47

Journal of Immunological Methods, 138 (1991) 47-56 © 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 002217599100125B JIM05874

Sensitive E L I S A for interleukin-6 Detection of IL-6 in biological fluids: synovial fluids and sera M a a r t e n Helle, L e o n i e Boeije, Els de G r o o t , Alex de Vos a n d Lucien A a r d e n Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and Laboratory for Experimental and Clinical Immunology, University of Amsterdam, Amsterdam, The Netherlands (Received 28 August 1990, revised received 29 October 1990, accepted 12 December 1990)

A monoclonal antibody and an affinity purified polyclonal antibody, both raised against recombinant human IL-6, have been employed in an ELISA procedure to quantitate human IL-6. Both antibodies were very potent in neutralizing the biological activity of recombinant as well as natural human IL-6. The monoclonal antibody was used as the capture antibody whilst the polyclonal antibody, in biotinylated form, was used as the detecting antibody in combination with a streptavidin horseradish peroxidase conjugate and a signal amplification system. The detection limit for natural as well as recombinant IL-6 was 1 pg/ml. A good correlation was found between the ELISA and the B9 biological assay when IL-6 was measured in crude culture supematants, in synovial fluids of rheumatoid arthritis patients and in the sera of patients with diverse diseases. Immunoprecipitation of IL-6, produced by different cell types, such as monocytes, endothelial cells and smooth muscle cells or derived from biological fluids, such as the serum of a patient with septic shock or the synovial fluid of a rheumatoid arthritis patient, revealed in every case only molecules in the molecular weight range of 21,000-26,000. Key words: ELISA; Interleukin-6; Serum; Amplification

Introduction

IL-6 is a major mediator of the inflammatory response which is involved in the induction of acute phase proteins (Gauldie et al., 1987; Le and

Correspondence to: L.A. Aarden, c / o Publication Secretariat, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, P.O. Box 9406, 1006 AK Amsterdam, The Netherlands. Abbreviations: BT, biotin-tyramine; HRP, horseradish peroxidase; Mab, monoclonal antibody; NSS, normal sheep serum; PBS, phosphate-buffered saline; PT, PBS, 0.02% (v/v) Tween 20; PTG, PT, 0.2% gelatin; PTA, PT, 0.1% NAN3; RA, rheumatoid arthritis; (r/n)IL, recombinant/natural interleukin

Vilcek, 1989; Heinrich et al., 1990) and the induction of fever (Helle et al., 1988a). A correlation between raised in vivo levels of IL-6 and inflammatory processes has been demonstrated in the synovial fluid and serum of rheumatoid arthritis (RA) patients (Houssiau et al., 1988; Swaak et al., 1988), in the serum of patient with burns (Nijsten et al., 1987), in the serum and urine of recipients of kidney transplants before rejection (Van Oers et al., 1988) and in the serum of septic shock patients (Hack et al., 1989; Waage et al., 1989). The most sensitive assay for IL-6 is the one based on the proliferation of B cell hybridomas, such as B9 or 7TD1 cells (Aarden et al., 1985; Van Snick et al., 1987; Helle et al., 1988b). However,

48 biological assays are, in general, quite sensitive to interfering substances such as drugs which may be present in biological fluids or added to the culture media. Therefore, assays based on the detection of antigen may be preferable to biological assays. The rapidity of immunoassay procedures is another advantage since the biological assay takes about 3 days. A major challenge in developing ELISA procedures for cytokines is the sensitivity required. The measurement of IL-6 in serum with the B9 assay has revealed that in a number of clinical conditions quantification of levels lower than 100 p g / m l is required (Nijsten et al., 1987; Swaak et al., 1988; Van Oers et al., 1988). Until now only two immunoassays for IL-6, both with moderate sensitivity, have been described. An ELISA with a sensitivity of 50 p g / m l (Matsuda et al., 1988) and an RIA with a sensitivity of 100 p g / m l rIL-6 (Schindler et al., 1990). Neither of these assays has been shown to detect IL-6 in sera. We have developed an ELISA with improved sensitivity that permits the detection of IL-6 in biological fluids and which correlates with the biological assay.

Materials and methods

Cytokines rIL-6 was isolated from E. coli containing the cDNA for human IL-6 (Brakenhoff et al., 1987) and purified to homogeneity by affinity chromatography on Sepharose CL-4B (Pharmacia, Uppsala, Sweden) to which purified polyclonal goat anti-IL-6 antibodies were coupled. Subsequently, gel filtration chromatography on an AcA 54 column (Pharmacia) was used to remove multimers. The specific activity of the rIL-6 was 109 U / m g as measured by the B9 assay. Purified murine IL-6 was a kind gift from R. Nordan (NIH, Bethesda). Human r I L - l a was provided by P.T. Lomedico (Hoffmann-La Roche, Nutley, N J) and had a specific activity of 5 x 10 6 U / r a g . Interleukin-6 production Natural IL-6 was derived from monocytes. Monocytes were isolated from citrated peripheral blood obtained from healthy volunteers by means

of elutriation as described previously (De Boer and Roos, 1986). Monocyte derived IL-6 was produced by incubating 105 monocytes/ml in 0.5 or 1 ml well flat bottom plates (Nunc, Roskilde, Danmark) in Iscove's modified Dulbecco's medium (IMDM) supplemented with 5% fetal calf serum (FCS, selected LPS free from Bodinco, Alkmaar, NL), 5 × 10-s M 2-mercaptoethanol (ME), penicillin and streptomycin (P/S), in the absence or presence of titrations of LPS (E. coli 055 :B5, Sigma). After 24 h the supernatants were harvested and tested in the ELISA a n d / o r the B9 assay or were used for immunoprecipitation. Endothelial cells were isolated from human umbilical cords and cultured as reported previously (Willems et al., 1982). The cells were identified by their typical characteristics and a positive immunofluorescence staining with antibodies against Von Willebrand factor (Willems et al., 1982). Smooth muscle cells were isolated from human umbilical cord vein as described (Willems et al., 1982). The cells stained negatively with anti-Von Willebrand factor antibodies as judged by indirect immunofluorescence. The smooth muscle cells could be distinguished from fibroblasts by morphology and prostaglandin secretion pattern (Giltay et al., 1989). Both cell types were cultured in medium M 1 9 9 / R P M I (1:1), supplemented with 2 mM glutamine and 20% pooled human serum. For IL-6 production the cells were cultured overnight in 0.5 ml well flat bottom plates (Nunc), in the presence of rIL-1 (5 ng/ml).

Production of anti-IL-6 rnonoclonal antibodies B A L B / c mice were immunized i.m. with 10/zg purified rIL-6 emulsified in complete Freund's adjuvant. Three subsequent immunizations with 10 btg rIL-6 were carried out with incomplete Freund's adjuvant at intervals of 4 - 8 weeks. 4 days after the last boost, spleen cells were fused with non-imrnunoglobulin secreting mouse myeloma cells Sp2/0. Since the proliferation of hybridomas, just after fusion, is dependent on hybridoma growth factor ( = IL-6) (Aarden et al., 1985), 100 U / m l of IL-6 were added. To avoid interference with the anti-human anti-IL-6 producing hybridomas, IL-6 of murine origin was used. Anti-IL-6 secreting hybridomas were selected by ELISA or RIA as described below. After

49 establishing the hybridomas, IL-6 independent clones were selected, so the bulk cultures of antiIL-6-producing cells were free of IL-6.

ELISA for screening of anti-IL-6 producing hybridomas Purified rIL-6 was coated overnight (O/N) at room temperature (0.5 /~g/ml) in phosphatebuffered saline (PBS), 100 /~l/well) on flat-bottomed microtiter plates (Microelisa, M129B, Dynatech, Plochingen, F.R.G.). The plates were washed with PBS, 0.02% (v/v) Tween 20 (PT, Baker Chemical Co., Philipsburg, NJ) and 1/2 dilutions of hybridoma culture supernatant in PT supplemented with 0.2% gelatine (PTG) were incubated for 2 h. After washing, the plate was incubated with HRP-conjugated rat monoclonal 226 anti-mouse x light chain in PTG (2 /~g/ml) for 1 h. Subsequently after washing the bound peroxidase was detected as described for the IL-6 ELISA.

RIA for screening of anti-IL-6-producing hybridomas Partially purified goat anti-murine Ig antibodies (GM17, this institute) were coupled to CNBractivated Sepharose CL-4B (Pharmacia). This Sepharose was suspended in PBS, 0.1% Tween 20, 0.1% NaN 3 (PTA) (10 mg/ml) to which the hybridoma supernatants were added in the presence of 125I-rIL-6. After head-over-head incubation for 6 h and extensive washing with PT, the bound radioactivity was measured by gammacounter. rIL-6 was labeled with 125I (Amersham, U.K.) using the Iodogen method (Pierce Chemical Co., Rockford, IL, U.S.A.) (Fraker and Speck, 1978) to a specific activity of + 4 × 10 7 cpm//~g.

Purification of monoclonal antibodies Hybridomas were grown in roller bottles in IMDM, supplemented with ME, P / S and 2% FCS. Supernatants were harvested from full-grown bulk cultures and concentrated 20-50-fold by means of a hollow-fiber dialyzer (Fresenius, Bad Homburg, F.R.G.). The concentrate obtained was dialysed against 3 M NaC1, 1.5 M glycine-NaOH, pH 8.9 (buffer A). Subsequently the dialysate was applied to a protein A-Sepharose CL-4B (Pharmacia) col-

umn, equilibrated in buffer A. Since all mAbs were of the IgG1 subclass, bound immunoglobulin was eluted with a solution of 0.1 M citric acid pH 6, following the manufacturer's instructions. Purity was checked by SDS-PAGE. Antibody concentration was determined by absorbance at 280 nm. The eluates were dialysed against PBS and frozen in aliquots at - 20 ° C.

Production and purification of polyclonal antibodies IL-6 specific polyclonal antiserum was obtained by immunizing a goat and a sheep (both i.m.), with 5 × 108 U purified rIL-6 (500/xg) mixed with an equal volume of complete Freund's adjuvant. Subsequently, each month a booster was given with 108 U rIL-6 (100 /~g) in incomplete Freund's adjuvant. 7 days after each booster injection serum was collected. Sheep and goat anti-IL-6 antibodies were purified by affinity chromatography, basically as described by Hack et al. (1981), using Sepharose CL-4B (Pharmacia) to which purified rIL-6 was coupled. Elution was performed with 0.1 M glycine-HC1 pH 2.5. Biotinylation of antibodies, using LC-biotin-N-hydroxysuccinimide ester (Pierce) was performed according to the manufacturer's instructions.

IL-6 ELISA The purified monoclonal antibody (mAb CLB.IL-6/8 (mAb8) or mAb CLB.IL-6/16 (mAb16)) was coated O / N at room temperature (1.5 # g / m l in PBS, 100/~l/well) on flat-bottomed microtiter plates (Microelisa, M129B, Dynatech, Plochingen, F.R.G.). All subsequent incubations were in 100 ~1 volumes at room temperature. The plates were washed (3 × PT) and serial dilutions of IL-6 containing samples diluted in PTG were added to the plates for 2 h. Without further washing an excess of biotinylated sheep antibodies were added in 10 #1 PTG (final concentration 0.6 /~g/ml) and incubated for another 1 h. If serum samples were to be tested, 1% normal sheep serum was added during the biotin-conjugate incubation to prevent false positive reactivity (heat inactivation of sera was not required). Plates were washed (3 × PT) and incubated with streptavidinhorseradish peroxidase (strep-HRP, Amersham), 1/1000 diluted in PTG, for 0.5 h, washed and

50 developed with a solution of 100 /~g/ml of 3,5,3',5'-tetramethylbenzidine (Merck, F.R.G.) with 0.003% H202 in 0.11 M sodium acetate, p H 5.5 (100/~l/well). The reaction was stopped by the addition of an equal volume of 2 M H2SO 4 to the wells. Plates were read at 450 nm in a Titertek Multiskan reader. Reactivity in the ELISA was compared to a titration of a standard preparation of natural IL-6 and expressed in p g / m l . If amplification was employed, using catalyzed reporter deposition (CARD) (Bobrow et al., 1989), an incubation of 15 min with biotin-tyramine (BT) (1/1000 of BT stock solution in 50 mM Tris pH 8.0 with 0.003% H202) was added after the strepH R P incubation and extensive washing (6 × PT). This was then followed after washing (6 × PT) by a second strep-HRP step of 15 rain and washing and development as described. The BT stock solution was prepared as described (Bobrow et al., 1989). Briefly, a solution of tyramine (Sigma), 40 m g / m l dimethyl-sulfoxide (DMSO, Baker Chemicals) was mixed with a solution of biotin-N-hydroxysuccinimide (Sigma), 100 m g / m l DMSO, overnight at room temperature.

Immunoprecipitation and blotting of IL-6 Sepharose CL-4B, to which affinity purified polyclonal goat anti-IL-6 IgG was coupled, was incubated with supernatants of monocytes, endothelial cells or smooth muscle cells or a synovial fluid sample from an RA patient or serum from a septic shock patient. Incubations were overnight at room temperature in 500 #1 PTA supplemented with 5% FCS (PTA-5% FCS) in Eppendorf tubes. Subsequently the Sepharose was washed twice with PTA-0.5% FCS and once with PBS. To elute Sepharose bound protein, the pellet was then boiled for 5 min in sample mix (50 m M Tris p H 6.8, 10 mM 1,4-dithioerythritol (Merck), 10% (v/v) glycerol, 1% ( w / v ) SDS) and the samples analysed by SDS-PAGE on 8-25% mini-gels (Pharmacia) followed by blotting according to the manufacturers description. The blotted IL-6 was visualized by means of biotinylated goat anti-IL-6 and streptavidin alkaline-phosphatase (Amersham), followed by a colour reaction with bromo chloro indolyl p h o s o p h a t e / n i t r o blue tetrazolium (BCIP/ N B T , Promega).

IL-6 biological assay The IL-6 assay was performed with B9 cells as previously described (Helle et al., 1988b). 1 p g / m l ( = 1 U / m l ) of IL-6 leads to half-maximal thymidine incorporation in B9 cells. The synovial fluid or serum samples to be tested were heated for 30 rain at 56 ° C to eliminate toxicity for the B9 cells (no heat inactivation was required when tested in the ELISA). IL-6 activity was not affected by this procedure. Inhibition of IL-6 activity by antibodies was tested by the addition of a two-fold titration of antibodies to 2 U / m l ( = 2 p g / m l ) of IL-6. Directly after mixing the IL-6 with the antibodies, B9 cells were added and the assay continued as usual. The resulting B9 cell proliferation was compared with a standard curve in order to derive the amount of free IL-6. As the antigen concentration is as low as 10 -13 M the dissociation constant (KD) can be estimated as the concentration of antibody required to obtain 50% inhibition. It should be realized that calculating affinity from the inhibiting capacity yields a minimum affinity since only the neutralizing antibodies are taken into account.

Results

Antibodies to IL-6 In order to detect IL-6 in the picomolar range by ELISA it would be beneficial to use antibodies with a K D in the picomolar range. In addition we decided to use antibodies which are able to neutralize the biological activity of IL-6 in the expectation that this would minimize the risk of detecting degradation products of IL-6 which are not biologically active. Finally the antibodies should not discriminate between recombinant IL-6 and natural IL-6 from different sources. In total 16 monoclonal antibodies to IL-6 were obtained from seven different fusions. From these antibodies, two with the above properties were selected. These antibodies, termed mAB CLB.IL-6/8 (mAb8) and CLB.IL-6/16 (mAbl6), were shown to be neutralizing antibodies. When tested in the B9 assay 50% inhibition of the biological activity of natural IL-6 was achieved at concentrations around 1 n g / m l or 30 n g / m l respectively for the two antibodies (Fig. 1). From this KD values of about 6 × 10 -12 M and 2 × 10-10 M respectively could be calculated.

51 100

and the polyclonal affinity purified sheep antibody as the detector antibody. Using this combination an IL-6 concentration of 30 p g / m l gave rise to a signal which was twice the background for natural as well as for recombinant IL-6 (Fig. 2).

80 E 0 ~G .13 _E E

6O

40"

o~ 20"

0 0.1

1

anti-lL6

10

100

(ng/ml)

Fig. 1. Inhibition of biological activity (B9 assay) of IL-6. A constant a m o u n t of natural IL-6 (2 p g / m l ) was incubated with various concentrations of mAb8 ( o o ) or m A b l 6 (O e). Background proliferation was 1300 cpm, maximal proliferation with 2 p g / m l niL-6 was 11000 cpm.

Both antibodies were able to neutralize recombinant and natural (monocyte or endothelial cell derived) IL-6 to the same extent (not shown). Affinity-purified polyclonal antibodies from different sources were selected for use as a second antibody. Goat as well as sheep antisera were purified by affinity chromatography on an IL-6 Sepharose column. Elution yielded a goat antibody preparation with a K D in the order of 6 × 10 -]2 M (50% inhibition at a concentration of 1 n g / m l ) and a sheep antibody preparation with a K D in the order of 1.2 x 10 -12 (50% inhibition at a concentration of 0.2 ng/ml). Both antibody preparations inhibited recombinant as well as natural IL-6 although slightly less efficiently in the case of natural IL-6 (data not shown). Subsequent experiments revealed that the affinity purified goat and sheep antibody preparations behaved in similar fashion in the ELISA. IL-6 E L I S A Antibodies were tested at various concentrations using either the monoclonal or the polyclonal antibody for coating the wells. It turned out that the most sensitive combination was obtained using a monoclonal antibody (mAb8) as capture

Correlation between the E L I S A and the bioassay Using natural IL-6 as a standard, IL-6 levels were measured in crude supernatants of resting and LPS-stimulated monocytes. It transpired that the ELISA and B9 assays showed a good correlation (Fig. 3A). When these experiments were extended using synovial fluids from rheumatoid arthritis (RA) patients a good correlation between the ELISA and B9 assays was again obtained (Fig. 3B). Measurement of IL-6 in sera Since the levels of IL-6 in most rheumatoid synovial fluids are above 1000 p g / m l , their quantification does not present a problem. However, the measurement of serum IL-6 levels over the range 10-1000 p g / m l is required in several disease states and the sensitivity of the ELISA had to

8 1.0 0 tO q
100 p g / m l ) in the ELISA with some appearing to contain more than 13,000 p g / m l of IL-6. The addition of normal sheep serum during the incubation with the affinity purified sheep antibodies abolished the false positive reactivity almost completely (Fig. 4), whereas neither the IL-6 standard nor true positive sera were influenced by the normal sheep serum. Comparing mAb8 with mAb16 at this stage it was obvious that both mAbs gave rise to a similar sensitivity, although m A b l 6 gave a lower number of false positive reactions with the sera (not shown). Thus the sensitivity of the ELISA, combining mAbl6 with the amplification system, was 1 p g / m l (Fig. 5). Using this improved ELISA, serum samples (n = 25) from patients with various inflammatory diseases were tested and the results of the ELISA procedure found to correlate very well with the B9 assay (Fig. 6).

Analysis of IL-6 from various sources by Western blotting It has been reported that a significant proportion of the IL-6 in biological fluids or in the supernatants of endothelial cells has an aberrant molecular weight. After immunopurification and

Discussion

Using the present ELISA procedure with m A b l 6 as the capture antibody and biotinylated affinity purified sheep antibodies during the detection step combined with H R P labeled streptavidin and catalyzed reporter deposition (Bobrow et al., 1989), it is possible to quantitate IL-6 with a detection limit of 1 p g / m l (Fig. 5). The amount of biologically active IL-6, whether measured in crude culture supernatants or in biological fluids, such as synovial fluids or sera, correlated with the amount of IL-6 detected in this ELISA (Fig. 3 and Fig. 6). There was no evidence that any cytokines other than IL-6 or that inactive forms of IL-6 were being recognized in this ELISA. The latter has been confirmed by mapping the antigenic site using IL-6 deletion variants which revealed that mAb8 and m A b l 6 recognize distinct structural determinants that are destroyed by deletions causing inactivation of the molecule (Brakenhoff et al., 1990). Moreover, there were no differences in reactivity between recombinant IL-6 and natural IL-6 (Fig. 2). Clearly it is important to be aware of the possibility that recombinant material might be recognized differentially such has been described in an IL-6 RIA (Schindler et al., 1990). Recombinant proteins often tend to aggregate during purification, which might influence reactivity in an immunological assay. In order to obtain pure monomeric rlL-6, gelfiltration was used as the final purification step for rlL-6. Its purity was

54

A

B

Mr 976642-31-21-14--

1

2

3

4

5

6

7

8

9

10

Fig. 7. Immunoprecipitation and Western blotting of IL-6 from several sources. Culture supernatants from monocytes (lane 1), smooth muscle cells (lane 2) and endothelial cells (lane 3) are depicted in panel A. Panel B shows precipitations from sera of three different patients with septic shock (lanes 5-7): one negative serum (lane 5) and two different positive sera (lanes 6-7), or from synovial fluids (SF) from R A patients (lanes 9-10): a negative SF (lane 9) and a positive SF (lane 10). Lane 4 is derived from a supernatant of LPS stimulated monocytes and lane 8 is identical to lane 4 but mixed with a normal control h u m a n serum.

checked in a symmetrical sandwich ELISA with m A b l 6 , in which the purified monomeric material was not reactive, whereas the aggregated form was (data not shown). When we first encountered positive reactions in almost half of the normal control sera which had been negative in the biological assay, we carried out a control ELISA using as a coat a monoclonal I g G ~ type anti-TNFa antibody combined with the sheep anti IL-6 conjugate. In this system none of the sera were positive suggesting that the material detected in normal sera and not active in the bioassay was antigenically related to IL-6. Subsequent experimentation however led us to conclude that the positivity was due to h u m a n antibodies that recognize a common determinant on murine and sheep Ig; analysis by gel filtration revealed that the ELISA reactive material migrated with a Mw of 160 kDa. Moreover, the fact that the addition of normal sheep serum to the biotin

conjugate eliminated the false positive reactions and settled this question (Fig. 4). However we were left with the results using the anti T N F a coating. We then used as a coat a variety of unrelated, protein A purified monoclonal antibodies, all of the same isotype (~,l-x) and tested all the IL-6 negative control sera (also used in Fig. 4). We observed that the different coatings combined with the affinity purified sheep anti IL-6 resulted in reactivities which ranged from completely negative to strongly positive. We have no explanation for this p h e n o m e n o n but it should be kept in mind that even an isotype-matched control coating could yield misleading results. IL-6 is a 21-26 k D a glycoprotein. As such it has been purified from monocytes and fibroblasts. Some others have shown that immunoaffinity purification followed by Western blotting of IL-6 derived from biological fluids or culture supernatants revealed an additional 45 k D a molecule.

55 This m o l e c u l e has b e e n p u r i f i e d f r o m sera ( F o n g et al., 1989; J a b l o n s et al., 1989), f r o m the synovial fluid o f R A p a t i e n t s (Bhardwaj et al., 1989) a n d f r o m e n d o t h e l i a l cell s u p e r n a t a n t s ( M a y et al., 1989). However, in a n o t h e r s t u d y it was s h o w n that n o n s t i m u l a t e d e n d o t h e l i a l cells p r o d u c e d as m u c h 45 k D a p r o t e i n s as IL-1 s t i m u l a t e d e n d o thelial cells, whereas o n l y in the IL-1 s t i m u l a t e d cells could IL-6 m R N A b e d e t e c t e d a n d biologically active IL-6 b e p r o d u c e d ( L o p p n o w a n d L i b b y , 1989). I n a d d i t i o n , the affinity p u r i f i e d g o a t p o l y c l o n a l a n t i b o d i e s d e s c r i b e d here were a b l e to b l o c k c o m p l e t e l y the b i o l o g i c a l activity of c r u d e IL-6 p r e p a r a t i o n s d e r i v e d f r o m a n y source, whereas they d i d n o t p r e c i p i t a t e a n y 45 k D a p r o tein (Fig. 7). This further indicates that there is n o evidence for a n y IL-6-1ike biological activity associated with this 45 k D a molecule. It is p o s s i b l e that a 45 k D a p r o t e i n is constitutively p r o d u c e d b y e n d o t h e l i a l cells ( L o p p n o w a n d L i b b y , 1989) a n d is antigenically r e l a t e d to IL-6. T h e E L I S A d e s c r i b e d in this p a p e r is a l m o s t as sensitive as the b i o l o g i c a l assay. G i v e n the correlation b e t w e e n the two assays a n d the s p e e d of the E L I S A we feel t h a t it c a n r e p l a c e the b i o a s s a y in m o s t instance. A n e x t r a a d v a n t a g e is that drugs or o t h e r agents which i n h i b i t the b i o a s s a y will n o t interfere w i t h results o b t a i n e d with the E L I S A procedure.

Acknowledgements T h e a u t h o r s are grateful to E d w a r d K n o l for isolating the m o n o c y t e s , to Betty H a k k e r t a n d J a e n e t t e R e n t e n a a r for isolating the e n d o t h e l i a l cells, a n d to H e r m - J a n B r i n k m a n for isolating the s m o o t h muscle cells. W e also wish to t h a n k T o m S w a a k for p r o v i d i n g the synovial fluid s p e c i m e n s a n d H a n H u i s m a n , Cees van K o o t e n a n d Paul P a r r e n for their c o m m e n t s on the m a n u s c r i p t . This s t u d y was s u p p o r t e d b y the N e t h e r l a n d s L e a g u e against R h e u m a t i s m ( G r a n t 8 8 / C R / 0 8 9 ) .

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Sensitive ELISA for interleukin-6. Detection of IL-6 in biological fluids: synovial fluids and sera.

A monoclonal antibody and an affinity purified polyclonal antibody, both raised against recombinant human IL-6, have been employed in an ELISA procedu...
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