JPS63158461A - Reagent for immunoassay - Google Patents

Abstract

PURPOSE:To realize a reagent for immunoassay capable of performing accurate quantification, by using phospholipid, glycolipid or fatty acid having an antigen or antibody immobilized thereon when a liposome of a multiple layer is formed. CONSTITUTION:A liposome is brought to a multiple layer consisting of two or more film layers and two or more of the film layers respectively contain phospholipid, glycolipid or fatty acid having an antigen or antibody immobilized thereon. By this method, the antigen or antibody is always immobilized not only in the film of the outermost shell layer of the multiple layer liposome but also in several inside layers thereof. Therefore, a functional group becoming the cause of non-specific destruction is not present in all of the films of the multiple layer liposome. As a result, for example, after the antigen-antibody reaction of the substance to be examined in serum being a specimen and the liposome is finished, the protein in serum or a very small amount of the substance therein is not reacted with the liposome and accurate quantification can be performed.

Description

Detailed Description of the Invention [Object of the Invention] (Industrial Application Field) The present invention relates to an immunoassay reagent for quantitatively analyzing a specific antigen or antibody present in a sample.

(Prior art) For quantitative analysis of specific antigens or antibodies present in a sample,
For example, radioimmunoassay (hereinafter referred to as RIA) is used. However, since RIA uses radioactive elements, it requires special equipment to be installed and operated by a qualified operator, and there are problems in that it requires care in the disposal of waste, etc.

In addition, other analytical methods, such as immunoelectrophoresis, are known. However, immunoelectrophoresis requires a long time for measurement, has low sensitivity, and cannot be applied when only a trace amount of a test substance is contained.

Therefore, the present inventors previously disclosed in Japanese Patent Application No. 58-224509 a liposome reagent in which a hydrophilic antibody or antigen is immobilized on the surface and a hydrophilic labeling substance is encapsulated inside.

This immunoassay reagent can be produced by the following method.

First, a desired lipid and a crosslinking agent are reacted in a solvent to introduce a functional group into a lipid molecule to form a functional lipid. This functional group acts as a functional group for immobilizing the antibody or part of the antibody or antigen on the liposome. Next, the obtained functional lipid and, if necessary, appropriate amounts of cholesterol and other lipids are placed in a flask, a solvent is added to dissolve and mix, and the solvent is removed by suction and dried. As a result, a thin lipid film is formed on the flask wall. Subsequently, a multilayer liposome suspension is prepared by adding an aqueous solution containing a labeling substance into the flask, sealing the flask, and shaking the flask.

On the other hand, if necessary, a crosslinking group is introduced into the antibody, a part of the antibody, or the antigen to be immobilized on the liposome by reaction with a crosslinking agent, and then, if necessary, the antibody is modified by treatment with a reducing agent.

Next, the ribbon-like suspension is reacted with the antibody, a portion of the antibody, or the antigen in a suitable buffer, thereby immobilizing the antibody, the portion of the antibody, or the antigen on the liposome.

Thereafter, the antibodies or antigens that are not immobilized with the buffer solution are washed and suspended in an appropriate amount of the buffer solution to prepare an immunoassay reagent consisting of multilayered liposomes.

However, in the case of multilayered liposomes obtained as described above, the functional groups of the functional lipids on the membrane surface of the outermost shell of the liposome bind to the antibody, a part of the antibody, or the antigen; The functional groups of the functional lipids in the several layers of membranes on the inner side exist in an unreacted state without immobilizing antibodies or antigens.

It has been found that when a serum or protein-containing sample is analyzed using a liposome reagent in such a state, a nonspecific reaction occurs in addition to the antigen-antibody reaction, and the liposome is destroyed due to this. This occurs after the outermost membrane of the multilamellar liposome, on which antibodies or antigens are immobilized, is destroyed by the antigen-antibody reaction, and then the several layers inside the multilamellar liposome are destroyed by the proteins in the sample. It is thought that they are sequentially destroyed by reactions with trace amounts of chemicals.

Therefore, since the membrane of the multilamellar liposome is destroyed by a reaction other than the antigen-antibody reaction, the labeling substance encapsulated in the liposome leaks out regardless of the concentration of the analyte, making stability difficult. there were.

(Problems to be Solved by the Invention) The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide an immunoassay reagent that allows precise and simple analysis.

[Structure of the invention]

(Means for Solving the Problems) The immunoassay reagent of the present invention comprises a liposome whose composition is a phospholipid or a glycolipid or a fatty acid on which an antigen or an antibody is immobilized with either a phospholipid or a glycolipid. An immunoassay reagent comprising a labeling substance encapsulated in a liposome, wherein the liposome is a multilayer liposome consisting of a membrane of 2M or more, and each of the two or more membranes immobilizes an antigen or an antibody. It is characterized by containing at least phospholipids, glycolipids, or fatty acids.

The immunoassay reagent of the present invention can be produced, for example, by the following method.

First, an appropriate amount of lipids and, if necessary, cholesterol are placed in a flask and a solvent is added to dissolve and mix them, and then the solvent is removed by suction and dried. As a result, a thin lipid film is formed on the flask wall.

On the other hand, an antigen or antibody immobilized on the phospholipid, glycolipid, or fatty acid derivative is prepared by chemically reacting the antigen or antibody with the phospholipid, glycolipid, or fatty acid derivative.

Next, a solution containing a labeling substance and an antigen or antibody immobilized on the phospholipid, glycolipid, or fatty acid derivative are added to the flask, which has a thin lipid film formed on the wall, and the flask is tightly capped and shaken. A suspension of multilamellar liposomes is prepared by:

The phospholipids and glycolipids used in the present invention are not limited to those having a small molecular weight, and may be of any kind.

For example, dipalmitoylphosphatidylcholine (DPP
C>, dipalmitoylphosphatidylethanolamine (DPPE>), dioleoylphosphatidylethanolamine (DOPE), simyristoylphosphatidylethanolamine (DMPE>), distearoylphosphatidylethanolamine (DSPE), and the like.

In the immunoassay reagent of the present invention, the liposome is composed of at least one of phospholipids and glycolipids. In addition, as mentioned above, 10 to 500 mol% of cholesterol may be added to the phospholipids and glycolipids as necessary, and thereby stable liposomes can be obtained.

Furthermore, the fatty acid carbon chain in phospholipids and glycolipids preferably has 12 to 18 carbon atoms, and more preferably an even number.

The phospholipids or glycolipids or fatty acid derivatives bound to antigens or antibodies used in the present invention refer to antigens or fatty acids such as tumor markers, immunoglobulins, hormones, and drugs that are bonded to the phospholipids and glycolipids or fatty acids via a crosslinking agent. It is conjugated with antibodies.

The crosslinking agent used in the present invention is, for example, N-succinimidyl 3-(2-pyridylthio)propionate (SP
DP), N-succinimidyl 4-(P-maleimidophenyl)butyrate (SMPB), N-(γ-maleimidobutyryloxy)succinimide (GMBS),
N-(ε-maleimidocapro)! (reoxy)succinimide (EMC3), N-hydroxysuccinimide, and the like.

The fatty acids used in the present invention include undecanoic acid, lauric acid, myristic acid, palmitic acid, heptadecanoic acid,
Examples include stearic acid.

In the immunoassay reagent of the present invention, the labeling substance encapsulated within the liposome is selected from a substance that is hydrophilic and can be quantified when it flows out of the liposome. Examples of such substances include fluorescent substances such as carboxyfluorescein, which does not exhibit fluorescence due to self-quenching at high concentrations, but emits very strong fluorescence at low concentrations (10-3 M or less); Luminescent substances such as luminol and luciferin that emit light; light-absorbing compounds (water-soluble dyes, etc.) that have a specific absorption band in the visible or ultraviolet region
: Sugars such as glucose and sucrose that are broken down by the action of oxidative enzymes, resulting in enzyme consumption or hydrogen peroxide production : Relatively large ionic compounds such as tetrabentylammonium : Such as nicotinamide adenine dinucleotide (NAD) coenzymes; radical compounds such as methyl viologen; and the like. These compounds are appropriately selected in consideration of factors such as detection method, sensitivity, and stability of liposomes.

The immunoassay reagent of the present invention is used as follows. That is, first, the immunoassay reagent of the present invention is added to a sample containing a test substance, and complement is added separately. Note that at this time, a method may be used in which a second antibody against the test substance is added to sandwich the test substance. As a result, due to the antigen-antibody reaction between the test substance and the antibody or part of the antibody or antigen immobilized on the liposome and the accompanying action of complement,
When the liposome is destroyed, the encapsulated labeling substance (for example, a fluorescent compound) flows out. Since there is a correlation between the amount of the leaked labeled substance and the amount of the analyte in the sample, it is possible to quantify the leaked labeled substance using an appropriate analysis method (e.g. fluorescence analysis). Substances can be quantified.

The test substance that can be quantified using the immunoassay reagent of the present invention is a tumor marker (AFPSBFP).

CEA, POA, etc.), immunoglobulin (IQG.

Examples include IQE, IQD, IgA, I (JM, etc.), hormones (insulin, ■3, etc.), and drugs, and the targets are wide-ranging.

(Function) The reagent for immunoassay of the present invention uses phospholipids, glycolipids, or fatty acids on which antigens or antibodies have been immobilized in advance when forming multilayer liposomes. Antigens or antibodies are always immobilized not only in the inner several layers of the membrane, but it is unlikely that any of the membranes of a multilamellar liposome has functional groups that could cause non-specific destruction. do not have.

Therefore, for example, after the antigen-antibody reaction between the liposome and the test substance in the serum sample is completed, accurate quantification can be performed without the liposome reacting with proteins or trace substances in the serum.

(Example) The present invention will be explained in detail below.

Example Measurement of human AFP ■ Modification of anti-human AFP antibody to functional fatty acid 1 nMa
The anti-human AFP antibody 2d of f was added to a phosphate buffer (E) H7,1 containing 2% deoxycholic acid (hereinafter referred to as PBS).
and add 50 μ9 of valmitic acid ester of N-human O-oximide. The mixture is incubated at 37° C. for 12 hours and then gel-filtered with o,is% deoxycholic acid using a Sephadex G-75 column. As a result, the antibody-modified valmitic acid and the unreacted valmitic acid ester are separated and collected.

(2) Preparation of liposomes All lipids used were dissolved in chloroform or a mixed solvent of chloroform/methanol (2/1).

5ff1M dipalmitoylphosphatidylcholine 20
0 ttl, 10mM cholesterol iooμm was placed in a 10rrdl capacity eggplant-shaped flask, and 2d of chloroform was added and mixed. Next, the solvent was removed using a rotary evaporator in a water bath at about 40° C. to form a lipid thin film on the wall of the flask. Subsequently, the flask was transferred to a desiccator and suctioned with a vacuum pump for about 1 hour to completely remove the solvent.

Then 0.2M carboxyfluorescein (E)H
7.4: Hereinafter referred to as CF > 100 μm and anti-human AFP antibody-immobilized valmitic acid (5+n as palmitic acid concentration) dissolved in PBS were added to a flask at 10 μ°C, the inside of the flask was replaced with nitrogen, and the cap was sealed. The sample was then immersed in a water bath at about 60°C for about 1 minute. Subsequently, the flask was vigorously shaken using a vortex mixer until the lipid thin film on the flask wall completely disappeared. Through this operation, a liposome suspension in which the antigen or antibody was immobilized on all membranes of the multilamellar liposome was prepared.

Furthermore, gelatin veronal buffer (
After adding CF and anti-human A (hereinafter referred to as GtJB-), the cells were transferred to a centrifugation tube and centrifuged several times at 15,000 rpm for 20 minutes at 4°C to remove unnecessary CF and anti-human A.
The FP antibody-immobilized palmitic acid was washed and removed.

Finally, the liposomes were suspended in 2d GVB-'' to prepare a reagent for immunoassay. FIG. 1 shows a conceptual diagram of the reagent for immunoassay of the present invention.

(2) Preparation of a standard curve for human AFP concentration using anti-human AFP antibody-immobilized liposome reagent The human AFP concentration was quantified by a sandwich assay using free rabbit anti-human AFP antibody as follows, and a standard curve was prepared.

GVB-G (GVB-G,
: 0.5mH VIQCf12 aqueous solution and 0.5m)
Add 5 μρ of liposome reagent diluted 30 times with CaC12 aqueous solution of
The reaction was allowed to proceed for 0 minutes. Next, 25μ of rabbit anti-human AFP antibody (manufactured by Dako) previously diluted 200 times with GVB
m was added and reacted for 5 minutes at 37°C. Subsequently, 25 μm of complement (5CH50) was added and reacted at 37° C. for 30 minutes.

Then 0.01 M EDTA-Veronal buffer 1
The reaction was stopped at 00 μm, and the amount of CF flowing out of the human AFP solution at each concentration was measured using an MTP-32 fluorescence spectrometer (manufactured by Corona Electric) under conditions of an excitation wavelength of 490 nm and a fluorescence wavelength of 520 nm.

Based on this measurement, the relative fluorescence intensity was calculated using the following formula in order to remove the influence of the stability of antibody-immobilized liposomes on complement.

Here, Fe: actually measured fluorescence intensity, FO: fluorescence intensity when the antigen is removed (when liposomes are not destroyed at all), Fa: fluorescence intensity when all liposomes are destroyed by adding deionized water. be. In addition, as a standard value, 10-7
The fluorescence intensities of CF solutions of M and 10 −8 M were used.

In this way, a calibration curve showing the relationship between human AFP concentration and relative fluorescence intensity was obtained.

(2) Actual measurement of human AFP concentration Using the calibration curve obtained in advance in (2), human AFP concentration was actually measured for 10 patient serum samples. The results are shown in Table 1. Note that in Table 1, reference examples are values measured by RIA.

Comparative Example A calibration curve for human AFP 11 degrees was created using the immunoassay reagent disclosed in the earlier Japanese Patent Application No. 58-224509 (briefly described in the prior art section above), and a calibration curve for the same sample as in the example was prepared. AFP concentration was actually measured. The results are shown in Table 1.

As is clear from Table 1, the immunoassay reagents of Examples provide measured values that closely match those measured by RIA. On the other hand, in most cases, measurement was impossible with the immunoassay reagent of the comparative example. This is because the immunoassay reagent of the comparative example causes a non-specific reaction.

FIG. 2 shows a conceptual diagram of a conventional immunoassay reagent.

Table 1 [Effects of the Invention] As detailed above, according to the immunoassay reagent comprising the multilamellar liposome of the present invention, samples such as serum containing test substances can be analyzed in their original form, and accurately. Moreover, it has remarkable effects such as being able to easily quantify the test substance.

[Brief explanation of the drawing]

FIG. 1 is a conceptual diagram of an immunoassay reagent comprising a multilamellar liposome according to the present invention, and FIG. 2 is a conceptual diagram of a conventional immunoassay reagent comprising a multilamellar liposome. Agent Patent Attorney Nori Chika Yudo Kikuo Takehana

Claims (3)

[Claims] (1) An immunoassay reagent comprising a liposome whose composition is a phospholipid or a glycolipid or a fatty acid on which an antigen or an antibody is immobilized with either a phospholipid or a glycolipid, and a labeling substance encapsulated within the liposome. , an immunoassay characterized in that the liposome is a multilayer liposome consisting of two or more membranes, and each of the membranes contains at least a phospholipid or a glycolipid or a fatty acid on which an antigen or antibody is immobilized. Reagent for use. (2) The immunization according to claim 1, wherein the liposome is composed of either a phospholipid or a glycolipid, a phospholipid or a glycolipid on which an antigen or an antibody is immobilized, or a fatty acid and cholesterol. Analytical reagents. (3) Claim 1, characterized in that the labeling substance is a fluorescent compound, a luminescent compound, a light-absorbing compound, a saccharide, an ionic compound, an enzyme, a coenzyme, or a radical compound.
Reagents for immunoassay described in section.