Reference typing report for complement component C3.

A comparison of five C3 variant samples has been performed by conventional high-voltage gel electrophoresis in three laboratories (Palermo, Berlin and Mainz). Local designation was shown within SD = +/-0.75 mm migration distance in the Mainz laboratory. Methodological modifications by laboratories were not accounted for (cooling temperature, relative mobilities between runs). In parallel, all reference samples were also sequenced after exon-specific amplification. As a result, two variants with identical final designations and two variants with different mobilities were shown to conform at the molecular basis exhibiting an amino acid exchange that causes the corresponding change in relative mobility as compared to normal C3F and C3S.

A modified competitive inhibition radioimmunoassay for the detection of C3a. Use of 125I-C3 instead of 125I-C3a.

Levels of C3a in plasma are currently measured by a competitive inhibition radioimmunoassay (RIA) in which 125I-C3a is used as a tracer. In this paper, we describe a modification of this RIA: 125I-C3 instead of 125I-C3a is used. The lower limit of detection of this modified RIA is 6 ng of C3a per ml of plasma (i.e. 0.66 nmol/l). This RIA, performed with polyclonal anti-C3a antibodies coupled to a solid phase, appeared to be 30 times more sensitive compared with an RIA in which a monoclonal antibody against C3a is used. In vitro activation of the complement system in serum by aggregated IgG, zymosan, and cobra venom factor resulted in the generation of significant amounts of C3a. Assessment of the C3a levels by the modified RIA in serial plasma samples from patients who underwent cardiopulmonary bypass, yielded results very similar to those described in the literature for the established C3a-RIA. Thus, the modified C3a-RIA offers a convenient alternative for the detection of C3a in plasma samples.

Quantification in enzyme-linked immunosorbent assay of a C3 neoepitope expressed on activated human complement factor C3.

A sensitive double antibody enzyme-linked immunosorbent assay (ELISA) for quantification of C3 activation products in human plasma, synovial fluid, and cerebrospinal fluid is described. The monoclonal antibody MoAb bH6, which is specific for a C3 neoepitope expressed on C3b, iC3b, and C3c, was used as capture antibody. Detection antibody was a polyclonal rabbit anti-human C3c followed by development with a peroxidase-conjugated anti-rabbit Ig antiserum. The activity in normal human EDTA plasma was 1.5% of that in zymosan-activated serum (ZAS). The interassay and intra-assay coefficients of variation were 15% and 3%, respectively. The lower detection limit was 0.0005% of the ZAS standard. Reference range (1.1-2.1% of ZAS) was obtained by measuring EDTA plasma from 40 healthy blood donors. A positive correlation rs = +0.92; P less than 0.0002) was found between the present assay and an already established C3'g' activation ELISA, when samples from 16 patients were examined in both assays simultaneously. The present assay and an assay detecting the terminal complement complex showed virtually identical activation patterns in consecutively drawn samples from a patient undergoing extracorporal circulation.

Quantification of the C3d split products of human complement by a sensitive enzyme-linked immunosorbent assay.

A double-antibody biotin-avidin enzyme-linked immunosorbent assay (ELISA) for quantification of the C3d split products is described. Polyethylene glycol 6000 was used to precipitate large C3 molecules, and the C3d-containing supernatant was used in the assay. C3d was measured in ethylenediaminetetraacetic acid plasma from 40 healthy blood donors, and the normal range was defined. Twenty-two patients were tested, and 12 of these had increased levels of C3d. No correlation was observed between total C3 and C3d in these patients. There was a close correlation between C3d measured by this method and by the double-decker rocket immunoelectrophoresis method. The C3d ELISA method is very sensitive, easy to perform, and time-saving and economical compared with most C3d methods already described. A procedure to define the lower detection limit and examine the reliability of an ELISA method in general is discussed.

The determination of the complement components C1q, C4 and C3 in serum and cerebrospinal fluid by radioimmunoassay.

Non-competitive 2-site radioimmunoassays (RIA) for the determination of the complement proteins C1q, C4 and C3 in cerebrospinal fluid (CSF) are described. The quantitative results of the RIAs were the same as those obtained by other assay methods: radial immunodiffusion and turbidimetry and, in the case of C4, the haemolytic assay. The concentrations of the complement proteins in paired CSF and serum samples from a group of 60 patients were measured, as well as those of albumin and IgG. The ratios (concentration in CSF)/(concentration in serum) of the complement proteins correlated poorly with that of albumin. In contrast, the ratio of IgG was significantly correlated with that of albumin. The ratios of the complement proteins were higher than might be expected on the basis of their molecular masses. This suggests that these proteins may be synthesized within the normal central nervous system.

Measurement of fragments of the third component of human complement on erythrocytes by a new immunochemical method.

This paper describes a new antiglobulin consumption method to quantitate the fragments of the third component of human complement (C3) on red blood cell (RBC) membranes. Zymosan-bound C3, which can be stored frozen at -80 degrees C for years, was used as a standard in these tests. Using anti-C3c antibody, zymosan-bound C3 could be calibrated against soluble converted C3 (beta 1A), but not against soluble, native C3 (beta 1C). Calibration with several commercial serum standards yielded virtually identical values. Approximately 79.8 +/- 28.2 C3d molecules (mean +/- 1 SD, n = 50) were detected on normal, freshly collected RBC by this method, whereas no C3c fragments were noted. EC43, prepared by dilution of blood samples with low ionic strength solution, had between 650 and 3,100 C3 molecules/RBC when measured with anti-C3c and between 1,140 and 6,500 C3 molecules when measured with anti-C3d. These data indicated that part of the C3b molecules on EC43 had cleaved to C3d. EC43 are reported to have up to 200,000 C3 molecules when measured by other techniques. To resolve this discrepancy, EC43 were prepared by dialysis of blood samples against low ionic strength solution. About 97.5% of C3 remained in plasma after dialysis supporting the results of our tests. The new assay is an accurate and sensitive method of quantitating C3 fragments bound to RBC in vivo and in vitro.

Reproducible in vitro preparation of intermediate C3d coated red blood cells.

As a standardizing reagent and/or as a positive control for the anti-C3d reactivity of antiglobulin reagents, test red blood cells (RBC) reproducibly coated with "C3d only" (i.e. lacking other complement components and immunoglobulins) are essential. We have prepared RBC coated by intermediate amounts of C3d. Two approaches to varying the amount of C3d bound to RBC were studied: a) variation in Mg++ concentration and b) dilution of donor plasma. The amount of C3d bound to RBC was assessed both by agglutination reactions with serial dilutions of a standard anti-C3d serum and by quantitation of bound anti-C3d with 125I-labeled anti-antiglobulin serum. Marked individual donor differences were encountered in response to varying Mg++ concentration and to dilution of donor plasma; no single set of conditions could be employed to produce a desired intermediate C3d-coated RBC from all donors. Examples of variations to be expected with both manipulations are illustrated, along with studies of conditions under which the standard deviation for bound C3d on intermediate C3d-coated RBC made from 5-donor pools was less than 10%.