Content uploaded by Jürgen Krücken
Author content
All content in this area was uploaded by Jürgen Krücken on Jun 23, 2015
Content may be subject to copyright.
Parasites & VectorsParasites & Vectors
This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted
PDF and full text (HTML) versions will be made available soon.
Development of a multiplex fluorescence immunological assay for the simultaneous
detection of antibodies against Cooperia oncophora, Dictyocaulus viviparus and
Fasciola hepatica in cattle
Parasites & Vectors Sample
doi:10.1186/s13071-015-0924-0
Sofia N. Karanikola (Sofia.Karanikola@fu-berlin.de)
Jürgen Krücken (Juergen.kruecken@fu-berlin.de)
Sabrina Ramünke (Sabrina.ramuenke@fu-berlin.de)
Theo de Waal (theo.dewaal@ucd.ie)
Johan Höglund (Johan.Hoglund@slu.se)
Johannes Charlier (johannes.charlier@UGent.be)
Corinna Weber (weber@laboklin.de)
Elisabeth Müller (mueller@laboklin.de)
Slawomir J. Kowalczyk (slawomirjankowalczyk@10g.pl)
Jaroslaw Kaba (jaroslaw_kaba@sggw.pl)
Georg von Samson-Himmelstjerna (gvsamson@fu-berlin.de)
Janina Demeler (j.demeler@fu-berlin.de)
Sample
ISSN 1756-3305
Article type Research
Submission date 1 May 2015
Acceptance date 1 June 2015
Article URL http://dx.doi.org/10.1186/s13071-015-0924-0
For information about publishing your research in BioMed Central journals, go to
http://www.biomedcentral.com/info/authors/
© 2015 Karanikola et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
(2015) 8:335
Development of a multiplex fluorescence
immunological assay for the simultaneous detection
of antibodies against Cooperia oncophora,
Dictyocaulus viviparus and Fasciola hepatica in cattle
Sofia N. Karanikola
1
Email: Sofia.Karanikola@fu-berlin.de
Jürgen Krücken
1
Email: Juergen.kruecken@fu-berlin.de
Sabrina Ramünke
1
Email: Sabrina.ramuenke@fu-berlin.de
Theo de Waal
2
Email: theo.dewaal@ucd.ie
Johan Höglund
3
Email: Johan.Hoglund@slu.se
Johannes Charlier
4
Email: johannes.charlier@UGent.be
Corinna Weber
5
Email: weber@laboklin.de
Elisabeth Müller
5
Email: mueller@laboklin.de
Slawomir J. Kowalczyk
6
Email: slawomirjankowalczyk@10g.pl
Jaroslaw Kaba
6
Email: jaroslaw_kaba@sggw.pl
Georg von Samson-Himmelstjerna
1
Email: gvsamson@fu-berlin.de
Janina Demeler
1*
*
Corresponding author
Email: j.demeler@fu-berlin.de
1
Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität
Berlin, Berlin, Germany
2
UCD School of Agriculture, Food Science and Veterinary Medicine, University
College Dublin, Dublin, Ireland
3
Department of Biomedical Sciences and Veterinary Public Health, Section for
Parasitology (SWEPAR), Swedish University of Agricultural Sciences, Uppsala,
Sweden
4
Department of Virology, Parasitology and Immunology, Faculty of Veterinary
Medicine, Ghent University, Ghent, Belgium
5
LABOKLIN GMBH & Co.KG, Bad Kissingen, Germany
6
Laboratory of Veterinary Epidemiology and Economics, Faculty of Veterinary
Medicine, Warsaw University of Life Science, Warsaw, Poland
Abstract
Background
A major constraint for the effective control and management of helminth parasites is the lack
of rapid, high-throughput, routine diagnostic tests to assess the health status of individual
animals and herds and to identify the parasite species responsible for these helminthoses. The
capability of a multiplex platform for the simultaneous detection of three pasture associated
parasite species was evaluated and compared to existing ELISAs.
Methods
The recombinant antigens 14.2 kDa ES protein for Cooperia oncophora, major sperm protein
for Dictyocaulus viviparus and Cathepsin L1 for Fasciola hepatica were recombinantly
expressed either in Escherichia coli or Pichia pastoris. Antigens were covalently coupled
onto magnetic beads. Optimal concentrations for coupling were determined following the
examination of serum samples collected from experimentally mono-infected animals, before
and after their infection with the target species. Absence of cross-reactivity was further
determined with sera from calves mono-infected with Haemonchus contortus, Ostertagia
ostertagi and Trichostrongylus colubriformis. Examination of negative serum samples was
characterised by low median fluorescence intensity (MFI).
Results
Establishment of the optimal serum dilution of 1:200 was achieved for all three bead sets.
Receiver Operating Characteristic analyses were performed to obtain cut-off MFI values for
each parasite separately. Sensitivity and specificity at the chosen cut-off values were close to,
or 100 % for all bead sets. Examination of serum samples collected on different days post
infection from different animals showed a high reproducibility of the assays. Serum samples
were additionally examined with two already established ELISAs, an in-house ELISA using
the recombinant MSP as an antigen and a DRG ELISA using Cathepsin L1 for liver fluke.
The results between the assays were compared and kappa tests revealed an overall good
agreement.
Conclusions
A versatile bead-based assay using fluorescence detection (xMAP® technology) was
developed to simultaneously detect antibodies against C. oncophora, D. viviparus and F.
hepatica in cattle serum samples. This platform provides rapid, high-throughput results and is
highly sensitive and specific in comparison to existing serological as well as coproscopical
diagnostic techniques.
Keywords
Parasitic gastroenteritis, Lungworm, Liver fluke, Luminex, Multiplex immunoassays, Serum,
Cattle, Diagnosis
Background
Nematode and trematode infections play an important role for animal welfare and are of great
concern for the economy of the global ruminant livestock industry today [1]. Constantly
increasing financial costs for anthelminthic prophylaxis and treatment due to the spread of
anthelmintic resistant parasite populations, as well as the often overlooked subclinical effects
of the helminth infections on animal productivity [2, 3] have led to the need of developing
new and sustainable strategies concerning the effective control of helminthoses. An important
step towards this end is the development of new, efficient and high-throughput diagnostic
techniques. Despite the development of more sensitive coproscopical methods [4], they often
target individual animals and are not suitable for high-throughput diagnosis. Serological
methods established so far appear to lack specificity, in particular when non-recombinant
antigens are used, and multiple tests have to be performed to detect mixed species infections.
Amongst the helminths responsible for pasture-borne parasitoses the liver fluke, lungworm as
well as gastrointestinal (GI) nematodes are the most important for cattle in temperate climate
regions. Cooperia oncophora, a parasite of the small intestine of cattle occurs worldwide
with high prevalence rates [5, 6]. It is usually associated with Ostertagia ostertagi,
parasitising the abomasum, and both contribute to the complex of parasitic gastroenteritis
(PGE) [1]. While O. ostertagi is known to be more pathogenic, C. oncophora is generally
considered as the dose-limiting species [7], particularly for the macrocyclic lactone (ML)
anthelmintics. Infections with these GI nematodes as well as with the bovine lungworm
Dictyocaulus viviparus cause considerable decrease in weight gain in calves during the first
grazing [8] season, can be responsible for significant reduction in milk yield [9, 10] and
impair animal welfare [11]. The economic importance of such parasitic diseases has been
repeatedly demonstrated [12], especially where intensive grazing management is practiced,
and cost-benefit evaluations have indicated that financial losses due to the presence of GI
nematodes can be high [2, 13]. Monitoring data provided by the Dutch Animal Health
Service for D. viviparus indicate that the incidences of parasitic bronchitis tended to increase
in the Netherlands [14]. In the same study, economic losses of approximately 160 € per cow
were calculated. The liver fluke Fasciola hepatica affects large and small ruminants. In
cattle, fasciolosis can appear as a chronic and subclinical form and is worldwide considered
as one of the most important parasitic diseases causing substantial economic losses, which are
estimated to be 2000 million $ (USA) per year in agriculture [15, 16]. Additionally, this
parasite has zoonotic potential and environmental contamination through infected animals
can be important for human health [17].
Diagnosis of these parasites is commonly based on coproscopical detection methods such as
sedimentation (liver fluke), flotation (GI nematodes) or baermannisation of larvae
(lungworm). Since eggs excreted by most GI nematodes are morphologically
indistinguishable, species identification can only be achieved following faecal culturing or
using molecular techniques [18–20]. The generally high handling costs as well as the
necessity to sample several animals led to the increased use of serological methods which can
be used for herd health monitoring. Serological diagnosis of F. hepatica has been described
in the literature using excretory/secretory (ES) products [21–23], a “f2” antigen (Fasciolosis
Verification Test, IDEXX, Hoofddorp, the Netherlands) and a recombinant Cathepsin L1
antigen [22]. The same applies for D. viviparus where the detection of antibodies in serum or
milk using ELISAs with either crude ES antigen [24–26] or recombinantly expressed major
sperm protein (MSP) [27–29] has been described. For the detection of C. oncophora, ELISA
using crude antigen [30], or a recombinantly expressed 14.2 kDa ES protein [31] were
reported.
All these ELISA only target a single species and in order to cover the spectrum of pasture-
borne helminthoses, multiple assays have to be conducted. Recent technical advances offer
the advantage of multiplex assays, resulting in higher throughput, increased flexibility,
reduced sample volume and lower costs [32–34]. A popular multiplex platform is the bead-
based Luminex® xMAP® technology (Luminex Corp., Austin, TX). The basis are different
polystyrene beads which are labelled with distinct ratios of two fluorescent dyes (red and
near-infrared), leading to more than 100 sets of distinguishable beads, which are also referred
to as microspheres in the literature. With each set, different analytes can be measured in
parallel in a single assay [35, 36]. Various ligands can be covalently conjugated to the surface
of these beads. If antigens are used as ligands, assays equivalent to ELISAs can be developed.
Interactions of the target analytes with antibodies is detected using biotinylated secondary
antibodies and streptavidin, labelled with the reporter fluorochrome phycoerythrin (SA-PE).
Fluorescence detection in the Luminex xMAP liquid suspension array system is achieved by
two-laser flow cytometry [37]. This technology is relatively widely used in the field of
human medical diagnosis [38] but only few reports have been published in the field of
veterinary diagnosis, particularly regarding serological assays [39–41].
The aim of this study was to develop a new, versatile diagnostic assay for the simultaneous
detection of specific antibodies against F. hepatica, D. viviparus and C. oncophora in cattle
serum samples. The performance of the Luminex® platform was evaluated through
comparison with already established ELISAs using the same or different antigens.
Methods
Serum samples and antigens
The standardisation of the assay was achieved by using control sera obtained from parasite
naïve animals before (negative control) and after experimental mono-infection with the target
parasites D. viviparus, (100 larvae over 5 consecutive days) C. oncophora (30,000–40,000
larvae) and F. hepatica (500 metacercariae). For testing specificity as well as cross reactivity,
sera from animals mono-infected with other important GI nematodes, Haemonchus contortus,
Trichostrongylus colubriformis and O. ostertagi were used. All animal experiments were
conducted in strict accordance with the respective local legislation and the European
guideline for animal experiments (2010/63/EU). They were approved by a) the Landesamt für
Gesundheit und Soziales, Berlin, Germany under the reference number L 0088/10, b) the
Ethical Commission of the Faculty of Veterinary Medicine, Ghent University, Belgium under
the reference number EC2009/086 and c) the Swedish Animal Ethics Committee under the
permission C4/2.
Additionally serum samples collected in Denmark (n = 39), Switzerland (n = 76) and Poland
(n = 367) were used. In Denmark and Switzerland, samples were taken from grazing young
cattle on randomly selected farms. The sampling in Poland took place on farms previously
identified for a cross-sectional survey using a two-stage sampling approach [42]. On a subset
of those farms, 10–15 first season grazing cattle were randomly sampled.
The antigen used for the detection of F. hepatica was a recombinant 37 kDa Cathepsin L1-
like protein [43] provided by ILDANA BIOTECH, UCD, Dublin. It is an active site
[Cys
26
Gly] mutant expressed in the yeast Pichia pastoris. For the detection of antibodies
against D. viviparus the recombinant 43 kDa MSP expressed as a glutathione-S-transferase
(GST) fusion protein in Escherichia coli BL21 (DE3) cells as previously described by
Gozdzik et al. [27] was used.
Production of recombinant C. oncophora ES 14.2 antigen
The protein used for the detection of antibodies against C. oncophora was a 14.2 kDa ES
protein described previously by Poot et al. [31]. A codon optimised (E. coli) version of the
open reading frame (ORF) was synthesised in vitro (SynthesisGene®; China). The ORF was
amplified using the forward primer (5′-CAC CAA TGA ATA TAC CGA TGC ACT GGC
AAA ATG TAC-3′) and reverse primer (5′-TTA TTC CCA ATA CAG ACA CAG AAC
TTT CAG TT-3′). PCR products were cloned into the pET151 TOPO expression vector (Life
Technologies). A Rosetta gami® (Novagene) E. coli clone containing the pET151/CoES14.2
was cultured at 37 °C until OD
600nm
reached 0.6. Synthesis of the ES14.2-V5-6 × His protein
was induced with 0.5 mM isopropylthio-galactoside (IPTG) at 37 °C for 4 h. The
recombinant ES14.2-V5-6 × His protein was purified from inclusion bodies using Protino®
Ni-IDA columns (Macherey-Nagel, Germany) according to the manufacturer’s protocol. An
additional wash step using a 50 mM concentration of imidazole and 2 % Tween20 was
conducted before elution with 250 mM imidazole. Purity of the eluted protein was analysed
on 12 % SDS–PAGE, stained with GelCode™ colloidal coomassie stain (ThermoFisher).
Western blotting using an anti-V5 antibody (Life Technologies) was carried out to confirm
that the target protein was obtained.
Antigen coupling to fluorescent beads
In order to remove sodium azide or imidazole, D. viviparus and C. oncophora recombinant
antigens were purified by gel filtration using Micro Bio-Spin 6 chromatography columns
(Bio-Rad, Germany) according to the manufacturer’s protocol. Concentrations of all antigens
were determined using the CB-X
TM
Assay (G-Biosciences, USA).
D. viviparus, C. oncophora and F. hepatica antigens were conjugated on the surface of
carboxylated magnetic beads (Bio-Plex Pro™ Magnetic COOH Beads – 1.25 × 10
7
beads/ml,
Bio-Rad) using the fluorescence regions 026, 062 and 065, respectively. Coupling reactions
were performed using the Amine Coupling Kit® (Bio-Rad), following a two-step
carbodiimide reaction protocol provided by the manufacturer. The stock suspension of
uncoupled beads was vortexed at high speed for 30 s followed by sonication for 15 s in order
to disperse bead aggregates. A 100 µl aliquot of monodisperse COOH beads (1.25 × 10
6
) was
transferred to one Bio-Plex coupling reaction tube and was placed into the magnetic separator
for 30 to 60 s before removal of the supernatant. The beads were washed once in 100 µl bead
wash buffer, followed by re-suspension in 80 µl bead activation buffer. Then 10 µl of 50
mg/ml N-Hydroxysulfosuccinimide sodium salt (S-NHS) (Sigma-Aldrich, Germany) and 10
µl of 50 mg/ml N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDAC)
(Sigma-Aldrich), which were prepared in bead activation buffer immediately prior to their
use, were added. The reaction tube was mixed gently, covered with aluminium foil and then
gently agitated on a shaker for 20 min at room temperature (RT). PBS (pH 7.4, 150 µl) was
added twice, always followed by vigorous vortexing. The recombinant protein was added and
total volume was brought to 500 µl with PBS (final antigen concentration 5–12 µg/500 µl).
Incubation was performed on a shaker at high speed (600–700 rpm) at RT for 2 h. In order to
achieve higher coupling yields, some alterations of the initial protocol were made. It was
observed that beads incubated at medium speed (500 rpm) as recommended had a tendency to
precipitate at the bottom of the coupling reaction tube. Therefore, speed was slightly
increased and the coupling reaction tube was vortexed once at high speed after 1 h to prevent
precipitation.
Initially, different concentrations were used for each of the three antigens and were separately
tested in order to determine the optimum antigen concentration. The amounts of the
conjugated protein were the following for each bead-set: for C. oncophora 7 µg, 5 µg, 3.5 µg,
2.5 µg, 1.75 µg, 1.25 µg, 0.9 µg and 0.45 µg, for D. viviparus 5 µg, 2.5 µg, 1.25 µg, 0.66 µg
and 0.45 µg, and for F. hepatica 5 µg, 2.5 µg and 1.25 µg. The coupled beads were placed
into a magnetic separator for 1 min and after removal of the supernatant they were washed
with 500 µl of PBS. The coupled beads were then re-suspended in 250 µl of blocking buffer
and gently agitated at RT in the darkness for 30 min. Finally, the beads were washed with
500 µl of storage buffer, re-suspended in 150 µl storage buffer and stored at 4 °C in the dark.
Beads were stored on the recommended conditions and always used within 4 months after
coupling since decreased performance was observed thereafter.
Luminex multiplex assay
The assays were conducted in 96-well polystyrene, round-bottom microplates (Greiner Bio-
One). The three bead-sets were initially tested in singleplex assays including negative
controls as well as the respective positive control sera. These were used in a two-fold eight
serial dilution series in PBS/Tween20 (0.05 %, pH 7.4) in order to identify the optimal
sample dilution. Cross-reactivity was assessed by running the assay with sera from calves
infected with non-target species. Then, the three bead-sets were combined in a bead-mix and
a multiplex assay was performed.
Prior to each examination, beads were re-suspended by vortexing and sonication for
approximately 20 s three times to avoid high numbers of aggregated beads. A 50 µl aliquot of
the working beadmixture (concentration 100 beads/µl) was transferred into the wells,
followed by the addition of 50 µl of diluted sera. The plate was incubated on a plate shaker
(800 rpm) in the dark at RT for 60 min. The plate was then placed into the magnetic separator
and left for separation for 60 s. The supernatant was carefully removed from each well by
manual inversion. Beads were washed 5 times by adding 100 µl PBS/Tween20 into each well
to ensure absence of any undesirable or non-specifically bound antibodies. The plate was then
removed from the magnetic separator and 100 µl of a biotinylated secondary antibody (goat
anti-bovine IgG, Dianova, Germany) diluted 1:1000 in PBS/Tween20 were added to each
well. Incubation was again conducted in darkness and at RT on a plate shaker (800 rpm) for
30 min before beads were washed as described above. Finally, 100 µl of streptavidin-
phycoerythrin (SA-PE, Millipore) at 2 µg/ml, diluted in assay buffer, were added to each
well. The plate was placed on the shaker, covered with aluminium foil and again incubated at
RT on a plate shaker (800 rpm) for 30 min. The supernatant was carefully removed after
magnetic separation of the beads by manual inversion and washing was performed as
previously described. Assay buffer (100 µl) was added into each well and the plate was
placed onto a plate shaker for approximately 15 s in order to achieve gentle agitation of the
beads.
The beads were analysed using the Bio-Plex 200 instrument following the manufacturer’s
instructions. A minimum of 100 events (beads) per well was read for every bead-set. All
samples were analysed in duplicates in each run. To investigate reproducibility of the assays,
several (between three and six) runs were performed using the sera from the same animals.
ELISA used for comparison
Result obtained using the Luminex® assay were compared to existing ELISAs. D. viviparus
antibodies were detected using either an in-house ELISA for lungworm based on the
recombinant MSP antigen as described in von Holtum et al. [29] or using the modification of
this ELISA as described by Gozdzik et al. [27]. Antibodies against F. hepatica were detected
using either an in-house ELISA for liver fluke based on crude ES antigen following the
method described by Salimi-Bejestani et al. [23] or the commercially available DRG liver
fluke ELISAs (using recombinant Cathepsin L1). For the detection of C. oncophora
antibodies no commercial ELISA is currently available and therefore no comparison was
conducted.
Statistical analysis
GraphPad Prism® software 5.04 was used for the statistical analyses. Five parameter logistic
regression curves were calculated in order to determine the optimal serum dilution for each
bead set separately. For differentiation, positive and negative samples as well as sera from
animals infected with non-target species were compared using box plots. Negative and
positive cut-off MFI values for each parasite specific assay were obtained using receiver
operating characteristics (ROC) analysis to determine at the same time sensitivity and
specificity. The respective values are automatically provided with the 95 % confidence
intervals in the software used.
For comparison with existing ELISA (F. hepatica and D. viviparus) two subsets of 39 and
370 serum samples, respectively, were examined and Kappa tests were performed.
Percentages with confidence intervals and differences between countries were calculated
using a Mid-P exact test in OpenEpi (http://www.openepi.com/Menu/OE_Menu.htm).
Results
Optimal amount of protein for coupling
The optimal amount of antigen identified for the target species were 0.45 µg for both, D.
viviparus and C. oncophora, and 2.5 µg for F. hepatica. These were determined based on the
amount of conjugated protein that would provide a reliable and reproducible MFI signal,
which enabled a clear differentiation between positive and negative serum control samples.
Optimisation of secondary antibody dilutions and SA-PE concentration
Using the optimal amount of antigen, four different secondary antibody dilutions (1:500,
1:1000, 1:2000 and 1:5000) and four SA-PE concentrations (0.5 µg/ml, 1 µg/ml, 2 µg/ml and
4 µg/ml) were tested. Optimal results were obtained for a 1:1000 dilution for the biotinylated
secondary antibody and a concentration of 2 µg/ml for SA-PE.
Optimal serum dilution
Determination of the optimal serum dilution was based on the examination of negative and
positive control sera in a two-fold dilution series ranging from 1:100 to 1:12,800 for the
separate coupled beadsets. The logistic regression curves of the MFI values for all three
target species enabled a clear differentiation between the target species and negative control
as well as non-target species (Fig. 1) with relatively low background MFI values. Multiplex
assays were also performed using an 82-fold dilution series and results were comparable to
those obtained in the singleplex assays (Fig. 2).
Fig. 1 Results for singleplex assays using serum dilutions of cattle infected with
Dictyocaulus, Fasciola and Cooperia. Five parameter logistic regression curves were
calculated based on median fluorescence intensity (MFI) values. a Cooperia oncophora
coupled beads using sera positive for C. oncophora (green), Dictyocaulus viviparus (black),
Fasciola hepatica (red) and negative control sera (blue). b D. viviparus coupled beads and c
F. hepatica coupled beads using the same sera. Dilutions are presented as 0.005 ≙ 1:200
Fig. 2 Results for the triplex assay using serum dilutions of cattle infected with Dictyocaulus,
Fasciola and Cooperia. Five parameter logistic regression curves were calculated based on
median fluorescence intensity (MFI) values. Beads are coupled with recombinant antigen for
the detection of Cooperia oncophora (green), Dictyocaulus viviparus (black) and Fasciola
hepatica (red). Artificial mixtures of sera from animals infected with the target species was
used. Dilutions are presented as 0.005 ≙ 1:200
For all assays R
2
values were close to 1. To achieve an optimal discrimination between
positive and negative sera for all three bead sets a dilution of 1:200 was chosen.
Assessment of cross-reactivity and cut-off determination
All bead sets were examined using positive and negative control sera as well as sera from
non-target species infections. Since no obvious differences were observed between singleplex
and multiplex assays, results were combined and are shown in Fig. 3. Box plots indicated
clear differentiation between positive and negative control serum samples for all three target
species. Regarding cross-reactivity antigens used for the detection of C. oncophora and F.
hepatica could clearly distinguish between infections with target and non-target species (H.
contortus, T. colubriformis, O. ostertagi, D. viviparus and F. hepatica or C. oncophora,
respectively). This was different for the recombinant MSP antigen, where cross-reactivity
was more pronounced for sera from C. oncophora and F. hepatica infected animals;
particularly for a few C. oncophora positive sera differences to the lowest observed MFI
value for D. viviparus were only minimal. Determination of the cut-off MFI values,
sensitivity and specificity was achieved by ROC analysis separately for each bead set. Since
serum samples were derived from experimentally infected animals and either clearly negative
(parasite naïve prior to infection) or positive, two cut-off values were defined, one
discriminating negative and one positive, leaving grey zone in between. For D. viviparus the
situation was slightly different with some cross-reactivity present particularly for the C.
oncophora coupled beads, so that for this assay only one cut-off value was determined. The
cut-off values with sensitivity and specificity including the 95 % confidence intervals are
presented in Table 1.
Fig. 3 Cross reactivity analysis using sera from target and non-target species. Results are
presented as box-plots showing the median fluorescence intensity (MFI) values obtained
from multiple testing of sera from negative and mono-infected animals. Bead set were
coupled with recombinant antigen for the detection of Cooperia oncophora (a), Dictyocaulus
viviparus (b) and Fasciola hepatica (c). Whiskers represent 5 % and 95 % percentage
quantiles and the mean is indicated by a +. Outliers are shown as individual dots
Table 1 Results of the Receiver Operating Characteristics analysis for negative (neg.) and
positive (pos.) cut-off values. Median fluorescence intensity (MFI), sensitivity and specificity
with confidence intervals (CI) are shown
MFI Specificity 95 % CI Sensitivity 95 % CI
C. oncophora
Neg. cut-off 379 99.84 % 99.11–100 % 100 % 98.17–100 %
Pos. cut-off 997 100 % 99.41–100 % 99.50 % 97.25–99.99 %
D. viviparus
Cut-off 950.5 100 % 99.32–100 % 100 % 97.93–100 %
F. hepatica
Neg. cut-off 340.8 99.46 % 97.01–99.99 % 100 % 98.02–100 %
Pos. cut-off 2670 100 % 98.02–100 % 99.46 % 97.01–99.99 %
Assay reproducibility
Serum samples were obtained from different animals on different days pre and post infection
and tested multiple times independently as well as in parallel on the same plate in order to
determine the reproducibility of the assay. The MFI values obtained indicated reproducible
results, which are shown in Table 2. The results obtained for the individual animals showed
distinct immune responses, resulting in different levels of mean MFI values. Although CV
were relatively high for some individuals when testing for antibodies against D. viviparus, all
individual values clearly identified the respective samples as positive.
Table 2 Results of technical reproducibility using serum from different experimentally
infected animals
Animal 1
Animal 2
Animal 3
Animal 4
Animal 5
C. oncophora
N 8 8 8 8 8
Mean MFI value 2236 3778 3870 3124 2876
CV 3.29 % 2.11 % 3.33 % 4.78 % 6.12 %
D. viviparus
N 12 8 6 6 6
Mean MFI value 2332 3738 2595 1923 1472
CV 4.47 % 5.32 % 17.24 % 24.88 % 9.58 %
F. hepatica
N 12 10 8
Mean MFI value 1583 1888 4770
CV 9.39 % 9.64 % 3.09 %
N number of replicates
MFI median fluorescence intensity
CV coefficient of variation
Comparison between multiplex assay and single ELISAs and field validation
The validation of this multiplex assay was performed by comparing the results obtained from
the examination of individual serum samples with already existing and established assays.
Initially, only positive and negative samples derived from experimentally infected animals
pre and post infection (F. hepatica and D. viviparus) were compared, using an in-house
ELISA for lungworm as described by von Holtum et al. [29] as well as an in-house ELISA
for liver fluke based on crude ES antigen. The results obtained were identical for both,
negative as well as positive control sera.
To increase the number of samples tested, additionally serum samples collected during a field
trial in Poland were used for comparison. Thirty-nine of these samples were analysed using
the commercially available DRG liver fluke ELISAs. For the detection of lungworm no
commercial ELISA is currently available. Serum samples were analysed in parallel in
Sweden using an in-house ELISA [27]. In the latter, all samples were negative for D.
viviparus antibodies while one sample was detected positive in the Luminex assay. Due to the
fact that almost all samples were negative, no kappa statistic could be calculated. The
comparison with the DRG liver fluke ELISA resulted in a kappa value of 0.37 (62.7 % of
0.60 maximum achievable). For this calculation the negative cut-off values for both assays
were used. A larger subset of 363 samples were analysed using the commercially available
SVANOVIR®F.hepatica-AbELISA (protocol identical to the in-house liver fluke ELISA as
mentioned above) in the laboratory in Ghent. The comparison resulted in a kappa value of
0.460 (74.4 % of 0.67 maximal achievable). While 67 samples were positive only in the
Luminex, there were also 21 samples which appeared positive in the ELISA but clearly
negative in the Luminex assay.
Finally the newly developed triplex Luminex assay was used to analyse field serum samples
collected in Denmark (n = 39), Switzerland (n = 76) and Poland (n = 367). The results
obtained show no or low rates of lungworm infection in all three countries. C. oncophora
appears in higher rates in Poland (73.8 %) in comparison to Denmark (28.2 %) and
Switzerland (48.7 %). Additionally in Poland, increased levels of liver fluke were detected.
Significant is the number of samples classified in the grey zone, which is relatively high in all
three sampled countries. The calculated percentages are presented in Table 3.
Table 3 Percentage of serum samples positive for antibodies against Cooperia oncophora,
Dictyocaulus viviparus and Fasciola hepatica. Results for the field samples are shown per
country and include 95 % confidence intervals (95 % CI)
Denmark (n = 39) Poland (n = 367) Switzerland (n = 76)
C. oncophora 28.21%
a
73.84%
b
48.68%
c
95 % CI 16.42–43.90 % 69.11–78.08 % 37.78–59.71 %
D. viviparus 0%
ab
3.82%
b
0%
a
95 % CI 0–10.68 % 2.23–6.34 % 0–5.77 %
F. hepatica 64.10%
ac
79.84%
b
67.11%
c
95 % CI 48.73–77.31 % 75.42–83.63 % 55.91–76.65 %
Percentages which do not share the same indices (
a, b, c
) are significantly different in a Mid-P exact test (p < 0.05)
Discussion
Pasture-borne parasitoses are highly prevalent in all grazing ruminants and have been
recognised as important issues for animal welfare and productivity [2, 44]. This is
particularly referring to the liver fluke, the lungworm and GI nematodes. Accelerated
problems regarding anthelminthic resistance, climate change, intensification of farming and
altered management practices have increased the need of development of new techniques in
order to accurately diagnose and monitor these diseases.
In the current study, the successful development of a triplex assay for the simultaneous
detection of antibodies against C. oncophora, D. viviparus and F. hepatica in serum from
cattle is reported. While widely used singleplex assays using recombinant antigens were
available for the latter two, expression of a C. oncophora antigen is described that was
previously only once described for use in an ELISA. The new triplex assay was established
using control sera from parasite naïve or controlled experimentally infected animals. Cross
reactivity was assessed for all three antigens using the target parasites and revealed reliable
detection with high sensitivity and specificity. Additionally, three non-target trichostrongylid
nematode species (H. contortus, O. ostertagi and T. colubriformis) have been included in
cross reactivity testing. Inclusion of Nematodirus, Paramphistomum and other important
pasture-borne parasites would have been desirable, however, infection doses or serum of
experimentally mono-infected animals were presently not available. Nevertheless, the
evaluation of cross-reactivity presented here includes more parasite species than evaluated for
many already commercialised assays.
Standardisation of any serological assay requires the use of defined negative and positive
control sera collected from parasite naïve animals before and after specific mono-infection
with the target parasites as well as non-target species. However, particularly regarding cross-
reactivity, this information is often not accessible for commercially available assays. Also for
the large number of developed and evaluated in-house ELISAs assessment of cross reactivity
has often not been performed or at least not been reported. The specificity of the newly
developed Luminex® assay was evaluated by testing the bead sets with sera obtained from
animals infected with other important species for livestock (H. contortus, O. ostertagi and T.
colubriformis). No cross-reactivity was obtained for all non-target species and all bead sets.
For C. oncophora and F. hepatica also no cross-reactivity was detected for the other target
species. For the detection of C. oncophora the 14.2 kDa recombinant antigen as reported by
Poot et al. [31] was used. These authors reported no cross reactivity when serum samples
from animals mono-infected with O. ostertagi or D. viviparus were used in their ELISA. This
mirrors the result obtained in the current study, where no cross reactions were observed for
any of the parasite species tested, leading to a sensitivity and specificity of 100 %.
For the detection of F. hepatica the recombinant Cathepsin L1 antigen was used. No cross
reactivity was observed for any of the other species tested, also resulting a 100 % sensitivity
and 100 % specificity. This is different to what has been reported in the literature regarding
the use of this antigen. Kuerpick and colleagues [22] observed two false positive results out
of 13 animals infected with D. viviparus and one out of four animals infected with C.
oncophora (sensitivity between 90–100 %, specificity 88.6 %). Cornelissen et al. [45]
obtained similar findings with five out of 191 animals infected with D. viviparus, one out of
31 animals infected with C. oncophora and one out of 55 animals infected with O. ostertagi
(sensitivity 99.1 %, specificity 98.5 %). The currently observed absence of cross reactivity
might not be confirmed in the Luminex® assay upon the use of significantly higher number
of animals infected with non-target species. However, a similar level of cross reactivity as
described above in the ELISAs would still be an improvement in comparison to systems
using complex antigens such as ES antigen with reported specificities between 83–96 % [44].
The D. viviparus bead set was coupled with the recombinant MSP antigen. Although no or
only minimal cross reactivity have been reported by von Holtum et al. [29] (sensitivity and
specificity >99 %) as well as Gozdzik et al. [27] (sensitivity 97.7 %, specificity 98.1 %) for
ELISAs using the same recombinant antigen, a few MFI values obtained for sera from
animals infected with C. oncophora were very close to the lowest values of D. viviparus
infected animals. However, no overlaps of MFI values occurred. In comparison to sera from
negative animals or animals infected with O. ostertagi or H. contortus, MFI values obtained
for sera from animals infected with F. hepatica and C. oncophora were substantially
elevated. This background prevented the definition of two cut-off values separating clearly
negative and clearly positive from the intermediate grey zone. The absence of two cut-off
values for D. viviparus might complicate interpretation of field data in future epidemiological
studies. In the field situation, some animals will be in the prepatency period or have already
cured the infection but still have elevated antibody titres. MFI values for such animals can be
expected to fall in the intermediate zone between negative and positive cut-off. Without such
an intermediate zone, it will become difficult to identify such animals. Regarding D.
viviparus it is also important to keep in mind that vaccination with attenuated larvae is
possible. A cross reactivity of serum from vaccinated cattle is not expected since MSP is not
expressed in premature stages and adult parasites do usually not develop. Additionally a sub-
unit vaccine showing partial protection has been described recently [46]. Here cross reactivity
should also not occur since entirely different recombinant antigens are used. However,
experimental evidence for this is not available yet.
Out of the currently available coproscopical, serological and molecular techniques for the
diagnosis and the identification of parasites only the first two are routinely used. The
sensitivity and specificity of the different techniques are extremely variable. Serological
assays have the advantage of their implementation as herd health monitoring tools. All
currently available assays are only capable of diagnosing antibodies against one parasite and
are often limited in their specificity due to the use of complex crude or ES antigens. Despite
the fact that for all three parasite species of interest, C. oncophora, D. viviparus and F.
hepatica, ELISAs using recombinant antigens have been described, only for the latter is a
commercial ELISA using recombinant antigen available. Multiplexing diagnostic systems are
well established in human medicine, but in veterinary medicine this technology has so far not
been extensively used. However, during recent years several commercial or “in-house” bead-
based multiplex assays have been developed, particularly in the field of virology [39, 47, 48],
where existing ELISAs were compared with the new multiplexing assays revealing generally
better performance for the latter. Additionally, xMAP technology has been used for the
detection of inflammatory markers, [49–52]. In the field of parasitology, serological
multiplex assays have only been reported for the simultaneous detection of Trichinella
spiralis and Toxoplasma gondii in pigs [40, 41] and for the detection of Plasmodium
falciparum in humans [53]. The Luminex® technology offers the advantage of reduced
volume needed for diagnosis [39]. When multi-plexing assays, the costs per analyte are
considerably lower in the Luminex® assay than in a standard ELISA, whereas the time
needed for the examination of the samples is generally similar but reduced labour time per
assay is achieved.
After the establishment of the Luminex assay, field samples obtained from naturally infected
grazing animals during a trial in Poland have been used for comparison of the new Luminex®
assay with already established ELISAs. In the case of multiplex bead-based immunoassays,
comparison of the Luminex is frequently made with serological assays, such as ELISA.
Correlation of these two methods varies among literature [34, 54–59]. In the current study,
agreement between the assays was generally good when the same (recombinant) antigen was
used. In case of the lungworm assay it was almost impossible to calculate kappa values since
only one animal out of the 39 samples tested appeared positive (only in the new assay). The
comparison of the DRG ELISA with Luminex was complicated by the fact that this ELISA
uses five different categories and it was unclear, how to handle samples negative or positive
in the Luminex assay and “questionable” or “low to moderate infection” in the other. Most of
the deviations in classification were due to the fact, that the number of positive samples was
higher in the Luminex assay but there were also one sample classified as negative in the
Luminex but positive with the ELISA. Comparison of the Luminex® data with the ELISA
using the ES antigen for F. hepatica detection had the advantage of a high number of samples
tested. This resulted in a better kappa value than that obtained for the first comparison,
however, there was a high number of samples positive in only one of the assays while being
clearly negative (ES ELISA) or in the grey zone (Luminex®) in the respective other one.
Since a recombinant antigen was used in the Luminex® assay a higher specificity can be
expected than using a complex ES antigen. At the same time, this might result in decreased
sensitivity since not all animals might develop antibodies against Cathepsin L1. However,
more samples were detected positive with the Luminex® assay (67 vs 21), so sensitivity
should not be of great concern here. Another explanation for the data obtained might be false
positive samples in the ES ELISA, for example due to infection with closely related
pathogens.
Conclusion
A multiplex fluorescence serum immunoassay was successfully developed for the
simultaneous detection of antibodies produced against C. oncophora, D. viviparus and F.
hepatica in serum samples from cattle. It is characterised by low cost and time, high
reproducibility and more importantly, by high sensitivity and specificity due to the use of
recombinant antigens. This platform provides rapid, high-throughput diagnostic results,
allowing the incorporation of further analytes and parameters in the future, such as hormones,
inflammatory biomarkers and additional parasites. In particular, inclusion of O. ostertagi
should be a major aim for the future, though recombinant antigens suitable for diagnosis are
currently not available.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SK, JD, GvS and JK participated in the design of the project. SK carried out the Luminex
experiments. SK and SR performed the protein expression. CW, EM, JC, JH contributed
ELISA data. EM, CW, SK and JaKa provided field samples. SK, JK and JD analysed the
data. SK, JD and JK drafted the manuscript. All authors contributed to finalisation of the
manuscript and read and approved the manuscript.
Acknowledgements
The financial support from the EU funded FP 7 project GLOWORM (KBBE 2011.1.3-04, No
288975) is gratefully acknowledged.
References
1. Morgan E, Charlier J, Hendrickx G, Biggeri A, Catalan D, von Samson-Himmelstjerna G,
et al. Global Change and Helminth Infections in Grazing Ruminants in Europe: Impacts,
Trends and Sustainable Solutions. Agriculture. 2013;3(3):484–502.
2. Charlier J, Hoglund J, von Samson-Himmelstjerna G, Dorny P, Vercruysse J.
Gastrointestinal nematode infections in adult dairy cattle: impact on production, diagnosis
and control. Vet Parasitol. 2009;164(1):70–9.
3. Alvarez Rojas CA, Jex AR, Gasser RB, Scheerlinck JP. Techniques for the diagnosis of
Fasciola infections in animals: room for improvement. Adv Parasitol. 2014;85:65–107.
4. Barda BD, Rinaldi L, Ianniello D, Zepherine H, Salvo F, Sadutshang T, et al. Mini-
FLOTAC, an innovative direct diagnostic technique for intestinal parasitic infections:
experience from the field. PLoS Negl Trop Dis. 2013;7(8):e2344.
5. Bisset SA. Helminth parasites of economic importance in cattle in New Zealand. N Z J
Zool. 1994;21(1):9–22.
6. Piekarska J, Ploneczka-Janeczko K, Kantyka M, Kuczaj M, Gorczykowski M, Janeczko K.
Gastrointestinal nematodes in grazing dairy cattle from small and medium-sized farms in
southern Poland. Vet Parasitol. 2013;198(1–2):250–3.
7. Coles GC. Cattle nematodes resistant to anthelmintics: why so few cases? Vet Res.
2002;33(5):481–9.
8. Dimander SO, Hoglund J, Sporndly E, Waller PJ. The impact of internal parasites on the
productivity of young cattle organically reared on semi-natural pastures in Sweden. Vet
Parasitol. 2000;90(4):271–84.
9. Gibb MJ, Huckle CA, Forbes AB. Effects of sequential treatments with eprinomectin on
performance and grazing behaviour in dairy cattle under daily-paddock stocking
management. Vet Parasitol. 2005;133(1):79–90.
10. Sanchez J, Dohoo I, Carrier J, DesCoteaux L. A meta-analysis of the milk-production
response after anthelmintic treatment in naturally infected adult dairy cows. Prev Vet Med.
2004;63(3–4):237–56.
11. Schunn A-M, Conraths FJ, Staubach C, Fröhlich A, Forbes A, Schnieder T, et al.
Lungworm Infections in German Dairy Cattle Herds — Seroprevalence and GIS-Supported
Risk Factor Analysis. PLoS ONE. 2013;8(9):e74429.
12. Perry BD, Randolph TF. Improving the assessment of the economic impact of parasitic
diseases and of their control in production animals. Vet Parasitol. 1999;84(3–4):145–68.
13. Nieuwhof GJ, Bishop SC. Costs of the major endemic diseases of sheep in Great Britain
and the potential benefits of reduction in disease impact. Anim Sci. 2005;81(01):23–9.
14. Holzhauer M, van Schaik G, Saatkamp HW, Ploeger HW. Lungworm outbreaks in adult
dairy cows: estimating economic losses and lessons to be learned. Vet Rec.
2011;169(19):494.
15. Schweizer G, Braun U, Deplazes P, Torgerson PR. The economic effects of bovine
fasciolosis in Switzerland. Vet Rec. 2005;157(7):188–93.
16. Spithill TW, Smooker PM, Sexton JL, Bozas E, Morrison CA, Creany J, et al.
Development of vaccines against Fasciola hepatica. In: Dalton JP, editor. Fasciolosis.
Wallingford, UK: CAB International Publishing; 1999. p. 377–410.
17. Fürst T, Keiser J, Utzinger J. Global burden of human food-borne trematodiasis: a
systematic review and meta-analysis. Lancet Infect Dis. 2012;12(3):210–21.
18. Bott NJ, Campbell BE, Beveridge I, Chilton NB, Rees D, Hunt PW, et al. A combined
microscopic-molecular method for the diagnosis of strongylid infections in sheep. Int J
Parasitol. 2009;39(11):1277–87.
19. Demeler J, Ramunke S, Wolken S, Ianiello D, Rinaldi L, Gahutu JB, et al. Discrimination
of gastrointestinal nematode eggs from crude fecal egg preparations by inhibitor-resistant
conventional and real-time PCR. PLoS One. 2013;8(4):e61285.
20. Roeber F, Jex AR, Campbell AJ, Campbell BE, Anderson GA, Gasser RB. Evaluation
and application of a molecular method to assess the composition of strongylid nematode
populations in sheep with naturally acquired infections. Infect Genet Evol. 2011;11(5):849–
54.
21. Charlier J, De Meulemeester L, Claerebout E, Williams D, Vercruysse J. Qualitative and
quantitative evaluation of coprological and serological techniques for the diagnosis of
fasciolosis in cattle. Vet Parasitol. 2008;153(1–2):44–51.
22. Kuerpick B, Schnieder T, Strube C. Evaluation of a recombinant cathepsin L1 ELISA and
comparison with the Pourquier and ES ELISA for the detection of antibodies against
Fasciola hepatica. Vet Parasitol. 2013;193(1–3):206–13.
23. Salimi-Bejestani MR, McGarry JW, Felstead S, Ortiz P, Akca A, Williams DJ.
Development of an antibody-detection ELISA for Fasciola hepatica and its evaluation
against a commercially available test. Res Vet Sci. 2005;78(2):177–81.
24. Boon JH, Ploeger HW, Raaymakers AJ. Sero-epidemiological survey of Dictyocaulus
viviparus infections in first-season grazing calves in The Netherlands. Vet Rec.
1986;119(19):475–9.
25. Bos HJ, Beekman J. Serodiagnosis of lungworm infection in calves using ELISA. Dev
Biol Stand. 1985;62:45–52.
26. Marius V, Bernard S, Raynaud JP, Pery P, Luffau G. Dictyocaulus viviparus in calves:
quantitation of antibody activities in sera and respiratory secretions by immuno-enzymatic
analysis. Ann Rech Vet. 1979;10(1):55–63.
27. Gozdzik K, Engstrom A, Hoglund J. Optimization of in-house ELISA based on
recombinant major sperm protein (rMSP) of Dictyocaulus viviparus for the detection of
lungworm infection in cattle. Res Vet Sci. 2012;93(2):813–8.
28. Schnieder T. Use of a recombinant Dictyocaulus viviparus antigen in an enzyme-linked
immunosorbent assay for immunodiagnosis of bovine dictyocaulosis. Parasitol Res.
1992;78(4):298–302.
29. von Holtum C, Strube C, Schnieder T, von Samson-Himmelstjerna G. Development and
evaluation of a recombinant antigen-based ELISA for serodiagnosis of cattle lungworm. Vet
Parasitol. 2008;151(2–4):218–26.
30. Ploeger HW, Kloosterman A, Rietveld FW, Berghen P, Hilderson H, Hollanders W.
Quantitative estimation of the level of exposure to gastrointestinal nematode infection in first-
year calves. Vet Parasitol. 1994;55(4):287–315.
31. Poot J, Kooyman FN, Dop PY, Schallig HD, Eysker M, Cornelissen AW. Use of cloned
excretory/secretory low-molecular-weight proteins of Cooperia oncophora in a serological
assay. J Clin Microbiol. 1997;35(7):1728–33.
32. Fulton RJ, McDade RL, Smith PL, Kienker LJ, Kettman Jr JR. Advanced multiplexed
analysis with the FlowMetrixTM system. Clin Chem. 1997;43(9):1749–56.
33. Carson RT, Vignali DAA. Simultaneous quantitation of 15 cytokines using a multiplexed
flow cytometric assay. J Immunol Methods. 1999;227(1–2):41–52.
34. Baker HN, Murphy R, Lopez E, Garcia C. Conversion of a capture ELISA to a Luminex
xMAP assay using a multiplex antibody screening method. J Vis Exp. 2012;6:65.
35. Kettman JR, Davies T, Chandler D, Oliver KG, Fulton RJ. Classification and properties
of 64 multiplexed microsphere sets. Cytometry. 1998;33(2):234–43.
36. Earley MC, Vogt RF, Shapiro HM, Mandy FF, Kellar KL, Bellisario R, et al. Report from
a workshop on multianalyte microsphere assays. Cytometry. 2002;50(5):239–42.
37. Dunbar SA. Applications of Luminex xMAP technology for rapid, high-throughput
multiplexed nucleic acid detection. Clin Chim Acta. 2006;363(1–2):71–82.
38. Tighe PJ, Ryder RR, Todd I, Fairclough LC. ELISA in the Multiplex Era; Potential and
Pitfalls. Proteomics Clin Appl. 2015;9(3-4):406–22.
39. Christopher-Hennings J, Araujo KP, Souza CJ, Fang Y, Lawson S, Nelson EA, et al.
Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories. J Vet
Diagn Invest. 2013;25(6):671–91.
40. Bokken GC, Bergwerff AA, van Knapen F. A novel bead-based assay to detect specific
antibody responses against Toxoplasma gondii and Trichinella spiralis simultaneously in sera
of experimentally infected swine. BMC Vet Res. 2012;8:36.
41. van der Wal FJ, Achterberg RP, Kant A, Maassen CB. A bead-based suspension array for
the serological detection of Trichinella in pigs. Vet J. 2013;196(3):439–44.
42. Ducheyne E, Charlier J, Vercruysse J, Rinaldi L, Biggeri A, Demeler J, et al. Modelling
the spatial distribution of Fasciola hepatica in dairy cattle in Europe. Geospat Health.
2015;9(2):261–70.
43. Collins PR, Stack CM, O’Neill SM, Doyle S, Ryan T, Brennan GP, et al. Cathepsin L1,
the Major Protease Involved in Liver Fluke (Fasciola hepatica) Virulence: propeptide
cleavage sites and autoactivation of the zymogen secreted from astrodermal cells. J Biol
Chem. 2004;279(17):17038–46.
44. Charlier J, Vercruysse J, Morgan E, van Dijk J, Williams DJ. Recent advances in the
diagnosis, impact on production and prediction of Fasciola hepatica in cattle. Parasitology.
2014;141(3):326–35.
45. Cornelissen JB, Borgsteede FH, van Milligen FJ. Evaluation of an ELISA for the routine
diagnosis of Dictyocaulus viviparus infections in cattle. Vet Parasitol. 1997;70(1–3):153–64.
46. Strube C, Haake C, Sager H, Schorderet Weber S, Kaminsky R, Buschbaum S, et al.
Vaccination with recombinant paramyosin against the bovine lungworm Dictyocaulus
viviparus considerably reduces worm burden and larvae shedding. Parasit Vector.
2015;8:119.
47. Go YY, Wong SJ, Branscum AJ, Demarest VL, Shuck KM, Vickers ML, et al.
Development of a fluorescent-microsphere immunoassay for detection of antibodies specific
to equine arteritis virus and comparison with the virus neutralization test. Clin Vaccine
Immunol. 2008;15(1):76–87.
48. Langenhorst RJ, Lawson S, Kittawornrat A, Zimmerman JJ, Sun Z, Li Y, et al.
Development of a fluorescent microsphere immunoassay for detection of antibodies against
porcine reproductive and respiratory syndrome virus using oral fluid samples as an alternative
to serum-based assays. Clin Vaccine Immunol. 2012;19(2):180–9.
49. Johannisson A, Jonasson R, Dernfalk J, Jensen-Waern M. Simultaneous detection of
porcine proinflammatory cytokines using multiplex flow cytometry by the xMAP technology.
Cytometry A. 2006;69(5):391–5.
50. Bjerre M, Hansen TK, Flyvbjerg A, Tonnesen E. Simultaneous detection of porcine
cytokines by multiplex analysis: development of magnetic bioplex assay. Vet Immunol
Immunopathol. 2009;130(1–2):53–8.
51. Wyns H, Croubels S, Demeyere K, Watteyn A, De Backer P, Meyer E. Development of a
cytometric bead array screening tool for the simultaneous detection of pro-inflammatory
cytokines in porcine plasma. Vet Immunol Immunopathol. 2013;151(1–2):28–36.
52. Bongoni AK, Lanz J, Rieben R, Banz Y. Development of a bead-based multiplex assay
for the simultaneous detection of porcine inflammation markers using xMAP technology.
Cytometry A. 2013;83(7):636–47.
53. Perraut R, Richard V, Varela ML, Trape JF, Guillotte M, Tall A, et al. Comparative
analysis of IgG responses to Plasmodium falciparum MSP1p19 and PF13-DBL1alpha1 using
ELISA and a magnetic bead-based duplex assay (MAGPIX(R)-Luminex) in a Senegalese
meso-endemic community. Malar J. 2014;13:410.
54. Klein DL, Martinez JE, Hickey MH, Hassouna F, Zaman K, Steinhoff M. Development
and characterization of a multiplex bead-based immunoassay to quantify pneumococcal
capsular polysaccharide-specific antibodies. Clin Vaccine Immunol. 2012;19(8):1276–82.
55. Dernfalk J, Persson Waller K, Johannisson A. The xMAP technique can be used for
detection of the inflammatory cytokines IL-1beta, IL-6 and TNF-alpha in bovine samples.
Vet Immunol Immunopathol. 2007;118(1–2):40–9.
56. Elshal MF, McCoy JP. Multiplex bead array assays: performance evaluation and
comparison of sensitivity to ELISA. Methods. 2006;38(4):317–23.
57. Lal G, Balmer P, Stanford E, Martin S, Warrington R, Borrow R. Development and
validation of a nonaplex assay for the simultaneous quantitation of antibodies to nine
Streptococcus pneumoniae serotypes. J Immunol Methods. 2005;296(1–2):135–47.
58. Pickering JW, Martins TB, Schroder MC, Hill HR. Comparison of a Multiplex Flow
Cytometric Assay with Enzyme-Linked Immunosorbent Assay for Quantitation of Antibodies
to Tetanus, Diphtheria, and Haemophilus influenzae Type b. Clin Diagn Lab Immunol.
2002;9(4):872–6.
59. Willman JH, Hill HR, Martins TB, Jaskowski TD, Ashwood ER, Litwin CM. Multiplex
analysis of heterophil antibodies in patients with indeterminate HIV immunoassay results.
Am J Clin Pathol. 2001;115(5):764–9.
