JPS60159652A - Reagent for immunological analysis - Google Patents

Abstract

PURPOSE:To widen the application range of a material to be detected by constituting a reagent in such a way that the 2nd antibody is deposited on a microcapsule. CONSTITUTION:This reagent 10 for immunological analysis consists of a microcapsule 11 consisting at least partly of a liquid film, the 2nd antibody 12, which is fixed onto the capsule 11, for the 1st antibody and a labeled material 13 sealed in said capsule 11. The reagent 10, a substantial amt. of the 1st antibody 14 and an antigen 15 which is the material to be detected are first mixed in a suitable buffer soln. to induce an antigen-antiboty reaction and to obtain an antigen-1st antibody-2nd antibody sensitized microcapsule 16. When a substantial amt. of complement 17 diluted with a suitable buffer is added thereto, the capsule 11 is broken by the effect of the complement 17 according to the quantity of the antigen 15, i.e., the antigen-(1st) antiboty reacting weight and the material 13 is released from the inside of the capsule 11. The quantity of the material 15 in the sample is measured by the analyzing method meeting the material 13.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field of the Invention] The present invention relates to an immunoassay reagent, and more particularly to an immunoassay reagent for quantitatively analyzing a specific antigen or antibody present in a sample.

[Technical background of the invention and its problems] As research on the four cancers progresses, various tumor markers have been discovered. Representative examples include α-fetoprotein (AFP), carcinoembryonic antigen (CEA), basic fetoprotein (RFP), and pancreatic carcinoembryonic antigen (POA). The concentration of these tumor markers is very low in normal people (for example, in the case of AFP: lOng/m or less)
. On the other hand, tumor patients often exhibit values that are about 10 times higher than normal people. In any case, extremely high detection sensitivity is required for the analysis and quantification of tumor markers.

To meet this demand, conventional radioimmunoassay methods (RI) using antigens or antibodies labeled with radioactive substances have been used.
A) was developed. However, R1 and A are difficult to handle and disposal is also a problem.

Therefore, immunoassay methods have been proposed that use antigens or antibodies labeled with various substances such as enzymes or fluorescent substances instead of radioactive substances, but even in these methods, free antibodies and bound antibodies must be separated in some way. It had the disadvantage that it did not. Also, Rosenthal A, F,
Vargas, M.G. and Klassc:,S.
.

(197B) Cl1n, Chem, 22.1899
The EMIT method announced in 2007 is an innovative method that allows measurement in a homogeneous system without the need for a separation step, but in principle it cannot be applied to high molecular weight protein antigens or antibodies.

By the way, Haxby, J.A., K1n5ky,
C, B, andKinsky S, C, (198B)
Biochemistry, fil 300 is a method for quantifying antibodies by preparing liposomes that incorporate lipid-soluble antigens into the membrane and encapsulating glucose, and measuring the amount of glucose flowing out as the liposomes are destroyed by antigen-antibody reactions. Announced. 1. - ゛ Additionally, as described in the patent application specification dated November 30, 1981, the present inventors have developed an immunoassay reagent in which a liposome carries a hydrophilic antigen or antibody, and We succeeded in easily quantifying antigens or antibodies using this system in a short time.

However, these immunoassay reagents that carry antigens or antibodies directly on liposomes have antigen-antibody specific reactivity, so one type of liposome can be used to
This method has the disadvantage that it can only be applied to different types of test substances, and liposomes sensitized with antigens or antibodies corresponding to each test substance must be prepared. Therefore, there has been a problem in that the productivity, versatility, and practicality of the reagent for immunoassay are low.

[Objective of the Invention] The present invention is an immunoassay reagent in which a labeling substance is encapsulated in complement-active microcapsules carrying an antigen or an antibody, and which improves the productivity, versatility, and practicality of an immunoassay reagent. The purpose is to improve.

[Summary of the Invention] As a result of extensive research in order to achieve the above object, the present inventors have discovered that without directly carrying an antigen or an antibody (first antibody) on complement active microcapsules, When an antibody (second antibody) against the antibody is immobilized on the microcapsule, various test substances can be immunoanalyzed using the same microcapsule, which improves the productivity, versatility, and practicality of immunoassay reagents. The present inventors have discovered that the present invention can be improved and have completed the present invention.

That is, the immunoassay reagent of the present invention comprises: a microcapsule at least partially made of a lipid membrane; a second antibody fixed on the microcapsule and directed against the first antibody; and a label encapsulated within the microcapsule. Characterized by being made of matter.

The present invention will be explained in more detail below.

Test substances that can be quantified using this analysis method include tumor markers (the aforementioned AFP, RFP, CEA, POA, ferritin, etc.), immunoglobulins (IgA, IgE, IgG, IgM, etc.), hormones (insulin, T3. T
4 and HCG) and drugs.

The microcapsules used in the immunoassay reagent of the present invention may be of any type as long as they exhibit complement activity, and in order to exhibit complement activity, at least a portion of the microcapsule membrane must be composed of a lipid membrane. There is a need. Examples of such microcapsules include liposomes, red blood cell ghost membranes, etc., and liposomes composed of phospholipids or glycolipids and cholesterol are particularly preferred. For example, when liposomes are synthesized from phospholipids and cholesterol, stable liposomes are likely to be obtained when the ratio of these is around 1:1. Moreover, it is preferable that the fatty acid residue in the phospholipid has 12 to 18 carbon atoms, and more preferably an even number.

The second antibody immobilized on the microcapsules is an antibody (for example, an anti-rabbit immunoglobulin obtained from a goat when the animal to be immunized with the antigen is a rabbit) of the antibody against the test substance (the first antibody). G (IgG) antibody).

These second antibodies are immobilized on the microcapsules by various covalent bonding methods used for enzyme immobilization. However, fixation methods using strong cross-linking agents such as glutaraldehyde.

This is undesirable because it significantly reduces the activity of the second antibody.

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 (lO-3
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; Absorption bands specific to the visual region or ultraviolet region light-absorbing compounds (water-soluble pigments, etc.) that have Amide Adenine Dinucleotide (NAD)
Coenzymes such as; radical compounds such as methyl viologen are desirable. 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 described above is produced, for example, by the following method. First, the desired lipid and N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(p-maleimidophenyl) phthalate (SMPH), N-succinimidyl 4-(p-maleimidophenyl) acetate A cross-linking agent such as (SMPA) (the case where this is used is called a cross-linking method) is reacted in a solvent (instead of a cross-linking agent, a lipid activator such as cyanogen promide (CNBr) is used).
, cyanuric chloride (CC), shrimp chlorohydrin (EH), o-bromoacetyl-N-hydroxysuccinimide, etc. (this method is referred to as the active lipid method), and immobilized on liposomes. A functional group capable of binding to a second antibody is introduced into a lipid molecule. Next, the obtained functional lipid, cholesterol and, if necessary, other lipids are placed in a flask, a solvent is added to cause a reaction, and then the solvent is distilled off and the mixture is suction-dried. Thereafter, an aqueous solution of a predetermined labeling substance is added into a flask with a thin film formed on the wall, the flask is tightly stoppered, and the flask is shaken to obtain a suspension of sensitized liposomes.

On the other hand, the second antibody to be immobilized on the liposome and a crosslinking agent are reacted in a buffer solution to introduce a crosslinking group, and then, if necessary, a reagent for reducing the crosslinking group (e.g. dithiothreitol, DTT) is used. ) to obtain a modified second antibody. Note that this step is not necessary if the lipid was treated with the activator in the above step.

Finally, the immunoassay reagent of the present invention is obtained by reacting the sensitized liposome with the modified second antibody (unmodified second antibody when using the active lipid method) in a buffer. Such a reagent is usually obtained as a microcapsule containing a labeling substance and carrying a second antibody immobilized on its surface.

In addition, the immunoassay reagent of the present invention first binds a lipid and a second antibody using a crosslinking agent or a lipid activator,
It is also possible to produce the conjugate by adding the obtained conjugate to water together with a surfactant to form micelles, and then removing the surfactant using dialysis or gel filtration.

Next, an analysis method using the reagent of the present invention will be explained with reference to FIG. 1 of the accompanying drawings.

FIG. 1 is a schematic diagram showing the principle of immunoassay using the reagent of the present invention.

First, an immunoassay reagent 10 of the present invention comprising a microcapsule 11 containing a second antibody 12 and a labeling substance 13 encapsulated therein, a sufficient amount of the first antibody 14, and an antigen 15 as a test substance. are mixed in an appropriate buffer (e.g., gelatin-veronal buffer) to cause an antigen-antibody binding reaction as indicated by arrow a in FIG.
Second antibody-sensitized microcapsules 16 are obtained. Next, a sufficient amount of complement 17 diluted in a suitable buffer is added thereto.

Then, according to the reaction indicated by the arrow, 15 amounts of antigen,
That is, depending on the amount of antigen-(first) antibody reaction, the microcapsule 11 is destroyed by the action of complement 17, and the labeling substance 13 is released from within the microcapsule 11. Next, quantification is performed using an analysis method depending on the labeling substance 13 (for example, fluorescence analysis if the labeling substance 13 is a fluorescent substance), and, for example, using a calibration curve prepared in advance, the amount of the analyte 15 in the sample is determined. Amounts can be measured.

Complement 17 used in the quantitative operation is not particularly limited, but guinea pig serum is usually used. However, rabbit, mouse, human serum, etc. may also be used.

[Effects of the Invention] The immunoassay reagent of the present invention has a second antibody supported on microcapsules, so compared to conventional reagents, it can be applied to a wider range of test substances, and is suitable for simultaneous measurement of multiple items. Easy to automate analysis. Furthermore, since there is no need to use different microcapsules for each measurement item, the productivity of immunoassay reagents is high, and analysis costs are low.

[Examples of the Invention] Hereinafter, the present invention will be explained in more detail with reference to Examples, but these Examples are not intended to limit the scope of the present invention in any way.

(A) Preparation of reagents and sensitized liposomes (1) Reagents dipalmitoylphosphatidylcholine (DPPC), cholesterol, dipalmitoylphosphatidylethanolamine (DPPE) and dithiothreitol (DT)
T) manufactured by Sigma was used. N-succinimidyl 3-(2-pyridyldithio)propionate (SPD
P) and Sephadex G-25 fine were purchased from Pharmacia. As for other reagents, commercially available products (special grade) were used without purification. Note that ion-exchanged water was used as water.

(2) Preparation of sensitized liposomes a) DPPE-dithiopyridinate (DPPE-DTP
) Preparation of 5m lomM DPPE (chloroform solution) in a test tube
Add jj and 50 mg (7) S P D P,
After purging with nitrogen gas, the reactor was sealed tightly and allowed to react at room temperature for 2 hours. (50 grams of triethylamine was added as a catalyst). After the reaction, it was extracted three times with 5 times the volume of physiological saline, the remaining chloroform phase was dried under reduced pressure, and finally 5 ml of chloroform was added and stored at 20°C in a tightly stoppered test tube.

b) Preparation of liposomes All lipids used were dissolved in chloroform or chloroform/methanol (2/1). First, 5mM
DPPC (200gl), 10mM cholesterol (
100gl) and 1mM DPPE-DTP (60p
-1> was added with 10 mM chloroform and mixed well. The solvent was removed on a rotary evaporator in a water bath (approximately 50° C.).

Add 2 ml of chloroform to the sardine again, stir thoroughly,
The solvent was evaporated again using a rotary evaporator. When this operation was repeated several times, a thin film was formed on the flask wall. The flask was placed in a desiccator and suctioned with a vacuum pump for about 1 hour to completely remove the solvent. Next, 10
0ILR 0.2M carboxylfluorescein (Eastman Kodak W.

PH7.4) was added (17), the inside of the flask was purged with nitrogen, the flask was tightly stoppered, and the flask was 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 wall surface completely disappeared. A liposome suspension was prepared by this operation.

A small amount of gelatin-veronal buffer (containing 0.1 mM MgC12 and 0.03 mM Caclt; hereinafter abbreviated as GVB2+) was added, and the liposome suspension was completely transferred to a centrifuge tube. Free carboxyl fluorescein was removed by centrifugation at 15,000 rpm for 20 minutes at 4°C.

This operation was repeated using GVB2+ until the supernatant became clear. Finally, add 2ml of GVB2+ and 5JLJl (
7) 10% NaN3 was added, suspended using a Vortex mixer, sealed with nitrogen, and stored in a refrigerator.

C) Modification of anti-horse heron IgG antibody (goat) 5 mg of anti-horse heron IgG antibody (goat) (manufactured by Miles) was added to 2 ml of 0.5 mg of anti-horse heron IgG antibody (goat). After dissolving in OIMHEPES buffer (pH 7, 45, 0, containing 85% NaCJl) and purging with nitrogen, 10#LJl (7) 10mM
5PDP (-1-tanol solution) was added, stirred thoroughly, and allowed to react at room temperature for 30 minutes. After the reaction, add Sephadex G-25 to which the reaction solution was saturated in advance with physiological saline.
Column packed with fine gel (gel volume: approx. 15 m)
Develop in 0.1M acetate buffer (P) 14.5, 0.85% Na
Cl-containing). First peak fraction (
Add another 2 ml of acetate buffer to the solution (approx.
) was added. After stirring thoroughly and reacting at room temperature for 20 minutes, the column was filled with Sephadex G-25 fine gel saturated with 0.01 M HEPES buffer (gel volume: approximately 30 m). The reaction was developed and eluted with HEPES buffer. The first peak fraction (approximately 2 mjL) was collected, purged with nitrogen, and stored in a refrigerator until use.

d) Preparation of anti-heron IgG antibody (goat) sensitized liposomes The liposome suspension prepared as described above and an equal volume of modified anti-heron IgG antibody (goat) solution were mixed, and the mixture was replaced with nitrogen and sealed. The mixture was allowed to react overnight at room temperature with slow shaking.

Unreacted antibodies were removed by washing with HEPES buffer and then GVB2+. 10% NaN3 of GVB2+ and 5#L corresponding to the amount of liposome suspension used in the reaction.
was added last, and after suspension and nitrogen substitution, the solution was stored in a refrigerator until use.

(3) Measurement of human IgG using anti-heron IgG antibody (goat) sensitized liposomes Pour an appropriate amount of GVB into a U-shaped plate (96 wells) manufactured by Tank Co., Ltd.
Human IgG diluted with 2+ was injected at 25p-1 doses.

Next, the above sensitized liposome suspension was diluted with GVBZ+ for 10
It was diluted 0 times and 5 copies were dispensed into each well. Furthermore, approximately 2 pg/ml of anti-human IgG antibody was added to the
Add Ogu to each well, and finally add 2 g of complement (derived from guinea pig) appropriately diluted with GVBZ+.
Added 5w sentences each. The reaction was carried out at a high temperature of 37°C.
Time went. After the reaction, add ioo to each -ell.
The reaction was stopped by adding tM EDTA-veronal solution, and the plate fluorescence spectrophotometer (manufactured by Corona Electronics, MTP) was used.
-12F), the fluorescence of each well was measured (Ex
: 490 nm, Em: 520 nm). Note that the measured values were obtained using 10% Triton X instead of the antibody.
The difference between the fluorescence of a well to which -100 was added for 25 days and the fluorescence of a well to which 25 JLI GVB2+ was added instead of the antibody was expressed as a relative value, with the difference being taken as 100%. The results obtained when a 400-fold dilution of complement was used are shown in FIG.

Human AFP (purchased from Japan Biotest Institute) was measured using the anti-heron IgG antibody (goat) sensitized liposome prepared in Example 1 and the anti-human IgG antibody (rabbit) [purchased from Maruzen Sekiyu]. The operating method was as shown in Example 1.

When using 800-fold diluted complement (guinea pig serum), human A within the range of 10-200 ng/ml
It was found that FP can be quantified.

Anti-heron IgG antibody (goat) sensitized liposome and anti-horse) -
Human ferritin (rabbit) using ferritin antibody (rabbit)
(purchased from Sigma) was measured.

The operating method is the same as in Example 1. When using 400-fold diluted complement (guinea pig serum), 5 to 500 ng/
Human ferritin was measurable within the mJl range.

The anti-heron IgG antibody (goat) sensitized liposomes prepared in Example 1 were stored in a refrigerator (4°C) and periodically immunized with human I
When gG was measured, no decrease in measurement sensitivity was observed even after 6 months, indicating that the liposome was stable for at least 6 months.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram showing the principle of immunoassay using the reagent of the present invention. Figure 2 shows the case where human IgG antibodies were measured using anti-heron IgG antibody (goat) sensitized liposomes. It is a correlation diagram between the dilution factor and relative fluorescence intensity of human IgG antibodies. lO... Reagent for immunoassay of the present invention II... Microcapsule 12... First antibody 13... Labeling substance 14... Second antibody 15... Antigen (test substance) 16...・Antigen-first antibody-second antibody sensitized microcapsule 17...complement

Claims (1)

[Scope of Claims] (]) A microcapsule at least partially composed of a lipid membrane; A second antibody fixed on the microcapsule and directed against the first antibody; A hydrophilic antibody encapsulated within the microcapsule; An immunoanalysis reagent comprising a labeling substance. (2) The immunoassay reagent according to claim 1, wherein the second antibody is covalently immobilized on the microcapsule. (3) Claim 1, wherein the labeling substance is at least one compound selected from the group of fluorescent compounds, luminescent compounds, light-absorbing compounds, sugars, ionic compounds, coenzymes, and radical compounds. Reagents for immunoassays. (4) The reagent for immunoassay according to claim 1, wherein the microcapsule is a liposome.