Research paper
EUVAC.NET collaborative study: Evaluation and standardisation of serology for diagnosis of pertussis

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Abstract

As part of the new EUVAC.NET contract with ECDC (Pertussis Work Area 4), a collaborative study was organised in July–December 2010. Two well-defined reference preparations with high and low IgG antibodies to pertussis toxin (PT), were sent to participants. The purposes of this study were to assess current laboratory performance of serological assays for pertussis; to compare in-house reference preparations that are currently used by participants for the serological assay; and to identify needs for standardisation of the serological assay. Reference Laboratories in Europe currently performing serological assays for the diagnosis of pertussis by measuring antibody to PT, were invited to participate in the study. A total of 17 laboratories/countries participated in this study. Results were reported from a total of 9 participants who used in-house ELISA assays and 10 participants who used commercial kits. All participants using in-house ELISA with purified PT coating plates distinguished the 2 preparations and gave results that were comparable to the expected values. A total of 6 commercial kits included in the study showed different results. The kits coated with mixture antigens did not appear to be able to give results that were correlated to the WHO reference preparations.

Highlights

► A EUVAX collaborative study was performed to assess serological tests for pertussis. ► Anti-pertussis toxin antibody in reference sera was measured. ► Both in-house ELISA and commercial kits were used by participants. ► All in-house ELISA assays gave results that were comparable to the expected values. ► Commercial kits coated with mixture antigens did not give satisfactory results.

Introduction

Pertussis remains endemic worldwide and is an important public health issue. In the past years, there has been a marked increase in reported pertussis cases in countries with high vaccination coverage. Laboratory diagnosis of pertussis is important for treatment, prevention and surveillance. It is noted that there have been wide variations in the reporting of laboratory-confirmed pertussis cases globally and one of the reasons could be the differences in the methods used for diagnosis.

Serological analysis by enzyme-linked immunosorbent assays (ELISAs) has been widely used for the evaluation of antibody responses to pertussis vaccination and infection. Serum Reference Standards are essential for comparison of both intra- and inter-laboratory estimates.

As part of the new EUVAC.NET contract with ECDC (Pertussis Work Area 4), a collaborative study to evaluate and standardise serology assay for diagnosis of pertussis was organised in July 2010.

Two coded anti-serum preparations with high and low anti-PT antibody concentrations, prepared in NIBSC from sera kindly donated by Dr Carl Heinz Wirsing von König, Institut für Infektiologie Krefeld GmbH, Krefeld, Germany and a purified pertussis toxin (PT) antigen preparation kindly donated by Statens Serum Institute (SSI), Denmark have been distributed to participants.

The aims of the study were:

  • Assess current laboratory performance of serological assays for pertussis

  • Compare in-house reference preparations that are currently used by participants for the serological assays

  • Identify needs for standardisation of the serological assay

Section snippets

Participants

Reference Laboratories in Europe currently performing serological assays for diagnosis of pertussis by measuring antibody to PT were invited to participate in the study. A total of 17 laboratories/countries participated in this study (Table 1).

Throughout this report, where assay results are attributed to the various laboratories, each laboratory is identified only by a randomly assigned code number from 1 to 17, so that they alone can identify which set of assay results they contributed.

Other materials

Participants were also encouraged to include their in-house reference sera and antigens/or reference sera/antigens provided by commercial kits in the assays.

Assay methods

The primary goal of the study was to evaluate the two serum reference preparations provided with respect to concentrations of IgG-anti-PT by ELISA.

Serological assay by in-house enzyme-linked immunosorbent assays (ELISAs) was used by 9 laboratories in the study and 10 laboratories used commercial ELISA kits in which 2 of the participants

Potency of preparation A relative to preparation B obtained by in-house ELISA

To ascertain how well the assays could distinguish between the low and the high samples (preparations A and B respectively), potency estimates were calculated for preparation A relative to preparation B where possible.

The four parameter logistic model was used for the following: laboratory 2 (assays 1, 2, 3, 4, 5, 6, 7, 8, 10, and 11), laboratory 4 (assays 1, 3, and 4), laboratory 10 (assays 1, 3, and 4), laboratory 11 (assays 2 and 3), laboratory 12 (all assays), laboratory 15 (all assays),

Discussion/conclusion

Harmonisation and standardisation of diagnostic methods are important for a successful laboratory-based surveillance system. ELISAs for measuring antibodies to pertussis antigens have been employed widely in acellular vaccine clinical trials and in sero-epidemiological studies, and it has became more popular for diagnostic purposes (De Melker et al., 2000). Purified or mixed pertussis antigens can be used for coating the ELISA plates, but only PT is specific for B. pertussis (Mattoo and Cherry,

Acknowledgements

We would like to thank the study participants for their contribution to this study. Grateful acknowledgements are due to Statens Serum Institute (SSI), Denmark for the provision of PT antigen. We gratefully acknowledge ECDC/EUVAC.NET for promoting this study and providing financial support, especially for Drs Sabrina Bacci and Steffen Glismann for their great support throughout this study.

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