Hrp conjugated anti human igg

96-well plates were coated with 10 µg/ml conformational mMOG or conformational hMOG in 1xPBS overnight. Diluted samples were added for 2 h. After washing, plate-bound Ab of murine samples and 8.18C5 Ab were detected with horseradish peroxidase (HRP)-conjugated anti-mouse IgG, directed against the Fc part of the bound Ab (1:6000; Sigma-Aldrich) or against the whole molecule (1:5000; Sigma-Aldrich). Anti-MOG Ab in human samples were detected with HRP-conjugated anti-human IgG (1:1000; Sigma-Aldrich). Plates were read at 450 nm wavelength by a Tecan Genios plate reader and analyzed using Magellan6 software or iMark microplate reader and software.

Purified human FH, FB, C3b, factor D, C1q, goat anti-human C1q antibody (Ab) and goat anti-human FH antibody (Ab) were purchased from Merck (Budapest, Hungary). Human serum albumin (HSA), bovine serum albumin (BSA), alpha1-antitrypsin, HRP-conjugated anti-human IgG, HRP-conjugated anti-human IgA, HRP-conjugated anti-human IgM, and monoclonal antibodies (mAbs) specific for IgG1, IgG2, IgG3, IgG4, Ig kappa and Ig lambda were purchased from Sigma-Aldrich (Budapest, Hungary). HRP-conjugated goat anti-mouse Ig and HRP-conjugated rabbit anti-goat Ig were purchased from DakoCytomation (Hamburg, Germany). HRP-conjugated goat anti-human C3 was from MP Biomedicals (Solon, OH). The anti-FH mAb A254 was purchased from Quidel (Biomedica, Budapest, Hungary), and the mAb C18 (40 (link)) was from Alexis Biochemicals (Lörrach, Germany). The anti-FH mAb IXF9 was described earlier (41 (link)).
Codon-optimized sequences of FHR-1, FHR-4B, FH SCRs 1-4, FH SCRs 8-14, FH SCRs 15-20 were synthesized (GenScript, Piscataway, NJ) and cloned into the pBSV-8His baculovirus expression vector, expressed in Spodoptera frugiperda Sf9 cells and purified by nickel affinity chromatography as described previously (42 (link), 43 (link)). FH SCRs 19-20 and mutant 19-20 fragments were expressed in E. coli (44 (link)).

The following IgG detection probes were used for immunoassays: HRP-conjugated Protein A (#95217)/ HRP-conjugated Protein G (#64256837) were purchased from Bio-Rad® (Hercules, CA, USA). HRP-conjugated Protein A (#101023)/HRP-conjugated Protein G (#101223) were purchased from Thermo-Fisher (Waltham, MA USA). HRP-conjugated anti-mouse IgG (#A4416, Sigma), HRP-conjugated anti-cattle IgG (#A5295, Sigma), HRP-conjugated anti-dog IgG (#A6792, Sigma), HRP-conjugated anti-rat IgG (#A9037, Sigma), HRP-conjugated anti-horse (#A6917, Sigma), HRP-conjugated anti-pig IgG (#A5670, Sigma) and HRP-conjugated anti-human IgG (#A170, Sigma) were used in this study. ELISA kits for visceral leishmaniosis (REF: #VET043-1) and equine infectious anemia (REF: #VET037-1) were kindly provided by Bioclin®. Scienco Biotech kindly provided tetramethylbenzidine (ONE STEP-TMB Linear, Scienco Biotech).

The purified recombinant protein was loaded onto 12% SDS-PAGE gels, fractionated and electrotransferred onto nitrocellulose membranes (Hybond ECL; GE Healthcare) on semidry equipment. A solution containing 10% non-fat dry milk in PBS 0.05% Tween 20 (PBS-T) was used to block unspecific sites in the membranes, which were then incubated with hamster serum for 1 h at room temperature. After extensive washing steps, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-hamster IgG (1:5000) in PBS-T 10% non-fat dry milk for 1 h. The transferred recombinant protein was also incubated with HRP-conjugated monoclonal anti-His tag antibodies (1:10,000) (Sigma, St. Louis, MO, USA). For the detection of human antibodies against epitopes contained in the chimeric protein in the leptospirosis serum samples, membranes were incubated with pooled MAT-positive, MAT-negative, or NHS samples (1:1000), and then, with HRP-conjugated anti-human IgG (1:10,000) or anti-human IgM (1:10,000) (Sigma). The negative control protein BSA was also included in these experiments. Detection was revealed using Super Signal West Dura Extended Duration Substrate (Thermo Fisher, Waltham, MA, USA).