PQDx 0198-071-00 WHO PQDx PR April/2016, version 2.

WHO Prequalification of In Vitro Diagnostics Programme

PUBLIC REPORT

Product: MP Diagnostics HIV Blot 2.2

Number: PQDx 0198-071-00

Abstract

MP Diagnostics HIV Blot 2.2 with product codes 11030-018 and 11030-036, manufactured
by MP Biomedicals Asia Pacific Pte. Ltd, CE marked regulatory version, was accepted for
the WHO list of prequalified in vitro diagnostics and was listed on 4 April 2016.

MP Diagnostics HIV Blot 2.2 kit is a qualitative enzyme immunoassay for the in vitro
detection of antibodies to HIV-1 and HIV-2 in human serum or plasma. It is intended for
use as a supplemental assay on human serum or plasma specimens found repeatedly
reactive using EIA. The separated specific HIV-1 viral antigens incorporate onto the strips
via electrophoretic and electrotransblot procedures combined with a specific HIV-2
synthetic peptide on the same strip allow for further delineation of the antibody responses
to specific viral proteins. Each strip also includes an internal specimen addition control to
minimize the risk of false negatives due to operations errors and to ensure the addition of
specimens.

The nitrocellulose strips are incorporated with separated bound antigenic proteins from
partially unpurified inactivated HIV-1 using electrophoretic blotting, plus a specific HIV-2
synthetic peptide on the same strips. Individual nitrocellulose strips are incubated with
diluted specimens and controls. Specific antibodies to HIV-1 and HIV-2 if present in the
specimens will bind to the HIV-1 proteins and HIV-2 peptide on the strips. The strips are
washed to remove unbound materials. Antibodies that bind specifically to HIV proteins can
be visualized using a series of reactions with goat anti-human IgG conjugated with alkaline
phosphatase and the substrate BCIP/NBT. This method has the sensitivity to detect
marginal amounts of HIV specific antibodies in serum or plasma.

The test kit contains:

Component 18 tests 36 tests
(product code (product code
11030-018) 11030-036)
Nitrocellulose Strips: 18 strips 36 strips
Incorporated with HIV-1 viral lysate, a
specific HIV-2 envelope peptide and
specimen addition control band.
Non-reactive control: 1 vial (80µl) 1 vial (80µl)

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PQDx 0198-071-00 WHO PQDx PR April/2016, version 2.0

Inactivated normal human serum non-

reactive for hepatitis B surface antigen
(HBsAg), antibodies to HIV-1/2, and anti-HCV.
Contains sodium azide and thimerosal as
preservatives.
Strong reactive control: 1 vial (80µl) 1 vial (80µl)
Inactivated normal human serum with high
titered antibodies to HIV-1 and HIV-2 and
non-reactive for HBsAg, and anti-HCV.
Contains sodium azide and thimerosal as
preservatives.
Weak reactive control: 1 vial (80µl) 1 vial (80µl)
Inactivated normal human serum with high
titered antibodies to HIV-1 ONLY and non-
reactive for HBsAg, anti-HIV-2 and anti-HCV.
Contains sodium azide and thimerosal as
preservatives.
Stock Buffer concentrate (10x): 1 bottle (20ml) 1 bottle (20ml)
Tris buffer with heat inactivated normal goat
serum. Contains thimerosal as presentative.
Wash buffer concentrate (20x): 1 bottle (70ml) 1 bottle (70ml)
Tris buffer with Tween-20. Contains
thimerosal as preservative.
Conjugate: 1 vial (160µl) 1 vial (160µl)
Goat anti-human IgG conjugated with
alkaline phosphate. Contains thimerosal as
preservative.
Substrate: 1 bottle (100ml) 1 bottle (100ml)
Solution of 5-bromo-4-chloro-3-indoyl-
phosphate (BCIP) and nitroblue tetrazolium
(NBT).
Blotting powder: 10 packets (1g 10 packets (1g
Non-fat dry milk each) each)
Incubation tray: 2 trays 4 trays
9 wells each
Instruction for use: 1 copy 1 copy
Forceps: 1 pair 1 pair

Storage:
The test kit should be stored at 2-8 °C.

Shelf-life:
24 months.

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PQDx 0198-071-00 WHO PQDx PR April/2016, version 2.0

WHO special warning:

WHO reviewed the instructions for use that were current at the time of WHO
prequalification, and a number of changes were suggested. Most but not all changes were
made by the manufacturer (outstanding comments relate to general layout and
nomenclature).

Summary of prequalification status for MP Diagnostics HIV Blot 2.2

Initial acceptance
Date Outcome
Status on PQ list 4 April 2016 listed
Dossier assessment 10 October 2014 MR
Inspection status 05 June 2015 MR
Laboratory evaluation N/A MR

MR: Meets Requirements

NA: Not Applicable

MP Diagnostics HIV Blot 2.2 was accepted for the WHO list of in vitro prequalified
diagnostics on the basis of data submitted and publicly available information.

Background information

MP Biomedicals Asia Pacific Pte. Ltd, submitted an application for prequalification of MP

Diagnostics HIV Blot 2.2. Based on the established prioritization criteria, MP Diagnostics
HIV Blot 2.2 was given priority for prequalification.

Product dossier assessment

MP Biomedicals Asia Pacific Pte. Ltd, submitted a product dossier for MP Diagnostics HIV
Blot 2.2 as per the Instructions for compilation of a product dossier (PQDx_018 v1). The
information submitted in the product dossier was reviewed by WHO staff and external
experts (assessors) appointed by WHO in accordance with the internal report on the
screening and assessment of a product dossier (PQDx_009 v2). Based on the product
dossier screening and assessment findings, a recommendation was made to accept the
product dossier for MP Diagnostics HIV Blot 2.2 for prequalification.

The manufacturer committed to amend and submit additional documentation on the

following issues:

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PQDx 0198-071-00 WHO PQDx PR April/2016, version 2.0

1. The Manufacturer will investigate the effect of transport conditions on HIV Blot 2.2
shelf life including a higher temperature range (expected completion date Q4 2017).
2. The manufacturer will commence testing all material used in the production of
positive and negative test kit controls using state-of-the-art methods (i.e. nucleic
acid detection).

Manufacturing site inspection

A comprehensive inspection was performed at the site of manufacture (2 Pioneer Place,

Singapore) of MP Diagnostics HIV Blot 2.2 in November 2014 as per the Information for
manufacturers on prequalification inspection procedures for the sites of manufacture of
diagnostics (PQDx_014 v1). The inspection found that the manufacturer had an acceptable
quality management system and good manufacturing practices in place that ensured the
consistent manufacture of a product of good quality. The manufacturer's responses to the
nonconformities found at the time of the inspection were accepted 14 July 2015.

The manufacturer committed conduct the following studies which will be reviewed at the
next re-inspection: drop and shock testing for HIV Blot 2.2.

Laboratory evaluation

The objective of the performance laboratory evaluation is to assess the performance and
operational characteristics of commercially available in-vitro diagnostics for the purpose of
advising the governments of WHO Member States on these issues. In particular, suitability
for use in resource-limited settings will be assessed.

Based on the risk level associated with the use of the supplemental assays, the known
general performance of supplemental assays and role of the supplemental assays in
patient care in resource-limited settings, it was decided that WHO will not conduct
performance evaluations of these assays as part of the prequalification assessment
process.

Consequently, laboratory evaluation of MP Diagnostics HIV Blot 2.2 was not conducted.

Page 4 of 5
PQDx 0198-071-00 WHO PQDx PR April/2016, version 2.0

Labelling

1. Labels
2. Instructions for use

Page 5 of 5
 LXX0311-1

LXX0611-1

LXX0211-1

LAX0711-2
2015-04-20 2015-04-20 2015-04-20 2015-04-20

Warning

LAX0311-2
P260
P501
H373 EUH210

2015-08-13

Warning
LAX0511-2

P260
P501
H373 EUH210

2015-08-13

Danger
LAX0211-2

LAX0911-1

P210
P280
H228 EUH210

2015-04-20

2015-08-05

HIV Blot 2.2

MP Diagnostics MP Diagnostics

18 36

LAE 0011-3
2015-07-22
 contains 100% contains 0.1%
Nitrocellulose Thimerosal

Danger Warning
H228 P210 EUH210 H373 P260 EUH210
P280 P501

HAZ LAE0011-0
HIV Blot 2.2

P485 Black size: 165mm(W)x83mm(H) 2015-08-13

 DESCRIPTION OF SYMBOLS USED KIT COMPONENTS Instructions For Use 1 copy gloves) should be disposed off as potentially biohazardous material.
Hazard Statements: H228 Flammable solid Do not autoclave material containing sodium hypochlorite.
The following are graphical symbols used in or found on MP Diagnostics Component Description Quantity PROTEIN FINDER 10. Autoclave all used and contaminated materials at 121°C at 15
Precautionary P210 Keep away from heat/sparks/
products and packaging. These symbols are the most common ones Provided The protein Finder shows an image of the 1 piece p.s.i. for 30 minutes before disposal. Alternatively, decontaminate
Statements: open flames/hot surfaces. – No smoking.
appearing on medical devices and their packaging. Some of the common assayed strip belonging to the same strip materials in 5% sodium hypochlorite solution for 30-60 minutes
P280 Wear protective gloves/protective
symbols are explained in more detail in the European and International NITROCELLULOSE STRIPS Available number found in this kit and the positions of before disposal in biohazard waste-bags.
clothing/eye protection/face protection. 11. Decontaminate all used chemicals and reagents by adding sufficient
Standard EN ISO 15223: 2012. Incorporated with HIV-1 viral lysate, a specific in 18 or 36 the specific HIV bands. It helps to locate the
HIV-2 envelope peptide and a serum addition strips HIV bands in the strip. volume of sodium hypochlorite to make a final concentration of at
Supplemental EUH210 Safety Data Sheet is available on
Use by In vitro diagnostic control band. Keep dry and away from light. least 1%. Leave for 30 minutes to ensure effective decontamination.
Statements: request
HIV BLOT 2.2 Synonym for this :
Expiry Date
medical device
NON-REACTIVE CONTROL 1 vial
Forceps 1 pair
Contains: 100% Nitrocellulose
12. We do not recommend re-use of incubation trays.

WESTERN BLOT ASSAY Catalogue Number Inactivated normal human serum non-reactive (80 Øl) Note : Volume of reagents provided are sufficient for 4 assay runs.
ANALYTICAL PRECAUTIONS
Instructions For Use Batch Code Synonyms
for this: for Hepatitis B surface antigen (HBsAg), 1. Optimal assay performance requires STRICT ADHERENCE to the
Synonyms for this are: Reference number Component: STOCK BUFFER CONCENTRATE (10x)
antibodies to HIV-1/2, and anti-HCV. Contains * Incubation trays provided but packed separately from the kit. assay procedure described in this Instructions For Use. Deviations
Lot Number Re-order WASH BUFFER CONCENTRATE (20x)
0.1% sodium azide and 0.005% thimerosal as from the procedure may lead to aberrant results.
0123 Batch Number number
preservatives. 2. DO NOT MODIFY OR SUBSTITUTE REAGENTS FROM ONE KIT
Signal Word: Warning
REVISION DATE 2015-10 LOT TO ANOTHER. Controls, conjugate and Western Blot strips are
Temperature Caution WARNINGS AND PRECAUTIONS matched for optimal performance. Use only the reagents supplied
MAE0011W-ENG-0 STRONG REACTIVE CONTROL 1 vial Pictogram:
Limitation Inactivated human serum with high titered (80 Øl) 1. For in vitro diagnostic use only. with the kit.
(18 tests kit) : 11030-018 Authorised antibodies to HIV-1 and HIV-2 and non- 2. For Professional use only. 3. Do not use kit components beyond the expiry date printed on the
(36 tests kit) : 11030-036 Manufacturer Representative in reactive for HBsAg & anti-HCV. Contains 3. Please refer to the product labelling for information on potentially kit box.
(WHO version)
the European Hazard Statements: H373 May cause damage to organs 4. Avoid microbial contamination of the reagents,when opening and
0.1% sodium azide and 0.005% thimerosal hazardous components.
NAME AND INTENDED USE Community through prolonged or repeated exposure removing aliquots from the original vials or bottles, as this will
Contains sufficient as preservatives. HEALTH AND SAFETY INFORMATION prematurely reduce the shelf life of the kits and give erroneous
The MP Diagnostics HIV BLOT 2.2 is a qualitative enzyme for <n> tests Precautionary P260 Do not breathe dust/fume/gas/mist/
Consult results. Use aseptic techniques including pipettes or disposable
immunoassay for the in vitro detection of antibodies to human WEAK REACTIVE CONTROL 1 vial CAUTION: This kit contains materials of human origin. No test Statements: vapours/spray.
Instructions for pipette tips when drawing aliquots from vials.
immunodeficiency virus type 1 (HIV- 1) and type 2 (HIV-2) in human Do not reuse Inactivated human serum with low titered (80 Øl) method can offer complete assurance that human blood products P501 Dispose of contents/container in
Use 5. The kit controls should be assayed concurrently with patients’
serum or plasma. It is intended for use as a more specific supplemental antibodies to HIV-1 ONLY and non-reactive will not transmit infection. HANDLE ASSAY SPECIMENS, accordance with local/regional/national/ samples for each test run.
assay on human serum or plasma specimens found repeatedly reactive Contents for HBsAg, anti-HIV-2 and anti-HCV. Contains STRONG REACTIVE, WEAK REACTIVE AND NON- international regulations. 6. Use a new pipette tip for each specimen aliquot to prevent cross
using screening assays such as the Enzyme-Linked Immunosorbent 0.1%sodium azide and 0.005% thimerosal as REACTIVE CONTROLS AS POTENTIALLY INFECTIOUS contamination.
Assay, Chemiluminescence Assay, and Point of Care Test. This assay preservatives. AGENTS. It is recommended that the components and test Supplemental EUH210 Safety Data Sheet is available on 7. For best results, dispense all reagents while they are freshly taken
is for use by trained professionals in the laboratory setting. specimens be handled using good laboratory working practices. Statements: request out from refrigeration and return to 2°C to 8°C storage as soon as
STOCK BUFFER CONCENTRATE (10x) 1 bottle They should be disposed of in accordance with established possible. This is to preserve the shelf-life of the reagents.
CHEMICAL & BIOLOGICAL PRINCIPLES OF THE PROCEDURE Tris buffer with heat inactivated normal (20 ml) Contains: 0.1% Thimerosal
safety procedures. 8. It is recommended that glassware to be used with the reagents
goat serum. Contains 0.1% thimerosal as should be washed with 2M hydrochloric acid and rinsed thoroughly
INTRODUCTION The nitrocellulose strips are incorporated with separated, bound antigenic 1. Avoid Microbial contamination of reagents when opening and
preservative. The Strong Reactive Control, Weak Reactive Control and Non- with distilled or deionised water prior to use.
proteins from partially purified inactivated HIV-1 using electrophoretic removing aliquots from the original vials or bottles.
Screening tests are widely available for detecting antibodies to both Reactive Control contain Thimerosal and Sodium azide while Stock 9. Use only reagent grade quality, deionised or distilled water to dilute
blotting, plus a specific HIV-2 synthetic peptide on the same strips. 2. Do not pipette by mouth.
HIV-1 and HIV-2, the etiologic agents of the Acquired Immunodeficiency WASH BUFFER CONCENTRATE (20x) 1 bottle Buffer Concentrate and Wash Buffer Concentrate contain Thimerosal reagents.
Individual nitrocellulose strips are incubated with diluted serum or plasma 3. Handle test specimens, nitrocellulose strips, Reactive, Weak
Syndrome (AIDS). Such tests can be extremely sensitive but have a Tris with Tween-20. Contains 0.1% thimerosal (70 ml) and Conjugate contains Sodium azide. Sodium Azide can react with 10. All reagents must be mixed well before use.
and controls. Specific antibodies to HIV-1 and HIV-2 if present in the Reactive and Non-Reactive Controls as potentially infectious agents.
potential for being less specific, leading to false positive interpretations. as preservative. copper and lead used in some plumbing systems to form explosive 11. Working Conjugate solution, Diluted Wash Buffer and Blotting Buffer
specimens will bind to the HIV-1 proteins and HIV-2 peptide on the strips. 4. Wear laboratory coats and disposable gloves while performing
Independent supplemental tests of high specificity are therefore salts. The quantities used in this kit are small, nevertheless when should be prepared fresh prior to use.
The strips are washed to remove unbound materials. Antibodies that bind the assay. Discard gloves in bio-hazard waste-bags. Wash hands 12. The Working Conjugate solution should be prepared using a
necessary to further confirm the presence of antibodies to HIV-1 and/ CONJUGATE 1 vial disposing of azide-containing materials they should be flushed away
specifically to HIV proteins can be visualized using a series of reactions thoroughly afterwards. polypropylene container or beaker.
or HIV-2. Goat anti-human IgG conjugated with alkaline (160 Øl) with relatively large quantities of water to prevent metal azide buildup
with goat anti-human IgG conjugated with alkaline phosphatase and 5. It is highly recommended that this assay be performed in a biohazard 13. Do not expose reagents or perform test in an area containing a high
phosphatase. Contains 0.1% sodium azide as in plumbing system.
the substrate BCIP/NBT. cabinet. level of chemical disinfectant fumes (e.g. hypochlorite fumes) during
The MP Diagnostics HIV BLOT 2.2 kit is intended for use as a more preservative. 6. Keep materials away from food and drink. storage or during incubation steps. Contact inhibits colour reaction.
specific supplemental test on human serum or plasma specimens Pursuant to EC regulation 1272/2008 (CLP), hazardous components 7. In case of accident or contact with eyes, rinse immediately with Also do not expose reagents to strong light.
found repeatedly reactive using ELISA. The separated specific HIV- SUBSTRATE 1 bottle are classified and labelled as follows: plenty of water and seek medical advice. 14. The assay should preferably be performed at room temperature
1 viral antigens incorporated onto the strips via electrophoretic and Solution of 5-bromo-4- chloro-3-indolyl- (100 ml) 8. Consult a physician immediately in the event that contaminated (25°C ± 3°C).
electrotransblot procedures, combined with a specific HIV-2 synthetic phosphate (BCIP) and nitroblue tetrazolium materials are ingested or come in contact with open lacerations, or
Component: Nitrocellulose strips 15. Make sure that the test strips are laid with the numbers on the strips
peptide on the same strip allow for further delineation of the antibody (NBT). other breaks in the skin. facing upwards.
responses to specific viral proteins. Each strip also includes an internal 9. Wipe spills of potentially infectious materials immediately with 16. For Western Blot Assay, it is important to use a rocking platform
BLOTTING POWDER 10 packets Signal Word: Danger
sample addition control to minimize the risk of false negatives due to absorbent paper and swab the contaminated area with 1% sodium shaker and not a rotary shaker. Otherwise, performance of the kit
operational errors and to ensure the addition of samples. Non-fat dry milk (1g each) hypochlorite solution before work is resumed. Sodium hypochlorite will be compromised. The recommended speed and tilt angle of
Pictogram:
Incubation Tray* should not be used on acid containing spills unless the area is wiped the shaker are 12 to 16 cycles per minute, and 5 to 10 degrees,
dry with absorbent paper first. Material used (including disposable respectively. The length of the strip must be placed in the same
direction as the rocking motion.
1 2 3

17. Ensure that automated equipment if used is validated before use. ADDITIONAL MATERIALS REQUIRED BUT NOT PROVIDED 4. Add 2 ml of BLOTTING BUFFER to each well. Tilt the 2 ml 3. Incubate the strips for 1 to 2 minutes at room 2 minutes
AMOUNT OF REAGENTS REQUIRED SUMMARY OF ASSAY PROTOCOLS
18. Add the specimens and controls directly to the buffer at the opposite FOR VARIOUS NUMBER OF STRIPS tray slightly and add the specimen(s) where the buffer temperature (25 ± 3°C) on a rocking platform (speed
end of the strip numbers; DO NOT add the specimens and controls is collected at the lower end of each well as per next of 12 to 16 cycles per minute). Remove buffer by Reagents Qty Room Temp Room Temp
directly to the strip, as this may cause the formation of dark spots. • Deionized or distilled water Reagents NUMBER OF STRIPS TO BE USED step. Ensure that the strip no. is at the higher end. aspiration. Rapid Assay Overnight Assay
Tilt the tray slightly and add the specimen(s) where the buffer is • Disposable gloves 5. Add 20 Øl each of specimens to their respective 20 Øl (Note: Do not allow the strips to dry. Failure may
3 6 9 15 20 27 36 Nitrocellulose strip 1 - -
collected at the lower end of each well. • Rocking platform (designed with a rocking speed range of 12 to 16 wells, and followed by 20 Øl each of Strong Reactive, result in watery marks on developed strips for some
19. Avoid the use of self-defrosting freezers for the storage of reagents cycles per minute, and which moves through a 5° to 10° tilt to wash Diluted Wash Buffer (ml) 60 100 140 240 300 400 600 Weak Reactive and Non-Reactive Controls to their specimens.) Wash Buffer 2 ml 1-2 mins 1-2 mins
and samples. membranes evenly) respective wells. The sequence of adding specimens 4. Add 2 ml of BLOTTING BUFFER to each well. Tilt the 2 ml
Blotting Buffer (ml) 20 40 60 80 100 120 160 Blotting Buffer 2 ml - -
20. We do not recommend the use of diluted or lyophilized samples, • Precision Pipettes ranging from 20Øl to 1000Øl dispensing volume or controls first is flexible. tray slightly and add the specimen(s) where the buffer
as they may give false results. If they form part of quality control and pipette tips of appropriate volume Blotting Powder (g) 1 2 3 4 5 6 8 6. Cover the tray with the cover provided and incubate for 60 minutes is collected at the lower end of each well as per next Specimen 20 Øl 60 mins Overnight
procedure, they should be validated by the user prior to use. • Aspirator with sodium hypochlorite trap (16 - 20 hours)
Working Conjugate Solution (ml) 7 13 19 31 41 55 73 1 hour at room temperature (25 ± 3°C) on the rocking step. Ensure that the strip no. is at the higher end.
• 56°C water bath (optional) platform. 5. Add 20 Øl each of specimens to their respective 20 Øl Wash Buffer 3 x 2 ml 3 x 5 mins 3 x 5 mins
STORAGE • Sodium hypochlorite for decontamination Conjugate (Øl), Rapid Assay 14 26 38 62 82 110 146 7. Carefully uncover the tray to avoid splashing or mixing wells, and followed by 20 ul each of Strong Reactive,
• Paper towel, adhesive tape, worksheet (non-absorbent white paper) Conjugate (Øl), Overnight Assay 7 13 19 31 41 55 73 of samples. Tilt the tray to aspirate the mixture from Weak Reactive and Non-Reactive Controls to their Conjugate 2 ml 60 mins 30 mins
1. Store MP Diagnostics HIV BLOT 2.2 kit and its components at 2-8°C
the wells. Change aspirator tips between samples to respective wells. The sequence of adding specimens Wash Buffer 3 x 2 ml 3 x 5 mins 3 x 5 mins
when not in use. Substrate (ml) 7 13 19 31 41 55 73
PREPARATION OF REAGENTS avoid cross- contamination. or controls first is flexible.
8. Add 2 ml of DILUTED WASH BUFFER. Wash each 3 x 2 ml 6. Cover the tray with the cover provided and incubate overnight Substrate 2 ml 15 mins 15 mins
2. All test reagents and strips when stored at 2°C to 8°C, are stable until 1. DILUTED WASH BUFFER (Ready to use) (or less) (or less)
ASSAY PROCEDURE - RAPID ASSAY strip 3 times with 5 minutes soak on the rocking overnight (16 - 20 hours) at room temperature (25 ±
the expiry date given on the kit. Do not freeze reagents. (a) DILUTED WASH BUFFER should be prepared fresh prior
to use. Note: a) Users can use either the rapid or overnight assay to run the platform between each wash. 3°C) on the rocking platform. Distilled Water 3 x 2 ml - -
(b) Dilute 1 volume of WASH BUFFER CONCENTRATE (20x) tests. Assayed strips from overnight assay may appear with (Note: Each wash cycle consists of dispensing 2 ml of 7. Carefully uncover the tray to avoid splashing or mixing
A. Antigen strips
with 19 volumes of reagent grade water. Mix well. additional and darker intensity bands, but the final results DILUTED WASH BUFFER, soaking time of 5 minutes, of samples. Tilt the tray to aspirate the mixture from We recommend that the Non-Reactive, Strong Reactive and Weak
• Avoid unnecessary exposure of antigen strips to light.
interpretation (positive/indeterminate/ negative) is the same. and aspiration.) the wells. Change aspirator tips between samples to Reactive controls be run with every assay regardless of the number of
2. BLOTTING BUFFER 9. Add 2 ml of WORKING CONJUGATE SOLUTION to 2 ml avoid cross-contamination. samples tested. In order for the results obtained from any assay to be
B. Reagents b) Aspirate all used chemicals and reagents into a trap
(a) BLOTTING BUFFER should be prepared fresh prior to each well. 8. Add 2ml of DILUTED WASH BUFFER. Wash each 3 x 2 ml considered valid, the following conditions must be met:
• Store reagents in their original vials or bottles, and they should use. containing 0.5% Sodium hypochlorite.
c) All incubations are to be carried out on a rocking platform. 10. Cover tray and incubate for 1 hour at room temperature 60 minutes strip 3 times with 5 minutes soak on the rocking
be capped for storage. (b) Dilute 1 volume of STOCK BUFFER CONCENTRATE (10x)
(25 ± 3°C) on the rocking platform. platform between each wash. 1. NON-REACTIVE CONTROL
• Dispense all reagents while they are freshly taken out from with 9 volumes of reagent grade water. Mix well.
(c) Add 1 g of BLOTTING POWDER to every 20 ml of the Caution: 11. Aspirate CONJUGATE from the wells. Wash as in step 3 x 2 ml (Note: Each wash cycle consists of dispensing 2ml of No HIV-1 and HIV-2 specific bands should be observed on the Non-
refrigeration and return to 2°C to 8°C storage as soon as possible.
diluted STOCK BUFFER prepared in step 2(b) above. Mix Some samples cause dark patches on the spot of the strip where they 8. DILUTED WASH BUFFER, soaking time of 5 minutes, Reactive control strips. The band for the serum control should be
Store reagents at refrigerated temperatures when not in use.
well by inversion or stir with a magnetic stirrer to ensure are added. To avoid this problem, one should ensure the following:- 12. Add 2 ml of SUBSTRATE SOLUTION to each well. 2 ml and aspiration.) visible (Fig 1c).
• Precipitates may form when the Substrate is stored at 2°C to 8°C.
powder dissolves completely. Stir again before dispensing. 13. Cover tray and incubate for 15 minutes on the rocking 15 minutes 9. Add 2 ml of WORKING CONJUGATE SOLUTION to 2 ml
This will not affect the performance of the kit.
(d) Stir again before dispensing. i. Sample should be added only after BLOTTING BUFFER is added. platform. each well. 2. STRONG REACTIVE CONTROL
ii. Tilt the tray slightly by elevating either the top or bottom end of the (Note: The reaction can be stopped before 15 minutes 10. Cover tray and incubate for 30 minutes at room 30 minutes All relevant molecular weight bands must be evident. Figure 1a
Caution: Avoid unnecessary exposure of substrate to light. 3. WORKING CONJUGATE SOLUTION
tray. The Blotting Buffer will flow to the lower end of the tray. Add if all the bands are visible for high anti-HIV titer temperature (25 ± 3°C) on the rocking platform. provides a guide to the relative positioning of bands visualized
Note : Prepare solution in polypropylene container / beaker. samples to avoid over development of bands and 11. Aspirate CONJUGATE from the wells. Wash as in step 3 x 2 ml
the sample where the Blotting Buffer is collected. When all the with the MP Diagnostics HIV BLOT 2.2 and permits identification
(a) WORKING CONJUGATE SOLUTION should be prepared
SPECIMEN COLLECTION, TRANSPORT AND STORAGE samples are added, return the tray back to its original flat position. difficulty in reading.) 8. of bands observed for the STRONG REACTIVE CONTROL. The
fresh prior to use.
(b) For RAPID ASSAY PROTOCOL, prepare WORKING Always ensure that the strips are kept wet during the process. 14. Aspirate the SUBSTRATE and rinse the strips at 3 x 2 ml 12. Add 2 ml of SUBSTRATE SOLUTION to each well. 2 ml bands are p17, p24, p31, gp41, p51, p55, p66, gp120/gp160.
Serum or plasma samples collected in EDTA, heparin or sodium citrate
CONJUGATE SOLUTION by diluting CONJUGATE iii. Alternatively, if tilting the tray is not desired, the samples may be least three times with reagent grade water to stop the 13. Cover tray and incubate for 15 minutes on the rocking 15 minutes Other bands associated with core antigens (p39, p42) may also be
may be used. Before storage, ensure that blood clot or blood cells have
at 1:500 into BLOTTING BUFFER, for example, 10 Øl added to the top or bottom end of the well. This way if dark patches reaction (A dark background can result if washing is platform. visible. Be careful not to misinterpret these as gp41. The envelope
been separated by centrifugation.
CONJUGATE to 5ml BLOTTING BUFFER. showed, the reading of the strip results will not be affected. insufficient at this step). (Note: The reaction can be stopped before 15 minutes antigens, gp41, gp120/gp160 appear as diffuse bands as they are
(c) For OVERNIGHT ASSAY PROTOCOL, prepare 15. Allow strips to dry in the wells of the tray. if all the bands are visible for high anti-HIV titer typical of glycoproteins; p55 viral band may appear faintly on the
Samples should be stored at 2°C to 8°C if the test is to be run within 7
WORKING CONJUGATE SOLUTION by diluting 16. Mount strips on worksheet (non-absorbent white samples to avoid over development of bands and actual Strong Reactive Control strip due to low titer of anti-p55 in
days of collection or frozen at -20°C or colder if the test is to be delayed Procedure:
CONJUGATE at 1:1000 into BLOTTING BUFFER, for paper). Do not apply adhesive tape over the developed difficulty in reading.) the Strong Reactive Control provided. The serum control band will
for more than 7 days. Clear, non-hemolyzed samples are preferred. example, 5 Øl CONJUGATE to 5ml BLOTTING BUFFER.
1. Add 2 ml of DILUTED WASH BUFFER to each well. 2 ml bands. Observe the bands (See Interpretation of 14. Aspirate the SUBSTRATE and rinse the strips at 3 x 2 ml be visible. The HIV-2 specific band should also be visible as shown
Lipemic, icteric or contaminated (particulate) samples should be filtered
2. Using forceps, carefully remove required number of Results) and grade the results. For storage, keep the least three times with reagent grade water to stop the in Figure 1a.
(0.45Øm) or centrifuged before testing. 4. SUBSTRATE SOLUTION (ready to use)
(a) Dispense directly the required volume from the bottle. STRIPS from the tube and place numbered side up strips in the dark. reaction (A dark background can result if washing is
Use a clean pipette. Cap tightly after use. into each well. Include strips for Strong Reactive, insufficient at this step). 3. WEAK REACTIVE CONTROL
Samples can be inactivated but this is not a requirement for optimal
Weak Reactive and Non-Reactive controls. ALTERNATIVE PROCEDURE - OVERNIGHT ASSAY 15. Allow strips to dry in the wells of the tray. The Weak Reactive control provides a measure of the sensitivity of
test performance.
3. Incubate the strips for 1 to 2 minutes at room 16. Mount strips on worksheet (non-absorbent white the kit. Weak bands at p24 and/or gp41 and gp120/gp160 should
2 minutes
temperature (25 ± 3°C) on a rocking platform (speed Procedure: paper). Do not apply adhesive tape over the developed appear. Some additional weak bands may or may not be present.
The use of inactivated specimens has not been validated.
of 12 to 16 cycles per minute). Remove buffer by 1. Add 2 ml of DILUTED WASH BUFFER to each well. 2 ml bands. Observe the bands (See Interpretation of The serum control band will be visible (Fig 1b).
aspiration. 2. Using forceps, carefully remove required number Results) and grade the results. For storage, keep the
Repeated freeze-thawing of sample is not recommended.
(Note: Do not allow the strips to dry. Failure may of STRIPS from the tube and place numbered side strips in the dark.
result in watery marks on developed strips for some up into each well. Include strips for Strong Reactive,
specimens.) Weak Reactive and Non-Reactive controls.

4 5 6
QUALITY CONTROL 3. p24 protein is abundant in HIV Blot 2.2 strip. For seroconverting Any viral specific bands present INDETERMINATE2 of primary and acute infection of HIV-1. Therefore, test algorithms Table 1: HIV-1 positive samples (209 samples) Panel HIV SFTS 94 from HIV-2 indicative = 6 HIV-2 positive = 7
specimens, it is well established that anti-p24 is the first to appear recommended by the US CDC (2001) and WHO (2004) are yet to
on Western Blot assays. Appearance of p24 band in HIV infected but pattern does not meet SFTS, France. Indeterminate = 1 Indeterminate = 0
NOTE: Developed strips must be completely dry to avoid misinterpretation. be updated, and NAT are yet to be included as methods for resolving Panel Name/ MP Diagnostics HIV-1
patients would fulfil the positive interpretation criteria for gag protein criteria for POSITIVE 7 members which were
by WHO, CDC and other international criteria. INDETERMINATE Western Blot results. Nevertheless, US CDC (2001) Source HIV Blot 2.2 Western blot
Any viral specific bands present INDETERMINATE 2 HIV-2 Western blot
The presence or absence of antibodies to HIV-1 sample is determined 4. The POL bands p66, p51 and p31 are generally detected acknowledged that when in consultation with clinical and infection status HIV Surveillance HIV-1 positive = 58 HIV-1 positive = 58 positive as indicated on
simultaneously. However the sensitivity of p66 and p31 are greater but pattern does not meet with among persons with an initial INDETERMINATE Western Blot.
by comparing each nitrocellulose strip to the assay control strips tested than that of p51. Panels from BioClinical Indeterminate = 1 Indeterminate = 1 the SFTS data sheet
criteria for POSITIVE but HIV-2 HIV-2 INDICATED Partners, Inc., USA
with the NON-REACTIVE, STRONG REACTIVE and WEAK REACTIVE were used. One of the
5. HIV-2 cross reactivity is variable but typically shows reactivity with specific band is visible. (BCP)
controls. GAG and/or POL antigens. However, there can be cross reactivity LIMITATION OF THE METHOD 7 members is a 1/10
with the gp160 band in some cases, but rarely with gp41. 5 panels (10 members diluted sample.
2
INTERPRETATION OF RESULTS FOR INDETERMINATE Detection of antibodies to HIV-1 does not constitute a diagnosis of each) and 1 panel (9
Figure 1a is suggested as an aid to identify the various bands developed (n = 7)
There is also a high molecular weight band around 160KD that is Acquired Immune Deficiency Syndrome (AIDS). A NEGATIVE BLOT members) with samples
on the STRONG REACTIVE Control strip. The Strong Reactive Control 6. presumed to be a GAG-POL precursor protein . This is seen with INDETERMINATE results should not be used as the basis for diagnosis HIV-2 positive samples HIV-2 indicative = 44 HIV-2 positive = 43
is not a guarantee that the causative agent for AIDS is not present. from USA, China,
as provided in the kit may contain relatively low titer of anti-p55 and some high titered HIV-2 or indeterminate (GAG Reactive Only) sera of HIV-1 infection. Based on the fact that most persons with an initial from Dr. Oliveiro Varnier, Indeterminate = 1 Indeterminate = 2
but the band pattern is a sharp discreet band which is different from Although a HIV-1 positive test result by Western Blot indicates infection Venezuela, Thailand,
anti-p39; as a result, p55 and p39 band for the Strong Reactive Control INDETERMINATE result who are infected with HIV-1 will develop Laboratory of Human
the diffuse band of ENV gp160. with the virus, a diagnosis of AIDS can only be made clinically if a Cameroon and India.
may appear faintly on the assayed strips. This has no impact on the detectable HIV antibodies within 1 month, US CDC (2001) recommended Retrovirology, Genova,
person meets the case definition of AIDS established by the Center (n = 59)
performance of HIV Blot 2.2 strips in detecting anti-p55 and anti-p39 7. Appearance of single band near p51/p55 is probably an HLA related such persons be re-tested for HIV-1 infection ≥1 month later. Persons for Disease Control (USA), the World Health Organization or other Italy
present in the specimens, as each lot of strip contains sufficient amount reactivity (p56), not specific for HIV-1. Panel HIV SFTS 94 from HIV-1 positive = 15 HIV-1 positive = 9 (n = 45)
with continued INDETERMINATE results after 1 month are unlikely to relevant authorities.
of p55 and p39 antigens. be HIV-infected unless recent HIV exposure is suspected. Sanguine Nationale Indeterminate = 0 Indeterminate = 6
8. Appearance of p39 and/or p42 without p24 or p17 should Transfusion Societes
HIV-2 positive samples HIV-2 indicative = 10 HIV-2 positive = 10
not be interpreted. It is known that persons who have recently seroconverted may display from BBI, BCP and Indeterminate = 0 Indeterminate = 0
PLEASE NOTE: The numbered end of the strips should be placed at Based on a recent study of Fiebig et al (2003), although the window (SFTS), France 15
9. Appearance of p66 alone is not HIV-1 specific, but is most likely a incomplete pattern but increase reactivity (both number and intensity Serologicals
period for Western Blot in the case of a primary HIV-1 infection could members which were HIV-
the bottom as shown in the Figure, i.e. the gp120/gp160 bands are the reactivity with the host cell proteins (p68). of bands) occurs when followed for a period of two to six months. Most (n = 10)
be as long as 22 days, the progression from an INDETERMINATE blot 1 Western blot positive as
furthest away from the numbered end. blots with POSITIVE results will have other viral specific bands present. HIV-2 Performance HIV-2 indicative = 25 HIV-2 positive = 21
The interpretation process involves the following:- to a full POSITIVE profile took no longer than 8 days. In addition, this indicated on the SFTS
data sheet were used. Panel PRF 201 (15 Indeterminate = 0 Indeterminate = 4
laboratory stage of having Western Blot INDETERMINATE was always INDETERMINATE results should not be used as the basis for diagnosis samples) and PRZ 202
MOLECULAR GENE ANTIGEN DESCRIPTION 1. Validate that the serum control band is visible. If the control is (n = 15)
accompanied with detectable RNA of HIV-1 with cases of true infection. of HIV-1 infection. It is recommended that all INDETERMINATE blots
WEIGHT negative, the results should be considered invalid as this indicates (10 samples) from BBI
Conversely, no seroconversion was evident in follow-up studies of be repeated using the original specimen and sequential samples. Blood HIV-1 positive samples HIV-1 positive = 38 HIV-1 positive = 38
gp 160 ENV Polymeric form of gp41 Broad diffuse glycoprotein a technical error such as not adding sample, conjugate or substrate. (n = 25)
individuals having screened positive and Western Blot INDETERMINATE donors with an INDETERMINATE blot should be re-tested using a fresh from Boston Biomedical Indeterminate = 0 Indeterminate = 0
gp 120 ENV Outermembrane Diffuse glycoprotein 2. Identify the molecular weight of each band of the test strip using results, once confirmed as negative by PCR methods (Sethoe et al, Inc., USA (BBI) and Total no. 107 107
specimen after one month (US CDC, 2001). In addition, antibodies to
the STRONG and/or WEAK REACTIVE Control strips as a guide. 1995). Therefore, it is reasonable to consider persons having Western Serologicals Inc., USA No. of true positives 102 93
p66 POL Reverse Transcriptase Discreet band p24 and p31 are known to decrease during the course of AIDS leading
3. Interpretation of the test strip is then based on the detection Blot INDETERMINATE results but additionally tested negative by a RNA (n = 38)
of specific band patterns as recommended by the appropriate to a shift in blot interpretation from POSITIVE to INDETERMINATE. No. of false negatives 0 0
p55 GAG Precursor protein Discreet band test as unlikely to be HIV-infected, especially when the tested individuals
authorities (i.e. Health Ministry, World Health Organization, etc.) Interpretation of results should then be based on subsequent blot testing HIV-1 positive plasma HIV-1 positive = 50 HIV-1 positive = 50 No. of indeterminates 5 14
p51 POL Reverse Transcriptase Discreet band just below are known as not having any risk factor associated with exposure. and clinical evaluations in such situations. from BBI Indeterminate = 0 Indeterminate = 0
p55 Sensitivity 95.33% (102/107); 86.92% (93/107);
Specific guidelines for interpretation may differ depending on the local (n = 50)
In particular, persons having Western Blot INDETERMINATE results 95% CI (89.43% - 95% CI (79.02% -
p39 GAG Fragment of p55 Discreet band policies. MP Diagnostics recommends following the accepted policy to Due to its highly specific nature , NON-REACTIVITY of samples with HIV-1 positive plasma HIV-1 positive = 47 HIV-1 positive = 47
derived from a test algorithm using fourth generation ELISAs as the 98.47%) 92.66%)
gp41 ENV Transmembrane Diffuse glycoprotein be in accordance with local regulations. HIV-2 specific envelope peptide on an Indeterminate viral blot, does not from LifeBiotech AG Indeterminate = 0 Indeterminate = 0
primary screen test should additionally be tested for viral RNA using exclude the possibility of infection with other strains of HIV-2.
p31 POL Endonuclease Doublet (n = 47) SEROCONVERSION
We recommended the following guidelines for the interpretation of the a molecular-base test such as RT-PCR with primer sets covering HIV-
p24 GAG Core protein Broad band MP Diagnostics HIV BLOT 2.2. Results should be recorded for each 1/2/O. If necessary, a follow-up should be conducted with an additional Total no. 209 209
Samples that are indicated as HIV-2 infections should be further tested
band detected, result should be interpreted as NEGATIVE, POSITIVE supplemental assay on a second specimen collected 1 month later. No. of true positives 208 202 This study was conducted by a third party institution, using a total
p17 GAG Core protein Broad band with specific HIV-2 supplemental assays.
or INDETERMINATE. The unique design of fourth generation ELISAs is for a simultaneous of 15 commercial seroconversion panels (SeraCare & Zeptometrix)
No. of false negatives 0 0
Some of the different antigens mentioned in the Table above are derived detection of both antigen and antibody. Consequently specimens which were qualified according to common technical specifications for
SPECIFIC PERFORMANCE CHARACTERISTICS No. of indeterminates 1 7 IVD medical devices (2009/886/EC). The seroconversion sensitivity of
from the same precursor protein and may have overlapping epitopes. identified as positive by a fourth generation ELISA should contain either
PATTERN INTERPRETATION Sensitivity 99.52% (208/209); 96.65% (202/209); MPD HIV Blot 2.2 and Chiron RIBA HIV-1/HIV-2 SIA are comparable
This should be considered when interpreting the pattern, for example:- antibody or antigen or both. Although more than 95% of those cases SENSITIVITY
No viral specific bands present NEGATIVE of true positive identified by a fourth generation ELISA were anti-HIV 95% CI (97.36% - 95% CI (93.22% - and both assays reacted similarly in the same panel follow up samples.
1. It is unlikely to detect gp41 in the absence of gp160 because the Detection of p17 antibodies NEGATIVE related and verifiable (confirmed) by Western Blot (Ly et al., 2000), 99.99%) 98.64%) See Table 3.
HIV POSITIVE SAMPLES
gp160 is the polymeric form of gp41 and the concentration of ONLY, no other bands a supplemental test using RT-PCR for viral RNA detection appeared
gp160 is higher than gp41 on the MP Diagnostics HIV BLOT 2.2. Table 2: HIV-2 positive samples (107 samples) Table 3: Performance of Kit based on Positives and/or Indeterminates
Detection of 2 ENV HIV-1 POSITIVE unavoidable for the small portion of reactivity relating to p24 antigen. The sensitivity of MP Diagnostics HIV Blot 2.2 was evaluated using 209
The gp41 appears as a diffuse band. Any sharp and discreet band detected in seroconversion panels
(gp160/gp41and gp120) and 1 Again, persons without any risk of exposure are unlikely HIV-infected, HIV-1 & 108 HIV-2 positive samples which were well characterized and Panel Name/ MP Diagnostics HIV-1
at the gp41 region should not be interpreted as gp41 band. Many
GAG (p17, p24, p55) or 1 POL if identified as positive by a fourth generation ELISA accompanied by commercially available. The HIV Blot 2.2 performance was evaluated Source HIV Blot 2.2 Western blot
non-HIV infected and normal specimens are found to be reactive
to this non-HIV antigen which is likely to originate from the human (p31, p51, p66) a Western Blot INDETERMINATE but the findings could not be further and compared with established Western blots for HIV-1 and HIV-2. Performance Panels No of Panels
supported by a POSITIVE result using a RNA test with primer sets HIV Surveillance Panels HIV-2 indicative = 17 HIV-2 positive = 12
cell line used to grow the HIV virus. MPD and Chiron have equal PRB965, PRB966, 10
Detection of 2 ENV HIV-1 POSITIVE from BCP Indeterminate = 3 Indeterminate = 8
2. p55 is the precursor for p24 and p17. covering HIV-1/2/O. detection of Positives and PRB968, PRB969,
(gp160/gp41 and gp120) and with 2 panels (10 members
The p55 band is generally detected when there is strong reactivity Indeterminates PRB970, ZMC6243,
1 GAG (p17, p24, p55) or 1 HIV-2 INDICATED However, nucleic acid tests (NAT) for HIV DNA or RNA were not each) with samples from
to p24 and/or p17, it normally appears as a thin band just above ZMC6245, ZMC6246,
POL (p31, p51, p66) and HIV-2 approved for diagnostic purpose by the relevant authorities (US CDC, Ghana and Nigeria
p51 band, sometimes these two bands are indistinguishable and ZMC9019, PRB9032
specific band is visible 2001; Constantine & Zink, 2005) until very recently. To date, only one (n = 20)
may appear as a single band. The bands seen as p42 and p39 are
both GAG fragments and should not be interpreted as gp41 (ENV). RNA qualitative assay has been approved by the US FDA for diagnosis

7 8 9

MPD detected Positives or PRB967, PRB972, 4 Lyme Disease Mixed HIV-1 negative = 8 HIV-1 negative = 1 Icteric samples from HIV-1 negative = 7 HIV-1 negative = 0 BIBLIOGRAPHY 17. Ming Guan, Frequency, causes and new challenges of indeterminate
Indeterminates earlier ZMC6240, Titer Performance HIV-1 positive = 0 HIV-1 positive = 0 BiosPacific HIV-1 positive = 0 HIV-1 positive = 0 results in Western Blot Confirmatory Testing for Antibodies to Human
ZMC12008 Panel PTL201 from Indeterminate = 2 Indeterminate = 9 (n = 10) Indeterminate = 3 Indeterminate = 10 1. V.C.W.Tsang, K. Hancock, M. Wilson. D.F. Palmer, S. Whaley, J.S. Immunodeficiency Virus. Clinical and Vaccine Immunology, June
Mc Dougal, and S. Kennedy. March 1985. Developmental Procedure 2007, Vol.14, No.6, p649-659.
Chiron detected Positives or PRB971 1 BBI Haemolysed samples HIV-1 negative = 4 HIV-1 negative = 0
: Enzyme-linked Immunoelectro-transfer Blot technique for HTLV-III/ 18. Reed DL, et al. HIV-1 Western Blot Standardization. European FIGURE 1
Indeterminates earlier (n = 10) from BiosPacific HIV-1 positive = 0 HIV-1 positive = 0
LAV antibodies; CDC, Altanta. Ligand Assay Society Sept 1995; 43-47.
Tuberculosis samples HIV-1 negative = 3 HIV-1 negative = 1 (n = 10) Indeterminate = 6 Indeterminate = 10
SPECIFICITY 2. H. Towbin, T. Staehlin, and J. Gordon. 1979. Electrophoretic transfer
from BCP (n = 9) HIV-1 positive = 0 HIV-1 positive = 0 Pregnant women HIV-1 negative = 30 HIV-1 negative = 4 of proteins from polyacrylamide gels to nitrocellulose sheets:
The specificity of MP Diagnostics HIV Blot 2.2 was evaluated using 200 Indeterminate = 6 Indeterminate = 8 samples from Biochrom, HIV-1 positive = 0 HIV-1 positive = 0 procedure and some applications. Proc. Natl. Acad. Sci.. USA 76: a b c d
negative blood donor samples, 81 clinical samples, and 167 potentially Anti-HCV Mixed Titer HIV-1 negative = 4 HIV-1 negative = 0 Germany Indeterminate = 3 Indeterminate = 29 4350-4354. MP Biomedicals Asia Pacific Pte Ltd.
interfering samples. The HIV Blot 2.2 performance was evaluated and Performance Panel HIV-1 positive = 0 HIV-1 positive = 0 (n = 33) 3. J. Schupbach, M. Popovic, R. V. Gilden. M.A. Gonda, M. G. 2 Pioneer Place
compared with established Western blot for HIV-1. (PHV 202) samples Indeterminate = 6 Indeterminate = 10 Total no. 81 81 Sarngadharan and R. C. Gallo. 1984. Serological Analysis of
from BBI Singapore 627885
No. of true negatives 61 4 subgroup of Human T-Lymphotropic retroviruses (HTLV-III)
Table 4: Normal blood donors (200 samples) Tel. No. : + 65 6775 0008 gp 160
(n = 10) No. of false positives 0 0 associated with AIDS. Science 224, 503-505.
Fax. No. : + 65 6774 6146 gp 120
SFTS 94, HTLV I/II HIV-1 negative = 9 HIV-1 negative = 2 No. of indeterminates 20 77 4. M. G. Sarngadharan, M. Popovic, L. Bruch, J. Schupbach and R.
Panel Name/ MP Diagnostics HIV-1 Email : [email protected]
positive panel from HIV-1 positive = 0 HIV-1 positive = 0 Specificity 75.31% (61/81); 4.94% (4/81); C. Gallo. 1984. Antibodies reactive with human T-Lymphotropic
Source HIV Blot 2.2 Western blot retroviruses (HTLV-III) in the serum of patients with AIDS. Science
SFTS, France Indeterminate = 1 Indeterminate = 8 95% CI (64.47% - 95% CI (1.36 % -
Normal human donor HIV-1 negative = 187 HIV-1 negative = 55 224, 506-608. p 66
(n = 10) 84.22%) 12.16%)
plasma (Lot VP8104) HIV-1 positive = 0 HIV-1 positive = 0 5. CDC. 1985. “Provisional public health service inter-agency Medical Technology Promedt p 55
HEV positive sera HIV-1 negative = 0 HIV-1 negative = 0
from Biomedical Indeterminate = 13 Indeterminate = 145 recommendations for screening donated blood and plasma Consulting GmbH p 51
from Armed Forces HIV-1 positive = 0 HIV-1 positive = 0 REPRODUCIBILITY
Resources (BMR) for antibody to the virus causing Acquired Immune Deficiency Altenhofstrasse 80
Research Institute Indeterminate = 9 Indeterminate = 9 gp 41
Lot VP8104 has 356 Using a HIV-1 Strong Positive sample (Accurun 24), Strong Reactive Syndrome” - United States Morbidity and Mortality Weekly Report
of Medical Sciences, D-66386 St. Ingbert p 39
individual units of blood Control, Weak Reactive Control and Non-Reactive Control, the 34 (1) :1-5.
Thailand 6. Proposed World Health Organization 1990 criteria for interpreting Germany
donations, 200 of assay reproducibility of the MP Diagnostics HIV Blot 2.2 assay was
(n = 9) results from Western blot assays for HIV-1, HIV-2, and HTLV-I/ Tel. No. : + 49 68 94 58 1020
which were randomly demonstrated with three technicians performing the assays (operator p 31
H. pylori positive HIV-1 negative = 4 HIV-1 negative = 1 HTLV-II, Weekly Epidemiological Record 65(37), 281-283. Fax No. : + 49 68 94 58 1021
selected for testing. All variability), on different days (environmental variability), on three different
the samples were tested samples from Dr HIV-1 positive = 0 HIV-1 positive = 0 strip lots (lot to lot variability) produced on two different production days 7. F. Clavel, D. Guetard., F. Brun-Vezinet, et al. 1986 Isolation of a new Email : [email protected]
negative for HBsAg, Roost Laboratory Indeterminate = 6 Indeterminate = 9 (batch variability). The various combinations and variations show that the human retrovirus from West African patients with AIDS. Science;
(n = 10) MP Diagnostics HIV Blot 2.2 is highly reproducible within a lot, across 233:343-346. p 24
HIV-1 antigen and
Dengue positive HIV-1 negative = 10 HIV-1 negative = 10 lots, batches and operators. 8. F. Clavel., 1987. HIV-2, the West African AIDS virus. AIDS 1:135-
antibodies for HIV-1/2,
samples from HIV-1 positive = 0 HIV-1 positive = 0 140. Regional Office:
HCV, and Syphilis by 9. R.S. Tedder, A. Hughes, T. Corrah et al 1988. Envelope cross-
FDA approved tests. Singapore General Indeterminate = 0 Indeterminate = 0
LIMITED EXPRESSED WARRANTY DISCLAIMER reactivity in Western Blot for HIV-1 and HIV-2 may not indicate dual
(n = 200) Hospital MP Biomedicals SAS p 17
The manufacturer makes no expressed warranty other than that the test infection. Lancet 11:927-930.
Total no. 200 200 (n = 10) Parc d’Innovation, BP 50067
kit will function as an in vitro diagnostic assay within the specifications 10. Bottiger B., A. Karlsson, F. Andreasson et al. 1990. Envelope cross-
No. of true negatives 187 55 Total no. 167 167 reactivity between Human Immunodeficiency Virus Type 1 and Type 67402 IIIkirch Cedex
No. of true negatives 117 31 and limitations described in the Product Instructions For Use when used
No. of false positives 0 0 in accordance with the instructions contained therein. The manufacturer 2 detected by different serological methods: Correlation between France
No. of indeterminates 13 145 No. of false positives 0 0 cross-neutralization and reactivity against the main neutralizing site. Tel No. : (33) 388 67 4607 Serum
disclaims any warranty, expressed or implied, including such expressed Control
Specificity 93.50% (187/200); 27.50% (55/200); No. of indeterminates 49 135 or implied warranty with respect to merchantability, fitness for use or J. Virol. 64(7):3492-3499. Fax No. : (33) 388 67 5420
95% CI (89.14% - 95% CI (21.44% - No. of true positives 1 1 implied utility for any purpose. The manufacturer is limited to either 11. Centers for Disease Control. 2001. Revised Guidelines for HIV Email: [email protected] HIV-2
Specificity 70.48% (117/166); 18.67% (31/166); replacement of the product or refund of the purchase price of the product. Counseling, Testing, and Referral and Revised Recommendations
96.49%) 34.24%)
95% CI (62.92 % - 95% CI (13.06 % - The manufacturer shall not be liable to the purchaser or third parties for for HIV Screening of Pregnant Women — United States, Morbid.
Table 5: Clinical samples (167 samples) 77.3 %) 25.45 %) any damage, injury or economic loss howsoever caused by the product Mortal. Weekly Rep. 50: RR-19.
12. Fiebig, E. W., D. J. Wright, B. D. Rawal, P. E. Garrett, R. T. * U.S. Patent 5,721,095
in the use or in the application thereof.
Panel Name/ MP Diagnostics HIV HIV-1 Table 6: Potential interfering and pregnant women samples (81 samples) Schumacher, L. Peddada, C. Heldebrant, R. Smith, A. Conrad,
Source Blot 2.2 Western blot S. H. Kleinman, and M. P. Busch. 2003. Dynamics of HIV viremia
TECHNICAL PROBLEMS / COMPLAINTS and antibody seroconversion in plasma donors: implications for
Chagas Panel (Panel HIV-1 negative = 16 HIV-1 negative = 0 Panel Name/ MP Diagnostics HIV-1
TC-6215) from BCP HIV-1 positive = 0 HIV-1 positive = 0 Should there be a technical problem / complaint, please do the following : diagnosis and staging of primary HIV infection. AIDS. 17:1871-1879.
Source HIV Blot 2.2 Western blot 13. Ly, T. D., C. Edlinger, and A. Vabret. 2000. Contribution of combined
(n = 25) Indeterminate = 9 Indeterminate = 25 Rheumatoid Factor HIV-1 negative = 15 HIV-1 negative = 0 1. Note the kit lot number, the expiry date and the strip lot number.
2. Retain the kits and the results that were obtained. detection assays of p24 antigen and anti-human immunodeficiency
Cross Reactivity HIV-1 negative = 63 HIV-1 negative = 16 samples from BCP HIV-1 positive = 0 HIV-1 positive = 0 a. Strong Reactive Control (Reactive for HIV-1 and HIV-2)
3. Contact the nearest MP Biomedicals office or your local distributor. virus (HIV) antibodies in diagnosis of primary HIV infection by routine
Panel (from BCP) HIV-1 positive = 0 HIV-1 positive = 0 Samples with RF values Indeterminate = 3 Indeterminate = 18 b. Weak Reactive Control (Reactive for HIV-1 only).
testing. J Clin Microbiol. 38:2459-2461.
comprising of HSV-1, Indeterminate = 10 Indeterminate = 57 (0-500), (501-999) & 14. Sethoe, S. Y., A. E. Ling, E. H. Sng, E. H. Monteiro, R. K. Chan. c. Non-Reactive Control.
HSV-2, Toxoplasma, True HIV-1 positive = 1 True HIV-1 positive (≥1000) are 2, 2 & 14, 1995. PCR as a confirmatory test for human immunodeficiency d. A typical HIV-2 seropositive serum.
Rheumatoid Arthritis, (Positive on both =1 respectively. virus type 1 infection in individuals with indeterminate western blot
SLE, Osteoporosis, devices and was (n = 18) (immunoblot) profiles. J Clin Microbiol. 33:3034-3036.
Sjogren’s Syndrome, not included in the Lipemic samples from HIV-1 negative = 5 HIV-1 negative = 0 15. Constantine, N. T. and H. Zink. 2005. HIV testing technologies after
UCTD/MCTD, calculations) BiosPacific HIV-1 positive = 0 HIV-1 positive = 0 two decades of evolution. Indian J Med Res. 121:519-538.
Scleroderma, VZV, (n = 10) Indeterminate = 5 Indeterminate = 10 16. World Health Organization. 2004. Guidelines for HIV Diagnosis and
Polycystic Ovary monitoring of antiretroviral therapy. Regional Office for South-East
samples Asia, New Delhi, India.
(n = 74) 10 11 12
 TROUBLE SHOOTING CHART

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

13 14 15

16 17 18