JPH06104071B2 - Factor IX Monoclonal antibody specific for conformation - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to blood coagulation factor IX monoclonal antibodies, and more particularly to human blood coagulation factor IX conformation specific monoclonal antibodies.

Blood coagulation factor IX is one of the vitamin K-dependent coagulation factors, and its congenital deficiency is known as hemophilia B. Diagnosis of hemophilia B and its carrier, clinical examination or clinical pathological elucidation of various diseases with acquired factor IX decrease, or method for separating and purifying factor IX from raw material liquid The immunoadsorption method, which is one of the methods, requires the use of an antibody against factor IX. However, the usual antibody against factor IX is a polyclonal antibody or an oligoclonal antibody and corresponds to several or more antigenic epitopes on the factor IX molecule. It has certain limits in the adsorption method.

Recently, various examples of preparation of a monoclonal antibody against this factor IX using the established hybridoma production technique such as Milstein have been reported, and its immuno / biochemical and clinicopathologic utility has been investigated. For example, Yoshioka et al., “Clinical Blood 26: 2, 153-158 and 159-165 (198
5) ”and“ Clinical Pathology XXXIV: 4, 469-474 (1986) ”, the Factor IX monoclonal antibody was prepared using the above hybridoma production technique, and its immunological properties, immunoassay using the same, immunoadsorption However, factor IX and other vitamin K-dependent factors (factor X, factor VII, prothrombin, protein C, protein S, protein Z, etc.) are prominent in their amino acid sequences. Due to the homology, it is said that conventional factor IX monoclonal antibodies recognize a small amount of vitamin K-dependent factors other than factor IX. Moreover, by immunoadsorption method using conventional factor IX monoclonal antibodies In the purification of factor IX, 6M guanidine,
Severe eluents accompanied by denaturation of the target such as 8M urea, low pH (eg pH = 2) acid solution must be used, resulting in large loss of purified factor IX bioactivity.

In addition, factor IX is one of the vitamin K-dependent proteins as described above, and includes Ca ⁺⁺ , for example, Sr ⁺⁺ , Mn ⁺⁺ , Co
It binds to metal ions such as ⁺⁺ and Mg ⁺⁺ and causes a more or less conformational change, that is, a change in three-dimensional structure. Tokushusho Sho 61-500226 is based on this conformation specificity IX.
Disclosed is a factor-specific monoclonal antibody, and in particular, when a conformation-specific factor IX monoclonal antibody that recognizes only factor IX bound to a metal ion is used as a factor IX adsorbent in an immunoadsorption method, It is extremely advantageous because it does not require non-specific elution conditions. That is, this conformation-specific monoclonal antibody was used as an immunoadsorbent to adsorb Factor IX in the presence of a metal ion, and then to adsorb the adsorbed Factor IX.
The bound metal ion is removed from the factor by a metal chelating agent such as EDTA. That way,
It is said that factor IX has a different conformation from that when it is bound to a metal ion, and it becomes nonspecific for the adsorbent, and factor IX can be easily purified and obtained without impairing its activity. However, this conformation-specific monoclonal antibody also recognizes Factor IX whose conformation is changed by a metal ion other than Ca ⁺⁺ , as also specified in Japanese Patent Publication No. 61-500226. If an immunoadsorbent is prepared using such a metal ion other than Ca ⁺⁺ , especially a heavy metal ion such as Mn ⁺⁺ or Co ⁺⁺ , or for some reason, the heavy metal ion may enter the purification system. When it is mixed, it is difficult to completely remove all the undesired metal ions even if the metal ion removing treatment is performed by using the metal chelating agent after the adsorption treatment. It is not preferable that trace amounts of such heavy metals remain in the purified Factor IX for any purpose such as therapeutic agents or assays. It is desirable for the purified factor IX to have only Ca ⁺⁺ necessary for its activity.

According to the present invention, blood coagulation Factor IX which recognizes only the conformation caused by the binding of Factor IX to Ca ⁺⁺ and does not recognize the conformation of Factor IX caused by the binding with other metal ions. Factor conformation specific monoclonal antibodies are provided.

The monoclonal antibody of the present invention is generally produced by a technique for producing a hybridoma such as Milstein described above, for example, "Natu
re256, 495- (1975) ”. More specifically, it is obtained by fusing spleen cells of mouse immunized with purified factor IX and mouse myeloma cells and further cloning. Among the monoclonal antibody-producing hybridomas, a hybridoma producing a monoclonal antibody having a specificity only for factor IX whose conformation has been changed by Ca ⁺⁺ is selected from among the hybridomas producing the monoclonal antibody, and the hybridoma is artificial medium. Alternatively, it can be obtained by growing the mouse in the abdominal cavity.

Hereinafter, the present invention will be specifically described with reference to examples.

[I] Purification of Factor IX Freshly frozen plasma was rapidly thawed at 37 ° C., and 1/10 volume of barium chloride was added dropwise with slow stirring at 4 ° C.
It was left for 2 hours.

Centrifuge at 4,000 rpm for 5 minutes at 4 ° C to collect the precipitate,
Suspend in Tris-buffer hydrochloric acid. 25-65% of this solution
Ammonium sulphate cut was performed, the obtained precipitate was resuspended, and DEAE-
Cellulose chromatography was performed. A fraction containing Factor IX was obtained with a concentration gradient of 0 → 0.4 M sodium chloride with a phosphate buffer of pH = 6.0. This fraction was further purified by conventional heparin sepharose chromatography and gel filtration, and concentrated on an Amicon ultrafiltration device (membrane having a molecular weight cut size of 10,000). All operations were performed at 4 ° C.

The purified Factor IX showed a single band on SDS-polyacrylamide gel electrophoresis, and was free from contamination with other coagulation factors.

[II] Preparation of monoclonal antibody 50 μg of the purified factor IX obtained above was dissolved in 0.05 ml of 0.9% physiological saline, and 0.1 ml of DIFCO's FCA as an adjuvant was added to make a water-in-oil type for basic immunization. It was used as an antigen.
Also, as a booster antigen, 50 μg of the above purified factor IX
Was dissolved in 0.05 ml of 0.9% physiological saline and used.

BALB / C mice 4 to 5 weeks old ♀ were inoculated with the antigen for basal immunization, and the mouse spleen cells obtained by twice immunizing with the booster antigen on the 15th day and the 45th day were subjected to the following conventional method, for example, "Takeshi Watanabe" Hybridoma Method and Monoclonal Antibody ", P.20-P.29, R & D Planning Company, 1982"
Mouse myeloma cells (P3-X63-A
It was fused with g8-U1) and cloned to obtain 8 hybridomas producing a Factor IX-specific monoclonal antibody. Each monoclonal antibody was obtained by growing each mouse intraperitoneally.

[III] Measurement of antigen specificity and identification of immunoglobulin subclass The antigen specificity of the monoclonal antibody produced by the eight hybridomas obtained in [II] above was measured as follows.

The measurement was performed by the ELISA method as follows.

1. Antigen coating To two microtiter plates, 50 μl of human factor IX (the same as the one purified above) prepared to 2 μg / ml with 50 mM Tris-hydrochloric acid (pH = 7.5) 0.1 M NaCl was added, Four
Incubated for 16 hours at ℃.

2. Washing _Two buffers containing 10 mM Tris-HCl (pH = 7.5), 0.5 M NaCl and 0.1% Tween 20 containing ₂ mM CaCl ₂ and 2 mM EDTA respectively were prepared, and one microtiter plate had 2 mM CaCl ₂ And the other plate was washed 4 times with a buffer containing 2 mM EDTA.

3. Blocking 50 mM Tris-HCl containing 1% bovine serum albumin (pH =
7.5) A 0.1 M NaCl solution was prepared, 200 μl of the solution was added to each plate, and incubated at 25 ° C. for 2 hours.

4. Addition of hybridoma supernatant 40 μl of hybridoma supernatant was added to each row of each plate and incubated at 4 ° C. for 16 hours.

5. Secondary antibody and color development Anti-mouse IgG with peroxidase bound to each plate
Rabbit antibody (50 μl) was added and incubated at 25 ° C. for 2 hours. After that, 100 μl of O-phenyldiamine solution was added and reacted at 25 ° C. for 30 minutes, and the absorbance at a wavelength of 492 nm was measured.

The results are shown in Table 1, and all are specific for factor IX. Among them, clones No. 3 to No. 8 are specific only to the conformation generated by binding to Ca ^++. I know there is.

The immunoglobulin subclass was identified for mouse α, γ1, γ2a, γ2b, γ3, μ, κ, and λ chains.
Each hybridoma culture supernatant was measured by the RIA method using each antiserum labeled with ¹²⁵ I.

[IV] Degree of Inhibition of Monoclonal Antibody on Factor IX Coagulation Activity The following experiment was conducted to examine the effect of factor IX on coagulation activity.

A hybrid of purified factor IX and clones No.1 to No.8
Monoclonal antibodies obtained from the mouse were mixed at a molar ratio of 1:20
And CaCl₂Is added to a final concentration of 5 mM, and the mixture is kept on ice for 10 minutes.
It was made to react. 50 μl of this mixture was added to 25 mM CaCl₂With 100 μl
Actin ; USA-made by Dade) 100 μl, commercially available
50 μl of factor IX-deficient plasma was added and coagulation time was measured.
It was The results are shown in Table 2 and the inhibitory activity is expressed in%.
is doing. That is, 0% means when there is no extension of coagulation time
However, the longer the coagulation time, the higher the percentage of inhibitory activity.

All show inhibitory activity, especially No. 1-7 is 84-99
%, Showing a strong inhibitory activity.

[V] Reactivity of Monoclonal Antibody Against Other Vitamin K-Dependent Coagulation Factors The antigen specificity of each monoclonal antibody prepared in [II] above was further confirmed as follows.

The experiment was performed by the ELISA method as described above. Prothrombin factor IX, protein C, protein S and bovine serum albumin were used as antigens to be confirmed. 5 μg / m of each purified antigen with 50 mM Tris-HCl (pH = 7.4) 0.1 M NaCl
It was adjusted to 1 and coated on a microtiter plate at 4 ° C. for 16 hours. Hereinafter, the procedures of blocking with bovine serum albumin, washing, addition of a monoclonal antibody, addition of a second antibody, and color development are the same as those in [III] above.

The results are shown in Table 3.

All clones responded only to Factor IX and not to each other coagulation factor and bovine serum albumin.

[VI] Measurement of Specificity of Monoclonal Antibody for Conformation with Other Metal Ions The experiment was carried out by the ELISA method as in the above [III].

The factor IX purified in [I] above was coated on 9 rows of microtiter plates. MgCl ₂ , MnCl ₂ , CuCl ₂ , N
Nine kinds of 10 mM Tris-HCl (pH is 2 mM) containing dCl ₂ , LaCl ₂ , ZnCl ₂ , FeCl ₂ , CoCl ₂ and CaCl ₂ (concentrations are all 2 mM)
= 7.5), 0.5M NaCl, 0.1% Tween20 was prepared, and each row of the microtiter plate was washed four times with each buffer containing the above 9 kinds of metal ions. Hereinafter, the procedure of blocking with bovine serum albumin, addition of the monoclonal antibody, addition of the second antibody, and color development is the same as in the above [III].

The results are shown in Table 4.

As is apparent from Table 4, the clone No. according to the present invention.
Monoclonal antibodies 4, 5, 6, and 8 specifically recognize only the factor IX conformation produced by Ca ⁺⁺ ions, and recognize the factor IX conformations produced by other metal ions. I know I won't.

Clone No. 2 is a positive control.

[VII] C when a monoclonal antibody recognizes factor IX
Effect of a ⁺⁺ ion concentration As described above, the monoclonal antibody of the present invention discriminates only the conformation of factor IX caused by Ca ⁺⁺ . At this time, it was examined how much mM Ca ⁺⁺ was required for the monoclonal antibody.

The experiment was performed by the ELISA method in the same manner as in [III] above.

The Factor IX purified in [I] above was coated on 9 rows of a microtiter plate. 0,0.1,0.2,0.4,0.6,0.
A buffer consisting of 9 kinds of 10 mM Tris-hydrochloric acid (pH = 7.5), 0.5 M NaCl, and 0.1% Tween 20 each containing 8,1.0, 1.5, and 2.0 mM CaCl was prepared. It was washed 4 times each with a buffer containing a concentration of Ca ⁺⁺ ions. Hereinafter, the procedure of blotting with bovine albumin, addition of the monoclonal antibody, addition of the second antibody, and color development was performed in the same manner as in the above [III].

The results are shown in Figure 1.

From Figure 1, clones Nos. 1 and 2 are the first regardless of Ca ⁺⁺ concentration.
It can be seen that it is associated with factor IX. On the other hand, clone No.
In 5,6, binding to factor IX increased with increasing Ca ⁺⁺ concentration. The concentration of CaCl ₂ at the time of 1/2 of the maximum amount of binding to clones No. 5 and 6 to factor IX was 0.5 to 1.0 mM, and
It was consistent with the amount required to change the conformation of the factor.

[Brief description of drawings]

FIG. 1 is a graph showing the specificity of the monoclonal antibody of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location G01N 33/53 L 8310-2J 33/577 B 8310-2J (C12P 21/08 C12R 1:91)

Claims (1)

[Claims] 1. A monoclonal antibody which recognizes only a conformation produced by binding of blood coagulation factor IX to Ca ^++, and which does not substantially recognize a factor IX conformation bound to another metal ion. .