JPH11290097A - Production of immunity-measuring reagent, immunity-measuring reagent, measurement of immunity and preservation of immunity-measuring reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a production method of an immunity-measuring reagent by which the reactivity of an antigen or an antibody is prevented from decreasing, and nonspecific autoagglutination is also prevented, and further to provide the immunity-measuring reagent, a method for measuring the immunity, and a method for preserving the immunity-measuring reagent. SOLUTION: This production method of an immunity-measuring reagent including an insoluble carrier carrying an antigen or an antibody comprises immobilizing the antigen or the antibody on the insoluble carrier in the presence of peptidylprolyl cis-trans isomerase prepared from bacteria belonging to the genus Methanococcus, or the peptidylprolyl cis-trans isomerase expressed by the DNA prepared from the bacteria belonging to the genus Methanococcus.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing an immunoassay reagent in which the reactivity of an antigen or an antibody is good and non-specific self-aggregation is prevented, an immunoassay reagent obtained thereby, and , An immunoassay method, and a method for storing an immunoassay reagent.

[0002]

2. Description of the Related Art Immunoassay reagents utilizing an antigen-antibody reaction with an insoluble carrier carrying an antigen or an antibody are widely used for measuring trace components in body fluids such as blood. As a method for producing such an immunoassay reagent, a method in which an antigen or an antibody is physically adsorbed on an insoluble carrier is generally used. That is, latex, a hydrophobic material such as a plastic plate, or an insoluble carrier such as erythrocytes treated with tannic acid, in an aqueous medium such as a buffer solution, physiological saline or purified water, and directly contact or adsorb the antigen or antibody. Then, the antigen or the antibody was supported on the insoluble carrier by immobilization by hydrophobic interaction.

[0003] However, when the immobilization of the antigen or antibody to the insoluble carrier is performed by a method utilizing such a hydrophobic interaction, the structure of the antigen or antibody may be changed depending on the conditions at the time of adsorption. In some cases, the reactivity of the antigen or the antibody is inactivated, or the insoluble carrier carrying the antigen or the antibody undergoes self-aggregation during storage, thereby significantly impairing the performance as a reagent.

[0004] In addition to the method utilizing hydrophobic interaction, there is also a method of immobilizing an antigen or an antibody on an insoluble carrier via a chemical bond or an affinity substance. , The reactivity may decrease, or the insoluble carrier may aggregate non-specifically in the suspension.

[0005] Japanese Patent Application Laid-Open No. 1-217266 discloses a method using casein or the like in order to prevent inactivation of antigen or antibody reactivity and to prevent nonspecific self-aggregation of an insoluble carrier. However, when casein is used as an inactivation inhibitor or a non-specific self-aggregation inhibitor, there is a problem that casein is adsorbed to endogenous peroxidase mixed into a body fluid, which is a sample, thereby hindering accurate measurement. there were. For example, when hemolyzed serum is used as a sample,
It sometimes adsorbed to glutathione peroxidase in red blood cells and interfered with the reaction.

[0006]

SUMMARY OF THE INVENTION In view of the above, the present invention provides a method for producing an immunoassay reagent in which a decrease in antigen or antibody reactivity and nonspecific self-aggregation are prevented, an immunoassay reagent obtained thereby, and It is another object of the present invention to provide an immunoassay method, and a method for storing an immunoassay reagent.

[0007]

The present invention relates to a method for producing an immunoassay reagent containing an insoluble carrier carrying an antigen or an antibody, wherein the antigen or the antibody is a peptidylprolyl prepared from a bacterium belonging to the genus Methanococcus. Cis-trans isomerase or D prepared from a bacterium belonging to the genus Methanococcus
This is a method for producing an immunoassay reagent to be carried on the insoluble carrier in the presence of peptidylprolyl cis-trans isomerase expressed from NA. Hereinafter, the present invention will be described in detail.

[0008] A protein such as an antigen or an antibody exerts its function by having a specific three-dimensional structure. However, when it is carried on an insoluble carrier, its steric structure may change depending on the interaction with the surface of the insoluble carrier or the bonding state with a functional group present on the surface of the insoluble carrier.
As a result, it is considered that the reactivity as an antigen or an antibody is reduced, and non-specific self-aggregation between insoluble carriers through a denatured portion of the antigen or the antibody is caused. The method for producing an immunoassay reagent of the present invention provides a peptidylprolyl cis-protein having an action of maintaining the three-dimensional structure of an antigen or antibody and / or an action of quickly repairing a structural change when the antigen or antibody is carried on an insoluble carrier. The use of trans isomerase (hereinafter also referred to as "PPIase") solves these problems.

The above-mentioned PPIase is an enzyme having an action of promoting the rotation of the N-terminal peptide bond of a proline residue which is a constituent amino acid in a polypeptide chain. The N-terminal peptide bond of this proline residue cannot rotate freely, and spontaneous cis-trans isomerization hardly occurs. As a result, the conversion of the protein chain in which the structural change has occurred to the normal structure is limited by the cis-trans isomerization reaction of proline and the like, and spontaneous regeneration is inhibited.

[0010] The PPIase promotes the rotation of the N-terminal peptide bond of the bromoline residue, so that it can promote the regeneration of the higher-order structure of the protein and can regenerate the denatured protein.

Examples of the PPIase include cyclophilin (protein that binds to cyclosporin A, which is an immunosuppressant) and FKB.
P (protein binding to FK506, an immunosuppressant)
Of the types, any PPIase can be used.

In the present invention, the PPIase is prepared from Methanococcus spp. Or expressed from DNA prepared from Methanococcus spp.

The above-mentioned genus Methanococcus belongs to the marine archaeal methane-producing bacterium and is a group of spheroid bacteria characterized by producing methane from hydrogen, carbon dioxide and formic acid. Based on their optimum growth temperature, they are classified into mesophilic and thermophilic bacteria, and the thermophilic bacteria include Methanococcus yannasii (Methanococcus).
ccus jannaschii, optimal temperature 85 ° C),
Methanococcus thermolystroficus (Metha
nococcus thermolithotroph
icus, optimal temperature 65 ° C; ATCC35097) 2
There are seeds.

The mesophilic bacteria whose optimum temperature is 20 to 40 ° C. include Methanococcus voltae (Methanococcus voltae).
ctus voltae; ATCC 33273), Methanococcus deltae (Methanococcus)
deltae; ATCC 35294), Methanococcus frisch (Methanococcus fr)
isius; ATCC 43340), Methanococcus maripardis (Methanococcus ma)
ripaludis; ATCC 43000), Methanococcus bannieri (Methanococcus)
vannielii; ATCC 35089).

The PPIase used in the present invention can be obtained by directly purifying the above-mentioned bacteria belonging to the genus Methanococcus, and the DNA encoding the PPIase of these organisms can be obtained.
It is preferable to clone the base sequence and prepare it by incorporating it into a fast-growing organism such as Escherichia coli or yeast, because mass production is possible.

The method for directly purifying the above-mentioned PPIase is not particularly limited, and for example, it can be obtained by the following method. The bacteria are cultured, for example, in a seawater-based medium (yeast extract 2 g, bactotryptone 2 g, sodium acetate 1 g, PIPES 1.7 g, DAB vitamin solution 10 mL / L seawater pH 6.8) under anaerobic conditions and hydrogen / carbon dioxide gas. 37 under aeration of a (4: 1) gas mixture.
Cultivate all day and night at ℃ to collect the cells. 25 collected cells
The cells were crushed by osmotic shock by suspending them in an mM phosphate buffer (pH 7.0), and the supernatant was collected by centrifugation.
Sepharose column: manufactured by Pharmacia, etc.), gel filtration column chromatography (Superrose)
A 12 HR column: manufactured by Pharmacia, etc.), an anion exchange column chromatography (MonoQ: manufactured by Pharmacia, etc.), etc., can collect the PPIase active fraction.

The obtained fraction can be confirmed to be a single fraction by SDS polyacrylamide gel electrophoresis. In the above purification, the PPIase activity can be confirmed by a coupling assay with an α-chymotrypsin reaction. For example, HEPE
A sample was injected into a cell using the peptide of SEQ ID NO: 1 in the sequence listing (final concentration: 17 μM) in S buffer (pH 7.8) as a substrate, incubated at 25 ° C., and the reaction was performed by adding a chymotrypsin solution. Start. The enzyme activity can be measured by monitoring the increase in absorption at 390 nm due to the release of pNA under stirring throughout. The peptide of SEQ ID NO: 1 in the above sequence listing was obtained under the trade name "N
-Suc-Ala-Ala-Pro-Phe-p-ni
A commercially available product of “troanilide” (manufactured by Sigma) is available.

D prepared from the above bacteria of the genus Methanococcus
As a method for expressing from NA, a conventional method such as a gene experiment protocol described in “Guide to Neoplastic Chemistry Experiment” (Chemical Doujinsha) or the like can be applied.

The DNA sequence of PPIase of the genus Methanococcus and the method for confirming the enzyme are described in, for example,
No. 91 and Japanese Patent Application No. 9-235999, which can be used. For example, a primer for PCR is prepared by synthesizing a partial base sequence so as to include an appropriate restriction enzyme site. Next, it is amplified by PCR using the genomic DNA of the genus Methanococcus as a template. As the genomic DNA, Japanese Patent Application No. Hei 9
-350623 prepared according to the method of Methanococcus voltae
itae) and the like. P obtained from PCR product
The PIase gene is inserted into a plasmid vector containing an appropriate resistance marker, and the resulting plasmid is used to transform fast-growing organisms such as Escherichia coli and yeast. The transformant is cultured and purified from the cultured cells by a conventional method such as hydrophobic chromatography and ion exchange chromatography.
You can get ase.

Among the above Methanococcus spp., Methanococcus voltae (Methanococcus) is preferred.
voltae) are preferred.
Methanococcus voltae (Methanococ)
cus voltae),
Other PPIases have not only the peptidylproline cis-trans isomerization action and the polypeptide chain folding action, but also the irreversible aggregation inhibitory action of chaperonin-like proteins. large. Methanococcus voltae (Meth
(Anococcus voltae) is also a mesophilic bacterium, and thus PPIase derived from the bacterium has an effect of stabilizing an antigen or an antibody in a wide range of 10 to 40 ° C.

The above Methanococcus spp., Especially Methanococcus voltae (Methanococcus vol.)
tae) has 7 to 8 types of PPIases like Escherichia coli and does not interact with each other in a complicated manner, and has only one type of PPIase, so that versatility to proteins is high. That is, it has the effect of stabilizing many types of antigens or antibodies.

The above-mentioned PPIase has been commercialized, or has been produced by Methanococcus voltae (Methanoc).
Occus voltae) -derived PPIase has established a production method by genetic recombination, and any of them can be stably supplied. The commercially available PPIases include calf thymus-derived and human-derived Cyclophilin (manufactured by Sigma), and human-derived FK-BINDING PROTEIN (manufactured by Sigma).
And the like.

In the present invention, in order to prevent the inactivation of antigen or antibody and to improve the rate of inhibiting non-specific self-aggregation of the insoluble carrier, it is also possible to combine protein disulfide-isomerase and the like in combination. . The protein disulfide-isomerase is an enzyme that performs a protein folding reaction by promoting the recombination of disulfide bonds of the protein.

The method for producing an immunoassay reagent of the present invention comprises carrying an antigen or antibody on an insoluble carrier in the presence of the above-mentioned PPIase. The insoluble carrier is not particularly limited as long as it has a hydrophobic surface in whole or in part, and examples thereof include materials made of synthetic polymers such as latex, plastic beads, and plastic plates; inorganic materials such as silica; Acid-treated red blood cells;
Polymer membranes such as nitrocellulose are exemplified. Especially,
Since it can be mass-produced with industrially stable quality, plastic molded products such as latex, plastic beads, plastic plates and the like are preferably used.

The insoluble carrier may have various functional groups for the purpose of chemical bonding on its surface. Further, it may have a spacer or the like for immobilizing a substance utilizing affinity of avidin / biotin or the like.

The insoluble carrier is preferably made of latex from the viewpoint that the inspection time can be reduced and labor can be saved by fully automatic processing of the inspection. The latex is not particularly limited, and examples thereof include polystyrene and synthetic rubber. More preferably, it is a polystyrene type. The average particle size of the insoluble carrier comprising the latex is preferably 0.05 to 2.0 μm. More preferably, it is 0.1 to 0.5 μm.

The method for producing an immunoassay reagent of the present invention is carried out, for example, as follows. First, an antigen or antibody is diluted with a suitable buffer, and this is used as an antigen or antibody solution. The antigen or antibody can be immobilized on the insoluble carrier by bringing the antigen or antibody solution and the insoluble carrier into contact with each other in an aqueous medium containing the PPIase and incubating for a predetermined time.

As the above-mentioned aqueous medium, an optimal buffer for the antigen or antibody to be used is appropriately selected. For example, a phosphate buffer, a Tris buffer, a Good buffer, a glycine-based buffer and the like are generally used. The buffer used is preferably used. The ionic strength of the buffer solution may be an optimal strength for the antigen or antibody used. The pH of the buffer is preferably 4.5 to 9.0.

The molar concentration of the PPIase in the aqueous medium is 0.05 to 5 times the molar concentration of the antigen or antibody.
00 times is preferred. If it is less than 0.05 times, the expected effect cannot be obtained, and if it exceeds 500 times, specific aggregation may be prevented.

The present invention 2 relates to an immunoassay reagent obtained by the method for producing an immunoassay reagent of the present invention 1. Since the immunoassay reagent of the present invention 2 is a highly sensitive reagent in which a decrease in antigen or antibody reactivity and nonspecific self-aggregation are prevented, for example, a radioimmunoassay (RIA), a fluorescence immunoassay (FI
A), enzyme immunoassay (EIA or ELISA), latex agglutination, hemagglutination, etc.

The present invention 3 relates to an immunoassay method utilizing an antigen-antibody reaction with an insoluble carrier carrying an antigen or an antibody, wherein the antigen-antibody reaction is carried out in the presence of PPIase. As the PPIase, those described in the present invention 1 can be suitably used.

As described in detail in the description of the present invention 1, the PPIase promotes the rotation of the peptide bond on the N-terminal side of the proline residue, and thus promotes the regeneration of the higher-order structure of the protein, It is an enzyme that can be regenerated. In the immunoassay method of the third invention, the antigen-antibody reaction is carried out in the presence of the PPIase, thereby quickly recovering the steric structural change of the antigen or antibody supported on the insoluble carrier, thereby improving the reactivity of the antigen or antibody. It is intended to prevent the reduction or non-specific self-aggregation of insoluble carriers through the denatured portion of these antigens or antibodies.

The immunoassay according to the third aspect of the present invention is performed, for example, by including the above-mentioned PPIase in a reaction solution in which an antigen-antibody reaction is performed. The aqueous medium exemplified in the present invention 1 can be used as the reaction solution.

The molar concentration of the PPIase in the reaction solution is preferably from 0.01 to 500 μM. 0.0
If it is less than 1 μM, the expected effect cannot be obtained and 50
If it exceeds 0 μM, specific aggregation may be prevented.

The present invention 4 relates to a method for storing an immunoassay reagent utilizing an antigen-antibody reaction with an insoluble carrier carrying an antigen or an antibody, and a method for storing an immunoassay reagent stored in the presence of PPIase. As the PPIase, those described in the present invention 1 can be suitably used.

The method for preserving the immunoassay reagent of the present invention 4 is a method for preserving an immunoassay reagent utilizing an antigen-antibody reaction with an insoluble carrier carrying an antigen or an antibody. By storing it under, by maintaining the three-dimensional structure of the antigen or antibody carried on the insoluble carrier or by quickly repairing the structural change, it is possible to reduce the reactivity of the antigen or antibody or to modify the denatured portion of these antigens or antibodies. This prevents non-specific self-aggregation of insoluble carriers through the carrier.

The method for storing the immunoassay reagent of the present invention 4 is, for example, the method of
It can be performed by storing in a storage solution containing Iase. The aqueous medium exemplified in the present invention 1 can be used as the preservation solution. The P in the storage solution
As the molar concentration of PIase, the molar concentration exemplified in the present invention 3 can be suitably used.

[0038]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Reference Example 1 Preparation of PPIase Methanococcus voltae prepared according to the method of Japanese Patent Application No. 9-350623.
PCR was performed using the genomic DNA of S. voltae) as a template and the primers of SEQ ID NOs: 2 and 3 in the Sequence Listing. PCR products were converted to pT7 blue vector (N
ovagen), transformed it into E. coli JM109,
g / mL ampicillin-containing LB medium plate (10 g of tryptone, 5 g of yeast extract, 10 g of NaCl)
/ LL (pH 7.0) all day and night at 37 ° C.
A library of R products was made. This is Hybond
Transferred to o-N ⁺ membrane (Amersham). DIG-DNA detection using the digoxigenin-labeled gene used in the PPIase gene screening described in Japanese Patent Application No. 10-91 as a probe.
kit (Boehringer Mannheim) using M. kit. vo
Itae-derived PPIase gene was screened. The plasmid was recovered from the obtained positive clone and
The inserted sequence was excised by col and BamHI and recovered by electrophoresis.

This was ligated to an expression vector pET-11d (manufactured by Stratigen) which had been treated with the same restriction enzymes. After that, this is BL21 (DE
3) Transformed into a strain (manufactured by Stratigen), and LB medium plate containing 100 μg / mL of ampicillin (10 g of tryptone, 5 g of yeast extract, 5 g of Na extract)
After culturing at 10 g / L of Cl (pH 7.0)), colonies were picked up and used as glycerol stock.

Preparation of PPIase from recombinant Escherichia coli An LB medium containing 100 μg / mL ampicillin (10 g of tryptone, 5 g of yeast extract, 10 g of NaCl / 1 L)
(PH 7.0) 200 mL was inoculated with one ear of BL21 (DE3) strain (recombinant Escherichia coli) carrying the above expression plasmid, and precultured at 37 ° C for 16 hours. This culture solution was inoculated into 6 liters of the above LB medium, and main culture was performed using a jar fermenter. Three hours after the start of the main culture, I
PTG (manufactured by Wako Pure Chemical Industries, Ltd.) was added to a concentration of 1 mM, and the culture was further continued for 4 hours. After completion of the culture, the bacteria were collected by centrifugation and stored frozen at -30 ° C. Next, the recovered recombinant Escherichia coli was suspended in a 50 mM phosphate buffer (pH 7.5) and disrupted by sonication. The supernatant was collected by centrifugation, and ammonium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the supernatant to 80% saturation, and salting out was performed. After 30 minutes, the precipitate collected by centrifugation was dissolved in a 50 mM phosphate buffer (pH 7.8) containing 1.8 M ammonium sulfate. This solution was subjected to separation and purification of PPIase using high performance liquid chromatography, and the eluate containing the enzyme was desalted by dialysis and used in the following Examples.

Example 1 1) Reagents and Materials Anti-human CEA monoclonal antibody; anti-human CEA monoclonal antibody (Clone No. CEA4-G67;
IgG-1) was used. Antibody dilution buffer: 50 mM sodium phosphate buffer (pH 7.4) was used. Latex; average particle size 0.3
0 μm polystyrene latex (solid content 10% (W /
V), manufactured by Sekisui Chemical Co., Ltd.). Latex dilution buffer: 50 mM sodium phosphate buffer (pH 7.50) was used. Blocking buffer; bovine serum albumin (Bovi) in 100 mM sodium phosphate buffer (pH 7.40)
ne serum albumin, Fraction
V, Reagent Grade, Miles Co
rp. 1% (W / V) and N
0.1% of aN ₃ (reagent grade, Nacalai Tesque)
(W / V) was used. CEA standard; CEA standard purified from human pooled serum (WHO standard, 90890 IU / mL, 11
0.0 μg / mL), 2000 in physiological saline,
They were used after being diluted to 500, 100, 20, 10, and 5 ng / ml, respectively. In addition, a physiological saline containing no CEA standard was set to 0 ng / ml. Diluent for sample dilution (R1 solution); polyethylene glycol 6000 (average molecular weight 75
00, manufactured by Wako Pure Chemical Industries, Ltd.) to a concentration of 3% (W / V).

2) Method (1) Preparation of CEA measurement reagent To 9 parts of a latex diluting buffer was added and diluted with 1 part of polystyrene latex having an average particle diameter of 0.30 μm (solid content: 10% (W / V)). A 1.0% latex solution was used. Anti-C
EA antibody (Clone No. CEA4-G67)
Protein concentration of 12.5 μg / ml (about 0.08 μM)
To obtain an antibody diluent. The PPIase shown in Reference Example 1 was prepared to a concentration of 260 μg / mL (about 8 μM) using a latex dilution solution, and 20 μL of this solution was added to 2 mL of the antibody diluent to prepare an antibody sensitizing solution (antibody: PPI The molar ratio of ase is 1: 1).

1.0% (W / V) latex liquid 600 μm
While stirring L in a 25 ° C. incubator with a magnetic stirrer, 1200 μl of the antibody sensitizing solution was quickly added, followed by stirring at 25 ° C. for 1 hour. Thereafter, 3.0 mL of a blocking buffer was added, and the mixture was continuously added at 25 ° C. for 2 hours.
Stirred for hours. Thereafter, the mixture was centrifuged at 15 ° C. and 15000 rpm for 15 minutes. 4.0 mL of a blocking buffer was added to the obtained precipitate, and the precipitate was washed by centrifugation in the same manner. The washing operation was performed three times. 1.8 mL of a blocking buffer was added to the precipitate, and the mixture was stirred well, and then subjected to a dispersion treatment with an ultrasonic crusher. Further, 1.8 mL of a blocking buffer was further added to obtain a CEA measurement reagent [1] having a solid content of 0.17% (W / V). CEA measurement reagent [1] thus prepared
Was stored at 4 ° C.

(2) Measurement of CEA amount The CEA amount was measured using an automatic analyzer for biochemistry Model 7150 (manufactured by Hitachi, Ltd.). The CEA measuring reagent [1] having a solid content of 0.17% (W / V) obtained in the above (1) was used as R2 solution (solid content 0.17% (W / V)). The measurement conditions were as follows.

Sample volume 20 μL Sample diluent (R1 solution) 210 μL Reagent (R2 solution) 30 μL Measurement wavelength 700 nm Measurement temperature 37 ° C.

The difference between the absorbance at about 80 seconds after the addition of the reagent (R2 solution) and the absorbance at about 320 seconds (ΔOD570)
Was measured, and a value obtained by multiplying the difference in absorbance by 10,000 times was defined as an absorbance change amount. The measurement was performed using a CEA standard product of a known concentration as a sample, and a calibration curve was prepared. The results are shown in Table 1 and FIG.

Example 2 A 2.6 m PPIase solution was added to 2 mL of the antibody diluent.
Preparation and measurement of the reagent were carried out in the same manner as in Example 1 except that g / mL (about 80 μM) and 20 μL were used (antibody: PPIase = 1: 10). The results are shown in Table 1 and FIG.

Example 3 2.6 mg / m of PPIase solution added to the antibody diluent
L, and the preparation and measurement of the reagent were performed in the same manner as in Example 1 except that L and 100 μL were used (antibody: PPIase =
1:50). The results are shown in Table 1 and FIG.

Comparative Example 1 A reagent was prepared and measured in the same manner as in Example 1 except that this solution was used as an antibody sensitizing solution without adding PPIase to the antibody diluent. The results are shown in Table 1 and FIG.

[0051]

[Table 1]

Examples 4 to 6 and Comparative Example 2 Storage stability of reagent In order to examine the storage stability of the prepared latex reagent,
The reagents prepared in Examples 1 to 3 and Comparative Example 1 were
A deterioration promotion test for storing for a week was performed, and measurement was performed using a CEA standard product of a known concentration to prepare a calibration curve. The respective results are shown in Table 2 and FIG.

[0053]

[Table 2]

Examples 7 to 9 and Comparative Example 3 Inhibition of Nonspecific Self-Aggregation In order to examine whether or not nonspecific self-aggregation was formed during storage of the prepared latex reagent, dispersibility was examined. After the preparation of the reagents in Examples 1 to 3 and Comparative Example 1, 3
The reagent stored at 7 ° C. for the period shown in Table 3 was measured at 610 nm / 700 nm using a spectrophotometer. The respective results are shown in Table 3 and FIG.

[0055]

[Table 3]

Examples 10 to 12 and Comparative Example 4 Specificity test In order to examine the specificity of the latex reagents prepared, the reagents prepared in Examples 1 to 3 and Comparative Example 1 were used to test the high RF serum and strong milk beads. The amount of CEA in the serum was measured. The calibration curves shown in Table 1 were used. For comparison, the same sample was measured by RIA using CEA rear beads (manufactured by Dynabot). Table 4 shows the results.

[0057]

[Table 4]

Example 13 1) Reagents and Materials The same reagents as in Example 1 were used.

2) Method (1) Preparation of Reagent for CEA Measurement To 9 volumes of a latex dilution buffer was added and diluted with 1 volume of polystyrene latex (solid content: 10% (W / V)) having an average particle size of 0.30 μm. A 1.0% latex solution was used. Anti-C
EA antibody (Clone No. CEA4-G67)
Protein concentration of 12.5 μg / mL (about 0.08 μM)
To obtain an antibody sensitizing solution. 600% of 1.0% (W / V) latex liquid
Was stirred in a 25 ° C. incubator with a magnetic stirrer, and 1200 μL of the antibody sensitizing solution was quickly added thereto, followed by stirring at 25 ° C. for 1 hour. Thereafter, 3.0 mL of a blocking buffer was added, and the mixture was continuously stirred at 25 ° C. for 2 hours. Then, at 15 ° C and 15000 rpm, 1
Centrifuged for 5 minutes. 4.0 mL of a blocking buffer was added to the obtained precipitate, and the precipitate was washed by centrifugation in the same manner. The washing operation was performed three times. 1.8 mL of a blocking buffer was added to the precipitate, and the mixture was stirred well, and then subjected to a dispersion treatment with an ultrasonic crusher. 0.32 mg of the PPIase prepared in Reference Example 1 was added to this solution.
/ ML (10 μM) was added to 1.8 mL of the solution added to the blocking buffer, and the solid content was 0.17% (W
/ V) was used as the CEA measurement reagent [2] (P in storage solution)
The PIase concentration will be 5 μM). The CEA measuring reagent [2] thus prepared was stored at 4 ° C.

(2) Measurement of CEA amount The CEA amount was measured using an automatic analyzer for biochemistry Model 7150 (manufactured by Hitachi, Ltd.). The CEA measuring reagent [2] having a solid content of 0.17% (W / V) obtained in the above (2) was used as R3 solution (solid content of 0.17% (W / V)). The measurement conditions were as follows.

Sample volume 20 μL Sample diluent (R1 solution) 210 μL Reagent (R3 solution) 30 μL Measurement wavelength 700 nm Measurement temperature 37 ° C.

The difference between the absorbance approximately 80 seconds after the addition of the reagent (R3 solution) and the absorbance approximately 320 seconds later ($ OD570)
Was measured, and a value obtained by multiplying the difference in absorbance by 10,000 times was defined as an absorbance change amount. The measurement was performed using a CEA standard product of a known concentration as a sample, and a calibration curve was prepared. The results are shown in Table 5 and FIG.

Example 14 Preparation and measurement of a reagent were performed in the same manner as in Example 13 except that the PPIase concentration of the PPIase solution added to the latex solution after the dispersion was changed to 1.6 mg / mL (50 μM). (PPIase concentration in the stock solution was 25 μM
Becomes). The results are shown in Table 5 and FIG.

Example 15 The concentration of the sample diluent (R1) was 0.064 mg / mL (2
The preparation and measurement of the reagent were carried out in the same manner as in Example 13 except that PPIase was added so that the concentration became (μM).
The results are shown in Table 5 and FIG.

Comparative Example 5 A reagent was prepared and measured in the same manner as in Example 13 except that PPIase was not added to the blocking buffer added to the latex solution after the dispersion treatment. The results are shown in Table 5 and FIG.

[0066]

[Table 5]

Examples 16 to 18 and Comparative Example 6 Storage Stability of Reagent The storage stability of the reagent prepared in Examples 13 to 15 and Comparative Example 5 was measured in the same manner as in Example 4. The results are shown in Table 6 and FIG.

[0068]

[Table 6]

Examples 19 to 21 and Comparative Example 7 Inhibition of Nonspecific Self-Aggregation The presence or absence of nonspecific self-aggregation of the reagents prepared in Examples 13 to 15 and Comparative Example 5 in the same manner as in Example 7. Was examined. The results are shown in Table 7 and FIG.

[0070]

[Table 7]

Examples 22 to 24 and Comparative Example 8 Specificity test The reagents prepared in Examples 13 to 15 and Comparative example 5 were subjected to a specificity test in the same manner as in Example 10. The results are shown in Table 8.

[0072]

[Table 8]

As is evident from FIGS. 1, 2, 4 and 5,
The calibration curve obtained according to the present invention shows good linearity from low to high concentrations of CEA, and the sensitivity hardly decreases even when a deterioration promotion test at 37 ° C. is performed.
It was confirmed that the decrease in the activity of the antigen-antibody reaction was suppressed. In addition, as is clear from FIGS. 3 and 6, it was confirmed that non-specific self-aggregation occurring during storage was suppressed. In addition, even in high RF serum or strong milk serum,
The measurement results were almost the same as those of the RIA method, and it was confirmed that the specificity was good.

[0074]

Since the method for producing an immunoassay reagent of the present invention has the above-mentioned constitution, it is possible to obtain an immunoassay reagent in which the reactivity of an antigen or an antibody is reduced and non-specific self-aggregation of an insoluble carrier is prevented. it can. Further, according to the method for determining the immunity of the present invention and the method for storing the immunoassay reagent of the present invention, it is possible to suppress the non-specific self-agglutination of the insoluble carrier and the decrease in the reactivity of the antigen or antibody, A highly sensitive measurement of an immune reaction can be performed.

[0075]

[Sequence list]

SEQ ID NO: 1 Sequence length: 4 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence characteristics Location: 1 Other information: Xaa = N-succinyl group at N-terminal (N-su
alanine modified by c) Location: 3 Other information: Xaa = Ala or Leu Location: 4 Other information: Xaa = C-terminal pNA (p-nitroanilide)
Phenylalanine sequence modified with Xaa Ala Xaa Xaa 1

SEQ ID NO: 2 Sequence length: 29 Sequence type: number of nucleic acid strands: single strand Topology: linear Sequence type: other nucleic acid, synthetic DNA sequence GSCCNWTGGB GATAANGCHA TCCGNCCGC 29

SEQ ID NO: 3 Sequence length: 30 Sequence type: number of nucleic acid strands: single strand Topology: linear Sequence type: other nucleic acid, synthetic DNA sequence ATGNCSTCCA TCCGYATGCG ATGCNGGTCW 30

[Brief description of the drawings]

1 is 4 ° C. after preparation in Examples 1 to 3 and Comparative Example 1.
FIG. 4 is a diagram showing the relationship between the concentration of a CEA standard and the amount of change in absorbance due to the reagent stored for one day. The vertical axis represents the amount of change in absorbance, and the horizontal axis represents the concentration of CEA (ng / mL).

FIG. 2 37 after preparation in Examples 4 to 6 and Comparative Example 2.
FIG. 4 is a diagram showing the relationship between the concentration of a CEA standard and the change in absorbance with a reagent stored at 2 ° C. for 2 weeks. The vertical axis represents the amount of change in absorbance, and the horizontal axis represents the concentration of CEA (ng / mL).

FIG. 3. 37 after preparation in Examples 7-9 and Comparative Example 3.
FIG. 4 is a diagram showing the dispersibility of a reagent stored at a temperature of ° C. The vertical axis is 6
The ratio between the absorbance at 10 nm and the absorbance at 700 nm is shown, and the horizontal axis is the number of days of storage at 37 ° C.

FIG. 4 is a graph showing the relationship between the concentration of a CEA standard and the amount of change in absorbance of a reagent stored at 4 ° C. for 1 day after preparation in Examples 13 to 15 and Comparative Example 5. The vertical axis represents the amount of change in absorbance, and the horizontal axis represents the concentration of CEA (ng / mL).

FIG. 5 is a graph showing the relationship between the concentration of a CEA standard product and the amount of change in absorbance of a reagent stored at 37 ° C. for 2 weeks after preparation in Examples 16 to 18 and Comparative Example 6. The vertical axis is the amount of change in absorbance, and the horizontal axis is the concentration of CEA (ng / mL).
It is.

FIG. 6 is a diagram showing the dispersibility of reagents stored at 37 ° C. after preparation in Examples 19 to 21 and Comparative Example 7. The vertical axis represents the ratio of the absorbance at 610 nm to the absorbance at 700 nm, and the horizontal axis represents the number of days stored at 37 ° C.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Mie Matsumoto 2-1 Hyakuyama, Shimamoto-cho, Mishima-gun, Osaka Sekisui Chemical Co., Ltd.

Claims (10)

[Claims] 1. A method for producing an immunoassay reagent comprising an insoluble carrier carrying an antigen or an antibody, wherein the antigen or the antibody is a peptidylprolyl cis-trans isomerase prepared from a bacterium belonging to the genus Methanococcus, A method for producing an immunoassay reagent, wherein the reagent is supported on the insoluble carrier in the presence of peptidylprolyl cis-trans isomerase expressed from DNA prepared from a bacterium belonging to the genus Coccus. 2. The method according to claim 1, wherein the genus Methanococcus is Methanococcus volta.
The method for producing an immunoassay reagent according to claim 1, which is e). 3. The method for producing an immunoassay reagent according to claim 1, wherein the insoluble carrier is latex. 4. An immunoassay reagent obtained by the method for producing an immunoassay reagent according to claim 1, 2 or 3. 5. An immunoassay method using an antigen-antibody reaction with an insoluble carrier carrying an antigen or an antibody, wherein the antigen-antibody reaction is performed using peptidylprolyl cis-trans isomerase prepared from a bacterium belonging to the genus Methanococcus, or
An immunoassay method, which is performed in the presence of peptidylprolyl cis-trans isomerase expressed from DNA prepared from a Methanococcus bacterium. 6. The bacterium belonging to the genus Methanococcus, Methanococcus voltae (Methanococcus volt).
The immunoassay according to claim 5, which is ae). 7. The immunoassay according to claim 5, wherein the insoluble carrier is latex. 8. A method for preserving an immunoassay reagent utilizing an antigen-antibody reaction with an insoluble carrier carrying an antigen or an antibody, comprising the steps of: peptidylprolyl cis-trans isomerase prepared from a bacterium belonging to the genus Methanococcus; A method for storing an immunoassay reagent, comprising storing in the presence of peptidylprolyl cis-trans isomerase expressed from DNA prepared from a bacterium. 9. The bacterium belonging to the genus Methanococcus, Methanococcus voltae.
The method for storing an immunoassay reagent according to claim 8, which is ae). 10. The method for storing an immunoassay reagent according to claim 8, wherein the insoluble carrier is latex.