Borrelia multiplex: a bead-based multiplex assay for the simultaneous detection of Borrelia specific IgG/IgM class antibodies

Abstract

Background: Lyme borreliosis (LB) is the most common tick-borne infectious disease in the northern hemisphere. The diagnosis of LB is usually made by clinical symptoms and subsequently supported by serology. In Europe, a two-step testing consisting of an enzyme-linked immunosorbent assay (ELISA) and an immunoblot is recommended. However, due to the low sensitivity of the currently available tests, antibody detection is sometimes inaccurate, especially in the early phase of infection, leading to underdiagnoses.

Methods: To improve upon Borrelia diagnostics, we developed a multiplex Borrelia immunoassay (Borrelia multiplex), which utilizes the new INTELLIFLEX platform, enabling the simultaneous dual detection of IgG and IgM antibodies, saving further time and reducing the biosample material requirement. In order to enable correct classification, the Borrelia multiplex contains eight antigens from the five human pathogenic Borrelia species known in Europe. Six antigens are known to mainly induce an IgG response and two antigens are predominant for an IgM response.

Results: To validate the assay, we compared the Borrelia multiplex to a commercial bead-based immunoassay resulting in an overall assay sensitivity of 93.7% (95% CI 84.8-97.5%) and a specificity of 96.5% (95%CI 93.5-98.1%). To confirm the calculated sensitivity and specificity, a comparison with a conventional 2-step diagnostics was performed. With this comparison, we obtained a sensitivity of 95.2% (95% CI 84.2-99.2%) and a specificity of 93.0% (95% CI 90.6-94.7%).

Conclusion: Borrelia multiplex is a highly reproducible cost- and time-effective assay that enables the profiling of antibodies against several individual antigens simultaneously.

Keywords: Borrelia; Immunoassay; Lyme Disease; Lyme borreliosis; Multiplex; Serology.

Fig. 1

Sample classification of Borrelia multiplex. Sample classification was done as follows. In the first step the MFI signal of each antigen was divided by the MFI signal of the corresponding CO samples. The obtained S/CO values were then scored in a point system for each antigen. For BmpA, DbpA, p83/100 and OppA-2, values from 0 to 2 were assigned. For VIsE and OspC, values from 0 to 3 were used. For DbpA, the antigens were considered a pair, with only the highest value used. For each antigen, samples with a S/CO ratio < 0.75 received 0 points, samples with a S/CO ratio between 0.75 and 1.00, received 1 point and samples with a S/CO ratio > 1.00 received 2 or 3 points, depending on the antigen. After the classification of all antigens, IgG and IgM scores were generated (“Sum of Points”). For IgG, a score ≥ 5 results in a sample classification as IgG positive. For IgM, a score ≥ 3 results in a sample classification as IgM positive. If either antibody class is positive, than the entire sample was classified as Borrelia positive

Fig. 2

Antibody response in Borrelia negative and positive samples. 341 serum samples from the MEMO study were measured with the Borrelia multiplex and a commercial bead-based immunoassay (recomBead Borrelia IgG/IgM 2.0). Based on the result of the recomBead Borrelia samples were categorized in Borrelia negative and positive. The corresponding MFI signals of the Borrelia multiplex were plotted in a Box-Whisker plot. Boxes include the median and the 25th and 75th percentiles. Whiskers are limited to 1.5 times IQR. Outliers are shown as depicted circles. A dashed line (S/CO = 1.0) indicates the determined MFI cut-offs. For the antigens DbpA–ZS7, DbpA–A14S and DbpA–PBi no cut-offs were needed. For statistical analysis Mann–Whitney test (two-tailed) was used. P values were classified as follows: > 0.05 (ns), ≤ 0.05 (*), ≤ 0.01 (**), ≤ 0.001 (***). A–F Box-Whisker plots for IgG dominant antigens. G, H Box-Whisker plots for IgM dominant antigens

Fig. 3

Adaption of Borrelia multiplex to the INTELLIFLEX platform. Adaption of the Borrelia multiplex to the new INTELLIFLEX platform was done in three steps. A, B 4 serum samples were measured for Borrelia specific IgG (A) and IgM (B) antibodies to compare the PE detection system on FLEXMAP 3D and INTELLIFLEX platform. C 12 serum samples were measured for IgG antibodies to compare a directly PE-labelled detection antibody with a biotinylated antibody in combination with BV421 labelled streptavidin. D, E 12 serum samples were measured for IgG (D) and IgM (E) antibodies separately (single detection) or in combination (dual detection). In each graph MFI signals were plotted against each other and analyzed by linear regression. A linear curve (x = y) shown as red dashed line indicates identical MFI signals for detection systems. Correlation analysis was performed after Spearman

Fig. 4

Manual and automated processing of Borrelia multiplex. 662 serum samples were measured with the Borrelia multiplex and a common 2-step diagnostics. For the Borrelia multiplex processing was either manually or by pipetting robot. A, B Correlation between manual and automated processing of Borrelia multiplex. S/CO values were plotted against each other. A red dashed line shows the curve for identical S/CO values. Correlation analysis was performed according to Spearman. C–F Box-Whisker plots of S/CO values for Borrelia negative and positive samples. Samples were categorized according to the result of the 2-step diagnostics. Boxes include the median and the 25th and 75th percentiles. Whiskers are limited to 1.5 times IQR. Outliers are shown as depicted circles. A dashed line (S/CO = 1.0) indicates the threshold between positive and negative signal. The grey area below the line gives the borderline area (0.75 < S/Co < 1.0). For statistical analysis Mann–Whitney test (two-tailed) was used. P values were classified as follows: > 0.05 (ns), ≤ 0.05 (*), ≤ 0.01 (**), ≤ 0.001 (***)

Fig. 5

Epidemiological analysis of 1,555 serum samples from MuSPAD study. 1555 serum samples from the MuSPAD study were measured with the automated Borrelia multiplex and analyzed regarding collection site (A), gender (B) and age (C). Additionally, seropositivity of men and women was compared over all age groups (D). For statistical analysis Fisher’s exact test was used. P values were classified as follows: > 0.05 (ns), ≤ 0.05 (*), ≤ 0.01 (**), ≤ 0.001 (***)