Establishment of Galectin-3 Time-resolved Fluoroimmunoassay and its Application in Idiopathic Membranous Nephropathy

The aim of this study was to establish a time-resolved fluorescent immunoassay (TRFIA) for the detection of serum Galectin-3 (Gal-3) and apply this method to evaluate the clinical significance of serum Gal-3 in predicting Idiopathic Membranous Nephropathy (IMN) progression. The Gal-3-TRFIA was established using the double antibody sandwich method, with the capture antibodies coated on a 96-well microplate and the detection antibodies chelated with Europium (III) (Eu3+). Serum Gal-3 was detected in 81 patients with IMN and 123 healthy controls to further evaluate the value of the Gal-3 in staging of IMN. The sensitivity of the Gal-3-TRFIA assay was 0.85 ng/mL, and the detection range was 0.85–1000 ng/mL. The Gal-3 intra-batch and inter-batch coefficients of variation were 3.45% and 5.12%, respectively. The correlation coefficient (R) between the Gal-3-TRFIA assay and commercially available enzyme-linked immunosorbent assay kits was 0.83. The serum Gal-3 concentration was higher in patients with IMN (65.57 ± 55.90 ng/mL) compared to healthy controls (16.29 ± 9.91 ng/mL, P < 0.0001). In this study, a wide detection range Gal-3-TRFIA assay was developed using lanthanide (Eu3+) chelates for the detection of Gal-3 concentrations in serum. Gal-3 concentration is elevated in patients with IMN.


Introduction
Galectin-3 (Gal-3), a member of β-galactoside-binding lectin family [1], is an important regulators of inflammatory responses, and it is expressed in many inflammatory cells, such as macrophages [2]. When the cell is at rest, Gal-3 is stored in the cytoplasm; however, in the event of tissue damage, activated cells secrete Gal-3 via nonclassical pathways [3]. This protein binds to the cell surface and extracellular matrix polysaccharides and influences a variety of physiological and pathological processes, including apoptosis, adhesion, migration, fibrosis [2], and inflammation [4,5]. The expression of Gal-3 is detected in many disease states, such as renal disease [6]. Gal-3 is a biomarker associated with the progression of renal functional decline [7]. Elevated serum Gal-3 levels are associated with a rapid decline in glomerular filtration rate and an increased risk of chronic kidney disease (CKD) [8,9]. However, the role of Gal-3 in MN has not yet been reported.
Membrane nephropathy (MN) is a unique glomerular lesion [10] and is the main cause of adult nephrotic syndrome [11]. Approximately 80% of cases are idiopathic membranous nephropathy (IMN) [10]. Approximately 20% of patients with IMN will progress to end-stage renal disease, and approximately 10% of them will die within 5-10 years [12,13].
Xiaomei Yu, Lingli Chen and Bo Lin this author contributed equally to this work, and should be considered as first authors Therefore, we conducted relevant studies to explore the value of Gal-3 in MN. In this study, an effective timeresolved fluoroimmunoassay (TRFIA) (Fig. 1) was established to detect the concentration of Gal-3 in serum with antibodies labeled with the lanthanide element Eu 3+ , and the fluorescence count of Eu detected was proportional to the amount of Gal-3 present in the binding reaction to achieve the quantification of Gal-3 in serum. This study also explored the diagnostic value of Gal-3 detection in patients with IMN. Moreover, this study was conducted to determine whether Gal-3 was associated with renal function impairment and disease progression in patients with IMN.

Samples
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.

Immobilization of Monoclonal Antibodies
Gal-3 antibody (10,301, 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 Eu 3+
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-50 K ultrafiltration tube and the labeling buffer was replaced by multiple centrifugation steps. The antibody was mixed with 60 μg Eu 3+ -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 Eu 3+ -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.

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 Eu 3+ -anti-Gal-3 antibody were added to obtain the optimal concentration of Gal-3 coated antibody.

Selection of the Dilution Ratio of Labeled Antibody
Eu 3+ -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 Eu 3+ -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.

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 log-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).

Precision
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
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.

Recovery Rate
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.

Statistical Analysis
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.

Selection of the Coated Antibody Concentration
Diluted anti-Gal-3 antibodies at 0.375, 0.75, 1.5, 3, and 6 μg/ mL were coated on 96-well microplates. After blocking, the Gal-3 standard and Eu 3+ -anti-Gal-3 antibodies were added to the plate. The counts per second (CPS) increased with an increase in the coated antibody concentration and leveled off when the coated concentration was 3 μg/mL ( Fig. 2A). Therefore, the optimal concentration of the coated antibody was determined to be 3 μg/mL.

Selection of the Labeled Antibody Dilution Ratio
Eu 3+ -anti-Gal-3 at 1:100 was considered the optimal because the background is low and the Gal-3 binding rate is high (Fig. 2B).

Assay Sensitivity
The linear results are shown in Fig. 3A. The fluorescence count of 10 wells of 0 ng/mL standard was substituted into the standard curve, and mean + 2 SD was calculated to obtain a sensitivity of 0.85 ng/mL. The sensitivity of TRFIA was better than that of ELISA (1.56 ng/mL). The Gal-3 detection range was 0.85-1000 ng/mL. The correlation coefficient of the Gal-3 standard curve was 0.996, indicating a good linear relationship between Gal-3 concentration and fluorescence count.

Assay Precision and Specificity
Precision was expressed as the coefficient of variation (CV%). The Gal-3 intra-and inter-batch coefficient of variation was 3.45% and 5.12%, respectively. All CV values were less than 10%, indicating that the precision of this experiment was acceptable. Studies of the specificity showed that the cross-reactivity with KIM-1 and Tim-3 were less than 1%, which would not affect the experimental results.

Assay Recovery Rate
The original concentration of the serum sample was 26.59 ng/mL. After the addition of the Gal-3 standard, the three measured concentrations of Gal-3 were 132.90, 124.79, and 127.70 ng/mL. The average recovery rate was 104%.

Correlation between Gal-3-TRFIA and ELISA
The established Gal-3-TRFIA and ELISA methods were used to simultaneously detect the serum Gal-3 concentration in patients. Linear regression analysis showed that the results of the two tests were well correlated (Fig. 3B, R 2 = 0.83086), indicating that the Gal-3-TRFIA detection method could be used for clinical detection of Gal-3 in human serum. The maximum detection limit of the TRFIA was 1000 ng/mL, which was higher than that of the ELISA (100 ng/mL).

Clinical Application
Eighty-one serum samples from patients with IMN and 123 serum samples from healthy controls were assayed using Gal-3-TRFIA methods to evaluate its clinical applicability. Serum Gal-3 concentration of patients with IMN was 65.57 ± 55.90 ng/mL, which was significantly higher than that of healthy controls (16.29 ± 9.91 ng/mL, P < 0.0001) (Fig. 4A). Receiver operating characteristic (ROC) curve analysis was performed based on the serum Gal-3 concentrations of patients with IMN and healthy controls (Fig. 4B). The area under ROC was 0.811 (P < 0.0001), indicating that Gal-3 was useful in the differentiation of IMN from healthy controls. Serum Gal-3 level had no significant correlation with PLA2R-Ab (Fig. 4C). However, staged Gal-3 concentrations by quartile showed that individuals with Gal-3 levels in the highest quartile had significantly higher levels of urea/ creatinine, urea, and serum creatinine and were related to a higher risk of developing proteinuria (Table 1).

Discussion
Gal-3 is a cytoplasmic protein synthesized in the cytoplasm [14]. Gal-3 is also released into the blood or urine from damaged cells and inflammatory cells under pathological conditions [8,15]. Many different blood assays have been used in Gal-3 clinical practice, with ELISA being the most commonly used [16]. However, some defects were observed in ELISA, such as unstable compounds labeled by the enzyme and narrow detection range. In ELISA technology, a horse radish peroxidase is used as a marker. Horse radish peroxidase is a large molecule and easily influenced by environmental factors. Horse radish peroxidase creates a spatial barrier during labeling, which affects labeling rate and binding to antibodies. Time-resolved immunofluorescence assay uses small molecule lanthanides for labeling, which can greatly improve the labeling rate without affecting the binding of antibodies. Gal-3 concentrations fluctuate widely in patients and are beyond the range of ELISA detection. Therefore, a method with a wider detection range should be developed to detect Gal-3 concentration in patients. TRFIA is widely used in many fields, such as clinical research [17]. The unique fluorescence properties of lanthanide chelates provide TRFIA with many advantages, such as a narrow emission peak and high quantum yield [18]. In addition, TRFIA has a wider detection range and less impact on the activity of the marker compared to ELISA; thus, it is suitable for the establishment of a wide detection range method for Gal-3.
In this study, the Eu 3+ -Gal-3 antibody was used as a tracer in the Gal-3-TRFIA to detect Gal-3 in the serum. The Gal-3-TRFIA assay showed a clear linear relationship between the concentration of the analyte and the fluorescence count over a wide range of detection. The detection range of the Gal-3-TRFIA assay was 0.85-1000 ng/mL. The Gal-3 intra-and inter-batch coefficients of variation were 3.45% and 5.12%, respectively. The correlation coefficient (R) between the Gal-3-TRFIA assay and commercially available enzymelinked immunosorbent assay kits was 0.83086. KIM-1 [19,20] and TIM-3 [21] were also found to be elevated in a variety of nephropathy and were selected as potential interferers for the test, which showed no effect on the measured Gal-3 concentration. Serum Gal-3 levels are negatively correlated with renal function [22]. However, the role of Gal-3 in IMN has not been reported yet. Therefore, the established Gal-3-TRFIA method was used to detect the levels of Gal-3 in patients with IMN and normal subjects. The results of the study showed that Gal-3 concentrations were higher in the serum of patients with IMN.
Idiopathic membranous nephropathy is the most common cause of nephrotic syndrome [10]. Early diagnosis of IMN mainly depends on renal biopsy [10]. Given that the PLA2R is now widely accepted as the primary target for IMN, [23,24] detection of anti-PLA2R antibodies in the blood and PLA2R antigen in renal biopsy is a definitive diagnosis [25]. PLA2R targets IMN with a positive rate of 70%-80% [10]. Other indicators are expected to achieve better assessment of disease progression. Therefore, we studied the value of Gal-3 in IMN and found that it clearly distinguish healthy subjects from patients with IMN, and the concentration of Gal-3 was correlated with the degree of kidney injury. In a study of the activation of acute inflammatory responses in patients with nephrotic syndrome, the level of CRP in patients with MN  (18) were higher than those in healthy controls [26]. However, our results showed that CRP concentration was only slightly higher in patients with IMN (4.14 mg/L) than in healthy subjects (0.68 mg/L). Moreover, CRP level was not associated with the degree of pathological injury as well as urinary protein and creatinine levels in patients with IMN. In our results, no significant correlation was found between Gal-3 and PLA2R-Ab levels. Different production mechanisms may be responsible for the uncorrelation between Gal-3 and PLA2R-Ab concentrations. PLA2R as a specific intrinsic podocyte antigen bind to its corresponding autoantibodies and leads to the formation of glomeruli subepithelial immune complex deposits in IMN [27]. Initial PLA2R-Ab production tends to precede proteinuria by weeks/months, and the disappearance of PLA2R-Ab antibodies also precedes clinical remission by weeks/months [28]. However, Gal-3 is widely involved in renal inflammation and fibrosis, and it is readily secreted from inflammatory cells and injured cells [2]. It is speculated that Gal-3 concentration will gradually increase with the severity of the disease after the occurrence of IMN. The results also confirmed that the concentration of Gal-3 increased with the stage of IMN.
To our knowledge, this is the first prospective study to examine the relationship between Gal-3 and IMN progression. Serum Gal-3 concentration gradually increased with the progression of IMN staging. This is of certain significance to judge the disease course of patients with IMN.

Conclusions
A reliable immunoassay with a wide detection range was established for Gal-3. Serum Gal-3 concentrations in patients with IMN were higher than those in healthy controls and had certain value in evaluating the renal function of patients with IMN.
Authors' Contributions Each of the authors has contributed to the article. Xiaomei Yu and lingli Chen were in charge of doing the experiment and completing the study. Xue Yang, Xiaobing Liu, Yueming Liu and Bo Lin were responsible for providing serum samples and clinical data. Xiumei Zhou, Yigang Wang, Pengguo Xia, Yuan Qin, liang Wang, Zhigang Hu and Biao Huang made many suggestions on the conception and revision of the article, and guided the experiment for many times. The writing of the article is mainly done by Xiaomei Yu.

Data Availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
Code Availability Not applicable.

Declarations
Ethical Approval The study was approved by the institutional research ethics committee of Jiangsu Institute of Nuclear Medicine. All enrolled subjects provided their written informed consent for study participation, and all methods were performed in accordance with the relevant guidelines and regulations. The research project was approved by Wuxi People's Hospital Affiliated to Nanjing Medical University. Ethic number is ky12016001.

Consent to Participate
Informed consent was obtained from all individual participants included in the study.

Consent for Publication
Patients signed informed consent regarding publishing their data and photographs.

Conflicts of Interest
Not applicable.