JP2930966B2 - Monoclonal antibody with polypeptide as antigen - Google Patents

Description

The present invention relates to a monoclonal antibody prepared using a polypeptide having an amino acid sequence corresponding to a retrovirus gene as an antigen, a hybridoma cell producing the monoclonal antibody, and a monoclonal antibody thereof. The present invention relates to a method for detecting unknown pathogenic retroviruses, and to agents for testing, diagnosing and treating diseases. Furthermore, the present invention relates to a drug for testing, diagnosing and preventing diseases using a polypeptide having an amino acid sequence corresponding to a retrovirus gene.

In the present specification, when amino acids, peptides and the like are indicated by abbreviations, they are based on commonly used abbreviations in the art, and examples thereof are shown in Table 1. In addition, when there is an optical isomer for an amino acid or the like, the L-form is indicated unless otherwise indicated.

Conventional technology Retroviruses contain RNA, not DNA, as a genetic information substance. When infected to animal cells, genomic RNA (RNA that carries the genetic information) is converted into both ends by the action of reverse transcriptase in virus particles. DNA with long repeat sequence (LTR)
Thus, the virus is characterized by being integrated into the cell chromosome (integration).

Since P. Rous discovered the Rous sarcoma virus, a virus that causes sarcomas in chickens, in 1911, retroviruses that cause sarcomas and leukemias have been increasingly found in various animals.

In humans, in 1980, Gallo et al.
VI was discovered and isolated, and in 1981, Hinuma and Yoshida et al. Revealed that HTLV-I infection has a close etiological role in the development of adult T-cell leukemia (ATL). Later, Golde et al. Released hairy cell leukemia (hai).
ry cell Leukemia) and identified it as "HTLV-II". In 1983, Montagier et al. Reported that acquired immunodeficiency syndrome (A
HIV was discovered as the causative virus of IDS).

Furthermore, since retroviruses that are responsible for sarcomas and leukemias in various animals have been discovered, it is possible that unknown retroviruses may cause other sarcomas and leukemias in humans. However, at present, it is difficult to detect and confirm the pathogenic retrovirus in humans, and it is considered that the incurable disease of unknown cause, that is, the etiology of cancer that causes changes in human DNA is caused by chemical substances.

On the other hand, SLE systemic lupus erythematosus (Systemic Iupus
erythematosus: In NZB and NZB / WF ₁ mice known as a model animal for SLE), C-type retroviruses S
It plays an essential role in the development of LE-like pathology.

Diagnosis of these retroviral infections usually involves using the virus itself or its constituent proteins to test for the presence of viral antibodies in the patient's serum, or vice versa by immobilizing the viral antibodies on a microtest plate or beads. And the reaction of the serum with the patient to check for the presence or absence of a virus in the serum. However, in these methods, false positives due to the antigenic diversity of the etiological virus,
It can give false-negative results and is a serious problem in the nature of the route of transmission and its impact on individuals and society.

In recent years, the involvement of retroviruses has been proposed in humans as the etiology of various autoimmune diseases. However, in the past, virological searches for such diseases have included confirming virus-like particles by electron microscopy, raising serum antibody titers against various viruses, or establishing cultured cell lines that are likely to produce viruses from patients. Serological reactions or nucleic acid hybridization experiments are being conducted. However, these are all very difficult and unreliable methods, and the etiology of these diseases has not yet been elucidated.

Problems to be Solved by the Invention As described above, in various diseases representing intractable diseases such as leukemia / cancer and autoimmune diseases in which retroviruses are involved in the onset of the disease, the cause of the onset, which is the first step of treatment, There remains an unsolved problem in the diagnosis and examination thereof, and the solution is strongly demanded by society.

Means for Solving the Problems The inventors of the present invention have been studying to solve this problem, and found that leukemia and / or leukemia related to retrovirus infection.
Departing from the idea of using antigens of individual pathogenic viruses that have been commonly used in serodiagnosis of cancer and autoimmune diseases, we have developed an epitope that is commonly conserved in retroviruses such as humans and primates. We focused on this and used it as an antigen that commonly exists among retroviruses, and succeeded in producing a new monoclonal antibody. Furthermore, the present inventors have succeeded in detecting various retroviruses such as humans and primates using the antibodies. In addition, the results strongly suggested that the synthetic peptide used when these monoclonal antibodies were prepared reacts with the antiretroviral antibody. That is, the present invention provides a monoclonal antibody prepared using a polypeptide containing a homologous portion between retroviruses in an amino acid sequence corresponding to a retrovirus gene as an antigen, a hybridoma cell producing the monoclonal antibody described above, A method for detecting a pathogenic retrovirus by causing an antigen-antibody reaction with an unknown pathogenic retrovirus using a monoclonal antibody, an agent for testing, diagnosing, and treating diseases caused by retrovirus infection comprising the monoclonal antibody described above, and The present invention relates to an agent for testing, diagnosing, and preventing diseases caused by retrovirus infection, which comprises a polypeptide containing a homologous portion between retroviruses in the amino acid sequence corresponding to the retrovirus gene.

When a retrovirus infects animal cells, the virus RN is activated by reverse transcriptase carried by the virus particles in the cytoplasm.
A copy of DNA is synthesized from A. Retrovirus linear DNA is detected in the cytoplasm, and linear and circular DNA is detected in the nucleus. This circular retroviral DNA is thought to be a direct precursor (provirus) that is incorporated into cellular DNA. The provirus integrated into the chromosome is 5 '
-LTR-gag-pol-env-LTR-3 'has gene regions arranged in this order. Here, the LTR does not encode a protein, but has a transcriptional regulatory sequence (enhancer or promoter) for a viral gene and is also a site for integration into a chromosome. Although gag is processed into several proteins and becomes a structure within the virus particle, its role in the virus synthesis process remains largely unclear. Pol is divided into three domains and encodes protease, reverse transcriptase, and endonuclease. env encodes a glycoprotein outside the virion and a region that penetrates the membrane. In addition to this,
Many retroviruses (cancer viruses) that cause cancer in animals have oncogenes (v-onc) in their genomes. H
V-onc for TLV-1, bovine leukemia virus (BLV), HIV, etc.
Although it does not have a gene, it is well known that it has a gene that encodes a protein that regulates the expression of the virus's own gene or cellular gene.

Among these genes, pol and gag are relatively hard to mutate. On the other hand, among the env proteins, the envelope glycoprotein is susceptible to mutation, but the transmembrane envelope protein (Transmembrane envelop
It is known that the gene encoding protein) is relatively hard to mutate, and there is a portion where amino acid homology is very well maintained between retroviruses. For some time, it has been known that when an animal is infected with a cancer virus, an immunodeficiency state often occurs with the retrovirus infection before a tumor is formed in the animal. In 1985, Cianciolo and colleagues showed that a peptide in this transmembrane envelope protein that had very good amino acid homology between retroviruses suppressed lymphocyte proliferation. This part was suggested to be related to immunodeficiency in animals. Focusing on these points, the present inventors have argued that, among retrovirus constituent proteins, reverse transcriptase (RT) and transmembrane envelope protein (TM) whose mutation is unlikely to occur and whose functions are characteristic of retroviruses ) Was compared between retroviruses.

Comparison of Amino Acid Sequences between Retroviruses The present inventors have previously elucidated the entire nucleotide sequence of BaEV (the baboon endogenous retrovirus strain M7) [Japanese Journal of Genetics (Jpn. J. Genet.) 62, 127
137 (1987)], deduced the amino acid sequences of reverse transcriptase and transmembrane envelope protein,
The homology, the secondary structure, and the degree of hydrophobicity / hydrophilicity with other retrovirus sequences were analyzed by the SDS-GENETYX program (Software Development Co., Ltd., Tokyo).

As a result, two sites (S0
1, S02), transmembrane envelope protein (TM)
One site (S03) was selected in the region, and the amino acid sequence of the corresponding portion was compared with other viruses,
The results are shown in FIGS. 1-1, 1-2, and 1-3. The retroviruses having the gene sequences elucidated so far were used as retroviruses for comparison. Ie, baboon endogenous retrovirus
M7 strain (BaEV), Moloney rodent leukemia virus (MML)
V), AKR rodent leukemia virus (AKV), human endogenous retrovirus (CL41), human T-cell leukemia virus type 1 (HTLV-I), Rous sarcoma virus (RSV), bovine leukemia virus (BLV), Mason・ Pfizer Monkey Virus (MPMV), Ape Acquired Immunodeficiency Syndrome Retrovirus (SRV-1), Human Endogenous Retrovirus (HERV), Human Immunodeficiency Virus (HIV), Jonai Type A Particles (IAP),
Moloney rodent tumor virus (MMTV), reticuloendothelioma virus A strain (REV-A), avian leukemia virus (FEL)
V), human foam retrovirus (HSRV) and the like.
In the figure, * mark shows the same amino acid as BaEV.

As a result, the amino acid sequences of S01 and S02
As shown in the figure and FIG. 1-2, it was presumed that the retrovirus was very rich in homology, had a turn structure sandwiched between β-sheet and α-helix, and was rich in hydrophilicity. In addition, as shown in FIG. 1-3, the amino acid sequence of S03 was highly homologous between the retroviruses, and was presumed to have an α-helical structure and to be highly hydrophilic.

Therefore, the present inventors synthesized three polypeptides having homology between these retroviruses, and observed the antigen-antibody reactivity of the monoclonal antibodies obtained using these polypeptides as antigens against various retroviruses.

Examples of the polypeptide include those having a homologous portion in the amino acid sequences corresponding to the genes of various retroviruses. For S01, SPWNTP (1-1)
Figure), LPQGFKNSPTLF (Figure 1-2) for S02 or
For S03, a polypeptide containing QNRRGLDLL (FIG. 1-3) is mentioned as an example. In order for the polypeptide to function as an antigen, it preferably has an amino acid sequence of about 20 to 30 amino acids, and those obtained by adding amino acids before and / or after the above amino acid sequence are preferably used.

Specific examples include, for example, baboon endogenous retrovirus peptide, and 1-1 corresponding to S01.
LELGVLRP, a peptide from amino acid position 200 to 221 of pol of the baboon endogenous retrovirus (BaEV) shown in the figure
CRSPWNTPLLPVKK is the equivalent of S02 to 1-2
QLTWTRLPQGFKNSPT, a peptide from amino acid positions 300 to 319 of pol of the baboon endogenous retrovirus shown in the figure
The LFDE also differs from the env amino acid position 434 of the baboon endogenous retrovirus shown in FIG.
SLAEVVLQNRRGLDLLTAEQGGI, a peptide from to 456
Is mentioned.

When performing immunization using the above-mentioned polypeptide as an antigen, it is necessary to add C (cysteine) to the 5 ′ or 3 ′ end of the polypeptide and bind it to KLH (keyhole limpet hemocyanin) to increase the antigen ability. In this case, C is often added at the 5 'or 3' end.
When C is present in the above-mentioned polypeptide in order to avoid reactions other than and KLH, it is preferable to use this C instead of S.

The peptide was chemically synthesized by a solid phase method and purified by reversed-phase high performance liquid chromatography (HPLC).

The polypeptide synthesized as described above was identified as the target by HPLC and an amino acid analyzer.

Next, animals were immunized using the synthetic peptide having the confirmed amino acid sequence as an antigen to prepare a monoclonal antibody. As antigens, KLH and BSA as described above
(Bovine serum albumin) or the like to which a high molecular weight protein is bound is preferably used.

When selecting an animal to be immunized here, the ability to respond to the animal species and antigen is important. Generally, it is desirable to immunize mice or rats as much as possible. This is because many of the myeloma cell lines that form stable antibody-producing hybridomas are derived from mice or rats. In particular, there are many cases in which a valve C (BALB / C) mouse is used.

Monoclonal antibodies can be produced by a general method. For example, cells that produce an antibody against the antigen can be obtained by frequently administering the antigen to an animal together with an appropriate adjuvant, for example, Freund's adjuvant. Also,
Since general F ₁ towards the mouse is higher compared to the antibody-producing ability to a parent mouse, it is preferable to use it. The antigen was suspended in complete Freund's adjuvant, injected intraperitoneally into mice, immunized for the first time, and then two weeks later, the antigen was again injected intraperitoneally into the animal with Freund's incomplete adjuvant. Perform immunization.
The objective is achieved by intraperitoneally injecting a physiological saline solution of the antigen 2 weeks after the booster immunization.

Next, lymph cells in the spleen are often used as a material for antibody-producing cells from an immunized animal, but lymph cells in a lymph node can also be used. For example, post-challenge, were excised mouse spleen highest antibody titer of the F ₁ male mice with Hanks solution to prepare a splenocyte suspension,
After washing the splenocytes with RPMI-1640 medium, the cell pellet is
It may be prepared by resuspending in I-1640 culture solution.

The myeloma cell line used for cell fusion was Valve C
S, a non-H, L chain-producing myeloma cell of mouse origin
P2 / 0.Ag14 (SP-2) or the like can be used.

The fusion agent used for cell fusion generally has an average molecular weight of about 1,
Polyethylene glycol (PEG) of 000 to 6,000 is conventionally used, and its concentration is 30% to 50%
(Weight / weight) in many cases. It is believed that the polyethylene glycol partially disrupts the lipid bilayer structure of the cell membrane to increase the fluidity of the cell membrane and cause membrane fusion between cells brought closer together by centrifugation. We placed 4 ml of 75 mM Hepes (p.
Boehringer Mannheim PEG 1,500 containing 2 g of molecular weight 1,500 PEG in H regulator) was used.

Various methods are generally used as the fusion conditions, but they can be roughly classified into two methods. That is, the first method is a method according to the method of Gulfre et al., In which a PEG solution is added to splenocytes and myeloma cells pelletized by centrifugation, and the cells are fused only by stirring and shaking. Usually, a 50% PEG solution is used. After adding about 1 ml of the solution over 1 minute, the solution and the cells are mixed for 1 minute, and then a serum-free medium is gradually added to add PE.
Dilute G. Since relatively many people have actually adopted this method, the method of the present inventor has followed this method.

The second method is a method according to the method of Gefter et al., In which splenocytes pelleted by centrifugation and myeloma are mixed with a PEG solution, and the cells are centrifuged at low speed to fuse the cells under gravity.

Here, there is a report that it is necessary to maintain the temperature at the time of fusion strictly at 37 ° C, but it is sufficient to consider that a PEG solution warmed to 37 ° C in advance should be added at room temperature. The present inventor used this because of reports. In addition, as for the ratio of the number of cells in which the spleen cells and the myeloma cells are fused, usually 1 to 2 × 10 ⁸ splenocytes are used, and the ratio of the number of splenocytes is 1: 1 to 10: 1 Is mixed with myeloma. Next, the cell suspension fused with PEG is centrifuged, and the pellet is suspended in a selective medium.
The sample was dispensed into a flat bottom microtest (III) plate (Falcon). In this case, the fused cells are at 37 ° C.
Incubate in an incubator under 5% CO ₂ humidity for about 10 days. About 5 × 10 ⁵ splenocytes / well were used.

Here, the following media were used as selective media. GI
T medium (manufactured by Nippon Pharmaceutical), 2-mercaptoethanol (2-M
E): 5 × 10 ⁻⁵ M, L-glutamine: 2 mM, penicillin: 100 U
/ ml, streptomycin: 100 μg / ml, insulin: 1
0 ^-3 U / ml and dihydroxyethylglycine (DHEG):
It was prepared by adding 1/50 volume of HAT (hypoxanthine, aminopterin and thymidine) 50-fold concentrated solution (Flow) to 1.8 mg / ml.

As the medium used for the mixed culture, any of various nutrient media used for normal cell culture can be used. For example, an RPMI-1640 medium, Eagle's minimum essential medium (MEM medium), or the like is modified using a serum replacement solution such as fetal calf serum (FCS) calf serum, and a medium to which penicillin, streptomycin, or the like is added, is exemplified. You can also.

Next, when sufficient hybridoma growth is observed, hybridomas producing the desired antibody are screened at appropriate times by various methods. As the measuring method, means such as an indirect fluorescent antibody method, a radioimmunoassay, and an enzyme immunoassay can be employed. Among them, the present invention generally employs an enzyme immunoassay (EI
A) was used. When an immunosorbent is used among EIAs, it is called an ELISA. In particular,
Each polypeptide used for immunization is adsorbed to an EIA plate, washed, coated with bovine serum albumin, washed again, and reacted with a hybridoma culture solution at room temperature for 2 hours.
After washing, horseradish peroxidase-labeled rabbit anti-mouse IgG (Capel) as an enzyme-labeled secondary antibody
Is reacted. After washing, 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) is used as a substrate.
When (ABTS) is added in the presence of hydrogen peroxide, the substrate is separated and colored by the action of horseradish peroxidase. The antibody titer in the hybridoma culture solution is measured based on the degree of the color development.

Currently, alkaline phosphatase (AP) or horseradish peroxidase (HRP) is exclusively used as a labeling enzyme, and a labeled animal species-specific immunoglobulin antibody is commercially available and easily available as a labeling antibody. As a substrate for HRP, hydrogen peroxide and a substance oxidized thereby are used, and in addition to ABTS, o-phenylenediamine and the like have high sensitivity and are often used.

The present inventors have proposed 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt:
Color development was performed using [ABTS- (NH ₄ ) ₂ ] and aqueous hydrogen peroxide as a substrate solution, and colorimetric determination was performed at 450 μm.

Next, the antibody-producing fusion cells are fractionated and cultured by an appropriate method to prepare a single clone cell line derived from a single cell. This operation is called cloning, and examples of the method include a limiting dilution method, a soft agar method, a fibrin gel method, and a cup method, and the limiting dilution method is preferable. For example, antibody positive wells were cloned by limiting dilution using mouse thymocytes (about 1 × 10 ⁶ cells / well) as feeder cells. Next, the culture supernatant of each well was screened again to select positive clones. In addition, the monoclonal antibody in the culture supernatant obtained by culturing this cell line in a serum-free medium was precipitated by the ammonium sulfate method, dialyzed with a phosphate buffer, and the amount of the antibody was determined using a Bio-Rad protein assay kit. Quantified.

In this way, hybridomas producing antibodies against the amino acid sequences of S01, S02, and S03 can be obtained by the specific antibody screening method for the synthetic peptide.

Next, immunoblotting was performed to determine the reactivity of the antibodies obtained from these hybridomas with the retrovirus (antigen).

Specific examples of various antigens (retroviruses) include A204 cells and BaEV-producing A204 cell lines and various retrovirus-producing cell lines such as HTLV-1-producing MT-2 cell line and MPMV-producing human lung adenocarcinoma. Cell line, SRV-1
Producing Raji cell line, MMLV producing D3hlg cell line, AKV producing AKR / J
SDS and mercaptoethanol were obtained by adding a buffer to a sediment obtained by centrifuging a culture solution of a mouse T lymphoma cell line, RSV-producing avian fibroblasts or the like, or by partially purifying these by equilibrium density gradient centrifugation using saccharose. And heated in hot water.

The various antigens described above were prepared by the method reported by Laemmli [Nature, 227, 680-685 (197
0)], and electrophoresis was performed on an SDS polyacrylamide gel. Next, the western blotting method is a method of transferring the antigen separated by SDS polyacrylamide gel from the gel surface to nitrocellulose, performing an antigen-antibody reaction on the membrane, and identifying the antigen. As a labeled secondary antibody, anti-mouse immunoglobulin or the like, which is labeled with an enzyme or a radioisotope, is used. In this case, ¹²⁵ I-labeled sheep anti-mouse IgG or the like is used.

First, the reactivity of each anti-peptide antibody to BaEV was assayed by immunoblotting, and the results are shown in FIGS. 2 and 3.

Specifically, when culture supernatants of BaEV-producing A204 cell line (A204BE) and non-producing A204 cell line (Cont) are used, antibodies against the amino acid sequences of S01 and S02, which are partial peptides of reverse transcriptase (2A each) , 2C, 1D, 1F and 11G, 3E) are BaEV
About 70,000 (Dalton, the same applies hereinafter) specific to the producer cell line
And two bands were detected at about 40,000. The band at about 70,000 is consistent with the molecular weight of BaEV reverse transcriptase. Also, about
The 40,000 band is not clarified unless the amino acid sequence is determined.However, since it is observed only in the culture supernatant of the BaEV-producing cell line, it may be a protein related to the enzyme, such as a reverse transcriptase of BaEV. is expected.

On the other hand, as shown in FIG. 3, an antibody (9G, 1C, 7A, 4G, 7H, 1G) against the amino acid sequence of S03 which is a partial peptide of the transmembrane envelope protein is about 2
0,000 bands were detected. This is consistent with the molecular weight of P20E, a protein associated with the transmembrane portion of the envelope protein of BaEV.

From the above, it was found that these antibodies are antibodies that actually react to the protein containing the peptide on which they are based.

Subsequently, the results of assaying the reactivity to various retroviruses by immunoblotting were shown in FIG.
6 to FIG. Specifically, one antibody is selected from the antibodies (FIGS. 2 and 3) that react with the amino acid sequence of each region (S01-1F).
Antibody, S02-11G antibody or S03-A7 antibody) The immunoblotting was performed using culture supernatants of various retrovirus-producing cell lines.

As a result, when the S01-1F antibody (antibody reacting with the amino acid sequence of S01) was used, as shown in FIG. , About 70,000 and about 40,000 bands that appear to be reverse transcriptase-related proteins were detected. Was detected.

Furthermore, SRV-1 producing cell lines and MPMV with a narrow correlation range
Approximately 90,000 and about 40,000 weak but likely reverse transcriptase-related proteins were also detected in the culture supernatant of the producer cell line.

No specific band was obtained for other viruses.

Therefore, this antibody was found to have a recognition site in SPWNTP in the amino acid sequence.

In addition, the human endogenous retrovirus (CL41) also preserves the amino acid sequence SPWNTP in the S01 region, which is likely to react with the antibody against S01. there is a possibility.

Next, as shown in FIG. 5, the S02-11G antibody had about 70,000 and about 40,000 reverse transcriptase-related protein bands in culture supernatants of MMLV-producing cell lines having exactly the same amino acid sequence as BaEV. Was detected, and about 70,000 bands considered to be reverse transcriptase-related proteins were detected in culture supernatants of AKV-producing cell lines having the same amino acid sequence. Furthermore, unlike the results at the time of S01, in S02, about 60,000 and about 40,000 reverse transcriptases of the culture supernatant of the HTLV-1-producing MT-2 cell line in which the amino acid sequence is substantially homologous to BaEV in Ba02. A band likely to be a related protein was detected. Therefore,
This antibody was found to have a recognition site at LPQGFKNSPTLF in the amino acid sequence.

Next, as shown in FIG. 6, the S03-A7 antibody had about 20,000 transmembrane proteins (T T) from the culture supernatants of the MPMV-producing cell line and the SRV-1-producing cell line having exactly the same amino acid sequence.
M) A band was detected at a position corresponding to P20E. further,
In culture supernatants of MMLV-producing cell lines and AKV-producing cell lines, which were homologous to a considerable extent, bands were observed at positions corresponding to transmembrane proteins (TM) P15E and P12E at around 15,000 and 12,000.

In addition, a band was observed at a position corresponding to about 20,000 of the transmembrane protein P20E in the culture supernatant of the HTLV-1-producing cell line in which 9 residues likely to be epitopes are common among the peptide portions. .

From the above, in the amino acid sequence corresponding to the retrovirus gene, the anti-S01 antibody was SPWNTP and the anti-S02 antibody was
LPQGFKNSPTLF and anti-S03 antibody were found to have recognition sites in QNRRGLDLL. In addition, it was found that a wide range of retroviruses can be detected among retroviruses that have been discovered by using these antibodies. Therefore, it is considered that a method for detecting an unknown pathogenic retrovirus can be obtained by performing an antigen-antibody reaction using these monoclonal antibodies.

Furthermore, as shown in Table 2 below, the synthetic peptides used in preparing these monoclonal antibodies reacted more strongly with goat anti-BaEV antiserum than with goat serum. It was strongly suggested that it has specific reactivity to antiretroviral antibodies. From the above results, this peptide has the potential to detect antiretroviral antibodies in the serum of patients infected with retrovirus, and represents intractable diseases such as leukemia, cancer and autoimmune diseases caused by retrovirus. It can be used as a test or diagnostic agent for various diseases. In addition, these synthetic peptides can be used for prophylactic drugs used as vaccines.

The new test method used in the present invention detects the most specific and basic common antigen of retrovirus and should be used as one of the test methods for obtaining a definitive diagnosis for retrovirus infection. It is also useful for solving personal and social problems of patients with these diseases.
Patients who have previously been clinically diagnosed with a retrovirus infection but who have been negative by serologic tests so far, if the test is positive, a new retrovirus with a different antigen specificity Infection by the strain will be detected. In addition, since this test method uses antigens and antibodies that react commonly to human retroviruses, it can be used as a test method to search for the relatedness of retroviruses for diseases of unknown etiology. it can. Of the so-called "intractable diseases", those for which retrovirus infection has been suggested by this test method can be used to pursue the etiology such as isolation of the virus, and these diseases can be used for medical diagnosis, treatment and prevention. Things. In addition, these antibodies may be used for therapeutic purposes, such as using them as neutralizing antibodies for retroviruses or binding to toxins for use in missile therapy.

Therefore, the present invention is rich in contribution to society and industrial use. Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

The three hybridoma cells producing these three antibodies were SP-2 S01-1F, SP-2 S02-11G and SP-2 S, respectively.
Under the designation of 03-A7, accession numbers No. 10538, 10
Nos. 539 and 10540 have been deposited and stored since February 15, 1999.

Example 1 Polypeptide Synthesis A polypeptide having the following amino acid sequence was synthesized by a solid phase method, and purified by reversed-phase high performance liquid chromatography (HPLC).

S01: LELGVLRPSRSPWNTPLLPVKK S02: QLTWTRLPQGFKNSPTLFDE S03: SLAEVVLQNRRGLDLLTAEQGGI The synthesized polypeptide was confirmed by HPLC and an amino acid analyzer. When used as an immunogen, a cysteine residue is added to the 5 'end of the
Bound with limpet hemocyanin (KLH). S01
As for, the ninth C from the 5 'end is represented by S in order to prevent the reaction with KLH.

Example 2 Preparation of Hybridoma Freund complete adjuvant (Freund complete adjuvant)
vant = FCA) mixed with each polypeptide (those bound to KLH) 20 [mu] g of 6-7 weeks old C ₃ CF ₁ mice (C3H × Balb /
C) was injected into F ₁ intraperitoneally male mice. Then two weeks later Freund incomplete adjuva
Boost was performed with a polypeptide (conjugated to KLH) mixed with nt = FIA).

As a result, the serum of the mouse that reacted best with the amino acid sequence of S01 after the second immunization reacted with the corresponding synthetic peptide up to 6,400-fold dilution by ELISA, and the amino acid sequence of S02 contained the following:
The serum of the mouse that reacted best was diluted up to 1,600-fold, and the serum of the mouse that reacted best to the amino acid sequence of S03 was diluted up to 102,400.

2 weeks after booster, polypeptide (KLH binding) 20μ
g saline solution intraperitoneally (final immunization), 3
The spleen cells were obtained a second time. Then, about 2 × 10 ⁸ spleen cells were added to SP-
2 About 4 × 10 ⁷ myeloma cells were added, followed by centrifugation, and 1 ml of PEG1,500 (manufactured by Boehringer Mannheim) was gradually dropped to perform cell fusion.

PEG1,500 was diluted with RPM1640 medium, centrifuged, then suspended in a selection medium, and dispensed into a flat bottom Mimic test (III) plate (Falcon) (splenocytes: about 5 × 10 ⁵ / well). Thereafter, the fused cells were cultured at 37 ° C. under 5% CO ₂ humidity for about 10 days.

The selection medium was GIT medium (Nippon Pharmaceutical Co., Ltd.) + 2
-Mercaptoethanol (2-ME): 5 x 10 ^-5 M + L-glutamine: 2 mM + penicillin: 100 U / ml + streptomycin: 100 µg / ml + insulin: 10 ^-3 U / ml + dihydroxyethylglycine (DHEG): 1.8 mg / ml It was prepared by adding 1/50 volume of HAT (hypoxanthine / aminopterin / thymidine) 50-fold concentrated solution (manufactured by Flow). As a result, hybridoma formation was observed in all wells. When the reactivity of the culture supernatant with the synthetic peptide was examined by ELISA, a large number of positive wells were obtained.

Example 3 Specific antibody screening by ELISA method
A method was used.

That is, 0.1 M containing 1 nM of each polypeptide used for immunization in Pro-Bind-assay-plate (manufactured by Falcon).
Sodium bicarbonate buffer (pH 9.6) was added at 50 μl / well and coated at 37 ° C. overnight. Each well contains 0.1% (V / V) Tween 20 [Tween 20 (trade name of surfactant)]
BS buffer [50 mM Tris-HCl (pH 8.2), 144 mM NaCl] (TBS
-Tween 4 times and 1% with TBS buffer, then 1%
(W / V) TBS containing bovine serum albumin (TBS-BSA) was added, left at room temperature for 60 minutes, and then washed similarly. 50 μl of the hybridoma culture supernatant was added to each well, and allowed to stand at room temperature for 120 minutes, followed by washing similarly.

Next, horseradish peroxidase-labeled rabbit anti-mouse IgG (manufactured by Capel) was used as an enzyme-labeled secondary antibody.
A solution diluted 800-fold with BS-BSA was added at 50 μg / well, and the mixture was allowed to stand at room temperature for 60 minutes. After washing, 1 μl / ml of 30% H ₂ O ₂
[2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt dissolved at a concentration of 1 mM in a 0.1 M citrate buffer (pH 5) containing ABTS- (N
H ₄ ) ₂ ] (Wako Pure Chemical Industries) 100 μl / well. 3 at room temperature
After standing for 0 minutes, the enzyme activity was measured with 405 mM excitation light.
As a result, 10 hybridomas stably producing specific antibodies were obtained for the amino acid sequence of S01, 3 for the amino acid sequence of S02, and 19 for the amino acid sequence of S03. Was.

Example 4 Cloning For antibody-positive wells, mouse thymocytes (about 1
(× 10 ⁶ cells / well) were used as feeder cells, and cloning was performed by the limiting dilution method. Next, the culture supernatant of each well was screened again to select positive clones.

Example 5 Purification of Monoclonal Antibody A hybridoma cell clone producing a specific antibody was grown in a complete serum-free medium [Cosmedium-001 (trade name)] without albumin, and the culture supernatant was filtered with a 0.22 μm filter (manufactured by Gelman). And filtered. The filtrate was salted out twice with 50% ammonium sulfate, dissolved in a phosphate buffer, and dialyzed. After removing the insoluble matter generated during the dialysis by centrifugation, the dialysate was dispensed and stored frozen at -20 ° C. The amount of antibody in the dialysate is BIO-
Quantification was performed using a RAD protein assay kit (manufactured by Bio-Rad Laboratories).

Example 6 Immunoblotting Various types of retrovirus-producing cell lines, including A204 cells and BaEV-producing A204 cell lines, HTLV-1-producing MT-2 cells, SR
V-1 producing Raji cell line, MPMV producing human lung adenocarcinoma cell line, MMLV producing D3hlg cell line, AKV producing AKR / J mouse T lymphoma cell line, or RSV producing avian fibroblast (fibrobl
ast), and when the stationary phase reaches about 10 ⁶ cells / ml, the culture medium is exchanged.
The mixture was centrifuged at 0 rpm for 5 minutes, and the supernatant was filtered with a 0.22 μm filter. 40 ml of the filtrate was centrifuged at 26,000 rpm for 2 hours at 4 ° C. using a Hitachi-SRP28SA rotor (manufactured by Hitachi Koki Co., Ltd.), and a TNE buffer [10 mM Tris-HCl (pH 7.6), 100 mM
NaCl, 1mM EDTA] or those partially purified by equilibrium density gradient centrifugation using saccharose.
400 μl of 3 times SDS sample buffer [(30% glycerol, 15% mercaptoethanol, 7% SDS, 0.19MT
ris-HCl (pH6.8), BPB (bromophenol blue) 0.
006%] and heated in boiling water for 5 minutes. 60 μl of the sample was subjected to SDS-polyacrylamide gel electrophoresis. Here, electrophoresis on SDS-polyacrylamide gel was performed by the method reported by Laemmli in 1970 [Nature, Vol. 227, 680-685 (197)
0)] in a 13.5% to 14.5% separation gel having a 5.7% concentration gel under a constant current of 20 mA. To determine the molecular weight, Bio-Rad molecular weight marker (LOW)
Was used. Next, after electrophoresis, the gel was blotted with a blotting solution [25
mM Tris-HCl (pH 8.3), 192 mM glycine, 20% methanol, 0.01% SDS], and then use a blotting device (manufactured by Nippon Aido) to remove the protein in the gel at 100 mA ON at 4 ° C. Transferred to nitrocellulose membrane. The blotted nitrocellulose membrane was treated with TBS [50 mM Tris-HCl (pH 8.
2), 144 mM NaCl] and blotted with TBS containing 4% skin milk for 2 hours at room temperature. After blotting, the nitrocellulose membrane was washed three times with TBS containing 0.1% Tween-20 (TBS-Tween) and containing 10% FBS containing a monoclonal antibody that specifically reacts with each synthetic peptide at a concentration of 5 μg / ml. The reaction was performed for 2 hours at room temperature with TBS-Tween (TBS-Tween-FBS).
After the reaction, the nitrocellulose membrane was washed four times with TBS-Tween, and ¹²⁵ I-sheep anti-mouse IgG (1
The reaction was carried out at room temperature for 2 hours with TBS-Tween-FBS containing 0.4 μCi / ml (4.8 μCi / μg) (ICN). Thereafter, the nitrocellulose membrane was washed four times with TBS-Tween and twice with TBS, dried, and then dried at -60 ° C using an intensifying screen (Kodak intensifying screen).
It was exposed to an HX-ray film (Fujifilm).

Example 7 Determination of Sub-Isotype of Monoclonal Antibody The determination of the sub-isotype of the monoclonal antibody was performed by ELISA using the mouse monoclonal sub-isotype kit of American Quorex Co., Ltd. of the culture supernatant of the hybridoma after cloning. The results are shown in the following table.

Example 8 Immunoblotting Assay for BaEV The reactivity of the anti-peptide antibody to BaEV was examined by immunoblotting of the culture supernatant of A204 cell line producing or not producing BaEV separated by SDS-polyacrylamide gel.

The culture supernatant of the A204 cell line producing or not producing BaEV was concentrated by ultracentrifugation, separated on a 14.5% SDS-polyacrylamide gel, and electroblotted on a nitrocellulose filter. Blots were cut longitudinally and assayed with different antibodies.

As shown in FIG. 2, 4 out of 10 antibodies and 2 out of 3 antibodies against the amino acid sequences of S01 and S02
Only when the culture supernatant of the EV-producing cell line was used, about 70,000 and 40,000 specific bands, which were considered to be BaEV reverse transcriptase-related proteins, were detected. On the other hand, about 6 antibodies out of 19 antibodies against the amino acid sequence of S03 detected about 20,000 specific bands only when the culture supernatant of the BaEV-producing cell line was used. this is
The protein was thought to be related to the transmembrane part (P20E) of the BaEV envelope protein (Fig. 3).

Example 9 Immunoblotting Assays for Various Retroviruses Antibodies that react with the amino acid sequence in each region were selected one by one showing strong reactivity (S01-1F antibody, S02-11
G antibody, or S03-7A antibody), and the reactivity of these antibodies to various retroviruses was determined by equilibrium density gradient centrifugation using saccharose with culture supernatants of various virus-producing cell lines separated by SDS-polyacrylamide gel. The partially purified product was examined by immunoblotting.

As a result, the S01-1F antibody detected about 70,000 and about 40,000 bands in the culture supernatant of the MMLV-producing cell line, and about 70,000 bands in the culture supernatant of the AKV-producing cell line.
Approximately 90,000 and 40,000 bands were detected in the culture supernatants of the SRV-1 producing cell line and the MPMV producing cell line.
Figure).

Next, in the S02-11G antibody, about 70,000 and about 40,000 bands were detected in the culture supernatant of the MMLV-producing cell line, and about 70,000 bands were detected in the culture supernatant of the AKV-producing cell line. . About 60,000 in culture supernatant of HTLV-1 producing cell line
And about 40,000 bands were detected (FIG. 5).

Furthermore, for the S03-7A antibody, about 20,000 bands were found in the culture supernatant of the SRV-1 producing cell line and the MPMV producing cell line, and 15,000 for the MMLV producing cell line and the AKV producing cell line. Two bands around 12,000, HTLV
20,000 bands were observed in the culture supernatant of the -1 producing cell line (FIG. 6).

Example 10 Reactivity of Synthetic Peptide with Goat Anti-BaEV Antiserum According to the method of Example 3 described in the section on specific antibody screening, the synthetic peptide of S01, S02 or S03 was adsorbed on a Pro-Bind-assay plate. , TBS-Tween-FB
ELISA for reactivity with goat anti-BaEV antiserum diluted 400 times with S
It was examined by the method. As a result, the absorbance was clearly higher at the excitation light of 405 nM as compared with the goat serum of the same dilution as a control, and it was confirmed that all of these peptides reacted with the goat anti-BaEV antiserum. Next, the results are shown.

[Brief description of the drawings]

FIGS. 1-1 and 1-2 show the amino acid sequences (S01 and S02) in the reverse transcriptase regions of various retroviruses. FIG. 1-3 shows the amino acid sequences (S03) of transmembrane envelope protein peptides of various retroviruses. FIGS. 2 and 3 show BaEV of the anti-peptide antibody of the present invention.
FIG. 2 shows the results of an assay of the reactivity with respect to the antibody by immunoblotting. FIG. 2 relates to the antibodies against S01 and S02, and FIG. 3 relates to the antibodies against S03. FIGS. 4, 5 and 6 are immunoblotting diagrams showing the reactivity of the S01 antibody, S02 antibody or S03 antibody with various retroviruses, respectively.

Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI G01N 33/569 G01N 33/577 B 33/577 C07K 14/15 // C07K 14/15 C12P 21/08 ZNA C12P 21/08 ZNA C12N 15 / 00 C Preliminary examination (56) References Biochem. Biophys. Res. Commun. Vol. 126, No. 1985. 2, P. 672-677 J.C. of Virology, 1984, Vol. 49, p. 471-478 Proc. Natl. Acad. Sc i. USA, 1983, Vol. 80, p. 3618-3622 Science, 1986, Vol. 231, p. 1567-1572 (58) Fields investigated (Int. Cl. ⁶ , DB name) C12P 1/00-41/00 C12N 15/00-15/90 C12N 1/00-7/08 WPI (DIALOG) BIOSIS (DIALOG) ) CAPLUS (STN)

Claims (4)

(57) [Claims] The present invention relates to SP-2 S01-1F (FER) which produces a monoclonal antibody that recognizes a human or primate retrovirus.
M P-10538), SP-2 S02-11G (FERM P-10539) or S
Hybridoma cells indicated by P-2 S03-A7 (FERM P-10540). 2. A monoclonal antibody that recognizes a human or primate retrovirus produced from the hybridoma cell according to claim 1. 3. A method for detecting a pathogenic retrovirus by reacting an antigen-antibody reaction with an unknown pathogenic retrovirus using the monoclonal antibody according to claim 2. 4. An agent for testing / diagnosing a disease caused by retrovirus infection, comprising the monoclonal antibody according to claim 2.