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 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 VT/VC 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 VR against the concentration using the equation: y = 0.0022x2 + 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 0.001μg/ml. After establishing the CM-EU-nanoparticle method, a lower LOD was recorded than the Alexa fluor-647 method (0.023µg/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, 10mm 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 1x 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.
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 intra-assay 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 4.53% to 6.89% (n = 10) and inter-assay CVs were from 5.93% to 8.83% (n = 30). All the obtained CVs were below 10%, which is in the acceptable precision region for the anti-Cys-C quantification.
Table 1. The intra- and inter-assay precision CV% values.
Samples (µg/ml)
|
Intra-assay precision (n=10)
|
Intra-assay precision (n=10)
|
|
Mean±SD (µg/ml)
|
CV%
|
Mean ±SD (µg/ml)
|
CV%
|
0.06
|
19.53±1.29
|
6.59
|
19.46±1.64
|
8.44
|
2
|
59.19±4.32
|
6.16
|
59.23±5.87
|
8.25
|
32
|
119.40±12.3
|
7.23
|
119.36±15.37
|
9.02
|
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).
Table 2. The Recovery percent of the spikes.
Spiked Concentration (µg/ml)
|
Observed Concentration (µg/ml)
|
Recovery (%)
|
0.06
|
0.01465
|
98
|
1
|
1.0645
|
107
|
16
|
16.05
|
100
|
32
|
32.5
|
102
|
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. For the optimization, the recombinant Cys-C standard sample was used at a 1, 10, and 32µg/ml concentration. It is also used to test the immunoreaction time of antibody-antigen interaction by evaluating the VR over a range of 3 to 20 minutes 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 VR ratio increased up to 10 min and then achieved the peak after 15 minutes. These results proved that the VR ratio is considered best to determine the concentration of Cys-C than utilizing the VT, and the VR ratio can eliminate the effects of kinetics in the immunoreactions and reduce the turnaround time. Finally, we used 15 minutes as the most appropriate response time for further research.
Sample volume
To eliminate the nonspecific adsorption of CM-EU conjugates, the amount of sample volume for the assay was optimized. Fluorescence signals with different sample volume were obtained using 100 µl (Fig. 6). As shown in Fig. S1, with the increase of sample volume, the VR ratio increases up to 85µl but decreases from the 85ul to 100µl (The results are shown in Figure 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 average temperature. The results are shown in Figure 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 with that of a commercially available ELISA assay kit (Abcam, UK). Seven different 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.7b); 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.