Murex HIV ELISA SOP
Author: Bonolo Khumotaka/Sikhulile
Moyo/Julita Magwenzi
Document Origin: Internal SOP
Review by
Heidi Hanes
Document Number:
Effective (or Post) Date:
Company:
SMILE Approved by:
Review date
Pro66-15
09 Feb 2009
BHHRL
Orlinda Maforo
7 Feb 2012
SMILE Comments: This document is provided as an example only. It must be revised to accurately reflect your lab’s
specific processes and/or specific protocol requirements. Users are directed to countercheck facts when considering
their use in other applications. If you have any questions contact SMILE.
Murex HIV ELISA SOP
Document No.: BHHRL/006TM-02
TEST METHODS MANUAL
Botswana-Harvard HIV
Reference Laboratory
(BHHRL)
Document
Title
Document No.: BHHRL/006/TM-02
MANUAL METHOD FOR HIV SCREENING USING MUREX
HIV-1.2.0 TEST KIT
Prepared by:
Name, Title
History
MANUAL METHOD FOR HIV
SCREENING USING MUREX
HIV-1.2.0 TEST KIT
Bonolo Khumotaka/Sikhulile Moyo/Julita Magwenzi
Current
Version
#
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04/Apr/2005
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Effective Date: 04/Apr/2005
Doc. No.: BHHRL/006TM-02
Page 2 of 14
Version 1.2
TEST METHODS MANUAL
Botswana-Harvard HIV
Reference Laboratory
(BHHRL)
006TM-02
02.1
02.2
02.3
02.4
02.5
02.6
02.7
02.8
02.9
02.10
02.11
02.12
02.13
02.14
02.15
02.16
Document No.: BHHRL/006TM-02
MANUAL METHOD FOR HIV
SCREENING USING MUREX
HIV-1.2.0 TEST KIT
MANUAL ELISA METHOD FOR MUREX HIV-1.2.0
Purpose
Application Scope
Personnel Responsibilities
Summary and Explanation of Test
Principle
Performance Characteristics
02.6.1 Local Performance Characteristics
02.6.2 Manufacturer Performance Characteristics
Specimen
02.7.1
Type/Stability/Storage
02.7.2
Specimen Rejection Criteria
02.7.3
Specimen Interferences
Equipment and Apparatus
Reagents
Safety Precautions
Procedure
Quality Control
02.12.1
Internal Quality Control
02.212
External Quality Control
Interpretation of Results
02.13.1
Negative Results
02.13.2
Reactive Results
Validation and Reporting of Results
Co-applicable Quality Management Documents
References
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Version 1.2
TEST METHODS MANUAL
Botswana-Harvard HIV
Reference Laboratory
(BHHRL)
02.1
Document No.: BHHRL/006TM-02
MANUAL METHOD FOR HIV
SCREENING USING MUREX
HIV-1.2.0 TEST KIT
PURPOSE
This chapter describes the Manual method of Murex HIV-1.2.0 assay used for the detection of
antibodies to human immunodeficiency virus types 1 (HIV-1, HIV-1 group O) and 2 (HIV-2) in
human serum or plasma.
02.2
APPLICATION SCOPE
This chapter applies to Botswana-Harvard HIV Reference Laboratory. This test method is part of
a double ELISA test algorithm with ORTHO HIV-1/HIV-2 Ab Capture (SOP # 006/TME-01).
02.3
PERSONNEL RESPONSIBILITIES
All Serology laboratory personnel are required to be knowledgeable of this procedure. New
employees are trained and assessed for competence before they can handle patient samples.
Documentation of training on this procedure can be found in the Personnel Files.
02.4
SUMMARY AND EXPLANATION OF THE TEST
Two types of human immunodeficiency virus, HIV-1 and HIV-2, have been described and
implicated as causative of the Acquired Immunodeficiency Syndrome (AIDS). Both are
retroviruses which are transmitted by exposure to certain infected body fluids, primarily blood
and genital secretions, and by transpalcental passage. Infection by HIV-1 has been reported
worldwide; HIV-2 infection has been reported as occurring mainly in West Africa and some
parts of European countries. The two types of virus show substantial antigenic cross reactivity in
their core proteins, but the envelope glycoproteins are less cross reactive. It is necessary for
screening purposes to use epitopes from the envelope proteins of both viruses in addition to the
major cross reacting core proteins to ensure detection of antibodies against both types of virus at
all stages following infection. Variants of HIV-1, classified together as group O, have been
identified in samples from Cameroon and Europe. Group O is highly divergent from the
originally known subtypes of HIV-1 (together classified as group M). Specific epitopes from the
envelope region of this virus can be used to detect antibody to group O in infected individuals;
reliance on cross reactions to the known subtypes of HIV is not satisfactory. The earliest specific
antibody response following infection by HIV may be of immunoglobulin M (IgM) followed by
a response in immunoglobulin G (IgG). Maximum sensitivity for detection of antiseroconversion is achieved by assays which respond to both IgM and IgG.
Effective Date: 04/Apr/2005
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Version 1.2
TEST METHODS MANUAL
Botswana-Harvard HIV
Reference Laboratory
(BHHRL)
Document No.: BHHRL/006TM-02
MANUAL METHOD FOR HIV
SCREENING USING MUREX
HIV-1.2.0 TEST KIT
Murex HIV-1.2.0 is expected to detect IgG and IgM and additionally IgA to the envelope
glycoproteins and the cross-reacting core proteins of HIV-1 potentially infectious samples of
serum. EDTA plasma or citrate plasma can be identified.
02.5
PRINCIPLE
Murex HIV-1.2.0 is based on microwells coated with a synthetic peptide representing an
immunodominant region of HIV-1 (O), recombinant protein derived from the envelope proteins
of HIV-1 and HIV-2 and an HIV core protein. The conjugate is a mixture of the same epitopes
all labelled with horseradish peroxidase.
Test specimens and control sera are incubated in microwells coated with a synthetic peptide and
antibodies to HIV in the sample or control sera bind to antigens on the microwell. Excess sample
and antibodies are then washed away. Subsequently, Conjugate is added which in turn binds to
any specific antibody already bound to the antigen on the well. Samples not containing specific
antibody will not cause the conjugate to bind to the well. Unbound Conjugate is washed away
and a solution containing 3, 3’, 5, 5’-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2)
is added to the wells. Wells with bound Conjugate develop a purple colour which is converted to
an orange colour when the reaction is stopped with sulphuric acid. After incubation the enzymic
reactions are terminated with sulphuric acid and the colour is read spectrophotometrically at
450nm. The amount of conjugate, and hence colour, in the wells is directly related to the
concentration of antibody to HIV in the sample.
02.6
PERFORMANCE CHARACTERICTICS
02.6.1
Local Performance Characteristics
Please refer to the paper referenced below:
Cheingsong, R., Gaolekwe, S., Kalake, W., Kenyon, T., Rayfield, M., Bond, K., Mei, J.,
and Xia, l. 2002. HIV Antibody Testing of Dried Blood Spot (Whole Blood): A
Preliminary Field Evaluation of HIV Kits in Botswana.
02.6.2
Manufacturer Performance Characteristics
The performance of Murex HIV-1.2.0 has been determined by testing samples from routine
blood donors, patients with AIDS diagnosed according to CDC criteria, patients with AIDS
Related Complex (ARC), other patients with known antibody to HIV-1 (including group O),
patients with confirmed HIV-2 infection and patients at risk of HIV infection or in other clinical
categories. In addition, its performance on commercially available seroconversion panels has
been evaluated.
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Version 1.2
TEST METHODS MANUAL
Botswana-Harvard HIV
Reference Laboratory
(BHHRL)
Document No.: BHHRL/006TM-02
MANUAL METHOD FOR HIV
SCREENING USING MUREX
HIV-1.2.0 TEST KIT
The specificity of Murex HIV-1.2.0 on presumed negative samples from European donors is
estimated to be 99.91% with a 95% confidence interval of 99.82% to 99.96% by the binomial
distribution.
Murex HIV-1.2.0 detected antibody to HIV in 100% of samples where antibody was
demonstrated by alternative format enzyme immunoassay and/or Western blot and 100% no
detection of antibody to HIV in samples from patients with other diseases unrelated to HIV.
02.7
SPECIMEN
02.7.1
Specimen Type/Stability/Storage
Serum, EDTA plasma or citrate plasma samples may be used. Blood collected by venepuncture
should be allowed to clot naturally. Care should be taken to ensure that the serum samples are
fully clotted. Any visible particulate matter in the sample should be removed by centrifugation.
Samples should be stored at 2 t0 8ºC. Samples not required for assay within 72 hours should be
removed from the clot or sell pellet and stored frozen at -15ºC or colder. Multiple freeze-thaw
cycles should be avoided.
02.7.2
Specimen Rejection Criteria
For specimen rejection criteria, refer to the following SOP:
*
Procedure for Specimen Rejection Criteria
02.7.3
Specimen Interferences
A specimen collected in citrate may give a raised absorbance.
Use of highly haemolysed samples, incompletely clotted sera, plasma samples containing fibrin
or samples with microbial contamination may give rise to erroneous results.
Erroneous results may be obtained with samples from cadavers.
02.8






EQUIPMENT AND APARRATUS
Multichannel micropipettes of appropriate volume (50 to 200μl).
Micropipettes to cover the range 50 to 1000μl.
37ºC ± 2ºC Thermostar Incubator.
Bo-Tek Instruments INC. ELX50 Auto Strip Washer.
Bio-Tek Instruments INC. ELX800 Universal Plate Reader.
Disposable Reagent Troughs
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TEST METHODS MANUAL
Botswana-Harvard HIV
Reference Laboratory
(BHHRL)
02.9
Document No.: BHHRL/006TM-02
MANUAL METHOD FOR HIV
SCREENING USING MUREX
HIV-1.2.0 TEST KIT
REAGENTS




MUREX HIV-1.2.0 Test Kit
Distilled/deionised water
Stop Solution (0.5M to2M Sulphuric Acid)
Sodium hypochlorite
02.10
SAFETY PRECUATIONS
All material of human origin should be considered as potentially infectious and therefore all
reagents and test specimen should be handled using the laboratory established universal safety
precautions.
Spillage of potentially infectious materials should be removed immediately with absorbent tissue
and the contaminated area swabbed with 1.0% sodium hypochlorite before work is continued.
Materials used to clean spills, including gloves, should be disposed of as potentially
biohazardous waste.
Do not pipette by mouth. Wear disposable gloves and eye protection while handling specimen
and performing the assay. Wash hands thoroughly when finished.
The following reagents contain low concentrations of harmful or irritant substances:


02.11
The conjugate Diluent and Sample Diluent contain ProClin 300 which can be
absorbed through the skin and is a sensitising agent. If any of the reagents come into
contact with the skin or eyes wash the area extensively with water.
Sulphuric acid required for the Stop Solution and hydrochloric acid used for washing
glassware are corrosive and should be handled with appropriate care. If they come
into contact with the skin or eyes, wash thoroughly with water.
PROCEDURE
NB: Use of a white background will aid visualisation of sample addition.
1.
Use only the number of wells required for the test. Avoid touching the tops or bottoms of
the wells.
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TEST METHODS MANUAL
Botswana-Harvard HIV
Reference Laboratory
(BHHRL)
Document No.: BHHRL/006TM-02
MANUAL METHOD FOR HIV
SCREENING USING MUREX
HIV-1.2.0 TEST KIT
2.
Reconstitute the conjugate by pouring the whole contents of the bottle of conjugate
diluent into the bottle of conjugate. Recap the latter and mix by gentle inversion – should
be red in colour.
3.
Add 50uL of sample diluent to each well using a multichannel pipette.
4.
Add 50uL of sample or control to the appropriate well. For each plate reserve wells A to
E in the first column for controls. Well F is left blank. This is an in-house arrangement to
allow easy comparison with ORTHO. Add controls to the designated wells after
dispensing the samples. Samples are added starting from well G1. Pipette 50uL of the
Negative Control into each of three wells A1 to C1and 50uL of the anti-HIV-1 and HIV2 Positive Controls into wells D1 and E1 respectively.
5.
Cover the plate with a lid and incubate at 37ºC for 30 minutes.
6.
Dilute Wash Fluid 1in 20 with either distilled or deionised water.
7.
At the end of incubation time, wash the plate using wash programme “Nunc Round” on
the automated washer.
8.
When washing is complete, invert the plate and firmly tap it on a clean paper towel to
remove excess Wash fluid.
9.
Add 50µL of Conjugate solution to each well using a multichannel pipette.
10.
Cover the wells and incubate as in 5 above.
11.
Repeat Steps 7 and 8 above.
12.
While incubating, prepare Substrate Solution by mixing equal volumes of substrate
diluent and the pink substrate concentrate in either a clean glass container or a new
polystyrene vessel – should be pink in colour. It is extremely important that this order of
addition is followed and that any pipettes and glassware used to prepare Substrate
Solution are clean. Keep away from light.
13.
Immediately add 100uL of substrate solution to each well using a multichannel pipette.
14.
Cover wells and incubate at 37ºC for 30 minutes. Keep away from direct sunlight. A
purple colour should develop in wells containing reactive samples.
15.
Add 50uL Stop Solution (0.5M to 2M sulphuric acid) to each well using a multichannel
pipette.
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TEST METHODS MANUAL
Botswana-Harvard HIV
Reference Laboratory
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16.
Document No.: BHHRL/006TM-02
MANUAL METHOD FOR HIV
SCREENING USING MUREX
HIV-1.2.0 TEST KIT
Within 15 minutes read the absorbance at 450nm using 620nm to 690nm as the reference
wavelength if available. Blank the instrument on air (no plate in the carriage). The reader
program is called “HIV 1.2.0”.
02.12
QUALITY CONTROL
Each plate must be considered separately when calculating and interpreting results of the assay,
regardless of the number of plates processed at the same time.
02.12.1
Internal Quality Control
Negative Control
Calculate the mean absorbance of the three Negative controls. If one of the control wells has an
absorbance more than 0.15 O.D. above the average of the three, discard it and calculate a new
mean from the two remaining controls.
Cut-off value
Cut-off value = Mean Negative Control + 0.2.
Results are valid only if the following criteria for controls are met:
*
Negative control
The mean must be less than 0.3.
*
Positive controls
The absorbance of each of the two Positive controls should be more than 0.8 above the mean
absorbance of the Negative control.
ASSAYS WHICH DO NOT MEET THESE CRITERIA SHOULD BE REPEATED!
Parallel Testing
Run two samples (negative and positive) from previous lot with each new lot.
Results (negative/positive) must match previous results.
02.12.2
External Quality Control
The Botswana-Harvard HIV Reference Laboratory is participating in the CAP Proficiency
Testing Program for this assay, which is performed three times in a year. The laboratory’s
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Document No.: BHHRL/006TM-02
TEST METHODS MANUAL
Botswana-Harvard HIV
Reference Laboratory
(BHHRL)
MANUAL METHOD FOR HIV
SCREENING USING MUREX
HIV-1.2.0 TEST KIT
performance in this program is reviewed by the quality manager and the personnel running the
test every time results are received from CAP and corrective measures implemented where
necessary, for purposes of continuous improvement.
02.13
INTERPRETATION OF RESULTS
02.13.1
Negative Results
Samples giving an absorbance less than the Cut-off value are considered negative (non-reactive
for HIV antibodies) in the assay.
02.13.2
Weak Reactivity
Samples giving an absorbance between the Cut-off value and 1.0 are considered weakly reactive
and therefore are repeated.
02.13.3
Reactive Results
Samples giving an absorbance equal to or greater than the Cut-off value are considered initially
reactive in the assay
02.14
VALIDATION AND REPORTING OF RESULTS
The results are valid per run only when both the positive and the negative controls have passed.
Positive specimen are marked on the Results (output) using symbol X with red pen.
Corresponding Lab numbers on the worksheet are assigned symbol P (Positive).
Requisition Forms are filled and stamped with appropriate stamps (i.e. Double ELISA Positive
or Double ELISA Negative), Staff Name and Date.
Test Algorithm
A1+A2+
Report as Positive
A1A2 (in parallel)
A1+A2A1-A2+
A1-A2Report as Negative
Indeterminate
Repeat A1A2
A1+A2+
Report as Positive
Effective Date: 04/Apr/2005
A1+A2- or A2+A1Indeterminate
Western Blot /DNA PCR
Doc. No.: BHHRL/006TM-02
Page 10 of 14
A1- A1Report as Negative
Version 1.2
TEST METHODS MANUAL
Botswana-Harvard HIV
Reference Laboratory
(BHHRL)
Document No.: BHHRL/006TM-02
MANUAL METHOD FOR HIV
SCREENING USING MUREX
HIV-1.2.0 TEST KIT
A1 = Murex HIV-1.2.0
A2 = Ortho HIV-1/HIV-2 Antibody Capture
Before the results can be released they must be double-checked (the Four-Eye Principle), verified
and signed-off by another competent personnel.
02.15
*
*
*
*
*
02.16
CO-APPLICABLE QUALITY MANAGEMENT DOCOMENTS
Manual Method for Ortho HIV-1/HIV-2 Ab-Capture
Waste Disposal Policy
Universal Precautions Policy
Operation and Maintenance Instructions for the Strip Washer ELX 800.
Specimen transport, reception, processing and storage
REFERENCES
Murex HIV-1.2.0 Enzyme immunoassay for the detection of antibodies to human
immunodeficiency virus types 1 (HIV-1 group O0 and 2 (HIV-2) in human serum or plasma.
ABBOTT murex. REF GE94/95 9E25-01/9E25-02. Sept. 2001.
Cheingsong, R., Gaolekwe, S., Kalake, W., Kenyon, T., Rayfield, M., Bond, K., Mei, J., and Xia,
l. 2002. HIV Antibody Testing of Dried Blood Spot (Whole Blood): A Preliminary Field
Evaluation of HIV Kits in Botswana.
Operational Characteristics of Commercially Available Assays to Determine Antibodies to HIV1 And/Or HIV-2 in Human Sera. Report 11. Geneva. January 1999. WHO/BTS/99.1.
UNAIDS/99.5.
Effective Date: 04/Apr/2005
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TEST METHODS MANUAL
Botswana-Harvard HIV
Reference Laboratory
(BHHRL)
Document No.: BHHRL/006TM-02
MANUAL METHOD FOR HIV
SCREENING USING MUREX
HIV-1.2.0 TEST KIT
This procedure has been read and understood by the undersigned:
Name of Officer
Effective Date: 04/Apr/2005
Signature
Doc. No.: BHHRL/006TM-02
Page 12 of 14
Initials
Date
Version 1.2
TEST METHODS MANUAL
Botswana-Harvard HIV
Reference Laboratory
(BHHRL)
MANUAL METHOD FOR HIV
SCREENING USING MUREX
HIV-1.2.0 TEST KIT
Name of Officer
Effective Date: 04/Apr/2005
Document No.: BHHRL/006TM-02
Signature
Doc. No.: BHHRL/006TM-02
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Initials
Date
Version 1.2
TEST METHODS MANUAL
Botswana-Harvard HIV
Reference Laboratory
(BHHRL)
MANUAL METHOD FOR HIV
SCREENING USING MUREX
HIV-1.2.0 TEST KIT
Name of Officer
Effective Date: 04/Apr/2005
Document No.: BHHRL/006TM-02
Signature
Doc. No.: BHHRL/006TM-02
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Initials
Date
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