JPS6354399A - Immobilization of antibody protein with lipid monomolecular film - Google Patents

Abstract

PURPOSE:To densely immobilize the titled substance useful as a diagnostic material or the like, by bringing the antibody protein dissolved in an aq. phase into contact with a long chain fatty acid monomolecular film developed on the surface of the aq. phase to form an antibody protein-monomolecular film complex at the interface and then transferring the formed complex to a solid substrate. CONSTITUTION:A 24-32C long chain fatty acid (e.g., lignoceric acid), a polyvalent metal salt thereof and/or an ester thereof are dissolved in a solvent such as chloroform and then gradually dripped on a water bath filled with distilled water to form a film developed on the surface of the water. A dividing plate placed on the water surface of the water bath is moved until the surface pressure becomes 20mN/m. Subsequently, an aq. soln. of a water-soluble antibody protein is poured into the water bath to bring the antibody protein into contact with the monomolecular film formed on the surface of the aq. phase to form an antibody protein-monomolecular film complex at the interface of the aq. phase. The formed complex is transferred to a solid substrate to immobilize the antibody protein with the lipid monomecular film.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a method for immobilizing antibody proteins. More specifically, the present invention relates to a method of immobilizing a water-soluble antibody protein at high density on a solid substrate while completely retaining its antigen-antibody reaction activity.

Background and Prior Art In diagnostic techniques that utilize antigen-antibody reactions of antibody proteins, many methods have been proposed to date for immobilizing antibody proteins on solid substrates. Some of these are
Functional groups that can react with or adsorb and bond with amino groups or carboxyl groups of antibody proteins are immobilized on the solid surface, but in this case, depending on the setting of reaction conditions for immobilization, denaturation or denaturation of the antibody proteins may occur. However, there was a problem in that the active site was easily deactivated due to non-specific reactions.

In addition, by incorporating antibody proteins into a hydrophilic gel,
Immobilization on a solid substrate is also a possible method, but in this case, the antigen protein approaches the antibody protein in the gel, making it difficult for it to be detected, resulting in a decrease in sensitivity as a diagnostic material using antigen-antibody reactions. In recent years, these methods have been developed by deploying a lipid membrane on the water surface and then introducing water-soluble enzymes (e.g., catalase, ferritin, hemiglobin) from the water.

glucose oxidase, etc.) and antibody proteins [
Immunogen [1 Purine G (HUG), IgE, etc.
] has been reported to be an attempt to adsorb and convert it into a solid charge (Biochem.
, Bio-pt)ys, Aeta,) 225ff
, 382 (1971) and Journal of Cellular Biochemistry] Lee (J, Ce1l.

Bioct+em, ) 29.239 (1985), etc.].

These are certainly excellent methods for immobilizing proteins while retaining their activity, but the lipid membranes conventionally used in this method are made of stearic acid, arachidic acid, and dipalmitoylphosphadecylcholine. (D P P C
> etc., t4. (At least 1μ
g/IJ2 water solubility), or those that easily form bilayer membrane vesicles in water (such as DPPC). Therefore, over time, the antibody protein adsorbed to the membrane on the water surface may be redissolved in the water together with the lipid membrane, or
- Even after being accumulated on the solid M plate, there was a problem in that it was easily dewitted when immersed in an aqueous antigen solution.

<Objective of the Invention> The present inventors have overcome the drawbacks of the prior art and have conducted extensive research to achieve a high retention rate of antigen-antibody reaction activity and immobilization of antibody proteins at high density. As a result, we have arrived at the present invention.

<Disclosure of the Invention> In other words, the present invention provides a monomolecular film of a long-chain fatty acid having 24 to 32 carbon atoms, a polyvalent metal salt thereof, and/or an ester thereof developed on the aqueous phase surface 1. By contacting the water-soluble antibody protein dissolved in the solution, an antibody protein-monolayer complex is formed at the aqueous phase interface, and this is layered on a solid substrate using a covering lipid medium molecule membrane. This is a method for immobilizing antibody proteins.

In the present invention, the antibody protein refers to a protein that causes an antigen-antibody reaction.
It is a general term for water-soluble proteins that contain an antigen recognition site (abbreviated as Fab) and a hydrophobic terminal site (FC and 11
8).

Specific examples of such antibody proteins include immunoglobulin G (abbreviated as IgG), gE, IgH and antibodies thereof, and chorionic gonadotropin ()-ICG.
> antibodies, carcinoembryonic antigen (CEA) antibodies, etc.

When immobilizing these antibody proteins, it is important to prevent the Fab portion from denaturing; however, as mentioned above, in the case of immobilization by conventional chemical reactions, the Fab portion also participates in the reaction and denatures the antibody protein. This resulted in a decrease in activity. In the present invention, the antibody protein is packed into a lipid monolayer (described later) at high density while interacting with the FC site hydrophobically, or the antibody protein is incorporated into the monolayer membrane while maintaining high immunoactivity due to the FC site. It is fixed by suction.

The lipid monomolecular film for immobilizing the antibody protein described above is preferably one that forms a solid condensed monomolecular film on the water surface and does not substantially dissolve in water. Specific examples of such substances include long chain fatty acids having 24 to 32 carbon atoms;
The polyvalent metal salt and/or the sprue are used.

Compounds with such properties include lignoceric acid (C23H47COOH) and cerotic acid (C25H5
1 COOH).

Montanic acid (C2□l-!55Goof-1>, Melisic acid (C29)-159COOI-1>, Laceronic acid (C31H63COOH) and C12n+I
They are polyvalent metal salts of long-chain fatty acids represented by C00M (n = 23-31, M - polyvalent metal ions such as alkaline earth metals, cadmium, aluminum, etc.) and esters of these fatty acids with methanol or ethanol.

These are dissolved, for example, in the form of carboxylic acid or its ester in an organic solvent such as benzene or chloroform.
After making a solution of 5 to 1.5 mmol/1, add distilled water or a solution containing polyvalent metal salts (e.g., barium chloride, cadmium chloride, aluminum chloride), pt16.5 to 7.
．． By spreading it on the surface of the aqueous solution of No. 5, a monomolecular film can be formed. Then, the monolayer was coated with a
After compressing to ~30 mN (milliN)/m, the above-mentioned antibody protein is injected into the aqueous phase below the membrane surface using the same compressed force f↑. Specified time (usually 30 minutes to 1 hour)
By bringing the antibody protein into contact with the monomolecular film on the water surface, the complexation between the protein and the monomolecular film is completed. At this point, the composite film of antibody protein and monolayer is compressed again at a surface pressure of 30 to 50 mmN/m, and then deposited on a solid substrate using the Langmuir-Brosietz method or the horizontal adhesion method (Details 1111). (Refer to Shin Jikken Kagaku Koko Volume 18, page 439), one or more layers can be laminated.

At this time, the amount of antibody protein immobilized on the solid substrate is calculated from the transfer ratio of the water surface spreading film to the substrate during lamination and the LJ V-- absorption spectrum intensity.

The complex on the substrate thus obtained (1) showed superior activity in terms of unity and activity determined by enzyme immunodiagnosis (EIA) for the anti-rabbit-antibody reaction activity of the antibody protein.

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

Example 1 Lignocerin W (C23H47COi41) 8
Dissolve J in 25 ml of distilled water and fill with distilled water. Place 100 μl of the above solution on the π-△ measurement water tank.
was gradually added dropwise. After dropping, the membrane was allowed to stand still for 5 minutes, and then the partition plate was moved until the surface pressure reached 20 mN/m.

Human-IgG aqueous solution in a water tank at a concentration of 0.03mM rnl
After injecting from water so that While compressing this water surface FI=(M membrane under a surface LF of 30 mN/m, it was placed on a quartz plate that had been subjected to hydrophobization treatment (silane coupling treatment) using the vertical immersion method (hereinafter referred to as l- B
The average cumulative ratio is 0 when two layers are accumulated due to the law and subversion.
．． It was 7. The amount of heat accumulated on the quartz plate determined from the ultraviolet absorption spectrum (the coverage of IG is 1.6 Xl0-
[It was 1 mol/butt. After applying a peroxidase-labeled anti-human 1 to IgG antibody (20 μm/d) solution to this accumulated membrane, the antigen activity in the accumulated membrane was examined by immersing it in a mixed solution of orthophenylenediamine and hydrogen peroxide. It was found that it retained its activity.

Example 2 In Example 1, instead of using lignoceric acid, a water surface-extending film was formed using luxelon v, (C311-163COO[-1) to absorb H-IgG from water. When accumulated on a quartz glass substrate, the average accumulation ratio was 0.82. In addition, when the antigenic activity of this product was investigated in the same manner as in Example 1, it was found that I (IG
It maintained the same high level of activity.

Comparative Example 1 A monomolecular film was spread on the water surface using a stearic acid solution in the same manner as in Example 1, and then Hi1-19G was injected into the water. After standing still for 5 minutes, the partition plate was moved to maintain a surface pressure of 30 mmN/m, and the area of the water surface membrane continued to decrease, indicating partial dissolution of stearic acid into the IC1G aqueous solution. When this water surface spread film was deposited in two -C layers on a quartz glass plate by the LB method, the cumulative ratio was as low as 0.41, and partial peeling of the film was observed during the accumulation. The antigen activity of this membrane was evaluated in the same manner as in Example 1, and it was found to have decreased to 173 to 1/4 of the rgG activity in an aqueous solution state.

Claims (1)

[Claims] By bringing a water-soluble antibody protein dissolved in the aqueous phase into contact with a monomolecular film of a long-chain fatty acid having 24 to 32 carbon atoms, its polyvalent metal salt, and/or its ester spread on the surface of the aqueous phase. A method for immobilizing an antibody protein using a lipid monolayer, which comprises forming an antibody protein-monolayer complex at the aqueous phase interface and stacking the complex on a solid substrate.