CN101451993B - Anti-Lp (alpha)antibody preparation and diagnosis kit for detecting Lp (alpha) - Google Patents
Anti-Lp (alpha)antibody preparation and diagnosis kit for detecting Lp (alpha) Download PDFInfo
- Publication number
- CN101451993B CN101451993B CN 200910000731 CN200910000731A CN101451993B CN 101451993 B CN101451993 B CN 101451993B CN 200910000731 CN200910000731 CN 200910000731 CN 200910000731 A CN200910000731 A CN 200910000731A CN 101451993 B CN101451993 B CN 101451993B
- Authority
- CN
- China
- Prior art keywords
- antibody
- serum
- antigen
- preparation
- salicylazosulfapyridine
- 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.)
- Expired - Fee Related
Links
- 238000002360 preparation method Methods 0.000 title claims abstract description 35
- 238000003745 diagnosis Methods 0.000 title abstract description 5
- 210000002966 serum Anatomy 0.000 claims abstract description 58
- 239000000427 antigen Substances 0.000 claims abstract description 42
- 102000036639 antigens Human genes 0.000 claims abstract description 42
- 108091007433 antigens Proteins 0.000 claims abstract description 42
- 102000004895 Lipoproteins Human genes 0.000 claims abstract description 9
- 108090001030 Lipoproteins Proteins 0.000 claims abstract description 9
- 230000000521 hyperimmunizing effect Effects 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 34
- DNIAPMSPPWPWGF-UHFFFAOYSA-N propylene glycol Substances CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 30
- 239000003153 chemical reaction reagent Substances 0.000 claims description 25
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 23
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 21
- 238000013016 damping Methods 0.000 claims description 20
- 239000012530 fluid Substances 0.000 claims description 20
- 238000001514 detection method Methods 0.000 claims description 19
- 230000003053 immunization Effects 0.000 claims description 17
- 125000003118 aryl group Chemical group 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 239000008213 purified water Substances 0.000 claims description 13
- KFSLWBXXFJQRDL-UHFFFAOYSA-N Peracetic acid Chemical compound CC(=O)OO KFSLWBXXFJQRDL-UHFFFAOYSA-N 0.000 claims description 12
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 11
- 239000007983 Tris buffer Substances 0.000 claims description 11
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 11
- 238000010790 dilution Methods 0.000 claims description 9
- 239000012895 dilution Substances 0.000 claims description 9
- FJKROLUGYXJWQN-UHFFFAOYSA-M 4-hydroxybenzoate Chemical compound OC1=CC=C(C([O-])=O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-M 0.000 claims description 8
- 238000009007 Diagnostic Kit Methods 0.000 claims description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 8
- 229960003511 macrogol Drugs 0.000 claims description 8
- 239000013049 sediment Substances 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 7
- NCEXYHBECQHGNR-QZQOTICOSA-N sulfasalazine Chemical compound C1=C(O)C(C(=O)O)=CC(\N=N\C=2C=CC(=CC=2)S(=O)(=O)NC=2N=CC=CC=2)=C1 NCEXYHBECQHGNR-QZQOTICOSA-N 0.000 claims description 7
- 229960001940 sulfasalazine Drugs 0.000 claims description 7
- 241001494479 Pecora Species 0.000 claims description 6
- 108010023197 Streptokinase Proteins 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 239000002504 physiological saline solution Substances 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 229960005202 streptokinase Drugs 0.000 claims description 6
- 238000001291 vacuum drying Methods 0.000 claims description 6
- 239000000284 extract Substances 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 4
- 230000006058 immune tolerance Effects 0.000 claims description 4
- 239000000376 reactant Substances 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 239000004094 surface-active agent Substances 0.000 claims description 4
- 101710145634 Antigen 1 Proteins 0.000 claims description 3
- 125000001931 aliphatic group Chemical group 0.000 claims description 3
- 238000005238 degreasing Methods 0.000 claims description 3
- 238000001502 gel electrophoresis Methods 0.000 claims description 3
- 229910052749 magnesium Inorganic materials 0.000 claims description 3
- 239000011777 magnesium Substances 0.000 claims description 3
- 238000000247 postprecipitation Methods 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 238000004945 emulsification Methods 0.000 claims 1
- 102100040214 Apolipoprotein(a) Human genes 0.000 abstract description 16
- 108010033266 Lipoprotein(a) Proteins 0.000 abstract description 12
- 241001465754 Metazoa Species 0.000 abstract description 11
- 230000036039 immunity Effects 0.000 abstract description 8
- 101710115418 Apolipoprotein(a) Proteins 0.000 abstract description 7
- 102000057248 Lipoprotein(a) Human genes 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 238000005199 ultracentrifugation Methods 0.000 abstract description 2
- 238000000034 method Methods 0.000 description 22
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 238000002347 injection Methods 0.000 description 7
- 239000000839 emulsion Substances 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 5
- 238000004879 turbidimetry Methods 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 230000000951 immunodiffusion Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N monobenzene Natural products C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 4
- -1 polyoxyethylene Polymers 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- WSWCOQWTEOXDQX-MQQKCMAXSA-M (E,E)-sorbate Chemical compound C\C=C\C=C\C([O-])=O WSWCOQWTEOXDQX-MQQKCMAXSA-M 0.000 description 3
- SPSPIUSUWPLVKD-UHFFFAOYSA-N 2,3-dibutyl-6-methylphenol Chemical compound CCCCC1=CC=C(C)C(O)=C1CCCC SPSPIUSUWPLVKD-UHFFFAOYSA-N 0.000 description 3
- 239000004255 Butylated hydroxyanisole Substances 0.000 description 3
- GHKOFFNLGXMVNJ-UHFFFAOYSA-N Didodecyl thiobispropanoate Chemical compound CCCCCCCCCCCCOC(=O)CCSCCC(=O)OCCCCCCCCCCCC GHKOFFNLGXMVNJ-UHFFFAOYSA-N 0.000 description 3
- 239000003508 Dilauryl thiodipropionate Substances 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- 102000007330 LDL Lipoproteins Human genes 0.000 description 3
- 108010007622 LDL Lipoproteins Proteins 0.000 description 3
- 102000013566 Plasminogen Human genes 0.000 description 3
- 108010051456 Plasminogen Proteins 0.000 description 3
- 239000004793 Polystyrene Substances 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 235000019282 butylated hydroxyanisole Nutrition 0.000 description 3
- CZBZUDVBLSSABA-UHFFFAOYSA-N butylated hydroxyanisole Chemical compound COC1=CC=C(O)C(C(C)(C)C)=C1.COC1=CC=C(O)C=C1C(C)(C)C CZBZUDVBLSSABA-UHFFFAOYSA-N 0.000 description 3
- 229940043253 butylated hydroxyanisole Drugs 0.000 description 3
- 235000010354 butylated hydroxytoluene Nutrition 0.000 description 3
- 235000019304 dilauryl thiodipropionate Nutrition 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 229920002223 polystyrene Polymers 0.000 description 3
- 238000003127 radioimmunoassay Methods 0.000 description 3
- 230000036647 reaction Effects 0.000 description 3
- 229940075554 sorbate Drugs 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- OWXMKDGYPWMGEB-UHFFFAOYSA-N HEPPS Chemical compound OCCN1CCN(CCCS(O)(=O)=O)CC1 OWXMKDGYPWMGEB-UHFFFAOYSA-N 0.000 description 2
- 239000007996 HEPPS buffer Substances 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 2
- YHSHAHLOHLZILM-UHFFFAOYSA-L S(=S)(=O)([O-])[O-].[Na+].C(C)[Hg].[Na+] Chemical compound S(=S)(=O)([O-])[O-].[Na+].C(C)[Hg].[Na+] YHSHAHLOHLZILM-UHFFFAOYSA-L 0.000 description 2
- 208000034953 Twin anemia-polycythemia sequence Diseases 0.000 description 2
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 2
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 150000005215 alkyl ethers Chemical class 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 238000004900 laundering Methods 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 229920000151 polyglycol Polymers 0.000 description 2
- 239000010695 polyglycol Substances 0.000 description 2
- 235000013824 polyphenols Nutrition 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 229960005356 urokinase Drugs 0.000 description 2
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- SGUSZVNJFNYDAM-UHFFFAOYSA-N 2-methoxyphenol propane-1,2-diol Chemical class C(C(C)O)O.COC1=C(C=CC=C1)O SGUSZVNJFNYDAM-UHFFFAOYSA-N 0.000 description 1
- 101150102415 Apob gene Proteins 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 239000008000 CHES buffer Substances 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
- 108010088842 Fibrinolysin Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108010008488 Glycylglycine Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- MKWKNSIESPFAQN-UHFFFAOYSA-N N-cyclohexyl-2-aminoethanesulfonic acid Chemical compound OS(=O)(=O)CCNC1CCCCC1 MKWKNSIESPFAQN-UHFFFAOYSA-N 0.000 description 1
- 239000007990 PIPES buffer Substances 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000002280 amphoteric surfactant Substances 0.000 description 1
- 239000003945 anionic surfactant Substances 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000002421 anti-septic effect Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- JXLHNMVSKXFWAO-UHFFFAOYSA-N azane;7-fluoro-2,1,3-benzoxadiazole-4-sulfonic acid Chemical compound N.OS(=O)(=O)C1=CC=C(F)C2=NON=C12 JXLHNMVSKXFWAO-UHFFFAOYSA-N 0.000 description 1
- CIZKTTKIKAXNBA-UHFFFAOYSA-N azane;cumene Chemical compound N.CC(C)C1=CC=CC=C1 CIZKTTKIKAXNBA-UHFFFAOYSA-N 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000012482 calibration solution Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000037029 cross reaction Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 229940001501 fibrinolysin Drugs 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 235000013905 glycine and its sodium salt Nutrition 0.000 description 1
- 229940043257 glycylglycine Drugs 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000010339 medical test Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000006318 protein oxidation Effects 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000001603 reducing effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229910052723 transition metal Inorganic materials 0.000 description 1
- 150000003624 transition metals Chemical class 0.000 description 1
- 238000002834 transmittance Methods 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relate to an anti-Lp(a) antibody preparation and a diagnosis kit for detecting Lp(a). The inventive antibody preparation comprises the steps of: after extracting lipoprotein in blood serum of healthy persons to prepare antigen, using immunity sentitive animals for a multiple times, obtaining the antibody having high idiocrasy with the Lp(a) in the blood serum of healthy persons contained in the hyper-immune serum, with the advantages of no ultracentrifugation process, short preparing time, high valence of the antibody in the preparation; the diagnosis kit prepared by the diagnosis kit matching with visible light/ultraviolet, full automatic biochemical analyzer to measure lipoprotein (a) in the blood serum of the persons has the advantages of high accuracy, good repeatability, strong antijamming capability, stable ground substance, and wide linear range and the like.
Description
[technical field]
The present invention relates to the preparation method of antiserum Lp (a) antibody, and be used for the external diagnosis reagent case that serum Lp (a) clinically detects with what this antibody was made, the invention belongs to laboratory medicine and biological technical field.
[background technology]
Lipoprotein (a) is that LP (a) is the lipid of a kind of similar low-density lipoprotein (LDL), a kind of special independently plasma lipoprotein mainly is secreted into blood after liver is synthetic, the variation of its content is at thrombotic diseases, kidney trouble has very important clinical meaning in the diseases such as diabetes.
Lp (a) is that Norway geneticist Berg in 1963 finds when the science of heredity variation of research low-density lipoprotein.The concentration individual difference of Lp among the crowd (a) is very big, concentration range can be at 0～1000mg/L, this species diversity is main to determine (" biological characteristics and the Clinical Application Analysis of serum lipoprotein a (Lp (a)) ", Shandong medical test, 2004 the 15th the 4th phases of volume by apo (a) gene loci.)。Lp (a) is a kind of lipoprotein that is rich in cholesterol, and the core is neutral lipid and apoB-100 molecule, and its periphery is holding hydrophilic apo (a), and the two is covalently bound with disulfide bond; Wherein apo (a) is the characteristic sugar protein ingredient of Lp (a), and mainly by a kind of Kringle that is called as, shape quite forms like the structure of Denmark's cake.Kringle has multiple, respectively called after K1, K2, K3, K4 and K5.Lp (a) only contains a K5, and the end of K5 is connected with 37 K4, contains an extra unpaired halfcystine among the 36th K4, infers it may is the position that apo (a) is combined with apoB100 with disulfide bond herein.K4 among the Lp (a) has 75%～85% amino acid identical with the amino acid residue of plasminogen (PG) Kringle 4, common epitope cluster is arranged, show both cross-immune reaction (" the research recent developments-molecular biology of lipoprotein (a) and the correlativity of disease ", 1999 the 22nd the 2nd phases of volume of foreign medical science science of heredity fascicle) is arranged.
To the clinical meaning of lipoprotein (a) content, early detection Lp (a) uses electrophoresis, but this method sensitivity is low, is used for qualitative detection more.Develop immunochemistry detection methods that some directly measure Lp (a) subsequently in succession, as radial immunodiffusion (RID), electroimmunodiffusion (EID), radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA), immune turbidimetry [comprising immune scattering turbidimetry (INA) and immune turbidimetry (ITA)], enhancing part fluorescence immunoassay (DELFIA) etc. dissociates.RID method and EID method do not need specific installation because of easy and simple to handle, still have some grass-roots units laboratories to adopt, but that shortcoming is sensitivity is low.The shortcoming of RIA method is complicated operation, and radionuclide contamination is arranged.The DELFIA method is because of the need specific apparatus, domestic also less application.It is close that the whole bag of tricks is measured Lp (a) gained reference value, and the domestic and international criterion that adopts is basic identical at present.It is generally acknowledged that 300mg/L is critical level, greater than being the pathology level more than the 300mg/L.The main method that adopts mainly is at present: enzyme-linked immunosorbent assay and immune turbidimetry (" assay method and the clinical meaning of lipoprotein (a) ", laboratory medicine Information Network.)
Enzyme-linked immunosorbent assay is not disturbed by Plg, and this method is to contain two kinds of antigen sites of apo (a), apoB according to Lp (a) to design, and its correlativity is good, but complex operation can't be used for the automated analysis instrument, is subject to outside contamination etc.
When immune turbidimetry is measured Lp (a) content, because the antibody generation cross-immune reaction in Plg and the reagent, thereby Lp (a) assay is produced interference, the main interference factors when this also is this method mensuration Lp (a) content.In addition, because Lp (a) has high homology with Plg, cause Lp (a) antibody and Plg that immunological cross-reaction takes place, and then the interference measurement result.This also is present topmost disturbing factor, the polymorphism of Lp (a) protein molecule grain size and the complicacy of structure, and consequently the result who measures is subjected to the influence of (a) molecular size of apo in the serum and antibody characteristic, causes between the chamber poor very big.
Simultaneously, the method for the preparation of the required antibody of detection Lp (a) and antigen extraction is directly connected to the height of antibody quality.Known method is to adopt the super centrifugation technique of density gradient of doubling dilution to collect other antigen of electrophoresis level, carries out the animal immune experiment again.The shortcoming of said method is that the super centrifugal used time is long, and the easy sex change of target antigen albumen and changed the immunogenicity of target antigen in leaching process, and the antibody serum that obtains is tired very low naturally.In addition, the easy and former albumen generation of the fibrinolysin cross-immune reaction of the antibody that adopts this kind method to obtain.
[summary of the invention]
[technical matters that will solve]
The objective of the invention is to: in order to overcome the deficiency of mentioned present detection Lp (a) method in the above-mentioned background technology, provide a kind of and have accuracy height, good reproducibility, antijamming capability is strong, matrix is stablized, be applicable to the kit for detection of Lp (a) of characteristics such as visible light/ultraviolet half, automatic clinical chemistry analyzer.
[technical scheme]
Be prepared into Lp (a) immunizing antigen of immune usefulness after extracting Lp (a) in the pooled serum by the many person-portions in the healthy population and adjuvant mixing, repeatedly inoculate healthy animal responsive to Lp (a) antigen but difficult generation immune tolerance, the hyper-immune serum that contains anti-Lp (a) antibody of acquisition;
Utilize this hyper-immune serum to be mixed with and contain anti-Lp (a) detection of antibodies reagent and Lp (a) titer;
The Lp (a) that the present invention describes detects the double reagent box, cooperates the automatic biochemistry analyzer with 340nm wavelength, 1cm optical path, 37 ℃ of thermostats to carry out the detection of human blood sample lipoprotein (a).
The basic operation of the anti-Lp of the specific embodiment of the present invention (a) Antibody Preparation as follows:
1. the preparation of antigen
(1) with aliphatics surfactant (as: cumene ammonium sulphonate, sheep oil etc.) in stirrer the mixing of 300ml pooled serum (mixing of the serum that obtains from the healthy population Different Individual) with 400ml, make it form surfactant-serum compound.
(2) phosphotungstic acid-magnesium 200ml of 38% is slowly joined on stirrer in above surfactant-serum compound, the lipoprotein post precipitation washs with physiological saline.
(3) the lipoprotein sediment after will washing joins in absolute ethyl alcohol/absolute ether (3: 2) mixed liquor of precooling, constantly stirs, after spending the night in 12 hours, 4 ℃ centrifugal, abandon supernatant, repeat once this operation behind the sediment that will collect.
(4) with the sediment after the above-mentioned degreasing with known SDS-PAGE gel electrophoresis, Lp (a) gel band is collected, smash to pieces, separate out Lp (a), after the vacuum drying, in-20 ℃ of preservations.
(5) preparation of immunizing antigen: vacuum drying Lp (a) antigen that will extract is diluted to liquid antigen by (W/V) with physiological saline, and (concentration is: 100mg/ml), mix through FCA (Freund's complete adjuvant) or FIA (incomplete Freund) equal proportion respectively again, place to make it thermal agitation 10min on the mixer and make antigen fully emulsified, obtain FCA immunizing antigen and FIA immunizing antigen.
2. the preparation of antibody is inoculated with immunizing antigen
Antibody preparation process is among the present invention:
(1) animal: the selection of the experiment immunization animal among the present invention can be: as long as healthy rabbit responsive to Lp (a) antigen but difficult generation immune tolerance, any animal preparation contains the hyper-immune serum of anti-Lp (a) antibody in sheep, the ox.Preferential selection is that same lineal rabbit is as immune object among the present invention.
(2) animal immune: that treats immunity moves into inoculation animal house with rabbit, probably needs four day laundering period, takes rabbit blood before immunity inoculation antigen, when extracting serum preparation and becoming to be used for antibody test as negative control sera.
Immunization method can adopt back multiple spot hypodermic injection, and dosage 0.4～0.6ml/ only annotates 0.1ml at every, and every interval 2 Zhou Houzai select difference injection (do not select to close on the site, otherwise ulcer healing being bad) in above-mentioned position.After immune 3～4 times, can obtain higher antibody of tiring, detect the examination blood of antibody titer for this reason after the 3rd immunity week, measure antibody titer (" practical clinical immunology check ", Jiangsu science and technology publishing house with simple immunodiffusion method.) meet the requirements (anti-human lipoprotein (a) antibody titer was at 1: 32～1: 64) as antibody titer, can be in last injection back one all bloodletting.
It is identical with program to prepare the hyper-immune serum immunization method with sheep, ox, but immunizing dose should increase by body weight, namely by every kg body weight 0.2～0.3ml, and every some injection 0.5～1.0ml.
(3) extraction of antibody:
Above-mentioned arteria carotis blood was placed 25～30 ℃ of conditions following 24 hours, and the centrifugal 30min of 3000r/min abandons precipitation, obtains to contain the serum of Lp (a) antibody.
Can be to adding non-reduced type sugar in the serum of Lp (a) antibody, glycerine, ethylenediamine tetraacetic acid two are received a kind of in the salt and are kept tiring of antibody stable, can use Sodium azide simultaneously, a kind of as anticorrosion in the ethyl mercury sodium thiosulfate etc.
3. the preparation of Lp (a) content kit in the detection human serum:
The principle of measuring the kit of Lp (a) content in the human serum is: in buffer system, and the Lp in the sample serum (a) and anti-Lp (a) antibody generation immune response, the immune complex of generation changes light intensity in transmission or scattering strength; Find the content of Lp in the detected sample (a) with the typical curve of the concentration of Lp (a) calibration object and light intensity in transmission correspondingly or scattering strength.
Because what the kit that the present invention relates to was used is the turbid detection method of immune transmittance, its significant characteristic selects lipoprotein (a) and plasminogen not to have the antibody combining site of homology exactly, the method of sheltering by antigenic determinant has been eliminated the interference of endogenous homologous protein (Plg), utilize streptokinase-polyxyethylated aryl oxide solution to eliminate shielding simultaneously, further reduce the interference of Plg and other foreign proteins.
The detection kit that is used for serum Lp (a) content, it is by reactant (reagent 1), antibody reagent (reagent 2) and titer composition.This kit must cooperate the automatic biochemistry analyzer with 340nm wavelength, 1cm optical path, 37 ℃ of thermostats to carry out the detection of human serum sample's lipoprotein (a) when using.
(1) reactant (reagent 1), it consists of:
Tris damping fluid (pH=7.2) 250～350ml
Sodium chloride 5～8g
Macrogol 8,000 50～70g
P-hydroxybenzoate 4～6g
Streptokinase-polyxyethylated aryl oxide solution
*80～120ml
BSA 60～80g
All the other supply 1000ml for purified water
*: streptokinase-polyxyethylated aryl oxide solution is that the 1000U streptokinase is dissolved in the polyxyethylated aryl oxide of 100ml, make solution, wherein streptokinase can substitute with urokinase, and polyxyethylated aryl oxide can substitute with polystyrene phenylate or polyoxyethylene alkyl ether.
The present invention chooses the Tris damping fluid, and damping fluid also can select phosphate buffer, Tris damping fluid, PIPES damping fluid, HEPES damping fluid, TAPS damping fluid, glycine buffer, borate buffer etc. to substitute, and the pH value is 7.0～10.0.
The accelerator of this kit adopts polyglycol 8000, but also can select in the molecular weight 1000～20000 other polyglycol for use.
The antiseptic of this kit adopts p-hydroxybenzoate.But also can adopt Sodium azide, ethyl mercury sodium thiosulfate.
More than the bSA in the prescription also can select for use EDTA-Na, magnesium chloride, bovine serum albumin(BSA), ethylene glycol, sweet mellow wine etc. to substitute.
This reagent is selected non-ionic surfactant for use: tween series, polyethenoxy ether class.Surfactant can also be selected anionic surfactant, amphoteric surfactant for use.
(2) antibody reagent (reagent 2), it consists of:
The serum dilution 1000ml of the anti-people Lp of rabbit (a) antibody
The serum of the anti-people Lp of rabbit (a) antibody (antibody titer 1: 32～1: 64) 1000ml
Salicylazosulfapyridine-propylene glycol solution
*150ml
*: salicylazosulfapyridine-propylene glycol solution: salicylazosulfapyridine 80g is dissolved in 50～150ml propylene glycol is made into.
Salicylazosulfapyridine in the prescription can adopt butylated hydroxy anisole, dilauryl thiodipropionate, dibutyl hydroxy toluene, benzene polyphenol, Radix Glycyrrhizae polyphenoils, phosphatide etc.
Wherein:
The serum dilution of the anti-people Lp of rabbit (a) antibody:
Tris damping fluid (pH=7.2) 200～250ml
Macrogol 8,000 30～50g
P-hydroxybenzoate 2～6g
BSA 50～70g
Purified water is added to 1000ml
(3) titer:
Calibration solution is one of key factor that determines the reagent accuracy.Lp (a) is under liquid condition, in external transition metal ionic oxide formation, chemical modification, cellular oxidation, can cause that all oxidative modification takes place Lp (a), makes the value of titer that very big decline take place at short notice, so slow down its oxidation rate with salicylazosulfapyridine-propylene glycol solution.And present most oxygenant is all according to the strong reducing property of its oxygenant self, thereby stops the Lp (a) in its titer oxidized, and therefore As time goes on anti-oxidant thinner effect descends.Discover to have the antioxidation material when being reduced, can produce a certain amount of free radical.In order to improve the stability of titer under liquid state, we have added an amount of citric acid therein, increase the stability of salicylazosulfapyridine-propylene glycol solution, thereby Lp (a) titer of the present invention's preparation keeps almost constant at 2～8 ℃ in 1 year.The definite value of Lp of the present invention (a) titer is: 10.0g/L, so titer is when preparing, purifying Lp (a) the antigen 1 0.0g/L that to add purity be 90% (high-pressure liquid phase chromatogram therapy determining), tire 1: 16～1: 32 (simple immunodiffusion method mensuration).
The main composition of its titer comprises:
PH 6～8Tris damping fluid 200ml
Lp (a) antigen (antibody titer 1: 16～1: 32) 10g
Salicylazosulfapyridine-propylene glycol solution
*90～150ml
Sorbate 0.1～0.5g
BSA 40～60g
Peracetic acid is received 4～7g
Citric acid 4～7g
Purified water is added to 1000ml
*: salicylazosulfapyridine-propylene glycol solution: salicylazosulfapyridine 60g is dissolved in the 150ml propylene glycol is made into.
This titer is chosen the Tris damping fluid.Also can adopt PBS, TAPS, HEPPS, CHES, glycocoll, glycylglycine etc. to substitute.Wherein salicylazosulfapyridine can use butylated hydroxy anisole, dilauryl thiodipropionate, dibutyl hydroxy toluene, Radix Glycyrrhizae polyphenoils etc. to substitute.
The antigen of bSA in can stabilized reference liquid prevents the antigen protein oxidation, and the activity that sex change etc. cause reduces.
4. the use of Lp (a) content kit in the detection human serum
(1) detecting instrument: the Lp that the present invention relates to (a) detects the double reagent box, must cooperate the automatic biochemistry analyzer with 340nm wavelength, 1cm optical path, 37 ℃ of thermostats to detect.
(2) preparation of test sample: sample is fresh serum, and LP in the serum (a) room temperature preservation can be stablized 1 day, can stablize 7 days freezing stablizing 3 months for 2～8 ℃.
(3) operation steps is as follows:
1) sets at automatic clinical chemistry analyzer: 37 ℃ of temperature, reaction time 10min, predominant wavelength 340nm, commplementary wave length 800nm.The volume ratio of measuring reagent and sample is 20: 1, and the end reaction thing is placed under the Biochemical Analyzer, detects the variation in predominant wavelength 340nm absorbance.
2) application of sample and record result:
Table 1 sample detection operation art formula
The result calculates: sample hose Lp (a) contains heavily (mg/L)=concentration of standard solution * Δ A
(sample hose)/ Δ A
(standard pipe)
The normal population reference value is 0～300mg/L.
(4) kit assay checking:
Specificity: by adopting kit of the present invention to detect respectively to contain Lp (a) and removed the human serum of Lp (a), show according to testing result, the recall rate that contains the human serum of Lp (a) is 99%, do not contain the recall rate 2% of the human serum of Lp (a), but detected content is very low.
Accuracy: get Lp (a) the numeraire serum of 710mg/L, under this kit condition that externally condition is identical that employing is same batch, measure measurement result such as following table 2 with automatic biochemistry analyzer:
Table 2 accuracy measurement result table
| Measure number of times | Result (mg/L) |
| 1 | 684 |
| 2 | 684.9 |
| 3 | 685.1 |
| 4 | 687.3 |
| 5 | 692.1 |
| 6 | 686.9 |
| 7 | 693.2 |
| 8 | 698.6 |
| 9 | 684.9 |
| 10 | 695.5 |
Accuracy is :-0.029%
Repeatability determination data such as following table 3:
The repeated determination data of table 3 is table as a result
Batch interpolation of the precision of this kit is CV≤8%.
Difference between batch is CV≤10%.
The relative deviation control of accuracy is in ± 10% scope.
Range of linearity control is at 0～1000mg/L.
Positive effect of the present invention is: Antibody Preparation of the present invention is after the lipoprotein (a) (Lp (a)) in the extraction healthy population serum is prepared into antigen, with its immune responsive animal repeatedly, contain in the hyper-immune serum that obtains with human serum in Lp (a) antigen the reactive antibody of high special is arranged, should have in the preparation process and need not handle by ultracentrifugation, preparation time is short, the high characteristics of tiring of antibody; Advantages such as the detection kit of preparing with this antibody cooperates visible light/ultraviolet half, automatic clinical chemistry analyzer to measure lipoprotein (a) in the human serum to have accuracy height, good reproducibility, antijamming capability is strong, matrix is stable, the range of linearity is wide.
Employed all ingredients and medicine are all purchased the pure or chemical pure level in the analysis in internal reagent company and medicine shop among the present invention.
Following examples are used for explanation the present invention, but are not used for limiting protection scope of the present invention.
The preparation of embodiment 1 antigen
Get aliphatics surfactant (sheep oil) mixing stirrer of 300ml pooled serum (mixing of the serum that obtains from the healthy population Different Individual) and 400ml, slowly add phosphotungstic acid-magnesium 200ml of 38% again, lipoprotein post precipitation to be formed washs it with physiological saline; Lipoprotein sediment after the washing is joined in absolute ethyl alcohol/absolute ether (3: 2) mixed liquor of precooling, constantly stirs, after spending the night in 12 hours, 4 ℃ centrifugal, abandon supernatant, repeat once this operation behind the sediment that will collect; Sediment after the above-mentioned degreasing with known SDS-PAGE gel electrophoresis, is collected Lp (a) gel band, smashed to pieces, separate out Lp (a), vacuum drying becomes Lp (a) freeze-dried products, is stored in-20 ℃.
Vacuum drying Lp (a) freeze-dried products that extracts is diluted to liquid antigen by (W/V) with physiological saline, and (concentration is: 100mg/ml), mix through FCA (Freund's complete adjuvant) or FIA (incomplete Freund) equal proportion respectively again, place to make it thermal agitation 10min on the mixer and make antigen fully emulsified, obtain FCA immunizing antigen emulsion and FIA immunizing antigen emulsion.
The preparation of embodiment 2 antibody
After choosing 20 healthy rabbits and moving into the rabbit room as immune animal, through 4 days laundering period, before immunity inoculation antigen emulsion, take rabbit blood, when extracting serum preparation and becoming to be used for antibody test as negative control.
For the first time immunity inoculation adopts FCA immunizing antigen emulsion, and immunization method can adopt back multiple spot hypodermic injection, and dosage 0.4～0.6ml/ only annotates 0.1ml at every, from the second time immunity inoculation use FIA immunizing antigen emulsion instead.Every interval 2 Zhou Houzai select the difference injection in above-mentioned position.One week is detected the examination blood of antibody titer in the 3rd immunity back, measures antibody titer with simple immunodiffusion method, reaches more than 1: 32 as antibody titer, can and extract serum in the 4th injection back one all arteria carotis bloodletting, is the serum that contains Lp (a) antibody.
Embodiment 3
The sheep of available health, ox and horse etc. need only rabbit responsive to antigen but that the difficult animal that produces immune tolerance replaces among the embodiment and prepare the serum that contains Lp (a) antibody.
Embodiment 4
The preparation of related solution in each component of diagnostic kit of detection Lp (a):
1. streptokinase-polyxyethylated aryl oxide solution
The 1000U streptokinase is dissolved in the polyxyethylated aryl oxide of 100ml, makes solution.
2. salicylazosulfapyridine-propylene glycol solution
Salicylazosulfapyridine 80g is dissolved in the 150ml propylene glycol and is made into.
3. the serum dilution of the anti-people Lp of rabbit (a) antibody
PH 7.2 Tris damping fluid 200ml
Macrogol 8000 50g
P-hydroxybenzoate 4g
BSA 60g
Purified water adds to 1000ml
Embodiment 5
Can select the polystyrene phenylate in the prescription of streptokinase among the embodiment 4-polyxyethylated aryl oxide solution, polyoxyethylene alkyl ether replaces polyxyethylated aryl oxide; Select for use urokinase to substitute streptokinase.
Embodiment 6
Salicylazosulfapyridine in embodiment 4 salicylazosulfapyridines-propylene glycol solution prescription can adopt butylated hydroxy anisole, dilauryl thiodipropionate, dibutyl hydroxy toluene, benzene polyphenol, Radix Glycyrrhizae polyphenoils, phosphatide etc. to substitute.
Embodiment 7
Detect the preparation of each component of diagnostic kit of Lp (a):
1. reagent I:
PH=7.2 Tris damping fluid 250ml
Sodium chloride 5g
Macrogol 8000 50g
P-hydroxybenzoate 4g
Streptokinase-polyxyethylated aryl oxide solution 80ml
BSA 60g
Purified water adds to 1000ml
2. reagent II:
The serum dilution 1000ml of the anti-people Lp of rabbit (a) antibody
The serum of the anti-Lp of rabbit (a) antibody (antibody titer 1: 32～1: 64) 1000ml
Salicylazosulfapyridine-propylene glycol solution 150ml
3. titer:
PH 7.2 Tris damping fluid 200ml
Lp (a) antigen (antibody titer 1: 16～1: 32) 10g
Salicylazosulfapyridine-propylene glycol solution 90ml
Sorbate 0.3g
BSA 40g
Peracetic acid is received 4g
Citric acid 4g
Purified water adds to 1000ml
Embodiment 8:
Detect the preparation of each component of diagnostic kit of Lp (a):
1. reagent I:
PH=7.2Tris damping fluid 300ml
Sodium chloride 7g
Macrogol 8000 60g
P-hydroxybenzoate 6g
Urokinase-polyxyethylated aryl oxide solution 90ml
BSA 60g
Purified water adds to 1000ml
2. reagent II:
The serum dilution 1000ml of the anti-people Lp of rabbit (a) antibody
The serum of the anti-people Lp of rabbit (a) antibody (antibody titer 1: 32～1: 64) 1000ml
Salicylazosulfapyridine-propylene glycol solution 150ml
3. titer:
HEPPS damping fluid 200ml
Lp (a) antigen (antibody titer 1: 16～1: 32) 10g
Butylated hydroxy anisole--propylene glycol solution 90ml
Sodium azide 0.5g
BSA 45g
Peracetic acid is received 7g
Citric acid 6g
Purified water adds to 1000ml
Embodiment 9:
Detect the preparation of each component of diagnostic kit of Lp (a):
1. reagent I:
Phosphate buffer 250ml
Sodium chloride 7g
Macrogol 8000 70g
Sodium azide 6g
Urokinase-polystyrene phenylate solution 100ml
BSA 70g
Purified water adds to 1000ml
2. reagent II:
The serum dilution 1000ml of the anti-Lp of rabbit (a) antibody
The serum of the anti-people Lp of rabbit (a) antibody (antibody titer 1: 32～1: 64) 1000ml
Salicylazosulfapyridine-propylene glycol solution 150ml
3. titer:
PBS damping fluid 200ml
Lp (a) antigen (antibody titer 1: 16～1: 32) 10g
Salicylazosulfapyridine-propylene glycol solution 100ml
Sodium azide 0.3g
BSA 50g
Peracetic acid is received 5g
Citric acid 5g
Purified water is added to 1000ml
Embodiment 10
1. reagent I
Tris damping fluid (pH=7.2) 300ml
Sodium chloride 8g
Macrogol 8000 70g
P-hydroxybenzoate 6g
Streptokinase-polyxyethylated aryl oxide solution 120ml
BSA 80g
Purified water is supplied 1000ml
2. reagent II:
The serum dilution 1000ml of the anti-people Lp of rabbit (a) antibody
The serum of the anti-people Lp of rabbit (a) antibody (antibody titer 1: 32～1: 64) 1000ml
Salicylazosulfapyridine-propylene glycol solution 150ml
(3) titer:
PBS damping fluid 200ml
Lp (a) antigen (antibody titer 1: 16～1: 32) 10g
Salicylazosulfapyridine-propylene glycol solution 150ml
Sorbate 0.5g
BSA 60g
Peracetic acid is received 7g
Citric acid 7g
Purified water is added to 1000ml
Claims (4)
1. the preparation method of an anti-Lp (a) antibody is characterized in that:
(1) preparation of antigen
1) potpourri and the aliphatics surfactant mixing of the serum that will obtain from the healthy population Different Individual make its formation surfactant-serum compound; Phosphotungstic acid-magnesium with 38% slowly joins in above surfactant-serum compound, and the lipoprotein post precipitation washs with physiological saline;
2) to join the volume ratio of precooling be in absolute ethyl alcohol/absolute ether mixed liquor of 3:2 to the lipoprotein sediment after will washing, constantly stir, after spending the night in 12 hours, 4 ℃ centrifugal, abandon supernatant, the volume ratio that the collecting precipitation thing joins precooling again is in absolute ethyl alcohol/absolute ether mixed liquor of 3:2, constantly stir, after spending the night in 12 hours, centrifugal at 4 ℃, abandon supernatant, the collecting precipitation thing;
3) with the sediment after the above-mentioned degreasing with known SDS-PAGE gel electrophoresis, Lp (a) gel band is collected, smash to pieces, separate out Lp (a), after the vacuum drying, in-20 ℃ of preservations;
Vacuum drying Lp (a) antigen that 4) will extract is diluted to the liquid antigen that concentration is 100mg/ml with physiological saline, respectively through the mixing of Freund equal proportion, emulsification, obtains FCA immunizing antigen and FIA immunizing antigen again;
(2) repeatedly inoculate Lp (a) antigen responsively but be difficult for producing the healthy rabbit of immune tolerance with immunizing antigen, any obtains to contain the hyper-immune serum of anti-Lp (a) antibody in sheep, the ox.
2. diagnostic kit that detects Lp (a), it is characterized in that this kit contains the formulated antibody reagent of hyper-immune serum that contains anti-Lp (a) antibody by claim 1 preparation, titer and the reactant of Lp (a) preparation that extracts in the pooled serum by many person-portions by claim 1, this reactant is by following one-tenth branch preparation: pH=7.2 Tris damping fluid 250～350ml, sodium chloride 5～8g, macrogol 8,000 50～70g, p-hydroxybenzoate 4～6g, streptokinase-polyxyethylated aryl oxide solution 80～120ml, bSA 60～80g, purified water is supplied 1000ml, wherein streptokinase-polyxyethylated aryl oxide solution is by following one-tenth branch preparation: the 1000U streptokinase is dissolved in the polyxyethylated aryl oxide of 100ml, makes solution.
3. the diagnostic kit of a kind of detection Lp as claimed in claim 2 (a), it is characterized in that antibody reagent is by following one-tenth branch preparation: the serum dilution 1000ml of the anti-people Lp of rabbit (a) antibody, tire and be the serum 1000ml of the anti-people Lp of the rabbit of 1:32～1:64 (a) antibody, salicylazosulfapyridine-propylene glycol solution 150ml, wherein salicylazosulfapyridine-propylene glycol solution is to be dissolved in the 150ml propylene glycol by salicylazosulfapyridine 80g being made into.
4. the diagnostic kit of a kind of detection Lp as claimed in claim 2 (a), it is characterized in that titer is by following one-tenth branch preparation: the Tris damping fluid 200ml of pH6～8, tire and be the Lp(a of 1:16～1:32) antigen 1 0g, salicylazosulfapyridine-propylene glycol solution 90～150ml, Sodium azide 0.5g, bSA 40～60g, Peracetic acid is received 4～7g, citric acid 4～7g, pure water adds to 1000ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910000731 CN101451993B (en) | 2009-01-09 | 2009-01-09 | Anti-Lp (alpha)antibody preparation and diagnosis kit for detecting Lp (alpha) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910000731 CN101451993B (en) | 2009-01-09 | 2009-01-09 | Anti-Lp (alpha)antibody preparation and diagnosis kit for detecting Lp (alpha) |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101451993A CN101451993A (en) | 2009-06-10 |
| CN101451993B true CN101451993B (en) | 2013-07-17 |
Family
ID=40734353
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200910000731 Expired - Fee Related CN101451993B (en) | 2009-01-09 | 2009-01-09 | Anti-Lp (alpha)antibody preparation and diagnosis kit for detecting Lp (alpha) |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101451993B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104059908A (en) * | 2014-03-10 | 2014-09-24 | 深圳市宝凯仑科技有限公司 | Primer and plasmid for preparing anti-Lp(a) polyclonal antiserum and application of the primer and the plasmid |
| CN104374927B (en) * | 2014-12-05 | 2016-03-02 | 重庆中元生物技术有限公司 | A kind of detection kit of quantitative detection LP(a) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0506523A2 (en) * | 1991-03-25 | 1992-09-30 | Daiichi Pure Chemicals Co. Ltd. | Monoclonal antibodies |
| CN101004421A (en) * | 2007-01-25 | 2007-07-25 | 兰州大学 | Test paper for semi quantitative detecting oxLDL |
| CN101140286A (en) * | 2007-10-16 | 2008-03-12 | 李红玉 | Colloid selenium test paper of semi-quantitative determination oxidized low density lipoprotein |
-
2009
- 2009-01-09 CN CN 200910000731 patent/CN101451993B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0506523A2 (en) * | 1991-03-25 | 1992-09-30 | Daiichi Pure Chemicals Co. Ltd. | Monoclonal antibodies |
| CN101004421A (en) * | 2007-01-25 | 2007-07-25 | 兰州大学 | Test paper for semi quantitative detecting oxLDL |
| CN101140286A (en) * | 2007-10-16 | 2008-03-12 | 李红玉 | Colloid selenium test paper of semi-quantitative determination oxidized low density lipoprotein |
Non-Patent Citations (2)
| Title |
|---|
| Jay M. Edelberg等.Lipoprotein a Inhibits Streptokinase-Mediated Activation of Human Plasminogen.《Biochemistry》.1989,第28卷(第6期), * |
| 许平等.脂蛋白(a)的分离纯化及其抗血清的制备.《生物化学与生物物理学进展》.1990,第17卷(第3期), * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101451993A (en) | 2009-06-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3040162B2 (en) | Methods for measuring analytes in biological fluids in the presence of substances that interfere with analyte assays | |
| CN102062777B (en) | Hepatopathy detecting test paper by colloidal gold chromatography and preparation method thereof | |
| CN106568978A (en) | Serum amyloid protein A detection method and reagent | |
| CA2765709A1 (en) | Method for measuring lipoprotein-specific apolipoproteins | |
| CN102507957A (en) | Method and kit for determining apolipoprotein A2 by using immunity transmission turbidimetric method | |
| JP4234207B2 (en) | Diagnosis method of allergic bronchopulmonary aspergillosis | |
| CN101403752A (en) | Chemical luminescence ELISA detection kit for gatifloxacin | |
| CN101451993B (en) | Anti-Lp (alpha)antibody preparation and diagnosis kit for detecting Lp (alpha) | |
| CN102621315B (en) | Colloidal gold chromatography anti-Ro52 antibody detection test paper and preparation method thereof | |
| KR910008637B1 (en) | Monoclonal antibody to cardlac myosin heavy chain | |
| CN104749371B (en) | People's nephroblastoma overepressed gene encoding proteins enzyme linked immunological kit | |
| CN102621311B (en) | Colloidal gold chromatography anti-SSB antibody detection test paper and preparation method thereof | |
| CN106568969B (en) | A kind of ELISA detection method of 129 phosphorylation alpha synuclein aggregation bodies of serine | |
| US6767709B1 (en) | Immunoassay of human medullasin and diagnosis of multiple sclerosis using the same | |
| CN202083693U (en) | Test paper for detecting anti-ribosomal P0 antibody by adopting colloidal gold chromatographic assay | |
| JPS5925183B2 (en) | Method for measuring elastase-1 | |
| CN202083695U (en) | Anti-nucleosome antibody detecting test paper adopting colloidal gold chromatography | |
| CN202141726U (en) | Anti-Jo-1 antibody test paper with colloidal gold immunochromatography | |
| CN102621312B (en) | Colloidal gold chromatography hepatopathy detection test paper and preparation method thereof | |
| CN202083696U (en) | Anti-U1-RNP antibody detecting test paper adopting colloidal gold chromatography | |
| CN202083698U (en) | Colloidal gold chromatographic Ro52 antibody resistance detection test paper | |
| CN202083697U (en) | Anti-SSB antibody detecting test paper adopting colloidal gold chromatography | |
| CA1193192A (en) | Method of determinination of human urine kallikrein and kit for the same | |
| CN102621310B (en) | Colloidal gold chromatography anti-U1-RNP antibody detection test paper and preparation method thereof | |
| CN202083694U (en) | Anti-Cenp-B antibody detecting test paper adopting colloidal gold immunochromatography |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130717 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |