Hrp labelled goat anti mouse antibody

IPTG (isopropyl-β-D-thiogalactopyranoside) and DTT were purchased from Sangon Biotech. Ni-nitrilotriacetic acid (Ni-NTA) was purchased from QIAGEN. Human plasminogen, ϵ-aminocaproic acid (ϵ-ACA), Creatinine Assay Kit, glass beads, urokinase-type plasminogen activator, anidulafungin, fluconazole, and DMEM medium were purchased from Sigma-Aldrich. Human Endothelial Serum Free Medium, HuMEC Basal Serum-Free Medium and Blood Urea Nitrogen Detection Kit were purchased from Thermo Fisher Scientific. Mouse nonspecific IgG2a was obtained from Invivogen. Cy3-labelled secondary antibody was purchased from Invitrogen. Chromogenic substrate D-Val-Leu-Lys-pNA·2HCl was purchased from Innovative Research. The LDH Cytotoxicity Assay Kit was obtained from Beyotime. PrimeScript ^TM RT Reagent Kit and the PrimeSTAR® Max DNA Polymerase were obtained from TaKaRa Bio. Rabbit anti-plasminogen antibody was obtained from Acris Antibodies. Anti-actin monoclonal antibody, and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG were obtained from Abcam. HRP-labelled goat anti-mouse antibody was purchased from Dingguochangsheng Biotechnology. pET-21a (+) was purchased from Novagen.

Recombinant C. albicans or non-albicans Candida spp. Eno1, indicated domains or mutants of C. albicans Eno1 were coated in 96-wells plates (Thermo Scientific, 442404) (0.25 μg/well) overnight and blocked with PBS containing 5% BSA. The plates were then subjected to a series dilution of anti-Eno1 antibody and incubated overnight. After incubation with an HRP-labelled goat anti-mouse antibody (Dingguochangsheng Biotechnology) for 1 h, TMB substrate was added and incubated for 15 min incubation, and then 1 M H2SO4 were added to stop the reaction. Finally, the absorbance of reaction mixture was measured at 450 nm using a multi-mode microplate reader. 96-well microplate coated with Bovine Serum Albumin (BSA) was applied as parallel control in the analysis of plasminogen binding to recombinant C. albicans proteins. The test data of parallel control was deducted to eliminate the non-specific binding of plasminogen or antibodies to the microplate.