JPH0712813A - Manufacture of reagent for measuring antigen or antibody - Google Patents

Abstract

PURPOSE:To provide the manufacturing method of a reagent for measuring antigens or antibodies having a wider measuring range while the increase of light absorbancy or intensity of scattered light against the concentration of an antigen or antibody to be measured due to the condensation of an insoluble carrier is maintained at a fixed rate. CONSTITUTION:In the manufacturing method of a reagent which is used for measuring the antigen or antibody corresponding to an antibody or antigen by detecting the degree of condensation of a carrier carrying the antibody or antigen due to an antigen-antibody reaction, the insoluble carrier is successively brought into contact with a nonionic or cationic surface active agent (e.g. Tween 20) after the carrier is made to carry the antibody or antigen and the concentration of the surface active agent in the reagent is controlled to 0.005-1mg/ml.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention is for measuring an antibody or an antigen corresponding to the antibody or the antigen by detecting the degree of aggregation of the carrier due to an antigen-antibody reaction by allowing the insoluble carrier to carry the antibody or antigen. And a method for producing the reagent.

[0002]

2. Description of the Related Art Conventionally, proteins and lipids in body fluids such as blood and urine such as hepatitis B virus, carcinoembryonic antigen, human immunodeficiency virus, C-reactive protein, hepatitis B virus antibody, human For measuring substances immunologically belonging to an antigen or antibody such as immunodeficiency virus antibody, rheumatoid factor, and anti-streptidine-O, an insoluble carrier such as a latex carrier is loaded with an antibody or an antigen corresponding to the antigen or the antibody. A reagent for measuring the antigen or antibody (hereinafter referred to as a substance to be measured) by detecting the degree of aggregation of the carrier due to an antigen-antibody reaction is generally used.

Conventionally, in a reagent for measuring a substance to be measured by detecting the agglutination of such a carrier, an increase in absorbance due to the agglomeration of an insoluble carrier in a reaction solution is usually an optical density (hereinafter referred to as "OD"). A calibration curve representing the relationship between the OD and the amount of the substance to be measured is prepared in advance, and an unknown amount of the substance to be measured is quantified using this calibration curve. This calibration curve is O
When D is plotted on the vertical axis and the concentration of the substance to be measured is plotted on the horizontal axis, it is usually not a straight line, and the OD is particularly low in the portion where the concentration of the substance to be measured is low. , A sigmoid-shaped S-shaped curve peculiar to the immune response.

Therefore, in order to perform accurate measurement, it is necessary to measure three or more different substances having different known concentrations or make a special correction when creating a calibration curve. However, the measurement of three or more points has a problem in that the operation becomes complicated and the cost is high, and particularly when measuring with an automatic analyzer, various restrictions are required on the device.

Therefore, even if the number of measurements is twice, two different amounts of the same antigen or antibody are carried in an attempt to bring the calibration curve close to a straight line so that accurate measurement can be performed. A method (JP-A-55-151264) of mixing and using latex particles having a particle size (hereinafter referred to as "latex carrier") has been proposed. In this proposal, when the latex carrier carrying the antigen or antibody corresponding to the substance to be measured and the substance to be measured undergo an antigen-antibody reaction, the latex carrier with a large particle size grows an agglomerate at an early stage, while the particle size With a small latex carrier, agglomerates grow slightly later. Therefore, the increase rate of the absorbance or scattered light intensity of the latex carrier in the low concentration and high concentration regions of the calibration curve approaches a certain level, and accurate measurement can be performed in a wider concentration range.

However, even this proposal is not sufficient because there is a limit to the concentration range of the substance to be measured that can be measured. Even if the increase in the absorbance of the reagent itself linearly extends with respect to the concentration of the substance to be measured, it is near the upper limit of measurement of the optical measuring device (the upper limit of measuring absorbance of a general optical measuring device is around OD = 3. Even if the concentration of the substance to be measured becomes high, the increase in absorbance due to it cannot be detected by the optical measuring instrument, the calibration curve reaches a peak, and the absorbance changes above the upper limit of measurement due to changes in absorbance. It is difficult to measure the concentration of the substance to be measured. That is, the maximum change width of the measurable absorbance is equal to the difference between the upper measurement limit of the instrument and the absorbance of the reagent blank (absorbance when the concentration of the substance to be measured is zero).

[0007] It is conceivable that the absorbance of the blank of the reagent may be lowered as much as possible when the maximum change range of the absorbance is widened so as to widen the concentration range of the substance to be measured. When the amount of latex carrier used in the reaction is reduced to reduce the absorbance of the blank of the reagent, the delay phenomenon occurs at a lower concentration than usual reagents, and the sigmoid of the calibration curve becomes more prominent and Accurate measurement cannot be made when a line is created.

[0008] As a proposal to broaden the maximum range of change in the absorbance so as to broaden the concentration range of the substance to be measured, an excess amount of the antigen is added to the reagent at the time of antibody measurement, and the free antigen not adsorbed on the carrier. In some cases, the amount of antibody can be measured up to a higher concentration by allowing the presence of the antibody (JP-A-57-9).
No. 723). However, in this method, the absorbance becomes too low when the concentration of the substance to be measured is low, and the calibration curve has a sigmoid shape. Therefore, there is a problem that accurate measurement cannot be performed if a calibration curve is created by two measurements.

[0009]

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and an object thereof is to make the increase rate of the absorbance or the scattered light intensity due to the insoluble carrier aggregation with respect to the concentration of the substance to be measured constant. An object of the present invention is to provide a method for producing a reagent for measuring an antigen or an antibody, which has a wider measurement range while keeping the same.

[0010]

As the insoluble carrier used in the present invention, any substance conventionally used to measure an antigen or an antibody by agglutination of the antibody or the insoluble carrier carrying the antigen is used. It is possible, and examples thereof include inorganic substance powder, organic polymer substance powder, microorganisms, blood cells, and cell membrane pieces. Examples of the inorganic substance include metals such as gold, titanium, iron and nickel; metal oxides such as alumina and titania; silica. The organic polymer is not particularly limited, but for example, styrene polymer, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride- Examples thereof include acrylic acid ester copolymers and vinyl acetate-acrylic acid ester copolymers. In particular, latex particles obtained by uniformly suspending these polymer powders in water are preferable. The average particle size of the latex particles is preferably 0.05 to 1.0 μm, and particularly preferably 0.05 to 0.5 μm.

Examples of the nonionic surfactant used in the present invention include polyoxyethylene-sorbitan-
Monolaurate (for example, product name Tween20; At
las Powder Co. Company), polyoxyethylene-sorbitan-monooleate (for example, trade name T
ween 80; Atlas Powder Co. Alcohol polyether-alcohol mixture (for example, trade name Triton; Rohm and Haas)
Co. Manufactured by the company), polyoxyethylene lauryl ether (for example, trade name Brij35; Atlas Powder)
er Co. Manufactured by the company), polyoxyethylene-octylphenol ether (for example, trade name Non-IdetP
-40), a polyethylene oxide-alkyl ether adduct (for example, trade name Lubrol PX; IC).
I. C-1 having 10 oxyethylene units
6, C-18 aliphatic alcohol (eg trade name, Be
rollEMU 043) and the like, and examples of the anionic surfactant include sodium dodecyl sulphate.

In the production method of the present invention, first, an antibody or an antigen is carried on the insoluble carrier in a suspension in which the insoluble carrier is dispersed. A known physical adsorption method or a chemical bonding method is used for this supporting. When carrying an antibody, a monoclonal antibody or a polyclonal antibody is preferably used as the antibody. These antibodies are usually purified by appropriately combining known antibody purification means such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography and affinity chromatography. Examples of the type of antibody include immunoglobulins such as IgG and IgM, and may be F (ab ′), if necessary.
² , Fab may be used. When carrying an antigen,
The type of antigen is not particularly limited, and for example, proteins, polypeptides, steroids, polysaccharides, lipids and the like are used depending on the measurement purpose.

In the present invention, following the above step of supporting the antibody or antigen, the supported carrier is brought into contact with a nonionic or anionic surfactant. After the completion of the supporting step, the time until contact with the above-mentioned surfactant becomes longer, so that the carrier agglomerates. Therefore, it is preferably within 5 hours immediately after the completion of the supporting step, and more preferably within 1 hour. If the carrier is brought into contact with the surfactant before the step of supporting the antibody or antigen, the supporting of the antibody or antigen on the carrier is hindered.

In the present invention, examples of the method of bringing the carrier carrying the antibody or antigen into contact with the nonionic or anionic surfactant include the following methods. A method in which only the carrier is separated from the solution containing the antibody or antigen solution used in the supporting step by means such as centrifugation or filtration, and the separated carrier is suspended in a buffer solution containing a surfactant. A method of adding a surfactant alone or a buffer solution containing a surfactant to a solution containing a carrier without separating the carrier from a solution containing the antibody or antigen solution used in the supporting step. In the above methods and, when the surfactant is dissolved in a buffer, the buffer usually has a pH of 5 to 1
A buffer solution of 0 is preferable, and examples thereof include a phosphate buffer solution, a Tris buffer solution, and a glycine buffer solution. After the step of loading the antibody or antigen, if the antibody or antigen-attachable site on the surface of the insoluble carrier still remains, such as bovine serum albumin is usually used to prevent nonspecific adsorption reaction when the carrier is used. An operation of saturating the active sites on the insoluble carrier with a protein (normally, this operation is called blocking) is carried out. By adding a surfactant to the solution used for this operation, the carrier is A method of suspending in a surfactant solution. In the case of blocking as described above, not only a method of suspending the carrier in the surfactant solution at the same time as the blocking as described above, but before or after the blocking, the carrier may be suspended in the surfactant solution. You may suspend.

In the production method of the present invention, the finally obtained reagent for measuring an antigen or antibody is a suspension of the insoluble carrier containing the nonionic or anionic surfactant. In the present invention, the concentration of the surfactant is 0.005 to 1 mg / ml in the suspension.
When the concentration of the surfactant is low, the effect of expanding the measurement range is small, and when it is high, the antigen-antibody reaction is inhibited during the use of the reagent.

The above-mentioned surfactant may coexist with the carrier when the carrier is used for the antigen-antibody reaction, or it may be present before the carrier is used for the antigen-antibody reaction. It may be separated.

The concentration of the insoluble carrier carrying the antibody or antigen present in the finally obtained reagent is
The amount in the above suspension is preferably 0.2 to 10 mg / ml, more preferably 1 to 5 mg / ml. When the concentration of the insoluble carrier is low, the effect of expanding the measurement range is small, and when it is high, the particles aggregate to inhibit the antigen-antibody reaction.

To measure the substance to be measured in the sample with the reagent for measuring an antigen or antibody produced according to the present invention, either an optical measuring method or a macroscopic measuring method may be used. For optical measurement, for example, first, a specimen is dispensed into a reaction container, and the reagent for measuring the antigen or antibody is dispensed and mixed therein to react. In this step, the sample and the reagent may be dispensed in the reverse order or at the same time as the above method. The pH in the reaction is preferably 5 to 10, more preferably 6 to 8. Examples of the buffer used include phosphate buffer, Tris buffer, glycine buffer and the like. The reaction temperature is preferably 0 to 50 ° C,
More preferably, it is 20-40 degreeC. The agglutination reaction time is
It is preferably 20 seconds to 30 minutes, more preferably 1 to 15 minutes.

The concentration of the substance to be measured in the sample is calculated by measuring the absorbance or the like of the production amount or production rate of the aggregate thus obtained. This includes, for example:
There are a method of measuring the absorbance once and a method of measuring the absorbance twice. A method of measuring scattered light instead of measuring absorbance can also be used. The measurement can be performed at an appropriate wavelength of 300 to 1000 nm, but it is preferable to use a wavelength sufficiently long with respect to the particle size of the latex. A known device can be used for such an optical measurement device. For example, a normal spectrophotometer for measuring absorbance, a device for measuring light scattering intensity, a device for measuring the number of particles and / or particle diameter, etc. are used. As a dedicated device for measuring the agglutination reaction, a biochemical automatic analyzer incorporating a spectrophotometer, a dedicated device for measuring the agglutination reaction by the immunoturbidimetric method, the agglutination reaction is measured using flow injection. There is a dedicated device for measuring the viscosity, a dedicated device for measuring the latex agglutination reaction, and the like.

The sample which can be measured by the reagent obtained according to the present invention is a sample containing an immunologically active substance such as an antigen or an antibody, particularly a biological sample (eg blood, pleural effusion, ascites fluid, lymph fluid, etc.). Body fluids, urine, feces, excrements such as sweat, or tissue extracts). The antigen or antibody that can be measured by the reagent obtained in the present invention generally includes all substances immunologically belonging to the antigen or antibody. For example, antithrombin III (AT III)
, Alphafetoprotein (AFP), rheumatoid factor (RF), anti-streptolysin-O (ASO), C-
Reactive protein (CRP), fibrinogen-fibrin degradation product (FDP), human chorionic gonadotropin (HC
G), proteins or polypeptides such as carcinoembryonic antigen (CEA), steroids, polysaccharides, lipids and the like.

[0021]

EXAMPLES Examples will be shown below to specifically explain the present invention. In the following Examples and Comparative Examples, a reagent for measuring antithrombin III (hereinafter referred to as “AT III”) was manufactured, and a calibration curve was prepared using the obtained reagent.

Example 1 (1) Preparation of Anti-AT III Antibody-Supporting Latex Reagent Retaining Antibody on Insoluble Carrier 2 mg of anti-human AT III goat-producing antibody (ATAB)
0.02M phosphate buffer (pH 6.5) at a concentration of 1 / ml
Into 5 ml of the solution dissolved in was added 500 μl of polystyrene latex (solid content 10%, Sekisui Chemical Co., Ltd.) having an average particle size of 0.1 μm, and the mixture was stirred at 18 ° C. for 2.5 hours. Step of suspending the carrier in the surfactant solution Next, containing Tween 20 as a surfactant at 0.0126 mg / ml, containing bovine serum albumin at a concentration of 2% by weight.
Dissolving phosphate buffer (pH 6.5, 0.35M) 5m
1 to the above-mentioned anti-human AT III goat-produced antibody-bearing latex suspension (after mixing, the final concentration of Tween 20 is 0.006 mg / ml), continue mixing at room temperature overnight, and continue stirring for AT III measurement The reagents were manufactured. It was then stored at 4 ° C.

(2) Optical measurement of AT III 200 μl of the above latex reagent was added to 1% by weight of bovine serum albumin, polyethylene glycol (polyethylene glycol 6000, average molecular weight 7500, manufactured by Wako Pure Chemical Industries, Ltd.)
Phosphate buffer (pH 6.
5, 0.35 M) diluted with 350 μl, and the sample (standard human plasma (Boehringer Mannheim Co., lyophilized product) reconstituted with pure water was diluted with a 5 wt% bovine serum albumin aqueous solution, or at the time of reconstitution Concentrate by reducing the amount of pure water added to 0, 25, 50, 7
Prepared concentration series of 5, 100, 150, 200%. Here, 100% is the AT III standard content concentration in normal human serum. ) Was added and stirred, and after 4 minutes, the absorbance at a wavelength of 570 nm was measured. The number of repeated measurements was 5 for each dilution series. The relationship between the obtained absorbance (average value of 5 measurements) and the AT III concentration is shown in FIG. In Figure 1, the measured absorbance is 1
The value is multiplied by 0000. In addition, in FIG. 1, what is shown by a dotted line is the upper limit value of the absorbance measurement of the device.

Examples 2 to 5 Tween 20 was added in an amount of 0.0126 mg / m 2 in the step of suspending the carrier in the surfactant solution in Example 1.
l Instead of using soluble phosphate buffer,
n20 is 0.105 mg / ml (Example 2),
1.05 mg / ml (Example 3), 1.68 mg / ml
(Example 4), 2.1 mg / ml (Example 5) Using a phosphate buffer solution that dissolves (final concentrations of Tween 20 were 0.05 mg / ml in Example 2 and 0.
5 mg / ml, Example 4 0.8 mg / ml, Example 5
Except that the concentration is 1.0 mg / ml), an AT III measuring reagent was produced in the same manner as in Example 1, and the relationship between the absorbance and the AT III concentration was determined in the same manner as in Example 1 and shown in FIG. Indicated.

Comparative Example 1 AT III was carried out in the same manner as in Example 1 except that a phosphate buffer containing no Tween 20 was used in the step of suspending the carrier in the surfactant solution in Example 1.
A measurement reagent was produced, and the relationship between the absorbance and the AT III concentration was determined in the same manner as in Example 1 and shown in FIG.

Comparative Example 2 Tween 20 was added at 0.0126 mg / m 2 in the step of suspending the carrier in the surfactant solution in Example 1.
l Instead of using soluble phosphate buffer,
The use of a phosphate buffer that dissolves n20 at 2.52 mg / ml (final concentration of Tween 20 is 1.2 mg
/ ML) and AT III in the same manner as in Example 1.
A measurement reagent was produced, and the relationship between the absorbance and the AT III concentration was determined in the same manner as in Example 1 and shown in FIG.

Comparative Example 3 The AT III measuring reagent obtained in Comparative Example 1 was a phosphate buffer (pH 6.5, 0.35M) containing 2% by weight of bovine serum albumin and containing no surfactant. And diluted 1.5 times to prepare an AT III measuring reagent in which the solid content concentration of the anti-AT III antibody-supported latex was reduced, and the relationship between the absorbance and the AT III concentration was determined in the same manner as in Example 1. It is shown in FIG.

Evaluation As can be seen from FIG. 1, the reagents obtained in Examples 1 to 5 were
Compared to the reagent of Comparative Example 1 obtained by the conventional method, a reagent blank (O at an AT III concentration of 0% in FIG.
The D value) is low, and the linear portion of the calibration curve extends to the high concentration region of AT III. Further, as can be seen from the calibration curve of Comparative Example 2, when the supported carrier is suspended in a surfactant solution having a higher concentration, the linear portion of the calibration curve is further extended, but the antigen-antibody reaction of the reagent itself does not occur. Since it is inhibited, the amount of change in absorbance per change in concentration is small. In addition, Comparative Example 3
When the latex concentration in the reagent is lowered like
It becomes a glyph.

Next, in Comparative Example 2 (Tween 20 final concentration 1.2 mg / ml) and Example 5 (Tween 20 final concentration 1.0 mg / ml), AT III concentrations of 0% and 2 were obtained.
Absorbance of 5% sample measured 5 times each,
The average value and the standard deviation thereof are shown in Table 1.

[0030]

[Table 1]

From Table 1, the surfactant concentration was 1.0 mg / m.
When it exceeds 1, the range of 2σ at the sample concentration of 25% and the range of 2σ at the sample concentration of 0% overlap, so that it is difficult to perform the measurement accurately.

[0032]

The composition of the method for producing the reagent for measuring an antigen or antibody of the present invention is as described above. According to this production method, the rate of increase in the absorbance or scattered light intensity due to the concentration of the insoluble carrier with respect to the concentration of the substance to be measured. Keeping constant,
A reagent for measuring an antigen or antibody having a wider measurement range can be manufactured. Since the rate of increase in the absorbance or the intensity of scattered light is constant, the measurement for preparing the calibration curve can be performed only twice, so the reagent obtained by the production method of the present invention is simple in operation and low in cost.

[Brief description of drawings]

FIG. 1 is a calibration curve of AT III measurement reagents obtained in Examples 1 to 5 and Comparative Examples 1 to 3.

Claims (1)

[Claims] 1. A suspension of an insoluble carrier in which the antibody or antigen is carried on the insoluble carrier, and the insoluble carrier carrying the antibody or antigen is then subjected to a nonionic or anionic interface. A method for producing a reagent for measuring an antigen or an antibody, which comprises contacting an active agent, wherein the concentration of the surfactant in the reagent is 0.005-1 mg / ml. Production method.