ELISA kits available from ADI (see details at the web site)
Instruction Manual No. M-400600-1
Catalog# ProdDescription
RV-400500-1
Recombivirus Swine/Pig Anti-Foot and mouth disease virus VP-1
(FMDV-VP1, Serotypes O+A+A1, Combo) Antibody ELISA kit, 96 tests
RV-400510-1
Recombivirus Swine/Pig Anti-Foot and mouth disease virus VP-1
(FMDV-VP1, Serotypes SAT1+2+3, Combo) Antibody ELISA kit, 96 tests
RV-400520-1
Recombivirus Swine/Pig Anti-Foot and mouth disease virus FMDV (all
serotypes O/A/A1/SAT1-3) 3ABC Antibody DIVA ELISA kit, 96 tests
RecombiVirus Bovine Anti-FMD VP1
(Serotypes O+A+A1) IgG)
ELISA KIT Cat. # RV-400600-1, 96 tests
RV-400530-RT-50 Swine Anti-Foot and mouth disease virus VP-1 (FMDV-VP1,
Serotypes O+A+A1, Combo) Antibody rapid tests (results in 2-10 min), 50 cards/pk
RV-400540-RT-50 Swine Anti-Foot and mouth disease virus VP-1 (FMDV-VP1,
Serotypes SAT1-3, Combo) Antibody rapid tests (results in 2-10 min), 50 cards/pk
For Qualitative Determination of FMDV (O+A+A1) VP1
Combo IgG In Bovine/Cow Serum or plasma
RV-400550-RT-50 Swine Anti-Foot and mouth disease virus FMDV (all serotypes
O/A/A1/SAT1-3) 3ABC Antibody rapid tests (results in 2-10 min), 50 cards/pk
RV-400600-1
Recombivirus Bovine/Cow Anti-Foot and mouth disease virus VP-1
(FMDV-VP1, Serotypes O+A+A1, Combo) Antibody ELISA kit, 96 tests
For In Vitro Research Use Only
RV-400610-1
Recombivirus Bovine/Cow Anti-Foot and mouth disease virus VP-1
(FMDV-VP1, Serotypes SAT1+2+3, Combo) Antibody ELISA kit, 96 tests
RV-400620-1
Recombivirus Bovine/Cow Anti-Foot and mouth disease virus FMDV
(all serotypes O/A/A1/SAT1-3) 3ABC Antibody DIVA ELISA kit, 96 tests
RV-400630-RT-50 Bovine/Cow Anti-Foot and mouth disease virus VP-1 (FMDV-VP1,
Serotypes O+A+A1, Combo) Antibody rapid tests (results in 2-10 min), 50 cards/pk
RV-400640-RT-50 Bovine/Cow Anti-Foot and mouth disease virus VP-1 (FMDV-VP1,
Serotypes SAT1-3, Combo) Antibody rapid tests (results in 2-10 min), 50 cards/pk
RV-400650-RT-50 Bovine/Cow Anti-Foot and mouth disease virus FMDV (all serotypes
O/A/A1/SAT1-3) 3ABC Antibody rapid tests (results in 2-10 min), 50 cards/pk
6203 Woodlake Center Drive • San Antonio• Texas 78244 • USA.
RV-400700-1
Recombivirus Goat/Sheep Anti-Foot and mouth disease virus VP-1
(FMDV-VP1, Serotypes O+A+A1, Combo) Antibody ELISA kit, 96 tests
Phone (210) 561-9515 • Fax (210) 561-9544
RV-400710-1
Recombivirus Goat/Sheep Anti-Foot and mouth disease virus VP-1
(FMDV-VP1, Serotypes SAT1+2+3, Combo) Antibody ELISA kit, 96 tests
Toll Free (800) 786-5777
RV-400720-1
Recombivirus Goat/SheepAnti-Foot and mouth disease virus FMDV
(all serotypes O/A/A1/SAT1-3) 3ABC Antibody DIVA ELISA kit, 96 tests
Email: [email protected]
Web Site: www.4adi.com
Note: All ELISA kits are also available in 5x96 (480 tests) and rapid cards in bulk package
of 1000.
DRAFT MANUAL: PLEASE CONSULT THE MANUAL SUPPLIED
WITH THE KIT FOR ANY LOT SPECIFIC CHNAGES.
Alpha Diagnostic Intl. (www.4adi.com ) RV-400600/141218A
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Bovine Anti FMDVP1 (O+A+A1) IgG ELISA KIT Cat. # RV-400600-1 (96 tests)
Bovine Anti FMDVP1 (O+A+A1) IgG ELISA KIT Cat. # RV-400600-5 (5x96 tests)
Kit Contents:
Items
(VP1-4) are not as well conserved within the serotypes but the Non-structural
(NSPs such as 2ABC or 3ABC) are highly conserved across the FMD serotypes.
VP1 proteins also show some variations within the strains of a given serotype.
1x96
tests
5x96
tests
FMD Serotypes
1
plate
5
plates
Bovine Anti-FMD VP1 (O+A+A1) IgG
Negative control A (ready to use)
clear cap #400602NC (1 U/ml)
0.5
ml
2 ml
Bovine Anti-FMD VP1 (O+A+A1) IgG
Cut-off control B (ready to use)
Blue cap #400602CO (5 U/ml)
0.5
ml
2 ml
O
A
Asia-1
C
SAT-1
SAT-2
SAT-3
Within serotype
Strain variations
Bovine Anti-FMD VP1 (O+A+A1) IgG
High Positive control C (ready to use)
Red cap #400602PC (100 U/ml)
0.5
ml
2 ml
Sample Diluent (20x, green color
solution, dilute 1:20 before use), 1 bottle
#SD20TG
6 ml
30 ml
Wash Buffer Concn. (50X), clear
bottle/blue cap (dilute 1:50 before use)
#WB50
Anti-bovine IgG HRP-Conj.(clear bottle
and pink soln) #400603
12 ml
60 ml
6 ml
30 ml
HRP Substrate Solution, Brown
bottle/brown cap (ready to use) #80091
Stop Solution (white bottle/red cap)
#80101
Instruction manual #RV-400600-1
6 ml
30 ml
6 ml
30 ml
1
1
Recombinant FMDO VP1 (O+A+A1)
antigen Coated microwell (8 x 12 strips),
Ready-to-use #400601
Intended Use
ADI Foot and Mouth Disease Virus (FMDV) IgG Combo ELISA kits is designed
to detect the presence of FMD Viral Protein 1 (FMDVP1 Serotypes O, A, and
Asia-1) IgG in bovine or cow serum, plasma or other biological fluids. This kit
employs a mixture a highly purified recombinant VP1s from serotypes O, A, and
Asia-1. Therefore, antibodies to all 3 serotypes are detected in a single test.
This kit can be used to determine the FMDVP1 (serotypes O, A, and A-1)
antibody status in vaccinated or naturally infected animals. FMD serotype
specific SAT1-3 VP1 antibody ELISA are also available.
FMD Protein sequence conservations
VP1
2ABC
3ABC
3D
100%
100%
100%
71%
98%
98%
92
68%
97%
99%
|
70%
98%
99%
100%
44%
85%
93%
47%
91%
96%
50%
85%
93%
97-100%
ADI ELISA kit #RV-400600-1 is coated with a mix of recombinant purified VP1
from Serotype O, A, and Asia-1 to allow detection of these antibodies.
Crossreactivity of the VP1 IgG was investigated by testing IAEA reference sera
from 6 FMDV serotypes: A (24 Cruceiro, 28 dpi), O (363, 28dpi), A-1 (348-1, 28
dpi), SAT1 (8001, 42 dpi), SAT2 (Uga2, 28 dpi), and SAT3 (102, 28 dpi). ADI
combo O+A+A-1 detected not only O, A, and A-1 but also SAT1-3. However, we
recommend using #RV-400610-1 that utilizes recombinant VP1 from SAT1-3 for
the more specific detection of SAT1-3 antibodies.
Specificity & Species Reactivity
Bovine antibody conjugate used in the kit detects only the IgG type and not IgM
or IgA FMDV antibodies. This kit is designed for bovine samples only. ADI has
similar kits for swine, goat, and other species.
LIMITATIONS
This kit should be used for research use only (RUO). A positive VP1 antibody
test alone is not diagnostic or indicative of the FMDV diseases. All positive tests
must be verified by other techniques and DIVA tests.
This ELISA kit is not to be used for serotyping of FMDV.
This kit has no FMD viral proteins or inactivated virus. FMDV antigens are
purified recombinant proteins. This kit is for in vitro research use only (RUO).
Alpha Diagnostic Intl. (www.4adi.com ) RV-400600/141218A
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TEST PROCEDURE (ALLOW ALL REAGENTS TO REACH ROOM TEMP).
Materials Required But Not Provided:
Pipettors and pipettes that deliver 100 ul and 1-10ml. A multi-channel
pipettor is recommended.
Disposable glass or plastic 5-15ml tubes for diluting HRP Concentrate.
Stock bottle to store diluted Wash Solution; 200ml to 1L.
Distilled or deionized water to dilute reagent concentrates.
ELISA reader at 450 nm and ELISA Washer (optional).
STORAGE AND STABILITY
The microtiter well plate and all other reagents are stable at 2-8oC until the
expiration date printed on the Kit box. The whole kit stability is usually 6-12
months from the date of shipping under appropriate conditions. Do not
contaminate the bottles. Withdraw solutions in a separate clean tube or
dispensing trays. Any unused solution should be discarded and not returned to
the bottle. Do not use HRP substrate solution if this solution is blue. Do not
expose these solutions to strong light.
REAGENTS PREPARATION
Prepare 1X wash buffer and 1X sample diluent.
Dilute
serum/plasma samples (see page 3). Controls are supplied ready
to use (do not dilute supplied controls). Remove required
number of coated strips and arrange them on the plate. Store
unused strips in the bag.
For Qualitative Test: Run the supplied –Ve, Cut-off and high
positive controls, blank, and samples in duplication. Samples are
compared to a cut-off control and results expressed as –ve or +ve.
1.
Pipet 50 ul of 1x diluent (for use as blank), negative, and
positive controls (or 100-12.5 U/ml calibrators), and diluted
samples (1:100 or higher) into appropriate wells in duplicate. Mix
gently, cover the plate and incubate for 60 minutes at room temp
(25-28oC).
2.
Aspirate and wash the wells 3 times with 200-300 ul of 1x
wash buffer. We recommend using an automated ELISA plate
washer for better consistency. Failure to wash the wells
properly will lead to high blank values. If washing manually,
plate must be tapped over paper towel between washings to
ensure proper washing.
3.
Add 50 ul of antibody enzyme conjugate into each well. Mix
gently. Cover the plate and incubate for 30 minutes at room
temp.
4.
Aspirate and wash the wells 4 times as above. After the last
wash, tap the plate over fresh paper towel to remove any traces
of conjugate.
5.
Add 50 ul of TMB substrate into each well. Mix gently. Cover
the plate and incubate for 15 minutes at room temp. Blue
color develop in +ve controls and samples. Note: It is possible
to increase or decrease the incubation time + 5 min if there is too
little to too much bluer color development.
6.
Stop the reaction by adding 50 ul of stop solution to all wells.
Mix gently. Blue color turn yellow. Measure the absorbance at
450 nm using an ELISA reader within 15 min.
1x WASH BUFFER preparation: Dilute 50X stock 1:50 in distilled water (10 ml
stock in 490 ml water. Keep 50X stock at 4oC and 1X stock at room temp.
Unused 1X stock should also be stored at 4oC when not in use.
1X sample Diluent: Dilute 1:20 sample diluent (green color) with distilled water
(1 ml stock in 19 ml water). Prepare as necessary. 1x diluent is used for sample
dilution.
Sample Dilutions
1.
Collect serum, plasma or other biological samples and store frozen or
use fresh samples.
2.
Prepare 1:10 initial stock of all samples by diluting in green sample
diluent (10 ul sample in 90 ul diluent). The presence of dye does not
interfere with the ELISA. Antibodies are stable in this diluent and can
be kept at 4oC for weeks. It also avoids freezing and thawing of the
original samples. All subsequent test dilutions (1:100 or more) of
samples should be made fresh on the day of the test. For example,
samples that will be tested at 1:100 will be diluted another 1:10 fold
from the 10X sample stocks. High antibody samples should be diluted
more (e.g. for 1:500 dilution, dilute initial 1:10 stock another 50 fold or
10 ul in 490 ul of 1x diluent).
3.
Users may choose to test their samples at a dilution different than
1:100 recommended in this manual (e.g. 1:50 or 1:200 etc) and
compare the control groups with experimental group or vaccinated vs
non-vaccinated or normal Vs infected. A difference in the A450 values
of one group Vs another should be indicated of the FMDV antibody.
Alpha Diagnostic Intl. (www.4adi.com ) RV-400600/141218A
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Alpha Diagnostic Intl. (www.4adi.com ) RV-400600/141218A
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CALCULATION OF RESULTS
INTRODUCTION
Typical ELISA Set Up
Wells
A1, A2
Stds/Samples
B1, B2
Blank (1X
diluent)
-ve control
C1, C2
D1, D2
E1, E2
F1, F2
G1, G2
H1, H2
Average
Values
450nm
0.100
Net A450
Results
-
0.200
0.100
Cut-off control
0.590
0.459
High Positive
control
Sample 1
Sample 2
Sample 3
Sample 4
1.940
1.85
0.450
0.870
1.55
0.200
0.350
0.77
1.45
0.100
-
-ve
+ve
+ve
-ve
Establish FMD IgG ELISA Cut-off values
The ELISA cut-off values for each FMD VP1 antibody (serotypes ), A, and Asia1) have been tested with IAEA infected with each serotype (O, A or Asia-1) at
28dpi after infection. It is possible that a given population of animals have
different basal values in vaccinated or non-vaccinate animals. We strongly
recommend testing in-house demographic and age specific animals –ve and cutoff values to establish antibody status of vaccinated for non-vaccinated animals.
INTERPREATION OF RESULTS
Negative
Positive
A450 values less than or equal to Cut-off values
A450 values higher than Cut-off values
Quality Control
Negative control values must be less than the cut-off control. Positive control
must be higher than Cut-off and at least A450=0.800 or higher. Repeat the
controls if values are not met; repeated failure may indicate serious technical
error or deterioration or expiration of the kit components.
Blanks values are typically in the range of 0.100-0.200. High blanks (0.300 or
higher) are indicative of improper washing. Check the washing protocol and
increase number of washing.
Specificity of the FMDV Serotype VP1 IgG Detection
Of the seven FMD serotypes of the virus, A, C, O, Asia 1 and SAT3 appear to be
distinct lineages; SAT 1 and SAT 2 are unresolved clades. Structural proteins
Alpha Diagnostic Intl. (www.4adi.com ) RV-400600/141218A
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FMD affects millions of farm animals (cow, buffalo, pigs, sheep, goat, etc) in the
world that are of great economic concerns. Current FMD vaccines and
diagnostic tests (antigen and antibody) needs to be carefully matched in
sensitivity, specificity, and the ability to distinguish infected from vaccinated
animals (DIVA tests). All currently approved FMD Vaccines employ killed FMD
viruses (Monovalent or a combination of various serotypes); FMD virus grown in
BHK-21 (baby hamster kidney cells (BHK) or epithelial cell culture and then
killed using chemicals (binary ethylamine or BEI). Most vaccines contain Alum
or oil adjuvants. Worldwide, tens of billion doses of FMD vaccine are
administered every year. Endemic Asian countries like India manufacture mono,
bi and trivalent vaccines BioFMD-Oil™ (Biovet), FUTVAC™ (Brilliant
Biopharma). Raksha (Indian Immunological) against strains O, A, Asia 1. Other
manufacturers of FMDV vaccine are Bayer, Merial, and Intervet.
ADI offers the most comprehensive selection of individual FMD recombinant
proteins (structural and non-structural), antibodies, and FMD antibody ELISA
detection tests for various animals (Cow, Buffalo/Bison, Pig, Sheep, Goat, and
Deer). NSPs (2A, 2B, 2C, 3A, 3B, 3C, and 3D proteins are highly conserved
(95-100% in various FMD serotypes). New DIVA tests based upon 3ABC, 3D,
2ABC have also been developed to offer an expanded testing of animals for
DIVA tests. These NSP DIVA tests is applicable to all FMD serotypes.
PRINCIPLE OF THE TEST
Anti-FMD IgG ELISA kit is based on binding of FMD antibody (IgG/IgM) from
samples to FMD antigens immobilized on microtiter wells. After a washing step,
antibody-HRP conjugate (species and antibody type specific) is added. After
another washing step, to remove all the unbound enzyme conjugate,
chromogenic substrate (TMB) is added and color developed. The enzymatic
reaction (color) is directly proportional to the amount of FMD IgG present in the
sample. Adding stopping solution terminates the reaction. Absorbance is then
measured on a microtiter well ELISA reader at 450 nm and the concentration of
anti-FMD antibody (IgG or IgM) in samples is calculated on the basis of the A450
of the negative, positive, and, calibrator standards.
MATERIALS AND EQUIPMENT REQUIRED
Adjustable (5-100 µl) and multichannel pipet with disposable plastic tips. Reagent troughs,
wash bottle or ELISA plate Washer and Reader.
PRECAUTIONS
Applicable MSDS, if not already on file, for the following reagents can be obtained from
ADI or the web site.
TMB (substrate), H2SO4 (stop solution), and Prolcin-300 (0.1% v/v in standards, sample
diluent and HRP-conjugates).
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Alpha Diagnostic Intl. (www.4adi.com ) RV-400600/141218A
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