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Measurement of biologically active interleukin-1 by a soluble receptor binding assay
185,206-212
ANALYTICALBIOCHEMISTRY
(1990)
Measurement of Biologically Active Interleukin-1 by a Soluble Receptor Binding Assay Frank Riske,’ Richard Chizzonite, Perla Nunes, and Alvin S. Stern* Departments Hoffmann-La
Received
Molecular Genetics and *Protein Biochemistry, Roche Inc., Nutky, New Jersey 0711 O-l 199
of
September
26,1989
A soluble receptor binding assay has been developed for measuring human interleukin-la (IL-la), human IL- 18, and mouse IL- la. The assay is based on a competition between unlabeled IL-1 and ‘a51-labeled mouse recombinant IL- la for binding to soluble IL- 1 receptor prepared from mouse EL-4 cells. The assay measures only biologically active IL- 1 folded in its native conformation. The ratio of human IL-la! to human IL-18 can be measured in the same sample by a pretreatment step which removes human IL- 1~9from samples prior to assay. This technique has been used to monitor the purification of recombinant IL- 1, and may be utilized to specifically and accurately measure bioactive IL-1 in human serum and cell culture supernatants. o 1990 Academic
Press,
Inc.
Interleukin-1 (IL-l): a product of macrophages and other cell types, was originally described as a lymphokine capable of stimulating murine thymic cells following mitogenic activation (1). However, in the last several years it has become apparent that in addition to the augmentation of cell-mediated and humoral responses (2), IL-l is also involved in proinflammatory responses and has catabolic effects on certain tissues. This lymphokine can stimulate fibroblast and neutrophil proliferation, induce muscle proteolysis and the release of prostaglandin Ez, stimulate the synthesis and production of collagenase and hepatic acute-phase protein, and induce fever i To whom correspondence should be addressed. ’ Abbreviations used: IL-l, interleukin-1; rIL-1, recombinant interleukin-1; PBS, Dulhecco’s phosphate-buffered saline (without calcium chloride or magnesium chloride); CHAPS, 3-[ (3-cholamidopropyl)dimethylammoniol-1-propanesulfonate; PEG, polyethylene glycol; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; PMSF, phenylmethylsulfonyl fluoride; PBS-Tw, PBS containing 0.5 mM NaCl, 0.05% Tween-20; DTT, dithiothreitol. 206
Roche Research Center,
(3). IL-l may also be involved in some pathological conditions associated with immune and inflammatory responses such as joint erosion in rheumatoid arthritis. Therefore, a method is needed to detect and measure bioactive IL-l, especially in tissue or blood fluids which contain components that interfere with conventional bioassays. The recent cloning of IL-l genes and advances in the purification of the IL-l proteins (4-8) have had a major impact on the study of IL-l. Two species of IL-l have been identified and have been distinguished as human IL-la! and human IL-l& Although there is no more than 25% sequence homology between the two proteins, both human IL-la and human IL-l/3 have similar, if not identical, activities. The expression of the two genes in Escherichia coli provided sufficient material for the development of assays for measuring these cytokines. Currently, IL-l is measured by immunoassay (9-16) or bioassay (1,17-20). Immunological assays employ anti-IL-l antibodies to capture and measure IL-l. In bioassays, a measurable response by a cell line to IL-l is equated with lymphokine amount. Unfortunately, both methodologies suffer from drawbacks. Bioassays generally require several days to complete and are nonspecific since the cells respond to other lymphokines and substances found in serum or bodily fluids. Immunoassays measure both biologically active and inactive molecules. This study describes the development of a soluble receptor assay for the quantification of biologically active IL-l. The assay is based on a competition between radiolabeled mouse rIL-la and IL-l in the sample for binding to soluble IL-l receptor. The assay is rapid and simple and can be used to detect and measure biologically active human IL-la, human IL-l/?, and mouse IL-lo. MATERIALS AND METHODS Preparation of Soluble IL-l Receptor
Mouse EL-4.IL-2 thymoma cells (ATCC No. TIB181) were grown in a l-liter bioreactor containing 5% Copyright 0 1990 All rights of reproduction
by
0003-2697/90 $3.00 Academic Press, Inc. in any form reserved.
SOLUBLE
RECEPTOR
BINDING
horse serum in Iscove’s modified Dulbecco’s medium. The cells were harvested, washed three times with cold (4°C) Dulbecco’s phosphate-buffered saline without calcium chloride or magnesium chloride (PBS), and solubilized by resuspension in 5X vol of 8 mM CHAPS, 20 mM NaPO,, 5 mM EDTA, 40 pg/ml PMSF, and 0.05% NaN3 for 1 h at 4°C (21). The cell detergent suspension was stirred for 60 min at 4°C and insoluble material was removed by centrifugation at 120,OOOgfor 1 h. The supernatant solution was removed, divided into 3-ml aliquots, and stored at -70°C. The soluble preparation is stable for at least 3 months when stored at -70°C.
Proteins
and Antibodies
Human rIL-la, human rIL-l& and mouse rIL-la were purified as previously described (8) except that the proteins were prepared in 25 mM Tris-HCl, pH 8.1, containing 0.4 M NaCl. The purified proteins were tested in the mouse DlO.G4.1 cell proliferation assay (20) to determine biological activity. Human rIL-la, human rIL-l@, and mouse rIL-la had specific activities of 5.7 X lOa, 6.1 X 107, and 5.1 X 10’ units/mg, respectively. Antibodies specific for human rIL-la and human rILl/3 were produced by immunizing goats with 100 pg of human rIL-1 protein mixed in a 1:l ratio with Freund’s complete adjuvant. The goats were subsequently injected every 28 days with 10 pg of human IL-l mixed 1:l with Freund’s incomplete adjuvant. Serum was collected every 14 days after the third immunization, filtered, and stored at -70°C. Alternately, the serum was diluted fourfold with PBS containing 0.5 M NaCl, 0.05% Tween20 (PBS-Tw), filtered, and applied to affinity columns containing human rIL-la or human rIL-l/3 immobilized on a silica matrix (22). The columns were washed extensively with PBS-Tw and PBS, and antibodies specific for human rIL-1 were eluted with 0.2 M acetic acid, 0.2 M NaCl, pH 2.8, and immediately neutralized with 3 M Tris. The anti-human IL-l activity of the purified antibody preparations was evaluated by determining the concentration of antibody necessary to block the lz51rIL-1 (20 fmol) binding to EL-4 cells. Recombinant human interferon-a-2a, interleukin-2, and interferon-y were produced at Hoffmann-La Roche, Inc.
Radiolabeling
of Mouse rIL-la
and Human
rIL-la
Human rIL-la (15 pg) and mouse rIL-la (5.4 pg) were radiolabeled with 1.0 mCi Na1251(Amersham Corp., Arlington Heights, IL) as previously described (23). Briefly, IL-l was diluted with 25 mM Tris-HCl, 0.4 M NaCl, pH 7.5, to a volume of 70 ~1. Enzymobead reagent (50 ~1 in distilled water; Bio-Rad Laboratories), 0.2 M sodium phosphate buffer, pH 7.5 (50 pl), and 1.0 mCi Na1251were added to the IL-l. The reaction was initiated at room temperature with 1% ~-D-glucose and termi-
207
ASSAY FOR INTERLEUKIN-1
nated after 20 min. The labeled protein was separated on a 1 X 20-cm Bio-Gel P-6DG column equilibrated with 25 mM Tris-HCl buffer, containing 0.4 M NaCl, pH 7.5. The peak fractions of ‘251-IL-1 were pooled and diluted to a concentration of 1 X lo6 cpm/ml with 25 mM TrisHCl containing 1 mM EDTA and 1% BSA, pH 7.5. Each of the radiolabeled IL-l proteins contained a single labeled polypeptide of approximately 17 kDa which comigrated with unlabeled IL-l when analyzed by SDSPAGE and autoradiography. The labeled proteins are fully active in the DlO.G4.1 mouse T-cell proliferation assay when compared to the unlabeled proteins. The specific activities in each case ranged from 22 to 57 &i/ pg protein. The radioligand preparations were also tested for binding activity by increasing the number of EL-4 cells which were added to a fixed concentration of labeled IL-l. In each case, the maximum amount of radioligand bound by the EL-4 cells was approximately 80%. Furthermore, the binding of the radioligand to the cell surface binding site occurred in a specific and saturable manner and could be inhibited by adding 50 nM unlabeled human rIL-la, human rIL-lb, or mouse rIL-la.
Soluble Receptor Binding
Assay for Measuring
IL-l
The receptor binding assay is based on competition between IL-l in a sample and radiolabeled mouse rILla for soluble IL-l receptor. Solutions of human rIL-la (in 50 mM phosphate buffer with 0.1 M NaCl, pH 6.5), human rIL-1B (in 10 mM phosphate buffer with 0.45 mM DTT, pH 7.2), and mouse rIL-la (in the same buffer as human rIL-la) were diluted with binding buffer (PBS, 8 mM CHAPS, 0.25 M NaCl, 5 mM EDTA, and 0.1% BSA) to concentrations of 58.8, 470, and 1.6 nM, respectively. These concentrations of IL-l were sufficient for complete inhibition of 1251-labeledmouse rIL-la binding to receptor. The initial concentration of each IL-l was serial diluted in binding buffer to set up nine additional concentrations for quantification in the assay. Twentyfive microliters from the ten samples was pipetted in duplicate into 12 X 75-mm tubes. In addition, two tubes each were set up to determine total and nonspecific binding of 1251-labeledmouse rIL-la to the soluble receptor. Total binding was determined by adding 25 ~1of binding buffer to the tubes while nonspecific binding was measured by adding 50 to 500 nM unlabeled IL-1 to the tubes. Radiolabeled mouse rIL-la (25 ~1 containing 1.5 X lo4 cpm) was then added to each tube. Soluble IL-l receptor (10 ccl), diluted lo-fold in binding buffer, was added and the tubes were incubated for 1 h at 37°C. Fifty microliters of a human IgG solution (14 mg/ml in PBS) followed by 3 ml of 10% polyethylene glycol (PEG) 6000 in PBS (Sigma Chemical Co.) was added and the tubes incubated for an additional 30 min at 4°C. The contents of each tube were vacuum filtered through Whatman GF/C filters (Fisher Scientific). The filters were washed
208
RISKE
twice with 4 ml of the 10% PEG solution and counted in an LKB 1282 Compugamma gamma counter. Activity was determined as percentage inhibition of specific binding of labeled mouse rIL-la by IL-l in the test sample. The percentage inhibition was calculated by subtracting from 1 the difference between sample binding and nonspecific binding (cpm) divided by the difference between total binding and nonspecific binding (cpm). This number was then multiplied by 100 to obtain the percentage inhibition value. The percentage inhibition for each sample was plotted as a function of the concentration of IL-l expressed in mass (pmol) or biological activity (units). Units of IL-l activity were determined using the DlO.G4.1 cell proliferation assay (20). IL-l samples were also tested in Iscove’s modified Dulbecco’s medium with 10% fetal bovine serum and human sera (Gibco Labs, Grand Island, NY) diluted 1:l with PBS. Sample Pretreatment IL-l a and Human
to Measure Mixtures IL-l /3
of Human
To selectively measure human IL-la! when present along with human IL-l& samples were pretreated with goat anti-human IL-l@ antibody preparations. In 1.5-ml Eppendorf tubes, anti-human IL-lfl antibody was added (50 pg in 25 ~1) to 250 ~1 of a 2% suspension of Pansorbin (Calbiochem, La Jolla, CA) in PBS-Tw. An additional 200 ~1 of PBS-Tw was added and the tubes were gently mixed for 2 h at room temperature. Following centrifugation at 2000g for 3 min, the supernatant solutions were aspirated and the pellets washed three times with 1 ml of PBS-Tw. The tubes were firmly tapped to break the pellets of Pansorbin saturated with anti-human IL-l@ antibody. Duplicate samples of unknown or standards were added (25 ~1) to the pellets. After vortexing, the tubes were placed on a mixer overnight at 4”C, or 2 h at room temperature. Tubes were then centrifuged at 2000g for 3 min. The supernatant solution (25 ~1) was removed from each and placed in a 12 X 75 mm-tube, and the IL1 concentration was measured by the soluble receptor assay. Purification
of Mouse rIL-1 a
Mouse rIL-la was extracted from an E. coli clone containing an expression plasmid encoding for the protein (5). One hundred grams of a frozen E. cob paste was suspended in 500 ml of 30 mM Tris-HCl (pH 8.0), 5 mM EDTA, and 1 mM PMSF. The cells were lysed with a Manton-Gaulin cell homogenizer and the lysate was centrifuged at 27,000g for 30 min at 4°C. Solid ammonium sulfate was added to the supernatant solution to 60% saturation and stirred overnight at 4°C. The resultant pellets were dissolved in 100 ml PBS containing 0.4 M NaCl. This solution was applied directly to a column
ET
AL.
“1
. / ..... .-F.--r.
o!&wd--./. 1CP
10-Z
10"
100
10'
lo2
IL-1 (pmdhssay) FIG. 1. Measurement of mouse rIL-la (a), human rIL-la human rIL-I@ (A) by soluble receptor binding assay. Serial dilutions starting at 0.235,8.8, and 70.6 pmol of the respective were added to duplicate tubes and the assay was performed scribed under Materials and Methods.
(B), and twofold ligands as de-
(12 X 120 cm) of Sephacryl S-200 (Pharmacia LKB Biotechnology, Inc.) equilibrated in PBS containing 0.4 M NaCl and run at a flow rate of 240 ml/h. Aliquots were assayed by the receptor binding assay as described above and fractions with the highest specific activity were pooled. The protein solution was dialyzed (Spectra/Par 1, Fisher Scientific) against 100 vol 25 mM Tris-HCl, pH 8.1, and applied to a column (5 X 100 cm) of DE-53 cellulose (Whatman Biosystems Inc.) equilibrated with 25 mM Tris-HCl, pH 8.1. The mouse rIL-la was eluted with a linear gradient of 0 to 800 mM NaCl (in 25 mM Tris-HCl, pH 8.1) at a flow rate of 240 ml/h. Aliquots were assayed in the receptor binding assay as described above. RESULTS
The measurement of IL-l is based on competition between radiolabeled mouse rIL-la and unlabeled IL-l for binding to soluble mouse IL-l receptor. Total counts of 1251-labeled mouse rIL-lcr bound to receptor ranged between 2500 and 3000 cpm; nonspecific binding ranged between 7 to 10% of these counts. Mouse rIL-la, human rIL-la, and human rIL-l/3 at 0.235, 8.8, and 70.6 pmol, respectively, effectively inhibited 100% of 1251-mouse rIL-la (1.5 X lo4 cpm) binding to soluble receptor (Fig. 1). The linear range of the assays was 5 to 120 fmol for mouse IL-la, 60 fmol to 1.1 pmol for human IL-la, and 2.0 to 35 pmol for human IL-l@. Fifty percent displacement of total binding (I&) occurred with additions of 22 fmol, 234 fmol, and 6.0 pmol of mouse rIL-lcr, human
SOLUBLE
RECEPTOR
BINDING
ASSAY
FOR 120
INTERLEUKIN-1
209
1
%o05 p760g+6o 25 z
/ /* 0I e-04 :’ lo’
,// / ’
E 5" -I
,=ci=-. .,
IO2
40
103
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..... I
10'
106
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IL-1 BIOACWITY
FIG. 2. Measurement of mouse rIL-la (O), human rIL-la (m), and human rIL-10 (A) by soluble receptor binding assay. Protocol was exactly the same as that described in Fig. 1 except that IL-l is expressed in biological units rather than picomoles of protein. Duplicate samples were assayed.
FIG. 3. Measurement of human rIL-lcz in binding buffer (a), human sera diluted 1:l with PBS (@ , and in Iscove’s modified Dulbecco’s medium with 10% fetal bovine serum (A). Serial twofold dilutions of human rIL-la (8.8 pmol to 17.2 fmol) were tested. Protocol was that described in Fig. 1.
rIL-la, and human IL-l& respectively (Fig. 1). Expressing the I&, in terms of biological units rather then in concentration resulted in values for human rIL-lcu and human rIL-la of 2246 and 7060 units, respectively, while mouse rIL-la exhibited a much lower IC& of 191 units (Fig. 2). The sensitivity of the receptor assay for human rIL-la and human rIL-l@ was not increased by using ‘251-labeled human rIL-la in place of 1251-labeledmouse rIL-la in the assay (data not shown). The receptor binding assay specifically measures the interaction of IL-1 with its receptor. Other lymphokines such as interferon-a, interferon-y, and interleukin-2 do not compete with ‘251-labeled mouse rIL-1cY for receptor binding at a concentration of 80 pmol (Table 1). Fur-
thermore, samples of denatured mouse rIL-lcr, human rIL-la, and human rIL-1P (80 pmol) did not reduce the total specific binding of 1251-labeledmouse rIL-lcu (Table l), indicating the specificity of the assay for native, biologically active IL-l. Finally, substances present in human sera or culture media did not interfere with the measurement of IL-1 in this receptor assay. The curves generated from spiking human rIL-la into these solutions were essentially the same as that for human rILla in binding buffer (Fig. 3). To selectively measure human rIL-lcu in the presence of human IL-l& samples were pretreated with immobilized anti-human IL-l@ antibodies. Initial studies had attempted to add antibodies directly to the assay mixture. However, the antibodies and IL-1 formed high-molecular-weight crosslinked complexes which were precipitated by the 10% PEG in PBS. This resulted in exceptionally high nonspecific backgrounds. A protocol which utilized a pretreatment with immobilized antibody allowed for human IL-la to be measured specifically. Antibody-treated and untreated samples (17 fmol to 8.8 pmol) of human rIL-la generated similar inhibition of binding curves (Fig. 4). In addition, the anti-human rIL-16 antibodies effectively removed human rILl/3, and showed no cross-reactivity with human rIL-la. Assay of pretreated human rIL-la (8.8 pmol) and human IL-l@ (70.6 pmol), at saturating concentrations resulted in complete inhibition of labeled ligand with human rIL-la but no inhibition of labeled ligand with human rIL-l@ (data not shown). Furthermore, the inhibition of binding curves for samples of human rIL-la alone (17.2 fmol to 8.8 pmol) was identical to that of pretreated samples containing both human rIL-la (17.2
TABLE
Effect of Other Lymphokines Binding of lz51-Labeled Mouse Receptor
a Proteins
and Denatured IL-l on the rIL-lol to the Soluble IL-l
Amount bnol)
Factor None Mouse rIL-la Human rIL-lol Human rIL-l/3 Denatured mouse Denatured human Denatured human Interferon-cy-2a Interferon-y Interleukin-2
1
0 0.24 9 71 80 80 80 80 80 80
rIL-101” rIL-la” rIL-lj3”
were denatured
% Inhibition
by incubation
at 80°C
0 100 100 100 3 0 0 4 0 0 for 15 min.
210
RISKE
HUMAN IL-la. (pmol/assay) FIG. 4. Comparison of pretreated and untreated samples by soluble receptor binding assay. Pretreatment involved the addition of each sample to tubes containing 250 ~1 of Pansorbin previously saturated with anti-human rIL-lj3 antibodies. After 2 h at room temperature, or overnight at 4”C, the 25 pl of sample was removed and radioassayed. The protocol for assay was as described for Fig. 1. Duplicate serial twofold dilutions of human rIL-la (17.2 fmol to 8.8 pmol) were tested with pretreatment (m) and without pretreatment (0). Duplicate samples containing human rIL-lo! (17.2 fmol to 8.8 pmol) and human rIL18 (2.2 pmol to 70.6 pmol) were tested following pretreatment (A). The pretreatment depleted 100% of the human rIL-la.
fmol to 8.8 pmol) and human rIL-l/3 (2.2 pmol to 70.6 pmol) (Fig. 4). A major purpose in developing the soluble receptor binding assay was to monitor the purification of rIL-1 proteins. A scheme employing gel filtration and ion-exchange chromatography has been successfully used for the isolation of IL-l (8). The soluble receptor assay allowed the facile analysis of fractions collected during chromatographic separations, to determine the elution position and quantity of biologically active IL-l present in each. Fractions from the separation of a crude E. coli lysate (as described under Materials and Methods) by gel filtration were assayed (Fig. 5A). The fractions with the highest specific activity (fractions 200 through 300) were pooled and then chromatographed on an anion-exchange resin (Fig. 5B). The column was eluted with a salt gradient and fractions were assayed for IL-l. Fractions containing the highest specific activity were pooled. Quantification of the purified mouse rIL-la by receptor binding assay or DlO.G4.1 cell proliferation assay gave identical results (i.e., 1.8 X lOa units/ml). DISCUSSION A rapid, simple assay has been developed to detect and quantitate biologically active IL-l. The assay utilizes the
ET
AL.
sensitivity and selectivity of the interaction of IL-1 with its receptor. The range of sensitivity of the assay is 5 to 120 fmol for mouse IL-la, 60 fmol to 1.1 pmol for human IL-la, and 2.0 to 35 pmol for human IL-l@ As little as 21 units of mouse IL-la and 637 units of human IL-la can be detected. Expressed in concentrations, the assay of human IL-lp had 30-fold lower sensitivity as compared to human IL-la. However, comparisons based on units of biological activity reveal only a 3- to lo-fold difference in the sensitivity of the assay. This apparent discrepancy is due to an approximate lo-fold lower specific activity of human rIL-lb versus human rIL-la (6.1 X lO’units/mgvs 5.7 X lO’units/mg). Since both human IL-la! and human IL-l@ can be present in the same biological sample, a simple method was devised to pretreat the samples so that the level of human IL-la, could be measured. Bioassays based on cell proliferation are extremely sensitive, capable of detecting less than 5 pg/ml (0.294 fmol/ml) of human IL-l@ (12) and 2 pg/ml(O.l18 fmol/ ml) of human IL-la (W. Benjamin, unpublished work). However, the cells used in these assays can often be inhibited or stimulated by other substances present in serum or bodily fluids. For example, interleukin-2 and interleukin-4 can directly stimulate T cells or T-cell clones (24), thereby leading to enhanced IL-1 estimates. Tumor necrosis factor (25) and interleukin-6 (26,27) may also interfere with specific estimation of IL-1 activity. Adjuvants and mitogens inhibit T cells leading to lower IL-1 estimates, and prostaglandins and arachidonic acid metabolites can have either a positive effect (28) or a negative effect (29) on cell growth. Furthermore, Mochizuki et al. (30) reported that IL-1 and colony-stimulating factors act synergistically on some cells. Although synovial and endothelial cells have been suggested as an alternative to T cells for use in the assay of IL-l, these cells are also stimulated by tumor necrosis factor (31). The problems associated with nonspecificity suggest that bioassays may be impracticable for determining IL-l in serum and other bodily fluids. Enzyme immunoassays and radioimmunoassays have been used for the measurement of IL-1 in complex solutions. These systems most commonly are competitive inhibition assays (32) or solid-phase assays in which a first antibody is coupled either to wells in polystyrene plates (10,14) or to polystyrene balls (11). A second antibody labeled with 1251or an enzyme such as horseradish peroxidase is then used to detect IL-1 previously captured by the first antibody. Monoclonal antibodies, polyclonal antibodies, or both are employed as first and second antibodies in these systems. The sensitivity of some enzyme immunoassays and radioimmunoassays can approach that of bioassays. Ferrua et al. (9) and Kenney et al. (12) reported the detection of 10 pg/ml(O.588 fmol/ml) of human IL-l& and 15 pg/ml (0.882 fmol/ml) of human IL-l& The antibodies
SOLUBLE
RECEPTOR
Fraction
BINDING
ASSAY
FOR
211
INTERLEUKIN-1
Number
FIG. 5.
(A) Gel filtration on a Sephacryl S-200 column (12 X 120 cm) of cytosolic fraction from lysed E. coli (100 g) producing mouse rIL-la. Aliquots (1 ~1) of fractions (24 ml) were assayed by the receptor binding assay as described under Materials and Methods. (--) Absorbance monitored at 280 nm, ( mouse rIL-la (mouse IL-l). (B) Anion-exchange chromatography of mouse rIL-la. Molecular-size fractions containwere pooled and dialyzed against 2X 100 vol of 25 mM Tris-HCl, pH 8.1. This material was applied onto a DE-53 ing IL-1 binding activi column (5 X 100 cm) and proteins were eluted with a linear NaCl gradient to 800 mM (in 25 mM Tris-HCl, pH 8.1). Aliquots (5 ~1) of fractions (16 ml) were assayed by the receptor binding assay as described under Materials and Methods. (-) Absorbance monitored at 280 nm, ( mouse rIL-la (mouse IL-I).
used in these studies were specific for one form of human IL-l and did not cross-react with other tested lymphokines. However, a major drawback to these systems is that they cannot distinguish between active and inactive IL-l. The IL-l soluble receptor binding assay combines the advantages of the IL-l bioassays and immunoassays while eliminating their disadvantages. The receptor binding assay, similar to an IL-l bioassay, will only detect protein which is correctly folded and can interact with its receptor and which, therefore, retains biological activity. However, in contrast to a bioassay which often requires incubation periods of several days, results can be obtained in several hours. In addition, the assay has been designed to differentiate human IL-lo! and human IL-l@. Some immunoassays have been demonstrated to have a higher sensitivity than the receptor binding assay. This may necessitate the concentration of certain dilute samples before quantitation in the receptor binding assay. However, Thorpe et al. (14) have reported, for example, that joint fluid from arthritic patients had as much as 25 rig/ml of human IL-la, an amount detected by the receptor binding assay. Most important is that the assay
has the distinct advantage over immunoassays in only detecting active IL-l. Until recently, the use of receptors for analytical and preparative techniques was limited because sufficient quantities of purified receptors were not available. Recently, recombinant DNA technology has facilitated the large-scale production of soluble receptors (33). This has made receptor-affinity chromatography practical. This type of affinity chromatography has been demonstrated to have many advantages over classical immunoaffinity chromatography, e.g., binding only the fully active biomolecule in its native conformation (34). Similarly, receptor binding assays, as has been demonstrated in the case of IL-l, will prove a useful tool in the determination of levels of biologically active lymphokines. ACKNOWLEDGMENTS The authors thank Dr. W. Benjamin for performing T-cell proliferation assays and Ms. M. Miedel for amino of IL-l.
the DlO.G4.1 acid analysis
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128,892-897. J., and Hopper,
and Tushinski,
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75-82.
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19. Dayer, J.-M., Robinson, D. R., and Krane, S. M. (1977) J. Exp. Med. 145,1399-1404. 20. Kaye, J., Parelli, S., Tite, J., Jones, B., and Janeway, C. A. (1983) J. Exp.Med. 158,836-856.
30. Mochizuki,
13. Tanaka, K., Ishikawa, E., Ohmoto, J. Zmmunol. 17,1527-1530.
S., Damais, C., Lando, Res. 8,129-139.
17. Bevilacqua, M. P., Pober, J. S., Majeau, G. R., Cotran, Gimbrone, M. A. (1984) J. Erp. Med. 160,618-623. 18. Conlon, P. J. (1983) J. Zmmunol. 131,1280-1282.
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Med.
12. Kenney, J. S., Masada, M. P., Eugui, E. M., Delustro, kins, M. A., and Allison, A. C. (1987) J. Zmmunol.
15. Sunahara, Yamada,
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D. Y., Eisenman, J. P., Conlon, P. J., Larsen, A. D., P. J. (1987) Proc. Natl. Acad. Sci. USA 64,5267-
5271. 31. Saklatvala, J. (1986) Nature (London) 322.547-549. 32. Lisi, P. J., Chu, C.-W., Koch, G. A., Endres, S., Lonnemann, and Dinarello, C. A. (1987) Lymphokine Res. 6,229-244.
33. Hakimi,
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J., Seals, C., Anderson, L. E., Podlaski, F. J., Lin, P., Danho, W., Jenson, J. C., Perkins, A., Donadio, P. E., Familletti, P. C., Pan, Y.-C. E., Tsien, W.-H., Chizzonite, R. A., Casabo, L., Nelson, D. L., and Cullen, B. R. (1987) J. Biol. Chem. 262,1733617341.
34. Bailon, 840.
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335,839-
ANALYTICALBIOCHEMISTRY
(1990)
Measurement of Biologically Active Interleukin-1 by a Soluble Receptor Binding Assay Frank Riske,’ Richard Chizzonite, Perla Nunes, and Alvin S. Stern* Departments Hoffmann-La
Received
Molecular Genetics and *Protein Biochemistry, Roche Inc., Nutky, New Jersey 0711 O-l 199
of
September
26,1989
A soluble receptor binding assay has been developed for measuring human interleukin-la (IL-la), human IL- 18, and mouse IL- la. The assay is based on a competition between unlabeled IL-1 and ‘a51-labeled mouse recombinant IL- la for binding to soluble IL- 1 receptor prepared from mouse EL-4 cells. The assay measures only biologically active IL- 1 folded in its native conformation. The ratio of human IL-la! to human IL-18 can be measured in the same sample by a pretreatment step which removes human IL- 1~9from samples prior to assay. This technique has been used to monitor the purification of recombinant IL- 1, and may be utilized to specifically and accurately measure bioactive IL-1 in human serum and cell culture supernatants. o 1990 Academic
Press,
Inc.
Interleukin-1 (IL-l): a product of macrophages and other cell types, was originally described as a lymphokine capable of stimulating murine thymic cells following mitogenic activation (1). However, in the last several years it has become apparent that in addition to the augmentation of cell-mediated and humoral responses (2), IL-l is also involved in proinflammatory responses and has catabolic effects on certain tissues. This lymphokine can stimulate fibroblast and neutrophil proliferation, induce muscle proteolysis and the release of prostaglandin Ez, stimulate the synthesis and production of collagenase and hepatic acute-phase protein, and induce fever i To whom correspondence should be addressed. ’ Abbreviations used: IL-l, interleukin-1; rIL-1, recombinant interleukin-1; PBS, Dulhecco’s phosphate-buffered saline (without calcium chloride or magnesium chloride); CHAPS, 3-[ (3-cholamidopropyl)dimethylammoniol-1-propanesulfonate; PEG, polyethylene glycol; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; PMSF, phenylmethylsulfonyl fluoride; PBS-Tw, PBS containing 0.5 mM NaCl, 0.05% Tween-20; DTT, dithiothreitol. 206
Roche Research Center,
(3). IL-l may also be involved in some pathological conditions associated with immune and inflammatory responses such as joint erosion in rheumatoid arthritis. Therefore, a method is needed to detect and measure bioactive IL-l, especially in tissue or blood fluids which contain components that interfere with conventional bioassays. The recent cloning of IL-l genes and advances in the purification of the IL-l proteins (4-8) have had a major impact on the study of IL-l. Two species of IL-l have been identified and have been distinguished as human IL-la! and human IL-l& Although there is no more than 25% sequence homology between the two proteins, both human IL-la and human IL-l/3 have similar, if not identical, activities. The expression of the two genes in Escherichia coli provided sufficient material for the development of assays for measuring these cytokines. Currently, IL-l is measured by immunoassay (9-16) or bioassay (1,17-20). Immunological assays employ anti-IL-l antibodies to capture and measure IL-l. In bioassays, a measurable response by a cell line to IL-l is equated with lymphokine amount. Unfortunately, both methodologies suffer from drawbacks. Bioassays generally require several days to complete and are nonspecific since the cells respond to other lymphokines and substances found in serum or bodily fluids. Immunoassays measure both biologically active and inactive molecules. This study describes the development of a soluble receptor assay for the quantification of biologically active IL-l. The assay is based on a competition between radiolabeled mouse rIL-la and IL-l in the sample for binding to soluble IL-l receptor. The assay is rapid and simple and can be used to detect and measure biologically active human IL-la, human IL-l/?, and mouse IL-lo. MATERIALS AND METHODS Preparation of Soluble IL-l Receptor
Mouse EL-4.IL-2 thymoma cells (ATCC No. TIB181) were grown in a l-liter bioreactor containing 5% Copyright 0 1990 All rights of reproduction
by
0003-2697/90 $3.00 Academic Press, Inc. in any form reserved.
SOLUBLE
RECEPTOR
BINDING
horse serum in Iscove’s modified Dulbecco’s medium. The cells were harvested, washed three times with cold (4°C) Dulbecco’s phosphate-buffered saline without calcium chloride or magnesium chloride (PBS), and solubilized by resuspension in 5X vol of 8 mM CHAPS, 20 mM NaPO,, 5 mM EDTA, 40 pg/ml PMSF, and 0.05% NaN3 for 1 h at 4°C (21). The cell detergent suspension was stirred for 60 min at 4°C and insoluble material was removed by centrifugation at 120,OOOgfor 1 h. The supernatant solution was removed, divided into 3-ml aliquots, and stored at -70°C. The soluble preparation is stable for at least 3 months when stored at -70°C.
Proteins
and Antibodies
Human rIL-la, human rIL-l& and mouse rIL-la were purified as previously described (8) except that the proteins were prepared in 25 mM Tris-HCl, pH 8.1, containing 0.4 M NaCl. The purified proteins were tested in the mouse DlO.G4.1 cell proliferation assay (20) to determine biological activity. Human rIL-la, human rIL-l@, and mouse rIL-la had specific activities of 5.7 X lOa, 6.1 X 107, and 5.1 X 10’ units/mg, respectively. Antibodies specific for human rIL-la and human rILl/3 were produced by immunizing goats with 100 pg of human rIL-1 protein mixed in a 1:l ratio with Freund’s complete adjuvant. The goats were subsequently injected every 28 days with 10 pg of human IL-l mixed 1:l with Freund’s incomplete adjuvant. Serum was collected every 14 days after the third immunization, filtered, and stored at -70°C. Alternately, the serum was diluted fourfold with PBS containing 0.5 M NaCl, 0.05% Tween20 (PBS-Tw), filtered, and applied to affinity columns containing human rIL-la or human rIL-l/3 immobilized on a silica matrix (22). The columns were washed extensively with PBS-Tw and PBS, and antibodies specific for human rIL-1 were eluted with 0.2 M acetic acid, 0.2 M NaCl, pH 2.8, and immediately neutralized with 3 M Tris. The anti-human IL-l activity of the purified antibody preparations was evaluated by determining the concentration of antibody necessary to block the lz51rIL-1 (20 fmol) binding to EL-4 cells. Recombinant human interferon-a-2a, interleukin-2, and interferon-y were produced at Hoffmann-La Roche, Inc.
Radiolabeling
of Mouse rIL-la
and Human
rIL-la
Human rIL-la (15 pg) and mouse rIL-la (5.4 pg) were radiolabeled with 1.0 mCi Na1251(Amersham Corp., Arlington Heights, IL) as previously described (23). Briefly, IL-l was diluted with 25 mM Tris-HCl, 0.4 M NaCl, pH 7.5, to a volume of 70 ~1. Enzymobead reagent (50 ~1 in distilled water; Bio-Rad Laboratories), 0.2 M sodium phosphate buffer, pH 7.5 (50 pl), and 1.0 mCi Na1251were added to the IL-l. The reaction was initiated at room temperature with 1% ~-D-glucose and termi-
207
ASSAY FOR INTERLEUKIN-1
nated after 20 min. The labeled protein was separated on a 1 X 20-cm Bio-Gel P-6DG column equilibrated with 25 mM Tris-HCl buffer, containing 0.4 M NaCl, pH 7.5. The peak fractions of ‘251-IL-1 were pooled and diluted to a concentration of 1 X lo6 cpm/ml with 25 mM TrisHCl containing 1 mM EDTA and 1% BSA, pH 7.5. Each of the radiolabeled IL-l proteins contained a single labeled polypeptide of approximately 17 kDa which comigrated with unlabeled IL-l when analyzed by SDSPAGE and autoradiography. The labeled proteins are fully active in the DlO.G4.1 mouse T-cell proliferation assay when compared to the unlabeled proteins. The specific activities in each case ranged from 22 to 57 &i/ pg protein. The radioligand preparations were also tested for binding activity by increasing the number of EL-4 cells which were added to a fixed concentration of labeled IL-l. In each case, the maximum amount of radioligand bound by the EL-4 cells was approximately 80%. Furthermore, the binding of the radioligand to the cell surface binding site occurred in a specific and saturable manner and could be inhibited by adding 50 nM unlabeled human rIL-la, human rIL-lb, or mouse rIL-la.
Soluble Receptor Binding
Assay for Measuring
IL-l
The receptor binding assay is based on competition between IL-l in a sample and radiolabeled mouse rILla for soluble IL-l receptor. Solutions of human rIL-la (in 50 mM phosphate buffer with 0.1 M NaCl, pH 6.5), human rIL-1B (in 10 mM phosphate buffer with 0.45 mM DTT, pH 7.2), and mouse rIL-la (in the same buffer as human rIL-la) were diluted with binding buffer (PBS, 8 mM CHAPS, 0.25 M NaCl, 5 mM EDTA, and 0.1% BSA) to concentrations of 58.8, 470, and 1.6 nM, respectively. These concentrations of IL-l were sufficient for complete inhibition of 1251-labeledmouse rIL-la binding to receptor. The initial concentration of each IL-l was serial diluted in binding buffer to set up nine additional concentrations for quantification in the assay. Twentyfive microliters from the ten samples was pipetted in duplicate into 12 X 75-mm tubes. In addition, two tubes each were set up to determine total and nonspecific binding of 1251-labeledmouse rIL-la to the soluble receptor. Total binding was determined by adding 25 ~1of binding buffer to the tubes while nonspecific binding was measured by adding 50 to 500 nM unlabeled IL-1 to the tubes. Radiolabeled mouse rIL-la (25 ~1 containing 1.5 X lo4 cpm) was then added to each tube. Soluble IL-l receptor (10 ccl), diluted lo-fold in binding buffer, was added and the tubes were incubated for 1 h at 37°C. Fifty microliters of a human IgG solution (14 mg/ml in PBS) followed by 3 ml of 10% polyethylene glycol (PEG) 6000 in PBS (Sigma Chemical Co.) was added and the tubes incubated for an additional 30 min at 4°C. The contents of each tube were vacuum filtered through Whatman GF/C filters (Fisher Scientific). The filters were washed
208
RISKE
twice with 4 ml of the 10% PEG solution and counted in an LKB 1282 Compugamma gamma counter. Activity was determined as percentage inhibition of specific binding of labeled mouse rIL-la by IL-l in the test sample. The percentage inhibition was calculated by subtracting from 1 the difference between sample binding and nonspecific binding (cpm) divided by the difference between total binding and nonspecific binding (cpm). This number was then multiplied by 100 to obtain the percentage inhibition value. The percentage inhibition for each sample was plotted as a function of the concentration of IL-l expressed in mass (pmol) or biological activity (units). Units of IL-l activity were determined using the DlO.G4.1 cell proliferation assay (20). IL-l samples were also tested in Iscove’s modified Dulbecco’s medium with 10% fetal bovine serum and human sera (Gibco Labs, Grand Island, NY) diluted 1:l with PBS. Sample Pretreatment IL-l a and Human
to Measure Mixtures IL-l /3
of Human
To selectively measure human IL-la! when present along with human IL-l& samples were pretreated with goat anti-human IL-l@ antibody preparations. In 1.5-ml Eppendorf tubes, anti-human IL-lfl antibody was added (50 pg in 25 ~1) to 250 ~1 of a 2% suspension of Pansorbin (Calbiochem, La Jolla, CA) in PBS-Tw. An additional 200 ~1 of PBS-Tw was added and the tubes were gently mixed for 2 h at room temperature. Following centrifugation at 2000g for 3 min, the supernatant solutions were aspirated and the pellets washed three times with 1 ml of PBS-Tw. The tubes were firmly tapped to break the pellets of Pansorbin saturated with anti-human IL-l@ antibody. Duplicate samples of unknown or standards were added (25 ~1) to the pellets. After vortexing, the tubes were placed on a mixer overnight at 4”C, or 2 h at room temperature. Tubes were then centrifuged at 2000g for 3 min. The supernatant solution (25 ~1) was removed from each and placed in a 12 X 75 mm-tube, and the IL1 concentration was measured by the soluble receptor assay. Purification
of Mouse rIL-1 a
Mouse rIL-la was extracted from an E. coli clone containing an expression plasmid encoding for the protein (5). One hundred grams of a frozen E. cob paste was suspended in 500 ml of 30 mM Tris-HCl (pH 8.0), 5 mM EDTA, and 1 mM PMSF. The cells were lysed with a Manton-Gaulin cell homogenizer and the lysate was centrifuged at 27,000g for 30 min at 4°C. Solid ammonium sulfate was added to the supernatant solution to 60% saturation and stirred overnight at 4°C. The resultant pellets were dissolved in 100 ml PBS containing 0.4 M NaCl. This solution was applied directly to a column
ET
AL.
“1
. / ..... .-F.--r.
o!&wd--./. 1CP
10-Z
10"
100
10'
lo2
IL-1 (pmdhssay) FIG. 1. Measurement of mouse rIL-la (a), human rIL-la human rIL-I@ (A) by soluble receptor binding assay. Serial dilutions starting at 0.235,8.8, and 70.6 pmol of the respective were added to duplicate tubes and the assay was performed scribed under Materials and Methods.
(B), and twofold ligands as de-
(12 X 120 cm) of Sephacryl S-200 (Pharmacia LKB Biotechnology, Inc.) equilibrated in PBS containing 0.4 M NaCl and run at a flow rate of 240 ml/h. Aliquots were assayed by the receptor binding assay as described above and fractions with the highest specific activity were pooled. The protein solution was dialyzed (Spectra/Par 1, Fisher Scientific) against 100 vol 25 mM Tris-HCl, pH 8.1, and applied to a column (5 X 100 cm) of DE-53 cellulose (Whatman Biosystems Inc.) equilibrated with 25 mM Tris-HCl, pH 8.1. The mouse rIL-la was eluted with a linear gradient of 0 to 800 mM NaCl (in 25 mM Tris-HCl, pH 8.1) at a flow rate of 240 ml/h. Aliquots were assayed in the receptor binding assay as described above. RESULTS
The measurement of IL-l is based on competition between radiolabeled mouse rIL-la and unlabeled IL-l for binding to soluble mouse IL-l receptor. Total counts of 1251-labeled mouse rIL-lcr bound to receptor ranged between 2500 and 3000 cpm; nonspecific binding ranged between 7 to 10% of these counts. Mouse rIL-la, human rIL-la, and human rIL-l/3 at 0.235, 8.8, and 70.6 pmol, respectively, effectively inhibited 100% of 1251-mouse rIL-la (1.5 X lo4 cpm) binding to soluble receptor (Fig. 1). The linear range of the assays was 5 to 120 fmol for mouse IL-la, 60 fmol to 1.1 pmol for human IL-la, and 2.0 to 35 pmol for human IL-l@. Fifty percent displacement of total binding (I&) occurred with additions of 22 fmol, 234 fmol, and 6.0 pmol of mouse rIL-lcr, human
SOLUBLE
RECEPTOR
BINDING
ASSAY
FOR 120
INTERLEUKIN-1
209
1
%o05 p760g+6o 25 z
/ /* 0I e-04 :’ lo’
,// / ’
E 5" -I
,=ci=-. .,
IO2
40
103
....
..... I
10'
106
Ulassav)
IL-1 BIOACWITY
FIG. 2. Measurement of mouse rIL-la (O), human rIL-la (m), and human rIL-10 (A) by soluble receptor binding assay. Protocol was exactly the same as that described in Fig. 1 except that IL-l is expressed in biological units rather than picomoles of protein. Duplicate samples were assayed.
FIG. 3. Measurement of human rIL-lcz in binding buffer (a), human sera diluted 1:l with PBS (@ , and in Iscove’s modified Dulbecco’s medium with 10% fetal bovine serum (A). Serial twofold dilutions of human rIL-la (8.8 pmol to 17.2 fmol) were tested. Protocol was that described in Fig. 1.
rIL-la, and human IL-l& respectively (Fig. 1). Expressing the I&, in terms of biological units rather then in concentration resulted in values for human rIL-lcu and human rIL-la of 2246 and 7060 units, respectively, while mouse rIL-la exhibited a much lower IC& of 191 units (Fig. 2). The sensitivity of the receptor assay for human rIL-la and human rIL-l@ was not increased by using ‘251-labeled human rIL-la in place of 1251-labeledmouse rIL-la in the assay (data not shown). The receptor binding assay specifically measures the interaction of IL-1 with its receptor. Other lymphokines such as interferon-a, interferon-y, and interleukin-2 do not compete with ‘251-labeled mouse rIL-1cY for receptor binding at a concentration of 80 pmol (Table 1). Fur-
thermore, samples of denatured mouse rIL-lcr, human rIL-la, and human rIL-1P (80 pmol) did not reduce the total specific binding of 1251-labeledmouse rIL-lcu (Table l), indicating the specificity of the assay for native, biologically active IL-l. Finally, substances present in human sera or culture media did not interfere with the measurement of IL-1 in this receptor assay. The curves generated from spiking human rIL-la into these solutions were essentially the same as that for human rILla in binding buffer (Fig. 3). To selectively measure human rIL-lcu in the presence of human IL-l& samples were pretreated with immobilized anti-human IL-l@ antibodies. Initial studies had attempted to add antibodies directly to the assay mixture. However, the antibodies and IL-1 formed high-molecular-weight crosslinked complexes which were precipitated by the 10% PEG in PBS. This resulted in exceptionally high nonspecific backgrounds. A protocol which utilized a pretreatment with immobilized antibody allowed for human IL-la to be measured specifically. Antibody-treated and untreated samples (17 fmol to 8.8 pmol) of human rIL-la generated similar inhibition of binding curves (Fig. 4). In addition, the anti-human rIL-16 antibodies effectively removed human rILl/3, and showed no cross-reactivity with human rIL-la. Assay of pretreated human rIL-la (8.8 pmol) and human IL-l@ (70.6 pmol), at saturating concentrations resulted in complete inhibition of labeled ligand with human rIL-la but no inhibition of labeled ligand with human rIL-l@ (data not shown). Furthermore, the inhibition of binding curves for samples of human rIL-la alone (17.2 fmol to 8.8 pmol) was identical to that of pretreated samples containing both human rIL-la (17.2
TABLE
Effect of Other Lymphokines Binding of lz51-Labeled Mouse Receptor
a Proteins
and Denatured IL-l on the rIL-lol to the Soluble IL-l
Amount bnol)
Factor None Mouse rIL-la Human rIL-lol Human rIL-l/3 Denatured mouse Denatured human Denatured human Interferon-cy-2a Interferon-y Interleukin-2
1
0 0.24 9 71 80 80 80 80 80 80
rIL-101” rIL-la” rIL-lj3”
were denatured
% Inhibition
by incubation
at 80°C
0 100 100 100 3 0 0 4 0 0 for 15 min.
210
RISKE
HUMAN IL-la. (pmol/assay) FIG. 4. Comparison of pretreated and untreated samples by soluble receptor binding assay. Pretreatment involved the addition of each sample to tubes containing 250 ~1 of Pansorbin previously saturated with anti-human rIL-lj3 antibodies. After 2 h at room temperature, or overnight at 4”C, the 25 pl of sample was removed and radioassayed. The protocol for assay was as described for Fig. 1. Duplicate serial twofold dilutions of human rIL-la (17.2 fmol to 8.8 pmol) were tested with pretreatment (m) and without pretreatment (0). Duplicate samples containing human rIL-lo! (17.2 fmol to 8.8 pmol) and human rIL18 (2.2 pmol to 70.6 pmol) were tested following pretreatment (A). The pretreatment depleted 100% of the human rIL-la.
fmol to 8.8 pmol) and human rIL-l/3 (2.2 pmol to 70.6 pmol) (Fig. 4). A major purpose in developing the soluble receptor binding assay was to monitor the purification of rIL-1 proteins. A scheme employing gel filtration and ion-exchange chromatography has been successfully used for the isolation of IL-l (8). The soluble receptor assay allowed the facile analysis of fractions collected during chromatographic separations, to determine the elution position and quantity of biologically active IL-l present in each. Fractions from the separation of a crude E. coli lysate (as described under Materials and Methods) by gel filtration were assayed (Fig. 5A). The fractions with the highest specific activity (fractions 200 through 300) were pooled and then chromatographed on an anion-exchange resin (Fig. 5B). The column was eluted with a salt gradient and fractions were assayed for IL-l. Fractions containing the highest specific activity were pooled. Quantification of the purified mouse rIL-la by receptor binding assay or DlO.G4.1 cell proliferation assay gave identical results (i.e., 1.8 X lOa units/ml). DISCUSSION A rapid, simple assay has been developed to detect and quantitate biologically active IL-l. The assay utilizes the
ET
AL.
sensitivity and selectivity of the interaction of IL-1 with its receptor. The range of sensitivity of the assay is 5 to 120 fmol for mouse IL-la, 60 fmol to 1.1 pmol for human IL-la, and 2.0 to 35 pmol for human IL-l@ As little as 21 units of mouse IL-la and 637 units of human IL-la can be detected. Expressed in concentrations, the assay of human IL-lp had 30-fold lower sensitivity as compared to human IL-la. However, comparisons based on units of biological activity reveal only a 3- to lo-fold difference in the sensitivity of the assay. This apparent discrepancy is due to an approximate lo-fold lower specific activity of human rIL-lb versus human rIL-la (6.1 X lO’units/mgvs 5.7 X lO’units/mg). Since both human IL-la! and human IL-l@ can be present in the same biological sample, a simple method was devised to pretreat the samples so that the level of human IL-la, could be measured. Bioassays based on cell proliferation are extremely sensitive, capable of detecting less than 5 pg/ml (0.294 fmol/ml) of human IL-l@ (12) and 2 pg/ml(O.l18 fmol/ ml) of human IL-la (W. Benjamin, unpublished work). However, the cells used in these assays can often be inhibited or stimulated by other substances present in serum or bodily fluids. For example, interleukin-2 and interleukin-4 can directly stimulate T cells or T-cell clones (24), thereby leading to enhanced IL-1 estimates. Tumor necrosis factor (25) and interleukin-6 (26,27) may also interfere with specific estimation of IL-1 activity. Adjuvants and mitogens inhibit T cells leading to lower IL-1 estimates, and prostaglandins and arachidonic acid metabolites can have either a positive effect (28) or a negative effect (29) on cell growth. Furthermore, Mochizuki et al. (30) reported that IL-1 and colony-stimulating factors act synergistically on some cells. Although synovial and endothelial cells have been suggested as an alternative to T cells for use in the assay of IL-l, these cells are also stimulated by tumor necrosis factor (31). The problems associated with nonspecificity suggest that bioassays may be impracticable for determining IL-l in serum and other bodily fluids. Enzyme immunoassays and radioimmunoassays have been used for the measurement of IL-1 in complex solutions. These systems most commonly are competitive inhibition assays (32) or solid-phase assays in which a first antibody is coupled either to wells in polystyrene plates (10,14) or to polystyrene balls (11). A second antibody labeled with 1251or an enzyme such as horseradish peroxidase is then used to detect IL-1 previously captured by the first antibody. Monoclonal antibodies, polyclonal antibodies, or both are employed as first and second antibodies in these systems. The sensitivity of some enzyme immunoassays and radioimmunoassays can approach that of bioassays. Ferrua et al. (9) and Kenney et al. (12) reported the detection of 10 pg/ml(O.588 fmol/ml) of human IL-l& and 15 pg/ml (0.882 fmol/ml) of human IL-l& The antibodies
SOLUBLE
RECEPTOR
Fraction
BINDING
ASSAY
FOR
211
INTERLEUKIN-1
Number
FIG. 5.
(A) Gel filtration on a Sephacryl S-200 column (12 X 120 cm) of cytosolic fraction from lysed E. coli (100 g) producing mouse rIL-la. Aliquots (1 ~1) of fractions (24 ml) were assayed by the receptor binding assay as described under Materials and Methods. (--) Absorbance monitored at 280 nm, ( mouse rIL-la (mouse IL-l). (B) Anion-exchange chromatography of mouse rIL-la. Molecular-size fractions containwere pooled and dialyzed against 2X 100 vol of 25 mM Tris-HCl, pH 8.1. This material was applied onto a DE-53 ing IL-1 binding activi column (5 X 100 cm) and proteins were eluted with a linear NaCl gradient to 800 mM (in 25 mM Tris-HCl, pH 8.1). Aliquots (5 ~1) of fractions (16 ml) were assayed by the receptor binding assay as described under Materials and Methods. (-) Absorbance monitored at 280 nm, ( mouse rIL-la (mouse IL-I).
used in these studies were specific for one form of human IL-l and did not cross-react with other tested lymphokines. However, a major drawback to these systems is that they cannot distinguish between active and inactive IL-l. The IL-l soluble receptor binding assay combines the advantages of the IL-l bioassays and immunoassays while eliminating their disadvantages. The receptor binding assay, similar to an IL-l bioassay, will only detect protein which is correctly folded and can interact with its receptor and which, therefore, retains biological activity. However, in contrast to a bioassay which often requires incubation periods of several days, results can be obtained in several hours. In addition, the assay has been designed to differentiate human IL-lo! and human IL-l@. Some immunoassays have been demonstrated to have a higher sensitivity than the receptor binding assay. This may necessitate the concentration of certain dilute samples before quantitation in the receptor binding assay. However, Thorpe et al. (14) have reported, for example, that joint fluid from arthritic patients had as much as 25 rig/ml of human IL-la, an amount detected by the receptor binding assay. Most important is that the assay
has the distinct advantage over immunoassays in only detecting active IL-l. Until recently, the use of receptors for analytical and preparative techniques was limited because sufficient quantities of purified receptors were not available. Recently, recombinant DNA technology has facilitated the large-scale production of soluble receptors (33). This has made receptor-affinity chromatography practical. This type of affinity chromatography has been demonstrated to have many advantages over classical immunoaffinity chromatography, e.g., binding only the fully active biomolecule in its native conformation (34). Similarly, receptor binding assays, as has been demonstrated in the case of IL-l, will prove a useful tool in the determination of levels of biologically active lymphokines. ACKNOWLEDGMENTS The authors thank Dr. W. Benjamin for performing T-cell proliferation assays and Ms. M. Miedel for amino of IL-l.
the DlO.G4.1 acid analysis
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B. H. (1972)
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6. March,
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