JP3287249B2 - Immunoassay method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a fibrous protein and a solid phase which are bound by using a linker reagent, and then the fibrous protein and an antigen or an antibody are further bound by using a linker reagent different from the linker reagent. The present invention relates to an immunoassay method for reacting the sample with a specimen and a labeled antigen or antibody, and then reacting with a fibrous protein degrading enzyme.

[0002]

2. Description of the Related Art In recent years, hormones, enzymes, serum proteins, tumor antigens, DNA-binding proteins, cytokines, cells, viruses, protozoa and the like contained in living organisms or antibodies against these have been measured, and the disease has been measured early in the disease. It is used for diagnosis and monitoring of treatment. In this measurement method, a solid phase to which an antigen or antibody is bound, a labeled antigen or antibody, and a sample are mixed to form an immune complex on the solid phase, and then a labeled substance bound to the solid phase is measured. Labeled immunoassays for detecting an object to be measured in a sample are widely used. A measurement method using a solid phase is known as a highly operable and highly sensitive measurement method because only a labeling substance bound to the solid phase by the bound / free (B / F) separation is selectively measured.

However, in this method, even if the solid phase is washed, the labeling substance non-specifically adsorbed on the solid phase cannot be removed, and the labeling substance must be distinguished from the labeling substance bound by forming an immune complex. Can not. As a result, the labeling substance due to non-specific adsorption raises the background during the measurement, lowering the S / N (signal / noise) ratio, and is a satisfactory result especially in the measurement of a low-concentration analyte. Was not obtained. Therefore, in order to selectively measure only a labeling substance bound by forming an immune complex on a solid phase, a reagent in which a bond that is cleaved by reduction, oxidation, or exchange reaction is formed in the immune complex is used. A method has been found in which only the labeling substance bound by the immune complex is released from the solid phase for measurement. Examples of the measuring method include a method of providing a disulfide bond (SS bond) in a reagent for forming an immune complex, a method of cross-linking dextran using a disulfide bond, and a method of biotin-binding.
A method of providing an avidin bond (JP-A-53-1313)
0424, JP-A-2-222837).

[0004]

In the method for cleaving the disulfide bond formed in the immune complex on the solid phase, it is difficult to selectively and completely decompose it because the cleavage is carried out by a reduction reaction. It can also have a profound effect on substances. In addition, in the method of decomposing dextran with a glycolytic enzyme, the reaction rate for decomposing modified sugar residues is slow, and a long reaction time is required for complete dissociation. Further, a method using a biotin-avidin bond is as follows.
An excess of biotin or avidin is added to the solution in which the immune complex has been formed, and the equilibrium reaction is shifted to one side and biotin-
In order to dissociate the avidin bond, it takes a long time to completely cleave, and there is a problem that the dissociation cannot be performed completely.

[0005]

Means for Solving the Problems Accordingly, as a result of intensive studies, the present inventors have found that after binding a fibrous protein and a solid phase using a linker reagent, the fibrous protein is further combined with an antigen or an antibody. After binding using a linker reagent different from the linker reagent and reacting the same with a specimen and a labeled antigen or antibody, a labeled substance released from the solid phase by reacting with a fibrous protein degrading enzyme is measured. An immunoassay method was found, and the present invention was completed.

In the measurement of the present invention, after the fibrous protein and the solid phase are bound using a linker reagent, the fibrous protein and the antigen or antibody are further bound using a linker reagent different from the linker reagent. A solid phase that has been used can be used. This fibrous protein includes animal connective tissue, bone,
For example, collagen present in teeth, gin zones, tendons, dermis, etc.
Gelatin and the like can be mentioned. The collagen and gelatin are not limited in molecular weight and properties as long as they are enzyme-degradable collagen or gelatin, and may be obtained from any animal (such as mammals, birds, and fish).
As gelatin, heat-treated collagen, for example, acid-treated gelatin produced by chemical treatment with acid or alkali,
Examples thereof include alkali-treated gelatin. Further, a collagen derivative or a gelatin derivative obtained by chemically modifying the collagen or gelatin by introducing a functional group such as an amino group, an imino group, a carboxyl group, a mercapto group, or a hydroxyl group using a known method can also be used.

[0007] Examples of the solid phase include various solid phases for immunoassay, for example, the inner wall of a plastic test tube, the inner wall of a microplate well, glass beads, plastic beads manufactured from polystyrene, cellulose, cellulose, and the like. Membrane such as nitrocellulose, ferrite particles (for example, JP-A-3-115862 and JP-A-7-921)
No. 68).

[0008] The antigen or antibody to be bound to the solid phase may be an antibody, an antigen or a derivative thereof against an object to be measured. The measurement object of the present invention includes, for example, drugs such as theophylline, phenytoin, valproic acid, low molecular weight antigens such as calcitonin, thyroxine, estrogen, elastradiol, CEA, AFP, ferritin, CA1
9-9, tumor-associated antigens such as CA125, HIV, ATL
A, HBV, HCV, viral antigens such as TP, TSH,
High molecular hormones such as insulin, IL-1, IL-2,
In addition to cytokines such as IL-6, various close factors such as EGF and PDGF, and the like, there are antibodies against the measurement object, antibodies against viral DNA and RNA, and the like.
Antibodies to be bound to the solid phase include polyclonal antibodies and monoclonal antibodies, as well as Fab, Fab ′, F (a) produced by enzymatic and / or reduction treatment of these antibodies.
b ′) It may be an antibody fragment such as ₂ .

Examples of the linker reagent include a maleimide reagent, glutaraldehyde, cyanuryl chloride, carbodiimide reagent and the like used for producing a reagent for immunoassay (Peptide synthesis method, Maruzen Co., 1975); Enzyme immunoassay, Kyoritsu Shuppan (1987); Protein nucleic acid enzyme, Supplement No. 31 (19
1987)). As the maleimide reagent, for example, N-
Succinimidyl maleimide acetic acid, N-succinimidyl
4-maleimidobutyric acid (GMBS), N-succinimidylmaleimidohexanoic acid, N-succinimidyl 4- (N
Carbodiimide reagents such as -maleimidomethyl) cyclohexane-1-carboxylic acid, N-sulfosuccinimidyl 4-maleimidomethyl-cyclohexane-1-carboxylic acid and the like, for example, dicyclohexylcarbodiimide (DCC), 1- (3-dimethylaminopropyl) -3
-Ethylcarbodiimide (EDC) and the like. In the carbodiimide reagent, a functional group can be condensed to form, for example, an amide group, an ester group, and the like, and bonded.

[0010] First, the fibrous protein and the solid phase are bound to the solid phase by a linker reagent. Subsequently, a solid phase used for measurement can be produced by reacting the antigen or antibody with a linker reagent different from the linker reagent. Further, the fibrous protein is reacted with a linker reagent having a divalent functional group to introduce a functional group, and then reacted with a solid phase using a linker reagent that does not react with the functional group.
It can be produced by reacting a functional group previously introduced into a fibrous protein with an antigen or an antibody. According to this method, the antigen or antibody can be selectively bound to the solid phase via the fibrous protein. In this production method, for example, after reacting a maleimide reagent with a fibrous protein, an amino group remaining in the fibrous protein and a carboxyl group of the solid phase are bound to form an amide group with a carbodiimide reagent, A method in which a maleimide group remaining in the dendritic protein is bonded to a thiol group of the antibody. Since the solid phase obtained by this method has a specific ratio of antigen or antibody bound to the solid phase, the solid phase is easily cleaved by a fibrous protein-degrading enzyme, and can be decomposed in a short time, thereby shortening the measurement time. Can be shortened.

On the other hand, a labeled antigen or antibody includes a labeling reagent used in a labeled immunoassay, and can be produced by binding a labeled substance to an antigen or antibody according to a well-known method. Examples of the label include an enzyme, a radioisotope, a fluorescent substance, a luminescent substance and the like used in the label immunoassay. Examples of the enzyme include peroxidase (POD), alkaline phosphatase (Alp), β-galactosidase and the like. As the radioisotope, for example, iodine 125,
As a fluorescent substance such as tritium, for example, rhodamine,
Examples of the luminescent substance such as umbelliferone include a luminol or isoluminol derivative, an acridinium ester derivative, and the like. In addition, examples of the antigen or antibody include an antigen, an antibody, or a derivative thereof corresponding to a measurement method to be performed or an object to be measured.
The labeled antigen or antibody can be produced using a method for forming a covalent bond or a non-covalent bond according to the method for producing the antigen or antibody-bound solid phase. Bolton-Hunter reagent can be used to label radioisotopes. Examples of the non-covalent bonding method include a physical adsorption method.

The immunoassay method of the present invention can be performed by a well-known one-step method, a one-step delay method, a two-step method or the like using the antigen- or antibody-bound solid phase and the labeled antigen or antibody, or a competitive method. It can be carried out by measuring only the label of the immune complex formed on the solid phase by the immune reaction. For example, to detect antigen 2
In the step method, after the solid phase and the sample containing the antigen are incubated in a buffer solution (for example, at 5 to 50 ° C. for 5 minutes to 1 day), the solid phase is washed with a washing solution. Next, the solid phase is transferred into a buffer containing a labeled antibody, and after further incubation (for example, 5 to 50 ° C., 5 minutes to 1 day), the solid phase is washed again. Next, a fibrous protein degrading enzyme is allowed to act on the immune complex formed on the solid phase to separate the solid phase, and then the label released in the solution is measured. This decomposition reaction can be carried out under the reaction conditions of the enzyme, usually at 4 ° C. to 42 ° C. for about 30 seconds to 10 minutes.

As the enzyme for decomposing a fibrous protein of the present invention, any enzyme that does not affect the label can be used, and examples thereof include collagenase and gelatinase. This degrading enzyme can be used alone or in combination.

In the measurement of a labeled substance, a radioactive isotope is measured by a radiation measuring device using the labeled substance, and luminescence,
Fluorescence, color development, and the like can be measured visually or by a measuring instrument such as a colorimeter, a fluorometer, a photon counter, a photosensitive film, or the like. Furthermore, the labeled substance is peroxidase (P
OD), an enzyme such as alkaline phosphatase (Alp), β-galactosidase (β-Gal), and the like, the enzyme activity of which is added to a luminescent substrate, a fluorescent substrate, a chromogenic substrate, and the like, and a reaction is performed, and the measurement is performed by the above-described measuring instrument. (See, for example, Eiji Ishikawa, “Enzyme Immunoassay” published by Medical Shoin). Examples of the sample used in the immunoassay of the present invention include:
Examples include body fluids such as whole blood, serum, plasma, urine, and lymph.

[0015]

EXAMPLES The present invention will be described in more detail with reference to Examples, Examples and Comparative Examples.

Reference Example 1 Preparation of CT02 Antibody (IgG) -Binding Particles 5 mg of 5% carboxylated ferrite particles obtained according to the method described in Example 4 of JP-A-3-115862 in 500 μl of 20 mM phosphate buffer (pH 4.5) Was dispersed, and 5 mg of a water-soluble carbodiimide was added thereto. After reacting at room temperature for 20 minutes, the supernatant was removed, and a CT-02 antibody (1 mg / ml, 20 mM
(Phosphate buffer, pH 4.5) (300 μl) was added and stirred. After 2 hours, the particles were washed with a 2% BSA solution (0.1 M Tris-HCl, 1 mM magnesium chloride, 0.1 mM zinc chloride, pH 7.5), dispersed in the BSA solution, and CT02 antibody (IgG) Bound particles were obtained.

Reference Example 2 Preparation of maleimidated gelatin A 6 mg / ml gelatin solution (manufactured by Miyagi Chemical Co., Ltd.) (0.1
M-phosphate buffer (pH 7.0) (1.3 ml) was added with N-succinimidyl-4-maleimidobutyric acid (GMBS, Dojindo Chemical) solution (20 mg / ml) (22 μl), and allowed to react at room temperature for 1 hour. .1M phosphate buffer (pH 4.
Unreacted GMBS was removed using a PD-10 column equilibrated in 5), and 1.5 ml of a maleimidated gelatin solution was removed.
1 was obtained.

Reference Example 3 Preparation of Maleimidated Gelatin Cross-Linked Particles 150 μl of the maleimidated gelatin solution prepared in Reference Example 2
and 5.0 mg of the above-mentioned carboxylated ferrite particles were suspended in the suspension and added to a water-soluble carbodiimide (Nacalai Co., Ltd., 1
50-22) 107 μl of an aqueous solution (10 mg / ml) was added. After stirring for 1 hour, 50 mM phosphate buffer (p
H7.0) three times to obtain maleimidated gelatin crosslinked particles.

Reference Example 4 Gelatin-crosslinked CT02 antibody (I
gG) Preparation of binding particles 1 mg of anti-calcitonin antibody (CT02 antibody) was added to 50 mM
The buffer was exchanged using a PD-10 column equilibrated with a sodium bicarbonate buffer (pH 8.0), and 1.4 m
18 μl of a g / ml iminothiolane solution was added. After reacting for 30 minutes at room temperature, 50 mM phosphate buffer (pH
Buffer exchange was performed using PD-10 equilibrated in 4.5) to remove unreacted iminothiolane to obtain iminothiolanated CT02 antibody. Iminothiolanated CT02 was added to 5 mg of the maleimidated gelatin crosslinked particles prepared in Reference Example 3.
300 μg of the antibody was added and stirred. After 2 hours, the particles were washed with a 2% BSA solution (0.1 M Tris-HCl, 1 mM magnesium chloride, 0.1 mM zinc chloride, pH 7.5), dispersed in the BSA solution and mixed with gelatin crosslinked C
The particles were T02 antibody (IgG) -bound particles.

Reference Example 5 Preparation of CT02 Antibody (Fab ') 20 mg of pepsin (Boehringer Mannheim, 108057) was added to 1 mg / ml of CT02 antibody (0.2 M sodium acetate buffer (pH 4.2)) and 1 ml, and the mixture was heated at 37 ° C. 1
Incubated for 0 hours. Adjust the pH to 7.0 beforehand with 0.1 M sodium phosphate, 1 mM EDT
A, Spadex G-200 equilibrated with 2Na buffer (pH 7.0), gel filtration column (Pharmacia, 1
The fraction eluted from 63 ml to 79 ml in 6/60) was concentrated by Centprep 3000 (Amicon), and concentrated.
(Ab ') _Two fractions each of 300 µg were obtained. 2-mercaptoethanolamine was added to 300 μg of this antibody to give 1
The mixture was incubated at 37 ° C for 2 hours at 0 mM. 0.1M sodium phosphate, 1mM EDTA,
2 Mercaptoethanolamine was removed with a PD-10 column equilibrated with 2Na buffer (pH 7.0) to remove CT0.
260 μg of Fab ′ fraction of two antibodies was obtained.

Reference Example 6 Gelatin cross-linked CT02 antibody (F
ab ') Preparation of binding particles Maleimidated gelatin crosslinked particles 5 m prepared in Reference Example 3
g in CT02-Fab 'fraction 100 prepared in Reference Example 5.
μg was added and stirred for 2 hours. Then the particles are reduced to 2%
Wash with BSA solution (0.1 M Tris-HCl, 1 mM magnesium chloride, 0.1 mM zinc chloride, pH 7.5)
This was dispersed in the BSA solution and gelatin-crosslinked CT02 was added.
Antibody (Fab ')-bound particles were obtained.

Reference Example 7 Alp-labeled CT08 / 0CT1
Preparation of Antibody Anti-calcitonin Antibody OCT Reacting with Human Calcitonin
2 mg of 1 or CT08 antibody 1 mg / ml (200 mM sodium acetate buffer (pH 4.2), Kansai New Technology Laboratory Co., Ltd.) was reacted with pepsin 40 μg at 37 ° C. for 10 hours, and purified by gel filtration column chromatography. As a result, 730 μg and 670 μg of the F (ab ′) ₂ fraction of the OCT1 or CT08 antibody were obtained. Further, each of the fractions was reduced with 2 mercaptoethanolamine in the same manner as in Reference Example 5, and Fab ′ of the OCT1 or CT-08 antibody was obtained.
Fractions were made. Next, alkaline phosphatase (Al
p) and GMBS were reacted to remove unreacted GMBS to obtain maleimidated alkaline phosphatase. This maleimidated alkaline phosphatase was added to the mixed solution of the Fab 'fractions of the OCT1 and CT-08 antibodies, and the mixture was reacted. The mixture was purified by gel filtration column chromatography to obtain an Alp-labeled CT08 / OCT1 antibody. It can be seen that the obtained Alp-labeled CT08 / OCT1 antibody has a molecular weight of 340,000 and is bound at a ratio of Alp: Fab ′ antibody = 1: 4.

Example 1 Measurement of Calcitonin by Collagenase Degradation 70 μl of the Alp-labeled CT08 / OCT1 antibody prepared in Reference Example 7 and 70 μl of a BSA solution containing 1 ng / ml calcitonin were mixed in a cartridge and incubated at 37 ° C. for 10 minutes. . After the reaction, 10
Reference Example 4 in which 0 μl was suspended at 0.06% in a BSA solution
37, and 62.5 μl of the gelatin-crosslinked CT02 antibody (Fab ′)-bound particles of Reference Example 6 or 62.5 μl of the CT02 antibody (IgG) -bound particles prepared in Reference Example 1. 10 at ℃
Incubated for minutes. The cartridge was brought into contact with a magnet to collect the particles, the supernatant was discarded, and the cartridge was washed. Then, 20 mM bistris-hydrochloric acid was added to the cartridge,
After adding 40 μl of 120 μg / ml collagenase (manufactured by Wako Pure Chemical Industries) diluted with 0.05% sodium azide and 1 mM calcium chloride (pH 8.0) buffer, the mixture was stirred and left at room temperature for 2 minutes. Further, a magnet was brought into contact with the cartridge to collect the particles, the particles and the supernatant were separated, and the supernatant was transferred to another cartridge. The luminescent substrate, 3- (4-methoxyspiro [1,2′-dioxetane-3,2′-), was added to each of the particles and the supernatant transferred to another cartridge.
Substrate solution containing (5′-chloro) tricyclo [3.3.1.1 ^3,7 ] decane] -4-yl) phenyl phosphate disodium salt (CSPD) at 200 μg / ml (0.1 M DEA-hydrochloric acid, 1 mM magnesium chloride,
(pH 10.0) was added, and the mixture was reacted at 37 ° C. for 5 minutes and measured with a photon counter. Blank value and S
Figures 1 to 3 show the results of measurement of labeled substances in the supernatant or particles together with the results of / N. In addition, FIG. 4 shows the results of measuring the rate at which Alp is released into the solution by reacting the above three types of particles with collagenase.

Reference Example 8 Preparation of SPDP-Conjugated Dextran 20 m diluted with 0.2 M phosphate buffer (pH 4.5)
125 μl of a 7.4 mg / ml sodium metaperiodate solution was added to 1 ml of the g / ml dextran solution, and the mixture was reacted at room temperature for 30 minutes while shielding light. Ethylene glycol 1
After adding 25 μl and further reacting at room temperature for 30 minutes,
Buffer exchange was performed using PD-10 equilibrated with 0.1 M sodium bicarbonate, 5% ethylenediamine buffer (pH 9.5). Next, sodium borohydride is added to a concentration of 1.2 mg / ml, and the mixture is reacted at 4 ° C. overnight. Next, acetic acid was added at a volume ratio of 1/65, and after stirring, the buffer was exchanged using PD-10 equilibrated with a 0.1 M phosphate buffer (pH 7.0), and 5.6 mg.
/ Ml of aminodextran / ml was obtained. 20 g of this aminodextran dissolved in DMF
g / ml of SPDP (444 μl) was added and reacted for 1 hour at room temperature. The buffer was exchanged using PD-10 equilibrated with 0.1 M phosphate buffer (pH 7.0), and unreacted SPD was added.
P was removed to obtain 4 mg / ml and 1.5 ml of SPDP-modified dextran.

REFERENCE EXAMPLE 9 Preparation of SPDP Dextran Crosslinked Particles 5 described in Examples of JP-A-3-115862 suspended at 0.5% in 0.1 M phosphate buffer (pH 7.0).
50 ml of a 20 mg / ml SPDP solution dissolved in DMF was added to 1 ml of the% aminosilanated ferrite particles, and the mixture was reacted at room temperature for 2 hours. After washing twice with ethanol and 0.1 M phosphate buffer (pH 7.0), 0.1 MDTT
Was added at 1/5 volume ratio and reacted at room temperature for 30 minutes.
After washing with a 0.1 M phosphate buffer (pH 7.0), 750 μl of SPDP-dextran prepared in Reference Example 6 was added, and the mixture was further reacted at room temperature for 30 minutes to prepare SPDP dextran crosslinked particles.

Reference Example 10 Preparation of AFP Antibody (Fab ') 20 μg of pepsin (Boehringer Mannheim, 108057) was added to 1 mg / ml anti-AFP antibody (0.2 M sodium acetate buffer (pH 4.2)) and 1 ml.
Incubated for 10 hours at ° C. The pH was adjusted to 7.0, and 0.1 M sodium phosphate, 1 mM E
The fraction eluted from 63 ml to 79 on a Superdex G-200, gel filtration column (Pharmacia, 16/60) equilibrated with DTA, 2Na buffer (pH 7.0) was concentrated by Centprep 3000 (Amicon). . F (ab ') ₂ fractions of anti-AFP antibody
0 μg was obtained. Furthermore, F (ab ') ₂ fraction of the anti-AFP antibody was added to 300 μg of 2 mercaptoethanolamine to make 10 mM and incubated at 37 ° C. for 2 hours. 0.1M sodium phosphate, 1mM EDT
A, PD-1 equilibrated with 2Na buffer (pH 7.0)
0 excludes 2 mercaptoethanolamine and Fab '
260 μg of the fraction was obtained.

Reference Example 11 Preparation of Dextran-Crosslinked Anti-AFP Antibody-Bound Particles 5 m of SPDP dextran crosslinked particles prepared in Reference Example 9
100 g of the anti-AFP antibody Fab 'fraction prepared in Reference Example 10 was added to the resulting mixture, and the mixture was stirred and reacted at room temperature for 2 hours. The particles were added to a 2% BSA solution (0.1 M Tris-HCl, 1 m
M magnesium chloride, 0.1 mM zinc chloride, pH7.
After washing in 5), this was dispersed in the BSA solution to obtain dextran-crosslinked anti-AFP antibody-bound particles.

REFERENCE EXAMPLE 12 Preparation of Gelatin Cross-Linked Anti-AFP Antibody-Bound Particles
100 g of the Fab 'fraction of the anti-AFP antibody prepared in Reference Example 10 was added to g, and the mixture was stirred for 2 hours. Thereafter, the particles were washed with a 2% BSA solution (0.1 M Tris-HCl, 1 mM magnesium chloride, 0.1 mM zinc chloride, pH 7.5), dispersed in the BSA solution, and gelatin-crosslinked anti-AFP antibody-bound particles. I got

Example 2 Measurement of AFP 0.015% gelatin cross-linked anti-AF prepared in Reference Example 12
Mix 10 μl of a sample containing 30 ng / ml AFP with 250 μl of P antibody-bound particles and place in a cartridge at 37 ° C.
For 10 minutes. Thereafter, a magnet was brought into contact with the cartridge to collect the particles, the supernatant was discarded, and the cartridge was washed.
Next, Reference Example 7 diluted to 0.3 μg / ml with a BSA solution
Alp-labeled anti-AFP antibody 250 prepared in the same manner as
μl was mixed and incubated at 37 ° C. for 10 minutes. The cartridge was brought into contact with a magnet to collect particles, the supernatant was discarded, and the cartridge was washed. Then, 40 μl of 120 μg / ml collagenase (Wako) diluted with 20 mM bistris-hydrochloric acid, 0.05% sodium azide, and 1 mM calcium chloride (pH 8.0) buffer was added to the cartridge.
Was added and, after stirring, left at room temperature for 2 minutes. Further, a magnet was brought into contact with the cartridge to collect the particles, the particles and the supernatant were separated, and the supernatant was transferred to another cartridge. A substrate solution (0.1 M) containing 200 µg / ml of CSPD as a luminescent substrate was added to each of the particles and the supernatant transferred to another cartridge.
DEA-HCl, 1 mM magnesium chloride, pH10.
0) was added and reacted at 37 ° C. for 5 minutes, and measured with a photon counter. The results are shown in FIGS. FIG. 8 shows the release ratio of Alp.

Reference Example 13 Measurement of AFP (Conventional Method) 30 ng / ml AF was added to 250 μl of the 0.015% dextran-crosslinked anti-AFP antibody-bound particles prepared in Reference Example 11.
Mix 10 μl of sample containing P
The reaction was performed at 7 ° C. for 10 minutes. Thereafter, a magnet was brought into contact with the cartridge to collect the particles, and the supernatant was discarded and washed. Next, 250 μl of the Alp-labeled AFP antibody prepared by the above method, diluted to 0.3 μg / ml with a BSA solution, was mixed and incubated at 37 ° C. for 10 minutes. The cartridge was brought into contact with a magnet to collect particles, the supernatant was discarded, and the cartridge was washed. Then, 20 mM bistris-hydrochloric acid, 0.05% sodium azide buffer (pH
After adding 40 μl of 120 μU / ml dextranase (manufactured by Wako Pure Chemical Industries, Ltd.) diluted in 6.5), and stirring, the mixture was stirred at room temperature for 2 hours.
Let stand for minutes. Further, a magnet was brought into contact with the cartridge to collect the particles, the particles and the supernatant were separated, and the supernatant was transferred to another cartridge. Each of the particles and the supernatant transferred to a separate cartridge was loaded with 200 μg / m of luminescent substrate CSPD.
250 µl of a substrate solution (0.1 M DEA-hydrochloric acid, 1 mM magnesium chloride, pH 10.0) containing
The reaction was performed for 5 minutes, and the measurement was performed using a photon counter. The results are shown in FIGS. Further, the release ratio of Alp is shown in FIG.
Shown in

[0031]

According to the immunoassay of the present invention, it is possible to perform measurement with a lower blank as compared with the conventional method, and a large improvement in S / N can be achieved. As a result, in the present invention, a low-concentration measurement target contained in a specimen can be measured with high sensitivity, and can be used for early diagnosis of various diseases, monitoring of treatment, and the like. In addition, since enzymatic degradation is performed in a shorter reaction time than the conventional method of crosslinking using dexlan,
Measurement in a short time is achieved.

[Brief description of the drawings]

FIG. 1 is a diagram showing signal values when calcitonin is measured using gelatin-crosslinked CT02 antibody (IgG) -bound particles, gelatin-crosslinked CT02 antibody (Fab ′)-bound particles, and CT02 antibody (IgG) -bound particles.

FIG. 2 is a diagram showing blank values when calcitonin is measured using gelatin-crosslinked CT02 antibody (IgG) -bound particles, gelatin-crosslinked CT02 antibody (Fab ′)-bound particles, and CT02 antibody (IgG) -bound particles.

FIG. 3 is a graph showing S / N values when calcitonin was measured using gelatin-crosslinked CT02 antibody (IgG) -bound particles, gelatin-crosslinked CT02 antibody (Fab ′)-bound particles, and CT02 antibody (IgG) -bound particles.

FIG. 4 shows the release ratio (release rate) of a labeled product when calcitonin is measured using gelatin-crosslinked CT02 antibody (IgG) -bound particles, gelatin-crosslinked CT02 antibody (Fab ′)-bound particles, and CT02 antibody (IgG) -bound particles. FIG.

FIG. 5 is a diagram showing a signal value when AFP is measured using gelatin-crosslinked anti-AFP antibody-bound particles and dextran-crosslinked anti-AFP antibody-bound particles.

FIG. 6 is a view showing blank values when AFP is measured using gelatin-crosslinked anti-AFP antibody-bound particles and dextran-crosslinked anti-AFP antibody-bound particles.

FIG. 7 is a graph showing S / N values when AFP is measured using gelatin-crosslinked anti-AFP antibody-bound particles and dextran-crosslinked anti-AFP antibody-bound particles.

FIG. 8 is a diagram showing the release ratio (release rate) of a labeled substance when AFP is measured using gelatin-crosslinked anti-AFP antibody-bound particles and dextran-crosslinked anti-AFP antibody-bound particles.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-63-24155 (JP, A) JP-A-4-204379 (JP, A) JP-A-55-156865 (JP, A) JP-A-5-205 43600 (JP, A) Special Table Hei 6-505802 (JP, A) (58) Fields surveyed (Int. Cl. ⁷ , DB name) G01N 33/547

Claims (4)

(57) [Claims] 1. A fibrous protein and a solid phase are bound using a linker reagent, and the fibrous protein and an antigen or antibody are further bound using a linker reagent different from the linker reagent. An immunoassay method comprising reacting a sample and a labeled antigen or antibody, and then reacting with a fibrous protein degrading enzyme. 2. The method according to claim 1, wherein the fibrous protein is collagen or gelatin. 3. The method according to claim 1, wherein the linker reagent is a maleimide reagent, glutaraldehyde, cyanuric chloride or carbodiimide reagent. 4. The method according to claim 1, wherein the degrading enzyme is gelatinase or collagenase.