JPS63253098A - Monoclonal antibody against human antibody, its production and use thereof - Google Patents

Abstract

NEW MATERIAL:An antibody exhibiting extremely high specific reactivity to either of IgG and IgM of human antibody. USE:Useful for the remedy of human disease and diagnosis by imaging. A reagent useful for the removal or recovery of human antibody from human serum. PREPARATION:A small experimental animal is immunized with a mixed material containing human antibody (e.g. human gamma-globulin). A lymphocyte obtained from the immunized animal is fused with a myeloma cell to obtain a hybridoma. The hybridoma is cloned and the established hybridoma strain is cultured in a peritoneal cavity of a small experimental animal to produce the objective antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a monoclonal antibody that has extremely high specific reactivity to human antibodies and can be used in the production of pharmaceuticals and reagents.

Furthermore, the present invention provides the above-mentioned hybridoma strain, which is produced from a hybridoma line established by cloning a hybridoma obtained by cell fusion of lymphocytes and myeloma cells of a small experimental animal obtained by immunization with a human antibody mixture. The present invention relates to a method for producing the monoclonal antibody by culturing a hybridoma strain that produces the monoclonal antibody.

The present invention also relates to a method for removing or recovering human antibodies, which comprises separating a complex consisting of a human antibody and the monoclonal antibody from a human antibody-containing solution using the monoclonal antibody described above. be.

[Description of prior art]

Human monoclonal antibodies are considered to be the most preferable antibodies used for human treatment, imaging diagnosis, etc., considering antigen specificity, measurement sensitivity, reproducibility, antigenicity for humans, etc.

Therefore, attempts have been made to produce human monoclonal antibodies against cancer cells, cancer-related antigens, and other antigens that serve as diagnostic markers.

The method for producing human monoclonal antibodies requires experimental operations to obtain the desired human monoclonal antibody-producing hybridoma strain from fused cells obtained by cell fusion of appropriate mouse myeloma cells or human myeloma cells and human lymphocytes. It is.

In this experimental procedure, antiserum against human antibodies is used as the reagent for antibodies against human antibodies, but recently monoclonal antibodies have been shown to be superior to antiserum in terms of quality. Since human antibodies are thought to be more common, monoclonal antibodies or mixtures of monoclonal antibodies directed against human antibodies are also being used.

Therefore, when using a monoclonal antibody against a human antibody as a reagent in this experimental procedure, first
There is a complicated problem in that it is necessary to obtain several monoclonal antibody-producing strains with high specificity for G, IgM, etc., and then to mix and use the monoclonal antibodies obtained by culturing these strains.

On the other hand, pharmaceutical antibodies used in antitoxin therapy include antisera made by immunizing foreign animals such as horses with diphtheria and tetanus toxins. This antiserum contains many foreign proteins that can cause serum sickness, so this antiserum is further purified into an antibody fraction and used. However, this antibody fraction itself also It is a foreign protein award because it is a horse antibody.

Therefore, recently, allogeneic serum with high antibody titer obtained by immunization with allo (other person) is also used. Even in this case, antibodies used for human treatment should be considered as the Hazardous Substances in Human Serum Award (
It is preferable to use antibodies that are free from contamination with viruses, pathogenic stones, denatured proteins, etc. In order to efficiently obtain such antibodies, antibodies with extremely high specific reactivity against human antibodies are preferred. monoclonal antibodies are required.

In addition, when obtaining physiologically active substances other than antibodies contained in human serum, it is recommended to remove antibodies, which are major contaminants, from the bloodstream in advance to facilitate subsequent recovery of the desired physiologically active substances. It becomes easier.

At this time as well, monoclonal antibodies with extremely high specific reactivity to human antibodies are required.

[Problem that the invention seeks to solve]

An object of the present invention is to provide reagents necessary for producing human monoclonal antibodies for use in human treatment, imaging diagnosis, and the like.

That is, instead of the conventional mixture of two types of monoclonal antibodies, one that is reactive with human IgG and one that is reactive with human IgM, which was used when producing human monoclonal antibodies, these It is an object of the present invention to provide one type of monoclonal antibody that exhibits reactivity with all (more preferably, all of human IgA, IgG, and IgM).

Another object of the present invention is to provide a method for producing a monoclonal antibody by culturing a monoclonal antibody-producing strain.

Furthermore, the present invention provides reagents necessary for recovering antibodies in human serum or removing antibodies as unnecessary components in human serum that can be used for human therapy. That is, the objective is to provide reagents necessary for removing or recovering human antibodies from human serum.

[Means for solving problems]

As a result of intensive research to solve the above-mentioned problems, the present inventors have discovered that lymphocytes and myeloma cells of small experimental animals obtained by immunization with a mixture of human antibodies (e.g., human γ-globulin, etc.) Hybridoma strains obtained by cloning hybridomas produced by cell fusion can produce monoclonal antibodies that exhibit extremely high specific reactivity to human antibodies IgG, IgM, and IgA. They discovered this and completed the present invention.

Furthermore, we discovered that the monoclonal antibody-producing strain can produce monoclonal antibodies that exhibit extremely high specific reactivity to human antibodies not only in flask culture but also in intraperitoneal culture of small experimental animals, and completed the present invention. did.

Furthermore, the inventors discovered that human antibodies in human serum can be removed or recovered using this monoclonal antibody, thereby completing the present invention.

That is, the present invention provides human antibodies produced from a hybridoma strain established by cloning a hybridoma obtained by cell fusion of lymphocytes and myeloma cells of a small experimental animal obtained by immunization with a mixture containing human antibodies. Ig
The present invention relates to a monoclonal antibody against human antibodies, which is characterized by having extremely high specific reactivity to both G and IgM.

In addition, the present invention provides a hybridoma strain established by cloning a hybridoma obtained by cell fusion of lymphocytes and myeloma cells of a small laboratory animal obtained by immunization with a mixture of human antibodies, and injected intraperitoneally into the peritoneal cavity of a small laboratory animal. By culturing, 1. The present invention relates to a method for producing a monoclonal antibody against human antibodies, which is characterized by producing a monoclonal antibody that has extremely high specific reactivity to both gG and IgM.

Furthermore, the present invention enables contact reaction between a carrier to which a monoclonal antibody having extremely high specific reactivity to both IgG and IgM of human antibodies is bound and a human antibody-containing solution, and the monoclonal antibody The present invention relates to a method for removing or recovering human antibodies, which is characterized by separating a complex consisting of a human antibody and a human antibody.

The present invention will be explained in more detail below.

In the method for producing monoclonal antibodies against human antibodies of the present invention, hybridomas are produced by conventionally known methods, for example, the method of Milstein and Kholer (Nat.
ure, 256.495 (1975)]. Preferred methods for producing such hybridoma strains will be outlined below.

i) Preparation of immunized animal lymphocytes The method for immunizing small experimental animals (e.g., mice, rats, hamsters, etc.) is to use a mixture of human antibodies, such as human γ-globulin, dissolved in PBS (neutral phosphate buffer). (1 to 400 μg) is administered to small experimental animals several times at several intervals. The first immunization is preferably administered as an emulsion with a complete adjuvant (immune stimulant). Lymphocytes can be obtained from the blood, lymph nodes, lungs, etc. several days after the final immunization, but from the viewpoint of experimental procedures, it is preferable to obtain them from the lungs.

ii) Preparation of myeloma cells The myeloma cells of small experimental animals used for cell fusion include mouse-derived NPC-11, P3-X63-Ag8.
653 (653), P3-X63-Ag8-[11
(P2O3), P3-MS-1(NS-1), SF
310-Ag14 (SF310), etc., and rat-derived 210. RCY3. AgL, 2.3. (Y3), but in the present invention, 653, P2O3, NS
Mouse myeloma cells such as -1,5P210 are preferred.

1ii) 1! In cell fusion, the number of lymphocytes and myeloma cells of a small experimental animal immunized as described above was reduced (5 to 10):
Mix well using a commonly used lymphocyte culture medium component solution at a ratio of 1:1 and pellet after centrifugation (
PEG (average molecular weight; 1000-6000)
solution (30-60% by weight of polyethylene glycol)
) and mix well.

Myeloma cells used for cell fusion can be derived from animals different from the immunized experimental small animal, but considering the stability of the monoclonal antibody production ability of the resulting hybridoma strain, The immunized small experimental animal is preferably of the same species, and more preferably of the same species.

iv) Selection of hybridomas Selection of hybridomas consists of H
The cells are cultured in AT medium (a medium containing hypoxanthine, aminopterin, thymidine, and fetal bovine serum). Cells are cultured in an appropriate number on culture plates, and feeder cells are used as necessary. After culturing the selected hybridomas in HT medium (a medium containing hypoxanthin, thymidine, and fetal bovine serum) for several days, Culture in a commonly used lymphocyte culture medium containing fetal bovine serum.

■) Selection of antibody-producing hybridomas To test whether hybridomas obtained by selection in HAT medium produce antibodies against human T-globulin, for example, ELISA (enzyme-linked immunosorbent assay) can be used. Follow the same procedure. That is, a hybridoma culture supernatant is added to an ELISA plate on which human T-globulin is immobilized, and the plate is left standing. Then, an antibody bound to a labeling substance capable of reacting with the bound antibody derived from a small experimental animal is added to these washed wells, and the wells are allowed to stand still. These wells are washed, substrate solution is added and enzyme activity is measured.

If enzyme activity is observed, it is known that a hybridoma producing the antibody of interest was present in the well from which the culture supernatant was collected.

vi) Hybridoma cloning: Hybridomas in wells in which antibody production has been observed are cloned using limiting dilution or single cell manibration methods (one hybridoma per well under an inverted microscope). Can be cloned. At this time, using the culture supernatant of the well in which hybridoma growth was observed,
) Antibody-producing wells are assayed in the same manner as in the selection of antibody-producing hybridomas, and the supernatants of wells in which antibody production is observed are further assayed for reactivity with other antigens. Then, a hybridoma strain that is highly specific to human γ-globulin and has a high antibody titer is selected.

Furthermore, the method for producing monoclonal antibodies against human antibodies by culturing monoclonal antibody-producing strains of the present invention is carried out as follows.

Using the hybridoma strain selected in the above vi) hybridoma line cultivation, culture in flasks or in small experimental animals II! Monoclonal antibodies are produced by culturing within the cavity.

In flask culture, for example, the cells are cultured in a lymphocyte culture medium containing 0 to 20% fetal bovine serum until the cell concentration reaches the upper limit. The monoclonal antibody is contained in the culture supernatant obtained by centrifugation.

On the other hand, in order to obtain a larger amount of antibodies in consideration of production costs, it is possible to use animals of a different species than those used for cell fusion, but it is preferable to use animals of the same species. Preferably, animals of the same breed are used.

Monoclonal antibodies can be produced by intraperitoneally administering mineral oil such as Blistane to suitable small experimental animals, and a few weeks later,
'~10? This can be done in a few weeks by administering several hybridoma line cells and allowing the hybridoma line cells to grow to high density intraperitoneally.

The antibody concentration in the animal ascites thus obtained is 10 to 1000 times higher than the culture supernatant obtained by flask culture.

The monoclonal antibody is purified by a salting-out method, an ion exchange method, etc., which are applied to the immediate purification of proteins, and becomes a highly pure monoclonal antibody.

The human antibody stand obtained as described above was inserted into the proboscis (for example, human T
Monoclonal antibodies against human antibodies such as IgA, IgG, and IgM (globulin, etc.) are human antibodies.

It has extremely high specific reactivity against all of the F(ab')z portion of IgE and human IgG (the portion containing only the variable site obtained by pepsin digestion of gc), No reactivity was observed with antibodies from livestock such as cows, horses, goats, and rabbits, and with all human IgG Fc portions (parts that do not contain the variable region obtained by pepsin digestion of IgG).

Furthermore, the method for removing or recovering human antibodies from human serum components according to the present invention involves combining the monoclonal antibody-binding carrier obtained by binding the monoclonal antibody to a facial carrier and the human antibody-containing carrier. This can be carried out by causing a contact reaction with a liquid (human blood, plasma, serum, etc.) and then separating the complex consisting of the monoclonal antibody-bound carrier and human antibody using filtration, centrifugation, a column, etc. can.

It is preferable to use an insoluble solid phase carrier as the solid phase carrier to which the monoclonal antibody is bound.

The solid phase carrier that binds to the monoclonal antibody may be in the form of a film, particles, or fibers. Each of these solid phase carriers can be used alone or in combination.

Such solid phase carriers include a cross-linking agent and a suitable blocking agent (a substance used to prevent human antibodies from non-specifically binding to the solid phase carrier, such as proteins such as bovine serum albumin and ovalbumin). For example, insoluble substances such as minerals, resins, sugars, and proteins can be used.
It is preferable to use the polysaccharide Sepharose (Pharmacia).

The method of binding the monoclonal antibody to the solid phase carrier is performed using a coupling buffer (for example, 0.1M NaHCO3 and 0.1M NaHCO3).
．． This can be carried out using a solution containing 5 M NaCl (pH 8.0).

Removal or recovery of human antibodies using the monoclonal antibody-binding carrier can be carried out in batches or in a continuous manner using a column or the like.

The binding between the human antibody and the monoclonal antibody on the solid phase carrier is preferably carried out at around neutrality using PBS (phosphate buffer), Tris-HCl buffer, or the like.

Removal of the human antibody can be performed by removing the complex consisting of the monoclonal antibody and human antibody on the solid phase carrier using filtration, centrifugation, a column, or the like.

Recovery of human antibodies is performed by adjusting the pH of the solution containing this complex (
pH5-9), (pH2-4) or (pH10-1)
2), the monoclonal antibody and human antibody can be dissociated, and further separated using filtration, centrifugation, a column, or the like. Human antibodies dissociated in this way are contained in the flow-through portion in filtration or columns, and are contained in the supernatant in centrifugation.

Furthermore, dissociation of the complex can be further enhanced by concurrently using an operation to increase the ionic strength of the solution containing the complex consisting of the monoclonal antibody and human antibody on the solid phase carrier.

On the other hand, the monoclonal antibody-binding carrier remaining after collecting the human antibody can be reused.

(Examples) Examples of the present invention will be specifically described below.These Examples do not limit the scope of the present invention.

Example 1 [Preparation of hybridoma strain producing monoclonal antibodies against human antibodies] (a) Immunization of mice and preparation of lung lymphocytes 5 μg of human T-globulin (antigen) was dissolved in PBS (phosphate buffer, pH 7,4) 0 .25 ml and 0.25 ml of Freund's complete adjuvant are mixed to make an emulsion, and the emulsion is 0.5 pF! BAL is a small experimental animal.
It was administered intraperitoneally to B/e mice (male, 4 weeks old).

After 4 weeks, 5 μg of the above antigen was dissolved in PBS0.2.
5mf and incomplete Freund's adjuvant 0.25
ml to make an emulsion, and the emulsion is 0.5 m
1 was administered intraperitoneally to the mice.

Furthermore, after 3 weeks, as a final immunization, 5μ of the above antigen was administered.
PBSO, 5mj2 in which 100 g of PBSO was dissolved was administered into the tail vein of the mice.

From mice immunized in this way, 4
The mouse lungs removed on day 1 were placed on ice under RPM.
The cells were washed in a petri dish containing RPM I 1640 solution (lymphocyte culture medium powder dissolved in distilled water), transferred to newly prepared RPM I 1640 solution, and loosened using a bottle set.

The floating lymphocytes thus obtained were collected using RPM11640.
Suspend in liquid and centrifuge at rotation speed: 1000 rp
m, time; 5 minutes) and resuspended in RPM 11640 solution to obtain mouse lung lymphocytes used for cell fusion.

(b) Cell fusion 8-azaguanine-resistant mouse myeloma cells (MS-
1) to 2X10? and the mouse lung lymphocytes l
Xl0'' were placed in a 50 m2 plastic conical centrifuge tube, mixed, and the supernatant was centrifuged.
The pellet was loosened by tapping the same centrifuge tube at a rotation speed of 1000 rpm and a time of 5 minutes.

1 m2 of 50% PEG (37°C) was placed into this pellet for 1 minute while shaking vigorously, and the pellet was shaken vigorously for 1 minute. Furthermore, while gently shaking the centrifuge tube, R
Gradually add PM I 1640 solution (37"C), and finally centrifuge at room temperature (rotation speed: 10 m2).
1000 rpm, time: 5 minutes), and the supernatant was removed by suction.

Gently tap the centrifuge tube to loosen the pellet, add HAT medium (IXIO-'M hyboxanthin, 4 x 10-'M 7!J 7, 1.6 R P M I 164 containing
0 medium) 2'0mff1, dispense 100μi into each well of two 96-well culture plates, and
Culture was performed at 37°C using an Oz incubator.

(C) Selection of hybridomas Over a period of 1 to 3 weeks from the start of the culture described above (°C), whether or not the culture supernatant of each well of the culture plate in which cell proliferation was observed contains antibodies against human γ-globulin. This was investigated using the ELISA method shown below.

First, human γ-globulin solution (10 μg/mj!, P
Dissolved in BS) was dispensed in 50 μi portions and left to stand at 4°C overnight.Next, each well of the ELISA plate was washed with a washing solution (PBS containing 0.1% 'l'ween20), and the The culture supernatant of each well of the culture plate was
0μ2 was dispensed into each well of an ELISA plate and allowed to stand at room temperature for 2 hours. (For the supernatant as a negative control, a mixture of mouse lung lymphocytes and mouse myeloma cells before fusion was similarly cultured. The obtained supernatant was used).

Next, each well of the ELISA plate was washed, and 50 μl of a horseradish oxidase-labeled antibody solution against mouse immunoglobulin was dispensed into each well and allowed to stand at room temperature for 2 hours. After washing each well of the ELISA plate, the substrate solution (0-), 20 mg of unilene diamine, and 35% H20! Solution 10al! , p
0 of H5,0. (dissolved in 50ml of IM citrate buffer) to 100μ! The solution was dispensed into each well and allowed to stand at room temperature for 30 minutes in the dark.

Finally, add 50μ of 2N sulfuric acid to each well. After the enzyme reaction was stopped, the absorbance of each well at 500 nm was measured using an absorbance measuring device for microplates.

It was confirmed that a hybridoma producing a monoclonal antibody against human γ-globulin was present in each well of the culture plate from which the supernatant positive for enzyme activity was collected.

As a result of examining the antibody production in the supernatant of the wells of the culture plate used for hybridoma culture as described above, (number of antibody producing wells/number of ELISA wells)
The ratio was 96/102.

(d) Hybridoma line creation (cloning) Using RPM11640 medium containing 15% fetal bovine serum, hybridomas in the antibody-producing wells shown in step c3 were cloned using the single cell cloning method (under an inverted microscope). Cloning was performed using a method in which one hybridoma was placed in one well.

For culture, a 96-well culture plate was used, and a suspension of BAL B/c mouse thymocytes (
10? using (1 hybridoma)
/(ff'4 glandular cell suspension 100g/2)/well.

In the above culture, the supernatant of the well of the culture plate observed as a single colony from around the 10th day was collected,
ELISA method using human T-globulin (as described above (C
) Screening of antibody-producing wells was performed using the same method as in Step 2), and for the wells in which antibody production was observed, other antigens (human serum albumin, IgG, IgM,
and IgA).

As described above, human IgC, IgM, and IgA
One strain that showed reactivity with any of the antibodies but no reactivity with human serum albumin was re-cloned [this strain is shown in Table 1 as the HGG-2 strain].

HGG-2 strain; applied for deposit with the Agency of Industrial Science and Technology on November 21, 1985, and received a notice of refusal of deposit (61 micro-request letter No. 1564) on December 1, 1985].

The class/subclass and type of L chain of the monoclonal antibody contained in the HGG-2 strain culture plate supernatant were determined in the following measurement test I, and the reactivity to various antigens was examined in measurement test (2).

[Determination of classes and subclasses of monoclonal antibodies against human antibodies] Classes and subclasses of immunoglobulins produced by the HG G-2 strain
Determination of subclasses is performed using a peroxidase-labeled antibody solution (i.e., r
gG+ , I gGz a , I gGzb
, IgG□, IgM, IgA, horseradish peroxidase-labeled antibodies against uniform chain or λ-shape chain, etc.), and the same ELISA method as in step (C) above was used.

As a result, the monoclonal antibody produced by this strain was
gG+, and the type of L chain was type (shown in Table 1).

Table 1: HOG 2 Gix [Examination of the reactivity of monoclonal antibodies against human antibodies to various antigens] Regarding the specificity of the monoclonal antibodies produced by the HOG-2 strain, the substances shown in the antigen type column of Table 2 The reactivity was investigated using the following method.

First, various antigen solutions (10 g g/m
1, dissolved in PBS) were dispensed in 100 μf portions and left to stand overnight at 4°C.

Next, after washing each well of the ELISA plate with a washing solution, 100μβ of the culture supernatant of HGG-2 strain diluted in multiple stages with PBS was dispensed into each well of the ELISA plate and incubated at room temperature for 2 hours. I sieved 1. And EL
Wash each well of the ISA plate with washing solution, and add peroxidase-labeled antibody solution against mouse immunoglobulin.
100μ2 portions were dispensed into each well and allowed to stand at room temperature for 2 hours. The subsequent operations were performed in the same manner as the ELISA method in step (C) described above (results are shown in Table 2).

As shown in Table 2, the reactivity of the monoclonal antibody produced by the HGG-2 strain is highly species-specific with respect to livestock antibodies (reactivity with cattle, horse, goat, and rabbit antibodies). However, the class specificity for each class of human antibodies is low (human IgG, jgM, IgM
It reacts well with A and IgE) and shows no reactivity with albumin, a major component in human serum.

Antibodies of HGG-2 strain type of antigens in Table 2 From the above, monoclonal antibodies produced by HGG-2 strain have different antibodies against human IgG, IgM, and IgA, which were used in the production of conventional human monoclonal antibodies. It can be used in place of a reagent mixed with antibodies, and can also be used to recover physiologically active substances in human serum that can be used in human therapy.

Example 2 [Production of monoclonal antibodies in flask culture] Monoclonal antibodies were produced in flask culture.

Hybridoma cell line (HGG-2 strain described above) obtained by culturing in RPM11640 medium containing 15% fetal bovine serum
The cells were transferred to RPM11640 solution (ie, fetal bovine serum was 0%) and cultured until just before death.

The monoclonal antibody was contained in the supernatant obtained by centrifuging the culture solution 12mj2 (rotation number: 3000 rpm, time: 5 minutes) at a concentration of 35 μg/m2 (shown in Table 3).

Example 3 [Production of monoclonal antibodies in mouse peritoneal cavity] In order to obtain a large amount of monoclonal antibodies against human antibodies, hybridoma line cells (the above HGC-2
strain) was cultured.

B ALB/c 7 mice (male, 6 doses, 0.5 m 1. intraperitoneal administration of Bristan 2 weeks ago)
5 x 106 cells of the HGG-2 strain cultured above suspended in RPMI 1640 were intraperitoneally administered to the mice.

The weight of this mouse showed a remarkable increase from around the first week, and ascites fluid (8 mf/mouse) was collected at the second week. This ascites was centrifuged (rotation speed: 3000 rpm).

time: 5 minutes) to obtain ascites supernatant. The monoclonal antibody was contained in the ascites supernatant at 8 mg/ml.

Table 3 shows the production status of "monoclonal antibodies when flasks or mice were used in Examples 2 and 3 above" and their reactivity to each antigen (the measurement method was in accordance with measurement test ■). show.

As shown in Table 3, the reactivity of monoclonal antibodies produced by the HGG-2 strain with various antigens was the same as shown in Table 2 in both flask culture and mouse intraperitoneal culture. there were.

The monoclonal antibody was purified from the monoclonal antibody-containing solution thus obtained by dialyzing the precipitate with 50% saturated ammonium sulfate against PBS, and then obtaining a fraction that passed through a column packed with DEAE-cellulose. The purification purity was confirmed to be high by a slab gel electrophoresis experiment using SDS polyacrylamide.

Table 3 Example 4 [Method for removing or recovering human antibodies] A monoclonal antibody-binding carrier for human antibodies was prepared as follows.

First, 20 mg of a monoclonal antibody of the HGG-2 strain obtained by purification from the ascites of a BALB/c mouse and CNBr-activated Sepharose 4, which is an insoluble solid phase carrier, were used.
B (manufactured by Pharmacia) Coupling buffer (0.1M NaHCO3, 0.5M NaCff1) with L mβ
．． The mixture was reacted with rotational shaking for 2 hours at room temperature in pH 8.3).

Next, blocking buffer (0.2M glycine.

After treatment with pH 8.0 at room temperature for 2 days, adsorption buffer (pH 8.0)
mM Tris-HCl, 0.15M NaCl 1° pH 8,
The blocking buffer was removed by washing with 0) to prepare the monoclonal antibody-bound carrier.

The monoclonal antibody-binding carrier prepared as described above was packed into a column, and the bed volume was
Flow the adsorption buffer into the same column to equilibrate, and flow the same column with a mixed solution of 50 μ2 of human serum and 1 ml of the adsorption buffer (L 5 ml/hr).
While washing until absorbance at 0 nm is no longer observed,
The solution that passed through was fractionated into 1 mfl portions.

Next, in order to dissociate and separate the human antibody from the complex consisting of the monoclonal antibody-binding carrier and the human antibody, an elution buffer (0.2M Nax Cox,
The adsorption elution solution was fractionated into 1 mff1 portions while flowing P Hll, 0) until absorbance at 280 nm was no longer observed.

After each column operation, the results of removal or recovery of human antibodies from human serum were examined using each fraction that passed through the column and each fraction that had an absorbance at 280 nm from the adsorbed elution fraction. .

That is, in order to increase the concentration of each percentage of protein with absorbance at 280 nm to a concentration that can be detected in slab gel electrophoresis using SDS polyacrylamide, an ultrafiltration membrane (such as protein) was used. (substances with a molecular weight of 10,000 or more do not pass through), 25μ2 of each concentrated solution was taken, reduced with β-mercaptoethanol, and each was used to perform slab gel electrolysis using SDS polyacrylamide. A migration experiment was conducted.

As a result, in the fraction of human serum that passed through the monoclonal antibody-binding column, almost no bands corresponding to the H chain and L chain portions of human IgG were observed (that is, there was no human antibody in each fraction of these). was not included).

-4. In the adsorbed elution fraction of human serum using a monoclonal antibody-binding column, almost no other bands were observed other than the band corresponding to the H chain of human IgG, 1 (partially). each portion contained only human antibodies).

From these results, it was found that the monoclonal antibody of the HGG-2 strain can specifically react with human antibodies in human serum and recover highly pure human antibodies (or remove human antibodies). ).

Furthermore, even if the same experiment as above was performed again using the column filled with the antibody-immobilized carrier that had been dissociated as described above, the same results were obtained, indicating that the same column can be reused. .

〔Effect of the invention〕

The monoclonal antibody of the present invention, which has extremely high specific reactivity to the human antibody, can be used in the production of pharmaceuticals, reagents, and the like.

Claims (4)

[Claims] (1) IgG and Ig of human antibodies produced from hybridoma lines established by cloning hybridomas obtained by cell fusion of lymphocytes and myeloma cells of small experimental animals obtained by immunization with a human antibody mixture.
A monoclonal antibody against a human antibody characterized by having extremely high specific reactivity with any of M. (2) Cultivating a hybridoma strain established by cloning a hybridoma obtained by cell fusion of lymphocytes and myeloma cells of a small experimental animal obtained by immunization with a mixture containing human antibodies in the peritoneal cavity of a small experimental animal. A method for producing a monoclonal antibody against a human antibody, which is characterized by producing a monoclonal antibody having extremely high specific reactivity with both IgG and IgM of human antibodies. (3) A carrier to which a monoclonal antibody against a human antibody having extremely high specific reactivity with both IgG and IgM of human antibodies is bound is brought into contact with a human antibody-containing solution, and the monoclonal antibody A method for removing or recovering a human antibody, which comprises separating a complex consisting of a human antibody and a human antibody. (4) The method for removing or recovering human antibodies according to claim 3, which comprises dissociating the complex into a carrier to which the monoclonal antibody is bound and a human antibody.