0095-1137/82/030456-09$02.00/0
Solid-Phase Clq-Binding
Fluorescence Immunoassay for
Detection
of
Circulating Immune Complexes
MICHAELEEN M. COLLINS, CONRAD H. CASAVANT,* AND DANIEL P. STITES Departmentof Laboratory Medicine, University of California at San Francisco, San Francisco, California
94143
Received 2 July 1981/Accepted 7 October 1981
A fluorescence immunoassay for detection of immune complexes bound to
solid-phase Clq was developed. The method was standardized by using human
aggregated immunoglobulin G (IgG) to simulate immune complexes. A linear
relationship existed between the concentrations of the aggregated IgG standards
and theresulting fluorescent intensity. The methodwasfoundtobereproducible and capable of detecting as little as 10 ,g of aggregated IgG per ml of
heat-inactivated human serum. Antigen-antibody complexes prepared in vitro were
detectablefrom equivalencetomoderateantigenexcess. EndogenousserumClq inhibited thebinding of aggregated IgGto solid-phase Clq. Pretreatment oftest serawith EDTAwasineffective in eliminating this competitive effect. Heatingthe sera at 56°C alleviated, but did not abolish, interference of endogenous Clq.
Elevated levelsof immune complexesweredetectable inserafromsevenofnine
patients with systemic lupus erythematosus, provided the samples were heat inactivatedbefore testing. Heparin andDNA werealsofoundtointerfere withthe
detection ofaggregated IgG added to human serum. Assay values were falsely decreased due to competitive inhibition by these anions. Lipopolysaccharides from a variety of bacterial preparations produced nodetectable interference. A
comparative studywasconductedonsamplesthat hadpreviouslybeen testedby fluid-phaseClq-bindingradioimmunoassay. Thetwomethodswereconcordantin assigningnormalorelevated levels of immunecomplexes in 70% of the samples tested. This solid-phase fluorescence immunoassay is proposed as a possible alternativetoradioimmunoassayforthedetection of circulating immune
complex-es.
Numerous non-antigen-specific methods for the detection of immune complexes have been described (12, 15). Many of these assaysutilize the ability of antigen-antibody complexes to
combine with thecomplement component Clq and have achievedwidespreaduse.Themajority ofClq-bindingassaysuseradio-labeled compo-nentsto quantitateimmunecomplexes (22, 26). Suchradioimmunologicalmethods are extreme-ly sensitive for detectionofbiologically impor-tant substances, but hazards exist in the
han-dling and disposal of radioactive materials.
Recent advances in fluoroimmunoassay (FIA) indicate that fluorescentlabels aresuitable
sub-stitutes for radiolabels in many immunoassays
(17, 20).
We have developed a fluorescence assay for the detection of immune complexes bound to
solid-phase Clq.Themethod has been adapted from the FIAX system described originally by International Diagnostic Technology (IDT) of
Santa Clara, Calif. Clq is adsorbed tothe
sur-face of the IDT StiQ sampler, which is then
incubated withthedilutedtestsample.
Antigen-antibody complexespresentin theserumbindto
Clq via theFcportionof theimmunoglobulinin thecomplex. Afterwashing, thebound compo-nents are then incubated with fluorescein-la-beled anti-immunoglobulin G (IgG).
Nonreac-tive substances are washed away, and the remaining fluorescence boundtothesolidphase is measured in the FIAX fluorometer. We pro-pose that FIA is an alternative to
radio-immunoassay (RIA) for the detectionofimmune
complexes and thus eliminates the use of
haz-ardousradioisotopes.
MATERIALS ANDMETHODS
Aggregated IgG (AGG). Human IgG (Gammagee; Merck,Sharp & Dohme,WestPoint,Pa.)wasdiluted to aconcentration of10mg/ml inphosphate-buffered saline(PBS), pH7.2.Thepreparationwasaggregated by heating for20min at63°C and then centrifugedat
1,500 x gfor20minto removeinsoluble aggregates. Preparationofsolid-phaseClq(SP-Clq).StiQ sam-plers (IDT)wereusedasthesolid-phase matrix. The samplerswereplacedhorizontally inamoist chamber with the polymeric surface upward. Purified Clq, purchased from DavidBing at the Center for Blood 456
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457
Research, Boston, Mass.,wasdilutedtoa
concentra-tion of 50 ,g/ml in PBS. Thirty microliters of the diluted Clqwasplacedonthecellulose disk of each sampler, using a hand-held pipetter. The samplers
wererefrigerated overnight in the moistchamber. Fluorescent-antibody reagent. Purified F(ab')2 goat anti-humanimmunoglobulin conjugated with fluores-ceinisothiocyanate (FITC)waspurchasedfrom Kal-lestad Laboratories, Chaska, Minn. The antiserum
was diluted to a concentration of 8 ,ug/ml in PBS containing 0.125% bovineserumalbumin(BSA).
Reaction buffer and wash buffer. PBS, 0.05 M, containing 0.1% Tween 20 and 0.5% BSA, pH 6.4 (PBS-BSA-Tw),wasusedassamplediluent and initial washsolution for solid-phasecomponents.
Immunecomplexassay.Assays werecarriedoutin glasstubes (12 by75 mm), and all incubations were allowedtoproceedonanautomatic horizontal shaker.
IDTsamplerscoated with 1.5FgofClqwere individ-uallywashed for 30minin tubescontaining0.5 ml of
PBS-BSA-Twbuffer.Blank controlsamplersforeach specimenwerealso washed in this bufferto coat the exposed polymericsurface with BSA andwerecarried throughtheprocedure concurrentlywith thesamplers coatedwith immobilizedClq.Thesamplerswerethen transferred to tubes containing 50
RI
of AGGstan-dards, controls, or unknown samples and 500 ,ul of PBS-BSA-Tw buffer and incubated for 2 h atroom temperature. Allsamplerswerewashed for30minin PBS-BSA-Tw and then immersed for 45minin 0.5 ml ofFITC-F(ab')2goatanti-humanIgG. Unreacted
ma-terialswerewashedawayby transferringthesamplers
totubes containing 0.5 ml ofPBS with 2.0o BSA. After this final30-min wash, each sampler wasthen inserted into thesamplestageof theFIAX fluorome-ter,andthefluorescentintensitywasmeasured.
Quantitationof fluorescence. TheFIAXfluorometer measures the fluorescence ofcomponents bound to thesampleratawavelengthof 540nmwhen excitedby lightatawavelengthof475nm(11). The fluorometer
wasadjustedtozerowithoutasampler in thesample
stage.The200-Rg/mlAGG standardwastheninserted,
and the fluorometerwasmanuallyadjustedtoread190
± 5 fluorescent signal units (FSU). The remaining
standards and unknownsamplersaswellastheblanks for eachspecimenweretheninserted into the fluorom-eter,andthe FSU ofeachwasrecorded. TheFSUof the blank samplerfor each specimenwassubtracted
from theFSUofthe samplercoated withClqtoobtain
aAFSU. On lineargraphpaper,microgramsof
aggre-gated IgGpermilliliterwasplottedonthexaxis,and
the AFSUvalue of theAGG standardswereplottedon they axis. The degree of fluorescence was directly
proportionaltotheconcentrationofaggregates pres-ent.Control andpatient samplerswerecomparedwith thestandard calibration curve, andthe results were
expressedasmicrogram equivalents of AGGper
milli-liter.
Sera. Normal human sera (NHS) were collected from apparently healthyhospital personnel after ob-taininginformedconsent.Controlsera wereprepared byadding AGG to heat-inactivated (30min at 56°C) NHS. Patients' sera that had been tested ina
fluid-phase [12-I]Clq-binding assay (16) were kindly
sup-plied by Scripps-Miles ImmunologyReference Labo-ratory, LaJolla,Calif.Allspecimensweredivided into
equal portions, stored at -70°C, and thawed just beforetesting.
Preparation ofIgG-anti-IgG complexes. Chromato-graphicallypurifiedhumanIgGwassupplied byTago Laboratories,Burlingame,Calif. Thepreparationwas centrifuged inaBeckman Airfuge (Beckman Instru-ments, Inc.,PaloAlto, Calif.) for 20 minat165,000x g toremovesoluble aggregates.Goat anti-humanIgG waspurchasedfrom MeloyLaboratories(Springfield, Va.),and thespecific antibody contentwasprovided by the company. IgG-anti-IgG complexes were pre-pared byincubatinga constant amountof goat anti-human IgG with increasing amounts of monomeric humanIgG for 1 hat37°C followedbya48-h incuba-tionat4°C. Theprecipitincurve wasdetermined (9), and the antigen/antibody ratio at equivalence was foundtobe 1:2.5. Immunecomplexeswere prepared atfour concentrations of goatanti-human IgG: 20, 50, 100,and 500,ug/ml. Increasingamountsof humanIgG wereadded to constant amounts of antibody to pro-ducecomplexes rangingfrom 8 timesantibodyexcess to 15 times antigen excess as compared with the equivalence ratio. Both the antigen and theantibody werediluted in PBS and incubated togetherfor 1 h at 37°C followed by a 48-h incubation at 4°C. After centrifugation for 20 minat1,000 x g, the superna-tantsweretested in the immunecomplex assay.
Preparation oftetanustoxoid-antitetanus toxoid com-plexes. Purified tetanus toxoid was purchased from LederleLaboratories, PearlRiver, N.Y. The undilut-ed preparationwasdialyzed against PBS for 24 hat 4°C. Proteincontentasdetermined by the method of Lowry etal. (13) was 165,ug/ml. The immunoglobulin fraction ofahumanantitetanus antiserum (Hypertet) was purchased from Cutter Laboratories, Berkeley, Calif. This antiserum wasdiluted in PBS and centri-fuged for 60 minat130,000 x gin a Beckman LS-65 ultracentrifuge to remove aggregated immunoglob-ulins. Soluble immune complexes were prepared by usinga1:10 and 1:20dilution of the tetanusantisera. Increasingamounts of toxoid (10to 165 ,ug/ml) were added to tubes containing constant amounts of the antibody-containingfraction. Both theantigen and the antibodywerediluted inPBS and incubated together for1 h at 37°Cfollowed byincubation for 4 days at 4°C. The tubes were thencentrifuged for 30 min at 1,000 x g, and the supernatants were testedfor the presenceofantigenorantibody by Ouchterlony analy-sis andvisuallyby the Swift-Wilson-Lancefield capil-lary tube test (14). Equivalence was determined as that tube in which neitherantigen norantibody could be detected. The supernatants were then tested in the immunecomplex assay.
Potential interfering substances. Double-stranded (ds) DNA(calfthymus DNA, type I)waspurchased from Sigma Chemical Co., St. Louis, Mo. Single-stranded(ss) DNAwasprepared by heating the native DNA at100°C for10min at aconcentration of 0.5 mg/ ml in PBS, followed by immediate cooling in an ice bath.Liquaeminsodium heparin was purchased from Organon, Inc.,WestOrange, N.J.
Lipopolysaccharides (LPS) from the following bac-terialpreparations were purchased from Sigma: phe-nol extract ofSalmonella minnesota, S. typhosa, S. typhimurium, and Escherichia coliO111:B4; trichloro-acetic acidextractof S. enteritidis, S. typhosa, and S. typhimurium. The trichloroacetic acid extracts of S. VOL. 15,1982
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80
40
0 A
10 25 50 100 200
AGGREGATED IgG(Cug/ml)
FIG. 1. Linearity and precision of the standard curve.Eachpointrepresentsthemeanof 30 separate determinations of each AGG standard. Vertical bars representstandarddeviation from the mean.
minnesota 9700 and ofE. coli 0111:B4 were pur-chasedfrom Difco Laboratories, Detroit, Mich. The lyophilizedpreparations were diluted in normal saline and heatedtoboiling for 10 minto ensuresolubility.
RESULTS
Linearityandprecision ofthe standard curve.
Standard curves were constructed by assaying AGG at concentrations of 10, 25, 50, 100, and 200 ,ug/ml in PBS. To determine linearity and reproducibility the AFSU values of 30 separate curves were examined. The results shown in Fig.1illustrate themeanandstandard deviation of the fluorescence obtained with each AGG standard. Least-squares linearregression
analy-IL
sis resulted in a correlation coefficient of 0.996,
and the slope of the curve was 0.93. These results indicated that a nearlyperfect relation-ship existed between the concentration of AGG
and the degree of fluorescence produced, and that the binding of AGG to SP-Clq followed a linear dose-response curve.
Assay of complexes formed in vitro. IgG-anti-IgG complexes were prepared by using human
monomeric IgG and goat anti-human IgG anti-body. Werecognized that quantitation ofthese prepared complexes would be based on the
binding ofFITC-anti-human IgG to the antigen
(human IgG) bound in the complex. Therefore,
inaddition toverifying the detection of antigen-antibody complexes, weassessed the adaptabil-ity of this assay as anantigen-specific method. Controls which contained antibody or antigen alone were included. The goat anti-human IgG antibody alone produced fluorescence
equiva-lent to thatofthebuffer blank.Thefluorescence of the human monomeric IgG alonewasminimal at all concentrations tested (Fig. 2). Soluble complexes were detected from equivalence to
five timesantigen excess atall antibody
concen-trations.Complexesinantibodyexcess were not
detected.
Soluble tetanus toxoid-antitoxoid complexes
were tested in a similar manner, and controls
wereincluded which contained antigenor anti-body alone. The tetanus toxoid antigen alone producedfluorescencenohigher than that of the buffer blank, and fluorescenceof the antitetanus antiserawasminimal(Fig.3). Soluble
complex-es were detected from equivalence to three timesantigenexcess. Again, complexes in anti-body excess were notdetected.
.05 .2 .4 .6 .8 2 4 6
equivalence
(A, E
Z-
32u P
3e
UJ
1--ag/ab RATIO
FIG. 2. Detection of solubleIgG-anti-IgG complexespreparedin vitro.Complexeswereformed with 20(A), 50(A), 100 (0), and 500(0) ,ug of goat anti-humanIgG antibody/ml andvarying concentrations of human monomericIgG.Theantigen-antibodyratioatequivalencewasapproximately0.4.The control valuesshownare
theresultingFSU of human monomericIgGaloneatthehighestconcentration used in each assay: 120(A),300 (A),600(0),and3,000(0) ,ug/ml.
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FLUOROIMMUNOASSAY FOR IMMUNE COMPLEXES 459
ii
LI
0.5 1 2 3 4
equivalence
TETANUS TOXOID EQUIVALENT AMOUNT
FIG. 3. Detection of soluble tetanus toxoid-anti-toxoid complexes preparedin vitro. The complexes
wereformed witha1:10(0) and 1:20 (A) dilution of
human antitetanus antiserumandvarying
concentra-tions of purifiedtetanustoxoid.The concentration of antigen compared with equivalence is expressed as
equivalent amount. For the 1:10 and 1:20 antibody dilutions, this was40 and25 ,goftoxoid/ml,
respec-tively. The control values shownarethe resultingFSU of the 1:10(0)and 1:20(A) antibodydilutions alone.
Effects of EDTA and56°C heat inactivation on
Clq binding of AGG in human serum. Prelimi-nary investigations demonstrated that addition
ofexogenousClqtoseracontainingAGG inhib-itedbinding of AGGtoSP-Clq. Itwasapparent that test samples must be treated to eliminate this competitive effect. The commonly used techniques areadditionof EDTAorheating the
testseraat56°C for 30min. An experimentwas
designed similar to one described by Zubleret al. (26) to compare the effects of EDTA and heatingonthebinding of AGGtoSP-Clq andto determine theappropriate handling oftest
sam-ples. The experimental designwas asfollows. (i) AGG was added to untreated NHS at a
concentration of 200,ug/ml.Aftera30-min
incu-bation at 37°C, portions were diluted 1:2 in either PBS or 0.3 M EDTA, pH 7.5. The final concentrationof AGGin the NHSwasthen 100 ,ug/ml. Each samplewasagain divided into
por-tions and incubated for 30 min at either 37 or
560C.
(ii) NHSwas diluted 1:2in0.3 MEDTA, pH
7.5 (NHS/EDTA). Then AGG was added at a
final concentration of 100 ,ug/ml followed by incubation for 30 min at 37°C. Portions were
furtherincubatedfor 30 minateither 37or560C.
(iii) NHSwas heat inactivated for 30 min at 56°C (NHS-560C). AGGwas addedata
concen-tration of 200 ,ug/ml, and the samplewas
incu-bated for30minat37°C. Portions were diluted
1:2 in either PBSor0.3 M EDTA, pH 7.5. The
final concentration of AGGpresentin the
NHS-56°Cwasthus 100,LgIml. Each portionwasthen incubatedfor 30 minat37°C.
(iv) Each of the abovesampleswastested in
theimmune complexassay. For allofthe condi-tions, controls without AGGwerealsotested.
The results are shown in Fig. 4. Addition of EDTAtoNHSeither beforeorafter theaddition of AGG didnoteliminate theinhibitory effect of endogenous serum Clq. After incubation of AGG with NHSorNHS/EDTA, heatingat56°C partially restored thebinding of AGG with SP-Clq. It isapparent thatheatingdid not destroy
all complex-boundClqbut did allow for
signifi-cantlygreaterbinding. Detection of AGG added
toNHS-56°Cwasessentially thesamewhen the samplewasdiluted in PBSorEDTA,suggesting thatEDTAitselfwasnotadversely affecting the reaction. The Clq-binding activity ofthe
nega-tive controls is shown for each sample. Heating NHSat56°C resulted in onlyaslightincrease in Clq binding in thisassay.Therefore, it appeared that theincreased detection of AGG after heat inactivation of positive sampleswas due tothe release of bound endogenous Clq rather than nonspecific aggregationof immunoglobulin.
Effect of 56°C heat inactivation on the detection of immunecomplexesinseraof SLEpatientsand normal controls. Serum samples from nine
sys-temic lupus erythematosus (SLE) patients and nine normal individuals were tested in the,
im-munecomplexassaybefore andafterheatingfor 30 min at 56°C (Fig. 5). All sera from patients with SLE contained significant levels of anti-bodytodsDNA(>25ngof DNA bound/25,ulof
serum) whentestedbyRIA. Ameanincreaseof 6.0,ugequivalents (eq) of AGG/mlwasobserved
in the sera of normal individuals after heat
treatment.Themeanincrease after heatingsera
of SLEpatientswas16.4
jig
eqofAGG/ml. This increase was more than 2 standard deviations above the mean increase seen in the normal controls, suggesting that the increased Clq-binding activity was not due merelytononspe-cificaggregation alone.
Subsequently, 54 human serum samples ob-tained from apparently healthy donors were
heated for 30min at56°C and assayed in
tripli-cate.Themeanvalueobtainedwas15.98, and1
standard deviation was 5.85. To determine the
upper limit of normal, the data were ranked from low tohigh, and the95% confidence value was
calculated. The normal range wasfoundtobe 1 to 25
jig
eq ofAGG/ml.Detection ofAGG inthe presence of NHS. The accuracy and precision of this method were studied by adding AGG to heat-inactivated NHS. Controls were prepared to contain
ap-proximately 10, 50, and 150 ,ug ofAGG/mland were tested in triplicate in 10 separate runs.
Serum without added aggregates was included VOL. 15,1982
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INCUBATION AND PRETREATMENT OFSPECIMENS
0
0)
CD a
w
I-0
0
0
z
a
0)120
rAGGREGATEDIgG
NHSN NHSE NHSEDTA NH56C
I
I
Ie
PBS EDTA 37°C PBS EDTA
37C 56C 37t'C 37576iC 37C 3C
Wu L] 00
L~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~..
100o
801-60
-40
-
201-OL
FIG. 4. Effects of EDTA and56°CheatinactivationonClqbindingof AGG in NHS.Symbols: O,Specific Clq-binding activityof eachsample;*, Clq-binding activityofserumcontrols without AGG.
witheachdetermination,andthefluorescenceof
thisnegative samplewassubtractedfromthatof the serum containing AGG. There was good
agreementbetween the measured andexpected concentrations of AGG at all levels, and the resultswerereproducible (Table 1).
Intra-assayprecisionwasassessedby
prepar-ing controls to contain 25, 100, and 150 ,ug of AGG/ml. Table2illustratesgoodreproducibility
when 20replicates of each controlwere tested. Effectsofpotentialinterferingsubstances. The susceptibility of this method to interference by Clq-reactive materialswasassessed byaseries
of competitive inhibition experiments. Fixed volumes ofpotential interfering substanceswere added in various concentrations to portions of controls containing known amounts of AGG. Percentageof inhibitionwascalculatedby
com-paring the amount of AGG detected in the presence of each substance with the amount
detectedwhenonly PBS had been added to the
positivecontrol.
(i) Bacterial LPS. Five different types ofLPS
prepared by two different purification proce-dures were examined. Each preparation was added at a final concentration of 200 ,Ig/ml to
sera containing AGG. In all instances, 95% or moreof the AGG could be detectedonly in the
presenceof200 ,ug of LPSper ml.
(ii)ds and ss DNA. Increasingconcentrations
of dsDNA(1.0to300 pug/ml)or ssDNA(0.5to 100 ,ug/ml) were added to portions of serum
containing50,ugofAGG/ml.Aslittleas5 ,ugof
ssDNAand 30,ugof ds DNApermlinterfered
inthis assay(Fig. 6).
(iii) Heparin. Increasing concentrations of heparin (1.0 to 200 U/ml) were added to sera
containing 70 or 150 ,ug ofAGG/ml. Inhibition
wasdetectableatheparin concentrations of5U/ ml (Fig. 7). Extremely high concentrations of
heparin were required to totally abolish the
binding ofAGG toSP-Clq.
Comparisonofthesolid-phase FIA witha fluid-phase RIA. Sixty-eightserumsampleswerefirst tested for immune complexes at Scripps-Miles Immunology Reference Laboratory by the
[1251I]Clq-binding
assayas describedbyNydeg-ger et al. (16).The specimens were heat inacti-vated, tested,andfrozen at -70°Cbeforebeing shipped to this laboratory. Upon receipt, the
specimensremained frozenat -70°C untilbeing
tested in the solid-phase FIA. On the basis of
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0)
uu
0
cn
1--z
LU
-I
a wU
70
1-60
F-
501-40 30
0
20
1o0-I
0
0 0
r
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0
UNTREATED 56C UNTREATED NORMAL SLE PATIE
FIG. 5. Effect of 56°C heat inacti detection of immune complexes in se tientsandnormal controls. Serum samp in thenativestateand afterheatingfor Normal, borderline,andpositiveranges edafterestablishingimmunecomplexIt healthyindividuals.
positive and negative results, the showed70%agreementandnosigr enceatthe80% confidence intervE
DISCUSSION Results of this study suggest t] useful alternative method for qu
immunecomplexes boundtosp-Cl
systemused in thisinvestigation is the plastic samplers used as the
matrix. In addition, the system re
cialized fluorometer to accommod pler. Nevertheless, the techniques this study should be readily adapt
* solid-phase surfaces, allowing forquantitation in * conventionalfluorometers.
As withmostClq-bindingassays, AGG prep-arations were used to standardize the method. The aggregates could bestoredat-70°C forup to4weeks and consistentlygaveahighly
repro-ducible standard curve. The curve was linear
* (slope, 0.93), and the fluorescent intensity
corre-latedwellwith theconcentrationof AGG intro-duced into the system(r = 0.996).
* POSITIVE Thereproducibility and sensitivity ofthis
as-say were assessed by adding AGG to heat-inactivated NHS. There was good agreement
ft INEbetween the measured and expected
concentra-BRDERLINE
tionsof AGGatalllevels,and thisassayprovedcapable of detecting immunoglobulin aggregates at concentrationsaslowas10,ug/ml. The
abso-NORMAL luteamountof AGGdetectable in the volumeof
serumselected for the testwas 0.5 ,ug.
The FIA was applied to the detection of in 5ec vitro-formed
IgG-anti-IgG
and tetanus toxoid-antitoxoid soluble complexes. Detection ofENTS thesecomplexes wasvery efficientin therange
ivation on the ofequivalence tomoderate (three to five times) ra of SLE pa- antigenexcess butappeared less sensitive with
les weretested smallcomplexes formed inextremeantigen
ex-30minat56C. cess. Complexes in antibody excess were not sweredelineat- detected. Thus, this method may be applied to
evelspresentin the detection of clinically significant immune
complexes. The antigen-antibody ratio is an
important factor in determining the fate and biological activities of immune complexes. twomethods Slight to moderate antigen excess results in iificant differ- complexes of intermediate size which arelarge
al (Table 3). enough to activate complement but are not readilyrecognized and cleared from the circula-tion by phagocytic cells (25). Therefore, these complexes may have the greatest pathogenic hat FIA is a potential, and we have demonstrated that they
Lantitation of aredetectable with the FIA.
Iq. The FIAX Theflexibility of this methodwas
demonstrat-costly dueto ed byassaying preformed IgG-anti-IgG immune e solid-phase complexes. Quantitation of these complexes quiresa spe- was basedon the binding ofFITC-anti-IgG to late the sam- the antigen (human IgG) bound in thecomplex.
described in This immunoassay may thus be adapted to an
table to other antigen-specific method by using the appropriate
TABLE 1. Accuracy and interassayprecision
Expected Detected(tjg/mI)b
(IWg/ml)a
Mean Range SD CV(%)10 13 9-18 2.7 20.9
50 64 59-70 3.7 5.8
150 152 136-171 11.9 7.8
a The indicated concentrations ofaggregated IgG wereadded to heat-inactivated normal human serum. b Values listedaretheresults of 10 determinations on10separatedays. CV, Coefficient of variation.
TABLE 2. Intra-assayprecision
Expected Detected(qg/ml)b
(,Lg/ml),
Mean Range SD CV (%)25 25 21-31 2.7 10.8
100 92 87-100 4.3 4.7
150 164 127-202 20.9 12.7
aThe indicated concentrations of aggregated IgG wereadded toheat-inactivated normal human serum. bValues listed are the results of 20 replicates as-sayed in thesamerun. CV, Coefficient of variation. VOL. 15,1982
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I-z 0-0
DNA ADDED (,ug/ml)
FIG. 6. Effect of ss and ds DNAonthebinding of AGG to sp-Clq.Varying concentrations of ss(@)or ds (A) DNAwereadded to serumcontaining 50p.gof AGG/ml. Percentage of inhibition was calculated by comparison with aggregated IgG detected in the absence of inhibitors.
fluorescein-labeled antibody. Similarly, other
classes or subclasses of antibodies bound in immune complexesmaybeidentified by altering the FITC-labeled probe accordingly.
Endogenous serum Clq wasfound to inhibit thebinding of immune complexes and AGG to
SP-Clq. This isnotsurprising, since most Clq-binding assays are similarly affected (19, 21). Pretreatment oftest sera with EDTAis a tech-nique used by others (8, 26)toeliminate
endoge-nous Clq interference, but was found to be ineffective inour assay. EDTAchelates calcium
necessary for integrity of the Clqrs complex. However, calcium is notneeded for binding of Clq with complexed immunoglobulin. Appar-ently, addition of EDTA in this solid-phase
assay will not "unmask" immune complexes which have boundcomplement in vivo.
10
8
6 4 2
1U_
10
_
v
!0 -}7
0^
I
0
I~~~~~~~~~~~~~~~~~~1
1 5 10 25 50 100 200
HEPARIN (units/ml)
FIG. 7. Effect ofheparinonthebindingofAGGto
solid-phase Clq. Various concentrations of heparin
wereaddedtoserumcontaining 70(0)or150(A) Fgof
AGG/ml. Percentage of inhibitionwas calculated by
comparisonwith theamountofAGG detectedin the absence ofheparin.
Heating the sera at56°C before testing under thedescribed conditionswasfoundtobe
essen-tial, even though we recognize that there are
problemscreated by thistreatment. Heat inacti-vation of the test sera after addition of AGG partially destroyed the inhibitory effect of
en-dogenous Clq. However, the levels of detection
were somewhat lower than when AGG was
added to preheated sera. These findings are
consistentwith those of Teppo and Wager (24), who reported that free Clqis more heat labile than Clq boundto immunecomplexes. There-fore, thesensitivityof the FIA fordetecting low levels of immune complexes which have fixed complement in vivo may be somewhat dimin-ished.
Heatingsera at56°C before detecting immune complexes has often been criticized. Some in-vestigators havefoundfalse-positive results due
to heat-induced aggregation ofserum immuno-globulins (5). Other studies have shown that heating test sera may decrease the ability of preexisting complexes to bind exogenous Clq (19, 26). When sera from normal individuals
weretested in the FIA, no significant evidence
TABLE 3. Comparison of the solid-phaseFIAanda fluid-phaseRIAfor the detection of immune
complexesa
Assay No. No: Total
positive negative
Solid-phaseFIA 25 43 68
Fluid-phaseRIA 39 29 68
Total 64 72 136
a Positiveindicates abnormal levels of immune com-plexes for agiven assay. Negative indicates normal levels of immunecomplexes foragiven assay. -x2
5.0;P<0.025
0
zO
m
I1
,0%
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463
for heat-induced aggregation was observed. However, when sera from patients with SLE weretested,thisassay was ableto detect
com-plexes insevenof ninepatients ifthe sera were heat inactivated before testing. The increased levels of complex detection were more than 2
standard deviationsgreaterthan that seen with
normal seraand thus couldnotbeexplained by nonspecific aggregation. No decrease in Clq bindingwasobserved inanyof the samples after heating, which indicates that complexes were notdestroyed by this treatment.
The specificity of this assay wasinvestigated by competitive inhibition studies. Since this method uses a fluorescein-labeled anti-IgG probe, non-immunoglobulin substances that bind Clq willnotproducefalse-positive results. However, iftest sera contain antigen-antibody complexes aswellascertainotherClq-reactive substances, assay values may be falsely
de-creasedduetocompetitive inhibition.
Although LPS has been showntoactivateClq (4), concentrations ashighas200,ug/ml didnot
interfere with the detection of AGG.
Single-stranded DNAand,to alesserextent,
ds DNA were foundto inhibit binding of AGG with SP-Clq. This was probably the result of a
firm ionic interaction between the positively charged Clq molecule and these anions (3). In
addition, the strength ofbindingtoClq may be dependentonmolecular size(10). High-molecu-lar-weight substances such as DNA may be capable ofinteracting with several Clq-binding sitessimultaneouslyormaysimply induce steric hindrance. Thereaction conditions of thisassay
included low-ionic-strength buffer to obtain maximalsensitivity for complexes of IgG. Most
anionic substances also bindmoststrongly with Clq under this condition (5, 10). Possibly, the
inhibitory capacity of anionic substancesmaybe decreased by performing this assay at higher ionicstrength.
Undoubtedly, interference by such anions isa
limitationtothespecificity of this method, but it isone that is shared by mostimmune complex
assaysusingClq. One major difference,
howev-er,is that in otherClq-bindingassays, withthe
exception ofthe solid-phase method described by Hayetal. (8), theinterference byDNAmay be manifested as falsely elevated levels of im-mune complexes (12). Therefore, these
tech-niques might not discriminate between free DNAantigenaloneandimmune complexes
con-taining DNA. Of the existing methodologies using Clq, it is possible that only direct
Clq-binding assays using labeled
anti-immunoglob-ulin canaccurately distinguish clinically signifi-cant immune complexes from DNA. This
suggestion may partially explain the finding of
Abrassetal.(1)thatthesolid-phase Clq-binding
RIA correlated better than the fluid-phase Clq assay with clinical activity in SLE. However,
the fact that low levels of complexes may go
undetected in the presence ofDNA cannot be
ignored. Detailed clinical studies were not con-ducted in ourlaboratory,but sera from seven of nine SLE patients tested were shown to have
elevated levels of immune complexes. These results areconsistent with those found in
exten-sive studies of SLE (1, 16, 18). In pathological conditions, antigen-antibody ratiosmay change daily (7). Interpretation of immune complex assay results must necessarily take all these
factors into consideration.
Heparinwas also foundtoinhibit the binding of AGGtoSP-Clq. As withDNA, the effect of thispolyanion is probably relatedto negatively chargedgroupsinteracting with the cationic Clq (2). Competitive inhibition studies demonstrated that approximately 5 U of heparin per ml of
serumwas required tointerfere with the detec-tion ofAGG. The primary significance of this
finding is that samples to be tested by this method must not be collected in heparinized tubes. Although it has not been verified, sera
from patients undergoing carefully controlled heparin therapy may still be suitablefor testing by this method. Patients receiving therapeutic heparin doses generally have plasma levels of 0.1 to 0.5 U of heparin/ml (6, 23). However, heparinabsorption and excretion ratesvaryfor each individual, and again, potential
interfer-ence mustbe considered when thisClq-binding
assayis used fordetection of immune
complex-es.
A comparative study was performed on 68 samples that hadpreviouslybeen testedby
fluid-phaseClq-binding RIA. The two methodswere
concordant in assigningnormal orelevated lev-elsofimmune complexes in 70% of the samples tested. Our data indicate thatthe fluorescence method is subjecttothesametypesofproblems
commonto mostClq-bindingassaysbutsuggest
thatthetechniquemaybeasreliable in quantita-tion of immune complexes asother assays
cur-rently available. Investigationsarein progressto
find alternatives to heat inactivation and to
minimize the effects of interfering substances. However, theprimarysignificance of thisreport isthat FIApresents analternativeto RIA for the
detection ofimmunecomplexes.
ACKNOWLEDGMENTS
WeareindebtedtoMarciaWileyandLorineAsamotoof
Scripps-MilesImmunology ReferenceLaboratory for provid-ingserumsamples.WealsothankDavidMilichforvaluable advice andCharles CannadyandSusanPappasfortyping the manuscript.
Thisstudywassupported inpartby Public HealthService grantHD-03939 from the National InstitutesofHealth. VOL.15,1982
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