JPH0823969A - Cell clone having human abortion-associated fetal antigen derived from b-lymphocyte strain originating from patient with bloom's syndrome and diagnosis by measuring abortion-relating fetal antibody in serum of abortion patient at early stage of pregnancy using those cell membrane proteins and abortion suppressing agent - Google Patents

Abstract

PURPOSE:To obtain a cell clone having a human abortion-associated feral antigen derived form a B-lymphocyte strain originating form a patient with Bloom's syndrome and to provide a diagnostic method for measuring the abortion-associated fetal antibody relating to the abortion at early stage of pregnancy, the judgment of the effect after immunological therapy with husband lymphocytes, and an abortion suppressing agent. CONSTITUTION:A cell clone having a human abortion-associated fetal antigen obtained by TPA treating by a B-lymphocyte from the blood of a Bloom syndrome patient with EB virus-sensitized high frequency sister chromatid exchanged character, cell surface IgM and Pre-B lymphocyte plasma cell a diagnostic method for examining the presence of the human abortion-associated fetal antibody in the serum of a woman experiencing an early-stage abortion through a fluorescent antibody technique by determining a product from the antigen- antibody reaction between this cell clone and her serum specimen, a diagnostic reagent therefrom for this antibody by allowing the cell clone to react with the serum positive to this antibody, selectively collecting only a large amount of the antigen-positive cells by the panning method to recover the cell membrane proteins, solubilzing the proteins and allowing this antigen to react with a serum to be examined according to the WB method, and a therapeutic agent based on this diagnostic method.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a cell clone having a human abortion-related fetal antigenicity derived from a B lymphocyte cell line derived from a patient with Bloom syndrome, and a method for diagnosing a fetal antibody associated with early pregnancy and abortion using the cell membrane protein thereof. The present invention relates to a method for determining an effect after immunotherapy of humoral lymphocytes and a therapeutic drug.

[0002]

2. Description of the Related Art Early abortion miscarriage (including habitual miscarriage) often occurs not only once but also in several pregnancies, and is a socially important issue including infertility. is there. It goes without saying that for almost all young men and women who get married, habitual miscarriage and infertility are very sick in terms of family design, even considering the desire to have a child. So far, the causes of this have been considered to be early pregnancy (splitting after fertilization, implantation into the uterine wall at the blastocyst stage), implantation failure due to lack of luteinizing hormone (embryonic adhesion hormone), etc. Approximately 10 to 15% of cases of premature abortion are considered to be due to lack of luteinizing hormone, and hormone treatment is also performed. However, as a result of detailed examination of 85 women who experienced premature abortion (repeated 2 to 3 times), the inventor showed that in about 70% or more cases, abortion was caused by antibodies to the fetus produced in the mother's serum. It was found to eliminate the fetal villi (placenta) that connect the uterus to the fetus.

As a method for detecting a miscarriage-related fetal antibody, the B lymphocyte cell line of a patient with Bloom syndrome established by the present inventor was treated with the humor promoter (TPA), and then the miscarriage-related fetal antigenicity that strongly appeared on the cell surface was used in the mother serum. Of the abortion-related fetal antibody and the antigen-antibody reaction, and the fluorescent antibody method for measuring the antibody in serum and the Western blot (WB) method.

[Problems to be Solved by the Invention]

[0004] In previous studies, this antigen did not react at all with normal humans, normal pregnant female serum, and cancer patient serum. Therefore, human embryonic antigenass
ociated with abortion, HEAAb). In the previous studies, 300 sera of pregnant women were examined, and as a result, in the case of normal pregnancy, there are only cases where a positive reaction by the fluorescent antibody method with fetal antigen cells derived from a patient with Bloom syndrome established by the present inventor (hereinafter referred to as BS) is observed. Since this was not done, the BS-derived fetal antigen referred to here is one that reacts only with the antibody present in the serum of a woman who has experienced post-pregnancy abortion. Of course, some miscarriages occur only once, and some are called so-called habitual miscarriages that occur repeatedly. However, one of the differences between normal pregnancy and these habitual miscarriages is chromosomal abnormality (trisomy, Cases of miscarriage caused by monosomy, abnormal translocation), other causes of miscarriage are due to blood group mismatch of parents, ie,
After the husband's genetic traits with the wife's ovum and after fertilization some kind of immune incompatibility causes the serum of the mother to act as an anti-fetal antibody against the fetus, or the chromosome is not abnormal, but the germ cells (sperm / ovum) and after fertilization The mother's B lymphocyte system was recognized because the maternal immune system recognized as a kind of strong fetal antigenicity as a kind of foreign substance in a situation where a gene in a fetal cell had some abnormality and could not occur in normal pregnancy due to it. It is thought that fetal antibodies will be produced by this, and as a result, the fetal villi that connect the mother and fetus will be eliminated, and the fetus (embryo) will not be able to feed nutrients and oxygen, resulting in fetal death (miscarriage) (placenta). To be

[0005] In this regard, the inventors of the present invention have described the abortion-experienced female serum which showed a positive reaction with BS-derived fetal antigen cells,
Immediately, aborted fetal villi (placenta) (shred as fine as possible with scissors, scalpel) and fluorescent antibody reaction (Imm-unoflu)
orescence, IF). In conclusion, in all cases where the BS-derived fetal antigen cells and serum showed a positive reaction,
A positive reaction could be confirmed between the aborted fetal villi and the serum of the person (or other aborted female serum). However, with this direct reaction method, it is very difficult to separate the cells from the villous tissue in pieces and to try the IF method without pretreatment with trypsin or the like, and it was impossible to put it into practical use. In this respect, the BS-derived fetal antigen cell according to the present invention is a free cell, and the operation in the IF method is simple and practical.

[0006] Further, in the study of treatment of miscarriage and infertility in the obstetrics and gynecology field for the last 5 to 10 years, in the case of habitual miscarriage (wife), the lymphocyte immunity of the husband (separating lymphocytes from peripheral blood, It is known to be effective in about 70% of cases of miscarriage, and the wife is known to reach normal pregnancy (childbirth) again, and is used for treatment.

In this respect also, the present inventor followed up on 30 pairs of habitual abortion couples. That is, the serum of a woman who has experienced a miscarriage is reacted with the BS-derived fetal antigen established by the inventor, and immediately after the miscarriage, after husband lymphocyte immunization (usually 2 to 3 times), 2
The same patient antibody was examined at weekly intervals. The results show that even in cases where strong antibodies were detected before immunization with husband lymphocytes, disappearance (or drastic decrease) of antibodies was confirmed after immunization with husband lymphocytes (2 to 3 times), and in 22 cases out of 30 cases. It became possible to become pregnant again and safely delivered in 15 cases (the remaining cases are currently pregnant). But,
Eight cases of antibody disappearance (or cases without drastic reduction to normal level) have not been effectively seen, and have not yet become pregnant (this is because the antibody has not completely disappeared and immunity is not yet sufficient). It is also possible that the fetal antibody disappeared after immunization of husband's lymphocytes. The aborted fetus genetically contained half of her husband's genetic traits, and some of her husband's genetic traits had an antigenic effect. In these cases, it is considered that the husband's lymphocytes were repeatedly immune-stimulated and the antibody-producing ability was lost. In the case of allergies such as hay fever, a type of immunological tolerance (immunologi) (immunologi)
It is interpreted in the same way as causing cal tolerance).

Therefore, the therapeutic method of the present invention not only serves as a standard for determining the efficacy of the conventional husband lymphocyte immunotherapy and the husband lymphocyte immune effect, but also the BS-derived abortion-related fetal antigen and maternal serum. It strongly supports the abortion-related fetal antibody. As a matter of course, the present inventor's future plan is to use an effective antigen component obtained by purification of BS-derived abortion-associated fetal antigen cell membrane protein as an immunogen, in place of the husband lymphocyte that is currently generally used for treatment. It can be considered to be applied to a treatment method for women who have experienced habitual miscarriage. If these effective antigen proteins are mass produced,
It is possible to omit the troublesome operation of separating husband lymphocytes as before. Furthermore, at the present time, only the antigen-positive cells are collected from the BS-derived abortion-related fetal antigen cells by the panning method using the latest molecular biology techniques, and the nucleic acid (DN
A), ribonucleic acid-m ribonucleic acid (RNA-mRNA) is extracted, and oligo dT primer and reverse transcription oxygen reaction are used.
The purpose of the present invention is to synthesize a S-derived abortion-related fetal antigen gene cDNA and provide a BS-derived abortion-related fetal antigen gene clone.

The present invention relates to Bloom syndrome patients (BS patients)
The B lymphocyte cell line was obtained from the blood of Escherichia coli by EB virus sensitization and TPA treatment, and as a result of diligent research on the antigenicity of the cell membrane surface, a cell clone having human abortion-related fetal antigenicity was successfully isolated. It came to completion. Accordingly, the present invention provides blood cells of BS patients with 100 high frequency SCE traits obtained by EB virus sensitization and TPA treatment.
% Cell, IgM-positive cells on a part of the cell surface, and a cell line exhibiting strong human abortion-related fetal antigenicity on the cell surface. Furthermore, the present invention uses such a cell line clone to react with an antibody in the serum of a female who has experienced early pregnancy abortion, means for diagnosing by qualitatively and quantitatively determining the antibody, and husband lymphocytes. The present invention provides a method for determining the therapeutic effect by the serum abortion-related fetal antibody titer assay after the immunotherapy using.

If a purified protein can be obtained by isolating the above-mentioned cell clone surface protein, identifying the antigen molecular weight and the amino acid sequence, it can be used as a more effective therapeutic method in place of the lymphocytes of the husband. Furthermore, it was clarified that after extracting RNA-mRNA from these cell clones, a cDNA for cloning a miscarriage-related fetal antigen gene can be synthesized by using a reverse transcriptase and an oligo dT antisense primer.

[0011]

The present invention has been made to solve the above problems, and one of the main objects of the present invention is to provide EB virus to B lymphocytes obtained from blood of patients with Bloom syndrome. Cells having the high-frequency sister chromatid exchange trait and IgM and Pre-B cell traits on the cell surface, which were obtained by sensitizing
It is to provide a cell clone characterized by having human abortion-related fetal antigenicity, which is isolated from a cell line (group) exhibiting human abortion-related fetalal antigenicity obtained by treatment with A).

Another object of the present invention is a high-frequency sister chromatid exchange trait obtained by sensitizing B lymphocytes obtained from blood of patients with Bloom syndrome with EB virus, IgM on the cell surface, and A cell clone having a miscarriage-related fetal antigenicity isolated from a cell line (s) exhibiting a human miscarriage-related fetal antigenicity obtained by treating a cell having a Pre-B cell trait with a Tumor promoter (TPA) An antigen-antibody reaction is caused by contacting serum, and the reaction product is measured by a fluorescent antibody method or the like to verify the presence of a fetal antibody associated with abortion in the serum of a woman who experienced early pregnancy abortion. The present invention provides a method for diagnosing a miscarriage-related fetal antibody.

Still another object of the present invention is to provide a high frequency sister chromatid exchange trait obtained by sensitizing B lymphocytes obtained from blood of patients with Bloom syndrome with EB virus and IgM on the cell surface. And a cell having the Pre-B cell trait as a tumour promoter (TP
A miscarriage-related fetal antibody such as female serum that has undergone early pregnancy abortion of a cell clone having a miscarriage-related fetal antigenicity isolated from a cell line (s) exhibiting human miscarriage-related fetal antigenicity obtained by treatment with A) Characterized by reacting with positive serum, collecting a large amount of only antigen-positive cells by the panning method, recovering cell membrane protein, solubilizing, and reacting the antigen obtained by the Western blotting (WB) method with the serum of the sample To provide a method for diagnosing a miscarriage-related fetal antibody.

Still another object of the present invention is to provide a high frequency sister chromatid exchange trait obtained by sensitizing EB virus to B lymphocytes obtained from the blood of patients with Bloom syndrome and IgM on the cell surface. And a cell having the Pre-B cell trait as a tumour promoter (TP
Addition of an IgM block in the fluorescent antibody method and the WB method using a cell clone having a miscarriage-related fetal antigenicity isolated from a cell line (group) exhibiting a human miscarriage-related fetal antigenicity obtained by treatment with A) To provide a diagnostic method.

Still another object of the present invention is to find a high-frequency sister chromatid exchange trait obtained by sensitizing B lymphocytes obtained from the blood of patients with Bloom syndrome with EB virus and IgM on the cell surface. And a cell having the Pre-B cell trait as a tumour promoter (TP
Antigen-antibody reaction which reacts the serum of a female who has experienced a miscarriage with a cell clone having a miscarriage-associated fetal antigenicity isolated from a cell line (group) exhibiting a human miscarriage-related fetal antigenicity obtained by treatment with A). Thus, there is provided a method for determining the effect of immunotherapy, which comprises determining the effect after immunotherapy using the lymphocytes of the husband.

Still another object of the present invention is to provide a high frequency sister chromatid exchange trait obtained by sensitizing B lymphocytes obtained from blood of patients with Bloom syndrome with EB virus and IgM on the cell surface. And a cell having the Pre-B cell trait as a tumour promoter (TP
A miscarriage-related fetal antigen cell clone isolated from a cell line (s) exhibiting human miscarriage-related fetal antigenicity obtained by treatment with A) reacts with a miscarriage-related fetal antibody-positive serum or a female serum that has experienced early pregnancy abortion RNA-m was obtained from cells in which only antigen-positive cells were separated by the panning method.
RNA is extracted and reacted with oligo dT antisense primer in the presence of reverse transcriptase to synthesize cDNA of abortion-related fetal antigen cell clone. Based on the obtained cDNA, abortion-related fetal antigen gene is analyzed and the gene is analyzed. It is to provide a diagnostic treatment method characterized by obtaining the same.

Still another object of the present invention is to incorporate the cDNA obtained by synthesizing the cDNA of the above-mentioned abortion-related fetal antigen cell clone into Taiyo bacterium and propagate it to carry the above gene. Another object of the present invention is to provide a diagnostic treatment method for analyzing a miscarriage-related fetal antigen gene, which is characterized by isolating Taiyobacterium.

Still another object of the present invention is a cell clone having a miscarriage-related fetal antigenicity derived from a B lymphocyte cell line derived from a patient with Bloom syndrome, which is located at the 9th and 16th positions. The present invention provides a remedy for abortion having the following amino acid sequence with specificity.

[0019]

[Table 2]

[0020]

The present invention will be described in detail with reference to the following examples. However, the present invention is not limited to these examples. Example 1 Obtaining B Lymphocyte Cell Line and Detecting Abortion-Related Fetal Antigenicity from Blood of Bloom Syndrome Patient; -Blood Syndrome Patient's Peripheral Blood with Peparin Blood
Aseptically assembling 15 ml in a test tube 5 ml of Ficoll pack solution
Place the sample on a plate and centrifuge at 1600 rpm for 30 minutes at room temperature to collect the band-separated mononuclear cell population (mostly lymphocytes) in a test tube. The separated mononuclear cells are subjected to R by centrifugation.
Aseptically wash with PMI 1640 medium.

For sensitization with EB virus, the isolated BS mononuclear cell population (5 × 10 ⁷ cells) was evenly divided into 2 cells.
EB for 2.5 × 10 ⁷ cells on one side
3 ml of culture supernatant of virus producer cell strain 95-8
EB virus is absorbed into B lymphocytes derived from BS patients at 37 ° C. for about 2 hours, and then EB is collected by centrifugation.
The virus supernatant is removed, and the cell pellet is mixed with RPMI 1640 medium containing 20% fetal calf serum (FCS) (total volume adjusted to 10 ml) and cultured at 37 ° C. in a 5% CO incubator.

On the other hand, the same was applied to 2.5 × 10 ⁷ cells.
B virus producer cell (B95-8) culture supernatant 3m
20% FCS + RPMI 1640 after sensitizing for 2 hours with EB virus.
When culturing in a medium (10 ml); culturing is performed while adding a chumore promoter (TPA) (12-0-tetradecanoyl phyllball-13 acetate, 10 ng / ml) to the medium and culturing for about 30 days. -B lymphocyte cell line (BS-SHY) was obtained. In establishing a cell line, it was always very difficult to establish a cell line only by sensitizing EB virus by the first method, but EB virus + T
In the PA group, the establishment of the strain was relatively quick, and as a result of intensive studies, the expression of human abortion-related fetal antigenicity described below was observed.

(A) Characteristics of cells 1. Ibnoglobulin IgM positivity is recognized on some cell surfaces. The cell surface IgM is J. Immunol. 114, 105.
8-1063, 1975, Platts-Mills et al. And J. Ex.
p. Med. 138, 1365 -1378, 1975. Human IgM, IgD, IgG,
It was examined by direct fluorescence with a FITC-labeled goat antiserum specific for IgA.

2. As is clear from the part shown in FIG.
By incorporating dR into the 2 cell generation, 100% of cells (BS-SHY, BS-SHY-HEAAb) exhibit high frequency SCE. FIG. 1 shows BS-SHY-HEAAb.
Bromodeoxyuridine (BrdU) (10 μg / m
1) Incorporation of 2) into 2 cell generation, and then preparation of chromosome sample and staining with Hoechst (2 μg / ml) (20 minutes, room temperature)
It is a micrograph figure which shows the high frequency sister chromatid exchange (SCE) (70 or more SCE is counted.) Observed by light irradiation and heat processing Giemsa method after that.

(B) Cultured cell density 5 × 10 ⁶ cells / 10 ml medium The medium is RPMI1640 culture medium containing 10% FCS after establishment.

(C) Subculture Subculture is possible without limitation. Usually 5 × 10 ⁶ cells / 10
When the cells are cultured in a ml medium at 37 ° C in a 5% CO incubator, subculture is performed every 1 to 2 days.

(D) Storage 3 × 10 ⁷ (or more) cells were sufficiently suspended in a culture medium prepared by adding 10% methyl sulfoxide (DMSO) to the medium described in (B) above, and then placed in a deep freeze at -80 ° C. After freezing for 1 day at -190
Can be stored permanently in liquid nitrogen. This BS-B lymphocyte cell line is stable in cell growth under culture conditions, and is a cell line capable of easily producing large numbers of cells.

Example 2 Cloning of BS-B lymphocytes having abortion-related fetal antigenicity by panning; -1. Fluorescent antibody method (Immunofluorescence: IF);-A method for detecting cell surface antigens, which will be described below. The detection method will be described. Pre-cultured BS-derived abortion-related fetal antigen cells (BS-SHY) (RPMI16
The number of viable cells (40% + 10% fetal bovine serum) (1.5% trypanble-staining) was counted, centrifuged (2000 rpm, 5 minutes), and the supernatant was discarded. 500 μl goat serum (5% PBS) was added to the cell pellet at 5 × 10 ⁶ cells. ), Pipette well, and suspend the suspension (500 μm).
Dispense l into plastic tubes (determine the number depending on the serum antibody to be examined) (goat blocking). After treatment with Goat Serum Blockin Cog for 30 minutes, 5 ml PBS (pH 7.2) is added to each tube, and after pipetting, centrifugation (200 rpm, 5 minutes) is performed. This operation is repeated once again and washing is performed twice. After centrifuging again (2000 rpm, 5 minutes), the supernatant was completely removed, and the patient serum prepared in advance (miscarriage patient, normal pregnancy,
Healthy people, sick people, etc. 300 μl (10 μl patient serum + 290 μl
Suspend in 1PBS, heat treatment inactivated at 56 ℃ for 30 minutes) and react at room temperature for 30 minutes (or 4 ℃ for 60 minutes) (1
Next reaction). Next, after 30 minutes, 5 ml PBS is added, well suspended, and centrifuged (2000 rpm, 5 minutes). This operation is repeated once again, and after washing twice, centrifugation (2000 rpm, 5 minutes) is performed again, the supernatant is completely removed, and 100 μl of the secondary antibody FITC joint antibody goat antihuman IgG (1 / 160 diluted PB
Suspend to S), room temperature 30 minutes (or 4 ℃, 60 minutes)
(Secondary reaction) React. Next, after 30 minutes, 5 ml PBS is added, well suspended, and centrifuged (2000 rpm, 5 minutes). After removing the supernatant, the cells are suspended with 30 μl of non-fluorescent glycerin (PBSI: glycerin 1), dropped on a slide glass, covered with a cover glass, and observed under an epifluorescence microscope.

2. Separation of abortion-related fetal antigen cells by panning method; -Panning method is a method of focusing on certain antigenicity on the cell membrane and collecting only cells having a specific antigenicity from a mixed cell population. In principle, it is almost the same as the above-mentioned fluorescent antibody method, and BS-derived abortion-related fetal antigen cells (BS-SH
Y-HEAAb) is firstly reacted with the antibody in the serum of a miscarriage patient, and then secondarily reacted on a polystyrene petri dish previously coated with IgG. Since it is the antigenic positive cells that attach, remove the non-adherent cells (cathode cells), remove the positive cells (fetal antigenicity associated with miscarriage) with a capillary pipette (the operation is performed aseptically), and after washing with PBS, It can be cultured again in RPMI 1640 + 19% FCS medium. Non-adherent cells are usually completely negative,
60% positive cells for the adherent cell population
If you expect more than above, you should pan twice (see Figures 2 and 3). FIG. 2 is a characteristic diagram showing a positive cell rate by separation of abortion-related fetal antigen cells obtained by a panning method from a TPA-treated BSB lymphocyte cell line (BS-SHY). As shown in the figure, in the cell line before panning (BS-SHY-HEAAb), 30 cases of miscarriage (immediately after) showed positive results at a frequency of about 2 to 17% with the female serum. The cell line obtained by panning once (BS-SHY-HEAAbAb ₁ increased the positive rate of 23 abortion-experienced female sera to 20-40%, and BS-SHY-obtained by panning twice). HEAAbAb ₁ Ab
_The positive rate increased by 40-60% in the _two cell lines. The sera of the remaining 7 women who had miscarriage had a positive rate of 5% or less even after panning once or twice, and did not change at all. Furthermore, healthy people (Non-Pregnancy, Non-P), normal pregnancy (Normal) Pregn
ancy, NP) Cancer patients (Cancer, Can) also had a low positive rate and the average ± 2SD level was 5% or less, so 5% or less was regarded as negative in this measurement method. Therefore,
For the following measurements, BS-SHY-HEAAbAb ₁ Ab ₂
Cells shall be used.

FIG. 3 shows BS-SHY-HEAAbAb ₁
Ab ₂ cells were blocked with 5% goat serum and then aborted female serum (Ab ₁ ) (1/30 PBS diluted, room temperature, 30
Minute) and then FITC Goat Anti-Human Ig
It is a microscope picture figure observed by the fluorescence microscope after G staining.

Example 3 Fluorescent antibody method using IgG block; -In the above-mentioned fluorescent antibody method (IF), when Goat Anti-human IgG is used as the secondary antibody,
Goat as first blocking After blocking,
Primary antibody (patient serum), secondary antibody (FITC-IgG)
Then, the fluorescent color is emitted at the end, but as a result of intensive research under various conditions so as to effectively eliminate the background occasionally seen in healthy people (especially drug users),
FIT of the same type as used for secondary antibody after goat treatment
As a result of blocking with C-IgG (X20) (concentration is preferably 8 times) and then reacting with primary antibody (patient serum) and secondary antibody (FITC-IgG (X160)), miscarriage-related Although there was no effect on fetal antibody, false positives due to some adsorbed substance in serum disappeared surprisingly, and it was established as a highly sensitive antibody detection method (see FIG. 4). New fluorescent antibody (IF) (C) method by block method and conventional IF method (A),
It is a comparison symmetry figure of the measurement result shown by comparing with (B). It is considered that this is because if there is a substance that easily adsorbs IgG contained in human serum (especially drug users, blood pressure lowering agents, etc.), it will appear as a false positive. In any case, it is a diagnostic method that overcomes the difficulties inherent in testing a complex sample of serum.

Example 4 B in the serum of a woman who has experienced a miscarriage
Measurement of S-related abortion-related fetal antibody; -BS-SHY-HE obtained by the above panning method
Using AAb ₁ Ab ₂ cells, the antibodies in the serum of miscarriage-experienced women, normal pregnant women, various cancer patients, etc. were searched by the fluorescent antibody method (IF). As shown in FIG. 2, in healthy people, normal pregnant women and cancer patient serum (1/30 PBS dilution,
As a general rule, no positive reaction was observed in the heat treatment at 56 ° C for 30 minutes). Regarding the positive range in the IF method, as shown in FIG. 2, 5% or less (mean ± 2SD) was regarded as the negative level. As shown in Figure 2, 30
Twenty-three of the aborted female sera (immediately after abortion) showed strong positive levels, while 7 showed negative levels in the cell clones after panning. For the 7 cases with negative levels, we are currently searching for other hormones, etc., and it is possible that they are due to some other cause. In any case, the fact that serum immediately after miscarriage (both of which are recurrent miscarriage) shows a positive rate of 70% or more in the IF reaction with BS-SHY-HEAAb ₁ Ab ₂ cells supports the importance of this assay. To do.

Further, 27 abortion-experienced patients (30 out of 30) who had been immunized with husband's lymphocytes were continuously examined for abortion-related fetal antibody by the IF method at intervals of 2 weeks after immunization. As shown in FIG. 5, 4 out of 27 cases showed that the postnatal abortion-related fetal antibody disappeared to a negative level, and the other showed a very low positive rate of 5.2%. In 5 of these cases, pregnancy became possible without subsequent immunization, and in subsequent investigations also gave birth by normal delivery. In addition, as shown in FIG. 5, after 6 weeks, 14 cases have reached a negative level and 15 cases have become pregnant after 6 weeks of husband lymphocyte immunization. However, 8 cases still showed a positive level (though the antibody level is decreased but the husband's lymphocyte immunity is considered to be insufficient) after 3 times of immunization, and these cases are not yet pregnant. In addition, it is noteworthy that 2 months after the miscarriage without husband lymphocytes, the antibody spontaneously disappeared 6 months later, leading to pregnancy. In these 3 cases, we tried to avoid the antigen stimulation by the sperm of the husband as much as possible by using a condom in sexual life, and this fact is also pointed out by the present inventor. Does not seem to contradict.

With the husband lymphocyte immunization method described above, 7 × 10 ⁷ lymphocytes are separated from about 30 ml of paring blood to a husband of a couple who has had a miscarriage several times, and intracutaneous inoculation of the forearm of the wife is performed. The serum was collected at 2-week intervals and tested by the IF method.

Example 5 Separation of Antigen Protein from BS-SHY-HEAAb Cell Line Having Abortion-Related Fetal Antigenicity by Panning Method and Identification of Molecular Weight of Antigen Protein by Western Blot Method; -BS-Derived Miscarriage Obtained by Panning Method Cells with related fetal antigenicity (BS-SHY-HEAAbAb ₁ Ab
₂ ) was accumulated (1 × 10 ⁸ cells), and the cells were thoroughly washed with PBS, and then Tris-Triton buffer [10 mM
Tris- Hcl, pH7.4, 150 mM NaCl, 0.5% Tr
Iton X-100, 0.2 mM phenyl methyl sulfonyl fluoride (PMSF)] is used to solubilize cell membrane surface components by treating them at room temperature for 10 minutes to 4 ° C for 30 minutes (however, the nuclear fraction is removed by high-speed centrifugation). After electrophoresis on a 4-20% gradient acrylamide gel, transfer to the Immobilon membrane by the semi-dry Western blotting (WB) method, part of which was confirmed by Coomassie staining and part of the band was confirmed on the membrane. Blocking, abortion patient serum antibody (1 /
(10, diluted with PBS) After reaction, avidin biocin (ABC)
A secondary reaction was performed with the kit, and a miscarriage-related fetal antigen band site was searched by benzidine staining. (See Figure 6).

FIG. 6 shows BS-SHY-HEAAbAb ₁
After the Ab ₂ cell membrane protein was electrophoresed and transferred to the immobilon membrane,
It is an electrophoretic (Western blotting, WB) photograph which shows WB analysis made to react with patient serum. As is clear from FIG. 6, since the 77KD band strongly reacts only with the serum of the miscarriage patient and does not react with the serum of the normal pregnant person, it was assumed that the 77KD band might be the antigen band. However, to conclude that this is really the same antigen that was found to be fluorescent in the IF method, the following absorption test is required.

In examining the specificity of the reaction between the antibody in the serum of the abortion patient and the BS-derived abortion-related fetal antigenicity, the serum antibody of the abortion patient was absorbed with the BS-SHY-HEAAb ₁ Ab ₂ antigen cells (antigen-antibody reaction). It is necessary to test the presence or absence of the antibody by the IF method, and further, in the WB method, it is necessary to examine whether or not the assumed 77KD band disappears completely or whether it has any effect on other bands. First, BS-SHY
-HEAAbAb ₁ Ab ₂ cells (5 × 10 ⁷ cells) in PBS
Rinse 3 times with P.C., completely remove fetal bovine serum and the like from the culture medium, accumulate 5 × 10 ⁷ cells in a Subitz test tube, and
The cells were suspended in a patient serum diluted to 1/10 with BS (female who had a miscarriage) at a volume ratio of 1: 1 and incubated at 37 ° C for 1 hour and at 4 ° C for 24 hours, followed by centrifugation (2000 rp).
m, 10 minutes, 4 ° C) separation and collect the supernatant (serum).
The recovered serum was assayed by the IF method and the WB method, but it showed no fluorescence by the IF method as compared with the untreated serum,
By law, only the 77KD band disappeared. Since the other bands were not affected as shown in FIG. 6, it was concluded that the 77-KD WB band was a miscarriage-related fetal antigen. In addition, as an absorption test, 5 × 10 ⁷ normal serum B lymphoid cell lines derived from BS (without any miscarriage-related antigenicity) were collected from patient serum, and absorption was performed under the same conditions as above. It was confirmed that the serum remained positive by the IF method and the 77KD band was not affected by the WB method.

Example 6 Amino Acid Sequence of Abortion-Related Fetal Antigen; -The 77KD antigen band site identified by the WB analysis in the preceding paragraph was cut out from the immobilon membrane and subjected to a microsequencer to examine the amino acid sequence. The results were able to identify 16 amino acid residues (N-terminal) as shown in FIG.
As a result of searching homology (Homology) of these 16 residues using literatures and computers, it was found that the end (Frame work) and No. 9 of human immunoblobulin VHIII.
Except for 16 amino acids and others were found to be homologous,
It was found to be a point mutation of No. 9 and 16 amino acids. This fact suggests that recently, instead of the husband's lymphocyte immunity, mixed intravenous immunoglobulin large dose method extracted from blood products is effective for preventing miscarriage (M
uller-Eckhardt G. et al. (1991) Lancet. 337: 424-42
There is 5.), but this is a state in which immunoglobulin is mixed, and it is completely unknown which component. further,
Since it is taken out from a large amount of blood, it is expensive and there is concern about infection such as AIDS. Considering these points, the present invention seems to be important. That is, immunoglobulins from blood products are not pure products but are mixed products. In this respect, the present inventors' invention is IgVHIII.
It has been discovered that this is a point mutation only for framework Nos. 9 and 16, which will open the way for future diagnosis and therapeutic drug development.

Example 7: Miscarriage-related fetal antibody homogeneity; -It is necessary to describe the homogeneity of antibodies in the serum of women who have experienced miscarriage. As mentioned in the previous section, in WB analysis, 24/32 cases
Considering that there is commonality in the band region of one 77KD band, and that after the absorption reaction between the patient serum and BS-derived fetal antigen cells, only the antibody reactive with the 77KD band disappeared from these positive sera The antibody in the serum can be considered to be the same even if the difference is different. However, to further confirm this fact, as shown in FIG.
The BS-derived fetal antigen cells were reacted with serum antibodies once, further washed with PBS, and then the same serum or different patient sera was subjected to so-called antigen-antibody reaction twice to perform a detailed search. As shown in the first column of FIG. 8, when the BS fetal antigen cells were reacted once with different patient sera, a positive reaction was observed by the fluorescent antibody method, but as shown in the second column, the antibody-positive serum was detected. After reacting once with IgG, after blocking IgG, the positive serum was reacted again, and FITC-anti-humma
As a result of searching by the IF method with n IgG, when the completely positive serum was reacted in the first reaction, all were negative even when the same or different positive serum was reacted in the second reaction. Meanwhile, initially subjected to husband lymphocytes immunized Ab ₁ patients were potent positive, after IgG block reacted not yet completely negative way immunizing (weakly positive sera) Ab ₁ the first time, two more time Positive serum (Ab
₁ Ab _{2 ---} ), a positive reaction was shown this time. From these results, it was confirmed by the present inventors that the antibodies detected by the IF method and the WB method from the serum of women who have experienced miscarriage are the same.

Example 8 cDNA from BS-B lymphocyte cell line having abortion-related fetal antigenicity by panning method
Synthetic (for miscarriage-related fetal antigen gene clone);-Cells (BS-SHY-HEAAb) having BS-derived miscarriage-related fetal antigenicity obtained by the panning method are accumulated (5 x 10 cells), and cells and PBS are accumulated. After washing twice with
RNA separation kit (Toyobo) and mRNA separation kit
(Toyobo) is used to extract mRNA from total RNA. The total amount of RNA obtained by these kits was 173 μg /
It was 100 μl.

Next, dNTP was added to this mRNA (3 μl).
(DGTB, dATP, dTTP, dCTP (350 μ
m), rTth DNA polymerase (2 μl, 5 Un
Its / 20 μl) and oligo dT primer (1 μl) are mixed and a PCR (Polymerase chain reaction) reaction (25 ° C, 10 minutes, -42 ° C, 10 minutes, -70 ° C, 2 minutes 30 seconds) is performed to detect miscarriage-related antigen genes. The cDNA was synthesized.

[Brief description of drawings]

BRIEF DESCRIPTION OF THE FIGURES FIG. 1. Brom in BS-SHY-HEAAb cells.
Incorporation of odeoxyuridine (BrdU) (10 μg / ml) into 2 cell generations, preparation of chromosome samples, staining with Hoechst (2 μg / ml) (20 min, room temperature), light irradiation, heat treatment Giemsa method Chromatid exchange (SC
E) (SCE of 70 or more are counted).

FIG. 2 is a characteristic diagram showing the positive cell rate by separation of abortion-related fetal antigen cells obtained by panning from a TPA-treated BSB lymphocyte cell line (BS-SHY).

FIG. 3 shows BS-SHY-HEAAbAb ₁ Ab.
After blocking 5% goat serum on ₂ cells, reacting with aborted female serum (Ab ₁ ) (1/30 PBS diluted, room temperature, 30 minutes), and then FITC goat anti-human IgG
It is a microscope picture observed by the fluorescence microscope after dyeing.

FIG. 4 is a new fluorescent antibody (IF) (C) method based on the IgG block method and conventional IF methods (A) and (B).
It is a comparison symmetry diagram of the measurement result which compared and was shown.

FIG. 5 shows BS-SHY-HEAAbAb ₁ Ab.
_{Using 2} cell lines, 27 out of 30 cases of abortion-experienced patients (female) were immunized intra-dermally with the husband's lymphocytes 2-3 times every two weeks, and abortion-related fetal anti-serum in serum was immunized. It is a characteristic view which measured the antibody titer.

FIG. 6 shows BS-SHY-HEAAbAb ₁ Ab.
₂ is an electrophoretic (Western blotting, WB) photograph showing WB analysis in which a cell membrane protein was electrophoresed and then transferred to an immobilon membrane and then reacted with patient serum.

FIG. 7: 77KD on the immobilon membrane in FIG.
It is a HEAAb antigen amino acid sequence diagram which cut out only the band part of a band part, and analyzed the HEAAb antigen amino acid sequence with the microsequencer.

FIG. 8 shows the same antigen cells BS-SHY-HEAA.
It is a schematic diagram which shows that the antibody in the serum of women who have experienced a miscarriage is the same due to the two reactions in which b is subjected to the antigen-antibody reaction twice.

 ─────────────────────────────────────────────────── ─── Continuation of front page (54) [Title of the Invention] Cellular clones with abortion-related fetal antigenicity derived from B lymphocyte cell lines derived from patients with Bloom syndrome and early pregnancy abortion using those cell membrane proteins Diagnostic method and abortion remedy by measuring fetal antibody related to abortion in patient serum

Claims (8)

[Claims] 1. B obtained from blood of a patient with Bloom syndrome
High-frequency sister chromatid exchange trait obtained by sensitizing EB virus to lymphocytes, IgM and Pre on the cell surface
-Human abortion-related fetal antigenicity isolated from a cell line (s) exhibiting human abortion-related fetal antigenicity obtained by treating cells having a B-cell trait with the Tumor promoter (TPA) Cell clone. 2. A high frequency sister chromatid exchange trait obtained by sensitizing EB virus to B lymphocytes obtained from blood of a patient with Bloom syndrome, IgM and P on the cell surface.
A cell clone having a miscarriage-related fetal antigenicity isolated from a cell line (s) exhibiting a human miscarriage-related fetal antigenicity obtained by treating a cell having a re-B cell trait with a Tumor promoter (TPA) An antigen-antibody reaction is caused by contacting serum, and the reaction product is measured by a fluorescent antibody method or the like to verify the presence of a fetal antibody associated with abortion in the serum of a woman who experienced early pregnancy abortion. Method for diagnosing abortion-related fetal antibody. 3. A high-frequency sister chromatid exchange trait obtained by sensitizing EB virus to B lymphocytes obtained from the blood of a patient with Bloom syndrome and IgM on the cell surface.
And a cell clone having a miscarriage-related fetal antigenicity isolated from a cell line (s) exhibiting a human miscarriage-related fetal antigenicity obtained by treating a cell having a Pre-B cell trait with a Tumor promoter (TPA) Reacted with abortion-related fetal antibody-positive sera such as female sera that experienced premature abortion, and collected a large amount of only antigen-positive cells by the panning method to recover cell membrane proteins, solubilized, and obtained by Western blotting (WB) method. A method for diagnosing a miscarriage-related fetal antibody, which comprises reacting the antigen with the serum of a specimen. 4. A high frequency sister chromatid exchange trait obtained by sensitizing B lymphocytes obtained from blood of a patient with Bloom syndrome with EB virus, and IgM on the cell surface.
And a cell clone having a miscarriage-related fetal antigenicity, which is isolated from a cell line (s) exhibiting a human miscarriage-related fetal antigenicity, which is obtained by treating a cell having a Pre-B cell trait with a Tumor promoter (TPA) The diagnostic method according to claim 2 or 3, wherein an IgG block is added to the fluorescent antibody method and the WB method. 5. A high-frequency sister chromatid exchange trait obtained by sensitizing B lymphocytes obtained from blood of a patient with Bloom syndrome with EB virus, and IgM on the cell surface.
And a cell clone having a miscarriage-related fetal antigenicity isolated from a cell line (s) exhibiting a human miscarriage-related fetal antigenicity obtained by treating a cell having a Pre-B cell trait with a humor promoter (TPA) and a miscarriage A method for determining the effect of immunotherapy, which comprises determining the effect after immunotherapy using the lymphocytes of the husband by an antigen-antibody reaction that reacts with the serum of a woman who has undergone. 6. A high-frequency sister chromatid exchange trait obtained by sensitizing EB virus to B lymphocytes obtained from blood of a patient with Bloom syndrome, and IgM on the cell surface.
And a miscarriage-related fetal antibody cloned from a miscarriage-related fetal antigen cell clone isolated from a cell line (s) exhibiting human miscarriage-related fetal antigenicity obtained by treating cells having the Pre-B cell trait with a humor promoter (TPA) Reactive with positive sera or female sera that experienced early pregnancy abortion, RNA-mRNA was extracted from cells in which only antigen-positive cells were separated by the panning method, and reacted with oligo dT antisense primer in the presence of reverse transcriptase to cause miscarriage. A diagnostic and therapeutic method comprising synthesizing a cDNA of a fetal antigen cell clone, analyzing a miscarriage-related fetal antigen gene based on the obtained cDNA, and obtaining the gene. 7. A method wherein the cDNA obtained by synthesizing the cDNA of the abortion-related fetal antigen cell clone is incorporated into Taiyo bacterium and propagated to isolate Taiyo bacterium having the gene. The diagnostic treatment method for analyzing the abortion-related fetal antigen gene according to claim 6. 8. A cell clone having a miscarriage-related fetal antigenicity derived from a B lymphocyte cell line derived from a patient with Bloom syndrome, which has the following amino acid sequence with specificity at positions 9 and 16: A miscarriage remedy. [Table 1]