JPH02156156A - Method for bonding antibody to ribosome - Google Patents

Abstract

PURPOSE:To make it possible to measure immunity of antibody-bonded ribosome accurately at high sensitivity by oxidizing an antibody under a mild condition. CONSTITUTION:At first an antibody is oxidized by using an oxidizing agent or reaction with oxygen under a mild condition, i.e. the condition wherein the hydroxyl group in sugar chain in the antibody is oxidized into formyl group but other amino acids are hardly oxidized. Then, the oxidized antibody is bonded to ribosome incorporating a compound having a group which is selected among hydroxyamino group, hydrazino group, ureid group and amino group. The formyl group of the oxidized antibody is bonded to the ribosome by forming reaction of Schiff base.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a method for binding antibodies to liposomes, and more particularly, to antibody-binding liposomes used in diagnostic reagents that utilize complement-dependent liposome membrane damage reactions. This invention relates to a method for binding antibodies to liposomes that can prepare liposomes.

[Conventional technology]

Immunoassay methods that utilize antigen-antibody antibodies, 2, have become indispensable in the clinical diagnosis of various endocrine diseases, and these methods can be broadly divided into labeled methods and non-labeled methods. .

Among these, various labeling substances (markers), such as radioisotopes, fluorescent substances, enzymes, or enzyme-related substances, have been used to carry out labeled immunoassay methods, which are superior in terms of sensitivity. Conventionally, radioisotopes have been widely used as labeling substances due to their sensitivity, but radioisotope reagents have a short half-life and are unstable, causing problems with radiation damage and the use of expensive facilities. In addition, measurement methods that use fluorescent substances or enzymes as labels are attracting even more attention in conjunction with research into improving their sensitivity.

Immunoassay methods that use fluorescent substances or enzymes as labeling substances currently use a heterogeneous system that requires a step to separate labeled substances that are bound by antigen-antibody reactions from those that are not. There are two methods: one method uses a homogeneous system that does not require such a separation step.

Immunoassay methods that use a heterogeneous system include the two-antibody method, which is also used in radioisotope labeling immunoassay, and the solid phase method, but these methods require separation of unreacted substances and reacted substances. However, this separation process is complicated to carry out, and therefore has the drawback that it is difficult to speed up quantitative determination. On the other hand, immunoassay methods that use a homogeneous system were proposed with the aim of simplifying and speeding up quantification by eliminating the need for separation steps, but in reality they have low sensitivity and a limited measurement range. The reality is that it has not been put into practical use for quantifying antigens or antibodies because of its narrowness.

In recent years, as a method to overcome such problems, liposomes encapsulating markers are sensitized with antigens or antibodies, and the sensitized liposomes are allowed to coexist with the specimen so that the antigens or antibodies covalently bound to the liposomes and antibodies in the specimen are sensitized. The same sensitized liposomes as above were reacted with various known amounts of antigen or antigen to form an antigen-antibody complex, and this complex was specifically destroyed to measure the marker flowing out from the liposome. A method is proposed in which antibodies or antigens are quantified by coexisting with antibodies and preparing a standard calibration curve in advance by measuring the outflow marker in the same manner as above, and comparing the above measurement results regarding the specimen with the above standard calibration curve. has been done.

According to this immunoassay method, the above-mentioned problems are solved,
It becomes possible to easily quantify the test substance in a homogeneous system in a short time.

(Problem to be solved by the invention) However, the liposome reagents conventionally provided are
The drawback was that the sensitivity was still not sufficient. That is,
Specific antibodies against antigens are mainly bound to liposomes via cross-linking agents, but conventionally widely used N
-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(p
-maleimidophenyl)butyrate (SMPB), N-
Succinimidyl 4-(p-maleimidophenyl) acetate (SMP^), N-succinimidyl 4-(p-
maleimidophenyl) propionate (SMPP), N
-(γ-maleimidobutyryloxy)succinimide (
Cross-linking agents such as GMBS) and N-(epsilon-maleimidocaproyloxy)succinimide (EMC5) covalently link antibodies to the liposome surface by cross-linking the amino residues of the antibody with, for example, phosphatidylethanolamine in the liposomes. It uses action. but,
Because the amino groups of the antibodies that bind to the crosslinking agent cannot be specified, it was a stroke of luck that the antibodies bound to the liposomes in different directions.

Conventionally, as a technique for imparting directionality to antibody binding, there is a method of treating the antibody (IgG) with pepsin and then reducing it to utilize the thiol group in the antibody hinge, but this method is not only complicated, but also requires two steps. Monovalent antibody (Fab')
Therefore, there were problems such as a decrease in the binding strength with the antigen.

(Means for Solving the Problems) Under such circumstances, the present inventor conducted intensive research and found that the present inventor oxidizes the sugar chain present in the cl-+2 domain of an antibody (IgG) and oxidizes the formyl group produced thereby. The present invention was completed based on the discovery that antibodies can be oriented and bound to liposomes by using the following methods.

That is, the present invention provides a liposome characterized in that an antibody is oxidized under mild conditions and then bound to a liposome containing a compound having a group selected from a hydroxyamino group, a hydrazino group, a ureido group, and an amine group. This is an antibody binding method.

To carry out the method of the present invention, it is first necessary to oxidize the antibody under mild conditions. The mild conditions mentioned here are conditions in which the hydroxyl groups of sugar chains in the antibody are oxidized to formyl groups, and other amino acids are hardly oxidized.Although it can be determined experimentally, it is usually conditions such as periodic acid, etc. The reaction may be carried out at about 25 to 30° C. for about 15 to 60 minutes using an oxidizing agent or by oxygen oxidation.

The oxidized antibody is then combined with the liposomes.

In this method, the formyl group of an oxidized antibody is bound to a ribosome by a Schiff base-forming reaction, so a group capable of forming a Schiff base must be present in the liposome. For this purpose, it is necessary that the liposome contains a compound having a group selected from a hydroxyamino group, a hydrazino group, a ureido group, and an amino group (hereinafter abbreviated as "Schiff binding substance"). Although such liposomes may exist naturally, they can preferably be prepared by adding a Schiff binding substance that can be a liposome component during the production of synthetic liposomes.

Examples of Schiff-binding substances that can be added to liposomes include phosphatidylethanolamine, hydrazine having -NH2 at the end, hydroxylamine, hydrazone, etc. More specifically, difatty acid phosphatidyl such as dipalmitoylphosphatidylethanolamine Ethanolamine is preferred, and other known liposome constituents such as phospholipids and cholesterol can be used. Among these, as for the phospholipid, it is preferable that the number of carbon atoms in the fatty acid residue is 12 to 18, and more preferably an even number.

Liposomes that can be used in the present invention can be produced, for example, as follows. For example, when liposomes are synthesized from phospholipids and cholesterol, it is known that stable liposomes are easily obtained when the ratio of these is around 1:1. The ratio is 0. It is desirable to add a Schiff binding substance so that O2N2.5, especially about 0.1, and synthesize liposomes according to a conventional method.

Since the binding of the oxidized antibody to the liposome is a Schiff base formation reaction as described above, it may be carried out according to the general conditions for this method.

For example, oxidized antibody (1trrg/!lft:
0. OIM carbonate buffer p) 19.2) solution 1 ta
This is carried out by suspending a liposome pellet prepared by a conventional method in il and mixing by inverting the mixture overnight at 4 to 10°C.

The liposomes obtained as described above can be used as diagnostic reagents that utilize complement-dependent liposome membrane damage reactions. Specifically, after encapsulating a labeling substance in an antibody-bound liposome, when this is reacted with a sample and complement, the liposome is destroyed depending on the amount of antigen in the sample, and the labeling substance flows out in proportion to this. Therefore, the amount of antigen in the sample can be measured from the amount of this labeled substance.

The labeling substance encapsulated within the liposome must be hydrophilic and capable of being quantified when eluted outside the liposome. For example, such substances do not exhibit fluorescence due to self-quenching at high concentrations, but at low concentrations (10-'
Fluorescent compounds such as carboxyl fluorescein that emit very strong fluorescence at (below M); Luminescent compounds such as luminol and luciferin that emit light by oxidation reaction outside the liposome; have a specific absorption band in the visible or ultraviolet region Light-absorbing compounds (water-soluble dyes, etc.); Sugars such as glucose and sucrose that are broken down by the action of oxidative enzymes, resulting in oxygen consumption or hydrogen peroxide production; Relatively large ionic compounds such as tetrabentylammonium; Nicotinamide adenine dinucleotide (NAD)
Coenzymes such as ); radical compounds such as methyl viologen are desirable.

〔Effect of the invention〕

The present invention utilizes the 'm chain of the C82 portion on antibodies (IgG) to bind antibodies to liposomes with good orientation, so that antibodies that work more effectively than methods using conventional cross-linking agents can be used. There are many. Therefore, the sensitivity of the antibody-bound liposome as a reagent is high, and more accurate immunoassays are possible.

(Example) Next, the present invention will be explained in more detail with reference to Examples.

Example 1 (1) Preparation of marker-encapsulated liposome Dipalmitoylphosphatidylcholine (DPPII: : 10
mM, 100μ℃), cholesterol (Chol;
10mM, 100μfL) and dipalmitoylphosphatidylethanolamine (DPPE; 1
mM, 100 u 11 ) in a molar ratio of 1:1:0.1, and using chloroform as a solvent, put it in a 10 mu flask and stir well. When the solvent chloroform is evaporated with an evaporator, a film-like substance is formed on the inner surface of the flask. This film-like material is
After vacuum drying for 2 hours, 0.0% of carboxyfluorescein (CF) used as a marker was removed. lN N
Add 100 μl of aOH solution and stir vigorously with a portex mixer for 5-10 minutes.

Then, excess carboxyfluorescein was added to 10,000
By centrifuging at XG, a liposome (MLV type) in which carboxyfluorescein is encapsulated as a marker is obtained.

This liposome contains 0.15M NaClt.
Centrifugal washing was performed three times using 01M carbonate buffer (pH 9,2) to obtain a pellet.

(2) Antibody oxidation treatment: Goat IgG (10l11g/mJl!) 1 mλ
Buffer exchange was performed by gel filtration on Sephadex G25 using 0.1M acetate buffer (pH 4,0). The resulting protein fraction 1.6 mJ2 (4,3
mg protein/m℃) was collected, sodium periodate was added to it to give O, QIM, and the mixture was left in a water bath at 25℃ for 30 minutes.Then, the reaction product was diluted to 0.15M
0.01M carbonate buffer (pH 9,
2) and gel filtration with Sephadex G25 to obtain a protein fraction of 1 mL (approximately 1.9+1 protein/IIIft).

(3) Binding of oxidized antibody to liposomes = The liposome pellet obtained in (1) was added to the protein fraction (oxidized antibody) obtained in (2), and mixed by inverting overnight at 4°C. , GVB- (0,
(containing 1% NaN3) to obtain goat IgG-conjugated liposomes.

Example 2 96-well U-shaped microplate and Goat I obtained in Example 1
The following people can use gG-conjugated liposomes! Released in the past
Assay (LIL8) was performed. In addition, GVB was used for diluting each reagent in the following. First, 100
Goat IgG sensitized liposomes diluted 1:0, 25 μm, 1
Rabbit anti-goat IgG antibody (MB
(manufactured by L) 25 μk.

10CHso complement 25μ! and GVB 25 μm were mixed and incubated for 1 h at 37 °C, then 10
The reaction was stopped by adding 100 μm of mMEDTA-VBS (pH 7.5), and the fluorescence intensity was measured. Fluorescence intensity measurement was performed using Corona Electric's MTP-32 with an excitation wavelength of 49.
2 na+, and the fluorescence measurement wavelength was 530 r+m.

In addition, the CF leakage rate (%) from the liposome is determined by assuming that the fluorescence intensity when no rabbit anti-goat I or gG antibody is added is 0.
Fluorescence intensity when adding 10% Triton X-100 is 1
It was determined from the relative intensity, which is set to 00. The results are shown in FIG.

From FIG. 1, marker release was found to be dependent on the rabbit anti-goat IgG antibody concentration. This clearly shows that goat IgG was bound to the liposome.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the results of a release assay using goat IgG-sensitized liposomes of the present invention. that's all

Claims (1)

[Claims] 1. An antibody to a liposome, which is characterized by oxidizing the antibody under mild conditions and then binding it to a liposome containing a compound having a group selected from a hydroxyamino group, a hydrazino group, a ureido group, and an amino group. combination method.