JPH01321362A - Sample adjustment for implementing laser magnetic immunological measurement - Google Patents

Abstract

PURPOSE:To achieve reduction in non-specific reaction by causing an antigen- antibody reaction after a centrifugal sedimentation of a magnetically labelled antibody and a sample. CONSTITUTION:In a first process, an antigen-antibody reaction is caused between a magnetically labelled antibody wherein a magnetic fine particle is added to a specified antigen or antibody and an antigen or antibody as sample. Then, in a second process, a magnetic field is made to act on a solution containing a magnetically labelled sample composite as combination of the magnetically labelled antibody and the sample following the first process. With such an arrangement, the magnetically labelled sample composite is induced and concentrated in a laser light irradiation area. Here, before the antigen-antibody reaction, the magnetically labelled antibody and the sample are settled by a centrifugal operation to concentrate and a processing is performed for a higher probability of reaction. Thus, only a limited amount of the magnetically labelled antibody is enough even for a very small amount of the sample thereby making this method effective for reduction in non-specific reaction based on an unreacted magnetically labelled antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION "Field of Industrial Application" The present invention relates to an immunoassay method using antigen-antibody reactions. More specifically, the present invention relates to a sample preparation method for carrying out a laser magnetic immunoassay method capable of quantitatively detecting a specific antibody or antigen from an extremely small sample.

``Conventional technology'' The development of immunoassay methods that utilize antigen-antibody reactions as an early detection method for new viral diseases such as acquired immunodeficiency syndrome, adult T-cell leukemia, etc., and various cancers is currently facing a hurdle. It is being promoted on a large scale.

As conventionally known microimmunoassay methods, radioimmunoassay (hereinafter referred to as RIA method), enzyme immunoassay (EIA), fluorescence immunoassay (F'lA) method, etc. have already been put into practical use. These methods use an antigen or antibody labeled with an isotope, an enzyme, or a fluorescent substance, respectively, and detect the presence or absence of an antibody or antigen that specifically reacts with the antigen or antibody.

RIA quantifies the amount of specimen contributing to an antigen-antibody reaction by measuring the radiation dose of a labeled isotope, and is currently the only method capable of measuring ultra-trace amounts on the order of picograms. However, since this method uses radioactive substances, it requires special equipment, and the attenuation of the labeling effect due to half-life, etc. must be taken into consideration, so there are significant restrictions on its implementation. Furthermore, considering the current situation where radioactive waste disposal has become a social issue, its implementation is naturally limited.

On the other hand, a method using an enzyme or a fluorophore as a label is a method of detecting the specimen m that has contributed to the antigen-antibody reaction by observing color development or luminescence, and there are no restrictions on implementation as in the RIA method. However, the detection limit is on the order of nanograms.

As mentioned above, in conventional immunoassay methods, the RIA method, which has a high detection sensitivity, has many restrictions due to the use of radioactive substances. ) method, fluorescence immunoassay (F'lA) method, etc. have low sensitivity and have been mainly applied to antibody tests. Since antibody testing is a method of detecting antibodies produced by the human body's immune response, it is theoretically impossible to directly detect the virus in the blood immediately after infection.

Further, as a method for detecting the presence or absence of an antigen-antibody reaction using laser light, for example, there is a method using APP (alpha-fetoprotein), which was developed mainly for the purpose of detecting liver cancer. The fundamental drawback of the conventional laser light scattering method is that only a portion of the solution in which the analyte is dispersed is irradiated with a laser for detection, so it is theoretically undesirable to further increase the detection sensitivity. Furthermore, due to these essential drawbacks, a large amount of specimen was required.

In response, the present inventors conducted research on an immunoassay method that differs in principle from conventional methods, and previously filed a patent application for
No. 4567, Patent Application No. 1982-252427, Patent Application No. 1983
-254164, Japanese Patent Application No. 62-22062, Japanese Patent Application No. 1982-22063, Japanese Patent Application No. 152791-1982, Japanese Patent Application No. 152792-1982, Japanese Patent Application No. 184902-1983
No., Patent Application No. 1982-264319, Patent Application No. 62-267
No. 481, he has filed a patent application for an invention relating to a laser magnetic immunoassay method and a measuring device. These new immunoassay methods are characterized by the use of magnetic particles as labeling materials, and are capable of detecting ultra-trace amounts of picograms without using isotopes. Based on the above-mentioned patent, the present inventors labeled magnetic fine particles with antigens or antibodies and detected viruses for the first time. It is being confirmed that this new laser magnetic immunoassay method has a higher detection sensitivity than the conventional RIA method, which is said to have a high detection sensitivity. For example, as the present inventors announced at the 35th general meeting of the Japanese Society of Virology (November 1988, lecture number 4011r, "Virus detection experiment using a newly developed immunoassay device"),
When a virus detection experiment was conducted using inactivated influenza virus type A%B as a virus model, it was possible to detect even when about one virus was present in 1 mQ. The method of irradiating the a-shrinked specimen with a laser beam and detecting the light emitted from the specimen has extremely high detection sensitivity as described above.

"Problems to be Solved by the Invention" By the way, in the above-mentioned labeling methods such as the RIA method, EIA method, and EIA method, each labeling reagent such as an isotope, an enzyme, or a fluorescent dye is added in excess to a sample, and the labeling reagents are added to the sample in excess. A method of removing unreacted labeling reagents by washing is used after causing an antigen-antibody reaction between the two. As the amount of specimen decreases, the labeling reagent becomes larger in excess of the specimen, so non-specific reactions of the residual labeling reagent that cannot be removed by washing have become an important problem. Such a problem is also a problem in the previously filed laser magnetic immunoassay method.

An object of the present invention is to provide a sample preparation method for reducing non-specific reactions in the laser magnetic immunoassay method previously invented by the present inventors.

"Means for Solving Imposition" A sample preparation method for carrying out the laser magnetic immunoassay method according to the present invention involves centrifuging a sample and a magnetically labeled antibody formed by binding an antibody to magnetic fine particles. After that, an antigen-antibody reaction is performed.

Further, as an embodiment of the present invention, the magnetic fine particles may be used as superparamagnetic ultrafine particles. This makes it possible to quantify the specimen even if unreacted magnetically labeled antibodies are present.

In another embodiment of the present invention, the specimen is captured in non-magnetic microspheres having a sufficiently larger mass or volume than the magnetically labeled antibody before the centrifugation step. This simplifies the centrifugation step and makes it easy to separate unreacted magnetically labeled antibodies from the magnetically labeled sample complexes that have reacted with the sample. As a means of separation, centrifugation or a filter can be used.

"Operation" The sample preparation method for carrying out the laser magnetic immunoassay method according to the present invention is particularly effective in detecting trace amounts of virus at the initial stage of infection. If there are only a few viruses,
In order to label a virus, it is necessary to add a large excess of labeling reagent to increase the chance of encountering the virus. but,
If a large excess of the labeling reagent is added, a large amount of the labeling reagent that does not bind to the virus will remain during measurement, resulting in an increase in background due to non-specific reactions. The present invention is characterized by performing a process that increases the probability of reaction by concentrating the magnetically labeled antibody and the specimen by precipitating them by centrifugation. Therefore, even if the amount of the sample is minute, only a small amount of magnetically labeled antibody is required, which is effective in reducing non-specific reactions caused by the unreacted magnetically labeled antibody. In this way, it has become possible to significantly reduce the amount of labeling reagent that was conventionally added in large excess.

After specimen preparation according to the present invention, the precipitate is dispersed in a buffer solution, and the magnetically labeled antibody that has reacted with the specimen can be collected into a test container. The magnetically labeled antibodies recovered by such sample preparation methods include some that have not reacted with the sample. The so-called I3/
When F separation is performed, separation can be easily performed by centrifugation by introducing a process in which the specimen is captured with microbeads, which the present inventors have applied for a patent.

That is, after capturing the specimen on the surface of non-magnetic particles having a mass sufficiently larger than that of the magnetically labeled antibody obtained by binding the antibody to the magnetic fine particles, the magnetically labeled antibody and the specimen are combined with the antigen. Make antibody reaction.

The non-magnetic particles for capturing the analyte must not be magnetic. This is because there is no point in labeling the specimen with a magnetically labeled antibody after capturing it. The non-magnetic fine particles preferably have an average particle diameter of about 0.1 to 10 μm. The non-magnetic particles are, for example, plastic microspheres made of acrylic polymer resin or polystyrene resin,
Alternatively, inorganic colloid particles such as phosphoric acid and alumina are preferable.

One method for capturing a specimen on the surface of a non-magnetic particle is to activate the surface of the non-magnetic particle to non-specifically adsorb the specimen. This method is effective for screening tests and for detecting influenza virus from patient's gargle fluid. There are only a few hundred viruses at most in gargle fluid,
Furthermore, since there are multiple mutant strains such as type A and type B, it is suitable for the purpose of reliably capturing the virus on the non-magnetic particles without first identifying the virus. Another method is to immobilize a known antibody or antigen on the surface of non-magnetic particles in advance and specifically bind the sample through an antigen-antibody reaction. This method is suitable for detailed inspections that eliminate non-specific reactions as much as possible and reliably detect only specific viruses.

The specimen collected by the specimen preparation method of the present invention is preferably subjected to B/F separation by centrifugation, but it is also possible to separate it by using a filter. As an example of centrifugation, for example, when 1 μm acrylic polymer resin is used as the non-magnetic particles, the antigen-antibody complex captured by the non-magnetic particles is precipitated by low-speed centrifugation at +50 Orpm for 5 minutes.

On the other hand, unreacted magnetically labeled antibodies do not precipitate and remain as a supernatant. The precipitate is collected and the antigen-antibody complexes are dispersed in a buffer such as, for example, HEPES.

In addition to the method of performing B/F separation described above, if a method using superparamagnetic ultrafine particles, which the present inventors previously invented and applied for a patent, is applied, the unreacted Since it is possible to quantify the sample even when magnetically labeled antibodies are present, it is possible to directly quantify the sample prepared using the laser magnetic immunoassay method of the present invention using the laser magnetic immunoassay device. I can do it.

The specimen of the present invention is carried out as described above.

After applying the sample preparation method of the present invention, a minute amount of antigen or antibody can be detected by the method disclosed in the above-mentioned laser magnetic immunoassay method and measuring device. This new laser magnetic immunoassay method is sensitive enough to detect the virus even if it is on one side.

"Example" and Yuko Otori This example was carried out for the purpose of investigating the detection limit of the sample preparation method of the present invention using an inactivated influenza virus that is highly safe for experiments.

'=V Hyperimmune serum against influenza virus obtained by immunizing a rabbit is in-phase with the antibody on the surface of non-magnetic particles made of acrylic polymer having an average particle diameter of 1 μm. The non-magnetic particles stored by freeze-drying are placed in a reaction container and dispersed in a PBS buffer. Influenza virus (A/Ishikawa 83 N 2 ) at a known concentration as a model specimen was added to the non-magnetic particle dispersion obtained in the above step, and the influenza virus was captured on the surface of the non-magnetic particles. In this example, in order to investigate the detection limit of viruses, the concentration was 10 to 10 million cells/
Virus solutions of various concentrations were used, which were serially diluted lθ times over a range of mQ.

The magnetically labeled antibody is coated with dextran and has an average particle size of 40
r+I! IgG antibodies isolated from antiserum obtained by immunizing ferrets are covalently bonded to magnetic fine particles made of positive magnetite.

The specimen captured by the non-magnetic particles and the magnetically labeled antibody IXlO-''mg were placed in a centrifuge tube, and the mixture was heated at 3000 r.
After centrifugation at pm for 15 minutes, incubation was performed at 35° C. for 2.5 hours. Through this operation, the specimen was magnetically labeled.

Next, in order to separate the antigen-antibody complex obtained in the above step from the unreacted magnetically labeled antibody, the precipitate is dispersed in a buffer solution and centrifuged at 1500 rpm for 5 minutes to separate the antigen-antibody complex. was precipitated, and unreacted magnetically labeled antibody was obtained as a supernatant. The precipitate obtained in this step was collected and added to 1 mf2 of HE P E S buffer solution, and the interferometry method (Japanese Unexamined Patent Application Publication No. 62-118) invented by the present inventors was applied.
84902) for virus detection. As a result, it was possible to detect the degree of virus 1 side.

In the case of the present invention, on the surface of nonmagnetic particles with a large surface area, 3
Since the specimen is captured dimensionally and the antigen-antibody reaction is performed with the magnetically labeled antibody, the reaction time can be shorter than the conventional two-dimensional reaction method. This feature also makes it advantageous for capturing extremely small amounts of analytes. If a stirring process is used in combination with the capture/reaction process, the time required for the capture/reaction process can be further shortened.

When the example of the present invention was applied, the amount of magnetically labeled antibody was able to be reduced by more than one order of magnitude compared to the case where it was not applied.

Further, if B/F separation is performed by centrifugal force as in this embodiment, separation is more reliable than washing.

Optimal centrifugation conditions are determined depending on the difference in specific gravity between the nonmagnetic particles and the magnetically labeled antibody used.

At this time, by performing density gradient centrifugation with albumin and Percoll added, non-magnetic particles and magnetically labeled antibodies can be separated more reliably.

Example 2 This example is an experimental example of detecting influenza virus from a person's gargling fluid. 30m12 of patient's gargling fluid
First, as a pretreatment, 3000 rpm, I
Foreign matter was precipitated and removed by centrifugation for 0 minutes, and this supernatant was used as a sample.

The magnetically labeled antibody is a magnetite superparamagnetic ultrafine particle with an average particle size of 5 nm, the surface of which is coated with dextran, and the IgG antibody isolated from the antiserum obtained by immunizing ferrets is covalently coated with the dextran. combined.

The sample and the magnetically labeled antibody made of the superparamagnetic ultrafine particles were placed in a centrifuge tube, and the mixture was heated at 35° C. and 20,000 rpm.
Both were precipitated by ultracentrifugation for 60 minutes, and an antigen-antibody reaction was caused. The precipitate In( thus obtained) was collected and
Measurements were made using the laser magnetic immunoassay device described below.

In the measurement step of this example, the present inventors used superparamagnetic ultrafine particles described in the currently pending laser magnetic immunoassay method as a label. In this method, the test container is inserted into a gradient magnetic field generator consisting of an electromagnet and a magnetic pole piece placed opposite the electromagnet, and the sample concentrated on the water surface directly below the magnetic pole piece of the test container is collected. This method detects emitted light such as scattered light, and does not require so-called 13/F separation. In other words, the volume of the magnetically labeled antibody that has reacted with the antigen-antibody with the sample increases more than that of the unreacted magnetically labeled antibody, and the Brownian motion becomes inactive. This is a method that takes advantage of the fact that it is easier to concentrate. For example, it is possible to distinguish between reacted and unreacted light from the temporal change pattern of scattered light.

When the sample preparation method described above was applied to a model experiment assuming the detection of influenza virus in gargling fluid, it was found that the time required from sample preparation to measurement was 3
It was possible to detect it in about an hour. The conventional method of culturing the influenza virus in gargling fluid in chicken eggs and observing the hemagglutination reaction with the naked eye took several weeks.

"Effects of the Invention" As detailed above, the method for preparing a sample for carrying out the laser magnetic immunoassay method according to the present invention makes it possible to reduce the amount of magnetically labeled antibodies used. It is extremely effective in reducing non-specific reactions caused by body-labeled antibodies. As a result, the S/N ratio of measurement is improved, which is extremely effective in improving detection sensitivity. Moreover, since expensive monoclonal antibodies can be used effectively, economical efficiency can be achieved. Furthermore, since the specimen can be reacted continuously, it is advantageous for detecting viruses that are only present in a small amount in a large amount of specimen such as gargling fluid.

In this way, since it has a detection sensitivity higher than that of the RIA method, it is possible to conduct an antigen test immediately after infection, which was previously impossible. Furthermore, non-magnetic particles and magnetic ultrafine particles used as labels do not pose any problems in terms of radiation or toxicity, and are stable against specimens that can be easily prepared manually.

The present invention is not limited to the detection of viruses as mentioned in the examples, but also the early diagnosis of cancer, allergy, bacterial testing, various hormones such as peptide hormones, various enzymes, vitamins, It can also be applied to the measurement of drugs, etc. Therefore, it becomes possible to widely carry out precise measurements in a general environment, which could previously only be carried out in limited facilities using the RIA method. If accurate measurements such as screening tests for various viruses and cancers can be carried out in general situations such as mass medical examinations, it will be possible to make early diagnosis of cancer or viral diseases, and provide effective early treatment. It becomes possible to implement it accurately. As described above, the effects of the present invention in the medical and medical fields are immeasurable.

Applicant: Nippon Telegraph and Telephone Corporation

Claims (3)

[Claims] (1) A first step of causing an antigen-antibody reaction between a predetermined antigen or antibody labeled with magnetic fine particles as a label and an antibody or antigen serving as a specimen, and a step of causing an antigen-antibody reaction between the magnetically labeled material and the specimen after the first step. a second step of inducing and concentrating the magnetically labeled analyte complex in a laser beam irradiation area by applying a magnetic field to a solution containing a magnetically labeled analyte complex that is a complex with a laser-magnetic immunoassay. A method for preparing a specimen for carrying out the method, the laser magnetic immunoassay comprising: centrifugally precipitating a specimen with a magnetically labeled antibody formed by binding an antibody to the magnetic fine particles, and then causing an antigen-antibody reaction. Sample preparation method for carrying out the method. (2) A specimen preparation method for carrying out the laser magnetic immunoassay method according to claim 1, wherein the magnetic fine particles are superparamagnetic ultrafine particles. (3) The laser magnetic immunotherapy according to claim 1 or 2, wherein the specimen is captured in non-magnetic microspheres having a sufficiently larger mass or volume than the magnetically labeled antibody before the centrifugation step. Sample preparation method for carrying out the measurement method.