JPH04503600A - Anti-interleukin-1α and -1β monoclonal antibodies, methods for producing the same, and application of the antibodies to detection and treatment of interleukin-1α and -1β - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Anti-interleukin-1α and -1β monoclonal antibodies, their production method, and Application of the above antibodies to detection and treatment of interleukin-1α and -1β This invention provides anti-interleukin, especially anti-interleukin-1α and -1β monoclonal antibodies, their production methods, anti-interleukin-1α and -1β monoclonal antibodies, Clonal antibody/acetylcholinesterase conjugate, said antibody and/or conjugate Enzyme immunoassay that detects and quantifies interleukin-1α and -1β using the body testing method (EIA), and various other applications, specific on therapeutic applications.

Lymphokines are the strong molecules produced by the interaction between lymphocytes and specific antibodies that sensitize them. It is a powerful drug that can modify, induce or suppress the function of many types of cells, and can be used during inflammatory responses. , and plays an essential role in the phenomena of bone resorption, fibrogenesis and chemotaxis. In addition , which is involved in the control of hematopoiesis and the regulation of immune responses. Lymphokine is produced by the 1976 bulb, but like interleukin-1, It is known that some can be produced by non-white blood cells.

Interleukin-1 (IMMUNOLOGIE, Jean-Francqui s　BACH.

Chapter xm, pages 425-42G, No. 3 [1986 Medical Examination] I! , related antigens to tissues It is a mesoietor that constitutes the biological signals presented, and is a monocyte T lymphocyte cooperative. This is what characterizes the same action. I L-1 activity is PHA (I u9/1l l) in the presence of C3H/HeJY mouse thymocyte proliferation test (LPS-tolerance). The classic one is If IL-1 is absent, stimulated with PHA pedicle cells are blocked in the Go/Gl phase. Only interleukin When IL-1 is added, the main action of IL-1 is interleukin-2. This suggests that it induces synthesis.

h) I L-1 has an estimated molecular weight of 18'KDa and a main impurity of +5KDa. It is a glycoprotein. Furthermore, the molecular weight that indicates the physiological activity of IL-1 is 2 KD8. Two peptide fractions, 1 and 4 Da, were isolated from normal urine.

TI, -1, excluding T lymphocytes, mononuclear cells, macrophages, keratinocytes, such as astrocytes, melanocytes, mesangial cells and B-lymphoblasts. It appears to be produced by many types of cells.

The biological effects of IL-1 are not limited to the induction of IL-2 synthesis.

TL-1 is involved in the activation of B cells and is involved in the activation of fibrotic non-cells and dinobioxide (5i stimulates the proliferation of cells (noviocyto) and increases body temperature by acting on the thermoregulatory center , induces the production of inflammatory proteins by liver cells. 270 amino acid precursor The lL-1 gene encoding the gene was recently cloned. Contains this gene The plasmid contains a carboquine terminal consisting of +56 amino acids with IL-1 activity. Induces the production of terminal peptides. In the above cited document, a group of A hypothesis about molecules has been proposed (DINA). RELLO, J, CLIN, IMMUNOL, (1985), 5°285 ~297 pages) and Oppenheim et al. LINOL.

TODAY (198B), pp. 9.45-46) shows that IL-1 is involved in the inflammatory response. It is a large group of bioactive proteins derived from mononuclear cells that are involved in immune responses. Announced. Two distinct species of human IL-1 have been identified. That is, IL-1a IL-1β (MARCHEt al, NATtlRE (1985), ■, 6 (pp. 41-647), these two species have the same molecular weight, similar physiological effects, and The presence of IL-1 in the presence of the same receptor on target cells and in physiological fluids is mainly due to In addition, studies such as the mouse leg line cell co-stimulation test and the mouse thymoma cell line EL4 test Measured as described above using a bioassay. However, IL-1's link These methods that take advantage of lymphocyte-activating properties have limitations, and IL-1α and IL -1β and other active substances such as IL-2, lectins and growth factors The presence of quality inhibits the bioassay.

[FERRUA at al, J, OF IMMUNOI,, METH, (+9118).

114, pp. 41-48], the existence of recombinant IL-1 and hybridoma technology Due to the development, several teams of researchers [GAFFNEY eL al., 1987 : KASAHARA et al, +9'87: KENNEY et , 19g7; KOICHIROeLal', 1987), but most raw Detects IL-1α and IL-1β with a detection limit comparable to that of a physical test. developed an isotope-free sandwich immunoassay and then used IL It is pointed out that a detection method for IL-1α and IL-1β can be provided. this The method allows detection of IL-1α and 11°-1β at levels below 1 pmol. Two separate colorimetric sandwich enzyme immunoassays are used. However, that The antibodies used here were rabbit or sheep polyclonal antibodies.

However, the properties of monoclonal antibodies, especially their great specificity and sensitivity, With issue α! It is clear that it would be advantageous if the method could be used to separately measure Ru.

Yamanaka et al. [TANAKA et at, EUR, J, IMMUMOL, (1 978), 17. pp. 1527-1530 are derived from peripheral blood mononuclear cells in vitro. The produced IL-1α and IL-1β were treated with polyclonal antibodies and monoclonal antibodies. We proposed to measure it by a sandwich enzyme immunoassay using the body.

That is, rabbit anti-human 11. , -1α monoclonal IgG1 or anti-human I Polystyrene beads coated with 1β IgG1 were added to recombinant human 11. − 1α, recombinant human It, -1β or culture supernatant of mononuclear cells. After washing, anti-human 1-1α Fab”/peroxidase-conjugated body or affinity-purified anti-human 1βFib'/peroxider Incubate with enzyme. The second antibody used was rabbit anti-IL-Lα or is an anti-IL-1β polyclonal antibody.

Anti-IL-1α antibodies and/or anti-IL-1β antibodies have also been described in other patent applications. Ru.

Otsuka Taiyaku's European Patent Application No. 267゜611 describes anti-IL-1α monochrome monoclonal antibodies and anti-1-1β monoclonal antibodies have been described, and the affiliates of these antibodies have been described. The affinity constant is on the order of 3.6×10″@M/Q.

IlllMUNEX (D Yo) (7 brim patent application No. 245, No. 052 specifically states (a) samples of biological fluids believed to contain IL; and (b) the reaction between the first antibody specific for the first antigen site characteristic of IT and -1. It consists of detecting the reaction between the first antibody and IL-1! Inflammation involving Shi-1 A detection method is described. The antibodies specifically described in this Immunex patent application are , a monoclonal antibody (named 15A4) that specifically reacts with IL-1α. clonal antibody) and a monoclonal antibody (7B) that specifically reacts with IL-1β. 4).

However, the antibodies described in this application have an interleukin of less than 1 nanogram/R (l). concentration cannot be measured.

European Patent No. 220,063 (Dainippon) includes in part IT, -1. Antibodies have been described that are specifically used in competitive or immunoassays. . In this assay, using this antibody, the sensitivity was 0.1βg/mQ in the competitive method. In the order of magnitude and immunoassay, it is only on the order of O, lng/x12.

Generally speaking, □Prior art antibodies are highly sensitive to IL-1 assays (1 pin). (order of cograms).

In French Patent No. 83713,389, published by Ishimoto Honjo, an enzyme immunoassay was performed. The antibody, antigen, or hapten is covalently bound to acetylcholinesterase to We have proposed a conjugate that allows the new conjugate to Enables small-scale enzyme immunoassays on the order of nanograms43 pages 6-450] consists of the antigen labeled with acedercholinesterase. The patented conjugate was synthesized into a membrane complex, Photosystem I (PS I) Application to screening of monoclonal antibodies against peripheral proteins It states that. The authors tested this method in an orthopedic immunoassay and allowing highly accurate quantification of PSl polypeptides in physical extracts. There is. This immunoenzymatic test method detects two groups of polypeptides that make up Psl. Screening a series of monoclonal antibodies that can recognize different polypeptides This test method consists of the following three steps.

- The anti-PSI monoclonal antibody present in the hybridoma culture supernatant is and indirectly immobilize the corresponding biodenylated antigen of PSL. -This biodenylated antigen strongly binds to avidin, and avidin itself is biotin. The activity of knee-bound AChE that binds to labeled AChE is measured by Ellman colorimetry. Measure using standard method. The authors reported that the Chi system can detect monoclonal antibodies. It is shown that the limit is significantly improved. This screening method is a competitive method, with different It is well suited for constant stirring measurement of PSI polypeptides.

Therefore, this invention provides a highly specific method for detecting and quantifying IL-1α and IL-1β. The aim is to provide a suitable means to enable sensitive enzyme immunoassays in Moreover, this means is also possible for therapeutic applications and for in-vivo diagnostics.

The object of this invention is to transform mammals, particularly rodents, more specifically mice, into IL- IL-1α and IL obtained by immunization with 1α or IL-1β -1β specific monoclonal antibody, optionally natural or recombinant specifically recognizes IL-1α or natural or recombinant IL-1β, In fact, these two 11. , -1 monoclonal anti- IL-1α or I It is composed of several groups that recognize different regions of L-1β, and several groups of antibodies can bind compatiblely with all antibodies of other groups, i.e. anti! L- Three groups of 1α antibodies recognize three different regions of interakin-1α (A, B and C), in which all antibodies from one group are matched with all antibodies from the other two groups. Can bind together but not with other antibodies of the same group: Anti-tt, -iβ antibodies are composed of four groups (A, B, C and D), of which The first two groups (A and B) behaved similarly to anti-IL-1α antibodies; The antibody is compatible with only one antibody from each of the first two groups, of which the fourth group (D) The antibody is incompatible with any of the first three groups of antibodies and is not compatible with recombinant native IL-1β. These antibodies recognize modified interleukin-1β and do not bind to IL -1β/AChEt! l! 1 conjugate: as well as the anti-IL-1α antibody and the anti-IL-1α antibody. IL-1β antibody is it, -iα or iris L-1β/acetylcholinesterer A monoclonal antibody that recognizes AChE conjugate. Ru.

These antibodies show virtually no cross-reactivity (<0.01%) between the two IL-1 does not indicate.

According to this invention, interleukin-1α and interleukin-1β-specific Monoclonal antibodies are used specifically in mammals, such as rodents, and more specifically in mice. obtained from Interleukin-1α or -1β when mice are injected appropriately. The immunized mammal is immunized by I? work cells in an appropriate manner , fused with myeloma cells from the same species of mammal, and cultured the resulting hybridoma. , if necessary, anti-interleukin-1α or anti-interleukin-1β Hybridomas in the supernatant in which antibodies were detected are subcloned to correspond to Depending on the interleukin, anti-I-1α or anti-IL-1β monoclonal A cell line is obtained that secretes the antibody.

The interleukin-1α or interleukin-1b used to immunize the mammal is It may be a manufactured natural interleukin or a recombinant interleukin. stomach.

Invention anti-IL-1α and anti-11. -1β monoclonal antibody The hybridoma is a Pasteur Instice hybridoma (PASTEURllll5T). ITUT [:) (, - belongs to 4 COLLECTION IIATIOIIAL E DE CtlLTtlREs DE MICROOROAITSMES (CNCII) Deposited on December 8, 1988. Deposited c The ibridomas are numbered 1823, l-824, l-825, l-826, irises. -827, I -828 and 1, -829.

In addition, the problem of this invention is to treat acetylcholinesterase and anti-11, -1α or It, -1β monoclonal antibodies or fragments of these antibodies with Fab' It is a conjugate with a fragment.

Furthermore, an object of the present invention is to provide anti-interleukin-1α or -11 This is a method for producing a Fab' fragment of a β monoclonal antibody. According to this method, The monoclonal antibody was treated with pepsin in an acidic medium to obtain F(ab') fragments. obtained and isolated by molecular sieve chromatography, and then these P(ab') the fragment, especially with a suitable reduction such as β-mercaptoethylamine (β-MEA). Fab' fragments are obtained by subjecting them to a reduction reaction controlled by a chemical agent, and then passed through a molecular sieve, Chromag. Purify by passing through a Raffy column.

Another object of the present invention is to use AChE and anti-11. -1α or anti! L-1β model A conjugate with a monoclonal antibody, and a conjugate with AChE and a conjugate of the monoclonal antibody. A method for producing a conjugate with a Fab' fragment, in particular a Fab' fragment, which comprises the above-mentioned antibody or a fragment thereof. at least one thiol group incorporated or naturally occurring in one of the AChE incorporates a small number of maleimide groups, especially tosuccinimide groups. Zyl-4-(tomaleimidomethyl)cyclohexane-1-carboxylade (SM Coupling with a suitable coupling agent such as a tosuccinimide ester such as CC) This manufacturing method is characterized by combining.

Furthermore, the problem of this invention is to improve the relationship between IL-1α in cell culture supernatant and biological fluid. ! This is a mirror table immunoenzymatic test method for detecting Shi-1β, and this method is used as the first antibody. The anti-IL-1α or anti-! L-1β monoclonal antibody, IgG supported on a suitable solid support, used as a second antibody, is interleukin-1 Following α or β, ChE activity should be visualized by appropriate means. This method is characterized by

A subject of the invention is also the anti-IL-1α and anti-IL-1α of the invention carried on a suitable support. And/or against! IL-1β monoclonal antibody (n+Ab) or TL-1β and +sAb/^ChE conjugate, followed by AChE. Cell culture, characterized in that the activity is made visible by any suitable means. Advanced Detection of IL-1α and IL-1β in Nutrient Supernatants and Biological Fluids This is a specific and sensitive enzyme immunoassay method.

According to aspects of these enzyme immunoassays, IL-1α or IL-1β, b/^ChE conjugates were added to the antibodies at the same time, and the two monoclonal antibodies were Reacts simultaneously with L-1α or IL-1β.

According to other embodiments of these enzyme immunoassays, IL-1α or IL-1β first introduced and reacts with the antibody, then the Sho^b/^ChE conjugate is subsequently introduced and make the ChE activity visible by appropriate means.

Suitable supports are in particular microtitration plates or particles to which the antibody can be bound. It is a support in the form of

Furthermore, the problem of this invention is to A series of microtiter plates coated with rabbit anti-(mouse) IgG antibodies. To; Proper dosage of anti! L-1α monoclonal antibody and/or anti-IL-1β antibody At least one IL-1α and AChE conjugate containing a noclonal antibody. at least one bottle containing and/or a conjugate of IL-1β and AChE. at least one bottle containing; Appropriate amount or dose of substance that makes AChE visible! L-1α and and/or an appropriate amount or dosage of IL-1β. This is a kit for performing a mirror table immunoenzyme test according to the invention.

- Also, the subject of this invention is an anti-IL-1α monoclonal antibody and/or an antibody A series of microtiter plates coated with IL-1β monoclonal antibodies: Conjugation of AChE with anti-IL-1α and/or anti-IL-1β monoclonal antibodies At least one bottle containing the body: Suitability of the substrate to make ChE visible IL-1α and/or IL-1α in an appropriate amount or dose A kit for carrying out the enzyme immunoassay method of the present invention, which is characterized by comprising lβ. It is a cut.

The object of the present invention is to further provide an anti-IL-1α monoclonal compound of the present invention as an active ingredient. null antibody and/or anti-I-1β antibody and/or fragment thereof alone, or or a conjugate or hybridizer or conjugate with pond material; It is a therapeutic reagent characterized by containing or containing these substances.

According to this invention, the anti-IL'-1α and the anti-! Shiichi 1β monoclonal antibody A particularly advantageous therapeutic use of is its 1-1 inhibitory activity.

The object of the invention is also to use antibodies and/or hebutan and/or other antibodies. or in combination with antibody fragments and/or labeled with stable or radioactive labels. anti-IL-1α antibody and/or anti-IL-1β antibody or a fragment thereof This is a diagnostic agent that can be used for in-vivo diagnosis.

This invention includes aspects other than those described above, which will be made clear in the description below. It will be something like that.

The present invention refers to the accompanying drawings (Figs. 1 to 7) which graphically illustrate embodiments of the invention. It will be more easily understood from the description given below with reference to .

Figure 1: Illustration of IL-1α when comparing two different pairs of monoclonal antibodies. Nonspecific effects of serum on the immunoassay standard curve.

Figure 2: Changes in the calibration curve for IL-1β and time due to enzymatic reactions (eye visualization stage).

Figure 3: ``Error profile'' in the IL-1β and time test due to enzymatic reactions. Changes in lofil° (visualization stage).

Figure 4; Human mononuclear cells, lymphocytes and neutrophils not stimulated with LPS. (mouth, -) and those stimulated with IPS (◆, ◇) were purified by centrifugal water straining method. IL-1α (mouth, ◆) and IL-1 performed with specific E1^ in the obtained supernatant In vitro release of β (■, ◇).

Figure 5: Superose 12” (SUPERO3E) of culture supernatant or plasma 12) Distribution of rL-1α immunoreactivity by molecular sieve chromatography.

FIG. 6: Detection limits of prior art kits using competitive assays for IL-1α.

FIG. 7: Detection limits of prior art kits using the 1L-1β immunoradiometric assay.

However, these embodiments and drawings only illustrate the subject matter of this invention, and are not limited to this invention. It should be clearly understood that this is not intended to limit the scope of the invention.

Example 1: Conducting tests of the invention: Preparation of reagents (monoclonal antibodies and conjugates) Parameters for preparation and reagent selection [Purification and acetylcholinesterase] Controlled Electric Eel [Electrophorus Electricus] Acetylcholinesterase (ACh Recite E) ＾5 SOUL I E and BON reported the method [Eur, J, Bi (1976), pp. 68.531-539] Purified by stomatography. The tetrameric form of the enzyme or the G4 form can be interleukinized. It was used to label proteins and antibodies. ^ChE activity was determined by ELLMAN et al. Colorimetric method [BIOCIIEM, PHARM, (1961), 7.88 = 95 pages ]. ! The Elman unit is 2 with a medium of 1112 and an optical path length of Lcs. It is defined as the amount of enzyme that causes an increase in absorbance of 100 in 1 minute at 5°C.

This unit corresponds to approximately 8 ng enzyme and 7.32 enzyme units (1 enzyme unit is the amount of enzyme that can hydrolyze 1 ulol of acetylcholine per minute at 25℃ ). The concentration of AChE was 4.4X lo'mol/h of catalyst per site. It was determined enzymatically using a constant and a molecular weight of 80 KDa for the catalytic subunit.

From these values, a detection limit of 1.8 atmol (101 mol) of the enzyme was determined for form 64. (i.e., 0.5 cm in 20 μe Ellman medium) amount of AChE that results in an increase in absorbance of 0.010 D in 1 hour with a path length of ).

2. Immunization and hybridoma production method Anti-interleukin-1α and anti-interleukin-1β antibodies are - Biozzi H4gh Re5ponder (HR) mouse It was produced by At zero 0, 15μ9 recombinant interleukin-1α or 1β was emulsified in Freund's complete undivanted solution and added to the sole of the mouse's foot. injected into the thick wall of This mouse was then aspirated to avoid side effects such as hyperthermia. Treated with phosphorus (0, lu/day). First booster injection (thick sole of foot) It was performed on day 21, and blood was collected one week later. Corresponding mouse anti-interleukin antibody The presence in the serum of the antibody is determined by the binding of the antibody to the interleukin-1/AChE conjugate. The performance was monitored by testing. These tests are then hybrid Utilizing the same method used to screen the supernatants of tumor cultures, Second lft (vtx) Performed on microtiter plate coated with IgG antibody did. For each interleukin-1, the map showing the highest titer in the enzyme immunoassay mice were selected for production of monoclonal antibodies. At this stage, 1/1. A titer of 0.000 to 1/10.000 was observed. Intravenous injection into selected mice. -Star injection (7 μ2 interleukin) was performed 2-3 days before performing the fusion described below. Ta. The corresponding mouse evaluation as described in the report of GRASSE et al. Cells were fused with NSI mouse myeloma cells.

3. Labeling of interleukins with AChE interleukin-1β, bovine acidic growth factor (aPGF) and rhabdoprolactin. Using known techniques for labeling heterobifunctional reagents, i.e. Succinimidyl 4-(tomaleimidomethyl)cyclohexane-1-carmeboxy It was covalently linked to AChE by sylate (SMCC).

This method uses a thiol group (previously introduced into interleukins) and 5v It consists of a reaction with a maleimide group incorporated into the enzyme by reaction with cc. stomach Primary amino group of interleukin and tosuccinimidyl S-acetylthioacetate interleukin C5ATA) in an alkaline medium. Kin was thiolated.

Incorporation of monothiol groups into interleukins: 2, including 5ATA of 'lu/touge. lOμQ of anhydrous dimethylformamide (DMF) solution with 0.1M boric acid Interleukin-1α or Add to 100 μ9 of Fuki β. This mixture was allowed to react for 30 minutes at 25°C and then Add 200 μQ of a 1M solution of hydroxylamine (pi(7); After reacting at 25°C for minutes, excess reagents (5ATA and hydroxylamine) were removed. Equilibrate with lO-IM phosphate equilibration solution (pH 6) containing 5xlO-”M EDTA Molecular sieve chromatography on Sephadex G-25 column (15X1cm+) Remove with graphics. Before and after chromatography, the eluent removes dissolved oxygen. removed and kept under continuous nitrogen flow to avoid possible oxidation of the thiol groups.

Both parts containing the thiolated interleukin are combined. at 280ns Is the extinction coefficient I? -'Lee-' has a molecular weight of 17. Assuming that it is Goo, the ultraviolet component Interleukin concentrations were measured by photometry. Its thiol group content is 0. After reacting with 5mM 3.5°-dithiobis(nitrobenzoic acid) (DTMB) , measured by colorimetric method (412 nm). These measurements show that interleukin It was found that approximately one thiol group was incorporated per molecule.

Incorporation of monomaleimide groups into AChE and thiolated interleukins The combination of: A maleimide group was introduced into AChE (G4 form) by reaction with SMCC. , the resulting ChE-8MCC formulation should be frozen at -80°C if not used immediately. It retains its reactivity for at least several weeks. Thiolated in Tarleukin and AChE-8MEC (G4 type) preparation are molecular sieve chromatographs. Mix the two as soon as possible, either immediately after isolation or after thawing. The two were combined by At this stage (SH-interleukin/G4 -SMCC) molecular ratio of 50:1 was used. After reacting at 30°C for 3 hours, unreacted Interleukin was added to a 0.5M Biogel A column (9 Removed by chromatography using 0x1.5c). The obtained conjugate was 12 Store frozen at 0°C.

No loss of enzymatic activity was observed during the entire conjugation process.

Under these storage conditions for several years, no significant immunological or enzymatic properties of the conjugate were observed. No changes occurred at all.

4. Monoclonal anti-AChE labeling monoclonal antibody (*Ab ) is precipitated with caprylic acid and ammonium sulfate by methods described in the prior art. It was purified from ascites fluid by The purity of the resulting formulation is determined by denaturing conditions and reducing Tested by polyechrylamide gel electrophoresis under the following conditions.

Two labeling methods were used to link purified sAb to AChE.

4 a) Labeling of g+Ab with biotin Activation of biotin N-hydroxy Succinimide ester (IBF, France) was reacted with the primary amino group of the antibody. Biotin was covalently bound to so^b as described above.

The above activated ester is dissolved in anhydrous dimethylformamide and labeled with the antibody. of alkaline solution. D containing 5 mg/mQ biotin ester 40μe of the solution with MF was dissolved in 2zQ of 0.1M borate buffer (pH 8°5). 11119 antibody. After 30 minutes at room temperature, the 2zQ EIA slow A buffer solution (defined below) was added. The resulting formulation was purified without further purification. It was used to determine the "binding incompetence" of various -Abs. This preparation is frozen and stored at -20"C.

4b) Covalent labeling with Fab’ab’AChE Labeling of antibody fragments with other enzymes The method announced by HIKAIA et al. (J, IMMUNOAssAY , 4@, pp. 209-327, 1983). ,+nAb was covalently linked to AChE by Fab'ab'SMCC. use The principle of Tako’s method is that the thiol group involved in the binding reaction )Fab'ab'obtained by reduction of the s fragment, except that naturally occurring It is very similar to the method used to label interleukins described above. Ru.

・Production of F(ab’) fragments The F(ab')y fragment was obtained from mAb in acidic medium (acetate buffer, pH 4.3). Obtained by treatment with pepsin. 0,1 containing 5xlO-'M EDT^ 0,5+*Biogel A equilibrated with M phosphate solution (p) (6) Molecular sieve chromatography using gel A) column (30X 1.5cm) F(ab')1 fragments were isolated from the crude pepsin-treated preparations described above. F( ab’) fractions were determined by polyacrylamide gel electrophoresis under non-reducing denaturing conditions. Checked with motion method.

These measurements indicate that more than 80% of the total protein content is composed of F(ab')y fragments. It was shown that

・Fab’ab’ manufacturing The F(ab’)* fragment was treated with O, 1M β-mercaptoethylamine (βMEA). Reduction was carried out for 1 hour at 37°C in the presence of As mentioned earlier about thiolated interleukins, Solid, Sephadex G25 column (30x 1.5c+e) molecular sieve Excess β-MEA was removed by chromatography. Fab'ab' concentration, 28 The extinction coefficient at 0 nm is t, 4ag-', Lca+-', and the molecular weight is 46. OO It was measured by ultraviolet spectroscopy assuming that the temperature was high. The thiol group content of Fab' drug is As in the case of thiolated interleukin, it was measured by reacting with DTNB.

Depending on the mAb used, a value of Sl+ groups of -4 Fab' molecules was obtained.

・Incorporation of maleimide group into AChE and coupling of Fab'ab' The maleimide group was converted to AChE (G 4) was introduced. Coupling enzyme and Fab'ab', ^ChE-3M CC formulation (immediately after isolation of this formulation by molecular mesh chromatography or thawing) ) and excess Fab'ab'. molar ratio of 5o At this stage, a commonly used L''le (tNawachi Fab'+7) thiol group/G4-S MCC). After reacting at 30°C for 3 hours, use a 0.5m Biogel^ column. G4/Fab'ab' was isolated by molecular sieve chromatography. conjugate was eluted in the form of a single homogeneous peak. Collect both corresponding aliquots and freeze at -20°C. It was stored in a frozen state. No significant loss of enzyme activity was observed during the entire conjugation step . The stability of the conjugate was proven to be excellent. The reason is that the conjugate is 120' The enzyme properties of C. Or because it could be stored without losing its immunological properties.

5. Screening of culture supernatant The presence of anti-interleukin-1 antibodies in the supernatant of hybridoma cultures was previously demonstrated. was detected using a method similar to the immunoenzymatic test. This culture supernatant was added to the second antibody ( Mouse) coated with gG antibody and interleukin-1/AChE conjugate The cells were cultured in microtiter plates. During this step, the anti-infusion present in the supernatant Tarleukin-1 mAb binds simultaneously to IL-1/AChE conjugate and solid phase thus resulting in indirect immobilization of ChE activity to the plate. Furthermore, in the solid phase The presence of AChE is determined by adding substrate and measuring colorimetrically. (after washing) could be visualized. The manufacturing method and characteristic values of the second homologous antibody are known 50μQ of each supernatant present in a 96-well microtiter plate was added to the second Transfer under sterile conditions to an identically sized microtiter plate coated with antibody. Ta. Interleukin-1/AChE conjugate dissolved in EIA II buffer (1 hour 50 μC of Mann units IRQ) was added and the resulting mixture was allowed to react overnight at +4°C. Ta. After completing this first reaction phase, carefully wash the plate and add 20 0 μg of Ellman's reagent was added. At this stage, the ChE activity bound to the solid phase is The wells containing IL-1 develop a deep yellow color, indicating that anti-IL-1 antibody is present in the corresponding supernatant. It was shown that there is a The absorbance of each well at 414 nm was measured using an automated plate reader. - (T ITERTEK, Finland) for 30 minutes or 1 hour. was measured. This screening method requires mAb amounts on the order of 1 nanogram. Hybridomas can be selected very quickly and effectively if they are easily detected. Can be done. One person is required to screen 2,000 supernatants. This requires at most 4 hours of work by the measurer.

6. Determination of binding compatibility of each pair of mAbs The binding compatibility of two different mAbs to one molecule of interleukin-1 was One mAb is immobilized on a solid phase and the other mAb is labeled with a biotin molecule. Measured by test method. These tests were conducted as follows.

All dilutions were made in E1^ buffer, followed by recombinant interleukin-1α or is 100μ of a solution containing 100nf/i+e of recombinant interleukin-1β. g was added to the wells of one of the transfusion b-coated microtitration plates. stirring After incubating for 1 hour at room temperature with stirring, add the other biotin-labeled mAb. 100 μg of a solution containing lθμ9/mQ was added. At room temperature with further stirring After 3 hours of reaction, the plates were carefully washed. With biotin on a solid phase, The presence of labeled antibodies is detected by adding a mixture of avidin and biotinylated ChE. This made it visible. Non-specific binding of AChE to the surface was inhibited by in Control experiments using 100 μQ of buffer instead of tarleukin-1 were evaluated.

■^b immobilized on the solid phase and the biotin-labeled mAb were bound simultaneously. In some cases, a deep yellow color developed upon addition of Ellman's reagent.

? , immunoenzymatic assay for detecting IL-1α and IL-2β interleukin-1α We developed two types of EIA for 1β and 1β.

Monoclonal antibodies derived from culture supernatant or ascites are first used as tracer. was tested in a competitive immunoassay using the jL-1/^ChE conjugate as a conjugate. 2nd stage In step 1, the anti-interleukin was coated with a solid phase or labeled with AChE. An immunoassay using mAb and mAb simultaneously was performed. Reagents used for this immunoassay All drugs were diluted in E1^ buffer as described above. The composition of this buffer is 0゜4M ) fact, 10-”M EDTA, 0.1% BSA and 0.01% azyl This is a 0.1 M phosphate buffer (pH 7.6) containing sodium chloride.

All concentrations mentioned for the immunoassays above are based on the initial volume before mixing other reagents. ” (50 μQ for competitive assays, 100 μQ for immunoassays) Refers to the concentration of reagent.

Competitive immunoassay using -It, -1/AChE conjugate.

Combination of quotient^b as the first antibody and IL-1/^ChE as the tracer Conventional competitive immunoassays using It was carried out in the same way. These assays use rabbit anti-(mouse) IgG The test was carried out on a 96-well microtitration plate coated with a secondary antibody consisting of: this The second solid phase antibody separates the bound and free fractions of the tracer during a specific immunoreaction step. separation. The total reaction volume was 150 μQ, but each component (tracer, m Ab and recombinant IL-1) were added in a volume of 50 μe. IL-1/^Ch The conjugate of E was used at a concentration of 1 Leman unit/MQ. Working dilution of seagull 1) can be predicted by working with antibody dilution curves from culture supernatants or ascites fluid. It has been decided. The sensitivity of this test method is characterized by the dose of IL-1; Is the bond observed in the absence of coalescence reduced by 50% (B / B o = 50) or a statistically significant reduction in the binding observed in the absence of competitors. [i.e. Bo-3fi standard deviation (99% confidence limits)].

- Immunoassay using 5Ab/^ChE conjugate.

Immunoassay for 96-well microtitration coated with one of the monoclonal antibodies It was carried out on a plate. This coating is described above for the second solid phase antibody. It was carried out exactly as described above. In summary, the wells of the microtitration plate are , 5 x 10-1M phosphate buffer (pH 7,4), : Dissolved 10u9/112 (7) Filled with 200 μQ of solution containing mAb. - after reacting at room temperature overnight 101 phosphate buffer (pH 7,4) containing Tween 20, O,OS% , the plates were carefully washed. Next, 300 μe of E1 at room temperature for at least 4 h. The solid phase was saturated by adding ^buffer to each well. the resulting play The samples were stored in this saturated buffer at 4°C until use. stored under these conditions No change in binding properties was observed for at least 4 months. Exemption The disease test was performed using two different methods.

The first protocol (named “simultaneous protocol”) is a 100μQ interface. - Leukine-1 solution (introduced in the form of a recombinant standard or sample) ,! 00 μQ of 1^b/^ChE conjugate (usually at a concentration of 0 Ellman units IRQ) The protocol consists of adding the microtiter to the wells of a microtiter plate.

In this method, two antibodies (a solid-phase antibody and an AChE-labeled antibody) are used to interact with each other. -Reacts simultaneously with leukin-1. Depending on the nature of the test sample, standard recombinant Interleukin-1 can be added to the corresponding diluent, i.e. culture medium, plasma or serum. Dissolve. All diluents used should contain as much interleukin-1α as possible. Alternatively, it must lack -1β. This means that plasma or serum Accordingly, immunoassays showed that significant amounts of 11. -1α or ■L-1β is detected. This is possible by selecting individual liquids that are not included. EIA in these media When performing the test, add mouse immunoglobulin (1/20 to Ehrlich's ascites fluid). (introduced at a dilution rate) are used and added during the immune reaction to increase the amount of human present in these fluids. Neutralize anti-mouse antibodies. Non-specific binding was measured using 100 μQ of buffer or Test in separate wells where appropriate diluent was added in place of recombinant interleukin-1. I valued it. Then, at various times and at several temperatures, the reaction is I let it happen. After this immunoreaction step, the plate was washed with phosphate buffer/Tweet. Cleaned carefully using a water bottle. The AChE activity bound to the solid phase was It was measured by adding Luman's reagent.

The second protocol (named 'sequential protocol') -1 and the mAb/AChE conjugate separately. be. During the first incubation period, EIA buffer or other suitable diluent (medium, plasma Interleukin-1 (introduced in standard or sample form) dissolved in interleukin-1 (e.g.) 200 µQ of the sample) is reacted with the solid phase. After this first incubation, play Washed and E! mAb/ChE conjugate dissolved in buffer (usually 10 min. Add 20h12 (concentration in unit of ion/Iff). After the second reaction period, remove the plate. Wash again and visualize ChE activity as described above.

The “error profile” of the standard curve was calculated for all measurements (non-specific binding and standard measurements). 8 times. For each dose, the precision was calculated by the coefficient of variation (CV) %) in the form of a curve as a logarithmic function of dose.

The sensitivity of an immunoassay is characterized by the “minimum detectable concentration” (MPC), which The concentration is standard recombinant! The observed connectivity in the absence of shi−1 is calculated statistically. This corresponds to a concentration of IL-1 that significantly increases. MDC has non-specific binding +3 Calculated as the dose of IL-1 corresponding to x standard deviation (99% confidence limits) Ta.

8.FPLC test Detected by immunoassay in biological media (culture supernatant, plasma) Immunoreactive substances are transferred to the FP, LC device, and a superfluous solution equilibrated with El8. Fractionate the sample by molecular sieve chromatography using a Rose 12 column The characteristics were determined by The standard recombinant IL-1 and the sample were mixed at 500 Inject with a volume of μQ and a flow rate of 24 m12/hr, and collect fractions of 0.8 mQ each. I met. When the chromatography was completed, 100 μe of each fraction was Una IL-1α and/or! It was tested by carrying out the 1β test.

- Eluted cell supernatant Mononuclear leukocytes (lymphocytes and monocytes) and peripheral blood polymorphonuclease morphonuclear) white blood cells (neutrophils), F IC0LLPAQUE Centrifuge using (PHARMACI^FINE C) IEMICALS) Pure lymphocytes, monocytes and and neutrophils were obtained. After this, mononuclear cells or polymorphonucleas - Cells were placed in the separation chamber of a Beckman (JE-6) elution rotor at 8 or 20xQ. Each injection was performed at a constant rotor speed of 2.50 Orpm at a flow rate of /min. elution The buffer was Dulbecco's saturated P supplemented with 0.25% BS8 and ZmMEDT^. It consisted of BS solution. The plate was inserted 1 hour and 30 minutes after elution. Washed at flow rate. Fractionation of mononuclear leukocytes was performed as follows. pure lymphocytes is to increase the flow velocity from 8 shoulders Q / m i n to 20xf) / n + in So I got it. 50m+! of media was collected at each stage and centrifuged (350 x g, l0m1n), then the cell pellet was washed once in an isotonic solution and finally Medium (L-glutamine, 75% fetal bovine serum and penstrep (pens RPMI 1640) containing L rep). pure lymphocytes Collect the monocyte fraction (monocytes <0.5%) or the enriched monocyte fraction (lymphocytes <20%). and adjusted to 10×IO@ or lXl0@cells/jlQ, respectively. flow rate From 201112/sin to 25xQ/uln by 0.25t12/sin By elevating and collecting the SaQ fraction, neutrophils are free from contamination by monocytes. is gone. The rotor was then stopped and pure neutrophils were collected in a single fraction. . Both parts of the neutrophils were washed once with isotonic solution and finally washed with 20X 10@cells/pass. Resuspend in medium. Culture in 12x 72 mm propylene culture tubes for 37 The experiment was carried out at 5% CO in a humidified atmosphere at 10°C. How to follow a differential leukocyte count It was done by

(1) Flow cell measurement method using dispersion characteristic values of acute-angle light and right-angle light (2) Esterase Non-specific staining method using Wright's dye (3) Monochrome conjugated with fluorescein Immunofluorescence using null antibodies, and (4) An analysis method using a phase-shifting microscope for platelet contamination.

Cell viability was determined as usual for each experiment using the Trivand Blue exclusion test. I went to the store and it was greater than 95%. Cells were treated with 5 μv/xQ ribopolysaccharide (LPS). ) was cultured both in the absence and presence of 0.1 μQ i Performed in the presence of domethacin. At different time intervals, centrifuge the medium for 60 min. 0x9°110 min, 4°C), immediately frozen at -20°C, and using the selected E1^ ! The cells were stored as they were until IL-1α and IL-1β were measured.

Example 2: Anti-11. ,-1 Characterization of monoclonal antibodies and implementation of immunoassays For each interleukin, the serum had the highest titer in the immunoenzymatic assay. selected mice were used for fusion. The corresponding 4° visceral cells were prepared as described above. fused with NSI myeloma cells. One pharyngeal period after fusion, approximately 500 wells are There were hybridomas that proliferated rapidly against both interleukins. . The presence of mouse anti-IL-1 antibodies indicates that their corresponding IL-1 culture supernatants by testing their ability to bind /AChE conjugates. All confirmed. - In the next screening, 90 and 116 strongly positive cultures The nutrient supernatant liquid is ■1, -. 1α and IL-1β were detected. dilution Subcloning the corresponding hybridoma and distributing the antibody by restricting the We obtained a secreting cell line that is stable and unique to each culture. -ta. Finally, cell lines 36 and 11 were prepared for TL-1α and 1C1β, respectively. It has stabilized. Clones are derived from inter (α or α or All chronos were characterized by the number before the letter β. Clone as ascites 11 pus monoclonal cells by culturing and precipitation with capric acid and ammonium sulfate. The antibody was purified from ascites fluid. By Ophterlony's double diffusion method, the isotai 1soLype was measured from ascites fluid. All mAbs were 1 (zero IgG with light chain) However, Subclass II Table: Results obtained by using anti-IL-1α (primary antibody) Compatibility test results MAI3 with tag Binding compatibility testing was performed as described in Example 1.

The black circle indicates that the 1^b immobilized on the solid phase and the floating 1^b combine at the same time. A pair in which this was observed is shown. For clarity of presentation, ^b with the same definition is They were grouped into three categories (8-C).

Iris gG, 餉^b璽00 (Δ group), and 5Ab50 and 176 (13 groups All rings are sAb of subclass IgG1, except for

Table 11: Results of binding compatibility test for anti-IL-1β monoclinal antibody Mackerel with a tag ^B Binding compatibility tests were performed as described in Example 1. A pair in which a defined -Ah and a labeled audience b are observed to bind simultaneously. show. For clarity of presentation, mAbs exhibiting the same type of binding are grouped into four categories. They were divided into groups (8-D). The 1gG subclass for age 1) is shown in the last column. It is.

Both culture supernatant and olfactory fluid were conjugated with II, -I/^ChE as tracer. The body was used in the 1° ring gold immunoenzyme assay. In most cases, (except for β2G and β54), the standard curve was calculated using the recombinant mouth, −1 It could be created using α and IL-1β. Define B/+30=50% In fact, the sensitivity of these assays is 4-100nf/ml! It is. In the best case, It was possible to calculate the minimum assayable concentration of Inf/112. The gold award^b is this showed that they are very specific to the corresponding inter (Zikin). It is highly unlikely that there is any cross-reactivity between IL-1α and IL-1β. This is because we were able to measure that it exists in a much weaker manner. These for 1 and 1α Some features of competitive assays and the use of sheep or rabbit polyclonal antibodies The results obtained in the body are shown in Table 2 below for guidance.

Table ■: Monoclonal antibodies and IL-1α/^ that appear to be Voriclor antibodies Use ChE conjugates, 11. Main features of -1α ring immunoenzyme assay method Umbrella 11/Ro: 50% is the trace observed when there is no competitive expression. It is equivalent to a dosing sentry that reduces the binding of sir by 50%.

The essential aspects of this invention (1-) are 11. −1 α and 1 sea lβ. The first step was to develop an immunoassay method. In other words, at the same time as each interleukin It is appropriate to determine which pairs of mAbs can bind. Complementarity of binding These tests involved immobilizing one of the -Abs on a solid phase and immobilizing the other mAb with biodin. The test was carried out by children with tags attached. Labeling with biotin was chosen at this stage. . The reason is that it labels a large number of mAbs (over 40) in minutes. This is because the second method is the preferred and simplest method. In addition, avidin and biotin The next step is to use a mixture of ACh labeled with sensitive detection of fluorinated antibodies can be performed.

Tables 1 and 0 above show these values for IT, -1α and irises 1β, respectively. The results of the offset test are shown. In the case of wealth 1, - Iku α, there are three different regions of the ■shi 1 α molecule. It can be seen that there are three groups of -^b that recognize epitopes occurring in the region. these groups are called 8, B and Cn, but they are black and white of 10.25 and l, respectively. It has an adult body. All Iit mAbs are compatible with all other 2n mAbs. Although it shows combinable binding properties, it appears that the compound binds simultaneously with mAbs from the same group. It never matches. 11. For −1β, the situation is less clear. actually Four different groups are observed. The first 2ffl (called A and B, respectively 4 and 3), similar to that observed for IL-1α The logical behavior of and two mAbs that are compatible only with group B mAbs (β28 and β38, respectively). It's okay. Finally, I) Itl is a two-component compound that cannot be immobilized by any other solid phase-Ab. We have determined all the phantom mAbs that can exhibit ^b, ,'a combined binding properties. Later, the optimal properties were determined from the perspective of developing sensitive, specific, and reliable immunoassays. There is a 4 degree group 1 problem in which the pairs shown are selected for each interface. This selection is Shared-^b/^ChE conjugates (and bi1,000) are not TFI-recognized mAbs ) is the reason these conjugates interlock This is because it is possible to more sensitively detect Ikin. In the case of IL-1β, 29Pp, This selection is easy to implement since it has only one oily property. and the corresponding of 9-2 It was suggested to choose to produce a ^h/^ChE conjugate. But this This proved difficult in the case of fL-1α. That means there are 570 possible species. It has been confirmed that sex exists! In addition, 36 I^b/^chE conjugates were produced. This is because building a zuroko is a considerable amount of work. For this reason, the method used has been changed. That is, in the first stage, several types of +aAh/^ChF and foam coalescence were produced. were constructed and then tested on the entire relevant solid phase. Taking into consideration the corresponding results, the best Results (1^b/^ChE retention for 1 Ikin) Identify the mAb that yielded the highest binding binding capacity and conjugate it to the corresponding immobilized ) and continued to play 3 pieces with them. Therefore, antibody α5. α29. αl　 110, α1/l7 (group A), α176, α39 (BI′IT) and α+ o + (cltT) was tested with continuous I identification.

By this method, each 11. It is possible to show a small number of pairs for which −1 can be determined more sensitively. Now I can do it. , l[, -1α, the best result is αI (l I <as a tracer in the 11th edition), Mat 2 is a tracer of the Δ revision and a tracer of the 13th group. In the case of 6 T+, -1β, which is 1v# by holding the fixed tll, the fixed 1 Ill with β79 as 1 and β70/^ChE as a tracer! +c was found to be significantly superior to all other pairs. optimal The final selection of pairs is determined not only by the strength of the signal but also by the the lack of cross-reactivity with related substances, the level of non-specific binding, and the Including non-specific effects brought about by the biological medium (likely serum) This was carried out taking into account the standards of the pond. Specificity criteria are not mandatory. That's called The reason is that all of the pairs tested are similar to other interleukins (+Liα and rL− 1β and! This is because it shows very weak cross-reactivity with L-2). However, weak It is important to select pairs that exhibit specific binding.

The reason is that this connectivity has a significant impact on the test sensitivity 1f. in the same way , in biological media to enhance the validity of measurements made in these media. It is necessary to minimize non-specific effects. In the end, we selected the following pairs.

α185-)+1 on Zunawara solid phase. , α29-^ChE for Iα, and Solid phase, β70/^ChE for β79+IL-1β above. IL-1β In particular, this pair is characterized by very weak non-specific binding. The above selection was made because it shows very little sensitivity to quarter effects. rear Figure 1 shows the characteristics of the samples, and Table 8 shows the characteristics of the simultaneous method, which was cultured overnight at 1°C. The characteristics of the 4° filtration test, which detects ■+, -1α and II, -1β, are summarized. ing.

These two assays appear to be very sensitive. If so, 4βg/llQ. A small minimum detectable concentration was added to each batch using 30 minutes for enzyme measurements. [This is because we were able to calculate for Ikin. Enzyme reaction (III visualization film formation stage long If continued, an increase in sensitivity was observed. In such a case, a minimum lower than 2βg/*C Small detectable concentrations should be obtained (see Table 1 above).

(Margin below) The change over time of the calibration curve due to enzyme measurement is shown in the wtz diagram.

This increase in sensitivity is due to the increase in measurement accuracy at the bottom of the calibration curve, as shown in Figure 4. Therefore, the profile of the error is changed over the course of 1 hour. Results (for sensitivity) were obtained using overnight immunoreactions at +4°C. But good Calibration curves were obtained using short reaction times (3-5 hours) at +4°C.

In all cases, a minimum detectable concentration of less than lOpq/*I2 was observed. . Room temperature too! jPerforming the assay at 37°C has no effect on these results. stomach.

Example 3; 11. in culture medium. Measurement of -1α and IL-1β in stimulated medium: Measurements of two interleukins in the RN fluid were performed using a calibration curve with J≦. , The presence of 10% fetal bovine serum in the medium adversely affects the slope of the calibration curve. It should be noted that non-specific binding is significantly reduced without any loss.

These test methods have shown that these assays can detect eye 11-like immunity in the supernatant of cultures. The presence of immune-reactive substances can be detected, and the appearance of immunoreactivity can be used as a stimulus for a small amount. Seki i! ! It turned out that it would work. A typical example is shown in Figure 4. U9/I (!) ribopolysaccharide-stimulated, eluted non-human white L1L cells (monocytes, lymphocytes or neutrophils) in the supernatant, at concentrations of -1α and II, -1β. This shows the variation over time.

To confirm the validity of the measurements performed, the same sample was tested by mouth.

Azusa @# stimulated by both -1α and mouth, -1β, P G F by blast cells! * was tested using a bioassay based on measuring the release of Corresponding school A positive curve is shown in FIG. The results obtained are shown in Figure 6, and these results are The immunoassay method was qualitatively confirmed and the specificity of the immunoassay method was demonstrated.

Substances detected in culture supernatant ei (presumably intracellular cell extract) and calibration The identity of the recombinant IL-1 used to establish the curves indicates that 3 This has been confirmed in a controlled type of experiment. The first experiment consisted of Although this was an experiment in which the sample was diluted, a flat i'r dilution curve was given to the calibration curve. This conclusion The results are shown in Table 7 below, and the results are shown in Table 7 below. Showing the results of two 1+'4 tests for 4°. Similar results were obtained for the samples tested Obtained for all.

Table v - Illustration of the similarity between the calibration curve and the curve for diluting test t1 and l2 Dilute B to the sample as shown in Table 7, and use the ElA method to mix IL-1α and 11. -1 A test was conducted to detect β. Both the dilution curve and its calibration curve are based on the medium (rtr’M +110% fetal bovine serum). Calibration curve and sample dilution To clearly show the similarity to the curves, the raw data were multiplied by the dilution factor and shown in the table above. did.

Sample Δ: supernatant of alveolar macrophages in culture stimulated with 'rNFα liquid.

Sample B: Intracellular extract of alveolar macrophages in culture.

Although these test results have been confirmed by recovery tests, This shows that known recombinant IL-1 can be effectively measured.

Recovery levels were between 86% and 93% throughout the tests performed in culture.

The third test was carried out using the culture supernatant after fractionation by molecular sieving (l-madography). This is a test consisting of IF. This analysis shows that immunoreactive substances are As shown, the peak is eluted in the form of a single homogeneous peak; This exactly matches the peak observed for the prey interleukin. This is 8th, 7th As shown in the figure, this figure shows 2. A test to detect 2+α and a test to detect It, -1β. In both experiments, we obtained information on stimulated alveolar macrophages and histolymph fluid. Also The profiles of different C17 tographs are shown. Similar j sword results, other Observed in culture supernatants or intracellular extracts. Looking at it as a whole, All of the data from these authors, also by two test methods, were This clearly shows that interleukin-1 can be effectively quantitatively measured.

Example 4: Measurement of IL-1α and IL-1β in plasma and 1111 serum A different E1^ can be used to test healthy donors or patients with rheumatoid disorders. A test was conducted to measure IL-1α and IL-1β in human plasma or serum. Ta. These measurements, as described above, are based on the determination of the amount of human anti- mouse)+1 (G) in the presence of G to also neutralize antibodies.

This caution is very necessary. This is because according to Ike's experiment, there was no mouse IgG. Measurements carried out in particular in body fluids of patients suffering from rheumatoid disorders. -1 This is because it has been found that the concentration of can be significantly overestimated.

Plasma from healthy volunteers is detectable by tests performed on serum. The ability to select fluids that do not contain active heavy IL-1a or II-1β I found out. These collected plasma or serum samples are used to establish an appropriate calibration curve. It can be used as a diluent for Once we establish a curve like this, we get this The measurement of 4% of II, -1 in the fluid of patients and both apparently healthy donors and patients. Measurement of interleukin concentration of 5 pq/t12 to several 1+g/ytQ in humans It is now possible to do this. The immunoreactive substance is transferred to the recombinant body 11,1. Molecules much higher than the corresponding molecular weight! Eluted at R (>500.Goo) Therefore, the results shown in Fig. , although not sufficient, it is nevertheless possible to measure II,−1. The test method is suitable for the assay of +fl plasma and +Iurn.

Example 5: Effects of the present invention on the biological activity (neutralizing activity) of IL-1 Therefore, the inhibitory activity of the anti-1 of this invention against the biological activity of 11,-1 1. -2 series of test methods to determine the inhibitory activity conferred by monoclonal antibodies. It was shown using

1. Test for inhibition of IL-2 receptor induction This test method was developed by KAY ['; et al. , Journal or Immunology, 133@, No. 3 +339- 1345! , 19R4 Comb.

-Principle: Jurka L cells, e.g. (a cell line derived from human T lymphoma), are phytohemagglutinated. In the presence of tinin (1) IIΔ), 11. Stimulated by -1α or 1-1β and secretes 1 and -2. Some anti-IL-1α or anti-IL-1β antibodies can inhibit the effect of II, -1. This is because these antibodies A complex of 1 or 1 antibody that recognizes the site involved in binding to the protein This is because they form and inhibit biological processes.

- Experimental method: Anti-+1 of this invention. -1α or anti-Iris-1β antibody, IL-1α or IL-! Cultured for 18 hours at 4°C in the presence of β (10n9/ml). Control test , IL-1α or 1β (10n9/true 0), the same except that there is no antibody. This is done by culturing under the following conditions.

Next, Jurk*L cells (5XIO' cells/xi cells were 9/RQ') in a 5% CO2 atmosphere at 37°C for 24 hours with the various formulations listed above. Stimulate below. Under these experimental conditions, the concentration of ``Shi1'' at the time of stimulation is IB/mQ It is. In the supernatant of cell culture! The concentration of Sea 2 is based on the immune system developed at SPI. It is measured using an assay method, the principle of which is the same as that developed for IL-1. It is one.

The results obtained are expressed as percentage inhibition relative to two controls.

One was performed without I-1 (only to P11) and showed 100% inhibition. other It was performed without the other antibody and showed 0% inhibition.

(Margin below) One test result wealth 2. Inhibitory assays for binding of 1+, -1 to cellular receptors, especially rsm cells. Experimental method.

Test methods of this type are described in particular in the documents listed below.

-LowenLhal et al., J, 1x1), IE0.164Q, 1060 pages , 1986 and 13eckinger et al., The Jour+I or l+++munology, volume 139, pages 1546-1549, 1987 One principle: 11, -1α acts on cells through membrane receptors, and IL-1α acts on cells through membrane receptors. Labeled with +tsl, which specifically binds to! ! , -1α binding is tested. You can do something like a dragon. IL- whose monoclonal r1-nal antibody is included in the complex If it recognizes the part of the 1α molecule, this monoclonal antibody recognizes II, -1α. It can be inhibited from binding to receptors. inhibits binding to receptors By doing so, these antibodies inhibit biological effects.

- Experimental protocol: ``'It? [II with m, -1, with antibody at a concentration of 2G0n9/112 Both were incubated for 1 hour at 4°C. When the reaction was complete, the resulting mixture was incubated at +4°C. 8 hours, E! , 4 cells (mouse leg nematode cell line).

The cells are then washed with the appropriate treatment agent, and the bound radioactivity is removed with solid scintillary silica. Measure with Unku (1, KB multigrisma).

The results show that IIJ-labeled II,-1α was not incubated with the antibody. It is expressed as a percentage of light (100%) binding to light (100%) binding.

-Test results Example 6: Demonstration of the sensitivity of the monoclonal antibody of the present invention; using the previously described antibody Comparison test with the trial drive.

Example 2 above shows that in the optimal reaction of the test method of the second invention, the minimum detectable concentration is 2p9/RQ (see especially Table 2).

1. Comparison test: Comparison with the Mersham test method for the +17 test.

As mentioned in Example 1, the first sentence of the test method is observed when there are no competitors. Look! 1, a dose of 1-1 that statistically significantly decreased binding (+(o) [i.e. Bo-3e1 standard deviation (99% confidence eye circle)]1. .

The assay was carried out under conditions defined by AllershA- (Radioimmunoassay 1α). The curve shown in Figure 8 was obtained, but this In the figure, the horizontal axis is 11. -1α concentration (p9/stomach 0, vertical axis is B/rso (%), and this figure shows that the detection limit is on the order of 80 py-/glyceride. are doing. Therefore, this is the feeling of the test method of this invention described in -1-: The sensitivity is extremely low.

2. Immunoassay method: Comparative assay with MEIHI: NIX test, ODG): III X (immunoradiometric assay, 'IfJ-labeled anti-1C1β antibody) The test was carried out under the following conditions. The curve shown in Fig. 9 is obtained, and the horizontal axis is 1 seal lβ The concentration (pv/mQ) and the vertical axis is the bound radioactivity (cps). This diagram is It shows that the detection limit is on the order of 15 p97MQ. As shown in Example 1 The sensitivity of the immunoassay is equivalent to 3 standard deviations (99% confidence limit) for nonspecific binding. -1 dose type. This is the result of 2p9/”Q as mentioned above. This corresponds to a significantly lower sensitivity than that of the inventive test method of the order of magnitude.

These two test methods are based on the monoclonal antibodies of this invention (1 release is 5 x 1g). Unexpectedly, these antibodies were used by This clearly shows that the sensitivity of the assay test method can be increased.

As is clear from the above, the present invention is based on the implementation of the invention described in more detail. It is not intended to focus on the first invention and the method of implementing and applying it, but on the second invention. , any operation that the presenter could think of without departing from the scope or limits of this invention. Include changes.

Envy of art Tao Yasu Marrow hours Hiroshi international search report 11111-1-PCT/FR89100634 International Search Report FRB900634 SA　33301 -11-□1 heart l 89100634

Claims (13)

[Claims] 1. Mammals, especially rodents, more specifically mice, are treated with IL-1α or IL-1α. -1β, optionally natural or recombinant specifically recognizes IL-1α or natural or recombinant IL-4β, and shows virtually no cross-reactivity between IL-1 of IL-1α and IL-1β. a monoclonal antibody specific for It is composed of several groups that recognize different regions of IL-1α or IL-1β. , antibodies from some of these groups are able to bind compatibly to all antibodies from other groups. i.e. Anti-IL-1α antibodies recognize three different regions of inkleukin-1α3 It consists of groups (A, B and C), and all antibodies in one group are compatible with all antibodies in the other two groups. other antibodies of the same group are incompatible; Anti-IL-1β antibodies are composed of four groups, of which the first two groups (A and B) are anti-IL-1β antibodies. It behaves similarly to the 1α antibody, with group C antibodies being compatible with only one antibody from each of the first two groups. antibodies of group 4 (D) are incompatible with any of the antibodies of the first three groups and Rather than binding to recombinant native IL-1β, these antibodies recognizes IL-1β and reacts with IL-1β/AChE conjugate; and The anti-IL-1α and anti-IL-1β antibodies are IL-1α or IL-1β/α A model characterized in that it recognizes a cetylcholinesterase (AChE) conjugate; Noclonal antibodies. 2. Acetylcholinesterase and anti-IL-1α or anti-IL-1β mono Conjugates with clonal antibodies or fragments of these antibodies, especially Fab' fragments. 3. The anti-interleukin-1α or anti-interleukin-1β model of claim 1 A method for producing a Fab' fragment of a monoclonal antibody, wherein the monoclonal antibody is F(ab')2 fragments were obtained by treatment with pepsin in acidic medium, and these molecular sieve chromatography These F(ab')2 fragments were isolated by chromatography and Subjected to a controlled reduction step with a suitable reducing agent such as ethylamine (βMEA). This fragment was then passed through a molecular sieve chromatography column. A method for producing a Fab' fragment, the method comprising purifying it by filtration. 4. Conjugation of AChE with whole anti-IL-1α or anti-IL-1β monoclonal antibody and AChE and anti-IL'-1α or anti-IL-1β monoclonal A method for producing an antibody, in particular a conjugate with the Fab' fragment of claim 2, comprising; at least one component incorporated into one of the above fragments or naturally occurring; If the ol group is in particular a suitable cup such as N-hydroxysuccinimide ester, Linking with at least one maleimide group incorporated into AChE by a ring agent A method for producing a conjugate, characterized by: 5. Competition for detecting IL-1α and IL-1β in cell culture supernatants and biological fluids An immunoenzymatic test method; an appropriate anti-IL-1α or anti-IL-1α used as the first antibody; is used as an anti-IL-1β monoclonal antibody and a second antibody, and is IL-1/AChE conjugate that acts as a tracer to 18G retained in the carrier. and interleukin-1α or interleukin-1β, and characterized in that the activity of AChE is made visible by any suitable means. Test method. 6. Detection of IL-1α and IL-1β in cell culture supernatants and biological fluids. A highly specific and sensitive enzyme immunoassay method comprising; Anti-IL-1α and/or anti-IL-1β models carried by a suitable support Noclonal antibodies were combined with IL-1α or IL-1β and mAb/AChE conjugates. and then making the AChE activity visible by suitable means. Characteristic test method. 7. IL-1α or IL-1β at the same time as the mAb/AChB conjugate The two monoclonal antibodies are simultaneously added to IL-1α or IL-1α. 7. A method according to claim 6, characterized in that it reacts with 1β. 8. IL-1α or IL-1β is first introduced, and then the antibody conjugated to the support and reaction and then introduce the mAb/AChE conjugate to determine AChE activity by appropriate means. 7. The test method according to claim 6, wherein the test method is made visually visible. 9. A series of microscopic antibodies coated with antibodies consisting of rabbit anti (mouse) 1gG Titration plate; Anti-IL-1α monoclonal antibody and/or anti-IL-1β monoclonal antibody at least one bottle containing the appropriate dosage for the body; at least one bottle containing a conjugate of IL-1α and AChE; or at least one bottle containing a conjugate of IL-1β and AChE; Appropriate amount or dosage of substrate that makes AChE visible; a dose of IL-1α and/or IL-Iβ; Carrying out the competitive immunoenzyme test method according to claim 5, which comprises: kit for. 10. Anti-IL-1α monoclonal antibody and/or anti-IL-1β monoclonal antibody A series of microtiter plates coated with null antibodies; Combination of AChE and anti-IL-1α and/or anti-IL-1β monoclonal antibodies at least one bottle containing foam; a suitable substrate that makes the AChE visible; Amount or dosage; appropriate amount or dosage of IL-1α and/or IL- The immunoassay according to any one of claims 6 to 8, characterized in that it consists of 1β. A kit for carrying out the test method. 11. As an active ingredient, anti-IL-1α monoclonal antibody and/or IL-1α monoclonal antibody and/or IL-1α monoclonal antibody 1β antibodies and/or their fragments alone or conjugated or mixed with other components. It is characterized by containing or consisting of speciation or combination. A drug that has specific therapeutic uses. 12. antigens and/or haptens and/or other antibodies or antibody fragments. Anti-IL combinations and/or labeled with stable or radioactive labels -1α antibody and/or anti-IL-1β antibody or fragment thereof A diagnostic agent that can be used for in-vivo diagnosis. 13. Collection Nationale belonging to Pasteur Institut Culture de Microorganismus (CNCM) in December 1988 I-823, I-824, I-8 in this collection 25, I-826, I-827, I-828 and I-829. The anti-IL-1α and anti-IL-1β monochromes according to claim 1 or 2, A hybridoma used to produce local antibodies.