CN111562385A - Lateral antibody chip for detecting diclofenac and indometacin by marker antibody probe - Google Patents
Lateral antibody chip for detecting diclofenac and indometacin by marker antibody probe Download PDFInfo
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- CN111562385A CN111562385A CN202010310898.8A CN202010310898A CN111562385A CN 111562385 A CN111562385 A CN 111562385A CN 202010310898 A CN202010310898 A CN 202010310898A CN 111562385 A CN111562385 A CN 111562385A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9486—Analgesics, e.g. opiates, aspirine
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
Abstract
The invention relates to a lateral antibody chip for detecting diclofenac and indometacin by a marker antibody probe, which comprises a lining plate, and a bifunctional antigen release pad, a universal fluorescent probe release pad, a chromatographic membrane and a water absorption pad, wherein the bifunctional antigen release pad, the universal fluorescent probe release pad, the chromatographic membrane and the water absorption pad are sequentially adhered to the lining plate, and the two adjacent layers are partially overlapped; the bifunctional antigen release pad comprises a specific marker coupled on the basis of a detection antigen formed by coupling ovalbumin with diclofenac and indometacin respectively; the universal fluorescent probe release pad comprises a universal fluorescent probe labeled with an anti-specific marker antibody; the chromatographic membrane is provided with a detection line A, a detection line B and a quality control line, and an anti-diclofenac antibody, an anti-indometacin antibody and an anti-specific marker antibody are respectively fixed on the chromatographic membrane. The invention can solve the production of all detection reagents by using a universal fluorescent probe, thereby reducing the cost and improving the quality control reliability; but also increases the signal intensity and stability of detection, and improves the detection sensitivity and the quantitative precision.
Description
Technical Field
The invention relates to the field of food and drug safety detection, in particular to a lateral antibody chip for detecting diclofenac and indometacin by using a marker antibody probe.
Background
Diclofenac and indometacin belong to nonsteroidal anti-inflammatory drugs and have the effects of resisting inflammation, relieving pain and relieving fever. Because the two types of analgesic effects are rapid and low in price, illegal manufacturers illegally add diclofenac and indomethacin in health-care food (mainly health-care wine) aiming at rheumatic diseases to enhance curative effect and gain improper benefits. The two nonsteroidal anti-inflammatory drugs belong to prescription drugs, and improper administration of the two nonsteroidal anti-inflammatory drugs can cause damage to the digestive tract, the liver and the kidney, and severe even cause anaphylactic shock and acute renal failure. The hazard of patients unconsciously taking health food containing diclofenac and indometacin for a long time is very serious, and the supervision and inspection of illegal addition of diclofenac and indometacin products must be enhanced.
At present, the confirmation method for detecting the illegal addition of diclofenac and indometacin is high performance liquid chromatography and liquid chromatography-mass spectrometry detection. However, the methods have the disadvantages of large equipment investment, high operating cost, complex sample pretreatment, incapability of carrying out field detection and difficulty in large-scale screening of illegal addition phenomena. The existing rapid detection method for diclofenac and indometacin mainly comprises a chemical detection method and a thin-layer chromatography detection method, and the detection sensitivity and the anti-interference capability are required to be improved.
In addition, immunological detection methods are also widely used in the field of food safety detection because of their sensitivity, specificity, rapidity and low cost. The fluorescence immunoassay method for small molecule compounds reported at present is to label quantum dot microspheres on a detection antibody to prepare a fluorescence probe, and form an immune complex with a detection antigen fixed on a detection line to generate a detection signal. However, this method has the following disadvantages: 1. when different target substances are detected, corresponding detection antibodies are required to be connected with the quantum dot microspheres to prepare fluorescent probes, so that the workload is high, the yield is low, and the quality is unstable; 2. the detection antibody is used for preparing a fluorescent probe through chemical reaction, and the structure and the immunoaffinity of the antibody can be influenced; 3. and a signal amplification mechanism is lacked, the detection signal strength is weak, and the sensitivity is low.
The existing patent technology utilizes the principle of sandwich immune complex to detect food additives, namely, an antibody aiming at a detection antigen carrier protein is used as a binding site of a universal antibody to mediate a universal probe to identify a detection antigen, and a sandwich immune complex is formed to generate a detection signal, so that the detection aim is fulfilled. However, the immunogenicity of the 'detection antigen carrier protein' is unstable, and slight changes of protein conformation can cause the reduction of the affinity of the 'universal antibody' and the 'detection antigen', weaken the formation of the 'sandwich immune complex' and influence the generation of detection signals.
Disclosure of Invention
Based on the disadvantages of the detection method, the invention aims to provide a lateral antibody chip for detecting diclofenac and indomethacin by using a marker antibody probe.
The technical scheme adopted by the invention is as follows:
a lateral antibody chip for detecting diclofenac and indomethacin by a marker antibody probe comprises a lining plate, and a bifunctional antigen release pad, a universal fluorescent probe release pad, a chromatographic membrane and a water absorption pad which are sequentially adhered to the lining plate and partially overlapped between the adjacent lining plate; the bifunctional antigen release pad comprises a specific marker coupled on the basis of a detection antigen formed by coupling ovalbumin with diclofenac and indometacin respectively; the universal fluorescent probe release pad comprises a universal fluorescent probe labeled with an anti-specific marker antibody; the chromatographic membrane is provided with a detection line A, a detection line B and a quality control line, wherein the detection line A and the detection line B are respectively fixed with an anti-diclofenac antibody and an anti-indometacin antibody, and the quality control line is fixed with an anti-specific marker antibody.
Preferably, the specific marker is 7-amino-4-hydroxy-2-naphthalenesulfonic acid.
Compared with the traditional binary system immune competition method, the detection card provided by the invention utilizes the principle of the sandwich immune competition method, and adds a universal antibody aiming at the bifunctional antigen marker 7-amino-4-hydroxy-2-naphthalenesulfonic acid in addition to two immune detection elements of the bifunctional antigen and the detection antibody. The detection substance diclofenac and indomethacin are respectively connected with carrier protein (ovalbumin) in a covalent mode to synthesize a bifunctional antigen, a universal antibody (an anti 7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody) is marked on a quantum dot, a detection antibody 1 (an anti diclofenac antibody) is fixed on a detection line A of a chromatographic membrane, a detection antibody 2 (an anti indomethacin antibody) is fixed on a detection line B of the chromatographic membrane, so that the detection substance coupled on the bifunctional antigen is captured by the detection antibody, the universal antibody is combined with the 7-amino-4-hydroxy-2-naphthalenesulfonic acid coupled on the bifunctional antigen to form a sandwich immune complex, and a fluorescence detection signal is generated on the corresponding detection line. When the detection antibody is bound by the free detection substance, the fluorescence intensity on the corresponding detection line will be suppressed, thereby generating an suppressed detection signal.
Specifically, a sample solution is dripped on the bifunctional antigen release pad during detection, the bifunctional antigen and the universal fluorescent probe are dissolved in the movement process of the sample solution and are carried to a detection line A, a detection line B and a quality control line, and an anti-7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody and the bifunctional antigen which are marked by the carried universal fluorescent probe form a sandwich immuno conjugate with an anti-diclofenac antibody on the detection line A and an anti-indomethacin antibody on the detection line B respectively, so that a fluorescence signal is generated in the detection line; the carried general fluorescent probe labeled antibody of 7-amino-4-hydroxy-2-naphthalenesulfonic acid is combined with the antibody of 7-amino-4-hydroxy-2-naphthalenesulfonic acid fixed on the quality control line, and is accumulated on the quality control line to form a fluorescent signal. When free diclofenac and indometacin in the sample reach a certain concentration, immunoreaction on the detection line A and the detection line B can be competitively blocked by corresponding detection products, and a fluorescence signal is inhibited; while the fluorescence signal on the quality control line is generated based on the 7-amino-4-hydroxy-2-naphthalenesulfonic acid and is not influenced by the concentration of diclofenac and indometacin.
Because the marker molecules are introduced into the bifunctional antigens and the anti-marker molecule antibody is used for constructing the universal fluorescent probe, the production of all detection reagents can be realized by using one universal fluorescent probe, so that the cost is reduced and the quality control reliability is improved; and the signal intensity and stability of detection can be increased, and the detection sensitivity and the quantitative precision are improved.
Further, the bifunctional antigen release pad is prepared by absorbing PBS solution containing two bifunctional antigens, a surfactant, mannitol and sucrose by adopting a glass cellulose membrane. Preferably, PBS solutions containing two concentrations of 20. mu.g/mL labeled diclofenac detection antigen, 25. mu.g/mL labeled indomethacin detection antigen, 50. mu.g/mL surfactant, 30mg/mL mannitol, 50mg/mL sucrose, respectively, are well absorbed by a 0.85mm thick glass cellulose membrane. Wherein, mannitol is used as a freeze-drying bracket for ensuring that the bifunctional antigen is rapidly dissolved in the detection process; the sucrose is used for adjusting the viscosity of the detection solution and controlling the chromatographic development speed; the surfactant is used for eliminating non-specific adsorption in the detection process, and preferably polyethylene glycol octyl phenyl ether (Triton X-100); the two bifunctional antigens are respectively synthesized by OVA/diclofenac and OVA/indometacin through covalent coupling, and then are marked with 7-amino-4-hydroxy-2-naphthalenesulfonic acid to become the bifunctional antigen. The detection antibody can be combined on the membrane, and can also be combined by the universal antibody on the quantum dot.
Furthermore, the universal fluorescent probe release pad is prepared by absorbing PBS (phosphate buffer solution) containing the quantum dot fluorescent probe, polyethylene glycol, mannitol, sucrose and glycine by adopting a cellulose membrane. Preferably, a PBS solution containing 30. mu.g/mL universal quantum dot fluorescent probe, 60. mu.g/mL polyethylene glycol (PEG-500), 10mg/mL mannitol, 60mg/mL sucrose, 10mg/mL glycine is absorbed by a cellulose-based membrane with a thickness of 0.34 mm. Wherein, the mannitol is used as a freeze-drying bracket for ensuring that the universal fluorescent probe is quickly dissolved in the detection process; the polyethylene glycol and the sucrose are used for adjusting the viscosity of the detection solution and controlling the chromatographic development speed; glycine is used for eliminating nonspecific adsorption in the detection process; the quantum dot fluorescent probe is formed by labeling an anti-7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody on the surface of a quantum dot, and can be combined with a bifunctional antigen captured on two detection lines and the anti-7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody fixed on a quality control line to respectively generate fluorescent detection signals.
Further, the chromatographic membrane adopts a nitrocellulose membrane, and the detection line A is prepared by spraying a PBS solution containing the anti-diclofenac antibody and sucrose on the nitrocellulose membrane. Preferably, a 0.05M PBS solution (pH 7.4) containing 0.3mg/mL of anti-diclofenac antibody and 10mg/mL of sucrose is sprayed onto a nitrocellulose membrane in an amount of 3.0. mu.g/cm. The basis for the formation of the fluorescence signal of the detection line is the immunoreaction of the antibody and the diclofenac coupled with the bifunctional antigen, and free diclofenac in the detected product can compete for inhibition. Similarly, the detection line B is prepared by spraying a PBS solution containing an indometacin antibody and sucrose on a nitrocellulose membrane. Preferably, a 0.05M PBS solution (pH 7.4) containing 0.5mg/mL anti-indomethacin antibody and 10mg/mL sucrose is sprayed onto a nitrocellulose membrane in an amount of 2.0. mu.g/cm. The basis for the detection of the formation of the fluorescence signal is the immunoreaction of the antibody and indomethacin coupled with the bifunctional antigen, and the indomethacin free in the detected product competes for inhibition.
Further, the control line was prepared by spraying a PBS solution containing an anti-7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody and sucrose on a nitrocellulose membrane. Preferably, a 0.05M PBS solution (pH 7.4) containing 0.3mg/mL 7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody and 10mg/mL sucrose is sprayed on a nitrocellulose membrane in an amount of 1.0 to 3.0. mu.g/cm. The basis of the formation of the fluorescence signal of the quality control line is that the quantum dots and the quality control line both contain the anti-7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody, and respectively generate immunoreaction with the 7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody on the detection antigen to form a sandwich immune complex, so that the detection signal generated on the quality control line is irrelevant to a target detection product and cannot compete and inhibit free diclofenac and indomethacin in the detection product.
The invention also comprises a detection method for simultaneously detecting the diclofenac and the indomethacin, which comprises the following steps:
s1: preparing a bifunctional antigen, a general fluorescent probe, a detection line and a quality control line; the two bifunctional antigens are respectively coupled with diclofenac and indometacin by ovalbumin and then labeled with 7-amino-4-hydroxy-2-naphthalenesulfonic acid, the universal fluorescent probe is labeled with an anti-7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody, the detection line A is used for immobilizing an anti-diclofenac antibody, the detection line B is used for immobilizing an anti-indometacin antibody, and the quality control line is used for immobilizing an anti-7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody;
s2: mixing the sample solution, the two bifunctional antigens and the universal fluorescent probe, conveying the mixture to a detection line A, a detection line B and a quality control line together, and judging whether the sample contains diclofenac and indomethacin according to fluorescent signals of the two detection lines and the quality control line.
The diclofenac and indometacin medicine is a small molecular compound, the immunodetection principle of the conventional detection of the small molecular compound is a 'binary system immune competition method' formed by a 'bifunctional antigen' and a 'detection antibody', aiming at the defects of the method in quantum dot lateral immunochromatography, the invention adds a 'universal antibody' to form a 'ternary system immune competition method' on the basis of the 'bifunctional antigen' and the 'detection antibody', adjusts the combination of various immunoreaction elements in a chromatographic system, fixes a diclofenac capture antibody on a detection line A, marks an anti-7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody on a quantum dot to form a fluorescent probe, and forms a 'sandwich' immune complex with the 'bridging' of the bifunctional antigen 1 (diclofenac-OVA-7-amino-4-hydroxy-2-naphthalenesulfonic acid), the fluorescent probes accumulate to form a detection signal. When the detection antibody is bound by free diclofenac, the fluorescence intensity will be suppressed, thereby generating an inhibition detection signal.
Similarly, the indomethacin antibody/detection antigen 2 (indomethacin-OVA-7-amino-4-hydroxy-2-naphthalenesulfonic acid)/fluorescent probe forms a 'three-element system immune competition method', and a fluorescent signal capable of being inhibited by free indomethacin is generated in the detection line B.
The minimum detection limit of the detection method of the invention on diclofenac and indometacin medicines in a sample solution is 2.0ng/mL, the minimum detection limit on illicitly adding diclofenac and indometacin medicines in health products and Chinese patent medicines is 2.0 mug/kg, and the rapid detection can be completed within 5 minutes.
Further, in step S2, the determination is made by the following method: the detection line A, the detection line B and the quality control line all display fluorescence signals, the result is judged to be negative, and the sample does not contain diclofenac and indometacin medicine; only the detection line A does not display a fluorescence signal, the quality control line displays a fluorescence signal, and the diclofenac acid in the sample is judged; only the detection line B does not display a fluorescence signal, the quality control line displays a fluorescence signal, and the indometacin in contained in the sample is judged. The quality control line is set for checking whether the method is effective or not, the quality control line displays a fluorescent signal to indicate that the method is effective, and the quality control line does not display the fluorescent signal to indicate that the method is ineffective.
Further, the universal fluorescent probe generates 630nm emitted light under the excitation of a 365nm light source.
Compared with the prior art, the invention has the following advantages:
1. the invention introduces a small molecular marker 7-amino-4-hydroxy-2-naphthalenesulfonic acid as an immune recognition site for detecting the antigen, and ensures the stability of the affinity of the universal antibody and the detection antigen. The labeled molecule has strong immunogenicity, high coupling efficiency and good hydrophilicity, can not appear in a sample, and is very suitable for the detection requirement of the invention. The 4-hydroxy-2-naphthalenesulfonic acid is used as an epitope, has super-strong immunogenicity and small steric hindrance, is easy to induce and obtain a high-affinity antibody, and is combined with a marker antibody to guide a universal probe to generate a detection signal, so that the detection signal strength and sensitivity are improved;
2. the quantum dots marked by the anti-7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody are uniformly used for generating detection signals, and the production of reagents of different varieties and different batches can be met only by uniformly preparing the universal probes, so that the scale effect not only improves the production efficiency, but also is beneficial to ensuring the uniformity of the quality of products of different batches;
3. the detection antibody is fixed on the chromatographic membrane, and the activity protection effect on the antibody is more favorable by sealing and drying the membrane, so that the stability of the antibody is improved;
4. the specificity is strong, the detection time is short (5-10 minutes), the field operation can be realized, the fluorescent signal can be excited by means of a portable 360nm light source, the result can be read by naked eyes, the detection cost is low, the operation is simple and convenient, and the kit is suitable for basic level detection personnel to operate.
For a better understanding and practice, the invention is described in detail below with reference to the accompanying drawings.
Drawings
FIG. 1 is a schematic structural diagram of a lateral antibody chip for detecting diclofenac and indomethacin by using the marker antibody probe.
FIG. 2 shows the diazotization method used to label 7-amino-4-hydroxy-2-naphthalenesulfonic acid onto the carrier protein OVA of the test antigen.
FIG. 3 is a schematic diagram of the formation of immune complexes on the detection lines A and B and the generation of fluorescence signals.
FIG. 4 is a schematic diagram showing that the immune complexes in the detection lines A and B are blocked by competition and the fluorescence signal disappears.
FIG. 5 is a schematic diagram of the generation of fluorescence signal due to immunoreaction on the control line.
FIG. 6 shows the principle of generation of the fluorescence immunoassay and the principle of determination of the detection result.
Drawing notes: 1. the kit comprises a bifunctional antigen release pad 2, a universal fluorescent probe release pad 3, a nitrocellulose membrane 31, a detection line A311, an anti-diclofenac antibody 32, a detection line B321, an anti-indomethacin antibody 33, a quality control line 4, a water absorption pad 5, a lining plate 6, a sample solution 61, quantum dots 62, a ' 7-amino-4-hydroxy-2-naphthalenesulfonic acid ' marker 63, OVA 64, coupled diclofenac 641, free diclofenac 65, an anti-7-amino-4-hydroxy-2-naphthalenesulfonic acid ' antibody 66, coupled indomethacin 661, free indomethacin
Detailed Description
Example 1
A lateral antibody chip for detecting diclofenac and indometacin by a marker antibody probe comprises a lining plate 5, and a bifunctional antigen release pad 1, a universal fluorescent probe release pad 2, a nitrocellulose membrane 3 (chromatographic membrane) and a water absorption pad 4 which are sequentially adhered to the lining plate 5 and partially overlapped between the adjacent lining plate 5; the bifunctional antigen release pad 1 comprises a 7-amino-4-hydroxy-2-naphthalenesulfonic acid marker 62 which is coupled on the basis of a detection antigen formed by respectively coupling ovalbumin with diclofenac and indomethacin; the universal fluorescent probe release pad 2 comprises a universal fluorescent probe marked with an anti-7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody 65, and the universal fluorescent probe is a quantum dot 61; the nitrocellulose membrane 3 is provided with a detection line A, a detection line B and a quality control line 33, wherein the detection line A and the detection line B are respectively fixed with an anti-diclofenac antibody 311 and an anti-indomethacin antibody 321, and the quality control line 33 is fixed with an anti-7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody 65.
Referring to fig. 1, in this example, bifunctional antigens were synthesized by covalently linking detection substances diclofenac and indomethacin to a carrier protein (ovalbumin), respectively, and then 7-amino-4-hydroxy-2-naphthalenesulfonic acid was labeled to the detection antigens to prepare labeled detection antigens. Marking a universal antibody (an anti-marker 7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody 65) on a quantum dot 61, fixing an anti-diclofenac antibody 311 on a detection line A of a chromatographic membrane, fixing an anti-indometacin antibody 312 on a detection line B of the chromatographic membrane, so that the detection antibody captures a target hapten coupled on the bifunctional antigen, combining the universal antibody with the marker coupled on the bifunctional antigen to form a sandwich immune complex, and generating a fluorescence detection signal on the corresponding detection line. When the detection antibody is bound by the free detection substance, the fluorescence intensity on the corresponding detection line will be suppressed, thereby generating an suppressed detection signal.
Referring to FIG. 2, this example uses diazotization to label 7-amino-4-hydroxy-2-naphthalenesulfonic acid marker 62 onto the carrier protein OVA of the test antigen to construct the bifunctional antigen.
Referring to fig. 3, in the present embodiment, a sample solution is dropped onto the bifunctional antigen release pad during detection, two bifunctional antigens and a universal fluorescent probe are dissolved during movement of the sample solution and carried to the detection line a, the detection line B and the quality control line 33, and the carried anti-7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody 65 labeled by the universal fluorescent probe and the bifunctional antigen form a sandwich immunoconjugate with the anti-diclofenac antibody 311 on the detection line a and the anti-indomethacin antibody 321 on the detection line B, respectively, so that the detection line generates a fluorescent signal.
Referring to fig. 4, the basis of the formation of the fluorescence signal of the quality control line in this embodiment is that the quantum dots 61 and the quality control line 33 both contain the anti-7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody 65, and respectively generate immunoreaction with the 7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody on the detection antigen to form a "sandwich immune complex", so that the detection signal generated on the quality control line 33 is independent of the target detection product. When free diclofenac 641 or indomethacin 651 exists in the sample and reaches a certain concentration, immunoreactions on the detection line A and the detection line B are competitively blocked by corresponding detection objects, and a fluorescence signal is inhibited; the fluorescence signal on the quality control line 33 is not influenced by the concentration of diclofenac and indometacin.
Specifically, the preparation method of the bifunctional antigen release pad 1 comprises the following steps: a PBS solution containing 20. mu.g/mL of labeled diclofenac assay antigen, 25. mu.g/mL of labeled indomethacin assay antigen, 50. mu.g/mL of Triton X-100, 30mg/mL of mannitol and 50mg/mL of sucrose was sufficiently absorbed by a 0.85mm thick glass cellulose film, and was lyophilized for use.
Specifically, the preparation method of the universal fluorescent probe release pad 2 is as follows: a PBS solution containing 30. mu.g/mL universal fluorescent probe, 60. mu.g/mL PEG-500, 10mg/mL mannitol, 60mg/mL sucrose, 10mg/mL glycine was absorbed by a Whatman 85 cellulose membrane having a thickness of 0.34mm, and was lyophilized for use.
Specifically, the preparation method of the detection line is as follows: test line A was formed by spraying a 0.05M PBS solution (pH 7.4) containing 0.3mg/mL of anti-diclofenac antibody and 10mg/mL of sucrose on a nitrocellulose membrane in an amount of 3.0. mu.g/cm. Test line B was formed by spraying a 0.05M PBS solution (pH 7.4) containing 0.5mg/mL anti-indomethacin antibody and 10mg/mL sucrose on a nitrocellulose membrane in an amount of 2.0. mu.g/cm.
Specifically, the preparation method of the quality control line 33 is as follows: A0.05M PBS solution (pH 7.4) containing 0.3mg/mL of an anti-7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody and 10mg/mL of sucrose was sprayed on a nitrocellulose membrane in an amount of 1.0 to 3.0. mu.g/cm to form a quality control line.
Example 2
The detection method for simultaneously detecting diclofenac and indometacin comprises the following steps:
taking 0.2g (or 0.2mL) of sample, dissolving in 2.0mL of absolute ethyl alcohol to fully extract the target sample, standing the extract for 10 minutes to fully precipitate impurities, taking 0.2mL of supernatant, adding into 20mL of sample buffer solution, and fully shaking uniformly to obtain a sample solution; and dripping 3-4 drops of sample solution on the bifunctional antigen release pad by using a dropper, releasing the bifunctional antigen and the universal fluorescent probe in the process that the sample solution moves to the nitrocellulose membrane, sequentially crossing the detection line and the quality control line, and judging whether the sample contains diclofenac acid and indomethacin according to fluorescent signals on the detection line A, the detection line B and the quality control line.
See fig. 5. An anti-diclofenac antibody 311 fixed on a nitrocellulose membrane detection line A and an anti-7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody 65 marked on a general fluorescent probe are respectively combined with bifunctional antigens to form a sandwich immuno conjugate, so that the detection line generates a fluorescent detection signal. When free diclofenac acid in the sample reaches a certain concentration, the immunoreaction on the detection line is competitively blocked, and the fluorescence signal disappears. Similarly, in the detection line B for the indomethacin sample, the formation and determination of the detection signal are the same as those in the detection line a. The quality control line is set for checking whether the method is effective or not, the color development is effective, and the non-color development indicates that the method is ineffective.
Referring to FIG. 6, the universal fluorescent probe generates 630nm emission under 365nm light source.
In this embodiment, the principle of determining the detection result is as follows:
1) as shown in fig. 6, the detection result is schematically shown in fig. 6-1: the detection line A does not display a fluorescence signal, the detection line B and the quality control line display a fluorescence signal, the result is judged to be positive, and the sample contains diclofenac. Attention is paid to: when the concentration of diclofenac is higher than 2.0ng/mL, the fluorescence of the detection line A completely disappears, and the situation can be judged as strong positive; when the concentration of the diclofenac is in the range of 0-2.0 ng/mL, the detection line A is not completely blocked, but the fluorescence intensity is obviously weakened compared with the negative, and the situation can be judged to be weak positive.
2) As shown in fig. 6, the detection result is schematically shown in fig. 6-2: the detection line B does not display a fluorescence signal, the detection line A and the quality control line display a fluorescence signal, the result is judged to be indometacin positive, and the sample contains indometacin. Attention is paid to: when the concentration of the indometacin is higher than 2.0ng/mL, the fluorescence of the detection line B completely disappears, and the situation can be judged as strong positive; when the concentration of the indomethacin is in the range of 0-2.0 ng/mL, the detection line B is not completely blocked, but the fluorescence intensity is obviously weakened compared with the negative intensity, and the situation can be judged as weak positive of the indomethacin.
3) The detection result schematic diagram 6-3 is the same as the detection result schematic diagram 6-1 and the detection result schematic diagram 6-2, if neither the detection line A nor the detection line B shows fluorescence, but the quality control line shows fluorescence signals, the positive of diclofenac and indomethacin can be judged.
4) As shown in FIG. 6, when any of the cases where the control line does not show a fluorescent signal, i.e., 6-4, 6-5, and 6-6, appear, it indicates that the detection reagent has failed.
Compared with the prior art, the ternary system immune competition method adopted by the embodiment has the following advantages: 1. the general probe for resisting the mark of the marked object is uniformly used, only one general probe is required to be intensively prepared to be used as a fluorescent probe, and the scale and standardization of the detection work are facilitated; 2. the detection antibody is fixed on the chromatographic membrane, and the activity protection effect on the antibody is more favorable by sealing and drying the membrane, so that the stability of the antibody is improved; 3. when the antibody is used for immune binding, the steric hindrance is smaller, and the detection signal intensity and sensitivity are improved. The detection method of the invention has the minimum detection limit of 2.0ng/mL for diclofenac, 2.0ng/mL for indomethacin, 2.0 mug/kg for diclofenac and analogues illegally added into health products and Chinese patent medicines, and 2.0 mug/kg for indomethacin and analogues. Considering that the illegal addition of the two non-steroidal anti-inflammatory drugs in the health care products and the Chinese patent drugs is at the content level of more than 500 mug/kg, the embodiment can completely provide an effective detection means for the illegal addition of the two non-steroidal anti-inflammatory drugs.
In the above examples, the methods for inducing and generating specific antibodies of diclofenac and indomethacin, inducing and generating antibodies against 7-amino-4-hydroxy-2-naphthalenesulfonic acid, labeling antibodies against 7-amino-4-hydroxy-2-naphthalenesulfonic acid on quantum dots, and preparing quantum dot fluorescent probes were performed according to the prior art.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Claims (9)
1. A lateral antibody chip for detecting diclofenac and indomethacin by a marker antibody probe is characterized by comprising a lining plate, and a bifunctional antigen release pad, a universal fluorescent probe release pad, a chromatographic membrane and a water absorption pad which are sequentially adhered to the lining plate and partially overlapped between the adjacent lining plate; the bifunctional antigen release pad comprises a specific marker coupled on the basis of a detection antigen formed by coupling ovalbumin with diclofenac and indometacin respectively; the universal fluorescent probe release pad comprises a universal fluorescent probe labeled with an anti-specific marker antibody; the chromatographic membrane is provided with a detection line A, a detection line B and a quality control line, wherein the detection line A and the detection line B are respectively fixed with an anti-diclofenac antibody and an anti-indometacin antibody, and the quality control line is fixed with an anti-specific marker antibody.
2. The lateral antibody chip of claim 1, wherein said specific label is 7-amino-4-hydroxy-2-naphthalenesulfonic acid.
3. The lateral antibody chip of claim 1, wherein said bifunctional antigen releasing pad is made by absorbing a PBS solution containing two bifunctional antigens, a surfactant, mannitol, and sucrose with a glass cellulose membrane.
4. The lateral antibody chip of claim 1, wherein the universal fluorescent probe release pad is made of a cellulose-based membrane absorbing PBS solution containing quantum dot fluorescent probes, polyethylene glycol, mannitol, sucrose and glycine.
5. The lateral antibody chip of claim 1, wherein said chromatographic membrane is nitrocellulose membrane; the detection line A is prepared by spraying a PBS solution containing an anti-diclofenac antibody and sucrose on a nitrocellulose membrane; the detection line B is prepared by spraying a PBS solution containing an anti-indometacin antibody and sucrose on a nitrocellulose membrane.
6. The lateral antibody chip of claim 1, wherein said control line is prepared by spraying a PBS solution containing anti-7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody and sucrose on a nitrocellulose membrane.
7. The detection method for simultaneously detecting diclofenac and indometacin is characterized by comprising the following steps:
s1, preparing a bifunctional antigen, a general fluorescent probe, a detection line and a quality control line; the two bifunctional antigens are respectively coupled with diclofenac and indometacin by ovalbumin and then labeled with 7-amino-4-hydroxy-2-naphthalenesulfonic acid, the universal fluorescent probe is labeled with an anti-7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody, the detection line A is used for immobilizing an anti-diclofenac antibody, the detection line B is used for immobilizing an anti-indometacin antibody, and the quality control line is used for immobilizing an anti-7-amino-4-hydroxy-2-naphthalenesulfonic acid antibody;
s2: mixing the sample solution, the two bifunctional antigens and the universal fluorescent probe, conveying the mixture to a detection line A, a detection line B and a quality control line together, and judging whether the sample contains diclofenac and indomethacin according to fluorescent signals of the two detection lines and the quality control line.
8. The detection method according to claim 7, wherein the step S2 is determined by: the detection line A, the detection line B and the quality control line all display fluorescence signals, the result is judged to be negative, and the sample does not contain diclofenac and indometacin; only the detection line A does not display a fluorescence signal, the quality control line displays a fluorescence signal, and the sample is judged to contain only diclofenac acid; and only the detection line B does not display a fluorescence signal, the quality control line displays a fluorescence signal, and the sample is judged to contain only indomethacin.
9. The detection method according to claim 7, wherein the universal fluorescent probe generates 630nm of emission light under the excitation of 365nm light source.
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| CN202010310898.8A CN111562385A (en) | 2020-04-20 | 2020-04-20 | Lateral antibody chip for detecting diclofenac and indometacin by marker antibody probe |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1131463A (en) * | 1993-08-20 | 1996-09-18 | 博伊科德有限公司 | Marking of products to establish identity and source |
| CN104991076A (en) * | 2015-07-06 | 2015-10-21 | 同昕生物技术（北京）有限公司 | Lateral chromatography system and application thereof |
| CN108469528A (en) * | 2018-03-28 | 2018-08-31 | 韶关学院 | Indomethacin quantum dot immune chromatography detection card based on signal amplifying system and detection method |
-
2020
- 2020-04-20 CN CN202010310898.8A patent/CN111562385A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1131463A (en) * | 1993-08-20 | 1996-09-18 | 博伊科德有限公司 | Marking of products to establish identity and source |
| CN104991076A (en) * | 2015-07-06 | 2015-10-21 | 同昕生物技术（北京）有限公司 | Lateral chromatography system and application thereof |
| CN108469528A (en) * | 2018-03-28 | 2018-08-31 | 韶关学院 | Indomethacin quantum dot immune chromatography detection card based on signal amplifying system and detection method |
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