Feline Coronavirus (FCoV)
Development of an ELISA for detection of antibodies against
serotype I and II
Name: Leonie Tuinte
Student number: 3535800
Supervisors: Herman Egberink, Nancy Schuurman
With contributions of: Hui Hui Mou, Manifing Camara and Berend-Jan Bosch
Department: virology University of Utrecht
Date: 10 September 2012 – November/December 2012
Index
Summary ....................................................................................................................................3
1. Introduction ...............................................................................................................................4
1.1. Characteristics of coronaviruses ..........................................................................................4
1.2. Envelope proteins..................................................................................................................4
1.3. Serotypes ...............................................................................................................................5
1.4. Biotypes ................................................................................................................................5
1.5. Prevention .............................................................................................................................5
1.6. Diagnostic methods ...............................................................................................................6
1.7. Goal and strategy ..................................................................................................................6
2. Materials and methods..............................................................................................................7
2.1. IFA ........................................................................................................................................7
2.2. VNT ......................................................................................................................................8
2.2.1. FCWF cells................................................................................................................8
2.2.2. Virus titration ............................................................................................................8
2.2.3. Performing a VNT .....................................................................................................9
2.2.4. Fixation and coloration of the plates .........................................................................9
2.3. ELISA .................................................................................................................................10
2.3.1. Production of the S1 protein ....................................................................................10
2.3.2. ELISA S1-protein ....................................................................................................11
2.3.3. ELISA N-domain ....................................................................................................13
3. Results ......................................................................................................................................15
3.1. Results IFA .........................................................................................................................15
3.2. Results VNT........................................................................................................................16
3.3. Results ELISA ...................................................................................................................17
3.3.1. Results ELISA S1 protein .......................................................................................17
3.3.2. Results ELISA N-domain ........................................................................................22
4. Discussion and conclusion.......................................................................................................24
4.1. IFA ......................................................................................................................................24
4.2. VNT ....................................................................................................................................24
4.3. ELISA .................................................................................................................................24
4.4. Future experiments..............................................................................................................25
Appendix: ELISA results........................................................................................................27
2
Summary
The Feline Coronavirus (FCoV) belongs to the family Coronaviridae, order of Nidovirales. (Chang et
al., 2012). Antibodies against FCoV are found in 20-60% of pet cats and up to 100% in catteries or
multi-cat households. (Sharif et al., 2010) The FCoV contains three proteins in its viral envelope:
spike glycoproteins (S), transmembrane proteins (M) and the envelope protein (E). (Le Poder, 2011,
Belouzard et al., 2012). The S-protein is the most abundant protein. It consist of a S1 and S2 part of
which the S1 part is the most divergent. There are two serotypes (I and II) and 80-90% of FCoV
infections are caused by serotype I. Serotype II resulted from a recombination between serotype I
FCoV and CCoV. Serotype II obtained the S-protein from the CCoV and the rest of the genome from
serotype I FCoV. (Le Poder, 2011, Chang et al., 2012, Woo., et al. 2010). Classification based on
pathogenicity are the biotypes: Feline Enteric Coronavirus (FeCV) and feline infectious peritonitis
virus (FIPV). (Vogel et al., 2010). Only about 10% of the infections with FCoV results in the fatal
systemic disease FIP which is caused by a mutation in the FeCV that enables it to enter macrophages
and cause a systemic disease. Serotypes can be distinguished by RT-PCR and a virus neutralization
test (VNT) but these tests are expensive and take a lot of time. For this project an ELISA was
developed to distinguish serotype I and II infection based on antibody detection, which is fast and
cheap. With the ELISA the prevalence of serotype I and II was determined for a population of cats.
This is important for the sero-epidemiological features of the FCoV. The VNT was used as a gold
standard to compare the ELISA results with.
Sera were selected from the serum bank. Some of these sera were already screened in a VNT
against type 1 and 2 viruses in a previous study (A. blanken). All the sera with a unknown Immune
Fluorescence Assay (IFA) titer were screened for antibodies against the FCoV with the IFA. Samples
were considered to be seronegative with a titer of < 1:20. Most of the seronegative samples were
excluded from the rest of the project. IFA positive sera were subsequently screened in the VNT. Next
an ELISA was developed by coating the plates with the whole S1 protein of serotype I (UU23-S1) and
serotype II (SeroII-S1). First the sera with a known VNT titer were tested to validate the ELISA. Next
all the sera were screened with this ELISA. Followed testing whether the N-part was more
discriminative by coating the ELISA plates with serotype I-N (UU23-N-S1) and serotype II-N (SeroIIN-S1). Based on the results, which showed a high background, it was decided to optimize the ELISA
conditions by comparing two different block buffers: BSA1% and NGS10%.
The IFA results showed that 36.8% of the tested field samples were considered to be
seronegative. The ELISA results showed that it was able to discriminate between serotype I and II by
determining the ratio of antibody titers for type 1 and 2. All serotype I samples had a ratio I /II of ≥1
and all the type II samples a ratio I /II of <1. Of 80 field samples tested only one serum sample was
positive for serotype 2.The estimated seroprevalence of type II was 1.25% and 98.75% for type I for
80 field samples. There was no strong correlation between VNT titers and ELISA titers. After
performing ELISA with N-coated plates of serotype I and II, it appeared that the N-part was not more
discriminative than the whole S1 protein of both serotypes. However this was only tested for 11
samples. The overall conclusion is that plates coated with UU23-S1 and SeroII-S1 can distinguish
serotype I and II infections. The results of the optimization of the ELISA by comparing two block
buffers, showed that there was no significant difference between using BSA1% and NGS10% as a
block buffer.
In the future more information should be gathered about the tested samples to see whether a
correlation can be found between the results of the ELISA, symptoms and the strain that infected the
cat. Also the ELISA conditions could be optimized by increase the blocking time of the plates or
changing the substrate for instance. For this project, only the N-part of both serotypes was tested. The
C-part of both serotypes could also be included. Also more samples should be tested with the N- part
of serotype I and II to determine whether the N-part is more discriminative. One sample appeared to
be a lion sample, which indicated a negative result on the VNT but positive for serotype II in the
ELISA. More lion samples could be tested to see whether more lion samples give the same results.
3
1. Introduction
1.1.
Characteristics of coronaviruses
The Coronavirus belongs to the family Coronaviridae, order of Nidovirales. A Coronavirus is capable
of infecting mammals and birds. (Chang et al., 2012). Individual coronaviruses infect their hosts in a
species specific manner. (Masters, 2006). The coronavirus family is divided into three groups. Group 1
and 2 viruses have mammalian hosts, group 3 viruses have only been isolated from birds. The feline
coronavirus (FeCV and FIPV) belongs to group 1. Their genetic material is single stranded positive
RNA (+ssRNA). They are enveloped viruses with a genome of 28-32 kb. (Chang et al., 2012) (B.J.
Bosch, 2004). The canine coronavirus (CCoV) is known to infect dogs. FCoV is very common in cat
populations. Antibodies against FCoV are found in 20-60% of pet cats and up to 100% in catteries or
multi-cat households. About 75-100% of the cats in multi-cat environments shed the virus. (Sharif et
al., 2010).
1.2.
Envelope proteins
The Feline Coronavirus has three proteins in its viral envelope:
spike glycoproteins (S), transmembrane proteins (M) and an
envelope protein (E). (see fig. 1) (Le Poder, 2011, Belouzard et
al., 2012). The most prominent of these proteins are the Sproteins. The S-proteins are N-exo, C-endotransmembrane
proteins. The ectodomain makes up most of the molecule. (see
fig. 2). They are assembled in trimers. (Masters, 2006).
The S-protein is responsible for attachment and fusion with host
cells. (Le Poder, 2011, Chang et al., 2012). Other functions of
the S-proteins are: host range, pathogenesis and virulence of the
coronavirus. (B.J. Bosch, 2004)
Figure 1: Derived from Belouzard et al.
2012. A schematic view of the
Coronavirion. The RNA of the virus is
surrounded by the nucleocapsid protein
(N). The N-protein is surrounded by a
lipid bilayer where the spike (S,
membrane(M)) and envelope (E) protein
are situated. Coronaviruses of group 2
contain a fourth protein, the
heamglutinine esterase (HE) in their
envelope.
The S-protein can be divided in two parts by trypsin like host
proteases in the N-terminal/S1-protein and the C-terminal/S2protein. (see fig. 2). The S1 domain is the most divergent region
of the molecule, the sequence can vary extensively. (Masters,
2006). The S1 part is responsible for attachment of the virus to
the target cell and S2 for fusion of the viral membrane with the
host cell membrane by means of the fusion peptide (F).
(Belouzard et al., 2012). The S2 part also contains two
hydrophobic (heptad) repeat regions HR1 and HR2. (see fig. 2).
These hydrophobic parts can be problematic for producing the S-proteins during purification of the
proteins. (B.J. Bosch, 2004). The S1 protein can also be subdivided in a C- and N-terminal of which
the N-terminal is probably the most divergent because the N-terminal is less conserved than the Cpart.
4
Figure 2: Derived from (Masters, 2006). A schematic view of the S-protein. Close to the N-terminal (S1), the signal
sequence and receptor binding (RBDs)domains are situated. Close to the C-terminal (S2) the transmembrane domain,
heptad regions (HR1 and HR2) and the putative fusion peptide (F) are situated. The S1 and S2 part can be separated
because of non-covalent binding between them. This is the cleaving site (see arrow). The S2 protein also contains two HR
areas (HR1 and HR2). The left figure shows a model of the S-protein trimer.
1.3.
Serotypes
Two serotypes (I and II) are known based on difference in their sequence. Type I, which causes 8090% of the FCoV infections, is most common. Exact numbers of the prevalence of both serotypes in
The Netherlands are unknown.(Le Poder, 2011, Chang et al., 2012). Although type I is more common,
type II is used more often in vitro experiments because serotype I viruses are difficult to grow in cell
cultures and cause a slowly developing cytopathic effect. (Sharif et al., 2010). Serotype II resulted
from a recombination between serotype I FCoV and CCoV. Serotype II obtained the S-protein from
the CCoV and the rest of the genome from serotype I FCoV. (Le Poder, 2011, Chang et al., 2012,
Woo., et al 2010). In practice serotype I and II can be distinguished by means of a virus neutralization
test or PCR. (Vogel et al., 2010).
1.4.
Biotypes
Another classification of the FCoV is based on its pathogenicity. The first biotype is the Feline
Enteric Coronavirus (FeCV), the second is the feline infectious peritonitis virus (FIPV). (Vogel et al.,
2010). Only about 10% of the infections with FCoV results in the fatal systemic disease FIP which is
mainly seen in kittens. (Le Poder, 2011). The FIPV is in all probability resulted from a mutated FCoV
that, instead of reproducing in enterocytes, is able to leave the intestines and cause systemic disease
by entering and reproducing in the macrophages. (Myrrha et al., 2011). In all other cases FeCV stays
in the enterocytes and cause no or little problems like diarrhea. (Le Poder, 2011, Myrrha et al., 2011).
FeCV is spread by the fecal-oral route, the FIPV isn’t because it’s not shed in the feces. (Chang et al.,
2012).
1.5.
Prevention
To prevent FIP an intranasal, modified live vaccine is available, but the duration of the protection is
thought to be limited. As mentioned before only 10% of the cats develop FIP, thus in practice it’s not
of great value. The FeCV is spread by the fecal-oral route so, mainly in catteries, hygiene is a very
important measure. Other measures are early weaning, isolation of cats that are tested positive,
isolation and testing of cats after shows and immunization against other feline viruses. Also increasing
the amount of litter boxes will reduce the stress and decrease the FIP losses. The risk of developing
FIP is shown to have a genetic influence. Thus pedigree analysis is useful to make sure breeding is
done with FIP resistant breeds. (The Merck veterinary manual).
5
1.6.
Diagnostic methods
Because FeCV rarely causes severe disease, it’s not often required to diagnose the presence of the
virus in those cases. However the virus can be demonstrated in the feces of infected kittens by
electonmicroscopy (EM) and reverse transcriptase polymerase chain reaction (RT-PCR) assay or by
serological tests (testing for antibodies) on plasma samples. An immune fluorescence test (IFA) is
often used to screen samples for an FCoV infection. EM and serological tests are of limited value
because healthy kittens also shed the FCoV in their feces. (Sharif et al., 2010). But it can demonstrate
the presence of a FCoV infection in a colony of cats what can be good for management of a FCoV
infection for the creation of a FCoV free cattery for example. (Vogel et al., 2010). The sensitivity of
the commercially available test is 95% and the specificity is 83%. These test are not able to distinct
FIPV and FeCV. (Vogel et al., 2010, Sharif et al., 2010).
RT-PCR is able to distinguish an infection with serotype I and II. It demonstrates the presence
of the virus. RT-PCR is rapid and sensitive but must be interpreted in context of the clinical findings
and is expensive. (Sharif et al., 2010). The Virus Neutralization Test can also distinguish serotype I
and II. The VNT detects the antibodies against the virus, this might be antibodies from a former
infection. The disadvantage of the VNT is that is expensive and it costs a lot of time, for serotype I
about one week.
The diagnosis of FIP is based on history, haematology, serology, tissue biopsy and PCR. A
definitive diagnosis can only be established by histopathological examination of biopsies. (Sharif et
al., 2010).
1.7.
Goal and strategy
The goal of this project was to develop an Enzyme Linked Immunosorbent Assay (ELISA) that
can discriminate between antibodies against FCoV serotype I and II. An ELISA is a fast and relatively
cheap diagnostic method compared to other diagnostics methods that can discriminate between
serotype I and II infections. By means of the ELISA, the prevalence of serotype I and II was
determined in the selected population. This was established by screening large numbers of samples in
a relatively short period of time. The results of this project are important for the epidemiology of the
FCoV.
First the sera are selected and screened. The sera are available in the serum bank of the University of
Utrecht, department Virology. It contains sera from field cats and experimental infected cats.
Information about the sera is listed in a folder. From this list, sera with a unknown IFA titer or with a
titer >1:20 are selected. Some of the sera are derived from a former project in which the VNT titer was
determined.(A. blanken, jaartal). Those sera are important to validate the ELISA with.
The samples with a unknown IFA titer are screened for antibodies against FCoV by means of
an IFA. Sera with a titer ≥ 1:20 are considered to be seropositive and with a titer of < 1:20 the
samples are considered to be seronegative. This to prevent that time is wasted on samples that are not
seropositive after all. Most of the seronegative samples are excluded and with the seropositive
samples, the research is continued.
All samples with an unknown VNT titer are screened by means of a VNT performed with
TN406HP (serotype I) and FIPV 79-1146 (serotype II). The VNT is used as the gold standard. Thus
the results of the VNT are compared to those of the ELISA to validate the ELISA.
Next, a ELISA is developed with the S1 protein. The S1 protein is the most divergent region of the
spike protein. First the S1 protein is produced by means of transfection of plasmids in HEK293T cells
with the polyethylenimine complex (PEI). Next the optimal coating concentrations is determined, the
plates are coated with the S1 protein of both serotypes and control sera are tested on those plates. As a
positive control, experimental infected cats are used and as a negative control a specific pathogen free
(SPF) serum. Followed by testing all selected sera on S1 coated plates when the ELISA is valid.
The S1 protein can be subdivided in the C- and N-domain of which the N-domain is less
conserved. (see 1.2 envelope proteins). Thus the N-domain should be more discriminative. That is
tested by coating the plates with the N-domain of both serotypes.
6
2. Materials and methods
2.1.
Immune Fluorescence Assay
Megascreen 10 wells antigen glasses fixed with infected porcine kidney cells (PD5 cells) were used.
The cells were infected with the closely related Transmissible Gastro Enteric Virus (TGEV). The
antigen glasses were stored in the -20⁰C freezer. Before use, the glasses were rinsed with MilliQ
which removed the salts. Next they were dried by means of a cold hairdryer. The sera were twofold
diluted in PBS without magnesium and calcium (PBS0) starting from 1:20 to 1:2560. As a positive
control, ascites from a FIP cat (A40) was applied on one of the glasses. The serum dilutions were
applied with 20µl/well on each well. This was incubated for 1 hour at 37°C , 5% CO2 in a box with a
moist tissue at the bottom.
After the incubation period the glasses were rinsed three times for five minutes with PBS0 and once
with MilliQ. The glasses were dried by means of a cold hairdryer. Next the conjugate, Goat anti-Cat
IgG Fluorescein Isothiocyanate (FITC), was diluted. The conjugate was kept in the dark as much as
possible because otherwise the fluorescent label would fade. The conjugate was diluted 1:150**1 and
applied to the glasses with 20µl/well. This was to incubated for 1 hour at 37°C, 5% CO2 in a box with
a moist tissue at the bottom.
After the incubation period the glasses were rinsed again, three times for five minutes with PBS0 and
once with MilliQ. Next the glasses were dried again by means of a cold hairdryer. The last step was to
apply a drop of glycerol 50% in MilliQ on each well and an object glass of 24X50 mm was placed on
top of the glasses . By means of the fluorescence microscope the antibodies in the serum were
indirectly visualized by the fluorescent label (FITC). The titer was the last dilution that showed
fluorescence.
7
2.2.
Virus Neutralization Test
2.2.1. FCWF cells
The VNT was performed on cells that are sensitive for FCoV; the felis catus whole fetus cells (FCWF
cells). These cells were seeded overnight on 96-wells plates. The cells were contained in T75 flasks.
The medium consists of DMEM10% fcs, p/s. For passaging the cells, the supernatant was removed
from the flask and the cells were rinsed with 15 ml PBS0. The cells were detached by adding 5 ml
trypsin and were resuspended in DMEM 10% fcs, p/s. Than they were passed to new flask which was
filled with 18 ml DMEM 10% fcs, p/s. This was incubated at 37⁰C, 5% CO2 for a few days and passed
two times a week.
2.2.2. Virus titration
First a 96-wells plate was seeded with FCWF cells. To seed the 96-wells plate with FCWF cells, they
first have to grow for a few days as described in chapter 3.3.1.. When a solid monolayer was formed,
the supernatant was removed from the T75 flasks. Then the cells were rinsed with 15 ml PBS0 and
detached with 5 ml trypsin. The cells were resuspended in 30 ml DMEM 10% fcs, p/s and 1 ml was
put in a tube. Then 20µl was put in a counting chamber and the cells were counted by placing the
counting chamber in the light microscope. The cells of 3 boxes were counted and the average amount
of cells per box were calculated**1. The cell suspension was added with 100µl/well to the 96-wells
plate to seed the plates with 1*105 FCWF cells. The plate was incubated at 37⁰C, 5% CO2 overnight.
The next day, the virus titration was performed to
determine the amount of virus that’s present in the
virus stocks. The median tissue culture infective
dose (TCID50) was determined. Which is the
amount of pathogenic agent that will produce a
pathological change in 50% of cell cultures that
were inoculated. The following FCoV stocks were
used for inoculation: TN406HP for serotype I and
FIPV 79-1146 for serotype II. The virus was
tenfold diluted from 101 to 1012 in DMEM 2% fcs,
p/s. (see fig. 3). The 96-wells plates were removed
from the stove and emptied. Then the plates were flushed
Figure 3: Layout of the 96-wells plate for the
with PBS DEAE 50 mg/L which enables the virus to enter
virus titration. On the top of the plate, the
tenfold virus dilutions are displayed.
the cells by making the cells accessible. Next the virus
dilutions were added to the plates with 100µl/well except for
the last row. This is the “mock-row” where only DMEM 2% was added. The mock-row was used as
a cell control.(see fig. 3). The plates were incubated for 7 days (type I) or 6 days (type II). The
cytopathic effect (CPE) was checked every day.
8
2.2.3. Performing a VNT
First the sera were inactivated for 0.5 hours at 56 °C to disable the complement. A threefold serial
serum dilution was made from 1:5 to 1:10935 in 5% DMEM fcs, p/s (500µl) + gentamycin (500µl).
From the last tube 100µl had to be removed because each tube had to have the same volume before
adding the virus. The virus was added with 200µl 100TCID50/50µl to the serumdilutions.**1 This was
incubated for one hour at 37°C, 5% CO2 so when antibodies were present they would get the chance to
neutralize the virus. After the incubation period, the medium of the 96-wells plate was removed and
the plates were rinsed with PBS DEAE (100μl/well). Next the mixtures of sera and virus were added
to the 96-wells plates with 100μl/well in triplicate.(see fig. 4). The virus control was made by making
a tenfold serial dilution of 100TCID50 from 100TCID50 to 0.1TCID50. This was added with 50µl/well
together with 50µl DMEM5% with a total volume of 100μl/well. The cell control was made by adding
100µl DMEM5% per well. (see fig. 4). This was incubated for 2-3 days (type II) of 6-7 days (type I) at
37⁰C, 5%CO2.
Figure 4 : General layout of the 96-wells plate for
a VNT.
2.2.4. Fixation and coloration of 96-wells plates
After CPE was visible in the virus control, the plates were removed from the stove. They were colored
and fixated with kristalviolet 0.75% + formaldehyde 9% applied with 100µl/well. Kristalviolet colors
the proteins purple. That means that all intact cells were colored. Formaldehyde fixated the cells on the
plate.
9
2.3.
Enzyme Linked Immunosorbent Assay
2.3.1.
S1 protein production
Day 0 : Human Embryonic Kidney 293T cells (HEK293T cells)
For the production of the S1 protein, HEK293T cells were used for transfection. These cells were
maintained in T225 flasks. The medium contains Dulbecco's Modified Eagle Medium (DMEM) 10%
foetal calf serum inactivated for 0.5 hour at 56⁰C(fcs) and penicillin/streptomycin (p/s). New T225
flasks were plated with 1.0*107 HEK cells in 50 ml DMEM10+. The medium was removed and the
cells were rinsed with PBS0. Than the cells were detached by trypsin and resuspended in DMEM 10%
fcs, p/s. After counting the cells it was calculated how much of the cell suspension and medium should
be added in the new flasks. This was incubated overnight at 37⁰C, 5% CO2.
Day 1: Transfection
For the production of the S1 protein five plasmids were used:
 Type I
pCAGGS-UU23-S1-Fc
 Type I-N
pCAGGS-UU23-N-S1-Fc
 Type I-C
pCAGGS-UU21-S1-Fc
 Type II
pCAGGS -SeroII-S1-Fc
 Type II-N
pCAGGS -SeroII-N-S1-Fc
All the plasmids were coupled to the heavy chain of humane immunoglobulin, the Fragment
crystallizable (Fc-) part. For transfection the next reaction was set up: 25µg DNA, 2.24ml DMEM0
and 250µl PEI. DNA:PEI was used in 1:10. The five plasmids were diluted in DMEM without fcs and
p/s (DMEM0) because it’s not good for the formation of the PEI-complex to add undiluted plasmids to
the HEK cells. Then 1µg/µl PEI was added which condenses DNA to a positively charged particle
that will bind to a negatively charged cell. The DNA is brought into the HEK cells by endocytosis.
Once the vesicles enter the cells, the amine groups will be protonated. Because of the changed
potential, counter ions will enter the vesicles which causes the swelling and burst of the vesicles. Next
the DNA is free in the cytoplasm and will enter the nucleus of the HEK cells. After adding DMEM,
plasmids and PEI together the reactions were mixed and incubated for 15 minutes at room
temperature. After the incubation period, the reactions were added drop wise to the cells and incubated
at 37⁰C, 5% CO2 overnight.
Day 2: expression medium
The flasks, which were seeded with the HEK cells, were removed from the stove. Medium from the
transfections was aspirated and replaced by ~50ml expression medium.**1 This was incubated for 5
days at 37 ⁰C, 5% CO2. In these days the cells were able to produce the proteins.
Day 7: Purification of the proteins
The HEK cells produced the proteins and excreted them in the supernatant. The supernatant was
collected and divided over sixteen 50ml tubes. These tubes were cleared using centrifugation (D15P
centrifuge) 10 minutes for 1200 rpm. The supernatant was collected and centrifuged again for 15
minutes at 3500 rpm. Cleared medium was put in new 50 ml tubes and put on ice, 100µl aliquot from
the medium was collected and stored at -20⁰C. Next the A sepharose beads were added . The beads
contain protein A which the Fc-part has affinity for so the proteins will bind to the beads. The beads
were washed three times using 10 ml PBS0 and centrifuged for 2 minutes at 3000 rpm. Next the beads
were suspended in 1.4 ml PBS0 per tube (50% V/V, final volume was 2.8 ml). Then 0.5 ml beads
(50% V/V) and 1 ml 1M tris-HCl pH 8.0 (to increase the pH) were added to each 50 ml tube and
incubated overnight rotating at 4⁰C.
10
Day 8: removal of the Fc-parts
On day 8 the Fc-parts were removed from the proteins because they might give a higher background at
the ELISA. (S. van’t Ende, 2011). The tubes with the suspended beads were centrifuged for 15
minutes at 3000 rpm. The supernatant was discarded and transferred to 2 ml tubes. The Fc-parts were
cut off from the proteins by adding 1 ml Thrombin Cleaving buffer. This cleaving buffer was used to
wash the beads for five times. Followed by centrifuging for two minutes at 3000 rpm. The beads
were resuspended in 350µl Thrombin Cleaving buffer containing 20U thrombin and incubated for 16
hours at 22⁰C.
After 16 hours the beads were centrifuged and the supernatant was collected in a new 1.5 ml tube. To
get rid of the remaining beads the supernatant was centrifuged again and the supernatant was
transferred to a new tube. To determine the amount of protein at an optical density of 280nm (OD280),
the nanodrop was used. The S1 proteins were stored at -80⁰C in small aliquots.
**1
Content of the Expression Medium: 293SF II medium, +Glutamax, +0,3% Primatone, +0,2% Glucose, +0,37%
NaHCO3 and +1,5% DSMO.
2.3.2. ELISA S1-protein
The ELISA plates**1 were coated with the S1 proteins of both types, UU23-S1 for type I and SeroIIS1 for type II.
Optimal coating concentration
First the optimal S1-protein concentrations were determined. For the first coating the following protein
concentrations were used: 7.5 µg/ml, 1.5 µg/ml and 0.3 µg/ml. The amounts of the proteins were 1.16
mg/ml for UU23-S1 and 1.01 mg/ml for SeroII-S1.The S1 proteins were diluted in PBS with calcium
and magnesium (DPBS). From each dilution 100µl/well was added. Two plates were coated overnight
in the refrigerator. The next day the plates had to be at room temperature before use. The plates were
rinsed three times with PBS0. Next block buffer PBS0/Tween20 0.005% /1% Bovine Serum Albumin
(BSA**2 ) was added to the plates with 200µl/well. This was incubated for 45 minutes at 37°C, 5%
CO2.
During the incubation period the serum dilutions
were made in block buffer BSA 1%. As positive
controls cat 93**3 for type I and cat G317**4 for
type II were used. As negative control a SPF serum
from a cat was applied on one ELISA plate of each
serotype. The sera were threefold diluted from 1:15
to 1:32805 because this was done in former
projects. (S. van’t Ende, 2011). The serum dilutions
were added in duplicate with 100µl/well .(see figure
5). This was incubated for 1 hour at 37°C, 5% CO2.
After the incubation period the plates were rinsed
three times with wash buffer**5 and three times with PBS0.
Then the conjugate, Goat anti cat Horseradisch Protein
(HRP0), was diluted (1:2000) in block buffer BSA 1%
and added to the plates with 100µl/well. This was
incubated at 37°C, 5% CO2.
Figure 5: Lay out of the ELISA plate at the first
coating. Protein dilutions were added in
duplicate. On the left the positive controls were
added and on the right side the negative control
was added.
After the last incubation period the plates were rinsed again with wash buffer and PBS0, each three
times. Next 100µl Tetramethylbenzidine (TMB**6) was added to each well. To stop the substrate
11
reaction after 4 minutes, 2M H2SO4 was added with 100µl/well. The OD-values were determined with
the ELISA reader (Gen 5).
A second coating was performed based on the results.
(see results 3.3.1). The S1-protein concentrations of the
second coating were: 0.3 µg/ml, 0.06 µg/ml and 0.012
µg/ml. The proteins were tenfold diluted to increase the
amounts of plates that could be coated. The amounts of
proteins were 0.116 mg/ml for UU23-S1 and 0.101
mg/ml for SeroII-S1. The S1 proteins were diluted in
DPBS and added to the plates in 100µl/well. The sera
were fivefold diluted from 1:45 to 1:3515625. (see fig.
6). As control sera, the same sera were used as at the
fist coating. (For the protocol see 2.3.3. ELISA S1
protein, optimal coating concentration)
Figure 6: Lay out of the ELISA plate at the second
coating. Protein dilutions were added in duplicate.
On the left the positive controls were added and
on the right side the negative control was added.
Conjugate concentration
Conjugate was added in 1:2000 at the first two coatings
which was chosen based on results from a former project. (S. van’t Ende, 2011). To determine the
optimal conjugate concentration, different dilutions of the conjugate were added on one ELISA plate.
(see fig. 7). Two ELISA plates were coated, one with 0.3 µg/ml UU23-S1 and the other with 0.3
µg/ml SeroII-S1. As positive controls Cat 93 and 142 end serum**7 as a type I control and Cat G317
as a type II control were used. The sera were twenty-five fold diluted from 1:45 to 1:703125 and
added in duplicate on the plate. The conjugate was added in the following concentrations 1:2000,
1:4000 and 1:8000. (see fig. 7).
.
Figure 7: Lay out of an ELISA plate applied with the
following conjugate dilutions: 1:2000, 1:4000 and
1:8000. The twenty-five-fold serum dilutions were
made from 1:45 to 703125.
12
After determining the optimal coating concentration of UU23-S1 and SeroII-S1 (0.3 µg/ml) and the
optimal conjugate dilution (1:4000), all selected sera were tested according to the same protocol as
described in 2.3.3. ELISA S1 protein, optimal coating concentration.
All of the following ELISA plates were coated with 0.3 µg/ml UU23-S1 and 0.3 µg/ml SeroII-S1.
The conjugate was added with 1:4000. The fivefold serum dilutions were made from 1:45 to
1:3515625. For the general layout of the ELISA plates, see fig. 8.
Figure 8: Lay out of the ELISA plates coated with
UU23-S1 and SeroII-S1 to screen all the sera. The sera
are added to the plate in duplicate. On one plate of
each type a SPF serum was applied.
2.3.3. ELISA N-domain
As mentioned before in the introduction, the S1 protein can be subdivided in the C- and N-domain. Of
which the N-domain is less conserved and thus should be more discriminative than the whole S1
protein. To determine whether the N-part is more discriminative than the whole S1 protein, plates
were coated with UU23-N-S1 for serotype I and SeroII-N-S1. For both serotypes 0.3 µg/ml was
chosen to be the coating concentration. The amounts of proteins were 0.022 mg/ml for UU23-N-S1
and 0.043 mg/ml for SeroII-N-S1. Only 11 sera were tested on the N-coated plates of both serotypes.
The layout of the ELISA plates was similar to fig. 8. (For the protocol see 2.3.3. ELISA S1 protein,
optimal coating concentration)
Optimization of the ELISA conditions
Because the results of the N-coated ELISA plates showed a high background, the intention was to
optimize conditions of the ELISA to lower the background. Two plates were coated overnight with 0.3
µg/ml UU23-N-S1 and SeroII-N-S1 in the refrigerator. (see fig. 9). The ELISA was performed
according to the protocol mentioned in 2.3.3. (ELISA S1 protein, optimal coating concentration)
except for the block buffer . In this ELISA block buffer BSA1% was compared to Normal Goat Serum
10% (NGS10%) in tween20 0.005%. One plate was blocked with BSA 1% and the other with
NGS10%. Both for 45 minutes at 37 ⁰C, 5% CO2. The 5-fold serial serum dilutions were made in the
same buffer as the plates were blocked with.
Figure 9: Layout ELISA plate coated with 0.3 µg/ml UU23-N-S1
and SeroII-N-S1. With cat 93 as a positive control serum for type I
and G317 as a positive control for type II. On the left, the white
wells were coated with UU23-N-S1 and in the middle the green
wells were coated with SeroII-N-S1. The right wells (grey) were
empty.
13
**
1
**
2
**
3
Cat 93 = type I serum FeCV-RM, cat 93 trial V, 12-12-01.
**
4
Cat G317 = type II ascites exp. cat, 4-12-1996.
**
5
PBS0 + Tween20 0.005%
**
6
Substrate cat No TTMB-1000-01, TMB super slow, one component HRP, microwell substrate.
Lot. PBO1902, Exp/ 07-17-13, Store at 2°C to 8°C. Bio FX laboratories
**7
REF 655092, ELISA plate microloan, F-shape, Greiner bio one. Lot. E12Ø7ØF3. 2016-07. Raw
material batch: 38294575LO.
Details BSA: fraction V, pH 7, Cat. No K41-001, PAA the Cell Culture Company.
Cat 142 end serum: FeCV, UCD, experiment 8 group 6, 2006.
14
3. Results
3.1.
Results Immune Fluorescence Assay
Table 1 presents the results of the IFA perfomed on samples with an unkown IFA titer. For some sera
also the PCR, FeLV and/or FIV results are presented. However these tests were not performed in this
project. The first row presents the cat number, the second the material that was tested and the third the
IFA titer.
First the results of the experimental infected cats are presented at the top. Of the experimental infected
cats, only one showed a titer of <1:20. Next the results of the 38 tested field cats are presented in the
table. All samples that were considered to be seronegative are highlighted in orange. The results
showed that 14 out of the 38 tested field cats had an IFA titer of <1:20, which is 36.8%.
Exp. Type I
Exp. Type II
FIELD SERA
Cat nr.
91
93
95
115
Cat 142 end, D63
and D21
Kat 166 end, D61
and D63
G317
9912
C16235
C16289
C16298
C16299
C16301
C16305
Material
Serum
Serum
Serum
Serum
Serum
IFA titer
320
320
80
320
<20
PCR
n.p.
n.p.
n.p.
n.p.
n.p.
FeLV
n.p.
n.p.
n.p.
n.p.
n.p.
FIV
n.p.
n.p.
n.p.
n.p.
n.p.
Serum
160
n.p.
n.p.
n.p.
Serum
Serum
EDTA
EDTA
EDTA
EDTA
EDTA
640
2560
<20
160
<20
2560
320
<20
n.p.
n.p.
+
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
C16307
C16309
C16310
C16316
C16321
C16322
C16337(2003)
C16340
EDTA
EDTA
EDTA
EDTA
EDTA
EDTA
EDTA
EDTA
2560
320
80
1280
<20
<20
160
640
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
C16345
C16346
C16350
C16355
EDTA
EDTA
EDTA
EDTA
320
>2560
>2560
<20
+
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
-
n.p.
n.p.
n.p.
-
C16356
EDTA
<20
n.p.
-
-
C16365
C16367
EDTA
Heparin plasma
160
<20
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
C16369
C16371
C16372
C16373
C16374
C16378
EDTA
EDTA
EDTA
EDTA
EDTA
EDTA
>2560
<20
1280
1280
>2560
20
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
C16379
C16381
C16471
EDTA
EDTA
Plasma
<20
40
20
n.p.
n.p.
n.p.
n.p.
n.p.
-
n.p.
n.p.
-
15
C16472
C16473
C16475
C16476
C16477
C16478
C16479
C16684
Plasma
Plasma
Plasma
EDTA
EDTA
EDTA
EDTA
EDTA
<20
<20
640
<20
<20
160
80
1280
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
+
n.p.
n.p.
Table 1: Results of the IFA’s. The results of the PCR, FeLV and FIV are derived from the serum bank folder. Orange boxes
are considered to be seronegative. N.p. = not performed, + = positive, - = negative.
3.2.
Results Virus Neutralization Test
3.2.1.
Virus titration
Serotype I: TN406HP
These results were difficult to interpreted because many cells were already dead before they were
killed by the virus. The TCID50 was determined by the Spearmann-Kärber formula:
LogID50 / volume = (X0 – d/2) + (d/n * X1)
 X0 = -log of the highest dilution with positive wells.
 d = dose distance (log10 = 1)
 n = number of wells/dilution
 X1 = sum of all positive wells from X0
LogTCID50 / 100μl = (5 – ½) + (1/7 * 9)
LogTCID50 / 100μl = 4,5 + 1,29
LogTCID50 / 100μl = 105,79
LogTCID50 / ml = 106,79
106,79 TCID50/ml**1
**1 At first 106.79 TCID50/ml was used but this
appeared not to be enough. It was decided to use 106
TCID50/ml determined by A. blanken.
Figure 10: Layout 96 wells plate with
CPE (grey) after titration of TN 406HP.
Serotype II: FIPV 79-1146
LogTCID50 / 100μl = (6– ½) + (1/7 *10)
LogTCID50 / 100μl =5,5 + 1,43
LogTCID50 / 100μl = 106,93
LogTCID50 / ml = 107,93
Figure 11: Layout 96 wells plate with
CPE (grey) after titration of FIPV 791146 .
3.2.2. Results Virus Neutralization test
For results of the VNT see table 7 and 8.
16
3.3.
Results ELISA S1-protein
3.3.1.
Results S1 protein production
Protein
Whole type I-S1
Whole type II-S1
C-domain of type I-S1
N-domain of type I-S1
N-domain of typeII-S1
Name of the protein
UU23-S1
SeroII-S1
UU21-S1
UU23-N-S1
SeroII-N-S1
Concentration
1.16 mg/ml
1.01 mg/ml
1.76 ml/ml
0.20 mg/ml
0.43 mg/ml
Table 2: protein concentrations of the whole S1 serotype I and II + the N part of both and the C-part of type I.
3.3.2. Results ELISA S1-protein
Samples were considered to be seropositive when their mean OD-value was higher than two times the
mean OD-value of the SPF serum.
Optimal coating concentration
The results of the first coating are presented in table 3. This table shows the protein coating
concentrations 7.5, 1.5 and 0.3 µg/ml and the ELISA titer of a positive control serum on a
corresponding S1 coating. Thus a type I sample (cat 93) was tested on a serotype I S1 coating and a
type II sample (cat G317) was tested on a serotype II S1 coating. The table presents the titer of those
samples on plates coated with different concentrations of the S1 protein. The results showed that at all
concentrations, the samples had a titer of >32805, so the ending point of the ELISA titer was not yet
reached.
Protein concentration
Titer Cat 93 on Type I coating
7.5 µg/ml
1.5 µg/ml
0.3 µg/ml
>32805
>32805
>32805
Titer Cat G317 on type II
coating
>32805
>32805
>32805
Table 3: Titers of cat 93 (type I) and G317 (type II)at the first coating. Sample 93 was tested at a type I plate and G317 was
tested at a type II plate.
The results of the second coating showed, that when the plates were coated with lower concentrations
of the S1 protein, the ending point of the ELISA titer was reached. (see table 4). For the serotype I and
II samples 0.3 µg/ml showed the most favorable ratio between the signal and the background. (see
appendix: ELISA results second coating).
Protein concentration
Titer Cat 93 on Type I coating
0.3 µg/ml
0.06 µg/ml
0.012 µg/ml
1: 140625
1: 28125
1: 5625
Titer Cat G317 on type II
coating
1: 140625
1: 28125
1: 5625
Table 4: Titers of cat 93 (type I) and G317 (type II)at the second coating. Sample 93 was tested at a type I plate and G317
was tested at a type II plate.
17
ELISA S1-protein
Table 7 presents the ELISA titers of the experimental infected cats that were tested on S1 coated
plates. It also shows the VNT titer performed with type I (TN406HP) and type II (FIPV 79-1146) plus
the IFA titer.
The results show a higher titer of serotype I samples on a type I coated plate than on a type II coated
plate and vice versa. However, the results also show that there is cross reaction because type I samples
still had a high titer on type II plates and vice versa. That’s why the ELISA results are interpreted by
looking at the ratio between the titer on the type I plate / the titer on the type II plate. All type II sera
had a ratio of <1 and all type I titers a ratio of ≥1. All samples that were indicated as a type II sample
in the VNT were also indicated as a type II sample in the ELISA. Most samples that were indicated as
a type I sample in the VNT were also indicated as a type I in the ELISA except when both titers were
<45, then the ratio could not be determined.
Cat nr.
Exp. type II
Exp. type I
G317
C15809
9912
91
93
95
115
Cat 142 end
Cat 142 d63
Cat 142 d21
Cat 147
Cat 177
Cat 166 end
Cat 166 d63
Cat 166 d21
ELISA
titer type
I coating
<45
703125
1125
140625
28125
140625
140625
703125
>3515625
<45
>3515625
>3515625
140625
140625
28125
ELISA
titer type
II coating
28125
>3515625
703125
5625
45
1125
5625
<45
<45
<45
28125
140625
1125
5625
1125
Ratio
ELISA
TI/TII
<0.0016
<0.2
0.0016
25
625
125
25
>15625
>78125
c.b.d
>125
>25
125
25
25
VNT titer
TN406HP
40
<10
40
20
20
20
20
15
15
5
1215
3645
405
135
15
VNT titer
FIPV 791146
3645
3645
>1280
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
IFA titer
640
2560
2560
320
320
80
320
<20
<20
<20
>2560
2560
160
160
160
Table 5: overview of the results of the control sera. C.b.d. = cannot be determined.
18
Table 8 presents the results of the field sera that were tested in the S1 ELISA. Again, the results were
interpreted by means of the ratio between the ELISA titer on the type I coated plate/ ELISA titer on
the type II coated plate.
Only one serotype II sample (C16304) was found out of 80 samples, which is an estimated prevalence
of 1.25%. Three samples had a ratio of 1, which indicates that the ELISA was not able to discriminate
between serotype I and II. The rest of the samples all showed a ratio of >1 and thus were indicated as
serotype I samples. This was confirmed by the VNT results. (See table 8). One sample (C16251)
showed some remarkable results because the VNT titer for both serotypes was <5 but the ELISA
indicated that sample as a type II sample.
Cat nr.
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.
C15428
C15494
C15554
C15575
C15594
C15606
C15608
C15822
C15845
C15892
C15893
C15904
C15909
C15971
C15983
C16000
C16001
C16002
C16003
C16111
C16122
C16154
C16222
C16230
C16231
C16232
C16239
C16240
C16248
C16249
C16251
C16253
C16254
C16263
C16274
C16278
C16284
C16289
C16299
C16300
C16301
C16304
C16307
ELISA titer
type I
coating
140625
>3515625
5625
703125
>3515625
28125
>3515625
140625
1125
>3515625
703125
28125
>3515625
140625
>3515625
>3515625
>3515625
140625
140625
>3515625
140625
703125
28125
>3515625
28125
1125
140625
140625
140625
>3515625
<45
>3515625
28125
703125
140625
703125
>3515625
1406025
5625
140625
>3515625
5625
>3515625
ELISA
titer type
II coating
225
140625
225
5625
5625
1125
5625
1125
1125
5625
28125
1125
140625
5625
5625
140625
5625
5625
225
28125
225
5625
1125
140625
1125
1125
5625
1125
1125
5625
5625
28125
5625
5625
5625
5625
140625
1125
1125
28125
28125
>3515625
28125
Ratio
ELISA
TI/TII
625
>25
25
125
>625
125
>625
125
1
>625
25
25
>25
25
>625
>25
>625
25
625
>125
625
125
25
>25
25
1
25
125
125
>625
<0.008
>125
5
125
25
125
>25
125
5
5
>125
<0.0016
>125
VNT titer
TN406HP
3645
>10935
80
1280
≥1280
80
160
20
20
≥1280
1215
40
≥1280
80
160
1280
80
80
40
≤1280
80
40
20
≥1280
15
45
45
135
45
3645
<5 (tox)
3645
135
45
135
405
3645
405
10
45
≥1280
5 (tox)
3645
VNT titer
FIPV 791146
<10
<10
<10
<10
<10
<10
<5
<5
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<10
<5
<5
>10935
<5
IFA titer
320
2560
40
1280
640
320
640
1280
<20
2560
320
80
2560
320
1280
2560
320
40
160
2560
20
320
80
160
<10
20
1280
640
320
2560
<20
2560
640
320
2560
2560
2560
160
2560
160
320
1280
2560
19
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
C16309
C16310
C16316
C16337**
C16340
C16345
C16346
C16350
C16365
C16369
C16372
C16373
C16374
C16378
C16381
C16437
C16438
C16445
C16448
C16449
C16459
C16460
C16461
C16462
C16463
C16465
C16467
C16468
C16471
C16473
C16474
C16475
C16476
C16477
C16478
C16479
C16684
>3515625
>3515625
703125
140625
>3515625
28125
703125
>3515625
140625
>3515625
>3515625
>3515625
<45
5625
140625
>3515625
140625
703125
>3515625
>3515625
>3515625
140625
140625
140625
28125
28125
28125
703125
28125
<45
>3515625
28125
1125
<45
28125
703125
703125
5625
1125
28125
140625
28125
5625
28125
1125
1125
28125
5625
1125
<45
225
28125
140625
5625
5625
140625
28125
703125
1125
1125
5625
5625
225
1125
225
1125
<45
28125
5625
<45
<45
5625
5625
5625
>625
>3125
25
1
>125
5
25
>3125
125
>125
>625
>3125
c.b.d.
25
5
>25
25
125
25
>125
>5
125
125
25
5
125
25
3125
25
c.b.d.
125
5
>25
c.b.d.
5
125
125
135
45 (tox)
405
135
1215
45 (tox)
3645
3645
135
>10935
>10935
1215
40
45 (tox)
135
>10935
135
1215
>10935
1215
135
45
45
135
135
45
45
1215
45
15 (tox)
3645
135
15
15
135
135
>10935
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<10
<5
<5
<5
<5
<5
5
<5
135
<5
<5
5
5
<5
15
<5
<5
<5
5
<5
<5
<5
<5
<5
<5
320
80
1280
160
640
320
>2560
>2560
160
>2560
1280
1280
>2560
20
40
2560
160
320
>2560
80
640
160
160
320
640
160
40
2560
20
<20
>2560
640
<20
<20
160
80
1280
Table 6: overview of the results of the field sera. Grey = tested serotype II in the ELISA and/or VNT. C.b.d. = cannot be
determined.
20
Because the ELISA results were compared to the VNT titers it was decided to determine whether there
was a correlation between the ELISA and VNT titers. The titers of the ELISA and VNT of all the type
I samples, including the type I control samples were plotted against each other. The type II samples
were not included in the graph because only three samples were available. The correlation coefficient
was r2=0.53 which ranges between -1 to 1. The scatterplot shows that the samples which had a low
VNT titer, had divergent ELISA titers. However, the samples which showed a higher VNT titer also
showed high ELISA titer. The correlation between the ELISA and VNT titer is mainly due to those
samples.
ELISA vs. VNT titer
10000000
R² = 0.53
1000000
ELISA titer
100000
10000
1000
100
10
1
1
10
100
1000
10000
100000
VNT titer
Graph 1: scatterplot of the ELISA and VNT titer. The ELISA titer is displayed on the Y-axis in log, so is the VNT titer on the
X-axis.
21
3.3.3. Results ELISA N-domain
The results of the ELISA performed with the N-domain of both serotypes are presented in table 6. The
results of the ELISA performed with the whole S1 protein are presented on the left side (red) and the
results of the ELISA performed with the N-domain on the right (blue). Only cat C15809, G317 and 93
were experimentally infected. The rest were field cats that were selected based on the typeI/type II
ratio in the S1 ELISA.
Most of the titers of the serotype I samples were lower on the type I coated plates in the Ndomain ELISA than in the S1 ELISA. (see table 6). Some of them were higher but then the ratio was
unchanged or 1. One of the type II samples (C15809) did show improvement in the N-domain ELISA.
Because in the S1 ELISA it had a titer of 1:703125 on a type I coated plate, while it is a serotype I
sample, and in the N-domain ELISA it was negative on a type I coated plate.
In comparison with the coating with the whole S1 protein, cross-reactions from type I samples
on type II plates and vice versa were still present. But type I samples still had a higher titer on the type
I coated plates than on the type II coated plate and vice versa.
Type
II
Type
I
Lion
Cat nr.
UU23-S1
SeroII-S1
Ratio I/II
G317
C15809
C16304
93
C15845
C16232
C16374
C16002
C16254
C16300
C16251
<45
703125
5625
28125
1125
1125
<45
140625
28125
140625
<45
28125
>3515625
>3515625
45
1125
1125
<45
5625
5625
28125
5625
<0.0016
<0.2
<0.0016
625
1
1
C.b.d.
25
5
5
<0.008
UU23-NS1
<45
<45
1125
5625
1125
5625
225
5625
1125
1125
<45
SeroII-NS1
5625
140625
140625
<45
1125
5625
225
225
1125
1125
1125
Ratio
IN/IIN
<0.008
<0.00032
0.008
>125
1
1
1
25
1
1
<0.04
Table 7: Results of the ELISA with the N-part of serotype I and II. On the top the type II and on the bottom the type I titers.
On the left in the orange are the results of the coating with the whole S1 protein for comparison with the N-part on the right
(in the blue part). C.b.d. = cannot be determined.
22
Optimization of the ELISA conditions
To optimize the ELISA conditions, BSA block buffer was compared to NGS. Table 5 shows the titers
of experimental infected cats 93 (type I) and G317 (type II) on BSA block plates and NGS blocked
plates coated with the N-domain.
The results showed that the titer of both cats were the same for both block buffers.
Cat nr.
Cat 93 on type I coating
Cat G317 on type II coating
BSA (N-coating)
1:1125
1:28125
NGS (N-coating)
1:1125
1:28125
Table 8: titers of cat 93 (type I) on a type I-N coated plate and cat G317 (type II) on a type II-N coated plate blocked with
different block buffers (BSA and NGS).
The results of using NGS as a buffer compared to BSA are presented in graph 2 and 3. The results
show a minor difference in background. The BSA plate shows a higher background in the first
dilutions when the OD-values of the SPF sera of NGS and BSA are compared. The background on
the NGS plate is lower than on the BSA plate. However, when comparing the signal of the positive
control serum , the results show that the signal is lower on the NGS plate than on the BSA plate. In the
highest dilutions there is hardly any difference shown between BSA and NGS.
UU23-N-S1: BSA vs. NGS
SeroII-N-S1: BSA vs. NGS
1.5
1
SPF BSA
0.5
93 BSA
0
SPF NGS
OD-value
OD-value
2
5
4
3
2
1
0
SPF BSA
G317 BSA
SPF NGS
93 NGS
Titer
G317 NGS
Titer
Graph 2 and 3: on the left graph 2 and on the right graph 3. Graph 2 shows BSA Block buffer vs. NGS block buffer for a
UU23-N-S1 coated ELISA plate. The mean OD-values are displayed. Graph 3 shows BSA Block buffer vs. NGS block buffer
for a SeroII-N-S1 coated ELISA plate. The SPF OD values are mean*2.
23
4. Discussion and conclusion
4.1.
IFA
In the IFA, 38 field cats were screened for antibodies against the FCoV. Out of 38 samples, 14 had a
titer of <1:20 and were considered to be seronegative. That’s an estimated prevalence of 36.8%
seronegative and 63.2% seropositive samples. As mentioned in the introduction, antibodies are found
in 20-60% of pet cats (up to 100% in multicat households or catteries). Thus 63.2% is a good
representation, taken in consideration that the samples were derived from catteries and singlecat
households.
4.2.
VNT
The samples with an unknown VNT titer were tested with a VNT of both serotypes. All control sera
showed the same results, the type I samples had a titer of <5 in the type II VNT and were seropositive
in the type I VNT and the type II samples had a low titer in the type I VNT but a high titer in the type
II VNT. Most field samples showed the same results but there were a few that showed different
results. One sample (C16459) showed an equal titer (1:135) in the type I and II VNT. This sample
was indicated as a type I sample by the ELISA. This cat could be co-infected with both serotypes. As
mentioned in 3.2.2., one field sample (C16304) appeared to be a serotype II infected cat.
One of the difficulties of the VNT was, that some samples had a toxic effect on the cells which caused
cell death. This made the interpretation of the VNT results difficult. Another difficulty was that the
FCWF cells also died when the plates were incubated over a longer time frame. For the type II VNT
this was not a problem because these plates were incubated for 2-3 days but the type I VNT was
incubated for 5-6 days and that affected the quality of the cells which led to cell dead which was not
caused by the virus.
4.3.
ELISA
The results of table 7 shows that the ELISA was able to discriminate between infections with serotype
I and II because experimental infected cats with type I were indicated as a type I serum by the VNT
and by the ELISA. Because serotype I samples still gave a high signal on type II S1 coated plates (and
vice versa) the titer between the type I and II coated plates were necessary to determine what serotype
a cat was infected with. The ELISA plates cannot be used separately. The ratio of type I samples was ≥
1 and of type II samples <1. That means that there were some type I samples that had a ratio of 1 and
thus the serotype was not discriminated by the ELISA. (highlighted by blue boxes in table 8).
However, table 8 shows that those samples were considered to be seronegative in the IFA or had a
very low titer and the same goes for the VNT titers. Thus when the titers of the IFA and VNT are low
the ELISA is also less discriminative.
One sample (C16304) was indicated as a type II serum by the ELISA and the VNT. However,
another sample (C16251) was indicated as a type II serum by the ELISA but was seronegative (<5) for
both serotypes in the VNT. This appeared to be a lion serum. What the exact reason is for these
results, is unknown. One of the reasons could be that it’s a cross reaction of another coronavirus.
However, then a seropositive IFA was to be expected. A difficulty with this sample in the VNT was
that it had toxic effect on the cells, so the results were harder to be interpreted. More lion sera could be
tested to see whether they give the same results. Or this one could be tested again in the VNT, in the
ELISA this sample was tested twice and twice the sample was indicated as a type II.
Because the N-domain of the S1 protein is less conserved, ELISA’s performed with an N-coating
should be more discriminative than ELISA’s performed with a S1 coating. Only 11 samples were
tested on N-coated plates. Those results showed that the titer of type I samples on type I-N coated
plates were still higher than on the type II-N coated plates and vice versa. So the ratio of type I
samples was still ≥1 and of type II samples <1. This corresponds to the results of the S1 ELISA.
However, the ratio between the type I and II titer of the type I samples were unchanged for most of the
samples. Which indicates that the N-domain wasn’t more discriminative than the S1 protein.
24
For most of the type I samples the titer in the N-ELISA on the type I coated plates was lower than in
the S1 ELISA on the type I coated plates. For the type II samples it’s impossible to conclude whether
the N-domain is more discriminative because only three serotype II samples were tested. Of these
three samples, 1 sample showed improved results because it had a titer of 1:703125 in the S1 ELISA
type I coated plate and was negative (<1:45) in the N-domain ELISA type I coated plate. All the
results other samples of serotype I and II weren’t significantly changed because the ratio was
unchanged. Thus for these 11 samples the N-domain wasn’t more discriminative. However, this
should be tested on more samples to concluded whether the N-domain is really not more
discriminative than the whole S1 protein.
The results of the N-domain showed a high background. This can be due to conjugate that is
left on too long or is to strong, contaminants from laboratory glassware on non-specific antibody
binding that might be due to block buffer that is incubated too short of which is inaccurate. In this
project was tested whether a change of block buffer would lower the background. This was done by
comparing BSA1% to NGS10%. The results show that NGS10% actually caused a lower background
than BSA. But the signal of the positive control serum was also lower on the NGS10% block plates.
Thus the ratio between those two weren’t significantly different.
Because the VNT results were used as the gold standard for the ELISA results, it was decided to
compare those results and see whether there was a correlation between the two parameters. Graph 4
shows the scatterplot in which the VNT titers and ELISA titers are shown. The correlation coefficient
r2 = 0.53. The correlation coefficient ranges between -1 to 1 so 0.53 is not a very strong correlation
between the VNT and ELISA titers. The correlation was mainly seen in the samples with a high VNT
titer which also showed a high ELISA titer. The samples with a low VNT titer had divergent ELISA
titers. The difference in titer can be due to the fact that both tests demonstrate different antibodies. The
VNT only demonstrates the neutralization antibodies what makes the VNT more specific than the
ELISA .
4.4.
Future experiments
In this project the results of the ELISA, VNT and the IFA were not compared with the information
that is known about the samples that were used. The titers could be compared to the clinical signs to
determine whether there’s a correlation for an example.
This project can be used as a set up for other experiments because the ELISA appeared to be valid.
The whole serum bank could be screened to determine the seroprevalence of serotype I and II FCoV in
this population.
The different domains of the S1 protein could be tested. For this project the C-domain of serotype II
wasn’t available but that could be compared to the N-domain for an example. And more sera should be
tested on N-coated plates because only 11 samples were tested in this project.
For further optimization of the ELISA conditions other measures like changing the substrate or
increasing the blocking time, could be tested.
Because the lion sample (C16251) gave such remarkable results this one could be tested again or more
lion samples could be tested to determine whether they all give similar results.
25
References
Belouzard, S., Millet, J. K., Licitra, B. N., & Whittaker, G. R. (2012). Mechanisms of coronavirus
cell entry mediated by the viral spike protein. Viruses, 4(6), 1011- 1033.doi:10.3390/v4061011.
Website: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3397359/
Berend Jan Bosch. (2004). The Coronavirus spike protein: mechanisms of membrane fusion and virion
incorporation (25-11-2004). Promotor: Prof.dr. P.J.M Rottier. Printer: Wöhrmann Print Service, Zutphen. ISBN:
90-393-3847-7. Website: http://igitur-archive.library.uu.nl/dissertations/2005-0301-003047/full.pdf
Chang, H. W., Egberink, H. F., Halpin, R., Spiro, D. J., & Rottier, P. J. (2012). Spike protein fusion peptide
and feline coronavirus virulence. Emerging Infectious Diseases, 18(7), 1089-1095. doi:10.3201/eid1807.120143;
10.3201/eid1807.120143
Driciru, M., Siefert, L., Prager, K. C., Dubovi, E., Sande, R., Princee, F., et al. (2006). A serosurvey of viral
infections in lions (Panthera leo), from Queen Elizabeth National Park, Uganda. Journal of Wildlife Diseases,
42(3), 667-671.
Hofmann-Lehmann, R., Fehr, D., Grob, M., Elgizoli, M., Packer, C., Martenson, J. S., et al. (1996).
Prevalence of antibodies to feline parvovirus, calicivirus, herpesvirus, coronavirus, and immunodeficiency
virus and of feline leukemia virus antigen and the interrelationship of these viral infections in free-ranging
lions in east Africa. Clinical and Diagnostic Laboratory Immunology, 3(5), 554-562.
Kummrow, M., Meli, M. L., Haessig, M., Goenczi, E., Poland, A., Pedersen, N. C., et al. (2005). Feline
coronavirus serotypes 1 and 2: seroprevalence and association with disease in Switzerland. Clinical and
Diagnostic Laboratory Immunology, 12(10), 1209-1215. doi:10.1128/CDLI.12.10.12091215.2005
Le Poder, S. (2011). Feline and canine coronaviruses: common genetic and pathobiological features. Advances
in Virology, 2011, 609465. doi:10.1155/2011/609465
Masters, P. S. (2006). The molecular biology of coronaviruses. Advances in Virus Research, 66, 193
292. doi:10.1016/S0065-3527(06)66005-3
Myrrha, L. W., Silva, F. M., Peternelli, E. F., Junior, A. S., Resende, M., & de Almeida, M. R. (2011). The
paradox of feline coronavirus pathogenesis: a review. Advances in Virology, 2011, 109849.
doi:10.1155/2011/109849
Sharif, S., Arshad, S. S., Hair-Bejo, M., Omar, A. R., Zeenathul, N. A., & Alazawy, A. (2010). Diagnostic
methods for feline coronavirus: a review. Veterinary Medicine International, 2010, 809480.
doi:10.4061/2010/809480
The Merck veterinary manual. http://www.merckvetmanual.com/mvm/index.jsp?cfile=htm/bc/56900.htm
Blz. 354/355. ISBN 0-911910-93-X, ISSN 0076-6542.
Vogel, L., Van der Lubben, M., te Lintelo, E. G., Bekker, C. P., Geerts, T., Schuijff, L. S., et al. (2010).
Pathogenic characteristics of persistent feline enteric coronavirus infection in cats. Veterinary Research, 41(5),
71. doi:10.1051/vetres/2010043
Woo, P. C., Huang, Y., Lau, S. K., & Yuen, K. Y. (2010). Coronavirus genomics and bioinformatics
analysis. Viruses, 2(8), 1804-1820. doi:10.3390/v2081803.
26
Appendix: ELISA results
** samples are considered to be positive when their mean OD value is higher than 2*mean SPF
** grey = positive
** The second table shows the mean OD-values + mean OD value of the SPF serum*2
ELISA S1
First coating
Serotype I
Titer
7.5 µg/ml
1.5 µg/ml
0.3 µg/ml
7.5 µg/ml
1.5 µg/ml
0.3 µg/ml
1:15
4,20
3,37
4,33
4,43
4,44
4,22
0,74
0,87
0,95
0,99
1,33
1,54
1:45
4,35
4,33
4,44
4,42
4,36
4,31
0,39
0,36
0,49
0,43
0,61
0,68
1:135
4,30
4,40
4,50
4,46
4,27
4,13
0,24
0,25
0,38
0,27
0,30
0,31
1:405
4,35
4,40
4,41
4,36
4,02
3,75
0,15
0,14
0,15
0,15
0,15
0,09
1:1215
4,31
4,34
4,29
4,25
3,46
3,32
0,18
0,14
0,13
0,14
0,10
0,09
1:3645
3,96
4,12
3,74
3,83
2,51
2,38
0,16
0,13
0,12
0,15
0,09
0,07
1:10935
2,84
2,71
2,63
2,62
1,37
1,12
0,18
0,22
0,10
0,12
0,07
0,08
1:32805
1,32
1,42
1,43
1,32
0,61
0,60
0,19
0,16
0,10
0,13
0,07
0,07
Positive control 93
Negative control: SPF
Titer
7.5 µg/ml
1.5 µg/ml
0.3 µg/ml
7.5 µg/ml
1.5 µg/ml
0.3 µg/ml
1:15
3,78
4,38
4,33
1,61
1,94
2,87
1:45
4,34
4,43
4,34
0,75
0,92
1,29
1:135
4,35
4,48
4,20
0,48
0,65
0,61
1:405
4,38
4,38
3,88
0,29
0,31
0,25
1:1215
4,32
4,27
3,39
0,32
0,27
0,19
1:3645
4,04
3,79
2,45
0,29
0,26
0,16
1:10935
2,77
2,62
1,24
0,41
0,22
0,15
1:32805
1,37
1,37
0,60
0,35
0,22
0,14
Positive control: 93
Negative control: mean OD SPF*2
27
Serotype II
Titer
7.5 µg/ml
1.5 µg/ml
0.3 µg/ml
7.5 µg/ml
1.5 µg/ml
0.3 µg/ml
1:15
4,49
4,60
4,60
4,59
4,47
4,55
0,90
0,63
0,90
0,94
1,71
1,68
1:45
4,41
4,48
4,48
4,48
4,34
4,31
0,70
0,55
0,52
0,49
0,62
0,79
1:135
4,51
4,49
4,49
4,60
4,25
4,35
0,52
0,43
0,44
0,36
0,32
0,31
1:405
4,41
4,47
4,44
4,37
4,04
3,94
0,33
0,39
0,26
0,41
0,20
0,17
1:1215
4,29
4,41
4,46
4,42
3,79
3,77
0,27
0,40
0,34
0,21
0,10
0,10
1:3645
4,16
4,29
4,28
4,21
3,21
3,10
0,18
0,21
0,14
0,15
0,09
0,09
1:10935
3,72
3,87
3,62
3,55
2,24
2,26
0,17
0,15
0,10
0,14
0,07
0,08
1:32805
2,78
2,81
2,55
2,47
1,20
1,21
0,16
0,14
0,20
0,38
0,06
0,06
Positive control: G317
Negative control: SPF
Titer
7.5 µg/ml
1.5 µg/ml
0.3 µg/ml
7.5 µg/ml
1.5 µg/ml
0.3 µg/ml
1:15
4,54
4,60
4,51
1,53
1,84
3,39
1:45
4,45
4,48
4,32
1,25
1,02
1,41
1:135
4,50
4,54
4,30
0,95
0,79
0,62
1:405
4,44
4,40
3,99
0,72
0,67
0,36
1:1215
4,35
4,44
3,78
0,67
0,55
0,20
1:3645
4,22
4,24
3,16
0,39
0,29
0,18
1:10935
3,79
3,58
2,25
0,32
0,24
0,14
1:32805
2,79
2,51
1,21
0,30
0,58
0,13
Positieve controle: G317
Negative control: mean OD SPF*2
Second coating
Serotype I
Titer
0.3 µg/ml
0.06 µg/ml
0.012 µg/ml
0.3 µg/ml
0.06 µg/ml
0.012 µg/ml
1:45
4,20
4,21
3,16
3,22
1,46
1,34
0,32
0,25
0,31
0,33
0,41
0,41
1:225
4,15
4,02
2,50
2,57
0,85
0,86
0,11
0,11
0,11
0,12
0,12
0,12
1:1125
3,68
3,74
1,67
1,33
0,28
0,34
0,07
0,07
0,07
0,09
0,07
0,07
1:5625
2,03
2,09
0,70
0,57
0,14
0,12
0,06
0,06
0,06
0,06
0,06
0,06
1:28125
0,72
0,76
0,23
0,17
0,07
0,07
0,06
0,06
0,06
0,06
0,05
0,05
1:140625
0,22
0,20
0,09
0,08
0,06
0,05
0,06
0,06
0,06
0,05
0,05
0,06
1:703125
0,09
0,10
0,06
0,05
0,05
0,06
0,06
0,06
0,05
0,06
0,06
0,05
1:3515625
0,07
0,07
0,06
0,05
0,05
0,05
0,05
0,06
0,05
0,05
0,05
0,06
Positive control: 93
Negative control: SPF
28
Titer
0.3 µg/ml
0.06 µg/ml
0.012 µg/ml
0.3 µg/ml
0.06 µg/ml
0.012 µg/ml
1:45
4,21
3,19
1,40
0,57
0,64
0,83
1:225
4,08
2,53
0,85
0,22
0,22
0,24
1:1125
3,71
1,50
0,31
0,14
0,15
0,14
1:5625
2,06
0,63
0,13
0,12
0,13
0,11
1:28125
0,74
0,20
0,07
0,12
0,11
0,11
1:140625
0,21
0,08
0,06
0,11
0,11
0,11
1:703125
0,09
0,05
0,05
0,12
0,11
0,11
1:3515625
0,07
0,05
0,05
0,11
0,11
0,11
Positive control: 93 mean OD
Negative control: SPF mean*2
Serotype II
Titer
0.3 µg/ml
0.06 µg/ml
0.012 µg/ml
0.3 µg/ml
0.06 µg/ml
0.012 µg/ml
1:45
4,32
4,35
3,07
3,17
1,01
1,03
0,33
0,30
0,28
0,33
0,37
0,39
1:225
4,22
4,19
2,12
2,11
0,52
0,48
0,13
0,12
0,15
0,12
0,11
0,13
1:1125
3,88
3,51
1,26
1,39
0,27
0,26
0,08
0,08
0,06
0,06
0,07
0,07
1:5625
2,86
2,62
0,63
0,57
0,13
0,13
0,07
0,07
0,06
0,06
0,06
0,06
1:28125
1,06
1,04
0,22
0,21
0,08
0,08
0,08
0,06
0,06
0,06
0,05
0,06
1:140625
0,35
0,34
0,10
0,09
0,06
0,06
0,07
0,06
0,06
0,05
0,05
0,07
1:703125
0,11
0,11
0,07
0,06
0,06
0,06
0,07
0,08
0,06
0,06
0,06
0,06
1:3515625
0,07
0,07
0,06
0,05
0,05
0,06
0,07
0,06
0,06
0,06
0,06
0,07
Positive control: G317
Negative control: SPF
Titer
0.3 µg/ml
0.06 µg/ml
0.012 µg/ml
0.3 µg/ml
0.06 µg/ml
0.012 µg/ml
1:45
4,33
3,12
1,02
0,62
0,61
0,75
1:225
4,20
2,12
0,50
0,25
0,27
0,24
1:1125
3,69
1,32
0,26
0,15
0,13
0,13
1:5625
2,74
0,60
0,13
0,14
0,12
0,12
1:28125
1,05
0,21
0,08
0,14
0,11
0,11
1:140625
0,34
0,10
0,06
0,13
0,11
0,12
1:703125
0,11
0,06
0,06
0,15
0,11
0,12
1:3515625
0,07
0,05
0,05
0,13
0,11
0,13
Positive control: G317
Negative control: SPF
29
ELISA N-domain
Type IN (plate 1)
G317
C16304
93
C16251
C15809
SPF
1:45
0,28
0,28
1,05
1,04
1,73
1,72
0,83
0,82
0,96
0,71
0,44
0,42
1:225
0,12
0,11
0,41
0,41
0,84
0,78
0,29
0,30
0,38
0,32
0,16
0,14
1: 1125
0,07
0,07
0,20
0,19
0,30
0,32
0,17
0,14
0,15
0,15
0,08
0,07
1:5625
0,05
0,05
0,10
0,10
0,11
0,13
0,07
0,07
0,08
0,09
0,05
0,05
1:28125
0,05
0,05
0,06
0,07
0,08
0,06
0,05
0,05
0,07
0,06
0,05
0,04
1:140625
0,04
0,04
0,05
0,05
0,05
0,05
0,05
0,05
0,05
0,04
0,04
0,05
1:703125
0,05
0,04
0,05
0,05
0,05
0,05
0,05
0,05
0,05
0,05
0,05
0,05
1:3515625
0,04
0,04
0,07
0,04
0,06
0,06
0,04
0,04
0,05
0,04
0,05
0,04
G317
C16304
93
C16251
C15809
SPF
1:45
0,28
1,04
1,73
0,83
0,83
0,86
1:225
0,12
0,41
0,81
0,30
0,35
0,31
1: 1125
0,07
0,19
0,31
0,16
0,15
0,15
1:5625
0,05
0,10
0,12
0,07
0,08
0,11
1:28125
0,05
0,07
0,07
0,05
0,07
0,09
1:140625
0,04
0,05
0,05
0,05
0,05
0,09
1:703125
0,04
0,05
0,05
0,05
0,05
0,10
1:3515625
0,04
0,06
0,06
0,04
0,04
0,09
Type IN (plate 2)
C15845
C16232
C16374
C16002
C16254
C16300
1:45
1,22
1,12
1,93
1,74
1,15
1,22
2,37
2,33
1,85
1,87
2,09
2,03
1:225
0,49
0,52
0,83
0,77
0,36
0,38
1,40
1,27
0,79
0,78
0,93
0,95
1: 1125
0,22
0,20
0,25
0,28
0,14
0,15
0,56
0,46
0,28
0,27
0,34
0,36
1:5625
0,09
0,09
0,11
0,11
0,07
0,08
0,18
0,16
0,11
0,10
0,12
0,13
1:28125
0,06
0,06
0,07
0,09
0,05
0,05
0,08
0,07
0,07
0,06
0,06
0,06
1:140625
0,05
0,05
0,05
0,06
0,06
0,04
0,05
0,05
0,05
0,05
0,05
0,05
1:703125
0,05
0,05
0,04
0,07
0,05
0,05
0,05
0,05
0,05
0,05
0,05
0,05
1:3515625
0,04
0,05
0,04
0,04
0,05
0,05
0,04
0,05
0,05
0,05
0,05
0,04
C15845
C16232
C16374
C16002
C16254
C16300
SPF
1:45
1,17
1,83
1,19
2,35
1,86
2,06
0,86
1:225
0,50
0,80
0,37
1,34
0,79
0,94
0,31
1: 1125
0,21
0,26
0,14
0,51
0,27
0,35
0,15
1:5625
0,09
0,11
0,07
0,17
0,10
0,12
0,11
1:28125
0,06
0,08
0,05
0,07
0,06
0,06
0,09
1:140625
0,05
0,06
0,05
0,05
0,05
0,05
0,09
1:703125
0,05
0,06
0,05
0,05
0,05
0,05
0,10
1:3515625
0,04
0,04
0,05
0,04
0,05
0,04
0,09
30
Plaat IIN (plate 1)
G317
C16304
93
C16251
C15809
SPF
1:45
2,86
2,94
3,81
3,82
0,65
0,75
1,45
1,46
3,82
3,91
0,59
0,55
1:225
2,01
1,72
3,28
3,21
0,25
0,30
0,51
0,52
3,56
3,49
0,16
0,17
1: 1125
0,99
0,90
2,46
2,35
0,09
0,10
0,17
0,18
2,55
2,60
0,08
0,08
1:5625
0,38
0,47
1,59
1,51
0,07
0,07
0,14
0,09
1,49
1,43
0,06
0,06
1:28125
0,13
0,18
0,67
0,63
0,07
0,06
0,09
0,06
0,56
0,51
0,06
0,10
1:140625
0,09
0,07
0,20
0,24
0,05
0,06
0,06
0,05
,16
0,16
0,05
0,05
1:703125
0,07
0,06
0,10
0,09
0,05
0,05
0,05
0,06
0,07
0,07
0,04
0,04
1:3515625
0,05
0,05
0,06
0,06
0,05
0,06
0,05
0,05
0,06
0,05
0,05
0,06
G317
C16304
93
C16251
C15809
SPF
1:45
2,90
3,81
0,70
1,46
3,87
1,14
1:225
1,87
3,24
0,27
0,52
3,52
0,33
1: 1125
0,95
2,41
0,10
0,18
2,57
0,16
1:5625
0,42
1,55
0,07
0,11
1,46
0,12
1:28125
0,15
0,65
0,06
0,08
0,54
0,16
1:140625
0,08
0,22
0,05
0,05
0,16
0,10
1:703125
0,06
0,09
0,05
0,06
0,07
0,09
1:3515625
0,05
0,06
0,05
0,05
0,05
0,11
C16232
C16374
C16002
C16254
C16300
Type IIN (plate 2)
C15845
1:45
1,41
1,34
1,95
2,05
1,09
1,18
1,18
1,16
1,57
1,91
0,05
0,05
1:225
0,56
0,56
0,83
0,86
0,37
0,38
0,41
0,44
0,66
0,82
0,05
0,05
1: 1125
0,24
0,23
0,30
0,34
0,15
0,15
0,15
0,17
0,21
0,47
0,26
0,11
1:5625
0,10
0,10
0,13
0,14
0,08
0,08
0,09
0,08
0,10
0,10
0,07
0,06
1:28125
0,06
0,07
0,07
0,07
0,05
0,07
0,06
0,06
0,06
0,06
0,11
0,12
1:140625
0,05
0,06
0,07
0,05
0,05
0,05
0,05
0,05
0,05
0,05
0,26
0,29
1:703125
0,05
0,05
0,07
0,06
0,05
0,05
0,05
0,05
0,05
0,05
0,68
0,77
1:3515625
0,04
0,05
0,05
0,07
0,05
0,05
0,04
0,05
0,05
0,05
1,17
1,18
C15845
C16232
C16374
C16002
C16254
C16300
SPF
1:45
1,38
2,00
1,13
1,17
1,74
0,05
1,14
1:225
0,56
0,85
0,37
0,42
0,74
0,05
0,33
1: 1125
0,24
0,32
0,15
0,16
0,34
0,18
0,16
1:5625
0,10
0,14
0,08
0,09
0,10
0,07
0,12
1:28125
0,06
0,07
0,06
0,06
0,06
0,12
0,16
1:140625
0,05
0,06
0,05
0,05
0,05
0,27
0,10
1:703125
0,05
0,07
0,05
0,05
0,05
0,72
0,09
1:3515625
0,05
0,06
0,05
0,04
0,05
1,17
0,11
**C16300 was accidentally applied upside down.
31
Cat nr.
G317
C16304
93
C16251
C15809
C15845
C16232
C16374
C16002
C16254
C16300
ELISA
titer
type I
<45
5625
28125
<45
703125
1125
1125
<45
140625
28125
140625
ELISA
titer type
II
28125
>3515625
45
5625
>3515625
1125
1125
<45
5625
5625
28125
Type
I/II
ELISA titer type
IN
ELISA titer type IIN
Type IN/Type
IIN
<0.0016
<0.0016
625
<0.008
<0.2
1
1
c.b.d.
25
5
5
<45
1125
5625
<45
<45
1125
5625
225
5625
1125
1125
5625
140625
<45
1125
140625
1125
5625
225
225
1125
1125
<0.008
0.008
>125
<0.04
<0.00032
1
1
1
25
1
1
ELISA N-domain: optimization
NGS
Type I-N
SPF
Type II-N
93
SPF
G317
1:45
0,44
0,33
1,44
1,53
0,23
0,23
3,64
3,67
1:225
0,10
0,11
0,52
0,58
0,09
0,10
2,54
2,63
1: 1125
0,06
0,06
0,18
0,17
0,06
0,06
1,18
1,16
1:5625
0,05
0,05
0,07
0,08
0,05
0,05
0,33
0,38
1:28125
0,05
0,06
0,05
0,05
0,06
0,05
0,11
0,13
1:140625
0,05
0,05
0,05
0,05
0,05
0,05
0,06
0,05
1:703125
0,05
0,05
0,05
0,04
0,06
0,05
0,05
0,06
1:3515625
0,04
0,05
0,05
0,07
0,08
0,05
0,05
0,07
Type I-N
Type II-N
SPF
93
SPF
G317
1:45
0,77
1,48
0,46
3,66
1:225
0,21
0,55
0,20
2,58
1: 1125
0,12
0,18
0,11
1,17
1:5625
0,10
0,07
0,10
0,36
1:28125
0,10
0,05
0,11
0,12
1:140625
0,09
0,05
0,10
0,06
1:703125
0,10
0,05
0,11
0,05
1:3515625
0,10
0,06
0,12
0,06
32
BSA
Type I-N
SPF
Type II-N
93
SPF
G317
1:45
1:225
1: 1125
1:5625
1:28125
1:140625
1:703125
0,66
0,14
0,08
0,05
0,05
0,04
0,05
0,71
0,19
0,07
0,05
0,05
0,04
0,05
1,71
0,75
0,27
0,09
0,06
0,05
0,05
1,72
0,77
0,23
0,10
0,06
0,04
0,06
0,45
0,18
0,07
0,05
0,06
0,04
0,04
0,48
0,15
0,07
0,05
0,05
0,04
0,04
3,90
3,01
1,61
0,72
0,27
0,09
0,05
3,98
2,93
1,88
0,71
0,39
0,08
0,05
1:3515625
0,05
0,05
0,05
0,05
0,05
0,06
0,04
0,06
Type I-N
1:45
1:225
1: 1125
1:5625
1:28125
1:140625
1:703125
1:3515625
SPF
1,37
0,33
0,15
0,10
0,09
0,08
0,09
0,09
Type II-N
93
1,72
0,76
0,25
0,10
0,06
0,05
0,06
0,05
SPF
0,94
0,33
0,14
0,10
0,10
0,08
0,09
0,11
G317
3,94
2,97
1,75
0,71
0,33
0,08
0,05
0,05
33