Anti-γ-globulins in rheumatoid arthritis sera—III. The reactivity of anti-γ-globulin rheumatoid factors with heterologous γG-globulin

lmmunochemistry, 1972,Vol. 9, pp. 725-736. Pergamon Press. Printed inGreat Britain

ANTI-y-GLOBULINS IN RHEUMATOID ARTHRITIS SERA--III. THE REACTIVITY OF ANTI-y-GLOBULIN RHEUMATOID FACTORS WITH HETEROLOGOUS yG-GLOBULIN* DAVID E. NORMANSELL Department of Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia 22903, U.S.A. (First received 13 September 1971; in revisedform 20 November 1971) A b s t r a c t - T h e proportion of anti-,/-globulin rheumatoid factors, isolated from one

serum, showing specificity for either human, rabbit or both human and rabbit -/Gglobulin was determined by adsorption of the RF Onto columns of human or rabbit ,/G-globulin. The majority of the RF had specificity for both human and rabbit ,/Gglobulins, with smaller amounts specific for one or the other species of ,/G-globulin. The interaction between these RF species and human, rabbit and horse yG-globulins was examined in the ultracentrifuge. The measured binding constants were within the range 1-5 x liP L/M. INTRODUCTION Anit-y-globulin rheumatoid factors (RF) react with yG-globulins isolated from human and other animal sera. At least three species of RF may exist within a single rheumatoid arthritis serumi one species reactive only with human yG-globulin, one species reactive only with rabbit yG-globulin and another species reactive with both human and rabbit yG-globulins (Milgrom et al., 1962; Milgrom and T6nder, 1965; Skalba and Stanworth, 1969; T6nder and Natvig, 1966; Williams and Kunkel, 1963). Other investigators, however, consider that all RF in a given serum reacts with human yG-globulin, the reaction with heterologous yG-globulins representing a cross-reaction (Butler and Vaughan, 1964, 1965). To obtain further details of the properties of these various RF species, RF isolated from rheumatoid arthritic serum was purified by adsorption to insolubilized human or rabbit yG-globulin. The proportion of the total RF moiety reactive with each type of yG-globulin was measured, and the binding constants for these interactions obtained. Three types of RF were detected: RF reactive with human 7G-globulin, with rabbit 7G-globulin or with both human and rabbit 7G-globulin, with the majority of the RF reactive with both human and rabbit 7G-globulins. MATERIALS AND METHODS (a) Protein preparations RF was isolated as described previously (Normansell, 1971) from aliquots of one batch of serum from a patient (MB) with classical rheumatoid arthritis *Supported in part by Health Sciences Advancement Award No. FR06001; United States Public Health Service Research Grant AM 15248 from the National Institute of Arthritis and Metabolic Diseases; and the Virginia branch of the Arthritis Foundation. 725

IMM Vol. 9 No. 7 - D

726

DAVID E. NORMANSELL

by chromatography of the euglobulin fraction of the serum on G200 Sephadex at pH 4.5. RF solutions were stored in 0.01 M potassium phosphate buffer, pH 7.3 containing 0.15 M sodium chloride (PBS) at 4°C at 5-10 mg/ml, assuming an optical density of 1.2 at 280 nm for a 1 mg/ml solution (Miller and Metzger, 1965). RF solutions had strong reactivity with goat anti "/M-globulin and very weak reactivity with goat anti '/G-globulin (Kallestad Labs, Inc.). The '/Gglobulin contamination was almost completely removed by re-elution of the RF through G200 Sephadex at pH 4.5. The 7S fraction of human, rabbit and horse '/G-globulins was isolated from commercial Cohn Fn II preparations (Pentex, Mann Research Labs) as described previously (Normansell, 1970). T h e columns of cross-linked "/G-globulin were prepared from Fn II yG-globulin by the addition of glutaraldehyde (Eastman, 0.25% aqueous solution, 0.08 ml per mg yG-globulin) (Avrameas and Ternynck, 1969). The polymerized ,/G-globulin was homogenized, mixed with G25 Sephadex packed into a column (15 × 2 cm) and washed successively with PBS and 0-05 M glycine-HC1 buffer, pH 2.8, until the effluent was protein free. RF was applied to the columns, at 22°C, in PBS and the columns were eluted with PBS until the effluent, continuously monitored at 280 nm in a Gilford 240 spectrophotometer, was protein-free. Adsorbed RF was eluted with 0.05 M glycine-HCl buffer, pH 2.8; adjusted to pH 7-3 with concentrated dipotassium hydrogen phosphate and dialysed against PBS. In general, more than 80 per cent of the applied protein was recovered from the column. At least 30 mg RF could be adsorbed to the rabbit yG-globulin column without overloading; the capacity of the human yG-globulin column was somewhat lower, but at least 20 mg RF could be adsorbed without overloading. Re-elution of the eluate peaks on the column yielded similar elution patterns and agglutination titres, indicating that nonspecific adsorption was not occurring to any detectable extent. (b) Agglutination procedures Formalinized sheep erythrocytes were suspended to 4% suspension in PBS and activated by incubation with glutaraldehyde (0.5ml of 2.5% aqueous solution per 10 ml cell suspension) for 1 hr at 22°C, with stirring. The cells were spun down (1500 g, 10 min), the supernatant solution removed and the packed cells re-suspended, to 4% suspension, in protein solution (0.5 to 1.5 mg '/Gglobulin per ml PBS). The suspension was stirred for 1 hr at 22°C, the cells spun down and washed × 3 in PBS. The cells were re-suspended in PBS and incubated with excess L-lysine at 37 ° for 20 min to saturate any unreacted aldehyde groups. Finally, the coated cells were spun down, washed sequentially with PBS containing 1% BSA and PBS + 0" 1% BSA. The cells were stored as a 1% suspension at 4°C in PBS + 0" 1% BSA and used within 2 days. The level of coating achieved was checked by testing the cells with commercial antisera to human, rabbit and horse '/G-globulins. For agglutination tests, the RF fractions were adjusted to 1 mg/ml in PBS. Duplicate titrations were performed in microtiter plates (Bellco Glass Co.). (c) Measurement of binding constants Binding constants were measured in the ultracentrifuge as described

Anti-y-Globulins in Rheumatoid Arthritis Sera-III

727

previously (Normansell, 1970, 1971). Initially, RF and yG-globulin concentrations of 1.0 mg/ml were used, but this was changed to 0.5 mg/ml for the majority of the runs, in order to use the photoelectric scanning system of the Beckman Model E centrifuge in the 0 - 1 0 . D . range at 280 nm. The protein solutions were dialysed together against PBS in boiled dialysis tubing in order to equalize small ions. An ANFTi rotor was used, with Kel F or Aluminum double sector centerpieces, sapphire windows and wide aperture window holders. Runs were made at 60,000 rev/min and 22°C, three cells being scanned sequentially every 4 min. Protein concentrations were determined by comparison of pen deflections with the calibrating steps, corrected for sectorial dilution and averaged for each sample. For each binding constant determination, three concentrations each of RF and yG-globulin were examined, as controls, to determine their concentration. Mixtures o f RF and yG-globulin were then examined and the amount of yG-globulin bound to RF determined from the difference between the amount initially added (as determined from the control runs) and the amount remaining unbound. The binding constant was calculated from a conventional Scatchard plot as the free antigen (yG-globulin) concentration at half saturation of the antibody (RF) combining sites. It was assumed that all of the human yG-globulin was potentially reactive with RF. Previous studies (Normansell and Stanworth, 1968) have indicated that only approximately 67 per cent of rabbit yG-globulins and 33 per cent of horse yG-globulins react with RF in the ultracentrifuge. This is supported by the suggestion (Skalba and Stanworth, 1969) that one conformational form of rabbit yG-globulin may not react with a pooled RF preparation, and by the results of Henney and Stanworth (1964) where the amount of precipitation occurring between RF and heat aggregated yG-globulin varied according to the species of yG-globulin. Accordingly, in the results presented here, allowance was made for the presence of the inactive yG-globulin. For reactivity between RF and rabbit yG-globulin, one third of the yG-globulin was assumed inactive, and for the reactivity between RF and horse yG-globulin, two thirds of the yGglobulin was assumed inactive. RESULTS In preliminary experiments, about half of the applied RF was found to bind to either the human or the rabbit yG-globulin column at pH 7.3, and was subsequently eluted at pH 2.8. Examples of the elution profiles observed are shown in Fig. 1, which represents the separation of 40 mg RF on the rabbit yG-globulin column (Fig. 1A) and on the human yG-globulin (Fig. 1B). The yield and agglutination titres c f each eluate are shown in Table 1 and indicate that RF which did not adsorb, at pH 7.3, to one yG-globulin column did not agglutinate cells coated with that species of yG-globulin, but did agglutinate cells coated with the other species of yG-globulin. RF which bound to the column at pH 7.3 and which was eluted at pH 2"8, agglutinated cells coated with either species of yG-globulin. Several samples of this RF preparation were separated on these yG-globulin columns, with essentially similar results. In every case, protein which had adsorbed to the rabbit yG-globulin column at pH 7"3 was eluted as a compact peak at pH 2.8, whereas protein which had

728

DAVID E. NORMANSELL (a)

I

^ /, I /

g

~ ~

IO

pH 2"8

buffer 20

40

50

60

70

40

50

60

N

r'~ 0

(b)

PH 2-8

uifer Io

20

30

Fraction no. Fig. 1. Elution profiles of RF on human and rabbit 7G-globulin columns. (a) Starting RF preparation on rabbit yG-globulin column. (b) Starting RF preparation on human yG-globulin column. adsorbed to the human yG-globulin column at pH 7.3 was eluted as a much broader peak. The first part of this broad peak, fractions 33-40, Fig. 1B, was eluted between pH 6 and 7, while the remainder of the peak was eluted at pH 2.8. The pH 2.8 eluates from the human and rabbit 7G-globulin columns were examined for reactivity with human, rabbit and horse yG-globulin in the ultracentrifuge. The Scatchard plots obtained are shown in Fig. 2. RF purified on the human yG-globulin column had identical reactivity with the three yGglobulins (Fig. 2A), whereas RF purified on the rabbit yG-globulin column appeared to have stronger reactivity with rabbit yG-globulin than with human or horse 7G-globulins (Fig. 2B). Furthermore, the reactivity of both of these purified RF preparations with human and horse yG-globulin was identical. From these results, it appeared that rabbit-purified RF had stronger reactivity with rabbit "/G-globulin than did human-purified RF. However, the calculated binding constant for these interactions depended upon the value chosen for n, the number of binding sites per macromolecule, and this depended on the relative proportions of RF molecules specific for each type of 7G-globulin. RF purified by adsorption to the human yG-globulin column should be fully reactive with human yG-globulin, but the proportion reactive with rabbit yG-globulin should be lower, because the human yG-globulin purified RF contained some RF reactive only with human 7G-globulin and not with rabbit yG-globulin. To establish the proportion of RF having specificity for human or rabbit 7G-globulin, or both, the eluates obtained from one 7G-globulin column were

Human Rabbit

Column type

aNegative at 1/2 dilution.

35 40

Amount of RF added mg 19 20

Yield mg _ a 1/16

Anti-human yG-globulin 1/32 -

And-rabbit yG-globulin

pH 7-3 eluate titres

16 18

Yield mg

1/64 1/64

Anti-human 3K;-globulin

1/1024 1/1024

Anti-rabbit yG-globulin

pH 2-8 eluate titres

Table 1. Fractionation of 19S RF o n yG-globulin columns. Starting RF titres were: 1/128 anti-human yG-globulin, 1/1024 anti-rabbit yG-globulin. All titres were measured at a concentration of 1 mg/ml

I

t~

,,d,

g.

730

DAVID E. NORMANSELL (a)

4'01

(b) 5O

35

30

J

40

25 30 20 .J 0

15

20

x I0

05 I I

I 2

I 3

I 4

I 5

,°I 0

1 [

I 2

] 3

] 4

J 5

r

Fig. 2. Scatchard plots for the reactivity of RF purified by adsorption to, and elution from (a) human and (b) rabbit yG-globulin columns. O - - O reactivity with human yG-globulin. A--A reactivity with rabbit yG-globnlin. /N--V] reactivity with horse yG-globulin. The scale difference is due to the overall protein concentration. In (a) the total concentration was 0-5 mgm/0.5 ml, while in (b) it was 0"25 mgm/0"5 ml. further separated on the other yG-globulin column. Protein which had not adsorbed to the human yG-globulin column at pH 7-3 might contain both inactive material and RF with specificity directed towards rabbit yG-globulin but not human yG-globulin. Separation of this fraction on the rabbit yG-globulin column resulted in a pH 7.3 eluate devoid of RF activity and a pH 2.8 eluate reactive only with rabbit yG-globulin (Table 2, line 1). Protein eluted from the human yG-globulin column with pH 2.8 buffer, and reactive with both human and rabbit yG-globulins, also gave a pH 7.3 and a pH 2"8 eluate when separated on the rabbit yG-globulin column. The pH 7.3 eluate when separated on the rabbit yG-globulin column. The pH 7.3 eluate was reactive only with human yG-globulin, the pH 2.8 eluate reacted with both human and rabbit yG-globulins (Table 2, line 2). In a similar way, the eluates obtained from the separation of RF on the rabbit yG-globulin column were each separated on the human yGglobulin column (Table 2, lines 3 and 4) and again resulted in the separation of the various species of RF. These results indicated that several specifications exhibited by the RF in this serum could be clearly distinguished: RF showing only anti-hun}an yGglobulin reactivity adsorbed only to the human yG-globulin column, RF showing only anti-rabbit yG-globulin activity adsorbed only to the rabbit yG-globulin column, but RF showing both reactivities adsorbed to both columns. The results of several similar experiments enabled the specificity of about 90 per cent of the detectable RF in this preparation to be obtained. This is shown in Table 3. About 35 per cent reacted with both human and rabbit yG-globulins, about 18 per cent

25

25

30

31

Amount added mg

*Negative at 1/2 dilution.

H u m a n yG-globulin column p H 7.3 eluate H u m a n yG-globulin column p H 2.8 eluate Rabbit yG-globulin column p H 7"3 eluate Rabbit yG-globulin column p H 2.8 eluate

RF sub-fraction added

Rabbit yG-globulin Human yG-globulin Human yG-globulin

Rabbit yG-globulin

Column type

5

17

9

16

Yield mg

-

--

1/8

_ a

1/64

--

-

-

p H 7-3 eluate titres A n t i - h u m a n Anti-rabbit yG-globulin yG-globulin

20

12

13

9

Yield mg

1/512

1/32

1/512

-

1/2048

-

1/4096

1/2048

p H 2"8 eluate titres A n t i - h u m a n Anti-rabbit yG-globulin yG-globulin

Table 2. F u r t h e r fractionadon o f RF previously separated on h u m a n o r rabbit yG-globulin columns. All fractions tested at 1 mg/ml.

I

a

~o ¢b

=r

:t

t~ e~

=.

732

DAVID E. NORMANSELL

showed monospecific reactivity with h u m a n 7G-globulin and about 16 p e r cent showed monospecific reactivity with rabbit 7G-globulin. T h e RF preparations used in the e x p e r i m e n t s shown in Fig. 2 should thus consist o f approximately bi-specific RF and ½ monospecific RF, in each case, leading to a value o f 3-3 for n in the 'cross-reaction' ( h u m a n - p u r i f i e d RF reacting with rabbit 7G-globulin and vice versa)*. T h e binding constants calculated on this basis are shown in Table 4 and it is a p p a r e n t that although t h e r e is no large difference in the results, there is a t r e n d towards h i g h e r reactivity with rabbit 7G-globulin. T h e results p r e s e n t e d in Fig. 2 and T a b l e 4 reflect mainly the properties o f RF reactive with both h u m a n and rabbit 7G-globulins, since this RF species is the most a b u n d a n t in this serum. Several attempts were m a d e to isolate the bispecific RF in o r d e r to confirm its equal reactivity with h u m a n and rabbit yGglobulins. However, the p r o c e d u r e s involved, adsorption and acid elution f r o m each o f the two columns, a p p e a r e d to be too vigorous for the RF. Although RF a p p e a r e d able to withstand one e x p o s u r e to the acidic buffer, a second exp o s u r e always resulted in instability o f the RF molecules with the f o r m a t i o n of aggregates and fragments. Attempts were m a d e to measure the binding constants o f such preparations o f bi-specific RF in the ultracentrifuge but analysis o f the photoelectric scans was extremely difficult and inaccurate. H o w e v e r f r o m the results which could be obtained, no difference could be detected in the reactivity with h u m a n or rabbit 7G-globulins. F u r t h e r studies were directed towards those RF molecules showing specificity for h u m a n or rabbit 7G-globulin, but not both. RF which had not adsorbed at p H 7.3 to the h u m a n 7G-globulin c o l u m n was separated on the rabbit 7G-globulin column. About 30 per cent o f the protein adsorbed to the c o l u m n at p H 7.3 Table 3. RF specificities detected in serum MB Specificity

Average percentage found

Inactive Anti-human yG-globulin Anti-rabbit yG-globulin Anti-human and rabbit 7G-globulins

24 (18-31) 18 (14-24) 16(10-21) 35 (26-40)

Table 4. Binding constants of purified RF for rabbit and human yG-globulins -yG-globulin column used to purify RF Rabbit Human

Reactivity with rabbit 7G-globulin K0, L/M n 4 x l0 s 5 x l0 s

5 3-3

Reactivity with human yG-globulin K0, L/M n 4 x l0 s 2 x l0 s

3.3 5

*Only two-thirds of the RF reacts in the cross-reaction, hence the value of n is 5 x 3 = 3.3.

Anti,y-Globulins in Rheumatoid Arthritis Sera-II I

733

and was then eluted at pH 2"8. This acid eluate agglutinated sheep erythrocytes coated with rabbit yG-globulin to high titre (1/2048, see Table 2, line 1) but did not agglutinate cells coated with human yG-globulin. This fraction was concentrated and chromatographed on G200 Sephadex at pH 4-5. RF showing reactivity only with human yG-globulin was prepared by a similar procedure and the binding constants for the reactions of these RF species with their respective yG-globulins measured in the ultracentrifuge. The Scatchard plots obtained are shown in Fig. 3. Extrapolation of the curves indicated a binding constant of 1-2 × 105 L/M. The monospecific RF preparations obtained from this serum, and from a second serum (E.S.) were also tested against a commercial latex reagent (Hyland) and against agglutination systems involving aggregated human yG-globulin (Table 5). Both specific anti-rabbit yG-globulin preparations showed weak positive latex reactivity, but essentially no reactivity with heat-aggregated human yG-globulin. DISCUSSION It is now well established that at least two fractions of RF exist, one fraction reactive with human yG-globulin, another reactive with both human and rabbit yG-globulin. Strong evidence for the existence of a third fraction reactive with rabbit yG-globulin, but not with human yG-globulin, was obtained by Williams and Kunkel (1963), and T 6 n d e r and Natvig (1966). The results presented in the present paper add further evidence for the existence of the fraction specific for rabbit yG-globulin, and show that the majority of the RF isolated from this 5-0

4.0

3.0 --J ID t

o

2.0

X

0

I

I

I

I

2

3

I 4

I 5

r

Fig. 3. Scatchard plots for the reactivity of 'monospedfic' RF. C)~O human yG-globulin-RF anti-human yG-globulin. A--A rabbit yG-globulin-RF anti-rabbit yG-globulin.

734

DAVID E. NORMANSELL Table 5. Agglutination titres of monospecific RF preparations

Test System

Serum M.B. Serum E.S. Anti-human Anti-rabbit Anti-human Anti-rabbit yG-globulin yG-globulin yG-globulin yG-globulin

Commercial latex reagent SRCa coated with 7S human yG-globulin via glutaraldehyde SRC coated with 7S rabbit yG-globulin via glutaraldehyde SRC coated with heat-aggregated human yG-globulin via glutaraldehyde Tanned SRC coated with heat-aggregated human ,/G-globulin

1/64

1/4

1/64

1/32

_b

1/32

-

1/32

-

1/64

-

1/32

1/256

1/2

1/512

1/4

1/32

aSheep red cells. bNegative at 1/2 dilution. serum (M.B.) reacted with both h u m a n and rabbit yG-globulin. Early studies on the fractionation of RF employed either human Fn II coupled to tanned sheep erythrocytes, or a rabbit anti-sheep erythrocytessheep erythrocyte complex (Heimer et al., 1961; Heller et al., 1955). Subsequent studies have employed yG-globulin chemically coupled to sheep erythrocytes (Butler and Vaughan, 1964, 1965; Skalba and Stanworth, 1969) or to polyaminopolystyrene (Williams and Kunkel, 1963). In most instances, as in the present studies, the TG-globulin used was a Fn II preparation which would be expected to consist of up to 25 per cent aggregated material. Williams and Kunkel (1963) studied both insolubilized Fn II and heat-aggregated human yG-globulin columns as immunoadsorbants and did not find any significant differences in the ability of these columns to adsorb RF from serum. In all cases, local high densities of antigenic sites are present in the columns to which RF molecules bind, probably with multiple bond formation. There would not appear to be any significant difference in the properties of such columns due to the use of different insolubilization reagents. About 90 per cent of the RF could be accounted for in these experiments. The remainder could have been due to specificity for other yG-globulins not tested for, or to losses during the purification procedures. The agglutination titres of the bi-specific RF, for human or rabbit yG-globulin coated cells, were essentially the same as the titres exhibited by the starting RF preparation, whereas the titres of the monospecific RF preparations were often low. The reason for this difference is not clear at present. It is significant that the RF preparations showing monospecific reactivity with rabbit yG-globulin had a

Anti-y-Globulins in Rheumatoid Arthritis Sera- I I I

735

positive latex titre, although weak. This could, perhaps, explain some of the divergent results in the literature, since the latex test has been used extensively to screen the reactivity of RF and RF fractions. Such latex reactivity was not, however, observed by Williams and Kunkel (1963) in their preparations of RF specific for rabbit yG-globulin. Attempts to isolate bi-specific RF were not successful. The combination of two exposures to buffer at pH 2.8 followed by gel-filtration at pH 4.5 resulted in instability of the RF fraction, even though it had a high agglutination titre just prior to the final (pH 4.5) separation step. After such treatment, the amount of 19S material in these preparations was less than 30 per cent of the total, in each case. This instability was not, however, apparent after only one exposure to buffer at pH 2-8, although it is not known whether any molecular changes occurred within the immunoglobulin molecule. The binding constants observed for the interaction between this RF preparation and human yG-globulin: 2-5 x l0 s L/M, are the same as those observed previously (Normansell, 1970) for the interaction of both purified (elution from a human yG-globulin column at pH 2"8) and unpurified 19S RF with human yG-globulin. The Scatchard plots obtained always demonstrated curvature and the binding constants were calculated at half-saturation of antibody combining sites assuming a value of 5 for n (Chavin and Franklin, 1969; Metzger, 1967). This value was reduced in proportion to the average degree of contamination of the RF preparation by either inactive macroglobulin or RF of a different specificity to that under test. The reactivity with horse yG-globulin was difficult to measure, as can be seen from the scatter in Fig. 2. As n was not measured for this reaction, the binding constant could not be calculated. The monospecific RF preparations gave similar Scatchard plots. Since these monospecific RF preparations had been prepared by elution from appropriate yG-globulin columns, the value of n should be 5 (neglecting the possibility of 10 combining sites per RF pentamer), and since the RF had been exposed only once to buffer at pH 2"8, it should have been active. No evidence of instability was detected in these monospecific RF preparations, but their agglutination titres were often low. The bi-specific RF represents the major RF response in serum MB, in that it represents at least half of the total serum RF, and the monospecific RF fractions tested did not show stronger binding constants. Moreover, the agglutination titres of the whole RF preparation were reflected in the titres of the bi-specific RF rather than the monospecific RF. What is not clear, however, is why the RF in this serum shows a higher titre with rabbit yG-globulin coated cells than with human yG-globulin coated cells; it is not due to a large amount of high-titred RF monospecific for rabbit yG-globulin. The higher reactivity with rabbit yGglobulin was also reflected in the elution patterns from the yG-globulin columns and in the binding constants of the RF fractions. These results support the contention that RF consists of a set of separate antibody specificities, each specific for a part of a complex determinant, and that a unique part of the determinant is masked in each species tested. It is possible that monospecific RF may occur only in certain rheumatoid sera, perhaps only in very high-titred sera, and it may depend on the serum and on the test systems used whether such monospecific species are detected.

736

DAVID E. NORMANSELL

Acknowledgements- I thank Carol Lipton for excellent technical assistance, and Dr. Gerald Goldstein for helpful advice and review of the manuscript. The receipt of a travel grant from the Arthritis and Rheumatism Council, London, is gratefully acknowledged. REFERENCES Avrameas S. and Ternynck T. (1969) Immunochemistry 6, 53. Butler V. P. and VaughanJ. H. (1964) Proc. Soc. exp. Biol. Med. 116, 585. Butler V. P. and VaughanJ. H. (1965) Immunology 8, 144. Chavin S. and Franklin E. C. (1969)J. biol. Chem. 244, 1345. Heimer R., Schwartz E. R. and Freyberg R. H. (1961)J. Lab. din. Med. 57, 16. Heller G., Kolodny M. H., Lepow I. H., Jacobson A. S., Rivera M. E. and Marks G. H. (1955)J. #am. 74, 340. Henney C. S. and Stanworth D. R. (1964) Protides of the Biological Fluids, (Edited by Peeters H. p. 155. Elsevier, Amsterdam. Metzger H. (1967) Proc. hath. Acad. Sci. U.S.A. 57, 1490. Milgrom F., and TSnder O. (1965) Arthritis Rheum. 8, 203. Milgrom F., Witebsky E., Goldstein R. and Loza U. (1962),J. Am. med. Ass. 181,476. Miller F. and Metzger H. (1965)J. biol. Chem. 240, 3325. Normansell D. E. (1970) Immunochemistry 7, 787. Normansell D. E. (1971) Immunochemistry 8, 593. Normansell D. E. and Stanworth D. R. (1968) Immunology 15, 549. Skalba D. and Stanworth D. R. (1969) Immunology 16, 707. T6nder O. and NatvigJ. B. (1966) ,4cta path microbiol. Scand 68, 108. Williams R. C. and Kunkel H. G. (1963) Arthritis Rheum. 6, 665.