JPH0448265A - Immunoassay - Google Patents

Abstract

PURPOSE:To make simultaneous multiple item measurement within the same reaction vessel by continuously effecting a flocculation reaction based on antigens or antibodies of two items in the same reaction vessel using the same specimen. CONSTITUTION:The 1st flocculation reaction based on the antigens or antibodies of one item is first effected in the same reaction vessel, then the 2nd flocculation reaction based on the antigens or antibodies of another one item is effected, by which the presence or quantity of the desired antigens or antibodies of the two items is simultaneously determined with the one specimen. The antigens to be used are exemplified by all of the antigens having antigenicity, such as protein, bacteria, and the components thereof, polysaccharides, lipids, nucleic acids, hormones, vitamins, hapten of drugs, etc., allergen of pollen, etc. The antibodies are exemplified by antiserum contg. antibodies, polyclonal antibodies (IgG fractions) (excluding the antiserum in the case of sensitization to fine particle carriers). The antigens or antibodies of multiple items are simultaneously measured within the same reaction vessel in this way.

Description

[Detailed description of the invention] [Industrial application field] The present invention applies to body fluids (blood, serum, blood plasma, spinal fluid).

This relates to a method for measuring antigens or antibodies in urine, etc.).

Specifically, it relates to a simultaneous two-item immunoassay method for the same specimen.

[Conventional technology]

In recent years, immunoassay methods, especially turbidimetry, which measures agglutination based on antigen-antibody reactions as changes in optical turbidity, have rapidly become popular in the fields of medicine and clinical testing, along with advances in reagents and automatic analyzers. I've been doing it.

Conventionally, as well-known measurement methods, turbidimetry and nephelometry have been used to measure aggregates produced as a result of the reaction as changes in optical turbidity. The nephelometric method measures optical turbidity changes as changes in transmitted light [rclin, chem, vol.
Vol. 8, No. 1.0, pp. 2121-2124 (1982)
], the Hiro method measures optical turbidity changes as changes in scattered light (rAm, J, C11n, Pa
thol, Jw Vol. 76, p. 753 (1981)], and latex agglutination nephelometry, which uses a reagent in which antigens or antibodies are supported on microparticles such as latex to increase measurement sensitivity [rProtLdes of the Biology].
cal Fluids(PERGAMON PRESS
Inc.), 1st edition, pp. 589-593 (1973)].

[Problem to be solved by the invention]

Currently, such immunoassay methods are performed by measuring each single item. For example, in the diagnosis of patients with disseminated angiocoagulant syndrome (DIC), fibrinogen and fibrin degradation products (Fl)P, FDP-
E, FDP-D dimer, etc.) have been measured.
Currently, measurements are performed by collecting samples for each item and using separate dedicated reagents. Also, rheumatic fever, myocarditis,
For various inflammatory diseases such as glomerulonephritis, anti-streptolysin O titer (ASO) and C-reactive protein (CRP).

Multiple items are measured on the same patient, such as autoimmune diseases (detection of RF factors and anti-nuclear antibodies), but it is important to collect samples for each item and use separate dedicated reagents. This is the current situation. In addition to the above examples, multiple items are measured on the same patient for various diseases such as infectious diseases and malignant tumors. In addition, it is difficult to collect specimens from newborns and cancer patients in particular, and the burden on patients is large, so it is desired to further reduce the required amount of specimens. For this reason, simultaneous measurement of multiple items necessary for diagnosis is desired. However, simultaneous measurement of multiple items in the same reaction vessel was difficult due to the coexistence of multiple reactions.

The present invention provides an immunoassay that is easy to measure and rapid, with a minimum amount of specimen collected, by sequentially performing agglutination reactions based on two antigens or antibodies in the same reaction container using the same patient specimen. It provides law.

[Means to solve the problem]

The present invention is an immunoassay method in which the agglutination reaction between an antigen and an antibody is measured as a change in optical turbidity to determine the presence or amount of a target antigen or antibody in a specimen, in the same reaction vessel. By first performing a first agglutination reaction based on one antigen or antibody, and then performing a second agglutination reaction based on another antigen or antibody, two target antigens can be obtained simultaneously for one specimen. Or it relates to an immunoassay method characterized by determining the presence or amount of antibodies.

The present invention will be explained in detail below.

Immunoassay methods have been well known for a long time, in which the presence or amount of antigen or antibody in a specimen sample is determined by measuring the agglutination reaction between antigen and antibody as an optical turbidity change.

"Agglutination reaction between antigen and antibody" is a reaction in which antigen and antibody aggregate as a result of a specific binding reaction (antigen-antibody reaction) in an aqueous buffer. Usually, an antigen or antibody in a specimen to be measured is used as a sample, and an antibody or antigen against it is used as a reagent. The degree of progress of this reaction varies depending on the quantitative relationship between the antigen and antibody, the titer of the antibody, the type of antigen, the reaction temperature (usually from room temperature to 37° C.), the aqueous buffer solution used as the reaction medium, etc. Usually, as an aqueous buffer, 0.1 to 0.2 moQ/Q of 1-chloride is used.
Phosphate buffer with pH 5 to 10, preferably pH 6 to 8, containing tris[tris(hydroxymethyl)aminomethane]hydrochloric acid, glycine buffer, oxalic acid buffer, ammonia buffer.

A triethanolamine buffer or the like is used as a reaction medium, optionally along with additives such as stabilizers, preservatives, chelating agents, surfactants, and sensitizers (polyethylene glycol). Furthermore, the agglutination reaction between antigen and antibody is measured as an optical turbidity change.

Antigens used as reagents in the present invention include proteins, bacterial cells and their components, peptides, and polysaccharides.

lipids, nucleic acids, hormones, vitamins, steroids.

Examples include all things that have antigenicity, such as haptens such as drugs and allergens such as pollen. In addition, the antibodies used as reagents in the present invention include substances having affinity for antigens and antibodies obtained from the above-mentioned antigens by known methods, and antisera containing antibodies.

Polyclonal antibodies (IgG fraction), monoclonal antibodies, etc., and Fab +Fab derived from these antibodies
', F(ab'), etc. (however, antiserum is excluded for sensitization to particulate carriers).

In the present invention, a combination of two types of target measurement items is possible as long as the same component in the specimen is not the target.

In the present invention, "measuring changes in optical turbidity" refers to the nephelometric method, which measures changes in absorbance (transmitted light) using an ordinary spectrophotometer or automatic biochemical analyzer. is available. From a reagent perspective, there are reagents that use the antigen or antibody against the target antibody or antigen as is (i.e., without using a microparticle carrier), and sensitized microparticles in which the antigen or antibody is sensitized to an insoluble microparticle carrier such as latex. Reagents using carriers are available for water droplets.

The particulate carrier that can be used in the present invention includes organic polymerizable particulate carriers that are insoluble in the aqueous buffer used for measurement, such as polystyrene.

Examples include latex particles obtained by emulsion polymerization such as styrene-butadiene copolymer and styrene-styrene sulfonic acid, and spherical particles such as liposomes. The microparticle carrier is sensitized (bound) with an antigen or antibody and used as a measurement reagent, and the sensitization may be performed by physically adsorbing or chemically binding by a known method.

In general, with nephelometric reagents that do not use particulate carriers, the amount of change in absorbance is small when reacted as is, so a sensitizer such as polyethylene glycol 6000 is required. The presence of 4% polyethylene glycol 6000 is preferable, and in order to enable more sensitive measurement, dual-wavelength measurement with the main wavelength in a relatively short wavelength region of about 340 to 400 nm, or
- Wavelength measurements are preferred. Naturally, the amount of antigen or antibody used as a reagent should be appropriately determined, taking into account the measurement range of the antibody or antigen to be detected.

The sensitivity of measurements using nephelometric reagents using microparticle carriers is high, making it ideal for measuring trace components in specimens. When using a microparticle carrier, the particles themselves exhibit turbidity, block the transmission of light, and scatter light. However, on the other hand, the blank value before the reaction becomes high, and the area that can be used for the reaction is limited. In order to measure antigens or antibodies in a specimen with good precision and reproducibility over a wide concentration range, improvements can be made by selecting the measurement wavelength, the particle diameter of the microparticle carrier, and its concentration as necessary. For example, when the particle concentration of the microparticle carrier in the nephelometric method is the same, the blank value before the reaction becomes lower as the particle diameter of the microparticle carrier is smaller and the measurement wavelength is longer than the particle diameter. Conversely, the larger the particle diameter of the microparticle carrier and the closer the measurement wavelength is to the particle diameter, the higher the blank value before the reaction. The measurement system and reagent conditions are set as appropriate by those skilled in the art according to the measurement item, and the particle diameter and concentration of the microparticle carrier and measurement wavelength are set based on the balance between the sensitivity and measurement range required for the target item. Should.

In the immunoassay method detailed above, the present invention provides a first method based on one antigen or antibody in the same reaction vessel.
The presence or amount of two target antigens or antibodies is simultaneously determined for one specimen by performing an agglutination reaction based on one antigen or antibody and then performing a second agglutination reaction based on another antigen or antibody.

In that case, if you do the following, the second aggregation reaction will occur due to the first aggregation reaction.
This is preferable because it can reduce or eliminate the influence on the aggregation reaction.

(1) A sensitized fine particle carrier is not used in the first aggregation reaction, and a sensitized fine particle carrier is used in the second aggregation reaction.

(2) Sensitized fine particle carriers are used in both the first aggregation reaction and the second aggregation reaction.

In the case of the former combination (1), the first agglutination reaction measurement is
The second aggregation reaction is measured in a relatively short wavelength region of about 40 to 400 nm, and then in a longer wavelength region of 400 to 2400 nm.
It is particularly preferable to carry out the reaction at 8 nm, since this reduces the influence of the first aggregation reaction.

In the latter case (2), in the reagent for the second agglutination reaction,
It is particularly preferable to coexist an appropriate amount of the antigen or antibody used in the reagent for the first agglutination reaction, since the influence of the first agglutination reaction on the second agglutination reaction can be reduced or eliminated.

The turbidity change in each agglutination reaction can be measured using one point end measurement method (One Point End As5ay), which measures at one point after a certain period of time after the start of the reaction, or at two points, one before the start of the reaction and one after a certain period of time after the start of the reaction. Two Point End Measurement Method (Two Point Encl A)
s5ay), rate measurement measured at two points after the start of the reaction (
Tw.

Point Rate As 5ay) and multi-point measurement method that measures at two or more points.
1nt Rateassay). 1st
Although both the endpoint measurement method and the rate measurement method are suitable for measuring the second agglutination reaction, the rate measurement method is most suitable for measuring the second agglutination reaction. In the rate measurement method, it is preferable to perform measurement within a relatively short period of time, preferably within 5 minutes after the start of the reaction, because measurement over a too long period not only impairs the advantage of rapid measurement but also narrows the measurable range.

In addition, when measuring turbidity changes, the light actually measured is
It may be either so-called transmitted light (absorbance) or scattered light.

The wavelength of the immunoassay in the present invention is appropriately selected depending on the selection of the measuring device and measuring method, whether or not a particulate carrier is used, the particle size of the carrier, its concentration, and the like. Generally, in turbidimetry that does not use particulate carriers, the wavelength is 300 nm to 400 nm;
In the nephelometric method using a fine particle carrier, the wavelength is 400 nm to 24 nm.
00 nm monochromatic or polychromatic light is used.

The presence or amount of the target antigen or antibody is determined from the above measured values. For example, to determine the concentration, first measure a standard sample with a known concentration in the same way as the specimen and determine the relationship between the concentration of the target substance and the amount of change in turbidity. The concentration of can be converted.

〔Example〕

The present invention will be explained in more detail with reference to Examples below.
The invention is not limited by the following examples.

(Example 1) Amounts of fibrinogen and FDP-E in blood plasma 1) Reagent (1) Fibrinogen measurement reagent Rabbit antibody absorbed with excess amount of FDP-E antigen in 50mM phosphate buffer solution (pH 7.3) Contains 20 (v/v)% human fibrinogen antiserum, 3 (w/v)% polyethylene glycol 600° and 0.1M sodium chloride.

(2) FDP-E measurement reagent (anti-FDP-E antibody sensitized latex) 80mM of 50mM phosphate buffer solution (antibody concentration: 0.5mg/mQ) of anti-FDP-E antibody has an average particle size of 0. 0.5 mβ of 11 μm polystyrene latex (manufactured by Japan Synthetic Rubber Co., Ltd., solid content concentration: 10% by weight) was added, stirred at 10°C for 1 hour, and then centrifuged at 10°C for 30 minutes (20000 r, P, ■). . Decant the unreacted supernatant, and remove the remaining antibody-sensitized latex particles.
0mM phosphate buffer solution (containing 2 (w/v)% bovine serum albumin (BSA), 0.1M sodium chloride)8
It was redispersed to 0 mρ.

2) Measurement method Fibrinogen measurement reagent (first agglutination reaction reagent, hereinafter abbreviated as R) 350μQ and physiological saline 2oμQ
Add and mix into a reaction vessel (cell with optical path length 6m+), and
The reaction was carried out while keeping the temperature at 7°C, and after 5 minutes, the absorbance difference was measured at a main wavelength of 340 nm and a sub wavelength of 700 nm, and a reagent blank (Blk-AbsFg) for a fibrinogen measurement reagent was obtained.

Continuously, at the same time, 350 μQ of FDP・E measurement reagent (second agglutination reaction reagent, hereinafter abbreviated as R7) was added and mixed, and the mixture was heated at 37°C.
After 1 minute and 3 minutes, the wavelength was 700n.
Absorbance was measured at m. From this, calculate the amount of change in absorbance per unit time and use the reagent blank Bl of the FDP-Es constant reagent.
k-ΔabsFDP-E).

Next, in the same manner as above, the patient sample serum 2゜μQ and R
Add and mix 350 μQ into a reaction container, react at 37°C, and measure the absorbance at a wavelength of 340 nm after 5 minutes.
sFg). Continuously, at the same time, 350 μρ of R was added and mixed, and the mixture was allowed to react at 37° C., and the absorbance at a wavelength of 700 nm was measured after 1 minute and 3 minutes. From this, the amount of change in absorbance per unit time was determined and was defined as the amount of change in absorbance due to FDP-H in the sample (S・ΔAbsFDP-E), and the amount of change in absorbance due to fibrinogen in patient sample serum (Δ
AbsFg) and the amount of change in absorbance due to FDP-H in the sample (ΔAbsFDP-E) were calculated from equations 1 and 2.

ΔAbsFg=S-AbsFg-B1-AbsFg
-Formula 1%Formula %...Formula 2 In advance, a standard product containing fibrinogen and FDP-E of known concentrations was used instead of the patient sample serum, and the absorbance was measured in the same manner as above, and each absorbance was determined from Formulas 1 and 2. The amount of change was determined and a calibration curve was created for each. The concentrations of fibrinogen and FDP-E in the specimen were determined from this calibration curve and the amount of change in absorbance of fibrinogen ΔAbsFg and FDP-E (ΔAbsFg and ΔAbsFDP-E). The above measurements and calculations were automatically performed using a Hitachi 7050 automatic analyzer (manufactured by Hitachi, Ltd.).

Table 1 shows the results of fibrinogen and FDP-E patient specimens (5 specimens) measured by single item and the water droplet.

Table 1 Measuring method of fibrinogen and measured values (unit: m g
/ d Q) Book 2 FDP-E measurement method and measurement value (unit: ng/mQ)
Reagent is FDP-E measurement reagent) (Example 2) Amount of CRP and ASO 1) Reagent (1) CRP measurement reagent 50mM phosphate buffer solution of anti-human CRP antibody (antibody concentration; 0.5mg/m 12) 0.5 mu of polystyrene latex (manufactured by Tanesui Kagaku Co., Ltd., solid content concentration: 10% by weight) with an average particle size of 0.11 μm was added to 80 mQ of the solution, stirred at 10°C for 1 hour, and then stirred at 10°C for 30 minutes. Centrifugation (20.0OOr, 9.m) L. The unreacted supernatant was decanted, and the remaining antibody-sensitized latex particles were dissolved in 50 mM phosphate buffer solution [2 (W/V)
%BSA, 0.1M sodium chloride Contains sodium chloride (2) AS○ measurement reagent 80mQ of 50mM phosphate buffer solution of anti-human CRP antibody (antibody concentration: 0, 5 mg/mQ)
0 of polystyrene latex (manufactured by Tanesui Kagaku Co., Ltd., solid content concentration: 10% by weight) with an average particle size of 0.11 μm,
5 m fl was added, stirred at 10°C for 1 hour, and then centrifuged at 10°C for 30 minutes (20,0 OOr.p.
m. ) Shita. The unreacted supernatant was decanted and the remaining antibody-sensitized latex particles were dissolved in 50 mM phosphate buffer solution [2 (w/v)% BSA, 0.1 M sodium chloride and 0.05 (w/v)% anti- Contains human CRP antibody) 80m
It was redispersed in Q.

2) Measurement method 350μQ of CRP measurement reagent (first agglutination reaction reagent, hereinafter abbreviated as R1) and 3μQ of physiological saline were added and mixed in a reaction container (cell with optical path length 6m+), and reacted at 37°C. The absorbance at a wavelength of 570 nm was measured after 1 minute and 3 minutes. From this, calculate the amount of change in absorbance per unit time and use the reagent blank (Blk・ΔAbsCR) of the CRP measurement reagent.
P). Continuously, at the same time, 350 μQ of ASO measurement reagent (second agglutination reaction reagent, hereinafter abbreviated as R2) was added and mixed and allowed to react at 37°C.
The absorbance was measured at 00 nm. From this, calculate the amount of change in absorbance per unit time and use the reagent blank of the ASO measurement reagent (
Blk-ΔAbsASO).

Next, in the same manner as above, 3μi of patient sample serum and R,
350 μβ was added and mixed in a reaction container, and the reaction was kept at 37°C. The absorbance at a wavelength of 570 nm was measured after 1 minute and 5 minutes.
CRP). Subsequently, at the same time, 350 μa of R was added and mixed, and the mixture was reacted at 37° C., and the absorbance at a wavelength of 700 nm was measured after 1 minute and 3 minutes. From this, the amount of change in absorbance per unit time was determined and defined as the amount of change in absorbance due to ASO in the sample (S·ΔAbsASO). C of patient sample serum
The amount of change in absorbance due to RP (ΔAbsCRP) and the amount of change in absorbance due to ASO in the sample (ΔAbsASQ) are calculated using Equation 3.
Calculated from °4.

ΔAbsCRP = S・ΔAbsCRP−Blk・ΔA
bsCRP - Formula 3ΔAbsASO=S・ΔAbsA
SO-81? ΔAbsASO-Formula 4 In advance, a standard product containing known concentrations of CRP and ASO was used in place of the patient sample serum, and measurements were carried out in the same manner as above.The absorbance changes for each were determined from Formulas 1 and 2, and each calibration curve was calculated. I have created it.

The concentration of CRP and ASO in the sample is determined by using this standard line and CRP.
and ASO light absorbance change (ΔAbsCRP and ΔAb
The respective concentrations were determined from s/+SO). The above measurements and calculations were automatically performed using a Hitachi 7050 automatic analyzer.

Table 2 shows the measured values for single items and the measured values for CRP and ASO patient samples (5 samples).
RP measurement method and measured value (unit: mg/dΩ) Kyo2A S
Measuring method of O and measured value (unit: iu/mQ) Zero 3 Conventional method; Single item measurement (reagent is CRP measurement reagent) Traditional method;
Single item measurement (reagent is ASO measurement reagent, however.

No anti-human CRP antibody added. 〔Effect of the invention〕 As described above, according to the immunoassay method of the present invention.

Claims (1)

[Claims] 1. In an immunoassay method in which the agglutination reaction between an antigen and an antibody is measured as an optical turbidity change to determine the presence or amount of a target antigen or antibody in a specimen, in,
By first performing a first agglutination reaction based on one antigen or antibody, and then performing a second agglutination reaction based on another antigen or antibody, two target antigens can be obtained simultaneously for one specimen. or an immunoassay method characterized by determining the presence or amount of antibodies. 2. The immunoassay method according to claim 1, wherein the first agglutination reaction is a reaction that does not use a sensitized particulate carrier, and the second agglutination reaction uses a sensitized particulate carrier. 3. The immunoassay method according to claim 1, wherein the first agglutination reaction and the second agglutination reaction use a sensitized fine particle carrier.