Cenogenics
Febrile
AntigenDirectTest
INTENOED
USE
CENOGENICS'FEBRILEANTIGENS(bacterialagglutinationantigens)are
bacterialsuspensions
teststo detectthe
for usein eitherslideor tubeagglutination
presenceof bacterial agglutininsassociatedwith bacterial infectionor previous
exposureto a relatedorganism.
Two test proceduresars recommended,the rapid slide agglutinationtest and the
tube agglutinationtest. The rapid slide test is recommendedas a screening
procedureand should be used to establish the presence or absence of
homologousantibody.lf antiboclyis presentin the serum specimen,then the tube
procedureshouldbe used to establishantibodytiter.
PRINCIPLES
OF THE PROCEDURE
The principle of the test is an immunologic reaction between the antibodies
producedto viablebacteria(agglutinins)
febrile
and theirothervariouscounterpart
antigens.
REAGENTS
Brucellaaborlus
Brucellamelitensis
Salmonella
SomaticGroupAAntigen
Salmonella
SomaticGroupB Antigen
Salmonella
SomaticGroupC Antigen
Paratyphoid
A (SalmonellaFlagellara Antigen)
Paratyphoid
B (SalmonellaFlagellarb Antigen)
Paratyphoid
C (SalmonellaFlagellarc Antigen)
TyphoidH (SalmonellaFlagellard Antigen)
ProteusOX2
ProteusOX19
ProteusOXK
TyphoidO (Salmonella
SomaticGroupD Antigen)
PFESERVATIVES
Brucellaabortus,Brucellamelitensis,SalmonellaSomatic GroupsA, B, C, D,
ProteusOX2, OX19, and OXK are preservedwith 1.0olophenol.The flagellar
antigensincludingParatyphoidA,B, C and TyphoidH are preseruedwith 1.0ol"
formalin.
WARNING
For in vitrodiagnosticuse.
STORAGECONDITIONS
Antigensand controlserashouldbe storedin refrigerator(2'€'C)'
sTAEtLITY
Three yearsal refrigeratedtemperature.
SPECIMENS
Sera shouldbe clear and shouldnot be heated.
FEBRILE-DIRECT
PA6E1 OF 4
PROCEDURE
provided:
Materials
FEBRILE
ANTIGENS
of choice.
Materialsrequiredbutnotprovided:
plainglassringslide
Transparent
Serumpipettes
Applicator
sticks
TestTubes
NaClsolution,0.9%
Mechanical
rotator(optional)
TO ROOMTEMPERATURE
BRINGANTTGENS(S)
METHOD
A: RAPIDSLIDETEST
glassslideanddivideit into1112inchsquares
1. Obtaina cleartransparent
witha
waxpencilor a diamond
tippedpencil.A smallwindowpanecanbeusedforthis
purpose.
Theuseof ringslidesis alsorecommended.
pipette,
2. Usinga suitable
addthefollowing
amounts
of serumto betestedfrom
leftto rightto consecutive
squares
or rings:.08m1;
.04m1;
.0'lml;.005m1.
.O2ml;
Serumshouldbeclearandunheated.
Repeatthisprocedure
withpositive
and
negativecontrolsera.
3. Shakethe antigengentlyto insurea uniformsuspension.
just beloweach quantityof serum.
4. Add one dropof antigensuspension
5. Mix the serumand antigenwellusinga pieceof applicatorstick.Useseparate
applicatorsticksfor eachserumquantityor use the samestic{and proceedfrom
rightto left.Eachmixtureshouldform an area approximatelyr/2 inchby'l inch.
6. Rotateslideby hand or on a mechanicalshakerat 150 RPMfor 2-3 minutes.
7. Observefor agglutination
usingany good indirectlightagainsta dark
background.
8. A positiveserumof knowntiterand a negativeserumshouldbe includedas
controls.
RESULTS
The degreeof agglutination
is recordedas follows:
4+
- 100%of the organismsare agglutinated
3r
- 75o/"of the organismsare agglutinated
2+
of the organisrns
- 5Oo/"
are agglutinated
1+
- 25"/"of the organismsare agglutinated
+
- Lessthan 25%of the organismsare agglutinated
Negative - No agglutination
is observed
Althoughthe slidetest is not recommended
to establishtiter,the quantityof serum
giving507.agglutination
can be usedto establishthe approximate
to the
equivalent
tube testdilutionsshownbelow.
SerumVolume
.08m1
.04m1
.02m1
.01m1
.005m1
ApproximateTube Dilution
1:.20
1:40
1:80
1:160
1:320
METHODB: TESTTUBE TITRATIONTEST
1. Placeten 12x75mmtesttubesin a suitablerack.
2. Add 1.9mlof0.9%sodiumchtoridesolutionto the lirsttube.
3. Add 1.0m1
of 0.9% sodiumchloridesolutionto the remainingtubes.
4. Add 0.1mlof the serumto be testedto the firsttube.Mix weltandtransfer
1.0m1
of the dilutedserumfromthe firsttubeto the secondtube.Repeatthis
procedureuntilall ten tubescontainserialtwo-foldserumdilutionsof 1:20
through'l:'10,240.Remove1.0m1of the dilutedserumfrom tube 10 and
discard.TubeNo. 'l is considereda 1:20dilution.Repeatthisprocedurewith
positiveand negativecontrolsera.
5. Placeone tube at the end of the seriesof ditutiontubesand add 1.0mfof the
0.9%sodiumchloddesotutionusedto dilutethe sorum.Labelthistube
"salinecontrol".
FEBRILE-DIRECT
PACE2 OF 4
6. Mixth.eantigensuspension
weilby genilyshakingthe botile.Addonedrop
of antigen
to ebchtube.
7. shaketherackwellto mixtheantigenandserumand placein a waterbath.
Therecommended
timeandtemperature
of incubation'is
as foilows:
Time ol
Incubation
18 hours
18 hours
4 s ' -s 0 ' c
18hours
45' - S0' C
18hours
45'- 50'C
2 hours
45'- 50'C
2 hours
45'- 50'C
2 hours
45'- 50'C
2 hours
Brucellaabortusand Brucellamelitensis 37' C
48 hours
Proteus
OX2,OX19,& OXK
45'- 50'C
18hours
NOTE: TyphoidH and otherSalmonellaflagellarantigensshouldbe incubated
for 2 hoursat 45' - 50'C followedby 18 hoursat 2'-8'C beforefinalreading.
8. Afterincubation,
carefullyremovethe rack containingthe test tubesand
The useol an indirectlightsourceagainsta black
observefor agglutination.
backgroundwillgive optimalconditionsfor readingtube test.
9. Recordthetest resultsas follows:
4+ - All the organismsappearclumpedon the bottomof the tube and the
supemalantlluid is clear.
3+ - Approximately
75o/o
of the organismsare clumpedand the supernatant
is slightlycloudy.
2+ - Approximately
50o/o
of the organismsare clumpedand the supernatant
is moderately
cloudy.
1+ - Approximately
25a/"ol the organismsare clumpedand the supematurnt
is cloudy.
Negative- No agglutination
is observedand suspensionappearscloudy.
10. Recordtiterof reactiveserumas the last dilutionwhichgivesa 2+ reaction.
Antigen
Salmonella'O"
GroupA
"O"GroupI
Salmonella
"O'GroupC
Salmonella
Salmonella
"O"GroupD (Typhoid
O)
Salmonella'H"
a
Salmonella
"H"b
Salmonella
"H"c
Salmonella'H"
d
Temperature
45' . 50'C
45'.50'C
PRECAUTIONS
1. For greaterproficiency
in test interpretation,
alwaysincludepositiveand
negativeserumcontrols
as well as a salinecontrolin eachtest protocol.
2. All sera to be testedshouldbe clearand free from bacterialcontamination.
3. Do not heatserapriorto testing.
4. Shakeantigenvialwell beforeuse to insurea smooth,uniformsuspension.
5. Antigenshouldbe storedin refrigerator
at 2'-8'C when not in use.
6. Antigenshouldnot be frozen.
QUALITYCONTROLPROCEDURE
The use of positivecontrol sera tested in parallel with unkown test serum
specimensis recommendedlo assurethe laboratoryworker that the bacterial
antigenin use is capableof reactingwithits homologousantibody.CENOGENICS'
positive control sera have a titer of 1:80 or more with homologousantigens.
OF THE PROCEDURE
LIMITATIONS
1. Agglutininsare not alwaysproducedin bacterialinfections.
2. Cross reactionmay occurin certainpathologies.For instance,Tularemia
infectionsmay produceagglutininsto Brucellaanligens.
for severaldiseasesmay producecross reactingagglutinins.
3. Vaccinations
Typhusvaccinationmay
produceantibodies
to Proteusantigens.
NOTE
Accuratediagnosisof diseasedependson a close workingrelationshipbelween
the clinical laboratoryand the physician.A rise in antibodyliler betweenacute
phaseserumspecimensaccompanied
phase serum specimensand convalescent
by the usual signsand symptomsof a givendiseasein a patientis the best basis
for accuratediagnosis.
FEBRILE-DIRECT
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VALUES
EXPECTED
Whenviablebacleriaare introduced
intoa susceptiblehost.an immunergsponse
generallyoccutswhich is capableof prcducingantibodiescalled agglutinins.
These agglutininsare capable of reacting specillcally wilh suspenslonsof
salmonellaspeciesresponsiblefor he infection,causingthem to agglutinate.
Agglutininsare producedslowlyduringthe acutephaseof infectionandcontinue
phaseof infeclion.The titer of the concentration
to formduringthe convalescent
of antibody rises considerablybetweenacute infectionand convalescence.
Therefore,
a riseintiterbetweens€rumcollectedduringtheacutaor febrilestage
of infEctionand serum collectedduring the convaiescentstage can be bf
diagnosticsignificance.
The followingtable indicatesthe agglutinintiters
normallyexpectedduringthe courseof infectionwith severalbacterialspecies.
Peak
AnUbody
Titer usually
appoars Signiflcant
withln
Titer
Dlsease
Febrile
Antigen
used to lest
for agglutlnins
Brucellosis
Brucellaabortus
2-3weeks
3-5 weeks
1:g0or greater
Pa€lrphoid
Fever
Salmonella
Flagellara
2-3 weeks
4-5 weeks
1:80'
Paratyphoid
Fever
Salmonella
Flagellar
b
2-3 weeks
4-5 weeks
1:80'
RockyMountainProteus
OX19
SpottedFever
1-2weeks
2-3weeks
1:160
Tularemia
2 weeks
4-8 weeks
1:160
TyphoidFever Salmonella
Flagellard
fiyphoid H)
2-3weeks
4-5 weeks
1:80'
TyphoidFever Salmonella
GroupD
(TyphoidO)
1-2weeks
3-5 weeks
1:80'
Typhus
1-2 weeks
2-3 weeks
1:160
Francisella
tularensis
ProteusOX19
Antibodles
usually
appear
wlthln
'Significant
in non-vaccinated
individuals
BIBLIOGRAPHY
1. Dyer,R.E.:J.A.M.A.,
124:1165,
1944.
2. Felix,A.: Brit.Md.J., 11:597,
1942.
3. Spink,W.W.:Amer,J. Clin.path.,22:2O1,
1952.
4. Welch,H. & Stuart,
C.A.:J. Lab.Ctin.Med.,21:41.t.
1936.
CENOGENICS
CORPORATION
Morganville,N.J. 07751
Printedin U.S.A.
PG-06127-01
FEBRILE-DIRECT
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