CN110632297A - Immunoassay kit and determination method and preparation method thereof - Google Patents
Immunoassay kit and determination method and preparation method thereof Download PDFInfo
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- CN110632297A CN110632297A CN201910949006.6A CN201910949006A CN110632297A CN 110632297 A CN110632297 A CN 110632297A CN 201910949006 A CN201910949006 A CN 201910949006A CN 110632297 A CN110632297 A CN 110632297A
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention provides an immunoassay kit, which comprises 1) a conjugate carrier, a detection probe and a detection reagent, wherein the conjugate carrier comprises a ligand, a marker and a solid phase carrier, the ligand is used for carrying out a specific reaction with an analyte to be detected, the marker is used for signal detection, the ligand is connected with the marker, and the solid phase carrier is used for carrying the ligand and the marker; and 2) an immunochromatographic test strip; wherein, the conjugate carrier and the immunochromatographic test strip are arranged in a split mode. The kit has the characteristics of normal-temperature storage and transportation, good precision, simple and quick operation method and suitability for instant detection. When the kit is used for determination, the sample solution and the conjugate carrier are mixed uniformly and then added into the immunochromatography test strip for detection, so that the conjugate is released more fully. The kit is convenient and quick to prepare, low in manufacturing cost, and well compatible with the existing production line equipment, and complex processes and expensive equipment are not needed.
Description
Technical Field
The invention relates to the field of immunodetection, in particular to an immunodetection kit, and a determination method and a preparation method thereof.
Background
Immunochromatography is a method capable of qualitatively and quantitatively analyzing trace analytes in a short time using an antigen-antibody reaction, and has been used for diagnosing or detecting various diseases and a variety of fields including medicine, agriculture, animal husbandry, food, military industry, and environmental fields.
Immunochromatographic assays are generally performed using assay strips containing reactive substances that change in response to the analyte to be detected, or using assay devices that contain assay strips mounted in plastic housings. Conventional assay test strips include: the sample pad, the combination pad, the nitrocellulose membrane and the absorbent pad which are connected in sequence are sequentially overlapped and assembled on the bottom plate, wherein the sample pad is used for receiving a liquid sample, namely an analyte; the conjugate pad contains a conjugate, i.e., a detection reagent, to which the analyte can bind; a detection part is fixed on the nitrocellulose membrane, and an antibody or an antigen is fixed on the detection part, namely the detection part can perform specific reaction with the analyte combined with the conjugate; the absorbent pad is used to ultimately receive a liquid sample. In the immunochromatographic assay using the assay strip, when a liquid sample is dropped on a sample pad, it moves through a conjugate pad and a nitrocellulose membrane by capillary phenomenon, and is finally received in a water absorbent pad. The conjugate in the conjugate pad also moves as a mobile phase with the liquid sample, and if the analyte to be detected is present in the liquid sample, the conjugate will bind to the antigen or antibody on the nitrocellulose membrane by the analyte (generally referred to as a "sandwich reaction"), or the conjugate and the analyte compete for binding to the antigen or antibody (generally referred to as a "competition reaction"), and thus, the presence of the analyte in the sample can be visually perceived or perceived by the sensor.
However, the conventional assay strip has problems in that a liquid sample moving by capillary phenomenon is not uniformly bound to a dry conjugate immobilized on a conjugate pad and the conjugate is not completely released in the conjugate pad, and thus, there may be variations between assay strips, which may cause the accuracy and reproducibility of quantitative analysis of the sample to be lowered.
An immunochromatographic test strip, which can be constituted of a conjugate pad and an insoluble membrane support, is provided in patent CN103314297A, because a conjugate portion is formed in a linear shape and there is a certain positional relationship between the conjugate portion and the insoluble membrane support, to achieve excellent releasability of the conjugate from the conjugate pad, to complete the reaction in a shorter time, and also to achieve excellent sensitivity. However, the liquid sample moved by capillary phenomenon is still bound to the conjugate in the present patent, and thus effective release of the conjugate cannot be guaranteed.
Provided in patent CN104428675A are a lyophilized conjugate structure for point-of-care testing (POCT) immunochromatography, an immunoassay kit comprising the same, and a method for analysis using the kit, which can perform quantitative analysis with higher reproducibility and linearity depending on concentration, compared to a conventional assay method using an immunochromatographic test strip comprising a conjugate pad prepared by adsorbing a conjugate, because a sample is subjected to immunochromatography after uniformly reacting with the lyophilized conjugate structure prepared separately from the outside according to the present invention on the outside. However, the freeze-dried conjugate structure body needs to be obtained through a freeze-drying method, and the freeze-drying method has the advantages of long storage period, good uniformity, capability of storing the biological activity of the marker to the maximum extent, high cost and long production period, and the freeze dryer needs to be operated by professional personnel, and has high requirements on the components of the marker diluent.
In addition, the label bound with the antibody/antigen can be stored in a liquid reagent form to improve the accuracy and reproducibility of sample analysis, and the label is diluted with a diluent and then stored in a centrifuge tube. However, when the liquid reagent containing the antibody and the antigen is stored for a long time, the affinity of the antigen-antibody reaction is reduced, or the antigen and the antibody are denatured, the liquid reagent has a complicated process formula, the difficulty in maintaining good stability is high, the stability can be maintained only by cold storage and transportation at 2-8 ℃, and the production, transportation, storage and other costs are increased.
Based on the above situation, there is a need to design an immunoassay device that can solve the above problems.
Disclosure of Invention
The invention aims to provide an immunoassay kit, which comprises a conjugate carrier and an immunochromatographic test strip, has the characteristics of normal-temperature storage and transportation, good precision, simple and quick operation method, and suitability for instant detection, and solves the problem of inaccurate result caused by excessive retention and insufficient release of a conjugate on a binding pad in the conventional test strip.
The second purpose of the invention is to provide an immunochromatographic assay method, wherein the sample solution and the conjugate carrier are mixed uniformly and then added into an immunochromatographic test strip for detection, so that the conjugate is released more fully.
The third purpose of the invention is to provide a preparation method of the immunoassay kit, wherein the preparation of the conjugate carrier is convenient and rapid, the manufacturing cost is low, the immunochromatographic test strip keeps the original structure, and the immunoassay kit can be well compatible with the existing production line equipment without complex process and expensive equipment.
In order to achieve the above object, the present invention provides in a first aspect an immunoassay kit comprising:
1) the conjugate carrier comprises a ligand, a marker and a solid phase carrier, wherein the ligand is used for performing a specific reaction with an analyte to be detected, the marker is used for detecting a signal, the ligand is connected with the marker, and the solid phase carrier is used for carrying the ligand and the marker;
2) the immunochromatography test strip comprises a first sample pad, a nitrocellulose membrane, a water absorption pad and a bottom plate, wherein the first sample pad, the nitrocellulose membrane and the water absorption pad are sequentially arranged on the bottom plate along the liquid chromatography direction;
wherein, the conjugate carrier and the immunochromatographic test strip are arranged in a split mode.
Specifically, the ligand is linked to the label by chemical and/or physical force.
Specifically, the conjugate carrier is provided separately from the immunochromatographic strip, which means that the conjugate carrier is not contained on the immunochromatographic strip, for example, the conjugate carrier is packaged separately from the immunochromatographic strip. When the immunoassay kit is used for detection, a sample solution can be mixed with a conjugate carrier in a cavity, and then the mixed sample solution is added into an immunochromatography test strip to obtain a detection result, so that the release capacity of a conjugate can be improved, the reaction sufficiency of an analyte to be detected and a ligand can be improved, and the accuracy of the detection result can be further improved.
Preferably, the marker is any one of a time-resolved fluorescent microsphere, a common fluorescent microsphere, a magnetic microsphere, colloidal gold, a colored latex microsphere, a quantum dot, a time-resolved fluorescent dye, common fluorescein, an enzyme, biotin-avidin and a microsphere amplification system.
Preferably, the solid phase carrier is any one of glass fiber, polyester film and nonwoven fabric.
Preferably, the immunochromatographic test strip further comprises a second sample pad, and the second sample pad is arranged between the first sample pad and the nitrocellulose membrane.
Preferably, the conjugate carrier is stored and transported in a sealed chamber at normal temperature.
Preferably, the chamber is any one of a centrifuge tube, a plastic tube or a microplate.
Preferably, the diluent comprises buffer pairs, proteins, saccharides, polymers, blocking agents, preservatives, and surfactants.
The second aspect of the present invention provides an immunochromatographic assay method using the above immunoassay kit, comprising the steps of:
dissolving the conjugate: adding the sample solution into a conjugate carrier, dissolving the conjugate and fully reacting with the sample solution to obtain a mixed solution;
dropwise adding the mixed solution: taking the mixed solution fully reacted in the step, and dripping the mixed solution on a first sample pad in the immunochromatography test strip;
and (3) detection: and collecting signals of the T line and the C line by using an immunoassay instrument, calculating a T/C signal value, and obtaining the concentration of the analyte from the calibration curve.
Preferably, before the step of dissolving the conjugate, the method further comprises:
obtaining a calibration curve: and taking standard sample curves of the analyte with different concentrations to obtain corresponding T line and C line signals, establishing a standard curve of the analyte concentration corresponding to the T line and C line signals to obtain a calibration curve, and storing the calibration curve in the ID chip.
The third aspect of the invention provides a preparation method of an immunoassay kit, which uses the immunoassay kit and comprises the following steps:
s100, preparing a conjugate carrier;
s200, preparing immunochromatographic test paper;
s300, packaging immunoassay kit
In step S100, preparing a conjugate carrier includes:
s110, combining the ligand and the marker to obtain a conjugate;
s120, preparing a diluent, and dispersing the obtained conjugate in the diluent;
s130, fixing the diluent in a solid phase carrier in a spraying or smearing mode, and drying to obtain a conjugate carrier;
s140, cutting the conjugate carrier into fixed sizes and subpackaging the conjugate carrier in sealed chambers.
Preferably, the mass concentration of the conjugate in the diluent is 0.05-0.5 per mill; the diluent comprises buffer pairs, proteins, saccharides, high polymers, a blocking agent, a preservative and a surfactant.
The invention has the beneficial effects that:
1. the conjugate is treated by using a proper diluent, and is dispersed in a solid phase carrier, and the conjugate carrier is obtained after drying, so that the preparation method of the conjugate carrier is simple, the conjugate carrier can be stored and transported for a long time at normal temperature, and the cost is reduced;
2. the conjugate carrier and the immunochromatographic test strip are arranged in a split mode, so that the conjugate carrier can be independently packaged in a closed cavity, can be simply stored without pollution, and is easy to carry;
3. the conjugate can be quickly and fully dissolved in the sample solution to fully react with the analyte to be detected, the conjugate can not generate nonspecific aggregation after being redissolved, the releasing capacity of the conjugate and the reaction sufficiency of the analyte to be detected and the ligand can be effectively improved by redissolving the conjugate in the sample solution, and therefore the accuracy of the detection result is further improved;
4. compared with the conventional immunochromatographic test paper for solidifying the conjugate on the binding pad, the conjugate provided by the invention has stable titer, and shows better precision; compared with the immunochromatographic test paper for storing the marker in a liquid state or a freeze-dried state, the conjugate carrier is dried, is convenient and quick to prepare, has low manufacturing cost, and can be stored at normal temperature for a long time;
5. the immunoassay kit can keep the original chromatography test strip structure, can be well compatible with the existing production line equipment, and can not need complex processes and expensive equipment.
Drawings
FIG. 1 is a schematic diagram of an immunochromatographic test strip in the prior art;
FIG. 2 is a schematic view of an immunoassay kit of the present invention;
FIG. 3 is a schematic view of another configuration of the immunoassay kit of the present invention;
FIG. 4 is a flow chart of the assay method of the immunoassay kit of the present invention;
FIG. 5 is a flow chart of a method of making the immunoassay kit of the present invention;
FIG. 6 is a flowchart of the preparation of a conjugate carrier by the immunoassay kit of the present invention.
Wherein, in fig. 1: 1. a sample pad; 2. a nitrocellulose membrane; 21. detecting lines; 22. a quality control line; 3. a water absorbent pad; 4. a base plate; 5. a bonding pad;
in fig. 2 and 3: 1. a first sample pad; 2. a nitrocellulose membrane; 21. detecting lines; 22. a quality control line; 3. a water absorbent pad; 4. a base plate; 5. a second sample pad; 7. a chamber; 8 a conjugate carrier.
Detailed Description
(conventional immunochromatography test strip)
As shown in fig. 1, the conventional immunochromatographic test strip includes: the sample pad 1, the combination pad 5, the nitrocellulose membrane 2 and the absorbent pad 3 which are connected in sequence are sequentially overlapped and assembled on the bottom plate 4, the combination pad 5 contains conjugate, namely detection reagent, and analyte can be combined with the conjugate; the nitrocellulose membrane is coated with a detection line T line 21 and a quality control line C line 22, and the T line 21 is coated with a ligand which is specifically combined with an analyte to be detected.
(immunoassay kit)
The immunoassay kit provided by the present invention, as shown in fig. 2, comprises: conjugate carrier 8 and immunochromatographic test strip, wherein the conjugate carrier and the immunochromatographic test strip are arranged in a split mode.
1) The conjugate carrier 8 comprises a ligand, a marker and a solid phase carrier, wherein the ligand is used for carrying out specific reaction with a sample to be detected, the marker is used for signal detection, the ligand and the marker are connected through chemical force and/or physical force, and the solid phase carrier is used for carrying the ligand and the marker.
The label may produce a signal that is visually perceptible or perceptible using a sensor, may produce a signal by an inherent property of the label (e.g., luminescence), or may produce a signal by an external stimulus (e.g., fluorescence).
In some embodiments of the invention, the label is any one of a time-resolved fluorescent microsphere, a normal fluorescent microsphere, a magnetic microsphere, colloidal gold, a colored latex microsphere, a quantum dot, a time-resolved fluorochrome, a normal fluorescein, an enzyme, a biotin-avidin, and a microsphere amplification system.
The ligand may specifically bind to the analyte, and any substance may be used as a ligand in the present invention as long as it exhibits the property of specifically binding to a specific receptor. The ligand is bound to a label to form a conjugate, and the label and the ligand may be physically or chemically linked to each other.
In some embodiments of the invention, the ligand is an antibody that specifically binds to an antigen or an antigen that specifically binds to an antibody.
The solid phase carrier is used for bearing the conjugate, the solid phase carrier does not react with the conjugate, namely, the performance of the conjugate is not adversely affected, and long-term storage at normal temperature and the characteristics of the conjugate can be realized by preparing the conjugate into a solution with predetermined components and concentrations and then dispersing the solution on the surface of the solid phase carrier. At the same time, the solid support does not react with the sample solution or the analyte.
In some embodiments of the present invention, the solid support is any one of glass fiber, polyester film, and non-woven fabric.
In some embodiments of the invention, the conjugate carrier is stored separately and hermetically in chamber 7.
The chamber 7 is a space for storing the conjugate carrier, and the chamber 7 can be sealed to ensure that the storage process of the conjugate carrier is not polluted by external inappropriate environment to destroy the performance of the conjugate, so that the conjugate has a longer shelf life.
In some embodiments of the invention, the chamber 7 is any one of a centrifuge tube, a plastic tube, or a microplate.
In some embodiments of the invention, the chamber 7 comprises a lid. When storing, the chamber is sealed by a cover, and the conjugate carrier is hermetically stored in the chamber; when testing, the cover is opened and the sample solution is added to the chamber to dissolve the conjugate.
In some embodiments of the invention, the conjugate carrier is formed by: combining the ligand and the marker, diluting the marker combined with the ligand to a certain concentration by a diluent, uniformly distributing the marker on a solid phase carrier in a spraying or soaking mode, drying, cutting into a fixed size, and subpackaging in a sealed chamber, namely storing at normal temperature.
In some embodiments of the present invention, the dilution to a certain concentration means that the mass concentration of the conjugate in the diluent is 0.05% o to 0.5% o. In some embodiments of the invention, the mass concentration of the conjugate in the diluent is between 0.02% and 0.1%.
In some embodiments of the invention, the diluent comprises a buffer pair, a protein, a saccharide, a polymer, a blocking agent, a preservative, a surfactant.
The buffer pair in the diluent is Tris-HCl or PB, and the concentration is 0.01-0.05M; the protein is BSA or casein, and the concentration is 0.5 to 2 percent; the sugar is sucrose and/or trehalose, and the concentration is 5% -25%; the high polymer is any one or the combination of PVP, PVA or PEG, and the concentration is 0.5-2%; the blocking agent is Roche MAK series or Scantibodies HBR series, and the concentration is 1-5 mg/mL; the preservative is Proclin300 or sodium azide with the concentration of 0.05-0.1%; the surfactant is Tween-20, Triton-100 or S9, and the concentration is 0.1-1%.
The pH of the dilution is 7.0-9.0, preferably 9.0.
2) The immunochromatographic test strip comprises a first sample pad 1, a nitrocellulose membrane 2, a water absorption pad 3 and a bottom plate 4, wherein the first sample pad 1, the nitrocellulose membrane 2 and the water absorption pad 3 are sequentially arranged on the bottom plate 4 along the liquid chromatography direction.
Unlike the conventional immunochromatographic test strip, the present invention does not have a conjugate pad, but retains a sample pad, a nitrocellulose membrane and a water absorbent pad, and the structures thereof have been disclosed in the prior art and are not described herein.
Wherein, the cellulose nitrate membrane is coated with a detection line (T line) 21 and a quality control line (C line) 22, the T line 21 is coated with a ligand which is specifically combined with the analyte to be detected, and the final detection result can be judged by observing the change of the T line 21 and the C line 22. Further, the ligand coated on the T-line 21 may be the same as the ligand in the conjugate or may be different from the ligand in the conjugate.
As shown in fig. 3, in some embodiments of the present invention, the immunochromatographic test strip further comprises a second sample pad 5, and the second sample pad 5 is disposed between the first sample pad 1 and the nitrocellulose membrane 2.
In order to enable the sample solution to be uniformly transferred to the nitrocellulose membrane from the sample pad and facilitate the reduction of the influence of interference substances in the sample, two layers of sample pads can be arranged, a second sample pad is arranged between the first sample pad and the nitrocellulose membrane, and the material of the second sample pad can be the same as that of the first sample pad and can also be different from that of the first sample pad. Through this setting, the migration speed and the degree of consistency of steerable sample solution promote product property ability.
In some embodiments of the invention, the immunoassay kit further comprises a housing, the housing comprises an upper housing and a lower housing, the immunochromatographic test strip is mounted between the upper housing and the lower housing, the upper housing comprises a sample adding hole and an observation port, the sample adding hole corresponds to the first sample pad so as to facilitate the addition of a sample solution, and the observation port corresponds to the T line and the C line of the nitrocellulose membrane so as to facilitate the observation and the acquisition of the detection result. When the immunoassay kit is used for detection, a sample solution dissolved with a conjugate is added from a sample adding hole to carry out chromatography reaction, and signal acquisition is carried out at an observation port after a period of time.
The conjugate pad of the conventional immunochromatographic test strip is used for immobilizing a conjugate, wherein the conjugate is also stored in a solid state, when a sample solution migrates from the sample pad to the conjugate pad by capillary force, the immobilized conjugate is dissolved in the sample solution, while the amount of the sample solution dropped into the sample pad during detection is small, and meanwhile, the sample solution flows with the capillary force, so that the conjugate immobilized in all the conjugate pads cannot be completely released, namely, the conjugate in the conjugate pad remains after the detection is finished, and the accuracy of the final detection result is affected; meanwhile, after the detection of different immunochromatographic test strips is finished, the detection results of different immunochromatographic test strips are deviated due to different residual amounts of the conjugates in the binding pads.
In the invention, the conjugate is fixed in the conjugate carrier, and the conjugate carrier has two main advantages, namely 1) the conjugate is diluted and then uniformly distributed on the solid phase carrier in a spraying or soaking way, and the like, and can be stored at normal temperature after being dried and can also keep good stability after being stored for a long time; 2) the sample solution is firstly mixed with the conjugate carrier in the chamber, so that the releasing capacity of the conjugate can be improved, the reaction sufficiency of the analyte to be detected and the ligand can be improved, and the accuracy of the detection result can be further improved.
(immunochromatographic assay method)
The immunochromatographic assay method provided by the present invention, as shown in fig. 4, uses the above immunoassay kit, and comprises the following steps:
dissolving the conjugate: adding the sample solution into a conjugate carrier, dissolving the conjugate and fully reacting with the sample solution to obtain a mixed solution;
dropwise adding the mixed solution: taking the mixed solution fully reacted in the step, and dripping the mixed solution on a first sample pad in the immunochromatography test strip;
and (3) detection: and collecting signals of the T line and the C line by using an immunoassay instrument, calculating a T/C signal value, and obtaining the concentration of the analyte from the calibration curve.
In some embodiments of the invention, prior to the step of solubilizing the conjugate, further comprising:
obtaining a calibration curve: and taking standard sample curves of the analyte with different concentrations to obtain corresponding T line and C line signals, and establishing a standard curve corresponding to the concentration of the analyte and the T line and C line signals.
The invention can obtain the qualitative detection result and/or the quantitative detection result according to the signal intensity of the T line and the C line.
Specifically, when the analyte to be detected does not exist in the sample solution, the T line does not generate a signal, when the analyte to be detected exists in the sample solution, the T line generates a signal, the C line is a quality control line, and the C line generates a signal no matter whether the analyte to be detected exists in the sample, and the C line signal is a standard for judging whether the sample is enough or not and whether the chromatography process is normal or not. Therefore, the detection result can be qualitatively obtained.
Meanwhile, because the signal generated by the T line is in positive correlation with the concentration of the analyte to be detected in a certain range, according to the correlation curve of the signal generated by the T line and the concentration of the analyte to be detected, the invention can also carry out intensity analysis on the corresponding signals generated by the T line and the C line through auxiliary detection equipment, and then convert the signals into the concentration of the analyte to be detected through a certain corresponding relation, so that the detection result can be obtained quantitatively.
Of course, if the immunoassay instrument for auxiliary detection already has a corresponding calibration curve, or has already obtained a calibration curve once, the detection process can be completed without repeatedly obtaining the calibration curve in the subsequent detection process, and a quantitative detection result can be obtained.
An immunochromatographic assay method utilizing a combination of an immunoreaction principle based on an antigen-antibody reaction and an immunochromatographic principle in which a sample and a reagent move along a medium through a mobile phase. In the general sense, an immune response refers to an antigen-antibody reaction, but in the broad sense of the present invention it is meant to include not only antigen-antibody reactions, but also reactions between a receptor and a ligand to which it specifically binds. Further, without being limited thereto, it also includes a reaction between substances that specifically recognize each other, such as an enzyme-substrate reaction.
The analyte, also referred to as an analyte to be measured, includes viruses and physiologically active substances that can be generally measured by an antigen-antibody reaction.
In some embodiments of the invention, the virus comprises influenza viruses such as influenza a virus, influenza b virus, hepatitis c virus, human immunodeficiency virus, and the like.
In some embodiments of the present invention, the physiologically active substance includes human hemoglobin, hepatitis B antibody, hepatitis C antibody, human immunodeficiency virus antibody, tumor marker (AFP/CEA/PSA/CA199), infectious agent (CRP/PCT/SAA/IL6), and the like.
Sample, also called sample solution, refers to a sample that may contain an analyte, including all biological samples isolated from a mammal, preferably a human.
In some embodiments of the invention, the sample comprises whole blood, blood cells, serum, plasma, bone marrow spinal fluid, sweat, urine, tears, saliva, skin, mucous membrane, and hair. Preferably, the sample may be whole blood, serum, plasma.
In some embodiments of the invention, the sample may be buffered prior to being sufficiently reacted with the treated sample solution added to the conjugate carrier to sufficiently solubilize the conjugate to ensure complete dissolution of the conjugate and complete reaction with the analyte.
The immunoassay method of the present invention can be used for analyzing blood glucose levels and diagnosing diseases using serum as a sample. Examples of such diseases include malaria antigen (Ag), AIDS, hepatitis c, hepatitis b, syphilis, gastric ulcer-causing microorganisms, cancer markers (AFP, PSA, CEA), tuberculosis, SAS, dengue fever, and leprosy. Preferably, the analyte may be a cancer marker (AFP, PSA, CEA), but is not limited thereto.
(method of preparing immunoassay kit)
The method for preparing the immunoassay kit provided by the invention comprises the following steps as shown in figure 5:
s100, preparing a conjugate carrier;
s200, preparing immunochromatographic test paper;
s300, packaging the immunoassay kit.
As shown in fig. 6, the preparation of the conjugate carrier in step S100 includes:
s110, combining the ligand and the marker to obtain a conjugate;
s120, preparing a diluent, dispersing the obtained conjugate in the diluent, and diluting the conjugate to a certain concentration;
s130, fixing the diluent in a solid phase carrier in a spraying or smearing mode, and drying to obtain a conjugate carrier;
s140, cutting the conjugate carrier into fixed sizes and subpackaging the conjugate carrier in sealed chambers.
Wherein, in step S200, the preparation of the immunochromatographic test strip comprises:
s210, preparing a sample pad buffer solution, soaking the sample pad in the sample pad buffer solution in a distributed manner, taking out the sample pad, and drying the sample pad;
s220, coating a detection line T line and a quality detection line C line on the nitrocellulose membrane, and drying;
s230, sequentially arranging the sample pad, the nitrocellulose membrane and the water absorption pad on a bottom plate;
s240, clamping the immunochromatographic test strip between the upper shell and the lower shell to obtain the immunochromatographic test strip.
In some embodiments of the present invention, the dilution of the conjugate to a certain concentration in step S120 means that the mass concentration of the conjugate in the diluent is 0.05% o to 0.5% o. Preferably, the mass concentration of the conjugate in the diluent is 0.02% to 0.1%.
Wherein, in step S300, the packaged immunodetection kit comprises:
packaging the cavity containing the conjugate carrier obtained in the step S100, the immunochromatographic test strip obtained in the step S200 and a drying agent in an aluminum foil bag, sealing by heat sealing, and storing at normal temperature; or
And (4) respectively packaging the cavity containing the conjugate carrier obtained in the step (S100) and the immunochromatographic test strip obtained in the step (S200), respectively packaging the cavities and the immunochromatographic test strip together with a drying agent in an aluminum foil bag, sealing the aluminum foil bag in a heat sealing manner, and storing the aluminum foil bag at normal temperature.
Examples
Hereinafter, the present invention will be described in more detail with reference to examples. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1AFP fluorescent microsphere immunoassay kit
1. AFP fluorescent microsphere immunoassay kit structure:
the kit comprises an immunochromatographic test strip and a conjugate solid-phase carrier, wherein the immunochromatographic test strip comprises a base plate, a first sample pad, a second sample pad, a nitrocellulose membrane and a water absorption pad are sequentially connected to the base plate, an AFP antibody is coated at the T-line position of the nitrocellulose membrane, and a goat anti-mouse IgG is coated at the C-line position of the nitrocellulose membrane; the label in the conjugate solid phase carrier is europium ion fluorescent microspheres, the ligand is AFP antibody, the solid phase carrier is glass fiber, and the conjugate solid phase carrier is placed in a centrifuge tube.
2. Preparing an AFP fluorescent microsphere immunoassay kit:
reagents and consumables:
TABLE 1
Solution preparation:
0.025M MES buffer: weighing 5.33g of MES in 900mL of ultrapure water, adjusting the pH to 5.0 after completely dissolving, and metering the volume to 1L for washing and activating the microspheres;
EDC solution: 10mg/mL, dissolved in 0.25M MES buffer, pH 5.0;
NHS solution: 10mg/mL, dissolved in 0.25M MES buffer, pH 5.0;
0.01M PBS buffer: 0.24g KH was weighed out2PO4，1.44gNa2HPO48g of NaCl and 0.2g of KCl in 900mL of ultrapure water, and after complete dissolution, the pH was adjusted toThe required pH value is obtained, and then the volume is determined to be 1L by a volumetric flask;
0.02M PB buffer: first, 1L of 0.2M Na is prepared2HPO4·12H2O solution and 1L of 0.2M NaH2PO4Solution, 1L 0.2M Na2HPO4·12H2Solution O: 71.64g of Na were weighed2HPO4·12H2O, constant volume is 1L, 1L of 0.2M NaH is prepared2PO4Solution: weighing 24g NaH2PO4The volume is constant to 1L; according to different pH value requirements, two solutions with certain volumes are respectively taken, 800mL of ultrapure water is added, HCl or NaOH is used for fine adjustment to the required pH value, and the volume is adjusted to 1L;
0.01M Tris-HCl buffer: weighing 1.21g of the extract in 900mL of ultrapure water, adjusting the pH value to the required value after completely dissolving the extract, and then fixing the volume to 1L;
0.025M Tris-HCl buffer: weighing 3.03g of the solution in 900mL of ultrapure water, adjusting the solution to a required pH value after the solution is completely dissolved, and then fixing the volume to 1L;
microsphere coupling buffer: 0.02M PB, pH 7.0;
microsphere sealing liquid: 0.02M PB, 5% BSA, pH 7.4;
microsphere preserving fluid: 0.01M Tris-HCl, 0.9% NaCl, 0.2% Tween-20, 0.05% Proclin-300, pH8.0;
diluting the microspheres: 0.025M Tris-HCl, 1% BSA, 0.5% Tween-20, 0.5% PVP, 10% D-trehalose, 15% sucrose, 0.05% Proclin-300, 2mg/mL HBR-1 blocker, pH 9.0.
2.1 preparation of labeled antibody solid phase Carrier
Preparation of labeled antibody
Cleaning microspheres: taking 200 mu L of fluorescent microspheres (with solid content of 1%) in a 2mL centrifuge tube, washing three times by using 1mL MES buffer solution, and centrifuging at 15000rpm for 20 min;
activating the microspheres: adding 1mL MES buffer solution, carrying out ultrasonic resuspension, adding 10 and 20 mu L each of EDC and NHS, reacting at room temperature for 30min, centrifuging at 15000rpm for 20min, and removing supernatant;
labeling the antibody: adding 1mL of microsphere coupling buffer solution for ultrasonic resuspension, adding 200 μ g of Firoc organism 2AFP-28 antibody for room temperature oscillation reaction for 2h, centrifuging at 12000rpm for 20min, and removing supernatant;
sealing the microspheres: adding 1mL of microsphere sealing buffer solution, carrying out ultrasonic resuspension, carrying out oscillation reaction at room temperature for 2h, centrifuging at 15000rpm for 20min, and removing supernatant;
washing and preserving the microspheres: adding 1mL of microsphere preservation buffer solution for ultrasonic resuspension, centrifuging at 12000rpm for 20min, removing supernatant, washing for three times, finally adding 10mL of microsphere preservation solution for ultrasonic resuspension, and sealing and preserving at 4 ℃.
② solidification of antibody-labeled substance
Diluting the marked microspheres to 0.5 per mill (solid content of microspheres W/V) by using a diluent, spraying the microspheres onto 6mm multiplied by 300mm Oslon 8964 glass fibers according to the spraying amount of 4 muL/cm by using a Shanghai gold mark HM3230 film-cutting gold spraying instrument, drying the glass fibers at 37 ℃ for 4 hours, cutting the glass fibers into square small blocks of 4mm multiplied by 6mm, and packaging each small block into a centrifugal tube.
2.2 preparation of the first and second sample pad
First sample pad treatment solution: 0.2M PB, 0.5% S9, 2% BSA, 1.5% PVP, pH 7.4;
the second sample pad treatment solution was: 0.2M PB, 0.5% S9, 2% BSA, 1.5% PVP, 2% sucrose, 30ug/mL blocker, pH 7.4.
② the sample pad material is CB08, the first sample pad and the second sample pad are respectively soaked in the sample pad buffer solution for 1h at room temperature, and dried for 4h at 37 ℃.
2.3 preparation of coating film
Coating buffer solution: 0.01M PBS, 1.5% D-trehalose, pH 7.4;
coating buffer solution to dilute AFP antibody and goat anti-mouse IgG to 1 mg/mL;
thirdly, after the nitrocellulose membrane is adhered to a PVC bottom plate, a Shanghai gold mark HM3230 film scratching gold spraying instrument is used for coating the Fengcheng organism 2AFP-27 and goat anti-mouse IgG antibody solution on the positions of a T line and a C line on the nitrocellulose membrane, the spraying amount is 1 mu L/cm, the distance between the T line and the C line is 4mm, and the nitrocellulose membrane is dried for 4 hours at 37 ℃.
2.4, assembling an AFP fluorescent microsphere immunoassay kit:
the first sample pad, the second sample pad, the nitrocellulose membrane and the water absorption pad are sequentially arranged from left to right along the liquid chromatography direction to form the immunochromatography test strip, and the PVC bottom plate is arranged below the first sample pad, the second sample pad, the nitrocellulose membrane and the water absorption pad to support the first sample pad, the second sample pad, the nitrocellulose membrane and the water absorption pad.
(1) Sticking the water absorption pad: the bottom plate is flatly laid on the working table; tearing the protective film at the pasting position of the water absorption pad on the upper edge of the bottom plate, then pasting the water absorption pad on the protective film, pushing in a soft and uniform rolling way, and covering the water absorption pad on the nitrocellulose membrane by 2 mm.
(2) Sample pad pasting: cutting the first sample pad into pieces with width of 8mm and length of 300mm, tearing off the protective film at the lower edge of the nitrocellulose membrane, sticking the first sample pad on the protective film, pushing in a soft and uniform rolling manner, and covering the first sample pad on the nitrocellulose membrane by 2 mm; cutting the second sample pad into a size of 20mm in width and 300mm in length, tearing off the protective film at the lower edge of the nitrocellulose membrane, adhering the second sample pad to the lower part of the first sample pad, pushing in a soft and uniform rolling manner, and covering the second sample pad on the first sample pad by 2 mm;
(3) cutting and clamping the test strip: after the large plate is formed, a Shanghai gold mark ZQ2002 slitter is used for cutting the large plate into 4mm test strips, and each test strip is put into a plastic card shell to form a detection card, which is also called an immunochromatographic test strip.
(4) And (4) sealing and storing: and (3) placing each detection card and each centrifuge tube in an aluminum foil packaging bag, adding 1g of drying agent for 1 bag, sealing by heat seal, and storing at room temperature to complete the assembly of the AFP fluorescent microsphere immunochromatography kit.
2.5 preparation of AFP fluorescent microsphere immunochromatography kit standard curve
And testing an AFP work calibrator with gradient concentration by using the assembled AFP fluorescent microsphere immunochromatographic kit, establishing a T/C-concentration standard curve by using the obtained series T/C signal ratio and corresponding concentration, and storing the curve in an ID chip.
3. The using method of the AFP fluorescent microsphere immunochromatography kit comprises the following steps:
(1) starting up the detection instrument, inserting a chip with the same batch number as the reagent, and reading a standard curve in the chip;
(2) tearing the outer package of the kit, taking out the detection card and the centrifuge tube with the microsphere solid carrier, precisely sucking 120 mu L serum/plasma or whole blood sample by a liquid transfer device, adding the serum/plasma or whole blood sample into the centrifuge tube, slightly blowing and hitting the liquid transfer device up and down for several times, and then taking 70 mu L of the uniformly mixed sample solution into the sample adding hole of the detection kit.
(3) And after the reaction is carried out for 10min at room temperature, placing the detection card into a card slot of an instrument for testing, using an immunoassay instrument to calculate a T/C signal value by collecting strip fluorescence signals of a detection line (T) and a quality control line (C), and displaying a result.
The obtained kit is used for respectively detecting two sample solutions (respectively marked as a quality control product 1 and a quality control product 2) with different concentrations, and the results of imprecision tests are obtained after 10 times of tests, and are shown in table 2;
the stability of the obtained kit is accelerated at 37 ℃, one kit is periodically taken to respectively detect two sample solutions (respectively marked as a quality control product 1 and a quality control product 2) with different concentrations, and the stability test result is obtained, and the result is shown in table 3.
In order to prove the effect of the technical scheme provided by the invention, experimental comparison is carried out, and the precision and the stability are mainly compared.
Comparative example 1.1: the labeled antibody is sprayed on the bonding pad according to the same parameters as the example 1, the material of the bonding pad is the same as the material of the solid phase carrier in the example 1, and the bonding pad is dried for 4 hours at 37 ℃. The immunochromatographic test paper is prepared by a conventional structure, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on a PVC (polyvinyl chloride) bottom plate, two 70 microliter sample solutions (respectively marked as a quality control product 1 and a quality control product 2) with different concentrations are respectively added into a sample adding hole of a detection box for testing during testing, and imprecision results are obtained after 10 times of testing, and the results are shown in Table 1.
In comparison with example 1, comparative example 1.1 does not contain a conjugate solid phase carrier, but has a plurality of binding pads, wherein the conjugate solid phase carrier and the binding pads are prepared in the same manner, and the rest of the structure and the steps are the same as those of example 1.
Results of imprecision testing of example 1 and comparative example 1.1 are shown in table 2:
TABLE 2
It can be seen that the imprecision of example 1 is significantly reduced compared to comparative example 1.1, indicating that the assay results are more accurate using the protocol of example 1.
Comparative example 1.2: diluting the conjugate, namely the marked microspheres to the concentration of two hundred thousand parts by using a diluent, subpackaging each tube by adopting a centrifuge tube with 50 mu L, wherein the test strip is formed by combining a first sample pad, a second sample pad, a nitrocellulose membrane and a water absorption pad, firstly sucking two sample solutions (respectively marked as a quality control product 1 and a quality control product 2) with different concentrations with 50 mu L into the centrifuge tube containing the conjugate during testing, and respectively adding two sample solutions with 70 mu L after uniformly mixing into a sample adding hole of a detection box for testing after uniformly mixing. And the stability is accelerated at 37 ℃, and the stability result is obtained by periodic test, and the result is shown in table 2.
Compared with example 1, the conjugate in comparative example 1.2 is diluted and not sprayed on a solid phase carrier, but kept in a centrifuge tube in a liquid state, and the rest of the structure and the steps are the same as those in example 1.
The results of the stability tests of example 1 and comparative example 1.2 are shown in table 3:
table 3 units: ng/mL
It can be seen that the results of example 1 are substantially stable over time, while the deviation of the test results of comparative example 1.2 is more obvious, indicating that the kit of example 1 can maintain stable performance over long-term storage.
Example 2 CEA Quantum dot fluorescent microsphere immunoassay kit
1. The CEA quantum dot fluorescent microsphere immunoassay kit has the structure that:
the kit comprises an immunochromatographic test strip and a conjugate solid-phase carrier, wherein the immunochromatographic test strip comprises a bottom plate, a first sample pad, a second sample pad, a nitrocellulose membrane and a water absorption pad are sequentially connected to the bottom plate, a CEA antibody is coated on a T line position of the nitrocellulose membrane, and a goat anti-mouse IgG is coated on a C line position of the nitrocellulose membrane; the label in the conjugate solid phase carrier is quantum dot fluorescent microsphere, the ligand is CEA antibody, the solid phase carrier is glass fiber, and the conjugate solid phase carrier is placed in a plastic tube.
2. Preparing a CEA quantum dot fluorescent microsphere immunoassay kit:
reagents and consumables:
TABLE 4
Solution preparation:
0.025M HEPES buffer: 5.96g of HESPES is weighed into 900mL of ultrapure water, is completely dissolved and is adjusted to the required pH value, and then the volume is determined to be 1L to be used as microsphere coupling buffer.
0.025M MES buffer: weighing 5.33g of MES in 900mL of ultrapure water, adjusting the pH value to 5.0 after completely dissolving, and fixing the volume to 1L; the method is used for washing and activating the microspheres.
EDC solution: 10mg/mL, dissolved in 0.25M MES buffer, pH 5.0;
NHS solution: 10mg/mL, dissolved in 0.25M MES buffer, pH 5.0;
0.01M PBS buffer: 0.24g KH was weighed out2PO4，1.44gNa2HPO4After 8g of NaCl and 0.2g of KCl are dissolved completely in 900mL of ultrapure water, the pH value is adjusted to the required pH value, and then the volume is adjusted to 1L by using a volumetric flask.
0.02M PB buffer: first, 1L of 0.2M Na is prepared2HPO4·12H2O solution and 1L of 0.2M NaH2PO4Solution, 1L 0.2M Na2HPO4·12H2Solution O: 71.64g of Na were weighed2HPO4·12H2O, constant volume is 1L, 1L of 0.2M NaH is prepared2PO4Solution: weighing 24g NaH2PO4The volume is constant to 1L; according to different pH value requirements, two solutions with certain volumes are respectively taken, 800mL of ultrapure water is added, HCl or NaOH is used for fine adjustment to the required pH value, and the volume is adjusted to 1L.
0.01M Tris-HCl buffer: 1.21g of the suspension is weighed into 900mL of ultrapure water, completely dissolved, adjusted to the desired pH value, and then made to volume of 1L.
0.025M Tris-HCl buffer: 3.03g of the suspension is weighed into 900mL of ultrapure water, is adjusted to the required pH value after being completely dissolved, and then is added to 1L.
Microsphere sealing liquid: 0.02M PB, 5% BSA, pH 7.4;
microsphere preserving fluid: 0.01M Tris-HCl, 0.9% NaCl, 0.2% Tween-20, 0.05% Proclin-300, pH8.0;
diluting the microspheres: 0.025M Tris-HCl, 1% BSA, 0.5% Tween-20, 0.5% PVP, 10% D-trehalose, 15% sucrose, 0.05% Proclin-300, 2mg/mL HBR-1 blocker, pH 9.0.
2.1 preparation of labeled antibody solid phase Carrier
Preparation of microsphere labeled antibody
The microsphere labeling process was substantially the same as in example 1, except that the buffer used for coupling was HEPES buffer and the coupling antibody was CEA-labeled antibody.
② solidification of antibody-labeled substance
Diluting the marked microspheres to 0.04 per mill (solid content of microspheres W/V) with diluent, soaking the Oslon 6614 polyester film in the diluent microsphere solution at room temperature for 1h, drying at 37 ℃ for 4h, cutting into square blocks of 3mm × 3mm, and packaging each block in a plastic tube.
2.2 preparation of the first and second sample pad
The procedure was as in example 1.
2.3 preparation of coating film
Coating buffer solution: 0.01M PBS, 1.5% D-trehalose, pH 7.4;
② coating buffer solution to dilute the anti-CEA antibody and the goat anti-mouse IgG to 1.2 mg/mL;
thirdly, after the nitrocellulose membrane is adhered to a PVC bottom plate, a gold spraying instrument with a Shanghai gold mark HM3230 film scratching function is used for coating the CEA coated antibody and the goat anti-mouse IgG antibody solution on the nitrocellulose membrane at the positions of the T line and the C line, the spraying amount is 1 mu L/cm, the distance between the T line and the C line is 4mm, and the nitrocellulose membrane is dried for 4 hours at 37 ℃.
2.4 assembly of CEA quantum dot fluorescent microsphere immunochromatography kit
The procedure was as in example 1.
2.5 preparation of standard curve of CEA quantum dot fluorescent microsphere immunochromatography kit
The calibration process is basically the same as that of example 1, except that the working calibrator used for calibration is a CEA working calibrator.
3. The specific use method of the CEA quantum dot fluorescent microsphere immunochromatography kit comprises the following steps:
the procedure was as in example 1.
In order to prove the effect of the technical scheme provided by the invention, experimental comparison is carried out, and the precision and the stability are mainly compared.
Comparative example 2.1:
the procedure was as in comparative example 1.1.
In comparison with example 2, comparative example 2.1 does not contain a conjugate solid phase carrier, but has a plurality of binding pads, wherein the conjugate solid phase carrier and the binding pads are prepared in the same manner, and the rest of the structure and the steps are the same as those of example 2.
Results of imprecision testing for example 2 and comparative example 2.1 are shown in table 5:
TABLE 5
It can be seen that the imprecision of example 2 is significantly reduced compared to comparative example 2.1, indicating that the assay results are more accurate using the protocol of example 2.
Comparative example 2.2:
the procedure was as in comparative example 1.2.
Compared with example 2, the conjugate in comparative example 2.2 is diluted and not sprayed on a solid phase carrier, but kept in a centrifuge tube in a liquid state, and the rest of the structure and the steps are the same as those in example 2.
The results of the stability tests of example 2 and comparative example 2.2 are shown in table 6:
TABLE 6
It can be seen that the results of example 2 remained substantially stable over time, while the deviation of the test results of comparative example 2.2 was significant, indicating that the kit of example 2 maintained stable performance over long-term storage.
Example 3PCT fluorescent microsphere immunoassay kit
1. PCT colloidal gold immunoassay kit structure:
the kit comprises an immunochromatographic test strip and a conjugate solid-phase carrier, wherein the immunochromatographic test strip comprises a bottom plate, a first sample pad, a nitrocellulose membrane and a water absorption pad are sequentially connected to the bottom plate, a PCT antibody is coated on a T line position of the nitrocellulose membrane, and a rabbit anti-chicken IgY antibody is coated on a C line position of the nitrocellulose membrane; the label in the solid phase carrier of the conjugate is green fluorescent microspheres, the ligand is PCT antibody and chicken IgY, the solid phase carrier is glass fiber, and the solid phase carrier of the conjugate is placed in a centrifugal tube.
2. Preparing a PCT fluorescent microsphere immunoassay kit:
reagents and consumables:
TABLE 7
Solution preparation:
the solution formulation was the same as in example 2.
2.1 preparation of labeled antibody solid phase Carrier
Preparation of microsphere labeled antibody
The microsphere labeling procedure was substantially the same as in example 1, except that HEPES buffer was used as the buffer for coupling, and the coupling of the antibody PCT-mAb2 and the chicken IgY to the microspheres was performed separately.
② solidification of antibody-labeled substance
Diluting the marked PCT-mAb2 antibody-microsphere conjugate to 0.5 per thousand (microsphere solid content W/V) by using a microsphere diluent, diluting the marked chicken IgY-microsphere conjugate to 0.25 per thousand (microsphere solid content W/V), mixing the two according to a ratio of 1:1, spraying the mixture to glass fiber 8964 with the size of 6mm 300mm according to the spraying amount of 2 mu L/cm by using a Shanghai gold mark HM3230 film-cutting gold spraying instrument, drying the mixture for 4 hours at 37 ℃, cutting the mixture into 6mm 4mm square small blocks, and packaging each small block in a centrifugal tube.
2.2 preparation of the first sample pad
The first pad treatment solution was the same as in example 1, and the procedure was the same.
2.3 preparation of coating film
Coating buffer solution: 0.01M Na2HPO412H2O, 0.9% NaCl, 0.3% D-trehalose, 0.1% sodium azide, pH 7.4.
② coating buffer solution to dilute the PCT mAb1 antibody and the rabbit anti-chicken IgY antibody to 1 mg/mL.
Thirdly, after the nitrocellulose membrane is adhered to a PVC bottom plate, a PCT mAb1 antibody and rabbit anti-chicken IgY antibody solution are coated on the positions of a T line and a C line on the nitrocellulose membrane by using a Shanghai gold mark HM3230 film-cutting gold spraying instrument, the spraying amount is 1.0 mu L/cm, the distance between the T line and the C line is 4mm, and the nitrocellulose membrane is dried for 4 hours at 37 ℃.
2.4 PCT fluorescent microsphere immunoassay kit assembly
The assembly procedure was substantially the same as in examples 1 and 2, except that the PCT strip contained only the first sample pad, which was adhered under the nitrocellulose membrane for 2mm overlap.
2.5 preparation of standard curve of PCT fluorescent microsphere immunoassay kit
The calibration process is basically the same as that of embodiments 1 and 2, and the difference is that the working calibrator used for calibration is a CEA working calibrator.
3. The specific use method of the PCT fluorescent microsphere immunoassay kit comprises the following steps:
(1) starting up the detection instrument, and inserting a chip with the same batch number as the reagent;
(2) tearing the outer package of the kit, taking out the detection card and the plastic tube with the microsphere solid carrier, precisely sucking 100 mu L of sample solution by a liquid transfer device, adding the sample solution into the plastic tube, slightly blowing and striking the sample solution up and down by the liquid transfer device, uniformly mixing, and taking 70 mu L of the uniformly mixed sample solution into the sample adding hole of the detection card.
(3) And after the reaction is carried out for 10min at room temperature, placing the detection card into a card slot of an instrument for testing, using an immunoassay instrument to calculate a T/C signal value by collecting fluorescence signals of a detection line (T) and a quality control line (C), and displaying a result.
In order to prove the effect of the technical scheme provided by the invention, experimental comparison is carried out, and the precision and the stability are mainly compared.
Comparative example 3.1:
the procedure was as in comparative example 1.1.
In comparison with example 3, comparative example 3.1 does not contain a conjugate solid phase carrier, but has a plurality of binding pads, wherein the conjugate solid phase carrier and the binding pads are prepared in the same manner, and the rest of the structure and the steps are the same as those of example 3.
Results of imprecision testing for example 3 and comparative example 3.1 are shown in table 8:
TABLE 8
It can be seen that the imprecision of example 3 is significantly reduced compared to comparative example 3.1, indicating that the assay results are more accurate using the protocol of example 3.
Comparative example 3.2:
the difference from comparative example 1.2 is that the accelerated stability at 50 ℃ was compared and the other operation was the same as in comparative example 1.2.
Compared with example 3, the conjugate in comparative example 3.2 is diluted and not sprayed on a solid phase carrier, but kept in a centrifuge tube in a liquid state, and the rest of the structure and the steps are the same as those in example 1.
The results of the stability tests of example 3 and comparative example 3.2 are shown in table 9:
TABLE 9
It can be seen that the results of example 3 remained substantially stable over time, while the deviation of the test results of comparative example 3.2 was significant, indicating that the kit of example 3 maintained stable performance over long-term storage.
Example 4 folate fluoroimmunoassay kit
1. The folic acid fluorescence immunoassay kit comprises:
the kit comprises an immunochromatographic test strip and a conjugate solid-phase carrier, wherein the immunochromatographic test strip comprises a bottom plate, a first sample pad, a nitrocellulose membrane and a water absorption pad are sequentially connected to the bottom plate, a folate-BSA protein complex is coated on a T line position of the nitrocellulose membrane, and a rabbit anti-chicken IgY antibody is coated on a C line position of the nitrocellulose membrane; the label in the solid phase carrier of the conjugate is europium ion fluorescent microspheres, the ligand is a compound of goat anti-mouse IgG-mouse anti-folic acid monoclonal antibodies and chicken IgY, wherein the monoclonal antibodies are purified and mixed monoclonal antibodies and are derived from monoclonal antibody cell strains aiming at 2-6 different folic acid epitopes, the solid phase carrier is glass fiber, and the solid phase carrier of the conjugate is placed in a centrifugal tube.
2. Preparing a folic acid fluorescence immunoassay kit:
reagents and consumables:
watch 10
Solution preparation:
the solution formulation was the same as in examples 2 and 3.
2.1 preparation of labeled antibody solid phase Carrier
Preparation of microsphere labeled antibody
The labeling method of the chicken IgY antibody marker is the same as that of example 1.
The steps of the labeling method of the compound of goat anti-mouse IgG and mouse anti-folic acid monoclonal antibody are the same as example 1, but the difference is that in the labeling process, firstly, microspheres and goat anti-mouse IgG react according to the mass ratio of 5:1, after the microspheres react for 2 hours in a closed manner, the goat anti-mouse IgG and mouse anti-folic acid monoclonal antibody are added according to the mass ratio of 10:1, and the reaction is carried out for 30 minutes, so that the compound of the goat anti-mouse IgG and mouse anti-folic acid monoclonal antibody marked by the fluorescent microspheres is formed.
② solidification of antibody-labeled substance
Diluting the marked chicken IgY marker to 0.5 per mill (microsphere solid content W/V) by using a diluent, diluting the marker of the compound of goat anti-mouse IgG and mouse anti-folic acid monoclonal antibody to 1 per mill (microsphere solid content W/V), mixing the two at a ratio of 1:1, spraying the mixture onto 6mm x 300mm Oslon 8950 glass fiber according to the spraying amount of 3 mu L/cm by using a Shanghai gold mark HM3230 film-cutting gold spraying instrument, drying the mixture at 37 ℃ for 4h, cutting the dried mixture into 4mm x 6mm square small blocks, and respectively packaging each small block into a centrifuge tube.
2.2 preparation of the first sample pad
The first pad treatment solution was the same as in example one and prepared in the same manner as in example 1.
2.3 preparation of coating film
Coating buffer solution: 0.01M PB, 1.5% D-trehalose, pH 7.4.
② coating buffer solution to dilute the folic acid-BSA protein compound and the rabbit anti-chicken IgY antibody to 1 mg/mL.
Thirdly, after the nitrocellulose membrane is adhered to a PVC bottom plate, a Shanghai gold mark HM3230 membrane scratching and gold spraying instrument is used for coating the folic acid-BSA protein compound and the rabbit anti-chicken IgY antibody solution on the positions of the T line and the C line on the nitrocellulose membrane, the spraying amount is 0.5 mu L/cm, the distance between the T line and the C line is 5mm, and the nitrocellulose membrane is dried for 4 hours at 37 ℃.
2.4 Folic acid fluorescence immunoassay kit Assembly
The assembly procedure was the same as in example 3.
2.5 preparation of Standard Curve of Folic acid fluorescence immunoassay kit
The calibration process was substantially the same as in example 1, except that the working calibrant used for calibration was a folic acid working calibrant.
3. The specific use method of the folic acid fluorescence immunoassay kit comprises the following steps:
the procedure was as in example 1.
In order to prove the effect of the technical scheme provided by the invention, experimental comparison is carried out, and the precision and the stability are mainly compared.
Comparative example 4.1:
the procedure was as in comparative example 1.1.
In comparison with example 4, comparative example 4.1 does not contain a conjugate solid phase carrier, but has a plurality of binding pads, wherein the conjugate solid phase carrier and the binding pads are prepared in the same manner, and the rest of the structure and the steps are the same as those of example 4.
Results of imprecision testing for example 4 and comparative example 4.1 are shown in table 11:
TABLE 11
It can be seen that the imprecision of example 4 is significantly reduced compared to comparative example 4.1, indicating that the assay results are more accurate using the protocol of example 4.
Comparative example 4.2:
the procedure was as in comparative example 1.2.
Compared with example 4, the conjugate in comparative example 4.2 is diluted and not sprayed on a solid phase carrier, but kept in a centrifuge tube in a liquid state, and the rest of the structure and the steps are the same as those in example 1.
The results of the stability tests of example 4 and comparative example 4.2 are shown in table 12:
TABLE 12
It can be seen that the results of example 4 remained substantially stable over time, while the deviation of the test results of comparative example 4.2 was significant, indicating that the kit of example 4 maintained stable performance over long-term storage.
Claims (10)
1. An immunoassay kit, comprising:
1) the conjugate carrier comprises a ligand, a marker and a solid phase carrier, wherein the ligand is used for performing specific reaction with an analyte to be detected, the marker is used for signal detection, the ligand is connected with the marker, and the solid phase carrier is used for carrying the ligand and the marker; and the number of the first and second groups,
2) the immunochromatography test strip comprises a first sample pad, a nitrocellulose membrane, a water absorption pad and a bottom plate, wherein the first sample pad, the nitrocellulose membrane and the water absorption pad are sequentially arranged on the bottom plate along the liquid chromatography direction;
wherein, the conjugate carrier and the immunochromatographic test strip are arranged in a split mode.
2. The immunoassay kit of claim 1, wherein the label is any one of time-resolved fluorescent microspheres, ordinary fluorescent microspheres, magnetic microspheres, colloidal gold, colored latex microspheres, quantum dots, time-resolved fluorescent dyes, ordinary fluorescein, enzymes, biotin-avidin, and microsphere amplification systems.
3. The immunoassay kit according to claim 1, wherein the solid support is any one of a glass fiber, a polyester film and a nonwoven fabric.
4. The immunoassay kit of claim 1, wherein the immunochromatographic test strip further comprises a second sample pad disposed between the first sample pad and the nitrocellulose membrane.
5. The immunoassay kit of claim 1, wherein the conjugate carrier is stored and transported at ambient temperature in a sealed chamber.
6. The immunoassay kit of claim 5, wherein the chamber is any one of a centrifuge tube, a plastic tube, or a microplate.
7. An immunochromatographic assay method using the immunoassay kit according to any one of claims 1 to 6, comprising the steps of:
dissolving the conjugate: adding the sample solution into a conjugate carrier, dissolving the conjugate and fully reacting with the sample solution to obtain a mixed solution;
dropwise adding the mixed solution: taking the mixed solution fully reacted in the step, and dripping the mixed solution on a first sample pad in the immunochromatography test strip;
and (3) detection: and collecting signals of the T line and the C line by using an immunoassay instrument, calculating a T/C signal value, and obtaining the concentration of the analyte from the calibration curve.
8. The immunochromatographic assay method according to claim 7, further comprising, before the step of dissolving the conjugate:
obtaining a calibration curve: and taking standard sample curves of the analyte with different concentrations to obtain corresponding T line and C line signals, establishing a standard curve of the analyte concentration corresponding to the T line and C line signals to obtain a calibration curve, and storing the calibration curve in the ID chip.
9. A method of making an immunoassay kit according to any of claims 1 to 6, comprising the steps of:
s100, preparing a conjugate carrier;
s200, preparing immunochromatographic test paper;
s300, packaging an immunodetection kit;
in step S100, preparing a conjugate carrier includes:
s110, combining the ligand and the marker to obtain a conjugate;
s120, preparing a diluent, and dispersing the obtained conjugate in the diluent;
s130, fixing the diluent in a solid phase carrier in a spraying or smearing mode, and drying to obtain a conjugate carrier;
s140, cutting the conjugate carrier into fixed sizes and subpackaging the conjugate carrier in sealed chambers.
10. The method of preparing an immunoassay kit according to claim 9, wherein the mass concentration of the conjugate in the diluent is 0.05 to 0.5; the diluent comprises buffer pairs, proteins, saccharides, high polymers, a blocking agent, a preservative and a surfactant.
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