CN118275667A - Interference compensation reagent, preparation method, kit system, application and chemiluminescent analyzer thereof - Google Patents
Interference compensation reagent, preparation method, kit system, application and chemiluminescent analyzer thereof Download PDFInfo
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- G—PHYSICS
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
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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
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Abstract
The invention provides an interference compensation reagent, a preparation method, a kit system and application thereof, wherein the interference compensation reagent comprises a reagent 1 and a reagent 2; the interference compensation reagent comprises a reagent 1 and a reagent 2; the reagent 1 comprises luminescent particles coated by an antibody 1 without antigen binding activity; the reagent 2 comprises a biotin-labeled antibody 2 without antigen binding activity; the antibody 1 and the antibody 2 are inactivated antibodies capable of specifically recognizing different sites of the same antigen. The interference compensation reagent provided by the invention can be used for measuring the influence condition of an interfering substance on the whole signal value of a detection system, and the influence of the interfering substance on a detection result can be eliminated by detecting the interfering substance signal in a sample to be analyzed, and various interferences can be detected simultaneously, so that the step of analyzing the interfering substance is simplified.
Description
Technical Field
The invention relates to the technical field of chemiluminescent immunoassay, in particular to an interference compensation reagent, a preparation method, a kit system, application and a chemiluminescent analyzer.
Background
In the field of medical diagnostics, an appropriate therapeutic agent is selected by detecting one or more target analytes in a test sample and based on the concentration and/or presence of the target analytes in the detection result.
However, during the test, a variety of interfering substances may be present in the test sample, which are a significant source of clinical laboratory detection errors, and which to some extent appear to be a hazard to the patient, can affect the target analyte binding to its specific antigen/antibody, alter the structure of the target analyte or its specific antigen antibody, generate signals other than or in addition to the target analyte immune complex, and thereby affect the detection result of the actual target analyte content in the test sample, further affecting the result determination or subsequent diagnosis and treatment.
Disclosure of Invention
In order to solve the above problems in the prior art, the present invention provides an interference compensation reagent, which detects an interference signal in a sample to be analyzed by using the interference compensation reagent, and determines the influence of the interference on the signal value of the whole detection system, so as to eliminate the influence of the interference on the detection result. In addition, the interference compensation reagent can detect multiple kinds of interference at one time, and the interference substance analysis step is simplified. The invention also provides a preparation method of the interference compensation reagent, a kit system and application thereof.
The specific invention comprises the following steps:
in a first aspect, the present invention provides an interference compensation reagent comprising reagent 1 and reagent 2;
The reagent 1 comprises luminescent particles coated by an antibody 1 without antigen binding activity;
The reagent 2 comprises a biotin-labeled antibody 2 having no antigen binding activity.
Alternatively, the antibody 1 and the antibody 2 are inactivated antibodies capable of specifically recognizing different sites of the same antigen.
In a second aspect, the present invention provides a method for preparing the interference compensation reagent according to the first aspect, including:
S11, mixing the luminescent particles and carrying out a rotary reaction on the mixed luminescent particles and the antibody to obtain antibody-coated luminescent particles;
S12, mixing biotin and an antibody to perform a rotary reaction to obtain a biotin-labeled antibody;
s13, inactivating the antibody-coated luminous particles and the biotin-labeled antibody to obtain antigen-binding-activity-free antibody-coated luminous particles and biotin-labeled antigen-binding-activity-free antibodies;
S14, adjusting the concentration of the luminescent particles coated by the antigen-binding-free active antibody by using a luminescent reagent buffer solution to obtain a reagent 1; the concentration of the biotin-labeled antigen-binding inactive antibody was adjusted with a biotin reagent buffer to give reagent 2.
In a third aspect, the present invention provides a method for preparing the interference compensation reagent according to the first aspect, including:
S21, inactivating the antibody to obtain an antibody without antigen binding activity;
s22, mixing the luminous particles and reacting the luminous particles with the antibody without antigen binding activity to obtain luminous particles coated with the antibody without antigen binding activity;
s23, mixing biotin and reacting with the antigen-binding-free active antibody to obtain a biotin-labeled antigen-binding-free active antibody;
s24, adjusting the concentration of the luminescent particles coated by the antigen-binding-free active antibody by using a luminescent reagent buffer solution to obtain a reagent 1; the concentration of the biotin-labeled antigen-binding inactive antibody was adjusted with a biotin reagent buffer to give reagent 2.
In a fourth aspect, the present invention provides a kit system comprising a detection kit and an interference compensation kit comprising the interference compensation reagent according to the first aspect.
Optionally, the detection kit comprises a reagent 1 'and a reagent 2';
The reagent 1' comprises luminous particles coated by an antibody 1 with antigen binding activity;
The reagent 2' comprises a biotin-labeled antibody 2 having antigen binding activity.
Optionally, the mass concentration of the luminescent particles coated with the antibody 1 without antigen binding activity in the reagent 1 is the same as the mass concentration of the luminescent particles coated with the antibody 1 with antigen binding activity in the reagent 1';
the mass concentration of the biotin-labeled antigen-binding-inactive antibody 2 in the reagent 2 is the same as the mass concentration of the biotin-labeled antigen-binding-active antibody 2 in the reagent 2'.
In a fifth aspect, the present invention provides a kit system for detecting a target molecule content by chemiluminescent analysis, as described in the fourth aspect above; comprising the following steps:
Detecting a sample to be analyzed by using a detection kit in the kit system to obtain a detection light signal value;
detecting a sample to be analyzed by using an interference compensation kit in the kit system to obtain an interference optical signal value;
And carrying out mathematical operation on the detection optical signal value and the interference optical signal value of the same sample to be analyzed to obtain the target molecule concentration.
Alternatively, the volumes of the sample to be analyzed, reagent 1, reagent 2, reagent 1', reagent 2' in each reaction are the same at the time of detection.
Wherein the mass of the sample to be analyzed 1 and the mass of the sample to be analyzed 2 are equal.
In a sixth aspect, the present invention provides a chemiluminescent analyzer comprising at least the following:
A reagent module for storing the kit system according to the fourth aspect;
an incubation module for providing a suitable temperature environment for the chemiluminescent reaction;
The detection module is used for generating excitation light and obtaining a detection light signal value and an interference light signal value;
And the processor is used for judging whether the analysis target molecules exist in the sample to be analyzed or not and/or judging the concentration of the analysis target molecules in the sample to be analyzed according to the condition of the chemiluminescent signals detected by the detection module.
Compared with the prior art, the invention has the following advantages:
The invention provides an interference compensation reagent, which comprises a reagent 1 and a reagent 2; the reagent 1 comprises luminescent particles coated by an antibody 1 without antigen binding activity; the reagent 2 comprises a biotin-labeled antibody 2 having no antigen binding activity. The interference compensation reagent provided by the invention can be used for measuring the influence condition of an interference object on the whole signal value of the detection system, detecting the interference object signal in the sample to be analyzed so as to eliminate the influence of the interference object on the detection result, and simultaneously detecting various interferences, thereby simplifying the step of analyzing the interference object.
The kit system provided by the invention has the advantages that the interference compensation reagent in the interference compensation kit and the detection reagent in the detection kit have the same antibody, the antibody related in the interference compensation reagent is an inactivated antibody without antigen binding activity, and the antibody related in the detection reagent has biological activity. When the detection kit and the interference compensation kit are used for detecting the same sample to be analyzed, the detection kit is used for detecting the total signal value, namely, a specific optical signal generated by the immune complex formed by the specific binding of the antibody and the analyte, and an interference signal value except for the specific optical signal generated by the immune complex formed by the specific binding of the antibody and the analyte, the interference compensation kit detects the interference signal value except for the specific optical signal generated by the immune complex formed by the specific binding of the antibody and the analyte, and then the interference signal value is removed from the total signal value of the sample to be analyzed detected by the detection kit through mathematical operation, so that the interference-removed signal value of the analyte is obtained.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
Specific experimental steps or conditions are not noted in the examples and may be performed in accordance with the operation or conditions of conventional experimental steps described in the prior art in the field. The reagents used, as well as other instruments, are conventional reagent products available commercially, without the manufacturer's knowledge.
The technical principle of the photo-excitation chemiluminescence analysis technology is as follows: the sensitizer can excite oxygen molecules in the surrounding environment into singlet oxygen molecules under the irradiation of laser, and the singlet oxygen molecules can react with optical reaction substances with the distance of about 200nm to generate optical signals with certain wavelength; when the sample to be analyzed contains the antigen or antibody to be analyzed, the immune reaction of the antigen and the antibody can lead the photosensitive latex microsphere containing the sensitizer to be combined with the luminous latex microsphere containing the optical reaction substance, thereby generating a light signal with a specific wavelength, and detecting the light signal to detect the content of the antigen or antibody to be analyzed.
An "antigen" is a substance that stimulates the body to produce an immune response and binds to antibodies and sensitized lymphocytes, which are the products of the immune response, in vivo and in vitro, resulting in an immune effect. The antigen may be a fusion antigen and, in any desired case, the antigen may be further conjugated to other moieties, such as specific binding pair members, e.g. biotin or avidin (biotin-Avidin-specific binding pair member), and the like. When CEA antigen exists in a sample to be detected, the CEA antigen is combined with the luminous latex microsphere coated with the anti-CEA antibody and the biotin-marked anti-CEA antibody in a specific way, and a double-antibody sandwich compound is formed on the surface of the luminous latex microsphere; at this time, if light is added, the fluorescent groups on the same microsphere are further excited to generate cascade amplification reaction to generate fluorescence. At this time, the more CEA antigen marker is present, the stronger the fluorescence intensity.
"Sample to be analyzed" refers to a mixture that may contain an analyte. Typical samples to be analyzed that may be used in the methods disclosed herein include, but are not limited to: oropharyngeal swab, nasopharyngeal swab, saliva, nasal aspirate, serum, plasma, ascites fluid, hydrothorax, and the like.
The sample to be analyzed generally contains an interfering substance with opposite charges to the microsphere adopted by the photo-excitation chemiluminescence method, and the interfering substance can be combined with the microsphere through the attraction effect of positive and negative charges in a reaction system to generate an optical signal with a specific wavelength, so that a stronger interference effect is generated on a detection system. The presence of interfering substances can lead to false positive or false negative assay results, as well as high background signals in the assay, reduced assay sensitivity and reagent stability. In addition, signal generating components used in immunoassays, such as enzymes or labels that emit light based on fluorescence, colorimetry, chemiluminescence, or electrochemiluminescence principles, can also interfere with the detection system. To avoid interference based on the signal generating component, an excess of carrier protein (e.g., antibody) linked to the signal generating component is often added to the immunoassay mixture. Binding an interfering substance or agent to said signal generating component linked to the free carrier protein. Such measures are not sufficient to eliminate interference, and a class of substances similar to the actual signal generating component but which do not provide any signal themselves may also be added to the immunoassay mixture to allow the interference factor present in the immunoassay mixture to bind to an excess of the similar compound, but not to the actual target, i.e. the signal generating compound. The purpose of suppressing interference is achieved.
However, the above method for eliminating interference cannot be used for chemiluminescence analysis means, and different interference elimination modes need to be selected for different interference sources, so that the process is complicated, and all interference cannot be eliminated at one time.
Based on the above, a first object of the present invention is to provide an interference compensation reagent for detecting an interference substance signal in a sample to be analyzed to eliminate the influence of the interference substance on the detection result, wherein the interference substance signal measured by the interference compensation reagent provided by the present invention is the influence of the interference substance on the whole signal value of the detection system, so that the whole influence of multiple kinds of interference on the detection system can be determined by one detection, and the step of analyzing the interference substance is simplified.
In specific implementation, the interference compensation reagent provided by the invention needs to be combined with a detection kit to determine the influence condition of an interfering substance on the whole signal value of a detection system.
The term "antibody" as used herein is used in its broadest sense to 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 desired case, the antibody may be further conjugated to other moieties, such as specific binding pair members, e.g., biotin or streptavidin, and the like.
In particular embodiments, the interference compensation reagent comprises reagent 1 and reagent 2; reagent 1 comprises luminescent particles coated with antibody 1 without antigen binding activity; reagent 2 comprises biotin-labeled antibody 2 without antigen binding activity; antibody 1 and antibody 2 are inactivated antibodies capable of specifically recognizing different sites of the same antigen.
The interference compensation reagent provided by the invention can be used for measuring the influence condition of an interference object on the whole signal value of the detection system, detecting the interference object signal in the sample to be analyzed so as to eliminate the influence of the interference object on the detection result, and simultaneously detecting various interferences, thereby simplifying the step of analyzing the interference object.
A second object of the present invention is to provide a method for preparing the interference compensation reagent according to the first aspect, including:
S11, mixing the luminescent particles and carrying out a rotary reaction on the mixed luminescent particles and the antibody to obtain antibody-coated luminescent particles;
S12, mixing biotin and an antibody to perform a rotary reaction to obtain a biotin-labeled antibody;
s13, inactivating the antibody-coated luminous particles and the biotin-labeled antibody to obtain antigen-binding-activity-free antibody-coated luminous particles and biotin-labeled antigen-binding-activity-free antibodies;
S14, adjusting the concentration of the luminescent particles coated by the antigen-binding-free active antibody by using a luminescent reagent buffer solution to obtain a reagent 1; the concentration of the biotin-labeled antigen-binding inactive antibody was adjusted with a biotin reagent buffer to give reagent 2.
A third object of the present invention is to provide another method for preparing the interference compensation reagent according to the first aspect, comprising:
S21, inactivating the antibody to obtain an antibody without antigen binding activity;
s22, mixing the luminous particles and reacting the luminous particles with the antibody without antigen binding activity to obtain luminous particles coated with the antibody without antigen binding activity;
s23, mixing biotin and reacting with the antigen-binding-free active antibody to obtain a biotin-labeled antigen-binding-free active antibody;
s24, adjusting the concentration of the luminescent particles coated by the antigen-binding-free active antibody by using a luminescent reagent buffer solution to obtain a reagent 1; the concentration of the biotin-labeled antigen-binding inactive antibody was adjusted with a biotin reagent buffer to give reagent 2.
A fourth object of the present invention is to provide a kit system comprising a detection kit and an interference compensation kit, the interference compensation kit comprising the interference compensation reagent according to the first aspect. Wherein the detection kit comprises a reagent 1 'and a reagent 2'; reagent 1' comprises luminescent particles coated with antibody 1 having antigen binding activity; reagent 2' includes biotin-labeled antibody 2 having antigen binding activity.
In some embodiments, the invention can be directed to a detection function of a detection kit that matches different interference compensation reagents, e.g., a detection kit that detects CEA can be used in combination with an interference compensation reagent that detects CEA, and a detection kit that detects influenza b virus antigen can be used in combination with an interference compensation reagent that detects influenza b virus antigen. Among these kits that can be used in combination, the antibody involved in the interference compensation reagent is an inactivated antibody, and has no antigen binding activity, whereas the antibody involved in the detection reagent of the detection kit has biological activity. When the detection kit and the interference compensation kit are used for detecting the same sample to be analyzed, the detection reagent of the detection kit is used for detecting the total signal value, namely, the specific optical signal generated by the immune complex formed by the specific binding of the antibody and the analyte, and the signal value of the interfering object except the specific optical signal generated by the immune complex formed by the specific binding of the antibody and the analyte, while the antibody contained in the interference compensation kit has no antigen binding activity, so that only the signal value of the interfering object except the specific optical signal generated by the immune complex formed by the specific binding of the antibody and the analyte can be detected, and then the signal value of the interfering object is eliminated from the total signal value of the sample to be analyzed detected by the detection kit through mathematical operation, so that the signal value of the interfering object to be analyzed is obtained.
To ensure accuracy of the obtained interference-free analyte signal values, the detection process control of the detection kit and the interference compensation kit is done under single variable conditions, i.e. all other indexes are the same except that the activities of the antibodies involved in the detection kit and the interference compensation kit are different, the composition of the reagent in the interference compensation reagent and the content of each composition are required to be matched with the composition of the reagent in the detection kit and the content of each composition, which comprises that the mass concentration of the luminescent particles coated by the antibody 1 without antigen binding activity in the reagent 1 is the same as the mass concentration of the luminescent particles coated by the antibody 1 with antigen binding activity in the reagent 1'; the mass concentration of the biotin-labeled antibody 2 having no antigen binding activity in the reagent 2 is the same as the mass concentration of the biotin-labeled antibody 2 having antigen binding activity in the reagent 2'.
A fifth object of the present invention is to provide a use of the kit system according to the fourth aspect for detecting the content of a target molecule by a chemiluminescent assay; comprising the following steps:
Detecting a sample to be analyzed by using a detection kit in the kit system to obtain a detection light signal value;
detecting a sample to be analyzed by using an interference compensation kit in the kit system to obtain an interference optical signal value;
And carrying out mathematical operation on the detection optical signal value and the interference optical signal value of the same sample to be analyzed to obtain the target molecule concentration.
When the kit system provided by the invention is used for the photo-excitation chemiluminescence platform, only one tested interference amount, namely 10-50 mu L, is needed for analysis interference, and the sample is not required to be diluted or compared and tested by a plurality of platforms, so that the sample consumption is effectively saved; in addition, when a plurality of detection kits analyze the same sample, a plurality of known or unknown interferences can be detected only by one interference compensation reagent test (under the condition that the relation between a plurality of detection kit signals and interference signals is established), and the influence of the interferences on the detection result can be judged by taking the mixture of the detectable interferences and the plurality of interferences as the whole interferences.
In specific implementation, the kit system provided by the invention can be used for detecting the content of target molecules according to the following steps:
s31, mixing a sample 1 to be analyzed with the reagent 1 and the reagent 2 in equal volumes to obtain a mixed solution 1;
S32, mixing the sample 2 to be analyzed with the reagent 1' and the reagent 2' in equal volumes to obtain a mixed solution 1';
S33, carrying out warm bath on the mixed solution 1 and the mixed solution 1', then adding general liquid of the photo-excitation chemiluminescence analysis system, continuing the warm bath, and obtaining a detection light signal value and an interference light signal value of a sample to be analyzed through an automatic photo-excitation chemiluminescence detector.
Wherein the sample to be analyzed 1 and the sample to be analyzed 2 have the same volume and come from the same sample.
In the detection process, the volumes of the sample to be analyzed, the reagent 1, the reagent 2, the reagent 1 'and the reagent 2' used by the detection kit for detecting the sample to be analyzed and the interference compensation kit for detecting the sample to be analyzed are the same. As an example, when a detection kit is used for detecting a sample to be analyzed, 25 mu L of a reagent 1 and 25 mu L of a reagent 2 are added into 25 mu L of the sample to be analyzed to react, so that a first detection signal value of 1.85ng/mL is obtained; when the interference compensation kit is used for detecting the sample to be analyzed, 25 mu L of the sample to be analyzed is added with 25 mu L of the reagent 1 'and 25 mu L of the reagent 2' for reaction, and the obtained second detection signal value is 0.05ng/mL; subtracting the second detection value from the first detection value to obtain an interference-free analyte signal value of 1.80ng/mL.
In a sixth aspect, the present invention provides a chemiluminescent analyzer comprising at least the following:
A reagent module for storing the kit system according to the fourth aspect;
an incubation module for providing a suitable temperature environment for the chemiluminescent reaction;
The detection module is used for generating excitation light and obtaining a detection light signal value and an interference light signal value;
And the processor is used for judging whether the analysis target molecules exist in the sample to be analyzed or not and/or judging the concentration of the analysis target molecules in the sample to be analyzed according to the condition of the chemiluminescent signals detected by the detection module.
In order to make the present application more clearly understood by those skilled in the art, the following examples will illustrate the interference compensation reagent, the preparation method, the kit system, the application and the chemiluminescent analyzer according to the present application.
Example 1: preparation of reagents 1 and 2 in an interference compensation kit
1) Main experimental raw materials and equipment
Experimental raw materials:
1. An antibody for coating luminescent particles.
2. Antibodies for biotin labeling.
3. Luminescent particles.
4. Biotin (DMSO) solution.
5. Reagent buffer:
the reagent 1 buffer is: concentrated MES luminescent reagent solution (pH 6.0);
the reagent 2 buffer is: concentrated MES biotin reagent solution (pH 6.0); the buffer solution comprises buffer pairs, surfactants, preservatives and the like.
6. The fresh sample contains a control reagent measurement or target.
Experimental instrument: an automatic light activated chemiluminescent detector.
2) Experimental procedure
1. Preparation of a concentrated solution of reagent 1:
1.1, preparation of inactivated antibody coated luminescent microparticles:
1.1.1, CEA (Inactivated) (coated) antibody dialysis:
After FG-CEA antibody in the detection kit is inactivated (such as heat treatment and repeated freeze thawing), CEA (Inactivated) (coated) antibody is added into 0.05M CB buffer (pH9.6) for dialysis for 2 times at 2-8 ℃ and more than 4 hours each time, so as to obtain the treated antibody.
1.1.2 Treatment of luminescent particle suspensions:
The luminescent particle suspension is commercially available from Kemei diagnostic technology Co.Ltd, and the treated luminescent particles are obtained through centrifugation, ultrasonic treatment and concentration adjustment.
1.1.3, Reaction:
Treating the treated luminous particles and the treated antibody according to the weight ratio of 10: mixing at a ratio of 0.2 (mass ratio), and adding buffer solution to make the concentration of the microparticles in the reaction solution reach 25mg/mL, and performing rotary reaction at 37 ℃ and 25-40rpm for 16-18 hours. The weight ratio is as follows: naBH 4 solution is added in a proportion of 0.16, and the mixture is rotated at a temperature of between 2 and 8 ℃ at a speed of between 25 and 40rpm for 2 hours. And (3) measuring 75mg/mL of Gly electrophoresis buffer solution according to the requirement of 0.16 mL/mL of Gly electrophoresis buffer solution for each 10mg of particles, rapidly adding the Gly electrophoresis buffer solution into the reaction solution, and reacting at 25-40rpm and 2-8 ℃ for 1 hour to obtain CEA antibody coated luminescent particles.
1.1.4, Particle cleaning:
1.1.4.1, centrifuging:
The minimum centrifuge tube number was measured for washing (no more than 250mg per tube of microparticles) based on total preparation, and the coated FG microparticles were evenly distributed into the centrifuge tubes. And supplementing a luminous reagent cleaning solution, and weighing and balancing by an electronic balance. Centrifugation conditions: 2-8 ℃ (set at 4 ℃), 16000rpm, and centrifuged for 30min.
1.1.4.2, Ultrasonic:
The supernatant was discarded, luminescent reagent cleaning liquid was added to each of the separation tubes, and the mixture was put into an ultrasonic cleaner for ultrasonic dispersion 500W for 1 second/time for 30 times. The above centrifugation ultrasonic is repeated three times, and the last luminescent reagent cleaning solution is replaced by the reagent 1 buffer solution.
Note that: the preparation method of the luminous particles coated with the inactivated antibody can also carry out the cross-linking reaction of the FG-CEA antibody of the detection kit and the luminous particles to obtain the luminous particles coated with the CEA antibody, and then carry out the inactivation (heat treatment or repeated freeze thawing) treatment.
2. Preparation of concentrated solution of reagent 2
2.1, Preparation of biotin-labeled inactivated antibody:
2.1.1, CEA (Inactivated) (labeled) antibody dialysis:
after CEA-labeled antibody inactivation (e.g., heat treatment, repeated freeze thawing) of the detection kit, CEA (Inactivated) (labeled) is added to 0.1M NaHCO 3 buffer (pH 8.5) and dialyzed 2 times at 2-8deg.C for more than 4h each time.
2.1.2, Reaction: the dialyzed antibody was diluted to a reaction concentration of 1mg/mL with 0.1M NaHCO 3 buffer, biotin (DMSO) was added at a ratio of 1:15 of the molecular weight of the antibody to Biotin, and the Biotin (DMSO) was added rapidly to the centrifuge tube and mixed rapidly. The reaction is carried out for 14 to 18 hours at the temperature of 2 to 8 ℃ and at the speed of 25 to 40rpm by a vertical rotary mixer.
2.1.3, Dialysis: the biotin marks the dialysis buffer solution, and the dialysis is carried out for 2 times at the temperature of 2-8 ℃ for more than 4 hours each time.
3) Reagent 1 and reagent 2 preparation process:
the concentrated solutions of the above reagent 1 and reagent 2 were diluted to a fixed concentration (CEA detection kit reagent concentration) using a reagent 1 buffer and a reagent 2 buffer, respectively.
Example 2:
(1) Use of interference compensation kit
The CEA detection kit and the interference compensation kit are adopted for the selected sample at the same timeAnd (3) automatically detecting on an automatic light-activated chemiluminescence detector in a mode of adding 25 mu L of sample, 25 mu L of reagent 1 and 25 mu L of reagent 2, incubating, adding universal liquid of a light-activated chemiluminescence analysis system, and further incubating and reading to obtain a detection light signal value and an interference light signal value respectively.
(2) Processing of data
The detection kit obtains a signal plus or minus interference actual signal, and the signal plus or minus interference actual signal is brought into a calibration curve of the detection kit to obtain the final concentration.
(3) Experimental data
Table 1CEA detection kit measurements and interference compensation kit corrected measurements
As can be seen from table 1, the detection result of the detection kit is smaller in difference from the control reagent after the interference signal is eliminated by the matched interference compensation kit, and the result is more accurate, which indicates that the interference compensation kit can correct the interference detected by the detection kit.
The invention has been described in detail with reference to the above description of an interference compensation reagent, preparation method and kit system, application and chemiluminescent analyzer, and specific examples are used herein to illustrate the principles and embodiments of the invention, the above examples are only for aiding in understanding the method and core idea of the invention; meanwhile, as those skilled in the art will have variations in the specific embodiments and application scope in accordance with the ideas of the present invention, the present description should not be construed as limiting the present invention in view of the above.
Claims (10)
1. An interference compensation reagent, characterized in that the interference compensation reagent comprises reagent 1 and reagent 2;
The reagent 1 comprises luminescent particles coated by an antibody 1 without antigen binding activity;
The reagent 2 comprises a biotin-labeled antibody 2 having no antigen binding activity.
2. The interference compensating reagent of claim 1 wherein the antibody 1 and the antibody 2 are inactivated antibodies capable of specifically recognizing different sites of the same antigen.
3. A method of preparing an interference compensating reagent according to any of claims 1-2, comprising:
S11, mixing the luminescent particles and carrying out a rotary reaction on the mixed luminescent particles and the antibody to obtain antibody-coated luminescent particles;
S12, mixing biotin and an antibody to perform a rotary reaction to obtain a biotin-labeled antibody;
s13, inactivating the antibody-coated luminous particles and the biotin-labeled antibody to obtain antigen-binding-activity-free antibody-coated luminous particles and biotin-labeled antigen-binding-activity-free antibodies;
S14, adjusting the concentration of the luminescent particles coated by the antigen-binding-free active antibody by using a luminescent reagent buffer solution to obtain a reagent 1; the concentration of the biotin-labeled antigen-binding inactive antibody was adjusted with a biotin reagent buffer to give reagent 2.
4. A method of preparing an interference compensating reagent according to any of claims 1-2, comprising:
S21, inactivating the antibody to obtain an antibody without antigen binding activity;
s22, mixing the luminous particles and reacting the luminous particles with the antibody without antigen binding activity to obtain luminous particles coated with the antibody without antigen binding activity;
s23, mixing biotin and reacting with the antigen-binding-free active antibody to obtain a biotin-labeled antigen-binding-free active antibody;
s24, adjusting the concentration of the luminescent particles coated by the antigen-binding-free active antibody by using a luminescent reagent buffer solution to obtain a reagent 1; the concentration of the biotin-labeled antigen-binding inactive antibody was adjusted with a biotin reagent buffer to give reagent 2.
5. A kit system, characterized in that it consists of a detection kit and an interference compensation kit comprising the interference compensation reagent according to claim 1 or 2.
6. The kit system of claim 5, wherein the detection kit comprises reagent 1 'and reagent 2';
The reagent 1' comprises luminous particles coated by an antibody 1 with antigen binding activity;
The reagent 2' comprises a biotin-labeled antibody 2 having antigen binding activity.
7. The kit system according to claim 6, wherein the mass concentration of the antigen-binding-activity-free antibody 1-coated luminescent particles in the reagent 1 is the same as the mass concentration of the antigen-binding-activity-free antibody 1-coated luminescent particles in the reagent 1';
the mass concentration of the biotin-labeled antigen-binding-inactive antibody 2 in the reagent 2 is the same as the mass concentration of the biotin-labeled antigen-binding-active antibody 2 in the reagent 2'.
8. Use of a kit system according to any of the preceding claims 5-7 for detecting target molecule content by chemiluminescent analysis; comprising the following steps:
Detecting a sample to be analyzed by using a detection kit in the kit system to obtain a detection light signal value;
detecting a sample to be analyzed by using an interference compensation kit in the kit system to obtain an interference optical signal value;
And carrying out mathematical operation on the detection optical signal value and the interference optical signal value of the same sample to be analyzed to obtain the target molecule concentration.
9. The use according to claim 8, wherein the volumes of the sample to be analyzed, reagent 1, reagent 2, reagent 1', reagent 2' in each reaction are the same at the time of detection.
10. A chemiluminescent analyzer, wherein the chemiluminescent analyzer comprises at least the following:
a reagent module for storing the kit system according to any one of claims 5-7;
an incubation module for providing a suitable temperature environment for the chemiluminescent reaction;
The detection module is used for generating excitation light and obtaining a detection light signal value and an interference light signal value;
And the processor is used for judging whether the target molecule exists in the sample to be analyzed or not and/or the concentration of the target molecule in the sample to be analyzed according to the condition of the chemiluminescent signal detected by the detection module.
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