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ENMK07298
Mouse PGI2, PYCRL, RAG-1, RAG-2 Multiplex ELISA Kit
  • Assay Range：
    PGI2: 0.078-5 ng/mL; PYCRL: 0.156-10 ng/mL; RAG-1: 0.156-10 ng/mL; RAG-2: 0.156-10 ng/mL
  • Sensitivity：
    PGI2: 0.026 ng/mL; PYCRL: 0.052 ng/mL; RAG-1: 0.052 ng/mL; RAG-2: 0.052 ng/mL
  • Specification：
    96T
  • Shelf Life：
    12 months when stored at -20 ℃
  • Storage Conditions：
    -20 ℃; short-term storage at 2-8 ℃ is acceptable for up to 2 weeks
  • Purpose：
    Intended for the in vitro quantitative determination of Mouse PGI2, PYCRL, RAG-1, RAG-2 in liquid samples.
  • Application：
    Multiplex
Download The Manual
Kit Components Self-Prepared Equipment Sample Collection and Preparation Experimental Procedure Performance Metrics Result Determination Key Precautions
Kit Components
PART
96T
48T
STORAGE
Pre-Coated Plate
8×12
8×6
4 ℃ / -20 ℃
Pre-Mixed Standards
2 vials
1 vial
4 ℃ / -20 ℃
Pre-Mixed Biotinylated Antibody (1:100)
1 vial
1 vial
4 ℃ / -20 ℃
Enzyme Conjugate(1:100)
1 vial
1 vial
4 ℃ / -20 ℃
Enzyme Diluent
1 vial
1 vial
4 ℃ / -20 ℃
Antibody Diluent
1 vial
1 vial
4 ℃ / -20 ℃
Standard Diluent
1 vial
1 vial
4 ℃ / -20 ℃
Sample Diluent
1 vial
1 vial
4 ℃ / -20 ℃
Washing Buffer (1:25)
1 vial
1 vial
4 ℃ / -20 ℃
Color Reagent A
1 vial
1 vial
4 ℃ / -20 ℃
Color Reagent B
1 vial
1 vial
4 ℃ / -20 ℃
Color Reagent C
1 vial
1 vial
4 ℃ / -20 ℃
Manual
1 set
1 set
RT
Attention
RT: Room temperature; Mouse PGI2, PYCRL, RAG-1, RAG-2 Standard: Lyophilized; Color Reagent A: Store Away from Light
Self-Prepared Equipment
  • Microplate reader (450nm detection wavelength filter,with optional 570nm or 630nm correction wavelength filters).
  • Washer (adjustable injection volume to ensure that each well receives 350μL without overflow).
  • Clean benches,biological safety cabinets,fume hoods.
  • High-precision single-channel pipette (range 0.5-10μL,20-200μL,200-1000μL).
  • High-precision multi-channel pipette (8 or 12,the range of 50-300μL).
  • 37℃ incubator.
  • Low temperature centrifuge.
  • Refrigerators (4℃,-20℃,-86℃).
  • Analytical balance.
  • Scissors,tweezers,pliers,etc.
  • Plate mixer,low-frequency oscillator,etc.
Other Self-Provided Materials
  • Centrifuge tubes (capacity of 1.5 mL,5 mL,etc.).
  • Disposable pipette tips (range of 0.5-10 μL-20 μL,20-200 μL,200-1000 μL).
  • Pure water or distilled water.
  • Coordinate paper.
  • Absorbent paper.
Sample Collection and Preparation
Sample Preparation
01.Serum

Place collected whole blood in refrigerator at 4 ℃ overnight. Then centrifuge for 10mins at 1000-3000 rpm. Take supernatant and either test immediately or place samples at -20 ℃/-80 ℃(1-3 months) for storage.

02.Plasma

Take plasma where EDTA, sodium citrate, or heparin has been added as anticoagulant. Mix well. Centrifuge mixture for 10mins at 1000-3000 rpm. Take supernatant and test immediately or place samples at -20 ℃ /-80 ℃ (1-3 months) for storage.

03.Tissue Homogenate

Take tissue slices which have been washed in 0.01 M PBS and then a tissue protein extraction reagent has been added according to proportion of 1 g to 5-10 mL and then mixed on ice bath. After sufficient homogenization, please then centrifuge for 10mins at 5000-10000 rpm. Take supernatant for immediate testing, or place samples at -20 ℃/-80 ℃ (1-3 months) for storage.

04.Cell Culture

Centrifuge for 10 mins at 1000-3000 rpm. Take supernatant for immediate testing, or place samples at -20 ℃/-80 ℃ (1-3 months) for storage.

05.For Urine, Ascites, Cerebrospinal Fluid, etc

Centrifuge for 10mins at 1000-3000 rpm. Take supernatant for immediate testing, or place samples at -20 ℃/-80 ℃ (1-3 months) for storage.

Precautions
1. Blood collection tubes should be both pyrogen- and endotoxin-free. 2. Hemolyzed or hyperlipidemic specimens are not recommended to be used. 3. Samples ultimately should appear clear and mostly transparent. All particulates should be removed through centrifugation. 4. If collected samples are not used immediately, they should be divided according to single usage quantities and stored frozen at -20/-80℃, carefully avoiding repeated freeze-thaw cycles. 5. Sample dilution optimization is often necessary for proper sample resolution within the standard curve. Pre-experiments are always recommended to be performed prior to running the bulk of the samples, in order to determine if an optimizing dilution should be made to the samples. 6. It is recommended to collect sufficient samples for running multiple tests, since sometimes some users can experience issues that may result in a loss of data. 7. Always wear protective outerwear while collecting/handling samples (e. g. wearing gloves, coat, respirator, etc. ) and be aware of all potential risks involved in handling your specimens. Specimen processing should be conducted inside a properly-maintained biological safety cabinet.
Experimental Preparation
It is recommended that the user be well-informed regarding the general range of concentrations that are expected for their samples. We would suggest consulting the scholarly literature for references to similar samples' concentrations, and then to consider diluting your samples accordingly. 1. Please remove ELISA Kit from refrigerator 20 mins in advance, and begin test once it has been brought to room temperature. 2. Dilute the concentrated Washing Buffer with double distilled water (1:25). Return unused quantity back to the box. 3. Standard:Add 1. 0mL Standard Diluent to lyophilized standard vial and allow to sit for 30 mins. After the standard has completely dissolved, mix it slightly and mark with a label on the tube. 4. Procedure for standard sample dilution: Take 7 clean tubes and label them with their expected concentrations (STD2, STD3, STD4, STD5, STD6, STD7, STD8). Add 300 μL of Standard Diluent to each tube. Pipette 300 μL from the reconstituted STD1 into the tube labeled STD2 and mix well. Then pipette 300 μL from the STD2 tube into the STD3 tube and mix well. Repeat this procedure up to the STD7 tube. The Standard Diluent in the STD8 tube serves as the negative control. 5. Reconstituted standard stock solution cannot be reused. 6. Biotinylated Antibody: Remove the appropriate volume of Biotinylated Antibody solution for the quantity of wells intending to be assayed, and dilute with Antibody Diluent in a proportion of 1:100. This should be prepared 30mins in advance, and we would absolutely recommend not re-using for additional assaying. 7. Enzyme Conjugate: Remove the appropriate amount of Enzyme Conjugate solution for the quantity of wells intending to be assayed, and dilute with the Enzyme Diluent in a proportion of 1:100. This should be prepared 30mins in advance, and we would absolutely recommend not re-using for additional assaying. 8. Color Reagent: Prepare Color Reagent solution 30mins in advance by mix Color Reagent A and Color Reagent B at a volume ratio of 9:1.
Experimental Procedure
Experimental Procedure
Result Calculation
Take the concentration values of standards as the X-coordinates and OD readings as the Y-coordinates. Logarithmic, double-logarithmic, or semi-logarithmic plotting may also be used. Use a smooth line to connect each coordinate point of the standard values. The concentration of samples can be found by inputting the sample OD values into the line equation for the standard curve. It is recommended to employ professional curve software (e.g. curve expert 1.3) to analyze and compute the results.
Performance Metrics
Mouse PGI2, PYCRL, RAG-1, RAG-2 Multiplex ELISA Kit Typical Data
TYPICAL DATA
Reference STD1
Reference STD2
Reference STD3
Reference STD4
Reference STD5
Reference STD6
Reference STD7
Reference STD8
PGI2, ng/mL
5
2.5
1.25
0.625
0.313
0.156
0.078
0
PGI2, OD.
2.739
1.521
0.837
0.465
0.245
0.142
0.079
0.054
PYCRL, ng/mL
10
5
2.5
1.25
0.625
0.313
0.156
0
PYCRL, OD.
3.571
2.106
1.113
0.637
0.358
0.197
0.11
0.073
RAG-1, ng/mL
10
5
2.5
1.25
0.625
0.313
0.156
0
RAG-1, OD.
2.622
1.441
0.834
0.459
0.25
0.142
0.077
0.05
RAG-2, ng/mL
10
5
2.5
1.25
0.625
0.313
0.156
0
RAG-2, OD.
2.462
1.354
0.776
0.435
0.244
0.133
0.072
0.049
Mouse PGI2, PYCRL, RAG-1, RAG-2 Multiplex ELISA Kit Standard Curve

STANDARD CURVE

Mouse PGI2, PYCRL, RAG-1, RAG-2 Multiplex ELISA Kit Linear

To assess linearity of the assay, samples were spiked with high concentrations of Mouse PGI2, PYCRL, RAG-1, RAG-2 and diluted with the appropriate calibrator diluent to produce samples with values within the dynamic range of the assay.

Mouse PGI2, PYCRL, RAG-1, RAG-2 Multiplex ELISA Kit Recovery Rate
The recovery of Mouse PGI2, PYCRL, RAG-1, RAG-2 was evaluated by spiking at three different levels within the assay range in various matrices.
Mouse PGI2, PYCRL, RAG-1, RAG-2 Multiplex ELISA Kit Recovery Rate
Matrix
Scope %
Average % Recovery Rate
serum(n=5)
85-91
88
EDTA plasma(n=5)
94-96
95
Cell Culture Media(n=5)
94-100
97
Mouse PGI2, PYCRL, RAG-1, RAG-2 Multiplex ELISA Kit Precision
Intra-assay precision (within-assay precision): Three samples of known concentration were tested 20 times on one plate to assess intra-assay precision. Inter-assay precision (between-assay precision): Three samples of known concentration were tested in 20 separate assays to assess inter-assay precision. The experiments were performed by at least two technicians using two lots of reagent components.
Within-Run Precision
Inter-Batch Precision
Sample
1
2
3
1
2
3
n
20
20
20
20
20
20
CV%
6.4
6.5
6
8.9
7.5
8.9
Result Determination
1.The OD values of each sample and standard should be corrected by subtracting the OD value of the blank well. 2.Draw the standard curve manually: Plot the standard curve using the standard concentrations as the X-axis and the corresponding OD values as the Y-axis. Connect the plotted points with a smooth curve. The concentration of samples can be calculated by substituting the sample OD values into the standard curve equation. It is recommended to use professional curve-fitting software (e.g., Curve Expert 1.3) for result analysis and calculation. 3.If the sample OD value is higher than that of the highest-concentration standard in the standard curve, the sample should be diluted (or further diluted) and reassayed. Multiply the measured concentration by the dilution factor when calculating the final concentration.
Precautions
  • 01
    The re-dissolved standard cannot be stored again once prepared, so please do not attempt to re-freeze it once it has been reconstituted.
  • 02
    Due to shaking/inversion during transport, centrifuging of the tubes/bottles of the kit might be necessary to consolidate the material contained within. Tubes should be shaken manually or centrifuged for 1 min at 1000 rpm to pool all material to the bottom.
  • 03
    Concentrated Washing Buffer might crystallize slightly. Use a water bath to help the dissolution during diluting process. The crystals must be totally dissolved when preparing the Washing Buffer.
  • 04
    The prepared standard is intended to only be a single-use aliquot, so please do not try to re-use the standard that has already been tested. Please use the second vial provided if you run the assay again.
  • 05
    Only use reagents/components that came directly with this kit. Do not mix batches/lots from other orders of this kit, or from different kits.
  • 06
    Ensure the reagents are well mixed. For the reagents in the microplate, adequate mixing is particularly important for accurate test results. It is recommended to employ a micro-oscillator (at the lowest frequency). If a micro-oscillator is not available, please slightly shake the microplate manually for 1 min, in a circular motion in order to make sure the wells are sufficiently mixed.
  • 07
    Please ensure the kit has been brought to room temperature prior to beginning the assay.
  • 08
    Standards are always recommended to be tested in duplicate or triplicate.
  • 09
    Place the unused microplate strips into the foil bag at 2-8 ℃ for storage (if you intend to use the strips within a relatively short timeframe).
  • 10
    The chromogen reagent is sensitive to light, therefore please avoid exposing to light.
  • 11
    Kits that have passed their expiration date should not be used.
  • 12
    When using dual-wavelength, the wavelengths should be set at 450nm and 630nm.
  • 13
    All the samples, Washing Buffer and wastes should be treated as biowaste. Color Reagent C is 1M sulfuric acid, so please pay close attention to safety when it is used.
  • 14
    Sample addition should always be done via pipette or similar instrument. Calibrate the instrument prior to running the assay in order to avoid experimental errors. Please add samples to the wells quickly, as it is recommended to control the sample addition time to less than ~5 mins. You might want to consider multi-pronged pipettes if this helps with the loading time.
  • 15
    Do not re-use the adhesive strips. Cut them to size if you only use part of the plate at a time. Always discard after use.
  • 16
    New standard curves should be made for every new run of the assay. If the observed concentrations of test samples are too high (OD value of the sample is higher than that of standard well maximum concentration), dilute by a certain factor, and correct for said factor in the end calculations.
  • 17
    Samples containing NaN3 cannot be tested due to NaN3 inhibiting the activities of horseradish peroxidase (HRP).
  • 18
    When washing plate via plate washer, the volume of buffer injected into each well should be slightly more than 350μL. Make sure the sampling head is not jammed or blocked. Also, if washing by hand, please take care when using an absorbent material to remove excess water – make sure this absorbent material wasn’t used to clean any of the other reagents to prevent contamination.
  • 19
    After the coloration reaction termination by Color Reagent C, please read OD within 10 mins.
  • 20
    If duplicate wells were performed, the mean value of the wells should be used.
  • 21
    Hemolyzed samples may cause false positive results, so we consider these samples to be incompatible with this kit.
  • 22
    During the assay, please try to control the humidity to ~60%.
  • 23
    We recommend regularly checking the thermostat and calibration in order to confirm the incubation temperature remains at a stable 37 ℃.