The Ability of Antibody against Truncated Matrix Metalloproteinase 9 Peptide to Evaluate the Native Protein on Tear Drops of Dry Eye Disease Patients by a Point-of-Care Diagnostic System

Zahra Afshari; Mohammad Javad Rasaee; Maliheh Paknejad; Saeed Khalili; Alireza Baradaran-Rafii

doi:10.4103/joco.joco_320_22

The Ability of Antibody against Truncated Matrix Metalloproteinase 9 Peptide to Evaluate the Native Protein on Tear Drops of Dry Eye Disease Patients by a Point-of-Care Diagnostic System

J Curr Ophthalmol. 2023 Dec 21;35(2):170-176. doi: 10.4103/joco.joco_320_22. eCollection 2023 Apr-Jun.

Authors

Zahra Afshari¹, Mohammad Javad Rasaee¹, Maliheh Paknejad², Saeed Khalili³, Alireza Baradaran-Rafii⁴

Affiliations

¹ Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
² Department of Clinical Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
³ Department of Biology Sciences, Faculty of Sciences, Shahid Rajaee Teacher Training University, Tehran, Iran.
⁴ Department of Ophthalmology, Labbafinejad Medical Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

Abstract

Purpose: To obtain a reactive and specific antibody against truncated matrix metalloproteinase 9 (MMP-9), that has reactivity toward the native protein. Precision, accuracy, specificity, and sensitivity were evaluated using a point-of-care test.

Methods: An in silico study was used to confirm the anti peptide truncated MMP-9 is against native MMP-9. After an antibody titer assessment, purification, and characterization, the anti MMP-9 was assessed. The cut-off value was determined using the purified gelatinases of the supernatant HCT 116 cell line. The supernatant was purified by preparative native-polyacrylamide gel electrophoresis based on charge and size of the proteins. Furthermore, quality control (QC) of the results were performed following standard densitometry methods.

Results: A truncated MMP-9 is the major epitope peptide that can trigger the immune system to scavenge for a specific and reactive antibody against the native MMP-9. The MMP-9 native protein is purified from the supernatant of the HCT 116 cell line and the commercially available, full-length MMP-9. The cut-off value was estimated at 30 μg/mL. QC results indicated that the specificity was 80%, sensitivity was 96.7%, accuracy was 91%, and precision was 91.66%. The area under curve was 0.827 (P < 0.001). The positive predictive value was 83%, and the negative predictive value was 96%.

Conclusions: The antibody against the synthetic epitope peptide can detect the native MMP-9. Native MMP-9 is considered the main biomarker in an immunoassay POCT and is used to diagnose dry eye disease (DED). In accordance with QC results, MMP-9 point of care test can be utilized for screening patients suffering from DED.

Keywords: Dry eye disease; Epitope peptide; In silico analysis; Matrix metalloproteinase 9 point-of-care test; Polyclonal antibody.