EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 12 to 16 October 2015 · Geneva, 12 to 16...

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

study (seeTable A4).

*(corresponding author, E-mail address: [email protected])

NOTE:

This document has been prepared for the purpose of inviting comments and suggestions on the

proposals contained therein, which will then be considered by the Expert Committee on Biological

Standardization (ECBS). Comments MUST be received by 14 September 2015 and should be

addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention: Technologies,

Standards and Norms (TSN). Comments may also be submitted electronically to the Responsible

Officer: Dr M Nübling at email: [email protected]

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WHO/BS/2015.2266

Summary

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

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

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

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

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

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

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


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.’


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

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

toxoplasma infections. J Clin Microbiol 1981;14:486-491.

5) EDQM – Council of Europe. CombiStats v5.0. www.combistats.eu.

6) 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

7) Hedman K, Lappalainen M, Seppaia I, Mäkelä O. Recent primary toxoplasma infection

indicated by a low avidity of specific IgG. J. Infect. Dis. 1989;159:736–740.

8) Herbrink P, van Loon AM, Rotmans JP, van Knapen F, van Dijk WC. Interlaboratory

evaluation of indirect enzyme-linked immunosorbent assay, antibody capture enzyme-linked

immunosorbent assay, and immunoblotting for detection of immunoglobulin M antibodies to

Toxoplasma gondii. J Clin Microbiol. 1987;25:100-105.

9) Jones JL, Lopez A, Wilson M, Schulkin J, Gibbs R. Congenital toxoplasmosis: a review.

Obstet Gynecol Surv 2001;56:296–305.

10) Kirkwood TBL. Predicting the stability of biological standards and products. Biometrics,

1977;33:736-742.

11) Naot Y, Barnett EV, Remington JS. Method for avoiding false-positive results occurring

in immunoglobulin m enzyme-linked immunosorbent assays due to presence of both

rheumatoid factor and antinuclear antibodies. J Clin Microbiol 1981;14:73-78.

12) Payne RA, Joynson DHM, Balfour AH, Harford JP, Fleck DG, Mythen M, Saunders

RJ. Public Health Laboratory Service enzyme-linked immunosorbent assay for detecting

Toxoplasma specific IgM antibody. J Clin Pathol 1987;40:276-281.

13) Reiter-Owona I, Petersen E, Joynson D, Aspock H, Darde ML, Disko R et al. The past

and present role of the Sabin-Feldman dye test in the serodiagnosis of toxoplasmosis. Bull

WHO 1999;77:929-35.

14) Rigsby P, Rijpkema S, Guy EC, Gaines Das R. International collaborative study:

evaluation of a candidate replacement international standard preparation for anti-toxoplasma

serum, human. 2003. WHO/BS/03.1971.

http://www.combistats.eu/

WHO/BS/2015.2266

15) 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

16) Sharma SD, Mullenax J, Araujo FG, Erlich HA, Remington JS. Western blot analysis of

the antigen of Toxoplasma gondii recognized by human IgM and IgG antibodies. J Immunol

1983;131:977-983.

17) Torgerson PR, Mastroiacovo P. The global burden of congenital toxoplasmosis: a

systematic review. Bull World Health Organ 2013;91:501-508.

18) Villard O, Breit L, Cimon B, Franck J, Fricker-Hidalgo H, Godineau N, Houze S, Paris

L, Pelloux H, Villena I, Candolfi E; French National Reference Center for

Toxoplasmosis Network. Comparison of four commercially available avidity tests for

Toxoplasma gondii-specific IgG antibodies. Clin Vaccine Immunol 2013;20:197-204.

19) WHO Expert Committee on Biological Standardization. WHO Technical Report Series,

54th

report 2003;927:17.

WHO/BS/2015.2266

Table 1: Characteristics of samples used in this study Study

code

NIBSC

code

Description of source material Results of native material Results of freeze dried material

Dye test1

(IU mL-1

)

IgM ELISA2

(EIU mL-1

)

Dye test

(IU mL-1

)

IgM ELISA

(EIU mL-1

)

TOXM TOXM 3rd

IS for anti-Toxoplasma Ig Human not done not done 10003

not done

A 01/600 1st IS for anti-Toxoplasma IgG Human not done not done 20

3 not done

B 01/576 Pool of seven normal human sera not done not done <23 not done

C and E 13/132 Candidate IS for anti-Toxoplasma IgM Human

from a pool of 6 plasma donations4

794 (500-1000) 101±12 561 (500-1000) 98±7

D 174 Anti-Toxoplasma plasma from one donor5 1000 105±1 1000 125±7

F 637 Anti-Toxoplasma plasma from one donor5 250 94±6 500 101±7

1: Lab code 6.1. Results given as geometric mean titre (range).

2: Lab code 6.2. Results given as geometric mean Enzyme Immunossay Unit ± SD: > 40: positive; >100: strongly positive (12)

3: Taken from Rigsby et al. 2004 (14)

4: Taken from 6 measurements over three days; differences in IU for native and freeze dried samples are not significant.

5: Taken from 2 measurements over two days.

WHO/BS/2015.2266

Table 2: Assays used in this study

Type of test Manufacturer and name of the test,

antibody specificity (n)

Participant

code

Lab code

Titration assays (8)

Agglutination

assay

Dye test

IFA3

In-house, HSDA1, IgG (2)

bioMérieux ISAGA2, IgM (4)

In-house, ISAGA, IgA (1)

In-house, ISAGA, IgM (2)

MAST Latex toxoreagent, IgG/IgM (1)

In-house, Ig (6)

In-house, IgG (1)

In-house, IgM (1)

5,9

4,5,7,11

9

2,9

4

1,2,3,6,10,14

7

7

5.1,9.1

4.1,5.2,7.1,11.1

9.2

2.1, 9.3

4.2

1.1,2.2,3.1,6.1,10.1,14.1

7.2

7.3

Enzyme immunoassays and enzyme linked fluorescent assays (15)

ELFA4

ELISA5

Immunoblot

bioMérieux VIDAS Toxo Competition (1)

bioMérieux VIDAS Toxo IgG II (9)

bioMérieux VIDAS Toxo IgG Avidity (1)

bioMérieux VIDAS Toxo IgM (10)

Abbott ARC Toxo IgG (2)

Abbott ARC Toxo IgG Avidity (1)

Abbott ARC Toxo IgM (2)

Bio Rad Platelia Toxo IgM (1)

Diasorin Liasion Toxo IgG (1)

Diasorin Liasion Toxo IgM II (1)

In-house, IgG (2)

In-house, IgG avidity (1)

In-house, IgM capture (2)

In-house, IgM (1)

LDBio Diagnostics IgG (1)

13

1,2,5,7,8,10,

12,13,16

16

1,2,5,7,8,10,

12,13,15,16

11,12

11

11,12

4

11

11

4,15

15

3,6

4

11

13.1

1.1,2.2,5.3,7.4,8.1,10.2,

12.3,13.2,16.1

16.2

1.2,2.3,5.4,7.5,8.2,10.3,

12.4,13.3, 15.3,16.3

11.2,12.1

11.3

11.4,12.2

4.3

11.5

11.6

4.4,15.1

15.2

3.2,6.2

4.5

11.7

1: High sensitivity direct agglutination assay (3)

2: Imunosorbent agglutination assay (4)

3: Immunofluorescence assay

4: Enzyme linked fluorescent assay

5: Enzyme linked immunosorbent assay

WHO/BS/2015.2266

Table 3: Summary of results from titration assays for samples A to F relative to TOXM, 01/600 and 13/132

Test ID Test sample A (01/600) C/E (13/132) D F

Reference TOXM TOXM 01/600 (A) TOXM 01/600 (A) 13/132 (C/E) TOXM 01/600 (A) 13/132 (C/E)

Dye test (Ig) Lab code 1.1 0.02 0.54 28.8 0.77 40.7 1.41 0.24 12.8 0.45

Lab code 2.2 0.03 0.28 8.8 0.50 15.6 1.78 0.25 7.8 0.89

Lab code 3.1 0.02 0.33 21.1 0.23 14.9 0.71 0.20 12.5 0.59

Lab code 6.1 0.02 0.32 19.7 0.63 39.4 2.00 0.25 15.6 0.79

Lab code 10.1 0.03 0.21 6.7 0.35 11.3 1.68 0.31 9.8 1.46

GM1 0.02 0.32 14.78 0.46 21.15 1.43 0.25 11.40 0.77

GCV2 42.7% 41.0% 85.9% 60.7% 81.4% 51.2% 17.3% 30.7% 56.1%

Latex MAST

(IgM, IgG)

Lab code 4.2 0.55 0.42 15.4 0.50 18.3 1.19 0.35 13.0 0.84

IFA (IgM) Lab code 7.3 Not done 0.50 Not done 0.42 Not done 0.84 0.42 Not done 0.84

ISAGA

(IgM)

Lab code 2.1 Not done 0.26 Not done 0.26 Not done 1.00 0.26 Not done 1.00

IFA (IgG) Lab code 7.2 0.01 0.42 67.3 0.50 80.0 1.19 0.25 40.0 0.59

HSDA (IgG) Lab code 5.1 0.06 0.50 8.0 0.50 8.0 1.00 0.50 8.0 1.00

Lab code 9.1 0.02 0.13 6.7 0.36 19.0 2.83 0.25 13.5 2.00

GM1 0.03 0.25 7.3 0.42 12.3 1.68 0.35 10.4 1.41

1GM: geometric mean

2GCV: geometric coefficients of variation

WHO/BS/2015.2266

Table 4: Summary of IgM results in ELISA and ELFA for samples A to F relative to TOXM, 01/600 and 13/132



bioMérieux

VIDAS Toxo

IgM

Lab code 1.2 0.01 0.76 53.0 0.72 50.0 0.94 0.85 59.3 1.12

Lab code 2.3 0.02 0.79 48.7 0.76 46.3 0.95 0.87 53.1 1.09

Lab code 5.4 0.02 0.77 45.0 0.75 43.6 0.97 0.99 21.5 1.01

Lab code 7.5 0.02 0.76 49.6 0.73 47.7 0.96 0.87 50.8 1.13

Lab code 8.2 0.02 0.80 53.9 0.81 52.9 0.98 0.95 63.0 1.17

Lab code 10.3 0.01 0.69 61.4 0.66 59.8 0.97 0.79 69.3 1.13

Lab code 12.4 0.01 0.76 51.7 0.75 51.0 0.99 0.84 57.4 1.11

Lab code 13.3 0.01 0.74 50.8 0.72 49.2 0.97 0.85 58.2 1.15

Lab code 15.3 0.01 0.74 57.1 0.73 55.1 0.97 0.85 64.6 1.13

Lab code 16.3 0.01 0.72 49.5 0.72 50.2 1.01 0.80 55.2 1.11

GM1 0.01 0.75 51.9 0.73 50.4 0.97 0.85 58.6 1.13

GCV2 12.3% 4.5% 9.1% 5.0% 9.3% 2.0% 5.1% 9.8% 2.0%

Abbott ARC

Toxo IgM

Lab code 11.4 0.01 0.53 79.2 0.45 68.0 0.86 0.67 100.3 1.27

Lab code 12.2 0.01 0.45 65.6 0.36 52.7 0.80 0.57 82.7 1.26

GM1 0.01 0.49 72.1 0.41 59.9 0.83 0.62 91.1 1.26

Biorad

Platelia Lab code 4.3 0.05 0.98 21.3 0.97 21.0 0.99 0.99 21.5 1.01

IgM capture

ELISA

Lab code 3.2 Negative 0.25 Negative 0.20 Negative 1.02 0.25 Negative 0.93

Lab code 6.2 Negative 0.67 Negative 0.70 Negative 1.04 0.64 Negative 0.95

GM1 - 0.41 - 0.37 - 1.03 0.40 - 0.94

1GM: geometric mean


WHO/BS/2015.2266

Table 5: Summary of IgG results in ELISA and ELFA for samples A to F relative to TOXM, 01/600 and 13/132



bioMérieux

VIDAS Toxo

IgG II

Lab code 1.1 0.20 0.36 1.82 0.39 1.98 1.09 0.46 2.37 1.30

Lab code 2.2 0.36 0.58 1.64 0.63 1.77 1.08 0.98 2.75 1.67

Lab code 5.3 0.20 0.41 2.01 0.49 2.38 1.18 0.77 3.77 1.87

Lab code 7.4 0.46 0.67 1.46 0.72 1.57 1.08 0.91 1.98 1.36

Lab code 8.1 NNV1 NNV

1 1.74 NNV1 2.29 1.32 NNV

1 3.29 1.36

Lab code 10.2 0.34 0.69 1.83 0.72 1.95 1.06 0.88 2.59 1.41

Lab code 12.3 0.40 0.67 1.68 0.68 1.70 1.01 0.94 2.34 1.40

Lab code 13.2 0.10 0.49 5.05 0.57 5.94 1.18 0.73 7.60 1.50

Lab code 16.1 0.36 0.61 1.72 0.58 1.61 0.94 0.97 2.72 1.58

GM2 0.27 0.55 1.95 0.59 2.14 1.10 0.81 3.01 1.49

GCV3 68.2% 28.5% 44.4% 24.1% 50.5% 10.3% 28.4% 48.5% 12.5%

Abbott ARC

Toxo IgG

Lab code 11.2 0.04 0.23 5.84 0.28 7.35 1.26 0.26 6.85 1.17

Lab code 12.1 NNV1 NNV

1 5.75 NNV

1 7.14 1.24 NNV

1 6.68 1.16

GM2 5.80 7.24 1.25 6.77 1.17

Diasorin

Liaison Toxo

IgG

Lab code 11.5 0.29 0.53 1.83 0.53 1.83 1.00 0.66 2.28 1.25

IgG ELISA Lab code 4.4 0.84 0.62 0.74 0.55 0.65 0.88 1.54 1.83 2.48

Lab code 15.1 0.42 0.75 1.79 0.73 1.74 0.97 0.88 2.10 1.18 1NNV: TOXM result fell outside assay range and no numerical value was given

2GM: geometric mean


WHO/BS/2015.2266

Figure 1: The ratio of the relative results of coded duplicate samples C and E representing candidate IS 13/132 is presented for various

assays. Most ratios fall within the twofold range (50-200%) relative to a C/E ratio of 1. Only participant 10 reported results for the dye test

that exceeded this range. In general, the highest precisions were achieved by commercial ELISAs and ELFAs.

WHO/BS/2015.2266

Table 6: Results of avidity IgG ELISAs for samples A to F and TOXM

Test ID Abbott ARC Toxo

IgG Avidity (%)a

Lab code 11.3

bioMérieux VIDAS Toxo

IgG avidity (OD)

b

Lab code 16.1

IgG avidity ELISA (%)c

Lab code 15.2 Sample

tested

TOXM 22.7 (Low) 0.079 (Low) 27 (Low)

A (01/600) 82.4 (High) 0.496 (High) 59 (High)

C/E (13/132) 34-38.4 (Low) 0.139-0.149 (Low) 42 (Borderline)

D 30.1 (Low) 0.119 (Low) 35 (Borderline)

F 51.9 (Gray zone) 0.218 (Intermediate) 47 (High) a: Avidity index: Low < 50.0 %; Gray zone 50.0 – 59.9 %; High ≥60.0 %

b: Avidity index: Low < 0.200; Intermediate ≥0.200 & <0.300; High ≥0.300

c: Avidity index: Low <30; Borderline 30-40; High > 40

Table 7: Results of qualitative assays for TOXM and samples A to F Lab

code

Method Antibody

detected

Test sample

A B C D E F TOXM

4.1 bioMérieux ISAGA IgM - - + + + + +

4.5 Immunoblot IgM - - + + + + +



9.2 ISAGA IgA - - + + + + +

9.3 ISAGA IgM - - + + + + +


11.7 LDBio Diagnostics Immunoblot IgG + - + + + + +

13.1 bioMérieux VIDAS Toxo Competition Ig + - + + + + +

WHO/BS/2015.2266

Figure 2: IgM immunoblot of duplicates of TOXM (Tx) and coded samples A to F, with T. gondii

strain RH as antigen (lab code 4.5). The presence of the 6 kD band denotes an IgM positive sample

(8). The numbers in subscript indicate the duplicates per sample pack.

WHO/BS/2015.2266

Table 8: Test results for samples of candidate IS 13/132 stored at elevated temperatures

for approximately

15.3 months

Lab code

And Test

Storage

temperature

Result1 Range Geometric

mean titre

Geometric mean titre

relative to -200C Day 1 Day 2 Day 3

6.1

Dye test

(IU mL-1

)

-200C 500 500 500 500 500 -

+40C 500 500 500 500 500 1.00

+200C 500 375 500 250-500 445 0.89

+370C 375 250 250 250-500 281 0.56

+450C 125 125 188 125-250 140 0.28

6.2

IgM

capture

ELISA

(EIU mL-1

)

-200C 91 98 94 91-101 94 -

+40C 96 94 96 92-101 95 1.01

+200C 85 85 88 81-90 86 0.92

+370C 51 52 60 50-62 55 0.58

+450C 5

2 18 22 4-23 12 0.13

1: Average result of two vials is given.

2: Reconstituted material stored at +45

0C was highly viscous on day 1 prohibiting accurate pipetting of volumes.

The viscosity decreased after a 24 hour incubation at +40C, allowing accurate pipetting of the sample on day 2 and

3.

WHO/BS/2015.2266

Appendices

Pg

Appendix 1:

Table A1: Results of the relative potency for Ig in samples A to F by Latex agglutination

assay and Sabin-Feldman dye test…..…………………………...…………………….22

Table A2: Results of the relative potency for IgM in samples A to F by agglutination assays,

ELISA and IFA……………………...…………………………………………………24

Table A3: Results of the relative potency for IgG in samples A to F by agglutination assays,

ELISA and IFA……………...…………………………………………………………29

Appendix 2:

Table A4: Participants of the collaborative study…………………………………………...…34

Appendix 3:

Instructions for Use and Material Safety Data Sheet for NIBSC 13/132………………………35

WHO/BS/2015.2266

Appendix 1

Table A1: Results of the relative potency for Ig in samples A to F by Latex

agglutination assay and Sabin- Feldman dye test1

Sample Lab

code

Method Unit Assay 1 Assay 2 Assay 3 Assay 4 GM GCV

Ref Test

A 13/132 1.1 Dye Test IU 22.85 23.03 45.25 28.77 48%

A 13/132 2.2 Dye Test IU 10.70 10.70

A 13/132 3.1 Dye Test Titer 22.63 19.60 21.06

A 13/132 4.2 Latex MAST Titer 1.00 1.00 0.47 0.71 0.76 43%

A 13/132 6.1 Dye Test IU 22.10 22.10 15.63 19.69 22%

A 13/132 10.1 Dye Test IU 9.88 4.61 6.75

A D 1.1 Dye Test IU 32.32 32.59 64.00 40.70 48%

A D 2.2 Dye Test IU 15.63 15.63

A D 3.1 Dye Test Titer 8.00 28.00 14.97

A D 4.2 Latex MAST Titer 1.00 1.00 0.67 1.00 0.90 22%

A D 6.1 Dye Test IU 62.50 31.25 31.25 39.37 49%

A D 10.1 Dye Test IU 16.13 7.98 11.34

A F 1.1 Dye Test IU 8.11 16.27 16.00 12.83 49%

A F 2.2 Dye Test IU 9.77 9.77

A F 3.1 Dye Test Titer 8.00 20.00 12.65

A F 4.2 Latex MAST Titer 1.00 1.00 0.33 0.50 0.64 72%

A F 6.1 Dye Test IU 15.63 15.63 15.63 15.63 0%

A F 10.1 Dye Test IU 12.10 7.98 9.82

13/132 D 1.1 Dye Test IU 1.41 1.42 1.41 1.41 0%

13/132 D 2.2 Dye Test IU 1.46 1.46

13/132 D 3.1 Dye Test Titer 0.35 1.43 0.71

13/132 D 4.2 Latex MAST Titer 1.00 1.00 1.41 1.41 1.19 22%

13/132 D 6.1 Dye Test IU 2.83 1.41 2.00 2.00 41%

13/132 D 10.1 Dye Test IU 1.63 1.73 1.68

13/132 F 1.1 Dye Test IU 0.35 0.71 0.35 0.45 49%

13/132 F 2.2 Dye Test IU 0.91 0.91

13/132 F 3.1 Dye Test Titer 0.35 1.02 0.60

13/132 F 4.2 Latex MAST Titer 1.00 1.00 0.71 0.71 0.84 22%

13/132 F 6.1 Dye Test IU 0.71 0.71 1.00 0.79 22%

13/132 F 10.1 Dye Test IU 1.22 1.73 1.46 1: results of samples C and E are represented by sample 13/132

WHO/BS/2015.2266

Table A1: Continued

Sample Lab

code

Method Unit Assay 1 Assay 2 Assay 3 Assay 4 GM GCV

Ref Test

TOXM A 1.1 Dye-test IU 0.02 0.02 0.02 0.02 17%

TOXM A 2.2 Dye Test IU 0.03 0.03

TOXM A 3.1 Dye Test Titer 0.02 0.02 0.02

TOXM A 4.2 Latex MAST Titer 0.50 0.50 0.75 0.50 0.55 22%

TOXM A 6.1 Dye Test IU 0.02 0.02 0.02 0.02 0%

TOXM A 10.1 Dye Test IU 0.02 0.05 0.03

TOXM 13/132 1.1 Dye Test IU 0.43 0.51 0.72 0.54 30%

TOXM 13/132 2.2 Dye Test IU 0.27 0.27

TOXM 13/132 3.1 Dye Test Titer 0.35 0.31 0.33

TOXM 13/132 4.2 Latex MAST Titer 0.50 0.50 0.35 0.35 0.42 22%

TOXM 13/132 6.1 Dye Test IU 0.35 0.35 0.25 0.32 22%

TOXM 13/132 10.1 Dye Test IU 0.20 0.22 0.21

TOXM D 1.1 Dye Test IU 0.61 0.72 1.02 0.77 30%

TOXM D 2.2 Dye Test IU 0.40 0.40

TOXM D 3.1 Dye Test Titer 0.13 0.44 0.23

TOXM D 4.2 Latex MAST Titer 0.50 0.50 0.50 0.50 0.50 0%

TOXM D 6.1 Dye Test IU 1.00 0.50 0.50 0.63 49%

TOXM D 10.1 Dye Test IU 0.33 0.38 0.35

TOXM F 1.1 Dye Test IU 0.15 0.36 0.26 0.24 53%

TOXM F 2.2 Dye Test IU 0.25 0.25

TOXM F 3.1 Dye Test Titer 0.13 0.31 0.20

TOXM F 4.2 Latex MAST Titer 0.50 0.50 0.25 0.25 0.35 49%

TOXM F 6.1 Dye Test IU 0.25 0.25 0.25 0.25 0%

TOXM F 10.1 Dye Test IU 0.25 0.38 0.31 1: results of samples C and E are represented by sample 13/132

WHO/BS/2015.2266

Table A2: Results of the relative potency for IgM in samples A to F by

agglutination assay, ELISA, ELFA and IFA1

Sample Lab

cod

e

Method Unit Assay

1

Assay

2

Assay

3

Assay

4

GM GCV

Ref Test

A 13/132 1.3 VIDAS Toxo IgM Index 51.75 52.50 50.13 58.10 53.04 7%

A 13/132 2.1 ISAGA Titer 64.00

64.00

A 13/132 2.3 VIDAS Toxo IgM Index 48.70

48.70

A 13/132 4.3 Platelia OD 24.31 21.78 18.98 20.50 21.30 11%

A 13/132 5.4 VIDAS Toxo IgM Index 43.44 46.52

44.95




61.39

A 13/132 11.4 ARC Toxo IgM Index 79.24

79.24

A 13/132 12.2 ARC Toxo IgM Index 65.59

65.59

A 13/132 12.4 VIDAS Toxo IgM Index 51.72

51.72


50.81


57.10


49.53

A D 1.3 VIDAS Toxo IgM Index 48.10 49.45 48.55 54.00 49.97 5%

A D 2.1 ISAGA Titer 64.00

64.00

A D 2.3 VIDAS Toxo IgM Index 46.27

46.27

A D 4.3 Platelia OD 23.88 21.08 18.99 20.51 21.04 10%

A D 5.4 VIDAS Toxo IgM Index 41.75 45.55

43.61




59.84

A D 11.4 ARC Toxo IgM Index 68.03

68.03

A D 11.6 Diasorin Toxo IgM Index >53.3 -

A D 12.2 ARC Toxo IgM Index 52.70

52.70

A D 12.4 VIDAS Toxo IgM Index 51.00

51.00


49.22


55.11


50.16

1: results of samples C and E are represented by sample 13/132

Results presented in shaded cells could not be transformed to a numerical value and were not used to calculate a

unitage.

WHO/BS/2015.2266

Table A2: Continued

Sample Lab

code

Method Unit Assay

1

Assay

2

Assay

3

Assay

4

GM GCV

Ref Test

A F 1.3 VIDAS Toxo IgM Index 58.10 59.55 57.00 62.50 59.25 4%

A F 2.1 ISAGA Titer 64.00

64.00

A F 2.3 VIDAS Toxo IgM Index 53.09

53.09

A F 4.3 Platelia OD 24.60 22.27 18.96 20.48 21.48 12%

A F 5.4 VIDAS Toxo IgM Index 48.54 53.18

50.81




69.35

A F 11.4 ARC Toxo IgM Index 100.27

100.27

A F 11.6 Diasorin Toxo IgM Index >30.3 -

A F 12.2 ARC Toxo IgM Index 82.73

82.73

A F 12.4 VIDAS Toxo IgM Index 57.40

57.40


58.25


64.61


55.17

13/132 D 1.3 VIDAS Toxo IgM Index 0.93 0.94 0.97 0.93 0.94 2%

13/132 D 2.1 ISAGA Titer 1.00

1.00

13/132 D 2.3 VIDAS Toxo IgM Index 0.95

0.95

13/132 D 4.3 Platelia OD 0.98 0.97 1.00 1.00 0.99 2%

13/132 D 5.4 VIDAS Toxo IgM Index 0.96 0.98

0.97

13/132 D 6.2 IgM ELISA EIU 1.09 1.10 0.94

1.04 9%


13/132 D 7.3 IgM IFA Titer 1.00 1.00 0.50 1.00 0.84 41%



0.97

13/132 D 11.4 ARC Toxo IgM Index 0.86

0.86

13/132 D 11.6 Diasorin Toxo IgM Index >1.37

13/132 D 12.2 ARC Toxo IgM Index 0.80

0.80

13/132 D 12.4 VIDAS Toxo IgM Index 0.99

0.99


0.97


0.97


1.01



unitage.

WHO/BS/2015.2266

Table A2: Continued

Sample Lab

code

Method Unit Assay

1

Assay

2

Assay

3

Assay

4

GM GCV

Ref Test

13/132 F 1.3 VIDAS Toxo IgM Index 1.12 1.13 1.14 1.08 1.12 3%

13/132 F 2.1 ISAGA Titer 1.00

1.00

13/132 F 2.3 VIDAS Toxo IgM Index 1.09

1.09

13/132 F 4.3 Platelia OD 1.01 1.02 1.00 1.00 1.01 1%

13/132 F 5.4 VIDAS Toxo IgM Index 1.12 1.14

1.13

13/132 F 6.2 IgM ELISA EIU 0.95 0.95 0.95

0.95 0%


13/132 F 7.3 IgM IFA Titer 1.00 1.00 0.50 1.00 0.84 41%



1.13

13/132 F 11.4 ARC Toxo IgM AU 1.27

1.27

13/132 F 11.6 Diasorin Toxo IgM AU 0.78

0.78

13/132 F 12.2 ARC Toxo IgM Index 1.26

1.26

13/132 F 12.4 VIDAS Toxo IgM Index 1.11

1.11


1.15


1.13


1.11

TOXM A 1.3 VIDAS Toxo IgM Index 0.02 0.01 0.01 0.01 0.01 5%

TOXM A 2.1 ISAGA Titer 0.00

0.00

TOXM A 2.3 VIDAS Index 0.02

0.02

TOXM A 4.3 Platelia OD 0.04 0.04 0.05 0.05 0.05 14%

TOXM A 5.4 VIDAS Toxo IgM Index 0.02 0.02

0.02

TOXM A 7.5 VIDAS Toxo IgM Index 0.02 0.02 0.02 0.01 0.02 5%

TOXM A 7.3 IgM IFA Titer 0.00 0.00 0.00 0.00 0.00

TOXM A 8.2 VIDAS Toxo IgM Index

0.02 0.02 0.02

TOXM A 10.3 VIDAS Toxo IgM Index 0.01

0.01

TOXM A 11.4 ARC Toxo IgM Index 0.01

0.01

TOXM A 11.6 Diasorin Toxo IgM Index <0.02 -

TOXM A 12.2 ARC Toxo IgM Index 0.01 0.01

0.01


0.01


0.01


0.01


0.01



unitage.

WHO/BS/2015.2266

Table A2: Continued

Sample Lab

code

Method Unit Assay

1

Assay

2

Assay

3

Assay

4

GM GCV

Ref Test

TOXM 13/132 1.3 VIDAS Toxo IgM Index 0.77 0.77 0.72 0.78 0.76 4%

TOXM 13/132 2.1 ISAGA Titer 0.26

0.26

TOXM 13/132 2.3 VIDAS Toxo IgM Index 0.79

0.79

TOXM 13/132 4.3 Platelia OD 0.95 0.97 1.00 1.00 0.98 3%

TOXM 13/132 5.4 VIDAS TXM Index 0.78 0.76

0.77

TOXM 13/132 6.2 IgM ELISA EIU 0.69 0.62 0.70

0.67 7%

TOXM 13/132 7.5 VIDAS Toxo IgM Index 0.74 0.76 0.76 0.77 0.76 1%

TOXM 13/132 7.3 IgM IFA Titer 0.50 0.50 0.50 0.50 0.50 0%

TOXM 13/132 8.2 VIDAS Index

0.77 0.83 0.80

TOXM 13/132 10.3 VIDAS Index 0.69

0.69

TOXM 13/132 11.4 ARC Toxo IgM Index 0.53

0.53

TOXM 13/132 11.6 Diasorin Toxo IgM Index <0.73 -

TOXM 13/132 12.2 ARC Toxo IgM Index 0.45

0.45


0.76

TOXM 13/132 13.3 VIDAS Toxo IgM Index 0.77 0.72

0.74


0.74

TOXM 13/132 16.3 VIDAS Toxo IgM Index 0.74 0.69

0.72

TOXM D 1.3 VIDAS Toxo IgM Index 0.72 0.73 0.70 0.72 0.72 2%

TOXM D 2.1 ISAGA Titer 0.26

0.26

TOXM D 2.3 VIDAS Toxo IgM Index 0.76

0.76

TOXM D 4.3 Platelia OD 0.93 0.94 1.00 1.00 0.97 4%

TOXM D 5.4 VIDAS TXM Index 0.75 0.75

0.75

TOXM D 6.2 IgM ELISA EIU 0.75 0.68 0.66

0.70 7%

TOXM D 7.5 VIDAS Toxo IgM Index 0.73 0.75 0.71 0.73 0.73 2%

TOXM D 7.3 IgM IFA Titer 0.50 0.50 0.25 0.50 0.42 41%

TOXM D 8.2 VIDAS Toxo IgM Index

0.78 0.83 0.81


0.66

TOXM D 11.4 ARC Toxo IgM Index 0.45

0.45

TOXM D 11.6 Diasorin Toxo IgM Index n/a -

TOXM D 12.2 ARC Toxo IgM Index 0.36

0.36


0.75

TOXM D 13.3 VIDAS Toxo IgM Index 0.74 0.70

0.72


0.73

TOXM D 16.3 VIDAS Toxo IgM Index 0.73 0.72

0.72



unitage.

WHO/BS/2015.2266

Table A2: Continued

Sample Lab

code

Method Unit Assay

1

Assay

2

Assay

3

Assay

4

GM GCV

Ref Test

TOXM F 1.3 VIDAS Toxo IgM Index 0.87 0.88 0.82 0.84 0.85 3%

TOXM F 2.1 ISAGA Titer 0.26

0.26

TOXM F 2.3 VIDAS Toxo IgM Index 0.87

0.87

TOXM F 4.3 Platelia OD 0.96 0.99 1.00 1.00 0.99 2%

TOXM F 5.4 VIDAS Toxo IgM Index 0.88 0.87

0.87

TOXM F 6.2 IgM ELISA EIU 0.65 0.59 0.67

0.64 7%

TOXM F 7.5 VIDAS Toxo IgM Index 0.87 0.89 0.86 0.87 0.88 2%

TOXM F 7.3 IgM IFA Titer 0.50 0.50 0.25 0.50 0.42 41%

TOXM F 8.2 VIDAS Toxo IgM Index

0.93 0.96 0.95


0.79

TOXM F 11.4 ARC Toxo IgM Index 0.67

0.67

TOXM F 11.6 Diasorin Toxo IgM Index <0.57 -

TOXM F 12.2 ARC Toxo IgM Index 0.57

0.57


0.84


0.85


0.85


0.80



unitage.

WHO/BS/2015.2266

Table A3: Results of the relative potency for IgG in samples A to F by

agglutination assays, ELISA, ELFA and IFA1

Sample Lab

code

Method Uni

t

Assay

1

Assay

2

Assay

3

Assay

4

GM GCV

Ref Test

A 13/132 1.2 VIDAS Toxo IgG II IU 2.15 1.87 1.63 1.66 1.82 14%

A 13/132 2.2 VIDAS Toxo IgG II IU 1.64

1.64

A 13/132 4.4 IgG ELISA

Tite

r 0.50 0.42 1.41 1.00 0.74 77%

A 13/132 5.3 VIDAS Toxo IgG II IU 2.36 1.71

2.01

A 13/132 5.1 HSDA IgG IU 8.00 8.00

8.00


A 13/132 7.2 IgG IFA IU 80.00 56.57 80.00 56.57 67.27 22%


A 13/132 9.1 HSDA IgG

Tite

r 8.94 5.48

7.00


1.83

A 13/132 11.2 ARC Toxo IgG IU 5.84

5.84

A 13/132 11.5 Diasorin Toxo IgG IU 1.83

1.83

A 13/132 12.1 ARC Toxo IgG IU 5.75

5.75

A 13/132 12.3 VIDAS Toxo IgG II IU 1.68

1.68


5.05

A 13/132 15.1 IgG ELISA OD 2.46 1.51 1.60 1.17 1.62 36%


1.72

A D 1.2 VIDAS Toxo IgG II IU 2.46 1.82 1.83 1.86 1.98 16%

A D 2.2 VIDAS Toxo IgG II IU 1.77

1.77

A D 4.4 IgG ELISA

Tite

r 0.50 0.50 1.00 0.71 0.65 39%

A D 5.3 VIDAS Toxo IgG II IU 2.98 1.91

2.38

A D 5.1 HSDA IgG IU 8.00 8.00

8.00


A D 7.2 IgG IFA IU 80.00 80.00 80.00 80.00 80.00 0%


A D 9.1 HSDA IgG

Tite

r 28.00 14.00

19.80


1.95

A D 11.2 ARC Toxo IgG IU 7.35

7.35

A D 11.5 Diasorin Toxo IgG IU 1.83

1.83

A D 12.1 ARC Toxo IgG IU 7.14

7.14

A D 12.3 VIDAS Toxo IgG II IU 1.70

1.70


5.94

A D 15.1 IgG ELISA OD 2.30 1.49 1.67 1.28 1.65 28% 1: results of samples C and E are represented by sample 13/132

WHO/BS/2015.2266

Table A3: Continued

Sample Lab

code

Method Unit Assay

1

Assay 2 Assay

3

Assay 4 GM GCV

Ref Test

A D 16.1 VIDAS Toxo IgG II IU 1.48 1.75 1.61

A F 1.2 VIDAS Toxo IgG II IU 3.15 2.28 2.43 1.79 2.37 26%

A F 2.2 VIDAS Toxo IgG II IU 2.75 2.75

A F 4.4 IgG ELISA Titer 2.00 2.00 2.00 1.41 1.83 19%

A F 5.3 VIDAS Toxo IgG II IU 3.80 3.73 3.77

A F 5.1 HSDA IgG IU 8.00

8.00

A F 7.4 VIDAS Toxo IgG II IU 1.82 1.86 2.00 2.28 1.98 11%

A F 7.2 IgG IFA IU 40.00 40.00 40.00 40.00 40.00 0%

A F 8.1 VIDAS Toxo IgG II IU >3.13 3.29 >3.13 >2.97 3.29

A F 9.1 HSDA IgG Titer 24.00 8.00

13.86


2.59

A F 11.2 ARC Toxo IgG IU 6.85

6.85

A F 11.5 Diasorin Toxo IgG IU 2.28

2.28

A F 12.1 ARC Toxo IgG IU 6.68

6.68

A F 12.3 VIDAS Toxo IgG II IU 2.34

2.34


7.60

A F 15.1 IgG ELISA OD 2.94 1.92 1.79 1.60 2.01 30%


2.72

13/132 D 1.2 VIDAS Toxo IgG II IU 1.14 0.97 1.12 1.12 1.09 8%

13/132 D 2.2 VIDAS Toxo IgG II IU 1.08

1.08

13/132 D 4.4 IgG ELISA Titer 1.00 1.19 0.71 0.71 0.88 30%

13/132 D 5.3 VIDAS Toxo IgG II IU 1.26 1.11

1.18

13/132 D 5.1 HSDA IgG IU 1.00 1.00

1.00


13/132 D 7.2 IgG IFA IU 1.00 1.41 1.00 1.41 1.19 22%


13/132 D 9.1 HSDA IgG Titer 3.13 2.56

2.83


1.06

13/132 D 11.2 ARC Toxo IgG IU 1.26

1.26

13/132 D 11.5 Diasorin Toxo IgG IU 1.00

1.00

13/132 D 12.1 ARC Toxo IgG IU 1.24

1.24

13/132 D 12.3 VIDAS Toxo IgG II IU 1.01

1.01


1.18

13/132 D 15.1 IgG ELISA OD 0.93 0.99 1.04 1.10 1.01 7%


0.94


Results presented in shaded cells could not be transformed to a numerical values and were not used to calculate a

unitage.

WHO/BS/2015.2266

Table A3: Continued Sample Lab

code

Method Unit Assay 1 Assay 2 Assay

3

Assay 4 GM GCV

Ref Test

13/132 F 1.2 VIDAS Toxo IgG II IU 1.46 1.22 1.50 1.08 1.30 17%

13/132 F 2.2 VIDAS Toxo IgG II IU 1.67 1.67

13/132 F 4.4 IgG ELISA Titer 4.00 4.76 1.41 1.41 2.48 92%

13/132 F 5.3 VIDAS Toxo IgG II IU 1.61 2.18 1.87

13/132 F 5.1 HSDA IgG IU 1.00 1.00 1.00

13/132 F 7.4 VIDAS Toxo IgG II IU 1.40 1.21 1.39 1.44 1.36 8%

13/132 F 7.2 IgG IFA IU 0.50 0.71 0.50 0.71 0.59 22%

13/132 F 8.1 VIDAS Toxo IgG II IU >1.97 1.36 >1.83 >2.11 1.36

13/132 F 9.1 HSDA IgG Titer 2.68 1.46

1.98


1.41

13/132 F 11.2 ARC Toxo IgG IU 1.17

1.17

13/132 F 11.5 Diasorin Toxo IgG IU 1.25

1.25

13/132 F 12.1 ARC Toxo IgG IU 1.16

1.16

13/132 F 12.3 VIDAS Toxo IgG II IU 1.40

1.40


1.50

13/132 F 15.1 IgG ELISA OD 1.20 1.27 1.12 1.37 1.24 9%


1.58

TOXM A 1.2 VIDAS Toxo IgG II IU 0.17 0.21 0.19 0.21 0.20 11%

TOXM A 2.2 VIDAS Toxo IgG II IU 0.36

0.36

TOXM A 4.4 IgG ELISA Titer 1.41 1.00 0.50 0.71 0.84 56%

TOXM A 5.3 VIDAS Toxo IgG II IU 0.21 0.20

0.20

TOXM A 5.1 HSDA IgG IU 0.06 0.06

0.06

TOXM A 7.4 VIDAS Toxo IgG II IU 0.47 0.48 0.49 0.41 0.46 9%

TOXM A 7.2 IgG IFA IU 0.01 0.01 0.01 0.01 0.01 0%

TOXM A 8.1 VIDAS Toxo IgG II IU <0.32 <0.34 -

TOXM A 9.1 HSDA IgG Titer 0.02 0.02

0.02


0.34

TOXM A 11.2 ARC Toxo IgG IU 0.04

0.04

TOXM A 11.5 Diasorin Toxo IgG IU 0.29

0.29

TOXM A 12.1 ARC Toxo IgG IU <0.11 -


0.40


0.10

TOXM A 15.1 IgG ELISA OD 0.35 0.42 0.44

0.40 13%


0.36



unitage.

WHO/BS/2015.2266


code


3

Assay 4 GM GCV

Ref Test

TOXM 13/132 1.2 VIDAS Toxo IgG II IU 0.37 0.39 0.31 0.36 0.36 10%

TOXM 13/132 2.2 VIDAS Toxo IgG II IU 0.58

0.58

TOXM 13/132 4.4 IgG ELISA Titer 0.71 0.42 0.71 0.71 0.62 30%

TOXM 13/132 5.3 VIDAS Toxo IgG II IU 0.49 0.34

0.41

TOXM 13/132 5.1 HSDA IgG IU 0.50 0.50

0.50

TOXM 13/132 7.4 VIDAS Toxo IgG II IU 0.61 0.74 0.71 0.65 0.67 9%

TOXM 13/132 7.2 IgG IFA IU 0.50 0.35 0.50 0.35 0.42 22%

TOXM 13/132 8.1 VIDAS Toxo IgG II IU <0.55 <0.47 -

TOXM 13/132 9.1 HSDA IgG Titer 0.14 0.11

0.13


0.69

TOXM 13/132 11.2 ARC Toxo IgG IU 0.23

0.23

TOXM 13/132 11.5 Diasorin Toxo IgG IU 0.53

0.53

TOXM 13/132 12.1 ARC Toxo IgG IU <0.63


0.67


0.49

TOXM 13/132 15.1 IgG ELISA OD 0.86 0.64 0.71

0.73 17%


0.61

TOXM D 1.2 VIDAS Toxo IgG II IU 0.42 0.38 0.35 0.40 0.39 8%

TOXM D 2.2 VIDAS Toxo IgG II IU 0.63

0.63

TOXM D 4.4 IgG ELISA Titer 0.71 0.50 0.50 0.50 0.55 19%

TOXM D 5.3 VIDAS Toxo IgG II IU 0.62 0.38

0.49

TOXM D 5.1 HSDA IgG IU 0.50 0.50

0.50

TOXM D 7.4 VIDAS Toxo IgG II IU 0.70 0.78 0.77 0.66 0.72 9%

TOXM D 7.2 IgG IFA IU 0.50 0.50 0.50 0.50 0.50 0%

TOXM D 8.1 VIDAS Toxo IgG II IU <0.77 <0.75 -

TOXM D 9.1 HSDA IgG Titer 0.44 0.29

0.36


0.72

TOXM D 11.2 ARC Toxo IgG IU 0.28

0.28

TOXM D 11.5 Diasorin Toxo IgG IU 0.53

0.53

TOXM D 12.1 ARC Toxo IgG IU <0.78 -


0.68


0.57

TOXM D 15.1 IgG ELISA OD 0.81 0.63 0.74

0.72 13%


0.58



unitage.

WHO/BS/2015.2266


code


3

Assay 4 GM GCV

Ref Test

TOXM F 1.2 VIDAS Toxo IgG II IU 0.54 0.48 0.47 0.39 0.46 15%

TOXM F 2.2 VIDAS Toxo IgG II IU 0.98

0.98

TOXM F 4.4 IgG ELISA Titer 2.83 2.00 1.00 1.00 1.54 68%

TOXM F 5.3 VIDAS Toxo IgG II IU 0.80 0.74

0.77

TOXM F 5.1 HSDA IgG IU 0.50

0.50

TOXM F 7.4 VIDAS Toxo IgG II IU 0.85 0.89 0.99 0.93 0.91 7%

TOXM F 7.2 IgG IFA IU 0.25 0.25 0.25 0.25 0.25 0%

TOXM F 8.1 VIDAS Toxo IgG II IU n/a n/a -

TOXM F 9.1 HSDA IgG Titer 0.38 0.17

0.25


0.88

TOXM F 11.2 ARC Toxo IgG IU 0.26

0.26

TOXM F 11.5 Diasorin Toxo IgG IU 0.66

0.66

TOXM F 12.1 ARC Toxo IgG IU <0.73


0.94


0.73

TOXM F 15.1 IgG ELISA OD 1.03 0.81 0.79

0.87 16%


0.97

1: Results of samples C and E are represented by sample 13/132


unitage.

WHO/BS/2015.2266

Appendix 2

Table A4: Participants of the collaborative study

Name Affiliation Country D. Dickeson MPh Pathology West ICPMR Westmead, Centre for Infectious Diseases &

Microbiology Laboratory Services, Westmead Hospital, Westmead,

New South Wales

Australia

Dr H. Vedel Nielsen Unit of Mycology and Parasitology, Department of Microbiology and

Infection Control, Statens Serum Institute, 5 Artillerivej, Copenhagen

Denmark

Professor E. Candolfi Institut de Parasitologie et de Pathologie Tropicale de Strasbourg,

Laboratoire Associé Centre National de Référence de la Toxoplasmose,

Université de Strasbourg, 3 rue Koeberlé, Strasbourg

France

Professor M.–L. Dardé Parasitoloy department, Centre Hospitalier Universitaire Dupuytren, 2

Avenue M Luther King, Limoges

France

Professor H. Pelloux and

Dr H Fricker-Hidalgo

Parasitologie-Mycologie, Département des Agents Infectieux, Institut

de Biologie et de Pathologie, Centre Hospitalier Universitaire de

Grenoble, Grenoble

France

Professor I. Villena and

Dr C. Chemla

Laboratoire de Parasitologie-Mycologie, Hôpital Maison Blanche, 45

Rue Cognac-Jay, Reims

France

Dr. I. Reiter-Owona Institute of Medical Microbiology, Immunology and Parasitology

(IMMIP) University Clinic Bonn, Sigmund-Freud-Str. 25, 53127 Bonn

Germany

Dr C. Talbot National Virus Reference Laboratory, University College Dublin,

Belfield, Dublin

Ireland

Dr I. Riklis Toxoplasma National Laboratory, Public Neath Laboratory, Ministry

of Health, Abu-Kabir, Tel Aviv

Israel

Dr L. M. Kortbeek and

D. Hoek

Department of Bacteriology and Parasitology Center of Disease

Control, National Institute of Public Health and Environment,

Bilthoven

The Netherlands

Dr O. Djurković-

Djaković

National Reference Laboratory for Toxoplasmosis, Institute for

Medical Research, University of Belgrade, Bul. Oslobodjenja 18,

Belgrade

Serbia

Dr. A. Mahittikorn Department of Protozoology, Faculty of Tropical Medicine, Mahidol

University, 420/6 Ratchawithi Road, Ratchathewi, Bangkok

Thailand

Professor Dr. A. Y.

Gürüz and Prof. Dr. M.

Korkmaz

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

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.

WHO/BS/2015.2266

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.


http://www.who.int/biologicals/en/

http://www.bipm.org/en/committees/jc/jctlm/

http://www.nibsc.ac.uk/products/biological_reference_materials/frequently_asked_questions/how_are_international_units.aspx

http://www.nibsc.ac.uk/products/biological_reference_materials/frequently_asked_questions/how_are_international_units.aspx

http://www.nibsc.ac.uk/products/ordering_information/frequently_asked_questions.aspx

http://www.nibsc.ac.uk/terms_and_conditions.aspx


WHO/BS/2015.2266

14. MATERIAL SAFETY SHEET

Physical and Chemical properties

Physical appearance: Dry powder

Corrosive: No

Stable: Yes Oxidising: No

Hygroscopic: No Irritant: No

Flammable: No Handling:See caution, Section 2

Other (specify):

Contains human plasma

Toxicological properties

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.

http://www.nibsc.org/About_Us/Terms_and_Conditions.aspx

WHO/BS/2015.2266

Attached: No

17. CERTIFICATE OF ANALYSIS

NIBSC does not provide a Certificate of Analysis for WHO Biological Reference Materials because

they are internationally recognised primary reference materials fully described in the instructions for

use. The reference materials are established according to the WHO Recommendations for the

preparation, characterization and establishment of international and other biological reference

standards

http://www.who.int/bloodproducts/publications/TRS932Annex2_Inter_biolefstandardsrev2004.pdf

(revised 2004). They are officially endorsed by the WHO Expert Committee on Biological

Standardization (ECBS) based on the report of the international collaborative study which

established their suitability for the intended use.

http://www.who.int/bloodproducts/publications/TRS932Annex2_Inter_biolefstandardsrev2004.pdf

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