Journal: bioRxiv

Article Title: Lipoproteome screening of the Lyme disease agent identifies novel inhibitors of antibody-mediated complement killing

doi: 10.1101/2021.09.23.461563

Figure Lengend Snippet: (A) 1×10 strain B31-e2 producing one of 80 B. burgdorferi surface lipoproteins (; Table S1 ), as well as a periplasmic lipoprotein (BB0460) to serve as a negative control, were applied to microtiter wells coated with human C1 complex in duplicate. After washing, bound bacteria were quantitated by the change in OD 405nm over time by ELISA using an anti- Bb antibody (Abcam, ab20118). The clones are sorted in order of binding signal. Error bars indicate SEM. ( A, inset ) Binding of clones producing the indicated B. burgdorferi Elp protein, along with a positive control (BBK32) or a periplasmic negative control (BB0460), to immobilized human C1 complex or BSA was quantitated as described above. Error bars indicate SEM. ****, p<0.0001; *, p<0.05; ns, not significant using Student’s t test to compare mean values. ( B) Binding of the indicated GST-fusion proteins to wells coated with the indicated concentration of human C1 complex was quantitated. The experiment was performed six times (GST-ErpB) or nine times (GST-BBK32 and GST-ErpQ) at each concentration and error bars indicate SEM. Affinity analysis was performed with Prism GraphPad software, using a non-linear regression analysis. ( C) The ability for GST-ErpB (left) GST-ErpQ (right) to bind human C1 complex was evaluated by SPR. A two-fold dilution series (0.6 - 150 nM) of C1 complex was injected over GST-ErpB and GST-ErpQ biosensors and steady-state affinity analysis was carried out with T200 Evaluation Software. Each SPR experiment was performed in triplicate. Equilibrium dissociation constants ( K D ) calculated from ELISA-type and SPR binding assays are shown in .

Article Snippet: Kinetic analyses were performed on each sensorgram series using the Biacore T200 Evaluation Software 3.1 (GE Healthcare) and a 1:1 (Langmuir) binding model.

Techniques: Negative Control, Enzyme-linked Immunosorbent Assay, Clone Assay, Binding Assay, Positive Control, Concentration Assay, Software, Injection

Journal: bioRxiv

Article Title: Lipoproteome screening of the Lyme disease agent identifies novel inhibitors of antibody-mediated complement killing

doi: 10.1101/2021.09.23.461563

Figure Lengend Snippet: A) Extracts from untreated (“-“) or pronase-treated (“+”) 1×10 7 strain B31-e2 spirochetes that ectopically produce the indicated surface lipoproteins were separated by SDS-PAGE and transferred to PVDF membranes. The filters were probed with purified C1 complex (top), C1r enzyme (middle) or C1r proenzyme (bottom), and bound probe revealed by anti-C1r antibody, followed by HRP-conjugated anti-mouse antibody. Shown is a representative of 3 experiments. B) Filters prepared identically to panel A were probed with purified C1 complex (top), C1s enzyme (middle) or C1s proenzyme (bottom), and bound probe revealed by anti-C1s antibody, followed by HRP-conjugated anti-mouse antibody. Shown is a representative of 3 experiments. C and D) Biosensors immobilized with GST-ErpB (top) or GST-ErpQ (bottom) were tested by SPR for binding to the indicated concentrations of the enzyme or proenzyme forms of C1r (C) or C1s (D) Injection series were each performed in triplicate. For both panels C) and D), steady-state affinity fits were determined by T200 Biacore Evaluation software and K D values are reported in .

Article Snippet: Kinetic analyses were performed on each sensorgram series using the Biacore T200 Evaluation Software 3.1 (GE Healthcare) and a 1:1 (Langmuir) binding model.

Techniques: SDS Page, Purification, Binding Assay, Injection, Software