CN113009144B - Antibody detection kit and detection method based on microfluidic technology - Google Patents
Antibody detection kit and detection method based on microfluidic technology Download PDFInfo
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- CN113009144B CN113009144B CN202010553012.2A CN202010553012A CN113009144B CN 113009144 B CN113009144 B CN 113009144B CN 202010553012 A CN202010553012 A CN 202010553012A CN 113009144 B CN113009144 B CN 113009144B
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention discloses an antibody detection kit and a detection method based on a microfluidic technology, wherein the kit comprises a double-layer microfluidic chip; a monoclonal detection antibody; fluorescent-labeled IgG concentrate; working fluid and antibody diluent; the double-layer microfluidic chip comprises a reaction layer substrate, a microcavity sample adding layer and a sealing cover layer from bottom to top in sequence, wherein the reaction layer substrate comprises a glass slide substrate and glutaraldehyde assembled on the glass slide substrate, and a purified antigen bar code is printed on the glutaraldehyde. The kit and the method disclosed by the invention are simple to operate, stable in performance, low in cost, high in detection sensitivity and good in application value.
Description
Technical Field
The invention relates to the technical field of antibody detection, in particular to an antibody detection kit and a detection method based on a microfluidic technology.
Background
Along with the remarkable increase of patients with tumor and blood system diseases and the rapid development of clinical transfusion medicine, the transfusion dosage rises year by year. Blood transfusion is taken as an effective measure for improving platelet count, preventing bleeding and guaranteeing life safety of patients, and safe, scientific and effective infusion has become a necessary requirement for clinical blood transfusion technology development. However, most blood transfusion still adopts random infusion at present, and incompatible blood is destroyed and removed by the immune system of the organism rapidly after being infused into the patient, so that ineffective blood transfusion frequently occurs, precious blood resources are wasted, the optimal treatment opportunity of the patient is delayed, and pain and economic burden of the patient are increased. In order to improve the current situation and improve the blood transfusion effect of patients, a part of clinical hospitals have newly developed blood antibody detection projects. The existing blood group detection technologies at present are as follows: immunofluorescence assay, enzyme-linked immunosorbent assay; the mixed passive blood coagulation technology, the mixed cell adhesion test (solid phase technology), the specific monoclonal antibody to antigen fixing test and the like, but the operation procedures of the tests are complex, the time is long, and some required equipment and instruments are expensive, so blood typing is not a conventional detection project for clinical blood transfusion at present, and part of projects cannot meet the requirement of safe blood transfusion due to accuracy and sensitivity in the clinical development process, are excessively complicated in operation and are gradually eliminated clinically. The principle of the SPRCA technology is that an anti-blood monoclonal antibody is coated in a reaction plate, and a blood monolayer can be formed at the bottom of a reaction hole after blood suspension is centrifugally washed. After incubation in wells, serum or plasma is added, and if the serum or plasma contains antibodies, the antibodies bind to the blood monolayer in the reaction wells and unbound components are removed by washing. Anti-human IgG and human IgG sensitized erythrocytes (indicating erythrocytes) are added, and after centrifugation, the indicating erythrocytes are combined with antibodies on a blood monolayer through bridging of the anti-human IgG, so that the positive reaction is that the indicating erythrocytes are spread on the bottom surface of the reaction hole. The negative reaction indicates that red blood cells are gathered at the center of the bottom of the reaction hole under the action of centrifugal force; the MAIPA technology principle is that blood binds to human alloantibodies first and then incubates with different murine anti-human monoclonal antibodies against blood membrane glycoproteins (anti-GPIb/CD 42b, GPIIb/CD41a, GPIIIa, GPIX, HLA, etc.). After washing, blood is cracked, the product is transferred into a coated goat anti-mouse IgG micro-pore plate, goat anti-human IgG is marked by horseradish peroxidase, and the alloantibody specific to the membrane glycoprotein can be detected through enzyme substrate color development.
When the method is used for qualitatively detecting the antibody, the sensitivity and the specificity of the antibody are only 30-40%, the result is judged to have human errors, the detection can be completed in about several hours, the quick requirement of a clinician can not be met, and meanwhile, the technology does not have a corresponding quality control technology. In order to solve the practical problems of low sensitivity, poor specificity, complex clinical operation, overlong detection time, difficult result interpretation, lack of corresponding reagent quality control and the like of the antibody detection method in clinic; the existing antibody detection method of microcolumn gel indicates that red blood cells are generally subjected to hydroformylation treatment by adopting chemical reagents (such as glutaraldehyde), and then anti-human IgG is coated to indicate a reaction result. Thus, there is a need to establish an antibody detection system that is sensitive, fast, low cost and easy to operate.
Disclosure of Invention
In order to solve the technical problems, the invention provides an antibody detection kit and a detection method based on a microfluidic technology, so as to achieve the purposes of rapidness, low cost, simple operation and accurate detection of antibodies.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
an antibody detection kit based on a microfluidic technology comprises a double-layer microfluidic chip and a monoclonal detection antibody; fluorescent-labeled IgG concentrate; working fluid and antibody diluent; the double-layer microfluidic chip comprises a reaction layer substrate, a microcavity sample adding layer and a sealing cover layer from bottom to top in sequence, wherein the reaction layer substrate comprises a glass slide substrate and glutaraldehyde assembled on the glass slide substrate, and a purified specific antigen bar code is printed on the glutaraldehyde.
In the above scheme, the microcavity sample adding layer is provided with a plurality of through holes which are penetrated up and down, and the through holes and the antigen bar codes are in one-to-one correspondence to form a plurality of reaction tanks; the bottom of the sealing cover layer is provided with a plurality of micro-channel grooves, the micro-channel grooves correspond to the reaction tanks one by one, and two ends of each micro-channel groove are respectively provided with an inflow hole and an outflow hole which vertically penetrate through the sealing cover layer.
In the scheme, the working solution is a bovine serum albumin solution with the mass concentration of 0.5-5% prepared by taking phosphate buffer solution DPBS as a solvent.
In the scheme, the antibody diluent is a bovine serum albumin solution with the mass concentration of 0.5-5% prepared by taking phosphate buffer solution DPBS as a solvent.
In the scheme, the pH of the DPBS solution is 7.2-7.4, and the concentration is 0.01-0.1mol/L.
In the above protocol, the monoclonal antibody concentration is greater than 100ug/ml.
In the scheme, the concentration of the IgG concentrate is more than 100ug/ml, and the dilution multiple of the working concentration of the IgG concentrate is 1:100-1:500.
The antibody detection method based on the microfluidic technology adopts the antibody detection kit based on the microfluidic technology, and comprises the following steps:
(1) Injecting different monoclonal detection antibodies of a sample to be detected from the inflow hole respectively, incubating for 10-40min, enabling the antibodies in the sample to be detected and the monoclonal detection antibodies to be fully combined with specific antigens on the reaction layer substrate, draining redundant liquid from the outflow hole after incubation is completed, flushing with BSA, and flushing the antibody to be detected which is not adsorbed on the reaction layer substrate;
(2) Injecting fluorescent-labeled IgG concentrate from the inflow holes, incubating for 10-40min to enable the fluorescent-labeled IgG concentrate to be fully combined with antibodies in a sample to be detected on the reaction layer substrate, draining redundant liquid from the outflow holes after incubation is completed, flushing with BSA, and flushing the fluorescent-labeled IgG concentrate which is not adsorbed on the reaction layer substrate;
(3) And taking down the reaction layer substrate, and detecting the intensity of the optical signal and the detection signal of the target monoclonal detection antibody, thereby realizing the fluorescence detection of the antibody.
Through the technical scheme, the antibody detection kit and the detection method based on the microfluidic technology provided by the invention have the following beneficial effects:
the kit disclosed by the invention uses a double-layer microfluidic chip, self-assembled glutaraldehyde is integrated with the microfluidic chip, purified specific antigen bar codes are printed on glutaraldehyde, through holes on a microcavity sample adding layer are in one-to-one correspondence with the specific antigen bar codes to form a plurality of reaction tanks, so that a plurality of biochemical or chemical indexes of a sample can be detected at the same time, the consumption of working solution and detection samples in the traditional quantitative analysis process of the antibody is greatly reduced, the steps are simplified, and the sensitivity is high. The invention has simple structure, stable performance and low cost, and has remarkable progress and good popularization and application value. When the chip is used, only the outflow hole and the inflow hole are communicated with the outside, most of the reaction processes are completed in a closed micro-channel system, and the interference of the external environment to the reaction and detection processes is effectively reduced; the chip is simple in structure and easy to integrate and combine with matched automatic equipment to realize automatic detection.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
Fig. 1 is a schematic structural diagram of a dual-layer microfluidic chip according to an embodiment of the present invention;
FIG. 2 is a schematic view of a substrate structure of a reaction layer according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of a microcavity sample-loading layer according to an embodiment of the present invention;
FIG. 4 is a schematic view of the back side structure of a capping layer according to an embodiment of the present invention;
FIG. 5 is a standard curve of an embodiment of the present invention;
FIG. 6 is a bar graph of serum CD41a measurements from 5 patients with hematological disorders and 5 healthy controls.
In the figure, 1, a reaction layer substrate; 2. a microcavity sample addition layer; 3. a capping layer; 4. an antigen barcode; 5. a through hole; 6. a microchannel groove; 7. an inflow hole; 8. and an outflow hole.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
The embodiment of the invention provides a platelet antibody detection kit based on a microfluidic technology, which comprises a double-layer microfluidic chip, a CD41a monoclonal detection antibody, a CD42b monoclonal detection antibody, a GPIIa monoclonal detection antibody, a GPIX monoclonal detection antibody and an HLA monoclonal detection antibody; fluorescence labeled goat anti-mouse IgG concentrate; working fluid and antibody diluent.
As shown in fig. 1, the double-layer microfluidic chip sequentially comprises a reaction layer substrate 1, a microcavity sample adding layer 2 and a cover sealing layer 3 from bottom to top, wherein as shown in fig. 2, the reaction layer substrate 1 comprises a glass slide substrate and glutaraldehyde assembled on the glass slide substrate, and purified mouse anti-human recombinant proteins CD41a, CD42b, GPIIIa, GPIX and HLA specific antigen bar codes 4 are printed on the glutaraldehyde.
As shown in fig. 3, the microcavity sample adding layer is provided with a plurality of through holes 5 which are communicated up and down, and the through holes 5 and the antigen bar codes 4 are in one-to-one correspondence to form a plurality of reaction tanks; as shown in fig. 4, a plurality of micro-channel grooves 6 are arranged at the bottom of the sealing cover layer 3, the micro-channel grooves 6 are in one-to-one correspondence with the reaction tanks, and inflow holes 7 and outflow holes 8 which penetrate through the sealing cover layer up and down are respectively arranged at two ends of the micro-channel grooves 6.
In this example, the working solution was a 3% strength by mass bovine serum albumin solution prepared using phosphate buffer DPBS as the solvent.
In this example, the antibody diluent was a 1% strength by mass bovine serum albumin solution prepared using phosphate buffer DPBS as a solvent.
In this example, the DPBS solution had a pH of 7.2-7.4 and a concentration of 0.05mol/L.
In this example, the monoclonal antibody concentration was greater than 100ug/ml.
In this example, the concentration of Alexa Fluor 488-sheep anti-mouse IgG concentrate was greater than 100ug/ml, and the working concentration dilution of Alexa Fluor 488-sheep anti-mouse IgG concentrate was 1:200.
The embodiment of the invention also provides a platelet antibody detection method based on the microfluidic technology, which adopts the platelet antibody detection kit based on the microfluidic technology and comprises the following steps:
(1) Different samples to be tested and monoclonal detection antibodies are respectively injected from the inflow hole 6, incubation is carried out for 20min, so that the antibodies in the samples to be tested and the monoclonal detection antibodies are fully combined with specific antigens on the reaction layer substrate, after incubation is finished, redundant liquid is pumped out from the outflow hole 7, and the BSA with the mass concentration of 1% is used for washing, so that the antibodies to be tested which are not adsorbed on the reaction layer substrate are washed cleanly;
(2) Injecting the fluorescence-labeled goat anti-mouse IgG concentrate from the inflow hole 6, incubating for 20min to enable the fluorescence-labeled goat anti-mouse IgG concentrate to be fully combined with the antibody in the sample to be detected on the reaction layer substrate, pumping redundant liquid from the outflow hole 7 after incubation is completed, flushing with BSA with the mass concentration of 1%, and flushing the fluorescence-labeled goat anti-mouse IgG concentrate which is not adsorbed on the reaction layer substrate;
(3) The reaction layer substrate 1 is removed, and the intensity of an optical signal and a signal detected by a target monoclonal detection antibody are detected by a microarray scanner, so that the fluorescence detection of the platelet antibody is realized. The detection wavelength is 488nm, the total detection time is 30min, each standard substance is measured by 3 reaction tanks for 3 times, the average value of the results is obtained, and a standard curve is drawn.
(4) Result judgment
(1) The concentration is prepared by taking the base of 10 as the abscissa, the fluorescence value is plotted by taking the base of 10 as the ordinate, a standard curve is drawn, taking CD41a as an example, and the fitting formula is lg [ f (x) ]=1.07252+0.8233 lgx, and the standard curve is shown in fig. 5.
(2) And selecting a sample and checking a sample detection hole, obtaining a sample threshold fluorescence value and a checking sample fluorescence value by using random software of a real-time fluorescence quantitative GenePix 4400 microarray scanner, and carrying in the measured fluorescence value according to a relation between the fluorescence value and the concentration obtained by a standard curve to obtain the content of CD41 a. The results of serum CD41a measurements of 5 patients with hematological disorders and 5 healthy controls are shown in FIG. 6.
The method uses 5 blood system disease patients as a blood system disease group, 5 healthy control patients as a non-blood system disease group, draws a bar graph, and results show that the lowest detection limit of platelet antibody concentration detection by adopting the kit is 5pg/mL, and the detection range is 5-50000pg/mL. By comparing the detection result with the detection result of the existing commercial detection method (electrochemical method), the detection result obtained by using the kit disclosed by the invention has high consistency with the detection result of the existing commercial detection method (electrochemical method), and the detection result obtained by using the kit disclosed by the invention has accuracy. Meanwhile, the coefficient of variation among the obtained detection results is smaller than 10% by carrying out multiple (10 times are adopted in the embodiment) measurement on the same serum sample, so that the detection kit has good repeatability and can be further used as a reference for medical diagnosis of blood system diseases.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (1)
1. The platelet antibody detection kit based on the microfluidic technology is characterized by comprising a double-layer microfluidic chip, a CD41a monoclonal detection antibody, a CD42b monoclonal detection antibody, a GPIIa monoclonal detection antibody, a GPIX monoclonal detection antibody and an HLA monoclonal detection antibody; alexa Fluor 488-goat anti-mouse IgG concentrate; working fluid and antibody diluent;
the double-layer microfluidic chip sequentially comprises a reaction layer substrate (1), a microcavity sample adding layer (2) and a sealing cover layer (3) from bottom to top, wherein the reaction layer substrate 1 comprises a glass slide substrate and glutaraldehyde assembled on the glass slide substrate, and purified mouse anti-human recombinant proteins CD41a, CD42b, GPIIIa, GPIX and HLA specific antigen bar codes (4) are printed on the glutaraldehyde;
a plurality of through holes (5) which are vertically communicated are arranged on the microcavity sample adding layer, and the through holes (5) and the antigen bar codes (4) are in one-to-one correspondence to form a plurality of reaction tanks;
the bottom of the sealing layer (3) is provided with a plurality of micro-channel grooves (6), the micro-channel grooves (6) are in one-to-one correspondence with the reaction tanks, and two ends of the micro-channel grooves (6) are respectively provided with an inflow hole (7) and an outflow hole (8) which vertically penetrate through the sealing layer;
the working solution is a bovine serum albumin solution with the mass concentration of 3% prepared by taking phosphate buffer solution DPBS as a solvent;
the antibody diluent is bovine serum albumin solution with the mass concentration of 1% prepared by taking phosphate buffer solution DPBS as a solvent;
the pH of the DPBS solution is 7.2-7.4, and the concentration is 0.05mol/L;
monoclonal antibody concentrations greater than 100ug/ml;
the concentration of the AlexaFluor 488-sheep anti-mouse IgG concentrate is more than 100ug/ml, and the working concentration dilution of the AlexaFluor 488-sheep anti-mouse IgG concentrate is 1:200.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105063168A (en) * | 2015-07-17 | 2015-11-18 | 上海交通大学 | Method and device for detecting free epithelial cell organ sources in blood |
| CN106148187A (en) * | 2016-07-20 | 2016-11-23 | 国家纳米科学中心 | For expressing unicellular sorting and the micro-fluidic chip of polygenic locus detection of EGFR |
| CN109655611A (en) * | 2018-12-20 | 2019-04-19 | 天津瑞普生物技术股份有限公司 | Micro-fluidic chip immunodiagnosis kit and preparation method thereof |
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| EP2888349B1 (en) * | 2012-08-24 | 2022-02-09 | Yale University | System, device and method for high-throughput multi-plexed detection |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105063168A (en) * | 2015-07-17 | 2015-11-18 | 上海交通大学 | Method and device for detecting free epithelial cell organ sources in blood |
| CN106148187A (en) * | 2016-07-20 | 2016-11-23 | 国家纳米科学中心 | For expressing unicellular sorting and the micro-fluidic chip of polygenic locus detection of EGFR |
| CN109655611A (en) * | 2018-12-20 | 2019-04-19 | 天津瑞普生物技术股份有限公司 | Micro-fluidic chip immunodiagnosis kit and preparation method thereof |
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