84 zyxwvutsrqpo B. Teisner, N. E. Petersen, J. Folkersen zyxwvutsrqp and J. Hau Electrophoresis 1984,5,84-87 Barge Teisner' Niels E. Petersen' Jmgen J. Folkersen' Jann Hau2 'Institute of Medical Microbiologyand 2Biomedical Laboratory, University of Odense Crossedimmunoelectrophoretic analysis of split products of the third complement factor (C3d) following zy in zy vivo activation of the complement system Split products of the third complement factor expressing D but not C epitopes (C3d) were analysed by crossed immunoelectrophoresis using the intermediate gel tech- nique. Four molecular forms of C3d were identified and designated 1,2,3 and 4 ac- cording to their electrophoretic migration velocity. Whereas the relative concentra- tion of molecular forms 2 and 3 were increased followingacute in vivo activation and nephritic factor (NeF)-mediated in vitro activation, the relative concentration of mo- lecular form 1 was found to be increased in patients with chronic activation of the complement system. The results obtained by the addition of anti-C3c antibodies to the first-dimensional gel suggestedthat form 4 was a C3d-likemolecule split from the native C3 during electrophoresis. By the addition of anti-albumin antibodies to the first-dimensionalgel it was demonstrated that molecular form 1 of C3d was a com- plex between C3d and albumin. The immunoprecipitation patterns of molecular forms 2 and 3 was unaffected by anti-albumin and anti-C3c antibodies added to the first-dimensionalgel. These data suggestthat molecular forms 2 and 3 of C3d are the primary split products of C3b and these molecules are able to form a stable complex with human albumin. 1 Introduction Activation of the complement system by the classical as well as the alternative pathway results in activation of the third complement factor (C3) and generation of C3 split products. Measurements of C3d, one of the final split products of C3, have been demonstrated to be a useful and reliable indicator of complement activation and superior to C3c measurements due to the shorter half-life ofthe latter component zyxwvut [ 1-41. How- ever, molecules immunochemically identifiable as C3d, i. e. molecules expressing D but not C epitopes,have been found zyxwvu to exhibit molecularheterogeneityfollowing in vitro degradation of C 3 and more recently a similar heterogeneityhas been dem- onstrated following in vivo activation [5-71. These molecular forms of C3d differ with respect to size, charge, lectin affmity and polyethylene glycol precitability [5-81. The aim of the present study was to compare the immunoelectrophoretic precipitation pattern and composition of molecules im- munologically identifiable as C 3d in samples from patients ex- pressing acute and chronic complement activation. Further- more, the precipitation pattern following in vitro activation of normal human serum by serum containing nephritic factor was examined. 2 Materials and methods 2.1 Patients and blood samples Blood samples were obtained from 10 apparently healthy in- dividuals (normal human serum-NHS), from a patient suffer- ing from acute activation ofthe complementsystem caused by an anaphylactoid reaction to transfusion with leucocyte Correspondence: Dr. B. Teisner, Institute of Medical Microbiology, Uni- versity of Odense, J. B. Winslewsvej 19, DK-5000 Odense C, Denmark Abbreviations: C3d: Fragments of the third complement factor expressing D but not C epitopes; C3c: Fragment@) of the third complement factor ex- pressing C but not D epitopes; NHS: Normal human serum; SLE: Systemic lupus erythematosus; RA: Rheumatoid arthritis; NeF: Nephritic factor depleted blood [31, from patients suffering from systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and from a patient with NeF activity causing chronic activation of the C3. Serum was separated within 2 h of collection and stored at -70 zyxw "C until analysis. 2.2 Electroimmunoassays Crossed immunoelectrophoresis with intermediate gel was performed in 1 % w/v agarose (Indubiose A-37, L'Industrie Biologique Franqaise, Clichy, France) in 0.02 M Tris-barbital buffer, pH 8.6. The same buffer was used as electrophore- sis buffer. The first-dimensional electrophoresis (4 or 2 ~1 samples) was performed at 10 V/cm until a Bromophenol Blue-stained albumin marker had migrated 5.0 cm. The first- dimensional gels (7 x 1.5 cm) were transferred to 7 x 8 cm glass plates. The second-dimensionalgels consisted of (see Fig. la): (A) a 1.5 x 7 cm gel containing 10 pl rabbit anti-C3c (Dakopatts, Copenhagen, Denmark, code no. 10-062) per cm2 agarose; (B)a 0.5 x 7 cm neutral spacer gel, and (C) a 4.5 x 7 cm gel containing 0.5 pl rabbit anti-C3d (Dakopatts, code no. A062) per cm2 agarose and 4 % w/v polyethylene glycol 6000. In some experimentsthe first-dimensional gel contained antibodies (rabbit anti-human albumin or anti-C3c, Dako- patts) at a concentration of 75 pl per cm2 agarose. In this series a neutral gel (1.0 x 7 cm) was interposed between the first- dimensional gel and gel A (I x 7 cm) and the antibody con- centration in gel A was increased to 15 @/cm2. 3 Results The analysis of C3d by crossed immunoelectrophoresis is shown in Fig. 1. The precipitation pattern of serum obtained from an apparently healthy donor (NHS) shows relative high concentrations of molecular forms 1 and 4 in the prealbumin and beta-region, respectively. Only trace amounts of molec- ular forms 2 and 3 present in the alpha region was seen in NHS (Fig. la). The analysis of a sample obtained 2 h after an an- 0173-0835/84/0204-0084S02.50/0 0 Verlag Chemie GmbH, D-6940 Weinheirn, 1984