Development and Evaluation of Europium-Based Quantitative Lateral Flow Immunoassay for the Chronic Kidney Disease Marker Cystatin-C

This study aimed to establish a Europium label time-resolved fluorescence immunoassay (TRFIA) to detect the chronic kidney disease (CKD) biomarker Cystatin-C. An Europium based Time resolved fluorescence immunoassay was developed to detect the concentration of Cystatin-C in a urine sample to increase the sensitivity with captured anti-Cystatin-C antibodies immobilized on nitrocellulose membrane and then bonded with detection anti-Cystatin-C labelled with CM-EU, followed by fluorescence measurement using time-resolved fluorometry in 15 min. The performance of this TRFIA was evaluated using the clinical urine serum and compared with the ELISA assays. The linear calibration range was 0.015–32 µg/ml, and the limit of detection (LOD) quantified was 0.0001 µg/ml. This current work has improved the LOD of our previous work from 0.013 µg/ml to 0.001 µg/ml. These results indicated that the CM-EU nanoparticle-based LFIA is rapid, more sensitive, reliable, and reproducible for point-of-care testing of Cys-C concentrations in urine.


Introduction
Glomerular filtration rate (GFR) is the most valuable estimate of kidney function and is crucial for the detection and treatment of acute renal failure (ARF) and chronic kidney disease (CKD). Kidney function loss due to decreased estimated glomerular filtration rate (eGFR) is related to short survival. Cystatin-C (Cys-C) is an important biomarker for eGFR determination that is not dependent on muscle mass and relatively constant with age and gender [1]. Cys-C is a 13 kDa protein. It is excreted in only small amounts in urine, filtered by the glomeruli, and absorbed and catabolized by the epithelial cells present in the proximal tubule [2]. The range for Cys-C is 0.67-6.15 mg/l in urine [3]. Serum levels are 0.80 ± 0.21 (range from 5.7-11.2 µg/ml) while it is increased up to 21 ± 1.1 µg/ml in patients in kidney diseases [2,4]. Cys-C is measured in urine using particle-supported turbidimetric or nephelometric immunoassay, photoelectrochemical, fluorescence, near-infrared spectrometry proteomics-based methods, and microfluidics [5][6][7][8][9][10][11][12][13]. Lateral flow immunoassay is the most rapid test for detecting Cys-C levels, the organic dye-based lateral flow assay. The organic dye Alexafluor-647 often had very high background fluorescence measured due to the membrane scattering. The small stokes shifts of Alexafluor worsen the interferences coming from light scattering and sample matrices. Although the technology has a remarkable performance, it still has some disadvantages, such as low detection sensitivity [14].
The time-resolved fluorescence (TRF) detection method shows the capacity to offer higher than conventional organic dye-based fluorescence because of its low background noise [15,16]. Dye-based methods utilize optical filters to dissociate the fluorescence from background light using wavelength differences. The specific fluorescence properties of TRF show high sensitivity, no background fluorescence, significant stokes shift, dissociation enhancement. This method excites a fluorescent label with a short pulse of light and then waits for a while for the background and other membrane fluorescence to get off a low level and then collects the fluorescent signal. This method eliminates the scattered light signals from the blood and can read only the fluorescence from the label. In addition, by removing the autofluorescence from the membrane, time-resolved methods increase the detection sensitivity over conventional fluorescence [17].
The time-resolved fluorescence detection technique is mainly utilized for biological assays using lanthanide chelates Europium (EU) as probes [18] due to its photostability. In addition, carboxylic acid groups on the EU nanoparticle enhance the conjugation efficiency of the protein by increasing the stability of the reporter. To date, EU (III) chelateddyed nanoparticles are widely used in various immunoassays and lateral flow immunoassays [19]. However, using EU (III) polystyrene chelate microparticles, all quantitative results in LFIA were based solely on the fluorescence signals of the test line. Therefore, this study used carboxylate-modified polystyrene (CM-EU) microparticles to label Cys-C-LFIA fluorescence in urine samples.
This article reports a Europium label time-resolved fluorescence immunoassay (TRFIA) to detect the chronic kidney disease (CKD) biomarker Cystatin-C. We have used commercial Europium particles as probes to demonstrate the uniqueness of the technology. The technology should perform several applications, ranging from clinical diagnostics, food and environmental monitoring. Therefore, our strategy offers excellent potential for further development for rapid detection at the treatment site and clinical application. Labelling of the Europium with the Biomolecules 2 mg of CM-EU was dissolved in 1 ml of activating buffer (25 mM MES, pH 6.1) consisting of 10 mM of sulfo-NHS and 1.25 mM of EDC. Mixtures were shaken at 250 rpm for 60 min at room temperature. After that, the solution was centrifuged at 14,000 rpm for 25 min to remove the excess NHS and EDC. The activated CM-EU was washed and resuspended with 25 mM Phosphate Buffer (PB) using the water bath sonicator. For the conjugation, 50 µg/ml of anti-Cys-C mAb was gently mixed with the activated CM-EU, overnight. The unconjugated antibody was removed by centrifugation at 14,000 rpm for 15 min at 8 °C. After washing thrice, 1 ml of blocking buffer (25 mM PB, 2% casein, pH 7.4) was added and incubated for one hour. After incubation, the conjugate was washed thrice. Finally, the conjugates were suspended in 25 mM PB, 0.5% BSA, 2.5% Trehalose, and 10% sucrose) to the concentration of 10 µg/ml and stored at 4 °C.

Preparation of Europium Based LFIA Strip
The Europium-based test strip consists of a sample, conjugate, nitrocellulose membrane (NC), and absorbent pad. The sample and conjugate pad were pre-treated with sample pad and conjugate pad treatment buffer (1 × Tris-Casein + 0.25% Tween-20). The conjugates were diluted and mixed in the 1 × Tris-Casein to the final concentrations of 0.2, 0.3, 0.4, 0.5 ng/ml. Then the mixture of conjugates was dispensed onto the pre-treated conjugate pad, and the casein with CM-EU was used at 1 mg/ml and then dried at 37 °C overnight. Next, anti-Cys-C (0.5 mg/ml) and anti-mouse IgG (1 mg/ml) were dispersed on an NC membrane on the test and control line. After that, the NC membrane was then dried at 37 °C for two hours and stored at the desiccator. Finally, the whole assembly was cut into a 3.1 mm-wider and placed in cassettes. The strips were stored in a desiccator at room temperature until use.

Fluorescence Lateral Flow Assay Procedure
First of all, 80 μl of the different concentrations of standards in the chase buffer (0.1 m Tris-Casein + 0.125% Tween-20) was incubated at room temperature for 15 min. Then the mixture is dropped in the sample port after 15 min reaction. Next, the test strip cartridge was inserted and scanned using the IQuant TRF reader. Finally, the V T (Volume Test line ) and V C (Volume Control line ) were measured, and the ratio of V T / V C was calculated. A schematic illustration of the EU (III) chelate nanoparticle-based LFIA procedure is shown in (Fig. 1).
Quantitative data was obtained from the IQuant-TRF [19] Immunoanalyzer (developed by Healthcare Technology Innovation Center, IIT Madras). The instrument scanned the intensity of the test and control line NC to calculate the volume ratio. The device was developed by Karthik et al. [19].

Accuracy Assay
The assay precision was done by analyzing the low, medium, and high concentrations of specimens. In addition, intraand interassay variations were obtained from 10 independent experiments.

Recovery Assay
Recovery was made by adding various concentrations of Cys-C to three clinical urine specimens. The recovery was expressed as a percentage of the amount added. The equation was as follows: Recovery (%) = (measured concentration / enriched concentration) × 100.

Statistical Analysis
The anti-Cys-C analytical curve was obtained by plotting the V R vs. the concentration of Cys-C. The curves were obtained using Sigmaplot best fitting curve results. Microsoft Excel was utilized to calculate sample means and the standard deviations (SD). We used the mean concentration plus 3 standard deviations (SD) of the blank assayed in duplicate in 20 independent measurements as the definition of the limit of blank (LOB). The sensitivity was determined by using both the measured LOB and test replicates of a sample known to contain a low concentration of Cys-C (limit of detection [LOD]: LOD = LOB + 1.645 [SD low concentration sample ]).

Principle of the Method
The developed CM-EU-based LFA to determine anti-Cys-C levels was performed as a sandwich-based lateral flow fluorescent immunoassay, as illustrated in (Fig. 1). First, the assay buffer containing Cys-C protein was added to the sample pad. Then, the CM-EU -anti-Cys-C conjugates bind to the Cys-C protein and flow across the NC membrane, which was reacted with the anti-Cys-C capture antibody on the test line, resulting in a fluorescent band on the test, and for the control line, anti-mouse IgG was coated, which binds with the remaining conjugates with the Europium binds to give The LFA strip photograph for the 0.06, 0.12, 1, 4, 8, 16, and 32 µg/ml of Cys-C the fluorescent in the control lines, respectively. Once the reaction is completed, the test strip data was acquired from the IQuant TRF reader by measuring the peak volume of the test line and the control line (Fig. 1). As it is a sandwich assay system, anti-Cys-C mAb with CM-EU conjugates form a sandwich with Cys-C in the sample and the anti-Cys-C, coated in the test line. Therefore, the fluorescence intensity at the test line is directly proportional to the concentration of anti-Cys-C in the sample. Next, the V T /V C ratio was used for the measurements and made it more reliable for the analytical sensitivity of clinical applications.
The analytical curve for the LFIA was plotted by a series of different concentrations of anti-Cys-C standards (0.06, 0.12, 1, 4, 8, 16, and 32 µg/ml) in sample buffer. A standard curve was obtained after recording the fluorescence intensities plotting the V R against the concentration using the equation: y = 0.0022x 2 + 0.1928x + 0.0478, (r = 0.9952) and in (Fig. 2). For each concentration, the coefficients of variation (CVs) recorded were less than 10%. The mean of five replicates was recorded using 10 blanks to estimate the LOD. The LODs were recorded from 0.001 μg/ml. After establishing the CM-EU-nanoparticle method, a lower LOD was recorded than the Alexa fluor-647 method (0.023 µg/ml).

Optimization of the Conjugation of CM-EU Nanoparticle with the Anti-Cys-C Antibody
CM-EU is used as the carrier for the conjugation of mAb against Cys-C. Typical EDC and Sulfo-NHS conjugation methods were used. After optimization, 10 mm of EDC and 1.25 mm of Sulfo-NHS were used to activate a 2 mg CM-EU nanoparticle solution. The mAb was then conjugated to the surface of the CM-EU nanoparticle. After optimization, 50 ng of anti-Cys-C mAb were conjugated to 2 mg of CM-EU nanoparticle, the solution was added to the strip, and the strip observed higher fluorescence intensity. The CM-EU antibody conjugate is diluted to a concentration of 0.2, 0.3, 0.4, 0.5 ng/ml to optimize the amount of conjugate. Finally, 0.3 ng/ml was chosen for further experiments shown in (Fig. 3).

Optimization of the LFIA Strip
Before producing the LFIA strip, non-specific protein adsorption was prevented by using an optimized blocking buffer containing 1 × Tris-casein, 1.0% casein, 5% sucrose, 1.5% trehalose, and 1.25% Tween 20. The casein prevented non-specific protein adsorption, and sucrose and Trehalose aided the conjugate movement in the conjugation pad. In addition, Tween 20 enhanced the specificity of interaction between the mAb and the antigen illustrated in (Fig. 4).

Selection of Conjugation Pad
For the experimental purpose, we used a Conjugation pad from a Cellulose conjugation pad from Merck (CFSP223000), a Glass fiber conjugation pad (GFCP203000), and a Glass fiber diagnostic Pad (GFDX203000). The proper release of the conjugation material from the above conjugation pad resulted from the Glass fiber Diagnostic Pad (GFDX203000), so we used this pad for all the experiments. Data not shown. Fig. 2 The standard curve of CM-EU-based LFIA strips for anti-Cys-C is obtained from standards from 0.015 to 32 µg/ ml by determining fluorescence peak heights on the readout curve

Precision Assay
The intra-assay and inter-assay precision were calculated to show the reproducibility of the developed assay. Three concentrations (low, medium, high) of anti-Cys-C in spiked urine samples were quantified 10 times per day to determine intraassay precision, and 10 replicates were performed on 3 continuous days the evaluate inter-assay precision. The results are shown in Table 1. The intra-assay CVs were from 6.59% to 7.23% (n = 10) and inter-assay CVs were from 8.44% to 9.02% (n = 10). All the obtained CVs were below 10%, in the acceptable precision region for the anti-Cys-C quantification.

Recovery Study
The LFIA recovery percentage of the assay was quantified by dividing the spiked concentration of Cys-C with the observed concentration multiplied by 100. Control urine (no Cys-C) samples were spiked with four different concentrations of Cys-C standard samples (0.06, 1, 16, 32 µg/ml). The experiment's recovery rates of the four selected samples showed 98, 107, 100, and 102%, respectively ( Table 2).

Optimization of the Immunoreaction Time
The immunoreaction time of the LFIA is the most significant parameter that can influence the fluorescence intensity development in the Lateral flow strip. The recombinant Cys-C standard sample was used for the optimization at a 1, 10 and 32 µg/ml concentration. It is also used to test the immunoreaction time of antibody-antigen interaction by evaluating the V R over a range of 3 to 20 min incubation, as illustrated in (Fig. 5). Each value was calculated in triplicate, and the scale of error represented the standard deviation of the experiment. The experiment recorded the increase in the V R ratio increased up to 10 min and then achieved the peak after 15 min. These results proved that the V R ratio is considered best to determine the concentration of Cys-C than utilizing the V T , and the V R ratio can eliminate the effects of kinetics in the immunoreactions and reduce the turnaround time. Finally, we used 15 min as the most appropriate response time for further research.

Sample Volume
The amount of sample volume for the assay was optimized to eliminate the non-specific adsorption of CM-EU conjugates. Fluorescence signals with different sample volumes were obtained using 100 µl (Fig. 6). As shown in Fig. S1, with the increase of sample volume, the V R ratio increases up to 80 µl but decreases from 80 µl to 100 µl (The results are shown in Fig. S1 (Supplementary Materials).

Stability
The stability study of the CM-EU labelled LFIA was conducted. The strips were preserved in the airtight aluminum foil at 4 °C, and the room temperature was assayed at different time points (months 0, 1, 2, 3). The standard solutions were prepared to contain various concentrations of Cys-C (1, 16, 32 µg/ml). The relative standard deviation is a relative percentage between the standard deviation and the mean value. It is found that the strip still functions well for detection after 3-month airtight preservation at an average temperature. The results are shown in Fig. S2 (Supplementary Materials).

Method Comparison with Standard ELISA
To evaluate the potential clinical application of our CM-EU-based LF assay, we compared its analytical performance Fig. 3 The concentration of the Dab in the conjugation pad for the non-specific binding with that of a commercially available ELISA assay kit (Abcam, UK). Seven concentrations (from 0 to 20 µg/ml) of Cys-C protein were prepared and tested in both ELISA and the LFIA. As shown in (Fig. 6), the minimum detectable concentration of Cys-C using the commercial assay kit is 0.3 µg/ml (Fig. 6b); this is significantly compared with the CM-EU-based lateral flow assay. Furthermore, it is also possible to carry out a highly sensitive quantitative assay of Cys-C in the lower concentration range. These results mean that the CM-EU labeled LFA method for Cys-C has a good performance compared with other widely commercialized methods.

Discussion
Cys-C is a valuable biomarker in GFR measurement as the non-GFR factors do not influence Cys-C. Therefore, a rapid, cost-effective, and quantitative Europium-based Cys-C detection to meet the vast application in the clinical application is of urgent need. Therefore, we developed a Europium-based lateral flow kit for the Cys-C determination, as the organic dye has low stokes shift and photobleaching, and we selected Europium as the fluorosphere to overcome the above disadvantages.
A previous study reported developing a user-friendly Alexafluor-647-LF assay to detect Cys-C in the urine samples [14]. The assay was optimized for its sensitivity and allowed detection of Cys-C in the urine sample up to 0.013 µg/ml [14]. Next, we developed an aptamer-based LFA with an increased LOD of 0.0001 µg/ml [20]. This study describes a distinct CM-EU-LFA format for Cys-C to increase the sensitivity. The newly developed kit was a Europium-based sandwich lateral flow assay that uses Europium as the reporter in the LFA kit. The results were obtained within 15 min which provided quantitative results from a portable IQuant-TRF reader. The gold-standard method for quantifying the Cys-C is nephelometric and turbidimetric methods, which require larger analytical instruments [5]. Fig. 4 The concentration of the Tris-Casein for the non-specific binding Table 1 The intra-and inter-assay precision CV% values Samples (µg/ml) Intra-assay precision (n = 10) Inter-assay precision (n = 10)  On the other hand, the LFA is rapid and easy to use onsite. However, photobleaching, low stokes shift is the issue due to the organic dye reporter used in the development of the kit, which limited its usage in the development of the lateral flow strips [12]. So we selected an alternative to organic dye as Europium to overcome the limitations faced in the organic-based kit development. The strategy for the work is as followed.
First, we selected the correct nitrocellulose membrane through the specifications given by the company [7]. The lateral flow character of the NC membrane is influenced by the chase buffer ingredient, sensitivity, and consistency of the test line, which are mainly considered. We selected HF70, as the CM-EU particle size is 100 nm. Second, the concentration of the antibodies on the lines and the conjugates on the conjugation pad were optimized. Lastly, the volume ratio against the concentration of Cys-C was plotted to get the analytical value. The recovery percent was found between 90-100% for this assay. The LOD of the aptamer LFIA kit was 0.0001 µg/ml [20]. The observed LOD is low than our previously developed antibody-based LFA kit, which showed 0.023 µg/ml [14]. The lower LOD can be due to the significant stokes shift and low background noise. Therefore, the developed kit is highly acceptable for the clinical detection of Cys-C.

Conclusion
A lateral flow immunoassay based on Cys-C antibodies has been developed, optimized, and successfully validated. The assay is rapid (15 min), affordable, and needs only 80 µl of the sample. It provides a quantitative result that was used to determine the Cys-C concentration in the sample. Under the optimal conditions, the biosensor could be used to detect as low as 1 ng/ml of Cys-C. The sensitivity was comparative with that of organic dye-based fluorescent lateral flow immunoassay. The assay precision is less than 10% below the LFIA acceptance level as determined by the within-assay and inter-assay CV with the stability recorded for one year at 4 °C. The stability test experiment showed that by keeping the strips at one week at 37 °C, the volume ratio did not change significantly. Thus, the Cys-C Rapid Test kit fulfils the demands of clinical practice.
Authors' Contributions SN, EB, and JJ designed the experiment. SN implemented the experiment. SN, EB, and JJ analysed the data, prepared the manuscript. All authors read and approved the final manuscript.
Funding No funding was received for this article.

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The data provided is transparent.

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Conflict of Interest
The authors declare that the study has not received any funding, and there are no conflicts of interest.