Human Immunology ELISA Kits 1
Human HMGB-1 (High mobility group protein B1) CLIA Kit (HUES00881)
- SKU:
- HUES00881
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 7.5pg/mL
- Range:
- 12.5-800pg/mL
- ELISA Type:
- Sandwich
- Synonyms:
- HMGB1, HMG1, HMG3, SBP-1, Amphoterin
- 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: | 12.50-800 pg/mL |
| Sensitivity: | 7.50 pg/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Human HMGB-1 in samples. No significant cross-reactivity or interference between Human HMGB-1 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 HMGB-1. 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 HMGB-1 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 HMGB-1, 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 HMGB-1. The concentration of Human HMGB-1 in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | Multifunctional redox sensitive protein with various roles in different cellular compartments. In the nucleus is one of the major chromatin-associated non-histone proteins and acts as a DNA chaperone involved in replication, transcription, chromatin remodeling, V(D)J recombination, DNA repair and genome stability. Proposed to be an universal biosensor for nucleic acids. Promotes host inflammatory response to sterile and infectious signals and is involved in the coordination and integration of innate and adaptive immune responses. In the cytoplasm functions as sensor and/or chaperone for immunogenic nucleic acids implicating the activation of TLR9-mediated immune responses, and mediates autophagy. Acts as danger associated molecular pattern (DAMP) molecule that amplifies immune responses during tissue injury (PubMed:27362237). Released to the extracellular environment can bind DNA, nucleosomes, IL-1 beta, CXCL12, AGER isoform 2/sRAGE, lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and activates cells through engagement of multiple surface receptors. In the extracellular compartment fully reduced HMGB1 (released by necrosis) acts as a chemokine, disulfide HMGB1 (actively secreted) as a cytokine, and sulfonyl HMGB1 (released from apoptotic cells) promotes immunological tolerance (PubMed:23519706, PubMed:23446148, PubMed:23994764, PubMed:25048472). Has proangiogdenic activity. May be involved in platelet activation. Binds to phosphatidylserine and phosphatidylethanolamide. Bound to RAGE mediates signaling for neuronal outgrowth. May play a role in accumulation of expanded polyglutamine (polyQ) proteins such as huntingtin (HTT) or TBP (PubMed:23303669, PubMed:25549101). |
| NCBI Summary: | This gene encodes a protein that belongs to the High Mobility Group-box superfamily. The encoded non-histone, nuclear DNA-binding protein regulates transcription, and is involved in organization of DNA. This protein plays a role in several cellular processes, including inflammation, cell differentiation and tumor cell migration. Multiple pseudogenes of this gene have been identified. Alternative splicing results in multiple transcript variants that encode the same protein. [provided by RefSeq, Sep 2015] |
| UniProt Code: | P09429 |
| NCBI GenInfo Identifier: | 123369 |
| NCBI Gene ID: | 3146 |
| NCBI Accession: | P09429. 3 |
| UniProt Secondary Accession: | P09429,Q14321, Q5T7C3, Q6IBE1, A5D8W9, |
| UniProt Related Accession: | P09429 |
| Molecular Weight: | 24,894 Da |
| NCBI Full Name: | High mobility group protein B1 |
| NCBI Synonym Full Names: | high mobility group box 1 |
| NCBI Official Symbol: | HMGB1 |
| NCBI Official Synonym Symbols: | HMG1; HMG3; HMG-1; SBP-1 |
| NCBI Protein Information: | high mobility group protein B1 |
| UniProt Protein Name: | High mobility group protein B1 |
| UniProt Synonym Protein Names: | High mobility group protein 1; HMG-1 |
| Protein Family: | High mobility group protein |
| UniProt Gene Name: | HMGB1 |
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 (pg/mL) | RLU | Average | Corrected |
| 800 | 50035 55249 | 52642 | 52616 |
| 400 | 21479 21989 | 21734 | 21708 |
| 200 | 10340 9076 | 9708 | 9682 |
| 100 | 4353 4751 | 4552 | 4526 |
| 50 | 2204 2172 | 2188 | 2162 |
| 25 | 1140 980 | 1060 | 1034 |
| 12.50 | 474 544 | 509 | 483 |
| 0 | 25 27 | 26 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human HMGB-1 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human HMGB-1 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 (pg/mL) | 38.94 | 71.20 | 397.79 | 37.00 | 72.83 | 379.26 |
| Standard deviation | 4.74 | 8.16 | 27.92 | 3.48 | 5.48 | 28.71 |
| C V (%) | 12.17 | 11.46 | 7.02 | 9.41 | 7.52 | 7.57 |
Recovery
The recovery of Human HMGB-1 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) | 99-111 | 104 |
| EDTA plasma (n=5) | 94-111 | 102 |
| Cell culture media (n=5) | 86-99 | 91 |
Linearity
Samples were spiked with high concentrations of Human HMGB-1 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 (%) | 93-109 | 103-118 | 103-120 |
| Average (%) | 101 | 109 | 109 | |
| 1:4 | Range (%) | 95-111 | 94-107 | 85-98 |
| Average (%) | 103 | 99 | 90 | |
| 1:8 | Range (%) | 93-109 | 86-100 | 101-114 |
| Average (%) | 99 | 93 | 108 | |
| 1:16 | Range (%) | 88-101 | 85-99 | 88-101 |
| Average (%) | 95 | 91 | 94 |
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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