Anti rabbit igg ap conjugate

N. gonorrhoeae 1291 and clinical isolates grown under oxygen-limited conditions or N. gonorrhoeae 1291 aniA::kan cells grown under standard conditions were adjusted to an OD_600nm of ~2.5 in PBS for SDS-PAGE using NuPAGE 4–12% Bis-Tris polyacrylamide gels (Life Technologies). Pre-immune or post-immune sera were diluted appropriately in 5% skim milk powder in TBS-T as the primary antibody. Secondary antibodies used were anti-mouse IgG AP-conjugate or anti-rabbit IgG AP-conjugate (Sigma-Aldrich) at a 1: 10 000 in 5% skim milk powder in TBS-T. Antibody binding was detected with SigmaFAST NBT/BCIP tablets (Sigma-Aldrich) according to the manufacturer’s instructions.

pET-BL21 and AniA-BL21 cells were inoculated into LB broth supplemented with ampicillin at a concentration of 100μg ml^-1 from a mid-log phase culture (OD_600nm = ~ 0.4) and grown at 37°C for 1 hour before protein expression was induced with 0.1mM IPTG at 22°C for 3 hours. Cells were harvested, washed twice in PBS and adjusted to give a final OD_600nm of 1. 100μl of cell suspension was combined with 0μg or 2μg of trypsin (Mass Spectrometry Grade, New England Biolabs) and incubated at 37°C for 60 mins. Samples of cell suspensions at t0 and at 60 mins (t60) were taken in triplicate for the determination of CFUs/ml. This was conducted to ensure that cell lysis was not occurring over the course of the assay, which would be indicated by a reduction in CFUs/ml. Differences between CFUs/ml at t0 and at t60 for each sample were assessed for statistical significance with Prism 5 software (GraphPad Software, Inc.) using a two-tailed unpaired Student’s t-test. P values <0.05 were considered significant. Trypsin digest reactions were terminated by the addition of SDS-PAGE sample buffer and incubation at 95°C for 5 mins. The susceptibility of recombinant AniA to proteolytic digestion with trypsin was assessed by western blot analysis with anti-AniA polyclonal rabbit serum as the primary antibody (1: 20 000) and anti-rabbit IgG AP-conjugate (Sigma-Aldrich) as the secondary antibody (1: 10 000).

The following antibodies were used: rabbit polyclonal anti-ACE2 (#SAB2100025), anti-mouse polyclonal Immunoglobulins (IgG, IgA, IgM) alkaline phosphatase (AP)-conjugate and anti-rabbit IgG AP-conjugate were purchased from Sigma-Aldrich (St. Louis, MO, USA); rabbit polyclonal anti-TMPRSS2 (#PA5-116054), rabbit monoclonal anti-CD147 (#MA5-29060), anti-mouse IgG Alexa Fluor^® 488-conjugate, Alexa Fluor^® 568 phalloidin (#A12380), and V5 Tag Monoclonal Antibody Alexa Fluor 488-conjugate (#37-7500-A488) were bought from Invitrogen; SARS-CoV/SARS-CoV-2 (COVID-19) spike antibody (#GTX632604) was purchased from GeneTex, while anti-SARS/SARS-CoV-2 Coronavirus Nucleocapsid Antibody (#MA1-7403) was from Thermo Fisher Scientific. SARS-CoV-2 Spike protein S1 (aa 1-674) Recombinant antigen was bought from Merck Millipore (Darmstadt, Germania). All other chemicals were purchased from Sigma-Aldrich.