EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 12 to 16 October 2015 · Geneva, 12 to 16 October 2015 ... recent infection (7,18). ... residual moisture level in 12 ampoules
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WHO/BS/2015.2266
ENGLISH ONLY
EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION
Geneva, 12 to 16 October 2015
A WHO collaborative study to evaluate candidate International
Standard 13/132 for anti-Toxoplasma Ig (Human)
as a replacement for TOXM
Sjoerd Rijpkema
1*, Jason Hockley
2, Peter Rigsby
2, Edward C Guy
3
and the Toxoplasma study group4
1Division of Bacteriology and
2Biostatistics Section, National Institute for Biological Standards and
Control, Potters Bar, Hertfordshire, EN6 3QG, 3National Reference Centre for Toxoplasmosis,
Singleton Hospital, Sgeti, Swansea SA2 8QA, United Kingdom; 4partcipants of the collaborative
Candidate International Standard (IS) 13/132 is a freeze dried preparation of 0.5 mL pooled human
plasma, taken from 6 donors, who experienced a recent Toxoplasma gondii infection. It was
assessed for its suitability as an IS for anti-Toxoplasma Ig, IgA, IgM and IgG in a collaborative
study (CS) with 16 laboratories from 12 countries. The potency of candidate IS 13/132 was
compared with TOXM (3rd
IS for anti-Toxoplasma Serum, Human) in agglutination assays for IgA,
IgM and IgG, IgM (n=3) and IgG (n=2); enzyme linked immunosorbent assays and enzyme linked
fluorescent assays for Ig, IgM (n=6), IgG (n=4) and avidity of IgG (n=3); immunofluorescence
assays for IgG and IgM; immunoblots for IgM and IgG and the Sabin-Feldman dye tests (n=6). For
continuity, IS 01/600 (1st IS for anti-Toxoplasma IgG, Human) was also included in the CS.
The assays showed candidate IS 13/132 to be strongly positive for Ig, IgA, IgG and IgM. . Within-
and between-laboratory repeatability was generally very good. Candidate IS 13/132 contains high
levels of anti-Toxoplasma IgG and IgM thus allowing calibration in terms of IgG and IgM and its
potency falls between TOXM and 01/600. The unitage assigned to candidate IS 13/132 for Ig by
dye test relative to TOXM was calculated as 320 IU mL-1
with a GCV of 41.0% (n=5). The avidity
of IgG from candidate IS 13/132 was found to be low, similar to the avidity of IgG from TOXM
and considerably lower than the avidity of IgG from 01/600.
Candidate IS 13/132 was stable at the temperature used for storage (-20°C) and 3695 ampoules are
available for distribution by NIBSC. Results from accelerated thermal degradation studies at 15
months indicate that candidate IS 13/132 is stable for long-term use. Candidate IS 13/132 should be
a useful addition for standardisation of the serology for toxoplasmosis and support appropriate
clinical management of this disease. Candidate IS 13/132 is proposed as the 4th
IS for anti-
Toxoplasma Ig (Human) with a unitage of 160 IU per ampoule, to replace TOXM.
Introduction
Toxoplasmosis is caused by the parasite Toxoplasma gondii. Congenital transmission of T. gondii
remains a considerable burden on global health, with the highest incidence, 3.4/1000 births, reported
for South America (17). The main objective of screening programs is to prevent infection of the
foetus by the parasite during pregnancy, and serology is widely used to diagnose Toxoplasmosis
during pregnancy (9). In addition, toxoplasmosis is a major cause of mortality among transplant
patients (2). The provision of appropriate antibody standards enables diagnostic laboratories and
manufacturers of diagnostic tests to validate serologic assays to diagnose this infection. In 1994,
TOXM was validated by Hansen et al. and establish as the 3rd
International standard (IS) by the
Expert Committee on Biological Standards (ECBS) of the World Health Organization (6). TOXM
was used by manufacturers of in vitro diagnostic tests, national reference laboratories and hospital
laboratories. Since 2000, stocks of TOXM have been low and these have now been exhausted. In
2003, originally intended to replace TOXM, 01/600 was established by the ECBS as the 1st IS for
anti-Toxoplasma IgG of 20 international units (IU) mL-1
. The unitage is based on the results of the
Sabin-Feldman dye test relative to TOXM (14,15).
WHO/BS/2015.2266
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The dye test is a complement-mediated cell killing assay, utilising toxoplasma tachyzoites and does
not distinguish between immunoglobulin classes that bind complement (1). Although the assay is
now carried out by fewer laboratories, the dye test is still considered a reference test and a
confirmatory assay to validate commercial assays (13). The unitage of 01/600 can be traced back to
the 2nd
IS TOXS (6,14,15). Therefore the dye test remains an important assay for the standardisation
of anti-toxoplasma Ig levels in individuals suspected of toxoplasmosis. With its low level of IgG, the
reactivity of 01/600 falls within the linear range of most commercially available immunoassays,
which are now widely used, and enable diagnostic laboratories to distinguish between historic,
background and diagnostic levels of IgG. These assays are sufficiently sensitive to quantify and
monitor the IgG response associated with acute toxoplasmosis and this information is important to
support appropriate clinical management.
The ECBS did not consider IS 01/600 a suitable replacement for TOXM because of the low levels of
specific IgG and absence of specific IgM. The committee decided that a replacement for TOXM
should contain high levels of IgM and IgG (15).
Recently, NIBSC acquired plasma samples from acute cases of Toxoplasmosis, which all contained
high levels of specific IgM and IgG, the latter with low to borderline avidity. Specific IgG of high
avidity is seen as a marker of latent toxoplasmosis, whereas IgG of low avidity can be indicative of a
recent infection (7,18).
The plasma donations were pooled, filled and freeze dried and the final preparation was labelled
NIBSC 13/132. The levels of specific IgM and Ig in candidate IS 13/132 were analysed before and
after freeze drying by dye test and IgM capture enzyme linked immunosorbent assays (ELISA)
respectively (see Table 1). The results indicate that freeze drying did not affect levels of IgM and Ig.
The avidity of IgG in the native sample was found to be borderline (results not shown).
To establish if candidate IS 13/132 is fit for purpose a collaborative study (CS) was designed. The
primary aim is to assign a unitage to candidate IS 13/132 based on the potency relative to TOXM in
the dye test. In support of this aim, the CS will:
assess the suitability of the candidate IS 13/132 as an IS for human anti-Toxoplasma Ig.
compare the reactivity of the candidate IS 13/132 relative to TOXM in the dye test.
compare the reactivity of the candidate IS 13/132 relative to TOXM and 01/600 in
immunoassays for IgM and IgG, including avidity assays.
assess the reactivity of the candidate IS 13/132 in agglutination assays, immunoassays and in
other titration assays currently in use.
Materials and Methods
Participating laboratories and assay codification
Sixteen laboratories from 12 countries, including national reference laboratories, took part in the CS.
Details are given in Table A4 of Appendix 2. Throughout the study, participating laboratories are
identified by a randomly assigned code number to maintain confidentiality. Data were collected and
analysed at NIBSC. Each participant received two sets of samples comprising coded ampoules A to
WHO/BS/2015.2266
Page 4
F including 01/600 (A), duplicates of candidate IS 13/132 (C and E), and one ampoule of TOXM
(see Table 1).
Samples
Samples were distributed as lyophilized preparations by courier at room temperature. Samples A to
F and TOXM were reconstituted as described in the ‘instructions for use’. Samples that were
exposed to an elevated temperature range for the analysis of the stability of candidate IS 13/132
were distributed on dry ice. A brief characterisation of the samples, CS codes, NIBSC codes and
their reactivity in the dye test and the IgM capture ELISA are given in Table 1.
Characterization of the candidate international standard 13/132
Plasma samples were donated with consent by 6 female individuals of 21-33 years of age and
obtained from Cerba Specimen Services (Saint-Ouen l'Aumône, France). At NIBSC, all samples
tested negative for antibodies to HIV1 and HIV2, Hepatitis C RNA and Hepatitis B surface antigen.
Samples were stored at -80 oC until further use. Prior to pooling, samples were defrosted and stored
at 2-8oC overnight. The next day, samples were pooled (volume appr. 3 L) during which clotting
occurred. Clots were removed by a filtration step using Whatman filter paper (1001-150). The
filtrate of the pool was stored at 2-8oC overnight, and dispensed in 0.5 ml aliquots into glass
ampoules coded 13/132 the following day. The mean fill weight for 123 ampoules was 0.5156 g
(CV of 0.16 %). On the same day, freeze-drying under vacuum was started and completed after four
days. Ampoules were back filled with pure N2 and the mean O2 content of 12 ampoules was 0.17 %
(CV of 53.71 %). This implies ampoules passed the test for integrity, because the presence of cracks
would be associated with an O2 level of 21% similar to that found in the atmosphere. The mean
residual moisture level in 12 ampoules was 0.6608 % (CV of 18.89 %). One hundred and sixty
ampoules were rejected during the production process, 50 ampoules were held for accelerated
degradation studies and 3695 ampoules were stored at -20oC. These are available for distribution by
NIBSC.
Native and freeze-dried samples of candidate IS 13/132 and samples 637 and 174 were tested in the
dye test and IgM capture ELISA to determine the effect of freeze drying on specific Ig and IgM
respectively. The results are given in Table 1. No significant differences in the mean values for
levels of specific IgM and Ig were found before and after freeze-drying. Differences in unitage,
observed by dye test following freeze drying, were found for candidate IS 13/132 (C, E) and
samples D and F. These changes fall within the four fold range and are therefore not considered
significant.
Test methods and procedures
An overview of the 24 assay formats used for the detection of anti-T. gondii antibodies, including
participant and laboratory codes, is given in Table 2. Titration methods were represented by seven
assay formats. Five assays were developed in-house: the dye test for Ig (n=6), the high sensitivity
direct agglutination assay (HSDA [n=2]) for IgG (3), in-house immunofluorescence assays (IFA) for
IgG and IgM, and the imunosorbent agglutination assay (ISAGA) for IgA and IgM ([n=2], 4). Two
commercially available agglutination assays were used: the Toxoreagent kit for IgG/IgM (Mast) and
the ISAGA for IgM (bioMérieux, [n=4] ).
WHO/BS/2015.2266
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Six ELISA and enzyme linked fluorescent assay (ELFA) formats were used to detect IgM; four
commercially available assays (Abbot [n=2], bioMérieux [n=10], Biorad, Diasorin) were used,
capture ELISAs [n=2] and one IgM immunoblot were developed in-house (8,11,12). One IgG
immunoblot (LDBio Diagnostics) and 7 ELISAs and ELFAs, including avidity assays, were used to
detect specific IgG: five commercially available assays (Abbot [n=3], bioMérieux [n=10], Diasorin)
and in-house ELISAs [n=3] were used. A competition ELFA (BioMérieux) was used to detect Ig.
All samples were tested in duplicate on two different days. The data sets containing raw data,
transformed data and operating procedures were submitted to NIBSC for analysis.
Data analysis
For the majority of laboratories and methods, reported results (endpoint titres, potencies in IU or EU
etc.) were converted directly into relative potencies by dividing by the result obtained for the
appropriate standard. For IgM data, relative results (given as index, signal/cut off ratio etc) are
shown from ELFAs and ELISAs, but these cannot be directly interpreted as a relative potency.
ELISA data from lab 3 were analysed by parallel line bioassay comparing assay response to log
concentration in a four-parameter logistic model using version 5.0 of EDQM’s CombiStats software
(5). The final estimate in each assay for candidate IS 13/132 was taken as the geometric mean (GM)
of the two coded duplicates (C and E).
All mean estimates shown in this report are unweighted GM estimates. Variability between
laboratories has been expressed using geometric coefficients of variation (GCV = {10s-1}×100%
where s is the standard deviation of the log10-transformed estimates.
Results and Discussion
Titration assays Titration assays were carried out by 11 laboratories and all participants reported results for TOXM
and samples A to F. All participants correctly identified sample B. The potencies of the coded
positive samples relative to ISs TOXM and 01/600, and candidate IS 13/132 are summarised in
Table 3. Participant 14 did not identify TOXM and 01/600 as positive by dye test and the data of lab
code 14.1 were not included in calculations to determine the relative potency of candidate IS 13/132.
Individual test results of samples are given in Tables A1 to A3 (Appendix 1). Data sets which did
not contain numerical values for TOXM or 01/600, or which qualified samples A to F as positive or
negative only were not included in the calculation of the relative potency of candidate IS 13/132
presented in Table 3. Analysis of the results for duplicates C and E, representing candidate IS
13/132, in the dye test and IFA showed that for most lab codes, with one exception, the potency of C
relative to E fell within a two-fold difference relative to 1 (0.5 to 2.0), an indication of adequate
diagnostic precision among participating laboratories. Commercial and in house agglutination assays
performed better than the dye test in this respect (Fig. 1).
Hansen et al. assigned a unitage of 1000 IU mL
-1 of TOXM for Ig (6). The potency of anti-
Toxoplasma Ig in sample A (representing 01/600) relative to TOXM is reported as 0.02, which is
equal to a unitage of 20 IU mL-1
. This value is identical to the unitage assigned to 01/600 previously
(14,15,19). Candidate IS 13/132 (represented by samples C and E) had a GM potency of 0.32 with a
WHO/BS/2015.2266
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GCV of 41.0% in the dye test relative to TOXM and a potency of 15.4 with a GCV of 79.3%
relative to 01/600 (Table 3). The calculated unitage of candidate IS 13/132 for Ig is therefore 320 IU
mL-1
and 308 IU mL-1
relative to TOXM and 01/600, respectively.
The dye test results given in Table 3, show that the potency value of sample D (relative to candidate
IS 13/132 and other ISs) was nearly twice that of sample F. If the relative potencies derived from
Table 3 (1.43 for D and 0.77 for F) are used to calculate the actual unitages for D and F, based on
the unitage of candidate IS 13/132 given in Table 1, then the calculated unitages are similar to those
presented in Table 1. The unitage for sample D is 1000 and 802 in Table 1 and Table 3 respectively;
the unitage for sample F is 500 and 432 in Table 1 and Table 3 respectively.
The relative potency of candidate IS 13/132 compared to TOXM varied in other agglutination
assays. Compared to the dye test, the Toxoreagent kit (Mast) gave the closest results for candidate
IS 13/132 and for samples D and F. This assay detects both IgG and IgM, whereas IFA and ISAGA
specifically detect either IgG or IgM. Thus the differences in unitage for the latter two assays reflect
differences in the antibody class detected and in assay procedures.
Enzyme immunoassays and enzyme linked fluorescent assays All participants who carried out ELISAs and ELFAs to detect IgM or IgG reported results for
TOXM and samples A to F. Sample B was reported as negative for IgM and IgG. Sample A
(01/600) was reported as negative for IgM, but in two commercial assays a very low value for IgM
relative to TOXM was obtained and a GM could be calculated (see Table 4). The relative results of
candidate IS 13/132 and samples D and F for IgM relative to IS TOXM, IS 01/600 and candidate IS
13/132 are summarised in Table 4 and Table A2 of Appendix 1. As mentioned above, due to low
values for IgM of IS 01/600, high values for IgM were obtained for candidate IS 13/132 and
samples D and F. These should be considered for information only. The potencies of candidate IS
13/132 and samples D and F for IgG relative to IS TOXM, IS 01/600 and candidate IS 13/132 are
summarised in and Table 5 and Table A3 of Appendix 1. The results of qualitative assays and quantitative assays, which failed to assign a numerical value to
TOXM (irrespective of whether numerical values were given for other samples) were not used to
assign a unitage and are excluded from Tables 4 and 5. These results are presented in Table 7 or in
Tables A2 and A3 of Appendix1 (shaded cells). The ratio of the results of coded duplicate samples C and E in various ELFAs, ELISAs and
agglutination assays are shown in Figure 1. These showed that commercial ELFAs and ELISAs
have a high level of precision and reproducibility compared to in-house ELISAs. All assay results
fell within a two-fold difference relative to 1 (0.5 to 2.0), an indication of good diagnostic precision
among participating laboratories.
Based on 10 data sets generated by the bioMérieux VIDAS Toxo IgM ELFA, candidate IS 13/132
had a GM relative result of 0.75 with a GCV of 4.5% relative to TOXM (Table 4). The relative
results for samples D and F for IgM were close to that of candidate IS 13/132. Two data sets from
the Abbott ARC Toxo IgM ELISA and in-house IgM capture ELISAs gave a lower GM values for
candidate IS 13/132 of 0.49 and 0.41 respectively. The relative results for samples D and F for IgM
WHO/BS/2015.2266
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were also close to candidate IS 13/132 in this assay. The GCVs of the results of samples D and F in
the bioMérieux VIDAS Toxo IgM ELFA were lower when candidate IS13/132 was used as
reference compared to TOXM.
Based on 9 data sets from the bioMérieux VIDAS Toxo IgG II ELFA, candidate IS 13/132 had a
GM potency value of 0.55 with a GCV of 28.5% relative to TOXM and a GM potency value of
1.95 with a GCV of 44.4% relative to IS 01/600 (Table 5). The relative potency of sample D for
IgG was close to that of candidate IS 13/132. The Diasorin Toxo IgG ELISA and an in-house IgG
ELISA gave comparable GM potency value relative to TOXM for candidate IS 13/132 of 0.29 and
0.40 respectively. The relative potencies of sample F for IgG was considerably higher than those for
candidate IS 13/132 in these assays (Table 5). The GCVs of the results of samples D and F in the
bioMérieux VIDAS Toxo IgG II ELFA were lower when candidate IS13/132 was used as reference
compared to TOXM.
Hansen et al. estimated that the unitage of TOXM for IgG to be 1000 IU mL-1
, therefore the
calculated unitage of candidate IS 13/132 for IgG varies from 230-730 IU mL-1
(6). However if the
potency of candidate IS 13/132 for IgG is estimated relative to IS 01/600 than the GM unitage is
considerably lower at 32-39 IU mL-1
(Table 5). This difference is likely to be caused by the presence
of IgM and IgA in TOXM and in candidate IS 13/132. Both Ig classes will compete with IgG for
binding to exposed epitopes in ELISA but not in the dye test, whereas in IS 01/600 these Ig classes
are absent thus allowing a relative high proportion of specific IgG to bind in ELISAs (15).
Results of IgG avidity by ELISA and ELFA are presented in Table 6. The avidity of IgG in
candidate IS 13/132 was assessed by three assays (lab codes 11.3, 15.2 and 16.2). The avidity of IgG
in samples C to Fwas found to be low in 2 out of 3 assays and similar to the avidity of IgG from
TOXM. Indeed, Hansen et al. postulated that IgG from TOXM was of low avidity. By contrast the
avidity of IgG in IS 01/600 (A) is considerably higher, pointing to a historic infection.
Qualitative assays were used to detect total Ig, IgA, IgG or IgM, and these include capture ELISAs,
competition ELFA, immunoblot assays and ISAGAs. The results are presented in Table 7 and
Figure 2. Results of qualitative assays for Ig, IgG and IgM are in agreement with the outcome of
quantitative ELISAs and agglutination assays. For example, the immunoblot for IgG (lab code 11.7)
confirmed the presence of IgG in TOXM, samples A and C to F by ELISA, HDSA and IFA. The
immunoblot for IgM (lab code 4.5) confirmed the presence of IgM in TOXM and samples C to F.
Specific IgM bound to a 6 kilo Dalton (kD) antigen of T. gondii (see Fig 2). Previous work by
Sharma et al. demonstrated that IgM but not IgG from patients with toxoplasmosis reacts with the 6
kD antigen. Hence this reactivity is deemed a diagnostic marker of acute infection (16). Herbrink et
al. showed that the IgM immunoblot can be used to confirm results of IgM capture ELISAs (8). Our
data extend this to IgM detected by IFA and ISAGA.
Stability studies Samples of candidate IS 13/132 were stored for 481 days (appr. 15.3 months) at -20
oC and at
elevated temperatures +4oC, +20
oC, +37
oC and +45
oC. Two samples exposed to each temperature
were tested in duplicate in the dye test (lab code 6.1) and in-house IgM ELISA (lab code 6.2). The
potency of the samples, subjected to accelerated thermal degradation, was calculated relative to the
samples stored at -20oC and is given in Table 8.
WHO/BS/2015.2266
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These results were used to fit Arrhenius equations relating the degradation rate to absolute
temperature assuming first-order decay and hence predict the degradation rates when stored at -20°C
(10). Data from the dye test showed predicted losses of potency of 0.004%, 0.10%, 0.65% and
3.77% per month and 0.05%, 1.16%, 7.27% and 36.0% per year at storage temperatures of -20oC,
+4oC, +20
oC and +37
oC respectively. Data from the in-house IgM ELISA are given in Enzyme
Immunossay Units (EIU) and are shown for information only (12). Results given in EIU are relative
to an internal standard and these cannot be directly interpreted as relative potency.
Commutability studies The results of individual donor sera (D and F) in this study show a close correlation with candidate
IS 13/132 in titration assays, IgM and IgG ELISAs and ELFAs (see Tables 3, 4 and 5). The
commutability of candidate IS 13/132 remains to be determined; we have planned to use the
candidate alongside patient sera in routine assays carried out by participant 6.
Recommendations
Candidate IS 13/132 contains of 0.5 mL freeze dried pooled plasma per ampoule. The unitage
assigned to candidate IS 13/132 for Ig by dye test relative to TOXM was calculated as 320 IU mL-1
or 160 IU per ampoule with a GCV of 41.0%. The avidity of IgG from candidate IS 13/132 is low
and comparable to the avidity of IgG from TOXM.
Candidate IS 13/132 contains high levels of anti-Toxoplasma IgG and IgM thus allowing calibration
in terms of IgG and IgM and its potency falls between TOXM and 01/600. Therefore candidate IS
13/132 meets the requirements for a replacement of TOXM as set out by the ECBS in 2003 (14).
Candidate IS 13/132 will be a useful addition for the standardisation of Toxoplasma serology and
support appropriate clinical management of this disease. We propose candidate IS 13/132 as the 4th
IS for anti-Toxoplasma Ig (Human) to replace TOXM.
Replies from participants
Replies were received from 9 out of 16 participants, all of whom approved the report. Queries are
given in italics, and where appropriate the précis of the reply is given.
Participants 2, 3, 4, 7, 9, 10, 13, 14 and 15 approved the report.
Participant 2 approved the report and asked to:
1) introduce the term ELFA instead of ELISA for all VIDAS assays
2) change the name of the test represented by lab codes 1.1, 2.2, 5.3, 7.4, 8.1, 10.2, 12.3, 13.2
and 16.1in Table A3 to ‘bioMérieux VIDAS Toxo IgG II’
The term ELFA was introduced in Table 2 and throughout the report. Because of limited space in
Table A3, assays were renamed ‘VIDAS Toxo IgG II’ instead of ‘bioMérieux VIDAS Toxo IgG II’.
Participant 3 approved the report and asked to amend the affiliation and correct three typos.
WHO/BS/2015.2266
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Participant 4 approved the report and asked to:
1) change the name of the test represented by lab code 4.1 to bioMérieux ISAGA and change
the tables 2 and 7 accordingly
2) add data and lab code of the in-house IgG ELISA and change tables 2 and 5 accordingly
3) explain abbreviations for GM and GCV in tables
The report was amended as indicated.
Participant 7 approved the report but noted that the presentation of the results in tables and text is
complex and sometimes difficult to follow, and asked to:
1) amend affiliation and change ‘BioMérieux’ to ‘bioMérieux’
2) change Pg4 ln 1-2 to emphasise that high avidity excludes recent infection, but that more
studies are needed to confirm that low avidity is an indicator of recent infection
3) clarify authorship rule.
The name of the manufacturer and the affiliation of the participant were amended. On Pg 4 the
sentence was modified to ‘IgG of high avidity is seen as a marker of latent infection, whereas IgG of
low avidity can be indicative of a recent infection.’
Participant 9 approved the report and asked to:
1) change the test format of lab code 9.2 and 9.3 from ELISA to ISAGA to better represent these
assays
2) explain why only one set of ISAGA results was included to calculate the relative potency of
candidate IS 13/132
3) queried the calculation of HSDA results in Table 3 by transformation of assay data as
"geometric mean"
The report was amended and ISAGA instead of ELISA was used for lab codes 9.2 and 9.3. All but
one set of ISAGA results were given as indices and hence results of these could not be used to
determine relative potencies.
We investigated the transformation of the HSDA data in Table 3 (lab code 9.1). The participant was
informed that all data were calculated as ‘geometric mean’ and only this outcome was used to
determine the relative potencies. The participant offered an alternative calculation to determine the
potency of samples relative to TOXM as a ratio log transformed data. We explained that we adhered
to our formula for data transformation since this method had been used previously to calculate the
potency of 01/600 relative to TOXM (14,15).
Acknowledgements
We gratefully acknowledge the staff of the participating laboratories (see Table A4, Appendix 2) for
their important contributions, time, expertise and effort, which were indispensable for the validation
WHO/BS/2015.2266
Page 10
of candidate IS 13/132. We are grateful to Dr G Carrard and Mr C Bena of Cerba Specimen Services
for organising the transfer and documentation of the source material for candidate IS 13/132. We
would also like to thank Mr M Harris and Mr G Divall and their colleagues at the Centre for
Biological Reference Materials for processing of candidate IS 13/132, coding and packaging of
samples and organising the distribution of sample packs for the CS.
References
1) Beverley JK, Beattie CP. Standardization of the dye test for toxoplasmosis. J Clin Pathol
1952;5:350-353
2) Derouin F, Pelloux H. Prevention of toxoplasmosis in transplant patients. Clin Microbiol
Infect. 2008;14:1089-1101.
3) Desmonts G, Remington JS. Direct agglutination test for diagnosis of Toxoplasma
infection: method for increasing sensitivity and specificity. J Clin Microbiol 1980;11:562-
568.
4) Desmonts G, Naot Y, Remington JS. Immunoglobulin M immunosorbent agglutination
assay for diagnosis of infectious diseases: diagnosis of acute congenital and acquired
Department of Parasitology, Ege University Medical Faculty, Bornova-
Izmir
Turkey
Professor E. C. Guy National Reference Centre for Toxoplasmosis, Singleton Hospital,
Sgeti, Swansea
United
Kingdom
Dr J. G. Montoya, R.
Ramirez and C. Press
Toxoplasma Serology Laboratory, Palo Alto Medical Foundation,
Ames Building, 795 El Camino Real, Palo Alto, California
United States
Professor M. Golightly Immunology Laboratory, Stony Brook University, Stony Brook, New
York
United States
WHO/BS/2015.2266
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Appendix 3
WHO International Standard 4th IS for anti-Toxoplasma Ig (Human)
NIBSC code: 13/132 Instructions for use
(Version 1.00, Dated dd/mm/yyyy)
N/A
1. INTENDED USE International standard (IS) 13/132 is suitable for standardisation of the Sabin-Feldman dye test. A collaborative study compared the potency of IS 13/132 for Ig in the dye test relative to TOXM (3rd IS for anti-Toxoplasma Serum, Human) and 01/600 (1st IS for anti-Toxoplasma IgG, Human). IS 13/132 is suitable for use in ELFAs and ELISAs for Ig, IgA, IgM or IgG, IgG avidity assays, IFAs, agglutination assays and immunoblots for IgG and IgM [1]. IS 13/132 was strongly positive for Ig, IgA, IgG and IgM in these assays [1]. The potency of IS 13/132 falls between TOXM and 01/600 and the avidity of IgG is low [1,2]. More information on the reactivity of the standard in various immunoassays is given in [1]. 2. CAUTION This preparation is not for administration to humans. Human source material 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 should include the wearing of protective gloves and avoiding the generation of aerosols. Care should be exercised in opening ampoules or vials, to avoid cuts. 3. UNITAGE A unitage of 320 IU per mL (GCV of 41.0%, n=5) was assigned to IS 13/132 for Ig by dye test relative to TOXM. The avidity of IgG from IS 13/132 was low; similar to the avidity of IgG from TOXM and considerably lower than the avidity of IgG from 01/600 [1,2]. The unitages for IgM by ELFA and capture ELISAs relative to the estimated IgM content of 3000 IU per mL in TOXM ranged from 1230-2940 IU per mL depending on the assay of choice. For IgG by ELFAs and ELISAs the unitages relative to estimated IgG content of 1000 IU per mL in TOXM ranged from 230-730 IU per mL depending on the assay of choice [1,3]. 4. CONTENTS Country of origin of biological material: France. IS 13/132 is a freeze dried preparation of 0.5 mL pooled human plasma, taken from 6 donors, who experienced a recent Toxoplasma gondii infection. The material is free from antibodies to HIV1 and HIV2, Hepatitis C RNA and Hepatitis B surface antigen. 5. STORAGE On receipt, store ampoules at -20C. It is recommended that reconstituted material is held for no longer than
one week at 4⁰C. Unused contents should be frozen. Please note: because of the inherent stability of lyophilized material, NIBSC may ship these materials at ambient temperature. 6. DIRECTIONS FOR OPENING Din Ampoule 7. USE OF MATERIAL No attempt should be made to weigh out any portion of the freeze-dried material prior to reconstitution Samples should be reconstituted with 0.5 ml distilled water immediately before use.
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8. STABILITY Reference materials are held at NIBSC within assured, temperature-controlled storage facilities. Reference Materials should be stored on receipt as indicated on the label. NIBSC follows the policy of WHO with respect to its reference materials. 9. REFERENCES 1) Rijpkema S, Hockley J, Rigsby P, Guy EC. A WHO collaborative study to evaluate candidate International Standard 13/132 for anti-Toxoplasma Ig (Human) as a replacement for TOXM. WHO/BS/15.xxxx 2) Rigsby P, Rijpkema S, Guy EC, Francis J, Gaines Das R. Evaluation of a candidate international standard preparation for human anti-Toxoplasma IgG. J Clin. Microbiol. 2004; 42: 5133-5138 3) Hansen GA, Lyng J, Petersen E. Calibration of a replacement preparation for the second international standard for anti-Toxoplasma serum, Human. 1994; WHO/BS/94.1761 10. ACKNOWLEDGEMENTS We are grateful to Dr G Carrard and Mr C Bena of Cerba Specimen Services (France) for organising the transfer and documentation of the source material for IS 13/132. 11. FURTHER INFORMATION Further information can be obtained as follows; This material: [email protected] WHO Biological Standards: http://www.who.int/biologicals/en/ JCTLM Higher order reference materials: http://www.bipm.org/en/committees/jc/jctlm/ Derivation of International Units: http://www.nibsc.org/products/biological_reference_materials/frequently_asked_questions/how_are_international_units.aspx Ordering standards from NIBSC: http://www.nibsc.org/products/ordering_information/frequently_asked_questions.aspx NIBSC Terms & Conditions: http://www.nibsc.org/terms_and_conditions.aspx 12. CUSTOMER FEEDBACK Customers are encouraged to provide feedback on the suitability or use of the material provided or other aspects of our service. Please send any comments to [email protected] 13. CITATION In all publications, including data sheets, in which this material is referenced, it is important that the preparation's title, its status, the NIBSC code number, and the name and address of NIBSC are cited and cited correctly.
Effects of inhalation: Not established, avoid inhalation
Effects of ingestion: Not established, avoid ingestion
Effects of skin absorption:
Not established, avoid contact with skin
Suggested First Aid
Inhalation: Seek medical advice
Ingestion: Seek medical advice
Contact with eyes:
Wash with copious amounts of water. Seek medical advice
Contact with skin:
Wash thoroughly with water.
Action on Spillage and Method of Disposal
Spillage of ampoule contents should be taken up with absorbent material wetted with an appropriate disinfectant. Rinse area with an appropriate disinfectant followed by water. Absorbent materials used to treat spillage should be treated as biological waste.
15. LIABILITY AND LOSS
In the event that this document is translated into another language, the English language version
shall prevail in the event of any inconsistencies between the documents.
Unless expressly stated otherwise by NIBSC, NIBSC’s Standard Terms and Conditions for the Supply
of Materials (available at http://www.nibsc.org/About_Us/Terms_and_Conditions.aspx or upon request by the Recipient) (“Conditions”) apply to the exclusion of all other terms and are hereby incorporated into this document by reference. The Recipient's attention is drawn in particular to the provisions of clause 11 of the Conditions. 16. INFORMATION FOR CUSTOMS USE ONLY
Country of origin for customs purposes*: United Kingdom * Defined as the country where the goods have been produced and/or sufficiently processed to be classed as originating from the country of supply, for example a change of state such as freeze-drying.
Net weight: 0.04 g
Toxicity Statement: Non-toxic
Veterinary certificate or other statement if applicable.