ELISA plates were coated with 10μg/mL mannan or 10μg/mL LPS then blocked as described above. WT mouse serum or NHS were diluted in BBS buffer, added to the plate and incubated for 1h at 37°C. Binding of C3b or C4b were detected using either rabbit anti-C3c (Dako) or rabbit anti-C4c (Dako), mouse anti-human C5b-9 (Dako) or rat anti mouse C5b-9, respectively, followed by HRP conjugated goat anti-rabbit IgG, rabbit anti-human or rabbit anti-rat antibodies. To assess complement C3b deposition via the AP, serum samples were diluted in EGTA buffer (4 mM barbital, 145 mM NaCl, 20mM EGTA, 2mM MgCl2, pH 7.4), a buffer that prevents C3b deposition via the CP or LP while leaving the AP unaffected.
Mouse anti human c5b 9
Manufactured by Agilent Technologies
Mouse anti-human C5b-9 is a laboratory reagent used to detect the membrane attack complex (MAC) formed during the complement activation cascade. It specifically recognizes the C5b-9 complex, which is a key component of the terminal pathway of the complement system.
Automatically generated - may contain errors
2 protocols using mouse anti human c5b 9
1
Complement Activation via ELISA
2
Complement Deposition Assay Using Modified IgG
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Maxisorp plates (Nunc) were randomly coated with 10 µg/ml (ca‐)IgG in coating buffer (0·1 M Na2CO3, 0·1 M NaHCO3, pH 9.6) In mixing experiments the final concentration was 10 µg/ml with different ratios between the ca‐IgG and non‐modified IgG, unless indicated otherwise. Plates were blocked with PBS/1% BSA and incubated with 1% normal human serum (NHS, pooled from four healthy donors) or heat‐inactivated NHS as a control (diluted in GVB++; 0·1% gelatin, 5 mM Veronal, 145 mM NaCl, 0·025% NaN3, 0·15 mM CaCl2, 0·5 mM MgCl2, pH 7.3). Complement binding or deposition was analyzed with rabbit anti‐human C1q (Dako; cat. no. A0136), goat anti‐human C4 (Quidel, San Diego, CA, USA; cat. no. A305), rabbit anti‐human C3c (Dako; cat. no. A0062) and mouse anti‐human C5b9 (Dako; cat. no. M0777) with corresponding HRP‐labelled secondary antibodies in PBS/1% BSA/0·05% Tween. Finally, the substrate was added using ABTS and absorbance measured at 415 nm using the Biorad iMark Microplate Absorbance Reader.
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