Description
The purified I52-1486 antibody reacts with the mouse C5a protein. Anaphylatoxin C5a is a bioactive cleavage product released from plasma component C5 during complement activation and is involved in mediation of a variety of cellular immune responses, as well as being potent pro-inflammatory agents. The release of this cleavage product is a reliable indicator of in vivo or in vitro complement activation. Clone I52-1486 hybridoma was generated by the fusion of spleen cells from rats immunized with recombinant mouse C5a protein. The I52-1486 antibody is specific for a neoepitope exposed in mouse C5a/C5adesArg and does not cross-react with mouse C5. This capacity makes antibody I52-1486 a preferential capture antibody for direct detection of mouse C5a from plasma or serum samples.
Recommended Assay Procedure: The purified I52-1486 antibody is useful as a capture antibody for a sandwich ELISA that measures mouse C5a protein levels in plasma or serum samples. Purified I52-1486 as capture antibody can be paired with the biotinylated I52-278 anti-mouse C5a (Cat. No. 558028) as the detection antibody, using recombinant mouse C5a protein (Cat. No. 622597) as the standard. Addition of FUT-175 (Futhan, Cat. No. 552035) to plasma samples at the time of sample collection provides additional protection from ex-vivo activation, and therefore ensures more accurate measurements that reflect the circulating levels of complement activation products. The purified I52-1486 antibody should be titrated between 1-4 µg/ml, diluted in carbonate buffer pH=9.5, to determine the optimal ELISA plate-coating concentration. For blocking buffer, PBS containing 10% FCS is recommended (Cat. No. 555213). To obtain linear standard curves, doubling dilutions of recombinant mouse C5a protein ranging from 156 pg to 10 ng/ml are recommended for inclusion in each ELISA plate. For specific methodology and buffer recipes, see Chapter 7: ELISA for specifically measuring the levels of cytokines, chemokines, and inflammatory mediators and their receptors. 2003. Techniques for Immune Function Analysis Application Handbook 1st Edition. BD Biosciences. This ELISA antibody pair shows no cross-reactivity with the following: recombinant mouse C3a, native human, and rat C5a.
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. Store undiluted at 4°C.
1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
3. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
