JPH066068B2 - Monoclonal antibody against human IgG subclass, method for producing the same, and monoclonal antibody-producing cell line - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION The present invention relates to a monoclonal antibody HG against human IgG ₂ .
2-56F, a hybridoma producing the monoclonal antibody, and a method for producing the monoclonal antibody. The monoclonal antibody of the present invention is useful as a clinical test agent or a ligand for affinity chromatography.

2. Description of the Related Art IgG of human serum immunoglobulin has a variable region with almost constant amino acid sequence and a constant region with almost constant amino acid sequence.
The variable region is involved in binding to various antigens, and the constant region is involved in various biological actions. The amino acid sequence in the constant region is almost constant, but it can be reduced to 4
Species subclass IgG1, IgG2, IgG3 and I
It is classified as gG4. Concentration in normal adult serum (mg / ml)
Is 5-12 for IgG1, 2-6 for IgG2, IgG3
Is 0.5-1 and IgG4 is 0.2-1.

Examples of reports on physicochemical properties of human IgG subclasses are as follows. (Table 1).

IgG has low electric mobility and is difficult to be decomposed by pepsin.
IgG2 has four S—S bonds between H chains and is resistant to degradation by proteolytic enzymes. Since IgG3 has a repeated structure in the hinge region, it has as many as 11 S—S bonds between H chains, has a short half-life in serum, and is easily affected by proteolytic enzymes.

IgG4 has the highest electric mobility.

The biological properties of the human IgG subclass also differ depending on the subclass, as shown in Table 2 as a report example.

That is, IgG4 does not bind complement C ₁ q and IgG4
Rheumatoid factor and protein A do not bind to 3. In addition, the binding to human or xenogeneic cells also differs.

In addition, a deviation in subclass distribution has been reported for IgG as an antibody against various antigens (Table 3).

HL Spregelberg, Advances in Immunology (Adv. Immunol.) 19 , 259 (1974), that is, the distribution of IgG subclasses for complex protein antigens such as tetanus toxoid, diphtheria toxoid, and thyroglobulin in serum abundance ratios. Although similar, only IgG2 was obtained for the antigens of carbides such as dextran and teichoic acid, and Ig2 was also obtained for self-antigens.
A bias is seen in the distribution of the G subclass.

As described above, it is expected that the subclasses of IgG having different constant regions involved in various biological actions are involved in various biological functions and diseases.

Problems to be Solved by the Invention So far, human IgG subclasses have been studied using sera of myeloma patients belonging to any IgG subclass, but IgG subclasses obtained from a single sera of myeloma patients In addition to its subclass nature, its IgG
Since it also has properties peculiar to the molecule (variable range and allotype), not only properties common to subclasses can be obtained. The same applies to Ig obtained from a single myeloma patient serum
The same can be said of the specificity of the antibody obtained by immunizing the G subclass. In addition to the specific antibody activity of the subclass, the antibody activity specific to the variable region also coexists.

To solve this problem, a large number of myeloma patient sera belonging to the same IgG subclass are used, but this is possible only in a limited number of facilities. Robe et al., Monoclonal anti-human IgG subclass antibody [J. Lowe et al., Immunology 47 , 329 (1982)]
Although the monoclonal antibody of the present invention has been prepared,
At least their reactivity is different. “Means for Solving Problems” In the present invention, an object of the present invention is to obtain 4 types of polyclonal human IgG subclasses from normal human pool serum, and to use them to obtain monoclonal antibodies specific to each subclass. By making the best use of the differences in the physicochemical properties of IgG subclasses reported up to now, polyclonal IgG subclasses were separated from human pool serum as much as possible, and each IgG was enriched in each subclass.
Get subclass crude product. Next, immunize the crude product, prepare a large number of monoclonal antibodies by the cell fusion method, select clones that strongly bind to each subclass or do not bind to a certain subclass, and immobilize those monoclonal antibodies. To make an affinity column,
This time, by combining this affinity column, Ig
Recognize the antigenic determinants unique to each subclass by further purifying the G subclass and using the obtained polyclonal human IgG subclass purified product to more strictly test the subclass specificity of the already obtained monoclonal antibody. This is a selection of monoclonal antibodies. In the present invention, several kinds of monoclonal antibodies against the same IgG subclass can be obtained, but as shown in the following Examples and Reference Examples, at least their reactivity is different.

By using the monoclonal antibody obtained this time,
Since IgG subclass in human serum can be detected, it is useful as a clinical test drug, and the relationship between various diseases and IgG subclass can be studied. Furthermore, by separating and purifying the polyclonal human IgG subclass by affinity chromatography using this monoclonal antibody, their physicochemical and physiological properties can be studied in more detail.

1) Separation of 4 kinds of polyclonal human IgG subclass 1st method (preparation of crude product) Human pool serum is salted out to collect a γ-globulin fraction.
The γ-globulin fraction was mixed with 0.01M phosphate buffer (pH 8.
When it is passed through a DEAE ion exchange column in 0), IgG4 is adsorbed. The flow-through fraction of the DEAE column was added to the Protein A- Sepharose column, Duhamel et al's method [RC Duhamel (Duhamel), et al., The Journal of Immunological Mesozzu (J.Immunol.Methods) 31, 21
1 (1979)], polyclonal human IgG1, IgG
A crude product of 2 and IgG3 is obtained. That is, the crude polyclonal human IgG3 product is 0.1M phosphate buffer (pH
Eluted as the flow-through fraction at 7.0), then 0.1
Gradient elution with M citrate buffer from pH 5.0 to 3.0 elutes IgG1 slightly later than IgG2. The IgG1 and IgG2 fractions are again chromatographically eluted under the same conditions to obtain crude polyclonal human IgG1 and IgG2 products. The adsorbed component of the DEAE column is eluted with 0.02M phosphate buffer (pH 6.0). Monoclonal antibody HG2-1 that does not bind IgG4, but does bind all other IgG subclasses
I already had 9 [S. Hosoi et al., The Journal of Allergy and Clinical Immunology (J. Allergy Clin. Immunol.) 75 , 320 (1985)], so
Anti-human Ig bound to cyanogen bromide activated sepharose
G1, 2, 3 affinity columns were prepared and DEAE
Subclasses other than IgG4 mixed in the adsorbed components of the column are absorbed and removed to obtain a polyclonal human IgG4 crude product.

Second Method (Preparation of Purified Product) Monoclonal antibody that reacts only with human IgG4, monoclonal antibody that reacts only with human IgG3, and monoclonal antibody that reacts with a subclass other than human IgG4 are immobilized on cyanogen bromide-activated sepharose. , Anti-human IgG3 and anti-human IgG1, respectively,
Prepare a few affinity columns. Human γ-globulin fraction was added to 0.01M phosphate buffer saline (pH
Since the human IgG4 is adsorbed when it is passed through the anti-human IgG4 affinity column in 7.4), after elution with a buffer containing low pH or a denaturing agent, anti-human IgG1,
Subclasses other than human IgG4 are adsorbed and removed through a few affinity columns to give polyclonal human Ig
Obtain G4 purified product. When the flow-through fraction of the anti-human IgG4 column is passed through an anti-human IgG3 affinity column, human IgG3 is adsorbed, so after elution with a buffer containing a low pH or a denaturant, it is further passed through a protein A-Sepharose column. , A subclass other than human IgG3 is adsorbed and removed to obtain a purified polyclonal human IgG3 product. The flow-through fraction of the anti-human IgG3 column contained human IgG1 and I
Since gG2 was contained, the gradient elution with the protein A-Sepharose column was repeated twice as in the first method. Obtain purified products of polyclonal human IgG1 and IgG2.

2) Preparation of hybridoma An animal such as a mouse or a rat is treated with polyclonal human Ig.
Immunize with G subclass. Obtain antibody-producing cells from the immunized animal and fuse them with myeloma cells. From the hybridomas obtained, select wells of hybridomas that strongly react with the immunized human IgG subclass but not with other subclasses. To do. The selected hybridoma is cloned, and the resulting hybridoma clone is subjected to a detailed assay for antibody production ability against 4 polyclonal human IgG subclasses. A clone that strongly reacts with the immunized IgG subclass but does not react with other subclasses. Is selected, and this is cultured to collect the monoclonal antibody. The immunization method, fusion method, selection of fused cells and the like can be carried out by known ordinary methods.

More specifically, the monoclonal antibody of the present invention can be produced, for example, as follows.

First, mice are immunized with the polyclonal human IgG subclass. The animal to be immunized is not limited to a mouse, but a rat such as a rat, an animal or other animals may be used.
Usually, it is preferable to use a mouse. The polyclonal human IgG subclass may be used after being thermally aggregated (63 °, 30 minutes). For example, BALB / c mice are each provided with 100 to 200 μg of polyclonal human IgG subclass or a thermal aggregate thereof in Freund (Freun).
d) Administered intraperitoneally or subcutaneously with complete adjuvant.
After about 3 weeks, 10 to 50 μg of polyclonal human IgG subclass or its thermal aggregate together with alum adjuvant was used once or daily for 2 to 4 times.
Administered once, intraperitoneally or intravenously. After 1 to 3 days, the spleen is removed to obtain a cell suspension, which is used as a fusion antibody-producing cell.

Since this cell does not have the ability to grow, it is fused with a cell that has the ability to self-proliferate. Myeloma cells are particularly preferable as the cells having the self-proliferating ability. The myeloma cell is preferably a hypoxanthine / guanine / phosphoribosyltransferase-deficient strain which does not produce an antibody and is derived from the same species of animal. The fusion antibody-producing cells and myeloma cells were combined with oi and Herzenberg (H
Erzenberg) [Selected Methods in Cellular Immunology]
Immunology), 351-372 (1980), San Francisco (San F
ransisco), WH Freeman & Company (WHF
Reeman and Co.)] and fusion in the presence of polyethylene glycol. The ratio of antibody-producing cells to myeloma cells used is preferably 2: 1 to 10: 1 in terms of cell number ratio. RPMI-1640 medium containing 15% fetal bovine serum (complete RPMI medium) is used as a basal medium, and HAT medium containing hypoxanthine, aminopterin and thymidine and HT medium containing only hypoxanthine and thymidine are prepared. Only hybridomas are selected by culturing the fused cells in HAT medium for about 2 weeks. Incubate in HT medium for about 2 weeks, then complete R
Culture in PMI medium.

3) Selection of hybridoma Polyclonal human IgG subclass is physically adsorbed in a well of a polystyrene microtiter plate to be solid-phased and reacted with a culture supernatant of hybridoma. After washing, an oxygen-labeled anti-mouse immunoglobulin is reacted, and after washing, a substrate is added and the enzyme activity is measured to determine the antibody activity in the culture supernatant. Hybridoma wells that respond strongly to the immunized human IgG subclass and less to other IgG subclasses are selected. The selected hybridoma is cloned by the limiting dilution method or the like, and the resulting hybridoma clone is subjected to polyclonal human I
The production of antibodies against gG subclass 4 will be examined in more detail. A clone is selected that responds strongly to the immunized human IgG subclass but not to the other subclasses.

4) Preparation of monoclonal antibody Selected hybridomas are cultured in vitro or in vivo.
A monoclonal antibody is obtained by proliferating by a culture method. in
The in vitro culture method can be performed, for example, as follows. That is, the hybridoma is cultured in complete RPMI medium until the growth limit is reached, and the culture supernatant is collected.
The in vivo culture method can be performed, for example, as follows. That is, 5 × 10 ⁶ to 10 ⁷ hybridomas were intraperitoneally transplanted into BALB / c mice treated with pristene intraperitoneally in advance, and after about 3 weeks, the abdominal hypertrophy of the mice was confirmed and the ascites was collected. To do.

Example 1 (1) Separation of 4 types of crude human IgG subclass crude products 800 ml of human pool serum was salted out three times with 12% sodium sulfate to collect a γ-globulin fraction, and the fraction was subjected to DEA.
Add to E-Sepharose column (2.6 x 40 cm),
It was separated into a component I passing through with a 0.01 M phosphate buffer (pH 8.0) and a component II eluted with a 0.02 M phosphate buffer (pH 6.0). Ingredient I is protein concentration 20mg / ml
0.1M phosphate buffer solution (pH 7.0) was added to the protein A-Sepharose column (1.6 × 10 cm) in an amount of 5 to 10 ml, and a 0.1M phosphate buffer solution (pH 7.0) was added. 0) Polyclonal human I from the flow-through fraction
A crude gG3 product was obtained. Then with 0.1M citrate buffer
IgG1 when gradient eluted from pH 5.0 to 3.0
Elutes slightly later than IgG2. IgG1 and I
The fractions of gG2 were again chromatographically eluted under the same conditions to obtain polyclonal human IgG1 and Ig.
A crude product of G2 was obtained. In addition to IgG4, component II contains I
Since gG1, IgG2 and IgG3 are also mixed,
Anti-human IgG1, 2, 3 affinity column (1.5
X12 cm) to remove the mixed subclass other than IgG4 by absorption to obtain a crude polyclonal human IgG4 product.

The obtained 4 types of polyclonal human IgG subclass crude products were porcine anti-human I from Nordic.
An immunodouble diffusion method using the gG subclass was performed to identify the subclass and confirm the purity of the subclass.

(2) Immunization Three 8-week-old female BALB / c mice were each treated with 160
μg of polyclonal human IgG1 crude product was intraperitoneally administered with Freund's complete adjuvant, and 3 weeks later,
Further, 20 μg of the polyclonal human IgG1 crude product was intraperitoneally administered as an alum precipitation suspension for 3 consecutive days.

(3) Cell fusion On the day after the final immunization, 3 spleens were extracted and a cell suspension was prepared using RPMI medium. The 3 × 10 ⁸ splenocytes and 9 × 10 ⁷ myeloma cells NS-1 were mixed,
After centrifugation, 1 ml of 50% polyethylene glycol (average molecular weight 4000) was added to the precipitate with gentle stirring, and the mixture was further stirred for 1 minute. 1 ml of RPMI medium was added over 1 minute, and 1 ml was added in the same manner, and then 7 ml was added over 3 minutes. After centrifugation, the sediment was suspended in 60 ml of RPMI medium (complete RPMI medium) containing 15% fetal bovine serum,
96-well microculture plate 0.1 ml in each well of 6 plates
Each was inoculated and cultured at 37 ° C. in the presence of 7% carbon dioxide. 24 hours later, complete RPM containing hypoxanthine 100 μM, aminopterin 0.4 μM, thymidine 16 μM
Add 0.1 ml of I medium (HAT medium). After culturing,
On days 2, 3, 5 and 8, discard 0.1 ml of culture supernatant,
0.1 ml of HAT medium was added. On days 11 and 14,
Throw away 0.01 ml of culture supernatant and remove hypoxanthine 100μ
0.1 ml of complete RPMI medium (HT medium) containing M and 16 μM of thymidine was added. Hybridoma colonies appeared from all of the 576 wells.

(4) Selection of hybridoma The hybridoma in the well was cultured in complete RPMI medium, and the presence or absence of specific antibody production in the culture supernatant was detected as follows.

A solution (10 μg / ml) of a polyclonal IgG1 crude product dissolved in 0.05M carbonate buffer (pH 9.1) was added to each well of a polystyrene microtiter plate [Immulon2, Dynatech] 50.
After adding 1 μl and leaving it at 37 ° C. for 1 hour, 0.01 M phosphate buffer (pH 7.4) containing 10% fetal bovine serum 10
0 μl was added and blocked at 37 ° C. for 20 minutes. 0.0
0.2M phosphate buffer containing 5% Tween 20 (pH 7.
It wash | cleaned by 4) and produced the crude IgG1 solid phase well. Coarse I
Add 50 μl of culture supernatant to the solid well of gG1 and incubate at 37 ℃
And incubated for 1 hour. After washing in the same manner as above, 50 μl of horse radish oxidase-labeled goat anti-mouse IgG (H + L) antibody that does not react with human IgG was added as the second antibody, and the mixture was incubated at 25 ° C. for 1 hour.
Similarly, after washing, 1.1% hydrogen peroxide solution and 150 μg / ml
50 μl of 50 mM citrate buffer (pH 4.5) containing azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) was added, and color was developed at 25 ° C. for 30 minutes. 50 μl of 2 mM sodium azide was added as a stop solution, and the absorbance at 450 nm was measured by a spectrophotometer for a microplate, and 137 wells of hybridoma with an absorbance of 0.5 or more were selected.

With respect to the hybridoma producing the antibody against the crude IgG1, the presence or absence of the reaction with other IgG subclasses was examined by the ELESA method as described above. That is, the crude products of polyclonal human IgG2, IgG3, and IgG4 are similarly solid-phased in the wells of a microtiter plate to prepare respective subclass solid-phase wells,
The reaction of the hybridoma culture supernatant was examined. As a result, hybridoma 5 wells that strongly react with crude IgG1 and hardly with other subclasses were selected. The selected hybridomas were cloned by the limiting dilution method, and the resulting culture supernatants of the hybridoma clones were assayed by the ELISA method for the antibody productivity with respect to the four purified human IgG subclass purified products obtained in Example 7.

In this way, it reacts with human IgG1 but
Two clones of a hybridoma producing a monoclonal antibody that does not react with the gG subclass were obtained, and each of the monoclonal antibodies was labeled with HG1-1E and HG1-16.
It was named D.

(5) Preparation of Monoclonal Antibody (a) In Vitro Culture Method The hybridoma was grown in a complete RPMI medium at the growth limit (1 × 1).
Cultivation was carried out until the amount reached to ⁶⁰ cells / ml, and the culture supernatant was recovered.

(B) In vivo culture method B treated with 0.5 ml of pristene by intraperitoneal injection
ALB / c mouse was intraperitoneally transplanted with 5 × 10 ⁶ hybridomas. After about 3 weeks, the abdominal hypertrophy of the mouse was confirmed, and the ascites was collected.

(6) Monoclonal antibodies HG1-1E and HG1-
Characteristics of 16D (a) Reactivity with human IgG subclass Monoclonal antibody HG1-obtained by in vitro culture method
The reactivity of 1E and HG1-16D with the four purified human IgG subclass purified products obtained in Example 7 was examined by an ELISA method. As shown in Table 4, the HG1-1E and HG1-16D monoclonal antibodies reacted with human IgG1 but not with other IgG subclasses.

(B) Determination of Isotype Rabbit anti-mouse IgG1, rabbit anti-mouse IgG2a, rabbit anti-mouse IgG2b, rabbit anti-mouse IgG3, rabbit anti-mouse IgGM, rabbit anti-mouse Ig from Miles
A, double anti-diffusion method using rabbit anti-mouse L (k) chain and rabbit anti-mouse L (λ) chain was performed. Monoclonal antibodies HG1-1E and HG1-16D are both mouse I
It belonged to gG1 (k).

(C) Reactivity with animal IgG Monoclonal antibody HG1-obtained by in vitro culture method
The reactivity of 1E and HG1-16D to various animal IgGs was examined by the ELISA method (Table 5). Monoclonal antibody HG1-1E reacts with porcine, dog and monkey IgG, but not chicken, cow, goat,
It did not react with sheep, cat, rabbit and rat IgG. Monoclonal antibody HG1-16D reacted with monkey IgG but not chicken, cow, pig, goat, sheep, cat, dog, rabbit and rat IgG.

Example 2 (1) Immunization Two female BALB / c mice aged 8 weeks were each administered with 200
μg of heat-aggregated (63 ° C., 30 minutes) polyclonal human IgG2 crude product was intraperitoneally administered with Freund's complete adjuvant, and 3 weeks later, 20 μg of heat-aggregated polyclonal human IgG2 crude product was added to an alum sedimentation suspension. Was intraperitoneally administered for 3 consecutive days.

(2) Cell fusion In Example 1, 3 × 10 ⁸ splenocytes and 9 × 1 of 3 splenocytes
0 ^seven instead fused with NS-1, 2 mice spleen cells 2 ×
Fusion of 10 ⁸ and 6 × 10 ⁷ NS-1
Cell fusion was performed in exactly the same manner as in Example 1 except that 40 ml of the suspension was inoculated to 4 plates instead of inoculating the suspension of 60 ml of complete RPMI medium to 6 plates. Hybridoma colonies appeared from 211 wells out of a total of 384 wells.

(3) Selection of hybridoma In exactly the same manner as in Example 1, EL using a microtiter well in which a crude polyclonal human IgG2 product was immobilized
A total of 20 welloma hybridomas producing antibodies against crude IgG2 were selected by the ISA method.
Hybridoma 5 wells that responded strongly to gG2 but not to other subclasses were selected. Next, Example 1
The selected hybridomas were cloned by the limiting dilution method and polyclonal human IgG
ELISA for antibody production against 4 subclass purified products
It was tested by the method.

In this way, it reacts with human IgG2, but with other I
Three clones of hybridomas producing monoclonal antibodies that did not react with the gG subclass were obtained, and the respective monoclonal antibodies were designated as HG2-6A, HG2-30F, and HG2-56F.

(4) Preparation of Monoclonal Antibody Monoclonal antibodies were prepared by the in vitro and in vivo culture methods in the same manner as in Example 1.

(5) Monoclonal antibodies HG2-6A, HG2-30
Properties of F and HG2-56F (a) Reactivity with human IgG subclass It was examined by the same ELISA method as in Example 1. As shown in Table 4, HG2-6A, HG2-30F and HG2
The -56F monoclonal antibody reacted with human IgG2 but not with other IgG subclasses.

(B) Determination of isotype As in Example 1, it was examined by the immunodiffusion double diffusion method. Monoclonal antibodies HG2-6A, HG2-30F and HG
All of 2-56F belonged to mouse IgG1 (k).

(C) Reactivity with animal IgG The same as in Example 1 was examined by the ELISA method (Table 5).
Monoclonal antibodies HG2-6A and HG2-56F
Did not react with chicken, cow, pig, goat, sheep, cat, dog, rabbit, rat and monkey IgG. Monoclonal antibody HG2-30F reacts against goat and sheep IgG but against goat and sheep IgG, but not chicken, cow, pig, cat, dog, rabbit, rat and monkey IgG. Did not react.

Example 3 (1) Immunization To one 8-week-old female BALB / c mouse, 160 μg of a crude polyclonal human IgG3 product was intraperitoneally administered together with Floyd's complete adjuvant.
Crude polyclonal human IgG3 product was administered intraperitoneally as an alum precipitation suspension.

(2) Cell fusion In Example 1, the day after the final immunization, 3 splenocytes 3
Instead of fusing × 10 ⁸ and 9 × 10 ⁷ NS-1, one splenocyte was fused 1 × 10 ⁸ and 3 × 10 ⁷ NS-1 3 days after the final immunization. That and complete RP
Cell fusion was carried out in exactly the same manner as in Example 1 except that 20 ml of the suspension was inoculated to the two plates instead of inoculating the suspension of 60 μl of MI medium to the six plates. 198 in total
Hybridoma colonies appeared from 98 of the wells.

(3) Selection of hybridoma In exactly the same manner as in Example 1, EL using a microtiter well in which a crude polyclonal human IgG3 product was immobilized
By the ISA method, a total of 15 wells of hybridomas producing an antibody against crude IgG3 were selected, and further, crude Ig
Hybridoma 3 wells that responded strongly to G3 but not to other subclasses were selected. Then, in exactly the same manner as in Example 1, the selected hybridomas were cloned by the limiting dilution method, and the antibody-producing ability to 4 polyclonal human IgG subclass purified products was assayed by the ELISA method.

In this way, it reacts with human IgG3, but with other I
One clone of a hybridoma producing a monoclonal antibody that does not react with the gG subclass was obtained, and the monoclonal antibody was named HG3-7C.

(4) Preparation of Monoclonal Antibody Monoclonal antibodies were prepared by the in vitro and in vivo culture methods in the same manner as in Example 1.

(5) Characteristics of monoclonal antibody HG3-7C (a) Reactivity with human IgG subclass It was examined by the same ELISA method as in Example 1. As shown in Table 4, the HG3-7C monoclonal antibody reacted with human IgG3 but not with other IgG subclasses.

(B) Determination of isotype As in Example 1, it was examined by the immunodiffusion double diffusion method. The monoclonal antibody HG3-7C belonged to mouse IgG1 (k).

(C) Reactivity with animal IgG The same as in Example 1 was examined by the ELISA method (Table 5).
Monoclonal antibody HG3-7C responded to cat and dog IgG but not chicken, cow, pig, goat, sheep, rabbit, rat and monkey IgG.

Example 4 (1) Immunization One female BALB / c mouse at 8 weeks of age was given 160
μg of the polyclonal human IgG3 crude product was intraperitoneally administered together with Floyd's complete adjuvant, and 3 weeks later, 20 μg of the polyclonal human IgG3 crude product was further intraperitoneally administered as an alum precipitation suspension for 3 consecutive days.

(2) Cell fusion Cell fusion was carried out exactly as in Example 1, and hybridoma colonies appeared from 504 wells out of a total of 576 wells.

(3) Selection of hybridoma Hybridomas were selected in exactly the same manner as in Example 3, and 4 clones of hybridomas producing a monoclonal antibody that reacts with human IgG3 but does not react with other IgG subclasses were obtained. The antibodies were named HG3-3B, HG3-32C, HG3-45B and HG3-49D.

(4) Preparation of Monoclonal Antibody Monoclonal antibodies were prepared by the in vitro and in vivo culture methods in the same manner as in Example 1.

(5) Monoclonal antibodies HG3-3B, HG3-32
Characteristics of C, HG3-45B, and HG3-49D (a) Reactivity with human IgG subclat The same as in Example 1, the ELISA method was examined. As shown in Table 4, HG3-3B, HG3-32C, HG3-4
The 5B and HG3-49D monoclonal antibodies reacted with human IgG3 but not with other IgG subclasses.

(B) Determination of isotype As in Example 1, it was examined by the immunodiffusion double diffusion method. Monoclonal antibodies HG3-3B, HG3-32C, HG3-
Both 45B and HG3-49D are mouse IgG1
Belonged to (k).

(C) Reactivity with animal IgG The same as in Example 1 was examined by the ELISA method (Table 5).
Monoclonal antibodies HG3-3B, HG3-32C, H
G3-45B and HG3-49D did not react with chicken, cow, pig, goat, sheep, cat, dog, rabbit, rat and monkey IgG.

Example 5 (1) Immunization Two female BALB / c mice aged 8 weeks were each treated with 175
μg of the polyclonal human IgG4 crude product was intraperitoneally administered together with Floyd's complete adjuvant, and 3 weeks later, 20 μg of the polyclonal human IgG4 crude product was further intraperitoneally administered as an alum precipitation suspension for 4 consecutive days.

(2) Cell fusion Cell fusion was carried out in exactly the same manner as in Example 2, and hybridoma colonies appeared in all 384 wells in total.

(3) Selection of hybridoma As in Example 1, EL using a microtiter well in which a polyclonal human IgG4 crude product was immobilized
A total of 233 wells of hybridomas producing an antibody against crude IgG4 were selected by the ISA method.
Hybridoma 3 wells that responded strongly to gG4 but not to other subclasses were selected. Next, in exactly the same manner as in Example 1, the selected hybridomas were cloned by the limiting dilution method to give polyclonal human Ig.
ELISA for antibody production against 4 G subclass purified products
It was assayed by Method A.

In this way, it reacts with human IgG but not other Ig
One clone of a hybridoma producing a monoclonal antibody that does not react with the G subclass was obtained, and the monoclonal antibody was named HG4-3F.

(4) Preparation of Monoclonal Antibody Monoclonal antibodies were prepared by the in vitro and in vivo culture methods in the same manner as in Example 1.

(5) Characteristics of monoclonal antibody HG4-3F (a) Reactivity with human IgG subclass It was examined by the same ELISA method as in Example 1. As shown in Table 4, the HG4-3F monoclonal antibody reacted with human IgG4 but not with other IgG subclasses.

(B) Determination of isotype As in Example 1, it was examined by the immunodiffusion double diffusion method. Monoclonal antibody HG4-3F belonged to mouse IgG1 (k).

(C) Reactivity with animal IgG The same as in Example 1 was examined by the ELISA method (Table 5).
Monoclonal antibody HG4-3F reacts against pig and monkey IgG, but chicken, cow, goat, sheep, cat, dog, rabbit, rat and monkey IgG.
Did not respond to.

Example 6 (1) Immunization Three female BALB / c mice of 8 weeks of age each received 175
μg of polyclonal human IgG4 crude product was intraperitoneally administered with Freund's complete adjuvant, and 3 weeks later,
Further, 20 μg of polyclonal human IgG4 crude product was intraperitoneally administered as an alum sedimentation suspension.

(2) Cell fusion Cell fusion was performed in the same manner as in Example 1 except that the spleen was extracted 3 days after the day after the final immunization instead of the day after the final immunization. Hybridoma colonies appeared from 320 wells out of a total of 576 wells.

(3) Selection of hybridoma Hybridomas were selected in exactly the same manner as in Example 5, and two clones of hybridomas producing a monoclonal antibody that reacts with human IgG4 but does not react with other IgG subclasses were obtained. The antibodies were named HG4-31C and HG4-38B.

(4) Preparation of Monoclonal Antibody Monoclonal antibodies were prepared by the in vitro and in vivo culture methods in the same manner as in Example 1.

(5) Monoclonal antibodies HG4-31C and HG4
Characteristics of -38B (a) Reactivity of human IgG subclass It was examined by an ELISA method as in Example 1. As shown in Table 4, the HG4-31C and HG4-38B monoclonal antibodies reacted with human IgG4 but not with other IgG subclasses.

(B) Determination of isotype As in Example 1, it was examined by the immunodiffusion double diffusion method. Monoclonal antibodies HG4-31C and HG4-38B belonged to mouse IgG1 (k).

(C) Reactivity with animal IgG The same as in Example 1 was examined by the ELISA method (Table 5).
Monoclonal antibodies HG4-31C and HG4-38
B responded to porcine IgG but not chicken, cow, goat, sheep, cat, dog, rabbit, rat and monkey IgG.

Example 7 (1) Preparation of anti-IgG subclass affinity column Monoclonal antibody HG4-53G [Japanese Patent Application No. 60-127844], which reacts with human IgG4 but not with other IgG subclasses, but reacts with human IgG3, etc. Monoclonal antibody HG3-7C that does not react with the IgG subclass of E. coli and monoclonal antibody HG that does not react with human IgG4 but does react with other IgG subclasses
3-13F (obtained separately in Example 3) was added to cyanogen bromide activated sepharose (Pharmacia) to immobilize each. In each case, 5 mg of the monoclonal antibody was reacted with 1 ml of Sepharose. The obtained monoclonal antibody-immobilized sepharose was packed in a column, and an anti-human IgG4 affinity column and an anti-human IgG were prepared, respectively.
3 affinity column and anti-human IgG1, 2, 3
An affinity column was prepared.

(2) Preparation of 4 kinds of polyclonal human IgG subclass purified products 20 g of human γ-globulin (Cohn Fr. II) was added to 0.01 M
A solution prepared by dissolving 200 ml of phosphate buffered saline (pH 7.4) was first passed through an anti-human IgG4 affinity column (2.5 × 9.5 cm), and the adsorbed component was 0.01M phosphoric acid containing 3M potassium thiocyanate. After elution with a buffered saline solution (pH 7.4) and dialysis with a 0.01 M phosphate buffered saline solution (pH 7.4), anti-human IgG1, 2,
73 mg of polyclonal human IgG4 purified product was obtained in the flow-through fraction through 3 affinity columns (2.5 × 6.0 cm). Next, permeate fraction of the anti-human IgG4 affinity column was passed through an anti-human IgG3 affinity column (2.5 × 6.5 cm) to collect 3 M of adsorbed component
After elution with 0.01 M phosphate buffered saline (pH 7.4) containing potassium thiocyanate and dialysis with 0.01 M phosphate buffered saline (pH 7.4), protein A-
Sepharose column (Pharmacia, 2.5 × 14.
5 cm) and the polyclonal human I
53 mg of gG3 purified product was obtained. The flow-through fraction of the anti-human IgG3 affinity column is further passed through an anti-human IgG4 affinity column and an anti-human IgG3 affinity column to obtain a polyclonal human IgG and IgG2.
17.5 g of a mixture of 500 μg of this mixture was added to a Protein A-Sepharose column, passed through 0.1 M phosphate buffer (pH 7.0), and then gradient eluted with 0.1 M citrate buffer to pH 5.0 to 3.0. IgG1 elutes slightly later than IgG2. I
The fractions of gG1 and IgG2 were again chromatographically eluted under the same conditions to give polyclonal human IgG1
355 and 53 mg of purified product of IgG2 and IgG2, respectively
Obtained.

(3) Evaluation of 4 kinds of polyclonal human IgG subclass purified products Obtained polyclonal human IgG subclass purified products 4
Regarding species, sheep anti-human I from Nordic
gG subclass 4 species and porcine anti-human IgG subclass 4 species and sheep anti-human IgG1 and sheep anti-human IgG2 from Miles and Serotec were used for immune double diffusion, and the corresponding subclass antibodies were obtained. The formation of a sedimentation line was observed between and. At this time, the agar plate is Difco's Agar Noble 1.2%
And 2% polyethylene glycol (average molecular weight 40
00) is included.

A monoclonal anti-human IgG subclass [J. Robe (Lowe), et al., Immunology (Immunology) 47, 3
29 (1982)], four types of monoclonal antibodies were confirmed by Reimer et al. Using a large number of myeloma serum-derived human IgG subclasses for the reaction specificity for each subclass [C.
B. Reimer (Reimer) et al., Were purchased from the hybridoma (Hybridoma) 3, 263 (1984 )] Yunibasu (Unipath) Inc., was used to polyclonal human IgG subclasses purified product four ratings. 10 µg / ml or 2 µg of polyclonal human IgG subclass purified product
Immobilization was performed in a well of a microtimer plate at a concentration of / ml, and ascites of each monoclonal antibody was diluted to an optimal multiple for reaction, and then evaluated by an ELISA method as in Example 1. As shown in Table 6, each polyclonal human IgG subclass purified product reacted particularly strongly with the corresponding monoclonal anti-human IgG subclass, and the reaction with the monoclonal antibody to the other subclass was sufficiently small.

Furthermore, using the quantitative immunodiffusion kit “human IgG subclass” from Miles, polyclonal human I
As a result of quantifying the gG subclass, human IgG1 was 90.
%, Human IgG2 108%, human IgG3 100
%, Human IgG4 was 85%, and both were close to 100%.

Reference Example 1 Reactivity of Unibus Monoclonal Antibody to Animal IgG According to Example 1, JL-512 ascites was diluted 1000 times and G was added.
OM1 ascites 2700 times, ZG4 ascites 8100 times, R
ELIS of J4 ascites diluted 8100 times
By method A, the reactivity to animal IgG was examined. The results are shown in Table 7.

Reference Example 2 a) Affinity constant for solid-phase human IgG subclass In each well of a polystyrene microtiter plate, four polyclonal human IgG subclass purified products were dissolved in 0.05 M carbonate buffer (pH 9.1) (2).
50 μl of μg / ml) was added, the mixture was allowed to stand at 37 ° C. for 1 hour, 100 μl of 0.01 M phosphate buffer (pH 7.4) containing 10% fetal bovine serum was added, and the mixture was blocked at 37 ° C. for 20 minutes. The cells were washed with 0.01 M phosphate buffer (pH 7.4) containing 0.05% Tween 20 to prepare human IgG subclass solid phase wells. ¹²⁵ I-labeled monoclonal antibody HG4-3 was added to human IgG subclass solid phase wells.
F, HG4-31C or HG4-38B 240 ~
Dilute with 0.01 M phosphate buffer (pH 7.4) containing 10% fetal bovine serum to a concentration range of 0.24 mg / ml, add 50 μl, incubate at 25 ° C. for 1 hour, and wash similarly , Γ-counter. The affinity constant was determined by the Scatchard method.

b) Affinity constant for liquid-phase human IgG subclass 0.01 M containing 10% fetal bovine serum in a polystyrene tube
100 μl of phosphate buffer (pH 7.4) and ¹²⁵ I-labeled monoclonal antibody HG4-3F dissolved in the same buffer,
HG4-31C or HG4-38B (40 mg / ml) 5
0 μl and the monoclonal antibody HG4 diluted with the same buffer to a concentration range of 2.5 to 0.005 μg / ml
-3F, HG4-31C or HG4-38B (50μ
1) and 50 μl of 2.5 μg / ml human IgG subclass purified product were added and incubated at 25 ° C. for 16 hours. An appropriate amount (600 μg / ml, 100 μl) of Sephadex-conjugated monoclonal anti-human L chain antibody was added to this reaction solution.
Was added, and the mixture was incubated at 25 ° C. for 1 hour, washed 3 times with physiological saline, and counted with a γ-counter. The affinity constant was determined by the Scatchard method.

The above results are shown in Table 8, and even monoclonal antibodies against the same IgG subclass have different affinities.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location G01N 33/53 N 8310-2J 33/577 B 9015-2J (C12P 21/08 C12R 1:91) (56) References Immunology, 47 (2), 329-336 (1982) Hybridoma, 3 (3), 263-276 (1984) Immunol. Lett. , 10 (3-4), 223-252 (1985) Monogr. Allergy, 19, 71-85 (1986)

Claims (1)

[Claims] 1. Binding to human IgG2, but other human Ig
Substantially non-binding to the G subclass, chicken, bovine,
Substantially no binding to pig, goat, sheep, cat, dog, rabbit, rat and monkey IgG, mouse IgG1
Monoclonal antibody HG2-56F belonging to (k).