Abstract Book of EAVLD2012 - eavld congress 2012

S4 - P - 10
ID SCREEN ® INTERFERON GAMMA ELISA : IMPROVEMENT OF ANALYTICAL SENSITIVITY AND
INTRODUCTION OF A STANDARD REFERENCE CONTROL TO IMPROVE RESULT INTERPRETATION
Pourquier, P. 1 , Loic, C. 1 , Olagnon, L. 1 , Marion, S. 1,
1
IDvet, Montpellier, France
Interferon gamma, diagnostics, ELISA
Introduction
Detection of Interferon gamma (IFN-g) by the sandwich ELISA
method is widely used to detect the cellular response to
pathogens such as Mycobacterium avium subsp paratuberculosis
(Map) or Mycobacterium bovis by measuring the difference
between activated and non-activated whole blood or peripheral
Blood Mononuclear Cells (PBMC) IFN-g signals. Results,
expressed as the difference between raw optical densities, are
not generally linked to a stable reference control.
The ID Screen ®
Ruminant IFN-g ELISA, contrary to other
commercial ELISAs, expresses the level of IFN-g with respect
to a standardized, freeze-dried positive reference control.
This relative expression of the measured quantity of IFN-g
guarantees the standardization of results between runs and kit
batches.
Results are expressed as sample / positive reference control
(S/P) ratios.
Materials & methods
ID Screen ® Interferon gamma Capture ELISA: Wells are coated
with an anti-IFN-γ monoclonal antibody (Mab). The conjugate is
anti-IFN-γ–HRP conjugate monoclonal antibody.Creation of
standard reference controls: A freeze-dried positive control
containing native bovine IFN-g was created and introduced onto
each plate. All results were calculated with respect to this
standard. A standard positive plasma reference sample was also
created.
Results
Test baseline: Bovine and ovine sera and PBS-activated bovine
and caprine plasmas were tested and found to have a low
baseline with few heterophilic reactions.
Analytical sensitivity: 5 bovine, 1 caprine and 1 ovine blood
sample were non-specifically activated with 5 µg/ml of lectin, 24h
at 37°C. Plasma was collected and each sample was serially
diluted in non-activated plasma. Samples were then tested in
parallel with the ID Screen ®
ELISA and another commerciallyavailable
IFN-g sandwich ELISA (kit A).
- The ID Screen ® ELISA and Test A showed similar analytical
sensitivity for ovine IFN-g, while the ID Screen ELISA
demonstrated superior analytical sensitivity for bovine and
caprine IFN-g. In addition, the ID Screen ® ELISA showed clearer
separation of positive and negative samples.
Multi-site field validation: 3 French laboratories tested previouslyactivated
samples (n=216) using the ID Screen ®
ELISA and
another commercial ELISA.
- Excellent correlation (98.6%) was found between the two tests,
with the ID Screen ® test giving higher OD values for the positive
samples.
Field data from a Map-infected goat herd: 180 caprine blood
samples from herd with a history of clinical paratuberculosis were
activated with 9 different antigens, including 3 different Map
extracts from a field caprine isolate, as well as PPD avium.
- One of the IDvet caprine Map antigen preparations gave a
similar result profile to the PPD avium antigen tested.
Discussion & conclusions
The new ID Screen® Ruminant IFN-g ELISA has improved
upon current IFN-g ELISA technology by incorporating a standard
positive reference control for standardized result calculation and
interpretation.
The improved analytical sensitivity allows for the efficient
detection of small quantities of native IFN-g (threshold dilution on
other techniques), giving optical densities clearly distinct from the
test baseline, thus avoiding false positive results. The very low
level of heterophilic reactions and the low baseline guarantee that
result interpretation is possible for all samples to be tested.
In conclusion, the ID Screen ® Ruminant IFN-g ELISA is a reliable
tool for the measurement of ruminant interferon gamma.
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