S erological tests for measurement of antibodies in the vaccinated animals are the suitable alternative of challenge test for determination of efficacy of several veterinary and human vaccines. Detection of antibody is also used to certify individual animals prior to import or export (i.e. for trade), to confirm suspected cases of disease, and to substantiate absence of infection. The common antibody detection assays are haemagglutination inhibition (HAI), the complement fixation test (CFT), virus neutralization test (VNT), enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) etc. Among these assays, ELISA is the widely used test for sero-monitoring and sero-surveillance of the particular disease. The assay has shown great potential in measuring humoral immunity of the body after infection and after vaccination to demonstrate the efficacy of vaccination. ELISA is also an approved test by the Word Organization for Animal Health (OIE) for several infectious agents /diseases such as African horse sickness, Aujeszky's disease, Bluetongue, Foot and mouth disease, Peste des petits ruminants, Infectious bovine rhinotracheitis, Swine vesicular disease, Bovine brucellosis, Brucella abortus, Contagious bovine pleuropneumonia, Enzootic bovine leukosis, Ovine and caprine brucellosis etc. The most commonly used ELISA formats for detection of antibody are indirect and competitive.

1. Vikramaditya Upmanyu
Scientist
Division of Biological Standardization
Enzyme Linked Immuno-Sorbent Assay(ELISA

2. Most common prescribed and alternative diagnostic test for OIE
listed diseases
Prescribed test Alternative test
Pseudo rabies, BTV, FMD,
rabies , VS, Bovine
brucellosis, CBP, IBR, CAE,
Masdi-Visna, Caprine and
ovine brucellosis AHS, CSFV
Paratuberculosis ,RVF,
Trichinellosis, Bovine
babesiosis PPR,
Dourine, Equine
infectious anemia,
SVD, TGE, AIB, AILT,
IBD
Enzyme Linked Immuno-Sorbent Assay(ELISA)

3. 4. Optimisation
Study Design
2. Study design
and
protocol
2. Reagents and controls
1. Intended purpose
of the assay
Development Pathway of Antibody ELISA

4. Assays characteristics Determinants for fitness of the test
1 2 3 4 5 6
a b
Diagnostic sensitivity (DSe) +++ +++ +++ +++ +++ + +
Diagnostic specificity (DSp) + + + + +++ + +++
Reproducibility and repeatability +++ +++ +++ +++ +++ +++ +++
Intended purpose of the assay
1. Demonstration of freedom from infection in a defined population.
2. Certify freedom from infection of animal or products for trade/movement
purposes
3. Eradication of disease or elimination of infection from defined populations
4. Confirmatory diagnosis of clinical cases
5. Estimate prevalence of infection or exposure to facilitate risk analysis
6. Determine immune status in individual animals or populations

5. 2. Test materials, reagents and controls
 Antigens
 Monoclonal antibody
 Reference standards- Antisera directed against reference
pathogen or OIE reference standard
 Positive and negative reference samples
1. Experimental infected or vaccinated reference animals
2. Samples from animals of unknown status- reference test
3. Reference animal populations (positive and negative)

6. Number of reference samples required
Known positive (279) Known negative (95)
Positive 270 (TP) A 7 (FP) B
Negative 9 (FN) C 88 (TN) D
Diag. sensitive (DSe)
A/A+C
TP/(TP+FN)=96.8%
Diag. specificity (DSp)
D/D+B
TN/(TN+FP)=92.6%
Number of reference samples required to achieve
an anticipated diagnostic sensitivity & specificity
C-confidence interval (95%), e- percentage of error (expressed in decimal )
N= 92 (1-92) (1.96)2
= 707
(0.02)2
N= (Dse) (1 - DSe) (C2)
e2

8. Design of the test methods
• Availability of reagents and continuous supply- large scale application
• Monoclonal antibody
• Crude antigen/ purified antigen
• Multispecies disease
• Antibody isotypes
Commonly used format for ELISA for detection of antibody
A. Indirect ELISA
B. Competitive ELISA
1. Direct
2. Indirect
3. Sandwich-solid phase and liquid phase

9. INDIRECT ELISA
Coated antigen either recombinant antigen or purified virus

10. COMPETITIVE ELISA
Competitive ELISA
1. Indirect-solid phase
2. Direct- solid phage
3. Sandwich-solid phase and liquid phase
Sample Ab
MAb
Ag

11.  Most of the commercially available cELISA
 Specific monoclonal antibody linked with HRPO
 Further reduction in duration
 Most of the ELISA of livestock diseases manufactured by IDEXX
and Prionics are using this format
Competitive ELISA- Direct
Sample Ab
MAb
Ag

12. Competitive ELISA- Sandwich type
Sample Ab
2nd MAb
1st MAb
Ag in solid phase
Rabbit Ab
Guinea pigs

13. Sample Ab
2nd MAb
1st MAb
Competitive ELISA- Sandwich type
Ag in solid phase

14. Expression of results
 The OD values at specific wavelength of all samples are
expressed as percentage inhibition relative to the OD
value of negative control.
PI = (100 – OD value of test sample)x 100
OD value of negative sample
 In most of the commercial kits, PI value above 40 % is
considered as positive for antibody.
 The antibody titre in the serum samples is directly
correlated with the PI value.
 Higher the PI values, higher the antibody titre.
 For FMD
 purposes of demonstrating freedom from infection in a
naïve population, greater than 60% inhibition is considered
positive
 certifying individual animals for international trade, an
inconclusive range may be set between 40 and 60%
.

15. LPB ELISA
Sandwich type competitive ELISA
LPB ELISA for FMD Prescribed test As per OIE
Commonly used test in India for detection serotype Specific Abs
Two fold dilutions of serum samples are tested
in-house LPB ELISA kit developed by PDFMD, Mukteswar.
Ag Ag
Rabbit (serotype specific)
G. pig Ab
Color
Test serum
50 µl
Ag
Known serotype Ag
50 µl
Mix & incubate
HRP
HRP HRP
HRP
Substrate
ELISA Plate
Carrier plate
Herd-based
serosurveillance a cut-
off of 1/90 may be
appropriate

16. OIE certified test kit
Disease
Name of the
Diagnostic kit
Name of the
Manufacturer
Type of kit
Avian Influenza Avian Influenza Disease
Antibody Test Kit
BioChek UK Ltd ELISA
White spot disease IQ 2000TM WSSV GeneReach Bio. PCR
BSE Prionics® - Check Prionics Western Blot
TSE TeSeETM Western Blot Bio-Rad Western Blot
Salmonellosis Check&Trace Salmonella Check-Points B.V. Multiplex followed
micro array
Bovine tuberculosis Mycobacterium bovis
Antibody Test Kit
IDEXX Laboratories Indirect ELISA
Newcastle disease Newcastle Disease Virus
antibody detection ELISA
BioChek UK Ltd ELISA
Bovine tuberculosis BOVIGAM® - Prionics Sandwich ELISA
Contagious equine
metritis
Pourquier® IIF Taylorella
equigenitalis
IDEXX Laboratories Indirect immuno
fluorescence test
Middle East
Respiratory
Syndrome
BIONOTE® Rapid MERS-
CoV Ag Test
BioNote, Inc Immuno
chromatographic
assay

17. Thanks