European Sero-Epidemiology Network: standardisation of the results of diphtheria antitoxin assays

doi:10.1016/S0264-410X(00)00125-0

Vaccine

Volume 18, Issue 28, 1 August 2000, Pages 3287-3296

https://doi.org/10.1016/S0264-410X(00)00125-0 Get rights and content

Abstract

A European Sero-Epidemiological Network (ESEN) was established with the aim to co-ordinate and harmonise serological surveillance of immunity to communicable diseases in Europe. In this study the inter-laboratory standardisation of diphtheria toxin antibody measurements is reported. A standard panel of 162 sera was tested by the participating laboratories using an in vitro assay of their choice: VERO cell toxin neutralisation assay (NT), double-antigen delayed time-resolved fluorescence immuno-assay (DA-DELFIA), double-antigen enzyme-linked immunosorbent assay (DAE), toxin binding inhibition test (ToBI) and an indirect enzyme-linked immunosorbent assay (ELISA). The results were standardised using regression against the NT. The variations due to inter-laboratory and inter-assay variation, which would otherwise make it difficult directly to compare the main serum bank results by the different laboratories and the various assays were successfully minimised by the standardisation. The regression equations obtained will be used to transform the respective local results of testing the main serum bank into the reference test unitages. This study also gave the opportunity to compare the various assays within and between laboratories. This demonstrated a very high correlation between DA-DELFIA, DAE, ToBI and the NT. The ELISA showed a good correlation, too, however sera below some 0.1 IU/ml were overestimated.

Introduction

The European Sero-Epidemiological Network (ESEN) was established in 1996 with funding from the European Union. The aim of the project was to co-ordinate and harmonise serological surveillance of immunity to five vaccine preventable diseases, including diphtheria, pertussis, measles, mumps and rubella, in the population of eight collaborating countries [1]. Thus, national banks of several thousand age and sex stratified serum samples have been collected in each participating country.

To ensure direct comparability of the measured antibody levels with various in vitro serological techniques, a process of standardisation was developed [2]. This involved the establishment of a reference centre for each of the five diseases and the creation and distribution of a panel of selected sera to be tested by each of the participating laboratories for calibration against the chosen reference laboratory. The present study outlines the mathematical procedure and the results of the standardisation of diphtheria toxin antibodies among geographically widely separated laboratories using either the same or different tests. Furthermore, in addition to the standardisation process, the results of testing the standard panel were also used for an inter- and intra-laboratory comparison of various in vitro techniques used in this study.

Section snippets

Participants

The participating countries and the respective laboratories involved in the study were: Denmark (DK), Statens Serum Institut (SSI), Copenhagen; Finland (FI), National Public Health Institute (KTL), Helsinki; France (FR), Pasteur-Merieux Connaught, Val De Reuil; Germany (DE), Robert Koch Institute, Berlin; Italy (IT), Istituto Superiore di Sanità, Roma; The Netherlands (NL), National Institute of Public Health and Environment, Bilthoven; Sweden (SE), Swedish Institute for Infectious Diseases

Repeat panel testing

DE, IT, UK and FI tested the panel twice 6 months apart, DK tested the panel twice at the beginning, the NL tested it twice at the end. There was no evidence of any assay drift for DE or UK, however for IT and FI there were small differences (average differences were 0.06 and 0.08 log₁₀ units, respectively). The second tests of the panel were used for standardisation against the reference test (FI-NT test).

Comparison of tests

The majority of the participating laboratories performed one of the double-antigen

Discussion

Diphtheria toxin antibodies can successfully be measured by simple in vitro techniques like DA-DELFIA, DAE and ToBI, which may be suitable alternatives to the VERO cell assay in large serosurveillance studies. However, antibody levels measured against international standard preparations and thus expressed in IU/ml show some variations due to inter-laboratory and inter-assay variation, making it difficult to compare directly the results of one laboratory or one assay with another. In the present

Acknowledgements

We gratefully acknowledge for excellent technical assistance: Giovanna Alfarone (Italy), Halina Pedersen (Denmark), Paivi Paalanen, Kaija Vuontela (Finland), Edelgard Kriebel and Ingrid Deitemeier (Germany), Ton Marzec (The Netherlands), K. Batey (UK), The Unit of Virology and Immunochemistry (Pasteur-Merieux Connaught, France). We also thank Richard G. Pebody (PHLS, Communicable Disease Surveillance Centre and Central Public Health Laboratory, London, UK) for advice and critical reading of the

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Concentrations ≥1 IU/mL indicate a good long-term protection. The value 0.01 IU/mL is often used as a cut off value indicating protection when functional assays, e.g., the toxin neutralising assay in Vero cells or the DAE assay are used, because a high correlation between these two assays has been shown also in low concentrations [17]. Consequently the value 0.01 IU/mL was used as a cut off value for protection for the results obtained in this study.
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^☆: On behalf of the ESEN project. The members of the European Sero-Epidemiology Network: Denmark: Henrik Aggerbeck, Blenda Böttiger, Per Chr. Grauballe, Anne-Marie Plesner, Helle Rieck, Tove Rønne, Nils Strandberg Pedersen, Henrik Wachmann. England: Nick Andrews, David Brown, Paddy Farrington, Nigel Gay, Louise Hesketh, Chris Maple, Elizabeth Miller, Peter Morgan-Capner, Kate Osborne, Richard Pebody, Mary Ramsay. Finland: Irja Davidkin, Mervi Eerola, Pauli Leinikki, Rose-Marie Ölander, Martti Valle. France: Sabine Baron, Christine Blondeau, Olivier Chappey, Françoise Fievet-Groyne, Maurice Raux, Daniel Lévy-Bruhl, Anne-Sophie Malet, Isabelle Rebière. Germany: Doris Altmann, Edith Gerike, Annette Siedler, Sonja Swidsinski, Annedore Tischer. Italy: Alessandra Anemona, Pietro Crovari, Giovanni Gabutti, Anna Giammanco, Cristina Giordano, Maria Cristina Rota, Stefania Salmaso, Christina von Hunolstein. Netherlands: Guy Berbers, Marina Conyn-van Spaendonck, Hester de Melker, Mirjam Kretzschmar, Joop Schellekens. Sweden: Eivor Bonin, Hans Hallander, Johan Lindback, Margaretha Ljungman, Lars Magnius, Patrick Olin, Ulla Rude. Australia: Geoffrey Hogg, Diane Tallent.

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European Sero-Epidemiology Network: standardisation of the results of diphtheria antitoxin assays☆

Abstract

Introduction

Section snippets

Participants

Repeat panel testing

Comparison of tests

Discussion

Acknowledgements

J. Immunol. Methods

J. Biol. Stand.

J. Biol. Stand.

Biologicals

J. Biol. Stand.