Goat anti human igg peroxidase conjugate

A total of 10 peptides containing the chosen sequences of SAG1, GRA6, and GRA7 (Figure 1) were commercially synthesized by Invitrogen (Texas, USA) or Biosynthesis (Texas, USA) with 95–99% purity, as demonstrated by mass spectroscopy. The lyophilized peptides were stored at −80°C until use.
For ELISA, Thermo Scientific Immobilizer Amino plates (Nunc Co, Denmark) were sensitized with 100 μL/well of the peptides at 10 μg/mL of 15 mM sodium carbonate buffer, pH 9.6 and incubated at 4°C overnight. Among steps, wells were washed five times with 200 μL/well of 10 mM phosphate buffered, 0.15 M saline, pH 7.2, plus 0.05% Tween 20 (PBS-T). Non-specific binding sites were blocked with 200 μL/well of 0.3% bovine serum albumin in PBS-T for 1 h at 37°C. The serum samples and the goat anti-human IgG-peroxidase conjugate (Sigma-Aldrich Corp., St Louis, MO, USA) were diluted 1:1,000 in PBS-T, and incubated for 2 h at 37°C. A solution of Ortho-phenylendiamine (5 mg) /30% H₂O₂ (4 μL) in 10 mL of 0.1 M sodium citrate/citric acid buffer, was used to develop the reaction, which was stopped by addition of 50 μL/well of 0.1 N sulfuric acid. Absorbance values were obtained in an ELISA reader (Turner Biosystems 9300-010, Sunnyvale, California) at 490 nm and captured with the Modulus TM Microplate Reader program.

SDS-PAGE was run using a 12% Mini-PROTEAN^® TGX™ precast gel (Bio-Rad Laboratories, Inc., Hercules, CA, USA). For immunoblotting, a goat anti-human IgG peroxidase conjugate (Sigma-Aldrich, St. Louis, MO, USA) was used as a detection antibody at a 1:10,000 dilution. TMB Stabilized Substrate (Promega, Madison, WI, USA) was used for color development. Samples were concentrated using Amicon^® Ultra 0.5 mL centrifugal filter units with a 10 kDa membrane (MilliporeSigma, Burlington, MA, USA). Prior to concentration, samples were filtered using a 0.2 μm syringe filter (MilliporeSigma, Burlington, MA, USA).