Development of a Quantification Method to Specific Anti-NS3 Antibodies against BVDV using a Blocking ELISA - PowerPoint PPT Presentation

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Development of a Quantification Method to Specific Anti-NS3 Antibodies against BVDV using a Blocking ELISA

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Development of a Quantification Method to Specific Anti-NS3 Antibodies against BVDV using a Blocking ELISA Stephan Guillossou1,2, Daniel Thomson1, Cindy Thomson2 – PowerPoint PPT presentation

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Title: Development of a Quantification Method to Specific Anti-NS3 Antibodies against BVDV using a Blocking ELISA


1
  • Development of a Quantification Method to
    SpecificAnti-NS3 Antibodies against BVDV using a
    Blocking ELISA
  • Stephan Guillossou1,2, Daniel Thomson1, Cindy
    Thomson2
  • 1Kansas State University, College of Veterinary
    Medicine, Department of Clinical Science,
    Manhattan, KS, USA
  • 2Synbiotics Corporation, Manhattan, KS, USA

2
Introduction
BVD Ab
  • Controlling BVDV
  • Identify the best cost effective model
  • European epidemiological models for BVDV control
  • Identify herds harboring PI animals (sentinel
    animals)
  • Inside these herds, Identify and Remove PI
    animals
  • Difficulties to adapt in area with use of
    vaccination
  • needs differentiation between vaccinated induced
    Abs and virus infection induced Abs(Presence of
    PI animal)

3
Introduction
BVD Ab
  • Envelop
  • External glycoprotein
  • Strain variability (Except Erns better
    stability
  • but not perfect!)
  • Important neutralizing properties (Mainly for
    the gp53 E2)
  • Capsid
  • protein
  • internal
  • weak variability
  • Non protected Ab
  • Non Structural proteins
  • ex NS 2-3 (p80/125)
  • Highly antigenic without generating immunity(? no
    protection)
  • Highly conserved among strains
  • Production occurs during viral replication inside
    the cell

4
Objective
BVD Ab
  • Sero Neutralizing Test (SNT) measures only
    antibodies with seroneutralizing properties
  • Western blot are specific to antibody
    subpopulations
  • Currently, no existing standardized method to
    quantify subpopulation of antibodies without SN
    properties
  • Objective of the study
  • Development of a quantification method to titer
    anti-NS3 antibody subpopulation
  • Commercial ELISA
  • SERELISA BVD p80 Ab Mono Blocking
  • Synbiotics Europe, France

5
Diagnostic Ab Mono Blocking ELISA
BVD Ab
Negative sample
Positive sample
6
Objectives limits/opportunities
BVD Ab
  • Objectives
  • to develop a quantitative serum antibody test for
    BVDV for a subpopulation of antibodies with a
    commercially available test
  • Limits/opportunities
  • Blocking ELISA are specific of only a sub
    population of antibody targeting a specific
    epitope of an antigen
  • But blocking ELISA usuallyhave less
    linearitythan indirect ELISA

blocking
indirect
7
Material and Methods 1/2
BVD Ab
  • Samples positive reference serum (Sbio) was
    used to establish a reference panel
  • Labs Synbiotics Corporation, Manhattan, KS,
    USA
  • Kansas State University, College of Veterinary
    Medicine, Department of Clinical Science,
    Manhattan, KS, USA
  • OD results Results are expressed as a function
    of OD obtained with the ELISA (SN, SNc, or PI)
    that includes correction with the controls
    (positive and negative controls)
  • Model selection Eight models have been
    investigated

T fn(OD) 1/T fn(1/OD) T fnLog(OD) T fnlogit(OD)
LogT fn(OD) Log(1/T) fn(1/OD) LogT fnLog(OD) L
ogT fnlogit(OD)
8
Material and Methods 2/2
BVD Ab
  • Methods Model development and selection
  • For each of the previous models, graphical and
    mathematical pertinence of the models are
    assessed by interpretation of coefficient of
    determination R2 and residual analysis
  • Sample dilution protocol
  • Reference serum was used at the following
    dilutions (final dilutions into wells 1/10,
    1/50, 1/100, 1/500, 1/1000, 1/5000,
    1/10000 Measures were repeated four times
  • Statistical analysis R 2.7.2
  • SPSS for Windows ver.16.0
  • Excel ver2003

9
Results model selection
BVD Ab
10
Results model selection
BVD Ab
Analysis of coefficient of determination
(R2) T fn(OD) R20.8842 1/T fn(1/OD)
R21 T fnLog(OD) R20.7045 T fnlogit(OD)
R20.9167 LogT fn(OD) R20.9899 Log(1/T) fn
(1/OD) R20.8979 LogT fnLog(OD)
R20.9766 LogT fnlogit(OD) R20.9835
11
Results model selection
BVD Ab
Analysis of residual dispersion
12
Results selected model
BVD Ab
  • Best fited model
  • Best linearity achieved with SNc between 0.11 and
    0.93
  • With
  • Slope
  • Intercept

13
Results final model
BVD Ab
As all models are valid within its limits and to
ensure a quantification from very low to very
high titers, samples were diluted in three sample
wells and logit model applied inside each of
these wells A Excel worksheet is available upon
request from the authors
cSN
1/10,000
1/1,000
1/100
Titer
14
Discussion/Conclusion
BVD Ab
Innovative quantitative method for specific
detection of anti-NS3 (p80) antibodies against
BVDV using latest quantitative model from human
medicine/biostatistics Excellent
linearity Quantitative method for a specific
antibody subpopulation using a blocking ELISA
This standardized quantitative test is a tool
that will lead to a breakthrough in the
understanding of the BVDV epidemiology by
monitoring antibodies populations and be utilized
to assess BVDV control measures.
15
Acknowledgments
BVD Ab
  • This project was funded through the Kansas State
    University College of Veterinary Medicine and the
    Beef Cattle Institute in conjunction with
    Synbiotics Corporation
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