JPS5917779B2 - Method for producing specific antibody against HBsAg - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing specific antibodies against HB and Ag.

More specifically, plasma or serum immunoglobulin IgG,
Or from purified globulin obtained by fractionating these, B
Related to type hepatitis ■) Specific antibodies against HB and Ag (
Anti-HB below.

This invention relates to the selective acquisition of an electrophoretically slow (s10w) immunoglobulin fraction, which has been newly discovered to be concentrated in electrophoretically (denoted as s10w), using known fractionation techniques. HBsAg exists in human serum, etc.
It is considered to be the so-called transfusion hepatitis virus or a substance closely related to the same virus, and as a preventive measure against transfusion hepatitis, detecting the presence or absence of HBsAg in the blood to be transfused is an important clinical test method that is currently widely used. It is.

Conventionally, HBA in serum has been determined using the Ouchterlony method using anti-HB serum, countermeasures, etc.
・Electrophoresis (Counter-ele)
ctrophoresis), complement fixation reaction, sensitive hemagglutination inhibition reaction, etc.

However, these conventional methods require a long time to make a determination;
The procedure was also complicated. Recently, I took anti-HB globulin and
By mixing the coated latex with the serum to be tested and gently shaking it for several minutes, agglutination of the latex occurs in the serum containing HBA, resulting in HB.

It has been reported that HBsAg detection does not occur in serum that does not contain HBsAg, so it is possible to distinguish between the two, and a reagent for detecting HBsAg prepared based on this principle is commercially available. However, this reagent also has the following drawbacks, and it is considered that it cannot at least replace the various testing methods described above7). 1 It is not particularly sensitive than other methods, and even serum that is clearly determined to contain HBA20 by other methods is often determined to be negative.
Human serum that does not contain this substance may be determined to be positive.

As described above, there is still no detection method that can simply, quickly, and accurately detect HBA, and there has been a desire to establish such a method and a detection agent for detecting HB and Ag.

Based on this technical background, the present inventors investigated a method for obtaining high-concentration anti-HB globulin, and as a result, obtained the target specific antibody, anti-HB, and sensitized it to a granular carrier. It has been found that the reagent obtained in this manner is an HB and Ag detection reagent for reverse passive carrier agglutination reactions with less nonspecific aggregation than ever before.

The present invention is characterized in that Slowe 8G gold belonging to the γ-position 35 (particularly preferably the highly basic γ-position) is fractionated electrophoretically from immunoglobulin obtained from anti-HBs-positive plasma or serum. A specific antibody against HBsAg (anti-HB).

).

In general, immunoglobulin IG electrophoretically belongs mainly to the γ position, but some extend to the α position.
Those belonging to the α and β positions are “Fast IG”
G)", those belonging to the Ggγ position are "slow IgG (Sl
OwlgG)".

For example, according to the findings of the present inventors, it has been found that the tetanus antibody has the highest anti-g titer in the basic part of slow IG (Presentation at the 23rd Society of Blood Transfusion).

In this way, even when viewed electrophoretically, antibodies are localized specifically depending on their type, and some antibodies have antibody activity concentrated in Fast IG. The electrophoretic distribution of this substance was unknown, and in particular, the distribution from the viewpoint of specificity in antigen-antibody reactions was completely unknown.

In order to solve this problem, the inventors developed hyperimmune anti-HBs,
Through repeated research using positive human plasma, it was discovered for the first time that anti-HB was localized in slow IG, and was particularly concentrated at the γ position, which is electrophoretically highly basic.

Furthermore, when a particulate carrier is sensitized with this antibody fraction and an antibody agglutination reaction is performed through an antigen-antibody reaction, the reaction is extremely highly specific compared to sensitization with antibodies obtained by conventional methods. I found out. As is well known, immunoglobulin can be produced using plasma or serum as a starting material, using ethanol fractionation, ammonium sulfate fractionation, ribanol fractionation, ion exchange chromatography, or isoelectric focusing, as well as these methods. obtained by a combination of methods 7).

In general, immunoglobulins are divided into fractions with different electrophoretic behavior.
, γ1, γ2, and other fractions are known.

As a fractionation method for each of these α-, β-, and γ-positions, cation exchange chromatography (ImmunOchemistry: A, 1Ol) is used.
1967)], Anion Exchange Chromatography Method [TransfussiOn] 6,
146 (1966)] and Prock electrophoresis method [Electrophoresis Experimental Methods, p. 303, edited by the Electrophoresis Society (Bunkodo)] and isoelectric focusing method [Biophysical Chemistry 14 (3) 21, 209, (
1969)] are publicly known. In the present invention, human anti-HB positive plasma)
Immunoglobulin obtained from S or serum according to a conventional method is fractionated using a known method, and the γ1 position (γ1, γ2
) fraction, preferably a more basic fraction consisting of γ2.

Among the above-mentioned known fractionation methods, the most preferred fractionation method in terms of the reagents, operations, and conditions used is ion exchange chromatography.

However, immunoadsorption methods can also be used, provided one uses the highly purified HBAlSg required and anticipates some specificity of the product and attenuation of the antibody titer.

The conditions for fractionation by ion exchange chromatography are as follows:
In any case, although selective for each carrier,
This can be done by separating the fraction in which anti-HB is concentrated and Sg is electrophoretically localized in slow IG from the fractionated fraction.

For example, when using a cation exchanger, 0.2 to 0.0
05M. Equilibrate with H4-7 phosphate buffer and transfer
Immunoglobulin is adsorbed to the solution, and the immunoglobulin is eluted while increasing the salt concentration.

From the eluate, the fraction containing the above-mentioned target antibody is examined and separated. Also, when using an anion exchanger, 0
．． The carrier is p-equilibrated with a phosphate buffer of 1 to 0.005 M, H7 to 10, and immunoglobulin is adsorbed and fractionated and eluted while increasing the salt concentration.

In this way, a slow fraction of γ-globulin is obtained. EXAMPLES Below, the present invention will be explained in more detail with reference to Examples.

Example Starting from anti-HB positive human plasma 11, crude immunoglobulin fractions 8.
I got 29.

Anti-H by PHA method (passive hemagglutination assay) of this product
The antibody titer of B8 is 1: when measured with a 1(:fl) solution.
It was 8000. Next, this immunoglobulin 19 was added to 0.
After overnight dialysis against 01M phosphate buffer (H6.O),
Equilibrate with the same pH buffer and add CM-cellulose (Serv
It was adsorbed on a column (diameter: 3CTIL, length: 30?) manufactured by Company A), used for equilibration, and thoroughly washed with the same buffer solution as that used for the column.

Next, 0.01M, H6.0 phosphate buffer and
P.O. OlM, H6. Na in phosphate buffer of O
CllcpO. A friend who performs concentration gradient elution using a solution containing 4M.

It was confirmed that the early eluting fraction is the fast IG fraction, the late eluting fraction is the slow I, G fraction, and the middle one is the middle IG fraction.

After each boule, concentrate under reduced pressure as necessary.
When we standardized the concentration to 1% and measured the anti-HB antibody titer using the PHA method, it was only 1:64 in the FAST IG fraction, but 1:64,000 in the SLOW IG fraction, and 8%.
Purified by 2 times g.

When immunoelectrophoresis was performed using a 1% solution of each fraction, it was found that Slow IG (Sg) with concentrated anti-HB activity
The fraction migrates toward the cathode side among immunoglobulins,
The first IG fraction migrates toward the anode side)
g was given.

The middle IG fraction was migrated at 0g at a position intermediate between the two.

Test Examples Slow IG fraction fractionated with CM-cellulose,
Using the Fast IG fraction and the crude immunoglobulin shown in Example 1gSg as starting materials, an affinity chromatography method [OxSang.
29, 319, (1975)] was used to sensitize sheep red blood cells treated with glutar agluthehyde with a certain amount of IgG [Affinity IG], and the specificity of the obtained reagent was tested. compared.

That is, sheep red blood cells are washed four times with phosphate buffered saline and made into a 5% suspension.

A 2.5% solution of glutaraldehyde was added to this at a ratio of 20%, the reaction was carried out at room temperature for 60 minutes, and then the unreacted glutaraldehyde was removed by washing four times with the above physiological saline and fixed. erythrocytes are obtained. The fixed red blood cells were made into a 5% suspension and sensitized with various immunoglobulins according to the method of Imai et al. (Igaku no Ayumi, 78, 759760 (1970)).

Table 1 summarizes the presence or absence of positive sera from 2,605 adult sera and the presence or absence of false positives by agglutination inhibition reaction using purified erythrocytes to examine the specificity of antibody-sensitized red blood cells.

There were 707 false positives in those sensitized with the first IgG fraction.
It is extremely high in humans, and is the first I among immunoglobulins.
It can be seen that components that cause this unspecific reaction are collected in g in the G fraction.

On the other hand, in those sensitized with IG purified by conventional Affinity chromatography, there were 11 false positives;
When slow IG was used, this process was even more significantly improved, and was reduced to about 1/3.

The fast IG fraction removed in this way has a high non-g-specific reaction, while the slow IG fraction has a high non-specific reaction).
Since the g response is approximately 1/57 of that, the effectiveness of the method of fractionating immunoglobulin into a slow IG fraction and a fast IGgSg fraction is clear.

Furthermore, anti-HB fraction S highly purified using conventional technology
Fractionated into slow IG and fast IG
This suggests the effectiveness of Gg.

Claims (1)

[Claims] 1. A specific antibody against HB_sA_g (anti-H
A method for producing anti-HB_s, which comprises separating "slow I_gG" belonging to the γ-position, which is electrophoretically more basic.