DRG

Astrovirus Ag ELISA (EIA-4456)

RUO in the USA
Revised 1 De!" #1 !! ($ers" %"#)

Please use only the valid version of the package insert provided with the kit.
1 I&'RODU('IO&
Astrovirus was firstly described in 1975 and named according to its star-shaped structure visible under the electron
microscope.
Astrovirus belongs to the family Astroviridae. Human Astroviruses are subdivided into 7 serotypes (1.
!ogether with "otavirus and Adenovirus Astrovirus is one of the most common causes of non-bacterial gastroenteritis in
children under 5 years of age all over the world. !hus #$% of children between 5 and 1$ years of age are anti-Astrovirus-
antibody positive. Astrovirus caused gastroenteritis in adults and nosocomial infections are observed as well (&.
!he course of the disease is usually self-limiting and of short duration. After the incubation time of 1-& days a 1-' days
lasting gastroenteritis develops accompanied by vomiting( diarrhea( fever and abdominal pain finally causing dehydration.
Although occurring all over the year Astrovirus infections are mainly observed during the winter months ()('.
Astrovirus infections are spread via faecal-oral transmission from person to person or via contaminated things or food.
*nfected persons e+crete high amounts of Astrovirus particles with their faeces (1(&.
!he detection of Astrovirus may be performed by electron microscopy or by molecular biology techni,ues such as
polymerase chain reaction (-.". /eanwhile immunological methods li0e en1yme immunoassay have established as
preferential methods for routine laboratory diagnosis since these methods are fast( economical and automation is possible
I&'E&DED USE
Astrovirus Ag 23*4A is an in-vitro-diagnostic device for direct detection of Astrovirus in faecal samples.
) *RI&(I*LE O+ ',E 'ES'
Astrovirus A5 23*4A is a one-stepen1yme immunoassay on the basis of polyclonal and monoclonal antibodies against
Astrovirus antigens.
6iluted stool specimens and horseradish pero+idase (H"- labelled monoclonal anti-Astrovirus-antibodies are dispensed
simultaneously into the wells of a microtitration plate coated with polyclonal anti-Astrovirus-antibodies.
After an incubation time of 7$ min at room temperature ("! unbound components are removed by a washing step.
H"- converts the subse,uently added colorless substrate solution of )()8(5(58-!etramethylben1idine (!/9 within a 1$
min reaction time at room temperature protected from light into a blue product. !he en1yme reaction is terminated by
sulphuric acid dispensed into the wells turning the solution from blue to yellow.
!he optical density (:6 of the solution read at '5$;< 7&$ nm is directly proportional to the specifically bound amount of
Astrovirus.
DRG Intern-tion-./ In!"
USA +-01 (2%)) 564-%556 e-3-i.1 !or45drg-intern-tion-."!o3 1
DRG

Astrovirus Ag ELISA (EIA-4456)

RUO in the USA
Revised 1 De!" #1 !! ($ers" %"#)

4 *RE*ARA'IO& A&D S'ORAGE O+ SA6*LES
4"1 (o..e!tion -nd stor-ge
4tool samples should be stored at & =. > # =. immediately after collection and processed within 7& hours.
3onger storage is possible at -&$=.. "epeated free1ing and thawing of samples should be avoided.
4tool samples already diluted with the sample diluent ?6*3@ can be stored for up to 7& h at & =. - # =. before testing in
the 23*4A.
4" *re4-r-tion
Auic0ly thaw fro1en samples. Barm samples to room temperature and mi+ well.
!he Astrovirus 23*4A (2*A-''57 can be performed with 1C7 or 1C11 diluted specimens. *n case of additional testing of
the same sample in the .lostridium difficile !o+in AD9 23*4A (2*A-'''# the 1C7 dilution is recommended.
*re4-r-tion o7 - 1111 s-34.e di.ution1
-ipette 1### 8L of sample diluent into a clean tube.
Esing a disposable stirring rod transfer about 1## 3g (diameter about &-) mm of faeces if solid or pipette 1## 8L if
li,uid into the tube and suspend thoroughly.
*f necessary( sediment floating particles by a centrifugation step with a micro centrifuge for one min. at ma+imum speed.
*re4-r-tion o7 - 116 s-34.e di.ution1
-ipette 1### 8L of sample diluent into a clean tube.
Esing a disposable stirring rod transfer about ## 3g (diameter about '-7 mm of faeces if solid or pipette ## 8L if
li,uid into the tube and suspend thoroughly.
*f necessary( sediment floating particles by a centrifugation step with a micro centrifuge for one min at ma+imum speed.
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DRG

Astrovirus Ag ELISA (EIA-4456)

RUO in the USA
Revised 1 De!" #1 !! ($ers" %"#)

5 'ES' (O6*O&E&'S +OR 26 9ELLS
1. 9ELLS
6i:rotitr-tion 4.-te/ 1 sing.e ;re-:-;.e
<-=e.. stri4s (in -.. 26 =e..s) !o-ted =ith
4o.>!.on-. -nti-Astrovirus--nti;odies
(r-;;it)
1
v-:uu3 se-.ed
=ith desi!!-nt
2. 9AS,?U+
(O&( 1#@
9-sh ;u77er( 1#-7o.d
for 1$$$ m3 solution
1$$ m3
concentrate
white cap
3. DIL S-34.e di.uent
1$$ m3
ready to use
coloured yellow
blac0 cap
4. (O&'ROL A
*ositive !ontro.
Astrovirus 23*4A reactive sample
1.5 m3
ready to use
red cap
5. (O&'ROL B
&eg-tive !ontro.
Astrovirus negative sample
1.5 m3
ready to use
coloured blue
green cap
6. (O&C ,R*
,R*-!onDug-teE
H"--labelled( monoclonal anti-Astrovirus-
antibodies (mouse
1& m3
ready to use
brown cap
7. SU?S'R '6?
Su;str-te
)()8(5(58-!etramethylben1idine and hydrogen
pero+ide
15 m3
ready to use
blue cap
8. S'O*
Sto4 so.ution
$.&5 / 4ulphuric acid
15 m3
ready to use
yellow cap
6 6A'ERIALS REFUIRED ?U' &O' *RO$IDED
micropipettes
multi-channel pipette or multi-pipette
reagent container for multi-channel pipette
#-channel wash comb with vacuum pump and waste bottle or microplate washer
microplate reader with optical filters of '5$ nm for measurement and < 7&$ nm for reference
distilled or de-ioni1ed water
glassware
tubes (& m3 for sample preparation
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DRG

Astrovirus Ag ELISA (EIA-4456)

RUO in the USA
Revised 1 De!" #1 !! ($ers" %"#)

% *RE*ARA'IO& A&D S'ORAGE O+ REAGE&'S
%"1 Git siHe -nd e04ir>
:ne 0it is designed for 97 determinations.
!he e+piry date of each component is reported on its respective label( of the complete 0it on the outer bo+ label.
Epon receipt( all test components have to be 0ept at & =. - # =.( preferably in the original 0it bo+.
After opening all 0it components are stable for at least & months( provided proper storage.
!his ready to use wash buffer solution is stable for at least )$ days when stored at & =. - # =..
%" Re-gent 4re4-r-tion
Allow all components to reach room temperature prior to use in the assay.
!he microtitration plate is vacuum-sealed in a foil with desiccant. !he plate consists of a frame and strips with brea0able
wells. Allow the sealed plate to reach room temperature before opening. Enused wells should be stored refrigerated and
protected from moisture in the original cover carefully resealed.
-repare a sufficient amount of wash solution by diluting the 1$fold concentrated wash buffer 1 D 9 with distilled or de-
ioni1ed water.
For 2+ampleC 1$ m3 Wash Buffer concentrate D 9$ m3 distilled water.
< ASSAI *RO(EDURE
6ilute samples with Sample Diluent () 1111 or 116/ e.g.
1$$ mg or 1$$ G3 stool D 1.$ m3 (1C11 sample diluent () or
&$$ mg or &$$ G3 stool D 1.$ m3 (1C7 sample diluent ()
Avoid any time shift during dispensing of reagents and samples.
/a0e sure that the soa0 time of the wash buffer in the wells is at least 5 seconds per wash cycle and that the remaining
fluid is completely drained in every single wash cycleH
Avoid light e+posure of the !/9 substrate solutionH
<"1 9or:ing ste4s
1. Barm all reagents to room temperature ("! before use. /i+ gently without causing foam.
&. 6ispense
dro4s (or %5 8L) CONJ HRP (6 and
). *i4ette %5 8L CON!RO" # -ositive control ($
CON!RO" % Iegative control (&
5# 8L diluted sample( mi+ gently
'. .over plate and incubate for 6# 3in at "!.
5. 6ecant( then wash each well 50 with )## 8L wash solution (diluted from (' and tap dry onto absorbent paper.
7. 6ispense
dro4s (or %5 8L) ()*(!R !+* (,.
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DRG

Astrovirus Ag ELISA (EIA-4456)

RUO in the USA
Revised 1 De!" #1 !! ($ers" %"#)

7. *ncubate for 1# 3in at "! protected from light.
#. 6ispense
dro4s (or %5 8L) (!OP (-( mi+ gently.
9. "ead :6 at 45# n3 (reference filter < 7&$ nm with a microplate reader within )$ min after reaction 4top
2 RESUL' I&'ER*RE'A'IO&
Fu-.it-tive ev-.u-tion
(ut-o77 deter3in-tion1 OD neg-tive !ontro. A #"1#
4amples with :6 values e,ual with or higher than the cut-off are considered 4ositive.
4amples with :6 values below the cut-off are considered neg-tive for Astrovirus antigen.
1# RE+ERE&(E $ALUES
Astrovirus Antigen
&eg-tive J .ut-off
*ositive < .ut-off
*t is recommended that each laboratory establishes its own normal and pathological reference ranges as usually done for
other diagnostic parameters too. !herefore( the above mentioned reference values provide a guide only to values which
might be e+pected.
1#"1 'est v-.idit>
!he test run is valid ifC
the mean :6 of the negative control is K $.15 (manual performance
K $.)$ (automatic performance using 23*4A processor
the mean :6 of the positive control is < 1.$$
*f the above mentioned ,uality criteria are not met( repeat the test and ma0e sure that the test procedure is followed
correctly (incubation times and temperatures( sample and wash buffer dilution( wash steps etc.. *n case of repeated failure
of the ,uality criteria contact your supplier.
1#" Li3it-tions o7 the 4ro!edure
!here is no correlation between measured absorbance and seriousness of the infection.
*t is also not allowed to correlate absorbances of the samples with that of the positive control.
.ross contamination of reagents and samples can produce false positive results. *ncorrect dilutions( not sufficiently
homogeni1ed samples or solid particles after centrifugation of the suspension can cause false negative as well as false
positive results.
A negative test result not necessarily e+cludes an Astrovirus infection.
*nhomogeneous virus distribution in the sample can cause false negative results.
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DRG

Astrovirus Ag ELISA (EIA-4456)

RUO in the USA
Revised 1 De!" #1 !! ($ers" %"#)

!he investigation of samples that were ta0en beyond the acute phase of the disease can cause false negative results(
because the number of virus particles has decreased under the detection limit of the test. *t is therefore recommended to
ta0e samples within the acute phase of the disease where a ma+imum number of e+creted virus particles are to be
e+pected.
A final interpretation of the test results should consider clinical findings as well.
11 AU'O6A'I( *RO(ESSI&G
-erforming the Astrovirus Ag 23*4A on fully automated microplate processors (e.g. 64&( 64L or -ersonal3ab may
cause elevated absorbances in comparison to the manual procedure due to individual differences concerning wash
procedures and general technical specifications of the e,uipment. *n these cases a ma+imum value of $.) absorbance units
is permissible for the negative control.
*t is recommended to use a wash procedure including 1$ seconds soa0 time per strip and wash step followed by a wash
step with distilled or deioni1ed water with 1$ seconds of soa0 time after the final wash step of each wash cycle. *f
necessary the number of washing steps can be enhanced from 5+ to 7+-#+.
.orrelationC /anual > automatic processing
A panel of 97 stool specimens was investigated in parallel by manual and automatic processing method resp. !he
correlation was calculated with r M $.99).
1 *ER+OR6A&(E (,ARA('ERIS'I(S
1"1 *re!ision
*ntra-assay coefficient of variation (.N in the Astrovirus Ag ELISA calculated from #-fold determination of samplesC
S-34.e 6e-n OD St-nd-rd devi-tion ($ (J)
1 1.777 $.1'# #.9
& $.99' $.$7) 7.'
) $.'') $.$&7 7.1
' $.1#5 $.$1# 9.#
*nter-assay coefficient of variation (.N in the Astrovirus Ag ELISA from 7 different test runs from #fold determination of
samplesC
S-34.e 6e-n OD St-nd-rd devi-tion ($ (J)
1 1.#5) $.$71 ).#
& 1.$19 $.$59 5.#
) $.5#) $.$79 11.9
' $.)5$ $.$)' 9.7
1" Lo=er dete!tion .i3it
!he lower detection limit of Astrovirus antigen in the Astrovirus Ag ELISA was determined by titration of purified
Astrovirus-antigen.
3ower detection limitC 7 ng;m3.
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DRG

Astrovirus Ag ELISA (EIA-4456)

RUO in the USA
Revised 1 De!" #1 !! ($ers" %"#)

1") S4e!i7i!it> -nd Sensitivit>
A total of 9# stool samples were investigated in parallel in the Astrovirus Ag ELISA and in another commercially available
23*4A.
!o34-r-tive ELISA 4ositive !o34-r-tive ELISA neg-tive
DRG 23*4A positive '9 $
DRG 23*4A negative & '7
4pecificityC 1$$ %
4ensitivityC 97 %
1"4 (ross re-!tivit>
4tool samples positive for one of the subse,uent pathogens have been tested with the Astrovirus Ag 23*4A (2*A-''57
and showed no cross reactivityC
"otavirus (nM1$( Adenovirus (nM&$( Astrovirus (nM#( Iorovirus (nM)1( .lostridium difficile (nM11( .ampylobacter
OeOuni (nM7( .ampylobacter coli (nM1( 4almonella enteritidis (nM1#( 5iardia lamblia (nM1
Iegative stool specimens have been spi0ed with < 1$# colony forming units of the following microorganisms and tested
negative with the Astrovirus Ag 23*4A (:6'5$;7&$ nm J .ut-:ffC
Aeromonas hydrophila (A!.. 7977 Klebsiella pneumoniae (A!.. 1)##)
Baillus ereus (A!.. 1177# !eptostreptoous anaerobius (A!.. &7))7
Baillus subtilis (A!.. 77)) !roteus vulgaris (A!.. #'&7
Bateroides fragilis (A!.. &5&#5 !seudomonas aeruginosa (A!.. 1$1'5
"andida albians (A!.. 1$&)1 Salmonella enteria Serovar enteritidis (A!.. 1)$77
"ampylobater oli (A!.. ))559
Salmonella enteria Serovar
typhimurium
(A!.. 1'$&#
"ampylobater #e#uni (A!.. ))&91 Shigella fle$neri (A!.. 1&$&&
"itrobater freundii (A!.. #$9$ Shigella sonnei (A!.. &59)1
"lostridium sordellii (A!.. 971' Staphyloous aureus (A!.. &59&)
Enterobater
aerogenes
(A!.. 1)$'# Staphyloous epidermidis (A!.. 1&&&#
Enterobater loaae (A!.. 1)$'7 %ibrio parahaemolytius (A!.. 17#$&
Enteroous faealis (A!.. &9&1& %ibrio holerae .linical isolate
Esherihia oli (A!.. &59&& &ersinia enteroolitia Serotyp '() '* .linical isolates
1) (O66O& AD$I(ES A&D *RE(AU'IO&S
'his :it is 7or in vitro use on.>"
Follow the wor0ing instructions carefully. !he 0it should be performed by trained technical staff only.
!he e+piration dates stated on the respective labels are to be observed. !he same relates to the stability stated for
reconstituted reagents.
6o not use or mi+ reagents from different lots e+cept for sample diluent( wash buffer( !/9;substrate solution and stop
solution.
6o not use reagents from other manufacturers.
Avoid time shift during dispensing of reagents.
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DRG

Astrovirus Ag ELISA (EIA-4456)

RUO in the USA
Revised 1 De!" #1 !! ($ers" %"#)

All reagents should be 0ept at & =. - # =. before use.
4ome of the reagents contain small amounts of !himerosal (J $.1 % w;v and Pathon (1.$ % v;v as preservative. !hey
must not be swallowed or allowed to come into contact with s0in or mucous membranes.
Handle all components and all patient samples as if potentially ha1ardous.
4ince the 0it contains potentially ha1ardous materials( the following precautions should generally be observedC
6o not smo0e( eat or drin0 while handling 0it material(
Always use protective gloves(
Iever pipette material by mouth(
Iote safety precautions of the single test components.
Re7eren!es1
1. "ohwedder( A. (&$$$C QNirale 5astroenteritiden( 2rreger und 6iagnosti0R(
/i0robiologe( 1$. Sg. -.1&1-1&7.
&. -alombo( 2. A. and 9ishop( ". F. (1997C QAnnual *ncidence( 4erotype 6istribution and 5enetic 6iversity of Human
Astrovirus *solates from Hospitali1ed .hildren in /elbourne( AustraliaRT
Sournal of .linical /icrobiology( Nol. )'( Io. 7( p. 175$-175).
). .u0or( 5. and 9lac0low( I. ". (19#'C QHuman Niral 5astroenteritisR(
/icrobiological "eviews( Sune( Nol. '# Io. &( p. 157-179.
'. 5aggero( A.T :U"yan( /. et al. (199#C Q-revalence of Astrovirus *nfection among .hilean .hildren with Acute
5astroenteritisR( Sournal of .linical /icrobiology( Nol. )7 Io. 1&( p. )791-)79).
SI6?OLS USED 9I', DRG ASSAIS
S>3;o. Eng.ish Deuts!h +r-nK-is Es4-Lo. It-.i-no
.onsult instructions for use
5ebrauchsanweisung
beachten
.onsulter les instructions
d8utilisation
.onsulte las instrucciones de
uso
.onsultare le istru1ioni per
l8uso
2uropean .onformity
.2-PonfirmitVts-
0enn1eichnung
.onformitW au+ normes
europWennes
.onformidad europea .onformitX europea
*n vitro diagnostic device *n-vitro-6iagnosti0um
Esage 6iagnostic
in vitro
-ara uso 6iagnYstico in vitro -er uso 6iagnostica in vitro
RUO For research use only Iur fZr Forschungs1wec0e
4eulement dans le cadre de
recherches
4Ylo para uso en
investigaciYn
4olo a scopo di ricerca
.atalogue number Patalog-Ir. IumWro de catalogue I[mero de cat\logo Iumero di .atalogo
3ot. Io. ; 9atch code .hargen-Ir. IumWro de lot I[mero de lote Iumero di lotto
.ontains sufficient for Jn]
tests;
Ausreichend fZr RnR AnsVt1e
.ontenu suffisant pour RnR
tests
.ontenido suficiente para
Jn] ensayos
.ontenuto sufficiente per RnR
saggi
4torage !emperature 3agerungstemperatur !empWrature de conservation !emperatura de conservaciYn !emperatura di conserva1ione
2+piration 6ate /indesthaltbar0eits-datum 6ate limite d8utilisation Fecha de caducidad 6ata di scaden1a
3egal /anufacturer Hersteller Fabricant Fabricante Fabbricante
6istributed by 6istributor Nertreiber 6istributeur 6istribuidor 6istributore
.ontent .ontent *nhalt .onditionnement .ontenido .ontenuto
Nolume;Io. Nolume ; Io. Nolumen;An1ahl Nolume;AuantitW Nolumen;I[mero Nolume;AuantitX
Rev. 12/6/12cc
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