40

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29 30 31

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KEY -
81383 INNO-
UPA HCAHEBVNM - d
vl
2011
p T Q
.NNOGENEJICS
,-rorffK H O t O'* ’
8

INNO-UPA HBV
Multi-DR
English
1
8
2
9
[TVD1C ) 459 15 16
22 23
Manufactured^ N. V
INNOGENETICS 6
29 30
Technologiepark
9052 Gent
SSS» »» 1
m
BTW BE 0427.550 .660
RPR Gent
Distributed by:
EMBB
INNOGENETICS s .a .r .l.
INNOGENETICS GmbH Les Conquerants , Bat . Le
Kilimandjaro
Hans-Bockler-Allee 20 8/ 10 , avenue des Tropiques T C
30173 Hannover 91940 Les Ulis
Germany Franrp
J + 49-511-8573931
1 1
-
GD+33 1 69 07 48 34
INNOGENETICS S.r.l. INNOGENETICS Diagnostica Iberia , S.L.U 4 c
Calle Tarragona 161 , Planta 14
6
Via Vaccareccia 39/A
n 00040 Pomezia (Roma) 08014 Barcelona
V;
Italy Spain 11 1
. 3 + 39-06 965 28 700 <J>+34-93 270 53 00
INNOGENETICS N. V. 18 1
Technologiepark 6
9052 Gent 25 2
Belgium
JJ+32-9 329 13 29
Other languages see / Autres langues voir / Andere Sprachen siehe / Altre lingue
vedere / Ver otros idiomas / Outras linguas ver:
www.e-labeling.eu/INX38790 ® EUROPE +800 135 79 135
GR 00800 161 2205 7799
8:00 - 17:00 GMT+1 IS 800 8996
MTWTFSS LT 8800 30728
(3 (H (3 H H
RO

70
0800 895 084
SK 0800 606 287
LI +31 20 796 5692
MT +31 20 796 5693

© 2011 Innogenetics 70
i -
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k
81383 JNNayPAHBVMull ,-OR 2 ^/ emi
The INNO-LiPA HBV
be lines ^
^' KEWCODEMNXJS^- 0;

^
DR
maSfel
The „
16

Reagents
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Deseript -OH, preparation for use and
recommended storage conditions c
® ®

CL
JV
^- ,
The an pllflcatl0n and LiPA reagents should be
placed in storage at the s
^
emperatUre n amva : the amplification reagents
\ °
ana LiPA reagents in the post-amplification
area.
in the pre-amplification area n
I stored at 2 8 C , opened or unopened, the reagents
are stable until the expiry date
Do not use the reagents beyond the expiry date.
In order to avoid contamination, it is recommended to dispense amplification
reagents into aliquots at the first use. s
- The reagents should be stored protected from any source of contaminating DMA,
especially amplified products. Disposable tubes and pipette tips (preferably cotton R
plugged) should be used.
- All reagents should be brought to room temperature (20-25 C ) approximately

.
immediately after

. .*** -
30 minutes before use and should be returned to the refrigerator
all reagents before opening the vials. Close the
use Briefly vortex and spin down

ationsln physical*appearance , *reacts

. Alt
^
- ?o miSthe possibility
.
tube horizontally
that
o he ay inhtcat

strips curt before rrse, it is recomraerrrteb

»
to store the
IT
F
C

Reagents supplied
Qusrm B§L Descngtion
. . --
Component
WUl Iiyv
—
Amplification reagents1: ,
1 0 04ml 59209 Contains
biotin land P , «s v
* 0.05%
,,
N h as
Primer Mix preservative.
^^
LiPA reagents :
Strips
1x 20 59208 1 plastic tube containing INNO-LiPA
blue marker line.
marked with acontaining
HBV DR vTstrip

INNO-LiPA HBV DR v3
strips
^ ]

1 plastic tube
1x 20 60245 marker line. \
marked with a yellowish
containing
vial
EDTA. Caution: Theclosed *t
Alkaline solution should be
1x 1 ml 56718 containing the Denaturation Solution of this solution to
Denaturation immediately after use ; prolonged exposure
Solution deterioration.
air leads to rapid preservatives
with detergents and warmed to a
SSC -buffer - r

Hybridization
Contains
of at least 37
temperature
°C and
be pre
1x 80 ml 57420 The Hybridization Solution shouldmust not exceed 49
^

preservatives
and
-
with detergents pre-warmed to a I
Solution Containing SSC-buffer
Stringent Wash
200 ml 57421 The Stringent Wash
temperature of at least
be
Solution should not exceed •
37°C and must
-
Solution

• x
- .
 30 31 5
29

^ 1 xi

/ tOBY-
COOf^
90
., -
^J£^
5 nilU nt
Tr rr
^
vi 0
28101
Mu/ti -DR / STSSS working solution 1
4/17 l3S
Laa** 7
3 INNO
-LIPA HBV
£jfg“5
,
FM&SOlW *****
" *•
Tpreserv Prepare 2m o. > ^ 19 ate
AutO-LiPA
, make
,ines . and
^ use ^ ^^
***
rne/ yei OWish before

--
| | <
nd th
eaen

~~~ z
/100 in
. to be diluted 1 working
dimethyif0rmamide 2 ml substrate
I the NBT 1X 0.8
ml 569 fJazoljumBuffer before use. Prepare
jn
2 ml in excess
.
Su0s/ra
(e BCIP
/
Substratefor each test trough + excess. The substrate
ition area j00x solution make . 10 ml in at room
temperature
-
For Auto LiPA is stable for 24 hours
working solution in the dark . %
*Piry date .
) when stored
(20-25°C containing NaCI MgCI2 and . 0.01% MIT /0.1
Tns-buffer .
Substrate Buffer
lx 180 ml
56953
CAA as preservatives
buffer containing
. 0.01%
NaCI Triton® and 1/5 (1 part +
diluted
)NA . 56721 Phosphate as preservative. To
be Prepare
otton 5x U 80 ml MIT /0.48% CAA or deionized water before use ,
Rmse Solution 4 pads ) in distilled ,
0n f r 6aCh t6
St rou9h + 10 ml in

LTJSSFO7
excess. For M Auto-UPA prepare
SpAt
in
. °
12 ml Rinse workino
excess p:nco
9, •

after trough + 20 ml
solution per test C-
8‘
312‘ t
^
„,, ^^
***- . , ,
pM u no provided
Disposable gloves
< +*» - OOtton plogged .

-
ips

•
SSS. * "
Microtube racks
Microtube centrifuge

^- 2ooMland 200.1000
3 ^
1 pi
SS
/
jpspsSSasRasr-- rase (Cat no:

ni
a
25 mM)
pr
: 9en® i0x

Aspiration apparatus.
Calibrated thermometer.
rs . .
Creeps
jraduated cylinders
(10, 25, 50, and 100
ml).
 V

Vortex mixer or equivalent 813

- Water bath with shaking . « S 5 IHei
platform (fan
8 ,
°
m
minimum 49°C ± 0.5"C). Pm' Incl neu lld: temperature pie
adjustable to infc
iSSSST 1
ver
:
-
aSSJsssa--.
Warnings and precautions
For professional use only
.
All pipette tips and tubes
for the amplification process should
be
ips with cotton plugs
are recommended. Use only disposable labautoclaved. Pipette
Use a new sterile pipette tip materials.
Do not mix reagents from different specimen aliquot.
for each
- kits unless the components have identical lot
R20
R 34
numbers. R 36
- To prevent PCR contamination, maximize the physical
separation of the pre- and
R 42
post -amplification steps. Do not return samples, equipment, or reagents R 61
to the area S9
where you performed the previous step. If you need to return to a previous work S2C
area , first perform the appropriate decontamination steps. S22
S24
- Use forceps to handle strips and wear disposable gloves . Do not touch strips with S2t
your bare hands. S36
- Use only pencil to write on the strips. The assay reagents may remove ink from the S3
S4
*
‘

"assessSTJS5,
• S5.
. S6(

Wa W
,
. c^'isa'SKSSsrsisrissss
. maximize
wash by the orbital
Srtpwithoot splashing solutionsbeW eentroughsm
.
the movement o the reagents over
ray

^ ^
—
m .
^^

:S aSSfessS-
-
.
Asp

^ “* s
D
P
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29 30

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_ X 38790
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label *!
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81383J
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6/17
Ith an
to the

stable to

sstfSSS* SASH
SU8
^ **
- iPA 48).

X SSSJ"*"
. “ SSSfKT
!l 3
^ soln,

ved. Pipette
als.
^ sive! hvdroxide id water .

hums
tical lot

*6? cause f8 ? .
"'
^ .ventilated place.

— —- —
pre- and
:o the area s’ May

vapour/spray 1
^1 -ssss r, zisss&z
f3
is work Do not breathe
224 Avoid contact with Skin'

f^ KSSfflS, .- -
In case
rips with protective clothing . 9 mmediately (show the label
where
/37/39 Wear suitable
aw
k from the B
possible)
>s and S53 Avoid exposure
r. Use a $60
all blood
5 close the
low, the
ay bs"Stnd1 ed as such. Only adequately trained
personnel should
. -
bath with
. MMMDMMSSENSAILD biological materials should be disposed of in accord
?n <

stringent
djust the
gents over
.•
with established safety procedures.
Autoclave for at least 15 minutes at 121°C.
Incinerate disposable material
• liquid waste with sodium hypochlorite so that the final concentration is ± 1°/0J
Mix
‘vel in the sodium hypochlorite. Allow to stand overnight before disposal.
y . Prevent Caution: Neutralize liquid waste that contains acid before adding sodium
hypochlorite.
Use of personal protective equipment is necessary: gloves and safety spectacles
when manipulating dangerous or infectious agents.
3Spirator ). )ul be ha
AIMOHASH! ? led according to the institution's waste disposal guidelines

Specimen collection, ^
r s a * e . and local environmental
handling and storage
regulations should also be observed

-
- - an be extracted

,
25?£!! ,
from human serum or plasma .
’
s ora9e or P|asma samples
o :
Stenie tubes containing EDTA.
Do not use heparin as an anticoagula ? -
m

li
 -

JJ383
3 INN0-LiP UHB / ultm
r/
^ ^^
.. 81Q1 v1 / KEY-CODE: INX 38790 p 8 / 17

i / Sturers
inStmct 0ns '
-
•
Store plasma at or below -70°C. Thaw only
Preparation_ and storage
I
x I I

*leCuibl° '
, .
^ for

^
serum. .samples
, ,
1
n st erile tubes with
^ A
I

no
^' i
:
anticoagulant
A ow blood to clot at room temperature , centrifuge ,
O C4 I
^
or in serum separator tubes.
and separate serum from cells
'

according to the manufacture's instructions.

Store serum at or below -7u C Thaw only for use.
DNA extracted from HBV-positive samples should be used immediately or frozen at or
below -20°C, preferably never thawed and refrozen. Amplified material of the HBV -pol
gene domain A to F can then be generated.
Use 10 pi of amplified product for the LiPA procedure.
Amplification test procedure
For the DNA extraction, Qiagen QIAamp® DNA Blood Mini Kit (Cat no: 51104) is ;
recommended. See protocol and please follow instructions for use of the extraction
method. When using other commercially available extraction methods, the method
should be validated in your laboratory .
Amplification protocol
The following protocol is designed for optimal amplification, using
- Qiagen HotStarTaq® polymerase (5 U/pl).
SO, , 15 mM MgCl2, pH 8.7 ).
- Qiagen® 10x PCR buffer (containing Tris-HCI, KCI, (NH Biosystems
- MicroAmp® PCR tubes (0.2 ml thin-walled tubes; Applied * ^
Cat. no:
N8010540).
- GeneAmp ® PCR system PE-9700 thermal cycler. (Applied Biosystems Cat. no:
N8050200) .
This protocol can be used with most commercial types of thermal cyclers , but these
may require some modifications provided by the manufacturer of the cycler.
, which
Take the following necessary precautions to avoid non-specific amplification
may impair the results:
- Thaw the components listed below and place delaythem on ice.
- Perform all pipetting steps on ice and without .
1 Determine the number of vials to be prepared (N) as: N
= Number of samples + 1
+ 1 Positive Control +
Negative Control for extraction + 1 Negative Control for PCR
1 extra
Master Mix in an autoclaved nuclease -
2. Using cotton-plugged pipette tips, prepare a
free microcentrifuge tube.
3. Prepare a Master Mix containing:
b (N pi) autoclaved distilled water
WO jY , [ ^J
£ 10x PCR buffer
w w pl
Nx 5.0
\ \("
+ ~r •)/ w
-
Qiagen
•
) dNTP mix (25 mM)
«v x
u — i

yJ ~( N x pi
Primer Mix
(N x 1.0
+ IN
T AI *)
. pi t
f W <m

+ (N x 1.0 pi) 25 mMMgCI2

.
HotStarTaq® DNA Polymerase 5 U/pl
of this Master Mix into ( N - 1 ) autoclaved
+ (N x 0 4 pi) Qiagen
dispense 30 pi aliquots
Vortex briefly and
amplification tubes

cf '
 1 .
"MM
INX 38790 9/ 17
1H1 1 KFY -CODE:
p 8/17
81383 INNO-LiPA HBV Multi-D
to the Negate
4 . Pipette 20 pi of p pi of distilled water (no DNA
Control). Remark: Mix briefA spin the mixture to bring the
liquid to the bottom of
the tubes.
m

7 '~ hv
5. Place the samples into the calibrated thermal block (see instructions^
Pr - _
° , . J

> es. prograi

r \ cells the manufacturer of the thermal cycler ). Start the amplification
the INNO-LiPA HBV Multi-DR amplification.
cycler
INNO-LiPA HBV Multi-DR amplification profile for a PE-9700 thermal

P
in at or Step Temp Time
BV-pol 1 Denature 95°C 15 min.
2 Denature 94 UC 30 sec. repeat cycle «

3 Anneal primers 55 °C 30 sec. step 2 - 4

4 Extend primers 72°C 40 sec. 50 times
5 Elongate 72°C 10 min.
is 6 Cool to 4°C
ion 6. After the amplification process, use the samples immediately with the INNO-LiPA
od HBV Multi-DR test strip or store them at -15/-25°C.
7. After this process, amplification may be checked by analyzing 5 pi of the amplified
product on 2% agarose gel or store at -15/-25°C. Note: Do not store the amplified
DNA products with amplification reagents or extracted DNA.
Amplification results
•»
- J
pH 8.7). Visualization 35
- The presence of the amplified product can be checked on a 2% agarose gel.
- Load 5 pi of amplified product per slot . 361
>: - The amplicon should appear as a band with a length of 867 bp.
Quality control 37
se - Include at least one blank for extraction.
:h
- Include at least one positive and one negative control each time amplification is 38 k
performed. As with any new laboratory procedure , the inclusion of additional positive
and negative controls should be considered until a high degree of confidence is 39
reached in the ability to correctly perform the procedure. If the addition of a positive
control is desirable, use a known HBV positive sample.
•1 - If a positive band is obtained in the gel for the negative control for extraction, it is
ol + advised to discard the results of the run, and to repeat and the procedure from
extraction.
3 se- LiPA manual test procedure
rl
^J »
Denaturing the samples
Caution: Using a calibrated thermometer , ensure that the temperature of the shaking
water bath is 49°C ± 0.5°C, and adjust the temperature if necessary before adding a
sample tray.
1. Equilibrate a shaking water bath to 49°C ± 0.5°C.
2 Place the Hybridization Solution and Stringent Wash Solution in a water bath at
37 = C to 49°C to dissolve all crystals. Ensure that the water bath does not exceed
d 49°C ± 0.5°C. Mix by shaking the bottle before use.
Besides a positive control sample, it is advised always to include a negative contr:
sample for the extraction step, a negative control sample for the PCR step , and a
 83 INNO-LiPA HBV Multi-DR /
,or , 28101 v1 / KEY-CQPF -

sasrsrsr
4. For each sample,
HBV DR v3 Strip remove one INNO-LiPA HBV
from the
,
he LIPA s ep (add
INX3879D
10 / 17

Note: Do not place the

respective tubes
identification number above the
using forceps
marker line on each strip wttK 006 lNN1 °write
3 penci
-LiPA
strips in the troughs until . ’ an
5. Place one trough step 2 in Hybridizing the
for each sample/strip(s)
6. Add 10 pi
Denaturation Solution to the
in the tray.
upper
•
sampies.
Note: Close the vial
containing the Denaturation
corner of each trough
Prolon9ed exPosure to air Solution immediately after each
AHH
dd causes the solution
'

^ „ !°
Ml Sample ( r contro1 sample) to
°
Carefully mix by pipetting up
8. Allow and down.
the
denaturation to proceed for 5 minutes at
Hybridizing
to deteriorate.
Denaturation Solution in each trough.
room temperature.
I
1
the samples I
1. Carefully add 2 ml Hybridization I
Solution to each trough, taking care not
contaminate other troughs. Gently shake the trough to mix reagents.
to
2. For each sample,
immediately place both strips (INNO-LiPA HBV DR
INNO-LiPA HBV DR v3) into the trough v 2 and
with the marker line facing up for each
Note: Wear disposable gloves and use strip.
3. Ensure that the strips are completely forceps when handling the strips.
submerged in the solution. Do not cover the
troughs with microplate sealers as this could
4. Place the tray into the 49°C ± 0.5°C shaking result in cross-contamination.
water bath and immobilize the tray
between two heavy weights. Set the water bath to approximately
80 rpm, close the
lid, and incubate the tray for 60 minutes. Ensure that each
strip remains completely
submerged and floats freely.
from .the waterr .
, .„
5. When hybridization incubation is complete, remove the tray . 4 ,
bath.
Washing the strips
-
caution Hold the tray at a low angle so the liquid accumulates at one end of a trough,
above the marker line on the strips, for easy removal. Do not damage the surface of

TZpm
the strips Avoid splashing or transferring solutions between troughs.
the solution from the trough using a pipette, preferably attached to a

|, "PS ^ ***
in
I sssss ;" ,
3

*
,
he ay or 1 m nute at 20'25°c '
^
,
316 the solu on rom
"
,
Astringent »
i
m
4. Add 2 Wash Solution
shaking water bath and immobilize
tj w ''efghts
. SeUhe water 1
m bath to approximately 80 rpm, close e and incubate the tray for 30 minutes
5. Prepare the rinse working solution and conjugate working solution (See Reagents).
A
Color development
"
of a trough
- a so the liquid accumulates at one end
Caution: Hold the tray at a low angle
—
above the marker line on the strips
trips, for easy removal. Do
uo noi
, aamayt the surface of
not damage
the strips. Avoid splashing or transferring solutions
4Unc Kof iGon troughs
between
i
* trminhs
\

f
-
3
 ns zo ZsO 4/

29 30 31 5

38790 p 11/ 17
0 / 17 INNO - LiPA HBV Multi-DR / 28101 v 1 / KEY - COPEJNX
81383
1 Aspirate the solution from tin r(gh using a
pipette .
*
and wash the strips by rocking the
2. Add 2 ml rinse working sol B . each trough
^
'

tray for 1 minute at room temperate . Aspirate the solution

from eac roug
1 Repeat this step once . , .
to each trough place the tray on as a e
3 Add 2 ml conjugate working solution ,
for 30 minu es
es. (orbital at 160 rpm or rocker at 50 rpm) at 20- 25° C , and incubate
the en o^
Note: Prepare the substrate working solution about 10 minutes before
the conjugate incubation ( See Reagents ).
the
h 4 . When the incubation is complete , remove the tray from the shaker and aspirate
solution from each trough using a pipette .
Jh.
iI 5. Add 2 ml rinse working solution to each trough and wash the strips by rocking the
tray for 1 minute at 20- 25°C . Aspirate the solution from each trough . Repeat this
step once .
f 6 . Add 2 ml Substrate Buffer to each trough and wash the strips by rocking the tray for
1 minute at 20- 25°C . Aspirate the solution from each trough.
7 . Add 2 ml substrate working solution to each trough , place on shaker at 20-25° C ,
and incubate for 30 minutes .
strip . 8 . When the incubation is complete , stop the color development by washing the strips:
remove the tray from the shaker, aspirate the solution from each trough , and then
le add 2 ml distilled water to each trough and place the tray on the shaker for at least
3 minutes . Repeat this step once.
9. Using tweezers , remove each strip from its trough and place the strip with the
the marker line facing up on absorbent paper .
tely 10.Dry the strips completely before reading the results. Store the developed and dried
strips in the dark .
LiPA automated test procedure: Auto-LiPA and Aufo-LiPA 48
The LiPA test procedure is extremely well suited for automation . Therefore , the Auto-
LiPA and Aufo-LiPA 48 are designed to fully handle hybridization , stringent wash and
color development steps . The Auto-LiPA and Au/o-LiPA 48 are featured as a walk-
away system with automated heating and cooling, and with automated aspiration and
pipetting. For more information and specific protocols on Auto- LiPA and Auto- LiPA 48 .
please contact your local distributor .
L i P A results
the
Reading
Figure 1 illustrates the position of the different oligonucleotide probes on the INNO-LiPA
sing HBV DR v2 strip. A line is considered positive when a visible purple/brown band

I appears at the end of the test procedure.

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Location of the blue
colored line marker
control line Conj .
the conjugate , ( . control), the amp if cat
control Sne (Amp. control), and the 32 probe lines on the INNO-LiPA HBV DR v2 strip.
I