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Enzygnost® HBsAg 6.0

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Intended UseEnzygnost® HBsAg 6.0 is an enzyme immunoassay for the qualitative detection of hepatitis B(surface) antigen in human serum or plasma.The enzyme immunoassay can be processed using the ELISA processors, BEP® III System,BEP® 2000 System or BEP 2000 Advance® System, as well as the Quadriga® and Quadriga®BeFree Systems. A non-automated processing of the test is also possible.

Summary and ExplanationType B viral hepatitis is usually accompanied by the appearance of hepatitis B surface antigen(HBsAg) in the serum1,2. Normally, HBsAg can be detected in the serum as early as 2 or3 weeks before the onset of the illness and reaches a peak titer at the time when thecharacteristic symptoms appear (jaundice, changes in the concentrations of liver-specificenzymes)3. This is normally followed by a gradual elimination of the antigen. In some casesand in an unknown percentage of subclinical hepatitis B infections, the antigen can bedetected in the serum for years, if not for life4.As transfusions of blood containing HBsAg have often resulted in hepatitis B infection of theperson receiving the transfusion5, the special importance of testing for HBsAg relatesparticularly to the screening of blood donations as a means of reducing the incidence of post-transfusion hepatitis B6. In addition to the detection of the eight previously known genotypesof the hepatitis B virus (A to H)7,8, the detection of mutated forms of HBsAg has becomeincreasingly important in recent years9 to prevent the transmission of diagnostic escapemutants by way of transfusion.In addition, the HBsAg test is also used for the diagnosis of acute or chronic hepatitis Binfection5. Despite the high sensitivity of HBsAg assays, a risk of the transmission of hepatitis Bby a HBsAg-negative sample cannot be totally ruled out.

Principle of the MethodEnzygnost® HBsAg 6.0 is an enzyme immunoassay based on a two-step method. In the firststep, the HBsAg contained in the sample reacts simultaneously with the polyclonal anti-HBsantibodies attached to the wells of the microtitration plates and with Conjugate 1 (Anti-HBs/Biotin, colored blue). In the second step, after removing the unbound reactants, Conjugate 2(Streptavidin/POD, colored yellow) then reacts with Conjugate 1.After removing the unbound reactants, the enzyme activity of the bound Conjugate 2 isdetermined (blue color reaction). The enzymatic conversion of the chromogen is terminatedby the addition of Stopping Solution POD (yellow color reaction). The color intensity isproportional to the concentration of antigen in the sample.

ReagentsSymbols Materials provided Enzygnost® HBsAg 6.0 2 x 96 10 x 96 10 x 96 (Q)MTP Enzygnost® HBsAg 6.0 test plate 2 pcs. 10 pcs. 10 pcs.

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Symbols Materials provided CONJUGATE 1 Conjugate 1 (Anti-HBs/Biotin) 2 x 5 mL 10 x 5 mL 3 x 15 mLCONJUGATE 2 Conjugate 2 (Streptavidin/POD) 2 x 12.5 mL 10 x 12.5 mL 2 x 75 mLCONTROL − HBsAg Control Serum, negative 2 x 2.5 mL 4 x 2.5 mL 3 x 2.5 mLCONTROL + HBsAg Control Serum, positive 2 x 1.5 mL 3 x 1.5 mL 3 x 1.5 mL Label "Empty bottle for conjugate 1" --- 1 pc. --- Label "Empty bottle for conjugate 2" --- 1 pc. --- Instructions for Use 1 pc. 1 pc. 1 pc. Barcode table of values 1 pc. 1 pc. 1 pc. Polyethylene bag 1 pc. 1 pc. 1 pc.The test plate, Conjugate 1, Conjugate 2, as well as HBsAg Control Serum, negative and HBsAgControl Serum, positive must be used in the given combination of 6-digit lot numbers printedon the package, respectively stated in the enclosed barcode table of values.

Materials required but not providedSupplementary Reagents for Enzygnost®/TMB ( REF OUVP)The reagents Chromogen TMB and Buffer/Substrate TMB must be used only in thecombination of lots stated for the Supplementary Reagents kit. The applicable lot numbers arethe 6-digit lot numbers listed on the package.

CompositionEnzygnost® HBsAg 6.0 test plate: Microtitration plate coated with sheep antibodies toHBsAg.Conjugate 1 (Anti-HBs/Biotin): Monoclonal anti-HBs (mouse), biotin-conjugated, coloredblue.Preservative: phenol (≤ 1 g/L)Conjugate 2 (Streptavidin/POD): Streptavidin, peroxidase (POD)-conjugated, colored yellow.Preservative: phenol (≤ 1 g/L)HBsAg Control Serum, negative: Stabilized human serum, colored green, nominalabsorbance: ≤ 0.150.Preservative: phenol (≤ 1 g/L)HBsAg Control Serum, positive: Stabilized human serum: 0.15 to 0.32 U/mL, target value:0.25 U/mL (traceable to the reference material HBsAg Ad of the Paul Ehrlich Institute), coloredred, nominal absorbance ≥ 0.7.Preservative: phenol (≤ 1 g/L)

Warnings and PrecautionsFor in-vitro diagnostic use only.The test was developed for testing individual samples, not for pooled samples.CAUTION! POTENTIAL BIOHAZARDEach donor or donor unit used for manufacturing the HBsAg Control Serum, negativewas tested and found to be negative for human immunodeficiency virus (HIV) 1 and2, hepatitis B virus (HBV) and hepatitis C virus (HCV) using either tests found to be inconformance with the In Vitro Diagnostic Directive in the EU or FDA approved tests.Because no known test can offer complete assurance of the absence of infectiousagents, all human derived products should be handled with appropriate caution.


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It is advisable to wear protective gloves throughout the entire test procedure. Pleasefollow the recommendations of the manufacturer concerning the compatibilitybetween gloves and exposed materials.For disposal, it is recommended that solid infectious materials should be autoclavedfor at least 1 hour at 121 °C. All aspirated liquids should be collected in tworeceptacles connected in series. Both should contain a disinfectant suitable forinactivating human pathogens. The concentrations and reaction times specified by themanufacturer must be observed.Buffer/Substrate TMB, Chromogen Working Solution and Stopping Solution POD mustnot be allowed to come into contact with heavy metal ions or oxidizing substances (donot use pipettes with metal parts which are in direct contact with the liquid). Thesubstrate reaction steps must not be performed in the vicinity of disinfectantscontaining hypochlorite. If the Chromogen Working Solution has spontaneouslydeveloped a blue color before being transferred into the test plate, this indicates thatthe solution is contaminated; in such cases, prepare a fresh solution in a cleancontainer. Skin contact with the above mentioned solutions is to be avoided.

Preparation of the ReagentsBring all reagents and test samples to 15 to 25 °C before starting with the test. Do not removethe foil pouch from the test plate during this step. Before starting the test processing, removenot required strips from the holder and store these in the enclosed polyethylene bag for lateruse (see Table 1). If reagents or working reagent solutions need to be mixed, avoid foamformation.To avoid a frequent change of syringes when processing large series of samples on the BEP® IIISystem, the kit 10 x 96 (Q) is recommended. When using kit variant 10 x 96, several vials ofthe conjugates can be transferred into larger bottles (e.g. fresh, surplus empty bottles suppliedfor the Chromogen Working Solution from the kit Supplementary Reagents REF OUVP 17),which are placed in the reagent rotor with the respective label "EMPTY VIAL CONJUGATE…" forConjugate 1 (Anti-HBs/Biotin) or Conjugate 2 (Streptavidin/POD).For each test plate, dilute 20 mL of Washing Solution POD from the Supplementary ReagentsKit for Enzygnost®/TMB with distilled or deionized water to 400 mL.For each test plate, dilute 1 mL of Chromogen TMB with 10 mL of Buffer/Substrate TMBfrom the kit Supplementary Reagents for Enzygnost®/TMB using the supplied empty plasticbottle (Chromogen Working Solution). Store protected from light. After use, carefully rinse thebottle with distilled or deionized water.It is also permissible to pour together the full contents of the Chromogen TMB vial and theBuffer/Substrate TMB vial into the empty bottle.When using Supplementary Reagents for Enzygnost®/TMB, REF OUVP 27, the completecontents of the Chromogen TMB vial have to be transferred into the barcode labeled Buffer/Substrate TMB vial.

Storage and StabilityStored unopened at the stated temperature all components of the Enzygnost® HBsAg 6.0 kitmay be used up to the expiry dates given on the labels. For complete stability and storage dataof the open reagents, see Table 1. Table 2 contains information on the on-board stability ofreagents on the various analyzer systems.

Equipment RequiredBEP® III: For automated processing of the test after dispensing the

samples as well as for evaluation.BEP® 2000/BEP 2000 Advance®: For fully automated processing and evaluation of the test.Quadriga®: For fully automated processing of the test and evaluation

in conjunction with BEP® III.


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Pipettes: Piston-type pipettes with fixed or variable volumes, orsingle- and multi-channel pipettes with adjustablevolumes.

The following items are required additionally if the test is not processed automatically:Incubator: Covered thermostatic water bath (37 ±1 °C) or a similar

incubation method.Washing device: Microtitration plate washer.Photometer: Photometer suitable for microtitration plates, measuring

wavelength 450 nm, reference wavelength 650 nm(between 615 nm and 690 nm as appropriate). For SUREmeasurements, wavelength 405 nm is also required.

All the equipment used in the test must have been validated.

SpecimensSuitable specimens are individual samples (human sera or CPDA/EDTA/heparinized/citratedplasma) obtained by standard laboratory techniques.CPDA, heparinized and citrated plasma specimens should be stored for no more than 3 days at2 to 8 °C, whereas serum and EDTA plasma can be used up to 8 days under these storageconditions. If samples are frozen within this period, they can be stored at below -20 °C for atleast 2 years and 8 months if repeated freeze-thaw cycles are avoided.

ProcedureNon-automated Test Procedure

1. Assay scheme: The necessary number of test plate wells is given by the number ofsamples plus the number of determinations (n = 5) for HBsAg Control Serum, negative andpositive.

2. Dispense samples: Dispense 100 µL HBsAg Control Serum, negative into each of 3 wells(A1-C1), 100 µL HBsAg Control Serum, positive into one well (D1) and 100 µL of undilutedsample into each of the subsequent wells. At the end of the series respectively test platefill one further well with 100 µL HBsAg Control Serum, positive.Important:It is not permitted to first pipette the HBsAg Control Serum, positive into the wells at thestart and end of the sample series, and then put the samples in-between.Alternative pipetting scheme: Dispense 100 µL HBsAg Control Serum, negative into eachof 3 wells (A1-C1), 100 µL HBsAg Control Serum, positive into each of 2 wells (D1-E1), and100 µL of undiluted sample into each of the subsequent wells.Each sample must be pipetted with its own pipette tip. The pipetting steps must becompleted within 30 minutes per test plate.

Pipetting Control (optional):The correct pipetting of the controls and samples can easily be checked visually. The differencebetween HBsAg Control Serum, negative (green), HBsAg Control Serum, positive (red), sample andempty well can be clearly differentiated. The pipetting of the controls and samples can also bechecked qualitatively by photometric measurement at 405 nm against 650 nm (the so-called SUREfunction).For a SURE measurement, a photometric measurement at 405 nm and 650 nm is carried out usingthe test plate loaded with controls and samples. The SURE value (ASURE) is calculated as follows:ASURE = A405 nm - A650 nmIf ASURE ≥ 0.100, the well in question was not empty. If ASURE < 0.100, it is questionable whetherthe sample or control has been correctly pipetted into the well in question.

Immediately after dispensing the samples respectively completing the SURE measurement,proceed with dispensing Conjugate 1.


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3. Dispense Conjugate 1: Pipette 25 µL of Conjugate 1 (Anti-HBs/Biotin) into each well. Thenseal with foil and, immediately after completion of the conjugate dispensing step, placethe plate into the incubator.

4. Incubate: Incubate for 60 ± 2 minutes at 37 ±1 °C, then proceed immediately to the washstep.

5. Wash: Remove the foil, aspirate all wells and wash each well 4 times with approx. 0.3 mLwashing solution with a 10-second standing time between each cycle. After completingthe wash cycles, proceed immediately to the Conjugate 2 dispensing step in order toprevent drying out of the wells.

6. Dispense Conjugate 2: Dispense 100 µL Conjugate 2 (Streptavidin/POD) into each well.Then seal with foil and, immediately after completion of the conjugate dispensing step,place the plate into the incubator.

7. Incubate: Incubate for 30 ± 2 minutes at 37 ±1 °C, then proceed immediately to the washstep.

8. Wash: Remove the foil, aspirate all wells and wash each well 4 times with approx. 0.3 mLwashing solution with a 10-second standing time between each cycle. After completingthe wash cycles proceed immediately to the substrate dispensing step in order to preventdrying out of the wells.

9. Dispense substrate: Pipette 75 µL of the Chromogen Working Solution into each wellthen seal the microtitration plate with fresh foil.

10. Incubate substrate: Incubate protected from light at 15 to 25 °C for 30 ± 2 minutes.11. Stop reaction: Remove the foil. Add 75 µL of Stopping Solution POD to each well, keeping

to the same timing as during the substrate dispensing step.12. Measure: Read the test plate at 450 nm within one hour. The recommended reference

wavelength is 650 nm, or where appropriate between 615 and 690 nm.If zeroing of the photometer is required, Stopping Solution POD should be used for this. Followthe guidelines of the manufacturer.

Procedure for the BEP® III SystemWhen using the BEP® III, the test plates must be prepared up to the sample dispensing step(steps 1 and 2 in the section "Non-automated Test Procedure"). Ensure that partially loadedtest plates are supplemented with water-filled strips to at least half plates (6 test strips).Immediately afterwards place the uncovered test plates, i.e. not covered with foil, into theBEP® III.All subsequent processing steps are performed fully automatically by the instrument (seeBEP® III Instruction Manual). The settings for the incubation times in the BEP® III software maydiffer from the times in the section "Non-automated Test Procedure" for technical reasons(system speed) but have been validated for Enzygnost® on the BEP® III System.

Procedure for fully automated systems (BEP® 2000 and Quadriga®)The sample dispensing steps and subsequent processing of the test are performed fullyautomatically by the analyzer (see respective instruction manual). Ensure that partially loadedtest plates are supplemented with water-filled strips to at least half plates (6 test strips).For technical reasons (system speed) the settings for the incubation times in the software ofthese analyzers may differ from the times specified under "Non-automated Test Procedure" buthave been validated for the analyzer and the kit.

Internal Quality ControlValidation Criteria

Validations are performed automatically with the BEP® III, BEP® 2000 and Quadriga® Systems.Please consult the relevant Instruction Manual. The individual absorbance values of HBsAgControl Serum, negative are used to calculate the mean value if both of the following criteriaare satisfied:


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Criterion A: -0.010 ≤ Aneg. ≤ 0.150Criterion B: Aneg. < cut-off

When using the analyzers, a maximum of one out of three absorbance values for HBsAg Control Serum,negative, can be eliminated if it does not satisfy both criteria. Alternatively, when the fully automatedBEP® 2000 System is used, a value lying outside criterion B can be eliminated if the following applies:Cut-off < Aneg. ≤ 0.150For reasons of required pipetting safety during manual dispensing of the controls and samples, this latteroption is not available for the BEP® III System; the test run is invalid.

Both individual absorbance values of HBsAg Control Serum, positive must meet the followingspecification:Apos. ≥ 0.700If this specification is not met, the test must be repeated.

ResultsThe evaluations are performed automatically with the BEP® III, BEP® 2000 and Quadriga®Systems. Please consult the relevant Instruction Manual. The following sections must be takeninto account when performing measurements without software support.To calculate the cut-off, use the mean of the valid absorbance values of HBsAg Control Serum,negative and add a value of 0.050.Āneg. + 0.050 = cut-offBased on the criteria of the test, the samples are classified as follows:HBsAg negative: Asample < cut-offHBsAg reactive: Asample ≥ cut-offAn interpretation of the test results is also possible by forming the quotient of Asample and cut-off (Ratio):

Ratio =Asample cut-off

When the BEP® III, BEP® 2000 and Quadriga® Systems are used, the ratio is calculatedautomatically by the analyzer software. With this method results from different runs can bestandardized and made comparable with each other. The test result must be interpreted asfollows:HBsAg negative: Ratio < 1HBsAg reactive: Ratio ≥ 1Samples with absorbance values equal to or above the cut-off respectively a ratio equal to orgreater than 1.0 must be tested again in duplicate. If the results of both duplicate testsproduce absorbance values below the cut-off or ratios less than 1.0, the sample is to beconsidered as antigen-negative according to the test criteria.A sample is considered repeatedly reactive if at least one of two retest absorbance values areequal to or higher than the cut-off or if ratios are equal to or above 1.0. All reactive samplesmust be clarified according to a recognized confirmation method.

Limitations of the Procedure1. Anticoagulants such as heparin, EDTA, citrate and CPDA do not interfere with the test.2. Lipemic samples do not have any adverse effect on the test.3. When testing icteric samples (tested concentrations: up to 600 mg/L bilirubin), no

interference with the test has been observed.4. Samples from pregnant women and samples containing the following potentially

interfering substances were investigated: ANA, HAMA, as well as antibodies to HIV, HCV,CMV, HAV, EBV, HSV and parvovirus. These samples did not affect the test results.


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5. Heat-treated samples (30 minutes, 56 °C) may exhibit increased non-specific reactivity inthe Enzygnost® HBsAg 6.0.

6. An increased hemoglobin concentration (up to a concentration of 6 g/L) caused nointerference with the test result.

7. A raised biotin content (checked up to 2.6 µg/L) in the samples has no effect on the testresult.

8. Co-infections with HIV, HCV, CMV, HDV, EBV, VZV, HAV, HSV, Toxoplasma gondii,Treponema pallidum (syphilis) and parvovirus do not affect the test result.

9. Tests on blood samples from deceased people are not part of the intended use ofEnzygnost® HBsAg 6.0 and are not supported by Siemens Healthcare Diagnostics.

10. Incompletely coagulated sera and microbially contaminated samples should not be used.Any particulate components in the sample (e.g. fibrin clots, erythrocytes) should beremoved before the test.

11. When using thawed samples, ensure good homogenization of the material prior to use.12. Previously frozen samples and samples that have been stored for long periods above the

blood clot may exhibit increased non-specific reactivity in the Enzygnost® HBsAg 6.0.13. Rheumatoid factors in the sample can cause increased non-specific reactivity in the

Enzygnost® HBsAg 6.0, although they do not have any negative impact on the sensitivityof the test.

14. In case of manual testing do not touch the edge of the well with the pipette tip duringconjugate dispensing in order to avoid false reactive results.

15. The test plate should remain fixed during incubation (e.g. placed on a secured floatationaid, or in a non-circulating water-bath); the wells of the plate must be in contact with thetemperature-controlled water. If stabilizers are used to prevent microbial contamination ofthe water, care must be taken that neither the surface of the test plate nor the wells comeinto contact with the liquid since such contamination can lead to unspecific reactions.

16. Highly reactive samples may cause a precipitation of the dye during the stopping reaction.This does not interfere with the photometric evaluation.

17. The control sera were produced using native human sera. Therefore, turbidity may occurbut does not impair the test result.

18. If a dilution of standards to determine the test’s analytical sensitivity is carried out, onlythe HBsAg Control Serum, negative included in the kit should be used as dilution medium.

19. If the blood was donated not long after vaccination against hepatitis B, the test’s highsensitivity can lead to results that may be interpreted as reactive. A prior vaccinationagainst hepatitis B can exhibit transient HBsAg in the sample, since the vaccine containsHBsAg as a protective immunogen which, in view off the high sensitivity of Enzygnost®HBsAg 6.0, still remains detectable for a while after vaccination.

20. Siemens has validated use of these reagents on various analyzers to optimize productperformance and meet product specifications. User defined modifications are notsupported by Siemens as they may affect performance of the system and assay results. It isthe responsibility of the user to validate modifications to these instructions or use of thereagents on analyzers other than those included in Siemens Application Sheets or theseInstructions for Use.

21. Results of this test should always be interpreted in conjunction with the patient’s medicalhistory, clinical presentation and other findings.

Performance CharacteristicsSensitivity and Specificity

The results of the sensitivity and specificity studies are shown in Tables 3 and 4 (Appendix).The diagnostic sensitivity was determined using 607 positive samples. All samples were testedas positive.


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The analytical sensitivity of the assay with reference to the PEI reference materials HBsAg,subtype Ad and HBsAg, subtype Ay was below 0.020 U/mL for each standard. Using the 2ndInternational Standard for HBsAg (HBsAg subtype adw2, genotype A; NIBSC code number:00/588) of the WHO10, the analytical sensitivity of Enzygnost® HBsAg 6.0 was below0.032 IU/mL.The reactivity of the test in respect to seroconversion samples was investigated using 39seroconversions. It was found that Enzygnost® HBsAg 6.0 exhibited a sensitivity in detectingseroconversions which is comparable to or better than similar tests. Nevertheless, it cannot beruled out that isolated samples may escape detection when the test is used on a large scale.For the determination of specificity, 28,253 HBsAg-negative sera were investigated at 2evaluation sites and a specificity of 99.89 % (initial testing) and 99.91 % after retesting wasobtained (see Table 4). For the determination of specificity in plasma, 11,369 EDTA plasmaswere investigated at 3 sites and a specificity of 99.79 % (initial testing) and 99.84 % afterretesting was obtained (see Table 4). The centrifugation conditions used at the various testingcenters are also listed in Table 4 for reference purposes. In relation to sample population, testprocedure, and other factors different values may be obtained, which however have to be inaccordance with the Common Technical Specifications (CTS).According to current knowledge, a positive result cannot determine with certainty that there isinfectious HBsAg in the blood, nor can a negative test result exclude for certain the presenceof HBsAg. All reactive samples must therefore be clarified according to a recognizedconfirmation method11,12.

Detection of HBsAg MutantsDuring the validation studies 272 HBsAg mutants were tested at different sites withEnzygnost® HBsAg 6.0. The sample collective consisted of native patient samples (n = 148)and recombinant HBsAg samples (n = 124). The mutated variants of HBsAg contained both,single amino acid exchanges versus the wild type sequence (e.g. G145R or T123 N) andmultiple exchanges up to 25 within the amino acid positions 100 to 160 of the small HBsAg(sHBsAg).All studied HBsAg variants were detected by Enzygnost® HBsAg 6.0

PrecisionThe results of intra-/interassay precision are listed in Table 5 (in the Appendix). These resultsare for illustrative purposes only. Differing values may well be obtained depending on the testprocedure.

Bibliography1. Blumberg BS, Alter HJ, Visnich S. A „new“ antigen in leukemia sera. JAMA 1965;191:541-6.2. Dane DS, Cameron CH, Briggs M. Virus-like particles in serum of patients with Australia-

antigenassociated Hepatitis. Lancet 1970;295:695-8.3. Koff RS. Hepatitis B today: clinical and diagnostic overview. Pediatr Infect Dis J

1993;12:428-32.4. Hoofnagle JH, Shafritz DA, Popper H. Chronic type B hepatitis and the „healthy“ HBsAg

carrier state. Hepatology 1987;7:758-63.5. Frösner G. Viral hepatitis. In: Thomas L, ed. Clinical Laboratory Diagnostics. Frankfurt:

THBooks Verlagsgesellschaft, 1998;1260-87.6. Purcell RH. Hepatitis viruses: Changing patterns of human disease. Proc Natl Acad Sci USA

1994;91:2401-6.7. Norder H, Couroucé AM, Coursaget P, et al. Genetic diversity of hepatitis B virus strains

derived worldwide: genotypes, subgenotypes, and HBsAg subtypes. Intervirology2004;47:289-309.

8. Bartholomeusz A, Schaefer S. Hepatitis B virus genotypes: comparison of genotypingmethods. Rev Med Virol 2004;14:3-16.


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9. Levicnik-Stezinar S. Hepatitis B surface antigen escape mutant in a first time blood donorpotentially missed by a routine screening assay. Clin Lab 2004;50:49-51.

10. WHO Expert Committee on Biological Standardization. Fifty-forth Report. Geneva: WorldHealth Organization. Tech Rep Ser 927 (ISBN 9241209275):27-9.

11. Mitteilungen des Arbeitskreises Blut des Bundesministeriums für Gesundheit. Verfahrenzur Rückverfolgung (Look Back) (gemäß § 19 Transfusionsgesetz). Bei der 62. Sitzung desArbeitskreises Blut am 14.6.2006 wurde folgendes Votum (V 34) verabschiedet.Bundesgesundheitsbl - Gesundheitsforsch - Gesundheitsschutz 2006;49:940–957.

12. Mitteilungen des Arbeitskreises Blut des Bundesministeriums für Gesundheit. Ergänzungzum Votum 34 „Verfahren zur Rückverfolgung (Look Back) (gemäß § 19Transfusionsgesetz)“ vom 14. 6. 2006. Bei der 63. Sitzung des Arbeitskreises Blut am11.10.2006 wurde folgendes Votum (V35) verabschiedet. Bundesgesundheitsbl -Gesundheitsforsch - Gesundheitsschutz 2007;50:246–247.

Definition of Symbols

Do not reuse Use By

LOT Batch Code REF Catalogue Number

Caution, consult accompanyingdocuments Manufacturer

EC REPAuthorized representative in theEuropean Community Contains sufficient for tests

Biological Risks IVD In Vitro Diagnostic Medical Device

Temperature Limitation Consult instruction for Use

Non-sterile CE mark

CONTENTS Contents Reconstitution volume

LEVEL Level Keep away from sunlight and heat

BEP®, BEP 2000 Advance®, Enzygnost® and Quadriga® are trademarks ofSiemens Healthcare Diagnostics.

© 2010 Siemens Healthcare Diagnostics Products GmbH.

All rights reserved.

2014-05

Siemens Healthcare Diagnostics Products GmbHEmil-von-Behring-Str. 7635041 Marburg/Germanywww.siemens.com/diagnostics


OPFMG03C11 Rev. 11 – EN 2014-05 9/13

Tab. 1 Storage and StabilityMaterial/reagent State Storage Stabilitya

Enzygnost® HBsAg 6.0 testplate, remaining strips

once opened 2–8 °C15–25 °Cin the bag withdesiccant

4 weeks6 x 6– 8 hoursb

Conjugate 1(Anti-HBs/Biotin)

once opened 2–8 °C15–25 °C


Conjugate 2(Streptavidin/POD)

once opened 2–8 °C15–25 °C


HBsAg Control Serum,negative

once opened 2–8 °C15–25 °C≤ -15 °C

4 weeks6 x 6– 8 hoursb3 months

HBsAg Control Serum,positive

once opened 2–8 °C15–25 °C≤ -15 °C

4 weeks6 x 6– 8 hoursb3 months

Chromogen TMB once opened 2–8 °C expiry date

Buffer/Substrate TMB once opened 2–8 °C expiry date

Chromogen WorkingSolution

diluted 1+10 2–8 °C15–25 °Cclosed container,protected from light

5 days8 hours

Washing Solution POD once openeddiluted 1+19

2–8 °C2–8 °C18–25 °C

expiry date1 week1 day

Stopping Solution POD once opened 2–8 °C expiry datea use each component by the expiry date at the latest.b Number of cycles of standing time opened on the laboratory bench (within 4 weeks), closed in-between at

2–8 °C.

Tab. 2 Details regarding On-board StabilityMaterial/reagent Storage Stabilitya

Conjugate 1(Anti-HBs/Biotin)

BEP® III, Quadriga® 48 hours opened (pooled in plasticbottle or original container)

BEP® 2000 24 hours in original container

BEP® III, BEP® 2000, Quadriga® 6 x 6– 8 hoursc

Conjugate 2(Streptavidin/POD)

BEP® III, Quadriga® 48 hours opened (pooled in plasticbottle or original container)

BEP® 2000 24 hours in original container

BEP® III, BEP® 2000, Quadriga® 6 x 6– 8 hoursc


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Material/reagent Storage Stabilitya

HBsAg Control Serum,negative

BEP® 2000, Quadriga® 6 x 6– 8 hoursd

HBsAg Control Serum,positive

BEP® 2000, Quadriga® 6 x 6– 8 hoursd

c Number of cycles of standing time open in the BEP® III, BEP® 2000 Systems, or in the Quadriga® Systems(within 4 weeks after first opening), closed in-between at 2–8 °C.

d Number of cycles of standing time open in the BEP® 2000 Systems or in the pipettor area of the Quadriga®Systems (within 4 weeks after first opening), closed in-between at 2–8 °C.

Tab. 3 SensitivityWhen evaluating the sensitivity, 607 HBsAg positive samples were tested with the following results:

Sample population Number of samples Reactive samples

HBsAg positive samples, genotype A 31 31

HBsAg positive samples, genotype B 8 8

HBsAg positive samples, genotype C 12 12

HBsAg positive samples, genotype D 74 74

HBsAg positive samples, genotype E 14 14

HBsAg positive samples, genotype F 12 12

HBsAg positive samples, genotype G 4 4

HBsAg positive samples, genotype H 1 1

Tab. 4 SpecificityThe following data was obtained when testing HBsAg negative samples to determine the specificity:

Site Specimen type Number ofsamples

Centrifugationconditions

Initial reactivesamples

(specificity in %)

Retest reactivesamples

(specificity in %)

1 serum 22,802 4 minutes at3,500 x g

27(99.88 %)

22(99.90 %)

2 serum 5,451 10 minutes at3,000 x g

3(99.94 %)

3(99.94 %)

3 EDTA plasma 5,188 4 minutes at3,500 x g

13(99.75 %)

11(99.79 %)

4 EDTA plasma 5,101 4 minutes at3,200 rpm

11(99.78 %)

7(99.86 %)

5 EDTA plasma 1,080 10 minutes at2,500 x g

0(100 %)

0(100 %)

Tab. 5 PrecisionFive test samples with different concentrations of HBsAg were tested to determine the intra- andinterassay variation coefficients (CV) (8-fold replicates in 5 runs). The calculations were performed byanalysis of variance. Exemplary results obtained from two studies on the BEP® III are summarizedbelow:


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Study 1Sample Status Mean absorbance value

(A)Intraassay CV (%) Interassay CV (%)

FP1 Negative 0.017 10.9 39.8

FP3 Borderlinepositive

0.139 6.1 13.0

FP4 Positive(lower range)

0.614 4.7 10.2

FP5 Positive(mid-range)

1.308 5.0 9.9

FP6 Positive(upper range)

1.992 2.9 6.3

Study 2Sample Status Mean absorbance value

(A)Intraassay CV (%) Interassay CV (%)

FP1 Negative 0.019 16.9 2.3

FP3 Borderlinepositive

0.186 3.0 6.7

FP4 Positive(lower range)

0.697 3.7 6.3

FP5 Positive(mid-range)

1.411 4.1 6.7

FP6 Positive(upper range)

2.121 2.7 5.5


12/13 2014-05 OPFMG03C11 Rev. 11 – EN

Tab. 6 Test Procedure

Prepare reagents

measurement:

450 nm vs. 650 nm

Test result

75 µL Stopping Solution

Incubation (protected from light):

30 ±2 minutes(15 to 25 °C)

75 µL Chromogen WorkingSolution

Wash 4 x, standing time:at least 10 seconds

Wash 4 x, standing time: at least 10 seconds

Incubation:60 ±2 minutes (37 ±1 °C)

BEP® IIIautomated test

processing

Incubation:30 ±2 minutes (37 ±1 °C)

3 x 100 µL HBsAg Control Serum, negative1 x 100 µL HBsAg Control Serum, positive100 µL undiluted sample each1 x 100 µL HBsAg Control Serum, positive

In the case of partially filled plates: Add "water-filled

strips" to half fill the plates

Optional SURE measurement:405 nm vs. 650 nm

SURE result ≥ 0.1 A: Well is not empty

BEP® 2000- /Quadriga® Systems fully automated test

processing

25 µL Conjugate 1

100 µL Conjugate 2

(after max. 60 minutes)


OPFMG03C11 Rev. 11 – EN 2014-05 13/13

■ ENIntended UseSummary and ExplanationPrinciple of the MethodReagentsMaterials required but not providedCompositionWarnings and PrecautionsPreparation of the ReagentsStorage and StabilityEquipment Required

SpecimensProcedureNon-automated Test ProcedureProcedure for the BEP® III SystemProcedure for fully automated systems (BEP® 2000 and Quadriga®)Internal Quality ControlValidation Criteria

ResultsLimitations of the ProcedurePerformance CharacteristicsSensitivity and SpecificityDetection of HBsAg MutantsPrecision

BibliographyDefinition of SymbolsStorage and Stability Details regarding On-board Stability SensitivitySpecificityPrecisionTest Procedure

enzygnost® hbsag 6.0 5peramed.com/peramed/docs/opfm03-opfm05_en.pdf · (between 615 nm and 690 nm...

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