Horseradish peroxidase conjugated goat anti rabbit igg antibody

We performed ELISAs in order to determine the presence or otherwise of antifibrinogen IgA antibody and the IgA-bound fibrinogen in the patient's plasma. Normal plasma with an equivalent IgA concentration to the patient's plasma was used as the control. For the detection of anti-fibrinogen IgA antibody, recombinant fibrinogen (1.2 µg/well) was coated as the solid phase, and diluted plasmas were reacted. After washing, rabbit anti-human IgA (α-chain) antibody was reacted as a capture antibody and followed horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Medical and Biological Laboratories Ltd) as the second antibody. For the detection of IgA-bound fibrinogen, goat anti-human fibrinogen antibody (Cosmobio, Co., Ltd, Tokyo, Japan) was coated as a solid phase, and diluted plasma with fibrinogen equivalent to 500 ng/mL was reacted. After washing, the IgA-bound fibrinogen was reacted as described above. Finally, a TMB Microwell Peroxidase Substrate System (KPL, Gaithersburg, MD) was used as a substrate, and the absorbance at 450 nm was measured using an ELISA reader (BioRad, Tokyo, Japan). The reactions were performed in triplicate, and the statistical significances of differences among groups were determined using a oneway ANOVA with Dunnett post-hoc test. A difference was considered significant when the p value was lower than 0.05.

Patient plasma fibrinogen was analyzed by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) in reducing conditions (10% polyacrylamide gel) and immunoblotting using a rabbit-anti-human fibrinogen antibody (DAKO, Carpinteria, CA, USA) or rabbit anti-human Bβ-chain antibody (Chemicon International, Temecula, CA, USA) [9] (link). Reacting species were visualized with a horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Medical and Biological Laboratories Ltd., Nagoya, Japan) and enhanced chemiluminescence detection reagents (Amersham Pharmacia Biotech, Buckinghamshire, UK). Blot were then exposed using a ChemiDoc XRS+ System (Bio-Rad Laboratories, Hercules, CA, USA).