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

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.

Figure 1

Titration curve of normal rabbit and immunized rabbit serum. BSA: Bovine serum albumin, CT: Control serum (nonimmunized), MMP-9: Matrix metalloproteinase 9

Figure 2

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two bands 50 kD H-chains and 25 kD L-chains related to immunoglobulin G (s) in fraction 2 (F2). Lane 1, 2, 4: Fraction 1, 2, and 3 of purified pAb. F2 was selected for further experiments

Figure 3

Reaction of immunoglobulin G purified antibody fraction 2 with synthetic peptide, bovine serum albumin, native commercial matrix metalloproteinase 9, OD value of control serum (1/500) about 0.32. MMP-9: Matrix metalloproteinase 9, BSA: Bovine serum albumin

Figure 4

(a) Native polyacrylamide gel electrophoresis was performed on HCT 116 supernatant. 72/82 kD extracted proteins are located in lane 2. (b) Titration curve of purified immunoglobulin G fraction 2 against 82 kD matrix metalloproteinase 9 (MMP-9), 72 kD MMP-2 and a mixture of 72/82 kD proteins. (c) Western blot analysis of purified immunoglobulin G against gelatinases (from HCT 116 cell supernatant). BSA: Bovine serum albumin

Figure 5

Titration curve of mixture of purified immunoglobulin G fraction 2 and inhibition peptides (20aa, FPFIFQGQSYSACTTDGRSD; 12aa, MHPN AGHGSLMR) against synthetic peptide. All performed in triplicates. BSA: Bovine serum albumin

Figure 6

The diagnostic abilities were evaluated by receiver operating characteristic curve as 96.7% sensitivity, 80% specificity and area under curve with P < 0.001. MMP-9: Matrix metalloproteinase 9. AUC: Area under curve