDTA) |
0 | 11.6 | - | - |
| 10 | 20.8 | 9.20 | 92.0 | |
| 50 | 54.4 | 42.8 | 85.6 | |
| 100 | 100 | 88.4 | 88.4 | |
| 500 | 564 | 552 | 110 | |
| Average | 94.0 | |||
Rat
| Amount spiked (pg/mL) |
Measured value (pg/mL) |
Recovery volume (pg/mL) |
Recovery rate (%) |
|
|---|---|---|---|---|
| Serum | 0 | 4.52 | - | - |
| 10 | 14.5 | 9.98 | 99.8 | |
| 50 | 52.7 | 48.2 | 96.4 | |
| 100 | 93.7 | 89.2 | 89.2 | |
| 500 | 572 | 567 | 113 | |
| Average | 99.6 | |||
| Plasma (EDTA) |
0 | 18.0 | - | - |
| 10 | 27.8 | 9.80 | 98.0 | |
| 50 | 65.0 | 47.0 | 94.0 | |
| 100 | 112 | 94.0 | 94.0 | |
| 500 | 520 | 502 | 100 | |
| Average | 96.5 | |||
Dilution Linearity Test
(1) Human
(2) Mouse
(3) Rat
Application Data
Measurement of human samples
Vasopressin in human saliva, urine, and serum was measured using this product.
FAQ
About sample
- What types of samples can be used?
- The assay can be performed with human saliva/serum/plasma/urine, mouse serum/plasma, and rat serum/plasma.
- What is the required sample volume?
- The minimum sample volume required is 50 µL (n=2).
- How should samples be stored?
- For long-term storage of samples, add the protease inhibitor aprotinin at 100 KIU/mL (example: Product Number 018-18111) and the preservative Proclin™ 950 at 1/750 volume to the samples, and store at -80°C or lower. Repeated freezing and thawing should be avoided whenever possible. Frozen specimens should be thawed immediately before measurement and thoroughly mixed.
- Which anticoagulants can I use when using plasma as sample?
- Anticoagulants such as EDTA do not significantly influence the assay when used in normal amounts. Heparin- and citrate-plasma have not been tested.
About pretreatment
- Is there any point to pay special attention to during pretreatment?
- Since the gel contained in Sample Pretreatment Solution 3 settles over time after stirring, stir again occasionally and pay attention to settling while adding. If the gel is not sufficiently suspended and the amount of added gel is insufficient, an abnormal value (RLU of B0 < RLU of sample) may result due to insufficient removal of reaction inhibitors. An abnormal value will also result if gel of Sample Pretreatment Solution 3 is mixed in the measurement sample. Also, please be careful not to aspirate the gel when separating the supernatant.
- How should I pretreat highly viscous saliva samples?
- Highly viscous saliva may not yield expected measurement values if it is only pretreated following the procedure specified in the package insert of this kit. In this case, spike the saliva with aprotinin and Proclin™ 950, freeze and thaw the mixture once, and use the supernatant after centrifugation (e.g., 5000 g, 10 minutes, 4°C) as the sample for the pretreatment procedure specified in the package insert of this kit.
About analyte
- What is the degree of cross-reactivity with oxytocin?
- Cross-reactivity with oxytocin is about 4.7%.
- Can mesotocin, isotocin, and vasotocin be measured?
- No data are available for mesotocin, isotocin, and vasotocin.
About the kit
- Is it possible to purchase the antibodies provided in this kit?
- These antibodies are not sold separately.
- Can I divide plates?
- Since the plate in this kit are separable and each row (8 wells) is detachable, divided use of the kit is possible. Once opened, reagents should be used promptly, as their performance may be affected by storage conditions. Use scissors or other tool to cut the necessary portion of the plate seal to cover the wells to be used.
About kit usage
- What reagents, instruments, and equipment are required for the assay using this kit?
- The reagents, instruments, and equipment required for the use of this kit are listed below. This kit uses a highly sensitive competitive ELISA and requires a plate shaker and a plate reader for luminescence measurement.
- Purified water (distilled water)
- Standard solution, tubes for sample dilution
- Glassware for dilution of washing solution (graduated cylinders, beakers)
- Pipettes with disposable tips (disposable tips for 10 µL and 200-500 µL)
- Repeating pipette to dispense 100 µL
- Paper towel or other absorbent material (to remove liquid left on the plate after washing)
- Mixer (Vortex type) Microplate shaker (about 500-800 rpm)
- Plate washer for 96-well plates (preferred) or wash bottles
- Plate reader for 96-well plates (for luminescence measurement)
- Software for data analysis
- What type of plate readers can be used?
- A plate reader for luminescence measurement is required for this kit.
- Please let me know the measurement parameters for the plate reader.
- As an example, the parameters when using Infinite M200 PRO (Tecan) are listed below.
Equipment: Infinite M200 PRO (Tecan)
Software: Plate Manager V5
Measurement type: Endpoint measurement
Measurement mode: Emission intensity
Plate size: 12 x 8
Well scan size: 1 x 1
Number of wavelengths: 1
Conversion of measurement values: W1
Units of measurement values: RLU
Attenuation 1: AUTOMATIC
Integration time 1: 500 ms
Settle time 1: 300 ms
- What type of plate shaker should be used?
- A capacity to shake the ELISA plates at about 500 rpm is required.
- Is it possible to provide contract services using this kit?
- You can provide contract services using this kit.
Trouble Shooting
- What should I do if the calibration curve is not good?
- Competitive reactions are reversible, but if it takes too long to add standards or samples after the addition of biotinylated vasopressin solution, the desired calibration curve slope may not be achieved due to increased variability or base emission intensity. As a rough standard, addition of samples or standards should be completed within 10-15 minutes (within approximately 1/10 of the competitive reaction time). If the number of samples is large, it is efficient to dispense the samples into flexible plates, etc. and transfer the musing a multi-channel pipette.
- What should I do if the measurement value of the sample is higher than that of the background?
- A higher RLU value for a sample than that of background may occur when reaction inhibitors remain in the sample after pretreatment(RLU of B0 < RLU of sample in competitive ELISA). In particular, reaction inhibitors may remain if the amount of gel contained in Sample Pretreatment Solution 3 is insufficient. As the gel settles over time after stirring, stir again occasionally and pay attention to settling while adding. An abnormal value will also result if gel of Sample Pretreatment Solution 3 is mixed in the pretreated sample. Also, please be careful not to aspirate the gel when separating the supernatant.
- What should I do if a low concentration range cannot be measured well?
- Perform sufficient stirring in each step, and pay special attention to the following steps:
■Immediately after addition of samples or standards Sufficient stirring is required. More homogeneous mixing can be achieved by stopping and starting the stirring intermittently as compared with continuous stirring. As a rough standard, it is recommended to perform the procedure [stirring at 600-800 rpm for 10 seconds with a microplate shaker → stopping once → stirring again; 3 times].
■During competitive reaction A stirring speed of 500 rpm is recommended. Insufficient stirring speed or intensity may result in decreased reactivity in a low concentration range. Immediately after addition of peroxidase-conjugated streptavidin solution
■Sufficient stirring is required in the same manner as for immediately after the addition of samples or standards. As a rough standard, it is recommended to perform the procedure [stirring at 600-800 rpm for 10 seconds with a microplate shaker → stopping once → stirring again; 3 times].
- What should I do if bubbles remain in wells during cleaning?
- For manual cleaning using a wash bottle, etc., try the method to avoid bubbles by overflowing the cleaning fluid only in the last round of cleaning. Use the cleaning fluid provided with the kit. Do not use ultrapure water for cleaning. Ultrapure water is also called "hungry water": since it has a strong propensity to incorporate materials coming in contact with it, there is concern about effects on antigen-antibody conjugates or other materials.
- What should I do if the luminous intensity is too high?
- Please check the following two points:
1. Is it taking a long time after the addition of biotin-labeled vasopressin solution before the next standard solution or sample is added?
2. Is the luminescent reagent allowed to warm up to room temperature before use?
If the above does not improve the situation, consider shortening the integration time (measurement duration) or using an attenuation filter.
For research use or further manufacturing use only. Not for use in diagnostic procedures.
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