Preliminary Assessment of Burn Depth by Paper-Based ELISA for the Detection of Angiogenin in Burn Blister Fluid-A Proof of Concept

Abstract

Rapid assessment of burn depth is important for burn wound management. Superficial partial-thickness burn (SPTB) wounds heal without scars, but deep partial-thickness burn (DPTB) wounds require a longer healing time and have a higher risk of scar formation. We previously found that DPTB blister fluid displayed a higher angiogenin level than SPTB blister fluid by conventional ELISA. In this study, we developed a paper-based ELISA (P-ELISA) technique for rapid assessment of angiogenin concentration in burn blister fluid. We collected six samples of SPTB blister fluid, six samples of DPTB blister fluid, and seven normal healthy serum samples for analysis. We again chose ELISA to measure and compare angiogenin levels across all of our samples, but we developed a P-ELISA tool and compared sample results from that tool to the results from conventional ELISA. As with conventional ELISA, DPTB blister fluid displayed higher angiogenin levels than SPTB in P-ELISA. Furthermore, our P-ELISA results showed a moderate correlation with conventional ELISA results. This new diagnostic technique facilitates rapid and convenient assessment of burn depth by evaluating a key molecule in burn blister fluid. It presents a novel and easy-to-learn approach that may be suitable for clinically determining burn depth with diagnostic precision.

Keywords: P-ELISA; angiogenin; burn blister fluid; burn wound healing; partial-thickness burn injury.

Figure 1

Procedure of paper-based ELISA (P-ELISA) for angiogenin analysis in burn blister fluid. Three microliters of burn fluid was loaded onto the test zone, and the test paper was incubated for 10 min. Three microliters of 1% BSA was then added for blocking, and the test paper was incubated for 10 min. The first image was recorded after loading 3 μL primary antibody, and the test paper was incubated for 10 min. The test paper was washed with 5 μL and then 10 μL TBST (Tris-buffered saline + 0.05% Tween 20) before adding 3 μL of horseradish peroxidase (HRP)-conjugated secondary antibody. The final procedure was to wash again with TBST and add 3 μL of substrate solution. After 10 min of incubation, we recorded the second image.

Figure 2

Analysis of angiogenin levels from superficial partial-thickness burn (SPTB) and deep partial-thickness burn (DPTB) blister fluids using P-ELISA. (a,b) Clinical pictures of superficial partial-thickness burn (SPTB, a) and deep partial-thickness burn (DPTB, b) wounds. (c) Comparison of angiogenin levels from two different burn fluids and healthy human blood serum as the control (n = 6 in SPTB and DPTB, n = 7 in control, mean ± S.D, ** p < 0.01).

Figure 3

Analysis of angiogenin and VEGF (Vascular endothelial growth factor) concentrations in SPTB and DPTB blister fluids with conventional plate ELISA. A trend toward higher angiogenin concentration in DPTB fluids was detected, compared to SPTB (n = 4 in SPTB, n = 6 in DPTB, mean ± S.D.; p = 0.07). No significant difference in VEGF levels was observed between two different blister fluids (n = 4 in SPTB, n = 5 in DPTB, mean ± S.D.; p = 0.26).

Figure 4

Correlation of the angiogenin detection between conventional plate ELISA and paper-based ELISA in burn blister fluids. The data show a moderate correlation between the results of P-ELISA and conventional plate ELISA (r = 0.5906, p = 0.0722).

Figure 5

Clinical examination of angiogenin concentration by paper-based ELISA. Test paper was designed to absorb blister fluid from burn patients. Angiogenin signals were captured and analyzed. The mean intensity of detected angiogenin in burn blister fluid was significantly higher than that in normal serum control (mean ± S.D.; **** p < 0.0001, n = 6).