Development of an enzyme-linked immunosorbent assay (ELISA) for determining neutrophil elastase (NE) - a potential useful marker of multi-organ damage observed in COVID-19 and post-Covid-19 (PCS)

Abstract

Background: The ongoing post-COVID-19 syndrome (PCS) epidemic, causing complications of diverse etiology, necessitates the search for new diagnostic markers and the development of widely accessible methods for their detection. This would enable the prognosis of PCS progression and faster implementation of targeted treatments. One potential marker is neutrophil elastase (NE), whose elevated levels in the blood during PCS may result from organ damage caused by increased secretion of severe inflammatory mediators or amyloidosis resulting from the interaction of NE with SARS-CoV-2. The aim of this publication is to present a step-by-step method for designing an enzymatic ELISA test, enabling the quantitative assessment of NE in the blood serum of patients.

Methods: NE was measured using the designed ELISA test.

Results: The study outlines all the steps necessary for designing and optimizing the ELISA test, including the selection of standards, primary and secondary antibodies, and their dilutions. Using the test, elevated NE levels were demonstrated in patients with advanced-stage diabetic nephropathy after symptomatic COVID-19, compared to a relative group of patients sampled before COVID-19.

Conclusion: The undertaken efforts enabled the development of a test with high performance parameters (initially set sensitivity: ≥40 pg/μL; intra-assay precision: 7%; inter-assay precision <20%). No significant cross-reactivity with other tested proteins was observed. Serial dilution of plasma samples resulted in a proportional decrease in signal intensity.

Keywords: COVID-19; ELISA; long-COVID biomarkers; neutrophil elastase 1; post-COVID-19-syndrome (PCS).

FIGURE 1

Scheme of the designed ELISA assay for the quantitative assessment of neutrophil elastase in blood serum.

FIGURE 2

The first standard curve for the quantitative assessment of neutrophil elastase, which required optimization.

FIGURE 3

The absorbance values obtained in the custom-designed ELISA test for NE determination for four patient sera in dilutions: (A) 5x, (B) 10x, (C) 25x, (D) 50x, treated with IAb and IIAb (dark blue color) and IIAb (light blue color) – serving as the control for IIAb, BLANK (black color).

FIGURE 4

Absorbance values for the individual concentrations of the standard (human native neutrophil elastase) – samples 1-22, and for the background (BLANK); standard number: 1-22 - in the order of increasing standard dilutions. The main steps of the ELISA test were carried out under the following conditions: I) Incubation with AbI: 440x, 2.5 h, 37°C; II) blocking solutions: 5% skimmed milk, overnight, 4°C; III) AbII: 5000x, 1.5 h, 37°C.

FIGURE 5

Standard curve for quantitative assessment of neutrophil elastase in blood serum (for concentration range of standard: 20 pg/mL to 300 pg/mL) under the given ELISA test conditions of the main steps: I) Incubation with AbI: 440x, 2.5 h, 37°C; II) blocking solutions: 5% skimmed milk, overnight, 4°C; III) AbII: 5000x, 1.5 h, 37°C.

FIGURE 6

The absorbance values of the ELISA tests conducted with three different blocking agents at various concentrations (marked in light blue), alongside the control reaction (i.e., the plate without primary antibody solution–secondary antibody control), are as follows: (A) Powdered skimmed milk at concentrations of 5%, 7.5%, 10%, and 12.5%; (B) Bovine serum albumin solutions at concentrations of 0.5%, 1%, 1.5%, and 2%; (C) Casein solutions at concentrations of 0.5%, 1%, and 1.5%.

FIGURE 7

The absorbance values obtained in the ELISA test conducted with two different blocking agents at various concentrations: (A) powdered skimmed milk at concentrations of 5%, 7.5%, 10%, 12.5%; (B) bovine serum albumin solutions at concentrations of 0.5%, 1%, 1.5%, 2%, using three different dilutions of the primary antibodies (RAbI): 440x (green), 880x (yellow), 2200x (blue).

FIGURE 8

Standard curve for quantitative assessment of neutrophil elastase, corrected with control values, in the concentration range of standard: 1.5 pg/μL to 50 pg/μL. The main steps of the ELISA test were carried out under the following conditions: I) Incubation with AbI: 440x, 2.5 h, 37°C; II) blocking solutions: 7.5% skimmed milk, overnight, 4°C; III) AbII: 5000x, 1.5 h, 37°C.

FIGURE 9

The absorbance values (corrected for the corresponding control absorbance values) obtained for the serum samples from four patients with advanced diabetic nephropathy at four different dilutions: 5x (blue), 10x (pink), 25x (gray), and 50x (yellow).

FIGURE 10

The absorbance values obtained for: the blood sera of four patients with advanced diabetic nephropathy at four different dilutions: 5x, 10x, 25x, and 50x, their corresponding controls, and BLANK (dark blue: 5x test sample; light blue: 5x control; dark green: 10x test sample; light green: 10x control; dark yellow: 25x test sample; light yellow: 25x control; black: 50x test sample; gray: 50x control; black: test control).

FIGURE 11

Standard curves for human native neutrophil elastase determined based on absorbance values obtained using primary antibodies diluted 220-fold and 440-fold.

FIGURE 12

The values (corrected for control values) of absorbance for selected blood serum samples diluted 2-fold, 4-fold, and BLANK treated with primary antibody solutions diluted 220 times (RAbI:220x) or 440 times (RAbI:440x). The absorbance values for the 2-fold diluted serum are marked in light blue, and for the 4-fold diluted serum in green.

FIGURE 13

Standard curve for human native neutrophil elastase determined based on absorbance values obtained using primary antibodies diluted 440 times, in the concentration range of standard: 14 pg/μL to 200 pg/μL. The main steps of the ELISA test were carried out under the following conditions: I) Incubation with AbI: 440x, 2.5 h, 37°C; II) blocking solutions: 7.5% skimmed milk, overnight, 4°C; III) AbII: 5000x, 1.5 h, 37°C.

FIGURE 14

The mean absorbance values of fourteen randomly selected serum samples from patients with advanced diabetic nephropathy (stage IV or V), who had recovered from COVID-19, and the background (BLANK) obtained in the designed ELISA test for quantitative assessment of NE in blood serum.