The NS1 ELISA has been tested on the TBE virus

Department of Virology at the Norwegian Institute of Public Health (NIPH) has been the National Reference lab for Tick-borne encephalitis virus (TBE virus) since 2008. The reference lab is divided into a routine and a research lab group. The NS1 TBE research group consists of Maria Juul Diekmann (Master student, in collaboration with Roskilde University), Katrine M Paulsen (Chief engineer), Alaka Lamsal (PhD student) and Rose Vikse, Åshild K Andreassen (senior scientists/Professors) as well as external Professor Karen A. Krogfelt (Roskilde University, Denmark).

Our gold standard method for TBE detection and confirmation is serology with the Enzygnost anti-TBE/FSME/ETG (IgG, IgM) kits (Siemens, Marburg, Germany). Due to the discontinued production of these kits, NIPH had to evaluate and validate other commercial methods. The main aim was to find a method with similar sensitivity and specificity as the Enzygnost anti-TBE/FSME/ETG (IgG, IgM) method. In addition, we wanted to find a method that could distinguish between vaccinated and infected TBE cases. The recombinant NS1 protein in the TBF NS1 ELISA assay (developed by ICGEB) is designed to detect flavivirus antibodies with reduced cross-reactivity and high sensitivity and detection of vaccine breakthrough cases. Detection of the difference in the TBE immune response between infected and vaccinated cases is important and we decided to test the non-commercial TBE NS1 ELISA assay together with the commercial ELISA tests available on the market.

Our material consists of 146 random IgM/IgG serum and spinal fluid samples from previously examined cases including quality control samples known to cross-react with the TBE antibodies.

In addition, we have 12 patient serum samples from a collaborative study with the University of Oslo analysed by the same method (Enzygnost) and confirmed by virus neutralization test (VNT) at the Veterinary Research Institute in the Czech Republic. Some of these sera may be from individuals who are vaccinated by cross-reacting agents i.e. other flaviviruses than TBE.

Preliminary results:

Our master student has performed several initial tests to customize the TBE NS1 ELISA. There has been some trouble in finding the best replacement reagent available since the HRP-linked goat antibodies anti-human IgG from Sigma (A6029-1ml) was not available at the time we started testing. Some strong TBE positive, vaccinated and negative samples were tested with different blocking solutions of 2% BSA and different dilutions of the new secondary antibody HRP-linked goat antibodies anti-human IgG (Fc specific) from Sigma (A0170-1ml). We experienced some high background problems that were solved by a fresh 2% BSA and therefore the new secondary antibody was titrated. In our hands, the dilution suggested from the protocol was too high with the new secondary antibody. New tests will be performed shortly in order to establish the best dilution concentration when using our chosen secondary antibody.

Thanks to this work, we are now able to distinguish between the vaccinated, infected and negative samples and can start to validate the test samples.