Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1998-04-28
2003-08-12
Devi, S. (Department: 1645)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S388100, C530S387100, C530S809000, C435S070210, C435S326000, C435S346000, C435S975000, C436S548000
Reexamination Certificate
active
06605705
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a monoclonal antibody specific to human L-PGDS present predominantly in cerebrospinal fluid (CSF), a hybridoma producing said monoclonal antibody, methods for detection of L-PGDS or diseases by said monoclonal antibody, and a kit for detection of L-PGDS by said monoclonal antibody.
BACKGROUND OF THE INVENTION
Prostaglandin D is a biologically active substance synthesized in animal tissues from arachidonic acid released from a biomembrane upon stimulation, and it is produced from prostaglandin H (a common precursor of prostaglandin families produced by cyclo-oxygenase) by prostaglandin D synthase (PGDS).
The presence of two types of PGDS, glutathione-independent L-PGDS and glutathione-dependent spleen type PGDS, is known (Shimizu et, al., J. Biol. Chem., 254, 5222-5228 (1979); Urade et al., J. Biol. Chem.,
260
, 12410-12415 (1985); Christ-Hazelhof and Nugteren, Biochim. Biophys. Acta, 572, 43-51 (1979); Urade et al., J. Biol. Chem., 262, 3820-3825 (1987). The former is known to be predominantly located in the central nervous system such as brain, epididymis, spinal cord, retina, and inner ear (Urade et al., J. Biol. Chem., 260, 12410-12415 (1985); Ueno et al., J. Neurochem., 45, 483-489 (1985); Urade et al., J. Biol. Chem., 262, 3820-3825 (1987); Goh et al., Biochim. Biophys. Acta, 921, 302-311 (1987); and Tachibana et al., Proc. Natl. Acad. Sci. USA, 84, 7677-7680 (1987)), and the latter is known to be distributed broadly in almost all peripheral organs including spleen, bone marrow, digestive organs, thymus, and skin (Ujihara et al., Arch. Biochem. Biophys., 260, 521-531 (1988); and Ujihara et al., J. Invest., Dermatol., 90, 448-451 (1988)).
On the other hand, a protein called &bgr;-trace was observed to be present specifically in human CSF, but its physiological function remained unrevealed (Causen, J. Proc. Soc. Exp. Biol. Med., 107, 170-172 (1961)).
A certain correlation between &bgr;-trace known as a protein specific to CSF and severe brain disorders or certain diseases (multiple sclerosis, brain tumors, Meckel's syndrome and paraproteinemia) was noted from the observation that &bgr;-trace levels depend on such disorders (Ericsson et al., Neurology, 19, 606-610 (1969); Olsson et al., J. Neurol. Neurosurg. Psychiat. 37, 302-311 (1974); Link, J. Neurol. Sci., 16, 103-114 (1972); Whistsed and Penny, Clinica Chimica Acta, 50, 111-118 (1974); and Chemke et al., Clinical Genetics, 11, 285-289 (1977)). However, the exact correlation between &bgr;-trace and such disorders could not be determined because the physiological function of &bgr;-trace still remained unrevealed and because there was no tool available for determining the exact amount (concentration) of &bgr;-trace.
Recently, the nucleotide sequence of cDNA coding for L-PGDS was reported (Nagata et al., Proc. Natl. Acad. Sci. USA, 88, 4020-4024 (1991)), and production of L-PGDS by genetic recombination became feasible. The L-PGDS thus produced was examined and its amino acid sequence was estimated and compared with an N-terminal partial amino acid sequence of human &bgr;-trace in searching for their homology (Kuruvilla et al., Brain Research, 565, 337-340 (1991), Zahn et al., Neuroscience Letters, 154, 93-95 (1993)) or with the amino acid sequence of purified human &bgr;-trace (Hoffmann et al., J. Neurochem., 61(2), 451-456 (1993)), and further immunological examination was made using polyclonal antibodies (Watanabe et al., Biochem. Biophys. Res. Communication, 203, 1110-1116 (1994)). These studies revealed that &bgr;-trace was identical with L-PGDS.
Prostaglandin D occurring abundantly in the central nervous system functions, in one physiological action, as a neuromodulator of several central actions including sleep promotion. Prostaglandin D synthase is considered as a key enzyme for sleep-wake activities (Hayashi, FASEB J., 5, 2575-2581 (1991)), and it is believed that at least a part of the L-PGDS secreted from competent cells is accumulated in CSF (Watanabe et al., Biochem. Biophys. Res. Communication, 203, 1110-1116 (1994)).
Accordingly, the analysis of L-PGDS distribution etc. in the central nervous system is useful for detection of diseases in the central nervous system, and it is expected that L-PGDS levels in CSF or humor can also be used as an indicator in early diagnosis and prognostic observations for other diseases caused by abnormalities in the central nervous system. It is further expected that L-PGDS (or &bgr;-trace) can be used for examination of a reproduction ability, diagnosis of fetal growth, etc. because this enzyme is distributed in such humors derived from genital organs, as semen, oviduct fluid and amniotic fluid, as well. For such applications, there is demand for antibodies specifically recognizing L-PGDS.
Nevertheless, such antibodies have still not been established with high specificity to meet such demand.
DISCLOSURE OF THE INVENTION
The object of the present invention is to provide a monoclonal antibody specifically recognizing L-PGDS, a hybridoma producing said antibody, methods for detection of L-PGDS or diseases by said monoclonal antibody, and a kit for detection of L-PGDS by said monoclonal antibody.
As a result of their eager researches, the present inventors have successfully obtained a monoclonal antibody specifically recognizing L-PGDS from a hybridoma prepared by cell fusion between myeloma cells and antibody-producing cells from an animal immunized with L-PGDS which is a major protein in human CSF, and they thereby arrived at the present invention.
That is, the present invention relates to a monoclonal antibody specifically recognizing L-PGDS. The subclass of such monoclonal antibody includes immunoglobulin G1 or G2.
Further, the present invention relates to a hybridoma producing said monoclonal antibody by cell fusion between myeloma cells and antibody-producing cells from an animal immunized with L-PGDS.
Further, the present invention relates to methods for detection of L-PGDS or diseases by said monoclonal antibody. An example of such diseases is oligospermia.
Further, the present invention relates to a kit for detection of L-PGDS, which is selected from a reagent containing said monoclonal antibody labeled with an enzyme and a substrate solution, and a reagent containing said monoclonal antibody obtained by biotination, enzyme-labeled avidin, and a substrate.
Further, the present invention relates to a method for detection of diseases by said kit for detection of L-PGDS. An example of such diseases is oligospermia.
Hereinafter, the present invention is described in detail.
1. Production of the Monoclonal Antibody
Production of the present monoclonal antibody against L-PGDS consists of the steps of:
(1) Preparation of an antigen;
(2) Immunization and preparation of antibody-producing cells;
(3) Establishment of an antibody-titration system;
(4) Cell fusion;
(5) Selecting and cloning hybridomas; and
(6) Isolation of the monoclonal antibody.
Hereinafter, each step is described.
(1) Preparation of an Antigen
L-PGDS can be produced in large amounts in a usual manner by
E. coli
and CHO cells etc. with its known cDNA (Nagata et al., Proc. Natl. Acad. Sci. USA, 88, 4020-4024 (1991)). In this production of L-PGDS, a recombinant DNA containing the cDNA for L-PGDS is constructed and transformed into a microorganism, which is then cultured to produce the enzyme. The L-PGDS thus produced can be purified from the culture by conventional means.
Then, an immunogen is prepared by dissolving the resulting L-PGDS in a buffer and then adding an adjuvant to it. Examples of such adjuvants are Freund complete adjuvant, Freund incomplete adjuvant, BCG, Hunter's Titermax (CytRx Corporation), key hole limpet hemocyanin-containing oil, etc., and any of them can be mixed.
(2) Immunization and Preparation of Antibody-producing Cells
The immunogen thus obtained is administrated as antigen into mammals such as horse, monkey, dog, pig, cow, goat, sheep, rabbit, guinea pig, hamster and mouse, or birds such as pigeon and chicken. In particul
Nishikawa Masazumi
Oda Hiroshi
Saji Fumitaka
Sato Nobuyuki
Seiki Kosuke
Devi S.
Fish & Richardson P.C.
Maruha Corporation
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