JPH0318149B2 - - Google Patents

Description

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

[Technical background of the invention and its problems]

Conventional immunoassay methods utilize, for example, antigen-antibody reactions between radioisotope-labeled antibodies (or antigens) and antigens (or antibodies) in samples collected from living organisms to identify specific antigens (or antibodies) in samples. ), or by an antigen-antibody reaction between an oxygen-labeled antibody (or antigen) and an antigen (or antibody) in a sample collected from a living body, an antigen-antibody complex is obtained, and the antigen-antibody combination is obtained. There is an enzyme immunoassay method that quantitatively analyzes a specific antigen (or antibody) in a sample using an oxygen reaction caused by oxygen labeled with a bound substance.

However, the radioimmunoassay method has the disadvantage that the equipment is large-scale because it uses radioactive substances, and both the radioimmunoassay method and the enzyme immunoassay method do not reach sufficient detection sensitivity. The drawback is that the analysis takes a long time, from several hours to several tens of hours.

〔Effect of the invention〕

This invention has been made in view of the above circumstances, and provides an immunoassay reagent that can analyze antigens and antibodies in a sample in an extremely short analysis time, and an immunoassay method using the immunoassay reagent. The purpose is to

[Summary of the invention]

To achieve the above object, the present invention is summarized as follows:
Red blood cells that bind antigens or antibodies to the cell membrane of red blood cells that undergo cytolytic action due to complement activation and contain enzymes or substrates that specifically react with enzymes, and substrates that specifically react with enzymes or substrates within red blood cells. It is characterized by having an enzyme and a complement, and also binds an antigen or antibody to the cell membrane of red blood cells that undergo cytolytic action due to complement activity, and also contains an enzyme or a substrate that specifically reacts with the enzyme inside. A reagent containing a substrate or an enzyme and complement that specifically reacts with the red blood cells contained in the red blood cells, enzymes or substrates in the red blood cells, and a sample containing antibodies or antigens is mixed, and the complement activated by the antigen-antibody reaction that occurs is mixed. This method is characterized by quantifying antigens or antibodies in a sample by quantifying the product of an enzymatic reaction produced by dissolving the cell membrane of red blood cells by cytolytic action.

[Embodiments of the invention]

First, the immunoassay reagent according to the present invention binds an antibody to the cell membrane of red blood cells that undergo cytolytic action due to complement activation, and reacts specifically with red blood cells that contain enzymes inside and with enzymes stored within red blood cells. Has substrate and complement.

As cells capable of binding antigens and accommodating enzymes, red blood cells of animals such as sheep can be suitably used. The cell membrane of red blood cells contains lipids, carbohydrates,
Since cells are made of proteins, there are many types of antibodies that bind to cell membranes. On the other hand, since the membrane of liposome is made of lipid, the types of antibodies that can bind to the membrane are limited. Therefore, red blood cells are superior to liposomes. Note that even red blood cells from other animals can be used as cells in the present invention as long as they can bind antibodies to their cell membranes and accommodate enzymes within the cells.

The antibody that binds to the cell membrane is appropriately selected from one that induces a specific antigen-antibody reaction with the antigen in the sample used in the immunoassay method, which is another invention related to this application, which will be described later. For example, if the antigen in the sample is α-
When it is fetoprotein, an α-fetoprotein antibody can be mentioned.

The enzymes contained within the cell may also include, for example, oxidizing enzymes such as glucose oxidase, uricase, glycerol oxidase, and cholesterol oxidase, dehydrogenases such as lactate dehydrogenase, glucose dehydrogenase, and glutamate dehydrogenase, decarboxylases such as glutamate dehydrogenase, etc. Examples include carboxidase, urease, pyruvate decarboxidase, and a mixture of the various enzymes mentioned above. Further, as an embodiment of accommodating an enzyme within a cell, a microorganism containing such an enzyme may be accommodated within the cell. Instead of accommodating the enzymes in the cells, a substrate that specifically reacts with the various enzymes described above may be accommodated. However, if immunoassay reagents that contain enzyme-containing cells are stored for a long period of time, the enzymes may become inactivated, so it is better to store the substrate rather than the enzyme in the cells. is preferable.

The amount of enzyme to be accommodated in cells is 1 ng/ml to 1 μ of antigen that reacts with antibodies that bind to cell membranes.
An amount sufficiently in excess of g/ml, for example, 100 U/ml or more in terms of enzyme activity value, preferably 500 U/ml
~1000U/ml. As described above, since the amount of intracellular enzyme is greatly in excess of the amount of antibody, immunoassay measurements using the immunoassay reagent of the present invention can be carried out in a short period of time.

The preparation of cells in which an antibody such as an α-fetoprotein antibody is bound to the cell membrane of sheep red blood cells and an enzyme such as glucose oxidase is contained within the red blood cells can be carried out, for example, as follows.

First, when α-fetoprotein is subcutaneously injected into animals such as rabbits, goats, mice, rats, etc., α-fetoprotein is sensitized in the animal body.
- Fetoprotein antibodies are produced. Approximately one week after the subcutaneous injection, a predetermined amount of blood is collected from the animal, and the supernatant of the obtained blood is separated to obtain antiserum containing α-fetoprotein antibodies. Note that, in order to improve the specificity of the antigen-antibody reaction, the antiserum may be further purified. On the other hand, a predetermined amount of blood is collected from another animal, such as a sheep, and purified, and an isotonic solution, such as a sheep, is purified.
An isotonic solution in which red blood cells are suspended is obtained by mixing with a buffer (PH 7) containing a salt at a concentration of 0.15M. Next, when the isotonic solution in which sheep red blood cells were suspended and the antiserum containing the α-fetoprotein antibody were mixed, the α-fetoprotein antibody was adsorbed onto the cell membrane of the sheep red blood cells.
An isotonic solution is obtained with red blood cells binding alpha-fetoprotein antibodies to cell membranes. Since the red blood cells suspended in this isotonic solution contain cell fluid, mitochondria, etc., the cell fluid and the like are expelled from the red blood cells using the standard dialysis method, and glucose, an enzyme, is added to the red blood cells. Contains oxidase. The amount of glucose oxidase accommodated is 500 to 1000 U/ml per whole red blood cell,
It is not limited to this, and can be appropriately determined according to the needs of the immunoassay method described below.

Complement contained in the blood of animals can be used; for example, guinea pig serum can be used as it is as a complement-containing fluid.

As the substrate in the immunoassay reagent, the above-mentioned substrates that specifically enzymatically react with the enzyme accommodated in the cells can be used. The amount of substrate may be an amount corresponding to the enzyme contained within the cell.

When the buffer solution containing cells that binds antibodies to cells and contains enzymes therein, prepared as described above, is mixed with the serum and substrate containing complement, the immunization according to the present invention can be performed. Analytical reagents can be obtained.

Next, an immunoassay method using the immunoassay reagent obtained as described above will be described.

In the analysis procedure, first, an immunoassay reagent and a sample, such as a blood sample collected from a patient, are mixed. As shown in FIG. 1, when the antibody 3 bound to cells in the reagent for immunoassay and the antigen 4 in the blood sample cause an antigen-antibody reaction, the complement in the reagent for immunoassay causes the antigen-antibody reaction. The cell membrane 1 is dissolved by the cytolytic action of complement activity, and the enzyme 2 contained within the cell is released outside the cell. A reaction product is obtained by an enzymatic reaction between the released enzyme 2 and the substrate 5 in the immunoassay reagent. This reaction product is then quantified by an appropriate means. As a quantitative means, for example, when the reaction product is an ion, an ion electrode is used.
When the reaction product is hydrogen peroxide, electrochemical means using a hydrogen peroxide electrode can be used, or the reaction product is reacted with other reagents to color the reaction solution. Spectroscopic means of measuring the absorbance of the liquid may also be employed. By quantifying the reaction product, it is possible to quantify the antigen contained in the blood sample. In other words, the amount of antibody bound to cells and the amount of enzyme contained within the cells in the immunoassay reagent must be known at the time of reagent preparation, and the amount of antigen bound to cells and antigen in the sample must be quantitatively determined. Because the enzyme released extracellularly reacts quantitatively with the substrate pre-existing outside the cell, it is possible to quantify the products of the enzymatic reaction using a calibration curve obtained experimentally in advance. This allows the antigen to be quantified. Moreover, since the amount of enzyme accommodated within the cells is much larger than the amount of antibodies bound to the cells, the immunoassay method of the present invention allows analysis to be carried out, which would take several tens of hours using conventional methods. It can be done in about 5 to 10 minutes.

Experimental example of immunoassay method A pre-prepared immunoassay reagent containing α-FP antibody and a blood sample known to contain α-FP are mixed according to the following conditions, and as a result of an antigen-antibody reaction, the immunoassay is released outside the cells. Hydrogen peroxide produced when glucose in the immunoassay reagent was oxidized by the action of glucose oxidase was quantified using a hydrogen peroxide electrode.

FIG. 2 shows the relationship between the current increase value output from the hydrogen peroxide electrode and the amount of α-FP in the blood sample.
As shown in Figure 2, there is a linear correlation between the current increase value and the amount of α-FP.
As shown in FIG. 3, there is an extremely good correlation between the FP quantification results and the α-FP quantification results obtained by the immunoassay method according to the present invention (referred to as the present method). Therefore, using the graph shown in Figure 2 as a calibration curve, α-
This proves that α-FP can be accurately and rapidly quantified by the immunoassay method of the present invention in blood samples with unknown FP content.

〔Effect of the invention〕

According to this invention, an antigen or antibody in a sample is detected by combining an antigen-antibody reaction and an enzyme reaction.
Quick and accurate quantification can be achieved simply by quantifying the enzymatic reaction product. Furthermore, since antibodies that bind to the cell membrane of red blood cells can be arbitrarily selected and bound thereto, it is possible to quantify over a wide range of antigens that specifically react with the bound antibodies.

Furthermore, its properties are stable and it can be stored for a certain period of time.

[Brief explanation of the drawing]

Fig. 1 is an explanatory diagram showing the principle of the immunoassay method according to the present invention, and Fig. 2 shows the increased value of the current output from the electrode for detecting the amount of enzyme reaction product produced in the immunoassay method according to the present invention. A graph showing the correlation with the amount of antigen in the sample, and FIG. 3 are graphs showing the correlation between the analysis results of the radioimmunoassay method and the analysis results of the immunoassay method according to the present invention. 1... Cell membrane, 2... Enzyme, 3... Antibody, 4...
...antigen, 5...substrate, 6...product.

Claims (1)

[Scope of Claims] 1. A red blood cell that has an antibody bound to its cell membrane and contains an enzyme inside, a substrate that reacts with the enzyme in the red blood cell, and a complement that causes lysis of the cell membrane through an antigen-antibody reaction. An immunoassay reagent comprising: 2. An immunoanalytical reagent comprising red blood cells that have antibodies bound to their cell membranes and contain enzymes inside them, a substrate that reacts with the enzymes in the red blood cells, and complement that causes lysis of the cell membranes through an antigen-antibody reaction. , an enzymatic reaction between the enzyme and the substrate occurs by mixing a sample having an antigen that reacts with the antibody, and lysing the cell membrane of the red blood cell by the cell membrane lytic action of complement activated by the antigen-antibody reaction. An immunoassay method, characterized in that the antigen is quantified by quantifying the product.