Part Ⅰ Development Of A Europium Nanoparticles Lateral Flow Immunoassay For NGAL Detection in Urine And Diagnosis Of Acute Kidney Injury

Abstract

1. Background

AKI is related to severe adverse outcomes and mortality in Coronavirus Disease 2019 (COVID-19) patients, so early diagnosis and intervention are imperative. Neutrophil gelatinase-associated lipocalin (NGAL) is one of the most promising biomarkers for the detection of acute kidney injury (AKI), but current detection methods are inadequate, so more rapid, convenient, and accurate methods are needed to detect NGAL for early diagnosis of AKI. Herein, we established a rapid, reliable, and accurate lateral flow immunoassay (LFIA) based on europium nanoparticles (EU-NPS) for the detection of NGAL in human urine specimens.

2. Methods

A double-antibody sandwich immunofluorescent assay using europium doped nanoparticles were employed and the NGAL monoclonal antibodies (MAbs) conjugate as labels were generated by optimizing electric fusion parameters. Eighty-three urine samples were used to evaluate the clinical application efficiency of this method.

3. Results

The quantitative detection range of NGAL in AKI was 1-3000 ng/mL, and the detection sensitization was 0.36 ng/mL. The coefficient of variation (CV) of intra-assay and inter-assay were 2.57-4.98 % and 4.11-7.83 %, respectively. Meanwhile, the correlation coefficient between europium nanoparticles-based lateral fluorescence immunoassays (EU-NPS-LFIA) and the ARCHITECT analyzer was significant (R2 = 0.9829, n = 83, p < 0.01).

4. Conclusions

Thus, a faster and easier operation quantitative assay of NGAL for AKI has been established, which is very important and meaningful to diagnosing early AKI, suggesting that the assay can provide an early warning of the outcome of the disease.

Keywords

neutrophil gelatinase-associated lipocalin (NGAL), monoclonal antibody, lateral flow immunoassay, acute kidney injury (AKI), Cistanche's effects.

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Introduction

Coronavirus Disease 2019 (COVID-19) has widely spread on a worldwide scale with serious disasters [1]. Acute kidney injury (AKI) has a higher rate of morbidity and mortality in common complications for critical illnesses and counted about 5-7 % of hospitalized patients in the world[2].- Several studies have evaluated the development of AKI as more strongly related to worse outcomes and mortality rates of COVID-19, describing the 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 containing the progression of COVID-19 hospitalized patients[4]. Among them, neutrophil gelatinase-associated lipocalin (NGAL) has been recognized as one of the promising biomarkers candidates for the detection of AKI. NGAL is a 25 kDa glycoprotein associated with gelatinase from neutrophil and usually exist at a lower level in human tissues such as the stomach, colon, and kidney, but its expression is dramatically increased in serum and urine when the kidney was with ischemic or nephrotoxic injury[5].

Lateral flow immunoassays (LFIA) have been regarded as desired screening assays on account of their simplicity, in-situ analysis, and ease of work[6]. The LFIA with fluorescent microparticles has already been used for the 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 nano labels, 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 is a long fluorescence lifetime and is also available with an average particle size of 75–100 nm range, the large Stokes shift which is usually over 200 nm is conducive to avoiding the interference of scattered light caused by measuring excitation light. EU-NPS have a wide excitation band so 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 the urine based on two monoclonal antibodies (MAbs) 1G1 and 2F4 which are discovered by our lab. The method is a 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 the EU-NPS-1G1-antigen complex in T-line. Our results showed that EU-NPS-LFIA could be used for early NGAL detection in urine and allow improvement in the treatment of AKI patients.

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Methods

1. Expression and purification of NGAL

The human NGAL gene sequence from Genbank (NP_ 005555.2) was synthesized by the 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 to 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).

2. 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 intraperitoneal injections spaced 21 days. The serum samples were collected one week after the third injection, and the titer of the antiserum was determined with an indirect enzyme-linked immunosorbent assay (ELISA)[12]. Three days before fusion, mice performed 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 in the literature[13]. Briefly, the mixed cell was washed twice with 10 mL electrofusion buffer (0.3 M mannitol, 0.1 mM CaCl2, 0.1 mM MgCl2, pH 7.2), and re-suspended at a concentration of 2 × 107 cells/ mL. The fusion was completed using an alternating current voltage of 50 V at 0.8 MHz for 20 s, a 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 % CO2 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 of fusion, and the calculated number of hybridoma clones was. The BALB/c mice which were injected with paraffin oil in advance were inoculated with 1 × 106 of NGAL hybridoma cells, the ascites were purified by Protein A column.

3. 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 was determined with the 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 was 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 from 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 were analyzed in 12 % SDS-PAGE under non-reducing conditions and reducing conditions. Briefly, the 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 a PVDF membrane that was activated by soaking in methanol for 15 s, and then subjected to the electrophoresis conditions at 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, the 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).

4. Competitive enzyme-linked immunosorbent assays (ELISA)

Two MAbs were tested for the ability to recognize unique epitopes on NGAL by cELISA. MAb was conjugated with HRP by using the HRP Antibody Labeling Kit (Shanghai YSRIBIO industrial co., LTD), the working concentration of which was tested through direct ELISA. 96-well Microtitre plates were coated with 2 µg/ mL of NGAL antigen 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 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 a Bio-Rad microplate reader (Bio-Rad Laboratories, Inc). The blocking effects of MAbs were calculated by using the following equation: 100×[1-OD_{450 nm} of (HRP-MAb + MAb)/OD450 nm of HRP-MAb]. Two MAbs recognized different epitopes if blocking effects were observed in more than 40 %.

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5. Conjugation of EU-NPS

The Anti-NGAL monoclonal antibodies 1G1 and 2F4 were covalently conjugated to EU-NPS with the standard procedure of Bangs Laboratories. Briefly, 100 µL EUNPS 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-ethyl carbodiimide hydrochloride (EDC). The activated EUNPS was 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 europiumconjugated 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℃.

6. The development of LFIA

The LFIA strips consisted of a 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 was dried for 48 h at 45℃ before being tested. The different EU-NPS-1G1 and EU-NPS-2F4 conjugate particles were coated on the conjugate pad, and the fluorescence signal was measured by an immunofluorescent analyzer (Guangzhou Labsim Biotech Co., Ltd).

7. Urine sample collection and patients

A total of 83 Human urine samples from AKI patients were harvested from the Affiliated Hospital of Jilin Medical University. The ethical guidelines were strictly complied with in the experiment and were 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 by the Declaration of Helsinki Ethical Principles. The average age of patients 62 years ranged from 20 to 80 years who were not infected by COVID-19, and AKI stage 1–3 were 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). To minimize potential confounding factors, urine samples were analyzed rapidly at the 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 sample 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 an ARCHITECT urine NGAL reagent Kit (Lisnamuck, Longford, Co. Longford, Ireland) utilizing a non-competitive, sandwich format with chemiluminescent signal detection. Urinary NGAL was recognized by an antifungal antibody which was covalently attached to paramagnetic particles in microparticle reagent, and the conjugate of a 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.

8. 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 reactions of bioconjugate, each concentration did 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) differences and math sample standard matrix effects[15]. The standard curve was plotted against each concentration of NGAL and HT/HC ratio.

9. Statistical analysis

The Passing-Bablok regression analysis and BlandAltman plot were performed by analysis of variance (ANOVA) of MedCalc and SPSS 17.0 software. All data were shown as mean values with standard deviation (mean ± S.D. ).

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Moli Yin1 , Yuanwang Nie2 , Hao Liu2 , Lei Liu1 , Lu Tang1 , Yuan Dong2 , Chuanmin Hu1 and Huiyan Wang1

1 Jilin Collaborative Innovation Center for Antibody Engineering, Jilin Medical University, 132013 Jilin, PR China.

2 Academy of Laboratory, Jilin Medical University, 132013 Jilin, PR China.