JPH0752194B2 - Immunoassays using antibodies against solid antigens - Google Patents

Description

TECHNICAL FIELD The present invention relates to an immunoassay in which a solid substance powder is administered to an animal as an antigen to induce an immune response and the produced antibody is used as a reagent.

[Prior Art] The conventional analytical methods in the fields of medicine, food, etc. are based on the principle of chromatography and the reaction of complementary ligands (eg, antigens) and receptors (eg, antibodies) with each other. Based (so-called immunoassay).

However, the method based on the principle of chromatography has a drawback that it is not suitable for detecting a trace amount of substance because it is not sufficiently sensitive, and the procedure is complicated and requires skill, so that it cannot be performed by an unskilled person at any time.

On the other hand, the immunoassay is based on a specific reaction between a ligand and a receptor, so that it can detect an extremely small amount of substance, and once an antibody is prepared and a protocol is completed, it can be easily performed by an unskilled person. There are advantages.

As described above, it is premised that the antibody is obtained in the immunoassay, but in the conventional case, to obtain the antibody, a pure product of the compound to be analyzed is dissolved in water and administered to an animal to induce an immune response. To produce antibodies, and the antibodies were collected from body fluids (eg blood).

However, when trying to apply an immunoassay to a natural drug such as a crude drug in medicine, it is necessary to extract an appropriate active ingredient from the crude drug, purify it, and administer it as described above. However, since a variety of similar compounds are contained in crude drug components, mutual separation is complicated, and the content of one compound is very small, so it is not easy to collect it as a pure product. Another problem is whether the type and amount of the crude drug can be confirmed simply by analyzing one component of the crude drug.

[Problems to be solved by the invention] Prices of crude drugs fluctuate depending on quality, and similar products (counterfeit) are on the market for expensive crude drugs. , It is necessary to know the grade and content correctly. However, as mentioned above, there was no simple and accurate measurement method for crude drugs, so
Its development was strongly desired.

[Means for Solving the Problems] In the present inventor, it was common knowledge that the compound was conventionally administered in the form of an aqueous solution in order to induce an immune response.
An attempt was made to powderize the crude drug and directly dissolve it in water for administration. As a result, they have found that an immune response can be induced by such a method, and a specific antibody to the whole tissue of the administered crude drug can be obtained. Then, it was found that an immunoassay for the crude drug itself can be carried out by using this specific antibody, and that a specific component can be detected by selecting a specific ligand or a group of ligands for reacting with the antibody. It has been found that such a method can be widely applied not only to crude drugs but also to all plant products, animal products, food products, textile products and the like.

[Constitution of Invention] The present invention binds a label capable of generating a measurable signal or a precursor thereof to at least a part of a ligand or its ancillary receptor to competitively or non-competitively bind the ligand and the receptor. In a labeled immunoassay method of analyzing a ligand or a receptor by reacting and converting to a label when a precursor is used and measuring the generated signal, a ligand mixture containing at least a water-insoluble solid antigen as a solid is used as a receptor. The present invention provides a method comprising using an antibody produced in a body fluid by contacting the body fluid containing an antibody production system of an animal while containing the above.

The above method can be carried out by competitively reacting a free ligand and a solid phase ligand obtained by binding a ligand mixture containing at least a water-insoluble solid antigen on a solid support with a receptor. In this case, the ligand mixture bound to the solid-phase ligand may be different from the ligand mixture used for producing the antibody, but the antibody having cross-reactivity with both may be used.

Furthermore, the present invention is an antibody produced by contacting a ligand mixture containing at least a water-insoluble solid antigen with a body fluid containing an animal antibody production mechanism while containing a solid,
It is intended to provide an immune response inducer comprising a solid phase ligand obtained by binding a ligand mixture containing at least a water-insoluble solid antigen to a solid carrier and a ligand mixture containing at least a water-insoluble solid antigen.

[Definition] The terms used in the present invention are explained below.

Labeled immunoassay: A ligand or a precursor thereof capable of generating a measurable signal is bound to at least a part of a ligand or a receptor complementary thereto, and the ligand and the receptor are reacted competitively or non-competitively, and the precursor is reacted. Included are all methods of analyzing the ligand or receptor by converting it to a label, if used, and measuring the signal generated. This is due to differences in measurement targets (ligands, receptors), differences in labels (enzymes, radionuclides, spins, fluorescence, chemiluminescence, etc.), reaction formats (competitive, non-competitive), and systems (homogeneous, heterogeneous). Various methods are included based on differences and the like. That is, first, they are classified into the homogeneous method and the heterogeneous method depending on the difference in the system. The homogeneous method is a method that does not separate the ligand-receptor binding type and the non-binding type, and the heterogeneous method is a method that separates both. Each includes a competitive method and a non-competitive method depending on whether or not the labeled substance and the unlabeled substance are reacted competitively. Heterogeneous competition law
A liquid phase 2 antibody method using a second antibody (that is, an antibody against an antibody) in a liquid phase method, a solid phase 1 antibody method, a solid phase 2 antibody method, a method using a solid phase antigen and a labeled antibody, a sandwich method And a method using two types of antibodies against a titer antigen and an immunoenzymometric method (a method of removing unreacted labeled antibody with a solid phase ligand). Taking the case where an enzyme is used as a label, these are briefly described as follows.

Homogeneous competitive method: A method in which an enzyme-labeled ligand and an unlabeled ligand are competitively reacted with a receptor (antibody), and whether the enzyme that had an activity before binding to the receptor loses its activity by the binding, The opposite phenomenon is used. In the former case, the enzyme activity increases with an increase in the amount of unlabeled ligand (analyte), and in the latter case, it decreases on the contrary. This method includes modified methods such as using an enzyme bound with an inhibitor, a coenzyme, or a substrate instead of the enzyme.

Homogeneous non-competitive method: Receptor (antibody) enzyme-labeled with β-D-galactosidase, a ligand, and o-nitrophenyl-β-D-galactoside are reacted, and then a succinylated antibody is reacted, and the presence of a succinylated antibody Turbidity is measured by causing light scattering of the enzymatic reaction product.

Heterogeneous liquid phase 2 antibody method: In the liquid phase, an enzyme-labeled ligand and an unlabeled ligand are allowed to competitively react with a first antibody, and then a second antibody (for example, when the first antibody is rabbit IgG, goat anti-rabbit IgG) And immunoprecipitated to react with the conjugate (B)
And the non-bound body (F) (supernatant) are separated (S / F separation), and the enzyme activity of B or F is measured.

Heterogeneous solid phase 1 antibody method: The first antibody is bound to a solid support (hereinafter referred to as “solid phase”), and an enzyme-labeled ligand and an unlabeled ligand are allowed to undergo a competitive reaction, and after B / F separation, they are solid phased. The bound enzyme activity is measured.

Heterogeneous solid phase 2 antibody method: In the above liquid phase 2 antibody method, a solid phased second antibody is used as the second antibody.

Method using solid-phase ligand and labeled antibody ... Solid-phased ligand and non-solid-phased ligand (specimen) compete with enzyme-labeled receptor (antibody) for B / F separation, and then enzyme activity bound to solid phase To measure.

Sandwich method: A ligand (specimen) is bound to a solid-phased receptor (antibody), B / F separation is performed, and then an enzyme-labeled antibody is bound to a solid, B / F separation is performed, and the enzyme activity bound to the solid phase is detected. taking measurement. This sequence of reactions is called the forward method. As a modified method, the reverse method in which the reactions are performed in the reverse order of the above,
And there is simultaneous law. There is also a modified method in which a biotinylated antibody is used instead of the labeled antibody and detection is performed with labeled avidin or avidin + labeled biotin.

Immunoenzymometric method: A ligand (sample) is reacted with an enzyme-labeled receptor (antibody), unreacted receptor is removed by reaction with a solid-phased ligand, and the enzyme activity of the supernatant is measured.

The above-mentioned methods can be carried out in the same manner as known methods.

Ligand: an organic compound capable of producing a corresponding receptor, and when it is used in the production of an antibody in the present invention, in particular, animal or botanical herbs, dried plants, dried foods, vegetable seasonings, natural fibers, etc. Such a mixture containing powders of animal or vegetable substances. A typical crude drug is rhubarb,
Nikuzuku, cinnamon, bushi, bird head, yellow lichen, spicy, peony skin,
Includes opium, licorice, yellow oak, quintuplets, carrots, bamboo-bushi dolls, medicinal products, radishes, kisage, half-summer, buricho, juko, beef yellow, bear gall, etc. Foods include bonito, sardines, hamachi, daizu, beef, pork, sheep, chicken and the like. Fibers include cotton, wool, cashmere, alpaca and the like.

Receptor: A compound capable of recognizing and binding to a particular spatial and electronic structure of a molecule, such as an epitope or antigenic determinant. Receptors are usually antibodies or immunoglobulins and are obtained as antisera. Antibodies are produced by suspending the antigen in a suitable medium (eg, buffer), and optionally contacting the antibody production system with an adjuvant.

Label: A substance that produces a measurable signal, and includes an enzyme (in combination with a substrate), a spin compound, a radionuclide, a fluorescent substance, a chemiluminescent substance, a light absorbing substance, and the like.
A preferred label is an enzyme. Examples of the enzyme include peroxidase, β-galactosidase, glucose oxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase and the like. The methods for measuring enzyme activity include an absorbance method, a fluorescence method and a chemiluminescence method. For example, peroxidase uses a substrate such as 2,2'-azinodi (3-ethylbenzthiazoline) -6'-sulfonic acid (ABTS) as a chromogen, and the resulting dye is measured by the absorbance method. Since glucose oxidase produces hydrogen peroxide using glucose as a substrate, it can be coupled with peroxidase and measured in the same manner as above. β-galactosidase can be measured using o-nitrophenyl-β-D-galactoside as a substrate. Examples of the fluorescence method include peroxidase using p-hydroxyphenylpropionic acid as a substrate, β-galactosidase using fluorescein-β-D-galactoside or 4-methylumbelliferyl-β-D-galactoside as a substrate, and phosphatase. Can be measured using umbelliferyl phosphate as a substrate. As the chemiluminescence method, for example, peroxidase can be measured using luminol and hydrogen peroxide as substrates. To attach a label (eg enzyme) to an antigen or antibody, for example, bis [2- (succinimidooxycarbonyloxy) ethyl] sulfone (BSOCOES), bis (sulfosuccinimidyl) suberate (BS3), 1,5- Difluoro-2,4-dinitrobenzene (DFDNB), 4,4'-diisothiocyano-2,2'-disulfostilbene disodium salt (CDIDS), adipic acid dimethyl dihydrochloride, N- (γ-maleimide) Butyryloxy) succinimide (GMBS), N- (4-azidophenylthio) phthalimide (APTP), N-succinimidyl-6- (4′-azido-phenyl) -1,3 ′
-Use a crosslinking reagent such as dithiopropionate (SADP), pyridine disulfide, thiophthalimide.

Label precursor: A substance that can be converted into a label, such as a label blocked with an inhibitor.

Signal: visible light, fluorescence, radioactivity, other electromagnetic waves, etc., which can correlate the measured value with the amount of the substance that generated it.

Antibody production system: A system containing cells (eg, spleen cells, lymphocytes) or a set of components thereof necessary for recognizing an antigen and producing an antibody based on the antigen. Cattle, horses, sheep, goats, rabbits, rats, mice, etc. Excluding persons other than preserved organs.)
Is. The antibody is separated and collected from a body fluid (for example, blood).

Solid carrier: A substance capable of physically or chemically binding to or encapsulating a ligand or a receptor (antigenic determinant or a group capable of binding thereto) while maintaining the properties thereof, usually glass, natural or synthetic polymer. , Fungus body,
Plates or grains of red blood cells. Commonly used are plastic test tubes such as polystyrene, polyethylene, polypropylene and acrylic resin, microtiter plates, beads, and beads such as glass, sepharose and cellulose. Immobilization of the ligand or receptor is usually performed by a crosslinking agent, but the surface of the carrier may be silylated before that. As the cross-linking agent, for example, glutaraldehyde, triene diisocyanate, etc. are used.

Embodiments According to one embodiment of the present invention, dried plants, such as crude drugs, are powdered, preferably in an aqueous medium, for example by an ultrasonic disintegrator. Freund's complete adjuvant is mixed and emulsified in this suspension, and the mixture is subcutaneously administered to a mammal animal. When booster is required, it is preferably 2-8.
Repeat, for example, 5 times to collect blood and separate the specific antiserum. An enzyme such as β-D-galactosidase is bound to the antibody obtained from the antiserum with a crosslinking reagent, such as GMBS. An antigen (crude drug) is immobilized on a solid carrier such as aminodilac using a crosslinking agent such as glutaraldehyde. The sample and the solid-phased antigen are allowed to undergo a competitive reaction with the antiserum, and the antibody bound to the solid phase after B / F separation is measured by labeling the labeled antibody. A labeled curve is created using a known amount of sample and compared to this to measure an unknown amount of sample.

[Effect] According to the present invention, the antibody in the labeled immunoassay is produced by administering the whole tissue such as a crushed product of a crude drug to an animal as an antigen containing solids. Therefore, it is not necessary to extract and purify the active ingredient, which is troublesome. Can be omitted. In addition, since the antibody is against the whole tissue, the whole tissue can be used as an antigen in the assay. Furthermore, since the antibody has a wide range of specificity, it can be reacted not only with the tissue itself used to produce the antibody, but also with its components or similar crude drugs. As its application, for example, the sensitivity can be increased by competing with a solid-phase antigen having relatively low specificity for an antibody. Moreover, since the method of the present invention is based on the simple principle that an antibody is produced using the tissue as it is as an antigen, it can be widely used for authenticity determination of crude drugs, foods, fibers and the like, content measurement and the like.

[Examples] Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1 (Jako) [Method] Preparation of antiserum: 1 mg / ml of Jako was sensitized subcutaneously on the dorsal skin of a rabbit together with an equal amount of adjuvant, preferably Freund's adjuvant, and a 1/4 dose of booster immunization. Each of them was similarly sensitized three times in total, and blood was collected to obtain antiserum.

Preparation of solid phase: The musk was sonicated, and this was bound to the solid support, aminodilark, via glutaraldehyde.

Assay method: Solid phase antigen, specimen musk, antiserum 3
Anti-rabbit IgG-
After labeling with β-D-galactosidase and reacting with a fluorescent substrate,
The fluorescence value was measured. The measurement was performed according to the following procedure.

0.1 ml of serum and 0.1 ml of sample were incubated at room temperature for 1 hour, Amino Dailark-bound antigen was added, and the mixture was incubated at room temperature for 1 hour with shaking. After B / F separation and washing, 0.2 ml of anti-rabbit IgG-β-D-glucosidase was added to the solid phase and incubated at room temperature with shaking for 3 hours. After separation and washing, the solid phase is placed in a separate container with 0.2 ml of substrate and 30
Incubate for 1 hour at ℃, 0.2M-glycine-NaOH buffer (p
H: 10.6) 2 ml, excitation light 365 nm, fluorescence 448 nm It measured.

[Results] Examination of antiserum: As a result of performing a titer test on the antiserum obtained by sensitization, the highest titer was obtained at 8 weeks after the final sensitization. As a result of various studies, a 10 ⁴ -fold dilution of this antiserum was used for the subsequent measurements.

Measurement standard curve: The musk was measured using a solid phase antigen prepared with a 10 μg / ml solution of the sensitized musk. The measuring range of the standard curve is 10 −10 ⁴ ng / ml. (Fig. 1) Table 1 shows the measurement accuracy and reproducibility in this case. The coefficient of variation was less than 18.6% for diurnal variation and less than 23.2% for diurnal variation.

Examination of cross-reactivity: Table 2 shows the cross-reactivity when 50% inhibition of musk was taken as 100%. From this, it was revealed that the measurement method had specificity for the musk.

Example 2 (Takebushi ginseng) [Method] Preparation of antiserum: An antiserum was obtained by sensitizing a rabbit obtained by sonicating Takebushi ginseng powder as a sensitizing antigen.

Measurement method: Measurement was performed in the same manner as in Example 1 using a solid phase antigen in which sonicated Takebushi ginseng was bound to aminodilark as a solid phase support and an oxygen-labeled goat anti-rabbit IgG antibody.

[Results] Examination of standard curve: Fig. 2 shows the standard curve of Takebetsu ginseng obtained using an antiserum against Takebushi ginseng. This measurement method
Since ginseng can be measured, its standard curve is also shown in FIG. The measuring range of Takebushi ginseng is 100 ng-μg, and the measuring range of ginseng is 10 ng-3 μg. The accuracy and reproducibility of the measurement are shown in Table 3.

Examination of cross-reactivity: Table 4 shows the cross-reactivity when 50% inhibition of Takebushi ginseng is taken as 100%.

The strongest cross-reactivity was found in all ginseng herbs, especially ginseng. No cross-reactivity was observed with other herbal medicines such as musk and half-summer.

Application: The method of measuring ginseng extract in Kampo preparations was examined. As a result of various studies, a standard curve of ginseng extract as shown in FIG. 3 was obtained when ginseng extract was used as the solid phase antigen instead of sonicated bamboo ginseng powder.

This result is contrary to the conventional wisdom in immunology,
It is a completely new standard test method for ginseng preparations that had no conventional measurement method.

Example 3 (half summer) [Method] Preparation of antiserum: 1 mg / ml half summer was obtained by sensitizing the back of a female rabbit with an equal amount of Freund's complete adjuvant. Additional rabbits were done in the same way with the previous half amount, and a total of 5 times.

Preparation of solid-phase antigen: The crushed half-summer was sonicated and fixed with 1% glutaraldehyde solution on the aminated Dilark of the solid-phase carrier.

Oxygen immunoassay: After the primary reaction between standard half-summer and the antibody, the remaining free antibody was allowed to react with the solid antigen, and the amount of the antibody reacted with the solid antigen was measured as the anti-rabbit IgG antibody-β-D-galactosidase complex. After labeling with the body, it was reacted with a fluorescent substrate and the resulting fluorescent product was measured with a fluorescence spectrophotometer.

[Results] Preparation of standard curve: FIG. 4 shows the standard curve obtained using the anti-half-summer antiserum. The measurement range was 0.1-10 μg / ml. Table 5 shows the measurement accuracy and reproducibility of this method.

Examination of cross-reactivity: Half-summer sensitizing antigen (Sichuan 501-C09
Table 6 shows the cross-reactivity when the 50% inhibition of 1) was taken as 100. Although it was possible to measure in the summer of different production areas using this method,
No cross-reactivity was observed with other herbal medicines, musk and ginseng.

Example 4 (Ginseng) [Method] Preparation of antiserum: After sonicating 1 mg of ginseng powder, the back of a female rabbit was sensitized with an equal amount of Freund's complete adjuvant. 1/2 booster every 2 weeks
The amount was similarly sensitized and this was done twice. From the day of the first sensitization, blood was collected from the ear vein every week to obtain antiserum.

Preparation of solid-phase antigen: Ginseng powder was sonicated and fixed with 1% glutaraldehyde solution on aminated Dilerk solid-phase carrier.

Assay method: According to the basic assay method, goat anti-rabbit IgG antibody-GMBS-enzyme β-D-galactosidase was used as an enzyme label, and after reacting with a substrate, immunological activity was measured by measuring the fluorescence value of saw.

[Results] Examination of antiserum: The results of examination of the titer of the antiserum obtained by sensitization are shown in FIG. For the measurement, an antiserum 9 weeks after the first sensitization having the highest titer was used.

Preparation of marker curve: Fig. 6 shows a standard curve of ginseng prepared according to the measuring method. Measuring range is 100ng-10μg
Table 7 shows the measurement accuracy and reproducibility in this case.

Cross-reactivity study: Cross-reactivity of other herbal medicines, ginseng and ginseng extract 10% of ginseng with 50% inhibition
It is shown in Table 8 as 0%.

[Brief description of drawings]

FIG. 1 is a standard curve in Example 1, FIGS. 2 and 3
The figure shows the standard curve in Example 2, FIG. 4 shows the standard curve in Example 3, and FIGS. 5 and 6 show the standard curve in Example 4.

Claims (6)

[Claims] 1. A precursor or a precursor thereof capable of generating a measurable signal is bound to at least a part of a ligand or a receptor complementary thereto, and the ligand and the receptor are reacted competitively or non-competitively to give a precursor. When the body is used, it is converted into a label, and the generated signal is measured to analyze the ligand or the receptor. In a labeled immunoassay method, a ligand mixture containing at least a water-insoluble solid antigen as a receptor is used as an animal antibody production system. A method comprising using an antibody produced in a body fluid by contacting the body fluid containing the body fluid. 2. The method according to claim 1, wherein a free ligand and a solid phase ligand obtained by binding a ligand mixture containing at least a water-insoluble solid antigen to a solid support are competitively reacted with the receptor. 3. The method according to claim 2, wherein the ligand mixture bound to the solid phase ligand is different from the ligand mixture used for producing the antibody, but the antibody has cross-reactivity with both. . 4. An antibody produced by bringing a mixture of ligands containing at least a water-insoluble solid antigen into contact with a body fluid containing an animal antibody production mechanism. 5. A solid-phase ligand obtained by binding a ligand mixture containing at least a water-insoluble solid antigen to a solid carrier. 6. An immune response inducer comprising a ligand mixture containing at least a water-insoluble solid antigen.