This kit was based on sandwich ELISA method. The experiment lasted 120 minutes. Capture antibody was conjugated to an affinity tag that was recognized by a specific antibody coated on the QuickTest plate. Add the Cap/Det Ab working solution into each well, then add the standards and pilot samples into individual wells. If the sample contains PYCARD, a capture antibody-PYCARD-biotin-detection antibody complex was formed. After incubation, unbound conjugates were removed by wash buffer. HRP-Streptavidin was added. After washing, TMB substrates were added to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that turned yellow after adding a stop solution. Read the O.D. absorbance at 450nm in a microplate reader. The concentration of PYCARD in the sample was calculated by drawing a standard curve. The concentration of the target substance is proportional to the OD450 value.
Alternative Names
Apoptosis-associated speck-like protein containing a CARD ELISA Kit, hASC ELISA Kit, Caspase recruitment domain-containing protein 5 ELISA Kit, PYD and CARD domain-containing protein ELISA Kit, Target of methylation-induced silencing 1 ELISA Kit, PYCARD ELISA Kit, ASC ELISA Kit, CARD5 ELISA Kit, TMS1 ELISA Kit
Reactivity
Human
Detection Method
Double Antibody
Detection Range
78.125-5000pg/ml
Assay Type
Sandwich
Assay Duration
120 minutes
Kit Components
Kit Components
Item
Size(48T)
Size(96T)
Storage Condition for Opened Kit
E001
ELISA Microplate(Dismountable)
8x6
8x12
Put the rest strips into a sealed foil bag with the desiccant. Stored for 1 month at 2-8°C; Stored for 6 month at -20°C
E002
Lyophilized Standard
1vial
2vial
Put the rest standards into a desiccant bag. Stored for 1 month at 2-8°C; Stored for 6 month at -20°C
E054
Cap/Det Ab (Ready to use, blue)
3ml
6ml
2-8°C (Avoid Direct Light)
E053
HRP-Streptavidin (Ready to use, orange)
5ml
10ml
E024
TMB Substrate
5ml
10ml
E039
Sample Dilution Buffer
20ml
20ml
2-8°C
E026
Stop Solution
5ml
5ml
E038
Wash Buffer(25X)
15ml
30ml
E006
Plate Sealer
3 pieces
5 pieces
E007
Product Description
1 copy
1 copy
Recovery
Add a certain amount of PYCARD into the sample. Calculate the recovery by comparing the measured value with the expected amount of PYCARD in the sample.
Sample Type
Recovery Range(%)
Average(%)
serum(n=10)
93-105
100
EDTA plasma(n=10)
91-101
96
Heparin plasma(n=10)
93-105
98
Linearity
Dilute the sample with a certain amount of PYCARD at 1:2, 1:4 and 1:8 to get the recovery range.
Sample Type
1:2
1:4
1:8
serum(n=10)
86-99%
82-100%
84-99%
EDTA plasma(n=10)
91-105%
83-97%
80-94%
Heparin plasma(n=10)
85-95%
83-100%
80-96%
Sensitivity
46.875pg/ml
Specificity
Specifically binds with PYCARD , no obvious cross reaction with other analogues.
Standard Curve
This product is detected by QC department and meets performance required in the manual. (Laboratory Humidity: 20%-60%; Temperature: 18°C -25°C; Equilibrate TMB substrate to 37°C before staining. After adding into the ELISA wells, incubate for 15min at 37°C in dark.) Due to different assay environments and operations, assay data below and standard curve are provided for reference. Experimenters should establish standard curve according to their own assay.
STD.(pg/ml)
OD-1
OD-2
Average
0
0.066
0.068
0.067
78.125
0.134
0.132
0.133
156.25
0.218
0.213
0.216
312.5
0.466
0.462
0.446
625
0.877
0.87
0.832
1250
1.061
1.069
1.091
2500
1.601
1.616
1.574
5000
2.238
2.184
2.2
Assay Procedure Summary
Step 1: Take out the required plate wells, add 50ul Cap/Det Ab into each well, then add 50ul Standard or Sample into individual well. (When adding standard or sample, the disposable tip lightly touches the liquid level. Change the disposable tips for different samples and standards.) Gently tap the plate for 10s to ensure thorough mixing then static incubate for 60 minutes at 37°C.
Washing: Wash the plate twice without immersion.
Step 2: Add 100ul HRP-Streptavidin (orange) into each well, seal the plate and static incubate for 30 minutes at 37°C.
Washing: Wash the plate five times without immersion.
Step 3: Add 90ul TMB substrate solution, seal the plate and static incubate for 10-20 minutes at 37°C. (Accurate TMB visualization control is required.)
Step 4: Add 50ul stop solution. Read at 450nm immediately and calculate.
Stability
Perform the stability test for the sealed kit at 37°C and 2-8°C and get relevant data.
ELISA kit(n=5)
37°C for 1 month
2-8°C for 6 months
Average(%)
80
95-100
UniProt ID
Q9ULZ3
Antigen
PYCARD
Usage Notes
Microplate reader (wavelength: 450nm) 37°C incubator (CO2 incubator for cell culture is not recommenced.) Automated plate washer or multi-channel pipette/5ml pipettor (for manual washing purpose) Precision single (0.5-10uL, 5-50uL, 20-200uL, 200-1000uL) and multi-channel pipette with disposable tips(Calibration is required before use.) Sterile tubes and Eppendorf tubes with disposable tips Absorbent paper and loading slot Deionized or distilled water
Intra-assay Precision: samples with low, medium and high concentration are tested 20 times on the same plate. Inter-assay Precision: samples with low, medium and high concentration are tested 20 times on three different plates.
Item
Intra-assay Precision
Inter-assay Precision
Sample
1
2
3
1
2
3
n
20
20
20
20
20
20
Mean (pg/ml)
158.5
588.98
2568.36
153.08
629.23
2442.16
Standard deviation
6.89
24.32
88.87
6.75
25.42
103.06
CV(%)
4.35
4.13
3.46
4.41
4.04
4.22
Storage
2-8°C(Sealed), Don't cryopreserve.
Reviews of Human PYCARD (Apoptosis-associated speck-like protein containing a CARD) QuickTest ELISA Kit