Monoclonal antibody recognizing membrane phospholipase A2 and immunoassay of membrane phospholipase A2

Abstract: The present invention first provides monoclonal antibodies recognizing membrane phospholipase A₂, namely, monoclonal antibodies PL-49, PL-71, PL-76, and PL-78, hybridomas producing them, methods for producing them, and immunoassays of membrane phospholipase A₂ using them. The immunoassay of PLA₂M is useful for the diagnosis of articular rheumatism, cancers, and a wide variety of inflammatory states.

Description: The present invention relates to monoclonal antibodies recognizing membrane phospholipase A₂; hybridamas producing said monoclonal antibodies: a method for producing said monoclonal antibodies: and immunoassays using said monoclonal antibodies. Phospholipase A₂ (PLA₂) (EC 3.1.1.4) is an enzyme which can hydrolyze the fatty acyl ester bond at the sn -2 position of glycerophospholipids. It is well known that the enzyme is present in the pancreas or snake venom, and it has been observed that the level of pancreatic PLA₂ in blood increases in patients suffering from pancreatitis (Ogawa et al., Gendai Iryou, 20 (1988) 3013-3017). The PLA₂, however, is not only present in the external secretion system, but also found in almost all of the cells in a living body, although the amount thereof is very small (Van den Bosch, H. in Phospholipids (Hawthrone, J. N. and Ansell, G. B. eds.) (1982) pp. 313-357, Elsevier/North-Holland Biomedical Press, Amsterdam). It is believed that the enzyme would play an important role in the metabolic regulation of membrane phospholipids and in the eicosanoide biosynthesis through arachidonic acid (Van den Bosch, H., Biochim. Biophys. Acta (1980) 604 , 191-246), and would relate to inflammation and cellular injury through the direct action or through its metabolites such as lysophospholipids, leukotrienes, platelet-activating factor and lipid peroxides (Vadas, P. et al., Lab Invest. (1986) 55 , 391-404). By analysis of the protein primary structure it was revealed that membrane PLA₂ (PLA₂M) isolated from human splenic membrane fraction was a type of PLA₂ which is different from pancreatic PLA₂ (Kanda, A. et al., Biochem. Biophys. Res. Commun. (1989) 163 , 42-48), and it was also found that PLA₂M was induced by an inflammatory mediator such as IL-1 and TNF and secreted out of the cells (Nakano, T. et al, FEBS Lett. (1990) 261 , 171-174). Moreover, a comparison between PLA₂M and PLA₂ purified from rheumatoid arthritic synovial fluid showed that they are identical in their structure and reactivity (Kramer, R. M. et al., J. Biol. Chem. (1989) 264 , 5768-5775). DeJong, et al. in European J. Biochem. 164 (1987), 129-135, describes monoclonal antibodies which have been raised against membrane-associated phospholipase A₂ from rat liver mitochondria. The monoclonal antibodies show cross-reactivity with rat liver cytosolic and solubilized rat platelet phospholipase A₂. From a clinical point of view, an increase of the PLA₂ enzymatic activity was found in the blood of patients with an infectious disease such as septicemia, pustular psoriasis, Crohn's disease, and rheumatoid arthritis. Further, it was found that the PLA₂ enzymatic activity is induced by intracutaneous injection of bacteria, viruses, or other inflammatory inducers into an animal (Vades, P. et al., supra ). To date, however, no report has appeared concerning the assay of PLA₂M, and it has not yet been shown whether an increase of the PLA₂ enzymatic activity accompanying the aforesaid diseases is caused by PLA₂M or not. As described above, because the PLA₂ enzymatic activity in blood is increased when suffering from rheumatism, septicemia, pustular psoriasis, Crohn's disease, or the like, it has been expected that the diagnosis of these diseases can be realized from the measurement of PLA₂M and an assay of PLA₂M has been desired. Therefore, it is the object of the present invention to provide monoclonal antibodies which can be used in an immunoassay of membrane phospholipase A₂. This object is achieved by the provision of monoclonal antibodies recognizing membrane phospholipase A₂, namely, monoclonal antibodies PL-49, PL-71, PL-76, and PL-78. The membrane phospholipase A₂ is preferably derived from human spleen. The present invention further provides hybridomas producing the monoclonal antibodies. The monoclonal antibodies can be produced by growing the hybridomas in the abdominal cavity of a mouse and separating the monoclonal antibodies from the ascitic fluid accumulated in the abdominal cavity. The present invention also provides an immunoassay of membrane phospholipase A₂ using the monoclonal antibodies. The preferred immunoassay is a radioimmunoassay. In a sandwich immunoassay, an enzyme immunoassay is preferred. The immunoassay of PLA₂M using the monoclonal antibodies is useful not only for the diagnosis of articular rheumatism but also for the diagnosis of cancers and a wide variety of inflammatory states including an external wound. Brief Description of the Drawings Figure 1 shows a calibration curve for PLA₂M in the sandwich assay using PL-78 and PL-71-peroxidase conjugate. Figure 2 shows a chromatogram obtained in the gel filtration of ¹²⁵I-labeled solution of PLA₂M. Figure 3 shows a standard curve in the RIA of PLA₂M. Figure 4 shows a dilution curve for human sera. Figure 5 shows the results of ion exchange chromatography of the standard PLA₂M and sera from patients with articular rheumatism. Figure 6 shows the concentrations of PLA₂M in sera from normal individuals, patients with articular rheumatism, patients with cancer, and patients with an external wound. The present invention provides monoclonal antibodies recognizing membrane phospholipase A₂. The membrane phospholipase A₂ is preferably derived from human spleen, as described in Biochem. Biophys. Res. Commun. Vol. 163, No. 1, 1989, pp. 42-48. In the present invention, monoclonal antibody PL-49, monoclonal antibody PL-71, monoclonal antibody PL-76, and monoclonal antibody PL-78 were obtained as detailed in Examples below. The present invention further provides hybridomas producing the corresponding monoclonal antibodies described above. The hybridoma PL-49, hybridoma PL-71, hybridoma PL-76, and hybridoma PL-78 can produce monoclonal antibody PL-49, monoclonal antibody PL-71, monoclonal antibody PL-76, and monoclonal antibody PL-78, respectively. These hybridoma PL-49, hybridoma PL-71, hybridoma PL-76, and hybridoma PL-78 were deposited on May 9, 1990, under the terms of the Budapest Treaty with Fermentation Research Institute, Agency of Industrial Science and Technology, 1-3, Higashi 1 chome, Tsukuba-shi, Ibaraki-ken, 305 Japan, and have been assigned Mouse hybridoma PL-49 with the Accession No. FERM BP-2891, Mouse hybridoma PL-71 with the Accession No. FERM BP-2892, Mouse hybridoma PL-76 with the Accession No. FERM BP-2889, and Mouse hybridoma PL-78 with the Accession No. FERM BP-2890, respectively. The aforesaid monoclonal antibodies can be produced by growing the corresponding hybridomas described above in the abdominal cavity of a mouse, and separating the monoclonal antibodies from the ascitic fluid accumulated in the abdominal cavity. The present invention also provides an immunoassay of membrane phospholipase A₂ using the aforementioned monoclonal antibodies. The preferred immunoassay is a radioimmunoassay. In the immunoassay where the membrane phospholipase A₂ is sandwiched between two different species of the aforesaid monoclonal antibodies, an enzyme immunoassay is preferred. Example 1 Preparation of monoclonal antibodies against membrane PLA₂ (PLA₂M) (1) Immunization The method for preparing PLA₂M used in the immunization and the assay of antibodies was as described in Biochem. Biophys. Res. Commun. Vol. 163, No. 1, 1989, pp. 42-48. First immunization: a solution of PLA₂M in phosphate-buffered saline (PBS) was mixed with Freund's Complete Adjuvant (FCA) to form an emulsion in a ratio of 1:1. The emulsion was administered subcutaneously to eight mice (Balb/C, female, 12 weeks old) at a dose equivalent to 2 µg of protein for each mouse. Second immunization: it was carried out 23 days after the first immunization in the same manner as that of the first immunization. Third immunization: it was carried out 44 days after the second immunization in the same manner as that of the first immunization. Fourth immunization: it was carried out 81 days after the third immunization in the same manner as that of the first immunization. (2) Determination of serum titer The level of anti-PLA₂M antibody in the blood of the immunized animals was determined by ELISA. That is, 0.5 ng of PLA₂M in 0.1 ml of 0.1 M NaHCO₃ was added to each well of a microtiter plate and left overnight at 4°C to coat the plate, after which 0.3 ml of 1% bovine serum albumin (BSA) in PBS was added thereto and the mixture was incubated at 37°C for 1 hour to block the plate. Then, 0.05 ml of a sample was added and allowed to react at 37°C for 1 hour, followed by an assay with a Vectastain® ABC kit (mouse IgG kit, Vector Laboratories, Inc.) according to its protocol. As a color former, 1 mg/ml of ortho-phenylenediamine (OPD) was used and the difference in absorbances at 492 nm and at 660 nm was determined by a Corona microplate photometer MTP-22. The mice exhibiting a high PLA₂M antibody titer were used for hybridoma preparation. (3) Preparation of hybridomas (3.1) Experiment 1 Thirty-one days after the fourth immunization, a fifth immunization was carried out. Four micrograms of PLA₂M were dissolved in 0.2 ml of PBS and the resulting solution was administered intraperitoneally to the mice. Three days after the fifth immunization, a cell fusion was carried out. The method for the cell fusion was substantially the same as that of Galfre and Milstein (Methods Enzymol. 73 , 46 (1981)). That is, mouse myeloma cells (P3X63-Ag8.653) were cultured in an RPMI1640 medium (90% RPMI1640; 10% fetal calf serum; 0.15 mg/ml sodium pyruvate; 0.15 mg/ml oxaloacetic acid; and 0.1 mg/ml kanamycin). From the resulting culture, 4.67 x 10⁷ mouse myeloma cells were harvested and mixed with 9.33 x 10⁷ spleen cells of the immunized mice, and the combined cells were pelleted by centrifugation in a centrifugation tube and 1 ml of 48% polyethylene glycol 4000 solution was added dropwise over 1 minute. The mixture was then stirred for 1.5 minutes, followed by slow addition, in a dropwise fashion with stirring, of a serum-free RPMI1640 medium in an amount of 2 ml over 2 minutes, 2 ml over 1 minute, and 6 ml over 2 minutes. Finally, 15 ml of the same medium was gently added and the mixture was pelleted by centrifugation. The pellets obtained were suspended in an HAT medium (70% RPMI1640; 10% NCTC109; 20% fetal calf serum; 10⁻⁴M hypoxanthine; 4 x 10⁻⁷M aminopterin; 1.6 x 10⁻⁵M thymidine; 0.15 mg/ml sodium pyruvate; 0.15 mg/ml oxaloacetic acid; 0.2 IU/ml insulin; 2.5 x 10⁻⁴M 2-mercaptoethanol; 5 x 10⁻³M HEPES; 0.1 mg/ml kanamycin; and nonessential amino acids). The suspension was adjusted to have a concentration of 0.75 x 10⁶ spleen cells per milliliter and then dispensed as 0.2 ml portions into each well of 96-well plates. The plates were incubated in 95% air-5% CO₂ at a temperature of 37°C and humidity of 95% or more, and the medium was replaced, if necessary, half by half with a fresh HAT medium. (3.2) Experiment 2 Thirty-five days after the fourth immunization, a fifth immunization was carried out in the same manner as in Experiment 1. Three days after the fifth immunization, a cell fusion was carried out. As the mouse myeloma cell strain, 8.89 x 10⁷ P3X63-Ag.8.653 cells were used for the cell fusion with 1.7 x 10⁸ mouse spleen cells in the same manner as in Experiment 1. The cells subjected to the cell fusion were suspensed in an HAT medium to have a concentration of 0.63 x 10⁵ cells/ml, and the suspension was then dispensed as 0.2 ml portions into each well of 96-well plates, followed by incubation in the same manner as in Experiment 1. (4) Screening of hybridomas After about 2 weeks the culture supernatant of hybridomas which grew was examined whether anti-PLA₂M antibodies were produced therein or not. The assay was carried out in the same manner as that of Item (2). Four hybridomas (PL-49, PL-71, PL-76 and PL-78) each of which stably produced an antibody having a specific reactivity with PLA₂M were obtained by the screening. (5) Cloning of hybridomas and storage in frozen state The aforesaid four hybridoma cells were cloned by a limiting dilution technique. That is, each of the hybridomas was suspended in an RPMI1640 medium and the suspension was added to a 96-well plate to have a concentration of 0.3 cells in 0.2 ml for each well, followed by incubation. The anti-PLA₂M antibody titer of the culture supernatant was determined in the same manner as that of Item (4). The anti-PLA₂M antibody-producing hybridomas were selected and grown, after which they were stored under freezing in a freezing solution (90% fetal calf serum and 10% dimethylsulfoxide). (6) Preparation of ascitic fluid To each group of mice (Balb/C, female, 10-15 weeks old) to which 0.5 ml of pristane had been administered intraperitoneally 7-10 days earlier, a suspension of each of the hybridoma cells in PBS (2-5 x 10⁶ cells/ml) was administered intraperitoneally at a dose of 0.5 ml for each mouse. After about 1 week, an ascitic fluid accumulated in the respective mice which was collected by a tapping technique. From the ascitic fluid collected, precipitate was removed by centrifugation using a Separapid tube (SEKISUI KAGAKU). The ascitic fluid so treated was dispensed and stored under freezing. (7) Determination of antibody class and subclass The immunoglobulin class and subclass of the monoclonal antibodies produced by the respective hybridomas were determined by ELISA. A mouse MonoAb-ID-EIA kit (Zymed Co., Ltd.) was used for the determination. In all cases of the four hybridomas, PL-49, PL-71, PL-76 and PL-78, immunoglobulins produced thereby were identified as IgG₁ (γ1, κ). (8) Purification of antibodies The monoclonal antibodies were purified from the ascitic fluid by the use of an Affigel® protein A MAPS II kit (Bio-Rad Co., Ltd.) according to its protocol. (9) Preparation of peroxidase conjugate According to the method of Nakane et al. (J. Histochem. Cytochem., 22 , 1084 (1974)), a conjugate of PL-71 with horseradish peroxidase was prepared. First, 0.1 ml of 0.1 M NaIO₄ was mixed with peroxidase (2 mg/0.5 ml in water) and the mixture was allowed to react at room temperature for 20 minutes. The reaction mixture was dialyzed overnight at 4°C against 1 mM sodium acetate buffer and then adjusted to pH 9.5 with 0.2 M Na₂CO₃, followed by addition of PL-71 (4 mg/ml in 0.01 M NaHCO₃). The mixture was then stirred at room temperature for 2 hours and 50 µl of NaBH₄ (4 mg/ml in water) was added thereto. The mixture was also stirred at 4°C for 2 hours and dialyzed overnight at 4°C against PBS to give the desired conjugate. (10) Sandwich assay of PLA₂M A solution of monoclonal antibody (PL-78) was added to each well of a microtiter plate (at a concentration of 0.1 µg in 0.1 ml of NaHCO₃ for each well). After standing overnight at 4°C, 0.3 ml of PBS containing 1% BSA was added to the plate, which was then incubated at 37°C for 1 hour, thereby blocking the plate. Thereafter, 0.05 ml of PLA₂M solution (1% BSA in PBS) was added to the plate and allowed to react at 37°C for 1 hour. Then, 0.05 ml of an 1,000-fold dilution of the peroxidase conjugate with monoclonal antibody PL-71 was added to the plate and allowed to react at 37°C for 2 hours. Thereafter, ortho-phenylenediamine (at a concentration of 1 mg in 0.1 ml of 0.1M citrate buffer, pH 4.2) and H₂O₂ (at a final concentration of 0.03%) were added to the plate and allowed to react at room temperature for 30 minutes. Finally, the reaction was stopped by adding 0.1 ml of 1 N H₂SO₄ and the difference in absorbances at 492 nm and at 660 nm was measured. Figure 1 shows a calibration curve of PLA₂M in the sandwich assay using PL-78 and the peroxidase conjugate with PL-71. The sensitivity for detecting PLA₂M by the present method was approximately 0.01 ng/well (see Figure 1). Example 2 Radioimmunoassay (RIA) of membrane phospholipase A₂ (PLA₂M) (1) Preparation of ¹²⁵I-labeled PLA₂M The ¹²⁵I-labeled PLA₂M was obtained by a chloramine T technique in accordance with the Hunter-Greenwood method (Nature, 194 , 495-496 (1962)). 1 ○ Reagents PLA₂M solution: 111 µg/ml in 0.5 M phosphate buffer (PB) (pH 7.4), Na¹²⁵I solution: 3.7 GBq/ml in dilute NaOH solution (pH 7-11) Chloramine T solution; 2 mg/ml in 0.5 M PB (pH 7.4), Sodium pyrosulfite solution: 2.5 mg/ml in 0.1 M PB (pH 7.4), BSA: 10 mg/ml in 0.1 M PB (pH 7.4) 2 ○ Procedure First, 25 µl of 0.5 M PB (pH 7.4) and 22.5 µl of the PLA₂M solution were put in a polypropylene tube, after which 2.5 µl of the Na¹²⁵I solution were added and the contents were mixed well together. Then, 2.5 µl of the chloramine T solution were also added and the contents were stirred at room temperature for 50 seconds. To this tube, 12.5 µl of the sodium pyrosulfite solution were further added and the contents were stirred, after which 2.5 µl of BSA and 2.5 µl of potassium iodide solution were added and the contents were mixed well together. The resulting mixture was then subjected to gel filtration (the column was PD-10 (manufactured by Pharmacia Co., Ltd.); the eluent was 0.1 M PB (pH 7.5) containing 0.5M sodium chloride, 0.5% BSA, and 0.05% sodium azide), and 1 ml-fractions of the eluate were collected. The radioactivity of each fraction was measured by means of a well-type scintillation counter. The chromatograms obtained are shown in Figure 2. Fraction No. 4 in this figure was identified as ¹²⁵I-labeled PLA₂M. (2) RIA of PLA₂M 1 ○ Reagents PLA₂M standard solution: 0.2 - 200 ng/ml assay buffer, Ascitic dilutions: 680,000-, 470,000-, 860,000- and 2,300,000-fold dilutions of PL-49, PL-71, PL-76 and PL-78, respectively, with the assay buffer, ¹²⁵I-labeled PLA₂M solution: a dilution of the labeled solution as described in Item (1) with the assay buffer (2 x 10⁶ cpm/ml), Immunobead liquid: a suspension of rabbit anti-mouse immunoglobulin-bound polyacrylamide gel (manufactured by Bio-Rad Co., Ltd.) in the assay buffer (1 mg/ml), Assay buffer: 0.1 M PB (pH 7.5) containing 0.5 M sodium chloride, 1 mM ethylenediaminetetraacetic acid, 0.5% BSA, and 0.02% sodium azide. 2 ○ Procedure First, 100 µl of the PLA₂M standard solution or a serum sample were put in a polypropylene tube, after which 275 µl of the assay buffer and 25 µl of the ¹²⁵I-labeled PLA₂M solution were added and the contents were mixed well together. Then, 100 µl of the ascitic fluid dilution were also added and the contents were incubated at room temperature for 16 hours. To this tube 100 µl of the immunobead liquid were further added and the contents were incubated at room temperature for 1 hour, followed by centrifugation (2000 x g, 10 min). After the removal of the supernatant by suction, the radioactivity of the precipitate was measured by means of a well-type scintillation counter. Based on the readings from a standard curve obtained by the use of the PLA₂M standard solution, the concentration of PLA₂M in the serum sample was determined. 3 ○ Standard curve and sensitivity Figure 3 shows a standard curve obtained in the RIA when PL-49 was used as an ascitic fluid. Other ascitic fluids gave similar competition curves. The sensitivity (i.e., concentration for 90% inhibition) was 0.3 ng/ml for PL-49, 1.1 ng/ml for PL-71, 0.9 ng/ml for PL-76 and 0.2 ng/ml for PL-78, all of which were considered to be highly sensitive. 4 ○ Specificity As shown in Figure 4, all the dilution curves for human sera obtained by the present method exhibited a good linear regression and there appeared no influence of sera on the curves. Moreover, no cross reaction of human pancreas PLA₂ was observed in the present method. In addition, standard PLA₂M and sera from patients with articular rheumatism were subjected to ion exchange chromatography (column was S-Sepharose® Fast Flow Type (manufactured by Pharmacia Co., Ltd.): eluent was 50 mM Tris buffer (pH 7.0) with a linear gradient containing from 0.2 to 1M sodium chloride and 0.1% CHAPS (manufactured by Dotite Co., Ltd.)). The eluate was fractionated and PLA₂M present in each fraction was measured by the present method. The results provided a chromatogram as shown in Figure 5, indicating that the pattern of the chromatogram obtained by the standard PLA₂M had a good fit to that obtained by the sera of patients. These results revealed that the present method is applicable to a specific assay of PLA₂M present in human sera. 5 ○ Assay of human sera According to the present method, various sera from normal individuals, patients with articular rheumatism, patients with cancer, and patients with an external wound were applied to the assay of PLA₂M in an undiluted or appropriately diluted form with the assay buffer. The results are shown in Figure 6. As seen from this figure, many of serum samples from patients with articular rheumatism, patients with cancer, and patients with an external wound exhibited a higher level of PLA₂M than that of the serum sample from normal individuals, indicating that the present method is useful for the diagnosis of articular rheumatism and cancers.

Claims: Monoclonal antibodies recognizing membrane phospholipase A₂ obtainable from the hybridoma cell lines having the deposit accession numbers FERM BP-2891, FERM BP-2892, FERM BP-2889 or FERM BP-2890. The monoclonal antibodies according to claim 1, wherein said membrane phospholipase A₂ is derived from human spleen. Hybridoma cell lines producing the monoclonal antibodies of any one of claims 1 to 2 and having the deposit accession numbers FERM BP-2891, FERM BP-2892, FERM BP-2889 or FERM BP-2890. A method for producing a monoclonal antibody of any one of claims 1 to 2, which comprises the steps of: growing a hybridoma of claim 3 in the abdominal cavity of a mouse; and separating said monoclonal antibody from the ascitic fluid accumulated in said abdominal cavity. A method for preparing a hybridoma cell line producing a monoclonal antibody of any one of claims 1 to 2, which comprises immunizing mice with membrane phospholipase A₂, fusing cells capable of producing antibodies obtained from the immunized mice with myeloma cells and screening the resulting hybridoma cells for production of one of said monoclonal antibodies. An immunoassay of membrane phospholipase A₂ using a monoclonal antibody of any one of claims 1 to 2. The immunoassay according to claim 6, which is a radioimmunoassay. The immunoassay according to claim 6, wherein said membrane phospholipase A₂ is sandwiched between two different species of said monoclonal antibodies. The immunoassay according to claim 8, which is an enzyme immunoassay.

Inventors: IDE MISAO, KANDA AKIKO, KONO MASAO, YOSHIDA NOBUO, IDE, MISAO, KANDA, AKIKO, KONO, MASAO, YOSHIDA, NOBUO
Applicants: SHIONOGI & CO, SHIONOGI SEIYAKU KABUSHIKI KAISHA TRADING UNDER THE NAME OF SHIONOGI & CO. LTD.
Application year: 1991
Main classification: C12P21
IPC codes: G01N33, C12N5, C07K16, A61K39, C12R1, C12N15, C12P21