Human Immunology ELISA Kits 1
Human C4a (Complement Component 4a) CLIA Kit (HUES00163)
- SKU:
- HUES00163
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 0.19ng/mL
- Range:
- 0.31-20ng/mL
- ELISA Type:
- Sandwich
- Synonyms:
- C4AD, C4S, CO4, CPAMD2, RG
- Reactivity:
- Human
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Immunology
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection method: | Chemiluminescence |
| Detection range: | 0.31-20 ng/mL |
| Sensitivity: | 0.19 ng/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Human C4a in samples. No significant cross-reactivity or interference between Human C4a and analogues was observed. |
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 C4a. 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 C4a 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 C4a, 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 C4a. The concentration of Human C4a in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | C4A: C4 plays a central role in the activation of the classical pathway of the complement system. It is processed by activated C1 which removes from the alpha chain the C4a anaphylatoxin. The remaining alpha chain fragment C4b is the major activation product and is an essential subunit of the C3 convertase (C4b2a) and the C5 convertase (C3bC4b2a) enzymes of the classical complement pathway. Defects in C4A are the cause of complement component 4A deficiency (C4AD). A rare defect of the complement classical pathway associated with the development of autoimmune disorders, mainly systemic lupus with or without associated glomerulonephritis. Defects in C4A are a cause of susceptibility to systemic lupus erythematosus (SLE). A chronic, inflammatory and often febrile multisystemic disorder of connective tissue. It affects principally the skin, joints, kidneys and serosal membranes. It is thought to represent a failure of the regulatory mechanisms of the autoimmune system. Interindividual copy- number variation (CNV) of complement component C4 and associated polymorphisms result in different susceptibilities to SLE. The risk of SLE susceptibility has been shown to be significantly increased among subjects with only two copies of total C4. A high copy number is a protective factor against SLE. |
| UniProt Protein Details: | Protein type:Inhibitor; Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 6p21. 3 Cellular Component: extracellular region; extracellular space; plasma membrane Molecular Function:complement component C1q binding; endopeptidase inhibitor activity Biological Process: complement activation; complement activation, classical pathway; inflammatory response; innate immune response; regulation of complement activation Disease: Blood Group, Chido/rodgers System; Complement Component 4a Deficiency |
| NCBI Summary: | This gene encodes the basic form of complement factor 4, part of the classical activation pathway. The protein is expressed as a single chain precursor which is proteolytically cleaved into a trimer of alpha, beta, and gamma chains prior to secretion. The trimer provides a surface for interaction between the antigen-antibody complex and other complement components. The alpha chain may be cleaved to release C4 anaphylatoxin, a mediator of local inflammation. Deficiency of this protein is associated with systemic lupus erythematosus. This gene localizes to the major histocompatibility complex (MHC) class III region on chromosome 6. Varying haplotypes of this gene cluster exist, such that individuals may have 1, 2, or 3 copies of this gene. In addition, this gene exists as a long form and a short form due to the presence or absence of a 6. 4 kb endogenous HERV-K retrovirus in intron 9. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P0C0L4 |
| NCBI GenInfo Identifier: | 476007827 |
| NCBI Gene ID: | 721 |
| NCBI Accession: | P0C0L4. 2 |
| UniProt Secondary Accession: | P0C0L4,P01028, P78445, Q13160, Q13906, A6H8M8, A6NHJ5 A7E2V2, B0QZR6, B0V2C8, B2RUT6, B7ZVZ6, |
| UniProt Related Accession: | P0C0L5 |
| Molecular Weight: | 187,704 Da |
| NCBI Full Name: | Complement C4-A |
| NCBI Synonym Full Names: | complement component 4B (Chido blood group) |
| NCBI Official Symbol: | C4B |
| NCBI Official Synonym Symbols: | CH; C4F; CO4; C4B1; C4B2; C4B3; C4B5; C4BD; C4B12; C4B_2; CPAMD3 |
| NCBI Protein Information: | complement C4-B |
| UniProt Protein Name: | Complement C4-A |
| UniProt Synonym Protein Names: | Acidic complement C4; C3 and PZP-like alpha-2-macroglobulin domain-containing protein 2 |
| Protein Family: | Complement C4 |
| UniProt Gene Name: | C4A |
| UniProt Entry Name: | CO4A_HUMAN |
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 |
| 20 | 31423 36541 | 33982 | 33950 |
| 10 | 12117 12823 | 12470 | 12438 |
| 5 | 5461 5039 | 5250 | 5218 |
| 2.5 | 2490 2558 | 2524 | 2492 |
| 1.25 | 1490 1276 | 1383 | 1351 |
| 0.63 | 894 840 | 867 | 835 |
| 0.31 | 588 658 | 623 | 591 |
| 0 | 31 33 | 32 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human C4a were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human C4a 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) | 0.93 | 1.76 | 6.74 | 0.89 | 1.75 | 6.60 |
| Standard deviation | 0.10 | 0.14 | 0.45 | 0.10 | 0.20 | 0.61 |
| C V (%) | 10.75 | 7.95 | 6.68 | 11.24 | 11.43 | 9.24 |
Recovery
The recovery of Human C4a 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) | 89-104 | 97 |
| EDTA plasma (n=5) | 100-115 | 108 |
| Cell culture media (n=5) | 101-116 | 107 |
Linearity
Samples were spiked with high concentrations of Human C4a 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 (%) | 101-117 | 96-109 | 87-98 |
| Average (%) | 107 | 103 | 93 | |
| 1:4 | Range (%) | 103-118 | 91-103 | 86-100 |
| Average (%) | 109 | 98 | 91 | |
| 1:8 | Range (%) | 86-98 | 86-97 | 90-108 |
| Average (%) | 92 | 91 | 98 | |
| 1:16 | Range (%) | 100-118 | 100-111 | 88-103 |
| Average (%) | 108 | 105 | 95 |
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 ×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 A | 1 vial, 5 mL | 4°C (shading light) |
| Substrate Reagent B | 1 vial, 5 mL | 4°C (shading light) |
| 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 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.
- Determine the RLU value of each well immediately.
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