HMGB1 Detection Kit

HMGB1 Detection Kit - Cat Number: 6010 From Chondrex.

Research Field: Inflammation

Clonality: N/A

Cross-Reactivity:

Host Origin: N/A

Applications: N/A

Isotype: N/A

Detection Range: 50 ng/ml-0.78 ng/ml

Sample Type: Serum, Plasma, Culture Media

Concentration: N/A

Immunogen:

PRODUCT SPECIFICATIONS

DESCRIPTION: ELISA kit to quantify HMGB1
FORMAT: 96-well ELISA Plate with removeable strips
ASSAY TYPE: Sandwich ELISA
ASSAY TIME: 2 hours*
STANDARD RANGE: 50 ng/ml to 0.8 ng/ml
NUMBER OF SAMPLES: Up to 40 (duplicate) samples/plate
SAMPLE TYPES: Cell culture, Serum, and Plasma
RECOMMENDED SAMPLE DILUTIONS: 1:1 (at least)
CHROMOGEN: TMB (read at 450 nm)
STORAGE: -20°C
VALIDATION DATA: Human Serum: Intra-Assay (2.7-3.6%)/Inter-Assay (1.3-5.1%)/Recovery (101+/-25.1%)
Mouse Serum: Intra-Assay (1.4-8.1%)/Inter-Assay (0.9-4.5%)/Recovery (104+/8.3%)
NOTES: *This kit has an overnight incubation step

INTRODUCTION

HMGB1 (high mobility group box 1) (1) was recently rediscovered as a late lethal mediator of endotoxin (2) and is currently considered a
pro-inflammatory cytokine that plays crucial roles in a variety of acute and chronic inflammatory diseases. HMGB1 contains 216 amino acids
(3) and maintains 99% of its sequence identity among mice (4), rats (5), bovines (6), and humans (7). HMGB1 consists of three structural
domains (8), termed “A box (9-85)”, “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-inflammatory activity whereas the A box acts as an antagonist of HMGB1 (9,10).

Several lines of evidence highlight the significance of HMGB1 in the immune inflammatory response. For example, it has been shown that
HMGB1 is actively released by a variety of cells such as macrophages when stimulated by lipopolysaccharides (LPS), TNF-α, and IL-1 (2),
and is passively released by injured or necrotic cells associated with collapsing cell structures. In fact, patients who died from septic shock
had higher serum HMGB1 levels than surviving sepsis patients (11). Similarly, high serum HMGB1 levels are observed in sepsis animal
models and in collagen-induced arthritis animal models (12). With regard to the function of the protein itself, HMGB1 has also been shown
to stimulate the release of TNF-α and IL-1 (13,14), as well as bind LPS and synergistically increase peripheral blood mononuclear cell IL-6
production (15). Together, these observations demonstrate that HMGB1 plays important roles in the inflammatory cascade.

Chondrex, Inc. provides an HMGB1 Detection ELISA kit (Cat # 6010) to determine HMGB1 levels in cell culture media and sera. This kit
contains enough reagents to measure 40 samples in duplicate together with standards.

KIT COMPONENTS 

ASSAY OUTLINE

PLATE LAYOUT

NOTES BEFORE USING ASSAY

NOTE 1: It is recommended that the standard and samples be run in duplicate.
NOTE 2: Warm up all buffers to room temperature before use.
NOTE 3: Crystals may form in Wash Buffer, 20X when stored at cold temperatures. If crystals have formed, warm the wash buffer by placing
the bottle in warm water until crystals are completely dissolved.
NOTE 4: Measure exact volume of buffers using a serological pipet, as extra buffer is provided.
NOTE 5: Cover the plate with plastic wrap or a plate sealer after each step to prevent evaporation from the outside wells of the plate.
NOTE 6: For partial reagent use, please see the assay protocol’s corresponding step for the appropriate dilution ratio. For example, if the
protocol dilutes 50 µl of a stock solution in 10 ml of buffer for 12 strips, then for 6 strips, dilute 25 µl of the stock solution in 5 ml of buffer.
Partially used stock reagents may be kept in their original vials and stored at -20⁰C for use in a future assay.
NOTE 7: This kit contains animal components from non-infectious animals and should be treated as potential biohazards in use and for
disposal.
NOTE 8: This kit can be used to determine HMGB1 levels in sera and cell culture media samples. However, special concern should be
considered for assaying HMGB1 in human serum because autoantibodies to HMGB1 are determined in 9-89% of sera from patients with
autoimmune and inflammatory diseases (16-19). These reports indicate that human serum polyclonal antibodies to HMGB1 might mask the
epitopes recognized by the capture and detection antibodies used in this kit, resulting in interference against the assay.