Peroxidase conjugated goat anti human igg

Ten µl aliquots of recombinant FcεRIαproteins (30 µg/ml in PBS) were dotted onto the centers of individual squares made on a nitrocellulose membrane (NC) and air-dried. Unoccupied sites on the NC were blocked with 0.15% casein in PBS (PBSC) for 10 min. After washing, individual patient serum was diluted 1∶4 in PBSC and incubated for 10 min. Bound IgG were detected using peroxidase-conjugated goat anti-human IgG (Zymed). The reaction was developed using a chemiluminescent substrate solution (Applied Biosystem, Bedford, MA, USA) and the signals were recorded by exposure to an X-ray film. Quantitative analysis of the spots was conducted by video densitometer using the software, Kodak Analyzer.

Purified recombinant FcεRIαproteins in 0.1 M sodium carbonate, pH 9.6 were used to coat a 96-well plate (Nunc, Denmark) in duplicate and were incubated overnight at 4°C. After the ELISA, the plate was washed 3 times with PBST and then blocked with 0.15% casein for 2 hours at room temperature. After washing, the serum samples were added at a 1∶100 dilution with PBS to each well, which were then incubated for 2 h at room temperature. After washing, peroxidase-conjugated goat anti-human IgG (Zymed, CA, USA) was added to each well (1∶10,000 dilution), which were then incubated for 1 h at room temperature. The reaction was visualized using 2,2′-azino-bis(3-ethylbenzthiazoline-sulfonic acid (ABTS, Sigma) as substrate for 10 min and read at 415 nm with a Sunrise Absorbance Reader (TECAN, Austria).
A standard curve was generated using the polyclonal human IgG (Octagam, Austria) concentrations in ng/ml and its corresponding absorbance values measured at 415 nm by a logistic regression algorithm. The linear region of the standard curve was used to quantify the anti- FcεRIαantibodies in the serum samples.