JP3007120B2 - Monoclonal antibody against reverse transcriptase of human immunodeficiency virus, its production method and its antibody producing strain - Google Patents

Description

The present invention relates to a reverse transcriptase of human immunodeficiency virus (hereinafter abbreviated as HIV), which is considered to be a cause of AIDS (acquired immunodeficiency syndrome). RT) (abbreviated as RT).

[Conventional technology]

Conventionally, various antibodies have been used for diagnosis of AIDS or basic research of HIV.

From previous studies, in order for HIV to infect humans, gene products of the pol region of the HIV gene (eg, each gene product such as a reverse transcriptase region, a protease region, and an endonuclease region) are essential. It is believed that.

And, regarding RT of these gene products,
The amount of polyclonal antibody that inhibits the enzyme activity of RT
It has been shown to decrease or disappear as the stage of DS progresses (Laurence, J., Saunders, A. and Kulkosky,
J .: Science, 256 , 1501-1504 (1987), Sano, K. et al .:
J. Clin. Microbiol. 25 , 2415-2417 (1987)].

Therefore, to produce a monoclonal antibody that has reactivity to a conventionally unknown RT and can inhibit the RT activity has a very important significance in grasping the pathology of AIDS. Things.

[Problems to be solved by the invention]

An object of the present invention is to provide a method for producing a monoclonal antibody having reactivity to HIV RT and capable of inhibiting its RT activity.

[Means for solving the problem]

The present inventors have conducted intensive studies to solve the above problems, and as a result, immunized mice with a vaccinia virus capable of expressing HIV RT, and fused lymphocytes obtained from the mice with mouse myeloma cells. By culturing the hybridoma strain obtained in this manner, it has been found that a monoclonal antibody having reactivity to HIV RT and capable of inhibiting the RT activity can be obtained, and to complete the present invention. Reached.

That is, the present invention relates to (1) both reactive with p66, which is an RT of HIV, and p51 formed by processing thereof, and
A monoclonal antibody having inhibitory activity.

(2) The method as described above, wherein a mouse is immunized with a vaccinia virus capable of expressing RT of HIV, and a hybridoma strain obtained by fusing lymphocytes obtained from the mouse with mouse myeloma cells is cultured. Method for producing monoclonal antibody (3) The present invention relates to a hybridoma strain producing the above monoclonal antibody.

 Hereinafter, the present invention will be described in detail.

The monoclonal antibody of the present invention comprises HIV (for example, HIV-
1, HIV-2, etc., but preferably H
IV-1 is preferred. ) Is reactive to RT and
It is a monoclonal antibody that can inhibit RT activity. And such a monoclonal antibody, HIV R
It can be produced by immunizing a mouse with a vaccinia virus capable of expressing T as an immunogen, and culturing a hybridoma strain obtained by fusing lymphocytes obtained from the mouse with mouse myeloma cells.

When a vaccinia virus capable of expressing HIV RT is used as an immunogen as the mouse immunogen in the present invention, for example, a recombinant vaccinia virus expressing HIV RT can be mentioned.

The monoclonal antibody of the present invention has an HIV RT of about 65K.
Protein and about 51 formed by its processing
It is reactive with any of the K proteins and its R
An extract of Escherichia coli that can inhibit T activity but does not express HIV RT (supernatant obtained by crushing Escherichia coli with ultrasonic waves and centrifuging) has no reactivity.

Monoclonal antibodies having such characteristics are known as HIV
Can be produced by culturing a hybridoma strain (for example, 7C4 strain (Microtechnical Research Laboratories No. 3012) or the like) prepared using a vaccinia virus capable of expressing RT as an immunogen. The degree of inhibiting the RT activity of the thus obtained monoclonal antibody of the present invention is determined, for example, by the method of Jeffrey Laurence et al. [Science: 235 ,
1501-1504 (1987)], the method of Anthony D. Hoffman et al. [V
irology: 147 , 326-335 (1985)].
% Or more.

Such hybridomas are produced, for example, by Milste
It can be performed according to the method of in & Khler [Nature, 256 , 495 (1976)]. An outline of a preferred method for producing such a hybridoma strain will be described below in order.

Preparation of Monoclonal Antibody-Producing Hybridoma Strain (i) Preparation of Lymphocytes from Immunized Animals The method of immunizing mice was performed by vaccinia virus inserted with a gene encoding the RT region of HIV [for example, 1 to 5 ×
Buffer containing 10 ⁸ PFU / ml (for example, a solution containing a medium component such as MEM)] or RT of HIV isolated from transformed bacteria
[For example, a recombinant obtained by expressing in Escherichia coli
A buffer solution containing 10 to 800 μg / ml of RT or the like (for example, a solution containing a medium component such as MEM)] as an immunogen,
It can be performed by administering once or several times to mice at μ intervals.

Lymphocytes can be obtained from blood, lymph nodes, spleen, etc. several days after the final immunization after confirming a sufficient antibody titer of the immunized mouse, but it is preferable to obtain them from the spleen.

(Ii) Preparation of myeloma cells For cell fusion, mouse-derived MPC-11, P3-X63-Ag8
・ 653 (653), P3-X63-Ag8-U1 (P3U1), P3-NS-1
(NS-1), SP2 / 0-Ag14 (SP2 / 0) and the like, and rat-derived Emiroma cells such as 210.RC Y3.Ag1.2.3 (Y3) can be used, but 653, P3U1, NS- 1, It is preferable to use myeloma cells that do not produce and secrete antibodies outside the cell, such as SP2 / 0.

(Iii) Cell fusion Cell fusion is a cell suspension that does not interfere with cell fusion by increasing the cell number between lymphocytes and myeloma cells of the mouse immunized as described above at a ratio of (5-20): 1. Solutions, for example, commonly used media components for lymphocyte culture (IMDM,
Mix well using MEM, DMEM, McCoy, RPMI1640, etc.) solution, isotonic buffer, etc., and centrifuge the pellet (cell mass) with HVJ (Sendai virus) or P
It can be carried out by adding an EG (polyethylene glycol) solution, preferably a PEG solution, more preferably an average molecular weight of 1,000 to 8,000.
It is preferred to use a 30-60% by weight PEG solution. At this time, colchicine, dimethyl sulfoxide, poly-L-arginine and the like can be added to promote cell fusion.

As the myeloma cells used for cell fusion, those derived from an animal different from the immunized animal can be used.However, in view of the amount of antibody production and the stability of the obtained monoclonal antibody-producing hybridoma strain, It is better to use the same type of myeloma cells as the animal, and more preferably to use the same type of myeloma cells.

(Iv) Selection of hybridoma Selection of hybridoma involves selection of cells after cell fusion.
The culture can be carried out by culturing in a HAT medium (a medium containing hypoxanthine, aminopterin, thymidine, fetal bovine serum, and a commonly used medium component for lymphocyte culture).

Hybridomas are cultured by adding the number of cells suitable for searching for antibody-producing wells to each well (culture well) of a culture plate. At this time, a hybridoma growth-promoting substance or cells producing the same (eg, thymus, spleen) , Lymph node-derived lymphocytes, etc.) can be used as feeder cells, if necessary, or a lymphocyte culture medium containing IL-6 can be used.

Hybridomas selected by growing in HAT medium are HT medium (medium containing hypoxanthine, thymidine, fetal calf serum, commonly used as a medium component) until the number of cells suitable for searching antibody production wells is reached. The culture medium can be used for several days, and further cultured in a generally used lymphocyte culture medium containing fetal bovine serum.

(V) Selection of antibody-producing hybridoma The assay for determining whether the hybridoma obtained in the above (iv) is producing the desired antibody is performed by, for example, LEISA (enzyme-linked immunosorbent assay), immunoblotting, plaque Search antibody-producing wells by the formation method, agglutination method, RIA (radioisotope-based method), indirect fluorescent antibody method (IFA), etc., and examine whether the antibody inhibits HIV RT activity However, in the case of searching for antibody-producing wells when the number of assays is very large, it is preferable to first search by ELISA.

 This ELISA method is performed as follows.

The RT of HIV (eg, RT isolated from HIV, RT isolated from a transformant that produced RT (eg, Escherichia coli RT described above), a peptide partially synthesized from RT, etc.) was immobilized ( When an antibody having reactivity with HIV RT is present, the amount required for immobilization may be such an amount that the presence of the antibody can be confirmed by this ELISA.) Each well (measurement well) of the ELISA plate , A hybridoma culture supernatant is added thereto, and the mixture is allowed to stand for a certain time.

An enzyme-labeled antibody capable of reacting with and binding to an animal-derived antibody bound to each of the washed measurement wells (enzymes required for labeling include, for example, peroxidase, alkaline phosphatase, β-galactosidase, etc.) The antibody to be labeled is not particularly limited as long as it can react and bind only to the antibody derived from the hybridoma culture supernatant bound to the measurement well.
Examples thereof include serum obtained from mice, rats, rabbits, goats and the like, and monoclonal antibodies produced by hybridoma strains prepared using mouse cells and the like. ) Is added to these measurement wells and allowed to stand for a certain period of time.

Next, these measurement wells are washed, and a substrate solution corresponding to the enzyme used is added to measure the enzyme activity. And
When the enzyme activity is observed, it is understood that a hybridoma producing an antibody having reactivity with HIV RT was present in the culture well in which the culture supernatant was taken.

The search for hybridomas that are reactive with HIV RT and produce antibodies that inhibit HIV RT activity is described in Jeffrey
Laurence et al. [Science: 235 , 1501-1504 (198
7)], Anthony D.Hoffman et al's method [Virology: 147, 326
~ 335 (1985)], and [ ³ H] Deoxythymidi to DNA
It can be known by measuring the amount of uptake of ne triphoshate and searching for culture wells that inhibit the uptake.

In this way, a hybridoma that has cell proliferation, is reactive with HIV RT, and produces an antibody that inhibits HIV RT activity can be obtained.

For wells in which hybridomas producing antibodies reactive with HIV RT were present, in addition to ELISA, the HIV R was further analyzed by Western blotting.
It is preferred to reconfirm the reactivity with T.

(Iv) Straining (cloning) of hybridomas Hybridomas in culture wells in which antibody production was observed were determined by limiting dilution, single-cell manipulation (a method in which one hybridoma was placed in one well under an inverted microscope). ), A method of picking up colonies using soft agar, or a method using FACS (Fluorecent Activated Cell Sorter). At this time,
The antibody-producing hybridoma found in (v) is cultured by any one of the above-mentioned cloning methods, and the supernatant is used in the culture well in which the proliferation was observed. The same as the ELISA method performed in the selection of the antibody-producing hybridoma in (v). The antibody production well is searched by the method described above.

In this way, it is highly specific for HIV and
A hybridoma strain that produces a monoclonal antibody having a high antibody titer that inhibits the RT activity of can be selected.

Production of Monoclonal Antibody The production of a monoclonal antibody having a high specificity for HIV and a high antibody titer that inhibits the RT activity of HIV is described in (v.
It can be carried out by culturing the hybridoma strain obtained in i) in a flask or culturing intraperitoneally of an animal.

The production of the monoclonal antibody in the culture of the hybridoma strain obtained in (vi) in a flask is, for example, 0 to 20%
This can be done by culturing cells in a commonly used lymphocyte culture medium containing fetal bovine serum (eg, a medium containing medium components such as IMDM, MEM, DMEM, McCoy, and RPMI1640) until the cell concentration reaches the upper limit. it can. At this time, the monoclonal antibody is contained in the culture supernatant obtained by centrifugation.

On the other hand, the production of the monoclonal antibody in the intraperitoneal culture of the hybridoma strain obtained in the above (vi) can be performed using an animal different from the animal from which the cells used for cell fusion are derived. The method is preferably performed using an animal of the same type, and more preferably performed using an animal of the same type.

By such a method, the production of the monoclonal antibody having a high specificity for HIV RT and a high antibody titer that inhibits HIV RT activity can be performed intraperitoneally in a suitable animal such as a mouse, rat, or hamster. A substance that reduces the immunity of the animal, for example, a mineral oil such as pristane, is administered, and several weeks later, 5 × 10 ⁵ to 10 ⁷ hybridoma cell lines obtained in the above (vi) are administered, and the peritoneal cavity is intraperitoneally administered. This can be done by growing this cell line in high density in a few weeks.
At this time, the monoclonal antibody is contained in the ascites supernatant obtained by centrifugation. The antibody concentration is 10 to 1000 times the antibody concentration of the culture supernatant obtained by culturing in the flask.

The monoclonal antibody obtained by culturing the hybridoma strain in a flask or in the animal intraperitoneal cavity is subjected to salting out, dialysis, ion exchange chromatography, affinity chromatography, etc., which are applied to a general method for purifying proteins. To obtain a high-purity monoclonal antibody.

The monoclonal antibody obtained as described above is used for HIV.
Highly specific reactivity to RT of HIV (High specific reactivity to both about 65K of HIV RT protein and about 51K of protein formed by its processing) ) And inhibits HIV RT activity, but HIV RT
Does not react with an E. coli extract (supernatant obtained by crushing E. coli with ultrasonic waves and centrifuging) that does not express the E. coli.

〔Example〕

Hereinafter, the present invention will be described specifically with reference examples and examples. Note that these examples do not limit the scope of the present invention.

Reference Example 1 [Transformed vaccinia virus expressing WRT of HIV (WRR
T) and Preparation of Transformed Bacteria (ECRT)] A rabbit kidney cell line (RK13) cultured in a monolayer was inoculated with WRRT (moi: 1 PFU / cell or more), cultured for 2 days, and the cells were collected. Suspended in 1% fetal calf serum)
After thawing and thawing multiple times, sonicate and centrifuge (1500
G, 10 minutes) to obtain an immunogen supernatant. Plasmid clone pBH10 containing the HIV-1 genome [Ra
TTNER et al .: AIDS Res. Hum. Retro. 3 , 57-69 (1987)] and the following two synthetic DNA fragments: Using the method of Flexner et al. [Virology 166 , 339-349 (19)
88)] to prepare 1.68 Kb of an HIV-1 RT domain DNA fragment.

This fragment was introduced into the vaccinia virus thymidine kinase (TK) gene by the method of Flexner et al. To produce a recombinant vaccinia virus (WRRT).

The HIV-1 RT domain DNA fragment was ligated to the EcoRI I plasmid of Escherichia coli expression plasmid pKK223-3 (Pharmacia).
A plasmid pKKRT inserted into the site was prepared, and the transformed Escherichia coli having this plasmid was named ECRT.

Reference Example 2 [Preparation of ELISA antigen] The transformed Escherichia coli (ECRT) of Reference Example 1 was mixed with 250 ml of LB medium (10 g / trypton, 5 g / yeast extract, 10 g /
After shaking culture (37 ° C.) with NaCl, 100 μg / ml ampicillin) for 6 hours, isopropil-β-D was adjusted to 1 mM.
-Thio-Galactopyranoside (IPTG) and 6 more
Culture was continued for hours. Centrifuge this culture (7000G, 10
Min), collect the E. coli, wash it three times with PBS, and add 4 ml
Suspended in PBS, frozen and thawed three times, then sonicated,
By centrifuging (1500 G, 10 minutes), a supernatant as an antigen solution for ELISA was obtained. In ELISA, the supernatant was used in a carbonate buffer (1.6 g / Na ₂ CO ₃ , 2.96 g / NaHCO ₃ , 0.16 g
/ NaN ₃ ) diluted 300 times before use.

Example 1 [Preparation of a Hybridoma Strain Producing a Monoclonal Antibody Against HIV RT] (a) Immunization of Mice and Preparation of Spleen Lymphocytes The vaccinia virus WRRT of Reference Example 1 was prepared at 1.5 × 10 ⁸ PFU / ml.
Containing 50% of MEM solution (containing 2% fetal calf serum)
c mice (♀, 7 weeks old) were administered subcutaneously to both hind toes.

Six weeks after this initial immunization, a solution of vaccinia virus WRRT prepared in the same manner as above was injected into the tail vein of the same mouse at 10 minutes.
0 μ was administered.

The spleen excised from the mouse immunized as described above (excluded on the third day after the final immunization) was dissolved in a MEM solution (a lymphocyte culture medium powder in distilled water, 10 mM
HEPES (including HEPES) in a Petri dish, transfer into a freshly prepared MEM solution, and add M
Perfused with EM.

The floating lymphocytes thus obtained were suspended in a MEM solution, centrifuged (350 G, 5 minutes), resuspended in the MEM solution, and used as mouse spleen lymphocytes for cell fusion.

(B) Cell fusion A 50 ml volume of 3.75 × 10 ⁷ 8-azaguanine-resistant mouse myeloma cells (SP2 / 0) in logarithmic growth phase and 1.5 × 10 ⁸ mouse spleen lymphocytes of (a) above After placing in a plastic conical centrifuge tube, mixing, and then centrifuging the supernatant (350 G, 6 minutes), the pellet was gently tapped to loosen the pellet.

While shaking the pellet vigorously,
1 ml of a% PEG 4000 solution (37 ° C.) was added over 1 minute, and the mixture was shaken vigorously for 1 minute.

MEM solution (37 ° C) while gently shaking the centrifuge tube
Was gradually added over several minutes, finally to 10 ml, centrifuged at room temperature (120 G, 6 minutes), and the supernatant was aspirated off.

Tap the centrifuge tube lightly to loosen the pellet, and add 70 ml of HA.
T medium (1 × 10 ⁻⁴ M hypoxanthine, 4 × 10 ⁻⁷ M aminopterin, IMDM medium containing 1.6 × 10 ⁻⁵ M thymidine and 20% fetal calf serum, kept at 37 ° C.) 100 μl was dispensed into each culture well of 19 culture plates of 96 wells and cultured using a CO ₂ incubator (5% CO ₂ ,
95% air, 37 degrees, humidity 100%).

(C) Selection of hybridoma It was determined whether the antibody to HIV was contained in the culture supernatant of each well of the culture plate in which cell proliferation was observed over 2 to 4 weeks from the start of culture in (b) above. The following ELISA method was used.

First, 50 μl of the solution of Reference Example 2 was dispensed into each analysis well of a 96-well ELISA plate and allowed to stand at 4 ° C. overnight.

Then, each assay well of the ELISA plate was washed with a washing solution (0.0
After washing twice with PBS containing 5% Tween 20, 0.5% BS
A solution (dissolved in PBS) was dispensed at 100 μl into each analysis well and allowed to stand at room temperature for 30 minutes, and each of these analysis wells was washed twice with a washing solution. Was dispensed into each of the analysis wells in an amount of 50 μl and allowed to stand at room temperature for 2 hours (a negative control was obtained by culturing a mixture of mouse spleen lymphocytes and mouse myeloma cells before fusion in the same manner. On the other hand, as a positive control, a serum obtained by diluting the serum of the mouse used for cell fusion in the present invention to 10 times with a washing solution was used.)

Next, each analysis well of the ELISA plate was washed four times with a washing solution, and an alkaline phosphatase-labeled antibody solution against mouse immunoglobulin was dispensed at 5 μl into each analysis well and allowed to stand at room temperature for 1 hour. Then, after washing each analysis well of the ELISA plate four times with a washing solution, sodium p-nitrophenylphosphate · 6H ₂ O solution (1 mg / ml was added to 100 μl)
Each well was allowed to react at room temperature for 30 minutes.
The absorbance at 5 nm was measured.

As a result of these studies, production of antibodies to HIV RT was observed in 14 out of 658 culture wells in the culture plate.

For the 14 culture wells that produced these antibodies,
Immunoblotting (WB) was performed.

As a result, 10 out of 14 culture wells containing antibodies reacted with HIV RT were observed. That is, it was confirmed that hybridomas producing antibodies that react with HIV RT were present in these 10 culture wells.

(D) Hybridoma cell line formation (cloning) Using IMDM medium containing 20% fetal bovine serum, the antibody production shown in the case of the above-mentioned step (c) was confirmed.
Hybridomas were cloned by limiting dilution in eight of the ten culture wells.

For the hybridoma culture, a 96-well culture plate was used, and (1 hybridoma) / feeder cells (1 × 10 6
⁷ / ml) and 100 μl / well of lymphocyte culture medium.

In the cloning of each of the eight culture wells, the supernatant of the culture well observed as a single colony from around day 10 was collected, and ELISA using the solution of Reference Example 2 was performed.
The antibody-producing wells were screened by the method (the method similar to the above-mentioned step (c)), and at least two hybridoma strains were obtained in each cloning, and these were recloned.

Using the thus obtained culture supernatants of 14 recloned strains, it was determined whether or not those antibodies inhibit HIV RT activity by the method of Jeffrey Laurence et al. [Science: 235 , 1501].
1501504 (1987)], the method of Anthony D. Hoffman et al. [Virol
ogy: 147 , 326-335 (1985)].

RT solution (supernatant containing RT-13 of HIV-1 obtained by sonication of RK-13 cells infected with WRRT,
% Triton X-100 diluted in PBS) 10μ
And the test monoclonal antibody-containing solution (PBS) 10μ
And allowed to react at 4 ° C. for 30 minutes, followed by RT buffer [100 mM Tris (pH 8.0), 10 mM dithiothreitol,
10 mM MgCl ₂ , 100 mM KCl, 0.05 Triton X-100, 0.
6mM glutathione, 1mM ethylene glycol-bis-tetraacetic acid,] 25μ added, 2.mu. a ^{15nM 3 H-dTTP, 175μg /} m
2 μl of polyA (dT) ₁₅ , add and mix at 37 ° C.
Let it react for hours, drop it into filter paper (made of DEAE-cellulose), infiltrate it, dry, wash with 4 × SSC, wash with ethanol, dry and dry the filter paper for radiation dose (CPM)
Was measured with a liquid scintillation counter. Assuming that the radiation dose when a solution containing no antibody was added was taken as 100% RT activity (inhibition activity 0%), the degree of inhibition of the RT activity of the antibody of the present invention was measured. There were two clones, of which one clone (7C4 strain) accounted for 90% or more.

The strain obtained in this manner was changed to 7C4 strain (Microtechnical Lab.
No. 2), and the monoclonal antibody produced by this strain
Called C4.

The class, subclass, and type of L chain of the monoclonal antibody contained in the culture supernatant of this strain were determined in the following assay I, and the reactivity to various compounds was examined in assay II.

Measurement Test I [Determination of Class and Subclass of Monoclonal Antibody Against HIV RT] The determination of the class and subclass of the immunoglobulin produced by the 7C4 strain was performed by using a peroxidase-labeled antibody solution (IgG ₁ , IgG ₂ a, IgG
_{2 b, IgG 3, IgM,} IgA, κ type L-chain or λ L chain similar to the ELISA method as described above in step (c) using an antibody) labeled with horseradish peroxidase for such, and the murine antibody Antibody solution (Ig
_{G 1, IgG 2 a, IgG} 2 b, IgG 3, IgM, IgA, κ type L chain or λ
(Antibody against a type L chain or the like).

As a result, the monoclonal antibody (7C4
4) an antibody belonging to IgG ₁ (L chain was found to be kappa).

Measurement test II [Examination of reaction of monoclonal antibody to HIV RT] Regarding the reaction specificity of monoclonal antibody (7C4),
The reactivity of the IV RT with the approximately 65K protein, the approximately 51K protein produced by its processing, and the approximately 65K and 51K proteins of Escherichia coli RT were examined by Western blotting.

As a result, this monoclonal antibody was reactive with all proteins.

Example 3 Production of Monoclonal Antibody Against HIV RT in Flask Culture 7C4 obtained by culturing in IMDM medium containing 15% fetal bovine serum
The cultured cells of the strain were transferred to 10 ml of IMDM solution (without fetal bovine serum) and cultured until just before death.

Monoclonal antibody against HIV RT (7C4) was found in the supernatant obtained by centrifuging the culture (1500G for 5 minutes).
5 μg / ml (measured by ELISA).

Example 4 Production of Monoclonal Antibody Against HIV RT in Mice Intraperitoneally In order to obtain a large amount of monoclonal antibody against HIV RT, cells of the 7C4 strain were cultured in the mouse intraperitoneally.

BALB / c mice (♀, 6 weeks old, 2 weeks ago with pristane
1 × 10 ⁶ cells of the 7C4 strain suspended in PBS were administered intraperitoneally (0.5 ml intraperitoneally administered).

The weight of this mouse showed a remarkable increase from around the first week, and ascites (10 ml / animal) was collected at the second week. This ascites was centrifuged (1500G, 5 minutes) to obtain ascites supernatant.

A monoclonal antibody against HIV RT (7C4) was contained in the ascites supernatant at 8.0 mg / ml (determined by ELISA).

〔The invention's effect〕

The HIV RT of the present invention has reactivity with HIV RT
Monoclonal antibodies that can inhibit the activity
It can be expected to be used as a reagent for basic research on V, a clinical test measurement reagent for simple, rapid, and accurate diagnosis of AIDS, and a therapeutic drug.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁷ Identification code FIC12R 1:91) (72) Inventor Taizo Uda 1978 Ogushi, Obe City, Ube City, Yamaguchi Prefecture 5 Ube Industries, Ltd. Ube Research Laboratories (72 Inventor Toshiaki Kobayashi 5-1978 Kogushi, Ube City, Ube City, Yamaguchi Prefecture Inside Ube Research Institute, Ube Industries, Ltd. (72) Inventor Yasuda Yasuda 4-25-1, Shichirigahama, Kamakura City, Kanagawa Prefecture Takanori 2-26 Kajiwara, Kamakura-shi, Kanagawa Japan Zeon 2-305 Kajiwara Company Housing (56) References Vol. 7 [1] (1988) p. 239-243 Science, Vol. 235 (1987), p. 1501-1504 Aids Res. Hum; Refoviruses, Vol. 5 [1] (1989) p. 51-60 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 15/00 C12P 21/08 WPI (DIALOG) BIOSIS (DIALOG)

Claims (3)

(57) [Claims] 1. A monoclonal antibody having reactivity with p66, which is a reverse transcriptase (RT) of human immunodeficiency virus (HIV), and p51 formed by processing thereof, and having an RT inhibitory activity. 2. A monoclonal antibody is immunized by immunizing a mouse with a vaccinia virus capable of expressing HIV RT, fusing lymphocytes obtained from the mouse with mouse myeloma cells, and culturing the obtained hybridoma strain. The method for producing a monoclonal antibody according to claim 1, wherein the monoclonal antibody is obtained. 3. A hybridoma strain producing the monoclonal antibody according to claim 1.