Description
Human C4BP (C4 Binding Protein) ELISA Kit
The Human C4BP (C4 Binding Protein) ELISA Kit is specifically designed for the precise measurement of C4BP levels in human serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit ensures dependable and consistent results, making it a valuable tool for various research purposes.C4BP is a vital protein that plays a key role in the regulation of the complement system, a part of the immune system responsible for defending the body against infections and removing damaged cells.
Dysregulation of the complement system and C4BP levels have been implicated in autoimmune diseases, inflammatory disorders, and infections, making C4BP an important biomarker for studying these conditions and potential therapeutic interventions.Overall, the Human C4BP ELISA Kit offers researchers a reliable method for accurately quantifying C4BP levels, providing valuable insights into the intricate mechanisms of the complement system and its implications in various health conditions.
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 0.63-40 ng/mL |
| Sensitivity: | 0.38 ng/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human C4BP in samples. No significant cross-reactivity or interference between Human C4BP 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 Human C4BP. 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 Human C4BP 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 C4BP, 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 Human C4BP. The concentration of Human C4BP in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | C4BPB: Controls the classical pathway of complement activation. It binds as a cofactor to C3b/C4b inactivator (C3bINA), which then hydrolyzes the complement fragment C4b. It also accelerates the degradation of the C4bC2a complex (C3 convertase) by dissociating the complement fragment C2a. It also interacts with anticoagulant protein S and with serum amyloid P component. The beta chain binds protein S. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 1q32 Cellular Component: extracellular space; plasma membrane Molecular Function:protein binding Biological Process: blood coagulation; negative regulation of complement activation, classical pathway; positive regulation of protein catabolic process; regulation of complement activation |
| NCBI Summary: | This gene encodes a member of a superfamily of proteins composed predominantly of tandemly arrayed short consensus repeats of approximately 60 amino acids. A single, unique beta-chain encoded by this gene assembles with seven identical alpha-chains into the predominant isoform of C4b-binding protein, a multimeric protein that controls activation of the complement cascade through the classical pathway. C4b-binding protein has a regulatory role in the coagulation system also, mediated through the beta-chain binding of protein S, a vitamin K-dependent protein that serves as a cofactor of activated protein C. The genes encoding both alpha and beta chains are located adjacent to each other on human chromosome 1 in the regulator of complement activation gene cluster. Alternative splicing gives rise to multiple transcript variants. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P20851 |
| NCBI GenInfo Identifier: | 115213 |
| NCBI Gene ID: | 725 |
| NCBI Accession: | P20851. 1 |
| UniProt Secondary Accession: | P20851,Q5VVR0, Q9BS25, A5JYP8, D3DT81, |
| UniProt Related Accession: | P20851 |
| Molecular Weight: | 28,286 Da |
| NCBI Full Name: | C4b-binding protein beta chain |
| NCBI Synonym Full Names: | complement component 4 binding protein beta |
| NCBI Official Symbol: | C4BPB |
| NCBI Official Synonym Symbols: | C4BP |
| NCBI Protein Information: | C4b-binding protein beta chain |
| UniProt Protein Name: | C4b-binding protein beta chain |
| Protein Family: | C4b-binding protein |
| UniProt Gene Name: | C4BPB |
| UniProt Entry Name: | C4BPB_HUMAN |
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 |
| 40 | 2.383 2.435 | 2.409 | 2.352 |
| 20 | 1.686 1.7 | 1.693 | 1.636 |
| 10 | 0.958 0.92 | 0.939 | 0.882 |
| 5 | 0.481 0.489 | 0.485 | 0.428 |
| 2.5 | 0.258 0.236 | 0.247 | 0.19 |
| 1.25 | 0.173 0.145 | 0.159 | 0.102 |
| 0.63 | 0.108 0.112 | 0.11 | 0.053 |
| 0 | 0.052 0.062 | 0.057 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human C4BP were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human C4BP 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) | 1.97 | 5.15 | 19.82 | 1.83 | 5.10 | 18.73 |
| Standard deviation | 0.11 | 0.30 | 1.00 | 0.13 | 0.27 | 0.94 |
| C V (%) | 5.58 | 5.83 | 5.05 | 7.10 | 5.29 | 5.02 |
Recovery
The recovery of Human C4BP 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) | 88-103 | 96 |
| EDTA plasma (n=5) | 93-109 | 101 |
| Cell culture media (n=5) | 92-105 | 99 |
Linearity
Samples were spiked with high concentrations of Human C4BP 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 (%) | 87-99 | 83-95 | 85-97 |
| Average (%) | 94 | 90 | 91 | |
| 1:4 | Range (%) | 92-106 | 83-94 | 87-97 |
| Average (%) | 98 | 88 | 92 | |
| 1:8 | Range (%) | 88-99 | 83-94 | 87-100 |
| Average (%) | 93 | 89 | 93 | |
| 1:16 | Range (%) | 90-106 | 86-97 | 87-97 |
| Average (%) | 97 | 92 | 92 |
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 100 µL of standard solutions into the standard wells.
- Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
- Add 100 µL 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 100 µL 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 350 µL 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 100 µL 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 90 µL 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.
