JP2677753B2 - Aggregation immunoassay - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for immunologically measuring an antigenic substance in a fluid such as a biological sample by utilizing an agglutination reaction (aggregation immunoassay method).

[0002]

2. Description of the Related Art Recently, in hospitals, test centers, etc., automation of various tests such as clinical tests and / or due to lack of manpower, cost reduction, or request for large sample processing,
Alternatively, the measurement time has been shortened. As a method suitable for such automation, an agglutination method in which an antigenic substance is qualitatively or quantified by utilizing an agglutination reaction of insoluble carrier particles has been attracting attention.

In the immunochemistry field, a latex agglutination immunoassay method has been mainly used as an agglutination method. The latex agglutination method known in the art means, for example, as described in JP-B-58-11575, when an antigenic substance in a fluid (test sample) such as a biological sample is measured, the test A sample is mixed with a latex carrying an antibody or a fragment thereof that specifically binds to the antigenic substance, and the degree of aggregation of the latex is measured to detect or quantify the antigenic substance. is there.

In this case, a polyclonal antibody is usually used as the above-mentioned antibody. Since a monoclonal antibody reacts only with a specific epitope on the antigen molecule, even if the antigenic substance is a multivalent antigen, the specific epitope is not always multivalent. Therefore, when a monoclonal antibody is used as the above antibody, the antigen is more likely to be a monovalent antigen with respect to the epitope corresponding to the specific monoclonal antibody.

When the antigenic substance to be measured is a monovalent antigen, even if the antibody carried on the latex reacts with or binds to the antigenic substance in the sample, aggregation usually does not occur. In this case, the latex carrying the antibody, an agglutinin capable of aggregating the latex (for example, polyhapten), and the test sample are mixed and reacted,
A method (aggregation inhibition method) for detecting or quantifying the antigenic substance in a test sample by measuring the extent to which the antigenic substance inhibits latex aggregation is known in the art.

Therefore, in the case of a protein antigen which is a multivalent antigen,
In the agglutination method using a monoclonal antibody, there is a possibility that the antigen epitope will be monovalent for the above-mentioned reason.Therefore, when measuring such a protein antigen, the agglutination inhibition using a polyhapten containing the antigen epitope portion is used. You will have to rely on measurement by the method.

[0007] Therefore, when a monovalent antigen is measured using a monoclonal antibody, polyhapten as an agglutinin is newly required, but it is guaranteed that the polyhapten exhibits the same reactivity as the native antigenic substance in the biological sample. There is no.

[0008] Comparing the ordinary latex agglutination method using a polyclonal antibody with the latex agglutination inhibition method using a monoclonal antibody, it is difficult to always obtain a polyclonal antibody of the same quality, and it is purified to a specific antibody. While the loss of the antibody at the stage of preparation is large, the monoclonal antibody is suitable for mass production of an antibody of constant quality. The monoclonal antibody is more advantageous in terms of such stability of quality, but when the monoclonal antibody is used, there is a drawback that the latex aggregation inhibition method has to be used as described above. In consideration of such a point, the usual latex agglutination method using a polyclonal antibody is currently used.

When a monoclonal antibody is used, it goes without saying that a monoclonal antibody that reacts with polyhapten as an agglutinin is used, but there are cases where the monoclonal antibody does not react with the native antigenic substance. In such a case, it is considered that some treatment is required to bring the reactivity of the native antigenic substance closer to the reactivity of the polyhapten.

From the above-mentioned drawbacks, the limits of the latex aggregation method (or latex aggregation inhibition method) known in the art are clear.

More specifically, in the conventional latex agglutination method, a monoclonal antibody that recognizes only a specific epitope is generally used, except in the case of sugar chain antigens containing a plurality of the same epitope in the molecule. Can not. In this case, the possibility of applying the latex agglutination inhibition method remains, but even if the polyhapten, which is an agglutinin, reacts with the monoclonal antibody, the monoclonal antibody does not react with the native antigenic substance, and some treatment is required. Is likely to be needed. This is because in the case of a protein antigen, the higher order structure of the protein antigen plays an important role in the reactivity with the antibody, whereas the polyhapten which is an agglutinin is a polymer (or oligomer) of a peptide, and It is thought that this is due to the difference in the three-dimensional structure from the protein antigen itself.

Therefore, one of the objects of the present invention is to provide a new agglutination immunoassay method for antigenic substances.

More specifically, an object of the present invention is to provide a simpler agglutination immunoassay method that uses a monoclonal antibody and does not require agglutinin.

Another object of the present invention is to provide an agglutination immunoassay method using a more stable and simple agglutination reagent.

[0015]

Means for Solving the Problems As a result of earnest research, the present inventors have found that when measuring an antigenic substance in a test sample, rather than using an insoluble carrier particle carrying an antibody specific to the antigenic substance, It was found that the insoluble carrier particles are selectively aggregated by adsorbing the antigenic substance itself as a measurement sample on the insoluble carrier particles and reacting with an antibody or antibody complex specific to the antigenic substance.

The latex agglutination immunoassay method of the present invention is based on the above findings. More specifically, the antigenic substance in a test sample is adsorbed or bound to insoluble carrier particles and specifically reacted with the antigenic substance. The insoluble carrier particles are selectively aggregated by reacting the antibody or antibody complex to be reacted.

In the present invention, the concentration of the biological sample, the buffer concentration of the suspension of insoluble carrier particles, and the like depend on the abundance of the antigenic substance in the biological sample from the viewpoint of facilitating the securing of the quantitative property. It is preferable to select the conditions according to the characteristics of the measurement items so that the particles are proportionally adsorbed to the insoluble carrier particles.

In the present invention, it does not matter whether the antigenic substance to be adsorbed on the insoluble carrier particles is a polyvalent antigen or a monovalent antigen. However, it is not always easy to apply the present invention to the measurement of an antigenic substance that is difficult to be adsorbed on a trace amount of biological components or insoluble carrier particles.

When an insoluble carrier particle is adsorbed with an antigenic substance in a sample to be reacted with a monoclonal antibody which specifically reacts with the antigenic substance, the antigen remaining in the liquid phase without being immobilized. The presence of an active substance (that is, an unadsorbed antigenic substance) may affect the measured value.

More specifically, when the above-mentioned monoclonal antibody reacts not only with the antigenic substance adsorbed on the insoluble carrier particles but also with the unadsorbed antigenic substance, the aggregation of the insoluble carrier particles is inhibited. , Inconvenience occurs. This inconvenience can be solved by adsorbing the antigenic substance to the insoluble carrier particles and then removing the unadsorbed antigenic substance by washing the insoluble carrier. The agglutination immunoassay of the invention may reduce the practicality or speed.

In such a case, by increasing the ratio of the antigenic substance adsorbed on the insoluble carrier particles, the amount of the non-adsorbed antigenic substance can be treated as a substantially non-problematic amount. . For example, in the case of adsorbing IgG (immunoglobulin G) on latex, under certain conditions, even if the amount of IgG remaining in the liquid phase is 1/1000, the amount of IgG immobilized on the latex will be It is known that it decreases only to one fifth (Reference name: No. 1
See Chapter 6, Medical Latex, pages 230-244 (Mr. Kuge).

As a result of further research in consideration of the above points, the present inventor has found that in the present invention, the above-mentioned monoclonal antibody does not substantially react with the antigenic substance in the liquid phase and It was found that it is possible to selectively react with the antigenic substance adsorbed and solid-phased on. Therefore, according to a preferred embodiment of the present invention, in the above-mentioned agglutination immunoassay method, after adsorbing the antigenic substance in the test sample to the insoluble carrier, the antigenic substance is removed without washing the test sample. It becomes possible to react with a monoclonal antibody against.

That is, in a more preferred embodiment of the present invention, the monoclonal antibody used reacts with the antigen adsorbed on the insoluble carrier particles, but substantially does not react with the unadsorbed antigenic substance in the liquid phase. By selecting the one that does not react, it becomes possible to completely eliminate the aggregation inhibitory action by the unadsorbed component.

(Antigenic substance) The antigenic substance to be measured by the agglutination immunoassay method of the present invention is capable of adsorbing (or binding) to an insoluble carrier and is a polyclonal antibody or monoclonal antibody corresponding to the antigenic substance. It is not particularly limited as long as at least one of them can be prepared or obtained, but from the viewpoint of easy adsorption to the insoluble carrier, 100 μg / mL or more (further 1 mg or more) in the biological sample.
/ ML or more) or 1% or more (of the total protein weight) is preferable. From the viewpoint of easy production of the polyclonal antibody or the monoclonal antibody, the antigenic substance is preferably a substance having a molecular weight of 10,000 or more (protein or the like).

The "antigenic substance" to be measured by the agglutination immunoassay method of the present invention may be any one which can produce or obtain at least one of a polyclonal antibody and a monoclonal antibody corresponding thereto.

(Antibody Specific to Antigenic Substance) In the present invention, as the “antibody that specifically reacts with the antigenic substance”, either a polyclonal antibody and / or a monoclonal antibody can be used.

Such a monoclonal antibody is available from Koe.
hler & Milstein (Nature, 25
6, 495-497, 1975) and the like. This method itself is a conventional method and need not be described again, but in this method, it is necessary to efficiently select the hybridoma cells that produce the desired monoclonal antibody. Since a large amount of sample can be easily treated, a 96-well plate is immobilized with a predetermined antigen, and the culture supernatant of hybridoma cells is reacted therewith, and further enzyme-labeled anti-mouse immunoglobulin is reacted with the so-called ELISA method (enzyme Screening is often performed by immunoassay). At this time, since the antigen is solid-phased, when an assay system for reacting the antibody in the state where the antigen is solid-phased is used, this selection method is sufficient, but it was obtained by selection by the ELISA method. It may be found that some monoclonal antibodies produced by hybridoma cells do not react in an assay system in which an antigen and an antibody react in a liquid phase, such as radioimmunoassay (RIA).

Although such a phenomenon per se is well known or well known in the art, the use of such a monoclonal antibody in the agglutination immunoassay method of the present invention causes inhibition of the antigenic substance in the liquid phase. Instead, it becomes possible to specifically react with the antigenic substance immobilized on the insoluble carrier particles.

Now, let us consider a case where an antigenic substance having a molecular weight smaller than that of an ordinary antibody molecule IgG, for example, a molecular weight of about 70,000 is measured. According to the conventional technique, an antibody against the antigenic substance is adsorbed on the latex surface. At this time, the antibody molecule is adsorbed to the latex at the Fc portion and exists with the Fab portion facing outward, and it is known that the antibody molecule has a fairly orderly orientation (for example, literature name: Chapter 16, Medical Latex). , 230-24
See page 4 (Mr. Kuge). Therefore, according to the study by the present inventor, in the prior art, I
The gG molecule stretches out its arm, and the antigen molecule binds to each IgG molecule in the middle of the latexes. The distance between the latexes is 2 IgG molecules with a molecular weight of 150,000 and the antigen molecule with a molecular weight of 70,000. That's one.

On the other hand, in the method according to the present invention, the antigenic substance is adsorbed on an insoluble carrier (for example, latex), and the antibody that specifically reacts with the antigen molecule adsorbed on the latex has a Fab portion. It is thought to take the form of spreading and joining. Therefore, the distance between the latexes is one IgG molecule having a molecular weight of 150,000 and two antigen molecules having a molecular weight of 70,000. In the present invention, as compared with the prior art, in the case of such measurement, It is believed that it is important to bring the latex particles closer together to agglomerate the latex.

Since latex particles usually repel each other due to negative surface charges, it is important to bring the latexes closer to each other than the conventional method when measuring a molecule having a relatively small molecular weight. Then, it is considered preferable to generate a stronger cohesive force. Therefore, in the present invention, it is preferable to use an antibody having a stronger cohesive force.

However, when the antigenic substance to be measured in the present invention is larger than the IgG molecule (molecular weight of about 150,000), the opposite (the antibody with weaker cohesive force is sufficient) may be valid. .

In the agglutination immunoassay method according to the present invention, it is considered that the latex may not agglutinate even after adsorbing the antigenic substance on the insoluble carrier such as latex and then reacting with the monoclonal antibody, for the reason described above. However, even in such a case, it is possible to cause aggregation by the following means.

The repulsive force between the latex particles due to the negative charge on the surface decreases as the distance between the latexes increases. Therefore, by increasing the three-dimensional structure of the monoclonal antibody, the same antibody can cause aggregation.
More specifically, the monoclonal antibody is reacted with a second antibody that selectively reacts with the monoclonal antibody to form a monoclonal antibody complex, and the antibody complex is reacted with a latex having an antigen adsorbed thereon. For example, it is thought that the latex particles can bind to each other at a distance (that is, with a smaller force) to cause aggregation.

In addition, as a method for forming an antibody complex, a method in which a monoclonal antibody is labeled with biotin and avidin is added to form a complex, and a method by a chemical bond (for example, used when enzyme-labeling an antibody is used. Method) and the like.

The antibody complex used in the present invention is
It is preferable that two or more molecules of IgG type antibody molecule (molecular weight of about 150,000) are contained, and the molecular weight thereof is preferably three times or more the molecular weight of IgG type antibody molecule.

In the agglutination immunoassay method of the present invention, it is preferable to use an IgM type monoclonal antibody, which is a pentameric antibody, as an antibody that specifically reacts with an antigenic substance, for the reason described above. Is preferable from the viewpoint of strong cohesive force.

When a rheumatoid factor (autoantibody against the Fc portion of denatured IgG) is detected in the latex agglutination reaction, it is known in the art that it is IgM type rheumatoid factor that causes aggregation (eg, , Literature name:
Clinical Immunity, 21 (Suppl. 14) 80-84, 19
89). That is, in the art, it is said that when the antigen-sensitized latex is reacted with IgM type rheumatoid factor, it is agglutinated, but it is not aggregated even when the IgG type rheumatoid factor is reacted.

Although the monoclonal antibody has been mainly described above, the agglutination immunoassay method of the present invention can be carried out using a polyclonal antibody. However,
In the case of a polyclonal antibody, unlike the case of a monoclonal antibody that recognizes only a single epitope, it is not easy to select an antibody that reacts with the immobilized antigen but not with the antigen in the liquid phase. Not considered. Therefore, when a polyclonal antibody is used, unadsorbed antigenic substance as described above becomes substantially negligible at the stage of adsorbing (or binding) the antigenic substance in the test sample to the insoluble carrier. As described above, it is preferable to select a condition with good adsorption efficiency.

Since there are a plurality of reaction sites on the antigen that react with the polyclonal antibody, it is generally considered that the polyclonal antibody has a stronger cohesive force than the monoclonal antibody. That is, in the present invention, when the antigenic substance in the test sample is adsorbed on an insoluble carrier such as latex, if the antigenic substance remains in the liquid phase, the added polyclonal antibody is solid phase or liquid phase antigen. Therefore, it is considered that the polyclonal antibody reacts with the antigen in the liquid phase and crosslinks the insoluble carrier particles to increase the cohesive force.

As described above, according to the present invention, the insoluble carrier is selected by reacting the antigenic substance adsorbed or bound to the insoluble carrier particles with the antibody or antibody complex specific to the antigenic substance. Agglomeration can occur.

As described above, according to the present invention, since the production of the reagent is simple and easy, it is possible to provide the method using the reagent having high storage stability.

More specifically, latex is generally used as an insoluble carrier in the art, but a latex reagent (latex carrying antigens, antibodies, etc.) of the same quality is always prepared. However, it is not easy to store in a stable state while preventing non-specific aggregation and precipitation.

On the other hand, according to the present invention, since the antigen or antibody is not substantially sensitized to the insoluble carrier (latex etc.), it is possible to use commercially available unsensitized latex as it is. It will be possible. Furthermore, the antibody does not necessarily have to be a purified product, and since the reagent is simple, it is possible to maintain high storage stability, which is an advantageous method for manufacturers.

Hereinafter, the present invention will be described in detail with reference to the drawings as necessary.

(Insoluble Carrier Particles) In the process of developing the agglutination immunoassay method of the present invention, the present inventor has observed that the time for immobilizing the antigenic substance on the insoluble carrier is extremely short. Therefore, the present inventor has tried to immobilize a test sample of an aqueous solution containing an antigenic substance on an insoluble carrier as it is, and completed the present invention.

The insoluble carrier used in the present invention is, for example, fine particles of an organic polymeric substance, fine particles of an inorganic oxide, or a core thereof as described in the prior art (Japanese Patent Publication No. 58-11575). Fine particles whose surface is treated with an organic substance or the like are preferably used, but are not limited thereto. In the present invention, latex such as polystyrene latex is particularly preferably used as the insoluble carrier. Originally latex is an emulsion that exudes when a rubber tree is damaged,
The latex in the present invention means a suspension or emulsion in which discontinuous fine particles are suspended in an aqueous liquid.

Except when used in a qualitative reaction, when performing immunoassay quantitatively, usually, uniformity of particle size, control of surface state, selection of internal structure, etc. are required in a high degree of dimension. Good latex particles for such a reagent can be selected from commercial products and used.

For example, the latex particles have an average particle size of 0.04 to 0.8 micron (further 0.08 to 0.2).
Those of about micron size are preferably used.

The material of the latex is not particularly limited,
Styrene latex such as polystyrene latex, acrylic acid latex and the like are preferably used. It is preferable to use a latex having a strongly hydrophobic surface (for example, polystyrene latex) for smooth adsorption of proteins or peptides. Furthermore, various modified latices (for example, carboxylic acid modified latices), magnetic latices (latex containing magnetic particles) and the like may be used as necessary.

As the latex particles, polystyrene particles obtained by emulsion polymerization without using an emulsifier are particularly preferably used. Such a latex can stably exist even without an emulsifier due to the repulsion between the negative charges on the surface.

In order to adsorb the antigenic substance in the test sample to the latex particles, the buffer solution for suspending the latex particles is basically the same technique as when the antigen is immobilized on the plate by ELISA or the like. Can be used. Some latex particles tend to spontaneously aggregate, but in such a case, it is preferable to suspend them in a weakly alkaline glycine buffer solution or borate buffer solution from the viewpoint of stability. Regarding latex concentration,
It is preferably used as a 0.05 to 1 weight percent suspension.

(Antigen-antibody reaction) In the present invention, after immobilizing the antigenic substance in the test sample, an antibody or antibody complex that specifically reacts with the antigenic substance is reacted, An insoluble carrier such as latex is aggregated.
The aqueous solution used at this time contains a surfactant such as Tween 20 in an amount of 0.1 to 0.1% in order to prevent the antibody or antibody complex from adsorbing to an insoluble carrier such as latex.
It is preferable to contain about 3%.

(Agglutination Reaction) The container for carrying out the agglutination reaction of the present invention is not particularly limited, but a normal tube (test tube; made of polystyrene, for example) container can be used. Considering the fact that simultaneous processing of many specimens is easy, an ELISA plate having a plurality of wells (holes) (for example, a 96-well ELISA plate (NUNC-I
MMUNO LATE etc.) can be used. As described later, it is preferable to carry out the reaction using a substantially transparent container from the viewpoint of facilitating the measurement of latex agglomeration by an optical method. When an automatic analyzer as described below is used, the agglutination reaction is usually carried out in the reaction tank of the analyzer.

(Measurement of Aggregation) The method for measuring the extent of aggregation of the insoluble carrier particles is not particularly limited. For example, in the case of qualitatively or semi-quantitatively measuring aggregation, the degree of aggregation of the above insoluble carrier particles is visually determined by comparison with the degree of turbidity of a known sample. It is also possible. In the case of quantitatively measuring the aggregation, it is preferable, for example, to optically measure from the viewpoint of simplicity.

A known method can be used as an optical measuring method for the aggregation of the insoluble carrier particles made of latex or the like. More specifically, for example, so-called nephelometry (capture agglomerate formation is regarded as an increase in turbidity), particle size distribution measurement method (agglomerate formation is regarded as a change in particle size distribution or average particle size), integrating sphere Various methods such as the turbidity method (the change of the forward scattered light due to the formation of aggregates is measured using an integrating sphere and the ratio to the transmitted light intensity is measured) can be used. For each of these measurements, a rate test (rate assay; taking at least two measurements at different time points,
The degree of aggregation is determined based on the increment (that is, the rate of increase) of the measured value between these time points, and the end point test (end point assay; one time point (usually considered to be the end point of the reaction)) It is possible to obtain a measured value and determine the degree of aggregation based on this measured value).
From the viewpoint of simplicity and quickness of measurement, it is preferable to perform a velocity test using a nephelometry.

In the optical measurement of the agglomeration of the insoluble carrier particles, when the average particle diameter of the insoluble carrier particles made of latex or the like is about 0.04 to 0.8 μm,
It is preferable to measure using light having a wavelength of about 1400 nm.

(Example of measurement of hemoglobin A1c) As one embodiment showing the features of the agglutination immunoassay method of the present invention, an example of measurement of hemoglobin A1c (HbA1c) will be described below. It is not limited to measurement.

In the above "hemoglobin A1c", glucose non-enzymatically binds to the α-amino group of valine, which is the amino acid residue at the β chain N-terminus of hemoglobin (Hb),
It becomes glycosylated hemoglobin, and the blood level of this hemoglobin A1c reflects the relatively long-term blood glucose control state of diabetes. Therefore, this HbA1c
It is clinically very meaningful to know the glycemic control state (for example, Japanese clinical, 48,
Special Issue, 315-322 (1990)).

Hemoglobin is a heterotetramer consisting of two α-chains and two β-chains, and hemoglobin A1c is characterized in that one N-terminal α-amino group of two β-chains is glycosylated. Yes, therefore hemoglobin A1c
There is one reaction site characteristic of. In other words, hemoglobin A1c functions as a monovalent antigen in terms of reactivity with a monoclonal antibody specific for hemoglobin A1c.

When measuring hemoglobin A1c,
It is conceivable to perform the measurement by a latex aggregation inhibition method using a conventional technique, but according to JP-A-1-150857, the monoclonal antibody carried on the latex reacts with the polyhapten which is an agglutinin to cause aggregation. However, the native hemoglobin A1c in the biological sample does not react with the monoclonal antibody. Therefore, pretreatment such as adding guanidine hydrochloride to the test sample and heating it at 56 ° C. for 15 minutes is required.

There was a further peculiar problem regarding the measurement of hemoglobin A1c. That is, hemoglobin A1c has been conventionally measured by high performance liquid chromatography (HPLC), and the fraction% of hemoglobin A1c in the total hemoglobin has been determined. Therefore, the above-mentioned JP-A-1-150
According to Japanese Patent No. 857, it is essential to separately quantify hemoglobin in order to determine the fraction% of hemoglobin A1c by performing the latex aggregation inhibition method.

According to the agglutination immunoassay method of the present invention,
Regarding the measurement of hemoglobin A1c, for example, a test sample (for example, hemolyzed whole blood added with purified water) is adsorbed on latex (insoluble carrier), and anti-hemoglobin A1c is adsorbed.
By reacting the monoclonal antibody complex, selective aggregation of latex can be caused, and by measuring the degree of this selective aggregation, hemoglobin A
1c can be quantified. A standard sample with a known hemoglobin A1c% value measured by HPLC was quantified at the same time by the agglutination immunoassay method of the present invention to prepare a calibration curve. Based on this calibration curve, the fraction% value of hemoglobin A1c of an unknown sample was determined. Can be asked.

(Anti-HbA1c Monoclonal Antibody) Anti-Hb
As an A1c monoclonal antibody, HbA1c which is substantially immobilized by reacting with HbA1c adsorbed or immobilized
Anti-Hb that does not substantially react with bA0 (preferably, it also does not substantially react with HbA1c and HbA0 in the liquid phase)
Any A1c monoclonal antibody can be used without particular limitation. According to the study of the present inventors, generally, such a monoclonal antibody can be obtained by preparing a monoclonal antibody using a glycated peptide as an immunogen. Here, the reactivity to HbA1c and HbA0 is
For example, it can be measured as follows.

For example, by reacting with immobilized HbA1c,
The fact that it does not react with HbA0 can be measured as follows.

96-well (well) ELISA plate (for example, NUNC; NUNC-IMMUNO PLA)
TE, MAXISORP F96 (4-42404))
HbA1c (eg, EXOCELL; purified HbA
1c) 0.05 so that the concentration becomes 5 μg / mL
After diluting with M citrate buffer (pH 5.6), dispense 50 μL of each well. This plate is left overnight at 4 ° C. to immobilize HbA1c on an ELISA plate.

This plate was replaced with PBS-T (0.01M phosphate buffer, pH 6.8 / 0.15M NaCl / 0.1%).
After washing four times with Tween 20), 50 μL of a sample containing anti-HbA1c monoclonal antibody (for example, hybridoma culture supernatant) is added and left to react at room temperature (about 25 ° C.) for 2 hours. After washing the plate 4 times with PBS-T, POD (peroxidase) labeled anti-mouse IgG
(For example, 3711-0081 manufactured by CAPPEL)
Dilute 5000-fold with BS-T, add 50 μL, and add 1 at room temperature.
Let react for hours.

After washing four times with PBS-T, the substrate solution (0.
1% o-phenylenediamine (manufactured by Sigma) / phosphate-citrate buffer (7.3 g of citric acid monohydrate / 23.9 g of disodium phosphate dodecahydrate / 23.9 g of purified water / 1 L) /
0.003% H2O2) 100 μL was added, and the mixture was stirred at room temperature for 30
After reacting for 2 minutes, 50 μL of 2N-sulfuric acid is added to stop the enzyme reaction. Then, the absorbance (OD) at 490 nm of the solution after the enzyme reaction is determined using an immunoplate reader (MR5000 manufactured by Dynatech).

Instead of HbA1c used above, HbA0
The reactivity between the monoclonal antibody and HbA0 can be similarly determined by the OD value by carrying out the same procedure as described above except using (for example, EXOCELL; purified HbA0).

In the present invention, the anti-HbA1c monoclonal antibody preferably exhibits a reactivity with HbA1c of 1.0 or more (further 2.0 or more) on the scale of the above immunoplate reader, and HbA0 is 0 or less. It is preferable to exhibit a reactivity of 0.1 or less (further, 0.05 or less).

On the other hand, the fact that the above-mentioned monoclonal antibody reacts with HbA1c immobilized on a solid phase but does not react with HbA1c or HbA0 in the liquid phase can be measured as follows.

100 μL of 0.2 M glycine buffer (pH 2.5) was added to 100 μL of HbA1c, and the mixture was left at room temperature for 5 minutes. Add 800 μL of PBS-T,
After stirring, 100 μL of anti-hemoglobin A1c monoclonal antibody (hybridoma culture supernatant, diluted 50 times) is added to 100 μL, and the mixture is left at room temperature for 30 minutes. Then, 100 μL of that was solidified with HbA1c in advance (5 μg / mL, 100 μL was added to a 96-well plate and left at 4 ° C. overnight to be solid-phased, and PBS-T was used.
(Washed 4 times with 4), and incubate for 2 hours at room temperature.

After washing four times with PBS-T, 100 μL of POD-labeled anti-mouse IgG antibody (diluted 5000 times) was used.
In addition, it is made to react at room temperature for 1 hour. After washing 4 times with PBS-T, the substrate reaction was carried out at room temperature for 30 minutes, and a stop solution was added to add 49
The OD value at 0 nm is measured. The reactivity in the liquid phase between the monoclonal antibody and HbA0 can be determined in the same manner as above except that HbA0 is used instead of HbA1c used above.

The above is the case of denaturing hemoglobin. As a control, the same procedure as above was carried out except that 100 μL of glycine buffer was added to 100 μL of pure water in order to react HbA1c and HbA0 without denaturation. Then, the reactivity with the monoclonal antibody in the liquid phase can be determined.

In the present invention, the anti-HbA1c monoclonal antibody is used in combination with native HbA1c, HbA0, and denatured HbA0 at 10 μg / mL in the liquid phase.
(Even 20 μg / mL) does not show reactivity, while
Denatured HbA1c is 1 μg / mL (more 0.5
(μg / mL) or less is also preferable to show reactivity.

In this measurement, HbA1c and HbA1c
0 is preferably purified before use. This purified HbA1c
As HbA0 and HbA0, commercially available products can be used, but if no suitable commercial products are available, they may be purified by HPLC before use. In this purification, for example, 50 μL of whole human blood is centrifugally washed with 2 mL of PBS, and the supernatant is discarded.
2 mL of pure water is added to the red blood cell pellet to cause hemolysis. This is fractionated by HPLC to separate the HbA1c and HbA0 peaks.

As the separation conditions of HPLC, for example, the following conditions are preferably used.

Analytical instrument: Shimadzu HPLC system Column: High resolution column for separation of hemoglobin MICROPEA
RL (Sekisui Chemical Co., Ltd.) Eluent A: Low Phosphate, (Auto A1c, P type
(Kyoto Daiichi Kagaku) Eluent B: High Phosphate, (1 g of phosphoric acid 8.5 g,
2 potassium phosphate 22.5 g, potassium chloride 3.5 g, purified water 1 L) Mobile phase: A + B 0 to 3.5 min B concentration 0% 3.5 to 4.0 min B concentration 0% to 80% gradient 4 0 to 17.0 min B concentration 80% Flow rate: 1 mL / min Monitor: Absorbance 415 nm Sample amount: 20 μL (Antibody that binds to anti-HbA1c monoclonal antibody) The above-mentioned anti-HbA1c monoclonal antibody (hereinafter, the monoclonal antibody is also referred to as “MAb”). The antibody that selectively binds to (described below) (hereinafter, referred to as “second antibody”) is an antibody that is substantially reactive with the purified immunoglobulin of the animal species from which the MAb is derived in the Ouchterlony method. , Can be used without particular limitation.

In the present invention, the reactivity between the purified immunoglobulin and the second antibody can be evaluated by the following method, for example.

Agarose was added to physiological saline so as to be 1%, heated and dissolved to prepare an agarose plate having a thickness of 1.5 mm, and holes having a diameter of 3 mm and intervals of 2 mm (7 holes) were prepared. ) With a puncher.

Purified mouse IgG (eg, ZYMED
Laboratories Inc. Product number 02-6502) 0.5
mg / mL, 0.125 mg / mL prepared with 0.15 M sodium chloride, and 10 μL of the second antibody (for example, anti-mouse IgG goat serum) in the center hole of 7 holes at intervals of 2 mm was 0.15 M sodium chloride. 4 times, 8 times, 16
10 times diluted to 32 times, 32 times, 62 times, 128 times
Add μL to the surrounding holes and allow it to diffuse in the agarose by leaving it wet to form a sedimentation line. 4 at room temperature
After -5 hours, it is visually observed up to the fold dilution of the antiserum against the purified mouse IgG to form a sedimentation line.

The second antibody used in the present invention preferably forms a sedimentation line with purified mouse IgG up to at least 8-fold dilution, and can form a sedimentation line up to at least 32-fold dilution (further, 64-fold dilution). More preferable.

Next, a preferred embodiment for measuring HbA1c in the present invention will be described.

In the present invention, each tube is usually
1 to 20 μL of hemolyzed blood (further 2 to 5 μL) as a test sample
L) Collect approximately. As the hemolyzed blood to be actually used, a test sample (for example, 50 μL of whole blood plus 1 mL of pure water) may be diluted about 5 to 10 times with a glycine buffer or the like.

Next, one latex suspension (for example, 0.12 μm latex, 0.2% concentration) was added to each tube.
About 100 to 300 μL (further 150 to 200 μL) is added and left at 37 C for about 1 to 30 minutes (further 3 to 10 minutes) to adsorb HbA1c in the sample to the latex. The latex suspension used at this time is preferably used by diluting the latex stock solution with a glycine buffer solution or the like.

Next, HbA1c adsorbed on the latex
And a solution containing a complex of an anti-HbA1c monoclonal antibody (for example, a mouse ascites-derived monoclonal antibody) and a second antibody in an amount of 100 to 300 μL (further, 150 to 200 μL).
L) about 2 to 30 minutes at 37 ° C (further 3 to 10)
After leaving (incubation) for about 10 minutes, HbA1c reacts with the monoclonal antibody. The concentration of the monoclonal antibody complex solution used at this time is, for example, anti-Hb.
When using the A1c monoclonal antibody and the anti-mouse IgG antibody, it is preferable to make a dilution series of each antibody and obtain the optimum concentration. The buffer used for dilution is, for example, 0.05-0.1 M glycine buffer (GBS;
ine buffered saline; pH 8.2-8.5, 0.1
(Including 5M NaCl) and the like. It is recommended that a surfactant (Tween 20, etc.) be added to the buffer solution at this time in an amount of 0.1 to 0.5% (further, 0.2 to 0.3%). It is preferable from the viewpoint of preventing physical adsorption.

In the actual measurement, when using a biochemical automatic analyzer (for example, Hitachi model 7150 automatic analyzer), for example, a sample amount of 3 to 15 μL,
When a reagent amount of about 1 to 350 μL is suitably used, for example, the following amount ratio and reaction time are preferably used.

Test sample: 4 μL Latex suspension: 200 μL (reaction time 5 minutes) Monoclonal antibody complex: 200 μL (reaction time 5
Minutes)

[0089]

EXAMPLES The agglutination immunoassay method of the present invention will be described in more detail below with reference to production examples and examples.

Production Example 1 (Preparation of Monoclonal Antibody against Hemoglobin A1c β-Chain N-Terminal Glycopeptide Epitope) A peptide corresponding to the N-terminal amino acid sequence of hemoglobin β-chain was synthesized, and α of Val of the N-terminal amino acid residue was synthesized. -Glucose was non-enzymatically bound to the amino group to synthesize a glycopeptide, which was further bound to a carrier protein via a spacer to obtain a monoclonal antibody.

Specifically, it was carried out as follows.

Fmoc-L-Valine, Nα-Fmoc-Nim
-Trityl-L-Histidine, Fmoc-L-Leucine, Fm
oc-Ot-Butyl-L-Threonine, Fmoc-LP
Val-His-Leu using roline and Fmoc-s-trityl-L-Cysteine (both manufactured by Peptide Institute)
-Thr-Pro-Cys and Val-His-Leu-Cys peptides were synthesized (the Cys (cysteine) is a part of the spacer). The synthesis was performed using a commercially available peptide synthesizer (Applied Biosystems, Model 430A). After peptide synthesis, the peptide was released from the solid phase resin to obtain a crude peptide.

The crude peptide was dissolved in purified water to 1 mg / mL and cooled to 4 ° C. 1.5 times the molar ratio of 2,2'-dithiodipyridine was added dropwise with stirring to give 1
The reaction was carried out for 0 minutes to protect the SH group of cysteine. After freeze-drying, it was dissolved in 1% acetic acid, the insoluble matter was removed by centrifugation, and the peptide fraction obtained by gel filtration with Sephadex G25 (15 × 900 mm column) was freeze-dried. This peptide was dissolved in acetic acid, glucose was added in a molar ratio of 2 times in the presence of pyridine, and the mixture was stirred at room temperature for about 10 days. When the peptide is saccharified, the retention time (retention time) in HPLC becomes shorter.
Checked by PLC.

The analysis conditions by HPLC are as follows.

Column: TSKgel, ODS-120A
(4.6 × 250 mm, manufactured by Tosoh Corporation) Instrument: Shimadzu HPLC system Mobile phase: Linear gradient from 10% acetonitrile / 0.1% trifluoroacetic acid to 60% acetonitrile / 0.1% trifluoroacetic acid Flow rate: 0 0.8 mL / min Time: 25 min Monitor: Absorbance 280 nm This glycated peptide was finally purified by HPLC and freeze-dried.

The separation conditions are as follows.

Column: TSKgel, ODS-120A
(21.5 × 300 mm, manufactured by Tosoh Corporation) Mobile phase: linear gradient from 10% acetonitrile / 0.1% trifluoroacetic acid to 60% acetonitrile / 0.1% trifluoroacetic acid Flow rate: 5 mL / min Monitor: Absorbance 280 nm Lyophilized peptide was dissolved in 0.1 M potassium phosphate buffer (pH 8.5), and 3 times amount of DTT (Dithiotreitol) was added to remove the cysteine protecting group, and nitrogen gas was filled for 24 hours. It was made to react. And p with hydrochloric acid
After reducing H to 5, it was purified by HPLC as described above and freeze-dried to finally obtain a glycated peptide. However, 28
The absorbance at 215 nm was measured instead of the absorbance at 0 mm.

E was used as a spacer for this glycated peptide.
Using MCS (N-succinimidyl 6-maleimidocaproate), a carrier protein (as a water-soluble protein, Th
yroglobulin, Edestin as insoluble protein (both Fl
Uka) and used as an immunogen.

100 μg of the above immunogen was mixed with Complete Freund's Adjuvant at a w / o (w) level.
Ater in oil) type micelles were injected into the abdominal cavity of Balb / c mice (8 weeks old). Booster immunization uses Incomplete Freund's Adjuvan
In t), w / o micelles were used and the test was performed twice at intervals of one month. The immunogen in saline (physiological saline) was subcutaneously injected 3 days before cell fusion, and this was used as a booster.

Cell fusion was performed by a conventional method. That is, splenocytes were taken from the spleen and used as mouse / myeloma cell lines (PAI, provided by Tokyo University of Science, Arai Laboratory; available at Cancer Research Source Bank (JCRB)).
Mix at a ratio of 0: 1, polyethylene glycol 400
Cells were fused at 0. Suspend the cells in HAT medium,
The cells were seeded on a 96-well plate, and the fused cells were allowed to grow clonally. Pre-purified Hb
It was carried out by reacting the culture supernatant with a 96-well ELISA plate on which A1c or HbA0 was immobilized. P
After washing with BS-T, POD-labeled anti-mouse IgG was reacted, and after washing, color was developed with o-phenylenediamine.

More specifically, the screening was carried out as follows.

96-well ELISA plate (NUN
C company made; NUNC-IMMUNOPLATE, MAXI
SORP F96 (4-42404)) with HbA1c (B
(purified product by ioRex70), the concentration is 5 μg / mL
0.05M citrate buffer (pH 5.6)
After diluting with, each well was dispensed in 50 μL aliquots. This plate was left overnight at 4 ° C. to immobilize HbA1c on an ELISA plate.

This plate was added to PBS-T (0.01 M phosphate buffer, pH 6.8 / 0.15 M NaCl / 0.
After washing 4 times with 1% Tween 20), culture supernatant 50μ
L was added, and the mixture was allowed to react at room temperature (about 25 ° C.) for 2 hours. After washing 4 times with PBS-T, POD (peroxidase) -labeled anti-mouse IgG (CAPPEL; 37
11-0081) was diluted 2500 times with PBS-T, and 5
0 μL was added, and the mixture was reacted at room temperature for 1 hour. 4 with PBS-T
After washing twice, the substrate solution (0.1% o-phenylenediamine / phosphate-citrate buffer (citrate monohydrate 7.3
g, disodium phosphate dodecahydrate 123.9 g, purified water 1 L / 0.003% H2 O2 100 μL were added and reacted at room temperature for 30 minutes, and then 2N-sulfuric acid 50 μL was added to stop the enzyme reaction. .

Then, the absorbance (OD) at 490 nm of the solution after the above enzyme reaction was determined using an immunoplate reader (MR5000 manufactured by Dynatech).

Instead of HbA1c used above, HbA0
The reactivity between the culture supernatant and HbA0 could be similarly determined by the OD value by the same procedure as above except that (purified product by BioRex 70) was used.

A hybridoma producing a monoclonal antibody which specifically reacts with HbA1c is present in a well which reacts with HbA1c but does not react with HbA0.

Hybridomas in wells that reacted with HbA1c but not HbA0 were cloned by the limiting dilution method. After mass-culturing a clone, Balb / c mice (9 to 10 weeks old), which had been intraperitoneally injected with 1 mL of pristane (P1403, manufactured by Sigma) 1 week before, had the hybridoma 1
× 10 7 cells were intraperitoneally injected with saline (physiological saline), and when the ascites was filled in the abdominal cavity, a monoclonal antibody was collected as ascites.

Purified HbA1c used in the above ELISA,
HbAo was obtained as follows.

That is, HbA1c is high (10% or more)
Was removed by centrifugation at 2500 rpm for 20 minutes to remove plasma proteins, then physiological saline in an amount 5 times the amount of the red blood cell pellet was added, and centrifugal washing was performed twice. An equal amount of pure water was added to the erythrocyte pellet after centrifugation to cause hemolysis. 1
After high-speed centrifugation at 5,000 rpm for 45 minutes to remove the precipitate, the supernatant was added to Bio Rex 70 (Bio Rad, 20
(0 to 400 mesh, column size 28 x 255 mm)
Was used to fractionate HbA1c and HbA0 (Trivelli, L.
A., et al., New Engl. J. Med. 284, 353-357,
(1971)). When the purity of each fraction was checked by HPLC, HbA1c was 80 to 90% and HbA0 was 9%.
9% or more. The protein concentration was measured by a protein quantification reagent (BCA Protein Assay Reagent manufactured by Pierce).
) Was used according to the attached manual.

Production Example 2 (Preparation of anti-mouse IgG) Anti-mouse IgG as second antibody
Was commissioned to an external company. That is, purified mouse IgG (ZYMED Laboratories Inc. Product No. 02
6502) was immunized subcutaneously with goats together with Freund's complete adjuvant. All were collected based on the fact that the antibody titer was 64 times or more.

Example 1 2 μL of purified water was added to 50 μL of whole blood collected from a human to cause hemolysis, which was used as a test sample.

In a polystyrene tube, 4 μL of the above-mentioned test sample was taken, and a latex having a particle size of 0.12 μm (Sekisui Chemical Co., Ltd., “latex for reagent, product number N-10” was used.
0 ") to 12.5 mM glycine buffer pH 8.2 (1
0.2% diluted with 8.8 mM sodium chloride)
Add 200 μL of latex suspension at the specified concentration, 37 ℃
And leave it in the latex for 20 minutes to
bA1c was adsorbed.

Next, to the above-mentioned latex suspension, the anti-HbA1c monoclonal antibody derived from mouse ascites obtained in Production Example 1 and the anti-mouse IgG goat serum obtained in Production Example 2 were GBS.
200 μL of diluted with -T (0.3% of Tween 20 dissolved in GBS) and mixed to form a complex
In addition, the mixture was left at 37 ° C. for 20 minutes to carry out a selective aggregation reaction of latex.

An agglutination reaction was similarly carried out using a sample with a known HbA1c value as a standard, and a spectrophotometer (Beckman,
DU-640) to measure the absorbance at 660 nm,
A standard curve was determined. The standard curve of HbA1c thus obtained is shown in the graph of FIG. From this standard curve, the percentage value of A1c in the unknown sample was determined. As a control method of the latex method, an automatic measuring device, Hi-Auto A1c
HbA1c was measured by the HPLC method using HA-8121 type (manufactured by Kyoto Daiichi Kagaku Co., Ltd.) to determine the correlation between the measured values of both the latex method and the HPLC method. The results are shown in the graph of FIG.
In FIG. 2, HPLC measurement data is labile HbA.
A1c% including 1c was shown. As shown in FIG. 2, a good correlation was found between the latex method and the HPLC method.

Example 2 A serial dilution series of the anti-HbA1c monoclonal antibody obtained in Production Example 1 was prepared from × 1 to × 256 dilutions, and the anti-mouse IgG serum (second antibody) obtained in Production Example 2 was multiplied by ×. The same volume of 10 dilutions was added, and the mixture was stirred and allowed to stand at room temperature to form a monoclonal antibody complex. From the observation after 4 hours, the largest amount of immunoprecipitate was observed at the monoclonal antibody × 32 dilution and × 64 dilution. Therefore, the amounts of the monoclonal antibody and the second antibody at the dilution ratios were It was found that the quantitative ratio (ie, the optimum ratio) was suitable for forming the immunoprecipitate of.

HbA1c 0%, 8%, 16% hemolyzed 4
μL was adsorbed on 200 μL of latex in the same manner as in Example 1, and each of the above-mentioned monoclonal antibody complexes was subjected to GBS-T.
Then add 200 μL of the solution diluted x16 times with
The reaction was carried out at 0 ° C for 20 minutes to aggregate the latex. The results are shown in the graph of FIG. Suitable monoclonal antibody conjugates for the agglutination immunoassay according to the invention were those in which the monoclonal antibody was present in approximately 8-fold excess over the optimal ratio.

Example 3 The concentration of the anti-HbA1c monoclonal antibody obtained in Production Example 1 was kept constant at a × 8-fold dilution, and the concentration of the second antibody was doubled from a × 2-fold dilution to a × 512-fold dilution. A monoclonal antibody complex was formed by adding by volume, stirring and allowing to stand at room temperature. The optimal ratio in which the highest amount of immunoprecipitate was observed was at the secondary antibody × 2-fold dilution, × 4-fold dilution.

Each of the above-mentioned monoclonal antibody complexes was subjected to GBS
The latex was agglomerated in the same manner as in Example 2 except that a solution further diluted with T by × 8 was used. Fig. 4 shows the results.
Is shown in the graph. Suitable monoclonal antibody complexes for the agglutination immunoassay method according to the present invention were those in which the monoclonal antibody was present in approximately 8-fold excess over the optimal ratio.

Example 4 The case where the complex of the anti-HbA1c monoclonal antibody obtained in Production Example 1 was in the form of a complex of the monoclonal antibody and the anti-mouse IgG antibody (second antibody), and the biotin-labeled monoclonal antibody was used. The correlation with the case of avidin D complex was determined.

As a biotinylation reagent, NHS-LC-B
Using iotin (PIERCE, No. 21335), the anti-HbA1c monoclonal antibody was biotinylated according to the manual attached to the reagent. Unreacted biotin was removed by repeating dialysis. Avidin D
As the above, A-2000 manufactured by VECTOR was used, and a complex was formed by adding the biotin-labeled monoclonal antibody.

After adsorbing HbA1c in the test sample onto the latex as in Example 1, 200 μL of a complex of anti-HbA1c monoclonal antibody and anti-mouse IgG goat serum (second antibody) was added, and the mixture was further incubated at 37 ° C. Let react for 20 minutes,
A selective flocculation reaction of latex was performed.

As a control, the same as above except that the complex of the biotin-labeled HbA1c monoclonal antibody and avidin D obtained above was added in place of the complex of the anti-HbA1c monoclonal antibody and the anti-mouse IgG goat serum. A selective flocculation reaction of latex was performed.

The results are shown in the graph of FIG. As a method for conjugating anti-HbA1c monoclonal antibody, anti-mouse Ig
A good correlation was observed between the A1c% values obtained when the G antibody was used and when the biotin-avidin-based method was used.

Example 5 A lower red blood cell layer was obtained by gently pipetting plasma, which is a supernatant of a normal whole blood sample left at 4 ° C. overnight. This red blood cell layer is regarded as a whole blood sample with excess red blood cells, and is serially diluted with the previously separated plasma (× 1, × 2, ×
4, x8, x16) were made. These were regarded as whole blood samples having different hemoglobin concentrations, and 0.5 mL, 1 mL, and 2 mL of purified water were added to 25 μL of each to cause hemolysis. The selective agglutination reaction of the latex was performed in the same manner as in Example 1 except that 4 μL of each of the thus hemolyzed samples was used as the test sample, and the OD value at 660 nm was measured.

The obtained results are shown in the graph of FIG. In the case of the test sample of 4 μL, hemolyzed whole blood with 20 times purified water was added according to the decrease in hemoglobin concentration 6
The OD value increased by 60 nm, whereas when hemolyzed by adding 40 times of purified water, it was almost constant at 660 nm.
The OD value is shown, and it was found that the difference in hemoglobin concentration did not significantly affect the measured value.

In addition, the influence of interfering substances (interference check A,
When I checked the International Reagents Co., product number 79290),
Neither chyle nor bilirubin affected the agglutination immunoassay method of the present invention.

Example 6 A selective agglutination reaction of latex was carried out in the same manner as in Example 1 except that the reaction time of the first reaction, that is, the test sample and the latex suspension was 2 to 30 minutes, and 660 nm. Was measured. The results are shown in the graph of FIG. It was found that the adsorption of hemoglobin A1c on the latex was carried out in a short time.

The second reaction was carried out in the same manner as in Example 1 except that the time for reacting the complex of the anti-hemoglobin A1c monoclonal antibody and the anti-mouse IgG antibody (second antibody) was 2 to 30 minutes. The selective agglutination reaction was performed and the OD value at 660 nm was measured. The obtained results are shown in the graph of FIG. It was found that the latex aggregates in a short time by using the monoclonal antibody complex.

Example 7 HbA1c and HbA0 were respectively fed with saline (saline).
Diluted in fold to prepare a dilution series.

For each 100 μL of these dilution series, 0.2 M glycine buffer (pH 2.5) was added to 100 μL.
μL was added and left at room temperature for 5 minutes.

PBS-T (0.01 M phosphate buffer, pH 6.8 / 0.15 M NaCl / 0.1% Tw) was added thereto.
een 20), add 800 μL and stir, then 1
100 μL of anti-hemoglobin A1c monoclonal antibody (culture supernatant, diluted 50 times) was added to 00 μL, and the mixture was left at room temperature for 30 minutes. Next, 100 μL of this was added to a 96-well plate (5 μg) on which HbA1c was immobilized in advance.
/ ML, 100 μL, add to 96 well plate, 4 ℃
It was solidified by standing overnight, washed with PBS-T four times), and reacted at room temperature for 2 hours.

After washing four times with PBS-T, POD was performed.
(Peroxidase) labeled anti-mouse IgG antibody (CAP
PEL, 3711-0081) (5000-fold dilution)
Was added at 100 μL and reacted at room temperature for 1 hour.

Subsequent operations were carried out in the same manner as the screening at the time of preparing the monoclonal antibody. That is,
Substrate reaction is performed at room temperature for 30 minutes, and 490 by adding stop solution
nm O. The D value was measured. The above operation was for denaturing HbA1c or HbA0 to determine whether or not they inhibit the reaction of the anti-HbA1c monoclonal antibody with the immobilized HbA1c.

As a control, in order to react HbA1c and HbA0 in an undenatured state, 100 μL of glycine buffer was added instead of 100 μL of pure water. The D value was measured.

The results are shown in the graph of FIG. As shown in FIG. 9, when HbA1c was denatured, it reacted with the anti-HbA1c monoclonal antibody in a liquid phase and inhibited the reaction with immobilized HbA1c. On the other hand, such inhibition was not observed in HbA1c that was not denatured, and it was revealed that it did not react with the anti-HbA1c monoclonal antibody in the liquid phase. Also, H
bA0 did not react with anti-HbA1c monoclonal antibody with or without denaturation.

[0136]

As described above, according to the present invention, the antigenic substance in the test sample is adsorbed on the insoluble carrier particles, and the antibody or antibody complex that specifically reacts with the antigenic substance is reacted. An agglutination immunoassay method is provided, which comprises selectively aggregating insoluble carrier particles.

According to the agglutination immunoassay method of the present invention,
Since the antigenic substance in the test sample can be simply and quickly measured without performing complicated pretreatment, it is extremely easy to simultaneously process many samples in parallel. In addition, since such an assay method can be suitably applied to a biochemical automatic analyzer, the measurement can be automated and a large amount can be processed.

Further, according to the present invention, there are advantages not only in the assay method itself but also in the point of manufacturing reagents. That is, in general, it is not always easy to manufacture latex reagents (latex having antigens, antibodies, etc. bound thereto) of the same quality. In addition, know-how is required to prevent aggregation and precipitation during storage. In general, the cost of the material latex is low in the price of the latex diagnostic agent, and it is the cost required for the biological material and the process for coating the latex particles with the biological material, which accounts for the majority of the cost of the reagent.

On the other hand, according to the present invention, the antigen or antibody is not substantially sensitized to the insoluble carrier such as latex, and the commercially available insoluble carrier for the reagent (latex etc.) may be used as it is. It is not necessary to manufacture a new "latex reagent" because it is possible. In addition, in the present invention, the antibody does not necessarily have to be a purified product, nor does it require an operation such as enzyme labeling.
This facilitates the production of the measurement reagent and is extremely advantageous in terms of storage stability.

[Brief description of the drawings]

FIG. 1 shows HbA1c by the agglutination immunoassay method of the present invention.
It is a graph which shows the standard curve of.

FIG. 2 is a graph showing the correlation between the measured values of HbA1c by the HPLC method and the agglutination immunoassay method of the present invention.

FIG. 3 is a graph showing the reactivity of the monoclonal antibody complex when the amount of the monoclonal antibody is variable and the amount of the second antibody is constant. The arrow in the figure indicates the optimum ratio region.

FIG. 4 is a graph showing the reactivity of the monoclonal antibody complex when the amount of the monoclonal antibody is constant and the amount of the second antibody is variable. The arrow in the figure indicates the optimum ratio region.

FIG. 5 shows agglutination immunoassays according to the present invention in the case where an anti-HbA1c monoclonal antibody was conjugated with anti-mouse IgG (second antibody) and when the biotin-labeled anti-HbA1c monoclonal antibody was conjugated with avidin D. It is a graph which shows correlation with a measured value.

FIG. 6 is a graph for explaining the influence of the amount of red blood cells in a sample (that is, the amount of hemoglobin) and the amount of purified water when the sample is hemolyzed, on the values measured by the agglutination immunoassay method of the present invention.

FIG. 7 is a graph for explaining the influence of the time for which HbA1c is adsorbed on latex on the value measured by the agglutination immunoassay method of the present invention.

FIG. 8 is a graph for explaining the influence of the reaction time of the anti-HbA1c monoclonal antibody complex on the measured value by the agglutination immunoassay method of the present invention.

FIG. 9 is a graph showing that an anti-HbA1c monoclonal antibody reacts with HbA1c immobilized on an ELISA plate, but does not react with HbA1c in a liquid phase but with denatured HbA1c in a liquid phase. Is.

Claims (9)

(57) [Claims] 1. An antigenic substance in a test sample is adsorbed or bound to insoluble carrier particles in a state in which an antigen and / or an antibody is not substantially adsorbed, and the antigenic substance is specifically adsorbed on the insoluble carrier particle. An agglutination immunoassay method for reacting a reacting antibody or antibody complex to selectively agglomerate insoluble carrier particles, wherein the antibody or antibody complex is native to an antigenic substance present in a liquid phase Is an antibody or antibody complex which does not substantially react and specifically reacts with an antigenic substance immobilized on an insoluble carrier, which is an agglutination immunoassay method. 2. An antibody or antibody complex that specifically reacts with an antigenic substance without removing the test sample by washing after adsorbing or binding the antigenic substance in the test sample to the insoluble carrier particles. The agglutination immunoassay method according to claim 1, wherein the body is reacted. 3. The agglutination immunoassay method according to claim 1, wherein the antibody is a monoclonal antibody. 4. The agglutination immunoassay method according to claim 1, wherein the antibody is a polyclonal antibody. 5. The agglutination immunoassay method according to claim 1, wherein the antibody complex comprises an antibody that specifically reacts with an antigenic substance and a second antibody that selectively reacts with the antibody. 6. The agglutination immunoassay method according to claim 1, wherein the antibody complex comprises a biotin-labeled antibody of an antibody that specifically reacts with an antigenic substance, and avidin. 7. The fine particles of an organic polymer substance, wherein the insoluble carrier particles are substantially insoluble in an aqueous liquid medium, and / or
Alternatively, the agglutination immunoassay method according to claim 1 or 2, which comprises fine particles of an inorganic substance. 8. The agglutination immunoassay method according to claim 1 or 2, wherein the insoluble carrier particles are surface-treated fine particles. 9. The insoluble carrier particles have an average particle size of 0.04 to
Latex particles in the 0.8 micron range.
Or the agglutination immunoassay method described in 2.