Human C3 (Complement Component 3) CLIA Kit (HUES00501)

SKU:
HUES00501
€599

Description

ELISA Kit Technical ManualMSDS

Human C3 (Complement Component 3) CLIA Kit

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human C3 . Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human 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 Human C3, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human C3. The concentration of Human C3 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection methodChemiluminescence
Detection range1.56-100 ng/mL
Sensitivity0.94 ng/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Human C3 in samples. No significant cross-reactivity or interference between Human C3 and analogues was observed.

Typical Data

As the RLU 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)
RLU Average Corrected
10033806
41302
3755437521
5014781
17701
1624116208
257808
7276
75427509
12.53388
3976
36823649
6.252007
1743
18751842
3.131036
966
1001968
1.56557
587
572539
032
34
33--

Precision

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

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

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)5.1213.5938.985.2412.5638.79
Standard deviation0.451.373.270.430.973.13
C V (%)8.7910.088.398.217.728.07

Recovery

The recovery of Human 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)92-110100
EDTA plasma (n=5)97-109103
Cell culture media (n=5)90-10095

Linearity

Samples were spiked with high concentrations of Human 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)
1:2Range (%)89-104101-112103-115
Average (%)96106109
1:4Range (%)99-11095-11099-115
Average (%)105101106
1:8Range (%)90-10193-10494-109
Average (%)969999
1:16Range (%)95-11094-10885-96
Average (%)10210190

Kit Components & Storage

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 CLIA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent A1 vial, 5 mL4'C (shading light)
Substrate Reagent B1 vial, 5 mL4'C (shading light)
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human C3 (Complement Component 3) CLIA Kit (HUES00501) Assay procedure

    1. 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.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids.) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. 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.
    7. 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.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
    11. Determine the RLU value of each well immediately.

Human C3 (Complement Component 3) CLIA Kit (HUES00501) Protein Information

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, signal peptide; Inhibitor; Secreted

Chromosomal Location of Human Ortholog: 19p13.3-p13.2

Cellular Component: extracellular space; extracellular region; plasma membrane

Molecular Function:protein binding; endopeptidase inhibitor activity; C5L2 anaphylatoxin chemotactic receptor binding; receptor binding

Biological Process: regulation of immune response; complement activation, alternative pathway; signal transduction; fatty acid metabolic process; complement activation; G-protein coupled receptor protein signaling pathway; positive regulation of angiogenesis; positive regulation of activation of membrane attack complex; positive regulation of type IIa hypersensitivity; positive regulation of G-protein coupled receptor protein signaling pathway; regulation of complement activation; innate immune response; immune response; positive regulation of protein amino acid phosphorylation; inflammatory response; complement activation, classical pathway

Disease: Complement Component 3 Deficiency, Autosomal Recessive; Hemolytic Uremic Syndrome, Atypical, Susceptibility To, 5; Macular Degeneration, Age-related, 9

NCBI Summary:Complement component C3 plays a central role in the activation of complement system. Its activation is required for both classical and alternative complement activation pathways. A peptide (C3a) derived from the encoded protein has antimicrobial activity, so people with C3 deficiency are susceptible to bacterial infection. [provided by RefSeq, Nov 2014]
UniProt Code:P01024
NCBI GenInfo Identifier:119370332
NCBI Gene ID:718
NCBI Accession:P01024.2
UniProt Secondary Accession:P01024,A7E236,
UniProt Related Accession:P01024
Molecular Weight:
NCBI Full Name:Complement C3
NCBI Synonym Full Names:complement component 3
NCBI Official Symbol:C3  
NCBI Official Synonym Symbols:ASP; C3a; C3b; AHUS5; ARMD9; CPAMD1; HEL-S-62p  
NCBI Protein Information:complement C3; prepro-C3; C3a anaphylatoxin; complement component C3; complement component C3a; complement component C3b; acylation-stimulating protein cleavage product; epididymis secretory sperm binding protein Li 62p; C3 and PZP-like alpha-2-macroglobulin domain-containing protein 1
UniProt Protein Name:Complement C3
UniProt Synonym Protein Names:C3 and PZP-like alpha-2-macroglobulin domain-containing protein 1Cleaved 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_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
CLIA
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