Introduction
The BioQControl HBsAg is derived from the same source as has been used to prepare the second WHO HBsAg standard. The standard contains 8.25 IU/ml. Moreover the standard is calibrated against the Paul Erhlich standards. The Paul Ehrlich standards are calibrated in ng/ml and therefore relate to highest metrological level.
WHO HBsAg standard description
Background
The Working Group on Hepatitis and HIV Diagnostic Kits held at WHO in Geneva on 16 to 19 January 2000 agreed that the new WHO HBsAg Reference Standard and a series of dilutions would be of use to NRAs in evaluating kits. It was agreed that the range of dilutions should approximately encompass the range of approximately 25 IU to 0.1 IU/ml, as this range would encompass the sensitivity of the least and most sensitive kits available at present.
Production of dilutions
Plasma derived HBsAg was purified at the Central Laboratory of the Netherlands Red Cross by PEG precipitation and ultracentrifugation to remove Dane particles. The purified HBsAg was inactivated by heating at 101-103oC for 90 seconds followed by pasteurisation at 65 oC for 10h. This antigen preparation was diluted in recalcified plasma which has been shown to be negative for anti-HCV, anti-HIV 1+2, HBsAg as well as negative for HCV RNA, HBV DNA and HIV RNA negative. Merthiolate was added to the serum as preservative.
Five preparations containing four-fold dilutions of the HBsAg preparation were prepared along with a negative control containing the same plasma diluent. The preparations were filled as 1ml aliquots in rubber stoppered vials and were freeze-dried. The WHO Collaborating Centres for Biological Standards have proposed that the most concentrated panel member (panel member 1) be designated as a candidate replacement for the First International Standard for HBsAg (NIBSC Code 80/549). It will be calibrated in IU in this collaborative study.
Aim of the Collaborative studies
It is proposed that the collaborative studies be divided into two phases which will be undertaken concurrently. The aim of this collaborative study is to calibrate panel member 1 in International Units and ng/ml. The antigen content of this preparation will be calculated relative to the WHO International Standard, (subtype ad), the original Paul Ehrlich Institute standard, (the Thomssen-Gerlich native, non-inactivated HBsAg preparation), the current Paul Ehrlich Standard (heat inactivated), the AFSSAPS standard (formalin inactivated and the Abbott Standard. In this way, the relationships of the different units of activity assigned to these standards can be established. The remaining panel members (panel members 2-6) will be included in the study but only tested undiluted. The proposed WHO HBsAg reference panel would also be characterised by Professor Gerlich in physico-chemical tests.
In the second phase of the study it is proposed that the reference panel be characterised using a wide range of detection kits that are in use around the world and are accessible to members of the working group who would be asked to participate.
Design of the study - Calibration of panel member 1 - the 2nd WHO standard for HBsAg
Participants are requested to assay dilutions of the International Standard (80/549), panel member 1 (the candidate replacement International Standard), the original PEI standard ((Thomssen-Gerlich), the AFSSAPS standard, and the Abbott Diagnostics standard on three separate days, preferably at least one week apart. The unitage assigned to each preparation is given in Table 1 along with information on reconstitution and a suggested series of dilutions to be tested. Panel members 2-6 should be reconstituted and tested undiluted in each of the assays. A fresh vial of each sample should be used on each occasion.
Diluent (defibrinated delipidised plasma negative for HBsAg, anti-HCV, anti-HIV 1+2 and anti-HBs) will be supplied. Two replicate samples of each of these samples should be assayed in the same way as routine test samples would be used, i.e., according to the instructions for use of the kit being used. A fresh vial of each sample should be used on each occasion.
All samples should be tested in the previously agreed assay kits on 3 separate occasions so that at least 6 sets of data from the use of each kit are available. A series of dilutions can be used on as many assay kits as a laboratory can run concurrently. However, participants will be sent a number of aliquots/vials of each preparation so that the dilution series need be used on at most 2 assay kits.
The data will be analysed by parallel line analysis. The mean HBsAg content of each preparation will be calculated in IU/ml or ng/ml and relative to each other.
Table 1 Assigned unitage of study samples and suggested dilutions for assay in phase 1:
|
Preparation |
Assigned unitage |
Reconstitution and suggested first dilution to be assayed in duplicate |
Additional dilutions to be assayed in duplicate |
|
International Standard |
100IU/ampoule. |
Reconstitute in 1ml dH2O Prepare a 1 in 10 dilution (1 IU/ml) in the plasma diluent provided |
5 two-fold dilutions of the 1 in 10 pre-dilution in the diluent provided |
|
Panel member 1 |
Approximately 25 IU/ml |
Reconstitute in 1ml dH2O Prepare a 1 in 5 dilution in diluent provided |
5 two-fold dilutions of the 1 in 5 dilution in diluent provided |
|
First Paul Ehrlich Primary standard (50000PEI units/ml pre-diluted 1 in 10000 to 50PEI units) |
50 PEI units/ml |
Prepare a 1 in 5 dilution in diluent provided
|
Prepare 5 two-fold dilutions in diluent provided |
|
Current Paul Ehrlich Inst. standard 1000PEI units/ml (prediluted to 50PEI units) |
50 PEI Units/ml
|
Prepare a 1 in 5 dilution in diluent provided
|
Prepare 5 two-fold dilutions of in diluent provided |
|
AFSSAPS standard
|
5 French ng/ml |
Use as is |
Prepare 5 two-fold dilutions in diluent provided |
|
Abbott Standard |
Approximately 10 IU/ml |
Use as is |
Prepare 5 two-fold dilutions of (D) in diluent provided |
|
Panel member 2 |
1 in 4 dilution of panel member 1 |
Reconstitute in 1ml dH2O Undiluted |
Not applicable |
|
Panel member 3 |
1 in 16 dilution of panel member 1 |
Reconstitute in 1ml dH2O Undiluted |
Not applicable |
|
Panel member 4 |
1 in 64 dilution of panel member 1 |
Reconstitute in 1ml dH2O Undiluted |
Not applicable |
|
Panel member 5 |
1 in 256 dilution of panel member 1 |
Reconstitute in 1ml dH2O Undiluted |
Not applicable |
|
Panel member 6
|
Human re-calcified plasma, undiluted |
Undiluted |
Not applicable |
Table 2 List of HBsAg assays
|
Test method |
Testing laboratory |
|
|
1. |
Prism |
PEI, Sanquin, CBER |
|
2. |
Axsym |
PEI, Sanquin, |
|
3. |
Ausria II |
Abbott, CBER |
|
4. |
Auszyme |
NIBSC, Abbott, CBER |
|
5. |
Monolisa |
France, NIBSC |
|
6. |
Murex |
NIBSC, PEI |
|
7. |
Behring |
Sanquin,, PEI |
|
8. |
Biotest/Biokit |
PEI, France, Sanquin, |
|
9. |
Dia-Sorin |
PEI |
|
10. |
BioMerieux |
PEI, France |
|
11. |
Ortho |
France, Sanquin,, PEI, CBER |
