Enzygnost* Anti-HBc monoclonal - Medcorp

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Equipment required:BEP ® II: For automatic dispensing of reagent and washingBEP ® III: For automatic processing of the test after dispensing the samples as well as forevaluationBEP ® 2000: For fully automatic processing and evaluation of the testPipettes: piston-type pipettes: 25, 100 and 1000 µLIncubator: Covered water bath (+37 ± 1 °C) or comparable incubation methodsAll the equipment used in the test must have been validated.SpecimensSuitable specimens are individual samples (human sera or EDTA/ heparinized/citrated plasma)obtained by standard laboratory techniques.The samples should be stored for not more 3 days at +2 - +8 °C. If the samples are to be storedfor a longer period of time, they must be frozen.ProcedureProcedure using the BEP ® II1. Assay scheme: Ascertain the number of wells required (= number of samples to be testedplus 6 wells for controls). Strips not required for the test should be removed from the holderand stored for later use (See Table 1 for stability data).2. Dispense samples: Pipette 25 µL/well of negative Control, into 4 wells, 25 µL of positivecontrol into the next well and then fill the following wells with 25 µL/well of undiluted sample.At the end of the series / plate pipette 25 µL of positive control once more and proceed to ”3.Dispense conjugate“ as soon as possible, max. 15 min after completion of the sampledispensing step.As an alternative to the above pipetting scheme, it is also possible to pipette the positivecontrol twice at the start of the series.Pipetting scheme: Pipette 25 µL/well of the negative control into 4 wells, 25 µL/well of thepositive control into 2 wells, and fill the subsequent wells with 25 µL/well of undiluted sample.3. Dispense conjugate: Pipette 100 µL of the Working Conjugate Solution into each well, sealwith fresh foil and immediately place into the incubator.4. Incubate: Incubate at +37 °C ± 1 °C for 60 ± 5 min., then proceed immediately to the washstep.5. Wash: Remove the foil, aspirate all the wells and wash 4 times with approx. 0.3 mL/well ofWashing Solution POD.6. Add substrate: Pipette 100 µL of Working Chromogen Solution into each well and seal theplate with fresh foil.7. Incubate: Incubate protected from light at +18 to +25 °C for 30 ± 2 min.8. Stop reaction: Remove foil, and add 100 µL of Stopping Solution POD to each well, keepingto the same timing as in „6. Add substrate“.9. Read: Read at 450 nm within one hour.The use of a photometer with two wavelengths (measurement and reference beams) isrecommended.The absorbances of the control and patient samples are to be measured at a wavelength of 450nm. The wavelength recommended for the reference reading is 650 nm (if necessary between615 nm and 690 nm).OUWE G13 C0541 (879) CS/R 4
Test procedure using the BEP ® IIIBefore using the BEP ® III, prepare the test plates and sample dispensing steps (Section 1 and 2at "Test procedure with the BEP ® II"). Immediately afterwards place the uncovered test plates(i.e. not covered with adhesive foil) into the BEP ® III. Note that partially filled test plates need tobe made up to at least half plates (6 test strips) by adding "water-filled strips". The test is thenprocessed fully automatically (see BEP ® III instruction manual).The settings for the incubation times in the BEP ® III software may differ from the times on theBEP ® II for technical reasons (system speed) but have been validated for Enzygnost* on theBEP ® III.Test procedure using the BEP ® 2000The sample dispensing steps and subsequent processing of the test are performed fully automaticallyby the analyzer (see BEP ® 2000 instruction manual).Test validationThe individual values of the absorbances for the control sera are used to calculate the meanvalues if:A neg. ≥ 0.700-0.010 ≤ A pos. ≤ 0.100If one of the absorbance values of the Anti-HBc Control Serum, negative, is outside thespecification, this value can be neglected.Both absorbance values of the positive control must comply with the specification. If theseconditions are not fulfilled, the test is to be repeated.EvaluationThe evaluations are performed automatically if the BEP ® 2000 or BEP ® III is used. Pleaseconsult the relevant instruction manuals. The following sections apply if the measurements arecarried out without using a software.Calculate the mean absorbance value of the negative controls and then calculate the cut-offvalue by multiplying the mean value by a factor of 0.4:–A neg. x 0.4 = cut-off valueThe equivocal range is defined as:cut-off ± 10%Based on the criteria of the test, the samples are classed as follows:Test result:1. A sample > cut-off + 10% ^= negative2. A sample < cut-off – 10% ^= positive3. cut-off - 10% ≤ A sample ≤ cut-off + 10% ^= equivocalIf an equivocal result is obtained the sample is to be retested in dual determination. If, in theretest, both absorbance values are above or below the equivocal range, the initial equivocalresult can be ignored and the sample can be classed as negative or positive, respectively.However, if the sample again reacts equivocally in one or both of the determinations of the retest,to obtain final clarification it is recommended that a new sample should be tested which has beencollected 2 to 4 weeks after the first sample.Limitations of the Procedure1. Samples containing sodium azide must not be used!2. Anticoagulants such as heparin, EDTA and citrate do not affect the test result.3. No interferences were observed with heat-treated samples (60 min, +56 °C).4. Serum from insufficiently coagulated blood, and cellular blood components, may lead tounreliable results.5. Samples that are haemolytic or contain rheumatoid factors do not impair the test results.6. Samples containing antibodies to CMV, and samples which are positive for anti-HBs, do notinterfere with the test result.OUWE G13 C0541 (879) CS/R 5
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