HMGB1 Serum ELISA Assay

Additional Information

Assay Background

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 a 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. 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.

Assay Procedure

1. Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal it.
2. Add 50 µl of standards, samples and zero controls into appropriate wells.
3. Add 50 µl of Assay buffer into all wells immediately. Mix thoroughly by gently shaking or tapping the plate. Cover the plate and incubate for overnight (~16 hours) at 4°C.
4. Aspirate each well and wash, repeating the process 2 times for a total 3 washes. Wash by filling each well with 1X cold wash buffer (350 μl) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each is essential to good performance. After the last wash, remove any remaining buffer by aspirating, decanting or blotting against clean paper towels.
5. Add 100 µl 1X HRP-antibody conjugate into each well. Mix thoroughly by gently shaking or tapping the plate. Cover wells and incubate for 1 hour at 37°C.
6. Aspirate each well and wash as step 4.
7. Add 100 μl of TMB substrate to each well. Incubate for 10 minutes at RT in dark. Substrate will change from colorless to different strengths of blue.
8. Add 50 μl of Stop solution to each well. The color of the solution should change from blue to yellow. Mix thoroughly by gently shaking the plate.
9. Read the OD with a microplate reader at 450 nm immediately.

Standard Curve