1. Ingeniería

Project Directorate on Foot and Mouth Disease
(FAO Regional Centre for South Asia)
Mukteswar India 263138
Indian Council of Agricultural Research
Instruction manual
|Liquid Phase Blocking ELISA Kit (LPBE - FMD Kit)|
Application: Estimation of antibodies against structural proteins
of FMD virus serotypes O, A and Asia1 in all FMD susceptible species
Reagents for testing minimum of 165 serum samples
Store at; -20ºC
Expiry: Use within 6 months after date of manufacture
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CONTENTS
S.
No
Title
1
Page No.
02
Background
2
03
Kit Components
4
04
Other reagents/ equipments required (not provided with kit)
5
05
Before start of test
6
06
Precautions
7
07
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Liquid phase blocking ELISA
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Background
Quantification of protective antibody levels against FMD in animals following vaccination is
essential for effective implementation of vaccination based FMD control programme in the country.
Generally protective antibody level is expressed as log10 titer and titer of 1.8 is accepted as the
protective level. To determine the titers of antibodies, conventionally serial two fold dilution method
of test sera is performed and named as liquid phase blocking ELISA. In this assay, test serum sample
is mixed with equal volume of a constant dose of viral inactivated antigen in a liquid medium and
allowed to react. The antigen-antibody reaction is carried out in a suspension (or liquid medium), and
the antigen is blocked by the homologous antibodies, if present, in the test serum for subsequent
detection by guinea pig serum. The antigen molecules which are not completely blocked by the
antibodies in the test serum are trapped to the wells of the ELISA plates with the pre-coated typespecific rabbit antibodies. Antigen and background controls are added containing antigen without
serum and blocking buffer respectively as described in plate layout. Subsequently, the bound antigen
is traced by sero-type specific guinea pig serum and anti-guinea pig-HRPO conjugate and substrate
reaction is followed in a standard ELISA procedure. Color reaction is stopped and measured in terms
of optical density (OD) at 492 nm wavelength with reference to 620nm. OD of reference antigen
controls are used for normalization of data in terms of percentage inhibition and titer is calculated as
the log10 of the inverse dilution at which percentage inhibition is 50%. With this kit 11 serum samples
(in duplicate) can be tested in an ELISA plate for one serotype. Software for titer estimation is
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included in the kit.
Kit Components for 165 serum samples
Reagents
S No
1
96-well Nunc Maxisorp ELISA plates
Quantity
Number of
vials/ kit
15
-
Lyophilized ready-to-use Type specific
anti-146S FMDV Anti-FMDV coating
serum.
One vial for
three plates
5 vials/ serotype
3
Lyophilized ready-to-use Type specific
anti-146S FMDV Anti-FMDV tracing
serum.
One vial for
three plates
5 vials/ serotype
4
Lyophilized ready-to-use inactivated
Type O, A, and Asia-1 antigens.
One vial for
three plates
5 vials/ serotype
5
Rabbit/ goat anti-guinea pig
immunoglobulin HRPO conjugate
(DAKO)
One vial for
three plates
15 vials
6
Substrate: OPD (Orthophenylenediamine
dihydrochloride, Sigma)
One vial for
three plates
15 vials
7
Lyophilized
ready-to-use
specific Serum Controls
Set of eight
vials for each
serotype
24 vials
8
Lyophilized Blocking Buffer
One vial in a
kit
One vial
9
Phosphate buffer saline
One sachet of
one liter
One sachet
10
Tween-20
10ml
10ml
serotype
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1
(Nunc cat. No. 442404)
Other reagents/ equipments required (not provided with kit)
1. Precision pipettes: multi channel pipettes variable range from 50 to 200µl & Single channel
pipettes variable range from 1 to 20µl, 20 to 100µl, 50 to 200µl and 200 to 1000µl along
with disposable tips.
2. Double Distilled water
3. 1M H2SO4
4. 30% Hydrogen Peroxide
5. Wash bottle fitted with Immunowasher
6. 1 container: 1 to 2 liters
7. ELISA plate reader, 492nm filter attached with computer
8. Refrigerator: range of -20ºC to +4ºC
9. Incubator: warm wall incubator maintained at +35ºC to 37ºC.
10. pH meter: with accuracy of 0.01pH units or good quality pH strips with varying range from
2 to 10 can be used.
11. Glassware/ plastic ware: flasks (50-5000ml), graduated cylinders (10-2000ml), graduated
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pipettes (1-100ml).
Before start of the test
Note: Equilibrate all the reagents provided in the kit at room temperature
1. Preparation of solutions/ reagents
S No
Reagents
Reconstitute
with
Reconstitute
Volume
1
Lyophilized ready-to-use Type specific
anti-146S FMDV Anti-FMDV coating
antibodies.
DDW
18ml/ Vial
2
Lyophilized ready-to-use Type specific
anti-146S FMDV Anti-FMDV tracing
serum.
PBST-0.1%
18ml/ Vial
3
Lyophilized ready-to-use inactivated
Type O, A, and Asia-1 antigens.
PBST-0.05%
18ml/ Vial
4
Lyophilized ready to use Rabbit/ goat
anti-guinea pig immunoglobulin HRPO
conjugate (DAKO)
PBST-0.1%
18ml/ Vial
5
Lyophilized ready-to-use Substrate: OPD
(Orthophenylenediamine
dihydrochloride, Sigma)
DDW
18ml/ Vial
6
Lyophilized
ready-to-use
specific Serum Controls
DDW
100µl/ Vial
7
Lyophilized Blocking buffer
PBST-0.05%
10ml
serotype
2. Washing buffer: dissolve one PBS capsule in 1000ml of distilled water, check pH and
if necessary adjust to pH 7.4 with 3M NaOH or 1M HCl. Add tween-20 to final
concentration of 0.1% and mix it well.
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3. PBS 0.05% tween: add tween-20 in PBS to final concentration of 0.05%.
Precautions:
1. Use the prescribed format for ELISA only
2. Do not intermix the reagents from different batches
3. All the glass wares to be used should be clean and sterile.
4. Slight drop in pH (less than 7.2) in washing buffer can adversely affect antigen antibody
reaction. Ensure correct pH every day before use of all buffers.
5. Microbial contamination/ precipitation formation of any degree in any of the reagents/
buffer will very much reduce the specificity of the test.
6. Mark the plate orientation properly with water proof marker before starting the test to avoid
any confusion which may arise later.
7. Do not stack plates one over other in incubator.
8. Prepare fresh substrate solution every time.
9. Do not store prepared buffers more than 15 days.
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10. Check pH of every buffer every day before starting the test.
Liquid phase blocking ELISA
Procedure:
1. Coating of ELISA plates: Reconstitute the ready-to-use Lyophilized serotype specific
coating antibodies with 18ml of DDW and coat three plates by adding 50µl of the
reconstituted solution in each wells of ELISA plate. Incubate the ELISA plates at 37ºC for 1
hour followed by incubation at 4ºC overnight.
2. Preparation of Ag-Ab mixture: Prepare two-fold dilutions (4 dilutions starting from 1:16)
of test serum samples in a low binding Perspex plate with PBS containing 0.05% Tween-20.
Distribute 75 µl each dilutions of the serum sample to four separate (labeled O, A, and
Asia1) Perspex plates. Reconstitute the antigen vial with 18ml of PBST0.05% and add equal
volume of diluted antigen (75µl) to all the dilutions of test serum samples. For antigen
control, add equal volume of PBS-Tween 20 (0.05%) to the already diluted antigen. The
dilution of each antigen and antibody in this step will be strictly made with PBS containing
tween-20 (not the PBS containing 0.1% tween-20 as used as washing buffer for washing of
the plate). Incubate the diluted antigen and antibody mixture at 4ºC overnight without
disturbance.
3. Washing of ELISA plates: Wash the wells of ELISA plates by adding washing buffer
(200-300µl) using immune-wash and discard the contents of wells by abrupt downward
hand motion. Repeat the washing 3 times with 3 minutes of hold period between each wash.
Slap the inverted microplate 3-4 times onto a dust free absorbent pad to remove all residual
contents in the wells.
4. Dispensing of antigen-antibody (Ag-Ab) mixture: Dispense 50µl of the overnight
incubated Ag-Ab mixture (Step 2) in duplicate to the ELISA plates as instructed in the plate
lay out (Figure 1). Incubate the plates at 37°C for 1 hr. Add 50µl of the reconstituted
blocking buffer to the wells of background control.
5. Wash the plate as in the step 3.
6. Tracing: Reconstitute the tracing serum vial with 18ml PBST0.1% and add the 50µl of
reconstituted serotype specific tracing serum to each serotype specific plate. Incubate at
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37°C for 1 hr. Wash the plate as in the step 3.
7. Conjugate: Reconstitute each vial of anti-guinea pig-HRPO conjugate with 18ml (for nine
plates use three vials) PBST0.1% and add 50µl to all the wells. Incubate at 37°C for 1 hr.
Wash the plate as in step 3.
8. Substrate: Reconstitute substrate vial just before use with 18ml DDW (for nine plates use
three vials) and add 9.6µl of 30%H2O2 solution (or 28.8µl in 54ml for 3 vials) and add 50µl
to each well of the plate and incubate at 37°C for 15 minutes.
9. Stop the color reaction by adding 50µl of stopping solution. Read the OD in the wells at 492
nm.
10. Transfer/ convert the OD sheet data into MS Excel format and analyze and interpret the
results using PDFMD ELISA Analyst v2.0 as per the instructions.
Figure 1: LPBE plate Format for addition of diluted test serum samples in ELISA plate (TS* Test
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sample)
Trouble shooting:
1. Invalid Antigen control:
•
Check the batch and expiry
•
Cross mixing of serotype specific reagents
•
Check the washing buffer and PBST0.05% for pH and contamination
•
Cross check the temperature of incubator
2. Invalid Background control
•
Improper washing
Cross contamination of tips used for conjugate and substrate solution.
•
Not using the blocking buffer provided.
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3. Even OD in all the wells
Notes
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Contact:
Project Director
Project Directorate on Foot and Mouth Disease
Mukteswar, Nainital Uttarakhand 263138
India
Email: [email protected], [email protected]
Phone: 05942286004; Fax: 05942-286307
Website: www.pdfmd.ernet.in
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