Enzygnost lyme link vlse igg

Serological tests for B. burgdorferi consisted of an IgM and IgG ELISA, followed by an immunoblot. In case only an ELISA was performed and no immunoblot, the ELISA result was leading. If both an ELISA and immunoblot were performed, the immunoblot result was leading [6 ]. An indeterminate ELISA without an immunoblot was considered negative, as well as an indeterminate immunoblot. The ELISAs used were the Enzygnost Lyme Link VlsE/IgG and the Enzygnost Borreliosis IgM by DADE Behring, Marburg, Germany [15 (link)]. The immunoblot used was the recomLine Borrelia IgG and IgM immunoblot of Mikrogen, Neuried, Germany [16 ]. Pleocytosis was defined as leucocyte cell count in CSF >5 per μl or >15 per 3 µl [17 (link)]. Intrathecal Borrelia antibody production was detected by analysing a serum and CSF pair with the IDEIA Lyme neuroborreliosis OXOID, Hampshire, UK [18 ]. Diagnostic tests were performed as indicated by the manufacturers.

Blood from healthy volunteers without (n = 8) or with (n = 1) specific anti-Borrelia antibodies was collected in 6-mL Vacutainer plastic tubes (BD Bioscience, Plymouth, UK) with the addition of the specific thrombin inhibitor hirudin (Refludan, Pharmion Ltd, Cambridge, UK), at a final concentration of 50 µg/mL blood. This study, using blood from healthy blood donors given their written consent, was performed with consent of the Ethical Committee of the University Hospital of Linköping, Sweden (#03-520). Plasma was collected by centrifugation at 3000 g for 20 min and stored at −80°C. For viability studies, aliquots of plasma were heat inactivated by incubation at 56°C for 30 min. For the phagocytosis experiments and cytokine release assays, blood was collected as described above and used within 30 min. Anti-Borrelia antibodies were measured in serum using the commercially available enzyme-linked immunosorbent assay (ELISA) kits Enzygnost Lyme link VlsE/IgG and Enzygnost Borreliosis IgM (DADE Behring, Marburg, Germany) on a BEP 2000 Advance System (Siemens Healthcare, Erlangen, Germany), according to the instructions from the manufacturer.

Serum samples were tested for IgG at the National Reference Centre for Borrelia using same guidelines, assays and approach for all study groups (including the studies in adults [17 (link)]), which is also the standard for clinical diagnostics [6 (link),8 (link),19 ]. We used a two-step approach involving a screening with an enzyme-linked immunosorbent assay (ELISA) (Enzygnost Lyme link VlsE/IgG, Siemens Healthcare Diagnostics GmbH, Eschborn, Germany), followed by a confirmatory immunoblot (line blot) (Borrelia Europe plus TpN17 LINE, IgG, Virotech, Rüsselsheim, Germany) test in case of a positive or borderline ELISA result. The immunoblot test covered a range of specific Bb antigens and was considered positive if reactive to at least two bands. Potential cross-reactions were accounted for by including the antigen TpN17, testing a reaction specific to Treponema pallidum (causative agent of syphilis), the most relevant potential cross-reaction in Germany. More details on the test systems and test result categorisation are provided in the Supplement. Serum samples that tested either positive or borderline with the ELISA and positive with the immunoblot were considered seropositive. We refer to Supplementary Figure S1 for a scheme displaying this categorisation.