CN111398580B - Method for detecting target IgM antibody in sample - Google Patents
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- CN111398580B CN111398580B CN202010123237.4A CN202010123237A CN111398580B CN 111398580 B CN111398580 B CN 111398580B CN 202010123237 A CN202010123237 A CN 202010123237A CN 111398580 B CN111398580 B CN 111398580B
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
Abstract
The invention relates to a method for detecting target IgM antibodies in a sample, which comprises the following steps: first preparing a complex formed by a C1 q-donor-IgM antibody-known antigen-acceptor; then, the compound is treated by energy or active compound to excite the donor to generate singlet oxygen, and the acceptor reacts with the singlet oxygen to generate a detectable chemiluminescent signal; and finally, analyzing the chemiluminescent signal condition, and judging whether the target IgM antibody and the content of the target IgM antibody exist in the sample to be detected. The method for detecting the target IgM antibody in the sample solves the problem of influence of the nonspecific IgM antibody on detection.
Description
The application is a divisional application of Chinese patent application with the application date of 2017, 11, 3, 201711069928.5 and the name of 'a homogeneous immunoassay kit for detecting target IgM antibodies in samples, and a using method and application thereof'.
Technical Field
The invention belongs to the technical field of biomedical detection, and particularly relates to a homogeneous phase immunoassay kit for detecting target IgM antibodies in a sample, and a use method and application thereof.
Background
Detection of IgM-type antibodies is of great importance in medical testing. The pathogen first infects the body, and the first produced antibodies are always IgM antibodies, with the progress of the immune response of the body, an antibody class transition occurs, producing IgG, igM or IgA antibodies. Thus, positive IgM antibody detection is a hallmark of an symptomatic infection in the diagnosis of infectious diseases.
The currently used method for detecting IgM antibodies is a capturing method, wherein anti-human IgM (mu chain) antibodies are coated on a solid phase carrier in a solid phase, after a serum/plasma sample reacts with the solid phase carrier, all IgM antibodies in the sample are captured on the solid phase carrier, at the moment, interference of specific IgG antibodies can be removed through washing, and labeled antigen is added to generate a detectable signal through reaction. The method has the following two disadvantages: firstly, the anti-human IgM antibody is taken as a capture antibody, the specific IgM antibody and the non-specific IgM antibody cannot be distinguished, and when the concentration of the specific IgM antibody is low and the concentration of the non-specific IgM antibody is particularly high, the specific IgM antibody can be washed and removed, so that a false negative result is caused. Secondly, the detection process needs to be incubated and washed in two steps, the washing efficiency of washing equipment is very dependent in the washing process, the problems of poor repeatability and the like easily occur, the incubation steps in two steps are complicated, the time consumption is long, and the working efficiency is reduced.
In addition, the detection method of IgM antibody uses a homogeneous one-step method to detect, and bioactive substances for removing specific IgG interference are added in the detection process, so that the problems of low two-step washing efficiency and long time consumption of the capture method are solved. However, this method adds a new bioactive substance, and there is excessive protein component in the reaction system, which may cause false positives. In addition, non-specific IgM antibodies in homogeneous reaction systems consume large amounts of anti-human IgM antibodies, and there is the potential for false negatives when the specific IgM antibody concentration is low, but not particularly high.
Disclosure of Invention
The invention aims to solve the technical problem of providing a kit and a method for detecting target IgM antibodies in a sample aiming at the defects of the prior art. The complement C1q contained in the kit is used for replacing an anti-human IgM antibody as a capture object or a tracer, and then the target IgM antibody is detected, and the nonspecific IgM antibody does not react with a known antigen and is in a free state, so that the nonspecific IgM antibody cannot be combined with the complement C1q, and the interference of the nonspecific IgM on detection is removed.
To this end, a first aspect of the present invention provides a homogeneous immunoassay kit for detecting IgM antibodies of interest in a sample, comprising:
a reagent a comprising a known antigen capable of specifically binding to a target IgM antibody in a sample to be tested to form a first complex;
an agent b comprising complement C1q, the complement C1q being capable of specifically binding to the first complex to form a second complex, and the complement C1q being incapable of binding to free IgM antibodies;
wherein either of the known antigen and complement C1q is linked to a receptor; the acceptor is capable of reacting with singlet oxygen received thereby to produce a detectable chemiluminescent signal;
reagent c comprising a donor capable of generating singlet oxygen in an excited state.
In other embodiments of the invention, the kit comprises:
a reagent a comprising a known antigen capable of specifically binding to a target IgM antibody in a sample to be tested to form a first complex;
an agent b comprising complement C1q, the complement C1q being capable of specifically binding to the first complex to form a second complex, and the complement C1q being incapable of binding to free IgM antibodies;
wherein either of the known antigen and complement C1q is linked to a receptor microsphere and the other is linked to a first label; the receptor microsphere can react with the singlet oxygen received by the receptor microsphere to generate a detection signal;
a reagent c comprising donor microspheres bound to a second label, the donor microspheres being capable of generating singlet oxygen in an excited state; the second label is capable of specifically binding to the first label.
In some embodiments of the invention, the known antigen in the reagent a is linked to a first label and the complement C1q in the reagent b is linked to a receptor microsphere. In some specific embodiments, the concentration of the known antigen linked to the first label in reagent a is 1-100 μg/ml and the concentration of complement C1q linked to the receptor microsphere in reagent b is 0.1-10 μg/ml.
In other embodiments of the invention, the known antigen in the reagent a is linked to a receptor microsphere and the complement C1q in the reagent b is linked to a first label. In some specific embodiments, the concentration of known antigen associated with the receptor microsphere in reagent a is 0.1-10ug/ml and the concentration of complement C1q associated with the first label in reagent b is 1-100 ug/ml.
In some embodiments of the invention, the concentration of the donor microsphere bound to the second label in the reagent c is 1-100 μg/ml.
According to the invention, the first label may be biotin and the second label may be streptavidin. The biotin is widely existing in animal and plant tissues, and has two cyclic structures on the molecule, namely an imidazolone ring and a thiophene ring, wherein the imidazolone ring is a main part combined with streptavidin. Activated biotin can be coupled to almost all known biomacromolecules, including proteins, nucleic acids, polysaccharides, lipids, and the like, mediated by protein cross-linking agents. "streptavidin" is a protein secreted by Streptomyces and has a molecular weight of 65kD. Streptavidin molecules consist of 4 identical peptide chains, each of which is capable of binding to one biotin. Thus, each antigen or antibody can be conjugated to multiple biotin molecules simultaneously, thereby producing a "tentacle effect" that enhances assay sensitivity.
In some embodiments of the invention, a blocking agent is also included in the kit, which blocking agent blocks binding of the known antigen to IgG antibodies in the sample.
According to the invention, the blocking agent consists of polyclonal antibodies and/or antibody fragments, preferably monovalent antibody fragments, such as Fab fragments, which can be obtained by cleavage of the antibodies with papain and subsequent cleavage of the cleavage products.
In some preferred embodiments of the invention, the molar amount of blocking agent is greater than the molar amount of IgG antibody in the sample; preferably, the molar amount of the blocking agent is more than 5 times the molar amount of IgG antibodies in the sample.
In a second aspect, the invention provides a method for detecting IgM antibodies of interest in a sample using the kit of the first aspect of the invention, comprising the steps of: first preparing a complex formed by a C1 q-donor-IgM antibody-known antigen-acceptor; then, the compound is treated by energy or active compound to excite the donor to generate singlet oxygen, and the acceptor reacts with the singlet oxygen to generate a detectable chemiluminescent signal; and finally, analyzing the chemiluminescence signals, and judging whether the target IgM antibody exists in the sample to be detected or not and the content or the concentration of the target IgM antibody.
In some embodiments of the invention, the method specifically comprises the steps of:
s1, adding a reagent a and a reagent b into a sample to be detected to obtain a first mixture;
s2, adding the reagent c into the first mixture to obtain a second mixture;
s3, treating the second mixture with energy or an active compound to excite the donor to form singlet oxygen, and enabling the acceptor to react with the singlet oxygen received by the acceptor to generate a detection signal;
s4, analyzing the chemiluminescence signals, and judging whether the target IgM antibody and the content of the target IgM antibody exist in the sample to be detected.
In some embodiments of the present invention, in step S1, reagent a is added to the sample to be tested before reagent b is added.
In other embodiments of the present invention, in step S1, reagent a and reagent b are added to the sample to be tested simultaneously.
According to the invention, step S0 is further included before step S1, and the sample to be tested is added into the solution containing the IgG antibody blocking agent for blocking, so as to obtain the blocked sample to be tested. The IgG antibodies in the blocked test sample cannot form antigen-IgG antibody complexes with known antigens.
According to the invention, when the chemiluminescent signal value of the second mixture is more than or equal to the chemiluminescent signal value of the qualitative reference, the sample to be detected is a positive sample; and when the chemiluminescent signal value of the second mixture is less than the chemiluminescent signal value of the qualitative reference, the sample to be tested is a negative sample.
In the invention, the term "chemiluminescent signal value of a qualitative reference" refers to the chemiluminescent signal value of a calibrated target IgM antibody critical value when detected under the same conditions.
In a third aspect, the invention provides the use of a kit according to the first aspect of the invention or a method according to the second aspect of the invention for detecting IgM antibodies of hepatitis virus.
The beneficial effects of the invention are as follows: the complement C1q contained in the kit is used as a capture object or a tracer for detecting the target IgM antibody. By utilizing the characteristic that complement C1q does not react with free antigen and free antibody and only reacts with IgM antibody and antigen-antibody complex formed by some subclasses of IgG antibody, an IgG antibody blocking agent is added into a reaction system to block the formation of antigen-IgG antibody complex, thereby effectively detecting the existence of target IgM antibody, effectively removing the interference of nonspecific IgM antibody on detection, and solving the influence of nonspecific IgM antibody on detection in the traditional IgM antibody detection.
Detailed Description
In order that the invention may be readily understood, the invention will be described in detail.
As previously mentioned, existing techniques for detecting IgM antibodies are accomplished by binding anti-human IgM antibodies to IgM antibodies. Anti-human IgM antibodies as capture antibodies cannot distinguish between specific IgM antibodies (target IgM antibodies) and non-specific IgM antibodies, and when the target IgM antibody concentration is low but the non-specific IgM antibody concentration is particularly high, false negative results can be caused. The inventor of the application uses the characteristic that complement C1q can only react with an antigen-antibody complex, and can effectively remove the interference of non-specific IgM antibodies on detection by taking the complement C1q as a capture object or a tracer, and effectively detect target IgM antibodies.
Thus, the present invention relates in a first aspect to a homogeneous immunoassay kit for detecting IgM antibodies of interest in a sample, comprising:
a reagent a comprising a known antigen capable of specifically binding to a target IgM antibody in a sample to be tested to form a first complex;
an agent b comprising complement C1q, the complement C1q being capable of specifically binding to the first complex to form a second complex, and the complement C1q being incapable of binding to free IgM antibodies;
wherein either of the known antigen and complement C1q is linked to a receptor; the acceptor is capable of reacting with singlet oxygen received thereby to produce a detectable chemiluminescent signal;
reagent c comprising a donor capable of generating singlet oxygen in an excited state.
"complement C1q" is an important component constituting complement C1, and is composed of 6 identical subunits to form a symmetrical hexamer. Complement C1q binds only to antigen-antibody complexes formed by IgM antibodies, igG1, igG2, and IgG3 antibodies. Since non-specific IgM antibodies cannot react with known antigens to produce antigen-antibody complexes, complement C1q does not bind to non-specific IgM antibodies (C1 q binds only IgM bound to antigen, and does not bind free IgM (unbound to antigen), and IgG has a weaker binding capacity than IgM, with a significant difference). If a blocking agent for IgG antibody is added into a sample to be tested, the formation of antigen-IgG antibody complex is blocked, and only antigen-target IgM antibody complex reacts with complement C1 q. One of the known antigen and complement C1q is linked to the receptor, the other is linked to biotin, and the C1 q-target IgM antibody-antigen complex is bound to the added streptavidin-bound donor, and the distance between the receptor and the donor is pulled. Upon photoexcitation, the acceptor reacts with singlet oxygen emitted from the nearby donor, and the energy generated is emitted in the form of light, producing a chemiluminescent signal value.
The term "donor" as used herein refers to a sensitizer that upon activation of energy or an active compound is capable of generating an active intermediate such as singlet oxygen that reacts with the acceptor. 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, porphyrin, phthalocyanine, and chlorophyll as disclosed in U.S. Pat. No. 5709994 (which is incorporated herein by reference in its entirety), and derivatives of these compounds having 1-50 atom substituents for making these compounds more lipophilic or hydrophilic, and/or as linking groups to specific binding pair members. Examples of other photosensitizers known to those skilled in the art may also be used in the present invention, such as described in U.S. patent No. 6406913, 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. Examples of other donors include: 1, 4-dicarboxyethyl-1, 4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10-endoperoxide, and the like, and singlet oxygen is released by heating these compounds or by direct absorption of light by these compounds.
The "acceptor" as used herein refers to a compound that is capable of reacting with singlet oxygen to produce a detectable signal. The donor is induced to activate by energy or an active compound and releases singlet oxygen in a high energy state which is captured by the acceptor in close proximity, 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 may decompose while or subsequently emit light. Typical examples of such substances include, but are not limited to: enol ethers, enamines, 9-alkylidene xanthan, 9-alkylidene-N-alkyl acridines, arylvinyl ethers, bisoxyethylene, dimethylthiophene, aromatic imidazoles or gloss concentrates. In other embodiments of the invention, the acceptor is an olefin capable of reacting with singlet oxygen to form a hydroperoxide or dioxetane that can decompose to a ketone or carboxylic acid derivative; stable dioxetanes that can be decomposed by the action of light; acetylenes that can react with singlet oxygen to form diketones; hydrazones or hydrazides of azo compounds or azocarbonyl compounds, such as luminol, may be formed; and aromatic compounds which can form endoperoxides. A specific, non-limiting example of a receptor that can be utilized in accordance with the present disclosure and claimed invention is described in U.S. patent No. US5340716 (which is incorporated herein by reference in its entirety). In other embodiments of the present invention, the acceptor comprises an olefinic compound and a metal chelate that is non-particulated and soluble in an aqueous medium, such acceptor being described in PCT/US2010/025433 (which is incorporated herein by reference in its entirety)
In some preferred embodiments of the invention, the acceptor comprises an olefin compound and a metal chelate.
In some preferred embodiments of the present invention, the olefinic compound is represented by formula (I):
wherein X' is a sulfur atom or NR, said R being an alkyl group having 1 to 10 carbon atoms or an aryl group having 6 to 18 carbon atoms; r' is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms; d and D' are each independently a hydrogen atom or an alkyl group having 1 to 20 carbon atoms.
In other preferred embodiments of the invention, the metal chelate is selected from the group consisting of chelates of europium, terbium, dysprosium, samarium, osmium, or ruthenium; preferably, the metal chelate is selected from chelates of europium; further preferably, the metal chelate is selected from the group consisting of chelates of europium with a bis-butanedione compound as shown in formula (II) as a ligand;
wherein: x is a linear alkylene group having 1 to 5 carbon atoms; or is arylene, which is
In the present invention, the "donor" and/or "acceptor" may be coated on a substrate by a functional group to form a "donor microsphere" and/or "acceptor microsphere". The "matrix" according to the invention is a microsphere or microparticle, known to the person skilled in the art, which may be of any size, organic or inorganic, expandable or non-expandable, porous or non-porous, of any density, but preferably has a density close to that of water, preferably is floatable in water, and is composed of transparent, partially transparent or opaque material. The matrix may or may not be charged 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 acceptor 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, hydroxyl, mercapto, and the like. One non-limiting example of a matrix suitable for use in the present invention is a carboxyl modified latex particle. Details of such substrates can be found in U.S. Pat. nos. 5709994 and 5780646 (both of which are incorporated herein by reference in their entirety).
In some embodiments of the invention, the kit specifically comprises:
a reagent a comprising a known antigen capable of specifically binding to a target IgM antibody in a sample to be tested to form a first complex;
an agent b comprising complement C1q, the complement C1q being capable of specifically binding to the first complex to form a second complex, and the complement C1q being incapable of binding to free IgM antibodies;
wherein either of the known antigen and complement C1q is linked to a receptor microsphere and the other is linked to a first label; the receptor microsphere can react with the singlet oxygen received by the receptor microsphere to generate a detection signal;
a reagent c comprising donor microspheres bound to a second label, the donor microspheres being capable of generating singlet oxygen in an excited state; the second label is capable of specifically binding to the first label.
In some embodiments of the invention, the first label is biotin and the second label is streptavidin.
In other embodiments of the invention, a blocking agent is also included in the kit that blocks binding of the known antigen to IgG antibodies in the sample. The blocking agent consists of polyclonal antibodies or/and antibody fragments, preferably monovalent antibody fragments, such as Fab fragments, which can be obtained by cleavage of the antibodies with papain and subsequent cleavage of the cleavage products. Further preferably, the blocking agent is used in a molar amount of 5 times or more the molar amount of the IgG antibody to be blocked.
The second aspect of the present invention relates to a method for detecting an IgM antibody of interest (for non-disease diagnosis purposes) in a sample using the kit of the first aspect of the present invention, comprising the steps of: first preparing a complex formed by a C1 q-donor-IgM antibody-known antigen-acceptor; then, the compound is treated by energy or active compound to excite the donor to generate singlet oxygen, and the acceptor reacts with the singlet oxygen to generate a detectable chemiluminescent signal; and finally, analyzing the chemiluminescence signals, and judging whether the target IgM antibody exists in the sample to be detected or not and the content or the concentration of the target IgM antibody.
In some embodiments of the invention, the method comprises the steps of:
s0, adding the sample to be tested into a solution containing the IgG antibody blocking agent, and incubating for 5-10min at 35-40 ℃ to block formation of antigen-IgG antibody complex, thereby obtaining the blocked sample to be tested.
S1, adding the reagent a into a sample to be detected, incubating for 5-10min at 35-40 ℃, then adding the reagent b, and incubating for 5-10min at 35-40 ℃ to obtain a first mixture. The known antigen in the reagent a is linked to a first label, and the complement C1q in the reagent b is linked to a receptor microsphere. Specifically, the concentration of the known antigen linked to the first label in the reagent a is 1-100. Mu.g/ml, and the concentration of the complement C1q linked to the receptor microsphere in the reagent b is 0.1-10. Mu.g/ml. Alternatively, the known antigen in the reagent a is linked to a receptor microsphere and the complement C1q in the reagent b is linked to a first label. Specifically, the concentration of the known antigen linked to the receptor microsphere in the reagent a is 0.1-10ug/ml, and the concentration of the complement C1q linked to the first marker in the reagent b is 1-100 ug/ml.
S2, adding the reagent c into the first mixture, and incubating for 5-10min at 35-40 ℃ to obtain a second mixture. The concentration of the donor microsphere bound to the second label in the reagent c is 1-100 μg/ml.
S3, treating the second mixture with energy or an active compound to excite the donor microspheres to generate singlet oxygen, and reacting the acceptor microspheres with the singlet oxygen to generate a detectable chemiluminescent signal;
s4, analyzing the chemiluminescence signals, and judging whether the target IgM antibody and the content of the target IgM antibody exist in the sample to be detected. If the signal value of the second mixture is more than or equal to the signal value of the qualitative reference, the sample to be tested is a positive sample; and if the signal value of the second mixture is smaller than the signal value of the qualitative reference, the sample to be tested is a negative sample.
In other embodiments of the invention, the method comprises the steps of:
s0, adding the sample to be tested into a solution containing the IgG antibody blocking agent, and incubating for 5-10min at 35-40 ℃ to block formation of antigen-IgG antibody complex, thereby obtaining the blocked sample to be tested.
S1, adding the reagent a and the second mixture into a sample to be detected at the same time, and incubating for 5-10min at 35-40 ℃ to obtain a first mixture. The known antigen in the reagent a is linked to a first label, and the complement C1q in the reagent b is linked to a receptor microsphere. Specifically, the concentration of the known antigen linked to the first label in the reagent a is 1-100. Mu.g/ml, and the concentration of the complement C1q linked to the receptor microsphere in the reagent b is 0.1-10. Mu.g/ml. Alternatively, the known antigen in the reagent a is linked to a receptor microsphere and the complement C1q in the reagent b is linked to a first label. Specifically, the concentration of the known antigen linked to the receptor microsphere in the reagent a is 0.1-10ug/ml, and the concentration of the complement C1q linked to the first marker in the reagent b is 1-100 ug/ml.
S2, adding the reagent c into the first mixture, and incubating for 5-10min at 35-40 ℃ to obtain a second mixture. The concentration of the donor microsphere bound to the second label in the reagent c is 1-100 μg/ml.
S3, treating the second mixture with energy or an active compound to excite the donor microspheres to generate singlet oxygen, and reacting the acceptor microspheres with the singlet oxygen to generate a detectable chemiluminescent signal;
s4, analyzing the chemiluminescence signals, and judging whether the target IgM antibody and the content of the target IgM antibody exist in the sample to be detected. If the signal value of the second mixture is more than or equal to the signal value of the qualitative reference, the sample to be tested is a positive sample; and if the signal value of the second mixture is smaller than the signal value of the qualitative reference, the sample to be tested is a negative sample.
The third aspect of the invention relates to the use of a kit according to the first aspect of the invention or a method according to the second aspect of the invention for detecting IgM antibodies of hepatitis virus.
Examples
In order that the invention may be more readily understood, the invention will be further described in detail with reference to the following examples, which are given by way of illustration only and are not limiting in scope of application. The starting materials or components used in the present invention may be prepared by commercial or conventional methods unless specifically indicated.
Example 1: detection of HBc IgM antibodies
The reagents used include:
r1: a complement C1q solution at a concentration of 25ug/ml coupled to the receptor microsphere;
r2: a concentration of 1ug/ml of HBcAg solution linked to biotin;
r3: a diluent comprising an IgG antibody blocking agent;
r4: a solution of Streptavidin (SA) -linked donor microspheres at a concentration of 25 ug/ml.
The preparation method, composition structure and content of the receptor microsphere used in the invention can be seen in example 1 of Chinese patent CN100429197C, and then C1q complement is coated on the surface of the receptor microsphere.
The donor microspheres were prepared by placing 200g of chlorophyll-a into 200nm carboxyl-modified latex particles and coating the surface with streptavidin according to the method described in the examples of patent US5780646 to form the donor microspheres according to the invention.
The detection steps are as follows:
(1) Adding 100ul of reagent R3 into the reaction hole, then adding 10ul of sample to be tested, and incubating for 5min at 37 ℃;
(2) Adding 25ul of each of the reagent R1 and the reagent R2 into the reaction hole, and incubating for 10min at 37 ℃ to obtain a first mixture;
(3) Adding 25ul of reagent R4 into the reaction well, and incubating for 10min at 37 ℃ to obtain a second mixture;
(4) The second mixture was placed in a photo-activated chemiluminescent detection instrument and the chemiluminescent signal values were read, the results of which are shown in Table 1.
Table 1: chemiluminescent signal values read for each sample.
Example 2: detection of HEV IgM antibodies
The reagents used include:
r1: HEV Ag solution with concentration of 25ug/ml and connected with receptor microsphere;
r2: a 1ug/ml complement C1q solution coupled to biotin;
r3: a diluent comprising an IgG antibody blocking agent;
r4: a solution of streptavidin-linked donor microspheres at a concentration of 25 ug/ml.
The detection steps are as follows:
(1) Adding 100ul of reagent R3 into the reaction hole, then adding 10ul of sample to be tested, and incubating for 5min at 37 ℃;
(2) Adding 25ul of each of the reagent R1 and the reagent R2 into the reaction hole, and incubating for 10min at 37 ℃ to obtain a first mixture;
(3) Adding 25ul of reagent R4 into the reaction well, and incubating for 10min at 37 ℃ to obtain a second mixture;
(4) The second mixture was placed in a photo-activated chemiluminescent detection instrument and the chemiluminescent signal values were read, the results of which are shown in Table 2.
Table 2: chemiluminescent signal values read for each sample.
Example 3: detection of HBc IgM antibodies
The reagents used include:
r1: a complement C1q solution associated with the receptor at a concentration of 25 ug/ml;
r2: a concentration of 1ug/ml of HBcAg solution linked to biotin;
r3: a diluent comprising an IgG antibody blocking agent;
r4: a concentration of 25ug/ml of Streptavidin (SA) -linked donor solution.
Wherein the receptor-linked complement C1q used in the present invention is prepared according to the protocol described in PCT/US2010/025433, and has a structure composed of C1q-BSA- (dimethylthiophene) (BHHCT) and is completely soluble in an aqueous solution.
The donor was prepared by placing 200g of chlorophyll a into 200nm carboxyl-modified latex particles and coating the surface with streptavidin according to the method described in the examples of patent US5780646 to form donor microspheres according to the invention.
The detection steps are as follows:
(1) Adding 100ul of reagent R3 into the reaction hole, then adding 10ul of sample to be tested, and incubating for 5min at 37 ℃;
(2) Adding 25ul of each of the reagent R1 and the reagent R2 into the reaction hole, and incubating for 10min at 37 ℃ to obtain a first mixture;
(3) Adding 25ul of reagent R4 into the reaction well, and incubating for 10min at 37 ℃ to obtain a second mixture;
(4) The second mixture was placed in a photo-excited chemiluminescent detection instrument and chemiluminescent signal values were read, the results being shown in Table 3.
Table 3: chemiluminescent signal values read for each sample.
It should be noted that the above-described embodiments are only for explaining the present invention and do not constitute any limitation of the present invention. The invention has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined in the appended claims, and the invention may be modified without departing from the scope and spirit of the invention. Although the invention is described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, as the invention extends to all other means and applications which perform the same function.
Claims (14)
1. A method for detecting IgM antibodies of interest in a sample for non-disease diagnostic and therapeutic purposes, comprising the steps of: first preparing a complex formed by a C1 q-donor-IgM antibody-known antigen-acceptor; then, the compound is treated by energy or active compound to excite the donor to generate singlet oxygen, and the acceptor reacts with the singlet oxygen to generate a detectable chemiluminescent signal; finally, analyzing the chemiluminescent signal condition, and judging whether the target IgM antibody and the content of the target IgM antibody exist in the sample to be detected;
the method further comprises adding an IgG antibody blocking agent to the sample to be tested, blocking the formation of antigen-IgG antibody complexes.
2. The method of claim 1, wherein the kit used to prepare the complex formed by the C1 q-donor-IgM antibody-known antigen-acceptor comprises: a reagent a comprising a known antigen capable of specifically binding to a target IgM antibody in a sample to be tested to form a first complex; an agent b comprising complement C1q, the complement C1q being capable of specifically binding to the first complex to form a second complex, and the complement C1q not binding to free IgM antibodies; wherein either of the known antigen and complement C1q is linked to a receptor; the acceptor is capable of reacting with singlet oxygen received thereby to produce a detectable chemiluminescent signal; reagent c comprising a donor capable of generating singlet oxygen in an excited state.
3. The method of claim 2, wherein complement C1q in reagent b is linked to a receptor.
4. A method according to claim 2 or claim 3, wherein the kit further comprises a blocking agent which blocks binding of the known antigen to IgG antibodies in the sample.
5. The method of claim 4, wherein the blocking agent consists of polyclonal antibodies and/or antibody fragments.
6. The method of claim 5, wherein the blocking agent consists of monovalent antibody fragments.
7. The method of claim 4, wherein the blocking agent is present in a molar amount greater than the molar amount of IgG antibodies in the sample.
8. The method according to claim 1 or 2, characterized in that it comprises in particular the following steps:
s1, adding a reagent a and a reagent b into a sample to be detected to obtain a first mixture;
s2, adding the reagent c into the first mixture to obtain a second mixture;
s3, treating the second mixture with energy or an active compound to excite the donor to generate singlet oxygen, and reacting the acceptor with the singlet oxygen to generate a detectable chemiluminescent signal;
s4, analyzing the chemiluminescent signal condition, and judging whether the target IgM antibody and the content of the target IgM antibody exist in the sample to be detected.
9. The method according to claim 8, wherein in step S1, reagent a is added to the sample to be tested before reagent b is added.
10. The method according to claim 8, wherein in step S1, reagent a and reagent b are added to the sample to be tested simultaneously.
11. The method according to claim 8, wherein step S1 is preceded by a step S0 of adding the sample to be tested to a solution containing an IgG antibody blocking agent for blocking, thereby obtaining a blocked sample to be tested.
12. The method of claim 8, wherein the sample to be tested is a positive sample when the chemiluminescent signal value of the second mixture is greater than or equal to the chemiluminescent signal value of the qualitative reference; and when the chemiluminescent signal value of the second mixture is less than the chemiluminescent signal value of the qualitative reference, the sample to be tested is a negative sample.
13. The method of any one of claims 1-2, 5-7, 9-12, wherein the acceptor comprises an olefin compound and a metal chelate.
14. The method according to any one of claims 1-2, 5-7, 9-12, wherein the reagents used are non-particulated reagents and are soluble in an aqueous medium.
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| CN110531073B (en) * | 2018-05-23 | 2024-03-15 | 科美诊断技术股份有限公司 | Method for in vitro assessment of the presence or absence of rheumatoid arthritis by biomarker joint detection |
| CN110579600B (en) * | 2018-05-23 | 2023-12-26 | 科美诊断技术股份有限公司 | Method for in vitro assessment of the presence or absence of rheumatoid arthritis by biomarker joint detection |
| CN110579601B (en) * | 2018-05-24 | 2023-12-26 | 科美诊断技术股份有限公司 | Method for in vitro assessment of the presence or absence of rheumatoid arthritis by biomarker joint detection |
| CN110579602B (en) * | 2018-05-24 | 2024-03-15 | 科美诊断技术股份有限公司 | Method for in vitro assessment of the presence or absence of rheumatoid arthritis by biomarker joint detection |
| CN116429758A (en) * | 2018-08-13 | 2023-07-14 | 科美博阳诊断技术(上海)有限公司 | Homogeneous chemiluminescence detection kit and application thereof |
| CN111122852A (en) * | 2018-10-31 | 2020-05-08 | 博阳生物科技（上海）有限公司 | Homogeneous phase chemiluminescence analysis method and application thereof |
| CN111521779B (en) * | 2019-02-01 | 2024-02-23 | 科美诊断技术股份有限公司 | Combined detection method and kit for hepatitis C virus antigen and antibody |
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