lmmunochemistry, 1975, Vol. 12, pp. 6574162. Pergamon Press. Printed in Great Britain
THE USE OF BABOON IgG-SENSITIZED SHEEP ERYTHROCYTES AS AN ALTERNATIVE INDICATOR SYSTEM IN THE STUDY OF THE INTERACTION OF RHEUMATOID FACTOR WITH HUMAN IgG G. A. STEWART,* I. M. HUNNEYBALL and D. R. STANWORTH Department of Experimental Pathology, The Medical School, University of Birmingham, Birmingham BI5 2TJ, England (First received 10 May 1974; in revised.form 11 November 1974) Abstract--A haemagglutination-inhibition assay for studying the interaction of rheumatoid factor with monomeric human IgG has been developed. The indicator system employed consists of sheep red blood ceils actively sensitized with baboon (Papio cynocephalus) IgG. Studies revealed that sub-classes 1, 2 and 4 of human IgG were capable of inhibiting the agglutination of baboon IgG-sensitized sheep red blood cells by rheumatoid arthritic sera, irrespective of Gm phenotype or the anti-Gm specificity of the rheumatoid arthritic sera employed. The pFc', F(ab')z and Fab fragments from pooled human IgG lailed to inhibit the reaction, in contrast to the Fc fragment. The agglutination titres of a panel of rheumatoid arthritic sera from baboon IgG-sensitized sheep red blood cells were compared with their titres in a conventional Rose-Waaler system (i.e. rabbit IgG-sensitized sheep red blood cells). Such a comparison revealed a close similarity in the titres recorded for each serum examined irrespective of the assay system employed. The significance of these findings is discussed. INTRODUCTION
Rheumatoid factor (R.F.) is a collective term for a group of 19S IgM immunoglobulins found in the sera of most patients with rheumatoid arthritis which are distinguished by their reactivity with 7S IgG. Much experimental work has indicated that there are two broad groups of rheumatoid factors, characterized by their specificity for certain regions of the IgG molecute. The first group consists of rheumatoid factors with a specificity for antigenic determinants resident within the Fc region of the whole molecule, and includes antibodies directed against known heavy chain allotypes such as Gm determinants (Grubb, 1956; Henney and Stanworth, 1964); the second group consists of rheumatoid factors with a specificity for determinants present within the F(ab')2 fragment, which are normally 'hidden' but are exposed by peptic digestion of the whole molecule (Osterland et al., 1963; Natvig, 1966). It is highly unlikely that a particular serum contains a rheumatoid factor population possessing only one of these types of specificity. Indeed, it is more probable that rheumatoid factors with both types of specificity are present (Kunkel and Tan, 1964). Such multispecificity obviously interferes with studies aimed at the location of just one group of IgG determinants. To overcome this difficulty a haemagglutinationinhibition assay, employing sheep red blood cells (SRBC) sensitized with a sub-agglutinating dose of baboon anti-sheep red blood cell antibody has been developed. Baboon (Papio cynocephalus) IgG was used as a coating agent for two reasons: (1) it has been shown to possess a maximum cross-reactivity with human IgG heavy chain (Shuster et al., 1969); (2) not all the Gm allotypes expressed on human IgG are expressed on baboon IgG (van Loghem et al., * All correspondence to G. A. Stewart.
1968). Those that are include (s), (b°), (c ~) and (z) allotypes, but the interference of these allotypes in the test system is unlikely since (s), (b °) and (c 5) have only been detected by employing Snagg antisera--from immunized or normal persons--(Natvig and Kunkel, 1968); and Gm (z) has only been detected with antisera raised in rabbits against the Fab fragment (Litwin and Kunkel, 1966). Initially, studies were undertaken to compare directly the use of baboon IgG-coated SRBC with that of rabbit IgG-coated SRBC in the detection of rheumatoid factor in the sera of patients with rheumatoid arthritis. Afterwards the capacity of various myeloma IgG sub-classes and their fragments to inhibit the reaction between R.F. and baboon IgG was investigated. Finally, the capacity of heat aggregated IgG from baboons, humans and rabbits to inhibit the reaction between R.F. and baboon IgG and R.F. and rabbit IgG was studied. MATERIALS AND METHODS
Preparation of baboon anti-sheep red blood cell antiserum The antiserum to sheep red blood cells (SRBC) was raised in the yellow baboon (Papio cynocephalus). SRBC (Burroughs Wellcome Ltd.) were washed twice with 0.970 saline, and one volume of 3070 SRBC in saline was emulsified with an equal volume of complete Freunds adjuvant (4 ml total volume). Equal volumes were then injected intramuscularly into each hind limb. Fourteen days after the initial injection a booster injection of 1.2 ml of 30% SRBC (containing no adjuvant) was administered intradermally in 0.2ml aliquots into different sites. This dosage was repeated after a further 7 days and the animal was exsanguinated 8 days after the final injection. The rabbit antiSRBC antiserum was raised in a similar manner.
Haemagglutination and haemagglutination-inhibition techniques 1. Direct haemagglutination. The rheumatoid arthritic sera used in this study were hospital specimens from con657
658
G.A. STEWART, I. M. HUNNEYBALL and D. R. STANWORTH
firmed or suspected cases of rheumatoid arthritis. The nor-, mal sera were obtained from normal blood donors. T h e s e sera were tested for their capacity to agglutinate SRBC coated with either rabbit or baboon IgG. Prior to testing, the sera were heated at 56°C for 20 min to destroy complement activity and then incubated at 37°C with an equal volume of packed SRBC for 1 hr to absorb non-specific haemagglutinin activity. Suspensions of SRBC were sensitized with a sub-agglutinating dose of the appropriate antiserum and adjusted to a final cell concentration of 1 per cent. Aliquots (0.025 ml) of the cell suspensions were added to equal volumes of serial doubling dilutions of the test sera in the wells of a Takatsy tray. They were covered to avoid evaporation and incubated for 1 hr at 37°C. Agglutination was characterized by an even distribution of cells over the bottom of the well, whereas a negative reaction was indicated by a central compact button. 2. Haemagflutination-inhibition. For inhibition studies, selected rheumatoid sera were diluted to give an agglutination titre of ¼. Aliquots (0.025 ml) of this were added to equal volumes of serial doubling dilutions of the inhibitor and incubated for 1 hr at 37°C. Following this, aliquots (0'025 ml) of baboon IgG-sensitized SRBC were added to the wells and the inhibition titres were read after incubation for 1 hr at 37°C.
Preparation qf IgG and subJ~agments Human IgG was prepared from pooled human serum by precipitation with 33 per cent saturated ammonium sulphate and then purified by batch chromatography (Stanworth, 1960), employing Whatman DEAE-Cellulose equilibrated with 0.01 M phosphate buffer (pH 6"5). Purity was assessed by immunoelectrophoresis and analytical ultracentrifugation. Human myeloma IgG1, IgG2, IgG3, baboon IgG and rabbit IgG were prepared in an identical manner. Human myeloma IgG4 was isolated by ionexchange chromatography on a column of DEAE cellulose (90 x 2.2cm) equilibrated with 0.01 M phosphate buffer (pH 6.3) by elution with a sodium chloride gradient (0.0010.05 M). IgG4 eluted at a position corresponding to 0.02 M sodium chloride. The sub-class specificities of the myeloma proteins were determined by immunodiffusion analysis using specific sheep antisera. The Fc and Fab fragments of the human IgG were prepared by papain digestion of pooled IgG in the presence of cysteine. IgG at a concentration of 10 mg/ml in 0'075 M phosphate buffer (pH 7.0) containing 0-075M NaC1, 0.002M EDTA and 0.01 M cysteine was digested with papain (Sigma Chemical Co., London) at an enzyme-substrate ratio of 1:100 for 1.5 hr at 37aC. Digestion was terminated by addition of N-ethyl maleimide to give a final concentration of 0.01 M. Undigested material was removed by gel filtration on a Sephadex G-150 column (90 x 3.2cm) equilibrated with 0'05 M ammonium carbonate buffer (pH 8.6). The Fc and Fab fragments were separated by ion-exchange chromatography (CM sephadex and DEAE cellulose) by the method of Franklin and Prelli (1960) using stepwise elution. The purified Fc and Fab fractions were concentrated by .ultrafiltration and their purity assessed by immunoelectrophoresis and analytical ultracentrifugation at a concentration of 10 mg/ml. The F(ab')2 and pFc' fragments were prepared from pooled human IgG by peptic digestion. IgG, at a concentration of 20 mg/ml in 0.1 M sodium acetate buffer (pH 4.5), was digested with pepsin (Sigma Chemical Co., London) at an enzyme-substrate ratio of 1:100 for 10hr at 37°C. The resulting fragments were immediately separated by gel filtration on a Sephadex G-150 column (90 x 3.2 cm) equilibrated with 0.05 M ammonium carbonate buffer (pH 8.6). Those fractions containing F(ab')2 and pFc' fragments were concentrated by ultrafiltration. The crude pFc' fraction was purified by recycling gel filtration on a Sephadex G-75 column (90 x 2.2cm) equilibrated in
0"05M ammonium carbonate buffer (pH 8.6). The crude F(ab')z fraction was separated from undigested IgG by recycling gel filtration on a Sephadex G-200 column (120 x 2.2cm) equilibrated in 0'05M ammonium carbonate buffer (pH 8.6). The purified pFc' and F(ab')2 fractions were concentrated by ultrafiltration and their purity assessed by immunoelectrophoresis and analytical ultracentrifugation at a concentration of 10 mg/ml.
Heat aggregation of IgG Native IgG preparations were purified by Sephadex G200 gel filtration. Any polymeric forms of IgG eluting in the void volume were discarded. Aggregation of IgG was achieved by heating a 5 mg/ml solution of the appropriate protein at 63°C for 20 min in a thermostatically controlled water bath. Any insoluble aggregates were removed by centrifugation at 3000 rev/min for 10min in a MSE minor centrifuge. The aggregated protein was purified by centrifugation at 65,000 rev/min (300,000 g) for 30 min in a Beckman Model L2-65 ultracentrifuge. The supernatant was discarded and the pellet washed once with 0.01 M phosphate buffer (pH 7.2) containing 0.15 M NaCI (PBS). The pellet of aggregate was finally resuspended in 0.5 ml PBS and the concentration adjusted to 5 mg/ml. RESULTS
1. Comparison of baboon IgG- and rabbit loG-coated SRBC in the detection of rheumatoid factors A comparison of the agglutination titres obtained using sera from suspected or confirmed cases of rheum a t o i d arthritis (R.A.) a n d SRBC sensitized with either b a b o o n IgG or r a b b i t IgG (as used in the conventional Rose--Waaler test for r h e u m a t o i d arthritis) was undertaken. A typical set of results on a group of R.A. sera together with a series of normal sera (blood donors) is shown in Fig. 1. O f the 405 confirmed or suspected R.A. sera tested, 270 were found to give
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Fig. 1. Comparison of the capacity of various R.A. sera and normal sera to agglutinate SRBC sensitized with baboon IgG or rabbit IgG. Each point refers to the titre of an individual serum but, for the sake of clarity of presentation, the number of sera shown has been reduced proportionally in each group (by a factor of four).
659
Reactivity of Baboon IgG With Rheumatoid Factor Table 1. The capacity of various rheumatoid arthritic sera of known Gm specificity to agglutinate SRBC sensitized with either baboon or rabbit IgG Reciprocal agglutination titre Rheumatoid arthritic serum
Anti-Gm specificity (a, x, f, b, fl, b4, and InV)
SRBC sensitized with baboon IgG
SRBC sensitized with rabbit IgG
Ha Hu FI Po Wi Si Je
a b~ a x a -a, non a
512 512 256 256 64 1024 2048
2048 1024 128 1024 128 256 4096
an agglutination titre in one or both test systems; whereas the remainder were unreactive. It was found that of the 270 reactive sera, 232 (86 per cent) gaze an agglutination titre in both test systems. Of the remaining 38 sera, 23 were positive (i.e. gave a titre greater than ¼) in the rabbit IgG-sensitized system and negative in the baboon IgG-sensitized SRBC system; 15 were positive in the baboon IgG-sensitized SRBC system, and negative in the rabbit IgG-sensitized SRBC system. The majority (89 per cent) of the normal sera tested gave similarly low (i.e. less than titres in both the rabbit and the baboon systems. Frequency distribution charts for the titres obtained in each system (Fig. i) revealed that the range of titres was almost identical, and the frequency distributions were similar. Statistical analysis revealed a correlation coefficient of 0'65 (P < 0.001) for the two sets of titres. The capacity of R.A. sera of known Gm specificity to agglutinate SRBC sensitized with either baboon or rabbit IgG was investigated. It can be seen, from Table 1, that the sera agglutinated both the baboon and rabbit IgG-sensitized SRBC irrespective of antiGm specificity [which was anti-Gin (a), (non a), (x) or (b)] in the rheumatoid sera employed.. Further experiments of this nature were not performed due to lack of rheumatoid sera demonstrating other anti-Gm specificities. The baboon IgG was typed for Gm specificity using anti-human IgG antisera and found to possess neither (a), (x), (f), (b) nor (g) specificities.
2. Inhibition of agglutination of baboon loG-sensitized SRBC and rabbit IgG-sensitized SRBC by various native and heat-aggregated loG preparations The capacity of native and aggregated IgG from human, rabbit and baboon to inhibit the agglutina-
tion by R.A. serum of SRBC sensitized with either baboon IgG or rabbit IgG was investigated. It was found (Table 2) that the agglutination of SRBC sensitized with rabbit IgG could be inhibited by: native rabbit IgG, aggregated rabbit IgG, aggregated human IgG and aggregated baboon IgG whereas native human IgG and baboon IgG were unreactive. The agglutination of SRBC sensitized with baboon IgG could be inhibited by native human IgG, native baboon IgG, the aggregated forms of human IgG, baboon IgG and rabbit IgG, but not by native rabbit IgG.
3. Inhibition of agglutination of baboon lgG-sensitized SRBC by human myeloma IgG sub-classes and IgG cleavage fragments The capacity of the four human IgG sub-classes to inhibit the reaction between baboon IgG-sensitized SRBC and a rheumatoid arthritic serum with no significant anti-Gm specificity was investigated. It can be seen from Table 3 that IgG sub-classes I, 2 and 4 inhibited the reaction whereas native IgG3 did not. However, all four sub-classes were found to inhibit after heat aggregation at 63°C for 20min. Similar results were obtained with a rheumatoid arthritic serum demonstrating anti-Gm(a) specificity. Sub-fragments of normal pooled human IgG produced by pepsin and papain digestion were also investigated for their capacity to inhibit the haemagglutination reaction between SRBC sensitized with baboon IgG and R.A. serum (demonstrating no significant antiGm activity). It was found (Table 4) that the Fc fragment was the only fragment which retained the capacity to inhibit the reaction. The pFc', Fab and F(ab')2 fragments failed to exhibit any inhibitory activity.
Table 2. The capacity of native and aggregated IgG from humans (Hu), baboons (Bb), and rabbits (Rb) to inhibit the reaction of rheumatoid serum with SRBC sensitized with either baboon IgG or rabbit IgG. All inhibitors were tested at an initial concentration of 10 mg/ml prior to dilution
Inhibitor Native pooled Hu IgG Native Bb IgG Native Rb IgG Heat aggregated Hu IgG Heat aggregated Bb IgG Heat aggregated Rb IgG IMM. 12/g
I~
Reciprocal inhibition haemagglutination titre (using SRBC sensitized with Bb IgG)
Reciprocal inhibition haemagglutination titre (using SRBC sensitized with Rb IgG)
128 256 2 256 512 64
0 0 64 128 128 1024
660
G.A. STEWART, I. M. HUNNEYBALL and D. R. STANWORTH Table 3. The capacity of native and aggregated human myeloma IgG sub-classes to inhibit the reaction between rheumatoid arthritic serum (with no significant anti-Gm activity) and SRBC?sensitized with baboon IgG. Initial concentration of inhibitor = 5 mg/ml
Inhibitor
Gm phenotype (a, x, f, b, and g)
Reciprocal inhibition-haemagglutination titre
Native lgGl (Jen) Heat aggregated IgG1 (Jen) Native IgG2 (Pe) Heat aggregated IgG2 (Pe) Native IgG3 (O'R) Heat aggregated IgG3 (O'R) Native IgG4 (Re) Heat aggregated IgG4 (Re)
f f --b, g~ b, g ---
64 256 64 128 0 64 128 256
"The demonstration of both Gm(b) and Gm(g) in this preparation is probably due to contamination of the myeloma protein by normal IgG3. Table 4. The capacity of normal human IgG sub-fragments to inhibit the reaction between rheumatoid serum (with no significant anti-Gin activity) and SRBC sensitized with baboon IgG. Initial concentration of inhibitor = 5 mg/ml Inhibitor
Reciprocal inhibitionhaemagglutination titre
Hu IgG Fc Fab F(ab')2 pFc'
32 32 0 0 0
DISCUSSION The investigations described have established that the baboon IgG-sensitized SRBC reagent is capable of detecting the existence of rheumatoid factors present within rheumatoid arthritic sera. This reactivity is consistent with the previous findings of Schur and Kunkel (1965), who demonstrated that baboon IgG reacted with rheumatoid factor in free solution to produce a 22S complex demonstrable in the analytical ultracentrifuge, and Penttinen and Wager (1973) who demonstrated the reactivity of baboon IgG with rheumatoid factor employing a haemagglutination technique. A comparison of the agglutination titres shown by a large panel of rheumatoid arthritic sera on measurement with this indicator system with those obtained employing the conventional rabbit IgG sensitized SRBC (Rose-Waaler) reagent revealed a similarity in the degree of agglutination recorded in the two test systems for each rheumatoid serum assayed, which was confirmed by statistical analysis. A correlation coefficient of 0.65 (P < 0.001, n = 270) between the two sets of titres was obtained. Analysis of the frequency distributions for each set of data again revealed similarities, both in the range of titres obtained and also in the general shape of the frequency distribution curves. Both reagents detected a small percentage of significant agglutination titres (i.e. titres of ~2 and above) within the panel of normal controls (blood donors) tested, i.e. 5 per cent for the baboon IgG-sensitized SRBC reagent, and 9 per cent for the rabbit IgG-sensitized SRBC reagent.
Certain of the sera of patients in which R.A. was suspected or confirmed were found to give an agglutinatiori titre with only one of the haemagglutination reagents. However, the incidence of this was low, i.e. about 9 per cent of the total number of sera tested, and the significance of these results was not determined. There are several possible explanations for the degree of correlation which we have observed between the results obtained from the baboon and rabbit IgG-sensitized SRBC test systems. One is t h a t there exists within a rheumatoid serum just one population of rheumatoid factors directed against human IgG determinants, which is strongly cross-reactive with rabbit IgG and is reflected as such in the similarity in titres obtained with each cell coat. However, we feel that this is unlikely since it has been demonstrated (Milgrom et al., 1962; Williams and Kunkel, 1963; Normansell, 1972) that there exists three groups of rheumatoid factor populations; one specific for human IgG, one specific for rabbit IgG and one reacting with both. In view of this data, the alternative interpretation of our findings is that each SRBC indicator system (sensitized with either baboon IgG or rabbit IgG) is detecting a specific rheumatoid factor population, i.e. one which is specific for human IgG (and reflected by its cross-reactivity with baboon IgG) and one which is specific for rabbit IgG. The third population of rheumatoid factors would be expected to react with both SRBC indicator systems. A more detailed quantitative interpretation of our data concerning the two sets of agglutination titres obtained using baboon IgG-sensitized SRBC and rabbit IgG-sensitized SRBC with the panel of sera should be approached with caution, since Normansell (1972) has shown that high titres obtained in a particular hoemagglutination system do not always reflect high concentrations of that particular rheumatoid factor. Indeed, it was found that the rheumatoid factor population being studied gave a higher titre with rabbit IgG-coated SRBC than with human IgG-coated SRBC. This phenomenon was not related to anti-IgG concentration, but was thought to be associated with a higher binding constant. In our studies the capacity of various rheumatoid sera of known anti-Gm specificities to agglutinate SRBC sensitized with baboon IgG was determined.
Reactivity of Baboon IgG With Rheumatoid Factor It was found that there was no demonstrable correlation between the anti-Gin reagent employed and the titre obtained. It would appear, therefore, that the baboon lgG-sensitized SRBC indicator system is not detecting anti-allotypic antibodies but rather another population of antibodies, which is directed against another determinant on the IgG molecule and probably corresponds to the population of antibodies termed 'general' rheumatoid factor by Aho et al. (1964). Our panel of rheumatoid anti-Gm reagents was small and therefore some specificities were not included, but such a conclusion is supported by the data of van Loghem et al. (1968) who demonstrated that baboon IgG expresses the (s), (b°), (c 5) and (z) allotypes only. The interference of these allotypes in the test system is unlikely since it has been shown (Natvig and Kunkel, 1968) that the anti-Gm (s), (b °) and (c 5) specificities occur only in 'Snagg' sera and the Gm (z) specificity is only detectable with heterologous antisera (Litwin and Kunkel, 1966). The inability of monomeric human IgG to inhibit the agglutination of rabbit IgG-sensitized SRBC in the conventional Rose-Waaler system has been a serious handicap in studies directed towards the location of rheumatoid factor reactive determinants on human IgG cleavage fragments. In our studies it was found that the monomeric sub-classes 1, 2 and 4 of human IgG were capable of inhibiting the reaction between baboon IgG-sensitized SRBC and rheumatoid arthritic sera, irrespective of the IgG Gm phenotype or the anti-Gm specificity of the rheumatoid sera employed. On the other hand, it was found that IgG3 inhibited only after aggregation. Such a pattern of reactivity between rheumatoid factor and the IgG sub-classes has also been demonstrated in other types of reaction systems (Schur and Kunkel, 1965; Normansell and Stanworth, 1968). The pattern of reactivity of the four human IgG sub-classes (in the native state) with 'general' rheumatoid factor, as detected by the baboon IgG indicator system, parallels the reported reactivity of these proteins with antibodies directed against the iso-allotypic 'Ga' antigen (Allen and Kunkel, 1966; Natvig and Turner, 1970). Thus, both the 'Ga' and 'general' rheumatoid factor-reactive determinants appear to be present on molecules of the same sub-class. The high frequency of agglutination of baboon IgG-sensitized SRBC by the panel of rheumatoid sera also parallels the high frequency of occurrence of anti-'Ga' antibodies in the 19S fraction of rheumatoid sera detected by Gaarder and Natvig (1970). The capacity of purified heat-aggregated preparations of all four sub-classes of human IgG to inhibit the agglutination of baboon IgG-sensitized SRBC by rheumatoid arthritic sera was also investigated. It was found that all four sub-classes inhibited the reaction. However, the aggregated IgG3 preparation gave slightly lower inhibitory titres. In similar experiments the capacity of both native and aggregated forms of human, baboon, and rabbit IgG to inhibit the reaction of rheumatoid arthritic sera and SRBC sensitized with either baboon or rabbit IgG was studied. It was found that in the baboon IgG-sensitized SRBC system only native human and baboon IgG inhibited whereas rabbit IgG did not. However, upon aggregation all three species of IgG inhibited. In the rabbit IgG-sensitized SRBC system,
661
native human and baboon IgG were found not to inhibit, whereas native rabbit IgG did. Upon aggregation all three species of IgG inhibited the reaction. Gaarder (1973) has reported similar findings for the inhibitory capacity of human IgG1 myeloma proteins employing rabbit IgG-sensitized human erythrocytes. Such results suggest that the antigens being studied in heterologous systems are present only on aggregated forms of the IgG. The capacity of various proteolytic cleavage fragments of pooled human IgG and human IgG1 myeloma proteins to inhibit the baboon IgG-sensitized SRBC system has been investigated. It was found that the peptic pFc' and F(ab')2 fragments together with the papain Fab fragment were non-inhibitory, whereas the papain Fc fragment retained the activity of the whole molecule. Similar results were obtained for the capacity of proteolytic cleavage fragments of rabbit IgG to inhibit the agglutination of rabbit IgGsensitized SRBC by rheumatoid sera (Stewart et al., 1973). This pattern of reactivity suggests that the 'general' rheumatoid factor-reactive determinants are present within the N-terminal half of the Fc region of human IgG. These findings are consistent with the proposed location of the 'Ga' antigenic determinant in the N-terminal half of the Fc region (Gaarder and Natvig, 1970). It would appear, therefore, that the 'Ga' and 'general' rheumatoid factor-reactive determinants are similarly located within IgG molecules of sub-classes 1, 2 and 4 and may correspond to the same determinant. Our studies have established that the anti-allotypic specificity of rheumatoid factor is no longer a problem in the study of those Fc determinants to which a proportion of rheumatoid factors are directed. Consequently the haemagglutination-inhibition technique described is now being used to investigate such determinants; the ability to inhibit the agglutination of baboon IgG-sensitized SRBC by non-aggregated human IgG and Fc subfragments proving an obvious advantage. Acknowledgements--The Gm typing of the myeloma pro-
teins and the rheumatoid arthritic sera was kindly performed by Dr. K. Goldsmith (M.R.C. Blood Group Reference Laboratory) and Miss D. F. Barr (Regional Blood Transfusion Service). The myeloma proteins were isolated and sub-typed by Miss H. Evans. Generous financial support from the Medical Research Cohncil is gratefully acknowledged.
REFERENCES
Aho K., Harboe M. and Leikola J. (1964) Immunology 7, 403. Allen J. C. and Kunkel H. G. (1966) Arthritis Rheum. 9, 758. Franklin E. C. and Prelli F. (1960) J. clin. Invest. 39, 1933. Gaarder P. I. (1973) Scand. d. lmmun. 2, 444. Gaarder P. I. and Natvig J. B. (1970) d. Immun. 105, 928. Grubb R. (1956) Acta path. microbiol, scand. 39, 195. Henney C. S. and Stanworth D. R. (1964) Nature, Lond. 201, 511.
Kunkel H. G. and Tan M. (1964) Adv. Immunol. 4, 351. Litwin S. D. and Kunkel H. G. (1966) Nature, Lond. 210, 866. van Loghem E., Shuster J. and Fudenberg H. H. (1968) Vox Sang. 14, 81.
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Milgrom F., Witebsky E., Goldstein R. and Loza V. (1962) J. Am. reed. Ass. 181, 476. Natvig J. B. (1966) Acta path. microbiol, scand. 66, 369. Natvig J. B. and Kunkel H. G. (1968) Ser, Haematol. I, 66. Natvig J. B. and Turner M. W. (1970) Nature, Lond. 225, 855. Normansell D. E. (1972) Immunochemistry 9, 725. Normansell D. E. and Stanworth D. R. (1968) Immunology 15, 549. Osterland C. K., Harboe M. and Kunkel H. G. (1963) Fox Sang. 8, 133.
Penttinen K. and Wager O. (1973) Scand. J. Immun. 2, 443. Shur P. H. and Kunkel H. G. (1965) Arthritis Rheum. 8, 468. Shuster J., Warner N. L. and Fudenberg H. H. (1969) Ann. N,Y. Acad. Sci. 162, 195. Stanw0rth D. R. (1960) Nature, Lond. 188, 156. Stewart G. A., Smith A. K. and Stanworth D. R. (1973) lmmunochemistry 10, 755. Williams R. C., Jr and Kunkel H. G. (1963) Arthritis Rheum. 6, 665.
THE USE OF BABOON IgG-SENSITIZED SHEEP ERYTHROCYTES AS AN ALTERNATIVE INDICATOR SYSTEM IN THE STUDY OF THE INTERACTION OF RHEUMATOID FACTOR WITH HUMAN IgG G. A. STEWART,* I. M. HUNNEYBALL and D. R. STANWORTH Department of Experimental Pathology, The Medical School, University of Birmingham, Birmingham BI5 2TJ, England (First received 10 May 1974; in revised.form 11 November 1974) Abstract--A haemagglutination-inhibition assay for studying the interaction of rheumatoid factor with monomeric human IgG has been developed. The indicator system employed consists of sheep red blood ceils actively sensitized with baboon (Papio cynocephalus) IgG. Studies revealed that sub-classes 1, 2 and 4 of human IgG were capable of inhibiting the agglutination of baboon IgG-sensitized sheep red blood cells by rheumatoid arthritic sera, irrespective of Gm phenotype or the anti-Gm specificity of the rheumatoid arthritic sera employed. The pFc', F(ab')z and Fab fragments from pooled human IgG lailed to inhibit the reaction, in contrast to the Fc fragment. The agglutination titres of a panel of rheumatoid arthritic sera from baboon IgG-sensitized sheep red blood cells were compared with their titres in a conventional Rose-Waaler system (i.e. rabbit IgG-sensitized sheep red blood cells). Such a comparison revealed a close similarity in the titres recorded for each serum examined irrespective of the assay system employed. The significance of these findings is discussed. INTRODUCTION
Rheumatoid factor (R.F.) is a collective term for a group of 19S IgM immunoglobulins found in the sera of most patients with rheumatoid arthritis which are distinguished by their reactivity with 7S IgG. Much experimental work has indicated that there are two broad groups of rheumatoid factors, characterized by their specificity for certain regions of the IgG molecute. The first group consists of rheumatoid factors with a specificity for antigenic determinants resident within the Fc region of the whole molecule, and includes antibodies directed against known heavy chain allotypes such as Gm determinants (Grubb, 1956; Henney and Stanworth, 1964); the second group consists of rheumatoid factors with a specificity for determinants present within the F(ab')2 fragment, which are normally 'hidden' but are exposed by peptic digestion of the whole molecule (Osterland et al., 1963; Natvig, 1966). It is highly unlikely that a particular serum contains a rheumatoid factor population possessing only one of these types of specificity. Indeed, it is more probable that rheumatoid factors with both types of specificity are present (Kunkel and Tan, 1964). Such multispecificity obviously interferes with studies aimed at the location of just one group of IgG determinants. To overcome this difficulty a haemagglutinationinhibition assay, employing sheep red blood cells (SRBC) sensitized with a sub-agglutinating dose of baboon anti-sheep red blood cell antibody has been developed. Baboon (Papio cynocephalus) IgG was used as a coating agent for two reasons: (1) it has been shown to possess a maximum cross-reactivity with human IgG heavy chain (Shuster et al., 1969); (2) not all the Gm allotypes expressed on human IgG are expressed on baboon IgG (van Loghem et al., * All correspondence to G. A. Stewart.
1968). Those that are include (s), (b°), (c ~) and (z) allotypes, but the interference of these allotypes in the test system is unlikely since (s), (b °) and (c 5) have only been detected by employing Snagg antisera--from immunized or normal persons--(Natvig and Kunkel, 1968); and Gm (z) has only been detected with antisera raised in rabbits against the Fab fragment (Litwin and Kunkel, 1966). Initially, studies were undertaken to compare directly the use of baboon IgG-coated SRBC with that of rabbit IgG-coated SRBC in the detection of rheumatoid factor in the sera of patients with rheumatoid arthritis. Afterwards the capacity of various myeloma IgG sub-classes and their fragments to inhibit the reaction between R.F. and baboon IgG was investigated. Finally, the capacity of heat aggregated IgG from baboons, humans and rabbits to inhibit the reaction between R.F. and baboon IgG and R.F. and rabbit IgG was studied. MATERIALS AND METHODS
Preparation of baboon anti-sheep red blood cell antiserum The antiserum to sheep red blood cells (SRBC) was raised in the yellow baboon (Papio cynocephalus). SRBC (Burroughs Wellcome Ltd.) were washed twice with 0.970 saline, and one volume of 3070 SRBC in saline was emulsified with an equal volume of complete Freunds adjuvant (4 ml total volume). Equal volumes were then injected intramuscularly into each hind limb. Fourteen days after the initial injection a booster injection of 1.2 ml of 30% SRBC (containing no adjuvant) was administered intradermally in 0.2ml aliquots into different sites. This dosage was repeated after a further 7 days and the animal was exsanguinated 8 days after the final injection. The rabbit antiSRBC antiserum was raised in a similar manner.
Haemagglutination and haemagglutination-inhibition techniques 1. Direct haemagglutination. The rheumatoid arthritic sera used in this study were hospital specimens from con657
658
G.A. STEWART, I. M. HUNNEYBALL and D. R. STANWORTH
firmed or suspected cases of rheumatoid arthritis. The nor-, mal sera were obtained from normal blood donors. T h e s e sera were tested for their capacity to agglutinate SRBC coated with either rabbit or baboon IgG. Prior to testing, the sera were heated at 56°C for 20 min to destroy complement activity and then incubated at 37°C with an equal volume of packed SRBC for 1 hr to absorb non-specific haemagglutinin activity. Suspensions of SRBC were sensitized with a sub-agglutinating dose of the appropriate antiserum and adjusted to a final cell concentration of 1 per cent. Aliquots (0.025 ml) of the cell suspensions were added to equal volumes of serial doubling dilutions of the test sera in the wells of a Takatsy tray. They were covered to avoid evaporation and incubated for 1 hr at 37°C. Agglutination was characterized by an even distribution of cells over the bottom of the well, whereas a negative reaction was indicated by a central compact button. 2. Haemagflutination-inhibition. For inhibition studies, selected rheumatoid sera were diluted to give an agglutination titre of ¼. Aliquots (0.025 ml) of this were added to equal volumes of serial doubling dilutions of the inhibitor and incubated for 1 hr at 37°C. Following this, aliquots (0'025 ml) of baboon IgG-sensitized SRBC were added to the wells and the inhibition titres were read after incubation for 1 hr at 37°C.
Preparation qf IgG and subJ~agments Human IgG was prepared from pooled human serum by precipitation with 33 per cent saturated ammonium sulphate and then purified by batch chromatography (Stanworth, 1960), employing Whatman DEAE-Cellulose equilibrated with 0.01 M phosphate buffer (pH 6"5). Purity was assessed by immunoelectrophoresis and analytical ultracentrifugation. Human myeloma IgG1, IgG2, IgG3, baboon IgG and rabbit IgG were prepared in an identical manner. Human myeloma IgG4 was isolated by ionexchange chromatography on a column of DEAE cellulose (90 x 2.2cm) equilibrated with 0.01 M phosphate buffer (pH 6.3) by elution with a sodium chloride gradient (0.0010.05 M). IgG4 eluted at a position corresponding to 0.02 M sodium chloride. The sub-class specificities of the myeloma proteins were determined by immunodiffusion analysis using specific sheep antisera. The Fc and Fab fragments of the human IgG were prepared by papain digestion of pooled IgG in the presence of cysteine. IgG at a concentration of 10 mg/ml in 0'075 M phosphate buffer (pH 7.0) containing 0-075M NaC1, 0.002M EDTA and 0.01 M cysteine was digested with papain (Sigma Chemical Co., London) at an enzyme-substrate ratio of 1:100 for 1.5 hr at 37aC. Digestion was terminated by addition of N-ethyl maleimide to give a final concentration of 0.01 M. Undigested material was removed by gel filtration on a Sephadex G-150 column (90 x 3.2cm) equilibrated with 0'05 M ammonium carbonate buffer (pH 8.6). The Fc and Fab fragments were separated by ion-exchange chromatography (CM sephadex and DEAE cellulose) by the method of Franklin and Prelli (1960) using stepwise elution. The purified Fc and Fab fractions were concentrated by .ultrafiltration and their purity assessed by immunoelectrophoresis and analytical ultracentrifugation at a concentration of 10 mg/ml. The F(ab')2 and pFc' fragments were prepared from pooled human IgG by peptic digestion. IgG, at a concentration of 20 mg/ml in 0.1 M sodium acetate buffer (pH 4.5), was digested with pepsin (Sigma Chemical Co., London) at an enzyme-substrate ratio of 1:100 for 10hr at 37°C. The resulting fragments were immediately separated by gel filtration on a Sephadex G-150 column (90 x 3.2 cm) equilibrated with 0.05 M ammonium carbonate buffer (pH 8.6). Those fractions containing F(ab')2 and pFc' fragments were concentrated by ultrafiltration. The crude pFc' fraction was purified by recycling gel filtration on a Sephadex G-75 column (90 x 2.2cm) equilibrated in
0"05M ammonium carbonate buffer (pH 8.6). The crude F(ab')z fraction was separated from undigested IgG by recycling gel filtration on a Sephadex G-200 column (120 x 2.2cm) equilibrated in 0'05M ammonium carbonate buffer (pH 8.6). The purified pFc' and F(ab')2 fractions were concentrated by ultrafiltration and their purity assessed by immunoelectrophoresis and analytical ultracentrifugation at a concentration of 10 mg/ml.
Heat aggregation of IgG Native IgG preparations were purified by Sephadex G200 gel filtration. Any polymeric forms of IgG eluting in the void volume were discarded. Aggregation of IgG was achieved by heating a 5 mg/ml solution of the appropriate protein at 63°C for 20 min in a thermostatically controlled water bath. Any insoluble aggregates were removed by centrifugation at 3000 rev/min for 10min in a MSE minor centrifuge. The aggregated protein was purified by centrifugation at 65,000 rev/min (300,000 g) for 30 min in a Beckman Model L2-65 ultracentrifuge. The supernatant was discarded and the pellet washed once with 0.01 M phosphate buffer (pH 7.2) containing 0.15 M NaCI (PBS). The pellet of aggregate was finally resuspended in 0.5 ml PBS and the concentration adjusted to 5 mg/ml. RESULTS
1. Comparison of baboon IgG- and rabbit loG-coated SRBC in the detection of rheumatoid factors A comparison of the agglutination titres obtained using sera from suspected or confirmed cases of rheum a t o i d arthritis (R.A.) a n d SRBC sensitized with either b a b o o n IgG or r a b b i t IgG (as used in the conventional Rose--Waaler test for r h e u m a t o i d arthritis) was undertaken. A typical set of results on a group of R.A. sera together with a series of normal sera (blood donors) is shown in Fig. 1. O f the 405 confirmed or suspected R.A. sera tested, 270 were found to give
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Fig. 1. Comparison of the capacity of various R.A. sera and normal sera to agglutinate SRBC sensitized with baboon IgG or rabbit IgG. Each point refers to the titre of an individual serum but, for the sake of clarity of presentation, the number of sera shown has been reduced proportionally in each group (by a factor of four).
659
Reactivity of Baboon IgG With Rheumatoid Factor Table 1. The capacity of various rheumatoid arthritic sera of known Gm specificity to agglutinate SRBC sensitized with either baboon or rabbit IgG Reciprocal agglutination titre Rheumatoid arthritic serum
Anti-Gm specificity (a, x, f, b, fl, b4, and InV)
SRBC sensitized with baboon IgG
SRBC sensitized with rabbit IgG
Ha Hu FI Po Wi Si Je
a b~ a x a -a, non a
512 512 256 256 64 1024 2048
2048 1024 128 1024 128 256 4096
an agglutination titre in one or both test systems; whereas the remainder were unreactive. It was found that of the 270 reactive sera, 232 (86 per cent) gaze an agglutination titre in both test systems. Of the remaining 38 sera, 23 were positive (i.e. gave a titre greater than ¼) in the rabbit IgG-sensitized system and negative in the baboon IgG-sensitized SRBC system; 15 were positive in the baboon IgG-sensitized SRBC system, and negative in the rabbit IgG-sensitized SRBC system. The majority (89 per cent) of the normal sera tested gave similarly low (i.e. less than titres in both the rabbit and the baboon systems. Frequency distribution charts for the titres obtained in each system (Fig. i) revealed that the range of titres was almost identical, and the frequency distributions were similar. Statistical analysis revealed a correlation coefficient of 0'65 (P < 0.001) for the two sets of titres. The capacity of R.A. sera of known Gm specificity to agglutinate SRBC sensitized with either baboon or rabbit IgG was investigated. It can be seen, from Table 1, that the sera agglutinated both the baboon and rabbit IgG-sensitized SRBC irrespective of antiGm specificity [which was anti-Gin (a), (non a), (x) or (b)] in the rheumatoid sera employed.. Further experiments of this nature were not performed due to lack of rheumatoid sera demonstrating other anti-Gm specificities. The baboon IgG was typed for Gm specificity using anti-human IgG antisera and found to possess neither (a), (x), (f), (b) nor (g) specificities.
2. Inhibition of agglutination of baboon loG-sensitized SRBC and rabbit IgG-sensitized SRBC by various native and heat-aggregated loG preparations The capacity of native and aggregated IgG from human, rabbit and baboon to inhibit the agglutina-
tion by R.A. serum of SRBC sensitized with either baboon IgG or rabbit IgG was investigated. It was found (Table 2) that the agglutination of SRBC sensitized with rabbit IgG could be inhibited by: native rabbit IgG, aggregated rabbit IgG, aggregated human IgG and aggregated baboon IgG whereas native human IgG and baboon IgG were unreactive. The agglutination of SRBC sensitized with baboon IgG could be inhibited by native human IgG, native baboon IgG, the aggregated forms of human IgG, baboon IgG and rabbit IgG, but not by native rabbit IgG.
3. Inhibition of agglutination of baboon lgG-sensitized SRBC by human myeloma IgG sub-classes and IgG cleavage fragments The capacity of the four human IgG sub-classes to inhibit the reaction between baboon IgG-sensitized SRBC and a rheumatoid arthritic serum with no significant anti-Gm specificity was investigated. It can be seen from Table 3 that IgG sub-classes I, 2 and 4 inhibited the reaction whereas native IgG3 did not. However, all four sub-classes were found to inhibit after heat aggregation at 63°C for 20min. Similar results were obtained with a rheumatoid arthritic serum demonstrating anti-Gm(a) specificity. Sub-fragments of normal pooled human IgG produced by pepsin and papain digestion were also investigated for their capacity to inhibit the haemagglutination reaction between SRBC sensitized with baboon IgG and R.A. serum (demonstrating no significant antiGm activity). It was found (Table 4) that the Fc fragment was the only fragment which retained the capacity to inhibit the reaction. The pFc', Fab and F(ab')2 fragments failed to exhibit any inhibitory activity.
Table 2. The capacity of native and aggregated IgG from humans (Hu), baboons (Bb), and rabbits (Rb) to inhibit the reaction of rheumatoid serum with SRBC sensitized with either baboon IgG or rabbit IgG. All inhibitors were tested at an initial concentration of 10 mg/ml prior to dilution
Inhibitor Native pooled Hu IgG Native Bb IgG Native Rb IgG Heat aggregated Hu IgG Heat aggregated Bb IgG Heat aggregated Rb IgG IMM. 12/g
I~
Reciprocal inhibition haemagglutination titre (using SRBC sensitized with Bb IgG)
Reciprocal inhibition haemagglutination titre (using SRBC sensitized with Rb IgG)
128 256 2 256 512 64
0 0 64 128 128 1024
660
G.A. STEWART, I. M. HUNNEYBALL and D. R. STANWORTH Table 3. The capacity of native and aggregated human myeloma IgG sub-classes to inhibit the reaction between rheumatoid arthritic serum (with no significant anti-Gm activity) and SRBC?sensitized with baboon IgG. Initial concentration of inhibitor = 5 mg/ml
Inhibitor
Gm phenotype (a, x, f, b, and g)
Reciprocal inhibition-haemagglutination titre
Native lgGl (Jen) Heat aggregated IgG1 (Jen) Native IgG2 (Pe) Heat aggregated IgG2 (Pe) Native IgG3 (O'R) Heat aggregated IgG3 (O'R) Native IgG4 (Re) Heat aggregated IgG4 (Re)
f f --b, g~ b, g ---
64 256 64 128 0 64 128 256
"The demonstration of both Gm(b) and Gm(g) in this preparation is probably due to contamination of the myeloma protein by normal IgG3. Table 4. The capacity of normal human IgG sub-fragments to inhibit the reaction between rheumatoid serum (with no significant anti-Gin activity) and SRBC sensitized with baboon IgG. Initial concentration of inhibitor = 5 mg/ml Inhibitor
Reciprocal inhibitionhaemagglutination titre
Hu IgG Fc Fab F(ab')2 pFc'
32 32 0 0 0
DISCUSSION The investigations described have established that the baboon IgG-sensitized SRBC reagent is capable of detecting the existence of rheumatoid factors present within rheumatoid arthritic sera. This reactivity is consistent with the previous findings of Schur and Kunkel (1965), who demonstrated that baboon IgG reacted with rheumatoid factor in free solution to produce a 22S complex demonstrable in the analytical ultracentrifuge, and Penttinen and Wager (1973) who demonstrated the reactivity of baboon IgG with rheumatoid factor employing a haemagglutination technique. A comparison of the agglutination titres shown by a large panel of rheumatoid arthritic sera on measurement with this indicator system with those obtained employing the conventional rabbit IgG sensitized SRBC (Rose-Waaler) reagent revealed a similarity in the degree of agglutination recorded in the two test systems for each rheumatoid serum assayed, which was confirmed by statistical analysis. A correlation coefficient of 0.65 (P < 0.001, n = 270) between the two sets of titres was obtained. Analysis of the frequency distributions for each set of data again revealed similarities, both in the range of titres obtained and also in the general shape of the frequency distribution curves. Both reagents detected a small percentage of significant agglutination titres (i.e. titres of ~2 and above) within the panel of normal controls (blood donors) tested, i.e. 5 per cent for the baboon IgG-sensitized SRBC reagent, and 9 per cent for the rabbit IgG-sensitized SRBC reagent.
Certain of the sera of patients in which R.A. was suspected or confirmed were found to give an agglutinatiori titre with only one of the haemagglutination reagents. However, the incidence of this was low, i.e. about 9 per cent of the total number of sera tested, and the significance of these results was not determined. There are several possible explanations for the degree of correlation which we have observed between the results obtained from the baboon and rabbit IgG-sensitized SRBC test systems. One is t h a t there exists within a rheumatoid serum just one population of rheumatoid factors directed against human IgG determinants, which is strongly cross-reactive with rabbit IgG and is reflected as such in the similarity in titres obtained with each cell coat. However, we feel that this is unlikely since it has been demonstrated (Milgrom et al., 1962; Williams and Kunkel, 1963; Normansell, 1972) that there exists three groups of rheumatoid factor populations; one specific for human IgG, one specific for rabbit IgG and one reacting with both. In view of this data, the alternative interpretation of our findings is that each SRBC indicator system (sensitized with either baboon IgG or rabbit IgG) is detecting a specific rheumatoid factor population, i.e. one which is specific for human IgG (and reflected by its cross-reactivity with baboon IgG) and one which is specific for rabbit IgG. The third population of rheumatoid factors would be expected to react with both SRBC indicator systems. A more detailed quantitative interpretation of our data concerning the two sets of agglutination titres obtained using baboon IgG-sensitized SRBC and rabbit IgG-sensitized SRBC with the panel of sera should be approached with caution, since Normansell (1972) has shown that high titres obtained in a particular hoemagglutination system do not always reflect high concentrations of that particular rheumatoid factor. Indeed, it was found that the rheumatoid factor population being studied gave a higher titre with rabbit IgG-coated SRBC than with human IgG-coated SRBC. This phenomenon was not related to anti-IgG concentration, but was thought to be associated with a higher binding constant. In our studies the capacity of various rheumatoid sera of known anti-Gm specificities to agglutinate SRBC sensitized with baboon IgG was determined.
Reactivity of Baboon IgG With Rheumatoid Factor It was found that there was no demonstrable correlation between the anti-Gin reagent employed and the titre obtained. It would appear, therefore, that the baboon lgG-sensitized SRBC indicator system is not detecting anti-allotypic antibodies but rather another population of antibodies, which is directed against another determinant on the IgG molecule and probably corresponds to the population of antibodies termed 'general' rheumatoid factor by Aho et al. (1964). Our panel of rheumatoid anti-Gm reagents was small and therefore some specificities were not included, but such a conclusion is supported by the data of van Loghem et al. (1968) who demonstrated that baboon IgG expresses the (s), (b°), (c 5) and (z) allotypes only. The interference of these allotypes in the test system is unlikely since it has been shown (Natvig and Kunkel, 1968) that the anti-Gm (s), (b °) and (c 5) specificities occur only in 'Snagg' sera and the Gm (z) specificity is only detectable with heterologous antisera (Litwin and Kunkel, 1966). The inability of monomeric human IgG to inhibit the agglutination of rabbit IgG-sensitized SRBC in the conventional Rose-Waaler system has been a serious handicap in studies directed towards the location of rheumatoid factor reactive determinants on human IgG cleavage fragments. In our studies it was found that the monomeric sub-classes 1, 2 and 4 of human IgG were capable of inhibiting the reaction between baboon IgG-sensitized SRBC and rheumatoid arthritic sera, irrespective of the IgG Gm phenotype or the anti-Gm specificity of the rheumatoid sera employed. On the other hand, it was found that IgG3 inhibited only after aggregation. Such a pattern of reactivity between rheumatoid factor and the IgG sub-classes has also been demonstrated in other types of reaction systems (Schur and Kunkel, 1965; Normansell and Stanworth, 1968). The pattern of reactivity of the four human IgG sub-classes (in the native state) with 'general' rheumatoid factor, as detected by the baboon IgG indicator system, parallels the reported reactivity of these proteins with antibodies directed against the iso-allotypic 'Ga' antigen (Allen and Kunkel, 1966; Natvig and Turner, 1970). Thus, both the 'Ga' and 'general' rheumatoid factor-reactive determinants appear to be present on molecules of the same sub-class. The high frequency of agglutination of baboon IgG-sensitized SRBC by the panel of rheumatoid sera also parallels the high frequency of occurrence of anti-'Ga' antibodies in the 19S fraction of rheumatoid sera detected by Gaarder and Natvig (1970). The capacity of purified heat-aggregated preparations of all four sub-classes of human IgG to inhibit the agglutination of baboon IgG-sensitized SRBC by rheumatoid arthritic sera was also investigated. It was found that all four sub-classes inhibited the reaction. However, the aggregated IgG3 preparation gave slightly lower inhibitory titres. In similar experiments the capacity of both native and aggregated forms of human, baboon, and rabbit IgG to inhibit the reaction of rheumatoid arthritic sera and SRBC sensitized with either baboon or rabbit IgG was studied. It was found that in the baboon IgG-sensitized SRBC system only native human and baboon IgG inhibited whereas rabbit IgG did not. However, upon aggregation all three species of IgG inhibited. In the rabbit IgG-sensitized SRBC system,
661
native human and baboon IgG were found not to inhibit, whereas native rabbit IgG did. Upon aggregation all three species of IgG inhibited the reaction. Gaarder (1973) has reported similar findings for the inhibitory capacity of human IgG1 myeloma proteins employing rabbit IgG-sensitized human erythrocytes. Such results suggest that the antigens being studied in heterologous systems are present only on aggregated forms of the IgG. The capacity of various proteolytic cleavage fragments of pooled human IgG and human IgG1 myeloma proteins to inhibit the baboon IgG-sensitized SRBC system has been investigated. It was found that the peptic pFc' and F(ab')2 fragments together with the papain Fab fragment were non-inhibitory, whereas the papain Fc fragment retained the activity of the whole molecule. Similar results were obtained for the capacity of proteolytic cleavage fragments of rabbit IgG to inhibit the agglutination of rabbit IgGsensitized SRBC by rheumatoid sera (Stewart et al., 1973). This pattern of reactivity suggests that the 'general' rheumatoid factor-reactive determinants are present within the N-terminal half of the Fc region of human IgG. These findings are consistent with the proposed location of the 'Ga' antigenic determinant in the N-terminal half of the Fc region (Gaarder and Natvig, 1970). It would appear, therefore, that the 'Ga' and 'general' rheumatoid factor-reactive determinants are similarly located within IgG molecules of sub-classes 1, 2 and 4 and may correspond to the same determinant. Our studies have established that the anti-allotypic specificity of rheumatoid factor is no longer a problem in the study of those Fc determinants to which a proportion of rheumatoid factors are directed. Consequently the haemagglutination-inhibition technique described is now being used to investigate such determinants; the ability to inhibit the agglutination of baboon IgG-sensitized SRBC by non-aggregated human IgG and Fc subfragments proving an obvious advantage. Acknowledgements--The Gm typing of the myeloma pro-
teins and the rheumatoid arthritic sera was kindly performed by Dr. K. Goldsmith (M.R.C. Blood Group Reference Laboratory) and Miss D. F. Barr (Regional Blood Transfusion Service). The myeloma proteins were isolated and sub-typed by Miss H. Evans. Generous financial support from the Medical Research Cohncil is gratefully acknowledged.
REFERENCES
Aho K., Harboe M. and Leikola J. (1964) Immunology 7, 403. Allen J. C. and Kunkel H. G. (1966) Arthritis Rheum. 9, 758. Franklin E. C. and Prelli F. (1960) J. clin. Invest. 39, 1933. Gaarder P. I. (1973) Scand. d. lmmun. 2, 444. Gaarder P. I. and Natvig J. B. (1970) d. Immun. 105, 928. Grubb R. (1956) Acta path. microbiol, scand. 39, 195. Henney C. S. and Stanworth D. R. (1964) Nature, Lond. 201, 511.
Kunkel H. G. and Tan M. (1964) Adv. Immunol. 4, 351. Litwin S. D. and Kunkel H. G. (1966) Nature, Lond. 210, 866. van Loghem E., Shuster J. and Fudenberg H. H. (1968) Vox Sang. 14, 81.
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Milgrom F., Witebsky E., Goldstein R. and Loza V. (1962) J. Am. reed. Ass. 181, 476. Natvig J. B. (1966) Acta path. microbiol, scand. 66, 369. Natvig J. B. and Kunkel H. G. (1968) Ser, Haematol. I, 66. Natvig J. B. and Turner M. W. (1970) Nature, Lond. 225, 855. Normansell D. E. (1972) Immunochemistry 9, 725. Normansell D. E. and Stanworth D. R. (1968) Immunology 15, 549. Osterland C. K., Harboe M. and Kunkel H. G. (1963) Fox Sang. 8, 133.
Penttinen K. and Wager O. (1973) Scand. J. Immun. 2, 443. Shur P. H. and Kunkel H. G. (1965) Arthritis Rheum. 8, 468. Shuster J., Warner N. L. and Fudenberg H. H. (1969) Ann. N,Y. Acad. Sci. 162, 195. Stanw0rth D. R. (1960) Nature, Lond. 188, 156. Stewart G. A., Smith A. K. and Stanworth D. R. (1973) lmmunochemistry 10, 755. Williams R. C., Jr and Kunkel H. G. (1963) Arthritis Rheum. 6, 665.
