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This Package Insert is provided for product
evaluation purposes only and is
not intended to be used in place of the Package Insert shipped with the product.
The HTLV Western Blots are manufactured by Genelabs Diagnostics® and are
distributed exclusively in the U.S. and Caribbean by ZeptoMetrix Corporation.
HTLV BLOT 2.4
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For detection of antibodies to
HTLV-I and HTLV-II in serum or plasma samples. |
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The GENELABS DIAGNOSTICS (GLD)
HTLV BLOT 2.4 is a qualitative enzyme immunoassay for antibodies to
HTLV-I and HTLV-II in human serum or plasma samples. This test kit
is supplied for research purposes only. It is not intended for use
in the diagnosis or prognosis of disease. In particular, this test
cannot be used to evaluate blood specimens for the purposes of donor
screening or as a confirmatory diagnostic. |
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| The GLD HTLV Blot 2.4 is an
informational research test on serum or plasma samples. The GLD HTLV
Blot 2.4 incorporates MTA-1, a unique HTLV-I envelope recombinant
protein (rgp46-1), K55, a unique HTLV-II envelope recombinant protein
rgp 46-II and GD21, a common yet specific HTLV-I and HTLV-II epitope
recombinant envelope protein. Each strip also includes an internal
sample addition control to minimize the risk of false negatives due to
operational errors. |
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CHEMICAL & BIOLOGICAL
PRINCIPLES
OF THE PROCEDURE |
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| The nitrocellulose strips are
incorporated with HTLV-I viral proteins derived from native inactivated
disrupted viral particles and genetically engineered proteins.
Individual nitrocellulose strips are incubated with diluted serum or
plasma specimens and controls. Specific antibodies to HTLV-I/II, if
present in the specimen will bind to the HTLV-I/II proteins on the
strips. The strips are washed to remove unbound materials while
antibodies that bind specifically to the HTLV proteins can be visualized
using a series of reactions with goat anti-human IgG conjugated with
alkaline phosphatase and the substrate, BCIP/NBT. |
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1. NITROCELLULOSE STRIPS
Incorporated with HTLV-I viral lysate
and recombinant envelope antigens.
Keep dry and away from light. |
Available in
18 & 36 strips |
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2. NON-REACTIVE CONTROL
Inactivated normal human serum
non-reactive for anti-HCV, anti-HIV-
1/2, anti-HTLV-I/II and HBsAg.
Contains sodium azide
and thimerosal as preservatives. |
1 vial
(80ul) |
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3. STRONG REACTIVE CONTROL
I
Inactivated human serum with
high titered antibodies to
HTLV-I and non-reactive for anti-HCV,
anti-HIV-1/2 and HBsAg.
Contains sodium azide
and thimerosal as preservatives. |
1 vial
(80ul) |
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4. STRONG REACTIVE CONTROL
II
Inactivated human serum with
high titered antibodies to
HTLV-II and non-reactive
for anti-HCV, anti-HIV-1/2 and HbsAg
Contains sodium azide
and thimerosal as preservatives. |
1 vial
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5. LYOPHILIZED STOCK BUFFER
To be reconstituted in
reagent grade water.
Tris buffer with heat inactivated
animal and non-animal proteins.
Contains thimerosal as preservative. |
1 or 2 bottles
(each to be reconstituted to 100ml) |
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6. WASH BUFFER CONCENTRATE
(20X)
Tris with Tween‑20 and contains
thimerosal as preservative. |
1 bottle
(70ml) |
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7. CONJUGATE
Goat anti-human IgG conjugated
with alkaline phosphatase. |
1 vial
(120ul) |
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8. SUBSTRATE
Solution of 5-bromo-4-chloro
-3-indolyl-phosphate (BCIP) and
nitroblue tetrazolium (NBT). |
1 bottle
(100ml) |
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9. BLOTTING POWDER
Non-fat dry milk |
10 packets
(1g each) |
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10. Incubation trays, 9
wells each. |
2 or 4 trays |
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11. Instruction Manual
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1 copy |
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Volume of reagents provided are
sufficient for 4 runs. |
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CAUTION: Handle all assay
specimens, positive and negative controls
as potentially infectious agents. |
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| 1. Substituting reagents, even
between lots, may affect results. |
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| 2. FOR RESEARCH USE ONLY, NOT
FOR USE IN DIAGNOSTIC PROCEDURES. |
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| 3. Do not use kit components
beyond the expiry date. |
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| 4. Avoid microbial
contamination of reagents when opening and removing aliquots from
the original vials or bottles. |
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| 5. Gloves and lab coats
must be worn. |
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| 6. Do not pipette by
mouth. |
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| 7. Wipe spills quickly and
thoroughly with sodium hypochlorite solution. |
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| 8. Autoclave all used and
contaminated materials at 121°C at 15 p.s.i. for 30 minutes before
disposal. |
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| 9. It is highly
recommended that this assay be performed in a biohazard cabinet.
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| 10. Decontaminate all used
chemicals and reagents in sodium hypochlorite solution. |
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| 11. We do not recommend
re-use of incubation trays. |
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A. Antigen strips
Avoid unnecessary exposure of antigen strips to light.
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B. Reagents
Store all reagents at 2 - 8 °C. |
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| For best results, dispense
reagents while cold and return to 2 - 8 °C storage as soon as
possible. |
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| CAUTION: Avoid unnecessary
exposure of substrate to light. |
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MATERIALS REQUIRED
BUT NOT PROVIDED |
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| Rocking platform * |
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| Pipettor and tips |
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| Aspirator with sodium
hypochlorite trap * |
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| 56°C water bath [optional]
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| * Not required if using Autoblot
System 36. |
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SPECIMEN HANDLING
AND STORAGE (OPTIONAL) |
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| Sera can be inactivated but
this is not a requirement for optimal test performance. |
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Inactivated as follows:
1. Loosen caps of serum containers.
2. Heat serum at 56°C for 30 minutes in
a water bath.
3. Allow serum to cool before
retightening caps.
4. Serum can be stored frozen until
analysis.
We recommend that the sera should not undergo
repeated freeze-thaw cycles prior to testing. |
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| 1. DILUTED WASH BUFFER
(a) Dilute 1 volume of
WASH BUFFER CONCENTRATE (20X) with
19 volumes reagent grade water. Mix well.
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2. BLOTTING BUFFER
(a) Reconstitute each bottle of LYOPHILIZED
STOCK BUFFER
with 100ml reagent grade water. Mix well to
dissolve. This
RECONSTITUTED STOCK BUFFER is stable for 6
weeks if stored
at 2-80C
(b) BLOTTING BUFFER
should be prepared fresh prior to use.
Add 1 g of BLOTTING POWDER to every 20 ml of the
RECONSTITUTED STOCK BUFFER prepared in step 2(a)
above.
Mix well. |
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| 3. WORKING CONJUGATE
SOLUTION
(a) Prepare WORKING
CONJUGATE SOLUTION by diluting
CONJUGATE 1:1000 into BLOTTING BUFFER, for
example
10ul CONJUGATE to 10ml BLOTTING BUFFER.
(b) WORKING CONJUGATE
SOLUTION should be prepared fresh
prior to use. |
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| 4. SUBSTRATE SOLUTION
(ready to use)
(a) Dispense directly
the required volume from the bottle.
Use a clean pipette. Cap tightly after
use. |
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RECOMMENDED ASSAY PROCEDURE
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| Note: Aspirate all
used chemicals and reagents into trap containing sodium
hypochlorite. |
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| 1. Using
forceps, carefully remove required number of STRIPS from the tube
and place numbered side up into each well. Include strips for Strong
Reactive and Non-Reactive controls. |
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| 2. Add 2ml of
DILUTED WASH BUFFER to each well. |
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| 3. Incubate
the strips for at least 5 minutes at room temperature (25 + 3°C) on
a rocking platform. Remove buffer by aspiration. |
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| 4. Add 2ml of
BLOTTING BUFFER to each well followed by 20ul each of patients’ sera
or controls to appropriate wells. |
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| 5. Cover the
tray with the cover provided and incubate for 1 hour at room
temperature (25 + 3°C) on the rocking platform. |
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| 6. Carefully
uncover the tray to avoid splashing or mixing of samples . Aspirate
the mixture from the wells. Change aspirator tips between samples to
avoid cross-contamination. |
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| 7. Wash each
strip 3 times with 2ml of DILUTED WASH BUFFER allowing 5 minutes
soak on the rocking platform between each wash. |
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| 8. Add 2 ml of
WORKING CONJUGATE SOLUTION to each well. Cover tray and incubate for
1 hour at room temperature (25 + 3°C) the rocking platform.
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| 9. Aspirate
CONJUGATE from the wells. Wash as in step 7. |
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| 10. Add 2 ml of
SUBSTRATE SOLUTION to each well. Cover tray and incubate for 15
minutes on the rocking platform. |
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| 11. Aspirate the
SUBSTRATE and rinse the strips several times with reagent grade
water to stop the reaction. |
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| 12. Using
forceps, gently remove strips onto paper towels. Cover with paper
towels and dry. |
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| 13. Mount strips
on worksheet (non-absorbent white paper). Do not apply adhesive tape
over the developed bands. Observe the bands (see interpretation of
Results) and grade the results. For storage, keep the strips in the
dark. |
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AMOUNT OF REAGENTS REQUIRED FOR
VARIOUS NUMBER OF STRIPS
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| Reagents
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NUMBER OF STRIPS TO BE USED
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3 |
6 |
9 |
15 |
20 |
27 |
36 |
| 1X Wash Buffer (ml) |
60 |
100 |
140 |
240 |
300 |
400 |
520 |
| 1X Blotting Buffer (ml)
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20 |
40 |
60 |
80 |
100 |
120 |
160 |
| Conjugate (ul) |
11 |
17 |
23 |
35 |
45 |
59 |
77 |
| Substrate (ml) |
11 |
17 |
23 |
35 |
45 |
59 |
77 |
| Blotting Powder (g) |
1 |
2 |
3 |
4 |
5 |
6 |
8 |
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| We recommend that the
Non-Reactive Control and both Strong Reactive Controls be run with
assay regardless of the number of samples tested. |
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| 1. NON-REACTIVE CONTROL
No HTLV-I/II viral specific bands, rpg46-I,
rpg 46-II or GD21 should be observed on the Non-Reactive control
strip. The band for the serum control (anti-human IgG) should be
visible. |
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| 2. STRONG REACTIVE CONTROL
I
The serum control band and all
relevant HTLV-I/II molecular weight
bands must be evident . The relevant HTLV-I bands must be
present
are p19, p24, gp46, gp46-1 and GD21. Note that the gp46
band is diffused. |
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| 3. STRONG REACTIVE CONTROL
II
The serum control band and all
relevant HTLV-I/II molecular weight
bands must be evident . The relevant HTLV bands mist be
present
are p24, GD21 and rpg46-II. |
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| The serum control band serves as
a check for serum addition in the assay. Absence of this band
indicates that no test serum or conjugate or substrate has been
dispensed onto the test strip or other operational errors.
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| Locate and identify bands on the
strips run with Strong Reactive Controls. These strips are then used
to identify bands present on strips used with test specimens.
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| Serum with antibodies to both
viruses although rare, may occur and can also be differentiated
based on the above criteria. Banding patterns of such specimens will
indicate HTLV-I and HTLV-II positive. Available data demonstrates
that the seroreactivity to rgp46-I is specific for HTLV-I and
seroreactivity to rgp46-II is specific for HTLV-II. |
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LIMITATIONS OF THE PROCEDURE
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| Deviation from the recommended
procedure may lead to aberrant results. |
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LIMITED EXPRESSED
WARRANTY DISCLAIMER |
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| The manufacturer makes no
express warranty other than that the test kit will function as a
Research Use Only assay within the specifications and limitations
described in the product Instruction Manual when used in accordance
with the instructions contained therein. The manufacturer disclaims
any warranty expressed or implied, including such expressed or
implied warranty with respect to merchantability, fitness for use or
implied utility for any other purposes. The manufacturer is limited
to either replacement of the product or refund of the purchase price
of the product. The manufacturer shall not be liable to the
purchaser or third parties for any damage, injury or economic loss
however caused by the product in the use or in the application
thereof. |
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| 1.
Towbin H., Staehlin T. and Gordan J. Electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets:
procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 1976;
76 4350-4354. |
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2. Poiesz BJ., Ruscetti FW., Gazdar AF., Bonn PA.,
Minna JD. And Gallo RC. Detection and Isolation of type C retrovirus
particles from fresh and cultured lymphocytes of a patient with
cutaneous T-cell lymphoma. Proc. Natl. Acad. Sci. U.S.A. 1980;
77(12): 7415-7419. |
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3.
Kalyanaraman VS., Sarngadharan MG., Robert-Guroff M., Miyoshi I.,
Blayney D., Golde and Gallo RC. A new subtype of human T-cell
leukemia virus (HTLV-II) associated with a T-cell variant of hairy
cell leukemia. Science 1982; 218: 571-573.
|
| 4.
William AE., Fang CT., Slamon DJ. et al. Seroprevalence and
epidemiological correlates of HTLV-I infection in U.S. blood donors.
Science 1988; 240: 643-646. |
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| 5. Lee
H., Swanson P., Shorty VS., Zack JA., Roseblatt JD. and Chen ISY.
High rate of HTLV-II infection in seropositive IV drug abusers in
New Orleans. Science 1989; 244: 471-475. |
| |
| 6. Lipka
JJ., Bui K., Reyes GR, Moeckli R., Wiktor SZ., Blattner WA., Murphy
EL., Hanson CV., Shaw GM., Shinsky JJ. and Foung SKH. Determination
of a unique immunodominant epitope of HTLV-I. Infect Dis 1990; 162:
353-357 |
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| 7.
Wiktor SZ., Alexandra SS., Shaw GM. et al. Distinguishing between
HTLV-I and HTLV-II by Western Blot. Lancet 1990; 335: 1533.
|
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| 8.
Samuel KP., Lautenberger JA., Jorcyk CL., Josephs S., Wong Staal F.
and Papas TS. Diagnostic potential for human malignancies of
bacterially produced HTLV-I enevelope protein. Science 1984; 226:
1094-1097. |
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| 9.
Hadlock KG., Goh CJ., Bradshaw PA., Perkins S., Lo J., Habbaz RK.,
Kaplan J. and Foung SKH. Delineation of an immunodominant and highly
HTLV specific epitope within the HTLV-I transmembrane glycoprotein.
Blood 1995; 68(4): 1392-1399. |
| |
| 10. Varma
M., Rudolph D., Knuchel M., Switzer W., Hadlock KG., Velligan M.,
Chan L., Foung SKH., LaI RB. Enhanced specificity of truncated
transmembrane protein for serologic confirmation of HTLV-I and
HTLV-II infection by Western Blot assay containing recombinant
envelope |
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