JPH02189462A - Preparation of immobilized antibody - Google Patents

Abstract

PURPOSE:To enable such preparation of an immobilized antibody as not to impair the activity of an antibody by immobilize the antibody directly on an insoluble carrier at pH in a prescribed range. CONSTITUTION:An insoluble carrier is immersed in an antibody solution obtained by dissolving an antibody or an antibody fragment in a water solution of pH 2.0 to 4.5, so that the antibody or the antibody fragment be immobilized on the insoluble carrier. If pH of said solution is smaller than 2.0 or larger than 4.5, the activity of the antibody after immobilization is insufficient and sensitivity is low. The insoluble carrier whereon the antibody is to be immobilized is immersed in this solution and set stationary for about 1 to 24 hours at 4 deg.C or for several hours at a room temperature. Thereafter, the insoluble carrier whereon the antibody is immobilized is dried in air or centrifuged to remove water therefrom and preserved, or it is washed several times with PBS (phosphate buffer balanced saline solution), set stationary thereafter in a PBS solution of bovine serum albumin of about 1% for about 2 to 12 hours at the room temperature, washed further several times with the PBS and dipped in an appropriate buffer solution to be preserved.

Description

[Detailed description of the invention] a.Industrial application field The present invention relates to a method for producing immobilized antibodies.

b Problems to be solved by the prior art and the invention Enzyme immunoassay (EIA) and radioimmunoassay (RIA), which utilize immune reactions, are one of the methods for measuring trace amounts of substances present in living organisms, and they play an important role in clinical testing. In particular, EIA has been rapidly developed in recent years because it has high specificity and high sensitivity even without using radioactive substances.

In general, these measurement methods use antibodies immobilized on insoluble carriers (hereinafter referred to as "immobilized antibodies"), which must have high specificity, a large amount of immobilized antibody, and high activity. In addition, nonspecific adsorption of enzyme-labeled antibodies may be reduced.

Conventionally, methods for producing immobilized antibodies include a method of covalently bonding the antibody to an insoluble carrier via a crosslinking agent with an amino group, a carboxyl group, etc. of an insoluble carrier, a method of immobilizing the antibody in a matrix of an insoluble carrier, or a method of immobilizing the antibody on an insoluble carrier. There are methods such as physical adsorption. Covalent bonding has the advantage of firmly binding the antibody to the insoluble carrier, but it tends to have a negative effect on the antibody during the binding reaction and the procedure is cumbersome, so physical adsorption methods can obtain immobilized antibodies with simple procedures. is mainly used.

A general method for physical adsorption is, for example, immersing an insoluble carrier in an antibody solution and allowing the reaction to occur overnight at 4°C or for several hours at 25°C.

However, with the physical adsorption method, it is not always possible to immobilize a sufficient amount of antibody on an insoluble carrier. Attempts have been made to increase the adsorption m (Japanese Unexamined Patent Publication No. 60260857) by improving the adsorption m, but these have not always been sufficient in terms of sensitivity.

In addition, when immobilizing antibodies, the pH is adjusted to partially denature the antibodies.
2, 5 for a short time (E, I shtka
wa et at, J.

Immunoassay 1, 385-398.
1980), a method to improve measurement sensitivity by pretreating with a denaturing agent such as guanidine, thiocyanate ion, or urea and then fixing it (J, D, Gonnrad).
ie, J, I immunol.

Method 59. 289-299.1983> is reported.

However, in all of the above methods, antibodies are immobilized again after pretreatment, resulting in complicated operations. That is, in the method of Ishikawa et al., the antibody to be fixed is pi-12,5
After treatment in a solution, the acidic solution is neutralized, and then fixed on plastic beads. Also, the above J using urea.

[), Connradie et al.'s method requires one day of pretreatment with urea and then dialysis removal of the urea over several days.

In view of the shortcomings of the prior art, the present inventors conducted extensive research into methods for producing fixed antibodies without impairing antibody activity and using simple operations. We decided to directly immobilize the +r antibody on an insoluble carrier, that it is possible to produce the immobilized antibody without impairing the activity of the antibody, and that the immobilized antibody obtained by this manufacturing method is further heat-treated. The present invention was achieved by discovering that it is possible to produce a more activated immobilized antibody by using the method described above.

Means for Solving Problem C, namely, the present invention, immerses an insoluble carrier in 1 liter-12.0 to 4.5 of an antibody solution containing the antibody or antibody fragment, and prepares the antibody or antibody fragment +-. This is a method for producing an immobilized antibody that is immobilized on the insoluble carrier.

Antibodies used in the present invention include polyclonal antibodies,
Any monoclonal antibody or antibody derived from any animal may be used. Antibody fragments include F(all')2°Fab' and Facb. F(at)')z.

The preparation of the 7 fragments of antibodies such as Fab' and Facb can be obtained by enzymatically treating polyclonal antibodies or monoclonal antibodies using known methods. For example, an antibody can be treated with pepsin to obtain F(atl')2, and this fragment can be further treated with reduction to obtain Fab' (A).

N 1sonoHet at, Arch, Bioc
hem, B 1ophys, 4shi 230 (1960
), P., Parham, J., In+o.
nol,,dan.

2895 (1983), etc.).

The insoluble carrier of the present invention is polystyrene.

Polyester, polyethylene, polypropylene.

ABS, polyvinyl fluoride, polyamine methyl vinyl ether-maleic acid copolymer, 6-nylon, 6.6-
Examples include plastics such as nylon, amino W1 polymer, glass, and silica gel.

Such insoluble carriers may be in the form of beads, microspheres, etc.
, sticks, test tubes, films, microplates, membranes, etc., but are not particularly limited. Moreover, the surface may have any form such as a mirror surface or a rough surface.

In the present invention, the above-mentioned insoluble carrier is immersed in an antibody solution of p)l 2.0 to 4.5 containing the above-mentioned antibody or antibody fragment to immobilize the antibody and the like.

Such a solution can be obtained by dissolving antibodies and the like in an aqueous solution of pi-12.0 to 4.5.

Such an aqueous solution is not particularly limited, but a buffer solution is preferred. Buffers include glycine-hydrochloric acid buffer, sodium citrate-salt F[! [Liquid ii, potassium hydrogen phthalate-hydrochloric acid buffer, citric acid-potassium citrate buffer,
Citric acid-disodium phosphate buffer, sodium acetate-hydrochloric acid buffer, acetic acid-sodium acetate buffer, veronal! Any buffer solution may be used as long as it can satisfy a pH of 2.0 to 4.5, such as 1 buffer solution, and preferably a citric acid-disodium phosphate buffer solution. If the pH of the antibody solution is less than 2.0 or greater than 4.5, the antibody activity after fixation will be insufficient, resulting in low sensitivity, which is not preferred.

Among them, pl-12.5 to 3.5 is preferable. The salinity of the buffer solution is used in the range of 0.01 to 1M, preferably in the range of 0.1 to 0.5M.

Conditions such as the concentration of antibody, etc. in the antibody solution and fixation time vary depending on the type of antibody or antibody fragment, but generally a solution of 1 to 100 μg/d is used, and an insoluble carrier on which the antibody is to be immobilized is added to the solution. Soak for 1 to 24 hours at 4℃
or leave it at room temperature for several hours.

After standing, the insoluble carrier on which the antibody was immobilized was either air-dried, centrifuged to remove water, and stored, or washed several times with phosphate buffered saline (PBS). After that, add PBS' containing about 1% of bovine serum albumin.
m solution (hereinafter abbreviated as BSA-PBS) at room temperature.
Leave to stand for a while, wash several times with PBS, and store by immersing in an appropriate buffer.

The immobilized antibody prepared in this way has a stronger antigen binding ability than the immobilized antibody fixed at a pH around neutrality, and the final measurement sensitivity is greatly increased.

The present invention provides a method for producing a fixed antibody, which can obtain a fixed antibody with even higher sensitivity by further heat-treating the fixed antibody obtained by the above method in a solution. Such heat treatment may be performed directly after the immobilized antibody is taken out from the antibody solution, or after the immobilized antibody is taken out and washed and then immersed in an approximately 1% BSA-PBS solution and washed.

The humidity of the heat treatment for 2 hours varies depending on the antibody to be immobilized, but it is preferably carried out at a temperature of 30 to 80°C, and the period of time is 1 hour to several days, particularly preferably 2 to 24 hours.

The heat treatment is preferably performed in a solution. A preferable example of such a solution is a buffer solution having a pH of 8 to 8. Examples of such Wlil solution include phosphate buffer, hydrogen phthalate buffer, potassium dihydrogen phosphate-borax buffer, disodium phosphate buffer, and Benard's buffer.
Any am solution can be used as long as it is a buffer solution that can adjust the pH to 6 to 8, such as Ii buffer solution or Tris-HCl buffer solution.
Phosphate buffer is preferably used. The salt concentration of the buffer solution used is in the range of 0.01 to 1M.

Fixed antibodies heat-treated in this way have stronger antibody activity than fixed antibodies that are not heat-treated, and the final measurement sensitivity is significantly increased.

Method 'C of the invention - The obtained immobilized antibody is subjected to conventional EIA
Measurement kits, such as TSH, GH, LH.

Hormones such as FSH, HCG, prolactin, parathyroid hormone, insulin; e.g. ferritin, C
Veptides such as EA and alpha-fetoprotein; for example, protein C, protein S, plasmin/α2 plasminogen inhibitor complex 1 tissue plasminogen activator/blasminogen activator inhibitor complex, thrombin/antitumbin complex, thrombomodulin It can be used for fibrinolytic proteins such as, but not limited to.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 A monoclonal antibody against human chorionic gonadotropin (+-ICG) was prepared in different solutions of (+)8
1-9) 10μg/lR1! A stick of polystyrene was immersed in the solution and incubated at 4°C for 18 hours. This stick was then washed three times in 10 mM PBs (pH 7,2) to obtain an antibody-immobilized stick.
0.001 U/m of HCG O, 25 m and 0.75 d of peroxidase-labeled anti-HCG antibody were immersed in the incubation solution for 20 minutes. Next, wash the stick with tap water and add 7 parts (parts by volume) of 0.11% HzOz.
and 3 parts (by volume) of 0.08% 3.3',5.5'-tetramethylbenzidine. After 10 minutes, remove the stick and measure the absorbance of the remaining solution using a spectrophotometer at a wavelength of 650 nm.
It was measured with The results obtained in this way are shown in FIG.

As is clear from the figure, at pt-+ 2.5 to 4.0, the absorbance at 011 U/d did not increase and 1
It can be seen that the absorbance at 0IIIU/Id and 100m1U/d is significantly higher, and the sensitivity is increased after all.

Example 2 The same procedure as in Example 1 was carried out using a monoclonal antibody against LH instead of the monoclonal antibody against HCG. The results of measuring l-H instead of ICG are shown in FIG. l-I As in the case of CG, p! -12,
It can be seen that the upper limit of sensitivity is about 1.5 to about twice or more in the range of 0 to 4.0.

Example 3 In the same manner as in Example 1, in solutions with different p<pH 1~
9) Polystyrene beads (Building Chemical #80) were immersed in a solution containing a monoclonal antibody against protein C, a coagulation system protein, dissolved at a concentration of 10 μg/d, and incubated at 2 to 8°C for 20 ± 4 hours. did.

The beads were then washed with PBS. The beads thus obtained were used as antibody-immobilized beads. These antibody-immobilized beads were
After incubation in a mixture of standard human serum diluted 51 times with BS or PBS alone and peroxidase-labeled anti-protein C antibody and washing, color was developed in the same manner as in Example 1, and absorbance was measured. The results are shown in FIG. As is clear from the figure, an increase in sensitivity of about 4 to 5 points is observed in E) H2.0 to 3.0.

Example 4 Mouse monoclonal antibody LJTI-4) against human plasminogen activator inhibitor at 0.1
20 μg/M phosphate/citrate buffer (pH 3,0)
d, and add polystyrene beads (
Block Chemical Co., Ltd. #80) was immersed in oil and allowed to stand overnight at 4°C.

After washing with PBS, it was immersed in a 1% BSA-PBS solution at room temperature for 2 hours, and after washing with P[3S, it was soaked in PBS at 50°C.
of water volume for 1 to 6 hours and stored at 4°C.

The heat-treated beads were added to a PBS solution of human tissue plasminogen activator/plasminogen activator inhibitor complex (0.20 ng/td).
0.3ai! and incubated at 37°C for 2 hours. Pa with 0.05% T ween20 dissolved
After washing with PBS-T solution (0.37
) and incubated at 37°C for 30 minutes.

After washing with PBS, 2.5-M H202-0,025
% 3.3', 5.5' tramethylbenzidine solution (0.3d) was added, and color was developed at 37°C for 30 minutes. 1
The color reaction was stopped with N sulfuric acid (1d), and the absorbance at 450 nm was measured. The results thus obtained are shown in FIG.

As is clear from the figure, by heat treatment, OnM
II! The OD of 20 ng/mR was in the upper limit, and the OD of 20 ng/mR was about twice as high when the heating time was 2 hours or more.

The mouse anti-human plasminogen activator inhibitor monoclonal antibody (J'Tl-4) is
It was obtained by the method described in Japanese Patent Application No. 1981-81366 (title of the invention: "Monoclonal antibody against plasminogen activator inhibitor", filed on April 4, 1988). That is, VanMourich J, A
,et at,, J,Biol,Chem,.

259, 14914-14921 (1984), and isolated from the culture supernatant of human vascular wall endothelial cells.
nl obtained by immunizing mice with purified plasminogen activator inhibitor (FAI) as an antigen.
anti-FAI by cell fusion according to standard methods using 1 cell.
Four hybridoma clones (JTII-4) producing monoclonal antibodies were obtained. All JT11-4 are PA
JTl-4 recognizes t-PA-I and t-PA-PAI complexes, has a subclass of IgG+, has no effect on the neutralizing ability of PAtAt, and is a quiescent PA I, S.
It bound to both DS-activated PAI.

In addition, mouse anti-human tissue plasminogen activator monoclonal antibody (JTA-1) was obtained by patent application in 1983.
It was obtained by the method described in No.-81367 (title of the invention "Monoclonal antibody against plasminogen activator", filed on April 4, 1988). That is, from human melanoma cell culture supernatant, the method of R1jken D, C, et al. (R1jken D, C1etat J,
Biol, Chem, 256.7035
~7041 (1981)) to produce anti-tPA monoclonal antibodies by immunizing mice with anti-11JI and performing cell fusion using spleen cells obtained by immunizing mice using anti-11JI. The hybridoma was cloned and 4 clones producing anti-tPA monoclonal antibodies JTA1-4 were obtained.

JTA1-4 all bind to tPA and the tPA-PAI complex, JTA-1, 2, and 4 bind to the H chain of tPA, but JTA-3 does not, and all of them are of subclass IOG+. there were.

The above mouse anti-human tPA monoclonal antibody was
The F ab' fragment of the antibody was obtained by processing according to the method of Nisonoff et al.

Example 5 0.1M rabbit antibody against bovine chondrocalcin
20 μg in phosphate/citrate buffer (pi-13,0)
/d, and add polystyrene beads (
(Mizuzu Kagaku, #80) was immersed and left to stand at 4°C overnight.

After washing with PBS, the samples were immersed in a 1% [3SA-PBS solution for 2 hours at room temperature, then washed with PBS, heated in PBS in a 50°C water bath for 3 hours to 6 days, and stored at 4°C. did.

The heat-treated beads were mixed with bovine chondrocalcin in PBS.
solution (0.5 nl; l/#lf (0.2 me) and peroxidase-labeled rabbit antibody against bovine chondrocalcin (0.2 m) at 37°C.
It was left undisturbed for 2 hours. After washing with PBS-T, 2.5
mM H202-0,025% 3.3', 5.5
A mixed solution of 1-tetramethylbenzicine (0.4d) was added, and color was developed at 37° C. for 30 minutes. The color reaction was stopped with 1N sulfuric acid (1d), and the absorbance at 450na+ was measured.

The rabbit antibody against bovine chondrocalcin was prepared using the method of Choi et al. (H, U, Choi et al., J.

B iol, chem, 258. 655-66t
, 1983), bovine ]ndrocalcin was purified from the antiserum obtained by immunizing a domestic rabbit.

The results thus obtained are shown in FIG.

As is clear from the figure, 0n (1/d O
D did not change, and the OD of 5n (1/aj!) showed an OD of about 1.6 times due to heating.

[Brief explanation of the drawing]

FIG. 1 shows the results of HCG measurement using an immobilized antibody fixed in an HCG antibody solution at pH 1 to 9. FIG. 2 shows the results of LH measurement using an immobilized antibody fixed in an LH antibody solution at pH 1 to 9. FIG. 3 shows the measurement results of protein C in the case of t- using an immobilized antibody fixed in a protein C antibody solution at pH 1 to 9. Figure 4 shows the human tissue plasminogen activator using a fixed antibody fixed with a human plasminogen activator inhibitor MCΔ antibody solution at pH 3.0 and then heat-treated for 1 to 6 hours.・Showing the measurement results of blasminogen activator inhibitor complex. Figure 5 shows the results of using fixed antibodies fixed with a solution of anti-bovine chondrocalcin PCA antibody at pH 3.0, which was further heat-treated by varying the heating time.
The measurement results of chondrocalcin are shown. Patent applicant: Teijin Co., Ltd.

Claims (1)

[Claims] 1. Containing an antibody or antibody fragment at pH 2.0~
4. A method for producing an immobilized antibody, which comprises immersing an insoluble carrier in the antibody solution of 5 to immobilize the antibody or antibody fragment onto the insoluble carrier. 2. The method for producing a fixed antibody according to claim 1, which comprises immobilizing the antibody or the fragment on the insoluble carrier and then heat-treating the obtained fixed antibody in a solution. Production method.