JPH1142087A - Anti-human pre-b cell receptor antibody and its active fragment and detection of human pro-b cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject antibody (active fragment) having high specificity, capable of recognizing a human preB cell among lymphocytes, useful for clinical diagnosis of leukemia, etc., by immunologically dyeing the human preB cell and making the cell possess such a reactivity as to be observed by a microscope. SOLUTION: This antibody is reacted with a VpreB molecule of a substitute light chain constituting a human preB cell receptor expressing on the surface of the human preB cell and with a VpreB molecule expressing in a human pro-B cell but not with a λ 5 molecule of a substitute light chain. The reactivity of the antibody has such a degree as to be observed by a microscope by immunologically dyeing the human pre cell or human B cell by the antibody. Preferably the human preB cell receptor antibody (active fragment) is produced from a hybridoma HSL 96 (FERM P-16251) and the human pro-B cell in a specimen is detected by the antibody (active fragment).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a VpreB molecule of a light chain alternative to the human pre-B cell receptor expressed on the membrane surface of human pre-B cells and a VpreB molecule expressed in the cells of human pro-B cells. It relates to an antibody or an active fragment thereof. The present invention also relates to a detection method for detecting human pro-B cells using the above-mentioned antibody or an active fragment thereof using a saponin buffer. The antibody of the present invention can be applied to diagnosis of acute lymphocytic leukemia.

[0002]

2. Description of the Related Art It is known that B cells are differentiated from bone marrow hematopoietic stem cells into plasma cells through each stage of precursor cells, pro-B cells, pre-B cells, immature B cells, and mature B cells.

[0003] On the surface of B cells, immunoglobulin (hereinafter sometimes referred to as Ig) is used as an antigen recognition receptor.
Is expressed. This antigen-recognition receptor is composed of two types of polypeptides, a heavy chain and a light chain, and the composition changes as B cells are differentiated.

[0004] The differentiation of B cells and their antigen-recognition receptors will be described below using a mouse as an example.

[0005] In pro B cells, it is known that the pro B cell receptor is expressed on the cell surface. The pro-B cell receptor has an alternative light chain (SL chain) in which two polypeptides called λ5 and VpreB are associated with a heavy chain (H chain) called an alternative heavy chain (SH chain). The alternative light chain (surrogate light chain (SL chain)) is λ
It is so called because five molecules (hereinafter sometimes referred to as λ5) and VpreB molecules (hereinafter sometimes referred to as VpreB) associate and substitute for a normal light chain (L chain). λ5 corresponds to the constant region of the light chain, and VpreB corresponds to the variable region of the light chain.

[0006] In pre-B cells, it is known that the pre-B cell receptor is expressed on the cell surface. The pre-B cell receptor has a heavy chain (H chain) called μ chain,
And an alternative light chain (SL chain) in which two polypeptides called VpreB and VpreB are associated.

[0007] In immature B cells and mature B cells, a B cell receptor in which a normal L chain is bound to a μ chain is expressed as an antigen recognition receptor.

As can be seen from the above, λ5 and Vpr
eB is expressed on the membrane surface of cells only in pro-B cells and pre-B cells.

[0009] Similar findings as described above have been reported for human B cell differentiation and antigen recognition receptors.

Conventionally, human pre-B cell receptor, human V
The following are known as antibodies that react with preB or human λ5. Table 1 shows the reactivity etc.
Shown in Anti-human pre-B cell receptor antibody: SLC1, SLC
2, SLC3, SLC4 (Cell, Vol. 73, 73-8
6, p. 1993) Anti-human pre-B cell receptor antibody: 9C2 (Eur.
J. Immunolo. 24, 257-264, 1994) Anti-human VpreB antibody: 3C7, 6F6 (Eur.
J. Immunolo. , 26, 2172-2180
P. 1996) Anti-human VpreB antibody: B-MAD176, B-MA
D688, B-MAD792, B-MAD1112
(J. Exp. Med., 183, 2693-269)
8 pages, 1996)

[0011]

[Table 1]

[0012]

The abnormality of B cell differentiation is as follows.
Causes leukemia. Knowing at what stage of differentiation B cells abnormally proliferate is important in the treatment of leukemia. Particularly, acute lymphoblastic leukemia in children is often caused by abnormal proliferation of pro-B cells or pre-B cells. Currently, B cell surface markers generally used in diagnosis include anti-CD10 antibody, anti-CD19 antibody, anti-CD20
Antibodies, anti-CD21 antibodies. These antibodies are reacted with B cells, and as a result, the stage of the B cells is to be known. However, anti-CD19 antibody, anti-C
D20 antibody and anti-CD21 antibody are used for immature B cells or mature B cells.
It also reacts with cells, and the anti-CD10 antibody also reacts with T cells and epithelial cells. Therefore, it was impossible with these antibodies to specifically detect only human pro-B cells or human pre-B cells.

[0013] In the field of clinical diagnosis, observation of cells with a microscope is performed. This is because it is the simplest and fastest method, and the installation place of the equipment is minimized as compared with other methods. However, when the staining of the cells is very weak, even if the cells can be detected by a highly accurate detection method such as FACS, they cannot be observed with a microscope. the above~
All the antibodies against the human pre-B cell receptor and the like, in particular, the antibodies against VpreB mentioned above only show the results of FACS. In other words, it does not indicate whether the cells react with the antibody are reactive enough to be stained to the extent that they can be observed with a microscope. In addition, the reactivity with human pro-B cells only indicates the reactivity on the cell surface, but does not show intracellular staining.

[0014] The present invention relates to an antibody which specifically reacts with human pre-B cells and human pro-B cells, wherein the human pre-B cells expressing the human pre-B cell receptor on the membrane surface and the VpreB molecule are expressed in the cells. Human Pro B
An object of the present invention is to provide an antibody capable of immunostaining cells with the antibody and observing only the immunostained cells with a microscope.

[0015]

The antibody of the present invention is expressed in a VpreB molecule, which is a member of the SL chain of the human pre-B cell receptor expressed on the membrane surface of human pre-B cells, or in human pro-B cells. Reacting with the VpreB molecule, and not reacting with the alternative light chain λ5 molecule, and its reactivity is such that the human pre-B cell or human pro-B cell reacted with the antibody can be detected with a microscope. Antibodies that are reactive.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION

1. Active Fragments of Antibodies In the present invention, antibodies also include active fragments. An active fragment means a fragment of an antibody having antigen-antibody reaction activity, and specifically,
F (ab ') ₂ , Fab', Fab, Fv and the like can be mentioned. For example, when the antibody of the present invention is digested with pepsin, F (ab ') ₂ is obtained, and when digested with papain, Fab is obtained. Reduction of F (ab ') ₂ with a reagent such as 2-mercaptoethanol and alkylation with monoiodoacetic acid gives Fab'. Fv is a monovalent antibody active fragment in which a heavy chain variable region and a light chain variable region are linked by a linker.

A chimeric antibody can be obtained by retaining these active fragments and substituting other parts with fragments of another animal. As used herein, a “chimeric antibody” is an antibody in which the variable region or antigen-binding domain and the constant region or effector domain are each composed of immunoglobulin fragments belonging to genetically different species. For example, the variable (V) region fragment of a mouse monoclonal antibody is a human constant (C) region fragment, eg, γ
1 or γ4.

2. Genetic engineering method The antibody of the present invention can also be obtained using a genetic engineering method. For example, mR from spleen cells, lymphocytes or a hybridoma producing the monoclonal antibody of the present invention immunized with a substance that associates the μ chain and the SL chain of the pre-B cell receptor.
NA is collected, and a cDNA library is prepared based on the collected NA. Next, an antibody is expressed by the cDNA library. A clone that produces an antibody that reacts with an antigen is obtained from a cDNA library by screening, the obtained clone is cultured, and the target antibody is subjected to salting-out, ion-exchange chromatography, affinity chromatography, and other methods from the culture mixture. It can be purified in combination.

3. Detection Method Detection of human pre-B cells, human pro-B cells, pre-B cell receptors or VpreB molecules using the antibody of the present invention can be performed by any of ELISA, FACS, immunoprecipitation, or microscopic observation. In particular, the FACS method or microscopic observation not only detects pre-B cell receptors and VpreB molecules on the cell surface, but also mixes an antibody with a sample and reacts them by using a saponin buffer to reduce intracellular VpreB molecules. Can also be stained to detect the molecule or cells harboring the molecule.

4. Use as immunotoxin The production of an immunotoxin in which a toxin is bound to the antibody of the present invention is described in, for example, Proc. Natl. Acad.
Sci. USA 87 (1990) 8291-82
It is possible by the method described on page 95. By administering the immunotoxin to a pre-B cell or pro-B cell-derived leukemia patient, the toxin selectively acts on the pre-B cell or pro-B cell grown in the patient, killing the cell, Leukemia of origin can be treated.
Examples of toxins include exotoxin, diphtheria toxin and the like.

5. Use as a Complex The antibody or active fragment thereof of the present invention can be used alone, but can be used as a new complex by binding to a substance such as albumin or polyethylene glycol, if necessary. In general, such composites often exhibit their effects to the maximum without being decomposed for a long time in vivo. In the active fragment, albumin,
Methods for adding substances such as polyethylene glycol are described in, for example, Antibodies, A. Laboratory Manual (Antibodies, A. Laboratory).
y Manual) (Cold Spring Harbor Laboratory, 1988). Generally, when a bivalent reactive reagent such as SPDP (manufactured by Pharmacia) is used, the active fragment can be easily bound to albumin or the like.

[0022]

EXAMPLES Examples of the present invention will be described below to explain the present invention more specifically. However, the present invention is not limited to these examples, and includes animals to be immunized, target cell lines, labeled antibodies and the like. Can be appropriately used depending on the purpose.

Example 1 Preparation of Antibody Preparation of Antigen 1) Preparation of Recombinant Antigen Gene (1) Preparation of λ5 Gene and VpreB Gene For human λ5 gene and human VpreB gene, mRNA was prepared from human pre-B cell Nalm6 by an ordinary method,
For the mRNA, RT-PCR was performed using the following primers.
It was produced by performing the method. Each of the prepared mRNAs was sequenced, and human λ5 and human V
It was confirmed to have the base sequence of the preB gene.

Λ5 primer 5 ′ primer 5′-GGA ACT CGA GCC ACA AGG ACC TCT GAC CCT-3 ′ 3 ′ primer 5′-GGA AGC GGC CGC AGG CCT TTG GGT GGG GTC GG-3 ′ VpreB primer 5 ′ primer 5 '-GGA ACT CGA GGG AGT CAG AGC TCT GCA TGT-3' 3 'primer 5'-GGA AGC GGC CGC AGG GGA TGC GTG CCT CTG CT-3'

(2) Insertion into a Vector An expression vector BCMGSHyg for mammals having a cytomegalovirus promoter and an immunoglobulin μ chain enhancer (this method is described in J. Ex.
p. Med. 172, pages 969-972. ), The λ5 gene and the VpreB gene prepared in (1) were inserted by an ordinary method.

(3) Preparation of Transformant The host Ltk ⁻ (mouse μ chain) expressing the mouse μ chain was:
The vector prepared in (2) was introduced by an electroporation method (this method is described in J. Exp.
Med. 172 vol, see 969-972 pages), transformant Ltk express and secrete the chimeric pre-B cell receptor (mouse μ chain / human [lambda] 5 / person VpreB) ^- (mouse μ chain / human [lambda] 5 / person Vpre
B) was prepared.

[0027] (4) transformants screened transformant Ltk ^- (mouse μ chain / human [lambda] 5 / human V
preB) was cultured in a 96-well plate at 37 ° C. under 5% CO _{2 using} 10% FCS-RPMI1640 containing 0.2 mg / ml hygromycin and G418 as a medium. The culture supernatant was measured by the ELISA method under the following condition 1. Colonies were expressed in about 600 wells out of 1200 wells, of which 16 wells having a high absorbance at 405 nm were selected as positive.

ELISA condition 1 Coating of 96-well plate: goat anti-mouse IgM
(10 μg / ml). Sample: Transformants Ltk ^- (mouse μ chain / human [lambda] 5 / person VpreB) of the culture supernatant detection antibody: alkaline phosphatase-labeled goat anti-mouse IgM (Southern Biotechnology Associates (S
southern BiotechnologyAsso
substrate: PNPP (p-nitrophenyl phosphate,
Sigma)

The above ELISA was repeated several times, and the positive strain was cloned using the limiting dilution method to select a positive strain that highly expressed the pre-B cell receptor (mouse μ chain / human λ5 / human VpreB). . The positive strain was named Ltk ^- 17-72.

2) Mass cultivation of antigen protein The transformant Ltk ^- 17-72 obtained above was transformed with 5% F
The cells were cultured at 37 ° C. in CS-RPMI1640 medium in a plurality of 1-L roller bottles. 20 per roller bottle
0 ml of the medium was injected and cultured for 2 weeks. After 2 weeks of culture, the concentration of the pre-B cell receptor (mouse μ chain / human λ5 / human VpreB) in the medium was determined by ELISA as described above.
As a result of measurement under SA condition 1, the concentration was about 5 ng / ml.

3) Purification of antigen (1) Concentration 2 L of the culture supernatant obtained in 2) was collected and concentrated in a concentrator (MINITAN).
(Registered trademark, manufactured by Millipore Co.)).

(2) Purification A rat anti-mouse μ chain antibody was attached to beads (CNBr-activated Sepharose 4B, manufactured by Pharmacia) by a conventional method, and the mixture was packed in a column. The concentrated culture supernatant was circulated through this column for 2 weeks, and the pre-B cell receptor (mouse μ chain /
An antigen-bead conjugate was prepared by binding human λ5 / human VpreB) to beads as an antigen protein. The above-described concentration and purification were performed on about 20 L of the culture supernatant in total to prepare an antigen protein-bead conjugate in which 50 μg of the antigen protein was bound to 500 μl of antigen beads (the recovery rate of the antigen protein in the medium was 50%. there were.).

2. Immunization The antigen protein-bead conjugate prepared above was replaced with a female BA.
Subcutaneously at the base of limb of LB / c mouse, 1 μg per mouse
/ 10 μl of beads were immunized. For the second and subsequent immunizations, the antigen protein-bead conjugate was suspended in PBS, and 1 μg / 10 μl per animal was immunized every 7 days for a total of 7 times. After 8 weeks, the antigen protein solution (30 μg of antigen protein) eluted from the antigen protein-bead conjugate was intravenously injected into the tail to perform additional immunization. The antigen protein solution (0.1% of the antigen protein-bead conjugate)
SDS-P of M glycine (pH 2.7)
FIG. 1 shows the results of AGE electrophoresis. As a result of electrophoresis,
μ-chain, λ5, and VpreB bands were observed, and the antigen protein was composed of a secretory pre-B cell receptor (mouse μB).
Chain / human λ5 / human VpreB).

3. Preparation of Monoclonal Antibody 1) Preparation of Hybridoma Three days after booster immunization, 2.2 × 10 ⁸ spleen cells were removed from the spleen of the immunized mouse, and mouse myeloma cell P
AI and polyethylene glycol (manufactured by Boehringer) cells were fused at a ratio of 4: 1. The hybridoma thus obtained was spread on ten 96-well plates, and 100-fold diluted HAT (manufactured by Boehringer) and 500-fold diluted IL-6.
(Culture supernatant of the transformant X63BCMGNeo (refer to J. Exp. Med. Vol. 171: pp. 389-400 for the production method)) and 20% FCS-
The cells were cultured in IMDM (manufactured by Gibco).

2) Screening (1) ELISA Hybridomas were cultured for about 2 weeks. Colonies appeared in about 260 wells in 960 wells (10 plates). The culture supernatant of the hybridoma in which the colony was formed was measured by the ELISA method under the following condition 2.

ELISA condition 2 Coating of 96-well plate: The following four kinds of antibodies were coated on a 96-well plate, respectively. Each antibody used for coating was used at a concentration of 10 μg / ml, and 50 μl was added to each well of the plate at 4 ° C.
And left overnight. Then, 0.05% tween 20-
After washing with PBS, 4% BSA-0.2% tween
20-PBS was added as a blocking solution in an amount of 200 μl / well to each well, and left at 4 ° C. for 1 hour. A: After coating with a goat anti-mouse μ chain antibody (Southern Biotechnology Associates) and blocking, a 10-fold concentrated solution of Ltk ^- 17-72 culture supernatant was coated. B: Coated with goat anti-mouse μ chain antibody (Id.). C: Coated with human IgM (μ chain and λ chain) (Southern Biotechnology Associates). D: Coated with human IgM (μ chain and κ chain) (Southern Biotechnology Associates).

Antigen-antibody reaction: 0.05% of each plate
After washing with Tween 20-PBS, 4% was added to each well.
30 μl of BSA-0.2% tween 20-PBS was added as a diluent to each well, and the above-described hybridoma culture supernatant was added to each well in an amount of 30 μl, mixed, and allowed to stand at room temperature for 2 hours for antigen-antibody reaction. Was. Color reaction: 0.05% each plate after antigen-antibody reaction
After washing with Tween 20-PBS, 100 μl of an alkaline phosphatase-labeled goat anti-mouse IgG antibody (manufactured by Southern Biotechnology, diluted 500-fold) was added to each well as a detection antibody, and the mixture was allowed to stand at room temperature for 1 hour. Thereafter, each plate was treated with 0.05% tween 20-
After washing with PBS, 100 μl of each was added to each well using PNPP as a substrate, and allowed to stand at room temperature for 30 minutes to develop color.

Thereafter, the absorbance at 405 nm was measured. Hybridomas producing a culture supernatant which became positive when the absorbance at 405 nm increased and became positive when coated with A and negative when coated with B, C and D were selected. As a result, a colony 2
Fourteen well hybridomas were selected from the 60 well hybridomas.

(2) Cell Surface Staining From the 14 clones selected as positive strains above, a clone positive for reaction with the pre-B cell receptor expressed on the cell surface of the human pre-B leukemia cell line Nalm6 is selected. For this, cell surface staining was performed.
Specifically, 1 × 10 6 was added to each well of a 96-well plate.
^Six Nalm6 cells were added, and 50 μl of the culture supernatant of each clone was added to each, and reacted at room temperature for 30 minutes.
Thereafter, 100 μl of PE-labeled goat anti-mouse κ chain antibody (2 μg / ml) was added to each well as a secondary antibody.

This sample was placed in a fax caliber (FA).
CS Calibur, manufactured by Becton Dickinson). As a target of cross-reactivity, mature B cells Da
Udi and LBW-4 were similarly stained on the cell surface and measured by FACS Calibur. As a result, 1
Of the 4 clones, 4 clones were positive for Nalm6,
Daudi and LBW-4 were negative.

(3) Immunoprecipitation 1 ml of the culture supernatant of the above 4 clones was mixed with 20 μl of Protein G Sepharose (Pharmacia) (50% V /
V) at 4 ° C overnight. 10,000 r of this mixture
protein G to which antibody was bound by centrifugation at pm for 5 minutes
Sepharose was set aside. The protein G Sepharose is dissolved (1% NP-40, 150 mM NaCl)
And washed.

1 × 10 ⁵ pre-B cells Nalm6 internally labeled with isotope ³⁵ S-methionine cysteine were mixed with a lysate (1% NP-40, 150 mM NaCl) to obtain a cell lysate. Antibody-bound protein G Sepharose was suspended in the cell lysate and mixed for 2 hours. This mixture was centrifuged at 10,000 rpm for 5 minutes to obtain an immunoprecipitate (Nalm bound to Protein G Sepharose as described above).
6 derived proteins).

The immunoprecipitate was washed with a washing solution (1% NP-40, 6%).
Washing with about 1 ml of 50 mM NaCl) was repeated four times to obtain a sample for electrophoresis. The immunoprecipitate was reduced at 95 ° C. for 5 minutes in the presence of 2-mercaptoethanol, and then developed on a 13% acrylamide gel. Pre-B cell receptor (human μ / human λ5 / human VpreB) 3
The band was confirmed in one of the four clones. FIG. 2 shows the results of electrophoresis of the clone.
As controls, a culture supernatant containing mouse IgG (control) and a culture supernatant containing anti-human μ-chain antibody were used.

3) Cloning The hybridoma of one clone finally remaining was spread on a 96-well plate at 1 cell / well by a limiting dilution method, and then cultured for 10 days. 10 colonies with 1 colony / well were selected, and ELISA conditions 2
The absorbance at the coating of A was measured by the ELISA method. The hybridoma having the highest absorbance was designated as HSL96, and the hybridoma was deposited with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology on May 30, 1997 (Accession No. FERM P-16251). In addition, the antibody produced by the hybridoma HSL96 is called an HSL96 antibody.

4. Mass production of monoclonal antibodies ICRnu / nu mice at 7 weeks of age (Charles
0.5 ml of pristane was injected into the peritoneal cavity of
Five days later, each mouse was injected with 1 × 10 ⁷ hybridoma HSL96. After about two weeks, the ascites was collected, and the caprylic acid method (Antibodies A Labora) was used.
tory Manual Chapter 8, 300 pages, 198
8 years Cold Spring Harbor Lab
Purification was carried out according to the method described in Oral Publication, and a monoclonal antibody was obtained. Monoclonal mouse Ig isotyping kit (Pharmingen)
Was used to confirm that the subclass of the antibody was IgG ₁ κ.

Example 2 Confirmation of Specificity of Antibody 1-EL
ISA method Under the same conditions as in ELISA condition 2, the following samples were reacted with the following coated antigen proteins, and the absorbance at 405 nm was measured. Coated antigen protein: 10-fold concentrated solution of Ltk ^- 17-72 culture supernatant (pre-B cell receptor) Human IgM (human μ chain and human κ chain) (10 μg /
ml) human IgM (human μ chain and human λ chain) (10 μg /
Sample): Culture supernatant of HSL96 (HSL96 antibody) Culture supernatant of mouse hybridoma 141PF11 (anti-human κ chain antibody) Culture supernatant of mouse hybridoma HP6054 (anti-human λ chain antibody) Antibody for detection: alkaline phosphatase-labeled goat anti- Mouse IgG antibody 500-fold diluted solution Substrate: PNPP

FIG. 3 shows the results. Only the HSL96 antibody reacts with the pre-B cell receptor and HSL9
6 shows that the antibody 6 does not react with the human μ chain, κ chain or λ chain.

<Example 3> Confirmation of antibody specificity 2-FA
CS method 1. Cell surface immunostaining The following cell lines are reacted with the HSL96 antibody to perform cell surface staining, and then performed by FACSCalibur.
bur, manufactured by Becton Dickinson). Cell line: Pre-B leukemia cell line Nalm6 Pre-B leukemia cell line SMS-SB Pre-B leukemia cell line 697 B lymphoma cell line Daudi T leukemia cell line Jurkat

Specifically, 2% with 0.1% BSA-PBS
Each washed cell was added to buffer 0.1% BSA-PBS for 2 times.
The cells were suspended at a density of 10 ⁷ cells / ml and left on ice for 10 minutes. 50 μl of each cell was added to each well of a 96-well plate
l (1 × 10 ⁶ ), each containing biotin-labeled HS
Add 100 μl of 1 μg / ml of L96 antibody, and add
The reaction was performed for 0 minutes. Thereafter, 100 μl of PE-labeled streptavidin (2 μg / ml) was added to each well to develop color. This sample was sent to a fax caliber (FACS).
Calibur, manufactured by Becton Dickinson).

FIG. 4 shows the results. It was confirmed that pre-B leukemia cells were immunostained by the HSL96 antibody, but B lymphoma cells and T leukemia cells were not immunostained by HSL96.

2. Intracellular immunostaining In the same manner as in the cell surface staining described above, a buffer for suspending cells and antibodies was added to 0.1% BSA-0.05% saponin
As 10 mM Hepes (pH 7.3) -PBS,
The cells were stained intracellularly and measured with a fax caliber. FIG. 5 shows the results. Pre-B by HSL96 antibody
It was confirmed that leukemia cells were also immunostained in the cells, but B lymphoma cells and T leukemia cells were not immunostained in the cells.

For pro-B cell line Nalm16, 0.1% BSA-
0.05% saponin-10 mM Hepes (pH 7.
3) Using -PBS buffer, intracellular staining was performed with the HSL96 antibody, and measurement was performed using a fax caliber. . FIG. 6 shows the results. The HSL96 antibody showed that intracellular immunostaining was possible using saponin buffer even for pro-B cells.

3. Intracellular immunostaining of transformant X63-Ag8.653 Mouse myeloma cells X63-Ag8.653 (J.I.
mmunol. , 123, p. 1548). Human V5 gene or human Vp
The reB gene was introduced by the following three methods by an electroporation method to prepare a transformant. Introduce only λ5 (X63 / λ5) Introduce only VpreB (X63 / VpreB) Introduce λ5 and VpreB (X63 / λ + V)

Each of the above transformants was subjected to intracellular immunostaining using a saponin buffer in the same manner as described above, and was measured using a fax caliber. FIG. 7 shows the results. H
It was confirmed that the SL96 antibody reacts with the recombinant VpreB molecule but does not react with the recombinant λ5 molecule.

Example 4 Confirmation of Specificity of Antibody 3-Immunoprecipitation The culture supernatant of the hybridoma HSL96 was used in Example 1.
In the same manner as in (2) (3) of immunoprecipitation, Nalm6 and the three types of X63-Ag8.653 prepared in Example 3 were used.
The cells were reacted with the transformants and immunoprecipitated. As a control, protein G sepharose reacted with a culture supernatant containing an antibody having the same isotype was reacted with a lysate of each cell, and centrifuged. In addition, centrifugation was performed with A8 (an antibody that reacts with human λ light chain and human λ5) as a positive control. Fig. 8 shows the results.
11 to FIG. In each figure, the left lane shows the result of immunoprecipitation with A8, the center shows the result of control, and the right end shows the result of immunoprecipitation with the culture supernatant of hybridoma HSL96. By comparing the band density with the control in each figure, the HSL9
It was confirmed that Antibody 6 reacted with the recombinant VpreB molecule but did not react with the recombinant λ5 molecule. In addition, as in the results of the ELISA method, Nalm6 and HSL9 were used.
It was also confirmed that 6 antibodies reacted.

Example 5 Microscopic Observation of Immunostained Cells Microscopic observation of immunostained pre-B cells The pre-B leukemia cell line Nalm6 was prepared as described in Example 3. In the same manner as described above, the cells were reacted with a biotin-labeled HSL96 antibody to immunostain the cells. FITC-labeled streptavidin was reacted with a biotin-labeled HSL96 antibody, and the fluorescence was observed with a fluorescence microscope. The result is shown in FIG. The cell where the fluorescence of FITC is observed is Nalm6. As a result, the intracellular immunostaining was performed by reacting the HSL96 antibody. As a result, it was confirmed that pre-B cells could be easily detected by microscopic observation.

2. Microscopic observation of immunostained pro-B cells Pro-B leukemia cell line Nalm27 In the same manner as described above, the cells were reacted with a biotin-labeled HSL96 antibody using a saponin buffer to immunostain the cells. FIT
C-labeled streptavidin was reacted with a biotin-labeled HSL96 antibody, and the fluorescence was observed with a fluorescence microscope. FIG. 13 shows the results. FIG. 13 (A) is a photomicrograph showing the result of the reaction with the HSL96 antibody, and FIG. 13 (B) is a photomicrograph when the mouse IgG ₁ κ was reacted instead of the HSL96 antibody as a control. The cell in which the fluorescence of FITC is observed is Nalm27. With this, HSL
As a result of intracellular immunostaining by reacting 96 antibodies with pro-B cells, it was confirmed that pre-B cells could be easily detected by microscopic observation.

The results of the specificities of Examples 2 to 4 are shown in Table 2.

[0059]

[Table 2]

The HSL96 antibody of the present invention is a recombinant Vp
Since it has been confirmed that it reacts with the reB molecule alone, it is conceivable that it reacts with the VpreB molecule in pro-B cells and intracellularly. The HSL96 antibody of the present invention is capable of intracellular staining of both pre-B cells and pro-B cells, but there is no such finding with conventional antibodies. Further, the HSL96 antibody of the present invention was prepared in Example 5
As shown in the above, it is possible to observe the immunostained cells with a microscope, but there is no such finding with a conventional antibody.

[0061]

The antibody of the present invention reacts with VpreB, a light chain alternative to the human pre-B cell receptor, and does not react with λ5, but VpreB corresponds to the variable region of the light chain, and λ5
Corresponds to the constant region, and thus the antibody of the present invention has high specificity among antibodies that react with human pre-B cells.

The antibodies of the present invention enable human pre-B cells or human pro-B cells to be distinguished from human lymphocytes. Lymphocytes are separated from a sample of blood or the like of a patient suspected of having leukemia, and the lymphocytes are immunostained with the antibody of the present invention and observed with a microscope. At this time, if it is observed that the cell surface is stained, the cells can be identified as human pre-B cells, and it can be diagnosed that the patient is suffering from leukemia due to abnormal proliferation of human pre-B cells. It is possible. As described above, the antibody of the present invention enables diagnosis by observing a sample with a microscope, and thus dramatically improves the efficiency of clinical diagnosis.

When the inside of lymphocytes is immunostained with the antibody of the present invention using a saponin buffer and observed under a microscope, if the cells are observed to be It can be identified as B cells or human pro-B cells, and the patient can be diagnosed as suffering from leukemia due to abnormal proliferation of human pre-B cells or human pro-B cells.

[0064]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid sequence GGAACTCGAG CCACAAGGAC CTCTGACCCT 30

SEQ ID NO: 2 Sequence length: 32 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid sequence GGAAGCGGCC GCAGGCCTTT GGGTGGGGTC GG 32

SEQ ID NO: 3 Sequence length: 30 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid sequence GGAACTCGAG GGAGTCAGAG CTCTGCATGT 30

SEQ ID NO: 4 Sequence length: 32 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid sequence GGAAGCGGCC GCAGGGGATG CGTGCCTCTG CT 32

[Brief description of the drawings]

FIG. 1. Chimeric pre-B cell receptor (mouse μ chain / human λ5) used as an immunogen to produce the antibodies of the present invention.
/ Human VpreB).

FIG. 2 is an electrophoresis photograph showing the results of electrophoresis of an immunoprecipitate using a culture supernatant of a hybridoma selected by screening by immunoprecipitation.

FIG. 3: HSL96, anti-human κ or anti-human λ chain antibodies of the invention against a pre-B cell receptor, human IgM (human μ and human κ chains) or human IgM (human μ and human λ chains) It is a figure which shows the result of each ELISA.

FIG. 4 is a diagram showing the results of immunostaining the cell surface of each cell with the HSL96 antibody of the present invention and measuring by FACS.

FIG. 5 is a diagram showing the results of immunostaining of the inside of each cell with the HSL96 antibody of the present invention and measurement by FACS.

FIG. 6 shows the results of immunostaining of pro-B cells with the HSL96 antibody of the present invention and measurement by FACS.

FIG. 7 is a graph showing the results of immunostaining of the transformants into which the human λ5 gene or the human VpreB gene has been introduced and the human pre-B cells with the HSL96 antibody of the present invention, and the results measured by FACS.

FIG. 8 is an electrophoretic photograph showing the result of immunoprecipitation of human pre-B cells with the HSL96 antibody of the present invention and electrophoresis of the immunoprecipitate.

FIG. 9 is an electrophoretic photograph showing the results of immunoprecipitation of a human λ5 gene-introduced transformant and human pre-B cells with the HSL96 antibody of the present invention and electrophoresis of the immunoprecipitate.

FIG. 10 shows that the HSL96 antibody of the present invention provides for human Vpr
5 is an electrophoretic photograph showing the result of immunoprecipitation of a transformant into which an eB gene has been introduced and human pre-B cells and electrophoresis of the immunoprecipitate.

FIG. 11 is an electrophoretic photograph showing the result of immunoprecipitation of a transformant transfected with the human λ5 gene and human VpreB gene and human pre-B cells by the HSL96 antibody of the present invention, and electrophoresis of the immunoprecipitate.

FIG. 12. Pre-B leukemia cell line Nalm6 was transformed into HSL96
It is a microscope photograph which shows the result of having carried out the immunostaining of the inside of a cell by making it react with an antibody, and making it develop with FITC.

FIG. 13. Pro-B leukemia cell line Nalm16 was transformed into HSL9
6 is a photomicrograph showing the result of immunostaining the inside of a cell by reacting the antibody 6 with a saponin buffer and developing the color with FITC. FIG. 13 (A) shows the result of the reaction with the HSL96 antibody, and FIG. 13 (B) shows the mouse IgG as a control.
_This is the result of reacting ₁ κ.

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[Procedure amendment]

[Submission date] July 30, 1997

[Procedure amendment 1]

[Document name to be amended] Drawing

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[Correction contents]

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Claims (3)

[Claims] 1. Vpre, an alternative light chain constituting the human pre-B cell receptor expressed on the surface of human pre-B cells
B molecule and Vpre expressed in human proB cells
Reacting with the B molecule, not reacting with the alternative light chain λ5 molecule, and the reactivity of the antibody is such that human pre-B cells or human pro-B cells can be immunostained with the antibody and observed with a microscope Or an active fragment thereof. 2. Hybridoma HSL96 (Accession No. FERM P-1625 from Institute of Biotechnology, Industrial Science and Technology)
2. The antibody or active fragment thereof according to claim 1, which is a monoclonal antibody or an active fragment thereof produced by the hybridoma deposited in 1. 3. A method for detecting human pro-B cells by immunostaining human pro-B cells in a sample with the antibody or the active fragment thereof according to claim 1, wherein the human pro-B cells are detected. A method for detecting human pro-B cells, comprising using a saponin buffer when immunostaining VpreB molecules in the cells.