JPH0459879B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to monoclonal anti-G3m(21) antibodies. Research on the Gm type antigen, which is an allotype of the H chain of human IgG, has become active in recent years.
Since the discovery of G1m (1) by Grubb, there have been 20
More than one type of Gm type antigen has been discovered. this
Among blood genetic markers, the Gm type is the most useful for individual identification, paternity testing, and population genetics, and recently, the relationship between diseases and Gm has attracted attention. However, it relies on the hemagglutination inhibition reaction.
Gm type determination is not widely used in practice because it is extremely difficult to obtain specific anti-Gm serum derived from humans and blood cell-sensitizing anti-D antibodies, which are IgG bearing various Gm antigens. Recently, rabbit anti-Gm has been used to replace human-derived antiserum for routine Gm typing.
Attempts have been made to produce antiserum, but it takes a lot of time and money to produce antiserum, and there are various antigens that can be used as antigens.
Because it is difficult to collect Gm type human serum, there is a limit to the ability to reproduce antiserum, and the antibody titer of antiserum is low, so there are still problems with specificity, so it has not been put into practical use. do not have. Therefore, the present inventors developed an antibody that is specific, has a high antibody titer, and is easy to reproduce.
In order to obtain Gm antibodies, as a result of intensive research on the production of monoclonal anti-Gm antibodies produced by hybridomas, we found that monoclonal anti-G3m antibodies were produced by hybridomas.
(21) An antibody was created. Table 1 shows the results of examining the specificity and antibody titer of the antibodies obtained in the present invention.

[Table] In the presence of Gm standard serum lacking G3m (21), the antibody of the present invention strongly agglutinates red blood cells sensitized with anti-D antibody having G3m (21), and the antibody titer shows that the antibody contains When expressed as a dilution factor of mouse ascites,
It showed a large value of 1:16,384. Also,
In the presence of standard serum containing G3m (21), agglutination between the antibody of the present invention and red blood cells sensitized with anti-D antibody containing G3m (21) was completely inhibited. This result indicates that this antibody specifically reacts with G3m (21) and G1m.
(1), G1m (2), G1m (3), G3m (5), G3m
(13), G3m (15), and G3m (16). Antibodies of the present invention, i.e. [-] monoclonal anti-
The method for producing a hybridoma that produces G3m(21) antibody usually consists of the following five steps. 1. Antigen preparation 2. Immunization 3. Cell fusion 4. Selection of antibody-producing hybridomas 5. Monocloning The immunizing antigen used for the production of hybridomas producing the antibodies of the present invention is IgG3 obtained from normal human serum with G3m (21). do. IgG3 is preferably dissolved in physiological saline or a buffer solution, and administered at a dose of 1 μg to 300 μg per mouse or rat at a time. Immunization is carried out in several parts, but the first immunization is generally carried out together with an adjuvant. Freund's adjuvant, aluminum hydroxide, etc. are used as the adjuvant. Immunity is 1~
Usually, the administration is carried out at three-week intervals, and the adjuvant is not used except for the first time, and the solution is dissolved in a buffer solution, physiological saline, etc., and administered intraperitoneally or intravenously. The immunized animal is determined by the tumor cell line used for cell fusion, but rats and mice are generally used. Tumor cell lines that do not produce immunoglobulin have been established in mice.
BALB/c is often used. Two to four days after the final immunization, the lymph nodes or spleen are removed and the obtained lymphocytes are subjected to cell fusion. On the other hand, the tumor cell line used for cell fusion is P3-X63-8AZ, which does not produce immunoglobulin.
-U1, P3-NS-1, etc. are used. During cell fusion, 5 to 20 times more lymphocytes than tumor cells are used. Cell fusion is carried out using HVJ (Sendai virus) or polyethylene glycol (PEG), but generally a 40-60% solution of PEG with an average molecular weight of 1,000-8,000 is used because it is convenient to handle. In order to promote fusion, colchicine, dimethyl sulfoxide, poly-L-arginine, etc. may be added, but this is not essential. As the feeder cells, syngeneic rat or mouse thymocytes, splenocytes, etc. are used, and are added at a concentration of 0.5 to 2×10 ⁶ /ml. Antibody-producing hybridomas are selected by performing photogenic screening in the culture solution using a hemagglutination inhibition reaction or the like several weeks after cell fusion. Once a hybridoma is obtained, cloning is performed, but the cloning method is as follows:
Methods include using FACS (Fluorescent Activated Cell Sorter), picking up colonies using Soft Agar, and the commonly used limiting dilution method. Regardless of the method used, cloning is repeated two or more times to ensure a completely single clone. Monoclonal anti-G3m (21) antibodies can be produced using hybridomas obtained through these steps by either in vitro or in vivo methods, but in vivo methods are preferred. is preferable because the antibody titer is much higher. Next, the method for producing the monoclonal antibody of the present invention will be described in detail with reference to Examples. Example [1] Method for producing hybridomas producing monoclonal anti-G3m (21) antibodies 1 Preparation of antigen 35
The crude globulin was precipitated with % saturated ammonium sulfate and purified by DEAE cellulose column chromatography to obtain IgG. Furthermore, it was purified by protein A column chromatography to obtain IgG3, which was used as an antigen. 2 Immunization Add Freund's Complete Adjuvant to 100 μg of antigen and use BALB/c
The mice were injected intraperitoneally. Thereafter, 100 μg of antigen dissolved in phosphate buffer was intraperitoneally injected twice at 10-day intervals. 3 Cell Fusion Four days after the final immunization, the spleen of the immunized mouse was removed and a single cell suspension was prepared. This 1×
10 ⁸ splenocytes and 1×10 ⁷ 8-azaguanine-resistant myeloma cells NS-1 were fused using 50% polyethylene glycol (average molecular weight 6,000). Syngeneic mouse thymocytes were added to the fused cells as feeder cells, suspended in RPM1640 medium containing 15% fetal bovine serum, and placed in a 24-well tissue culture plate. After 24 hours, half of the culture supernatant was replaced with HAT medium (hypoxanthine 1×10 ⁻⁴ M, aminoprithene 4×10 ⁻⁷ M, thymidine 1.6×10 ⁻⁵ M). Until day 12 after cell fusion, 2-3
The culture medium was replaced in the same manner at daily intervals. After that, HT double base (HAT) was applied three times at intervals of 3 to 5 days.
The culture medium was replaced with a medium (without aminopuritene). 4. Selection of antibody-producing hybridomas Three weeks after cell fusion, antibody production in the culture solution was screened by hemagglutination inhibition reaction. First, add 1 drop of each culture supernatant and 1 drop of Gm standard serum to the hole of a microflocculation slide and mix.
Additionally, one drop of a 0.2% suspension of red blood cells sensitized with anti-D antibody bearing G3m (21) was added. Shake the slide at room temperature for 30 minutes.
After reacting for some time, the presence or absence of red blood cell agglutination was observed. Among all the culture supernatants, only one supernatant agglutinated sensitized red blood cells in the presence of Gm standard serum that does not contain G3m (21) and
When used in the presence of standard serum containing (21),
Aggregation of sensitized red blood cells was prevented. Therefore, it was confirmed that this culture supernatant contained anti-G3m (21) antibody. Therefore, we selected a hybridoma that produced this antibody. 5 Monocloning The selected hybridomas were cloned by limiting dilution method, dispensed into a 96-well tissue culture plate, and placed in RPMI1640 containing 15% fetal bovine serum.
Cultured in medium. Cloning was performed twice. [2] Production of monoclonal antibodies (a) In vitro method The hybridoma clone obtained in [1] was cultured in RPMI1640 medium containing 15% fetal bovine serum, and the culture supernatant was obtained. (b) In vivo method BALB/c mice were injected with pristane (2,6,10,14-tetramethylpentadecane) in advance (7 to 14 days before transplantation). Next, approximately 1×10 ⁷ hybridoma clones obtained in [1] were intraperitoneally injected into each mouse. Ascites fluid was collected 7 to 14 days after transplantation. [3] Confirmation of specificity of monoclonal antibodies and measurement of antibody titer in mouse ascites Serially dilute mouse ascites containing monoclonal antibodies, add one drop of the diluted solution to the hole of a microcuration slide, and add Gm standard serum. Add 1 drop, mix, and then add 1 drop of red blood cells sensitized with anti-D antibody containing G3m (21).
One drop of 0.2% suspension was added. After reacting for 30 to 2 hours while shaking the slide, the presence or absence of agglutination of red blood cells was observed. The results are shown in Table 1. That is, in the presence of Gm standard serum that does not contain G3m (21), sensitized red blood cells are agglutinated up to a dilution of 1:16384, and in the presence of Gm standard serum that contains G3m (21), the agglutination of sensitized red blood cells is completely inhibited. was prevented. Therefore, this monoclonal antibody is G3m (21)
G1m (1), G1m (2), G1m
(3), G3m (5), G3m (13), G3m (15), G3m
(16) was proven not to react. Also,
The antibody titer in mouse ascites was 1:16384.

Claims (1)

[Scope of Claims] 1 Produced by a hybridoma formed by fusion of mouse splenocytes previously immunized with G3m (21) type human IgG3 and cells from a mouse myeloma cell line. , a monoclonal antibody with the following characteristics: Reacts specifically with G3m (21), G1m (1), G1m
(2), G1m (3), G3m (5), G3m (13), G3m
(15), does not react with G3m (16).