Validation of a multi-analyte panel with cell-bound complement activation products for systemic lupus erythematosus.

BACKGROUND: We describe the analytical validation of an assay panel intended to assist clinicians with the diagnosis of systemic lupus erythematosus (SLE). The multi-analyte panel includes quantitative assessment of complement activation and measurement of autoantibodies. METHODS: The levels of the complement split product C4d bound to erythrocytes (EC4d) and B-lymphocytes (BC4d) (expressed as mean fluorescence intensity [MFI]) are measured by quantitative flow cytometry, while autoantibodies (inclusive of antinuclear and anti-double stranded DNA antibodies) are determined by immunoassays. Results of the multi-analyte panel are reported as positive or negative based on a 2-tiered index score. Post-phlebotomy stability of EC4d and BC4d in EDTA-anticoagulated blood is determined using specimens collected from patients with SLE and normal donors. Three-level C4 coated positive beads are run daily as controls. Analytical validity is reported using intra-day and inter-day coefficient of variation (CV). RESULTS: EC4d and BC4d are stable for 2days at ambient temperature and for 4days at 4°C post-phlebotomy. Median intra-day and inter-day CV range from 2.9% to 7.8% (n=30) and 7.3% to 12.4% (n=66), respectively. The 2-tiered index score is reproducible over 4 consecutive daysupon storage of blood at 4°C. A total of 2,888 three-level quality control data were collected from 6 flow cytometers with an overall failure rate below 3%. Median EC4d level is 6 net MFI (Interquartile [IQ] range 4-9 net MFI) and median BC4d is 18 net MFI (IQ range 13-27 net MFI) among 86,852 specimens submitted for testing. The incidence of 2-tiered positive test results is 13.4%. CONCLUSION: We have established the analytical validity of a multi-analyte assay panel for SLE.

Identification of complement receptor type 1-related proteins on primate erythrocytes.

The purpose of this study was to characterize the structure and function of the immune adherence receptor (CR1, CD35, C3b/C4b receptor) of primates. Western blotting, immunoprecipitation, ELISA, and affinity chromatography with homologous C3b and C4b were utilized. The major cross-reactive E membrane protein of ten species of primates tested was lower in m.w. than was human CR1 and fell into two size groups of 55 to 75 and 130 to 165 kDa. There was 10- to 100-fold more CR1 per primate E than human E. Five species also expressed lesser quantities of a protein similar in m.w. (approximately 200 kDa) to human CR1. In contrast to E, the major cross-reactive protein on PBMC was similar in size to human CR1. Four species also expressed lesser amounts of a lower m.w. protein on their PBMC of the same M(r) as that found on their E. Affinity chromatography demonstrated that the approximately 200-kDa form, if present, was recovered with a similar efficiency to that of human CR1. Three patterns of binding, however, were identified among the lower m.w. proteins: 1) C3b > or = C4b; 2) C4b > C3b; and C3b only or predominantly. The fact that these E proteins cross-react with Ab to human CR1, bind homologous C3b and, in most cases, C4b, and for some species represent the only such protein expressed on their E identifies them as immune adherence receptors. The 70-kDa CR1 of the chimpanzee E seems to arise by alternative splicing of the mRNA encoding the 200-kDa protein. These data raise interesting questions relative to the evolution of CR1 in primates and provide a basis for analysis of structure-function relationships among these size forms of CR1.

Separation of different forms of the fourth component of human complement by fast protein liquid chromatography.

Disruption of the thiolester in native C4 yields a 'C4b-like C4' molecule (iC4) that functionally resembles C4b and is therefore probably accompanied by conformational changes in the C4 molecule. In most purified C4 preparations, iC4 and C4b are present to a variable extent. In this study we evaluated the use of fast protein liquid chromatography (FPLC) to resolve and isolate these various forms of C4. C4 was purified from fresh human plasma in a 4-step procedure that included barium citrate adsorption, polyethylene glycol 6000 (PEG) precipitation, Q-Sepharose Fast Flow and mono Q ion exchange chromatography. The final preparation appeared to be homogeneous on SDS-PAGE and under reducing conditions consisted of three bands that corresponded to the intact alpha, beta and gamma chains of C4. In some preparations the alpha' chain of C4b was also observed. On a Mono Q column the purified C4 preparations could be separated into three peaks that by hemolytic assay and SDS-PAGE were characterized as representing native C4, and monomeric and dimeric iC4 (or monomeric and dimeric C4b). Finally, the apparent KA of the various forms of C4 for C4b-binding protein (C4BP) was investigated. The monomeric iC4 and C4b species demonstrated similar C4BP binding affinity with an apparent KA of 5.6-6.4 x 10(8) M-1, whereas their dimeric forms demonstrated a higher affinity for C4BP with an apparent KA: 0.9-2.3 x 10(9) M-1. Binding of native C4 to C4BP was undetectable.

Structural differences between the two human complement C4 isotypes affect the humoral immune response.

An animal model has been used to address the question of the biological importance of the known structural difference between the two isotypes of human C4, i.e., C4A and C4B. Guinea pigs deficient in C4 were reconstituted transiently with either human C4A or C4B protein and immunized with the bacteriophage phi X174. Results from this study showed that C4A-reconstituted animals made a secondary response, i.e., switch from IgM to IgG; whereas the C4B-reconstituted animals did not.

Localization of the covalent C3b-binding site on C4b within the complement classical pathway C5 convertase, C4b2a3b.

C5 convertase of the classical complement pathway is a trimolecular protein complex consisting of C4b, C2a, and C3b. In the complex there is an ester bond between C3b and C4b. We analyzed the C5 convertase formed on erythrocytes and localized the covalent binding site of C3b to a small region on C4b. The covalently linked C4b.C3b complex was purified from a detergent extract of the erythrocytes and digested with lysyl endopeptidase. An Mr 17,000 fragment containing the ester linkage between C4b and C3b was purified and its amino-terminal sequence was examined. Two amino acids were obtained at each cycle and identified with those in the sequences of C3 and C4. The sequence derived from C3 corresponded to the thioester region. The sequence derived from C4 started at Ala-1186. Alkali treatment of the fragment yielded an Mr 7,000 peptide derived from C4, which thus appeared to span the region of C4 from Ala-1186 to Lys-1259. Therefore, the covalent C3b-binding site on C4b is located within a 74-residue region of the primary structure. This finding supports the notion that after cleavage of C3 by the C4b2a complex, the covalent binding of metastable C3b to C4b is a specific reaction to form a trimolecular complex with a defined quaternary structure.