JCM Accepts, published online ahead of print on 10 December 2014 J. Clin. Microbiol. doi:10.1128/JCM.02735-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Non-structural protein 1-specific immunoglobulin M and G antibody-capture
2
enzyme-linked immunosorbent assays in diagnosis of flaviviral infections in
3
humans
4 5
Day-Yu Chao1#, Jedhan Ucat Galula2, Wen-Fan Shen3, Brent S. Davis4,
6
Gwong-Jen J. Chang4#
7
1. Graduate Institute of Microbiology and Public Health, College of Veterinary
8 9
Medicine, National Chung-Hsing University, Taichung, Taiwan (402) 2. Department of Veterinary Medicine, College of Veterinary Medicine, National
10 11
Chung-Hsing University, Taichung, Taiwan (402) 3. Ph.D. Program in Microbial Genomics, National Chung-Hsing University,
12 13
Taichung, Taiwan (402) 4. Division of Vector-Borne Diseases, Centers for Disease Control and Prevention,
14
Fort Collins, Colorado 80521, USA
15 16
#
17
Day-Yu Chao, Ph.D.
18
Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine
19
National Chung-Hsing University
20
Taichung, Taiwan (401)
21
TEL:886-4-22840694
22
FAX:886-4-22852186
23
EMAIL: [email protected]
: Corresponding authors
24 25
Gwong-Jen J Chang, PhD
26
Arboviral Diseases Branch, Division of Vector-borne Diseases, Centers for Disease
27
Control and Prevention
28
Fort Collins, Colorado, USA
29
EMAIL: [email protected] 1
30 31
Keywords: Flavivirus; anti-NS1 antibody; MAC-ELISA ; GAC-ELISA
32
Running headline: Novel approach for anti-NS1 MAC-ELISA
33
Word counts: 244 (abstract); 3568 (text)
34
Conflict of Interests: We declare no conflict of interests.
35 36
2
37
Abstract
38
IgM antibody- and IgG antibody-capture ELISAs (MAC/GAC-ELISA) targeted
39
at envelope protein (E) of dengue viruses (DENV), West Nile virus and Japanese
40
encephalitis virus (JEV) are widely used as sero-diagnostic tests for presumptive
41
confirmation of viral infection.
42
nonstructural protein-1 (NS1) have been proposed as serological markers of natural
43
infections among vaccinated populations. The aim of the current study is to optimize
44
an IgM and IgG antibody-capture ELISAs (MAC/GAC-ELISA) to detect anti-NS1
45
antibodies and compare it with anti-E MAC/GAC-ELISA.
46
premembrane/envelope (prM/E) or NS1 proteins of six medically important
47
flaviviruses, including dengue (DENV-1 to DENV-4), West Nile (WNV) and
48
Japanese encephalitis (JEV) viruses, were constructed.
49
for the productions of prM/E containing virus-like particles (VLPs) and secreted NS1
50
(sNS1) from COS-1 cells.
51
confirmed DENV, JEV, WNV infections, along with naive sera, were subjected to
52
NS1-MAC/GAC-ELISAs before or after depletion of anti-prM/E antibodies by
53
pre-absorption with or without VLPs. Human serum specimens from previously
54
confirmed DENV infections showed significantly enhanced P/N ratios for
55
NS1-MAC/GAC-ELISAs after the depletion of anti-prM/E antibodies. No statistical
Antibodies
directed
against
the
flavivirus
Plasmids to express
These plasmids were used
Archived clinical specimens from patients with
3
56
differences in sensitivities and specificities were found between the newly developed
57
NS1- and VLP-MAC/GAC-ELISAs.
58
JEV infected serum panels showed similar results.
59
MAC/GAC-ELISAs for NS1 antibody detection was successfully developed with
60
great potential to differentiate antibodies elicited by the tetravalent chimeric yellow
61
fever-17D/dengue vaccine or DENV infection.
Further application of the assays to WNV and
62 63
4
A novel approach to perform
64
INTRODUCTION
65
Flaviviruses are associated with a number of mosquito-transmitted diseases
66
worldwide and are serologically divided into several complexes including medically
67
important members of the Japanese encephalitis (JEV) and dengue (DENV) and
68
yellow fever virus (YFV) serocomplexes (1). The DENV serocomplex, comprised
69
of four antigenically distinct serotypes (DENV-1 to -4), is the most important and
70
rapidly emerging group of arthropod-borne pathogens imposing a great deal of
71
economic and public health burden, especially in tropical and subtropical countries
72
(2-5).
73
worldwide with 500,000 severe cases and 25,000 deaths, mostly affecting children
74
(6).
75
vaccine in development is the tetravalent chimeric YF-17D dengue (TCYD) vaccine.
76
Currently, the TCYD vaccine is in a large scale human efficacy trial and the vaccine
77
has been estimated to achieve 56% efficacy based on the primary endpoint calculation
78
(7).
79
future.
80
vaccination, and be used as a surveillance tool to monitor vaccine effectiveness, is
81
urgently needed.
A recent study estimates that annually 390 million DENV infections occur
Currently there is no DENV vaccine available.
The most advanced dengue
Optimistically, the TCYD vaccine could be available globally in the very near Thus, a serological assay that can differentiate natural infection from
5
82
Conventional
serological
tests
such
as
neutralization
(Nt),
83
hemagglutination-inhibition (HAI), and IgM and IgG antibody-capture enzyme-linked
84
immunosorbent assays (MAC- and GAC-ELISA), which exclusively measure
85
antibodies to the premembrane (prM) and envelope (E) structural proteins of
86
flaviviruses, cannot be used to determine natural infections among vaccinated
87
populations (8). Antibodies to nonstructural protein 1 (NS1) have been proposed as
88
serological markers of natural infection among inactivated Japanese encephalitis virus
89
(JEV) vaccinated populations and a NS1-ELISA has been developed (9-11).
90
However, this NS1-ELISA required the microplates to be sensitized with either
91
purified NS1 or NS1 antigen be captured by NS1-specific murine monoclonal
92
antibodies, before adding test human serum specimens.
93
sensitivity of NS1-specific IgM or IgG isotyping might be reduced due to the
94
competition between two antibody isotypes for the same antigenic sites.
95
Isotype-specific antibody-capture ELISA eliminates the isotype competition for the
96
same antigenic sites and, thus enhances the sensitivity of the assay.
97
commercially available anti-prM/E ELISA kits for both IgM and IgG detection are
98
commonly used for DENV clinical sero-diagnosis and results from comprehensive
99
evaluations suggest a good correlation of these assays with the gold standard Nt test
100
(12-14). 6
We speculate that the
A number of
101
Our long term goal is to develop anti-NS1 MAC- and GAC-ELISAs that can be
102
used as tools to differentiate the immune responses elicited by vaccinations versus
103
those elicited by natural infections. We initiated this study to develop and optimize
104
the NS1-MAC- and GAC-ELISAs using recombinant NS1 antigens.
105
archived flavivirus-infected clinical serum panel, the objectives of this initial study
106
are: (1) to determine the sensitivity and specificity of the NS1-MAC/GAC-ELISAs as
107
compared to the standard E-MAC/GAC-ELISAs, and (2) to determine the
108
cross-reactivity of NS1-MAC/GAC-ELISA between different sero-complexes of
109
flaviviruses.
110
reported in previous studies(9, 11), is due to a relative abundance of anti-prM/E
111
antibodies in the serum of infected individuals.
112
procedure to deplete anti-prM/E antibodies using prM/E-containing virus-like
113
particles (VLPs) can improve the sensitivity of NS1-ELISA.
114
DENV-infected serum pairs collected during the acute and convalescent phases were
115
used for extensive comparison of the correlation and agreement of the assays by
116
testing simultaneously with NS1-, VLP-MAC/GAC-ELISAs and InBios DENV
117
Detect IgM capture ELISA.
118
collection panel for the test validation by calculating the sensitivity and specificity of
Using an
We hypothesize that lower sensitivities of NS1 antibody detection, as
In this study, we propose that a
Furthermore, panel of
The test algorithm was expanded to the single serum
7
119
the assay performance since a single time-point clinical is the most common practice
120
in the field study.
121
8
122
Materials and Methods
123
Cell culture, plasmid construction and soluble protein expression
124
COS-1 cells (ATCC CRL 1650; Manassas, VA) were grown at 37°C, with 5%
125
CO2 in Dulbeco's modified Eagle's minimal essential medium (DMEM, GIBCO,
126
Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS,
127
Hyclone Laboratories, Inc., Logan, UT), 110 mg/L sodium pyruvate, 0.1 mM
128
nonessential amino acids, 2 mM L-glutamine, 20 ml/L 7.5% NaHCO3, 100 U/mL
129
penicillin, and 100 ug/mL streptomycin.
130
optimized eukaryotic cell expression plasmid was used as the backbone to express
131
premembrane/envelope (prM/E) or NS1 protein of six medically important
132
flaviviruses including DENV-1 strain 16007, DENV-2 strain 16681, DENV-3 strain
133
C0331/94, DENV-4 strain H241, West Nile virus (WNV) strain NY99, and Japanese
134
encephalitis virus (JEV) strain SA14-14-2 for prM/E and strain CH1392 for NS1.
135
Molecular cloning procedures to construct these plasmids were described previously
136
(15, 16).
137
electroporated with recombinant expression plasmids encoding the prM/E and NS1
138
genes, respectively, using the protocol described previously (17, 18).
139
cells were recovered in 50 ml DMEM, seeded into three separate 75 cm2 culture
A transcriptional and translational
The VLPs and soluble NS1 protein (sNS1) were expressed by COS-1 cells
9
Electroporated
140
flasks for VLP/sNS1 expression and incubated at 28°C with 5% CO2.
The secretion
141
of recombinant antigens was determined from tissue culture media harvested 4–5 days
142
post transformation for VLP and sNS1, respectively.
143
144
Antigen titration and VLP- and NS1-specific MAC/GAC-ELISAs
145
Antigen-capture ELISAs (Ag-ELISAs) were used to detect and quantify secreted
146
antigens from the prM/E- and NS1-gene encoded plasmid transformed COS-1 cells as
147
described previously (17, 18).
148
polyclonal rabbit sera were used to capture the antigens. Murine hyper-immune
149
ascetic fluid (MHIAF) specific for DENV-1 to 4, WNV or JEV were diluted 1:2,000
150
for detection.
151
immunoglobulin recognizing all potential antigenic epitopes were generated at the
152
Centers for Disease Control and Prevention, U.S.A. (CDC).
153
were standardized at an optical density 450nm (OD450) of 1.4, within the region of
154
antigen excess near the upper asymptote of the sigmoidal antigen dilution curve
155
because antigens are considered to be the limiting factor relative to the high
156
concentration of polyclonal rabbit serum and MHIAF antibodies used for capture and
157
detection, respectively.
Anti-DENV-1 to 4, WNV or JEV VLP and NS1
Both the polyclonal capture and detector sera containing high-titer
10
Antigen concentrations
158
Human sera were assayed for the presence of prM/E- and sNS1-specific
159
antibodies using both MAC- and GAC-ELISAs as previously described (18).
160
Briefly, 96-well plates were coated with goat anti-human IgM or IgG (Kirkegaard &
161
Perry Laboratories, Gaithersburg, MD) that was diluted 1:2,000 in PBS buffer and
162
incubated overnight at 4°C.
163
and negative control sera were diluted 1:2,000 in wash buffer (PBS with 0.05%
164
Tween-20), added to wells, and incubated at 37°C for 120 min.
165
JEV VLPs and sNS1 were diluted appropriately in wash buffer and tested against
166
each serum sample in triplicate.
167
detectors.
168
used as negative antigen controls for the standard quantification titration curve and for
169
the MAC/GAC-ELISA, respectively.
170
were determined according to the procedure described previously (18) using internal
171
positive and negative serum controls included in each 96-well plate. Positive (P)
172
values for each specimen were determined as the average OD450 for the patient serum
173
sample reacted with positive viral antigen. Negative (N) values were determined for
174
individual 96-well plates as the average OD450 for the normal human serum control
175
reacted with the positive viral antigen.
176
the diagnostic lab in CDC to interpret test results.
DENV, WNV or JEV infected patient sera and positive
DENV, WNV or
MHIAFs against homologous antigens were used as
The mock-transfected cell culture supernatants or 5% milk/PBS were
The positive-to-negative ratio (P/N) values
This P/N ratio is the current standard used in
11
177
To deplete anti-prM/E antibodies, the same procedure as described for the
178
Ag-ELISA was used to capture secreted VLP antigens over the 96-well plates. The
179
patients’ or negative control sera were diluted 1:2,000 in PBS as previously suggested
180
(19), pre-mixed with VLP antigens, added immediately to wells and incubated at
181
37°C for 60 min.
182
to the pre-coated anti-human IgM or IgG plates for MAC/GAC-ELISA as described
183
above.
A total of 50 L of prM/E antibody-depleted sera were transferred
184
185
186
Human serum panel
Archived serum specimens were obtained from Arboviral Diseases Branch,
187
Division of Vector-Borne Diseases, CDC.
Clinical diagnostic serum specimens
188
available from 1999 to 2008 were assembled and the status of infection was
189
confirmed by 90% focus-reduction microneutralization test (FRµNT).
190
serum panel included 54 acute and convalescent presumptive DENV-infected patient
191
serum pairs from Brazil and 97 presumptive WNV-infected patient serum specimens
192
from South Dakota.
193
a control panel including IgG-positive yellow fever-17D (YF-17D) post-vaccinated
194
sera (n=10), acute phase patient serum specimens from St. Louis encephalitis virus
This archived
Thirty non-DENV patient serum specimens were assembled as
12
195
(SLEV) (n=2), Zika virus (n=1), chikungunya virus (n=10), and other viral (n=7)
196
infections. Thirty-six normal human sera from CDC blood bank were included as
197
the naïve serum control. Among 54 paired serum specimens from Brazil, 36 pairs
198
showed a four-fold increase of Nt antibodies by FRµNT90 and were further classified
199
as taken from individuals with recent DENV infections.
200
suspected DENV-infected patient sera, collected from October 2008 to February 2009,
201
originally sent to ARUP Laboratories (Salt Lake City, UT), and obtained through
202
InBios (Seattle, Washington), were used as a testing panel for validation of the assay.
203
The serum panels with evidence of JEV (n=16) infection were assembled from
204
Taiwanese residents and provided by the Center for Disease Control in Taiwan
205
(Taiwan-CDC) and the JEV infection status was determined by VLP-MAC- and
206
GAC-ELISA.
207
was approved by CDC-human studies review board.
Seventy-four travel-related,
An institutional review board (IRB) waiver to use this serum panel
208 209
Statistical analysis
210
Student’s t test for comparisons between normally distributed continuous
211
variables and Mann-Whitney U test for comparisons between continuous variables
212
that are not normally distributed were used.
213
represented the linear relationship between the P/N (OD450 of test specimen divided 13
The Pearson correlation of coefficient
214
by OD450 of negative control specimen) ratios obtained from two methods of VLP and
215
NS1-MAC-ELISA or VLP and NS1-GAC-ELISA. Its value ranges from -1 to +1,
216
with 1 representing the perfect correlation of the two methods. A significance level
217
=0.05 was obtained under two-tailed p value. P
1
Non-structural protein 1-specific immunoglobulin M and G antibody-capture
2
enzyme-linked immunosorbent assays in diagnosis of flaviviral infections in
3
humans
4 5
Day-Yu Chao1#, Jedhan Ucat Galula2, Wen-Fan Shen3, Brent S. Davis4,
6
Gwong-Jen J. Chang4#
7
1. Graduate Institute of Microbiology and Public Health, College of Veterinary
8 9
Medicine, National Chung-Hsing University, Taichung, Taiwan (402) 2. Department of Veterinary Medicine, College of Veterinary Medicine, National
10 11
Chung-Hsing University, Taichung, Taiwan (402) 3. Ph.D. Program in Microbial Genomics, National Chung-Hsing University,
12 13
Taichung, Taiwan (402) 4. Division of Vector-Borne Diseases, Centers for Disease Control and Prevention,
14
Fort Collins, Colorado 80521, USA
15 16
#
17
Day-Yu Chao, Ph.D.
18
Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine
19
National Chung-Hsing University
20
Taichung, Taiwan (401)
21
TEL:886-4-22840694
22
FAX:886-4-22852186
23
EMAIL: [email protected]
: Corresponding authors
24 25
Gwong-Jen J Chang, PhD
26
Arboviral Diseases Branch, Division of Vector-borne Diseases, Centers for Disease
27
Control and Prevention
28
Fort Collins, Colorado, USA
29
EMAIL: [email protected] 1
30 31
Keywords: Flavivirus; anti-NS1 antibody; MAC-ELISA ; GAC-ELISA
32
Running headline: Novel approach for anti-NS1 MAC-ELISA
33
Word counts: 244 (abstract); 3568 (text)
34
Conflict of Interests: We declare no conflict of interests.
35 36
2
37
Abstract
38
IgM antibody- and IgG antibody-capture ELISAs (MAC/GAC-ELISA) targeted
39
at envelope protein (E) of dengue viruses (DENV), West Nile virus and Japanese
40
encephalitis virus (JEV) are widely used as sero-diagnostic tests for presumptive
41
confirmation of viral infection.
42
nonstructural protein-1 (NS1) have been proposed as serological markers of natural
43
infections among vaccinated populations. The aim of the current study is to optimize
44
an IgM and IgG antibody-capture ELISAs (MAC/GAC-ELISA) to detect anti-NS1
45
antibodies and compare it with anti-E MAC/GAC-ELISA.
46
premembrane/envelope (prM/E) or NS1 proteins of six medically important
47
flaviviruses, including dengue (DENV-1 to DENV-4), West Nile (WNV) and
48
Japanese encephalitis (JEV) viruses, were constructed.
49
for the productions of prM/E containing virus-like particles (VLPs) and secreted NS1
50
(sNS1) from COS-1 cells.
51
confirmed DENV, JEV, WNV infections, along with naive sera, were subjected to
52
NS1-MAC/GAC-ELISAs before or after depletion of anti-prM/E antibodies by
53
pre-absorption with or without VLPs. Human serum specimens from previously
54
confirmed DENV infections showed significantly enhanced P/N ratios for
55
NS1-MAC/GAC-ELISAs after the depletion of anti-prM/E antibodies. No statistical
Antibodies
directed
against
the
flavivirus
Plasmids to express
These plasmids were used
Archived clinical specimens from patients with
3
56
differences in sensitivities and specificities were found between the newly developed
57
NS1- and VLP-MAC/GAC-ELISAs.
58
JEV infected serum panels showed similar results.
59
MAC/GAC-ELISAs for NS1 antibody detection was successfully developed with
60
great potential to differentiate antibodies elicited by the tetravalent chimeric yellow
61
fever-17D/dengue vaccine or DENV infection.
Further application of the assays to WNV and
62 63
4
A novel approach to perform
64
INTRODUCTION
65
Flaviviruses are associated with a number of mosquito-transmitted diseases
66
worldwide and are serologically divided into several complexes including medically
67
important members of the Japanese encephalitis (JEV) and dengue (DENV) and
68
yellow fever virus (YFV) serocomplexes (1). The DENV serocomplex, comprised
69
of four antigenically distinct serotypes (DENV-1 to -4), is the most important and
70
rapidly emerging group of arthropod-borne pathogens imposing a great deal of
71
economic and public health burden, especially in tropical and subtropical countries
72
(2-5).
73
worldwide with 500,000 severe cases and 25,000 deaths, mostly affecting children
74
(6).
75
vaccine in development is the tetravalent chimeric YF-17D dengue (TCYD) vaccine.
76
Currently, the TCYD vaccine is in a large scale human efficacy trial and the vaccine
77
has been estimated to achieve 56% efficacy based on the primary endpoint calculation
78
(7).
79
future.
80
vaccination, and be used as a surveillance tool to monitor vaccine effectiveness, is
81
urgently needed.
A recent study estimates that annually 390 million DENV infections occur
Currently there is no DENV vaccine available.
The most advanced dengue
Optimistically, the TCYD vaccine could be available globally in the very near Thus, a serological assay that can differentiate natural infection from
5
82
Conventional
serological
tests
such
as
neutralization
(Nt),
83
hemagglutination-inhibition (HAI), and IgM and IgG antibody-capture enzyme-linked
84
immunosorbent assays (MAC- and GAC-ELISA), which exclusively measure
85
antibodies to the premembrane (prM) and envelope (E) structural proteins of
86
flaviviruses, cannot be used to determine natural infections among vaccinated
87
populations (8). Antibodies to nonstructural protein 1 (NS1) have been proposed as
88
serological markers of natural infection among inactivated Japanese encephalitis virus
89
(JEV) vaccinated populations and a NS1-ELISA has been developed (9-11).
90
However, this NS1-ELISA required the microplates to be sensitized with either
91
purified NS1 or NS1 antigen be captured by NS1-specific murine monoclonal
92
antibodies, before adding test human serum specimens.
93
sensitivity of NS1-specific IgM or IgG isotyping might be reduced due to the
94
competition between two antibody isotypes for the same antigenic sites.
95
Isotype-specific antibody-capture ELISA eliminates the isotype competition for the
96
same antigenic sites and, thus enhances the sensitivity of the assay.
97
commercially available anti-prM/E ELISA kits for both IgM and IgG detection are
98
commonly used for DENV clinical sero-diagnosis and results from comprehensive
99
evaluations suggest a good correlation of these assays with the gold standard Nt test
100
(12-14). 6
We speculate that the
A number of
101
Our long term goal is to develop anti-NS1 MAC- and GAC-ELISAs that can be
102
used as tools to differentiate the immune responses elicited by vaccinations versus
103
those elicited by natural infections. We initiated this study to develop and optimize
104
the NS1-MAC- and GAC-ELISAs using recombinant NS1 antigens.
105
archived flavivirus-infected clinical serum panel, the objectives of this initial study
106
are: (1) to determine the sensitivity and specificity of the NS1-MAC/GAC-ELISAs as
107
compared to the standard E-MAC/GAC-ELISAs, and (2) to determine the
108
cross-reactivity of NS1-MAC/GAC-ELISA between different sero-complexes of
109
flaviviruses.
110
reported in previous studies(9, 11), is due to a relative abundance of anti-prM/E
111
antibodies in the serum of infected individuals.
112
procedure to deplete anti-prM/E antibodies using prM/E-containing virus-like
113
particles (VLPs) can improve the sensitivity of NS1-ELISA.
114
DENV-infected serum pairs collected during the acute and convalescent phases were
115
used for extensive comparison of the correlation and agreement of the assays by
116
testing simultaneously with NS1-, VLP-MAC/GAC-ELISAs and InBios DENV
117
Detect IgM capture ELISA.
118
collection panel for the test validation by calculating the sensitivity and specificity of
Using an
We hypothesize that lower sensitivities of NS1 antibody detection, as
In this study, we propose that a
Furthermore, panel of
The test algorithm was expanded to the single serum
7
119
the assay performance since a single time-point clinical is the most common practice
120
in the field study.
121
8
122
Materials and Methods
123
Cell culture, plasmid construction and soluble protein expression
124
COS-1 cells (ATCC CRL 1650; Manassas, VA) were grown at 37°C, with 5%
125
CO2 in Dulbeco's modified Eagle's minimal essential medium (DMEM, GIBCO,
126
Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS,
127
Hyclone Laboratories, Inc., Logan, UT), 110 mg/L sodium pyruvate, 0.1 mM
128
nonessential amino acids, 2 mM L-glutamine, 20 ml/L 7.5% NaHCO3, 100 U/mL
129
penicillin, and 100 ug/mL streptomycin.
130
optimized eukaryotic cell expression plasmid was used as the backbone to express
131
premembrane/envelope (prM/E) or NS1 protein of six medically important
132
flaviviruses including DENV-1 strain 16007, DENV-2 strain 16681, DENV-3 strain
133
C0331/94, DENV-4 strain H241, West Nile virus (WNV) strain NY99, and Japanese
134
encephalitis virus (JEV) strain SA14-14-2 for prM/E and strain CH1392 for NS1.
135
Molecular cloning procedures to construct these plasmids were described previously
136
(15, 16).
137
electroporated with recombinant expression plasmids encoding the prM/E and NS1
138
genes, respectively, using the protocol described previously (17, 18).
139
cells were recovered in 50 ml DMEM, seeded into three separate 75 cm2 culture
A transcriptional and translational
The VLPs and soluble NS1 protein (sNS1) were expressed by COS-1 cells
9
Electroporated
140
flasks for VLP/sNS1 expression and incubated at 28°C with 5% CO2.
The secretion
141
of recombinant antigens was determined from tissue culture media harvested 4–5 days
142
post transformation for VLP and sNS1, respectively.
143
144
Antigen titration and VLP- and NS1-specific MAC/GAC-ELISAs
145
Antigen-capture ELISAs (Ag-ELISAs) were used to detect and quantify secreted
146
antigens from the prM/E- and NS1-gene encoded plasmid transformed COS-1 cells as
147
described previously (17, 18).
148
polyclonal rabbit sera were used to capture the antigens. Murine hyper-immune
149
ascetic fluid (MHIAF) specific for DENV-1 to 4, WNV or JEV were diluted 1:2,000
150
for detection.
151
immunoglobulin recognizing all potential antigenic epitopes were generated at the
152
Centers for Disease Control and Prevention, U.S.A. (CDC).
153
were standardized at an optical density 450nm (OD450) of 1.4, within the region of
154
antigen excess near the upper asymptote of the sigmoidal antigen dilution curve
155
because antigens are considered to be the limiting factor relative to the high
156
concentration of polyclonal rabbit serum and MHIAF antibodies used for capture and
157
detection, respectively.
Anti-DENV-1 to 4, WNV or JEV VLP and NS1
Both the polyclonal capture and detector sera containing high-titer
10
Antigen concentrations
158
Human sera were assayed for the presence of prM/E- and sNS1-specific
159
antibodies using both MAC- and GAC-ELISAs as previously described (18).
160
Briefly, 96-well plates were coated with goat anti-human IgM or IgG (Kirkegaard &
161
Perry Laboratories, Gaithersburg, MD) that was diluted 1:2,000 in PBS buffer and
162
incubated overnight at 4°C.
163
and negative control sera were diluted 1:2,000 in wash buffer (PBS with 0.05%
164
Tween-20), added to wells, and incubated at 37°C for 120 min.
165
JEV VLPs and sNS1 were diluted appropriately in wash buffer and tested against
166
each serum sample in triplicate.
167
detectors.
168
used as negative antigen controls for the standard quantification titration curve and for
169
the MAC/GAC-ELISA, respectively.
170
were determined according to the procedure described previously (18) using internal
171
positive and negative serum controls included in each 96-well plate. Positive (P)
172
values for each specimen were determined as the average OD450 for the patient serum
173
sample reacted with positive viral antigen. Negative (N) values were determined for
174
individual 96-well plates as the average OD450 for the normal human serum control
175
reacted with the positive viral antigen.
176
the diagnostic lab in CDC to interpret test results.
DENV, WNV or JEV infected patient sera and positive
DENV, WNV or
MHIAFs against homologous antigens were used as
The mock-transfected cell culture supernatants or 5% milk/PBS were
The positive-to-negative ratio (P/N) values
This P/N ratio is the current standard used in
11
177
To deplete anti-prM/E antibodies, the same procedure as described for the
178
Ag-ELISA was used to capture secreted VLP antigens over the 96-well plates. The
179
patients’ or negative control sera were diluted 1:2,000 in PBS as previously suggested
180
(19), pre-mixed with VLP antigens, added immediately to wells and incubated at
181
37°C for 60 min.
182
to the pre-coated anti-human IgM or IgG plates for MAC/GAC-ELISA as described
183
above.
A total of 50 L of prM/E antibody-depleted sera were transferred
184
185
186
Human serum panel
Archived serum specimens were obtained from Arboviral Diseases Branch,
187
Division of Vector-Borne Diseases, CDC.
Clinical diagnostic serum specimens
188
available from 1999 to 2008 were assembled and the status of infection was
189
confirmed by 90% focus-reduction microneutralization test (FRµNT).
190
serum panel included 54 acute and convalescent presumptive DENV-infected patient
191
serum pairs from Brazil and 97 presumptive WNV-infected patient serum specimens
192
from South Dakota.
193
a control panel including IgG-positive yellow fever-17D (YF-17D) post-vaccinated
194
sera (n=10), acute phase patient serum specimens from St. Louis encephalitis virus
This archived
Thirty non-DENV patient serum specimens were assembled as
12
195
(SLEV) (n=2), Zika virus (n=1), chikungunya virus (n=10), and other viral (n=7)
196
infections. Thirty-six normal human sera from CDC blood bank were included as
197
the naïve serum control. Among 54 paired serum specimens from Brazil, 36 pairs
198
showed a four-fold increase of Nt antibodies by FRµNT90 and were further classified
199
as taken from individuals with recent DENV infections.
200
suspected DENV-infected patient sera, collected from October 2008 to February 2009,
201
originally sent to ARUP Laboratories (Salt Lake City, UT), and obtained through
202
InBios (Seattle, Washington), were used as a testing panel for validation of the assay.
203
The serum panels with evidence of JEV (n=16) infection were assembled from
204
Taiwanese residents and provided by the Center for Disease Control in Taiwan
205
(Taiwan-CDC) and the JEV infection status was determined by VLP-MAC- and
206
GAC-ELISA.
207
was approved by CDC-human studies review board.
Seventy-four travel-related,
An institutional review board (IRB) waiver to use this serum panel
208 209
Statistical analysis
210
Student’s t test for comparisons between normally distributed continuous
211
variables and Mann-Whitney U test for comparisons between continuous variables
212
that are not normally distributed were used.
213
represented the linear relationship between the P/N (OD450 of test specimen divided 13
The Pearson correlation of coefficient
214
by OD450 of negative control specimen) ratios obtained from two methods of VLP and
215
NS1-MAC-ELISA or VLP and NS1-GAC-ELISA. Its value ranges from -1 to +1,
216
with 1 representing the perfect correlation of the two methods. A significance level
217
=0.05 was obtained under two-tailed p value. P
