The inhibitory capacity of borrelial proteins on the CP, LP or the AP was analysed by a microtiter-based approach as described previously59 (link). Briefly, microtiter plates were coated with either human IgM (30 ng/ml) (Merck, Darmstadt, Germany) for the CP, mannan (1 µg/ml) (Merck, Darmstadt, Germany) for the LP or LPS (100 ng/ml) (Hycult Biotech, Beutelsbach, Germany), for the AP at 4 °C overnight. Following blocking, NHS (1% for the CP, 2% for the LP, and 15% for the AP) pre-incubated with His6-tagged proteins (10 µg each or increasing concentrations thereof) were added to initiate complement activation. Formation of the MAC was detected by using an anti-C5b-9 antibody (1:500) (Quidel, Athens, USA) and antigen–antibody complexes were visualized by applying HRP-conjugated anti-mouse immunoglobulins (1:1000). The reactions were developed by adding o-phenylenediamine (Merck, Darmstadt, Germany) and measuring the absorbance at 490 nm.
Anti c5b 9 antibody
Manufactured by Quidel
Sourced in United States
The Anti-C5b-9 antibody is a laboratory reagent designed to detect the presence of the C5b-9 complex, also known as the membrane attack complex (MAC). The C5b-9 complex is a key component of the complement system, which plays a crucial role in the immune response. The Anti-C5b-9 antibody can be used to analyze and quantify the levels of this complex in various biological samples, providing researchers and scientists with a tool for studying the complement system and its involvement in different physiological and pathological processes.
Automatically generated - may contain errors
Lab products found in correlation
Human igm, by Merck Group (2 mentions)
O phenylenediamine, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Hycult Biotech (1 mentions)
Mannan, by Merck Group (1 mentions)
Mouse anti his antiserum, by GE Healthcare (1 mentions)
Trypsin, by Merck Group (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Factor h, by Complement Technology (1 mentions)
Horseradish peroxidase hrp conjugated immunoglobulins, by Agilent Technologies (1 mentions)
Urokinase plasminogen activator, by Merck Group (1 mentions)
Human glu plasminogen, by Prolytix (1 mentions)
Factor b, by Complement Technology (1 mentions)
4 protocols using anti c5b 9 antibody
1
Complement Inhibition by Borrelial Proteins
2
Serological Analyses of Anti-Borrelia Antibodies
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Non-immune human serum (NHS) collected from healthy blood donors was initially tested for the presence of anti-Borrelia IgM and IgG antibodies as previously described (29 (link)). Only sera considered to be negative were combined to form a serum pool. All complement components were purchased from Complement Technology (Tyler, TX, USA). Polyclonal anti-FH and anti-C3 antisera were obtained from Merck Biosciences (Bad Soden, Germany) and the neoepitope-specific monoclonal anti-C5b-9 antibody was purchased from Quidel (San Diego, CA, USA). Purification of FHL-1, FH fragments containing different CCP domains, and the anti-CCP1-4 antiserum has been described previously (39 (link)). The monoclonal antibody L41 1C11 was used to detect the borrelial FlaB protein (40 (link)). Proteinase K and trypsin were purchased from Merck (Darmstadt, Germany). For the detection of His-tagged proteins, a mouse anti-His antiserum was used (GE Healthcare, Munich, Germany), and all horseradish peroxidase-conjugated immunoglobulins were purchased from Dako (Hamburg, Germany).
3
Plasminogen Activation and Complement Regulation
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Human serum (NHS) was collected from healthy blood donors as described previously50 (link). Human glu-plasminogen was purchased from Haematologic Technologies (Essex Junction, VT, USA) and urokinase plasminogen activator (uPA) (Merck, Darmstadt, Germany) were used for the activation of plasminogen to plasmin. The chromogenic substrate S-2251 (D-Val-Leu-Lys p-nitroanilide dihydrochloride) were from Sigma-Aldrich (Steinheim, Germany). Factor H, Factor B, C3b, and C5 were purchased from Complement Technology (Tyler, TX, USA). Polyclonal anti-plasminogen antibody was purchased from Acris Antibodies (Herford, Germany), and the monoclonal anti-plasminogen antibody (clone 10-V-1) was from Calbiochem, Merck, Darmstadt, Germany). The polyclonal anti-FH and anti-C3 antibody were obtained from Merck Biosciences (Bad Soden, Germany) and the polyclonal anti-C5, anti-Factor B antibody as well as the neoepitope-specific monoclonal anti-C5b-9 antibody was from Quidel (San Diego, CA, USA). The mouse anti-His antiserum was obtained from Novagen (Merck Darmstadt, Germany) and Qiagen (Hilden, Germany) and the horseradish peroxidase (HRP)-conjugated immunoglobulins were purchased from Dako (Hamburg, Germany).
4
Quantifying Complement Inhibition by Orthologs
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The inhibitory capacity of CihC/FbpC orthologs on the classical (CP) and alternative pathway (AP) was assessed by employing an ELISA-based approach as described previously (26 (link), 27 (link)). Microtiter plates were coated with either human IgM (30 ng/ml) (Merck, Darmstadt, Germany) for the CP and Zymosan A (10 µg/µl) (Merck, Darmstadt, Germany) for the AP at 4°C overnight. Following three wash steps with TBS containing 0.05% Triton X-100 (TBS-T), wells were blocked with PBS containing 0.05% Tween20 and 1% BSA for 1 h at RT. NHS (1% for the CP and 15% for the AP) was pre-incubated with increasing concentrations of His-tagged proteins for 15 min at 37°C before added to the wells to initiate complement activation. After washing with TBS-T, the anti-C5b-9 antibody (1:500) (Quidel, Athens, OH; USA) was added to determine assembly of the pore-forming membrane attack complex. Following incubation for 1 h at RT, wells were washed thoroughly with TBS-T and incubated with HRP-conjugated anti-mouse IgG (1:1000) at RT for 1 h. After adding o-phenylenediamine, the absorbance values were measured at 490 nm.
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