Detection of C-reactive Protein by Background Fluorescence Quenching-Immunochromatography†

doi:10.7503/cjcu20170494

Abstract

Abstract: Background

fluorescence immunochromatography assay(bFQICA) has been established, which has the advantages of simple operation, high sensitivity, strong anti-interference and rapid quantitative detection of C-reactive protein(CRP). This method of using double antibody sandwich method principle, the appropriate concentration of capture antibody and tracer antibodies are respectively fixed on the test strip and the micropore. During chromatography, antibodies were captured, while tracer antibodies and samples were inserted to form a sandwich structure of antibody antigen antibody. The method has a good correlation with fluorescence signal values(F₁/F₂) in the range of 0.0—100.0 ng/mL concentration in CRP, the minimum detection limit was 0.0939 ng/mL, and the recovery rate was 87.69%—111.0%, three batches of reagents and inter and intra assay relative standard deviation was less than 15%, and procalcitonin(PCT, 20.0 ng/mL), serum amyloid A like protein(SAA, 10.0 μg/mL) had not cross reaction with this method and the immune turbidimetric method for the simultaneous determination of 41 serum samples, the detection results had a good correlation(r=0.9585, P<0.01), there was no significant difference between the two methods(P>0.05). This method has high sensitivity, simple operation, and can be used for rapid and accurate quantitative detection of CRP.

Key words: Background fluorescence quenching-immunochromatography, C-Reactive protein, Colloidal gold

TrendMD:

CHEN Junlei, ZHANG Wei, LI Xinxia, LI Jiutong, GUAN Ming. Detection of C-reactive Protein by Background Fluorescence Quenching-Immunochromatography^†[J]. Chem. J. Chinese Universities, 2018, 39(1): 41.

Figures/Tables 10

Scheme 1 Schematic diagram of reaction principle on background fluorescence quenching-immunechromatographyBackground fluorescence quenching-immunochromatography paper strip(herein after referred to as the paper strip) consists of 6 parts: 1. PVC plate: used to attach all membrane materials; 2. sample pad: used to carry sample test solution; 3. test line: used to display the results, above contains “capture antibody of tested”; 4. control line: used to show whether the test strip is valid, the above contains a “goat anti-mouse IgG”; 5. nitrocellulose membrane: used to carry T/C lines and background fluorescence; 6. absorbent filter paper.

Fig.1 Absorption spectrum(A) of colloidal gold particles, excitation(a) and emission(b) spectra of rhodamine B(B)

Fig.2 Optimal concentrations of the marker antibody and the capture antibody system under the CRP standard solution measurements

Fig.3 Fluorescence diagram of test strip(A) and the C-reactive protein CRP standard curve(B)

Table 1 Repetitive determination(n=10)

Concentration/(ng·mL^-1)	F₁/F₂	Measured value/(ng·mL^-1)	CV(%)
5.0	1.546	5.599	7.70
30.0	4.619	32.60	7.32

Table 2 Results of precision test(n=10)

Project	Concentration/(ng·mL^-1)	F₁/F₂	Measured value/(ng·mL^-1)	CV(%)
The first batch	5.0	1.545	5.588	9.89
	30.0	4.317	29.10	11.20
The second batch	5.0	1.573	5.815	7.94
	30.0	4.390	29.94	10.72
The third batch	5.0	1.479	5.043	9.42
	30.0	4.212	28.02	13.96
Inter-assay	5.0	1.532	5.482	9.08
	30.0	4.306	29.02	11.96

Table 3 Determination of spiked recovery(n=3)

Sample	Addition amount/ng	F₁/F₂	Measured value/ng	Recovery(%)
1	2.000	3.572	1.754	87.69
2	0.200	1.180	0.222	111.0

Fig.4 Correlation of the two test methods

Table 4 Results of sensitivity contrast experiment(n=20)

Method	Concentration/(ng·mL^-1)	Signal value	Standard deviation	Minimum detection limit/(ng·mL^-1)
bFQICA	0.0	1.011	0.0067	0.1297
GICA	0.0	36.35	7.673	0.8021

Table 5 Results of repetitive contrast experiment(n=10)

Method	Concentration/(ng·mL^-1)	Signal value	Measured value/(ng·mL^-1)	CV(%)
bFQICA	5.0	1.473	5.599	5.17
	30.0	3.854	32.19	10.8
GICA	5.0	224.1	5.470	14.2
	30.0	598.8	26.22	16.7

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