Introduction

Peste des petits ruminants (PPR) is an acute, highly contagious disease caused by Peste des petits ruminant virus (PPRV), a member of the genus Morbillivirus, family Paramyxoviridae. PPRV is a negative-sense, single-stranded RNA virus [1]. PPR has been listed as one of the notifiable terrestrial animal infectious diseases by the World Organization for Animal Health (OIE), and it is required to inform the OIE of the occurrence of the disease (OIE 2016). To date, only one PPRV serotype has been identified. No human infection has been reported yet. The PPRV epidemic has been found in more than 70 countries, ranging from the Middle East, Africa to Asia. In China, the disease was first identified in 2007 in the Tibet Autonomous Region. As an animal infectious disease crossing geographical borders, PPR has had severe consequences, especially on the development of animal husbandry in the developing countries [2]. Given the severe situation of the widespread disease, the OIE and the Food and Agriculture Organization of the United Nations (FAO) launched the Global Strategy for the Control and Eradication of PPR (GCES) in 2015. One of the key conditions for the success of the PPR eradication program was the use of live attenuated PPR vaccines that were highly efficacious in protecting small ruminants against the disease.

The PPRV antigen-detection methods comprised the virus neutralization test (VN), agar gel immunodiffusion (AGID), immunofluorescent antibody test (IFAT), counterimmunoelectrophoresis (CIEP), enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) [3–9]. In addition, the rapid gold-immunochromatographic assay for PPRV antigen was established by J Baron et al. [10]. With respect to the evaluation of PPRV prevalence of infection or surveillance, immune status in individual animals or populations post vaccination, competitive enzyme-linked immunosorbent assay (c-ELISA) was recommended by OIE [11]; however, professional detection instruments and staff were necessary for this test, and the whole test procedure takes 2.5 h at least, which are usually not available in remote areas for an onsite test. Previous studies have neglected this. The quantum dot-based lateral-flow immunoassay system (QD-LFIAS) has several merits, such as high sensitivity, rapid results, easy operation, and high feasibility, which could provide timely onsite evaluation on the immune effects to achieve PPRV eradication.

Immunochromatography (EICA) was developed in the 1980s based on ELISA technology, monoclonal technology, and new material technology [12–15]. In ELISA, the solid-phase carrier is the polystyrene plate. In Dot-ELISA and EICA, the nitrocellulose membranes (NC) replaces the polystyrene plate [16, 17]. When the enzyme markers in EICA or ELISA are replaced by ultrasensitive fluorescent dye, the substrate of the enzyme is no longer needed in the reaction; therefore, the detection period is greatly reduced, which makes for easy access in the field for antibody detection and post vaccination evaluation (PVE).

In this study, the N gene of PPRV was expressed to establish the ultrasensitive fluorescence QDs-based immunochromatography for serum antibody detection. The water-soluble carboxyl-functionalized QDs were used as fluorescent labels to conjugate the streptococcus G protein (SPG) through an amide bond. Once the PPRV antibody (immunoglobulin G [IgG]) was added to the QD-LFIAS detection system, the QD-labeled SPG could bind to PPRV IgG and react with PPRV N protein in the detection zone, which is visualized as a highlighted fluorescent band by ultraviolet (UV) excitation at the wavelength of 365 nm. The fluorescence intensity, which is proportional to the antibody concentration, can be measured by a low-cost and easily maintained instrument. In the current study, QD-LFIAS was evaluated for use in detecting serum antibody against PPRV with regard to specificity, sensitivity, and the detection limit. A fast and accurate detection assay for PPRV antibody was established.

Materials and methods

Materials

SPG, rabbit anti-SPG IgG, N-hydroxy-succinamide (NHS), and 1-(3-dimethylamino propyl) ethyl-3-ethyl carbon imine hydrochloride (EDC) were purchased from Sigma-Aldrich Corp., St. Louis, MO, USA. NC, glass wool, plastic boxes, water-absorbent paper, and supporting plates were purchased from Millipore (Merck Millipore, Billerica, MA, USA); the water-soluble carboxyl-functionalized QDs were purchased from Najing Technology Corporation, Hangzhou, China; PPRV control sera, the plasmid DNA pBluescriptsk-PPRV-N and pFastBacHTA, expression hosts TOP10 Chemically Competent Escherichia coli, and DH10BAC E. coli were provided by the animal quarantine laboratory of the Yunnan Entry-Exit Inspection and Quarantine Bureau (China). PPRV antibody immuno chromatographic colloidal gold test strips (ICS) were provided by the animal quarantine laboratory of the Shenzhen Entry-Exit Inspection and Quarantine Bureau (China). The PPRV antibody c-ELISA kit was purchased from Pirbright Institute (Woking, UK).

Preparation of recombinant PPRV N protein

The open reading frame of the N gene encoding the PPRV nucleocapsid protein was synthesized and the specific primers for the N gene were synthesized according to the published genome of PPRV strain Nigeria 75/1 from the National Center for Biotechnology Information (GenBank accession number X74443). The primers were as follows:

  • PPRV-F: 5′-GGGAATTCATGGCTACTCTCCTTAAAAG-3′;

  • PPRV-R: 5′-TTGGTACCTTTCAGCTGAGGAGATCCTTGTG-3′;

The EcoR I and Kpn I restriction sites were introduced into the N gene by PCR amplification and subcloned into the baculovirus expression vector pFastBacHTA. The recombinant vector was named “pFastBacHTA-PPRV-N” with an N gene fragment of 1578 bp. The transfer vector Bacmid-PPRV-N was generated by transforming pFastBacHTA-PPRV-N into a competent host cell of DH10Bac. Bacmid-PPRV-N was then infected into Sf9 insect cells through lipid transfection reagent to obtain the recombinant baculovirus. The recombinant protein was purified by ultrasound lysis and ultracentrifugation. The activity and concentration of the recombinant protein was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, and indirect ELISA.

Sera samples

PPRV-positive sera

There were 60 PPRV-positive samples of goat serum, comprising 20 from Tibet Entry-Exit Inspection and Quarantine Bureau (abbreviated as Tibet serum 1-20), 20 from the Xinjiang Entry-Exit Inspection and Quarantine Bureau (abbreviated as Xinjiang serum 1-20), and 20 from the Yunnan Entry-Exit Inspection and Quarantine Bureau (abbreviated as Yunnan serum 1-20). Twenty-two convalescent sera were taken from sheep that were naturally infected with PPRV and provided by the Tibet Entry-Exit Inspection and Quarantine Bureau. All sera were inactivated at 56 °C for 30 min and stored at −70 °C until required.

Control sera samples

Rinderpest (RPV)-positive serum was provided by the Key Laboratory of Tropical and Subtropical Animal Viruses of the Ministry of Agriculture, People’s Republic of China; bluetongue virus (BTV)-positive serum was provided by the British Institute of Health; canine distemper virus (CDV)-positive serum was provided by the Veterinary Research Institute of the Chinese Academy of Military Medical Sciences; goat pox virus (GPV)-positive and foot-and-mouth disease virus (FMDV)-positive sera were provided by the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences.

Field sera samples

A total of 506 sera samples comprising 120 sheep and 386 goat sera were collected from October 2014 to December 2015 in Tibet, China. Among the 386 goat sera, 296 were taken from the Ali district, where PPR first broke out in 2007 and caused a clinical disease. The other 90 goat and 120 sheep sera were kindly provided by six veterinary hospitals in different counties in Tibet. All sera were inactivated at 56 °C for 30 min and stored at −70 °C until required.

Fluorescence CdSe/ZnS QDs conjugated with SPG markers

Two milligrams carboxyl-functionalized CdSe/ZnS QDs was dispensed into 0.1 M MES buffer at pH 4.7, after which 2.0 mM NHS and 5.0 mM EDC were added and the solution activated for 30 min to obtain activated QDs. The solution was centrifuged at 15,000×g for 15 min, the precipitates were resuspended in 50 mM borate buffer (pH 8.5), and 0.1 mg SPG was added. After mixing well, the solution was incubated at 37 °C for 3 h followed by blocking with 5.0 % bovine serum albumin for 30 min. The QDs were centrifuged at 15,000×g for 15 min and resuspended with phosphate buffer solution (PBS). This process was repeated three times. The labeled SPG QDs were stored at 4.0 °C.

Establishment of the quantum dot-based lateral-flow immunoassay strip

The CdSe/ZnS QDs conjugated with SPG were sprayed onto the front end of a sample pad at 12 μL/cm; the pads were dried at 37 °C for 4 h. The recombinant N protein was diluted with 20 mM PBS to a concentration of 1.0 mg/mL and sprayed onto a nitrocellulose membrane at the detection zone. Rabbit anti-protein G was diluted to a concentration of 1.0 mg/mL and sprayed onto the control zone of the nitrocellulose membrane and the membrane was dried at 37 °C for 4 h. The NC membrane, sample pad, absorbent pad, and backings were assembled and cut into 3.5 mm strips using a CM4000 cutter (BioDot Co., Irvine, CA, USA) (Fig. 1).

Fig. 1
figure 1

Working mechanism of quantum dot-based lateral-flow immunoassay system

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Analysis procedure

One hundred microliters of serum diluted 10 times with 50 mM PBS (pH 7.2) was added to the QD-LFIAS sample pad to form a QD-SPG-IgG complex, which could specifically bind to PPRV N protein and precipitate to be visualized as a bright fluorescent band after excitation by UV at a wavelength of 365 nm. If the sample did not contain PPRV antibody, the QD-SPG complex would not bind to PPRV N protein at the detection zone, but would, instead, bind to rabbit anti-SPG IgG at the control zone to form a fluorescent band; the fluorescent intensity was positively correlated with the amount of captured QD-SPG-IgG complex at the detection zone. Fluorescence intensity of 620 nm emission wavelength excited by 365 nm wavelength could be visually observed with UV light and also could be detected by a fluorescence spectrometer instrument. Test results were obtained within 15 min reaction after sample addition.

Performance evaluation in real serum samples

The limit of detection (LOD) was estimated by serial dilution from 1:5 of the diluted positive standard serum with QD-LFIAS. The applicability and ruggedness were shown by carrying out the analysis on different days and by different technicians.

c-ELISA is regarded as the gold standard method which is the most commonly used in the detection of PPRV antibody. To evaluate the diagnostic performance of the proposed QD-LFIAS, we compared the results of the proposed method with c-ELISA by 60 positive serum samples, 22 healthy serum samples, and 8 positive/negative standard sera. The positive serum of the other ruminant animal viruses, such as PRV, BTV, CDV, GPV, and FMDV, was also used for a cross-reaction test to evaluate the analytical specificity.

To compare the detection limits among QD-LFIAS, immune colloidal gold assay (ICS), and c-ELISA, 12 PPRV serum samples (including 6 hyper-immune which were provided by Yunnan Entry-Exit Inspection and Quarantine Bureau and 6 convalescent sera) were serially diluted with 20 mM PBS (pH 7.2) from 1:5 to 1:2560 and tested using the three detection methods.

Practical field sample tests

Five hundred and six serum samples (120 sheep and 386 goat serum samples) were tested by QD-LFIAS, and c-ELISA was used as the reference method (gold method); the detection results were evaluated using a two-sided contingency table. The diagnostic specificity was expressed as the percentage of the number of negative results in both QD-LFIAS and c-ELISA tests to the number of negative results in c-ELISA. The diagnostic sensitivity was expressed as the percentage of the number of positive results in both tests to the number of positive results in c-ELISA [18]. The agreement between QD-LFIAS and c-ELISA was evaluated by kappa statistic [19].

Results

PPRV N protein characterization and the selection of the concentration used for the antibody detection zone

The PPRV N protein had a satisfactory bioactivity of approximately 61.3 kU assessed using SDS-PAGE, indirect ELISA, and western blot (Fig. 2). The recombinant PPRV N protein was diluted to a concentration of 0.5, 1.0, and 2.0 mg/mL and sprayed onto a Millipore HF1800425NC membrane using XYZ3050 dispense workstation (BioDot, Irvine, CA, USA) to obtain an optimal immune chromatography system for the detection of standard positive serum, weakly positive serum, and negative serum, respectively. No visible band was present on the detection zone for the weakly positive serum when the concentration of PPRV N protein was 0.5 mg/mL, indicating that the sensitivity of the strip was not high enough. A weak, non-specific band could be visualized on the detection zone for the negative serum when PPRV N protein was at a concentration of 2.0 mg/mL. The expected results could be visualized for strong positive serum, weak positive serum, and negative serum when PPRV N protein was at a concentration of 1.0 mg/mL; therefore, the optimal concentration for PPRV N protein was 1.0 mg/mL.

Fig. 2
figure 2

SDS-PAGE (a) and western blot (b) analysis of expressed N protein. a (1, 2) Extract from recombinant baculovirus expressed in sf9 cells, (3) extract from sf9 cells; b (1) extract from sf9 cells, (2, 3) extract from recombinant baculovirus expressed in sf9 cells, M, molecular mass standard in kilo units

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Formation of water-soluble carboxyl-functionalized QDs and SPG

The average size of QDs was 20 nm, determined by transmission electron microscopy with a high dispersion effect (Fig. 3a). QDs have a wide range of excitation wavelength, and the maximum emission peak at 620 nm can be achieved by excitation at a wavelength of 365 nm (Fig. 3b). The water-soluble carboxyl-functionalized QDs were covalently conjugated with SPG through EDC activation to form a stable labeled complex. The presence of sulfo-NHS can reduce QD self-cross linking. The diameter of carboxyl-functionalized water-soluble QDs as measured by dynamic light scattering (DLS) was 57.8 nm (Fig. 3c), and the diameter of the hydrated particle increased to 117 nm after SPG covalent conjugation, indicating that the antibody was successfully conjugated to QDs.

Fig. 3
figure 3

Characterization of quantum dots (QDs). a Transmission electron microscope of QD; b excitation and emission spectra of QD; and c dynamic light scattering analysis of QDs before and after antibody labeling

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Detection limit of QD-LFIAS

The LOD of QD-LFIAS for PPRV serum antibody was 1:320 dilution fold. Compared with ICS, and c-ELISA, the results indicated that the detection limit of QD-LFIAS for PPRV antibody was superior than ICS and c-ELISA, which was a dilution one- to twofold lower than that of c-ELISA, and two- to threefold lower than that of ICS (Fig. 4).

Fig. 4
figure 4

Antibody titers tested by quantum dot-based lateral-flow immunoassay system (QD-LFIAS), colloidal gold immunochromatographic strip (ICS), and competitive enzyme-linked immunosorbent assay (c-ELISA). Sera were diluted twofold from 1:5 to 1:640. The number (1, 2…n) in the y-axis represents that the sample was diluted 1:(5 × 2n)

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Specificity of QD-LFIAS

The positive serum of PPRV, FMDV, CDV, GPV, RPV, BTV and the negative serum were tested by QD-LFIAS, the results showed that only PPRV-positive serum was tested as positive result (Fig. 5), indicating that QD-LFIAS had no cross reaction with the other antigen-positive serum and the test was specific to only PPRV antibody. The high consistency (100 %) between c-ELISA antibody detection and QD-LFIAS was demonstrated, suggesting that QD-LFIAS was an accurate assay for PPRV antibody detection (Table 1).

Fig. 5
figure 5

Result of the specificity test

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Table 1 Analytical specificity of quantum dot-based lateral-flow immunoassay system (QD-LFIAS)
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Field sample tests

Among all 506 serum samples, QD-LFIAS showed 128 positive and 378 negative samples, and c-ELISA showed 129 positive and 377 negative samples. Both methods showed 126 positive and 375 negative samples (Table 2). Compared with c-ELISA, the specificity and sensitivity of QD-LFIAS were 99.47 and 97.67 %, respectively. There was excellent agreement between the results obtained by QD-LFIAS and c-ELISA (kappa = 0.974).

Table 2 Results of antibody detection in field sera samples using the quantum dot lateral-flow immunoassay strip (QD-LFIAS) and competitive enzyme-linked immunosorbent assay (c-ELISA)
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Discussion

There were several improvements over conventional antibody detection methods for antibody detection in the current study as follows:

First, for selection of antigens, the recombinant virus protein was preferred over the virus antigen in pathogen antibody detection by immune chromatography because of its superior safety and stability. Second, between the prokaryotic and eukaryotic expression systems, we chose the latter for the satisfactory activeness of protein it could achieve despite its low yield, high cost, and complicated procedures [20–22]. Third, based on the particular optical property (i.e., wide excitation spectra and narrow emission spectra[23–25]) and the successful application of QDs for rapid diagnosis in the field, especially for immunolabeling technology, and its use in the detection of antigens, antibodies, feature proteins, and nucleic acids with the advantages of high sensitivity, quantifiable results, and good repeatability[26, 27], we established a rapid immunochromatography detection strip composed of the ultrasensitive fluorescent water-soluble carboxyl-functionalized QDs, SPG, and the recombinant PPRV N protein produced by the baculovirus expression system. It is estimated that QDs are 20 times brighter and 100 times more stable than traditional fluorescent reporters [28]. Fourth, SPG and staphylococcal protein A (SPA) are different bacterial antibody (Ab)-binding proteins that are widely used as immunological tools. SPG consists of nearly 600 amino acid residues. The carboxyl-terminal half contains three IgG-binding domains—I, II, and III. SPG has a broader specificity than SPA for IgGs from different sources [29]. In addition, SPG and SPA, mostly coupled with horseradish peroxidase, fluorescein isothiocyanate, and colloidal gold particles, are often used as antibody detectors; however, the binding capacity of SPA and SPG with IgG varies among mammalian animal species. For goats, sheep, and cattle antibodies, SPG has a greater capacity for binding to IgG than that of SPA; therefore, SPG is the better choice for coupling with QDs as an antibody detection reagent [30].

There were multiple merits for using QD-LFIAS for field applications as follows: (1) rapid detection, (2) good repeatability, (3) relatively low cost, and (4) little interference based on immune chromatography [31]. The SPG-conjugated carboxyl-functionalized QDs were used in the current study to capture the antibody and react specifically with recombinant PPRV-N protein, which overcame the disadvantage of having to use a large amount of colloidal gold to be visualized. The sensitivity of QD-LFIAS was significantly increased by a special signal amplification system with reduced basal interference and incomparable advantages compared with conventional label technology.

Conclusion

The rapid detection system of ultrasensitive fluorescent QD immunochromotography in combination with recombinant PPRV N protein was established in this study to detect PPRV antibody in serum. The excellent monochromaticity of carboxyl-functionalized QDs with the full bandwidth at half maximum less than 30 nm was demonstrated, and the excellent biocompatibility and optimized surface carboxyl domain could be widely used for the establishment of a biological molecular conjugation and immunochromatography system. QD-LFIAS was superior to ICS and c-ELISA for detecting PPRV serum antibody, which was more specific, sensitive, rapid, and accurate, making it more suitable for onsite inspection of PPRV antibodies and epidemiological surveillance on PPR.