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#6010 Chicken Egg Ovalbumin ELISA Kit, 96 tests, Quantitative

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Product name : Chicken Egg Ovalbumin ELISA Kit, 96 tests, Quantitative

Catalog number : 6010

Quantity: 1 kit

Availability: Yes

Supplier name : Alpha Dia

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Chicken Egg Ovalbumin ELISA Kit, 96 tests, Quantitative
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Chicken Egg Ovalbumin ELISA Kit, 96 tests, Quantitative antibody storage GENTAUR recommends for long therm storage to freeze at -24 C. For short time storage up to 30 days we suggest fridge storage at 1 to 10 C. Prevent multiple freeze taw cycles of Chicken Egg Ovalbumin ELISA Kit, 96 tests, Quantitative.

More Details about

HMGB1 Detection Kit
Catalog # 6010

For Research Use Only - Not Human or Therapeutic Use

HMGB1 (high mobility group box 1) (1) was recently rediscovered as a late lethal mediator of endotoxin (10) and currently considered as a pro-infl ammatory cytokine that plays crucial roles in a variety of acute and chronic infl ammatory diseases. HMGB1 contains 216 amino acids (6) that share more than 99% sequence identity in mice (2), rats (3), bovines (4), and humans (5). HMGB1 consists of three structural domains (7), termed “A box (9-85)” and “B box (88-162)” and a negatively charged carboxyl terminus (186-216). Moreover, it has been previously shown that the B box recapitulates the pro-infl ammatory activity whereas the A box acts as an antagonist of HMGB1 (8, 9). Several lines of evidence indicate the signifi cance of HMGB1 in the immune infl ammatory response. For example, it has been shown that HMGB1 is actively released from a variety of cells such as macrophages stimulated by lipopolysaccharides (LPS), TNF and IL-1 (10), and is passively released by injured or necrotic cells associated with collapsing cell structures. In fact, deceased patients with septic shock have higher serum HMGB1 levels than surviving patients from sepsis (13). Similarly, high serum HMGB1 levels are observed in sepsis animal models and in collagen-induced arthritis animal models (14). With regard to the function of the protein itself, HMGB1 has also been shown to stimulate the release of TNF and IL-1 (11, 12), as well as the ability to bind LPS and synergistically increase peripheral blood mononuclear cell IL-6 production (19). Taken together, these observations demonstrate that HMGB1 plays important roles in the infl ammatory cascade. Chondrex provides a capture ELISA kit to determine HMGB1 levels in cell culture medium and mouse sera. This kit contains enough reagents
to measure 40 samples in duplicate together with standards.



HMGB1 Assay Summary



Note 1: It is recommended that the standard and samples be run in duplicate.
Note 2: Partially used diluted capture, detection and streptavidin peroxidase reagents may be kept at –20°C.
Note 3: Crystals may form in the 20X wash buffer when stored at cold temperatures. If crystals have  ormed, it is necessary to warm the wash buffer by placing the bottle in warm water until crystals have dissolved completely.
Note 4: Measure exact volume of buffers using a serological pipette prior to diluting. Extra buffer is provided.
Note 5: This kit can be used to determine HMGB1 in mouse serum and cell culture medium. However, special concern should be considered for assaying HMGB1 in human serum, because it has been reported that autoantibody to HMGB1 is determined in 9 to 89% of sera from patients with autoimmune and infl ammatory diseases (15-18). If it is true, polyclonal antibodies in human sera might mask the epitopes recognized by the capture and detection antibodies used in this kit, and interfere with the assay. Therefore, it is important to use this kit with background knowledge of patients.

All reagents must be at room temperature before use.

1. Add Capture Antibody: Dilute one vial of Capture Antibody with 10 ml of Capture Antibody Dilution Buffer (Solution A). Add 0,1ml of capture antibody solution to each well and incubate at 4°C overnight.
2. Prepare Standard Dilutions: The recommended standard range is 1.6-100 ng/ml. Dilute one vial of HMGB1 Standard with 0,95m l of Sample/Standard Dilution Buffer (Solution B) - 100 ng/ml. Prepare serial dilutions of the standard by mixing 0,25 ml of the 100 ng/ ml standard with 0,25 ml of Solution B - 50 ng/ml. Then repeat this procedure to make fi ve more serial dilutions of standard - 25, 12.5, 6.25, 3.1 and 1.6 ng/ml solutions. The 100 ng/ml standard stock can not be stored for future assay. Discard unused extra standard solution. We recommend making fresh standard and serial dilutions for each assay.
3. Prepare Sample Dilutions: Centrifuge samples at 10,000 rpm at 4°C for 3 minutes to remove insoluble materials and lipids, and use the supernatant as samples. If the HMGB1 level is more than 100 ng/mL, re-assay the sample at a higher dilution.
4. Prepare Detection Antibody: Dissolve one vial of Detection Antibody in 5 ml of Detection Antibody Dilution Buffer (Solution C).
5. Dilute Wash Buffer: Dilute 50 ml of 20X wash buffer in 950 ml of distilled water (1X wash buffer). Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer. Empty the plate by inverting it and blot on a paper towel to remove excess liquid. Do not allow the plate to dry out.
6. Add Standards, Samples and Detection Antibody: Mix standards, samples and detection antibody tubes well. Add 0,05ml of Solution B (blank), standards and samples to appropriate wells (Figure 1). Add 0,05ml of diluted detection antibody solution to all wells. Mix detection antibody solution and sample/standard solutions in all wells by pipetting or using a plate shaker. Cover the plate with a plate sealer and incubate at 37°C for 1 hour, then settle at 4°C overnight.

7. Prepare Streptavidin Peroxidase: Dilute one vial of Streptavidin Peroxidase in 10 ml of Streptavidin Peroxidase Dilution Buffer (Solution D).
8. Wash: Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer. Empty the plate by inverting it and blot on a paper towel to remove excess liquid. Do not allow the plate to dry out.
9. Add Streptavidin Peroxidase: Add 100 l of streptavidin peroxidase solution to each well and incubate at room temperature for 30 minutes. Do not incubate the plate more than 60 minutes.
10. Wash: Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer. Empty the plate by inverting it and blot on a paper towel to remove excess liquid. Do not allow the plate to dry out.
11. TMB: Add 0,1ml of TMB Solution to all wells immediately after washing the plate. Incubate for 30 minutes at room temperature.
12. Stop: Add 0,05 ml of 2N sulfuric acid (Stop Solution) to each well.
13. Read Plate: Read the OD values within 30 minutes by dual wavelength at 450 nm (sample) and 630 nm (reference).

1. Average the duplicate OD values for the blank, standards and samples.
2. Subtract the averaged blank (B) OD value from the averaged standard and sample OD values.
3. Plot the OD values of standards against the amount of HMGB1 (ng/ml) using a log scale. Figure 2 shows a typical standard curve where the HMGB1 range is from 1.6 to 100 ng/ml.
4. The amounts of HMGB1 (ng/ml) in samples can be calculated using regression analysis.

Figure 2 - A typical standard curve


Intra-assay coeffi cient of variation is less than 4.2%. Inter-assay coeffi cient of variation is less than 7.6%.

Recovery of HMGB1 added to mouse serum is 87-137%. Recovery of HMGB1 added to culture medium is 113-118%.

Specifi city:
Cross reaction with bovine HMGB2 is the average 12.4%.

1. Einck L and Bustin M. The intracellular distribution and function of the high mobility group chromosomal proteins. Exp Cell Res.156:295-310 (1985).
2. Ferrari S et al. The mouse gene coding for high mobility group 1 protein (HMG1). J Biol Chem.269:28803-28808 (1994).
3. Paonessa G et al. Nucleotide sequence of rat liver HMG1 cDNA. Nucleic Acids Res. 15:9077 (1987).
4. Kaplan DJ and Duncan CH. Full length cDNA sequence for bovine high mobility group 1 (HMG1) protein. Nucleic Acids Res. 16 : 10375(1988).
5. Wen L et al. A human placental cDNA clone that encodes nonhistone chromosomal protein HMG-1. Nucleic Acids Res. 17:1197-1214(1989).
6. Parkkinen J et al. Amphoterin, the 30-kDa protein in a family of HMG1-type polypeptides. Enhanced expression in transformed cells,leading edge localization, and interactions with plasminogen activation. J Biol Chem. 268:19726-38 (1993).
7. Li J et al. Recombinant HMGB1 with cytokine-stimulating activity. J Immunol Methods. 289:211-23 (2004).
8. Bustin M. Regulation of DNA-dependent activities by the functional motifs of the high-mobility-group chromosomal proteins. Mol CellBiol. 19:5237-46 (1999).
9. Yang H et al. Reversing established sepsis with antagonists of endogenous high-mobility group box 1. Proc Natl Acad Sci U S A.101:296-301 (2004).
10. Wang H et al. HMG-1 as a late mediator of endotoxin lethality in mice. Science. 285:248-251 (1999).
11. Wang H et al. Proinfl ammatory cytokines (tumor necrosis factor and interleukin-1) stimulate release of high mobility group protein-1 by
pituicytes. Surgery. 126:389-392 (1999).
12. Abraham E et al. HMG-1 as a mediator of acute lung infl ammation. J Immunol. 165:2950-2954(2000).
13. Andersson U et al. High mobility group 1 protein (HMG-1) stimulates proinfl ammatory cytokine synthesis in human monocytes. J ExpMed. 192:565-570 (2000).
14. Kokkola R et al. Successful treatment of collagen-induced arthritis in mice and rats by targeting extracellular high mobility group box chromosomal protein 1 activity. Arthritis Rheum. 48:2052-2058 (2003).
15. Wittemann B et al. Autoantibodies to nonhistone chromosomal proteins HMG-1 and HMG-2 in sera of patients with juvenile rheumatoid arthritis. Arthritis Rheum. 33(9):1378-83 (1990).
16. Uesugi H et al. Prevalence and characterization of novel pANCA, antibodies to the high mobility group non-histone chromosomal proteins HMG1 and HMG2, in systemic rheumatic diseases. J Rheumatol. 25(4):703-9 (1998).
17. Sobajima J et al. High mobility group (HMG) non-histone chromosomal proteins HMG1 and HMG2 are signifi cant target antigens of perinuclear anti-neutrophil cytoplasmic antibodies in autoimmune hepatitis. Gut. 44(6):867-73 (1999).
18. Sobajima J et al. Novel autoantigens of perinuclear anti-neutrophil cytoplasmic antibodies (P-ANCA) in ulcerative colitis: non-histone chromosomal proteins, HMG1 and HMG2. Clin Exp Immunol. 107(1):135-40 (1997).
19. Hreggvidsdottir HS et al. The alarmin HMGB1 acts in synergy with endogenous and exogenous danger signals to promote infl ammation. J Leukoc Biol. 86(3):655-62 (2009).


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