21/09/2016

DogIgGRIDKit

CatalogNumber:646251

DogIgGRIDKit
FORTHEQUANTITATIVEDETERMINATIONOFDOGIgG
Fourantigenicallydistinctclassesofimmunoglobulins(IgA,IgG,IgE,andIgM)andfourtypesofIgGsubclasses(IgG1,IgG2a,
IgG2b,andIgG2c)havebeenidentifiedinthedog.Thedogsuffersfrombothimmunoproliferativeandimmunodeficiency
disorders.

ImmunoproliferativeDisorders
Anumberofdiseasesofthedogarecharacterizedbyabnormalproliferationofimmunoglobulinproducingcells(immunocytes)
andanexcessproductionofimmunoglobulins.Thegroupofdisordersarecalled"monoclonalgammopathies"andinclude
multiplemyelomaandmacroglobulinemia.

MonoclonalGammopathies
Theheterogeneityofnormalimmunoglobulinsresultsinpartfromheterogeneouscellpopulationproducingtheantibodiesof
differentspecificities.Ifonlyonecelloralimitednumberofcellswasselectedabnormallyasinmonoclonalgammopathies,a
greatexcessofhomogeneousimmunoglobulinwouldoccur.Theproteinthusformed,designatedamonoclonalimmunoglobulin
oran"Mcomponent",maybeofanyclass(IgG,IgM,IgA,orIgE).Theseproteinsareintactimmunoglobulinscomposedofheavy
andlightchains.Incertaininstancesfreelightchainsareproducedinexcessofheavychainsandbecauseoftheirsmallsizeare
rapidlyexcretedintheurine.ThesefreelightchainsarecalledBenceJonesproteins.
Themostcommonmonoclonalgammopathyinthedogisamalignantproliferationofplasmacellscalledmultiplemyeloma.
Numeroussystemsareaffectedtovaryingdegreesinthisdisorder.Notably,lesionstotheskeletalsystemmayrangefromnoneto
diffuseosteoporosisandpainfulcompressionfractures.Normalbonemarrowelementsareoftenreplacedinpartortotallybythe
malignantplasmacells.ThekidneymaybeaffectedbytheBenceJonesproteinemiaorbyplasmacellinfiltration.Otherorgans
includingtheliver,spleen,andlymphnodesmaybeheavilyinfiltratedbyplasmacells.Thecentralnervoussystemmaybe
affectedbytheextremebloodviscositythatoftenresultsfromthemassiveamountofproteinintheserumorbyrenalfailurefrom
kidneydamage.
MultiplemyelomaischaracterizedbyMcomponentintheserumand/orurine.TheMcomponentmaybeeitheraserum
myelomaglobulin(IgG,IgA,IgE),aBenceJonesprotein,orboth.
Treatmentincludeschemotherapywithvariouscytotoxicagents,plasmapheresistoreduceserumviscosity,andantibioticsto
preventbacterialinfection.Theincreasedsusceptibilitytonumerouspathogensresultsfromalackofnormalimmunoglobulins.
BydefinitionamonoclonalgammopathyoftheIgMclassiscalledmacroglobulinemia.Thisgammopathyislesscommonthan
multiplemyeloma.Becauseofthehighviscosityassociatedwithmacroglobulinemiathedisorderischaracterizedbysluggish
bloodflow,thromboses,centralnervoussystemdisturbances,andbleedingoftheskin,mucousmembranes,andgastrointestinal
tract.Treatmentincludesdrugstocontrolcellularproliferationandplasmapheresis.
NaturallyoccurringcasesofmultiplemyelomainvolvingIgG,IgA,andBenceJonesproteinsandmacroglobulinemiahavebeen
reportedinthedog.

PolyclonalGammopathy
IncontrasttothemonoclonalgammopathiescharacterizedbyincreasesinhomogeneousIg's,manydiseasesareassociatedwith
increasesofheterogeneousIg's.Suchdisordersaretermed"polyclonalgammopathies",whichimpliesthatmultiplecelllinesare
involvedintheIgincrease.Disorderswhicharefrequentlyassociatedwithpolyclonalgammopathiesincludeinflammatory
diseases,certainchronicinfections,autoimmunediseases,andneoplasticdiseasesofthedogandcat.

LaboratoryDiagnosisImmunoproliferativeDisorders
Thetechniquesofcelluloseacetateelectrophoresis,immunoelectrophoresis,radialimmunodiffusion(RID),bonemarrowsmear,
http://www4.mpbio.com/ecom/docs/proddata.nsf/1dc4471b047ea37e8525645d006ac0d5/d6b31e5172fcc8878525665f005cbe0f?OpenDocument

1/5

21/09/2016

DogIgGRIDKit

andthermaltestforBenceJonesproteinsinurine,arethelaboratorytestsmostfrequentlyusedtospecificallydiagnosemultiple
myeloma,macroglobulinemia(monoclonalgammopathies)orpolyclonalgammopathies.Inadditiontheroutinetechniquesto
determinetotalprotein(i.e.refractometer,Biuret)andA/Gratiosfrequentlyalertthecliniciantothepossibilityofagammopathy.
RIDisthemethodofchoicetocharacterizeandquantitatespecificimmunoglobulinselevatedintheimmunoproliferative
disordersdescribed.

ImmunodeficiencyDisorders
Immunodeficiencydisordersreflectanimpairmentinoneormoreofthemajorcomponentsofimmunity,including:Nonspecific
Immunity:1)phagocyticcells(i.e.,PMNandmacrophages,2)effectorsubstances(i.e.complement)and/orSpecificImmunity:1)
humoral(antibody),Bcellsystem,2)cellmediatedimmunity(CMI),Tcellsystem.Immunodeficienciesareclassifiedasprimary
orsecondarydeficiencies.Theprimarydeficiencydiseasesaregeneticallydefinedinbornerrorsofthebodilydefencemechanisms
andcanaffectnonspecificaswellasspecificcomponentsoftheimmuneresponse.Primarydeficienciesareveryuncommonin
thedog,butdooccuroccasionally.
Secondaryimmunodeficienciesareacquiredasaconsequenceofnumerousconditionsincluding:infectiousdisease,neoplasia,
aging,drugtherapy,failuretoreceivecolostrumandcertainnutritionaldeficiencies.Secondaryimmunodeficienciesaremore
commonthanprimarydeficiencies.Manyinvestigatorshavereportedsecondaryimmunodeficienciesorimmunosuppression
associatedwithcaninedistemperinfections,caninegeneralizeddemodecticmange,nutritionaldeficiencies,feline
panleukopenia,felineleukemia,ageandperhapsnumerousothersasyetunrecognizedconditions.
Cliniciansshouldconsiderimmunodeficiencyorimmunosuppressionwhenananimalispresentedwithahistoryofchronic
infection,autoimmunedisease,orneoplasticdisease.
Deficienciesofthehumoralimmunesystemwouldmostoftenbecharacterizedbyinfectionswithextracellularpyogenic
pathogens(i.e.streptococci).SelectiveIgAdeficienciesmaybecharacterizedbychronicrespiratoryorgastrointestinalinfections.
Thepresenceofmicrobesnormallynotpathogenicfortheanimalisoftenacluetoimmunodeficiencydisorders(i.e.,
Pneumocystiscariniiorstreptococciisolatedfromthelung,orGiardiainthegut).Deficienciesofcellmediatedimmunityare
associatedwithinfectionscausedbyfacultativeintracellularpyogenicpathogens(i.e.,acidfastorganisms,brucella,salmonella,
andentericpathogens,certainviruses,andfungi).

LaboratoryDiagnosisImmunodeficiencyDisorders
SpecificdeficienciesofthecanineimmunoglobulinIgG,IgM,orIgAmaybestbedeterminedbyradialimmunodiffusion.

NormalImmunoglobulinlevelsintheDog
Whileextensivestudiesonthenormalimmunoglobulinlevelsofthedoghavenotbeenconducted,theaccuracyofavailable
publisheddatahasbeenverifiedbytestingperformedinourlaboratoryandmajorveterinarylaboratoriesthroughoutthecountry.
Animmunoglobulindeficiencycouldbeconsideredifadog(olderthansixmonths)hasserumimmunoglobulinvaluesbelowthe
following(10):
IgG<700mg/100ml
IgM<75mg/100ml
IgA<40mg/100ml
PublishedNormalValues

PureBredSerum*
Mongrelserum
MongrelColostrum
*(FoxHoundsandCollies)

AdultMongrelSerum

TotalIgG
925
1445
1453

ReynoldsandJohnson(5)
MeanValues(mg/100ml)
IgG1
IgG2a+2b
300
512
562
771
694
670

HeddleandRowley(8)
MeanValues(mg/100ml)
TotalIgG
IgM
980
170

IgG2c
113
112
89

IgM
156
145
217

IgA
83
79
313

IgA
50

http://www4.mpbio.com/ecom/docs/proddata.nsf/1dc4471b047ea37e8525645d006ac0d5/d6b31e5172fcc8878525665f005cbe0f?OpenDocument

2/5

21/09/2016

DogIgGRIDKit

ParotidSaliva

AdultDogSerum
(MixedPureBreedand
Mongrels)

1.5

3.3

Schultz(10)
NormalRanges(mg/100ml)
TotalIgG
IgM
1500+500
150+50

52

IgA
100+60

AbouttheDogIgARIDKit
TheDogIgGKitprovidesarapid,quantitativeimmunochemicalresearchtoolforthedeterminationofcaninetotalIgGbasedon
theprincipleofradialimmunodiffusion.Eachplate,preparedbyincorporatingheavychainspecificantiIgGseruminbuffered
agarose,permitsthemeasurementofseventeen(17)sampleswithanaccuracyof+10%withinarangeof2502400milligrams
IgGper100milliliter.CanineIgGusedforimmunizationisisolatedfromserumbyammoniumsulfateprecipitationandcurrent
chromatographictechniques.Four(4)DogIgGreferencestandardsareprovidedinthisrange.Three(3)plates,enoughforfifty
one(51)determinationsareprovidedwitheachkit,alongwiththenecessaryaccessoriesforeachdetermination.

KitContents
3Immunodiffusionplatescontainingmonospecificantiserainbufferedagarose.
4ReferenceStandards(with0.02%Thimerosal).
1Instructionsheet.

PrinciplesofRadialImmunodiffusion
SingleradialimmunodiffusionmethodsarederivedprimarilyfromtheworksofFaheyandMcKelveyandMancini,et.al.These
methodsarespecificforthequantitativedeterminationofindividualproteingroupsinbiologicalfluids.Mancinidescribeda
linearrelationshipbetweenantigenconcentrationforequalvolumesoftestfluidsandthesquaresoftheircorrespondingring
diametersaftercompletediffusion.FaheyandMcKelveydiscoveredthatringdiameterscouldbemeasuredwellbeforeendpoint.
Thelogoftheproteinconcentrationwasfoundtobeproportionaltotheprecipitantringdiameterofequalvolumes.This
relationshipislinearwhenplottedonsemilogchartpaper.Themethodistimeandtemperaturedependentsincethediameterof
theprecipitantringwillcontinuetoincreaseuntilendpoint.
TheMPDogIgGKitisintendedprimarilyfortheFaheyandMcKelveytechniquetobereadafter1822hours.

SampleCollectionandPreparation
Thebloodsampleshouldbecollectedbyapprovedveterinarytechniques.Bloodshouldbeallowedtoclotandtheserum
collectedpromptlytoavoidhemolysis.Freshlycollectedserumshouldbestoredat+4 o Candtestedwithin24hours.Ifnotused
within24hourstheserumshouldbefrozenuntilreadyforassay.Repeatedfreezingandthawingoftheserumsampleistobe
avoided.

Storage
Storeat+4 o C.Platesshouldbestoredinaninvertedpositiontoinhibitmoisturecollectioninthewells.DoNotFreeze.Ifsome
wellsofindividualplatesareunusedafteropening,thecovermaybereplacedandtheplate(s)storedinvertedintheziplock
plasticbagforupto2weeks(providedprecautionsaretakenthatbacterialcontaminationdoesnottakeplaceduringuse).Shelf
lifeofunopenedplatesisoneyear.

Precaution
Forveterinarydiagnosticandresearchuse.Followstorageinstructions.
http://www4.mpbio.com/ecom/docs/proddata.nsf/1dc4471b047ea37e8525645d006ac0d5/d6b31e5172fcc8878525665f005cbe0f?OpenDocument

3/5

21/09/2016

DogIgGRIDKit

SpecificPerformanceCharacteristics
EachlotofDogIgGQuantitativeImmunodiffusionKitsmeetsourQualityAssurancespecificationsforspecificitytoDogIgGand
accuracywithintheacceptedlimitsoutlinedabove.

LimitationsoftheProcedure
Accurateresultsarelimitedbytheassayrangeofthekitwhichis2502400milligramsIgGper100mlsample.Forringdiameters
greaterthanthatofthehighestreferencestandard,dilutethesampleandrerun.Ifthediameteroftheringissmallerthanthatof
thelowestreferencestandard,concentratethesample(AmiconMiniconB125orequivalent)andrepeattheassay.

Procedure
1.Preparationofaninternalstandardcurve:Accuratelypipette5.0ulofeachreferencestandardintothewellsofRowBonthe
immunodiffusionplateasindicatedontheRecordSheetprovided.
2.AdditionofSample:Accuratelypipette5.0ulofthesampleintotheremainingwells.Atotalofseventeen(17)samplesmaybe
determinedononeplate.
3.Incubation:Pipetteafewdropsofwaterintothemoisturetroughborderingtheplate,replacethecoverandleaveundisturbedat
roomtemperatureovernight(1822hours).
4.Measurement:Precipitinringscanbedirectlymeasuredfromtheimmunodiffusionplateeachrulingontheplateisone
millimeter.ReadentirediameterofeachringandrecordonRecordSheet.
5.ConstructionofStandardCurve:Plottheringdiametersofthestandards(xaxis)againsttheknownconcentrationsofthe
standards(yaxis)onthesemilogarithmicgraphpaperprovided.
6.Interpretation:
Determinetheconcentrationofthesamplesbyreadingagainstthestandardcurve(step5)andrecordonthe
RecordSheet.ThevalueobtainedisequivalenttothemilligramsIgGper100mlsample.
Ifthediameterofthesampleislargerthanthediameterof2400mg/100mlreferencestandard,dilutethe
samplewith0.9%salinesolutionandrepeattheassay.
Ifthediameterofthesampleissmallerthanthediameterof250mg/100mlreferencestandard,concentrate
thesamplewithconcentrator(AmiconMiniconB125orequivalent)andrepeattheassay.
Theresultisthencorrectedbymultiplyingbytheconcentrationfactortoobtaintheactualvalue.

References
1.Fahey,J.L.,McKelvey,E.M.,"QuantitativeDeterminationofSerumImmunoglobulinsinAntibodyAgarPlates."J.
Immunology,v.94,84(1965).
2.Mancini,G.,Carbonara,A.O.,andHelremans,J.F.,"ImmunochemicalQuantitationofAntigensbySingleRadial
Immunodiffusion."Immunochemistry,v.2,235254(1965).
3.Johnson,J.S.,Vaughan,J.H.,andSwisher,S.N.,"CanineImmunoglobulins11:AntibodyActivitiesinSixImmunoglobulin
Classes."J.Immunology,v.98,935940(1967).
4.Patterson,R.,Roberts,M.,andPruzcnsky,J.J.,"TypesofCanineSerumImmunoglobulins."J.Immunology,v.101,687694
(1968).
5.Reynolds,H.Y.,andJohnson,J.S.,"QuantitationofCanineImmunoglobulins."J.Immunology,v.105,698703(1970).
6.Schultz,R.D.,"LaboratoryDiagnosisofImmunologicDisordersintheDogandCat."CornellVet.,v.68,235254(1978).
7.Schultz,R.D.,Bet.ClinicsofNorthAmerica,v.4,153(1974).
8.Heddle,R.J.andRowley,D.,"ImmunochemicalCharacterizationofDogSerumParotidSaliva,Colostrum,MilkandSamll
BowerFluid."Immunology,v.29,185(1975).
9.Berne,B.H.,"DifferingMethodologyandEquationsUsedinQuantitatingImmunoglobulinsbyRadialImmunodiffusion
Acomparativeevaluationofreportedandcommercialtechniques."ClinicalChemistry,v.20,6169(1974).
10.Schultz,R.D.,Vet.ClinicsofNorthAmerica,v.8,721753(1978).

KitForDeterminationofDogIgG
http://www4.mpbio.com/ecom/docs/proddata.nsf/1dc4471b047ea37e8525645d006ac0d5/d6b31e5172fcc8878525665f005cbe0f?OpenDocument

4/5

21/09/2016

DogIgGRIDKit

Date:
TestPerformedBy:
WellNo.

SampleCode
Diameter(mm)
Concentration(mg/100ml)
Diameter(mm)
Concentration(mg/100ml)

2400

1200

500

250

SampleCode
Diameter(mm)
Concentration(mg/100ml)
Date:
TestPerformedBy:
WellNo.

SampleCode
Diameter(mm)
Concentration(mg/100ml)
Diameter(mm)
Concentration(mg/100ml)

2400

1200

500

250

SampleCode
Diameter(mm)
Concentration(mg/100ml)
Date:
TestPerformedBy:
WellNo.

SampleCode
Diameter(mm)
Concentration(mg/100ml)
Diameter(mm)
Concentration(mg/100ml)

2400

1200

500

250

SampleCode
Diameter(mm)
Concentration(mg/100ml)
Allplotsshouldbeperformedonsemilogarithmicgraphpaper

http://www4.mpbio.com/ecom/docs/proddata.nsf/1dc4471b047ea37e8525645d006ac0d5/d6b31e5172fcc8878525665f005cbe0f?OpenDocument

5/5