CN113049835A - Combined detection kit, detection method and immunoassay system - Google Patents
Combined detection kit, detection method and immunoassay system Download PDFInfo
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- CN113049835A CN113049835A CN201911376725.XA CN201911376725A CN113049835A CN 113049835 A CN113049835 A CN 113049835A CN 201911376725 A CN201911376725 A CN 201911376725A CN 113049835 A CN113049835 A CN 113049835A
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/54—Interleukins [IL]
- G01N2333/5412—IL-6
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- G—PHYSICS
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- G01N2333/575—Hormones
- G01N2333/585—Calcitonins
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Abstract
The application discloses a combined detection kit, a detection method and an immunoassay system, wherein the combined detection kit comprises a capture antibody composition, a detection antibody composition and a marker, the capture antibody composition comprises a first solid phase carrier, a first capture antibody connected with the first solid phase carrier, a second solid phase carrier and a second capture antibody connected with the second solid phase carrier; the detection antibody composition comprises a first detection antibody and a second detection antibody; the marker comprises a first marker and a second marker; wherein the first solid phase carrier is different from the second solid phase carrier and is used for distinguishing a complex formed by connecting with the first solid phase carrier and a complex formed by connecting with the second solid phase carrier during detection. By means of the mode, the method and the device can realize simultaneous detection of procalcitonin and interleukin 6 in the sample to be detected, so that the detection efficiency is improved.
Description
Technical Field
The application relates to the technical field of immunoassay, in particular to a combined detection kit, a detection method and an immunoassay system.
Background
Healthy human blood only contains a small amount of Procalcitonin (PCT), and the PCT can be obviously increased after bacterial infection, and is mainly used for auxiliary diagnosis of bacterial infectious diseases clinically; interleukin-6 (IL-6) is a pleiotropic cytokine with many functions, after a single endotoxin stimulation, IL-6 is the marker of the earliest rising, can play a role in early warning to bacterial infection, mainly used for monitoring the immune state, inflammatory response, etc. of the organism clinically.
There are many ways for detecting PCT and IL-6, but the combined detection of two items cannot be realized in a single channel, if the results of two inflammation indexes of PCT and IL-6 are referred to, more sample size and detection time are consumed, the cost is increased, and simultaneously, a larger burden is brought to hospitals and patients.
Disclosure of Invention
The technical problem mainly solved by the application is to provide a joint detection kit, a detection method and an immunoassay system, which can realize the simultaneous detection of procalcitonin and interleukin 6 in a sample to be detected so as to improve the detection efficiency.
In order to solve the technical problem, the application adopts a technical scheme that: providing a procalcitonin and interleukin 6 combined detection kit, wherein the combined detection kit comprises a capture antibody composition, a detection antibody composition and a marker; the capture antibody composition comprises a first solid phase carrier and a first capture antibody connected with the first solid phase carrier, and a second solid phase carrier and a second capture antibody connected with the second solid phase carrier; the detection antibody composition comprises a first detection antibody and a second detection antibody; the label comprises a first label and a second label; wherein the first capture antibody is adapted to bind to a first epitope of procalcitonin in the test sample, the first detection antibody is adapted to bind to a second epitope of procalcitonin in the test sample, and the first label is adapted to label the first detection antibody to form a first test complex of first solid support-first capture antibody-procalcitonin-first detection antibody-first label; the second capture antibody is used for being combined with a first epitope of interleukin 6 in a sample to be detected, the second detection antibody is used for being combined with a second epitope of interleukin 6 in the sample to be detected, and the second marker is used for marking the second detection antibody so as to form a second complex to be detected of a second solid phase carrier, the second capture antibody, the interleukin 6, the second detection antibody and the second marker; wherein the first solid phase carrier is different from the second solid phase carrier and is used for distinguishing a complex formed by connecting with the first solid phase carrier and a complex formed by connecting with the second solid phase carrier during detection.
In order to solve the above technical problem, another technical solution adopted by the present application is: an immunoassay system is provided, the immunoassay system comprising an immunoassay device and a combined detection kit as described above.
In order to solve the above technical problem, another technical solution adopted by the present application is: the joint detection method for procalcitonin and interleukin 6 is provided, and comprises the following steps: adding a capture antibody composition into a sample to be detected, performing first incubation, and removing a supernatant, wherein the capture antibody composition comprises a first solid phase carrier, a first capture antibody connected with the first solid phase carrier, a second solid phase carrier and a second capture antibody connected with the second solid phase carrier, after the first incubation, at least part of the first capture antibody is combined with a first epitope of procalcitonin in the sample to be detected, and at least part of the second capture antibody is combined with a first epitope of interleukin 6 in the sample to be detected; adding a detection antibody composition to the sample to be detected after the first incubation, and removing the supernatant after a second incubation, wherein the detection antibody composition comprises a first detection antibody and a second detection antibody, at least part of the first detection antibody is combined with a second epitope of procalcitonin connected with the first capture antibody after the second incubation, and at least part of the second detection antibody is combined with a second epitope of interleukin 6 connected with the second capture antibody; adding a marker into the sample to be detected after the second incubation, performing a third incubation, and removing the supernatant to obtain a composition to be detected, wherein the marker comprises a first marker and a second marker, after the third incubation, at least a part of the first marker is connected with the first detection antibody, so as to obtain a first complex to be detected of the first solid phase carrier-the first capture antibody-procalcitonin-the first detection antibody-the first marker, and at least a part of the second marker is connected with the second detection antibody, so as to obtain a second complex to be detected of the second solid phase carrier-the second capture antibody-interleukin 6-the second detection antibody-the second marker; and detecting the composition to be detected so as to respectively obtain detection results of the procalcitonin and the interleukin 6 in a sample to be detected, wherein the first solid phase carrier is different from the second solid phase carrier so as to distinguish a complex formed by connecting with the first solid phase carrier and a complex formed by connecting with the second solid phase carrier during detection.
In order to solve the above technical problem, another technical solution adopted by the present application is: the joint detection method for procalcitonin and interleukin 6 is characterized by comprising the following steps: adding a capture antibody composition into a sample to be detected, performing first incubation, and removing a supernatant, wherein the capture antibody composition comprises a first solid phase carrier, a first capture antibody connected with the first solid phase carrier, a second solid phase carrier and a second capture antibody connected with the second solid phase carrier, after the first incubation, at least part of the first capture antibody is combined with a first epitope of procalcitonin in the sample to be detected, and at least part of the second capture antibody is combined with a first epitope of interleukin 6 in the sample to be detected; adding a detection composition into the sample to be detected after the first incubation, and removing the supernatant after the second incubation to obtain a composition to be detected, wherein the detection composition comprises a first detection antibody and a first marker connected with the first detection antibody, and a second detection antibody, namely a second marker connected with the second detection antibody, after the second incubation, at least part of the first detection antibody is combined with a second epitope of procalcitonin connected with the first capture antibody, so that a first complex to be detected of a first solid phase carrier, a first capture antibody, procalcitonin, the first detection antibody and the first marker is obtained, and at least part of the second detection antibody is combined with a second epitope of interleukin 6 connected with the second capture antibody, so that a second solid phase carrier, a second capture antibody, interleukin 6, a second detection antibody and a second marker are obtained A complex to be tested; and detecting the composition to be detected so as to respectively obtain detection results of the procalcitonin and the interleukin 6 in a sample to be detected, wherein the first solid phase carrier is different from the second solid phase carrier so as to distinguish a complex formed by connecting with the first solid phase carrier and a complex formed by connecting with the second solid phase carrier during detection.
The beneficial effect of this application is: different from the prior art, the first solid phase carrier and the second solid phase carrier are different in the application, so that when the detection composition is detected, a complex formed by connecting the first solid phase carrier and the second solid phase carrier is distinguished, and a signal of a first complex to be detected and a signal of a second complex to be detected are obtained by further combining the markers of the first marker and the second marker, so that the procalcitonin and the interleukin 6 in a sample to be detected can be detected simultaneously and accurately, and the detection efficiency is improved.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts. Wherein:
FIG. 1 is a schematic diagram of the structure of an embodiment of the immunoassay system of the present application;
FIG. 2 is a schematic flow chart of an embodiment of the combined detection method for procalcitonin and interleukin 6 of the present application;
FIG. 3 is a schematic flow chart of another embodiment of the combined detection method for procalcitonin and interleukin 6 according to the present application;
FIG. 4 shows the procalcitonin detection results of the Roche electrochemiluminescence method under the same conditions and the test kit of the first embodiment;
FIG. 5 shows the results of detecting interleukin 6 by Roche electrochemiluminescence under the same conditions and in the kit of the first embodiment;
FIG. 6 shows the procalcitonin detection results of the reagent kit of the second embodiment and the Roche electrochemiluminescence method under the same conditions;
FIG. 7 shows the results of detecting interleukin 6 by Roche electrochemiluminescence under the same conditions and the test kit of example II;
FIG. 8 shows the results of procalcitonin detection by Roche electrochemiluminescence under the same conditions in the test kit of the third example;
FIG. 9 shows the results of detection of interleukin 6 by Roche electrochemiluminescence under the same conditions in the third kit of example.
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
The application provides a procalcitonin and interleukin 6 joint detection kit.
Among them, procalcitonin consists of 116 amino acids, has a molecular weight of about 12.7kDa, and is expressed by neuroendocrine cells (including C cells of thyroid, lung and pancreatic tissues), and healthy human blood contains only a small amount of procalcitonin. The increased level of procalcitonin is seen in bacterial sepsis, especially severe sepsis and septic shock, and is mainly used for auxiliary diagnosis of bacterial infectious diseases clinically.
Interleukin-6 is a multifunctional pleiotropic cytokine, and is a protein composed of 184 amino acids, and has a molecular weight of 21.5-28 kDa. In acute inflammatory response to trauma, stress, infection, tumor and other diseases, rapid IL-6 production is induced. After single endotoxin stimulation, interleukin-6 is the earliest rising marker, can play a role in early warning to bacterial infection, can be used for evaluating the severity and prognosis of systemic inflammatory response syndrome, septicemia and septic shock, and is mainly used for monitoring the immune state, inflammatory response and the like of an organism clinically.
In one embodiment, the combination test kit comprises a capture antibody composition, a detection antibody composition, and a label.
The capture antibody composition may include a first solid phase carrier and a first capture antibody linked to the first solid phase carrier, and a second solid phase carrier and a second capture antibody linked to the second solid phase carrier, the detection antibody composition may include a first detection antibody and a second detection antibody, and the label may include a first label and a second label.
Specifically, the first capture antibody and the first detection antibody are both procalcitonin antibodies, such as at least one of a goat anti-human procalcitonin antibody, a rabbit anti-human procalcitonin antibody, and a mouse anti-human procalcitonin antibody, wherein the two antibodies can be specifically bound with different epitopes of procalcitonin respectively.
The second capture antibody and the second detection antibody are both interleukin 6 antibodies, such as at least one of goat anti-human interleukin 6 antibodies, rabbit anti-human interleukin 6 antibodies, and mouse anti-human interleukin 6 antibodies, wherein the two antibodies can be specifically bound with different epitopes of interleukin 6 in a sample to be detected respectively.
In one application scenario, the joint detection kit includes a first containing location, a second containing location, and a third containing location, wherein the capture antibody composition, the detection antibody composition, and the marker are respectively contained in the first containing location, the second containing location, and the third containing location.
When the combined detection kit is used for detecting a sample to be detected, the capture antibody composition contained in the first containing position is mixed with the sample to be detected, and incubation is carried out, so that the first capture antibody is specifically combined with a first epitope of procalcitonin in the sample to be detected, and the second capture antibody is specifically combined with a first epitope of interleukin 6 in the sample to be detected; after the incubation is finished and the supernatant is removed, further adding a detection antibody composition accommodated in a second accommodating position, and incubating so that the first detection antibody is specifically combined with a second epitope of procalcitonin and the second detection antibody is specifically combined with a second epitope of interleukin 6; after the incubation is finished and the supernatant is removed, the label accommodated in the third accommodating position is further added, the incubation is carried out, so that the first label is combined with the first detection antibody to form a first to-be-detected compound of a sandwich structure of the first solid phase carrier-the first capture antibody-procalcitonin-the first detection antibody-the first label, the second label is combined with the second detection antibody to form a second to-be-detected compound of a sandwich structure of the second solid phase carrier-the second capture antibody-interleukin 6-the second detection antibody-the second label, the to-be-detected composition is obtained, and the to-be-detected composition is further detected to obtain a detection result.
In another application scenario, the joint detection kit includes a fourth holding position and a fifth holding position, wherein the capture antibody composition is held in the fourth holding position, the detection antibody composition and the marker are held in the fifth holding position together, the first detection antibody is connected to the first marker, and the second detection antibody is connected to the second marker.
When the combined detection kit is used for detecting a sample to be detected, the capture antibody composition contained in the fourth containing position is mixed with the sample to be detected, and incubation is carried out, so that the first capture antibody is specifically combined with the first epitope of procalcitonin in the sample to be detected, and the second capture antibody is specifically combined with the first epitope of interleukin 6 in the sample to be detected; after the incubation is finished and the supernatant is removed, the detection antibody composition and the marker accommodated in the fifth accommodation position are further added, and the incubation is performed, so that the first detection antibody is specifically combined with the second epitope of procalcitonin to form a first to-be-detected compound of a sandwich structure of the first solid phase carrier, the first capture antibody, the procalcitonin, the first detection antibody and the first marker, and the second detection antibody is specifically combined with the second epitope of interleukin 6 to form a second to-be-detected compound of a sandwich structure of the second solid phase carrier, the second capture antibody, interleukin 6, the second detection antibody and the second marker, so that the to-be-detected composition is obtained, and the to-be-detected composition is further detected to obtain a detection result.
The first label and the second label may be the same or different, and specifically, a fluorescent label may be used, such as at least one of Phycoerythrin (PE), Fluorescein Isothiocyanate (FITC), Tetramethylrhodamine isothiocyanate (Tetramethylrhodamine), cyanine dyes (e.g., Cy2, Cy3), etc., although other labels may be used in other embodiments, which are not limited herein.
Because the first marker and the second marker adopt fluorescent markers, when the composition to be detected is detected, the fluorescent signals emitted by the corresponding fluorescent markers can be collected, and then the fluorescent signals are processed and analyzed.
It should be noted that the first solid phase carrier is different from the second solid phase carrier, so that when the detection composition is detected, the complex formed by connecting the first solid phase carrier and the complex formed by connecting the second solid phase carrier are distinguished, and the signals of the first complex to be detected and the second complex to be detected are further obtained by combining the labels of the first label and the second label, respectively, thereby being capable of realizing the simultaneous and accurate detection of procalcitonin and interleukin 6 in a sample to be detected, so as to improve the detection efficiency.
In an embodiment, the first solid phase carrier is a first fluorescence-encoded magnetic bead, and the second solid phase carrier is a second fluorescence-encoded magnetic bead, and specifically, both the first fluorescence-encoded magnetic bead and the second fluorescence-encoded magnetic bead may be a magnetic organic polymer nanoparticle, such as a magnetic polystyrene fluorescent nanoparticle. Further, the average particle size of the first fluorescent-encoded magnetic bead and the average particle size of the second fluorescent-encoded magnetic bead both satisfy 3 μm to 8 μm, specifically, 3 μm, 4 μm, 6 μm, 8 μm, etc., and it should be noted that the average particle size of the first fluorescent-encoded magnetic bead and the average particle size of the second fluorescent-encoded magnetic bead both satisfy the above range, but the average particle sizes are not limited to be the same.
Specifically, in one application scenario, the first fluorescent encoded magnetic bead and the second fluorescent encoded magnetic bead have the same average particle size, but have different fluorescent codes, respectively. Wherein, the difference of the fluorescence codes can be realized by coupling different kinds of fluorescent substances on the magnetic beads and/or coupling the fluorescent substances with different intensities.
When a sample to be detected is detected, the complex formed by connecting the first fluorescent encoding magnetic bead and the second fluorescent encoding magnetic bead and the complex formed by connecting the second fluorescent encoding magnetic bead can be distinguished through the difference of the fluorescent colors and/or the difference of the fluorescent intensities of the first fluorescent encoding magnetic bead and the second fluorescent encoding magnetic bead.
In another application scenario, the first fluorescent encoded magnetic bead and the second fluorescent encoded magnetic bead have the same fluorescent code but different average particle sizes.
In the application scenario, when a sample to be detected is detected, a complex formed by connecting the fluorescent coding beads with the first fluorescent coding beads and a complex formed by connecting the fluorescent coding beads with the second fluorescent coding beads can be distinguished by identifying the average particle size of the fluorescent coding beads.
Further, it should be noted that after the sample to be tested is processed by the reagent in the above-mentioned combined test kit and the composition to be tested is obtained, the test composition can be further tested by a fluorescence immunoassay device, such as a flow cytometer. Specifically, after the composition to be detected enters the flow chamber, further under the wrapping of the sheath fluid flow, the first compound to be detected, the second compound to be detected and the like in the composition to be detected, which are connected with the first fluorescent encoding magnetic bead and the second fluorescent encoding magnetic bead, can be singly arranged in a row and sequentially pass through the detection area, a fluorescent signal is generated under the irradiation of laser emitted by the laser, the fluorescent detector further collects the fluorescent signal and processes and analyzes the fluorescent signal, specifically, the fluorescent signal is divided into two paths, one path of the fluorescent signal is used for identifying the types of procalcitonin and interleukin 6 in the sample to be detected through the difference between the first fluorescent encoding magnetic bead and the second fluorescent encoding magnetic bead, the other path of the fluorescent signal is used for simultaneously detecting the intensities of the first fluorescent marker and the second fluorescent marker, and the content of procalcitonin and interleukin 6 in the sample to be detected is further obtained through integration treatment, thereby realizing the quantitative joint detection of procalcitonin and interleukin 6 in the sample to be detected.
In one embodiment, the first solid support has a first functional group on its surface and is covalently coupled to the first capture antibody via the first functional group; the second solid support has a second functional group on its surface and is covalently coupled to the second capture antibody via the second functional group.
Specifically, the first functional group and the second functional group may each include at least one of a carboxyl group, a hydroxyl group, an amino group, a tosyl group, a chloromethyl group, a mercapto group, an aldehyde group, a hydrazide, a silicon hydroxyl group, a succinimide ester, and an epoxy group, and are not particularly limited herein. The first functional group and the second functional group may be the same or different.
In one embodiment, the first detection antibody is a procalcitonin detection antibody modified with one of biotin and avidin and correspondingly the first label is a fluorescent label modified with the other of biotin and avidin.
Correspondingly, the second detection antibody is an interleukin 6 detection antibody modified by one of biotin and avidin, and correspondingly, the second marker is a fluorescent marker modified by the other of biotin and avidin.
It should be noted that avidin is a glycoprotein, which can be extracted from egg white, and each avidin molecule is composed of 4 subunits, which can be tightly bound to 4 biotin molecules. Although the combination of avidin and biotin is not immune reaction, it has strong specificity and high affinity, and once combined, the two are very stable. Since 1 avidin molecule has 4 binding sites for biotin molecules, more biotinylated molecules can be attached to form a lattice-like complex.
Therefore, after the first detection antibody, the second detection antibody, the first marker and the second marker are modified by biotin or avidin, the first marker and the first detection antibody, and the second marker and the second detection antibody can form stable complexes respectively, so that the first detection antibody is labeled by the first marker, and the second detection antibody is labeled by the second marker.
In one embodiment, the capture antibody composition further comprises a first buffer, the detection antibody composition further comprises a second buffer, and the label further comprises a third buffer.
The first buffer solution, the second buffer solution and the third buffer solution can comprise at least one of tris (hydroxymethyl) aminomethane-HCl buffer solution, 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution, Phosphate Buffer Solution (PBS), citric acid buffer solution, glycine buffer solution and barbiturate buffer solution.
Specifically, the pH of the first buffer, the second buffer and the third buffer can be 7.0-9.0, such as 7.0, 8.0, 9.0, etc., and the concentration can be respectively 10mmol/L-100mmol/L, such as 10mmol/L, 20mmol/L, 40mmol/L, 60mmol/L, 80mmol/L, 100mmol/L, etc. It should be noted that the components, pH values and concentrations of the first buffer, the second buffer and the third buffer may be the same or different, and are not limited herein.
Further, in one embodiment, the first buffer, the second buffer, and the third buffer may further include a stabilizer, an inorganic salt, a surfactant, a preservative, and the like.
Wherein, the stabilizer can comprise at least one of sucrose, trehalose, glycerol, mannitol, polyethylene glycol, sorbitol, ethylene diamine tetraacetic acid disodium, gelatin, bovine serum albumin, casein and the like, wherein the mass percentage of the stabilizer is 0.1-5%, specifically 0.1%, 0.5%, 1%, 3%, 5% and the like.
The inorganic salt may include at least one of sodium chloride, potassium chloride, calcium chloride, ammonium chloride, magnesium chloride, sodium sulfate, potassium sulfate, etc., wherein the inorganic salt is 0.5-2% by mass, specifically, 0.5%, 1%, 1.5%, 2%, etc.
The surfactant may comprise at least one of tween 20, polyvinylpyrrolidone, triton X-100, etc., wherein the surfactant is 0.05-0.2 wt%, specifically 0.05%, 0.1%, 0.15%, 0.2%, etc.
The preservative may comprise at least one of sodium azide, thimerosal, Proclin-300, etc., wherein the preservative is 0.05-0.2% by mass, such as 0.05%, 0.1%, 0.15%, 0.2% by mass, etc.
Referring to fig. 1, fig. 1 is a schematic diagram of an immunoassay system according to an embodiment of the present invention. In this embodiment, the immunoassay system 100 may include a combined detection kit 10 and an immunoassay device 20.
The combined detection kit 10 and the immunoassay device 20 in this embodiment are the same as those in the above-mentioned combined detection kit for procalcitonin and interleukin 6, and the details thereof are not repeated herein.
Referring to fig. 2, fig. 2 is a schematic flow chart of an embodiment of the method for jointly detecting procalcitonin and interleukin 6 according to the present application. In this embodiment, the joint detection method may include:
step S11: adding a capture antibody composition into a sample to be detected, performing first incubation, and removing supernatant, wherein the capture antibody composition comprises a first solid phase carrier, a first capture antibody connected with the first solid phase carrier, a second solid phase carrier and a second capture antibody connected with the second solid phase carrier, after the first incubation, at least part of the first capture antibody is combined with a first epitope of procalcitonin in the sample to be detected, and at least part of the second capture antibody is combined with a first epitope of interleukin 6 in the sample to be detected;
step S12: adding a detection antibody composition into the sample to be detected after the first incubation, and removing the supernatant after the second incubation, wherein the detection antibody composition comprises a first detection antibody and a second detection antibody, after the second incubation, at least part of the first detection antibody is combined with a second epitope of procalcitonin connected with the first capture antibody, and at least part of the second detection antibody is combined with a second epitope of interleukin 6 connected with the second capture antibody;
step S13: adding a marker into the sample to be detected after the second incubation, performing third incubation, and removing the supernatant to obtain a composition to be detected, wherein the marker comprises a first marker and a second marker, at least part of the first marker is connected with a first detection antibody after the third incubation, so that a first complex to be detected of a first solid phase carrier, a first capture antibody, procalcitonin, the first detection antibody and the first marker is obtained, and at least part of the second marker is connected with a second detection antibody, so that a second complex to be detected of the second solid phase carrier, the second capture antibody, interleukin 6, the second detection antibody and the second marker is obtained;
step S14: and detecting the composition to be detected to respectively obtain the detection results of procalcitonin and interleukin 6 in the sample to be detected, wherein the first solid phase carrier is different from the second solid phase carrier so as to distinguish a complex formed by connecting with the first solid phase carrier and a complex formed by connecting with the second solid phase carrier during detection.
Wherein, the supernatant liquid removing mode in each step can be carried out by a magnetic separation mode, and after the supernatant liquid is removed, a buffer solution can be further added for washing.
Further, before the test of the test composition in step S14, a certain amount of buffer solution may be added to the test composition, and the test composition may be shaken and then tested on an immunoassay device, such as a flow cytometer.
The various substances involved in the combined detection method for procalcitonin and interleukin 6 in the embodiment are the same as those in the embodiment of the combined detection kit for procalcitonin and interleukin 6 in the application, and for the relevant details, reference is made to the above embodiment, which is not described herein again.
It should be noted that, in the above manner, the content of procalcitonin and interleukin 6 can be detected simultaneously by using one sample to be detected, and the amount of the sample and the time required for detection can be saved, thereby improving the detection efficiency.
Referring to fig. 3, fig. 3 is a schematic flow chart of another embodiment of the method for jointly detecting procalcitonin and interleukin 6 according to the present application. In this embodiment, the joint detection method may include:
step S21: adding a capture antibody composition into a sample to be detected, performing first incubation, and removing supernatant, wherein the capture antibody composition comprises a first solid phase carrier, a first capture antibody connected with the first solid phase carrier, a second solid phase carrier and a second capture antibody connected with the second solid phase carrier, after the first incubation, at least part of the first capture antibody is combined with a first epitope of procalcitonin in the sample to be detected, and at least part of the second capture antibody is combined with a first epitope of interleukin 6 in the sample to be detected;
step S22: adding the detection composition into the sample to be detected after the first incubation, removing the supernatant after the second incubation to obtain the composition to be detected, wherein the detection composition comprises a first detection antibody and a first marker linked to the first detection antibody, and a second detection antibody, i.e. a second label linked to the second detection antibody, at least part of the first detection antibody binding to a second epitope of procalcitonin linked to the first capture antibody after the second incubation, thereby obtaining a first complex to be detected of the first solid support-the first capture antibody-procalcitonin-the first detection antibody-the first marker, at least part of the second detection antibody binding to a second epitope of interleukin 6 linked to the second capture antibody, thereby obtaining a second to-be-detected compound of a second solid phase carrier, a second capture antibody, interleukin 6, a second detection antibody and a second marker;
step S23: and detecting the composition to be detected to respectively obtain the detection results of procalcitonin and interleukin 6 in the sample to be detected, wherein the first solid phase carrier is different from the second solid phase carrier so as to distinguish a complex formed by connecting with the first solid phase carrier and a complex formed by connecting with the second solid phase carrier during detection.
Wherein, the supernatant liquid removing mode in each step can be carried out by a magnetic separation mode, and after the supernatant liquid is removed, a buffer solution can be further added for washing.
Further, before the test of the test composition in step S23, a certain amount of buffer solution may be added to the test composition, and the test composition may be shaken and then tested on an immunoassay device, such as a flow cytometer.
The various substances involved in the combined detection method for procalcitonin and interleukin 6 in the embodiment are the same as those in the embodiment of the combined detection kit for procalcitonin and interleukin 6 in the application, and for the relevant details, reference is made to the above embodiment, which is not described herein again.
It should be noted that, in the above manner, the content of procalcitonin and interleukin 6 can be detected simultaneously by using one sample to be detected, and the amount of the sample and the time required for detection can be saved, thereby improving the detection efficiency.
The above embodiments of the present application are specifically illustrated by the following specific examples.
Example one
Preparation of capture antibody composition
1) Selecting two microspheres with the particle sizes of 5.5 microns and 8 microns, wherein the 5.5 micron microspheres coat the mouse anti-human PCT antibody, and the 8 micron microspheres coat the mouse anti-human IL-6 antibody;
2) carrying out magnetic separation on 1mg of magnetic fluorescent coding microspheres by using a magnetic separation plate, and sucking supernatant;
3) 1mL of 2-morpholinoethanesulfonic acid (MES) buffer, 50mM, pH 6.0, was added, the microspheres were vortexed for 20s, magnetic separation was performed using a magnetic separation plate, and the supernatant was aspirated. This step was repeated three times;
4) adding 2mL of the MES buffer solution, performing vortex oscillation on the microspheres for 20s, adding 10 mu L of 50mg/mL N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution dissolved in MES into the microsphere solution, performing oscillation incubation for 20min at room temperature, performing magnetic separation by using a magnetic separation plate, and sucking a supernatant;
5) adding 20 mu g of corresponding antibody and 2mL of MES buffer solution, carrying out vortex oscillation on the microspheres for 20s, and then oscillating for 3.0 hours at room temperature;
6) performing magnetic separation by using a magnetic separation plate, and sucking supernatant;
7) taking 1mL of MES buffer solution, carrying out vortex oscillation on the microspheres for 20s, carrying out magnetic separation by using a magnetic separation plate, and sucking supernatant. This step was repeated three times;
8) adding 10 mu g of blocking agent Bovine Serum Albumin (BSA), vortexing the microspheres for 20s, and then vortexing at room temperature for 3.0 hours to block the microspheres;
9) magnetic separation was performed with a magnetic separation plate, and the supernatant was aspirated.
10) Washing with phosphate buffer (PBS 10mM, containing 0.05% Tween 20) of pH 8.0, vortexing for 20s, magnetic separation with magnetic separation plate, and aspirating the supernatant. This step was repeated three times;
11) adding the coated coded magnetic fluorescent microspheres into a container according to the proportion of 1:1, diluting with the phosphate buffer solution, wherein the final concentration of each microsphere is 0.2mg/mL, and storing at 2-8 ℃ in a dark place.
Preparation of detection antibody composition
1) Add 50. mu.g of the two detection antibodies to PCT and IL-6, respectively, to 100. mu.L of 50mM NaHCO3 solution, pH 8.3, vortexed for 20 s;
2) adding 5 μ L of 5mg/mL biotin succinimidyl ester (NHS-Biotion) to the antibody solution, vortexing for 20s, and incubating in an electric-heating incubator for 0.5 h;
3) the biotin-modified antibodies were added to a container at a ratio of 1:1, diluted with PBS-TBN buffer pH 8.0 to a final concentration of 0.5. mu.g/mL, vortexed for 5 seconds and stored at 2-8 ℃.
Preparation of the marker
SA-PE was diluted to 0.5. mu.g/mL with PBS-TBN buffer and stored at 2-8 ℃.
And respectively accommodating the prepared capture antibody composition, the prepared detection antibody composition and the prepared marker in different accommodating positions of the kit to obtain the procalcitonin and interleukin 6 combined detection kit.
Example two
Preparation of capture antibody composition
1) Selecting two coded magnetic fluorescent microspheres with different codes, and respectively coating a mouse anti-human PCT antibody and a mouse anti-human IL-6 antibody;
2) carrying out magnetic separation on 0.5mg of magnetic fluorescent coding microspheres by using a magnetic separation plate, and sucking supernatant;
3) 1mL of 20mM N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid (HEPES) buffer, pH 7.0 was added, the microspheres were vortexed for 20 seconds, magnetic separation was performed using a magnetic separation plate, and the supernatant was aspirated. This step was repeated three times;
4) adding 1mL of the HEPES buffer solution, performing vortex oscillation on the microspheres for 20s, adding 5 mu L of 50mg/mL N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution dissolved in the HEPES buffer solution into the microsphere solution, adding 20 mu g of corresponding antibody into the solution, performing oscillation incubation at room temperature for 3.0 hours, performing magnetic separation by using a magnetic separation plate, and sucking a supernatant;
5) washing was performed by adding 1mL of phosphate buffered saline (PBS 10mM, containing 0.05% Tween 20) at pH 8.0, vortexing the microspheres for 20s, magnetic separation was performed using a magnetic separation plate, and the supernatant was aspirated. This step was repeated three times;
6) adding 1mL of the phosphate buffer solution, adding 10 mu g of sealant casein, carrying out vortex oscillation on the microspheres for 20s, and then carrying out oscillation at room temperature for 6.0 hours for sealing;
7) magnetic separation was performed with a magnetic separation plate, and the supernatant was aspirated.
8) Washing with phosphate buffer (PBS 10mM, containing 0.05% Tween 20) of pH 8.0, vortexing for 20s, magnetic separation with magnetic separation plate, and aspirating the supernatant. This step was repeated three times;
9) adding the coated coded magnetic fluorescent microspheres into a container according to the ratio of 1:1, diluting with phosphate buffer solution (PBS 10mM, containing 0.05% Tween 20) with pH 7.4, wherein the final concentration of each microsphere is 0.2mg/mL, and storing at 2-8 ℃ in a dark place;
10) adding a certain amount of the phosphate buffer solution, and storing at 2-8 ℃ in a dark place.
Preparation of detection antibody composition
1) Mu.g of the two detection antibodies for PCT and IL-6 were added to 50. mu.L of 50mM NaHCO, pH 8.3, respectively3In the solution, vortex and shake for 20 s;
2) adding 3 μ L of 5mg/mL biotin succinimidyl ester (NHS-Biotion) into the antibody solution, vortexing for 20s, and standing and incubating for 0.5h in an electric-heating constant-temperature incubator;
3) the biotin-modified antibodies were added to a container at a ratio of 1:1, diluted with phosphate buffer pH 7.4pH 7.4 (PBS 10mM, containing 0.05% Tween 20) to a final concentration of 0.01mg/mL, vortexed for 5s, and stored at 2-8 ℃.
Preparation of the marker
SA-PE was diluted to 1. mu.g/mL with a citric acid buffer solution of pH 5.5 and stored at 2-8 ℃.
And respectively accommodating the prepared capture antibody composition, the prepared detection antibody composition and the prepared marker in different accommodating positions of the kit to obtain the procalcitonin and interleukin 6 combined detection kit.
EXAMPLE III
Preparation of capture antibody composition
1) Selecting two coded magnetic fluorescent microspheres with different codes, and respectively coating a sheep anti-human PCT antibody and a rabbit anti-human IL-6 antibody;
2) carrying out magnetic separation on 1.0mg of magnetic fluorescent coding microspheres by using a magnetic separation plate, and sucking supernatant;
3) add 1mL of 20mM, pH 8.0 boric acid (BS) buffer, vortex the microspheres for 20s, magnetically separate with a magnetic separation plate, and aspirate the supernatant. This step was repeated three times;
4) adding 2mL of the boric acid buffer solution, performing vortex oscillation on the microspheres for 20s, adding 5 mu L of 100mg/mL N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution dissolved in the boric acid buffer solution into the microsphere solution, adding 20 mu g of corresponding antibody into the solution, performing oscillation incubation at room temperature for 3.0 hours, performing magnetic separation by using a magnetic separation plate, and sucking a supernatant;
5) washing was performed by adding 1mL of phosphate buffered saline (PBS 10mM, containing 0.05% Tween 20) at pH 8.0, vortexing the microspheres for 20s, magnetic separation was performed using a magnetic separation plate, and the supernatant was aspirated. This step was repeated three times;
6) adding 1mL of the phosphate buffer solution, adding 10 mu g of sealant casein, carrying out vortex oscillation on the microspheres for 20s, and then carrying out oscillation at room temperature for 6.0 hours for sealing;
7) magnetic separation was performed with a magnetic separation plate, and the supernatant was aspirated.
8) Washing with phosphate buffer (PBS 10mM, containing 0.05% Tween 20) of pH 8.0, vortexing for 20s, magnetic separation with magnetic separation plate, and aspirating the supernatant. This step was repeated three times;
9) adding the coated coded magnetic fluorescent microspheres into a container according to the ratio of 1:1, diluting with phosphate buffer solution (PBS 10mM, containing 0.05% Tween 20) with pH 7.4, wherein the final concentration of each microsphere is 0.2mg/mL, and storing at 2-8 ℃ in a dark place;
10) adding a certain amount of the phosphate buffer solution, and storing at 2-8 ℃ in a dark place.
Preparation of detection antibody composition
1) Mu.g of the two detection antibodies for PCT and IL-6 were added to 50. mu.L of 50mM NaHCO, pH 8.3, respectively3In the solution, vortex and shake for 20 s;
2) adding 3 mu L of 5mg/mL biotin succinimidyl ester (NHS-Biotion) into the antibody solution, vortexing for 20s, and standing and incubating for 1h in an electric-heating constant-temperature incubator;
3) the biotin-modified antibodies were added to a container at a ratio of 1:1, diluted with phosphate buffer pH 7.4pH 7.4 (PBS 10mM, containing 0.05% Tween 20) to a final concentration of 0.01mg/mL, vortexed for 5s, and stored at 2-8 ℃.
Preparation of the marker
SA-FITC was diluted to 1. mu.g/mL with phosphate buffer pH 8.0 (PBS 10mM, containing 0.05% Tween 20), and stored at 2-8 ℃.
And respectively accommodating the prepared capture antibody composition, the prepared detection antibody composition and the prepared marker in different accommodating positions of the kit to obtain the procalcitonin and interleukin 6 combined detection kit.
The kit prepared by the above embodiments is used for detecting procalcitonin and interleukin 6 in a sample to be detected:
1) adding 50 μ L of capture antibody composition and 25 μ L of sample to be tested into a 96-well plate, shaking for 15s, incubating at 37 deg.C for 10min, performing magnetic separation with a magnetic plate, and removing supernatant.
2) An amount of phosphate buffer pH 8.0 (PBS 10mM, containing 0.05% Tween 20) was added, shaken for 15s, subjected to magnetic separation with a magnetic plate, and the supernatant was removed.
3) Adding labeled 50 μ L detection antibody composition, shaking for 15s, incubating at 37 deg.C for 6min, performing magnetic separation with magnetic plate, and removing supernatant.
4) Adding a certain amount of the phosphate buffer solution, shaking for 15s, performing magnetic separation by using a magnetic plate, and removing supernatant.
5) Adding 50 μ L of labeled substance, shaking for 15s, incubating at 37 deg.C for 6min, performing magnetic separation with magnetic plate, and removing supernatant.
6) Adding a certain amount of the phosphate buffer solution, shaking for 15s, performing magnetic separation by using a magnetic plate, and removing supernatant.
7) Adding a certain amount of the phosphate buffer solution, shaking for 15s, and detecting the median value of the PE-H channel on a flow cytometer. Calibration was performed using standards, two calibration curves were made for PCT and IL-6, and PCT and IL-6 concentrations were calculated from the standard curves.
The kits prepared in the above examples were compared with the kits PCT and IL-6 by roche, and specifically, the kits prepared in the above examples one, two, and three were tested on NovoCyte 2040R by the above detection method, respectively. Wherein, table 1 and fig. 4-5 are the results of the kit prepared in the first embodiment for multiple determination of procalcitonin and interleukin 6 in different samples to be tested, and the comparative analysis of the results of the determination by the roche electrochemical luminescence method under the same conditions; table 2 and FIGS. 6-7 show the results of the multiple measurements of procalcitonin and interleukin 6 in different samples and the comparative analysis of the results of the Roche electrochemiluminescence measurements under the same conditions with the kit prepared in example II; table 3 and FIGS. 8-9 show the results of the multiple measurements of procalcitonin and interleukin 6 in different samples and the comparative analysis of the results of the Roche electrochemiluminescence measurements under the same conditions with the kit prepared in the third example.
TABLE 1
TABLE 2
TABLE 3
From the above detection results, it can be seen that the two items detected by the combined detection kit for testing procalcitonin and interleukin 6 prepared according to the first, second and third embodiments of the present application have better correlation with the results of the Roche electrochemiluminescence detection, and the results of the three embodiments are r2The accuracy of the reagent kit in the first, second and third embodiments of the application is reliable, the correlation is good, and the reagent kit can be used for combined detection of procalcitonin and interleukin 6.
The above description is only for the purpose of illustrating embodiments of the present application and is not intended to limit the scope of the present application, and all modifications of equivalent structures and equivalent processes, which are made by the contents of the specification and the drawings of the present application or are directly or indirectly applied to other related technical fields, are also included in the scope of the present application.
Claims (14)
1. A procalcitonin and interleukin 6 joint detection kit is characterized in that the joint detection kit comprises a capture antibody composition, a detection antibody composition and a marker;
the capture antibody composition comprises a first solid phase carrier and a first capture antibody connected with the first solid phase carrier, and a second solid phase carrier and a second capture antibody connected with the second solid phase carrier; the detection antibody composition comprises a first detection antibody and a second detection antibody; the label comprises a first label and a second label;
wherein the first capture antibody is adapted to bind to a first epitope of procalcitonin in the test sample, the first detection antibody is adapted to bind to a second epitope of procalcitonin in the test sample, and the first label is adapted to label the first detection antibody to form a first test complex of first solid support-first capture antibody-procalcitonin-first detection antibody-first label; the second capture antibody is used for being combined with a first epitope of interleukin 6 in a sample to be detected, the second detection antibody is used for being combined with a second epitope of interleukin 6 in the sample to be detected, and the second marker is used for marking the second detection antibody so as to form a second complex to be detected of a second solid phase carrier, the second capture antibody, the interleukin 6, the second detection antibody and the second marker;
wherein the first solid phase carrier is different from the second solid phase carrier and is used for distinguishing a complex formed by connecting with the first solid phase carrier and a complex formed by connecting with the second solid phase carrier during detection.
2. The joint detection kit according to claim 1,
the first solid phase carrier is a first fluorescence coding magnetic bead, and the second solid phase carrier is a second fluorescence coding magnetic bead;
the first fluorescent encoding magnetic bead and the second fluorescent encoding magnetic bead have the same average particle size and different fluorescent codes respectively, or
The first fluorescent-encoded magnetic bead and the second fluorescent-encoded magnetic bead have the same fluorescent code and different average particle sizes.
3. The joint detection kit according to claim 2,
the first fluorescent coding magnetic bead and the second fluorescent coding magnetic bead are both magnetic polystyrene fluorescent nano-microspheres;
the average particle size of the first fluorescent coding magnetic bead and the average particle size of the second fluorescent coding magnetic bead are both 3-8 mu m.
4. The joint detection kit according to claim 1,
the first solid phase support has a first functional group on its surface and is covalently coupled to the first capture antibody via the first functional group,
the second solid phase support has a second functional group on the surface thereof and is covalently coupled to the second capture antibody via the second functional group,
wherein the first functional group and the second functional group each include at least one of a carboxyl group, a hydroxyl group, an amino group, a tosyl group, a chloromethyl group, a mercapto group, an aldehyde group, a hydrazide, a silicon hydroxyl group, a succinimide ester, and an epoxy group.
5. The joint detection kit according to claim 1,
the first capture antibody and the first detection antibody are at least one of a goat anti-human procalcitonin antibody, a rabbit anti-human procalcitonin antibody and a mouse anti-human procalcitonin antibody;
the second capture antibody and the second detection antibody are at least one of a goat anti-human interleukin 6 antibody, a rabbit anti-human interleukin 6 antibody and a mouse anti-human interleukin 6 antibody.
6. The joint detection kit according to claim 1, wherein the joint detection kit comprises a first holding position, a second holding position and a third holding position, wherein the capture antibody composition, the detection antibody composition and the label are respectively held in the first holding position, the second holding position and the third holding position.
7. The joint detection kit according to claim 6,
the first detection antibody is a procalcitonin detection antibody modified by one of biotin and avidin, and the first marker is a fluorescent marker modified by the other of biotin and avidin;
the second detection antibody is an interleukin 6 detection antibody modified by one of biotin and avidin, and the second marker is a fluorescent marker modified by the other of biotin and avidin.
8. The joint detection kit according to claim 7,
the fluorescent marker is at least one of phycoerythrin, fluorescein isothiocyanate, tetramethyl rhodamine isothiocyanate and cyanine dyes.
9. The joint detection kit according to claim 1, wherein the joint detection kit comprises a fourth holding position and a fifth holding position, wherein the capture antibody composition is held in the fourth holding position, the detection antibody composition and the label are held together in the fifth holding position, and the first detection antibody is linked to the first label and the second detection antibody is linked to the second label.
10. The joint detection kit according to claim 1,
the capture antibody composition further comprises a first buffer, the detection antibody composition further comprises a second buffer, and the label further comprises a third buffer;
wherein the first buffer solution, the second buffer solution and the third buffer solution respectively comprise at least one of tris (hydroxymethyl) aminomethane-HCl buffer solution, 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution, phosphate buffer solution, citric acid buffer solution, glycine buffer solution and barbiturate buffer solution,
the pH values of the first buffer solution, the second buffer solution and the third buffer solution are respectively 7.0-9.0, and the concentrations are respectively 10-100 mmol/L.
11. The joint detection kit according to claim 10,
the first buffer, the second buffer and the third buffer each further comprise at least one of a stabilizer, an inorganic salt, a surfactant and a preservative;
wherein the stabilizer comprises at least one of sucrose, trehalose, glycerol, mannitol, polyethylene glycol, sorbitol, disodium ethylene diamine tetraacetate, gelatin, bovine serum albumin and casein, and the mass percentage of the stabilizer is 0.1-5%;
the inorganic salt comprises at least one of sodium chloride, potassium chloride, calcium chloride, ammonium chloride, magnesium chloride, sodium sulfate and potassium sulfate, and the mass percentage of the inorganic salt is 0.5-2%;
the surfactant comprises at least one of Tween 20, polyvinylpyrrolidone and Triton X-100, and the mass percentage of the surfactant is 0.05% -0.2%;
the preservative comprises at least one of sodium azide, thimerosal and Proclin-300, and the mass percentage of the preservative is 0.05-0.2%.
12. An immunoassay system comprising an immunoassay device and a combined test kit according to any one of claims 1 to 11.
13. A combined detection method for procalcitonin and interleukin 6 is characterized by comprising the following steps:
adding a capture antibody composition into a sample to be detected, performing first incubation, and removing a supernatant, wherein the capture antibody composition comprises a first solid phase carrier, a first capture antibody connected with the first solid phase carrier, a second solid phase carrier and a second capture antibody connected with the second solid phase carrier, after the first incubation, at least part of the first capture antibody is combined with a first epitope of procalcitonin in the sample to be detected, and at least part of the second capture antibody is combined with a first epitope of interleukin 6 in the sample to be detected;
adding a detection antibody composition to the sample to be detected after the first incubation, and removing the supernatant after a second incubation, wherein the detection antibody composition comprises a first detection antibody and a second detection antibody, at least part of the first detection antibody is combined with a second epitope of procalcitonin connected with the first capture antibody after the second incubation, and at least part of the second detection antibody is combined with a second epitope of interleukin 6 connected with the second capture antibody;
adding a marker into the sample to be detected after the second incubation, performing a third incubation, and removing the supernatant to obtain a composition to be detected, wherein the marker comprises a first marker and a second marker, after the third incubation, at least a part of the first marker is connected with the first detection antibody, so as to obtain a first complex to be detected of the first solid phase carrier-the first capture antibody-procalcitonin-the first detection antibody-the first marker, and at least a part of the second marker is connected with the second detection antibody, so as to obtain a second complex to be detected of the second solid phase carrier-the second capture antibody-interleukin 6-the second detection antibody-the second marker;
and detecting the composition to be detected so as to respectively obtain detection results of the procalcitonin and the interleukin 6 in a sample to be detected, wherein the first solid phase carrier is different from the second solid phase carrier so as to distinguish a complex formed by connecting with the first solid phase carrier and a complex formed by connecting with the second solid phase carrier during detection.
14. A combined detection method for procalcitonin and interleukin 6 is characterized by comprising the following steps:
adding a capture antibody composition into a sample to be detected, performing first incubation, and removing a supernatant, wherein the capture antibody composition comprises a first solid phase carrier, a first capture antibody connected with the first solid phase carrier, a second solid phase carrier and a second capture antibody connected with the second solid phase carrier, after the first incubation, at least part of the first capture antibody is combined with a first epitope of procalcitonin in the sample to be detected, and at least part of the second capture antibody is combined with a first epitope of interleukin 6 in the sample to be detected;
adding a detection composition into the sample to be detected after the first incubation, and removing the supernatant after the second incubation to obtain a composition to be detected, wherein the detection composition comprises a first detection antibody and a first marker connected with the first detection antibody, and a second detection antibody, namely a second marker connected with the second detection antibody, after the second incubation, at least part of the first detection antibody is combined with a second epitope of procalcitonin connected with the first capture antibody, so that a first complex to be detected of a first solid phase carrier, a first capture antibody, procalcitonin, the first detection antibody and the first marker is obtained, and at least part of the second detection antibody is combined with a second epitope of interleukin 6 connected with the second capture antibody, so that a second solid phase carrier, a second capture antibody, interleukin 6, a second detection antibody and a second marker are obtained A complex to be tested;
and detecting the composition to be detected so as to respectively obtain detection results of the procalcitonin and the interleukin 6 in a sample to be detected, wherein the first solid phase carrier is different from the second solid phase carrier so as to distinguish a complex formed by connecting with the first solid phase carrier and a complex formed by connecting with the second solid phase carrier during detection.
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