Scand. J. Immttnot. 18, 319-328, 1983

An Extended Clq-Binding Assay UsingLactoperoxidase- and Chloramine-T-Iodinated Clq

Immediate Distinction between Immune-Aggregate-Mediatedand Non-Immune-Aggregate-Mediated Clq Binding

p. J^SPAETH. A. CORVETTA, U. E. NYDEGGER, M. MONTRONI &K.BUETLER

Central Laboratory of the Swiss Red Cross. Biood Transftision Service, Berne, Switzerland andInstitute of Medical Pathology. University of Ancona, Ancona. Italy

Spaeth. P. J.. Corvetta. A.. Nydegger, U. E., Montroni. M. & Buctler. R. An ExtendedClq-Bindmg Assay Usmg Lactoperoxidase and Chloramine-T-Iodinated Clq. ImmediateDistmction between Immune-Aggregate-Mediated and Non-Immunc-Aggregate-MediatedClq Binding. Scand. J. Immunot. 18, 319-328. 1983.

An extension of the Clq-binding assay for the detection of immune-aggregate-mediated andnon-immune-aggregate-mediated Clq binding is reported. The assay involves the use of twodifferent Clq preparations, one radioiodinated by means of lactoperoxidase (LPO-''-I-Clq)and the other hy means of chloramine-T (CT-'-'l-Clq). The treatment with CT for 21) min atroom temperature hetore iodination for I min led to abolishment of the C lq-binding capacitiesto comptexed IgG: approximately 5(Ff of LPO-'-^I-Clq but only 2<>; of CT-'''I-Clq bound to80 Atg/ml of IgG forming part of tetanus toxoid/anti-tetanus toxoid complexes or to 2()() ,:tj;/ml ofheat-aggregated human gamma globulin. Similar results were obtained with staphylococcalprotein-A-aggregated IgG. CT-treaied Clq was haemolyiicalty Inactive. In contrast to theresults with complexed IgG, CT treatment did not markedly reduce binding capacities of Clq toheparm: approximately 55% of LPO- and CT-'-n-Clq were hound by 127 U/ml of commercialhepann in normal human serum. Both Clq preparations bound to a comparable extent tofibroneetin, fibrinogen. and various bacterial endotoxins. When the LPO- and Cl-'-'l-Clq-bindmg patterns obtained on serum samples from patients with systemic lupus crytheniiitosus.rheumatoid arthritis, or essential mixed cryogtohulinaetnia were compared with bindingpatterns observed using laboratory reactants, an immediate deteetion of non-immune-aggregate-mcdiated Clq binding became possible.

P. Spaeth. Centrat Laboratory SRC, Blood Transfmioti Service Wankdorfstrasse 10 CH-1000Berne 22, Switzertand

The Clq-binding assay (Clq-BA) was one of thefirst of a new generation of sensitive tests for thedetection of Clq-binding material in serutnsamples [29. 41) and has now been widelyapplied. In a WHO collaborative sttidy theClq-BA wa.s most suitable to distinguish be-tween normal control serum samples andsamples from patients with various disea.sesassociated with circulating immune complexes(CIC). such as systemic lupus erythematosus.rheumatoid arthritis, vasculitis, Iymphoma, andleprosy [21],

The principal reaction in the detection of CICby the Clq-BA is thought to be the binding ofClq to the Cyl region of complcxed IgG [71.Binding sites on Clq for aggregated IgG arelocated on the six globular heads of the Clqmolecule, each of which is attached to onecollagenase-sensitive stalk, which are heldtogether at the base [18]. It was recentlydemonstrated that the collagen-like part of Clqcan interact with fibroneetin and native col-lagen, whereas native CI or Cl reconstitutedfrom Clq, Clr, and Cls displays little binding

0300-9475/83/1000-0319 $02.00 ©1983 Blackwell Scientific Publications 319

320 P. J. Spaeth et al.

activity for fibronectin |4, 16, 27. 28, 30]; itseems that Clr and Cls are masking somebinding sites of Clq in a native Cl complex.Under the conditions of the Clq-BA thecollagen-like part of the Ctq molecule isunmasked.

Besides fibronectin and native collagen,heparin. DNA and other polyanionic molecules,polysaccharides of the galactan type, fibrinogen,and bacterial or phage products can bind Clqunder certain conditions [1,3, II, 15, 22, 26, 34.35, 38, 40]. Binding of substances other thancomplexed IgG to the collagen-like part of Clqcan therefore result in complexes large enoughto be precipitated by 2.5% polyethyleneglycol(PEG). Such precipitating activity might bemisinterpreted as being mediated by CIC.

This study reports a possible extension of theconventional '^'I-Clq-BA that enables dif-ferentiation between binding of Clq to com-plexed IgG, on the one hand, and otherbiological material such as heparin, fibronectin.fibrinogen, and endotoxin, on the other. Dif-ferentiation is based on the measurement ofbinding activities of an enzymatically and achemically iodinated Clq preparation. Throughthe parallel use of the ''-^1-Clq preparations forthe assessment of Clq-binding material presentin scrum samples of patients with systemic lupuserythematosus (SLIZ). rheumatoid arthritis(RA), or essential mixed cryoglobulinaemia(EMC)., it has become possible to distinguishdifferent patterns of '"^l-Clq binding.

PATIENTS, MATERIALS ANDMETHODS

Patients. The study included I4i scrum siimplcsrrom 109 pitlieiils which were dividcJ inio threegroups; (I) 57 serum samples from M paticnis withSLE that h;id heen diagnosed in accordance wilh thecriteria of Ihe Ainericaii Rheumatism Asuocimion(ARA) [.S|, (2) ft5 serum samples from 6? palients whofulfilled the criteria of the .^RA for classical or definiteRA \y^\. and (3) IM serum samples from 12 patientswilh CMC |(*|. Patieius wiih SLE were treated wilhprcdnisonc (1.0 lo 1.5 mg/kg/ckty). and most of themwere in remission at ilie time scrum samples wereCDllecteil for ihis siudy. The main therapy lor RA wasnon-sicroid anli-inflamniatory drugs. Most ol thepatients with EMC were receiving therapy withprednisone (0.5 mg/kg^day).

Chemicals and standard preparations. All chemicalsused were of analytical grade. Protein A was obtainedfrom Pharmacia (Uppsala. Sweden). Standard prepa-

rations of aggregated human gamma globulin (AHG)and complement-reacied complexes of tetanus toxoid(Tc)/anii-Te human igG antibodies (Tc/aTe) fordeiection of CIC were obiained Irom stocks main-tained at the Central Laboratory ol the Swiss RedCross (SRC). Blood Tritnsfusion Service (Berne,Switzerland).

IgC-depteted serum. Nonnai human scrum (NHS)was obtained from the blood of hculihy donors. Toobtain IgG-depleled serum, NHS was applied lo aProtein A-Sepharose CL-4B eolumn (Pharmacia,Uppsala, Sweden). The breakthrough fraction wascollected, and the protein concentration was adjustedby ultracentrifugation to 85% of the initial serumconcenlration.

.Substances analysed for their Clij-binding activities.Lyophilizecl sodium salt of heparin with 127 USP'mg(units of United Slates Pharmacopoeia assay permilligram) and collagen type I were products of Sigma(Sl Louis. Mo.. USA). Gelaiin and high molecularweighl DNA from calf thymus was obtained IromServa (Heidelberg, FRG). Uiw molecular weighiDNA from herring was a produci of Fluka (Buchs,Switzerland). Liquid preparations of heparin werefrom I-Ioffmann-La Roche (Basle. Swiizerland) andfrom Kabi (Stockholm. Sweden). Bacterial endotox-ins tested were lipopoiysaccharide from Escherichiacoli OI1I:B4 (Difeo Laboratories, Detroit, Mich..USA), from £. ca//055: B5 (forming part of a test kitPyrogeni'", Mallinckrodt, St Louis, Mo., USA), fromSalmonella abortus equi (Novo-Pyrexal. Hermal-Chemie. Reinbck, FRG). and standard endoloxinpreparation EC-4 used for pyrogenicity lests (Bureauof Biologies, Food and Drug Administration. Bellies-da, Md.. USA). Sulphonated human gamma globulinwas a commercial preparation lor intravenous use(Venilon, Batch SO79.Teijin institute,Tokyo, Japan).Fibrinogen. eoagulable part over '45^^(, was purified asdescribed 15]. and fibronectin was a preparationpurified by gelatin affinity chromatography [10) (kindgifts of J,-J. Morgenthaler, Central Laboratory,

SRCVEnzymatic iodination of Clq. Clq was purified to

homogeneity by means of igG-Sepharose 4B col-umns [19|. The final concentration of purified Clq wasadjusted to 1 mg/ml by optical density, using a 0.3mol'l sodium chloride, 10 mmol/1 EDI A buffer. pH7.5. Aliquots were stored at -70°C. Radioiodinationwith lactoperoxidase (LPO; Calbioehem, Lucerne.Switzerland. Code 4274AA) was done as described [14].In brief, to 2(X1 ^l <>f « f 11 soUition (200 ^tg) wereadded6M.I of hydrogen peroxide (.1|)7( solution diluted1:10 •' with phosphate-buffered saline (PBS) wilh Smmol/l Na^HPO.,, I mtnol/l NaH.POj. and 140 mmol'lNaCI. pH 7.4), 2.5 /i.1 of LPO solution (.MK) IU/ml ofbarbiial (Veronal«')-buffered saline (VBS' ' ) with 3.3mmol/l barbital sodium salt. 150 nimoi/l NaCI. 0.5mmol'l MgCK, and0.15 mmol/l CaCh. pH 7.4), and 25fA of carrier-free Na'-^I solution (10 mCi/ml in 10mol/l sodium hydroxide). The reaction mixture wasallowed to stand for 20 min at 0"C. and iodination wasstopped by adding sodium azide.

Chemical iodination of Clq. If not otherwise stated,radioiodination of Clq by chloramine-T (CT) was

The Extended Clq-Himling Assay 321

done as follows: to 200 /xl of Clq solution .10 I of CTsolution (2.5 mg/ml PBS) were added, and the mixturewas incubated for 20 min at room temperature.Thereafier. 25 M1 of carrier-free Na'-'l (If) mCi/ml in10"' mol/l NaOH) were added. Aficr an additionalincubation for I min the reaction was stopped with 50All oi sodium melabisulphite (5 mg/ml PBS) and 10^1of O.i moi/l sodium iodide. The procedure forradiolitbelling by lodo-Cien (IN) was as follows: glasstubes were coated with 4f)0/xl of a solution of 1 mg IN(Pierce Chemical Co., Rockford, 111., USA) permillilitre of chloroform by evaporaiion of the solvent.To one coated tube 200 1 of C Iq solution were added.and the sample was allowed to stand for 211 min at roomtemperature. Thereafter 25 ij.\ of Na '-'I solution wereadded, and the substitution reaction was stopped after1 min by removing ihe incubation mixture from thecoated tubes. Protein-bound and free radioactivitywere separated on a PD-10 eolumn (Pharmacia.Uppsala. Sweden) pretreated with NHS. Alter sampleapplication, columns, equilibrated with VBS' * con-taining O.Hi human albumin (VBS-HA), were elutedwith VBS-HA. Fractions eontaining the majority ofthe labelled protein were pooled and adjusied to 2.5ml with VBS-HA; aliquots of 100 u.1 were .stored at

70»C

Clq-binding as.say. On the day of testing, an aliquoiof the enzymatically and an aliquot of the chemicallylabelled Clq were thawed and each diluted with . ml ofVBS' ' and 50^1 of hcal-inaeiivated serum (56°C for30 min). The solutions were then centrifuged at IH.(K)Og at 4X for 40 min (Sorvail RC-2B ccnirifuge, SS-34roior). The upper half of the siipernatants wereremoved carefully and were used for the Clq-BA. Thelesl was performed as described by Zuhler & Lambert[42|: 50;iI of Ihe serum sample to be tested were mixedwith lOf) 1 of 0.2 mol/l EDTA solution. pH 7.5. andwere incubated for M) min at 37T. Afler thisincubation iho tubes were transferred to an ice bath,and 50/il of one of the '"' I-Clq preparations and 1 mlof a solution of .l.d'i f PEG in borate buffer, pH S..1,were added. The tubes were allowed to stand for 1 h.whereafter Ihey were cenlrifuged at 1500 g at 4°C for20 min. The supcrnatants were discarded, atid theradioactivity of the precipitates was counted On eachday of testing reference curves of binding of thevarious '"!-Clq preparations to AHG and lo heparinsodium .salt, dissolved directly in NHS. were deter-mined. When patient serum samples were tested, thepercentage of '^^I-Clq precipitated in the presence ofNHS was subtracted [23|.

E.xperiments wiih non-iodinated Clq preparations.For some experiments Clq was treated with LPO inThe absence of iodide in accordance with Ihe aboveprocedure. CT and IN treatments were also repeated,but the substitution reaction wa.s omitted. Twopreparations of CT-Clq were tested: one with additionof sodium metabisulphite (CT-C!q) and one without(CT -Clq). For measurement of haemolytic activi-ties. Clq-depleted human serum was reconstituted bythe non-iodinated Ckj preparations [19]. The non-iodinatcd Clq preparations were also used for doubleimmutiodiffusion in accordance with standard labora-tory techniques, tor precipitation of AHG in agarose

[1| and for sodium dodecyi sulphate polyacrylamidegel electrophoresis (SDS-PAGE) analysis on linear4-30% acrylamide gradient slab gels [i"J).

Uttracentrifiigation. Four hundred microlitres ofpatient serum were loaded on a 4.(S-ml linear U)-M)%(w/v) sucro.sc gradieni in VBS' ' containing 30 mmol'lsodium azide Centriftigation was carried out for 18 hat room temperature in a Beekman SW ()5 rotor(Beekman Instruments. Palo Alto. Calif. USA) at34,lX)0 rpm. Gradients were fractionated wiih anISCO Density Gradient Fractionator. model fi40(Instrument Specialities Co., Lincoln. Nehr.. USA),Fractions of 0.." ml were eollected. and 5()-/u.l aliquotswere analysed for Clq-binding activities.

RESULTS

Effect of chloramine-T on purified Clq

Preliminary experiments were designed tostudy the effect of CT on the futictional andstructural integrity of Clq. ln a kinetic experi-ment an increase of the incubation time with CTbefore iodination resulted in a progressive lossof the binding capacity of '-'^[-Clq to AHG inthe presence of PEG. Preincubation for 30 mmwas long enough to abolish 85% of such abinding capacity (Table I),

To analyse whether CT or sodium metabisul-phite was responsible fur the changes of bindingof Clq to AHG. IN was used to ioditiate Clq, asit is a water-insoluble analogue to CT thatenables iodination to be stopped without using areducing agent f 12]. Incubation of Clq with INand ' I for more than 1 min at room tempera-

I, Effect of chloramine-T on the bindingcapacity of '-"^l-Clq to aggregated human gammaglobulin (AHG)

'i Ck] binding to AHG at conc.t of

Time* 3000 tg/ml 500/ig/ml 100 ,i*g/ml

30 sect10 min§30 minS

72.8(11)4R.4 (41111.2 (Kf.)

26.4 (23)8.7 (75)5.2 («5)

8.0 (13)1.7(81)1.3 (K6)

• At room temperature.t AHG prepared in accordance with Nydegger e/oA

[29]; the preparation contains a large amount ofmonomerie IgG but is not identical to the AHG dis-tributed by the Swiss Red Cross (Fig. 3).

t In accordance with McConahey & Dixon [251.§ Reaction lime with CT in the absence of ' ''I

followed by iodination for 1 min.Numbers in parentheses give percentage reduction inClq-binding activities with respect lo the mean valueobtained with 37 measurements using LPO-' ' I-CIq

322 P. J. Spaeth et ai.

ng Oq/ ml

FIG, 1. Haemolytic activity of non-iodinated but LPO-,CT-. or IN-treated Clq. Haemolysis of sensitizedsheep erythroeytes by increasing concentrations ofLPO-Clq ( • • ) or IN-Clq (x— • —x) andCT-Clq ( A - - - A ) was measured by reconstitu-tion of a Clq-depleted normal human serum. Thedotted line (• • • •) indicates percentage haemolysis bythe Clq-depleted NHS alone,

ture did not lead to a marked increase in theamount of '" 1 incorporated. A 1-min incubatiotireduced IN-'-^I-Clq-bitiding activity to AHGdowti to 62% of activity obtained with LPO-'-'l-Clq.

Sitnilarly to the treatment with CT, a prem-cubation of Clq with IN for 2(J min at roomtemperature, followed by a 1-min incorporationof '-^I, led to loss of Clq-binding activities toAHG in the presence of PEG.

To analyse selectively whether it is theincorporated ' I or the net effect of CT or INthat is responsible for the changes of Clqftinctional activities. Clq was subjected to LPO,CT, and IN treatment in the absence of '"M. Inagarose at pH 7,2 and in the absenee of PEG.CT-Clq did not precipitate AHG, whereasLPO-Clq did. CT- or IN-treated Clq lost itshaemolytic activity as assessed in a reconstitu-tion experiment involving an NHS that wasdepleted in Clq. whereas treatment with LPOdid not impair sueh activity (Fig. 1). Chemicalinfluence of CT and IN on the structure of Clqwas studied by itnmunoprecipitation and SDS-PAGE. It was observed in double immunodiffu-

sion experiments that the LFO-, the CT-, theCT -and the IN-Clq preparations were able toprecipitate with monospecific anti-human Clqantiserum. To elucidate the gross effect of CTand IN on the three polypeptide chains of Clq,all preparations were subjected to SDS-PAGE.As ean be seen in Fig. 2, the polypeptide chainsco-migrated for all Clq preparations to the sameposition of the gels.

Binding properties of LPO- and CT-treated Clqto various substances

The binding properties of LPO- and CT-'"l-Clq. as assessed with complexed IgG and withheparin, are compared in Fig. 3. When eitherTe/aTe complexes or AHG was used. LPO-'^^l-Clq bound in a dose-dependent manner to theacceptor complexes, whereas the binding activ-

FIC. 2. SDS-PAGE of non-iodinated but LPO-. CT:or IN-ireated Clq preparations. Isolated but notfurther treated Clq (track 2) was compared withLPO-treatcd (track 3), IN-treated (track 4), CT -treated (without adding sodium metabisulphite; track5). and CT-treated (with added sodium metabisul-phite; track 6) Clq preparations. Before elec-trophoresis all preparations were treated with 2-mercaptoethanol and SDS. and protein bands wereseparated on a linear 4-.10% aerylamide slab gel. TheIN-. CT- and CF -Clq preparation showed twoadditional protein bands wilh apparent molecularweights of about M.(KHI (H). Molecular weightmarkers were phosphorylase A, 94,fHH) (A); lac-toperoxidase. m.iWi.) (B); bovine serum albumin.67,(KX) (C); ovalbumin, 43,IXH) (D); carbonic anhyd-rase, 30,(KH) (E): trypsin inhibitor, 20,IW) (F), andalpha lactalbumin, 14.4(H1 (G). Serva blue R, umidoblack 10 stain.

The Extended Clq-Binding Assay 323

10 20 ^ug/mL

80 50 100 200 400ijg/ml

800 ai3 127USBml

1270

FIG. 3. Binding of LPO- and CT-'"I-Clq to complexed IgG and heparin. The extended Clq-binding assay wasapplied to detection of (A) eomplcment-reacted Te/aTe eomplexes and (B) heat-aggregated humangammaglobulin (AHG) using LPO- ( • • ) and CT-' ' I-Clq (A A). Te/aTe and AHG were standardpreparations distnbuted by the Swiss Red Cross. In X' binding to heparin in the presenee or absence of NHS isshown; ( • • ) and (A A) indicate binding of LPO- and CT-"-^I-Clq. respectively, to heparin in thepresenceofNHS.whereasin the absence ofNHSLPO-'~^l-Clq(O O)andCr- '"I-Clq(Q • • -Q) showedvirtually no binding to heparin. Mean values of six-monthly determinations are given (±1 SD).

ity of CT-'"l-Clq never exceeded backgroutidlevels. In contrast, binding activities in thepresence of increasing doses of heparin werefound with both '^^I-Clq preparations. In theabsence of NHS, heparin did not bind either Clqpreparation, and this is in accordance with aprevious report [3]. Binding patterns of IN-' "*!-Clq to AHG and heparin were identical to thatof CT-'^''I-Clq. We have checked preparationsof heparin from various manufacturers for theirClq-binding activities. All preparations avail-able in a crystalline form expressed similardose-dependent Clq-binding capacities in thepresence of NHS. Only fresh lots of heparinavailable in a liquid form, tested in the presenceof NHS, bound LPO- and CT-'"-Clq to thesame extent as the crystalline preparations did.It is therefore not recommended to use liquidheparin preparations for binding studies.

That the difference in binding of LPO- andCT-'"l-Clq to AHG and to heparin can be usedto assess a 'specific" Clq binding to complexedIgG became further apparent when it was seenthat only LPO-'"i-Clq and not CT-'"l-Clqbound to protein A cross-linked IgG; an NHSselectivity depleted of IgG but containing

protein A was unable to bind any Clq (Table 11).In addition, differential binding of LPO- andCT-' '*I-Clq was tested, using a sulphonatedIgG prepared for intravenous use, which wasincubated at 37''C for 30 min in the presence orabsence of NHS (incubation is part of theClq-BA). As shown in Table II. such chemicallymodified IgG showed strong Clq-binding capac-ity towards CT-'"l-Clq in the absence of NHS.The incubation at 3 7 ^ in the presence of NHSreduced CT-'"l-Clq binding to S-sulphonatedIgG markedly. Experiments analysing bindingof Clq to biological material other than com-plexed IgG were performed under the testconditions used for the Clq-BA in the presenceand absence of NHS (Table III). As can be seen,fibronectin. fibtinogen, and some endotoxinsbound both LPO- and CT-'^^I-Clq to variousextents, whereas DNA. collagen, and endotoxinfrom E. coli nill:B4 did not bind substantialamounts of either Clq.

Binding patterns of LPO- and CT-^^^l-Clq itiindividual serum satnples

The Clq-BA using LPO- and CT-'^^I-Clq

324 P. J. Spaeth et al.

il. Binding of LPO- and CT-'^^l-Clq to native and chemically modifiedhuman IgG

Sample

Protein A added to'NHSIgG-depleted NHS

S-sulphonated human IgG (10 mg/ml)in the absence of NHS

• Ready-for-inI usion'Hcat-aggregaiettt

S-sulphonated hunian IgG (lO mg/ml)in the presence ol NHS

• Ready-for-in fusion"Heat-aggregated-i-

'''< Binding of '"''!-

LPO

550.0

6182

9077

Clq labelled with

CT

2.20,7

10072

1327

' Resulting in a final conccnlration of 5 mg/nil and used to aggregate the nativeIgG.

t At 63'C for 10 min.

(the -extended Clq-BA') was applied to thestudy of 141 serum samples from 109 patientswith various chronic inflammatory diseases andto 50 serum samples from 50 heallhy blooddonors. The latter group bound 2.b''/f (upper 2SD limit) of LPO-'-^I-Ciq and 3.5% (upper 2SD limit) of CT-'"I-Clq.

When the pathological serum samples weretested with the extended Clq-BA. each of thethree diseases. SLE. RA and EMC, could besubdivided into categories of binding activities(Table IV), For category I no Clq-bindingcapacity with either Clq preparation was foutid.In serum samples of category 11 binding

activities towards LPO-'"l-Clq but not towardsCT-'-^I-Clq were observed. From the experi-ments reported above the Clq-binding materialin these serum samples is apparently of thecomplexed IgG type. Fifteen per cent of theSLE. 18% of the RA and 33 ^ of the EMCpatientswere in category II. In serum samples ofcategory III the Clq reactants ean hardly beregarded as complexed igC.-like or as heparin-like because LPO-'-^I-C!q-binding activitieswere lacking while CT-'-^l-Clq-binding activi-ties were present. Nine per cent of the SLEpatients. 29f of RA and none of the EMCpatients were in this category. The hallmark of

TABLE III. Binding of LPO- and CT-''^1-Clq to hiologieal substances other than eomplexed IgG in the presentsor absence of normal human serum

Substance

Fibroneetin

Fibrinogen

DNA. low m. wLDNA. high m. wt.Collagen type IGc'laiin

EndotoxinsE. £•(.»//Ul 11 : B 4

E. coti 055: B5S. aborttis equiEC-4 (BOB)

Control (NHS)

Cone.. mg/n:il

100.1

400.4

105

5x10-'10

U)2.5

10-"

91

LPO (-^NHS)

8812R2

4

9430

229

18

2.6

h Binding o( '-'

CT (-1-NHS)

77498319

6246

0217

10

3.5

'1-Clq labelled with

LPO (-NHS)

831

830

ND121

0111

CT(-NHS)

822

770

ND'03

Z*i

0I00

Not detennincti.

The Extended Clq-Binding Assay 325

TABLK IV. Various categories of '^'l-Clq binding ohiaincd with iheextended Clq-BA in individual serum samples'from patients withehronie inflammatary diseases

Binding aciiviiies of-'[-Clq labelled using

LPO/CT Category SLE' RA EMC

Absent/absentPresent/absentAb.seni/presenrPreseni.'presentTotal

III

IIIIV-IV

42537

57

(27)f(5)(3)(6)

(41)

36 (34)11(11)1 (1)

17(17)

ft5 (631

940A

ly

(5)(4)(0)O)

(12)

* Some patients have changed category during the study.t Number of saniple.s (number of palients),

category IV was the presence of bindingactivities towards both the LPO- and theCT-'-'l-CIq.

The serum of one patient with cryoglobuli-naemia and nephrotic syndrome wasselected for an uitracentrifugational study be-cause we observed high binding activities forboth LPO- and CT-'^^l-Clq in this material, andClq binding was therefore suspected not to bedue to cotTiplexed IgG. Indeed, there was nodistinct band of Clq-binding material at sedi-mentation rates ranging from 15 to 25 S (Fig. 4).it range in which CIC are usually found.

DISCUSSION

Since the original description ofthe complexedigG-Clq interaction for detection of CIC inclinical samples [1]. a large number of pro-cedures have been proposed to make such a testsystem more sensitive and reproducible, Amongthe fluid phase tests, the Clq-BA has now beenwidely applied, and the present paper, byintroducing the useofCT-'^'^I-Clq, could makethe Clq-BA more informative.

Radiolabelling by mean.s of CT is known tohave a deleterious effect on the haemolytic

a

Q.

ppt5 10 15Fraction Number

FIG. 4. Characterization of LPO- and CT-''^l-C 1 q-hinding material ol a paihol.igicalserum sample by uliraecntrifugation. The figure shows the oprical dcnsiiy patternand the various Clq-binding aciiviries in individual fractions as measured bv theextended Clq-BA alter ihe sucrose gradient separalion of a scrum sample from apatient with cryoglobuiinaemia and nephroiie syndrome. Material sedimcnled (ppt)was resuspendcd in 3UI ti\ of isotonic saline and was also subjected to extendedClq-BA. RefereneeSvaluesof4S-albumin(A),7S-IgG(B) and |yS-IfiM(C)areindicated.

326 P. J. Spaeth et al.

activity of Clq, but the effect on bindingcapacity to complexed IgG is not well describedWorking at (fC for 15 min with a 10- to 40-foldmolar excess of CT over Clq. Heusser et al. [ 14)found a reduction of Clq haemolytic activity,whereas Clq binding to immune complexes wasnot affected significantly. Tenner et al. [37]reported a M)% reduction of haemolytic activityof C Iq when iodination was performed at 0°C for31) min with a 3.3-fold molar excess of CT overClq; however, they did not analyse the bindingcapacity of their Clq to complexed IgG. Themodifications in haemolytic tiires were discus-sed in view of the degree of substitution (mol'"i/mol Clq), despite the fact that Heusser ff«/.114] suggested an effect of the oxidant alone onthe Clq functional activity. We have shownclearly that under the conditions used (66()-foldmolar excess of CT. 20 min at room tempera-ture) not only the haemolytic activity of Clq butalso the binding activity to complexed IgG wasalmost completely abolished in the absence ofany substitution reaction. The functional activi-ties of Clq can also be destroyed by using thesparingly soluble oxidant IN. We conclude that aprinciple common to CT and IN interacts withthe biological functions of Clq and that theincubation time and reaction temperature areimportant factors, which led to the destructionof Clq functiotial activities. A common principleof CTand IN would be their oxidation potential.Like others 114). we found that CT- orIN-treated Clq have overall physicocbemicalcharacteristics indistinguishable from those ofnative molecules when analysed by PAGE or bydouble immunodiffusion.

The binding pattern of LPO- and CT-''*^I-Clqwith complexed IgG represents the most in-teresting aspeet of the present work: it provedthat only LPO-'-M-Clq is capable of bindingsignificantly and dose-dependently to AHG,Te/aTe-comp!exes. and protein-A-aggregatedIgG. whereas no binding was observed usingCT- or IN-'"1-Clq. Testing a commerciallyavailable IgG preparation that was speciallyprepared for intravenous use by chemicalmodification through sulphonation. LPO-' M-Clq and CT-'-^l-Clq exhibited similar bindingactivities. In these experiments the unexpectedbinding of CT-'-^!-Clq to sulphonated IgGctjuld be due to excess charges generated in theIgG during chemical modification. It wasreported that such [gG gradually reconverts

upon reduction and that a full reconversion intoa state that is able to activate complement can beachieved within 24 h [24|. A 30-min incubationat 37°C in the presence of NHS indeed reducedthe unexpected CT-'~^I-Clq binding to sulphon-ated IgG. whereas LPO-'-'l-Clq binding by thesulphonated "ready-for-infusion' immunoglobu-lin preparation remained high (Table II).

The binding pattern of LPO- and CT-''^I-CIqobserved with heparin is of particular interest,since it has been reported that plasma obtainedfrom heparin-anticoagulated blood revealsabnormally high (falsely positive) Clq-bindingactivities [3. 11. 40]. In our study the two Clqpreparations bound equally well to heparin inthe presenee of NHS. Thus, the measurement ofbinding activities of plasma samples towardsLPO- and CT-'-^l-Clq could be of potentialinterest in the clinical situation of immunecomplex removal through plasma exchange thatis carried out with heparin as anticoagulant. Inthe absence of NHS heparin had no effect onLPO- and CT-''^I-Clq-binding activities, Themechanism that gives rise to this effect isunknown but was also observed recently byother authors [3]. For non-immune-aggregate-mediated Clq binding ofthe substances studied,the presence of serum factor(s) seems to be aprerequisite.

Attempts have been made to eliminate thebinding of heparin to Clq during the Clq-BA.Recently, the pretreatment of samples withexcess of protamine sulphate was recom-mended, since this compound antagonizes theinteraction of both heparin and fibrinogen withlabelled Clq without interfering with the in-teraction of ''-l-Clq and circulating immunecomplexes [11 j . It should be mentioned thatheparin-protamine sulphate interactions migbtresult in activation of the classical pathway ofcomplement [32]. which could interfere with im-mune complex determinations in pathologicalsera. The detection of non-immune-aggregate-mediated Clq binding is not possible with asolid-phase Clq-BA utilizing an IgG- and/orIgM specific indicator system [2, 9. 13, 36. 39].However, solid-phase Clq-BA fails to provideinformation about non-IgG-type Clq-bindingmaterial while the extended Clq-BA offers thispossibly important information.

In contrast to heparin. it is established that thepolyanionic DNA does not bind Clq under th'cconditions of the conventional Clq-BA (high

pH and low ionic strength) [20. 29. 34. 41], Inaddition to confirmation of these results, thepresent study also indicates that, under theconditions used, the CT-''^1-Clq does notinteract with DNA to form PEG-precipitablecomplexes.

The results of the clinical study performed onserum samples from patients with various typesof chronic inflammatory disorders revealed fourcategories of LPO- and CT^^^I-Clq-bindingactivities that can be compared with the bindingpatterns obtained when using complexed IgG.heparin. and other non-IgG Clq reactants. Infact, category I corresponds to the Clq-BA-negative NHS. and a relatively large number ofserum samples from patients with SLE werefound here. This i.s in contrast to some otherreports [29, 41] but may be due to steroidtherapy. Binding activities of serum samples incategory II are consistent with those found withcomplexed IgG. and category IV reflects Clqbinding to heparin. We suggest that serumsamples belonging to category II contain solublecomplexed IgG. interacting only with LPO-'^^l-Clq. Of course, such a binding may also behidden in category IV sera, and the relativecontribution of either reactant could be calcu-lated by deduction of the CT-' ' I-Clq fromLPO-'*'I-C]q-bindingactivities. Category IIT ofreactivity was only observed when using re-latively low concentrations of fibronectin andfibrinogen, and the sole presence of CT-'-^I-Clq-binding activity in absence of LPO-''^I-Clq-binding activity could mean that treatmentwith CT leads tci generation of binding sites fornon-IgG compounds in the collagen-Hke part ofthe Clq molecule.

ACKNOWLEDGMENTS

The support of G. Danieli and A. Haessig isgratefully acknowledged. We thank Mrs L.Meyer-Haenni and Mi.ss S. Braegger for excel-lent technical help and Miss S. Thueler and V.Haeuptii for secretarial assistance. H. Engleroffered helpful comments during the prepara-tion of the manuscript,

Part of these studies has been presented inabstract form: Spaeth. P. j . & Buetler, R.Experientia 3S, 1375, 1982.

23

The Extended Clq-Binding Assay 327

REFERENCES

1 Agneilo. V.. Winchester. R.J. & Kiinkel. H.G.Precipititi reactions of the Clq component ofcomplement with aggregated y-gkibulin nmi im-mune complexes in gel diffusion. Imtnunologv 19,909, 1970.

2 Ahistedt. S.. Hanson. L.A. & Wadsworth, C. AClq immunosorbent assay compared with thin-layer gel (iltration for measuring igCi aggregates.Scand. J. Immunot. 5. 293. 1976.

3 Baldwin, R.W.. Byers. V.S. & Robins. K.A.Circulating itnmune complexes in cancer: charac-terization and potential as lumour markers.Behring Inst. Res. Comtnun. 64. 63, 197 .

4 Bing. D.H., Aimcda. S.. Isliker. H.. Lahav, J. &Hynes. R.O. Fibronectin binds to the Clqcomponent of complement. Proc. natn. Acad. Sci.USA 79. 4198, 19«2.

5 Biomback. B, & Blomback. M. Purification ofhuman and bovine fibrinogen. Arkiv for Kemi 10,415. 195ft.

f> Brotiet.J.-C.,Clauvei,J.-P.,Danon.F.. Klein. M.& Seligmann, M. Biologic and clinieal significanceof cryoglobulins. A report of 86eases. Am. J. Med.57. 115. 1974.

7 Burton. D.R.. Boyd. J . Bramplon. A.D . Easter-brook-Smiih. S.B.. Ematiucl. E.J-. Novotny. J..Rademjicher. T.W., van Schravendijk. M.R..Sternberg. M..I.E. & Dwek. R.A. The Clqreceptor site on immunoglobulin G. Nature 288,338. 1980.

8 Cohen, A.S.. Reynolds. W.E.. Franklin, E.G..Kulka. J.P., Ropes, M.W.. Shulman. L.E. &Wallace. S.L. Preliminary erileria lor ihc classi-fication of systemic lupus eryihematosus- Bidt.Rheutn. Dis. 21. 643. 1971.

V Collins. M.M.. Casavam. C.H. & Stites. D.P.Solid-phase Clq-binding lluorcscence immunoas-say for detection of circulatinii immune complexes.J. ctin. Mikrobiol 15, 456. 1982.

10 Engvall, E. & Ruoslahti, E. Binding of solubleform of fibroblast surface protein, fibroneetin, tocollagen, hit. J. Cancer 20. 1, 1977.

It Faaber. P.. Capel. P.J.A. & Koene. R.A.P. Theinterference of fibrinogen and heparin with thedetermination oCeirculatinp immune complexes inthe Clq-binding assay. I'ttn. exp. tmmunol. 45.590. 1981,

12 Fraker. P.J. & Speck, J.C. Proiein and cellmembrane iodinations wilh a sparingly s<ilablechloroamide. l,3,4.6-tetrachloro-3a. fta diphcnyl-glycoluril. Rioctiem. hiophvs. Res. Commun. 80.849. 1978.

13 Hay, F.C.. Nineham. L.J. & Rtiiit. I.M. Routinea.ssay for the detection of immune complexes ofknown immunoglobutin class using solid phaseClq. Clin. exp. Immunol. 24. 396. 1976.

14 Heusser. C , Boesman, M., Nordin. J.H. &Isliker. H. Effect of chemical and enzymaticradioiodination on in vitro human Clq activities. /,tmmunol. 110.820. 1973.

15 Hughes-Jones, N.C. dt Gardner. B. The reactionbetween thecomplemeni subcomponent Clq, IgG

328 P. J. Spaeth et al.

complexes and polyionic molecules. Immunology34, 459. 1978.

lf> Isliker. H-. Bing. D.H,. Lahan. J, & Hyncs. R , 0 .Fibronectin interacts with Clq. a subcomponent ofthe first eomponent of complement, Immunot.Lett, 4, 39, 1982,

17 lUlS/WHO Working Group: Bentwich. Z,. Bian-co, N,. Jaeger, L,, Houba. V,. Lamhert. P,H.,Knapp, W., Rose, N.Sel igmann.M.. Thompson.R.,Torrigiani, G- & de Week. A, Use and abuse oflaboratory tests in clinical immunology: criticalconsiderations of eight widely used diagnosticprocedures. Clin, Immunot. Immunopathol. 24,122. 1982.

IK Knobel. H R . Villiger. W, & Isliker, H, Chemicalanalysis and electron microscopy studies of humanClq prepared by different methods. Eur. J.Immunol 5. 78. 1975,

14 Kolb. W,P,, Kolb. L,M, & Podack. E.R, Clq:isolation from human scrum in high yield by-affinity chromatography and development ot ;ihighly sensitive hemolytic assay, J. Immutwl. 122,2103.' 1979.

20 Korogodin, D.V, & Poverennyi. A.M, Interactionof the C lq component of eomplemeni and collagenwith nucleic acids and polyanions. Bull. e.xp. Biol.Med. (engl, transl,) 90. 1707, 1980.

21 Lambert. P.H,. Dixon. F.J., Zubler, R,H,.Agnello. V.,Cambiaso.C., Casali. P., Clarke. J..Cowdery, J.S,. McDuffie. F C , Hay. F.C.,MaeLennan. LC.M.. Masson. P.. Muller-Eberhard, H,J,, Penttinen. K,. Smith. M,. Tap-peiner. G,, Theofilopoulos. A.N, & Verroust. P.A WHO collaborative study for the evaluation ofeighteen methods for delecting immune complexesin serum. J. clin. Lab. Immunol. 1, 1, 1978,

22 Loos, M,. Bittcr-Suermann. D, & Dierich. M,Interaction of the first (CI). the second (C2) andthe fourth (C4) component of complement withdifferent preparationsof baeterial lipopolysacchar-ides and with lipid A. J. Immunol, 112, 935. 1974.

23 Mahieu. P.. Lambert, P H . & Mieseher, P A ,Detection of anli-glomerular basement membraneantibodies by a radioimmunologieal technique.Clinical application in human nephropathies, J.ctin. Invest. 54. 128. 1974.

24 Masuho, Y,. Tomibe. K., Watanabe. T, &Fukumoto. Y, Development of an intravenousy-globulin with Fc activities, II, Reconversion ofS-sulphonated human y-globulin into the origmaly-globulin. Vox .Sang. 32. 290, 1977.

25 McConahey. P.J. & Dixon, F.J. A method of traceiodination of proteins for immunologie studies. Ini.Arch.s Allergy 29. 185. 1966,

26 McKay. E.J. A simple two-slep procedure for thepurification of plasma Clq from different animalspecies. Immunol. Lett. 3, 303. 19KI.

27 Menzel. E,-J., Smolen, J.S,. Liotta. L. & Reid.K,B,M, Interaction of fibronectin with Clq and itscollagen-like fragment (CLF), FEBS Lett. 129,188. 1981.

28 Menzel, E,-J., Smolen. J. & Reid. K, Interactionof collagen with Clq via its collagen-like portion,Biochim. biophys. acta 670, 265. 1981,

29 Nydegger. U,E., Lambert, P.H.. Gerher, H, &Mieseher. P.A, Circulating immune complexes inthe serum in systemic lupus erythematosus and incarriers of hepatitis B antigen, OuantitiUion bybinding to radiolabeled Clq, J. clin. Invest. 54,297, 1974.

30 Pearlstein. E., Sorvillo. J. & Gigli. 1, Theinteraction of human plasma fibronectin with asubunit of the first component of complement,Clq. 7- Immunol. 128. 2036, 1982.

31 Pohl. D,A..Tsai. C C . & Roodman. S,T, Detec-tion of immune complexes using a solid-phase Clqpolystyrene ball assay. J. Immunol, Meth. 40, 313,1981,

32 Rent, R., Ertel. N.. Eisenstein. R, & Gewurz, H.Complement activation by interaction of polyan-ions and polycations. 1. Heparin-protamine in-duced consumption of complement, J. Immunot.114, 120. 1975,

33 Ropes. M W . . Bennett, G.A.. Colb,S. .Jacox.R.& Jessar. R.A, Revision of diagnostic criteria forrheumatoid arthritis. Butl. Rheum. Dis. 9. 175,1958,

34 van Schravendijk, M.R. & Dwek. R.A. Interac-tion of Clq with DNA. Motec. Immunot. 19, 1179,1982.

35 Sobel. A.T,. Bokisch, V.A. & MuUer-Eberhard,H.J. Clq deviation test for the detection ofimmune complexes, aggregates of IgG. and bacte-rial products in Iiuman serum, J. exp. Med. 142,139, 1975,

36 Svehag, S.-E. A solid-phase radioimmunoassay forClq-binding immune complexes. .Scand. J. Im-munol. 4, 687. 1975.

37 Tenner, A,J,. Lesavrc, P H , & Cooper. N,R,Purification and nidiolabeiing of human Clq. J.Immunol. 127,648, 1981.

38 Uhlenbruck. G,. Karduek, D. . Haupt, H. &Schwick. G,H. C-reactive protein (CRP). 9,5 S"rglycoprotein and Clq: serum proteins withlectin properties? Z. Immunitaetsforsch. 155, 262.1979,

39 Wehler. C-. Andrews. J.M, & Bing. D,H, The useof solid phase Clq and horseradish peroxidase-conjugated goat anti-IgG for the detection ofimmune complexes in human serum. Molec.Immunoi. 18. 157. 1981,

40 Woodroffe. A.J,. Border, W.A.. Theofilopoulos.A.N.. Giitze. O,. Glassoek. R.J,. Dixon. F J , &Wilson. C,B. Detection of circulating immunecomplexes in patients with glomeruloncphritis.Kidney Int. 12. 268. 1977,

41 Zublgr. R H . . Lange. G.. Lambert. P.H, &Mieseher. P A , Detection of immune complexes inunheated sera by a modilied '-''1-Clq binduig tesi,Effeciof heating on tbe binding of Ciq by immunecomplexes and application of the test lo systemiclupus erythematosus, J. Immunol. 116, 2.'i2. 1976,

42 Zubler. R,H, & Lambert, P,H. Detection ofimmune complexes in human diseases. Progr.Altergy 24, 1. 1978.

Received 11 April 1983Reeeived in revised form 15 June 1983