Eur J Clin Microbiol Infect Dis DOI 10.1007/s10096-015-2353-6
ARTICLE
A multiplex ELISA-based protein array for screening diagnostic antigens and diagnosis of Flaviviridae infection D. Wang & Y. Zheng & X. Kang & X. Zhang & H. Hao & W. Chen & L. Liu & X. Li & L. Li & Q. Yuan & F. Chen & Y. Yang & Y. Jiang & H. Jiang
Received: 20 November 2014 / Accepted: 16 February 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Assays with the ability to detect multiple antibodies in parallel have a wide range of potential applications in epidemiologic research. Here, a multiplex enzyme-linked immunosorbent assay-based protein array (ELISA-array) was developed to simultaneously detect five Flaviviridae infections. The platform was based on an indirect ELISA and 15 antigens were constructed for specific antibody detection against five Flaviviridae viruses (Japanese B, tick-borne encephalitis, West Nile, dengue, and yellow fever viruses) and four serotypes of dengue virus. The specificity was evaluated by calculating the signal value cross-reacting with serum immunized with other viruses, and the sensitivity of antigens was compared with conventional ELISAs using immunized rabbit polyclonal antisera. IgG and IgM calibration curves were constructed to evaluate the reproducibility of the platform. Finally, 24 dengue fever (DF) infection and 15 tick-borne encephalitis (TBE) infection clinical sera were used to compare the D. Wang and Y. Zheng contributed equally to this work. D. Wang : Y. Zheng : X. Kang : X. Zhang : L. Li : Q. Yuan : Y. Yang (*) : Y. Jiang (*) : H. Jiang (*) State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing 100071, China e-mail: [email protected] e-mail: [email protected] e-mail: [email protected] H. Hao CAS Key Laboratory of Pathogenic Microbiology and Immunity, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China W. Chen : L. Liu : X. Li Genomics Institute in Shenzhen, Beishan Industry Beishan Road, Shenzhen 518083, China F. Chen College of Food Technology, Huazhong Agricultural University, Wuhan, China
advantage of ELISA-array to ELISA. After initial screening, 9 out of 15 antigens were chosen for ELISA-array printing. By using different virus-immunized rabbit antiserum, 7 out of 9 antigens showed good specificity in the ELISA-array. Eight out of 9 antigens showed four-fold greater sensitivity in ELISA-array compared to that in conventional ELISAs. The coefficients of determination (r2) close to 1 showed high reproducibility, and clinical sera test showed that ELISA-array had higher specificity and sensitivity than traditional ELISA. ELISA-array was a good platform for antigen screening and this multiplexed assay might be a useful and convenient tool for multiple immunological detection of infectious viral antibodies.
Introduction Viral antigen–antibody reactions assessed with conventional enzyme-linked immunosorbent assay (ELISA) requires each antigen to be tested separately and involves long incubation periods. It is labor-intensive and time-consuming, and requires significant volumes of antigen and serum or plasma. An immunoassay that simultaneously measures multiple antigen– antibody reactions would have significant clinical value, and such an ELISA-based microarray is emerging as a strong candidate platform for multiplex protein analysis due to its highthroughput potential, assay sensitivity and stringency, ease of handling, and its low sample volume demand. An ELISAbased microarray printed directly onto the bottom of a 96well glass plate was first reported by Genometrix [1] and can now also be printed directly onto the bottom of 384-well plastic microtiter plates (microELISA) [2, 3]. For example, SearchLight™ arrays (Pierce Biotechnology Inc., Rockford, IL, USA) have up to 16 assays per well in an indirect ELISA format [3]. The microELISA platform has been compared
Eur J Clin Microbiol Infect Dis
with routine identical conventional ELISAs and has been validated by tumor markers and cytokines [4, 5]. However, microELISA has not been reported for the detection of viral antibodies. With increasing human population growth and mobility, the emergence and spread of zoonotic and vector-borne diseases is a continual concern as air travel facilitates the global spread of infectious vector-borne diseases [6, 7]. Japanese B encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), West Nile virus (WNV), yellow fever virus (YFV), and dengue virus (DENV) are mosquito- or tick-transmitted viruses of the Flaviviridae family. They are causative pathogens of viral encephalitis or hemorrhagic fever. JEV, DENV, and TBEV are principal arboviruses of current public health importance in mainland China [8, 9]. Increasing evidence suggests that other arboviruses might be present and cause additional human infections in China [9, 10]. Thus, the early identification of highly pathogenic arboviruses is critical, and to achieve this, selection and validation of specific diagnostic antigens is a priority. Here, we depict a multiplex ELISA-based protein array (ELISA-array) platform to screen specific diagnostic antigens. This platform was based on an indirect ELISA consisting of antigens printed directly on a 96-well microtiter plate, which permitted 16 or 9 immunological assays per well. Compared to conventional ELISA, this multiplexed assay may offer better and more convenient immunological assay techniques for infectious viral agents.
Materials and methods Viruses JEV and DENV types 1–4 were grown in C6/36 cells in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 % fetal calf serum (FCS). TBEV, YFV, and WNV were grown in BHK21/23 cells in DMEM supplemented with 10 % FCS. Viruses were cultured at biosafety level 2 or 3 facilities. Virus titers were determined using a 50 % infectious dose in tissue culture (TCID50/mL) method. All viruses were inactivated prior to use with 1:4,000 β-propiolactone overnight at 4 °C. Viral antigen expression in Escherichia coli The primers and restriction sites are summarized in Table 1. Amplified fragments were cut with restriction enzyme and ligated into pET30a. Recombinants were transformed into E. coli BL21 (Novagen). Recombinant antigens were purified using nickel affinity chromatography. Protein purification was performed under nondenaturating conditions to maintain structures and purified proteins were stored at 4 °C and
analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) immediately prior to spotting to assess integrity, identity, and purity. Purified protein identified was confirmed based on expected molecular weight. Spotted antigens exceeded >90 % purity. Polyclonal antisera from immunized animals Rabbit polyclonal antiserum against WEEV, JEV, TBEV, WNV, DENV types 1–4, and YFV were prepared according to standard procedures. Briefly, the inactivated virus was purified by ultracentrifugation at 146,000×g at 4 °C for 2.5 h. The pellet was resuspended in phosphate-buffered saline (PBS) and quantified using a BCA assay (Pierce®, Thermo Scientific, Waltham, MA, USA). Female New Zealand rabbits weighing 1.5–2 kg and sera negatives for virus were subcutaneously inoculated with inactivated virus (200 μg–1 mg each) in Freund’s complete adjuvant (Sigma-Aldrich, St. Louis, MO, USA). The immunized rabbits received booster doses, which was 1/5-2/5 as the primary dose, at a time interval of 1– 2 weeks in Freund’s incomplete adjuvant (Sigma-Aldrich, St. Louis, MO, USA) for two times. Antisera were collected two weeks after the final injection and stored at −70 °C for further use. The specificity of immunized serum was confirmed with a standard immunofluorescence assay (IFA) and ELISA. Collection of human serum samples Serum samples of 15 clinically confirmed TBE cases were collected from Mudanjiang Forestry Hospital, and sera from 24 confirmed cases of dengue fever (DF) were collected from Guangzhou Hospital. TBE sera samples were collected 10–34 days after tick bites and 3–12 days after disease onset, and DF sera samples were collected 2–13 days after syndrome onset. An additional 30 healthy adult sera samples were collected from Beijing 301th Hospital without contact with TBEV and DENV. These healthy adults had no history of recent visits to TBEV or DENV epidemic regions, and no subject received TBEV/DENV vaccinations. All samples were collected at outpatient clinics and stored at −80 °C prior to use. ELISA-array printing Purified antigens were serially diluted with dilution buffer 0.02 mol L−1 PBS (pH 7.2) and printed in a 3×3 or a 4×4 grid in each well of a 96-well plate (Greiner Glasinstrumente GmbH, Nordrhein-Westfalen, Germany) (Fig. 1a) using a Bio-Dot Printing System (Beijing BGI-GBI Biotech Co. Ltd., Beijing, China). For rabbit antiserum testing, rabbit IgG was printed at the first spot in each well as a positive control (Fig. 1a). For clinical serum testing, human IgG and human IgM was printed at the first and last spots in each well as a positive control. The final concentration of antigen was 2–
Eur J Clin Microbiol Infect Dis Table 1
The primers and restriction sites used in this research
Virus
Genes
Sequences of primers (5′-3′)
Restriction sites
JEV
E-D3
JEV
PreM
TBEV
E-D3S
TBEV
E-D3I
TBEV
NS3
WNV
E-D3
WNV
NS1
DENV
E-D2
DENV
E-D3
DEN1
E-D3
DEN2
E-D3
F: 5′-CGCGGATCCGGTGGTGGTATGGACAAACTGGCTTTGAAGGGCACGAC-3′ R: 5′-CCGCTCGAGACCACCACCCGTGCTTCCAGCTTTGTGCCAGTGATGGT-3′ F: 5′-CGGGATCCATGAAGTTGTCAAATTTCCAGG-3′ R: 5′-CCGCTCGAGAGCATAGCCAGGATTCCTTAC-3′ F: 5′-ACGGCCATGGCGACATACACAATG-3′ R: 5′-CGTGGATATCTTTTTGGAACC-3′ F: 5′-CGCGGATCCGGTGGTGGTTTGACCTACACCATGTGCG-3′ R: 5′-CCGCTCGAGACCACCACCGATAGAAGAACCCTTCTGG-3′ F: 5′-CGGGATCCGGTAAGAGTGAACCCTTTCCG-3′ R: 5′-CCGCTCGAGTATTTGCGCCTCTTTCCATTGCACTAATCCACTGTCATC-3′ F: 5′-CGGGATCCGGTGGTGGTGGAACAACCTACGGCGTCTGT-3′ R: 5′-CCGCTCGAGTCCAGACTTGTGCCAATGGTGA-3′ F: 5′-CGGGATCCGACACTGGGTGTGCCATAGACATC-3′ R: 5′-CCCTCGAGTAGCATTCACTTGTGACTGCACG-3′ F: 5′-CGCGGATCCGGTGGTGGTAACCCTGCCGTCTTGCGCAAACT-3′ R: 5′-CCGCTCGAGACCACCACCTGCAAAGATTGTTGTCGTTCCT-3′ F: 5′-CGCGGATCCGGTGGTGGTATGTGCACAGGCTCATTTAAGCTAGA-3′ R: 5′-CCGCTCGAGACCACCACCTTTCCCTATGCTGCTTCCTTTCTTGA-3′ F: 5′-CGGGATCCGGTGGTGGTACGACAAAAATTTTTGCAGGACAC-3′ R: 5′-CCGCTCGAGTTTCCCTATGCTGCTTCCTTTCTTG-3′ R: 5′-CCCAAGCTTTGGGTGGTGGTGGATCCCAAGAAGGGGCCATG-3 R: 5′-CCGCTCGAGTCCTTTCTTAAACCAGTTGAGCTT-3′
BamHI Xho I BamHI Xho I Nco I EcoR V BamHI Xho I BamHI Xho I BamHI Xho I BamHI Xho I BamHI Xho I BamHI Xho I BamHI Xho I Hind III Xho I
DEN3
E-D3
DEN4
E-D3
YFV
NS3
YFV
E-D3
F: 5′-CGGGATCCTATGCAATGTGCTTGAATACCTTTG-3′ R: 5′-CCCTCGAGTCTTCCCAATCGAGCTTCCCTTC-3′ F: 5′-CGGGATCCGGTGGTGGTTGCAAAGTTCGCATGGAGAAA-3 R: 5′-GTGCTCGAGTCGCTTTGCGCCTCTGTATGTGGA-3′ F: 5′-CGGGATCCAACAGAGATGGAGACTCATACTAC-3′ R: 5′-CCGCTCGAGACTCTGGTCAGATGACACCC-3′ F: 5′-CGCGGATCCGGTGGTGGTACCTCTTACAAGATCTGTAC-3′ R: 5′-CCGCTCGAGACCACCACCTTCCTTGTGCCATTGGT-3′
BamHI Xho I BamHI Xho I BamHI Xho I BamHI Xho I
The underlined sequences are the restriction sites
600 mg L−1. Printing was performed in a cabinet at 55 % humidity and temperature 20±3 °C, and the conditions were monitored by a thermohygrometer. To avoid cross-contamination among spots, pins were washed with Milli-Q sterile water, 75 % ethanol, and then with 0.5 % sodium dodecyl sulfate (SDS). Printed plates were immediately removed from the cabinet and incubated for 1.5 h at 37 °C to secure antigen attachment to wells. After washing twice with phosphate buffered saline with 0.05 % Tween20 (PBST), the plates were incubated for 1 h at 37 °C with a solution of 10 % newborn calf serum in PBS to block nonspecific antibody binding. Finally, plates were inverted for 12 h on blotting paper at room temperature, and stored in boxes at 4 °C in the presence of silica gel bags as a desiccant, and used within 90 days of being printed. ELISA-array analysis Fifty microliters of rabbit antiserum diluted to various concentrations (dilution buffer 0.9 % NaCl and 0.1 % TritonX-100 with 5 % FCS) were added to the appropriate wells for 1 h at 37 °C.
Plates were washed five times in an ELx405™ microplate washer (BioTek Instruments, Winooski, VT, USA) with PBST, pH 7.2, which contained 0.05 % Tween20. Fifty microliters of horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibodies (Beijing CoWin Biotech Co. Ltd., Beijing, China) diluted in 0.02 mol L−1 PBS with 20 % FCS (1:4,000) were added to the plate for 40 min at 37 °C. Plates were washed five times with PBST as above. Forty microliters of SuperSignal ELISA Femto Stable Peroxide Solution (Pierce) diluted in distilled water (1:8) were added to produce a chemiluminescent response. The plate was imaged for 30 s using an AE1000 Cool CCD Image Analyzer (Beijing BGI-GBI Biotech Co. Ltd.) and analyzed by ELISA-Array software (provided by Beijing Genomics Institute, Shenzhen, China). The software calculated the pixel intensity for each spot. A ratio of immunized rabbit serum or patient sera intensity/mean value of unimmunized rabbit serum or healthy sera intensity ≥2.0 was regarded as positive. For clinical samples tested by ELISA-array, sera were diluted (1:600) and added to appropriate wells for 1 h at 37 °C.
Eur J Clin Microbiol Infect Dis Fig. 1 Array-ELISA 9-element array general design and schematic. a Arrays (9-element) were printed into each of the 96 wells on a polystyrene material array plate. Viral antigens were spotted as shown. Antigens were linked to the surface, and the measured antibody recognized the antigen. Next, the HRP-linked secondary antibody recognized bound measured antibody. Antisera against each virus were measured in duplicate in two rows. Antisera were added to each row at the same dilution. Finally, a luminol substrate was used to generate a chemiluminescent signal. b Columns 1 to 10: probed with sera from rabbit immunized with WEEV, DEN1, DEN2, DEN3, DEN4, TBEV, JEV, WNV, unimmunized (negative control), and blank, respectively. R1, R2: immunized rabbit antiserum (1:100) was tested in duplicate. ELISA: enzyme-linked immunosorbent assay; HRP: horseradish peroxidase
Fifty microliters of HRP-conjugated goat anti-human IgG antibodies (Beijing CoWin Biotech Co. Ltd.) or HRPconjugated goat anti-human IgM antibodies (Beijing CoWin Biotech Co. Ltd.) were added (1:4,000) and incubated for 40 min at 37 °C. For the detection of IgM antibodies, goat anti-human IgG serum (Beijing CoWin Biotech Co. Ltd.; 1:100) was added to sera sample diluent in advance to prevent IgG interference. Other protocols were identical to those depicted for the ELISA-array described above. Conventional ELISAs Recombinant antigens JEV-E-D3, TBEV-E-D3S, TBEV-ED3I, WNV-E-D3, and DENV-E-D3 types 1–4 used in the
ELISA-array were also tested by conventional ELISA. ELIS As were performed according to standard procedures: 96-well polystyrene round-bottom microtiter plates (MaxiSorp Nunc A/S, Roskilde, Denmark) were coated with antigens diluted in 0.02 mol L−1 PBS, and antigen binding was performed at 37 °C for 1.5 h. Optimal antigen concentrations were 6 mg L−1 for JEV-E-D3, 0.34 mg L−1 for TBEV-E-D3S, 6 mg L−1 for TBEV-E-D3I, 0.02 mg L−1 for WNV-E-D3, 0.2 mg L−1 for DENV-E-D3, 0.02 mg L−1 for DEN1-E-D3, 0.2 mg L−1 for DEN2-E-D3, 1.67 mg L−1 for DEN3-E-D3, and 0.12 mg L−1 for DEN4-E-D3, and each well was coated with 100 μL of diluted antigen. Unadsorbed material was removed with two washings of 0.05 % Tween 20 in PBS. Then, the plates were incubated for 1 h at 37 °C with a solution
Eur J Clin Microbiol Infect Dis
B
A JEV-E-D3
TBEV-E-D3S
5000
4000
Signal intensity(AU)
** *
3000 2000 1000
*** ***
4000 3000 2000 1000
0
Antiserum
N C Bl an k
V EN 1 D EN 2 D EN 3 D EN 4 TB EV JE V W N V D
PC
W
JE V W N V N C Bl an k
P W C EE V D EN D 1 EN D 2 EN D 3 EN TB 4 EV
0 EE
Signal intensity(AU)
5000
Antiserum
C
D **
WNV-E-D3
5000
***
4000 3000 2000 1000
***
ns
Signal intensity(AU)
Signal intensity(AU)
5000
TBEV-E-D3I
4000 3000 2000 1000
0
P W C EE V D EN D 1 EN D 2 EN D 3 EN TB 4 EV JE V W N V N C Bl an k
JE V N V N C Bl an k W
P W C EE V D EN D 1 EN D 2 EN D 3 EN TB 4 EV
0
Antiserum
Antiserum
Fig. 2 Specificity of the selected antigens detected by the ELISA-array platform. The immunized rabbit antiserum was diluted with the ratio of 1:100. The x-axis shows different virus-immunized rabbit antisera. NC represents preimmunized (negative control) rabbit antiserum. The ordinate is the mean chemiluminescence signal for specific IgG bound
to the printed antigens. a–i The result of different antigens tested. Each data point represents the mean signal intensity from triplicate spot measurement. The specificity the tested antigen was compared with the NC group and the group with the second highest signal. *p
ARTICLE
A multiplex ELISA-based protein array for screening diagnostic antigens and diagnosis of Flaviviridae infection D. Wang & Y. Zheng & X. Kang & X. Zhang & H. Hao & W. Chen & L. Liu & X. Li & L. Li & Q. Yuan & F. Chen & Y. Yang & Y. Jiang & H. Jiang
Received: 20 November 2014 / Accepted: 16 February 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Assays with the ability to detect multiple antibodies in parallel have a wide range of potential applications in epidemiologic research. Here, a multiplex enzyme-linked immunosorbent assay-based protein array (ELISA-array) was developed to simultaneously detect five Flaviviridae infections. The platform was based on an indirect ELISA and 15 antigens were constructed for specific antibody detection against five Flaviviridae viruses (Japanese B, tick-borne encephalitis, West Nile, dengue, and yellow fever viruses) and four serotypes of dengue virus. The specificity was evaluated by calculating the signal value cross-reacting with serum immunized with other viruses, and the sensitivity of antigens was compared with conventional ELISAs using immunized rabbit polyclonal antisera. IgG and IgM calibration curves were constructed to evaluate the reproducibility of the platform. Finally, 24 dengue fever (DF) infection and 15 tick-borne encephalitis (TBE) infection clinical sera were used to compare the D. Wang and Y. Zheng contributed equally to this work. D. Wang : Y. Zheng : X. Kang : X. Zhang : L. Li : Q. Yuan : Y. Yang (*) : Y. Jiang (*) : H. Jiang (*) State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing 100071, China e-mail: [email protected] e-mail: [email protected] e-mail: [email protected] H. Hao CAS Key Laboratory of Pathogenic Microbiology and Immunity, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China W. Chen : L. Liu : X. Li Genomics Institute in Shenzhen, Beishan Industry Beishan Road, Shenzhen 518083, China F. Chen College of Food Technology, Huazhong Agricultural University, Wuhan, China
advantage of ELISA-array to ELISA. After initial screening, 9 out of 15 antigens were chosen for ELISA-array printing. By using different virus-immunized rabbit antiserum, 7 out of 9 antigens showed good specificity in the ELISA-array. Eight out of 9 antigens showed four-fold greater sensitivity in ELISA-array compared to that in conventional ELISAs. The coefficients of determination (r2) close to 1 showed high reproducibility, and clinical sera test showed that ELISA-array had higher specificity and sensitivity than traditional ELISA. ELISA-array was a good platform for antigen screening and this multiplexed assay might be a useful and convenient tool for multiple immunological detection of infectious viral antibodies.
Introduction Viral antigen–antibody reactions assessed with conventional enzyme-linked immunosorbent assay (ELISA) requires each antigen to be tested separately and involves long incubation periods. It is labor-intensive and time-consuming, and requires significant volumes of antigen and serum or plasma. An immunoassay that simultaneously measures multiple antigen– antibody reactions would have significant clinical value, and such an ELISA-based microarray is emerging as a strong candidate platform for multiplex protein analysis due to its highthroughput potential, assay sensitivity and stringency, ease of handling, and its low sample volume demand. An ELISAbased microarray printed directly onto the bottom of a 96well glass plate was first reported by Genometrix [1] and can now also be printed directly onto the bottom of 384-well plastic microtiter plates (microELISA) [2, 3]. For example, SearchLight™ arrays (Pierce Biotechnology Inc., Rockford, IL, USA) have up to 16 assays per well in an indirect ELISA format [3]. The microELISA platform has been compared
Eur J Clin Microbiol Infect Dis
with routine identical conventional ELISAs and has been validated by tumor markers and cytokines [4, 5]. However, microELISA has not been reported for the detection of viral antibodies. With increasing human population growth and mobility, the emergence and spread of zoonotic and vector-borne diseases is a continual concern as air travel facilitates the global spread of infectious vector-borne diseases [6, 7]. Japanese B encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), West Nile virus (WNV), yellow fever virus (YFV), and dengue virus (DENV) are mosquito- or tick-transmitted viruses of the Flaviviridae family. They are causative pathogens of viral encephalitis or hemorrhagic fever. JEV, DENV, and TBEV are principal arboviruses of current public health importance in mainland China [8, 9]. Increasing evidence suggests that other arboviruses might be present and cause additional human infections in China [9, 10]. Thus, the early identification of highly pathogenic arboviruses is critical, and to achieve this, selection and validation of specific diagnostic antigens is a priority. Here, we depict a multiplex ELISA-based protein array (ELISA-array) platform to screen specific diagnostic antigens. This platform was based on an indirect ELISA consisting of antigens printed directly on a 96-well microtiter plate, which permitted 16 or 9 immunological assays per well. Compared to conventional ELISA, this multiplexed assay may offer better and more convenient immunological assay techniques for infectious viral agents.
Materials and methods Viruses JEV and DENV types 1–4 were grown in C6/36 cells in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 % fetal calf serum (FCS). TBEV, YFV, and WNV were grown in BHK21/23 cells in DMEM supplemented with 10 % FCS. Viruses were cultured at biosafety level 2 or 3 facilities. Virus titers were determined using a 50 % infectious dose in tissue culture (TCID50/mL) method. All viruses were inactivated prior to use with 1:4,000 β-propiolactone overnight at 4 °C. Viral antigen expression in Escherichia coli The primers and restriction sites are summarized in Table 1. Amplified fragments were cut with restriction enzyme and ligated into pET30a. Recombinants were transformed into E. coli BL21 (Novagen). Recombinant antigens were purified using nickel affinity chromatography. Protein purification was performed under nondenaturating conditions to maintain structures and purified proteins were stored at 4 °C and
analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) immediately prior to spotting to assess integrity, identity, and purity. Purified protein identified was confirmed based on expected molecular weight. Spotted antigens exceeded >90 % purity. Polyclonal antisera from immunized animals Rabbit polyclonal antiserum against WEEV, JEV, TBEV, WNV, DENV types 1–4, and YFV were prepared according to standard procedures. Briefly, the inactivated virus was purified by ultracentrifugation at 146,000×g at 4 °C for 2.5 h. The pellet was resuspended in phosphate-buffered saline (PBS) and quantified using a BCA assay (Pierce®, Thermo Scientific, Waltham, MA, USA). Female New Zealand rabbits weighing 1.5–2 kg and sera negatives for virus were subcutaneously inoculated with inactivated virus (200 μg–1 mg each) in Freund’s complete adjuvant (Sigma-Aldrich, St. Louis, MO, USA). The immunized rabbits received booster doses, which was 1/5-2/5 as the primary dose, at a time interval of 1– 2 weeks in Freund’s incomplete adjuvant (Sigma-Aldrich, St. Louis, MO, USA) for two times. Antisera were collected two weeks after the final injection and stored at −70 °C for further use. The specificity of immunized serum was confirmed with a standard immunofluorescence assay (IFA) and ELISA. Collection of human serum samples Serum samples of 15 clinically confirmed TBE cases were collected from Mudanjiang Forestry Hospital, and sera from 24 confirmed cases of dengue fever (DF) were collected from Guangzhou Hospital. TBE sera samples were collected 10–34 days after tick bites and 3–12 days after disease onset, and DF sera samples were collected 2–13 days after syndrome onset. An additional 30 healthy adult sera samples were collected from Beijing 301th Hospital without contact with TBEV and DENV. These healthy adults had no history of recent visits to TBEV or DENV epidemic regions, and no subject received TBEV/DENV vaccinations. All samples were collected at outpatient clinics and stored at −80 °C prior to use. ELISA-array printing Purified antigens were serially diluted with dilution buffer 0.02 mol L−1 PBS (pH 7.2) and printed in a 3×3 or a 4×4 grid in each well of a 96-well plate (Greiner Glasinstrumente GmbH, Nordrhein-Westfalen, Germany) (Fig. 1a) using a Bio-Dot Printing System (Beijing BGI-GBI Biotech Co. Ltd., Beijing, China). For rabbit antiserum testing, rabbit IgG was printed at the first spot in each well as a positive control (Fig. 1a). For clinical serum testing, human IgG and human IgM was printed at the first and last spots in each well as a positive control. The final concentration of antigen was 2–
Eur J Clin Microbiol Infect Dis Table 1
The primers and restriction sites used in this research
Virus
Genes
Sequences of primers (5′-3′)
Restriction sites
JEV
E-D3
JEV
PreM
TBEV
E-D3S
TBEV
E-D3I
TBEV
NS3
WNV
E-D3
WNV
NS1
DENV
E-D2
DENV
E-D3
DEN1
E-D3
DEN2
E-D3
F: 5′-CGCGGATCCGGTGGTGGTATGGACAAACTGGCTTTGAAGGGCACGAC-3′ R: 5′-CCGCTCGAGACCACCACCCGTGCTTCCAGCTTTGTGCCAGTGATGGT-3′ F: 5′-CGGGATCCATGAAGTTGTCAAATTTCCAGG-3′ R: 5′-CCGCTCGAGAGCATAGCCAGGATTCCTTAC-3′ F: 5′-ACGGCCATGGCGACATACACAATG-3′ R: 5′-CGTGGATATCTTTTTGGAACC-3′ F: 5′-CGCGGATCCGGTGGTGGTTTGACCTACACCATGTGCG-3′ R: 5′-CCGCTCGAGACCACCACCGATAGAAGAACCCTTCTGG-3′ F: 5′-CGGGATCCGGTAAGAGTGAACCCTTTCCG-3′ R: 5′-CCGCTCGAGTATTTGCGCCTCTTTCCATTGCACTAATCCACTGTCATC-3′ F: 5′-CGGGATCCGGTGGTGGTGGAACAACCTACGGCGTCTGT-3′ R: 5′-CCGCTCGAGTCCAGACTTGTGCCAATGGTGA-3′ F: 5′-CGGGATCCGACACTGGGTGTGCCATAGACATC-3′ R: 5′-CCCTCGAGTAGCATTCACTTGTGACTGCACG-3′ F: 5′-CGCGGATCCGGTGGTGGTAACCCTGCCGTCTTGCGCAAACT-3′ R: 5′-CCGCTCGAGACCACCACCTGCAAAGATTGTTGTCGTTCCT-3′ F: 5′-CGCGGATCCGGTGGTGGTATGTGCACAGGCTCATTTAAGCTAGA-3′ R: 5′-CCGCTCGAGACCACCACCTTTCCCTATGCTGCTTCCTTTCTTGA-3′ F: 5′-CGGGATCCGGTGGTGGTACGACAAAAATTTTTGCAGGACAC-3′ R: 5′-CCGCTCGAGTTTCCCTATGCTGCTTCCTTTCTTG-3′ R: 5′-CCCAAGCTTTGGGTGGTGGTGGATCCCAAGAAGGGGCCATG-3 R: 5′-CCGCTCGAGTCCTTTCTTAAACCAGTTGAGCTT-3′
BamHI Xho I BamHI Xho I Nco I EcoR V BamHI Xho I BamHI Xho I BamHI Xho I BamHI Xho I BamHI Xho I BamHI Xho I BamHI Xho I Hind III Xho I
DEN3
E-D3
DEN4
E-D3
YFV
NS3
YFV
E-D3
F: 5′-CGGGATCCTATGCAATGTGCTTGAATACCTTTG-3′ R: 5′-CCCTCGAGTCTTCCCAATCGAGCTTCCCTTC-3′ F: 5′-CGGGATCCGGTGGTGGTTGCAAAGTTCGCATGGAGAAA-3 R: 5′-GTGCTCGAGTCGCTTTGCGCCTCTGTATGTGGA-3′ F: 5′-CGGGATCCAACAGAGATGGAGACTCATACTAC-3′ R: 5′-CCGCTCGAGACTCTGGTCAGATGACACCC-3′ F: 5′-CGCGGATCCGGTGGTGGTACCTCTTACAAGATCTGTAC-3′ R: 5′-CCGCTCGAGACCACCACCTTCCTTGTGCCATTGGT-3′
BamHI Xho I BamHI Xho I BamHI Xho I BamHI Xho I
The underlined sequences are the restriction sites
600 mg L−1. Printing was performed in a cabinet at 55 % humidity and temperature 20±3 °C, and the conditions were monitored by a thermohygrometer. To avoid cross-contamination among spots, pins were washed with Milli-Q sterile water, 75 % ethanol, and then with 0.5 % sodium dodecyl sulfate (SDS). Printed plates were immediately removed from the cabinet and incubated for 1.5 h at 37 °C to secure antigen attachment to wells. After washing twice with phosphate buffered saline with 0.05 % Tween20 (PBST), the plates were incubated for 1 h at 37 °C with a solution of 10 % newborn calf serum in PBS to block nonspecific antibody binding. Finally, plates were inverted for 12 h on blotting paper at room temperature, and stored in boxes at 4 °C in the presence of silica gel bags as a desiccant, and used within 90 days of being printed. ELISA-array analysis Fifty microliters of rabbit antiserum diluted to various concentrations (dilution buffer 0.9 % NaCl and 0.1 % TritonX-100 with 5 % FCS) were added to the appropriate wells for 1 h at 37 °C.
Plates were washed five times in an ELx405™ microplate washer (BioTek Instruments, Winooski, VT, USA) with PBST, pH 7.2, which contained 0.05 % Tween20. Fifty microliters of horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibodies (Beijing CoWin Biotech Co. Ltd., Beijing, China) diluted in 0.02 mol L−1 PBS with 20 % FCS (1:4,000) were added to the plate for 40 min at 37 °C. Plates were washed five times with PBST as above. Forty microliters of SuperSignal ELISA Femto Stable Peroxide Solution (Pierce) diluted in distilled water (1:8) were added to produce a chemiluminescent response. The plate was imaged for 30 s using an AE1000 Cool CCD Image Analyzer (Beijing BGI-GBI Biotech Co. Ltd.) and analyzed by ELISA-Array software (provided by Beijing Genomics Institute, Shenzhen, China). The software calculated the pixel intensity for each spot. A ratio of immunized rabbit serum or patient sera intensity/mean value of unimmunized rabbit serum or healthy sera intensity ≥2.0 was regarded as positive. For clinical samples tested by ELISA-array, sera were diluted (1:600) and added to appropriate wells for 1 h at 37 °C.
Eur J Clin Microbiol Infect Dis Fig. 1 Array-ELISA 9-element array general design and schematic. a Arrays (9-element) were printed into each of the 96 wells on a polystyrene material array plate. Viral antigens were spotted as shown. Antigens were linked to the surface, and the measured antibody recognized the antigen. Next, the HRP-linked secondary antibody recognized bound measured antibody. Antisera against each virus were measured in duplicate in two rows. Antisera were added to each row at the same dilution. Finally, a luminol substrate was used to generate a chemiluminescent signal. b Columns 1 to 10: probed with sera from rabbit immunized with WEEV, DEN1, DEN2, DEN3, DEN4, TBEV, JEV, WNV, unimmunized (negative control), and blank, respectively. R1, R2: immunized rabbit antiserum (1:100) was tested in duplicate. ELISA: enzyme-linked immunosorbent assay; HRP: horseradish peroxidase
Fifty microliters of HRP-conjugated goat anti-human IgG antibodies (Beijing CoWin Biotech Co. Ltd.) or HRPconjugated goat anti-human IgM antibodies (Beijing CoWin Biotech Co. Ltd.) were added (1:4,000) and incubated for 40 min at 37 °C. For the detection of IgM antibodies, goat anti-human IgG serum (Beijing CoWin Biotech Co. Ltd.; 1:100) was added to sera sample diluent in advance to prevent IgG interference. Other protocols were identical to those depicted for the ELISA-array described above. Conventional ELISAs Recombinant antigens JEV-E-D3, TBEV-E-D3S, TBEV-ED3I, WNV-E-D3, and DENV-E-D3 types 1–4 used in the
ELISA-array were also tested by conventional ELISA. ELIS As were performed according to standard procedures: 96-well polystyrene round-bottom microtiter plates (MaxiSorp Nunc A/S, Roskilde, Denmark) were coated with antigens diluted in 0.02 mol L−1 PBS, and antigen binding was performed at 37 °C for 1.5 h. Optimal antigen concentrations were 6 mg L−1 for JEV-E-D3, 0.34 mg L−1 for TBEV-E-D3S, 6 mg L−1 for TBEV-E-D3I, 0.02 mg L−1 for WNV-E-D3, 0.2 mg L−1 for DENV-E-D3, 0.02 mg L−1 for DEN1-E-D3, 0.2 mg L−1 for DEN2-E-D3, 1.67 mg L−1 for DEN3-E-D3, and 0.12 mg L−1 for DEN4-E-D3, and each well was coated with 100 μL of diluted antigen. Unadsorbed material was removed with two washings of 0.05 % Tween 20 in PBS. Then, the plates were incubated for 1 h at 37 °C with a solution
Eur J Clin Microbiol Infect Dis
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Fig. 2 Specificity of the selected antigens detected by the ELISA-array platform. The immunized rabbit antiserum was diluted with the ratio of 1:100. The x-axis shows different virus-immunized rabbit antisera. NC represents preimmunized (negative control) rabbit antiserum. The ordinate is the mean chemiluminescence signal for specific IgG bound
to the printed antigens. a–i The result of different antigens tested. Each data point represents the mean signal intensity from triplicate spot measurement. The specificity the tested antigen was compared with the NC group and the group with the second highest signal. *p
