Ann. rheum. Dis. (1961), 20, 265.

IMMUNO-ELECTROPHORETIC ANALYSIS OF PROTEINSIN SERUM AND SYNOVIAL FLUID IN RHEUMATOID

ARTHRITIS AND ANKYLOSING SPONDYLITISBY

STEFAN MACKIEWICZ* AND WLADYSLAW FENRYCHFrom the Third Medical Clinic, Academy of Medicine, Posnan, Poland

Among the systemic manifestations of rheumaticdisease it is customary to include a number ofquantitative changes in the constituents of plasma,together with abnormalities in immunological andother properties of serum. Although most of thesechanges are neither uniquely nor constantly demon-strable in this group of diseases or in any memberof the group, it is likely that information on the truenature of the underlying changes in plasma con-stituents will contribute to an understanding of thefundamental significance of these phenomena.A complete list of all the changes so far recorded

is not relevant to this report, but among thosemore frequently demonstrable may be includedan increase in the total gamma globulin, and inalpha-l and alpha-2 glycoproteins as shown bypaper electrophoresis, elevation of the erythrocytesedimentation rate, and the presence of demon-strable amounts of one or more "rheumatoidfactors", having in common the property of reactingwith denatured gamma globulin.One or more fractions reacting in vitro with

nuclear material are demonstrable in sera from aminority of patients with rheumatoid arthritis,and also from the majority of patients with systemiclupus erythematosus, in whom a number of otherserum factors have been described which reactin vitro with more or less denatured constituents ofcells or organs.The immuno-electrophoretic technique of Grabar

and Williams (1953) provides an approach to theproblem of exploring the nature of abnormallyreactive components in serum and other bodyfluids, in that it permits the identification and separa-tion of a far larger number of distinct proteinfractions than is possible by any other method usedalone. The nature of the physiological properties

* Present address: Rheumatic Diseases Unit, Northern GeneralHospital, Edinburgh.

of many of these fractions remains unknown, andindeed it would be unreasonable to conclude atpresent that fractions identified from their asso-ciated electrophoretic and antigenic properties arenecessarily also functional entities. Nevertheless,it is likely that immuno-electrophoretic techniquescan now provide qualitative and semi-quantitativedata on the constituents of serum in health anddisease which may ultimately be integrated with theresults obtained by exploration of the functionalcharacteristics of whole serum and its constituentproteins.Schmid and MacNair (1956) were the first to use

immuno-electrophoretic techniques to study theproteins of serum and synovial fluid from patientswith rheumatic diseases; they confirmed that theindividual proteins from these two sources hadsimilar electrophoretic and antigenic properties.

Increased amounts of alpha-l and alpha-2 glyco-proteins and beta-2 macroglobulin were found insera from patients with rheumatoid arthritis orrheumatic fever by Cleve and Hartmann (1957)and by Cleve (1958). No correlation was apparentbetween the increase in beta-2 macroglobulin andthe Rose-Waaler haemagglutination titre in thesera from patients with rheumatoid arthritis, andthe protein changes in this disease and in rheumaticfever were interpreted as a manifestation of activeinflammation. The authors also investigated theproteins in synovial fluid where the concentration offractions of high molecular weight was lower than inserum.

Increased amounts of alpha-2 glycoproteins(i.e. haptoglobin and alpha-2 macroglobulin) in theserum of patients with rheumatoid arthritis werealso reported by Fenrych, Jazienicki, Mackiewicz,Mackiewicz, and Twardowski (1959). Moreover,the latter component was increased in twenty of the42 sera from patients with various forms of chronic

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ANNALS OF THE RHEUMATIC DISEASESarthritis examined by Francq, Eyquem, Podlia-chouk, and Jacqueline (1959). By specially preparedantisera, the authors demonstrated a correlationbetween the increase in beta-2 macroglobulin andgamma globulin, and the titre for agglutination ofsensitized sheep erythrocytes.From the foregoing it is evident that increased

amounts of certain fractions of the alpha and betaglobulins and of gamma globulins are frequentlydemonstrable in sera from patients with rheumatoidarthritis, although the relationship between thesechanges and other manifestations of the diseaseremains uncertain.The present report is concerned with further

attempts to characterize changes in protein fractionsin serum and synovial fluid associated with rheuma-toid arthritis.

Material and Methods

The sera investigated were obtained from 43 patientswith rheumatoid arthritis and 22 with ankylosingspondylitis and from twenty healthy individuals (with anormal paper electrophoresis pattern). The patientswith rheumatoid arthritis included eighteen men and25 women between the ages of 22 and 65 years, the dura-tion of disease being between 1 and 15 years. Thediagnosis was based upon typical clinical findings andconfirmed by radiography of the joints. Two patientsin this group also had chronic renal functional impair-ment with oedema and proteinuria, and in one of thetwo the presence of amyloidosis was demonstrated bya modification of the congo red test ofBennhold (Choderaand Mackiewicz, 1958), and by gum biopsy. Thehaemagglutination test of Rose and Heller was performedon sera from twelve of the patients with rheumatoidarthritis, and the titre was significantly raised in eachof them.The group of patients with ankylosing spondylitis

included twenty men and two women between the agesof 24 and 57, the duration of disease being between 2 and9 years. The diagnosis was made from clinical findingsand radiological evidence of bilateral sacro-iliitis; in twopatients there was a previous history of rheumatic feverand in one there were signs of mitral stenosis and incom-petence. Sera from eight of these patients were testedby the method of Rose and Heller and in all of themthe haemagglutination titre fell within the normal range.The erythrocyte sedimentation rate (E.S.R.) was

measured by the Westergren method in all patients inthe rheumatoid and spondylitic groups. In eleven itexceeded 100 mm./hr, in seventeen it was between60 and 99 mm./hr, and in the remaining 37 it was between40 and 59 mm. in the first hour. Thus all the patientsshowed a significant elevation, and from this and otherevidence it was concluded that they all had active disease.The haemoglobin and erythrocytes were determined inthe blood of these patients in an automatic apparatus.A normochromic or hypochromic anaemia was present

in nine patients with rheumatoid arthritis and in sixwith ankylosing spondylitis.

In the sera from patients and healthy subjects, totalprotein was determined by the biuret method and themajor protein fractions by filter paper electrophoresis.

All immuno-electrophoretic studies on sera in theseinvestigations were performed in duplicate. Synovialfluids obtained from the knee joints of a number of thepatients with rheumatoid arthritis were also subjectedto immuno-electrophoresis, as were samples of urinefrom the two patients with renal disease. In theimmuno-electrophoretic procedure the sera, synovialfluid, and urine were studied in terms of their antigenicconstituents. The antisera included polyvalent rabbitantihuman serum prepared by immunizing animals withwhole human serum either pooled or from individuals,following the procedure described by Cleve and Hartmann(1957). Two rabbits were immunized with serum fromnormal subjects, two with serum from the two patientswith renal disease, and eight with sera of a high haem-agglutination titre obtained from patients with rheumatoidarthritis. Two of the latter eight rabbits showed signssuch as loss of weight, roughening of the fur, andultimately paralysis of the hindquarters. Histologicalexamination of tissues obtained from those rabbits atautopsy showed widespread arteritis, together withdiffuse glomerulitis, the lesions being similar to thosepreviously observed in mice and rats after the intra-venous administration of soluble antigen-antibodycomplexes (McCluskey, Benacerraf, Potter, and Miller,1960; Benacerraf, Potter, McCluskey, and Miller, 1960).The remaining ten rabbits showed no adverse effects

from immunization. After immunization the rabbitswere bled from the carotid artery and the antiseraobtained were tested initially by double-diffusion in agarwith whole or fractionated human serum (Ouchterlony,1953). When used in immuno-electrophoresis the anti-sera produced at least fourteen and as many as nineteendistinct precipitin lines with a variety of human sera.There was no constant difference in the number of linesproduced with the antisera prepared against normalhuman serum, and the number produced with the anti-sera against serum from patients with rheumatoidarthritis.The known antigenic components of serum represented

by the precipitin arcs were identified by their character-istic distribution on immuno-electrophoresis, by meansof specific univalent antisera to individual componentsof serum (obtained from Behring and Co. Ltd., Marburg-Lahn), and by the use of differential staining techniquesfor lipoprotein. The following univalent antisera wereused: anti-prealbumin, anti-albumin, anti-alpha-2 macro-globulin, anti-alpha-2 lipoprotein, anti-beta-l lipo-protein, and anti-gamma globulin.

Sera obtained from the rheumatic patients werecompared with those from healthy subjects in terms ofthe number and intensity of precipitin lines apparentafter electrophoresis followed by double diffusion withparticular antisera. Two of the sera from healthyindividuals were chosen as standards of normality andall others were compared with these in terms of the

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IMMUNO-ELECTROPHORETIC ANAL YSIS OF PROTEINS

presence of particular immuno-electrophoretic com-ponents and on the basis of comparative quantitation ofthese components. To measure the relative concen-trations of a particular component in two different sera,aliquots of antiserum were absorbed with varyingamounts of one serum. The absorbed aliquots werethen tested in immuno-electrophoresis to determine theamount of the absorbing serum sufficient to preventformation of a precipitin line corresponding with thecomponent in question. Thus a relatively higher con-centration of a particular antigen in one of the two serawas reflected by the relatively smaller volume of serumrequired to absorb the specific antibody. The proceduresfollowed in the present studies were evolved on the basisof this principle, but were necessarily simplified in orderto permit the analysis of a large number of sera. As analternative and more satisfactory method for semi-quantitative determination of the relative concentrationsof components in two sera, polyvalent and univalentantisera were set up against several dilutions of both,and the dilution which prevented formation of the par-ticular precipitate was recorded for each serum. Theserum requiring more dilution for this effect was con-sidered to have a higher concentration of the componentin question.The immuno-electrophoretic technique is a modi-

fication, introduced by Fenrych and others (1959), of themicro-method described by Scheidegger (1955). Electro-phoretic separation of the sera was produced in 2 percent. agar-gel in barbiturate buffer pH 8-2; 0- 1 M. onmicroscope slides measuring 26 x 76 mm. The serumwas inserted in a round well (0 9 mm.) placed centrally.After electrophoretic separation, antisera were insertedinto longitudinal troughs (36 x 1 mm.) cut in the gelnear one or both sides of the slide and parallel to theaxis of electrophoretic migration. In this system linesof precipitate appear at the interfaces between com-ponents of serum diffusing outwards from the axis of

electrophoretic migration and their specific antibodiesdiffusing inwards from the troughs containing anti-serum. The position, shape, and density of the pre-cipitates so formed are determined by the interactionof factors, including electrophoretic mobility of antigen,the diffusion constants of antigen and antibody, therelative concentrations of these reagents, and theiroptimum combining ratios. The large number ofdistinct precipitates formed with a complex system ofantigens and antibodies depends upon the extremelylow probability that two or more antigen-antibodypairs in the system will be identical in so many differentrespects.

Results

The characteristic immuno-electrophoretic com-ponents of human serum are shown in Fig. 1, which,for clarity, is a diagram based upon the precipitinpatterns identified with this technique. In thisdiagram the components are named according to theterminology of Grabar (1960). The alpha-2 lipo-proteins are further subdivided into "fast" and"slow" components, and "alpha-I principle"(S20 = 3.5 Sv.) is designated "main alpha-l". Theprecipitates so far identified are labelled, and itshould be noted that the components do not formgroups limited to the major bands produced bysimple electrophoresis. This may result from thefree radial diffusion of all components occurringin immuno-electrophoresis during the phase neces-sary for formation of the specific precipitates.Such diffusion complicates the interpretation of theresults of immuno-electrophoresis in terms of thoseof simple electrophoresis, and the two methods ofanalysis are complementary rather than equivalent.

Siderophi inBe

Ro-2 lipoprotein Alpha-2 lipoprotein 'fast':

Seromucod acid !Alp ha-2 lipoprotein'sow

!Albumin:

::A '~~~~Cgorulop I

g oprotcin Alpha-2~macroglobulin

!(3-5 SvJ.]*usAk*

-A

ta-2 B

Beta-2 AIBcta-2

| macroglobulin' Gamma globulin* I I

II !I

Fig. I.-Immuno-electrophoretic components of serum. Diagram constructed from a number of studies and including the componentsso far identified.

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ANNALS OF THE RHEUMATIC DISEASESTABLE

COMPARISON OF RESULTS OF IMMUNO-ELECTROPHORESIS WITH THOSE OF PAPER ELECTROPHORESIS

Increase of Beta-2 macro-globulin and Gamma

globulinTr

Groups of Subjects

(1) Rheumatoid Grade IArthritis iGrade II

(Functional Grade IIICapacity) Grade IV

All Grades

No.ofIn-divi-

duals;

818134

43

(2) Ankylosing Spondylitis 22

(3) Normal Subjects 20

Immuno- Paper Immuno-electropnoresis raper Immuno- Paperelectro- Electro- Electro- electro- Electro-

phoresis hotali phoeta 2iphoresis Total Alpha2 Total phoresisphoresisAcid Alpl Haptoglo bin macro- Gamma

Seromcoid globulin globulinAlh- ar-

globulin

roobucon globulin globulin

No Per No Per No Per No Per No Per No Per No Percent. cent. cent. cent. cent. cent. cent.

4 50 2 25 5 63 4 50 5 63 5 63 5 6312 67 3 17 13 72 10 56 9 50 15 83 18 1007 54 2 16 10 77 6 46 6 46 11 85 10 771 25 0 0 2 50 2 50 1 25 3 75 4 100

24 56 7 16 30 70 22 51 21 47 35 81 37 86

6 27 3 14 13 59 10 45 5 23 15 68 17 77

2 10 0 0 0 0 2 10 0 0 3 15 0 0

For this reason data obtained by both the methodspresented in the Table are arranged to show thesemiquantitation of immuno-electrophoretic com-ponents alongside quantitation of the major electro-phoretic fractions likely to include part or all ofthese components.

Except in the case of albumin and gamma globulin,which are relatively homogenous, there was littleor no quantitative correlation between the immuno-electrophoretic components and the correspondingmajor protein fractions, at least in terms of theobserved differences between sera obtained fromhealthy individuals or patients, and the standardsera obtained from the other healthy individuals.Excessive amounts of individual components were

observed more frequently in the sera of rheumaticpatients than in those of healthy individuals, butthese changes were not clearly related either to otherfeatures of rheumatoid arthritis, such as elevationof the erythrocyte sedimentation rate or severityof the disease in terms of functional impairment,or to duration of the disease. No attempt will bemade to interpret the following changes observed inpresently identified immuno-electrophoretic com-

ponents of the major electrophoretic fractions.Pre-albumin. Ro-2 lipoprotein is a component

of pre-albumin. The latter fraction is usuallyfound in barely detectable quantities, if at all, innormal serum. The ro-2 lipoprotein was readilydemonstrated in the sera from four patients withrheumatoid arthritis, including the two with com-

plicating renal disease, and it was also demonstrablein the serum of one of the patients with ankylosingspondylitis. The fraction was more readily demon-strable with the antisera prepared against the serum

of patients with renal disease, because of the pre-sence of relatively greater amounts of the appropriateantibody.

Albumin. This was represented by a singleprecipitin line produced by all the sera tested, butthe density of the precipitate varied in the proportionto the concentration of albumin determined bypaper electrophoresis (Figs 2, 3, 4, opposite, andFig. 5, overleaf).

Alpha-i Globulin. Increased amounts of acidseromucoid were found in 24 (56 per cent.) of thesera obtained from the patients with rheumatoidarthritis, in six (27 per cent.) of those from thepatients with ankylosing spondylitis, and in two(10 per cent.) of those from healthy individuals.Although there was much variation in the densityof the precipitin lines of other components of thisfraction, no consistent pattern could be discerned(Fig. 2).

Alpha-2 Globulin. All the sera from the patientswith rheumatoid arthritis and sixteen (75 per cent.)of those from the patients with ankylosing spon-dylitis gave rise to precipitin lines of markedlyincreased density corresponding with componentsof alpha-2 globulin (Figs 2 and 4). On the basis ofthe semiquantitative procedures, thirty (70 per cent.)of the sera from patients with rheumatoid arthritiscontained increased amounts of the haptoglobincomponent and 22 (51 per cent.) of these sera alsocontained increased amounts of the alpha-2 macro-globulin component (Figs 2 and 4). The sera frompatients with rheumatoid arthritis containing in-creased amounts of alpha-2 macroglobulin included

268

Increase ofAlpha-i globulins

Increase ofAlpha-2 globulins

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Albumin

269IMMUNO-ELECTROPHORETIC ANALYSIS OF PROTEINS

Haptoglobin Beta-2macroglobulinBgtamg-B

macr~ohg- maglobulin

--x- Fig. 2-Increase of alpha-i globuflill. dLense band of haptoglobin. iilphl-2Alpha-I-globulIn itacroglobulin. beta-2 macroglobulin. and beta-I-H globulin.

Antiserum: Rabbit-antihuman-rheumatoid serum.Antigen: Serum from patient with rheumatoid arthritis (functional capacity Grade II, E.S.R. 110 to 127 mm./hr,

haemagglutination titre 1: 1,024).Paper Electrophoresis: Albumin 39 0 per cent., Alpha-I globulin 5 4 per cent., Alpha-2 globulin 12-3 per cent.,

Beta-l globulin 11-2 per cent., Gamma globulin 32-1 per cent.

Albumin Gamma qlobulinI

- - aI - -III

A c i d s e r o m u c o i d Fig. 3.-Well-niarked band of acid seroniucoid.Antiserum: Rabbit-antihuman-rheumatoid serum.Antigen: Synovial fluid from the same patient as in Figure 2.

HaptoglobinAlbumin Alpha-2 Gamma gio

IL macroglobulin% N

bulin

Fig. 4.-Dense band of haptoglobin, alpha-2 macroglobulin, and beta-2 macroglobulin.Antiserum: Rabbit-antihuman-rheumatoid serum.Antigen: Serum from patient with rheumatoid arthritis (functional capacity Grade III, E.S.R. 60 to 90 mm./hr,

haemagglutination titre 1: 64).Paper Electrophoresis: Albumin 48-0 per cent., Alpha-i globulin 4-7 per cent., Alpha-2 globulin 9-0 per cent.,

Beta globulin 11-8 per cent., Gamma globulin 26-2 per cent.

*w_.. ...._

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ANNALS OF THE RHEUMATIC DISEASES

Al bumirn Double arc ofgamma globulli.

I t

Fig. 5.-Double arc of gamma globulin.Antiserum: Rabbit-antihuman-rheumatoid serum.Antigen: Serum from patient with rheumatoid arthritis (functional capacity Grade Ill, E.S.R. 110 to 125 mm. hr,

haemagglutination titre 1: 1,024).Paper Electrophoresis: Albumin 40 5 per cent., Alpha-1 globulin 4 3 per cent., Alpha-2 globulin 9 9 per cent.,

Beta-globulin 1 1 3 per cent., Gamma globulin 34 0 per cent.

those from two patients with renal disease, in whosesera alpha-2 lipoprotein was also demonstrable.This component was subdivided into two moitiesdiffering slightly in their electrophoretic mobility(i.e. "slow" and "fast").

Beta-] Globulin.- A dense precipitate of beta-l-Bglobulin was produced with seven of the sera frompatients with rheumatoid arthritis by the rabbitantisera to serum from patients with renal disease(Fig. 2). Increased amounts of beta-I lipoproteinwere present in the serum of these two patients.

Beta-2 Globulin.-Three components of this frac-tion were identified as beta-2 A, beta-2 B, and beta-2M. There was evidence of a fourth componentin this fraction of the sera obtained from four of thehealthy individuals. This precipitate appeared onlywhen these were tested with rabbit antiserumprepared against serum from the patients with renaldisease. The component has not yet been identified.The precipitates attributed to the A and B com-

ponents varied too much to permit adequate descrip-tion, and only the M component will be considered.This was increased in 35 (81 per cent.) of the seraobtained from patients with rheumatoid arthritis,in fifteen (68 per cent.) of those from patients withankylosing spondylitis, and in three (15 per cent.)of those from healthy individuals (Figs 2 and 4).

Gamma Globulin. This fraction usually has asingle immuno-electrophoretic component. How-ever, eight sera from the patients with rheumatoidarthritis and two from patients with ankylosingspondylitis produced an additional precipitin line,parallel to the first, suggesting the presence of twodistinct antigenic components in the gamma globulinfraction (Fig. 5). This possibility is discussed later.

Synovial Fluid. Proteins in the synovial fluidsfrom eight patients with rheumatoid arthritis wereexamined by immuno-electrophoresis, and theconcentrations of several components were com-pared with those of the same components of serumproteins. In six of the eight synovial fluids theconcentration of alpha-2 and beta-l lipoproteinand of beta-2 macroglobulin was significantlylower than in the corresponding sera. A denseband of precipitate corresponding with acid muco-protein of serum was observed in the patternsproduced by the synovial fluids obtained from threerheumatoid patients with active inflammatoryfeatures of recent onset (Fig. 3).

Urine. In immuno-electrophoresis, urine fromthe two patients with rheumatoid arthritis and renaldisease produced seven or eight distinct precipitinlines (Fig. 6, opposite). The samples of urine con-tained 7 to 18 g. protein per litre, and the com-ponents producing the most dense precipitates wereidentified as albumin, alpha- 1 proteins, alpha-2proteins, siderophilin, and gamma globulin.

DiscussionIt is possible to measure the concentration of the

major electrophoretic fractions of serum as per-centages of the total protein in the sample, and tocalculate absolute concentration from these data.Similar quantitation of immuno-electrophoreticcomponents is not yet possible and although someinformation is provided by the intensity and positionof the precipitates, these characteristics are partlydetermined by the antigenic potency of individualproteins. A number of proteins may be present inonly small amounts (e.g. alpha-2 and beta-2 macro-globulins) and yet induce relatively large amounts

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IMMUNO-ELECTROPHORETIC ANAL YSIS OF PROTEINS

Siderophi linGamma globulin

Ik Alpha-2 globulinAlpha-I globulin

Fig. 6.-Upper: chronic nephrotic syndrome (proteinuria 18 g./litre). Lower: amyloidosis (proteinuria 15 g./litre).Antiserum: Rabbit-antihuman-nephrotic serum.Antigen: Urine from two patients with rheumatoid arthritis and renal disease.

of specific antibody in a polyvalent antiserum,with the consequent formation of dense precipitateson immuno-electrophoresis.

In the present investigations, quantitation ofindividual components relative to the same com-ponents in standard sera was attempted by methodsinvolving dilution or selective absorption. Thequantitative changes thus demonstrated in sera frompatients with rheumatic disease were in general notparalleled by comparable changes in the majorelectrophoretic fractions, and it seems likely thatpaper electrophoresis and immuno-electrophoresisare complementary rather than interchangeablemethods of analysis (Table).

All the abnormalities of serum proteins found onimmuno-electrophoresis occurred in both rheuma-toid arthritis and ankylosing spondylitis and withapproximately the same incidence. The possibilityremains that extended investigation may revealdifferences between the two diseases in terms ofserum proteins and their sub-fractions. However,the changes so far demonstrated appear to be non-specific manifestations of inflammation occurringin both diseases.The most frequently observed abnormality was

the presence of an increased amount of beta-2macroglobulin; this was demonstrated in 81 percent. of the sera from patients with rheumatoid

arthritis and in 68 per cent. of those from patientswith ankylosing spondylitis. This difference inincidence between the groups is not significant:

X2 =0-8; 0-5 > P> 03.

The abnormality appears to be associated withactive inflammation and has been previously ob-served in active rheumatoid arthritis by Cleve andHartmann (1957), Cleve (1958), Fenrych and others(1959), Mackiewicz and Fenrych (1959), and Francqand others (1959). Because increased amounts of thismacroglobulin were found in sera with a normalsheep cell agglutination titre and in those with a hightitre, it is evident that the amount of beta-2 macro-globulin was unrelated to the presence or absenceof rheumatoid factor. According to Burtin (1960),the beta-2 macroglobulin includes a number ofproteins differing in physiological properties buthaving identical basic antigenic structures. Thelack of correlation between the sheep cell titre andthe concentration of the macroglobulin is thereforenot evidence against the rheumatoid factor beinga member of this family of proteins.Many of the sera from rheumatic patients gave

rise to precipitates of increased density in the bandsidentified as alpha-i and alpha-2 glycoprotein,acid seromucoid, haptoglobin, and alpha-2 macro-globulin. Such changes were demonstrable with

Albumin

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ANNALS OF THE RHEUMATIC DISEASES

sera from rheumatoid patients in all functionalgrades, but were less frequent with sera from spon-dylitic patients; their significance remains unknown.The double precipitate of gamma globulin which

occurred with some sera has been reported pre-viously. While the double arc is unusual (Grabarand Williams, 1955) and may sometimes occur asan artefact due to temperature changes duringdiffusion, true duplication is a reproducible pheno-menon when particular antisera are used. A doubleband produced by sera from infants and childrenwas reported by Hitzig (1957) and by Giedion andScheidegger (1957). In adults the anomaly wasfound by Schultze and Schwick (1957) in chronichepatitis, chronic nephrosis, and reticulosis. Adouble gamma globulin precipitate was also pro-duced by sera from patients with reticulosis(Creyssel, Fine, and Morel, 1959) and by sera fromsome patients with rheumatoid arthritis (Fenrychand others, 1959). Recently also, Grabar and Burtin(1960) emphasized the immunological hetero-geneity of gamma globulin. The significance ofsuch heterogeneity remains unknown and requiresfurther investigation.

Summary

Immuno-electrophoretic analysis was performedon sera from 43 patients with rheumatoid arthritisand 22 patients with ankylosing spondylitis, onsynovial fluid from eight patients with rheumatoidarthritis, and on urine from two patients withrheumatoid arthritis complicated by renal disease.

In many sera from patients with rheumatoidarthritis and ankylosing spondylitis there wereincreased amounts of alpha-l and alpha-2 glyco-proteins.The most constant finding in both groups was

an increased amount of beta-2 macroglobulin. Insera from some patients with rheumatoid arthritisand ankylosing spondylitis, a double arc of gammaglobulin was found. The significance of thisfinding is discussed.

Immuno-electrophoretic patterns of synovialfluid and urine were compared with the patterns ofsera from the same patients.

We are much indebted to Dr. J. L. Potter and Dr.J. J. R. Duthie for their helpful advice. We wish tothank Dr. P. Burtin from the Institut Pasteur, Paris,for helping to interpret some of the immuno-electro-phoretic patterns.

REFERENCES

Benacerraf, B., Potter, J. L., McCluskey, R. T., andMiller, F. (1960). J. exp. Med., 111, 195.

Burtin, P. (1960). In "Ciba Foundation Symposiumon Cellular Aspects of Immunity", p. 213.Churchill, London.(1961). Personal communication.

Chodera, A. and Mackiewicz, S. (1958). Przegl. lek.,14, No. 4, p. 113.

Cleve, H. (1958). Z. Rheumaforsch., 17, 350.- and Hartmann, F. (1957). Klin. Wschr., 35, 334.

Creyssel, R., Fine, J. M., and Morel, P. (1959). Rev.Himat., 14, 238.

Fenrych, W., Jazienicki, B., Mackiewicz, S., Mackiewicz,U., and Twardowski, K. (1959). Pol. Tyg. kek.,14, 1937.

Francq, J., Eyquem, A., Podliachouk, L., and Jacqueline,F. (1959). Ann. Inst. Pasteur, 96, 413.

Giedion, A., and Scheidegger, J. J. (1957). Helv.paediat. Acta, 12, 241.

Grabar, P. (1960). Triangel, 4, 185.and Burtin, P. (1960). "L'analyse immuno-electrophoretique". Masson, Paris.and Williams, C. A. (1953). Biochim. biophys.Acta, 10, 193.

(1955). Ibid., 17, 67.Hitzig, W. H. (1957). Helv. paediat. Acta, 12, 596.Mackiewicz, S., and Fenrych, W. (1959). Postepy Hig.

Med. ddsw., 13, 809.McCluskey, R. T., Benacerraf, B., Potter, J. L., and

Miller, F. (1960). J. exp. Med., 111, 181.Ouchterlony, 0. (1953). Acta path. scand., 32, 231.Scheidegger, J. J. (1955). lnt. Arch. Allergy, 7, 103.Schmid, K., and MacNair, M. B. (1956). J. clin. Invest.,

35, 814.Schultze, H., and Schwick, J. (1957). Behringwerke

Mitteilungen, heft 33.

Analyse immuno-6lectrophoretique des proteines seriquesRf-sumf

On proceda a l'analyse immuno-electrophoretiquedes serums de 43 malades atteints d'arthrite rhumatismaleet de 22 malades atteints de spondylarthrite ankylosante.des liquides synoviaux de 8 malades atteints d'arthriterhumatismale et de l'urine de 2 malades atteints d'arthriterhumatismale compliqu&e d'une maladie renale.Dans le serum de beaucoup de cas d'arthrite rhumatis-

male et de spondylarthrite ankylosante on trouva destaux augments de glucoproteine alfa-1 et alfa-2.Le resultat le plus constant dans les deux groupes fut

une augmentation de la beta-2 macroglobuline. Dansles scrums des cas d'arthrite rhumatismale et de spondyl-arthrite ankylosante on trouva un double arc de globulingamma. On discute l'importance de ce resultat.

Les tableaux immuno-electrophoretiques du liquidesynovial et de l'urine furent compares a ceux offertpar les scrums des memes malades.

Analisis imuno-electroforetico de las proteinas sericasSUMARIO

Se llevaron a cabo analisis imuno-electroforeticosen sueros procedentes de 43 enfermos con artritis reuma-toide y 22 con espondilartritis anquilosante, en liquidosinovial de ocho enfermos con artritis reumatoide y enorina de dos enfermos con artritis reumatoide complicadacon enfermedad renal.

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IMMUNO-ELECTROPHORETIC ANALYSIS OF PROTEINS 273En el suero de muchos de los enfermos con artritis enfermos con artritis reumatoide y espondilartritis

reumatoide y espondilartritis anquilosante se encontr6 anquilosante se encontr6 un doble arco de gama globu-un aumento de la cantidad de alfa-l y alfa-2 gluco- lina. Se discute el significado de dicho hallazgo.proteinas. Los cuadros imuno-electroforeticos del liquido sinovial

El hallazgo mas constant en ambos grupos fue el y de la orina fueron comparados con los hallados en elaumento de beta-2 macroglobulina. En sueros de suero de los mismos enfermos.

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