Quantification by ELISA of erythrocyte-bound C3 fragments expressing C3d and/or C3c epitopes in patients with factor I deficiency and with autoimmune diseases.

A sensitive ELISA assay for quantifying erythrocyte (E) bound C3 fragments was developed. The assay employs a double-antibody sandwich technique, using polyclonal anti-C3d or anti-C3c antibodies to quantify C3 fragments, expressing C3d and/or C3c epitopes in washed, detergent-solubilized E. The assay detected 50-120 molecules of C3d per E in healthy individuals. Antigens reacting with anti-C3c antibodies were also detected on E from normal individuals, but the density of C3c-epitopes was 0.9-2.4 times lower than that of C3d-epitopes. In 2 patients with congenital factor I deficiency significantly increased density of E-bound C3c- as well as C3d-antigen was observed. Plasma infusion in one of the patients induced a loss of E-bound C3c-antigens, indicating cleavage of E-bound C3b to iC3b and further to C3c and E-bound C3d. Loss of C3c-antigens also occurred following in vitro treatment with normal human serum of E from one of the patients. Two thirds of 22 patients with systemic lupus erythematosus (SLE) and of 18 patients with rheumatoid arthritis had significantly increased density of E-bound C3d, the highest density being 490 C3d molecules/E in an SLE patient. The density of E-bound C3d correlated with the plasma-C3d concentration, indicating that the coating of E with C3d reflects the degree of complement activation.

Measurement of C3 conversion by ELISA estimation of neo-determinants on the C3d moiety.

An ELISA assay estimating neo-determinants on the C3d moiety is described. The C3d neo-determinants were detected by incubating the sample on F(ab')2 anti-C3d-coated plates followed by development with biotin-labelled anti-C3d and enzyme-labelled avidin. The assay was compared with rocket immunoelectrophoresis (IE) and crossed IE. Serum from a factor I deficient patient showed no detectable C3d when analysed by rocket IE, but activation was evident when analysing by crossed IE or ELISA. The results suggest that the neo-determinants detected by ELISA become exposed when C3 is split into C3a and C3b and this interpretation was supported by kinetic analysis of C3 activation by the three methods.

Relationship between levels of rheumatoid factor isotypes and complement component C3 conversion in blood from patients with rheumatoid arthritis.

Conversion of complement component C3 in plasma from rheumatoid arthritis patients was measured by two different methods. One of the methods gives an estimation of C3 conversion by ELISA measurement of neodeterminants present on the C3d moiety; the other method measures C3 split products expressing D, but not C, epitopes by rocket immunoelectrophoresis (RIE) with intermediate anti-C3c gel. Results from 20 RA patients obtained by the two methods did not correlate significantly (R = 0.52, 0.02 less than p less than 0.05). The results were compared to the level of rheumatoid factors (RFs) of IgG, IgM, and IgA class in serum. A significant correlation was found between the concentration of C3d measured by RIE and level of IgG RFs, whereas neither IgM nor IgA RFs showed correlation to complement activation. The results of the ELISA estimation of C3 activation showed no correlation with the RF level.

Human conglutinin-like protein.

The presence in human plasma of a molecule homologous to bovine conglutinin is indicated by the results of biological and immunochemical analysis. The human conglutinin-like protein shows calcium-dependent binding to complement-treated solid phase IgG and immunological cross-reaction with chicken anti-bovine conglutinin. The binding of the human protein to complement-treated IgG was inhibited by N-acetyl-D-glucosamine but not by other sugars. Analysis by SDS-PAGE and Western blotting showed reaction of anti-conglutinin with molecules of similar mobility to the monomer and hexamer of bovine conglutinin.

Increased non-specific binding of heat-treated proteins to plastic surfaces analyzed by ELISA and HPLC-fractionation.

Heat-treatment of proteins from human sera and bovine serum albumin caused an increase in their binding to microplates in a temperature-dependent manner. Quantitative data were obtained for 125I-labelled human IgG, with binding of up to 400 ng of 56 degrees-treated IgG per untreated well. However, only a minor increase in available antibody activity of adsorbed rabbit antibody was found upon pretreatment at 56 degrees. The increased binding to microplates as a result of pre-incubation at raised temperatures was by high pressure liquid gel permeation chromatography demonstrated to be accompanied by polymerization. Inhibition of the direct binding of the proteins to plastic surface was achieved most efficiently by coating with non-interfering proteins followed by non-ionic detergent (Tween 20), which should also be present in the wash buffer.

Semi-automatic analysis of proteins and protein complexes by automated enzyme immunoassay after separation by high-performance gel-permeation chromatography. Size distribution of C3-IgG complexes.

Enzyme immunoassay (ELISA) was applied in the establishment of an immunological method for the characterization of column effluents. Serum samples, containing aggregated immunoglobulin (IgG), were separated by high-performance gel-permeation chromatography on a TSK column and fractions were collected directly in 96-well ELISA microplates. IgG was determined on anti-IgG-coated plates, followed by development with biotin-labelled anti-IgG and enzyme-labelled avidin. Complement factor C3 was determined on anti-C3-coated plates, developed with biotin-labelled anti-C3 and enzyme-labelled avidin. Complexes between IgG and complement factor C3 were determined on anti-IgG-coated plates, developed with biotin labelled anti-C3 antibody and enzyme-labelled avidin. Complex formation between C3 and IgG after incubation of serum with aggregated IgG was demonstrated. The methodology described is generally applicable to the analysis of biological components and is uniquely useful for the analysis of complexes between different components.

Immune complex formation analysed by high-performance size exclusion chromatography (HPLC-SEC) using either 125I-labelled antigen or enzyme-linked immunosorbent assay (ELISA) for detection.

A physical/immunochemical method has been developed for the analysis of soluble immune complexes. Human serum with high amount of antibody against bovine serum albumin (BSA) was incubated at 37 degrees with a wide range of 125I-labelled BSA and separated according to size by high-performance size exclusion chromatography (HPLC-SEC) on a TSK G 6000 PW column. Fractions were collected onto microplates and analysed either by gamma counting or by an enzyme-linked immunosorbent assay (ELISA) technique detecting anti-BSA antibody. The size of the immune complexes were the same when analysed by the two methods. The heaviest immune complexes were seen in moderate antibody excess where the majority of the complexes were eluted in a broad peak around Mr of 7 X 10(6). In large antibody excess the immune complexes eluted at around 3 X 10(6). Around equilibrium two peaks were seen corresponding to 3 X 10(6) and 5 X 10(5). In antigen excess the peak at 3 X 10(6) is diminished and free antigen appears. No changes in the distribution of the complexes were observed when the serum was made 20 mM in EDTA before incubation with BSA.