JPS59141524A - Antibody adsorbent - Google Patents

Abstract

PURPOSE:An antibody adsorbent, prepared by immobilizing single-stranded DNA or IgG (antibody) on porous beads having specific properties, and capable of adsorbing and removing efficiently systemic lupus erythematosus (SLE) factor and rheumatic factor. CONSTITUTION:An antibody adsorbent obtained by immobilizing a single-stranded DNA or IgG on porous beads having many pores, having 900-2,200Angstrom , preferably 1,200-1,800Angstrom pore diameter, and passing therethrough and further 10- 30m<2>/g surface area. The particle diameter of the beads is preferably 70-200mu from the viewpoint of handling, flow velocity, clogging, etc. Glass beads, silica beads, plastic beads such as polyaminostyrene may be cited as the preferred beads, and the single-stranded DNA and IgG are immobilized on the beads by the well-known method. The antibody adsorbent is an adsorbent having a large surface area of the support for the antigen and improved adsorption efficiency capable of easily adsorbing systemic lupus erythematosus (SLE) factor and rheumatic factor.

Description

[Detailed description of the invention] 18 Background of the invention Technical field The present invention relates to a novel antibody adsorbent.

More specifically, the present invention is an antibody adsorbent in which single-stranded DNA or IgG is immobilized on porous beads having specific properties. Single-stranded DNA is known as an antigen for systemic lupus erythematosus (SLE) factor, and denatured IgG is known as an antigen for rheumatoid factor. Therefore, research is being conducted on a treatment method that removes the factors in the body fluids by guiding the body fluids of these patients outside the body and placing a device on the body fluid flow path of the extracorporeal circulation to adsorb and remove these factors (antibodies).

The present invention relates to an antibody adsorbent used for adsorbing and removing SLE factors or rheumatoid arthritis factors present in patient body fluids.

Prior Art Conventionally, as an antibody adsorbent, one in which an antigen is immobilized on a support such as polysaccharide or plastic has been used. For example, as an adsorbent for rheumatoid arthritis factor, one in which tryptophan is bonded to an insoluble polyvinyl alcohol support is known. However, the adsorption capacity of conventional antibody adsorbents is not necessarily high enough to be satisfactory, and there has been a desire for an antibody adsorbent with higher efficiency.

(1) Purpose of the Invention In order to develop an antibody adsorbent with excellent adsorption efficiency, the present inventors completed the present invention as a result of extensive research into the physical conditions that an antigen support should have.

The present invention firstly uses an antibody adsorbent in which single-stranded DNA or IgG is immobilized on porous beads having a large number of penetrating pores with a pore diameter of 900 to 2200× and a surface area of 10 to 30 m2/g. The purpose is to provide.

Second, the present invention provides that the porous beads have a particle diameter of 70 to 2.
The purpose of the present invention is to provide an antibody adsorbent having a molecular weight of 00μ.

Thirdly, the present invention provides that the porous beads have a pore diameter of 1,200 to 1,200.
The object of the present invention is to provide an antibody adsorbent that is 1sooX.

A fourth object of the present invention is to provide an antibody adsorbent in which the porous beads are glass beads, silica beads, or plastic beads.

The support on which NA or IgG (immunoglobulin G) is immobilized must have physical properties suitable for the purpose. That is, in order to immobilize as much of the antigen as possible, the surface area of the support is required to be large, and for this purpose beads having a large number of pores with as small a diameter as possible are desirable. However, on the other hand, the pores of the beads may have a pore size such that the antigenic substance enters and is immobilized, and the SLE factor or rheumatoid factor is easily adsorbed to the immobilized antigenic substance. required.

Furthermore, since the body fluid that has come into contact with the adsorbent is returned to the body, harmful substances must not be eluted from the adsorbent support, and fragments of the support must not be released. Furthermore, when the adsorbent is packed in a column and used, it is also required that the flow rate of liquid passing through the column is not reduced or that clogging does not occur, and that the column is easy to handle.

The adsorbent of the present invention is one that satisfies all of these requirements, and has single-stranded DNA or This is an antibody adsorbent made by immobilizing IgG. In the present invention, the surface area is measured by a nitrogen adsorption method.

In the present invention, the pore size of the bead pores is critical! l)
, 900 to 2200X is efficient, and more preferably 1200 to 1800X. It is necessary that these pores penetrate through the support particles, and a support having only closed pores has a low adsorption capacity. Further, the particle size of the beads is suitably 70 to 200 μm from the viewpoint of handling, flow rate, clogging, etc. when packed in a column and used.

There are no particular restrictions on the material of the beads, but those that elute harmful substances or those that are weak and break easily should be avoided as harmful substances and broken pieces may enter the body along with body fluids. . Examples of desirable particles include glass beads, silica beads, plastic beads such as polyaminostyrene, and the like. Methods known per se are used to immobilize single-stranded DNA or IgG on these beads. That is, in the case of glass or silica beads, the beads are aminopropylated, then activated by treatment with glutaraldehyde, and then reacted with single-stranded DNA or IgG. In the case of polyaminostyrene beads, they are activated with carbodiimide and then reacted with single-stranded DNA or IgG. Activation reactions and immobilization reactions are usually carried out in a buffer at room temperature. The DNA may be from any animal, but bovine and salmon DNA is preferred because it is easily available.

-IgG is used. Furthermore, denatured IgG is more preferable since it has a higher adsorption amount. When using the adsorbent of the present invention to remove SLE factor or rheumatoid factor from a patient's body fluid, a method known per se is used. That is, blood, lymph fluid, ascites, and other body fluids of a patient are led out of the body through extracorporeal circulation, passed through an antibody removal device containing the adsorbent of the present invention, and then returned to the patient's body, thereby eliminating the amount of fluid in the body fluid. Remove SLE factor or rheumatoid arthritis factor. When introducing body fluids to the removal device, lymph fluid, ascites, blood, etc. may be directly channeled, or the blood may be separated into blood cells and plasma using a filtration device or a centrifugal device in advance, and only the plasma may be guided to the removal device. EXAMPLES The present invention will now be described in more detail with reference to Examples.

EXAMPLE 25% glutaraldehyde in phosphate buffered saline (PBS) at pH 7.2 was added to each of the supports shown in Table 1.
ml, defoamed, stirred at room temperature for 1 hour, drained water and washed with PBS. Next, 1 my/me purified single-stranded DNA derived from calf thymus and 10 mg/ml human IgG.
Add 15 mJ't each and allow to react at 4°C overnight. After removing the supernatant, add 0.1 containing 0.2M glycine.
5M Tris buffer (pH 7,6) 507! Add and leave at room temperature for 2 hours. Then 0.5M NaCt was added.
1 M acetate buffer (pH 4,0) and 0.5 M N
Wash well alternately with 0.1M carbonate buffer (pH 8,0) containing aCt, and finally with PBS.

Next, collect SLE patient serum or rheumatoid arthritis patient serum 1' Q in a flask filled with the adsorbent obtained above.
ml is circulated and the DNA antibody titer and rheumatoid factor titer in the flask are measured before and after passing through the column for 30 minutes. Table 2 shows changes in the amount of DNA and IgG immobilized on each adsorbent, and the titer.

Table 1 *All support particle sizes are 120-200 mesh **Aminopropylated porous glass beads Table 2 Immobilization amount and adsorption results From the results shown in Tables 1 and 2, the pore size is 900-2200 mesh.
It will be clear that beads with X pores specifically adsorb large amounts of SLE factor or rheumatoid factor. On the other hand, the amount of the antibody adsorbed on beads having a pore diameter of 500X or less or larger than 2200X is significantly small.

(2) Effects of the Invention As detailed above, according to the present invention, an antibody adsorbent having an excellent ability to adsorb SLE factors or rheumatoid factors is provided.

If the antibody adsorption capacity is high, a small column can be used when making an extracorporeal circulation column using it, so the priming volume can be small, and costs can be reduced.

According to the present invention, there is further provided an IgG adsorbent that is easy to handle and can be easily handled, with a rapid flow of liquid through the column in successive layers and less clogging.

The porous beads used in the present invention have sufficient strength so that body fluids can be passed through the column under pressure, and the rate of fluid passage can be further increased.

The present invention further provides a safe antibody adsorbent that does not elute harmful substances or release bead fragments when it comes into contact with body fluids.

Patent applicant: Terumo Corporation

Claims (4)

[Claims] (1) An antibody adsorbent formed by immobilizing porous beads K single-stranded DNA ttaba IgG having a large number of penetrating pores with a pore diameter of 900 to 2200X and a surface area of 10 to 30 m2/L1. (2) The antibody adsorbent 0 according to claim 1, wherein the porous beads have a particle size of 70 to 200μ. (3) The antibody adsorbent according to claim 1 or 2, wherein the porous beads have a pore diameter of 1200 to 1800 holes. (4) The antibody adsorbent according to any one of claims 1 to 3, wherein the porous beads are glass beads, silica beads, or plastic beads.