Sustainable rFC Endotoxin Assay Kit - Reliable BET Testing from China Suppliers and Factory
Technical Information on Recombinant Factor C (rFC) Fluorometric Assay for Bacterial Endotoxin Detection
The rFC fluorometric assay is a horseshoe crab-free, in vitro method for quantitative bacterial endotoxin (lipopolysaccharide, LPS) detection, compliant with global pharmacopoeias (USP <85>, EP 2.6.14). Its core principle relies on recombinant Factor C (rFC)—a recombinant protein engineered from the LPS-binding domain of horseshoe crab Factor C—eliminating reliance on natural horseshoe crab blood and addressing sustainability concerns.
1. Assay Principle
Endotoxin LPS specifically binds to the LPS-recognition domain of rFC, triggering a conformational change that activates rFC’s serine protease activity. The intensity of AMC fluorescence is directly proportional to the endotoxin concentration in the sample, measurable via a fluorescence microplate reader.
2. Key Technical Parameters
- Sensitivity: Detects endotoxins at concentrations as low as 0.005 EU/mL, matching or exceeding traditional Limulus Amebocyte Lysate (LAL) assays.
- Linear Range: Typically covers 0.005–50 EU/mL, enabling quantitative analysis across a broad concentration spectrum.
- Specificity: Exhibits high specificity for LPS; it is not activated by (1→3)-β-D-glucans (a common interference in LAL gel-clot assays), reducing false-positive results.
- Incubation Conditions: Requires controlled temperature (37±1°C) and incubation time (60–120 minutes), with fluorescence measured at excitation/emission wavelengths of ~360 nm/~460 nm.
3. Workflow Overview
- 1Prepare samples (e.g., pharmaceuticals, medical devices, biological products) via dilution or extraction to remove matrix interference.
- 2Mix rFC reagent, fluorogenic substrate, and sample in a microplate well.
- 3Incubate at 37°C to allow LPS-rFC binding, activation, and substrate cleavage.
- 4Measure fluorescence intensity; calculate endotoxin concentration using a standard curve generated with known endotoxin standards (e.g., Escherichia coli O55:B5 LPS).
This assay combines sustainability (no horseshoe crab harvesting), reliability (low interference, high reproducibility), and efficiency, making it ideal for quality control in pharmaceutical and biotech industries.
Frequently Asked Questions (FAQ)
What is the Recombinant Factor C (rFC) assay?
The rFC assay is a modern, horseshoe crab-free, in vitro method used for the quantitative detection of bacterial endotoxins (lipopolysaccharides). It serves as an eco-friendly alternative to traditional LAL assays.
Is the rFC assay compliant with international pharmacopoeias?
Yes, the rFC fluorometric assay is recognized and compliant with major global pharmacopoeias, including USP <85> and EP 2.6.14, for endotoxin testing.
How sensitive is the rFC fluorometric assay?
The assay is highly sensitive, capable of detecting bacterial endotoxins at concentrations as low as 0.005 EU/mL, which matches or exceeds the performance of traditional LAL methods.
Does the rFC assay produce false positives from glucans?
No. Unlike traditional LAL assays, the rFC assay exhibits high specificity for LPS and is not activated by (1→3)-β-D-glucans, significantly reducing false-positive results.
What are the standard incubation parameters for the assay?
The assay requires incubation at a controlled temperature of 37±1°C for 60 to 120 minutes. Fluorescence intensity is subsequently measured at wavelengths of approximately ~360 nm (excitation) and ~460 nm (emission).