Journal oflmmunologicalMethods, 133 (1990) 181-190 Elsevier
181
JIM05710
Quantitation of components of the alternative pathway of complement (APC) by enzyme-linked immunosorbent assays Martin Oppermann, Horst Baumgarten *, Elke Brandt, Walter Gottsleben, Christian Kurts and Otto GStze Department of Immunology, University of GOttingen, 3400 GSttingen, F.R.G.
(Received 28 February 1990, revised received 14 May 1990, accepted 18 June 1990)
Sensitive enzyme-linked immunosorbent assays (ELISA) using monoclonal antibodies have been developed to specifically detect components of the alternative pathway of complement in human blood plasma. Normal values of the factor B split products Ba (1.01 + 0.30/~g/ml, mean + SD), Bb (0.65 + 0.23 /~g/ml), of the C3-fragments C3b/iC3b/C3dg (17.9 + 5.7 /~g/ml), native factor B (238 _+48 /~g/ml), factor D (1.05 + 0.27/~g/ml), and factor H (702 + 292 #g/ml) were determined in the EDTA-plasma of healthy probands (n = 55). The simultaneous quantitation of the main cleavage products and of control proteins in the plasma samples permits precise analysis of the activation of the alternative pathway of complement in various disease states. In addition, we describe a method for the specific depletion of factor B prior to fragment-specific assays utilizing monoclonal antibodies conjugated to paramagnetic beads. The latter should permit the quantitation of other complement split products. Key words: Alternative pathway of complement; Complement activation; ELISA; Paramagnetic particle
Introduction
The alternative pathway of complement (APC) represents an important humoral component of
Correspondence to: M. Oppermann, Department of Immunology, Kreuzbergring 57, 3400 GSttingen, F.R.G. * Present address: Boebringer Mannheim Research Center, D-8132 Tutzing, F.R.G. Abbreviations: ABTS, 2,2'-azino-di-(3ethyl-benzthiazolinesulfonate); actC3, activated C3; APC, alternative pathway of complement; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody; PBS, phosphate-buffered saline; PEG, polyethylene glycol; SD, standard deviation; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; VB-Mg2+, veronal buffer containing 5 mM Mg 2+.
natural defense against microbial attack (for review see GStze and Miiller-Eberhard, 1976; Pangburn and Mtiller-Eberhard, 1984). The interaction of the proteins C3, factor B, and factor D results in the formation of the alternative C3- and C5convertases, i.e., C3b,Bb and C3b,Bb,C3b(,). These multicomponent enzymes assemble on the surface of APC activators and are stabilized by properdin (P). The fluid-phase regulator proteins factors H and I strictly control APC activation. The participation of the alternative pathway of complement has been implicated in the pathogenesis of a wide variety of human diseases (Fearon et al., 1975; Perrin et al., 1975; Chenoweth, 1986). Elevated plasma levels of C3 and factor B splitproducts are taken as an indication of an accelerated turnover of the APC in vivo and may provide an estimate of the activity of ongoing
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
182 disease. Numerous assays have been developed for the quantitation of APC components in the past but these often lack specificity and sensitivity. In addition, as plasma levels of the individual components are affected by different rates of synthesis and degradation the evaluation of only a single parameter may not adequately reflect the degree of APC activation in vivo. We have therefore established sensitive enzyme-linked immunosorbent assay (ELISA) procedures for the quantitation of the native precursor protein factor B and its activation products Ba and Bb, the larger C3 fragments C3b/iC3b/C3dg, and the regulatory proteins factor D and factor H. The high specificity of the assays described in this paper is based on the use of monoclonal antibodies (mAb). Moreover, a method for the depletion of the precursor protein factor B by the use of mAb coupled to paramagnetic particles is described and shown to permit the quantitation of other complement protein split products when mAb with specificity for activation-dependent epitopes on complement components are not available.
Materials and methods
Buffers PBS: 9 mM phosphate buffer, pH 7.2, containing 140 mM NaC1. PBS-Tween: PBS containing 0.05% (v/v) Tween 20 (Serva, Heidelberg, F.R.G.). PBS-Tween-EDTA: PBS-Tween containing 20 mM sodium-ethylenediaminetetraacetic acid (EDTA, Serva, Heidelberg, F.R.G.). Coating buffer: 50 mM carbonate, pH 10.6. Blocking buffer: coating buffer containing 1% (w/v) gelatin (Difco, Hamburg, F.R.G.). Substrate buffer: 100 mM sodium-acetate, 50 mM sodium phosphate, pH 4.2.
Purification of complement components The complement proteins C3, factor B, factor D, and factor H were isolated from human serum according to published methods (G6tze and Mfiller-Eberhard, 1971; Tack and Prahl, 1976; Lesavre et al., 1979; Sim and DiScipio, 1982). Activation products of C3 and of factor B were generated by the incubation of 15 mg factor B, 15 mg C3, and 80/zg factor D in 5 ml VB-Mg 2+ for
60 min at 37°C. The complement split products Ba, Bb, and C3b were purified to homogeneity by anion-exchange chromatography on Q-Sepharose fast flow (Pharmacia, Freiburg, F.R.G.) at pH 7.7 followed by gel filtration on Sephadex G-75 superfine (Pharmacia, Freiburg, F.R.G.). 1 mg C3b was degraded by treatment with 100/zg factor H and 50 U factor I (Diamedix, Miami, U.S.A.) in 10 mM phosphate, pH 6.7, (4 h/37°C) to obtain iC3b. C3dg was isolated from human blood that had been activated using 7/~g cobra venom factor per ml (24 h/37°C) following a protocol that had been devised by Vik and Fearon (1985). Protein concentrations were determined using published extinction coefficients (Elc 1% m 280 nm) (Pangburn, 1988) when these were available or by the Folin assay using bovine serum albumin (Miles Lab., Munich, F.R.G.) as the protein standard (Lowry et al., 1951).
Production of monoclonal antibodies Monoclonal antibodies were produced by the immunization of BALB/c-mice with the purified complement components factor B, factor D, factor H, or C3dg and by the fusion of mouse spleen cells with X63-Ag8.653 myeloma cells following standard protocols (Peters et al., 1990). Positive clones were identified using an ELISA in which the isolated complement proteins were either directly bound to the solid phase or were presented by polyclonal rabbit antisera with specificities for the different proteins. In the case of factor B and C3b both human plasma and zymosan-activated serum were screened in addition to the purified immunogens in order not to miss monoclonal antibodies with specificities for activation-dependent epitopes (Mollnes, 1989). Positive clones were propagated as ascitic cells in BALB/c mice and antibodies were purified from the ascitic fluid by ammonium sulfate fractionation at 50% saturation followed by anion-exchange chromatography on TSK DEAE-650 (S) (Merck, Darmstadt, F.R.G.) or Mono Q (Pharmacia, Freiburg, F.R.G.). The specificities of the monoclonal antibodies obtained were analyzed by ELISA and immunoblotting studies.
Preparation of rabbit anti-Bb IgG Rabbits were immunized repeatedly by subcutaneous injections of purified Bb fragment.,
183
emulsified in Freund's adjuvant. Each animal received a total amount of 250 ~g Bb. IgG fractions of the immune sera were obtained by ammonium sulfate fractionation and affinity chromatography using Protein A-Sepharose (Pharmacia, Freiburg, F.R.G.). Biotinylation of antibodies Purified monoclonal antibodies as well as IgG fractions of polyclonal rabbit antisera were dialysed against 0.1 M sodium carbonate buffer, pH 7.8 and the protein concentration adjusted to 1 mg/ml. Biotinylation was performed by adding biotin-e-aminocaproic acid N-hydroxysuccinimide ester (Biotin-X-NHS, Calbiochem, Frankfurt, F.R.G.) to the antibody preparation at a molar ratio of 7 : 1 (biotin : mAb). After incubation at 37°C for 30 min any remaining free biotin-residues were blocked by the addition of glycine at a final concentration of 20 mM for an additional 30 rain followed by dialysis against PBS. CoupBng of monoclonal antibodies to paramagnetic particles The coupling of monoclonal antibodies to paramagnetic particles by carbodiimide was performed as described (Hebell and Gt~tze, 1989) with the following modifications: carboxylterminated paramagnetic particles (Advanced Magnetics, Cambridge, U.S.A.) were first suspended in 10 mM phosphate, pH 6.0, and activated by 1 mg/ml 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC, Bio-Rad, Munich, F.R.G.). After 4 min the pH was raised to pH 8.0 by the addition of an equal volume of monoclonal antibody (2 mg/ml) in 25 mM carbonate, pH 9.0. The efficiency of the coupling procedure was monitored by measuring the protein content in the supernatant. The incubation period was extended for up to 6 h and resulted in coupling efficiencies of 50-90%. The suspension was adjusted to 2 mg antibody per ml and stored at 4°C. Activation of human serum with zymosan Human serum was incubated for up to 6 h at 37°C in the presence of 0.1 mg zymosan/ml (Serva, Heidelberg, F.R.G.). The reaction was stopped by the addition of EDTA (final con-
centration 20 raM) and zymosan particles were removed by centrifugation (1 min/8000 × g). Plasma and serum samples Plasma samples containing 4 mM dipotassium-EDTA were obtained from 55 healthy blood donors by venipuncture. Plasma was separated from the blood cells by centrifugation (20 min/1000 × g) within 3 h and frozen in aliquots at - 7 0 ° C until use. Sera of patients with chronic renal failure were kindly provided by Dr. E. Quentin and were treated similarly. ELISA procedures The concentrations of the antibody preparations employed in the ELISA procedures for the quantitation of APC components are given in Table I. The ELISA for the quantitation of factor D is described in more detail as a prototypic assay for the determination of other complement components. The wells of microtiterplates (Nunc-Immunoplate Maxisorp, Nunc, Wiesbaden, F.R.G.) were filled with 100 /H of the mAb D10/4 (IgG2a/~) with specificity for factor D in coating buffer. After overnight incubation 200/xl of blocking buffer was added for 30 min to minimize non-specific binding during the further incubation steps. After rinsing once with PBS-Tween (250 td/well) 100 t~l sample diluted in PBS-Tween was added for 2 h. After two wash-cycles the second factor D-specific mAb 18/1 (IgG1/x) was applied in biotinylated form in PBS-Tween. An incubation period of one hour was followed by three washcycles and the addition of streptavidin-horseradish peroxidase conjugate (Amersham, Braunschweig, F.R.G.) in PBS-Tween for another hour. Determinations of factor D were performed by the colorimetric analysis of the peroxidase-mediated hydrolysis of 2 mM 2,2'-azino-di-(3-ethyl benzthiazolinesulfonate) (ABTS, Boehringer, Mannheim, F.R.G.) in substrate buffer containing 2.5 mM H202. Calculations were performed by evaluating the increase in absorbance at 410 nm (reference wavelength 490 nm) recorded using a microplate photometer (Dynatech MR600, Dynatech, Denkendorf, F.R.G.) over a 5-10 min reaction period. Factor D concentrations in the samples were calculated from calibration curves
184 TABLE I COMPOSITION AND SENSITIVITYOF ELISA PROCEDURES Complement component
1st antibody
2nd antibody a
Peroxidase conjugate
Detectionlimit b (ng/ml)
Intra-assay Inter-assay CV [%] ¢ CV (%) d
Factor B Factor D Factor H Ba Bb ActC3
P21/15 (10/tg/ml) D10/4 (10 ~tg/ml) C18/3 (40/~g/ml) P21/15 (10/tg/ml) M12/5 (10/~g/ml) I3/15 (5/tg/ml)
Anti-Bb(5/~g/ml) * I8/1 (1/tg/ml) L20/3 (2 ~g/ml) M20/6 (1/~g/ml) anti-Bb(10/tg/ml) * anti-C3d(5/tg/ml) *
1/1,000 1/1,000 1/1,000 1/2,1)00 1/250 1/500
40 0.5 4 1 1 40
8 3 4 6 6 5
15 5 11 10 13 11
a Applied in biotinylated form. b The detection limit is defined as the lowest concentration of the respective component that gives an absorption significantly (P < 0.005) different from background values. c The intra-assay coefficient of variation (CV) was calculated from 20 determinations of one plasma sample on the same ELISA plate. d The inter-assay coefficient of variation (CV) was calculated from determinations of one plasma sample in 15 consecutive assays. * Denotes polyclonal rabbit lgG preparations. All other antibodies used were mouse mAb.
obtained by plotting the absorbance values of a purified protein standard on a double log scale. The quantitation of factor B by ELISA followed the same procedure except for the following modifications. A monoclonal antibody P21/15 ( I g G 2 a / x ) with specificity for a common epitope on both factor B and the Ba-fragment of factor B was employed as the first antibody. Detection of bound factor B was achieved using a biotinylated rabbit anti-Bb IgG and streptavidin-peroxidase conjugate. The sample buffer was replaced by PBS-Tween-EDTA to prevent complement activation and consumption of factor B. The ELISA for the quantitation of factor H was based on two monoclonal antibodies (C18/3 and L20/3; IgG l/x) with specificity for factor H that by epitope analysis had been found to recognize different epitopes on factor H. This assay specifically detected native 150 kDa factor H since none of the mAb employed in the assay crossreacted with the truncated forms of factor H that have recently been reported in human serum (Misasi et al., 1989). Activated C3 was quantitated by an assay that used as the first antibody the monoclonal antibody 13/15 ( I g G 1 / r ) with specificity for a neo-antigenic determinant. The epitope recognized by mAb 13/15 is expressed on C3b, iC3b, and C3dg but not on native C3. Samples were diluted in PBS-Tween-EDTA and were directly applied to the wells of microtiter plates. Biotinylated rabbit anti-C3d IgG (Dakopatts,
Hamburg, F.R.G.) was used for the detection of bound C3 fragments. This well characterized commercially available antibody preparation has been used in a number of assays that have been devised for the quantitation of C3 fragments, usually necessitating the elimination of plasma-C3 by precipitation prior to the test (Peakman et al., 1987). As none of the monoclonal antibodies that were obtained by immunization with factor B exclusively recognized the activation products Ba or Bb the samples had to be depleted of factor B prior to the quantitation of Ba or Bb by ELISA. This was accomplished in the case of quantitation of the Ba fragment by incubating 100/~1 of plasma that had been diluted 1/200 in PBS-Tween-EDTA with the same volume of paramagnetic beads to which the anti-B/Bb mAb M 1 3 / 1 2 ( I g G 1 / x ) had been coupled. Similarly, particles that had been conjugated with the anti-B/Ba mAb P21/15 were used for the depletion of factor B from plasma prior to the quantitation of the Bb fragment. The optimum amount of antibody-conjugate was determined for each batch of paramagnetic particles and varied between 0.1-1.0 mg m A b / m l sample. The mixture was incubated in the wells of microtiter plates for 1 h with vigorous agitation. The suspension was then sedimented in a magnetic field by applying a samarium-cobalt magnet (Advanced Magnetics, Cambridge, U.S.A.). Factor Bdepleted plasma was taken from the supernatant and applied to the wells of ELISA plates that had
185
been coated with either the Ba-specific mAb P21/15 or the Bb-specific mAb M13/12. The detection of bound factor B-fragments was achieved using either biotinylated mAb M 2 0 / 6 ( I g G 1 / x ) that binds to an epitope on Ba distinct from the P21/15 determinant or of a biotinylated IgG-fraction that had been prepared from a polyclonal rabbit anti-Bb antiserum. Hemolytic assay for the quantitation of factor D A protocol devised by Lesavre et al. (1979) based on the hemolysis of rabbit erythrocytes by factor D depleted serum was used as a functional assay for the quantitation of factor D. Quantitation of factor B by nephelometry Concentrations of factor B in zymosanactivated sera were quantitated by rate nephelomerry using an ICS instrument and reagents provided by Beckman Instruments, Munich, F.R.G. SDS-PA GE and immunoblotting SDS-PAGE using linear polyacrylamide gradients from 3 to 20% T and immunoblotting were performed as described previously (Schulze and G~Stze, 1986). Molecular weights were determined by comparison with a commercially available calibration kit (Pharmacia, Freiburg, F.R.G.).
MW
[ko]
1
2
3
4
5
6
1
2
3
9467433020.1 14.4-
a
b
Fig. 1. a: Electrophoretic analysis of purified complement components (5/~g protein per lane) under reducing conditions. Staining was performed with CoomassieBrilliant Blue. Lane 1: Factor H. Lane 2: Factor B. Lane 3: Factor D. Lane 4: Bb fragment. Lane 5: Ba fragment. Lane 6: C3dg fragment, b: Immunoblot demonstrating the depletion of factor B from plasma by anti-B/Bb mAb coupled to paramagnetic beads. Factor B and the Ba fragment were visualized by the B/Baspecific mAb P21/15. Bound mAb was detected by a rabbit anti-mouse Ig-peroxidase-conjugate (Dakopatts, Hamburg, F.R.G.) and 4-chloro-l-naphthol (Merck, Darmstadt, F.R.G.) as peroxidase-substrate. Lane 1:500 ng purified factor B and 500 ng purified Ba. Lane 2:1.5 /~1 human plasma that was enriched with 100/~g Ba per ml. Lane 3: plasma treated as in lane 2 after incubation with anti-B/Bb mAb M13/12 coupled to paramagnetic beads.
Results
were depleted of the respective proteins restored their hemolytic activity (not shown).
Characterization of protein standards The complement components factor B, factor D, factor H, and the activation products C3dg, Ba, and Bb that were used as protein standards in the ELISA procedures were > 95% pure as demonstrated by SDS-PAGE and protein staining with Coomassie Brilliant Blue (Fig. la). The molecular weights of the purified proteins (factor B: 93 kDa; factor H: 150 kDa; factor D: 27 kDa; C3dg: 40 kDa; Ba: 33 kDa; Bb: 60 kDa) agreed well with p u b l i s h e d data. In a d d i t i o n to t y p i c a l physicochemical properties the identity of the purified preparations was confirmed by demonstrating their immunochemical reactivity with antisera of known specificity. Moreover, the addition of purified factor D or of factor B to sera that
Specificity and efficiency of factor B depletion by anti-B / Bb conjugated particles The efficiency of paramagnetic affinity supports that had been conjugated with factor B/Bb-specific mAb for the depletion of factor B was evaluated using immunoblotting and ELISA procedures. The specific removal of factor B from plasma was qualitatively demonstrated by SDSPAGE and immunoblotting of a sample that had been incubated with anti-B/Bb conjugates (Fig. lb). The analysis of residual factor B and of the Ba fragments by ELISA in the supernatant of plasma that had been depleted of factor B by various quantities of paramagnetic particles dem. onstrated the efficacy of this method in a quanti tative manner (Fig. 2).
186 2500
100
18
o - 0 : Piasma O - e : Plasma + l~g Ba/ml E
2000
80
~
y 1500
•1
=
60
,,
(~1 1000
40 •
Q.
i
0 I 0
• 500
7
.E
E x
E
o
T / / / 7 " / / / 7 / / / 7 / / / T Y / / / / / / / / / / / / / / / / / / I i r i i i
10'00500 2~0 125 ~2.5 31.3 1~.~ 7.~ 3:9
mAb [pg/ml]
c o n j u g a t e d t o paramagnetie
particles
Fig. 2 Efficiency and specificity of the depletion of factor B by the mAb M13/12 coupled to paramagnetic particles. EDTAplasma was diluted 1/200 with PBS-Tween-EDTA and incubated with increasing amounts of a suspension of the mAbconjugated paramagnetic particles. Residual factor B (A) in the supernatant and the apparent Ba concentrations in plasma (©) are shown. The detection limit of the assay for factor B (4 n g / m l ) is indicated by the hatched area. The determination of Ba remains unaffected by the removal of factor B as shown by the parallel shift of the curve obtained with plasma that had been substituted with 1 ~g B a / m l (e).
Specificity of the ELISA for the quantitation of
activated C3 (C3b / iC3b / C3dg) The ELISA for the quantitation of activated C3 (actC3) relies on a monoclonal antibody 0 3 / 1 5 ) with specificity for a neo-antigenic epitope that is absent from native C3 but is expressed on activated C3, i.e., C3b, iC3b, C3dg, and a 36 kDa fragment (C3d) obtained by trypsin digestion of iC3b (not shown). Purified C3 fragments were used to establish calibration curves for this ELISA (Fig. 3). Since purified C3 fragments were shown to give identical signals on a weight basis the amount of activated C3 in samples was expressed as 'Fg actC3 per ml'.
Correlation of factor D determinations by ELISA and by hemolytic titration Factor D concentrations in the sera of patients with chronic renal failure were determined using the established ELISA and a hemolytic assay in parallel (Fig. 4). Values obtained by both assays were highly significantly correlated (r = 0.948; P < 0.001) thereby demonstrating the specificity of the ELISA. The advantage of a hemolytic assay that specifically detects functionally active factor
14
o-o
C3dg
~-~
icsb
~'-~'
C3b
/f //
12 10
8
6
20
./ 0
o O ',x
16
4 2 0 0.08
0.16 0.31 0,63 1.25
2.5
5
C3 or C3-fragrnents
10
20
[pg/rnl]
Fig. 3. Calibration curves of the ELISA for actC3 obtained with purified C3 fragments. C3b, iC3b, and C3dg are detected with identical sensitivity on a weight basis. Native C3 is not recognized by the assay.
D is more than compensated by the higher sensitivity and better reproducibility of the ELISA procedure. Activation of factor B in zymosan activated serum The kinetics of factor B degradation in zymosan activated human serum was followed using the established ELISA procedures (Fig. 5). The gradual increase in levels of the factor B activation products Ba and Bb was accompanied by a concomitant loss of the intact precursor protein factor B. In contrast, nephelometric determinations of 20 n = 18 r = 0.948
E
a
15
u 113 " 10 •
-
'~ _.1 u.I
5 Q 0
. . . .
0
i .... 5
i .... 10
i .... 15
20
H e m o l y t i c a s s a y [pg F a c t o r D/ml] Fig. 4. Demonstration of a linear relationship between factor D levels in the sera of 18 patients with chronic renal failure as determined by a hemolytic assay and the ELISA.
187
50
300
~. 4o
250
EDTA-plasma. Normal values of APC components in EDTA-plasma were established by evaluating plasma samples from 55 healthy volunteers. These results are shown in Table II.
E
o
.m.
3O 4" I
*
~
150
I
Discussion
20 100 I
mm
'to
5o
o 0
J
i
,
r
~
60
120
180
240
300
Time of incubation
,
b"
o
360
[rain]
Fig. 5. Changes in factor B and its fragments in zymosanactivated serum. H u m a n serum was activated by the addition of 0.1 mg z y m o s a n / m l and incubated at 37°C for the indicated periods. Concentrations of factor B as determined by nephelometry ( o ) or by ELISA (/,) are shown. The generation of Ba (11) and of Bb (O) mirrors the degradation of native factor B.
factor B did not reflect the degree of activation in the samples.
Sensitivity and reproducibifity of ELISA determinations, effects of storage at room temperature, and normal values of APC components in EDTA plasma The detection limits as well as the intra-assay and interassay coefficients of variation of the different ELISA procedures that are described in this paper are given in Table I. Samples stored for up to 24 h at room temperature exhibited levels of the components between 85% and 115% when compared to determinations obtained with fresh
T A B L E II N O R M A L VALUES O F C O M P L E M E N T C O M P O N E N T S IN E D T A - P L A S M A a
Complement component
Mean + SD (/tg/ml)
Range (/~g/ml)
Factor B Factor D Factor H Ba Bb ActC3
238 + 48 1.05 + 0.27 702 -1-292 1.01+ 0.30 0.65 + 0.23 17.9 ± 5.7
125 - 38l 0.461.71 299 -1572 0.441.61 0.311.65 8.833.2
a n = 55. Values were obtained by applying the ELISA procedures described in the materials and methods section
Various techniques have been used for the assessment of alternative pathway activation in the past. Assays that are based on the hemolysis of rabbit erythrocytes by human serum are probably most often used as a measure of the functional integrity of the APC (Cooper et al., 1983). Levels of single complement components are determined either by hemolytic or immunochemical methods. The latter techniques generally do not discriminate between complement components in their activated vs. their native forms. The significance of determining native precursor proteins is limited by the fact that reduced levels may be caused by pronounced complement activation as well as by a decreased rate of synthesis. The wide normal ranges of complement components in human serum is a further disadvantage when attempting to interpret altered values. Complement split products are present in normal plasma in only small amounts and are therefore a more sensitive indicator of APC activation. As the formation of activation products and mechanisms involved in their elimination from plasma both influence the blood levels of complement components the quantitation of only a single activation product may not adequately reflect the degree of APC activation in vivo. In an attempt to delineate APC activation during hemodialysis with cuprophane dialyzer membranes we demonstrated that plasma levels of the different complement split products followed distinct patterns of generation and degradation (Oppermann et al., 1988). To quantitatively describe the APC in various disease states as precisely as possible we established ELISA procedures for the quantitation of the major breakdown products of C3 and of factor B, i.e., 'activated C3' (C3b/iC3b/C3dg), Ba, and Bb. In addition, assays were constructed for the specific determination of the precursor protein factor B and of the regulatory proteins factor D and factor H.
188 The C3 fragments C3b, iC3b, and C3dg are split products that are generated during in vivo activation and degradation of the third complement component (Davis et al., 1984). These fragments express a common neoepitope defined by the mAb I3/15 that is also present on C3d. Native C3 lacks this antigenic determinant and mAb I3/15 differs from C3-neoepitope specific mAb reported by others (Garred et al., 1989) in that it also recognizes C3b. An ELISA for the quantitation of activated C3 (actC3) was established using mAb I3/15 and normal plasma levels as defined by this assay were 17.9 _+ 5.7/~g actC3/ml which correlates well with values reported by Kanayama et al. (1986) and Mollnes and Lachmann (1987). The ELISA presented in this paper combines the advantages of both of the assays previously described in so far as it avoids the use of radiolabelled components and allows the results to be expressed in '~g/ml' rather than in arbitrary units. Monoclonal antibodies with specificities for neoepitopes on complement derived fragments are superior reagents for the quantitation of activation products compared to conventional immunochemical techniques (MoUnes, 1989). Nevertheless, most of the assays for the determination of complement fragments currently used still employ antibodies that react with both the split product and the respective precursor protein. In the past the elimination of interfering precursor proteins prior to the assay has generally been achieved on the basis of different physicochemical properties of the native complement protein and its fragments. Perrin et al. (1975) introduced the precipitation of plasma with 12% polyethylene glycol for the separation of complement factor B and the Ba fragment followed by radial immunodiffusion whereas CoUett et al., (1984) used nephelometry to measure Ba after PEG precipitation. The elimination of factor B by PEG precipitation is, however, neither specific nor complete (Perrin et al., 1975) and therefore may result in erroneous Ba determinations. In contrast, the removal of factor B using B/Bb-specific mAb that are coupled to paramagnetic beads utilizes specific immunologic properties of the complement component factor B and does not influence the quantitation of the Ba fragment. The detection of the Bb fragment has similarly been achieved by employing B/Ba-
specific mAb that were coupled to paramagnetic beads for the removal of factor B. Levels of the Ba and the Bb fragments in normal human plasma as defined by the ELISA procedures described in this paper compare favorably with values reported by Kolb et al. (1989) who employed assays that were based on neoepitope-specific mAb. The efimination of interfering complement components by antibodies that are coupled to paramagnetic beads may become more widely used for the quantitation of protein split products when antibodies with specificities for activation-dependent epitopes are not available. The serine protease factor D occurs in plasma as an active enzyme and is the rate-limiting protease of the APC. It has both the lowest plasma concentration and the lowest molecular weight of all complement components. Assays for the quantitation of factor D therefore need to be highly sensitive and specific. Functional assays have been reported that rely on the hemolysis of guinea-pig erythrocytes (Martin et al., 1976), or of rabbit erythrocytes (Lesavre et al., 1979). Previously factor D has been measured quantitatively by radioimmunoassay (Barnum et al., 1984) and by enzyme amplified electroimmunoassay (Truedsson and Sturfelt, 1983). In comparison to these procedures an ELISA based on two mAb with specificity for factor D, as described in this communication, is presently the most sensitive and specific assay for the quantitation of factor D. The regulatory protein factor H accelerates the decay of the alternative C3-convertase and possesses factor I cofactor activity. The quantitation of factor H by a conventional hemolytic assay employing sheep erythrocytes beating C3b,Bb,P (Weiler et al., 1976) is laborious and requires purified complement proteins. We have established a double mAb sandwich-ELISA that is sensitive and easily performed. Normal plasma levels of factor H as quantitated by this assay amounted to 702 + 292 /~g factor H per ml plasma (range: 299-1572 /~g/ml), thereby confirming earlier determinations (Weiler et al., 1976). It was the aim of this study to provide a methodological foundation for the analysis of APC activation rather than to present data on applications of the established assays to clinical research. The ELISA procedures described in this report are
189 c u r r e n t l y b e i n g e v a l u a t e d in clinical studies w i t h the o b j e c t i v e o f d e l i n e a t i n g t h e r o l e o f A P C a c t i v a t i o n in v a r i o u s disease states.
Acknowledgement T h e a u t h o r s w i s h to t h a n k Dr. E. Q u e n t i n , G/Sttingen, for p r o v i d i n g sera f r o m p a t i e n t s w i t h c h r o n i c r e n a l failure. T h i s w o r k was s u p p o r t e d b y t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t , S F B 236, a n d a f e l l o w s h i p f r o m the D F G ( O p 4 2 / 1 - 2 ) to M.O.
References Barnum, S.R., Niemann, M.A., Kearney, J.F. and Volanakis, J.E. (1984) Quantitation of complement factor D in human serum by a solid-phase radioimmunoassay. J. Immunol. Methods 67, 303-309. Chenoweth, D.E. (1986) Anaphylatoxin formation in extracorporeal circuits. Complement 3, 152-165. Collett, B., Alhaq, A., Abdullah, N.B., Korjtsas, L., Ware, R.J., Dodd, N.J., Alimo, E., Ponte, J. and Vergani, D. (1984) Pathways to complement activation during cardiopulmonary bypass. Br. Med. J. 289, 1251-1254. Cooper, N.R., Nemerow, O.R. and Mayes, J.T. (1983) Methods to detect and quantitate complement activation. Springer Semin. Immunopathol. 6, 195-212. Davis, A.E., Harrison, R.A. and Lachmann, P.J. (1984) Physiologic inactivation of fluid phase C3b: isolation and structural analysis of C3c, C3d, g (alpha2D), and C3g. J. Immunol. 132, 1960-1966. Fearon, D.T., Ruddy, S., Schur, P.H. and McCabe, W.R. (1975) Activation of the properdin pathway of complement in patients with gram-negative bacteremia. New Engl. J. Med. 292, 937-940. Garred, P., Mollnes, T.E. and Kazatchkine, M.D. (1989) Activation-dependent antigenic changes of human C3. Complement Inflamm. 6, 205-218. G/3tze, O. and Mtiller-Eberhard, H.J. (1971) The C3-activator system: an alternate pathway of complement activation. J. Exp. Med. 134, 90s-108s. G/Stze, O. and Mtiller-Eberhard, H.J. (1976) The alternative pathway of complement activation. Adv. Immunol. 24, 1-35. Hebell, T. and G/Stze, O. (1989) The isolation of B lymphocytes from human peripheral blood using antibodies coupled to paramagnetic particles and rosetting techniques. J. Immunol. Methods 123, 283-291. Kanayama, Y., Kurata, Y., McMillan, R., Tamerius, J.D., Negoro, N. and Curd, J.O. (1986) Direct quantitation of activated C3 in human plasma with monoclonal anti-iC3bC3d-neoantigen. J. Immunol. Methods 88, 33-36.
Kolb, W.P., Morrow, P.R. and Tamerius, J.D. (1989) Ba and Bb fragments of factor B activation: fragment production, biological activities, neoepitope expression and quantitation in clinical samples. Complement Inflamm. 6, 175-204. Lesavre, P.H., Hugli, T.E., Esser, A.F. and Mi~ller-Eberhard, H.J. (1979) The alternative pathway C3/C5 convertase: chemical basis of factor B activation. J. Immunol. 123, 529-534. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275. Martin, A., Lachmann, P.J., Halbwachs, L. and Hobart, M.J. (1976) Haemolytic diffusion plate assays for factors B and D of the alternative pathway of complement activation. Immunochemistry 13, 317-324. Misasi, R., Huemer, H.P., Schwaeble, W., S/51der, E., Larcher, C. and Dierich, M.P. (1989) Human complement factor H: an additional gene product of 43 kDa isolated from human plasma shows cofactor activity for the cleavage of the third component of complement. Eur. J. Immunol. 19, 17651768, Mollnes, T.E. (1989) Antigenic changes of complement components: relevance for the analysis of complement activation. Complement Inflamm. 6, 134-141. Mollnes, T.E. and Lachmann, P.J. (1987) Activation of the third component of complement (C3) detected by a monoclonal anti-C3'g' neoantigen antibody in a one-step enzyme immunoassay. J, lmmunol. Methods 101,201-207. Oppermann, M., Haubitz, M., Quentin, E. and G/3tze, O. (1988) Complement activation in patients with renal failure as detected through the quantitation of fragments of the complement proteins C3, C5, and factor B. Klin. Wochenschr. 66, 857-864. Pangburn, M.K. (1988) Alternative pathway of complement. Methods Enzymol. 162, 639-653. Pangburn, M.K. and Miiller-Eberhard, H.J. (1984) The alternative pathway of complement. Springer Semin. Immunopathol. 7, 163-192. Peakman, M., Lobo-Yeo, A., Senaldi, G., Nilsson, M., Tee, D.E. and Vergani, D. (1987) Quantification of C3d in biological fluids by an enzyme-linked immunosorbent assay. J. Immunol. Methods 104, 51-56. Perrin, L.H., Lambert, P.H. and Miescher, P.A. (1975) Complement breakdown products in plasma from patients with systemic lupus erythematosus and patients with membranoproliferative or other glomerulonephritis. J. Clin. Invest. 56, 165-176. Peters, J.H., Schulze, M. and Baumgarten, H. (Eds.) (1990) Monoklonale AntikOrper. Herstellung und Charakterisierung. Springer, Heidelberg. Schulze, M. and G~3tze, O. (1986) A sensitive ELISA for the quantitation of human C5a in blood plasma using a monoclonal antibody. Complement 3, 25-39. Sire, R.B. and DiScipio, R.G. (1982) Purification and structural studies on the complement system control protein beta~H (factor H). Biochem. J. 205, 285-293. Tack, B.F. and Prahl, J.W. (1976) Third component of human complement: purification from plasma and physicochemical characterization. Biochemistry 15, 4513-4521.
190 Truedsson, L. and Sturfelt, O. (1983) Human factor D of the alternative pathway: purification and quantitation by enzyme amplified electroimmunoassay. J. Immunol. Methods 63, 207-214. Vik, D.P. and Fearon, D.T. (1985) Neutrophils express a receptor for iC3b, C3dg and C3d that is distinct from CR1, CR2 and CR3. J. Imrnunol. 134, 2571-2579.
Weiler, J.M., Daha, M.R., Austen, K.F. and Fearon, D.T. (1976) Control of the amplification convertase of complement by the plasma protein BIH. Proc. Natl. Acad. Sci. U.S.A. 73, 3268-3272.
181
JIM05710
Quantitation of components of the alternative pathway of complement (APC) by enzyme-linked immunosorbent assays Martin Oppermann, Horst Baumgarten *, Elke Brandt, Walter Gottsleben, Christian Kurts and Otto GStze Department of Immunology, University of GOttingen, 3400 GSttingen, F.R.G.
(Received 28 February 1990, revised received 14 May 1990, accepted 18 June 1990)
Sensitive enzyme-linked immunosorbent assays (ELISA) using monoclonal antibodies have been developed to specifically detect components of the alternative pathway of complement in human blood plasma. Normal values of the factor B split products Ba (1.01 + 0.30/~g/ml, mean + SD), Bb (0.65 + 0.23 /~g/ml), of the C3-fragments C3b/iC3b/C3dg (17.9 + 5.7 /~g/ml), native factor B (238 _+48 /~g/ml), factor D (1.05 + 0.27/~g/ml), and factor H (702 + 292 #g/ml) were determined in the EDTA-plasma of healthy probands (n = 55). The simultaneous quantitation of the main cleavage products and of control proteins in the plasma samples permits precise analysis of the activation of the alternative pathway of complement in various disease states. In addition, we describe a method for the specific depletion of factor B prior to fragment-specific assays utilizing monoclonal antibodies conjugated to paramagnetic beads. The latter should permit the quantitation of other complement split products. Key words: Alternative pathway of complement; Complement activation; ELISA; Paramagnetic particle
Introduction
The alternative pathway of complement (APC) represents an important humoral component of
Correspondence to: M. Oppermann, Department of Immunology, Kreuzbergring 57, 3400 GSttingen, F.R.G. * Present address: Boebringer Mannheim Research Center, D-8132 Tutzing, F.R.G. Abbreviations: ABTS, 2,2'-azino-di-(3ethyl-benzthiazolinesulfonate); actC3, activated C3; APC, alternative pathway of complement; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody; PBS, phosphate-buffered saline; PEG, polyethylene glycol; SD, standard deviation; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; VB-Mg2+, veronal buffer containing 5 mM Mg 2+.
natural defense against microbial attack (for review see GStze and Miiller-Eberhard, 1976; Pangburn and Mtiller-Eberhard, 1984). The interaction of the proteins C3, factor B, and factor D results in the formation of the alternative C3- and C5convertases, i.e., C3b,Bb and C3b,Bb,C3b(,). These multicomponent enzymes assemble on the surface of APC activators and are stabilized by properdin (P). The fluid-phase regulator proteins factors H and I strictly control APC activation. The participation of the alternative pathway of complement has been implicated in the pathogenesis of a wide variety of human diseases (Fearon et al., 1975; Perrin et al., 1975; Chenoweth, 1986). Elevated plasma levels of C3 and factor B splitproducts are taken as an indication of an accelerated turnover of the APC in vivo and may provide an estimate of the activity of ongoing
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
182 disease. Numerous assays have been developed for the quantitation of APC components in the past but these often lack specificity and sensitivity. In addition, as plasma levels of the individual components are affected by different rates of synthesis and degradation the evaluation of only a single parameter may not adequately reflect the degree of APC activation in vivo. We have therefore established sensitive enzyme-linked immunosorbent assay (ELISA) procedures for the quantitation of the native precursor protein factor B and its activation products Ba and Bb, the larger C3 fragments C3b/iC3b/C3dg, and the regulatory proteins factor D and factor H. The high specificity of the assays described in this paper is based on the use of monoclonal antibodies (mAb). Moreover, a method for the depletion of the precursor protein factor B by the use of mAb coupled to paramagnetic particles is described and shown to permit the quantitation of other complement protein split products when mAb with specificity for activation-dependent epitopes on complement components are not available.
Materials and methods
Buffers PBS: 9 mM phosphate buffer, pH 7.2, containing 140 mM NaC1. PBS-Tween: PBS containing 0.05% (v/v) Tween 20 (Serva, Heidelberg, F.R.G.). PBS-Tween-EDTA: PBS-Tween containing 20 mM sodium-ethylenediaminetetraacetic acid (EDTA, Serva, Heidelberg, F.R.G.). Coating buffer: 50 mM carbonate, pH 10.6. Blocking buffer: coating buffer containing 1% (w/v) gelatin (Difco, Hamburg, F.R.G.). Substrate buffer: 100 mM sodium-acetate, 50 mM sodium phosphate, pH 4.2.
Purification of complement components The complement proteins C3, factor B, factor D, and factor H were isolated from human serum according to published methods (G6tze and Mfiller-Eberhard, 1971; Tack and Prahl, 1976; Lesavre et al., 1979; Sim and DiScipio, 1982). Activation products of C3 and of factor B were generated by the incubation of 15 mg factor B, 15 mg C3, and 80/zg factor D in 5 ml VB-Mg 2+ for
60 min at 37°C. The complement split products Ba, Bb, and C3b were purified to homogeneity by anion-exchange chromatography on Q-Sepharose fast flow (Pharmacia, Freiburg, F.R.G.) at pH 7.7 followed by gel filtration on Sephadex G-75 superfine (Pharmacia, Freiburg, F.R.G.). 1 mg C3b was degraded by treatment with 100/zg factor H and 50 U factor I (Diamedix, Miami, U.S.A.) in 10 mM phosphate, pH 6.7, (4 h/37°C) to obtain iC3b. C3dg was isolated from human blood that had been activated using 7/~g cobra venom factor per ml (24 h/37°C) following a protocol that had been devised by Vik and Fearon (1985). Protein concentrations were determined using published extinction coefficients (Elc 1% m 280 nm) (Pangburn, 1988) when these were available or by the Folin assay using bovine serum albumin (Miles Lab., Munich, F.R.G.) as the protein standard (Lowry et al., 1951).
Production of monoclonal antibodies Monoclonal antibodies were produced by the immunization of BALB/c-mice with the purified complement components factor B, factor D, factor H, or C3dg and by the fusion of mouse spleen cells with X63-Ag8.653 myeloma cells following standard protocols (Peters et al., 1990). Positive clones were identified using an ELISA in which the isolated complement proteins were either directly bound to the solid phase or were presented by polyclonal rabbit antisera with specificities for the different proteins. In the case of factor B and C3b both human plasma and zymosan-activated serum were screened in addition to the purified immunogens in order not to miss monoclonal antibodies with specificities for activation-dependent epitopes (Mollnes, 1989). Positive clones were propagated as ascitic cells in BALB/c mice and antibodies were purified from the ascitic fluid by ammonium sulfate fractionation at 50% saturation followed by anion-exchange chromatography on TSK DEAE-650 (S) (Merck, Darmstadt, F.R.G.) or Mono Q (Pharmacia, Freiburg, F.R.G.). The specificities of the monoclonal antibodies obtained were analyzed by ELISA and immunoblotting studies.
Preparation of rabbit anti-Bb IgG Rabbits were immunized repeatedly by subcutaneous injections of purified Bb fragment.,
183
emulsified in Freund's adjuvant. Each animal received a total amount of 250 ~g Bb. IgG fractions of the immune sera were obtained by ammonium sulfate fractionation and affinity chromatography using Protein A-Sepharose (Pharmacia, Freiburg, F.R.G.). Biotinylation of antibodies Purified monoclonal antibodies as well as IgG fractions of polyclonal rabbit antisera were dialysed against 0.1 M sodium carbonate buffer, pH 7.8 and the protein concentration adjusted to 1 mg/ml. Biotinylation was performed by adding biotin-e-aminocaproic acid N-hydroxysuccinimide ester (Biotin-X-NHS, Calbiochem, Frankfurt, F.R.G.) to the antibody preparation at a molar ratio of 7 : 1 (biotin : mAb). After incubation at 37°C for 30 min any remaining free biotin-residues were blocked by the addition of glycine at a final concentration of 20 mM for an additional 30 rain followed by dialysis against PBS. CoupBng of monoclonal antibodies to paramagnetic particles The coupling of monoclonal antibodies to paramagnetic particles by carbodiimide was performed as described (Hebell and Gt~tze, 1989) with the following modifications: carboxylterminated paramagnetic particles (Advanced Magnetics, Cambridge, U.S.A.) were first suspended in 10 mM phosphate, pH 6.0, and activated by 1 mg/ml 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC, Bio-Rad, Munich, F.R.G.). After 4 min the pH was raised to pH 8.0 by the addition of an equal volume of monoclonal antibody (2 mg/ml) in 25 mM carbonate, pH 9.0. The efficiency of the coupling procedure was monitored by measuring the protein content in the supernatant. The incubation period was extended for up to 6 h and resulted in coupling efficiencies of 50-90%. The suspension was adjusted to 2 mg antibody per ml and stored at 4°C. Activation of human serum with zymosan Human serum was incubated for up to 6 h at 37°C in the presence of 0.1 mg zymosan/ml (Serva, Heidelberg, F.R.G.). The reaction was stopped by the addition of EDTA (final con-
centration 20 raM) and zymosan particles were removed by centrifugation (1 min/8000 × g). Plasma and serum samples Plasma samples containing 4 mM dipotassium-EDTA were obtained from 55 healthy blood donors by venipuncture. Plasma was separated from the blood cells by centrifugation (20 min/1000 × g) within 3 h and frozen in aliquots at - 7 0 ° C until use. Sera of patients with chronic renal failure were kindly provided by Dr. E. Quentin and were treated similarly. ELISA procedures The concentrations of the antibody preparations employed in the ELISA procedures for the quantitation of APC components are given in Table I. The ELISA for the quantitation of factor D is described in more detail as a prototypic assay for the determination of other complement components. The wells of microtiterplates (Nunc-Immunoplate Maxisorp, Nunc, Wiesbaden, F.R.G.) were filled with 100 /H of the mAb D10/4 (IgG2a/~) with specificity for factor D in coating buffer. After overnight incubation 200/xl of blocking buffer was added for 30 min to minimize non-specific binding during the further incubation steps. After rinsing once with PBS-Tween (250 td/well) 100 t~l sample diluted in PBS-Tween was added for 2 h. After two wash-cycles the second factor D-specific mAb 18/1 (IgG1/x) was applied in biotinylated form in PBS-Tween. An incubation period of one hour was followed by three washcycles and the addition of streptavidin-horseradish peroxidase conjugate (Amersham, Braunschweig, F.R.G.) in PBS-Tween for another hour. Determinations of factor D were performed by the colorimetric analysis of the peroxidase-mediated hydrolysis of 2 mM 2,2'-azino-di-(3-ethyl benzthiazolinesulfonate) (ABTS, Boehringer, Mannheim, F.R.G.) in substrate buffer containing 2.5 mM H202. Calculations were performed by evaluating the increase in absorbance at 410 nm (reference wavelength 490 nm) recorded using a microplate photometer (Dynatech MR600, Dynatech, Denkendorf, F.R.G.) over a 5-10 min reaction period. Factor D concentrations in the samples were calculated from calibration curves
184 TABLE I COMPOSITION AND SENSITIVITYOF ELISA PROCEDURES Complement component
1st antibody
2nd antibody a
Peroxidase conjugate
Detectionlimit b (ng/ml)
Intra-assay Inter-assay CV [%] ¢ CV (%) d
Factor B Factor D Factor H Ba Bb ActC3
P21/15 (10/tg/ml) D10/4 (10 ~tg/ml) C18/3 (40/~g/ml) P21/15 (10/tg/ml) M12/5 (10/~g/ml) I3/15 (5/tg/ml)
Anti-Bb(5/~g/ml) * I8/1 (1/tg/ml) L20/3 (2 ~g/ml) M20/6 (1/~g/ml) anti-Bb(10/tg/ml) * anti-C3d(5/tg/ml) *
1/1,000 1/1,000 1/1,000 1/2,1)00 1/250 1/500
40 0.5 4 1 1 40
8 3 4 6 6 5
15 5 11 10 13 11
a Applied in biotinylated form. b The detection limit is defined as the lowest concentration of the respective component that gives an absorption significantly (P < 0.005) different from background values. c The intra-assay coefficient of variation (CV) was calculated from 20 determinations of one plasma sample on the same ELISA plate. d The inter-assay coefficient of variation (CV) was calculated from determinations of one plasma sample in 15 consecutive assays. * Denotes polyclonal rabbit lgG preparations. All other antibodies used were mouse mAb.
obtained by plotting the absorbance values of a purified protein standard on a double log scale. The quantitation of factor B by ELISA followed the same procedure except for the following modifications. A monoclonal antibody P21/15 ( I g G 2 a / x ) with specificity for a common epitope on both factor B and the Ba-fragment of factor B was employed as the first antibody. Detection of bound factor B was achieved using a biotinylated rabbit anti-Bb IgG and streptavidin-peroxidase conjugate. The sample buffer was replaced by PBS-Tween-EDTA to prevent complement activation and consumption of factor B. The ELISA for the quantitation of factor H was based on two monoclonal antibodies (C18/3 and L20/3; IgG l/x) with specificity for factor H that by epitope analysis had been found to recognize different epitopes on factor H. This assay specifically detected native 150 kDa factor H since none of the mAb employed in the assay crossreacted with the truncated forms of factor H that have recently been reported in human serum (Misasi et al., 1989). Activated C3 was quantitated by an assay that used as the first antibody the monoclonal antibody 13/15 ( I g G 1 / r ) with specificity for a neo-antigenic determinant. The epitope recognized by mAb 13/15 is expressed on C3b, iC3b, and C3dg but not on native C3. Samples were diluted in PBS-Tween-EDTA and were directly applied to the wells of microtiter plates. Biotinylated rabbit anti-C3d IgG (Dakopatts,
Hamburg, F.R.G.) was used for the detection of bound C3 fragments. This well characterized commercially available antibody preparation has been used in a number of assays that have been devised for the quantitation of C3 fragments, usually necessitating the elimination of plasma-C3 by precipitation prior to the test (Peakman et al., 1987). As none of the monoclonal antibodies that were obtained by immunization with factor B exclusively recognized the activation products Ba or Bb the samples had to be depleted of factor B prior to the quantitation of Ba or Bb by ELISA. This was accomplished in the case of quantitation of the Ba fragment by incubating 100/~1 of plasma that had been diluted 1/200 in PBS-Tween-EDTA with the same volume of paramagnetic beads to which the anti-B/Bb mAb M 1 3 / 1 2 ( I g G 1 / x ) had been coupled. Similarly, particles that had been conjugated with the anti-B/Ba mAb P21/15 were used for the depletion of factor B from plasma prior to the quantitation of the Bb fragment. The optimum amount of antibody-conjugate was determined for each batch of paramagnetic particles and varied between 0.1-1.0 mg m A b / m l sample. The mixture was incubated in the wells of microtiter plates for 1 h with vigorous agitation. The suspension was then sedimented in a magnetic field by applying a samarium-cobalt magnet (Advanced Magnetics, Cambridge, U.S.A.). Factor Bdepleted plasma was taken from the supernatant and applied to the wells of ELISA plates that had
185
been coated with either the Ba-specific mAb P21/15 or the Bb-specific mAb M13/12. The detection of bound factor B-fragments was achieved using either biotinylated mAb M 2 0 / 6 ( I g G 1 / x ) that binds to an epitope on Ba distinct from the P21/15 determinant or of a biotinylated IgG-fraction that had been prepared from a polyclonal rabbit anti-Bb antiserum. Hemolytic assay for the quantitation of factor D A protocol devised by Lesavre et al. (1979) based on the hemolysis of rabbit erythrocytes by factor D depleted serum was used as a functional assay for the quantitation of factor D. Quantitation of factor B by nephelometry Concentrations of factor B in zymosanactivated sera were quantitated by rate nephelomerry using an ICS instrument and reagents provided by Beckman Instruments, Munich, F.R.G. SDS-PA GE and immunoblotting SDS-PAGE using linear polyacrylamide gradients from 3 to 20% T and immunoblotting were performed as described previously (Schulze and G~Stze, 1986). Molecular weights were determined by comparison with a commercially available calibration kit (Pharmacia, Freiburg, F.R.G.).
MW
[ko]
1
2
3
4
5
6
1
2
3
9467433020.1 14.4-
a
b
Fig. 1. a: Electrophoretic analysis of purified complement components (5/~g protein per lane) under reducing conditions. Staining was performed with CoomassieBrilliant Blue. Lane 1: Factor H. Lane 2: Factor B. Lane 3: Factor D. Lane 4: Bb fragment. Lane 5: Ba fragment. Lane 6: C3dg fragment, b: Immunoblot demonstrating the depletion of factor B from plasma by anti-B/Bb mAb coupled to paramagnetic beads. Factor B and the Ba fragment were visualized by the B/Baspecific mAb P21/15. Bound mAb was detected by a rabbit anti-mouse Ig-peroxidase-conjugate (Dakopatts, Hamburg, F.R.G.) and 4-chloro-l-naphthol (Merck, Darmstadt, F.R.G.) as peroxidase-substrate. Lane 1:500 ng purified factor B and 500 ng purified Ba. Lane 2:1.5 /~1 human plasma that was enriched with 100/~g Ba per ml. Lane 3: plasma treated as in lane 2 after incubation with anti-B/Bb mAb M13/12 coupled to paramagnetic beads.
Results
were depleted of the respective proteins restored their hemolytic activity (not shown).
Characterization of protein standards The complement components factor B, factor D, factor H, and the activation products C3dg, Ba, and Bb that were used as protein standards in the ELISA procedures were > 95% pure as demonstrated by SDS-PAGE and protein staining with Coomassie Brilliant Blue (Fig. la). The molecular weights of the purified proteins (factor B: 93 kDa; factor H: 150 kDa; factor D: 27 kDa; C3dg: 40 kDa; Ba: 33 kDa; Bb: 60 kDa) agreed well with p u b l i s h e d data. In a d d i t i o n to t y p i c a l physicochemical properties the identity of the purified preparations was confirmed by demonstrating their immunochemical reactivity with antisera of known specificity. Moreover, the addition of purified factor D or of factor B to sera that
Specificity and efficiency of factor B depletion by anti-B / Bb conjugated particles The efficiency of paramagnetic affinity supports that had been conjugated with factor B/Bb-specific mAb for the depletion of factor B was evaluated using immunoblotting and ELISA procedures. The specific removal of factor B from plasma was qualitatively demonstrated by SDSPAGE and immunoblotting of a sample that had been incubated with anti-B/Bb conjugates (Fig. lb). The analysis of residual factor B and of the Ba fragments by ELISA in the supernatant of plasma that had been depleted of factor B by various quantities of paramagnetic particles dem. onstrated the efficacy of this method in a quanti tative manner (Fig. 2).
186 2500
100
18
o - 0 : Piasma O - e : Plasma + l~g Ba/ml E
2000
80
~
y 1500
•1
=
60
,,
(~1 1000
40 •
Q.
i
0 I 0
• 500
7
.E
E x
E
o
T / / / 7 " / / / 7 / / / 7 / / / T Y / / / / / / / / / / / / / / / / / / I i r i i i
10'00500 2~0 125 ~2.5 31.3 1~.~ 7.~ 3:9
mAb [pg/ml]
c o n j u g a t e d t o paramagnetie
particles
Fig. 2 Efficiency and specificity of the depletion of factor B by the mAb M13/12 coupled to paramagnetic particles. EDTAplasma was diluted 1/200 with PBS-Tween-EDTA and incubated with increasing amounts of a suspension of the mAbconjugated paramagnetic particles. Residual factor B (A) in the supernatant and the apparent Ba concentrations in plasma (©) are shown. The detection limit of the assay for factor B (4 n g / m l ) is indicated by the hatched area. The determination of Ba remains unaffected by the removal of factor B as shown by the parallel shift of the curve obtained with plasma that had been substituted with 1 ~g B a / m l (e).
Specificity of the ELISA for the quantitation of
activated C3 (C3b / iC3b / C3dg) The ELISA for the quantitation of activated C3 (actC3) relies on a monoclonal antibody 0 3 / 1 5 ) with specificity for a neo-antigenic epitope that is absent from native C3 but is expressed on activated C3, i.e., C3b, iC3b, C3dg, and a 36 kDa fragment (C3d) obtained by trypsin digestion of iC3b (not shown). Purified C3 fragments were used to establish calibration curves for this ELISA (Fig. 3). Since purified C3 fragments were shown to give identical signals on a weight basis the amount of activated C3 in samples was expressed as 'Fg actC3 per ml'.
Correlation of factor D determinations by ELISA and by hemolytic titration Factor D concentrations in the sera of patients with chronic renal failure were determined using the established ELISA and a hemolytic assay in parallel (Fig. 4). Values obtained by both assays were highly significantly correlated (r = 0.948; P < 0.001) thereby demonstrating the specificity of the ELISA. The advantage of a hemolytic assay that specifically detects functionally active factor
14
o-o
C3dg
~-~
icsb
~'-~'
C3b
/f //
12 10
8
6
20
./ 0
o O ',x
16
4 2 0 0.08
0.16 0.31 0,63 1.25
2.5
5
C3 or C3-fragrnents
10
20
[pg/rnl]
Fig. 3. Calibration curves of the ELISA for actC3 obtained with purified C3 fragments. C3b, iC3b, and C3dg are detected with identical sensitivity on a weight basis. Native C3 is not recognized by the assay.
D is more than compensated by the higher sensitivity and better reproducibility of the ELISA procedure. Activation of factor B in zymosan activated serum The kinetics of factor B degradation in zymosan activated human serum was followed using the established ELISA procedures (Fig. 5). The gradual increase in levels of the factor B activation products Ba and Bb was accompanied by a concomitant loss of the intact precursor protein factor B. In contrast, nephelometric determinations of 20 n = 18 r = 0.948
E
a
15
u 113 " 10 •
-
'~ _.1 u.I
5 Q 0
. . . .
0
i .... 5
i .... 10
i .... 15
20
H e m o l y t i c a s s a y [pg F a c t o r D/ml] Fig. 4. Demonstration of a linear relationship between factor D levels in the sera of 18 patients with chronic renal failure as determined by a hemolytic assay and the ELISA.
187
50
300
~. 4o
250
EDTA-plasma. Normal values of APC components in EDTA-plasma were established by evaluating plasma samples from 55 healthy volunteers. These results are shown in Table II.
E
o
.m.
3O 4" I
*
~
150
I
Discussion
20 100 I
mm
'to
5o
o 0
J
i
,
r
~
60
120
180
240
300
Time of incubation
,
b"
o
360
[rain]
Fig. 5. Changes in factor B and its fragments in zymosanactivated serum. H u m a n serum was activated by the addition of 0.1 mg z y m o s a n / m l and incubated at 37°C for the indicated periods. Concentrations of factor B as determined by nephelometry ( o ) or by ELISA (/,) are shown. The generation of Ba (11) and of Bb (O) mirrors the degradation of native factor B.
factor B did not reflect the degree of activation in the samples.
Sensitivity and reproducibifity of ELISA determinations, effects of storage at room temperature, and normal values of APC components in EDTA plasma The detection limits as well as the intra-assay and interassay coefficients of variation of the different ELISA procedures that are described in this paper are given in Table I. Samples stored for up to 24 h at room temperature exhibited levels of the components between 85% and 115% when compared to determinations obtained with fresh
T A B L E II N O R M A L VALUES O F C O M P L E M E N T C O M P O N E N T S IN E D T A - P L A S M A a
Complement component
Mean + SD (/tg/ml)
Range (/~g/ml)
Factor B Factor D Factor H Ba Bb ActC3
238 + 48 1.05 + 0.27 702 -1-292 1.01+ 0.30 0.65 + 0.23 17.9 ± 5.7
125 - 38l 0.461.71 299 -1572 0.441.61 0.311.65 8.833.2
a n = 55. Values were obtained by applying the ELISA procedures described in the materials and methods section
Various techniques have been used for the assessment of alternative pathway activation in the past. Assays that are based on the hemolysis of rabbit erythrocytes by human serum are probably most often used as a measure of the functional integrity of the APC (Cooper et al., 1983). Levels of single complement components are determined either by hemolytic or immunochemical methods. The latter techniques generally do not discriminate between complement components in their activated vs. their native forms. The significance of determining native precursor proteins is limited by the fact that reduced levels may be caused by pronounced complement activation as well as by a decreased rate of synthesis. The wide normal ranges of complement components in human serum is a further disadvantage when attempting to interpret altered values. Complement split products are present in normal plasma in only small amounts and are therefore a more sensitive indicator of APC activation. As the formation of activation products and mechanisms involved in their elimination from plasma both influence the blood levels of complement components the quantitation of only a single activation product may not adequately reflect the degree of APC activation in vivo. In an attempt to delineate APC activation during hemodialysis with cuprophane dialyzer membranes we demonstrated that plasma levels of the different complement split products followed distinct patterns of generation and degradation (Oppermann et al., 1988). To quantitatively describe the APC in various disease states as precisely as possible we established ELISA procedures for the quantitation of the major breakdown products of C3 and of factor B, i.e., 'activated C3' (C3b/iC3b/C3dg), Ba, and Bb. In addition, assays were constructed for the specific determination of the precursor protein factor B and of the regulatory proteins factor D and factor H.
188 The C3 fragments C3b, iC3b, and C3dg are split products that are generated during in vivo activation and degradation of the third complement component (Davis et al., 1984). These fragments express a common neoepitope defined by the mAb I3/15 that is also present on C3d. Native C3 lacks this antigenic determinant and mAb I3/15 differs from C3-neoepitope specific mAb reported by others (Garred et al., 1989) in that it also recognizes C3b. An ELISA for the quantitation of activated C3 (actC3) was established using mAb I3/15 and normal plasma levels as defined by this assay were 17.9 _+ 5.7/~g actC3/ml which correlates well with values reported by Kanayama et al. (1986) and Mollnes and Lachmann (1987). The ELISA presented in this paper combines the advantages of both of the assays previously described in so far as it avoids the use of radiolabelled components and allows the results to be expressed in '~g/ml' rather than in arbitrary units. Monoclonal antibodies with specificities for neoepitopes on complement derived fragments are superior reagents for the quantitation of activation products compared to conventional immunochemical techniques (MoUnes, 1989). Nevertheless, most of the assays for the determination of complement fragments currently used still employ antibodies that react with both the split product and the respective precursor protein. In the past the elimination of interfering precursor proteins prior to the assay has generally been achieved on the basis of different physicochemical properties of the native complement protein and its fragments. Perrin et al. (1975) introduced the precipitation of plasma with 12% polyethylene glycol for the separation of complement factor B and the Ba fragment followed by radial immunodiffusion whereas CoUett et al., (1984) used nephelometry to measure Ba after PEG precipitation. The elimination of factor B by PEG precipitation is, however, neither specific nor complete (Perrin et al., 1975) and therefore may result in erroneous Ba determinations. In contrast, the removal of factor B using B/Bb-specific mAb that are coupled to paramagnetic beads utilizes specific immunologic properties of the complement component factor B and does not influence the quantitation of the Ba fragment. The detection of the Bb fragment has similarly been achieved by employing B/Ba-
specific mAb that were coupled to paramagnetic beads for the removal of factor B. Levels of the Ba and the Bb fragments in normal human plasma as defined by the ELISA procedures described in this paper compare favorably with values reported by Kolb et al. (1989) who employed assays that were based on neoepitope-specific mAb. The efimination of interfering complement components by antibodies that are coupled to paramagnetic beads may become more widely used for the quantitation of protein split products when antibodies with specificities for activation-dependent epitopes are not available. The serine protease factor D occurs in plasma as an active enzyme and is the rate-limiting protease of the APC. It has both the lowest plasma concentration and the lowest molecular weight of all complement components. Assays for the quantitation of factor D therefore need to be highly sensitive and specific. Functional assays have been reported that rely on the hemolysis of guinea-pig erythrocytes (Martin et al., 1976), or of rabbit erythrocytes (Lesavre et al., 1979). Previously factor D has been measured quantitatively by radioimmunoassay (Barnum et al., 1984) and by enzyme amplified electroimmunoassay (Truedsson and Sturfelt, 1983). In comparison to these procedures an ELISA based on two mAb with specificity for factor D, as described in this communication, is presently the most sensitive and specific assay for the quantitation of factor D. The regulatory protein factor H accelerates the decay of the alternative C3-convertase and possesses factor I cofactor activity. The quantitation of factor H by a conventional hemolytic assay employing sheep erythrocytes beating C3b,Bb,P (Weiler et al., 1976) is laborious and requires purified complement proteins. We have established a double mAb sandwich-ELISA that is sensitive and easily performed. Normal plasma levels of factor H as quantitated by this assay amounted to 702 + 292 /~g factor H per ml plasma (range: 299-1572 /~g/ml), thereby confirming earlier determinations (Weiler et al., 1976). It was the aim of this study to provide a methodological foundation for the analysis of APC activation rather than to present data on applications of the established assays to clinical research. The ELISA procedures described in this report are
189 c u r r e n t l y b e i n g e v a l u a t e d in clinical studies w i t h the o b j e c t i v e o f d e l i n e a t i n g t h e r o l e o f A P C a c t i v a t i o n in v a r i o u s disease states.
Acknowledgement T h e a u t h o r s w i s h to t h a n k Dr. E. Q u e n t i n , G/Sttingen, for p r o v i d i n g sera f r o m p a t i e n t s w i t h c h r o n i c r e n a l failure. T h i s w o r k was s u p p o r t e d b y t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t , S F B 236, a n d a f e l l o w s h i p f r o m the D F G ( O p 4 2 / 1 - 2 ) to M.O.
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