JPH0387200A - Monoclonal antibody - Google Patents

Abstract

NEW MATERIAL:A monoclonal antibody specifically recognizing the propeptide (profragment) of human prorenin. USE:A reagent for immunologically measuring or purifying the human prorenin. PREPARATION:For example, a human prorenin propeptide synthesized by a solid phase method is mixed with a Freund's perfect adjuvant and administered into the abdominal cavity of a BALB/c mouse to immunize the mouse. Three days later, splenic cells are aseptically collected and subsequently fused with the myeloma cells of a mouse, followed by culturing the fused cells in a HAT medium to multiply only the hybridoma. The hybridoma is identified by an ELISA method to screen a clone producing the desired antibody, which is cloned, followed by culturing the single clone to provide the monoclonal antibody.

Description

[Detailed description of the invention] [Industrial application field]

The present invention relates to a novel monoclonal antibody useful for quantifying human prorenin or its pro-fragment and for producing these '4n.

[Prior Art and Problems to be Solved by the Invention] Prorenin is a precursor of the enzyme renin, which is involved in blood pressure regulation and electrolyte balance. The content of prorenin in the blood is
They are much more abundant than other tissues, and their biochemical and physiological functions have been of great interest. Using conventional techniques, it is extremely difficult to create antibodies that specifically recognize only prorenin, and until now it has not been possible to quantify only prorenin or its propeptide or to observe its presence in living tissues. It was impossible. Furthermore, since purification of prorenin requires complex biochemical operations, there has been a desire to develop a simple and rapid method.

[Means to solve the problem]

According to the present invention, a monoclonal antibody having specific reactivity to human prorenin is provided. Further, by using the antibody of the present invention, a means for immunologically purifying human prorenin and a means for immunologically measuring it are provided. The greatest feature of the antibody of the present invention is that it has specific reactivity to human prorenin, that is, it has specific binding property, and the antibody has 43+lII amino acids constituting the propeptide of human prorenin. Includes several monoclonal antibodies that specifically bind to different portions of. The method for producing the antibody of the present invention will be described in detail below. The antibody of the present invention is produced by fusing plasma cells (immune cells) of a mammal immunized with human prorenin propeptide consisting of 43 amino acids as an immunizing antigen with mammalian plasma cell lung cells.
), and from this, a clone that produces an antibody that specifically recognizes only the human prorenin is selected and obtained from this clone. In the production of the antibody of the present invention, it is not necessarily necessary to use a purified preparation of human prorenin propeptide.
It is also possible to use crude products containing this. In the above method, the immunizing antigen, namely human prorenin.
The borehole animal to be immunized with the propeptide is not particularly limited, but it is preferably selected in consideration of compatibility with the plasmacytoma cells used for cell fusion, and mice, rats, etc. are generally advantageously used. be done. Immunization is carried out by a conventional method, for example, by intraperitoneally or intravenously administering the human prorenin propeptide to the borer animal. More specifically, human prorenin propeptide was dissolved in physiological phosphate buffer (PBS).
) or physiological saline to an appropriate concentration, and administer this to animals several times, using a conventional adjuvant as necessary. As the immune cells, it is preferable to use tile cells extracted about 3 days after the final administration of the human prorenin propeptide. As the other parent mammalian plasmacytoma cells to be fused with the immune cells, various known cell lines, such as P3 in mice, can be used.
(P3/X63-8gB) (Nature256,49
5-497 (1975)), P3-Ul (Curren
Topics in Microbiology and
Tmmunology 81+1-7 (1987))
, NS] (Eur, J, Immu Shidan ■ 6.511-519 (1976)), SP 2/
O (Nature 276.269-270 (1978
)), FO (J. Immunoll, Meth, 35.1-21(1
980)), X63-6. 5. 3 (J, I
mmunol, 123154B-1550 (1979
)), S 194 CJ, Exp, Med, 1
48.313-323 (1978)], and R 210 in rats (Nature 277.131-
133 (1979)) are used. The fusion reaction between immune cells and plasma cells is basically carried out by known methods such as the method of Milstein et al.
thods Enzymo+, 73.3-46 (19
81)) etc. Specifically, the fusion reaction is carried out in a conventional nutrient medium, eg in the presence of a fusion promoter. Commonly used fusion promoters, such as polyethylene glycol (PEG) and Sendai virus (HVJ), are used, and auxiliary agents such as dimethyl sulfoxide (DMS○) are added to increase the fusion efficiency. You can also do that. The mixing ratio of immune cells and plasmacytoma cells is the same as in the usual method, for example, type 'f7a111 cell lI! For l111I cells,
Approximately 1 to 10 times as many immune cells may be used. The medium used for the fusion may be, for example, RPMI-1,640 medium or ME used for the proliferation of the plasmacytoma cell line.
Various media commonly used for culturing this type of cells, such as M medium, can be used, and it is usually advisable to exclude serum supplements such as fetal bovine serum (FO3). Fusion involves thoroughly mixing predetermined amounts of the immune cells and plasmacytoma cells in the medium;
This is carried out by adding a PEG78 solution preheated to about 37° C., for example, one having an average molecular weight of about 1000 to 6000, to a normal medium at a concentration of about 30 to 60% (w/v) and mixing. Thereafter, desired hybridomas are formed by repeating the steps of sequentially adding an appropriate medium, centrifuging, and removing the supernatant. Isolation of hybridomas is carried out by culturing them in a standard selection medium, such as a medium containing hypoxanthin, aminopterin, and thymidine (HAT medium). M
Culture in AT medium may be carried out for a sufficient period of time, usually from several days to several weeks, to kill cells other than the target hybridoma (unfused cells, etc.). Regarding the hybridoma thus obtained, a strain producing the antibody of interest is searched for and single cloned according to the usual limiting dilution method. The search for the producing strain is performed according to various methods generally used for detecting antibodies, such as ELISA method, plaque method, spot method, agglutination method, Ophthalony method, and radioimmunoassay method. It should be noted that a human prorenin propeptide preparation can be preferably used as the antigen used in the search for the antibody. The thus obtained hybridoma producing the desired monoclonal antibody can be subcultured in a conventional medium and can be stored for a long period of time in liquid nitrogen. The monoclonal antibody of the present invention can be collected from the hybridoma by culturing the hybridoma according to a conventional method and using the culture supernatant, or by administering the hybridoma to a compatible borer animal and growing it, and obtaining it as ascites. Masahiro et al. were hired. Furthermore, the antibody obtained as described above can also be purified by conventional purification means such as salting out method, gel filtration method, and affinity chromatography. Human prorenin can be easily and specifically purified by using the thus obtained antibody of the present invention in conventional immunological purification means such as immunoprecipitation or affinity chromatography. Furthermore, according to the use of the antibody of the present invention, radioimmunoassay (
Human prorenin can be easily measured with high sensitivity, high accuracy, and high specificity by conventional immunological means such as RIA), enzyme-linked immunosorbent assay (ELISA), and agglutination method.

【Effect of the invention】

According to the present invention, a specific antibody for human prorenin is provided. By using this antibody, it is possible to purify human prorenin by immunological purification means, and it is extremely useful in its production. Furthermore, according to the use of the antibody of the present invention,
An immunological measurement method for human prorenin is provided, which will clarify the relationship between human prorenin and various diseases, and provide clinically useful findings.

【Example】

Examples will now be described to explain the invention in more detail. 1) Synthesis of human prorenin propeptide Stewae
(Solid Phase Peptide 5y
nthesis (1984), Pierce Che
micalCo, Publ, Rockford,
IL (1984) ) and Yelton et al. (Cu
rrent Topics in Microbiolo
gy and Tmmunology 8L 1-7 (
1978)), eight pplied Blo
- Phenyl acetamido methyl (PAM) using a 5ystems 430A automatic peptide synthesis machine
The base was prepared using a solid phase method on a resin. The crude propeptide is
Purification was performed using a high performance liquid chromatograph equipped with a Vydac RP-C4 column. The amino acid sequence of the propeptide was confirmed by Edman degradation. 2) Production of hybridoma The human prorenin propeptide obtained by the above method was dissolved in physiological saline to a concentration of 0.1 mg/ml, mixed with an equal volume of Freund's complete adjuvant, and suspended. The human prorenin propeptide 0 of the resulting suspension
．． 005 ~ 0.0 T ■ content, 6 ~ 8 week old BALB
/c strain mice were intraperitoneally administered. Approximately 6 weeks later, the immunized animals were treated with human prorenin propbutide in saline at the same concentrations as above, 0.005-0.
The amount containing 01■ was administered intraperitoneally, and 3 days after administration, the lungs were aseptically removed from the immunized animals. Next, the cells were disaggregated into individual cells using a mesh and washed three times with RPMI-1640 medium. Mouse bone IF in the logarithmic growth phase, cultured for several days in the presence of 8-azaguanine to completely eliminate revertants! tumor cell line X63 Ag8 6. 5. 3 (J, Imm
unol. 123.1548-1550 (1979)) in the same manner as above, and 1 x 107 of the lung cells and 1 x 10' of the lung cells.
were mixed in a 50rd centrifuge tube. 200Xg, 5
After centrifugation, the supernatant was removed and PE015 was kept at 37°C.
40 (Wako Pure Chemical) RPMI-1640 containing 40%
Cell fusion was performed by adding 1 mB of medium. After removing PEG from the fused cells by centrifugation,
RPM containing hypoxanthine, aminopterin and thymidine (hereinafter abbreviated as HAT) using a well plate.
Only hybridomas were grown by culturing in L-1640 medium for 1 to 2 weeks. Thereafter, the clones were grown in a HAT-free medium, and after about 2 weeks, clones producing the desired antibody were selected from Test Example 1. A hybridoma producing the monoclonal antibody of the present invention having the desired reaction specificity was obtained. Rabbit anti-mouse Ig Eve antibodies (Miles)
The results of the isota monoclonal antibody 2-X-Cl, which is a monoclonal antibody produced by these hybridomas, are shown by the double immunodiffusion method using. Test example 1. Testing of antibody reactivity by ELISA The human prorenin propeptide used as the immunizing antigen was
20mM) Lis-HCl buffer containing 150mM sodium chloride and 0.02% sodium azide pJ (7,5(
TBS) to a concentration of 0. O]~0. 05mg
/d was added to each well of a 96-well plate and allowed to adsorb at room temperature for 1 hour. After the reaction, wash 3 times with TBS and add 1% TBS containing 1% bovine serum albumin (BSA).
Add 0.2 mR per well and treat at room temperature for 1 hour. Next, TBS (TT
0.05 mf of each of the crude or purified monoclonal antibodies diluted with BS) was added to the wells and allowed to react at room temperature for 1 hour. After the reaction, wash 3 times with TTBS, and then wash with TTB.
0.05 ml of alkaline phosphotase-conjugated anti-mouse IgG antibody (Jakson) diluted 1000 to 5000 times with S was added per well, and reacted at room temperature for 1 hour. Next, unreacted antibodies were thoroughly washed away with TTBS,
p-nitrophenyl phosphate solution (Bioland) 0.05 mj! Add anti-kon, sase, wave r:z 40
The absorbance at 5 tlm was measured to determine the reaction of the monoclonal antibody. Test example 2. Demonstration of antibody reaction specificity by Western blocking method This experiment was carried out by Towbin et al. (Proc. Na
tl, 8cad, sci, [ISA 76.435
0-4354 (1979)). That is, after electrophoresing the culture supernatant containing purified human renin or human prorenin by electrophoresis using sodium dodecyl sulfate (SDS-PAGE method), the gel is brought into close contact with a nitrocellulose membrane, and the gel side is A current was applied using the cellulose side as the cathode and the cellulose side as the anode, and the proteins migrated on the gel were transferred to the nitrocellulose membrane. After blocking and washing the transferred nitrocellulose membrane by immersing it in TBS containing 1% BSA overnight at room temperature, the monoclonal antibody prepared from the hybridoma obtained in 2) above was reacted as the primary antibody for another night. Ta. After the reaction, the membrane was thoroughly washed, an anti-mouse IgG goat antibody labeled with peroxidase was added as a secondary antibody, and the reaction was carried out for 2 hours in TBS containing 1% BSA. The nitrocellulose membrane was then washed with 0.2M sodium chloride solution and 0.2M sodium chloride solution was used.
1% 4-chloro-1-naphthol solution and 0.02%
Color was developed using an aqueous hydrogen peroxide solution. As a comparison,
Using a polyclonal antibody (rabbit) against human renin, in this case instead of anti-mouse IgG goat antibody,
An anti-heron IgG goat antibody was used. 3) Large amount of monoclonal antibody in gN The following method was used to prepare a large amount of monoclonal antibody from the hybridoma obtained in 2) above. (1.) Cell culture method The clones obtained in 2) above were incubated at 37°C in a 5% carbon dioxide incubator in RPMT-1640 medium.
Cultured for 8 hours. Next, the culture solution was incubated at 1500×g for 10
A culture supernatant containing the monoclonal antibody of interest was obtained by performing the following steps. (2) Method using mouse ascites From the clones obtained in 2) above, 1 x 106 cells were preliminarily transferred to Brisken (Aldrich Chemi).
Cals Inc.) was administered intraperitoneally to BALB/c strain mice that had been inoculated with the following. After 10 to 14 days, the accumulated ascites was collected to obtain ascites containing the antibody of the present invention. one

[Brief explanation of drawings]

Figure 1 is an explanatory diagram showing the amino acid sequence of the human prorenin propeptide (profragment) and the recognition site of the monoclonal antibody of the present invention.
FIG. 2 is an explanatory diagram showing the specificity of the present invention using a plotting method.

Claims (1)

[Claims] Human prorenin propeptide (profragment)
A monoclonal antibody that specifically recognizes.