C3a eia kit

Complement proteins FH (#341274), FB (#341262), C3b (#204860), C3 (#204885), factor I (FI) (#341280), C1q (#204876), goat anti-FB (#341272) and goat anti-FH (#341276) antiserum were obtained from Merck (Darmstadt, Germany; obtained via Merck Life Science Kft., Budapest, Hungary). FD (#A409), properdin (#A412), mouse anti-C5b-9 (#A239), anti-FB (Bb) (#A227), anti-FB (Ba) (#A225) and C3a EIA kit (#A032) were from Quidel (obtained via Biomedica, Budapest, Hungary). Purified human IgG (#I2511), human serum albumin (#A3782), human alpha-1 antitrypsin (#SRP6312), antibodies against C1q (#234390), human IgG (#A6029), IgM (#A0420), IgA (#A0295), IgG1 (clone HP-6001, #I9388), IgG2 (clone HP-6002, #I9513), IgG3 (clone HP-6050, #I7260), IgG4 (clone HP-6025, #I7385), IgGκ (clone KP-53, #K4377), IgGλ (clone HP-6054, #L6522), and avidin-HRP (#A7419) were purchased from Merck. Mouse IgG1 (clone MOPC-21, #BZ-400101) was obtained from BioLegend (San Diego, CA). MBL (#9086-MB) and biotin-conjugated goat anti-MBL (#BAF2307) were from R&D Systems (Minneapolis, MN), HRP-conjugated goat anti-mouse (#P0447), rabbit anti-goat (#P0449) and swine anti-rabbit (#P0217) antibodies were from DAKO (Hamburg, Germany). Goat anti-C3 F(ab’)₂ (#55062) and HRP-conjugated goat anti-C3 F(ab’)₂ (#55237) were from MP Biomedicals (Solon, OH).

Recombinant mouse FHR-B was generated using the pBSV-8His Baculovirus expression vector (40 (link)), expressed in Spodoptera frugiperda (Sf9) cells, and purified by nickel-affinity chromatography (40 (link)). Recombinant murine FH15–20, PTX3, CRP, and biotinylated goat anti-mouse PTX3 antibody were obtained from R&D Systems (Biomedica, Budapest, Hungary). The monoclonal rat anti-mouse FH antibody 5C2 (generated against mouse FH1–5) was previously described (39 (link)).
Purified human FH, C3, C3b, factor B (FB), factor D (FD), properdin [factor P (FP)], C1q, and goat anti-human FB antiserum were obtained from Merck Ltd. (Budapest, Hungary). The C3a EIA kit and the anti-human FH mAb A254 were from Quidel (Biomedica, Budapest, Hungary). Matrigel was from Sigma-Aldrich Ltd. (Budapest, Hungary). Horseradish peroxidase (HRP)-conjugated goat anti-human C3 was from MP Biomedicals (Solon, OH, USA). HRP-conjugated swine anti-rabbit immunoglobulins, rabbit anti-goat immunoglobulins and goat anti-mouse immunoglobulins were from Dako (Hamburg, Germany). The HRP- and FITC-conjugated anti-mouse C3 antibodies were kind gifts of Drs. Anna Erdei and József Prechl (Eötvös Loránd University, Budapest), respectively. Mouse serum was from PAA Laboratories (Pasching, Austria).

The solid phase AP C3 convertase was built up as described by Hourcade et al. in 2002 (37 (link)) with modifications. C3b was immobilized at 5 µg/ml on microplate wells (Maxisorp, NUNC). After washing, 2 µg/ml FB, 4 µg/ml properdin and 0.1 µg/ml FD were added in convertase buffer (DPBS containing 4% BSA, 2 mM NiCl₂, 0.1% Tween-20) for 1 hour at 37°C. Convertase formation was detected with goat anti-FB and HRP-conjugated rabbit anti-goat antibodies. Convertase activity was determined by adding 10 µg/ml C3 for 1 hour at 37°C. The generated C3a was measured by the C3a EIA kit (Quidel). In some assays, patient’s or control IgG was added together with convertase components to analyze their effect on convertase formation. Convertase decay was determined after incubation of the formed convertase with the isolated IgG fractions alone or in the presence of 1 µg/ml FH for 1 hour at 37°C. Remaining convertase was detected with goat anti-FB antiserum.