JP2683934B2 - Immunoassay method - Google Patents

Description

TECHNICAL FIELD The present invention relates to an immunoassay method in a clinical test, and more particularly to an immunoassay method by an antigen-antibody reaction using a solid phase and fluorescent particles.

(Prior art) As one of the immunoassay methods, the latex immunoassay method catches the aggregation caused by the antigen-antibody reaction as a change in turbidity in addition to the so-called slide glass method, which is a qualitative immunoassay method. Quantitative immunoassays that measure light optically (for example, LPIA (Latex Photometric Immu
noassay) "Inspection and Technology", Vol. 12, No. 7, p. 581 (198
4), JP-A-53-24015, JP-A-59-187264, etc.),
Furthermore, focusing on two or more aggregates of latex particles involved in antigen-antibody, a method of numerically measuring the degree of latex aggregation through an electrical resistance type or optical type particle counter (eg PACIA (Particle Coun
Ting Immunoassay), J. of Immuno., Meth., Vol. 18, p. 33 (1977), FCM (flow cytometry) JP-A-62-8156
No. 7) etc. In order to perform these quantitative measurements, various devices such as a special nephelometer, electric resistance type or optical type particle counter are used.

(Problems to be Solved by the Present Invention) The measurement method using the turbidimetric method requires a longer reaction time than enzyme immunoassay (EIA), which is one of the immunoassay methods currently widely used. Although it has features such as a short and highly automated device, it has a radioimmunoassay (RI
Compared to A) and EIA, it has lower sensitivity for immune reaction and is not suitable for detection of trace components that require high sensitivity. In addition, this measuring method has a drawback that coexisting substances in the sample that affect light scattering, such as milky serum and autoimmune complex, have a great influence on the measurement result because the turbidity is optically measured. . In order to avoid these effects, measures such as the method of measuring using near infrared rays and the so-called rate method of measuring the rate of change of turbidity with time have been adopted,
It cannot be said that sufficient sensitivity in the immune reaction has been obtained yet.

On the other hand, the measurement method using a particle counter is to measure the agglomerates of latex particles, and it is usually 1 μm due to the limitation of the performance of the particle counter.
Since latex particles of a certain degree are used, in principle, extremely high sensitivity can be expected. The smaller the latex particles, the faster the agglutination reaction proceeds, but the signal derived from the agglomerated particles is small, and in this case also coexisting substances other than latex, which are similar to the nephelometry, cause noise. Further, generally, in the apparatus for carrying out this method, it is necessary to flow the sample liquid through a nozzle having an extremely thin structure, so that the nozzle is apt to be clogged with dust or the like. Further, since the thin nozzle measures aggregation in a fluidized state, it has a drawback that the aggregate causes decomposition when passing through the nozzle.

In addition, the method of quantifying the agglutination image generated in a solvent by using fluorescent particles as particles by an image processing method through a fluorescence microscope / TV camera is extremely sensitive in principle, and the nozzle level is Although it can be made sufficiently low without being affected by fluctuations, etc., the particle counting speed is slow, so the number of particles involved in particle size distribution analysis is extremely small relative to the whole, and it is diluted so that only a small amount of aggregation occurs. There was a low probability that aggregation was detected in various solution states, and there was a practical limit to improvement in sensitivity.

The present invention provides a novel high sensitivity achieved by observing the binding of fluorescent particles, such as fluorescent latex, to a solid phase that completely avoids all of these conventional drawbacks. Furthermore, it is to provide a rapid immunoassay method.

Fluorescent particles sensitized with an antigen or antibody used in the present invention are disclosed in JP-A-62-81566, JP-A-62-81567 and JP-A-62-81567.
116264, etc., may be produced by the method disclosed in, for example, as the fluorescent particles such as fluorescent latex, in addition to polystyrene disclosed above, styrene-butadiene copolymer, styrene-acrylic acid copolymer. As the fluorescent substance such as coalesce, an organic fluorescent substance such as fluorescein or rhodamine, and an inorganic fluorescent substance such as platinum cyanide or a sulfide of an alkaline earth metal can be used alone or in combination.

Further, as the fluorescent particles, fine crystals of a naphthalimide-type or benzotriazole-type fluorescent dye can be used as they are as a carrier for sensitizing an antigen-antibody.

Further, as the solid phase in the present invention, a polystyrene-based flat-bottom microplate generally used for EIA ELISA, a flat-bottom microplate used for HLA typing, and the like are preferable. The flat bottom of these plates is convenient for observing the fluorescent particles bound to the plate surface by using an inverted microscope.

The antigen or antibody used for sensitization is not limited, and any antigen or antibody can be used. For example, those may be various tumor markers, antigens such as HBs, antibodies such as IgG and IgM, or degradation products thereof. As the sensitization method, physical adsorption or chemical reaction utilizing functional groups on the particles can be used.

The size of the fluorescent particles used is almost unlimited in the direction of the particle size normally used for latex agglutination reaction, for example, smaller than 1 μm. It is sufficient if the amount of fluorescence emitted by each particle can be measured, that is, irradiation with sufficiently strong excitation light, and further the particles emit sufficiently strong fluorescence,
Any light source that can be observed as a point light source regardless of its particle size may be used.

A simple measuring device for carrying out the present invention comprises a fluorescence microscope and a camera. In the measurement, the fluorescent particles bound to the solid phase by the antigen-antibody reaction are photographed with a camera and the point light sources on the photograph are counted. However, the preferred system consists of a fluorescence microscope, an additional TV camera and an image processing system combined therewith. FIG. 1 is a schematic diagram of an apparatus for carrying out the present invention.

An apparatus for carrying out the present invention comprises a fluorescence microscope 2 and an image processing apparatus 4 for processing the image data by taking an image with a TV camera 3 attached thereto, and the cuvette 1 for further reaction. , A microcomputer 5 for digitizing the measurement results, an ultraviolet light source for giving excitation light, for example, a high-pressure mercury lamp 6, a television for monitoring the image and displaying the calculation result, a printer for recording the calculation result, etc. are added. It is desirable to do.

To describe the apparatus for carrying out the present invention in more detail, the reaction cuvette used here is a flat-bottom ELISA microplate or HLA typing microplate, which is made of transparent plastic. In addition, it is convenient to use an instrument with an autofocus and an autostage for normal automatic measurement using a fluorescence microscope.

The image obtained in the state of using the reaction cuvette and the fluorescence microscope is the fluorescent particles bound to the bottom surface of the microplate and the fluorescent particles floating in the solution, but after the reaction,
By adding a black dye to the reaction solution, an image of the fluorescent particles bound to the microplate can be efficiently obtained.

The reaction solution in the cuvette containing the sensitized antigen and antibody is preferably stirred, so that the result can be obtained quickly and efficiently.

A high-pressure mercury lamp or the like is used as a device that gives excitation light. The selection of excitation wavelength and intensity can be changed depending on the fluorescent substance contained in the fluorescent particles. Further, the obtained image is sent to the image processing device via the TV camera. The image processing device is a video input unit that receives the output from the TV camera,
A digital image memory section for recording data of each screen and a data output section for sending the data to a computer as needed, and a microcomputer for processing the output is added.

Next, an example of the image processing method will be described. The images obtained are both an image of the fluorescent particles bound to the bottom of the plate and an image of the fluorescent particles not bound to the plate but accidentally located on the solid surface during image capture.

Therefore, at a certain position, a plurality of images are captured at different times. The image of the fluorescent particles bound to the plate is present in all the images captured in the meantime, and the fluorescent particle images that are accidentally located on the solid surface are captured differently from screen to screen. Therefore, when the average value of the brightness of each image of the plurality of pieces of image data is obtained, it is possible to virtually select only the fluorescent particle image bonded to the plate. Then, a calibration curve can be drawn by plotting the relationship between the titer of the immune component and the number of binding fluorescent particles.

(Operation) In the present invention, by using fluorescent particles, it is possible to avoid the influence of coexisting substances, which poses a problem in ordinary immunoassays, particularly latex agglutination.

These coexisting substances do not emit fluorescence having an intensity comparable to that of fluorescent particles upon irradiation with excitation light in this measuring method. In addition, the secondary effects that these coexisting substances scatter the fluorescence emitted by the fluorescent particles are theoretically considered, but these effects are measured in a microscope, and the portion near the solid surface is measured in layers. So it doesn't really matter at all. Therefore, information on the binding between the fluorescent particles and the solid surface of the plate can be selectively obtained by the image processing method.

Unlike the solid-phase method of RIA and EIA, the ordinary latex agglutination method cannot perform B / F separation, so it has the advantage of being easy to operate, but in the reaction with serum, for example, it is extremely difficult. The co-occurrence of aggregation due to non-specific reaction of complex components is unavoidable to some extent. For this reason, there is no choice but to devise such as diluting a serum sample to a large extent and using it to sacrifice the sensitivity to some extent to increase the stability. In contrast, in the present invention, after reacting the antigen or antibody sensitized on the plate surface with the corresponding antibody or antigen,
Since it is a method of binding the sensitized fluorescent particles in a sandwich type, it is possible to add a B / F separation (washing) operation if necessary. That is, for measurement items that have a high concentration in the sample and a sufficient margin with the measurement limit, it is possible to omit the washing operation and focus on the simplicity of the operation. A cleaning operation can be incorporated if low and sensitive measurements are required.

The reaction between the antigen and the antibody can be rapidly performed in a short time by stirring the reaction solution.

(Examples) The present invention will be described in more detail with reference to Examples below.

Example 1 Preparation of anti-human α-fetoprotein (AFP) monoclonal antibody-sensitized latex reagent (sensitized latex) Anti-human α-fetoprotein (AFP) monoclonal antibody tris (hydroxymethyl) aminomethane buffer (Tris buffer) ( Antibody concentration: 100 μg / ml) To 1 ml, 100 μ of fluorescent polystyrene latex with an average particle size of 0.3 μm (manufactured by Polyscience, solid content concentration: 2.5% by weight) was added, and the mixture was stirred at 37 ° C. for 1 hour and then centrifuged ( 12,000 rpm,
30 minutes).

Separate the anti-AFP monoclonal antibody-sensitized latex with Tris buffer containing 1.0% by weight of bovine serum albumin.
After washing twice, the sensitized latex was prepared by suspending the suspension so that the concentration was 0.002% by weight.

Example 2 Preparation of Anti-Human AFP Monoclonal Antibody Sensitization Plate Reagent (Sensitization Plate) The anti-human AFP monoclonal antibody used in Example 1 was recognized as an antigen on the flat-bottom plate (Nunc) used in the enzyme immunoassay. Phosphate buffer solution of anti-human AFP monoclonal antibody (antibody concentration: 10 μg / ml) 50 μ / we
ll was added and left at room temperature for 4 hours, then 0.01 wt% Tw
It was washed three times with phosphate buffered saline containing een20 (Tw-PBS).

After that, add 100 μ / well of phosphate buffer containing 1.0% by weight of bovine serum albumin, leave it at 4 ° C. overnight, and then add Tw.
-Washed 3 times with PBS to prepare a sensitized plate.

Example 3 Preparation of standard curve (1) To the sensitized plate prepared in Example 2, 40 μ / well of Tris buffer containing 1.0% by weight of bovine serum albumin was added,
Further, 10 μ / well of a standard AFP solution containing different concentrations of AFP was added, and an antigen-antibody reaction was performed at room temperature for 1 hour, and then 50 μ / wel of the sensitized latex prepared in Example 1 was added.
After the addition, l-antigen-antibody reaction was performed for 30 to 180 minutes at room temperature with stirring.

Then, add 200μ / well of black ink, set it on an inverted fluorescence microscope, shoot using an ISO 3200 ultra-sensitive film, and then measure the number of sensitized latex adsorbed on the sensitized plate. , The standard curve shown in FIG. 2 was obtained.

Example 4 Preparation of standard curve (2) To the sensitized plate prepared in Example 2, 50 μ / well of Tris buffer containing 1.0% by weight of BSA was added, and standard AFP solution containing human AFP of different concentration 5 μ / After adding well,
The antigen-antibody reaction was carried out at room temperature for 1 hour, then 50 μ / well of the sensitized latex prepared in Example 1 was added, and the antigen-antibody reaction was carried out at room temperature for 3 hours with stirring.

After that, 200 μ / well of black ink was added and set on a fluorescence microscope, and the number of sensitized latex fixed on the sensitized plate was measured by an image processing method using a computer, and the results shown in Table 1 were obtained. .

Example 5 Preparation of standard curve (3) To the sensitized plate prepared in Example 2, 50 μ / well of Tris buffer containing 1.0% by weight of BAS was added, and standard AFP solution containing human AFP of different concentration 5 μ / well and the sensitized latex prepared in Example 1 were added at 50 μ / well,
The antigen-antibody reaction was performed for 10 minutes at room temperature with stirring.

Then, black ink was added at 200 μ / well, set in a fluorescence microscope, and the number of sensitized latex fixed on the sensitized plate was measured by an image processing method using a computer, and the results shown in Table 2 were obtained.

Example 6 Measurement of AFP in whole blood and plasma To the sensitized plate prepared in Example 2, 50 μ / well of Tris buffer containing 1.0 wt% of BAS was added, and whole blood and plasma containing human AFP having different concentrations were added. 5 μ / well (whole blood and plasma to which purified AFP was added) and 50 μ / well of the sensitized latex prepared in Example 1 were added, and an antigen-antibody reaction was carried out for 10 minutes at room temperature with stirring.

Then, black ink was added at 200 μ / well, set in a fluorescence microscope, and the number of sensitized latex fixed on the sensitized plate was measured by an image processing method using a computer, and the results shown in Table 3 were obtained.

Example 7 Preparation of HBs Monoclonal Antibody Sensitized Latex (Sensitized Latex) Reagent Anti-HBs monoclonal antibody Tris buffer (antibody concentration:
200 μg / ml) 1 ml of fluorescent polystyrene latex with an average particle size of 0.3 μm (Polyscience, solid concentration: 2.5
100% by weight) was added, and the mixture was stirred at 37 ° C for 1 hour and then centrifuged (12,000 rpm, 30 minutes).

Separated anti-HBs monoclonal antibody-sensitized latex B
A sensitized latex was prepared by washing three times with a Tris buffer containing 1% by weight of SA, and then suspending the suspension to 0.002% by weight.

Example 8 Preparation of Anti-HBs Monoclonal Antibody Sensitization Plate (Sensitization Plate) Reagent A flat-bottom plate (Nunc) used in the enzyme immunoassay is defined as an antigen recognition site for the anti-HBs monoclonal antibody used in Example 7. Phosphate buffer solution (antibody concentration: 10 μg / ml) 50 μ / well of different anti-HBs monoclonal antibodies was added, and after standing at room temperature for 4 hours, 0.01 wt% Tw-PBS
It was washed 3 times.

After that, add 100 μ / well of phosphate buffer containing 1.0% by weight of bovine serum albumin, leave it at 4 ° C. overnight, and then add Tw.
-Washed 3 times with PBS to prepare a sensitized plate.

Example 9 Preparation of standard curve (4) To the sensitized plate prepared in Example 8, 50 μ / well of Tris buffer containing 1.0% by weight of BAS was added, and 5 μ / well of standard HBs solution containing HBs of different concentrations was added. After the addition, the antigen-antibody reaction was carried out at room temperature for 1 hour, 50 μl / well of the sensitized latex prepared in Example 7 was added, and the antigen-antibody reaction was carried out at room temperature for 3 hours with stirring.

After that, 200 μ / well of black ink was added, the mixture was set on a fluorescence microscope, and the number of sensitized latex fixed on the sensitized plate was measured by an image processing method using a computer, and the results shown in Table 4 were obtained. .

(Effect of the invention) In the present invention, in the antigen-antibody reaction, reaction images at different time points are photographed by a camera through a fluorescence microscope, both reaction images are compared, and fluorescent particles at different positions are imaged as solid phase unbound particles. It is an immunoassay method in which the number of solid-phase bound fluorescent particles is related to the amount of antigen or antibody by a treatment method. The present invention is a rapid and highly sensitive measurement method that eliminates the influence of coexisting substances, which has been a problem in the past, and greatly contributes to the field of immunoassay. In particular, it can sufficiently support the measurement of items that require high sensitivity, such as the measurement of HBs.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of an apparatus for carrying out the method of the present invention,
FIG. 2 is a graph showing an example of a standard curve for AFP measurement. 1: cuvette, 2: fluorescence microscope, 3: TV camera, 4: image processing device, 5: microcomputer, 6: UV source

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Takashi Sone 4-6-7 Shimochiai, Shinjuku-ku, Tokyo Fuji Rebio Co., Ltd. (72) Inventor Yosuke Kikuchi 4-6-7 Shimochiai, Shinjuku-ku, Tokyo Wealth Shirebio Co., Ltd. (72) Inventor Akiko Takayama 4-6-7 Shimochiai, Shinjuku-ku, Tokyo Fujishi Lebio Co., Ltd. Examiner Shoko Yamamura (56) Reference JP 59-160743 (JP, A) Special Kai 63-58237 (JP, A) JP 58-213253 (JP, A) JP 56-94264 (JP, A)

Claims (3)

(57) [Claims] 1. In an antigen-antibody reaction using a solid phase and fluorescent particles, reaction images at different time points are taken by a camera through a fluorescence microscope, both reaction images are compared, and fluorescent particles at different positions are solid phased. An immunoassay method characterized by eliminating unbound particles by image processing. 2. The method according to claim 1, wherein the fluorescent particles are fluorescent latex. 3. The method according to claim 1, wherein the camera is a TV camera.