Accepted Manuscript Title: Direct coating of culture medium from cells secreting classical swine fever virus E2 antigen on ELISA plates for detection of E2-specific antibodies Author: Ta-Chun Cheng, Chu-Hsiang Pan, Chien-Shu Chen, Kuo-Hsiang Chuang, Chih-Hung Chuang, Chien-Chaio Huang, Yu-Yi Chu, Ya-Chun Yang, Pei-Yu Chu, Chien-Han Kao, Yuan-Chin Hsieh, Tian-Lu Cheng PII: DOI: Reference:
S1090-0233(15)00068-4 http://dx.doi.org/doi:10.1016/j.tvjl.2015.02.007 YTVJL 4422
To appear in:
The Veterinary Journal
Accepted date:
4-2-2015
Please cite this article as: Ta-Chun Cheng, Chu-Hsiang Pan, Chien-Shu Chen, Kuo-Hsiang Chuang, Chih-Hung Chuang, Chien-Chaio Huang, Yu-Yi Chu, Ya-Chun Yang, Pei-Yu Chu, Chien-Han Kao, Yuan-Chin Hsieh, Tian-Lu Cheng, Direct coating of culture medium from cells secreting classical swine fever virus E2 antigen on ELISA plates for detection of E2-specific antibodies, The Veterinary Journal (2015), http://dx.doi.org/doi:10.1016/j.tvjl.2015.02.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
Short Communication
Direct coating of culture medium from cells secreting classical swine fever virus E2 antigen on ELISA plates for detection of E2-specific antibodies Ta-Chun Cheng a,1, Chu-Hsiang Pan b,1, Chien-Shu Chen c,1, Kuo-Hsiang Chuang a, Chih-Hung Chuang d, Chien-Chaio Huang d, Yu-Yi Chu e, Ya-Chun Yang d, Pei-Yu Chu d, Chien-Han Kao f, Yuan-Chin Hsieh f, Tian-Lu Cheng d,f,g,* a
Graduate Institute of Pharmacognosy, Taipei Medical University, 252 Wu Hsing Street, Taipei 110, Taiwan b Division of Hog Cholera Research, Animal Health Research Institute, Council of Agriculture, 376 Chung-Cheng Road, New Taipei City 251, Taiwan c School of Pharmacy, China Medical University, 91 Hsueh-Shih Road, Taichung 404, Taiwan d Department of Biomedical and Environmental Biology, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan e Institute of Bioinformatics and Biosignal Transduction, College of Bioscience and Biotechnology, National Cheng Kung University, 1 University Road, Tainan 701, Taiwan f Graduate Institute of Medicine, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan g Center for Biomarkers and Biotech Drugs, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan *Corresponding author. Tel.: +886 7 3121101 2360. E-mail address: [email protected] (T-L. Cheng). 1
These authors contributed equally to this study. Highlights 1. We generated a mammalian cell-based expression system for CSFV E2 protein. 2. The CSFV E2 protein is effectively secreted and well produced in the serum-free culture medium. 3. The mammalian cell-secreted CSFV E2 protein achieves complete post-translational modification. 4. The CSFV E2-containing medium can be directly coated on the ELISA plate without any adjustment and purification. 5. The CSFV E2-secreting medium-based ELISA can provide cheap, accurate and effective assessment of anti-E2 antibody in both vaccinated sera and field sera.
Page 1 of 10
45 46
Abstract The envelope glycoprotein E2 of classical swine fever virus (CSFV) is widely used as a
47
marker for measuring vaccine efficacy and antibody titer. The glycosylation profile of E2 may
48
affect the immunogenicity of the vaccine and the timing of re-vaccination. In this study, a
49
human embryonic kidney cell line was used to secrete fully-glycosylated CSFV E2, which was
50
then coated onto ELISA plates without purification or adjustment.
51 52
The resulting E2-secreting medium-direct-coating (E2-mDc) ELISA was successfully
53
used to measure anti-E2 antibody titers in vaccinated and field pig sera samples. Compared
54
with a virus neutralization test (as standard), the E2-mDc ELISA was found to be more
55
accurate (90%) than a commercial CSFV antibody diagnostic kit (62%). In conclusion, the
56
mammalian cell-secreted antigen can provide cheap, accurate and effective assays for vaccine
57
efficacy and disease diagnoses.
58 59
Keywords: Classical swine fever virus; E2; Glycosylation; Mammalian expression; Medium-
60
direct-coating ELISA
61
Page 2 of 10
62
Classical swine fever caused by the classical swine fever virus (CSFV) is a highly
63
contagious swine disease that results in serious financial losses. The envelope glycoprotein E2
64
of CSFV is a major determinant of CSFV virulence (Risatti et al., 2005) and can effectively
65
induce humoral neutralizing antibodies when used in sub-unit vaccines (Heldens et al., 2008).
66
The anti-E2 antibody has been developed as a commonly used diagnostic marker for evaluating
67
vaccination efficacy by detecting antibody titers and as an important index for determining
68
whether re-vaccination is necessary (Ganges et al., 2008).
69 70
Many commercial E2-based ELISA kits have been developed for neutralizing antibody
71
detection but to date most CSFV E2 glycoprotein has been produced by bacteria, yeast and
72
baculovirus expression systems. Producing E2 by these methods has two major drawbacks.
73
Firstly, E2 protein purification is necessary for antigen coating on the ELISA plate; this is
74
expensive and the process is time-consuming. Secondly, it has been reported that fully
75
glycosylated E2 induces neutralizing antibody titers and protection against CSFV challenge,
76
but un-glycosylated E2 does not (Gavrilov et al., 2011). The development of a fully
77
glycosylated CSFV E2-based ELISA may reduce the risks of inaccurate antibody titer
78
measurements and vaccination efficacy evaluations.
79 80
In this study, we fused the E2 extracellular domain with immunoglobulin kappa (Igκ)
81
signal peptide (Chuang et al., 2010) and Myc-His tags to generate a cell-secreting E2 (see
82
Appendix: Supplementary Fig. S1). In addition, we used a human embryonic kidney cell line
83
(HEK293) to secrete the glycoprotein E2 of CSFV (the major product comprising >90% of
84
total protein) (Fig. 1A) with concentrations as high as 130 μg/mL. Furthermore, we
85
demonstrated that the molecular weight of the HEK293-secreting E2 was 56 kDa while that of
86
the Escherichia coli-expressed E2 was 43 kDa (Fig. 1B). The 13.2 kDa shift in the molecular
Page 3 of 10
87
weight of the HEK293-secreted E2 indicated the fully glycosylated status of the HEK293-
88
based E2 (Montesino et al., 2009).
89 90
In order to develop an anti-E2 antibody diagnostic ELISA, the HEK293-secreting E2-
91
containing serum-free culture medium was directly coated onto ELISA plates to generate an
92
E2-secreting medium-direct-coating (E2-mDc) competitive ELISA. This system does not
93
require a protein purification and adjustment step (see Appendix: Supplementary Fig. S2), so it
94
may be used to develop low-cost diagnostic tests for commercial use. The advantages of using
95
HEK293-based protein-production are the serum-free culture conditions, a high level of
96
production, more complete post-translational modification, and facilitating Food and Drug
97
Administration (FDA)-approval for resulting products (Swiech et al., 2012). This HEK293-
98
secreted system may be adapted for other antigens (e.g. Erns and E1) for development of
99
commercial ELISA plates or vaccines.
100 101
The development of effective, diagnostic methods for field use is important in assessing
102
the efficacy of vaccination. CSFV E2 has been shown to effectively induce humoral
103
neutralizing antibodies and in the development of sub-unit vaccines (Heldens et al., 2008). In a
104
preliminary evaluation to detect anti-E2 antibody, we compared the respective capabilities of a
105
purified-E2-based competitive ELISA, an E2-mDc competitive ELISA and a commercial
106
CSFV antibody diagnostic kit (IDEXX) using two live attenuated CSFV vaccinated pig sera
107
(1825 and 1828) and two un-vaccinated sera (specific pathogen free and uninfected) to block
108
the binding of horseradish peroxidase-conjugated anti-CSFV antibodies (IDEXX).
109 110
The results were measured as: % inhibition =
Negative control - Sample 100% . Negative control
111
Page 4 of 10
112
The results indicated that the % inhibition values were similar in the purified-E2-based
113
competitive ELISA, the E2-mDc competitive ELISA and the commercial CSFV antibody
114
diagnostic kit (Fig. 2). We therefore concluded that the E2-mDc competitive ELISA can
115
effectively measure the anti-E2 antibody in both vaccinated serum and uninfected serum. In
116
future, the E2-mDc competitive ELISA could be combined with other sub-unit antigens of
117
CSFV (e.g. Erns and E1) to differentiate E2 subunit vaccinated pigs (with antibodies only
118
against the E2) from CSFV-infected pigs (with antibodies against several antigens of CSFV)
119
(Dong and Chen, 2007). Such a differentiation capacity could be very useful for disease
120
prevention in pigs.
121 122
The glycosylation status of an antigen is important to the immunogenicity and binding
123
efficacy of the antibody (Gavrilov et al., 2011). CSFV E2 has six N-linked glycosylation sites
124
(Montesino et al., 2009). In addition, the glycosylation profile of antigen is important in
125
antibody-antigen interactions and affects the binding affinity of antibody (Grinstead et al.,
126
2002). In the present study, we used fully glycosylated CSFV E2 to generate an anti-E2
127
antibody diagnostic ELISA plate as an alternative to a commercial CSFV antibody diagnostic
128
kit in which the E2 antigen was produced by a baculovirus system. To compare the respective
129
abilities of anti-E2 antibody detection, 25 field pig serum samples were identified by E2-mDc
130
competitive ELISA, commercial CSFV antibody diagnostic kit and virus neutralization test
131
(VNT) (as standard) (Li et al., 2013).
132 133
For the E2-mDc competitive ELISA and commercial CSFV antibody diagnostic kit, a
134
positive result was defined as % inhibition >30%. For the VNT, a neutralizing antibody titer
135
>16 was defined as a positive result (Li et al., 2013). Whereas measurement of anti-E2
136
antibody by E2-mDc competitive ELISA showed 90% agreement with a VNT, the commercial
137
CSFV antibody diagnostic kit showed only 62% agreement with the VNT (Table 1). We
Page 5 of 10
138
suggest that using the complete post-translationally modified antigen may improve the
139
accuracy of the diagnostic ELISA. According to the findings of this study, in the future it is
140
possible that a mammalian cell-secreted antigen-based ELISA may be suitable for use as a
141
substitute for the VNT.
142 143
In summary, we successfully generated a mammalian cell-based ELISA by directly
144
coating the medium onto the plates. The advantages of this test are: (1) complete post-
145
translational modification of the antigen; (2) effective and major production of antigens in the
146
culture medium; (3) no necessity for protein purification; (4) direct coating of the antigen on
147
the plate without any adjustment. These advantages should allow commercialization of this
148
ELISA kit at a low cost. Furthermore, this ELISA has been shown to be suitable for adaptation
149
for use with other antigens in a more effective assessment of vaccine efficacy and disease
150
diagnosis.
151 152 153 154
Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.
155 156 157
Acknowledgements This work was supported by grants from the National Research Program for
158
Biopharmaceuticals, Ministry of Science and Technology, Taipei, Taiwan (MOST 103-2325-
159
B-037-007, NSC 102-2320-B-038-043-MY2), the Ministry of Health and Welfare, Taiwan
160
(MOHW103-TD-B-111-05), the National Health Research Institutes, Taiwan (NHRI-EX103-
161
10238SC), 103NSYSU-KMU Joint Research Project (NSYSUKMU103 I-003) and the Grant
162
of Biosignature in Colorectal Cancers, Academia Sinica, Taiwan. This study was also
Page 6 of 10
163
supported partially by Aim for the Top 500 Universities grant, Kaohsiung Medical University,
164
Taiwan (KMU-TP103C00 and KMU-DT103005).
165 166 167 168
Appendix: Supplementary material Supplementary data associated with this article can be found, in the online version, at doi: …setters please insert doi number
169 170
References
171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203
Chuang, K.H., Wang, H.E., Cheng, T.C., Tzou, S.C., Tseng, W.L., Hung, W.C., Tai, M.H., Chang, T.K., Roffler, S.R., Cheng, T.L., 2010. Development of a universal antipolyethylene glycol reporter gene for noninvasive imaging of PEGylated probes. Journal of Nuclear Medicine 51, 933-941. Dong, X.N., Chen, Y.H., 2007. Marker vaccine strategies and candidate CSFV marker vaccines. Vaccine 25, 205-230. Ganges, L., Nunez, J.I., Sobrino, F., Borrego, B., Fernandez-Borges, N., Frias-Lepoureau, M.T., Rodriguez, F., 2008. Recent advances in the development of recombinant vaccines against classical swine fever virus: Cellular responses also play a role in protection. The Veterinary Journal 177, 169-177. Gavrilov, B.K., Rogers, K., Fernandez-Sainz, I.J., Holinka, L.G., Borca, M.V., Risatti, G.R., 2011. Effects of glycosylation on antigenicity and immunogenicity of classical swine fever virus envelope proteins. Virology 420, 135-145. Grinstead, J.S., Koganty, R.R., Krantz, M.J., Longenecker, B.M., Campbell, A.P., 2002. Effect of glycosylation on MUC1 humoral immune recognition: NMR studies of MUC1 glycopeptide-antibody interactions. Biochemistry 41, 9946-9961. Heldens, J.G., Patel, J.R., Chanter, N., Ten Thij, G.J., Gravendijck, M., Schijns, V.E., Langen, A., Schetters, T.P., 2008. Veterinary vaccine development from an industrial perspective. The Veterinary Journal 178, 7-20. Li, W., Mao, L., Yang, L., Zhou, B., Jiang, J., 2013. Development and partial validation of a recombinant E2-based indirect ELISA for detection of specific IgM antibody responses against classical swine fever virus. Journal of Virological Methods 191, 63-68. Montesino, R., Toledo, J.R., Sanchez, O., Zamora, Y., Barrera, M., Royle, L., Rudd, P.M., Dwek, R.A., Harvey, D.J., Cremata, J.A., 2009. N-glycosylation pattern of E2 glycoprotein from classical swine fever virus. Journal of Proteome Research 8, 546-555.
Page 7 of 10
204 205 206 207 208 209 210 211
Risatti, G.R., Borca, M.V., Kutish, G.F., Lu, Z., Holinka, L.G., French, R.A., Tulman, E.R., Rock, D.L., 2005. The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine. Journal of Virology 79, 3787-3796. Swiech, K., Picanco-Castro, V., Covas, D.T., 2012. Human cells: New platform for recombinant therapeutic protein production. Protein Expression and Purification 84, 147-153. Figure legend
212
Fig. 1. Characterization of HEK293-secreted classical swine fever virus E2. (A) The protein
213
loading of purified and unpurified HEK293/E2 medium was visualized by Coomassie brilliant
214
blue staining. (B) The molecular weight of HEK293- and E. coli-produced E2 was detected by
215
western blot using anti-E2 monoclonal antibody (WH303).
216 217
Fig. 2. Blocking assay of the purified E2-based competitive ELISA, E2-secreting medium-
218
direct-coating competitive ELISA and commercial classical swine fever virus (CSFV) antibody
219
diagnostic kit. The anti-E2 antibody positive serum (1825 and 1828), specific pathogen free
220
(SPF) serum and uninfected serum were 2-fold diluted and added to the plate of the purified
221
E2-based competitive ELISA (Purified E2), E2-secreting medium-direct-coating competitive
222
ELISA (E2-mDc) or commercial CSFV antibody diagnostic kit (Kit). The blocking abilities of
223
sera were determined by competing with horseradish peroxidase-conjugated anti-CSFV
224
antibodies of the commercial CSFV antibody diagnostic kit. The data are shown as %
225
inhibition (mean ± standard deviation).
226 227
Page 8 of 10
228
Table 1
229
Field pig serum samples were determined by the E2-secreting medium-direct-coating (E2-
230
mDc) completive ELISA, the classical swine fever virus (CSFV) antibody diagnostic kit and
231
virus neutralization test.
232 Sera 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Result d + + + + + + + + + + + + + + + + + +
Kit a % inhibition 13.0 47.7 62.7 29.5 23.3 36.3 38.7 37.7 54.7 60.9 57.1 60.8 73.2 86.4 52.3 56.3 68.4 71.5 73.8 78.7 78.9
E2-mDc b Result d % inhibition 20.2 27.7 29.3 20.0 11.6 22.2 14.4 24.9 28.5 24.2 30.4 + + 31.3 + 47.1 + 76.4 22.8 + 45.2 + 55.4 + 49.4 + 46.3 + 60.1 + 71.6
VNT c Result e Titer 8 8 8 16 16 16 16 16 16 16 16 + 32 + 32 + 32 + 64 + 64 + 64 + 64 + 64 + 128 + 128
233 234
a
Commercial CSFV antibody diagnostic kit.
235
b
E2-mDc completive ELISA.
236
c
Virus neutralization test.
237
d
The inhibition >30 % was defined as a positive result.
238
e
The neutralizing antibody titer >16 was defined as a positive result.
239
Page 9 of 10
240
Page 10 of 10
S1090-0233(15)00068-4 http://dx.doi.org/doi:10.1016/j.tvjl.2015.02.007 YTVJL 4422
To appear in:
The Veterinary Journal
Accepted date:
4-2-2015
Please cite this article as: Ta-Chun Cheng, Chu-Hsiang Pan, Chien-Shu Chen, Kuo-Hsiang Chuang, Chih-Hung Chuang, Chien-Chaio Huang, Yu-Yi Chu, Ya-Chun Yang, Pei-Yu Chu, Chien-Han Kao, Yuan-Chin Hsieh, Tian-Lu Cheng, Direct coating of culture medium from cells secreting classical swine fever virus E2 antigen on ELISA plates for detection of E2-specific antibodies, The Veterinary Journal (2015), http://dx.doi.org/doi:10.1016/j.tvjl.2015.02.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
Short Communication
Direct coating of culture medium from cells secreting classical swine fever virus E2 antigen on ELISA plates for detection of E2-specific antibodies Ta-Chun Cheng a,1, Chu-Hsiang Pan b,1, Chien-Shu Chen c,1, Kuo-Hsiang Chuang a, Chih-Hung Chuang d, Chien-Chaio Huang d, Yu-Yi Chu e, Ya-Chun Yang d, Pei-Yu Chu d, Chien-Han Kao f, Yuan-Chin Hsieh f, Tian-Lu Cheng d,f,g,* a
Graduate Institute of Pharmacognosy, Taipei Medical University, 252 Wu Hsing Street, Taipei 110, Taiwan b Division of Hog Cholera Research, Animal Health Research Institute, Council of Agriculture, 376 Chung-Cheng Road, New Taipei City 251, Taiwan c School of Pharmacy, China Medical University, 91 Hsueh-Shih Road, Taichung 404, Taiwan d Department of Biomedical and Environmental Biology, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan e Institute of Bioinformatics and Biosignal Transduction, College of Bioscience and Biotechnology, National Cheng Kung University, 1 University Road, Tainan 701, Taiwan f Graduate Institute of Medicine, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan g Center for Biomarkers and Biotech Drugs, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan *Corresponding author. Tel.: +886 7 3121101 2360. E-mail address: [email protected] (T-L. Cheng). 1
These authors contributed equally to this study. Highlights 1. We generated a mammalian cell-based expression system for CSFV E2 protein. 2. The CSFV E2 protein is effectively secreted and well produced in the serum-free culture medium. 3. The mammalian cell-secreted CSFV E2 protein achieves complete post-translational modification. 4. The CSFV E2-containing medium can be directly coated on the ELISA plate without any adjustment and purification. 5. The CSFV E2-secreting medium-based ELISA can provide cheap, accurate and effective assessment of anti-E2 antibody in both vaccinated sera and field sera.
Page 1 of 10
45 46
Abstract The envelope glycoprotein E2 of classical swine fever virus (CSFV) is widely used as a
47
marker for measuring vaccine efficacy and antibody titer. The glycosylation profile of E2 may
48
affect the immunogenicity of the vaccine and the timing of re-vaccination. In this study, a
49
human embryonic kidney cell line was used to secrete fully-glycosylated CSFV E2, which was
50
then coated onto ELISA plates without purification or adjustment.
51 52
The resulting E2-secreting medium-direct-coating (E2-mDc) ELISA was successfully
53
used to measure anti-E2 antibody titers in vaccinated and field pig sera samples. Compared
54
with a virus neutralization test (as standard), the E2-mDc ELISA was found to be more
55
accurate (90%) than a commercial CSFV antibody diagnostic kit (62%). In conclusion, the
56
mammalian cell-secreted antigen can provide cheap, accurate and effective assays for vaccine
57
efficacy and disease diagnoses.
58 59
Keywords: Classical swine fever virus; E2; Glycosylation; Mammalian expression; Medium-
60
direct-coating ELISA
61
Page 2 of 10
62
Classical swine fever caused by the classical swine fever virus (CSFV) is a highly
63
contagious swine disease that results in serious financial losses. The envelope glycoprotein E2
64
of CSFV is a major determinant of CSFV virulence (Risatti et al., 2005) and can effectively
65
induce humoral neutralizing antibodies when used in sub-unit vaccines (Heldens et al., 2008).
66
The anti-E2 antibody has been developed as a commonly used diagnostic marker for evaluating
67
vaccination efficacy by detecting antibody titers and as an important index for determining
68
whether re-vaccination is necessary (Ganges et al., 2008).
69 70
Many commercial E2-based ELISA kits have been developed for neutralizing antibody
71
detection but to date most CSFV E2 glycoprotein has been produced by bacteria, yeast and
72
baculovirus expression systems. Producing E2 by these methods has two major drawbacks.
73
Firstly, E2 protein purification is necessary for antigen coating on the ELISA plate; this is
74
expensive and the process is time-consuming. Secondly, it has been reported that fully
75
glycosylated E2 induces neutralizing antibody titers and protection against CSFV challenge,
76
but un-glycosylated E2 does not (Gavrilov et al., 2011). The development of a fully
77
glycosylated CSFV E2-based ELISA may reduce the risks of inaccurate antibody titer
78
measurements and vaccination efficacy evaluations.
79 80
In this study, we fused the E2 extracellular domain with immunoglobulin kappa (Igκ)
81
signal peptide (Chuang et al., 2010) and Myc-His tags to generate a cell-secreting E2 (see
82
Appendix: Supplementary Fig. S1). In addition, we used a human embryonic kidney cell line
83
(HEK293) to secrete the glycoprotein E2 of CSFV (the major product comprising >90% of
84
total protein) (Fig. 1A) with concentrations as high as 130 μg/mL. Furthermore, we
85
demonstrated that the molecular weight of the HEK293-secreting E2 was 56 kDa while that of
86
the Escherichia coli-expressed E2 was 43 kDa (Fig. 1B). The 13.2 kDa shift in the molecular
Page 3 of 10
87
weight of the HEK293-secreted E2 indicated the fully glycosylated status of the HEK293-
88
based E2 (Montesino et al., 2009).
89 90
In order to develop an anti-E2 antibody diagnostic ELISA, the HEK293-secreting E2-
91
containing serum-free culture medium was directly coated onto ELISA plates to generate an
92
E2-secreting medium-direct-coating (E2-mDc) competitive ELISA. This system does not
93
require a protein purification and adjustment step (see Appendix: Supplementary Fig. S2), so it
94
may be used to develop low-cost diagnostic tests for commercial use. The advantages of using
95
HEK293-based protein-production are the serum-free culture conditions, a high level of
96
production, more complete post-translational modification, and facilitating Food and Drug
97
Administration (FDA)-approval for resulting products (Swiech et al., 2012). This HEK293-
98
secreted system may be adapted for other antigens (e.g. Erns and E1) for development of
99
commercial ELISA plates or vaccines.
100 101
The development of effective, diagnostic methods for field use is important in assessing
102
the efficacy of vaccination. CSFV E2 has been shown to effectively induce humoral
103
neutralizing antibodies and in the development of sub-unit vaccines (Heldens et al., 2008). In a
104
preliminary evaluation to detect anti-E2 antibody, we compared the respective capabilities of a
105
purified-E2-based competitive ELISA, an E2-mDc competitive ELISA and a commercial
106
CSFV antibody diagnostic kit (IDEXX) using two live attenuated CSFV vaccinated pig sera
107
(1825 and 1828) and two un-vaccinated sera (specific pathogen free and uninfected) to block
108
the binding of horseradish peroxidase-conjugated anti-CSFV antibodies (IDEXX).
109 110
The results were measured as: % inhibition =
Negative control - Sample 100% . Negative control
111
Page 4 of 10
112
The results indicated that the % inhibition values were similar in the purified-E2-based
113
competitive ELISA, the E2-mDc competitive ELISA and the commercial CSFV antibody
114
diagnostic kit (Fig. 2). We therefore concluded that the E2-mDc competitive ELISA can
115
effectively measure the anti-E2 antibody in both vaccinated serum and uninfected serum. In
116
future, the E2-mDc competitive ELISA could be combined with other sub-unit antigens of
117
CSFV (e.g. Erns and E1) to differentiate E2 subunit vaccinated pigs (with antibodies only
118
against the E2) from CSFV-infected pigs (with antibodies against several antigens of CSFV)
119
(Dong and Chen, 2007). Such a differentiation capacity could be very useful for disease
120
prevention in pigs.
121 122
The glycosylation status of an antigen is important to the immunogenicity and binding
123
efficacy of the antibody (Gavrilov et al., 2011). CSFV E2 has six N-linked glycosylation sites
124
(Montesino et al., 2009). In addition, the glycosylation profile of antigen is important in
125
antibody-antigen interactions and affects the binding affinity of antibody (Grinstead et al.,
126
2002). In the present study, we used fully glycosylated CSFV E2 to generate an anti-E2
127
antibody diagnostic ELISA plate as an alternative to a commercial CSFV antibody diagnostic
128
kit in which the E2 antigen was produced by a baculovirus system. To compare the respective
129
abilities of anti-E2 antibody detection, 25 field pig serum samples were identified by E2-mDc
130
competitive ELISA, commercial CSFV antibody diagnostic kit and virus neutralization test
131
(VNT) (as standard) (Li et al., 2013).
132 133
For the E2-mDc competitive ELISA and commercial CSFV antibody diagnostic kit, a
134
positive result was defined as % inhibition >30%. For the VNT, a neutralizing antibody titer
135
>16 was defined as a positive result (Li et al., 2013). Whereas measurement of anti-E2
136
antibody by E2-mDc competitive ELISA showed 90% agreement with a VNT, the commercial
137
CSFV antibody diagnostic kit showed only 62% agreement with the VNT (Table 1). We
Page 5 of 10
138
suggest that using the complete post-translationally modified antigen may improve the
139
accuracy of the diagnostic ELISA. According to the findings of this study, in the future it is
140
possible that a mammalian cell-secreted antigen-based ELISA may be suitable for use as a
141
substitute for the VNT.
142 143
In summary, we successfully generated a mammalian cell-based ELISA by directly
144
coating the medium onto the plates. The advantages of this test are: (1) complete post-
145
translational modification of the antigen; (2) effective and major production of antigens in the
146
culture medium; (3) no necessity for protein purification; (4) direct coating of the antigen on
147
the plate without any adjustment. These advantages should allow commercialization of this
148
ELISA kit at a low cost. Furthermore, this ELISA has been shown to be suitable for adaptation
149
for use with other antigens in a more effective assessment of vaccine efficacy and disease
150
diagnosis.
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Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.
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Acknowledgements This work was supported by grants from the National Research Program for
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Biopharmaceuticals, Ministry of Science and Technology, Taipei, Taiwan (MOST 103-2325-
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B-037-007, NSC 102-2320-B-038-043-MY2), the Ministry of Health and Welfare, Taiwan
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(MOHW103-TD-B-111-05), the National Health Research Institutes, Taiwan (NHRI-EX103-
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10238SC), 103NSYSU-KMU Joint Research Project (NSYSUKMU103 I-003) and the Grant
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of Biosignature in Colorectal Cancers, Academia Sinica, Taiwan. This study was also
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supported partially by Aim for the Top 500 Universities grant, Kaohsiung Medical University,
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Taiwan (KMU-TP103C00 and KMU-DT103005).
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Appendix: Supplementary material Supplementary data associated with this article can be found, in the online version, at doi: …setters please insert doi number
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References
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Chuang, K.H., Wang, H.E., Cheng, T.C., Tzou, S.C., Tseng, W.L., Hung, W.C., Tai, M.H., Chang, T.K., Roffler, S.R., Cheng, T.L., 2010. Development of a universal antipolyethylene glycol reporter gene for noninvasive imaging of PEGylated probes. Journal of Nuclear Medicine 51, 933-941. Dong, X.N., Chen, Y.H., 2007. Marker vaccine strategies and candidate CSFV marker vaccines. Vaccine 25, 205-230. Ganges, L., Nunez, J.I., Sobrino, F., Borrego, B., Fernandez-Borges, N., Frias-Lepoureau, M.T., Rodriguez, F., 2008. Recent advances in the development of recombinant vaccines against classical swine fever virus: Cellular responses also play a role in protection. The Veterinary Journal 177, 169-177. Gavrilov, B.K., Rogers, K., Fernandez-Sainz, I.J., Holinka, L.G., Borca, M.V., Risatti, G.R., 2011. Effects of glycosylation on antigenicity and immunogenicity of classical swine fever virus envelope proteins. Virology 420, 135-145. Grinstead, J.S., Koganty, R.R., Krantz, M.J., Longenecker, B.M., Campbell, A.P., 2002. Effect of glycosylation on MUC1 humoral immune recognition: NMR studies of MUC1 glycopeptide-antibody interactions. Biochemistry 41, 9946-9961. Heldens, J.G., Patel, J.R., Chanter, N., Ten Thij, G.J., Gravendijck, M., Schijns, V.E., Langen, A., Schetters, T.P., 2008. Veterinary vaccine development from an industrial perspective. The Veterinary Journal 178, 7-20. Li, W., Mao, L., Yang, L., Zhou, B., Jiang, J., 2013. Development and partial validation of a recombinant E2-based indirect ELISA for detection of specific IgM antibody responses against classical swine fever virus. Journal of Virological Methods 191, 63-68. Montesino, R., Toledo, J.R., Sanchez, O., Zamora, Y., Barrera, M., Royle, L., Rudd, P.M., Dwek, R.A., Harvey, D.J., Cremata, J.A., 2009. N-glycosylation pattern of E2 glycoprotein from classical swine fever virus. Journal of Proteome Research 8, 546-555.
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Risatti, G.R., Borca, M.V., Kutish, G.F., Lu, Z., Holinka, L.G., French, R.A., Tulman, E.R., Rock, D.L., 2005. The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine. Journal of Virology 79, 3787-3796. Swiech, K., Picanco-Castro, V., Covas, D.T., 2012. Human cells: New platform for recombinant therapeutic protein production. Protein Expression and Purification 84, 147-153. Figure legend
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Fig. 1. Characterization of HEK293-secreted classical swine fever virus E2. (A) The protein
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loading of purified and unpurified HEK293/E2 medium was visualized by Coomassie brilliant
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blue staining. (B) The molecular weight of HEK293- and E. coli-produced E2 was detected by
215
western blot using anti-E2 monoclonal antibody (WH303).
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Fig. 2. Blocking assay of the purified E2-based competitive ELISA, E2-secreting medium-
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direct-coating competitive ELISA and commercial classical swine fever virus (CSFV) antibody
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diagnostic kit. The anti-E2 antibody positive serum (1825 and 1828), specific pathogen free
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(SPF) serum and uninfected serum were 2-fold diluted and added to the plate of the purified
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E2-based competitive ELISA (Purified E2), E2-secreting medium-direct-coating competitive
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ELISA (E2-mDc) or commercial CSFV antibody diagnostic kit (Kit). The blocking abilities of
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sera were determined by competing with horseradish peroxidase-conjugated anti-CSFV
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antibodies of the commercial CSFV antibody diagnostic kit. The data are shown as %
225
inhibition (mean ± standard deviation).
226 227
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Table 1
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Field pig serum samples were determined by the E2-secreting medium-direct-coating (E2-
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mDc) completive ELISA, the classical swine fever virus (CSFV) antibody diagnostic kit and
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virus neutralization test.
232 Sera 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Result d + + + + + + + + + + + + + + + + + +
Kit a % inhibition 13.0 47.7 62.7 29.5 23.3 36.3 38.7 37.7 54.7 60.9 57.1 60.8 73.2 86.4 52.3 56.3 68.4 71.5 73.8 78.7 78.9
E2-mDc b Result d % inhibition 20.2 27.7 29.3 20.0 11.6 22.2 14.4 24.9 28.5 24.2 30.4 + + 31.3 + 47.1 + 76.4 22.8 + 45.2 + 55.4 + 49.4 + 46.3 + 60.1 + 71.6
VNT c Result e Titer 8 8 8 16 16 16 16 16 16 16 16 + 32 + 32 + 32 + 64 + 64 + 64 + 64 + 64 + 128 + 128
233 234
a
Commercial CSFV antibody diagnostic kit.
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b
E2-mDc completive ELISA.
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c
Virus neutralization test.
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d
The inhibition >30 % was defined as a positive result.
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e
The neutralizing antibody titer >16 was defined as a positive result.
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