CN111272997A - Homogeneous phase immunoassay kit, homogeneous phase immunoassay method and application of homogeneous phase immunoassay kit - Google Patents

Abstract

The present invention relates to a homogeneous immunoassay kit and its application in the field of biotechnology. The kit includes Reagent I, which contains a first counterpart; the first counterpart is a known antigen or a known antibody that can specifically bind to the antigen to be tested in the sample to be tested; Reagent II, which contains a Singlet oxygen reacts to generate a signal-detectable receptor and a second counterpart bound to it, the second counterpart capable of specifically binding to the first counterpart; Reagent III, comprising a reagent capable of specifically binding to the first counterpart The bound third counterpart; Reagent IV, which comprises a donor capable of producing singlet oxygen in the excited state. The third counterpart is coated with biotin and the donor is coated with streptavidin. Using the homogeneous immunodetection method for combined detection of antigen and antibody by the kit reduces the difficulty of screening raw materials, and at the same time improves the detection sensitivity.

Description

A kind of homogeneous immunoassay kit, detection method and application thereof

This application is a divisional application of a Chinese patent application with an application date of November 27, 2017, an application number of 201711203146.6, and an invention title of "A homogeneous immunoassay kit, detection method and application thereof".

technical field

The invention belongs to the field of biotechnology, and in particular relates to a homogeneous immune kit and its application in the combined detection of the same virus antigen and antibody.

Background technique

In some in vitro diagnostic projects using chemiluminescence methods, it is necessary to detect both antigens and antibodies in the sample. For example, the detection of human immunodeficiency virus (Human Immunodeficiency Virus, HIV) has been developed from the third-generation reagents that only detect viral antibodies to the fourth-generation AIDS detection reagents that are more commonly used for combined detection of antigens and antibodies. In this way, it is possible to avoid the risk of missed detection in patients in the window period because they only have viral antigens but no corresponding antibodies or the corresponding antibody titers are not enough to be detected. Therefore, the establishment of an efficient and reliable method for combined detection of antigen and antibody is of great significance in the field of diagnostic reagent development and even in the medical and health industry.

The current chemiluminescence antigen-antibody joint detection method performs detection by labeling the antigen and the labeling antibody forming a sandwich complex with the antibody or antigen in the sample to be detected, and finally generating a signal for detection. Any one of the antigens or antibodies to be tested in the sample is reflected as an increase in signal. The disadvantage is that it cannot distinguish whether the positive signal value is caused by antigen or antibody, so it cannot accurately judge the course of disease, and cannot provide reliable information for clinical treatment. In addition, the labeled raw materials in the detection reagent need to be strictly screened, and the labeled antigen and antibody cannot cross-react, which increases the difficulty of reagent development and prolongs the development cycle. At the same time, since certain sites are artificially avoided, the sensitivity of some antigen and/or antibody detection is bound to be sacrificed.

SUMMARY OF THE INVENTION

Aiming at the deficiencies of the prior art, the present invention provides a homogeneous immune detection kit, and a homogeneous immune method for jointly detecting the same virus antigen and antibody by using the kit. The method integrates the competition method and the sandwich method, and only needs to label the antigen or the antibody, and does not need to label the antigen and the antibody at the same time, thereby avoiding potential cross-reaction, reducing the difficulty of screening raw materials, and improving the detection sensitivity. In addition, the method provided by the present invention has more clinical significance in the interpretation of the results. The results can determine whether the sample to be tested is positive for antigens or positive for antibodies, which provides a basis for the diagnosis of epidemic diseases and the formulation of treatment plans.

To this end, a first aspect of the present invention provides a homogeneous immunoassay kit, comprising:

Reagent I, comprising a first counterpart; the first counterpart is a known antigen or a known antibody that can specifically bind to the antigen to be tested in the sample to be tested; Reagent I can be used to dilute the sample to be tested;

Reagent II comprising a receptor capable of reacting with singlet oxygen to generate a detectable signal and a second counterpart bound thereto, the second counterpart being capable of specifically binding to the first counterpart;

Reagent III, which comprises a third counterpart capable of specifically binding to the first counterpart;

Reagent IV, which comprises a donor capable of producing singlet oxygen in the excited state.

In some embodiments of the present invention, the first counterpart is a known antibody that can specifically bind to the antigen to be tested in the sample to be tested, and both the second counterpart and the third counterpart are known antigen.

In some embodiments of the present invention, the known antibody is a monoclonal antibody or antibody positive serum.

In other embodiments of the present invention, the first counterpart is a known antigen, and both the second counterpart and the third counterpart are second antibodies capable of specifically binding to the known antigen.

In some embodiments of the present invention, the second antibody is a monoclonal antibody and/or a polyclonal antibody.

According to the present invention, the surface of the third counterpart is coated with biotin, while the surface of the donor is coated with streptavidin.

In some embodiments of the present invention, the acceptor comprises an olefin compound and a metal chelate and is soluble in an aqueous medium.

In other embodiments of the present invention, all reagents are non-particulate and soluble in aqueous media.

In the second aspect of the present invention, there is provided a homogeneous immunoassay method for combined detection of the same virus antigen and antibody using the kit according to the first aspect of the present invention, which comprises the following steps: The negative sample of the antibody is detected, and the obtained detection result is used as the background signal value; then the chemiluminescence signal value of the sample to be tested is detected, and the chemiluminescence signal value of the sample to be tested is compared with the background signal value. The comparison is performed to determine whether the antigen to be tested and/or the antibody to be tested exists in the sample to be tested.

In some embodiments of the present invention, the first counterpart contained in the reagent I is a known antibody that can specifically bind to the antigen to be tested in the sample to be tested, at this time:

If the detected chemiluminescence signal value of the sample to be tested is equal to the background signal value, then the sample to be tested does not contain the antigen to be tested and the antibody to be tested;

If the detected chemiluminescence signal value of the sample to be tested is greater than the background signal value, then the sample to be tested contains the antibody to be tested;

If the detected chemiluminescence signal value of the sample to be tested is less than the background signal value, the sample to be tested contains the antigen to be tested.

In other embodiments of the present invention, the first counterpart contained in the reagent I is a known antigen, and in this case:

If the detected chemiluminescence signal value of the sample to be tested is equal to the background signal value, then the sample to be tested does not contain the antigen to be tested and the antibody to be tested;

If the detected chemiluminescence signal value of the sample to be tested is greater than the background signal value, the sample to be tested contains the antigen to be tested;

If the detected chemiluminescence signal value of the sample to be tested is less than the background signal value, the sample to be tested contains the antibody to be tested.

According to the present invention, the method for detecting the chemiluminescence signal value of the sample to be tested comprises the following steps:

S1, mixing the sample to be tested and the reagent I to obtain a first mixture;

S2, adding reagent II, reagent III and reagent IV to the first mixture to obtain a second mixture;

S3, treating the second mixture with energy or an active compound, and detecting the chemiluminescence signal value of the second mixture;

S4, analyze the chemiluminescence signal value to determine whether the sample to be tested contains the antigen to be tested and/or the antibody to be tested.

In some embodiments of the present invention, in step S2, reagent II and reagent III are added first, and then reagent IV is added.

In other embodiments of the present invention, in step S1, the sample to be tested is added to the solution containing reagent I.

Here, it should be noted that the above method is not for the purpose of diagnosing diseases.

The third aspect of the present invention provides an application of the kit according to the first aspect of the present invention or the method according to the second aspect of the present invention in the combined detection of HCV antigen and antibody.

The term "known antigen" used in the present invention may be the standard of the antigen to be tested in the sample to be tested.

The term "a known antibody that specifically binds to the antigen to be tested in the sample to be tested" in the present invention may be a standard of the antibody to be tested in the sample to be tested.

The antigen to be tested and the antibody to be tested in the sample to be tested in the present invention may be antigens and antibodies against the same virus.

The term "the chemiluminescence signal value of the sample to be tested is equal to the background signal value" in the present invention means that the chemiluminescence signal value of the sample to be tested can be close to the local signal value by more than 90%, preferably more than 95%, more preferably above 99%.

The beneficial effects of the present invention are:

1. The components of the reagent are simple, only need to add labeled antigen or labeled antibody during the detection process, and it is not necessary to add both at the same time.

2. The screening of raw materials is easy, and there is no need to consider the cross-reaction between raw materials, the choice of raw materials is higher, and it is easier to find suitable raw materials.

3. The detection sensitivity is high, and there is no need to sacrifice some epitopes in the selection of raw materials in order to avoid cross-reaction, so that more epitopes can be combined with the analyte, and the detection sensitivity is correspondingly improved.

4. The interpretation of the results is accurate. The comparison of the signal value read during the test and the background signal value can not only determine whether the sample is positive, but also can distinguish whether the positive result is antigen-positive or antibody-positive, which is more meaningful than only general positive results. .

5. The ability to resist the hook effect is improved, and the samples with barbs caused by strong yang will be detected because the signal value read during detection is lower than the background signal value.

Description of drawings

In order to illustrate the technical solutions in the embodiments of the present invention more clearly, the following briefly introduces the drawings used in the description of the embodiments. Obviously, the drawings in the following brief description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained from these drawings without any creative effort.

FIG. 1 is a schematic diagram of the homogeneous immunization method for combined detection of the same virus antigen and antibody according to the present invention. The meanings of the reference numerals in the figure are as follows: 1. Known antibodies contained in reagent I that can specifically bind to the antigen to be tested in the sample to be tested; 2. Antibody to be tested in the sample to be tested; 3. Antibody to be tested in the sample to be tested test antigen; 4 known antigen-coated luminescent microspheres; 5 known antigen labeled with biotin.

FIG. 2 is a flow chart of the homogeneous immunization method for combined detection of antigen and antibody according to the present invention.

Figure 3 is a schematic diagram of the detection results of the hepatitis C core antigen in the embodiment.

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described in detail below. Before the present invention is described in detail, however, it is to be understood that this invention is not limited to the particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting.

Where a range of values is provided, it is understood that each intervening value between the upper and lower limits of the stated range and any other stated or intervening value in the stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

I. Terminology

The English definition of the term "homogeneous" in the present invention is "homogeneous", which means that the detection can be performed without separating the bound antigen-antibody complex and the remaining free antigen or antibody.

The term "sample to be tested" in the present invention refers to a mixture that may contain analytes, including but not limited to proteins, hormones, antibodies, antigens, and the like. Typical test samples that can be used in the methods disclosed herein include body fluids such as blood, plasma, serum, urine, semen, saliva, and the like.

The term "antibody" as used herein is used in the broadest sense to include antibodies of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies , humanized antibodies, single chain antibodies, bispecific antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. Where desired, the antibody can be further conjugated to other moieties, such as specific binding pair members, eg, biotin or streptavidin, and the like.

The term "monoclonal antibody" in the present invention refers to immunoglobulin secreted by monoclonal B lymphocytes, which can be prepared by methods known to those skilled in the art.

The term "polyclonal antibody" in the present invention refers to a collection of immunoglobulins produced by one or more clones of B lymphocytes, which can be prepared by methods known to those skilled in the art.

The term "antigen" in the present invention refers to a substance that can stimulate the body to produce an immune response, and can combine with the immune response product antibody and sensitized lymphocytes in vitro and in vivo to produce an immune effect.

The term "binding" as used herein refers to the direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and/or hydrogen bonding, including but not limited to interactions such as salt bridges and water bridges. .

The term "specific binding" in the present invention refers to the mutual discrimination and selective binding reaction between two substances, which is the conformational correspondence between the corresponding reactants from the perspective of steric structure.

The term "specific binding pair member" as used herein refers to a pair of molecules that are capable of specifically binding to each other, eg, enzyme-substrate, antigen-antibody, ligand-receptor. A specific example of a specific binding pair member pair is the biotin-streptavidin system, in which "biotin" is widely present in animal and plant tissues, and its molecule has two ring structures, namely the imidazolone ring. and thiophene rings, of which the imidazolone ring is the main site for binding to streptavidin. Activated biotin can be coupled with almost all known biological macromolecules, including proteins, nucleic acids, polysaccharides and lipids, under the mediation of protein cross-linking agents; while "streptavidin" is produced by Streptomyces sp. A secreted protein with a molecular weight of 65kD. The "streptavidin" molecule consists of 4 identical peptide chains, each of which can bind a biotin. Therefore, each antigen or antibody can be conjugated with multiple biotin molecules simultaneously, resulting in a "tentacle effect" to improve analytical sensitivity.

In any need, any reagents used in the present invention, including antigens, antibodies, acceptors or donors, can be conjugated with biotin or streptavidin according to actual needs, for example: the reagent III contains biotin Coated third counterpart; another example: Reagent IV comprises a streptavidin-coated donor.

The term "donor" as used herein refers to a sensitizer capable of generating reactive intermediates such as singlet oxygen that react with acceptors upon activation by energy or reactive compounds. Donors can be photoactivated (eg, dyes and aromatic compounds) or chemically activated (eg, enzymes, metal salts, etc.).

In some specific embodiments of the present invention, the donor is a photosensitizer, and the photosensitizer can be a photosensitizer known in the art, preferably a compound that is relatively photostable and does not react effectively with singlet oxygen, which is non-limiting Examples include compounds such as methylene blue, rose bengal, porphyrin, phthalocyanine, and chlorophyll as disclosed in U.S. Patent No. 5,709,994, which is incorporated herein by reference in its entirety, and derivatives of these compounds having 1-50 atomic substituents , the substituents are used to make these compounds more lipophilic or more hydrophilic, and/or as linking groups to members of specific binding partners. Examples of other photosensitizers known to those skilled in the art may also be used in the present invention, such as those described in US Pat. No. 6,406,913, which is incorporated herein by reference.

In other embodiments of the present invention, the donor is a chemically activated other sensitizer, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Some other examples of donors include: 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide, etc. Heat these compounds or Direct absorption of light by these compounds releases singlet oxygen.

The term "receptor" as used herein refers to a compound capable of reacting with singlet oxygen to generate a detectable signal. The donor is induced to activate by the energy or active compound and releases high energy singlet oxygen which is captured by the acceptor in close proximity, thereby delivering energy to activate the acceptor.

In some embodiments of the invention, the acceptor is a substance that undergoes a chemical reaction with singlet oxygen to form an unstable metastable intermediate that can decompose while simultaneously or glow afterwards. Typical examples of these materials include, but are not limited to: enol ethers, enamines, 9-alkylidene xanthan gum, 9-alkylidene-N-alkylacridan, vinyl arylene ether, diepoxyethylene, dimethyl Thiophene, aromatic imidazole or lucigenin.

In other embodiments of the present invention, the acceptor is an alkene capable of reacting with singlet oxygen to form hydroperoxides or dioxetanes that can be decomposed into ketones or carboxylic acid derivatives; Stable dioxetanes that decompose by the action of light; acetylenes that can react with singlet oxygen to form diketones; hydrazones or hydrazides that can form azo compounds or azocarbonyl compounds, such as luminol ; and aromatic compounds that can form endoperoxides. Specific, non-limiting examples of receptors that can be utilized in accordance with the present disclosure and claimed invention are described in US Pat. No. 5,340,716, which is incorporated herein by reference in its entirety.

In other specific embodiments of the present invention, the receptors comprise olefin compounds and metal chelates, which are non-particulate and soluble in aqueous media. The preparation method of such receptors can be found in patent PCT/US2010/ 025433 (this patent document is hereby incorporated by reference in its entirety).

In other specific embodiments of the present invention, the "donor" and/or "acceptor" can be coated on the substrate with functional groups to form "donor microspheres" and/or "acceptor microspheres" . The "matrix" of the present invention is a microsphere or particle known to those skilled in the art, which may be of any size, which may be organic or inorganic, which may be swellable or non-swellable, and which may be Porous or non-porous, of any density, but preferably of a density close to that of water, preferably capable of floating in water, and composed of transparent, partially transparent or opaque materials. The matrix may or may not be charged, and when charged, it is preferably negatively charged. The matrix can be solids (eg polymers, metals, glasses, organic and inorganic substances such as minerals, salts and diatoms), oil droplets (eg hydrocarbons, fluorocarbons, siliceous fluids), vesicles (eg Synthetic such as phospholipids, or natural such as cells, and organelles). The matrix can be latex particles or other particles containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, oil droplets, silica particles, metal sols, cells and microcrystalline dyes. The matrix is usually multifunctional or capable of binding to a donor or acceptor through specific or non-specific covalent or non-covalent interactions. Many functional groups are available or incorporated. Typical functional groups include carboxylic acid, acetaldehyde, amino, cyano, vinyl, hydroxyl, mercapto, and the like. A non-limiting example of a matrix suitable for use in the present invention is a carboxyl-modified latex particle. Details of such matrices can be found in US Pat. Nos. 5,709,994 and 5,780,646 (both of which are hereby incorporated by reference in their entirety).

II. Examples

As mentioned above, when the homogeneous immunoassay method is currently used for the combined detection of the same virus antigen and antibody, it is impossible to distinguish whether the positive signal value is caused by the antigen or the antibody, so the course of the disease cannot be accurately judged, and it cannot provide reliable information for clinical treatment. . In addition, the labeled raw materials in the detection reagents used in the detection process need to be strictly screened, and the labeled antigens and labeled antibodies cannot cross-react, which increases the difficulty of reagent development and reduces the detection sensitivity. The inventors of the present application found through research that a certain amount of known antibody or known antigen that specifically binds to the antigen to be tested in the sample to be tested is added to the reagent I (sample diluent), and then the competition method and sandwich method are used. The method is used to detect the same virus antigen and antibody in the sample to be tested. It only needs to add the labeled antigen or labeled antibody. Finally, the change of the signal value is used to determine whether the sample to be tested is antibody positive or antigen positive, which is more meaningful for clinical diagnosis. .

In the following, the detection principle of the present invention is described in detail by adding a certain amount of known antibody that specifically binds to the antigen to be detected in the sample to be detected in Reagent I (sample diluent), and the specific detection principle is shown in Figure 1 .

Since reagent I (sample diluent) is added with a known antibody that specifically binds to the antigen to be tested in the sample to be tested, reagent II includes receptors coated with known antigens, and reagent III includes biotin Labeled known antigens.

When the sample to be tested contains neither the antigen to be tested nor the antibody to be tested, the sample to be tested does not react with the known antibody in the sample diluent, and the receptors and biotin labels coated with the known antigen are subsequently added. After the known antigen, the known antigen coated on the receptor and the known biotin-labeled antigen react with the known antibody in the sample diluent to form a sandwich complex. After adding the streptavidin-labeled donor contained in reagent IV, the receptors in these sandwich complexes will react with the singlet oxygen emitted by the nearby donor by excitation with 680nm light, and the energy generated is 610nm wavelength. The form of light is emitted, and the signal value at the wavelength of 610nm is detected as the background signal value of the detection.

When the sample to be tested contains the antibody to be tested, after the sample to be tested is mixed with the sample diluent, the total amount of antibody increases, and the signal value of the final reaction will be higher than the background signal value. The detection method at this time is sandwich detection, the sandwich response is enhanced, and the response signal value is enhanced.

When the sample to be tested contains the antigen to be tested, after the sample to be tested is mixed with the sample diluent, the antigen to be tested in the sample to be tested reacts with the known antibody in the sample diluent, neutralizing and consuming part of the known antibody, so that Subsequently, the amount of antibodies that can react with known antigens coated on receptors and biotin-labeled known antigens decreases, and the final signal value after reaction will be lower than the local signal value. The detection method at this time is the competition method detection.

Correspondingly, if a certain amount of known antigen is added to reagent I (sample diluent), reagent II includes a second antibody-coated receptor that specifically binds to the known antigen, and reagent III includes biotin-labeled receptors. A secondary antibody capable of specifically binding to a known antigen.

At this time, when the sample to be tested contains the antigen to be tested, after the sample to be tested is mixed with the sample diluent, the total antigen content increases, and the signal value after the final reaction is higher than the local signal value. The detection method at this time is sandwich detection, and the stronger the sandwich reaction, the stronger the response signal value.

When the sample to be tested contains the antibody to be tested, after the sample to be tested is mixed with the sample diluent, the antibody to be tested in the sample to be tested reacts with the known antigen in the sample diluent, neutralizing and consuming part of the known antigen, so that Subsequently, the amount of antigen that can react with the second antibody coated on the receptor and the biotin-labeled second antibody decreases, and the signal value after the final reaction is lower than the local signal value, and the detection method is competitive detection.

Therefore, the homogeneous immunoassay kit involved in the first aspect of the present invention includes:

Reagent I, comprising a first counterpart; the first counterpart is a known antigen or a known antibody that can specifically bind to the antigen to be tested in the sample to be tested;

Reagent II comprising a receptor capable of reacting with singlet oxygen to generate a detectable signal and a second counterpart bound thereto, the second counterpart being capable of specifically binding to the first counterpart;

Reagent III, which comprises a third counterpart capable of specifically binding to the first counterpart.

Reagent IV, which comprises a donor capable of producing singlet oxygen in the excited state.

According to the present invention, the surface of the third counterpart is coated with biotin, while the surface of the donor is coated with streptavidin.

In some embodiments of the present invention, the kit specifically includes:

Reagent I, which comprises a known antibody that can specifically bind to the antigen to be tested in the sample to be tested;

Reagent II, which comprises known antigen-coated receptors;

Reagent III, which comprises a biotin-coated known antigen;

Reagent IV, which comprises a streptavidin-coated donor.

In other embodiments of the present invention, the kit specifically includes:

Reagent I, which comprises a known antigen;

Reagent II, which comprises a second antibody-coated receptor capable of specifically binding to a known antigen;

Reagent III, which comprises a biotin-coated secondary antibody that specifically binds to a known antigen;

Reagent IV, which comprises a streptavidin-coated donor.

The donor and acceptor of the above reagents can be coated on the matrix to form particled donor microspheres and acceptor microspheres, which can also be non-particulate reagents, soluble in an aqueous medium.

In some specific embodiments of the present invention, the kit specifically includes:

Reagent I, which comprises a known antibody that can specifically bind to the antigen to be tested in the sample to be tested;

Reagent II, which comprises receptor microspheres bound to known antigens;

Reagent III, which comprises a biotin-coated known antigen;

Reagent IV, which comprises streptavidin-coated donor microspheres.

In other specific embodiments of the present invention, the kit specifically includes:

Reagent I, which comprises a known antigen;

Reagent II, which comprises receptor microspheres coated with a second antibody capable of specifically binding to a known antigen;

Reagent III, which comprises a biotin-coated secondary antibody that specifically binds to a known antigen;

Reagent IV, which comprises streptavidin-coated donor microspheres.

In the second aspect of the present invention, the homogeneous immunoassay method for combined detection of the same virus antigen and antibody using the kit according to the first aspect of the present invention includes the following steps: The negative sample is detected, and the obtained detection result is used as the background signal value; then the chemiluminescence signal value of the sample to be tested is detected, and the chemiluminescence signal value of the sample to be tested is compared with the background signal value. , so as to determine whether the antigen to be tested and/or the antibody to be tested exists in the sample to be tested.

In some embodiments of the present invention, the first counterpart contained in the reagent I is a known antibody that can specifically bind to the antigen to be tested in the sample to be tested, at this time:

If the detected chemiluminescence signal value of the sample to be tested is equal to the background signal value, then the sample to be tested does not contain the antigen to be tested and the antibody to be tested;

If the detected chemiluminescence signal value of the sample to be tested is greater than the background signal value, then the sample to be tested contains the antibody to be tested;

If the detected chemiluminescence signal value of the sample to be tested is less than the background signal value, the sample to be tested contains the antigen to be tested.

In other embodiments of the present invention, the first counterpart contained in the reagent I is a known antigen, and in this case:

If the detected chemiluminescence signal value of the sample to be tested is equal to the background signal value, then the sample to be tested does not contain the antigen to be tested and the antibody to be tested;

If the detected chemiluminescence signal value of the sample to be tested is greater than the background signal value, the sample to be tested contains the antigen to be tested;

If the detected chemiluminescence signal value of the sample to be tested is less than the background signal value, the sample to be tested contains the antibody to be tested.

According to the present invention, the method for detecting the chemiluminescence signal value of the sample to be tested comprises the following steps:

S1, mixing the sample to be tested and the reagent I to obtain a first mixture;

S2, adding reagent II, reagent III and reagent IV to the first mixture to obtain a second mixture;

S3, treating the second mixture with energy or an active compound, and detecting the chemiluminescence signal value of the second mixture;

S4, analyze the chemiluminescence signal value to determine whether the sample to be tested contains the antigen to be tested and/or the antibody to be tested. The specific reaction flow diagram is shown in Figure 2.

In some embodiments of the present invention, in step S2, reagent II and reagent III are added first, and then reagent IV is added.

In other embodiments of the present invention, in step S1, the sample to be tested is added to the solution containing reagent I.

In the method of the present invention, after all the reagents are combined, they can be mixed and/or incubated as required. Specifically, the temperature of the incubation can be 35-45°C, and the time can be 10-20min; 41°C, 42°C, 43°C or 44°C; the incubation time can be selected from 12min, 15min, 16min or 18min.

In some embodiments of the present invention, light at a wavelength of 680 nm is used to excite the donor and/or donor microspheres to generate singlet oxygen, and the acceptor is capable of reacting with singlet oxygen to generate a chemiluminescent signal at a wavelength of 615 nm.

Specifically, in some specific embodiments of the present invention, the method for detecting the chemiluminescence signal value of the sample to be tested includes the following steps:

S1, add reagent I to the pre-dilution plate, then add the sample to be tested, shake and mix well, incubate at 35-40 °C for 10-15 min, to obtain the first mixture;

S2, add the first mixture into the detection area, then add reagent II and reagent III, react at 35-40°C for 10-15min, then add reagent IV, and incubate at 35-40°C for 10-15min to obtain the second mixture;

S3, treating the second mixture with light having a wavelength of 680 nm to excite the donor and/or donor microspheres therein to generate singlet oxygen, which combines with acceptor and/or acceptor microspheres to generate a wavelength of 615 nm chemiluminescence signal;

S4, detecting the chemical invention signal, and determining whether the antigen to be tested and/or the antibody to be tested exists in the sample to be tested according to the acquired chemiluminescence signal value.

According to the present invention, the detection sensitivity of the method is less than 10 ng/mL.

The third aspect of the present invention relates to an application of the kit according to the first aspect of the present invention or the method according to the second aspect of the present invention in the combined detection of HCV antigen and antibody.

In order to make the present invention easier to understand, the present invention will be further described in detail below with reference to the embodiments, which are only for illustrative purposes and do not limit the scope of application of the present invention. The raw materials or components used in the present invention can be obtained through commercial channels or conventional methods unless otherwise specified.

The photoexcited chemiluminescence analyzer used in the following examples is a fully automatic photoexcited chemiluminescence analyzer.

Example 1: Sensitivity test of a homogeneous immunoassay for single-well combined detection of human hepatitis C core antigen (HCV Core Ag)

1. Preparation of markers

The HCV antibody detection kit produced by Shanghai Boyang was used, batch number: 1606L. The specific labeling process of each reagent component is omitted.

2. Preparation of reagent I (sample diluent)

Use the sample diluent in the above-mentioned finished product kit to dilute 1 antibody-positive serum to 10-fold, 100-fold, 1,000-fold, 10,000-fold, 100,000-fold and 1 million-fold respectively. The solution with the above dilution is used as a sample for detection with the kit, and the dilution with a signal value of about 20,000 is found. Use this dilution as the ratio of adding antibodies to the sample diluent, and prepare 20ml of the sample diluent according to this ratio.

3. Detection of recombinant HCV core antigen concentration

Experiments were performed with recombinant HCV Core Ag. Antigens were diluted with phosphate buffered saline (PBS) to 2-fold, 5-fold and 10-fold, respectively, and then detected with BCA protein concentration detection kit. When testing, first use bovine serum albumin (BSA) calibration solution (0mg/ml, 0.1mg/ml, 0.25mg/ml, 0.5mg/ml, 0.75mg/ml and 1mg/ml total 6 points) for calibration , after operating according to the kit instructions, read at OD _562nm on a microplate reader. The test result is that the original antigen concentration is less than 1 mg/ml.

4. Detection of the sensitivity of the homogeneous immunoassay for the combined detection of human hepatitis C (HCV) core antigen

The antigens described in step 3 were serially diluted with PBS buffer, 10-fold, 1000-fold, 10,000-fold and 100,000-fold respectively.

Add sample diluent to the pre-dilution plate, 25ul per well, and then add the diluted HCV core antigen solution as the sample to be tested to 25ul per well of the sample diluent, shake and mix, and incubate at 37°C for 10min. first mixture.

Pipette 25ul of the first mixture into each well and add it to the detection well, then add 25ul each of the HCV core antigen-binding receptor microsphere solution and the biotin-coated HCV core antigen solution, react at 37°C for 15 minutes, and then add streptavidin The second mixture was obtained after 175ul of the donor microsphere solution coated with vegan, and incubated at 37°C for 10 min.

The second mixture was treated with light at a wavelength of 680 nm to excite the donor microspheres to generate singlet oxygen, which combined with the acceptor microspheres to generate a chemiluminescence signal at a wavelength of 615 nm. The chemical invention signal is detected, and the obtained chemical invention signal value is shown in FIG. 3 . It can be seen from Figure 3 that the detected signal value is no longer changed significantly after the HCV core antigen is diluted 100,000 times, indicating that the analytical sensitivity of this method for the detection of human hepatitis C core antigen is less than 10ng/ml.

It should be noted that the above-mentioned embodiments are only used to explain the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it is to be understood that the words used therein are words of description and explanation, rather than words of limitation. The present invention may be modified within the scope of the claims of the present invention as specified, and may be modified without departing from the scope and spirit of the present invention. Although the invention described herein refers to the specific methods, materials and embodiments, it is not intended to be limited to the specific examples disclosed therein, but rather, the invention extends to all other methods and applications having the same function.

Claims (12)

1. A homogeneous immunoassay kit comprising:

a reagent I comprising a first counterpart, said first counterpart being a known antigen;

a reagent II comprising a receptor capable of reacting with singlet oxygen to generate a detectable signal and a second counterpart capable of binding specifically to the first counterpart;

a reagent III comprising a third counterpart capable of specifically binding to the first counterpart;

a reagent IV comprising a donor capable of generating singlet oxygen in an excited state.

2. The kit of claim 1, wherein the second counterpart and the third counterpart are each a second antibody capable of specifically binding to a known antigen.

3. The kit according to claim 2, wherein the second antibody is a monoclonal antibody and/or a polyclonal antibody.

4. The kit of any one of claims 1 to 3, wherein the third counterpart is surface coated with biotin and the donor surface is surface coated with streptavidin.

5. The kit according to any one of claims 1 to 4, wherein the acceptor comprises an olefin compound and a metal chelate compound, and is soluble in an aqueous medium.

6. A kit as claimed in any one of claims 1 to 5, wherein all reagents are non-particulate and soluble in aqueous media.

7. A homogeneous immunoassay method for the combined detection of the same viral antigen, antibody for non-disease diagnostic purposes using the kit of any one of claims 1 to 6, comprising the steps of: firstly, detecting a negative sample without an antigen to be detected and an antibody to be detected, and taking an obtained detection result as a background signal value; and then detecting the chemiluminescence signal value of the sample to be detected, and comparing the chemiluminescence signal value of the sample to be detected with the background signal value, thereby judging whether the antigen to be detected and/or the antibody to be detected exist in the sample to be detected.

8. The method of claim 7, wherein if the detected chemiluminescent signal value of the test sample is equal to the background signal value, then the test sample is free of test antigen and test antibody;

if the detected chemiluminescence signal value of the sample to be detected is larger than the background signal value, the sample to be detected contains the antigen to be detected;

and if the chemiluminescence signal value of the sample to be detected obtained by detection is smaller than the background signal value, the sample to be detected contains the antibody to be detected.

9. The method according to claim 7 or 8, wherein the method for detecting the chemiluminescent signal value of the sample to be tested comprises the steps of:

s1, mixing the sample to be detected with the reagent I to obtain a first mixture;

s2, adding a reagent II, a reagent III and a reagent IV into the first mixture to obtain a second mixture;

s3, treating the second mixture with energy or active compound, and detecting the chemiluminescence signal value of the second mixture;

and S4, analyzing the chemiluminescence signal value, and judging whether the sample to be detected contains the antigen to be detected and/or the antibody to be detected.

10. The method of claim 9, wherein in step S2, reagent ii and reagent iii are added, followed by reagent iv.

11. The method according to claim 9 or 10, wherein in step S1, the sample to be tested is added to a solution containing the reagent i.

12. Use of a kit according to any one of claims 1 to 6 or a method according to any one of claims 7 to 12 for the combined detection of HCV antigens and antibodies.

Images