Abstract:
The present invention first provides monoclonal antibodies recognizing membrane phospholipase A 2 , namely, monoclonal antibodies PL-49, PL-71, PL-76, and PL-78, hybridomas producing them, methods for producing them, and immunoassays of membrane phospholipase A 2  using them. 
     The immunoassay of PLA 2  M is useful for the diagnosis of articular rheumatism, cancers, and a wide variety of inflammatory states.

Description:
This application is a continuation of application Ser. No. 07/697,008 filed on May 8, 1991, now abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to monoclonal antibodies recognizing membrane phospholipase A 2  ; hybridomas producing said monoclonal antibodies; a method for producing said monoclonal antibodies; and immunoassays using said monoclonal antibodies. 
     2. Description of the Prior Art 
     Phospholipase A 2  (PLA 2 ) (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 PLA 2  in blood becomes increased when suffering from pancreatitis (Ogawa et al., Gendai Iryou, 20 (1988) 3013-3017). The PLA 2 , 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 (Vades, P. et al., Lab Invest. (1986) 55, 691-404). 
     It was revealed that membrane PLA 2  (PLA 2  M) isolated from human splenic membrane fraction was the different type of PLA 2  from pancreatic PLA 2  by analysis of its protein primary structure (Kanda, A. et al., Biochem. Biophys. Res. Commun. (1989) 163, 42-48), and it was also found that PLA 2  M 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 PLA 2  M and PLA 2  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). 
     From a clinical point of view, an increase of the PLA 2  enzymatic activity was found in the blood of patients with an infectious disease such as septicemia, pustular psoriasis, Crohn&#39;s disease, and rheumatoid arthritis. Further, it was found that the PLA 2  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 PLA 2  M, and it has not yet been shown whether an increase of the PLA 2  enzymatic activity accompanying the aforesaid diseases is caused by PLA 2  M or not. 
     As described above, because the PLA 2  enzymatic activity in blood is increased when suffering from rheumatism, septicemia, pustular psoriasis, Crohn&#39;s disease, or the like, it has been expected that the diagnosis of these diseases can be realized from the measurement of PLA 2  M and an assay of PLA 2  M has been desired. 
     SUMMARY 
     The present invention first provides monoclonal antibodies recognizing membrane phospholipase A 2 , namely, monoclonal antibodies PL-49, PL-71, PL-76, and PL-78. The membrane phospholipase A 2  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 A 2  using the monoclonal antibodies. Preferred immunoassay is a radioimmunoassay. In a sandwich immunoassay, an enzymeimmunoassay is preferable. 
     The immunoassay of PLA 2  M 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 
     FIG. 1 shows a calibration curve for PLA 2  M in the sandwich assay using PL-78 and PL-71-peroxidase conjugate. 
     FIG. 2 shows a chromatogram obtained in the gel filtration of  125  I-labeled solution of PLA 2  M. 
     FIG. 3 shows a standard curve in the RIA of PLA 2  M. 
     FIG. 4 shows a dilution curve for human sera. 
     FIG. 5 shows the results of ion exchange chromatography of the standard PLA 2  M and sera from patients with articular rheumatism. 
     FIG. 6 shows the concentrations of PLA 2  M in sera from normal individuals, patients with articular rheumatism, patients with cancer, and patients with an external wound. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention provides monoclonal antibodies recognizing membrane phospholipase A 2 . The membrane phospholipase A 2  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 House hybridoma PL-49 with the Accession No. FERM BP-2891, House 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 A 2  using the aforementioned monoclonal antibodies. Preferred immunoassay is radioimmunoassay. In the immunoassay where the membrane phospholipase A 2  is sandwiched between two different species of the aforesaid monoclonal antibodies, an enzyme immunoassay is preferable. 
     EXAMPLE 1 
     Preparation of monoclonal antibodies against membrane PLA 2  (PLA 2  M) 
     (1) Immunization 
     The method for preparing PLA 2  M 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 PLA 2  M in phosphate-buffered saline (PBS) was mixed with Freund&#39;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-PLA 2  M antibody in the blood of the immunized animals was determined by ELISA. That is, 0.5 ng of PLA 2  M in 0.1 ml of 0.1 M NaHCO 3  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 PLA 2  M antibody titer were used for hybridoma preparation. 
     (3) Preparation of hybridomas 
     (3.1) Experiment 1 
     Thirty-one days after the fourth immunization, fifth immunization was carried out. Four micrograms of PLA 2  M was 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, 4.67×10 7  mouse myeloma cells were harvested and mixed with 9.33×10 7  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 -4  M hypoxanthine; 4×10 7  M aminopterin; 1.6×10 -5  M thymidine; 0.15 mg/ml sodium pyruvate; 0.15 mg/ml oxaloacetic acid; 0.2 IU/ml insulin; 2.5×10 -4  M 2-mercaptoethanol; 5×10 -3  M HEPES; 0.1 mg/ml kanamycin; and nonessential amino acids). The suspension was adjusted to have a concentration of 0.75×10 6  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% CO 2  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 HAI medium. 
     (3.2) Experiment 2 
     Thirty-five days after the fourth immunization, 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, 8.89×10 7  P3X63-Ag.8.653 cells were used for the cell fusion with 1.7×10 8  mouse spleen cells in the same manner as in Experiment 1. The cells subjected to the cell fusion were suspensed in an HAI medium to have a concentration of 0.63×10 6  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-PLA 2  M 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 PLA 2  M 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-PLA 2  M antibody titer of the culture supernatant was determined in the same manner as that of Item (4). The anti-PLA 2  M 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 before 7-10 days, a suspension of each of the hybridoma cells in PBS (2-5×10 6  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 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 four hybridomas, PL-49, PL-71, PL-76 and PL-78, immunoglobulins produced thereby were identified as IgG 1  (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 0.1M NaIO 4  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 0.2M Na 2  CO 3 , followed by addition of PL-71 (4 mg/ml in 0.01M NaHCO 3 ). The mixture was then stirred at room temperature for 2 hours and 50 μl of NaBH 4  (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 PLA 2  M 
     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 NaHCO 3  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 PLA 2  M 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 O.1M citrate buffer, pH 4.2) and H 2  O 2  (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 1N H 2  SO 4  and the difference in absorbances at 492 nm and at 660 nm was measured. FIG. 1 shows a calibration curve of PLA 2  M in the sandwich assay using PL-78 and the peroxidase conjugate with PL-71. The sensitivity for detecting PLA 2  M by the present method was approximately 0.01 ng/well (see FIG. 1). 
     EXAMPLE 2 
     Radioimmunoassay (RIA) of membrane phospholipase A 2  (PLA 2  M) 
     (1) Preparation of  125  I-labeled PLA 2  M 
     The  125  I-labeled PLA 2  M was obtained by a chloramine T technique in accordance with the Hunter-Greenwood method (Nature, 194, 495-496 (1962)). 
     1 Reagents 
     PLA 2  M solution: 111 μg/ml in 0.5M phosphate buffer (PB) (pH 7.4), 
     Na 125  I solution: 3.7 GBq/ml in dilute NaOH solution (pH 7-11) 
     Chloramine I solution; 2 mg/ml in 0.5M PB (pH 7.4), 
     Sodium pyrosulfite solution: 2.5 mg/ml in 0.1M PB (pH 7.4), 
     BSA: 10 mg/ml in 0.1M PB (pH 7.4) 
     2 Procedure 
     First, 25 μl of 0.5M PB (pH 7.4) and 22.5 μl of the PLA 2  M solution were taken in a polypropylene tube, after which 2.5 μl of the Na 125  I solution was added and the contents were mixed well together. Then, 2.5 μl of the chloramine T solution was also added and the contents were stirred at room temperature for 50 seconds. To this tube, 12.5 μl of the sodium pyrosulfite solution was 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 0.1M 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 FIG. 2. Fraction No. 4 in this figure was identified as  125  I-labeled PLA 2  M. 
     (2) RIA of PLA 2  M 
     1 Reagents 
     PLA 2  M 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, 
       125  I-labeled PLA 2  M solution: a dilution of the labeled solution as described in Item (1) with the assay buffer (2×10 6  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: 0.1M PB (pH 7.5) containing 0.5M sodium chloride, 1 mM ethylenediaminetetraacetic acid, 0.5% BSA, and 0.02% sodium azide. 
     2 Procedure 
     First, 100 μl of the PLA 2  M standard solution or a serum sample was taken in a polypropylene tube, after which 275 μl of the assay buffer and 25 μl of the  125  I-labeled PLA 2  M solution were added and the contents were mixed well together. Then, 100 μl of the ascitic fluid dilution was also added and the contents were incubated at room temperature for 16 hours. To this tube 100 μl of the immunobead liquid was further added and the contents were incubated at room temperature for 1 hour, followed by centrifugation (2000×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 PLA 2  M standard solution, a concentration of PLA 2  M in the serum sample was determined. 
     3 Standard curve and sensitivity 
     FIG. 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 FIG. 4, all the dilution curves for human sera obtained by the present method exhibited a good liner regression and there appeared no influence of sera on the curves. 
     Moreover, no cross reaction of human pancreas PLA 2  was observed in the present method. 
     In addition, standard PLA 2  M 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 PLA 2  M present in each fraction was measured by the present method. The results provided a chromatogram as shown in FIG. 5, indicating that the pattern of the chromatogram obtained by the standard PLA 2  M 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 PLA 2  M 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 PLA 2  M in an undiluted or appropriately diluted form with the assay buffer. The results are shown in FIG. 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 PLA 2  M than that of the serum sample from normal individuals, indicating that the present method is useful for the diagnosis of articular rheumatism and cancers.