J Clin Neurol.  2025 Mar;21(2):105-112. 10.3988/jcn.2024.0413.

Development and Application of a Cell-Based Assay for Detecting Anti-Agrin Antibodies Associated With Myasthenia Gravis

• Kim SH
• Chung HY
• Kim M
• Kim SW
• Shin HY

• KMID: 2565543
• DOI: http://doi.org/10.3988/jcn.2024.0413

Abstract

Background and Purpose
Anti-agrin antibodies (agrin Abs) have recently been identified in patients with myasthenia gravis (MG), sometimes in conjunction with antibodies (Abs) to the acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), or low-density lipoprotein receptor-related protein 4. This study aimed to develop an in-house cell-based assay (CBA) for detecting agrin Abs, and to test its application to serum samples collected from individuals diagnosed with MG.
Methods
Agrin complementary DNA as cloned into a pCMV6-AC-GFP vector, which was subsequently transfected into human embryonic kidney 293T (HEK293T) cells. Transfected HEK293T cells were incubated with patient serum and antihuman immunoglobulin G Ab conjugated with a red fluorescent dye. Agrin Ab levels were measured using the CBA in 389 serum samples: 340 from patients with MG, 36 from patients with other neuromuscular diseases, and 13 from healthy controls. The presence of agrin Ab was determined based on the fluorescence intensity and colocalization using fluorescence microscopy.
Results
The expression levels of agrin mRNA and protein in transfected HEK293T cells were confirmed using the reverse-transcription polymerase chain reaction and Western blotting, respectively. Agrin expression in cells was further confirmed by immunocytochemistry. Two (0.6%) of the 340 patients with MG tested positive for agrin Ab: 1 of 191 AChR-positive patients and 1 of 54 MuSK-positive patients.
Conclusions
We have developed and validated a novel CBA for detecting agrin Abs. This CBA was successfully applied to detect agrin Abs in serum samples obtained from individuals with MG.

Keyword

autoimmune diseases; neuromuscular junction; agrin; myasthenia gravis; immunocytochemistry