CN111426842A - Novel coronavirus IgM/IgG detection reagent, reagent card, kit and preparation method thereof - Google Patents
Novel coronavirus IgM/IgG detection reagent, reagent card, kit and preparation method thereof Download PDFInfo
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- CN111426842A CN111426842A CN202010110248.9A CN202010110248A CN111426842A CN 111426842 A CN111426842 A CN 111426842A CN 202010110248 A CN202010110248 A CN 202010110248A CN 111426842 A CN111426842 A CN 111426842A
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Abstract
The invention discloses a novel coronavirus (COVID-19) IgM/IgG detection reagent, a reagent card, a kit and a preparation method thereof, wherein the novel coronavirus (COVID-19) IgM/IgG detection reagent comprises a monoclonal anti-human IgM antibody, a monoclonal anti-human IgG antibody and fluorescent microspheres for marking and recombining novel coronavirus antigens. When a human body is exposed to an antigen (novel coronavirus COVID-19), IgM antibodies are directly secreted by a B cell surface receptor, IgM-producing B cells enter lymph nodes, and are stimulated by T cells and antigen-presenting cells at the occurrence center to further mature, differentiate into plasma cells, and produce IgG in a large amount. The increase of IgM occurs after about 3-7 days of acute infection, and the increase of IgG occurs after 7-21 days, so that the method can be used for reflecting whether the body is in an acute infection state or not and is used as a main index for early diagnosis.
Description
Technical Field
The invention belongs to the technical field of virus detection, and particularly relates to a novel coronavirus (COVID-19) IgM/IgG detection reagent, a reagent card, a kit and a preparation method thereof, aiming at the novel coronavirus (COVID-19).
Background
Coronaviruses (CoV) belong to the order nidoviridae, family coronaviridae, of three genera, α and β are only pathogenic to mammals CoV is also shown to be transmitted via the fecal oral route, primarily by direct contact with secretions or via aerosols, droplets.
Structural proteins of coronaviruses generally include S protein (spike-protein), HE protein, M protein, E protein, and N protein. Among them, the S protein (and HE protein) plays a key role in the binding of virus to host cell surface receptors and in the process of mediating the fusion of virus envelope and cell membrane and entering cells, and is also the main antigen protein of coronavirus.
The detection method commonly used in clinical kit laboratories includes colloidal gold method, immunofluorescence method and the like.
Disclosure of Invention
The invention aims to provide a novel coronavirus (COVID-19) IgM/IgG detection reagent.
In order to solve the technical problems, the technical scheme adopted by the invention is that the novel coronavirus (COVID-19) IgM/IgG detection reagent comprises a monoclonal anti-human IgM antibody, a monoclonal anti-human IgG antibody and fluorescent microspheres for marking and recombining novel coronavirus antigens.
When a human body is exposed to an antigen (novel coronavirus COVID-19), IgM antibodies are directly secreted by a B cell surface receptor, IgM-producing B cells enter lymph nodes, and are stimulated by T cells and antigen-presenting cells at the occurrence center to further mature, differentiate into plasma cells, and produce IgG in a large amount. The increase of IgM occurs after about 3-7 days of acute infection, and the increase of IgG occurs after 7-21 days, so that the method can be used for reflecting whether the body is in an acute infection state or not and is used as a main index for early diagnosis.
Preferably, the monoclonal anti-human IgM antibody is a mouse anti-human IgM monoclonal antibody-Hangzhou Longji organism-MS 00704; the monoclonal anti-human IgG antibody is mouse anti-human IgG monoclonal antibody-Nanjing Kinshire organism-V90401; the Recombinant novel coronavirus antigen is Recombinant 2019-nCoV N Protein (Recombinant 2019-nCoV Nucleocapsid Protein) -Nanjing Kirsi organism-T80103 and Recombinant 2019-nCoV S Protein (Recombinant 2019-nCoV Spike-RBD Protein) -Nanjing Kirsi organism-T80301.
Preferably, the novel coronavirus (COVID-19) IgM/IgG detection reagent is used in a chemiluminescent immunoassay.
Another technical problem to be solved by the present invention is to provide a novel coronavirus (COVID-19) IgM/IgG reagent card, which comprises a reagent strip and a card shell, wherein the reagent strip comprises the novel coronavirus (COVID-19) IgM/IgG detection reagent, wherein a nitrocellulose membrane is coated with a monoclonal anti-human IgM antibody, a monoclonal anti-human IgG antibody and goat anti-chicken IgY; fluorescent microspheres for marking the recombinant novel coronavirus antigen and fluorescent microspheres for marking the chicken IgY are adsorbed in the combination pads; the reagent strip further comprises a sample pad and absorbent paper, wherein the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are sequentially arranged.
The reagent card adopts an immunofluorescence method to detect the content of IgM/IgG of a new coronavirus (COVID-19). The monoclonal anti-human IgM antibody and the monoclonal anti-human IgG antibody are respectively coated on a test area on a nitrocellulose membrane, and the recombinant novel coronavirus antigen is marked into fluorescent microspheres which are fixed on a binding pad. Mixing a detection buffer solution with a sample, combining an IgM/IgG antibody in the sample with fluorescent microspheres marked by a recombinant novel coronavirus antigen in a combination pad, and capturing a complex by a monoclonal anti-human IgM antibody and a monoclonal anti-human IgG antibody which are fixed on a test area to form a fluorescent microsphere composite structure; the fluorescent particle compound marked by the chicken IgY in the combined pad is combined with the goat anti-chicken IgY fixed on the nitrocellulose membrane quality control area to form the quality control area. The complex is measured and analyzed by a matched instrument, and the condition of the new coronavirus IgM/IgG in human blood can be analyzed.
The kit is suitable for in vitro determination of the content of novel coronavirus (COVID-19) IgM/IgG in human serum, plasma and whole blood. The kit is stored at 4-30 ℃ with the validity period of 12 months; the reagent card is independently packaged, and the card strip is used within 1 hour after being unsealed.
Another technical problem to be solved by the present invention is to provide a novel coronavirus (COVID-19) IgM/IgG detection kit based on a microfluidic chip, which comprises a microfluidic chip and the novel coronavirus (COVID-19) IgM/IgG detection reagent, wherein fluorescent microspheres labeled with a recombinant novel coronavirus antigen and fluorescent microspheres labeled with chicken IgY are dried in a reaction chamber of the microfluidic chip; the monoclonal anti-human IgM antibody and the monoclonal anti-human IgG antibody are coated in the reaction cavity of the microfluidic chip.
The invention provides a novel coronavirus (COVID-19) IgM/IgG detection kit based on a microfluidic chip, which comprises the microfluidic chip and the novel coronavirus (COVID-19) IgM/IgG detection reagent, wherein the recombined novel coronavirus antigen is subjected to time-resolved fluorescence labeling and is dried in a reaction cavity of the microfluidic chip; the monoclonal anti-human IgM antibody and the monoclonal anti-human IgG antibody are fixed in the reaction cavity of the microfluidic chip through the magnetic beads.
Fixing the recombinant coronavirus antigen by using magnetic beads to form immunomagnetic beads; the monoclonal anti-human IgM antibody and the monoclonal anti-human IgG antibody are marked by time-resolved fluorescence to form a fluorescence marker, and the detection is carried out by a time-resolved fluorescence immunoassay method.
Preferably, the micro-fluidic chip is a closed micro-fluidic chip, and the closed micro-fluidic chip comprises a chip body, wherein the chip body sequentially comprises a lower chip, a middle chip and an upper chip from bottom to top; the chip body comprises a sample feeding cavity, a plurality of cavities and a micro-channel, and the middle-layer chip and the lower-layer chip are matched to define a closed micro-channel and a plurality of cavities; the sample introduction cavity is communicated with the plurality of cavities through the micro-channels; and a sealing cover is arranged on the sample injection cavity. Through set up the closing cap on advancing the appearance chamber, and not set up upper bleeder vent, the external introduction port of cooperation simultaneously seals and seals with the gas vent, makes the chip body seal to be applicable to and detect novel coronavirus, can not spill over the aerosol in the testing process, can not cause environment and personnel's virus infection risk, and the testing result rate of accuracy is high, and is with low costs, operates more portably.
Preferably, the closure cap is a one-way valve closure cap.
Preferably, the chip body further comprises an exhaust channel and a negative pressure air bag, the exhaust channel and the negative pressure air bag are arranged on the side face of the chip body, and the tail end of the exhaust channel is connected with the negative pressure air bag through a clamping joint.
Preferably, the exhaust port clamping joint is provided with a clamping joint puncturing part, an air inlet film is arranged at an air inlet of the negative pressure air bag, and the clamping joint puncturing part is used for puncturing the air inlet film of the negative pressure air bag when in use.
Preferably, the chamber comprises a reaction chamber and a waste liquid chamber; the reaction cavity comprises an upper reaction chamber arranged on the back surface of the middle chip and a lower reaction chamber arranged on the lower chip, and the position of the upper reaction chamber on the back surface of the middle chip is arranged corresponding to the position of the lower reaction chamber on the lower chip; the waste liquid cavity is arranged on the lower chip, and a middle-layer waste liquid cavity through hole is arranged on the middle chip in a penetrating manner at a position corresponding to the waste liquid cavity; and a waste liquid cavity cover plate is arranged on the back surface of the upper chip at a position corresponding to the waste liquid cavity.
Preferably, the microchannel comprises a reaction cavity input channel and a reaction cavity output channel, the reaction cavity input channel and the reaction cavity output channel are both arranged on the back surface of the middle-layer chip, the reaction cavity input channel is communicated with one end of the upper reaction chamber, and the reaction cavity output channel is communicated with the other end of the upper reaction chamber; the reaction cavity input flow channel comprises a reaction input flow channel a and a reaction input flow channel b, and the reaction input flow channel a is communicated with the sample injection cavity; a first backflow prevention structure is arranged between the reaction input flow channel a and the sample feeding cavity; an external liquid path interface is arranged on the chip body and is connected with the reaction input flow channel b through an external liquid path input flow channel; and a second backflow prevention structure is arranged between the external liquid path input flow channel and the reaction input flow channel b.
Preferably, an external liquid path sealing cover is arranged on the external liquid path interface.
Preferably, the external liquid path interface comprises an external liquid path through hole penetrating the upper chip and an external liquid path opening penetrating the front surface of the middle chip, the external liquid path input flow channel is arranged on the front surface of the lower chip, and the external liquid path opening is connected with the reaction input flow channel b through the external liquid path input flow channel.
Preferably, the chip body is provided with a groove at one side of the negative pressure air bag for embedding the negative pressure air bag. The arrangement ensures that the negative pressure air bag is directly clamped to the corresponding position of the chip when being installed.
Preferably, the sample injection cavity comprises a sample injection port arranged on the upper chip in a penetrating manner, a middle-layer liquid feeding through hole arranged at the corresponding position of the middle-layer chip in a penetrating manner, and a sample injection cavity body arranged on the front side of the lower-layer chip, wherein a middle-layer air hole is arranged above the middle-layer liquid feeding through hole in a penetrating manner.
Preferably, the first backflow prevention structure includes a first vertical flow channel a, a first vertical flow channel b and a first backflow prevention connection channel, the first backflow prevention connection channel is disposed on the back surface of the upper chip, and both the first vertical flow channel a and the first vertical flow channel b are disposed on the middle chip in a penetrating manner; an outlet of the sample feeding cavity is communicated with the reaction input flow channel a after passing through a first vertical flow channel a, a first backflow prevention connecting channel and a first vertical flow channel b of a first backflow prevention structure in sequence; the second backflow prevention structure comprises a second vertical flow channel a, a second vertical flow channel b and a second backflow prevention connecting flow channel; the second backflow-preventing connecting flow channel is arranged on the back surface of the upper chip, and the second vertical flow channel a and the second vertical flow channel b are arranged on the middle chip in a penetrating mode; and the external liquid path input flow channel is communicated with the reaction cavity input flow channel after sequentially passing through the second vertical flow channel a, the second backflow-preventing connecting flow channel and the second vertical flow channel b.
Preferably, the reaction chamber passes through in proper order reaction chamber output runner and waste liquid output microchannel with the waste liquid chamber is linked together, waste liquid output microchannel sets up the front of lower floor's chip, just waste liquid output microchannel with be equipped with the conductive rubber valve between the reaction chamber output runner, the conductive rubber valve is including setting up upper conductive rubber valve structure on the upper chip is in with setting up the middle level conductive rubber valve structure of the corresponding position department of middle level chip.
Preferably, the middle layer liquid adding through hole is fan-shaped, and a middle layer air vent is arranged above the middle layer liquid adding through hole in a penetrating manner; the appearance cavity of advancing is fan-shaped for the banana, including straining appearance pond and water conservancy diversion district, the liquid outlet setting in the narrow limit lateral wall in straining appearance pond, the water conservancy diversion district sets up strain the bottom in appearance pond, the top of advance appearance cavity is equipped with a plurality of bleeder vent, strain the position department of the bottom of the pool in appearance pond near wide lateral wall, correspond each the bleeder vent all is provided with a guide slot, guide slot with be provided with gaseous gathering area between the water conservancy diversion district.
Preferably, the flow guide area is at least divided into two flow guide areas at the bottom of the sample filtering pool according to the flow direction of the fluid, the two flow guide areas are communicated with each other, each flow guide area is provided with a plurality of flow guide strips distributed in a gathering manner, and the distribution density of the flow guide strips in the flow guide area at the front end of the fluid flow is smaller than that of the flow guide strips in the flow guide area at the rear end of the fluid flow.
Preferably, the bottom of the sample filtering pool is sequentially provided with a first flow guide area and a second flow guide area according to the flow direction of the fluid; the first diversion area comprises a plurality of first-level diversion bodies and a plurality of secondary diversion bodies, the first-level diversion bodies and the plurality of secondary diversion bodies are both ridge-shaped bulges, the cross section size of each first-level diversion body is larger than that of each secondary diversion body, the length of each first-level diversion body is consistent with that of each secondary diversion body, and the plurality of secondary diversion bodies are uniformly distributed between every two adjacent first-level diversion bodies.
Preferably, the second diversion area is arranged at a position corresponding to the first-stage diversion body and is provided with a diversion strip along the length extension direction of the first-stage diversion body, the diversion strip of the second diversion area is a rib protrusion, and the cross section size of the rib protrusion of the second diversion area is not larger than the cross section size of the first-stage diversion body.
Preferably, the number of the air guide grooves is 3, one end of each air guide groove, which is close to the side wall of the wide side of the sample filtering pool, is communicated with the air holes in a one-to-one correspondence manner, and a notch at the other end of each air guide groove is arranged and communicated with the gas polymer area.
Preferably, the number of the first flow guiding bodies in the first flow guiding zone is 3, and correspondingly, the number of the flow guiding bodies in the second flow guiding zone is 3, and the cross-sectional size of the ridge of the second flow guiding zone is the same as that of the first flow guiding bodies.
Compared with the prior art, the invention has the following advantages: adopt closing cap and external liquid circuit closing cap to seal introduction port and external liquid circuit interface, set up the gas that negative pressure gasbag collected the production in the testing process simultaneously to be applicable to novel coronavirus, make it can not spill over at the aerosol that the testing process produced.
Drawings
The following further detailed description of embodiments of the invention is made with reference to the accompanying drawings:
FIG. 1 is a schematic perspective view of a closed microfluidic chip according to the present invention;
FIG. 2 is a schematic perspective view of a closed microfluidic chip according to the present invention without a cap and a cap for an external fluid circuit;
FIG. 3 is a schematic diagram of the front structure of the lower chip of the closed microfluidic chip according to the present invention;
FIG. 4 is a schematic diagram of the back structure of the lower chip of the closed microfluidic chip according to the present invention;
FIG. 5 is a schematic diagram of the front side structure of the middle layer chip of the closed microfluidic chip according to the present invention;
FIG. 6 is a schematic diagram of the reverse structure of the middle layer chip of the closed microfluidic chip according to the present invention;
fig. 7 is a schematic structural view of the front surface of the upper chip of the closed microfluidic chip of the present invention;
fig. 8 is a schematic structural view of the reverse side of the upper chip of the closed microfluidic chip of the present invention;
FIG. 9 is a schematic diagram of the front side of the closed microfluidic chip of the present invention with the top chip mounted with a closure cap and an external liquid path closure cap;
fig. 10 is a schematic structural view of a closing cap of the closed microfluidic chip of the present invention;
fig. 11 is a schematic structural diagram of an external liquid path closing cap of the closed microfluidic chip of the present invention;
fig. 12 is a schematic structural view of a negative pressure air bag of the closed microfluidic chip of the present invention;
fig. 13 is a partial enlarged view of the closed microfluidic chip of the present invention;
wherein: 1-lower chip; 2-middle layer chip; 3-upper chip; 4-sample introduction cavity; 401-middle layer liquid adding through hole; 402-a closure cap; 403-sample introduction cavity; 404-a sample inlet; 405-middle layer air holes; 406-a sample filtration pool; 407-air holes; 5-a reaction chamber; 501-an upper reaction chamber; 502-a lower reaction chamber; 6-micro flow channel; 601-reaction chamber input runner; 6011-reaction input flow channel a; 6012-reaction input flow channel b; 602-a reaction chamber output flow channel; 603-external liquid path input flow channel; 604-waste liquid output micro flow channel; 7-negative pressure air bag; 701-a card connector; 702-a bayonet joint piercing section; 703-an air inlet film; 8-a first anti-backflow structure; 801-first anti-backflow connecting channel; 802-a first vertical flow channel a; 803-the first vertical flow channel b; 9-waste liquid chamber; 901-middle layer waste liquid cavity through hole; 902-waste chamber cover plate; 903-waste liquid backflow prevention structure; 904-middle layer conductive rubber valve structure; 905-upper conductive rubber valve structure; 10-a second anti-backflow structure; 1001-second anti-backflow connecting channel; 1002-a second vertical runner a; 1003-first vertical runner b; 11-external liquid path interface; 1101-external liquid circuit closing cover; 1102-external liquid path through hole; 1103-external liquid junction.
Detailed Description
The following is a more detailed description of embodiments of the invention:
the novel coronavirus (COVID-19) IgM/IgG detection reagent comprises a monoclonal anti-human IgM antibody, a monoclonal anti-human IgG antibody and fluorescent microspheres for marking and recombining novel coronavirus antigens.
When a human body is exposed to an antigen (novel coronavirus COVID-19), IgM antibodies are directly secreted by a B cell surface receptor, IgM-producing B cells enter lymph nodes, and are stimulated by T cells and antigen-presenting cells at the occurrence center to further mature, differentiate into plasma cells, and produce IgG in a large amount. The increase of IgM occurs after about 3-7 days of acute infection, and the increase of IgG occurs after 7-21 days, so that the method can be used for reflecting whether the body is in an acute infection state or not and is used as a main index for early diagnosis.
In this example, the monoclonal anti-human IgM antibody is murine anti-human IgM mab-hangzhou Longji biological-MS 00704; the monoclonal anti-human IgG antibody is mouse anti-human IgG monoclonal antibody-Nanjing Kinshire organism-V90401; the Recombinant novel coronavirus antigen is Recombinant 2019-nCoV N Protein (Recombinant 2019-nCoV Nucleocapsidprotein) -Nanjing Kisry organism-T80103 and Recombinant 2019-nCoV S Protein (Recombinant 2019-nCoVSpike-RBD Protein) -Nanjing Kisry organism-T80301.
The present example employs a novel coronavirus (COVID-19) IgM/IgG reagent card, which is composed of a reagent strip and a card shell, wherein the reagent strip has the aforementioned novel coronavirus (COVID-19) IgM/IgG detection reagent, and monoclonal anti-human IgM antibody, monoclonal anti-human IgG antibody and goat anti-chicken IgY are coated on a nitrocellulose membrane; fluorescent microspheres for marking the recombinant novel coronavirus antigen and fluorescent microspheres for marking the chicken IgY are adsorbed in the combination pads; the reagent strip further comprises a sample pad and absorbent paper, wherein the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are sequentially arranged.
The reagent card adopts an immunofluorescence method to detect the content of IgM/IgG of a new coronavirus (COVID-19). The monoclonal anti-human IgM antibody and the monoclonal anti-human IgG antibody are respectively coated on a test area on a nitrocellulose membrane, and the recombinant novel coronavirus antigen is marked into fluorescent microspheres which are fixed on a binding pad. Mixing a detection buffer solution with a sample, combining an IgM/IgG antibody in the sample with fluorescent microspheres marked by a recombinant novel coronavirus antigen in a combination pad, and capturing a complex by a monoclonal anti-human IgM antibody and a monoclonal anti-human IgG antibody which are fixed on a test area to form a fluorescent microsphere composite structure; the fluorescent particle compound marked by the chicken IgY in the combined pad is combined with the goat anti-chicken IgY fixed on the nitrocellulose membrane quality control area to form the quality control area. The complex is measured and analyzed by a matched instrument, and the condition of the new coronavirus IgM/IgG in human blood can be analyzed.
The kit is suitable for in vitro determination of the content of novel coronavirus (COVID-19) IgM/IgG in human serum, plasma and whole blood. The kit is stored at 4-30 ℃ with the validity period of 12 months; the reagent card is independently packaged, and the card strip is used within 1 hour after being unsealed.
The detection method comprises the following steps:
(1) preparing: before detection, the reagent card, the sample, the diluent and the like are restored to room temperature (15-30 ℃).
(2) Calibration: and opening the instrument, and importing a calibration curve in a mode of scanning the two-dimensional code of the kit to finish calibration work.
(3) And (2) adding a serum or plasma sample, namely sucking a 5 mu L sample, dropwise adding the sample to the sample adding position of the reagent card, immediately dropwise adding four drops of sample diluent (100 mu L-140 mu L), timing, adding the sample diluent into a sample diluent pipe, uniformly mixing, sucking 100 mu L, dropwise adding the sample diluent to the sample adding position of the reagent card, ensuring that no air bubbles are generated in the operation process, and timing.
(4) And (3) standing the reagent card after sample adding for 15 minutes at room temperature, and inserting the reagent card into an analyzer for detection.
(5) The analyzer performs analysis and detection and displays the result.
(6) And taking out the used reagent card.
(7) Quality control: the reagent card does not contain quality control product
The novel coronavirus (COVID-19) IgM/IgG detection reagent can also be used for chemiluminescence immunoassay.
The novel coronavirus (COVID-19) IgM/IgG detection reagent can also be used for a novel coronavirus (COVID-19) IgM/IgG detection kit based on a microfluidic chip, and comprises the microfluidic chip and the novel coronavirus (COVID-19) IgM/IgG detection reagent, wherein fluorescent microspheres for marking and recombining novel coronavirus antigens and fluorescent microspheres for marking chicken IgY are dried in a reaction cavity of the microfluidic chip; the monoclonal anti-human IgM antibody and the monoclonal anti-human IgG antibody are coated in the reaction cavity of the microfluidic chip.
The preparation method of the kit is approximately the same as that of the existing kit, and mainly comprises the following steps: (1) coating a microfluidic chip; (2) marking fluorescent microspheres; (3) assembling the microfluidic chip; (4) preparing a calibration material; (5) and obtaining the novel coronavirus (COVID-19) IgM/IgG detection kit based on the microfluidic chip.
The kit comprises a microfluidic chip and the novel coronavirus (COVID-19) IgM/IgG detection reagent, wherein the novel coronavirus (COVID-19) IgM/IgG detection reagent is recombined to obtain a novel coronavirus antigen, and the recombined novel coronavirus antigen is subjected to time-resolved fluorescence labeling and is dried in a reaction cavity of the microfluidic chip; the monoclonal anti-human IgM antibody and the monoclonal anti-human IgG antibody are fixed in the reaction cavity of the microfluidic chip through the magnetic beads.
The microfluidic chip detection method based on the magnetic bead technology comprises the following steps.
Step 5, fixing the fluorescent marker: and (3) after drying the confining liquid in the step (3), adding the fluorescent marker collected in the step (1) to a quantitative reaction chamber part of the lower chip, and then drying to fix the fluorescent marker and the lower chip.
Step 6, assembling the microfluidic chip: and sequentially assembling and combining the upper chip, the middle chip fixed with the immunomagnetic beads and the lower chip fixed with the fluorescent marker.
Step 10, developing color: starting an external liquid circuit device, entering a flow channel, entering a quantitative-reaction cavity, controlling the addition of quantitative enhancing liquid, and ultrasonically mixing for 3-8 minutes to perform color reaction.
Selecting a high-sensitivity time-resolved fluorescent substance as a marker, respectively marking the magnetic beads and the fluorescent substance with antibodies, and analyzing and detecting by using the immunoreaction of the antibodies, wherein the performance of the prepared reagent can reach the level of the same chemiluminescence reagent. Meanwhile, homogeneous reaction is adopted, and single-thread chromatographic flow reaction is avoided by ultrasonic mixing in the micro-fluidic chip cavity, so that the reaction efficiency is higher, and the reaction is more sufficient. After the immunoreaction, the immunomagnetic beads are fixed by an external magnetic field, and after the redundant components are completely removed by using cleaning solution, the developing solution is added for reading, so that the problem that the inside of the chip is difficult to clean or not thorough to clean is solved. The liquid phase reaction system makes the reaction more sufficient, and the rare earth europium fluorescent tracer and the magnetic bead amplification system are used, so that the detection sensitivity and the detection repeatability are far higher than those of a chromatography method.
The micro-fluidic chip is a closed micro-fluidic chip, and as shown in fig. 1-13, the closed micro-fluidic chip comprises a chip body, wherein the chip body sequentially comprises a lower chip 1, a middle chip 2 and an upper chip 3 from bottom to top; the chip body comprises a sample injection cavity 4, a plurality of cavities and a micro-channel 6, wherein the middle-layer chip 2 and the upper-layer chip 3 are matched to define a closed micro-channel 6 and a plurality of cavities; the sample introduction cavity 4 is communicated with a plurality of cavities through the micro-channel 6; a sealing cover 402 is arranged on the sample injection cavity 4, and the sealing cover 402 is a one-way valve sealing cover; the chip body further comprises an exhaust channel and a negative pressure air bag 7, the exhaust channel and the negative pressure air bag 7 are arranged on the side face of the chip body, and the tail end of the exhaust channel is connected with the negative pressure air bag 7 through a clamping joint 701; the clamping joint 701 is provided with a clamping joint puncturing part 702, an air inlet film 703 is arranged at an air inlet of the negative pressure air bag 7, and the clamping joint puncturing part 702 is used for puncturing the air inlet film of the negative pressure air bag 7 in use; the chamber comprises a reaction chamber 5 and a waste liquid chamber 9; the reaction chamber 5 comprises an upper reaction chamber 501 arranged on the back surface of the middle chip and a lower reaction chamber 502 arranged on the lower chip 1, and the position of the upper reaction chamber 501 on the back surface of the middle chip 2 corresponds to the position of the lower reaction chamber 502 on the lower chip 1; the waste liquid cavity 9 is arranged on the lower chip 502, and a middle waste liquid cavity through hole 901 penetrates through the middle chip 2 at a position corresponding to the waste liquid cavity 9; a waste liquid cavity cover plate 902 is arranged on the back surface of the upper chip 3 at a position corresponding to the waste liquid cavity 9; the micro flow channel 6 comprises a reaction cavity input flow channel 601 and a reaction cavity output flow channel 602, the reaction cavity input flow channel 601 and the reaction cavity output flow channel 602 are both arranged on the back surface of the middle layer chip 2, the reaction cavity input flow channel 601 is communicated with one end of the upper reaction chamber 501, and the reaction cavity output flow channel 602 is communicated with the other end of the upper reaction chamber 501; the reaction cavity input flow channel 601 comprises a reaction input flow channel a6011 and a reaction input flow channel b6012, and the reaction input flow channel a6011 is communicated with the sample injection cavity 4; a first backflow prevention structure 8 is arranged between the reaction input flow channel a6011 and the sample injection cavity 4; an external liquid path interface 11 is arranged on the chip body, and the external liquid path interface 11 is connected with the reaction input flow path b6012 through an external liquid path input flow path 603; a second backflow prevention structure 10 is arranged between the external liquid path input flow channel 603 and the reaction input flow channel b 6012; an external liquid path sealing cover 1101 is arranged on the external liquid path interface 11; the external liquid path interface 11 comprises an external liquid path through hole 1102 penetrating the upper chip 3 and an external liquid path port 1103 penetrating the front surface of the middle chip 2, the external liquid path input flow channel 603 is arranged on the front surface of the lower chip 1, and the external liquid path port 1103 is connected with the reaction input flow channel b6012 through the external liquid path input flow channel 603; the bottom of the external liquid circuit closing cover 1101 is a cone, and the top of the external liquid circuit closing cover is provided with a convex part in an outward protruding mode; a groove is formed in one side, provided with the negative pressure air bag 7, of the chip body and used for embedding the negative pressure air bag 7; the sample injection cavity 4 comprises a sample injection port 404 penetrating through the upper chip 3, a middle layer liquid adding through hole 401 penetrating through the corresponding position of the middle chip 2 and a sample injection cavity 403 arranged on the front surface of the lower chip 1, and a middle layer air vent 405 penetrates through the middle layer liquid adding through hole 401; the first backflow prevention structure 8 includes a first vertical flow channel a802, a first vertical flow channel b803, and a first backflow prevention connection channel 801, the first backflow prevention connection channel 801 is disposed on the back surface of the upper chip 3, and both the first vertical flow channel a802 and the first vertical flow channel b803 are disposed on the middle chip 2 in a penetrating manner; an outlet of the sample feeding cavity 4 is communicated with the reaction input flow channel a after passing through a first vertical flow channel a802, a first backflow-preventing connecting channel 801803 and a first vertical flow channel b of a first backflow-preventing structure in sequence; the second backflow prevention structure 10 includes a second vertical flow passage a1002, a second vertical flow passage b1003, and a second backflow prevention connection flow passage 1001; the second backflow-preventing connecting runner 1001 is arranged on the back surface of the upper chip 3, and both the second vertical runner a1002 and the second vertical runner b1003 penetrate through the middle chip 2; the external liquid path input flow channel 603 sequentially passes through the second vertical flow channel a, the second backflow-preventing connecting flow channel 1001 and the second vertical flow channel b and then is communicated with the reaction cavity input flow channel 601; the reaction chamber 5 is communicated with the waste liquid chamber 9 through the reaction chamber output flow channel 602 and the waste liquid output micro flow channel 604 in sequence, the waste liquid output micro flow channel 604 is arranged on the front surface of the lower chip 1, a conductive rubber valve is arranged between the waste liquid output micro flow channel 604 and the reaction chamber output flow channel 602, and the conductive rubber valve comprises an upper conductive rubber valve structure 906 arranged on the upper chip 3 and a middle conductive rubber valve structure 905 arranged at a position corresponding to the middle chip 2; a waste liquid cavity cover plate 902 is arranged on the back surface of the upper chip 3 at a position corresponding to the waste liquid cavity 9, and correspondingly, a waste liquid backflow prevention structure 904 is also arranged on the waste liquid output micro-channel 604; the middle layer liquid adding through hole 401 is fan-shaped, and a middle layer air vent is arranged above the middle layer liquid adding through hole 401 in a penetrating manner; the sample injection cavity 403 is fan-shaped, and comprises a sample filtering pool 406 and a flow guide area, wherein a liquid outlet of the sample filtering pool 406 is arranged on a narrow side wall, the flow guide area is arranged at the bottom of the sample filtering pool, a plurality of air holes 407 are formed in the top of the sample injection cavity 404, an air guide groove is formed in the position, close to a wide side wall, of the pool bottom of the sample filtering pool 406, corresponding to each air hole 407, and a gas gathering area is arranged between each air guide groove and the flow guide area; the flow guide area is at least divided into two flow guide areas at the bottom of the sample filtering pool 406 according to the flow direction of the fluid, the two flow guide areas are communicated with each other, each flow guide area is provided with a plurality of flow guide strips distributed in a gathering shape, and the distribution density of the flow guide strips in the flow guide area at the front end of the flow direction of the fluid is smaller than that of the flow guide strips in the flow guide area at the rear end of the flow direction of the fluid; the bottom of the sample filtering pool 406 is sequentially provided with a first flow guide area and a second flow guide area according to the flow direction of the fluid; the first diversion area comprises a plurality of first-stage diversion bodies and a plurality of second-stage diversion bodies, the first-stage diversion bodies and the second-stage diversion bodies are all rib protrusions, the size of the cross section of each first-stage diversion body is larger than that of the cross section of each second-stage diversion body, the length of each first-stage diversion body is consistent with that of each second-stage diversion body, and the plurality of second-stage diversion bodies are uniformly distributed between every two adjacent first-stage diversion bodies; the second diversion area is arranged at a position corresponding to the first-stage diversion body and is provided with a diversion strip along the length extension direction of the first-stage diversion body, the diversion strip of the second diversion area is a rib protrusion, and the cross section size of the rib protrusion of the second diversion area is not larger than that of the first-stage diversion body; the number of the gas guide grooves is 3, one end of each gas guide groove, which is close to the side wall of the wide edge of the sample filtering pool, is communicated with the air holes 407 in a one-to-one correspondence manner, and a gap at the other end of each gas guide groove is arranged and communicated with the gas polymer area; the number of the first-stage flow guiding bodies in the first flow guiding area is 3, correspondingly, the number of the flow guiding bodies in the second flow guiding area is 3, and the size of the cross section of the ridge of the second flow guiding area is the same as that of the cross section of the first-stage flow guiding body.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. A novel coronavirus (COVID-19) IgM/IgG detection reagent is characterized by comprising a monoclonal anti-human IgM antibody, a monoclonal anti-human IgG antibody and fluorescent microspheres for marking and recombining novel coronavirus antigens.
2. The novel coronavirus (COVID-19) IgM/IgG detection reagent of claim 1, wherein the monoclonal anti-human IgM antibody is murine anti-human IgM mab-Hangzhou Longji organism-MS 00704; the monoclonal anti-human IgG antibody is mouse anti-human IgG monoclonal antibody-Nanjing Kinshire organism-V90401; the Recombinant novel coronavirus antigen is Recombinant 2019-nCoVN Protein (Recombinant 2019-nCoV Nucleocapsid Protein) -Nanjing Kirsi organism-T80103 and Recombinant 2019-nCoV S Protein (Recombinant 2019-nCoV Spike-RBD Protein) -Nanjing Kirsi organism-T80301.
3. The novel coronavirus (COVID-19) IgM/IgG detection reagent of claim 1 or 2, wherein the detection reagent is for use in a chemiluminescent immunoassay.
4. A novel coronavirus (COVID-19) IgM/IgG reagent card, which comprises a reagent strip and a card shell, wherein the reagent strip comprises the novel coronavirus (COVID-19) IgM/IgG detection reagent of claim 1 or 2, wherein a nitrocellulose membrane is coated with a monoclonal anti-human IgM antibody, a monoclonal anti-human IgG antibody and goat anti-chicken IgY; fluorescent microspheres for marking the recombinant novel coronavirus antigen and fluorescent microspheres for marking the chicken IgY are adsorbed in the combination pads; the reagent strip further comprises a sample pad and absorbent paper, wherein the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are sequentially arranged.
5. A novel coronavirus (COVID-19) IgM/IgG detection kit based on a microfluidic chip comprises the microfluidic chip and is characterized by further comprising a novel coronavirus (COVID-19) IgM/IgG detection reagent disclosed in claim 1 or 2, wherein fluorescent microspheres for labeling and recombining novel coronavirus antigens are dried in a reaction cavity of the microfluidic chip; the monoclonal anti-human IgM antibody and the monoclonal anti-human IgG antibody are coated in the reaction cavity of the microfluidic chip.
6. A novel coronavirus (COVID-19) IgM/IgG detection kit based on a microfluidic chip comprises the microfluidic chip and is characterized by further comprising a novel coronavirus (COVID-19) IgM/IgG detection reagent disclosed in claim 1 or 2, wherein the recombined novel coronavirus antigen is subjected to time-resolved fluorescence labeling and is dried in a reaction cavity of the microfluidic chip; the monoclonal anti-human IgM antibody and the monoclonal anti-human IgG antibody are fixed in the reaction cavity of the microfluidic chip through the magnetic beads.
7. The novel coronavirus (COVID-19) IgM/IgG detection kit based on a microfluidic chip according to claim 6, wherein the microfluidic chip is a closed microfluidic chip and comprises a chip body, and the chip body sequentially comprises a lower chip, a middle chip and an upper chip from bottom to top; the chip body comprises a sample feeding cavity, a plurality of cavities and a micro-channel, and the middle-layer chip and the lower-layer chip are matched to define a closed micro-channel and a plurality of cavities; the sample introduction cavity is communicated with the plurality of cavities through the micro-channels; and a sealing cover is arranged on the sample injection cavity.
8. The microfluidic chip-based novel coronavirus (COVID-19) IgM/IgG detection kit according to claim 7, wherein the chip body further comprises an exhaust channel and a negative pressure air bag, the exhaust channel and the negative pressure air bag are arranged on the side surface of the chip body, and the tail end of the exhaust channel is connected with the negative pressure air bag through a clamping joint.
9. The novel coronavirus (COVID-19) IgM/IgG detection kit based on a microfluidic chip as claimed in claim 8, wherein a bayonet joint puncturing part is arranged on the exhaust port bayonet joint, an air inlet film is arranged at the air inlet of the negative pressure air bag, and the bayonet joint puncturing part is used for puncturing the air inlet film of the negative pressure air bag when in use.
10. The novel coronavirus (COVID-19) IgM/IgG detection kit based on a microfluidic chip according to claim 9, wherein an external fluid path interface is provided on the chip body, and the external fluid path interface is connected with the reaction input flow path b through an external fluid path input flow path; a second backflow prevention structure is arranged between the external liquid path input flow channel and the reaction input flow channel b; and an external liquid path sealing cover is arranged on the external liquid path interface.
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