One-step ultra-rapid immunoassay of calcitonin gene-related peptide for migraine diagnosis.

Migraine is known to be caused by calcitonin gene-related peptide (CGRP), prompting the need for quantitative analysis of CGRP for the clinical treatment of monoclonal antibodies targeting CGRP. Since CGRP is cleaved by proteolytic enzymes post-blood collection, rapid analysis methods are required. In this study, a one-step immunoassay for CGRP was developed using chemically mimicking peptides (mimotopes) with an analysis time of 32 min. Four clones from an Fv-antibody library were screened using two types of monoclonal antibodies against CGRP. Mimotopes for each monoclonal antibody were synthesized into peptides of 15 residues. The binding affinity (K) was estimated, and the interaction with monoclonal antibodies was analyzed using docking simulations. Finally, a one-step immunoassay for CGRP was demonstrated using migraine patient samples (n = 57) and healthy volunteer controls (n = 18). The limit of detection (LOD) of one-step immunoassay based on Fremanezumab (mimotope F1) was estimated to be 8.8 pg/mL with the limit of quantification (LOQ) of 125.9 pg/mL. And, the one-step immunoassay based on Galcanezumab (mimotope G7) showed the LOD of 9.4 pg/mL and the LOQ of 84.7 pg/mL. The total analysis time was estimated to be approximately 32 min and the assay results were estimated to be statistically consistent with conventional CGRP assay.