CN115524307A - Near-infrared gastric cancer detection kit and preparation method thereof - Google Patents
Near-infrared gastric cancer detection kit and preparation method thereof Download PDFInfo
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- CN115524307A CN115524307A CN202211162027.1A CN202211162027A CN115524307A CN 115524307 A CN115524307 A CN 115524307A CN 202211162027 A CN202211162027 A CN 202211162027A CN 115524307 A CN115524307 A CN 115524307A
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- 208000005718 Stomach Neoplasms Diseases 0.000 title claims abstract description 58
- 206010017758 gastric cancer Diseases 0.000 title claims abstract description 58
- 201000011549 stomach cancer Diseases 0.000 title claims abstract description 58
- 238000001514 detection method Methods 0.000 title claims abstract description 39
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 101100314454 Caenorhabditis elegans tra-1 gene Proteins 0.000 claims abstract description 52
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 47
- 239000000523 sample Substances 0.000 claims abstract description 29
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 14
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims abstract description 11
- 238000007112 amidation reaction Methods 0.000 claims abstract description 11
- MOFVSTNWEDAEEK-UHFFFAOYSA-M indocyanine green Chemical compound [Na+].[O-]S(=O)(=O)CCCCN1C2=CC=C3C=CC=CC3=C2C(C)(C)C1=CC=CC=CC=CC1=[N+](CCCCS([O-])(=O)=O)C2=CC=C(C=CC=C3)C3=C2C1(C)C MOFVSTNWEDAEEK-UHFFFAOYSA-M 0.000 claims abstract description 7
- 239000007987 MES buffer Substances 0.000 claims description 16
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 15
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 13
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 10
- 238000000502 dialysis Methods 0.000 claims description 9
- 239000011259 mixed solution Substances 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 230000008878 coupling Effects 0.000 claims description 7
- 238000010168 coupling process Methods 0.000 claims description 7
- 238000005859 coupling reaction Methods 0.000 claims description 7
- 210000002784 stomach Anatomy 0.000 claims description 7
- 206010061968 Gastric neoplasm Diseases 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 230000001464 adherent effect Effects 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 230000003698 anagen phase Effects 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- KDJUNNVUQBKNAY-UHFFFAOYSA-N 4-[(2E)-2-[(2E,4E,6E)-7-[3-[6-(2,5-dioxopyrrolidin-1-yl)oxy-6-oxohexyl]-1,1-dimethylbenzo[e]indol-3-ium-2-yl]hepta-2,4,6-trienylidene]-1,1-dimethylbenzo[e]indol-3-yl]butane-1-sulfonate Chemical compound CC1(C)\C(=C/C=C/C=C/C=C/C2=[N+](CCCCS([O-])(=O)=O)C3=C(C4=C(C=CC=C4)C=C3)C2(C)C)N(CCCCCC(=O)ON2C(=O)CCC2=O)C2=C1C1=C(C=CC=C1)C=C2 KDJUNNVUQBKNAY-UHFFFAOYSA-N 0.000 claims description 2
- 125000003277 amino group Chemical group 0.000 claims description 2
- 229960002317 succinimide Drugs 0.000 claims description 2
- 238000012360 testing method Methods 0.000 claims description 2
- 230000007717 exclusion Effects 0.000 claims 1
- 238000003384 imaging method Methods 0.000 abstract description 10
- 230000005284 excitation Effects 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 4
- -1 1-ethyl- (3-dimethylaminopropyl) carbonyl Chemical group 0.000 abstract 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 abstract 1
- RAABOESOVLLHRU-UHFFFAOYSA-N diazene Chemical compound N=N RAABOESOVLLHRU-UHFFFAOYSA-N 0.000 abstract 1
- 229910000071 diazene Inorganic materials 0.000 abstract 1
- 229960004657 indocyanine green Drugs 0.000 abstract 1
- 210000001519 tissue Anatomy 0.000 description 15
- 230000009435 amidation Effects 0.000 description 5
- 239000000975 dye Substances 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- IHWDSEPNZDYMNF-UHFFFAOYSA-N 1H-indol-2-amine Chemical compound C1=CC=C2NC(N)=CC2=C1 IHWDSEPNZDYMNF-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 210000001156 gastric mucosa Anatomy 0.000 description 1
- 239000001046 green dye Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000002271 resection Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/359—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using near infrared light
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/3577—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light for analysing liquids, e.g. polluted water
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Abstract
The invention relates to a near-infrared gastric cancer detection kit and a preparation method thereof, wherein under the action of 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride (EDC) and N-hydroxysuccinimide (NHS), a near-infrared light reagent is aminated to indocyanine green (ICG-NH) through amidation reaction 2 ) The kit is covalently coupled with a gastric cancer cell Her-2 monoclonal antibody (Her-2 mAb) to form a novel gastric cancer cell detection probe with near infrared light characteristics, the specific combination of the Her-2 mAb and gastric cancer cells is utilized to enable ICG to be combined with the surface of the gastric cancer cells or tumor tissue cells, a fluorescence microscope or a laser confocal microscope is utilized to realize rapid imaging detection on the gastric cancer cells and tumor tissue under the excitation of specific wavelength, and the kit has the characteristics of strong specificity, high sensitivity, high resolution and the like.
Description
Technical Field
The invention relates to a near-infrared gastric cancer detection kit and a preparation method thereof, belonging to the technical field of biology.
Technical Field
Stomach cancer is a common tumor recognized internationally, and is also the second largest tumor causing higher mortality of cancer patients at present. Although the treatment scheme of the gastric cancer is more and more, chemotherapy and surgical resection are the main means for treating the gastric cancer, and the recurrence rate is high. Therefore, the early screening and finding of the gastric cancer has a key significance for preventing gastric cancer diseases. At present, gastroscope detection and biopsy under the gastroscope are still the main ways for early diagnosis of gastric cancer, but the gastroscope not only needs to have high requirements on the specialty of a diagnostician so as to distinguish normal tissues from tumor tissues, but also brings strong discomfort to the stomach of the diagnostician due to a long detection process of a gastroscope instrument, and causes mechanical damage to the gastric mucosa of the diagnostician. However, in biopsy diagnosis of live tissue under gastroscope, sampling is difficult due to low resolution of gastroscope, or misjudgment or missed judgment of gastric cancer is caused due to poor specificity, unobvious early tumor and the like during detection. Therefore, a new method for diagnosing gastric cancer with strong specificity, high sensitivity and high imaging resolution is urgently needed. The existing fluorescent probe can identify gastric cancer cells to realize imaging detection, but has short luminescence wavelength, low resolution and low sensitivity because high-resolution imaging detection is difficult to realize. The invention provides a novel kit which is formed by coupling a gastric cancer cell specific recognition antibody Her-2 mAb with ICG and derivatives thereof to form a probe, so as to realize the imaging detection of gastric cancer cells or tumor tissues at near infrared wavelength.
Disclosure of Invention
The invention aims to provide a probe for near-infrared gastric cancer.
The invention aims to provide application of the probe in a near-infrared detection kit.
Yet another object of the present invention is to: provides a preparation method of the kit.
The purpose of the invention is realized by the following scheme: the invention provides a near-infrared gastric cancer probe, which is characterized in that 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are used for activating carboxyl on a gastric cancer cell Her-2 monoclonal antibody (Her-2 mAb) in an MES buffer solution system, and amino indole green dye (ICG-NH) is added 2 ) The near infrared light source of (a) so that the activated carboxyl group on Her-2 mAb is reacted with ICG-NH 2 Amidation reaction of the upper amino group to form the coupled probe.
Wherein, the antibody of the gastric cancer cell is Her-2 mAb, including but not limited to other gastric cancer cell antibodies and derivatives.
Further, the near-infrared light raw material is a derivative of indocyanine green, and further includes, but is not limited to, indocyanine green-carboxylic acid (ICG-COOH), indocyanine green-succinimide ester (ICG-NHS ester).
The invention also provides application of the probe in a near-infrared gastric cancer detection kit.
The near-infrared gastric cancer detection kit consists of reagents A, B and C, wherein 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are used for activating carboxyl on a gastric cancer cell Her-2 monoclonal antibody (Her-2 mAb) in an MES buffer solution system, and aminated indocyanine green (ICG-NH 2) is added, so that the activated carboxyl on the (Her-2 mAb) and amino on ICG-NH2 are subjected to amidation reaction to form a coupled probe, and a reagent A is obtained; then the buffer solution of the reagent B and the normal saline of the reagent C are matched to form the kit for imaging detection of gastric cancer cells or gastric tumor tissues.
In the kit, the concentration of Her-2 mAb in reagent A is 1-60. Mu.g/mL, preferably 40. Mu.g/mL.
In the kit, the concentration of ICG-NH2 in the reagent A is 1-80 mug/mL, and the optimal concentration is 60 mug/mL.
The invention also provides a preparation method of the near-infrared gastric cancer detection kit,
adding Her-2 mAb (final concentration of 40. Mu.g/mL) to 5 mL MES buffer (0.1 mol/L, pH 5.5) containing 50 mmol L-1 EDC and 25 mmol L-1 NHS, protecting from light at room temperature for 1 h to activate the carboxyl groups on Her-2 mAb, then adding ICG-NH2 (final concentration of 60. Mu.g/mL) to allow the activated Her-2 mAb to undergo amidation coupling with ICG-NH2 to form Her-2 mAb-ICG-NH2 probe conjugate, followed by further dialysis against 3500 Da dialysis bag in MES buffer (0.1 mol/L, pH 5.5) for 24 h to remove unconjugated ICG-NH2 residues, and storing the resulting probe conjugate Her-2 mAb-ICG-NH2 as reagent A at 4 ℃ for use;
reagent B in the kit is 500 mL10 mmol/L PBS buffer solution with pH 7.0;
reagent kit C:500 And mL medical normal saline.
The invention relates to a novel near-infrared gastric cancer detection kit, preparation and product application thereof, wherein the kit has the characteristics of simple preparation, convenient operation and the like, a novel gastric cancer cell detection probe with near-infrared light characteristics is formed by utilizing the specific combination of Her-2 mAb and gastric cancer cells, ICG is combined to the surface of the gastric cancer cells or tumor tissue cells, the imaging detection of the gastric cancer cells and tumor tissues is realized by utilizing a fluorescence microscope or a laser confocal microscope under the excitation of specific wavelength, and the probe has the characteristics of strong specificity, high sensitivity, high resolution and the like.
Drawings
FIG. 1 is a schematic representation of a detection technique;
FIG. 2 is an imaging diagram of MGC-803 under 780 nm excitation;
FIG. 3 is an image of mouse gastric tumor tissue under 780 nm excitation.
Detailed Description
In order to achieve the purpose, the invention adopts the following technical scheme:
the first embodiment is as follows:
a near-infrared gastric cancer detection kit comprises reagents A, B and C, and is prepared by the following steps:
reagent A: in 5 mL of a solution containing 50 mmol of L -1 EDC and 25 mmol L-1 NHS in MES buffer (0.1 mol/L, pH 5.5) Her-2 mAb was added to a final concentration of 40. Mu.g/mL, protected from light at room temperature reaction 1h activation of the carboxyl groups on Her-2 mAb, followed by addition of ICG-NH 2 To a final concentration of 60. Mu.g/mL, such that the activated Her-2 mAb is conjugated to ICG-NH 2 Amidation for coupling to form Her-2 mAb-ICG-NH 2 The probe conjugate was then dialyzed against a 3500 Da dialysis bag in 0.1 mol/L MES buffer pH 5.5 for a further 24 h to remove unconjugated ICG-NH 2 The residue, the resulting probe conjugate Her-2 mAb-ICG-NH 2 Recording as a reagent A, and storing at 4 ℃ for later use;
and (3) kit B:500 mL10 mmol/L PBS buffer, pH 7.0;
and (3) a kit C:500 mL medical normal saline.
Table 1 is the kit component composition:
the application method for detecting the gastric cancer cells comprises the following steps: in use, in a culture dish containing MGC-803 cells in the growth phase, reagent a: culturing the culture mixed solution with the culture medium volume ratio of 2% for 30 min in the dark, removing the culture mixed solution, washing adherent MGC-803 cells by using a reagent B, and removing the non-specifically adsorbed dye solution, thus realizing the detection of the gastric cancer cells. As shown in the detection technical route of figure 1, the detection of gastric cancer cells can be realized. FIG. 2 is an image of MGC-803 under 780 nm excitation, which shows that the kit can realize specific imaging detection of gastric cancer cells.
The second embodiment:
a near-infrared gastric cancer detection kit comprises reagents A, B and C, and is prepared by the following steps:
reagent A: in 5 mL of a solution containing 50 mmol of L -1 EDC and 25 mmol L-1 NHS in MES buffer (0.1 mol/L, pH 5.5) were added Her-2 mAb to a final concentration of 40. Mu.g/mL, and reacted at room temperature in the dark for 1 h to activate the carboxyl groups on Her-2 mAb, followed by addition of ICG-NH 2 To a final concentration of 60. Mu.g/mL, such that the activated Her-2 mAb is conjugated to ICG-NH 2 Amidation for coupling to form Her-2 mAb-ICG-NH 2 Probe conjugate, then at a concentration of 0.1 mol/L, pH 5.5Was further dialyzed against 3500 Da dialysis bag for 24 h to remove uncoupled ICG-NH 2 Residue, resulting Probe conjugate Her-2 mAb-ICG-NH 2 Recording as a reagent A, and storing at 4 ℃ for later use;
and (3) kit B:500 mL10 mmol/L PBS buffer pH 7.0;
and (3) kit C:500 mL medical normal saline.
Tumor tissue detection: preparing a mixed liquid from a reagent A and a reagent C, wherein the volume percentage of the reagent A is 1-20%, pouring 2 mL of the mixed liquid into a gastric tumor mouse, after 1 h, taking a small block of living stomach tissue, washing the small block of living stomach tissue by using a test C, and observing the small block of living stomach tissue under a laser confocal microscope, wherein as shown in a figure 3, normal tissues around the stomach of the mouse are not imaged, and the tumor tissue is clearly imaged, so that the kit can better meet the imaging detection of the gastric tumor tissue.
Example three:
a near-infrared gastric cancer detection kit comprises reagents A, B and C, and is prepared by the following steps:
reagent A: in 5 mL of a solution containing 50 mmol of L -1 EDC and 25 mmol L-1 NHS in MES buffer (0.1 mol/L, pH 5.5) were added Her-2 mAb (final concentration 1. Mu.g/mL), and reacted at room temperature in the dark for 1 h to activate the carboxyl groups on Her-2 mAb, followed by addition of ICG-NH 2 (final concentration 60. Mu.g/mL) so that the activated Her-2 mAb is conjugated to ICG-NH 2 Amidation for coupling to form Her-2 mAb-ICG-NH 2 The probe conjugate was then further dialyzed in MES buffer (0.1 mol/L, pH 5.5) against 3500 Da dialysis bag for 24 h to remove unconjugated ICG-NH 2 The residue, the resulting probe conjugate Her-2 mAb-ICG-NH 2 Recording as a reagent A, and storing at 4 ℃ for later use;
and (3) kit B:500 mL10 mmol/L PBS buffer pH 7.0;
and (3) kit C:500 And mL medical normal saline.
And (3) detecting gastric cancer cells: when the kit is used, a culture mixed solution with the volume ratio of the reagent A to the culture medium being 2% is added into a culture dish containing MGC-803 cells in a growth period, the culture mixed solution is cultured for 30 min in a dark place, then the culture mixed solution is removed, the adherent MGC-803 cells are washed by the reagent B), and the nonspecific adsorption dye solution is removed, so that the detection of the gastric cancer cells can be realized.
Example four:
a near-infrared gastric cancer detection kit comprises reagents A, B and C, and is prepared by the following steps:
reagent A: in 5 mL of a solution containing 50 mmol of L -1 EDC and 25 mmol L-1 NHS in MES buffer (0.1 mol/L, pH 5.5) were added Her-2 mAb (final concentration 40. Mu.g/mL), and reacted at room temperature in the dark for 1 h to activate the carboxyl groups on Her-2 mAb, followed by addition of ICG-NH 2 (final concentration 80. Mu.g/mL) so that the activated Her-2 mAb is conjugated to ICG-NH 2 Amidation for coupling to form Her-2 mAb-ICG-NH 2 The probe conjugate was then dialyzed further in MES buffer (0.1 mol/L, pH 5.5) against 3500 Da dialysis bag for 24 h to remove unconjugated ICG-NH 2 The residue, the resulting probe conjugate Her-2 mAb-ICG-NH 2 Recording as a reagent A, and storing at 4 ℃ for later use;
and (3) kit B:500 mL10 mmol/L PBS buffer, pH 7.0;
and (3) a kit C:500 mL medical normal saline.
And (3) detection of gastric cancer cells: in use, in a culture dish containing MGC-803 cells in the growth phase, reagent a: culturing the culture mixed solution with the culture medium volume ratio of 2% for 30 min in the dark, removing the culture mixed solution, washing adherent MGC-803 cells with a reagent B), and removing the non-specifically adsorbed dye solution, thus realizing the detection of the gastric cancer cells.
Claims (8)
1. A near-infrared gastric cancer probe is characterized in that carboxyl groups on a gastric cancer cell Her-2 monoclonal antibody (Her-2 mAb) are activated in an MES buffer system by using 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and then aminated indocyanine green (ICG-NH) is added 2 ) The near infrared light source of (a) so that the activated carboxyl group on Her-2 mAb is reacted with ICG-NH 2 Amidation reaction of the upper amino group to form the coupled probe.
2. The nir gastric cancer probe of claim 1, wherein the gastric cancer cell antibody is Her-2 mAb, including but not limited to other gastric cancer cell antibodies and derivatives.
3. The near-infrared gastric cancer probe of claim 1, wherein the near-infrared light source is a indocyanine green derivative, which further includes but is not limited to indocyanine green-carboxylic acid (ICG-COOH), indocyanine green-succinimide ester (ICG-NHS ester).
4. Use of a near-infrared gastric cancer probe according to any one of claims 1 to 3, characterized in that: is used for preparing a near-infrared gastric cancer detection kit.
5. The near-infrared gastric cancer detection kit according to claim 4, comprising reagents A, B and C, including the following,
reagent A Her-2 mAb was added to MES buffer (0.1 mol/L, pH 5.5) containing 50 mmol L-1 EDC and 25 mmol L-1 NHS to a concentration of 1-60. Mu.g/mL, and reacted at room temperature with exclusion of light for 1 h to activate the carboxyl group on the Her-2 mAb, followed by addition of ICG-NH 2 To a concentration of 1-80. Mu.g/mL, allowing the activated Her-2 mAb to undergo amidation reaction with ICG-NH2 to couple to form Her-2 mAb-ICG-NH 2 The probe conjugate was then further dialyzed in MES buffer (0.1 mol/L, pH 5.5) against 3500 Da dialysis bag for 24 h to remove unconjugated ICG-NH 2 The residue, the resulting probe conjugate Her-2 mAb-ICG-NH 2 Recording as a reagent A, and storing at 4 ℃ for later use;
reagent B is 10 mmol/L PBS buffer solution with pH 7.0;
and (3) a reagent C: is medical normal saline, and the volume ratio of the reagent A, the reagent B and the reagent C is 1:100:100.
6. the near-infrared gastric cancer detection kit according to claim 5, which comprises reagents A, B and C, and is prepared by the following steps:
reagent A: in 5 mL ofAddition of Her-2 mAb to MES buffer (0.1 mol/L, pH 5.5) with 50 mmol L-1 EDC and 25 mmol L-1 NHS to a final concentration of 40. Mu.g/mL, protection from light for 1 h at room temperature to activate the carboxyl groups on Her-2 mAb, followed by addition of ICG-NH 2 Coupling the activated Her-2 mAb with ICG-NH2 by amidation reaction to a final concentration of 60. Mu.g/mL to form a Her-2 mAb-ICG-NH2 probe conjugate, followed by further dialysis in MES buffer at a concentration of 0.1 mol/L, pH 5.5 for 24 h with 3500 Da dialysis bag to remove unconjugated ICG-NH2 residue, and storing the resulting probe conjugate Her-2 mAb-ICG-NH2 as reagent A at 4 ℃ for further use;
and (3) kit B:500 mL10 mmol/L PBS buffer, pH 7.0;
and (3) kit C:500 And mL medical normal saline.
7. A method of using the kit of claim 5 or 6, wherein:
in use, in a culture dish containing MGC-803 cells in the growth phase, reagent a: culturing the culture mixed solution with the culture medium volume ratio of 2% for 30 min in the dark, removing the culture mixed solution, washing adherent MGC-803 cells by using a reagent B, and removing the non-specifically adsorbed dye solution, thus realizing the detection of the gastric cancer cells.
8. A method of using the kit of claim 5 or 6, wherein:
for tumor tissue detection: preparing a mixed liquid from a reagent A and a reagent C, wherein the volume percentage of the reagent A is 1-20%, pouring 2 mL of the mixed liquid into a gastric tumor mouse, after 1 hour, taking a small tissue of a living stomach, washing the small tissue by using a test C, and observing the small tissue under a laser confocal microscope, wherein normal tissues around the stomach of the mouse are not imaged, and the tumor tissue is clearly imaged.
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| PCT/CN2022/139575 WO2024060432A1 (en) | 2022-09-23 | 2022-12-16 | Near-infrared gastric cancer test kit and preparation method therefor |
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| KR101847264B1 (en) * | 2016-03-29 | 2018-04-10 | 국립암센터 | Antigen-responsive antibody-fluorochrome conjugates and method detecting target-cells using fluorescence imaging by thereof |
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