Abstract Book of EAVLD2012 - eavld congress 2012

S4 - O - 06
15 MINUTE ELISA USING A LOW COST COMMERCIAL BIOSENSOR
Jason Sawyer 1 , Jennifer Cork 1 , Rebecca Jones 1
1
AHVLA (Weybridge), Technology Transfer Unit, New Haw, Addlestone, Surrey KT15 3NB, UK
ELISA, IBR, Biosensor, Penside testing, Serology, Vantix
Introduction
The use of biosensors for diagnostic testing offers several
potential advantages, but to date their use for routine diagnostic
testing has been limited to specific markets (e.g. glucose testing).
We evaluated the use of a commercially available potentiometric
biosensor platform - Vantix - to carry out a serological assay
for antibodies to infectious bovine rhinotracheitis (IBR), caused
by Bovine Herpes Virus 1 (BoHV-1).
The Vantix system 1
comprises a low cost reader unit and
disposable potentiometric biosensors. The biosensors consist of
a working and reference electrode coated in a conductive
polymer (polypyrrole) and covered in a protective plastic film.
Biochemical or enzymatic activity taking place on the surface of
the working electrode as a result of immunocomplexes built up on
the electrode, cause electrochemical changes in the conductive
polymer layer on and around the working electrode thus
generating a measurable change in electrical potential (measured
in millivolts) relative to the reference electrode. The signal
produced is proportional to the level of analyte under
investigation.
IBR is a highly contagious disease of cattle that is endemic in the
UK and many other parts of the world 2 . ELISA is commonly used
in the detection and control of this disease and AHVLA uses an
in-house indirect ELISA. In this assay, BoHV-1 antigens, bound
to the surface of a microtitre plate, capture BoHV-1 specific
antibodies present in test samples. Bound antibodies are then
detected using a horseradish peroxidase (HRP) labelled antibovine
immunoglobulin and subsequent HRP catalysed oxidation
of the chromogen substrate 3,3’,5,5’-tetramethylbenzidine (TMB)
produces a colour change. The in-house ELISA for BoHV-1 used
at AHVLA has a total assay time of over 3 hours which is typical
of commercial ELISA tests.
In this work, the well-characterised and established reagents
used in the parent ELISA were used to construct an assay on the
Vantix platform. A panel of serum samples, submitted for
routine ELISA testing, were then tested with the Vantix assay and
the results compared to the parent ELISA.
Table 1: Comparison of methodology of the IBR antibody biosensor
assay and parent ELISA.
Biosensor assay
Indirect ELISA
Step
Incubation
Incubation
Step
Time
Time
Biosensors and ELISA plates pre coated with BoHV1 antigen
and stored ready for use
Incubation with diluted
Incubation with diluted
5 min
sample
sample
2 h
8 Washes no soak 0.5 min
5 x washes 10 second
soak
5 min
Incubation with diluted
Rabbit anti bovine HRP 5 min
Incubation with diluted
Rabbit anti bovine HRP 1 h
Conjugate
Conjugate
8 Washes no soak 0.5 min
5 x washes 10 second
soak
5 min
Probes added to TMB
Plate incubated with
5 – 20 min
substrate and read
TMB substrate.
4 min
immediately. Potential
Addition of Stop 1 min
difference reading.
Colorimetric read 1 min
Total assay time 15 min Total assay time >3.25 h
Materials & methods
Table 1 summarises the steps and timings involved in the
performing the Biosensor assay compared to the ELISA.
Biosensor probes were prepared by coating with BoHV-1 antigen,
and were then stored at +4 o C for up to 12 weeks. To perform the
assay, the electrodes of the antigen coated biosensors were
briefly placed in diluted serum, washed, and briefly incubated
with conjugate. After a final wash step, the sensors were
inserted into the Vantix reader unit and the working and
reference electrode placed into TMB substrate. Changes in
electrochemical potential difference were observed over a period
of four minutes and recorded using Vantix software. The
biosensor assay was used to test 194 serum samples,
comprising 90 positive and 104 negative as designated by the in
house ELISA.
% signal relative to weak
positive control
250
200
150
100
50
0
IBR Biosensor
2 x 2 box analysis assay
Serum Samples Positive Negative Totals
IBR
ELISA
Negative
Positive 86 4 90
Negative 2 102 104
Totals 88 104 194
Positive
True positive rate
(Sensitivity)
0
0 25 50 75 100
False positive rate
(100 - Specificity)
Fig.1 Comparison of results obtained when testing serum
samples using the parent IBR antibody ELISA and the biosensor
assay. 2x2 table (Top); Box and whisker plot showing biosensor
results for ELISA negative and positive samples (bottom left) and
ROC analysis (bottom right).
Results
Fig 1 shows the resulting 2X2 Table, Box and Whisker plot and
ROC curve when the biosensor assay was compared to the
ELISA. Using the optimal cut-off value (determined by ROC
analysis and based on a percentage signal relative to the weak
positive control serum run alongside the test samples) the
biosensor assay had a sensitivity of 98% and a specificity of 96%
when compared to the indirect ELISA. Only 6 of 194 samples
resulting in a different categorisation (positive or negative)
compared to the ELISA results. Overall, the results demonstrate
that the biosensor assay produces test results that closely match
those obtained with the parent ELISA.
Discussion & conclusion
This work has shown that results equivalent to the ELISA can be
obtained using the Vantix biosensor platform in 15 min. The
relatively low cost of the biosensors (~ few Euro each) and reader
(~ few thousand euro) make this an interesting and promising
technology. The rapid and simple nature of the assays, and the
fact that established ELISA reagents can readily be converted to
the platform, suggest Vantix could be useful in a variety of
diagnostic testing scenarios. These may include rapid testing of
samples which require fast turnaround or testing in low tech
laboratories. The generation of an electronic signal offers the
potential for transmission across mobile networks. Vantix are
currently developing a hand held device which uses the same
biosensors but offers the potential for more rapid (