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Free Haemoglobin ELISA Kit (f-Hb)

Full Name: Free Haemoglobin ELISA Kit (f-Hb)
Reactivity: Human
Sample Type: Plasma, Tissue Homogenates, Serum, Biological Fluids
Sensitivity: 0.188 ng/ml

INTRODUCTION

Free haemoglobin (f-Hb) refers to the hemoglobin tetramer (α2β2) that is released from erythrocytes into plasma under hemolytic conditions. Unlike intracellular hemoglobin which transports oxygen, the cell-free form has reduced oxygen affinity and exerts toxicity due to oxidative side reactions. These stem largely from the heme cofactors’ ability to catalyze redox reactions which, though normally tempered by the globin chains, pose risks when uncontrolled upon hemolysis-induced release.

Once in the plasma, cell-free hemoglobin readily releases its heme prosthetic groups which then accumulate systemically. These free hemes fuel production of reactive oxygen species like hydroxyl radicals, superoxide, and hydrogen peroxide. By causing oxidative injury to lipids, proteins, and DNA, extracellular hemes trigger widespread damage and inflammatory signaling. These heighten endothelial activation and vascular permeability underlying conditions like transfusion-related acute lung injury. The globin portion likewise contributes via binding and subsequent scavenging of nitric oxide, a critical regulator of vascular homeostasis.

To mitigate toxicity from hemolysis-induced release, plasma proteins like haptoglobin and hemopexin scavenge f-Hb and free hemes respectively. Haptoglobin binds cell-free hemoglobin, targeting the complex for CD163-mediated endocytosis and degradation by macrophages. Hemopexin similarly sequesters heme, enabling hepatic clearance via LDL receptor related protein 1. These systems thereby counterbalance f-Hb’s oxidative and nitric oxide-depleting effects. Enhancing capacity of these scavenger pathways is emerging as a therapeutic strategy against hemolytic disorders like sickle cell disease and the side effects of blood transfusions.

INTENDED USE

Human free haemoglobin ELISA kit can measure concentrations of f-Hb (free haemoglobin, free Hb) present is plasma, serum, biological fluids or tissue homogenate samples.

CONTENT

All reagents supplied need to be stored at 2 °C – 8 °C, unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date.

  • One 96-Well Plate: Pre-coated with anti-free Hb antibody.
  • Standards: Lyophilized recombinant.
  • Sample/Standard Dilution Buffer.
  • Biotinylated-labelled Antibody.
  • Antibody Dilution Buffer.
  • HRP-Streptavidin Conjugate (SABC).
  • SABC Dilution Buffer.
  • TMB Substrate.
  • Wash Buffer (25x).
  • Plate Sealer.
  • Product Instructions.

TYPICAL RESULTS

For this free haemoglobin ELISA kit, it is recommended that a standard curve is generated for each assay carried out.

Standard Curve: 0, 0.312, 0.625, 1.25, 2.5, 5, 10, 20 ng/ml.
Reactivity: Human
Sensitivity: 0.188 ng/ml
Range: 0.313 – 20 ng/ml
Principle: Sandwich, Double Antibody
Application: Research Use Only.

ASSAY CHARACTERISTICS

– Specificity: Highly specific for free Hb, no cross reactivity or interference between free Hb and analogues was detected.
– Recovery: Serum (85 – 103%), EDTA Plasma (85 – 101%), Heparin Plasma (91 – 102%).
– Linearity: Serum (86 – 104%), EDTA Plasma (87 – 96%), Heparin Plasma (80 – 98%).
– Precison Intra-Assay: CV < 8%
– Precison Inter-Assay: CV < 10%

REFERENCES

  1. Free haemoglobin and pre-eclampsia. Lancet. (1990) 336 (8729): 1504. Woolfson R.G. and Williams D.J.
  2. Haemoglobin-based oxygen carriers. Expert Opin Investig Drugs. (2000) 9 (9): 1967-84. Vandegriff K.D.
  3. The functional role of the hemoglobin-water interface. Mol Aspects Med. (2022) 84: 101042. Meuwly M. and Karplus M.

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