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Oxytocin ELISA Kit Wako

for Immunochemistry
Manufacturer :
FUJIFILM Wako Pure Chemical Corporation
Storage Condition :
Keep at 2-10 degrees C.
GHS :
  • Structural Formula
  • Label
  • Packing
SDS
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Product Number
Package Size
Price
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Distributor
292-84401
Barcode No
4548995087829
96Tests
List Price
JPY 98,000

In stock in Japan

Document

SDS
Product Specification Sheet
Package Insert
Spectral Data
Certificate of Analysis
Calibration Certificate

Kit component

96 tests

Antibody-coated Plate 1 plate
Oxytocin Standard 100 μL
Buffer 60 mL
Biotin-conjugated Oxytocin 1 bottle
Peroxidase-conjugated Streptavidin Solution 100 μL
Luminescent Reagent 1 6 mL
Luminescent Reagent 2 6 mL
Wash Solution (10×) 100 mL
Sample Buffer 1 5 mL
Sample Buffer 2 12 mL
Plate Seal 4 sheets

Product Information

Oxytocin is a peptide hormone that comprises nine amino acids. It is often called the "happy hormone" or "love hormone", as it has stress-relieving, anti-anxiety, and fear-alleviating effects and plays a role in the development of maternal behavior. It is a molecule that has attracted attention in treating mental disorders such as depression and autism or developing functional food materials. However, the assay of oxytocin used to include the need for complicated pretreatment using a C18 column, and a large amount of sample.

Oxytocin ELISA Kit Wako is an ELISA kit that can determine the amount of oxytocin in a sample.It overcomes issues with conventional oxytocin assays, since sample pretreatment simply requires mixing, stirring, and centrifuging of reagents, and the minimum sample amount required is 50 μL.

Features

  • Easy-to-use pretreatment
    Sample pretreatment requires simply adding pretreatment solutions, stirring, and centrifuging. A C18 column and organic solvent are not required.
  • Can be assayed with a tiny sample volume
    Minimum sample amount required is 50 μL (n=1).
  • Short assay time
    Assay time is 2.5 hours. Pretreatment can be done in approximately 30 minutes.
  • Can be used with a variety of samples
    Human saliva/urine/serum/plasma
    Mouse or rat serum/plasma
[Requirements before your purchase]

As this product uses a competitive assay with high sensitivity, a plate shaker and a plate reader for luminescence detection are required.

  • Kit Performance

    The range of the calibration curve 4.00~1,024 pg/mL
    Assay target Oxytocin
    Sample Human saliva/urine/serum/plasma
    Mouse serum/plasma
    Rat serum/plasma
    The sample amount required 50 μL (Required amount for n=1)
    200 μL (Recommended amount for n=2)
    Assay time Approximately 2.5 hours
    Detection method Luminescence (a plate reader for luminescence detection is required)
  • Example of Calibration Curve

    04173912_img01.png
  • Assay Principles

    This kit is based on a competitive assay, and luminescence intensity decreases as the amount of oxytocin in the sample increases.

    04173912_img02.png
  • 1. In advance, anti-oxytocin antibody, rabbit polyclonal antibody, and anti-rabbit secondary antibody are solid phased in each well of the assay plate.
    2. Add oxytocin-biotin and the standard solution or the pretreated sample to wells of the assay plate. Allow them to react competitively.
    3. Allow biotin and HRP-conjugated streptavidin to react.
    4. Add a luminescent substrate for HRP.
    5. Assay the oxytocin concentration in the sample by measuring HRP activity (luminescence intensity) in the wells.

 

Sample Pretreatment Methods

  • Pretreatment method of FUJIFILM Wako
    (approximately 30 min, recovery rate: 90 – 120%)

    Sample (saliva/urine/serum/plasma)

    Add Sample Buffer 1

    After stirring, allow to stand at room temperature for 10 minutes (stir once after 5 minutes have elapsed)

    Add Sample Buffer 2

    After stirring, allow to stand at room temperature for 10 minutes (stir once after 5 minutes have elapsed)

    Centrifuge

    Samples to be measured consist of separated supernatant

  • Pretreatment method of competitive company E
    (approximately 2 – 4 hours, recovery rate: 42 – 110%)

    Sample (saliva/urine/serum/plasma)

    Add trifluoroacetic acid (TFA), and stir

    Centrifuge

    Add separated supernatant to a C18 column that was equilibrated with TFA and acetonitrile

    Wash with TFA and elute with acetonitrile

    Decompress and dry the elution fraction

    Dissolve with buffer to make samples to be measured

Concentration Method (Optional)

Samples can be concentrated by the method below. Try this for samples with low oxytocin concentrations. Note that a higher sample volume is the required.

Example of Concentration Protocol (3-fold concentration, duplicate)

Collect 300 μL of saliva/urine/serum samples (three times the volume of sample required for a standard duplicate measurement).
Add 30 μL of Sample Pretreatment Solution 1, mix, and allow to stand for 5 minutes. Mix again and allow to stand for another 5 minutes.
Add 225 μL of Sample Pretreatment Solution 2, mix, and allow to stand for 5 minutes. Mix again and allow to stand for another 5 minutes.
Mix and centrifuge (5,000-6,000 x g for 10 min at 4°C). Collect 360 μL of the supernatant.
Evaporate the supernatant to dryness under the following conditions:
 Equipment: miVac Duo LV (Cat No. DPP-10000-G00, SP SCIENTIFIC)
 Concentration Conditions: 30℃ for 3 hours
 Store dried samples at 4°C.
Add 120 µL of the buffer solution provided in the kit to the dried sample. (3-fold concentration by volume)

Comparison of Assay Results Before and After Concentration

ID Concentration (-) Concentration (+) Concentration Factor
Assay value (pg/mL) Assay value (pg/mL)
Saliva
No. 1 28.4 80.5 2.83
No. 9 15.1 40.3 2.67
No. 10 20.5 55.2 2.69
Urine
No. 1 36.8 105 2.85
No. 2 12.0 31.9 2.66
No. 3 44.5 151 3.39
Serum
No. 4 48.8 122 2.50
No. 5 31.9 99.7 3.13
No. 6 33.8 95.6 2.83

Concentration factors were calculated from the results before and after concentration. The concentration factors ranged from 2.67-2.83 for the saliva samples, 2.66-3.39 for the urine samples, and 2.50-3.13 for the serum samples, indicating that concentration was effective for all sample types, and the concentration factor was close to 3-fold.

Data

Spiked recovery test

  • Human

    Spiked Oxytocin
    (pg/mL)
    Assay value
    (pg/mL)
    Recovery volume
    (pg/mL)
    Recovery
    (%)
    Saliva 1 0 28.4 - -
    25 50.2 21.8 87.2
    500 505 477 95.4
    Saliva 2 0 20.7 - -
    25 46.4 25.7 103
    500 517 496 99.2
    Saliva 3 0 3.48* - -
    25 25.2 21.7 86.8
    500 444 440 88
    Saliva 4 0 9.29 - -
    25 35.8 26.5 106
    500 541 531 106
    Saliva 5 0 2.18* - -
    25 23.4 21.2 84.8
    500 444 442 88.4
    Saliva 6 0 45.1 - -
    25 66.5 21.4 85.6
    500 599 554 111
    Saliva 7 0 45.5 - -
    25 67 21.5 86
    500 474 429 85.8
    Saliva 8 0 1.07* - -
    25 24.4 23.3 93.2
    500 435 434 86.8
    Saliva 9 0 15.1 - -
    25 39.2 24.1 96.4
    500 506 491 98.2
    Saliva 10 0 20.5 - -
    25 44.7 24.2 96.8
    500 469 449 89.8
    Saliva 11 0 8.88 - -
    25 34.3 25.4 102
    500 449 440 88

    *The values are outside the standard curve (< 4.00 pg/mL) and are provided as supplementary data.

  • Mouse

    Spiked Oxytocin
    (pg/mL)
    Assay value
    (pg/mL)
    Recovery volume
    (pg/mL)
    Recovery
    (%)
    Serum 0 49.7 - -
    50 107 57.3 115
    100 152 102 102
    250 305 255 102
    500 586 536 107
    Average 107
    Spiked Oxytocin
    (pg/mL)
    Assay value
    (pg/mL)
    Recovery volume
    (pg/mL)
    Recovery
    (%)
    Plasma 0 61.8 - -
    50 110 48.2 96.4
    100 156 94.2 94.2
    250 320 258 103
    500 595 533 107
    Average 100

    Rat

    Spiked Oxytocin
    (pg/mL)
    Assay value
    (pg/mL)
    Recovery volume
    (pg/mL)
    Recovery
    (%)
    Serum 0.00 18.8 - -
    50.0 83.0 64.2 128
    100 136 117 117
    250 289 271 108
    500 535 516 103
    Average 114
    Spiked Oxytocin
    (pg/mL)
    Assay value
    (pg/mL)
    Recovery volume
    (pg/mL)
    Recovery
    (%)
    Plasma 0.00 60.1 - -
    50.0 110 49.9 99.8
    100 167 107 107
    250 335 274 110
    500 573 513 103
    Average 105

Dilution linearity test

Human Saliva

Oxytocin was added at 100 pg/mL to a pretreated passive drool saliva sample, and a test sample was prepared in accordance with the sample preparation method described in the instruction manual of the kit. Ten different dilutions of the test sample were prepared using the buffer provided in the kit and were subjected to the assay. (duplicate measurements)

  • 04173912_img03R.png
  • 04173912_img04R.png

References

Citation

Nakata, M. et al.: Int. J. Mol. Sci., 24(9), 8248(2023). [Mouse Plasma]
1,5-Anhydro-D-Fructose Exhibits Satiety Effects via the Activation of Oxytocin Neurons in the Paraventricular Nucleus.

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=1). Mouse serum/plasma samples can be diluted 2- to 4-fold with the buffer solution provided in the kit and used for the assay. A sample volume of 200 µL is recommended for duplicate measurements. The volume of 200 µL is necessary to separate the supernatant without disturbing the “gel-containing pellet” generated after adding Pretreatment Solution 2 and centrifugation. (In theory, if the gel is not disturbed, sample volumes less than 200 µL can be used.)
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.
For saliva samples, assay results did not change significantly after storage at room temperature for 6 hours, in a refrigerator for 24 hours at -20°C for 1 month or at -80°C for 6 months without adding aprotinin and Proclin™ 950. (Fluctuations in room temperature were within ±15% of the temperature at hour 0.) Repeated freezing and thawing should be avoided whenever possible. Fujifilm Wako has observed that assay results fluctuated 15% or more after four or more cycles of freezing and thawing.
What is the recommended method of collection of saliva samples?
The passive drool method is recommended for the collection of saliva samples. (the passive drool method requires subjects to salivate into a vial or tube.)
It has been confirmed that samples collected using cotton swabs or inert polymer swabs can be used similarly to samples collected by the passive drool method. However, caution should be taken because some swab materials may absorb target analytes.

About pretreatment

Is it possible to extend the standing time during sample pretreatment?
Do not extend the processing time for Sample Pretreatment Solution 1. Since Pretreatment Solution 1 contains acid, the sample becomes acidic on its addition. A prolonged standing period may cause denaturation of the sample matrix. A slight extension (approximately 1 to 4 minutes) of the processing time for Sample Pretreatment Solution 2 is acceptable. The processing time must not be shorter than the standard time of 5 minutes.
Is there any point to pay special attention to during pretreatment?
Since the gel contained in Sample Pretreatment Solution 2 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 2 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.
How should I process saliva samples with low oxytocin levels?
Try "Concentration procedure for saliva samples (optional)" for samples with low oxytocin levels. The required sample volume will be higher, but it is possible to concentrate samples for measurement.
Can I store pretreated samples?
Storage of pretreated samples is not recommended. If storage is unavoidable, store the sample in a polypropylene or polyethylene tube under refrigeration (2-10°C) and measure it on the following day. Any undissolved matter found at the time of measurement should be removed by centrifugation, etc., and the supernatant should be used as the sample for measurement.
When I add Sample Buffer 1 to the brain homogenate sample, the mixture becomes turbid. Can I proceed as is?
Turbidity may occur if the sample has a high protein concentration or contains a lot of cell debris, due to protein denaturation. If, after adding Sample Buffer 2 and centrifuging to remove the gel, you find the supernatant clear of insoluble turbid matter, you can use it as a processed sample for measurement. Should insoluble matter remain, either increase the centrifugal force to around 10,000 g or filter the sample through a 0.2-μm filter before using it as a processed sample.

About analyte

What is the degree of cross-reactivity with vasopressin?
Cross-reactivity with [Arg8]-vasopressin is about 2.9%.
Can mesotocin, isotocin, and vasotocin be measured?
No data are available for mesotocin, isotocin, and vasotocin.

About the kit

How is the standard (oxytocin) of this kit certificated?
The concentration of oxytocin is certificated using the WHO world standard product (OXYTOCIN 4th International Standard NIBSC code: 76/575).
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. By detaching the rows containing the portion to be used and store the remainder in a refrigerator. It will remain stable for the shelf life. 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 600-800 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: PlateManager V5
Measurement type: Endpoint measurement
Measurement mode: Emission intensity
Plate size: 12 × 8
Well scan size: 1 × 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.

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 oxytocin 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 them using 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 2 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 2 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:
  1. 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].
  2. 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.
  3. 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.

Overview / Applications

Outline Oxytocin is a peptide hormone consisting of 9 amino acids. It is produced in the hypothalamus and released mainly from the posterior pituitary gland in the wake of childbirth and lactation, promotes uterine contraction and milk production. Oxytocin is commonly called happiness hormone or love hormone because it is involved in behavior formation and anti-stress action. This product is an ELISA kit that can measure oxytocin in a sample. This kit can be measured by a simpler pretreatment without C18 column and organic solvent than conventionally pretreatment method.

Calibration curve range: 4-1,024 pg/mL
Sample: human saliva, urine, plasma, serum, mouse serum, plasma, rat plasma, serum
Sample volume: 50microL (minimum at n=1)
200microL (recommended at n=2)
Assay time: 2.5 hours
Detection method: luminescence detection

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For research use or further manufacturing use only. Not for use in diagnostic procedures.

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