Technical Brief Final - WordPress.com · 2017. 11. 30. · Collected milk samples must be...

Zoonoses&EmergingLivestockSystems

Using ELISA for the detection of

Brucella antibodies in milk

TECHNICAL BRIEF

UsingELISAfordetectionofBrucellaantibodiesinmilk:technicalbrief

ZELS-BRUCELLOSISPROJECTINWESTANDCENTRALAFRICA

Zoonoses&EmergingLivestockSystems(ZELS)isajointresearchinitiativebetweentheDepartment

forInternationalDevelopment(DFID)and:

• BiotechnologyandBiologicalSciencesResearchCouncil(BBSRC)

• EconomicandSocialSciencesResearchCouncil(ESRC)

• MedicalResearchCouncil(MRC)

• NaturalEnvironmentResearchCouncil(NERC)

• DefenceScienceandTechnologyLaboratory(DSTL)

TheZELSbrucellosisresearchproject inWestandCentralAfricafocusesondairyfarmsinthemain

peri-urbandairyproductionareasofWestandCentralAfricancountries,whicharemembersofthe

InterstateSchoolofVeterinaryScienceandMedicineofDakar(EISMV).

Oneoftheprojectobjectivesistoobtainestimatesoftheprevalenceofbrucellosisinperi-urbandairy

systemsinWestandCentralAfrica.Theseestimatesareneededtodesigncontrolprogramsthatare

appropriatefortheexistingbaselinelevelofinfection.Inpartnershipwithdiagnosticlaboratoriesfrom

differentcountries,cross-sectionalstudiesofdairyherdsareconductedusinganindirectELISAforthe

detectionofantibodiesinmilkprovidedbytheOIEReferenceLaboratoryforBrucellosisattheUK’s

Animal and Plant Health Agency (APHA). The high sensitivity of the assay allows identification of

infectedunvaccinatedherdsbytestingofbulkmilksamples,makingthemilkELISAausefultoolfor

brucellosis surveillance in disease-free countries such as the UK or as part of baseline surveys to

generateinitialprevalenceestimatesinareaswheretheyarelacking,suchasmanyWestandCentral

Africadairyproductionzones.

Inthistechnicalbrief,wesummarizelessonslearnedfromtheuseoftheindirectELISAassayaspart

ofcross-sectionalstudiesofbrucellosisindairyfarmscarriedoutacross10dairyproductionzonesof

7WestandCentralAfricancountries(Figure1).Wehighlighttheproblemsencounteredandhowto

avoidandovercometheminordertofacilitatetheadoptionofthistechniqueasadiagnostictoolin

otherlowandmiddle-incomecountries,wherethefacilitiesarenotoptimal.


Figure1.LaboratoriesfromsixWestandCentralAfricancountriesinvolvedinbulkmilksampletestingbyindirect

ELISAaspartoftheZELS-brucellosisprojectinWestandCentralAfrica.


WHATISELISA?

The Enzyme-linked immunosorbent assay (ELISA) is a test used to detect and quantify specific

antibodiesinhumansandanimals.Thetestconsistsofcapturingantigensorantibodiespresentina

sample using an enzyme-labelled immunoglobulin, which drives a colour change when a specific

chromogenic substrate is added2. This change is detected andquantified bymeasuring the optical

density.DifferenttypesofELISAhavebeendevelopedfordifferentpurposes,amongthemtheindirect

ELISA.InthistypeofELISA,thecorrespondingantigensarecoatedonthesurfaceoftheplateandused

tocapturethespecificantibodiespresentinthesample.Todetecttheboundantibodies,secondary

antibodiesthatareconjugatedtoanenzymesuchasperoxidaseoralkalinephosphataseareadded.

After an incubation period, the unbound secondary antibodies are washed off. When a suitable

substrateisadded,theenzymereactswithittoproduceacolour.Thiscolourproducedismeasurable

asafunctionorquantityofantibodiespresentinthegivensample(Figure2).

Figure2.IllustrationofthedifferentstepsoftheindirectELISAmethod3.

Incubateandwash Incubateandwash Incubate

SERASAMPLES CONJUGATE SUBSTRATE

READING


INDIRECTELISA

Indirect ELISA has been developed for the

detectionofantibodiesagainstBrucellaspp.andis

recommended by theOIE for screening of cattle

againstBrucellaspp.infection.IntheUK,quarterly

bulkmilktestingofdairyherdsbyindirectELISAis

oneof thekeyelementsof thecurrentlyapplied

brucellosis surveillance program. This technique

has thepotential tobeusedas an initial step to

ascertain the status of unvaccinated dairy herds

with regard to brucellosis and can be used to

produce baseline prevalence estimates in areas

where this information is lacking. It is with this

objectivethattheassayhasbeenused,sofar, in

10dairyproductionareasofBurkinaFaso,Burundi,

Cameroon,Mali,NigerandTogo.

INDIRECTELISAPROTOCOL

Toperformtheassay,laboratoriesshouldallbeprovidedwith:pre-coatedBrucellaabortusS99smooth

lipopolysaccharideantigenplateswithalid;conjugatestoredat-20°C;chromogensubstrate;diluting

buffer;stoppingsolution;andwashsolution.Additionally,thefollowinglaboratoryequipmentisalso

required:microtitreplatereaderwith405nmfilter;singleandmultichannelpipettesalongwithtips

andreagenttroughs;containers,bottles,tubesandbeakersfordistilled/deionisedwater,seraand/or

reagentstorage;refrigeratorsandfreezers;rotaryandmicrotitreplateshaker;andabsorbenttowels.

Collectedmilksamplesmustbeimmediatelyplacedinice,transportedtothelabassoonaspossible

andkeptinthefridgeuntiltesting.Curdledsamplesmustnotbeusedbecausethiswillaffectthetest

result.Onthedayofthetest,milksamplesshouldbecentrifugedat10,000xgfor2minat4°Candthe

cream layer shouldbe removed. It ishighly recommended todivide the supernatant intodifferent

aliquots and storedat -80°C.However, repeated thawingand freezing shouldbe avoidedas itwill

compromise the sample integrity and thus, the accuracy of the test result. Finally, It is also highly

recommendedtotesteachsampleinduplicateinordertoobtainreliableresults.


ThegeneralprotocoloftheindirectELISAassayincludesthefollowingmainsteps4: 

1.Preparetheplatebyadding50μlofdilutingbuffertoallwells. 

2. Add50μl of the samples and controls (positive,mediumandnegative) to thedesignatedwells

(Figure3). 

3. Incubateat roomtemperature for30minutesona rotaryshaker (orat37°C for1hourwithout

shaking). 

4.Washwithwashsolutionanddrybytappingwithabsorbentpapertowel. 

5.Add100μlofconjugatesolutionandincubatetheplateasmentionedisstep3. 

6.Washlikeinstep4. 

7.Add100μlofwellmixedsubstratesolutiontoallwellsandincubateatroomtemperaturefor10to

15minutes.

8.Add100μlofstoppingsolutionandreadtheplateat405nm(blankedattheblankwell).

Figure3.Designatedcontrolwells;positivecontrolsshouldbeplacedin wells A12 and B12 (red), intermediate controls in column 11(yellow),negativecontrolsonwellsC12,D12andE12,andnosample(blanks)shouldbeaddedinwellsF12,G12andH12.

Plateacceptancerequiresthecompliancetocertaincriteria,whichinclude:

a. Bindingratio(meanof8positivecontrolwells/meanof3negativecontrolwells)³10

b. Opticaldensity(OD)oftheblankwellandthemeanofthenegativeODs<0.100

c. MeanODofthe8positivecontrolwells>0.7000(optimal=1.000)

d. MeanODof3mediumcontrolwells>10%or


Figure4.Exampleofanacceptableplatewheresamplesdisplaydifferentreactionsand

the controls display as expected; strong colour in positive wells (A12 and B12),

intermediate colour in the medium controls (column 11) and clear negative wells in

negativewells(C12,D12,E12)andblankwells(F12,G12,H12).


ISSUESIDENTIFIEDDURINGPRACTICALUSEOFTHETEST

Despite its apparent simplicity and the precautions taken in the shipment and storage of assay

reagents,whichweredirectlyprovidedbythelaboratoryinchargeoftheirproduction,inspectionof

initialtestresultsrevealedanumberofissues.Wesummarizebelowtheproblemsencounteredand

makerecommendationstodetectandavoidtheseissues,whichmaybeusefulforotherlaboratories

interestedinmakinguseofthisorsimilarassays,inparticularinlow-resourcesettings:

1.ProblemswiththepHofthedilutingbuffer

Somesitesreporteddiscolourationoftheplates,which

can be explained by themisuse of the diluting buffer

wherethepHiseithertoohighortoolow.WhenthepH

ofthedilutingbufferis>8.2,phenolredindicatorwill

turnbrightpink(Figure5.A).Ontheotherhand,when

thepHofthedilutingbufferisbelow7.2,thecolourof

phenol red indicator turns yellow (Figure 4.B). If this

occursthebuffershouldbediscarded.Inordertoavoid

this problem, the diluting buffer should be prepared

following themanufacturer’s instructions and the pH

shouldbeadjustedbetween7.2and7.6.Thisissuewas

resolvedinthestudysiteswhenthetestwasrepeated

ensuring the pH of the diluting bufferwaswithin the

recommendedrange.

2.Problemswiththecontrols

Occasionally,controlwellsdidnotdeveloptheexpected

results and thus, compromised plate acceptance (see

aboveforplateacceptancecriteria).Infigure6thereis

aclearvisiblelackofcolourinthepositivecontrolwells

markedbyredarrows(A12andB12).Thiscouldbedue

to;(a)usingthewrongconcentrationofsomereagents,

(b)notdispensingthepositivecontrolinthedesignated

wellsor(c)pipettingerrors.

A.

Figure 5. Invalid plate due to thedilutingbufferbeing(A)>8.2,wherebyphenol red indicator turns bright pinkor (B) < 7.2, whereby phenol redindicatorturnsyellow.

B.

Figure6. ELISAplateswithmediumcontrolwellsnotdevelopingcolour.


3.ProblemswithreadingtheELISAplate

Inoneinstance,therewasanODreadingthatwascompletelydifferenttotheexpectedrangevalues

duetotheuseofaninappropriatefilter.Aftercarryingoutalltestprocedures,theELISAplateshould

bereadbyusingamicrotitreplatereaderwith405nmfilter.Usingafilterwithadifferentwavelength

resultsinincorrectODvaluesandwillcausemisinterpretationoftheresults.

SUMMARY

IndirectELISAinmilkisasimpleandpracticaltoolfortheserologicaldiagnosisofbrucellosisatherd

level. Validation studies carried out to assess its potential use as part of brucellosis surveillance

programsindisease-freesettingssuggesttheassayhasahighsensitivityallowingthedetectionofvery

lowproportionofseropositiveanimalsinthemilkingherd.Despiteitsapparentsimplicity,theuseof

thetest,inparticularinlaboratorieswithlimitedresourcesorwhereELISAtestingisnotcarriedout

routinelyposessomechallenges.Adherencetotheinstructionsiscriticalandcautionshouldbetaken

atallstages–fromsamplecollectionandstoragetointerpretationoftestresults.Mostimportantly,

samplesmustnotcurdle;thepHofthebuffermustbeadjusted;theinclusionofcontrolsismandatory;

opticaldensityreadingsmustbecarriedoutusingtheappropriatefilterandoverallvalidityofthetest

requiresthattheresultsofcontrolarewithintheexpectedranges.

Wehopethatthistechnicalbriefmaybeofassistancetolaboratorytechniciansinlowandmiddle-

incomecountrieswhoareinterestedinusingtheindirectELISAfortestingbulkmilksamples.


REFERENCES

1Available:https://www.zelsbrucellosis.wordpress.com. 

2WrightPF,etal.Standardisationandvalidationofenzyme-linkedimmunosorbentassay

techniquesforthedetectionofantibodyininfectiousdiseasediagnosis.RevSciTech1993;

435-450. 

3AdaptedfromSánchez-Vizcaínoetal.,2005.Available:

http://www.sanidadanimal.info/cursos/asf/caps/cap1.html.

4OIE.2017.Brucellosis(Brucellaabortus,B.melitensisandB.suis)(InfectionwithB.abortus,

B.melitensisandB.suis).In:ManualsofDiagnosticTestsandVaccinesforTerrestrialAnimals

2017(Chapter2.1.4).Available:

http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.01.04_BRUCELLOSIS.pdf.

FURTHERINFORMATION

Formoreinformationpleasecontact:

ImadiddenMusallam,RoyalVeterinaryCollege(UniversityofLondon),UK

[email protected]

JohnMcGiven,OIEReferenceLaboratoryforBrucellosis(Animal&PlantHealthAgency),UK

[email protected]

AyayiJustinAkakpoEcoleInter-EtatsdesSciencesetMédecineVétérinairesdeDakar(EISMV),

[email protected]

FormoreinformationontheZELSBrucellosisprojectinWestandCentralAfricapleasevisit:

https://zelsbrucellosis.wordpress.com/

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