CN112083165A - Application of human serum REG I alpha as detection target or standard substance in preparation of reagent or kit for predicting tumor - Google Patents
Application of human serum REG I alpha as detection target or standard substance in preparation of reagent or kit for predicting tumor Download PDFInfo
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- CN112083165A CN112083165A CN202010963089.7A CN202010963089A CN112083165A CN 112083165 A CN112083165 A CN 112083165A CN 202010963089 A CN202010963089 A CN 202010963089A CN 112083165 A CN112083165 A CN 112083165A
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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
- G01N33/574—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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Abstract
The invention discloses application of human serum pancreatic regeneration protein I alpha (REGI alpha) as a detection target or a standard substance in preparation of a reagent or a kit for predicting tumors. The method finds that the level of the REGI alpha protein in human serum can accurately predict the tumor. The human serum REGI alpha protein which is a brand new marker for predicting tumors and is provided by the invention can be used as a detection target spot for predicting or assisting in judging the occurrence and the development of the tumors of patients. The results of the clinical study showed that: the area under the ROC curve for the serum REGI α test for diagnosis of tumors was 0.764, with a sensitivity of 62.5% and a specificity of 87.5%. The optimal cutoff (cutoff) value for diagnosing tumor resistance was 46.97 ng/ml.
Description
Technical Field
The invention relates to the field of diagnosis and detection of tumors, in particular to application of a novel tumor biomarker.
Background
In the world, as the incidence of tumors is increasing, the tumors bear a great burden on the health and the economy of human beings, and therefore, the early diagnosis of tumors is always the focus and hot spot of global research. Tumor biomarkers refer to a substance present or secreted in tumor cells that can predict the development of tumors and monitor the response to tumor therapy, including tumor antigens, hormones, glycoproteins, enzymes, and oncogenes. With the development of molecular biological technology, the detection of tumor biomarkers plays an important role in various tumor diagnoses and treatments.
The REG gene, originally a gene isolated from pancreatic islets, plays an important role in the regeneration of beta cells. As one of the REG family genes, REG I α is located on chromosome 2p12, has an amino acid length of 166, and comprises 5 introns and 6 exons. Among malignant tumors, REG I alpha is found to be overexpressed in lung cancer, esophageal cancer, pancreatic cancer, salivary gland cancer, bladder cancer, stomach cancer, colorectal cancer and the like, and is closely related to the occurrence and development of tumors, however, detecting REG genes often requires pathological samples of patients, and is invasive and complex. REG I α encodes a protein, pancreatic regeneration protein I α, a polypeptide with a molecular weight of 16kDa, belonging to the superfamily of calcium-dependent lectin genes, which is rapidly elevated in acute or chronic pancreatic diseases, diabetes and impaired renal function.
Disclosure of Invention
The present invention aims to provide the use of human serum REG I alpha protein detection reagents in the preparation of reagents or kits for diagnosing tumors, in view of the above-mentioned deficiencies of the prior art.
Application of human serum pancreatic regeneration protein I alpha (REG I alpha) as a detection target or standard in preparation of a reagent or a kit for predicting tumors.
The application of a reagent for detecting the pancreatic regeneration protein I alpha in human serum in preparing a kit for predicting tumors.
A kit for predicting tumors comprising an antibody to detect REG I α protein in human serum.
The reagent for detecting human serum REG I α preferably comprises a reagent related to the detection of REG I α protein in human serum by ELISA.
Advantageous effects
The present invention found that the level of REG I alpha protein in human serum can accurately predict tumors (see FIG. 1). Based on the above findings, the present invention provides a novel marker human serum REG I alpha protein for tumor prediction, which can predict or assist in the determination of the occurrence and development of tumors in patients as a target for detection. The results of the clinical study showed that: the area under the ROC curve for the serum REG I α assay was 0.764 for diagnosis of tumors, with a sensitivity of 62.5% and a specificity of 87.5% (see FIG. 2). The optimal cutoff (cutoff) value for diagnosing tumors was 46.97 ng/ml.
Drawings
FIG. 1 human serum REG I α protein quantification and its difference between the normal and tumor groups (mean. + -. SEM; 32.93. + -. 1.52vs 60.60. + -. 6.87; normal vs tumor group; p < 0.01).
FIG. 2 human serum REG I α protein quantification and its differences between the normal group and the digestive, breast and respiratory tumor groups. (mean + -SEM; normal group of digestive tract tumor vs 77.41 + -16.92 vs 32.93 + -1.52; normal group of breast tumor vs 55.03 + -19.46 vs 32.93 + -1.52; normal group of respiratory tract tumor vs 41.92 + -12.64 vs 32.93 + -1.52; p < 0.05; p <0.01)
FIG. 3 ROC curve of human serum REG I α protein for tumor diagnosis.
Detailed Description
Examples
The invention discloses a reagent for preparing a predicted tumor by using human serum pancreatic regeneration protein I alpha (REG I alpha) as a detection target or a standard substance.
Experimental sample and reagent for detecting REG I alpha protein level in human serum
1. Collecting an experimental sample:
130 internal medical inpatients (subsidiary middle and large hospital of southeast university) were selected and the inclusion criteria were: patients with the age of more than or equal to 18 years and less than or equal to 90 years. Exclusion criteria were: pancreatitis; end stage renal disease; diabetes mellitus; active infection and trauma. Patients were divided into two groups, normal and tumor groups according to clinical diagnosis.
2. The main experimental reagents are as follows:
deionized water
1 × TBS (Triethanolamine buffered saline solution)
BSA (bovine serum albumin)
100 XTSST (TBS + Tween buffer)
96-well plate
Pore plate tectorial membrane
REG I alpha Standard (university of Zurich, Switzerland Hospital pancreas surgery laboratory)
Coating antibody (antibody 1) (BIOTEM Co.)
Biotinylated REG I alpha antibody (antibody 2) (BIOTEM Co.)
Extravavidin alkaline phosphatase (Sigma Co.)
Substrate solution (Sigma Co.)
Second, ELISA detection method
1. Serum collection and storage
Collecting 4mL of peripheral blood, standing, centrifuging for 5 minutes by a centrifuge at the rotation speed of 4000 revolutions, taking the upper layer serum, and freezing and storing in a sterile test tube at the temperature of-80 ℃.
2. Preparation of REG I α Standard Curve (prepared within 15min before use):
1) 1mL of 1% BSA (diluted 1 × TBS) was added to each bottle of REG I α standard, allowed to stand at room temperature for 10min while repeatedly inverting/rubbing to aid dissolution, and vortexed to mix well.
2) The dilution gradient is sequentially 4, 3.5, 2.5, 1.5, 1, 0.5, 0.1 and 0ng/mL for 8 standard product dilutions (see below), and a standard quantitative curve is established by a standard curve method.
3. Sample processing
1) Thawing the serum on ice, and slightly shaking and uniformly mixing;
2) adding 10 mu L of serum into 90 mu L of LTBS and mixing uniformly;
3) taking 10 mu L of the solution (2), adding 490 mu L of LPBS, and mixing uniformly;
4. human serum REG I alpha quantification
1) Antibody coating: antibody 1(1:360) was diluted with 1xTBS, 50ul per well and shaken overnight at room temperature.
2) Washing the plate: spin-drying the coated antibody, spreading absorbent paper on a laboratory bench, beating the ELISA plate downward, and washing the plate with 1xTBST for 5 minutes each time for 3 times. After the last washing, the washing liquid in the holes needs to be completely dried.
3) And (3) sealing: 1% BSA/1xTBS150ul was added to each well and blocked for 1h at room temperature.
4) Loading (standard or sample): and respectively arranging a standard sample hole and a sample hole to be detected. Discarding the blocking solution, adding 100 μ L of standard sample (see above) with different concentrations, and 100 μ L of sample to be tested, coating with enzyme linked plate, and incubating at 37 deg.C for 1 h.
5) Washing the plate: the step is the same as the step 2.
6) Antibody 2(1:830) was diluted with 1xTBS, 50. mu.L/well, coated, and incubated at 37 ℃ for 1 h.
7) Washing the plate: the step is the same as the step 2.
8) ExtrAvidin alkaline phosphatase (1:10000) was diluted with 1% BSA/1xTBS, and 50. mu.L of the diluted solution was added to each well, followed by coating, and the mixture was cooled at room temperature for 45 min.
9) Washing the plate: the step is the same as the step 2.
10) Adding 100 μ L of the substrate solution, covering with film, and developing at room temperature in dark for 8-20min (standard substance turns yellow).
11) The absorbance of each well was measured at a wavelength of 405nm using a TECAN microplate reader.
12) And calculating the REG I alpha concentration of each sample according to the standard curve and the absorbance of each sample hole to be detected.
Third, effectiveness
Differences in serum REG I α protein levels between the two groups were separately detected and compared (see FIG. 1); the difference in serum REG I α detection between the three major tumor groups, the digestive tract, breast and respiratory tract tumors, with the greatest distribution of tumor components, and the normal group (see FIG. 2). The area under the ROC curve for diagnosing tumors was 0.764, the sensitivity (true positive rate) was 62.5%, the specificity (true negative rate) was 87.5% (see FIG. 3), and the Youden (Youden) index J was 0.47. The optimal cutoff (cutoff) value for diagnosing tumors was 46.97 ng/ml.
Example 2
A kit for preparing a kit for predicting tumors by using human serum pancreatic regeneration protein I alpha (REG I alpha) as a detection target or a standard substance comprises the ELISA reagent for detecting the content of human serum pancreatic regeneration protein I alpha (REG I alpha) described in example 1.
Claims (1)
1. Application of human serum pancreatic regeneration protein I alpha (REGI alpha) as a detection target or standard in preparation of a reagent or a kit for predicting tumors.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115166260A (en) * | 2022-07-11 | 2022-10-11 | 东南大学 | Application of vitamin D binding protein in plasma brain cell source exosome in diagnosing depression |
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| US20060188889A1 (en) * | 2003-11-04 | 2006-08-24 | Christopher Burgess | Use of differentially expressed nucleic acid sequences as biomarkers for cancer |
| US20090150315A1 (en) * | 2005-07-29 | 2009-06-11 | Siemens Healthcare Diagnostics Inc. | Neoplastic Disease-Related Methods, Kits, Systems and Databases |
| US20130216545A1 (en) * | 2012-02-19 | 2013-08-22 | Claresa Levetan | Early Diagnosis and Novel Treatment of Cancer |
| CN111562395A (en) * | 2020-06-11 | 2020-08-21 | 青岛大学 | Marker of pancreatic cancer tumor and application and kit thereof |
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2020
- 2020-09-14 CN CN202010963089.7A patent/CN112083165A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20060188889A1 (en) * | 2003-11-04 | 2006-08-24 | Christopher Burgess | Use of differentially expressed nucleic acid sequences as biomarkers for cancer |
| US20090150315A1 (en) * | 2005-07-29 | 2009-06-11 | Siemens Healthcare Diagnostics Inc. | Neoplastic Disease-Related Methods, Kits, Systems and Databases |
| US20130216545A1 (en) * | 2012-02-19 | 2013-08-22 | Claresa Levetan | Early Diagnosis and Novel Treatment of Cancer |
| CN111562395A (en) * | 2020-06-11 | 2020-08-21 | 青岛大学 | Marker of pancreatic cancer tumor and application and kit thereof |
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| CN115166260A (en) * | 2022-07-11 | 2022-10-11 | 东南大学 | Application of vitamin D binding protein in plasma brain cell source exosome in diagnosing depression |
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