Quantitative data from five preparations of C3 by our newly developed and shortened small scale procedure demonstrates the efficiency of recovery and preparation as well as purity of C3 isolated in this manner. C3 is prepared as described within three days as a fully active, homogeneous protein with recovery of over 70%. We have also shown that C4, C5 and C9 as well are obtainable by this protocol. Goat polyclonal, monospecific antisera to complement components C1s, C1q and FB as well as HSA has been or is being produced. Anti-C1s IgG coupled Sepharose will be used to stabilize purified preparations of C4. Anti-C1-In contaminants IgG (including anti-albumin and alpha-lipoprotein) was used coupled to Sepharose to remove small amounts of these contaminants from C1-In prepared by our recently developed isolation scheme. These contaminants originally identified by radioautographic analysis of 125I labelled C1-In have been effectively removed as evidenced by their absence in readsorbed and radioiodinated C1-In preparations. C2 isolated by a recently developed, rapid, 3 step procedure involving PEG precipitation, DEAE-Sephacel chromatography and functional affinity chromatography on C4b-Sepharose only needs quantitative analysis for completion. Extension of our new procedure for C5a purification from citrated human plasma has allowed for the isolation of C3a as well. The procedure incorporates C3a immunoadsorption of C5a depleted plasma by use of anti-C3a Sepharose. The acid-glycine eluted C3a is further purified by gel filtration on ACA-44 and concentrated by use of carboxymethyl cellulose. Final characterization of C3a prepared by this method including determination of its biological potency is planned.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Intramural Research (Z01)
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