CN116655788B - 中性粒细胞明胶酶相关脂质运载蛋白(ngal)尿液检测试剂盒 - Google Patents
中性粒细胞明胶酶相关脂质运载蛋白(ngal)尿液检测试剂盒 Download PDFInfo
- Publication number
- CN116655788B CN116655788B CN202211126872.3A CN202211126872A CN116655788B CN 116655788 B CN116655788 B CN 116655788B CN 202211126872 A CN202211126872 A CN 202211126872A CN 116655788 B CN116655788 B CN 116655788B
- Authority
- CN
- China
- Prior art keywords
- ngal
- monoclonal antibody
- cancer
- antibody
- pad
- 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.)
- Active
Links
- 108010051335 Lipocalin-2 Proteins 0.000 title claims abstract description 76
- 102000013519 Lipocalin-2 Human genes 0.000 title claims abstract description 74
- 238000001514 detection method Methods 0.000 title claims abstract description 34
- 210000002700 urine Anatomy 0.000 title claims description 16
- 230000027455 binding Effects 0.000 claims abstract description 6
- 238000012360 testing method Methods 0.000 claims description 29
- 238000000034 method Methods 0.000 claims description 22
- 239000000020 Nitrocellulose Substances 0.000 claims description 13
- 229920001220 nitrocellulos Polymers 0.000 claims description 13
- 239000012528 membrane Substances 0.000 claims description 12
- 239000002250 absorbent Substances 0.000 claims description 6
- 238000010521 absorption reaction Methods 0.000 claims description 5
- 241000283707 Capra Species 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 230000002745 absorbent Effects 0.000 claims description 3
- 239000003365 glass fiber Substances 0.000 claims description 3
- 238000003908 quality control method Methods 0.000 claims description 3
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 238000009535 clinical urine test Methods 0.000 abstract 1
- 108090000623 proteins and genes Proteins 0.000 description 25
- 102000004169 proteins and genes Human genes 0.000 description 25
- 206010028980 Neoplasm Diseases 0.000 description 21
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 21
- 210000002966 serum Anatomy 0.000 description 10
- 239000000243 solution Substances 0.000 description 9
- 239000002671 adjuvant Substances 0.000 description 8
- 208000020832 chronic kidney disease Diseases 0.000 description 8
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 7
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 201000011510 cancer Diseases 0.000 description 7
- 238000003018 immunoassay Methods 0.000 description 7
- 238000002965 ELISA Methods 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 210000003734 kidney Anatomy 0.000 description 6
- 230000000087 stabilizing effect Effects 0.000 description 6
- 206010003445 Ascites Diseases 0.000 description 5
- 238000011725 BALB/c mouse Methods 0.000 description 5
- 206010008342 Cervix carcinoma Diseases 0.000 description 5
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 5
- 201000010881 cervical cancer Diseases 0.000 description 5
- 238000003745 diagnosis Methods 0.000 description 5
- 210000004408 hybridoma Anatomy 0.000 description 5
- 206010009944 Colon cancer Diseases 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 230000002163 immunogen Effects 0.000 description 4
- 230000009545 invasion Effects 0.000 description 4
- 201000007270 liver cancer Diseases 0.000 description 4
- 208000014018 liver neoplasm Diseases 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000007790 solid phase Substances 0.000 description 4
- 206010061218 Inflammation Diseases 0.000 description 3
- 206010027476 Metastases Diseases 0.000 description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 3
- 108010090804 Streptavidin Proteins 0.000 description 3
- 208000024770 Thyroid neoplasm Diseases 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000013399 early diagnosis Methods 0.000 description 3
- 210000001808 exosome Anatomy 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 230000004054 inflammatory process Effects 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 230000009401 metastasis Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- 201000002510 thyroid cancer Diseases 0.000 description 3
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 3
- 229940038773 trisodium citrate Drugs 0.000 description 3
- 230000002485 urinary effect Effects 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 208000009304 Acute Kidney Injury Diseases 0.000 description 2
- 206010061424 Anal cancer Diseases 0.000 description 2
- 208000007860 Anus Neoplasms Diseases 0.000 description 2
- 206010005003 Bladder cancer Diseases 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 201000009030 Carcinoma Diseases 0.000 description 2
- 208000017667 Chronic Disease Diseases 0.000 description 2
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 2
- 208000028399 Critical Illness Diseases 0.000 description 2
- 206010014733 Endometrial cancer Diseases 0.000 description 2
- 206010014759 Endometrial neoplasm Diseases 0.000 description 2
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 2
- 206010017993 Gastrointestinal neoplasms Diseases 0.000 description 2
- 102000019298 Lipocalin Human genes 0.000 description 2
- 108050006654 Lipocalin Proteins 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 208000032271 Malignant tumor of penis Diseases 0.000 description 2
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 2
- 206010033128 Ovarian cancer Diseases 0.000 description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 2
- 208000002471 Penile Neoplasms Diseases 0.000 description 2
- 206010034299 Penile cancer Diseases 0.000 description 2
- 206010060862 Prostate cancer Diseases 0.000 description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 2
- 208000015634 Rectal Neoplasms Diseases 0.000 description 2
- 208000033626 Renal failure acute Diseases 0.000 description 2
- 206010061481 Renal injury Diseases 0.000 description 2
- 208000004337 Salivary Gland Neoplasms Diseases 0.000 description 2
- 206010061934 Salivary gland cancer Diseases 0.000 description 2
- 206010039491 Sarcoma Diseases 0.000 description 2
- 206010040047 Sepsis Diseases 0.000 description 2
- 208000024313 Testicular Neoplasms Diseases 0.000 description 2
- 206010057644 Testis cancer Diseases 0.000 description 2
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 2
- 208000002495 Uterine Neoplasms Diseases 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 201000011040 acute kidney failure Diseases 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 201000011165 anus cancer Diseases 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000007910 cell fusion Effects 0.000 description 2
- 208000006990 cholangiocarcinoma Diseases 0.000 description 2
- 208000029742 colonic neoplasm Diseases 0.000 description 2
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000002357 endometrial effect Effects 0.000 description 2
- 201000004101 esophageal cancer Diseases 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- 208000005017 glioblastoma Diseases 0.000 description 2
- 201000010536 head and neck cancer Diseases 0.000 description 2
- 208000014829 head and neck neoplasm Diseases 0.000 description 2
- 238000003317 immunochromatography Methods 0.000 description 2
- 208000037806 kidney injury Diseases 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 210000000440 neutrophil Anatomy 0.000 description 2
- 201000002528 pancreatic cancer Diseases 0.000 description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 206010038038 rectal cancer Diseases 0.000 description 2
- 201000001275 rectum cancer Diseases 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 206010041823 squamous cell carcinoma Diseases 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 201000003120 testicular cancer Diseases 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 201000005112 urinary bladder cancer Diseases 0.000 description 2
- 206010046766 uterine cancer Diseases 0.000 description 2
- 206010046885 vaginal cancer Diseases 0.000 description 2
- 208000013139 vaginal neoplasm Diseases 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 208000003200 Adenoma Diseases 0.000 description 1
- 206010001233 Adenoma benign Diseases 0.000 description 1
- 108010032595 Antibody Binding Sites Proteins 0.000 description 1
- 102000000905 Cadherin Human genes 0.000 description 1
- 108050007957 Cadherin Proteins 0.000 description 1
- 208000001490 Dengue Diseases 0.000 description 1
- 206010012310 Dengue fever Diseases 0.000 description 1
- 208000007342 Diabetic Nephropathies Diseases 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 102000013382 Gelatinases Human genes 0.000 description 1
- 108010026132 Gelatinases Proteins 0.000 description 1
- 102000016761 Haem oxygenases Human genes 0.000 description 1
- 108050006318 Haem oxygenases Proteins 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 208000000172 Medulloblastoma Diseases 0.000 description 1
- 206010027406 Mesothelioma Diseases 0.000 description 1
- 108050000637 N-cadherin Proteins 0.000 description 1
- 206010052399 Neuroendocrine tumour Diseases 0.000 description 1
- 208000005890 Neuroma Diseases 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 201000000582 Retinoblastoma Diseases 0.000 description 1
- 206010040070 Septic Shock Diseases 0.000 description 1
- 206010041067 Small cell lung cancer Diseases 0.000 description 1
- 206010051379 Systemic Inflammatory Response Syndrome Diseases 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 201000000053 blastoma Diseases 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000035605 chemotaxis Effects 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 208000037893 chronic inflammatory disorder Diseases 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 229940109239 creatinine Drugs 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 208000025729 dengue disease Diseases 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 208000033679 diabetic kidney disease Diseases 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 201000008184 embryoma Diseases 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 208000015419 gastrin-producing neuroendocrine tumor Diseases 0.000 description 1
- 201000000052 gastrinoma Diseases 0.000 description 1
- 230000024924 glomerular filtration Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 201000010982 kidney cancer Diseases 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 150000002632 lipids Chemical group 0.000 description 1
- 206010024627 liposarcoma Diseases 0.000 description 1
- 201000002037 lung adenoma Diseases 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 206010027191 meningioma Diseases 0.000 description 1
- 238000010197 meta-analysis Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000000491 multivariate analysis Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 208000007538 neurilemmoma Diseases 0.000 description 1
- 208000016065 neuroendocrine neoplasm Diseases 0.000 description 1
- 201000011519 neuroendocrine tumor Diseases 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 201000002628 peritoneum cancer Diseases 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000000611 regression analysis Methods 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 206010039667 schwannoma Diseases 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000036303 septic shock Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 208000000587 small cell lung carcinoma Diseases 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 210000002437 synoviocyte Anatomy 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
-
- 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
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Genetics & Genomics (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Peptides Or Proteins (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本发明涉及中性粒细胞明胶酶相关脂质运载蛋白(NGAL)尿液检测试剂盒。本发明针对NGAL蛋白提供了特异性的单克隆抗体,所述抗体具有较好的特异性以及结合活性。将所述抗体用于提供对NGAL蛋白的检测,具有特异性强,灵敏度高,检测时间短,检测样品范围大的特点;本发明的检测方法不需要任何特殊仪器、设备,具有检测成本低的特点;本发明的检测试剂盒操作简便,不需由专业人员操作;本发明的试剂盒储存方便,具有较好的应用前景。
Description
技术领域
本申请涉及生物检测领域,具体的涉及中性粒细胞明胶酶相关脂质运载蛋白(NGAL)尿液检测试剂盒。
背景技术
中性粒细胞明胶酶相关脂质运载蛋白(NGAL)是lipocalin的一种,最初是在激活中性粒细胞中被发现的一种小分子量分泌性蛋白,现代研究表明,NGAL是诊断急性肾损伤的最有效生物学标志之一,也是早期糖尿病肾病的有效标志物之一。NGAL具有强大的功能,除了作为载脂家族成员具有结合并运输疏水性小分子的功能外,还与炎症、胚胎发育、免疫应答、趋化作用、信号转导以及多种肿瘤的发生与发展过程相关。NGAL可消炎、抗炎,可促进肾脏祖细胞向早期肾小管上皮细胞分化,可修复N-钙黏蛋白,上调保护酶血红素加氧酶和抑制细胞死亡。
研究发现,慢性肾病CKD患者的血清、尿NGAL水平显著高于正常人群,并与肾小球滤过率(GFR)呈密切负相关,而sCr与GFR的关联程度不及NGAL。因此,CKD患者NGAL水平不仅是优于sCr反映CFR下降的指标,更是评价CKD患者肾脏损伤程度的标志物。但CKD患者尿NGAL增高幅度不及AKI患者,可能与受到其他慢性疾病因素的干扰有关。欧洲一项CKD成年患者队列研究发现,血清NGAL>435ng/mL或尿NGAL>231ng/mL的患者更早达到随访终点事件;并且血清、尿液NGAL浓度每增高10ng/mL,CKD进展的风险分别提高了3%与2%,经多变量Cox比例风险回归模型分析,血清、尿NGAL是CKD的进展风险的独立预测因子。
NGAL已经被证实是急性肾损伤的标志物。早期诊断急性肾功能损伤(AKI)时血、尿NGAL浓度通常会迅速升高,2h最为明显(比临界值升高几十至几百倍),血清肌酐(sCr)、尿酶等传统指标往往要在24~72h才后明显升高,因而NGAL可用于AKI的早期诊断。Mishra等在体外循环术并发AKI患儿的队列研究中发现,2h尿NGAL诊断AKI的AUCROC为0.998,其临界值为50μg/L(ELISA法)时,敏感性、特异性分别为100%、98%,而血清NGAL诊断AKI的AUCROC为0.906,其临界值为25μg/L时,敏感性、特异性为70%、94%;多元回归分析显示,2h尿NGAL水平是预测AKI的强有力预测因子。也发现成年患者2h尿、血清NGAL水平是诊断AKI的可靠的早期诊断指标。meta分析表明,NGAL对AKI的诊断效能受到标本类型、年龄、检测方法等因素影响,但NGAL对儿童AKI的诊断效能优于成年人,可能是成年人受到其他慢性疾病等因素影响。值得注意的是,脓毒血症等危重疾病患者,因受到炎症反应等因素影响,NGAL可能并不适用于AKI的早期诊断,诊断特异性欠佳。北美地区研究发现,143名全身炎症反应综合征及脓毒性休克患儿入院后24h内,血清NGAL诊断AKI的AUCROC为0.677,在临界值为139ng/mL时,敏感性为86%,特异性仅为39%。经多变量分析后发现,血NGAL并不是发生AKI事件的预测指标。在成人患者研究中也得到类似结论。另有研究表明,危重患儿48h内尿液NGAL水平可以准确预测AKI的发生(AUCROC为0.79)。但当sCr升高后,AUCROC降至0.63。ICU中的AKI成人患者血浆NGAL水平在48h后与正常对照组亦无显著性差异。尿NGAL可作为脓毒血症后并发AKI的早期诊断标志物,准确性达0.968,这可能与种族间水平差异有关。
近年来NGAL基因的功能越来越受到人们的重视。研究表明NGAL与细菌感染性或非细菌性感染性疾病、一些慢性炎症、肿瘤的侵袭与转移以及与肿瘤细胞的调节与分化关系密切。而且NGAL与炎症的研究结果表明,NGAL在这些疾病中的表达特征趋于成熟,极有可能成为多种临床炎症相关性疾病诊断的标志物,同时NGAL在肿瘤的侵袭转移过程的功能与机制也趋于明朗,而NGAL与肿瘤细胞的调节和分化成为人们讨论的热点与研究的转折点。NGAL在多种肿瘤的发生及发展过程中发挥重要功能,是一种新的癌基因。研究发现,宫颈癌中NGAL的表达明显升高,与其发生及转移相关。NGAL在宫颈癌中相对于癌旁组织表达升高,甲状腺癌中抑制NGAL基因的表达可降低癌细胞的迁移和侵袭,而过表达可促进癌细胞的迁移和侵袭,NGAL表达的抑制后宫颈癌细胞增殖显著降低,凋亡增加。
基于NGAL作为标记的重要性,开发检测NGAL的检测试剂盒是研究的重要方向。生理情况下,NGAL在体内的一些器官的表达很低,包括肾脏、癌组织在内。因此,检测的敏感性要求很高。现有技术已有根据NGAL基因序列,设计并合成PCR引物,将PCR扩增的特异片段克隆入T载体,重组质粒经筛选、鉴定、定量后作为标准品。在上述NGAL扩增片段内部设计FQ-PCR引物,通过正交设计方式对PCR引物浓度、退火温度、SYBR greenI染料浓度等PCR反应条件进行优化,从而建立NGAL的检测方法。但是现有技术的方法主要是PCR的方法,对于仪器设备的要求比较高,而且检测不便利,急需一种可以便利化、不需要仪器即可检测的新方法。
发明内容
本发明根据现有技术的缺陷,提供了一种改进的方法。
一方面,针对NGAL,提供了特异性的单克隆抗体。
具体的,所述单克隆抗体为N-3A4,其轻链可变区的序列为SEQ ID NO:1和重链可变区的序列SEQ ID NO:2。
进一步的,本发明的单克隆抗体可以在轻链可变区和重链可变区中进行一个或二个或多个的保守氨基酸的突变,突变后的抗体仍然保持相应的抗体的活性。
进一步的,在捕捉目标蛋白时,可适宜地使用本发明的单克隆抗体。例如,将本发明的单克隆抗体生物素化并与外来体反应后,可通过使用链霉亲和素固相磁性珠进行分离。
更详细而言,首先依照公知的方法将本发明的单克隆抗体或其抗体片段生物素化。接着,将待检测蛋白与生物素化抗体混合,在4℃过夜反应。然后,添加链霉亲和素固相磁性珠,在4℃进一步反应2小时后,使用磁铁进行分离,可以回收结合于生物素化抗体的靶标蛋白。
具体地,使来源于受试者的机体试样与本发明的单克隆抗体或其抗体片段接触,依照前述蛋白的捕捉分离法分离靶标蛋白。
应予说明,在本说明书中,机体试样只要是选自血液、血清、和血浆的机体试样,则没有特别限定。(免疫测定法)使用本发明的单克隆抗体的套件进行免疫测定。作为免疫测定法,可举出:酶免疫测定法(EIA)、酶免疫测定法(ELISA)、荧光免疫测定法(FIA)、放射线免疫测定法(RIA)、发光免疫测定法、免疫印迹法、蛋白质印迹法等,从能够简便且灵敏度良好地检测抗体的角度出发,优选ELISA法。
ELISA法中可举出:通常的竞争法、夹心法等,由于可将本发明的单克隆抗体或其抗体片段用于夹心法中的固相抗体、或固相抗体和标记抗体两者,因而优选夹心法。接着,示出夹心ELISA法的一个方式。首先,使本发明的单克隆抗体或其抗体片段固相化,然后与含有外来体的被测试样接触,形成复合体。之后,对其添加将本发明的套件中的另一本发明的单克隆抗体或其抗体片段、疾病特异性蛋白抗体或其抗体片段分别进行了修饰的标记抗体,形成进一步的复合体,并检测标记,由此可分别测定来自试样中所含的外来体的信号量、试样中所含的疾病特异性蛋白量。因此,本发明还提供测定蛋白的存在量的方法,该方法包括使来源于受试者的机体试样与本发明的单克隆抗体的套件中所含的单克隆抗体接触而形成该复合体。
应予说明,在将本发明的单克隆抗体或其抗体片段固相化时,可以直接进行固相化,也可以经由公知的介质,例如,链霉亲和素来进行固相化。
本发明的单克隆抗体或者试剂盒可以用检测肾病或者癌症。
具体的癌症为“癌症”和“肿瘤”用于本文中时意指或描述哺乳动物中的生理学病症,其典型特征在于失控的细胞生长。癌症或肿瘤的实例包括,但不仅限于,癌、淋巴瘤、胚细胞瘤(包括成神经管细胞瘤和成视网膜细胞瘤)、肉瘤(包括脂肪肉瘤和滑膜细胞肉瘤)、神经内分泌肿瘤(包括良性肿瘤、促胃液素瘤、和岛细胞癌)、间皮瘤、神经鞘瘤(包括听神经瘤)、脑膜瘤、腺瘤、黑素瘤、和白血病或淋巴恶性肿瘤。所述癌症的更具体实例包括鳞状细胞癌(例如上皮鳞状细胞癌),肺癌包括小细胞肺癌,非小细胞肺癌,肺腺瘤和肺鳞状癌,腹膜癌,肝细胞癌,胃(gastric)或胃(stomach)癌包括胃肠癌,胰腺癌,成胶质细胞瘤,宫颈癌,卵巢癌,肝癌,膀胱癌,肝细胞瘤,乳腺癌,结肠癌,直肠癌,结肠直肠癌,子宫内膜或子宫癌,唾液腺癌,肾(kidney)或肾(renal)癌,前列腺癌,阴道癌,甲状腺癌,肝癌,肛门癌,阴茎癌,睾丸癌,食管癌,胆管肿瘤,以及头颈癌。优选地,所述癌症是实体瘤。术语“实体瘤”在用于本文中时指选自胃肠癌,胰腺癌,成胶质细胞瘤,宫颈癌,卵巢癌,肝癌,膀胱癌,肝细胞瘤,乳腺癌,结肠癌,直肠癌,结肠直肠癌,子宫内膜或子宫癌,唾液腺癌,肾(kidney)或肾(renal)癌,前列腺癌,阴道癌,甲状腺癌,肝癌,肛门癌,阴茎癌,睾丸癌,食管癌,胆管肿瘤,以及头颈癌的组的肿瘤。
进一步的,本发明还提供了一种检测用的试剂盒,所述试剂盒含有本发明的单克隆抗体。
进一步的,本发明提供一种检测试纸条。
所述的试纸条是免疫层析试纸条由样品垫、结合垫、硝酸纤维素膜及吸收垫4个部分组成。样品垫和结合垫用玻璃纤维,吸收垫用吸水滤纸。羊抗鼠多克隆抗体在硝酸纤维素膜上划线作为质控带;单克隆抗体在硝酸纤维素膜上划线作为检测带,参数0.1μL/mm划线,指控带与检测带间距约为7mm。划线后,将硝酸纤维素膜置于37℃培养箱中孵育。将吸收垫、样品垫及结合垫、硝酸纤维素膜叠加,组装成完整的层析条,然后切割层析条,宽度4mm/条,干燥避光保存。
有益效果
与现有技术相比本发明具有如下优点:
本发明针对NGAL蛋白提供了特异性的单克隆抗体,所述抗体具有较好的特异性以及结合活性。将所述抗体用于提供对肾损伤患者的检测,具有特异性强,灵敏度高,检测时间短(15~20分钟),检测样品范围大的特点;本发明的检测方法不需要任何特殊仪器、设备,具有检测成本低的特点;本发明的检测试剂盒操作简便,不需由专业人员操作;本发明的试剂盒储存方便,具有较好的应用前景。
附图说明
图1单抗识别表位鉴定结果
具体实施方式
本发明可通过后续对于本发明一些实施方案描述以及其中所包括的实施例的详细内容而更容易被了解。在进一步叙述本发明之前,应明了本发明不会被局限于所述特定实施方案中,因为这些实施方案必然是多样的。亦应明了本说明书中所使用的用语仅是为了阐述特定实施方案,而非作为限制,因为本发明的范围将会被仅仅界定在所附的权利要求中。
实施例1 NGAL单克隆抗体的制备
免疫BALB/c小鼠:将中性粒细胞明胶酶关联脂质运载蛋白(NGAL)重组蛋白(货号:kl-B388Ra01,KALANG)分别加入弗氏完全佐剂和弗氏不完全佐剂乳化制成弗氏完全佐剂免疫原和弗氏不完全佐剂免疫原,其中NGAL重组蛋白与弗氏完全佐剂、弗氏不完全佐剂体积比均为1:1;通过背部皮下多点注射的方法,用弗氏完全佐剂免疫原免疫8周龄的雌性BALB/c小鼠3只,100μl/只;在首次免疫14天和28天后分别用弗氏不完全佐剂免疫原以相同的方法和剂量对BALB/c小鼠进行加强免疫;免疫14d,尾部采血,测小鼠血清效价,选择效价最高的1号小鼠进行细胞融合;细胞融合前5天,通过腹腔注射的方法,用不含佐剂的NGAL蛋白对BALB/c小鼠进行超强免疫,免疫剂量是50μg/只。
经过超免5d后的1号BALB/c小鼠引颈致死,处死小鼠用常规方法进行融合。融合后的细胞转到半固体培养基中培养,生长的单克隆挑到96孔培养板中进行培养并采用ELISA法初筛获得了阳性反应最强的10株单抗,共筛到6株阳性杂交瘤,经过3次亚克隆和扩繁最终获得2株可稳定分泌抗体的杂交瘤细胞株,分别命名为N-3A4和N-5H16。
单抗腹水效价测定制备前1周,按照500μL/只向小鼠腹腔内注射石蜡,杂交瘤扩大培养后,每只小鼠按照106个细胞数腹腔注射200μL杂交瘤细胞悬液,6d后,小鼠腹围明显增大,采集腹水,用间接ELISA法测定腹水效价。结果显示N-3A4单克隆抗体腹水的效价为1.28×107,N-5H16单克隆抗体腹水的效价为5.12×107。利用Protein G亲和柱纯化二个抗体,SDS-PAGE蛋白电泳分析二个纯化后的抗体的纯度均达到99%以上。
实施例2 N-3A4单抗特性分析
(1)单体Ig亚类鉴定:单抗亚类的鉴定按照Sigma公司的鼠单抗亚型分型试剂鉴定试剂盒说明书进行。结果显示,根据抗体检测试剂盒单抗亚类检测方法,N-3A4株单抗的亚型为IgG1。
(2)单抗亲和力的测定:应用间接ELISA法,以1μg/mL的浓度用NGAL重组蛋白包被酶标板,封闭后加入倍比稀释的纯化单抗进行孵育,以HRP标记的山羊抗小鼠IgG为二抗,酶标仪读取OD450nm吸光值。连续几个稀释度的OD450nm读数不再增大时视为抗原抗体100%结合,以抗体浓度(mol/L)为横坐标,OD450nm吸光值为纵坐标做散点图,以读数最大值一半时抗原抗体结合率为50%,生成对数趋势线和公式。将OD450nm最大值的一半代入公式,求出此时的抗体浓度即为亲和力解离常数(Kd)。结果如下表1所示。
表1 N-3A4单抗的亲和力
抗体名称 | 解离常数(Kd,M) |
N-3A4单抗 | 8.52×10-10 |
(3)单抗特异性鉴定分别用BSA、PD-1、NGAL、大肠杆菌裂解液进行单抗特异性的鉴定,将不同蛋白稀释一定倍数后进行Western Blot,分别用稀释的单抗(1∶5000)作为一抗,检测所得单抗对常见的不同蛋白的交叉反应。结果显示,本发明的N-3A4单抗只与NGAL蛋白结合而不与其他蛋白结合,具有较好的特异性。
(4)通过抗体序列鉴定试剂盒,通过测序,获得N-3A4单抗的轻重链可变区序列分别如SEQ ID Nos:1-2所示。
实施例3 N-3A4单抗识别表位鉴定
根据NGAL序列共设计了4个NGAL截短体,分别命名为A(1-594bp)、B(1-477bp)、C(1-357bp)和D(1-237bp)。根据截短体分别设计引物(如表2所示)以人DNA为模板,扩增截短体DNA片段,在5′端和3′端分别引入NcoⅠ位点和EcoRⅠ位点.用扩增的截短体DNA片段构建重组PET32a-NGAL质粒并转化入大肠埃希菌中表达NGAL截短肽,分别命名为a1(aa1-aa198)、a2(aa1-aa159)、a3(aa1-aa119)、a4(aa1-aa79)。
表2扩增引物
分别以NGAL全蛋白、a1-a4为抗原,N-3A4单抗为一抗,进行Westernblot分析。经分析发现,N-3A4单抗与全蛋白以及a1和a2段短肽均可结合(图1),而与a3和a4都不结合,说明本发明的抗体结合位点在aa119-159之间。
实施例4胶体金免疫层析试纸条的制备
胶体金颗粒的制备:采用柠檬酸三钠还原法,制备25nm的胶体金。具体操作方法如下:取100mL双蒸水,加热至沸腾加入1mL1%的氯金酸,搅拌下准确加入1.5mL体积分数为1%的新鲜制备的柠檬酸三钠水溶液。此时可观察到淡黄色的氯金酸水溶液在柠檬酸三钠加入后很快变成灰色,续而转成黑色,随后逐渐稳定成酒红色,20min后停止加热。冷却至室温后4℃避光保存。
胶体金溶液的最佳PH值确定。取1mL的胶体金溶液8份,分别用0.2mol/LK2CO3将pH值调至5.0、6.0、6.5、7.0、7.5、8.0、8.5、9.0;分别加入0.2μg/mL N-3A4单克隆抗体100μL,混匀,静置10min后,加入100μL质量分数为10%为NaCl溶液,静置0.5h后观察胶体金颜色。胶体金颜色没有发生改变的最低pH值为胶体金溶液的最佳pH值。结果表明胶体金颜色未改变的最低pH值为7.5。
确定最低蛋白稳定量:在标记前,应首先测定能稳定一定量胶体金所需要的最小蛋白用量。将胶体金溶液调至最佳PH值7.5,抗原经高速离心除去大的聚集物,用5mmol/LPH8.0的PB溶液稀释至0.2mg/ml,将待标记N-3A4单克隆抗体逐级稀释后,各取等体积顺序加入一系列装有1ml胶体金的试管中,混匀;5min后,在上述各管内分别加入10%氯化钠溶液0.1ml,混匀,室温静置,用分光光度计OD580测定。未加蛋白及加入蛋白量不足以稳定胶体金的试管,即呈现由红变蓝的聚沉现象,而加入蛋白量达到或超过最低稳定量的试管则保持胶体金的红色不变。以此使胶体金红色不变而蛋白质含量最低的试管的蛋白量,即为稳定1ml胶体金的必需蛋白量,也即最低稳定量。在此基础上再加20%即为稳定1ml胶体金所需蛋白质的实际最适用量。结果本发明的N-3A4抗体即选择10μg/mL的N-3A4单克隆抗体加入量作为实际最小蛋白标记量较为合适。
将胶体金调至最适PH值7.5,加入最低蛋白稳定量的N-3A4单克隆抗体。静置5min后加入过量BSA封闭,离心,用0.01mol/LTris溶液重悬,低速离心去除聚集物,上清液即为胶体金标记的N-3A4抗体。
免疫层析试纸条由样品垫、结合垫、硝酸纤维素膜及吸收垫4个部分组成。样品垫和结合垫用玻璃纤维,吸收垫用吸水滤纸。以浓度为1mg/mL的羊抗鼠多克隆抗体在硝酸纤维素膜上划线作为质控带;以浓度为1mg/mL的N-3A4单克隆抗体在硝酸纤维素膜上划线作为检测带,参数0.1μL/mm划线,指控带与检测带间距约为7mm。划线后,将硝酸纤维素膜置于37℃培养箱中孵育2h。将吸收垫、样品垫及结合垫、硝酸纤维素膜叠加,组装成完整的层析条,然后切割层析条,宽度4mm/条,干燥避光保存。
检测及结果判读。在制备好的层析条的样品垫上手工加样100μL,15min后,检测带和指控带都出现红色为阳性,只有指控带出现红色为阴性,检测带和指控带均不显色,则为试剂失效,需换新的试纸条重测。
本试验例共收集尿液样本200例,其中阳性样本85例(肾损伤患者),阴性检测115例。与已上市的QuicKey-人中性粒细胞明胶酶相关脂质运载蛋白(NGAL)酶联免疫吸附测定试剂盒(货号:E-TSEL-H0003,Elabscience)对所述样本进行检测,结果阳性均检测为阳性,阴性检测结果均为阴性。用本发明实施例的检测试纸条对所述收集的200份尿液样本分别进行检测,汇总检测结果。用四格表分析135份数据的阴阳性符合率、约登指数,并进行Kappa检验,评价本发明实施例1的检测试纸条与对照试纸条的一致性。结果如表3所示。
表3临床性能验证结果
从表3,计算结果如下:
阳性样本符合率(真阳性率)=85/85×100%=100%
阴性样本符合率(真阴性率)=115/115×100%=100%
总符合率=200/200×100%=100%
约登指数=85/85+115/115-1=1
kappa值=1,说明本发明实施例1的检测试纸条与对照试纸条一致性很好。
检测结果符合率=(总样本数-差异样本数)/总样本数×100%=(200-0)/200×100%=100%
从以上数据可以看出,采用本发明实施例1的中性粒细胞明胶酶相关脂质运载蛋白(NGAL)检测试纸条(胶体金法)进行临床验证,以已上市的中性粒细胞明胶酶相关脂质运载蛋白测定试纸条(胶乳增强免疫比浊法)为对照,共检测尿液样本200例(其中阳性85例,阴性115例),整理并分析检测结果,评价试剂盒的临床性能,阳性符合率为100%,阴性符合率为100%,总符合率为100%,约登指数为1。Kappa值为1(Kappa检验,P<0.05),检测结果符合率为100%,本发明实施例1的检测试纸条与对照试纸条有很好的一致性。考核结果表明本发明实施例的检测试纸条与对照试纸条检测性能相仿,稳定性好,结果较为准确可靠。试纸条操作简单方便,且成本较进口试纸条价格低廉,具有很好的市场应用价值。
尽管本发明的内容是结合本实施例进行说明,但是不能认为是对本发明范围的限制,本发明的范围由所附权利要求书限定。另外,本领域的技术人员在所附权利要求书限定的范围内对本发明进行各种改动或修饰,这些改动或修饰形式同样落在本发明的保护范围内。
Claims (5)
1.一种特异性针对中性粒细胞明胶酶相关脂质运载蛋白的单克隆抗体,其特征在于所述单克隆抗体为N-3A4,单克隆抗体N-3A4其轻链可变区的序列为SEQ ID NO:1和重链可变区的序列为SEQ ID NO:2。
2.如权利要求1所述单克隆抗体N-3A4在制备用于检测尿液样本中NGAL蛋白含量的试剂盒中的用途。
3.如权利要求2所述的用途,其特征在于所述试剂盒中含有检测试纸条。
4.如权利要求3所述的用途,其特征在于所述试纸条中由样品垫、结合垫、硝酸纤维素膜及吸收垫4个部分组成。
5.如权利要求4所述的用途,其特征在于样品垫和结合垫用玻璃纤维,吸收垫用吸水滤纸;羊抗鼠多克隆抗体在硝酸纤维素膜上划线作为质控带;所述单克隆抗体N-3A4在硝酸纤维素膜上划线作为检测带。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211126872.3A CN116655788B (zh) | 2022-09-16 | 2022-09-16 | 中性粒细胞明胶酶相关脂质运载蛋白(ngal)尿液检测试剂盒 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211126872.3A CN116655788B (zh) | 2022-09-16 | 2022-09-16 | 中性粒细胞明胶酶相关脂质运载蛋白(ngal)尿液检测试剂盒 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116655788A CN116655788A (zh) | 2023-08-29 |
CN116655788B true CN116655788B (zh) | 2023-10-20 |
Family
ID=87724762
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202211126872.3A Active CN116655788B (zh) | 2022-09-16 | 2022-09-16 | 中性粒细胞明胶酶相关脂质运载蛋白(ngal)尿液检测试剂盒 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116655788B (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104181305A (zh) * | 2013-05-27 | 2014-12-03 | 中国科学院上海生命科学研究院 | 抗人中性粒细胞明胶酶相关脂质运载蛋白抗体及其用途 |
CN106645762A (zh) * | 2016-12-27 | 2017-05-10 | 菲鹏生物股份有限公司 | 中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒 |
CN112552396A (zh) * | 2020-12-30 | 2021-03-26 | 河南中泽生物工程有限公司 | 抗非洲猪瘟病毒p54蛋白单克隆抗体、制备方法及应用 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090123946A1 (en) * | 2007-10-19 | 2009-05-14 | Abbott Laboratories | Immunoassays and kits for the detection of ngal |
-
2022
- 2022-09-16 CN CN202211126872.3A patent/CN116655788B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104181305A (zh) * | 2013-05-27 | 2014-12-03 | 中国科学院上海生命科学研究院 | 抗人中性粒细胞明胶酶相关脂质运载蛋白抗体及其用途 |
CN106645762A (zh) * | 2016-12-27 | 2017-05-10 | 菲鹏生物股份有限公司 | 中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒 |
CN112552396A (zh) * | 2020-12-30 | 2021-03-26 | 河南中泽生物工程有限公司 | 抗非洲猪瘟病毒p54蛋白单克隆抗体、制备方法及应用 |
Non-Patent Citations (1)
Title |
---|
齐家龙等.中性粒细胞明胶酶相关脂质运载蛋白的单克隆抗体制备及化学发光免疫定量检测试剂研究.中国生化药物杂志.2015,35(04),5-9. * |
Also Published As
Publication number | Publication date |
---|---|
CN116655788A (zh) | 2023-08-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7090983B1 (en) | Methods for detecting early cancer | |
WO2017107974A1 (zh) | 血清psmd4蛋白的检测试剂盒及其检测方法与应用 | |
US7205118B2 (en) | Nicotinamide N-methyltransferase as a marker for colorectal cancer | |
US9097714B2 (en) | Method for diagnosing malignant tumor | |
RU2769987C2 (ru) | Анализ антител | |
US20060177880A1 (en) | Use of PRN3/ILEU as a marker for colorectal cancer | |
CN113248609B (zh) | 针对再生胰岛衍生蛋白1α的抗体组合以及包含其的检测试剂盒 | |
CN115991768B (zh) | 中性粒细胞明胶酶相关脂质运载蛋白(ngal)检测试剂盒 | |
US20060188949A1 (en) | Use of protein PLST as a marker for colorectal cancer | |
WO2011108628A1 (ja) | 胃癌検出用マーカー及び胃癌検出方法 | |
Wang et al. | Development of a fluorescent immnunochromatographic assay for the procalcitonin detection of clinical patients in China | |
US20060121540A1 (en) | Use of protein MASP as a marker for colorectal cancer | |
US7579158B2 (en) | Cellular retinoic acid binding protein II as a marker for breast cancer | |
CN116655788B (zh) | 中性粒细胞明胶酶相关脂质运载蛋白(ngal)尿液检测试剂盒 | |
CN107110848B (zh) | 以脱氧羟腐胺缩赖氨酸合酶基因作为指标使用的动脉硬化及癌的检测方法 | |
EP3129785B1 (en) | Diagnosis of cancer by detecting dimeric il-18 | |
CN111303289B (zh) | 抗人Tn型糖基化MUC1抗体及其用途 | |
JP2014115186A (ja) | 胃癌、肺癌及び/又は食道癌の検出方法 | |
US20100221742A1 (en) | Novel cancer associated antibodies and their use in cancer diagnosis | |
JP4292208B2 (ja) | 乳癌に対するマーカーとしてのタンパク質speeの使用 | |
WO2021246153A1 (ja) | 膵臓がんの検出方法及び検出試薬 | |
JP2014115188A (ja) | 胃癌、膵癌、肺癌及び/又は食道癌の検出方法 | |
JP2014115199A (ja) | 胃癌又は食道癌の検出方法 | |
US20060194266A1 (en) | Use of protein RLA-0 as a marker for colorectal cancer | |
WO2018034332A1 (ja) | EphA2 N末端フラグメント抗体 |
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 |