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\begin{document}

\title{Development of a competitive chemiluminescence immunoassay using a
monoclonal antibody recognizing 3B of foot-and-mouth disease virus for
differentiating infected from vaccinated animals}



\author[1]{Wei Liu}%
\author[1]{Guanglei Zhang}%
\author[1]{Sicheng Yang}%
\author[1]{Junhui Li}%
\author[1]{Sude Ge}%
\author[1]{Huihui Yang}%
\author[2]{Jun-Jun Shao}%
\author[1]{Hui-Yun Chang}%
\affil[1]{Affiliation not available}%
\affil[2]{Lanzhou Veterinary Research Institute}%


\vspace{-1em}



  
  \date{May 08, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Foot-and-mouth disease (FMD) is a devastating animal disease.
Differentiation of infected from vaccinated animals (DIVA) is very
important for confirming suspected cases, evaluating the prevalence of
infection, certifying animals for trade and controlling the disease. In
this study, a competitive chemiluminescence immunoassay (3B-cCLIA) for
DIVA was developed for the rapid detection of antibodies against
non-structural proteins (NSPs) in different species of livestock animals
using monoclonal antibody 9E2 as a competitive antibody that recognizes
NSP 3B, which only needs to be washed once and takes 15 min. The cut-off
(50\%), diagnosis sensitivity (97.20\%, 95.71\%, and 96.15\%) and
diagnosis specificity (99.51\%, 99.43\%, and 98.36) of the assay were
estimated by testing a panel of known background sera from swine, cattle
and sheep. The accuracy rate of 3B-cCLIA was also validated and compared
with that of two commercial diagnostic kits. The early diagnostic
performance showed that antibodies to NSPs occurred later (approximately
1--2 days) than antibodies to structural proteins. Furthermore, NSP
antibodies present in animals vaccinated multiple times
(false-positive), especially in cattle and sheep, were confirmed, and
the false-positive rate increased with the number of vaccinations. These
results indicated that 3B-cCLIA is suitable to rapidly detect antibodies
against FMDV NSP 3B in a wide range of species for DIVA.%
\end{abstract}%



\sloppy


\textbf{Development of a competitive chemiluminescence immunoassay using
a monoclonal antibody recognizing 3B of foot-and-mouth disease virus for
differentiating infected from vaccinated animals}

Running Title: Competitive CLIA for detection of 3B antibodies

Wei Liu, Guanglei Zhang, Sicheng Yang, Junhui Li, Sudan Ge, Huihui Yang,
Junjun Shao\textsuperscript{\#}, Huiyun Chang\textsuperscript{\#}

State Key Laboratory of Veterinary Etiological Biology, National
Foot-and-Mouth Diseases Reference Laboratory, Lanzhou Veterinary
Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou,
Gansu Province, China

\#Address correspondence to Huiyun Chang (\emph{changhuiyun@caas.cn)}and
Junjun Shao \emph{(shaojunjun@caas.cn)}

\subsection*{Summary}

{\label{summary}}

Foot-and-mouth disease (FMD) is a devastating animal disease.
Differentiation of infected from vaccinated animals (DIVA) is very
important for confirming suspected cases, evaluating the prevalence of
infection, certifying animals for trade and controlling the disease. In
this study, a competitive chemiluminescence immunoassay (3B-cCLIA) for
DIVA was developed for the rapid detection of antibodies against
non-structural proteins (NSPs) in different species of livestock animals
using monoclonal antibody 9E2 as a competitive antibody that recognizes
NSP 3B, which only needs to be washed once and takes 15 min. The cut-off
(50\%), diagnosis sensitivity (97.20\%, 95.71\%, and 96.15\%) and
diagnosis specificity (99.51\%, 99.43\%, and 98.36) of the assay were
estimated by testing a panel of known background sera from swine, cattle
and sheep. The accuracy rate of 3B-cCLIA was also validated and compared
with that of two commercial diagnostic kits. The early diagnostic
performance showed that antibodies to NSPs occurred later (approximately
1--2 days) than antibodies to structural proteins. Furthermore, NSP
antibodies present in animals vaccinated multiple times
(false-positive), especially in cattle and sheep, were confirmed, and
the false-positive rate increased with the number of vaccinations. These
results indicated that 3B-cCLIA is suitable to rapidly detect antibodies
against FMDV NSP 3B in a wide range of species for DIVA.

\textbf{Keywords:} chemiluminescence immunoassay, diagnosis,
foot-and-mouth disease virus, monoclonal antibody, non-structural
protein

\subsection*{1. INTRODUCTION}

{\label{introduction}}

Foot-and-mouth disease (FMD) is a highly infectious and economically
devastating viral disease of cloven-hoofed animals. The causative agent,
FMD virus (FMDV), has a positive-sense and single-stranded RNA that
codes for four structural proteins (SPs: VP4, VP2, VP3 and VP1) and 10
non-structural proteins (NSPs: L, 2A, 2B, 2C, 3A, 3B, 3C, 3D, 3AB and
3ABC) (Jaworski et al., 2011; W. Liu et al., 2019; Z. Z. Liu et al.,
2018).

To date, slaughtering infected and contacted animals together with
prohibiting the import of animal and animal products from FMD endemic
countries have been adopted to prevent the disease in FMD-free nations.
Considering the economic costs, a vaccination policy was adopted for
control and eradication of the disease in endemic countries (Sharma et
al., 2012; Uttenthal et al., 2010). However, vaccination with
inactivated vaccines raises other issues, such as the differentiation of
FMDV-infected from vaccinated animals (DIVA) and the production of
carrier animals to shed the virus (Sharma et al., 2012; Uttenthal et
al., 2010). Therefore, the detection of antibodies against NSPs is the
preferred diagnostic method for DIVA and helps to identify subclinical
infections, evaluate the prevalence of infection and control the disease
(Lu et al., 2010) because most NSPs are removed from the inactivated
vaccine by a series of purification methods during production (Fu et
al., 2014; W. Liu et al., 2019).

Thus far, there are three main diagnostic types of detecting antibodies
against NSPs for DIVA. One is indirect ELISA or LFIA based on
recombinant NSPs, such as recombinant fusion protein of 2B (Biswal,
Jena, Mohapatra, Bisht, \& Pattnaik, 2014), 2C (Y. Gao, Sun, \& Guo,
2016; Mackay et al., 1998; Meyer et al., 1997), 3A (Biswal, Ranjan, \&
Pattnaik, 2016; Kumar, Sharma, \& Kakker, 2007), 3B (A. K. Mohapatra,
Mohapatra, Pandey, Sanyal, \& Pattnaik, 2014), 3AB (Jaworski et al.,
2011; J. K. Mohapatra, Pandey, Sanyal, \& Pattnaik, 2011), 2C3AB (Lu et
al., 2010; Wu et al., 2011), or 3ABC (Brocchi et al., 2006; Bruderer et
al., 2004; De Diego, Brocchi, Mackay, \& De Simone, 1997; Hosamani et
al., 2015; Lu et al., 2007; Robiolo et al., 2006). In particular, 3ABC
is the most appropriate indicator because of its high immunogenicity and
its relatively low abundance in infected cell lysates (W. Liu et al.,
2019; Z. Liu et al., 2017; Lu et al., 2010; Robiolo et al., 2006; Van
Dreumel et al., 2015). Another is ELISA based on peptides or epitopes
derived from NSPs 2B, 2C, 3A, and 3B (M. Gao et al., 2012; H\selectlanguage{ngerman}öhlich,
Wiesmüller, Haas, et al., 2003; Höhlich, Wiesmüller, Schlapp, et al.,
2003; Inoue et al., 2006; Oem et al., 2005; Shen et al., 1999). Both
indirect ELISAs based on recombinant NSPs or epitopes that use
species-specific conjugates make simultaneous examination of sera from
different species difficult (Sørensen, de Stricker, Dyrting, Grazioli,
\& Haas, 2005). Block or competitive ELISA using polyclonal antibodies
(Chung, Sorensen, Liao, Yang, \& Jong, 2002; Sørensen et al., 1998; Van
Dreumel et al., 2015) or monoclonal antibodies (mAbs) (Fu et al., 2017;
Fu et al., 2014; Gelkop et al., 2018; Muller et al., 2010; Oem et al.,
2007; Sharma et al., 2012; Sørensen et al., 2005) has been widely used
because it can be used for each species that is susceptible to FMD.

In recent years, chemiluminescence immunoassay (CLIA) has gained
increasing attention in many fields, including clinical diagnosis, food
analysis, and environmental monitoring, because of its high sensitivity,
high signal-to-noise ratio, wide linear ranges and low cost (Wang, Jie,
Zong, Jie, \& Huang-Xian, 2012). In the DIVA test, indirect CLIAs based
on recombinant NSPs 2C and 3ABC (Z. Liu et al., 2017), as well as two
epitopes located in 3A and 3B (Z. Z. Liu et al., 2018), were developed.

In this study, we used mAb 9E2, which recognizes NSP 3B, to develop a
competitive CLIA (termed 3B-cCLIA) for the rapid detection of NSP
antibodies, which can be used for a wide range of species. The assay was
evaluated with sera collected from naïve, vaccinated and infected
animals, and the accuracy rate was validated and compared with that of
two commercial kits.

\subsection*{2. MATERIALS AND METHODS}

{\label{materials-and-methods}}

\subsubsection*{2.1 Serum samples}

{\label{serum-samples}}

Serum samples from naïve animals: Serum samples from clinically healthy
and unvaccinated animals, including 310, 175, and 61 samples from swine,
cattle, and sheep, respectively, were collected and tested using
liquid-phase blocking ELISA of FMDV O (O-LPBE) and A-LPBE (presented
negative results; titer, \textless{} 1:4). These samples were utilized
to estimate the cut-off value and diagnostic specificity (Dsp).

Serum samples from infected animals: A total of 107 serum samples were
collected from swine infected with FMDV O/Mya98 or A/GDMM/2013 at 7--25
days post infection (dpi) in the Animal Biological Safety Level 3
(ABSL-3) Laboratory at Lanzhou Veterinary Research Institute (Lanzhou,
China); 70 serum samples were collected from cattle infected with FMDV
(O/Mya98 or A/GDMM/2013) at 8--20 dpi; 52 serum samples were collected
from sheep during the early infection period. All of these samples were
used to estimate the cut-off value and diagnostic sensitivity (Dsn). In
addition, a total of 32 sera collected from four unvaccinated control
swine experimentally challenged with FMDV O/Mya98 at 0 dpi and 2--8 dpi
were used to compare the early diagnostic performance of 3B-cCLIA and
two commercial diagnostic kits (3ABC monoclonal antibody blocking ELISA
(3ABC-bELISA) and PrioCHECK FMDV NSP ELISA) and identify seroconversion
to FMDV SP and NSP.

Serum samples from vaccinated animals: One hundred serum samples were
collected at 21 days post vaccination (dpv) from swine vaccinated with
FMDV O univalent multiple-epitope recombinant vaccine developed by our
research group. These samples were utilized to estimate the cut-off
value and diagnostic specificity (Dsp). In addition, 120 serum samples
were collected from sows vaccinated 3--15 times with commercial O/A
divalent inactivated vaccines; 129 serum samples were collected from
dairy cows vaccinated with commercial O/A divalent inactivated vaccines
every four months for a total of 2, 5 or 10 vaccinations. Seventy-seven
serum samples were collected from 15 sheep vaccinated 1--3 times with
laboratory-made FMDV O/Mya98/BY/2010, FMDV A/AF72, or FMDV Asia 1/JS05
univalent inactivated vaccine. These samples were used to evaluate the
diagnostic performances of 3B-cCLIA and two commercial diagnostic kits
and verified the false-positive phenomenon with animals vaccinated
multiple times.

Serum samples collected in the field: 173 field sera collected from
swine suspected of FMDV infection during 2010--2018. These sera were
used to compare the coincidence rates between 3B-cCLIA and two
commercial diagnostic kits.

Serum samples from other virus-infected swine: One serum sample from
senecavirus A (SVA)-infected swine, one from classical swine fever virus
(CSFV)-infected swine, one from porcine reproductive and respiratory
syndrome virus (PRRSV)-infected swine, one from porcine parvovirus
(PPV)-infected swine, and two from porcine circovirus type 2
(PCV2)-infected swine were examined in this study.

Control sera: The serum sample derived from swine infected with FMDV
O/Mya98 at 25 dpi served as the standard positive serum (P51). The
percentage inhibition (PI) of the serum in the 3ABC-bELISA and PrioCHECK
FMDV NSP ELISA was 92\% and 93\%, respectively. The serum collected from
clinically healthy swine with no immunization against FMD served as the
standard negative serum sample (P734). The serum was tested with O-LPBE
and A-LPBE (produced negative result; titer, \textless{} 1:4),
3ABC-bELISA (produced negative result; PI = 1\%), and PrioCHECK FMDV NSP
ELISA (produced negative result; PI = -7\%).

\subsubsection*{2.2 Antigen and antibodies}

{\label{antigen-and-antibodies}}

The 3ABC coding region of FMDV A/GDMM/2013 mutated at positions 46 aa
and 163 aa was cloned into the pProEXHTB plasmid. The expression and
purification of the recombinant 3ABC protein has been described (Z. Liu
et al., 2017).

The mAbs against the 3ABC protein, designated 2G5 and 9E2, were obtained
at our laboratory, and their minimally identified epitopes are
``\textsuperscript{92}EYIEKA\textsuperscript{97}'', which is located in
3A, and ``\textsuperscript{23}EGPYAGPLE\textsuperscript{31}'', which is
located in 3B (W. Liu et al., 2019). MAbs 2G5 and 9E2 were largely
produced by injecting hybridomas into the peritoneal cavities of BALB/c
mice and purified by affinity protein G column chromatography. Then, the
mAbs 2G5 and 9E2 were conjugated with horseradish peroxidase (HRP). The
polyclonal antibody was obtained by inoculating rabbits with the
purified 3ABC protein.

\subsubsection*{2.3 Development of competitive CLIA using mAb against NSP
3B}

{\label{development-of-competitive-clia-using-mab-against-nsp-3b}}

The conditions of 3B-cCLIA were optimized by checkerboard titration.
Briefly, mAb 2G5 was coated onto 96-well white plates (Costar) at the
concentrations of 1, 0.5, 0.25, and 0.125 \selectlanguage{greek}μ\selectlanguage{english}g/mL in a 100-\selectlanguage{greek}μ\selectlanguage{english}L volume and
incubated overnight at 4 \selectlanguage{ngerman}°C. After washing, purified 3ABC protein was
diluted to 0.5, 0.25, and 0.125 \selectlanguage{greek}μ\selectlanguage{english}g/mL in PBST (PBS containing 0.05\%
Tween-20) and added to each well, and the plate was incubated for 1 h at
37 \selectlanguage{ngerman}°C. After three washes with PBST, 200 \selectlanguage{greek}μ\selectlanguage{english}L of blocking buffer was added
to each well and incubated for 2 h at 37 \selectlanguage{ngerman}°C. Then, standard positive
serum (P51) and standard negative serum (P734) were serially diluted
with dilution buffer (1\% casein, 10\% equine serum in PBST) at
1:2.5--1:20 dilutions, and 50 \selectlanguage{greek}μ\selectlanguage{english}L of the serum was transferred to each
well. Simultaneously, 50 \selectlanguage{greek}μ\selectlanguage{english}L of HRP-conjugated mAb 9E2 (9E2-HRP) at
concentrations of 0.08, 0.04, 0.02, 0.01, 0.005, and 0.0025 \selectlanguage{greek}μ\selectlanguage{english}g/mL was
added to each well, and the plate was incubated at ambient temperature.
Finally, after five washes, 100 \selectlanguage{greek}μ\selectlanguage{english}L of chemiluminescence (CL) substrate
(KEY-BIO, Beijing, China), including 50 \selectlanguage{greek}μ\selectlanguage{english}L of solution A (luminol and
luminous enhancer) and 50 \selectlanguage{greek}μ\selectlanguage{english}L of solution B (peroxide solution), were
added. After 5 min, the CL signals were measured using Varioskan lux
(Thermo Scientific, USA). The optimum mAb 2G5 concentration, 3ABC
protein concentration, serum dilution, 9E2-HRP concentration, and
incubation time were determined on the basis of the ratios of CL values
of standard negative serum to standard positive serum (N/P).

3B-cCLIA was performed under optimized condition. The PI was calculated
using the following formula:

PI\% = (1-CLs/CLn)\selectlanguage{ngerman}×100\%,

where CLs are the CL values of the test samples and CLn are the mean CL
values of the standard negative serum control.

A mean CLn \selectlanguage{english}[?] 6,000,000 and mean PI of the standard positive control
\textgreater{} 80\% indicate assay validity.

Similarly, 3A+3B-cCLIA was developed, and detailed information is
presented in the supplemental material.

\subsubsection*{2.4 Cut-off value, Dsn, and
Dsp}

{\label{cut-off-value-dsn-and-dsp}}

The cut-off value of 3B-cCLIA was determined by testing 875 serum
samples from different origins. NSP-negative sera from swine (n = 410),
cattle (n = 175), and sheep (n = 61) were used to estimate the Dsp of
each species, and NSP-positive sera from swine (n = 107), cattle (n =
70), and sheep (n = 52) were used to estimate the Dsn of each species
using MedCalc software.

\subsubsection*{2.5 Comparison of accuracy rates and diagnostic
performances}

{\label{comparison-of-accuracy-rates-and-diagnostic-performances}}

The accuracy rates of 3A+3B-cCLIA and two commercial diagnostic kits
were also evaluated using the abovementioned 875 NSP-negative and
positive serum samples. The diagnostic performances of the four assays
were compared by testing 430 serum samples, including 120, 60, and 77
samples from vaccinated swine, cattle, and sheep, respectively, and 173
samples from the field. In addition, serum samples from four swine
challenged with FMDV O/Mya98 collected at different times were utilized
to evaluate and compare the early diagnostic performance of 3B-cCLIA
with 3A+3B-cCLIA and two commercial diagnostic kits.

\subsubsection*{2.6 Estimation of the repeatability and stability of
3B-cCLIA}

{\label{estimation-of-the-repeatability-and-stability-of-3b-cclia}}

Three replicates of seven serum samples with different PI values were
examined on different days using plates coated in the same and different
batches to calculate the intra- and inter-batch repeatability
performances. After blocking, the coated plates were vacuum-packed and
stored for 15 days at 37 degC or for 1 year at 4 degC to identify the shelf
life of the plates.

\subsection*{3. RESULTS}

{\label{results}}

\subsubsection*{3.1 Optimization of
3B-cCLIA}

{\label{optimization-of-3b-cclia}}

The reaction conditions were optimized using checkerboard titration to
obtain high ratios of N/P.

In 3B-cCLIA, the coating concentrations of mAb 2G5 and 3ABC protein were
fixed at 0.25 \selectlanguage{greek}μ\selectlanguage{english}g/mL, serum dilution was 1:2.5, concentration of 9E2-HRP
was 0.005--0.02 \selectlanguage{greek}μ\selectlanguage{english}g/mL (0.01 \selectlanguage{greek}μ\selectlanguage{english}g/mL), and reaction time was 10 min.

\subsubsection*{3.2 Determination of cut-off value, Dsn, and Dsp of
3B-cCLIA}

{\label{determination-of-cut-off-value-dsn-and-dsp-of-3b-cclia}}

The cut-off values of 3B-cCLIA were determined using known background
sera from swine (NSP-negative sera, n = 410; NSP-positive sera, n =
107), cattle (NSP-negative sera, n = 175; NSP-positive sera, n = 70) and
sheep (NSP-negative sera, n = 61; NSP-positive sera, n = 52) by
analysing the receiver operating characteristic (ROC) and interactive
dot diagram (Figure 1 and Table 1). According to the ROC curve analysis,
when the cut-off of 3B-cCLIA was 50\%, the Dsn was 97.20\% (104/107),
95.71\% (67/70), and 96.15\% (50/52), and Dsp was 99.51\% (408/410),
99.43\% (174/175), and 98.36\% (60/61) in swine, cattle, and sheep,
respectively. Furthermore, evaluation of the cross-reaction of 3B-cCLIA
with sera from other virus-infected swine (SVA, CSFV, PRRSV, PPV, and
PCV2) indicated no cross-reaction (PI \textless{} 20\%).

\subsubsection*{3.3 Accuracy rates and diagnostic performances of the
four test
methods}

{\label{accuracy-rates-and-diagnostic-performances-of-the-four-test-methods}}

These serum samples (n = 875) were also tested by 3A+3B-cCLIA and two
commercial diagnostic kits to evaluate and compare the accuracy of the
test methods (Table 1). The accuracy rate of 3B-cCLIA was almost
equivalent to those of 3A+3B-cCLIA (98.13\%, 95.71\%, and 96.15\%) and
3ABC-bELISA (95.33\%, 94.29\%, and 98.08\%) and significantly better
than that of PrioCHECK FMDV NSP ELISA (85.05\%, 90.00\%, and 92.31\%)
for serum samples from infected animals. For the na\selectlanguage{ngerman}ïve serum samples,
the accuracy rate of 3B-cCLIA was equivalent to that of 3A+3B-cCLIA and
slightly higher than that of 3ABC-bELISA and lower than that of
PrioCHECK FMDV NSP ELISA. The accuracy rates of 3B-cCLIA and PrioCHECK
FMDV NSP ELISA in testing serum samples from sows inoculated 3--15 times
were almost equal to those in testing negative swine sera, and the
accuracy rate of 3ABC-bELISA was slightly lower. However, the accuracy
rates of the three assays in testing these sera from dairy cows
inoculated 2--10 times were significantly lower than those in testing
naïve cattle sera (namely, false-positive rate increase). Similarly, the
accuracy rates in testing sera from sheep vaccinated 1--3 times with
lab-made FMDV O, A, or Asia 1 univalent inactivated vaccine were lower
than those in testing naïve sheep sera. Furthermore, false positives
increased with the number of vaccinations in cattle and sheep (Table 2).
To address this problem, a competitive CLIA (3A+3B-cCLIA) using 3ABC
polyclonal antibody as the capture antibody to capture purified 3ABC
protein and two mAbs (2G5-HRP and 9E2-HRP) as the detection antibody was
developed to simultaneously detect antibodies against 3A and 3B (Figure
S1). However, the accuracy rate of 3A+3B-cCLIA in testing sera from
cattle and sheep vaccinated multiple times remained lower than that in
testing naïve sera (Table 1).

In addition, serum samples from 173 swine in the field were tested, and
the results showed that the coincidence rates of 3B-cCLIA with
3A+3B-cCLIA, 3ABC-bELISA, and PrioCHECK FMDV NSP ELISA were 99.42\%,
96.53\%, and 95.37\%, respectively.

\subsubsection*{3.4 Comparison of early diagnostic
performance}

{\label{comparison-of-early-diagnostic-performance}}

Seroconversion to NSP and SP was studied in four unvaccinated control
swine post-challenge using O-LPBE and ID Screen\textsuperscript{®} FMD
Type O Competition to detect the SP antibody and using 3B-cCLIA,
3A+3B-cCLIA, 3ABC-bELISA, and PrioCHECK FMDV NSP ELISA to detect the NSP
antibodies. Antibodies against SPs were first detected at 5 dpi, and all
the sera examined were positive at 7 dpi (Figure 2a). Antibodies against
NSPs were detected early at 7 dpi and identified as positive in all
swine at 8 dpi except for the PrioCHECK FMDV NSP ELISA (Figure 2b).
Therefore, seroconversion to NSP occurred approximately 1--2 days later
than that to SP.

\subsubsection*{3.5 Repeatability and stability of
3B-cCLIA}

{\label{repeatability-and-stability-of-3b-cclia}}

The assay was highly repeatable, as determined by four positive and
three negative serum samples tested on different days on same and
different batch plates (Figure 3). The standard positive serum (P51) and
standard negative serum (P734) were tested using 3B-cCLIA plates stored
for 15 days at 37 °C or for 1 year at 4 °C. The CL values of P734 were
\textgreater{} 6,000,000, and the PI value of P51 was \textgreater{}
80\%. The results indicated that the 3B-cCLIA plates can be stored for
up to 15 days at 37 °C and up to 1 year at 4 °C.

\subsection*{4. DISCUSSION}

{\label{discussion}}

DIVA tests are important for serological surveillance by providing
evidence of vaccinated herds infected with or freedom from FMD (W. Liu
et al., 2019; Z. Z. Liu et al., 2018; Van Dreumel et al., 2015). To
avoid omitting animals infected with different serotype isolates,
identifying conserved mAbs is important in developing a diagnostic
method for DIVA. The epitope
``\textsuperscript{23}EGPYAGPLE\textsuperscript{31}'' identified by mAb
9E2 is located in 3B and is well conserved among different serotypes of
FMDV, which has been verified in a previous study (W. Liu et al., 2019).
Therefore, in this study, a competitive CLIA (3B-cCLIA) using mAb 2G5 as
the capture antibody to capture purified 3ABC protein and 9E2-HRP as the
detection antibody was developed for DIVA. The cut-off PI value of
3B-cCLIA was determined to be 50\% by testing panels of serum samples
from different origins. Based on the cut-off PI of 50\%, diagnosis
sensitivity (97.20\%, 95.71\%, and 96.15\%) and diagnosis specificity
(99.51\%, 99.43\%, and 98.36) were determined in naïve and infected
swine, cattle and sheep, respectively (Figure 1). The PrioCHECK FMDV NSP
ELISA had good Dsp (accuracy rate 100\%) in testing naïve sera but had
low Dsn in testing infected sera when compared with 3B-cCLIA and
3ABC-bELISA.

Although most NSPs are removed from the inactivated vaccine, residual
NSPs are still present in the inactivated vaccine (W. Liu et al., 2019)
and can induce the corresponding antibodies after repeated vaccination,
especially in cattle (Lee, Jong, \& Yang, 2006; Mackay et al., 1998; A.
K. Mohapatra et al., 2014; J. K. Mohapatra et al., 2011; Wu et al.,
2011), which will interfere with the DIVA test based on detecting NSP
antibodies and affect the serological surveillance and evaluation of the
infection status (Fu et al., 2017). This phenomenon was also confirmed
in this study, in which dairy cows vaccinated multiple times with the
traditional inactivated vaccine produced a high false-positive rate
using 3B-cCLIA and two commercial diagnostic kits (Tables 1 and 2). In
addition, the false-positive rate in sheep vaccinated three times also
increased (Tables 1 and 2). However, the false positives in sows
vaccinated multiple times were almost equal to those in negative sera
(Tables 1 and 2), as found in previous reports (Chung et al., 2002; Wu
et al., 2011); this may be due to different immune systems in different
species. False positives increased with the number of vaccinations. To
address this problem, we attempted to use two mAbs recognizing NSP 3A
and 3B to develop 3A+3B-cCLIA. In theory, only when the two antibodies
were simultaneously present in serum, the serum was considered to be
positive, thereby using the assay to try to decrease the false-positive
rate in vaccinated animals. However, the results showed that 3A+3B-cCLIA
could not solve this false-positive problem (Tables 1 and 2) because
3AB\textsubscript{1}, 3AB\textsubscript{1}B\textsubscript{2}, and
3AB\textsubscript{1}B\textsubscript{2}B\textsubscript{3} are the main
forms of 3B-related proteins in the inactivated vaccine (data not shown)
and may induce the production of corresponding antibodies.

Usually, sera from animals vaccinated multiple times were faintly
positive. Therefore, it is an approach to increase the cut-off
(compromise true sensitivity) to decrease the false-positive rates in
testing vaccinated animals. Another approach is to establish the
correlation between residual NSPs in the inactivated vaccines with
different rounds of purification and the NSP antibody in different
species that were inoculated different times with the corresponding
inactivated vaccine, thereby conforming to a threshold value of residual
NSPs in the inactivated vaccine to guide vaccine manufacturers to
improve vaccine purity. In addition, the development and use of new FMD
vaccines that do not contain NSP components, such as marker vaccines (Li
et al., 2014; Uddowla, Hollister, Pacheco, Rodriguez, \& Rieder, 2012),
virus-like particle VLP vaccines (Guo et al., 2013), multiple-epitope
recombinant vaccines (Shao et al., 2011), and synthetic peptide
vaccines, could eliminate this barrier.

In this study, seroconversion to NSP occurred later than that to SP,
which is similar to previous reports (Mackay et al., 1998; A. K.
Mohapatra et al., 2014). The seroconversion time of the PrioCHECK FMDV
NSP ELISA was delayed compared with that of the other three methods,
which explains the lower Dsn of the PrioCHECK FMDV NSP ELISA in
detecting sera from infected animals.

In conclusion, a competitive CLIA that could rapidly detect antibodies
against FMDV NSP 3B was developed in this study. 3ABC-bELISA and
PrioCHECK FMDV NSP ELISA use the blocking ELISA format and need to be
washed twice in process, but 3B-cCLIA uses the competitive format, only
needs to be washed once and requires 15 min to obtain results. In
addition, the early diagnostic performance, repeatability, and stability
of 3B-cCLIA were also evaluated. These results indicated that 3B-cCLIA
can be a promising tool for large-scale serological surveys in
vaccinated areas for DIVA.

\subsection*{ACKNOWLEDGMENTS}

{\label{acknowledgments}}

This study was supported by grants from the National Key R\&D Program of
China (2017YFD0500902 and 2016YFE0204100).

\subsection*{CONFLICT OF INTEREST}

{\label{conflict-of-interest}}

None of the authors has any conflicts of interest.

\subsection*{ETHICS STAEMENT}

{\label{ethics-staement}}

All of the animal experiments were conducted in accordance with the
regulations for the administration of affairs concerning experimental
animals approved by the State Science and Technology Commission of the
People's Republic of China.

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Figure 1. Receiver operating characteristic (ROC) analysis for the
determination of the cut-off value of 3B-cCLIA. (A, C, E) Interactive
dot diagram of 3B-cCLIA in testing sera from swine, cattle, and sheep.
0, negative serum samples (n = 410, 175, and 61); 1, positive serum
samples (n = 107, 70, and 52). (B, D, and F) Each point on the ROC curve
represents a sensitivity-specificity pair in testing the sera from
swine, cattle, and sheep.

Figure 2. Early production of antibodies against FMDV SPs and NSPs in
sera from experimentally challenged swine. (A) Thirty-two serum samples
from four unvaccinated control swine after challenge with FMDV O/Mya98
were collected at 0 dpi and 2--8 dpi and tested using O-LPBE, ID Screen®
FMD Type O Competition to detect SP antibodies. The dashed
lines\textsubscript{\textbf{-- -- -- -- --}} and
\textbf{\ldots{}\ldots{}}represent the cut-off values of O-LPBE and ID
Screen\textsuperscript{®}FMD Type O Competition, respectively. (B)
Thirty-two serum samples were tested using 3B-cCLIA, 3A+3B-cCLIA, and
two commercial diagnostic kits (3ABC-bELISA and PrioCHECK FMDV NSP
ELISA) to detect NSP antibodies. The dashed line
(\textsubscript{\textbf{-- -- -- -- --}} ) represents the cut-off value
of 3B-cCLIA, 3ABC-bELISA, and PrioCHECK FMDV NSP ELISA, and the dashed
line (\textbf{\ldots{}\ldots{}} ) represents the cut-off value of
3A+3B-cCLIA. The bar represents the range of values obtained by
detecting sera from four swine at the given time.

Figure 3. Intra- and inter-batch repeatability performances of 3B-cCLIA
were assessed using four positive and three negative serum samples
tested on different days on same and different batch plates. The dashed
line represents the cut-off value of 3B-cCLIA.\selectlanguage{english}
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