Production of monoclonal antibodies against interleukin-1α and -1β

Journal of Immunological Methods, 123 (1989) 193-210 Elsevier

193

JIM 05315

Production of monoclonal antibodies against interleukin-la and -lfl Development of two enzyme immunometric assays (EIA) using acetylcholinesterase and their application to biological media Jacques Grassi i, Yveline Frobert 1, Philippe Pradelles 1 Francine Chercuitte 2 Dominique Gruaz 3, Jean-Michel Dayer 3 and Patrice E. Poubelle 2 t Section de Pharmacologie et d'immunologie, CEN-SACI_A Y, 91191 Gif-sur- Yvette Cedex, France, 2 Unit~ de Recherche Inflammation et lmmunologie.Rhumatologie, CHUL, Quebec, G1 V 4G2, Canada, and 3 Division d'lmmunologie et d'Allergologie, D~partement de M~decine, H~pital Cantonal Universitaire, GenOve 1211, Switzerland

(Received 27 February 1989, accepted 7 June 1989)

We describe two series of monoclonal antibodies (mAbs) directed against human interleukin-la (36 mAbs) and -lfl (11 mAbs). The binding compatibility of each of mAb was studied using biotin-labelled mAbs in immunometric tests. Among the different pairs of compatible mAbs, we selected one pair for each interleukin-1 (IL-1) with optimal properties for a two-site immunometric assay. In these assays, covalent conjugates of mAb coupled to the tetrameric form of acetylcholinesterase (mAb-AChE) were used as tracers. The tests were performed in 96-well microtiter plates coated with the complementary mAb. Both assays appeared sensitive and specific since minimum detectable concentrations as low as 1 pg/ml were determined for each IL-1 without any significant cross-reactivity ( < 0.01%). The intra-assay precision was also very good with a coefficient of variation of < 10% over a wide range (between 3 and 500 pg/ml depending on the time devoted to the enzymatic reaction). The high sensitivity and precision of the assays can be ascribed to the high affinities of the mAbs as well as the optimal catalytic properties of ACHE. The specificity of the determination performed in culture medium was demonstrated using different validation tests including a comparison with a bioassay and the fractionation of samples by molecular sieve chromatography. Evidence is presented that the assay could be used for the determination of IL-1 levels in biological media such as plasma or serum. Key words." lnterleukin-l; Cytokine; Enzyme immunoassay; Monoclonal antibody; Acetylcholinesterase; Inflammation

Introduction

The interleukin-1 (IL-1) species represent an important family of biologically active m o n o -

Correspondence to." J. Grassi, SPI-DB, Bat. 136. CENSACLAY, 91191 Gif-sur-Yvette Cedex, France.

nuclear cell-derived proteins which are involved in inflammatory reactions and in immune responses (Dinarello, 1985; Oppenheim et al., 1986). Two distinct human IL-1 species (1L-la and IL-lfl) have been identified (Auron et al., 1984; March et ai., 1985). They share similarities such as the same molecular weight, similar biological effects and the same receptors on target cells (Wood et al., 1985; Kilian et al., 1986).

0022-1759/89/$03.50 @~1989 Elsevier Science Publishers B.V. (Biomedical Division)

194

The presence of IL-1 in culture media or in biological fluids is usually assessed by means of bioassays which are based on the lymphocyteactivating properties of IL-1 such as the murine thymocyte co-stimulation assay or the mouse thymoma cell line EL4. The presence of IL-1 in biological media can also be estimated using specific target cells for IL-1 such as fibroblasts which release PGE 2 and collagenase in response to IL-1 (Mizel et al., 1981). These methods have limitations: they do not discriminate between ILl a and IL-lfl and the presence of other active substances can interfere with the bioassay (i.e., IL-2, TNFa, TNFfl, lectins, growth factors or IL-1 inhibitor) (Balavoine et al., 1986). In addition, these bioassays are cumbersome and not ideal for routine analysis. Even if bioassays provide an indication of the effective IL-1 activity in a particular milieu, a clear understanding of IL-1 synthesis and release in both in vitro and in vivo situations requires an assay which permits the specific determination of each IL-1 form. To this end several radio- and enzymoimmunoassays for IL-la and IL-1/3 have now been reported (Tanaka et al., 1987a, b, 1988; Gaffney et al., 1987; Lisi et al., 1987; Ferrua et al., 1988; Lonneman et al., 1988). In this paper we describe two series of monoclonal antibodies directed against IL-la and IL-lfl. Two pairs of these mAbs have been selected to set up two highly specific and sensitive enzyme immunometric assays for IL-la and IL-lfl. These assays are performed with mAb-acetylcholinesterase (mAb-AChE) conjugates, and we show that they can be applied to the measurement of both IL-ls in culture media.

Biological Standards and Controls, Potters Bar (England). Unless otherwise stated all reagents were of analytical grade and were purchased from Sigma (St. Louis, MO, U.S.A.).

Purification and assay of acetylcholinesterase Acetylcholinesterase (ACHE) from the electric eel Electrophorus electricus was purified by affinity chromatography as described by Massouli6 and Bon (1976). The tetrameric form of the enzyme (G 4 form) (for a review on molecular form of ACHE, see Massouli6 and Bon, 1982) was used for labelling interleukins and antibodies. The characteristics of this preparation have been described elsewhere (Pradelles et ai., 1985; Grassi et al., 1988). AChE activity was measured using the colorimetric method of Ellman (Ellman et al., 1961) as previously described (Pradelles et al., 1985). One Ellman unit was defined as the amount of enzyme producing an absorbance increase of 1 absorbance unit at 25 o C, during 1 min, in 1 ml of medium and for an optical path length of 1 cm; it corresponds to about 8 ng of enzyme and 7.32 enzyme units (1 enzyme unit corresponds to the amount of enzyme hydrolysing 1 ~moi of acetylcholine at 25 ° C during 1 min). AChE concentrations were determined enzymatically using a turnover number of 4.4 × 107 m o l - h -~ per site (Vigny et ai., 1978), and a molecular mass of 80 kDa for the catalytic subunit. According to these values a detection limit of 1.8 amol of enzyme was calculated for the G 4 form (i.e., the quantity of AChE producing an absorbance increase of 0.01 absorbance unit during 1 h, in 200 ~! Ellman medium, 0.5 cm path length) (Grassi et al., 1987).

Immunization and hybridoma procedure Materials and methods

Reagents Human recombinant IL-la and -/3 proteins from E. coli were kindly provided by Dr. A. Shaw (Biogen, Geneva, Switzerland). According to the manufacturer, this material was at least 98.6% pure with a specific activity of 1.3 × 10 7 U / m g as measured by the thymocyte co-stimulation assay. Rabbit and sheep antisera against IL-la and IL-lfl were a gift from Dr. S. Pool, National Institute of

Anti-interleukin-la and -lfl antibodies were raised in mice (Biozzi's High Responder (HR) strain) using the following immunization procedure: on day 0, 15/zg of recombinant interleukinl a or -lfl emulsified in complete Freund's adjuvant were injected into the foot pad. Mice were treated with aspirin (0.1 m g / d a y ) in order to avoid adverse effects such as hyperthermia and pain (Dinarelio, 1984). A first booster injection (in the foot pad) was given on day 21 and the mice were bled 1 week later. The presence of murine anti-interleukin antibodies in the corresponding

195 antisera was monitored by testing their capacity to bind interleukin-l-AChE conjugates (see below). These tests were performed in microtiter plates coated with anti-mouse IgG antibodies using the same procedure subsequently used for the screening of hybridoma culture supernatants (see below). At this step titers ranging from 1/1000 to 1/10000 were observed. For each interleukin-1 the mouse presenting the highest titer in the enzyme immunoassay was selected for the preparation of monoclonal antibodies. The animals received a booster i.v. injection (7 Fg of interleukin), 3 and 2 days before fusion. Spleen cells from the immunized mice were fused with NS1 mouse myeloma cells as described (Grassi et al., 1988).

Labelling of interleukins with AChE Interleukin-la and -lfl were covalently coupled to AChE by reaction with the heterobifunctional reagent N-succinimydyl-4-(N-maleimido-methyl)cyclohexane-l-carboxylate (SMCC) using the procedure previously described for the labelling of atriopeptin (McLaughlin et al., 1987) and bovine acidic fibroblast growth factor (Caruelle et al., 1988). This method involved the reaction of a thiol group (previously introduced into interleukins) with a maleimido group incorporated into the enzyme after reaction with SMCC. Interleukins were thiolated by reaction of their primary amino groups with N-succinimidyl-S-acetyl-thioacetate (SATA, Calbiochem. U.S.A.) in alkaline medium.

Incorporation of thiol groups into interleukins. To 100 /~g of interleukin-la or -lfl dissolved in 200 #1 of 0.1 M borate buffer, (pH 8.5) were added 10/~1 of a 2.7 mg/ml solution of SATA in anhydrous dimethylformamide (DMF). The mixture was allowed to react for 30 rain at 25°C before 200 #1 of a 1 M hydroxylamine solution (pH 7) were added. After a further 30 min reaction at 25 o C, excess reagents (SATA and hydroxylamine) were removed by molecular sieve chromatography on a Sephadex G-25 column (15 x 1 cm) equilibrated with a 100 mM phosphate buffer pH 6 containing 5 mM EDTA. Before and during the chromatography, the eluate was maintained under a continuous stream of nitrogen in order to eliminate dissolved oxygen and avoid possible oxidation of thiol groups. Fractions containing thiolated interleukins were pooled. The concentra-

tion of interleukins was evaluated by UV spectrometry assuming an extinction coefficient at 280 nm of 1 g - 1 . 1 - c m - 1 and a molecular weight of 17000. Their thiol content was determined colorimetrically (412 nm) after reaction with 0.5 mM 3,5'-dithiobis-nitrobenzoic acid (DTNB) as described by Ellman. These measurements revealed that about one thiol group was incorporated by one molecule of interleukin.

Incorporation of maleimido groups into AChE and coupling with thiolated interleukins. Maleimido groups were introduced into AChE (G 4 form) by reaction with SMCC and subsequently purified as previously described (McLaughlin et al., 1987; Caruelle et al., 1988). If not used immediately, AChE-SMCC preparations were frozen at - 80 ° C and kept without loss of their reactive properties for several weeks. Thiolated interleukins were coupled to SMCC-AChE (G 4 form) by mixing both reagents either immediately after their isolation by molecular sieve chromatography or thawed once. For this step a molecular ratio (SH-interleukin/ G4-SMCC) of 50/1 was used. After reaction for 3 h at 30 o C, unreacted interleukin was removed by chromatography on a Biogel A 0.5 m column (90 x 1.5 cm) as previously described (Pradelles et al., 1985). The conjugates were stored frozen at - 2 0 ° C. No loss in enzyme activity was observed during the overall coupling procedure. No significant modification of the immunological or enzymatic properties of the conjugates has been noted under these storage conditions over a 1 year period. We did not attempt to evaluate the exact stoichiometry (i.e., the interleukin/G 4 ratio) of the conjugates.

Labelling of monoclonal antibodies with AChE Monoclonal antibodies (mAbs) were purified from ascitic fluids using caprylic acid and ammonium sulfate precipitation methods as previously described (Reik et al., 1987). The purity of the preparation was checked by polyacrylamide gel electrophoresis using denaturing and reducing conditions (Laemli, 1970). Two different labelling methods were used for coupling purified mAbs with ACHE. (1) Labelling of mAbs with biotin. Biotin was covalently linked to the mAbs by reaction of an activated N-hydroxysuccinimide ester of biotin

196 (IBF, France) with the primary amino groups of the antibodies. The activated ester was dissolved in anhydrous dimethyl-formamide and added to an alkaline solution of the antibody to be labelled. Briefly, to 1 mg of antibody dissolved in 2 ml of 0.1 M borate buffer (pH 8.5) was added 40/tl of a 5 m g / m l solution of biotin ester in DMF. After 30 rain at room temperature, 2 ml of EIA buffer (see below) were added. This crude preparation was used to determine the 'binding compatibility' of the different mAbs. It was kept frozen at -20°C.

(2) Covalent labelling of Fab' fragments with ACHE. Fab' fragments of mAbs were covalently coupled to AChE by the intermediary of SMCC using a procedure derived from that described by Ishikawa (1983) for the labelling of antibody fragments with other enzymes. The principle of the method was very similar to the one used for labelling interleukins (see above), where thiol groups involved in the coupling reaction were naturally present in the Fab' fragments obtained by reduction of the corresponding F(ab') 2 fragment. Preparation of F(ab')2 fragments. F(ab')2 fragments were obtained by treatment of mAbs with pepsin in acidic medium (acetate buffer pH 4.3) as described (Lamoyi and Nisonoff, 1983). They were isolated from crude pepsin-treated preparations by molecular sieve chromatography on a Biogel A 0.5 m column (30 × 1.5 cm) equilibrated in 100 mM phosphate buffer pH 6 containing 5 mM EDTA. The purity of the F(ab')2 fraction was checked by polyacrylamide gel electrophoresis in non-reducing denaturing conditions. These measurements revealed that more than 80% of the total protein content was composed of F(ab') 2 fragments. The residual presence of intact antibody in this preparation was not important since such antibody was not coupled to AChE in the further steps of the coupling procedure. Preparation of Fab' fragments. F(ab') 2 fragments were reduced in the presence of 0.01 M fl-mercaptoethylamine (fl-MEA) at 37°C for 1 h. Excess /3-MEA was removed by molecular sieve chromatography on a Sephadex G-25 column (30 × 1.5 cm) as previously described for thiolated interleukins. The concentration of Fab' fragment was measured by UV spectrophotometry taking its

extinction coefficient at 280 nm and molecular weight to be 1.48 g- 1.1 • c m - 1 and 46 000 respectively. The thiol content of the Fab' preparation was determined by reaction with DTNB as for thiolated interleukins. Values ranging from one to four SH groups per molecule of Fab' were found depending on the mAb used.

Incorporation of maleimido groups into AChE and coupling with Fab' fragments. Maleimido groups were introduced into AChE (G 4 form) by reaction with SMCC and subsequently purified as previously described (McLaughlin et al., 1987; Caruelle et al., 1988). Coupling the enzyme to Fab' fragments was achieved by mixing the AChE-SMCC preparation (immediately after its isolation by molecular sieve chromatography or thawed once) with an excess of Fab' fragment. Usually a molecular ratio of 50 (i.e., thiol groups of Fab'/G4-SMCC ) was used at this step. After reacting for 3 h at 30 ° C, G4-Fab' conjugates were isolated by molecular sieve chromatography on a Biogel A 0.5 m column as previously described (Pradelles et al., 1985). The conjugate was eluted as a single homogeneous peak and the corresponding fractions were pooled and kept frozen at - 2 0 ° C . No significant loss in enzyme activity was observed during the overall coupling procedure. The stability of the conjugates proved to be very good since they could be kept frozen at - 2 0 ° C , either lyophilized or in liquid form at 4 ° C without losing their enzymatic or immunological properties.

Screening of culture supernatants The presence of anti-interleukin-1 antibodies in hybridoma culture supernatants was detected using an immunoenzymatic test as previously described (Grassi et al., 1988). In this method culture supernatants were incubated in microtiter plates coated with an anti-mouse IgG antibody together with interleukin-l-AChE conjugates. During this step, any anti-interleukin-1 mAbs present in the supernatants bound simultaneously to the IL-1-AChE conjugate and to the solid phase, thus leading to the indirect immobilization of AChE activity on the plates. The presence of AChE on the solid phase was further revealed (after a washing step) by addition of the substrate and colorimetric mea-

197

surement. The preparation and characteristics of the second antibody solid phase have been described elsewhere (Grassi et al., 1988). Briefly, 50 ~i of each supernatant from 96 well culture plates were transferred under sterile conditions into second antibody-coated microtiter plates of the same size. 50 /xl of interleukin-l-AChE conjugate (1 Ellman unit/ml) dissolved in EIA buffer were then added and the mixture allowed to react overnight at 4°C. At the end of this first reaction period the plates were extensively washed before the addition of 200 #1 of Ellman's reagent to each well. At this step, wells containing AChE solidphase-bound activity developed a strong yellow color indicating the presence of anti-IL-1 antibodies in the corresponding supernatant. The absorbance at 414 nm of each well was measured 30 rain or 1 h later using an automatic plate reader (Titertek, Finland).

Determination of binding compatibility for each pair

of mAb The compatible binding of two different mAbs to a single interleukin-1 molecule was determined in immunometric tests where one of the mAb was immobilized to the solid phase whilst the other was labelled with biotin molecules. These tests were carried out as follows: all dilutions were made in EIA buffer and 100 /zl of a 100 n g / m l solution of recombinant interleukin-la or -1/3 was added to microtiter plate wells coated with one of the rnAb. After a 1 h reaction at room temperature with continuous agitation, 100 /zl of a 10 ~ g / m l solution of another biotin-labelled mAb was added. After a further 3 h reaction period at room temperature with continuous agitation the plates were extensively washed. The possible presence of biotin-labelled antibody on the solid phase was further revealed by the addition of a mixture of avidin and biotinylated-AChE as previously described (Grassi et al., 1988). Non-specific binding of AChE on the solid-phase was evaluated in blank experiments where 100 #l of buffer were substituted for interleukin-1. When simultaneous binding of both solid-phase immobilized and biotin-labelled mAbs was possible, a strong yellow color was developed after addition of Ellman's reagent.

Enzyme immunoassays for IL-la and IL-lfl Two different types of EIA for interleukin-la and -1/3 were developed. Monoclonal antibodies from culture supernatants or ascitic fluids were first tested in a competitive immunoassay using IL-1-AChE conjugates as a tracer. In a second step, immunometric assays involving the simultaneous use of either solid-phase-coated or ACHElabelled anti-interleukin mAbs were developed. Unless otherwise stated all reagents used in the immunoassay were diluted in the following buffer (EIA buffer): 100 mM phosphate pH 7.4 containing 400 mM NaCI, 1 mM EDTA, 0.1% BSA and 0.01% sodium azide. All of the concentrations cited in the immunoassays refer to the concentration of the reagents in the 'initial volume' (50 #1 for competitive assay, 100 /~1 for immunometric assay) before mixing with the other reagents.

Competitive immunoassays using I L-1-A ChE conjugates. Conventional competitive immunoassays using mAbs as first antibodies and IL-1-AChE conjugates as tracer were performed exactly under the same conditions already described for other haptens or antigens (Pradelles et al., 1985; McLaughlin et ai., 1987; Caruelle et al., 1988). These assays were performed in 96-well microtiter plates coated with a rabbit anti-mouse IgG antibody (see above). This second antibody solid phase ensured a separation between the bound and free moieties of the tracer during the course of the specific immunoreaction. The total volume of reaction was 150 /xl, with each component (tracer, mAb and recombinant IL-1 standard) being added as a 50 /xl volume. IL-1-AChE conjugates were used at a concentration of 1 Ellman U / m l . The working dilutions for mAbs were previously determined by performing antibody dilution curves from culture supernatants or ascitic fluids. The sensitivity of the assays was characterized by the dose of IL-1 inducing a 50% lowering of the binding observed in the absence of competitor ( B / B o = 50%).

Immunometric assays" using mAbs-AChE conjugates. Immunometric assays were performed in 96-well microtiter plates coated with one of the mAbs. Coating was performed exactly as previously described for the second antibody solid phase (Pradelles et al., 1985; Grassi et al., 1988). Briefly, the wells of the microtiter plates were filled with 200 ~1 of a 10 # g / m l solution of mAb dissolved in

198 50 mM phosphate buffer pH 7.4. After overnight reaction at room temperature plates were extensively washed with 10 mM phosphate buffer pH 7.4 containing 0.05% Tween 20. The solid phase was then saturated by adding 300 ~1 of EIA buffer into each well for at least 4 h at room temperature. Plates were stored at 4 ° C in this saturation buffer until use. When stored under these conditions, we did not observe any modification of their binding properties over at least a 4 month period. Immunometric assays were alternatively performed using two different procedures. In the first procedure (named the 'simultaneous procedure'), 100 ~1 of interleukin-1 solution (introduced as a recombinant standard or as a sample) were added to the plate wells together with 100 /~1 of mAb-AChE conjugate (usually at a concentration of 10 Ellman U / m l ) . In this method both monoclonal antibodies (solid-phase-bound mAb and AChE-labelled mAb) reacted simultaneously with interleukin-1. Depending on the nature of the sample to be assayed, standard recombinant interleukin-1 was dissolved in the corresponding diluent, i.e., culture medium, plasma or serum. As far as possible, all the diluents used were free of interleukin-la or -1/3. For plasma or serum, this was made possible by selecting individual fluids for which the immunometric assays could not detect any significant amount of I L - l a or IL-1/3. When EIAs were performed in these media, mouse immunoglobulins (introduced as a 1 / 2 0 dilution of Erlich ascites in the tracer volume) were added during the immunoreaction in order to suppress the effect of human anti-mouse antibodies possibly present in these fluids (Bocasto and Stuart, 1986; Thompson et al., 1986). Non-specific binding was evaluated in separate wells where 100 /~1 of buffer or appropriate diluent were added instead of recombinant interleukin-1. Reactions were then allowed to proceed for various periods of time at various temperatures with or without agitation. At the end of this immunoreaction step, plates were extensively washed using phosphateTween buffer. Solid-phase bound AChE activity was determined by the addition of 200 ~1 of Ellman's reagent. In the second procedure the so-called 'sequential procedure', interleukin-1 and mAb-AChE were reacted separately. In a first incubation period,

200 ~1 of interleukin-1 (as standard or sample) dissolved in EIA buffer or any other appropriate diluent (culture medium, plasma, etc.) were reacted with the solid phase. At the end of this first incubation, plates were washed before 200 ~1 of mAb-AChE conjugate (usually at 10 Ellman U / m l ) dissolved in EIA buffer were added. After a second reaction period, plates were washed again and AChE activity revealed as usual. 'Imprecision profiles' of standard curves were established by performing all measurements (non-specific binding as well as standard point) eight times. For each point of the standard curve, the precision of the assay was defined by estimating the standard deviation of the eight determinations and expressed in terms of CV% (CV% = ( o / Z ) X 100). Curves were obtained by plotting CV% as a function of the logarithm of the dose. The sensitivity of immunometric assays was characterized by their 'minimum detectable concentration' (MDC) which corresponds to the concentration of IL-1 producing a statistically significant increase of the binding observed in the absence of standard recombinant IL-1. The MDC was calculated as the dose of IL-I corresponding to the non-specific binding plus three standard deviations (99% confidence).

Fast protein liquid chromatography (FPLC) experiments lmmunoreactive material detected by immunometric assays in biological media (culture supernatants, plasma... ) was characterized by fractionating these samples using FPLC equipment and a Superose 12 (HR 10/30) gel filtration column (Pharmacia, Sweden) equilibrated in EIA buffer. Standard recombinant IL-1 and samples (500 ~tl volume) were injected with a buffer flow rate of 24 m l / h and 0.8 mi fractions were successively collected. Each fraction was then tested with the IL-la a n d / o r IL-1/3 assay as described above. Recovery from the columns was estimated by injecting known amounts of recombinant I L - l a or IL-1/3, and found to range from 80 to 95%.

Preparation of samples to be assessed by EIAs for IL-lct and IL-lfl Supernatants of elutriated monocytes. Peripheral blood mononuclear (lymphocytes and mono-

199 cytes) leukocytes were obtained by centrifugation over Ficoll-Paque (Pharmacia Fine Chemicals) and were further subjected to counter flow centrifugal elutriation to give an enriched monocyte population (Poubelle et al., 1987). Briefly, mononuclear cells were injected into the separation chamber (in a Beckman JE-6 elutriator rotor) at a flow rate of 8 ml/min, with a constant rotor speed of 2500 rpm. The elutriation buffer consisted of Ca 2÷Mg 2+-free Dulbecco's phosphate-buffered saline (PBS) supplemented with 0.25% bovine serum albumin and 2 mM EDTA. Platelets were washed out after 0.5 h of elutriation at the injection flow rate. Fractionation of mononuclear leukocytes was achieved by increasing the flow rate from 8 to 24 ml/min. Lymphocytes were first eliminated between 8-20 m l / m i n (steps of 1 rnl/min) and monocytes were then obtained at flow rates from 20 to 24 m l / m i n (steps of 0.25 ml/min). 50 ml of medium were collected at each step, centrifuged (350 × g, 10 min) and the cell pellet washed once in isotonic solution before resuspension in the incubation medium (RPMI 1640 with L-glutamine, 7.5% fetal bovine serum (FBS)). Enriched monocyte fractions (lymphocytes < 20%) were pooled and adjusted to 1 × 10 6 cells/ml. Incubations were performed in 12 × 75 mm polypropylene culture tubes at 3 7 ° C in a humidified atmosphere with 5% CO 2. Differential cell counts of leukocytes were performed by (1) flow cytometry (EPIC-C, Coulter) using forward angle and right angle light scatter characteristics as described (Horan et al., 1986), (2) non-specific esterase and Wright's stains, (3) immunofluorescence analysis with specific fluorescein-conjugated monoclonal antibodies (anti-LeuM3 and anti-Leu4 from Becton Dickinson (Mountain View, CA)), and (4) phase-contrast microscopy analysis for platelet contamination. Cell viability was routinely assessed by trypan blue exclusion test in each experiment and was greater than 95%. Cells were cultured in the absence and presence of 5 / t g / m l lipopolysaccharide (LPS, phenol extract of E. coil, serotype 0127 : BS, Sigma Chemical Co. (St. Louis, MO)). At various time intervals, culture media were removed, centrifuged and supernatants immediately frozen at - 2 0 ° C until measurement of I L - l a and IL-lfl using selective EIAs. Each sample was assessed in duplicate.

Bioassay for IL-I: PGE 2 production by normal human fibroblasts Interleukin-1 has been shown to stimulate the synthesis of PGE z by fibroblasts in different experimental models (Mizel et al., 1981; Dayer et al., 1986) and fibroblast production of PGE 2 can be used as a bioassay to test media containing IL-1like material without discrimination between the two forms of IL-1 or other fibroblast stimulating factors. In an attempt to validate our EIAs for IL-1, we studied the production of PGE 2 by dermal fibroblasts in response to different concentrations of human recombinant I L - l a a n d / o r IL-lfl or in response to the various conditioned media from elutriated monocyte cultures. Fibroblast preparation. H u m a n fibroblasts were grown from dermis explants and passaged using trypsin-EDTA as required. The culture medium was Ham's F-10 (Flow Laboratories) supplemented with 20% fetal bovine serum (FBS, Sigma Chemical Co., St. Louis, MO) and antibiotics. For measurement of basal PGE 2 release, cells were used at passage 3 or 4 and were seeded in 12-well plates (Linbro, Flow Labs, Mississauga, Ont.) at a density of 5 × 104 cells/well. The ceils were cultured for 24 h in the medium described above. The culture medium was then removed, the wells were washed once and filled with 1 ml of medium containing equilibrated (37°C, 5% CO2) Earle's balanced salts solution (EBSS) with 5% heat-inactivated FBS and the conditioned medium from elutriated cell preparations at different dilutions. After a 6 h incubation period, supernatant aliquots were collected and immediately frozen at - 2 0 ° C until assayed for PGE z content. PGE 2 measurement. PGE 2 was directly assayed in incubation media with a specific enzymoimmunoassay (with acetylcholine esterase as a tracer) as previously described (Pradelles et al., 1985). The matrix correction was applied using 50 #l of EBSS with 5% FBS added to the standard curve reaction mixtures. Each determination was performed in duplicate. The assay significantly detected amounts of PGE 2 as low as 5 p g / m l . Calibration of bioassay with recombinant I L - l a or IL-lfl. H u m a n dermal fibroblasts (5 × 104/ml) were found to spontaneously release 420 ± 51 pg/rnl PGE 2 (n = 12) after a 6 h incubation period. When incubated with human recombinant

200 I L - l a , I L - l f l or I L - l a + I L - l f l , the release of P G E 2 by fibroblasts was d o s e - d e p e n d e n t . T h e conc e n t r a t i o n of a d d e d IL-1 which i n d u c e d a m o u n t s of P G E 2 significantly different from the basal P G E 2 release was f o u n d to be 0.1 p g / m l (800 + 52, 817 5 : 5 1 , 6 9 0 5:104 p g / m l P G E 2 for I L - l a , I L - l f l , I L - l a + I L - l f l respectively; in the latter case the c o n c e n t r a t i o n of each form of IL-1 was 50% of the final concentration). Each IL-1 c o n c e n t r a t i o n was tested in q u a d r u p l i c a t e on d e r m a l fibroblasts cultured from the s a m e donor. In these c o n d i t i o n s , it was possible to d r a w a s t a n d a r d curve of P G E 2 release in response to well-defined c o n c e n t r a t i o n s of IL-1. This s t a n d a r d curve was used to app r o x i m a t e the a m o u n t s of I L - l - l i k e m a t e r i a l s f o u n d in s u p e r n a t a n t s of elutriated cells (with or w i t h o u t LPS) when i n c u b a t e d with cultured dermal fibroblasts of the s a m e origin. In this system, LPS was f o u n d to have no effect on the g e n e r a t i o n of P G E 2 b y cultured fibroblasts ( d a t a n o t shown).

Results

Characterization of monoclonal anti-IL-1 antibodies and establishment of immunometric assays F o r each interleukin, the m o u s e whose serum possessed the higher titer in e n z y m e i m m u n o a s s a y (see materials a n d m e t h o d s section) was selected for fusion. The c o r r e s p o n d i n g spleen cells were fused with m y e l o m a NS1 cells as p r e v i o u s l y described ( G r a s s i et al., 1988). 1 week after fusion, a b o u t 500 wells p r e s e n t e d actively m u l t i p l y i n g hyb r i d o m a s for b o t h interleukins. T h e presence of m o u s e a n t i - i n t e r l e u k i n a n t i b o d i e s was c h e c k e d in all the culture s u p e r n a t a n t s b y testing their c a p a c ity to b i n d the c o r r e s p o n d i n g I L - 1 - A C h E conj u g a t e (see m a t e r i a l s and m e t h o d s section). As discussed elsewhere ( G r a s s i et al., 1988), this screening m e t h o d o l o g y p e r m i t t e d a very quick a n d efficient selection of h y b r i d o m a since n a n o g r a m a m o u n t s of m A b were easily d e t e c t e d , the screen-

TABLE I RESULTS OF BINDING COMPATIBILITY TESTS FOR ANTI-IL-la MONOCLONAL ANTIBODIES Binding compatibility tests were performed as described in the materials and methods section. Full dots indicate combinations for which simultaneous binding of solid-phase immobilized mAb and labelled mAb was observed. In order to clarify the presentation, mAbs presenting the same pattern were grouped in three categories (A-C). All mAbs were of IgG1 subclass, excepting mAb 100 (group A) and mAbs 50 and 176 (group B) which were lgG2. Labelled mAbs

Solid-phase immobilized mabs

137 5

310

186 231 267 275 294 296

124 153 159 176 177 185

137 147 184 230 260

5 29 35 73 100

44 48 50 71 82 117

18 22 30 33 34 39

147 29

184 35

230 73

260 100

310 186 124 44 18

231 153 48 22

267 159 50 30

275 176 71 33

294 177 82 34

296 185 117 39

101 A

m

C

101 A

B

201 TABLE II RESULTS OF B I N D I N G COMPATIBILITY TESTS FOR ANTI-IL-lfl MONOCLONAL ANTIBODIES Binding compatibility tests were performed as described in the materials and methods section. Full dots indicate combinations for which simultaneous binding of solid-phase immobilized mAb and labelled mAb was observed. In order to clarify the presentation, mAbs presenting the same binding pattern were grouped in four categories (A-D). lgG subclass of each mAb is listed in the last column. Solid-phase immobilized mAbs 20 28 44 79

Labelled mAbs 20

28

44

. . . .

79

. . . .

36

. . . .

61

70

. . . .

36 61 70 . .

26 54

. . A

.

.

. .

. .

.

.

.

.

. .

.

54

-

. .

.

26

. . . .

.

.

.

27

. . . .

. -

7 27

7

.

. .

B

ing of 2000 supernatants requiring no more than 4 h of work for one person. In a primary screening, 90 and 116 strongly positive culture supernatants were detected for IL-la and IL-lfl respectively. The corresponding hybridomas were subcioned by limiting dilutions to obtain a single, stable, antibody secreting cell line from each culture. Finally, 36 and 11 cell lines were stabilized for IL-la and IL-lfl respectively. All clones were characterized by a number preceded by the letter a or fl depending of the intedeukin against which they were raised. They were expanded as ascitic tumors, and monoclonal antibodies were purified from ascitic fluid using caprylic and ammonium sulfate precipitation (Reik et al., 1987). Isotypes were determined from ascitic fluid by the Ouchterlony double-diffusion technique as previously described (Grassi et al., 1988). All of the mAbs were IgG-possessing K light chains and most of them were of the IgG1 subclass (see Tables I and II). Culture supernatants as well as ascitic fluids were used in competitive enzyme immunoassays utilising IL-1-AChE conjugates as tracers. In most cases, (except using fl26 and fl54 clones), standard curves were generated using recombinant IL-la and IL-lfl as standards. When defined in

.

. .

. .

. .

C

-

lgGl lgGl IgG1 lgG1

A

IgG1 IgGl IgGl

B

IgG1 lgG2a

C

IgGl lgG1

D

D

terms of B / B o = 50% (see materials and methods section) the sensitivity of these assays ranged from 4 to 100 ng/ml. All rnAbs appeared to be very specific for the interleukin against which they

TABLE III MAIN C H A R A C T E R I S T I C S O F C O M P E T I T I V E ENZYMOIMMUNOASSAYS FOR I L - l a U S I N G EITHER POLYCLONAL OR M O N O C L O N A L A N T I B O D I E S A N D IL-I a-AChE CONJUGATES All mAbs tested in these experiments were from culture supernatants of hybridoma cells. Specific antibody

Sheep polyclonal antiserum Rabbit polyclonal antiserum mAb 39 mAb 260 mAb 29 mAb 185

Dilution of specific antibody 1 / 5 . l0 s 1/10 ~ 1/4000 1/1000 1/500 1/1000

Sensitivity a ( B / B o = 5070, ng/ml) 15 40 6 10 20 100

a B / B o = 5070 corresponds to the dose of standard inducing a 50% lowering of the tracer binding observed in absence of competitor.

202 were raised since either no or very low cross-reactivity could be measured between IL-la and IL-lfl (results not shown). As an indication, the characteristics of some of these competitive assays for IL-la are listed in Table III together with results obtained with polyclonal antibodies (sheep or rabbit). As the main purpose of this study was to develop two-site immunometric assays for both IL-la and IL-1/3, the next step was to determine which pairs of mAbs (one mAb immobilized on the solid phase, the other labelled with biotin molecules) could bind simultaneously to either IL-la or IL-1/3. These complementary binding studies were performed for all the possible combinations of mAb pairs produced in the present study. At this step, labelling with biotin was chosen because it is a very simple method which permits the labelling of a great number of mAbs (more than 40) in a small period of time (see materials and methods section). Furthermore, the subsequent use of a mixture of avidin- and biotinlabelled AChE allowed the sensitive detection of solid-phase immobilized biotin-labelled mAbs as previously described (Grassi et al., 1988). The systematic preparation of covalent mAb-AChE conjugates was not retained at this step essentially because this would have represented too much work. In addition, this would not have been relevant since it has been further demonstrated that owing to their size, the simultaneous binding of these conjugates together with solid-phase immobilized rnAbs is restricted in a certain number of cases. The results of these complementary tests for 1L-la and IL-lfl are presented in Tables I and II respectively. In the case of IL-la, a clear situation emerged. Three groups of mAbs recognizing epitopes located in three different regions of IL-la molecules could be characterized. These groups named A, B and C contained 10, 25 and 1 rnAb respectively. All of the mAbs of one group presented compatible binding with all of the mAbs of the two other groups whereas they did not bind simultaneously with themselves or mAbs of the same group. In the case of IL-1/3, a more complex situation occurred. In fact, four different patterns were observed. In the two first groups (named A and B containing four and three mAbs respec-

tively) behavior similar to that observed for IL-la was encountered (See Table II). The third group (C) contained two mAbs which were compatible with only one mAb of groups A and B (fl28 and /336 respectively). Finally, group D contained two mAbs which did not permit solid-phase immobilization of any of the other mAbs. A possible explanation for this somewhat puzzling situation will be presented in the discussion section. It is worth noting that these tests involving the use of biotin-labelled mAbs were not sensitive enough to detect interleukin concentrations below 1 ng/ml. Once all of the possible pairs of mAb presenting compatible binding were determined, the problem was to select the pair presenting the optimum properties in order to develop a sensitive, specific and reliable immunometric assay for each interleukin. This selection had to be made using covalent mAb-AChE conjugates (and not biotin-labelled mAbs) which had a greater specific activity and thus allowed a more sensitive detection of interleukins. In the case of IL-1/3 this was fairly easy since only 29 different possibilities existed. We chose to prepare the nine corresponding mAb-AChE conjugates (/326 and /354 were excluded), and all possibilities were tested. This strategy could not be applied to I L - l a because the preparation of 36 mAb-AChE conjugates as well as testing the 570 different existing possibilities would have represented too much work. We decided to use an alternative strategy. In a first step, only a few mAbs-AChE were prepared and subsequently tested against all the relevant solid phases. In view of the corresponding results, the mAbs providing the greater binding of mAb-AChE conjugates for a given dose of interleukin were labelled in turn and further tested with all the corresponding immobilized mAbs. During these experiments, mAbs a5, a29, al00, a147 (group A), a176, a39 (group B) and o301 (group C) were successively labelled and tested. Very significant differences were observed within the different groups (results not shown) indicating that most of the mAbs were derived from different cell lines. Final selection of the optimum pair was made taking into account not only the intensity of the signal but also other criteria including the absence of cross-reactivity with other interleukins or related substances, non-specific binding levels

203 1"fll

/ B

/ i.zl A b s o r b a n c e 1.01 Unit

/

0.8 t ~ 0.61 0.4 i ~/

0.0 t 0

160200300400500 ILia' ( p g / m l )

0

1 0 0 2 0 0 300 400 500 ILia" ( p g / r n l )

Fig. I. Non-specific effect of plasma on standard curves for IL-la assay. Comparison between two different combinations of monoclonal antibodies. Standard curves were established using recombinant IL-la diluted either in EIA buffer ( o ©) or in human plasma (* *) (a plasma containing no detectable amounts of IL-la was specifically selected for this experiment). EIA were performed using the simultaneous procedure with an immunoreaction period of 4 h at room temperature (each measurement in duplicate). The two antibody combinations tested were: (A) solid-phase a18; tracer G4-al01; (B) solid-phase a185; tracer G4-a29.

and non-specific influences exerted by biological media (plasma or serum). Finally, the following pairs were selected: solid-phase a185 + a29-AChE for IL-la and solid-phase ,879 +,836-ACHE for IL-1,8. In the case of IL-la this was essentially because this pair was characterized by a very low non-specific binding and was poorly sensitive to non-specific effects. This last feature is illustrated in Fig. 1. For IL-1,8 the pairing , 8 7 9 - ,836-ACHE was selected due to its markedly greater sensitivity. The main characteristics of IL-la and IL-1,8 assays per-

formed in EIA buffer when overnight incubation at 4 ° C and simultaneous procedure were used (see materials and methods section) are summarized in Table IV. Both assays appeared very sensitive since minimum detectable concentrations (see definition in materials and methods section) below 4 p g / m l could be calculated for each interleukin using a 30 min period for enzymatic measurement. An increased sensitivity was observed when the enzymatic reaction (revelation step) was allowed to proceed for a longer time. In this case minimum detectable concentrations below 2

TABLE IV MAIN CHARACTERISTICS OF THE IMMUNOMETRIC ASSAYS FOR IL-la AND IL-lfl PERFORMED IN EIA BUFFER WITH THE SELECTED COUPLES OF mAbs Results presented in this table have been obtained using the simultaneous procedure (see materials and methods section) and overnight reaction at 4 ° C.

lL-la IL-lfl

Non-specific binding (% total activity)

Sensitivity MDC a (pg/ml) 30rain

MDC (pg/ml) 2h

Specificity IL-la b (ng/ml)

IL-lfl b (ng/ml)

IL-2 b (ng/ml)

0.03 0.02

3 2

1.5 1

100

2000 -

2000 5000

a MDC = minimum detectable concentration, see definition in the materials and methods section. b Dose of interleukin producing a tracer binding significantly different of non-specific binding.

204 1.4 ¸

Absorbanee

2,t0 rain

20 18 16 14 12 CV (%) 10 8. 6. -t 2 0-

120 rrxin

0.8

nin

0.4 [ 0o

in ~

m

i

n

01; ................ o

xo

i _

"o

40

:~o

50

60

360 m i n

\

120 m i n 30 rain

'\ \. "

0.1

'k

\

l

t0

too

t000

ILl s ( p g / m l )

70

ILI/~ ( p g / m l ) Fig. 2. Evolution of standard curve for IL-1/~ as a function of the time devoted to the enzymatic reaction (indicator step). A standard curve was established as described in the materials and methods section using the simultaneous procedure and overnight incubation at 4 ° C . After washing the plates and addition of colorimetric substrate (Ellman's reagent), reading of absorbance was performed at time periods ranging from 15 to 360 rain. Experimental data presented in this figure are the mean of eight individual determinations.

Fig. 3. Evolution o f ' i m p r e c i s i o n profiles' for the IL-lfl assay as a function of the time of the enzymatic reaction (indicator step). Imprecision profiles were established as described in the materials and methods section using the data collected for the standard curves presented in Fig. 2. For each dose of IL-l,8 the precision of the measurement (expressed as a coefficient of variation in percent, see materials and methods section) was estimated and plotted as a function of the dose. Profiles obtained after 30, 120 and 360 rain of enzymatic reaction are compared.

p g / m l were obtained (see Table IV). The evolution of standard curves as a function of the time devoted to the enzymatic measurement is shown in Fig. 2. This increase in sensitivity was accompanied by an increase of measurement precision in the lower part of the standard curve as shown in Fig. 3 where the evolution o f ' i m p r e c i s i o n profiles' (Dudley et al., 1985) is presented as a function of time. The best results (in terms of sensitivity) were obtained using overnight immunoreaction at 4 o C, although very good standard curves were gener-

ated using shorter reaction periods (i.e., 3 - 5 h at 4°C). In every case, minimum detectable concentrations below 10 p g / m l were observed. Performing the assays at room temperature or at 3 7 ° C did not improve the performance of the tests. In the case of I L - l a this was due to an important increase of non-specific binding, whereas in the IL-lfl assay a significant lowering of the tracer binding was observed. As mentioned above very low cross-reactivities were observed between I L - l a , IL-1/3 and IL-2 in both assays (see Table IV).

1.8-

~.6:

A

1.4"

1.2: 1.008-

o.62 0.42 0.2-

0.0 0

!

1

!

1

!

r

i

50

100

150

200

250

0

100

ILlo'(pg/ml)

I

i

200 300 IL1B (pg/ml)

I

i

400

500

Fig. 4. Standard curves obtained with recombinant I L - l a or IL-lfl diluted in various media. Curves were established using the simultaneous procedure and using overnight reaction at 4 ° C (see materials and methods section). I L - l a or -fl were diluted in EIA buffer (D O), RPMI 1640 + 15% FBS (,', zx), h u m a n plasma ( + + ) and h u m a n serum ( ~ ~ ) . A : standard curves for IL-la. B: standard curves for IL-IB.

205

F o r b o t h interleukins it was possible to establish s t a n d a r d curves in different m e d i a including: R P M I 1640 c u l t u r e m e d i u m c o n t a i n i n g 15% FBS, h u m a n serum or p l a s m a (Fig. 4). Curves o b t a i n e d in R P M I + F B S were very similar to those o b s e r v e d in E I A buffer. In this m e d i u m nonspecific b i n d i n g was lower due to the presence of FBS. In p l a s m a or serum a significant decrease in b i n d i n g was noted. However, the sensitivity of b o t h assays was not d r a s t i c a l l y affected. C o m p a r e d to the results o b t a i n e d in E I A buffer ( T a b l e IV), the loss in sensitivity did not exceed 50%.

40000

A 30000

20000

101300

20(3000

/

150000

Measurements of IL-la and IL-lfl in biological media O n c e the basic characteristics of b o t h assays were established, the aim was to d e m o n s t r a t e that these assays were suitable for the d e t e r m i n a t i o n of I L - l a and I L - l f l in biological media. This was first investigated by analyzing b o t h interleukins in s u p e r n a t a n t s from s t i m u l a t e d cell cultures. Obviously for these experiments, s t a n d a r d curves were p e r f o r m e d in the c o r r e s p o n d i n g culture m e d i u m . These tests revealed that the assays actually perm i t t e d the d e t e c t i o n of I L - l - l i k e i m m u n o r e a c t i v e m a t e r i a l s in culture s u p e r n a t a n t , the a p p e a r a n c e of this i m m u n o r e a c t i v i t y being related to the s t i m u l a t i o n of the cells. A typical e x a m p l e is presented in Fig. 5A where the variations of b o t h I L - l a a n d I L - l f l c o n c e n t r a t i o n s in s u p e r n a t a n t s of h u m a n e l u t r i a t e d m o n o c y t e s s t i m u l a t e d with 5 # g / m l l i p o p o l y s a c c h a r i d e are plotted, as a function of time. In o r d e r to qualitatively ascertain the specificity o f these m e a s u r e m e n t s , the s a m e samples were assayed with a b i o a s s a y based on the m e a s u r e m e n t of P G E 2 release by fibroblasts s t i m u l a t e d b y b o t h IL-lct a n d I L - l f l (see materials a n d m e t h o d s section). As shown in Fig. 5B, results o b t a i n e d using s u p e r n a t a n t s of elutriated m o n o cytes tested in a fibroblast assay qualitatively correlated with the results of i m m u n o m e t r i c measurements, thus s u p p o r t i n g the specificity of the imm u n o a s s a y s . T h e i d e n t i t y between the m a t e r i a l d e t e c t e d in c u l t u r e s u p e r n a t a n t s (or i n t r a c e l l u l a r extracts o f cells) a n d r e c o m b i n a n t IL-1 used for the e s t a b l i s h m e n t of s t a n d a r d curves was ascert a i n e d by three types of control experiment. First, the dilutions of s a m p l e s in culture m e d i u m provided d i l u t i o n curves paralleling the s t a n d a r d

//yjb

B

uJ I-

100000

I.u =c .J

50000

o

,~"'P-,-,-,-,-,--~- i 6

12

.

,

18

24

TIME (hr) Fig. 5. In vitro release of IL-1 by human elutriated monocytes. A: Determination of IL-la and IL-lfl using immunometric assays: 1L-la (Eli) and lL-lfl (A,A) were assessed by the specific EIAs in supernatants of unstimulated (D,zx) and LPSstimulated (N,A) human leukocytes purified by centrifugal elutriation. Cell supernatants were stored at - 2 0 ° C until assayed for IL-1 (see materials and methods section for details). Results are the mean:t SEM (n = 6) corresponding to 1 × 10 6 cells. Suspensions of monocytes consisted of 80-85% Leu-M3* (or esterase + ) and 15-107o Leu-4 +. B: Assessment of IL-l-like material in supernatants of elutriated monocytes using fibroblasts as targets for IL-1. Human dermal fibroblasts (5× 104/well) were exposed to adequate dilutions of cell supernatants of human elutriated monocytes (control, ~ ; + LPS. , ) . Under these conditions, PGE2-induced release by fibroblasts was compared to a "standard curve" drawn from the PGE 2 response of fibroblasts to well-defined concentrations of IL-1 (see materials and methods section for details) leading to an estimation of IL-l-like material. Results are the mean of four experiments (corresponding to 1 × 106 monocytes).

curves. This is shown in T a b l e V where the results of two different assays for I L - l a a n d I L - l f l performed at different d i l u t i o n s a r e presented. Similar results were o b t a i n e d with all o f the s a m p l e s tested. This finding was also s u p p o r t e d b y recovery e x p e r i m e n t s which d e m o n s t r a t e d that k n o w n a m o u n t s of r e c o m b i n a n t 1L-1 i n t r o d u c e d into the s a m p l e s could be assayed. In all o f the

206 experiments performed in culture media, recovery ranged from 86% to 93%. Finally, the analysis of culture s u p e r n a t a n t s after fractionation by molecular sieve c h r o m a t o g r a p h y showed that imm u n o r e a c t i v e material was eluted as a single homogeneous peak c o r r e s p o n d i n g exactly to the peak observed with r e c o m b i n a n t interleukins. This is shown in Fig. 6 where two chromatographic profiles o b t a i n e d for s u p e r n a t a n t s of T N F a - s t i m u lated alveolar macrophages for both I L - l a a n d I L - l f l assays are presented. T a k e n together, all these data strongly indicate that both assays permitted a q u a n t i t a t i v e d e t e r m i n a t i o n of both forms of interleukin-1 in these media. U s i n g these specific EIAs, we attempted to measure I L - l a a n d I L - l f l levels in h u m a n p l a s m a a n d serum samples. Assays performed in plasma or serum of healthy volunteers revealed that it was possible to select fluids c o n t a i n i n g no detectable a m o u n t s of I L - l a or IL-lfl. These pooled plasma or sera were used as d i l u e n t to establish appropriate s t a n d a r d curves (see Fig. 4). W h e n rec o m b i n a n t I L - l a or -fl diluted in plasma or serum

TABLE V PARALLELISM BETWEEN STANDARD AND SAMPLE DILUTION CURVES Samples A and B were diluted as indicated in the table and assayed for IL-la and IL-lfl by EIA. Dilutions as well as standard curve were performed in culture medium (RPMI + 10% FBS). For each dilution of the sample the IL-1 content was determined by reference to the standard curve. These data were multiplied by the dilution factor listed in the table in order to make clear the parallelism between standard curve and sample dilution curves. Sample

Dilution

A~

1/1 1/2 1/4 1/8

Bb

1/2 1/4 1/8 1/ 16

IL-la (pg/ml)

IL-lfl (pg/ml)

160O 1760 1782

366 340 304 -

940 892 960 992

532 564 576 480

a Sample ,4: supernatant of alveolar macrophages in culture

stimulated by TNFa. b Sample B." intracellular extract of alveolar macrophages in

culture.

1

2

3 4 riLl

V't

300 ! 250 i I

ILl (pg/ml)

200 -

100 .

o

0

5

10

15 20 Fractions

25

30

Fig. 6. Distribution of IL-la immunoreactivity following Superose 12 molecular sieve chromatography of culture supernatants. Culture supernatants of TNF~t-stimulated alveolar macrophages were fractionated by molecular sieve chromatography on a Superose 12 (HR 10/30) column as described in the materials and methods section. IL-la (*) and IL-lfl (O) immunoreactivity of each fraction was measured by the immunometric assays. Arrows (1-4) indicate the position of different molecular weight markers: (1) lgM (MW: 900000); (2) acetylcholinesterase from Electrophorus electricus G4 form (MW: 330000); (3) bovine serum albumin (MW: 68000); (4) egg albumin (MW: 43000). Vt: total volume of the column was measured using potassium ferricyanide. Recombinant IL-la and IL-lfl were eluted as a single homogeneous peak in fraction 20 (see corresponding arrow).

was fractionated on a Superose 12 c o l u m n (see materials a n d m e t h o d s section), a single homogeneous peak was eluted at the v o l u m e c o r r e s p o n d ing to the r e c o m b i n a n t IL-1 dissolved in buffer (recovery greater t h a n 80%). C h r o m a t o g r a p h i c profiles o b t a i n e d were very similar to those presented in Fig. 6 for native IL-1. This indicated that s t a n d a r d IL-1 was not modified in these media a n d c o n f i r m e d the validity of the corres p o n d i n g s t a n d a r d curves.

Discussion

In this paper we describe two series of mouse m o n o c l o n a l a n t i b o d i e s directed against interl e u k i n - l a a n d -1ft. For I L - l a , 36 cell lines were stabilized. F r o m c o m p l e m e n t a r y b i n d i n g experim e n t s ( u n d e r t a k e n with biotin-labelled a n t i b o d ies), the c o r r e s p o n d i n g m A b s were classified into

207

three categories (A, B and C) recognizing three different parts of the interleukin-1 molecule. Within each category, significant differences between isotype subclass, binding efficiency or nonspecific binding were observed, indicating that most of mAbs were derived from different cell lines. When complementary binding experiments were performed with Fab'-AChE conjugates (data not shown) somewhat different results were obtained. In particular, binding of mAb a101 (the unique member of group C) was no more compatible with the few mAbs of group B (a39, a185) whilst binding compatibility was conserved with all the others. These results suggest that the epitope corresponding to mAb al01 is close to the region recognized by the antibodies of group B, and confirm that differences exist inside this group. A more complex situation was observed for IL-1/3 (see Table II). 11 cell lines were stabilized and the corresponding mAbs were classified into four categories (A-D). mAbs of groups A and B exhibited a behavior similar to that observed for IL-la whereas mAbs of group C (07 and /327) appeared to be compatible with only one mAb from the first two groups (/336 and ,828). This possibly reflects a particular location of mAbs f17 and /327 at the border of the two regions recognized by mAbs of groups A and B. In addition, mAbs fl36 and /328 may be located at the opposite extremity of each region. More puzzling was the behavior of mAbs /326 and fl54 which were not compatible with any of the other mAbs. In fact, it seems that these antibodies did not bind native recombinant IL-lfl whereas they reacted with the IL-1/3-AChE conjugate (a property by which they were selected during screening experiments, see materials and methods section). This was illustrated in competitive immunoassay experiments in which mAbs ,826 and fl54 were characterized by high titers but unlike all other mAbs were not inhibited by unlabelled recombinant IL-lfl up to a 10 /tg/ml concentration. We suggest that these mAbs have been produced against IL-lfl which was at least partly denatured during immunization. This interpretation is in agreement with recent results obtained using rabbit polyclonal antibodies (Ferrua et al., 1988). These authors observed a high reactivity with solid-phase immobilized IL-lfl which was not sui-

table for use in a two site immunometric assay unless recombinant IL-lfl was partly denaturated by heat treatment. For both interleukins, competitive immunoassays permitted detection in the nanomolar range (20 ng/ml, see Table III), thus indicating that the corresponding affinity dissociation constants were of the same order of magnitude. All of the mAbs tested appeared to be very specific since very low cross-reactivity ( < 0.01%) was observed between the two IL-I forms in immunometric as well as in competitive immunoassays. For each interleukin, a pair of antibodies showing optimal properties in the immunometric tests was selected (see Table IV). For IL-la, the critical selective factors were the value of nonspecific binding and resistance to the non-specific interference produced by plasma or serum (see Table IV and Fig. 1). For IL-1/3 pair of antibodies (solid-phase fl79, tracer fl36) provided standard curves significantly more sensitive than all other pairs and was thus selected. The assays performed with the selected couples were very sensitive since, under ideal conditions, minimum detectable concentrations close to 1 pg/ml (6 × 10 -14 M) were calculated. In absolute amounts, this represents a few attomoles of each IL-1 (for a 100 #1 assay volume). To the best of our knowledge only one assay of similar sensitivity has been described for IL-la (Tanaka et al., 1988) and no equivalent exists for IL-1/L This reflects both the high affinity of the mAbs and the optimal catalytic properties of AChE which permits the preparation of conjugates with high specific activity detectable in the attomole range (Grassi et al., 1987). The intra-assay precision of the assays was also very good since coefficients of variation lower than 10% were observed for IL-1 levels as low as 2-3 p g / m l (see Fig. 3). In addition to the high sensitivity of detection, AChE possesses other advantageous features. Firstly, it provides a continuous signal for many hours (at least 24 h under the conditions used). This offers the possibility of increasing both the sensitivity and the precision of the immunoassay by allowing the enzymatic reaction to proceed for longer periods. This point is clearly illustrated in Figs. 2 and 3 and in Table IV. Secondly, use of the colorimetric assay of Eilman permits the continu-

208

ous monitoring of the assay since it is not necessary to stop the enzymatic reaction. This allows the working range of the assay to be adapted to the level of antigen in the samples (high and low levels requiring short and long reaction times, respectively). Because of these features AChE appears to be superior to other enzymes currently used in EIA. In addition, this work demonstrates that ACHE, which has already proved to be useful in labelling haptens and antigens, is also very effective in the labelling of antibodies. The high versatility of AChE is clearly illustrated throughout this work, since the enzyme was used successfully at different stages of the assay development, i.e., as IL-1 conjugates (for the screening of mAbs or in competitive assays), as biotin-labelled AChE (for complementary binding experiments), as mAb-AChE conjugates (for immunometric assays). These immunometric assays appeared to be entirely suitable for the determination of both cytokines in different culture medium or cellular extracts and validation experiments performed in these media were satisfactory. When l L - l a and IL-1/3 were assessed in human plasma and serum samples, these measurements were performed in the presence of mouse lgG in order to neutralize human anti-mouse lgG antibodies possibly present in these fluids (Bocasto and Stuart, 1986; Thompson et al., 1986). This precaution appeared worthwhile since further experiments showed that measurements performed in the absence of mouse IgG could lead to a considerable over-estimation of IL-I levels. The first experiments performed in human plasma or serum indicated that the different validation criteria (parallelism, recovery, FPLC fractionation) were not always satisfied. Therefore, we conclude that the present status of IL-1 in these media is not clear. This point is still under examination in our laboratories and will be the scope of a future communication. The specificity of the assays for IL-la and IL-lfl was qualitatively ascertained by the results of a bioassay although quantitative differences were observed. This is not surprising since the two methods were based on very different principles. Compared with bioassays, the immunometric test appeared to be very versatile. Results were obtained within 24 h with a minimum of handling.

It also seems desirable to further characterize the specificity of the different mAbs by studying their capacity either to inhibit the biological effects of I1-1 or to bind IL-1 fragments or precursors. A better knowledge of this specificity might permit the selection of other mAb combinations more adapted to the determination of each interleukin in biological media.

Acknowledgements This work was supported by grants from the Arthritis Society of Canada (3-230-85), 'Le Fonds de ia Recherche en Sant~ du Quebec', 'Le Commissariat h l'Energie Atomique (CEA, France)', 'Le Fonds National Suisse de la Recherche Scientifique' (3.400.0.86), and by an Associateship of the Arthritis Society to P.E. Poubelle. The authors thank Dr. A. Shaw (Glaxo, Geneva) for providing recombinant IL-1, Dr. S. Pool (National Institute of Biological Standards and Controls, Potters Bar (England)) for polyclonal antiIL-I antibodies, Dr. F. Marceau (Unit¢ de Recherche IIR) for providing cultures of dermal fibroblasts, D. Harbour, P. Lamourette, M.C. Nevers and M. Plaisance for their technical excellence and Pierrette C6t6 for her secretarial expertise.

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