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The Challenge of False Positives in Endotoxin Detection Due to β-Glucan

Endotoxins can cause serious inflammatory responses in the body, leading to conditions like systemic inflammatory response syndrome (SIRS), blood hypercoagulation and organ dysfunction. Therefore, endotoxin testing is crucial in biomedical research and pharmaceutical manufacturing. Currently, the most common method for endotoxin detection is the Limulus Amebocyte Lysate (LAL) assay, which uses horseshoe crab blood cells to detect endotoxins through a specific reaction that causes aggregation. However, LAL assays are not entirely specific, as they can also react with other substances, leading to false-positive results. One such interfering substance is β-glucan, a polysaccharide that can interfere with endotoxin detection.

β-glucan shows therapeutic potential as an adjuvant in monoclonal antibody-based cancer immunotherapies by enhancing immune responses against tumors through complement receptor 3 (CR3) stimulation. When combined with antibodies like anti-PD-L1, β-glucan-conjugated antibodies (AGC) have achieved an impressive 86.7% tumor inhibition rate in mouse models.

Anti-PD-L1 antibody coupled with β-glucan to construct AGC

However, its interference with LAL-based endotoxin testing presents a challenge for antibody drug development, as β-glucan can cause false-positive results, potentially delaying drug approval and release. Moreover, its widespread presence in fungi, algae, and synthetic fibers makes it difficult to avoid in real-world applications.

Schematic diagram of false positive endotoxin test due to β-glucan

Overcoming False Positives with Recombinant Factor C Assays

Given the promising potential of AGC strategies in cancer immunotherapy, selecting an appropriate endotoxin detection method is critical during antibody drug development. Research has demonstrated that recombinant Factor C (rFC) assays offer significant advantages over traditional LAL assay in endotoxin detection. When tested for endotoxin contamination in β-glucan at concentrations of 10 μg/mL and 1 μg/mL, the rFC assay showed no non-specific signals, confirming its high specificity and ability to avoid interference from β-glucan. In contrast, the dynamic chromogenic LAL assay generated non-specific signals in the presence of β-glucan, highlighting its susceptibility to interference.

The recombinant Factor C assay for endotoxin is highly specific and unaffected by β-glucan.

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Using methods that are easily influenced by β-glucan interference may lead to inaccurate endotoxin detection, potentially affecting the quality and safety assessment of antibody drugs. In contrast, rFC assays, which are not affected by β-glucan, can provide precise endotoxin measurements, giving drug developers reliable data to ensure that antibody drugs meet the necessary quality standards. Ultimately, the use of rFC assays can improve the success rate of drug development and support the production of safe and effective antibody therapies.

Recombinant Factor C Endotoxin Detection Kit

ACROBiosystems has introduced the Recombinant Factor C Endotoxin Detection Kit (Cat. No. RES-A056), utilizing its proprietary platform for endpoint fluorescence measurement. This kit leverages recombinant Factor C technology to deliver rapid, precise endotoxin detection in samples, free from interference by β-glucan. It offers superior specificity, sensitivity, and batch-to-batch consistency, making it an ideal solution for meeting the stringent quality control standards required in pharmaceutical manufacturing.

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Protecting Horseshoe Crabs with ACROBiosystems

At ACROBiosystems, we are committed to protecting horseshoe crabs and advancing endotoxin detection technologies that contribute to the development of safer and more effective therapies while minimizing the impact on these vital marine species.

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Upcoming Endotoxin Detection Insights Series:

•  Part 1: The Role of Recombinant Factor C Endotoxin Detection in β-Glucan Conjugated Antibody Drugs

•  Part 2: Application of Recombinant Factor C Endotoxin Testing in Pharmaceutical Manufacturing

•  Part 3: Comparison of Recombinant Factor C and Other LAL Methods

•  Part 4: A Comprehensive Guide to Sample Dilution Calculations for Recombinant Factor C Endotoxin Testing

•  Part 5: Key Considerations for Endotoxin Testing Experimental Procedures

•  Part 6: Interfering Factors in Endotoxin Testing

•  Part 7: Endotoxin Detection Standards and Guidelines from a Pharmacopeial Perspective

Reference

1. The effects of β-glucan on human immune and cancer cells. Godfrey Chi-Fung Chan, Wing Keung Chan,　and Daniel Man-Yuen Sze.J. Hematol Oncol. 2009 Jun 10;2:25. doi: 10.1186/1756-8722-2-25

2. β-Glucan Functions as an Adjuvant for Monoclonal Antibody Immunotherapy by Recruiting Tumoricidal Granulocytes as Killer Cells.　Feng Hong, Richard D. Hansen, Jun Yan, and Gordon D. Ross.　CANCER RESEARCH 63, 9023–9031, 2003 Dec.

3. β-Glucan–conjugated anti–PD-L1 antibody enhances antitumor efficacy in preclinical mouse models. Qian Wang, Hao Jiang, Hongli Zhang, and Guangli Yu.　Carbohydrate Polymers 324 (2024) 121564.

4. https://www.dxy.cn/bbs/newweb/pc/post/37303996

5. Occupational endotoxin exposure and health effects