Using Synthetic Peptides in Next-Generation Inflammatory Disease Diagnostic Assays

Conditions like ulcerative colitis and Crohn's disease, which cause inflammation in the body, can be identified or tracked by measuring a protein called calprotectin. To do this, samples of stool are tested to determine the levels of calprotectin. Similarly, for rheumatoid arthritis, the inflammation status can be monitored by measuring calprotectin levels in the blood. Scientists use special antibodies that can detect and bind to calprotectin in patient samples. This method is similar to how home COVID-19 test kits work, using lateral flow assays to detect specific proteins.

Antibody-based calprotectin assays can have some limitations and inconsistencies in their results. The variations occur because different antibodies and assay methods may interact with calprotectin differently. Antibodies may bind to different regions of the protein or have varying compositions, leading to variations in the test outcomes. Additionally, over time, antibodies can become inactive or less effective due to unfolding or precipitation, further affecting the accuracy and reliability of the assay results.

A potential solution to address the limitations of antibody-based assays is the use of peptides instead. Peptides are short sequences of amino acids that can selectively and strongly bind to proteins, such as calprotectin, with high precision. Unlike antibodies, peptides can be chemically synthesized with consistent quality, purity, and stability. They are also more cost-effective to produce and exhibit lower variability between batches. Moreover, peptides can be specifically attached to surfaces, enabling the development of diagnostic assays with improved accuracy and control in detecting biomarkers. This approach simplifies the process of diagnostic assay development and enhances the detection of disease markers like calprotectin.

Under the collaboration of Christian Gerhold, CTO of BÜHLMANN, and Professor Christian Heinis at EPFL, a team of researchers worked on developing calprotectin ligands using peptides. By screening through a vast library of over 500 billion peptides, Cristina Diaz-Perlas, a postdoctoral researcher in Heinis's group, successfully identified several peptides that can bind to calprotectin. The team demonstrated that these peptides can be effectively used in simplified lateral flow assays for calprotectin quantification. The best-performing peptide exhibited a strong binding affinity to calprotectin, with a dissociation constant of 26 nM, indicating its potential suitability for diagnostic tests.

The developed peptide not only binds to a significant area of calprotectin but also specifically targets the form of calprotectin that is relevant in patient samples. Under the supervision of Benjamin Ricken at BÜHLMANN, the peptide was further evaluated in well-designed lateral flow cassettes, demonstrating its suitability for precise detection and measurement of calprotectin. In a preliminary study, this setup was used to determine the concentration of calprotectin in serum samples obtained from patients' blood.

The peptide developed in this study represents the first synthetic affinity reagent designed specifically for the biomarker calprotectin. The teams at EPFL and BÜHLMANN are currently conducting additional tests using this calprotectin-specific peptide. Their aim is to develop an assay that can elevate the diagnostic capabilities of this biomarker, ultimately benefiting patients who are dealing with inflammatory diseases. Christian Heinis expresses the importance of this work in advancing the understanding and management of such conditions.

This collaboration greatly benefited from BÜHLMANN's knowhow to produce and handle the biomarker, and expertise of the EPFL team to generate and screen large combinatorial libraries of peptides by phage display."

Christian Gerhold, CTO of BÜHLMANN