CN109212208B - Test strip for detecting tetanus antibody based on quantum dot fluorescent microsphere chromatography, and preparation method and use method thereof - Google Patents
Test strip for detecting tetanus antibody based on quantum dot fluorescent microsphere chromatography, and preparation method and use method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of transverse lateral flow chromatography and biological analysis, in particular relates to a test strip for detecting tetanus antibody based on quantum dot fluorescent microsphere chromatography, a preparation method and a use method thereof, and solves the technical problems in the rapid, low-consumption and portable detection of tetanus antibody content. The preparation method of the test strip comprises the following steps: the test paper strip is prepared by sealing a sample pad by using a TW20 solution with the mass concentration of 0.5-5%, sequentially marking a detection line and a quality control line on an NC film, drying the treated sample pad and the NC film at 37 ℃ in vacuum for 1h, sequentially adhering the sample pad, the NC film and a water absorption pad on a PVC bottom plate, overlapping the sample pad, the NC film and the water absorption pad in pairs for 2-3mm, and finally cutting the sample pad into the test paper strip with the width of 3.9 mm. Compared with the traditional colloidal gold immunochromatographic test strip, the quantum dot microsphere label has the advantages of good stability and high detection sensitivity, and can realize quantitative detection of low-concentration target objects.
Description
Technical Field
The invention belongs to the technical field of transverse lateral flow chromatography and biological analysis, and particularly relates to a test strip for detecting tetanus antibody based on quantum dot fluorescent microsphere chromatography, and a preparation method and a use method thereof.
Background
Tetanus is also known as ankylosing spondylitis, commonly known as "Koushuang". It is a specific infection, in which tetanus clostridium invades human body through skin or mucous membrane wound, grows and breeds under the anoxic environment, and after entering human body, it can act on central nervous system to produce strong neurotoxin to cause muscle spasm. After most patients suffer from the disease, the symptoms of muscular tension contraction and spasm are manifested, and the face is in a "smiling face". It also can block spinal cord inhibition on sympathetic nerve, and has symptoms of blood pressure increase, heart rate acceleration, and sweating. At the same time, it also causes local tissue necrosis and myocardial damage, acidosis, dehydration, and ultimately death. The disease has high fatality rate, and 80-100 million people die worldwide each year due to tetanus according to incomplete statistics after operation. Most cases of tetanus occur in some developing countries, and due to poor medical conditions, people cannot be well treated after tetanus infection, and the incidence rate of the tetanus is high for the middle-aged and old people, puerperae and neonate. Although vaccination with tetanus vaccine is the most effective means of preventing tetanus, the recently reported "Jilin growth sham vaccine" is well-received. The vaccine may have insufficient titer per se, or even if the vaccine is injected, the number of antibodies generated by immune reaction in a human body can reach the immune level, and in addition, the tetanus vaccine has a certain immune period and is not immune for the whole life, the antibodies generated after the immunization can be reduced to a certain period, and the antibodies can continue to reach the immune level after reaching a certain age, so that the problems are urgently needed to be solved. Therefore, the level of tetanus antibodies in human serum can be quickly and accurately known, and the method plays an important guiding role in preventing and treating tetanus.
At present, the in vitro detection methods of tetanus antitoxin mainly include enzyme-linked immunosorbent assay (ELISA), gel electrophoresis immunoassay, fluorescence immunoassay, radioimmunoassay, microfluidic platform method and the like. However, these methods require expensive instruments, complicated procedures, high cost, and unsatisfactory sensitivity. The ELISA method is a relatively commercial method, and has better specificity, but the difference between different batches is large, the detection time is long, and some false positive or false negative results are easy to occur. These detection methods are not well adapted when we encounter some emergencies or need to detect rapidly on site. Therefore, it is necessary to develop a method for rapidly detecting tetanus antibody. The novel fluorescence chromatography test strip prepared by the invention can be used for detecting the tetanus antibody content in serum with high sensitivity and high speed.
Disclosure of Invention
The invention provides a test strip for detecting tetanus antibody based on quantum dot fluorescent microsphere chromatography, a preparation method and a use method thereof, and solves the technical problems in the rapid, low-consumption and portable detection of tetanus antibody content.
The technical scheme of the invention is realized as follows:
the test strip for detecting the tetanus antibody based on the quantum dot fluorescent microsphere chromatography comprises a sample pad, an NC membrane and a water absorption pad, wherein a detection line and a quality control line are sequentially arranged on the NC membrane from the direction of the sample pad to the direction of the water absorption pad, the detection line is tetanus antigen, the quality control line is goat anti-human antibody, and the goat anti-human antibody is goat anti-human antibody coupled with carboxylated quantum dot microspheres.
The concentration of tetanus antigen is 0.5-2.8 mg/mL, and the concentration of goat anti-human antibody is 0.2-4 mg/mL.
The preparation method of the goat anti-human antibody coupled with the carboxylated quantum dot microspheres comprises the following steps:
a. centrifuging the carboxylated quantum dot microspheres, re-dissolving the carboxylated quantum dot microspheres in MES buffer solution, adding a certain amount of EDC, carrying out rotary reaction in a dark room for 20-60 min, activating the carboxylated quantum dot microspheres, centrifuging the reaction product, and discarding the supernatant to obtain activated carboxylated quantum dot microspheres;
b. ultrasonically redissolving the activated carboxylated quantum dot microspheres by using boric acid buffer solution, then adding goat anti-human antibody, carrying out a dark room rotation reaction for 30-120 min, then centrifuging, and removing the supernatant to obtain a coupling product;
c. adding the coupling product into a confining liquid for redissolution, carrying out rotary reaction in a dark room for 30-60 min, then carrying out centrifugal treatment, discarding the supernatant to obtain the goat anti-human antibody coupled with the carboxylated quantum dot microspheres, and storing in a boric acid buffer storage solution at the temperature of 2-8 ℃.
The carboxylated quantum dot microspheres in the step a are CdSe/ZnS nano microspheres coated with polystyrene with carboxylated surfaces; MES buffer solution with concentration of 10-100 mM and pH of 4.0-6.0.
The goat anti-human antibody in the step b is IgG, which is different proteins containing amino groups or a structural domain of the proteins, a monoclonal antibody, a polyclonal antibody or a section of polypeptide chain of the monoclonal antibody or the polyclonal antibody.
The mass ratio of the carboxylated quantum dot microspheres to the goat anti-human antibody in the step b is (2-10): 1; EDC at a final concentration of 1-2 mM; the concentration of the boric acid buffer solution is 20-100 mM, and the pH value is 7.0-7.5.
The blocking solution in the step c is 0.5 to 5 percent of bovine serum albumin, casein or glycine solution; the boric acid buffer preservation solution is boric acid buffer solution containing 0.01-0.1 wt% TW 20; the centrifugation conditions are 10000-15000 rpm and 10-30 min.
The preparation method of the test strip for detecting the tetanus antibody based on the quantum dot fluorescent microsphere chromatography comprises the following steps: the test paper strip is prepared by sealing a sample pad by using a TW20 solution with the mass concentration of 0.5-5%, sequentially marking a detection line and a quality control line on an NC film, drying the treated sample pad and the NC film at 37 ℃ in vacuum for 1h, sequentially adhering the sample pad, the NC film and a water absorption pad on a PVC bottom plate, overlapping the sample pad, the NC film and the water absorption pad in pairs for 2-3mm, and finally cutting the sample pad into the test paper strip with the width of 3.9 mm.
The using method of the test strip for detecting the tetanus antibody based on the quantum dot fluorescent microsphere chromatography comprises the following steps:
(1) preparing tetanus antibody standard solutions with gradient concentrations of 0, 0.2, 0.5, 1, 3, 5, 7, 9, 10, 12, 14, 18, 20 and 22mIU by using 50 mM of pH 7.4 boric acid buffer solution, mixing 10 mu L of tetanus antibody standard solutions with 1.5 mu L of goat anti-human antibody coupled with carboxylated quantum dot microspheres, buffering the mixed solution to 200 mu L by using boric acid, dripping 80-120 mu L of the mixed solution on a sample pad, recording fluorescence intensity detected when the concentration of the tetanus antibody standard solution is 0 and fluorescence peak intensity detected by the tetanus antibody standard solutions with different gradient concentrations after 5-30min, and calculating to obtain relative fluorescence intensity; and (3) drawing a Reader spectrogram and a linear relation graph of response by taking the concentration of the tetanus antibody as an abscissa and the relative fluorescence intensity as an ordinate, wherein the linear relation is y =118.34x +27.44, and R 2 =0.996；
(2) Mixing a 10 mu L tetanus antibody sample with unknown concentration with a goat anti-human antibody coupled with a carboxylated quantum dot microsphere, dripping 80-120 mu L mixed solution on a sample pad, and recording fluorescence intensity after 5-30 min;
(3) the concentration of tetanus antibody samples was calculated from the fluorescence intensity values and the linear relationship plot.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the method, the quantum dot fluorescent microspheres are used as signal labels, so that the interference of the external environment on the quantum dot fluorescent microspheres, such as quenching, can be overcome, and the stability of the quantum dot fluorescent microspheres is improved; more importantly, the quantum dot fluorescent microsphere has larger particle size, and a plurality of carboxylation sites are provided on the surface, so that more biological antibody molecules are connected on the surface of the microsphere, and the detection can be carried out only by a small amount of samples, thereby playing a role in signal amplification, greatly improving the detection sensitivity and reducing the nonspecific interference of other substances.
(2) Compared with other existing methods, such as ELISA, fluorescence immunoassay, microfluidic platform method and the like, the method greatly reduces the interference of other substances and eliminates false positive or false negative results by utilizing the high specificity between the tetanus antibody and the tetanus antigen, which is particularly needed for POCT detection. The method is simple to operate, does not need professional personnel to operate, does not need large-scale instruments, and is very high in detection speed and sensitivity. Meanwhile, compared with the traditional colloidal gold immunochromatographic assay test strip, the quantum dot microsphere test strip has the advantages of good stability of the quantum dot microsphere marker, high detection sensitivity, capability of realizing quantitative detection of a low-concentration target, and capability of improving the detection limit by 2-3 orders of magnitude compared with the colloidal gold method. Far below the international standard (0.01 IU) of tetanus antibody, the method is expected to become a new standard for detecting tetanus antibody, and only negative or positive detection results are given.
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 embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram of quantum dot fluorescent microsphere based immunochromatography for detecting tetanus antibodies.
FIG. 2 shows the preparation method of quantum dot fluorescent microsphere labeled protein and the selection of human IgG (Fc) amount in example 1.
FIG. 3 is a line graph and a photograph showing the detection of tetanus antibodies at different concentrations in the standard solution in example 2.
FIG. 4 is a bar graph of the test for tetanus antibody interference test strip-based assay in example 3.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
Example 1
The preparation method of the quantum dot fluorescent microsphere labeled protein comprises the following steps:
(1) mu.L of carboxylated quantum dot microspheres (QDNB) was first centrifuged for 15000, 30min and then reconstituted in 100. mu.L of 50 mM pH 6.0 MES buffer. Then 15 mu L of 8 mM N- (3-dimethylaminopropyl) -N-ethyl carbodiimide hydrochloride (EDC) is added, the reaction is rotated in a dark room for 30min, and the quantum dot microspheres are subjected to activation treatment and then centrifugal treatment for 15000 min and 30 min.
(2) Sonication was performed with 100. mu.L of 50 mM boric acid buffer pH 7.4, followed by addition of different volumes of 5mg/ml Fc, 2. mu.L, 3. mu.L, 5. mu.L, 7. mu.L, 10. mu.L, reaction by rotation in a dark room for 60 min, and centrifugation for 15000, 30 min.
(3) Adding 100 μ L of 2% BSA blocking solution for redissolution and blocking, rotating in dark room for 50 min, centrifuging for 15000 min and 30min, storing the final QDNB-Fc in 100 μ L boric acid buffer solution (containing 0.01% TW 20), and standing at 4 deg.C.
(4) And (3) mixing 20 mu L of 0.1 IU tetanus antibody with 1.5 mu L of QDNB-Fc with different coupling ratios, respectively taking out 90 mu L of the mixture, dripping the mixture onto the prepared test strip, running the sample, and recording a fluorescence intensity value after 10 min, wherein as shown in figure 2, the optimal effect of tetanus antibody detection can be achieved when the mass ratio of the quantum dot microspheres to the antibody Fc is 4: 1.
Example 2
The preparation method of the test strip for detecting the tetanus antibody based on the quantum dot fluorescent microsphere chromatography comprises the following steps: the test paper strip is prepared by sealing a sample pad by using a TW20 solution with the mass concentration of 0.5-5%, sequentially marking a detection line and a quality control line on an NC film, drying the treated sample pad and the NC film at 37 ℃ in vacuum for 1h, sequentially adhering the sample pad, the NC film and a water absorption pad on a PVC bottom plate, overlapping the sample pad, the NC film and the water absorption pad in pairs for 2-3mm, and finally cutting the sample pad into the test paper strip with the width of 3.9 mm.
Detection of tetanus antibody by fluorescent microsphere immunochromatographic test strip:
preparing a tetanus antibody standard solution (0-0.022 IU) with a certain concentration gradient from the test strip prepared in the steps, mixing the tetanus antibody standard solution with the concentration gradient with a certain amount of QDNB-Fc, dripping 90 mu L of the mixed solution on a sample pad of the tetanus antibody detection test strip, recording the fluorescence peak intensity corresponding to the tetanus antibodies with different concentrations after 10 min, and calculating to obtain the relative fluorescence intensity shown in 3 c. As shown in FIG. 3a, the fluorescence intensity on the detection line gradually increased with the increase of the concentration of tetanus antibodies, and a fluorescent photograph under an ultraviolet lamp was taken. Subsequent linear fitting of the fluorescence intensity to the concentration of tetanus antibodies revealed a good linear relationship between the fluorescence intensity and the concentration for tetanus antibodies ranging from 0.0002 to 0.018IU as shown in FIG. 3b, with the linear equation y =118.34x +27.44 (R =118.34x + 27.44) 2 =0.996) And has high sensitivity, and can be applied to the detection of tetanus antibody in actual serum samples.
Example 3
Interference of different classes of antibodies with tetanus antibody detection:
the compound of the quantum dot microsphere antibody prepared by the steps and an antigen-coated test strip are prepared, then a tetanus antibody standard solution with the concentration of 0.01 IU and an interference antibody with the concentration of 10 equivalents is prepared, for example, a pertussis antibody with the concentration of 0.1 IU, a rabies antibody with the concentration of 0.1 IU, a cytokeratin antibody with the concentration of 5 mug/ml, a thyroid transcription factor antibody with the concentration of 5 mug/ml, a pertussis antibody with the concentration of 0.1 IU and a tetanus antibody with the concentration of 0.01 IU are mixed, after the samples are mixed with a certain amount of QDNB-Fc, 90 muL of mixed solution is dripped on a sample pad of the tetanus antibody detection test strip, after 10 min, fluorescence peak intensities corresponding to different antibodies are recorded, and each group of experiments are repeated for 3 times in parallel. As shown in FIG. 4, the results show that the detection method has high selectivity on tetanus antibody, and the interference of other antibodies is small.
Example 4
The invention carries out a labeling recovery experiment on tetanus antibody in negative serum:
in order to further explore the application of the invention in complex samples, a standard recovery experiment of actual samples is designed and carried out. Negative serum was first diluted 100-fold, and different amounts of 5 mIU, 7.5 mIU, 10 mIU and tetanus antibodies were added, respectively, and the numbers thereof were 1.2.3, as shown in table 1, after tetanus antibodies of the above concentrations were mixed with 1.5 μ L QDNB-Fc, 90 μ L of the mixed solution was dropped on a sample pad of a tetanus antibody detection strip, and after 10 min, fluorescence peak intensities corresponding to different antibodies were recorded, and each set of experiments were repeated 3 times.
Table 1 based on the test strip in negative serum against tetanus antibodies labeled recovery
The fluorescence intensity value is brought into a linear relation graph, the standard adding recovery rate is 96.7-101.4% and the maximum RSD value is 2.35% through calculation, and the method has good precision and reliability in the detection of the tetanus antibody in an actual serum sample.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (5)
1. Test paper strip based on quantum dot fluorescence microsphere chromatography detects tetanus antibody, including sample pad, NC membrane and the pad that absorbs water, its characterized in that: a detection line and a quality control line are sequentially arranged on the NC membrane from the direction of the sample pad to the direction of the water absorption pad, the detection line is tetanus antigen, the quality control line is goat anti-human antibody, and the goat anti-human antibody is goat anti-human antibody coupled by carboxylated quantum dot microspheres; wherein the concentration of the tetanus antigen is 0.5-2.8 mg/mL, and the concentration of the goat anti-human antibody is 0.2-4 mg/mL;
the preparation method of the goat anti-human antibody coupled by the carboxylated quantum dot microspheres comprises the following steps:
a. centrifuging the carboxylated quantum dot microspheres, redissolving the carboxylated quantum dot microspheres in MES buffer solution, adding a certain amount of EDC, carrying out rotary reaction in a dark room for 20-60 min, activating the carboxylated quantum dot microspheres, centrifuging the activated carboxylated quantum dot microspheres, and discarding the supernatant to obtain the activated carboxylated quantum dot microspheres; the carboxylated quantum dot microspheres are CdSe/ZnS nano microspheres coated with polystyrene with carboxylated surfaces; the concentration of MES buffer solution is 10-100 mM, and the pH value is 4.0-6.0;
b. ultrasonically redissolving the activated carboxylated quantum dot microspheres by using boric acid buffer solution, then adding goat anti-human antibody, carrying out a dark room rotation reaction for 30-120 min, then centrifuging, and removing the supernatant to obtain a coupling product; the goat anti-human antibody in the step b is IgG, which is different proteins containing amino groups or a structural domain of the proteins, a monoclonal antibody, a polyclonal antibody or a section of polypeptide chain of the monoclonal antibody or the polyclonal antibody;
c. and adding the coupling product into a confining liquid for redissolution, carrying out rotary reaction in a dark room for 30-60 min, then carrying out centrifugal treatment, discarding the supernatant to obtain the goat anti-human antibody coupled with the carboxylated quantum dot microspheres, and storing in boric acid buffer storage solution at 2-8 ℃.
2. The test strip for detecting tetanus antibody based on quantum dot fluorescent microsphere chromatography as claimed in claim 1, wherein: the molar mass ratio of the activated carboxylated quantum dot microspheres in the step b to the goat anti-human antibody is (2-10): 1; EDC is used at a final concentration of 1-2 mM; the concentration of the boric acid buffer is 20-100 mM, and the pH is 7.0-7.5.
3. The test strip for detecting tetanus antibody based on quantum dot fluorescent microsphere chromatography as claimed in claim 1, wherein: the blocking solution in the step c is 0.5 to 5 percent of bovine serum albumin, casein or glycine solution; the boric acid buffer preservation solution is boric acid buffer solution with the mass concentration of 0.01-0.1 wt% TW 20.
4. The test strip for detecting tetanus antibody based on quantum dot fluorescent microsphere chromatography as claimed in claim 1, wherein: the centrifugation conditions were 10000-.
5. The preparation method of the test strip for detecting tetanus antibody based on quantum dot fluorescent microsphere chromatography, which is described in any one of claims 1 to 4, is characterized by comprising the following steps: the test paper strip is prepared by sealing a sample pad by using TW20 aqueous solution with the mass concentration of 0.5-5%, sequentially marking a detection line and a quality control line on an NC film, drying the treated sample pad and the NC film at 37 ℃ in vacuum for 1h, sequentially adhering the sample pad, the NC film and a water absorption pad on a PVC bottom plate, overlapping the sample pad, the NC film and the water absorption pad in pairs for 2-3mm, and finally cutting the sample pad into the test paper strip with the width of 3.9 mm.
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| CN111308067B (en) * | 2020-02-03 | 2022-09-09 | 江苏森信达生物科技有限公司 | Quantum dot microsphere immunochromatography test strip for quantitatively detecting fluoroquinolone drugs |
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| CN114167053B (en) * | 2021-12-06 | 2023-11-14 | 宁波慈溪生物医学工程研究所 | High-sensitivity quantitative detection method for carbon fluorescent microsphere lateral flow chromatography and application thereof |
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