CN116735888A - Indirect ELISA method for detecting COG5 by specific polyclonal antibody - Google Patents
Indirect ELISA method for detecting COG5 by specific polyclonal antibody Download PDFInfo
- Publication number
- CN116735888A CN116735888A CN202211447833.3A CN202211447833A CN116735888A CN 116735888 A CN116735888 A CN 116735888A CN 202211447833 A CN202211447833 A CN 202211447833A CN 116735888 A CN116735888 A CN 116735888A
- Authority
- CN
- China
- Prior art keywords
- cog5
- pbst
- elisa method
- indirect elisa
- washing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 102100036030 Conserved oligomeric Golgi complex subunit 5 Human genes 0.000 title claims abstract description 116
- 101000876001 Homo sapiens Conserved oligomeric Golgi complex subunit 5 Proteins 0.000 title claims abstract description 116
- 238000000034 method Methods 0.000 title claims abstract description 42
- 238000002965 ELISA Methods 0.000 title claims abstract description 41
- 210000002966 serum Anatomy 0.000 claims abstract description 48
- 239000011248 coating agent Substances 0.000 claims abstract description 45
- 238000000576 coating method Methods 0.000 claims abstract description 45
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims abstract description 37
- 239000007788 liquid Substances 0.000 claims abstract description 34
- 239000000243 solution Substances 0.000 claims abstract description 31
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims abstract description 28
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims abstract description 28
- 108090000790 Enzymes Proteins 0.000 claims abstract description 25
- 102000004190 Enzymes Human genes 0.000 claims abstract description 25
- 238000005406 washing Methods 0.000 claims abstract description 23
- 238000011161 development Methods 0.000 claims abstract description 19
- 238000007789 sealing Methods 0.000 claims abstract description 15
- 238000007865 diluting Methods 0.000 claims abstract description 14
- 238000006243 chemical reaction Methods 0.000 claims abstract description 10
- 239000012089 stop solution Substances 0.000 claims abstract description 8
- 239000013642 negative control Substances 0.000 claims abstract description 6
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 5
- 239000012895 dilution Substances 0.000 claims description 28
- 238000010790 dilution Methods 0.000 claims description 28
- 239000000427 antigen Substances 0.000 claims description 9
- 102000036639 antigens Human genes 0.000 claims description 9
- 108091007433 antigens Proteins 0.000 claims description 9
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 7
- 238000011534 incubation Methods 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000010009 beating Methods 0.000 claims description 2
- 239000002981 blocking agent Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 14
- 230000009977 dual effect Effects 0.000 abstract 1
- 210000004369 blood Anatomy 0.000 description 23
- 239000008280 blood Substances 0.000 description 23
- 201000000057 Coronary Stenosis Diseases 0.000 description 22
- 208000031481 Pathologic Constriction Diseases 0.000 description 22
- 230000036262 stenosis Effects 0.000 description 22
- 208000037804 stenosis Diseases 0.000 description 22
- 206010008479 Chest Pain Diseases 0.000 description 20
- 230000000903 blocking effect Effects 0.000 description 19
- 238000002474 experimental method Methods 0.000 description 16
- 206010003211 Arteriosclerosis coronary artery Diseases 0.000 description 15
- 208000029078 coronary artery disease Diseases 0.000 description 15
- 238000004458 analytical method Methods 0.000 description 14
- 238000002586 coronary angiography Methods 0.000 description 14
- 208000026758 coronary atherosclerosis Diseases 0.000 description 14
- 238000012360 testing method Methods 0.000 description 12
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 10
- 230000003902 lesion Effects 0.000 description 9
- 238000011084 recovery Methods 0.000 description 9
- 210000004351 coronary vessel Anatomy 0.000 description 8
- 206010011089 Coronary artery stenosis Diseases 0.000 description 7
- 108010023302 HDL Cholesterol Proteins 0.000 description 7
- 238000002835 absorbance Methods 0.000 description 7
- 230000000875 corresponding effect Effects 0.000 description 7
- 238000007477 logistic regression Methods 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 230000003143 atherosclerotic effect Effects 0.000 description 6
- 230000002596 correlated effect Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 229940109239 creatinine Drugs 0.000 description 5
- 238000012417 linear regression Methods 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 210000000440 neutrophil Anatomy 0.000 description 5
- 208000037260 Atherosclerotic Plaque Diseases 0.000 description 4
- 108010049003 Fibrinogen Proteins 0.000 description 4
- 102000008946 Fibrinogen Human genes 0.000 description 4
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 4
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 4
- 238000003745 diagnosis Methods 0.000 description 4
- 229940012952 fibrinogen Drugs 0.000 description 4
- 229940116269 uric acid Drugs 0.000 description 4
- 108010074051 C-Reactive Protein Proteins 0.000 description 3
- 102100032752 C-reactive protein Human genes 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 206010020751 Hypersensitivity Diseases 0.000 description 3
- 238000003657 Likelihood-ratio test Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000007717 exclusion Effects 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 210000001616 monocyte Anatomy 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 238000000611 regression analysis Methods 0.000 description 3
- 238000007619 statistical method Methods 0.000 description 3
- 201000001320 Atherosclerosis Diseases 0.000 description 2
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 241000282414 Homo sapiens Species 0.000 description 2
- 101001051093 Homo sapiens Low-density lipoprotein receptor Proteins 0.000 description 2
- 102100024640 Low-density lipoprotein receptor Human genes 0.000 description 2
- 238000010220 Pearson correlation analysis Methods 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000007505 plaque formation Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 230000000391 smoking effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 210000001685 thyroid gland Anatomy 0.000 description 2
- 150000003626 triacylglycerols Chemical class 0.000 description 2
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000019838 Blood disease Diseases 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 208000032928 Dyslipidaemia Diseases 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 238000001207 Hosmer–Lemeshow test Methods 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 206010023126 Jaundice Diseases 0.000 description 1
- 208000017170 Lipid metabolism disease Diseases 0.000 description 1
- 238000000585 Mann–Whitney U test Methods 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 206010062237 Renal impairment Diseases 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000010968 computed tomography angiography Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000010219 correlation analysis Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 238000013211 curve analysis Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 238000011984 electrochemiluminescence immunoassay Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 230000035931 haemagglutination Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000014951 hematologic disease Diseases 0.000 description 1
- 208000018706 hematopoietic system disease Diseases 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000010832 independent-sample T-test Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 210000004020 intracellular membrane Anatomy 0.000 description 1
- 230000005977 kidney dysfunction Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 230000005976 liver dysfunction Effects 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000008172 membrane trafficking Effects 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 210000002321 radial artery Anatomy 0.000 description 1
- 238000002601 radiography Methods 0.000 description 1
- 238000007430 reference method Methods 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Classifications
-
- 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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- 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
- G01N21/314—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths
- G01N21/3151—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths using two sources of radiation of different wavelengths
-
- 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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/20—Air quality improvement or preservation, e.g. vehicle emission control or emission reduction by using catalytic converters
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Toxicology (AREA)
- Cell Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses an indirect ELISA method for detecting COG5 by a specific polyclonal antibody, which comprises the following steps: coating the COG5 recombinant protein by using a coating liquid; pouring out the coating liquid in the plate holes the next day, and washing with PBST; sealing the coated ELISA plate by using a sealing agent; pouring off the sealing liquid and washing with PBST; diluting multiple antisera with PBST, setting blank control and negative control, and incubating in incubator; the immune serum was decanted and washed with PBST; diluting the enzyme-labeled secondary antibody by using PBST, incubating in a box, and washing the plate; adding a double-component color development solution A and B for warm bath; adding a stop solution to stop the color reaction; and (5) detecting the OD value of the dual wavelength. The method for measuring the serum COG5 has good detection performance and can preliminarily meet clinical requirements.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an indirect ELISA method for detecting COG5 by a specific polyclonal antibody.
Background
Cardiovascular disease is one of the diseases with higher social mortality rate in modern generation, is obviously higher than malignant tumor and other diseases, and is the first killer threatening the life safety and physical health of human beings. Studies have shown that dyslipidemia is a central factor in AS formation. Golgi is a central hub for lipid and protein transport/sorting, and is also the main site of protein and lipid glycosylation. The Conserved Oligomeric Golgi (COG) complex is reported to be located on the membranous surface of the golgi, consisting of eight subunits (COG 1-COG 8), distributed in two leaves. Among them, COG5 plays an important role in stabilizing the structure and function of golgi, and can affect intracellular membrane trafficking. Previous studies have found that knocking down COG5 results in intracellular LDLR accumulation. Further studies have found that COG5 expression is inversely correlated with LDLR expression in atherosclerotic plaques in humans. COG5 is therefore closely related to coronary atherosclerosis.
Thus, we speculate that COG5 may be released into the blood from coronary atherosclerotic plaques during the course of atherosclerotic cardiovascular disease. Whether or not a detection method can be used to determine the presence or absence and extent of stenosis of the coronary artery by detecting COG5 concentration in serum? The basic research is converted into clinical diagnosis application.
At present, the basic research on COG5 is more, no reference method for detecting serum COG5 exists at home and abroad, the detection results of all laboratories are not comparable, and the laboratory is not put into clinical application. Therefore, it is necessary to provide a novel indirect ELISA method for detecting COG5 by specific polyclonal antibodies.
Disclosure of Invention
In view of the above, the present invention provides an indirect ELISA method for detecting COG5 by using a specific polyclonal antibody.
In order to solve the technical problems, the invention discloses an indirect ELISA method for detecting COG5 by a specific polyclonal antibody, which comprises the following steps:
(1) Coating COG5 recombinant protein with coating solution, coating 100 μl/well on 96-well plate;
(2) Washing the plate: pouring out the coating liquid in the plate holes on the next day, washing with PBST three times, and taking 250 mu l of each hole for 3-5 minutes each time and taking the coating liquid as dry as possible;
(3) Sealing the coated ELISA plate with sealing agent, adding 200 μl into each hole, and performing warm bath at 37deg.C for 1 hr;
(4) Washing the plate: pouring out the sealing liquid in the plate hole, washing with PBST three times, and taking 250 mu l of each hole for 3-5 minutes each time as dry as possible;
(5) Adding primary immune serum, diluting multiple antisera with PBST, simultaneously setting blank control and negative control, adding 100 μl of each well, and incubating at 37deg.C for 1 hr;
(6) Washing the plate: pouring out immune serum in the plate holes, washing with PBST three times, 250 μl each time, 3-5 minutes each time, and beating as dry as possible;
(7) After the incubation of the primary antibody is finished and washed, diluting the secondary antibody of the enzyme label to 1:10000 dilution by using PBST, incubating 100 μl of each hole in a light-resistant incubator at 37 ℃ for 1h, and washing the plate;
(8) Adding 50 mu l of each hole of the two-component color development liquid A and B, placing the two-component color development liquid A and B into a constant-temperature water bath box, and performing light-proof operation in the whole process, wherein the temperature bath is not equal for 1-15min at 37 ℃;
(9) Adding 100 mu l of stop solution into each hole to stop the chromogenic reaction;
(10) And (3) detecting the double-wavelength OD value at 450-630nm by using a Thermo microplate reader.
Optionally, the amino acid sequence of the COG5 recombinant protein antigen is shown as SEQ ID NO. 1.
Optionally, the concentration of the COG5 recombinant protein in the step (1) is 0.0514ng/ml to 11.1111ng/ml.
Optionally, the coating solution in the step (1) and the COG5 recombinant protein antigen are diluted according to the ratio of 1:10; the coating conditions were 37℃for 1 hour, then placed in a refrigerator at 4℃for 24 hours overnight.
Optionally, the blocking agent of step (3) is 1% bsa.
Alternatively, the antisera of step (5) are diluted 1:5000 with PBST.
Optionally, the enzyme-labeled secondary antibody in the step (7) is an anti-rabbit secondary antibody labeled by horseradish peroxidase, and the dilution of the enzyme-labeled secondary antibody is 1:10000.
Optionally, the two-component color developing solutions A and B in the step (8) are TMB two-component color developing solutions PR1210-100ml respectively.
Optionally, the color reaction in the step (9) is 3min.
Optionally, the stop solution in the step (10) is C1058.
Compared with the prior art, the invention can obtain the following technical effects:
based on the specific polyclonal antibody of the COG5, an indirect ELISA method for detecting the COG5 by the specific polyclonal antibody is established. The method for measuring the serum COG5 has good detection performance and can preliminarily meet clinical requirements. COG5 has good diagnostic efficacy as an indicator for determining the presence or absence of coronary atherosclerotic plaques.
Of course, it is not necessary for any of the products embodying the invention to achieve all of the technical effects described above at the same time.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and do not constitute a limitation on the invention. In the drawings:
FIG. 1 is a graph of COG5 versus OD values according to the present invention;
FIG. 2 is a linear curve fitted with the COG5 recombinant protein of the invention;
FIG. 3 is a COG5 polyclonal antibody 1:5000 of the invention;
FIG. 4 is a COG5 polyclonal antibody 1:10000 according to the invention;
FIG. 5 is a COG5 polyclonal antibody 1:20000 of the present invention;
FIG. 6 is a selection of BSA blocking solutions of different concentrations according to the present invention;
FIG. 7 is a selection of color development time of the present invention;
FIG. 8 is a graph showing COG5 data distribution between the normal control and CAA groups according to the present invention;
FIG. 9 is a comparison of COG5 levels between the normal control and CAA groups of the present invention;
FIG. 10 is a ROC curve of COG5 of the invention for diagnosing CAA in a chest pain population in a local area;
FIG. 11 is a graph of the coronary atherosclerosis ROC of the chest pain diagnosing population according to models 1 and 2 of the present invention;
FIG. 12 is a correlation of COG5 with GENSINI scores of the present invention;
FIG. 13 is a GENSINI score inter-group COG5 level analysis according to the invention;
FIG. 14 is a calibration curve of the coronary stenosis model of the present invention;
FIG. 15 is a ROC curve for the coronary stenosis model differentiation of the present invention;
FIG. 16 is a graph of coronary stenosis degree alignment of the present invention.
Detailed Description
The following will describe embodiments of the present invention in detail by referring to examples, so that the implementation process of how to apply the technical means to solve the technical problems and achieve the technical effects of the present invention can be fully understood and implemented.
COG5 recombinant protein antigen was purchased from CUSABIO corporation, china, cat: CSB-EP890771HU, the expression host is Escherichia coli, the volume is 20 mu L/branch, the mass is 20 mu g/branch, the molecular weight is 33.8kDa, the split charging concentration is 1.0mg/ml, and the preservation conditions are as follows: cryopreservation at-20deg.C or-80deg.C, wherein the amino acid sequence is MGWVGGRRRDSASPPGRSRSAADDINPAPANMEGGGGSVAVAGLGARGSGAAAATVRELLQDGCYSDFLNEDFDVKTYTSQSIHQAVIAEQLAKLAQGISQLDRELHLQVVARHEDLLAQATGIESLEGVLQMMQTRIGALQGAVDRIKAKIVEPYNKIVARTAQLARLQVACDLLRRIIRILNLSKRLQGQLQGGSREITKAAQSLNELDYLSQGIDLSGIEVIENDLLFIARARLEVENQAKRLLEQGLETQNPT as shown in SEQ ID NO. 1;
Anti-COG5 antibody IgG (rabbit polyclonal antibody), cat No. ab229830, available from Abcam, usa, the immunogen is: recomtinant frogment coresponding to Human COG5s 1.300Database link:Q9UP83, the split charging concentration is 0.39mg/ml, and the volume is 50 mu L/branch.
The Goat anti-Human IgG Fc (HRP) Goat anti-rabbit HRP-labeled IgG (polyclonal antibody), cat# ab205718, available from Abcam, inc., U.S.A., the immunogens were: rabbt IgG, whole molecule, split concentration 2.0mg/ml, volume 500. Mu.L/branch.
All reagents and recombinant proteins are packaged and stored, wherein the recombinant protein is 2.0 mu l per tube, the Anti-COG5 polyclonal antibody is 2.5 mu l per tube, and the enzyme-labeled secondary antibody is 2.0 mu l per tube, and the recombinant protein are frozen and stored at-20 ℃ for one tube at a time so as to ensure the stable state of the packaged antibodies.
The main reagents are as follows in table 1:
TABLE 1 Main reagents
Example 1 Indirect ELISA method for detecting COG5 by optimizing specific polyclonal antibodies
1. Determination of linear concentration Range of COG5 recombinant protein antigen Standard
By establishing a relation model of COG5 recombinant protein antigens with different concentrations and OD values, selecting a part with better COG5 linearity from the relation model for R 2 Calculating the value, thereby narrowing the concentration range of COG5 detection, and detecting again until obtaining higher R 2 Values. The experimental method is specifically as follows
(1) Coating 100. Mu.l/well of COG5 recombinant protein (900 ng/ml, 300ng/ml, 100ng/ml, 33.3333ng/ml, 11.1111ng/ml, 3.7037ng/ml …, 0.0064 ng/ml) was coated on 96-well plates using a coating solution at a ratio of 37℃for 1 hour, then placed in a refrigerator at 4℃for 24 hours overnight.
(2) Washing the plate: the next day the plate wells were emptied of coating solution, washed three times with PBST, 250 μl each, 3-5 minutes each, and patted dry as much as possible.
(3) Sealing the coated ELISA plate with sealing agent, adding 200 μl of the sealing agent into each hole, and performing warm bath at 37deg.C for 1 hr. (blocking treatment with 1% BSA+PBST temporarily.)
(4) Washing the plate: the plate wells were emptied of blocking solution, washed three times with PBST, 250 μl each, 3-5 minutes each, and patted dry as much as possible.
(5) Immune serum was added to the dilution of polyclonal serum to a dilution of 1:5000 with PBST, with blank and negative controls, 100. Mu.l per well, and incubated for 1h at 37℃after addition.
(6) Washing the plate: the immune serum in the wells was decanted, washed three times with PBST, 250 μl each, 3-5 minutes each, and patted dry as much as possible.
(7) After the incubation of the primary antibody is completed, horseradish peroxidase (HRP) -labeled anti-rabbit secondary antibody (diluted 1:10000 with PBST) is added, and each well is incubated for 1h in a light-resistant incubator at 37 ℃ and washed.
(8) Adding 50 μl of each hole of the two-component color development liquid A and B, placing into a constant temperature water bath, and performing light-proof operation in the whole course, wherein the temperature bath is not uniform for 1-15min at 37deg.C.
(9) 100 μl of stop solution was added to each well to terminate the chromogenic reaction.
(10) And (3) detecting the double-wavelength OD value at 450-630nm by using a Thermo microplate reader.
(11) And carrying out result analysis by using corresponding statistical software.
Establishing a relation model of a gradient concentration COG5 recombinant protein standard substance and an absorbance value, wherein when the concentration of the COG5 recombinant protein is between 0.0514ng/ml and 11.1111ng/ml, the COG5 concentration is linearly related to an OD value, and the correlation coefficient R is shown in the figures 1 and 2 2 0.9910.
2. Optimizing dilution of COG5 polyclonal antibody and enzyme-labeled secondary antibody
Selecting the optimal dilution concentration of the COG5 polyclonal antibody and the enzyme-labeled secondary antibody through an orthogonal experiment, diluting the COG5 polyclonal antibody with the concentration of 0.39mg/ml by a multiple of 1:5000, 1:10000 and 1:20000, and using 2mg/ml of the enzyme-labeled secondary antibody to sample with the gradient of 1:10000, 1:20000 and 1:40000, detecting by ELISA, and comparing the difference of absorbance value in the detection result with R 2 Values. The experimental method is specifically shown in the following ratio
Coating liquid is used for mixing the COG5 recombinant proteins 11.1111ng/ml, 3.7037ng/ml … and 0.0514ng/ml with the coating liquid in the step (1);
in the step (3), blocking treatment is carried out by using 1% BSA+PBST temporarily;
in step (5), the polyclonal serum is diluted to a dilution of 1:5000, 1:10000, 1:20000 with PBST;
in the step (7), the PBST is diluted to 1:10000, 1:20000,1:40000;
the rest of the procedure is the same as in experiment 1 in example 1.
Diluting 0.39mg/ml of COG5 polyclonal antibody with 3 different concentrations (1:5000, 1:10000, 1:20000), sampling 2mg/ml of enzyme-labeled secondary antibody with 3 different concentrations (1:10000, 1:20000, 1:40000), respectively selecting the optimal concentrations of the COG5 polyclonal antibody and the enzyme-labeled secondary antibody by using orthogonal test, and detecting the concentrations as shown in Table 2 and figures 3-5, wherein when the concentration of the COG5 polyclonal antibody is 1:5000 and the sampling concentration of the enzyme-labeled secondary antibody is 1:10000, the correlation coefficient R 2 The value is 0.9935, the concentration of the polyclonal antibody with COG5 is 1:10000, the sample adding concentration of the enzyme-labeled secondary antibody is 1:10000, and the correlation coefficient R is 1:10000 2 The result 0.9946 is almost the same, but the result 2.137 of the difference in OD value at 1:5000 of the COG5 polyclonal antibody concentration is higher than the result 1.721 of the difference in OD value at 1:10000 of the COG5 polyclonal antibody concentration, so that the optimal dilution concentration of the two antibodies is selected, namely, the COG5 polyclonal antibody concentration is 1:5000, and the anti-enzyme-labeled secondary antibody loading concentration is 1:10000.
TABLE 2COG5 polyclonal antibody and enzyme-labeled secondary antibody orthogonal test results
3. Selection of a blocking fluid
The most suitable blocking mode was selected using 1% BSA and 5% BSA as blocking solutions, respectively. Fixing 0.39mg/ml COG5 polyclonal antibody to 1:5000 dilution, diluting 2mg/ml enzyme-labeled secondary antibody to 1:10000 times, and establishing R in mathematical relationship model 2 The value selects the optimal concentration of the blocking fluid. The specific experimental method is as follows
Coating liquid is used for mixing the COG5 recombinant proteins (11.1111 ng/ml, 3.7037ng/ml … and 0.0514 ng/ml) with the coating liquid in the step (1);
in the step (3), 200 mu l of 1% BSA and 5% BSA are used for sealing the coated ELISA plate;
in step (5), the polyclonal serum is diluted to a dilution of 1:5000 with PBST;
in the step (7), PBST is used for diluting the enzyme-labeled secondary antibody to 1:10000 dilution;
the rest of the procedure is the same as in experiment 1 in example 1.
After blocking with 2 kinds of blocking solutions (1% BSA and 5% BSA), the effect of blocking solutions at different concentrations was analyzed based on the experimental results, and the results are shown in Table 3, FIG. 6, the absorbance values and correlation coefficients R of the blocking solutions at 1% BSA and 5% BSA 2 The values were 1.544, 0.9920 and 0.661, 0.9840, respectively, with 1% BSA blocking solution resulting in better than 5% BSA blocking solution, so 1% BSA blocking solution was chosen as blocking solution for the present experiment.
TABLE 3 results of absorbance values for various concentrations of confining liquid
4. Color development time selection
After adding the color development A, B liquid in the color development step, one of 3 reaction times of 1min, 3min and 10min is selected as the optimal reaction time. Fixing 0.39mg/ml COG5 polyclonal antibody to a dilution of 1:5000, diluting 2mg/ml enzyme-labeled secondary antibody to 1:10000 times, performing ELISA detection by establishing standard curve, and comparing the difference of the detection result with R 2 Values. The specific experimental method is as follows
Coating liquid is used for mixing the COG5 recombinant proteins (11.1111 ng/ml, 3.7037ng/ml … and 0.0514 ng/ml) with the coating liquid in the step (1);
in the step (3), 200 mu l of 1% BSA per well is used for sealing the coated ELISA plate;
in step (5), the polyclonal serum is diluted to a dilution of 1:5000 with PBST;
in the step (7), PBST is used for diluting the enzyme-labeled secondary antibody to 1:10000 dilution;
the rest of the procedure is the same as in experiment 1 in example 1.
HRP can react with substrate liquid to form blue, and acid is added to stopThe liquid turned yellow and its OD was measured at a wavelength of 450 nm. The color development time and temperature influence the magnitude of the detection value. The negative control wells remained colorless for a period of time within a range, and the positive wells became darker in color with increasing development time. By comparing 3 different color development times (1 min, 3min, 10 min) of enzyme and substrate reaction, and judging the measurement effect under the conditions of different color development times according to ELISA test results, the results show (see Table 4 and FIG. 7), when the color development time is 3min, namely 50ml of each of the A and B color development solutions is added for reaction for 3min, 100ml of stop solution is added, color comparison is carried out within 5min after uniform mixing, and the OD value is not the highest, but the linear correlation coefficient R is the same 2 The highest value (0.9947) was found to be the color reaction time after the middle period was determined to be 3min.
TABLE 4 absorbance results at different development times
5. Selection of dilution factors for serum samples
A standard curve with gradient dilution concentration of 11.1111ng/ml, 3.7037ng/ml …, 0.0514ng/ml was constructed using COG5 recombinant protein, 10 blood samples were taken from the collected coronary atherosclerotic patient group and normal physical examination group blood samples for gradient dilution of 5%, 10%, 20%, 40%, 80% (i.e., 5. Mu.l serum+95. Mu.l coating solution, 10. Mu.l serum+90. Mu.l coating solution, 20. Mu.l serum+80. Mu.l coating solution, 40. Mu.l serum+60. Mu.l coating solution, 80. Mu.l serum+20. Mu.l coating solution), and blood sample COG5 indirect ELISA was performed to select the optimal blood sample dilution by comparing COG5 concentration in blood samples of different dilutions to the linear range. The specific experimental operation is as follows:
(1) Coating-construction of a standard curve with COG5 recombinant protein, ten blood samples from the group of coronary atherosclerotic patients and from the group of normal physical examination were each subjected to gradient dilution (5%, 10%, 20%, 40%, 80%) with a coating solution, and 100. Mu.l/well coating was performed on 96-well plates at 37℃for 1 hour, and then placed in a refrigerator at 4℃for 24 hours overnight.
In the step (3), 200 mu l of 1% BSA per well is used for sealing the coated ELISA plate;
in step (5), the polyclonal serum is diluted to a dilution of 1:5000 with PBST;
in the step (7), PBST is used for diluting the enzyme-labeled secondary antibody to 1:10000 dilution;
the rest of the procedure is the same as in experiment 1 in example 1.
Comparing 10 blood samples of the coronary atherosclerosis patient group and the normal physical examination group, respectively, carrying out gradient dilution for 5%, 10%, 20%, 40% and 80% (namely, 5 mu l of serum and 95 mu l of coating liquid, 10 mu l of serum and 90 mu l of coating liquid, 20 mu l of serum and 80 mu l of coating liquid, 40 mu l of serum and 60 mu l of coating liquid and 80 mu l of serum and 20 mu l of coating liquid), analyzing the measurement effect of different gradient serum dilutions according to ELISA test results, and determining that when the blood samples of the patient group and the normal group are diluted by 5% and 10%, the final concentration of COG5 in the blood samples is not greatly different, and is higher than the diluted concentration of 20%, 40% and 80%, which means that when the blood is diluted by 10%, the coating liquid can completely coat COG5 antigen to reach the optimal coating condition of the blood samples, so as to save the dosage of the coating liquid, thus determining that the diluted concentration of the blood samples is 10% (namely, diluted by 10 times).
TABLE 5 detection of serum samples at various dilution factors
Example 2 indirect ELISA method for detecting COG5 by specific polyclonal antibodies
The method comprises the following steps:
(1) Coating, namely constructing a standard curve by using the COG5 recombinant protein, diluting the COG5 recombinant protein by using coating liquid according to the ratio of 1:10, coating 100 μl/hole on a 96-well plate at the temperature of 37 ℃ for 1 hour, and then placing the mixture in a refrigerator at the temperature of 4 ℃ for 24 hours overnight; the concentration of the recombinant COG5 protein is 0.0514 ng/ml-11.1111 ng/ml.
(2) Washing the plate: the next day the plate wells were emptied of coating solution, washed three times with PBST, 250 μl each, 3-5 minutes each, and patted dry as much as possible.
(3) Blocking the coated ELISA plate with 200. Mu.l 1% BSA per well was subjected to blocking treatment at 37℃for 1h.
(4) Washing the plate: the plate wells were emptied of blocking solution, washed three times with PBST, 250 μl each, 3-5 minutes each, and patted dry as much as possible.
(5) The antiserum was diluted 1:5000 times with PBST, with blank and negative controls, 100. Mu.l per well, and incubated at 37℃for 1h after addition.
(6) Washing the plate: the immune serum in the wells was decanted, washed three times with PBST, 250 μl each, 3-5 minutes each, and patted dry as much as possible.
(7) After the incubation of the primary antibody is finished and washed, the enzyme-labeled secondary antibody is diluted to 1:10000 dilution by PBST, 100 μl of the enzyme-labeled secondary antibody is added to each well, incubated for 1h in a light-resistant incubator at 37 ℃, and the plates are washed.
(8) 50 μl of each well of the two-component color development liquid A, B (component color development liquid PR1210-100 ml) was added, and the mixture was placed in an incubator and subjected to a full-process light-shielding operation, and incubated at 37℃for 3min.
(9) The reaction was stopped using 100. Mu.l of stop solution per well.
(10) And (3) detecting the double-wavelength OD value at 450-630nm by using a Thermo microplate reader.
(11) And carrying out result analysis by using corresponding statistical software.
EXAMPLE 3 initial evaluation of Performance of an indirect ELISA method for detecting COG5 by specific polyclonal antibodies
1. Linearity:
linearity is defined as the linear dependence of the concentration of a substance on the absorbance value over a range of concentrations, generally defined as R 2 And (3) representing. R is R 2 Generally, the linear correlation is better as the linear correlation is closer to 1, and the linear correlation is more than 0.97. The specific test method was the same as that of example 1 in the test method 1.
When the concentration of the COG5 recombinant protein is between 0.0514ng/ml and 11.1111ng/ml, the concentration of the COG5 is linearly related to the OD value, and a linear regression equation fitted by an average slope method is as follows: y=0.09643+0.1446x, its correlation coefficient R 2 0.9910, which meets the technical specifications of ELISA method, and the result is shown in FIG. 2.
2. Recovery rate experiment:
the recovery rate can reflect the loss degree of the analyte in the sample analysis process, and the lower the loss is, the higher the recovery rate is. The experimental procedure is specifically as follows: a sample of 8 parts of the mixed serum was taken and its COG5 concentration was measured to be 2.121ng/ml, and further divided into 3 parts of 50. Mu.L each. And adding 50 mu L of three different concentrations of COG5 antigen recombinant proteins (3 ng/ml, 1.5ng/ml and 1.0 ng/ml) into the sample respectively, measuring the actual concentration of the three solutions, and obtaining the percentage of the corresponding recovery rate by using a recovery rate calculation formula. The formula is p=c2×v2—c1×v1/c0×v0×100, where V0 is the standard volume; c0 is the standard concentration; v1 is the sample volume; c1 is the sample concentration; v2 is (sample + standard) mixing volume; c2 is the (sample + standard) mixed concentration.
The higher the recovery, the better the reliability of the detection method. The recovery rate of the 3 samples is 90.8%, 99.8% and 88.5%, respectively, and the recovery rate is high. The experimental results are shown in the following table 6
TABLE 6 recovery experimental data
Note that: c0 is the standard concentration; c1 is the sample concentration; c2 is the (sample + standard) mixed concentration.
3. Within-batch and inter-batch repeatability experiments:
in the batch experiment, 3 serum samples with different concentrations (7.131 ng/ml, 6.362ng/ml and 4.140ng/ml respectively) were repeatedly measured on the same ELISA plate for 6 times, and CV values of 3 serum samples were detected. In the batch experiments, 3 serum samples with different concentrations (14.944 ng/ml, 9.691ng/ml and 8.373ng/ml respectively) were measured on 6 different ELISA plates, and CV values of 3 serum samples were also detected. Stability of the method was evaluated by analyzing intra-and inter-batch differences.
The results of the in-batch and inter-batch reproducibility analyses of 3 serum samples at different concentrations using the indirect ELISA method of example 2 are shown in Table 7, wherein the 3 serum samples have an in-batch variation coefficient of 10.12%, 6.43% and 11.61% respectively, and the inter-batch variation coefficients of 10.57%, 12.96% and 11.75% respectively, and the in-batch and inter-batch variation coefficients are less than 15% (CLIA-88), indicating good stability.
TABLE 7 within-and inter-lot repeatability test data
Example 4 clinical application of indirect ELISA method for detecting COG5 by specific polyclonal antibody
This example was performed by establishing a case control study of clinical coronary atherosclerotic patients, and using the indirect ELISA method established in example 2 to determine the difference in serum COG5 levels between coronary atherosclerotic patients identified by coronary angiography and non-coronary atherosclerotic patients of varying degrees of stenosis between 630 cases of intracardiac hospitalizations from a first affiliated hospital of the university of Kunming medical science, as confirmed by chest pain coronary angiography, and 315 cases of normal controls (including 264 cases of non-chest pain chest distress normal physical examination population and 51 cases of non-stenosed patient by chest pain coronary angiography). The clinical value of COG5 in diagnosing CAA occurrence in the chest pain population in the local area was studied and a study was conducted as to whether COG5 could be applied as a non-invasive means for clinical assessment of coronary artery stenosis severity.
1. Study object
1.1 selection of study objects
630 cases of coronary artery atherosclerosis patients which are taken from 6 months in 2020-12 months in the heart department ward of China and are subjected to coronary angiography are selected as case groups, and the ages are 18-90 years; 264 cases of the group of physical examination without chest distress and chest pain in the same period and 51 cases of the group without stenosis, which are clear from the coronary angiography of the department of cardiology, are selected as normal controls, and the ages are 18-90 years. The study received informed consent from the study subjects and kept the relevant information secret during the collection of clinical and analytical data.
1.2 inclusion criteria for study subjects
(1) Normal control person
No chest distress and chest pain, no plaque formation is found in the ultrasonic examination of the blood vessel, and no thickening of the layer thickness in the intima of the blood vessel wall is found; no plaque formation and coronary stenosis caused by it were examined via coronary angiography.
(2) Coronary atherosclerosis patient
Coronary atherosclerosis is defined as any stenosis or plaque found by performing a coronary angiography, with reference to Al-Mallah et Al (Al-Mallah, MH; qureshi, W; lin, FY; et Al Does coronary CT angiography improve risk stratification over coronary calcium scoring in symptomatic patients with suspected coronary artery diseaseResults from the prospective multicenter international CONFIRM registry [ J ]. Eur Heart J Cardiovasc imaging.2014,15 (3): 267-74).
1.3 exclusion criteria for study subjects
One of the following cases is excluded: (1) age <18 years or >90 years; (2) acute trauma or infection; (3) has severe liver and kidney dysfunction (blood ALT is 3 times higher than normal upper limit, blood creatinine)
133. Mu. Mol/L), severe hemagglutination dysfunction; (4) related medical history of malignant tumor, autoimmune disease, infectious inflammatory disease, blood disease, mental abnormality and the like; (5) the information data is imperfect; (7) there are other reasons why coronary examination cannot be tolerated.
2 Experimental methods
2.1 coronary angiography examination and Gensini integration
All patients were coronary angiographed by an experienced interventional cardiologist, and after puncturing the right radial artery with the Seldinger method, coronary angiography was performed using a Terumo common catheter. And two cardiac interventional professionals analyze the radiography results with unified standards and judge the coronary artery stenosis degree (expressed in%) according to the lumen diameter.
Based on the imaging results, the extent of stenosis of each coronary lesion was assessed using the Gensini scoring system (Gensini G G.A more meaningful scoring system for determining the severity of coronary heart disease. Am J Cardiol,1983,51 (3): 606.), which includes mainly two aspects: (1) firstly, determining a basic integral according to the coronary artery stenosis degree, wherein the lumen stenosis is 1 minute from 25% or more to 50% or more, 2 minutes from 50% or more to 50% or more, 4 minutes from 50% or more to 75% or more, 8 minutes from 75% or more to 90% or more, 16 minutes from 90% or more to 99% or more, and 32 minutes from 99% to 100% or less; (2) and then determining corresponding coefficients according to different coronary artery branches, wherein the corresponding coefficients are respectively as follows: left main branch (LM) lesion x 5; lesions of the anterior left descending (LAD): near segment x 2.5, middle segment x 1.5, far segment x 1; lesions of the diagonal branches: d1×1, d2×0.5; lesions of Left Circumflex (LCX): near segment x 2.5, blunt edge branch x 1, posterior descending branch x 1, far segment x 1, posterior side branch x 0.5; lesions of the Right Coronary Artery (RCA): near, middle, far and posterior descending branches are all multiplied by 1. The sum of the integral of each lesion vessel is the total Gensini score of the patient (table 8), which is the integral of the lesion vessel, by multiplying the integral of the stenosis of each coronary artery by the coefficient of the lesion site.
TABLE 8Gensini scoring System details Table
2.2 general clinical data and laboratory index collection for subjects
By consulting patient clinical data, it mainly includes basic demographics (age, sex, height and weight), basic vital signs (heart rate, respiration, blood pressure and body temperature), past medical history, smoking history and medication history. Laboratory related examination indexes of the study subject at this visit are also collected, and blood samples are collected intravenously within 24 hours after the study subject is admitted and after 12 hours of fasting. The test was performed using laboratory equipment from the first affiliated hospital clinical laboratory at the university of Kunming medical science. Comprises the following morning fasting biochemical indexes (bilirubin, creatinine and uric acid), the first blood cell analysis result of the hospital admission, fibrinogen and thyroid gland function examination and the like. The serum concentration of the biochemical index is measured by a Rogowski cobas8000 c701 full-automatic biochemical analyzer, the blood routine examination is measured by a SYSMEX XN-1000 blood cell analyzer, and the thyroid function detection is measured by a Rogowski cobas 601 full-automatic electrochemiluminescence immunoassay analyzer.
2.3 serum sample collection and COG5 concentration detection
2ml of venous blood is collected and is collected into a non-anticoagulation vacuum blood collection tube, serum is rapidly separated through centrifugation under the low-temperature condition (the rotating speed is 4000r/min, the time is 10 min), and the serum is frozen and stored in a refrigerator at the temperature of minus 80 ℃ for testing. Meanwhile, the collection of blood samples with serious lipid, hemolysis, jaundice and other interferences is avoided. The indirect ELISA established in the laboratory is used for detecting the serum COG5, and the U.S. Thermo FC semi-automatic enzyme-labeled instrument is used for detecting the absorbance value of the serum COG5, setting parameters strictly according to the requirements of instrument manufacturers and ensuring the daily quality control. The detection procedure is specifically as in example 2.
2.4COG5 tangential point for diagnosing coronary atherosclerosis of chest pain group in local area
And checking the COG5 concentration distribution characteristics of the included contrast negative group and the coronary atherosclerosis group, selecting a corresponding statistical method according to the distribution type of the data, and further analyzing and comparing the COG5 concentration difference between the groups. In the event of coronary atherosclerosis, a ROC curve was generated using SPSS 20 software to obtain specificity (Spe) and sensitivity (Sen) and calculate a boulder index (Youden index). The corresponding concentration of the maximum value of the Youden index is selected as a tangent point, the cut-off value of COG5 for diagnosing the coronary atherosclerosis of the local group is determined, and the diagnosis capacity is evaluated by calculating the positive likelihood ratio (positive likelihood ratio, +LR) and the negative likelihood ratio (negative likelihood ratio, -LR).
2.5 establishing two models to diagnose coronary atherosclerosis in chest pain crowd in local area
And (3) carrying out binary Logistic stepwise regression analysis on variables (neutrophil count, monocyte count, triglyceride, high density lipoprotein cholesterol, uric acid, creatinine, fasting blood glucose, hypersensitive C reactive protein and fibrinogen (P < 0.05)) with statistical differences in the analysis results of the baseline data through SPSS software, establishing a regression model 1, further, after adding a COG5 index, establishing a regression model 2 again, and finally, comparing the diagnosis efficacy of the two models by using a ROC curve with the coronary angiography stenosis of chest pain crowd as a positive event.
2.6 COG5 level analysis in coronary atherosclerotic patients
In total, 630 patients diagnosed with CAA by first coronary angiography were selected as subjects. The Pearson correlation between patient serum COG5 concentration and Gensini total score was first analyzed using simple linear regression. Coronary stenosis severity was then grouped according to Gensini total score: gensini score less than or equal to 25 is classified as mild stenosis, 25 score < Gensini score less than or equal to 75 is classified as moderate stenosis, and >75 is classified as severe stenosis, and thus the differences in concentration levels of COG5 in four groups of subjects with contrast negative, mild, moderate and severe stenosis are compared. The CAA patients were further divided into two groups: and (3) a light-medium stenosis group and a heavy stenosis group, discussing risk factors of the severity of the coronary artery stenosis by using binary Logistic regression analysis, and constructing a regression model. And evaluating the calibration degree and the distinguishing degree of the model by using the calibration curve and the ROC curve respectively. Finally, the advantage and the clinical application value of the model are shown by adopting an alignment chart.
3 statistical analysis
Data processing, mapping and statistical analysis were performed using SPSS 20, microsoft Excel 2016, graphPadprism 8.0 and R2.6.2 software. The characteristic of the data distribution is comprehensively analyzed by using Shapiro-Wilk normalization test and combining the skewness coefficient and the frequency distribution histogram. The normal distribution of measurement data is represented by mean value (+/-) (standard deviation), namely [ X (+/-) (S) ], the inter-group difference analysis uses independent sample t test, the correlation uses Pearson correlation analysis, the one-factor variance analysis, and the correlation coefficient is represented by r. The non-normally distributed metering data is represented by median (quartile spacing), namely [ M (IQR) ], and by using independent sample Mann-WhitneyU test, correlation is represented by Spearman rank correlation analysis, and correlation coefficient is represented by rs. The count data is expressed by relative numbers, and X is used for comparison between groups 2 And (5) checking. Establishing a Logistic regression model by using a forward stepwise regression method, wherein the inclusion level is0.05, with an exclusion level of 0.10. The difference of P <0.05 is statistically significant.
Results 4 results
4.1 inclusion of subject general clinical data and laboratory results comparisons
The subject was included in 945 subjects by rigorous screening and exclusion. Normal control 315, 51 in the contrast negative group, average age 55.41 (10.92) years, 25 in men (49%); CAA patient 630, average age 60.31 (10.18) years, male 441 (70%). The results show that the two groups of subjects were statistically significant (P < 0.05) with the exception of age, sex, smoking history, history of hypertension, history of diabetes, white blood cell count, neutrophil count, monocyte count, triglycerides, high density lipoprotein cholesterol, uric acid, creatinine, fasting blood glucose, hypersensitive C-reactive protein, fibrinogen and COG5 differences (P < 0.05). The basic clinical characteristics and laboratory data for all subjects are shown in Table 9.
TABLE 9 contrast negative and CAA group clinical data
/>
Note that: the normal distribution of the measurement data is represented by the average value ± (standard deviation), that is, (x±s), and the abnormal distribution of the measurement data is represented by the median (quartile spacing), that is, [ M (IQR) ].
4.2 inter-group COG5 level analysis
The serum COG5 of normal controls (including normal physical examination population and coronary angiography negative population) and CAA patients were both biased (fig. 8), and were compared pairwise using independent sample Mann-Whitney U test. The results show (FIG. 9) that the COG5 of the CAA group (coronary atherosclerosis group) is higher than that of the normal physical examination population, with concentrations of 62.68ng/ml [ IQR (56.31, 69.21) ] and 42.00ng/ml [ IQR (36.84, 48.36) ] (P < 0.001), respectively; and CAA group COG5 was higher than in the coronary negative population at concentrations of 62.68ng/ml [ IQR (56.31, 69.21) ] and 41.14ng/ml [ IQR (35.45, 45.18) ] (P < 0.001), respectively; there was no statistical difference between COG5 in normal physical examination population and coronary angiography negative population, and the concentrations were 42.00ng/ml [ IQR (36.84, 48.36) ] and 41.14ng/ml [ IQR (35.45, 45.18) ] (p=0.301), respectively.
4.3COG5 tangential point for diagnosing CAA of chest pain crowd in local area
Using CAA as a positive event, ROC curves were generated to yield Sen and Spe (FIG. 10). COG5 diagnosis of atherosclerosis of coronary artery with cut-off value 49.41ng/ml, AUC 0.913 (P < 0.05), sen and Spe 90.3% and 92% respectively, youden index 0.823, -LR and +LR 0.11 and 11.29 respectively.
Comparing with other statistically different indices of CAA, it was found that when diagnosing coronary atherosclerosis using a single index, the area under the curve (AUC) of COG5 was greater than the AUC of the other indices.
4.4 comparison of diagnostic efficacy of two models for diagnosing coronary atherosclerosis in the chest pain population in the local area
The variables with statistical differences in the analysis results of the above baseline data (neutrophil count, monocyte count, triglycerides, high density lipoprotein cholesterol, uric acid, creatinine, fasting blood glucose, hypersensitive C-reactive protein and fibrinogen (P < 0.05)) were subjected to binary Logistic stepwise regression analysis by SPSS software. The significance test of the regression coefficients was done using the likelihood ratio test and Wald statistics (see table 10). The results showed that neutrophil count was positively correlated with coronary stenosis in chest pain population (P < 0.05) and that high density lipoprotein cholesterol was negatively correlated with coronary stenosis in chest pain population (P < 0.05). The resulting regression model 1 was LogitP1=3.708+0.322×neutrophil-1.987 ×high density lipoprotein cholesterol. Further, the COG5 index was added and re-modeled with three indexes of neutrophil count, high density lipoprotein cholesterol and COG5, and the regression coefficient significance test was completed using the likelihood ratio test and Wald statistics as well (see table 11). The final regression model 2 was Logit P2= -7.414+0.207×COG5+0.509×neutrophil-2.446 ×high density lipoprotein cholesterol. Finally, the diagnostic efficacy of the two models was compared using ROC curves. The results showed that the ROC curve was plotted to give Sen and Spe (FIG. 11) with coronary stenosis in chest pain patients as a positive event. The cut-off value for model 1 was 2.66, AUC was 0.745 (P < 0.05), sen and Spe were 71.7% and 72.5%, respectively, and Youden index was 0.442; the cut-off value for model 2 was 1.97, AUC was 0.962 (P < 0.05), sen and Spe were 92.2% and 92.1%, respectively, and the Youden index was 0.844.
Comparing model 1 and model 2, it was found that when diagnosing coronary stenosis in chest pain patients with the model, the area under the curve (AUC) of model 2 was greater than the AUC of model 1.
Table 10 Logistic regression analysis data for model 1
Table 11 Logistic regression analysis data for model 2
Correlation of 4.5COG5 with Gensini score
630 patients diagnosed with coronary atherosclerosis by this admission coronary angiography were selected as subjects. The results of Pearson correlation analysis showed that Gensini score correlated positively with COG5 concentration (r=0.541, p<0.001). The scatter plot made with the Gensini score as X and COG5 concentration as Y was linearly trended (see fig. 12). Further, as a result of the linear regression analysis, it was revealed that as the coronary stenosis degree of the CAA patient increased, the serum COG5 concentration increased by b=0.297 (95% ci,0.26 to 0.34), and the coefficient R was determined 2 ＝0.293，P<0.001 (as in table 12). The linear regression of Gensini score with COG5 is shown in FIG. 12.
Table 12 results of parameters of the linear regression equation
4.6 Logistic regression analysis of coronary Severe stenosis risk factors
4.6.1 groups of coronary stenosis severity in CAA patients based on Gensini integration. The concentration of COG5 was increased (P < 0.001) in patients with mild stenosis (< 25), moderate stenosis (25-75), severe stenosis (> 75) compared to the contrast negative group 41.14ng/ml [ IQR (35.36, 45.72) ], see fig. 13, at concentrations of 54.23ng/ml [ IQR (44.61, 62.25) ], 62.52ng/ml [ IQR (56.40, 68.84) ] and 66.07ng/ml [ IQR (60, 78, 76.73) ], respectively.
4.6.2 are risk factors for coronary stenosis study, and CAA patients are further divided into two groups [ i.e., light and medium stenosis (Gensini score. Ltoreq.75 score, n=523) and severe stenosis (Gensini score >75 score, n=107) ]. Statistical variance analysis was performed on the two groups of patient baseline data. As a result, only sex, age, BMI, lymphocytes, platelets and COG5 were statistically different (P < 0.05). See table 13.
TABLE 13GENISINI score baseline data
/>
/>
Note that: the normal distribution of the measurement data is represented by the average value ± (standard deviation), that is, (x±s), and the abnormal distribution of the measurement data is represented by the median (quartile spacing), that is, [ M (IQR) ].
4.6.3 binary Logistic stepwise regression analysis was performed on the variables with statistical differences in the baseline data analysis results and COG5 by SPSS software. The significance test of the regression coefficients was done using the likelihood ratio test and Wald statistics (see table 14). The results showed that age and BMI had no statistical significance (P > 0.05) during modeling and were rejected. Finally, four variables of sex, lymphocyte, platelet and COG5 were kept for binary Logistic regression analysis (see table 15), and the results showed that sex (female), lymphocyte and platelet were negatively correlated with Log-GENISINI score (P < 0.05), COG5 levels were positively correlated with Log-GENISINI score (P < 0.05). The final regression model was Logit P= -4.191-0.770 Xgender (women) -0.001 Xlymphocytes-0.002 Xplatelets+0.050 XCOG 5.
TABLE 14 multifactor Logistic stepwise regression
TABLE 15 multifactor Logistic regression of coronary stenosis
4.6.4 by the use of Nagelkerke R 2 Coefficient (R) 2 The model was analyzed for goodness of fit by the misjudgment matrix (boundary value of 0.5, overall correct percentage of 82.9%) and the Hosmer-Lemeshow test (p=0.795), and the results showed that the model fit was good (fig. 14).
4.6.5 evaluation of the differentiation of the model using ROC curve analysis. The results showed that the area under the ROC curve (AUC) for the positive event of severe coronary stenosis was 0.667 (95% ci, 0.608-0.726) and the diagnosis was performed at the optimal threshold (p= -1.808), which model distinguishes between 80.5% Sen and 56.6% Spe for patients with severe coronary stenosis, respectively (fig. 15).
4.6.6 to more intuitively demonstrate the clinical application value of the predictive model, we have made a predicted nomogram of the degree of coronary artery stenosis (fig. 16). And assigning values to the risk factors according to the influence degree of the risk factors on coronary artery stenosis in the model, adding the integral of each index to obtain a total integral, and finally obtaining the possibility of occurrence of the coronary artery severe stenosis of different individuals through the conversion relation between the total integral and the occurrence frequency of the coronary artery severe stenosis. The alignment chart can convert a complex regression equation into a simple visual chart tool so as to clearly display the probability of predicting serious coronary artery stenosis, and the alignment chart has the characteristics of convenience, simplicity, accuracy and the like, and can provide a new auxiliary tool for clinical decision.
Accordingly, the serum COG5 level of CAA patients is higher than that of normal controls, and COG5 has better diagnostic ability as an index for distinguishing the presence or absence of coronary atherosclerotic plaques.
While the foregoing description illustrates and describes several preferred embodiments of the invention, it is to be understood that the invention is not limited to the forms disclosed herein, but is not to be construed as limited to other embodiments, and is capable of use in various other combinations, modifications and environments and is capable of changes or modifications within the spirit of the invention described herein, either as a result of the foregoing teachings or as a result of the knowledge or skill of the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims. Within the scope of the appended claims.
Claims (10)
1. An indirect ELISA method for detecting COG5 by using a specific polyclonal antibody, comprising the steps of:
(1) Coating COG5 recombinant protein with coating solution, coating 100 μl/well on 96-well plate;
(2) Washing the plate: pouring out the coating liquid in the plate holes on the next day, washing with PBST three times, and taking 250 mu l of each hole for 3-5 minutes each time and taking the coating liquid as dry as possible;
(3) Sealing the coated ELISA plate with sealing agent, adding 200 μl into each hole, and performing warm bath at 37deg.C for 1 hr;
(4) Washing the plate: pouring out the sealing liquid in the plate hole, washing with PBST three times, and taking 250 mu l of each hole for 3-5 minutes each time as dry as possible;
(5) Adding primary immune serum, diluting multiple antisera with PBST, simultaneously setting blank control and negative control, adding 100 μl of each well, and incubating at 37deg.C for 1 hr;
(6) Washing the plate: pouring out immune serum in the plate holes, washing with PBST three times, 250 μl each time, 3-5 minutes each time, and beating as dry as possible;
(7) After the incubation of the primary antibody is finished and washed, diluting the secondary antibody of the enzyme label to 1:10000 dilution by using PBST, incubating 100 μl of each hole in a light-resistant incubator at 37 ℃ for 1h, and washing the plate;
(8) Adding 50 mu l of each hole of the two-component color development liquid A and B, placing the two-component color development liquid A and B into a constant-temperature water bath box, and performing light-proof operation in the whole process, wherein the temperature bath is not equal for 1-15min at 37 ℃;
(9) Adding 100 mu l of stop solution into each hole to stop the color reaction;
(10) And (3) detecting the double-wavelength OD value at 450-630nm by using a Thermo microplate reader.
2. The indirect ELISA method of claim 1, wherein the amino acid sequence of the COG5 recombinant protein antigen is shown in SEQ ID NO. 1.
3. The indirect ELISA method according to claim 1, characterized in that the concentration of COG5 recombinant protein of step (1) is 0.0514ng to 11.1111ng/ml.
4. The indirect ELISA method according to claim 1, characterized in that the coating solution of step (1) is diluted 1:10 with COG5 recombinant protein antigen; the coating conditions were 37℃for 1 hour, then placed in a refrigerator at 4℃for 24 hours overnight.
5. The indirect ELISA method according to claim 1, characterized in that the blocking agent of step (3) is 1% bsa.
6. The indirect ELISA method according to claim 1, characterized in that the antisera dilution with PBST of step (5) is 1:5000.
7. The indirect ELISA method according to claim 1, characterized in that the enzyme-labeled secondary antibody of step (7) is an anti-rabbit secondary antibody added with horseradish peroxidase, and the dilution of the enzyme-labeled secondary antibody is 1:10000.
8. The indirect ELISA method of claim 1, wherein the two-component color-developing solutions A and B in the step (8) are TMB two-component color-developing solutions PR1210-100ml, respectively.
9. The indirect ELISA method according to claim 1, characterized in that the color reaction in step (9) is 3min.
10. The indirect ELISA method according to claim 1, characterized in that the stop solution of step (10) is C1058.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211447833.3A CN116735888B (en) | 2022-11-18 | 2022-11-18 | Indirect ELISA method for detecting COG5 by specific polyclonal antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211447833.3A CN116735888B (en) | 2022-11-18 | 2022-11-18 | Indirect ELISA method for detecting COG5 by specific polyclonal antibody |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116735888A true CN116735888A (en) | 2023-09-12 |
| CN116735888B CN116735888B (en) | 2024-01-12 |
Family
ID=87912019
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211447833.3A Active CN116735888B (en) | 2022-11-18 | 2022-11-18 | Indirect ELISA method for detecting COG5 by specific polyclonal antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116735888B (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008031288A1 (en) * | 2006-09-13 | 2008-03-20 | Beijing Hotgen Biotech Co., Ltd | Pre-filled centrifugal column for detecting hepatocellular carcinoma-specific alpha-fetoprotein variant and test kit containing the column |
| WO2010071405A1 (en) * | 2008-12-18 | 2010-06-24 | Erasmus University Medical Center Rotterdam | Markers for detecting predisposition for risk, incidence and progression of osteoarthritis |
| CA2814110A1 (en) * | 2011-06-20 | 2012-05-03 | Pbd Biodiagnostics, Llc | Methods, kits and arrays for screening for, predicting and identifying donors for hematopoietic cell transplantation, and predicting risk of hematopoietic cell transplant (hct) toinduce graft vs. host disease (gvhd) |
| US20120277999A1 (en) * | 2010-10-29 | 2012-11-01 | Pbd Biodiagnostics, Llc | Methods, kits and arrays for screening for, predicting and identifying donors for hematopoietic cell transplantation, and predicting risk of hematopoietic cell transplant (hct) to induce graft vs. host disease (gvhd) |
| CA2836750A1 (en) * | 2013-12-17 | 2015-06-17 | Agada Biosciences, LLC | Method and agents to quantify proteins from tissues |
| CN111122860A (en) * | 2019-12-31 | 2020-05-08 | 南京拂晓生物科技有限公司 | Indirect enzyme-linked immuno sorbent assay (ELISA) detection kit and detection method for detecting chlamydia pneumoniae antibody |
| CN111337670A (en) * | 2020-03-25 | 2020-06-26 | 河南农业大学 | Indirect ELISA detecting method for pilus subunit protein EbpA1 swine source enterococcus faecalis antibody |
| CN113325176A (en) * | 2021-06-02 | 2021-08-31 | 贵州大学 | Double-antibody sandwich direct ELISA (enzyme-Linked immuno sorbent assay) method for detecting avian leukosis P27 |
| CN114152748A (en) * | 2021-10-29 | 2022-03-08 | 浙江理工大学绍兴生物医药研究院有限公司 | Double-antibody sandwich ELISA diagnostic kit for detecting African swine fever virus and method thereof |
-
2022
- 2022-11-18 CN CN202211447833.3A patent/CN116735888B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008031288A1 (en) * | 2006-09-13 | 2008-03-20 | Beijing Hotgen Biotech Co., Ltd | Pre-filled centrifugal column for detecting hepatocellular carcinoma-specific alpha-fetoprotein variant and test kit containing the column |
| WO2010071405A1 (en) * | 2008-12-18 | 2010-06-24 | Erasmus University Medical Center Rotterdam | Markers for detecting predisposition for risk, incidence and progression of osteoarthritis |
| US20120277999A1 (en) * | 2010-10-29 | 2012-11-01 | Pbd Biodiagnostics, Llc | Methods, kits and arrays for screening for, predicting and identifying donors for hematopoietic cell transplantation, and predicting risk of hematopoietic cell transplant (hct) to induce graft vs. host disease (gvhd) |
| CA2814110A1 (en) * | 2011-06-20 | 2012-05-03 | Pbd Biodiagnostics, Llc | Methods, kits and arrays for screening for, predicting and identifying donors for hematopoietic cell transplantation, and predicting risk of hematopoietic cell transplant (hct) toinduce graft vs. host disease (gvhd) |
| CA2836750A1 (en) * | 2013-12-17 | 2015-06-17 | Agada Biosciences, LLC | Method and agents to quantify proteins from tissues |
| CN111122860A (en) * | 2019-12-31 | 2020-05-08 | 南京拂晓生物科技有限公司 | Indirect enzyme-linked immuno sorbent assay (ELISA) detection kit and detection method for detecting chlamydia pneumoniae antibody |
| CN111337670A (en) * | 2020-03-25 | 2020-06-26 | 河南农业大学 | Indirect ELISA detecting method for pilus subunit protein EbpA1 swine source enterococcus faecalis antibody |
| CN113325176A (en) * | 2021-06-02 | 2021-08-31 | 贵州大学 | Double-antibody sandwich direct ELISA (enzyme-Linked immuno sorbent assay) method for detecting avian leukosis P27 |
| CN114152748A (en) * | 2021-10-29 | 2022-03-08 | 浙江理工大学绍兴生物医药研究院有限公司 | Double-antibody sandwich ELISA diagnostic kit for detecting African swine fever virus and method thereof |
Non-Patent Citations (3)
| Title |
|---|
| PAESOLD-BURDA P 等: "Deficiency in COG5 causes a moderate form of congenital disorders of glycosylation", HUM MOL GENET, vol. 18, no. 22, pages 4350 - 4356 * |
| VAN DER LAAN SW 等: "Genetic Susceptibility Loci for Cardiovascular Disease and Their Impact on Atherosclerotic Plaques", CIRC GENOM PRECIS MED, vol. 11, no. 09, pages 002115 * |
| 孔云;张海龙;赵潇男;王明晶;李立新;: "COG拴留复合体对高尔基体功能的重要性", 基因组学与应用生物学, no. 03, pages 1096 - 1106 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116735888B (en) | 2024-01-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chaulin | Cardiac troponins: contemporary biological data and new methods of determination | |
| JP4523587B2 (en) | Method for distinguishing between type A and type B acute aortic dissection and acute myocardial infarction and kit for differentiation | |
| Sayre et al. | Measurement of cardiac troponin T is an effective method for predicting complications among emergency department patients with chest pain | |
| CN105092855A (en) | Kit for detecting liver fibration and liver cirrhosis | |
| CN103403549A (en) | Method for prediction of prognosis of sepsis | |
| Kavsak et al. | The potential role of a turbidimetric heart-type fatty acid-binding protein assay to aid in the interpretation of persistently elevated, non-changing, cardiac troponin I concentrations | |
| Agarwal et al. | Validation of the procalcitonin (PCT) assay: Experience in a pediatric hospital | |
| CN116735888B (en) | Indirect ELISA method for detecting COG5 by specific polyclonal antibody | |
| Nicoll et al. | Basic principles of diagnostic test use and interpretation | |
| Gosselin et al. | Evaluation of the Stratus® CS Acute Care™ D-dimer assay (DDMR) using the Stratus® CS STAT Fluorometric Analyzer: A prospective multisite study for exclusion of pulmonary embolism and deep vein thrombosis | |
| EP3495821B1 (en) | Diagnostic method for determining nox2 protein | |
| CN110596399B (en) | Application of substance for detecting content of angiopoietin-like protein8 | |
| Lewis Jr et al. | Clinical significance of low-positive troponin I by AxSYM and ACS: 180 | |
| Kline et al. | Method of using carbonic anhydrase to detect hemolysis | |
| Qiu | A Validation Study of Transferrin and I-FABP As Dried Blood Spot-Based Biomarkers of Environmental Enteric Dysfunction | |
| Pant et al. | Serum glucose, a cost-effective alternate of plasma glucose in diagnosing and monitoring diabetes mellitus | |
| Ritchie et al. | Laboratory assessment of the acute phase response: using CRP as a model | |
| DuBois | Point-of-Care Testing recognized as a distinct domain | |
| Blick | Economics of point-of-care (POC) testing for cardiac markers | |
| Shindelman et al. | Development of a High Sensitivity Immunoassay for C-reactive Protein on the Cholestech LDX: A Point of Care Analyzer | |
| CN113238063A (en) | Use of GDF15 to assess the progression of metabolic syndrome patients to cardiovascular disease | |
| Mansoor et al. | Urinary NMP-22 Plus Cytology: Diagnostic Synergy for Detecting Urothelial Carcinoma | |
| Kempe et al. | Promoting Improved Patient Care Through Informatics and an Insulin Infusion Protocol Project | |
| Lyon et al. | Can Chemistry and Hematology Laboratories Use a Common Blood Specimen? Genesis of Clinical Laboratory Integration | |
| Cembrowski et al. | Multicenter Evaluation of the Precision PCx Plus Glucose Test Strip with TrueMeasure Technology |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |