EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION

WHO/BS/07.2059
ENGLISH ONLY
EXPERT COMMITTEE ON BIOLOGICAL
STANDARDIZATION
Geneva - 8 to 12 October 2007
Evaluation of two Human plasma pools as candidate International Standard
Preparations for syphilitic antibodies
Peter Rigsby, Catherine Ison, Matthew Brierley, Ron Ballard, Hans-Jochen
Hagedorn, David Lewis, Daan W. Notermans, Jørn Riis, Peter Robertson,
Ilkka J.T. Seppälä and Sjoerd Rijpkema
© World Health Organization 2007
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WHO/BS/07.2059
Page 2
Evaluation of two Human plasma pools as candidate International Standard
Preparations for syphilitic antibodies
Peter Rigsby1, Catherine Ison2, Matthew Brierley1, Ron Ballard3, Hans-Jochen
Hagedorn4, David Lewis5, Daan W. Notermans6, Jørn Riis7, Peter Robertson8,
Ilkka J.T. Seppälä 9 and Sjoerd Rijpkema10
1
Biostatistics Section and 10Division of Bacteriology, National Institute for
Biological Standards and Control, South Mimms, Potters Bar and 2Sexually
Transmitted Bacteria Reference Laboratory, Health Protection Agency, Centre for
Infections, Colindale, United Kingdom; 3Laboratory Reference and Research
Branch, Division of STD Prevention, Centers for Disease Control and Prevention,
Atlanta, Georgia, USA; 4Labor Dr. Krone and Partner, Bad Salzuflen, Germany;
5
Sexually Transmitted Infections Reference Centre, National Institute for
Communicable Diseases (NHLS), Johannesburg, South Africa; 6Diagnostic
Laboratory for Infectious Diseases & Perinatal Screening Centre for Infectious
Disease Control, National Institute for Public Health and the Environment
(RIVM), Bilthoven, The Netherlands; 7Dept. of Clinical Biochemistry, Statens
Serum Institut, Copenhagen, Denmark; 8SEALS Area Serology Laboratory, Prince
of Wales Hospital, Randwick, New South Wales, Australia and 9HUSLAB, Unit
of Immunology, Clinical Microbiology, Helsinki University Hospital, Helsinki,
Finland
For correspondence: Division of Bacteriology, National Institute for Biological
Standards and Control, South Mimms, Potters Bar, EN6 3QG, United Kingdom
Tel: 44 (0)1707 641000; Fax: 44 (0)1707 641054; e mail: [email protected]
Summary
Two freeze dried human plasma preparations containing anti-Treponema pallidum
antibodies, 05/132 and 05/122, were assessed for their suitability as international
reference reagents for syphilis serology. A collaborative study was designed to
compare these preparations with the first international standard (IS) for syphilitic
serum antibodies, HS, by eight laboratories from eight countries. Samples were tested
in the T. pallidum passive particle agglutination assay (TPPA) and in non-treponemal
assays: the venereal disease research laboratory test (VDRL) and the rapid plasma
reagin test (RPR). In addition a range of other immunoassays was used. The outcome
of the collaborative study revealed that candidate standard 05/132 contains T.
pallidum-specific immunoglobulin G (IgG) and IgM and is reactive in VDRL or RPR.
On the basis of these results 05/132 is designated the 1st IS for human syphilitic plasma
IgG and IgM and assigned a unitage of 3 IU per ampoule relative to HS. Candidate
standard 05/122 contains T. pallidum specific IgG but is not reactive in either the
VDRL or RPR test. On the basis of these results, we propose to assign a unitage to
05/122 relative to 05/132 and designate 05/122 the 1st IS for human syphilitic plasma
IgG with a unitage of 300 mIU per ampoule.
WHO/BS/07.2059
Page 3
Introduction
Syphilis is a sexually transmitted disease caused by spirochetes of the species
Treponema pallidum subsp. pallidum. Recent reports show that the incidence of
syphilis has risen in both developed and developing countries [1, 2, 9-11]. In
Europe and USA, the rise in syphilis has largely been attributed to an increase in
unsafe sexual behaviour among men who have sex with men [1, 9], while in Africa
and Russia, the disease is usually heterosexually transmitted, often associated with
HIV infection and enhances its transmission, and untreated congenital syphilis
remains a major cause of perinatal morbidity and mortality [7, 9-11]. Therefore
worldwide, syphilis remains a significant public health problem, which has
additional implications for the safety of blood products and organ donations akin
to blood borne viruses.
Establishment of a rapid and accurate diagnosis remains an essential element in
the prevention and treatment of syphilis. A number of serological tests are
available to confirm a clinical diagnosis or screen for asymptomatic infection.
These tests can be divided in those assays that measure treponema-specific
antibodies, such as: the T. pallidum passive particle agglutination assay (TPPA),
the fluorescent Treponema antibody (FTA) assay and enzyme immunoassays
(EIAs) based on native or recombinant antigens; and assays which measure nonspecific antibodies: the venereal disease research laboratory test (VDRL) and the
rapid plasma reagin test (RPR) . The non-treponemal tests, which are relatively
inexpensive, have historically been used as screening tests and can be used to
monitor the efficacy of treatment. In contrast, the treponemal tests are more
expensive but significantly more specific than the non-treponemal tests.
Historically, they were used only as confirmatory assays; however with the advent
of automation, some tests (primarily EIAs) have been used as screening assays, but
this practice should only be adopted in low prevalence settings since samples
remain positive even after successful therapy [3].
Reference laboratories, diagnostic laboratories and test manufacturers need
international standards (ISs) to calibrate immunoassays assays. Indeed, a recent
study by Muller et al. highlighted the importance of standardisation and
proficiency testing for the maintenance of high standards in syphilis serology [8].
HS, the 1st IS for syphilitic serum antibodies, was established under auspices of
the WHO in 1957 and has a unitage of 24.4 IU ml-1 (49 IU per ampoule [12]). The
unitage was assigned on the basis of the reactivity in the cardiolipin and Kahn
assays, which were used routinely at that time and preceded the current
RPR/VDRL assays. Stocks of HS are now exhausted and a new preparation is
needed.
We selected two human plasma pools (05/122 and 05/132) as candidates to replace
HS and designed a collaborative study to assign a unitage to these preparations.
Preliminary analysis revealed that 05/122 contains specific immunoglobulin G
(IgG) but not IgM and is not reactive in the cardio-lipin based tests and that 05/132
contains specific IgG and IgM and is strongly reactive in the RPR test (Table 1).
WHO/BS/07.2059
Page 4
Therefore, 05/122 and 05/132 can be taken to represent the antibody response of
patients with previously treated early syphilis and active syphilis respectively [3].
Participating laboratories of the collaborative study were asked to analyse the
presence of syphilitic antibodies in 05/122 and 05/132 by TPPA and VDRL or
RPR. In addition participating laboratories were encouraged to use additional
assays that are part of their diagnostic routine. Our primary aims were to:
1) Assess the suitability of the two freeze-dried preparations 05/122 and 05/132
as candidate ISs for TPPA and RPR/VDRL assays and calibrate these in terms of
HS reactivity in the TPPA and RPR/VDRL.
2) To assess the reactivity of the two candidate ISs and HS in various immunoassays currently in use.
Materials and Methods
Participating laboratories. Eight laboratories from eight countries, including
national reference laboratories, tested the samples and supplied test data (see
author list for details). The participating laboratories were recruited from a
network of diagnostic reference laboratories for sexually transmitted diseases
maintained by one of the authors (CI). National blood banks or manufacturers of
diagnostic assays were not included in this study. We (CI, SR) lacked contacts
with representatives of these organisations and none were suggested to us. Data
were collected and analysed at the National Institute for Biological Standards and
Control (NIBSC), United Kingdom. Throughout the study, participating
laboratories have been identified by a randomly assigned code number to maintain
confidentiality.
Samples. Each participating laboratory received two sets of samples comprising
six coded ampoules and one ampoule of HS. The study codes, NIBSC codes, the
reactivity of the sample in RPR, TPPA, EIA and a brief characterisation are given
in Table 1. All samples tested negative for antibodies to HIV 1/2 and Hepatitis C,
and Hepatitis B surface antigen. Sample 05/142 was obtained from the Regional
Blood Transfusion Service (Leeds, UK) and permission for use was obtained at
source. Duplicates of sample 05/132 were included in the sample set to provide an
independent measure of within-laboratory variability. Processing of candidate ISs
and their use were approved by the local Human Materials Advisory Committee
(05/038 SR and 05/039 SR). All samples were distributed as lyophilized
preparations at room temperature by courier. Samples that were used to analyse
the stability of 05/132 and 05/122 were distributed on dry ice.
Characterisation of the proposed international standard 05/122. Three
samples (average volume: 281 ml) were obtained from the Regional Blood
Transfusion Service and permission for use was obtained at source. Samples tested
positive by EIA (Newmarket Laboratories, Newmarket, UK) and were negative in
the RPR (Axis Shield Diagnostics Ltd Cambridge, UK) and IgM Capture EIA
(Microgen Bioproducts Ltd, Camberley, UK). Samples were stored at -20 oC. At
WHO/BS/07.2059
Page 5
NIBSC, samples were thawed, pooled and dispensed in 1 ml aliquots into glass
ampoules coded 05/122. The pool was positive by EIA, negative in the RPR and
negative in the IgM capture EIA (Table 1). The mean fill weight for 13 ampoules
was 1.0096 g with coefficient of variation (CV) of 0.34 %. On the same day,
freeze-drying under vacuum was started and completed after four days. Ampoules
were back filled with pure N2 (moisture content <10 ppm). Residual moisture
measured by the Karl-Fischer method for 6 ampoules was 0.4066 % (CV of
9.44%). Twenty ampoules were rejected during the production process, 20
ampoules were held for accelerated degradation studies and 665 ampoules were
stored at -20 oC. These are available for distribution by NIBSC.
Characterisation of the proposed international standard 05/132. One litre of
pooled human plasma was collected in South Africa from 25 donors with active
syphilis and for this purpose local ethical permission was obtained (HD Supplies,
Aylesbury, UK). The pool tested positive by the TPPA, EIA, RPR and IgM
Capture EIA tests (Table 1). At NIBSC, samples were thawed and dispensed in 1
ml aliquots into glass ampoules coded 05/132. The mean fill weight for 12
ampoules was 1.0069 g (CV of 0.17 %). On the same day, freeze-drying under
vacuum was started and completed after four days. Ampoules were back filled
with pure N2 (moisture content <10 ppm). Residual moisture measured by the
Karl-Fischer method for 6 ampoules was 0.6941 % (CV of 26.09 %). Twelve
ampoules were rejected during the production process, 20 ampoules were held for
accelerated degradation studies and 929 ampoules were stored at -20 oC. These are
available for distribution by NIBSC.
Diagnostic assays. The assays used in this study are summarized in Table 2. Two
types of assay were distinguished: titration methods and EIAs. Titration methods
yield an end-point titre and participating laboratories that use this type of assay
were requested to perform four replicate tests in total for each sample. Since EIAs
produce a numerical response such as an absorbance or a fluorescence value,
participating laboratories were requested to report the results of four sequential
doubling dilutions (e.g. 1/2, 1/4, 1/8 and 1/16) for each sample. Participating
laboratories using in-house tests followed standard procedures, while laboratories
that used commercially available tests followed procedures described by the
manufacturer. Descriptions of the procedures were returned to NIBSC
accompanied by the raw data for analysis.
Data analysis. For the titration methods, relative endpoint titres were expressed as
potencies relative to HS. For the EIA methods, assays were analysed using the
principles of parallel line bioassay comparing transformed assay response to log
concentration [4]. All mean potencies shown in this report are unweighted
geometric mean potencies. Variability between estimates is shown using geometric
coefficients of variation (GCV).
WHO/BS/07.2059
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Results and Discussion
RPR and VDRL results. Eight laboratories reported positive non-treponemal test
results for samples HS, D and F, with one laboratory performing both RPR and
VDRL. All other samples were reported as negative. Results are summarised as
endpoint dilutions and as potencies relative to HS (see Tables 3 and 4). For
samples D and F, a geometric mean potency of 3.14 IU ml-1 (95% confidence
limits: 2.22 to 4.44; n=9) is calculated relative to HS (24.4 IU ml-1, [12]).
Laboratory 8 obtained identical relative potencies in RPR and VDRL for the same
set of ampoules. Inclusion of only one set of results from this participant 8 in the
calculation of the mean gives 3.02 IU ml-1 (95% confidence limits: 2.04 to 4.46;
n=8).
No significant difference between RPR and VDRL potencies was detected and no
laboratories were found to be outliers. Higher variability is noted for the endpoint
dilutions (GCV 90%; n=9) compared to the relative potencies (GCV 57%; n=9),
indicating that use of a common standard improves agreement between
laboratories. Variability within laboratories has been assessed using the potencies
of the coded duplicates (D and F) relative to one another. All potencies were
between 0.5 and 2.0 and the geometric mean of 1.09 shows agreement with its
expected value. The GCV of 50% indicates a comparable level of variability to
that observed between laboratories.
The unitage of HS was originally defined using cardiolipin antigen-based assays
such as the VDRL assay [12]. The reactivity of 05/132 falls within the range of the
RPR/VDRL and this preparation can be used as a reference standard in these
assays. Both HS and 05/132 represent the antibody response in patients with acute
syphilis prior to treatment with adequate antibiotic therapy. Relative to HS, 05/132
can be assigned a unitage of 3 IU per ampoule.
TPPA results. Seven laboratories reported results for samples HS, A, C, D and F.
All other samples were reported as negative. One participating laboratory did not
perform the TPPA. Results are summarised as endpoint dilutions and potencies
relative to HS, D & F or A (Tables 5 and 6). Results of participant 5 were
excluded as endpoint dilutions were obtained for sample A only. A further three
participating laboratories did not obtain a complete set of results as endpoint
dilutions for HS were outside of the assay range used. As a result of this, the
reactivity of 05/122 (A) cannot be compared to HS and is compared to 05/132
(Table 6). Taking the potency of 05/132 to be 3 IU ml-1, the potency of 05/122 in
the TPPA relative to 05/132 was calculated as 0.33 IU ml-1 (95% confidence
limits: 0.24 to 0.45; n=6). The GCV of 34% demonstrates good agreement
between laboratories.
In a recent study, Manavi et al. showed that TPPA is the most sensitive assay for
the detection of primary syphilis and was superior to RPR/VDRL [6]. Thus an IS
with relatively low antibody potency is better suited for the TPPA. The reactivity
of 05/132 and 05/122 falls within the range of the TPPA and both these
preparations can be used as reference standards in this assay. Relative to 05/132,
05/122 can be assigned a unitage of 300 mIU per ampoule.
WHO/BS/07.2059
Page 7
Ig(G) detection by EIA and FTA. EIA and FTA results are summarised in Tables
7 and 8 respectively. Samples B, E and G were negative in these assays. In most
cases, the EIA response for samples HS, D and F were located on a different
section of the response range to those for A and C, therefore potencies of D and F
relative to HS have been calculated separately from potencies of C relative to A.
Two participating laboratories reported qualitative EIA results and HS, A, C, D
and F were identified as positive in both tests (results not shown).
FTA results were obtained from three laboratories and these are in line with the
results of the Ig(G) EIAs. No false positive results were reported. We conclude
that the reactivity of 05/132 and 05/122 fall within the range of the Ig(G) EIAs and
FTAs and these preparations can be used as reference standards in these assays.
Detection of IgM by EIA and FTA in 05/132. The IgM capture ELISA detected
all IgM positive samples and returned both a positive and a negative result for
duplicates of sample C (Appendix 4). Samples B, E and G were reported as
negative. FTA with fractionated serum identified IgM positive samples correctly
with the exception of sample C (Table 8). Thus IgM capture EIA confirmed the
results of the 19S IgM FTA. It should be noted that sample C was unequivocally
positive by the IgM capture EIA prior to freeze drying (Table 1); therefore we
conclude that freeze drying of sample C may have decreased in the level of antitreponemal IgM further. One false positive result was reported for sample A by
19S IgM FTA (Table 5). All coded samples were reported negative by indirect
IgM EIA (Appendix 4). In the current study, the sensitivity of the 19S IgM FTA
was comparable to IgM capture EIA and superior to indirect IgM EIA. This
observation is in line with findings by Lefevre et al. who reported that for primary
syphilis the diagnostic sensitivity of 19S IgM FTA and the capture IgM EIA are
similar [5]. One laboratory performed blotting and identified IgM in HS, D and F
(Appendix 4) and one laboratory detected IgM detection by TPPA and reported
HS and D, but not F, as positive (results not shown).
We conclude that the reactivity of 05/132 falls within the range of the 19S IgM
FTA, IgM capture EIA and IgM blot, hence 05/132 can be used as an IgM positive
control in these assays. Due to the paucity of data we were not able to assign a
unitage for the IgM content of 05/132.
Stability studies. Preparations 05/132 and 05/122 were tested in one laboratory by
RPR and TPPA following storage at various temperatures for one year. Results are
shown in tables 9 and 10. Some loss of activity was seen at +37oC, but no loss was
seen at +4oC or +20oC, so no predicted loss of activity can be calculated. Our
findings strongly suggest that the unitage of 05/132 and 05/122 will remain stable
at -20oC.
Recommendation for adoption by the Expert Committee on Biological
Standardization. On the basis of the results from the RPR and VDRL assays, it is
recommended that 05/132 be established as the 1st IS for human syphilitic plasma
IgG and IgM with an assigned potency of 3 IU per ampoule relative to IS HS. On
the basis of the results from the TPPA assays, it is recommended that 05/122 be
established as the 1st IS for human syphilitic plasma IgG with an assigned potency
of 300 mIU per ampoule relative to 05/132.
WHO/BS/07.2059
Page 8
Comments from participating laboratories. All the representatives from
participating laboratories (see title page) have seen the original draft, were
consulted on their opinion and were able to provide comments and suggestions.
This is their right since they are quoted as authors and as such take responsibility
for the contents of this report. They have also been sent the version which is an
amalgamation of the first draft and their comments and is essentially the current
version. The comments of the authors were all of a technical nature (description
of disease and diagnostic tests) and grammatical nature and were mostly reported
as ‘track changes’. None of the comments and suggestions had any bearings on the
conclusions of the collaborative study. Hence the conclusions in this report are
supported by all authors.
Acknowledgements
We are grateful to Ms S Coughlan (Centre for Biological Reference Materials,
NIBSC) for coding and packaging of samples and organising their distribution.
The technical assistance from the following staff members is greatly appreciated:
Ms B Marsden and H Patel (Sexually Transmitted Bacteria Reference Laboratory,
Health Protection Agency Centre for Infections); Ms S Kikkert and P Thompson
(WHO Reference Center for Syphilis Serology, Centers for Disease Control and
Prevention); Ms S Jorde, B Hartmeier and I Pieper (Labor Dr. Krone and Partner);
Mr S Khumalo, Dr I Zietsman and Mr. F Radebe (STI Reference Centre, NICD,
NHLS); Mr M Mommers (Laboratory for Infectious Diseases & Perinatal
Screening, Centre for Infectious Disease Control, RIVM); Scientific and technical
staff (South Eastern Sydney Area (SEALS) Serology laboratory); Ms M Korpinen,
A Riutta and R Väisänen (HUSLAB, Helsinki University Hospital).
References
1) Centers for Disease Control and Prevention. Primary and secondary
syphilis--United States, 2003-2004. Morb Mortal Wkly Rep 2006; 55 (10):
269-273.
2) Chen ZQ, Zhang GC, Gong XD, Lin C, Gao X, Liang GJ, Yue XL,
Chen XS, Cohen MS. Syphilis in China: results of a national surveillance
programme. Lancet 2007; 369 (9556): 132-138.
3) Egglestone SI, Turner AJ. Serological diagnosis of syphilis. PHLS
Syphilis Serology Working Group. Commun Dis Public Health 2000; 3
(3): 158-162.
4) Finney, DJ. Statistical Method in Biological Assay. 3rd edition London:
Charles Griffin 1978.
5) Lefevre JC, Bertrand MA, Bauriaud R. Evaluation of the Captia enzyme
immunoassays for detection of immunoglobulins G and M to Treponema
pallidum in syphilis. J Clin Microbiol 1990; 28 (8): 1704-1707
WHO/BS/07.2059
Page 9
6) Manavi K, Young H, McMillan A. The sensitivity of syphilis assays in
detecting different stages of early syphilis. Int J STD AID 2006; 17 (11):
768-771.
7) Mullick S, Watson-Jones D, Beksinska M, Mabey D. Sexually
transmitted infections in pregnancy: prevalence, impact on pregnancy
outcomes, and approach to treatment in developing countries. Sex Transm
Infect 2005; 81(4): 294-302
8) Muller I, Brade V, Hagedorn HJ, Straube E, Schorner C, Frosch M,
Hlobil H, Stanek G, Hunfeld KP. Is serological testing a reliable tool in
laboratory diagnosis of syphilis? Meta-analysis of eight external quality
control surveys performed by the German infection serology proficiency
testing program. J Clin Microbiol 2006; 44 (4): 1335-1341.
9) Nicoll A, Hamers FF. Are trends in HIV, gonorrhoea, and syphilis
worsening in western Europe? BMJ 2002; 324: 1324-1327
10) Reynolds SJ, Risbud AR, Shepherd ME, Rompalo AM, Ghate MV,
Godbole SV, Joshi SN, Divekar AD, Gangakhedkar RR, Bollinger RC,
Mehendale SM. High rates of syphilis among STI patients are contributing
to the spread of HIV-1 in India. Sex Transm Infect 2006; 82 (2):121-126.
11) Salakhov E, Tikhonova L, Southwick K, Shakarishvili A, Ryan C,
Hillis S. Congenital syphilis in Russia: the value of counting epidemiologic
cases and clinical cases. Sex Transm Dis 2004; 31 (2): 127-32.
12) Weiss Bentzon M, Krag P. The international standard for human
Syphilitic serum. Bull. World Health Org 1961; 24: 257-264
Study
Code
HS
D,F
A
E
C
B
G
NIBSC
code
Sample description
HS
05/132
05/122
97/682
05/142
83/571
82/585
1st international standard
Candidate international standard ( pool of 25)
Candidate international standard (pool of 3)
Normal serum
Positive serum
Negative serum with high level of cortisol
Negative serum with high level of cortisol
RPR
64
8
neg
neg
neg
neg
neg
TPPA
>1280
1280
1280
neg
1280
neg
neg
EIA
Total Ig
IgM
pos
pos
pos
neg
pos
neg
neg
Pos
Pos
Neg
Neg
Pos
Neg
Neg
WHO/BS/07.2059
Page 10
Table 1: Samples used in this study
Table 2: Assays used in this study
Test format
Titration assay
Name of test
Rapid plasma reagin test
Immunoassay
Venereal disease research laboratory test
Fluorescent treponema antibody (FTA) assay
19S IgM FTA
T. pallidum passive particle agglutination test
Enzyme Immuno Assay Ig, IgG, IgM
Immunoblot IgM, IgG
Manufacturer
Axis Shield Diagnostics Ltd., BDMacro-Vue, Biokit, Wampole
Impact, In-house
Oxoid, Murex
BioMérieux/DIFCO, Zeus/BAG
BioMérieux/DIFCO, Zeus/BAG
Fujirebio Serodia-TP.PA
Abbott, Microgen bioproducts Ltd,
Mikrogen Gmbh, Newmarket
Laboratories, Radim SpA
Virotech, In-house
Laboratory codes
1,2,3,4,5,8
Total
6
6,7,8
2,6,7
2,6,7
1,2,4,5,6,7,8
1,2,3,5,7,8
3
3
3
7
6
2,7
2
WHO/BS/07.2059
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Lab
1
2
3
4
5
6
7
8
8
Method
Set
Endpoint Dilutions
HS
D
F
RPR
1
64
8
8
2
64
8
8
RPR
1
54
4
2
2
53.8
3.4
4
RPR
1
256
32
45.3
2
128
13.5
8
RPR
1
45.3
16
16
2
64
16
16
RPR
1
32
2.8
4
2
32
2.8
4
VDRL
1
90.5
9.5
13.5
2
90.5
11.3
11.3
VDRL
1
64
5.7
5.7
2
64
8
5.7
RPR
1
64
8
16
2
64
8
16
VDRL
1
32
4
8
2
32
4
8
Geometric Mean
95% Confidence Interval
% GCV
Geometric Mean
(D & F)
8.0
3.2
19.9
16.0
3.4
11.3
6.2
11.3
5.7
7.9
(4.8 - 13.0)
90
WHO/BS/07.2059
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Table 3: Results of RPR and VDRL expressed as average endpoint dilutions
Table 4: Results of RPR and VDRL expressed as relative potencies
Method
Set
Potency of F
relative to D
1.00
1.00
0.57
1.17
1.38
0.55
1.00
1.00
1.44
1.44
1.36
1.00
1.00
0.69
2.00
2.00
2.00
2.00
1.17
(0.95 - 1.43)
50
WHO/BS/07.2059
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Potency (IU ml-1
Geometric % GCV
Mean
calculated relative to HS)
D
F
1
RPR
1
3.05
3.05
3.05
0
2
3.05
3.05
2
RPR
1
1.81
0.90
1.46
39
2
1.54
1.81
3
RPR
1
3.05
4.32
2.68
54
2
2.57
1.53
4
RPR
1
8.62
8.62
7.25
22
2
6.10
6.10
5
RPR
1
2.14
3.05
2.55
22
2
2.14
3.05
6
VDRL
1
2.56
3.64
3.05
15
2
3.05
3.05
7
VDRL
1
2.17
2.17
2.37
19
2
3.05
2.17
8
RPR
1
3.05
6.10
4.31
49
2
3.05
6.10
8
VDRL
1
3.05
6.10
4.31
49
2
3.05
6.10
Geometric Mean
3.14
95% Confidence Interval
(2.22 - 4.44)
% GCV
57
Table 5: Results of TPPA expressed as geometric mean endpoint dilutions
Lab
2
4
6
7
8
Set
1
2
1
2
1
2
1
2
1
2
1
2
HS
163840
>28963
>20480
>28963
4305
3620
24355
28963
>26908
36204
20480
20480
F
28963
>14482
7241
13280
905
2560
14482
10240
5724
6400
2560
3620
WHO/BS/07.2059
Page 14
Lab
1
Endpoint Dilutions
A
C
D
1280
5120
14481
1810
3620
4482
1280
3948
12177
1280
3320
13280
320
453
1280
190
1076
3044
1810
6089
14482
1522
5120
14482
761
2862
5724
640
3200
6400
587
1174
10240
761
1396
10240
Potency relative to A
Individual
Results
C
4.00
2.00
3.08
2.59
1.42
5.66
3.36
3.36
3.76
5.00
2.00
1.83
Geometric
Mean
C
2.83
2.83
2.83
3.36
4.34
1.92
2.93
(2.21 - 3.88)
31
WHO/BS/07.2059
Page 15
Table 6: Results of TPPA expressed as relative potencies
Potency relative to 05/132 (D & F)
Lab Set
Individual
Geometric
Results
Mean
A
C
A
C
1
1
0.06
0.25
0.06
0.25
2
2
1
0.14
0.42
0.11
0.32
2
0.10
0.25
4
1
0.30
0.42
0.14
0.40
2
0.07
0.39
6
1
0.12
0.42
0.12
0.42
2
0.12
0.42
7
1
0.13
0.50
0.11
0.50
2
0.10
0.50
8
1
0.11
0.23
0.12
0.23
2
0.12
0.23
Geometric Mean
0.11
0.34
95% Confidence Interval
(0.08 - 0.15)
(0.25 - 0.47)
% GCV
34
36
Lab
Method
1
Ig EIA
2
IgG EIA
3
IgG EIA
5
Ig EIA
Potency (IU ml-1 calculated
relative to HS)
D
F
1
11.22
8.78
2
9.76
8.05
1
8.30
9.03
1
8.78
9.03
2
7.08
7.81
2
Non-parallel
Non-parallel
1
14.15
10.49
1
HS non-linear HS non-linear
2
9.27
9.27
2
D non-linear
15.13
1
9.03
11.22
1
8.78
8.54
2
9.03
10.98
2
8.30
8.54
Geometric Mean
Set
Geometric
Mean
Potency of C
relative to A
Geometric
Mean
9.27
0.31
0.30
C non-linear
C non-linear
0.71
0.72
0.34
0.44
0.31
0.40
0.48
0.44
0.48
0.46
0.30
8.30
11.47
9.27
9.52
0.71
0.37
0.47
0.44
WHO/BS/07.2059
Page 16
Table 7: Results of Ig(G) EIAs expressed as relative potencies
Table 8: Results of the FTA and 19S IgM FTA
Lab
2
End point dilution or titer for Ig(G)
HS
A
C
D
F
a
a
a
a
3520
160
80
960
880 a
2560 a
160 a
80 a
640 a
720 a
Set
1
2
6
1
++
++
+
++
2
++
++
+
++
7b
1/2
+++ a
+a
+a
+++ a
1/2
++++
++++
+
+++
Preparations B, E and G were reported as negative.
a
: IgG
b
: Only one sample of set 1 or 2 was tested
++
++
+++ a
++++
End point dilution or titer for IgM
HS
A
C
D
50
NEG
NEG
12
55
NEG
NEG
12
+
NEG
NEG
+
+
NEG
NEG
+
++++
++
NEG
+++
ND
ND
ND
ND
F
13
13
ND
ND
++
ND
WHO/BS/07.2059
Page 17
WHO/BS/07.2059
Page 18
Table 9: Results from RPR for 05/122 and 05/132 stored at elevated temperatures for one year
Sample
Storage
temperature
05/122
-20oC
+4oC
+20oC
+37oC
-20oC
+4oC
+20oC
+37oC
05/132
Assay 1 endpoint dilution
Duplicate 1 Duplicate 2
Geometric
Mean
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
1:8
1:8
1:8
1:8
1:8
1:8
1:8
1:8
1:8
1:4
1:4
1:4
Assay 2 endpoint dilution
Duplicate 1 Duplicate 2
Geometric
Mean
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
1:8
1:8
1:8
1:8
1:8
1:8
1:8
1:8
1:8
1:4
1:4
1:4
Geometric mean
potency
relative to -20oC
1.00
1.00
0.50
Table 10: Results from TPPA for 05/122 and 05/132 stored at elevated temperatures for one year
Sample
Storage
temperature
05/122
-20oC
+4oC
+20oC
+37oC
-20oC
+4oC
+20oC
+37oC
05/132
Assay 1 endpoint dilution
Duplicate 1 Duplicate 2
Geometric
Mean
1:640
1:640
1:640
1:640
1:640
1:640
1:640
1:640
1:640
1:320
1:320
1:320
1:5120
1:5120
1:5120
1:5120
1:5120
1:5120
1:5120
1:5120
1:5120
1:5120
1:5120
1:5120
Assay 2 endpoint dilution
Duplicate 1 Duplicate 2
Geometric
Mean
1:640
1:640
1:640
1:640
1:640
1:640
1:640
1:640
1:640
1:320
1:640
1:453
1:5120
1:5120
1:5120
1:5120
1:5120
1:5120
1:5120
1:5120
1:5120
1:2560
1:2560
1:2560
Geometric mean
potency
relative to -20oC
1.00
1.00
0.59
1.00
1.00
0.71
WHO/BS/07.2059
Page 19
WHO/BS/07.2059
Page 20
Appendix 1: Results from individual RPR and VDRL assays
Lab
1
2
Method
RPR
RPR
Assay
1
Set
1
1
HS
1:64
1:64
A
NEG
NEG
B
NEG
NEG
C
NEG
NEG
D
1:8
1:8
E
NEG
NEG
F
1:8
1:8
G
NEG
NEG
2
2
2
1:64
1:64
NEG
NEG
NEG
NEG
NEG
NEG
1:8
1:8
NEG
NEG
1:8
1:8
NEG
NEG
1
1
1
1
1
1:64
1:64
1:64
1:64
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
1:4
1:4
1:4
1:4
NEG
NEG
NEG
NEG
1:2
1:2
1:2
1:2
NEG
NEG
NEG
NEG
2
1
1
1
1
1:32
1:64
1:32
1:64
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
1:4
1:4
1:4
1:4
NEG
NEG
NEG
NEG
1:2
1:2
1:2
1:2
NEG
NEG
NEG
NEG
3
2
2
2
2
1:64
1:64
1:32
1:64
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
1:2
1:4
1:2
1:4
NEG
NEG
NEG
NEG
1:4
1:4
1:4
1:4
NEG
NEG
NEG
NEG
4
2
2
2
2
1:64
1:64
1:64
1:32
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
1:4
1:4
1:4
1:4
NEG
NEG
NEG
NEG
1:4
1:4
1:4
1:4
NEG
NEG
NEG
NEG
Appendix 1: Results from individual RPR and VDRL assays (continued)
Lab
3
4
RPR
RPR
Assay
1
2
3
4
Set
1
1
1
1
HS
1:256
1:256
1:256
1:256
A
NEG
NEG
NEG
NEG
B
NEG
NEG
NEG
NEG
C
NEG
NEG
NEG
NEG
D
1:32
1:32
1:32
1:32
E
NEG
NEG
NEG
NEG
F
1:64
1:64
1:32
1:32
G
NEG
NEG
NEG
NEG
5
6
7
8
2
2
2
2
1:128
1:128
1:128
1:128
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
1:16
1:16
1:8
1:16
NEG
NEG
NEG
NEG
1:8
1:8
1:8
1:8
NEG
NEG
NEG
NEG
1
2
3
4
1
1
1
1
1:32
1:32
1:64
1:64
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
1:16
1:16
1:16
1:16
NEG
NEG
NEG
NEG
1:16
1:16
1:16
1:16
NEG
NEG
NEG
NEG
5
6
7
8
2
2
2
2
1:64
1:64
1:64
1:64
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
1:16
1:16
1:16
1:16
NEG
NEG
NEG
NEG
1:16
1:16
1:16
1:16
NEG
NEG
NEG
NEG
1
2
1
1
1:32
1:32
NEG
NEG
NEG
NEG
NEG
NEG
1:4
1:2
NEG
NEG
1:4
1:4
NEG
NEG
3
4
2
2
1:32
1:32
NEG
NEG
NEG
NEG
NEG
NEG
1:2
1:4
NEG
NEG
1:4
1:4
NEG
NEG
WHO/BS/07.2059
Page 21
5
Method
RPR
Lab
6
7
Method
VDRL
VDRL
Assay
1
Set
1
1
1
1
HS
1:128
1:64
1:64
1:128
A
NEG
NEG
NEG
NEG
B
NEG
NEG
NEG
NEG
C
NEG
NEG
NEG
NEG
D
1:16
1:8
1:8
1:8
E
NEG
NEG
NEG
NEG
F
1:16
1:16
1:16
1:8
G
NEG
NEG
NEG
NEG
2
2
2
2
2
1:128
1:64
1:64
1:128
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
1:16
1:16
1:8
1:8
NEG
NEG
NEG
NEG
1:16
1:8
1:8
1:16
NEG
NEG
NEG
NEG
1
1
1
1
1
1:64
1:64
1:64
1:64
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
1:8
1:8
1:8
1:8
NEG
NEG
NEG
NEG
1:4
1:4
1:8
1:8
NEG
NEG
NEG
NEG
2
2
2
2
1:64
1:64
1:64
1:64
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
1:8
1:8
1:4
1:4
NEG
NEG
NEG
NEG
1:8
1:8
1:4
1:4
NEG
NEG
NEG
NEG
1
1
1:64
NEAT
NEG
NEG
1:8
NEG
1:16
NEG
2
2
1:64
NEAT
NEG
NEG
1:8
NEG
1:16
NEG
1
1
1:32
NEG
NEG
NEG
1:4
NEG
1:8
NEG
2
2
1:32
NEG
NEG
NEG
1:4
NEG
1:8
NEG
2
3
4
8
RPR
VDRL
WHO/BS/07.2059
Page 22
Appendix 1: Results from individual RPR and VDRL assays (continued)
Appendix 2: Results from individual TPPA assays
Lab
1
2
Method
TPPA
TPPA
Set
1
1
HS
1:327680
1:81920
A
1:2560
1:640
B
NEG
NEG
C
1:10240
1:2560
D
1:40960
1:5120
E
NEG
NEG
F
1:40960
1:20480
G
NEG
NEG
3
4
2
2
>1:20480
1:40960
1:2560
1:1280
NEG
NEG
1:5120
1:2560
1:20480
1:10240
NEG
NEG
>1:20480
1:10240
NEG
NEG
1
1
1
1
1
>=1:20480
>=1:20480
>=1:20480
>=1:20480
1:1280
1:1280
1:1280
1:1280
NEG
NEG
NEG
NEG
1:2560
1:5120
1:2560
1:5120
1:10240
1:20480
1:20480
1:20480
NEG
NEG
NEG
NEG
1:10240
1:10240
1:10240
1:10240
NEG
NEG
NEG
NEG
2
1
1
1
1
1:20480
1:20480
1:20480
1:20480
1:1280
1:1280
1:1280
1:1280
NEG
NEG
NEG
NEG
1:5120
1:2560
1:5120
1:5120
1:10240
1:5120
1:10240
1:10240
NEG
NEG
NEG
NEG
1:5120
1:5120
1:5120
1:5120
NEG
NEG
NEG
NEG
3
2
2
2
2
>=1:20480
>=1:20480
>=1:20480
>=1:20480
1:1280
1:1280
1:1280
1:1280
NEG
NEG
NEG
NEG
1:2560
1:2560
1:5120
1:2560
1:10240
1:10240
1:20480
1:10240
NEG
NEG
NEG
NEG
1:10240
1:10240
1:20480
1:10240
NEG
NEG
NEG
NEG
4
2
2
2
2
1:20480
1:40960
1:40960
1:81920
1:1280
1:1280
1:1280
1:1280
NEG
NEG
NEG
NEG
1:2560
1:5120
1:5120
1:2560
1:10240
1:20480
1:20480
1:10240
NEG
NEG
NEG
NEG
1:10240
1:20480
1:10240
1:20480
NEG
NEG
NEG
NEG
WHO/BS/07.2059
Page 23
Assay
1
2
Lab
4
5
6
Method
TPPA
TPPA
TPPA
Assay
1
2
3
4
Set
1
1
1
1
HS
1:5120
1:5120
1:2560
1:5120
A
1:320
1:320
1:320
1:320
B
NEG
NEG
NEG
NEG
C
1:1280
1:320
1:160
1:640
D
1:5120
1:640
1:640
1:1280
E
NEG
NEG
NEG
NEG
F
1:5120
1:320
1:640
1:640
G
NEG
NEG
NEG
NEG
5
6
7
8
2
2
2
2
1:2560
1:5120
1:2560
1:5120
1:80
1:160
1:640
1:160
NEG
NEG
NEG
NEG
1:320
1:2560
1:2560
1:640
1:640
1:5120
1:5120
1;5120
NEG
NEG
NEG
NEG
1:640
1:2560
1:5120
1:5120
NEG
NEG
NEG
NEG
1
2
1
1
>1:1280
>1:1280
1:1280
1:1280
NEG
NEG
>1:1280
>1:1280
>1:1280
>1:1280
NEG
NEG
>1:1280
>1:1280
NEG
NEG
3
4
2
2
>1:1280
>1:1280
1:1280
1:1280
NEG
NEG
>1:1280
>1:1280
>1:1280
>1:1280
NEG
NEG
>1:1280
>1:1280
NEG
NEG
1
1
1
1
1
1:40960
1:20480
1:20480
1:20480
1:2560
1:2560
1:1280
1:1280
NEG
NEG
NEG
NEG
1:10240
1:5120
1:5120
1:5120
1:20480
1:10240
1:10240
1:20480
NEG
NEG
NEG
NEG
1:20480
1:10240
1:20480
1:10240
NEG
NEG
NEG
NEG
2
2
2
2
2
1:40960
1:40960
1:20480
1:20480
1:2560
1:1280
1:1280
1:1280
NEG
NEG
NEG
NEG
1:5120
1:5120
1:5120
1:5120
1:20480
1:10240
1:10240
1:20480
NEG
NEG
NEG
NEG
1:20480
1:10240
1:5120
1:10240
NEG
NEG
NEG
NEG
WHO/BS/07.2059
Page 24
Appendix 2: Results from individual TPPA assays (continued)
Appendix 2: Results from individual TPPA assays (continued)
Lab
7
8
Method
TPPA
TPPA
Set
1
1
HS
>20000
>20000
A
1:640
1:640
B
<1:80
<1:80
C
1:2560
1:2560
D
1:5120
1:5120
E
<1:80
<1:80
F
1:5120
1:5120
G
<1:80
<1:80
2
2
2
1:51200
1:25600
1:1280
1:640
<1:80
<1:80
1:3200
1:3200
1:6400
1:6400
<1:80
<1:80
1:6400
1:6400
<1:80
<1:80
3
1
1
1:51200
1:51200
1:640
1:640
<1:80
<1:80
1:3200
1:3200
1:6400
1:6400
<1:80
<1:80
1:6400
1:6400
<1:80
<1:80
4
2
2
1:25600
1:25600
1:640
1:640
<1:80
<1:80
1:3200
1:3200
1:6400
1:6400
<1:80
<1:80
1:6400
1:6400
<1:80
<1:80
1
1
1
1
1
1:20480
1:20480
1:20480
1:20480
1:640
1:320
1:640
1:640
NEG
NEG
NEG
NEG
1:1280
1:640
1:1280
1:1280
1:10240
1:10240
1:10240
1:10240
NEG
NEG
NEG
NEG
1:2560
1:2560
1:2560
1:2560
NEG
NEG
NEG
NEG
2
1
1
1
1
1:20480
1:20480
1:20480
1:20480
1:640
1:640
1:640
1:640
NEG
NEG
NEG
NEG
1:1280
1:1280
1:1280
1:1280
1:10240
1:10240
1:10240
1:10240
NEG
NEG
NEG
NEG
1:2560
1:2560
1:2560
1:2560
NEG
NEG
NEG
NEG
3
2
2
2
2
1:20480
1:20480
1:20480
1:20480
1:1280
1:1280
1:640
1:640
NEG
NEG
NEG
NEG
1:1280
1:1280
1:1280
1:1280
1:10240
1:10240
1:10240
1:10240
NEG
NEG
NEG
NEG
1:5120
1:5120
1:5120
1:5120
NEG
NEG
NEG
NEG
WHO/BS/07.2059
Page 25
Assay
1
Lab
8
Method
TPPA
Assay
4
Set
2
2
2
2
HS
1:20480
1:20480
1:20480
1:20480
A
1:640
1:640
1:640
1:640
B
NEG
NEG
NEG
NEG
C
1:1280
1:2560
1:1280
1:1280
D
1:10240
1:10240
1:10240
1:10240
E
NEG
NEG
NEG
NEG
F
1:2560
1:2560
1:2560
1:2560
G
NEG
NEG
NEG
NEG
WHO/BS/07.2059
Page 26
Appendix 2: Results from individual TPPA assays (continued)
Appendix 3: Results from individual Ig(G) assays
Lab
1
2
Method
IgG EIA
(Ratio)
IgG EIA
(Ratio)
Set
1
1
HS
16.00
19.21
A
22.16
18.16
B
0.13 NEG
0.12 NEG
C
28.62
24.9
D
21.00
18.25
E
0.17 NEG
0.12 NEG
F
23.10
21.51
G
0.13 NEG
0.13 NEG
2
2
2
23.69
21.50
23.88
16.67
0.13 NEG
0.10 NEG
35.81
22.43
25.40
13.82
0.13 NEG
0.11 NEG
34.51
32.53
0.25 NEG
0.10 NEG
1
1
1
1
1
>150
>150
>150
>150
109
115
91
94
8 NEG
10 NEG
13 NEG
14 NEG
78
80
66
65
>150
>150
>150
>150
9 NEG
10 NEG
3 NEG
4 NEG
>150
>150
>150
>150
7 NEG
7 NEG
8 NEG
11 NEG
2
2
2
2
>150
>150
>150
>150
100
110
117
112
9 NEG
13 NEG
13 NEG
11 NEG
78
76
88
77
>150
>150
>150
>150
10 NEG
13 NEG
10 NEG
13 NEG
>150
>150
>150
>150
10 NEG
11 NEG
9 NEG
9 NEG
1
1
1
1
1
1:2560
1:2560
1:2560
1:5120
1:160
1:160
1:160
1:160
NEG
NEG
NEG
NEG
1:80
1:80
1:80
1:80
1:640
1:640
1:1280
1:640
NEG
NEG
NEG
NEG
1:640
1:640
1:1280
1:640
NEG
NEG
NEG
NEG
2
1
1
1
1
1:5120
1:5120
1:2560
1:2560
1:160
1:160
1:160
1:160
NEG
NEG
NEG
NEG
1:80
1:80
1:80
1:80
1:1280
1:1280
1:1280
1:640
NEG
NEG
NEG
NEG
1:1280
1:1280
1:640
1:640
NEG
NEG
NEG
NEG
2
1
2
FTA-abs
IgG
WHO/BS/07.2059
Page 27
Assay
1
Lab
3
Method
IgG EIA
(U)
Assay
1
Set
2
2
2
2
HS
1:2560
1:2560
1:2560
1:2560
A
1:160
1:160
1:160
1:160
B
NEG
NEG
NEG
NEG
C
1:80
1:80
1:80
1:80
D
1:640
1:640
1:640
1:640
E
NEG
NEG
NEG
NEG
F
1:640
1:640
1:640
1:640
G
NEG
NEG
NEG
NEG
2
2
2
2
2
1
1
1
1
1:2560
1:2560
1:2560
1:2560
343
338
1215
1123
1:160
1:160
1:160
1:160
64
56
220
231
NEG
NEG
NEG
NEG
28 NEG
24 NEG
82 NEG
68 NEG
1:80
1:80
1:80
1:80
28 NEG
25 NEG
102 NEG
110 NEG
1:640
1:640
1:640
1:640
203
238
1030
996
NEG
NEG
NEG
NEG
16 NEG
17 NEG
46 NEG
34 NEG
1:1280
1:640
1:640
1:640
166
235
725
1215
NEG
NEG
NEG
NEG
26 NEG
29 NEG
73 NEG
62 NEG
2
2
2
2
781
886
769
646
180
177
263
261
70 NEG
63 NEG
86 NEG
76 NEG
76 NEG
71 NEG
129 NEG
116 NEG
273
528
990
781
33 NEG
36 NEG
35 NEG
33 NEG
521
562
571
750
66 NEG
66 NEG
57 NEG
66 NEG
1
2
3
4
6
FTA-abs
Ig
1
2
1
2
++
++
++
++
NEG
NEG
+
+
++
++
NEG
NEG
++
++
NEG
NEG
IgG Blot
1
2
1
2
POS
POS
POS
POS
NEG
NEG
POS
POS
POS
POS
NEG
NEG
POS
POS
NEG
NEG
WHO/BS/07.2059
Page 28
Appendix 3: Results from individual Ig(G) assays (continued)
Appendix 3: Results from individual Ig(G) assays (continued)
Lab
7
Method
Ig EIA
Assay
1
Set
1
HS
3.58
3.63
4.01
4.02
A
2.81
2.56
3.15
3.17
B
0.05 NEG
0.05 NEG
0.06 NEG
0.06 NEG
C
2.58
2.59
2.55
2.53
D
2.99
3.01
3.23
3.27
E
0.06 NEG
0.05 NEG
0.06 NEG
0.08 NEG
F
3.05
3.14
3.36
3.45
G
0.06 NEG
0.05 NEG
0.06 NEG
0.06 NEG
2
2
FTA-abs
Ig
1
2
1
2
ND
++++
++++
ND
NEG
ND
+
ND
+++
ND
ND
NEG
ND
++++
ND
NEG
FTA-abs
IgG
1
2
1
2
ND
+++
+
ND
NEG
ND
+
ND
+++
ND
ND
NEG
ND
+++
ND
NEG
IgG Blot
1
2
1
2
++
++
++
++
NEG
NEG
++
++
++
++
NEG
NEG
++
++
NEG
NEG
WHO/BS/07.2059
Page 29
Lab
1
Method
IgM
capture
EIA
(ratio)
2
IgM EIA
(U ml-1)
Assay
1
Set
1
1
HS
1.12
0.86 NEG
A
0.46 NEG
0.37 NEG
B
0.23 NEG
0.17 NEG
C
0.80 NEG
0.58 NEG
D
1.14
0.92 EQ
E
0.13 NEG
0.12 NEG
F
1.14
0.95 EQ
G
0.17 NEG
0.19 NEG
2
2
2
1.48
1.54
0.68 NEG
0.65 NEG
0.34 NEG
0.30 NEG
1.12
1.10 EQ
1.50
1.57
0.29 NEG
0.21 NEG
1.63
1.68
0.28 NEG
0.27 NEG
1
1
1
1
1
102
91
60
63
5 NEG
4 NEG
5 NEG
6 NEG
4 NEG
4 NEG
5 NEG
5 NEG
2 NEG
2 NEG
3 NEG
4 NEG
12 NEG
14 NEG
12 NEG
13 NEG
3 NEG
3 NEG
3 NEG
4 NEG
15 NEG
17 NEG
14 NEG
12 NEG
4 NEG
4 NEG
3 NEG
3 NEG
2
2
2
2
>150
>150
>150
>150
7 NEG
8 NEG
5 NEG
5 NEG
6 NEG
8 NEG
3 NEG
3 NEG
4 NEG
3 NEG
2 NEG
4 NEG
22 EQ
23 EQ
13 NEG
14 NEG
5 NEG
4 NEG
2 NEG
4 NEG
19 NEG
21 EQ
11 NEG
13 NEG
4 NEG
5 NEG
3 NEG
5 NEG
1
1
1
1
1
1:40
1:40
1:80
1:40
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
1:10
1:10
1:20
1:10
NEG
NEG
NEG
NEG
1:10
1:10
1:10
1:20
NEG
NEG
NEG
NEG
2
1
1
1
1
1: 80
1:40
1:40
1:40
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
1:10
1:10
1:10
1:20
NEG
NEG
NEG
NEG
1:20
1:10
1:20
1:10
NEG
NEG
NEG
NEG
2
1
2
FTA-abs
IgM
WHO/BS/07.2059
Page 30
Appendix 4: Results from individual IgM assays
Appendix 4: Results from individual IgM assays (continued)
Lab
3
Method
IgM EIA
Set
2
2
2
2
HS
1:40
1:40
1:80
1:40
A
NEG
NEG
NEG
NEG
B
NEG
NEG
NEG
NEG
C
NEG
NEG
NEG
NEG
D
1:10
1:10
1:10
1:10
E
NEG
NEG
NEG
NEG
F
1:10
1:10
1:20
1:10
G
NEG
NEG
NEG
NEG
2
2
2
2
2
1: 80
1:40
1:40
1:80
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
1:20
1:10
1:10
1:20
NEG
NEG
NEG
NEG
1:20
1:10
1:10
1:20
NEG
NEG
NEG
NEG
1
1
1
2
2
377 NEG
371 NEG
218 NEG
214 NEG
307 NEG
312 NEG
159 NEG
127 NEG
385 NEG
365 NEG
189 NEG
107 NEG
281 NEG
266 NEG
127 NEG
122 NEG
481 NEG
450 NEG
316 NEG
321 NEG
309 NEG
306 NEG
148 NEG
140 NEG
569 NEG
434 NEG
296 NEG
330 NEG
305 NEG
357 NEG
159 NEG
150 NEG
2
2
2
2
662 NEG
643 NEG
445 NEG
412 NEG
439 NEG
536 NEG
242 NEG
443 NEG
581 NEG
569 NEG
449 NEG
441 NEG
475 NEG
455 NEG
413 NEG
387 NEG
754 NEG
762 NEG
488 NEG
489 NEG
481 NEG
529 NEG
335 NEG
377 NEG
525 NEG
718 NEG
472 NEG
470 NEG
576 NEG
603 NEG
348 NEG
350 NEG
1
2
+
+
NEG
NEG
NEG
NEG
NEG
NEG
+
+
ND
ND
ND
ND
ND
ND
1
2
+
+
NEG
NEG
NEG
NEG
NEG
NEG
+
+
NEG
NEG
+
+
NEG
NEG
2
3
4
6
FTA-abs
IgM
IgM Blot
1
WHO/BS/07.2059
Page 31
Assay
1
WHO/BS/07.2059
Page 32
Appendix 4: Results from individual IgM assays (continued)
Lab
7
Method
FTA-abs
IgM
Assay
Set
1
2
HS
ND
++++
A
++
ND
B
NEG
ND
C
NEG
ND
D
+++
ND
E
ND
NEG
F
ND
++
G
ND
NEG
WHO/BS/07.2059
Page 33
Appendix 5: Instructions for Use for 05/122
1st Human Syphilitic Plasma IgG International Standard
05/122
Version 1.0, 12th July 2007
1.
INTRODUCTION
The 1st IS for human syphilitic plasma IgG, 05/122, has been prepared from a
pool of 3 donations from individuals with previously treated early syphilis.
This preparation contains material of human origin, which has been tested and
found negative for HBsAg, HCV antibody and HIV antibody.
The material was calibrated in an international collaborative study involving
eight laboratories.
WHO/BS document reference to be added following ECBS meeting
2. UNITAGE
A unitage of 300mIU per ampoule has been assigned to this preparation
relative to the reactivity of 05/132 in the Treponema pallidum passive
particle agglutination test (TPPA). Each vial contains the equivalent of 1 ml
of plasma.
Uncertainty: the assigned unitage does not carry an uncertainty associated
with its calibration. The uncertainty may therefore be considered to be the
variance of the vial content and was determined to be +/- 0.34 %.
3. CONTENTS
Each vial contains 1 ml of lyophilized human plasma in a 5 ml ampoule.
4. CAUTION
THIS PREPARATION IS NOT FOR ADMINISTRATION TO HUMANS.
The preparation contains material of human origin. As with all materials of
biological origin, this preparation should be regarded as potentially hazardous to
health. It should be used and discarded according to your own laboratory's safety
procedures. Such safety procedures probably will include the wearing of
protective gloves and avoiding the generation of aerosols. Care should be
exercised in opening vials to avoid cuts.
This preparation contains material of human origin, which has been tested and
found negative for HBsAg, HCV antibody and HIV antibody.
WHO/BS/07.2059
Page 34
5. DIRECTIONS FOR OPENING THE AMPOULE
DIN ampoules have an ‘easy-open’ coloured stress point, where the narrow
ampoule stem joins the wider ampoule body.
Tap the ampoule gently to collect the material at the bottom (labelled) end.
Ensure that the disposable ampoule safety breaker provided is pushed down on
the stem of the ampoule and against the shoulder of the ampoule body. Hold the
body of the ampoule in one hand and the disposable ampoule breaker covering
the ampoule stem between the thumb and first finger of the other hand. Apply a
bending force to open the ampoule at the coloured stress point, primarily using
the hand holding the plastic collar.
Care should be taken to avoid cuts and projectile glass fragments that might
enter the eyes, for example, by the use of suitable gloves and an eye shield.
Take care that no material is lost from the ampoule and no glass falls into the
ampoule. Within the ampoule is dry nitrogen gas at slightly less than
atmospheric pressure. A new disposable ampoule breaker is provided with each
DIN ampoule.
6. USE OF MATERIAL
The vial should be stored at -20ºC on receipt. The vial should be opened as
directed in section 5 and then should be reconstituted in 1 ml milli-q water. Add
water and leave for 20-30 mins at RT to rehydrate. An appropriate dilution of
the standard can be used in agglutination assays or immunoassays for the
laboratory diagnosis of syphilis.
7. STABILITY
It is the policy of WHO not to assign an expiry date to their international
reference materials. They remain valid with the assigned potency and status
until withdrawn or amended.
Reference materials are held at NIBSC within assured, temperature-controlled
storage facilities. Reference Materials should be stored on receipt as indicated
on the label. Once reconstituted, diluted or aliquoted, users should determine the
stability of the material according to their own method of preparation, storage
and use.
NIBSC follows the policy of WHO with respect to its reference materials.
Users who have data supporting any deterioration in the characteristics of any
reference preparation are encouraged to contact NIBSC.
8. CITATION
WHO/BS/07.2059
Page 35
In any circumstance where the recipient publishes a reference to NIBSC
materials, it is important that the title of the preparation and any NIBSC code
number, and the name and address of NIBSC are cited correctly.
9. LIABILITY AND LOSS
Information provided by the Institute is given after the exercise of all reasonable
care and skill in its compilation, preparation and issue, but it is provided without
liability to the Recipient in its application and use.
It is the responsibility of the Recipient to determine the appropriateness of the
materials supplied by the Institute to the Recipient (“the Goods”) for the
proposed application and ensure that it has the necessary technical skills to
determine that they are appropriate. Results obtained from the Goods are likely
to be dependant on conditions of use by the Recipient and the variability of
materials beyond the control of the Institute.
All warranties are excluded to the fullest extent permitted by law, including
without limitation that the Goods are free from infectious agents or that the
supply of Goods will not infringe any rights of any third party.
The Institute shall not be liable to the Recipient for any economic loss whether
direct or indirect, which arise in connection with this agreement.
The total liability of the Institute in connection with this agreement, whether for
negligence or breach of agreement or otherwise, shall in no event exceed 120%
of any price paid or payable by the Recipient for the supply of the Goods.
WHO/BS/07.2059
Page 36
MATERIAL SAFETY SHEET
Physical properties (at room temperature)
Physical appearance
Freeze dried powder
Fire hazard
None
Chemical properties
Stable
Hygroscopic
Flammable
Other (specify)
Handling:
Yes
Corrosive:
No
Oxidising:
No
Irritant:
Contains material of human origin
See caution, section 1
No
No
No
Toxicological properties
Effects of inhalation:
Effects of ingestion:
Effects of skin absorption:
Not established, avoid inhalation
Not established, avoid ingestion
Not established, avoid contact with skin
Suggested First Aid
Inhalation
Ingestion
Contact with eyes
Contact with skin
Seek medical advice
Seek medical advice
Wash with copious amounts of water. Seek medical advice.
Wash thoroughly with water.
Action on Spillage and Method of Disposal
Spillage of ampoule contents should be taken up with absorbent material wetted with a
virucidal agent. Rinse area with a virucidal agent followed by water.
Absorbent materials used to treat spillage should be treated as biologically hazardous
waste.
WHO/BS/07.2059
Page 37
Appendix 6: Instructions for Use for 05/132
1st Human Syphilitic Plasma IgG and IgM International Standard
05/132
Version 1.0, 12th July 2007
1. INTRODUCTION
The 1st IS for human syphilitic plasma IgG and IgM, 05/132, has been
prepared from a pool of 25 donations from individuals with active syphilis.
This preparation contains material of human origin, which has been tested and
found negative for HBsAg, HCV antibody and HIV antibody.
The material was calibrated in an international collaborative study involving
eight laboratories.
WHO/BS document reference to be added following ECBS meeting
2. UNITAGE
A unitage of 3 IU per ampoule has been assigned to this preparation relative
to the reactivity of HS in the cardio-lipin based tests; the venereal disease
research laboratory test (VDRL) and the rapid plasma reagin test (RPR).
Each vial contains the equivalent of 1 ml of plasma.
Uncertainty: the assigned unitage does not carry an uncertainty associated
with its calibration. The uncertainty may therefore be considered to be the
variance of the vial content and was determined to be +/- 0.17 %.
3. CONTENTS
Each vial contains 1 ml of lyophilized human plasma in a 5 ml ampoule.
4. CAUTION
THIS PREPARATION IS NOT FOR ADMINISTRATION TO HUMANS.
The preparation contains material of human origin. As with all materials of
biological origin, this preparation should be regarded as potentially hazardous to
health. It should be used and discarded according to your own laboratory's safety
procedures. Such safety procedures probably will include the wearing of
protective gloves and avoiding the generation of aerosols. Care should be
exercised in opening vials to avoid cuts.
This preparation contains material of human origin, which has been tested and
found negative for HBsAg, HCV antibody and HIV antibody.
WHO/BS/07.2059
Page 38
5. DIRECTIONS FOR OPENING THE AMPOULE
DIN ampoules have an ‘easy-open’ coloured stress point, where the narrow
ampoule stem joins the wider ampoule body.
Tap the ampoule gently to collect the material at the bottom (labelled) end.
Ensure that the disposable ampoule safety breaker provided is pushed down on
the stem of the ampoule and against the shoulder of the ampoule body. Hold the
body of the ampoule in one hand and the disposable ampoule breaker covering
the ampoule stem between the thumb and first finger of the other hand. Apply a
bending force to open the ampoule at the coloured stress point, primarily using
the hand holding the plastic collar.
Care should be taken to avoid cuts and projectile glass fragments that might
enter the eyes, for example, by the use of suitable gloves and an eye shield.
Take care that no material is lost from the ampoule and no glass falls into the
ampoule. Within the ampoule is dry nitrogen gas at slightly less than
atmospheric pressure. A new disposable ampoule breaker is provided with each
DIN ampoule.
6. USE OF MATERIAL
The vial should be stored at -20ºC on receipt. The vial should be opened as
directed in section 5 and then should be reconstituted in 1 ml milli-q water. Add
water and leave for 20-30 mins at RT to rehydrate. An appropriate dilution of
the standard can be used in agglutination assays or immunoassays for the
laboratory diagnosis of syphilis.
7. STABILITY
It is the policy of WHO not to assign an expiry date to their international
reference materials. They remain valid with the assigned potency and status
until withdrawn or amended.
Reference materials are held at NIBSC within assured, temperature-controlled
storage facilities. Reference Materials should be stored on receipt as indicated
on the label. Once reconstituted, diluted or aliquoted, users should determine the
stability of the material according to their own method of preparation, storage
and use.
NIBSC follows the policy of WHO with respect to its reference materials.
Users who have data supporting any deterioration in the characteristics of any
reference preparation are encouraged to contact NIBSC.
8. CITATION
WHO/BS/07.2059
Page 39
In any circumstance where the recipient publishes a reference to NIBSC
materials, it is important that the title of the preparation and any NIBSC code
number, and the name and address of NIBSC are cited correctly.
9. LIABILITY AND LOSS
Information provided by the Institute is given after the exercise of all reasonable
care and skill in its compilation, preparation and issue, but it is provided without
liability to the Recipient in its application and use.
It is the responsibility of the Recipient to determine the appropriateness of the
materials supplied by the Institute to the Recipient (“the Goods”) for the
proposed application and ensure that it has the necessary technical skills to
determine that they are appropriate. Results obtained from the Goods are likely
to be dependant on conditions of use by the Recipient and the variability of
materials beyond the control of the Institute.
All warranties are excluded to the fullest extent permitted by law, including
without limitation that the Goods are free from infectious agents or that the
supply of Goods will not infringe any rights of any third party.
The Institute shall not be liable to the Recipient for any economic loss whether
direct or indirect, which arise in connection with this agreement.
The total liability of the Institute in connection with this agreement, whether for
negligence or breach of agreement or otherwise, shall in no event exceed 120%
of any price paid or payable by the Recipient for the supply of the Goods.
WHO/BS/07.2059
Page 40
MATERIAL SAFETY SHEET
Physical properties (at room temperature)
Physical appearance
Freeze dried powder
Fire hazard
None
Chemical properties
Stable
Hygroscopic
Flammable
Other (specify)
Handling:
Yes
Corrosive:
No
Oxidising:
No
Irritant:
Contains material of human origin
See caution, section 1
No
No
No
Toxicological properties
Effects of inhalation:
Effects of ingestion:
Effects of skin absorption:
Not established, avoid inhalation
Not established, avoid ingestion
Not established, avoid contact with skin
Suggested First Aid
Inhalation
Ingestion
Contact with eyes
Contact with skin
Seek medical advice
Seek medical advice
Wash with copious amounts of water. Seek medical advice.
Wash thoroughly with water.
Action on Spillage and Method of Disposal
Spillage of ampoule contents should be taken up with absorbent material wetted with a
virucidal agent. Rinse area with a virucidal agent followed by water.
Absorbent materials used to treat spillage should be treated as biologically hazardous
waste.
===