DESCRIPTION OF SYMBOLS USED

STRONG REACTIVE 1 vial WARNINGS AND PRECAUTIONS Precautionary Statements: P260 Do not breathe dust/ 7. For best results dispense all reagents while cold and return
CONTROL (80 Øl) to 2°C to 8°C storage as soon as possible.
fume/gas/mist/vapours/
The following are graphical symbols used in or found on Inactivated human serum with 1. For in vitro diagnostic use only. 8. It is recommended that glassware to be used with the
spray.
MP Diagnostics products and packaging. These symbols are high titered antibodies to HIV-1 2. For Professional use only. reagents should be washed with 2M hydrochloric acid and
P501 Dispose of contents/
the most common ones appearing on medical devices and and HIV-2 and non-reactive for 3. Please refer to the product labelling for information on rinsed thoroughly with distilled or deionised water prior to
container in accordance
their packaging. Some of the common symbols are explained HBsAg & anti-HCV. Contains potentially hazardous components. use.
with local/regional/national/
in more detail in the European and International Standard EN sodium azide and thimerosal 9. Use only reagent grade quality, deionised or distilled water
HEALTH AND SAFETY INFORMATION international regulations.
ISO 15223: 2012. as preservatives. to dilute reagents.
Supplemental Statements: EUH210 Safety Data Sheet is
CAUTION: This kit contains materials of human 10. All reagents must be mixed well before use.
Use by In vitro diagnostic WEAK REACTIVE 1 vial origin. No test method can offer complete available on request 11. Working Conjugate solution, Diluted Wash Buffer and
Synonym for this : medical device CONTROL (80 Øl) assurance that human blood products will not Blotting Buffer should be prepared fresh prior to use.
Contains: 0.1% Thimerosal
HIV BLOT 2.2
Expiry Date
Inactivated human serum transmit infection. HANDLE ASSAY SPECIMENS, 12. The Working Conjugate solution should be prepared using
Catalogue
Batch Code
with low titered antibodies to STRONG REACTIVE, WEAK REACTIVE AND 1. Avoid Microbial contamination of reagents when opening a polypropylene container or beaker.
Number
WESTERN BLOT ASSAY Synonyms for this are: Synonyms for this: HIV-1 ONLY and non-reactive NON-REACTIVE CONTROLS AS POTENTIALLY and removing aliquots from the original vials or bottles. 13. Do not expose reagents or perform test in an area containing
Lot Number Reference number for HBsAg, anti-HIV-2 and INFECTIOUS AGENTS. It is recommended that the 2. Do not pipette by mouth. a high level of chemical disinfectant fumes (e.g. hypochlorite
Instructions For Use anti-HCV. Contains sodium components and test specimens be handled using fumes) during storage or during incubation steps. Contact
Batch Number Re-order 3. Handle test specimens, nitrocellulose strips, Reactive,

number azide and thimerosal as good laboratory working practices. They should be Weak Reactive and Non-Reactive Controls as potentially inhibits colour reaction. Also do not expose reagents to
Temperature
0123 preservatives. disposed of in accordance with established safety infectious agents. strong light.
Limitation Caution procedures. 4. Wear laboratory coats and disposable gloves while 14. The assay should preferably be performed at room
REVISION DATE 2016-05 Note Changes Highlighted STOCK BUFFER 1 bottle temperature (25°C ± 3°C).
performing the assay. Discard gloves in bio-hazard waste-
MAE0011-ENG-5 Manufacturer Authorised CONCENTRATE (10x) (20 ml) The Strong Reactive Control, Weak Reactive Control 15. Make sure that the test strips are laid with the numbers
bags. Wash hands thoroughly afterwards.
Representative in
Tris buffer with heat inactivated and Non-Reactive Control contain Thimerosal and Sodium on the strips facing upwards.
the European 5. It is highly recommended that this assay be performed in
(18 tests kit) : 11030-018 Contains sufficient normal goat serum. Contains azide while Stock Buffer Concentrate and Wash Buffer 16. For Western Blot Assay, it is important to use a rocking

Community a biohazard cabinet.
(36 tests kit) : 11030-036 for <n> tests thimerosal as preservative. Concentrate contain Thimerosal and Conjugate contains platform shaker and not a rotary shaker. Otherwise,
6. Keep materials away from food and drink.
Sodium azide. Sodium Azide can react with copper and lead performance of the kit will be compromised. The
Consult 7. In case of accident or contact with eyes, rinse immediately
Do not reuse WASH BUFFER 1 bottle used in some plumbing systems to form explosive salts. recommended speed and tilt angle of the shaker are 12
NAME AND INTENDED USE
Instructions for with plenty of water and seek medical advice.
Use CONCENTRATE (20x) (70 ml) The quantities used in this kit are small, nevertheless when to 16 cycles per minute, and 5 to 10 degrees, respectively.
8. Consult a physician immediately in the event that
Tris with Tween-20. Contains disposing of azide-containing materials they should be flushed 17. Ensure that automated equipment if used is validated before
The MP Diagnostics HIV BLOT 2.2 is a qualitative enzyme contaminated materials are ingested or come in contact
Contents thimerosal as preservative. away with relatively large quantities of water to prevent metal use.
immunoassay for the in vitro detection of antibodies to human with open lacerations, or other breaks in the skin.
azide buildup in plumbing system. 18. Ensure that the specimens are added away from the strip.
immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) in 9. Wipe spills of potentially infectious materials immediately
human serum or plasma. It is intended for use as a more specific CONJUGATE 1 vial Pursuant to EC regulation 1272/2008 (CLP), hazardous with absorbent paper and swab the contaminated area with Tray can be tilted and specimen added where the buffer is
CHEMICAL & BIOLOGICAL PRINCIPLES OF THE
supplemental test on human serum or plasma specimens found Goat anti-human IgG (160 Øl) components are classified and labelled as follows: 1% sodium hypochlorite solution before work is resumed. collected at lower end. This prevents dark spot formation
PROCEDURE
repeatedly reactive using screening procedures such as the conjugated with alkaline Sodium hypochlorite should not be used on acid containing due to specimen addition on the strip.
Enzyme-Linked Immunosorbent Assay (ELISA). The nitrocellulose strips are incorporated with separated, bound phosphatase. Contains spills unless the area is wiped dry with absorbent paper 19. Avoid the use of self-defrosting freezers for the storage of
antigenic proteins from partially purified inactivated HIV-1 using sodium azide as preservative. Component: Nitrocellulose strips first. Material used (including disposable gloves) should reagents and samples.
electrophoretic blotting, plus a specific HIV-2 synthetic peptide Signal Word: Danger be disposed off as potentially biohazardous material. Do 20. We do not recommend the use of diluted or lyophilized
on the same strips. Individual nitrocellulose strips are incubated SUBSTRATE 1 bottle not autoclave material containing sodium hypochlorite. samples, as they may give false results. If they form part
INTRODUCTION with diluted serum or plasma and controls. Specific antibodies Solution of 5-bromo-4- chloro- (100 ml) Pictogram: or a whole QC panel, they should be validated.
10. Autoclave all used and contaminated materials at 121°C
to HIV-1 and HIV-2 if present in the specimens will bind to the 3-indolyl- phosphate (BCIP) at 15 p.s.i. for 30 minutes before disposal. Alternatively,
Screening tests are widely available for detecting antibodies
HIV-1 proteins and HIV-2 peptide on the strips. The strips are and nitroblue tetrazolium decontaminate materials in 5% sodium hypochlorite STORAGE
to both HIV-1 and HIV-2, the etiologic agents of the Acquired washed to remove unbound materials. Antibodies that bind
Immunodeficiency Syndrome (AIDS). Such tests can be (NBT). Hazard Statements: H228 Flammable solid solution for 30-60 minutes before disposal in biohazard
specifically to HIV proteins can be visualized using a series of waste-bags. 1. Store MP Diagnostics HIV BLOT 2.2 kit and its components
extremely sensitive but have a potential for being less reactions with goat anti-human IgG conjugated with alkaline Precautionary Statements: P210 Keep away from heat/
specific, leading to false positive interpretations. Independent BLOTTING POWDER 10 packets 11. Decontaminate all used chemicals and reagents by adding at 2-8°C when not in use.
phosphatase and the substrate BCIP/NBT. This method has the Non-fat dry milk (1g each) sparks/open flames/hot
supplemental tests of high specificity are therefore necessary sufficient volume of sodium hypochlorite to make a final
sensitivity to detect marginal amounts of HIV specific antibodies surfaces. – No smoking.
to further confirm the presence of antibodies to HIV-1 and/ concentration of at least 1%. Leave for 30 minutes to 2. All test reagents and strips when stored at 2°C to 8°C, are
in serum or plasma. P280 Wear protective
or HIV-2. Incubation Tray* ensure effective decontamination. stable until the expiry date given on the kit. Do not freeze
gloves/protective clothing/ reagents.
12. We do not recommend re-use of incubation trays.
KIT COMPONENTS Instructions For Use 1 copy eye protection/face
The MP Diagnostics HIV BLOT 2.2 kit is intended for use protection. A. Antigen strips
as a more specific supplemental test on human serum or ANALYTICAL PRECAUTIONS
Component Description Quantity 1 pair • Avoid unnecessary exposure of antigen strips to light.
plasma specimens found repeatedly reactive using ELISA. Forceps Supplemental Statements: EUH210 Safety Data Sheet is
Provided 1. O p t i m a l a s s a y p e r f o r m a n c e r e q u i r e s S T R I C T
The separated specific HIV-1 viral antigens incorporated onto available on request
ADHERENCE to the assay procedure described in this B. Reagents
the strips via electrophoretic and electrotransblot procedures, NITROCELLULOSE STRIPS Available Contains: 100% Nitrocellulose Instructions For Use. Deviations from the procedure may • Store reagents in their original vials or bottles, and they
combined with a specific HIV-2 synthetic peptide on the same Note : Volume of reagents provided are sufficient for 4 runs.
I n c o r p o r a t e d w i t h H I V- 1 in 18 or 36 lead to aberrant results. should be capped for storage.
strip allow for further delineation of the antibody responses viral lysate, a specific HIV-2 strips 2. DO NOT MODIFY OR SUBSTITUTE REAGENTS FROM • Dispense all reagents while cold and return to 2°C to
* Incubation trays provided but packed separately from the kit. Component: STOCK BUFFER
to specific viral proteins. Each strip also includes an internal envelope peptide and a serum ONE KIT LOT TO ANOTHER. Controls, conjugate and 8°C storage as soon as possible.
CONCENTRATE (10x)
sample addition control to minimize the risk of false negatives addition control band. Keep Western Blot strips are matched for optimal performance. • Precipitates may form when the Substrate is stored at
WASH BUFFER
due to operational errors and to ensure the addition of samples. dry and away from light. Use only the reagents supplied with the kit. 2°C to 8°C. This will not affect the performance of the kit.
CONCENTRATE (20x)
3. Do not use kit components beyond the expiry date printed
Signal Word: Warning
on the kit box. Caution: Avoid unnecessary exposure of substrate to
NON-REACTIVE CONTROL 1 vial Pictogram: 4. Avoid microbial contamination of the reagents,when light.
Inactivated normal human (80 Øl) opening and removing aliquots from the original vials or
serum non-reactive for bottles, as this will prematurely reduce the shelf life of the SPECIMEN COLLECTION, TRANSPORT AND STORAGE
Hepatitis B surface antigen kits and give erroneous results. Use aseptic techniques
(HBsAg), antibodies to HIV- Hazard Statements: H373 May cause damage to
including pipettes or disposable pipette tips when drawing Serum or plasma samples collected in EDTA, heparin or sodium
1/2, and anti-HCV. Contains organs through prolonged or
aliquots from vials. citrate may be used. Before storage, ensure that blood clot or
sodium azide and thimerosal repeated exposure
5. The kit controls should be assayed concurrently with blood cells have been separated by centrifugation.
as preservatives. patients’ samples for each test run.
6. Use a new pipette tip for each specimen aliquot to prevent Samples should be stored at 2°C to 8°C if the test is to be run
cross contamination. within 7 days of collection or frozen at -20°C or colder if the test
is to be delayed for more than 7 days. Clear, non-hemolyzed
1 2 3

samples are preferred. Lipemic, icteric or contaminated AMOUNT OF REAGENTS REQUIRED 7. Carefully uncover the tray to avoid 9. Add 2 ml of WORKING CONJUGATE 2 ml Strong Reactive Control strip due to low titer of anti-p55 in bands seen as p42 and p39 are both GAG fragments and
(particulate) samples should be filtered (0.45Øm) or centrifuged FOR VARIOUS NUMBER OF STRIPS splashing or mixing of samples. Tilt the SOLUTION to each well. the Strong Reactive Control provided. The serum control should not be interpreted as gp41 (ENV).
before testing. tray to aspirate the mixture from the wells. 10. Cover tray and incubate for 30 minutes 30 minutes band will be visible. The HIV-2 specific band should also be 3. p24 protein is abundant in HIV Blot 2.2 strip. For
Reagents NUMBER OF STRIPS TO BE USED Change aspirator tips between samples at room temperature (25 ± 3°C) on the visible as shown in Figure 1a. seroconverting specimens, it is well established that anti-p24
Samples can be inactivated but this is not a requirement for 3 6 9 15 20 27 36 to avoid cross- contamination. rocking platform. is the first to appear on Western Blot assays. Appearance
8. Wash each strip 3 times with 2 ml of 3 x 2 ml 11. Aspirate CONJUGATE from the wells. 3. WEAK REACTIVE CONTROL
optimal test performance. Diluted Wash Buffer (ml) 60 100 140 240 300 400 600 3 x 2 ml of p24 band in HIV infected patients would fulfil the positive
DILUTED WASH BUFFER allowing Wash as in step 8. The Weak Reactive control provides a measure of the
Blotting Buffer (ml) 20 40 60 80 100 120 160 sensitivity of the kit. Weak bands at p24 and/or gp41 and interpretation criteria for gag protein by WHO, CDC and
Inactivate as follows: 5 minutes soak on the rocking platform 12. Add 2 ml of SUBSTRATE SOLUTION to 2 ml other international criteria.
Blotting Powder (g) 1 2 3 4 5 6 8 between each wash. each well. gp120/gp160 should appear. Some additional weak bands
1. Loosen cap of sample container. may or may not be present. The serum control band will be 4. The POL bands p66, p51 and p31 are generally detected
2. Heat-inactivate sample at 56°C for 30 minutes in a water Working Conjugate 7 13 19 31 41 55 73 9. Add 2 ml of WORKING CONJUGATE 2 ml 13. Cover tray and incubate for 15 minutes 15 minutes simultaneously. However the sensitivity of p66 and p31 are
SOLUTION to each well. on the rocking platform. visible (Fig 1b).
bath. Solution (ml) greater than that of p51.
10. Cover tray and incubate for 1 hour at room 60 minutes (Note: The reaction can be stopped before
3. Allow sample to cool down before retightening cap. Conjugate (Øl), Rapid 14 26 38 62 82 110 146 INTERPRETATION OF RESULTS 5. HIV-2 cross reactivity is variable but typically shows reactivity
temperature (25 ± 3°C) on the rocking 15 minutes if all the bands are visible.)
4. Sample can be stored frozen until analysis. Assay with GAG and/or POL antigens. However, there can be cross
platform. 14. Aspirate the SUBSTRATE and rinse the 3 x 2 ml NOTE: Developed strips must be completely dry to avoid
Conjugate (Øl), Overnight 7 13 19 31 41 55 73 reactivity with the gp160 band in some cases, but rarely with
11. Aspirate CONJUGATE from the wells. 3 x 2 ml strips at least three times with reagent misinterpretation.
Repeated freeze-thawing of sample is not recommended. Assay gp41.
Wash as in step 8. grade water to stop the reaction (A dark
12. Add 2 ml of SUBSTRATE SOLUTION to background can result if washing is The presence or absence of antibodies to HIV-1 sample is 6. There is also a high molecular weight band around 160KD
Substrate (ml) 7 13 19 31 41 55 73 2 ml
ADDITIONAL MATERIALS REQUIRED BUT NOT each well. insufficient at this step). determined by comparing each nitrocellulose strip to the assay that is presumed to be a GAG-POL precursor protein . This
PROVIDED 13. Cover tray and incubate for 15 minutes 15 minutes 15. Using forceps, gently remove strips onto control strips tested with the NON-REACTIVE, STRONG is seen with some high titered HIV-2 or indeterminate (GAG
ASSAY PROCEDURE - RAPID ASSAY on the rocking platform. paper towels. Cover with paper towels and REACTIVE and WEAK REACTIVE controls. Reactive Only) sera but the band pattern is a sharp discreet
• Deionized or distilled water (Note: The reaction can be stopped before dry. Alternatively, allow strips to dry in the band which is different from the diffuse band of ENV gp160.
• Disposable gloves Note: a) Users can use either the rapid or overnight assay to 15 minutes if all the bands are visible.) wells of the tray. Figure 1a is suggested as an aid to identify the various bands
• Rocking platform (designed with a rocking speed range of run the tests. HIV bands are more developed and 14. Aspirate the SUBSTRATE and rinse the 3 x 2 ml 16. Mount strips on worksheet (non- developed on the STRONG REACTIVE Control strip. The The interpretation process involves the following:-
12 to 16 cycles per minute, and which moves through a 5° strips at least three times with reagent absorbent white paper). Do not apply Strong Reactive Control as provided in the kit may contain
more bands may appear with the overnight assay, 1. Validate that the serum control band is visible. If the control
to 10° tilt to wash membranes evenly) grade water to stop the reaction (A dark adhesive tape over the developed bands. relatively low titer of anti-p55 and anti-p39; as a result, p55 and
but the overall performance of the two assays is the is negative, the results should be considered invalid as
• Pipettors and tips of appropriate volume background can result if washing is Observe the bands (See Interpretation p39 band for the Strong Reactive Control may appear faintly on
same. this indicates a technical error such as not adding sample,
• Aspirator with sodium hypochlorite trap insufficient at this step). of Results) and grade the results. For the assayed strips. This has no impact on the performance of
b) Aspirate all used chemicals and reagents into a trap HIV Blot 2.2 strips in detecting anti-p55 and anti-p39 present in conjugate or substrate.
• 56°C water bath (optional) 15. Using forceps, gently remove strips onto storage, keep the strips in the dark.
containing Sodium hypochlorite. the specimens, as each lot of strip contains sufficient amount 2. Identify the molecular weight of each band of the test strip
• Sodium hypochlorite for decontamination paper towels. Cover with paper towels and
c) All incubations are to be carried out on a rocking of p55 and p39 antigens. using the STRONG and/or WEAK REACTIVE Control strips
dry. Alternatively, allow strips to dry in the
SUMMARY OF ASSAY PROTOCOLS as a guide.
PREPARATION OF REAGENTS platform. wells of the tray.
PLEASE NOTE: The numbered end of the strips should be 3. Interpretation of the test strip is then based on the detection
16. Mount strips on worksheet (non-absorbent Reagents Qty Room Temp Room Temp
1. DILUTED WASH BUFFER Caution: placed at the bottom as shown in the Figure, i.e. the gp120/ of specific band patterns as recommended by the appropriate
white paper). Do not apply adhesive tape Rapid Assay Overnight
(a) DILUTED WASH BUFFER should be prepared fresh Some samples cause dark patches on the spot of the strip Assay gp160 bands are the furthest away from the numbered end. authorities (i.e. Health Ministry, World Health Organization,
over the developed bands. Observe the
prior to use. where they are added. To avoid this problem, one should etc.)
bands (See Interpretation of Results) and Nitrocellulose strip 1 - -
(b) Dilute 1 volume of WASH BUFFER CONCENTRATE ensure the following:- grade the results. For storage, keep the MOLECULAR GENE ANTIGEN DESCRIPTION Specific guidelines for interpretation may differ depending on
(20x) with 19 volumes of reagent grade water. Mix strips in the dark. Wash Buffer 2 ml 1-2 mins 1-2 mins
i. Sample should be added only after BLOTTING BUFFER is WEIGHT the local policies. MP Biomedicals recommends following the
well. added. Blotting Buffer 2 ml - - accepted policy to be in accordance with local regulations.
gp 160 ENV Polymeric form of gp41 Broad diffuse
2. BLOTTING BUFFER ii. Tilt the tray slightly by elevating either the top or bottom end ALTERNATIVE PROCEDURE - OVERNIGHT ASSAY Specimen 20 Øl 60 mins Overnight Listed below are some of the criteria guidelines recommended
glycoprotein
(a) BLOTTING BUFFER should be prepared fresh prior of the tray. The Blotting Buffer will flow to the lower end of the (16 - 20 by different international organizations.
tray. Add the sample where the Blotting Buffer is collected. Procedure: gp 120 ENV Outermembrane Diffuse glycoprotein
to use. hours)
(b) Dilute 1 volume of STOCK BUFFER CONCENTRATE When all the samples are added, return the tray back to its 1. Add 2 ml of DILUTED WASH BUFFER to 2 ml p66 POL Reverse Transcriptase Discreet band ORGANIZATION CRITERIA FOR POSITIVE
original flat position. Always ensure that the strips are kept Wash Buffer 3 x 2 ml 3 x 5 mins 3 x 5 mins INTERPRETATION OF
(10x) with 9 volumes of reagent grade water. Mix well. each well. p55 GAG Precursor protein Discreet band
(c) Add 1 g of BLOTTING POWDER to every 20 ml of the wet during the process. 2. Using forceps, carefully remove required Conjugate 2 ml 60 mins 30 mins WESTERN BLOT TESTS
diluted STOCK BUFFER prepared in step 2(b) above. iii. Alternatively, if tilting the tray is not desired, the samples number of STRIPS from the tube and p51 POL Reverse Transcriptase Discreet band just below Association of State and Territorial Any two of p24, gp41, gp120/gp160
Wash Buffer 3 x 2 ml 3 x 5 mins 3 x 5 mins
Stir to ensure powder dissolves completely. may be added to the top or bottom end of the well. This place numbered side up into each well. p55 Public Health Laboratory Directors / bands
(d) Stir again before dispensing. way if dark patches showed, the reading of the strip results Include strips for Strong Reactive, Weak Substrate 2 ml 15 mins 15 mins Centers for Disease Control
p39 GAG Fragment of p55 Discreet band
will not be affected. Reactive and Non-Reactive controls. (Ready to use) (or less) (or less) (ASTPHLD1 /CDC), 1989 USA
3. WORKING CONJUGATE SOLUTION 3. Incubate the strips for 1 to 2 minutes at gp41 ENV Transmembrane Diffuse glycoprotein
2 minutes Distilled Water 3 x 2 ml - - Center Nationale Transfusion Sanguine Two ENV bands with GAG or POL
Note : Prepare solution in polypropylene container / beaker. Procedure: room temperature (25 ± 3°C) on a rocking p31 POL Endonuclease Doublet World Health Organization (WHO), Two ENV bands with or without GAG
(a) WORKING CONJUGATE SOLUTION should be platform (speed of 12 to 16 cycles per
prepared fresh prior to use. 1. Add 2 ml of DILUTED WASH BUFFER to 2 ml QUALITY CONTROL p24 GAG Core protein Broad band 1990 or POL
minute). Remove buffer by aspiration.
(b) For RAPID ASSAY PROTOCOL, prepare WORKING each well. Consortium for Retrovirus Serology One ENV band with p24 or p31
(Note: Do not allow the strips to dry. We recommend that the Non-Reactive, Strong Reactive and p17 GAG Core protein Broad band
CONJUGATE SOLUTION by diluting CONJUGATE 2. Using forceps, carefully remove required Standardization (CRSS), 1988 USA
Failure may result in watery marks on Weak Reactive controls be run with every assay regardless of
at 1:500 into BLOTTING BUFFER, for example, 10 Øl number of STRIPS from the tube and American Red Cross (ARC), 1988 One band each of GAG, POL and
developed strips for some specimens.) the number of samples tested. In order for the results obtained Some of the different antigens mentioned in the Table above
CONJUGATE to 5ml BLOTTING BUFFER. place numbered side up into each well. USA ENV
4. Add 2 ml of BLOTTING BUFFER to each from any assay to be considered valid, the following conditions are derived from the same precursor protein and may have
(c) For OVERNIGHT ASSAY PROTOCOL, prepare Include strips for Strong Reactive, Weak 2 ml
well. must be met: overlapping epitopes. This should be considered when Chinese Center for Disease Control Two ENV bands OR one ENV with
WORKING CONJUGATE SOLUTION by diluting Reactive and Non-Reactive controls.
5. Add 20 Øl each of patients’ sera or interpreting the pattern, for example:- and Prevention (CCDCP), 2004 PRC p24 band
CONJUGATE at 1:1000 into BLOTTING BUFFER, 3. Incubate the strips for 1 to 2 minutes at 2 minutes 20 Øl 1. NON-REACTIVE CONTROL
controls to appropriate wells. National and State Reference One ENV band with any three of GAG
for example, 5 Øl CONJUGATE to 5ml BLOTTING room temperature (25 ± 3°C) on a rocking No HIV-1 and HIV-2 specific bands should be observed on
6. Cover the tray with the cover provided 1. It is unlikely to detect gp41 in the absence of gp160 Laboratories (NRL) 1987, Australia or POL bands
BUFFER. platform (speed of 12 to 16 cycles per overnight the Non-Reactive control strips. The band for the serum
and incubate overnight (16 - 20 hours) because the gp160 is the polymeric form of gp41 and the German Association for Control of One ENV with at least one GAG or POL
minute). Remove buffer by aspiration. control should be visible (Fig 1c).
4. SUBSTRATE SOLUTION (ready to use) at room temperature (25 ± 3°C) on the concentration of gp160 is higher than gp41 on the MP Viral Diseases (DVV) band, see also DIN 58 969, part 41
(Note: Do not allow the strips to dry.
(a) Dispense directly the required volume from the bottle. rocking platform. Diagnostics HIV BLOT 2.2. The gp41 appears as a diffuse
Failure may result in watery marks on 2. STRONG REACTIVE CONTROL
Use a clean pipette. Cap tightly after use. 7. Carefully uncover the tray to avoid band. Any sharp and discreet band at the gp41 region should 1
ASTPHLD has been renamed Association of Public Health
developed strips for some specimens.) All relevant molecular weight bands must be evident. Figure
splashing or mixing of samples . Tilt the not be interpreted as gp41 band. Many non-HIV infected and Laboratories (APHL) in 1998.
4. Add 2 ml of BLOTTING BUFFER to each 2 ml 1a provides a guide to the relative positioning of bands
tray to aspirate the mixture from the wells. normal specimens are found to be reactive to this non-HIV
well. visualized with the MP Diagnostics HIV BLOT 2.2 and We recommended the following guidelines for the interpretation
Change aspirator tips between samples antigen which is likely to originate from the human cell line
5. Add 20 Øl each of patients’ sera or controls 20 Øl permits identification of bands observed for the STRONG of the MP Diagnostics HIV BLOT 2.2. Results should be
to avoid cross-contamination. used to grow the HIV virus.
to appropriate wells. Care should be REACTIVE CONTROL. The bands are p17, p24, p31, gp41, recorded for each band detected, result should be interpreted
8. Wash each strip 3 times with 2ml of 3 x 2 ml
taken to ensure specimens are not added p51, p55, p66, gp120/gp160. Other bands associated with 2. p55 is the precursor for p24 and p17. as NEGATIVE, POSITIVE or INDETERMINATE.
DILUTED WASH BUFFER allowing
directly on the strips. core antigens (p39, p42) may also be visible. Be careful not The p55 band is generally detected when there is strong
5 minutes soak on the rocking platform
6. Cover the tray with the cover provided and 60 minutes to misinterpret these as gp41. The envelope antigens, gp41, reactivity to p24 and/or p17, it normally appears as a thin
between each wash.
incubate for 1 hour at room temperature gp120/gp160 appear as diffuse bands as they are typical of band just above p51 band, sometimes these two bands are
(25 ± 3°C) on the rocking platform. glycoproteins; p55 viral band may appear faintly on the actual indistinguishable and may appear as a single band. The
4 5 6
 PATTERN INTERPRETATION However, nucleic acid tests (NAT) for HIV DNA or RNA were not Table 2: Specificity study of HIV-1 viral antigen reactivity A total of 15 commercial HIV-1 seroconversion panels were 8. F. Clavel., 1987. HIV-2, the West African AIDS virus. AIDS MP Biomedicals Asia Pacific Pte Ltd.
approved for diagnostic purpose by the relevant authorities (US with normal donor samples and sera with other viral tested with MP Diagnostics HIV Blot 2.2 and results showed that 1:135-140. 2 Pioneer Place
No viral specific bands present NEGATIVE CDC, 2001; Constantine & Zink, 2005) until very recently. To infections. the MP Diagnostics HIV Blot 2.2 was able to detect antibody to Singapore 627885
Detection of p17 antibodies NEGATIVE date, only one RNA qualitative assay has been approved by the HIV earlier or in the same sample in all the panels. 9. R.S. Tedder, A. Hughes, T. Corrah et al 1988. Envelope Tel. No. : + 65 6775 0008
ONLY, no other bands US FDA for diagnosis of primary and acute infection of HIV-1. HIV-1 REACTIVITY cross-reactivity in Western Blot for HIV-1 and HIV-2 may Fax. No. : + 65 6774 6146
Detection of 2 ENV HIV-1 POSITIVE Therefore, test algorithms recommended by the US CDC (2001) SAMPLE NUMBER LIMITED EXPRESSED WARRANTY DISCLAIMER not indicate dual infection. Lancet 11:927-930. Email : enquiry_ap@mpbio.com
POSITIVE
(gp160/gp41and gp120) and and WHO (2004) are yet to be updated, and NAT are yet to be TYPE TESTED INDETERMINATE3 NEGATIVE
GAG (p17, p24, p55) or POL included as methods for resolving INDETERMINATE Western The manufacturer makes no expressed warranty other than 10. Bottiger B., A. Karlsson, F. Andreasson et al. 1990. Envelope
(p31, p51, p66) Blot results. Nevertheless, US CDC (2001) acknowledged that Normal 208 0 11 197 that the test kit will function as an in vitro diagnostic assay cross-reactivity between Human Immunodeficiency Virus
Detection of 2 ENV HIV-1 POSITIVE when in consultation with clinical and infection status among Donors within the specifications and limitations described in the Type 1 and Type 2 detected by different serological
(gp160/gp41 and gp120) and with persons with an initial INDETERMINATE Western Blot. HTLV-1 5 0 0 5 Product Instructions For Use when used in accordance with methods: Correlation between cross-neutralization and MP Biomedicals Germany GmbH
GAG (p17, p24, p55) or POL HIV-2 INDICATED the instructions contained therein. The manufacturer disclaims reactivity against the main neutralizing site. J. Virol. Thüringer Straße 15
CMV 5 0 1 4
(p31, p51, p66) and HIV-2 LIMITATION OF THE METHOD any warranty, expressed or implied, including such expressed 64(7):3492-3499. 37269 Eschwege
specific band is visible EBV (IgM) 5 0 1 4 or implied warranty with respect to merchantability, fitness Germany
Any viral specific bands present INDETERMINATE 2 Detection of antibodies to HIV-1 does not constitute a V.zoster (IgG) 5 0 1 4 for use or implied utility for any purpose. The manufacturer 11. Centers for Disease Control. 2001. Revised Guidelines Tel. No. : +49 5651 921 204
but pattern does not meet diagnosis of Acquired Immune Deficiency Syndrome (AIDS). A Measles 6 0 2 4 is limited to either replacement of the product or refund of the for HIV Counseling, Testing, and Referral and Revised Fax No. : +49 5651 921 181
criteria for POSITIVE NEGATIVE BLOT is not a guarantee that the causative agent Rubella 5 0 1 4 purchase price of the product. The manufacturer shall not be Recommendations for HIV Screening of Pregnant Women Email : diagnostics@mpbio.com
Any viral specific bands present INDETERMINATE2 for AIDS is not present. Although a blot POSITIVE for antibodies liable to the purchaser or third parties for any damage, injury — United States, Morbid. Mortal. Weekly Rep. 50: RR-19.
Mumps 4 0 1 3
but pattern does not meet with to HIV-1 indicates infection with the virus, a diagnosis of AIDS or economic loss howsoever caused by the product in the use
criteria for POSITIVE but HIV-2 HIV-2 INDICATED can only be made clinically if a person meets the case definition Adenovirus 5 0 2 3 or in the application thereof. 12. Fiebig, E. W., D. J. Wright, B. D. Rawal, P. E. Garrett, R.
specific band is visible. of AIDS established by the Center for Disease Control (USA), HSV 5 0 0 5 T. Schumacher, L. Peddada, C. Heldebrant, R. Smith, A. Regional Office:
the World Health Organization or other relevant authorities. Dengue 5 0 1 4 TECHNICAL PROBLEMS / COMPLAINTS Conrad, S. H. Kleinman, and M. P. Busch. 2003. Dynamics
2
INTERPRETATION OF RESULTS FOR INDETERMINATE of HIV viremia and antibody seroconversion in plasma MP Biomedicals Germany GmbH
Total 258 0 21 237
It is known that persons who have recently seroconverted may Should there be a technical problem / complaint, please do donors: implications for diagnosis and staging of primary Thüringer Straße 15
INDETERMINATE results should not be used as the basis display incomplete pattern but increase reactivity (both number 3
All showed as a p24 or p17 band only. the following : HIV infection. AIDS. 17:1871-1879. 37269 Eschwege
for diagnosis of HIV-1 infection. Based on the fact that most and intensity of bands) occurs when followed for a period of 1. Note the kit lot number, the expiry date and the strip lot Germany
persons with an initial INDETERMINATE result who are two to six months. Most blots with POSITIVE results will have Table 3 : Sensitivity study of HIV-2 peptide band with HIV-2 number. 13. Ly, T. D., C. Edlinger, and A. Vabret. 2000. Contribution of Tel. No. : +49 5651 921 204
infected with HIV-1 will develop detectable HIV antibodies other viral specific bands present. seropositive samples. (Number of samples = 178) 2. Retain the kits and the results that were obtained. combined detection assays of p24 antigen and anti-human Fax No. : +49 5651 921 181
within 1 month, US CDC (2001) recommended such persons 3. Contact the nearest MP Biomedicals office or your local immunodeficiency virus (HIV) antibodies in diagnosis of Email : diagnostics@mpbio.com
be re-tested for HIV-1 infection ≥1 month later. Persons with INDETERMINATE results should not be used as the basis distributor. primary HIV infection by routine testing. J Clin Microbiol.
continued INDETERMINATE results after 1 month are unlikely HIV-2 Western Blot HIV-2 peptide Reactivity
for diagnosis of HIV-1 infection. It is recommended that 38:2459-2461.
to be HIV-infected unless recent HIV exposure is suspected. all INDETERMINATE blots be repeated using the original Serological profile@ Positive Negative BIBLIOGRAPHY
specimen and sequential samples. Blood donors with an 14. Sethoe, S. Y., A. E. Ling, E. H. Sng, E. H. Monteiro, R.
Based on a recent study of Fiebig et al (2003), although the GAG, POL and 2 ENV 160 0
INDETERMINATE blot should be re-tested using a fresh 1. V.C.W.Tsang, K. Hancock, M. Wilson. D.F. Palmer, S. K. Chan. 1995. PCR as a confirmatory test for human * U.S. Patent 5,721,095
window period for Western Blot in the case of a primary HIV-1 specimen after one month (US CDC, 2001). In addition, Whaley, J.S. Mc Dougal, and S. Kennedy. March 1985. immunodeficiency virus type 1 infection in individuals with
GAG, POL and 1 ENV 18 0
infection could be as long as 22 days, the progression from antibodies to p24 and p31 are known to decrease during the Developmental Procedure : Enzyme-linked Immunoelectro- indeterminate western blot (immunoblot) profiles. J Clin
an INDETERMINATE blot to a full POSITIVE profile took no course of AIDS leading to a shift in blot interpretation from @
Sera define as positive by results of Pasteur New LAV Blot 2. transfer Blot technique for HTLV-III/LAV antibodies; CDC, Microbiol. 33:3034-3036.
longer than 8 days. In addition, this laboratory stage of having POSITIVE to INDETERMINATE. Interpretation of results Data provided by Dr. Oliviero E. Varnier and Dr. Flavia Lillo. Altanta.
Western Blot INDETERMINATE was always accompanied should then be based on subsequent blot testing and clinical Laboratory of Human Retroviruses. University of Genoa. 15. Constantine, N. T. and H. Zink. 2005. HIV testing
with detectable RNA of HIV-1 with cases of true infection. evaluations in such situations. 2. H. Towbin, T. Staehlin, and J. Gordon. 1979. Electrophoretic technologies after two decades of evolution. Indian J Med
Conversely, no seroconversion was evident in follow-up Table 4 : Specificity study of HIV-2 peptide band with HIV-1 transfer of proteins from polyacrylamide gels to nitrocellulose Res. 121:519-538.
studies of individuals having screened positive and Western Due to its highly specific nature , NON-REACTIVITY of samples seropositive sera, normal donor samples and sera sheets: procedure and some applications. Proc. Natl. Acad.
Blot INDETERMINATE results, once confirmed as negative by with HIV-2 specific envelope peptide on an Indeterminate viral with other viral infections. Sci.. USA 76: 4350-4354. 16. World Health Organization. 2004. Guidelines for HIV
PCR methods (Sethoe et al, 1995). Therefore, it is reasonable blot, does not exclude the possibility of infection with other Diagnosis and monitoring of antiretroviral therapy. Regional
to consider persons having Western Blot INDETERMINATE strains of HIV-2. 3. J. Schupbach, M. Popovic, R. V. Gilden. M.A. Gonda, M. G. Office for South-East Asia, New Delhi, India.
SAMPLE NUMBER HIV-2 PEPTIDE REACTIVITY
results but additionally tested negative by a RNA test as unlikely Sarngadharan and R. C. Gallo. 1984. Serological Analysis
to be HIV-infected, especially when the tested individuals are TYPE TESTED POSITIVE NEGATIVE
Samples that are indicated as HIV-2 infections should be further of subgroup of Human T-Lymphotropic retroviruses (HTLV- 17. Ming Guan, Frequency, causes and new challenges of
known as not having any risk factor associated with exposure. tested with a HIV-2 Western Blot Kit. HIV-1 seropositive 197 16a 181 III) associated with AIDS. Science 224, 503-505. indeterminate results in Western Blot Confirmatory Testing
for Antibodies to Human Immunodeficiency Virus. Clinical
In particular, persons having Western Blot INDETERMINATE Normal Donors 208 0 208
4. M. G. Sarngadharan, M. Popovic, L. Bruch, J. Schupbach and Vaccine Immunology, June 2007, Vol.14, No.6, p649-
results derived from a test algorithm using fourth generation SPECIFIC PERFORMANCE CHARACTERISTICS HTLV-1 seropositive 5 0 5 and R. C. Gallo. 1984. Antibodies reactive with human 659.
ELISAs as the primary screen test should additionally be CMV 5 0 5 T-Lymphotropic retroviruses (HTLV-III) in the serum of
tested for viral RNA using a molecular-base test such as The performance of MP Diagnostics HIV BLOT 2.2 for the patients with AIDS. Science 224, 506-608.
RT-PCR with primer sets covering HIV-1/2/O. If necessary, detection of antibodies to HIV-1 or HIV-2 was evaluated in EBV (IgM) 5 0 5
a follow-up should be considered with any supplemental test clinical studies. V.zoster (IgG) 5 0 5 5. CDC. 1985. “ Provisional public health service inter-
1 month later. The unique design of fourth generation ELISAs agency recommendations for screening donated blood
Table 1 : Sensitivity study of HIV-1 viral antigen reactivity with Measles 6 0 6
is for a simultaneous detection of both antigen and antibody. and plasma for antibody to the virus causing Acquired
Consequently specimens identified as positive by a fourth HIV-1 seropositive samples. (Number of samples = Rubella 5 0 5 Immune Deficiency Syndrome” - United States Morbidity
generation ELISA should contain either antibody or antigen or 201) Mumps 4 0 4 and Mortality Weekly Report 34 (1) :1-5.
both. Although more than 95% of those cases of true positive
SEROLOGICAL HIV BLOT 2.2 DUPONT/ORTHO Adenovirus 5 0 5
identified by a fourth generation ELISA were anti-HIV related 6. Proposed World Health Organization 1990 criteria for
and verifiable (confirmed) by Western Blot (Ly et al., 2000), a PROFILE HIV-1 WB HSV 5 0 5 interpreting results from Western blot assays for HIV-1,
supplemental test using RT-PCR appeared unavoidable for GAG, POL and 97.5% 95.4% Dengue 5 0 5 HIV-2, and HTLV-I/HTLV-II, Weekly Epidemiological Record
the small portion of reactivity relating to p24 antigen. Again, ENV 65(37), 281-283.
persons without any risk of exposure are unlikely HIV-infected, if Total 455 16 439
identified as positive by a fourth generation ELISA accompanied p24, p31, gp41 94.9% 90.9% 7. F. Clavel, D. Guetard., F. Brun-Vezinet, et al. 1986 Isolation
and/or
a
When tested on the MP Diagnostics HIV-2 Western Blot, 6 of
by a Western Blot INDETERMINATE but the findings could not of a new human retrovirus from West African patients with
gp120/gp160 these samples had reactivity with ENV and GAG or POL, and 9
be further supported by a POSITIVE result using a RNA test AIDS. Science; 233:343-346.
of these samples had reactivity to only GAG and/or POL while
with primer sets covering HIV-1/2/O. ENV and GAG 100.0% 100.0% 1 sample was negative.
or POL

7 8 9

TROUBLE SHOOTING CHART

a b c d
Non-specific bands
develop and not HIV-2 Bands other than the Strips are Sharp, discrete band
indicative Serum Control band defective at gp41 region
Expected bands do not
develop or are of weak develops on negative
gp 160 Dark spots develop intensity. control
Strong Background develops on Watery marks on
gp 120 on strips strip in the absence or presence developed strips
of positive bands. Non-specific
White patches develop
bands and/or
on strips
dark background
p 66
develop on strips
p 55 Check positive control
p 51 1. Sample is too strong
Absence of Serum
and reacts with
gp 41 trace amounts of
Control Band
1. They are cracked.
p 39 1. Strips was flipped intermediates. 2. They contain air 1.Strips left to dry after
over during assay. 2. Sample cross reacts bubbles which cause pre-soaking step prior
2. Trays not properly with H-9 proteins the appearrance of to adding Blotting
p 31 washed before use. present in viral white spots in reactive Buffer.
3. Poor dissolution of preparation (eg. HLA, zones big enough
Blotting Powder. Tray wells or Control
ABC, DR) to prevent
4. Electrotransblot may have been crossed
3. Legitimate bands any detection.
interference during contaminated.
p 24 (deglycosylated 3. They show dark spots
manufacturing. envelope antigen) due to fungal growth
has been indentified upon initial opening
at around 80-90kD in of the strip tubes.
1. Bacterial or fungal some test samples. However, if dark spots
p 17 contamination of test 1. Overdeveloped strips develop sometime
sample. (stop reaction sooner). later after initial
2. Precipitation of 2. Incomplete washing. opening of the tube
immune complexes then the problem is
in aged test sample. Positive control weak Positive control OK
due to improper strip
3. Bacterial or fungal storage conditions at
Serum 1. Serum not added.
Control contamination on the user’s site.
strip due to improper 2. S trips flipped over
HIV-2 storage. during assay.
4. Strips physically 3. Conjugate not added.
1.This is not gp41 as
damaged, cracked or The problem is probably The problem is probably 4. Substrate not added.
gp41 is a diffuse band.
scratched. caused by the reagents. caused by test sample.
2. Do not interpret as
5. Strips not properly
gp41.
washed between 1.Reagents not properly 1. Wrong test sample
3. This is possibly a cell
assay steps. prepared. dilution.
line protein, p42.
2. Wrong conjugate 2. Test sample
1. Wrong test sample or
dilution. contaminated with
a. Strong Reactive Control conjugate dilution.
3. Unstable reagents conjugate.
(Reactive for HIV-1 and 2. Test sample/reagent
due to improper 3. Test samples severely
HIV-2) incubation too long.
temperature exposure. immune-complexed.
3. Incomplete washing
b. Weak Reactive Control 4. Conjugate contaminated 4. Test sample IgG
during assay.
(Reactive for HIV-1 only). with human IgG. deteriorated or
4. Incubation
c. Non-Reactive Control. 5. Incorrect substrate denatured due to
temperature greater
d. A typical HIV-2 seropositive pH due to exposure repeated freeze- thaw
than 30°C.
serum. to strong UV light or or improper storage.
5.Test sample reactive
reducing agent. 5. Rotary platform used
with non-viral proteins.
6. Trays, reagent(s) instead of Rocking
or water having platform.
high phosphate 6. Test sample may
concentration. be an ELISA “false”
7. Rotary platform used positive.
instead of Rocking
platform.

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