CN105004686A - Chromium-albumin chelate as well as preparation method and application thereof - Google Patents
Chromium-albumin chelate as well as preparation method and application thereof Download PDFInfo
- Publication number
- CN105004686A CN105004686A CN201510413563.8A CN201510413563A CN105004686A CN 105004686 A CN105004686 A CN 105004686A CN 201510413563 A CN201510413563 A CN 201510413563A CN 105004686 A CN105004686 A CN 105004686A
- Authority
- CN
- China
- Prior art keywords
- chromium
- albumin
- chelate
- sample
- chromatographic column
- 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
- 239000013522 chelant Substances 0.000 title claims abstract description 121
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 239000011651 chromium Substances 0.000 claims abstract description 131
- 229910052804 chromium Inorganic materials 0.000 claims abstract description 122
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 claims abstract description 119
- 102000009027 Albumins Human genes 0.000 claims abstract description 87
- 108010088751 Albumins Proteins 0.000 claims abstract description 87
- 238000001514 detection method Methods 0.000 claims abstract description 19
- 229910001430 chromium ion Inorganic materials 0.000 claims abstract description 14
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 102
- 238000000034 method Methods 0.000 claims description 100
- 239000013024 dilution buffer Substances 0.000 claims description 76
- 102000004190 Enzymes Human genes 0.000 claims description 58
- 108090000790 Enzymes Proteins 0.000 claims description 58
- 210000004369 blood Anatomy 0.000 claims description 45
- 239000008280 blood Substances 0.000 claims description 45
- 239000003480 eluent Substances 0.000 claims description 40
- 238000000746 purification Methods 0.000 claims description 31
- 239000000872 buffer Substances 0.000 claims description 29
- 238000006243 chemical reaction Methods 0.000 claims description 27
- 238000001962 electrophoresis Methods 0.000 claims description 27
- 239000000463 material Substances 0.000 claims description 27
- 102000004169 proteins and genes Human genes 0.000 claims description 26
- 108090000623 proteins and genes Proteins 0.000 claims description 26
- 239000003292 glue Substances 0.000 claims description 23
- 238000010790 dilution Methods 0.000 claims description 17
- 239000012895 dilution Substances 0.000 claims description 17
- 230000001900 immune effect Effects 0.000 claims description 17
- 230000009870 specific binding Effects 0.000 claims description 16
- 239000003153 chemical reaction reagent Substances 0.000 claims description 14
- 239000000945 filler Substances 0.000 claims description 14
- 238000000918 plasma mass spectrometry Methods 0.000 claims description 14
- 238000000862 absorption spectrum Methods 0.000 claims description 13
- 238000000605 extraction Methods 0.000 claims description 13
- 239000011248 coating agent Substances 0.000 claims description 12
- 238000000576 coating method Methods 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 229910019142 PO4 Inorganic materials 0.000 claims description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 9
- 239000010452 phosphate Substances 0.000 claims description 9
- 238000010612 desalination reaction Methods 0.000 claims description 8
- 238000011068 loading method Methods 0.000 claims description 8
- 238000003556 assay Methods 0.000 claims description 6
- 239000003547 immunosorbent Substances 0.000 claims description 6
- 229920002401 polyacrylamide Polymers 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- 238000012797 qualification Methods 0.000 claims description 5
- 238000001042 affinity chromatography Methods 0.000 claims description 4
- 238000004090 dissolution Methods 0.000 claims description 4
- 238000011010 flushing procedure Methods 0.000 claims description 4
- 239000002504 physiological saline solution Substances 0.000 claims description 4
- 238000010170 biological method Methods 0.000 claims description 2
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 claims 1
- 210000002966 serum Anatomy 0.000 abstract description 5
- 229940088598 enzyme Drugs 0.000 description 54
- 239000000523 sample Substances 0.000 description 45
- 239000007788 liquid Substances 0.000 description 43
- 238000005406 washing Methods 0.000 description 26
- 230000000694 effects Effects 0.000 description 20
- 239000013642 negative control Substances 0.000 description 19
- 239000000758 substrate Substances 0.000 description 18
- 238000011534 incubation Methods 0.000 description 15
- 239000000499 gel Substances 0.000 description 13
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 12
- 239000013641 positive control Substances 0.000 description 12
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 11
- 229940098773 bovine serum albumin Drugs 0.000 description 11
- 229910052751 metal Inorganic materials 0.000 description 11
- 239000002184 metal Substances 0.000 description 11
- 235000018102 proteins Nutrition 0.000 description 11
- 210000002381 plasma Anatomy 0.000 description 10
- 238000001556 precipitation Methods 0.000 description 10
- 238000012360 testing method Methods 0.000 description 9
- 239000007790 solid phase Substances 0.000 description 8
- 108090000526 Papain Proteins 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 238000010828 elution Methods 0.000 description 6
- 229910001385 heavy metal Inorganic materials 0.000 description 6
- 239000012160 loading buffer Substances 0.000 description 6
- 235000020183 skimmed milk Nutrition 0.000 description 6
- 229910021642 ultra pure water Inorganic materials 0.000 description 6
- 239000012498 ultrapure water Substances 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 230000005469 synchrotron radiation Effects 0.000 description 5
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 4
- 239000004365 Protease Substances 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000002523 gelfiltration Methods 0.000 description 4
- 229940055729 papain Drugs 0.000 description 4
- 235000019834 papain Nutrition 0.000 description 4
- 229920000151 polyglycol Polymers 0.000 description 4
- 239000010695 polyglycol Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000000108 ultra-filtration Methods 0.000 description 4
- 238000005199 ultracentrifugation Methods 0.000 description 4
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical group O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- -1 acidulant Substances 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 238000001426 native polyacrylamide gel electrophoresis Methods 0.000 description 3
- 229910017604 nitric acid Inorganic materials 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- GIJIKFKGLZGENN-UHFFFAOYSA-N (4-ethoxyphenyl) 4-butoxycarbonyloxybenzoate Chemical compound C1=CC(OC(=O)OCCCC)=CC=C1C(=O)OC1=CC=C(OCC)C=C1 GIJIKFKGLZGENN-UHFFFAOYSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 2
- 229940038476 chelated chromium Drugs 0.000 description 2
- 238000013016 damping Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 230000035622 drinking Effects 0.000 description 2
- 239000003344 environmental pollutant Substances 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000012921 fluorescence analysis Methods 0.000 description 2
- 235000013905 glycine and its sodium salt Nutrition 0.000 description 2
- 238000000673 graphite furnace atomic absorption spectrometry Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 231100000719 pollutant Toxicity 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 210000002345 respiratory system Anatomy 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- ALLIZEAXNXSFGD-UHFFFAOYSA-N 1-methyl-2-phenylbenzene Chemical group CC1=CC=CC=C1C1=CC=CC=C1 ALLIZEAXNXSFGD-UHFFFAOYSA-N 0.000 description 1
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 208000031404 Chromosome Aberrations Diseases 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108010044467 Isoenzymes Proteins 0.000 description 1
- VHJLVAABSRFDPM-IMJSIDKUSA-N L-1,4-dithiothreitol Chemical compound SC[C@H](O)[C@@H](O)CS VHJLVAABSRFDPM-IMJSIDKUSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 239000006035 Tryptophane Substances 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000002441 X-ray diffraction Methods 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 239000000956 alloy Substances 0.000 description 1
- 229910045601 alloy Inorganic materials 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 235000012206 bottled water Nutrition 0.000 description 1
- 229910052793 cadmium Inorganic materials 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000003115 checkerboard titration Methods 0.000 description 1
- 230000009920 chelation Effects 0.000 description 1
- 231100000005 chromosome aberration Toxicity 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 230000002016 colloidosmotic effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 235000019621 digestibility Nutrition 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000005670 electromagnetic radiation Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 235000012631 food intake Nutrition 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 231100000025 genetic toxicology Toxicity 0.000 description 1
- 230000001738 genotoxic effect Effects 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 231100000567 intoxicating Toxicity 0.000 description 1
- 230000002673 intoxicating effect Effects 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229910052745 lead Inorganic materials 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000011785 micronutrient Substances 0.000 description 1
- 235000013369 micronutrients Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000003973 paint Substances 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 230000008288 physiological mechanism Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 230000005783 single-strand break Effects 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- UCIYAWPLDHZJJL-UHFFFAOYSA-M sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;hydrogen carbonate Chemical compound [Na+].OC([O-])=O.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O UCIYAWPLDHZJJL-UHFFFAOYSA-M 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229910052723 transition metal Inorganic materials 0.000 description 1
- 150000003624 transition metals Chemical class 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 229960004799 tryptophan Drugs 0.000 description 1
- 238000004876 x-ray fluorescence Methods 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a chromium-albumin chelate and a preparation method and application thereof, wherein the chromium-albumin chelate is prepared by chelating chromium ions and albumin through sulfydryl or/and cysteine residues. The invention establishes a qualitative and quantitative detection method of chromium-albumin chelate so as to quantitatively detect the application of the chromium-albumin chelate in evaluating the chromium pollution degree of a region. The condition of chromium pollution of people in a region can be indirectly reflected by quantitatively detecting the chromium-albumin chelate in the serum of people in the region, so that the chromium pollution degree of the region is indirectly reflected. The accuracy of the quantitative detection method of the chromium-albumin chelate is greatly improved, and the repeatability of detection is greatly improved.
Description
Technical field
The present invention relates to detection field, more particularly, relate to a kind of chromium-albumin chelate and its preparation method and application.
Background technology
Seralbumin (HSA) is the most rich in protein of content in mammalian plasma, and it can store and transport numerous endogenouss and exogenous material, and this and its specificity structure are closely related.Seralbumin is a kind of simple protein synthesized by liver, is only made up of amino acid, does not have modification group and other adjuncts.According to data information display, bovine serum albumin(BSA) (BSA) molecule is made up of 583 amino acid residues.And human serum albumins (HSA) 2 amino acid more than BSA, i.e. 585 amino acid residue compositions.Both difference is that BSA lacks the residue of HSA amino acid sequence 116 and 585.The most significant feature of albumin aminoacid ingredient is the tryptophane of low content, respectively containing 1 or 2 residue in HSA and BSA per molecule.Phenylalanine and isoleucine content are also lower.Halfcystine, leucine, glutamic acid and lysine content are all very high.A large amount of ionization residues provides the very high electric charge of albumin, and under the condition of PH7, per molecule albumin, with 185 ions, so just forms albumin ease of solubility.
The unique design feature of albumin is disulfide bond pattern.BSA sequence comprises 35 cysteine residues, forms 17 disulphide bridgeses, and residue nearest on adjacent halfcystine pair and chain links, and forms a pair ring of 10-47 residues in length.S-S key crossover adds rigidity and the stability of albumin structure.
Albuminous tertiary structure is considered to a kind of molecule of high degree of spiral, and learn that the residue of in HCR crystal about 67% participates in defining 28 α mono-conveyor screws by X-ray diffraction, 10% residue participates in forming β-bend, and 23% residue forms extended chain.
On physiological mechanism, albumin maintains 80% of human plasma colloid osmotic pressure.In addition, albumin is also the main carriers of various macromolecular substances in blood, plays an important role in metabolin transhipment, biological conversion.It is also the component regulating coagulation function and inflammatory reaction simultaneously.Human serum albumins is mainly used in research that is clinical, metabolic and genetic aspect.
Chromium (chromium, Cr) be a kind of transition metal in the periodic table of elements VI race, its quality is hard, surface has gloss, there is higher fusing point, there is great economic worth in the industrial production, be widely used since the century, annual global chromium turnout is up to 107 tons, and thus chromium is a kind of genuine important meals pollutant.At present, chromium is widely used in the various aspects such as plating, tanning, pigment, paint, alloy, printing and dyeing, closely bound up with the life of people, but equally also has an immense impact on to the healthy of the mankind.In the U.S., Cr and Cr, Cd, Pb are called as four overall situation pollutants.According to statistics, have the common people of tens hundred million every year all at the water source drinking pollution of chromium, and markon's welfare Asia have up to 30% water resource all by pollution of chromium, thus pollution of chromium drastically influence the healthy of people.
Occurring in nature chromium mainly exists with the form of trivalent and sexavalence, and it is generally acknowledged, trivalent chromium (Cr
3+) be the trace element of necessary for human, and sexavalent chrome (Cr
6+) be only obvious toxicant.Because sexavalent chrome easily enters cell by cell membrane, and can series reaction be passed through, produce strong cytotoxicity and genotoxicity, thus generally believe that chromic toxicity is higher than trivalent chromium, reach 10-100 doubly.Scholar thinks when namely the content of 6-valence Cr ions in water is poisonous more than 0.05mg/L.Sexavalent chrome can be drunk by skin contact, food intake, potable water and enter human body into approach such as, respiratory tract suctions, mainly sucking sexavalent chrome dust by respiratory tract for occupational exposed population group, is then take in sexavalent chrome by drinking into polluted source, the modes such as contaminated food, suction of eating for general population.Sexavalent chrome enters after in body, can initiated oxidation stress, chromosome aberration, Apoptosis, multiple DNA damage (comprising single-strand break, DNA-protein cross, DNA-amino acid crosslinks, the formation of Cr-DNA complex etc.), thus cause body many places tissue, organ, system injury.Moreover, sexavalent chrome is also considered to a kind of strong carcinogenic substance, as far back as nineteen ninety international cancer research organization.
In close relations between chromium and multiple proteins, can be combined with multiple proteins, suppress the activity of multiple protein enzyme.Along with going deep into chromium toxicity research, people recognize that the interaction of chromium and albumen plays important role in its intoxicating process gradually, be considered to the key point of toxicity mechanism understanding chromium, therefore, the research of chromium and protein interaction just seemed ever more important.And the present mechanism about chromium and protein effect is only tip of the iceberg, also has the relation between a lot of protein and chromium not clear, thus strengthen for the relation between chromium and protein most important.
Summary of the invention
For the problem that pollution of chromium is serious, the object of the present invention is to provide a kind of chromium-albumin chelate and preparation method thereof, and set up the qualitative and quantitative analysis method of chromium-albumin chelate, quantitatively to detect the application of chromium-albumin chelate in the regional pollution of chromium degree of evaluation one.Indirectly can reflect the situation of this regional crowd by pollution of chromium by quantitatively detecting chromium-albumin chelate in regional crowd's serum, thus indirectly reflect this regional pollution of chromium degree.
The technical solution adopted for the present invention to solve the technical problems is: provide a kind of chromium-albumin chelate, chromium ion and albumin by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides a kind of preparation method of above-mentioned chromium-albumin chelate, comprises the following steps:
A) chromium and albuminous chelatropic reaction: add chromium ion in the albumin coming from human body or the albumin of recombinating according to biological method and carry out chelatropic reaction purifying, obtain reaction solution;
B) extraction of purifying chromium-albumin chelate: adopt immune-affinity chromatography, removes unreacted albumin and unnecessary chromium ion in reaction solution, obtains chromium-albumin chelate.
Wherein, step B described in external synthetic method) specific as follows:
(1) sample dissolution: to through steps A) add physiological saline in the reaction solution that obtains chromium-albumin chelate is redissolved;
(2) balance chromatographic column: use dilution buffer to rinse the pipeline of chromatographic column, load in chromatographic column can with the filler of albumin specific binding, after dress post, continue to use dilution buffer balance chromatographic column;
(3) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (1), albumin and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the phosphate solution of 0.05-0.1mol/L to carry out wash-out, obtain eluent I;
(5) collect: collect eluent I;
(6) dialyse: by the eluent of the collection in step (5), dress bag filter, uses ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatographic column: adopt new chromatographic column, use dilution buffer flushing line, in this chromatographic column load can with the filler of chromium specific binding, dress post after balance chromatographic column by dilution buffer again;
(8) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out, obtain eluent II;
(10) collect: collect eluent II;
(11) dialyse: by the eluent of the collection in step (10), dress bag filter, uses ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain chromium-albumin chelate.
Wherein, the preparation method of above-mentioned chromium-albumin chelate, also comprises step C): to the qualification of chromium-albumin chelate;
Wherein, step C) in specific as follows:
(1) glue bed is prepared: prepare glue bed using the one in Ago-Gel, polyacrylamide gel as medium;
(2) application of sample: get step B) in chromium-albumin chelate of obtaining of extraction purification, add dilution buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: on glue bed, find out the protein band containing chromium, this protein band is taken out and dissolves, and then adopt ELISA method or ICP-MS method or AAS method to detect the content whether containing chromium and detection chromium.
The present invention also provides the application of a kind of chromium described above-albumin chelate in preparation human body in the reagent of chromium-albumin chelate or kit.
The present invention also provides a kind of and at least comprises the kit of chromium described above-albumin chelate as standard items.
Preferably, also comprise coating buffer in this kit, this coating buffer contains the material of catching albuminous material or catching chromium.
The present invention also provides a kind of method of quantitative detection chromium-albumin chelate, using the above-mentioned chromium-albumin chelate of known content as standard items, a pair sample of following methods is adopted to detect: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification chromium-albumin chelate and enzyme linked immunological combined techniques, purification chromium-albumin chelate and atomic absorption spectrum combined techniques, purification chromium-albumin chelate and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
Implement chromium of the present invention-albumin chelate and its preparation method and application, there is following beneficial effect:
1. the present invention's external synthesis chromium-albumin chelate first;
2. the present invention proposes chromium-albumin chelate first and can be used for preparing application in the reagent or kit detecting chromium-albumin chelate in blood sample.
3. the present invention establishes the method for qualitative and quantitative detection of chromium-albumin chelate, quantitatively to detect the application of chromium-albumin chelate in the regional pollution of chromium degree of evaluation one.Indirectly can reflect the situation of this regional crowd by pollution of chromium by quantitatively detecting chromium-albumin chelate in regional crowd's serum, thus indirectly reflect this regional pollution of chromium degree.Chromium-its accuracy of albumin chelate quantitative detecting method that the present invention sets up greatly improves, and the repeatability of detection is greatly enhanced.
Accompanying drawing explanation
Fig. 1 is the non denatured electrophoretic band figure of chromium of the present invention-albumin chelate;
Fig. 2 is the synchrotron radiation X line fluorescence analysis figure of the electrophoretic band of chromium of the present invention-albumin chelate.
Embodiment
Below, in conjunction with embodiment, the present invention is described further:
Unless otherwise indicated, the laboratory operating procedures related to is the step of this area routine in the present invention, and agents useful for same, material are as cited by following, and the reagent being this area conventional not enumerating out in the present invention maybe can be obtained by commercial mode:
Phosphate solution is 0.05-0.1mol/L Na
2hPO
4solution or 0.5-1mol/L Na
2hPO
4solution;
Extracting reagent is PEG solution, borate buffer solution etc. (adopting PEG method);
Glue bed medium is the one in Ago-Gel, polyacrylamide gel;
Catch the human albumin antibody that the purchase of albuminous material is A-0577 from Novus company model; Wherein, be of the present inventionly catch albuminous material with the material of albumin specific binding, anti-HSA antibody, anti-albumin antibodies;
The material purchase of catching chromium is the anti-chromium antibody of RK10641 from the model of Ran Ke bio tech ltd, Guangzhou; Wherein, of the present inventionly resist with the material of chromium specific binding, anti-Cr antibody, anti-chromium antibody, chromium the material being and catching chromium;
Enzyme labelled antibody is the one in the antibody containing the enzyme labeling such as horseradish peroxidase, alkaline phosphatase;
Substrate is methyl biphenyl amine (TMB) solution;
Cleansing solution is for containing KH
2pO
40.2mg/ml, Na
2hPO
412H
2the pH of O 2.90mg/ml, NaCl8.0mg/ml, KCl 0.2mg/ml, 0.5%Tween-20 is the 0.15M PBS solution of 7.4;
Confining liquid is 1%-5% bovine serum albumin(BSA) or skimmed milk power;
Dilution buffer is for containing 1.5mg/mL Na
2cO
3, 2.93mg/ml NaHCO
3pH be 9.6 0.05M carbonate buffer solution;
Enzyme labelled antibody is HRP enzyme labelled antibody;
Stop buffer is: by the 2M H of 21.7ml
2sO
4be settled to the ddH of 200ml
2in O;
Eluent be containing the PH of papain be 8.0 0.1mol/L Tris-HCL damping fluid;
Acidulant is nitric acid;
Sample-loading buffer is for containing 1M Tris-HCl (pH 6.8) 15.5mL, 1% bromophenol blue 2.5mL, ddH
2the Sample buffer (5X) of O7mL, glycocoll 25mL;
Electrophoretic buffer be containing Tris 3mg/ml, glycocoll 14.4mg/ml PH be the ddH of 6.8
2o solution;
The invention provides a kind of chromium-albumin chelate, chromium ion and albumin by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides the preparation method of a kind of chromium-albumin chelate, comprises the following steps:
A) chromium and albuminous chelatropic reaction: add chromium ion and carry out chelatropic reaction in the albumin in people source, obtain reaction solution;
B) extraction of purifying chromium-albumin chelate: adopt immune-affinity chromatography, remove unreacted albumin and unnecessary chromium ion in reaction solution, obtain chromium-albumin chelate, concrete steps are as follows:
(1) sample dissolution: to through steps A) add physiological saline in the reaction solution that obtains chromium-albumin chelate is redissolved;
(2) balance chromatographic column: use dilution buffer to rinse the pipeline of chromatographic column, load in chromatographic column can with the filler of albumin specific binding, after dress post, continue to use dilution buffer balance chromatographic column; Described can be the silica gel or the resin that contain anti-albumin antibodies with the filler of albumin specific binding;
(3) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (1), albumin and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the phosphate solution of 0.05-0.1mol/L to carry out wash-out, obtain eluent I;
(5) collect: collect eluent I;
(6) dialyse: by the eluent of the collection in step (5), dress bag filter, uses ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatographic column: adopt new chromatographic column, use dilution buffer flushing line, in this chromatographic column load can with the filler of chromium specific binding, dress post after balance chromatographic column by dilution buffer again; Described can be the silica gel or the resin that contain anti-chromium antibody with the filler of chromium specific binding;
(8) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out, obtain eluent II;
(10) collect: collect eluent II;
(11) dialyse: by the eluent of the collection in step (10), dress bag filter, with 2 liters of ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain chromium-albumin chelate;
C) to the qualification of chromium-albumin chelate, concrete steps are as follows:
(1) glue bed is prepared: prepare glue bed using the one in Ago-Gel, polyacrylamide gel as medium;
(2) application of sample: get step B) in chromium-albumin chelate of obtaining of extraction purification, add dilution buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: on glue bed, find out the protein band containing chromium, this protein band is taken out and dissolves, and then adopt ELISA method or ICP-MS method or AAS method to detect the content whether containing chromium and detection chromium.
The present invention also provides a kind of and at least comprises the kit of chromium described above-albumin chelate as standard items.
Preferably, also comprise coating buffer in this kit, this coating buffer contains the material of catching albuminous material or catching chromium.
In the present invention, the kit that can realize the object of the invention can be listed following several, but is not limited to this.
Detect a kit for chromium in blood sample-albumin chelate, comprise containing the coating buffer of catching albuminous material, confining liquid, cleansing solution, material, enzyme labelled antibody, substrate, stop buffer, dilution buffer, positive control, negative control etc. as two anti-caught chromium.
Detect a kit for chromium in blood sample-albumin chelate, comprise containing the coating buffer of catching albuminous material, confining liquid, cleansing solution, eluent, positive control, negative control etc.
Detect a kit for chromium in blood sample-albumin chelate, comprise containing the coating buffer of catching albuminous material, confining liquid, cleansing solution, eluent, acidulant, hydrogen peroxide, positive control, negative control etc.
Detect a kit for chromium in blood sample-albumin chelate, comprise as extracting reagent in purification whole blood needed for albumin, redissolve liquid, containing the coating buffer, confining liquid, cleansing solution, enzyme labelled antibody, substrate, stop buffer, dilution buffer, positive control, negative control etc. of material of catching chromium.
Detecting a kit for chromium in blood sample-albumin chelate, comprising as extracting reagent, redissolution liquid, positive control, negative control etc. in purification whole blood needed for albumin.
Detecting a kit for chromium in blood sample-albumin chelate, comprising as extracting reagent, redissolution liquid, acidulant, hydrogen peroxide, positive control, negative control etc. in purification whole blood needed for albumin.
Detect a kit for chromium in blood sample-albumin chelate, comprise as extraction reagent albuminous in purification whole blood, glue bed medium, redissolve liquid, sample-loading buffer, on dissolving glue bed containing liquid needed for the protein band of chromium, containing the coating buffer, confining liquid, cleansing solution, enzyme labelled antibody, substrate, stop buffer, dilution buffer, positive control, negative control etc. of material of catching chromium.
Detect a kit for chromium in blood sample-albumin chelate, comprise as extracting reagent, glue bed medium in purification whole blood needed for albumin, redissolve liquid, sample-loading buffer, on dissolving glue bed containing liquid, positive control, negative control etc. needed for the protein band of chromium.
Detect a kit for chromium in blood sample-albumin chelate, comprise as extracting reagent, glue bed medium in purification whole blood needed for albumin, redissolve liquid, sample-loading buffer, on dissolving glue bed containing liquid, acidulant, hydrogen peroxide, positive control, negative control etc. needed for the protein band of chromium.
In above-mentioned several kit, described positive control is standard items, is namely chelated with the albumin chelate of heavy metal chromium or is chelated with the BSA chelate of heavy metal chromium; Described negative control is the dilution buffer not containing standard items.
Mentioned reagent box is for detecting the albumin of chelated chromium, to improve the accuracy of detection, repeated, and makes it to be promoted in clinical.
The present invention also provides a kind of method of quantitative detection chromium-albumin chelate, using the above-mentioned chromium-albumin chelate of known content as standard items, a pair sample of following methods is adopted to detect: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification chromium-albumin chelate and enzyme linked immunological combined techniques, purification chromium-albumin chelate and atomic absorption spectrum combined techniques, purification chromium-albumin chelate and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.In the present invention, following several with having of detecting that the method for chromium-albumin chelate can list, but be not limited to following several.
Wherein, the reagent adopted in the method for above-mentioned quantitative detection chromium-albumin chelate is as follows:
method one:euzymelinked immunosorbent assay (ELISA) (ELISA method) detects chromium-albumin chelate, detects in accordance with the following steps:
1) albuminous material can be caught, as anti-albumin antibodies (anti-HSA Ab) is coated on solid phase carrier: by dilution buffer by anti-HSA Ab extremely dilution 1000-8000 times, add in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
2) get whole blood from the circulation system, make measuring samples, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with cleansing solution, after washing completes, add using 1%-5% bovine serum albumin(BSA) or skimmed milk power as confining liquid, place 1 hour, remove confining liquid, and wash with cleansing solution for 37 DEG C, after having washed, elisa plate places 1-2 hour in 36.5-37.5 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard items with the chromium of known content-albumin chelate; By dilution buffer, measuring samples is diluted to 10-40 doubly, adds in micropore, 37 DEG C of effect 1-2 hour;
5) material can catching chromium is added, and incubation: remove measuring samples, and wash with cleansing solution, after washing completes, add and be diluted to 5000-40000 anti-Cr Ab doubly by dilution buffer, 37 DEG C of effect 1-2 hour, make the crome metal on anti-Cr Ab and albumin react and form antigen antibody complex;
6) enzyme conjugates incubation: remove anti-chromium antibody, and wash with cleansing solution, after washing completes, add the enzyme labelled antibody with dilution buffer dilution, make the concentration of the enzyme labelled antibody of dilution be 2 μ g/mL, 37 DEG C of effect 1-2 hour, make itself and enzyme labelled antibody react;
7) substrate incubation: remove enzyme labelled antibody, and wash with cleansing solution, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
8) cessation reaction: stop buffer is dropped to each micropore;
9) wavelength 405nm is got, after adding stop buffer, elisa plate is placed in microplate reader and detects, read the OD value of measuring samples and standard items respectively, by drawing standard curve, try to achieve the content (also microplate reader can not be used, directly carry out qualitative detection by colour developing situation) of measuring samples.
The method utilizes ELISA principle, specificity albumin in whole blood can be extracted, the albumin upper part extracted is chelated with heavy metal chromium, and the chromium on this part albumin catch by anti-chromium antibody, afterwards can again by horseradish peroxidase, the antibody of the enzyme labelings such as alkaline phosphatase is caught (this antibody nonrecognition coating protein), the antibody of catching is under the effect of developer and stop buffer, OD value can be read under instrument, and the albumin not containing chelated mineral chromium, then can not catch by the specific antibody of anti-chromium, also can not with horseradish peroxidase, the antibody of the enzyme labelings such as alkaline phosphatase caught, and also not containing crome metal (negative control group result is negative) in agents useful for same, thus when read OD value result is shown as the positive, the i.e. provable crome metal detecting chelating on albumin.
method two:enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) detect chromium-albumin chelate and detect in accordance with the following steps:
1) albuminous material can be caught, as anti-albumin antibodies (anti-HSA Ab) is coated on solid phase carrier: by dilution buffer, anti-HSA Ab is diluted to 1000-8000 doubly, add in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
2) get whole blood from the circulation system, make measuring samples, the centrifugal 5-8 minute of 1000rpm, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with cleansing solution, after washing completes, add using 1%-5% bovine serum albumin(BSA) or skimmed milk power as confining liquid, place 1 hour, remove confining liquid, and wash with cleansing solution for 37 DEG C, after having washed, elisa plate places 1-2 hour in 36.5-37.5 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard items with the chromium of known content-albumin chelate; By dilution buffer, measuring samples is diluted to 10-40 doubly, adds in micropore, 37 DEG C of effect 1-2 hour;
5) wash-out: remove measuring samples, and wash with cleansing solution, after washing completes, add eluent, wash-out 1-3 hour at 37 DEG C.
6) detect: sample from the micropore of elisa plate, detect the chromium of chelating on albumin in Atomic Absorption Spectrometer, and drawing standard curve, readout value;
In conjunction with atomic absorption spectrum (AAS) principle on the basis that the method utilizes ELISA principle, Atomic Absorption Spectrometer is utilized to detect the chromium of chelating on albumin, owing to only containing albumin in solution, and not containing any heavy metal (negative control group result is negative) in agents useful for same, interference can not be caused to result, thus when read result is shown as the positive, i.e. the provable crome metal detecting chelating on albumin.
method three:enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) detect chromium-albumin chelate and detect in accordance with the following steps:
1) albuminous material can be caught, as anti-albumin antibodies (anti-HSA Ab) is coated on solid phase carrier: by dilution buffer, anti-HSA Ab is diluted to 1000-8000 doubly, add in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
2) get whole blood from the circulation system, make measuring samples, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with cleansing solution, after washing completes, add using 1%-5% bovine serum albumin(BSA) or skimmed milk power as confining liquid, place 1 hour, remove confining liquid, and wash with cleansing solution for 37 DEG C, after having washed, elisa plate places 1-2 hour in 36.5-37.5 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard items with the chromium of known content-albumin chelate; By dilution buffer, measuring samples is diluted to 10-40 doubly, adds in micropore, 37 DEG C of effect 1-2 hour;
5) wash-out: remove measuring samples, and wash with cleansing solution, after washing completes, add eluent, wash-out 1-3 hour at 37 DEG C.
6) acidifying: in step 5) in solution in add acidulant acidifying carried out to solution, sealing is spent the night, and thorough acidifying, adds hydrogen peroxide, and acid is caught up with in heating;
7) detect: from step 6) elisa plate solution get 0.5ml liquid, under icp ms, detect chelating in albuminous chromium, and drawing standard curve, readout value.
The method is on the basis utilizing ELISA principle, in conjunction with sense coupled plasma mass spectrometry (ICP-MS) principle, the chromium of chelating on albumin is detected with icp ms, owing to only containing albumin in solution, and not containing any heavy metal (negative control group result is negative) in agents useful for same, interference can not be caused to result, thus when read result is shown as the positive, i.e. the provable crome metal detecting chelating on albumin.
method four:purification chromium-albumin chelate and enzyme linked immunological combined techniques (method of purification+ELISA method) detect chromium-albumin chelate, detect in accordance with the following steps:
1) from whole blood, albumin is extracted: adopt the polyglycol PEG precipitation method or ultracentrifugation or the method such as molecule ultrafiltration or gel filtration from the albumin whole blood extraction purification blood, and redissolve in solution by albumin, obtain albumin solution;
2) anti-Cr Ab is coated on solid phase carrier: by dilution buffer, anti-Cr Ab is diluted to 5000-40000 doubly, adds in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
3) close: remove dilution buffer, and wash with cleansing solution, after washing completes, add using 1%-5% bovine serum albumin(BSA) or skimmed milk power as confining liquid, place 1 hour, remove confining liquid, and wash with cleansing solution for 37 DEG C, after having washed, elisa plate places 1-2 hour in 36.5-37.5 DEG C;
4) add measuring samples, and incubation: from step 1) solution sample, make measuring samples; Standard items are made with the chromium of known content-albumin chelate; By dilution buffer, measuring samples is diluted to 10-40 doubly, adds in micropore, 37 DEG C of effect 1-2 hour;
5) enzyme conjugates incubation: remove step 4) in measuring samples, and to wash with cleansing solution, after washing completes, add the enzyme labelled antibody with dilution buffer dilution, make the concentration of the enzyme labelled antibody of dilution be 2 μ g/mL, 37 DEG C of effect 1-2 hour, make itself and enzyme labelled antibody react;
6) substrate incubation: remove enzyme labelled antibody, and wash with cleansing solution, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
7) cessation reaction: stop buffer is dropped to each micropore;
8) wavelength 405nm is got, after adding stop buffer, elisa plate is placed in microplate reader and detects, read the OD value of measuring samples and standard items respectively, by drawing standard curve, try to achieve the content (also microplate reader can not be used, directly carry out qualitative detection by colour developing situation) of measuring samples.
method five:purification chromium-albumin chelate and atomic absorption spectrum combined techniques (method of purification+AAS method) detect chromium in blood sample-albumin chelate, detect in accordance with the following steps:
1) from whole blood, albumin is extracted: adopt the polyglycol PEG precipitation method or ultracentrifugation or the method such as molecule ultrafiltration or gel filtration from the albumin whole blood extraction purification blood, and redissolve in solution by albumin, obtain albumin solution;
2) detect: from step 1) sample the solution that obtains, detect the chromium of chelating on albumin in Atomic Absorption Spectrometer, and drawing standard curve, readout value.
method six:purification chromium-albumin chelate and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ICP-MS method) detect chromium-albumin chelate, detect in accordance with the following steps:
1) from whole blood, albumin is extracted: adopt the polyglycol PEG precipitation method or ultracentrifugation or the method such as molecule ultrafiltration or gel filtration from the albumin whole blood extraction purification blood, and redissolve in solution by albumin, obtain albumin solution;
2) acidifying: from step 1) sample the solution that obtains, add acidulant (as nitric acid) in the solution and carry out acidifying to solution, sealing is spent the night, and thorough acidifying, adds hydrogen peroxide, and acid is caught up with in heating;
3) detect: from step 2) get 0.5ml liquid the solution that obtains, under icp ms, detect the chromium of chelating on albumin, and drawing standard curve, readout value.
method seven:electrophoresis and euzymelinked immunosorbent assay (ELISA) or atomic absorption spectrography (AAS) or inductivity coupled plasma mass spectrometry combined techniques (electrophoresis-ELISA method/AAS method/ICP-MS method) detect chromium-albumin chelate, detect in accordance with the following steps:
1) from whole blood, albumin is extracted: adopt the polyglycol PEG precipitation method or ultracentrifugation or the method such as molecule ultrafiltration or gel filtration from the albumin whole blood extraction purification blood, and redissolve in solution by albumin, obtain albumin solution;
2) glue bed is prepared: select Ago-Gel or polyacrylamide gel or SDS-polyacrylamide gel etc. as medium as required, conventionally prepare glue bed;
3) application of sample: get step 1) the solution 8 μ L that obtains adds 2 μ L sample-loading buffers, mixing, of short duration centrifugal; (noticing that step can not be boiled herein)
4) electrophoresis: connect electrophoresis plate, carry out electrophoretic separation;
5) detect: on glue bed, find out the protein band containing chromium, this band is taken out, after treatment this protein band is dissolved among liquid, and then utilize ELISA or ICP-MS or AAS to detect the chromium content be dissolved in liquid respectively.In addition, the albuminous isoelectric point of the method detection chelated chromium, molecular weight and content etc. can also be utilized.
Albumin in method seven can with multiple Methods For Purification out (such as supercentrifugation or HPLC or gel-filtration chromatography or gel electrophoresis or ELISA method etc.), the albumin of purifying out is redissolved in solution, get a certain amount of albumin, utilize electric charge shifting principle, carry out electrophoresis (electrophoresis, EP), can according to molecular weight on gel slab (different medium can be adopted as required), the difference such as isoelectric point runs out of different bands, find out the respective strap being rich in chromium, protein in gel is redissolved in solution, namely relevant albuminous content can be detected at a particular wavelength, also ELISA can be utilized, AAS, the principles such as ICP-MS detect the chromium content of chelating on albumin, owing to only containing albumin in solution, and not containing any heavy metal (negative control group result is negative) in agents useful for same, interference can not be caused to result, thus when read result is shown as the positive, the i.e. provable crome metal detecting chelating on albumin.
embodiment 1: the preparation method of chromium-albumin chelate, comprises the following steps:
A) chromium and albuminous chelatropic reaction: add chromium ion and carry out chelatropic reaction in the albumin in people source, obtain reaction solution;
Preparation of reagents:
1) borate buffer solution (0.01M): take 0.31g boric acid and be dissolved in 400ml ultrapure water, regulates pH to 9.0 with the NaOH of 0.1M, is settled to 500mL.
2) albumin solution: take 4.0mg albumin and be dissolved in 4.0mL 0.01M pH9.0 borate buffer solution, dissolving of fully vibrating, is mixed with the protein solution of 1.0mg/mL;
3) EDTA-NaHCO3 mixed solution: take EDTA2H
2o 1.86g, NaHCO
316.8g is dissolved in 900mL ultrapure water, adjusts pH to 8.0 be settled to 1000ml, autoclaving, room temperature preservation with 1.0M NaOH;
4) ITCBE buys from Japanese colleague's chemistry institute, and article No. is M030;
5) bag filter is purchased from Bioshop Inc, molecular cut off 14000.
The chelation step of chromium-albumin chelate:
1) process of bag filter: EDTA-NaHCO bag filter being put into 500ml
3in mixed solution, boil 10min; Tipping EDTA/NaHCO
3liquid, with ultrapure water rinsing gently, then boils 10min with the 5mmol/LEDTA solution of 500ml; Discard boiling liquid, thoroughly clean with ultrapure water, add ultrapure water immersion bag filter 4 DEG C and spend the night.During use, put on one's gloves, take out bag filter, with its surfaces externally and internally of ultrapure water cleaning down;
2) getting 2.0mg ITCBE is dissolved in 2ml DMSO;
3) getting 4.0mg albumin is dissolved in 4.0ml borate buffer solution;
4) slowly by step 2) liquid prepared adds step 3) in the albumin solution prepared, dropping limit, limit is shaken, and being placed in temperature is 25 DEG C, and rotating speed is react 24h in the shaking table of 100r/min, then to dialyse 24h with bag filter, remove the ITCBE be not combined with albumin;
5) by step 4) the liquid 1mol/L HCl adjust ph to 7.0 of gained, then slowly drip the 1mmol/L chromium ion solution of 80 μ l gradually, dropping limit, limit vibrates, in order to avoid chromium ion makes albuminous degeneration precipitate;
6) by step 5) to be placed in temperature be 25 DEG C for the solution of gained, rotating speed is react 2h in the shaking table of 100r/min, carries out dialysis 24h with bag filter;
7) by step 6) dialysis after liquid preserve in-20 DEG C of packing, obtain reaction solution.
B) extraction of purifying chromium-albumin chelate: adopt immune-affinity chromatography, remove reaction solution (the i.e. step 7) reaction solution that obtains) in unreacted albumin and unnecessary chromium ion, obtain chromium-albumin chelate, concrete steps are as follows:
(1) sample dissolution: to through step 7) add physiological saline in the reaction solution that obtains chromium-albumin chelate is redissolved;
(2) balance chromatographic column: use dilution buffer to rinse the pipeline of chromatographic column, load in chromatographic column can with the filler of albumin specific binding, after dress post, continue to use dilution buffer balance chromatographic column;
(3) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (1), albumin and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the phosphate solution of 0.05-0.1mol/L to carry out wash-out, obtain eluent I;
(5) collect: collect eluent I;
(6) dialyse: by the eluent of the collection in step (5), dress bag filter, uses ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatographic column: adopt new chromatographic column, use dilution buffer flushing line, in this chromatographic column load can with the filler of chromium specific binding, dress post after balance chromatographic column by dilution buffer again;
(8) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out, obtain eluent II;
(10) collect: collect eluent II;
(11) dialyse: by the eluent of the collection in step (10), dress bag filter, with 2 liters of ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain chromium-albumin chelate;
C) to the qualification of chromium-albumin chelate, concrete steps are as follows:
(1) glue bed is prepared: prepare glue bed using Ago-Gel as medium;
(2) application of sample: get step B) in chromium-albumin chelate of obtaining of 8 μ L extraction purification, add 2 μ L sample-loading buffers, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, add electrophoretic buffer and carry out electrophoresis; In electrophoresis process, electric current is 22mA constant current, and environment temperature is 4 DEG C; Electrophoresis is stopped when electrophoresis to bromophenol blue moves to bottom glue;
(4) detect: on glue bed, find out the protein band containing chromium, this protein band is taken out and dissolves, and then adopt ELISA method or ICP-MS method or AAS method to detect the content whether containing chromium and detection chromium.
D) testing result
(1) content of beary metal in GFAAS (graphite furnace atomic absorption spectrometry) (AAS) Preliminary Determination HSA: the results are shown in Table 1
Table 1 is content of beary metal in albumin (μ g/L)
Sample name | Cr(μg/L) |
HSA | 227.098 |
(NH 4) 2SO 4 | 0.054 |
N.S | 0.412 |
N.S | 0 |
(2) electrophoresis result:
Wherein, Fig. 1 is the non denatured electrophoretic band figure of chromium of the present invention-albumin chelate.
Native polyacrylamide gel electrophoresis (Native-PAGE) or be called that active electrophoresis is under the condition not adding the denaturant such as SDS and thin base ethanol, to keeping active protein to carry out polyacrylamide gel electrophoresis, be usually used in the qualification of enzyme, Isozyme Analysis and purification.The native polyacrylamide gel electrophoresis not adding SDS can make biomacromolecule in electrophoresis process, keep its natural shape and electric charge, their separation is the molecular sieve effect of difference according to its electrophoretic mobility and gel, thus higher resolution can be obtained, especially after electrophoretic separation, still can keep the biologically active of the biomacromolecule such as protein and enzyme, therefore the right swimming lane M is sex change Marker, only for detecting, do not mark effect, swimming lane 1 is albuminous band.
(3) Synchrotron Radiation X-Ray Fluorescence Anal ysis:
In protein band, the SRXRF of micronutrient levels analyzes and " synchrotron radiation bunch completes at the 4W1 of BEPC (BEPC).In storage rings, beam current energy is 2.2GeV, beam intensity 100mA.Sample transfer table (TSA200 type, Beijing stand upright Han Guang company) can move up to change launching spot position along X, Y two-dimensional square under computer-controlled step motor drives, and moving step length is 0.0025mm.The X ray gone out from electromagnetic radiation is detected by Si (Li) detector (PGT Inc.LS 30143-DS), probe is mutually vertical with incident SR line copline, apart from sample irradiation point 20mm, signal obtains with PGT MCA (MCA4000) and exports.By the monochromatic synchrotron radiation light excited sample of 11.5keV, launching spot (1mmx3mm) position is regulated to make it to be in band one end, in the minute of 300s, hot spot is always along band evenly slowly movement, and at the end of counting, hot spot moves on to this band other end.A spectrum is got along the every 1mm in electrophoresis direction.Adopt AX IL software data processing, and with deriving from air and the Ar signal peak of content constant carries out normalization to other element peak, to offset the impact that beam intensity change produces signal power.Measure the fluorescence Spectra of the dry glued membrane of quantitative criterion at identical conditions in the same way.
Fig. 2 is the synchrotron radiation X line fluorescence analysis figure of the electrophoretic band of chromium of the present invention-albumin chelate, and in figure, horizontal ordinate is protein band position, and ordinate is each metal energy of this band (content) value.
the determination of the testing conditions of the method for a kind of quantitative detection chromium-albumin chelate of the present invention:
1. the determination of the optimum diluting multiple of anti-albumin antibodies, anti-chromium antibody best effort concentration and blood plasma
Step is as follows:
(1) be that 1:1000,1:2000,1:4000,1:8000 dilute by anti-HSA Ab dilution buffer according to the mass volume ratio of anti-HSA Ab and dilution buffer, add in elisa plate micropore, anti-HSA Ab is coated on solid phase carrier, each concentration bag is by three rows, and 4 DEG C are spent the night 18 hours;
(2) remove dilution buffer, and wash with cleansing solution, after washing completes, with 2% bovine serum albumin(BSA) as confining liquid, place 1 hour, remove confining liquid, and wash with cleansing solution for 37 DEG C;
(3) measuring samples is that 1:20,1:40,1:80 dilute by the mass volume ratio of measuring samples and dilution buffer, add in micropore, according to the anti-HSA Ab concentration of above-mentioned bag quilt, the anti-HSAAb of same concentration adds different dilutability blood plasma respectively, 37 DEG C of effects 1 hour;
(4) measuring samples is removed, and wash with cleansing solution, after washing completes, add anti-Cr Ab, anti-Cr Ab is that 1:5000,1:10000,1:20000,1:40000 dilute by the mass volume ratio of anti-Cr Ab and dilution buffer, and according to each identical anti-HSA Ab, whole blood dilute concentration, the anti-Cr Ab of variable concentrations respectively adds 2 holes, 37 DEG C act on 1 hour, the crome metal on anti-Cr Ab and HSA are reacted;
(5) enzyme labelled antibody selects the suitableeest working concentration, i.e. 2 μ g/mL, removes anti-Cr antibody, and wash with cleansing solution, after washing completes, add with dilution buffer dilution HRP enzyme labelled antibody, 37 DEG C act on 1 hour, HRP enzyme labelled antibody and chromium-albumin chelate are reacted;
(6) remove enzyme labelled antibody, and wash with cleansing solution, after washing completes, add substrate, 37 DEG C of lucifuge effects 30 minutes;
(7) stop buffer is dropped to each micropore;
(8) get wavelength 405nm, after adding stop buffer, elisa plate is placed in microplate reader and detects, read each hole OD value respectively.According to each hole OD value numerical value, select anti-HSA Ab, the best effort concentration of anti-Cr Ab and the optimum diluting multiple of blood plasma.
Simultaneously using made standard items as positive control in test, select anti-HSA Ab+ close+anti-Cr Ab+ enzyme mark+substrate (namely not adding measuring samples) is as negative control 1; Anti-HSA Ab+ closes+and blood plasma+enzyme mark+substrate (namely not adding anti-Cr Ab) is as negative control 2; Anti-HSA Ab+ closes+and enzyme mark+substrate (namely not adding measuring samples and anti-Cr Ab) is as negative control 3; Anti-HSA Ab+ closes+and blood plasma+anti-Cr Ab+ substrate (i.e. not enzyme-added mark) is as negative control 4; Closed+blood plasma+anti-Cr Ab+ enzyme mark+substrate (namely do not add anti-HSA Ab and catch HSA) is as blank 1; Only add substrate as blank 2 and PBS as blank 3.Testing result is in Table 2-3.
Table 2: Checkerboard titration method determines the data (each data are all mean value) of anti-HSA Ab, the best effort concentration of anti-Cr Ab and the optimum diluting multiple of blood plasma
Table 3:ELISA positive control and negative control ELISA testing result
Shown by table 2-3 data, we can find out, and the dilutability as anti-HSA Ab is 1:2000, whole blood dilutability is when be the anti-dilutability of 1:20, Cr being 1:10000, OD value is maximum, so select concentration corresponding to this value as best effort concentration.
2. quantitatively detect the determination of eluent best effort concentration and elution time in method two, three in the method for chromium-albumin chelate
Step is as follows: (1) bag quilt: anti-Cr Ab dilution buffer be diluted to 10000 times (mass volume ratios), add in elisa plate micropore, 37 DEG C of water-baths 3 hours, stores refrigerator;
(2) close: remove dilution buffer, and wash with cleansing solution, after washing completes, with 2% bovine serum albumin(BSA) as confining liquid, place 1 hour, remove confining liquid, and wash with cleansing solution for 37 DEG C;
(3) remove confining liquid, and wash with cleansing solution, after washing completes, add the enzyme labelled antibody with dilution buffer dilution, it is 2 μ g/mL that enzyme labelled antibody is diluted to concentration, and 37 DEG C act on 2 hours, itself and anti-Cr Ab are reacted;
(4) prepare eluent: papain pH 8.0,0.1mol/L Tris-HCI buffer is become 100ng/ml, then add 1mmol/L dithiothreitol (DTT) (DTT) 37 DEG C and hatch 30min;
(5) enzyme labelled antibody is removed, by dilution buffer, eluent is diluted, make the concentration of the papain in eluent and the ratio of the concentration of enzyme labelled antibody be 1:80,1:40,1:20,1:10,1:5 (wherein each concentration does 3 multiple holes), be positioned over wash-out 1h, 2h, 3h at 37 DEG C of temperature respectively;
(6) remove eluent, and wash with cleansing solution, after washing completes, add substrate, 37 DEG C of lucifuge effects 30 minutes;
(7) stop buffer is dropped to each micropore;
(8) get 405nm wavelength, after adding stop buffer, be placed in by elisa plate in microplate reader and detect, read respectively and often organize OD value, by comparing with PBS damping fluid group, compare optimum concentration and the elution time of eluent, concrete outcome is see table 4.
Table 4:ELISA eluent best effort concentration and elution time are determined
From table 4, we can find, as the ratio=1:20 of the concentration of the papain in eluent with the concentration of enzyme labelled antibody, each group of OD value is all lower than other several groups, this concentration elution effect optimum (reach maximum by the anti-Cr Ab-enzyme mark compound wash-out degree that ELISA hole wall combines, thus OD value is minimum) is described; And no matter elution time is 1h, 2h, 3h, the change of each group OD value is all little, as seen along with the prolongation of time, enzyme activity weakens gradually, when enzyme concentration is constant, extends digestion time and can not improve digestibility, so the elution time of eluent is that 1-3h all can in this test.
application Example 1
Get and adopt ELISA method to detect chromium-albumin chelate in 100 parts of sample blood plasma, detect according to following steps: euzymelinked immunosorbent assay (ELISA) (ELISA method) detects chromium-albumin chelate, detect in accordance with the following steps:
1) anti-HSA Ab is coated on solid phase carrier: by dilution buffer, anti-HSA Ab is diluted to 2000 times, adds in elisa plate micropore, 37 DEG C of water-baths 1 hour, store refrigerator;
2) get whole blood from the circulation system, make measuring samples, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with cleansing solution, after washing completes, add 2% bovine serum albumin(BSA) as confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with cleansing solution, after having washed, elisa plate is placed 1 hour in 37 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard items with the chromium of known content-albumin chelate; By dilution buffer, measuring samples is diluted to 20 times, adds in micropore, 37 DEG C act on 1 hour;
5) material can catching chromium is added, and incubation: remove measuring samples, and wash with cleansing solution, after washing completes, add and dilute 10000 times by dilution buffer, 37 DEG C act on 1 hour, the crome metal on anti-Cr Ab and albumin are reacted;
6) enzyme conjugates incubation: remove anti-chromium antibody, and wash with cleansing solution, after washing completes, adds the HRP enzyme labelled antibody with dilution buffer dilution, and 37 DEG C act on 1 hour, itself and enzyme labelled antibody are reacted;
7) substrate incubation: remove enzyme labelled antibody, and wash with cleansing solution, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
8) cessation reaction: stop buffer is dropped to each micropore;
9) getting wavelength is 405nm, after adding stop buffer, is placed in by elisa plate in microplate reader and detects, and read measuring samples OD value (also can not use microplate reader, directly carry out qualitative detection by colour developing situation) respectively, concrete detected value is as shown in table 5.
The ELISA testing result of chromium-albumin chelate in table 5:100 part blood sample
application Example 2
Employing enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) detect the chromium-albumin chelate in 100 points of sample blood samples, detect in accordance with the following steps:
1) albuminous material can be caught, as anti-albumin antibodies (anti-HSA Ab) is coated on solid phase carrier: by dilution buffer, anti-HSA Ab is diluted to 2000 times, adds in elisa plate micropore, 4 DEG C are spent the night 18 hours;
2) get whole blood from the circulation system, make measuring samples, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with cleansing solution, after washing completes, add 2% bovine serum albumin(BSA) or skimmed milk power as confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with cleansing solution, after having washed, elisa plate is placed 1 hour in 37 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard items with the chromium of known content-albumin chelate; With dilution buffer dilution, measuring samples is diluted to 20 times, adds in micropore, 37 DEG C act on 1 hour;
5) wash-out: remove measuring samples, and wash with cleansing solution, after washing completes, add the eluent that Papain enzyme concentration is 100ng/ml, wash-out 1-3 hour;
6) detect: sample from the micropore of elisa plate, detect the chromium of chelating on albumin in Atomic Absorption Spectrometer, detected value is as shown in table 6.
The AAS testing result of chromium-albumin chelate in table 6:100 part blood sample
application Example 3
Employing enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) detect the chromium-albumin chelate content in 100 points of sample blood samples, detect in accordance with the following steps:
1) albuminous material can be caught, as anti-albumin antibodies (anti-HSA Ab) is coated on solid phase carrier: by dilution buffer, anti-HSA Ab is diluted to 2000 times, adds in elisa plate micropore, 4 DEG C are spent the night 18 hours;
2) get 100 parts of standard blood samples as measuring samples, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with cleansing solution, after washing completes, add 2% bovine serum albumin(BSA) as confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with cleansing solution, after having washed, elisa plate is placed 1 hour in 37 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard items with the chromium of known content-albumin chelate; By dilution buffer, measuring samples is diluted to 20 times, adds in micropore, 37 DEG C act on 1 hour;
5) wash-out: remove measuring samples, and wash with cleansing solution, after washing completes, add the eluent that Papain enzyme concentration is 100ng/ml, wash-out 1-3 hour;
6) acidifying: in step 5) in solution in add nitric acid acidifying carried out to solution, sealing is spent the night, and thorough acidifying, adds hydrogen peroxide, and acid is caught up with in heating;
7) detect: from step 6) sample the solution that obtains, under icp ms, detect chelating in albuminous chromium, detected value is as shown in table 7.
The ICP-MS testing result of chromium-albumin chelate in table 7:100 part blood sample
To one skilled in the art, according to technical scheme described above and design, other various corresponding change and deformation can be made, and all these change and deformation all should belong within the protection domain of the claims in the present invention.
Claims (8)
1. chromium-albumin chelate, is characterized in that, chromium ion and albumin by sulfydryl or/and cysteine residues chelating forms.
2. a preparation method for chromium as claimed in claim 1-albumin chelate, is characterized in that, comprise the following steps:
A) chromium and albuminous chelatropic reaction: add chromium ion in the albumin coming from human body or the albumin of recombinating according to biological method and carry out chelatropic reaction purifying, obtain reaction solution;
B) extraction of purifying chromium-albumin chelate: adopt immune-affinity chromatography, removes unreacted albumin and unnecessary chromium ion in reaction solution, obtains chromium-albumin chelate.
3. preparation method according to claim 2, is characterized in that,
Described step B) specific as follows:
(1) sample dissolution: to through steps A) add physiological saline in the reaction solution that obtains chromium-albumin chelate is redissolved;
(2) balance chromatographic column: use dilution buffer to rinse the pipeline of chromatographic column, load in chromatographic column can with the filler of albumin specific binding, after dress post, continue to use dilution buffer balance chromatographic column;
(3) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (1), albumin and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the phosphate solution of 0.05-0.1mol/L to carry out wash-out, obtain eluent I;
(5) collect: collect eluent I;
(6) dialyse: by the eluent of the collection in step (5), dress bag filter, uses ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatographic column: adopt new chromatographic column, use dilution buffer flushing line, in this chromatographic column load can with the filler of chromium specific binding, dress post after balance chromatographic column by dilution buffer again;
(8) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out, obtain eluent II;
(10) collect: collect eluent II;
(11) dialyse: by the eluent of the collection in step (10), dress bag filter, uses ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain chromium-albumin chelate.
4. the preparation method of chromium according to claim 2-albumin chelate, is characterized in that, also comprise step C): to the qualification of chromium-albumin chelate;
Wherein, step C) in specific as follows:
(1) glue bed is prepared: prepare glue bed using the one in Ago-Gel, polyacrylamide gel as medium;
(2) application of sample: get step B) in chromium-albumin chelate of obtaining of extraction purification, add dilution buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: on glue bed, find out the protein band containing chromium, this protein band is taken out and dissolves, and then adopt ELISA method or ICP-MS method or AAS method to detect the content whether containing chromium and detection chromium.
5. the application of chromium as claimed in claim 1-albumin chelate in the reagent or kit preparing to detect chromium-albumin chelate in blood sample.
6. one kind at least comprises the kit of chromium as claimed in claim 1-albumin chelate as standard items.
7. kit according to claim 6, is characterized in that, also comprises coating buffer, and this coating buffer contains the material of catching albuminous material or catching chromium.
8. one kind is quantitatively detected the method for chromium-albumin chelate, it is characterized in that, using the chromium according to claim 1-albumin chelate of known content as standard items, a pair sample of following methods is adopted to detect: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification chromium-albumin chelate and enzyme linked immunological combined techniques, purification chromium-albumin chelate and atomic absorption spectrum combined techniques, purification chromium-albumin chelate and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510413563.8A CN105004686B (en) | 2015-07-14 | 2015-07-14 | Chromium-albumin chelate as well as preparation method and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510413563.8A CN105004686B (en) | 2015-07-14 | 2015-07-14 | Chromium-albumin chelate as well as preparation method and application thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN105004686A true CN105004686A (en) | 2015-10-28 |
CN105004686B CN105004686B (en) | 2018-01-05 |
Family
ID=54377439
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201510413563.8A Expired - Fee Related CN105004686B (en) | 2015-07-14 | 2015-07-14 | Chromium-albumin chelate as well as preparation method and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN105004686B (en) |
-
2015
- 2015-07-14 CN CN201510413563.8A patent/CN105004686B/en not_active Expired - Fee Related
Non-Patent Citations (5)
Title |
---|
何春燕、喻红: "《医学生物化学实验指导》", 30 September 2010, 湖北科学技术出版社 * |
孔涛: ""重金属铜、镉快速免疫检测技术研究"", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
赵丽: ""三种重金属单抗的制备与抗体基因序列分析及其免疫检测方法的建立"", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
邹军辉: ""六价铬间接竞争 ELISA 检测试剂盒的研制及新型一步法竞争 ELISA的建立"", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
郭明等: ""重金属Cr(Ⅵ)与血清蛋白质组的结合反应性能研究"", 《无机化学学报》 * |
Also Published As
Publication number | Publication date |
---|---|
CN105004686B (en) | 2018-01-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN105004686A (en) | Chromium-albumin chelate as well as preparation method and application thereof | |
CN104987390A (en) | Cadmium-albumin chelate as well as preparation method and application thereof | |
CN105004683A (en) | chromium-IgE chelate as well as preparation method and application thereof | |
CN105021549A (en) | Nickel-albumin chelate as well as preparation method and application thereof | |
CN105004684A (en) | Nickel-IgE chelate as well as preparation method and application thereof | |
CN105021548B (en) | Arsenic-fibrinogen chelate as well as preparation method and application thereof | |
CN104987400A (en) | Mercury-hemoglobin chelate as well as preparation method and application thereof | |
CN105001324A (en) | Chromium-hemoglobin chelate as well as preparation method and application thereof | |
CN104987391B (en) | Mercury-albumin chelate and preparation method and application thereof | |
CN104987387A (en) | Chromium-fibrinogen chelate as well as preparation method and application thereof | |
CN105044009B (en) | Chromium-very low density lipoprotein chelate and preparation method and application thereof | |
CN105001321A (en) | Mercury-fibrinogen chelate and preparation method and application thereof | |
CN105044005A (en) | chromium-IgA chelate as well as preparation method and application thereof | |
CN104961824A (en) | Lead and high-density lipoprotein chelate as well as preparation method and application thereof | |
CN105021551A (en) | chromium-IgG chelate as well as preparation method and application thereof | |
CN104987412A (en) | Arsenic-IgE chelate as well as preparation method and application thereof | |
CN104987415A (en) | Chromium-IgM chelate as well as preparation method and application thereof | |
CN104987388A (en) | Lead-albumin chelate as well as preparation method and application thereof | |
CN104987389A (en) | Arsenic-albumin chelate as well as preparation method and application thereof | |
CN104987401A (en) | Cadmium-hemoglobin chelate as well as preparation method and application thereof | |
CN104987403A (en) | Nickel-hemoglobin chelate as well as preparation method and application thereof | |
CN105001322A (en) | Chromium-high density lipoprotein chelate as well as preparation method and application thereof | |
CN104987398A (en) | Chromium-low density lipoprotein chelate as well as preparation method and application thereof | |
CN104987402A (en) | Lead-hemoglobin chelate as well as preparation method and application thereof | |
CN104987404A (en) | Nickel-IgG chelate as well as preparation method and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
TR01 | Transfer of patent right |
Effective date of registration: 20210416 Address after: Room 3787, 3rd floor, No.22 GUANGTANG West Road, Tianhe District, Guangzhou, Guangdong 510630 Patentee after: Guangzhou Jinhai Health Technology Co.,Ltd. Address before: 200000, Shanghai, Pudong New Area, China (Shanghai) free trade zone, new Jinqiao Road, No. 13, building 27, 2 floor Patentee before: SHANGHAI BAIHAO BIOTECHNOLOGY Co.,Ltd. |
|
TR01 | Transfer of patent right | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180105 Termination date: 20210714 |
|
CF01 | Termination of patent right due to non-payment of annual fee |