MSACL 2024 Abstract

INTRODUCTION: Immunoprecipitation-based mass spectrometry (IP-MS) has played a key role in antigen discovery for decades. Despite its success, this technique has several limitations. First, IP-MS is very labor intensive. Further, IP-MS does not enable epitope mapping during the discovery of the antigen. In contrast, phage immunoPrecipitation sequencing (PhIP-Seq) technique enables simultaneous screening and mapping of antigens. This method also enables whole proteome wide screening and is amenable for high throughput.

OBJECTIVE: To utilize PhIP-Seq and screen for presence of novel antigen in immune-mediated rippling muscle disease (iRMD). A serological biomarker of this disease is lacking.

METHODS: First, a 150-mer oligo library representing ~650k epitopes covering the entire human proteome was synthesized. This library was inserted into T7-phage and resulting phage library was amplified. Amplified library was analyzed to ensure representation of >90% of the targeted antigens. Next, human serum and cerebospinal fluid (CSF) based phage library immunoprecipitation protocols were optimized and implemented for high-throughput screening using a liquid handler. This process starts by exposing patient serum/CSF to the phage library and isolating the antibody-phage complexes. Isolated complexes are analyzed to detect potential antigens found in each sample.

RESULTS: We evaluated a total of 10 patients diagnosed with iRMD based on the presence of electrically silent percussion- or stretch-induced muscle rippling and percussion-induced rapid muscle contraction with or without muscle mounding and an autoimmune basis. Sera were evaluated for a common biomarker using PhIP-Seq. An IgG autoantibody specific for caveolae-associated protein 4 (cavin-4) was identified in serum of patients with iRMD. PhIP-Seq also enabled mapping of the epitope in the patients. This information was utilized to develop immunoassays and confirmed cavin-4 IgG seropositivity in 8 of 10 patients with iRMD. Results for healthy and disease-control individuals (n = 241, including myasthenia gravis and immune-mediated myopathies) were cavin-4 IgG seronegative. Muscle biopsy was performed in seven of the eight patients with cavin-4 IgG. All six specimens analyzed immunohistochemically revealed a mosaic pattern of sarcolemmal cavin-4 immunoreactivity. In a separate study, we also utilized the phage library to map the epitopes of Leucine Zipper Protein 4 (LUZP4) antigen present in certain germ cell tumors and associated paraneoplastic syndromes. Even though LUZP4 was discovered by our group via IP-MS, the epitope of the antigen remained a mystery. We applied PhIP-Seq to successfully map the epitope, which enables development of better immunoassays for clinical screening.

CONCLUSION: PhIP-Seq has shown great promise in enabling simultaneous detection and epitope mapping of novel antigens. This technology is also highly scalable and enables screening of hundreds of samples in a very economical fashion. We utilized this technology to not only discover novel antigens but also successfully epitope map the antigens to enable more efficient translation of the antigens into clinical laboratory for routine screening.