CN114217076A

CN114217076A - Homogeneous phase immunoassay kit for detecting target anti-Carp antibody and application thereof

Info

Publication number: CN114217076A
Application number: CN202111529223.3A
Authority: CN
Inventors: 饶星; 刘宇卉; 李临
Original assignee: Chemclin Diagnostics Corp
Current assignee: Chemclin Diagnostics Corp
Priority date: 2018-02-11
Filing date: 2018-02-11
Publication date: 2022-03-22
Also published as: CN114217075A; CN108445223A; CN114217077A

Abstract

The invention relates to a homogeneous immunoassay kit for detecting a target anti-Carp antibody and application thereof. The detection kit comprises: a component a comprising an antigen that binds to a receptor; component b comprising a second anti-Carp antibody; the second anti-Carp antibody is a biotinylated rabbit anti-carbamoylated protein antibody; the second anti-Carp antibody binds to biotin; a component c comprising a donor capable of producing singlet oxygen in an excited state; the donor binds to streptavidin. The kit adopts a one-step competition method reaction mode, and the reaction mode can specifically detect anti-Carp Ab such as IgG, IgA and IgM, so that the washing process in the traditional indirect method is omitted, and the one-step method is formed to complete the whole reaction. Compared with the traditional indirect method, the method saves time, is free from cleaning and is simple and convenient to operate.

Description

Homogeneous phase immunoassay kit for detecting target anti-Carp antibody and application thereof

The application is a divisional application with the application number of 201810142718.2, the application date of 2018, 02/11, and the invention name of 'homogeneous immunoassay kit for detecting target anti-Carp antibody and application thereof'.

Technical Field

The invention belongs to the technical field of immunoassay, and particularly relates to a homogeneous immunoassay kit for detecting a target anti-Carp antibody, and a preparation method and a use method thereof.

Background

anti-Carbamylated protein antibodies (anti-Carp Ab) are a novel biomarker that has been discovered in recent years to be closely related to the diagnosis and course monitoring of Rheumatoid Arthritis (RA) disease. Carbamylation is a non-enzyme-mediated post-translational modification of proteins, in which lysine residues of the proteins are converted into homocitrulline residues by the action of urea derivatives, cyanate or cyanate. This process breaks human immune tolerance and induces the production of the autoimmune antibody anti-Carp antibody (anti-Carp Ab).

The anti-Carp Ab detection methods disclosed in WO2012/105838a1 and WO2016/014612a2 are methods in which carbamylated fetal calf serum (Car-FCS) and carbamylated human α 1 antitrypsin (Car-hAlAT) are used as antigens, and the detection is performed by Immunoblotting (Immunoblotting) and enzyme-linked immunosorbent assay (ELISA), respectively.

The detection method in patent WO2012/105838a1 has the following drawbacks: 1. anti-Carp Ab in human body is an antibody generated by carbamylated protein in human body, while carbamylated fetal calf serum (Car-FCS) is used as an antigen of animal origin and has an epitope which is not completely identical to that of human self carbamylated protein; 2. carbamylated fetal calf serum contains a large amount of proteins and other complex components which can non-specifically interact with anti-Carp Ab antibodies and other immunoglobulins in a human serum sample, so that the background value of the experiment is high, and the result analysis is interfered; 3. carbamylated fetal calf serum is difficult to prepare repeatedly, has batch difference and may influence the reproducibility of experimental results; 4. the subtype of the anti-Carp Ab to be detected is only IgA and IgG, and IgM and IgD antibodies are not detected; 5. when the Immunoblotting (Immunoblotting) is used for detection, the detection steps are complex, the experimental operation standards of different detection mechanisms are different, the time consumption is long, the full-automatic high-throughput analysis cannot be realized, and the interference of nonspecific detection bands generated on the blotting membrane on the experimental result when the animal serum is used as a secondary antibody for detection is avoided.

The detection method in WO2016/014612A2 has the defects that an ELISA indirect method is adopted to detect the anti-Carp Ab, and the detection background value is high, the sensitivity is low, the linear range is narrow, and false positive experiment results are easy to generate; meanwhile, for detecting antibodies of different subtypes, corresponding enzyme-labeled secondary antibodies are required to be adopted for detection, and the experiment operation is relatively complex. Meanwhile, in order to avoid interference of nonspecific Immunoglobulin G (IgG) (nonspecific IgG refers to IgG except for the carbamoylated protein antibody) in a sample on a test result in the detection process, the addition amount of the enzyme-labeled secondary antibody needs to be increased, and washing is performed after the reaction is completed to remove the enzyme-labeled secondary antibody bound with the nonspecific IgG.

In addition, the two methods have the common defect that the two detection means are heterogeneous reaction systems, and compared with homogeneous reaction systems, the selectivity, sensitivity, reaction speed and the like of the two methods are not the same as those of the latter methods.

Therefore, in order to overcome the above defects in the prior art, a homogeneous immunoassay kit for detecting anti-Carp Ab, which has the advantages of strong antigen specificity, good signal amplification effect, high sensitivity, no need of washing, wide linear range, simple operation and more stable test, needs to be researched and developed.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a homogeneous immunoassay kit for detecting a target anti-Carp antibody and application thereof aiming at the defects of the prior art, wherein the kit adopts a competition method reaction mode, the reaction mode can specifically detect anti-Carp abs such as IgG, IgA and IgM, and the like, and a washing process in the traditional indirect method is omitted. Compared with the traditional indirect method, the method saves time, is free from cleaning and is simple and convenient to operate.

To this end, the present invention provides in a first aspect a homogeneous immunoassay kit for the detection of an anti-Carp antibody of interest, comprising:

a component a comprising an antigen capable of specifically binding to an anti-Carp antibody of interest and a second anti-Carp antibody;

component b comprising a second anti-Carp antibody.

In the present invention, said antigen or said second anti-Carp antibody binds to a receptor; the receptor is capable of reacting with singlet oxygen to generate a detectable chemiluminescent signal.

In some preferred embodiments of the invention, the antigen binds to a receptor.

In some embodiments of the invention, the acceptor comprises an olefinic compound and a metal chelate, which is in non-particulate form and soluble in an aqueous medium; and/or the acceptor is polymer particles filled with a luminescent compound and lanthanide.

According to the invention, the reagent set also comprises a component c comprising a donor capable of generating singlet oxygen in the excited state.

In some embodiments of the invention, the donor is bound to one member of a specific binding partner, and the other member of the specific binding partner is bound to the antigen or the second anti-Carp antibody.

In some particularly preferred embodiments of the invention, the donor is bound to streptavidin and the antigen or the second anti-Carp antibody, respectively, is bound to biotin.

In some embodiments of the invention, the donor is a photoactivated or chemically activated sensitizer, which is in non-particulate form and soluble in an aqueous medium; and/or the donor is polymer particles filled with photosensitive compounds and can generate singlet oxygen under the excitation of light.

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

In some preferred embodiments of the invention, the second anti-Carp antibody is a monoclonal antibody.

In some embodiments of the invention, the antigen is selected from the group consisting of a synthetic carbamylated peptide, a polypeptide formed on a peptide chain by synthesis of at least 2 single carbamylated peptide, a mixture of carbamylated peptide containing at least 2 single carbamylated peptide, and a carbamylated protein.

In some preferred embodiments of the invention, the antigen is selected from the group consisting of a synthetic carbamylated peptide, a polypeptide formed on one peptide chain by synthesis of at least 2 single carbamylated peptide, and a mixture of carbamylated peptide containing at least 2 single carbamylated peptide.

In some preferred embodiments of the invention, the antigen is a polypeptide synthesized on one peptide chain from 2-4 carbamylated peptide fragments or a carbamylated peptide fragment mixture containing 2-4 single carbamylated peptide fragments; preferably, the carbamylated peptide fragment is selected from SEQ ID Nos. 2-5.

In some further preferred embodiments of the invention, the antigen is bound to the receptor via an intermediate, which is a hydrophilic polymeric substance.

In some preferred embodiments of the invention, the intermediate is a protein, preferably selected from the group consisting of hemocyanin, ovalbumin, bovine serum albumin, and bovine thyroglobulin.

In other preferred embodiments of the present invention, the intermediate is selected from the group consisting of dendrimers, polycarboxylates, polymercaptans, and polyethylene glycols.

In some embodiments of the invention, the total concentration of the antigen and the receptor bound thereto is 0.005-0.1. mu.g/mL.

In some embodiments of the invention, the total concentration of the second anti-Carp antibody, and the member of the specific binding partner to which it binds, is from 0.025 to 0.1. mu.g/mL.

In some embodiments of the invention, the total concentration of the donor and the other member of the specific binding pair bound thereto is from 5 to 20. mu.g/mL.

In a second aspect, the present invention provides a homogeneous immunoassay kit for the detection of a target anti-Carp antibody comprising a homogeneous immunoassay kit according to the first aspect of the present invention.

In a third aspect, the present invention provides an immunoassay method for detecting a target anti-Carp antibody in a sample to be detected, wherein the immunoassay method comprises the following steps:

m1, allowing the second anti-Carp antibody and the target anti-Carp antibody in the sample to compete for binding with the epitope of the antigen, and forming a first immune complex composed of the antigen-second anti-Carp antibody and a second immune complex composed of the antigen-anti-Carp antibody through the epitope and the antigen, respectively;

m2, detecting the presence or absence of a second immune complex; if the second immune complex is present, it indicates the presence of anti-Carp antibody in the test sample.

According to some embodiments of the present invention, the step M1 includes mixing the second anti-Carp antibody, the sample to be tested and the antigen uniformly, reacting, allowing the second anti-Carp antibody and the target anti-Carp antibody in the sample to compete for binding with the epitope of the antigen, and forming a first immune complex composed of the antigen-second anti-Carp antibody and a second immune complex composed of the antigen-target anti-Carp antibody with the antigen through the epitope of the antigen, respectively.

In some embodiments of the invention, the step of uniformly mixing the second anti-Carp antibody, the sample to be tested and the antigen comprises simultaneously uniformly mixing the second anti-Carp antibody, the sample to be tested and the antigen.

In other embodiments of the present invention, the step of uniformly mixing the second anti-Carp antibody, the test sample, and the antigen comprises mixing the second anti-Carp antibody with the test sample to form a1 st mixture, and then mixing the antigen with the 1 st mixture.

According to further embodiments of the present invention, the step M1 includes the steps of:

p1, mixing and reacting the antigen with the sample to be detected, and combining the target anti-Carp in the sample to be detected with the epitope of the antigen to form a second immune complex formed by the antigen-target anti-Carp antibody, so as to obtain a 11 th mixture;

p2, mixing the second anti-Carp antibody with the 11 th mixture, reacting the mixture to allow the second anti-Carp antibody to bind to the epitope of the antigen in the 11 th mixture that is not bound to the target anti-Carp antibody in the sample to be tested, thereby forming a first immune complex composed of the antigen-the second anti-Carp antibody.

According to the method of the present invention, the presence or absence of the second immune complex is detected by chemiluminescence in step M2.

In some embodiments of the invention, step M2 comprises comparing the chemiluminescent signal value of the mixture 1 to the chemiluminescent signal value of a control immunocomplex formed from an equivalent amount of antigen and second anti-Carp antibody as in step M1, thereby determining whether a second immunocomplex is present.

In some embodiments of the invention, the antigen is bound to a receptor and the second anti-Carp antibody is bound to biotin, and the receptor is capable of reacting with singlet oxygen to generate a detectable chemiluminescent signal.

According to further embodiments of the present invention, step M0 is further included before step M1, the sample to be tested is diluted with the sample diluent according to a volume ratio of 1 (4-20); preferably, the sample to be tested is diluted by the sample diluent according to the volume ratio of 1 (6-16); more preferably, the sample to be tested is diluted with the sample diluent according to the volume ratio of 1 (8-12).

In some specific embodiments of the present invention, the method comprises the steps of:

r1, mixing the second anti-Carp antibody combined with biotin with the sample to be tested to form a2 nd mixture;

r2, mixing the antigen bound with the receptor with the mixture of item 2, allowing the second anti-Carp antibody bound with biotin and the target anti-Carp antibody in the sample to compete for binding to the epitope of the antigen bound with the receptor, and forming a third immune complex composed of receptor-antigen-second anti-Carp antibody-biotin and a fourth immune complex composed of receptor-antigen-anti-Carp antibody with the antigen bound with the receptor through the epitope, respectively, thereby forming an item 3 mixture;

r3, mixing the streptavidin-bound donor with the 3 rd mixture, and allowing the streptavidin-bound donor to bind to biotin in the third immune complex to form a fifth immune complex consisting of receptor-antigen-second anti-Carp antibody-biotin-streptavidin-donor, thereby forming a4 th mixture;

r4, exciting the donor with energy or an active compound to produce singlet oxygen, said acceptor reacting with the singlet oxygen to generate a detectable chemiluminescent signal; detecting the presence or absence of a fourth immune complex; if the fourth immune complex is present, the anti-Carp antibody of interest is present in the test sample.

In some embodiments of the invention, the method further comprises the step of preparing a standard working curve of the anti-Carp antibody of interest prior to step R1.

In some embodiments of the invention, at step R4, the intensity of the chemiluminescent signal is detected and the amount of anti-Carp antibody in the test sample is determined based on an anti-Carp antibody standard working curve.

In some embodiments of the present invention, the 4 th mixture is irradiated with the excitation light with a wavelength of 600-700nm to excite the donor in the fifth immunocomplex to generate singlet oxygen, the acceptor reacts with the contacted singlet oxygen to generate the emission light with a wavelength of 520-620nm, the signal value of the emission light of the 4 th mixture is detected and compared with the chemiluminescence signal value of the control immunocomplex formed by the equal amount of the antigen and the second anti-Carp antibody in the step R2, thereby determining whether the anti-Carp antibody and/or the anti-Carp antibody is/are present in the test sample.

t1, mixing the antigen bound with the receptor with the sample to be tested, reacting, and binding the target anti-Carp in the sample to be tested with the epitope of the antigen bound with the receptor to form a fourth immune complex composed of receptor-antigen-anti-Carp antibody, to obtain a 12 th mixture;

t2, mixing the biotin-bound second anti-Carp antibody with the 12 th mixture, reacting the mixture to compete with the biotin-bound second anti-Carp antibody for binding to an epitope of the receptor-bound antigen not bound to the anti-Carp in the sample to be tested in the 12 th mixture, and forming a third immune complex composed of the receptor-bound antigen-second anti-Carp antibody-biotin, thereby forming a 13 th mixture;

t3, mixing the streptavidin-bound donor with the 13 th mixture, and allowing the streptavidin-bound donor to bind to biotin in the third immune complex to form a fifth immune complex consisting of receptor-antigen-second anti-Carp antibody-biotin-streptavidin-donor, thereby forming a 14 th mixture;

t4, exciting the donor with energy or an active compound to produce singlet oxygen, the acceptor reacting with the singlet oxygen to generate a detectable chemiluminescent signal; detecting the presence or absence of a fourth immune complex; if the fourth immune complex is present, then an anti-Carp antibody is present in the test sample.

In some embodiments of the invention, the method further comprises the step of preparing a standard working curve of the anti-Carp antibody of interest prior to step T1.

In some embodiments of the present invention, at step T4, the intensity of the chemiluminescent signal is detected and the amount of anti-Carp antibody in the test sample is determined based on an anti-Carp antibody standard working curve.

In some embodiments of the present invention, the 14 th mixture is irradiated with 600-700nm excitation light to excite the donor in the fifth immunocomplex to generate singlet oxygen, the acceptor reacts with the contacted singlet oxygen to generate 520-620nm emission light, and the signal value of the emission light of the 14 th mixture is detected and compared with the chemiluminescence signal value of a control immunocomplex formed by an equal amount of the antigen in step T1 and an equal amount of the second anti-Carp antibody in step T2, thereby determining whether the anti-Carp antibody and/or the anti-Carp antibody is present in the test sample.

In a fourth aspect, the present invention provides the use of a homogeneous immunoassay kit according to the first aspect of the present invention or a homogeneous immunoassay kit according to the second aspect of the present invention or a method according to the third aspect of the present invention for detecting the presence and/or amount of a target anti-Carp antibody in a test sample, wherein the test sample is selected from the group consisting of blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, saliva, synovial fluid and emphysema fluid.

In a fifth aspect, the invention provides the use of a kit of reagents according to the first aspect of the invention in the preparation of a kit for detecting an anti-Carp antibody of interest in a test sample of a subject suspected of suffering from rheumatoid arthritis, thereby determining the level of the anti-Carp antibody of interest in said test sample and correlating the level thus determined with the presence, risk, potential or predisposition of rheumatoid arthritis in the subject.

The invention has the beneficial effects that:

1) the detection technology adopted by the kit is a homogeneous immunoassay method, and compared with the existing detection method of a heterogeneous reaction system, the kit has the advantages of no need of cleaning, no influence of a coating/labeling process on the activity of an antigen and an antibody, high specificity, good sensitivity, wide linear range, small background interference, high reaction speed, simplicity and convenience in operation, low technical requirement and the like, and can realize full-automatic high-throughput test.

2) In the present application, it was surprisingly found that the use of carbamylated human serum albumin as antigen has significant advantages in antigen specificity, antigen purity, and batch-to-batch variation compared to carbamylated fetal bovine serum. Namely, the specificity of the human self-carbamylated protein as an antigen is stronger, and the detection rate of the anti-Carp Ab in a human serum/plasma sample is obviously improved. Moreover, the selected human serum albumin is a purified product, the purity of the human serum albumin is more than 95 percent, the human serum albumin does not have nonspecific interaction with other substances in a human serum/plasma sample, and the detection is less interfered. In addition, the extraction and purification process of the human serum albumin is mature and reliable, has small batch difference and can be stably obtained.

3) The method can specifically detect anti-Carp Ab such as IgG, IgA and IgM, and omits the washing process in the traditional indirect method. Compared with the traditional indirect method, the method saves time and avoids complex and fussy processes of cleaning steps.

Drawings

For the present invention to be readily understood, the following description is made with reference to the accompanying drawings.

FIG. 1 shows a comparison of the distribution of positive and negative results in the homogeneous immunoassay of anti-Carp antibodies using the one-step competition method of the present invention.

FIG. 2 shows a comparison of the distribution of positive and negative results when a homogeneous immunoassay of an anti-Carp antibody was performed on a specimen by the two-step competition method of the present invention.

FIG. 3 shows a characteristic curve (ROC) of a subject with elevated diagnostic levels of anti-Carp Ab as RA disease when tested on clinical serum samples using the kit prepared in example 1.

FIG. 4 shows a comparison of the distribution of anti-Carp Ab levels in non-RA patients and RA patients when tested on clinical serum samples using the kit prepared in example 1.

FIG. 5 shows the receiver operating characteristic curve (ROC) of RA disease with elevated diagnostic levels of anti-Carp Ab when tested on clinical serum samples using the kit prepared in example 2.

FIG. 6 shows a comparison of the distribution of anti-Carp Ab levels in non-RA and RA patients when tested on clinical serum samples using the kit prepared in example 2.

Fig. 7 shows the sensitivity and specificity of the detection method of anti-Carp Ab for RA diagnosis in comparative example using the kits prepared in example 1 and example 2 for clinical serum sample testing, compared to the detection method of anti-Carp Ab in patent WO2012/105838a1 and patent WO2016/014612a 2.

Detailed Description

In order that the invention may be readily understood, a detailed description of the invention is provided below. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Where a range of values is provided, it is understood that each intervening value, to the extent that there is no stated or intervening value in that stated range, to the extent that there is no such intervening value, 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 a specified 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.

Term of

"subject", "subject" and "patient" are used interchangeably and, without particular reference or limitation, refer to mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs and other mammalian species.

The term "homogeneous" as used herein is defined in english as "homogeneous" and means that the bound antigen-antibody complex and the remaining free antigen or antibody are detected without separation.

The term "test sample" as used herein refers to a mixture that may contain an analyte, including but not limited to a protein, hormone, antibody or antigen. Typical test samples that may be used in the disclosed methods include body fluids such as blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, saliva, synovial fluid, emphysema fluid, and the like. The sample to be tested may be a solution obtained by diluting a sample that may contain an analyte with a diluent or a buffer solution as needed before use. For example, to avoid the HOOK effect, the analyte may be diluted with a sample diluent before the on-line detection, and then the detection may be performed on the detection apparatus, in which case the diluted solution that may contain the analyte is collectively referred to as the sample to be detected.

The terms "antibody" and "immunoglobulin" are used in the broadest sense of the invention and include antibodies or immunoglobulins 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 an antigen-binding portion of an antibody and a non-antibody protein. In any case desired, the antibody may be further conjugated to other moieties, such as a specific binding partner, e.g., biotin or streptavidin (a member of a biotin-streptavidin specific binding partner), and the like.

The term "second anti-Carp antibody" as used herein refers to a substance that competes with the anti-Carp antibody in the sample to be tested for binding to the antigen.

The term "monoclonal antibody" as used herein refers to an immunoglobulin secreted from a monoclonal B lymphocyte, which can be prepared by methods known to those skilled in the art.

The term "polyclonal antibody" as used herein refers to a collection of immunoglobulins produced by more than one B lymphocyte clone, which may be prepared by methods well known to those skilled in the art.

The term "antigen" as used herein refers to a substance that stimulates the body to produce an immune response and that binds to the immune response product antibodies and sensitized lymphocytes in vitro and in vivo to produce an immune effect. For example, the antigen of the present invention is an antigen capable of specifically binding to an epitope-binding site of an anti-Carp antibody.

The term "binding" as used herein refers to 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 and water bridges.

The term "specific binding" as used herein refers to the mutual discrimination and selective binding reaction between two substances, and is the conformation correspondence between the corresponding reactants in terms of the three-dimensional structure.

The term "specific binding partner" as used herein refers to a pair of molecules which are capable of specifically binding to each other, e.g., enzyme-substrate, antigen-antibody, ligand-receptor. An example of a specific binding partner pair is the biotin-streptavidin system, where "biotin" is widely present in animal and plant tissues and has two cyclic structures on the molecule, an imidazolone ring and a thiophene ring, respectively, where the imidazolone ring is the main site for binding to streptavidin. Activated biotin can be conjugated to almost any biological macromolecule known, including proteins, nucleic acids, polysaccharides, lipids, and the like, mediated by a protein cross-linking agent; "streptavidin" is a protein secreted by Streptomyces and has a molecular weight of 65 kD. The "streptavidin" molecule consists of 4 identical peptide chains, each of which is capable of binding a biotin. Thus, each antigen or antibody can be conjugated to multiple biotin molecules simultaneously, thereby creating a "tentacle effect" that increases assay sensitivity. Any reagent used in the present invention, including antigens, antibodies, acceptors or donors, may be conjugated to any member of the biotin-streptavidin specific binding partner, as desired.

The term "donor" as used herein refers to a sensitizer capable of generating a reactive intermediate such as singlet oxygen that reacts with an acceptor upon activation by energy or an active compound. The donor may be photoactivated (e.g., dyes and aromatic compounds) or chemically activated (e.g., enzymes, metal salts, etc.). In some embodiments of the invention, the donor is a photosensitizer which may be a photosensitizer known in the art, preferably a compound that is relatively light stable and does not react efficiently with singlet oxygen, non-limiting examples of which include compounds such as methylene blue, rose bengal, porphyrins, phthalocyanines, and chlorophylls disclosed in, for example, U.S. Pat. No.5,5709994, which is incorporated herein by reference in its entirety, as well as derivatives of these compounds having 1 to 50 atom substituents that serve to render these compounds more lipophilic or more hydrophilic, and/or as a linker group for attachment to a specific binding partner. 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 patent No. US6406913, which is incorporated herein by reference. In other embodiments of the 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. Other examples of donors include: 1, 4-dicarboxyethyl-1, 4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10-endoperoxide, etc., which are heated or directly absorb light to release singlet oxygen.

The term "acceptor" as used herein refers to a substance capable of reacting with singlet oxygen to produce a detectable signal. The donor is induced by energy or an active compound to activate and release singlet oxygen in a high energy state that is trapped by a close proximity acceptor, thereby transferring 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 with or subsequently emit light. Typical examples of such substances include, but are not limited to: enol ether, enamine, 9-alkylidene xanthan gum, 9-alkylidene-N-alkyl acridin, aromatic vinyl ether, diepoxy ethylene, dimethyl thiophene, aromatic imidazole or lucigenin. In other embodiments of the invention, the acceptor is an alkene capable of reacting with singlet oxygen to form a hydroperoxide or dioxetane that can be decomposed into ketones or carboxylic acid derivatives; a stable dioxetane which can be decomposed by the action of light; acetylenes which can react with singlet oxygen to form diketones; hydrazones or hydrazides which 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 disclosed and claimed invention are described in U.S. patent No. US5340716, which is incorporated herein by reference in its entirety. In other embodiments of the invention, the receptor comprises an olefinic compound and a metal chelate, which is non-particulated and soluble in an aqueous medium, as in the case of the receptor described in patent PCT/US2010/025433 (which is incorporated herein by reference in its entirety).

The 'donor' can be polymer particles filled with photosensitive compounds formed by coating functional groups on a substrate, and can generate singlet oxygen under the excitation of light; and/or the acceptor can be polymer particles filled with the luminescent compound and the lanthanide element formed by coating the functional groups on the substrate.

In the invention, the donor can be polymer particles filled with photosensitive compounds formed by coating functional groups on a substrate, and can generate singlet oxygen under the excitation of light, at the moment, the donor can also be called photosensitive microspheres or photosensitive particles, and the solution containing the photosensitive microspheres or photosensitive particles can be called photosensitive solution or general solution; and/or the receptor can be a polymer particle which is coated on the substrate through a functional group to form a luminescent compound and lanthanide elements filled in the polymer particle, and the polymer particle is called a luminescent microsphere or a luminescent particle. In the application, the system is based on that a luminescent substance coated on the surface of a substrate induces a luminescent signal through optical excitation and energy transfer, and the energy transfer is realized by leading a photosensitive microsphere and a luminescent microsphere to be close to each other depending on antigen-antibody combination. Thus eliminating the need for a separation process. The diameter of the nano microsphere is smaller, the suspension performance is stronger, and meanwhile, a three-level amplification luminescent system is adopted, so that the nano microsphere has higher analysis sensitivity; the whole detection process does not need cleaning, namely, the binding label and the binding label do not need to be separated, so the reaction time is shorter; the tracer substances (photosensitizer and luminous agent) are marked on the substrate instead of the biomolecule, so that the activity of the biomolecule is not influenced, and the substrate has a large specific surface area, so that more tracer substances and biomolecules can be coated on the surface of the substrate, and the performance of the substrate in the aspects of effective concentration and sensitivity of the reagent, detection background and the like is better.

The "matrix" according to the invention is microspheres or microparticles known to the person skilled in the art, of any size, preferably of nanometric size, organic or inorganic, expandable or non-expandable, porous or non-porous, of any density, but preferably of a density close to that of water, preferably capable of floating in water, and consisting of a transparent, partially transparent or opaque material. The substrate may or may not have a charge, and when charged, is preferably negatively charged. The matrix may be a solid (e.g., polymers, metals, glass, organic and inorganic substances such as minerals, salts and diatoms), oil droplets (e.g., hydrocarbons, fluorocarbons, siliceous fluids), vesicles (e.g., synthetic such as phospholipids, or natural such as cells, and organelles). The matrix may 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 generally multifunctional or capable of binding to a donor or recipient by 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, hydroxy, mercapto, and the like. One non-limiting example of a matrix suitable for use in the present invention is a carboxyl or aldehyde modified latex particle. Details of such substrates can be found in U.S. patent nos. US5709994 and US5780646 (both of which are incorporated herein by reference in their entirety).

The term "epitope" as used herein refers to any protein determinant capable of specifically binding to an immunoglobulin or T cell receptor. In some embodiments of the invention, an epitope is a region of the antigen surface that can be specifically assembled by an antibody. Epitope determinants may generally include chemically active surface groups of the molecule such as, but not limited to: amino acids, sugar side chains, phosphoryl groups and/or sulfonyl groups. In other embodiments of the invention, epitopes may be characterized by specific three-dimensional structural features as well as specific charge characteristics.

The term "homogeneous immunoassay kit" as used herein refers to all reagents or combinations of reagents necessary for homogeneous immunoassays.

The term "carbamylated peptide mixture" as used herein refers to a mixture of at least 2 individual carbamylated peptides.

II, embodiments

The invention is based on the principle that: a certain amount of carbamylated antigen coated on a receptor and biotin-labeled rabbit anti-carbamylated protein antibody are added into a reaction system, the biotin-labeled rabbit anti-carbamylated protein antibody and a specific antibody in the sample compete for binding with the carbamylated antigen coated on the receptor to form an antigen antibody-immune complex, then an excessive streptavidin-labeled donor is added, the biotin on the immune complex and the streptavidin-labeled donor are specifically bound, under the excitation of laser, the energy transfer of ionic oxygen between particles occurs, further high-level red light is generated, and the number of photons is converted into the concentration of a target molecule through a single photon counter and mathematical fitting. The kit is not interfered by non-specific antibodies in the detection process, so that the washing process in the traditional indirect method can be omitted, the kit has high specificity, good sensitivity, wide linear range, high reaction speed and simple and convenient operation, and can realize full-automatic high-throughput test.

Accordingly, in a first aspect the present invention provides a homogeneous immunoassay kit for the detection of anti-Carp antibodies comprising:

component b comprising a second anti-Carp antibody;

a component c comprising a donor capable of producing singlet oxygen in an excited state.

In some preferred embodiments of the present invention, the acceptor is a polymer particle filled with a luminescent compound and a lanthanide.

In some preferred embodiments of the present invention, the donor is a polymer particle filled with a photosensitive compound.

In some embodiments of the invention, the second anti-Carp antibody is a polyclonal antibody and/or a monoclonal antibody. For example, in some preferred embodiments of the invention, the second anti-Carp antibody is a monoclonal antibody.

The method for producing a polyclonal antibody in the present invention is not particularly limited, and can be produced by a method generally used in the art, for example, a method for producing a polyclonal antibody comprising: immunizing an animal with a carbamylated antigen to obtain animal serum containing the polyclonal antibody; and purifying the animal serum by affinity chromatography to obtain the polyclonal antibody for specifically recognizing the carbamylated antigen.

The method for producing the monoclonal antibody in the present invention is not particularly limited, and can be produced by a method generally used in the art, for example, a method for producing the monoclonal antibody includes: the preparation method of the monoclonal antibody comprises the following steps: fusing spleen cells of rabbits immunized by carbamylated antigens with rabbit osteoma cells, culturing, detecting cell culture supernatant, and reserving positive cell strains.

According to the invention, the antigen is a carbamoylated antigen.

In some preferred embodiments of the invention, the antigen is selected from the group consisting of a synthetic carbamylated peptide, a polypeptide formed on one peptide chain by synthesis of at least 2 single carbamylated peptide, and a carbamylated peptide mixture comprising at least 2 single carbamylated peptide; preferably, the antigen is a polypeptide synthesized on one peptide chain by 2-4 carbamylated peptide fragments or a carbamylated peptide fragment mixture containing 2-4 single carbamylated peptide fragments.

In some preferred embodiments of the invention, the carbamoylated peptide fragment is selected from SEQ ID Nos. 2-5.

In some embodiments of the invention, the molar ratio of the plurality of different peptide fragments in the first antigen to each other is the same for a polypeptide formed on one peptide chain synthesized from at least 2 single carbamylated peptide fragments.

In other embodiments of the invention, the mass ratio of the plurality of different peptidyl fragments in the first antigen to each other is the same for a carbamylated peptidyl fragment mixture containing at least 2 single carbamylated peptidyl fragments.

It will be appreciated by those skilled in the art that for reducing steric hindrance, a first antigen selected from the group consisting of a synthetic carbamylated peptide, a polypeptide synthesized on one peptide chain from at least 2 single carbamylated peptides and a mixture of carbamylated peptide containing at least 2 single carbamylated peptides is preferably bound to the receptor via an intermediate which is a hydrophilic polymeric substance.

In some embodiments of the invention, the intermediate is a protein, preferably selected from the group consisting of hemocyanin, ovalbumin, bovine serum albumin, and bovine thyroglobulin.

In other embodiments of the present invention, the intermediate is selected from the group consisting of dendrimers, polycarboxylates, polymercaptans, and polyethylene glycols.

The mode of coupling the first antigen of the carbamylated peptide fragment mixture containing at least 2 single carbamylated peptide fragments to the intermediate is not particularly limited in the present invention, and the carbamylated peptide fragment mixture can be formed by coupling the intermediate separately from each single carbamylated peptide fragment and then mixing the coupled intermediate; or a formylated peptide fragment mixture formed by mixing single formylated peptide fragments and then coupling an intermediate to form a coupled intermediate; preferably, the individual formylated peptide fragments are mixed to form a mixture of formylated peptide fragments, which are then coupled to an intermediate to form a mixture of carbamylated peptide fragments coupled to an intermediate.

In some embodiments of the invention, the total concentration of the antigen and the receptor bound thereto is 0.005-0.1 μ g/mL; the total concentration of said second anti-Carp antibody, and of one of the specific binding partners bound thereto, is from 0.025 to 0.1. mu.g/mL; the total concentration of the donor and the other member of the specific binding pair bound thereto is 5-20. mu.g/mL.

In a second aspect, the present invention provides a homogeneous immunoassay kit for the detection of a target anti-Carp antibody comprising a homogeneous immunoassay kit according to the first aspect of the present invention. The kit can be prepared by putting the homogeneous phase immunoassay reagent kit for detecting the target anti-Carp antibody into a kit.

Specifically, the homogeneous immunoassay kit for detecting the target anti-Carp antibody provided by the invention comprises an anti-Carp Ab calibrator, a carbamylated antigen coated on a receptor (luminescent particles), a biotin-labeled rabbit anti-carbamylated protein antibody, a donor (light-sensitive liquid) and a sample diluent.

In a specific embodiment, the carbamylated antigen is carbamylated human serum albumin as shown in SEQ ID No. 1.

In one embodiment, the carbamoylated antigen is a plurality of carbamoylated peptide sequences synthesized on a single peptide chain or coupled to Bovine Serum Albumin (BSA). The peptide segment and BSA are shown in SEQ ID No.2-SEQ ID No. 5.

In one embodiment, the receptors (luminescent particles) are selected from aldehyde-containing reactive groups.

In one embodiment, the marker is selected from biotin.

In one embodiment, the donor (the photoactive fluid) comprises the following components: the surface is coated with photosensitive substances phthalocyanine and streptavidin.

In one embodiment, the sample diluent comprises the following components: 0.02M phosphate, 0.15M sodium chloride, 0.5% Tween-20 and 2% bovine serum albumin fragment 5.

In a specific embodiment of the present invention, the present invention provides a method for preparing the above kit, comprising the steps of:

1) preparing an anti-Carp Ab calibrator;

2) coating the receptor with carbamylated antigen (luminophore);

3) labeling biotin with a rabbit anti-carbamylated protein antibody;

4) and preparing a sample diluent.

In one embodiment, the step of carbamoylating the antigen is as follows:

I) performing in-vitro carbamylation modification on the antigen by adopting a KOCN solution;

II) dialyzing the carbamoylated modified antigen to remove residual KOCN.

Wherein, in the step I), 1-10mg of the antigen is added into 0.5-2M KOCN to react for 24-36h at 36-38 ℃. For example, 1mg of human serum albumin was added to 1M KOCN (prepared with 0.2M PB buffer, pH 7.2) and reacted at 37 ℃ for 24 hours.

In step II), dialysis is performed at 2-8 deg.C for 48-72h with 0.1-0.25M PB buffer to remove residual KOCN. For example, dialysis against 0.2M PB buffer at 2-8 ℃ for 48h removed residual KOCN.

In a specific embodiment, the anti-Carp Ab calibrator, the carbamylated antigen-coated receptor (luminescent particles), the carbamylated antigen-labeled biotin and the sample diluent can be dispensed in step 5), and finally the components are assembled into a finished product.

In one embodiment, the steps for synthesizing the plurality of carbamylated peptide stretches on one peptide chain or coupling to Bovine Serum Albumin (BSA) are as follows:

TABLE 1

Serial number	Sequence of
		SEQ ID No.2	HQCHQEST-Hcit-GKSKGKCGKSGS
SEQ ID No.3	CKAAATQ-Hcit-KVERCARRR
		SEQ ID No.4	NEAN-Hcit-YQISVN-Hcit-YRG
SEQ ID No.5	NEEGFFSA-Hcit-GHRPLDKK

The four peptide fragments are synthesized and prepared into the carbamylated antigen by adopting the following method:

1. the four peptides are independently synthesized, are obtained according to a peptide fragment synthesis method, are synthesized by certain biotechnology limited company in Shanghai, and have the purity of more than 90 percent through high performance liquid chromatography-mass spectrometry (HPLC-MS).

2. Synthesizing the four peptide segments into one peptide to obtain the synthetic polypeptide. Obtained by peptide fragment synthesis method, synthesized by certain Biotech limited of Shanghai, with purity of over 90% by HPLC-MS detection.

3. Synthesizing the four peptide segments into one peptide to obtain the synthetic polypeptide. Obtained by peptide fragment synthesis method, synthesized by certain Biotech limited of Shanghai, with purity of over 90% by HPLC-MS detection. Coupling it with BSA by the following steps:

(1)4-8mg of sulfonic acid-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate sodium salt (Sulfo-SMCC) was dissolved in 640. mu.L of dimethyl sulfoxide (DMSO) to give a final concentration of 20 mM.

(2)1-5mg of the synthetic polypeptide in dry powder form was dissolved in 1000. mu.L of purified water to a final concentration of 5 mg/ml.

(3)10-50mg BSA was dissolved in 200-1000. mu.L purified water to a final concentration of 50mg/ml

(4) Fully mixing the synthetic polypeptide solution and the BSA solution according to the mass ratio of 1:1, dissolving the mixture in 5 times of PBS-EDTA solution, and standing the mixture at room temperature for 1 hour.

(5) To the above mixed solution, 100. mu.L of SMCC solution was added and the reaction was carried out overnight at room temperature.

(6) The reaction solution after the completion of the above reaction was transferred to a cross-linked dialysis buffer (0.1M PBS pH7.4) and dialyzed to remove free polypeptide, followed by purification to obtain carbamylated synthetic polypeptide-BSA.

In one embodiment, the preparation of an anti-Carp Ab calibrator comprises the steps of:

A1) preparation of calibrator buffer: accurately weighing 4.77g HEPES and 1.7g NaCl, adding 160mL purified water, mixing for 30min, adjusting pH to 7.4 + -0.2, and adding Proclin 3000.1 g, BSA 30g and 1M MgCl₂ 0.5mL、0.1M ZnCl₂0.1mL, stirring for 30min, adding purified water to constant volume of 200mL, after the pH value is measured again, 2-8C is reserved;

B1) preparing a calibration product: adding anti-Carp Ab with known active concentration into the buffer solution in the step A1), and preparing a standard substance with a certain concentration gradient. At least 3 concentrations are prepared, preferably 5 concentrations.

In one embodiment, carbamoylating antigen-coated receptors (luminescent microparticles) comprises the steps of:

carbamoylated human serum albumin coated receptors (luminophores):

A2) taking carbamylated human serum albumin for dialysis, adopting 1L of cross-linked dialysis buffer solution for dialysis at the temperature of 2-8 ℃, wherein the dialysis time is not less than 5h, and the dialysate is replaced every 2h (the formula is: 1.54gNa₂CO₃、2.94g NaHCO₃Dissolving in 1L purified water, adjusting pH to 9.0 + -0.05), and changing solution for 2-3 times;

B2) sucking out the dialyzed carbamylated human serum albumin, transferring the dialyzed carbamylated human serum albumin into a clean centrifugal tube, sampling and determining the protein concentration, wherein the determination method of the protein concentration is an ultraviolet spectrum absorption method or a BCA protein quantitative analysis kit;

C2) a certain amount of the receptor (luminescent particle) is added into a centrifuge tube, and the receptor (luminescent particle) is washed. The washing method comprises centrifuging at 12000rpm for 10min, discarding supernatant, adding 10-20 times volume of cross-linked dialysis buffer (1.54 g Na as formulation)₂CO₃、2.94g NaHCO₃Dissolving in 1L purified water, adjusting pH to 9.0 + -0.05), and cleaning with ultrasonic wave for 5 min. Centrifuge again and repeat the above washing step 2 times.

D2) Placing the washed receptor (luminous particles) in the step C2) on an analytical balance for zero setting, adding the carbamylated human serum albumin with the mass of 0.01-0.1 time of the receptor (luminous particles) dialyzed in the step A2) into the washed receptor (luminous particles) and calculating the volume (the density is calculated according to 1 g/mL), and supplementing a certain volume of cross-linked dialysis buffer solution into the receptor (luminous particles) to ensure that the total volume is 200 muL. Carbamylated human serum albumin and receptor (luminescent particles) were mixed well and placed on a vertical rotary mixer at 37 ℃ for overnight reaction at 25-40 rpm.

E2) Cooling the centrifugal tube after the reaction in the step D2) at 2-8 ℃ for 10min, and taking NaBH with the mass of 0.1-0.5 time that of the receptor (luminescent particle)₄Adding the mixture into a centrifuge tube and mixing the mixture evenly, and then placing the centrifuge tube on a vertical rotary mixer at the temperature of 2-8 ℃ for reaction for 2 hours at the speed of 25-40 rpm.

F2) Adding Gly 1-3 times of the mass of the acceptor (luminescent particles) into the centrifugal tube after the reaction in the step E2), and reacting for 1 hour at 25-40rpm on a vertical rotary mixer under the condition of room temperature.

G2) Washing step F2) carbamylated human serum albumin coated receptors (luminophores) by centrifugation at 12000rpm for 10min, discarding the supernatant, and adding 200 μ L of washing buffer (formulation: 2.90g Na₂HPO₄·12H₂O、0.296g NaH₂PO₄·2H₂O was dissolved in 1L of purified water), and washed with ultrasonic waves for 5 min. Centrifuging again, repeating the above washing steps for 2 times, and washing with microparticle preservation solution (formula: 2.5g HEPES, 17.5g NaCl, 1.0g Tween-20, 10g bovine serum albumin fragment 5 dissolved in 1L purified water) once.

H2) Add 200. mu.L of microparticle storage solution to store carbamylated human serum albumin-coated receptor (luminescent microparticles) and store at 2-8 deg.C for use.

Carbamoylation of synthetic polypeptide-BSA coated receptor (luminescent microparticles):

A3) taking carbamylation synthetic polypeptide-BSA for dialysis, adopting 1L cross-linked dialysis buffer solution for dialysis at the temperature of 2-8 ℃, wherein the dialysis time is not less than 5h, and the dialysate is replaced every 2h (the formula is: 1.54gNa₂CO₃、2.94g NaHCO₃Dissolving in 1L purified water, adjusting pH to 9.0 + -0.05), and changing solution for 2-3 times;

B3) sucking out the dialyzed carbamylated synthetic polypeptide-BSA, transferring the dialyzed carbamylated synthetic polypeptide-BSA into a clean centrifugal tube, sampling and determining the protein concentration, wherein the determination method of the protein concentration is an ultraviolet spectrum absorption method or a BCA protein quantitative analysis kit;

C3) a certain amount of the receptor (luminescent particle) is added into a centrifuge tube, and the receptor (luminescent particle) is washed.The washing method comprises centrifuging at 12000rpm for 10min, discarding supernatant, adding 10-20 times volume of cross-linked dialysis buffer (1.54 g Na as formulation)₂CO₃、2.94g NaHCO₃Dissolving in 1L purified water, adjusting pH to 9.0 + -0.05), and cleaning with ultrasonic wave for 5 min. Centrifuge again and repeat the above washing step 2 times.

D3) Placing the washed receptor (luminous particles) in the step C3) on an analytical balance for zero setting, adding the carbamation synthetic polypeptide-BSA with the mass of 0.01-0.1 time of the receptor (luminous particles) dialyzed in the step A2) into the washed receptor (luminous particles) and calculating the volume (the density is calculated according to 1 g/mL), and supplementing a certain volume of cross-linked dialysis buffer solution into the receptor (luminous particles) to ensure that the total volume is 200 muL. The carbamylation synthetic polypeptide-BSA and the receptor (luminescent particle) are evenly mixed and then the centrifugal tube is placed on a vertical rotary mixer at 37 ℃ for overnight reaction at 25-40 rpm.

E3) Cooling the centrifugal tube after the reaction in the step D3) at 2-8 ℃ for 10min, and taking NaBH with the mass of 0.1-0.5 time that of the receptor (luminescent particle)₄Adding the mixture into a centrifuge tube and mixing the mixture evenly, and then placing the centrifuge tube on a vertical rotary mixer at the temperature of 2-8 ℃ for reaction for 2 hours at the speed of 25-40 rpm.

F3) Adding Gly 1-3 times of the mass of the acceptor (luminescent particles) into the centrifugal tube after the reaction in the step E3), and reacting for 1 hour at 25-40rpm on a vertical rotary mixer under the condition of room temperature.

G3) Washing step F3) of the formylated synthetic polypeptide-BSA coated receptors (luminescent particles), centrifugation at 12000rpm for 10min, discarding the supernatant, and adding 200 μ L of washing buffer (formulation: 2.90g Na₂HPO₄·12H₂O、0.296g NaH₂PO₄·2H₂O was dissolved in 1L of purified water), and washed with ultrasonic waves for 5 min. Centrifuging again, repeating the above washing steps for 2 times, and washing with microparticle preservation solution (formula: 2.5g HEPES, 17.5g NaCl, 1.0g Tween-20, 10g bovine serum albumin fragment 5 dissolved in 1L purified water) once.

H3) Adding 200 μ L of microparticle preservation solution to preserve carbamylated synthesized polypeptide-BSA coated receptor (luminescent microparticle), and preserving at 2-8 deg.C for use.

In one embodiment, the labeling of biotin by a rabbit anti-carbamoylated protein antibody comprises the steps of:

A4) dialyzing a certain amount of rabbit anti-carbamoylation protein antibody, dialyzing with 1L cross-linked dialysis buffer solution at 2-8 deg.C for not less than 5h, and replacing dialysate every 2h for 2-3 times.

B4) And (3) sucking out the dialyzed rabbit anti-carbamylation protein antibody, transferring the antibody into a clean centrifugal tube, sampling and determining the protein concentration, wherein the determination method of the protein concentration is an ultraviolet spectrum absorption method or a BCA protein quantitative analysis kit.

C4) Taking a certain amount of rabbit anti-carbamylation protein antibody into a centrifuge tube, adding biotin according to the labeled molecular mass ratio of the rabbit anti-carbamylation protein antibody to the centrifuge tube of about 1:30, quickly and uniformly mixing after adding the biotin, and supplementing a certain volume of labeled buffer solution to ensure that the total volume is 200 mu L. The centrifuge tube was placed on a 2-8 ℃ vertical rotary mixer for overnight reaction at 25-40 rpm.

D4) Dialyzing the biotinylated rabbit anti-carbamoylation protein antibody marked in the step C4), dialyzing by adopting 1L of marked dialysis buffer solution at the temperature of 2-8 ℃, wherein the dialysis time is not less than 5h, and the dialysate is replaced every 2h for 2-3 times.

E4) Transferring the biotinylated rabbit anti-carbamoylated protein antibody obtained in the step D4) into a clean centrifuge tube, sampling, determining the protein concentration, and storing at 2-8 ℃ for later use.

The preparation of the sample diluent comprises the following steps:

accurately weighing 2.90g of Na by using a precision balance₂HPO₄·12H₂O、0.296g NaH₂PO₄·2H₂And O, adding 800mL of purified water, mixing uniformly for 30min, adjusting the pH value to 7.2 +/-0.2, continuously adding 8.5g of NaCl, 5g of Tween-20 and 20g of bovine serum albumin fragment 5, stirring for 30min, adding the purified water to a constant volume of 1L, and measuring the pH value again to obtain 2-8C for later use.

In a third aspect of the present invention, the homogeneous immunoassay method for detecting a target anti-Carp antibody in a sample to be tested comprises using the homogeneous immunoassay kit provided in the first aspect of the present invention to determine whether the target anti-Carp antibody is present in the sample to be tested and/or to determine the content of the target anti-Carp antibody.

Similarly, the homogeneous immunoassay method for detecting a target anti-Carp antibody in a test sample according to the present invention further comprises using the homogeneous immunoassay kit according to the second aspect of the present invention to determine whether the target anti-Carp antibody is present in the test sample and/or to determine the content of the target anti-Carp antibody.

In some embodiments of the present invention, the homogeneous immunoassay method for detecting a target anti-Carp antibody in a test sample comprises:

m1, allowing the second anti-Carp antibody and the target anti-Carp antibody in the sample to compete for binding with the epitope of the antigen, and forming a first immune complex composed of the antigen-second anti-Carp antibody and a second immune complex composed of the antigen-target anti-Carp antibody through the epitope and the antigen, respectively;

According to some embodiments of the present invention, when the homogeneous immunoassay of the target anti-Carp antibody is performed by using the one-step competition method, the step M1 includes mixing the second anti-Carp antibody, the sample to be tested and the antigen uniformly, reacting the mixture, allowing the second anti-Carp antibody and the target anti-Carp antibody in the sample to be tested to compete with the epitope of the antigen for binding, and forming a first immune complex composed of the antigen-second anti-Carp antibody and a second immune complex composed of the antigen-anti-Carp antibody through the epitope and the antigen, respectively.

In the present invention, when the homogeneous immunoassay of the target anti-Carp antibody is performed by the one-step competition method, there is no particular limitation on the manner of adding the second anti-Carp antibody, the sample to be detected, and the antigen, for example, in some embodiments of the present invention, the step of uniformly mixing the second anti-Carp antibody, the sample to be detected, and the antigen may include uniformly mixing the second anti-Carp antibody, the sample to be detected, and the antigen at the same time; in other embodiments of the present invention, the step of uniformly mixing the second anti-Carp antibody, the sample to be tested, and the antigen comprises mixing the second anti-Carp antibody with the sample to be tested to form a mixture 1, and then mixing the antigen with the mixture 1.

In some preferred embodiments of the present invention, when the homogeneous immunoassay of the target anti-Carp antibody is performed by the one-step competition method, the step of uniformly mixing the second anti-Carp antibody, the sample to be tested, and the antigen comprises mixing the second anti-Carp antibody and the sample to be tested to form a1 st mixture, and then mixing the antigen and the 1 st mixture.

According to other embodiments of the present invention, the homogeneous immunoassay of the anti-Carp antibody of interest is performed using a two-step competition method, wherein the step M1 comprises the steps of:

p1, mixing the antigen with the sample to be tested, reacting, combining the antigen with the target anti-Carp in the sample to be tested to form a second immune complex composed of antigen-target anti-Carp antibody, obtaining the 11 th mixture;

p2, mixing the second anti-Carp antibody with the 11 th mixture, reacting to allow the second anti-Carp antibody to compete for binding to the antigen in the 11 th mixture that is not bound to the target anti-Carp in the sample to be tested, thereby forming a first immune complex composed of antigen-second anti-Carp antibody.

In the present invention, the presence or absence of the second immune complex is detected by chemiluminescence in step M2.

In some specific embodiments of the invention, the homogeneous immunoassay of the anti-Carp antibody of interest is performed using a one-step competition method, said method comprising the steps of:

r2, mixing the antigen bound with the receptor with the 2 nd mixture, allowing the second anti-Carp antibody bound with biotin and the target anti-Carp antibody in the sample to compete for binding with the antigen bound with the receptor, to form a third immune complex composed of receptor-antigen-second anti-Carp antibody-biotin and a fourth immune complex composed of receptor-antigen-anti-Carp antibody, respectively, thereby forming a3 rd mixture;

In some embodiments of the invention, at step R4, the intensity of the chemiluminescent signal is detected and the amount of the target anti-Carp antibody in the sample to be tested is determined based on the anti-Carp antibody standard working curve.

In some preferred embodiments of the present invention, before step R1, there is further included step R0 of diluting the sample to be tested with the sample diluent in a volume ratio of 1 (4-20); preferably, the sample to be tested is diluted by the sample diluent according to the volume ratio of 1 (6-16); more preferably, the sample to be tested is diluted with the sample diluent according to the volume ratio of 1 (8-12).

In some embodiments of the present invention, the 4 th mixture is irradiated with the excitation light with a wavelength of 600-700nm to excite the donor in the fifth immunocomplex to generate singlet oxygen, the acceptor reacts with the contacted singlet oxygen to generate the emission light with a wavelength of 520-620nm, the signal value of the emission light of the 4 th mixture is detected and compared with the chemiluminescence signal value of the control immunocomplex formed by the equal amount of the antigen and the second anti-Carp antibody in the step R2, thereby determining whether the target anti-Carp antibody and/or the concentration of the target anti-Carp antibody is present in the test sample.

In some preferred embodiments of the present invention, the 4 th mixture is irradiated with excitation light having a wavelength of 680nm to excite the donor in the fifth immunocomplex to generate singlet oxygen, the acceptor reacts with the contacted singlet oxygen to generate emission light having a wavelength of 612nm, and a signal value of the emission light of the 4 th mixture is detected and compared with a chemiluminescence signal value of a control immunocomplex formed by equal amounts of the antigen and the second anti-Carp antibody in step R2, thereby determining whether the target anti-Carp antibody and/or the concentration of the target anti-Carp antibody is present in the sample to be tested.

In some embodiments of the invention, the homogeneous immunoassay of anti-Carp antibodies is carried out using a two-step competition method, comprising the steps of:

t1, mixing the antigen bound with the receptor with the sample to be tested, reacting to combine the antigen bound with the receptor with the target anti-Carp in the sample to be tested to form a fourth immune complex composed of the receptor-antigen-target anti-Carp antibody, and obtaining a 12 th mixture;

t2, mixing the biotin-bound second anti-Carp antibody with the 12 th mixture, reacting the mixture to compete the biotin-bound second anti-Carp antibody for binding to the receptor-bound antigen that is not bound to the target anti-Carp in the sample to be tested in the 12 th mixture, and forming a third immune complex composed of the receptor-bound antigen-second anti-Carp antibody-biotin, thereby forming a 13 th mixture;

In some embodiments of the present invention, at step T4, the intensity of the chemiluminescent signal is detected and the amount of the target anti-Carp antibody in the sample to be tested is determined based on the anti-Carp antibody standard working curve.

In some preferred embodiments of the present invention, step T0 is further included before step T1, the sample to be tested is diluted with the sample diluent according to a volume ratio of 1 (4-20); preferably, the sample to be tested is diluted by the sample diluent according to the volume ratio of 1 (6-16); more preferably, the sample to be tested is diluted with the sample diluent according to the volume ratio of 1 (8-12).

In some embodiments of the present invention, the 14 th mixture is irradiated with 600-700nm excitation light to excite the donor in the fifth immunocomplex to generate singlet oxygen, the acceptor reacts with the contacted singlet oxygen to generate 520-620nm emission light, and the signal value of the emission light of the 14 th mixture is detected and compared with the chemiluminescence signal value of a control immunocomplex formed by an equal amount of the antigen in step T1 and an equal amount of the second anti-Carp antibody in step T2, thereby determining whether the target anti-Carp antibody and/or the concentration of the target anti-Carp antibody is present in the test sample.

In some preferred embodiments of the present invention, the excitation light with a wavelength of 680nm is used to irradiate the second wavelength mixture, so as to excite the donor in the fifth immunocomplex to generate singlet oxygen, the acceptor reacts with the contacted singlet oxygen to generate emission light with a wavelength of 612nm, and the signal value of the emission light of the first immunocomplex is detected and compared with the chemiluminescence signal value of a control immunocomplex formed by an equivalent amount of the antigen in step T1 and an equivalent amount of the second anti-Carp antibody in step T2, so as to determine whether the target anti-Carp antibody and/or the concentration of the target anti-Carp antibody is present in the test sample.

In the present application, the system is based on the induction of a luminescent signal by a luminescent substance coated on the donor-acceptor surface via photoexcitation and energy transfer, which is dependent on antigen-antibody binding leading to the proximity of the donor-acceptor. Therefore, a separation process is not needed, the diameter of the nano particles is smaller, the suspension performance of the nano particles is stronger, and meanwhile, a three-level amplification luminescent system is adopted, so that the nano particles have higher analysis sensitivity; the whole detection process does not need cleaning, namely the binding label and the binding label do not need to be separated, so the reaction time is shorter; the tracer substances (photosensitizer and luminescent agent) are marked on the solid-phase microsphere instead of the biomolecule, so that the activity of the biomolecule is not influenced, and the solid-phase microsphere has a larger specific surface area, so that more tracer substances and biomolecules can be coated on the surface of the solid-phase microsphere, and the performance of the solid-phase microsphere in the aspects of effective concentration and sensitivity of a reagent, detection background and the like is better.

The inventors further investigated and studied the homogeneous immunoassay method for detecting the target anti-Carp antibody in the sample to be tested. In one example, homogeneous immunoassay of the target anti-Carp antibody was performed using a one-step competition method, in which a carbamylated BSA-coated receptor, a sample and a biotin-labeled rabbit anti-carbamylated lysine antibody were added together to the reaction system. At this point, the target anti-Carp antibody in the sample competes simultaneously with the biotin-labeled rabbit anti-carbamylated lysine antibody for binding to a limited amount of carbamylated BSA. The inventors tested a total of 6 negative samples and 16 positive samples. B/B0<0.8 is used as the judgment standard of yin and yang (B is the test luminescence value after adding the sample, B0 is the luminescence value of the control group without adding the sample, and when the luminescence value of B/B0 is reduced, the existence of carbamylated BSA with limited competitive binding of the target anti-Carp antibody and the rabbit anti-carbamylated lysine antibody in the sample is indicated). As shown in FIG. 1, it can be seen from FIG. 1 that all negative samples were judged to be negative, and 13 of 16 positive samples were judged to be positive. This indicates that the one-step competition method can clearly distinguish the negative sample from the positive sample, but the sensitivity is low, and part of the positive sample cannot be detected.

In another embodiment, homogeneous immunoassay of the target anti-Carp antibody is performed by a two-step competition method, the receptor coated with carbamylated BSA and the sample are added into the reaction system for 5 minutes in advance, and the rabbit anti-carbamylated lysine antibody labeled with biotin is added after the reaction is completed. In this reaction mode, the target anti-Carp antibody in the sample binds to a portion of the epitopes on carbamylated BSA first, and then the biotin-labeled rabbit anti-carbamylated lysine antibody binds to the remaining epitopes on carbamylated BSA. The inventors tested a total of 7 negative samples and 16 positive samples. B/B0<0.8 is used as the judgment standard of yin and yang. As a result, as shown in FIG. 2, it can be seen from FIG. 2 that all negative samples were judged to be negative, and all positive samples were judged to be positive. This shows that the test result of the two-step competition method is obviously superior to that of the one-step competition method, and the sensitivity is better.

In a fourth aspect of the present invention, the use of the homogeneous immunoassay reagent kit according to the first aspect of the present invention for detecting the presence and/or amount of a target anti-Carp antibody in a sample to be tested is understood as a method for determining the presence and/or amount of a target anti-Carp antibody in a sample to be tested by using the homogeneous immunoassay reagent kit according to the second aspect of the present invention, wherein the sample to be tested is selected from blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, saliva, synovial fluid and emphysema fluid, preferably the sample to be tested is selected from blood, plasma and serum, and more preferably the sample to be tested is serum.

Similarly, the use of the homogeneous immunoassay kit provided by the second aspect of the present invention in detecting the presence and/or content of an anti-Carp antibody in a sample to be tested can be understood as a method for determining the presence and/or content of a target anti-Carp antibody in a sample to be tested by using the homogeneous immunoassay kit provided by the second aspect of the present invention, wherein the sample to be tested is selected from blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, saliva, synovial fluid and emphysema fluid, preferably the sample to be tested is selected from blood, plasma and serum, and further preferably the sample to be tested is serum.

Similarly, the application of the homogeneous immunoassay method provided by the present invention according to the third aspect of the present invention to detecting the presence and/or amount of a target anti-Carp antibody in a sample to be tested can be understood as a method for determining the presence or absence of a target anti-Carp antibody in a sample to be tested and/or determining the amount of a target anti-Carp antibody by using the homogeneous immunoassay method according to the first aspect of the present invention, wherein the sample to be tested is selected from blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, saliva, synovial fluid and emphysema, preferably the sample to be tested is selected from blood, plasma and serum, and further preferably the sample to be tested is serum.

Similarly, the application of the homogeneous immunoassay method provided by the present invention in detecting the presence and/or amount of the target anti-Carp antibody in the sample to be tested can be understood as a method for determining the presence or absence of the target anti-Carp antibody in the sample to be tested and/or determining the amount of the target anti-Carp antibody by using the homogeneous immunoassay method provided by the second aspect of the present invention, wherein the sample to be tested is selected from blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, saliva, synovial fluid and emphysema, preferably the sample to be tested is selected from blood, plasma and serum, and further preferably the sample to be tested is serum.

A fifth aspect of the present invention relates to the use of a kit of reagents according to the first aspect of the present invention in the preparation of a kit for detecting an anti-Carp antibody of interest in a test sample of a subject suspected of suffering from rheumatoid arthritis, thereby determining the level of the anti-Carp antibody of interest in said test sample and correlating the level thus determined with the presence, risk, potential or predisposition of rheumatoid arthritis in the subject, comprising:

step N1, providing a sample to be tested from a main body to be tested;

step N2, judging whether the target anti-Carp antibody exists in the sample to be detected and/or determining the content of the target anti-Carp antibody;

step N3, comparing the content of said anti-Carp antibody in a normal control sample, a rheumatoid arthritis control sample, or a pre-treatment sample from the same subject to be tested;

wherein the sample to be tested is selected from blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, saliva, synovial fluid and emphysema effusion.

In some embodiments of the invention, the presence of the anti-Carp antibody of interest in the test sample is a diagnostic indicator of rheumatoid arthritis in the test subject, as compared to a normal control sample.

In other embodiments of the invention, an increase in the amount of the anti-Carp antibody of interest in the test sample as compared to a normal control sample is a diagnostic indicator of rheumatoid arthritis in the test subject.

In still other embodiments of the present invention, a content of the target anti-Carp antibody of not less than 123.5U/mL in the test sample compared to a normal control sample is a diagnostic indicator of rheumatoid arthritis in the test subject.

In the present invention, the relative amount of the target anti-Carp antibody in the test sample as compared to the rheumatoid arthritis control sample is a prognostic indicator of rheumatoid arthritis in the test subject.

In some embodiments of the invention, the relative amount of the anti-Carp antibody of interest in the test sample, as compared to a pre-treatment sample from the same test subject, is indicative of the efficacy of the treatment regimen.

According to the method of the present invention, in step N2, the method according to the fourth aspect of the present invention is used to determine whether the target anti-Carp antibody is present in the test sample and/or to determine the content of the target anti-Carp antibody.

Example III

In order that the present invention may be more readily understood, the following detailed description will proceed with reference being made to examples, which are intended to be illustrative only and are not intended to limit the scope of the invention. The starting materials or components used in the present invention may be commercially or conventionally prepared unless otherwise specified.

Example 1:

reagents and experimental materials:

the reagent I is: carbamylated human serum albumin coated with receptors (luminescent particles) having aldehyde-based reactive groups on their surface;

the reagent II is: the biotin-labeled rabbit anti-carbamoylated protein antibody.

The various buffers were prepared as follows:

calibrator buffer: accurately weighing 4.77g of HEPES and 1.7g of NaCl by using a precision balance, adding 160mL of purified water, uniformly mixing for 30min, adjusting the pH value to 7.4 +/-0.2, and continuously adding 3000.1g of Proclin, 30g of BSA and 1M MgCl₂0.5ml、0.1M ZnCl₂0.1ml, stirring for 30min, adding purified water to a constant volume of 200g, and measuring pH value again to obtain a solution of 2-8C for later use.

KOCN solution: KOCN 8.112g and Na were precisely weighed using a precision balance₂HPO₄·12H₂O 5.9g、KH₂PO₄0.488g and purified water to 100mL, and adjusting the pH value to 7.2 +/-0.05.

Cross-linked dialysis buffer 1: accurately weighing Na by using a precision balance₂CO₃1.54 g、NaHCO₃2.94g, adding purified water to a constant volume of 1L, and adjusting the pH value to 9.0 +/-0.05.

Cross-linked dialysis buffer 2: accurately weighing 4.875g of MES by using a precision balance, dissolving the MES in 1L of purified water, and adjusting the pH value to 5.0 +/-0.05.

Washing buffer solution: accurately weighing 2.90g of Na by using a precision balance₂HPO₄·12H₂O、0.296gNaH₂PO₄·2H₂And O, adding purified water to the volume of 100 mL.

Microparticle preservation solution: precisely weighing 2.5g of HEPES, 17.5g of NaCl, 1.0g of Tween-20 and 10g of bovine serum albumin fragment 5 by using a precision balance, and adding purified water to the volume of 100 mL.

Preparation of carbamylated human serum albumin comprising the steps of:

1) 1mg of human serum albumin was added to a 1M solution of KOCN to conduct a reaction at 37 ℃ for 24 hours.

2) After the reaction is finished, dialyzing with ultrapure water for 48h at the temperature of 2-8 ℃ to remove residual KOCN, and storing at the temperature of 2-8 ℃ for later use.

In the above carbamylated human serum albumin, there are one or more lysine residues carbamylated, corresponding to the presence of one or more anti-Carp Ab binding sites.

The preparation method of the anti-Carp Ab light-activated chemiluminescence immunoassay detection kit comprises the following operation steps:

firstly, preparing a working solution of a calibrator

1. Preparation of calibrator buffer: accurately weighing 4.77g HEPES and 1.7g NaCl, adding 160mL purified water, mixing for 30min, adjusting pH to 7.4 + -0.2, and adding proclin 3000.1g, BSA 30g and 1M MgCl₂ 0.5ml、0.1M ZnCl₂0.1ml, stirring for 30min, adding purified water to weight of 200g, and measuring pH value again, and keeping at 2-8C for later use.

2. Preparing a calibration product: an anti-CarpAb with a concentration of 500U/mL was prepared as a 40U/mL solution, which was subsequently diluted sequentially to 1, 2.5, 8, 20U/mL, plus a 40U/mL concentration point and a 0U/mL concentration point (buffer), to give A: 0U/mL, B: 1U/mL, C: 2.5U/mL, D: 8U/mL, E: 20U/mL, F: 40U/mL, 6 concentrations of the calibrator were combined.

Preparation of carbamylated human serum Albumin-coated receptor (luminescent particles) (reagent I)

Carbamylated human serum albumin coated receptors (luminescent microparticles) containing aldehyde-reactive groups:

1. dialyzing 0.2mg carbamylated human serum albumin with 1L cross-linked dialysis buffer solution 1at 2-8 deg.C for at least 5 hr, and changing the dialysate every 2 hr for 2-3 times.

2. And (3) sucking out the carbamylated human serum albumin dialyzed in the step (1), transferring the carbamylated human serum albumin into a clean centrifugal tube, and sampling to determine the protein concentration, wherein the determination method of the protein concentration is an ultraviolet spectrum absorption method or a BCA protein quantitative analysis kit.

3.2 mg of the acceptor (luminophore) was added to the centrifuge tube and the acceptor (luminophore) was washed. The washing method comprises centrifuging at 12000rpm for 10min, discarding the supernatant, adding 200 μ L cross-linked dialysis buffer solution into the centrifuge tube, and washing with ultrasonic wave for 5 min. The supernatant was centrifuged again and the washing step was repeated 2 times.

4. And (3) placing the washed receptor (luminescent particle) in the step (3) on an analytical balance for zero setting, adding 0.1mg of carbamylated human serum albumin dialyzed in the step (1) into a centrifuge tube filled with the receptor (luminescent particle), calculating the volume (the density is calculated according to 1 g/mL), and supplementing a certain volume of cross-linked dialysis buffer solution into the receptor (luminescent particle) to ensure that the total volume is 200 mu L, wherein the concentration of the particle is 10 mg/mL. The two were mixed well in a centrifuge tube and placed on a vertical rotary mixer at 37 ℃ for overnight reaction at 25-40 rpm.

5. Accurately weighing 8mg of KBH₄Dissolved in a labeling buffer to a final concentration of 8 mg/mL.

6. Cooling the centrifuge tube after the reaction in the step 4 at 2-8 ℃ for 10min, and taking 4 mu L of NaBH in the step 5₄The solution is added into a centrifuge tube and mixed evenly, and then the centrifuge tube is placed on a vertical rotary mixer at the temperature of 2-8 ℃ for reaction for 2 hours at the speed of 25-40 rpm.

7. To the centrifugal tube in which the reaction was completed in step 6, 32. mu.L of 75mg/mL glycine solution (75 mg of glycine was accurately weighed and dissolved in purified water to a final concentration of 75mg/mL) was added and reacted at room temperature for 1 hour at 25 to 40rpm on a vertical rotary mixer.

8. Washing the carbamylated human serum albumin coated receptor (luminescent particles) in step 7 by centrifugation at 12000rpm for 10min, discarding the supernatant, adding 200. mu.L of washing buffer to the centrifuge tube, and washing with ultrasound for 5 min. Centrifuging again and discarding the supernatant, repeating the above cleaning steps for 2 times, and finally cleaning once with the microparticle preservation solution.

9. Adding particle preservation solution (2.5g HEPES, 17.5g NaCl, 1.0g Tween-20, 10g bovine serum albumin fragment 5, adding purified water to constant volume to 100mL) to preserve carbamylated human serum albumin-coated receptor (luminescent particles), sampling to determine protein concentration, making its working concentration be 0.1 μ g/mL, and preserving at 2-8 deg.C for use.

Thirdly, preparation of biotinylated Rabbit anti-carbamoylated protein antibody (second anti-Carp antibody) (reagent II)

1. Dialyzing 0.2mg rabbit anti-carbamoylated protein antibody with 1L cross-linked dialysis buffer (Na)₂CO₃1.54g、NaHCO₃2.94g, adding purified water to a constant volume of 1L, adjusting the pH value to 9.0 +/-0.05), dialyzing at the temperature of 2-8 ℃, wherein the dialysis time is not less than 5h, and the dialysate is replaced every 2h for 2-3 times.

2. And (3) sucking out the rabbit anti-carbamylation protein antibody dialyzed in the step (1), transferring the rabbit anti-carbamylation protein antibody into a clean centrifugal tube, sampling and determining the protein concentration, wherein the determination method of the protein concentration is an ultraviolet spectrum absorption method or a BCA protein quantitative analysis kit.

3.5 mg biotin was weighed out accurately and dissolved in DMSO to a final concentration of 5 mg/mL.

4. Adding 0.1mg of rabbit anti-carbamoylated protein antibody into a centrifuge tube, adding 3 μ L of biotin solution (labeled molecular mass ratio between the two is about 1:30) obtained in step 3, adding the biotin solution, rapidly mixing, and supplementing a certain volume of cross-linked dialysis buffer solution to make the total volume 200 μ L. The centrifuge tubes were then placed on a 2-8 ℃ vertical rotary mixer for overnight reaction at 25-40 rpm.

5. Dialyzing the biotinylated rabbit anti-carbamoylation protein antibody marked in the step 4, dialyzing by adopting 1L of cross-linked dialysis buffer solution at the temperature of 2-8 ℃, wherein the dialysis time is not less than 5h, and changing the dialysate every 2h for 2-3 times.

6. And (3) transferring the biotinylated rabbit anti-carbamylation protein antibody obtained in the step (5) into a clean centrifuge tube, sampling to determine the protein concentration, wherein the working concentration is 0.1 mu g/mL, and storing at 2-8 ℃ for later use after sampling to determine the protein concentration.

Fourthly, preparing sample diluent

Accurately weighing 2.90g of Na by using a precision balance₂HPO₄·12H₂O、0.296g NaH₂PO₄·2H₂Adding 800mL of purified water, mixing for 30min, adjusting the pH value to 7.2 +/-0.2, continuously adding 8.5g of NaCl, 5g of Tween-20 and 20g of bovine serum albumin fragment 5, stirring for 30min, adding purified water to a constant volume of 1L, and measuring the pH value again and keeping at 2-8 ℃ for later use.

Fifthly, preparation of donor (sensitization liquid)

(1) Donor (photosphere) suspension treatment

Sucking a certain amount of photosensitive microspheres in a high-speed refrigerated centrifuge for centrifugation, discarding supernatant, adding a certain amount of MES buffer solution, performing ultrasound on an ultrasonic cell disruptor until the particles are resuspended, and adding MES buffer solution to adjust the concentration of the photosensitive microspheres to 100 mg/ml.

(2) Preparation of streptavidin solution

A certain amount of streptavidin was weighed and dissolved in MES buffer to 8 mg/ml.

(3) Mixing

Mixing the processed photosensitive microsphere (donor) suspension, 8mg/ml Avidin and MES buffer solution in a volume ratio of 2: 5: 1, and quickly mixing to obtain a reaction solution.

(4) Reaction of

Preparing 25mg/ml NaBH by MES buffer solution₃CN solution is added according to the volume ratio of 1: 25 to the reaction solution and is rapidly and evenly mixed. The reaction was rotated at 37 ℃ for 48 hours.

(5) Sealing of

MES buffer solution is prepared into 75mg/ml Gly solution and 25mg/ml NaBH₃CN solution, according to

Adding the mixture into the solution according to the volume ratio of the mixture to the reaction solution of 2: 1:10, uniformly mixing, and carrying out rotary reaction at 37 ℃ for 2 hours. Then, 200mg/ml BSA solution (MES buffer) was added thereto at a volume ratio of 5: 8, and the mixture was rapidly mixed and subjected to a rotary reaction at 37 ℃ for 16 hours.

(6) Cleaning of

Adding MES buffer solution into the reacted solution, centrifuging by a high-speed refrigerated centrifuge, discarding the supernatant, adding fresh MES buffer solution, resuspending by an ultrasonic method, centrifuging again, cleaning for 3 times, finally suspending by a small amount of photosensitive reagent buffer solution, measuring the solid content, adjusting the working concentration to 20 mu g/mL by using the photosensitive reagent buffer solution, and using the solution as a universal solution.

Sixthly, semi-finished products and finished product composition

Subpackaging the obtained product to obtain semi-finished product, performing spot inspection to obtain qualified product, assembling into finished product, and storing at 2-8 deg.C.

Seventh, clinical serum sample testing

Collecting serum: there were 419 cases of clinically elevated serum anti-Carp Ab levels collected in SJ and Jilin KZ, including 177 patients with confirmed RA and 242 non-RA. Determining yin-yang judgment standard according to the working characteristic curve of the subject.

The kit prepared in this example was applied to the detection step of a full-automatic light-activated chemiluminescence immunoassay analyzer LICA500 (manufactured by Shanghai Boyang).

1) Diluting the sample in a pre-dilution hole position according to a ratio of 1:10, and uniformly mixing for 20 seconds;

2) a sample adding Tip sucks 10 mu L of diluted sample or calibrator into a reaction microplate;

3) a reagent sample adding Tip absorbs 25 mu L of carbamylated human serum albumin coated receptor (luminescent particles) to a reaction microporous plate;

4) absorbing 25 mu L of biotinylated rabbit anti-carbamylation protein antibody into a reaction microplate by a reagent sample adding Tip;

5) mixing for 20 s, and incubating at 37 deg.C for 17 min;

6) absorbing 175 mu L of donor (photosensitive solution with working concentration of 20 mu g/mL) into a reaction micropore plate by a reagent sample adding Tip;

7) mixing for 20 s, and incubating at 37 deg.C for 15 min;

8) under the irradiation of the excitation light generated by the instrument, the photosensitive particles in the donor are induced to be activated, and active oxygen ions in high energy states are released. The active oxygen ions in the high energy state are captured by the acceptor (luminescent particle) at a close distance, thereby transferring energy to activate the luminescent compound in the acceptor (luminescent particle). After a few microseconds, the luminescent compound in the receptor (luminescent particle) will release high-level red light, and the high-level photons are measured by a single photon counter;

9) respectively testing the luminous values of the standard substances with different concentrations according to the steps 1) to 8), and drawing a standard curve according to a five-parameter fitting method to obtain a relational expression between the luminous values and the anti-Carp Ab concentration; and respectively testing the luminous values of the samples to be tested according to the steps 1) -8), and calculating the concentration of the anti-Carp Ab in the samples to be tested according to the relational expression.

The result of the detection

Calibration curve:

TABLE 2

Point of concentration	Luminous value
		0U/mL	121407
1U/mL	104039
		2.5U/mL	82153
8U/mL	70119
		20U/mL	45012
40U/mL	21513

And (4) conclusion: the standard curve degree fitting equation R2 is more than 0.99, and meets the requirement of clinical quantitative determination.

Serum testing: the kit of this example 1 diagnoses elevated anti-Carp Ab levels as RA sensitivity and specificity of 52% and 96.3% respectively, using 123.5U/ml as a cut-off value, as shown in FIG. 3:

for all patients with elevated serum anti-Carp Ab concentrations, the true negative rate for RA diagnosis was 73.27%, the true positive rate was 91.09%, and the total accuracy was 77.56% at the cutoff value of 123.5U/ml, as shown in table 3 and fig. 4:

TABLE 3

Example 2

In contrast to example 1, the carbamylated antigen in reagent I is carbamylated synthetic polypeptide-BSA, reagent I is: carbamoylation of synthetic polypeptide-BSA coated receptors (luminescent microparticles).

First, preparing carbamylation synthetic polypeptide-BSA

1.5 mg of sulfonic succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate sodium salt (Sulfo-SMCC) was dissolved in 800. mu.L of dimethyl sulfoxide (DMSO) to give a final concentration of 20 mM.

2. 3mg of the synthetic polypeptide in a dry powder form was accurately measured and dissolved in 600. mu.L of purified water to a final concentration of 5 mg/ml.

3. 40mg of BSA was accurately measured and dissolved in 800. mu.L of purified water to a final concentration of 50 mg/ml.

4. The synthetic polypeptide solution and BSA solution were mixed well at a mass ratio of 1:1 (2 mg each) and dissolved in 2.25ml of 0.01M PBS buffer, and the mixture was allowed to stand at room temperature for 1 hour.

5. To the above mixed solution was added 100. mu.L of Sulfo-SMCC solution, and the reaction was carried out overnight at room temperature.

6. Transferring the reaction solution to cross-linked dialysis buffer (0.1M PBS pH7.4), dialyzing to remove free polypeptide, purifying to obtain carbamylated synthetic polypeptide-BSA, and storing at 2-8 deg.C.

Secondly, carbamylation synthesis of polypeptide-BSA coated receptor (luminescent particle) (reagent I)

1. Dialyzing 0.2mg carbamylation synthetic polypeptide-BSA, dialyzing with 1L cross-linked dialysis buffer solution 1at 2-8 deg.C for not less than 5h, and replacing dialysate every 2h for 2-3 times.

2. And (3) sucking out the carbamylated synthetic polypeptide-BSA dialyzed in the step (1) and transferring the carbamylated synthetic polypeptide-BSA into a clean centrifugal tube, and sampling to determine the protein concentration, wherein the determination method of the protein concentration is an ultraviolet spectrum absorption method or a BCA protein quantitative analysis kit.

4. And (3) placing the washed receptor (luminescent particle) in the step (3) on an analytical balance for zero setting, adding 0.1mg of carbamylated synthetic polypeptide-BSA dialyzed in the step (1) into a centrifuge tube filled with the receptor (luminescent particle), calculating the volume (the density is calculated according to 1 g/mL), and supplementing a certain volume of cross-linked dialysis buffer solution into the receptor (luminescent particle) to ensure that the total volume is 200 mu L, wherein the concentration of the particle is 10 mg/mL. The two were mixed well in a centrifuge tube and placed on a vertical rotary mixer at 37 ℃ for overnight reaction at 25-40 rpm.

6. Placing the centrifuge tube with the reaction completed in the step 4 in a range of 2-8Cooling at 10 deg.C for 10min, and collecting 4 μ L of NaBH from step 5₄The solution is added into a centrifuge tube and mixed evenly, and then the centrifuge tube is placed on a vertical rotary mixer at the temperature of 2-8 ℃ for reaction for 2 hours at the speed of 25-40 rpm.

8. Washing the receptor (luminescent particle) coated by the carbamylation synthetic polypeptide-BSA in the step 7, centrifuging the receptor (luminescent particle) at 12000rpm for 10min, discarding the supernatant, adding 200 mu L of a washing buffer solution into a centrifuge tube, and washing the centrifuge tube for 5min by adopting ultrasonic waves. Centrifuging again and discarding the supernatant, repeating the above cleaning steps for 2 times, and finally cleaning once with the microparticle preservation solution.

9. Adding particle preservation solution (2.5g HEPES, 17.5g NaCl, 1.0g Tween-20, 10g bovine serum albumin fragment 5, adding purified water to constant volume to 100mL) to preserve carbamylated synthetic polypeptide-BSA coated receptor (luminescent particles) to make its working concentration be 0.1 μ g/mL, and preserving at 2-8 deg.C for use.

Otherwise the same as example 1

The result of the detection

Calibration curve:

TABLE 4

Point of concentration	Luminous value
		0U/mL	130743
1U/mL	110167
		2.5U/mL	98671
8U/mL	76854
		20U/mL	42678
40U/mL	19963

Serum testing: the kit of this example 2 diagnoses elevated anti-Carp Ab levels as 52% sensitivity and 96.3% specificity of RA, respectively, with a cut-off of 123.5U/ml, as shown in fig. 5:

for all patients with elevated serum anti-Carp Ab concentrations, the true negative rate for RA diagnosis was 73.27%, the true positive rate was 91.09%, and the total accuracy was 77.56% at the cutoff value of 123.5U/ml, as shown in table 5 and fig. 6:

TABLE 5

RA sample (example)	177
		non-RA sample (example)	242
Negative test (example)	318
		True negatives (example)	233
True negative rate (%)	73.27
		Positive test (example)	101
True positive (example)	92
		True Positive Rate (%)	91.09
Total accuracy (%)	77.56

The above test results show that the clinical application effect of the kit prepared in example 2 is at the same level as that of the kit prepared in example 1.

The results of investigation experiments on the amount of carbamylated peptide fragments which are connected in series to form the polypeptide show that, for the polypeptide which is synthesized on one peptide chain by at least 2 single carbamylated peptide fragments, the molar ratio of a plurality of different peptide fragments is the same, and the activity of the prepared polypeptide is the highest.

Comparative example 1

The performance of the anti-Carp Ab kit prepared in example 1 and example 2 of the present invention was compared with the performance of the detection reagent for anti-Carp Ab reported in the prior published patent literature, and the results are shown in fig. 7.

As can be seen from fig. 7, the sensitivity and specificity of detecting anti-Carp Ab by ELISA indirect method using carbamylated fetal calf serum (Ca-FCS) as antigen in WO2012/105838a1 are 31.3% and 98.8%, respectively; in WO2016/014612A2, carbamylated human alpha 1 antitrypsin (Ca-A1AT) was used as an antigen, and the sensitivity and specificity of anti-Carp Ab detected by ELISA indirect method were 35% and 98.8%, respectively. The specificity for RA diagnosis is far from the specificity for RA diagnosis in the methods of example 1 and example 2, and the sensitivity is significantly lower than that of example 1 and example 2. The Anti-Carp Ab kit prepared by the invention has obvious advantages in the index of sensitivity compared with Anti-Carp Ab kits prepared in patents WO2012/105838A1 and WO2016/014612A2 in the aspect of diagnosis of RA.

By examining the amount of individual carbamylated peptides forming a mixture of carbamylated peptides, the results show that for a mixture of carbamylated peptides comprising at least 2 individual carbamylated peptides, the mass ratio of the different peptides to each other is the same and the resulting mixture of carbamylated peptides has the highest activity.

The carbamylated peptide fragment mixture is coupled with BSA to prepare the kit for clinical serum sample test, and the detection result shows that the clinical application effect of the kit is in the same level as that of the kit prepared in the embodiment 1.

Antigen-coated receptors prepared according to the preparation method in the examples described in patent PCT/US2010/025433, with the structure carbamylated human serum albumin-BSA- (dimethylthiophene) - (BHHCT), in non-particulate form, and soluble in aqueous media, were used. The kit prepared by the antigen-coated receptor and other corresponding reagents in the embodiment 1 is used for clinical serum sample testing, and the detection result shows that the clinical application effect of the kit is in the same level as or even better than that of the kit prepared in the embodiment 1.

It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.

Sequence listing

<110> Koume diagnostic technology GmbH

<120> homogeneous immunoassay kit for detecting target anti-Carp antibody and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 609

<212> PRT

<213> (human serum albumin)

<400> 1

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala

20 25 30

His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu

35 40 45

Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val

50 55 60

Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp

65 70 75 80

Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp

85 90 95

Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala

100 105 110

Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln

115 120 125

His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val

130 135 140

Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys

145 150 155 160

Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro

165 170 175

Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys

180 185 190

Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu

195 200 205

Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys

210 215 220

Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val

225 230 235 240

Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser

245 250 255

Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly

260 265 270

Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile

275 280 285

Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu

290 295 300

Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp

305 310 315 320

Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser

325 330 335

Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly

340 345 350

Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val

355 360 365

Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys

370 375 380

Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu

385 390 395 400

Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys

405 410 415

Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu

420 425 430

Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val

435 440 445

Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His

450 455 460

Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val

465 470 475 480

Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg

485 490 495

Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe

500 505 510

Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala

515 520 525

Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu

530 535 540

Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys

545 550 555 560

Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala

565 570 575

Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe

580 585 590

Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly

595 600 605

Leu

Claims

1. A homogeneous immunoassay kit for the detection of a target anti-Carp antibody comprising:

a component a comprising an antigen that binds to a receptor; the antigen is capable of specifically binding to both the anti-Carp antibody of interest and the second anti-Carp antibody; wherein the antigen is a carbamylated synthetic polypeptide-BSA in which a polypeptide formed on one peptide chain is synthesized from at least 2 single carbamylated peptide fragments and the polypeptide is coupled to the BSA;

component b comprising a second anti-Carp antibody; the second anti-Carp antibody is a biotinylated rabbit anti-carbamoylated protein antibody; the second anti-Carp antibody binds to biotin;

a component c comprising a donor capable of producing singlet oxygen in an excited state; the donor binds to streptavidin.

2. The kit of claim 1, wherein the antigen comprises a plurality of different peptide fragments in a molar ratio relative to each other; and/or

The carbamylated peptide fragment is selected from SEQ ID No. 2-5.

3. The kit of claim 1, wherein the total concentration of the antigen and the receptor bound thereto is 0.005-0.1 μ g/mL; and/or the total concentration of the second anti-Carp antibody and biotin bound thereto is 0.025-0.1. mu.g/mL; and/or the total concentration of the donor and the streptavidin combined with the donor is 5-20 mug/mL.

4. The kit of any one of claims 1 to 3, wherein the method for preparing the carbamylated synthetic polypeptide-BSA comprises the steps of:

s1, synthesizing at least 2 single carbamylation peptide segments on one peptide chain to obtain the polypeptide;

s2, dissolving the polypeptide to obtain a polypeptide solution; dissolving BSA to obtain a BSA solution;

s3, mixing the polypeptide solution and the BSA solution in a PBS solution to obtain a mixed solution;

s4, reacting the mixed solution with an SMCC solution to obtain a reacted system;

s5, dialyzing and purifying the reacted system to obtain the carbamylated synthetic polypeptide-BSA.

5. The kit according to claim 4, wherein in step S1, the polypeptide has a purity of 90% or more; and/or

In the step S2, the concentration of the polypeptide solution is 4-6 mg/ml; the concentration of the BSA solution is 45-55 mg/ml; and/or

In step S3, the mass ratio of the polypeptide solution to the BSA solution in the mixed solution is 1 (0.9-1.1); the volume of the PBS solution is 4-6 times of the total volume of the polypeptide solution and the BSA solution; and/or

In step S4, the concentration of the SMCC solution is 18-22 mg/ml.

6. An immunoassay method for detecting a target anti-Carp antibody in a test sample using the homogeneous immunoassay kit according to any one of claims 1 to 5, comprising the steps of:

7. The method according to claim 6, characterized in that it comprises the steps of:

r4, exciting the donor with energy or an active compound to produce singlet oxygen, said acceptor reacting with the singlet oxygen to generate a detectable chemiluminescent signal; detecting the presence or absence of a fourth immune complex; if the fourth immune complex is present, then an anti-Carp antibody is present in the test sample.

8. The method according to claim 6, characterized in that it comprises the steps of:

t1, mixing the antigen bound with the receptor with the sample to be tested, reacting, and binding the target anti-Carp in the sample to be tested with the epitope of the antigen bound with the receptor to form a fourth immune complex composed of the receptor-antigen-target anti-Carp antibody, to obtain a 12 th mixture;

9. Use of a homogeneous immunoassay kit according to any one of claims 1 to 5 or a method according to any one of claims 6 to 8 for detecting the presence and/or amount of a target anti-Carp antibody in a test sample selected from the group consisting of blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, saliva, synovial fluid and emphysema fluid.

10. Use of a kit according to any one of claims 1 to 5 in the preparation of a kit for the detection of a target anti-Carp antibody in a test sample of a subject suspected of having rheumatoid arthritis, whereby the level of the target anti-Carp in the test sample is determined and the level thus determined is correlated with the presence, risk, potential or predisposition of rheumatoid arthritis in the subject.