Pro65-04 Murex Hep HBsAg SOP

Non-Smile ResourceAuthor: Bonolo Khumotaka /Sikhulile Moyo/Julita Magwenzi

Document Number: Pro65-04Effective (or Post) Date: 15 Aug 2005

Document Origin Company: BHHRLSMILE Approved by: Penny Stevens

Review by Heidi Hanes Review date 7 Feb 2012SMILE 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.

Botswana-Harvard HIVReference Laboratory

(BHHRL)

TEST METHODS MANUAL

Enzyme Immunoassay for the Detection of Hepatitis B Surface Antigen in Human Serum or Plasma Using Murex HBsAg

Version 3 Test Kit

Document No.: BHHRL/006TM-03

Document Title

Document No.: BHHRL//006TM-03

ENZYME IMMUNOASSAY FOR THE DETECTION OF HEPATITIS B SURFACE ANTIGEN IN HUMAN SERUM OR

PLASMA USING MUREX HBSAG VERSION 3 TEST KIT

Prepared by:Name, Title Bonolo Khumotaka/Sikhulile Moyo/Julita Magwenzi

History

Current Version #

Supersedes Version #

Effective Date(dd/mm/yy)

Description Notes

1.0 0.0 Sept/2..2 First issue1.1 1.0 09/Sept/20031.2 1.1 11/Apr/2005

Annual Review:

Review Date Revision Date Signature

Approved by:

Name, Title Signature DateLaboratory Director (MOH)Laboratory Director (HSPH)

Distributed to:

Name/Location # of Copies

Name/Location # of Copies

Effective Date: 11/Apr/2005 Doc. No.: BHHRL/006TM-03 Version 1.2

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006TM-03 Enzyme Immunoassay for the Detection of Hepatitis B Surface Antigen in Human Serum or Plasma – Murex HBsAg Version 3.

03.1 Purpose03.2 Application Scope03.3 Personnel Responsibilities 03.4 Summary and Explanation of the Test03.5 Principle of the Test03.6 Performance Characteristics

03.6.1 Manufacturer Performance Characteristics03.7 Specimen

03.7.1 Specimen Type/Stability/Storage03.7.1 Specimen Rejection Criteria03.7.1 Specimen Interferences

03.8 Equipment and Apparatus03.9 Reagents03.10 Safety Precautions03.11 Procedure

03.11.1 Preparation of Reagents03.11.2 Test Procedure

03.12 Quality Control03.12.1 Internal Quality Control03.1.2 External Quality Control

03.13 Interpretation of Results03.13.1 Negative Results03.13.2 Reactive Results

03.14 Validation of Results03.15 Co-applicable Quality Management Documents03.16 References


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03.1 PURPOSE

This chapter describes the manual method of Murex HBsAg Version 3 assay for the detection of antibodies to Hepatitis B virus surface antigen (HBsAg) in human serum or plasma.

03.2 APPLICATION SCOPE

This chapter applies to Botswana-Harvard HIV Reference Laboratory.

03.3 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.

03.4 SUMMARY AND EXPLANATION OF THE TEST

The causative agent of serum hepatitis is hepatitis B virus (HBV) which is an enveloped DNA virus. During infection, HBV produces an excess of hepatitis B surface antigen (HBsAg), also known as Australia antigen, which can be detected in the blood of infected individuals. HBsAg is the first serological marker after infection with HBV appearing one to ten weeks after exposure and two to eight weeks before the onset of hepatitis. HBsAg persists during this acute phase and clears late in the convalescence period. Failure to clear HBsAg within six months indicates HBsAg carrier state. Blood from individuals in the acute or chronic state is potentially infectious to recipients and should not be transfused. Consequently, potentially infectious samples of serum, EDTA plasma can be identified.

03.5 PRINCIPLE OF THE TEST

In the Murex HBsAg version 3.0 the sample is pre-incubated in microwells coated with a mixture of mouse monoclonals specific for different epitopes on the a determinant of HBsAg. Affinity purified goat antibody to HBsAg conjugated to horseradish peroxidase is then added to the sample in the well. During the two incubation steps any HBsAg


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present in the sample is bound to the well in an antibody-antigen-antibody-enzyme complex. In the absence of HBsAg no conjugate will be bound. After washing to remove sample and unbound Conjugate, a solution containing 3, 3’, 5, 5’-tetramethylbenzidine (TMB) and hydrogen peroxide is added to the wells. Wells that contain HBsAg and hence bound conjugate will develop a purple colour which is converted to orange when the enzyme reaction is terminated with sulphuric acid. The amount of colour can be determined spectrophotometrically and is directly proportional to the amount of Conjugate bound and hence the concentration of HBsAg in the sample.

03.6 PERFORMANCE CHARACTERISTICS

03.6.1 Manufacturer Performance Characteristics

The specificity of Murex HBsAg Version 3 on presumed negative samples from donors is estimated to be 99.97% with a 95% confidence interval of 99.92% to 99.99% by the binomial distribution.

A total of 630 samples from patients suffering from acute and chronic hepatitis B infection were tested with Murex HBsAg version 3. All 630 samples were confirmed with an alternative immunoassay for HBsAg.

A further six samples from patients infected with mutant forms of hepatitis B infection, confirmed by DNA sequencing, were also tested by Murex HBsAg version 3 and were all detected successfully.

In addition 998 potentially cross-reactive samples from patients with conditions unrelated to hepatitis B infection were tested with Murex HBsAg version 3. A total of 996 of these samples were non-reactive with Murex HBsAg version 3. Of the two repeatedly reactive samples, one was false reactive and showed no other hepatitis markers, the remaining sample was anti-HBc positive.

03.7 SPECIMEN

03.7.1 Specimen Type/Stability/StorageSerum, 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.


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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.

03.7.2 Specimen Rejection CriteriaFor specimen rejection criteria, refer to the following SOP:

* Procedure for Specimen Rejection Criteria

03.7.3 Specimen InterferencesA 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.

03.8 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

03.9 REAGENTS

MUREX HBsAg Version 3 Test Kit Distilled/deionised water Stop Solution (0.5M to2M Sulphuric Acid) Sodium hypochlorite

Reagents Storage ConditionsAll kit components must be stored at 2 to 8ºC, unless otherwise stated, under these conditions they will retain activity until the expiry date of the kit.


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03.10 SAFETY PRECAUTIONS

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 0.5% sodium hypochlorite before work is continued. Materials used to clean spills, including gloves, should be disposed of as potentially biohazardous waste.

Neutralised acids and other liquid waste should be decontaminated by adding a sufficient volume of sodium hypochlorite to obtain a final concentration of 0.5%. A 3minute exposure to 0.5% sodium hypochlorite is recommended to ensure effective decontamination.

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:

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.

03.11 PROCEDURE

NotesAllow the wells and the reagents to equilibrate to room temperature. All the reagents should be prepared as stipulated and thereafter allowed to equilibrate to room temperature (18 to 30 0C).All the reagents should be stored at the recommended temperature after use.

03.11.1 Preparation of Reagents


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Substrate SolutionTo prepare the Substrate Solution, add a volume of colourless Substrate Diluent to an equal volume of pink Substrate Concentrate in either a clean glass container or a new polystyrene vessel. It is extremely important that this order of addition is followed and that any pipettes and glassware used to prepare Substrate Solution are clean.

Wash FluidDilute the Wash Fluid 1 to 20 with distilled or deionized water to give the required volume or dilute the entire contents of one bottle of Wash Fluid to a final volume of 2500ml. Store the working strength Wash Fluid at room temperature (18 to 30ºC) in a closed vessel under which conditions it will retain activity for one month.

03.11.2 Test ProcedurePrepare Substrate Solution and Wash Fluid.Use only the number of wells required for the test.

1. Add 25µl of Sample Diluent to each well. 2. Add 75µl of Samples or Controls to the wells

To each plate add 75µl of the Negative Control to wells A1 and B1 and 75µl of Positive Control to well C1.

3. Add the Controls to the designated wells after dispensing the samples.

4. Cover the plate with a lid and incubate for 60 minutes for at 37oC.

5. Add 50 µl of Conjugate to each well.

6. Shake the plate using a plate shaker for 10 seconds or manually agitate by gently tapping the sides for 10 seconds.

7. Cover the plate with the lid and incubate for 30 minutes at 37oC.

8. At the end of the incubation time wash the plate 5 times using the automated strip washer programme.

10. After washing is completed invert the plate and tap out any residual wash fluid onto absorbent paper.

11. Immediately after washing the plate, add 100µl of Substrate Solution to each well.


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12. Cover the plate with the lid and incubate for 30 minutes at 37oC while colour develops. A purple colour should develop in wells containing reactive samples.

13. Add 50µl Stop Solution to each well.

14. Within 15 minutes read the absorbance of each well at 450 nm using 620 to 690 nm as the reference wavelength if available.

15. Blank the instrument on air (no plate in carriage).

03.12 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.

03.12.1 Internal Quality Control

Negative controlCalculate the mean absorbance of the replicates of the Negative Control. If one of the Negative Control wells has an absorbance more than 0.03 above the other discard the higher value. The results of an assay run are valid if the following criteria for the Negative Control are met:

The mean A450/690 of the Negative Control must be less than 0.15 or the mean A450 of the Negative Control is less than 0.2.

Cut-off valueCalculate the Cut-off Value by adding 0.05 to the mean of the Negative Control replicates

HBsAg Positive controlThe results of an assay run are valid if the following criteria for the Positive Control are met:

The A450/690 or A450 of the positive control must be more than 0.8 above the mean A450/690 or A450 of the Negative Control.

If the above requirements are not met by the Negative and Positive control the assay run is unsatisfactory and should be repeated.


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Parallel Testing

Run two samples (negative and positive) from previous lot with each new lot.Results (negative/positive) must match previous results.

03.12.2 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 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.

03.13 INTERPRETATION OF RESULTS

Negative Results

Samples giving an absorbance less than the Cut-Off Value are considered non-reactive in Murex HBsAg Version 3.

Reactive Results

Samples giving an absorbance equal to or greater than the Cut-Off Value are considered initially reactive in the assay.

03.14 VALIDATION OF RESULTS

The results are valid only when both the positive and the negative controls have passed.

Before the results can be released they have to be double-checked by a second person authorised to release results.


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03.15 CO-APPLICABLE QUALITY MANAGEMENT DOCUMENTS

* An Enzyme immunoassay for the detection of antibodies to hepatitis C virus (HCV) in human serum or plasma. SOP #: 006/TME-04.

* 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.

03.16 REFERENCES

Enzyme Immunoassay for the Detection of Hepatitis B Surface Antigen in Human Serum or Plasma. Murex HBsAg Version 3. ABBOTT murex. Ref: GE34/36 9F80-01/9F80-05. D02GE34GB. May 2002.


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This procedure has been read and understood by the undersigned:

Name of Officer Signature Initials Date


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