M&c&r Immunology, Vol. 24, No. 11, pp. 121 l-1211, 1987 Printed in Great Britain.

0161-5890/87 $3.00 +O.OO 0 1987 Pergamon Journals Ltd

THE EFFECTS OF IMMUNOGLOBULIN ISOTYPE AND ANTIBODY AFFINITY ON COMPLEMENT-MEDIATED

INHIBITION OF IMMUNE PRECIPITATION AND SOLUBILIZATION

ANN JOHNSON,* SUSANNAH HARKIN,* M. W. STEWARDS and K. WHALEY*$ *University Department of Pathology, Western Infirmary, Glasgow Gl 1 6NT, U.K.; and tImmunology Unit, Department of Medical Microbiology, London School of Hygiene and Tropical Medicine, Keppel

Road, London WCIE 7HT, U.K.

(First received I1 February 1987; accepted in revised form 18 May 1987)

Ah&act-We have studied complement-mediated prevention of precipitation (PIP) and solubilization of immune complexes (IC) formed with DNP,,-BSA and murine monoclonal antibodies (MCAs) of different isotypes and affinities. PIP was effective for IC formed with IgGl and IgM antibodies, but not for IgA MCA. For IgGl MCAs, affinity appeared to exert a minor effect on PIP, and IC formed in antibody excess or at equivalence were retained in solution more readily than those formed in antigen excess. For IgM MCAs affinity and antigen-antibody ratio did not affect PIP. As PIP did not occur in Mg-EGTA, it was concluded that PIP was entirely classical pathway dependent. Solubilization of IC containing IgGl MCAs occurred more rapidly and to a greater extent with low affinity antibodies and an inverse relationship between affinity and the extent of solubilization was observed. Complexes formed with IgGl MCAs were solubilized relatively poorly when formed in antigen excess. In contrast, affinity and antigen-antibody ratio did not influence the rate and extent of solubilization of IC containing IgM MCAs. IC formed with IgG2b were solubilized rapidly whereas those formed with IgG2a or IgA were solubilized poorly. The relative contributions of the classical and the alternative pathways to solubilization varied with each antibody and the effect of antigen-antibody ratio on these relative contributions was inconsistent.

INTRODUCITON

When insoluble antigen-antibody complexes (IC) are incubated with serum they become soluble (Czop and Nussenzweig, 1976). Solubilization is complement- dependent, displaying an absolute requirement for the alternative pathway, but being most efficient in the presence of an intact classical pathway (Takahashi et al., 1977, 1978). The critical step in the solubilization process is the binding of C3b to the complex with subsequent disruption of the antigen- antibody lattice (Takahashi et al., 1977). In contrast to solubilization, when IC are formed in the presence of serum, their precipitation is prevented by com- plement activation (Schifferli et al., 1980).

Complement-mediated prevention of immune pre- cipitation (PIP) displays an absolute dependency on an intact classical pathway (Schifferli et al., 1982; Naama et al., 1984) with the alternative pathway appearing to play a secondary role (Naama et al., 1985), perhaps helping to retain IC in solution once

SAuthor to whom correspondence should be addressed. Abbreviations: BSA, bovine serum albumin; DNP, dinitro-

phenol; DNP,,-BSA, dinitrophenylated bovine serum albumin containing 19 molecules of DNP per molecule BSA; EDTA, ethylenediamine tetraacetic acid; IC, antigen-antibody complex; MCA, monoclonal anti- body; Mg-EGTA, ethylglycol tetraacetic acid contain- ing MgCl,; PIP, prevention of immune precipitation.

they have been rendered soluble (Schifferli and Pe- ters, 1983). As in solubilization, the critical step in PIP appears to be the binding of C3b to the IC lattice (Naama et al., 1984, 1985). In their original paper, Czop and Nussenzweig (1976) reported that proper- ties of both antigen and antibody itiJenced solu- bilization. In particular, they showed that antibody affinity played a significant role; complexes formed with antibodies of low affinity were solubilized more readily than those formed with antibodies of high affinity. However, other authors have shown that antibody affinity does not influence the solubilization process (Stassen and Bell, 1979).

Since the influence of antibody affinity and isotype on PIP have not been investigated, we have studied the effect of these immunoglobulin characteristics on PIP and solubilization by using a panel of murine monoclonal anti-dinitrophenol (anti-DNP) anti- bodies of different affinities and isotype.

MATERIALS AND METHODS

Monoclonul antibodies (MCAs)

Four IgGl, three IgM, one IgG2a, one IgG2b and one IgA (MOPC-315) murine MCAs were studied. Details of the preparation, characterization and mea- surement of the affinity of the IgGl and IgM anti- bodies have been published elsewhere (Stanley et ul., 1983). The IgG2a and IgG2b antibodies were gifts

1211

1212 ANN JOHNSON et al.

from Dr G. G. B. Klaus (N.I.M.R., London) and IgA MOPC-315 was a gift from Dr M. Browning (De- partment of Bacteriology and Immunology, Univer- sity of Glasgow).

Antigen

Dinitrophenylated bovine serum albumin contain- ing 19 molecules of DNP per molecule of BSA (DNP,,-BSA) was prepared by the method of Eisen et al. (1953). The degree of substitution was deter- mined by sp~trophotometry, and the conjugate ra- diolabelled with ‘25I by the chloramine T procedure (McConahey and Dixon, 1966).

The equivalence point for each antibody was deter- mined by quantitative preciptin analysis in which 20 pg antibody was added to each tube together with varying quantities of “‘1-DNP-BSA.

Kinetics of immune precipitation

One hundred microlitres (100 ~1) PBS were added to a series of microcap tubes containing 2Opg anti- body in 20 ~1 PBS. Sufficient ‘251-DNP,,-BSA in 5 ~1 PBS was added to each tube to ensure that antigen-antibody ratios of 2-fold antibody-excess, equivalence and 2-fold antigen-excess were achieved. The tubes were incubated at 4”C, and after 30 min, 1, 2, 4 and 24 hr, 10 1.11 of each reaction mixture were transferred to a microcap tube containing 1 ml ice- cold PBS. The tubes were centrifuged and the quan- tity of solubie ‘*‘-DNP,,BSA measured.

Prevention of immune precipitation (PIP)

A pool of normal human serum was frozen in aliquots at -70°C. Each aliquot was thawed only once, immediately prior to use in the assay. For experiments in which the level of PIP at different serum dilutions (1: 5, 1: 10, 1: 15, 1:20) was measured, the serum was diluted in isotonic Verona1 buffered saline (VBS, pH 7.4) containing CaCl, (150 pmol/l), MgCl, (1 mmol/l) and gelatin 0.1% (w/v). One hun- dred microlitres (100 ~1) of each dilution were added to a series of t&t tubes containing 20 pg of antibody in 20~1 VBS and, after warming the mixtures to 37”C, sufficient ‘2SI-DNP,,-BSA was added to each tube so that antigen-antibody ratios of 2-fold antibody-excess, equivalence and 2-fold antigen ex- cess were achieved. After 1 hr at 37°C the tubes were centrifuged (Beckman Microfuge, 10,000g x 5 mins) and the amount of non-precipitated antigen deter- mined. Controls included antigen and antibody with 100 ~1 VBS in place of serum (maximum precipi- tation) and antigen in 105 ~1 VBS in the absence of antibody (antigen input). The proportion of precip itated antigen which remained soluble in the presence of serum was calculated as follows:

% IC soluble (PIP) =

cpm serum supernatant - cpm buffer supernatant

total cpm/tube - cpm buffer supernatant x 100.

All assays were performed in triplicate (Naama et al., 1983). For the studies of the kinetics of PIP, 100 ~1 of serum diluted 1:2 with VBS and 20 pg of antibody in 20 ~1 VBS were added to a series of test tubes which were then warmed to 37°C. At time zero, 5~1 VBS containing sufficient ‘2SI-DNP,9-BSA was added to each tube to ensure antigen-antibody of 2-fold antibody-excess, equivalence and 2-fold anti- gen-excess were achieved. At 15, 30, 60 and 120 min, 20 ~1 were removed from each reaction mixture and the amount of non-precpitat~ antigen determined. Kinetic assays were also performed using serum which was diluted 1:2 in VBS which contained EDTA (2Ommol/l) but no added cations, or EGTA (20 mmol/l) and MgCl, (10 mmol/l) but no added CaCl, (Mg-EGTA). Serum was diluted in these re- agents so that complement activation was prevented completeiy (EDTA) or only the alternative pathway was able to function (Mg-EGTA).

Solubilization

IC were formed by mixing 20 @g antibody in 20 1.11 VBS with 5 ~1 VBS containing sufficient ‘2S-I-DNP,,-BSA to achieve 2-fold antibody-excess, equivalence and 2-fold antigen-excess, in a series of microcap tubes containing 50 ~1 VBS. After incu- bation for 1 hr at 37”C, serum (final dilution 1:2; 1:4; 1:8; 1: 16; 1: 32) was added following mixing and the incubation was continued for 30min at 37°C. The amount of soluble antigen was calculated as de- scribed for PIP. The control for the assay was a tube containing IC at the appropriate antigen-antibody ratio to which 50 ~1 VBS had been added in place of serum. The results were calculated as follows:

% Solubilization

cpm assay tube x 100

= cpm input - cpm buffer control’

The kinetics of solubilization were studied by sampling reaction mixtures at 15, 30, 60 and 120 min following the addition of serum diluted I:2 in VBS.

As the rates of precipitation of IC prepared with IgG2a and IgG2b antibodies were extremely slow, these immune precipitates were formed by incubating antigen and antibody at 4°C for 24 hr.

RESULTS

The affinities of the monoclonal antibodies studied, their equivalence points and maximal amount of antigen precipitated by 20 pg of antibody are shown in Table 1. For each isotype the affinity did not directly affect the equivalence point or the amount of antigen precipitated. The rates of precipitation of antigen by IgGl, IgM and IgA antibodies were rapid and usually reached a maximum level by 1 hr, al- though one of the IgM antibodies precipitated more slowly, reaching its maximum after 2 hr. In contrast, the IgG2a (7) and IgG2b (8) antibodies precipitated

Immunoglobulin isotype and aflinity and solubilization 1213

Table 1. Characteristics of monoclonal anti-DNP antibodies investigated

Antibody Isotype Affinity” (M-l)

pg Antigen added

Equivalence pain? cg antigen precipitated

Wl IgGl IgGl IgGl IgG2a IgG2b IgM IgM IgM

7.8 x lo6 41.3 x 106 16.5 x 10“ 0.4 x 106 0.4 x 106 0.5 x 106 2.2 x 10’ 3.2 x lo6 2.2 x 106

2.5 1.8 (78%)d 2.3 1.2(51%) 2.3 1.6 (69%) 0.3 0.2 (67%) 3.1 1.6 (50%) 1.6 1 .O (63%) 3.1 2.3 (74%) 0.5 0.4 (80%) 1.6 1.3(81%)

10 (Mig315) 2.4 x IO6 1.6

“Affinity determined by equilibrium dialysis (Stanley et al., 1983). *Determined with 20 pg antibody.

1.0 (63%)

‘Micrograms of antigen precipitated at equivalence point. dFigure in brackets represents the percentage of added antigen precipitated.

antigen extremely slowly with maximum precip- itation occurring after 24 hr.

Prevention of immune precipitation (PZP)

IC formed in serum with any of the IgGl or IgM MCAs were more soluble than those formed in buffer (Figs 1 and 2). IC formed with IgGl antibodies were

% IC SOLUBLE

a.

0 15 30 60 120 0 15 30 60 120 0 15 30 60 120

MINS

Fig. 1. Kinetics of precipitation of murine IgGl monoclonal anti-DNP antibodies in buffer (closed symbols) or in serum (open symbols). Immune complexes were formed with antibodies 1 (0, O), 2 (a, a), 3 (A, A), and 4 (v, V) at (a) 2-fold antigen excess, (b) equivalence, or (c) 2-fold antibody excess.

b

more efficiently kept in solution when formed in antibody excess or at equivalence than those formed in antigen excess. No consistent pattern was observed with IC formed with IgM antibodies. IC formed with the IgA antibody did not become soluble when formed in serum (data not shown).

The dose-response curves showed that, although

c.

a

% IC SOLUBLE

60

b.

1 , , I 1

0 15 30 60 120 0 15 30 60 120 0 15 30 60 120

MINS

Fig. 2. Kinetics of precipitation of murine IgM monoclonal anti-DNP antibodies in buffer (closed symbols) or in serum (open symbols). Immune complexes were formed with antibodies 7 (0, O), 8 (A,

A), and 9 (m, q ) at (a) 2-fold antigen excess, (b) equivalence, or (c) 2-fold antibody excess.

1214 ANN JOHNSON et al.

a. b. c. % IC

SOLUBLE

loo- v-0-v-v -

EO-

60-

Fig. 3. Kinetics of solubilization of immune complexes formed with IgGl murine monoclonal anti-DNP antibodies, at (a) 2-fold antigen excess, (b) equivalence, or (c) 2-fold antibody excess. Antibodies 1 (O),

2 (O), 3 (A), and 4 (77) are depicited by the symbols shown.

there was no close relationship between affinity and PIP within the IgGl group of antibodies, IC formed with the two antibodies of lower affinity (1 and 4) tended to remain more soluble when formed in serum than the two of higher affinity (2 and 3, data not shown), particularly in antigen excess. This re- lationship was not seen with IC formed with IgM antibodies.

Kinetic studies showed that IC formed in serum from MCAs containing high affinity IgGl or IgM tended to precipitate on prolonged incubation (Figs 1 and 2). This late phase of precipitation was marked with IC formed in antigen-excess. Antibody 4 (IgGl which had a very low affinity) consistently remained the most soluble whereas three IgGl antibodies of higher affinity precipitated more in antigen-excess and in antibody-excess (Fig. 1). At equivalence, the highest affinity antibody (2) precipitated even less

than antibody 4. When IC were formed in serum containing EDTA or Mg-EGTA, the rates of precipi- tation were the same as those for IC formed in buffer. Because of their extremely slow rates of precipitation, PIP was not studied with IC formed with either of the two IgG2 MCAs.

Solubilization

Solubilization of immune precipitates formed with IgGl antibodies was dependent upon the input of serum, and was clearly more efficient with IC formed in antibody-excess and least efficient with those formed in antigen-excess (Fig. 3). These experiments also showed an inverse relationship between antibody affinity and solubilization (Fig. 4). Kinetic studies showed that maximum solubilization was achieved rapidly for IC formed with lower affinity antibody (within 15 min), whereas those formed with antibody

b. c.

O-AI 10 20 30 LO 10 20 30 co 10 20 30 co

AFFINITY IMOi'x1tj6j

Fig. 4. Relationship between affinity of IgGl murine monoclonal anti-DNP antibodies and solubilization, of immune complexes formed at 2-fold antigen excess (left hand panel), equivalence (centre panel), or 2-fold antibody excess (right hand panel). Immune complexes were formed with antibodies 1 (O), 2 (m), 3 (A), and 4 (0) and solubilization is represented as the percentage of immune complex soluble after

incubation of immune precipitate with serum for 2 hr.

Immunoglobulin isotype and affinity and solubilization 1215

a. % IC

SOLUBLE

loo-

A' A-AAA

80-

b. t.

*-*-A 1,

A Ao-m-m 1 II

0 a-o-a a

o/o-o 0 I’ 1 OY O-------O

0’

0 15 30 60 120 0 15 30 60 120 0 15 30 60 120

MINS

Fig. 5. Kinetics of solubilization of immune complexes formed with IgM murine monoclonal anti-DNP antibodies at (a) 2-fold antigen excess, (b) equivalence, or (c) 2-fold antibody excess. Antibodies 7 (O),

8 (A), and 9 (V) are depicted by the symbols shown.

of high affinity were solubilized more slowly (Fig. 3). Immune precipitates formed with IgM antibodies were solubilized by serum. However, in contrast to precipitates formed with IgGl antibodies, the effect of antigen-antibody ratio was not pronounced, al- though two (those formed with antibodies 8 and 9) were solubilized somewhat better in antibody-excess than in antigen-excess (Fig. 5). Another point of difference between IC formed with IgM and IgGl antibodies was the apparent lack of any effect of affinity to influence solubilization of the former type of IC. Kinetic studies of solubilization showed that affinity of IgM antibodies did not affect the rate of solubilization. Immune precipitates formed with IgG2a, IgG2b or IgA antibodies were solubilized poorly with diluted serum compared with IC formed with IgGl or IgM antibodies. IgG2b-containing IC were quite well solubilized with undiluted serum (Fig. 6) and again IC formed in antigen-excess were poorly solubilized compared with those formed at equiv-

% IC SOLUBLE

100

1

a.

60

alence or in antibody-excess. In contrast, the small degree of solubilization produced with IgG2a and IgA containing IC was greatest in antigen-excess. Kinetic studies showed that maximum solubilization of IC formed with IgG2a or IgG2b was achieved rapidly while solubilization with IgA-containing IC was an extremely slow process (Fig. 6).

Kinetic studies showed that solubilization did not occur in the presence of EDTA, and occurred at a slower rate and to a lesser extent in the presence of Mg-EGTA (data not shown). The final extent of solubilization in Mg-EGTA was dependent upon the antigen-antibody ratio and immunoglobulin isotype (Table 2). In general, the alternative pathway played a significant role in the solubilization of IC formed with IgGl and IgM antibodies. In contrast, solu- bilization of IC formed with the IgG2a and IgG2b, even the IgA antibodies, appeared to be mainly dependent upon the classical pathway at all three antigen-antibody ratios.

b. c.

o-n-a

I

0 15 30 60 120 0 15 30 60 120 0 15 30 60 120

MINS

Fig. 6. Kinetics of solubilization of immune complexes formed with murine monoclonal IgG2a (5; O), IgG2b4 (6; 0) or IgA (MOPC-315; 10; A) anti-DNP antibodies at (a) 2-fold antigen excess, (b)

equivalence or (c) 2-fold antibody excess.

1216 ANN JOHNSON et al.

Table 2. The role of the classical and alternative complement pathways in the solu- bilk&ion of different immune precipitates by human serum

Two times antigen Two times antibody excess Equivalence excess

Antibody Tot.’ Class! Alt.c Tot. Class. Alt. Tot. Class. Alt.

IgGl 1 34 35 65 62 89 11 100 66 34 2 18 72 28 15 80 20 51 78 22 3 32 50 50 35 74 26 91 85 15 4 66 56 44 67 58 42 45 55 45

IgM 1 61 43 51 64 42 58 59 37 63 8 94 84 16 100 69 31 100 52 48 9 30 43 57 82 70 30 LOO 75 25

IgG2a 5 31 90 10 27 89 11 5 100 0 IgG2b 6 45 94 6 98 96 4 100 93 I IgA 10 15 80 20 15 100 0 5 100 0

‘Total percentage of precipitate solubilized by incubation in whole serum for 2 hr at 37°C.

bathe percentage reduction in solubilization occurring in Mg-EGTA-treated serum is considered to be that fraction which is dependent upon the classical pathway.

‘Percentage of solubilization occurring in Mg-EGTA-treated serum so that the classical pathway is inhibited.

DISCUSSION

The high incidence of immune complex diseases in patients with inherited deficiences of the classical pathway complement components and C3 (Schifferli and Peters, 1983) suggests that failure of PIP and low solubilization may play an important role in these diseases. Although the complement requirements for both of these phenomena have been well documented (Takahashi et al., 1977; Fujita et al., 1981; Schifferli et al., 1982; Naama et al., 1984, 1985; Groaski et al.,

1985), the effects of variations in the antibody mole- cule have not been determined. In this paper we have begun to examine this problem by measuring PIP and solubilization of IC formed with monoclonal anti- DNP antibodies of different isotypes and affinities. In preliminary studies, it was obvious that with the possible exception of IgG2a and IgG2b antibodies, both the rate and extent of precipitation of antigen were not clearly affected by isotype or affinity, which has been shown previously (Nimmo et al., 1984). IC formed in serum with IgGl or IgM antibodies were readily retained in solution, without any obvious significant isotype differences. Similarly immune pre- cipitates formed with IgGl or IgM antibodies were solubilized equally well at low serum dilutions. Both PIP and solubilization of IC formed with IgGl or IgM antibodies generally occurred more efficiently with complexes formed in antibody-excess or at equivalence, compared with those formed in antigen- excess. Previously PIP has been shown to be more efficient for IC formed in antibody excess with a polyclonal rabbit antiserum (Schifferli et al., 1980). Previous data on the effect of antigen-antibody ratio on solubilization have been conflicting. Czop and Nussenzweig (1976) showed that IC formed with polyclonal IgG anti-DNP antibodies were solubilized better in antigen-excess, while Genin and Lesavre (1983) using human polyclonal anti-tetanus toxoid

IgG found that solubilization was more efficient when IC were formed in antibody-excess. The explanations for this discrepancy are unclear, but could be related to species of antibody, differences in affinity hetero- genity, or to the different antigens. In contrast to IC formed with IgGl and IgM antibodies, those formed with IgA precipitated even when formed in serum, and solubilization of immune precipitates formed with IgG2a and IgA antibodies was extremely lim- ited. The greater amounts of IC formed with IgG2b were only solubilized efficiently at low serum dilu- tions, and at equivalence or in antibody excess.

The observations that complexes formed with IgGl or IgM antibodies precipitated in Mg-EGTA-treated sera show that PIP is entirely dependent upon the classical pathway (Naama et al.,

1984). The findings that solubilization of immune precipitates formed with these antibodies was partly reduced in the presence of Mg-EGTA confirm that this function is dependent upon the alternative path- way, but requires an intact classical pathway for optimal expression. Although these data do not provide direct evidence of complement activation in relation to PIP or solubilization, in another series of similar experiments we have measured the generation of C4a and C3a. These results are too extensive to be published in detail here, but they do confirm our conclusions of the role of the classical and alternative pathways in PIP and solubilization based on obser- vations of these processes in Mg-EGTA-treated sera. They also show that IC formed with MOPC-315 do not activate the classical or alternative pathways, which explains why these IgA-containing immune precipitates are not solubilized by complement (Stew- art et al., manuscript in preparation).

There was an inverse relationship between affinity and solubilization of IgGl containing IC. The expla- nation of this finding is unknown but possibly infers that some disruption of antigen-antibody bonds oc-

Immunogiobulin isotope and affinity and solubilization 1217

curs during the solubili~tion process. As low affinity IgG antibody activates complement less effectively than high affinity antibody (Fauci et al., 1970), these differences are unlikely to be due to differences in the efficiency of complement activation produced by different antibodies. During complement activation by IC, some C3b binds to both the antigen and antibody (Takata et al., 1984); the former could play a role in dissociating antigen-antibody bonds. In PIP, complement activation occurs as soon as antigen and antibody combine, before extensive crosslinking oc- curs. In this situation, C3b bound to the antigen could prevent antibody binding and thus limit lattice formation. As less energy is required to prevent the formation of antiaen-antibodv bonds. than to dis-

Genin C. and Lesavre P. (1983) Immune-complexes solu- bilization: effect of antigen-antibody ratio and the rela- tive role of alternative and classical pathway. MO&. Immun. 20, 10691072.

Groaski P., Bodenbender L., Kanzy E. J., Loos M. and Seiler F. R. (1985) The modulation of immune complex aggregation by classical pathway mediated reactions. Immunobiology 169, 346361.

McConahey P. J. and Dixon F. J. (1966) A method of trace iodination of proteins for immunological studies. Int. Archs Allergy appl. Immun. 29, 185-189.

Naama J. K., Hamilton A. O., Yeung-Laiweh A. C. and Whaley K. (1984) Prevention of immune precipitation by purified classical pathway complement components. C&z. exp. Immun. 58, 48-92.

Naama J. K., Holme E., Hamilton E. and Whaley K. (1985) Prevention of immune pr~pi~tion by purified com- ponents of the alternative pathway. C&n. exp. Immun. 60,

surmising that antibodv affinitv did not avvear to

rupt them, it is possible to argue that it is easier for C3b to prevent the formation of antigen-antibody bonds, than to disrupt them. Thus it would not be

diseases. LClin.* exp. Immui. 51, 292-298.

169-177. Naama J. K., Mitchell W. S., Zoma A., Veitch J. and

Whaley K. (1983) Complement-mediated inhibition of immune nrecioitation in natients with immune comolex

1 1 11

influence PIP. The failure to find any relationship Nimmo G. R., Lew A. M., Stanley C. M. and Steward M.

between affinity and solubilization of IgM-containing W. (1984) Influence of antibody affinity on the per-

IC is probably due to the high functional affinity of formance of different antibody assays. J. Immun. Meth. 72. 111-187.

IgM antibodies (even though they have relatively low ._, _.. __

Schifferli J. A., Bartolotti S. R. and Peters D. K. (1980) intrinsic affinities) arising from the polyvalent inter- Inhibition of immune precipitation by complement. C&z. action between IgM antibody and the antigen. exp. Znzmun. 42, 387-394.

In summary, our data show that properties of the Schifferli J. A. and Peters D. K. (1983) Complement, the

antibody moiety of IC including isotype and affinity ~mune-complex lattice, and the pathophysiology of complement deficiency syndromes. Lancet ii, 957-959.

may influence PIP and solubilization. How these Schifferli J. A., Woo P. and Peters D. K. (1982) properties of antibodies influence these two poten- Complement-mediated inhibition of immune pre-

tially important biological activities is the subject of cpitation. 1. Role of the classical and alternative path-

ongoing investigations. ways. Clin. exp. Immun. 47, 555-562.

Stanley C., Lew A. M. and Steward M. W. (1983) The

Acknowledgements-The authors would like to thank the Arthritis and Rheumatism Council for Research for financial support.

measurement of antibody affinity: a comparison of five different techniques utilising a panel of monoclonal anti- DNP antibodies and the effect of high affinity antibody on the measurement of low affinity antibody. Immun. Meth. 64. 119-132.

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