JP3333569B2 - Immunoassay method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoagglutination assay which is frequently used in clinical test reagents and the like.

[0002]

2. Description of the Related Art Immunoturbidimetry, which is one of immunoassays, optically detects and measures the change in turbidity when an antigen and an antibody bind by an antigen-antibody reaction to form an immune complex. How to When an antigen or antibody to be measured is continuously quantified using an automatic analyzer by immunoturbidimetry, an interval phenomenon occurs in which non-specific agglutination becomes stronger when measurement is interrupted and then resumed. Therefore, an incorrect measurement value may be given.

[0003] In order to prevent non-specific agglutination in immunoturbidimetry, a method of chemically modifying an antibody against the antigen to be measured with an acylating agent (Japanese Patent Publication No. 2-166161) has been proposed. There is known a method of binding proteins (JP-A-3-14865) and a method of adjusting the pH of a reaction solution to 8.5 or more (JP-A-1-15857).

[0004]

[Problems to be solved by the invention]

[0005] An object of the present invention is to react non-specific agglutination, especially continuous antibody, by reacting an antibody to the antigen to be measured or an antigen to the antibody to be measured with the antigen to be measured or the antibody to be measured in a negatively charged state in the reaction solution. It is an object of the present invention to provide a simple immunoagglutination measurement method without any interval phenomenon during the measurement.

[0006]

According to the present invention, an antibody to a measurement antigen or an antigen to a measurement target antibody is negatively charged and reacted with a measurement target antigen or a measurement target antibody in a sample. The present invention relates to an immunoagglutination assay for an antigen or an antibody, which comprises measuring a change in the aggregation of an antibody.

[0007] The immunoagglutination assay in the present invention may be any method in which an antigen molecule and an antibody molecule bind by an antigen-antibody reaction to form a large immune complex and the complex is quantified. In which an antibody to the antigen to be measured or an antigen to the antibody to be measured is bound to a carrier. Examples of the method of binding to a carrier include a latex agglutination turbidimetric method and a hemagglutinating method.

The antigen to be measured or the antibody to be measured in the present invention includes carcinoembryonic antigen (CEA), immunoglobulin (IgG, IgA, IgM, IgD, IgE), complement (C3, C4, C5, C1q). , C-reactive protein (CR
P), α ₁ -antitrypsin, α ₁ -microglobulin, β ₂ -microglobulin, haptoglobulin,
Transferrin, ceruloplasmin, ferritin, albumin, hemoglobin A ₁ , hemoglobin A _1C , myoglobin, myosin, dupan-2, α-fetoprotein (AFP), tissue polypeptide antigen (TPA), apolipoprotein A ₁ , apolipoprotein B, apolipoprotein Protein C ₂ ,
Apolipoprotein C ₃ , Apolipoprotein E, Rheumatoid factor, Anti-streptolysin O (ASO), Fibrin degradation product (FDP), Fibrin degradation product D fraction (FDP-
D), fibrin degradation product DD fraction (FDP-DD)
imer), fraction of fibrin degradation product E (FDP-
E), proteins such as antithrombin-III (AT-III), amylase, prostatic acid phosphatase (PA
P), nerve-specific enolase (NSE), fibrinogen, elastase, plasminogen, enzymes such as creatine kinase myocardium (CK-MB), insulin, thyroid stimulating hormone (TSH), 3,5,3'-triiodosilo Hormones such as nin (T ₃ ), thyroxine (T ₄ ), adrenocorticotropic hormone (ACTH), growth hormone (GH), luteinizing hormone (LH), hepatitis B virus-related antibodies, hepatitis B virus-related antigens, Hepatitis C virus antibody, HTLV (adult T cell leukemia virus) antibody, H
Antibodies to various infectious disease-causing viruses, such as an IV (AIDS virus) antibody, chlamydia antibody, syphilis antibody, and toxoplasma antibody.

Antibodies to the antigen to be measured include antibodies capable of reacting to normal antigens, such as polyclonal antibodies to the antigen to be measured and monoclonal antibodies to the antigen to be measured [“Single Coulomb Antibody Experiment Manual”, edited by Sakuji Toyama et al. , Published by Kodansha Scientific, 1987
Year: Shinsei Kagaku Jikken Koza Vol. 12, "Molecular Immunology III Antigen, Antibody, Complement", edited by The Biochemical Society of Japan, Tokyo Chemical Dojinsha, 1992]. The antibody may be composed of a plurality of antibodies, or may be one obtained by subjecting the antibody to limited decomposition.

The antigen for the antibody to be measured may be an antibody-binding site of a natural antigen, or may be artificially prepared by genetic manipulation or the like. For example, when the antibody to be measured is an antibody against a virus causing various infectious diseases, an antigen against the antibody to be measured may be a marker protein or the like of the above-mentioned virus of infectious disease. The antigen may be composed of a plurality of antigen molecules, or may be one obtained by limitedly decomposing the antigen molecules.

When the quantification method of the present invention uses a carrier, for example, in the case of a latex agglutination method, an antibody to the antigen to be measured bound to latex or an antigen to the antibody to be measured may be used. Is
An antibody to the antigen to be measured or an antigen to the antibody to be measured may be used by adsorbing it on the surface of red blood cells.

As a method for negatively charging an antibody to the antigen to be measured or an antigen to the antibody to be measured in the reaction solution, the antibody or antigen is purified so that the isoelectric point is within a certain range, and then the isoelectric point is reduced. The reaction may be carried out in a reaction solution having a high pH, or after changing the isoelectric point of the antibody or antigen by chemical modification or the like, and then reacting in a reaction solution having a pH higher than the isoelectric point.

The method for purifying the antibody or antigen so that the isoelectric point is within a certain range may be any method as long as the isoelectric point is within a certain range. For example, ion exchange chromatography , Isoelectric focusing,
Isoelectric focusing, isoelectric focusing, chromatofocusing and the like may be mentioned, and these may be used in combination.

The chemical modification method for changing the isoelectric point of the antibody or antigen is a chemical modification method for changing the isoelectric point without decreasing the binding activity of the antibody or antigen [Biochemical Experimental Method 12].
And Volume 13, Chemically Modified Proteins, First and Second Publications, 19
1986, published by Gakkai Shuppan Center]. Any method may be used, for example, a method of binding to bovine serum albumin (BSA) by using a bivalent cross-linking agent, or a method in which a lysine residue of an antigen or antibody is dehydrated. An acylation method such as succinylation with succinic acid and the like can be mentioned.

Hereinafter, the method of the present invention will be described in detail. In ion exchange chromatography, an antibody to the antigen to be measured or an antigen to the antibody to be measured is about 10 mg to 2 mg.
g was dissolved in a buffer solution having a pH of 6.5 to 9.0, separated by anion exchange chromatography or the like, and then a fraction having a desired isoelectric point was collected to obtain an antibody against the antigen to be measured having a constant isoelectric point. Alternatively, an antigen for the antibody to be measured is prepared.

Examples of the buffer include a phosphate buffer, a Tris-HCl buffer, an acetate buffer, a glycine buffer, a barbital buffer, a borate buffer, and the like. The anion exchange chromatography includes DEAE-Sepha. Dex,
DEAE-Sephacel, DEAE-Sephacryl exchange chromatography and the like.

As the acylation method in the chemical modification method, an antibody to the antigen to be measured or about 1 to 100 mg of the antigen to the antibody to be measured is added to the antigen or antibody in a buffer solution at pH 6.0 to 9.0. It is reacted with an acylating agent in an amount of 1 to 10,000 times at 0 to 30 ° C for 0.5 minutes to 12 hours.
The obtained acylated antigen or antibody is separated by a conventional method such as column chromatography. Examples of the acylating agent include succinic anhydride, acetic anhydride, N-acetylsuccinimide, N-hydroxysuccinimide acetate, N-acetylimidazole and the like.

When a BSA complex is prepared as a chemical modification method, an antibody to the antigen to be measured or about 1 to 500 mg of the antigen to the antibody to be measured in a buffer is added to the antigen or antibody at a 2- to 10,000-fold amount of N- [ (E) - maleimidocaproyloxy] Sukushi two bromide (EMCS), etc. in the chemical modifying reagent and 0 to 40 ° C. to react for 0.5 to 120 minutes.
On the other hand, BSA 1 to 500 mg and BSA 2 to 500 mg
0 times the amount of S- acetyl mercapto succinic anhydride, N- scan <br/> comb two Mijiru 3- (2'-pyridyldithio) propionic acid, the chemical modification reagents such as iminothiolane in buffer 0
React at 40 ° C. for 0.5-120 minutes. The obtained chemically modified antibody or antigen is reacted with an equivalent amount of chemically modified BSA in a buffer at 0 to 40 ° C. for 0.5 to 120 minutes to obtain a complex of the antigen or antibody and BSA.

In the above chemical modification reaction, the buffer is as defined above, and if desired, a stabilizer such as albumin,
Ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis (β-aminoethyl ether) -N, N, N ′
_, N ' A chelating agent such as tetraacetic acid (EGTA); In addition, intermediates and products in the above-mentioned chemical modification reaction may be purified and used by column chromatography or the like, if necessary, for example, by gel filtration.

The immunoagglutination assay of the antigen or antibody in the present invention is carried out under the conditions of a general antigen-antibody reaction, and the antigen to be measured and the antibody to the antigen to be measured or the antibody to be measured and the antibody to be measured are With an antigen, a neutral, weakly basic or weakly adjusted phosphate buffer, phthalate buffer,
The reaction is carried out at 4 to 40 ° C. for 30 seconds to 72 hours in the presence of a buffer such as a good buffer or a Tris-HCl buffer.

In the reaction system, polyethylene glycol (P
EG) aggregation promoters such as 6000, polyvinylpyrrolidone, dextran, dextran sulfate, protein components such as gelatin and casein, surfactants such as polyoxyethylene (10) octyl phenyl ether (PMG50),
Salts such as sodium chloride and calcium chloride, metal ions such as magnesium, and stabilizers such as albumin can be appropriately added. Any of these additives can be used as long as they do not inactivate the test substance and do not inhibit the antigen-antibody binding reaction and the immunoagglutination reaction.

Any known means for measuring the change in aggregation of the substance after the reaction can be applied, and the change in the aggregation rate after the start of the reaction is measured by a turbidimeter or an absorptiometer.

[0023]

The present invention will be described below with reference to examples.

Example 1 a, Purification of anti-human CRP-rabbit IgG 20 ml of anti-human C-reactive protein (CRP) rabbit serum (manufactured by Oriental Yeast) was mixed with 1.5 M glycine buffer (pH 8) containing 3 M sodium chloride. .9, protein A-diluted 3-fold with buffer A) and equilibrated with buffer A
This was added to a column packed with 20 ml of Sepharose 4B gel (Pharmacia). After the addition, the column was washed with a phosphate buffer solution containing physiological saline (PBS), and then IgG was eluted with a 50 mM glycine / hydrochloric acid buffer solution (pH 2.5). The eluted IgG solution was immediately neutralized with 1 M phosphate buffer (pH 7.5), dialyzed against 1 L of 10 mM phosphate buffer (pH 8.5) at 4 ° C. overnight, and repeated three times. Was. The collected IgG was SDS-
PAGE electrophoresis showed a single band. The obtained purified anti-human CRP-rabbit IgG was used in the following experiments.

B, Selection of negatively charged antibody in the reagent D equilibrated with 10 mM phosphate buffer (pH 8.5)
EAE-Sephacel column (Pharmacia, 2.5c
m × 20 cm), the IgG fraction obtained in section a was added,
Elution was performed with a linear concentration gradient of 0 to 0.5 M sodium chloride, and the eluate was collected as a 10 ml fraction. The IgG in each fraction was subjected to isoelectric focusing using Pharmacia's Fast System and IEF gel, and the isoelectric point of IgG contained in each fraction was determined based on the isoelectric marker manufactured by Bio-Rad. Was determined, and a fraction containing IgG having an isoelectric point of 7 or less was selected.

C, CR by measuring reagent using selected antibody
Immunoturbidimetric measurement of P Using a direct photometric biochemical automatic analyzer (Hitachi 736 automatic analyzer, manufactured by Hitachi Koki Co., Ltd.) with a multi-item selection function using a transparent synthetic resin as a reaction vessel And the CRP concentration in the sample was measured. In the reaction vessel of this automatic analyzer, a number of cells are individually collected in a disk shape, and this disk-shaped reaction vessel rotates intermittently, and each cell is moved to a first position while passing one point at a time. During the return, one measurement operation and one washing operation are completed.

The anti-human-C having an isoelectric point of 7 or less obtained in section b.
The following operation was performed with an automatic analyzer using RP rabbit IgG and a reaction solution of pH 7.2.

At the first point, 10 μl of CRP standard (manufactured by Oriental Yeast) was added to the cell from the sampler. The cell was transferred to a second point. Here, polyethylene glycol (PEG) 6000 (50 m
g / ml), 100 mM sodium chloride, PGM50
(1 mg / ml) and sodium azide 1 mg / ml
20 mM Good buffer (pH 7.2) containing 350 μl
1 was added as the first reagent and pre-reacted at 37 ° C. for 5 minutes until transferred to the third point. At the third point, the sorted anti-human CRP-rabbit IgG (6.5 mg /
ml), 50 μl of 20 mM Good's buffer (pH 7.2) containing 100 mM sodium chloride and 1.0 mg / ml sodium azide as a second reagent and transferred to the fourth point for 5 minutes at 37 ° C. This reaction was performed.
The reaction was terminated at the fourth point. The turbidity of the solution was measured by a spectrophotometric method (340n) for 20 times (20 points) at intervals of about 20 seconds in a total reaction time of 7.5 minutes in the pre-reaction time.
m, 700 nm) and the change in absorbance was recorded.
The results of the change in absorbance in the preliminary reaction and the change in absorbance due to the antigen-antibody reaction (the main reaction) after the addition of the second reagent are shown in the first column.
It is shown in the table.

[0029]

[Table 1]

The CRP standard was diluted with the above-mentioned phosphate buffer (0.5, 1, 3, 6, 9, 12, 15 m).
g / dl) and the obtained diluent was measured by the above-mentioned method to prepare a calibration curve. The results are shown in FIG.

Example 2 Of the IgG obtained in Example 1, section b, the same procedure was carried out except that the antibody having an isoelectric point of 7.5 to 8.0 was used, and the pH of the reaction solution was changed to 8.5. In the same manner as in Example 1, the change in absorbance in the preliminary reaction and the change in absorbance due to the antigen-antibody reaction (the main reaction) after the addition of the second reagent were determined. The results are shown in Table 2.

[0032]

[Table 2]

The CRP standard was diluted with the above phosphate buffer (0.5, 1, 3, 6, 9, 12, 15 m).
g / dl) and the obtained diluent was measured by the above-mentioned method to prepare a calibration curve. The results are shown in FIG.

Example 3 a, Preparation of succinylated IgG Anti-human CRP-rabbit IgG purified in section a of Example 1
Was replaced with a 0.1 M borate buffer (pH 8.0) by dialysis, and then succinic anhydride / 25 times the IgG molecular weight was used.
A 0.1 M borate buffer (pH 8.0) solution was added, and 15
The reaction was carried out at 45 ° C for 45 minutes. After the completion of the reaction, the mixture was applied to a G-25 column equilibrated with PBS to collect an IgG fraction, which was used as a succinylated anti-human CRP-rabbit IgG. The isoelectric point of the succinylated anti-human CRP-rabbit IgG was measured in the same manner as in the isoelectric focusing performed in section b of Example 1, and it was confirmed that the isoelectric point was 7 or less.

B, Succinylated anti-human CRP-rabbit I
Immunoturbidimetric measurement of CRP using gG In the same manner as in Example 1 except that succinylated anti-human CRP-rabbit IgG was used, the absorbance change in the preliminary reaction and the antigen-antibody reaction after addition of the second reagent (this reaction) ) Was determined. The results are shown in Table 3.

[0036]

[Table 3]

The CRP standard was diluted with the above-mentioned phosphate buffer (0.5, 1, 3, 6, 9, 12, 15 m).
g / dl) and the obtained diluent was measured by the above-mentioned method to prepare a calibration curve. The results are shown in FIG.

Example 4 a, Preparation of IgG-BSA conjugate The external solution of IgG obtained in section a of Example 1 was subjected to dialysis to give 0.1 g.
After replacement with 1M phosphate buffer (pH 7.0), IgG
An EMCS (Dodojin Chemical Co., Ltd.) / DMF solution having a molecular weight of 25 times was added with stirring, and reacted at 30 ° C. for 30 minutes to obtain IgG.
A maleimide group was introduced into. IgG was separated from Sephadex G-equilibrated with 0.1 M phosphate buffer (pH 6.5).
The mixture was applied to 25 columns to remove unreacted reagents, and maleimide group-introduced IgG was recovered. 1 g of BSA was dissolved in a 0.1 M borate buffer (pH 8.0), and an iminothiolane (Pierce) 50 times the BSA molecular weight / 0.1 M borate buffer (pH 8.0) was added thereto. After reacting for 30 minutes, the mixture was applied to a Sephadex G-25 column (Pharmacia) equilibrated with a 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA to remove unreacted reagents and remove SH groups. The introduced BSA was recovered. The maleimide group-introduced IgG and the SH group-introduced BSA were mixed at the same molar ratio, reacted at 30 ° C. for 30 minutes, and then equilibrated with PBS using an AcA22 column (LKB).
(3cm × 100cm) to purify and collect the IgG-BSA complex. The recovered complex was subjected to isoelectric focusing electrophoretic analysis in the same manner as in Example 1, section a, and it was confirmed that the isoelectric point had changed to 7 or less.

B, anti-human CRP-rabbit IgG-BSA
Immunoturbidimetric measurement of CRP using conjugate Except for using anti-human CRP-rabbit IgG-BSA conjugate, the absorbance change in the preliminary reaction and the antigen-antibody after addition of the second reagent were performed in the same manner as in Example 1. The change in absorbance due to the reaction (this reaction) was determined. The results are shown in Table 4.

[0040]

[Table 4]

The CRP standard was diluted with the above-mentioned phosphate buffer (0.5, 1, 3, 6, 9, 12, 15 m).
g / dl) and the obtained diluent was measured by the above-mentioned method to prepare a calibration curve. The results are shown in FIG.

Hereinafter, the effect of the present invention on the interval phenomenon will be described with reference to test examples. Test Example 1 Using the automatic analyzer used in Example 1, the anti-human CRP-rabbit IgG having an isoelectric point of 7 or less used in Example 1
As a comparison control, purified anti-human CRP-rabbit IgG before separation by isoelectric point (Comparative Control 1) or anti-human CRP-rabbit IgG having an isoelectric point of 8 or more obtained in the same manner as in Example 1, section b ( Using Comparative Control 2), continuous measurement was carried out as follows in accordance with the method of Example 1 while the pH of the reaction solution was 7.2.

After continuously measuring 10 samples of physiological saline, 6 samples of CRP standard used in Example 1, and 20 samples of physiological saline, measurement was interrupted for 2 hours. After the measurement was restarted, the turbidity of 50 samples of physiological saline was continuously measured. The results are shown in FIG.

According to FIG. 5, the control 1 and the control 2 showed an interval phenomenon, but the anti-human-rabbit IgG used in Example 1 did not show the interval phenomenon.

Test Example 2 Using the automatic analyzer used in Example 1, the anti-human CRP-rabbit IgG used in Example 2
(Isoelectric point 7.5 to 8.0) and the same pH as in Example 2.
8.5 reaction solution or pH 7.2 reaction solution (comparative control)
, And continuous measurement was performed as follows according to the method of Example 1.

After continuously measuring 10 samples of physiological saline, 6 samples of CRP standard used in Example 1, and 20 samples of physiological saline again, the measurement was interrupted for 2 hours. After the measurement was restarted, the turbidity of 50 samples of physiological saline was continuously measured.
The results are shown in FIG.

According to FIG. 6, although the control showed an interval phenomenon, the anti-human CRP-rabbit IgG used in Example 2 did not show the interval phenomenon.

Test Example 3 Using the automatic analyzer used in Example 1 and the succinylated anti-human CRP-rabbit IgG used in Example 3 or the purified non-succinylated anti-human-rabbit IgG used as a control, the method of Example 1 was used. The continuous measurement was performed as follows according to the following.

After continuously measuring 10 samples of physiological saline, 6 samples of CRP standard used in Example 1, and 20 samples of physiological saline, measurement was interrupted for 2 hours. After the measurement was restarted, the turbidity of 50 samples of physiological saline was continuously measured. The results are shown in FIG.

According to FIG. 7, the succinylated anti-human CRP-rabbit IgG used in Example 3 showed no interval phenomenon, while the non-succinylated control showed an interval phenomenon.

Test Example 4 Using the automatic analyzer used in Example 1, the BSA-conjugated anti-human CRP-rabbit IgG used in Example 4 or a purified anti-human CRP-rabbit IgG not bound to BSA as a control was used. The continuous measurement was performed as follows according to the method.

After continuously measuring 10 samples of physiological saline, 6 samples of CRP standard used in Example 1, and 20 samples of physiological saline, measurement was interrupted for 2 hours. After the measurement was restarted, the turbidity of 50 samples of physiological saline was continuously measured. The results are shown in FIG.

According to FIG. 8, although the control showed an interval phenomenon, the BSA-conjugated anti-human C used in Example 4 was used.
RP-rabbit IgG did not show interval phenomena.

[0054]

According to the present invention, there is provided an immunoagglutination quantification method which does not show an interval phenomenon even in continuous measurement and which is free from nonspecific agglutination.

[Brief description of the drawings]

FIG. 1 is a calibration curve obtained in Example 1.

FIG. 2 is a calibration curve obtained in Example 2.

FIG. 3 is a calibration curve obtained in Example 3.

FIG. 4 is a calibration curve obtained in Example 4.

FIG. 5 shows non-specific aggregation in the continuous measurement system of Example 1.

[Description of Signs]-● -Comparative Control 1 (Antibody with non-uniform isoelectric point)-■ -Comparative Control 2 (Antibody with isoelectric point of 8 or more)-−-Example 1 (Antibody with isoelectric point of 7 or less)

FIG. 6 shows non-specific aggregation in the continuous measurement system of Example 2.

[Explanation of symbols]

 -● -Comparative control (pH 7.2 of reaction solution)-−-Example 2 (pH 8.5 of reaction solution)

FIG. 7: Non-specific aggregation in the continuous measurement system of Example 3

[Explanation of symbols]

 -● -Comparative control (non-succinylated antibody)-○ -Example 3 (succinylated antibody)

FIG. 8 shows non-specific aggregation in the continuous measurement system of Example 4.

[Explanation of symbols]

 -● -Comparison control (BSA non-binding antibody)-○ -Example 4 (BSA binding antibody)

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 33/536 G01N 33/531 G01N 33/543

Claims (17)

(57) [Claims] 1. An antibody to an antigen to be measured or an antigen to an antibody to be measured is prepared so as to be negatively charged in a reaction solution, and the antigen or antibody to be measured in a sample is reacted with the antigen or antibody in the reaction. immunoagglutination Determination how the characterized antigens or antibodies to measure the aggregation change. (2) Antibody or anti-antibody to the antigen to be measured
A method for negatively charging an antigen against the body in a reaction solution is described in US Pat.
Purify the antibody or antigen so that its isoelectric point is within a certain range.
And then react in a reaction solution with a higher pH than the isoelectric point.
2. The method according to claim 1, which is a method. (3) Antibody or anti-antibody to the antigen to be measured
A method for negatively charging the body antigen in the reaction solution has been developed.
After changing the isoelectric point of the antibody or antigen by chemical modification,
It is a method to react in a reaction solution with a pH higher than the electric point.
The quantification method according to claim 1. (4) Chemical modification using bovine cross-linking agent
Claim: A method for binding to serum albumin (BSA)
Item 3. The quantification method according to Item 3. (5) 4. The chemical modification is an acylation method.
Quantitation method as described. 6. The measured antigen is C-reactive protein, 請
6. The quantification method according to any one of claims 1 to 5 . 7. Antibody or anti-antibody to the antigen to be measured
An antigen for the body and the antibody or the antigen
An antigen containing a buffer having a pH such that the
Is a reagent for immunoagglutination determination of antibodies. Claim 8. Antibody or anti-antibody to the antigen to be measured
The antigen to the body is a chemically modified antibody or antigen.
The reagent according to claim 7, wherein 9. If the chemically modified antibody or antigen is
An antibody or antigen bound to serum albumin.
8. The reagent for quantification according to 8. 10. Antibodies or antigens that have been chemically modified
The quantification according to claim 8, which is a silylated antibody or antigen.
For reagents. 11. The antigen to be measured is a C-reactive protein
A reagent for quantitative determination according to any one of claims 7 to 10. 12. Antibody to the antigen to be measured or measured
Prepare antigen for antibody to be negatively charged in reaction solution
A method for suppressing an interval phenomenon. Claim 13 Antibody to the antigen to be measured or measured
The method of negatively charging the antigen for the antibody in the reaction solution,
Purify the antibody or antigen so that its isoelectric point is within a certain range
After that, the purified antibody or antigen is
13. The method according to claim 12, which is a method of containing the compound in a high buffer solution.
Suppression method. 14. Antibody to the antigen to be measured or measured
The method of negatively charging the antigen for the antibody in the reaction solution,
After changing the isoelectric point of the antibody or antigen by chemical modification, etc.
The antibody or antigen whose electric point has been changed is brought to a pH higher than the isoelectric point.
13. The method according to claim 12, which is a method of containing a high buffer.
Suppression method. 15. Chemical modification is performed using a bivalent crosslinking agent.
A method for binding to serum albumin (BSA)
15. The suppression method according to claim 14. 16. The chemical modification is an acylation method.
15. The method according to 14 above. 17. The antigen to be measured is a C-reactive protein;
The suppression method according to claim 12.