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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.
SIV
Western Blot Assay
FOR
RESEARCH USE ONLY. NOT FOR in vitro DIAGNOSTIC OR CLINICAL USE.
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ZMC Catalog # :
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0801500 (10 Strip Kit)
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ZMC Catalog # : |
0801501 (30 Strip Kit) |
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ZMC Catalog # : |
0801502 (10 Strips) |
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INTENDED
USE |
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Simian Immunodeficiency Virus (SIV) is the most closely related
lentivirus to Human Immunodeficiency Virus (HIV), therefore, SIV
infection in monkeys has become the best animal model for studying
the pathogenesis of and efficacy of vaccines against HIV infection
in humans. (1-7,10-12)
The ZeptoMetrix SIV Western Blot Assay is a
qualitative enzyme immunoassay for the in vitro detection of
antibodies to SIV in serum or plasma. It is supplied for research
purposes only. It is not intended for use in the diagnosis or
prognosis of disease, or for screening and may not be used as a
confirmatory test in diagnostic situations |
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PRINCIPLE
OF
THE TEST |
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Nitrocellulose strips contain SIVMAC
viral proteins resulting from electrophoresis and electroblotting of
detergent disrupted virus particles.(8,9)
A serum control band is
included on each strip to minimize the risk of false negative
results due to operational error.
Individual strips are reacted with diluted serum or plasma
specimens. If specific SIV antibodies are present, they will bind to
the SIV proteins on the nitrocellulose. The strips are washed to
remove unbound material. The bound antibodies are visualized by
subsequent reaction with goat anti-human IgG conjugated with
alkaline phosphatase, followed by BCIP/NBT substrate.
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REAGENTS |
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MATERIALS SUPPLIED
- SIV
Nitrocellulose Strips (10 or 30 strips): Contains
electrophoretically separated SIV proteins. Also contains bovine
proteins as a blocking agent.
- Non-Reactive
Control (1 vial, 100 µl): Serum or plasma non-reactive for HTLV,
HCV, HIV, SIV and Hepatitis B surface antigen. Contains 0.1%
sodium azide as a preservative.
- Reactive Control
(1 vial, 100 µl): Heat inactivated serum or plasma containing a
high titer of antibodies to SIV. Contains 0.1% sodium azide.
- Blotting Buffer
(1 bottle, 125 ml): Contains Tris, NaCl, non-immune goat serum
and Tween-20®. Contains 0.1% sodium azide as a preservative.
- 20X Wash Buffer
(1 bottle, 100 ml): Contains Tween-20®, Tris, and NaCl. Contains
0.1% sodium azide when diluted as a preservative.
- Conjugate (1
vial, 100 µl): Goat anti-human IgG (heavy and light chain)
antibodies. Contains Tris, NaCl and non-immune goat serum.
Contains 0.1% sodium azide as a preservative.
- Substrate (1
bottle, 75 ml): 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and
nitroblue tetrazolium (NBT) in a buffered solution.
- Blotting Powder
(1 packet, 10 g): Nonfat dry milk
- Incubation
Trays: 1 or 3, 10 well trays.
® Tween- 20 is a registered trademark of Imperial Chemical
Industries
MATERIALS
REQUIRED BUT NOT SUPPLIED
• Rocking platform
• Aspirator with disinfectant trap
• Disposable gloves
• Calibrated adjustable micropipettes
• Graduated cylinders and assorted beakers
• Timer
• 1% sodium hypochlorite as disinfectant. May be prepared from
household
bleach
• Distilled or deionized water
• Tweezers or forceps
STORAGE AND PROPER KIT HANDLING
• To avoid microbial contamination store kit reagents tightly capped
and at 2-8 C when not in use
• Do not use kit beyond its expiration date
• Use reagent grade water for diluting kit reagents
• Reusing trays is not recommended
• Keep all reagents tightly capped and refrigerated when not in use
• Allow strips to reach room temperature before opening tube. Close
tube immediately after removing strips for use.
• Allow reagents to reach room temperature before use
• Do not touch strips with fingers. Use forceps when removing strips
from tube |
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PRECAUTIONS |
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• Prior to performing
the assay, carefully read all instructions.
• Handle assay specimens, strips, non-reactive and reactive controls
as if capable of transmitting infectious agents. Although the virus
proteins on the nitrocellulose strips have been inactivated, the
reactive control has been heat inactivated and the non-reactive
control has been found negative for HIV, HCV and Hepatitis B surface
antigen, no test can offer assurance that products derived from
human or non-human primate blood will not transmit infectious
agents.
• Use Universal precautions when performing this assay (MMWR, June
24, 1988, Vol. 37 No. 24, pp. 377-382, 387-388).
• Disposal: When testing potentially-infectious specimens, adhere to
all applicable local, state and federal regulations regarding the
disposal of biohazardous materials.
• Kit components contain sodium azide as a preservative. If disposed
of through a sink or other plumbing system, flush with generous
amounts of water to prevent accumulation of potentially explosive
compounds. See “Safety Management No. CDC-22, “Decontamination of
Laboratory Sink Drains to Remove Azide Salts” (Centers for Disease
Control, Atlanta, GA, April 30, 1976). |
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PREPARATION
OF REAGENTS |
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Assay may be performed
as either an overnight or a rapid procedure (3.5 hour total time).
The overnight protocol provides additional sensitivity.
1.
1X WASH BUFFER
Dilute 1 volume of 20X WASH BUFFER with 19 volumes of distilled or
deionized water. Mix well. Diluted wash buffer may be stored at room
temperature for 1 month.
2. 1X WORKING BLOTTING BUFFER
1X WORKING BLOTTING BUFFER should be prepared fresh prior to use.
Dissolve 1.0 g of NONFAT DRY MILK in 20 ml of BLOTTING BUFFER. Mix
thoroughly to dissolve the powder. Total volume required listed in
table below.
3. 1X WORKING CONJUGATE
1X WORKING CONJUGATE should be prepared fresh prior to use.
Dilute CONJUGATE 1:1000 in 1X WORKING BLOTTING BUFFER. Example; Add
10 µl conjugate to 10 ml of 1X WORKING BLOTTING BUFFER.
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Amount of
Reagents Required (ml) for Various Number of Strips
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No. of
Strips: |
3
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5 |
10 |
15 |
20 |
30 |
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1X Wash
Buffer |
36
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60
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120
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180
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240
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360
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1X Working
Blotting Buffer |
12
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20
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40
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60
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80
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120
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1X Working
Conjugate |
6
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10
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20
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30
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40
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60
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TEST
PROCEDURE |
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Allow all reagents to
reach room temperature before use. Avoid splashing while handling
incubation tray. Do not handle samples or sample loaded pipette tips
over uncovered trays. Splashing or aerosols can result in cross
contamination of sample wells. Remove lid carefully to avoid
condensate from falling into sample wells.
1. Using forceps, remove enough strips from the tube for each sample
and for reactive and non-reactive controls. Place numbered side up
into each well.
2. Add 2.0 ml 1X WORKING BLOTTING BUFFER to each well, followed by
20 µl of sample or control sera.
3. Cover the tray and incubate 16-20 hrs (overnight method) at room
temperature (25±3C) or two hours at room temperature (rapid method)
on a
rocking platform.
4. Carefully remove cover avoiding splashing or mixing of samples.
Aspirate each well into disinfectant trap.
5. Wash each strip 3 times with 2.0 ml 1X WASH BUFFER allowing 5
minutes on the rocking platform between each wash.
6. Add 2.0 ml 1X WORKING CONJUGATE to each well. Cover tray and
incubate on a rocking platform at room temperature for 60 minutes
(±5 minutes).
7. Aspirate each well. Wash strips as described in Step 5.
8. Add 2.0 ml of SUBSTRATE to each well and incubate at room
temperature on a rocking platform for up to 15 minutes.
9. Aspirate SUBSTRATE from each well and stop the reaction by
rinsing each strip several times with distilled or deionized water.
10. Using forceps, place the strips onto absorbant paper towel,
cover with another towel and allow to dry. Score strips as soon as
possible after the assay is complete. Strips do not have to be dry
prior to scoring. Strips will eventually fade with exposure to light
and air. For best results, strips can be mounted and stored between
clear plastic sheets. Do not place adhesive tape over bands as this
will cause them to fade. |
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TEST
VALIDITY |
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The
REACTIVE and NON-REACTIVE CONTROL must be included in
each run.
1. NON-REACTIVE CONTROL: Viral specific bands should not be
observed on the nitrocellulose strip used to test the NON-REACTIVE
CONTROL. The serum control band should be visible as in Figure
1b. The test should be considered invalid and repeated if the
serum control band is not visible on the strip.
2. REACTIVE CONTROL: All major molecular weight bands must be
visible on the nitrocellulose strip used to test the REACTIVE
CONTROL. These bands are p17, p27, p31, gp41, p55, p66, gp120 and
gp160. Other virus specific bands may also be present. The serum
control band should be visible. Figure 1a provides a guide to
the relative positioning of bands observed with the REACTIVE CONTROL
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INTERPRETATION
OF RESULTS |
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The presence or absence
of antibodies to SIV in a specimen, and the identity of such
antibodies is determined by comparison of each nitrocellulose strip
to the REACTIVE CONTROL AND NON-REACTIVE CONTROL tested with that
run.
The interpretation process is three-step:
1. Establish that the serum control band is present, assuring the
sample was
added to the well and that the assay was performed properly. The
test should be considered invalid and repeated if the serum control
is not visible.
2. Assign each band present a molecular weight based on its
position. Use the
REACTIVE CONTROL strip in side by side comparison with the sample
strip
to facilitate this step. Use the illustration on page 5 to aid in
band identification
and assignment.
3. Interpretation is based on the combination of bands present.
The major SIV gene products used in the interpretation are:
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GENE
PRODUCTS |
May also be
known as: |
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gag
(core antigens) |
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p17
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p15, p16
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p27
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p24, p26, p28
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p55
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p56, p58
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pol
(endonuclease polymerase antigens)
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p31
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p34, p36
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p66
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p68
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env
(envelope antigens)
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gp41(transmembrane)
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gp36, gp34
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gp80 (dimer of gp41)
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gp120(surface)
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gp105, gp125
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gp160(oligomer* of gp41)
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gp140
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*Trimers
and/or tetramers(13)
Expected
Values
In studies evaluating the ZeptoMetrix Western Blot, a full spectrum
of reactivity was observed against gag, pol and env proteins with
non-human primate serum or plasma. Specimens containing antibodies
to SIV strains mac, agm, sm, mne and sykes as well as human and
non-human primate serum and plasma containing antibodies to HIV-2
were reactive. Reactivity was observed with macaques infected with
SHIV strains SF, IIIB, 229, and 89.6p. |
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LIMITATIONS
OF THE PROCEDURE |
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Optimal
assay performance requires adherence to the procedures described in
this insert. Deviations from the procedure may cause aberrant
results.
Highly reactive specimens may cause overdevelopment of the strip
making interpretation of the bands difficult due to strong
background staining. Such specimens may be diluted and retested.
A negative blot result does not exclude infection with SIV.
Occasionally, reactivity of specimens to bands that do not
correspond to SIV antigens may occur. This may be due to antibodies
cross-reactive with cellular proteins found in the host cells used
to propagate the SIV virus. |
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REFERENCES |
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1. Beer BE, Bailes E, Sharp PM, Hirsch VM, 1999.
Diversity and Evolution of Primate Lentiviruses. WWW document. URL
http//:hiv-web.lanl.gov/REVIEWS/Beer99/ Beer.html
2. Robertson DL, 1998. The Evolution of the AIDS Viruses. WWW
document. URL http/:igs-server.cnrs-mrs.fr/anrs/phylogenetics/
hiv_evolution.doc.html
3. Chen Z, Luckay A, Sodora DL, Telfer P, Reed P, Gettie A, Kanu JM,
Sadek RF, Yee J, Ho DD, Zhang L, Marx PA, 1997. Human
Immunodeficiency Virus Type 2 (HIV-2) Seroprevalence and
Characterization of a Distinct HIV-2 Genetic Subtype from the
Natural Range of Simian Immunodeficiency Virus-Infected Sooty
Mangabeys. Journal of Virology, 71(5):3953-3960.
4. Chen Z, Telfer P, Gettie A, Reed P, Zhang L, HO DD, Marx PA,
1996. Genetic Characterization of New West African Simian
Immunodeficiency Virus SIVsm: Geographic Clustering of
Household-Derived SIV Strains with Human Immunodeficiency Virus Type
2 Subtypes and Genetically Diverse Viruses from a Single Feral Sooty
Mangabey Troop. Journal of Virology, 70(6):3617-3627.
5. Marx PA, Li Y, Lerche NW, Sutjipto S, Gettie A, Yee J, Brotman
BH, Prince AM, Hanson A, Webster RG, Desrosiers RC, 1991. Isolation
of a Simian Immunodeficiency Virus Related to Human Immunodeficiency
Virus Type 2 from a West African Pet Sooty Mangabey. Journal of
Virology, 65(8):4480-4485.
6. Gao F, Yue L, Robertson DL, Hill SC, Hui H, Biggar RJ, Neequaye
AE, Whelan TM, Ho DD, Shaw GM, Sharp PM, Hahn BH, 1994. Genetic
Diversity of Human Immunodeficiency Virus Type 2: Evidence for
Distinct Sequence Subtypes with Differences in Virus Biology,
Journal of Virology, 68(11):7433-7447.
7. Yee JL, Jennings MB, Lowenstine LJ, Lerche NW, 1993. Performance
Characteristics of HIV-2 Serology Reagents on Blood From SIV/HIV-2
Infected Monkeys. Abstract 52 presented at the 11th Annual Symposium
on Non-human Primate Models for AIDS, September 19-22, 1993.
8. Tsang VC, Peralta JM, and Simons, 1983. Enzyme-linked
Immunoelectrotransfer Blot Techniques (EITB) for Studying the
specificities of Antigens and Antibodies Separated by Gel
Electrophoresis. Methods in Enzymology 92:377-391.
9. Towbin H, Staehlin T, and Gordon J, 1979. Electrophoretic
Transfer of Proteins from Polyacrylamide Gels to Nitrocellulose
Sheets: Procedure and Some Applications. Proc. Natl. Acad. Sci.,
76:4350-4354.
10. Desrosiers RC, 1990. The Simian Immunodeficiency Viruses. Annual
Review of Immunology, 8(2):557-78.
11. Kanki PJ, McLane MF, King NW, Letvin N L, Hunt RD, Sehgal P,
Kanki Daniel MD, Desrosiers RC, Essex M, 1985. Serologic
Identification and Characterization of a Macaque T-Lymphotropic
Retrovirus Closely Related to HTLV-III.
Science 228(4704):1199-201.
12. Daniel MD, Letvin N, King NW, Kannagi M, Sehgal PK, Hunt RD,
Kanki PJ,
Essex M., Desrosiers RC. 1985. Isolation of T-Cell Tropic
HTLV-III-Like Retrovirus from Macaques. Science 228(4704):1201-4.
13. Parekh BS, Pau C-P, Granade TC, Rayfield M, De Cock KN, Gayle H,
Schochetman G, George JR (1991) Oligomeric nature of transmembrane
gylcoproteins of HIV-2 : procedures for their efficient dissociation
and preparation of Western blots for diagnosis. AIDS 5:1009-1013
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PROCEDURAL
FLOW CHART |
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OVERNIGHT METHOD
RAPID METHOD
INCUBATE OVERNIGHT AT INCUBATE 2 HOURS AT
ROOM TEMPERATURE ROOM TEMPERATURE
WASH STRIPS 3 TIMES
PIPET WORKING CONJUGATE
INCUBATE 1 HOUR AT ROOM TEMPERATURE
WASH STRIPS 3 TIMES
PIPET SUBSTRATE
INCUBATE UP TO 15 MINUTES AT ROOM TEMPERATURE
STOP REACTION WITH WATER RINSE
READ STRIPS |
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