Journal of Immunological Methods, 29 (1979) 207--220 © Elsevier/North-Holland Biomedical Press
207
AN ENZYME-IMMUNOASSAY METHOD FOR DETECTING C I R C U L A T I N G I M M U N E C O M P L E X E S BY I N H I B I T I O N O F POLYCLONAL RHEUMATOID FACTOR
L. RODA, R. MAIOLINI, B. FERRUA and R. MASSEYEFF INSERM FRA 12, Laboratoire d'Immunologie, CHU de Nice, 06031 Nice Cedex, France
(Received 30 November 1978, accepted 29 March 1979)
An enzyme-immunoassay for circulating immune complexes (IC-EIA is described which depends on inhibition of binding of rheumatoid factor (RF) to aggregated IgG. The assay involves 3 steps: (1) sera are pretreated with an aggregated IgG sorbent in order to remove any endogenous RF; (2) absorbed sera are then incubated with a polyelonal RF of known titre and an aggregated IgG sorbent; (3) after washing, the amount of RF on the solid phase is revealed by an IgG-glucose oxidase conjugate. Results are expressed as per cent inhibition of RF fixation. The assay range extends from 1 to 100 mg/l. Intra- and inter-assay coefficients were 6 and 9% respectively. Specificity has been assessed by various tests: dose-response curve with aggregated monomerie or denatured IgG and human albumin localization of the inhibitory fraction of sera after Sephadex G-200 chromatography, absence of interference of IgG, and use of artificial IC. Poor correlation was obtained between IC-EIA and other techniques. The method demonstrated the occurrence of IC in a large proportion of patients with viral hepatitis, schistosomiasis and rheumatoid arthritis and their absence in normal subjects. INTRODUCTION A v a r i e t y o f t e c h n i q u e s f o r t h e assay of c i r c u l a t i n g i m m u n e c o m p l e x e s (IC) have b e e n d e s c r i b e d . T h e y t a k e a d v a n t a g e o f t h e s p e c i f i c p h y s i c o - c h e m ical p r o p e r t i e s o f IC, a n d / o r o f t h e i r a b i l i t y t o b i n d e i t h e r t o t h e s u r f a c e o f s o m e cells s u c h as p l a t e l e t s ( P e n t t i n e n e t al., 1 9 7 1 ) , m a c r o p h a g e s ( O n y e w o t u e t al., 1 9 7 4 ) , R a j i cells ( T h e o p h i l o p o u l o s e t al., 1 9 7 6 ) , or t o m o l e c u l e s such as C l q ( L u r h u m a e t al., 1 9 7 6 ) , c o n g l u t i n i n (Casali e t al., 1 9 7 7 ) , m o n o c l o n a l p r o t e i n s w i t h r h e u m a t o i d f a c t o r ( R F ) a c t i v i t y ( W i n c h e s t e r et al., 1 9 7 1 ; L u t h r a et al., 1 9 7 5 ; G a b r i e l a n d A g n e l l o , 1 9 7 7 ) , or h u m a n p o l y c l o n a l R F ( C o w d e r y e t al., 1 9 7 5 ; L u r h u m a e t al., 1 9 7 6 ) . All t h e s e t e c h n i q u e s have diff e r e n t s p e c t r a o f s p e c i f i c i t y a n d a p p e a r t o be c o m p l e m e n t a r y . M e t h o d s util i z i n g m o l e c u l a r l i g a n d s are m o r e c o n v e n i e n t since t h e r e a g e n t s a r e e a s i e r t o p r e p a r e a n d t o s t o r e . H o w e v e r , since 12SI-labelled r e a g e n t s h a v e a l i m i t e d shelf-life, we s t u d i e d t h e p o s s i b i l i t y o f u t i l i z i n g a s t a b l e e n z y m e l a b e l l e d reagent. T h e p r e s e n t p a p e r d e s c r i b e s an e n z y m e - i m m u n o a s s a y f o r i m m u n e corn-
208 plexes (IC-EIA) based upon inhibition of binding of a polyclonal R F (PRF) to an IgG-solid phase by IC present in biological fluids. This technique derives from the R F assay described by Maiolini et al. (1978) and comprises 3 sequential steps depicted in Fig. 1 : (1) absorption of endogenous R F in sera by an IgG-Sepharose sorbent; (2) a known a m o u n t of P R F is incubated with the absorbed samples in the presence of an IgG-cellulose sorbent which competes with IC for binding sites of the P R F reagent; (3) P R F bound to IC is removed by washing whereas P R F linked to the solid phase is allowed to react with an IgG-glucose oxidase conjugate. The enzyme activity bound to the solid phase is assayed and results are expressed as percentage inhibition of P R F fixation. MATERIAL AND METHODS
Human IgG, gamma-globulins and albumin IgG-solid phase and aggregated IgG were prepared using purified IgG purchased from the Centre National de Transfusion Sanguine de Paris (CNTS). Monomeric and denatured IgG were prepared from the same batch of protein. IgG was aggregated by heating a 30 g/1 stock solution in phosphate-buffered saline (PBS) 0.01 M pH 7.2, at 63°C for 20 min. The solution was then cooled in an ice bath and monomeric IgG separated from aggregated IgG by gel filtration on a Sephadex G-200 column (2.6 cm X 100.0 cm), equilibrated with the same buffer. Denatured IgG was prepared by heating the stock solution at 100°C for 5 min. IgG was purified by DEAE cellulose chromatography according to Kapusta and Halberstam (1964), before labelling with glucose oxidase. Human gamma-globulin was also obtained from the CNTS and purified albumin from Behring laboratories. IgG-solid phase Aggregated IgG was either linked to CNBr-activated cellulose (microcrystalline cellulose, Merck A.G.) as previously described by Wide et al. (1967) or to periodate activated cellulose (Ferrua et al., 1979). No difference in activity of solid-phase preparations was noted between IgG and gamma-globulin. In some experiments, IgG-coated polystyrene tubes were used as the solid phase. IgG immunosorbent To remove endogenous RF, two different sorbents were successfully employed: human gamma-globulin was linked either to Sepharose 6B according to Cuatrecasas et al. (1968) or activated glass powder according to the procedure of Jungfer (1975) which gave 22 pg of protein b o u n d per mg of glass
209 powder. Both sorbents were suspended in dilution buffer containing 0.05% NAN3, (usually 1 : 25) and stored at 4°C until used.
Labelled IgG Pure IgG was conjugated to glucose oxidase of funsal origin by the one step glutaraldehyde technique (Avrameas, 1969). After concentration to 0.75 g/l, labelled IgG was diluted in glycerol (1 : 1) and stored at --20°C until used. Rheumatoid factor The serum from a rheumatoid arthritis patient was used as a source of RF. The antiglobulin activity was shown to be polyclonal and of IgM and IgG classes as determined by EIA. Buffers Incubation buffer: IgG i m m u n o s o r b e n t was suspended in PBS 0.05 M pH 7.2 containing 0.4% Na2-EDTA, 0.05% Tween-20, 0.05% NaN3 and 1% ovalbumin or h u m a n albumin. Washing buffer: washing steps were performed with PBS 0.05 M pH 7.2 containing 0.05% Tween-20. Dilution buffer: dilutions of protein solutions and sera were made in washing buffer containing 1% ovalbumin or h u m a n albumin. Ser~ Forty-eight sera from healthy adults were obtained from blood donors and laboratory workers. Sera containing rheumatoid factor (RF) or alpha-fetoprotein (AFP) were routinely titrated in our laboratory by EIA techniques (Maiolini et al., 1975, 1978). HBs antigen was identified in 60 sera by the Hepanostika EIA test (Organon Teknika S.A.). A semi-quantitative positivity index (+ to +++), was used. All sera were kept frozen (--20 ° C) until tested. In vitro preparation of immune complexes Tetanus toxoid-human anti-tetanus toxoid immune complexes were prepared by mixing a constant a m o u n t of specific human gamma-globulin (Gamma TS, 125 IU/ml, Centre de Transfusion Sanguine) to increasing amounts of toxoid (gift of Prof. Relyveld, Institut Pasteur de Paris, batch no. TC 1 4 1 , 4 2 5 FU/ml). The mixture was incubated for 1 h at 37°C and then overnight at 4°C. After centrifugation, supernatants were tested to determine the equivalence point by gel diffusion. Artificial IC from antibody and antigen excess zones were assayed in the inhibition test. For each determination non specific inhibition due to identical concentrations of a n t i b o d y or antigen tested separately was subtracted.
210
IC-EIA procedure The assay was performed at room temperature in disposable polystyrene tubes; all reagents were distributed by an automatic sampler-diluter (Gilson Base Analymat). Absorption of endogenous R F (where present). 100 pl of serum was incubated for 20 min with 100 pl of a 0.2 M pH 7.6 Na2-EDTA solution. After incubation, 800 pl of IgG sorbent suspension was added and the mixture rotated for 2 h. After centrifugation (10 min at 3000 X g), duplicate aliquots of the supernatants were tested by IC-EIA. Immune complexes assay. 200 pl of absorbed sera were added to 500 pl of IgG-coupled cellulose suspension and 200 pl of PRF diluted 1 : 50. Tubes were rotated for 3 h. After incubation, the tubes were filled with 4 ml of washing buffer and centrifuged for 30 sec at 2000 X g. The supernatant was then removed by suction so that about 0.4--0.5 ml of buffer was left above the pellet. This washing step was repeated once. 200 pl of glucose-oxidase-labelled IgG (diluted 1 : 200) were then added. The tubes were stoppered and rotated for 18 h. The solid phase was washed again 3 times in the same way. Enzymatic activity linked to the solid phase was measured as described by Maiolini et al. (1978). Under these conditions, maximal O.D. corresponding to no inhibition was in the range 0.800--0.850. Each series included 4 controls where the test sample was replaced by 200 /~l of dilution buffer to evaluate maximal binding of labelled IgG (O.D.m~×), and 4 other controls where PRF was replaced by the dilution buffer in order to determine non-specific absorption of labelled IgG on the solid phase (O.D.blank) to zero the photometer. Other tests for IC Polyethylene glycol precipitation of IC (PEG test) was performed according to Digeon et al. (1977). Precipitated C4 (C4 test) was quantified by laser i m m u n o n e p h e l o m e t r y (Hyland PDQ system). [121I]Clq binding activity (Clq-BA) was kindly performed on 65 sera from hospital patients by Prof. J.P. Revillard and Dr. C. Vincent (INSERM U 80, Lyon) and on 27 sera from schistosomiasis patients by Prof. Capron and Dr. Santoro (Centre d'Immunologie et de Biologie Parasitaire, Institut Pasteur, Lille), according to the technique of Zubler and Lambert (1976). Expression and treatment of results Automatic processing of data was achieved with a mini-computer Wang 2200. Since the binding modes of aggregated IgG and IC to RF were clearly different, they could n o t be expressed in the same weight equivalents. Thus the percentage of inhibition (% INH) defined as: 100X(1--
O.D-sample ~ )wascalculated.
211 RESULTS
Absorption of endogenous RF The binding capacity of IgG-Sepharose or IgG-glass powder sorbents was checked by measuring the RF level in rheumatoid arthritis sera before and after absorption. The RF remaining was always close to background levels for sera, the original titre of which was up to about 300 IU/ml. Non-specific protein absorption on the sorbent was estimated by titrating RF in the same sera before and after absorption with albumin-Sepharose or albumin-glass powder sorbent. Although slightly less potent, Sepharose sorbent was chosen for routine use because it was more readily and evenly suspended.
Binding inhibition of PRF A typical standard dose-response curve with aggregated IgG as IC equivalent is shown in Fig. 2. With this reagent, the assay range lies between 1 and 100 rag/1. Lower values (0.5 mg/1) could be detected but with insufficient precision.
Specificity The assay specificity was studied in the following experiments.
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212
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Fig. 3. Lack of correlation between inhibiting activity and IgG levels in 44 hepatitis sera.
213
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100/1 Fig. 4. Per c e n t of i n h i b i t i o n b y soluble IC p e r f o r m e d in vitro as a f u n c t i o n of antigena n t i b o d y ( A g / A b ) ratio. EP = e q u i v a l e n c e p o i n t .
-- The inhibitory effects of different monomeric IgG concentrations were measured. Fig. 2 shows t h a t a 0.5% 1NH was given by a 100 mg/1 solution of m o n o m e r whereas 91% INH was obtained with the same concentration of aggregated IgG. -- Indirect evidence that mon~meric IgG gives no significant interference was noted by measuring IgG levels in 44 hepatitis sera assayed in the IC-EIA. No correlation was seen between IgG levels and % INH (Fig. 3). -- No noticeable inhibition was produced either by h u m a n albumin or by denatured IgG (Fig. 2). -- Aggregated IgG was mixed in various final concentrations with human sera and % INH was measured. The inhibitory effect of the sera alone was subtracted. Taking into account non specific absorption, the observed inhibition was close to that expected. -- Fig. 4 shows the % INH given by preformed tetanus toxoid-anti-toxoid IC as a function of antigen/antibody ratio. Equivalence was reached with a ratio of I : 10 (v/v). Inhibiting activity was seen only in the antigen excess zone.
IC levels in h u m a n sera (Fig. 5) With 48 sera from healthy adults assayed in the IC-EIA, the % INH ranged from 0 to 15.3%, irrespective of age and sex, with a mean value of 4.8 + 4.4. The upper limit of normal was 14% INH. Sixty hepatitis sera: positivity indexes determined with the Hepanostika test, were distributed as follows: 27 +++, 18 ++ and 15 +. Highest inhibition levels were f o u n d in the (+) group. The mean inhibition was 18.5% and 65% of sera were considered positive. Of 15 sera from primary liver cancer patients with alpha-fetoprotein levels ranging from 479 to 100,000 pg/1, one was positive. Of 63 sera from hospital patients suffering from various diseases with a positive I~F test, 32 were positive. RF values ranged from 10 to 245 IU/ml and were distributed independently of IC levels.
214
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Of 32 sera from patients with rheumatoid arthritis (according to American Rheumatism Association criteria), 18 were positive. Correlations with clinical staging, radiographic involvement, time of duration and therapeutic response will be reported elsewhere.
Chromatography of positive sera Two positive sera were chromatographed on a Sephadex G-200 column. Eluted volumes were pooled in 10 different fractions and each was measured for % INH and immunoglobulin levels. In the first patient (IgA multiple myeloma), inhibiting activity was located in the excluded peak (Fig. 6). In a second patient (psoriasis), the bulk of the inhibiting activity was in the ascending slope of the second peak. Reproducibility Intra-assay variation was calculated for 10 determinations on 6 different aggregated IgG concentrations. Table 1 shows that for a 1 mg/l IgG solution (which corresponds to the threshold sensitivity of the assay), the coefficient of variation was less than 9%. Inter-assay reproducibility was assessed on 5 successive days in various
215 T A B L E 1_ I N T R A - A S S A Y P R E C I S I O N O F THE IC-EIA E S T I M A T E D F O R 10 D E T E R M I N A TIONS AT 6 C O N C E N T R A T I O N S O F A G G R E G A T E D IgG P E R F O R M E D ON ONE OCCASION IgG (rag/l)
Mean % INH -+ S.D.
C o e f f i c i e n t o f variation in %
0.5 1.0 10.0 100.0 200.0
6.4 10,9 59.5 89.0 91.9
15.1 8.9 3.6 2.0 2.4
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TABLE 2 I N T E R - A S S A Y P R E C I S I O N O F THE IC-EIA P E R F O R M E D ON THE S A M E S E R A ON 5 D I F F E R E N T DAYS N u m b e r o f sera t e s t e d
M e a n % INH -+ S,D.
C o e f f i c i e n t o f variation in %
5 6 5 5
20.6 7.7 73.0 43.4
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216
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Fig. 7. a: P E G t e s t in 13 c o n t r o l s , 17 R F p o s i t i v e sera a n d 17 h e p a t i t i s sera. b: lack o f c o r r e l a t i o n b e t w e e n i n h i b i t o r y a c t i v i t y a n d P E G t e s t in t h e s a m e sera.
sera stored at --20°C and covering the assay range (Table 2). With IgG-coated polystyrene tubes as the solid phase, intra-assay variations were only slightly greater, but inter-assay reproducibility was unacceptably worse. This simpler procedure was therefore discarded. Correlation with differen t IC assay systems Agreement between different tests for IC varied according to disease. Two groups of sera previously assayed by Clq--BA were examined. -In the first, originating from 65 miscellaneous hospital patients, 30 were C l q positive and 35 negative. The IC-EIA results did not discriminate between the two, and only 3% were found positive (r --- 0.23, P < 0.01). -- The second group consisted of 26 sera from schistosomiasis patients, all C l q positive. Of these, 88% were positive in the IC-EIA (r = 0.47, P < 0.01). -PEG test and C4 test: 13 normal sera showing no significant % INH and 34 considered positive (17 RF and 17 HBs antigen positive sere ) were assayed. Although significantly different mean O.D. values were obtained in the two groups, no correlation was found between PEG and IC-EIA results (r = 0.33, P < 0.01), (Fig. 7a, b). Discrepant results were also obtained with the C4 test (r = 0.25, P < 0.01). DISCUSSION
This paper describes an IC assay which combines the specific reactivity of a c o m m o n biological material (polyclonal RF) with the advantage of EIA, and avoids use of radionucleides. It is suitable for laboratories possessing only non-isotopic routine equipment.
217
Choice of PRF Criteria of choice of the PRF used in this assay were operational: its reactivity towards aggregated IgG and in vitro formed IC showed it competed for both. Moreover, previous purification was not necessary as whole serum could be used in a high, non-interfering dilution. A possible drawback might by the presence of anti-Gm activity in the PRF. However, no anti-Gin activity was found when the PRF used in this assay was tested against a panel of known Gm allotypes, kindly provided by Dr. L. Rivat (INSERM, U. 78). Conversely, several other sera, with high RF activity could n o t be used in this assay, due to anti-GM or Inv activity. Absorption of endogenous RF: EDTA treatment This procedure was found to be useful before performing the IC assay for several reasons. The titrated quantity of PRF required would be doubled with serum samples containing, for example, 200 IU/ml. Furthermore, RF positive unabsorbed sera gave inhibition values consistently different from absorbed ones. Cowdery et al. (1975), in another test using polyclonal RF, found that endogenous RF interfered. However, the reverse was observed in IC assays using monoclonal RF (Luthra et al., 1975; Gabriel and Agnello, 1977). Some workers find it sufficient to treat sera with dithiothreitol (Lurhuma et al., 1976). We have also tried another technique, in which PRF was linked to the solid phase and thus able to fix IC w i t h o u t interference from endogenous RF. Free remaining PRF binding sites were quantified with an aggregated IgG-conjugate. Results (not shown) were quite satisfactory but the procedure required purification of PRF and the yield was too low. Differing results regarding interference by C1 of C l q have been reported (Gabriel and Agnello, 1977; Levinsky and Soothill, 1977). In our experiments the mean % INH of 30 hepatitis and 10 normal sera was similarly 6% lower when EDTA pretreatment was omitted. IC and aggregated IgG Several biological properties are shared by IC and aggregated IgG, particularly reactivity with RF (Henney and Stanworth, 1965; Hirose and Osler, 1965). Nevertheless, the similarity of conformational changes between heated IgG and antigen-antibody (Ag-Ab) aggregates has been questioned and some comparisons of various techniques of IC assay show no correlation between ability to detect aggregated IgG and IC in pathological sera (C. Vincent and J.P. Revillard, unpublished). The relative binding of aggregated IgG and IC to RF may vary with the Ag/Ab ratio in the latter (Cowdery et al., 1975), and, in addition to this, aggregated IgG is usually prepared from pooled polyclonal IgG preparations and possesses most of the iso- and allotypic specificities. Such an 'antigenic
218 display' is unlikely to occur with Ag/Ab complexes. For these reasons, the inhibition curve with aggregated IgG should not be interpreted as it currently is, as an IC sensitivity index, nor as a reference comparison between various techniques, but rather as an intra- and inter-assay control. For the same reasons, we have discarded aggregated IgG equivalents as reference values and calibrate our results in arbitrary % INH units. Specificity RFs may display specificity for certain IgG subclasses (Normansell and Young, 1975) and this may endow them with selective sensitivity. However, this is dependent on the quality of the PRF chosen, since some, in IC assay, are inhibited by all human IgG subclasses (Gabriel and Agnello, 1977). It is generally assumed that RF react with both native and altered IgG. Whether the substrate for preferential reactivity with the latter has special conformational determinants, or only polyvalent interaction with antigens shared by both (Normansell, 1971; Eisenberg, 1976), is still undecided. In the IC-EIA, monomeric IgG did not appreciably interfere. To inhibit RF Gabriel and Agnello (1977) found it necessary to add monomeric IgG concentrations 100 times greater than aggregated IgG but even such a high ratio may be insufficient. We do n o t find it useful to dilute all sera to a known final concentration as proposed by Luthra et al. (1975). The standard dilution used in this assay was adequate. IC prepared in vitro Size and Ag-Ab ratio have been shown to be determining factors in recognition of IC by RF (Lightfoot et al., 1969). This sensitivity to Ag-Ab ratio could be usefully exploited to identify the Ag involved in the complex (Delire and Masson, 1977). Maximum reactivity varies with the different RF used and shifts from equivalence (Cowdery et al., 1975) towards slight or marked antigen excess (Lurhuma et al., 1976; Gabriel and Agnello, 1977). Our study confirms this tendency of RF to detect IC formed mainly in Ag excess. Normal and pathologic human sera A puzzling problem in IC assay is the definition of a normal population. Several authors found significantly elevated activity in up to 14% of a blood donor population (Lurhuma et al., 1976). Several factors could account for this. For example, antiglobulin factors have been detected in normal subjects (Torrigiani and Roitt, 1967) and might be involved in formation of complexes, and IC resulting from mild inapparent infections or from uptake of food antigen have also been incriminated (Lurhuma et al., 1976). It is possible that IC are a physiological constant in healthy individuals. In the hepatitis cases we studied, an apparent inverse relationship was observed between the level of antigenemia and the occurrence of IC. Such a
219 relationship need confirmation on a larger series. In rheumatoid arthritis, we found no correlation between RF titre and IC levels, although there was an association between the occurrence of IC and RF (Fig. 5), as previously suggested (Lurhuma et al., 1976). RF production is presumably stimulated by Ag-Ab complexes. Chromatography o f IC positive sera The presence of Ca 2÷ during isolation procedures may prevent Ag-Ab complexes dissociating (Lurhuma et al., 1976). The chromatography buffer we used in this work did n o t contain Ca 2÷ and this may account for the diminished inhibiting activity of fractions as compared to whole serum. In the first chromatogram discussed above, inhibitory activity was located in excluded fractions corresponding to high molecular weight material, as reported by L u r h u m a et al. (1976) and L y e t et al. (1974); but in our second chromatogram, inhibitory material was unexpectedly detected also in lighter fractions. This may indicate that RF assay is able to detect IC as light as 8S (Gabriel and Agnello, 1977) and is sensitive to complexes produced in marked antigen excess. Correlation with other IC assays Previous attempts to compare results obtained with RF and C l q reagents have shown poor agreement (Winchester et al., 1971; Luthra et al., 1975; Gabriel and Agnello, 1977) and this suggests that they probably detect IC differing either in the size or in nature of the antibody or at least that different determinants in ICs are being quantified. Our own results show in most cases a similar lack of correlation. It thus seems unwarranted to consider any of these assays as a reference technique. Comparisons of their sensitivities probably have little significance and the contribution of each assay is rather to widen the panel of specificities detected. Conclusions (1) The advantage of the assay described is highly dependent upon the PRF used. The serum we used was exceptional in possessing all the required qualities. However, PRF are reagents of human origin and their supply is unpredictable. We have not so far succeeded in preparing xenogeneic antisera with properties comparable to RF. (2) The need to eliminate endogenous RF complicates the test. (3) Each PRF used has its own peculiarities so that results with different PRFs are hardly comparable. Reagents with more defined specificities are needed. RF-like antiglobulins prepared in animals may answer these objections. Techniques of production are reported (Milgrom and Witebsky, 1960; Abruzzo and Christian, 1961) and attempts have been made to use them in IC assay (Levinsky and Soothill, 1977). Suitable RF-like antiglobulins would render the assay described operative and provide abundant, exchangeable, standardizable reagents.
220
ACKNOWLEDGEMENTS
We are grateful to Professor Capron and to Professor Revillard, and to Doctors Santoro and Vincent who kindly supplied C l q assayed sera and to Professor Relyveld who provided tetanus toxoid solution. We are also grateful to L. Rivat for providing a Gm allotype panel. This work was supported by Contract No. 77.7.1875. DGRST, Paris. REFERENCES Abruzzo, J. and C. Christian, 1961, J. Exp. Med. 114, 791. Avrameas, S., 1969, Immunochemistry 6, 43. Casali, P., A. Bossus, N.A. Carpentier and P.H. Lambert, 1977, Clin. Exp. Immunol. 29, 342. Cowdery, J., P. Treadwell and R. Fritz, 1975, J. Immunol. 114, 5. Cuatrecasas, P., M. Wilchek and C.B. Anfinsen, 1968, Proe. Natl. Acad. Sci. U.S.A. 61, 636. Delire, M. and P.L. Masson, 1977, Clin. Exp. Immunol. 29, 385. Digeon, M., M. Laver, J. Riza and J.F. Bach, 1977, J. Immunol. Methods 16, 165. Eisenberg, R., 1976, Immunochemistry 13, 355. Ferrua, B., R. Maiolini and R. Masseyeff, 1979, J. Immunol. Methods 25, 49. Gabriel, A. and V. Agnello, 1977, J. Clin. Invest. 59, 990. Henney, C.S. and D.R. Stanworth, 1965, Immunology 9, 139. Hirose, S.I. and A.G. Osler, 1965, J. Immunol. 91,927. Jungfer, H., 1975, *rztl. Lab. 21, 80. Kapusta, M.A. and D. Halberstam, 1964, Biochim. Biophys. Acta 93, 657. Levinsky, R.J. and J.F. Soothill, 1977, Clin. Exp. Immunol. 29,428. Lightfoot, R.W., D.E. Drusin and C.L. Christian, 1969, Ann. N.Y. Acad. Sci. 168, 105. Lurhuma, A.Z., C.L. Cambiaso, P.L. Masson and J.F. Heremans, 1976, Clin. Exp. Immunol. 25, 212. Luthra, J.H., F.C. McDuffie, G.G. Hunder and E.A. Samayoa. 1975, J. Clin. Invest. 56, 458. Lyet, J.P. and D.E. Normansell, 1974, Immunochemistry 11,417. Maiolini, R. and R. Masseyeff, 1975, J. Immunol. Methods 8, 223. Maiolini, R., B. Ferrua, J.F. Quaranta, A. Pinoteau, L. Euller, G. Ziegler and R. Masseyeff, 1978, J. Immunol. Methods 20, 25. Milgrom, F. and E. Witebsky, 1960, Fed, Proc. 19, 197. Normansell, D.E., 1971, Immunochemistry 8, 593. Normansell, D.E. and C.W. Young, 1975, Immunochemistry 12, 187. Onyewotu, I., E.J. Holborow and G.D. Johnson, 1974, Nature 248, 156. Penttinen K., A. Vaheri and G. Myllyla, 1971, Clin. Exp. Immunol. 8, 389. Theophilopoulos, A., C. Wilson and F. Dixon, 1976, J. Clin. Invest. 57, 169. Torrigiani, G. and I.M. Roitt, 1967, Ann. Rheum. Dis. 26, 334. Wide, L., R. Axen and J. Porath, 1967, Immunochemistry 4, 381. Winchester, R.J., H.G. Kunkel and V. Agnello, 1971, J. Exp. Med. 134,286. Zubler, R.H. and P.H. Lambert, 1976, in: In Vitro Methods in Cell-mediated and Tumor Immunity, eds. B.R. Bloom and J.R. David (Academic Press, New York) p. 565.
207
AN ENZYME-IMMUNOASSAY METHOD FOR DETECTING C I R C U L A T I N G I M M U N E C O M P L E X E S BY I N H I B I T I O N O F POLYCLONAL RHEUMATOID FACTOR
L. RODA, R. MAIOLINI, B. FERRUA and R. MASSEYEFF INSERM FRA 12, Laboratoire d'Immunologie, CHU de Nice, 06031 Nice Cedex, France
(Received 30 November 1978, accepted 29 March 1979)
An enzyme-immunoassay for circulating immune complexes (IC-EIA is described which depends on inhibition of binding of rheumatoid factor (RF) to aggregated IgG. The assay involves 3 steps: (1) sera are pretreated with an aggregated IgG sorbent in order to remove any endogenous RF; (2) absorbed sera are then incubated with a polyelonal RF of known titre and an aggregated IgG sorbent; (3) after washing, the amount of RF on the solid phase is revealed by an IgG-glucose oxidase conjugate. Results are expressed as per cent inhibition of RF fixation. The assay range extends from 1 to 100 mg/l. Intra- and inter-assay coefficients were 6 and 9% respectively. Specificity has been assessed by various tests: dose-response curve with aggregated monomerie or denatured IgG and human albumin localization of the inhibitory fraction of sera after Sephadex G-200 chromatography, absence of interference of IgG, and use of artificial IC. Poor correlation was obtained between IC-EIA and other techniques. The method demonstrated the occurrence of IC in a large proportion of patients with viral hepatitis, schistosomiasis and rheumatoid arthritis and their absence in normal subjects. INTRODUCTION A v a r i e t y o f t e c h n i q u e s f o r t h e assay of c i r c u l a t i n g i m m u n e c o m p l e x e s (IC) have b e e n d e s c r i b e d . T h e y t a k e a d v a n t a g e o f t h e s p e c i f i c p h y s i c o - c h e m ical p r o p e r t i e s o f IC, a n d / o r o f t h e i r a b i l i t y t o b i n d e i t h e r t o t h e s u r f a c e o f s o m e cells s u c h as p l a t e l e t s ( P e n t t i n e n e t al., 1 9 7 1 ) , m a c r o p h a g e s ( O n y e w o t u e t al., 1 9 7 4 ) , R a j i cells ( T h e o p h i l o p o u l o s e t al., 1 9 7 6 ) , or t o m o l e c u l e s such as C l q ( L u r h u m a e t al., 1 9 7 6 ) , c o n g l u t i n i n (Casali e t al., 1 9 7 7 ) , m o n o c l o n a l p r o t e i n s w i t h r h e u m a t o i d f a c t o r ( R F ) a c t i v i t y ( W i n c h e s t e r et al., 1 9 7 1 ; L u t h r a et al., 1 9 7 5 ; G a b r i e l a n d A g n e l l o , 1 9 7 7 ) , or h u m a n p o l y c l o n a l R F ( C o w d e r y e t al., 1 9 7 5 ; L u r h u m a e t al., 1 9 7 6 ) . All t h e s e t e c h n i q u e s have diff e r e n t s p e c t r a o f s p e c i f i c i t y a n d a p p e a r t o be c o m p l e m e n t a r y . M e t h o d s util i z i n g m o l e c u l a r l i g a n d s are m o r e c o n v e n i e n t since t h e r e a g e n t s a r e e a s i e r t o p r e p a r e a n d t o s t o r e . H o w e v e r , since 12SI-labelled r e a g e n t s h a v e a l i m i t e d shelf-life, we s t u d i e d t h e p o s s i b i l i t y o f u t i l i z i n g a s t a b l e e n z y m e l a b e l l e d reagent. T h e p r e s e n t p a p e r d e s c r i b e s an e n z y m e - i m m u n o a s s a y f o r i m m u n e corn-
208 plexes (IC-EIA) based upon inhibition of binding of a polyclonal R F (PRF) to an IgG-solid phase by IC present in biological fluids. This technique derives from the R F assay described by Maiolini et al. (1978) and comprises 3 sequential steps depicted in Fig. 1 : (1) absorption of endogenous R F in sera by an IgG-Sepharose sorbent; (2) a known a m o u n t of P R F is incubated with the absorbed samples in the presence of an IgG-cellulose sorbent which competes with IC for binding sites of the P R F reagent; (3) P R F bound to IC is removed by washing whereas P R F linked to the solid phase is allowed to react with an IgG-glucose oxidase conjugate. The enzyme activity bound to the solid phase is assayed and results are expressed as percentage inhibition of P R F fixation. MATERIAL AND METHODS
Human IgG, gamma-globulins and albumin IgG-solid phase and aggregated IgG were prepared using purified IgG purchased from the Centre National de Transfusion Sanguine de Paris (CNTS). Monomeric and denatured IgG were prepared from the same batch of protein. IgG was aggregated by heating a 30 g/1 stock solution in phosphate-buffered saline (PBS) 0.01 M pH 7.2, at 63°C for 20 min. The solution was then cooled in an ice bath and monomeric IgG separated from aggregated IgG by gel filtration on a Sephadex G-200 column (2.6 cm X 100.0 cm), equilibrated with the same buffer. Denatured IgG was prepared by heating the stock solution at 100°C for 5 min. IgG was purified by DEAE cellulose chromatography according to Kapusta and Halberstam (1964), before labelling with glucose oxidase. Human gamma-globulin was also obtained from the CNTS and purified albumin from Behring laboratories. IgG-solid phase Aggregated IgG was either linked to CNBr-activated cellulose (microcrystalline cellulose, Merck A.G.) as previously described by Wide et al. (1967) or to periodate activated cellulose (Ferrua et al., 1979). No difference in activity of solid-phase preparations was noted between IgG and gamma-globulin. In some experiments, IgG-coated polystyrene tubes were used as the solid phase. IgG immunosorbent To remove endogenous RF, two different sorbents were successfully employed: human gamma-globulin was linked either to Sepharose 6B according to Cuatrecasas et al. (1968) or activated glass powder according to the procedure of Jungfer (1975) which gave 22 pg of protein b o u n d per mg of glass
209 powder. Both sorbents were suspended in dilution buffer containing 0.05% NAN3, (usually 1 : 25) and stored at 4°C until used.
Labelled IgG Pure IgG was conjugated to glucose oxidase of funsal origin by the one step glutaraldehyde technique (Avrameas, 1969). After concentration to 0.75 g/l, labelled IgG was diluted in glycerol (1 : 1) and stored at --20°C until used. Rheumatoid factor The serum from a rheumatoid arthritis patient was used as a source of RF. The antiglobulin activity was shown to be polyclonal and of IgM and IgG classes as determined by EIA. Buffers Incubation buffer: IgG i m m u n o s o r b e n t was suspended in PBS 0.05 M pH 7.2 containing 0.4% Na2-EDTA, 0.05% Tween-20, 0.05% NaN3 and 1% ovalbumin or h u m a n albumin. Washing buffer: washing steps were performed with PBS 0.05 M pH 7.2 containing 0.05% Tween-20. Dilution buffer: dilutions of protein solutions and sera were made in washing buffer containing 1% ovalbumin or h u m a n albumin. Ser~ Forty-eight sera from healthy adults were obtained from blood donors and laboratory workers. Sera containing rheumatoid factor (RF) or alpha-fetoprotein (AFP) were routinely titrated in our laboratory by EIA techniques (Maiolini et al., 1975, 1978). HBs antigen was identified in 60 sera by the Hepanostika EIA test (Organon Teknika S.A.). A semi-quantitative positivity index (+ to +++), was used. All sera were kept frozen (--20 ° C) until tested. In vitro preparation of immune complexes Tetanus toxoid-human anti-tetanus toxoid immune complexes were prepared by mixing a constant a m o u n t of specific human gamma-globulin (Gamma TS, 125 IU/ml, Centre de Transfusion Sanguine) to increasing amounts of toxoid (gift of Prof. Relyveld, Institut Pasteur de Paris, batch no. TC 1 4 1 , 4 2 5 FU/ml). The mixture was incubated for 1 h at 37°C and then overnight at 4°C. After centrifugation, supernatants were tested to determine the equivalence point by gel diffusion. Artificial IC from antibody and antigen excess zones were assayed in the inhibition test. For each determination non specific inhibition due to identical concentrations of a n t i b o d y or antigen tested separately was subtracted.
210
IC-EIA procedure The assay was performed at room temperature in disposable polystyrene tubes; all reagents were distributed by an automatic sampler-diluter (Gilson Base Analymat). Absorption of endogenous R F (where present). 100 pl of serum was incubated for 20 min with 100 pl of a 0.2 M pH 7.6 Na2-EDTA solution. After incubation, 800 pl of IgG sorbent suspension was added and the mixture rotated for 2 h. After centrifugation (10 min at 3000 X g), duplicate aliquots of the supernatants were tested by IC-EIA. Immune complexes assay. 200 pl of absorbed sera were added to 500 pl of IgG-coupled cellulose suspension and 200 pl of PRF diluted 1 : 50. Tubes were rotated for 3 h. After incubation, the tubes were filled with 4 ml of washing buffer and centrifuged for 30 sec at 2000 X g. The supernatant was then removed by suction so that about 0.4--0.5 ml of buffer was left above the pellet. This washing step was repeated once. 200 pl of glucose-oxidase-labelled IgG (diluted 1 : 200) were then added. The tubes were stoppered and rotated for 18 h. The solid phase was washed again 3 times in the same way. Enzymatic activity linked to the solid phase was measured as described by Maiolini et al. (1978). Under these conditions, maximal O.D. corresponding to no inhibition was in the range 0.800--0.850. Each series included 4 controls where the test sample was replaced by 200 /~l of dilution buffer to evaluate maximal binding of labelled IgG (O.D.m~×), and 4 other controls where PRF was replaced by the dilution buffer in order to determine non-specific absorption of labelled IgG on the solid phase (O.D.blank) to zero the photometer. Other tests for IC Polyethylene glycol precipitation of IC (PEG test) was performed according to Digeon et al. (1977). Precipitated C4 (C4 test) was quantified by laser i m m u n o n e p h e l o m e t r y (Hyland PDQ system). [121I]Clq binding activity (Clq-BA) was kindly performed on 65 sera from hospital patients by Prof. J.P. Revillard and Dr. C. Vincent (INSERM U 80, Lyon) and on 27 sera from schistosomiasis patients by Prof. Capron and Dr. Santoro (Centre d'Immunologie et de Biologie Parasitaire, Institut Pasteur, Lille), according to the technique of Zubler and Lambert (1976). Expression and treatment of results Automatic processing of data was achieved with a mini-computer Wang 2200. Since the binding modes of aggregated IgG and IC to RF were clearly different, they could n o t be expressed in the same weight equivalents. Thus the percentage of inhibition (% INH) defined as: 100X(1--
O.D-sample ~ )wascalculated.
211 RESULTS
Absorption of endogenous RF The binding capacity of IgG-Sepharose or IgG-glass powder sorbents was checked by measuring the RF level in rheumatoid arthritis sera before and after absorption. The RF remaining was always close to background levels for sera, the original titre of which was up to about 300 IU/ml. Non-specific protein absorption on the sorbent was estimated by titrating RF in the same sera before and after absorption with albumin-Sepharose or albumin-glass powder sorbent. Although slightly less potent, Sepharose sorbent was chosen for routine use because it was more readily and evenly suspended.
Binding inhibition of PRF A typical standard dose-response curve with aggregated IgG as IC equivalent is shown in Fig. 2. With this reagent, the assay range lies between 1 and 100 rag/1. Lower values (0.5 mg/1) could be detected but with insufficient precision.
Specificity The assay specificity was studied in the following experiments.
(1) S
+ ~1~
(2) C
i
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+
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3--
---), PRF ( ~ ) , endogenous RF ( l ) , cellulose-IgG sorbent (C~>-), immune complexes ( ~ ) , Labelled IgG (>~-). (1) pretreatment of sera; (2)competition step; (3)enzymatic activity determination.
212
100- % IINIH
/
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t
/
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/
/ / Z /
/ / /
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Fig. 2. I n h i b i t i n g a c t i v i t y o f aggregated (o . . . . . . e), m o n o m e r i c (m) a n d d e n a t u r e d (A) IgG a n d h u m a n a l b u m i n (•). T h e vertical bars r e p r e s e n t ± 1 S.D.
~olNH 40-
30-
20°o
10-
•
r = 0.33
Fig. 3. Lack of correlation between inhibiting activity and IgG levels in 44 hepatitis sera.
213
1
I:~:t':'::: : :.'~. , ~ :: :.:.:.:.
~: :: ::.:::~ :J
!iiiiiii!i!!!i i !iiiii!!iiii~~ ~.i:?i!i:i:2:!:5:!::ii:i:i'i:i:?:.~ EP ,: :::: :::::::::: ::::::::: %
-t i:::i;i:::/;!:::i:::::~::{:i:i:i:i:i:i:!:i~. Ag/Ab ratio I
'
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' I
100/1 Fig. 4. Per c e n t of i n h i b i t i o n b y soluble IC p e r f o r m e d in vitro as a f u n c t i o n of antigena n t i b o d y ( A g / A b ) ratio. EP = e q u i v a l e n c e p o i n t .
-- The inhibitory effects of different monomeric IgG concentrations were measured. Fig. 2 shows t h a t a 0.5% 1NH was given by a 100 mg/1 solution of m o n o m e r whereas 91% INH was obtained with the same concentration of aggregated IgG. -- Indirect evidence that mon~meric IgG gives no significant interference was noted by measuring IgG levels in 44 hepatitis sera assayed in the IC-EIA. No correlation was seen between IgG levels and % INH (Fig. 3). -- No noticeable inhibition was produced either by h u m a n albumin or by denatured IgG (Fig. 2). -- Aggregated IgG was mixed in various final concentrations with human sera and % INH was measured. The inhibitory effect of the sera alone was subtracted. Taking into account non specific absorption, the observed inhibition was close to that expected. -- Fig. 4 shows the % INH given by preformed tetanus toxoid-anti-toxoid IC as a function of antigen/antibody ratio. Equivalence was reached with a ratio of I : 10 (v/v). Inhibiting activity was seen only in the antigen excess zone.
IC levels in h u m a n sera (Fig. 5) With 48 sera from healthy adults assayed in the IC-EIA, the % INH ranged from 0 to 15.3%, irrespective of age and sex, with a mean value of 4.8 + 4.4. The upper limit of normal was 14% INH. Sixty hepatitis sera: positivity indexes determined with the Hepanostika test, were distributed as follows: 27 +++, 18 ++ and 15 +. Highest inhibition levels were f o u n d in the (+) group. The mean inhibition was 18.5% and 65% of sera were considered positive. Of 15 sera from primary liver cancer patients with alpha-fetoprotein levels ranging from 479 to 100,000 pg/1, one was positive. Of 63 sera from hospital patients suffering from various diseases with a positive I~F test, 32 were positive. RF values ranged from 10 to 245 IU/ml and were distributed independently of IC levels.
214
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Fig. 5. Inhibiting activity of various sera: controls (T), alpha-fetoprotein positive sera (AFP), rheumatoid factor positive sera (RF), r h e u m a t o i d arthritis sera (RA) and hepatitis sera (H 2+, H 2+, H3+).
Of 32 sera from patients with rheumatoid arthritis (according to American Rheumatism Association criteria), 18 were positive. Correlations with clinical staging, radiographic involvement, time of duration and therapeutic response will be reported elsewhere.
Chromatography of positive sera Two positive sera were chromatographed on a Sephadex G-200 column. Eluted volumes were pooled in 10 different fractions and each was measured for % INH and immunoglobulin levels. In the first patient (IgA multiple myeloma), inhibiting activity was located in the excluded peak (Fig. 6). In a second patient (psoriasis), the bulk of the inhibiting activity was in the ascending slope of the second peak. Reproducibility Intra-assay variation was calculated for 10 determinations on 6 different aggregated IgG concentrations. Table 1 shows that for a 1 mg/l IgG solution (which corresponds to the threshold sensitivity of the assay), the coefficient of variation was less than 9%. Inter-assay reproducibility was assessed on 5 successive days in various
215 T A B L E 1_ I N T R A - A S S A Y P R E C I S I O N O F THE IC-EIA E S T I M A T E D F O R 10 D E T E R M I N A TIONS AT 6 C O N C E N T R A T I O N S O F A G G R E G A T E D IgG P E R F O R M E D ON ONE OCCASION IgG (rag/l)
Mean % INH -+ S.D.
C o e f f i c i e n t o f variation in %
0.5 1.0 10.0 100.0 200.0
6.4 10,9 59.5 89.0 91.9
15.1 8.9 3.6 2.0 2.4
_+ 0.9 -+ 1.9 -+ 2.2 -+ 1.8 -+ 2.1
TABLE 2 I N T E R - A S S A Y P R E C I S I O N O F THE IC-EIA P E R F O R M E D ON THE S A M E S E R A ON 5 D I F F E R E N T DAYS N u m b e r o f sera t e s t e d
M e a n % INH -+ S,D.
C o e f f i c i e n t o f variation in %
5 6 5 5
20.6 7.7 73.0 43.4
7.4 9.0 6.4 7.4
-+ 1.5 + 0.7 -+ 4.6 + 3.2
immunoglobulin g/~
~INH
/ 20
•
oO•
•
/ •
•
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\
s
\ \
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Fig, 6. S e p h a d e x G-200 c h r o m a t o g r a p h y o f an IgA m y e l o m a p r o t e i n . I n h i b i t i n g activity o f the w h o l e s e r u m was a b o u t 75%. H a t c h e d area = inhibiting activity, IgG (o • • ) . IgA (. . . . . . ), IgM ( - - - - - - . . - - ) , O.D. 280 ( ). .
.
.
.
.
.
216
(a) (b) 1.0-
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.
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.:
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110 00280
Fig. 7. a: P E G t e s t in 13 c o n t r o l s , 17 R F p o s i t i v e sera a n d 17 h e p a t i t i s sera. b: lack o f c o r r e l a t i o n b e t w e e n i n h i b i t o r y a c t i v i t y a n d P E G t e s t in t h e s a m e sera.
sera stored at --20°C and covering the assay range (Table 2). With IgG-coated polystyrene tubes as the solid phase, intra-assay variations were only slightly greater, but inter-assay reproducibility was unacceptably worse. This simpler procedure was therefore discarded. Correlation with differen t IC assay systems Agreement between different tests for IC varied according to disease. Two groups of sera previously assayed by Clq--BA were examined. -In the first, originating from 65 miscellaneous hospital patients, 30 were C l q positive and 35 negative. The IC-EIA results did not discriminate between the two, and only 3% were found positive (r --- 0.23, P < 0.01). -- The second group consisted of 26 sera from schistosomiasis patients, all C l q positive. Of these, 88% were positive in the IC-EIA (r = 0.47, P < 0.01). -PEG test and C4 test: 13 normal sera showing no significant % INH and 34 considered positive (17 RF and 17 HBs antigen positive sere ) were assayed. Although significantly different mean O.D. values were obtained in the two groups, no correlation was found between PEG and IC-EIA results (r = 0.33, P < 0.01), (Fig. 7a, b). Discrepant results were also obtained with the C4 test (r = 0.25, P < 0.01). DISCUSSION
This paper describes an IC assay which combines the specific reactivity of a c o m m o n biological material (polyclonal RF) with the advantage of EIA, and avoids use of radionucleides. It is suitable for laboratories possessing only non-isotopic routine equipment.
217
Choice of PRF Criteria of choice of the PRF used in this assay were operational: its reactivity towards aggregated IgG and in vitro formed IC showed it competed for both. Moreover, previous purification was not necessary as whole serum could be used in a high, non-interfering dilution. A possible drawback might by the presence of anti-Gm activity in the PRF. However, no anti-Gin activity was found when the PRF used in this assay was tested against a panel of known Gm allotypes, kindly provided by Dr. L. Rivat (INSERM, U. 78). Conversely, several other sera, with high RF activity could n o t be used in this assay, due to anti-GM or Inv activity. Absorption of endogenous RF: EDTA treatment This procedure was found to be useful before performing the IC assay for several reasons. The titrated quantity of PRF required would be doubled with serum samples containing, for example, 200 IU/ml. Furthermore, RF positive unabsorbed sera gave inhibition values consistently different from absorbed ones. Cowdery et al. (1975), in another test using polyclonal RF, found that endogenous RF interfered. However, the reverse was observed in IC assays using monoclonal RF (Luthra et al., 1975; Gabriel and Agnello, 1977). Some workers find it sufficient to treat sera with dithiothreitol (Lurhuma et al., 1976). We have also tried another technique, in which PRF was linked to the solid phase and thus able to fix IC w i t h o u t interference from endogenous RF. Free remaining PRF binding sites were quantified with an aggregated IgG-conjugate. Results (not shown) were quite satisfactory but the procedure required purification of PRF and the yield was too low. Differing results regarding interference by C1 of C l q have been reported (Gabriel and Agnello, 1977; Levinsky and Soothill, 1977). In our experiments the mean % INH of 30 hepatitis and 10 normal sera was similarly 6% lower when EDTA pretreatment was omitted. IC and aggregated IgG Several biological properties are shared by IC and aggregated IgG, particularly reactivity with RF (Henney and Stanworth, 1965; Hirose and Osler, 1965). Nevertheless, the similarity of conformational changes between heated IgG and antigen-antibody (Ag-Ab) aggregates has been questioned and some comparisons of various techniques of IC assay show no correlation between ability to detect aggregated IgG and IC in pathological sera (C. Vincent and J.P. Revillard, unpublished). The relative binding of aggregated IgG and IC to RF may vary with the Ag/Ab ratio in the latter (Cowdery et al., 1975), and, in addition to this, aggregated IgG is usually prepared from pooled polyclonal IgG preparations and possesses most of the iso- and allotypic specificities. Such an 'antigenic
218 display' is unlikely to occur with Ag/Ab complexes. For these reasons, the inhibition curve with aggregated IgG should not be interpreted as it currently is, as an IC sensitivity index, nor as a reference comparison between various techniques, but rather as an intra- and inter-assay control. For the same reasons, we have discarded aggregated IgG equivalents as reference values and calibrate our results in arbitrary % INH units. Specificity RFs may display specificity for certain IgG subclasses (Normansell and Young, 1975) and this may endow them with selective sensitivity. However, this is dependent on the quality of the PRF chosen, since some, in IC assay, are inhibited by all human IgG subclasses (Gabriel and Agnello, 1977). It is generally assumed that RF react with both native and altered IgG. Whether the substrate for preferential reactivity with the latter has special conformational determinants, or only polyvalent interaction with antigens shared by both (Normansell, 1971; Eisenberg, 1976), is still undecided. In the IC-EIA, monomeric IgG did not appreciably interfere. To inhibit RF Gabriel and Agnello (1977) found it necessary to add monomeric IgG concentrations 100 times greater than aggregated IgG but even such a high ratio may be insufficient. We do n o t find it useful to dilute all sera to a known final concentration as proposed by Luthra et al. (1975). The standard dilution used in this assay was adequate. IC prepared in vitro Size and Ag-Ab ratio have been shown to be determining factors in recognition of IC by RF (Lightfoot et al., 1969). This sensitivity to Ag-Ab ratio could be usefully exploited to identify the Ag involved in the complex (Delire and Masson, 1977). Maximum reactivity varies with the different RF used and shifts from equivalence (Cowdery et al., 1975) towards slight or marked antigen excess (Lurhuma et al., 1976; Gabriel and Agnello, 1977). Our study confirms this tendency of RF to detect IC formed mainly in Ag excess. Normal and pathologic human sera A puzzling problem in IC assay is the definition of a normal population. Several authors found significantly elevated activity in up to 14% of a blood donor population (Lurhuma et al., 1976). Several factors could account for this. For example, antiglobulin factors have been detected in normal subjects (Torrigiani and Roitt, 1967) and might be involved in formation of complexes, and IC resulting from mild inapparent infections or from uptake of food antigen have also been incriminated (Lurhuma et al., 1976). It is possible that IC are a physiological constant in healthy individuals. In the hepatitis cases we studied, an apparent inverse relationship was observed between the level of antigenemia and the occurrence of IC. Such a
219 relationship need confirmation on a larger series. In rheumatoid arthritis, we found no correlation between RF titre and IC levels, although there was an association between the occurrence of IC and RF (Fig. 5), as previously suggested (Lurhuma et al., 1976). RF production is presumably stimulated by Ag-Ab complexes. Chromatography o f IC positive sera The presence of Ca 2÷ during isolation procedures may prevent Ag-Ab complexes dissociating (Lurhuma et al., 1976). The chromatography buffer we used in this work did n o t contain Ca 2÷ and this may account for the diminished inhibiting activity of fractions as compared to whole serum. In the first chromatogram discussed above, inhibitory activity was located in excluded fractions corresponding to high molecular weight material, as reported by L u r h u m a et al. (1976) and L y e t et al. (1974); but in our second chromatogram, inhibitory material was unexpectedly detected also in lighter fractions. This may indicate that RF assay is able to detect IC as light as 8S (Gabriel and Agnello, 1977) and is sensitive to complexes produced in marked antigen excess. Correlation with other IC assays Previous attempts to compare results obtained with RF and C l q reagents have shown poor agreement (Winchester et al., 1971; Luthra et al., 1975; Gabriel and Agnello, 1977) and this suggests that they probably detect IC differing either in the size or in nature of the antibody or at least that different determinants in ICs are being quantified. Our own results show in most cases a similar lack of correlation. It thus seems unwarranted to consider any of these assays as a reference technique. Comparisons of their sensitivities probably have little significance and the contribution of each assay is rather to widen the panel of specificities detected. Conclusions (1) The advantage of the assay described is highly dependent upon the PRF used. The serum we used was exceptional in possessing all the required qualities. However, PRF are reagents of human origin and their supply is unpredictable. We have not so far succeeded in preparing xenogeneic antisera with properties comparable to RF. (2) The need to eliminate endogenous RF complicates the test. (3) Each PRF used has its own peculiarities so that results with different PRFs are hardly comparable. Reagents with more defined specificities are needed. RF-like antiglobulins prepared in animals may answer these objections. Techniques of production are reported (Milgrom and Witebsky, 1960; Abruzzo and Christian, 1961) and attempts have been made to use them in IC assay (Levinsky and Soothill, 1977). Suitable RF-like antiglobulins would render the assay described operative and provide abundant, exchangeable, standardizable reagents.
220
ACKNOWLEDGEMENTS
We are grateful to Professor Capron and to Professor Revillard, and to Doctors Santoro and Vincent who kindly supplied C l q assayed sera and to Professor Relyveld who provided tetanus toxoid solution. We are also grateful to L. Rivat for providing a Gm allotype panel. This work was supported by Contract No. 77.7.1875. DGRST, Paris. REFERENCES Abruzzo, J. and C. Christian, 1961, J. Exp. Med. 114, 791. Avrameas, S., 1969, Immunochemistry 6, 43. Casali, P., A. Bossus, N.A. Carpentier and P.H. Lambert, 1977, Clin. Exp. Immunol. 29, 342. Cowdery, J., P. Treadwell and R. Fritz, 1975, J. Immunol. 114, 5. Cuatrecasas, P., M. Wilchek and C.B. Anfinsen, 1968, Proe. Natl. Acad. Sci. U.S.A. 61, 636. Delire, M. and P.L. Masson, 1977, Clin. Exp. Immunol. 29, 385. Digeon, M., M. Laver, J. Riza and J.F. Bach, 1977, J. Immunol. Methods 16, 165. Eisenberg, R., 1976, Immunochemistry 13, 355. Ferrua, B., R. Maiolini and R. Masseyeff, 1979, J. Immunol. Methods 25, 49. Gabriel, A. and V. Agnello, 1977, J. Clin. Invest. 59, 990. Henney, C.S. and D.R. Stanworth, 1965, Immunology 9, 139. Hirose, S.I. and A.G. Osler, 1965, J. Immunol. 91,927. Jungfer, H., 1975, *rztl. Lab. 21, 80. Kapusta, M.A. and D. Halberstam, 1964, Biochim. Biophys. Acta 93, 657. Levinsky, R.J. and J.F. Soothill, 1977, Clin. Exp. Immunol. 29,428. Lightfoot, R.W., D.E. Drusin and C.L. Christian, 1969, Ann. N.Y. Acad. Sci. 168, 105. Lurhuma, A.Z., C.L. Cambiaso, P.L. Masson and J.F. Heremans, 1976, Clin. Exp. Immunol. 25, 212. Luthra, J.H., F.C. McDuffie, G.G. Hunder and E.A. Samayoa. 1975, J. Clin. Invest. 56, 458. Lyet, J.P. and D.E. Normansell, 1974, Immunochemistry 11,417. Maiolini, R. and R. Masseyeff, 1975, J. Immunol. Methods 8, 223. Maiolini, R., B. Ferrua, J.F. Quaranta, A. Pinoteau, L. Euller, G. Ziegler and R. Masseyeff, 1978, J. Immunol. Methods 20, 25. Milgrom, F. and E. Witebsky, 1960, Fed, Proc. 19, 197. Normansell, D.E., 1971, Immunochemistry 8, 593. Normansell, D.E. and C.W. Young, 1975, Immunochemistry 12, 187. Onyewotu, I., E.J. Holborow and G.D. Johnson, 1974, Nature 248, 156. Penttinen K., A. Vaheri and G. Myllyla, 1971, Clin. Exp. Immunol. 8, 389. Theophilopoulos, A., C. Wilson and F. Dixon, 1976, J. Clin. Invest. 57, 169. Torrigiani, G. and I.M. Roitt, 1967, Ann. Rheum. Dis. 26, 334. Wide, L., R. Axen and J. Porath, 1967, Immunochemistry 4, 381. Winchester, R.J., H.G. Kunkel and V. Agnello, 1971, J. Exp. Med. 134,286. Zubler, R.H. and P.H. Lambert, 1976, in: In Vitro Methods in Cell-mediated and Tumor Immunity, eds. B.R. Bloom and J.R. David (Academic Press, New York) p. 565.
