Background
Coronavirus Disease 2019 (COVID-19) has widely spread in the worldwide scale with serious disaster[
1].Acute kidney injury (AKI) has a higher rate of morbidity and mortality in common complication for critical illnesses and counted about 5-7 % of hospitalized patients in world[
2].Several studies have evaluated the development of AKI is more strongly related to worse outcomes and mortality rates of COVID-19, described incidence of AKI that ranges widely from 0.5 to 36.6 % in COVID-19 patients[
3].Early detection and precise treatments of AKI can implement better preventive strategies and prevent deterioration of renal function and renal failure, effectively contain progression of the COVID-19 hospitalized patients[
4]. Among them, neutrophil gelatinase-associated lipocalin (NGAL) has been recognized as one of the promising biomarkers candidate for detection of AKI. NGAL is a 25 kDa glycoprotein associated with gelatinase from neutrophil and usually exist at lower level in human tissues such as stomach, colon and kidney, but its expression is dramatical increased in serum and urine when the kidney was with ischemic or nephrotoxic injury[
5].
Lateral flow immunoassays (LFIA) has been regard as desired screening assays on account of simplicity, in-situ analysis and easy to work[
6]. The LFIA with fluorescent microparticles have already been used for detection of various microbial pathogens and several inflammation markers[
7,
8]. Several novel nanoparticles have been generally applied to improve the sensitivity of LFIA, including carbon nanoparticles, quantum dots, fluorescent dyes, magnetic nanolabels and europium nanoparticles (EU-NPS)[
9]. EU-NPS as carriers can improve 100-fold sensitivity contrast with colloidal gold nanoparticles labeled in LFIA[
10]. EU-NPS are long fluorescence lifetime and also available with an average particle size of 75–100 nm range, the large Stokes shift of which is usually over 200 nm is conducive to avoid the interference of scattered light caused by measuring excitation light. EU-NPS have wide excitation band so that it is beneficial to increase the excitation energy, the sharp emission peak, low background and high resolution, used in sandwich-type immunoassays of medical diagnostics recently[
11].
Here, we established a new method with EU-NPS as labels of LFIA for the rapid, sensitive and early measurement of NGAL in urine based on two monoclonal antibodies (MAbs) 1G1 and 2F4 which are discoveried by our lab. The method is double-antibody sandwich immunofluorescent assay using EU-NPS and MAbs conjugate as labels. The mAb 1G1 was conjugated with EU-NPS and the mAb 2F4 was used to capture EU-NPS-1G1-antigen complex in T-line. Our results showed that EU-NPS-LFIA could be used for the early NGAL detection in urine and allow improvement in the treatment of AKI patients.
Methods
Expression and purification of NGAL
The human NGAL gene sequence from Genbank (NP_005555.2) was synthesized by Beijing Institute of Genomics (BGI) and added restriction enzymes HindIII and XhoI at both ends. The plasmids were digested with HindIII and XhoI, and then cloned into the pSecTag2A vector. The constructed plasmids were sequenced to confirm without mutation, and then transformed into Chinese Hamster Ovary (CHO) cells by the LipofectamineTM2000. After 8 days, the cell supernatant was collected after filtering 0.45 μm filter. The expressed NGAL-6×His protein was purified with the Nickel Nitrilotriacetic Acid (Ni-NTA) column, and the different fractions were collected and appraised by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bicinchoninic acid (BCA) Protein Quantification Kit measured the concentration of protein.
The purified recombinant protein was subjected in 12 % SDS-PAGE, then adsorbed onto a polyvinylidene fluoride (PVDF) membrane. After the membrane was blocked in tris buffer with 1 % Tween-20 (TBST) solution containing 5 % skimmed milk at room temperature (RT) for 2 h, incubated with HRP-conjugated anti-6×His tag antibody (1:5000) in the dark for 1 h at RT, visualized with Benzidine after washed with TBST and visualized by Bio-Rad Western blotting detection system (DNR Bio-Imaging Systems Ltd., Israel).
Generation and purification of MAbs
In the first immunization, the female BALB/c mice (age of 6 weeks, a total of 10) were immunized with 50 µg NGAL-6×His recombinant protein emulsified in equal dosage of complete Freund’s adjuvant, and accessional immunized were accomplished with protein emulsified in incomplete Freund’s adjuvant. Two hypodermic injections on the back of mice and subsequent were intraperitoneal injections spaced 21 days. The serum samples were collected one week after the third injection, and the titre of antiserum was determined with indirect enzyme-linked immunosorbent assay (ELISA)[
12]. Three days before fusion, mice were performed to booster immunization with 50 µg of NGAL-6×His diluted with 0.9 % NaCl.
The isolated immune mice spleen cells and the SP2/0 myeloma cells mixed at a ratio of 3:1 to fuse in a platinum electrode LF498-3 fusion chamber (BEX Co., Ltd, Japan) as described literature[
13]. Briefly, the mixed cell was washed twice with 10 mL electrofusion buffer (0.3 M mannitol, 0.1 mM CaCl
2, 0.1 mM MgCl
2, pH 7.2), re-suspended at a concentration of 2 × 10
7 cells/mL. The fusion was completed using an alternating current voltage of 50 V at 0.8 MHz for 20 s, direct current pulse voltage of 450 V of 2 repetitions for 0.5 s, and post-fusion was 50 V at 0.8 MHz for 7 s. Finally, the electric-treated cell suspension was moved from the fusion chamber into 4.5 mL of preheated RPMI 1640 (20 % fetal bovine serum) for 30 min at 37℃, then cultured in 96-well plates and incubated with 5 % CO
2 at 37℃. After 24 h, hypoxanthine-aminopterin-thymidine (HAT) was supplemented to each well. The Cell culture supernatants were screened by ELISA after 9 days fusion, and calculated number of hybridoma clones. The BALB/c mice which injected with paraffin oil in advance were inoculated with 1 × 10
6 of NGAL hybridoma cells, the ascites were purified by Protein A column.
Identification of MAbs
The immunoglobulin subclasses of antibodies were analyzed using the antibody subclass identification kit. The indirect ELISA screened the specific MAbs by using purified recombinant NGAL-6×His protein and PCT-6×His protein. The interaction between antigen and antibodies were determined with BIAcore T200 system (GE Healthcare, Stockholm, Sweden) in HBS-EP buffer (0.005 % surfactant P20, 10 mM Hepes, pH 7.4, 3 mM EDTA, 150 mM NaCl). The NGAL antigen was adsorbed on CM5 biosensor chips reaching 400–480 response units (RU) by an amine coupling kit. The antibodies (2F4 and 1G1) were diluted in HBS-EP buffer were slowly passed over the chip with 50 µL/min for 5 min, respectively, and subsequently HBS-EP buffer injected over the chip to monitor the dissociation phase for 4 min. The sensor chips were regenerated with Glycine solution (pH 3.0) following the dissociation phase. For each analyte passed over the chip, the specific responses from the antigen flow channel could subtract non-specific responses for the control flow channel. The fitted saturation binding curves were plotted based on concentrations of analyte for equilibrium binding responses to calculate KD.
Purified anti-NGAL MAbs was analyzed in 12 % SDS-PAGE under non-reducing conditions and reducing conditions. Briefly, sample was mixed with 5 x non-reducing buffer or 5 x protein loading buffer and loaded onto 12 % SDS-PAGE. The specificity of anti-NGAL MAbs was determined by Western Blot. The NGAL proteins were done to 12 % SDS-PAGE, then adsorbed onto PVDF membrane that activated by soaking in methanol for 15 s, and then subjected to the electrophoresis conditions in 100 V for 2 h. After blocking in Tris-HCl buffer with 1 % Tween-20 solution (TBST) containing 5 % skimmed milk at RT for 2 h, the membrane was incubated with mouse anti-NGAL MAbs as the primary antibodies at 4 °C. The next day, membrane was washed with TBST and incubated with anti-mouse conjugated HRP IgG in the dark for 1.5 h at RT. Finally, the membrane was visualized by the Western blot detection system of enhanced chemiluminescence (ECL).
Competitive enzyme-linked immunosorbent assays (cELISA)
Two MAbs were tested for the ability to recognize to unique epitopes on NGAL by cELISA. MAb was conjugated with HRP by using HRP Antibody Labeling Kit (Shanghai YSRIBIO industrial co., LTD), and the working concentration of which was tested through direct ELISA. 96-well Microtitre plates were coated with 2 µg/mL of NGAL antigenin overnight at 4 °C, then unlabeled MAb (0.2 µg/ well, 2 µg/ well) was competitively bound with the optimal dilution ratio of HRP labeled MAb and incubated for 1 h at 37 °C. The enzymatic reaction was appeared with hydrogen peroxide by substrate 3,3’,5,5’-Tetramethylbenzidine (TMB) and stopped by 2 M sulfuric acid to all wells. The absorbance (OD450 nm) was determined by Bio-Rad microplate reader (Bio-Rad Laboratories, Inc). The blocking effects of MAbs was calculated by using the following equation: 100×[1-OD450 nm of (HRP-MAb + MAb)/OD450 nm of HRP-MAb]. Two MAbs recognized different epitopes if blocking effects observed more than 40 %.
Conjugation of EU-NPS
The Anti-NGAL monoclonal antibody 1G1 and 2F4 were covalently conjugated to EU-NPS with standard procedure of Bangs Laboratories. Briefly, 100 µL EU-NPS were added to 900 µL 0.05 M MES (pH 7.0) and dispersed by ultrasound, vibrated for 15 min at RT in the presence of 0.08 M N-hydroxysulfosuccinimide (NHS) and 0.05 M 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). The activated EU-NPS ware washed with coupling buffer (0.05 mM H3BO3, 0.04 mM Na2B4O7, pH 7.5) and reacted with 0.3 mg antibody for 2.5 h at RT. The europium-conjugated compound was incubated in 1000 µL blocking buffer (10 % BSA, 20 % tween-20, 0.05 M Tris-HCl) for 1 h, added to 1000 µL stock solutions (10 % BSA, 20 % trehalose, 20 % tween-20, 0.05 M Tris-HCl ) to store at 4℃.
The development of LFIA
The LFIA strips were consisted of nitrocellulose membrane, conjugate pad, sample pad and absorbing pad. The glass fiber membranes were soaked in the blocking buffer (20 % trehalose, 10 % BSA, 20 % tween-20, 0.05 M Tris, 3.2 mM EDTA.Na2, pH 8.6) for 1.5 h. The concentration of 1 mg/mL MAbs (2F4 or 1G1) was coated on the test line (TL) of nitrocellulose membrane, and goat-anti-mouse IgG 1 mg/mL was coated on the same NC membrane at a distance of 4 mm to form a quality control line (CL), the spray volume of dispenser instrument was set at 1 µL/cm. The membrane and glass fiber mat were dried for 48 h at 45℃ before tested. The different EU-NPS-1G1 and EU-NPS-2F4 conjugate particles were coated on the conjugate pad, and fluorescence signal was measured by immunofluorescent analyzer (Guangzhou Labsim Biotech Co., Ltd).
Urine sample collection and patients
The total of 83 Human urine samples from AKI patients were harvested from Affiliated Hospital of Jilin Medical University. The ethical guidelines were strictly complied in the experiment, was provided by the Affiliated Hospital of Jilin Medical University (No.2018-LW029). All subjects received oral and in written informed consent in Chinese for the study of urine samples. All experiments were performed in accordance with the Declaration of Helsinki Ethical Principles. The average age of patients was 62 years ranging from 20 to 80 years who were not infected by COVID-19 ,and AKI stage 1–3 was classified according to Kidney Disease Improving Global Outcomes AKI criteria[
14]. Urine samples were collected for NGAL analysis up to 12 h before AKI was diagnosed and at frequent intervals after operation at various time points (12, 24, 48 h). For minimize potential confounding factors, urine samples were analyzed rapidly at clinical chemistry laboratory including urine biochemistry (total protein concentrations < 1 g/dL, urea < 12 g/dL, glucose < 1 g/dL, Urine Creatinine < 1 g/dL, albumin < 2.5 g/dL, pH 4.5-9.0) and carried out in duplicates. Within 30 min of samples collection, they were centrifuged at 3000 rpm at 4℃ for 15 min. A minimum of 100 µL of supernatant was dispensed into sterile containers and stored at -80℃ for further analyses, to avoid repeated freeze-thaw cycles.
The measurement of NGAL levels was performed using ARCHITECT urine NGAL reagent Kit (Lisnamuck, Longford, Co. Longford, Ireland) utilized a non-competitive, sandwich format with chemiluminescent signal detection. Urinary NGAL was recognized by anti-NGAL antibody which was covalently attached to paramagnetic particles in microparticle reagent, and the conjugate of second anti-NGAL antibody associated with acridinium. Following the manufacturer’s instructions, the calibration assay was carried out in the range of 0-1500 ng/mL and the concentration of NGAL was measured.
Clinical sample testing and analysis
The serial concentrations of NGAL standards antigen (10, 50, 100, 200, 400, 800, 1500, 2000 and 3000 ng/mL) were prepared by using FBS to strengthen specific reaction of bioconjugate, each concentration done three replicates. After the clinical samples were added onto the sample pads, the results of fluorescence intensity on the T line (HT) and the C line (HC) were recorded by the reader. Quantitative detection was completed by the HT/HC ratio to effectively eliminate strips (T and C) difference and matrsample standard matrix effects[
15]. The standard curve was plotted against each concentration of NGAL and HT/HC ratio.
Statistical analysis
The Passing-Bablok regression analysis and Bland-Altman plot were performed by analysis of variance (ANOVA) of MedCalc and SPSS 17.0 software. All data were showed as mean value with standard deviation (mean ± S.D. ).
Discussion
The EU-NPS-LFIA for detection of NGAL in human urine and diagnosis of AKI has been developed in our study. Current diagnosis of AKI is confirmed by the concentration of serum creatinine (sCr), which is steady unless at least 50 % of damaged kidney function[
19]. AKI developed in 89 % of severe patients with COVID-19, and a majority of patients had predominantly oliguria when appears earlier than plasma Creatinine[
20]. Biomarkers increased elevated levels at admission, which associated with increased mortality. NGAL is one of these biomarkers, levels of which may positive correlation with risk for mortality in COVID-19 hospitalized patients[
21]. In AKI, several studies have discovered NGAL concentration in urine is significantly associated with sCr concentration[
22]. In recent years, various electrochemical and immunological methods have been developed for the detection of NGAL, instance of electrochemical determination, solid-phase proximity ligation assay and enzyme-free electrochemical immunoassay[
23,
24]. However, these techniques require precise equipment, specialized personnel and professional interpretation of the results. Therefore, to develop an effective and convenient detection assay for NGAL concentration in the urine is critical for this disorder monitoring. The low efficiency of polyethylene glycol (PEG) fusion in conventional methods to causes difficulties in obtaining functional antibodies, we optimized electric fusion parameters that enabled enhancement of fusion efficiency to prepare of viable hybridomas, and obtained two anti-NGAL MAbs. MAbs are not only high affinity, but also directed towards different epitopes, used to establish high quality of diagnostic assays.
In recent years, many studies have been reported to develop several NGAL rapid diagnostic immunoassays, including the photoelectrochemical immunosensor and three electrochemical immunosensors, the solid-phase proximity ligation assay and lateral flow assay[
25]. In one study, immunosensor has been made by NGAL capture antibodies immobilized to screen-printed-modified carbon electrode and labeled addition of secondary antibody against NGA to PB-NP-decorated g-C
3N
4 nanosheets forming a sandwich on the SPCE[
24]. The other study developed antibody against NGAL was immobilized on a screen printed electrode (SPCE) modified with electropolymerized aniline deposited on top of an electrosprayed graphene/poly-aniline (G/PANI)[
25]. Two reports showed the LOD varied widely from 0.6 pg/mL to 21.1 ng/mL, depending on the g-C
3N
4 nanosheets with N element enhanced electrocatalytic efficiency of the nanohybrids than graphene nanosheets, and the LOD of the NGAL assay varies depending on immunosensor, conjugated-complex and antibodies[
26]. According to those studies, we enlightened that sensitivity of LFIA could be improved by basing on different fluorescence nanoparticles mentioned in the preamble section.The high sensitivity nanoparticles labeled in LFIA as fluorescent probes has been continually researched over past decades, gold nanoparticles are the most widely used, but the application of the lateral flow tests based on traditional labels is limitedfor there poor identification and weaker the signal[
27]. Currently there are antibodies available for the development of NGAL have been applied to different platforms for NGAL detection, but not available in LFIA based on EU-NPS labels experimented on the detection of NGAL in addition to UCP technology-based lateral flow assay [
28,
29].
Fluorescence immunoassays have advantange in various detection fields, especially in mature quantum dots, that sensitivity was influenced by the MAbs and fluorescent materials, and could greatly improve by EU-NPS and specific MAbs to use the fluorescent probes[
30]. The EU-NPS were applied to fluorescence LFIA strips had matured in the detection field, and enhanced several advantages of LFIA for assay sensitivity, specificity and stability[
31]. Due to excellent properties of the EU-NPS that the limit of detection for NGAL in this method was 0.36 ng/mL, while the mean urine NGAL levels was about 7.0 ng/mL in healthy individuals[
32]. The developments and application about the EU-NPS-LFIA of NGAL are still in initial phase, this assay need to test NGAL of serum and urine samples with COVID- 19 patients for which improvement in the treatment in the future of works.
Conclusions
In the study, we successfully got two mouse anti-NGAL MAbs (2F4, 1G1) and were labeled with EU-NPS to establish the lateral flow immune technique. The EU-NPS-LFIA was found to detect NGAL in a wide range of 1-3000 ng/mL within 15 min, the detection sensitivity reached 0.36 ng/mL. These anti-NGAL MAbs could be reliability utilized in fluorescence LFIA of NGAL detection in the urine samples, so that it should be applicable in the AKI diagnosis.
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