JPH0192661A - Immunoassay method - Google Patents

Abstract

PURPOSE:To suppress nonspecific reaction and to make precise quantitative analysis by allowing a nonspecific dissolution preventive material of liposome to co-exist in a reaction liquid and cause the ensuing immune reaction at the time of bringing an immunoassay reagent and specimen into contact with each other. CONSTITUTION:Desired lipid and crosslinking agent are brought into reaction in a solvent to introduce a functional group into the lipid molecule so that the functional lipid is prepd. This functional group acts as the functional group to immobilize the antibody or part of the antibody or antigen on the liposome. The liquid suspension of the liposome is then prepd. by adding a solvent to the resultant functional lipid, and dissolving and mixing the lipid, then sucking away the solvent to dry the lipid, forming the thin film of the lipid on the wall surface of a flask, adding an aq. soln. contg. a labeling material, tightly plugging the flask and boiling the liquid. Such liquid suspension and the antibody or part of the antibody or the antigen are brought into reaction in a suitable buffer son. and are thereby immobilized.

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the Invention] (Industrial Application Field) The present invention relates to an improvement in an immunoassay method 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, there are problems in that special equipment must be installed and operated by a qualified operator, and that care must be taken in the disposal of waste.

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. The immunoassay method using this reagent is as follows. That is, when the liposome reagent is added to a sample containing an antigen or antibody and complement is added separately, the liposome is destroyed by the antigen-antibody reaction and the accompanying action of complement, and the encapsulated label substance ( e.g. fluorescent chemicals). There is a correlation between the amount of the labeled substance that has leaked out and the amount of the analyte in the sample, so by quantifying the labeled substance that has leaked out using a predetermined analysis method (e.g. fluorescence analysis), it is possible to Substances can be quantified. If this reagent is used, problems such as those of RIA will not occur, and immunoassays can be expected to be simplified.

However, when analyzing serum or protein-containing samples using this liposome reagent, it has been found that non-specific reactions occur in addition to the antigen-antibody reaction, resulting in liposome destruction. This is thought to be due to the reaction between proteins and trace chemicals in the sample and liposomes. For this reason, conventionally, serum or protein-containing samples have been diluted for analysis.

For example, anti-human α-fetoprotein antibody (
When analyzing AFP in human serum using a reagent immobilized with anti-human AFP antibody (hereinafter referred to as anti-human AFP antibody), the human serum was diluted 100 times in order to eliminate the influence of non-specific reactions. However, in the serum of normal people, AFP or 10-89/
It contains only mJ or less. Therefore, if normal human serum is diluted 100 times, the AFP concentration will be 10-10
There was a problem in that the measurement (for example, fluorescence analysis) corresponds to less than g/mA, making precise quantification difficult.

(Problems to be Solved by the Invention) As described above, conventional immunoassay methods have problems such as destruction of liposomes due to non-specific reactions and inability to perform precise and simple quantification.

Therefore, an object of the present invention is to provide an immunoassay method that suppresses non-specific reactions and allows precise and simple analysis.

[Structure of the Invention] (Means for Solving the Problems) The immunoassay method of the present invention comprises a liposome whose composition is at least one of phospholipids and glycolipids, and a labeling substance encapsulated in the liposome. When using an immunoassay reagent consisting of an antibody, a part of an antibody, or an antigen immobilized on the liposome by a cross-linking method, when the immunoassay reagent and the specimen are brought into contact, non-specific dissolution of the liposome occurs in the reaction solution. A preventive material (hereinafter referred to as a "blocking material") is allowed to coexist, and the subsequent immune reaction is caused to occur.

(Function) According to the immunoassay method of the present invention, the immobilization functional group remaining on the ribosome after immobilizing the antibody or a part of the antibody or the antigen is absorbed by the blocking agent in the immunoreaction mixture. For example, non-specific reactions with proteins or trace substances in the serum sample will not occur.

Therefore, there is no need to excessively dilute the test substance,
Precise quantitative analysis is possible and operation is simple.

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

In the present invention, the blocking material is derived from the composition of the liposome, particularly the functional group for antibody immobilization present in the lipid membrane of the liposome, and is derived from a sample (for example, plasma).

The aim is to prevent non-specific dissolution of liposomes caused by contact with serum, body cavity fluids, urine, etc.).

The immunoassay reagent used in the present invention can be produced, for example, 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, a part of the antibody, or the 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 was formed on the flask wall. Subsequently, an aqueous solution containing a labeling substance is added into the flask, the flask is tightly stoppered, and the flask is boiled to prepare a liposome suspension.

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 liposome suspension and the antibody, a part of the antibody, or the antigen are reacted in a suitable buffer to immobilize the antibody, part of the antibody, or the antigen on the liposome.

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, phospholipids,
10 to 500 mol% cholesterol based on glycolipids
may also be added, whereby 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.

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, calcein, and rhodamine B, which do not exhibit fluorescence due to self-quenching at high concentrations, but emit very strong fluorescence at low concentrations (10-3 M or less): Luminescent substances such as luminol and luciferin that emit light through an oxidation reaction outside liposomes; Light-absorbing compounds (water-soluble pigments, etc.) that have a specific absorption band in the visible or ultraviolet range; Decomposed by the action of oxidative oxygen, Sugars such as glycose and sucrose that lead to consumption or hydrogen peroxide production; relatively large ionic compounds such as tetrapentylammonium; supplementary oxygens such as nicotinamide adenine dinucleotide (NAD); radical compounds such as methyl viologen. etc. These chemicals are appropriately selected in consideration of factors such as detection method, sensitivity, and stability of liposomes.

In the immunoassay reagent of the present invention, the antibody or part of the antibody or antigen immobilized on the liposome is a protein or peptide (eg, ICIG).

ICIE, ■Immunoglobulins such as gD, ICIA, ICIM; Enzymes such as transaminase, lactate dehydrogenation, taleatin phoskinase; Cancer markers such as α-fetoprotein, carcinoembryonic antigen (CEA); Insulin;
peptide hormones such as growth hormone), carbohydrates (e.g. various Lewis antigens, Forsmann antigens, microbial cell wall polysaccharides), nucleic acid-related substances (e.g. polynucleotides, nucleotides, nucleosides, etc.), lipids (e.g. lipoproteins,
Calciolipin, etc.) and other low-molecular substances (steroid hormones, thyroxine, various drugs, etc.) can be arbitrarily selected depending on the object to be measured. At this time, it is desirable to immobilize antibodies or parts of antibodies, such as IgG or ICIM, corresponding to each antigen on the liposome, except for low-molecular-weight antigens (haptens) for which it is difficult to induce antibodies alone.

In the immunoassay reagent of the present invention, a cross-linking agent is used which reacts with a lipid molecule in order to introduce a functional group for immobilization into liposomes, and if necessary, a cross-linking agent is introduced into an antibody or a part of an antibody or an antigen. Examples of crosslinking agents for this purpose include N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl
- (p-maleimidophenyl)butyrate (SMPB
), N-succinimidyl 4-(1)-maleimidophenyl) acetate (SMPA), N-succinimidyl 4-(p-maleimidophenyl) propionate (
SMPP), N-(γ-maleimidobutyryloxy)
Succinimide (GMBS), N-(εmaleimidocaproyloxy)succinimide (EMC8), disuccinimidyl sperate (DSS), glutaraldehyde (GA), phthalaldehyde, terephthalaldehyde, phthalic acid, terephthalic acid, HOOC-( CH2) n -COOH (n = 1 to 10
Aliphatic dicarboxylic acids represented by the chemical formula ) are hetero-2° functional reagents for producing intermolecular (crosslinked) conjugates.

Note that when a crosslinking group is introduced by reacting an antibody, a part of the antibody, or an antigen with a crosslinking agent, it may be necessary to modify the crosslinking group by reduction treatment as described above.

When 5PDP is used as a crosslinking agent, the dithiopyridyl group (DTP) remaining on the liposome side causes a nonspecific reaction with the analyte. The blocking agent used here is DTP.
A chemical substance that has a reducing effect that cleaves the disulfide bonds of . As the reducing chemical substance, SH
The chemical substance having a group may be used, and specifically, it is selected from dithiothreitol, 2-mercaptoethanol, cysteine, cysteamine, glutathione, and the like. In this case, 2
Mercaptoethanol, cysteine, etc. are more suitable as blocking agents in the present invention. The concentration of the blocking agent can be 0.1 to 10 .mu.M, preferably 1 to 2 .mu.M in the case of 2-mercap-1-ethanol and cysteine. Various methods of adding the present blocking agent may be considered; however, the sample may be directly diluted with the present blocking agent, or it may be added separately when the sample and liposome are brought into contact with each other.

The immunoassay method of the present invention is carried out as follows. That is, first, a sample (specimen) containing a test substance is mixed with an isotonic solution containing a blocking agent. At this time, the dilution rate of the specimen is selected depending on the measurement limit of the substance to be measured.

An immunoassay reagent consisting of liposomes is added to the sample containing the specimen and blocking agent, and complement is added separately. At this time, a method may be used in which a second antibody against the test substance is added to sandwich the test substance. The second antibody and/or complement can be diluted with an isotonic solution containing a blocking agent, if necessary. As a result, the liposome is destroyed and the labeled substance encapsulated (e.g. Fluorescent compounds) flow out. Since there is a correlation between the amount of the labeled substance that has leaked out and the parent of the test substance in the sample, the amount of the labeled substance that has leaked out can be quantified using an appropriate analytical method (e.g., fluorescence analysis). Substances can be quantified.

In actual quantitative analysis, a calibration curve is created in advance using a test substance with a known concentration, and based on this, quantitative analysis is performed by reacting with a test substance of unknown concentration under the same conditions. I do.

The time required for a sufficient reaction between the immunoassay reagent of the present invention and the test substance depends on the type of test substance, the characteristics of the liposome, the reaction conditions, and the amount of antibody or part of the antibody or antigen immobilized on the liposome. Varies depending on type, purity, immobilization form, etc. Therefore, by performing a preliminary measurement using a sample containing the same kind of substance as the test substance that has been prepared in advance to a specific concentration depending on each individual case, the optimal reaction time between the immunoassay reagent and the test substance can be determined. It is desirable to set this.

Test substances that can be quantified by the immunoassay reagent of the present invention include tumor markers (AFP (α-fetoprotein), B
FP (basic fetoprotein).

CEA (@embryonic antigen), POA (hepatic carcinoembryonic antigen)
etc.), immunoglobulin (1ΩG, ICIE.

Examples include IgD, IgA, IgM, etc.), hormones (insulin, IgM, etc.), drugs, etc., and the targets are wide-ranging.

An experimental example will be explained below.

Experimental Example 1 Measurement of Human AFP Among the reagents used in this practical example, dipalmitoylphosphatidylethanolamine (DPPE>, dipalmitoylphosphatidylcholine (DPPC), cholesterol, and dithiothreitol (DTT>) were manufactured by Sigma. In addition, N-cycsinimidyl 3-(
2-pyridyldithio)propionate (SPDP) and Sephadex G-25 fines manufactured by Pharmacia were used. Other reagents were commercially available products (special grade) that were used without purification. In addition, all water used was ion-exchanged water.

■Functional phospholipid: Preparation of DPPE-dithiopyridylpropionic acid amide (DPPE-DTP) 70mff of DPPE was collected in an Erlenmeyer flask with a tight stopper, and chloroform/
It was dissolved in 25 mA of methanol (5:1) mixed solvent. Next, 60μ of triethanolamine and 50μ of SPDP
After the mixture was replaced with nitrogen, the mixture was reacted for 1 hour at ¥ temperature to introduce dithiopyridylpropionic acid amide (DTP). Subsequently, the solvent was removed using a rotary evaporator. Next, the dried product was dissolved in chloroform/methanol (1
After dissolving in a 0:1) mixed solvent, it was purified using a silica gel column and a DPPE-DTP fraction was collected. Furthermore, it was concentrated to about 5 ml using a rotary evaporator. The yield of DPPE-DTP was 80-95%.

This was sealed with nitrogen and stored at -20°C.

The dithiopyridyl group introduced into DPPE by this reaction becomes a functional group for immobilization.

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

200μp of 5mM DPPC, 10mM cholesterol 1001, and 1n+H DPPE-DTP (functional phospholipid >50μ obtained in ■) were placed in a 10ml pear-shaped flask, and 2ml of chloroform was added and mixed well. Next, The solvent was removed by rotary evaporation in a water bath at approximately 40°C.
After adding J2 and stirring thoroughly, the solvent was removed again using a rotary evaporator. When this operation was repeated several times, a thin lipid film was formed on the flask wall. Subsequently, the flask was transferred to a desiccator and suctioned with a vacuum pump for about 1 hour to completely remove the solvent.

Next, 100 μl of 0.2M carboxyfluorescein (manufactured by Eastman Kodak Company, pH 7.4; hereinafter referred to as CF) was added, and after purging the inside of the flask with nitrogen, the flask was tightly capped and placed in a water bath at approximately 60°C. It was immersed for about 1 minute. Subsequently, the flask was vigorously shaken using a VorteX mixer until the lipid thin film on the flask wall was completely wetted. A liposome suspension was prepared by this operation.

Furthermore, 0.1M HEPES buffer (
0.85% NaCj! After adding a small amount of HBS (containing, pH 7.5 or less), transfer everything to a centrifuge tube,
Centrifugation at 15,000 rpm for 20 minutes at °C was repeated several times. Finally, it was suspended in 1 mJ2 of l-IBs.

■Modification of anti-human AFP antibody 2 ml of 1 myimp anti-human AFP antibody was diluted with HBS, 10 μl of 10+nH SPDP ethanol solution was added, the air was replaced with nitrogen, and then reacted at the lowest temperature for 30 minutes. introduced a group. Next, in advance, 0.1M acetate buffer (containing 0.85% NaCjl!),
Sephadex G-25 equilibrated with pi-14.5)
Purification was performed by removing unreacted SPDP by gel filtration using a fine column (gel volume: approximately 15 m), and only the protein fraction was collected.

Next, add about 20 m of DTT to this fraction! After adding J and replacing with nitrogen, the reaction was carried out for 20 minutes at the lowest temperature to convert the dithiopyridyl group into S.
Modified by substitution with H group. Subsequently, unreacted DTT was removed and purified by gel filtration using a Sephadex G-25 fine column equilibrated with HBS, and only the protein fraction was collected.

■Preparation of anti-human AFP antibody-immobilized liposomes ■Pomme suspension 1rr+j! t5 at 4°C
．． Mix the liposome precipitate centrifuged at 00 Orpm for 20 minutes with 2mj2 of the O, 1g protein/ml modified anti-human AFP antibody solution obtained in ①, replace the mixture with nitrogen, seal it, and shake slowly at 20°C. The reaction was allowed to take place overnight. Next, unreacted antibodies were removed by washing with HBS.

Gelatin veronal buffer (pH 7.4 or less GVB-) was added to the liposome reagent thus obtained.
After adding 10% NaN320111 and stirring thoroughly, the mixture was purged with nitrogen and stored at 4°C.

■ Examination of blocking agents Using the liposomes obtained in (before antibody sensitization), we investigated the effects of blocking agents on nonspecific lysis of liposomes induced in the presence of serum (final addition concentration 1
mM＞.

Gelatin veronal buffer containing 3.2mM each of dithiothreitol, 2-mercaptoethanol, cysteine, glutathione, ascorbic acid, methionine, and sodium glutamate? (pH 7,4゜0.5mMMCI
Cj! 2. 0.15mHCaCj! 3 content, hereinafter referred to as GV
Fresh normal human pool serum (hereinafter NH8>) was diluted 10 times with GVB'+ or GVB'+.Next, each diluted NH
325'-1 was dispensed into a 96-well microtiter, 5 μm of liposome reagent (30-fold diluted with GVB") was added thereto, and the mixture was stirred at 37°C.

The reaction was allowed to proceed for 10 minutes. Next, complement (5CH50) 50mj
! and reacted at 37°C for 30 minutes.

Then 0.01 M EDTA-Veronal buffer 1
The reaction was stopped with a 00μ acetate, and the amount of CF effluent was measured using an MTP-32 fluorescence spectrometer (
(manufactured by Corona Electric) under the conditions of an excitation wavelength of 490 nm and a fluorescence wavelength of 5201 m.

Based on this measurement, relative fluorescence intensity was calculated using the following formula.

Here, Fe: actually measured fluorescence intensity, Fo: fluorescence intensity derived from a buffer solution or plug body, Fa: fluorescence intensity when all liposomes were destroyed by adding deionized water. Note that the fluorescence intensities of 10-7M and 10-8M CF solutions were used as standard values.

Table 1 shows the effect of the coexisting blocking agent on the nonspecific dissolution of the liposomes thus obtained.

(Left below) The background of liposomes in Table 1 is 8.5% based on GVB, whereas N1-18 (1/10
When diluted) was added instead of GVB''', the increase increased to 34.8%. This is due to the non-specific lysis mentioned above. Addition of the 8H compound clearly prevented non-specific lysis and returned to the same level as GVB''. Among SH compounds, glutathione had a weak effect. Simple reducing agents such as ascorbic acid and amino acids such as methionine and monosodium glutamate contain 5
No substitution effect of the t-1 compound was observed.

■Effect of the amount of cysteine added on serum AFP measurement■Using the anti-human AFP antibody sensitizing pomme obtained in ■
The effect of cysteine addition on serum-based AFP measurements was investigated.

GVB containing NH3 from O to 6mM cysteine”
Dilute 10 times with 0.1 to 8 1. oo.

AFP derived from human placenta was added to give ml/mA.

Next, 25μ of each sample was dispensed into a 96-well microtiter, and 5μ of liposome reagent (anti-AFP antibody sensitized product, diluted 30 times with GVB4+) was added thereto. After stirring, the mixture was reacted at 37°C for 10 minutes. .Next, O to 311
A second antibody (anti-human AFP rabbit antiserum A2l110-0.1) diluted with 1 M cysteine-containing GVB4+ and complement (10 CHso/mj) were added at 25 μA each, stirred, and reacted at 37° C. for 30 minutes. Finally 0.01
MEDTA-Veronalol Buffer-(1) H7,4
> The reaction was stopped with a 100 μl spatula, and the fluorescence intensity of the reaction solution was measured in the same manner as in ①.

The results are shown in the drawing. As is clear from the figure, significant non-specific lysis is observed in the cysteine-free system, whereas non-specific lysis is clearly suppressed under a cysteine concentration of 0.7m)1 or more, and AFPl ng/mA to 1
, 000 nc+/mA.

■Human A using anti-human AFP antibody-immobilized liposome reagent
FP concentration measurement (cystine collection concentration 1.5mM).

Human AFP concentration was measured on an NHS basis using a method similar to (2), and a calibration curve was created. Next, using this calibration curve, human APF! The degree was actually measured. The results are shown in Table 2. In addition, in Table 2, the reference example is R
This is a value measured by IA.

As is clear from Table 2, the immunoassay method according to this experimental example yielded measured values that were in good agreement with the measured values by RIA.

Experimental Example 2 Measurement of Human CEA Anti-human C prepared based on methods ① to ② of Example 1
Using EA monoclonal antibody-sensitized liposomes, human CEAm
The degree was actually measured. At this time, the final concentration of cysteine added was 2 m)
It was set as f. The results are shown in Table 3. Note that the reference examples in Table 3 are also measured values by RIA.

Table 3 - 25 = As is clear from Table 3, in the immunoassay method according to the present experimental example, measured values were obtained in CEA measurement that were in good agreement with the measured values by RIA, similar to AFP.

[Effects of the Invention] As detailed above, according to the immunoassay method of the present invention, a sample such as serum containing a test substance can be analyzed while suppressing non-specific reactions and without excessively diluting it. This method has remarkable effects such as being able to accurately and easily quantify test substances.

[Brief explanation of the drawing]

The figure is a graph showing the liposome dissolution rate according to the amount of cysteine added in Examples of the present invention. Agent: Patent Attorney: Nori Chika Ken, Canshu Dai, Ko Norifu 261 Procedural Amendment April 7, 1988

Claims (1)

[Scope of Claims] (1) A liposome comprising at least one of phospholipids and glycolipids, a labeling substance encapsulated in the liposome, and an antibody or antibody immobilized to the liposome by a crosslinking method. An immunoassay method using an immunoassay reagent consisting of a part of an antibody or an antigen, characterized in that a substance for preventing non-specific dissolution of liposomes is allowed to coexist in the reaction solution. (2) Claims characterized in that the substance for preventing non-specific dissolution of liposomes that is caused to coexist in the reaction solution is derived from the composition of the liposomes and prevents non-specific dissolution of liposomes that is caused in the coexistence with a sample. The immunoassay method according to item 1. (3) The non-specific dissolution inhibiting substance is disulfide (-S.
3. The immunoassay method according to claim 2, which is a chemical substance having a reducing action that cleaves S-) bonds. (4) The immunoassay method according to claim 1, wherein the liposome comprises at least one of phospholipids and glycolipids and cholesterol.