Pt-Pd合金纳米颗粒标记口蹄疫A型病毒抗体检测的免疫层析方法

doi:10.3864/j.issn.0578-1752.2021.03.018

Abstract

Abstract:

【Objective】A novel immunochromatographic test strip with peroxidase-like activity of Pt-Pd bimetal nanoparticles (NPs) as a marker (shortened as NPs test strip) was developed. The signal amplification was based on Pt-Pd bimetal NPs possessing high peroxidase-like activity toward 3, 3, 5, 5＇-tetramethylbenzidine, which could produce characteristic colored bands. It could not only improve the sensitivity of detection, but also completed a quantitative analysis of results with reader. This method provided a rapid and effective screening means for monitoring.【Method】Pt-Pd bimetal NPs were prepared by K₂Ptcl₄ and Na₂Pdcl₄, which were reduced by ascorbic acid (AA). The mixture was continuously sonicated in water bath. The purified antigen of FMDV serotype A was coupled with Pt-Pd bimetal NPs. The purified 146S particles of FMDV serotype A (FMDV-146S) and the antibodies against FMDV-146S (FMDV-146S-Ab) were blotted on nitrocellulose membrane as test line and control line, respectively. The sensitivity, specificity and stability of this NPs test strip were assessed by testing known positive and negative FMDV serum and positive serum against other animals’ disease. Parallel tests of the positive FMDV serum by LPB-ELISA were performed.【Result】This established method could be accomplished qualitatively as well as semi-quantitative detection in 10 minutes, the sensitivity was up to 1:2⁸/test, and the linear range was from 1:2⁶/test-1:2⁹/test. The detection sensitivity for the serum positive for FMDV type A was 98%, and the detection sensitivity for FMDV-negative serum, serum positive for FMDV type O and type Asia 1 as well as sera positive for other common viral diseases in animals was 94.8%. The differences between batches were small. Test serum samples showed that the strip results were coincident with LPB-ELISA, R²=0.9112. 【Conclusion】An immunochromatographic assay based on Pt-Pd bimetal nanoparticles was established innovatively and applied to the detection of FMDV antibodies. The results demonstrated its high sensitivity, which was a 2⁴-fold increase as compared with the colloidal gold. In addition, this novel assay also displayed excellent maneuverability and reproducibility. The established method represented a new attempt in fast immunochromatographic assay and probably predicted an alternative and highly promising application of the alloy nanoparticles.

Key words: immunochromatographic assay, Pt-Pd nanoparticles, peroxidase, foot-and-mouth disease virus (FMDV) type A

SUN YanYan,LI Xin,LIN Mi,LI FengSong,Bao YanFang,CHEN XiaHui,YANG Guang,ZENG QiaoYing,JIANG Tao. Establishment of a Novel Immunochromatographic Assay Based on Foot-and-Mouth Disease Virus Serotype A Labeled by Pt-Pd Bimetal Nanoparticles[J].Scientia Agricultura Sinica, 2021, 54(3): 653-661.

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References 32

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T/C值 T/C	血清效价 Serum titer（ST）	结果判定 Result
T/C<0.05	ST ≤1:64	±/-
0.05≤T/C<0.15	1:64<ST≤1:128	+
0.15≤T/C<0.40	1:128<ST≤1:256	+
0.40≤T/C<0.70	1:256<ST≤512	+

血清稀释倍数 Sera dilution	T/C平均值 T/C	标准差 Standard deviation	变异系数 Variation coefficient (%)
1:2	0.637	0.021	3.4
1:2²	0.386	0.018	4.7
1:2³	0.136	0.007	5.1
1:2⁴	0.035	0.005	14.3
1:2⁵	0.030	0.002	6.7

血清性质 Serum	血清稀释倍数 Sera dilution		1:2²	1:2⁴	1:2⁶	1:2⁸	1:2¹⁰
口蹄疫A型免疫血清 Positive sera of FMDV type-A	Pt-Pd合金纳米颗粒试纸条（定量） Pt-Pd bimetal nanoparticles test strip	T/C值 T/C	0.627	0.396	0.138	0.047	0.029
	Pt-Pd合金纳米颗粒试纸条（定量） Pt-Pd bimetal nanoparticles test strip	结果判定 Results	+	+	+	±	-
	胶体金试纸条（定性）Gold test strip		+	+	-	-	-

Establishment of a Novel Immunochromatographic Assay Based on Foot-and-Mouth Disease Virus Serotype A Labeled by Pt-Pd Bimetal Nanoparticles

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