JPH079428B2 - Immunoassay method - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION The present invention relates to an immunoassay method using a liposome immunoassay reagent for specifically quantitatively analyzing a trace amount of a test substance present in a sample.

[Technical background of the invention and its problems]

In recent years, various tumor markers have been found as researches on cancer have progressed. For example, α-fetoprotein (AFP), carcinoembryonic antigen (CEA), basic fetoprotein (BFP) and pancreatic carcinoembryonic antigen (P
OA) can be mentioned as a typical example. The concentrations of these tumor markers are very low in normal individuals (eg CEA: several ng / ml or less). On the other hand, a tumor patient often shows a value about 10 times or more that of a normal person. In any case, a very high detection sensitivity is required for analysis and quantification of tumor markers.

To meet this demand, conventionally, a radioimmunoassay (RIA) using an antigen or antibody labeled with a radioactive substance has been developed. However, the RIA is troublesome to handle, and disposal also becomes a problem. Therefore, an immunoassay method using antigens or antibodies labeled with various substances such as enzymes and fluorescent substances instead of radioactive substances was proposed, and in these methods, free antibody and bound antibody must be separated by some method. It had the drawback that it had to be. Also, Rosent-thal
A ・ F, Vargas, MGand Klass CS (1976) Clin.Chem.2
The EMIT method, which was published in 2,1899, is an epoch-making method that allows measurement in a homogeneous system that does not require a separation step, but in principle cannot be applied to high molecular weight protein antigens or antibodies.

By the way, Haxby, JA, Kinsky, CBand kinsky SC (19
68) In Biochemistry, 61 300, quantification of the antibody is performed by preparing a liposome in which a lipid-soluble antigen is incorporated into a membrane and encapsulating glucose, and measuring the number of glucose efflux associated with the destruction of the liposome due to an antigen-antibody reaction. The method was announced.

However, in order to measure tumor markers, the markers themselves or antibodies against these markers, that is, immunoglobulins that are proteins, must be carried on liposomes. However, until now, it was possible to use a liposome carrying a lipophilic protein, but no immunoassay method for an antigen or antibody using a liposome carrying a hydrophilic protein has been reported. This is because a technique for supporting hydrophilic proteins on liposomes has not been established.

Further, JP-A-56-132564 "Product and Method for Immunoassay" discloses a method for immunoassay using a liposome carrying an antigen or an antibody and encapsulating an enzyme therein. Proposes a method of using a bifunctional crosslinking reagent such as glutaraldehyde as a method for supporting proteins. According to the research conducted by the present inventors, it has been found that when an antibody is loaded on a liposome with such a crosslinking reagent, the activity of the antibody is generally lowered and destruction of the liposome due to an antigen-antibody reaction is not caused.

Further, the conventional immunoassay techniques generally have a drawback that the analysis takes a long time and a large amount of sample cannot be automatically measured.

[Object of the Invention]

An object of the present invention is to provide an immunoassay method for analyzing a trace amount of a test substance in a sample with high accuracy and high sensitivity.

[Outline of Invention]

The present inventors have conducted extensive studies to achieve the above object, and as a result, immobilized at least a part of the first antibody against the test substance without lowering the activity on the liposome that is lysed by the complement activity. By further reacting a liposome immunoassay reagent obtained by further encapsulating a labeling substance in a liposome with a sample containing a test substance for a predetermined period of time, and then allowing a second antibody and a complement to the test substance to act. It has been found that the object of the present invention can be achieved. In this case, the first antibody is placed by removing the supplemental binding site by enzymatic treatment.

Hereinafter, the present invention will be described in more detail.

The liposome in the liposome immunoassay reagent used in the immunoassay method of the present invention is composed of at least one of phospholipid and glycolipid and cholesterol. The composition ratio of these is preferably 10 to 500 mol% of cholesterol with respect to the phospholipids and glycolipids in order to obtain stable liposomes. The fatty acid residue in the phospholipid preferably has 12 to 18 carbon atoms, and more preferably has an even number.

The labeling substance encapsulated in the liposome must be hydrophilic and can be quantified when eluted outside the liposome. As such a substance, for example, at a high concentration, no fluorescence is shown due to self-quenching, but at a low concentration (10 ⁻³ M
Fluorescent compounds such as carboxyfluorescein that emit very strong fluorescence in the following); luminescent compounds such as luminol and luciferin that emit light by an oxidation reaction outside the liposomes; a specific absorption band in the visible or ultraviolet region Absorbing compounds (water-soluble dyes, etc.) possessed; sugars such as glucose and sucrose that are decomposed by the action of an oxidase to cause oxygen consumption or hydrogen peroxide production; relatively large ionic compounds such as tetrapentylammonium; nicotinamide Coenzymes such as adenine dinucleotide (NAD); radical compounds such as methyl viologen are preferable. These compounds are appropriately selected in consideration of factors such as the detection method, sensitivity and liposome stability.

The liposome immunoassay reagent used in the immunoassay method of the present invention is produced from such liposomes and a labeling substance by, for example, the following method. First, by reacting a desired lipid with a crosslinking agent in a solvent, a functional group capable of binding to an antibody or a part of the antibody immobilized on a liposome is introduced into a lipid molecule to form a functional lipid. Then, add the obtained functional lipid and cholesterol and, if necessary, an appropriate amount of other lipids to a flask and add a solvent to dissolve them.
After mixing, the solvent is distilled off and dried by suction. After that, an aqueous solution of a predetermined labeling substance is added into a flask having a thin film formed on the wall surface, the container is sealed and shaken to obtain a liposome suspension.

On the other hand, if necessary, the antibody or a part of the antibody immobilized on the liposome is modified by introducing a cross-linking group with a cross-linking agent and then treating with a reducing agent (for example, dithiothreitol; DTT). A liposome immunoassay reagent used in the immunoassay method of the present invention can be obtained by reacting the liposome suspension with an antibody or a part of the antibody optionally modified, in an appropriate buffer.

Examples of the cross-linking agent in the above production method include N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), N-succinimidyl 4- (p-maleimidophenyl) butyrate (SMPB), N-succinimidyl 4- (p). -Maleimidophenyl) acetate (SMP
A), N-succinimidyl 4- (p-maleimidophenyl) propionate (SMPP), N- (γ-maleimidobutyryloxy) succinimide (GMBS), N- (ε-
Maleimidocaproyloxy) succinimide (EMCS)
And disuccinimidyl sperate (DSS).

For example, SPDP has the following formula: Is a cross-linking agent that reacts under mild conditions to bond compounds having a primary amino group.

SMPB has the following formula: , The antibody can be immobilized by the same reaction as SPDP.
The final product does not contain a -S-S-bond (only -S-bond) and is stable even in a reducing atmosphere such as serum.

The complement used in the immunoassay method of the present invention is not particularly limited, but guinea pig serum having a high complement value is usually preferable. However, depending on the case, serum of rabbit, mouse, human or the like may be used.

The animal species of the second antibody against the test substance used in the present invention is not particularly limited, but a rabbit that easily takes up complement is more preferable, but this second antibody must have a shape having a complement binding site. I won't. On the other hand, as for the antibody immobilized on the liposome, the animal species of the antibody is not particularly limited when the complement fixing site is removed by enzyme treatment, but if not removed, a goat stable to guinea pig complement is obtained. Antibody is preferred.

The test substance that can be quantified by the immunoassay method according to the present invention using the thus-obtained liposome immunoassay reagent is a tumor marker (the aforementioned AFP, BFP, CEA, and POA).
Etc.) Immunoglobulins (IgA, IgE, IgG and IgM etc.), hormones (insulin, T ₃ etc. and antigens such as drugs,
Furthermore, the antibodies corresponding to them are also test substances to which the immunoassay method of the present invention can be applied, and the subject thereof covers a wide range. These test substances are quantitatively analyzed in the following process.

First, the test substance is appropriately diluted to obtain a sample. By adding a liposome immunoassay reagent to this sample, a test substance is obtained by further adding the liposome immunoassay reagent in which at least a part of the antibody against the test substance is immobilized and the second antibody against the test substance added separately. Was caught in
When the complement acts there, the labeling substance encapsulated in the liposome immunoassay reagent migrates. Since the migration of the labeling substance occurs corresponding to the concentration of the test substance,
Quantitative analysis of the test substance can be performed by measuring the amount of the labeled substance that has migrated.

And in the actual quantitative analysis, a calibration curve was prepared in advance using a known concentration of the test substance, and based on this, the label that flowed out due to the reaction with the unknown concentration of the test substance under the same conditions. The substance is measured and quantitative analysis is performed.

In the analysis process as described above, the present inventors have made the sensitivity of the analysis reagent dramatically by adding the second antibody and complement after the liposome immunoassay reagent is sufficiently reacted with the test substance in advance. We have found that it can be improved several hundred times.

From this, the labeling substance is effluent and quantified by adding the second antibody and complement almost simultaneously with the liposome immunoassay reagent without allowing the reaction between the liposome immunoassay reagent and the test substance to proceed sufficiently. The analysis method of the present invention enables the detection and quantification of a trace amount of a test substance, which was impossible by the method, for the first time. The time required for a sufficient reaction between the liposome immunoassay reagent and the test substance depends on the concentration of the test substance, the characteristics of the liposome, the reaction conditions,
Furthermore, it depends on the type, purity, immobilized form, etc. of the antibody immobilized on the liposome immunoassay reagent. Therefore,
The optimal reaction time between the reagent for liposome immunoassay and the test substance can be determined by carrying out preliminary measurements using samples containing substances of the same type as the test substance prepared in advance to a specific concentration depending on the individual case. It is specified as appropriate.

Example of Invention

Hereinafter, the present invention will be described in more detail with reference to Examples, but these Examples do not limit the scope of the present invention in any way.

Example 1. Human IgG was measured using a liposome immunoassay reagent on which goat anti-human immunoglobulin IgG antibody was immobilized.

First, a goat anti-human IgG antibody-immobilized liposome immunoassay reagent was prepared. The reagents used at this time were dipalmitoylphosphatidylcholine (DPPC), cholesterol, dibalmitoylphosphatidylethanolamine (DPPE).
And dithiothreitol (DTT) manufactured by Sigma, N-succinimidyl 3- (2-pyridyldithio)
Propionate (SPDP) and Sephatex G-25 Fine were manufactured by Pharmacia. Other reagents were used as commercial products (special grade) without purification. All water used was ion-exchanged water.

Then, a liposome immunoassay reagent was prepared by the following steps.

a) Preparation of dithiopyridyl-DPPE (DTP-DPPE) 70 mg of DPPE was dispensed into an Erlenmeyer flask with a sealed stopper, dissolved in 25 ml of chloroform / methanol (5: 1) solution, 60 μl of triethanolamine and 50 mg of SPDP. Was replaced with nitrogen. After reacting for 1 hour at room temperature, the solvent was removed by a rotary evaporator. The dried product was dissolved in 5 ml of chloroform / methanol (10: 1) and purified using a silica gel column. Collect the product fractions and use an evaporator to transfer about 5 ml.
Concentrated to. The yield was 80-95%. Storage was carried out at -20 ° C under nitrogen.

b) Preparation of liposomes All the lipids used were dissolved in chloroform or chloroform / methanol (2/1). First, 5mM DPPC (200
μl), 10 mM cholesterol (100 μl) and 1 mM DTP-D
Add PPE (50 μl) to a 10 ml pear-shaped flask and add 2 ml.
Chloroform was added and mixed well. In the water bath (about 50
The solvent was removed by rotary evaporation at (° C.). Chloroform (2 ml) was added again, and after sufficient stirring, the solvent was evaporated again by using a rotary evaporator.
When this operation was repeated several times, a thin film was formed on the wall surface of the flask. The flask was transferred into a desiccator, and the solvent was completely removed by suction with a vacuum pump for about 1 hour. Next, 100μ
l 0.2M carboxyfluorescein (CF; manufactured by Eastman Kodak Co., pH 7.4) was added, the inside of the flask was replaced with nitrogen, and the flask was sealed and immersed in a water bath at about 60 ° C. for about 1 minute. Then, the flask was vigorously shaken using a Vortex mixer until the lipid thin film on the wall surface completely disappeared. By this operation, a liposome suspension was prepared.
Gelatin - veronal buffer (hereinafter, GVB ^- abbreviated) was added a small amount was transferred to a fully centrifugal Chupu a liposome suspension. Free CF was removed by centrifugation at 15,000 rpm for 20 minutes at 4 ° C. It was repeated by using the ^- supernatant GVB until clear. Finally, 2 ml of GVB ⁻ and 5 μl of 10% NaN ₃ were added, suspended in a Vortex mixer, filled with nitrogen, and stored in a refrigerator.

c) Modification of goat anti-human IgG antibody 2 ml of goat anti-human IgG antibody (about 15 mg / ml) was added with 10 μl of 10 m.
M SPDP (ethanol solution) was added, and the mixture was sufficiently stirred and allowed to react at room temperature for 30 minutes. After the reaction, the reaction solution was developed on a column (gel volume: approx. 15 ml) packed with Sephadex G-25 Fine gel, which had been saturated with physiological saline in advance, and the volume was adjusted to 0.1M.
The acetate buffer (pH 4.5, containing 0.85% NaCl) was eluted. 2 ml of acetate buffer was added to the first protein fraction (about 2 ml), and after nitrogen substitution, dithiothreitol (about 30 mg)
Was added. The mixture was thoroughly stirred and reacted at room temperature for 20 minutes.
After the reaction, the reaction solution was developed on a column (gel volume: about 30 ml) packed with Sephadex G-25 Fine gel saturated with 0.01 M HEPES buffer solution in advance and eluted with the HEPES buffer solution. The first protein fraction (about 2 ml) was collected, replaced with nitrogen, and stored in a refrigerator until use.

d) Preparation of goat anti-human IgG antibody-immobilized liposomes (reagent for immunoassay) Mix the liposome suspension prepared as described above with an equal amount of the modified antibody, replace with nitrogen, stopper tightly, and gently shake at room temperature. The reaction was continued overnight. Then, it was washed with the HEPES buffer and then GVB ⁻ to remove unreacted antibody and leaked CF. Thus goats were prepared anti - GVB corresponds to the amount of liposome suspension was used in the reaction in the human IgG antibody immobilized liposome immunoassay reagent ^- and 10μl of 10% Na
After adding N ₃ , suspending and replacing with nitrogen, the mixture was stored in a refrigerator until use.

Using the goat anti-human IgG antibody-immobilized liposome immunoassay reagent thus prepared,
IgG was measured.

An appropriate amount of GVB ⁺ (0.1m
GVB containing the M ngCl ₂ and 0.03 mM CaCl ₂ ^-) a human IgG diluted as a sample in which the U-shaped microplates (96 wells each 25 [mu] l; was injected into well made Nunc). Next, 5 μl each of the goat anti-human IgG antibody-immobilized liposome immunoassay reagent prepared by the above method was injected into the sample, and the mixture was allowed to react at 37 ° C. for 30 minutes. Thereafter, a rabbit anti-human IgG antibody (manufactured by Miles; diluted 400-fold) as a second antibody and 25 μl each of guinea pig serum (0.5 CH ₅₀ ) as a complement were added, and the mixture was allowed to act at 37 ° C. for 1 hour.

After the reaction, 100 μl of 0.01 M EDTA-Veronal buffer was added to each well to stop the reaction, and the fluorescence of each well was measured with a plate spectrophotometer (MTP-12F manufactured by Corona Electronics Co., Ltd.) ( Ex: 490nm, Em: 520nm). The measured value was 25 μl of 10% Triton X-100 and GVB ⁻ instead of the second antibody and complement.
The difference between the fluorescence of wells to which 50 μl each was added and the fluorescence to which 25 μl GVB ⁺ was added instead of human IgG was expressed as a relative emigration rate with 100%.

The result is shown by the curve a in FIG. As you can see from the figure, 10 ^-3
Emigration of the labeling substance was observed in the concentration range of 10 ^-8 (g / ml). Then, by using a curve showing the characteristics in the concentration range of 10 ^{-6 to} 10 ^-8 (g / ml) as a calibration curve, quantitative analysis of a sample of unknown concentration within such a minute range is possible. You can see that That is, the immunoassay method of the present invention was able to achieve extremely high sensitivity as compared with conventional assay methods.

Comparative Example 1 In the same manner as in Example 1, human IgG diluted with an appropriate amount of GVB + to a known concentration in advance was used as a sample, and 25 μl of this was injected into the well of a U-shaped microplate.

Then, a goat anti-human IgG antibody-immobilized liposome immunoassay reagent (GVB ⁺ 1 was prepared by the same method as that used in Example 1).
(Diluted 00 times) 5 μl, and rabbit anti-second antibody
Human IgG antibody (manufactured by Miles; 400-fold diluted) was added in an amount of 25 μl, and complement (guinea pig serum; 0.5CH ₅₀ ) was added in an amount of 25 μl for 5 minutes. The reaction was carried out for 1.5 hours while kept at 37 ° C. After that, the same treatment as in Example 1 was performed, and the relative emigration rate was measured by the same method. The result is shown by the curve b in FIG.
As can be seen from the figure, migration was observed in the range of about 10 ^{-1 to} 10 ^-7 (g / ml), but compared with the case of Example 1, the quantifiable concentration was about 10 ^{-5 to} 10 ^-6. Since it was on the higher concentration side and the measurable range was narrow, it did not have the detection at the low concentration, that is, the high sensitivity as shown in Example 1.

Example 2 Human IgG at a concentration of 10 ^-7 g / ml was used as a sample, and in Example 1, it was 30 minutes. The effect was investigated by increasing or decreasing the contact reaction time between the reagent for liposome immunoassay and the test substance in the sample before adding the second antibody and complement. The reaction temperature at this time was maintained at 37 ° C. After the reaction, the second antibody and complement were added, and the mixture was reacted at 37 ° C. for 1 hour, and then treated in the same manner as in Example 1 to measure the labeled substance flowing out. The results are shown in FIG. As can be seen from the figure, even in the case of the sample having the same analyte concentration of 10 ^-7 g / ml, the reaction time between the sample before the addition of the second antibody and complement and the liposome immunoassay reagent Due to the length, there is a great difference in the relative migration rate of the labeling substance. That is, when the liposome immunoassay reagent and the second antibody and the complement are simultaneously added to the sample containing the test substance, the relative migration rate is about 3%, whereas the test substance and the liposome immunoassay are used. If the reaction time between the test substance and the reagent for liposome immunoassay is 15 minutes or more when the reagent and the second antibody are allowed to react after the contact reaction with only the reagent, the labeling substance is approximately 20 times as much. Outflow, which provides a significant improvement in accuracy. Of course, in the case of this example, the time required for a sufficient reaction between the liposome immunoassay reagent and the test substance was about 15 minutes. This time is appropriately selected depending on the characteristics and the like.

Example 3 Human CEA was measured using a liposome immunoassay reagent in which a rabbit anti-human CEA antibody (Fab ′) that had been partially immobilized by removing the complement binding site was immobilized.

(A) Based on the conventional method (immunity experiment operation method p1166,1974),
F (ab ′) ₂ of the antibody was prepared by removing the complement binding site of rabbit anti-human CEA antibody (manufactured by Dako) by treatment with pepsin. The concentration was about 2 mg / ml. ² ml of this F (ab ′) 2
Then add 10 mg of 2-mercaptoethylamine,
The reaction was carried out for 90 minutes under a nitrogen atmosphere while maintaining the temperature at 37 ° C. After the reaction, it was eluted with 0.01M HEPES buffer (pH7.45) using Sephadex G-25fine (Pharmacia) column to obtain Fab ′ of the antibody. About 100 μg of Fab ′ of this antibody was mixed with 1 ml of the liposome suspension prepared in the same manner as in Example 1, and 1
Rabbit anti-partialized by shaking overnight at room temperature
A reagent for immunoassay of liposomes on which human CEA antibody (Fab ′) was immobilized was obtained.

(B) Measurement of human CEA Using the liposome immunoassay reagent prepared in (a),
Human CEA was measured by the same method as shown in Example 1. That is, 25 μl of human CEA diluted with an appropriate amount of GVB ⁺ to a known concentration was used as a sample.
Each was injected into the well of a U-shaped microplate. Then
The above-mentioned reagent for liposome immunoassay on which the partially rabbit anti-human CEA antibody (Fab ′) was immobilized was diluted 100-fold with GVB ⁺ , and 5 μl of each was injected into the sample and reacted at 37 ° C. for 30 minutes. After that, 25 μl each of rabbit anti-human CEA / antibody (manufactured by Dako; 100-fold diluted) as a second antibody and guinea pig serum (0.5 CH ₅₀ ) as a complement were added, and the mixture was reacted at 37 ° C. for 1 hour. After the reaction, the relative elution rate of carboxyfluorescein as a labeling substance was measured in the same manner as in Example 1.
Results are shown in FIG. As can be seen in the figure, 0.1-20ng /
It was confirmed that human CEA can be quantified in the range of ml.

〔The invention's effect〕

In the immunoassay according to the present invention, first, a sample containing a test substance is sufficiently reacted with a liposome immunoassay reagent, and then a second antibody and a complement against the test substance are allowed to act to allow the test to be performed. The accuracy and sensitivity in quantitative determination of substances can be greatly improved.

[Brief description of drawings]

FIG. 1 shows the relationship between the concentration of a test substance and the relative migration rate of a labeling substance in the immunoassay method of the present invention (curve a), and the concentration of the test substance and the labeling substance when the immunoassay method of the present invention is not used. FIG. 2 is a characteristic diagram showing the relationship (curve b) with the relative emigration rate of the labeled substance, and FIG. 2 is a characteristic showing the change in the relative elution rate of the labeled substance with respect to the reaction time between the test substance and the reagent for liposome immunoassay. FIG. 3 and FIG. 3 are characteristic diagrams showing the relationship between the concentration of a test substance and the relative migration rate of a labeling substance when another liposome immunoassay reagent is used in the immunoassay method of the present invention.

Claims (6)

[Claims] 1. A liposome comprising at least one of phospholipids and glycolipids and cholesterol, on which at least a part of an antibody to a test substance is immobilized by a crosslinking method, and further, a hydrophilic labeling substance is present inside the liposome. In the immunoassay method in which the encapsulated liposome immunoassay reagent is brought into contact with a sample containing the test substance in advance to cause a reaction, and then a second antibody and complement to the test substance act, the immobilization The immunoassay method is characterized in that the antibody to which the complement-fixing site has been removed by an enzyme treatment, and the time for which the reagent is brought into contact with the sample in advance is a certain time or more. 2. The immunoassay method according to claim 1, wherein a rabbit antibody is used as the second antibody. 3. A cross-linking agent used in the cross-linking method is N-succinimidyl 3- (2-pytodyldithio) propionate (SPDP), N-succinimidyl 4- (p-maleimidophenyl) butyrate (SMPB), N-succinimidyl 4-. (P-maleimidophenyl) acetate (SMPA),
N-succinimidyl 4- (p-maleimidophenyl)
The invention is characterized in that it is selected from propionate (SMPP), N- (γ-maleimidobutyryloxy) succinimide (GMBS) and N- (ε-maleimidocaproyloxy) succinimide (EMCS). The immunoassay method according to the item. 4. The immunoassay method according to claim 1, which contains 10 to 500 mol% of cholesterol based on the phospholipids and glycolipids constituting the liposome. 5. Activity of complement used in analysis (complement value)
Is 0.1 to 10 CH ₅₀ , The immunoassay method according to claim 1, wherein 6. The labeling substance is a fluorescent compound, a luminescent compound,
The immunoassay method according to claim 1, which is a light-absorbing compound, a saccharide, an ionic compound, an enzyme, a coenzyme, or a radical compound.