Hrp conjugated anti mouse igg1

CpG 1018 was custom-synthesized by Trilink Biotechnologies (San Diego, CA, USA). AddaVax was obtained from InvivoGen (vac–adx–10, San Diego, CA, USA). Reagents used in molecular cloning, such as Phusion DNA polymerase and restriction enzymes, were purchased from New England Biolabs (NEB, Ipswich, MA, USA). Fluorescence-conjugated antibodies used in immunostaining and flow cytometry were obtained from BioLegend (San Diego, CA, USA). TMB substrate was purchased from Thermo Fisher Scientific (34028, Waltham, MA, USA). Horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG secondary antibody was purchased from Cytiva (NA931, Marlborough, MA, USA). HRP-conjugated anti-mouse IgG1 was purchased from Invitrogen (046120, Waltham, MA, USA). HRP-conjugated anti-mouse IgG2c was purchased from Bethyl Laboratories (A90–136P, Montgomery, TX, USA).

rSmLEV1.3 was coated onto Immunlon 4HBX 96 well plates at a concentration of 6.25 μg/ml as before. Experimental mouse sera were used to probe rSmLEV1.3 at a dilution 1:100 in wash buffer (PBS containing 0.05% Tween-20) with 1% bovine serum albumin (BSA). HRP-conjugated anti-mouse IgG₁, anti-mouse IgG_2b (Invitrogen) and anti-mouse IgE (ThermoFisher) were diluted 1:1,000 in wash buffer with 1% BSA. As a negative control, the secondary antibody alone was included on every plate. Development was achieved with 2,2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS) substrate solution (Sigma Aldrich); the reaction was stopped after 15 min with 1% sodium dodecyl sulphate (SDS) and the optical density (OD) read at 405 nm, using a Polarstar Omega Plate reader (BMG Labtech, Offenburg, Germany). Statistical analysis of unmatched groups (vaccinated vs controls) was conducted using a Student’s t-test post-hoc analysis (least significant difference). Longitudinal statistical analysis of the same individuals (i.e., pre-, and post-vaccination) was analysed using a Wilcoxon signed-rank test.