WO2001077362A1 - Immunoassay of anti-hm1.24 antibody - Google Patents

Immunoassay of anti-hm1.24 antibody Download PDF

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Publication number
WO2001077362A1
WO2001077362A1 PCT/JP2001/002964 JP0102964W WO0177362A1 WO 2001077362 A1 WO2001077362 A1 WO 2001077362A1 JP 0102964 W JP0102964 W JP 0102964W WO 0177362 A1 WO0177362 A1 WO 0177362A1
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Prior art keywords
antibody
hm1
anti
soluble
antigen
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PCT/JP2001/002964
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French (fr)
Japanese (ja)
Inventor
Yasuko Kinoshita
Yuji Ishikawa
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Chugai Seiyaku Kabushiki Kaisha
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Priority to JP2000-105423 priority
Application filed by Chugai Seiyaku Kabushiki Kaisha filed Critical Chugai Seiyaku Kabushiki Kaisha
Publication of WO2001077362A1 publication Critical patent/WO2001077362A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Abstract

A process whereby a highly purified soluble HM1.24 antigen protein can be produced at a high efficiency. Namely, a process for producing a soluble HM1.24 antigen extracellular domain characterized by comprising culturing animal cells transformed by an expression vector which contains an (A1)EF1a promoter and a gene encoding soluble HM1.24 antigen lacking the intracellular domain ligated downstream of the promoter, and isolating and purifying the soluble HM1.24 antigen from the culture.

Description

Field of immunoassay invention a light fine manual anti HM 1. 2 4 antibody

The present invention relates to immunochemical measurement method of anti-HM1.24 antibody. The invention also relates to an immunochemical method for measuring the soluble HM1.24 antigen protein. The invention further relates to DNA encoding soluble HM1.24 antigen protein and the same. BACKGROUND

Goto, T Rawahi preparative plasma cells obtained by immunizing a molecular weight expressed singular manner the B cell lineage have reported Mausumonoku polyclonal antibody 1.24 antibody recognizing the 22 to 39 kDa antigen (Blood (1994) 84, 1922-1930). The mouse anti-HM1.24 antibody as well as human plasma cells and have you in the transplanted mice in vivo anti-tumor effect, ADCC against human plasma cells (an t lbody- dependent cellular cytotoxicity) Ϊ tongue 个生 [Koyoru in vitro to 佘 tumor effect (0zaki, S et al., Blood, (1997) 90, 3179-3186) also chimeric antibodies and reconstituted human antibody of the murine anti HMl.24 antibodies have been produced (Ono Koichiro et al second 0 times Japan annual meeting of the molecular Biology Society (1997) abstracts general presentations 3- 501-P- 478; W0 98/14580).

On the other hand, these mice HM1.24 antibody, a chimeric antibody, reconstituted human activity measurement of the antibody Cell- ELISA using human plasma cell line RPMI8226 (Goto, T, et al., Blood (1994) 84, 1922-1930) by has done, the high measurement method with good Ri accuracy has been demanded. Disclosure of the Invention

Anti HML. For 24 antibody and HML. 24 antigenic proteins that are expressed on the cell membrane is an antigen has been reported as described above. While only soluble highly purified HM1. Fabrication method of 24 antigen is not known, therefore with soluble HM1. 24 antigen protein is highly purified, low concentrations of soluble HM1. 24 antigen protein or method of detecting or measuring anti-HM1. 24 antibody was not known.

The invention therefore firstly provides a method capable of producing with high efficiency a highly purified soluble HM1. 24 antigen protein.

That is, the present invention is transformed with an expression vector comprising a the (A l) EF1 shed promoter, the linked downstream, Ru soluble HM1. 24 antigen co one dos lacking intracellular domain gene It has been cultured animal cells, and provides soluble HM1. 24 manufacturing method of an antigen extracellular domain, wherein the soluble negation 1.24 antigen child isolated and purified from the culture.

The present invention also includes, (A 2) wherein the soluble HM1 24 antigen SEQ ID NO:. 5 or having an amino acid sequence shown in 16, to provide the method.

The present invention also includes, (A 3) said soluble HM1 24 antigen SEQ ID NO:. 7 or has a § Mi acid sequence shown in 17, to provide the method.

The present invention also provides the method in the form of a fusion protein with (A 4) said soluble HM1. 24 antigen influenza agglutinin (HA). The present invention is also, (A 5) wherein the fusion protein SEQ ID NO: having the amino acid sequence set forth in 10 or 18 to provide the method.

The present invention is al, (A 6) the fusion protein SEQ ID NO: 11 or 19 having an amino acid sequence set forth to provide the method.

The present invention further, (A 7) wherein said animal cell is a CH0 cell to provide the method. The present invention is al, in which were gene amplification in the presence of (A 8) transformed animal cells in a concentration of 100 nmo l / L Main preparative Torekise Ichito (MTX), said method I will provide a.

The present invention is al, provides a convenient method of detecting or measuring anti-HM1. 24 antibody using a highly purified soluble HM1. 24 antigen protein. That is, the present invention, (B 1) soluble HM1 24 antigen protein and the test -.... Anti-HM1 contained in the sample 24 and the antibody are reacted, soluble HM1 24 anti bound to antigenic proteins HM1 24 step a including detecting or measuring antibody provides anti HM1. 24 antibody immunochemical assay methods. Soluble HM1. 24 antigen protein are preferably combined fusion with another peptide or polypeptide. Soluble HM1. 24 antigen protein is preferably bound to a support.

The support is preferably beads or plates. Soluble HM1. 24 antigen protein is preferably bound to soluble HM1. 24 antigen protein or the soluble HM1. Supported by 24 antibody against other peptides or Poripe petit de fused to an antigen protein bodies.

The present invention also provides, (B 2) soluble HM1. 24 anti HM1 bound to an antigen protein. The 24 antibody, anti-HM1. Above, wherein the detecting or measurement Ri by the primary antibody against 24 antibody (B 1 providing immunochemical assay method described in).

The present invention also provides, (B 3) soluble HM1. 24 anti HM1 bound to an antigen protein. The 24 antibody, anti-HM1. 24 to by Ri detected or measured in a secondary antibody against the primary antibody and primary antibody against antibodies providing immunochemical assay method according to above, wherein the (B 1) or (B 2). In the (B 1) or (B 2), the primary antibody or secondary antibody is preferably a radioactive isotope, an enzyme, is Rishirube identified by the Piochin / avidin or a fluorescent substance. The present invention also provides, (B 4) anti-HM1.24 soluble HM1.24 antigen protein bound to the antibody, the first antibody or to soluble HM1.24 antigen protein soluble HM1.24 antigen protein fused with other peptides or above, wherein the detecting or determining by the primary antibody against the polypeptide (1) provides an immunochemical measurement method according to (3).

The present invention also provides, (B 5) anti-HM1.24 antibody soluble HM1.24 antigen protein bound to the soluble HM1.24 primary antibody or to an antigen protein other fused to soluble HM1.24 antigen protein peptide or Poribe above, wherein the by Ri detect or measure child secondary antibody against the primary antibody and primary antibody against peptide (B 1) to provide a seismic 疫化 biological measurement method according to ~ (B 4). In the (B 1) ~ (B 4), the primary or secondary antibody is preferably labeled radioisotopes, enzymes, by bi O Chin / avidin or a fluorescent substance. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram showing the sandwich ELISA system using HA tagged soluble antigen.

Figure 2 is a diagram HA-tagged soluble HM1.24 antigen-producing cell lines generated were compared the amount of soluble antigens that are produced during the culture supernatant. If the dilution factor is high it is a high production.

Figure 3 is HA-tagged soluble HM1.24 antigen-producing cells 4 strains (164-2-1, 16 4-2-13, 164-2-17, and 164-2- 31) reduced state culture on brass of after S DS- polyacrylamide Riruami Dogeru electrophoresis on performs western blot with a mouse anti-HM1.24 antibody, it is a photograph substituted for a drawing, showing the result of the detection of HM1.24 antigen.

Figure 4 is a view to graph a standard curve of human-type anti-HM1.24 antibody in Sa Ndi Tutsi ELISA system using the purified antigen 5 'serially diluted 3-fold dilutions from 750-fold.

Figure 5 is a graph showing a standard curve of human type anti-HM1. 24 antibody in San de I Tutsi ELISA system using purified antigen 5000 fold dilution (about 76. 4 ng / mL).

Figure 6 is a diagram showing the HM1. 24 amino acid sequence of the antigenic protein co cDNA nucleotide sequence and corresponding to one mode.

Figure 7 is a diagram showing the HM1. 24 nucleotide sequence and the corresponding amino acid sequence of the antigen protein code sul cDNA.

Figure 8 is a schematic diagram of a clone P3 was isolated using the Panning technique. 19 and five clones isolated by immune scan cleaning method (I S1 ~I S5).

9, anti-HM1 24 antibody is a diagram showing (A; CH0 / HM '; CH0 / NE0, B) the result of full low side tome Application Benefits An Analysis Using.. Anti HM1. 24 arsenide scan chromatogram of antibodies by solid lines, histograms isotype matched control antibody (UPC10) is indicated by a wavy line. The horizontal axis represents the fluorescence intensity, and the vertical axis represents the cell number.

Figure 1 0 is substituted for drawing showing the HM1. 24 the expression of antigen anti HM1. 24 immunoprecipitation with antibodies. Result detected Ri by the Western Bro Tsu Te queuing method in various cell lines and HM1. 24 expressing CH0 cells is a photograph. Anti HM1. 24 antibody binding Sepharose 4B (lanes 1-6) or after immunoprecipitation with unbound sepharose low scan 4B (lanes 7 and 8), anti-HM1. Western Bro with 24 antibody performs a Tti ring, were detected HM1. 24 antigen (displayed on the right-hand side). (*;. Anti HM1 24 antibody H-chain) embodiment of the invention

. As a soluble HM1 24 antigen protein of the present invention, SEQ ID NO: has an amino acid sequence consisting of Gin amino acid position 132 of the Amino acid positions 1-position of Asn in 5 indicates to § Mi acid sequence, and if soluble HM1. 24 protein having the biological activity of the antigen protein may be of any type. Soluble HM1. The biological activity of 24 antigen protein, is specifically bound to the anti-HM1. 24 antibody, cell membrane soluble liberated from the cell membrane not bonded, is and dimer.

Soluble HM1 24 antigen protein of the present invention, soluble HM1 24 has antigen protein biological activity, and SEQ ID NO:.. 1 for amino acid sequence shown in 5 or a plurality of § Mi Amino Acids substitution of residues, may be soluble HM1. 24 antigen protein having a deletion and / or by Ri modified § Mi acid sequence in addition. . Soluble HM1 24 antigen protein of the present invention, as long as the good Ri specifically to have a biological activity of soluble HM1 24 antigen protein, SEQ ID NO:. In amino acid sequence shown in 5, 1 or 2 or more , preferably 1 or 24 or less, good Ri preferably may have a § Mi Roh acids 1 or 12 or less § Mi Roh acid residue has been substituted.

Or, SEQ ID NO: Yes in the amino acid sequence shown in 5, one or two or more on, preferably 1 or 42 or less, more preferably amino acids 1 or 17 or less amino acid residues have been deleted it may have. Or, SEQ ID NO: Yes in amino acid sequence shown in 5, 1 or 2 or more, preferably 1 or 50 or less, more preferably amino acids 1 or 14 or less Amino acid residues are added it may have. Soluble HM 1. 24 antigen protein used in the present invention are also substituted in the amino acid modification by deletion and / or addition may be made at the same time.

Soluble negation 1.24 antigenic protein is SEQ ID NO: to exhibit 5 in position 1 § Mi Roh from acid Asn is have an amino acid sequence up to 90th Amino acid Arg if its biological activity It is obviously summer. Te the month, soluble invention HM1 24 antigen protein, SEQ ID NO:. 5 force has the amino acid sequence of the odor Te from position 1 amino acid Asn until 9 0 position amino acids Arg or 1-position, the amino acid Asn 9 0 position 1 or against § Mi acid sequence up to amino acid Arg of a plurality of § Mi acid residue of substitution, deletion and / or by Ri modified § in addition it may be the soluble HM1.24 antigen protein having the Mi acid sequence.

Soluble HM1.24 antigen protein so long as it has the biological activity of SEQ ID NO: force having an amino acid sequence from amino acids Arg 9 0 position in 5 to 132 of the amino acid Gin or the amino acid, substitution of one or a plurality of § Mi acid residue for the sequence may be soluble negation 1.24 antigenic protein with a deletion and or by Ri modified amino acid sequence in addition.

SEQ ID NO: substitution of one or a plurality of § Mi acid residue for § Mi acid sequence shown in 5, the soluble HM1.24 antigen protein having a deletion and / or by Ri modified Amino acid sequence in addition to, SEQ ID NO: 7 or 17, 10 or 18, or the soluble HM1.24 antigen protein and the like having the amino acid sequence shown in 11 or 19.

Substitution of one or more Amino acid residues for a amino acid sequence, the protein having a deletion and / or § Mi acid sequence which is modified by the addition to maintain its biological activity already known (Ma rk, DF et al., Proc. Natl. Acad. Sci. USA (1984) 81, 566 2-5666, Zoller, MJ & Smith, M. Nucleic Acids Research (1 982) 10, 6487 -6500, Wang, A. et al., Science 224, 1431-1433, Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. US A (1982) 79, 6409-6413).

Soluble negation 1.24 antigenic protein of the present invention, derived species, they Ri by the host and / or purification methods to produce the amino acid sequence, molecular weight, isoelectric point, presence or absence and position of glycosylation glycosylation, sugar structure of the chain, the presence of Li phosphorylation state and Z or disulfide bond differs. However, as long as it can be suitably used in the present invention, it may be a protein having any structure. As a species in which the protein is derived from the human is preferred. Soluble HM1 24 antigen protein DNA encoding the present invention, SEQ ID NO:. Consisting of base position 1 of the nucleotide sequence shown in 5 bases adenine 396 of the base Guanin nucleotide sequences. The present onset Ming soluble HM1 is the 24 antigen protein and DNA encoding SEQ ID NO:. If DNA having the nucleotide sequence of 5 may be DNA of any origin. Such as a DNA, such Jieno Mi click DNA, cDNA, synthetic DNA and the like. These various cells, tissues or organs or human non cDNA library obtained from seeds, may be a DNA obtained from Jieno mission-Clive rally, be they a commercially available DNA library good. Is a vector et is used for these libraries, plasmid, Park bacteriophage one like YAC base Kuta may be of any type.

. Soluble HM1 of the present invention also has a 24 antigen protein as co sul DNA, SEQ ID NO:. Hybridized Dizu to a nucleotide sequence shown in 5, and soluble HM1 code for a polypeptide having the biological activity of 24 antigen protein it may be a sul DNA. Soluble HM1. The conditions for 24 antigen protein co one de to DNA hybridizes, is in such a hybridizing conditions include hybridizing DNA in moderate be sampled Li Njie solvency conditions, such as low It is sampled Li Nje Nshi one condition, and the like. The Teisu Application Benefits Njiwenshi in a flat condition, for example, 4 2 ° C, 5 X SSC, 0. 1% dodecyl sulfate isocyanatomethyl Li um, a washing conditions by conferred is 50% e Rumuami de. Good Ri preferably include the high-be sampled Li Njie solvency conditions. Is a Kosu Application Benefits Njienshi conditions, for example, 60 ° (:., 0. 1 SS C, 0. is a washing condition provided by 1% dodecyl sulfate isocyanatomethyl Li © abasic sequence encoding a certain protein contrast, protein DNA that hybridizes Dizu at moderate condition code is already known to have the same biological activity as the protein.

Accordingly, the soluble HM1.24 antigen protein of the present invention is encoded Ri by the "high Buri Dizu to DNA" described above, also encompasses proteins having the biological activity of the soluble HM1.24 antigen protein.

Incidentally, amino acid sequence SEQ ID NO of human HM1.24 antigen protein expressed on the cell membrane: shown in 15 or 23. SEQ ID NO: E. coli containing the plasmid pRS38-pUC19 which holds the heat Totanpaku quality DNA encoding between Xb al cleavage site of pUC base compactors having the Amino acid sequence of 15 or 23 Escherichia coli DH5 Fei (PRS38- named pUC19), 1993 year 1 October to 5 days with at the Agency of life Institute of Advanced industrial Science and technology (thorn castle Tsukuba Higashi 1-chome, 3) as the accession number FERM BP-4434 Te, it has been an international deposit based on Budapest, Treaty.

Soluble HM1.24 antigen protein of the present invention may also be in the protein fused with other peptides or polypeptides as long as they have the biological activity of the soluble HM1.24 antigen protein. Methods of making these fusion proteins, can already be used a known method. Other peptides or polypeptides to be fused with the protein may be any peptide or polypeptide as long as it is effectively used in the present invention.

For example, as a peptide, FLAG (Hopp, TP et al., BioTec hnology (1988) 6, 1204-1210), 6 XHis, lOXHis, influenza agglutinin of six His (histidine) residues (HA), fragments of human c-myc, fragments of VSV-GP, fragments of pl8HIV, T7_tag, HSV-tag, E - tag, fragments of SV40T antigen, lck tag, a- tubulin fragment, B- tag, Pr ote in C fragments such as, peptides are used already known. Further, for example, is a polypeptide de, GST (Darutachion · S · trans Hue hydrolase), HA, Imunoguroburi emissions constant region, b-galactosidase, MBP (maltose-binding protein), and the like. These are commercially available ones can and Mochiiruko.

Protein DNA encoding the present invention, described above DNA can and by connexion building child in kit Ya methods known city sales a. For example, restriction enzyme digestion, addition of linker one can start codon (ATG) and Bruno or preoccupied codon and by Ri build child to insertion of (ATT, TGA or TAG) is.

Expression vectors protein of the present invention may be any expression vector as long as expression vectors that will be suitable for use in the present invention. Is an expression vector, expression vectors derived from mammals, for example pEF, pC DM8, expression vectors derived from insect cells such pBacPAK8, expression vectors derived from plants such Romyuita1, pMH2, Kuta one base expression from animal viruses, for example P HSV, pMV, expression vectors derived from yeast, e.g. PNVl l, expression vectors derived from Bacillus subtilis, e.g. pPL608, Rokappatauitaderutaomikuron, expression vectors derived from Escherichia coli, for example pGEX, pGEMEX, include pMAL P 2.

The expression vector of the protein of the present invention, for example, a soluble HM1. 24 antigen protein DNA encoding ligated downstream of the promoter, and by Ri manufacturing child in the child introduced Re this to expression vector can. Is a flop opening motor Z Enhansa one, promoter derived from a mammal

/ Enhansa, eg EF1 - a promoter / Enhansa, gamma one § actin promoter / Enhansa, promoter one coater Ζ Enhansa insect Uinoresu, for example a polynuclear (Porihedori down) Wirusupu port motor ζ Enhansa, flop port plant-derived motor Bruno enhancer one, for example, Tano Komozaiku Ui Roh-less promoter ζ Enhansa, animal Uinoresu promoters from / Enhansa, such as SV40-flop opening motor / Enhansa, human CMV promoter Z Enhansa, yeast-derived promoter / Enhansa, such as alcohol dehydrogenase promoter Roh enhancer, Escherichia coli promoters from Z E enhancer, e.g. Lac promoter Z Enhansa, Trp promoter one coater / Enhansa, the Tac promoter / Enhansa one mentioned It is.

The expression of the protein of the present invention may be used by adding host signal sequences suitable for use in the expression. As a signal sequence, for example, the signal sequence of a secreted protein and the like. It is a signaling null sequence of secretory protein, for example, the signal sequence of mammalian-derived secretory protein, for example Imunoguroburi down signal sequences. As the signal sequence of secretory protein, a signal sequence of E. coli-derived secretory protein, for example, expression vectors peri Burazumu secretory signal sequence thus produced good urchin like such as OmpA can be introduced into the host Ri by the known methods can. Is a method of introducing into a host, for example, elect Roporeshiyo emissions, calcium-phosphate method, Ru ribosome methods are exemplified.

Protein used in the present invention can be obtained as a recombinant protein was produced using gene recombination technology as described above. For example, recombinant protein, cells expressing them the nucleotide sequence of the genes described herein, tissue, or organ Karak cloned, incorporated into a suitable vector, which is introduced into a host to produce. The present invention can be used with this recombinant protein.

Specifically, cells expressing the protein used in the present invention, organization, or from the organ, isolating mRNA encoding the gene. MRNA is isolated by a known method, for example, guanidine ultracentrifugation method (Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299), AGPC method (Ch omczynski, P. and Sacchi, N ., Anal. Biochem., 1987) lb2, 15 6-159) total RNA was prepared Ri by the like, mRNA purification Kit (using Pharma cia) and the like to purify mRNA from the total RNA. Further, the by Ri mRNA to be used QuickPrep m RNA Purification Kit (Pharmacia) can also direct preparation child.

From the obtained mRNA using reverse transcriptase to synthesize cDNA of genes. Synthesis of cDNA can also be carried out using AMV Reverse Transcriptase First-strand cDNA S ynthesis Kit (Seikagaku Corporation) and the like. Alternatively, for the synthesis and amplification of cDNA is Marathon cDNA Amplification kit (manufactured by CLONTECH) and poly main chain reaction. (Polymerase chain react ion; PCR) 5 '-RACE method using an (Frohman, MA et al, Proc .... Natl Acad Sci USA (1988) 85, 8998-9002;.. Belyavsky, A. et al, Nucleic Acids Res (1989) 17, it is a Turkey to use 2919-2932).

The resulting DNA fragment of interest is prepared from the PCR products and ligated with a vector DN A. Et al is to prepare a recombinant vector Ri Kitareyo, was introduced into E. coli etc., from which colonies are selected to prepare a desired 耝換 Ebe compactors. Known method the nucleotide sequence of the DNA of interest, for example, to check the options by the Jidokishinu Kureochi de chain terminating cane down method. As long obtained shall be the objective DNA, which is then integrated into an expression vector. The yo Ri specifically, good urchin was constructed DNA Said, yo is urchin expression below, it is possible to obtain the protein.

When using mammalian cells, a useful promoter regularly used Z E enhancer, the gene to be expressed, be expressed by a vector comprising operably coupled to form a DNA or it poly A signal on the 3 'side downstream it can. For example, as a promoter z Enhansa and foremost, a human Tosai cytomegalovirus early promoter enhancer (huma n cytomegalovirus immediate early promoter / enhancer) can Kobushike Rukoto.

In addition, as a promoter / E down Hansa one that can be used in other protein expression, Les collected by filtration virus, Porio one Mawirusu, Adenou-virus, simian virus 40 (SV 40) c I Angeles promoter / Enhansa Ya human Terongeshi Yo such as Nfa click ter 1 a may be used promoter Z Enhansa from mammalian cells (HEF1 a). For example, when using the SV 40 promoter / Enhansa, Mu Lligan's method (Nature (1979) 277, 108), also when using the HEF1 shed promoter one coater Z Enhansa, of Mizushima et al. Method (Nucl eic Acids Res . (1990) 18, it can be easily carried out according to the 5322).

For E. coli, a commonly used useful promoter, a signal sequence for protein secretion, the gene to be expressed operably coupled to form can be revealed by calling. As the promoter include a lacZ promoter one coater, the araB promoter. When using the lacZ promoter one, Ward's method (Nature (1098) 341, 544-546; FASEB J. (1992) 6, 2422-2427), when using the araB promoter, Better et al's method (Science (1988) 240, may follow in 1041-1043).

As a signal sequence for protein secretion, when produced in the E. coli Bae re-flop Razumu, pelB signal sequence (Lei, SP et al J. Bacteriol. (1987) 169, 4379) may be employed.

Is the origin of replication, SV 40, polio one Mawirusu can be used those derived from such Adenowiru scan, © shea papillomavirus (BPV). Et al is, to increase the gene copy number in the host cell system, expression vectors as a selection marker, Aminoguri Koshidohosuho trans luciferase (APH) gene, the thymidine kinase (TK) gene, E. coli xanthine guaiacolsulfonate Nin phosphoryl ball transferase (Ecogpt) gene, may include the dihydrofolate reductase (dhfr) gene, and such leave in.

In the present invention, for the production of proteins, it is possible to use any production system. Production systems for protein production, there are the production systems in vitro and in vivo. As the in vitro production system include a production system that uses the production system and prokaryotic cells using MakakuHoso cells.

When eukaryotic cells are used, there are animal cells, plant cells, production systems Ru using fungal cells. Is an animal cell, (1) mammalian cells such as CH0

(J. Exp. Med. (1995) 108, 945), COS, myeloma, BHK (ba by hamster kidney), HeLa, Vero, (2) amphibian cells, for example, § unfavorable mosquito laevis oocytes (Valle, et al., Nature (1981) 291, 35 8-340), or (3) insect cells, for example sf9, sf21, Tn5 is known. Is a CH0 cells, particularly in CH0 cells deficient in DHFR gene dhfr- CHO (Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4 220) and CHO Kl (Proc. Natl. Acad. Sci. USA (1968) 60, 1275) can be suitably used.

It is a plant cell, Nicotiana 'Tapakumu (Nicotiana tabacum) and cells derived from known, which is subjected to callus culture. It is a fungal cell, a yeast, for example Saccharomyces Mrs (Saccharomyces) genus, for example the Saccharomyces Mrs' cerevisiae (Saccharomyces cerevisiae), filamentous fungi such Asuperugiusu genus (Aspergillus) genus, for example Asupe Noregiusu and secondary Gar (Aspergillus niger ) force has been known S. When the prokaryotic cells are used, there are the production systems which employ bacterial cells. Is a bacterial cell, Escherichia coli (E. coli), and Bacillus subtilis are known.

These cells were DNA by Ri transformed for the purpose of, by Ri protein is obtained by culturing was transformed cells in vitro. Culture is carried out according to known methods. For example, it can be used as the culture liquid, DMEM, ME M, RPMI1640, IMDM. At that time, it can either be a serum supplement such as fetal calf serum (FCS), may be serum-free culture. Preferably, pH of the culture is about 6-8. The cultivation is usually carried out at about 3 0 to 4 0 ° C for about 1 5-2 0 0 hours, replacement of the medium, if necessary, aeration and stirring.

On the other hand, as a production system in vivo is, production systems, and the like to use the production systems and plants to use the animal. Introducing DN A shall be the object into these animals or plants, proteins are produced in such animals or plants, and recovered.

When animals are used, there are the production systems which employ mammals and insects. Mammals, catcher formic, pigs, Hijji, mice can Turkey and force S using © Shi (Vicki Glaser, SPECTRUM Biotechnology Applica tions, 1993). Alternatively, the mammals may be used transgenic click animals.

For example, by inserting the protein produced in the middle of the gene coding for the preparation as a fused gene specific to DNA catcher formic] 3 casei good of emissions Una milk a of interest. The DNA is injected DNA fragments containing the fusion gene inserted in catcher formic into the embryo, and the embryo is introduced into a female catcher formic. Trans Jie nick catcher formic or its progeny born from catcher formic who received the embryo is obtained a protein from milk that eggplant production. To increase the amount of milk containing the protein to be produced from trans-diethyl nick catcher formic, it may also be used as appropriate hormones to trans Jie nick catcher formic Rere. (Ebert, KM et al., Bio / Technology (1994) 12, 699-702). Further, as the insects, it can be used, for example silkworms. When using a chi co, it was infected with Pakyurowirusu inserting the DNA of interest to the force I co, to obtain the desired protein Ri by the body fluid of the silkworm (Susumu, M. et al., Nature (1985) 315, 592-594).

When using plants, for example, tobacco may be used in is al. When using a Tapako, insert the DNA of interest into a plant expression vector, the pMON 530 if example embodiment, the vector is introduced into good Unano Kuteri authors Aguropakuteri ©-time Tsu main Fashiensu (Agrobacterium tumefaciens). The Pakuteria tobacco, for example, by infecting Nicotiana Tapaku arm (Nicotiana tabacum), from the leaves of the tobacco to obtain the desired protein (Julian, K. -C. Ma et al., Eur. J. Immunol. ( 1 994) 24, 131-138).

Incidentally, as a method for introducing the expression vector into a host, a known method, for example-phosphate calcium method (Virology (1973) 52, 456-467) Yae Lek Toroporesho down method (EMBO J. (1982) 1 , 841-845), and the like can be used. In consideration of codon usage frequency in the host used for expression, it is possible to design a high sequence of more expression efficiency (Grantham, R. et al., Nucleic Acids Research (1981) 9, r43- r74) .

To introduce these animals or above of good sea urchin gene in plants, the protein is produced in the body of the animal or plants, and recovered. Expressed and produced proteins as described above can be purified to homogeneity separated inside or outside of the cell, from the host. Separation of protein used in the present invention, refining separation used in conventional protein, Bayoku Using purification method, not intended to be limiting in any way.

For example, Afi two Teak chromatography etc. chromatograph Ikaramu, filter, ultrafiltration, salting out, dialysis, SDS poly Ata Li Ruami Dogeru electrophoresis, appropriately selecting the isoelectric focusing and the like, the combination lever, proteins separation, can be purified (new experimental biochemistry Lecture 1 (1990), Tokyo Kagaku Dojin).

Is a chroma Togurafi one, for example, Afi two tea chroma toggle roughy, ion exchange chroma preparative chromatography, hydrophobic chromatograph I over, gel filtration, reverse-phase chroma preparative chromatography, eclipsed adsorption chroma Togurafi Chief force 牵 (Strategies for Protein Purification and then ha racterization:.. A Laboratory Course Manual Ed Daniel R. Mar shak et al, Cold Spring Harbor Laboratory Press, 1996). Chroma preparative chromatography of these can be carried out using HPLC, the liquid phase chroma Togurafi one such FPLC.

Proteins, can measure the concentration using known methods. For example, it may be used measurement or Bradford methods absorbance.

The present invention includes a step of the anti-HM1.24 antibody contained in the soluble HM1.24 antigen protein and the test sample is reacted, to detect or measure the anti-HM1.24 antibody bound to the soluble HM1.24 antigen protein , immunochemical measurement method of anti-HM1.24 antibody; and

And the soluble HM1.24 antigen protein reacted contained in the anti-HM1.24 antibody and the test sample, comprising detecting or measuring the soluble HM1.24 antigen protein bound to the anti-HM1.24 antibody, on the immunochemical method of measuring the soluble HM1.24 antigen protein.

Immunochemical measurement method provided in the present invention is carried out as § Ssi system in vitro. It is shown schematically an example of the method in FIG. Atsusi system of in vitro is carried out in a non-cellular system. Specifically, by binding soluble HM1.24 antigen protein to a support, adding a test sample containing Ko匪 1.24 antibody to the protein, soluble HM1.24 antigen bound to the support is washed after incubation it may be detected or measured binding of anti-HM1.24 antibody to the protein. Or anti-that specifically, to bind the anti-HM1. 24 antibody to a support, the protein soluble HM1. The test sample containing 24 antigen protein was added and bound to the support by washing after the Lee Nkyube bets HM1. soluble HM1 for 24 antibodies. 24 may be detected or measured antigen binding protein.

Soluble HM1. 24 antigen protein or anti-HM1. 24 antibody, cells expressing them uniquely, they were introduced DNA encoding cells, proteins produced from them was introduced DNA encoding an animal or plant and Ru can be used in the form of or in crude state purified.

Purified or crude purified soluble HM1, 24 antigen protein or anti-HM1. 24 is attached to a support either a protein antibody. It can be immobilized on the supporting lifting body protein in a standard way in binding the protein to the support. It is as a support to bind proteins, such as insoluble polysaccharides, for example Agarosu, dextrin preparative run-cellulose, synthetic resins such as Po Li styrene, poly accession Li Ruami de Siri co emissions, and the like.

The good Ri specific commercially available beads, which are produced by them as a raw material, flop rate is used. In the case of beads, it may also be using a column such as these have been filled with records,. For plates, multiwell plates (96 Anama Ruchi well plates, etc.) or a biosensor chip and the like.

Binding of the protein and the support, a chemical bond, physical adsorption or the like, may be coupled Ri by the method to be had through conventional. Further, the protein specifically recognizing antibody allowed bind to by Ri advance support to the method described above, Ru can also bind Ri by the possible to connect the this antibody and protein. Et al is soluble HM1 can be attached via avidin / Piochin. 24 antigen protein and an anti-HM1. 24 binding antibodies is usually carried out in a buffer. Is a buffer, for example-phosphate buffer, Tr IS buffer solution is used. Further, as the condition of i Nkyube metropolitan, conditions that have been used already well, for example, Lee Nkyubesho down from 1 hour to 2 4 hours at 4 ° Celsius to room temperature is carried out. Lee Nkyube after preparative washing, the soluble HM1. 24 antigen protein and an anti-HM1. Well anything as long as it does not interfere with the binding of the 24 antibodies, for example, a buffer containing a surfactant is used. As a surfactant, for example, 0. 05% Tween 20 is used.

Soluble HM1 to be determined in accordance with the present invention. Is a test sample containing 24 antigen protein or anti-HM 1. 24 antibodies, human body fluids (blood, serum, urine, Takashi Seki solution, etc.), cell culture supernatant, animal secretions (milk, etc.), may be mentioned pharmaceutical preparations and the like.

Soluble HM1 contained in these test sample. 24 when detecting or measuring the binding of anti-HM1. 24 antibody or soluble HM1. 24 antigen protein to an antigen protein or an anti-HM1. 24 antibody, Inkyupe bets and washed under appropriate conditions Ri by the and the child, the specific binding and non-specific binding can and child separation. The soluble HM1. 24 antigen protein and an anti-HM1. May be evaluated state of bonding between 24 antibody.

In immunochemical assay method of the present invention may be installed control group with the group contacting a test sample to the protein. Is the Control group, soluble HM1 which is negative control group or purifying containing no test sample. 24 antigen protein or anti-HM1. Positives controls opening Lumpur group containing 24 antibodies of preparation or the two groups it can be placed.

Ri by the immunochemical assay method of the present invention, the bound protein can and the detection child. Or binding proteins can also child quantitatively measured. In these cases, the results obtained with negative control group containing no test sample, standard soluble HM1. 24 antigen protein or Ko匪 1.24 antibody results and or refining obtained in the group containing test sample goods to by comparing the results obtained in including positive control group, it is a child detecting binding of the soluble HM1. and 24 antigen protein anti HM1. 24 antibody.

Further, obtained as numerical results of their detection, Ri by the comparing child their numbers, soluble HM1 contained in the test sample. 24 antigen protein or anti-HM1. Quantitatively measuring 24 antibody It can also be. If for quantitatively measuring, comparing the values ​​obtained in the group to which the test sample containing numbers and soluble HM1. 24 antigen protein or anti-HM1. 24 antibody obtained in the negative control group containing no test sample I Ri, it is possible to quantify the amount of binding between the soluble HM1. 24 antigen protein and Ko匪 1-24 antibody. If soluble HM1. 24 antigen protein or anti-HM1. 24 antibody contained in the test sample, the soluble HM1 Ri by that protein bound exists. 24 antigen protein or anti-HM1. Detecting or measuring 24 antibody be able to.

In the case of quantitative determination, the soluble HM1. 24 antigen protein or anti-HM1. 24 antibody can be quantitated based on a standard curve Ri created by the values ​​obtained by the positive control group containing known amounts.

In immunochemical assay method of the present invention, it is possible to use a biosensor using the surface Burazumon resonance phenomenon as a means for detecting or measuring soluble HM1. 24 antigen protein or anti-HM1. 24 antibody in the test sample . Surface plasmon resonance phenomenon biosensor utilizing the interaction between proteins one protein without whether One labeled child using protein traces, be observed in Li Alta I beam as a surface plasmon resonance signal It can be (e.g. B IAcore; manufactured Pharmac ia). Therefore, it is possible to detect or measurement of binding of the B soluble Ri by using a biosensor such as IAcore HM1. 24 antigen protein and an anti-HM1. 24 antibody. Specifically, the soluble HMl.24 antigen protein or anti HMl.24 antibody immobilization to a sensor chip, contacting the test sample containing anti-HM1.24 antibody or soluble HM1.24 antigen protein, soluble HM1 .24 Ru can anti-HM1.24 antibody or soluble HM1.24 antigen protein binds to an antigen protein or anti-HM1.24 antibody is detected or measured as a change in the resonance signal.

The good Ri specifically, may be carried out as follows. A sensor chip CM5 (Biosensor, Inc.) are activated to immobilize the soluble HM1.24 antigen protein or anti-HM1.24 antibody on the sensor chip in the beginning. That, ED CI NHS solution (200 mM EDC (N-Echiru _Ν '- (3- dimethylaminopyridine Bruno flop port pills) Kabone preparative hydrochloride), 50mM NHS (N - arsenate Dorokishisakushin Lee Mi de)) in I Ri sensor chip activity after ized, HBS puffer (10mM HEPES pH7., 150mM NaCl, 3.4m MEDTA, 0.05% Tween20) washing the more sensor chips.

Then a test sample containing an appropriate amount of anti-HM1.24 antibody or soluble HM 1.24 antigenic protein dissolved in HBS puffer one in contact with the sensor chip immobilized. After washing the by Ri sensor chip in HBS buffer one, ethacrylic Noruami emissions solution (1M Etanoruami down hydrochloride, pH 8.5) to block the remaining active groups on the sensor chip Ri by the. Used for washing bound evaluate the sensor chip Ri by the HBS buffer one again.

Then injecting a test sample containing an appropriate amount of anti-HM1.24 antibody or soluble HM 1.24 antigenic protein dissolved in HBS puffer scratch. Immobilized amount of anti-HM1.24 antibody or soluble HM1.24 antigen protein of the soluble HM1.24 antigen protein or the test sample bound to the anti-HM1.24 antibody resonance signal values ​​to sensor-chip at this time It is observed as the increase in the.

Et al is also with the group containing the test sample, may be installed control group. The control group, negative con preparative port Lumpur group containing no test sample, be placed a known amount of soluble HM1. 24 antigen protein or anti-HM1. Positive controls opening Lumpur group or its two groups containing 24 antibody it can . Bound protein can be measure quantitatively as a variation of the resonance signal value. In this case, the results obtained with negative controls opening Lumpur group containing no test sample, obtained as a result and / or in the group containing the test sample a known amount of a soluble HM1. 24 antigen protein or anti-HM1. 24 antibody by the comparing the results obtained with the positive properties con preparative rolls comprising Ri, it is possible to detect or measure the protein of interest in a test sample.

In immunochemical assay method of the present invention, as a means of detecting or measuring a protein in the test sample bound, soluble HM1. 24 antigen protein or anti-HM1. Primary antibody that specifically recognizes 24 antibody it can be used.

For example, soluble HM1. Contacting the 24 antigen protein or Ko丽 1. Test specimen 24 antibody is by Ri detecting or measuring a protein washed and bound to primary antibody that specifically recognizes the protein. That is, preferably contacting a test sample containing protein while power sale to one of the proteins obtained by binding to a support. After Incubate, with washing, it may be specifically by Ri detect or measure the primary antibody recognizing the protein of protein binding. The primary antibody, rather than are labeled Ri by the labeling substance preferred.

Soluble HM1. 24 antigen protein may be fused with another peptide or Poripe Petit de. Therefore, soluble HM1 contained in the test sample. 24 to the Ko匪 1.24 antibody to detect antigen protein can that you use, the soluble HM1. 24 Other peptidyl de or poly fused to an antigen protein it is possible to use an antibody against the base peptide. It is also possible to use the anti-HM1. 24 antibody that specifically recognizes the antibody to detect anti-HM1. 24 antibody contained in the test sample. If anti-HM1. 24 antibody is a mouse antibody, it may be used an anti-HM1. As an antibody that specifically recognizes 24 antibody anti Mausui Munoguroburi down antibodies. Moreover, anti-HM1. If 24 antibody is a chimeric antibody or a human type antibody can be used anti-HM1. As an antibody that specifically recognizes 24 antibody anti human Toimunoguropuri down antibodies.

Protein can be by Ri labeling to the normal methods known. As the labeling substance, for example, radioisotope, enzyme, fluorescent substance, bi Ochin / avidin, and the like. These labeling substances may be commercially available labeling substances. It is to radioisotopes, for example 3 2?, 3 3 P, 1 3 1 I, 1 2 5 I, 3 H, 1 C, 3 5 S and the like. Is an enzyme, for example alkaline phosphatase, horseradish Pas Okishidaze, beta - galactopyranoside DOO oxidase, beta - Darukoshidaze the like. Is used as the fluorescent substance, for example furo O b fluorescein isothiocyanate Shi Aneto (FITC), include Rodami down. These can be obtained commercially, it is labeled by known methods.

Specifically, it can be carried out as follows. That solution was added to plates to containing the soluble negation 1.24 antigenic protein or anti-HM1. 24 antibody, fixed to the plate and left overnight. Soluble HM1. 24 antigen motor damper click proteins or anti-HM1. When fixing the 24 antibodies, to fix the antibody to each beforehand Me plates, soluble HM1 the immobilized antibody. 24 antigen protein or anti-HM1. 24 antibody coupled to form it may be. After plate washing, pro Kkingu with, for example, BSA to prevent non-specific binding of other emissions Park protein. Washed again, anti-HM1. Add 24 antibodies or soluble HM1. 24 antigen protein including a test sample to the plate. At the same time puts the group containing no test sample (Negative Control port Lumpur) and or a known concentration of anti-HM1. 24 antibody or soluble Itamyu 1. group plus 24 antigen protein (positive control), which Raoi Nkyube DOO to. After Lee Nkyube bets, washed added antibody against the test sample. After the proper degree of I Nkyubesho down, detecting or measuring a protein Ri by the primary antibody that specifically recognizes the protein was washed plates. The detection or measurement is more detected or measured when a liquid scintillation one to emissions of radioactive isotopes. For enzyme that substrate was added, the enzymatic change of the substrate, for example, detecting or measuring a color by absorbance meter. In the case of the fluorescent substance is detected or measured Ri by a fluorometer. These results can be used to determine the test sample containing the inhibitor by comparing values ​​obtained by con preparative port Lumpur group.

In immunochemical assay method of the present invention, soluble in the test sample HM1. 24 antigen protein or anti-HM1. As a means for detecting or measuring the 24 antibodies, soluble HM1. 24 antigen protein or anti-HM1. 24 antibody specificity can be used specifically recognizes secondary antibody to the primary antibody and primary antibody you recognized.

For example, in immunochemical assay methods described above, soluble HM1. 24 antigen protein or anti-HM1. 24 antibody contacted with the test sample, after Incubate, singular manner the protein protein washing to bound I Ri detected or measured specifically recognizes secondary antibody primary antibody and primary antibody recognized the. That, in particular soluble HM1. 24 antigen protein or anti-HM1. 24 antibody is immobilized to a support, contacting a test sample. After incubate, and washed, may be detected or measured by specifically recognizing the secondary antibody that specifically recognizes the primary antibody and primary antibody the protein protein bound. The secondary antibody is preferably labeled with a labeling substance. Antibodies can be labeled Ri by the usual known Ru aforementioned method.

Specifically, it can be carried out as follows. That solution was added to plates to containing the soluble HM1. 24 antigen protein or anti-HM1. 24 antibody, fixed overnight allowed to the plate. When fixing to the plate, in advance soluble HM1. Antibodies were fixed to the plate for 24 antigen protein or anti-HM1. 24 antibody, soluble HM1 to the immobilized antibody. 24 antigen protein or anti-HM1. To bind the 24 antibody it may be. After the plates wash, Bro Kkin grayed with, for example, BSA to prevent non-specific binding of proteins. It washed again, adding a test sample to the plate. At the same time the group does not contain the test specimen (negative con preparative roll) and and Z or a known concentration of anti-HM1. Place the 24 antibodies or soluble HM1. 24 group plus antigen protein (positive controls) and incubated them.

After Lee Nkyube bets, anti HM1 contained in the washed test sample. 24 antibody also added soluble HM1. 24 primary antibody against the antigen protein. After appropriate Lee Nkyubesho down, the plates were washed and then added singular recognizes the secondary antibody primary antibody. After appropriate Lee Nkyubesho down, washed to detect or measure the protein specifically specifically recognizes secondary antibody by Ri protein primary antibody recognizing contained in the test sample. The detection or measurement, to detect or measure when Ri by the liquid thin Chireshiyo emissions of radioactive isotopes. For enzyme that substrate was added, the enzymatic change of the substrate, for example, detected or measured by an absorbance meter coloration. In the case of the fluorescent substance is detected or measured Ri by the fluorometer.

These results can be used to determine the test sample containing the inhibitory substance by comparing the value obtained in control group. Soluble HM1. 24 antigen protein, but it may also be fused with other peptides or polypeptides. Therefore, soluble HM1 contained in the test sample. 24 and temporary antibodies to detect antigen protein anti HM1. It can be used 24 antibodies, soluble HM1. Other Bae fused with 24 antigen protein peptide or may be used an antibody against the polypeptide. It is also possible to use the anti-HM1. To detect 24 antibody anti HM1. 24 antibody singular recognize antibodies contained in the test sample.

If anti-HM1. 24 antibody is a mouse antibody, anti-HM1. It can be used an anti-mouse I Takeno Glo purine antibody 24 antibodies as specifically recognizes the primary antibody. Also, if the anti negation 1.24 antibody is a chimeric antibody or a human type antibody can be used anti-HM1. Anti human Toimunoguropuri emissions antibody 24 antibodies as specifically recognizes the primary antibody. Further, as the secondary antibody, as possible out appropriately selecting the antibody that specifically recognizes the primary antibody. For example, if the primary antibody is a Hijji antibodies can be used anti Hijjiimuno Guropuri down antibodies. Further, if the primary antibody is Usagi antibodies can be used anti-© heron I Takeno Glo purine antibody.

Yo Ri particular, the present invention is particularly preferably this and force S performed ELI SA (Enzyme-l inked Immunosorbent Assay) good by Ri follows Unishi. That is, soluble HM1. 24 antigen protein and fused HA antibody immobilized buffer against (Flu Enza agglutinin) (0. 1 M NaHC0 3, 0. 02% NaN 3, pH9. 6) Ri is diluted by the . Adding an appropriate amount of this solution was diluted to each well of a 96-well Imunopure bets (manufactured by Nunc), 4 ° C De 晚I Nkyupe reports open to immobilize.

After washing 3 times each well with wash buffer (PBS to 0, 05% Tween20 become as those prepared), (manufactured by S IgM a) 5% BSA in PBS solution 20 0 beta 1 was added, at room temperature for 2 hours to Bro Kkingu.

Then washed three times each well with wash buffer, dilution buffer (1% Β SA, 0. 5% Tween20, PBS) the soluble HM1 fused to HA diluted in. In one 24 antigen protein was added 4 ° C evening incubated reports open soluble fused with anti-HA antibody and HA HM1. binding the 24 antigen protein. After washing with wash puffer one three times, human I gG antibody constant region chimeric anti HM1 having a (C region). The test sample containing 24 antibody constant amount added and Bok Kyube 1 hour fin at room temperature.

Each well is washed three times with wash buffer, 5000-fold alkaline phosphatase diluted labeled catcher formic anti human I g G antibody (manufactured by IB I) 100 mu 1 was added to each well at a dilution buffer, for 1 hour at room temperature incubated. Each well is washed five times with wash puffer developing solution; the (substrate puffer. 50 mM NaHC0 3, 10mM MgCl 2, pH9 8 to Sigma 104 dissolved in a concentration of 1 mg / ml) 100 μ 1 to each well in addition, measured using the absorbance microphone Ropure bets leader in 405 eta m after reacting at room temperature (Model3550, BI O- manufactured RAD). These results by comparing the values ​​obtained by the negative con preparative rolls and Roh or positive control group, it is possible to detect or measure the chimeric anti-HM1. 24 antibody. Further, Ri by the same method, it is possible to detect or measure the soluble HM1. 24 antigen protein.

Disk-di-learning method of the present invention can also be used for High Throughput Screening (HTS). Specifically, carried out until Bro Kkingu manually, subsequent reaction is O over Bok Meshiyo Ni spoon by made by a mouth pots, can be force s to achieve a High Throughput screening.

That is, the antibody-immobilized buffer against HA (0. 1M NaHC0 3, 0. 02% NaN 3, pH9. 6) Ri is diluted by the. 96 to immobilize and one 晚 Lee Nki Yubeto qs added 4 ° C the solution was diluted to each well of Imunopure bets (manufactured by Nunc) of the hole.

After washing 3 times each well with wash buffer (PBS to those prepared to be a 0. 05% Twe en20), (manufactured by SI GMA) 5% BSA in PBS solution 20 0 mu 1 added, 2 at room temperature time to blow Kkingu. Then washed three times each well with wash buffer and foremost, dilution buffer (1% BSi, 0. 5% Tween20, PBS) soluble fusion with diluted HA in HML. At 4 ° C overnight added 24 antigen protein Lee Nkyube reports open soluble fused with anti-HA antibody and HA HM1. binding the 24 antigen protein.

In the next Rere, Iretsue iiB iomek2000 HTS syst em (manufactured by Beckman) ί to set the individual I beam Bruno plate, chimeric anti-HM1. Test sample containing 24 antibody, chimeric anti-HM1. One for 24 antibody primary antibody and executes by Uni system control program of the addition of the secondary antibody against the primary antibody.

In this case, the dispenser as the Bi omek 2000 dispensing machine (manufactured by Beckman) there have the Mul t ipi P et t e96 Penetration dispenser of the solution Imuno plates to each well by using the (Sagian Ltd.) it is possible to perform the removal of the dispensing or solution. Also, the cleaning of each well of Imunopure bets can be used EL404 microplate © O Sshiya (Bi o Tek Co.). Also, the measurement of the absorbance can have use the SPECTRAmax250 plate reader (manufactured by Mo l ecular Devi ces).

Program is set to perform the following operation. That was washed 3 times each well with wash puffer, the test sample and the dilution buffer (1% BSA, 0. 5% Tween20, PBS) chimeric anti HM1 diluted in. Add a fixed amount of test specimen comprising 24 antibodies. At the same time the group containing no test sample (negative con trolls) and a known concentration of chimera anti HM1. 24 antibody Place the group (positive con preparative port Lumpur) was added, 1 hour Incubate these at room temperature. In the cleaning path Ffa washed three times each well, Usagi was diluted 5000-fold the anti-human I gG antiserum (manufactured by New England Bio labs) 100 μ 1 was added to each well in dilution buffer and incubated 1 hour at room temperature . Each well is washed three times with wash buffer, added Al force was diluted 5000-fold re off Osufataze labeled catcher formic anti Usagi I gG antibody (manufactured by TAG0) to 100 mu 1 each well at a dilution puffer, 1 hour incubation at room temperature Tosuru.

Each well is washed five times with washing buffer, color development solution (substrate buffer;. 50 mM NaHC0 3, 10 mM MgCl 2, pH9 of 8 to lmg / ml concentration this溶角Army the p- nits Roff enyl phosphate ( added S-made i gma)) to each well ΙΟΟ μ Ι, absorbance microplate rate Bok di one goo at 405 nm after reaction at room temperature, B i omek play Bok]) one coater (be ckman / Mo le cular It measured using a D-made evi ces). Ri by the comparing the values ​​obtained these results con preparative rolls, it is possible to detect or measure the chimeric anti-HM1. 24 antibody contained in the test sample. Further, Ri by the same method, it is also possible to detect or measure the soluble HM1. 24 antigen protein.

• immunochemical measurement method that is provided by the present invention, it is possible to measure the soluble HM1. 24 antigen protein or anti-HM1. 24 antibody to a concentration of 500 pg / ml.

Antibodies used in the present invention, it is possible to use antibody that is part of the commercially available antibodies and a commercially available kit, it can be obtained as monoclonal antibodies or polyclonal antibodies using known means .

Monoclonal antibody, using the desired sensitizing antigen and is immunized in the conventional method of immunization, immune cells thus obtained are fused with known parent cells in the conventional cell fusion legal, conventional screening method I Ri, the mono-click polyclonal antibody-producing cells can be by connexion prepared to be screened in.

Specifically, the following procedures may be used to prepare a monoclonal antibody or poly-click polyclonal antibodies.

For example, the sensitizing antigen for obtaining antibody can be obtained, but are not limited to animal species from which it is derived, actually mammal a - derived peptides or polypeptides for use in the present invention, for example human, mouse or rat those derived from the good Masui. Of these, especially sensitizing antigen derived from human is preferred. For example, human soluble HM1. 24 When using as a sensitizing antigen antigenic proteins, their nucleotide sequences and Amino acid sequence can be obtained using the gene sequence heritage disclosed herein . In the case of using the other peptide or Poribe Petit de to be fused to the soluble Yuzuru 1.24 antigenic protein as a sensitizing antigen, chemically synthesis their peptide or polypeptide or, it can be obtained by genetic engineering techniques.

Protein used as a sensitizing antigen, peptide or Poribe Petit de may be used to its full length, also it can be used fragments thereof. Is a fragment, Ru include for example C-terminal fragment or N-terminal fragment. Alternatively, the protein to be used as a sensitizing antigen, peptide or as possible out also be used as a sensitizing antigen cells expressing Poribe peptide.

Is a mammal to be immunized with the sensitizing antigen are not particularly limited, but is preferably you selected in consideration of compatibility with the parent cell used for cell fusion, generally bald One tooth th Usagi eyes, primates are used.

Is the animal order Rodentia, such as mice, rats, hamsters and the like. Is a Usagi th animal, for example, Usagi is used. As the primates of animals, such as monkeys are used. It is a monkey, a narrow nose under the eyes of monkeys (Old World monkeys), for example, force two Kuizaru, Akagezaru, sacred baboons, chimpanzees and the like are used.

Immunization of animals with a sensitizing antigen, Ru is carried out in accordance with known methods. For example, as a general method, the sensitizing antigen intraperitoneally mammalian or carried out Ri by the fact that subcutaneous injection. Specifically, conventional adjuvants sensitizing antigen diluted in PBS (Phosphat e- Buffe red Sal ine) and appropriate amount of physiological saline etc. is mixed, as desired, which was suspended, for example, an appropriate amount mixed Freund's complete Ajupanto and, after emulsification, preferably administered several times 4-2 1 daily to the mammal. It is also possible to use a suitable carrier at the time of immunization of the sensitizing antigen. Thus immunization, the desired antibody levels confirmed by a conventional method to increase in the serum.

Here, to obtain a polyclonal antibody, after the desired antibody levels in the serum was confirmed to be elevated, taking out a mammal sensitized with the blood antigens. This separates the by Ri serum to known methods from the blood. Use may be made of a serum containing polyclonal antibodies to a polyclonal antibody, a fraction containing the polyclonal antibodies from the serum may be further isolated if necessary.

To obtain monoclonal antibodies, after the desired antibody levels in the serum of mammals sensitized with the antigen was confirmation of the increase, it removed immune cells from a mammal, may lie down to cell fusion. In this case, as a preferred immune cells used for cell fusion, in particular a mammalian myeloma cells as the other parent cells which splenocytes are fused with the immune cells and the like, already various known cell lines, for example, P3 (P3x6 3Ag8.653) (Kearney, JF et al., J. Immunol. (1979) 123, 154 8-1550), P3x63Ag8. Ul (Yelton, DE et al., Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS - 1 (.. Kohler, G. and Milstein, C., Eur J. Immunol (1976) 6, 511-519), MPC - 11 ( . Margulies, DH et al, Cell (1976) 8, 405 - (. Shulman, M. et al, Nature (1978) 415), SP 2/0 276, 269-270), F0 (de St. Groth, SF and Scheidegger, D., J. Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, IS, J. Exp. Med. (197 8) 148, 313-323), R210 (Galfre, G. et al., Nature (1979) 277, 131-133) or the like is preferably used.

The immune cells and cell fusion of myeloma port Ichima cells are basically methods who is known to, for example, the method of Mirusuti Nra (Galfre, G. and Milstein, in, Methods Enzymol. (1981) 73, 3-46 ) Ru can be carried out in a manner similar to like.

The yo Ri specifically, the above cell fusion, for example, carried out in the presence of a cell fusion promoter in a conventional nutrient culture medium. As the cell fusion accelerator, for example

, Polyethylene glycol (PEG), it is used Sendai virus (HVJ) or the like, may be added using an auxiliary agent such as dimethylsulfoxide in order to enhance further desired fusion efficiency.

The ratio of immune cells to myeloma cells is preferably, for example, immune cells 1 to 1 0 times the myeloma cells. Is the culture medium used for the cell fusion, for example, suitable RPMI 1640 culture medium 增 ingrowth of the myeloma cell line, MEM culture medium, and other conventional culture medium used for this type of cell culture is used is possible, furthermore, it may be a serum supplement fetal calf serum (FCS) or the like.

Cell fusion, the mixed well with immune cells and the culture solution in a predetermined amount of the myeloma cells, to which a PEG solution previously heated to about 37 ° C, for example, a PEG solution having an average molecular weight 1000 to 6000 a , it is added at a concentration of 30~ & 0% (w / v), to obtain the desired fusion cells by mixing child (high pre dormer) is formed. Subsequently, sequential addition of a suitable culture liquid, can be removed by centrifugation in growth by Ri High Priestess dormer repeats the operation to remove the supernatant, cell fusion agents etc. which are undesirable.

The High Priestess dormer is standard selection medium, for example HAT medium is selected Ri by to culture with (a culture solution containing hypoxanthine, Aminoputeri emissions and thymidine). The culture in the HAT culture medium is sufficient time for the cells other than High Priestess dormer of interest (non-fused cells), to die continues usually several days to several weeks. Then, the conventional limiting dilution method is disk renin grayed and cloning of High Priestess dormer producing the antibody of interest.

Further, in addition to Ru give the High Priestess dormer by immunizing animals with antigens other than human, human lymphocytes, for example human lymphocytes and in vi t ro with peptide or Poripe infected with EB virus peptide and sensitized with lysates of those expressing cells or their, myeloma cells having a sensitized lymphocytes with ability to divide permanently from human, for example, fused with U266, peptide or Poribe binding activity to Petit de it is also possible to obtain a High Priestess dormer producing the desired human antibody having (JP 63-17688).

Furthermore, transgenic an antigen click animal peptide or polypeptide having Repato Li one human antibody genes, their expression fine 胞又 acquires immunity to antibody-producing cells and the lysate which Mie port It may be acquired human antibodies against a peptide or polypeptide used in the present invention by using a High Priestess dormer fused to Ichima cells (international Patent application Publication No. Keio 2 - 03918, W093-2227, easy 4 - 02602, W094-2 5585, image 6 - 96/33735 and field 6 - 96/34096; see).

Hype Li dormer producing mono click polyclonal antibody prepared in this way, it is possible to subcultured in a conventional culture medium, or can be long-term storage in liquid nitrogen .

To obtain a mono-click polyclonal antibody from the hybridoma is a person the High Priestess dormer is cultured in the conventional method, the method may be a culture supernatant or High Priestess dormer mammal compatible therewith and, transplanted to proliferate, such as the Ru is employed a method of obtaining as its ascites. The former method is suitable for obtaining highly pure antibody, while the latter method is suitable for mass production of antibodies.

In addition to producing antibodies with High Priestess dormer, it may be used cells immortalized by immune cells oncogene sensitized lymphocytes, etc. to produce antibodies (onc ogene).

Thus obtained monoclonal antibodies can also be obtained as a recombinant antibody produced using genetic recombination technology. For example, recombinant antibody by cloning the antibody gene from the immune cells of the sensitized lymphocytes, etc. to produce High Priestess dormer or antibody, incorporated into a suitable Betata one, which was introduced into a host to produce. In the present invention, the recombinant antibody of this can and Mochiiruko (for example, Borrebaeck, see C. A. K. and Larrick, JW, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990) .

Antibodies used in the present invention, as long as they have the desired binding activity may be an antibody fragment or modified antibody of it. For example, antibody fragments, Fab, F (ab ') 2, Fv or Shinguruchiwei down Fv obtained by linking Fv of H chain and L chain with a suitable linker one (scFv) can be mentioned. Specifically, enzyme antibody, for example, Papai down, or to produce antibody fragments with pepsin, or the antibody fragment to construct a gene encoding, after introducing an expression vector so, for example the expression (in a suitable host cell, Co, MS et al, J. Immunol (1994) 152, 2968-2976;... be tter, M. and Horwitz, AH, Methods Enzymol (1989) 178, 47 6-496;. Pluckthun, A. and Skerra, A., Methods Enzymol (1989) 178, 497-515;. Lamoyi, E., Methods Enzymol (1986) 121, 65 2-663; Rousseau, J. et al ., Methods Enzymol (1986) 121, 6 63-669;.. Bird, RE and Walker, BW, Trends Biotechnol (1991) 9, reference 132 - 137). The present invention can be used a chimeric antibody or a human-type antibody Ru produced Ri by the known techniques.

Moreover, antibody that is by Ri detected or measured in immunochemical assay methods of the present invention, the aforementioned antibody, antibody produced in example High Priestess dormer, recombinant antibodies, chimeric antibodies and human-type antibody It may be any of.

The yo urchin expression, antibodies produced can be purified to homogeneity separated inside or outside of the cell, from the host. Separation of the antibody used in the present invention, purification separation that is conventionally used for proteins can be used purification method, not intended to be limiting in any way.

For example, Afi two Teak Roma Togurafi Chief of click Loma chromatograph Ikaramu, filter, ultrafiltration, salting out, dialysis, SDS poly accession Li Ruami Dogeru appropriately selected electrophoresis, isoelectric focusing, etc., the combination lever, antibody the can be separated, and purified child is (Antibodies:. a Labora tory Manual Ed Harlow and David Lane, Cold spring Harbor La boratory, 1988).

Is a column with the Afi two Teak chromatography, flop port tee down A column include Purotei emissions G column. For example, as a column using flop Roti down A column, Hyper D, POROS, Sephar ose FF (Pharmacia) and the like.

Is the Afi two Teak Loma preparative except chromatography clauses Roma Togurafi one, for example, ion exchange click chromatography, hydrophobic click chromatography, gel filtration, Gyakushoku chromatography, adsorption click Loma toggle Rafi Chief like force S (Strategies for Protein Purification a nd Character izat ion:... A Laboratory Course Manual Ed Daniel Mar shak et al, Cold Spring Harbor Laboratory Press, 1996). These click chromatography can and this done using HPLC, the liquid phase click Roma toggling Rafi such FPLC.

Densitometry or activity confirmation of the antibody obtained above, a known method, eg if ELISA, EIA (enzyme immunoassay), RIA (radioimmunoassay) Oh Rui can and Mochiiruko immunofluorescence.

High Priestess Doma HM1.24 that produces the anti-HM1.24 antibody, the Agency of Industrial Science and Technology in the raw life of Industrial Science and Research Institute (if arrive Ibaraki Prefecture city Higashi 1-Chome, 3), 1995 September 14, FERM It was deposited when on the basis of countries Putapesu Treaty as a BP-5233. Another aspect of the present invention, an anti-HM1 contained in the test sample. 24 to measure the antigen binding activity of the antibody is a way to control the quality of the anti-HM1. 24 antibody with this. In the medicament comprising the antibody as an active ingredient, simply, not only the amount of antibodies, and this biological activity of the antibody is appropriately held is important. Biological activity is often an antibody, a binding activity to the antigen, the antibody to retain binding activity to antigen is confirmed whether the degree Te in the pharmaceutical composition odor, active ingredient high body it is essential to the quality control of pharmaceutical products containing as. The method of the present invention, anti negation 1. Method person to appropriately control the quality of the medicament comprising as an active ingredient 24 antibodies, and anti-HM1 were properly control the quality. 24 antibodies, and anti-HM1. 24 It provides a pharmaceutical composition comprising as an active ingredient the antibody.

In the manufacturing process of pharmaceuticals, it is necessary to manage the quality of pharmaceuticals or pharmaceutical compositions. Accordingly, suitable clauses quality control method is part of a manufacturing process of pharmaceuticals or pharmaceutical compositions. The method of the present invention, by providing the appropriate quality control methods, anti-HM1. The method of manufacturing 24 antibodies and provides a manufacturing method of an anti-HM1. Pharmaceutical compositions containing as an active ingredient 24 antibody it is intended.

Example

EXAMPLES Hereinafter the present invention will be described by Ri in detail but are not intended to limit the scope of the present invention.

Example 1. Construction of a soluble human HM1. 24 antigen for the expression plasmid

Ec oR I (Takara Shuzo Co., Ltd.) and No tl HEF expression vector containing the EF1 a promoter Ri prepared by the digestion with (Takara Shuzo Co., Ltd.) (International Patent Publication No. W092 - 19759) and the I g leader sequence Coat the HA tag dos genetic Napea the (Ame r sham Pharmac ia Co.), 50 mmo l / L Tr is -. HC 1, pH7 6, 10 mM MgC l 2, 10 mmo l / L Jichiosurei torr, 1 mmo l / L ATP, 50 mg / mL of poly ethylene glycol and 10 Interview two Tsu preparative T4 DNA ligase reaction mixture containing (manufactured T0Y0B0 companies) were ligated and reacted for 3 hours at 16 ° C.

The inserted Ig leader sequence and HA tag as genes co one de, Ec oRI, Kpnl (Takara Shuzo Co., Ltd.) and Notl synthetic gene pair showing a restriction enzyme recognition site in SEQ ID NO: 1 and 2 connected by a linker one It was used. Then ligation mixture was added to E. coli DH5 a combi competent cells (manufactured by GIBC0-BR L Inc.) on ice for 30 minutes this 1 minute at 42 ° C, and allowed to stand again on ice for 1 minute.

Then, 00 S0C medium μ L (Molecular Cloning: A Laborato ry kanua 丄, Sambrook et al., And old Spring Harbor Laboratory Press, (1989)) was added and after 1 hour incubation at 37 ° C, 5 0 μ LB agar medium containing ampicillin in g / mL (Molecular Clonin g a Laboratory Manual, Sambrook b, Cold Spring harbor Lab oratory Press, (1989)) on the seeded and then the E. coli overnight I Nkyube preparative at 37 ° C to obtain an E. coli transformant was.

The E. coli transformant was cultured overnight in LB medium containing ampicillin of 50 / xg / mL at 37 ° C, from the culture, the alkali method (Mole cular loning: A Laboratory Manual, Sambrook et al., and old ipr in g Harbor Laboratory Press, was prepared plasmid DNA according to (1989)).

On the other hand, the gene of the extracellular region of HM1.24 antigen was amplified by PCR method using Thermal Cycler (Perk in Elmer Cetus Corp.). Of HM1.24 antigen cDNA (SEQ ID NO: 15) as the 铸型, primers shown in SEQ ID NO: 3 and 4 of 100 pmol, 10 mmol / L Tris- HC1, pH8.3, 50 mmol / L KC 1, 0.1 mmol / L dNTPs (dATP, dGTP, dCTP, dTTP), 1.5 mmol / LM gCl 2 and 5 Yuni' bets DNA poly main hydrolase Ampli Taq (Perkin Elm er Cetus, Inc.) containing a mixture initially 94 ° C after an initial denaturation at 1 min at 94 ° C, for 1 minute, 30 cycles of 1 minute at 72 ° C at 55 ° C, was finally Inkyubesho down for 10 minutes at 72 ° C .

The PCR product as a gene of the extracellular region of HM1.24 antigen (SEQ ID NO: 5), Kpnl and BamHI digested the plasmid DNA and 50 mmol / L Tris_HCl, pH7.6, 10 mmol / L MgCl 2, 10 mM Jichiosurei toe Le, with 1 mmol / L ATP, 50 mg / mL polyethylene glycol and 1 Interview Knitting bets T4 DNA ligase reaction mixture containing (manufactured T0Y0B0 companies), reacted for 3 hours at 16 ° C It was consolidated. In the same manner as described above, the ligation mixture was added to E. coli DH5o! Konbiten DOO cells, to obtain an E. coli transformant was prepared from plasmid DNA it. The plasmid DNA HA tagged soluble antigen expression plasmid, was PsHM.

Moreover, using the primers shown in SEQ ID NO: 3 and 6, emitting the extracellular region of HM1.24 antigen deleted C-terminal in the same manner as (SEQ ID NO: 7) - current to plasmid was prepared PsHM164.

Sequencing

Sequencing psHM and psHM164 is Te automated DNA Shikuensa (manufactured by Ap plied Biosystem Inc. Corp.) Single Oyohi aq Dye terminator was ycle Seq uenc ing kit (.Applied Biosystem Inc. Corp.) for Rere, manufacturers specify pro tocol which was carried out according to the. Using primers (Sawaditekuno Logistics Ltd. one company) shown in SEQ ID NO: 8 and 9. As a result, it was confirmed that the the soluble antigen was connected to HA Tagupe peptide fusion protein (SEQ ID NO: 10 及 beauty 11) has a structure expressed.

Establishment of Example 2. Soluble human HM1.24 antigen high expression cell

(1) Toransufu Kushiyo in to the CH0 cell

To establish the HA-tagged soluble HM1.24 antigen stable production system, Ereku preparative Roporeshiyo down method the expression vector linearized obtained by digesting (PsHM and PsHM164) with Pvul (manufactured GIBC0-BRL Co.) It was transfected into by Ri CH0 cell M Bll strain (given Medical Research Council collaboration Center by Rikyo). Vector 1 PBS (-) in addition to 0.8 mL § Li Coat of 1.1 X10 7 cells / mL, the pulse at the capacity of 1.5 kV, 25 M F using Gene Pulser apparatus (Bio- manufactured Rad) Gave.

After a recovery period of 10 minutes at room temperature, the electronics Toro poration cells, (manufactured by GIBC0- BRL Co.) 100 mL of 10% FCS, 1% penicillin - be sampled replica Tomai Shin (GIBC0- BRL Co. ) containing non -MEM (suspended in j click Reoshi de-free) selection medium (manufactured by GIBC0-BRL, Inc.), 100 mu L / Ueru (1 X10 4 cells / Ueru) in flat-bottom 96-well plates (FALCON Corp.) They were seeded in. After incubation overnight at 37 ° C, 5% C0 2 incubator one further 100 μ ΐ7 Ueru added selection culture areas, was carried out selector tion. Performed Atsusi by Sa Ndoi pitch ELISA (see section Selection of cell line) On day 14, the HA-sHM or HA-SHM164 select 24 clones with high expression, the expansion at 24 © E Le plate (l mL / Ueru) was. After clones selected by these nucleic acid-free medium is confirming stable growth, further have rows Atsusi, focused on ten clones, respectively.

(2) selection of cell lines

ELISA (described later) of soluble human HM1.24 was carried out following good Unishi. The production amount of a soluble antigen anti-HA antibody (Boehring er Mannheim Co., Ltd.) and human-type anti-HM1.24 antibody (Ono et al. Koichiro 20th Japan Annual Meeting of the Molecular Biology Society in general in order to select the strain of high production Title 3-501- P- 478) by comparison with Sandi Tutsi ELISA, were selected cell lines. Since fried antigen concentrations has been obtained purified antigen is not known, comparison of concentration considering the cell number at the time of performing ELISA.

In the present embodiment, a light chain Pajiyo down a heavy chain Pajo emissions s according reconstructed human anti-HM1.24 antibody (human-type anti-HM1.24 antibody) to the W098 / 14580 . E. coli with a plasmid containing the light chain Pajiyo down a can, as an Escherichia coli DH5 a (P UC19- VLa -AHM-gK), the Industrial Technology Institute Life Institute of Advanced Industrial Science and Technology, 1996 (1997) on August 29, it has been an international deposit based on Budapest, Treaty as FERM BP- 5645. Furthermore, E. coli having a plasmid containing the heavy chain Pajiyon s of human-type anti-HM1.24 antibody, Escherichia coli DH5 a (pUC19- RVHs - AHM- gy 1) as, Agency of Industrial Science Technology Institute to, 1997

In (1997) September 29, it has been an international deposit-out based on the Budapest, Treaty with the FERM BP-6127.

Anti-HA antibody (Boehringer Mannheim Co.) Coating Buffer: those (CB 0.1 mol / L bicarbonate isocyanatomethyl Li © beam buffer, pH 9.6, 0.02% diisocyanato Li um azide de) was prepared in 1 mu g / mL at was added to flat bottom 96 well plate (Nunc Co.) in 100 L / well, and over 晚 Koti ring at 4 ° C.

Using plates washer, 300 mu L / Ueru of 0.05% Tween 20 and including PBS (-) dilution buffer with 200 / L / Ueru the anti-HA antibody coated plates were washed 3 times with (50 mmol / L Tris- HC1, pH 8.1, 1 mmol / L

MgCl 2, 0.15 mol / L NaCl , 0.05% Tween 20, 0.02% diisocyanato Li Umua disilazide, 1% BSA) were added thereto to carry out 2 hours Bro Kkingu at room temperature. After discarding the dilution buffer, CH0 cells by a material obtained by diluting 100 mu! 7 Ueru added culture supernatant as such or in an appropriate dilution buffer and 2 hours reaction at room temperature.

As a positive control using the CGM / sHM (OYadoriki Kyoko et al 60 times Japan Society of Hematology general abstract 690). Then, similarly washed plates for dilution human-type anti-HM1.24 antibody (Ono Koichiro et 20th Japan Annual Meeting of the Molecular Biology Society one 般演 entitled 3-501-P-478) in 1 mu g / mL those prepared in buffer was added 100 mu L / © Le reacted at room temperature for 1 hour. After washing Similarly, those diluted 5000-fold with dilution buffer to alkaline phosphatase-labeled Hijji Kohi preparative IgG antibody (BI0S0URCE Inc.) was added in 100 Mi7 © Engineering Le, and reacted at room temperature for 1 hour.

Finally, it washed five times, Sigma 104 (p-nitro-off We sulfonyl phosphate Ninato Li © unsalted hexahydrate: SIGMA Co.) substrate buffer (SB: 0.05 mol / L bicarbonate isocyanatomethyl Li © beam buffer, pH 9.8, 10 mmol / L MgCl 2) in and developed added in 100 mu L / Weru what was 1 mg / mL, MICR0PLATE READER ( BIO - manufactured by RAD) at 405 nm-655 nm absorbance It was measured.

A. 10nmol / L MTX by gene amplification

And an HA tag added respectively, in DXB11 cells transfected with expression vectors sHM164 lacking C-terminal of the soluble HM1.24 antigen lacking the transmembrane region of HM1.24 antigen (sHM) and sHM, by the 10 strains ( sHM producing strain:: -! 1, 8-2, 9-3, 11-4, 14-5, - 16, -17, -22, -23, -24, sHM164 producing strain: 164-1, -2 one 3, one 5, -6, one 7, one 8, one 10, one 13, -16) (Te trick Rere, 25 cm 2 flasks at 10 nmol / L main Bok Bok Rekise one Bok (Methotrexate) ( M TX) containing medium (a -MEM (manufactured by GIBC0-BRL, Inc.), 10% FCS (GIBC0 - BRL Co., 1 percent Bae Nishiri emissions - made be sampled Reputomai Shin (GIBC0- BRL Co.), 10 0 nmol / L They were cultured in MTX (SIGMA Co., Ltd.)).

After 8 days, the antigen production in the culture supernatant (3-day culture) was measured by ELISA. The expression level is high and the cells are sHM-producing strain is growing well 11 - 4 as well as in sHM164 producing strains 164- 2 and 164- 13 was Gene amplification by 100 nmol / L MTX for (rear group B see. Section). Since the remaining strains has failed are adapted to fully 10 nmol / L MTX, the culture was continued at 10 nmol / L MTX medium further.

After 11 days, the antigen production in the culture supernatant (3-day culture) was measured by ELISA, sHM producing strain 8 2 showing high expression level, 9-3, 14-16 and 14 24 and SHM164 producing strain 164 - 1, 164- (see the rear group B Section.) for 5 and 164- 8 ί tips Rere be performed 100 nmol / L MTX this by gene amplification. This most production amount of higher was 164- 13 at the time showed antigen production of about 10 times the CGM / sHM (OYadoriki Kyoko et 60th Japan Society of Hematology general abstract 690).

B. Gene amplification by 100 nmol / L MTX

For sHM-producing strains and sHM164 producing strains, 10 nmol / L MTX medium at each respective 5 strains showing high antigen production amount (sHM-producing strain 8 2, 9-3, 11-4, 14 - 1 6 and 14 24 and sHM164 producing strains 164- 1, 164-2, 164-5, 164- about 8 and 164-13) were gene amplification by 100 nmol / L MTX.

10 nmol / L MTX medium in accordance with the number of cells in order from an adaptation to 1/15, were passaged 1/10 or 1/4 amount 25 cm 2 flasks. After one day culture at 10 nmol / L MTX culture area, 100 nmol / L MTX medium (α - MEM (manufactured by GIBC0-BRL, Inc.), made by 10% FCS (GIBC0- BRL Co., Ltd.), 1% penicillin - be sampled Lev Tomai Thin (GIBC0- BRL Co., Ltd.), was replaced with 100 nmol / L MTX (SIGMA Co., Ltd.)), were cultured in 100 nmol / L MTX medium or later. sHM-producing strain 11 4, 19 days after sHM164 producing strain 164- 2 and 164-13 as well, sHM producing strain 8 2, 9-3, 14-16 and 14 - 24 and sHM164 producing strains 164-1, 164 - 5 and 164-8 after 8 days, the antigen production in the culture supernatant (2-day culture) was measured by ELISA. Even higher production amount, or becomes higher potential sHM-producing strain 8-2 and sHM164 producing strain 164- 2 and 164- 13 Nitsu Rere cultivation continued at 100 nmol / LM TX medium Te, after 15 days, the culture It was measured again with ELISA antigen production amount in the supernatant (3 days of culture). Initially, most production amount of the higher was 164-2 strain showed the antigen production volume of more than five times the CGM / sHM (OYadoriki Kyoko et al. 60th Annual Meeting of the Japanese Society of Hematology general abstract 690). However, higher was 164- 2 shares of most production amount and overlaying the passage indicates the antigen production amount of slightly inferior Ri by CGM / sHM, tended to production amount is reduced. It supersedes any, it was decided to carry out the lysine of Arc loan reduction by the limiting dilution method.

C. Shindaruku port Ichin reduction by limiting dilution

For sHM producing strain 8- 2 and sHM164 producing strains 164-2 and 164- 13, it was subjected to single-cloning by limiting dilution. 8-2, 164- 2 and 164- 13 was prepared in each 100 nmol / L MTX medium at 1.7 cells / DIL, 0.99 mu! 7 Ueru (0.25 cells / Ueru) was dispensed into each three 96 Uwerupureto. After 13 days of culture, © Ell formation of colonies was observed (8-2: 13 Ueru, 164-2: 36 Weru, 164-13: 23 Ueru) antigen production in the culture supernatants (day 4 of culture) It was measured by ELISA.

Production amount of higher was Ueru (8-2: 6 Ueru, 164-2: 15 Ueru, 164-13: 9 Uweru) cells were passaged to 24 Uwerupureto from. We prepared two plates for measurement and for passage plates for measurement the medium was replaced when it becomes configurator Ruento, 3 days were cultured and measured antigen production in the culture supernatant by ELISA .

96 Uweru from 164- 2, finally CGM / sHM (OYadoriki Kyoko et 60th Japan Society of Hematology general presentations 690) 4 strain showing a production of about 100 times that produced in (164-2-1 , 164-2-13, 164-2-17 and 164-2- 31) was obtained (FIG. 2).

D. Western blots

164-2-1, 164-2-13, 164-2-1 and 164 - 2 - 31 1 day cell lines in 25 cm 2 flasks / 5 mL of medium, 3 days, and 5 days of culture and the culture supernatant and subjected to Western blots attached to.

Culture supernatants 5 μ ΐ PBS (-) by preparing a total volume 10 L, and each SDS- sample buffer (manufactured by reducing TEFC0 companies)) was added an equal amount. After heating 5 min at them in 100, it was performed SDS- PAGE (18 mA, 1.5 hours) a. And 伹, gel 12.5% ​​separating gel and a stack Gel 4.5% Minisurabu ten thousand method Laemi (Current Protocols in Molecular Biology 10.2.6 - 10.2.6) was prepared according to. After electrophoresis, the gel PVDF main Nburen (produced by millimeter pore Co., Ltd.)) in the capital La Nsupuro Tsu was collected (10 V, 30 minutes). The surface Ren Tris buffer containing 5% FBS (TBS (Takara Shuzo)) and earthenware pots preparative shaking for 1 hour at 25 ° C in was performed Bro Kkingu. 'After rinse in TBS (TBS-T) containing 0.05% T een 20, 50 μ g / mL mouse anti-HM1.24 antibody (Blood (1994) 84, 1922-1930) was added, vibration = 25 ° C in while cormorants door was allowed to react for 1 hour. Replace the six buffer at 10-minute intervals with the Hare preparative shaking at room temperature by the addition of TBS-T, was washed main Nburen. Then, while cormorants door shaking in alkaline phosphatase labeled turbocharger formic anti-mouse IgG antibody (Zy med Co., Ltd.)) in the same way as a secondary antibody which was diluted 2000 times with TBS-T 25 ° C It was allowed to react for 30 minutes.

After the reaction, washing the main Nburen repeated for 10 minutes oice cormorants six times = 25 ° C in the addition of TBS-T. The main Nburen the BCIP / NBT chromogenic substrate (Prom ega Ltd.)) Two Toroburute Torazori © beam of 33 mu L using (ΝΒ Τ) and 16.5 mu L of 5-blanking opening mode - 4-click furnace opening - 3 - Lee emissions Dorinore Hosufue one preparative western detection buffer containing (BCIP) (0.1 mol / L NaCl, 5 mmol / L MgCl 2 0.1 mol / L Tris-HCl buffer containing, pH 9.5) and immersed film coloration It was.

After the pack ground was shown develop to the extent that not rise, and washed with distilled water to detect HM1.24 antigen. 4 clones obtained as shown in FIG. 3 (164-2-1, 164-2-13, 164-2-17 and 164- 2 31) in both the reduction state, terrorism to by glycosylation soluble antigen was detected as a blowing de of Pando of 23-28 kDa, which is believed generator tea. However, 18 kDa

Since the acknowledged terrorist Pando to the derived HM antigen protein around 14 kDa, by performing the click Roma bets, those except which was decided to soluble antigen.

Example 3. Purification of soluble human HM1.24 antigen

Ri supernatant soluble human HM1.24 antigen expression CH0 cell culture, purified soluble human HM 1.24 antigen. The soluble human HM1.24 antigen expression CH0 cell culture

[(Manufactured M0REGATE Co.) 10% FBS, 1% penicillin Ichisu Torebutomaishi emissions (GIBC0- BRL Co.), alpha ME M medium containing 500 nmol / L MTX (Sigma Co.) (GIBCO-BRL, Inc.) a medium, were cultured in C0 2 the presence 37 ° C, 5%. By centrifugation of the culture supernatant of about 2 L, it was recovered.

AHM Con to Jiyu gate § affinity chromatography (the AHM about 300 mg co Njuge you encountered a CNBr- activated Sepharose 4FF), the culture supernatant was applied, PBS (10XPBS) those were diluted 10-fold: washed with Nacalai) after One was eluted with 0.2 mol / L Glycine buffer (pH 2.48). This fraction by reverse-phase click Kuchma Togurafi one using Vydac C4 force ram, 0.880 § Se Toni preparative drill, to obtain a its purified product.

Furthermore, the its purified product, similar in anti-phase chroma preparative chromatography was purified by performing a re chroma preparative chromatography twice. The purified product

, Diluted 5-fold with PBS, and using a Fast Desalting HR10 / 10 column was subjected to buffer replacement to PB S. From 280 nm of the absorption, the concentration of soluble human HM1.24 antigen obtained is estimated to be about 0.382 mg / mL, purified product of the total 42 mL was obtained. The degree of purification, the peak area ratio of Gyakushoku chromatography was greater than 95% purity.

Example 4. Purification Soluble human ELISA system constructed anti-HA antibody using HM1.24 antigen (Boehringer Mannheim Co.) coated Buffer (CB: 0.1 mol / L bicarbonate isocyanatomethyl Li © beam buffer, pH 9.6 , those prepared to 1 mu g / mL in 0.02% isocyanatomethyl Li Umua disilazide) were added to flat-bottom 96-well plates (Nunc Co.) in 100 mu! 7 Ueru was over 晚 coated with 4 ° C. 0.05% including Tween 20 PBS (-)! Dilution buffer with 200 mu 7 Uweru the anti-HA antibody Koti Ngupu rate was washed with (50 mmol / L Tris- HC1, pH 8 .1, 1 mmol / L MgCl 2, 0.15 mol / L NaCl, 0.05% Tween 20, 0.02% diisocyanato Li Umuaji de, 1% BSA) were added thereto to carry out 2 hours Bro Kkingu at room temperature.

After discarding the dilution buffer, 750-fold purification HM1.24 antigen in dilution buffer, 2250 times, 6750 times, 20250 times, or those diluted 100 mu 1 / Ueru added 60,750 times, reacted at room temperature for 1 hour It was. Then, similarly diluted and washed with pre over Tonihi preparative anti-HM1.24 antibody (the 20th Japan Molecular Biological Society Annual General Ono Koichiro et Title 3- 501-P-478) in 1 g / mL those prepared in buffer was added 100 Mr / Ueru reacted at room temperature for 1 hour. After washing Similarly, alkaline phosphatase labeled hits di anti - a a human IgG antibody (BI0S0URCE Co.) which was diluted 5000-fold with the dilution buffer was added by 100 / xL / © El, was reacted for 1 hour at room temperature .

Finally, washed five times, Sigma 104 substrate buffer as a (SIGMA Co.) Substrate: In (SB 0.05 mol / L bicarbonate Na Application Benefits um buffer, pH 9.8, 10 m mol / L MgCl 2) 1 mg / mU elasticity and the added by 100 μ ΐ7 Ueru is by color ones, the absorbance was measured 405 nm-655 nm in a microplate reader (manufactured by BI0-RAD Co.), the Ri Contact to that shown in FIG. 4 arsenide preparative of a standard curve of anti-HM1.24 antibody was obtained, using a 5000-fold dilution Ri Kitareyo determines that Ru reasonable der. Further, although the standard 'quasi curve of human-type anti-HM1.24 antibody in ELISA systems when using the purified antigen 5000 fold dilution in 5, measurement limit of about 500 pg / mL .

Preparation of Reference Example 1. mouse anti-HM1.24 mono-click polyclonal antibody-producing hybrid Dorchester Ma

Goto, T. et al., Blood (1994) 84, similar method described in 1992-1930, was prepared mouse anti-HM1.24 mono click polyclonal antibody production High Priestess dormer.

Human multiple myeloma patient bone marrow-derived plasma cell line KPC-32 (7 pieces lxlO) (Goto, T. et al ., Jpn. J. Clin. Hematol. (1991) 32, 1400) and BALB / C mice It was injected twice every six weeks intraperitoneally (Chiya made one Rusuripa).

In order to increase the 3 antibody production titer mouse days before slaughter the mouse is in La, 1.5Xl0 six KPC- 32 were injected into the spleen of mice (Goto, T. et al., Tokushima J. Exp. Med. (1990) 37, 89). The spleen was removed after slaughter the mouse, Groth, the de St. & Schreidegger methods (Cancer Research (1981) 41, 3465) spleen cells with myeloma cells SP2 / 0, which was removed in accordance with and subjected to cell fusion.

KPC - 32 the Cell ELISA using (.. Posner, MR et al, J. Immunol Methods (1982) 48, 23) by were antibody of screening in High Priestess dormer culture supernatant. 5Xl0 4 pieces of KPC-32 were suspended in 50 ml of PBS, 96-well plates (U-bottomed, Corning, Iwaki, Ltd.) was dried dispensed 37 ° C De晚風to. After pro click with PBS containing 1% © shea serum albumin (BSA), and 2 hours ink Yupeto at 4 ° C was added a High Priestess dormer culture supernatant. Then reacted for 1 hour Peruokishidaze labeled anti-mouse IgG catcher formic antibodies at 4 ° C (manufactured by Zymed), 30 minutes in the cleaning room temperature after 0 - reacting a phenylene Renjiami emission substrate solution (manufactured by Sumitomo Bakelite) It was.

1 mol / L and the reaction was stopped with sulfuric acid, the absorbance was measured at 492nm with ELISA reader (manufactured by Bio-Rad). To remove the High Priestess dormer which produce antibody against human immune Guropuri down, positive High Priestess dough Ma culture supernatant was pre-adsorbed to human serum, and screened for reactivity to other cell lines by ELISA. Selecting positive of High Priestess dormer were examined for reactivity to various cells in Furosai Tome Application Benefits scratch. Last selected High Priestess dormer clones were cloned twice, which were injected into the peritoneal cavity of BALB / C mice treated with pristane were obtained ascites.

Monoclonal antibodies were purified Ri I'm Ri mouse ascites in precipitation and Puroti down A Afi two Tikuroma door chromatography kit with sulfuric acid Anmoniumu (Ampure PA, made Amersh am). Purified antibody was F ITC labeling Ri due to the use of the Quick Tag FI TC-binding kit (Beri Ngamanhai made-time).

As a result, 30 hybridized Domaku loan reacted with monochromator Lorna Honoré antibody force S KPC- 32 and RPMI 8226 produced. After Kuroyungu were examined for reactivity with these culture supernatants of hybridoma and other cell lines or peripheral blood mononuclear cells.

Of this, three clones were monoclonal antibodies that specifically react with plasma cells. These 3 Tsunoku loan sac Chi, are useful in most flow one site Tome preparative Lee analysis, and select the High Priestess dormer clones having CDC activity against RPM I 8226, was named HM1. 24. The subclass of mono click polyclonal antibodies the hybridomas are produced, was determined by EL I SA using subclass-specific anti Mausuusagi antibody (manufactured by Zymed). Anti HM1. 24 antibody had a subclass of I g G2a kappa. High Priestess Doma HM1 to produce an anti-HM1. 24 antibody. 24, an industrial technology Institute of Industrial Science Research Institute (Tsukuba, Ibaraki Prefecture Higashi 1-Chome, 3), and FERM BP 5233 to September 14, 1995 It was deposited when on the basis of countries Butapesu Treaty to.

Reference Example 2. Human-type anti-HM1. 24 production of the antibody

The human-type anti-HM1. 24 antibody was obtained Ri by the following methods.

From the High Priestess Doma HM1. 24, which is prepared in Reference Example 1, total RNA was prepared Ri by the conventional method. The cDNA Po Li isomerase chain reaction co one de mouse antibody V region Ri Kitareyo (PCR) method and the 5 '- Ri by the RACE method, synthesized and amplified. To obtain a DNA fragment containing the gene encoding a mouse V region, respectively a DNA fragment of these concatenates the plasmid pUC-based click low training vector of all, to obtain an E. coli transformant was introduced into E. coli competent cells It was. The obtained from the transformant the above plasmid was determined in a conventional manner a nucleotide sequence of the cDNA co one de region in plasmid, to determine the phase complementarity determining regions of each V region (CDR) in further. To generate base compactors expressing chimeric anti ΗΜ1 · 24 antibodies, their respective the cDNA co one de the V region of the mouse anti-HM1.24 antibody L chain and Η chain HEF vector 揷. Was input. Further, in order to produce a human-type anti-HM1.24 antibody, a V region CDR of a mouse anti-HM1.24 antibody were transplanted into human antibodies by CDR grafting. Using human antibody REI Works chain and the L chain of human antibody, for FR4 using FR1-3 of human antibody HG3 for the framework region (FR) 1-3 as a human antibody H chain was on use the FR4 of human antibody JH6. It was replaced § Mi Bruno acid FR of H chain V region as antibody CDR grafted forms an appropriate antigen-binding site.

Thus human type genes of L chain and H chain of anti-HM1.24 antibody prepared by a for expression in mammalian cells, the HEF vector, introducing each gene separately, human-type anti a vector expressing the L chain or the H chain of HM1.24 antibody was produced.

These two expression vector Ri by the introducing simultaneously CH0 cells were established cell line producing human-type anti-HM1.24 antibody. The antigen binding activity and binding inhibition activity to human amnion-derived cell line WISH of human-type anti-HM1.24 antibody obtained by culturing this cell line were investigated by Cell ELISA. As a result, human-type anti-l.24 antibody has the same antigen binding activity and the chimeric antibody, for the further binding inhibitory activity using Piochin mouse anti-HM1.24 antibody, a chimeric antibody or a mouse antibody It had the same activity with.

Incidentally, E. coli having an L chain V region and H chain V region of chimeric anti-HM1.24 antibody plasmid containing the DNA encoding each Escherichia coli DH5 a (pUC19-l.24L- g κ) and Escherichia coli DH5 as the a (pUC19-1.24H- gy 1), the Agency life Institute of Advanced industrial Science and technology (if arrive Ibaraki Prefecture city Higashi 1-chome, 3), on August 29, 1996, each has been an international deposit based on Budapest, Bokujo yarn ladle as a 々_FERM BP-5646 * 5 jt / FERM BP- 5644.

The ratio of preparative anti-HM1.24 antibody L chain V region a Pajiyo down (SEQ ID NO: 12) and H chain V region Pajo down (SEQ ID NO: 13) E. coli carrying a plasmid containing DNA encoding each Escherichia co li DH5a (pUC19- RVLa- AHM- g k) and Escherichia coli DH5 a (PUC19- RVHr- a丽- gyl) as, Agency of industrial Science technology Research Institute (Higashi, Tsukuba, Ibaraki, 1 chome No. 1 No. 3), on August 29, 1996, they were each FERM BP- 5645 and FERM BP- 5643 and was based on Hazuki in Budapest, Treaty in international deposit.

Furthermore, H chain V region s Pajiyo emissions of human-type anti-HM1.24 antibody (SEQ ID NO: 14) E. coli carrying a plasmid containing DNA encoding the, cherichis coli DH5 a (pUC19- RVHs - AHM - g γ 1) and to, in industrial technology Institute of life Institute of Advanced industrial Science and technology (Tsukuba, Ibaraki Prefecture Higashi 1-chome, 3), FERM BP in 1997 (1997) September 29 - and 6127 It has been an international deposit based on Budapest, Treaty Te.

Cloning of Reference Example 3. HM1.24 antigen co one cDNA encoding

1) cell lines

The cells were cultured at human myeloma cell line RPMI8226, U266 is RPMI1640 medium supplemented with 10% © Shi fetal serum (FBS) (GIBC0- BRL), human myeloma cell line KPMM2 (Japanese Unexamined 7- 236475) is They were cultured in the added RP MI1640 medium 20% © shea fetal serum.

2) Construction of cDNA library one

Total RNA was isolated by the 1X10 8 pieces of KPMM2 cell by Ri Chioshian acid Guanin chloride Seshiu beam method, it was subjected to purification of mRNA Te use Rere the Fast Track mRNA Isolation Kit (Invi trogen ). 10 g mRNA from Not I of, oligo_dT 18; After cDNA was synthesized using (Time Saver cDNA synthesis Kit Pharmacia Biot ech), was ligated with EcoR I adapter. Fractionated using 0.7kbp or more cDNA 1.0% low melting Agarosugeru (Sigma), digested with pCOSl expression vector or by Notl; inserted into EcoR l ZNot I site of L ExCell vector one (Pharmacia Biotech), directly expression cloning library (library a) for use in (screening by panning) and library for immunization screening the (library B) were constructed, respectively.

Incidentally, PCOSl expression vector constructed by ligating HEF- PMh- from GYL (see W092-19759), delete the gene contained Ri by the EcoRI and Sinai digested, EcoRI-Notl-BamHI Adapter (Takara Shuzo) did.

3) Panning

The library A Ri by the electronics Toroporeshiyo down method COS- 7 was introduced into the cells. That, 20 mu (including 5X10 5 independent clones) § of plasmid DNA were combined 0.8 ml of cells (1 X10 7 cells / ml in PBS) and mixed, Rere use a Gene Pulser (Bio- Rad) the 1.5 kV, 25 μ Έ conditions ί trowel Electrabel Toroporeshiyo emissions of went Te. After Kiyoshi置 1 0 minutes at room temperature, the cells 10 ° /. FBS added DMEM (GIBC0- BRL) suspended cultured for 7 for 2 hours at 3 7 ° C divided into four 100 mm culture de I Mesh on.

After culturing the cells were washed with-phosphate buffer (PBS), the Karoe cells PBS were S Appendix (1 force containing 5 mM EDTA, 1-2 at 5% FBS, 0.02% NaN 3 added Caro PBS the cell suspension X10 6 cells / ml was prepared. Subsequently cells were 2 hours Kiyoshi置on panning plates were Koti bridging the anti-HM1.24 antibody (see below), the plates 5% FBS, 0.02 % after gently washing three times with wash with 3 ml of PBS containing NaN 3, from cells bound to the on plates, a solution of Hirt. (Hitt J., Mol Biol 26:.. 365-369, 1983) ( 0.6% SDS, was recovered plasmid DNA using 10 mM E DTA). the recovered plasmid DN a was amplified in E. coli, it was used for the next panning. panning plates of preparation Te next good Unishi at went. 3 ml ¾HMl. 24 antibody solution (lOii g / ml in 50mM Tris-HCl, pH 9.5) was added to 6 0 mm di Mesh (Falcon), and 2 hours Kiyoshi置 at room temperature, 0.15 M NaCl 3 washes After, 3 ml of 5% FBS, 1 mM EDTA, and 0.02% NaN 3添力Pairobeta S addition, panning plate was removed was subjected to 2 hours Kiyoshi置to blow Kkingu at room temperature. Bro Tsu key ring solution It was saved at 20 ° C - until use.

By the This is repeated three times panning 5 X10 5 amino plasmid drive rally containing clone (library A) a as a starting material, plasmid DNA with cDNA of about 0.9 kb P as Insato enriched. Dye Terminator Cycle sequencing Kit (Applied Biosystems) result of the determination of the more basic sequences Te use ヽ to 373A Moshiku ί or 377DNA Sequencer (Applied Biosystems) and cloned P3.19 comprises l, the cD NA of 012Bp, 180 having an open Riden Gouf rate arm (23-549) encoding amino acids revealed (FIGS. 6 and 7) (SEQ ID NO: 15). The cDNA by Ri expected amino acid sequence showed a characteristic structure in the membrane protein type II, had a N-type sugar chain binding site of the two places.

4) immune scan cleaning

Raipurari B were subjected to immune screening using anti-HM1.24 antibody. That is, the heavy layer on agar with E. coli NM522 (Pharmacia Biotech) to Fajirai Burari containing 1.5 × 10 5 independent clones were cultured for 3.5 hours at 42. After incubation, repeated two Torosenorero Sufuinoreta pretreated with 10 mM IPT G on the plate (Schleicher & Schue 11), and cultured for 3 hours at 37 ° C in further. Filter the 0.05% (v / v) Tween-20 added TBS (20mM Tris- HC1, pH 7.4, 150 mM NaCl) was washing with 1% of (w / v) BSA added TBS was added, 1 at room temperature It was Bro Kkingu in time Lee Nki Yube door.

After Bro Kkingu, anti-HM1.24 antibody solution (10 μ g / ml bromide Kkingu buffer) was added, and 1 hour Lee Nkyube preparative at room temperature, after washing 5,000 BaiNozomi dilution was alkaline phosphatase-conjugated anti-mouse I g anti serum; the (picoBlue Immunoscreening kit Stratagene) force];! ^, and incubated for 1 hour at room temperature to Ri. The spot that reacted with antibody 0 3 m g / ml- preparative Robusurete Torazori © beam, 0.15mg / ml 5 -. Bromo-4 Ichiku B row 3 Lee emission chromogenic solution containing indolyl phosphate (lOOmM Tris-HCl , pH 9.5, were developed in lOOmM NaCl, 5 mM MgCl 2) .

5 positive clones by immunological risk cleanings have been isolated, it al all matches the partial sequence of P3.19 (Figure 8). Homology one search result, P3.19 the BST-2 (Ishikawa J. et al., Genomics, 26; 527 - 534, 1995) expressed on bone marrow or Nameramakusu bets Rome cells bases Rooster those yourself columns and same it became clear it. It Ri same molecule in the screening method of the two ways is obtained, suggesting strongly that P3.19 is the co-sul membrane protein is an HM1. 24 antigen molecule.

Incidentally, E. coli containing the plasmid PRS38- pUC19 which insert the human protein DNA encoding between Xbal cleavage site pUC Betata having the same sequence as the human HM1.24 antigen protein Escherichia co li DH5 a (P RS38-pUC19) and is named, in 1993 years deposited on October 5, Agency of life Institute of Advanced industrial Science and technology (Tsukuba, Ibaraki Prefecture Higashi 1-chome, 3) number FERM BP- 4434 as a, it has been an international deposit based on Budapest, Treaty.

5) FACS analysis

Et al is, in order to confirm whether the protein is code indeed binds to anti-HM1.24 antibody by P3.19, were established CH0 transformed cell lines introduced with P3.19. That is, after the introduction of the P3.19 clone to by Ri CHO cells electronics Toropo Reshi Yo down method, 500 g / ml of G418 and cultured in the presence of (GI BC0-BRL), HM1.24 antigen-expressing CH0 cells to obtain a stock.

1 X10 6 pieces of cultured cells of FACS buffer (PBS (-) / 2% FCS / 0.1% NaN 3) were suspended in, it added HM1.24 antibody was reacted in ice for 30 minutes. After washing with FACS buffer, GAM- FITC solution (25 / zg / ml in FACS buffer; Becton Dickinson) and resuspended in, and reacted for 30 minutes more in ice. After washing twice with FACS buffer solution was measured at resuspended FACScan (Becton Dickinson) in FACS buffer 600 mu 1.

Incidentally, with UPC10 as a negative control antibody.

Results of FACS analysis, CH0 cells transfected with P3.19 whereas reacted strongly with anti-HM1.24 antibody, Control This setup role of the expression vector alone introduced CH0 cells (CH0 / NE0) the significant binding was not observed (Fig. 9). Thus, proteins code by P3.19 was confirmed to bind to anti-HM1.24 antibody.

6) immunoprecipitation

Cells PBS (-) was washed twice with lysis buffer method (50 mM Moesan'na Application Benefits um, 150 mM NaCl, 0.5% Dokishikoru acid isocyanatomethyl Li um, 1% No nidet P_40, 0. lmg / ml Fueninoreme Chino Les Norre e Nino reflex Honoré cage de, subjected to ultrasonic disruption in protease inhibition Hitoshi IJ Kakuteniru [Boehringer Mannheim]) to obtain a solubilized fraction. Solubilized fraction was added to Sepharose 4B beads anti-HM1.24 antibody and Con Juge bets. After centrifugation, the precipitate is separated Ri by the SD S-PAGE (12% gel), transferred to PVDF membrane. PVDF membranes anti HM 1.24 antibody, is reacted with a POD-anti- mouse IgG Te continued Rere were detected using the ECL kit (Amersham).

KPMM2, RPMI8226, and various myeloma cell lines U266 is strongly expressed HM1.24 antigen and intends row immunoprecipitation of these cell lysates with anti-HM1.24 antibody, the molecular weight of the protein of approximately 29~33kDa specific It was detected (Figure 10). P3. 19 the introduced CHO cell line (CHO / HM) result of the experiment of same in, immunoprecipitates as with Myeroma cell lines in CH0 / HM cells is confirmed (FIG. 10, lane 4) control cells (CH0 / NE0) in such immunoprecipitation Fubutsu introduced only the expression vector pCOSl was not observed (Figure 10, lane 5).

P3. 19 is present is 180 amino consisting acid predicted molecular weight 19. has code a protein of 8 kDa, 2 N-type sugar chain binding motif Chikarasho (Figure 6). Thus, the presence of those having different molecular weights observed by immunoprecipitation was believed to be due to modification of the difference in the N-type sugar chains. In fact, it immunoprecipitates is bound to several lectins has been confirmed. Industrial Applicability

丽 1. To obtained to establish 24 antigen high producing strain, was obtained from the culture supernatant 16 mg purified antigen of 2 L. Since establishing the ELI SA system using purified antigens, as compared to the ELI SA system 4 ° C De 晚 no longer need to incubating with culture supernatants, rather I is reacted at room temperature for 1 hour, the reaction time reduction of was done. Further, eliminates the involvement by that ELI SA system components contained in the culture supernatant, anti-HM1 was administered serum. Also available stably as the concentration measurement system 24 antibody.

Mentioned and international depositary authority to Deposited Microorganism prescribed in PCT Rule first Rule 3

International depositary authority

Name National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Center Athena, Japan Higashi, Tsukuba, Ibaraki Central 6, 1-1-1

(1) Name Es cher i chia co li DH5 a (pRS38- P UC19)

Accession number FERM BP-4434

The preferred surrender date 1993, October 5 (2) Name Mouse-mouse hybridoma HMl.24

Accession number FERM BP - 5233

The preferred surrender date September 14, 1995

(3) Name Escherichia coli DH5 a (pUC19-RVHr -AHM-g 7 1) accession number FERM BP-5643

August 29 曰 nearest surrender date 1996

(4) Name Escherichia coli DH5 Fei (pU19-l.24H-g γ 1)

Accession number FERM BP - 5644

The preferred surrender August 29, 1996

(5) Name Escherichia coli DH5 a (pUC19-RVLa-AHM-gk) accession number FERM BP - 5645

The preferred surrender August 29, 1996

(6) Name Escherichia coli DH5 Fei (pUC19-l.24L-gk)

Accession number FERM BP - 5646

The preferred surrender August 29, 1996

(7) Name Escherichia coli DH5 a (pUC19- RVHs- AHM-g γ 1) accession number FERM BP - 6127

The preferred surrender date September 29, 1997

Claims

The scope of the claims
1. Cultivated EF1 alpha and promoter, the linked downstream, soluble HM1. 24 animal cells transformed by calling the current base restrictor comprising a gene encoding the antigen lacking the intracellular domain, the soluble HM1 from the culture. soluble HM1, which comprises 24 antigens isolated and purified. 24 manufacturing method of an antigen extracellular domain.
.. 2 wherein the soluble HM1 24 antigen SEQ ID NO: having an amino acid sequence shown in 5 or 16, method according to claim 1.
.. 3 wherein the soluble HM1 24 antigen SEQ ID NO: 7 or having an amino acid sequence shown in 17, The method of claim 1.
4. The soluble HM1. 24 The method of claim 1 antigen is in the form of a fusion protein with influenza agglutinin (ΗΑ).
. 5 The fusion protein SEQ ID NO: 10 or 18 having the Amino acid sequence according The method of claim 4.
. 6 The fusion protein SEQ ID NO: 11 or 19 having the Amino acid sequence according The method of claim 4.
7. The animal cell is a CH0 cells, The method according to any one of claims 1-6.
8. In which it transformed animal cells was carried out gene amplification in the presence of 100 nmo l / L of the concentration of main ingestion Rekise over preparative (MTX), to any one of claims 1 to 7 the method described.
9. Is reacted with anti-HM1. 24 antibody contained in the soluble HM1. 24 antigen protein and the test sample which Ri produced by the method according to any one of claims 1-8, soluble HM1. 24 anti HM1 bound to an antigen protein. comprising detecting or measuring the 24 antibodies, anti-HM1. 24 immunochemical measurement method of an antibody.
10. The soluble HM1. 24 antigen protein, characterized in that it is bound to the support, immunochemical measurement method of claim 1.
11. The soluble HM1. 24 antigen protein, characterized in that it is fused to another peptide or polypeptide peptide, according to claim 9 or Ki载 immunochemical assay method in 10.
12. support is characterized that it is a bead or plate, immunochemical measurement method of claim 10.
13. soluble HM1. 24 anti bound to the antigen protein HM1. 24 antibody or an anti-HM1. 24 Soluble bound to the antibody HM1. 24 antigen protein, to anti-HM1. 24 primary antibody or soluble HM1 for antibody. 24 antigen protein immunochemical assay method according to claim 9 to 12, whichever is the paragraph (1), characterized by detecting or measuring Ri by the primary antibody.
14. soluble HM1. 24 anti HM1 bound to an antigen protein. The 24 antibody or an anti-HM1. 24 Soluble bound to the antibody HM1. 24 antigen protein, to anti-HM1. 24 primary antibody or soluble HM1 for antibody. 24 antigen protein immunochemical assay method according to any one of claims 9-12, characterized in that by Ri detected or measured in a secondary antibody against the primary antibody and the primary antibody.
15. Primary or secondary antibody is a radioisotope, enzyme, Piochin / avidin or immunochemical assay according to any one of claims 9 to 14, characterized in that it is by Ri labeled fluorescent substance Method.
Quality control method using the method according to 16. Claim 9-15.
17. Anti-HM1 has been controlling the quality by the quality control method of claim 16.24 antibody.
Anti HM1. A pharmaceutical composition containing as an active ingredient 24 antibody according to 18. Claim 17. -
19. including quality control method according to claim 16, anti-HM1. 24 The method of manufacturing the antibody.
20. including quality control method according to claim 16, anti-HM1. Process for preparing a pharmaceutical composition containing 24 antibody as an active ingredient.
PCT/JP2001/002964 2000-04-06 2001-04-05 Immunoassay of anti-hm1.24 antibody WO2001077362A1 (en)

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