Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Mouse
Detection Method:	Colormetric
Detection Range:	3.13-200 ng/mL
Sensitivity:	1.88 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Mouse C3 in samples. No significant cross-reactivity or interference between Mouse C3 and analogues was observed.

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Mouse C3. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Mouse C3 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Mouse C3, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Mouse C3. The concentration of Mouse C3 in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	C3: C3 plays a central role in the activation of the complement system. Its processing by C3 convertase is the central reaction in both classical and alternative complement pathways. After activation C3b can bind covalently, via its reactive thioester, to cell surface carbohydrates or immune aggregates. Defects in C3 are the cause of complement component 3 deficiency (C3D). A rare defect of the complement classical pathway. Patients develop recurrent, severe, pyogenic infections because of ineffective opsonization of pathogens. Some patients may also develop autoimmune disorders, such as arthralgia and vasculitic rashes, lupus-like syndrome and membranoproliferative glomerulonephritis. Genetic variation in C3 is associated with susceptibility to age-related macular degeneration type 9 (ARMD9). ARMD is a multifactorial eye disease and the most common cause of irreversible vision loss in the developed world. In most patients, the disease is manifest as ophthalmoscopically visible yellowish accumulations of protein and lipid that lie beneath the retinal pigment epithelium and within an elastin- containing structure known as Bruch membrane. Defects in C3 are a cause of susceptibility to hemolytic uremic syndrome atypical type 5 (AHUS5). An atypical form of hemolytic uremic syndrome. It is a complex genetic disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, renal failure and absence of episodes of enterocolitis and diarrhea. In contrast to typical hemolytic uremic syndrome, atypical forms have a poorer prognosis, with higher death rates and frequent progression to end-stage renal disease. Susceptibility to the development of atypical hemolytic uremic syndrome can be conferred by mutations in various components of or regulatory factors in the complement cascade system. Other genes may play a role in modifying the phenotype. Increased levels of C3 and its cleavage product ASP, are associated with obesity, diabetes and coronary heart disease. Short-term endurance training reduces baseline ASP levels and subsequently fat storage.
UniProt Protein Details:	Protein type:Secreted; Inhibitor; Secreted, signal peptide Cellular Component: extracellular space; extracellular region Molecular Function:protein binding; endopeptidase inhibitor activity; cofactor binding; lipid binding; C5L2 anaphylatoxin chemotactic receptor binding Biological Process: positive regulation of developmental growth; immune system process; complement activation, alternative pathway; fatty acid metabolic process; complement activation; positive regulation of angiogenesis; positive regulation of activation of membrane attack complex; positive regulation of phagocytosis; positive regulation of G-protein coupled receptor protein signaling pathway; positive regulation of type IIa hypersensitivity; innate immune response; positive regulation of protein amino acid phosphorylation; lipid metabolic process; blood coagulation; inflammatory response; complement activation, classical pathway
NCBI Summary:	This gene encodes complement protein C3 which plays a central role in the classical, alternative and lectin activation pathways of the complement system. The encoded preproprotein undergoes a multi-step processing to generate various functional peptides. Mice deficient in the encoded protein fail to clear bacteria from the blood stream upon infection, display diminished airway hyperresponsiveness and lung eosinophilia upon allergen-induced pulmonary allergy, and develop severe lung injury after deposition of IgG immune complexes. Deficiency of the homolog of the encoded protein in humans was found to be associated with increased susceptibility to infections, age-related macular degeneration, and atypical hemolytic uremic syndrome. [provided by RefSeq, Mar 2015]
UniProt Code:	P01027
NCBI GenInfo Identifier:	126518317
NCBI Gene ID:	12266
NCBI Accession:	NP_033908. 2
UniProt Secondary Accession:	P01027,Q61370, Q80XP1,
UniProt Related Accession:	P01027
Molecular Weight:	60,952 Da
NCBI Full Name:	complement C3
NCBI Synonym Full Names:	complement component 3
NCBI Official Symbol:	C3
NCBI Official Synonym Symbols:	ASP; Plp; HSE-MSF; AI255234
NCBI Protein Information:	complement C3; complement factor 3; complement component 3d; acylation stimulating protein
UniProt Protein Name:	Complement C3
UniProt Synonym Protein Names:	HSE-MSFCleaved into the following 12 chains:Complement C3 beta chain; C3-beta-c; C3bc; Complement C3 alpha chain; C3a anaphylatoxin; Acylation stimulating protein; ASPAlternative name(s):C3adesArg
Protein Family:	Complement C3
UniProt Gene Name:	C3
UniProt Entry Name:	CO3_MOUSE

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration (ng/mL)	O.D	Average	Corrected
200	2.31 2.346	2.328	2.246
100	1.491 1.513	1.502	1.42
50	0.918 0.9	0.909	0.827
25	0.453 0.475	0.464	0.382
12.5	0.246 0.22	0.233	0.151
6.25	0.19 0.162	0.176	0.094
3.13	0.127 0.135	0.131	0.049
0	0.075 0.089	0.082	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse C3 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse C3 were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (ng/mL)	10.66	20.07	84.42	9.88	20.58	79.18
Standard deviation	0.74	1.14	3.29	0.64	1.20	2.99
C V (%)	6.94	5.68	3.90	6.48	5.83	3.78

Recovery

The recovery of Mouse C3 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type	Range (%)	Average Recovery (%)
Serum (n=5)	88-101	93
EDTA plasma (n=5)	91-107	99
Cell culture media (n=5)	91-106	99

Linearity

Samples were spiked with high concentrations of Mouse C3 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

		Serum (n=5)	EDTA plasma (n=5)	Cell culture media (n=5)
1:2	Range (%)	92-104	99-112	95-108
1:2	Average (%)	99	106	102
1:4	Range (%)	88-102	87-97	87-102
1:4	Average (%)	95	92	93
1:8	Range (%)	89-104	88-98	87-102
1:8	Average (%)	96	93	93
1:16	Range (%)	85-99	86-98	86-100
1:16	Average (%)	92	93	91

An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item	Specifications	Storage
Micro ELISA Plate(Dismountable)	8 wells ×12 strips	-20°C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100×)	1 vial, 120 µL
Concentrated HRP Conjugate (100×)	1 vial, 120 µL	-20°C(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4°C, 6 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25×)	1 vial, 30 mL
Substrate Reagent	1 vial, 10 mL	4°C(shading light)
Stop Solution	1 vial, 10 mL	4°C
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
Aliquot 100µl of standard solutions into the standard wells.
Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.