JPH06148188A - Immunological reinspecting method - Google Patents

Abstract

PURPOSE:To easily perform the same inspection of plural times or multiple item inspection to the same analite, or the inspection to a plurality of analites in one reaction vessel by use of the same sample. CONSTITUTION:On a solid phase carrier to which a ligand corresponding to a target analite, analite in a sample to be inspected is fixed, and a first marker particle to which the ligand corresponding to the analite is bonded to the analite fixed on the solid phase carrier. A first judgment is conducted on the basis of this result, and the first marker particle is separated and removed from the bonded body. A second marker particle on which the ligand corresponding to the analite is fixed is bonded to the analite fixed on the solid phase carrier, and a second judgment is conducted on the basis of the result.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a test method for immunologically detecting an antibody present in a sample, and more particularly to a test method capable of specifying the type of antibody present in the sample. Pertain.

[0002]

2. Description of the Related Art When a substance that is recognized as a foreign substance by a living body, such as a bacterium or a virus, enters the living body, an antibody is produced in the living body through several reactions. By reversely utilizing this, by determining the presence or absence of this antibody, it is possible to test whether or not a bacterium or virus has entered the living body.

As a method for testing such an antibody, EIA (Enzyme Immuno Assay) using an enzyme-labeled antibody, RIA (Radio Immuno Assay) labeled with a radioisotope,
FIA (Fluorescence Immuno Assay) using a fluorescent substance
) And other agglomeration methods are commonly used, among which E
IA is the most widely used method.

Generally, EIA is performed as follows. First, a carrier (for example, a well or a test tube) on which an antigen that reacts with a target antibody is solid-phased is prepared, and a sample is added to the carrier and reacted. As a result, the target antibody is captured on the carrier surface. Next, the carrier surface is washed to remove unbound mixed components, and then an enzyme-labeled antibody is added to bind to the target antibody captured on the carrier. Further, the carrier surface is washed to remove the unbound enzyme-labeled antibody, and then the enzyme substrate reaction is performed to indirectly determine the presence or absence of the desired antibody.

[0005]

By the way, when a foreign body invades a living body, it is generally said that the IgM antibody appears first and then the IgG antibody appears. Therefore, it is possible to estimate the degree of disease progression by discriminating the type of antibody, which is considered to be clinically significant. However, it is difficult to determine the type of antibody by the conventional antibody testing method.

For example, in EIA, the type of enzyme-labeled antibody must be selected according to the type of antibody to be detected. That is, the antibody to be detected is I
In the case of gG antibody, anti-IgG antibody enzyme-labeled,
In the case of IgM antibody, it is necessary to use enzyme-labeled anti-IgM antibody. However, in many cases, an enzyme-labeled blend of an anti-IgG antibody and an anti-IgM antibody is used because of the necessity of widely detecting the presence of the antibody. Therefore, even if the presence of the antibody is detected, it is impossible to determine whether the anti-IgG antibody or the anti-IgM antibody has reacted, and the type of antibody present in the sample cannot be determined. If not only the presence of an antibody but also the type of the antibody is to be determined, the same test must be repeated using a single enzyme-labeled antibody such as an anti-IgG antibody and an anti-IgM antibody.

As an antibody test against red blood cells, a Coomass test is widely performed in which a specimen is added to a screening blood cell, and after reaction / washing, Coomass serum is added to determine the presence or absence of aggregation. However, also in this method, reaction components such as anti-IgG antibody, anti-IgM antibody, and complement are added to Coomass serum for the purpose of widely detecting antibodies. Therefore, even if aggregation occurs, it is not possible to determine which antibody is present.

The same applies to the agglomeration method. For example, in the ATLA test, a sample is added to gelatin particles to which ATLV antigen is adsorbed, and the determination is made based on the presence or absence of aggregation. Even if the aggregation occurs, it is not possible to determine the type of antibody present in the sample. It is impossible. In addition, in the agglutination method, it is more difficult to determine the type of antibody after causing the agglutination. As described above, in EIA, the type of antibody in a sample can be determined by properly using several types of enzyme-labeled antibodies. However, in the case of the agglutination method, the elimination method such as the reactivity test after the sample absorption operation, the decomposition of IgM by 2-mercaptoethanol (2-ME), etc. I have no choice. Therefore, an object of the present invention is to provide a test method capable of detecting the presence of an antibody in a sample and easily discriminating the type of the antibody.

[0009]

Means and Actions for Solving the Problems
As a result of intensive research in view of the above circumstances, the present invention has been completed. That is, the immunological retest method of the present invention is
A first reaction step of reacting a test sample with a ligand corresponding to the target analyte immobilized on a solid phase carrier to immobilize the analyte in the test sample on the solid phase carrier; First marker with a ligand corresponding to the light fixed
Contacting the particles with an analyte immobilized on a solid support,
A second reaction step of forming a combined body of the solid phase carrier and the first marker particles through the analyte, and a first determination step of making a determination based on the reaction result in the second reaction step,
A step of releasing the first marker particles from the combined body, and the absence of the released first marker particles,
Second marker with immobilized ligand corresponding to analyte
Contacting the particles with an analyte immobilized on a solid support,
It is characterized by comprising a re-reaction step of forming a combined body of the solid phase carrier and the second marker particles through the analyte, and a re-judgment step of making a judgment based on the reaction result in the re-reaction step. And

In the re-inspection method of the present invention, after the re-judgment step, the marker particles used in this re-reaction step are separated from the binder formed in the immediately preceding re-reaction step to leave the reaction system. The removal step of removing, and the marker particles to which the ligand corresponding to the analyte is fixed are brought into contact with the analyte fixed to the solid phase carrier, and the solid phase carrier and the marker particles via the analyte The series of steps including the re-reaction step of re-forming the conjugate of 1. and the re-judgment step of making a judgment based on the reaction result in this re-reaction step may be further performed once or more. Hereinafter, the immunological retesting method of the present invention will be described in detail.

In the present invention, the analyte means a test substance to be analyzed, and specifically, physiological substances such as globulin, hormones and enzymes in a specimen sample composed of body fluid such as blood and urine. , Bacteria, virus, allergic
It refers to various antigens such as immunological foreign substances such as substances, or their antibodies.

[0012] The term "ligand" refers to a substance that exhibits an immunologically specific binding reactivity to the analyte, that is, an antibody or an antigen. In the present invention, the ligand is used in a state of being immobilized on the solid phase carrier and the particle marker. As a result, one or more layers of the ligand coating surface are formed on the surfaces of the solid phase carrier and the particle marker. Therefore, the ligand exhibits a property as a solid phase. The immobilized ligand specifically reacts only with the analyte in the test sample, and plays a role of capturing the analyte on the solid phase carrier and the particle marker. The solid phase carrier and the ligand immobilized on each particle marker used in the present invention may be the same or different.

The solid phase carrier may function as a reaction container itself, or may be put in a reaction container together with a test sample, a reagent and the like. When it functions as a reaction container itself, it is necessary to have a sufficient space to accommodate the required amount of test sample, reagent, etc., and it has a tubular or concave shape. Things are used. Further, when used in a reaction container, it is necessary to have a surface area capable of coming into contact with a sufficient amount of test sample, reagent, etc., and it may be in the form of particles, plates, etc. What is done is used. Which carrier is used is arbitrary, and its shape and size may be appropriately selected according to the measurement principle to be used and the convenience of operation. As a method for immobilizing a ligand on a solid phase carrier, various known methods such as a physical adsorption method, a covalent bond method, and a biotin-avidin method can be used. Further, the use of a chemical or biological adhesive such as polyethyleneimine, a charged dye, or a plant agglutinin is preferable because the immobilization of the ligand becomes easier and the adjustment becomes easier. The density, area, etc. of the solid phase ligand can be appropriately determined according to the reaction conditions with the analyte.

Thus, a predetermined amount of the sample to be tested is supplied to the solid-phase carrier on which the ligand is solid-phased, for example, the surface on which the ligand is solidified on the inner wall of the reaction vessel, using a dispensing nozzle or the like, and under appropriate conditions. For example, the analyte is allowed to react with the ligand by incubating for a certain period of time to immobilize the analyte on the solid phase carrier. At this time, antigens, antibodies, etc. other than the analyte theoretically do not bind to the ligand and are not immobilized on the solid phase carrier. However, in the test sample, substances other than the analyte that have immunogenicity and can bind nonspecifically to the ligand due to the adsorption ability between the proteins are present, and the reactivity of the analyte may decrease. is there. Therefore, it is preferable to remove the substance that non-specifically binds by appropriate washing to prevent the decrease in reactivity. For such washing and removal, purified water containing no non-specifically adsorbed components, buffer solution, physiological saline, etc. is used as a washing solution, the inner wall of the reaction vessel is rinsed and centrifuged, and then the supernatant is removed by suction. A method of performing decanting or decanting is generally used. It is also preferable to perform a blocking treatment in order to prevent undesired non-specific adsorption to a site other than the site where the ligand is immobilized. For the blocking treatment, an immunologically inactive protein such as non-human-derived serum albumin or gelatin may be contained in the test sample or the washing solution in advance.

In the present invention, the separation of the marker particles from the bound substance formed on the solid phase carrier is preferably carried out by a liquid flow generated by washing or a vibration corresponding thereto. For this reason, the marker particles may be of any type as long as they have a resistance surface that receives sufficient resistance to separate from the combined body by these liquid flows, and the material, specific gravity, type, shape and size. Etc. are not limited. Specifically, the particle size is 0.3 to 100 μm, preferably 3
Known immunological carrier particles of -15 μm can be used. In addition, as a detachment method utilizing other than liquid flow or vibration, particularly as a detachment method from the inner wall of the reaction vessel, a method using magnetic substance-containing particles excellent in magnetic response or charged particles having electrophoretic ability can be mentioned. it can. The plurality of marker particles used in the present invention (the first marker particle and the second marker particle, and optionally the plurality of marker particles used thereafter) are respectively The same or different ones can be used.

The measurement principle that can be used in the retesting method of the present invention is not particularly limited as long as it utilizes an immune reaction. For example, enzyme immunoassay (EI)
A), chemiluminescent immunoassay (CLIA), fluorescent immunoassay (FIA), radioimmunoassay (RIA), etc. The principle of a test method using an antigen or antibody labeled with a detectable tracer, and immunology. The agglutination reaction can be mentioned. However, when carrying out an analysis using a marker substance that requires a chemical reaction, it is necessary to add a reaction terminator for a certain period of time for measurement, such as a color development reaction or a luminescence reaction, Many of the reaction terminators used at this time have the effect of inactivating the immunological reaction as well. Therefore, as the measurement principle used in the second reaction step,
The principle of immunological testing with a marker substance that can be detected without going through a chemical reaction is preferred. However, this does not apply when a reagent such as water that does not affect the reactivity immunologically is used as the reaction terminator.

The most preferable measurement principle used in the second reaction step is an immunological agglutination reaction. The carrier particles widely used in the immunological agglutination reaction have specifications such as particle size that meet the above-mentioned release characteristics, and can be used in the second reaction step without any change. On the other hand, E
Unlike IA, CLIA, FIA, and RIA, the marker substance is not fixed to carrier particles, but is used in the reaction in the state of being bound only to a ligand such as an antibody. Since the properties are not satisfied, it cannot be used as it is for the second reaction step. Therefore, when utilizing these measurement principles, carrier particles having the marker substance and ligand used in each measurement principle immobilized should be prepared in advance and used in the form of marker particles. Are required, which is also a feature of the present invention. In general, individual measurement principles usually have different advantages. Therefore, in the process of re-reaction, by appropriately combining marker particles used in various measurement principles and / or using them in an appropriate order,
It becomes possible to extract a plurality of useful information from one test sample.

In the detachment step, the marker particles are detached from the ligand-analyte-marker particle conjugate formed on the solid phase carrier in the second reaction step, and the detached marker is further detached. -To remove particles from the ligand-immobilized region. As means for releasing the marker particles, any method may be used as long as it applies a force enough to separate the marker particles from the solid phase carrier. For example, by adding a washing solution to a reaction vessel and using a liquid flow when removing it by suction, or by adding or not adding a washing solution, the reaction vessel is stirred or shaken to remove the marker particles. A method in which the solid phase carrier is physically dissociated, and then the liquid in the container is removed by suction or decantation can be mentioned. However, it is a violent one that uses a washing solution that inhibits the subsequent immunological reaction or inactivates the reagent, and releases the ligand immobilized on the carrier and the analyte bound to this ligand. must not. Examples of the washing solution that can be used in this removal step and which does not inhibit the subsequent reaction include water, buffer solution, physiological saline and the like.

When the marker particles are minute, the marker particles are not easily dissociated by means such as the flow of the cleaning liquid or vibration. Therefore, a certain degree of vigorous treatment is required, but for this reason, there is a risk that the ligands and analytes fixed on the carrier will also be released. For this reason,
It is necessary to devise a method such as appropriately repeating the detaching process in which a relatively gentle treatment is performed. In a container with a shallow liquid surface such as a microplate or a slide glass, even if the container is shaken so that the solid phase surface on which the ligand is immobilized is inverted, only the marker particles are detached. Therefore, it can be preferably used as a solid-phased carrier.

When the marker particles include a magnetically responsive magnetic substance, a magnet having a magnetic force sufficient to overcome the binding of the marker particles in the container is provided outside the reaction container. The particles can be collected and removed in one place by means of the washing machine without adding washing liquid or stirring.
It becomes possible to separate the car particles. Also, so that the marker particles gather outside the solid-phased region of the ligand, a magnet is applied from the outside of the reaction vessel to separate the marker particles from the solid-phased surface and move away from this state. While maintaining
By using as the marker particles for the subsequent re-reaction, those having no magnetic response, it is possible to shift to the re-reaction step without removing the marker particles detached in the detachment step from the reaction vessel. It will be possible. In this case, it is not necessary to use a large member such as a cleaning system, especially a waste liquid tank,
The apparatus can be downsized and simplified, and the marker particles that are no longer needed can be discarded or collected together after the re-reaction, which improves the processing efficiency.
However, even in this case, when the detached marker particles are particles that reduce the measurement sensitivity by the marker particles in the subsequent re-reaction, for example, particles in which the same substance or a similar substance is solid-phased, Again, it is preferable to remove it from the reaction vessel.

When the carrier to be used by adding it to the reaction container in the form of granules, plates, etc. is used as the solid phase carrier, it has a size that can sufficiently resist the shaking or movement of the bound marker particles. Or it is necessary to have the weight. If the size and weight of the solid phase carrier are too small, the solid phase carrier moves together with the marker particles in the separation step, and it becomes difficult to separate the marker particles. When the solid phase carrier has a particle shape, the particle size is usually 1 mm or more, preferably 5 to 10 mm or more.

Such a granular or plate-like solid phase carrier can be easily recovered or discarded by an appropriate means such as gripping or suction holding after the completion of a predetermined process. Therefore, it becomes possible to reuse the reaction container containing these solid phase carriers by a simple process. Further, the recovered solid-phase carrier can be transferred to another reaction container to perform different analysis. At that time, the analysis can be always performed optimally by using a container that is suitably formed according to the principle of the analysis.

After the marker particles are detached and removed from the ligand-immobilized region of the carrier, new marker particles are added to carry out a re-reaction. This re-reaction step can be repeated substantially as many times as desired by sufficiently removing the marker particles in the previous step. If the marker particles are not sufficiently desorbed, the sensitivity will decrease each time the re-reaction is repeated, but the extent of the decrease is measured in advance by repeating the re-reaction process using a reference sample. It is possible to Therefore, based on this measured value, the re-inspection can be performed up to the maximum allowable number of times in which the detection error can be avoided in the same reaction container. In a usual operation, the reactivity itself is not lost even if the re-reaction step is repeated 6 times or more for the agglutination reaction using the microtiter plate.

It is also possible to store the reaction vessel used for one analysis once for a certain period of time, and then further subject it to the re-reaction step. However, in this case, it is necessary to store under conditions that do not inactivate the immunological reactivity. Further, the above-mentioned leaving operation may be performed before or after the saving process.

The re-inspection method of the present invention can be applied not only to re-inspection for the same analyte but also to multi-item inspection for changing the analyte to be analyzed. The re-inspection for the same analyte can be performed in the same manner whether it is a reproducibility test in which the same type of inspection is repeated or a confirmation test in which inspections with different measurement principles are performed.

An example of the multi-item test is a test having test items for determining the presence (positive) or absence (negative) of the analyte. In such tests,
By setting a high cut-off value that defines the boundary between the positive reaction and the negative reaction or the determination holding range in advance, the re-reaction can be performed a desired number of times. Examples of such test items include blood type tests that are comprehensively determined by the presence or absence of three or more types of analytes, and specifically include ABO type, HLA type, and platelet antigen type. In the case of these inspections, since the type determination can be carried out in one reaction container, various kinds of different samples can be simultaneously inspected with a small amount and a small space, which can be an extremely effective means.

[0027]

EXAMPLES Example 1 Detection of Anti-Red Blood Cell Antibodies Anti-red blood cell antibodies were detected according to the immunological measurement method using magnetic particles described in JP-B-2-124464. 1) Preparation of blood cell binding plate

Wheat germ lectin (hereinafter abbreviated as WGA) was dissolved in 0.01 M PBS, pH = 7.0 to obtain 10 μg /
Prepare a ml solution and use the Nunc U-bottom plate (Maxisor).
50 μl / well was dispensed into each well of p). This is 5 ℃
After incubating overnight in PBS, the plate was washed with 0.01 M phosphate buffered saline (PBS) and blocked with PBS containing 0.1% bovine serum albumin (BSA) to obtain a blood cell binding plate. 2) Solid phase of red blood cells

Selectogen (D (+), E (-), Le ^a produced by Ortho Co., which is an O-type red blood cell with a known antigen. (+), M (+))
To prepare a suspension of about 1%. Then, 25 μl of this suspension was dispensed into the well of the blood cell-binding plate prepared in 1) above, and left standing for 10 minutes to immobilize the blood cells on the plate. Then, the cells were washed three times with physiological saline to remove unbound blood cells. 3) Addition of anti-red blood cell antibody

In each well of the plate on which blood cells were immobilized,
LISS (Low Ionic Strength Medium) was dispensed in 75 μl aliquots, and human serum containing irregular antibodies (anti-D, anti-E,
Anti Le ^a , Anti Le ^{a + b} , Anti-M) and irregular antibody-negative human serum (25 μl each) were dispensed into separate wells.
Incubated at 10 ° C for 10 minutes. Then, using physiological saline as a washing solution, washing was performed 6 times with a microplate washer (Autotech washer manufactured by Biotech, AMW-2). 4) Detection of irregular antibody by anti-IgG + anti-IgM antibody sensitized particles

Goat anti-human IgG antibody and goat anti-human I
A suspension of magnetic substance-encapsulated particles (manufactured by Olympus Optical Co., Ltd.) sensitized with gM antibody was dispensed at 25 µ / well into the plate prepared in 3) above, and disclosed in JP-B-2-124464. According to the method described, it was left on the magnet for 3 minutes to form a pattern. The judgment results of the formed patterns are shown in Table 1 below. In Table 1, (++) means positive,
(-) Represents negative, respectively. 5) Determination of antibody type by anti-IgG antibody sensitized particles and anti-IgM antibody sensitized particles

The pattern formed in each well in 4) above.
After deciding the level, the cells were washed once with physiological saline. afterwards,
A suspension suspension of goat anti-human IgG antibody-sensitized magnetic substance-encapsulated particles and goat anti-human IgM antibody-sensitized magnetic substance-encapsulated particles (both manufactured by Olympus Optical Co., Ltd.) was dispensed into separate wells at 25 μl / well. The pattern was formed by leaving it on the magnet for 3 minutes in the same manner as in 4) above. Table 1 also shows the determination results of the formed patterns.

[0033]

[Table 1]

As shown in Table 1, anti-IgG antibody + anti-Ig
When M antibody-sensitized particles were used, those to which anti-E antibody and negative serum were added were determined to be negative, and other anti-D and anti-Le ^a , Anti Le ^{a + b} And the anti-M antibody was determined to be positive. This is the red blood cell antigen (D
(+), E (-), Le ^a (+), M (+)).

Anti-IgG antibody + anti-IgM antibody sensitized granules
Anti-IgG antibody sensitized particles and anti-IgM after removal of offspring
In pattern formation using antibody-sensitized particles, anti-D, anti-Le
^{a + b} For anti-M antibody, anti-IgG antibody sensitized particles
Anti-Le when used^a For anti-IgM antibody sensitized granules
Each was positive when the offspring were used. Therefore,
Anti-D, Anti-Le^{a + b} And the anti-M antibody is an IgG antibody
Anti-Le^a We know that the antibody is an IgM antibody.
It Example 2 Determination of IgG antibody and IgM antibody of irregular antibody

In the same manner as in Example 1, O-type red blood cells (Ortho
75 μl / well of LISS was dispensed into each well of a U-bottomed plate (Maxuncorp, manufactured by Nunc Co., Ltd.) on which immobilized Selectgen) was immobilized. Next, irregular antibody-positive specimens, ie, anti-D (2 cases), anti-E (3 cases), anti-Le ^a (4 cases), anti-M
(1 case) and anti-Fy ^a Each of the 11 kinds of antibodies (1 example) was added to a separate well at 25 μl / well and incubated at 37 ° C. for 10 minutes. After that, fill each well with saline.
After washing 6 times, 25 μl of a suspension suspension of anti-IgG antibody-sensitized magnetic substance-encapsulated particles (manufactured by Olympus Optical Co., Ltd.) was dispensed into each well to form a pattern on the magnet for 3 minutes. . Table 2 shows the determination results of the formed patterns.

Then, each well was washed once with physiological saline to remove the anti-IgG antibody-sensitized particles and to remove the anti-Ig antibody.
25 μl of a suspension suspension of M antibody-sensitized magnetic substance-encapsulated particles (manufactured by Olympus Optical Co., Ltd.) was dispensed to form a pattern on the magnet for 3 minutes. Table 2 also shows the determination results of the formed patterns.

[0038]

[Table 2]

As is clear from Table 2, the anti-D antibody of 2 cases and the anti-E antibody of 3 cases are all IgG-type, and anti-Le ^a Antibody (I) is IgG type, anti-Le ^a Antibody (II) is I
Both gG and IgM, anti-Le ^a Antibodies (III) and (IV) were IgM-based. Furthermore, anti-M antibody and anti-Fy ^a It was also possible to determine that they were IgG-type. Example 3 Qualitative and IgG properties of HBs antibody, IgM
Sex determination

HBs antigen (manufactured by Meiji Dairy Co., Ltd.) was used in PBS 8
The solution diluted to μg / mol was dispensed into U-bottom microplate wells (Nunc, Maxisorp) at 50 μl / well and incubated at room temperature for 60 minutes. Then, the wells were washed 3 times with PBS, and 0.01M containing 1.0% BSA was added.
Blocking treatment was performed with PBS.

75 μl of 3% BSA / PBS was dispensed into each well of the HBs antigen solid phase plate thus prepared, and 4 types of HBs antibody positive sera and negative sera were placed in separate wells as specimens. 25 μl / well, 37
Incubated at 10 ° C for 10 minutes. Then, the wells were washed 6 times with physiological saline, and anti-IgG antibody-sensitized particles (manufactured by Olympus Optical Co., Ltd.) were added to each well to form patterns. Table 3 below shows the determination results of the formed patterns.

[0042]

[Table 3]

From Table 3, among the four HBs antibody-positive sera, HBs-positive sera (I) and (II) contain both IgG-type and IgM-type antibodies, and HBs-positive sera (III).
It can be seen that and (IV) contain only IgG antibodies. Example 4

Using the anti-D serum dilution series as a sample sample, the sensitivity changes were compared when the re-reaction step was performed twice or more.
Further, the removal and removal operations of carrier particles are performed by the following operations a to d.
A comparison was also made in the case of changing as described in.

Operation a) Anti-IgG antibody of step 4) + anti-Ig
The irregular antibody detection by the M antibody-sensitized magnetic substance-encapsulated particles was performed in the same manner as in Example 1, and the first determination was performed. Next, the plate was washed once with a microplate washer, and a suspension of anti-IgG antibody + anti-IgM antibody-sensitized magnetic substance-encapsulated particles was dispensed into each well at 25 μl / well. . Thereafter, a magnet was brought close to each well and left standing for 3 minutes to form a pattern, and the second determination was performed. Thereafter, the same operation was repeated, and the pattern formation and judgment up to the sixth time were performed.

Operation b) Instead of washing with the microplate washer in the above operation a, the microplate is shaken to suspend the particles in a suspended state, and immediately after that, the plate is inverted. The water was drained and particles were removed by quickly descending. After that, new anti-Ig
The suspension suspension of the G antibody + anti-IgM antibody-sensitized magnetic substance-encapsulated particles was dispensed in an amount of 25 μl / well, and a pattern was formed in the same manner as in the above operation a, and the second determination was carried out. Thereafter, the same operation was repeated, and the pattern formation and judgment up to the sixth time were performed.

Operation c) The particles were suspended and suspended by shaking the microplate without washing with the microplate washer in the above operation a, and then the pattern was directly removed without removing the particles. Formation was repeated.

Operation d) Instead of washing with the microplate washer in the above operation a, the microplate was shaken to suspend the particles in a suspended state, and the washing solution was gently added to each well. Draining was performed. After that, the particles in the suspended state were newly added and the pattern formation was repeated.

The results of these judgments are shown in Table 4 below. In Table 4, (++) is a strong positive pattern spread over the entire surface of the microplate, and (+) is a positive or weak positive, which is smaller than (++) but spread relatively large in the well. , (-) Is a negative pattern in which particles are gathered in the center of the well, and (±) is a pattern showing the middle between (+) and (-), indicating the suspension of determination.

[0050]

[Table 4]

Further, a dilution series of anti-E serum was used as a sample sample, and the particles were removed by suspending the particles by shaking the plate without washing with a microplate washer. The sixth and subsequent determinations were performed in the same manner as in the above operation a, except that the pattern formation was repeated as it was. The results are shown in Table 5. The symbols used in the table have the same meanings as in Table 4 above.

[0052]

[Table 5] As is clear from Tables 4 and 5, according to the retesting method of the present invention, the anti-D serum and the anti-E serum could be repeatedly retested several times.

[0053]

INDUSTRIAL APPLICABILITY As described above, according to the immunological retesting method of the present invention, retesting for a specific analyte, for example, reproducibility test using the same type of test method, measurement method of different principle is used. The confirmation test can be performed easily and accurately.

In addition, the presence or absence and the type of existence of a plurality of types of analytes can be individually inspected for each analyte. Therefore, it is possible to more reliably and easily make a determination for each analyte, as compared with the case where multiple items are tested at the same time.

Further, even when a plurality of tests are carried out, only one reaction container is used and the sample is dispensed once and repeatedly used, so that the reaction container and the sample can be economically used. . Particularly, it is useful for a sample in which the usable amount is limited or only a very small amount can be obtained.

Claims (2)

[Claims] 1. A reaction between a test sample and a ligand corresponding to an analyte of interest immobilized on a solid phase carrier,
The first reaction step of immobilizing the analyte in the test sample on the solid phase carrier, and the first marker immobilizing the ligand corresponding to the analyte.
Contacting the particles with an analyte immobilized on a solid support,
A second reaction step of forming a conjugate between the solid phase carrier and the first marker particle via an analyte, a first determination step of performing determination based on a reaction result in the second reaction step, and the above-mentioned binding A step of removing the first marker particles from the body to remove them from the reaction system, and a second marker having a ligand corresponding to the analyte immobilized thereon
Contacting the particles with an analyte immobilized on a solid support,
Immunology comprising a re-reaction step of forming a conjugate between a solid-phase carrier and a second marker particle via an analyte, and a re-determination step of making a determination based on the reaction result in the re-reaction step Re-examination method. 2. A separation step of, after the re-determination step, separating the marker particles used in the re-reaction step from the binder formed in the immediately preceding re-reaction step to remove it from the reaction system. Marker particles to which a ligand corresponding to light is immobilized are brought into contact with analytes immobilized on a solid phase carrier to form a conjugate between the solid phase carrier and the marker particles via the analyte. The series of steps comprising a re-reaction step and a re-determination step for making a determination based on the reaction result in the re-reaction step are further performed once or more.
The immunological retest method described.