JPH0278946A - Immunoassy and chemical amplifier type immune sensor - Google Patents

Abstract

PURPOSE:To make measurement with ultra-high sensitivity without using a special labeling compd. by bringing an antigen and antiserum into reaction in the presence of a complement on a lipid film electrode having an antigen- contg. lipid film and detecting the reaction as microcurrent. CONSTITUTION:The serum corresponding to the antigen on the lipid film 3 is charged into a cell 9 and a specified voltage is impressed thereto via a counter electrode 7 from a constant voltage power source 13. The antibody (a) in the antiserum makes immune reaction with the antigen (c) on the lipid film 3 and this immune complex activates the complement (b) to induce the complement reaction. The local collapse of the lipid film 3 is induced by this reaction and a film channel (d) is formed on the lipid film 3. An ion (e) enters the inside of the lipid film electrode 1 through this film channel (d). The transfer and reception of the electron generated between this electrode and the electrode 5 are measured as the microcurrent value by a high-speed current amplifier 15, a storage oscilloscope 17 and an X-Y recorder 19. The measurement with the ultra-high sensitivity is enabled in this way without using the special labeling compd.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for measuring an immune reaction and a chemically amplified immunosensor.

Conventional techniques and their problems Immune reactions caused by antigens and antibodies play an important role in the body's defense mechanism. In such an immune response,
Because highly selective complex formation occurs, the resulting complex is characterized by having high molecular discrimination ability. Detection of biological components using immune reactions has become extremely important in the biomedical field in general since the latex agglutination reaction was proposed by Singer, Piotz, and others in 1956. Particularly, in the field of clinical medicine, it is widely used including cancer markers. However, conventional biological component detection methods that utilize immune reactions require extremely step-by-step reactions, or are impossible to measure in 5 in situ.
This method has the problem of high cost because it requires special equipment.

Recently, attempts have been made to chemically amplify immune reactions and detect them quickly and with high sensitivity. Examples of chemical amplification methods include:
A method that utilizes the lipid membrane breakdown reaction of liposomes by the complement reaction system (antiserum is reacted with liposomes that have incorporated an antigen in the lipid membrane and encapsulated a labeling substance to cause the lipid membrane of the liposome to collapse, thereby releasing the A method for quantifying labeled substances is known. For example, Yasuda et al. have proposed a method using carboxyfluorescein, a fluorescent substance, as a label, and Umezawa et al. have developed a potential measurement method using an electrolyte as a label. However, these methods require 1) indirect methods that require labeled compounds labeled with fluorescence, spin, etc.;
2) Since it is a relative concentration detection method, it is essential to create a standard curve; and 3) Liposomes are unstable, resulting in high background noise, which limits the ability to increase sensitivity. There are some problems.

Problems to be Solved by the Invention It is known that membrane attack complexes (MACs) consisting of C3b-9, which form channels in lipid membranes, are generated with complement activation initiated by immune reactions. There is. The purpose of the present invention is to rapidly and superimpose an immune response without using liposomes or special labeling compounds by generating MAC on a lipid membrane and detecting the current flowing through the membrane channel formed thereby. It is an object of the present invention to provide a method and apparatus capable of direct amperometric measurement with high sensitivity.

Means for Solving the Problems The objects of the present invention are achieved by the following immune reaction measuring method and chemically amplified immunosensor.

(2) A method for measuring an immune reaction, which comprises reacting the antigen with an antiserum in the presence of complement on a lipid membrane electrode having an antigen-containing lipid membrane, and detecting this reaction as a microcurrent.

■A chemically amplified immunosensor comprising a lipid membrane electrode having an electrode inside and an antigen-containing lipid membrane provided on the gate surface, and an electrode opposite to the internal electrode of the lipid membrane electrode. .

According to the present invention, extremely sensitive antibodies can be detected even at extremely low concentrations of sub-picomolar/ITIQ.
It can provide a new method for measuring immune reactions.

The antigen-containing lipid membrane in the present invention refers to a known lipid bilayer membrane such as black membrane, membrane filter, or screen that is impregnated with an antigen-containing lipid as a support to form an antigen-containing lipid membrane on the support. mean something

Antigen-containing lipids can be produced according to known methods. For example, an antigen-containing compound and a lipid may be reacted in a suitable solvent. As the antigen, known antigens can be used, such as antigens consisting of haptens, sugars, peptides, etc., having functional groups such as dinitrophenyl groups (DNP). The lipids are not particularly limited as long as they are conventionally used in the preparation of liposomes, such as egg yolk lecithin (EYL), silauroyl phosphatidylcholine (DLPC), simyristoyl phosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine ( Phospholipids such as DPPC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylethanolamine (DPPE), simyristoylphosphatidylserine (DMPS), and dipalmitoylphosphingomyelin (DPSM), cholesterol and its derivatives, etc. be able to. Lipids can be used alone or in combination of two or more. The ratio of antigen and lipid to be used is not particularly limited and can be appropriately selected from a wide range, but usually the former should be used in an amount of about 1 to 10 times the molar amount per 1 mole of the latter. The reaction temperature may normally be about 5 to 45°C. Examples of the solvent include hydrocarbons such as n-octane, halogenated hydrocarbons such as chloroform, and lower alcohols such as methanol and isopropanol.

The antigen-containing lipid membrane used in the present invention can be obtained by making the antigen-containing lipid thus obtained into a bimolecular membrane or impregnating it into a membrane filter or screen according to a conventional method. In this case, the background current is 1O- for a potential gradient of 20mV.
It is preferable to set it to about IO to 10-'IA. If the current value is larger than that, a lipid membrane sensitive to complement reaction may not be formed. The membrane filter or screen is not particularly limited as long as it has been conventionally used for retaining lipids, and examples include polycarbonate membranes, cellulose ester membranes, Teflon membranes, and the like. Among these, those with a diameter of 0.1 to 1
Preferably, the material has pores of about 0 μm.

The method of the present invention comprises a lipid membrane electrode having an electrode therein and a gate surface provided with the antigen-containing lipid membrane obtained as described above, and an electrode opposite to the internal electrode of the lipid membrane. It is carried out using a sensor and based on counter-electrode constant potential coulometric analysis. The internal electrode of the lipid membrane electrode and the counter electrode thereof are not particularly limited, and those conventionally used for current measurement may be appropriately selected and used. Specific examples include Ag-AgCQ electrodes, pt electrodes, and the like.

The method of the present invention will be explained in more detail below with reference to the drawings. FIG. 1 is a schematic diagram showing an example of the immunosensor of the present invention. In the immunosensor, a lipid membrane electrode (1
) Other known devices can be used. Lipid membrane electrode (
1) includes an antigen-containing lipid membrane (3) on the gate surface and an electrode (5) inside the membrane. (7) is the opposite of (5). Furthermore, (9) is a cell, (11) is a water jacket, (13) is a constant voltage power supply, (15) is a high-speed current amplifier, (17) is a storage oscilloscope, and (19) is an XY recorder.

First, antiserum corresponding to the antigen on the lipid membrane (3) is applied to the cell (
9). The antiserum used here is not particularly limited, and antisera prepared by known methods can be appropriately selected and used. The cell (9) is normally filled with a suitable buffer that does not affect the immune reaction. A constant voltage is applied to this immune reaction system from a constant voltage power source (13) via a counter electrode (7). Although the voltage is not particularly limited, it is not preferable that the voltage is too high, and it is usually about 0 to 100 mV. Further, the reaction temperature can be appropriately selected by adjusting the temperature of the water in the water jacket (11), but is usually about 5 to 45°C.

As shown in Figure 2, antibodies in the input antiserum (a)
causes an immune reaction with the antigen (c) on the lipid membrane (3).

The immune complex thus formed activates complement (b), causing a complement reaction.

This complement reaction causes local collapse of the lipid membrane (3), and membrane channels (d) are formed in the lipid membrane (3).

Through this membrane channel (d), -ions (e) enter into the lipid membrane electrode (1).

This ion (e) exchanges electrons with the electrode (5), and this electrochemical reaction causes a high-speed current amplifier (15).
, a storage oscilloscope (17) and an X-Y recorder (19) to measure and detect the minute current value.

Effects of the Invention According to the method of the present invention, it is possible in principle to detect the presence of even one molecule of antibody as a microcurrent without using liposomes or special labeling compounds.
Ultra-high sensitivity measurement of sub-picomolar/some antibody concentration is possible. Therefore, the method of the present invention has significantly superior sensitivity compared to conventional immunoreaction measurement methods using liposomes. In addition, the method of the present invention enables the detection of immune reactions in a modinian system, and can be applied not only to biological measurements but also to a wide range of applications such as food fermentation processes and environmental measurements.
It can also be used for complement assays.

Example Examples will be given below to further clarify the present invention.

Example 1 Immune reactions were measured using the immunosensor of the present invention shown in FIG.

As the lipid membrane electrode (1), the tip of a glass tube with a diameter of 2 mm was made of polycarbonate impregnated with an n-octane solution containing DNP-cap-DPPC1 mmolar-1 egg yolk phosphatidylcholine 10 mmolar and cholesterol 10 mmolar. A membrane [(3) having pores with a pore diameter of 1 to 5 μm] was inserted, and an Ag/AgCR electrode (5) was inserted inside the membrane. As the opposite pole (7),
A g/A g CQ electrode was used. In addition, as an antibody, rabbit anti-DN made by DNP-globulin disease was used.
P antiserum was used.

20 μQ of the above rabbit anti-DNP antiserum was put into a cell (9) containing GVB buffer, the voltage of the counter electrode was varied between 0 and 100 mV, and the temperature was 37.
Conductance was measured over time by immunoreaction at ℃.

Comparative Example 1 Measurement was carried out in the same manner as in Example 1, except that rabbit anti-DNP antiserum in which complement had been immobilized by heating was used.

Comparative Example 2 O, in place of rabbit anti-DNP antiserum, O in INKCR solution
．． 20 μQ of 1% palinomycin was added, and the lipid mixture was 2% EYL and 1% cholesterol.
Measurement was carried out in the same manner as in Example 1 except for using an n-hebutane solution containing .

FIG. 3 shows the change in conductance over time in Example 1 and Comparative Example 1, and FIG. 4 shows the change in conductance over time in Comparative Example 2.

As is clear from Figure 3, when the reaction was carried out by adding rabbit anti-DNP antiserum (Example 1...◆), 1
After a time lag of about 5 minutes, an increase in conductance was observed at a rate of 17 nS/min. ), no reaction was observed. According to the method of Example 1, it was possible to detect antibodies at a concentration of 1 subpicomole/m12.

Furthermore, in FIG. 4, when palinomycin was added (Comparative Example 2), the conductance increased without any time lag, and it is clear that a conductive lipid film was formed.

From the above results, it can be concluded that the current in Example 1 is due to channel formation on the lipid membrane associated with complement activation, and that the measurement method of the present invention has extremely excellent sensitivity for detecting immune reactions. I understand.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram showing an example of an apparatus for carrying out the method of the present invention. FIG. 2 is a diagram schematically showing the reaction according to the measurement method of the present invention. Figure 3 shows implementation F! 711
11 is a graph showing changes in conductance over time in reactions of No. 11 and Comparative Example 1. FIG. 4 is a graph showing the conductance change over time in the reaction of Comparative Example 2. (1) Lipid membrane electrode (3) Antigen-containing lipid membrane (5) Electrode (7) Counter electrode (9) Cell (11) Water jacket (13 )...Constant voltage power supply (15)...High speed current amplifier (17)...Storage oscilloscope (19)
)...X-Y recorder (a)...Antibody (b)...Complement (c)...
・Antigen (d)...Membrane channel (e)...Ion (more) Kaya 1 figure

Claims (1)

[Claims] (1) On a lipid membrane electrode having an antigen-containing lipid membrane,
A method for measuring an immune reaction, which comprises reacting the antigen with an antiserum in the presence of complement, and detecting this reaction as a microcurrent. (2) A chemical amplification type characterized by comprising a lipid membrane electrode having an electrode inside and an antigen-containing lipid membrane provided on the gate surface, and an electrode opposite to the internal electrode of the lipid membrane electrode. Immune sensor.