CN107664696B - double-antibody sandwich ELISA kit for adult glioma diagnosis - Google Patents
double-antibody sandwich ELISA kit for adult glioma diagnosis Download PDFInfo
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Abstract
the invention discloses application of serum CCKBR as a glioma diagnosis marker and a double-antibody sandwich ELISA diagnosis kit for glioma. The invention discloses a novel biomarker for diagnosing glioma, which has particularly important function and clinical application value for early warning and early diagnosis of glioma. Meanwhile, an enzyme-linked immunosorbent assay (ELISA) diagnosis system based on a double-antibody sandwich principle is established and developed into a glioma diagnosis kit, so that CCKBR can be rapidly and accurately detected, rapid auxiliary diagnosis can be performed on primary glioma of each stage (particularly early stage), and the ELISA diagnosis kit has important clinical application value. The kit has the advantages of simple operation, stable reagent, good repeatability, strong specificity, high sensitivity and the like, and is easy to popularize and apply in a large range.
Description
Technical Field
The invention belongs to the technical field of biological medicines. More particularly, relates to a double-antibody sandwich ELISA detection method and a kit for adult glioma diagnosis.
background
The term "glioma" refers to a tumor of glial cell origin, including astrocytoma, oligodendroglioma, oligoastrocytoma, and ependymoma, among others. Gliomas are the most common primary intracranial tumors, and the classification of WHO central nervous system tumors classifies gliomas into I-IV grades, wherein the I and II grades are low-grade gliomas, and the III and IV grades are high-grade gliomas. Gliomas can be pathologically diagnosed and classified by tumorigenic sites, cell morphology, arrangement, differentiation characteristics, immunophenotype, ultrastructural and molecular genetic alterations.
Histomorphometric observation remains the basis of pathological diagnosis, but molecular biological labeling of gliomas is an important advance in modern diagnostic pathology. In 2016, the latest version of WHO (white central nervous system) tumor classification changes the traditional glioma morphological typing method subversively, molecular typing is used as the core basis of tumor typing for the first time, and the new genotyping method combining genotype and appearance increases the diagnosis accuracy, can better judge the prognosis of patients and accurately guide treatment. Certainly, we must also clear that the existing biological markers are not specific or specific to a certain kind of tumor, and as science and technology develops, more biological markers are needed, so that in clinical diagnosis, multiple biological markers should be used for comprehensive evaluation.
In addition, the diagnosis of the glioma at present mostly depends on the imaging technology, and early warning cannot be realized.
Disclosure of Invention
the invention aims to overcome the defects and shortcomings of the existing glioma diagnosis technology and provides a novel glioma diagnosis marker, namely a human cholecystokinin B receptor (CCKBR). The research of the invention finds that the serum CCKBR level is a better detection index of glioma, can be used as a clinical auxiliary diagnosis means of glioma, and particularly has important functions and clinical application values for early warning and early diagnosis of early stage (I-grade glioma) of glioma.
the invention aims to provide application of serum CCKBR as a glioma diagnosis marker.
The invention also aims to provide a double-antibody sandwich ELISA detection method of serum CCKBR and a kit thereof.
The above purpose of the invention is realized by the following technical scheme:
the human cholecystokinin B receptor (CCKBR) is located on chromosome 11p15.4, has the total length of 12454bp, has the mRNA length of 2206bp, and has 5 exons and 4 introns. The receptor is mainly present in the central nervous system and the digestive system, regulates dopamine and influences the transmission of brain neurotransmitters, regulates mental anxiety, exercise and food intake. The inventor finds that the serum CCKBR level is a better detection index of the glioma by detecting the content of CCKBR in the serum of normal people and glioma patients and analyzing the correlation between the content of CCKBR and the glioma by statistics, and particularly finds a new biomarker for clinical serological early diagnosis of the disease for early warning and early diagnosis of the glioma.
therefore, the application of the serum CCKBR as a glioma diagnosis marker and the application of the serum CCKBR as an early warning and/or early diagnosis marker of glioma onset are both within the protection scope of the invention.
The invention provides a glioma diagnostic kit which comprises a human CCKBR recombinant monoclonal antibody.
Preferably, the kit comprises the following components:
(1) a human CCKBR recombinant monoclonal antibody coated on a solid-phase 96-well enzyme label plate;
(2) enzyme conjugate working solution: horse Radish Peroxidase (HRP) marked human CCKBR recombinant monoclonal antibody reaction solution;
(3) developing solution ABTS substrate: ABTS 1mg + 0.1M pH5.0 citric acid buffer 2ml + 3% H2O2 (ready for use);
(4) Washing solution (0.05% Tween 20-PBS);
(5) conjugate dilution: 0.1% BSA-PBS;
(6) Stopping liquid: 1 to 2M H2SO 4.
More preferably, the kit further comprises:
(7) Positive control: human CCKBR standard protein;
(8) Negative control: standard negative sera.
preferably, the kit refers to a glioma diagnosis kit and an early warning diagnosis kit.
the invention also provides a double-antibody sandwich ELISA detection method of serum CCKBR, and the kit is used for carrying out double-antibody sandwich ELISA detection.
preferably, the steps of the double-antibody sandwich ELISA detection method for serum CCKBR are as follows:
S1, coating: diluting the CCKBR monoclonal antibody to 0.5-2 mu g/ml by using a coating buffer solution (0.05M, pH9.6 Na2CO3-NaHCO3 buffer solution), adding the diluted CCKBR monoclonal antibody into a 96-well plate, placing the diluted CCKBR monoclonal antibody at 90-110 mu l/well for 30-40 hours at 0-10 ℃;
s2, sealing: washing the 96-well plate for several times by using a washing solution (0.05% Tween 20-PBS), adding 180-220 mu l/well of a conjugate diluent (0.1% BSA-PBS), and standing at 35-38 ℃ for 1.5-2.5 hours;
S3, adding a serum sample to be detected: washing the 96-well plate for several times by using a washing solution, adding 90-110 mu l/well of a standard substance (the standard substance is the positive control, human CCKBR standard protein) diluted by the serum sample to be detected and the conjugate diluent, and standing for 1.5-2.5 hours at 35-38 ℃;
S4, adding an enzyme-labeled antibody: the 96-well plate is washed several times with washing solution, and the enzyme-labeled antibody is diluted with conjugate diluent to 1: 1000, adding a 96-well plate, 90-110 mu l/well, and standing for 1.5-2.5 hours at 35-38 ℃;
S5, color development: washing the 96-well plate for several times by using washing liquid, adding a developing liquid ABTS substrate into the 96-well plate, adding a stopping liquid into the 96-well plate at 90-110 mu l/well, and developing for 15-30 minutes at room temperature or 35-38 ℃;
S6, standard curve: and drawing a standard curve according to the OD value of the standard substance, checking the content of the sample to be detected on the standard curve, and counting the content of serum CCKBR according to data.
more preferably, as a preferred embodiment, the method for detecting serum CCKBR by double antibody sandwich ELISA comprises the following steps:
S1, coating: diluting CCKBR monoclonal antibody to 1 μ g/ml with coating buffer solution, adding into 96-well plate, 100 μ l/well, standing at 4 deg.C for 36 hr;
S2, sealing: washing 96-well plate with washing solution for 5 times, adding conjugate diluent 200 μ l/well, standing at 37 deg.C for 2 hr;
S3, adding a serum sample to be detected: washing 96-well plate with washing solution for 5 times, adding 100 μ l/well of standard substance diluted by serum sample to be detected and conjugate diluent, and standing at 37 deg.C for 2 hr;
s4, adding an enzyme-labeled antibody: wash washes 96-well plates 5 times, diluting enzyme-labeled antibody with conjugate diluent to 1: 1000, adding a 96-well plate, 100 mu l/well, and standing at 37 ℃ for 2 hours;
S5, color development: washing a 96-well plate for 5 times by using washing liquid, adding 100 mu l/well of color development liquid ABTS substrate into the 96-well plate, adding stop solution, and developing for 15-30 minutes at room temperature or 37 ℃;
s6, standard curve: and drawing a standard curve according to the OD value of the standard substance, checking the content of the sample to be detected on the standard curve, and counting the content of serum CCKBR according to data.
The invention has the following beneficial effects:
the invention discloses a novel biomarker for diagnosing glioma, which has particularly important function and clinical application value for early warning and early diagnosis of glioma.
meanwhile, the invention also establishes an enzyme-linked immunosorbent assay system chicken based on the double-antibody sandwich principle, develops the enzyme-linked immunosorbent assay system chicken into a glioma diagnosis kit, can quickly and accurately detect CCKBR, does not need to dilute a serum sample, and obviously improves the sensitivity; therefore, the rapid auxiliary diagnosis can be made on the primary glioma in each stage (particularly in the early stage), and the method has important clinical application value.
The detection kit has the advantages of simple operation, stable reagent, good repeatability, strong specificity, high sensitivity and the like, and is easy to popularize and apply in a large range, thereby having wide market prospect.
drawings
FIG. 1: t test results of samples of serum CCKBR concentration box plots of normal adults (70), all gliomas (121), grade I (20), grade II (27), grade III (43), and grade IV (31), and serum CCKBR concentration of each glioma cohort patients, independent of normal human serum CCKBR concentration. Mapping and statistics were done with statistical software SPSS 22.0.
Detailed Description
the invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1
1. Experimental serum sample materials (see table 1):
(1) 70 normal adult samples: the serum samples of normal physical examination population without metabolic diseases (diabetes, nephropathy), infectious diseases (HBV, HCV and HIV) and any tumor were tested by clinical laboratory.
(2) 121 parts glioma patient serum sample: which comprises 20 parts of phase I, 27 parts of phase II, 43 parts of phase III and 31 parts of phase IV.
table 1: demographic and clinical data for normal adults and glioma patients
2. double-antibody sandwich ELISA (enzyme-Linked immuno sorbent assay) detection kit for CCKBR (complementary continuous polymerase chain reaction)
The kit is internally provided with:
(1) a human CCKBR recombinant monoclonal antibody coated on a solid-phase 96-well enzyme label plate;
(2) the enzyme conjugate working solution is Horse Radish Peroxidase (HRP) marked human CCKBR recombinant monoclonal antibody reaction solution;
(3) The positive control is human CCKBR standard protein, and the negative control is standard negative serum;
(4) Developing solution ABTS substrate: ABTS 1mg + 0.1M, pH5.0 citric acid buffer 2ml + 3% H2O 24 μ l (for use);
(5) Washing solution (0.05% Tween 20-PBS);
(6) Conjugate dilution: 0.1% BSA-PBS (for dilution of specimens, standards and binders);
(7) A stop solution (1-2M H2SO 4).
3. detection method
(1) Coating: the crude protein of CCKBR monoclonal antibody was diluted to 1. mu.g/ml with coating buffer (0.05M, pH9.6 Na2CO3-NaHCO3 buffer), added to a 96-well plate at 100. mu.l/well, and left at 4 ℃ for 36 hours.
(2) And (3) sealing: the washing solution was washed 5 times on the 96-well plate, and 200. mu.l/well of the conjugate dilution was added thereto, and the mixture was left at 37 ℃ for 2 hours.
(3) Adding a sample to be detected: washing 96-well plate with washing solution for 5 times, adding 100 μ l/well of standard substance diluted by sample to be detected and conjugate diluent, and standing at 37 deg.C for 2 hr.
(4) adding an enzyme-labeled antibody: washing 96-well plate with washing solution for 5 times, diluting enzyme-labeled antibody with conjugate diluent at a ratio of 1: 1000, adding into 96-well plate at a concentration of 100 μ l/well, and standing at 37 deg.C for 2 hr.
(5) color development: washing the 96-well plate for 5 times by using washing liquid, adding 100 mu l/well of a developing liquid ABTS substrate into the 96-well plate, and developing for 15-30 minutes at the temperature of a stop liquid or at 37 ℃.
(6) standard curve: and drawing a standard curve according to the OD value of the standard substance, and checking the content of the sample to be detected on the standard curve.
P P P(7) And (3) data statistics: all statistics were expressed as mean ± S using the SPSS 22.0 software Package ((statistical Package for the Social Sciences, Chicago, IL, USA), all P values were two decimal places, P <0.05 was considered statistically significant, and P <0.01 was considered significantly significant.
4. The results are shown in Table 2 and FIG. 1.
(1) FIG. 1: t test results of samples of serum CCKBR concentration box plots of normal adults (70), all gliomas (121), grade I (20), grade II (27), grade III (43), and grade IV (31), and serum CCKBR concentration of each glioma cohort patients, independent of normal human serum CCKBR concentration. Mapping and statistics were done with statistical software SPSS 22.0.
Pall gliomas therein (not grouped): normal, CCKBR mean ratio, P = 6.498X 10-12
PGrade I glioma: normal, CCKBR mean ratio, P =0.023
PGrade II glioma: normal, CCKBR mean ratio, P =0.000049
PGrade III glioma: normal, CCKBR mean ratio, P =5.567 × 10-7
PGrade IV glioma: normal, CCKBR mean ratio, P =0.000010
PNote: when the P value is less than 0.05, the comparison of the mean values of the two is proved to have statistical significance;
PWhen the P value is less than 0.01, the comparison of the two means has significance statistics.
(2) Table 2: normal adult, all glioma, grade I glioma, grade II glioma, grade III glioma, and grade IV glioma patients have mean values of serum CCKBR concentrations and their standard deviations. Statistics are done by the statistical software SPSS 22.0.
TABLE 2
(3) Analysis of results
Mean concentrations of CCKBR (13.29 pg/ml) in the sera of patients with gliomas (not grouped) were higher than the mean concentration of CCKBR (5.18 pg/ml) in the sera of normal humans (table 2) and were statistically significant (P =6.498 × 10-12, fig. 1).
PAfter the grouping of gliomas, the statistical significance was observed in comparison with the normal group (FIG. 1). Wherein the mean concentration of CCKBR in serum from grade I glioma patients (8.78pg/ml) was slightly higher than that of normal group (5.18 pg/ml) (table 2), with statistical significance (P =0.023, fig. 1); other groups, including grade II, III and IV glioma patients all had significantly higher mean serum CCKBR concentrations than normal (P values <0.01, table 2 and figure 1).
the experimental result proves that the serum CCKBR level is a better detection index of glioma; especially, the reagent has diagnostic value for early patients (I-grade glia), plays a role in early warning and has potential clinical application value.
Claims (5)
1. The application of the detection reagent of serum CCKBR in the preparation of glioma diagnostic reagent kit.
2. The application of the detection reagent of the serum CCKBR in the preparation of the glioma early warning and/or early diagnosis kit.
3. The use of claim 1 or 2, wherein the detection reagent comprises a human CCKBR recombinant monoclonal antibody.
4. the use of claim 3, wherein the detection reagent further comprises the following components:
(1) A human CCKBR recombinant monoclonal antibody coated on a solid-phase 96-well enzyme label plate;
(2) enzyme conjugate working solution: horse radish peroxidase-labeled human CCKBR recombinant monoclonal antibody reaction solution;
(3) Developing solution ABTS substrate: ABTS + 0.1M citric acid buffer pH5.0 + 3% H2O 2;
(4) washing liquid: 0.05% Tween 20-PBS;
(5) Conjugate dilution: 0.1% BSA-PBS;
(6) stopping liquid: 1 to 2M H2SO 4.
5. the use of claim 4, wherein the detection reagent further comprises the following components:
(7) positive control: human CCKBR standard protein;
(8) Negative control: standard negative sera.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1255860A (en) * | 1997-05-12 | 2000-06-07 | 埃弗顿有限公司 | Immunogenic compsns to CCK-b/gastrin-receptor and methods for treatment of tumors |
| CN101514989A (en) * | 2008-12-19 | 2009-08-26 | 中山大学 | Application of serum HSP27 in preparing diagnostic reagent kit for primary hepatocellular carcinoma |
| CN105734159A (en) * | 2016-04-29 | 2016-07-06 | 北京泱深生物信息技术有限公司 | Molecular marker of esophageal squamous carcinoma |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1255860A (en) * | 1997-05-12 | 2000-06-07 | 埃弗顿有限公司 | Immunogenic compsns to CCK-b/gastrin-receptor and methods for treatment of tumors |
| CN101514989A (en) * | 2008-12-19 | 2009-08-26 | 中山大学 | Application of serum HSP27 in preparing diagnostic reagent kit for primary hepatocellular carcinoma |
| CN105734159A (en) * | 2016-04-29 | 2016-07-06 | 北京泱深生物信息技术有限公司 | Molecular marker of esophageal squamous carcinoma |
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