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Mouse HMGB-1(High Mobility Group Protein B1) ELISA Kit

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Price:   $609
Cat Number:  E-EL-M0676
Lead Time:   7~10 days

Reactivity:  Mouse

Detection Range:  15.63~1000 pg/mL

Sensitivity:  9.38 pg/mL

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Intended use

This ELISA kit applies to the in vitro quantitative determination of Mouse HMGB-1 concentrations in serum, plasma and other biological fluids.

Test principle

This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Mouse HMGB-1. Standards or samples are added to the micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Mouse HMGB-1 and Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Mouse HMGB-1, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of 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 HMGB-1. You can calculate the concentration of Mouse HMGB-1 in the samples by comparing the OD of the samples to the standard curve.

Assay type Sandwich-ELISA
Format 96T/48T
Assay time 3.5h
Reactivity Mouse
Detection Method Colormetric
Detection Range 15.63—1000 pg/mL
Sensitivity 9.38 pg/mL
Sample Volume Required Per Well 100μL
Sample Type serum, plasma and other biological fluids

Specificity

This kit recognizes Mouse HMGB-1 in samples. No significant cross-reactivity or interference between Mouse HMGB-1 and analogues was observed.

Typical data

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
(pg/mL)
O.D Average Corrected
1000 2.453
2.489
2.471 2.405
500 1.573
1.591
1.582 1.516
250 0.889
0.883
0.886 0.82
125 0.448
0.454
0.451 0.385
62.5 0.279
0.263
0.271 0.205
31.25 0.175
0.157
0.166 0.1
15.63 0.115
0.121
0.118 0.052
0 0.056
0.076
0.066 --

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse 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 Mouse 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) 50.50 98.56 334.18 52.21 95.09 337.87
Standard deviation 2.65 4.51 17.78 3.53 5.30 13.18
C V (%) 5.25 4.58 5.32 6.76 5.57 3.90

Recovery

The recovery of Mouse 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) 92-105 99
EDTA plasma (n=5) 88-103 94
Cell culture media (n=5) 83-98 90

Linearity

Samples were spiked with high concentrations of Mouse 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 (%) 91-104 96-110 87-97
Average (%) 97 103 92
1:4 Range (%) 90-104 84-98 85-98
Average (%) 97 90 91
1:8 Range (%) 90-101 84-96 82-95
Average (%) 95 89 88
1:16 Range (%) 90-106 81-95 82-94
Average (%) 98 87 87

Kit components & Storage

An unopened kit can be stored at 4℃ 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℃, 6 months
Reference Standard 2 vials
Concentrated Biotinylated Detection Ab (100×) 1 vial, 120 μL
Concentrated HRP Conjugate (100×) 1 vial, 120 μL -20℃(shading light), 6 months
Reference Standard & Sample Diluent 1 vial, 20 mL 4℃, 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℃(shading light)
Stop Solution 1 vial, 10 mL 4℃
Plate Sealer 5 pieces
Product Description 1 copy
Certificate of Analysis 1 copy

Note: All reagent bottle caps must be tightened to prevent evaporation and microbial pollution.
The volume of reagents in partial shipments is a little more than the volume marked on the label, please use accurate measuring equipment instead of directly pouring into the vial(s).

Other supplies required

  • Microplate reader with 450 nm wavelength filter
  • High-precision transfer pipette, EP tubes and disposable pipette tips
  • Incubator capable of maintaining 37℃
  • Deionized or distilled water
  • Absorbent paper
  • Loading slot for Wash Buffer

Assay Procedure

1.Add 100 μL standard or sample to each well. Incubate for 90 min at 37℃.

2.Remove the liquid.

3.Add 100 μL Biotinylated Detection Ab. Incubate for 1 hour at 37℃. Aspirate and wash 3 times.

4.Add 100 μL HRP Conjugate. Incubate for 30 min at 37℃. Aspirate and wash 5 times.

5.Add 90 μL of Substrate Reagent. Incubate for 15 min at 37℃.

6.Add 50 μL Stop Solution.

7.Read at 450 nm immediately. Calculation of results.

Citations

  1. Publication: Li Y H , Zhang X , Wan X Y , Liu X H , et al. Inducing Endoplasmic Reticulum Stress to Expose Immunogens: A DNA Tetrahedron Nanoregulator for Enhanced Immunotherapy[J]. Advanced Functional Materials, 2020, 00532.
    Sample Type: Serum
  2. Publication: Xu C F , Yu Y L , Sun Y , et al. Transformable Nanoparticle-Enabled Synergistic Elicitation and Promotion of Immunogenic Cell Death for Triple-Negative Breast Cancer Immunotherapy[J].Advanced Functional Materials, 2019, 1905213.
    Sample Type: Cell supernatant
  3. Publication: Xu C F , Yu Y L , Sun Y , et al. Transformable Nanoparticle-Enabled Synergistic Elicitation and Promotion of Immunogenic Cell Death for Triple-Negative Breast Cancer Immunotherapy[J].Advanced Functional Materials, 2019, 1905213.
    Sample Type: Cell culture supernatant
  4. Publication: Yuan S , Xu Y H , Wang C , et al. Doxorubicin-polyglycerol-nanodiamond conjugate is a cytostatic agent that evades chemoresistance and reverses cancer-induced immunosuppression in triple-negative breast cancer[J]. Journal of Nanobiotechnology, 2019, 17: 110.
    Sample Type: Cell culture supernatant
  5. Publication: Liu Q, Xie W, Wang Y, et al. JAK2/STAT1-mediated HMGB1 translocation increases inflammation and cell death in a ventilator-induced lung injury model[J]. Laboratory Investigation, 2019, 99(12): 1810-1821.
    Sample Type: Cell lysate,Bronchoalveolar Lavage Fluid,Tissue ho
  6. Publication: Stojanovic B, Milovanovic J, Arsenijevic A, et al. Galectin-3 deficiency facilitates TNF-α-dependent hepatocyte death and liver inflammation in MCMV infection[J]. Frontiers in microbiology, 2019, 10.
    Sample Type: Tissue homogenate
  7. Publication: Huang X, Hou X, Chuan L, et al. miR-129-5p alleviates LPS-induced acute kidney injury via targeting HMGB1/TLRs/NF-kappaB pathway[J]. International Immunopharmacology, 2020, 89: 107016.
    Sample Type: Serum
  8. Publication: Ma X F, Qin J, Guo X H. miR-181-5p protects mice from sepsis via repressing HMGB1 in an experimental model[J]. European Review for Medical and Pharmacological Sciences, 2020, 24: 9712-9720.
    Sample Type: Serum
  9. Publication: Zhan L, Peng X, Lin J, et al. Honokiol Reduces Fungal Load, Toll-Like Receptor-2, and Inflammatory Cytokines in Aspergillus fumigatus Keratitis[J]. Investigative Ophthalmology & Visual Science, 2020, 61(4): 48-48.
    Sample Type: Cell culture supernatant
  10. Publication: Wei S, Gao Y, Dai X, et al. SIRT1-mediated HMGB1 deacetylation suppresses sepsis-associated acute kidney injury[J]. American Journal of Physiology-Renal Physiology, 2018, 316(1): F20-F31.
    Sample Type: Serum
  11. Publication: Taylor O J, Thatcher M O, Carr S T, et al. High-Mobility Group Box 1 Disrupts Metabolic Function With Cigarette Smoke Exposure In A Ceramide-Dependent Manner[J]. International Journal of Molecular Sciences, 2017.
    Sample Type: Serum,Cell culture supernatant
  12. Publication: Liu Y, Dong F, Guo R, et al. Hydrogen-rich saline ameliorates experimental autoimmune encephalomyelitis in C57BL/6 mice via the Nrf2-ARE signaling pathway[J]. Inflammation, 2018: 1-12.
    Sample Type: Tissue homogenate
  13. Publication: Liu X X, Yu D D, Chen M J, et al. Hesperidin Ameliorates Lipopolysaccharide-Induced Acute Lung Injury In Mice By Inhibiting Hmgb1 Release[J]. International Immunopharmacology, 2015, 25(2): 370-376.
    Sample Type: Bronchoalveolar Lavage Fluid
  14. Publication: Li G, Chen M J, Wang C, et al. Protective Effects Of Hesperidin On Concanavalin A-Induced Hepatic Injury In Mice[J]. International Immunopharmacology, 2014, 21(2): 406-411.
    Sample Type: Serum
  15. Publication: Zou J, Qi F, Ye L, et al. Protective Role Of Grape Seed Proanthocyanidins Against Ccl4 Induced Acute Liver Injury In Mice[J]. Medical Science Monitor, 2016, 22: 880-889.
    Sample Type: Serum
  16. Publication: Le Y, Wang Y, Zhou L, et al. Cigarette smoke‐induced HMGB1 translocation and release contribute to migration and NF‐κB activation through inducing autophagy in lung macrophages[J]. Journal of Cellular and Molecular Medicine, 2020, 24(2): 1319-1331.
    Sample Type: Serum,Cell culture supernatant
  17. Publication: Zeng X, Li M, Fan X, et al. Hypothermic Oxygenated Machine Perfusion Alleviates Donation after Cardiac Death Liver Injury Through Regulating P-Selectin-Dependent and-Independent Pathways in Mice[J]. Available at SSRN 3237696, 2018.
    Sample Type: Tissue homogenate
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