Comparison of a fluorometric method with radial immunodiffusion assays for determination of complement components C3 and C4

JOURNAL MICROBIOLOGY,Aug. 1982, 0095-1137/82/080271-05$02.00/0

Comparison

of

a

Fluorometric Method with Radial

Immunodiffusion Assays for

Determination of Complement

Components C3 and C4

MARGOTKOELLEANDWILLIAM R. BARTHOLOMEW*

Division ofClinical Microbiology andImmunology,Erie CountyLaboratory, Erie CountyMedical Center,

Buffalo,New York 14215, and Department of Microbiology, School of Medicine, andDepartmentofMedical

Technology, School ofHealth RelatedProfessions, State University ofNew YorkatBuffalo, Buffalo, New

York14214

Received 23 November1981/Accepted 29 April1982

Measurementsof patientserumcomplementcomponentsC3 and C4areuseful

indicators ofcomplement consumption in immune complex diseases. A fluoro-metric quantitative immunofluorescence system was evaluated in terms of

mea-suring these complementcomponents,and the resultswere compared with those

of radial immunodiffusionassays.Forcomparison of thetwosystems,232patient

sera wereevaluated for C3, and 202 specimensweretested for C4.Analysis of the

data by linearregression indicatedaproportional difference between the methods.

C3 andC4 concentrations measuredby the fluorometric method werelowerthan

those measured by radial immunodiffusion, especially concentrations exceeding thenormalranges.Indetecting lower concentrations (<120mg/dlfor C3 and <25

mg/dlforC4), thetwomethodsshowed betteragreement. Eachassaysystemwas

reproducible and could be usedtoevaluate changes thatoccurinconcentrations ofcomplementcomponents during therapeutic treatment. However, the ease in processingalarge volume ofspecimens and the short time neededtocomplete the

assay areadvantages that make the fluorometric methodmoresuitable thanradial

immunodiffusion foruseinalarge clinical laboratory.

Concentrations of

complement

components

C3 and C4 in serum are used as indicators of

complement

consumption

in immune complex diseases. Concentrations of C3 and C4 have

beenofparticular valueasanaid in thediagnosis and management of

systemic

lupus

erythmato-sis.Monitoring the concentration of theseserum

complement

componentshas beenusedto deter-mine the effectiveness oftherapeutic treatment

of this disease (1,

7).

The quantitation ofserumcomplement com-ponentsis

usually

performed by

radial immuno-diffusion (RID). RID assays are based on an

antigen-antibody precipitation reaction. Antigen

is added to wellscut in anagarose matrix which

contains anoptimal concentration of

monospe-cific antiserum. In the method of Fahey and

McKelvey(6), theprecipitin ringsaremeasured within aspecifictimeperiod;complete diffusion

is notnecessary. Theprotein concentrations of

the specimens are determined by interpolation fromthe standard curve onwhichthediameters oftheprecipitinringsareplottedagainstthelog

of concentrations ofthe standards. One ofthe more recently developed test systems for the

quantitation of complement components and otherserum

proteins

isasolid-phase fluoromet-ricassay (3, 9).

Thepurposeof this studywastoevaluatethe

quantitative immunofluorescence (QIF) system

forthe

quantitation

of

complement

components

C3 and C4 and tocompare this

technique

with traditionalassays.TheQIFassayis basedonthe reaction ofserumcomplementcomponentC3or

C4 with fluorescein

isothiocyanate

(FITC)-la-beled

monospecific

antibodies in a buffer solu-tion. After a 20-min

incubation,

the unreacted antibodiesareadsorbedontosmalldisks coated withC3 orC4. The disks are 6mmin diameter

and attached to the lower portion of plastic

probes, whichcan easilybeinsertedinto 75-by

100-mm test tubes.After adsorptionofthe

resid-ual labeledantibodies,theprobesarewashedin

buffer,and the amount of fluorescence

emanat-ing from the disk is determined by insertion of

theprobe intothe specifically designed

fluorom-eter. The amount offluorescence, measured in

fluorescentsignal units (FSU), is inversely

pro-portional to the concentration of the

comple-mentcomponent in the serum.

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The precision of the assay systems, as wellas

the total time for performing each assay, is discussed in this study.

MATERIALS AND METHODS

Serum samples. We evaluated the QIF system by

testing 232 patient serum samples for complement

component C3and 202samplesforC4, usingboth the

fluorometric and the RID methods. The sera were

randomly selected from hospital and clinic samples

submittedforcomplement assays. Many ofthe

speci-mens from clinic patients were from systemic lupus

erythmatosispatients who werebeingmonitored.

Normal rangesfor complement componentsC3and C4 wereestablished bytheQIFsystem,usingseraof

102 healthy adults. These sera were donated by

healthylaboratory personnel.

Bloodspecimenswereheldatroomtemperature for

1 hafterbeingdrawn toallowaclottoformand then

heldat4°C for1 h sothat theclotwould retract. At this

point, thespecimens wereimmediately centrifugedat

500 xg for 10min,divided intoportions,and stored at

-70°C until assayed.

Fluorometric analysis. (i) Instrumentation. The

FIAXfluorometer(InternationalDiagnostic

Technolo-gy,SantaClara,Calif.)wastheinstrumentusedforthe

QIF assays. Itisequippedwith atungstenlightsource.

Theexcitationwavelengthis 475nm, and the emission

is readat 540 nm. Aspecifically designed stage holds

theplasticprobes, which haveattached, cellulose-like

disksonwhichthereactions takeplace. A

microcom-puter interfaced with the fluorometer was used to

obtain a standard curve and calculate the values for

each specimen. A horizontal shaker was utilized to

agitate the tubes during the washing and various

incubation periods. It is designed to hold test tube

racks at a450 angleand operatesat200oscillationsper min at a distance of2.5 cm. All dilutions were

per-formed with a pipette diluter (model 1500; Cavro

Instruments, Los Altos, Calif.). This instrument can

beadjusted from5to50,ul for thedeliveryofreagents

orserumsamples,and thediluentmaybesetfrom0.25 to 1.0ml.

(ii) Reagents and method. Testkits forthe

determi-nations of C3 (3C/431A) and C4 wereobtained from

International Diagnostic Technology. The procedure

described by the manufacturer was followed without

modification.

To startthecomplementassayforC3,we mixed50 ,ul of a 1:101 dilution of standard or patient serum with 50 u1l ofFITC-labeled monospecific antiserum in 500

p.l ofphosphate-buffered saline (pH 7.4). Themixture

was incubated for 20 min. The tubes were placed on the horizontal shaker at the beginningof the

incuba-tion period. During the same time, thereaction

sur-faces of the disks were coated with the C3complement component by insertion of the plastic probes with attached disks into 500 p.l of the component reagent supplied with the kit. After 10 min, the probes were removed and placed into a second row of tubes

containing phosphate-buffered saline (pH 7.4) for a

wash. The disks were then ready to be used in the

assay and were placed into the incubated

serum-antiserum mixture. Residual, monospecific,

FITC-la-beled antibodies to C3 were absorbed by the C3-coated disks during a 20-min incubation. The disks

were then washed in phosphate-buffered saline (pH

7.7), and the amount offluorescence attributable to residual labeled antibody was determined with the

fluorometer;theintensitywasexpressedinFSU.The standardcurve wasbasedonthe FSU of four standard sera suppliedin thekit, FSU being inversely propor-tionaltothe concentrationsof C3in thestandardsera. The levels of C3 in patient serum samples were

determinedby interpolation from the standardcurve.

For the C4 assay, 20 p.l of a 1:13.5 dilution of standardorpatientserumwasmixed with 20p.l ofthe

FITC-labeled monospecific antiserum in 500 p.l of

phosphate-buffered saline (pH 7.4). The incubation

periods,aswellastheprocedureforcoatingthedisks withC4,werethesameasthose used for the C3 assay.

The determination ofFSU and standard curves were

similartothe determinationofC3.

RID. Test kits wereobtained from Meloy

Labora-tories, Inc., Springfield, Va. Theprocedure employed

wasthatgiven onthe insertincluded in each kit. The

incubation time for the C3determinationswas 18 hat

4°C, and that for the C4determinations was 22 h at 37°C. The antiserum used in the agarose for the measurement of serum C3byRID wasshown to react

with

P3IC

and ,1A. The diameters of the precipitin

rings were measured with a vernier caliper and a

viewer-magnifier (National Instrument Laboratories,

Inc., Rockville, Md.). To obtainastandard curve,we

plottedtheconcentration ofeach serumstandard (log

[ordinate]) againstthediameterofeachprecipitinring

(abscissa) on semilog paper. For each new lot, we

establisheda standard curveby plotting threevalues

for each control serum. Each time the assay was

performed, high and low standards were included on

each plate and checked against the values of the

standard curve toensurethat the kitwasfunctioning

satisfactorily. Patientand standardsera wereassayed

in the same manner. After diffusion, the precipitin rings were measured, and the concentration of

com-plementcomponentin each patientserum was

deter-minedby interpolation from the standardcurve.

RESULTS

Complement concentrations found in sera of healthy individuals. The normal ranges for the complement components C3 and C4, as

mea-sured by thefluorometric system,werebased on the analysis of 102 sera. Thefrequency distribu-tion curves, plotted in increments of 0.5 stan-dard deviation, showed a bimodal distribution. The Kolmogorov-Smirnov test, used to deter-mine whether the observed data followed a

normaldistribution, indicated no significant de-viation for C3 (P>0.05) butdid show significant deviation for C4 (0.01 < P <0.05). On the basis of the mean ± two standard deviations, we calculated the normal range for C3 to be 72 to 170mg/dl (mean, 121 mg/dl), and thatfor C4 to be 12 to 46mg/dl (mean, 29mg/dl). The range for C4, calculated fromnonparametric data (exclud-ing 2.5% of the highestvalues and 2.5% of the lowest), ranged from 13 to45 mg/dl.

Comparison of the fluorometric system with

RID. The fluorometric system was compared

J.CLIN. MICROBIOL.

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FIG. 1. Quantitation of C3: comparison of QIF andRID. Thestatisticalanalysis is shown in Table1.Line of

best fit:y= 45.7 +0.421x;n = 232; Sy.x= 19.2. Thehighest pointnotplottedbutincluded incalculationswas

137,368 (y, x).

with RIDin terms ofmeasuring C3 and C4. A

total of 232 serum samples, 91 ofwhich were

from

patients

with

systemic

lupus erthymatosis,

were analyzed for C3. For the C4 evaluation, 202 serum samples were analyzed, 91 ofwhich

werefrom patients withsystemic lupus

erythma-tosis.

The results forC3 obtained bythe

fluoromet-ric assay wereplotted againstRID results (Fig. 1),using theRIDassay as thereference method. The data for C4 are

plotted

in Fig. 2. The

statistical analyses of C3 and C4

by

linear

re-gressionare summarized in Table 1.

For C3, the range of concentrations plotted varied from 25 to 368 mg/dl. The slope of the

regression line for C3 was 0.421, and the

inter-cept was45.7.

Considering

thataslope of1.0, an

intercept of0, and minimal random error

indi-cate anideal bivariate relationship, the data for C3 show a

proportional

as well as a constant

difference

between the twoassay systems. Re-sults ofQIF were lower than those of RID. The random error was 19.2 mg/dl, asdefined by the standard deviation for distribution of the ob-servedy values above and below theregression

line

(syJ.)

The coefficient of correlation was 0.788.

ForC4,theplottedvaluesrangedfrom 4 to 73

mg/dl(Fig. 2).Theline ofbest fit was y = 5.3 +

0.694x. Theslope of0.694indicates

proportional

difference,

and the intercept (5.3) relates to

somedegree ofconstant difference in C4

mea-surement between the two assay systems. At

levels of25 mg/dl, both methods showed good

agreement. The random error, sy., was6.2 mg/ dl; the correlation coefficient, which relates to

the fit ofthe values to the regression line, was

0.855. Three outliers were excluded from the

data (4

syJ.)

Considering the large number of

data, the elimination of obvious outliers is

ap-propriate (4).

Precision of QIF and RID assays. (i) RID plates. The run-to-run precision of the RID

method for quantitating C3 and C4 was deter-minedby inclusion of high and low standards for

C3andC4 in eachassay.Thesestandards hadto

give ring diameters that fell within a predeter-minedrange tobe considered ashaving

accept-able

sensitivity

andprecision.Thecoefficientof

variation (CV) wascalculated from the

concen-trations (mg/dl) obtained for the high and low standards for C3 and C4. The CVfor C3, deter-minedby

using

thehigh standard in 61

determi-nations, was 8.24%. The CV forC3, based on resultsof the low standard in 59determinations,

was4.27%. In termsoftherun-to-runprecision

of the C4assay,the CV for thehighstandard(58 determinations)was6.74%,and thatforthelow standard (56determinations) was8.76%.

(ii) QIF assay. The within-runprecision ofthe C3 assay was determined by evaluation of a

standard serumeight timeswithin a test run. The CV was 3.1%. In terms of the C4 within-run precision, theCV was7.4%, determined on the

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FIG. 2. Quantitation of C4: comparison ofQIF and RID. The statistical analysis is shown inTable 1.Line of best fit: y =5.3 + 0.694x; n= 202;sY.X =6.2. Three outlierswereexcluded from data(>4

syJ.)

basisof 10 determinations. Therun-to-run

preci-sion for the C3 and C4 determinations was

determined with a freshly thawed portion ofa

serumpool (stored at -70°C) in each run. The

CVswerecalculated from atotalof 38

determi-nationsfor each C3 and C4. the CVfor C3 was

9.3%, and that for C4was9.7%.

TimecomparisonofQIFandRID assays. The

quantitation of C3 and C4in 10 serum samples

by the QIF system was accomplished in 3 h,

includingdilution of thesera,performanceof the

assay (which includes four standards and two

dilutionsof the control serumpertestrun),and

readingand reportingtheresults.

The RID assay of C3 and C4 in 10 serum

specimens tookapproximately 1 h. After 18hof

incubation for C3 and22h forC4,theprecipitin

ringsareread,theconcentrationsarecalculated,

and the results are reported. The latter three

tasks took a total of 2 h; thus, the total time

needed to quantitate C3 and C4 in 10 serum

specimens byRIDwasapproximately3 h.

How-ever, the time from receipt of the specimen to

submission ofa report wouldbe aminimum of

21to25 h forRID,whereas the total time for the

QIF systemwould be approximately 3 h.

DISCUSSION

TheQIF and RID systems werecompared in

termsof thequantitationofcomplement compo-nents C3 and C4. A total of 232 clinical

speci-mens were analyzed for C3,and 202 specimens were analyzed for C4. Analysis of the data by

linear regression indicated a significant

propor-tional difference forboth C3 and C4 (slopes of

0.421 and 0.694, respectively) and some degree

ofconstantdifference.QIFvaluesfor C3and C4

tended to be lower than RID values. This was

especially true for C3 in concentrations above 160 mg/dl: the concentrations determined by

RID were almost twofold greater than those

determinedby QIF.The correlation coefficients

were 0.788 for C3 and 0.855 for C4, which

TABLE 1. Quantitation ofcomplementcomponents C3 and C4: statistical comparison of theQIFand RID assaysbylinearregression analysis

Component No.ofspecimens Concn QIFy RIDx Correlation

ComPonent

tested

(mg/dl)

(mg/dl)

(mg/dl)

Slope Intercept Sy coefficient

C3 232 25-368 114.6 163.6 0.421 45.7 19.2 0.788

C4 202 4-73 25.7 29.2 0.694 5.3 6.2 0.855

0 0 0

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COMPLEMENT COMPONENT QUANTITATION 275

indicates that the correlations were 62.1 and

73.1%, respectively.

For the determination of serum complement component C3, RID and QIF are designed to measure serum fractions 131C and 13A, 31A beingabreakdownproduct of

P1C.

Of the three antigenic determinants present on

P1C,

only the A determinant is shared by

13A.

It has been

found that aged serum samples deplete more monospecific antibodies to the A determinant than are depleted by the amount of

PjC

in fresh serum(5,10).Davisetal. (5) have reported that when serum samples are held at 37°C for up to 7

days, the ratio of the A determinant in aged versus fresh sera is 1.56. These researchers

found no appreciable difference when serum

samples were held at 5°C. An autoanalyzer system was used for the determination of

j1C

and

P1A.

The fact that the serum specimens assayedinourstudy were stored at -70°C until

assayed rulesoutthe possibility ofany

apprecia-ble breakdown of

P13C

fromthe time the speci-men was received to the time the assay was

performed.

Bruver and Salkie (2) compared C3 values

obtained byusingtheHyland model PDQ

neph-elometerand the RIDmethod of Mancini et al.

(8). Forthe RIDmethod, the researcherschose ashortened

incubation

of16hat room tempera-ture, after which the diffusion rings were

mea-sured.Twokitswereused for the comparison of nephelometry and RID. In the analysis of the

data by linear regression, theresearchers

report-ed higher values obtained by nephelometric analysis than by RID. However, we observed that values forC3 obtained by the fluorometric method werelowerthanthose obtained by RID.

The correlation coefficient reported by Bruver

and Salkie (nephelometric assay versus RID) was similar to the value we obtained for the

fluorometric assay versus RID, 0.79 (Fig. 1).

Whenmonitoring

patients

for C3 and

C4,

both

thefluorometric andRID assays werecapable of detecting a change in complement component

concentration.

In terms ofthe within-run

precision

of

QIF,

the CV for C3 was

3.1%,

and that for C4 was

7.4%. Values for within-run precision ofRID have beenreported by Stevensetal.(9).TheCV

for the run-to-run precision ofthe RIDassays, based on the concentrations (milligrams per

deciliter)

ofhighandlowstandards, rangedfrom 4.27to

8.24%

forC3 and from 6.74 to

8.76%

for C4. Intermsof the run-to-run reproducibilityof

QIF, theCVs, based on a serumpool,were9.3 and

9.7%

for C3 and

C4, respectively.

TheQIF methodis

relatively

easyto

perform,

and valuesmay bereported withinthesame

day

that the specimen isreceived. The

QIF

system

providesahigh degree of

precision

in the deter-mination ofserumcomplement components C3

andC4.

Both methods are suitable for

quantitating

patientserumcomplementcomponents in clini-cal laboratories. The QIF system,

however,

provides a reliable alternative method for the

quantitation of C3 and C4. The results may be

reported on the same day that the specimen is received, whereas the RID method

requires

a

minimum of 2 days before the results can be reported.

ACKNOWLEDGMENT

Wethank Helen Lees, Department of MedicalTechnology, State University of New York at Buffalo, Buffalo, for her guidance with the statisticalanalysis.

LITERATURE CITED

1. Appel, A. E., L. B. Sablay, R. A. Golden, P. Barland, A.I. Grayzel,and N. Bank. 1978.Theeffect of normaliza-tion of serum complement and anti-DNAantibodyonthe courseof lupusnephritis.Am.J. Med. 64:274-283. 2. Bruver, R. M., and M. L. Salkie. 1978. Acomparative

study of three approachestotheroutinequantitationof human serumproteins.Clin. Biochem. 11:112-116. 3. Casavant, C. H., A.C. Hart, and D. P. Stites. 1979.

Comparison ofasemiautomated fluorescent immunoas-say system and indirectimmunofluorescence for detection ofantinuclear antibodies in humanserum.J.Clin. Micro-biol. 10:712-718.

4. Cornbleet,P.J.,and N.Gochman. 1979. Incorrectleast squares regression coefficients in method comparison analysis. Clin. Chem. 25:432-438.

5. Davis,N.C., C.D.West,and M. Ho.1972.Effect of aging

ofserum onquantitation of complement component C3.

Clin. Chem. 18:1485-1487.

6. Fahey, J. L., and E. M. McKelvey. 1965. Quantitative determinations of serum immunoglobulins in antibody agarplates. J. Immunol. 94:84-90.

7. Garin,E.H.,W. H.Donnelly,S. T.Shulman,R. Fernan-dez, C. Finton,R. L.Williams,andG.A.Richard. 1979. Thesignificance of serialmeasurementsofserum comple-mentC3 and C4 components and DNAbinding capacityin patients with lupus nephritis. Clin. Nephrol. 12:148-155. 8. Mancini, G., A.0. Carbonara, and J.F.Heremans.1965. Immunochemical quantitation of antigens by single radial immunodiffusion.Immunochemistry 2:235-254. 9. Stevens, R.W., D. Elmendorf, M. Gourlay, E. Strobel,

and H. A.Gaafar. 1979. Applicationof

fluoroimmunoas-saytocerebrospinalfluidimmunoglobulinG and albumin. J.Clin. Microbiol.10:346-350.

10. West, C. D., S. Winter, J. Forristal, and N. C. Davis. 1968. Effect ofaging of serumonconsumption of antibody by P3IC-globulin determinants; evidence for circulating

breakdown products in glomerulonephritis. Clin. Exp.

Immunol.3:57-62.

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