Reagents and instruments
Two monoclonal antibodies against different epitopes of Gal-3, the capture antibody (10301) and detection antibody (10302), were purchased from Medix (Kauniainen, Finland). The Gal-3 standard (abs 01295) was purchased from Absin Bioscience Inc (Shanghai, China). The enzyme-linked immunosorbent assay (ELISA) kit was purchased from Sino (Beijing, China). Eu3+-N1‐(p‐isothiocyanatobenzyl)‐diethylenetriamine‐N1, N2, N3, and N4‐tetra acetic acid (DTTA) and enhancement solution were purchased from Zhejiang Boshi Biological Technology Co., Ltd (Zhejiang, China). The Sephadex-G50 column was purchased from Seebio Biotech Co (Shanghai, China). Ninety-six-well plates were purchased from Xiamen Yunpeng Technology Development Co., Ltd (Xiamen, China). The time-resolved immunofluorescence analyzer was purchased from Foshan Da'an Medical Instrument Co., Ltd., and an electric thermostatic incubator was purchased from Shanghai Jinghong Laboratory Instrument Co., Ltd. (Shanghai, China).
Serum samples were collected from 81 patients with IMN diagnosed by pathology and from 123 healthy subjects in the Department of Nephrology, Wuxi People’s Hospital Affiliated to Nanjing Medical University. All serum samples were collected using a separation gel-sampling tuble. After centrifugation, the samples were stored in a refrigerator at -20°C and returned to room temperature before detection. Informed consent was obtained from all participants. The research project was approved by the Wuxi People’s Hospital, Affiliated to Nanjing Medical University.
The buffers included a coating buffer (50 mM Na2CO3-NaHCO3, pH 9.6), analysis buffer (50 mmol/L Tris-HCl, containing 0.01% Tween-20, 20 µM diethylenetriamine pentaacetate, 0.9% NaCl, 0.2% bovine serum albumin (BSA), and 0.05% NaN3, pH 7.8), labeling buffer (50 mmol/L Na2CO3-NaHCO3, pH 9.0), blocking buffer (50 mmol/L Tris-HCl, containing 0.9% NaCl, 1% BSA, and 0.05% NaN3, pH 7.8), washing solution (50 mmol/L Tris-HCl, containing 0.9% NaCl, 0.05% proclin-300, pH7.8) and elution buffer (50 mmol/L Tris-HCl, containing 0.2% BSA, 0.05% Proclin-300, pH 7.8). Gal-3 standard (1000 ng/mL) was diluted with analysis buffer to final concentrations of 0, 12.5, 25, 50, 100, and 1000 ng/mL.
Immobilization of monoclonal antibodies
Gal-3 antibody (10301, capture antibody) was diluted to 3 µg/mL in coating buffer at pH 9.6. Then, 100 µL of the diluted antibody was added to each well and incubated overnight at 4°C. The plates were washed with washing solution, and 150 µL of blocking buffer containing 1% BSA was added to each well. The coated antibody were blocked at room temperature for 2 h, dried thoroughly, and stored at − 20°C.
Labeling monoclonal antibodies with Eu3+
Anti-Gal-3 antibody labeling was performed according to the manufacturer’s instructions. A total of 0.3 mg anti-Gal-3 antibody was added to the Ultracel-50K ultrafiltration tube and the labeling buffer was replaced by multiple centrifugation steps. The antibody was mixed with 60 µg Eu3+-DTTA chelate and incubated overnight at 28°C. The labeling solution was injected onto a Sephadex-G50 column chromatography balanced with 0.2% BSA eluent, and the fluorescence count of the collected proteins was detected by time-resolved immunofluorescence analyzer. The first elution peak was collected, and the labeled antibodies were packaged and stored at -20°C.
Gal-3-TRFIA assay procedure
A two-step method was used for detection to avoid the influence of ethylenediaminetetraacetic acid in the serum. Standard or serum samples (50 µL) were added to 96-well microplates. Subsequently, 50 µL of the analysis buffer was added to each well. The samples were then incubated at 37°C for 2 h and washed with washing solution twice. Thereafter, 100 µL diluted Eu3+-anti-Gal-3 antibody was added to each well, and the plate was incubated at 37°C for 2 h. After washing with washing solution six times, 100 µL enhancement solution was added to each well. After shaking for 3 min, the fluorescence count of each well was measured using a time-resolved immunofluorescence analyzer. Finally, the Gal-3 concentration in serum samples was calculated according to the standard curve of the Gal-3-TRFIA.
Optimization of the experimental reaction conditions
Selection of the coated antibody concentration
The Gal-3 coated antibody was diluted to different concentrations (0.375, 0.7, 1.5, 3, and 6 µg/mL). After coating and blocking, Gal-3 standards and the Eu3+-anti-Gal-3 antibody were added to obtain the optimal concentration of Gal-3 coated antibody.
Selection of the dilution ratio of labeled antibody
Eu3+-anti-Gal-3 was diluted at ratios of 1:25, 1:50, 1:100, 1:200, 1:400 and 1:800. Different dilution ratios of Eu3+-anti-Gal-3 were tested with 100 ng/mL Gal-3 standard or analytical buffer to determine the fluorescence counts or background counts, respectively. The optimal dilution ratio of labeled antibodies was selected when both high fluorescence counts and low background counts were obtained.
Evaluation of the Gal-3-TRFIA method
Sensitivity and linearity
The Gal-3 standard curve was determined by plotting the corresponding fluorescence counts across a serial of concentrations (0, 12.5, 25, 50, 100, and 1000 ng/mL) of the Gal-3 standard and analyzing it logit-log linear regression fitting. Based on the standard curve, the assay sensitivity was determined by the concentrations of Gal-3 which corresponded to the fluorescence of 10 wells of zero calibrators (mean + 2SD).
The precision of the method was evaluated using two Gal-3 standards. Two Gal-3 standards with low (12.5 ng/mL) and high (100 ng/mL) Gal-3 concentrations were selected. Meanwhile, 10 independent experiments were conducted on each of the three samples to obtain intra-batch precision. The inter-batch precision was repeated three times with ten independent experiments each time.
Specificity was assessed using 1000 ng/mL Kidney injury molecule 1 (KIM-1) and 1000 ng/mL T cell immunoglobulin-3 (TIM-3) as a potential interferers. The ratio of the actual measured KIM-1/TIM-3 concentration to the theoretical concentration was calculated as the specificity.
The Gal-3 standard at a concentration of 1000 ng/mL was mixed with serum sample with low concentrations of Gal-3 in a ratio of 1:9. The ratio of the actual measured Gal-3 concentration to the theoretical concentration of the mixed serum was calculated as the recovery rate using the following formula: recovery (%) = (measured concentration/theoretical concentration) × 100. The ratio of each measurement to the theoretical value was then calculated.
Correlation between Gal-3-TRFIA and ELISA
Gal-3 serum levels were measured using the established Gal-3-TRFIA in 28 patients with IMN and compared to those detected using commercially available ELISA kits, serums whose Gal-3 concentration exceeded the ELISA kit's range were diluted and remeasured.
Clinical application of Gal-3-TRFIA
Gal-3 serum levels in 81 patients with IMN and 123 healthy controls were measured using the established Gal-3-TRFIA. The patients’ urea, uric acid, urine protein,serum albumin, serum creatinine, and glucose levels were obtained from database of the hospital.
The data are presented as the mean ± SD. Median and quartile ranges were used if the data distribution was skewed. Results were analyzed using SPSS (version 24.0, Chicago, IL, USA), and charts were plotted using PRISM 7.00 (San Diego, CA, USA) and Origin 2018 (Origin Lab, USA). The Pearson correlation coefficient (R) or Spearman correlation coefficient (ρ) was used to evaluate whether the Gal-3 concentration was correlated with each parameter. A Mann-Whitney U-test was used to analyze whether a significant difference existed between the two groups.