CLIN. CHEM. 32/1, 124-129 (1986)

124 CLINICAL CHEMISTRY, Vol. 32, No. 1, 1986

Turbidimetry of Rheumatoid Factor in Serum with a Centrifugal AnalyzerLuis Borque, Martin Yago, Carmen Mar, and Carmen Rodriguez

We describe the simple, rapid turbidimetry of 1gM rheumatoidfactor in human serum by use of the Cobas-Bio centrifugalanalyzer. Heat-aggregated human IgG is used as the anti-gen. The immunoturbidimetric reaction is monitored at 340nm for 300 s, and the changes in absorbance after theantigen is added are used to prepare the standard curve.Test results are calculated from the stored curve and report-ed in mt. units/mL, based on comparison with the WHO

reference serum for rheumatoid factor. There is no interfer-ence from bilirubin(up to 340 pmol/L) or hemoglobin (up to5600 mg/L). Serum samples with a triglyceride concentration>2.20 mmol/L must be cleared of lipids before analysis. Thestandard curve is linear from 30 to 500 mt. units/mL. Preci-sion, accuracy, linearity, and sensitivity are quite acceptable.The CV was generally <5% for different concentrations ofrheumatoid factor. Results agree well with those by a rate-nephelometnc procedure on the Beckman ICS system (r5 =

0.932). However, both correlate poorly with a modified classi-cal Waaler-Rose test. Of 47 patients with rheumatoid arthri-tis, 34 had 1gM rheumatoid factor in their serum, but themeasured value did not reflect the activity of the disease.

AddItIonal K.yphrases: immunoglobulin rheumatoid arthritisimmurioassay

The most consistent serological feature of rheumatoidarthritis is the increased concentration of autoantibodiesdirected against antigenic sites in the Fc region of humanand animal IgG, namely rheumatoid factors (RFs) in theblood and joint fluid (1, 2).’ The potential role of thesefactors in the pathogenesis of this disease has been studiedextensively (3-5), with the finding that both environmentaland genetic factors affect production of RFs, which arecomposed of a heterogeneous population of immunoglob-ulins having a wide spectrum of biological properties (6-8).RF determinations are clinically important for diagnosis (9),prognosis (10), and assessment of therapeutic efficacy (11) ofrheumatoid arthritis. Although RFs may be found in allimmunoglobulin classes (12, 13), the RF most frequentlydetected in the laboratory is 1gM type, present in about 75%of adult patients with rheumatoid arthritis (14) but in about10% of children with juvenile rheumatoid arthritis (15).

The conventional tests used in routine estimation of RFare based on its ability to agglutinate erythrocytes, latex, orsimilar passive particles attached to IgG (16-18). Theseassays have important disadvantages in routine clinical use,however, being difficult to quantify (at best being onlysemiquantitative), and yielding large variations in titers forthe same serum samples-not only among different labora-tories but also within laboratories (19, 20). In an effort toovercome these problems, several techniques have beendeveloped-including quantitative immunodiffusion (21),

radioimmunoassay (22,23), enzyme immunoassay (24,25),

Laboratorio de Bioquunuica, Hospital #{176}Ortizde Z#{225}rate,”01009Vitoria, Alava, Espa#{241}a.

1 Nonstandard abbreviations: Ig, immunoglobulin; RF, rheuma-toid factor; HA-IgG, heat-aggregated IgG; PEG, polyethylene gly-col.

Received August 30, 1985; accepted October 7, 1985.

and nephelometry (26, 27)-that offer the advantages ofhaving standardized reagents and being reproducible andquantitative. However, because these procedures generallyrequire specialized reagents and equipment, long reactiontimes, and multiple incubation and washing steps, and

because they are not easily automated, they are seldom usedin routine practice. With the introduction of automatednephelometers in the last few years, some of the aboveshortcomings have been overcome (28,29) and quantiflca-tion of RF has become more widespread.

Here we describe the use of the Cobas-Bio centrifugalanalyzer for turbidimetric measurement of 1gM RF as asimple alternative to the automated nephelometric proce-dure. The method is fast and reliable, it takes advantage ofthis analyzer’s sensitive detection system and its wideapplicability in routine clinical chemistry, and it obviatesthe need for expensive special apparatus.

Materials and Methods

ApparatusWe used a Cobas-Bio centrifugal analyzer (Hoffmann-La

Roche, Basel CH-4002, Switzerland) equipped with a DENS

(Data Reduction for Non-linear Standard Curves) program(version 8326).

Reagents

Bovine serum albumin (Cohn Fraction V) was obtainedcommercially (J. T. Baker Chemical B.V., Deventer, TheNetherlands); polyethylene glycol (PEG; M� 5000-7000) was

purchased from E. Merck, Darmstadt, D-6100 F.R.G.; DL-

dithiothreitol from Sigma Chemical Co., St. Louis, MO63178; and “Lipoclean” clearing agent from Behring Insti-tute (Behringwerke AG, Marburg, F.RG.). All other re-agents were analytical grade.

All solutions were prepared in distilled water, and filteredthrough 0.45-�zni (av. pore diameter) filters (Millipore Corp.,Bedford, MA 01730). They were stable for at least sixmonths when stored in well-stoppered flasks at 4#{176}C.

Cobas-Bw buffer. Borate buffer (50 mmol/L, pH 7.6)

containing 0.20 mol of NaC1, 15 g of PEG, and 15 mmol ofNaN3 per liter, with the pH adjusted to 7.6 with a 0.2 moIfLsolution of NaOH.

Antigen diluent. Borate buffer (50 mmol/L pH 7.6) con-

taining 0.15 mol of NaCl and 15 mmol of N3Na per liter, pHadjusted as above.

Cobas-Bio ‘�start” reagent. The working antigen reagentwas prepared just before use by diluting the heat-aggregat-ed IgG (HA-IgG, see below) 25-fold with antigen diluent.

Isotonic saline. NaC1, 0.15 moL’L (8.7 g/L), plus N3Na, 1g/L.

RF calibrator. The RF-positive control serum usedthroughout this study was pooled from 20 sara with titersgreater than 1/160 as determined by the modified Waaler-Rose test (30). Its potency (in international units per millili-ter) was established by the turbidimetric procedure outlinedbelow, with reference to World Health Organization (wHo)1st International Reference Preparation (IRP) of humanrheumatoid arthritis serum as standard. The RF content ofthe control serum was adjusted to 500 hit. unita/mL with thesaline and stored at -70 #{176}C.

1 Units2 Calculation factor3 Std I concn

Std II concnStd Ill concnStd IV concnStd V concn

6 LimIt7 Temp, #{176}C8 Type of analysis9 Wavelength, nm

10 Sample (undiluted) vol, �L11 Diluent (water) vol, �L12 Reagent vol, j�L13 Incubationtime,s14 Start reagent (antigen) vol, 1L15 Time of first reading, S16 Time Interval, s17 No. of readings18 Blanking mode19 Printout mode

in units/mL1000

31.362.5

125250500500

25.07.6

3403010

1001050

1.0300

2

Methods

CLINICAL CHEMISTRY, Vol. 32, No. 1, 1986 125

Collection. Blood was sampled into red-top VacutainerTubes (Becton Dickinson, Rutherford, NJ), allowed to clot,and the serum was removed for immediate assay or forstorage (at -20 #{176}C)ifanalysis was to be done later.

Isolation and aggregation of IgG. Human IgG was isolatedfrom a large poo1 of normal human sera by precipitationwith 45% saturated ainmomum sulfate solution, followed byelution from diethylainunoethyl-cellulose with sodium phos-

phate buffer (25 mmoJJL, pH 8.0). The IgG so collected was

concentrated by precipitation with 140 g/L sodium sulfate,dialyzed extensively against saline until the dialysates weresulfate free, and stored in aliquots at -70 #{176}C.Preparationsof IgG were tested for purity by immunoelectrophoresisagainst antisera to whole human serum; only a singleprecipitation line formed. Turbidimetry (31) of these prepa-rations in all cases yielded values for IgG exceeding 98%.

To IgG in saline, 50 g/L, was added bovine albumin to givea final concentration of 5 g/L. This mixture was heated for30 mm at 63 ± 1#{176}Cin a water bath. This heat-aggregatedpreparation (HA-IgG) was stable for at least three monthswhen stored at 4#{176}C.

Turbidimetiy. The present assay is based upon the turbid-ity formed from the reaction between the antigen (solubleHA-IgG), and the multivalent IgM-RF antibody in the sara.

The rapid generation of large complexes so formed is detect-ed spectrophotometrically at 340 nm. To accelerate theformation of the complexes we find it convenient to add asmall amount of PEG to the reaction mixture. Complementis inactivated by heating sample aliquots in a water bath at56 ± 1 #{176}Cfor 30 miii. Lipemic sara are delipidated bytreatment with “Lipoclean.”

Table 1 lists the instrument settings for the Cobas-Bio.The use of five standards extends the useful working rangefor the assay from 30 to 500 int. units/mL. Samples withmore RF than this must be diluted eightfold in saline andre-run. The notation “Type of analysis 6” corresponds to anendpoint method involving two reagents. Introducing thisprocedure into the present setup as a 7.6 code allows datareduction by the DENS program, in accord with the chosenprintout mode “2”: logit transformation.

To perform the assay, 30 �tL of heat-inactivated BYcalibrators, controls, or patients’ sara is added automaticallyto the appropriate wells of the rotor, along with 100 �L

Table 1. Cobas-Slo Settings for the Determinationof gM RF In Serum

of reagent buffer and 10 pL of water diluent. The contents ofcuvettes on the rotor are mixed by centrifugation andincubated for lOs at 25#{176}C,after which the first absorbancereading at 340 nm is taken, the “sample blank.” Then, theinstrument adds 50 yL of start reagent and 20 �L of waterdiluent and monitors the reaction via the absorbance at 340ma. The final reading is made 3008 after mixing. Each runincludes a cuvette containing only the reagent. The analyzercalculates the increase in absorbance, corrects for the rea-gent blank, and estimates unknown concentrations (in int.units/mL) by comparison with the concurrently analyzed BYcalibrators.

Rate -nephelometry. For comparison, we assayed the sam-ple by an automated rate-nephelometric assay for BY quan-tification (auto ICS-il; Beckman Instruments, Inc., Fuller-ton, CA 92634), following the instructions of the manufac-turer. The nephelometer measures the changes in light-scatter when serum containing IgM BY is added to partlyaggregated human IgG. The rate of formation of the com-plexes is estimated by reference to a precalibrated standardcurve, and the value is displayed as int. units/mL. Results<60 mt. unIts/mL were considered negative.

Waaler-Rose test. We also performed a modified Waaler-Rose test procedure (“Rheuniaton”; Wampole Laboratories,Cranbury, NJ 8512) as described by the manufacturer.Agglutination of sheep erythrocytes that have been sensi-tized with rabbit gamma-globulin, when mixed with the 10-fold diluted serum, was considered positive.

Analytical Variables

Assay optimization. We prepared a series of standardcurves by twofold serial dilution of the BY calibrator insaline to give BY concentrations from 30 to 500 int. urn-ita/mL. The instrument was programed to acquire ab-sorbance readings at 30-s intervals from 0 to 900 s.

To investigate the optimized conditions for the assay wevaried several analytical factors. We evaluated the effect ofpH by using 50 mmolfL phosphate buffers (pH ranging from

5.7 to 7.8) and borate buffers (pH ranging from 7.4 to 9.0),each containing 0.20 mol of NaC1 per liter. The PEGconcentration in the reagent buffer was varied from 0 to 30g/L. To establish the effect of antigen concentration in thestart reagent, we prepared HA-IgG solutions in diluentbuffer, at concentrations of 0.75, 1.5, 2.0, 3.0, and 6.0 mg/L.The effect of temperature on the assay was examined at25.0, 30.0, and 37 #{176}C.

Interfering substances. We assessed the effect of bilirubinand hemoglobin by adding known amounts of these sub-stances to a serum pool containing 250 int. units of RF permilliliter. Interference from endogenous triglycerides (tri-acylglycerols) was assessed by adding to the same poolvarious amounts of a seronegative lipemic serum and evalu-ating BY recovery. The triglyceride concentration was deter-mined by a standard enzymatic colorimetric method.

Detection of RF excess. The prozone phenomenon wasstudied by analyzing five patients’ samples for which theWaaler-Rose titers ranged from 1/1280 to 1/5 120.

Precision. Three serum pools with different BY contentswere assayed repeatedly. Within-run precision was assessedwith a single rotor load of 20 replicates of each pool. Day-to-day precision was evaluated by analyzing one replicate ofeach concentration daily over a 10-day period.

Limit of detection. The sensitivity of the turbidimetricassay was taken as the lowest BY concentration for whichthe ratio of absorbance to noise, in terms of the CV, for 100sara analyzed without added antigen was 2 or greater.

Linearity. To evaluate assay linearity, we made serialtwofold dilutions with saline of 20 patients’ samples for

ljfl,#{149}. nn#{232}��

Uz4

2

022

4

FIg. 1. Effect of PEG 6000 on the reactIon rate of the RF assayPEG ccncentratlon (gIL): 0, 15; V, 10; 4, 5; U, 0

out the addition of the antigen. A 15 g/L PEG concentrationdid not produce high absorbances for reagent blanks, andnonspecific precipitation in serum samples was avoided; athigher concentrations there was substantial precipitation ofantigen. We therefore used a final concentration of PEG inthe reagent buffer of 15 g/L.

Effect of antigen concentration. Figure 2 shows the influ-ence of varying the antigen concentration. Two oppositeeffects are produced when antigen concentration is in-creased: an important increase in absorbance for the highBY values, thus extending the assay range, and a markeddecrease in absorbance for the low BY values, thus decreas-ing sensitivity. As a compromise between these effects weused an antigen concentration of 2 mg/mL in the startreagent.

Method Performance

The turbidimetric method performance is demonstratedin several areas:

Kinetics of the immunocomplex formation. The turbidi-metric curves of absorbance vs time (Figure 3) all show asimilar shape: an initial rapid increase in absorbance whenantigen is added, then a leveling off. There was littleincrease in absorbance after 300 a, and the fit of thestandard curve was the best for values taken at this time;therefore we used this reaction time in all further work,which allowed analysis of more than 100 specimens perhour.

500

00

9 400.

UU

a300

0UU

200

100

0

0 2 S

A NT ICE N #{149}mg / ml

FIg. 2. Effect of antigen concentration on absorbance changes for threeRF calibrators: 500 (#{149}),250 (V), and 62.5 mt unlts/mL (A)

4 2

126 CLINICAL CHEMISTRY, Vol. 32, No. 1, 1986

which BY content exceeded 100 mt. units/mL, then analyzedthese in duplicate. Values exceeding the limit of detectionwere multiplied by the appropriate dilution factor and

compared with the expected value.

Clinical Studies

The study population consisted of nine men and 38 womendiagnosed by members of the rheumatology staff as havingrheumatoid arthritis according to criteria of The American

Rheumatism Association (32). Their average age was 56years (range 27-75), and the average duration of the diseasewas 10.5 years (range 1-36). Rheumatoid arthritis activitywas assessed and an index of disease activity was calculated

based on a multivariate analysis (33) of two subjectivefactors (morning stiffness and pain scale); two semi-objectivefactors (grip strength and articular index), and two objectivedeterminations; hemoglobin concentration and erythrocytesedimentation rate, both measured by standard methods.

The control group was 260 apparently healthy adults.

Results and Discussion

Analytical Variables

Effect of sodium chloride concentration. The ionic strengthof the reaction medium and the charge density of the ionspresent significantly affect the antigen-antibody reaction(34). Because the concentration of sodium chloride canprofoundly affect the reaction rate (35), its effect should beinvestigated in each antigen-antibody system. We haveobserved that sodium chloride affects the solubility of theantigen; consequently, a low concentration of sodium chlo-ride in the reagent buffer will correspond to increasedabsorbance in the reagent blank. This effect is negligiblewhen the sodium chloride concentration in the reactionmixture exceeds 0.15 mol/L. However, higher concentra-tions of sodium chloride have a marked chaotropic effect(34-36) on the reaction rate; therefore, we chose a concen-tration of 0.20 moIIL for NaC1 in the reagent buffer, tocompensate for the water added as diluent.

Effect of pH and temperature. The immunoprecipitin reac-tion is relatively independent of temperature and pH, with-in reasonable limits (34,36,37). We found the most sensi-tive pH range to be pH 6.5 to 8.5, with the maximumabsorbance response at pH 7.6. Outside this pH range, thereaction was appreciably inhibited. We adopted borate buff-er for all further work, because the turbidimetric responsewas slightly superior to that with the phosphate buffers atpH 7.6.

There were no substantial differences in the reaction rateat the three reaction temperatures we examined, although

we noted a slight decrease in absorbance at higher tempera-tures. We therefore assayed at 25#{176}C.

Effect of PEG 6000. Polymer enhancement of immune-chemical reactions was described by Heilsing (38), whofound PEG 6000 to be the most suitable. As Figure 1 shows,increased concentrations of PEG in the reagent buffer,markedly enhance the reaction rate and increase the ab-sorbance changes, which improves assay sensitivity andshortens analysis time. However, mixing antigen or serumspecimens with buffers containing PEG may cause thesesolutions to become turbid, because PEG decreases thesolubility of serum proteins and HA-IgG. Thus, in choosingthe optimum working concentration of PEG, we want to usethe maximum PEG concentration that does not lead tononspecific precipitations. To determine this concentration,we tested the effect of various concentrations of PEG in thereagent buffer on the solubility of the antigen and on 25serum samples analyzed under routine conditions but with-

§x

LU

Tin’.

�1�.fl

xw

.

00

9 300

wU21ffaa0aa1ff

-o

200

100 -

10000 #{149}0000

Fig. 5. Immunoprecipitation curve for a hlgh-RF serum sample

0 100 200 300 400 500

P F iflt.uflitS/ mL

Fig. 4. RF calibration curve for the turbidimetric assay

FIg. 3. Progress of the turbidimetric reaction at RF values of calibrationcurveRFconoentration (mt units/mL): *, 500; *, 250; Y, 125; U, 62.5;#{149},31.3

Standard curve. Figure 4 illustrates a typical standardcurve for BY determination. The range covered by this curve

suffices to provide an excellent signal-to-noise ratio foraccurate determination of BY. The change in the responsefrom 30 to 500 mt. units/rnL is more than 0.300 A; anabsorbance change (M) of 0.015 is equivalent to 30 int.units/mL.

Interferences. Hemoglobin (up to 5600 mg/L), bilirubin (upto 340 �zmol/L), and tryglycerides (up to 2.20 mmol/L) do notinterfere with either immunocomplex formation or theturbidimetric reading. Icteric and hemolyzed samples as-sayed by this method gave results similar to those obtainedby the rate-nephelometric procedure compared. However,lipemic samples gave different results by each method.

Table 2 summarizes the results we obtained for fivelipemic sera before and after delipidation. Both the turbidi-metric and the nephelometric method are affected by lipe-mia, but the effect on the turbidimetric procedure wasstatistically significant (r = 0.900, p <0.001). We concludethat all samples with a triglyceride concentration exceeding2.20 mmol/L should be clarified before assaying.

Detection of RF excess. The problem of “antigen excess” isa difficult one in immunoprecipitation. In any immunopre-

400

Table 2. Results for RF in Five Patients’ LipemicSamples before (L) and after (DL) Deilpldatlon

RF, Int. unfts/mL

--

TriglycerIde In Turbldlmsti#{149}y (ICS)serum, mmol/L L DL L DL

2.22 179 270 60 3182.26 128 142 142 1042.43 130 184 165 1253.55 61 2019 60 33903.80 828 2016 60 2985

Sera were delipidated by treating 1.5 mL of asia with 1.0 mL of Lipoclean.

cipitin method one should be able to distinguish whenantigen or antibody is in excess, to facilitate the accuratemeasurement of the amount of precipitin formed. Figure 5illustrates the response curve of a specimen with a Waaler-Rose test of 1/5 120 and an estimated BY content about30000 int. units/mL. Similar results were obtained for fourother high-BY sera analyzed.

The course of the precipitation pattern in the BY excesszone does not follow the classicial Heidelberger curve. Veryhigh BY concentrations do not produce a decrease in mea-sured absorbances; on the contrary, there is a tendency toplateau, at least up to the concentration assayed. Thus, therisk of misinterpreting a high value ofRF is very smallindeed. This is in contrast to the fluid-phase immunopre-cipitin reaction of proteins, but it has been observed byothers in the nephelometi-y of BY (39).

Precision. Table 3 shows the precision of the method atvarious BY concentrations. The precision and reproducibili-ty of the turbidimetric assay are excellent, with CVs of lessthan 5%, even for relatively low concentrations of BY. The

Table 3. pRhsu

reclsionmatold factor

Data

, mt.untts/mL

CV,%Mean SD

Wit hin-run�Sample I 69 1.6 2.3Sample II 132 1.6 1.2Sample IIIDay..to.dayb

261 3.1 1.2

Sample I 61 2.9 4.9Sample II 126 4.3 3.4Sample III 250

= 20. bfl = 10.6.0 2.4

CLINICAL CHEMISTRY, Vol. 32, No. 1, 1986 127

- SO

�0S10

a

a

n�107

501

0

Y:X

0

0

0

0

0

00

0

800

0

0 00

C

C

00

00

o 0o o0

0

2500

2000

1500

1000

300

S

0 1000 2000 300010 50 150 #{149}40 25S0

Methed

Waaler-Rose testRate-nephelometryTurbidimetry

Sensitivity, %

92.399.398.7

30000000

128 CLINICAL CHEMISTRY, Vol. 32, No. 1, 1986

0

0

8 80

0

§0 0

00 8

Oo

0�00��o

0

0

0

0

0

RF by rate- flephelornel ry fir sf1151 roL Reciprocal of Waler - Rose litre

FIg. 6. Correlation of results for RF samples assayed by turbidimetiy and rate-nephelometmy (len) and by turbidimetry and Waaler-Rose (light)

Table 4. RelatIve Sensitivities and Specificitles ofThree Methods for RF

Specificity, %

96.897.596.6

error of this method is comparable with that of otherquantitative techniques for HF quantification (25,28).

Specificity for pentameric 1gM RF. This method appears tobe specific for pentameric 1gM HF. Treatment of RF-positivesamples with equal volumes of a 10 mmolJL dithiothreitolsolution for 30 miii completely abolished their BY activity asdetected by the turbidimetric assay (data not shown).

Linearity. I�ost samples diluted serially showed somenonlinearity at the HF values measured. The differencebetween the extreme values for each sample was always lessthan 40%. The most severe departure from linearity normal-ly occurred with undiluted sample. This effect can not beattributed to endogenous interference from lipids, becausedelipidating the same samples before diluting them gavesimilar nonlinear results. This lack of linearity is probably

related to interference from endogenous IgG (40), a phenom-enon also noted by others for the ICS BY assay (28).

Method comparison. Sera from 296 patients, submittedfrom the rheumatology unit, were analyzed for BY activityby the Waaler-Rose test, the BY ICS rate nephelometry,and the turbidimetric method. Analytical sensitivity andspecificity obtained are summarized in Table 4. We consid-ered as true positive or true negative those specimens thatwere positive or negative for at least two of the three assays.The frequency of “false-positive” results appears to be thesame for all three methods, but the Waaler-Rose testshowed the highest incidence of”false-negative” results. Thesensitivity of the other two methods was excellent andsimilar to each other.

The concentrations of 1gM BY in pathological sera asmeasured by the turbidimetric assay were compared withthose obtained by the other two methods. Correlations weredetermined by calculating Spearman’s rank correlationcoefficients (r.). The overall correlation coefficient between

the turbidimetric assay and the ICS method was excellent(r8 = 0.932) and better than others reported between quanti-

tative assays (25). However, as Figure 6 shows, nephelome-try overestimates HF values that exceed 400 int. units/niL.The poor linearity of the nephelometric method (28) mayexplain this phenomenon, because the ICS system requireshigher dilutions of sera in the reaction mixture to measureHF exceeding 400 mt. units/mL. Results by turbidimetry (r1= 0.601) and nephelometry (r� = 0.522) correlated poorlywith those by the modified Waaler-Rose test, presumablybecause of the inherent subjectivity and lack of precision ofslide-agglutination tests (29).

Clinical Studies

Serum samples were positive for BY in 34 of 47 patients(73%) who had definite or classical rheumatoid arthritis, butin only nine (3.4%) of 260 controls. Thus the clinicalspecificity and sensitivity of the turbidimetric assay issimilar to that obtained with slide-agglutination or nephelo-metric techniques (25, 26, 29). However, there was nosignificant correlation between actual HF values and theindividual activity index (r8 = 0.282). This confirms others’reports (11,41,42) that the concentration of serum 1gM HFdoes not reflect the activity of rheumatoid arthritis.

We conclude that turbidimetry provides an attractivealternative to other methods of HF estimation. Althoughturbidimetry does not increase the diagnostic specificity orsensitivity of BY detection, it is easier to perform, less timeconsuming, very inexpensive, and more precise than previ-ous tests.

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3. Reyes PA, Maluf JG, Curd JG, Vaughan JH. Association ofrheumatoid factor with complement activation in rheumatoid ar-thritis and other diseases. Cliii Exp Immunol 1983;53:391-6.

4. Mannik M. Chap 34. In: Hollander FC, McCarthy DS, eds.Rheumatoid factors, arthritis and allied conditions. Philadelphia:Lea and Febiger, 1979;504-12.

CLINICAL CHEMISTRY, Vol. 32, No. 1, 1986 129

5. Duc Dodon M, Gazzolo L, Quash GA. Cellular myeloperoxidaseactivity in human monocytes stimulated by hyposialylated immu-noglobulins and rheumatoid factors. Immunology 1984;52:291-7.6. Van Smck JL, Coulie PG, Stevens M. Genetic control of rheuma-toid factor production in the mouse. Arthritis Rheum 198326: 1085-90.7. Theofflopoulos AN, Burtonboy G, Lospalluto JJ, Ziff M. 1gMrheumatoid factor and low molecular weight 1gM: an associationwith vasculitis. Arthritis Rheum 1974;17:272-4.

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14. Fye KH, Mantsopoulos HM, Talal N. Basic and clinical immu-nology. Los Altos, CA: Lange, 1978:422.15. Speiser JC, Moore TL, Weiss TI), Baldas8are AR, Ross SC,Osborn TO, et al. Hidden 19S 1gM rheumatoid factor in adults withjuvenile rheumatoid arthritis onset. Ann Rheuxn Dis 1985;44:294-8.

16. Wader E. On the occurrence of a factor in human serumactivating the specific agglutination of sheep blood corpuscles. ActsPathol Microbiol Scand 1940;17:172-8.17. Rose kiM, Ragan C, Pearce E, Lipman MO. Differential aggluti-nation of normal and sensitised sheep erythrocytes by sera ofpatients with rheumatoid arthritis. Proc Soc Exp Biol Med1948;68:1-6.18. Singer JM, Plot.z CM. The latex fixation test. Am J Med195621:888-92.

19. Performance of latex-fixation kits used for serologic diagnosis ofrheumatoid factor in rheumatoid arthritis sera. Am J Clin Pathol1979;72:597-603.20. Rippey JH, Beisecker JL. Results of tests for rheumatoid factoron CAP survey specimens. Am J Cliii Pathol 1983;80:599-602.21. Torrigiani G, Roitt IM, Lloyd KN, Corbett M. Elevated IgGantiglobuluns in patients with seronegative rheumatoid arthritis.Lancet 1970;i:14-6.22. Koopman WJ, Schrohenloher RE. A sensitive radioim-munoassay for quantitation of 1gM rheumatoid factor. ArthritisRheum 198023:302-8.

23. Nordfang 0, H$ier-Hansen M, Danielsen A. Immunopre-cipitates used as a reagent for rheumatoid factor. Scand J Rheuma-tel 1984;13:257-64.24. Nishimalci I, Watenabe S, Yoshida H, Kasukawa R. Immuno-globulin class of rheumatoid factors detected by enzyme-linked

immunosorbent assay. Clin Rheumatol 19832:145-51.

25. Bampton JLM, Cawston TE, Kyle MV, Hazleman BL Mea-surement of rheumatoid factors by an enzyme-linked immunosor-bent assay (EU8A) and comparison with other methods. Ann RheumDis 1985;44:13-9.26. Weinblatt ME, Schur H. Rheumatoid factor detection by nephe-lometry. Arthritis Rheum 1980;23:777-9.27. Jones CE, Rousseau RJ, Maxwell KW. Quantitation of rheuma-toid factor activity by nephelometry. Am J Clin Pathol1979;72:432-6.

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