Human Haemophilus Influenza B IgG ELISA Kit

Kit Components

• Each kit typically includes pre-coated microplates, standards, controls, enzyme conjugates, substrates, stop solutions, and wash buffers. Specific components may vary by manufacturer but generally adhere to the following:
• Pre-coated microplate: Wells coated with purified Hib antigens (e.g., capsular polysaccharide polyribosylribitol phosphate, PRP) to capture anti-Hib IgG antibodies from samples.
• Hib IgG Standards: Serial dilutions of purified human anti-Hib IgG with known concentrations for standard curve generation.
• Controls: Positive and negative controls to validate assay performance (e.g., positive control: serum with confirmed high anti-Hib IgG; negative control: serum with undetectable anti-Hib IgG).
• Enzyme Conjugate: Horseradish peroxidase (HRP)-labeled anti-human IgG antibody, which binds to captured Hib-specific IgG and catalyzes substrate reactions.
• Substrate Solution: Chromogenic substrate that produces a color change proportional to the amount of bound enzyme.
• Stop Solution: Acidic solution to terminate the enzyme reaction, stabilizing the color for absorbance reading.
• Wash Buffer: Phosphate-buffered saline with Tween-20 to remove unbound components and reduce non-specific binding.

Specifications

• Sample Type: Serum, plasma (EDTA or citrate anticoagulated), or other biological fluids (validated by manufacturer).
• Sensitivity: Typically 0.1–0.5 μg/mL (varies by kit), ensuring detection of low antibody levels post-vaccination or mild infection.
• Specificity: High specificity for Hib IgG, with minimal cross-reactivity to other Haemophilus strains (e.g., non-typeable H. influenzae) or unrelated pathogens.
• Assay Range: 0.5–20 μg/mL (extendable via sample dilution).
• Incubation Time: 2–3 hours (total assay time, including washing and detection steps).
• Storage: Components stored at 2–8°C (or -20°C for long-term storage, per manufacturer instructions) to maintain stability.

Assay Principle

• The kit uses a sandwich enzyme-linked immunosorbent assay (ELISA) format, leveraging antigen-antibody specificity and enzyme-mediated colorimetry:
• Capture: Samples (or standards/controls) are added to pre-coated wells. Hib-specific IgG antibodies in the sample bind to the immobilized Hib antigens (PRP) on the well surface.
• Washing: Unbound proteins and sample components are removed to eliminate background noise.
• Detection: HRP-labeled anti-human IgG conjugate is added, binding to the captured Hib-specific IgG, forming a "Hib antigen-IgG-enzyme conjugate" complex.
• Substrate Reaction: TMB substrate is added, and HRP catalyzes its conversion to a blue product. The intensity of the color is directly proportional to the concentration of Hib IgG in the sample.
• Termination & Reading: Stop solution halts the reaction, turning the color yellow. Absorbance is measured at 450 nm (with a reference wavelength of 630 nm) using a microplate reader. A standard curve is generated from the known standards, and sample concentrations are calculated against this curve.

Sample Collection & Preparation

• Proper sample handling is critical for accurate results:
• Serum: Collect whole blood in serum separator tubes (SST), allow clotting at room temperature (30–60 minutes), then centrifuge at 1,000–1,500 × g for 10–15 minutes. Transfer serum to clean tubes; avoid hemolysis (red blood cell rupture can interfere with absorbance).
• Plasma: Use EDTA or citrate as anticoagulants. Centrifuge whole blood immediately at 1,000 × g for 10 minutes to separate plasma.
• Storage: Fresh samples are ideal, but samples can be stored at 2–8°C for up to 48 hours or frozen at -20°C (avoid repeated freeze-thaw cycles, which degrade antibodies).