CN116223800A - Human papilloma virus E6/E7 colloidal gold detection kit and application thereof - Google Patents
Human papilloma virus E6/E7 colloidal gold detection kit and application thereof Download PDFInfo
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
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Abstract
The invention relates to the field of immunoassay detection, in particular to a human papillomavirus E6/E7 colloidal gold detection kit and application thereof. The device comprises a packing box, a detection reagent card, a sample extracting solution, a sample extracting tube, a sterile sampling swab, a straw and a piece of instruction. The colloidal gold technology is applied to the detection of HPVE6E7, and the detection of the E6E7 of HPV virus in a sample is realized by coating anti-HPVE 6E7 antibody protein on a detection band of a binding pad or an analysis membrane, so that an HPVE6E7 positive specimen is rapidly detected, and a rapid auxiliary diagnosis mode is provided for clinical screening and treatment of cervical cancer. The detection kit has strong specificity and high sensitivity, has high detection speed of 10-20 minutes, does not need special equipment or professional operation, and can be applied to preliminary screening of various places such as medical institutions, communities, airports, even families and the like.
Description
Technical Field
The invention relates to the field of immunoassay detection, in particular to a human papillomavirus E6/E7 colloidal gold detection kit and application thereof.
Background
E6/E7 is an oncogene, encodes viral oncoproteins, plays a vital role in cell transformation and maintenance of the malignant phenotype of transformed tissue, and E6/E7 is a direct marker of HPV oncogene (genetic material) expression (initiation of oncoprotein synthesis), is a template for oncoprotein synthesis, is a necessary pathway for cervical lesions to begin and progress, and its expression level represents the activity of the oncogene.
The current primary detection method is detection of E6/E7 by reverse transcription PCR amplification or by branched DNA signal amplification.
Whichever type of detection has higher requirements on equipment, the detection can be performed in a laboratory with relatively perfect conditions, the average detection time is more than 4 hours, the operation is complex, and the detection depends on a real-time quantitative PCR instrument and high-level technicians; therefore, the existing detection technology is complicated and the detection cost is high. The detection also requires a long time, and therefore, a rapid, convenient and low-cost detection mode is needed for improvement.
Disclosure of Invention
In order to solve the problems, namely the problems proposed by the background technology, the invention provides a human papillomavirus E6/E7 colloidal gold detection kit and application thereof, and the specific technical scheme is as follows:
a human papilloma virus E6/E7 colloidal gold detection kit is prepared by adopting a colloidal gold immunochromatography technology.
The kit comprises: the packaging box, and a detection reagent card, a sample extracting solution, a sample extracting tube, a sterile sampling swab, a suction tube and a piece of instruction which are placed in the box body.
The detection card comprises a card shell and a test strip, wherein the test strip comprises a sample pad, a combination pad, a detection line TE6 line, a detection line TE7 line quality control line C line, a water absorption pad nitrocellulose membrane and a PVC bottom plate. The binding pad is coated with a colloidal gold-labeled mouse anti-human HPV E6 protein monoclonal antibody and a mouse anti-human HPV E7 protein monoclonal antibody.
The nitrocellulose membrane is respectively coated with an anti-human HPV E6 protein monoclonal antibody detection line TE6 line, an anti-human HPV E7 protein monoclonal antibody detection line TE7 line and a quality control line C line formed by anti-mouse HPV E6 and HPV E7 IgG antibodies.
The front of the clamping shell is provided with an observation window and a sample adding hole, and the observation window corresponds to the nitrocellulose membrane. The suction heads of the sample extraction tube and the suction tube are made of polystyrene plastic.
A kit is used for rapid detection of HPV virus antigen-typed human papilloma virus E6/E7.
The specific method comprises the following steps:
1. sample collection: cervical cell collection:
1) After the vaginal speculum expands the vagina, the longer brush wire in the center of the cervical brush head is inserted into the cervical canal, and the shorter brush wires at the two ends prop against the mucous membrane surface of the external orifice of the cervix until the bristles of the two wings are closely contacted with the two sides of the cervical orifice;
2) Pinching the brush handle and taking the cervical outer opening as the center of a circle, always rotating at least 3-5 circles towards one direction, taking down the brush head by forceps or scissors, putting into sample extracting solution collected by a sample, screwing the bottle cap, shaking for a plurality of times, and taking out the brush head. 2. Sample inspection:
1) Before testing, the instruction manual must be read completely and operated strictly according to the instruction manual;
2) Taking out the detection card and putting the detection card flat;
3) Sucking 2-3 drops of the treated sample (60-90 mu L) at one third of the depth of the liquid in the sample extracting solution from the bottom of the bottle by using a suction pipe, and vertically dripping the sample into a sample adding hole of the detection card;
4) And (5) standing at room temperature for 10-20 minutes, and then observing the detection result.
The beneficial technical effects of the invention are as follows: in order to overcome the defects of the existing method, the colloidal gold technology is applied to the detection of HPV E6E7, and the detection of the HPV E6E7 positive specimen in a sample is realized by coating anti-HPV E6E7 antibody protein on a detection band of a binding pad or an analysis membrane, so that a rapid auxiliary diagnosis mode is provided for clinical screening and treatment of cervical cancer. The detection kit has the advantages of strong specificity, high sensitivity, high detection speed of 10-20 minutes, simple and convenient operation, no special equipment or professional operation, and can be applied to preliminary screening of various places such as medical institutions, communities, airports, even families and the like.
Drawings
FIG. 1 is a schematic diagram of the structure of a test strip in the kit of the invention.
Detailed Description
Preferred embodiments of the present invention are described below. It should be understood by those skilled in the art that these embodiments are merely for explaining the technical principles of the present invention, and are not intended to limit the scope of the present invention. A human papilloma virus E6/E7 colloidal gold detection kit is prepared by adopting a colloidal gold immunochromatography technology. Comprising the following steps: the packaging box, and a detection reagent card, a sample extracting solution, a sample extracting tube, a sterile sampling swab, a suction tube and a piece of instruction which are placed in the box body. The sample extraction tube and the suction head of the suction tube are both made of polystyrene plastic.
The detection card comprises a card shell 1 and a test strip 2, wherein the test strip 2 comprises a sample pad 1, a combination pad 2, a detection line TE6 line 3, a detection line TE7 line 4 quality control line C line 5, a water absorption pad 6 nitrocellulose membrane 7 and a PVC bottom plate 8.
The binding pad 2 is coated with a colloidal gold-labeled mouse anti-human HPV E6 protein monoclonal antibody and a mouse anti-human HPV E7 protein monoclonal antibody.
The nitrocellulose membrane 7 is respectively coated with an anti-human HPV E6 protein monoclonal antibody detection line TE6 line 3, an anti-human HPV E7 protein monoclonal antibody detection line TE7 line 4 and a quality control line C line 5 formed by anti-mouse HPV E6 and HPV E7 IgG antibodies.
The front of the cartridge 1 is provided with an observation window 11 and a sample application hole 12, and the observation window 11 corresponds to the nitrocellulose membrane 7.
The preparation process of the double-marked colloidal gold immunochromatographic test strip is as follows:
1. preparing colloidal gold: preparing colloidal gold according to a trisodium citrate reduction method, taking 50ml of 0.01% chloroauric acid solution, heating and stirring until boiling, rapidly adding 2ml of 1% trisodium citrate solution after boiling, rapidly and uniformly stirring for 15min until the liquid becomes red, naturally cooling at room temperature, supplementing ultrapure water to 50ml, and preserving at 4 ℃ for later use.
2. Preparing gold-labeled protein: the gold-labeled proteins to be labeled include, but are not limited to, mouse anti-human HPV IgG antibodies, rabbit anti-human HPV IgG antibodies and sheep anti-human HPV IgG antibodies, adjusting the pH of colloidal gold to 7.9-8.5 by using 0.1M sodium carbonate, slowly adding 5-25 ug of the proteins to be labeled into each milliliter of colloidal gold solution, uniformly mixing, standing for 10min, then adding BSA to the final concentration of 0.5-1%, uniformly mixing, standing for 10min, centrifuging at 9000rpm for 2min, removing precipitates, transferring the upper layer solution to a new tube, centrifuging at 12500 for 10min, removing supernatant, adding a heavy suspension to the original amount, transferring the solution to the new tube, centrifuging at 12500rpm for 10min again, adding a preservation solution with one tenth of the initial volume, and re-suspending the precipitate at 4 ℃ for later use.
Preparing a colloidal gold test strip: and diluting with a coating buffer solution, detecting the protein to be coated to a concentration of 0.5-1.5 mg/mL, and spraying the streptavidin and the protein to be coated on a membrane at a distance of 0.5-1.5 uL/cm from a position 1cm away from the bonding pad by using a membrane-drawing gold spraying instrument to form a detection belt 4, a quality control belt 5, wherein the distance between the detection belt and the quality control belt is about 5mm, and the distance between the quality control belt and the water absorption pad is about 1cm. The analytical membrane is dried at 37 ℃ and packaged for standby.
5. The coating buffer solution is prepared from borate, carbonate, phosphate, tris-HCl or Tris-phosphate, etc., and the pH value of the buffer solution is 7.0-7.5, and the TE6 line is detected: the murine anti-human HPVE6 monoclonal antibody was mixed with 10mM PBS buffer solution at pH 7.2 to prepare a mixed solution at a concentration of 0.4mg/mL, which was sprayed onto nitrocellulose membrane at a coating weight of 1uL/cm.
Detection zone TE7 line: the murine anti-human HPVE7 monoclonal antibody was mixed with 10mM PBS buffer solution at pH 7.2 to prepare a mixed solution at a concentration of 0.4mg/mL, which was sprayed onto nitrocellulose membrane at a coating weight of 1uL/cm.
And C line of the quality control belt: the goat anti-mouse IgG polyclonal antibody is mixed with 10mM PBS buffer solution with pH of 7.5 to prepare mixed solution with concentration of 0.5mg/mL, and the mixed solution is sprayed on a nitrocellulose membrane, and the kit with coating amount of 1uL/cm is used for rapid detection of HPV virus antigen typed human papilloma virus E6/E7.
The specific method comprises the following steps:
1. sample collection: cervical cell collection:
1) After the vaginal speculum expands the vagina, the longer brush wire in the center of the cervical brush head is inserted into the cervical canal, and the shorter brush wires at the two ends prop against the mucous membrane surface of the external orifice of the cervix until the bristles of the two wings are closely contacted with the two sides of the cervical orifice;
2) Pinching the brush handle and taking the cervical outer opening as the center of a circle, always rotating at least 3-5 circles towards one direction, taking down the brush head by forceps or scissors, putting into sample extracting solution collected by a sample, screwing the bottle cap, shaking for a plurality of times, and taking out the brush head. 2. Sample inspection:
1) Before testing, the instruction manual must be read completely and operated strictly according to the instruction manual;
2) Taking out the detection card and putting the detection card flat;
3) Sucking 2-3 drops of the treated sample (60-90 mu L) at one third of the depth of the liquid in the sample extracting solution from the bottom of the bottle by using a suction pipe, and vertically dripping the sample into a sample adding hole of the detection card;
4) And (5) standing at room temperature for 10-20 minutes, and then observing the detection result.
3. Interpretation of the test results:
1. positive: the two mauve strips, the detection line (T16 line) and the quality control line (C line) are both developed; or the detection line (T18 line) and the quality control line (C line) are developed;
2. negative: one purple red strip, only the quality control line (C line) develops color;
3. invalidation: the quality control line (C line) has no color development, the detection is invalid, and the detection card is recommended to be taken for re-detection.
In an embodiment of the present invention, in the present invention,
the method has higher clinical significance and value through repeated comparison of experiments.
The terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, article, or apparatus/means that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, article, or apparatus/means.
While the preferred embodiments have been described herein with reference to the preferred embodiments, those skilled in the art will readily appreciate that the scope of the present invention is not limited to such specific embodiments. Equivalent modifications and substitutions for related technical features may be made by those skilled in the art without departing from the principles of the present invention, and such modifications and substitutions will fall within the scope of the present invention.
Claims (9)
1. A human papilloma virus E6/E7 colloidal gold detection kit is characterized in that: a colloidal gold immunochromatography technology is adopted to prepare a rapid detection kit for HPV virus E6E 7.
2. The human papillomavirus E6/E7 colloidal gold assay kit according to claim 1, wherein: the kit comprises: the packaging box, and a detection reagent card, a sample extracting solution, a sample extracting tube, a sterile sampling swab, a suction tube and a piece of instruction which are placed in the box body.
3. The human papillomavirus E6/E7 colloidal gold assay kit according to claim 2, wherein: the detection card comprises a card shell (1) and a test strip (2), wherein the test strip (2) comprises a sample pad (1), a combination pad (2), a detection line TE6 line (3), a detection line TE7 line (4) quality control line C line (5), a water absorption pad (6) nitrocellulose membrane (7) and a PVC bottom plate (8).
4. The human papillomavirus E6/E7 colloidal gold assay kit according to claim 3, wherein: the binding pad (2) is coated with a colloidal gold-labeled mouse anti-human HPV E6 protein monoclonal antibody and a mouse anti-human HPV E7 protein monoclonal antibody.
5. The human papillomavirus E6/E7 colloidal gold assay kit according to claim 3, wherein: the nitrocellulose membrane (7) is respectively coated with an anti-human HPV E6 protein monoclonal antibody detection line TE6 line (3), an anti-human HPV E7 protein monoclonal antibody detection line TE7 line (4) and a quality control line C line (5) formed by anti-mouse HPV E6 and HPV E7 IgG antibodies.
6. The human papillomavirus E6/E7 colloidal gold assay kit according to claim 3, wherein: the front of the clamping shell (1) is provided with an observation window (11) and a sample adding hole (12), and the observation window (11) corresponds to the nitrocellulose membrane (7).
7. The human papillomavirus E6/E7 colloidal gold assay kit according to claim 2, wherein: the suction heads of the sample extraction tube and the suction tube are made of polystyrene plastic.
8. A method of detecting the kit of claim 1, wherein: the rapid detection of human papilloma virus E6/E7 for HPV virus antigen typing.
9. The method for detecting a kit according to claim 8, wherein: the specific method comprises the following steps:
1. sample collection: cervical cell collection:
1) After the vaginal speculum expands the vagina, the longer brush wire in the center of the cervical brush head is inserted into the cervical canal, and the shorter brush wires at the two ends prop against the mucous membrane surface of the external orifice of the cervix until the bristles of the two wings are closely contacted with the two sides of the cervical orifice;
2) Pinching the brush handle and taking the cervical outer opening as the center of a circle, always rotating at least 3-5 circles towards one direction, taking down the brush head by forceps or scissors, putting into sample extracting solution collected by a sample, screwing the bottle cap, shaking for a plurality of times, and taking out the brush head.
2. Sample inspection:
1) Before testing, the instruction manual must be read completely and operated strictly according to the instruction manual;
2) Taking out the detection card and putting the detection card flat;
3) Sucking 2-3 drops of the treated sample (60-90 mu L) at one third of the depth of the liquid in the sample extracting solution from the bottom of the bottle by using a suction pipe, and vertically dripping the sample into a sample adding hole of the detection card;
4) And (5) standing at room temperature for 10-20 minutes, and then observing the detection result.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211589582.2A CN116223800A (en) | 2022-12-12 | 2022-12-12 | Human papilloma virus E6/E7 colloidal gold detection kit and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211589582.2A CN116223800A (en) | 2022-12-12 | 2022-12-12 | Human papilloma virus E6/E7 colloidal gold detection kit and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116223800A true CN116223800A (en) | 2023-06-06 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN118243929A (en) * | 2024-05-30 | 2024-06-25 | 弗雷米德生物医药技术（天津）有限公司 | Combined detection kit for detecting precancerous lesions and cancerous lesions of cervical cancer as well as preparation method and application of combined detection kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101576561A (en) * | 2009-06-16 | 2009-11-11 | 重庆理工大学 | Immune colloidal gold test card for testing high-risk type human papillomavirus and test method thereof |
| CN103865883A (en) * | 2014-03-26 | 2014-06-18 | 重庆理工大学 | Monoclonal antibodies for resisting high-risk human papillomavirus proteins and application of monoclonal antibodies |
| CN111394460A (en) * | 2020-04-02 | 2020-07-10 | 安徽科技学院 | Colloidal gold lateral chromatography test strip for breast cancer detection and colloidal gold lateral chromatography test strip for simultaneous detection of breast cancer and cervical cancer |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101576561A (en) * | 2009-06-16 | 2009-11-11 | 重庆理工大学 | Immune colloidal gold test card for testing high-risk type human papillomavirus and test method thereof |
| CN103865883A (en) * | 2014-03-26 | 2014-06-18 | 重庆理工大学 | Monoclonal antibodies for resisting high-risk human papillomavirus proteins and application of monoclonal antibodies |
| CN111394460A (en) * | 2020-04-02 | 2020-07-10 | 安徽科技学院 | Colloidal gold lateral chromatography test strip for breast cancer detection and colloidal gold lateral chromatography test strip for simultaneous detection of breast cancer and cervical cancer |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118243929A (en) * | 2024-05-30 | 2024-06-25 | 弗雷米德生物医药技术（天津）有限公司 | Combined detection kit for detecting precancerous lesions and cancerous lesions of cervical cancer as well as preparation method and application of combined detection kit |
| CN118243929B (en) * | 2024-05-30 | 2024-08-13 | 弗雷米德生物医药技术（天津）有限公司 | Combined detection kit for detecting precancerous lesions and cancerous lesions of cervical cancer as well as preparation method and application of combined detection kit |
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