CN114236138A - Preparation method of fucose glycoprotein fluorescent color development carrier, preparation method of fluorescent intensity reference carrier and application - Google Patents
Preparation method of fucose glycoprotein fluorescent color development carrier, preparation method of fluorescent intensity reference carrier and application Download PDFInfo
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- CN114236138A CN114236138A CN202111562311.3A CN202111562311A CN114236138A CN 114236138 A CN114236138 A CN 114236138A CN 202111562311 A CN202111562311 A CN 202111562311A CN 114236138 A CN114236138 A CN 114236138A
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- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 title claims abstract description 87
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 title claims abstract description 82
- 108090000288 Glycoproteins Proteins 0.000 title claims abstract description 79
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- 239000000243 solution Substances 0.000 claims description 45
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- HOVAGTYPODGVJG-ZFYZTMLRSA-N methyl alpha-D-glucopyranoside Chemical compound CO[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HOVAGTYPODGVJG-ZFYZTMLRSA-N 0.000 claims description 3
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- FYCGGEVYPRTFDS-UHFFFAOYSA-M 3-amino-6-azaniumylidene-9-[2-carboxy-4-(2,5-dioxopyrrolidin-1-yl)oxycarbonylphenyl]xanthene-4,5-disulfonate Chemical compound C=12C=CC(=[NH2+])C(S([O-])(=O)=O)=C2OC2=C(S([O-])(=O)=O)C(N)=CC=C2C=1C(C(=C1)C(O)=O)=CC=C1C(=O)ON1C(=O)CCC1=O FYCGGEVYPRTFDS-UHFFFAOYSA-M 0.000 description 1
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Images
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
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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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57423—Specifically defined cancers of lung
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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
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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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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Abstract
The invention discloses a preparation method of a fucose glycoprotein fluorescent color development carrier, which comprises the following steps: covalently binding lectin to the surface of the carrier; extracting the body fluid glycoprotein and labeling the glycoprotein with fluorescence; binding the fluorescent-labeled glycoprotein to a carrier having lectin immobilized thereon to form lectin-fucose affinity binding on the surface thereof. The method can also be used for making a fluorescence intensity reference carrier and diagnosing lung cancer.
Description
Technical Field
The invention belongs to the technical field of biomolecule analysis reagents, and particularly relates to a preparation method of a fucose glycoprotein fluorescent developing carrier, a manufacturing method of a fluorescent intensity reference carrier and application of the fluorescent intensity reference carrier.
Background
Lung cancer is the leading cause of death among cancer patients worldwide, with 180 million deaths annually due to lung cancer accounting for 25% of all cancer deaths. The high mortality rate of lung cancer is usually due to the fact that at the time of diagnosis, the patient is already in an advanced stage of cancer, since lung cancer is asymptomatic before its tumor spreads, so that it is difficult to detect. Definitive diagnosis of lung cancer can greatly reduce the survival chances of patients and increase medical costs. Statistics indicate that non-small cell lung cancer (NSCLC) patients have a 5-year survival rate of approximately 60% if the cancer is localized to only a single lung; if the cancer has spread to the extrapulmonary or lymph nodes (regional lung cancer), 5-year survival rate drops to 33%; if the cancer has moved, i.e. metastasized to the lungs and further organs including the brain, bones, etc., the 5-year survival rate is only 6%. Early diagnosis is therefore one of the most effective means to improve survival and reduce medical costs. Current methods of detecting lung cancer include chest X-ray (CXR), Computed Tomography (CT), Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET), sputum analysis, and lung biopsy. Despite the advances in current technology and cancer research, 57% of lung cancer patients are diagnosed after metastasis to different organs.
Therefore, it is necessary to develop a preparation method of a fucose glycoprotein fluorescence developing carrier, a preparation method of a fluorescence intensity reference carrier and an application thereof to solve the problems that the diagnosis of most lung cancers is difficult to be confirmed in the existing early lung cancer screening technology, and the confirmed cancers caused by early asymptomatic diseases belong to late stage and cannot be treated.
Disclosure of Invention
The invention aims to provide a preparation method of a fucose glycoprotein fluorescence color development carrier, a preparation method of a fluorescence intensity reference carrier and application.
The invention has a technical scheme that:
a preparation method of a fucose glycoprotein fluorescence color development carrier comprises the following steps:
1) covalently binding lectin to the surface of the carrier;
2) extracting the body fluid glycoprotein and labeling the glycoprotein with fluorescence;
3) binding the fluorescent-labeled glycoprotein to a carrier having lectin immobilized thereon to form lectin-fucose affinity binding on the surface thereof.
Further, in step 1), the covalently binding lectin to the surface of the carrier includes:
soaking a carrier into a buffer solution containing an oxidant, and reacting at room temperature to oxidize ortho-dihydroxy in glucose on the carrier to form two aldehyde groups;
taking out the carrier, soaking the carrier in a NaCl solution for oscillation cleaning, taking out the carrier, soaking the carrier in deionized water for oscillation cleaning, and finally taking out the carrier;
reacting the carrier in a PBS solution containing lectin;
adding sodium cyanoborohydride into the PBS solution containing the lectin, slightly oscillating, and reacting at room temperature to obtain a lectin carrier;
and sequentially immersing the lectin carrier into a NaCl solution, an ACN solution and deionized water for cleaning to obtain the carrier with the fucose lectin on the surface.
Further, the carrier is a test paper or a spherical resin, the test paper has cellulose when the test paper is selected as the carrier, and the spherical resin has an aldehyde group when the spherical resin is selected as the carrier.
Further, the oxidizing agent is sodium periodate, the concentration of the oxidizing agent in the buffer solution is 5-20mM, and the buffer solution is 90-100mM sodium acetate.
Further, the lectin is any one or more of LCA, UEAI or AAL.
Further, in step 2), the extracting and fluorescent labeling of the body fluid glycoprotein comprises the steps of:
collecting body fluid, and placing the body fluid in a centrifugal tube;
adding ethanol into the body fluid, and extracting proteins in the body fluid by a precipitation method;
re-dissolving the protein in diisopropylethylamine, and uniformly mixing to obtain a sample;
adding a fluorescent labeling reagent with the weight ratio equal to the weight ratio of the protein into the sample, and reacting at room temperature to obtain a glycoprotein solution with a fluorescent label.
Further, in step 3), the binding of the fluorescent-labeled glycoprotein to the lectin-immobilized carrier to form an affinity binding of the lectin to fucose on the surface thereof comprises the steps of:
(1) soaking the carrier with the fucose agglutinin on the surface by the glycoprotein solution with the fluorescent label, and incubating at room temperature to ensure that the agglutinin is fully combined with fucose;
(2) when the carrier with the fucose agglutinin on the surface has one agglutinin, washing with an agglutinin buffer solution; eluting the lectin-binding glycoprotein with at least one eluent when the carrier having the fucose lectin on the surface has a plurality of lectins, so that the at least one lectin-binding glycoprotein remains on the carrier having the fucose lectin on the surface;
(3) sequentially immersing the carrier with the fucose agglutinin on the surface into a NaCl aqueous solution, an ACN solution and deionized water, and slightly oscillating to elute the non-fucose glycoprotein which is not hydrophilically combined;
(4) and (3) placing the carrier with the fucose agglutinin on the surface in a fluorescence detection system, and carrying out quantitative analysis.
Further, in the step (2), the lectin buffer comprises: any one or more of LCA buffer solution, UEAI buffer solution and AAL buffer solution, wherein the LCA buffer solution is 10mM HEPES buffered saline, pH 8.5, 0.1mM CaCl2The UEAI buffer solution is 10mM HEPES buffered saline, pH 8.5, 0.1mM CaCl2The AAL buffer solution is 10mM HEPES buffered saline, pH 8.5, 0.1mM CaCl2(ii) a The eluent comprises: an LCA elution solution, a UEAI elution solution and an AAL elution solution, wherein the LCA elution solution is a mixture of 200mM alpha-methyl glucoside and 200mM alpha-methyl mannoside; the UEAI elution solution is 50mM-100mM L-fucose; the AAL elution solution is 50mM-100mM L-fucose.
The other technical scheme of the invention is as follows: a method for manufacturing a fluorescence intensity reference carrier comprises the following steps:
collecting body fluids of healthy and early patients, middle and late patients and patients with malignant lung cancer, and obtaining the fucose glycoprotein fluorescent color development carrier by using the preparation method of the fucose glycoprotein fluorescent color development carrier;
and (3) performing fluorescence detection, quantifying fluorescence intensity, and combining with a pathological result to obtain a fluorescence intensity reference carrier with gradually increased fluorescence intensity.
The third technical scheme of the invention is as follows: a method for preparing fucose glycoprotein fluorescence color development carrier is used in preparing test paper of fucose glycoprotein fluorescence color development carrier for diagnosing lung cancer.
The invention provides a preparation method of a fucose glycoprotein fluorescence color development carrier and a preparation method of a fluorescence intensity reference carrier, which can detect lung cancer in early stage. For healthy people, under the condition of no symptoms and no need of operation, the carrier can be used very conveniently to test the possibility of having lung cancer, and the method can be applied to various scenes, such as:
(1) testing whether saliva has the lung cancer characteristic marker by using the carrier at home;
(2) a marker characteristic different from that of a healthy population can also be detected by a vector in the case of a non-cancer;
(3) several carriers can be used, each of which binds to a single lectin, to improve the accuracy of detection.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein the content of the first and second substances,
FIG. 1 is a schematic diagram of the molecular structure of carrier cellulose and the process for oxidizing covalently bound lectin in the present invention;
FIG. 2 is a schematic diagram of the principle of covalent reaction for fluorescent labeling of proteins in the present invention;
FIG. 3 is a schematic diagram showing the affinity binding of fucose lectin to a fluorescent label having fucose glycoprotein in the present invention;
FIG. 4 is a flow chart of lectin carrier detection of the salivary fucose glycoprotein marker in the present invention;
FIG. 5 shows fluorescence intensity reference carrier prepared by carrier detection of saliva of different populations according to the present invention.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are further described below. The invention is not limited to the embodiments listed but also comprises any other known variations within the scope of the invention as claimed.
First, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The present invention is described in detail by using the schematic structural diagrams, etc., and for convenience of illustration, the schematic diagrams are not enlarged partially according to the general scale when describing the embodiments of the present invention, and the schematic diagrams are only examples, which should not limit the scope of the present invention. In addition, the actual fabrication process should include three-dimensional space of length, width and depth.
Human body fluid contains glycosylated glycoprotein with high quantity and abundance, and the glycosylation abnormality of glycoprotein shows the occurrence and development of tumors, so that the body fluid screening of the tumor specific protein glycosylation is an effective means for screening early tumor markers. By analyzing the pathology of saliva and the proteomics of glycoproteins, it can be found that the glycoproteins in lung cancer patients are abnormally fucosylated, i.e. have fucose on their glycoproteins that is very different from that of healthy or non-cancer people, with core fucose (α 1,6 linkages) and branched fucose (α 1,2 and α 1,3 linkages), respectively. It has been found that the relative abundance of these fucose sugars is low or undetectable in early stages of the disease, and increases progressively with worsening. Therefore, lung cancer can be screened at an early stage by quantifying fucose eggs.
Detection of fucose glycoproteins can be achieved by lectin binding to fluorescent labels. Several fucose lectins are commonly used, including lentil Lectin (LCA) or pea lectin (PSA) (α 1,6 linkage), negundo chastetree lectin (UEA I) (α 1,2 linkage), and Aleurosporium Aurantiaca Lectin (AAL) (α 1,3 linkage). Through the affinity binding of the lectins and the fucose glycoprotein, the protein in the saliva is fluorescently labeled, and the fluorescence detection and the quantification of the fucose glycoprotein with the specificity of the lung cancer are carried out, so that the possibility of the lung cancer of the people is screened.
Example 1
Referring to FIG. 4, FIG. 4 is a flow chart of lectin carrier detection of the fucose glycoprotein marker in saliva. As shown in FIG. 4, a method for preparing a fucose glycoprotein fluorescence color development carrier comprises the following steps:
1. oxidation of carrier material and covalent binding of lectin
If a carrier (such as test paper, spherical resin, etc.) is selected, and the test paper must have ortho-dihydroxy (cis or trans), the structure can be oxidized by a chemical reagent, so that the next covalent binding can be realized. When spherical resins are selected, they must have aldehyde groups. The main component of the carrier (test paper) is Cellulose (Cellulose), its main chemical structure is shown in fig. 1, fig. 1 is a flow chart of the molecular structure of the carrier Cellulose and the oxidized covalent binding lectin in the invention. As shown in FIG. 1, the monosaccharide of the repeating unit of cellulose is Glucose (. beta.1, 4-Glucose), and the number of the repeating unit is generally more than 1 ten thousand (n > 10000). Glucose (Glucose) has a trans-ortho-dihydroxy (OH) and can be oxidized with 5-20mM sodium periodate (NaIO 4).
For ease of understanding, the support used in this example was a commercial grade 1 chromatographic strip (1.5 m long, 19 mm wide, 0.16 mm thick) cut to the desired chromatographic support size (60 mm long, 10mm wide).
Immersing the chromatographic carrier in a solution containing an oxidizing agent (sodium periodate (NaIO)4) In a buffer solution of 90-100mM sodium acetate (sodium acetate) at a pH of 5-6 at a concentration of 5-20mM, and reacting at room temperature for 1-2 hours, so that the ortho-dihydroxy group in the glucose (glucose) on the chromatographic carrier is oxidized to form two aldehyde groups.
Taking out the chromatographic carrier, soaking in 1M NaCl solution, shaking for 5-6min, taking out, soaking in deionized water, shaking for 5-6min, and taking out the chromatographic carrier.
Dissolving one or more of lentil Lectin (LCA), vitellus negundo agglutinin (UEAI), and alexinella Aurantiaca Agglutinin (AAL) with 1 × PBS buffer, reacting the chromatographic carrier in the PBS solution containing lectin with lectin concentration of 10-20 μ g/mL, and shaking at room temperature for 2-3 hr, wherein the volume of the PBS solution containing lectin can be used to fully immerse the chromatographic carrier to obtain the carrier with covalently bound lectin; or partially immersing the chromatographic carrier to obtain covalent binding of the lectin to the carrier only in the immersed portion, so as to reduce lectin usage.
To further stabilize the covalent binding of the lectin to the carrier, sodium cyanoborohydride (NaCNBH) was added to the lectin PBS solution to a final concentration of 48-52mM3) The reaction was carried out at room temperature for 2 to 3 hours with gentle shaking. This step eventually forms stable covalently bound lectins on the chromatographic support. As shown in FIG. 1, lectin1 represents a lectin, lectin2-4 represents several other lectins, and a lectin carrier can be obtained by a single lectin reaction, i.e., using one of LCA, UEAI or AAL; if binding is achieved by reaction using multiple lectins, a mixed lectin carrier is obtained.
The lectin-bound chromatographic carrier is washed by sequentially immersing the lectin-bound chromatographic carrier in three solutions including 1M NaCl, 10% ACN (by volume) and deionized water, each in an amount corresponding to a volume capable of completely soaking the lectin carrier, and washing 2 to 3 times with each solution to obtain a carrier having fucose lectin on the surface.
2. Fluorescent-labeled fucose glycoprotein (fluorescence treatment of saliva)
Collecting body fluid (such as saliva, urine sample, etc.), and placing in a 1-2mL centrifuge tube;
adding 0.5-1.5mL of ethanol into 0.1-0.3mL of body fluid to make the volume ratio of the body fluid to be 1:5, and extracting protein in the body fluid by a precipitation method;
redissolving the protein in 0.10-0.15mL of 10mM Diisopropylethylamine (N, N-Diisopropylethylamine ═ DIEA), and mixing well to obtain a sample;
adding a fluorescent labeling reagent (AF488 NHS ester) (Thermo Fisher or Promega) in an equal weight ratio to the protein to the sample;
the sample is left to react for 2-4 hours at room temperature or 37 ℃. Referring to fig. 2, fig. 2 is a schematic diagram illustrating a principle of covalent reaction for fluorescent labeling of protein in the present invention, and as shown in fig. 2, NHS ester of AF488 reacts with amino group or lysine of protein in a sample to form a covalent bond, i.e., a stable amide bond (amide) is formed, and a glycoprotein with fluorescent label is obtained, so that the protein has fluorescent property.
3. Affinity binding of lectins to fucosyl proteins, markers for humoral lung cancer
The glycoprotein obtained in step 2 is a fluorescent label and can be used in combination with the carrier having the fucose lectin on the surface in step 1. Referring to FIG. 3, FIG. 3 is a schematic diagram of the affinity binding between fucose agglutinin and a fluorescent label having fucose glycoprotein in the present invention. The method comprises the following steps:
(1) taking a fluorescence labeling protein solution by using a pipette, soaking the carrier combined with the lectin, and incubating at room temperature or 37 ℃ for 30-60 minutes to ensure that the lectin is fully combined with fucose;
(2) if 1 lectin is used on the carrier, it is first washed 3-5 times with lectin buffer, 10mM HEPES buffered saline, pH 8.5, 0.1mM CaCl2(LCA, UEAI or AAL use the same buffer); if multiple lectins are used on the carrier, but only one or two of the lectin affinity binding glycoproteins need to be retained, then the desired lectin binding glycoproteins need to be eluted using 1 or 2 of the 3 lectins. Namely: if only LCA binding glycoprotein is required, UEAI eluate and AAL eluate; if it is desired to retain LCA and UEAI binding glycoproteins, an AAL eluate is used; the following are the buffer solution and elution solution for affinity binding of LCA, UEAI and AAL lectins to fluorescently labeled fucoglycoprotein, respectively: LCA buffer solution includes 10mM HEPES buffered saline, pH 8.5, 0.1mM CaCl2(ii) a The LCA elution solution comprises a mixture of 200mM alpha-methyl glucoside and 200mM alpha-methyl mannoside; UEAI buffer solution includes 10mM HEPES buffered saline, pH 8.5，0.1mM CaCl2(ii) a The UEAI elution solution comprises 50mM-100mM L-fucose; the AAL buffer solution comprises 10mM HEPES buffered saline, pH 8.5, 0.1mM CaCl2(ii) a The AAL elution solution comprises 50mM-100mM L-fucose;
(3) immersing the carrier in an aqueous 1M NaCl solution with gentle shaking in order to elute non-hydrophilically bound non-fucose glycoproteins so that only affinity bound fucose glycoproteins remain on the carrier;
(4) soaking the carrier into 10% ACN solution, slightly oscillating, and further cleaning the non-glycoprotein on the surface of the carrier;
(5) the carrier is immersed in deionized water, and the non-fucose glycoprotein or the non-glycoprotein is further eluted, so that the specificity of the carrier for fucose combination is improved, and the fluorescence interference of non-specific protein is reduced.
4. The carrier is placed in a fluorescent detection system and the fucose glycoprotein has core alpha 1,6 fucose, and/or alpha 1,2 fucose, and/or alpha 1,3 fucose. Wherein LCA is combined with alpha 1,6 fucose, UEAI is combined with alpha 1,2 fucose, AAL is combined with all linked fucose, the N-end of the protein of fucose glycoprotein or Lysine (Lysine) is covalently combined with a fluorescent marker, the glycoprotein which is combined with lectin affinity on the surface of the carrier is fucose modified protein, and the possibility that the tested human group has cancer can be determined by fluorescence detection.
Example 2
Referring to FIG. 5, FIG. 5 is a diagram illustrating the fluorescence intensity reference carrier prepared by carrier detection of saliva of different populations according to the present invention. As shown in fig. 5, the prepared lectin carrier is used for saliva test of healthy and different-period lung cancer patients respectively to obtain a standard reference, and the standard carrier is used for quantitative analysis of a population to be tested, and specifically comprises the following steps:
collecting saliva of healthy and early patients (local tumor cells, stage 1 or 2), middle and late patients (stage 3 or 4) and patients with malignant lung cancer (metastasis), and extracting protein and fluorescent markers;
titrating the fluorescence labeled protein onto a fucose carrier, and carrying out affinity binding with the fucose agglutinin fixed on the carrier;
eluting the non-fucose glycoproteins and retaining the fucose glycoproteins on the carrier;
fluorescence detection is used to quantify the fluorescence intensity, and the intensity is combined with the pathological result to obtain a vector with gradually increased fluorescence intensity as shown in fig. 5, wherein higher intensity indicates higher probability of lung cancer.
Example 3
Fucose glycoprotein fluorescent color development vehicle use test procedure:
collecting saliva (HC) of healthy people, saliva (OD) of non-lung cancer patients and saliva (LC) of non-small cell lung cancer patients;
HC, OD and LC population saliva were processed and tested as described in example 1;
combining HC, OD and LC population salivary glycoprotein with carrier lectin, and imaging by fluorescence detection;
pure green (0,255,0) is set to 100, white (0,0,0) is set to 0, AFF488 green fluorescence yields between 0-100 (color pictures can be analyzed with software, such as Photoshop or ImageJ, etc.) according to the RGB color specification;
the fluorescence intensity of Healthy (HC) people and the fluorescence intensity of malignant tumor caused by lung cancer patients are maximum;
TABLE 1
Table 1 shows the fluorescence labeling intensity of lectin in healthy and patients and the comparison table of medical detection, and the comparison of fluorescence intensity and patient detection result is detailed in Table 1. As can be seen from Table 1, the fluorescence intensity of the LC population is obviously higher than that of the HC population and that of the OD population, and the difference of the fluorescence intensities is large, so that the fucose glycoprotein fluorescent color development carrier can be used for diagnosing lung cancer. And can be diagnosed in patients with early stage (stage II), middle and late stage (stage IV) or malignant lung cancer.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (10)
1. A preparation method of a fucose glycoprotein fluorescence color development carrier is characterized by comprising the following steps:
1) covalently binding lectin to the surface of the carrier;
2) extracting the body fluid glycoprotein and labeling the glycoprotein with fluorescence;
3) binding the fluorescent-labeled glycoprotein to a carrier having lectin immobilized thereon to form lectin-fucose affinity binding on the surface thereof.
2. The method for preparing a fucose glycoprotein fluorescence developing carrier as claimed in claim 1, wherein in the step 1), the covalent binding of lectin on the surface of the carrier comprises:
soaking a carrier into a buffer solution containing an oxidant, and reacting at room temperature to oxidize ortho-dihydroxy in glucose on the carrier to form two aldehyde groups;
taking out the carrier, soaking the carrier in a NaCl solution for oscillation cleaning, taking out the carrier, soaking the carrier in deionized water for oscillation cleaning, and finally taking out the carrier;
reacting the carrier in a PBS solution containing lectin;
adding sodium cyanoborohydride into the PBS solution containing the lectin, slightly oscillating, and reacting at room temperature to obtain a lectin carrier;
and sequentially immersing the lectin carrier into a NaCl solution, an ACN solution and deionized water for cleaning to obtain the carrier with the fucose lectin on the surface.
3. The method for preparing a fluorescent chromogenic carrier for fucose glycoprotein according to claim 2, wherein the method comprises the steps of: the carrier is test paper or spherical resin, the test paper has cellulose when the test paper is selected as the carrier, and the spherical resin has aldehyde groups when the spherical resin is selected as the carrier.
4. The method for preparing a fluorescent chromogenic carrier for fucose glycoprotein according to claim 2, wherein the method comprises the steps of: the oxidant is sodium periodate, the concentration of the oxidant in the buffer solution is 5-20mM, and the buffer solution is 90-100mM sodium acetate.
5. The method for preparing a fluorescent chromogenic carrier for fucose glycoprotein according to claim 2, wherein the method comprises the steps of: the lectin is one or more of LCA, UEAI or AAL.
6. The method for preparing a fluorescent chromogenic carrier for fucose glycoproteins as claimed in claim 2, wherein in step 2), said extracting and fluorescently labeling the glycoproteins from the body fluid comprises the steps of:
collecting body fluid, and placing the body fluid in a centrifugal tube;
adding ethanol into the body fluid, and extracting proteins in the body fluid by a precipitation method;
re-dissolving the protein in diisopropylethylamine, and uniformly mixing to obtain a sample;
adding a fluorescent labeling reagent with the weight ratio equal to the weight ratio of the protein into the sample, and reacting at room temperature to obtain a glycoprotein solution with a fluorescent label.
7. The method for preparing a fucose glycoprotein fluorescence developing carrier according to claim 6, wherein in the step 3), the binding of the fluorescence labeled glycoprotein to the carrier immobilized with the lectin and the affinity binding of the lectin and fucose formed on the surface thereof comprises the steps of:
(1) soaking the carrier with the fucose agglutinin on the surface by the glycoprotein solution with the fluorescent label, and incubating at room temperature to ensure that the agglutinin is fully combined with fucose;
(2) when the carrier with the fucose agglutinin on the surface has one agglutinin, washing with an agglutinin buffer solution; eluting the lectin-binding glycoprotein with at least one eluent when the carrier having the fucose lectin on the surface has a plurality of lectins, so that the at least one lectin-binding glycoprotein remains on the carrier having the fucose lectin on the surface;
(3) sequentially immersing the carrier with the fucose agglutinin on the surface into a NaCl aqueous solution, an ACN solution and deionized water, and slightly oscillating to elute the non-fucose glycoprotein which is not hydrophilically combined;
(4) and (3) placing the carrier with the fucose agglutinin on the surface in a fluorescence detection system, and carrying out quantitative analysis.
8. The method for preparing a fluorescent chromogenic carrier for fucose glycoprotein according to claim 7, wherein in the step (2), the lectin buffer comprises: any one or more of LCA buffer solution, UEAI buffer solution and AAL buffer solution, wherein the LCA buffer solution is 10mM HEPES buffered saline, pH 8.5, 0.1mM CaCl2The UEAI buffer solution is 10mM HEPES buffered saline, pH 8.5, 0.1mM CaCl2The AAL buffer solution is 10mM HEPES buffered saline, pH 8.5, 0.1mM CaCl2(ii) a The eluent comprises: an LCA elution solution, a UEAI elution solution and an AAL elution solution, wherein the LCA elution solution is a mixture of 200mM alpha-methyl glucoside and 200mM alpha-methyl mannoside; the UEAI elution solution is 50mM-100mM L-fucose; the AAL elution solution is 50mM-100mM L-fucose.
9. A method for manufacturing a fluorescence intensity reference carrier is characterized by comprising the following steps:
collecting body fluid of healthy, early stage patients, middle and late stage patients and patients with malignant lung cancer, and obtaining the fucose glycoprotein fluorescent color developing carrier by using the preparation method of the fucose glycoprotein fluorescent color developing carrier of any one of claims 1-8;
and (3) performing fluorescence detection, quantifying fluorescence intensity, and combining with a pathological result to obtain a fluorescence intensity reference carrier with gradually increased fluorescence intensity.
10. Use of the method of any one of claims 1 to 8 for preparing a test strip of a fucose glycoprotein fluorescent color developing carrier for diagnosing lung cancer.
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| GB2048896A (en) * | 1979-04-28 | 1980-12-17 | Prirodovedecka Fakulta Univers | Sorbents for Saccharides, Glycoproteins and Saccharide- containing Polymers and a Method of Their Manufacture |
| CN105652002A (en) * | 2016-01-07 | 2016-06-08 | 西北大学 | Lectin microarray for detecting carbohydrate chain marker based on sialoprotein and detection method of carbohydrate chain marker using same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| GB2048896A (en) * | 1979-04-28 | 1980-12-17 | Prirodovedecka Fakulta Univers | Sorbents for Saccharides, Glycoproteins and Saccharide- containing Polymers and a Method of Their Manufacture |
| CN105652002A (en) * | 2016-01-07 | 2016-06-08 | 西北大学 | Lectin microarray for detecting carbohydrate chain marker based on sialoprotein and detection method of carbohydrate chain marker using same |
| JP2021038980A (en) * | 2019-09-02 | 2021-03-11 | 富士レビオ株式会社 | Afp-l3 measurement method and afp-l3 measurement kit, and blocked labeled lectin used for these |
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