Human Carboxyhemoglobin (HbCO) ELISA Kit

The Human HbCO ELISA Kit is a high-sensitivity, sandwich-based immunoassay designed for the quantitative measurement of carboxyhemoglobin (HbCO) levels in human serum, plasma, cell culture supernatants, tissue homogenates, and other biological fluids. HbCO is formed when hemoglobin binds to carbon monoxide (CO), making it a key biomarker in studies related to carbon monoxide poisoning, hypoxia, and environmental toxicology. This assay provides a reliable and efficient method to monitor HbCO levels in biological systems.

Key Features

• Assay Type: Sandwich ELISA
• Detection Range: 6 – 400 ng/mL
• Sensitivity: 1.2 ng/mL
• Sample Volume Required: 10 μL
• Detection Wavelength: 450 nm
• Storage Conditions: 2–8 °C
• Shelf Life: 6 months
• Precision:
  • Intra-assay CV <10%
  • Inter-assay CV <12%

Assay Principle

This ELISA kit uses a double-antibody sandwich method. The microplate is pre-coated with an anti-human HbCO antibody. Following sample or standard addition, HRP-conjugated anti-HbCO detection antibodies are applied to bind to the captured antigen. After washing, TMB substrate is added, producing a blue color that turns yellow after addition of the stop solution. Absorbance is read at 450 nm, and the concentration of HbCO is determined using a standard curve.

Kit Components

Sample Types & Preparation

• Serum / Plasma: Collect and centrifuge at 2,000–3,000 rpm for 20 min.
• Urine / CSF / Pleural Fluids: Collect, centrifuge, and isolate the supernatant.
• Cell Culture Supernatants: Collect, centrifuge to clarify.
• Cell Lysates: Lyse cells in PBS (pH 7.2–7.4) using freeze-thaw cycles; centrifuge and collect supernatant.
• Tissue Homogenates: Homogenize tissue in PBS (pH 7.4), centrifuge at 4 °C, and collect supernatant.

Note: Avoid sodium azide (NaN₃), which inhibits HRP. Minimize freeze-thaw cycles. Store extracted samples at -20°C if not used immediately.

Assay Procedure Overview

1. Prepare standard dilutions (540 → 30 ng/mL).
2. Add 40 μL sample diluent + 10 μL sample to each well (dilution factor = 5).
3. Incubate for 30 minutes at 37 °C, sealed with plate membrane.
4. Wash plate 5× using diluted wash buffer.
5. Add 50 μL HRP-conjugated antibody, incubate again.
6. Wash and add 50 μL Chromogen A + 50 μL Chromogen B, incubate in dark for 15 minutes.
7. Add 50 μL stop solution to each well.
8. Measure OD at 450 nm within 15 minutes.

Performance Summary

A standard curve is plotted (log OD vs. log concentration) to determine HbCO levels. Multiply final results by the dilution factor.

The Human HbCO ELISA Kit (SL0404Hu) offers a robust and sensitive method for quantifying carboxyhemoglobin, a critical marker in hypoxia, environmental exposure to CO, and toxicology research. With validated performance in a wide variety of sample types and easy-to-follow protocols, this kit enables high-confidence detection of HbCO in both clinical and environmental health research settings.