JP3185215B2 - Immunoassay - Google Patents

Description

The present invention relates to a luminescence immunoassay using insoluble carrier particles.

[Prior Art] Conventionally, an immunoassay utilizing an antigen-antibody reaction has been known as a method for early detection of various diseases and a method for detecting trace amounts of substances. There are various high-sensitivity immunoassays, such as radioimmunoassay (RIA), which uses an antibody or antigen bound with a radioisotope (RI), an enzyme, a fluorescent substance, a luminescent substance, etc. as a labeling substance, and an enzyme immunoassay ( EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA)
And so on. Also, a method of reacting an antibody or antigen carried on insoluble carrier particles such as latex with a corresponding antigen or antibody and measuring the speed of the antigen-antibody reaction from the change in the transmitted light of the reaction mixture accompanying the reaction (LP
IA) is known.

[Problems to be Solved by the Invention] However, such conventional techniques have various problems. For example, RIAs have restrictions on handling and disposal of RIs. Although LPIA is a method excellent in simplicity and operability, improvement of detection sensitivity is desired. EIA, FIA, etc. are advantageous in handling safety, but the detection sensitivity is slightly inferior to RIA. Also, EIA involves an enzymatic reaction and is not as easy to handle as FIA.

As a method of improving detection sensitivity in FIA, JP-A-59-122
No. 950 describes an immunoassay using fluorescent fine particles. That is, since the number of fluorescent substances can be increased by loading the antigen or antibody on colloid particles containing a fluorescent substance instead of the labeled antigen or labeled antibody usually used in FIA, high-sensitivity detection is expected. Is done.

However, it is known that so-called particle agglutination reactions such as latex agglutination reaction, gelatin agglutination reaction, and the like, particularly non-specific reactions involving non-specific agglutination are particularly frequently observed. That is, a phenomenon in which the antigen or antibody to be measured is observed to be present even though it is not present at all in the sample liquid, or to be observed to be present more than actually exists. Known causes include non-specific adsorption between particles and solid phase, non-specific aggregation of particles, and reaction by a so-called non-specific sample containing a non-specifically reacting substance. Therefore, as long as the particles are used without using a method utilizing particle aggregation,
Attention must be paid to these non-specific reactions.

For example, as an example of a non-specific sample, a substance called rheumatoid factor is present in the blood of many patients with interstitial rheumatism. This is a kind of autoantibody, which is an IgM or IgG type antibody having the property of reacting with the IgG fraction of immunoglobulin, and shows a cross-reactivity with IgG of other species. Therefore, in the latex carrying IgG, non-specific aggregation due to the rheumatoid factor occurs.
Therefore, in the reagents that utilize the latex agglutination method that is currently in practical use, the non-specific reaction is reduced by removing the Fc portion that reacts with the rheumatoid factor from the IgG molecule by treatment with pepsin. Further, 'since the analyte also present in minor amounts to react with _2, in this case the reaction system F prepared from rabbits not immunized (ab F (ab)' a) ₂ was added, the latex reagent F (ab ') ₂ It is dealt with by absorbing before reacting. (Mitsubishi Kasei R & D Re
view, 2 , 105 (1988)).

Alternatively, there is a method in which non-specific reactions are removed by removing unnecessary proteins by pretreating the sample with an acetate buffer or the like.

Although the occurrence of non-specific reactions can be considerably reduced by the above method, it is still impossible to completely suppress non-specific reactions, and adsorption caused by the particles themselves cannot be avoided. Also,
No method has been developed to measure nonspecific reactions. Therefore, those which dissociate compared to the measured values obtained by a method such as RIA or EIA, which is said to have relatively few nonspecific reactions, are called nonspecific reactions.

Therefore, in order to commercialize an immunoassay using fluorescent fine particles as described in JP-A-59-122950 as a highly sensitive detection method, a method for suppressing or detecting these nonspecific reactions has been developed and used in combination. Is an important requirement.

[Means for Solving the Problems] The present inventors have conducted intensive studies to solve the above problems, and as a result, can detect non-specific reactions of particles while labeling the insoluble carrier particles with high sensitivity. A new luminescent immunoassay was developed.

That is, the gist of the present invention is as follows: (1) (a) (i) a first dye for emitting fluorescence or phosphorescence and (ii) a first dye for immunoassay comprising an antibody or antigen carried on insoluble carrier particles. (B) (i) the first dye is different from the first dye in terms of emission wavelength, excitation wavelength or emission lifetime, and emits fluorescence or phosphorescence; and (ii) is supported on the first particle. A second dye-labeled particle for detecting non-specific reaction or adsorption, wherein the antibody or the antigen contains a substance not containing a site that specifically binds to the antigen or the antibody in the sample, and is supported on the insoluble carrier particles. c) reacting with a sample solution containing the antigen or antibody to be measured, and measuring the change in signal due to the first dye-labeled particle accompanying the antigen-antibody reaction and the change in signal due to the second labeled particle due to the non-specific reaction, Non-specific during antigen-antibody reaction An immunoassay method characterized by detecting a different reaction (excluding a method using a reaction vessel in which a substance that specifically binds to the antigen or antibody to be measured is immobilized).

 Hereinafter, the present invention will be described in detail.

The labeling dye used in the present invention is a dye that emits fluorescence or phosphorescence. In the present invention, two types of dyes are respectively supported on insoluble carrier particles, and the first dye particles are used for immunoassay.
Since the second dye-labeled particles are used for detecting a non-specific reaction or adsorption, it is necessary that the first and second dyes can be independently measured in a coexisting system. Therefore, the conditions required for the first and second dyes used in the present invention are, for example, 1) that the emission peak wavelengths are separated from each other.

2) The wavelengths of the excitation lights are separated from each other.

3) The lifetimes of the light emission are separated from each other.

And the like.

As an example of 1), for example, when europium (Eu) and samarium (Sm) are used as rare earth chelate compounds as elements, two kinds of antigens or antibodies can be measured simultaneously.
As suggested by Clinical Chemistry, 33 , 48 (1987), the combination of Eu (maximum emission: 615 nm) and terbium (Tb) (maximum emission: 545 nm) is farther away from the peak wavelength of emission (about 70 nm). Rare earth chelate compounds are suitable for separation measurement because of their sharp emission spectrum, but other combinations of fluorescent and phosphorescent dyes that can be measured by compensating for the effects of background and coexistence of dyes But no problem. Usually, a combination of fluorescent dyes such as fluorescein isothiocyanate (emission maximum 520 nm), tetramethylrhodamine isothiocyanate (emission maximum 570 nm), and phycobiliprotein (phycocyanin: emission maximum 650 nm, phycoerythrin: emission maximum 580 nm) can be used. Many.

In the case of 2), many luminescent dyes having different excitation lights are known, but the excitation light generally shows a broad spectrum similarly to the emission. Many can be observed. Therefore,
Actually, more accurate measurement can be performed by a combination of 1) and 2), that is, a combination of dyes in which emission peak wavelengths are separated from each other and excitation light wavelengths are also separated from each other. These combinations include
Examples include a combination of Eu and Tb.

Examples of 3) include combinations of ordinary fluorescent dyes such as fluorescein and rhodamine (lifetime number—several tens of nanoseconds) and rare earth chelate compounds (lifetime number of 10 μsec—several milliseconds),
A combination of a usual short-lived fluorescent dye such as fluorescein or rhodamine and a phosphorescent dye such as eosin, or a combination of an Eu chelate compound (lifetime of 100 μsec) and a Tb chelate compound (lifetime of 10 μsec to several msec) can be considered.

The insoluble carrier particles that carry out the dye labeling are substantially suitably insoluble in the liquid medium used in the reaction and are 0.01 to 10 μm.
m, preferably those having an average particle size of 0.05-3 μm.

The material of the carrier particles is a polystyrene, a latex of an organic polymer obtained by emulsion polymerization such as styrene-butadiene copolymer, or silica, an inorganic oxide such as silica-alumina, a lipid polymer such as liposome,
Biological components such as red blood cells, metal colloid particles such as gelatin or gold colloid, and the like are used.

The method of supporting the labeling dye on the insoluble carrier particles includes a method of chemically binding, a method of adding a dye when polymerizing the particles and confining the inside of the particles, and a method of binding the dye to a protein or peptide in advance. After that, there is a method of supporting the protein or peptide on the particles. For example, Japanese Patent Application Laid-Open No. 54-101439 describes a method in which a rare-earth chelate is confined inside a latex of an organic polymer using a cooperative extraction method with TOPO (tri-n-octylphosphine oxide). . The labeled particles prepared according to the method had good labeling strength and stability.

The first labeled carrier particles of the present invention carry the above-mentioned first labeled dye and an antigen or antibody which is the same or opposite to the antigen or antibody to be measured. The second labeled carrier particles carry a substance that does not include a site that specifically binds the second dye and the antigen or antibody to be measured, which can be measured in the presence of the first dye.

Many methods have been proposed for supporting (sensitizing) a substance that does not contain a site that specifically binds to the antigen or antibody or the antigen or antibody to be measured on the insoluble carrier particles. For example, it is well known that a substance to be sensitized to a carrier is physically adsorbed, a method of chemically modifying a substance after being chemically modified with a coupling agent, and a method of bonding with a spacer molecule interposed therebetween. . After sensitizing the antibody, the antigen may be bound to the antigen-sensitized particles. In addition, there are a method of incorporating an antigen into a particle at the time of preparing a particle by polymerization, a method of physically or chemically sensitizing the protein after binding to another protein by a chemical bonding method, and the like.

Similar to sensitizing insoluble carrier particles with a substance that does not contain a site that specifically reacts with the antigen or antibody or the antigen or antibody to be measured, the method for supporting the antigen and antibody on the solid phase is also applied to each solid phase. In addition, various methods have been applied. Also in this case, there are a method by physical adsorption and a method by chemical bonding.

As the antibody, IgG is usually used. However, using an enzyme such as pepsin or papain or a reducing agent such as dithiothreitol or mercaptoethanol, F
(Ab ') ₂ , Fab, Fab' and the like, which have a reduced molecular weight, may be used. In addition, not only IgG but also IgM or IgG
A fragment whose molecular weight has been reduced by the same treatment as described above may be used. Further, both polyclonal antibodies and monoclonal antibodies can be applied.

For application of monoclonal antibodies, one or more monoclonal antibodies can be used for proteins having a repeating structure, such as hepatitis B virus surface antigen. Further, even if there is no repeating structure, the present invention can be applied if two or more monoclonal antibodies having different recognition epitopes are used in combination.

On the other hand, examples of antigens include various antigens such as proteins, polypeptides, steroids, polysaccharides, lipids, pollen, recombinant proteins produced by genetic engineering, and drugs.

Those sensitized to the second labeled particles include immunoglobulin fractions that do not contain a site that specifically reacts with the antigen or antibody to be measured, derivatives thereof or other proteins, or stabilization of carrier particles. Surfactants and polymers that cover the particle surface. For example, F purified from antiserum obtained by immunizing a rabbit with the antigen to be measured is applied to the first labeled particle.
(Ab ') ₂ the case of sensitization, the second label particle, F (ab purified from rabbits not immunized' it is desirable to sensitize) _2. Alternatively, when the first labeled particle is sensitized with an antigen produced by using a genetic recombination technique, the second labeled particle is sensitized with another protein present in the culture solution, and the second labeled particle is present in the antigen. It is possible to detect a non-specific reaction originating from the impurities.

The order in which the substance to be sensitized with the labeling dye is carried on the insoluble carrier particles is not particularly limited, but it is preferable to carry the substance to be sensitized after carrying the labeling dye.

The amounts of the first labeled particles and the second labeled particles are not particularly limited, but are generally 0.0001-1% by weight in the reaction solution.
Preferably, 0.001-0.1% by weight is used. There is no particular limitation on the ratio between the first labeled particles and the second labeled particles, but the second particles may also function to suppress non-specific reactions of the first particles. It is preferable to use the same amount or more as one particle. When the amount of the first and second carrier particles is different from each other, the amount is usually converted into the surface area.

A known method can be used to measure a change in a signal caused by the first dye-labeled particle accompanying a specific immune reaction and a change in a signal caused by a second dye-labeled particle accompanying a non-specific immune reaction. For example, reaction tubes, glass beads,
Fine particles such as latex and gelatin, fine particles such as polystyrene and cellulose containing magnetic substances, membranes,
Using a filter or the like as a solid phase, a method of measuring an increase in signal by measuring each dye-labeled particle bound via the antigen or antibody to be measured by a sandwich method, an inhibition method, or the like, The decrease in signal may be measured by measuring each of the remaining dye-labeled particles that did not bind.

When measuring, SDS or Tween
The measurement can also be performed in a state where the labeled particles bound by using a surfactant such as en20 are separated from the solid phase and suspended in a liquid.

In the case of measuring the supernatant, the amount of decrease in the fluorescence intensity of the first labeled particle represents the sum of the amount of specific reaction with the antigen to be measured and the amount of nonspecific reaction with the solid phase, and the intensity of fluorescence of the second labeled particle. Represents the amount of non-specific reaction with the solid phase.

In the case of measuring the amount of solid phase binding, the amount of increase in the fluorescence intensity of the first labeled particle represents the sum of the specific reaction amount due to the binding to the antigen to be measured and the non-specific reaction amount with the solid phase, and the second The amount of increase in the fluorescence intensity of the labeled particles indicates the amount of non-specific reaction with the solid phase.

As a method of measuring a signal change, instead of measuring the one bound to a solid phase as described above, aggregated particles obtained by separating unreacted particles through dye-labeled particles aggregated by an antigen-antibody reaction May be measured.

When separating the labeled particles bound to the solid phase from the unbound labeled particles, the washing is usually repeated several times as described above, but in the case of a solid phase containing a magnetic substance, If magnetic force is used, separation can be performed relatively easily. There is also known a method of separating easily by passing through a filter or the like.

An antigen measurement using a solid phase containing a magnetic substance will be described below as an operation example.

After mixing and suspending a solid phase containing a magnetic substance (hereinafter, referred to as a Mg solid phase) on which a primary antibody against the antigen to be measured is immobilized, a first labeled particle, and a second labeled particle, A standard solution or a sample solution containing the antigen to be measured and a reaction buffer are added and incubated for a certain period of time. The Mg solid phase and the reaction aggregate containing the Mg solid phase are precipitated using a magnet, and separated into a supernatant sample and a precipitated sample.

From the supernatant sample, the signal of each of the remaining dye-labeled particles that did not react with the solid phase is obtained, and the change in the signal is measured as a decrease in the fluorescence intensity.

From the precipitated sample, a signal of each dye-labeled particle that has reacted with the solid phase is obtained, and a change in the signal is measured as an increase in fluorescence intensity.

In addition, Japanese Patent Application No. 1-329093 describes a method for detecting aggregation of fine particles carrying a long-lived luminescent dye according to an immune reaction as a change in the degree of emission polarization. According to this method, the measurement can be easily performed without separating the reaction solution.

[Example] Hereinafter, a more detailed description will be given based on an example, but the present invention is not measured in the example unless it exceeds the gist.

Example 1 A rare earth chelate Eu-TTA thenoyltrifluoroacetone compound (1 × 10 ⁻⁴ mol) and TOPO (described above) (2 × 10 ⁻⁴ mol) were dissolved in 40 g of acetone, and then a polystyrene latex having a particle size of 0.22 μm was dissolved. (Dow) 3 g suspended in 40 ml of water were mixed, and acetone was removed by an evaporator. Thus, an Eu chelate compound was co-extracted with the TOPO to latex particles to produce Eu-labeled particles.

Similarly, a rare earth chelate compound Tb-PTA pivaloyl trifluoroacetone compound 5 × 10 ⁻⁵ mol and TOPO 11 ×
10 ^-4 mol of polystyrene latex having a particle size of 0.497 μm (D
ow Co.) to prepare Tb-labeled particles.

After rabbit anti-HBs (hepatitis B virus surface) antigen F (ab ') ₂ was sensitized to Eu-labeled latex, the particles were stabilized by treatment with BSA.

After sensitizing normal rabbit F (ab ') ₂ to Tb-labeled latex, the particles were stabilized by treatment with BSA.

Rabbit anti-HBs antigen F (ab ') ₂ is sensitized to a magnetic material-containing polystyrene texex (hereinafter, referred to as Mg latex, Rhone Poulin) having an average particle size of 1.5 μm, and then treated with BSA to stabilize the particles. I let it.

Eu-labeled latex 0.02%, Tb-labeled latex 0.2%, M
A mixed suspension of 0.2% g latex was prepared and used as a latex reagent.

As an antigen, HBs antigen produced by a genetic recombination method was diluted with Tris buffer to obtain 0, 17.5, 35, 70, 140, 280 U /
A standard solution of ml was prepared.

After adding 60 μl of the antigen solution, 460 μl of the BSA-containing Tris buffer, and 80 μl of the above latex reagent, the mixture was stirred and an immunoreaction was performed for 10 minutes.

Next, using a magnet, Mg latex and a reaction aggregate containing Mg latex are precipitated to obtain a supernatant sample. The remaining precipitate is washed several times with water, and finally dispersed in a 0.1% SDS solution to obtain a precipitate sample.

Using the Hitachi Fluorescence Spectrophotometer F-4010, the excitation light 345 nm, the fluorescence 616 nm (Eu), the excitation light 296 nm, and the fluorescence 546 nm (Tb) were measured for both the above-mentioned sample and the precipitate, and the results are shown in Table 1. , Shown in FIG.

It can be seen that as the antigen concentration in the antigen solution increases, the fluorescence intensity of Eu decreases in the supernatant sample and increases in the precipitate sample. On the other hand, it can be seen that non-specific agglutination rarely occurs in a standard solution such as the above-mentioned antigen solution, and the fluorescence intensity of Tb always shows a substantially constant small value.

[Example 2] The latex reagent (Eu label, Tb label,
Mg latex mixed suspension).

Normal human serum (HBs antigen negative confirmed by EIA method), non-specific sample (Non-specific agglutination by LPIA method and HBs antigen negative confirmed by EIA method) as sample solution ), HBsAg positive sample (Midori Cross HBsAg. LP
Positivity was confirmed by the IA method).

The same reaction and separation operation as in Example 1 were performed, and the supernatant,
Both samples of the precipitate were prepared, and the fluorescence concentrations of Eu and Tb were measured respectively. The results are shown in Table 2 and FIG.

Assuming that the cut-off value of HBs antigen is 10 U / ml, Example 1
As a result, the measured value of Eu is 136.9 or less in the supernatant, and 7.404 or more in the precipitate is HBs antigen-positive. Similarly, from the results of Example 1, it is considered that the measured value of Tb, which is a monitor for nonspecific reaction, is 45.0 or less in the supernatant and 5.0 or more in the precipitate.

Therefore, when the measured value of Eu becomes HBs antigen positive, T
By considering the measurement of b, whether it is true positive,
Judge whether to suspend the judgment. In the case of suspension of the determination, it is usually performed to determine whether the antigen is positive by an operation of absorbing the antigen in the sample solution with the confirmation antibody.

When the above determination was applied to the various specimens of this example, one of the non-specific specimens was determined to be HBs antigen-positive in the precipitated sample, but the remaining specimens were positive for HBs antigen. It can be seen from the measurement of Eu that the antigen is positive and that the measurement of Tb indicates that there is no non-specific reaction. In the case of a negative sample, the measured value of Eu indicates negative, or when the measured value of Eu enters a positive region, the measured value of Tb also indicates a non-specific reaction.
Therefore, when it is determined to be negative from the Eu measurement, the true positive can be more accurately determined by considering the measured value of Tb together. (See Table 3) [Effects of the Invention] According to the method of the present invention, not only high-sensitivity immunoassay can be performed, but also nonspecific reaction of particles can be detected.

[Brief description of the drawings]

FIG. 1 is a diagram showing the change in the fluorescence intensity with respect to the HBs antigen concentration. In the figure, ○ indicates the fluorescence intensity of the Eu chelate compound in the supernatant sample, ● indicates the fluorescence intensity of the Eu chelate compound in the precipitate sample, and Δ indicates the supernatant. The fluorescence intensity of the Tb chelate compound of the sample, and the triangle indicates the fluorescence intensity of the Tb chelate compound of the precipitated sample. FIG. 2 is a diagram showing the fluorescence intensities of a supernatant sample and a precipitate sample using normal human serum, a non-specific (HBs antigen-negative) sample and an HBs antigen-positive sample as a sample solution. Has the same meaning as in FIG.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-62-81566 (JP, A) JP-A-3-216554 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 33/543 501 G01N 33/531

Claims (3)

(57) [Claims] (1) A first dye-labeled particle for immunoassay, comprising (a) (i) a first dye which emits fluorescence or phosphorescence and (ii) an antibody or an antigen carried on insoluble carrier particles. b) (i) a second dye which emits fluorescence or phosphorescence and has a different emission wavelength, excitation wavelength or emission lifetime from the first dye, and (ii) an antibody or antigen carried on the first particles. A second dye-labeled particle for detecting a non-specific reaction or adsorption, comprising a substance which does not contain a site that specifically binds to an antigen or an antibody therein, and (c) an antigen or antibody to be measured React with a sample solution containing
By measuring the change in the signal due to the first dye-labeled particle due to the antigen-antibody reaction and the change in the signal due to the second dye-labeled particle due to the non-specific reaction, it is possible to detect the non-specific reaction during the antigen-antibody reaction. A characteristic immunoassay method (excluding a method using a reaction vessel in which a substance that specifically binds to the antigen or antibody to be measured is immobilized). 2. The method according to claim 1, wherein the second dye-labeled particles are (i) to (ii)
The immunoassay according to claim 1, wherein the substance selected from (i) is carried on an insoluble carrier. (I) an immunoglobulin fraction and a derivative thereof that do not contain a site that specifically reacts with the antigen or antibody to be measured; and (ii) a case in which the first dye-labeled particle carries an antigen produced using a genetic recombination technique. Are other proteins present in the culture, and (iii) surfactants and polymers that cover the particle surface for stabilization of the insoluble carrier particles. 3. A microparticle containing a magnetic substance specifically reacting with an antigen or an antibody to be measured is used as a solid phase, and a dye-labeled particle bound to the solid phase and a dye-labeled particle not bound are used by magnetic force. 2. The method according to claim 1, wherein a change in signal of the supernatant sample or the precipitate sample is measured.
Or the immunoassay according to 2.