Rat C3a (Complement Component 3a) QuickTest ELISA Kit

Mfr.No.

QT-ER0782

Sensitivity

0.375ng/ml

Detection Range

0.625-40ng/ml

Reactivity

Rat

Assay Duration

120 minutes

Assay Type

Sandwich

Description

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 C3a, a capture antibody-C3a-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 C3a in the sample was calculated by drawing a standard curve. The concentration of the target substance is proportional to the OD450 value.

Alternative Names

C3a ELISA Kit, C3a-desArg ELISA Kit, ASP ELISA Kit, Anaphylatoxin ELISA Kit, C3 ELISA Kit

Detection Method

Double Antibody

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 C3a into the sample. Calculate the recovery by comparing the measured value with the expected amount of C3a in the sample.

Sample Type	Recovery Range(%)	Average(%)
serum(n=10)	87-100	95
EDTA plasma(n=10)	86-105	91
Heparin plasma(n=10)	87-98	91

Linearity

Dilute the sample with a certain amount of C3a at 1:2, 1:4 and 1:8 to get the recovery range.

Sample Type	1:2	1:4	1:8
serum(n=10)	88-105%	87-100%	81-100%
EDTA plasma(n=10)	86-102%	87-100%	82-100%
Heparin plasma(n=10)	87-104%	86-100%	80-98%

Specificity

Specifically binds with C3a , 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.(ng/ml)	OD-1	OD-2	Average
0	0.138	0.142	0.14
0.625	0.183	0.181	0.182
1.25	0.299	0.292	0.295
2.5	0.435	0.432	0.417
5	0.652	0.646	0.619
10	0.895	0.902	0.92
20	1.71	1.727	1.682
40	2.382	2.326	2.342

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.

Precision

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 (ng/ml)	1.16	5.08	21.4	1.21	4.96	19.28
Standard deviation	0.04	0.21	0.77	0.05	0.2	0.75
CV(%)	3.61	4.08	3.61	3.84	4	3.88

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

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

Protocol

QT-ER0782.pdf
MSDS

Storage	2-8°C(Sealed), Don't cryopreserve.

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SKU	Supplier No.	Size	Your price	Quantity
BHE10810453	QT-ER0782	48T	$315		Add to Cart
BHE10810453	QT-ER0782	96T	$450		Add to Cart

Rat C3a (Complement Component 3a) QuickTest ELISA Kit

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