JPS63201568A - Method of immune analysis - Google Patents

Abstract

PURPOSE:To make easy and exact analysis in an immune analysis utilizing the agglutination reaction of microcapsules by discriminating the agglutinated microcapsules and non-agglutinated microcapsules by a filter. CONSTITUTION:The microcapsules which are conjugated with an antibody and contain a determinable marker therein or in the constituting film and the serum to be examined are first brought into reaction for 5-10min; thereafter, the liquid is filtered by using, for example, 0.45mumHa filter to capture the agglutinated microcapsules. The antigen, etc., in the serum to be examined are measured by directly measuring the fluorescent intensity, absorbancy, etc., of the agglutinated microcapsules or adding a hypotonic liquid as well as a surfactant and org. solvent to dissolve the same, then measuring the marker material in the supernatant liquid by, for example, fluorescent intensity, absorbancy, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention relates to an immunoassay method using microcapsule agglutination reaction.

(B) Conventional techniques Conventional immunoassay methods include, for example, radioimmunoassay (RIA) and enzyme immunoassay (EIA).
There is a law). Although the RIA method is quantitative, these measurement methods use radioactive isotopes, which limits the places where they can be used and requires large-scale equipment.

The EIA method is complicated to operate, and all measurement methods require a long time for analysis, ranging from several hours to several tens of hours. There is also an agglutination method using microcapsules (mainly red blood cells), but it is not a common method because it relies on visual judgment or uses radioactive substances.

(c) Problems to be Solved by the Invention The purpose of the present invention is to solve problems that can be solved by simple operation in immunoassays.
The object of the present invention is to provide a novel analysis method that can be processed in a short time of ~10 minutes, and can quantify amounts with high sensitivity and accuracy.

(d) Means and action for solving the problems The present inventors have conducted various studies in view of the above-mentioned circumstances, and have found that a marker substance that binds antibodies or antigens and can be quantified inside or in the constituent membranes has been developed. It has been found that the above object can be achieved by using microcapsules and combining them with a filter.

The microcapsules used in the present invention include red blood cells,
Various types including liposomes can be used, but spherical and uniform shapes are preferable. The appropriate pore size of the filter used in combination with this is from the same diameter to about 5 times the diameter of the microcapsules used. From the viewpoint of processing a large amount of specimen at once, it is preferable to use, for example, a Millipore millititer plate, which has a filter attached to the bottom of a 966 microplate.

As the quantifiable marker substance contained inside the microcapsule or in the constituent membrane, suitable for the purpose of the present invention are:
There are no particular restrictions as long as it has such functionality.
In the case of red blood cells, it is hemoglobin, and in the case of liposomes and chemically synthesized microcapsules by interfacial polymerization, various substances can be used, but fluorescent substances are preferable from the viewpoint of high intensity.

The antibody or antigen to be bound to the microcapsules is appropriately selected depending on the purpose of the test. For example, antibodies corresponding to the antigens listed below are exemplified.

The antibody is produced by immunization of animals, conventional techniques for producing monoclonal antibodies, and the like.

Binding of antibodies to microcapsules is performed by known methods. When the target is red blood cells, antibody binding is performed by the chromium chloride method. The binding of antibodies to microcapsules, such as liposomes, can also be accomplished by the method of Leserman et al. (Nature, 288.604-606°19).
80), the method of Martin et al. (Biochemis
try, 2o.

4229-4238.1981).

The analysis method of the present invention is carried out as follows (see FIG. 1). First, antibody-bound microcapsules and test serum (for example, human serum) are reacted for 5 to 10 minutes, and then the reaction solution is filtered using a filter to capture aggregated microcapsules. Next, the fluorescence intensity or absorbance of the aggregated microcapsules is directly measured, or a hypotonic solution, surfactant, or organic solvent is added to dissolve them. After dissolution, hemoglobin or marker substances in the supernatant are measured, for example, by fluorescence intensity or absorbance, and antigens and the like in the test serum are measured. In other words, if there is no antigen to be measured in the test serum, the microcapsules will not aggregate and will not be captured by the filter (and no fluorescence will be detected, resulting in a negative result. If there is an antigen present, the microcapsules aggregate with the presence of the antigen, and the aggregated microcapsules are captured by a filter.Then, the fluorescence intensity, etc., can be measured either directly or by dissolving the captured microcapsules. Quantification is performed by

In the final method, if the degree of aggregation of microcapsules is low even though a large amount of the target antigen is present in the test serum, if the antibody used is a monoclonal antibody, another type of monoclonal antibody with a different recognition epitope may be used. It is possible to increase the degree of aggregation and sharpen the sensitivity by reacting microcapsules with an antigen and then reacting with a secondary antibody.

If so-called mediastinal agglutination occurs, in which microcapsules aggregate even in the absence of antigen, a buffer containing an antibody or serum of the same type as the antibody bound to the microcapsules (but does not react with the antigen) and the test sample are used. The extent of this can be suppressed by mixing it with serum.

Targets of analysis include various cancer antigens such as AFP, CEA, β2-microglobulin, and CA19-9, and HBsAg.
, HBcAg, Ant 1HBs, HTLV-I, HT
Preferred examples include virus-related antigens and antibodies such as LV-III, as well as various hormones in the blood and plasma proteins such as IgG.

(E) Effects of the Invention According to the immunoassay method of the present invention, processing can be performed in a short time with simple operations, and the amount can be quantified accurately over a high range.

Antibody-sensitized microcapsules are agglomerated by the target antigen, and can be processed in a short time because they are captured by a filter without forming a sedimentation image over a long period of time.Also, as a marker contained in the microcapsules, Since non-radioactive materials, such as fluorescent materials, are used instead of radioactive materials, measurement equipment has become cheaper and can be placed anywhere.

(f) Examples Examples will be given to explain the present invention in more detail.
The present invention is not equally limited by these.

Example 1 An attempt was made to detect the gamma marker AFP.

As microcapsules, liposomes (reverse phase evaporative vesicles) containing two types of monoclonal antibodies with different recognition epitopes and containing a fluorescent substance (carboxyfluorescein) inside are used.
It was used after being passed through a filter with a pore size of 0.2 μm.

A 96-hole millititer plate manufactured by Millipore (equipped with a 0.45 μm HA filter) was used as a reaction vessel.

50 μl of normal human serum containing various concentrations of AFP
50 μl of the above liposome suspension was added to the solution and allowed to react at room temperature for 10 minutes. Next, the reaction solution was removed by suction, and free liposomes were removed by washing three times with Hepes buffer. Next, 200 .mu.i of water was added to each well to dissolve the liposomes captured on the filter, and the fluorescence intensity was measured. The results are shown in FIG. 2, and human AFP can be quantified based on the linear relationship between human AFP concentration and fluorescence intensity.

Example 2 An attempt was made to detect the gamma marker CEA.

A liposome (conjugated with one type of anti-CEA monoclonal antibody and containing rhodamine B as a marker) prepared in the same manner as in Example 1 was used.

Millibore 966mm titer play as a reaction vessel!
- (equipped with a 0.45 μm HA filter) was used.

50 μl of normal human serum containing various concentrations of human CEA
25μ2 of the above liposome suspension was added and reacted at 37°C for 5 minutes, and further anti-CEA rabbit polyclonal antibody was added and reacted at 37°C for 5 minutes.

Next, the reaction solution was removed by suction, and free liposomes were removed by washing three times with Hepes buffer. Then, the fluorescence intensity was measured as it was. The results are shown in FIG. 3, and h) human CEA can be quantified based on the linear relationship between CEA concentration and fluorescence intensity.

[Brief explanation of the drawing]

FIG. 1 is a conceptual explanatory diagram of the immunoassay method of the present invention. FIG. 2 is an explanatory diagram of the relationship between the concentration of human AFP and fluorescence intensity in the immunoassay method of the present invention. Figure 3: Concentration of CEA in the immunoassay method of the present invention (Idiot 3)
Figure 1

Claims (1)

[Claims] In an immunoassay method that uses microcapsules that bind an antigen or antibody and contain a quantifiable marker substance inside or in a constituent membrane, the microcapsules are aggregated by an immune reaction, and the corresponding antigen or antibody is quantified. , an immunoassay method characterized in that quantitative determination is performed by separating aggregated microcapsules and non-aggregated microcapsules.