sop fmd lpbe-sadc-final 8-11-10

SOP FMD 5: Detection of Antibodies against Foot-and-Mouth Disease Virus (FMDV) in a Liquid Phase Blocking ELISA (LPBE)

SOP FMD Compiled by: G.Thobokwe and R. Dwarka Issued by:

Revision Authorised by: Issue date: June 2010

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SADC harmonized SOP for the detection of foot

and mouth disease virus (FMDV) antibodies using

liquid phase blocking ELISA

Version 1.0 Reviewed by G. Thobokwe1 and R. Dwarka2

Effective date: June 2010

Edited by:

E.M. Fana3 and S. Dibe4

This Standard Operating Procedure (SOP) is available at:

http://www.fao-ectad-gaborone.org

_______________ 1: C.VO- Botswana Vaccine Institute (BVI): 2: Head: ARC-OVI, South Africa: 3: Research Supervisor, BVI-SSRL Botswana; 4:

Scientist, BVI-SSRL, Botswana.




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DISTRIBUTION LIST

This SOP has been prepared for use by National Veterinary Laboratories of the following SADC

member states:

o Botswana

o Lesotho

o Malawi

o Mozambique

o Namibia

o Swaziland

o Tanzania

o Zambia

o Zimbabwe




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Contents:

1. Purpose

2. Principle

3. Materials and equipment

3.1 Chemicals

3.1.1 Coating buffer

3.1.2 Washing buffer (PBS)

3.1.3 Diluent

3.1.4 Blocking buffer

OPD OPTION

3.1.5 Phosphate citrate buffer

3.1.6 Chromogen

TMB OPTION

3.1.7 Blue substrate buffer

3.1.8 Chromogen solution

3.1.9 Substrate

3.1.10 Substrate/chromogen

3.1.11 Stopping solution

3.2 Reagents

3.2.1. Trapping antibody (Rabbit serum)

3.2.2 Detecting antibody (Guinea Pig serum)

3.2.3 Antigen stock

3.2.4 Anti-species conjugate

3.2.5 Positive and negative control sera

3.3 Consumables

3.3.1 Pipettes

3.3.2 Glassware/plastic ware

3.3.3 Reagent troughs

3.3.4 Micro plates

3.3.5 Tupperware containers

3.4 Apparatus

3.4.1 ELISA reader

3.4.2 Computer

3.4.3 Orbital Shaker




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3.4.5 Refrigerator

3.4.6 Freezer

3.4.7 Incubator

3.4.8 pH meter

3.4.9 Balance

3.4.10 Timer

4. Method

4.1 Reagent and sample preparation

4.1.1 Trapping antibody stocks

4.1.2 Coating of micro titre plates

4.1.3 Detecting antibody stocks

4.1.4 Anti-species conjugate stocks

4.1.5 Antigen stocks

4.1.6 Dilution of test sera

4.2 Dilution of reagents

4.3 Blocking ELISA: Assay procedure

4.3.1 Plate layout + Addition of the control/test serum to a carrier plate

4.3.2 Mix antigen and sera

4.3.3 Addition of detecting antibodies

4.3.4 Addition of conjugate

4.3.5 Addition of substrate/chromogen and stopping solution

4.4 Recording of results

4.5 Calculation of results

4.6 Interpretation of results

Apendix

References




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1. Purpose

The aim of the LPBE is to test sera of cloven-hoofed animal species for the presence of antibodies

against the structural proteins of FMDV. Antibodies directed against one or more type of FMDV can

either be identified (screening test) or quantified (titration test). The test has a very wide application.

Serum antibodies are induced against the outer capsid structural proteins following both vaccination

(domestic animals) against and infection (both domestic animals and game) with FMDV. Furthermore

it can be used to establish FMD free status in animals destined for import and export into the region

2. Principle

The test is a LPBE for the detection of FMDV antibodies in serum. The test is based upon specific

blocking of liquid phase FMD antigen by antibodies in the test serum sample. ELISA plates are coated

with anti-FMD antibody. Sera premixed with FMD antigen is then added to the coated plates. If

antibodies are present in the test sera, they will block the antigen and prevent it from binding to the

coating antibody. If there are no specific antibodies in the test sera then the antigen will be available

to be trapped on the plate, this will be detected by a positive colour indicating negative test results.

3. Materials and equipment

3.1 Chemicals

3.1.1 Coating buffer (Carbonate/bicarbonate buffer (0.05M))

To prepare 10x concentrated stocks

Sodium Hydrogen Carbonate (NaHCO3 0.5M; Merck,Sigma) ...................................................... 1.47g

Sodium Azide ............................................................................................................................. .1g

Sodium Carbonate anhydrous (Na2CO3 0.5M; Merck, Sigma) ...................................................... 0.79g

Distilled water ........................................................................................................................1 litre

Store at 4ºC+ 2ºC

pH9.6 + 0.05

3.1.2 Washing buffer (PBS)

To prepare concentrate:

Sodium chloride (NaCl; Merck, Sigma) ..................................................................................... 1600g

Potassium chloride (KCl; Merck, Sigma) ..................................................................................... 40g

Potassium dihydrogen orthophosphate (KH2PO4; Merck, Sigma) ........................................................... 40g

Disodium hydrogen phosphate dodecahydrate (Na2HPO4.12H2O; Merck, Sigma) ..................... 574,48g

Distilled water (up to) ..............................................................................................................6 litre




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Dissolve completely

Set pH 7.2 – 7.4 (use HCl/NaOH)

Dispense in 175ml aliquots, store at +4ºC + 2ºC

To prepare washing buffer for use: dissolve 175ml in 5 litre distilled water

3.1.3 Diluent (PBST)

PBS ……………………………………………………………………………………………………………..…………………500ml

Tween-20 ………………………………………………………………………………………………………………..………250ul

Store at 4°C and use within 24hrs

3.1.4 Blocking buffer

Normal rabbit sera ……………………………………………………………………………………………………………...5ml

Normal Bovine sera ……………………………………………………………………………………………….…………...10ml

PBS ………………………………………………………………………………………………………..…………………………85ml

Store at -20°C

PBS-T milk

Skim milk (Merck,Unilab) .......................................................................................................... 100g

PBST ......................................................................................................................................2 litre

The pH must be constantly reset to neutral by adding drops of 0.5M NaOH.

Set pH 7.0-7.6

OPD OPTION

3.1.5 Phosphate citrate buffer

To Prepare 0.05M solution

Phosphate citrate buffer tablet (Sigma)………………………………..1

Distilled water……………………………………………………………………100ml

Set pH 5.0 (can be stored at room temperature for 1 month)

3.1.6 Chromogen

OPD tablet…………………………………………………………………………1

Phosphate citrate buffer----------------------------------------------55ml

Hydrogen Peroxide………………………………………………………………1/2000dilution i.e. 27.5µl




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TMB OPTION

3.1.7 Blue substrate buffer

Solution A

Citric Acid Monohydrate (Merck, Sigma, Fluka) ............................................................................ 21g

Distilled water .........................................................................................................................1 litre

Solution B

Tri-Potassium Citrate (Merck, Sigma, Fluka) .............................................................................. 32,4g

Distilled water ........................................................................................................................1 litre

Mix solution A and solution B to obtain a pH 4

Store at 4ºC

3.1.8 Chromogen solution

N, N- Dimethylacetamide (DMA) (Merck, Sigma,Fluka) ............................................................. 100ml

Tetrabutylammonium borohydride (TBABH) (Merck, Sigma, Fluka) ............................................. 0,21g

3, 3’, 5, 5’-Tetramethylbenzidine (TMB) (Sigma, Merck, Fluka) ............................................... 0,9853g

Store in the dark at 2 - 8ºC

3.1.9 Substrate

H2O2 30% m/v (Merck analar)

Store at 4ºC

3.1.10 Substrate/Chromogen

The substrate/chromogen must be freshly prepared every time an ELISA is performed, just before use:

H2O2 ......................................................................................................................................... 30ul

Chromogen solution ................................................................................................................... 1ml

Blue substrate buffer ............................................................................................................. 100ml

Dissolve completely

3.1.11 Stopping solution

1.25M H2SO4 (Merck, Sigma, Fluka)

H2SO4 (98%) Merck, Sigma, Fluka) ............................................................................................ 67ml

Water with crushed ice ........................................................................................................... 933ml

To prepare stopping solution: add the concentrated acid very slowly to the water.




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NOTE: Always add sulphuric acid to water - never add water to sulphuric acid

3.2 REAGENTS

3.2.1 Trapping antibody

The trapping antibody used is rabbit antiserum (whole serum) specific for the serotype of FMD.

3.2.2 Detecting antibody

The detecting antibody used is guinea-pig antiserum (whole serum) of the same specificity as the

trapping antibodies.

3.2.3 Control antigen stocks

FMD antigens used in the sandwich ELISA as positive control antigens are supernatant derived from

infected cell culture which has been inactivated with binary ethyleneimine (BEI). Negative control

antigens are prepared in a similar way as the positive antigens but from uninfected cell cultures.

3.2.4 Anti-species Conjugate

Rabbit anti guinea-pig IgG conjugated to horseradish peroxidase (Chemicon-Millipore- AQ108P).

3.2.5 Positive and negative control sera

3.3 Consumables

3.3.1 Pipettes

Single channel pipettes, variable ranges from 20-200 and 200-1000ul

Digital 12 Channel pipette, variable ranges from 50-300ul

combipette

Tips: blue and yellow tips

Combitips

3.3.2 Glassware/plasticware

A selection of beakers (25-1000ml), flasks (50-1000ml), graduated cylinders (50-1000ml), graduated

pipettes (1-10ml), bottles with screw caps 1-2000ml, dilution tubes (2-10ml) and suitable test tube

racks




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3.3.3 Reagent Troughs

Reagent troughs suitable for simultaneous, multi-channel pipetting of a single reagent

3.3.4 Microplates

NUNC-Immuno Plate F96 Maxisorp (flat bottom)

NUNC-96 well Tissue culture plates (V/U shape) carrier plates

3.4 Apparatus

3.4.1 Photometer

Microplate reader (or similar) with an interference filter of 450/490nm. The photometer must be

interfaced with a PC

3.4.2 Computer (PC)

The appropriate software necessary for the calculation of ELISA results must be installed with Excel

3.4.3 Orbital Shaker

Orbital shaker (or similar) capable of rotating between 40 – 100rpm

3.4.4 Plate Washer optional (Hand wash)

Automated Microtitre plate washer. The inlet (of the plate washer) must be connected to two 20 litre

bottles containing washing buffer and distilled water respectively. The outlet of the plate washer must

be connected, via a waste collecting bottle, to a suitable vacuum source.

3.4.5 Refrigerator

Any type with a range between 2ºC - 6ºC

3.4.6 Freezer

-20ºC + 2ºC freezer

Ultrafreezer -70ºC + 5ºC

3.4.7 Incubator

Walk-in warm wall incubator with a range between 35ºC - 39ºC




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3.4.8 pH meter

With an accuracy of 0.1 pH units

3.4.9 Balance

Analytical with sensitivity of 0,01g

3.4.10 Timer

An electronic timer with countdown capabilities and an audible alarm

4. Method

4.1 Plate layout + Addition of the control/test serum to a carrier plate

Prior to test, the type of test (screening/titration) and plate lay-out must be decided. Half titrations are

routinely used for screening to determine the immune status of the samples and full titrations are

done when the samples gives a reaction with the screening test.

Note: - The working concentrations of all produced reagents need to be optimized before a

new virus/antigen or trapping/detecting antibody or conjugate is introduced into the test.

When new control sera are introduced, they need to be tested several times until a good

range of titres is obtained. The running mean may then be accepted as early results may be

unrepresentative.

Pos

Ag

Tcc

Pos

Ag

Tcc

Pos

Ag

Tcc

Pos

Ag

Tcc

Pos

Ag

Tcc

Pos

Ag

Tcc

Serum Dil

1 2 3 4 5 6 7 8 9 10 Ca Ca

A S1 S1 S2 S2 S3 S3 S4 S4 S5 S5 Ca Ca 1/20

B S1 S1 S2 S2 S3 S3 S4 S4 S5 S5 Ca Ca 1/40

C S1 S1 S2 S2 S3 S3 S4 S4 S5 S5 Ca Ca 1/80

D S1 S1 S2 S2 S3 S3 S4 S4 S5 S5 Ca Ca 1/160

E S6 S6 S7 S7 S8 S8 S9 S9 S10 S10 C++ C++ 1/20

F S6 S6 S7 S7 S8 S8 S9 S9 S10 S10 C++ C++ 1/40

G S6 S6 S7 S7 S8 S8 S9 S9 S10 S10 neg neg 1/80

H S6 S6 S7 S7 S8 S8 S9 S9 S10 S10 neg neg 1/160




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Plate layout for screening test

Pos Ag Positive antigen colum (50ul)

Tcc Negative antigen column (background - 50ul)

1 – 12 Column number

A – H Row number

C++ Strong control positive serum

Neg negative control

Ca Control antigen (Max OD – PBS/casein)

S1 – S10 Test serum

1/20 – 1/160 Serum dilution

Reference sera and test sera are titrated in duplicate wells against a reference antigen/virus in a

carrier plate starting with an initial dilution of 1/20. Add 50ul PBST to all wells then an extra 40ul PBST

to rows A 1-12, E1-12 and G11 & 12. Dilute by adding 10ul of the sample to 90ul of PBST. Referring

to the plate layout, make a twofold dilution series by transferring 50ul from row to row consecutively

discarding 50ul from the last dilution. Positive and negative sera are treated same way as samples and

diluted two fold.

4.1.1 Mix antigen and serum

Dilute pos antigen in PBST as per validated reagent dilution. Add 50ul of the diluted pos antigen to

columns 1, 3, 5, 7, 9, & 11 and Tcc (50ul PBST) to columns 2, 4, 6, 8, 10, & 12 of the carrier plate

with diluted serum. Incubate for 1h at 37oC + 2ºC on an orbital shaker or over night at 4oC + 2ºC.

4.1.2 Coating of microtitre plates

Microtitre plates are coated in bulk and then stored at -20ºC + 2ºC. Plates coated and stored in this

way can be used for both the sandwich and LPB ELISA for a period of one year.

Microtitre plates must be labeled according to date and the type of FMD virus with which they were

coated.

Trapping antibody stocks

For each strain of FMD virus used in the LPBE, prepare a coating serum stock dilution (rabbit

antiserum) of 1/100 to provide dilutions in carbonate buffer. This is aliquoted (5ml/ bottle) or less

depending on volume required per test. Store at -20ºC + 2ºC.




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The stocks of the 1/100 diluted coating sera must be thawed and diluted just prior to use. Dilute

coating sera stocks in carbonate buffer pH 9.6 + 0.05. Thus, all coating sera are used at a

predetermined final dilution. The diluted coating serum is poured in a reagent trough and 100ul

dispensed to all the wells of a microtitre plate using a multi-channel pipette.

The plates are covered with plastic lids or plate sealers and incubated at 37oC+ 2ºC for 1h on a plate

shaker or overnight at 4oC+ 2ºC.

Plates are washed in the automate microplate washer with PBS (three cycles) then blot dried on tissue

paper or cloth.

The coated microtitre plates are now ready for use or it can be sealed in small plastic bags (two plates

per bag) and stored at -20oC+ 2ºC until needed.

Transfer 50ul of the antigen/serum mixture from the carrier plates to the corresponding wells of the

coated ELISA plates. For titrations, transfer row A first, followed by subsequent dilutions.

Incubate 1h/37oC + 2ºC on an orbital shaker. Wash the plate (3 times). Blot dry.

4.1.3 Detecting antibody stocks

The guinea-pig sera used as detecting antibody are pre-blocked with normal bovine serum (50% v/v).

This is done by mixing 10ml of guinea-pig sera and 10 ml of bovine sera. This is aliquoted in 10ml

volumes and stored at -20ºC + 2ºC. This is known as the ‘treated working stocks’ of the typing sera.

Addition of detecting antibody

The guinea-pig detecting (typing) serum for each type must be of the same specificity as the rabbit

coating sera for each microplate. Dilute the guinea-pig serum in PBST-milk as per validation report

and add 50ul to all wells of all plates. Plates are sealed and incubated for 1h at 37oC + 2ºC on an

orbital shaker. Wash plates three times with a plate washer and blot dry.

4.1.4 Anti-species conjugate stocks

Reconstitute according to supplier’s instructions then dilute it with PBS to 1/100. Before using the

conjugate it is blocked at 1:1 with blocking buffer making final concentration of 1/200. Store in

aliquots of 1.0 ml in -70ºC + 2ºC. Just before use an ampoule is thawed and diluted as per validation

report.




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Addition of conjugate

Prepare a working dilution as described above of the conjugate in PBST-milk in a volume sufficient for

all microplates (5ml/plate) just before use. Pour conjugate in a reagent trough and distribute 50ul to

the wells of all the plates with a multi channel pipette. Plates are incubated for 1h at 37oC + 2ºC on an

orbital shaker as before. Plates are washed three times with plate washer and blot dried.

4.2.5 Substrate/chromogen and stopping solution stocks

Prepare chromogen solution as described in 3.1.10 in a volume sufficient for the number of plates

being used (10ml/plate) just before use. Prepare stopping solution as described in 3.1.11.

Addition of substrate/chromogen and stopping solution

A clean microtitreplate will be used for blanking the photometer. After washing the plates, pour

substrate/chromogen into a reagent trough. Start electronic timer. Distribute 100ul to all the wells of

the microtitre plates, including the blanking plate, with a multi-channel pipette. After incubation at

room temperature for 15 + 5 minutes, the reaction (colour development) is stopped by the addition of

50ul of stopping solution to all the wells of the plates.

4.3 Recording of results

Plates are read with the Thermo Multiskan EX at 450nm if using blue substrate buffer chromogen

(TMB option) or 492nm if using phosphate citrate buffer chromogen (OPD option). Blank the plate

reader as appropriate. Place an ELISA plate in the reader and initiate the reading sequence.

The Multiskan interfaces with a PC which receives readings from the Multiskan. The readings are

transferred to a spreadsheet which contains statistical formulae which perform calculations according

to specifications, presenting results in an acceptable format. The formulae are described in 4.4.

4.4 Calculation of test sera percentage inhibition for LPBE

The serum titre is calculated as that dilution where a 50% reduction of the colour reaction is recorded.

Antibody titres are expressed as the 50% end-point titre, i.e. the dilution at which the reaction of the

test sera results in an optical density of the reaction (antigen) control wells (Kärber method).

Titres > 1/40 should be retested using the VN test.




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Calculation of ODCa / Max OD

ODCa = [Average (A11, B11, C11, D11)] – [Average (A12, B12, C12, D12)]

(Note: A11 to D11 is applicable for screening tests, full titrations will go down to H11)

Calculation of OD’s for each sample dilution

Corrected OD = OD Pos Ag – OD Tcc

e.g. Corrected OD’s for sample 1:

corrected OD s1 1st dilution = A1 – A2

corrected OD s2 1st dilution = B1 – B2

corrected OD s3 1st dilution = C1 – C2

corrected OD s4 1st dilution = D1 – D2

Calculation of percentage inhibition for each sample dilution

Percentage inhibition = 100-[(Corrected OD sample / Corrected OD Ca ) x 100]

Average of Median OD of antigen control (Max OD)

The Percentage Inhibitions for the four dilutions of each sera (PI 1, PI2, PI3, PI4) are used to

calculate the serum titre.

NOTE

If % Inhibition < 50 Then = Negative

If % Inhibition ≥ 50 Then = Positive

Calculation of serum titres

Log titre = [((ΣPI 1+ PI 2 + PI 3 + PI 4)/100) – 05]*Log serial dilution (2) + Log starting

dilution (1)

Serum titre = Antilog of TCID 50

Acceptance of assay

The assay is accepted if the results meet the following criteria:

a. The titer of the reference sera should not be more than 2 fold of the running mean.

b. The negative control should be <50% inhibition

c. The titer of the strong positive sera should be >128.

d. The titer of the weak positive should be in the range of 45-128.

Note: Test results may still be acceptable when the positive control falls out of the range of criteria,

on the condition that all other controls pass and at the discretion of the line manager or an

experienced, competent operator.




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4.5 Interpretation of results

a. Imported/exported/post vaccination samples

Titre of a sample ≥45 Positive

Titre of a sample <45 Negative

b. suspected FMD cases

Titre of a sample ≥90 Positive

Titre of a sample <64 Negative

The titre of a sample in the range of 64-90 is inconclusive

Note: Positive samples or inconclusive results are confirmed by VNT.

Appendix 1

LAF-cab laminar air flow cabinet

FMDV foot-and-mouth disease virus

FMD antigen inactivated FMD virus used in ELISA

ELISA Enzyme linked immuno sorbent assay

SAT Southern Africa type

PBS Phosphate Buffered Saline

H2O2 Hydrogen peroxide

H2SO4 Sulphuric acid

BEI Binary ethyleneimmine

LPBE Liquid Phase Blocking ELISA

Appendix 2 diagrammatic representation of Liquid Phase Blocking Elisa (LPBE)




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IRS = immune rabbit serum IGPS = immune guinea pig serum (antibody) HRPO = horseradish peroxidase (enzyme conjugated antibody) OPD = substrate

References

Hamblin, C., Barnett, I.T.R. and Hedger, R.S. (1986) A new enzyme-linked immunosorbent assay

(ELISA) for the detection of antibodies against foot-and-mouth disease virus. I Development. J.

Immun. Meth. 93, 115-121

Hamblin, C., Barnett, I.T.R. and Hedger, R.S. (1986) A new enzyme-linked immunosorbent assay

(ELISA) for the detection of antibodies against foot-and-mouth disease virus. II Application. J. Immun.

Meth. 93, 123-129

Karber, G (1931) 50% end-point calculation. Arch. Exp. Pathol. Pharmak., 162, 480-48.

Manual of standards for diagnostic tests and vaccine. Office International des Epizooties.

http://www.oie.int/eng/normes/mmanual/2008/pdf/2.01.05_fmd.pdf

IRS

FMD Antigen

IGPS

HRP Substrate

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