Annals of the Rheumatic Diseases, 1985, 44, 13-19

Measurement of rheumatoid factors by an

enzyme-linked immunosorbent assay (ELISA) andcomparison with other methodsJ L M BAMPTON, T E CAWSTON, M V KYLE, AND B L HAZLEMAN

From the Rheumatology Research Unit, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ

SUMMARY IgM rheumatoid factor (RF) was measured in the sera of 48 rheumatoid patients andof 48 age and sex-matched normal controls by the Rose-Waaler and latex agglutination tests, a

rate nephelometer, and an enzyme-linked immunosorbent assay (ELISA). Good correlation wasobtained between all assays. The rate nephelometer assay was the easiest and quickest to performand gave results in international units/ml. The Rose-Waaler was the least sensitive assay and themost difficult to perform and interpret. Both the latex agglutination and the ELISA were

sensitive, though some overlap of patient and control sera was seen with all the assays. Inaddition to IgM RF the ELISA was used to measure IgG RF and IgA RF in both rheumatoid andcontrol sera. Although some normal sera had detectable amounts of IgG and IgA RF, the levelsof both were significantly raised in the rheumatoid sera. IgG RF levels were lower after pepsindigestion of the sera, suggesting that IgM RF interfered with the assay for IgG RF unless thistreatment was included.

Key words: arthritis rheumatoid, Rose-Waaler test, latex fixation test, nephelometry.

Rheumatoid factors (RF) are anti-IgG autoanti-bodies with specificity directed against antigenicdeterminants in the Fc region of IgG.' They arepresent in the large majority of patients withrheumatoid arthritis and have long been found to beassociated with the more severe forms of rheuma-toid disease. The measurement of RFs is importantin the diagnosis of rheumatoid arthritis and indetermining prognosis, especially in high titres, asthese patients tend to develop extra-articular com-plications. In addition it has been proposed that RFsare important in the pathogenesis of the disease.2There is considerable evidence that RF contributesextensively to the immunopathology of RA, as it isthe predominant antibody known to initiate immunecomplex formation and complement activation inthe peripheral circulation and in tissue sites.2 3The majority of routine laboratory tests for the

detection of rheumatoid factor measure IgM RF byits ability to agglutinate sheep red blood cells, latex,or similar particles coated with IgG.4 However,recent publications have suggested that both IgGAccepted for publication 18 July 1984.Correspondence to Dr J L M Bampton.

13

RF2 7 and IgA RF are also raised in seropositiveRA, and IgG RF may be associated with the moresevere extra-articular complications of the diseasesuch as vasculitis.3 8The majority of hospital laboratories still rou-

tinely measure IgM RF by either the Rose-Waaler or latex agglutination test. These methodsare both difficult to quantitate, and we have foundthat there is a large variation in titres for thesame sera between different laboratories. In addi-tion it is not possible to measure either IgG RF orIgA RF by these methods. Although procedureshave been described for the measurement ofdifferent classes of RF, specialised reagents andequipment are generally required. More recentlysuitable ELISA methods have been reported whichcan be routinely used in clinical laboratories.9 10

In this study we have compared four methods forthe estimation of IgM RF and developed an ELISAmethod that can also measure IgG RF and IgA RF.

Materials and methods

All reagents were of analytical grade and were

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obtained from Sigma Ltd, Poole, UK, unless other-wise indicated.

Patients and controls. Sera were obtained from 48rheumatoid patients with active disease attendingroutine rheumatology outpatient clinics and from 48age and sex matched normal healthy blood donors.All sera were stored at -70°C, thawed before useand heated at 56°C for 30 min before use toinactivate complement.Development of ELISA. Rabbit IgG (R IgG) and

human Fc were both tested as antigen in an ELISA.No significant difference was found between them,and R IgG was used in all further tests.

Evaluation of microtitre plates. Microtitre plateswere obtained from four manufacturers: Nunc(Gibco Ltd, Paisley, Scotland), Falcon (BectonDickinson UK Ltd, Cowley, Oxford), Linbro (FlowLaboratories Ltd, Irvine, Ayrshire, Scotlaind), andDynatech (Dynatech Laboratories, Billinghurst,Surrey) and tested by ineasuring the amount of125I-labelled R IgG bound to each well. AlthoughFalcon plates bound slightly greater amounts of IgGthan Linbro or Dynatech, no significant differencein final sensitivity was found, and the lower-costLinbro plates were subsequently used. No signifi-cant difference was found between the amount of RIgG bound by wells at the centre and wells at theedge of any plate.

Binding ofR IgG to microtitre plates. The amountof 125I-labelled R IgG bound to microtitre plates wasestimated under different conditions of time (2-24h), temperature (4°C, 22°C, 37C), pH (2 0, 7 2,9.6), and concentration (10-500 ,ug/ml). The opti-mum conditions chosen for binding of R IgG werean overnight treatment at 37°C of 50 RI of a 100 [tg/mlsolution in 50 mM sodium carbonate buffer pH 9-6.

Blocking of non-specific antibody adsorption afterIgG coating. No significant difference in titre wasfound between wells treated with bovine serumalbumin (100 Rtg/ml) in either phosphate bufferedsaline (PBS) or sodium carbonate buffer (pH 9-6)for 1 hour at 37°C after R IgG adsorption and wellswhen this treatment was omitted.

Investigation of temperature and time of incuba-tion. Various incubation times and temperatureswere investigated for each stage of the ELISA assay.The optimum conditions chosen for a one-day assaywere incubation of the plate with serum for 3 h at4°C; incubation with enzyme conjugate for 2 h at4°C, and incubation with enzyme substrate for 30min at 37°C.

Investigation of enzyme substrate. The substrateso-phenylenediamine (OPD) and 2,2'-azino-di(3-ethyl-benzthiazoline-6-sulphonic acid) for perox-idase and p-nitrophenyl phosphate for alkalinephosphatase were investigated. The most sensitive

substrate was OPD, and so horseradish peroxidaseimmunoglobulin conjugates were subsequentlyused.

Final ELISA protocol. R IgG (50 Rl) at aconcentration of 100 [ig/ml in 50 mM sodiumcarbonate buffer pH 9-6 was added to Linbro ElAmicrotitre plates. Wells containing sodium carbon-ate buffer (50 il) only were prepared as controls.The plates were sealed with Nescofilm (Jeuicons SciLtd, Leighton Buzzard, UK), incubated overnightat 37°C, then washed three times with PBS + 0.05%Tween 20 (PBST), soaking for 3 min between eachwash. Serum was diluted 1:10 and in a further seven1:4 dilutions across the plate, so that each serum wastested in a dilution range of 1:10 to 1:138 240.Aliquots (50 RI) of each dilution were added to testand control wells. The plates were incubated for3 hours at 4°C and then washed three times aspreviously described. The peroxidase-conjugatedsecond antibody was diluted 1:1000 in PBST.Aliquots (50 pl) were added to each well and theplates incubated at 40C for 2 h. OPD (20 mg) wasprepared immediately before use in citric-phosphatebuffer pH 5*0 (20 ml) and then H202 (8 pl of 30%solution) was added. Aliquots (50 RI) of OPD inbuffer were added to each well and the platesincubated in the dark at 37°C for 30 min. Thereaction was stopped by the addition of 2 M H2SO4(50 tl) to all wells and the absorbance of each wellread at 492 nm with a Titertek multiscan (FlowLaboratories, Irvine, UK). The results were ex-pressed as log,() titre at an absorbance value of 0 4.Measurement of RF immunoglobulin class. IgM

RF and IgA RF were detected with peroxidaseconjugated rabbit antihuman p chain and antihumana chain immunoglobulins (Dako, Mercia BrocadesLtd, Weybridge, UK). IgG RF was detected bymeans of a rabbit antihuman IgG/Fab antibody(Nordic Immunological Labs Ltd, Maidenhead,Berks), which was conjugated to horseradish perox-idase by the method of Wilson and Nakane 1978.11

Treatment of sera with dithiothreitol and pepsin.Sera were reduced with an equal volume of 10 mMdithiothreitol in PBS at 37°C for 1 h12 and thenassayed by the procedure outlined above.

Sera were pepsin digested by a modification of themethod of Faith et al.' The concentration of pepsinchosen was that which destroyed all IgM RF asdetected by our ELISA. Sera were diluted 1:20 inpepsin-acetate buffer 150 pg/ml pepsin in 100'mMacetate buffer (pH 4.4) incubated for 20 h at 370Cand then neutralised with an equal volume of 0-564M disodium hydrogen phosphate in PBST. Controlsera were incubated in 100 mM acetate buffer pH4-4 foi 20 h at 37°C and then neutralised as above.The sera were then assayed by the ELISA with the

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Measurement of rheumatoid factors by an enzyme-linked immunosorbent assay 15

lowest dilution being 1:40. IgG RF and IgM RFlevels were measured in sera before and afterreduction with DTT and before and after pepsindigestion.

OTHER RF ASSAYSRose-Waaler test. The sera were adsorbed by adding0*1 ml of concentrated washed sheep red blood cells(SRBC) to 0.5 ml of serum and incubated at 4°Covernight. Sensitised SRBC were prepared byadding a 2% (v/v) solution of washed SRBC insaline to an equal volume of rabbit antisheep cellserum (Wellcome Reagents Ltd, Beckenham, Eng-land) at its sensitising dilution. The inactivatedadsorbed sera were titrated against the sensitisedcells in microtitre plates: 40 [tl of saline was added toall wells. 40 pl of inactivated adsorbed serum wasadded to the first well, mixed, and 40 ,ul transferredto the next well. In this way the serum was dilutedacross the plate. 40 [il of sensitised cells was thenadded to each well, and the plates incubated at 37°Cfor 1-5 h. Cells which had settled into a completelynegative button were reported as negative, andthose sera with a titre of <1:16 were considerednegative.

Latex agglutination. The test was carried out withthe Mercia Brocades latex kit (Mercia Brocades,Weybridge, UK). Titres of <1:20 were considerednegative.

Rate nephelometry. A rate nephelometric assayfor RF was performed with the Beckman Im-munochemistry System (Beckman R11C, HighWycombe, UK). The nephelometer measures thechanges in light scatter when serum containing IgMRF is added to partially aggregated human IgG. Therate of formation of the complexes is estimated byreference to a precalibrated standard curve, and thevalue is displayed as international units/ml. Levelsof <60 IU/ml were considered negative.The Beckman ICS was also used to measure IgG,

IgA, and IgM levels in all sera and results wereexpressed as mg/100 ml.

Statistical analysis. The results from the fourassays were analysed by the Mann-Whitney U testand the effect of pepsin and DIT on IgG RF levelsanalysed by the Wilcoxon rank sum test. Correla-tions were determined by calculating Spearman'srank correlation coefficient.

Results

COMPARISON OF ASSAYS FOR IgM RFThe IgM RF levels of both normal and pathologicalsera as measured by each assay are shown in Fig. la.The results are shown as loglo titre for the Rose-Waaler, latex agglutination, and ELISA assays and

loglo IU/ml for the nephelometer. The levels of IgMRF were significantly raised (p<0.0001) in RApatients' sera compared with normal sera in allassays. IgM RF levels correlated well between allassays as shown in Table 1. The strongest correla-tion was found between the latex agglutination assayand the ELISA, though all correlations were>0-7000 and were significant at p<0-0001.

Results were considered negative if the titre was<1:16 (Rose-Waaler test) or <1:20 (latex aggluti-nation test). All normal samples were negative forthese two tests, while 60% of RA patients' sera hada titre of >1:16 in the Rose-Waaler test and 90% ofRA patients had a titre of >1:20 in the latexagglutination test. A value of 60 IU/ml was consi-dered to be negative in the nephelometer method,and 98% of the normal sera were below this level;69% of the RA patients had values above thisfigure. A value of 1-75 (logl0 titre) was chosen forthe IgM RF ELISA, which corresponded to themean +2SD of the normal sera. With this figure 92%of RA patients had elevated levels of RF while 94%of the normal persons were negative.

MEASUREMENT OF IgA RF AND IgG RFDifferent immunoglobulin classes of RF were de-tected with the ELISA protocol, and secondantibody-enzyme conjugates specific for IgG andIgA were used.The levels of IgA RF in patients' serum was

measured by a rabbit antihuman a chain-peroxidaseconjugate. The levels of IgA RF in RA patients'serum were significantly raised (p<O-OOOT) relativeto those in normal sera (Fig. lb) and 88% wereabove the mean + 2 standard deviations of normalsera (1.211 loglo titre). The majority (94%) ofnormal sera were below this level. The levels of IgGRF measured in RA patients and control sera with arabbit antihuman IgG/Fab antibody-peroxidasecomplex are also shown in Fig. lb. The levels of IgGRF were significantly raised (p<O0OOO1) in RApatients relative to normal sera. However, althoughthe majority of RA patients sera (85%) were abovethe mean + 2 standard deviations of normal sera(1-903), and 96% of normal sera had IgG RF levelsbelow this value, owing to the large range of normalvalues there was considerable overlap with the lowerpathological sera values. For a more accurateestimate of IgG RF both normal and RA patientsera were treated with pepsin. After pepsin diges-tion IgG RF activity in all normal sera was reducedto below a titre of 1:40 and was significantly reduced(p<O0001) in RA patients sera (Fig. lc). (Themethod for pepsin digestion did not allow IgG RFtitres to be measured below a value of 1:40.) Allvalues for normal sera were <1:40 after pepsin

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RA N RA N RA N RA N

Rose-Waaler Latex Nephelornetry ELISA

agglutination IgM RF

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Fig. 1 Estimation ofrheumatoid factor in rheumatoid control sera-comparison ofassays. (a) The levels ofIgM RF in 48rheumatoid and 48 age and sex matched controls were measured by the Rose-Waaler, latex agglutination, rate nephelometricassays and ELISA as described in the 'Methods' section. (b) The levels ofIgA RFand IgG RF were measured in rheumatoidand control sera by the ELISA. (c) The level ofIgG RF was measured in rheumatoid and control sera after pepsin digestion.This treatment destroys IgM RF, which can interfere with the measurement ofIgG RF. The horizontal bars indicate themean values for each group.

Table 1 Correlation coefficients of the levels of 1gM RFbetween assays

Latex agglutination Nephelometry ELISA

Rose-Waaler 0-8178 0 6998 0-8029Latex agglutination - 0-7635 0-8733Nephelometry - - 0 8121

The correlation coefficients of the levels of IgM RF between thefour assays were measured by calculating Spearman's rank correla-tion coefficient. All correlations were significant at p<0-0001.

digestion, while 46% of the RA patients sera were

above this value. There was a high degree ofcorrelation between all classes of RF measured byELISA in RA patients sera (Table 2).The total immunoglobulin levels for both normal

and RA patients' sera were measured and are shownin Fig. 2. The levels of all immunoglobulins were

significantly raised in RA patients' sera whencompared with the levels in normal sera (IgG andIgM at p<O.0001 and IgA at p<O-0002). Table 3

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Measurement of rhteumatoid factors by an enzyme-linked immunosorbent assay 17

Table 2 Correlation between RFs of differentimmunoglobulin class in rheumatoid patients' sera

IgA RF IgM RF

IgG RF 0-7701 0-9184Pepsin IgG RF 0-7403 0-7224IgA RF - 0-7329

The correlation coefficients between the levels of IgG, IgA, andIgM RFs in rheumatoid patients' sera were measured by calculatingthe Spearman's rank correlation coefficient. All correlations weresignificant at p<-0001.

illustrates the correlations obtained between thedifferent classes of RF with the total immunoglobu-lin content of RA patients' sera. Good correlationwas found between the levels of IgG and IgA in RApatients' sera, but correlation between other im-munoglobulin classes in RA and normal sera werelow.

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Table 3 Correlation between immunoglobulin levels inrheumatoid and normal sera

IgA lgM

RA patients:IgG 0-6799 0 4111IgA - 0-3442

Normals:IgG 0-1880 0-2798IgA - 0-0515

The correlation coefficients between the levels of IgG, IgA, andIgM in rheumatoid and normal sera were calculated.

Discussion

At present there is considerable interest in ELISAassays in the clinical laboratory. These assaysrepresent a quick and cost-effective way of proces-sing large numbers of samples while using simple

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t

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and relatively inexpensive equipment. A number ofELISA methods have been published for measuringRF,9 10 13-15 but they have had a restricted routineusage, and the more common Rose-Waaler andlatex agglutination assays are still performed in themajority of hospital service laboratories. If theELISA assay is to gain general acceptance, it mustbe carefully compared with other methods. In thepresent study we have compared the two traditionalmethods with a rate nephelometric assay and anELISA for rheumatoid factor. There was goodcorrelation for IgM RF values between all assays.Although the Rose-Waaler and latex agglutinationtests are relatively easy to perform, the end points ofboth assays are difficult to determine withoutexperience, and both assays are prone to observervariability. Consistent results can be obtained withcare in one laboratory if the same personnel repeatthe test from week to week, but interlaboratoryvariation for the same sera is high. The ratenephelometric assay was the easiest and quickesttest to perform and gave quantitative results in avery short time. It is useful for following patientsserially over a period of time, and consistent resultsare obtained from serum samples when assayed atdifferent times. Of the four assays tested the ratenephelometric assay gave the most consistent re-sults. Other assays had to be run with a standard andthe titres adjusted up or down for all samplesaccording to the result obtained for the standard.The ELISA was quick and easy to perform andwould suit a routine laboratory. It was the mostsensitive assay and recognised 92% of rheumatoidpatients' sera as positive. The corresponding figuresfor the other assays were latex agglutination test90%, rate nephelometric assay 69%, and Rose-Waaler 60%.The ELISA was also able to measure IgG and IgA

RF in addition to IgM RF. The results were quotedin titres for the ELISA, but it can be adapted byincorporating a standard curve to give quantitatiVeresults in ,ug IgM RF/mll' or in internationalunits.13 15 We are currently evaluating quantitationof the assay using these methods and interfacing theELISA reader to a computer to automate theestimations and allow the results to be calculated byreference to a standard curve.The classical tests for RF usually mea'sure only

IgM RF, but in recent years increasing attention hasfocused on the role of both IgG and IgA RF. IgGRF was first measured quantitatively by Torrigianiand Roitt.16 Since that time many different assayprotocols for the measurement of IgG RF and IgARF have been published, and the most widely usedare radioimmunoassay,2 7 8 17-19 immunofluorescenttechniques,20 21 or ELISA.9 10 13 14 March et al.22

have published a simple microtitre plate assay usingimmunoglobulin coupled to red blood cells.The significance of the raised levels of IgG RF in

rheumatoid disease is not clear. Early reportssuggested that IgG RF was significantly raisedin both seropositive and seronegative arthri-tides.'6 23-25 However, it is now thought that theseearly results were incorrect, and subsequent studiesusing more sensitive and specific methods of detec-tion have shown that significant levels of IgG RF arenot associated with seronegative diseases.' 2 17 IgMRF can interfere with the measurement of IgG RF insolid phase assays, as it can bind to the immobilisedtarget IgG and to IgG in the serum. The boundserum IgG is then detected with the antibodyconjugate as IgG RF. Two methods have been usedto destroy IgM in the test serum and so preventthese interactions. These are either pepsindigestionl0 17 26 or treatment with a reducingagent.7 21 In this study we used pepsin digestion andadapted the method of Faith et al.10 to adjust thelevel of pepsin so that all serum IgM was destroyed.We found that the results obtained after reductionand alkylation of sera were variable and that higherlevels of IgG RF were present in control sera afterthis treatment. Similar findings have been reportedby Carson et al.7 and Wernick et al. 17 and so pepsindigestion was considered to be the method ofchoice.

High titres of IgG RF are associated with themore severe forms of rheumatoid disease, andpatients with vasculitis or subcutaneous noduleshave high levels of serum IgG RF.3 8 25 Immunecomplexes containing IgG RF have been detected inthe serum, synovial fluid, and synovium of patientswith rheumatoid arthritis,2 and the levels of IgG RFare higher in synovial fluid than in the correspond-ing serum sample.25 This suggests that locallyproduced IgG RF may be important in thepathogenesis of the disease maintaining the rheuma-toid synovitis through its ability to activate comple-ment by self-associating27 or in combination withIgM RF. The vasculitic lesions probably result fromthe deposition of pathogenic immune complexes inthe microvasculature of the skin and other targetorgans, and IgG RF appears to be present in manyof these complexes.20 In addition it is possible thatIgG RF may aggregate platelets in vivo and sopromote vascular damage.8

Relatively little interest has been shown in thefinding that the levels of IgA RF are raisedin rheumatoid patients, though this finding hasbeen previously reported. 2 29 Dunne etal.28 demonstrated that IgA RF was present in notonly the serum but also the saliva of rheumatoidpatients and patients with Sjogren's syndrome. The

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Measurement of rheumatoid factors by an enzyme-linked immunosorbent assay 19

IgA RF showed a pattern of reaction with humanIgG subclasses similar to that of IgM RF from thesame patients. Koopman et al.29 detected signifi-cantly raised levels of IgA RF in rheumatoid seraand synovial fluid compared with serum frompatients with seronegative disease or normal controlserum. In the present study we obtained similarresults, finding significantly raised levels of IgA RFin rheumatoid sera, but their significance in theimmunopathogenesis of the disease is unknown.Rheumatoid factor measurement by the conven-

tional tests of Rose-Waaler and latex agglutinationare difficult to quantify. We found that the neph-elometric assay is rapid, reproducible, and allowsresults to be presented as international units/mi. TheELISA for rheumatoid factor is simple to performand interpret and uses equipment which is present inthe majority of routine hospital laboratories. Weaim to develop a screening test to distinguish quicklybetween positive and negative sera and also use astandard curve in each assay so that results arequoted in either international units/ml or ig RF/ml.

We thank Ms Sarah Roberts (Department of Community Medi-cine, University of Cambridge) for advice and help with thestatistical analysis and Mrs Jane McAusland for typing themanuscript. This work was generously supported by the Arthritisand Rheumatism Council and the East Anglian Regional HealthAuthority.

References

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2 Pope R M, McDuffy S. IgG rheumatoid factor. Relationship tosero-positive rheumatoid arthritis and the absence in sero-negative disorders. Arthritis Rheum 1979; 22: 988-98.

3 Scott D G I, Bacon P A, Allen C. Elson C J, Wallington T. IgGrheumatoid factor, complement and immune complexes inrheumatoid synovitis and vasculitis: comparative and serialstudies during cytotoxic therapy. Clin Exp Immunol 1981; 43:54-63.

4 Waaler E. On the occurrence of a factor in human serumactivating the specific agglutination of sheep blood corpuscles.Acta Pathol Microbiol Scand 1940; 17: 172.

5 Rose H M, Ragan C, Pearce E, Lipman M 0. Differentialagglutination of normal and sensitised sheep erythrocytes bysera of patients with rheumatoid arthritis. Proc Soc Exp BiolMed 1948; 68: 1-6.

6 Singer J M, Plotz C M. The latex fixation test. Am J Med 1956;21: 888-92.

7 Carson D A, Lawrance S, Cataland M A, Vaughan J H,Abraham G. Radioimmunoassay of IgG. IgM rheumatoidfactors reacting with human IgG. J Immunol 1977; 119:295-300.

8 Allen C, Elson C J, Scott D G 1, Bacon P A, Bucknall R C. IgGantiglobulins in rheumatoid arthritis and other arthritides:relationship with clinical features and other parameters. AnnRheum Dis 1981; 40: 127-31.

9 Nishimaki 1, Watenabe S. Yoshida H. Kasukawa R. Immuno-globulin class of rheumatoid factors detected by enzyme-linkedimmunosorbent assay. Clin Rheumatol 1983; 2: 145-51.

10 Faith A, Pontesillia G, Unger A, Panayi G S, Johns P. ELISAassays for IgM and IgG rheuimatoid factors. J Immunol Meth1982; 55: 167-77.

11 Wilson W B, Nakane P K. Immunofluorescence and relatedtechniques. In: Knapp W. Holnbark, Wick G. Amsterdam:Elsevier Holland, 1978: 215.

12 Palosuo T, Milgram F. IgG RF detection by EIA in RA andnormal subjects. J Immunol Meth 1981; 41: 171-81.

13 Karsh J, Halbert S P, Klima E, Steinberg A D. Quantitativedetermination of rheumatoid factor by an enzyme-linkedimmunosorbent assay. J Inmunol Meth 1980; 32: 115-26.

14 Vejtorp M, Hoier-madsen M, Halberg P. Enzyme-linkedimmunosorbent assay for determination of IgM rheumatoidfactor. Scand J Rheumatol 1979; 8: 59-63.

15 Zoila B, Tuokko H. Solid-phase enzyme immunoassay of1gM-class rheumatoid factor. Acta Pathol Aficrobiol Scand SectC 1980: 88: 127-30.

16 Torrigiani G, Roitt I M. Antiglobulin factors in sera frompatients with rheumatoid arthritis and normal subjects AnnRheum Dis 1967; 26: 334-4(.

17 Wernick R, Lospalluto J J, Fink C W, Ziff M. Serum IgG andIgM rheumatoid factors by solid-phase radio-immunoassay.Arthritis Rheum 1981; 24: 1501-11.

18 Nordfang D, Hloier-Madsen M, Halberg P, Lieberkin J. A newradioimmunoassay for IgM and IgG rheumatoid factors basedon a double antibody method.]ImJmunol Meth 1981; 47: 87-97.

19 Yamagata J, Barnett E V, Knutson D W, Nasu H, Chia D.Characterisation and measurement of anti-lgG antibodies inhuman sera by radioimmunoassay. J Immunol Meth 1979; 29:43-56.

20 Quismorio F P, Beardmore T, Kaufmnan R L, Mongan E S. IgGrheumatoid factors and anti-nuclear antibodies in rheuimatoidvasculitis. Clin Exp Immunol 1983; 52: 333-40.

21 Kallerup H E, Egeskjold E M, Graudel H. IgG, IgM and IgArheumatoid factors in healthy adults and rheumatoid patientsdetermined by an indirect immunofluorescence method. ScandJ Rheumatol 1979; 8: 1-9.

22 March R E, Reeback J S, Holborow E J, Coombs R R A. Mrs.P A H, a simple microtitre plate test for rheumatoid factors ofdifferent classes. J Immunol Meth 1981; 42: 137-46.

23 Torrigiani G, Ansell B M, Chown E E A, Roitt I M. RaisedIgG antiglobulin factors in Still's disease. Ann Rheu,n Dis 1969;28: 424-7.

24 Howell F A, Chamberlain M A, Perry R A, Torrigiani G, RoittI M. IgG antiglobulin levels in patients with psoriatic arthro-pathy, ankylosing spondylitis and gout. Anti Rheum Dis 1972;31: 129-31.

25 Hay C, Neneham L J, Percival R, Roitt I M. Intra-articular andcirculating immune complexes and antiglobulins (IgG and IgM)in rheumatoid arthritis, correlation with clinical features. AnnRheum Dis 1979; 38: 1-7.

26 Theofilopoulos A N, Burtonboy G. Lospalluto J J. Ziff M. 1gMrheumatoid factor and low molccular weight 1gM An associa-tion with vasculitis. Artlhritis Rheum 1974; 17: 272-X4.

27 Pope R M, Mannik M. Gilliland B C. Tellcr D C. Thehyperviscosity syndromc in rheumatoid arthritis due to in-termediate complexes formed by the self-association of IgGrheumatoid factors. Arthritis Rheu,n 1975; 18: 97- l)6.

28 Dunne J V, Carson D A. Spiegelberg H L, Alspaugh M A.Vaughan J H. IgA rheumatoid factor in the sera and saliva ofpatients with rheumatoid arthritis and Sjogren's syndrome. AnnRheum Dis 1979; 38: 161-5.

29 Koopman W J, Schrohenloher R E. Solomon A. A quantitativeassay for IgA rheumatoid factor. J Imtnunol Methods 1982; 50:89-98.

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