JPH0711522B2 - Chemically amplified chemiluminescence immunoassay - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION The present invention relates to a novel chemo-amplified chemiluminescent immunoassay.

2. Description of the Related Art Conventional technology and its problems Radioimmunoassays, enzyme immunoassays, etc. have been developed as methods for analyzing and quantifying substances such as antigens and antibodies that are present in trace amounts in clinical samples such as blood and urine. However, in the former case, radioactive compounds are used, so there are problems in management such as storage, handling, and disposal, and because reagents with short-lived isotopes must be used frequently, reagents must be adjusted frequently. There is a problem that it takes time. Further, the latter has problems that the measurable substances are limited and the sensitivity is insufficient, and that it is necessary to synthesize a compound in which an expensive enzyme is labeled with an antigen or an antibody. In addition, both of them have a problem that a heterogeneous method is mainly used and a complicated separation operation is required.

In addition, glucose, electrode active materials, fluorescent dyes, etc. are embedded in liposomes and released according to the amount of antibody by utilizing the phenomenon that membrane destruction is caused by activation of complement following immune reaction. A liposome immunoassay method is known in which the content is measured and the amount of antibody is quantified. However, this method has a problem that the liposome is unstable and leaks easily or the reproducibility of the liposome production is poor, and the sensitivity is not sufficient because the embedding amount is small.

As described above, no method has been established for homogeneously measuring a small amount of a substance to be measured such as an antigen and an antibody easily, rapidly and with high sensitivity, and there is a demand for such a method.

Means for Solving the Problem The present inventor, as a result of earnest research to meet the above-mentioned demand,
In the liposome immunoassay, erythrocytes are particularly used instead of liposomes, and the complement hemolysis reaction between the substance to be measured and erythrocytes is further found by combining the luminol reaction with hemoglobin released in the reaction, thereby achieving the object, The present invention has been completed.

That is, the present invention is a chemical amplification chemiluminescence immunoassay characterized in that a substance to be measured is subjected to a complement hemolysis reaction with erythrocytes, and then chemiluminescence is caused by a luminol reaction by hemoglobin released from erythrocytes, and the luminescence amount is measured. Pertaining to the law.

According to the present invention, the complement hemolysis reaction, that is, the presence of an antigen-antibody complex due to an immune reaction on the erythrocyte cell membrane, causes activation of complement, and a membrane-injured complex is constructed on the membrane so that the contents of hemoglobin Due to the reaction that causes release, it is said that this release is sufficiently caused by the formation of one complex. Therefore, one antigen-antibody complex exerts a chemical amplification effect by the large release of hemoglobin. Furthermore, since hemoglobin, which is the content, is a catalyst for the luminol reaction, the chemical amplification effect is also exhibited by this chemiluminescence reaction.

In the method of the present invention, since the antigen, the antibody, the antigen-antibody complex and the complement are essential for the complement hemolysis reaction, any of these can be quantitatively measured as a substance to be measured, and various kinds of qualitative measurements can be performed. Further, the method of the present invention can be directly applied to various conventionally known immunoassay methods such as competitive reaction, sandwich method, homogeneous method, and heterogeneous method.

In the present invention, by using stable red blood cells that are obtained in large quantities and homogeneously, and because the luminol reaction is highly sensitive, stability, reproducibility, immunoassay excellent in sensitivity, etc. It can be done easily. The red blood cells to be used are not particularly limited, but examples thereof include red blood cells of sheep, rabbit, mouse, rat, guinea pig, dog and the like. Red blood cells themselves have membrane antigens, but they may also have the antigen, antibody or antigen-antibody complex to be measured on their surface. The method for preparing red blood cells carrying an antigen, an antibody or an antigen-antibody complex is not particularly limited, and various conventionally used methods can be used. As the antigen, any of various low molecular weight substances and high molecular weight substances can be used.

The measurement procedure of the present invention will be described below by taking the measurement of complement as an example. To the sensitized red blood cells in which a fixed amount of the antigen-antibody complex is formed on the red blood cells, a solution to be measured containing a complement such as serum is added and reacted at a constant temperature and time as appropriate. The presence of the antigen-antibody complex results in activation of complement, building a membrane-damaged complex on the erythrocyte membrane, and releasing the contents. Red blood cells contain a high concentration of hemoglobin, which catalyzes the chemiluminescence of luminol. After this,
Stop the complement fixation reaction by adding ice-water, adding EDTA, etc. The luminol reaction can be performed by a conventional method. That is, usually, a certain amount of luminol and hydrogen peroxide are added to the reaction liquid which has been subjected to the above stop operation under a constant temperature in the dark to cause chemiluminescence. The amount of chemiluminescence can be measured by a conventional method such as a photon counting method. For the amount of luminescence, either the total amount (integrated value) or the maximum value for a certain period of time after the reaction may be used, and a calibration curve is prepared using a standard sample with a known concentration, and measurement such as quantification of an unknown sample is performed. .

Non-leaked hemoglobin has no contact action for the luminol luminescence reaction, and non-hemolytic erythrocytes do not interfere with the reaction. Therefore, in the method of the present invention, the measurement can be carried out by the homogeneous measurement system as it is, without separating the non-hemolytic erythrocytes.

EFFECTS OF THE INVENTION As described above, according to the method of the present invention, an immune reaction on erythrocytes can be theoretically performed without using a special labeling compound.
If molecules are generated, it is possible to perform immunoassay in a homogeneous system without separation of blood cells, which is significantly amplified and has excellent stability and reproducibility.

Therefore, according to the present invention, a novel effect is provided that a novel chemical amplification type chemiluminescence immunoassay capable of simply and rapidly measuring with high sensitivity a small amount of a substance to be measured such as an antigen, an antibody and a complement is provided. Is played.

EXAMPLES The present invention will be described in more detail with reference to the following examples.
The present invention is not limited to the following examples.

Example 1 Measurement of anti-sheep erythrocyte antibody was performed.

Sheep red blood cells were added to a gelatin veronal buffer (hereinafter referred to as GVA ⁺⁺ ) having a pH of 7.4 containing Ca ⁺⁺ and Mg ⁺⁺ ,
The cells were suspended at a rate of × 10 ⁹ cells / ml. To this suspension, 5 µl of a rabbit serum sample containing the above antibody was added in an equal amount, immunoreacted at 37 ° C for 10 minutes, and then guinea pig serum was diluted 500-fold with GVB ⁺⁺ to obtain a complement solution. 12.5 μl and GVB
⁺⁺ 20 μl was added, and the complement fixation reaction was carried out at 37 ° C. for 60 minutes, followed by cooling with ice to stop the reaction. GVB ⁺⁺ 37.5μ in the reaction solution after reaction
l, and then 50 μl of 1 mM luminol solution, put the sample cup in the dark box, and add 50 μl of 5 mM hydrogen peroxide solution.
Was added to cause a chemiluminescent reaction at 25 ° C. The chemiluminescence measurement was performed by the photon counting method (using the Hamamatsu Photonics Universal Photon Counting System). First
The figure is a plot of the maximum number of photon counts when the dilution ratio of the serum sample (antibody) is changed, and it is clear that the target antibody can be accurately measured.

Example 2 Measurement of human complement was performed.

Commercially available at a rate of 5 × 10 ⁸ cells / ml in a GVB ⁺⁺ diluted solution 162 to 608 times higher than that of normal human serum at low temperature (Ishizu Pharmaceutical Co., Ltd.)
Suspension) of sheep sensitized red blood cells (produced by the antigen-antibody complex) prepared in (1) were mixed at a ratio of 26: 4, incubated at 37 ° C. for 2 hours with shaking, and then ice-cooled with EDTA. GVB was added to stop the complement fixation reaction. 1 μm for 60 μl of this reaction solution
50 μl of M luminol solution was added, the sample cup was put in a dark box, 50 μl of 5 mM hydrogen peroxide solution was added, and chemiluminescence reaction was caused at 37 ° C. The chemiluminescence measurement was performed by the photon counting method (using the Hamamatsu Photoix Universal Photon Counting System). FIG. 2 is a plot of the maximum number of photon counts against the dilution ratio of complement, and it is clear that the target complement can be accurately measured.

On the other hand, for comparison, 2.8 ml of the reaction solution after the above reaction was stopped and the supernatant obtained by centrifugation at 3000 rpm for 5 minutes was used, and the optical path length was 1 cm and the wavelength was 541 nm according to the conventional absorbance measurement method.
Absorbance was measured with a Hitachi 557 type dual wavelength spectrophotometer.

FIG. 3 is a plot of the maximum count of photon counts measured by the method of the present invention and the total count within the measurement time, and the absorbance measured by the conventional method for the same sample. The correlation coefficient between the absorbance and the amount of luminescence is 0.99, and it can be seen that according to the method of the present invention, even a low complement sample can be measured with a small amount of sample and high sensitivity without separation of blood cells.

Example 3 Dinitrophenylated bovine γ-globulin (hereinafter referred to as DN
The measurement was performed using P-GGB) as an antigen.

2.5 μl of a sheep red blood cell suspension (1 × 10 ⁹ cells / ml GVB ⁺⁺ ) in which dinitrophenyl (hereinafter referred to as DNP) groups were retained on the membrane surface with dinitrofluorobenzene, was added with GVB ⁺ of DNP-GGB at various concentrations. 1.25 μl of sample solution dissolved in ⁺ and anti-DN
Rabbit sera 1.25μl added containing P antibody, is 10 minutes competition immunoreaction at 37 ° C., then guinea pig serum was added to complement solution 12.5μl and GVB ⁺⁺ 20 [mu] l diluted 10-fold in GVB ^++, 3
After performing a complement fixation reaction at 7 ° C for 60 minutes, the reaction was stopped by cooling with ice. This reaction solution is diluted 2-fold and 1 mM luminol solution 50
μl was added, the sample cup was placed in a dark box, 50 μl of 5 mM hydrogen peroxide solution was added, and chemiluminescence reaction was caused at 25 ° C. The chemiluminescence measurement was performed by the photon counting method (using the Hamamatsu Photonics Universal Photon Counting System).

FIG. 4 is a plot of the maximum number of photon counts plotted against the amount of DNP-GGB in the sample solution.
It is shown that the greater the amount of B, the less red blood cells participate in the reaction in the competitive immune reaction, and as a result, the maximum count decreases. It is clear that the target antigen can be accurately measured by this.

[Brief description of drawings]

FIG. 1 is a graph showing the results of Example 1. 2 and 3 are graphs showing the results of Example 2. FIG. 4 is a graph showing the results of Example 3.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-60-363 (JP, A) JP-A-60-364 (JP, A) JP-A-60-29665 (JP, A) JP-A-56- 96249 (JP, A) "Chemical Dictionary 4", Chemical Dictionary Editorial Committee, edited by Kyoritsu Publishing, issued on July 31, 1937 P. 862 "Luminol" "Luminol Luminescence Test"

Claims (3)

[Claims] 1. A chemically amplified chemiluminescence immunoassay characterized in that a substance to be measured is subjected to a complement hemolysis reaction with erythrocytes, and then chemiluminescence is caused by a luminol reaction by hemoglobin released from erythrocytes, and the amount of luminescence is measured. Law. 2. The assay method according to claim 1, wherein the red blood cells have an antigen, an antibody or an antigen-antibody complex on the surface. 3. The measuring method according to claim 1, wherein the substance to be measured is an antigen, an antibody, an antigen-antibody complex or a complement.