JPS628054A - Immobilized antigen - Google Patents

Abstract

PURPOSE:To enable manufacturing by a simple method and to attain to enhance storage stability by strongly fixing an antigen to a support, in regenerating crystalline fibroin from an aqueous fibroin solution, by including an antigen in the fibroin. CONSTITUTION:An antigen is prepared in a state included in crystalline fibroin when said fibroin is regenerated from an aqueous fibroin solution. The form of the immobilized antigen for immunoassay is pref. a film form or the membrane applied to a support in order to simplify and facilitate operation for separating an immobilized antigen-antibody composite from the free antibody in a specimen or washing and removing the same. Because the immobilized antigen is generally used in an aqueous solution, it is required that the regenerated fibroin of a carrier has water resistance. Therefore, fibroin receives regeneration treatment so that the crystallization degree thereof is at least 20% or more.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention particularly relates to an immobilized antigen suitable as an immobilized antigen for solid-phase immunoassay.

(Prior art) The reaction in which an antigen and an antibody specifically bind to form a complex and the radioactivity of a radioisotope labeled on the antigen or antibody are measured, or Analytical methods that combine this with a highly sensitive measurement method by measuring elementary reaction rates are called radioimmunoassay (RIA) or enzyme immunoassay (EIA), respectively. These analytical methods are widely used in the fields of biochemistry research and clinical testing as methods for quantifying trace amounts of components in living organisms.

In both RIA and EIA, it is necessary to separate the antigen/antibody conjugate generated in the sample from unbound antigen or antibody, and in order to facilitate the separation operation, the antigen or antibody is generally placed on a carrier. (immobilized antigen or antibody) is used.

Methods for immobilizing antigens or antibodies on carriers include methods of bonding them with chemical bonds to carriers such as dextran (crosslinked), agarose gel, glass beads with organic functional groups on the surface, porous glass, and plastics. A method of physically adsorbing and bonding onto a piece of silicone rubber, a method of enclosing it in a polyacrylamide gel [C11nical Chemistry % vo
I, 19, NO, 12, 1889 (1978)] are known.

It has been known that fibroin is permeable to low-molecular-weight enzyme substrates and can be used as a carrier for enzymes that support such substrates, and that enzyme activity is maintained fairly stably in the carrier. [Published 52-57892,
Agric, Biol, Chem,, 42(9),
1661 (1978)].

However, no attempt has been made to apply the above-mentioned fuveloin as a carrier for immobilized antigen.

(Problems to be Solved by the Invention) The present inventors have conducted two studies on the use of fibroin as an antigen carrier, and have found that various antigens can be firmly and stably encapsulated in fibroin in a state where antigen-antibody reactions can occur. We have completed the present invention by discovering that non-specific adsorption of antibodies to the fibroin carrier (which becomes a factor interfering with analysis in immunoassay or affinity chromatography) can be easily eliminated. be.

(Means for solving the problems) The present invention is an immobilized antigen using crystallized fibroin as a carrier.

The fibroin-immobilized antigen of the present invention is produced by entrapping the antigen in fibroin during regeneration of crystalline fibroin from an aqueous fibroin solution.

The above fibroin aqueous solution is prepared from silk raw materials (raw silk,
Purified fibroin fibers obtained from raw silk scraps, kiki, bis, scrap cocoon, etc.) are treated with hydroxide steel-7 ammonia aqueous solution,
It is obtained by dissolving in a copper hydroxide-ethylenediamine aqueous solution, a rhodanate aqueous solution, a lithium bromide aqueous solution, a calcium chloride aqueous solution, a calcium nitrate aqueous solution, a magnesium nitrate aqueous solution, etc., and dialysis-desalting the salts from these solutions.

The immobilized antigen of the present invention can be made into a film, powder, granule, T&fiber, etc. by changing the fibroin regeneration method depending on the purpose.

The form of immobilized antigen for immunoassay is immobilized antigen-
In order to simplify and facilitate the separation of the antibody complex from the free antibody in the sample or the washing and removal thereof, it is particularly preferable to form the antibody in the form of a film or a thin film coated with a support.

The film-like immobilized antigen is preferably a fibroin aqueous solution (the concentration of fibroin is preferably 2 to 20% by weight).
), the solution is cast on a flat plate such as a glass plate, a Teflon plate, an acrylic plate, etc., and dried to regenerate a crystallized buproin film and then peeled off. More preferably, the antigen is uniformly attached. A fibroin aqueous solution (the concentration of fibroin is preferably 2) is placed on a flat plate or a flat plate on which ultra-thin fibroin films containing high concentrations of antigens are evenly layered.
~20% by weight) is cast and dried to regenerate a crystallized buproin film, which is then peeled off. It can be manufactured by a transfer method or the like.

The above antigen-attached plate is prepared by adding an antigen aqueous solution or a fibroin aqueous solution (the fibroin aqueous solution is preferably 40 to 10% fibroin to the antigen).
%) on a flat plate and drying.

The support coating thin film is coated on a flat plate surface such as a glass plate or acrylic plate or on the inner surface of an analytical test tube with an antigen-containing aqueous fibroin solution (fibroin concentration is preferably 0.1 to 5.
% by weight) and drying to regenerate a thin fibroin film on those surfaces.

It can be used in the form of powder, granules, or fibers, and the powder or granules are preferably prepared by mixing an antigen-containing fibroin aqueous solution (fibroin concentration preferably 2 to 20% by weight) with stirring in an ammonium sulfate aqueous solution, sodium acetate aqueous solution, or sodium sulfate. A method in which solid-phase fibroin is precipitated by adding it to an aqueous solution, separated, and dried. More preferably, glass beads or a silica carrier with a large pore size are impregnated with an antigen-containing aqueous fibroin solution (fibroin concentration is preferably 0.1 to 5% by weight). It can be produced by a method of regenerating a buproin membrane on a carrier by evaporating the water. Fibrous forms can be produced by spinning an antigen-containing aqueous fibroin solution (fibroin concentration preferably 15 to 20% by weight) into the same aqueous fibroin coagulating inorganic salt solution as described above.

Immobilized antigens in the form of powders, granules, or fibers can be used not only in analytical methods, but also as carriers for chromatography, so-called affinity chromatography, for separating and purifying specific components from biological components.

Since immobilized antigens are usually used in aqueous solutions, the regenerated fibroin carrier is required to be water resistant. Therefore, the regenerated fibroin in the present invention is regenerated so that its crystallinity is at least 20% or more, preferably 80% or more. Therefore, when solid-phase fibroin is regenerated by water evaporation, water evaporation is preferably performed by air drying in an atmosphere of room temperature to 60% relative humidity.In this case, crystallization is promoted or quality is stabilized. Therefore, preferably 2 to 4
Alcohols such as ethyl alcohol, ethylene glycol, glycerin or preferably 2 to 20% by weight
It is desirable to add 1% coagulating salt such as sodium sulfate, magnesium chloride, ammonium sulfate, etc. When regenerating fibroin powder or granules by precipitation, an aqueous fibroin solution in which an antigen is dissolved is added to an aqueous solution of a coagulating salt that is 80% or more saturated, and the mixture is precipitated.

The crystallinity of fibroin was reduced to 20% by the above regeneration treatment method.
% or more, about 80 to 50%. In addition,
Here, the degree of crystallinity refers to a crystalline film or a film that can be considered to be almost amorphous obtained by air-drying a crystalline fibroin powder and a fibroin aqueous solution on a flat plate at 4°C.
In a wide-angle X-ray diffraction chart (chart obtained by recording the diffraction intensity in the equator direction) as shown in Figure 1 using Kα rays, diffraction on a straight line connecting the diffraction intensity points of 2θ = 15° and 80° Let A and B be the areas under the intensity curve, respectively.
- defined as the percentage of B/A.

Antigens to be immobilized in the present invention include human chorionic gonadotropin, placental lactogen, luteinizing hormone, protein hormones such as insulin, serum proteins such as α-fetoprotein and haptocrobin, Escherichia coli toxin, and cholera toxin. , influenza virus, rubella virus,
Examples include toxins such as HBs antigen, viruses or virus components, steroid hormones, and various low molecular weight drugs bound to serum proteins.

The amount of antigen to be used can be appropriately selected depending on the purpose, but when regenerating films, powder granules, and fibers from an aqueous antigen-containing fibroin solution, it is usually 0.1 to 40% by weight, preferably 0.1 to 40% by weight, based on dry fibroin. 5
Up to 80% by weight, particularly preferably 1 to 20% by weight, of the antigen is added to an aqueous fibroin solution. When producing an antigen-immobilized film by the transfer method, the antigen is usually applied to the substrate at a rate of 0.2 to 20 μf/d1, preferably 0.5 μf/d1.
It is used by adhering it so that it becomes 15 .mu.I/d1, particularly preferably 1 to 10 .mu.f/c.

(Effects of the Invention) As is clear from the above description, the immobilized antigen of the present invention can be produced by an extremely simple method. As demonstrated by examples, antigens are firmly immobilized on carriers;
It is resistant not only to strong cleaning but also to falling off due to friction, and has excellent storage stability, with RIA%
It has excellent properties as an immobilized antigen for EIA analysis or affinity chromatography.

Hereinafter, the method for producing the immobilized antigen of the present invention and its characteristics will be clarified through Examples.

Example 1 Preparation of aqueous fibroin solution 100f of raw silk was mixed with 1.0% by weight Marcel soap aqueous solution 5
Fibroin raw material 71F was prepared by immersing it in 0.6% by weight Marcel soap 81 and scouring it for 8 hours to substantially remove sericin and the like. .

50f of water and 75% of ethyl alcohol in the kneader under driving.
g and calcium chloride 100jF were added, and after raising the temperature to 76°C, 5ON of the above-mentioned fibroin raw material was added, and 1 hour after the raw material was completely dissolved, 75°C water 2001 was added to dilute the fibroin solution. Obtained. This solution was poured into a hollow fiber dialyzer after bathing and desalted by dialysis with running water to obtain 880 ml of a 5.5% by weight fibroin aqueous solution (calcium chloride residual fi was 0.08% by weight).
were concentrated on a rotary evaporator, and then a portion was diluted with water to give a fibroin concentration of 10%.
A solution adjusted to 18% by weight was obtained.

(B) Preparation of human chorionic gonadotropin (hCG) immobilization film and symmetrical film.
Place a glass plate partitioned on all sides and apply hC of 1000:U.
8 ml of an aqueous solution containing G was cast into a partition and dried at 16°C for 10 hours.Then, the above fibroin aqueous solution (
Concentration is 10% by weight) 15f and glycerin 0.45
The mixed solution of gm was poured into the partition and heated for 15"
It was dried at C for 10 hours. Peel off the formed film,
A flexible film with a thickness of 120 ψm was obtained. Its crystallinity is 86%.

A glass plate with a thickness of 120 mm was prepared in the same manner as above without adhering hCG to the glass plate in advance. Hm1 is a film with a crystallinity of 87%.

(Measurement of effective immobilization amount of C1hCG and nonspecific adsorption amount of antibody to symmetrical fibroin thin film I X 0.5 a
After thoroughly washing the hCG-immobilized film piece cut into pieces with physiological saline, a bovine serum albumin labeled with peroxidase and containing 5 μf of mouse anti-hCG antibody, ton of 0.1
Immerse in 0.5 ml of physiological saline containing % commercially available water at 4°C for 24 hours.
I left it for a while. After thoroughly washing this film piece with physiological saline, it was washed with citric acid (pH 5.5) containing 4.65 mM of 0-phenylenediamine and 4.4 mM of hydrogen peroxide. After immersion in 4 ntl of buffer solution for 5 minutes, 2 Wll of 8.4 N sulfuric acid was added to stop the reaction, and 492 ml of reaction solution was added.
The absorbance at nm was measured. The absorbance is 1.024,
On the other hand, when a symmetrical fibroin film is subjected to the same reaction as described above, the absorbance is 0.018, and from this, the effective hCG immobilized on the film is about 0.018. I It
J/c-j (effective immobilization 2 x 1%), the apparent hCG immobilization rate due to non-specific adsorption was calculated to be 0.0012 IU/d.

(D) Measurement of immobilization strength of immobilized hCG Polyvinyl chloride film (thickness 120 μm) was
hCG 101U/ml phosphate buffer (p
It was immersed in H7,2) for 24 hours at 4°C.

After washing thoroughly with water, the adsorbed hC was removed in the same manner as in (C).
When the amount of G was measured, the absorbance was 1.242, which was about 0.12
It was 1 U/d. The hcc-immobilized fibroin film used in (C) and the above hCG-immobilized polyvinyl chloride film as a control were mixed at 0. Immersed in IM glycine-hydrochloric acid buffer (pH 2, 2) for 5 minutes, and load 1
For those that were rubbed between the filter papers 8 times with kli/cd,
(7) The amount of hCG immobilized was measured in the same manner as in 0. As a result, almost no decrease in the amount of immobilization was observed in the hCG-immobilized fibroin film of the present invention, but in the case of the control polyvinyl chloride film. Approximately 50% of the hCG was removed using the glycine-hydrochloric acid buffer, and approximately 80% was removed using the filter paper.

Example 2 In the same manner as in Example 1 (B), each of the antigens shown in Table 1 was attached to an acrylic plate at a rate of 6 μf/cd (drying was air-dried for 10'010 hours). 1. Next, a solution prepared by adding and mixing 4M amount of glycerin to the fibroin aqueous solution (A degree is 18% by weight) obtained in Example 1 (Al) was cast on it, and after drying at 15° C. for 10 hours, a mixture was formed. Peel off the film, wash thoroughly with water, and
The antigen-immobilized film shown in the table was obtained. The effective amount of immobilized antigen was measured in the same manner as in Example 1(C) using the corresponding mouse antibody labeled with peroxidase.

Table 1 Example 8 2 ml each of human albumin and human IgG.

2% fibroin aqueous solution (80% relative to fibroin)
A polyester film (with a thickness of 2
00 μm).

(The fibroin film was calculated to have a thickness of 2 μm.) After drying at 25° C. for 8 hours, the peroxidase of the antibody against each antigen was used in the same manner as in Example 1(c). When the amount of immobilized antigen was measured, it was 0.06 μg for human albumin and 4-human IgG.
It was 0.08 μf/d.

Example 4 'iN The human-α-fetoprotein-immobilized fibroin film obtained in Example 2 was cut into 0.7 x 1.2 cm,
One end is attached to a 2m x 103 polyester film (thickness 15
0 μm) to make it easier to handle.

A glass test tube with an outer diameter of 18 mm and a length of 90 cm contained θ~820 n/ml of anti-human α-fetoprotein antibody (immunized mouse, monoclonal antibody).
% Bovine Serum Albumin 0.5 ml of physiological saline solution was added, the above film was immersed in each, and reacted at 25"C for 2 hours. Next, after washing the film with water, 1μf7tnl was added.
anti-mouse IgG antibody (immunized animal, domestic rabbit, anti-blood/llI
0.5 ml of 0.1% bovine serum albumin saline containing horseradish peroxidase-labeled gG fraction)
was added and reacted at 26°C for 1 hour.

After taking out the film and washing it with water, it was placed in a glass sample T4Itf prepared separately, and 0-phenylenediamine 19.
1mM, hydrogen peroxide 2.45mM citric acid-2-sodium phosphate buffer (pH 5,5) 0.5 m
1 at a time, reacted for 80 minutes at room temperature in the dark, stopped the enzyme reaction with 2 ml of IN sulfuric acid, and added A to this reaction solution.
bs 492 was measured and a standard curve was obtained.

The results are shown in FIG. 2, and it was possible to measure 5 ng/me of anti-human α-fetoprotein antibody.

[Brief explanation of drawings]

Figure 1 shows an example of an X-ray wide-angle diffraction chart for measuring the crystallinity of fibroin. ), (a)
(b) is the diffraction intensity curve of the fibroin film and (b) of the amorphous fibroin film (control). FIG. 2 shows a standard curve of anti-AFP antibody. Figure 1

Claims (3)

[Claims] (1) Immobilized antigen immobilized with crystallized fibroin (2) The immobilized antigen according to claim 1, wherein the fibroin has a degree of crystallinity of at least 20% or more. (3) The immobilized antigen according to claim 1, wherein the immobilized antigen is in the form of a film.