and 2MRC Cellular Immunology Unit, Sir William Dunn. School of ..... We thank Dr. A. F. Williams for helpful discussion and Mrs. 3. Hancox and Miss B. Moffatt ...
II[9
Bioscience Reports 3, 1119-1131 (1983) Printed in Great Britain
Monoclonal antibodies against the complement control protein Factor H (81 H) E. SIMI, M. S. PALMER t, M. PUKLAVEC2, and R. B. SIM2 IMRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OXl 3QU, U.K.; and 2MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OXI 3RE, U.K. (Received 2 November 1983)
Two mouse monoclonal antibodies against the human complement c o n t r o l protein, Factor H (6tH) , are described. The antibodies are both IgG - Y I - subclass and are directed against different epitopes on the human Factor H molecule. One of the antibodies, MRC OX 24, increases the cofactor activity of Factor H in Factor I-mediated cleavage of soluble C3b. The second antibody, MRC OX 23, which has no effect alone, reduces the increase in cofactor activity observed in the presence of the first antibody. However, MRC OX 2# inhibits the binding of 1251-1abelled Factor H to surface-bound C3b (EAC3b). Again MRC OX 23 alone does not have any effect but decreases the inhibition in t251-Jabelled Factor H binding to EAC3b observed with MRC OX 2#. These studies show clearly that the interaction of Factor H with soluble C3b is different to its interaction with surface-bound C3b. In an indirect i m m u n o p r e c i p i t a t i o n system using these monoclonal antibodies, single-chain molecules of ]50 000 mol.wt. are specifically precipitated from human serum and also from the sera of other primates - rhesus monkey, cynomolgus monkey, and African green monkey. There was no precipitation from sera of cow, pig~ sheep, chick, or rabbit. Using a radioimmunoassay with radiolabelled monoclonal MRC OX 23, the concentration of Factor H in human plasma was determined.
When the complement cascade is activated, via either the classical or alternative pathways, C3 is cleaved to produce C3b. This proteolytic event is the central reaction of complement activation in that C3b, once generated, can participate in its own production via the alternative pathway C3 convertase, C3bBb. This step is controlled in Note: The nomenclature of the complement-system proteins used here is in accordance with the recommendations of the World Health Organization (1981). 01983
The Biochemical
Society
1120
SIM
ET
AL.
vivo by p r o t e o l y t i c c l e a v a g e ol C3b to iC3b, a p r o d u c t which can no l o n g e r f o r m a C3 c o n v e r t a s e (for review see Reid, 1983). The controlling e n z y m e which cleaves C3b is F a c t o r I (C3b I n a c t i v a t o r ) . It is essential t h a t C3b is a s s o c i a t e d with F a c t o r H b e f o r e C3b can be c l e a v e d by F a c t o r I (Whaley & Ruddy, 1976). F a c t o r H is thus an essential c o n t r o l protein of the c o m p l e m e n t s y s t e m . The c o m p l e m e n t r e c e p t o r for C3b, C R I , shares this c o f a c t o r function of F a c t o r H ( F e a r o n , 1979; E Sim & RB Sim, 1983) although the two proteins are not superficially similar in s t r u c t u r e . In order to investigate f u r t h e r the p r o p e r t i e s of F a c t o r H to serve as a m e a n s of c o m p a r i s o n with CRI, we have raised monoclonal a n t i b o d i e s a g a i n s t F a c t o r H. We r e p o r t on their e f f e c t s on the r e g u l a t o r y a c t i v i t y of F a c t o r H and also use these antibodies for q u a n t i f i c a t i o n of F a c t o r H in p r i m a t e sera. Materials and Methods Monoclonal antibodies
F a c t o r H was purified as described previously (RB Sim & DiScipio, 1982). Mice (Balb/c) were immunized with F a c t o r H the and sera t e s t e d for the p r e s e n c e of a n t i - F a c t o r - H antibodies according to the m e t h o d of Hsiung et al. (1982). The mouse with the highest serum t i t r e was r e - i n j e c t e d with 20 IJg of F a c t o r H and spleen ceils w e r e fused 4 days later with NS-O m y e l o m a cells as described previously ( G a l f r e et al., 1977). The c u l t u r e s u p e r n a t a n t s were t e s t e d for the p r e s e n c e of anti-bodies against F a c t o r H by radioimmunoassay, and two hybrid cultures producing antibodies' were cloned by limiting dilution. These clones are r e f e r r e d to as MRC OX 23 and MRC OX 24. For l a r g e - s c a l e production of monoclonal antibodies, 5 x 106 cells of each hybrid cell line were injected i n t r a p e r i t o n e a l l y into Balb/c m i c e which had been primed with pristane 3 weeks previously. Two weeks a f t e r i n j e c t i o n of cells, ascites fluid was c o l l e c t e d and antibodies were purified by a f f i n i t y c h r o m a t o g r a p h y on a g a r o s e - p r o t e i n A (Sigma) (Ey e t al., 1978). Purified antibodies were radiolabelled with 125I to a s p e c i f i c a c t i v i t y of 5 x 106 c.p.m./IJg using c h l o r a m i n e T, as described b e f o r e (RB Sim et al., 1981). F a c t o r H was radio-iodinated by the same m e t h o d to a specific a c t i v i t y of 2 x 105 c.p.m./lag. Radioimmunoassays
F o r d e t e c t i o n of a n t i - F a c t o r - H antibodies a solid-phase indirect radioimmunoassay was used as described by Hsiung et al. (1982), using 96-well vinyl m i c r o t i t r e plates c o a t e d with F a c t o r H, incubated with t e s t s o l u t i o n , and d e v e l o p e d with 125I-labelled rabbit a n t i - ( m o u s e im munoglobulin) (RAM). The a m o u n t of F a c t o r H in serum samples was d e t e r m i n e d by a d i r e c t r a d i o i m m u n o a s s a y by measuring the inhibition of binding of 125I-labelled MRC OX 23 antibody to F a c t o r - H - c o a t e d vinyl 96-well m i c r o t i t r e plates as follows: Samples of F a c t o r H (50 IJl) containing 50 I~g of F a c t o r H/ml in 20 mM potassium phosphate b u f f e r , pH 7.4, were placed in each well and incubated for 1 h at 22~ The solution was r e m o v e d , and to s a t u r a t e n o n - s p e c i f i c binding sites, 10 mM potassium phosphate p H 7.4/1~5 mM NaCl containing 1% ( w / v ) bovine serum albumin (PBS/BSA) was added to fill each well and the plate
MONOCLONAL
ANTIBODIES AGAINST FACTOR
H
iI21
was incubated for 10 min at 22~ and then washed 4 times with distilled water. The plate was drained, and 50 pl of the solution to be tested or serial dilutions in PBS/BSA were added. For each plate a standard curve was established with a series of dilutions in PBS/BSA of a s o l u t i o n of pur e F a c t o r H of known c o n c e n t r a t i o n . The c o n c e n t r a t i o n of F a c t o r H was d e t e r m i n e d from the extinction c o e f f i c i e n t (ElcmrlI% (w/v) = 14.2) (RB Sim & DiScipio, 1992). Finally to each L~v~ll i0 pl of li~SI-labelled MRC OX 23 antibody was added diluted in PBS/BSA to contain 50 000 c.p.m. The plate was incubated for 1 h at 22~ solutions were removed, and the plate was was h ed 4 times with distilled water. Radioactivity associated with each dried well was determined. A r a d i o i m m u n o a s s a y was established to det erm i ne the epitope specificity of the individual monoclonal antibodies. Vinyl m i c r o t i t r e plates coated with Fa c t or H and saturated with BSA as above were incubated (2.5 h, 22~ with 50 pl/well of serial dilutions of ascites fluid in PBS/BSA and 100 000 c.p.m./well of IzSI-labelled antibody. Solutions were removed and wells were washed 4 times with distilled water. Radioactivity associated with each well was determined in an LKB1270 Rackgamma counter. Immunoprecipitation
A n t i g e n , either I25I-Iabelled Fact or H (200 000 c.p.m.) or precleared serum was incubated (i h, z~~ with ascites fluid (1-10 pl) or nonspecific mouse IgG in a total vol. of 200-350 pl of 10% (v/v) foetal calf serum (Flow Laboratories, Irvine, Ayrshire, Scotland) in 100 mM potassium phosphate, 100 mM KCI, 5 mM KI, 5 mM EDTA, pH 8.1, containing 5 mg of BSA per ml. Then 200 pl of a suspension ( l / I ; v/v) of Sepharose-RAM in the same buffer without foetal calf serum was added and samples were incubated for 1 h at 4~ After centrifugation (2 min, 12 000 r.p.m.) the pellet was washed 5 times with 1 ml of PBS. A s a m p l e of t h e p e l l e t was counted for radioactivity. To r e l e a s e m o u s e antibody and antigen from the Sepharose-RAM, the pellet was incubated (4~ 30 min) in 0.9 ml of 0.1 M g l y c i n e - H C l , pH 2.2, and then centrifuged (2 min, 12 000 r.p.m.). The supernatant was removed and f r e e z e - d r i e d . The residue from the supernatant was resuspended in 100 pl of 0.1 M Tris/HC1; i% (w/v) sodium dodecyJsulphate (SDS); 4 M urea, pH 8.0, containing 3 mg /mI d i t h i o t h r e i t o l , and reduced and alkylated as described by Fairbanks e t al . ( 1 9 7 1 ) . S a m p l e s w e r e t h e n e x a m i n e d by SDS/polyacrylamide-gel electrophoresis. Sera were precleared before the initial incubation with ascites fluid by incubating (1 h, 4~ 2 vol. of each serum (made 5 mM EDTA) with 1 vol. of a l:I suspension of Sepharose-RAM. Preparation of mouse and rabbit immunoglobulin and of Sepharose-RAM
The gamma-globulin fraction was prepared from mouse serum by triple precipitation with 16% (w/v) sodium sulphate by a modification of the method of Prahl and P o r t e r (196g). Rabbits were immunized with mouse gamma globulin. The immunoglobulin fraction from the
1122
SIM ET AL.
r a b b i t antiserum was prepared by sodium sulphate precipitation as described above. The rabbit immunoglobulin fraction was coupled to C N B r - a c t i v a t e d Sepharose #B ( P h a r m a c i a ) ( C u a t r e c a s a s , 1970). Routinely, 350 mg of the rabbit immunoglobulin fraction in 100 mM potassium phosphate, 150 mM KCI, pH 7.5, was mixed with with 3 g (dry weight) of CNBr-activated Sepharose. Coupling efficiency was 75-80%. Excess reactive sites were blocked by incubation with 0.1 M ethanolamine-HCl at pH 8.5. The antibody affinity material prepared by this method (Sepharose-RAM) had a binding capacity of about 700 pg of mouse IgG per ml of packed Sepharose. The rabbit anti-(mouse-gamma-globulin) immunoglobulin fraction was further purified by passage on a column of mouse-gamma-globulin l i n k e d to C N B r - a c t i v a t e d Sepharose. Specific rabbit anti(mouse-gamma-globulin) a n t i b o d i e s w e r e e l u t e d with 0.1 M diethanolamine-HCI-0.2 M NaCl, pH 11.2, and rapidly readjusted to neutral pH. This fraction (affinity-purified RAM) was greater than 99% IgG, and was radio-iodinated for use in the indirect binding assays described above. Effect of antibodies
on the biological
activity of factor H
To determine the e f f e c t of monoclonaI antibodies on the cofactor activity of Factor H, samples of Factor H (2.5 pg) were incubated (I h, 22~ alone or with the purified monoclonal antibodies (12.5 pg) in a total vol. of 135 #1 of 20 mM NaCl. The cofactor activity was then determined by measuring the rate of conversion of tzSI-labeIled C3b to iC3b, as described previously (E Sim et al., 1981). The reaction was carried out in 20 mM potassium phosphate buffer, pH 7.4. To measure the e f f e c t of MRC OX 23 and MRC OX 2# on the binding of t25I-Iabelled Factor H to surface-bound C3b, sheep red blood ceils c o a t e d with a n t i b o d y , complement components C1, C#, and approx. 30 000 molecules of C3b per cell (EAC3b) were prepared as described previously (E Sim & RB Sim, 1981). Initially t251-1abelled Factor H (#-5 pg) was incubated (3 h, #~ in 270 pl of DVB++ (1#0 mM g l u c o s e / 7 l mM N a C I / 2 . 5 mM sodium 5 , 5 - d i e t h y l barbiturate/0.5 mM MgCI2/0.15 mM CaCl2, pH 7.5) containing 0.1% ( w / v ) BSA with monoclonaI antibodies (20-25 pg), polyclonal nonspecific mouse immunoglobulin (25 pg), or unlabelled Factor H (216 pg). Samples (120 pl) of these solutions were then mixed with either EAC3b or EA cells (7.5 x 107 cells) in 50 IJl of DVB++ containing 1% (w/v) BSA and incubated for 20 min at 37~ Samples (50 pl) were removed and centrifuged through oil (E Sim & RB Sim, 1981) and the pellet was counted for radioactivity. SDS/polyacrylamide-gel electrophoresis of the supernatant solutions was carried out to confirm that the Factor H was not proteolysed during the incubation procedures. Sera
Human outdated plasma was obtained from the National Blood Transfusion Service, 3ohn Radcliffe Hospital, Oxford. Sera from other animals were obtained from either Flow Laboratories, Irvine, A y r s h i r e , Scotland, or from Northeast Biomedical Labs, Uxbridge, England.
MONOCLONAL
ANTIBODIES
AGAINST
FACTOR
H
1123
Immunodiffusion A s s e s s m e n t of the class and subclass of m o n o c l o n a l a n t i b o d i e s was d o n e by d o u b l e radial i m m u n o d i f f u s i o n a g a i n s t s h e e p a n t i b o d i e s to m o u s e IgM, mouse IgA, mouse t o t a l IgG, m o u s e I g G l , m o u s e IgG2a, m o u s e I g G 2 b , and m o u s e IgG3. These antibody preparations were g e n e r o u s l y provided by Dr. A. R. Bradwell, I m m u n o d i a g n o s t i c R e s e a r c h L a b o r a t o r y , U n i v e r s i t y of B i r m i n g h a m , U.K. Results
and D i s c u s s i o n
During p u r i f i c a t i o n of MRC OX 23 and MRC OX 24 on P r o t e i n - A A g a r o s e " ( E y e t al., 1978), both antibodies w e r e e l u t e d f r o m the column at pH 6.0-5.5, and thus b e h a v e d in a way c h a r a c t e r i s t i c of the I g G l subclass, which is the m o s t a b u n d a n t of the m o u s e i m m u n o globulin subclasses (Ey et a l , 1978). This a s s i g n m e n t was c o n f i r m e d by double radial i m m u n o d i f l u s i o n of the purified a n t i b o d i e s a g a i n s t s h e e p a n t i s e r a s p e c i f i c for d i f f e r e n t h e a v y - c h a i n c l a s s e s and subclasses. P r e c i p i t a t i o n was found only with antibodies a g a i n s t mouse IgG and m o u s e IgG1. P o l y a c r y l a m i d e - g e l e l e c t r o p h o r e s i s of the r e d u c e d and a l k y l a t e d mouse a n t i b o d i e s a r e shown in Fig. 1. The m o b i l i t i e s of the h e a v y and light chains d i f f e r in the two m o n o c l o n a l antibodies. This m a y be due to d i f f e r e n c e s in g l y c o s y l a t i o n or to a d i f f e r e n c e in the l i g h t - c h a i n subclass. The two clones are t h e r e f o r e distinct. As well as being produced f r o m d i f f e r e n t hybrid clones, MRC OX 23 and MRC OX 24 a r e a g a i n s t d i f f e r e n t e p i t o p e s on the F a c t o r - H molecule. As shown in c o m p e t i t i o n binding studies to F a c t o r - H - c o a t e d vinyl p l a t e s , binding of 125I-labelled MRC OX 23 was r e d u c e d to 50% by a 7 000-fold dilution of the c o r r e s p o n d i n g a s c i t e s fluid w h e r e a s t h e r e was v i r t u a l l y no inhibition of 125I-labelled MRC OX 23 e v e n with undiluted a s c i t e s fluid f r o m MRC OX 24. In the r e c i p r o c a l e x p e r i m e n t in which the binding of 125I-labelled MRC OX 24 to F a c t o r - H - c o a t e d p l a t e s was m e a s u r e d , binding was inhibited to 50% by a 2 050-fold dilution of the a s c i t e s fluid f r o m MRC OX 24 w h e r e a s inhibition of binding was only seen with v e r y high c o n c e n t r a t i o n s of the a s c i t e s fluid f r o m the MRC OX 23 clone. T h e s e results a r e shown in Fig. 2 and c l e a r l y d e m o n s t r a t e t h a t the two m o n o c l o n a l a n t i b o d i e s a r e a g a i n s t d i f f e r e n t a n t i g e n i c d e t e r m i n a n t sites on the F a c t o r - H m o l e c u l e . Factor H in serum:
immunoprecipitation
and radioimmunoassay
In an indirect immunoprecipitation system, either MRC OX 23 or MRC OX 24 was found to precipitate specifically t25I-labelled F a c t o r H from solution. In studies of immunoprecipitation of Factor I4 from human serum, only a molecule of 170 000 apparent mol.wt, was specifically precipitated (Fig. 3). There are traces of a molecule of 140 000 apparent mol.wt. However, degradation of Factor 14 by serum proteases has been shown to produce a major fragment of 140 000 m o l . w t . (RB Sim & DiScipio, 1982) and this is the likely s o u r c e of this t r a c e c o m p o n e n t . In t e s t i n g for the p r e s e n c e of i m m u n o l o g i c a l l y c r o s s - r e a c t i v e F a c t o r H f r o m o t h e r sera, s p e c i f i c i m m u n o p r e c i p i t a t i o n oI a m o l e c u l e of 170 000 m o l . w t , was found in the c a s e of o t h e r p r i m a t e s e r a - ASrican g r e e n m o n k e y (Cercopithecus aethiops), c y n o m o l g u s m o n k e y (Macaca
112#
SIM ET AL.
Fig. I. SDS/polyacrylamide gel electrophoresis of monoclonal antibodies against Factor H. Purified MRC OX 23 (tracks 2 and 3) and MRC OX 24 (track 4) antibodies were electrophoresed on a 10% SDS/polya c r y l a m i d e gel and stained with Coomassie blue. Human IgG (track 5) was run as a standard. The position of the heavy (H) and light (L) chains are marked. The standards (track i) are as follows:212 000 mol.wt. A - myosin, 108 000 mol.wt. B - e' chain of C3b, 70 000 mol.wt. C - 8 chain of C3b, 60 000 mol.wt. D - eatalase, E - ovalbumin, 42 000 mol.wt. F - carboxypeptidase A, 25 000 mol.wt. All samples were reduced and alkylated as described in Materials and Methods.
fascicularis),
and rhesus m o n k e y (Macaca mulatta) - but not with the non-primate sera (pig~ cow, sheep, chick or rabbit) even though it has been shown that rat Factor H (Daha et al., 1982) and guinea-pig
Factor H (Bitter-Suermann et al., 1981) are structurally similar to h u m a n F a c t o r H. Although with all the primate sera the major c o m p o n e n t is the 170 000-apparent-mol.wt. species, in the case of cynomolgus monkey, a band of 140 000 apparent mol.wt, is also clearly seen (Fig. #). This may indicate that the Factor-H molecule from cynomolgus serum, like human and guinea-pig (Bitter-Suermann et al., 1981) Factor H, is susceptible to the same pattern of proteolysis. A direct radioimmunoassay measuring binding of 1251-labelled MRC OX 23 to h u m a n - F a c t o r - H - c o a t e d vinyl plates in the presence of c o m p e t i n g fluid-phase Factor H has been established to determine
MONOCLONAL ANTIBODIES
AGAINST FACTOR H
1125
O
x
2
"0 C 0
.o
I
E
Q. 0
I
1.0
2.0 3.0 log dilution
I
I
4.0
~0
Fig. 2. MRC OX 23 and MRC OX 24 antibodies are against different epitopes on Factor H. Vinyl plates were coated with Factor H and then incubated (2.5 h, 22~ with 125I-labelled monoclonal antibody (lO0 000 c.p.m./well) in the presence of serial dilutions in PBS/BSA of ascites fluid (60 ~l/well). The radioactivity associated with each well was determined after removal of solution and 4 washes with H20. 9 125I MRC OX 9 1251 MRC OX O 1251 MRC OX [] 1251 MRC OX The broken binding.
23 antibody + MRC 23 antibody + MRC 24 antibody + MRC 24 antibody + MRC line represents
OX 23 ascites fluid OX 24 ascites fluid OX 23 ascites fluid OX 24 ascites fluid 50% inhibition of
F a c t o r - H concentration (Fig. 5). Using this method, with human Factor H as the assay standard~ the concentration of Factor H in the sera Irom different primate species has been measured. The concentration of Factor H in human outdated plasma is 250 tJg/ml9 in agreement with earlier estimates (Whaley et al., 1978; RB Sire & DiScipio) 1982). The apparent concentration of Factor H in a single sample of rhesus-monkey serum (460 lag/ml) was of the same order of magnitude as the concentration in human serum. However, apparent concentrations determined in cynomolgus-monkey serum (2.63 mg/m]) and in African-green-monkey serum (3.03 mg/ml) were very high in comparison with human serum. Higher levels in these sera are also suggested by the intensities of immunoprecipitated materials examined on SDS/polyacrylamide gels (Fig. •). However~ it is possible that these high apparent concentrations could be a reflection of higher a f f i n i t y of t h e a n t i b o d y for cynomolgus or African-green-monkey F a c t o r H t h a n for human Factor H. The lack of immunological
1126
SIM ET AL.
Fig. 3. Immunoprecipitation of Factor H from human serum. Tracks 1-6 show immunoprecipitated material from O-, 3-, 30, I00-, 200-, and 300-~i samples, respectively, of precleared human serum by MRC OX 24. Tracks 7-12 show control samples after immunoprecipitation with non-specific mouse immunoglobulin. Tracks 13 and 14 are molecularweight markers as follows:A = myosin B = catalase C = ovalbumin D = carboxypeptidase E = Heavy chain of human Ig F = Light chain of human Ig All samples were reduced and alkylated, and gels (7.5% polyacrylamide) were stained with Coomassie blue.
cross-reactivity between MRC OX 23 and Factor H from non-primates, as above, was confirmed by radioimmunoassay. McMahan (1982) has shown that complement components C3 and C4 and Factor B from humans and the three monkey species discussed a b o v e also have common antigenic determinants but relative concentrations were not reported. In some monkey species, levels of whole-complement activity considerably higher than in human serum have been described (Rommel et al., 19g0). Effect of MRC O X 23 and MRC 24 on the c o f a c t o r a c t i v i t y of human factor H
Factor H participates in the physiological degradation of C3b a f t e r complement has been activated. C3 is initially activated to C3b by complex enzymes, C3 convertases. C3b consists of two disulphidelinked polypeptide chains ~' (10g 000 mol.wt.) and t3 (70 000 mol.wt.). It is during activation of C3 to C3b that C3b may become covalently
MONOCLONAL
ANTIBODIES AGAINST
FACTOR
H
1127
Fig. 4. Immunoprecipitation of Factor H from primate sera. Samples (300 ~i) of precleared sera from African green monkey (tracks 1 and 2), cynomolgus monkey (tracks 3 and 4), and rhesus monkey (tracks 5 and 6) were immunoprecipitated either with 5 U1 of a I:I (v/v) mixture of ascites fluid from MRC OX 23 and MRC OX 24 (tracks I, 3, and 5) or 5 U1 of nonspecific mouse IgG, as described in Materials and Methods. Human factor H is shown for comparison in track 7. The migration positions of the heavy (H) and light (L) immunoglobulin chains are indicated.
bound to surfaces on which a C3 convertase is assembled (Reid, 1983). C3B, both free in solution or bound to surfaces, is degraded i n i t i a l l y to iC3b by the protease Factor I. Factor H is an essential cofactor for this proteo]ytic reaction. It seems that fluid-phase C3b and C3b attached to surfaces may be recognized d i f f e r e n t l y by Factor I in the presence of Factor H (l-long et a l , 1982). T h e e f f e c t of the two monoc|onal antibodies on breakdown of fluid-phase C3b to iC3b is shown in Fig. 6. MRC OX 23 alone has only a slight e f f e c t on the cleavage reaction. However, MRC OX 24 increases the rate of the cleavage reaction 4-fold, and this increase is s i g n i f i c a n t l y Jess (only 2-fold) if both antibodies are present. To i n v e s t i g a t e the e f f e c t of the antibodies on the interaction between Factor H and bound C3b, the binding of t251-labelled Factor H to EAC3b cells has been measured. It is clear that MRC OX 23 has no e f f e c t on the binding of 1251-labelled Factor H to EAC3b cells (Fig. 7). MRC OX 2#, however, decreases the binding of t25J-labeHed Factor H to these cells. The result of having both antibodies present is again less than the e f f e c t of MRC OX 24 alone. Polyclonal non-specific mouse immunoglobulin has been found to have no e f f e c t on t251-labetled Factor H binding to EAC3b cells. The binding of t25I-JabeJled Factor H is specific, since unlabe]led Factor H reduces the binding to EAC3b to the background level.
l12g
SIM
ET
12000
800C
u
4000
0
i
,
I
1.0
~0
2~
"log [factor H]~.,,g/ml
Fig. 5. R a d i o i m m u n o a s s a y for quantification of Factor H. 96-well vinyl plates, coated with Factor H, were incubated (I h, 22~ with 125I-labelled MRC OX 23 and serial dilutions in PBS/BSA of a standard solution of Factor H. Each well contained a total vol. of 60 ~i with I00 000 c.p.m. Radioactivity associated w i t h each well was d e t e r m i n e d , as described in Materials and Methods, and is shown as a function of the log of the concentration of Factor H in ~g/ml. Only the linear portion is illustrated.
70 60 -u 5O > o
(D O
40
,c_30 [3
.o 20 "8
~10 I
0
10
20 30 Time {rain)
40
50
Fig. 6. Effect of MRC OX 23 and MRC OX 24 on c o f a c t o r a c t i v i t y of F a c t o r H for cleavage of soluble C3b by Factor I. The percentage cleavage of the ~' chain of soluble C3b by Factor I in the presence of Factor H ( 9 ), Factor H with MRC OX 23 ( O ), Factor H with MRC OX 24 ( 9 ), and Factor H w i t h both MRC OX 23 and MRC OX 24 ( [] ) was determined as described in Materials and Methods.
AL.
MONOCLONAL
ANTIBODIES
AGAINST
FACTOR
H
1129
12Q0C
900d
]
0
x
600
"0
7
S0 E o_ 3000 0
..,"1
A .,-'1 .-"1 /'1
P A
B
C
O
E
Fig. 7. Effect of MRC OX 23 and MRC OX 24 on the binding of 1251-1abelled Factor H to surface-bound C3b. E A C 3 b cells (open c o l u m n s ) or E A cells hatched columns) were incubate~ (20 min, 37~ with 25I-labelled Factor H (A), 125I-labelled Factor H and MRC OX 23 (B), 125I-labelled Factor H and MRC OX 24 (C), 125I-labelled Factor H and MRC OX 23 plus MRC OX 24 (D), or 125I-labelled Factor H and cold Factor H (E). The radioactivity bound to the cells was determined as d e s c r i b e d in M a t e r i a l s and Methods.
l
It has been reported that trypsinization of Factor H increases its cofactor a c t i v i t y towards fluid-phase C3b but decreases the cofactor a c t i v i t y towards surface-bound C3b (Hong et aJ., 1982). Since MRC OX 24 appears to have a similar effect to trypsinization, it was important to determine whether the effect of MRC OX 24 is due to p r o t e o l y s i s of F a c t o r H. However no proteolysis of 1251-JabeIIed Factor H was detectable by examination of SDS/polyacrylamide gels of the material used in these studies. Therefore the effect of MRC OX 2# on increasing the c o f a c t o r a c t i v i t y of F a c t o r H against fluid-phase C3b but d e c r e a s i n g the binding of F a c t o r H to bound C3b r e i n f o r c e s the c o n c e p t t h a t F a c t o r H i n t e r a c t s d i f f e r e n t l y with s u r f a c e - b o u n d and fluid-phase C3b. Although the MRC OX 23 and MRC OX 2# antibodies are d i r e c t e d against d i f f e r e n t antigenic sites on F a c t o r H (Fig. 2), the regions on the F a c t o r - H m o l e c u l e to which these antibodies bind are such t h a t the c a p a c i t y of F a c t o r H to a c t as a c o f a c t o r for soluble C3b is m u t u a l l y a f f e c t e d by the binding of both antibodies. T h a t the a c t i v i t y is increased argues against the antibodies occluding sites of i n t e r a c t i o n
1130
SIM
ET
AL.
with e i t h e r fluid-phase C3b or F a c t o r I. It is possible t h a t the region of the molecule which i n t e r a c t s with s u r f a c e - b o u n d C3b is masked by binding of MRC OX 2g. The F a c t o r H molecule is a s y m m e t r i c in t h a t it has a f r i c t i o n a l r a t i o of 2.11 (RB Sire & DiScipio, 1952). From c i r c u l a r - d i c h r o i s m studies, it has been suggested t h a t t h e molecule has neither alpha-helix nor b e t a - s h e e t s e c o n d a r y s t r u c t u r a l e l e m e n t s (DiScipio & Hugli, 1982) and t h a t a small c o n f o r m a t i o n a l Change occurs on binding of F a c t o r H to fluid-phase C3b. The monoclonal antibodies described h e r e are useful tools for f u r t h e r investigations of the d i f f e r e n c e in recognition of F a c t o r H for fluid-phase and s u r f a c e - b o u n d C3b.
Acknowledgements We thank Dr. A. F. Williams for helpful discussion and H a n c o x and Miss B. M o f f a t t for e x c e l l e n t t e c h n i c a l assistance.
Mrs.
3.
References B i t t e r - S u e r m a n n D, B u r g e r R & Hadding U (1981) A c t i v a t i o n o f t h e alternative pathway of complement: efficient fluid phase amplification by blockade of the regulatory complement protein, Bl H, through sulfated polyanions. Eur. J. Immunol. II, 291. Cuatrecasas P (1970) Protein purification by affinity chromatography. J. Biol. Chem. 245, 3059. Daha MR & Van Es LA (1982) Isolation, characterization and mechanism of action of rat Bl H. J. Immunol. 128, 1839. DiScipio RG & Hugli TE (1982) Circular dichroism of factor H a regulatory component of the complement system. Biochim. Biophys. Acta 709, 58. Ey PL, Prowse SJ & Jenkin CR (1978) Isolation of pure IgGl, IgG2a and IgG2b immunoglobulins from mouse serum using Protein A-Sepharose. Immunochemistry 15, 429. Fairbanks G, Steck TL & Wallach DFH (1971) Electrophoretic analysis of the major polypeptides of the human erthrocyte membrane. Biochemistry IO, 2606. Fearon DT (1979) Regulation of the amplification C3 convertase of human complement by an inhibitory protein isolated from the human erythrocyte membrane. Proc. Natl. Acad. Sci. U.S.A. 76, 5867. Galfre G, Howe SC, Milstein C, Butcher GW & Howard JC (1977) Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature, 266, 550. Hong K, Kinoshita T, Dohi Y & Inoue K (1982) Effect of trypsinization of the activity of factor H. J. Immunol. 129, 647. Hsiung L-M, Barclay AN, Brandon MR, Sim E & Porter RR (1982) Purification of human C3bINA by monoclonal antibody affinity chromatography. Biochem. J. 203, 293. McMahan MR (1982) Complement components C3, C4 and Bf in 6 nonhuman primate species. Lab. Animal Science 32, 57. Prahl JW & Porter RR (1968) Allotype-related sequence variation of the heavy chain of rabbit IgG. Biochem. J. IO7, 753.
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Reid KBM (1983) Proteins involved in the activation and control of the two pathways of human complement. Biochem. Soc. Trans. II, I. Rommel FA, Bendure DW & Kalter SS (1980) Haemolytic complement in nonhuman primates. Lab. Animal Science, 30, 1026. Sim E & Sim RB (1981) Binding of fluid phase complement component C3 and C3b to human lymphocytes. Biochem. J. 198, 509. Sim E & Sim RB (1983) Enzymic assay of C3b receptor on intact cells and solubilised cells. Biochem. J. 210, 567. Sim E, Wood AB, Hsiung L-M & Sim RB (1981) Pattern of degradation of human complement fragment C3b. FEBS Lett. 132, 55. Sim RB & DiScipio RG (1982) Purification and structural studies on the complement system control protein B1H (Factor H). Biochem. J. 205, 285. Sim RB, Twose TM, Paterson DS & Sim E (1981) The covalent binding reaction of complement component C3. Biochem. J. 193, ll5. Whaley K & Ruddy S (1976) Modulation of the alternative complement pathway by B1H globulin. J. Exp. Med. 144, I147. WhaleY K, Widener H & Ruddy S (1978) Modulation of the alternative pathway amplification loop in rheumatic disease, in: Clinical Aspects of the Complement System (Operferkuch W, Rother K & Schulz DR, eds), pp 99-112. G. Thieme Verlag, Stuttgart, W. Germany. World Health Organization (1981) Nomenclature for the components of the alternative pathway of complement. Bull. W.H.O. 59, 489.
