Purification of the rheumatoid factor

CLINICA

PURIFICATION

CHIMICA

ACTA

OF THE RHEUMATOID

(Received July zsth,

519

FACTOR

1963)

A preparation of rheamatoid factor was obtained from a pooled rheumatoid serum by absorption to diphtheria toxoid-human antitoxin precipitate, subsequent elution by lowering the pH, and gel filtration of the eluent. The isolated rheumatoid factor was homogeneous in the analytical ultracentrifuge and in immunoelectrophoresis. The corrected sedimentation coefficient was 19.3 S. The yield of rheumatoid factor as determined by serological and radioactivity techniques was 15-200/b.

INTRODUCTION

The rheumatoid factor (RF) is a ~~-macroglobulin having a sedimentation coefficient of 19 S. This serum fraction is known to contain a variety of antibodies, as well as the Waldenstrom type of macroglobufinsl. The RF is often found in the serum as a 22s complex composed of RF and 7s y-globulina, and the available evidence seems to indicate that the RF is an antibody against human y-globulin showing cross-reactivity with y-globulins of other speciess-6. Until now, it has not been possible to separate the RF from the other macrogIobulin antibodies by physicochemical fractionation methods. Therefore, advantage has been taken of its reactivity with y-globulins. HEIMER et aL7 used sheep erythrocyte stromata sensitized with rabbit anti-sheep erythrocyte amboceptor for absorption, and eluted the factor at PH 5.5. LOSPALLUTO AND ZIFF” and KUNKEL et at.% precipitated the RF by adding Cohn Fraction II and dissolved the complex either with 4 M urea or at pn 3. Further purification was achieved by density gradient ultracentrifugation’? %or by DEAE-cellulose chromatogrsphys. Differential absorption experiments have indicated that human immune precipitates wholly absorb the RF activity, measurable with any detector system, from rheumatoid sera, whereas immune precipitates with the antibody portion from other species completely remove only the activity measurable with the isologous systemB. It is possible to elute the absorbed RF from the washed precipitate by

K.

520

SIMONS et d.

lowering the PH. Purified RF products prepared under mild conditions are needed especially for studies of the possible biological effects of the RF. In the present paper, a simple purification method is described which is based on elution of the RF absorbed to immune precipitates and on gel filtration.

MATERIAL

ANDI\IETHODS

Immwae precipitates The production of human diphtheria antitosin has been described elsewhere”‘. Large amounts of diphtheria toxoid-human antitoxin precipitate were prepared in the zone of maximal precipitation to serve as a constant source of material. The thrice-washed precipitates were suspended in saline, and “Merthiolate” (trade-mark of IEli Lilly and Co. for Thimerosal) I : 5000 was added to the precipitate suspension. Rheumatoid

seyzlm

A pooled rheumatoid serum composed of eight high-titred sera was used in all experiments. Its Waaler-Rose titre was 2048 and SHC (Ripley) titre 8192-16384. It was stored at -20”. Iodi-nation of the rheumatoid

serum

The iodination of the rheumatoid serum was performed by the 13lICl method of McFARL~NE~~, adding 0.3 mg of ‘3IICl per milliliter of serum. The trace-labeled serum was immediately separated from the free iodine by gel filtration in Sephadex G-25 (AB Pharmacia). Radioactivity was counted in a well-type scintillation detector. Fyactionatiofl

method

In order to avoid spontaneous precipitation of the macroglobulin preparations, all purification procedures were performed at room temperature if not otherwise specified. A more effective absorption of the RF to human immune precipitates was accomplished by using diluted rheumatoid serum than by using the same serum undilutedlo. Therefore, the serum diluted I/O? with phosphate-buffered saline (pH 7.4) was mixed with the immune precipitate. After two hours the precipitate with the absorbed RF was washed three times with a large volume of chilled saline. It was then treated for one hour with acetate buffer pH 5.0. The precipitate was centrifuged and the supernate separated. The procedure was repeated twice. The three supernates were combined, neutralized with trishydroxymethylaminomethane and adjusted to ionic strength 0.15 with distilled water. After concentration by ultrafiltration to a volume of 5 ml, the resulting preparation was transferred to the top of a Sephadex G-zoo (free samples supplied by AB Pharmacia) column 3 x 45 cm. The optical density at 280 rnp was measured from the effluents with a Beckman DU spectrophotometer. Methods used for characterizatiolz

ofrkeumatoid factor

For measuring the RF activity, two techniques were used. In the Waaler-Rose test, the samples to be tested were first absorbed with normal sheep red cells. For sensitization of the cells, 113 of the minimum agglutinating dose (I/S -MAD) of homologous rabbit amboceptor was used. The SHC test was performed with the microscopic technique of HARBOEANDLUNDEVALL12.Anti-Rh serum Ripley (obtained (‘li)2.f.hiW..4&z, 9 (1964)519-52;

RHEUMATOID FACTOR

521

through the courtesy of Drs. MARION WALLER AND J. VAUGHAN) was used for coating the cells. The protein concentration of the fractions was determined by the method described by LOWRY et aZ.13. Immunoelectrophoresis was performed as described by SCHEXDEGGER~*.In addition to commercial antisera against human serum (Pasteur Institute and Behringwerke) and against human ~~-globulin (~ehringwerke), we used rabbit anti-human y-globulin serum and mouse anti-human ~~~-globulin serum prepared in our laboratory. The anti-immuno-globulin sera were unabsorbed. The sedimentation runs were done with Spinco Model E analytical ultracentrifuge at 59780 rev./min. at 20.0~. The samples were first dialyzed against 0.2 it;l NaCl in 0.01 fil phosphate buffer pH 7.1. The pooled rheumatoid serum was examined in the 3-mm cell at a concentration of 2.40/b ; the other runs were done in the rz-mm cell at concentrations of 0.03-0.08~,$. For the calculations of sedimentation coefficients, the value of 0.74 was used for the partial specific volume. RESULTS Fig. IA-C illustrates a puri~cation experiment. Fig. IA shows the sedimentati~~n diagram of the pooled rheumatoid serum, where a small macroglobulin peak isseen, totalling about 2yk of the serum proteins. The rheumatoid factor from IO ml of serum was then absorbed from the serum to diphtheria toxoid-human antitoxin precipitate. Based on preliminary experiments, the amounts of rheumatoid serum and immune precipitate were adjusted so that a marked RF titre decrease would be seen in the supernate after absorption. After washing of the precipitate, the RF was eluted at pn 5.0. The neutralized and concentrated product was subjected to ultracentrifugation. As seen in Fig. rB, three components were discernible. The relative concentrations and approximate sedimentation coefficients of these three peaks were as follows :

This product was further purified by gel filtration in Sephadex G-ZOO. Preliminary runs with dissolved rheumatoid cold precipitates had shown that the first protein peak contained all the RF activity and no y-globulin as measured by the Coombs inhibition technique. The elution diagram is shown in Fig. 2. Tubes 18-23 were pooled and concentrated by ultrafiltration and dialyzed. This preparation was subjected to analytical ultracentrifugation. As is seen in Fig. XC, one homogeneous peak with no impurities was detected. The sedimentation coefficient (So,,, W) of this peak was 19.3s. Fig. 3 shows an immunoelectrophoretic run of this final product with the commercial anti-human serum of ~ehringwerke. Only one Iine is seen in the @,-region. The same applies to all the antisera tested. In order to follow the purification procedure quantitatively, RF activity and protein determinations were supplemented by using Ia11 trace-labeled rheumatoid serum. The most satisfactory yield was obtained when a slight excess of rheumatoid serum was used for absorption. Under these conditions it was possible to recover in

K. SIMONS et ai.

522

Fig. I. ~?lt~acentrif~~~e patterns

of .X, pooled rheumatoid product.

__.!_.-,_..

20

_~...“._ .....J.__.___---__. 25

swum:

B, acid elucnt;

..-._A.._.

___ __

30 Tube

Fig.

2. Gel

filtratian

of the acid eluent.

35

number

C, tbal

purified

RHEUMATOID

523

FACTOR

the acid eluent about one-third of the radioactivity absorbable to a large excess of the immune precipitate. All the absorbed radioactivity could not be eluted from the immune precipitate at PH 5.0, and at lower PH levels the immune precipitate began to dissociate. !

Fig.

,/ ,-.

‘.-’ / \; ‘9

r-7

3. Immunoelectrophoresis

of the purified R F product.

TABLE PURIFICATION

_

OF THE

Initial rheumatoid serum Acid eluent from immune precipitate Final product from ultrafiltration and gel filtration

I

RHEUMATOID

FACTOR

2048

16384

2.0

512

4096

0.91

256

2048

290

* Titres are expressed in terms oi I ml of the original serum used for absorption. C&a.

Chim.

Acta,

g (1954)

srg-525

524

K. SIMONS

et al.

Table I is the protocol of a purification experiment. 4 ml of the pooled rheumatoid serum was absorbed to 13.6 mg of the immune precipitate. The Waaler-Rose and SHC (Ripley) titre decreases in the supernate after absorption were three twofold steps. The total volume of the acid eluent after neutralization was 15 ml and it contained 2.0 mg of protein. The final product had about 1/8 of the RF present in the original serum, and the amount of protein was 0.91 mg.

activity

Both analytical ultracentrifugation and the immunoelectrophoretic patterns indicate homogeneity of our RF preparation. However, there remains a theoretical possibility that the preparation might have been contaminated to some extent by other macroglobulins of the p2n serum fraction that had been coabsorbed to the immune precipitate and eluted together with the RF. The purification procedure described in the present paper appears to afford some advantages when compared with the methods previously employed. It is evident that the acid eluent from the immune

precipitate contains less impurities than that from the sensitized sheep erythrocyte stroma ‘. On the other hand, the use of 4 ,%I urea or pH 3 as a solvent for the RF-y-globulin complexs> 9 may be open to objection because of their pcssible denaturing effect on the RF. The low molecular weight impurities present in the RF preparation eluted from the immune precipitate were separated by us from the macroglobulin component by gel filtration on Sephades G-ZOO. This simple procedure gives a good separation of the macroglobulins from the other serum components15. The yield of RF obtained by the present method is about 15-ZOO;,. This must be considered satisfactory since no data on the recoveries of RF activity have been given in earlier reports where specific immunological methods were used. It is difficult to compare the present yield with the yields obtained in purifications of 7s antibodies, in which recoveries of even 90:;~ have been reported 16. Large manipulative losses are known to occur in the isolation of RF, which probably becomes more susceptible to denaturation as purification proceeds7, *f I’. The loss of RF activity encountered during the gel filtration and ultrafiltraticn steps was as high as 5000. For purposes in which highly purified RF is not necessary, use can be made of the acid eluent, omitting the last purification steps. By using immune precipitates with the antibody portion from different species, RF of different specificities can be isolated. We have successfully usedaneggalbumin rabbit anti-egg albumin precipitate for purifying RF reactive with rabbit y-globulin.

ACKNOWLEDGEMENTS

This investigation was supported by grants from the Sigrid JusClius Foundation, Finland, and in part by research grant A-3026 from the National Institute of Arthritis and Metabolic Diseases, U.S. Public Health Service. The authors gratefully acknowledge the competent technical assistance of Miss TUULA

KUMAJA.

Clin. Ckirn. Ada,

g

(1964)519-525

RHEUMATOID

FACTOR

REFERENCES 1 J. FAHEY, fl&m. ~ntrntc~o~., 2 jr962) 41. 2 E. FRANKLIN, N. HOLMAN, H. MULLER-EBERHARD

AND

5YJ

H. KUNKEL,

J. Exptl. &fed., Ioj

(1957) 425. 3 H. FUDENBERG AND H. KUNKEL,]. Exptl. Med., II.$ (1961) 257. 4 C. CHRISTIAN, Arthritis Rheumat., 4 (1061) 86. 5 J. VAUGHAN AND J. BUTLER, JR.,Anfz. InternalMed., 56 (1962) I. 6 Ii.AHO, _djtx.Med. Exptl.Biot.Fenrsiae [Hekinkij, 39 (rg@I) SxppE. 7. 7 R. HEIMER, Q. FEDERICO AND R. FRLWBERG, Proc. Sot. Expfl. Biof. filed., g9 (1958) 381. 8 J. LOSPALLUTO AND M. ZIFF, J. Exptl. Med., 110 (rg.jg) 169. 9 H. KUNKEL, E. FRANKLIN AND H. MULLER-EBBRHARU, J. CEirz. IRVFS~., 38 (1959) .&21. 1~ I<. .%i~o ANV K. SIMON& Avfhritis lihemnaf., in the press. I* X. MCFARLANE, ?~'ulure. 182 (1958) 53. 12 IN. HARBOE ANU J. LUNDEVALL, Acta Patfaol. Mirrohiol. Scmd., 45 (1959) 357. 13 0. LOWRY, PT.RO~EBROUGH, A. FARR AND R. RANDALL, J.Biol.Chenz., rqj (1951) 265. 14 JSCHEIDEGCER, Interra. .4rch._4&rg3~ Appt. Itimwol., 7 (1955) 103. 15 f-‘. F~onm AND J. KILLANDER, Biochi~. BiopRys. Act& 63 (1962) .ps. 16 E. K.&BAT AND M. MAYER, ~x~p~i~~en~~Zr~~~z~~~or~e~~sf~~, Charles C. Thomas, Sprin&itM,