CN113929776A - 抗真菌(1,3)-β-D葡聚糖单克隆抗体、其编码基因及其表达和应用 - Google Patents
抗真菌(1,3)-β-D葡聚糖单克隆抗体、其编码基因及其表达和应用 Download PDFInfo
- Publication number
- CN113929776A CN113929776A CN202111045511.1A CN202111045511A CN113929776A CN 113929776 A CN113929776 A CN 113929776A CN 202111045511 A CN202111045511 A CN 202111045511A CN 113929776 A CN113929776 A CN 113929776A
- Authority
- CN
- China
- Prior art keywords
- glucan
- monoclonal antibody
- antifungal
- seq
- beta
- 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
- 108010001062 polysaccharide-K Proteins 0.000 title claims abstract description 124
- 230000000843 anti-fungal effect Effects 0.000 title claims abstract description 64
- 229940121375 antifungal agent Drugs 0.000 title claims abstract description 63
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 26
- 241000233866 Fungi Species 0.000 claims abstract description 29
- 230000000295 complement effect Effects 0.000 claims abstract description 14
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 8
- 239000006249 magnetic particle Substances 0.000 claims description 40
- 238000000034 method Methods 0.000 claims description 26
- 238000002038 chemiluminescence detection Methods 0.000 claims description 21
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 18
- 239000013598 vector Substances 0.000 claims description 10
- 229960002685 biotin Drugs 0.000 claims description 9
- 235000020958 biotin Nutrition 0.000 claims description 9
- 239000011616 biotin Substances 0.000 claims description 9
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 108010047041 Complementarity Determining Regions Proteins 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 25
- 239000000126 substance Substances 0.000 abstract description 12
- 230000002452 interceptive effect Effects 0.000 abstract description 9
- 210000004027 cell Anatomy 0.000 description 55
- 239000000243 solution Substances 0.000 description 50
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 21
- 239000011534 wash buffer Substances 0.000 description 21
- 239000006285 cell suspension Substances 0.000 description 18
- 210000004408 hybridoma Anatomy 0.000 description 17
- 238000002156 mixing Methods 0.000 description 17
- 238000003259 recombinant expression Methods 0.000 description 16
- 241000699666 Mus <mouse, genus> Species 0.000 description 15
- 239000007788 liquid Substances 0.000 description 15
- 238000002360 preparation method Methods 0.000 description 14
- 239000002671 adjuvant Substances 0.000 description 12
- 239000007864 aqueous solution Substances 0.000 description 12
- 238000005406 washing Methods 0.000 description 12
- 238000011725 BALB/c mouse Methods 0.000 description 11
- 241000239218 Limulus Species 0.000 description 11
- 239000002158 endotoxin Substances 0.000 description 11
- 230000003053 immunization Effects 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 11
- 229910021642 ultra pure water Inorganic materials 0.000 description 11
- 239000012498 ultrapure water Substances 0.000 description 11
- 150000001413 amino acids Chemical group 0.000 description 10
- 239000003153 chemical reaction reagent Substances 0.000 description 10
- 238000002649 immunization Methods 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- 238000004140 cleaning Methods 0.000 description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 239000013642 negative control Substances 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 7
- 229920006008 lipopolysaccharide Polymers 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- OMDQUFIYNPYJFM-XKDAHURESA-N (2r,3r,4s,5r,6s)-2-(hydroxymethyl)-6-[[(2r,3s,4r,5s,6r)-4,5,6-trihydroxy-3-[(2s,3s,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]methoxy]oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)[C@H](O)[C@H](O)[C@H](O)O1 OMDQUFIYNPYJFM-XKDAHURESA-N 0.000 description 6
- 206010017533 Fungal infection Diseases 0.000 description 6
- 229920000926 Galactomannan Polymers 0.000 description 6
- 229920001503 Glucan Polymers 0.000 description 6
- 229920000057 Mannan Polymers 0.000 description 6
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 6
- 208000031888 Mycoses Diseases 0.000 description 6
- 108010013639 Peptidoglycan Proteins 0.000 description 6
- 108010040201 Polymyxins Proteins 0.000 description 6
- 108010090804 Streptavidin Proteins 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 239000011248 coating agent Substances 0.000 description 6
- 238000000576 coating method Methods 0.000 description 6
- 238000007865 diluting Methods 0.000 description 6
- 239000003085 diluting agent Substances 0.000 description 6
- 238000013399 early diagnosis Methods 0.000 description 6
- 230000002538 fungal effect Effects 0.000 description 6
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 6
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 6
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 210000004989 spleen cell Anatomy 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 5
- 206010035226 Plasma cell myeloma Diseases 0.000 description 5
- BDQRMEBGHYKVLA-UHFFFAOYSA-N acridine-1-sulfonamide Chemical compound C1=CC=C2C=C3C(S(=O)(=O)N)=CC=CC3=NC2=C1 BDQRMEBGHYKVLA-UHFFFAOYSA-N 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 238000010367 cloning Methods 0.000 description 5
- 230000037029 cross reaction Effects 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000005284 excitation Effects 0.000 description 5
- 239000013613 expression plasmid Substances 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 201000000050 myeloid neoplasm Diseases 0.000 description 5
- 239000002504 physiological saline solution Substances 0.000 description 5
- 239000011148 porous material Substances 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 239000000287 crude extract Substances 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- 239000012894 fetal calf serum Substances 0.000 description 4
- 238000007710 freezing Methods 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 150000004676 glycans Chemical class 0.000 description 4
- 229920001282 polysaccharide Polymers 0.000 description 4
- 239000005017 polysaccharide Substances 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 238000003908 quality control method Methods 0.000 description 4
- 210000000952 spleen Anatomy 0.000 description 4
- KQFCNGKUXYNDPF-UHFFFAOYSA-N 3-[9-[[4-(2,5-dioxopyrrolidin-1-yl)oxy-4-oxobutyl]-(4-methylphenyl)sulfonylcarbamoyl]acridin-10-ium-10-yl]propane-1-sulfonate Chemical compound C1=CC(C)=CC=C1S(=O)(=O)N(C(=O)C=1C2=CC=CC=C2[N+](CCCS([O-])(=O)=O)=C2C=CC=CC2=1)CCCC(=O)ON1C(=O)CCC1=O KQFCNGKUXYNDPF-UHFFFAOYSA-N 0.000 description 3
- 229920002498 Beta-glucan Polymers 0.000 description 3
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 3
- 229920002307 Dextran Polymers 0.000 description 3
- 229920001491 Lentinan Polymers 0.000 description 3
- 229930195725 Mannitol Natural products 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000012888 bovine serum Substances 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 239000003593 chromogenic compound Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 229940115286 lentinan Drugs 0.000 description 3
- 239000000594 mannitol Substances 0.000 description 3
- 235000010355 mannitol Nutrition 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- JJGWLCLUQNFDIS-GTSONSFRSA-M sodium;1-[6-[5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]hexanoyloxy]-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)CCCCCNC(=O)CCCC[C@H]1[C@H]2NC(=O)N[C@H]2CS1 JJGWLCLUQNFDIS-GTSONSFRSA-M 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- 239000012089 stop solution Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- FAEIQWHBRBWUBN-FXQIFTODSA-N Asp-Arg-Ser Chemical compound C(C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC(=O)O)N)CN=C(N)N FAEIQWHBRBWUBN-FXQIFTODSA-N 0.000 description 2
- YFSLJHLQOALGSY-ZPFDUUQYSA-N Asp-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)O)N YFSLJHLQOALGSY-ZPFDUUQYSA-N 0.000 description 2
- JXGJJQJHXHXJQF-CIUDSAMLSA-N Asp-Met-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(O)=O JXGJJQJHXHXJQF-CIUDSAMLSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- ZEXHDOQQYZKOIB-ACZMJKKPSA-N Cys-Glu-Ser Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O ZEXHDOQQYZKOIB-ACZMJKKPSA-N 0.000 description 2
- RJPKQCFHEPPTGL-ZLUOBGJFSA-N Cys-Ser-Asp Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O RJPKQCFHEPPTGL-ZLUOBGJFSA-N 0.000 description 2
- 108010090461 DFG peptide Proteins 0.000 description 2
- QKCZZAZNMMVICF-DCAQKATOSA-N Gln-Leu-Glu Chemical compound NC(=O)CC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O QKCZZAZNMMVICF-DCAQKATOSA-N 0.000 description 2
- AMHIFFIUJOJEKJ-SZMVWBNQSA-N Gln-Lys-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(=O)N)N AMHIFFIUJOJEKJ-SZMVWBNQSA-N 0.000 description 2
- RWQCWSGOOOEGPB-FXQIFTODSA-N Gln-Ser-Glu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O RWQCWSGOOOEGPB-FXQIFTODSA-N 0.000 description 2
- QXUPRMQJDWJDFR-NRPADANISA-N Glu-Val-Ser Chemical compound CC(C)[C@H](NC(=O)[C@@H](N)CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O QXUPRMQJDWJDFR-NRPADANISA-N 0.000 description 2
- NEDQVOQDDBCRGG-UHFFFAOYSA-N Gly Gly Thr Tyr Chemical compound NCC(=O)NCC(=O)NC(C(O)C)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 NEDQVOQDDBCRGG-UHFFFAOYSA-N 0.000 description 2
- HQRHFUYMGCHHJS-LURJTMIESA-N Gly-Gly-Arg Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N HQRHFUYMGCHHJS-LURJTMIESA-N 0.000 description 2
- VDCRBJACQKOSMS-JSGCOSHPSA-N Gly-Phe-Val Chemical compound [H]NCC(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O VDCRBJACQKOSMS-JSGCOSHPSA-N 0.000 description 2
- LCRDMSSAKLTKBU-ZDLURKLDSA-N Gly-Ser-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CN LCRDMSSAKLTKBU-ZDLURKLDSA-N 0.000 description 2
- FJWYJQRCVNGEAQ-ZPFDUUQYSA-N Ile-Asn-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N FJWYJQRCVNGEAQ-ZPFDUUQYSA-N 0.000 description 2
- AKOYRLRUFBZOSP-BJDJZHNGSA-N Ile-Lys-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)O)N AKOYRLRUFBZOSP-BJDJZHNGSA-N 0.000 description 2
- MLSUZXHSNRBDCI-CYDGBPFRSA-N Ile-Pro-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)O)N MLSUZXHSNRBDCI-CYDGBPFRSA-N 0.000 description 2
- QGXQHJQPAPMACW-PPCPHDFISA-N Ile-Thr-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)O)N QGXQHJQPAPMACW-PPCPHDFISA-N 0.000 description 2
- 208000037026 Invasive Fungal Infections Diseases 0.000 description 2
- 241000880493 Leptailurus serval Species 0.000 description 2
- MVJRBCJCRYGCKV-GVXVVHGQSA-N Leu-Val-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O MVJRBCJCRYGCKV-GVXVVHGQSA-N 0.000 description 2
- NRQRKMYZONPCTM-CIUDSAMLSA-N Lys-Asp-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O NRQRKMYZONPCTM-CIUDSAMLSA-N 0.000 description 2
- XDMMOISUAHXXFD-SRVKXCTJSA-N Phe-Ser-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O XDMMOISUAHXXFD-SRVKXCTJSA-N 0.000 description 2
- RMODQFBNDDENCP-IHRRRGAJSA-N Pro-Lys-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O RMODQFBNDDENCP-IHRRRGAJSA-N 0.000 description 2
- AIOWVDNPESPXRB-YTWAJWBKSA-N Pro-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2)O AIOWVDNPESPXRB-YTWAJWBKSA-N 0.000 description 2
- OJPHFSOMBZKQKQ-GUBZILKMSA-N Ser-Gln-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CO OJPHFSOMBZKQKQ-GUBZILKMSA-N 0.000 description 2
- JFWDJFULOLKQFY-QWRGUYRKSA-N Ser-Gly-Phe Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O JFWDJFULOLKQFY-QWRGUYRKSA-N 0.000 description 2
- ZWSZBWAFDZRBNM-UBHSHLNASA-N Ser-Trp-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CO)C(O)=O ZWSZBWAFDZRBNM-UBHSHLNASA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- DGDCHPCRMWEOJR-FQPOAREZSA-N Thr-Ala-Tyr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 DGDCHPCRMWEOJR-FQPOAREZSA-N 0.000 description 2
- KERCOYANYUPLHJ-XGEHTFHBSA-N Thr-Pro-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O KERCOYANYUPLHJ-XGEHTFHBSA-N 0.000 description 2
- FWTFAZKJORVTIR-VZFHVOOUSA-N Thr-Ser-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O FWTFAZKJORVTIR-VZFHVOOUSA-N 0.000 description 2
- RPECVQBNONKZAT-WZLNRYEVSA-N Thr-Tyr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H]([C@@H](C)O)N RPECVQBNONKZAT-WZLNRYEVSA-N 0.000 description 2
- DVAAUUVLDFKTAQ-VHWLVUOQSA-N Trp-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N DVAAUUVLDFKTAQ-VHWLVUOQSA-N 0.000 description 2
- LGEYOIQBBIPHQN-UWJYBYFXSA-N Tyr-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 LGEYOIQBBIPHQN-UWJYBYFXSA-N 0.000 description 2
- QKXAEWMHAAVVGS-KKUMJFAQSA-N Tyr-Pro-Glu Chemical compound N[C@@H](Cc1ccc(O)cc1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O QKXAEWMHAAVVGS-KKUMJFAQSA-N 0.000 description 2
- JIODCDXKCJRMEH-NHCYSSNCSA-N Val-Arg-Gln Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N JIODCDXKCJRMEH-NHCYSSNCSA-N 0.000 description 2
- PWRITNSESKQTPW-NRPADANISA-N Val-Gln-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CO)C(=O)O)N PWRITNSESKQTPW-NRPADANISA-N 0.000 description 2
- XWYUBUYQMOUFRQ-IFFSRLJSSA-N Val-Glu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C(C)C)N)O XWYUBUYQMOUFRQ-IFFSRLJSSA-N 0.000 description 2
- MIKHIIQMRFYVOR-RCWTZXSCSA-N Val-Pro-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C(C)C)N)O MIKHIIQMRFYVOR-RCWTZXSCSA-N 0.000 description 2
- DLRZGNXCXUGIDG-KKHAAJSZSA-N Val-Thr-Asp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N)O DLRZGNXCXUGIDG-KKHAAJSZSA-N 0.000 description 2
- ORILYTVJVMAKLC-UHFFFAOYSA-N adamantane Chemical compound C1C(C2)CC3CC1CC2C3 ORILYTVJVMAKLC-UHFFFAOYSA-N 0.000 description 2
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 2
- 108010092854 aspartyllysine Proteins 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- 238000009640 blood culture Methods 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 230000007910 cell fusion Effects 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 230000009260 cross reactivity Effects 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 2
- 108010062266 glycyl-glycyl-argininal Proteins 0.000 description 2
- 108010050848 glycylleucine Proteins 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 108010075702 lysyl-valyl-aspartyl-leucine Proteins 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 108010029020 prolylglycine Proteins 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000012064 sodium phosphate buffer Substances 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- PKOHVHWNGUHYRE-ZFWWWQNUSA-N (2s)-1-[2-[[(2s)-2-amino-3-(1h-indol-3-yl)propanoyl]amino]acetyl]pyrrolidine-2-carboxylic acid Chemical compound O=C([C@H](CC=1C2=CC=CC=C2NC=1)N)NCC(=O)N1CCC[C@H]1C(O)=O PKOHVHWNGUHYRE-ZFWWWQNUSA-N 0.000 description 1
- SADYNMDJGAWAEW-JKQORVJESA-N (2s)-2-[[(2s)-3-carboxy-2-[[(2s)-2-[[(2s)-2,6-diaminohexanoyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]-4-methylpentanoic acid Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN SADYNMDJGAWAEW-JKQORVJESA-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
- 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 1
- XQJAFSDFQZPYCU-UWJYBYFXSA-N Ala-Asn-Tyr Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N XQJAFSDFQZPYCU-UWJYBYFXSA-N 0.000 description 1
- WMYJZJRILUVVRG-WDSKDSINSA-N Ala-Gly-Gln Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O WMYJZJRILUVVRG-WDSKDSINSA-N 0.000 description 1
- MUGAESARFRGOTQ-IGNZVWTISA-N Ala-Tyr-Tyr Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N MUGAESARFRGOTQ-IGNZVWTISA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- UAOSDDXCTBIPCA-QXEWZRGKSA-N Arg-Ile-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CCCN=C(N)N)N UAOSDDXCTBIPCA-QXEWZRGKSA-N 0.000 description 1
- KMFPQTITXUKJOV-DCAQKATOSA-N Arg-Ser-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O KMFPQTITXUKJOV-DCAQKATOSA-N 0.000 description 1
- BLQBMRNMBAYREH-UWJYBYFXSA-N Asp-Ala-Tyr Chemical compound N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O BLQBMRNMBAYREH-UWJYBYFXSA-N 0.000 description 1
- RGKKALNPOYURGE-ZKWXMUAHSA-N Asp-Ala-Val Chemical compound N[C@@H](CC(=O)O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)O RGKKALNPOYURGE-ZKWXMUAHSA-N 0.000 description 1
- ZLGKHJHFYSRUBH-FXQIFTODSA-N Asp-Arg-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O ZLGKHJHFYSRUBH-FXQIFTODSA-N 0.000 description 1
- WSOKZUVWBXVJHX-CIUDSAMLSA-N Asp-Arg-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O WSOKZUVWBXVJHX-CIUDSAMLSA-N 0.000 description 1
- KFAFUJMGHVVYRC-DCAQKATOSA-N Asp-Leu-Met Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(O)=O KFAFUJMGHVVYRC-DCAQKATOSA-N 0.000 description 1
- QJHOOKBAHRJPPX-QWRGUYRKSA-N Asp-Phe-Gly Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=CC=C1 QJHOOKBAHRJPPX-QWRGUYRKSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- FWYBFUDWUUFLDN-FXQIFTODSA-N Cys-Asp-Arg Chemical compound C(C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CS)N)CN=C(N)N FWYBFUDWUUFLDN-FXQIFTODSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010015719 Exsanguination Diseases 0.000 description 1
- OAGVHWYIBZMWLA-YFKPBYRVSA-N Glu-Gly-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)NCC(O)=O OAGVHWYIBZMWLA-YFKPBYRVSA-N 0.000 description 1
- ZSWGJYOZWBHROQ-RWRJDSDZSA-N Glu-Ile-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ZSWGJYOZWBHROQ-RWRJDSDZSA-N 0.000 description 1
- MWTGQXBHVRTCOR-GLLZPBPUSA-N Glu-Thr-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(O)=O MWTGQXBHVRTCOR-GLLZPBPUSA-N 0.000 description 1
- YQAQQKPWFOBSMU-WDCWCFNPSA-N Glu-Thr-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O YQAQQKPWFOBSMU-WDCWCFNPSA-N 0.000 description 1
- JVZLZVJTIXVIHK-SXNHZJKMSA-N Glu-Trp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CCC(=O)O)N JVZLZVJTIXVIHK-SXNHZJKMSA-N 0.000 description 1
- ZYRXTRTUCAVNBQ-GVXVVHGQSA-N Glu-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N ZYRXTRTUCAVNBQ-GVXVVHGQSA-N 0.000 description 1
- FVGOGEGGQLNZGH-DZKIICNBSA-N Glu-Val-Phe Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 FVGOGEGGQLNZGH-DZKIICNBSA-N 0.000 description 1
- RPLLQZBOVIVGMX-QWRGUYRKSA-N Gly-Asp-Phe Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O RPLLQZBOVIVGMX-QWRGUYRKSA-N 0.000 description 1
- AQLHORCVPGXDJW-IUCAKERBSA-N Gly-Gln-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)CN AQLHORCVPGXDJW-IUCAKERBSA-N 0.000 description 1
- YWAQATDNEKZFFK-BYPYZUCNSA-N Gly-Gly-Ser Chemical compound NCC(=O)NCC(=O)N[C@@H](CO)C(O)=O YWAQATDNEKZFFK-BYPYZUCNSA-N 0.000 description 1
- ULZCYBYDTUMHNF-IUCAKERBSA-N Gly-Leu-Glu Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O ULZCYBYDTUMHNF-IUCAKERBSA-N 0.000 description 1
- JJGBXTYGTKWGAT-YUMQZZPRSA-N Gly-Pro-Glu Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O JJGBXTYGTKWGAT-YUMQZZPRSA-N 0.000 description 1
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical compound NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-N 0.000 description 1
- AFMOTCMSEBITOE-YEPSODPASA-N Gly-Val-Thr Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O AFMOTCMSEBITOE-YEPSODPASA-N 0.000 description 1
- QHUREMVLLMNUAX-OSUNSFLBSA-N Ile-Thr-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)O)N QHUREMVLLMNUAX-OSUNSFLBSA-N 0.000 description 1
- UGTHTQWIQKEDEH-BQBZGAKWSA-N L-alanyl-L-prolylglycine zwitterion Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UGTHTQWIQKEDEH-BQBZGAKWSA-N 0.000 description 1
- LXKNSJLSGPNHSK-KKUMJFAQSA-N Leu-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N LXKNSJLSGPNHSK-KKUMJFAQSA-N 0.000 description 1
- HDHQQEDVWQGBEE-DCAQKATOSA-N Leu-Met-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CO)C(O)=O HDHQQEDVWQGBEE-DCAQKATOSA-N 0.000 description 1
- IDGZVZJLYFTXSL-DCAQKATOSA-N Leu-Ser-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IDGZVZJLYFTXSL-DCAQKATOSA-N 0.000 description 1
- YNNPKXBBRZVIRX-IHRRRGAJSA-N Lys-Arg-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O YNNPKXBBRZVIRX-IHRRRGAJSA-N 0.000 description 1
- ULUQBUKAPDUKOC-GVXVVHGQSA-N Lys-Glu-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O ULUQBUKAPDUKOC-GVXVVHGQSA-N 0.000 description 1
- GQFDWEDHOQRNLC-QWRGUYRKSA-N Lys-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN GQFDWEDHOQRNLC-QWRGUYRKSA-N 0.000 description 1
- ZJWIXBZTAAJERF-IHRRRGAJSA-N Lys-Lys-Arg Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CCCN=C(N)N ZJWIXBZTAAJERF-IHRRRGAJSA-N 0.000 description 1
- PPNCMJARTHYNEC-MEYUZBJRSA-N Lys-Tyr-Thr Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H]([C@H](O)C)C(O)=O)CC1=CC=C(O)C=C1 PPNCMJARTHYNEC-MEYUZBJRSA-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
- YAWKHFKCNSXYDS-XIRDDKMYSA-N Met-Glu-Trp Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N YAWKHFKCNSXYDS-XIRDDKMYSA-N 0.000 description 1
- LUYURUYVNYGKGM-RCWTZXSCSA-N Met-Pro-Thr Chemical compound CSCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O LUYURUYVNYGKGM-RCWTZXSCSA-N 0.000 description 1
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- UEHNWRNADDPYNK-DLOVCJGASA-N Phe-Cys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC1=CC=CC=C1)N UEHNWRNADDPYNK-DLOVCJGASA-N 0.000 description 1
- RAGOJJCBGXARPO-XVSYOHENSA-N Phe-Thr-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H]([C@H](O)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 RAGOJJCBGXARPO-XVSYOHENSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- UIMCLYYSUCIUJM-UWVGGRQHSA-N Pro-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 UIMCLYYSUCIUJM-UWVGGRQHSA-N 0.000 description 1
- HATVCTYBNCNMAA-AVGNSLFASA-N Pro-Leu-Met Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(O)=O HATVCTYBNCNMAA-AVGNSLFASA-N 0.000 description 1
- QUBVFEANYYWBTM-VEVYYDQMSA-N Pro-Thr-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O QUBVFEANYYWBTM-VEVYYDQMSA-N 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- 239000012327 Ruthenium complex Substances 0.000 description 1
- WXUBSIDKNMFAGS-IHRRRGAJSA-N Ser-Arg-Tyr Chemical compound NC(N)=NCCC[C@H](NC(=O)[C@H](CO)N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 WXUBSIDKNMFAGS-IHRRRGAJSA-N 0.000 description 1
- ZIFYDQAFEMIZII-GUBZILKMSA-N Ser-Leu-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O ZIFYDQAFEMIZII-GUBZILKMSA-N 0.000 description 1
- VMLONWHIORGALA-SRVKXCTJSA-N Ser-Leu-Leu Chemical compound CC(C)C[C@@H](C([O-])=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]([NH3+])CO VMLONWHIORGALA-SRVKXCTJSA-N 0.000 description 1
- QJKPECIAWNNKIT-KKUMJFAQSA-N Ser-Lys-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O QJKPECIAWNNKIT-KKUMJFAQSA-N 0.000 description 1
- 208000032023 Signs and Symptoms Diseases 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- OHAJHDJOCKKJLV-LKXGYXEUSA-N Thr-Asp-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O OHAJHDJOCKKJLV-LKXGYXEUSA-N 0.000 description 1
- VUVCRYXYUUPGSB-GLLZPBPUSA-N Thr-Gln-Glu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N)O VUVCRYXYUUPGSB-GLLZPBPUSA-N 0.000 description 1
- ODXKUIGEPAGKKV-KATARQTJSA-N Thr-Leu-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)O)N)O ODXKUIGEPAGKKV-KATARQTJSA-N 0.000 description 1
- DXPURPNJDFCKKO-RHYQMDGZSA-N Thr-Lys-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)[C@@H](C)O)C(O)=O DXPURPNJDFCKKO-RHYQMDGZSA-N 0.000 description 1
- GVMXJJAJLIEASL-ZJDVBMNYSA-N Thr-Pro-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O GVMXJJAJLIEASL-ZJDVBMNYSA-N 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- KIMOCKLJBXHFIN-YLVFBTJISA-N Trp-Ile-Gly Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O)=CNC2=C1 KIMOCKLJBXHFIN-YLVFBTJISA-N 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- BARBHMSSVWPKPZ-IHRRRGAJSA-N Tyr-Asp-Arg Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O BARBHMSSVWPKPZ-IHRRRGAJSA-N 0.000 description 1
- UPODKYBYUBTWSV-BZSNNMDCSA-N Tyr-Phe-Cys Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CS)C(O)=O)C1=CC=C(O)C=C1 UPODKYBYUBTWSV-BZSNNMDCSA-N 0.000 description 1
- VENKIVFKIPGEJN-NHCYSSNCSA-N Val-Met-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N VENKIVFKIPGEJN-NHCYSSNCSA-N 0.000 description 1
- MNSSBIHFEUUXNW-RCWTZXSCSA-N Val-Thr-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N MNSSBIHFEUUXNW-RCWTZXSCSA-N 0.000 description 1
- -1 acridine ester Chemical class 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 108010050025 alpha-glutamyltryptophan Proteins 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 108010013835 arginine glutamate Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 210000001728 clone cell Anatomy 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000013024 dilution buffer Substances 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical compound CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 108010073628 glutamyl-valyl-phenylalanine Proteins 0.000 description 1
- 239000007973 glycine-HCl buffer Substances 0.000 description 1
- 108010078326 glycyl-glycyl-valine Proteins 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 210000003677 hemocyte Anatomy 0.000 description 1
- 229940000351 hemocyte Drugs 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 108010003700 lysyl aspartic acid Proteins 0.000 description 1
- 108010064235 lysylglycine Proteins 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 238000007885 magnetic separation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 210000003739 neck Anatomy 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000004768 organ dysfunction Effects 0.000 description 1
- 235000011837 pasties Nutrition 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000035778 pathophysiological process Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 229940066827 pertussis vaccine Drugs 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000002135 phase contrast microscopy Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 108010070643 prolylglutamic acid Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 229940037128 systemic glucocorticoids Drugs 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 108010038745 tryptophylglycine Proteins 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56961—Plant cells or fungi
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/583—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with non-fluorescent dye label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- G01N2400/12—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar
- G01N2400/24—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar beta-D-Glucans, i.e. having beta 1,n (n=3,4,6) linkages between saccharide units, e.g. xanthan
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Mycology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Botany (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明提供一种抗真菌(1,3)‑β‑D葡聚糖单克隆抗体、其编码基因及其表达和应用,属于生物和医疗检测技领域,所述抗体包括轻链可变区决定簇互补区,其氨基酸序列包括:SEQ ID NO:1所示的VL‑CDR1、SEQ ID NO:2所示的VL‑CDR2和SEQ ID NO:3所示的VL‑CDR3;所述抗体还包括重链可变区决定簇互补区,其氨基酸序列包括:SEQ ID NO:4所示的VH‑CDR1、SEQ ID NO:5所示的VH‑CDR2和SEQ ID NO:6所示的VH‑CDR3。该抗体与真菌(1,3)‑β‑D葡聚糖特异性结合,抗体亲和力高,不与干扰物质产生交叉反应。
Description
技术领域
本发明涉及一种抗体及其应用,尤其涉及一种抗真菌(1,3)-β-D葡聚糖单克隆抗体、其编码基因及其表达和应用。
背景技术
深部真菌感染(Invasive fungal infection,IFI)是指真菌侵入人体组织、血液或器官,并在其中生长繁殖引起组织损害、器官功能障碍、炎症反应的病理改变及病理生理过程。近年来,由于免疫抑制剂、广谱抗生素、糖皮质激素、抗肿瘤药物等的广泛使用及外科手术导管、置管的应用,侵袭性真菌感染的发病率和死亡率逐渐增多。又由于IFI的临床症状和体征缺乏特异性,导致目前缺少有效的早期诊断手段。
目前,检测真菌感染的主要方法有:组织活检法、血培养法、传统鲎试剂检测法、新一代鲎试剂检测法、分子生物学检测法等。其中,①组织活检属于有创性检查,临床标本很难获取。②血培养方法技术要求高,耗时长,培养阳性率低(有些真菌如曲霉菌几乎培养不出阳性结果),不能做到早期诊断。③鲎试剂检测法(即G试验)依赖海洋生物鲎的血细胞,但鲎试剂特异性较差,不同批次间试剂活性差异大,检测易受内毒素(脂多糖)和某些药物干扰,假阳性高,且鲎是濒危物种,属于国家二级保护动物,采血来源受限。④新一代G试验是利用基因工程技术重组鲎血G因子(即人工鲎试剂)来代替采集野生或人工养殖的鲎血而制成的传统鲎试剂,解决了鲎血来源和内毒素(脂多糖)干扰问题,但仍存在(1,3)-β-D葡聚糖类似物(半乳甘露聚糖、甘露聚糖、肽聚糖、纤维素)和某些药物(多粘菌素、香菇多糖等)干扰引起的检测假阳性的问题。⑤分子生物学检测是基于PCR法检测真菌核酸DNA,由于真菌细胞壁厚,很难使之破碎释放出DNA,因此PCR法检测真菌感染阳性率低,目前尚处于研发阶段。
以上各种方法的局限性导致目前临床上侵袭性真菌感染的早期诊断非常困难。因此,临床上十分需要一种灵敏度高、特异性好、干扰小且操作简单,适于真菌感染早期诊断的检测方法。
发明内容
针对上述问题,本发明提供一种抗真菌(1,3)-β-D葡聚糖单克隆抗体、其编码基因及其表达和应用。
为实现上述目的,本发明所采用的技术方案为:
一种抗真菌(1,3)-β-D葡聚糖单克隆抗体,包括轻链可变区决定簇互补区和重链可变区决定簇互补区;
所述轻链可变区决定簇互补区的氨基酸序列选自下组的CDR的氨基酸序列:SEQID NO:1所示的VL-CDR1、SEQ ID NO:2所示的VL-CDR2及SEQ ID NO:3所示的VL-CDR3;
所述重链可变区决定簇互补区的氨基酸序列选自下组的CDR的氨基酸序列:SEQID NO:4所示的VH-CDR1、SEQ ID NO:5所示的VH-CDR2及SEQ ID NO:6所示的VH-CDR3。
进一步的,所述抗真菌(1,3)-β-D葡聚糖单克隆抗体的轻链可变区决定簇互补区的氨基酸序列为SEQ ID NO:7;重链可变区决定簇互补区的氨基酸序列为SEQ ID NO:8。
一种编码上述抗真菌(1,3)-β-D葡聚糖单克隆抗体的基因,所述轻链可变区决定簇互补区的核苷酸序列为SEQ ID NO:9,所述重链可变区决定簇互补区的核苷酸序列为SEQID NO:10。
包含上述抗真菌(1,3)-β-D葡聚糖单克隆抗体的基因的重组载体;
所述重组载体为含有抗真菌(1,3)-β-D葡聚糖单克隆抗体的可变区的基因的真核重组表达质粒pCMVp-NEO-BAN-Anti-glucan。
包含上述抗真菌(1,3)-β-D葡聚糖单克隆抗体的基因的重组表达细胞;
所述重组表达细胞为抗真菌(1,3)-β-D葡聚糖单克隆抗体的重组表达中国仓鼠卵巢细胞;
所述重组载体及将重组载体导入细胞后所得的所述重组表达细胞都属于本发明保护范围,所述重组载体是指所有包含抗上述真菌(1,3)-β-D葡聚糖单克隆抗体的基因的重组载体(也可以称为重组质粒),所述重组表达细胞是指所有包含上述抗真菌(1,3)-β-D葡聚糖单克隆抗体的基因的重组表达细胞,并不限于本发明所指出的含有抗真菌(1,3)-β-D葡聚糖单克隆抗体的可变区的基因的真核重组表达质粒pCMVp-NEO-BAN-Anti-glucan及抗真菌(1,3)-β-D葡聚糖单克隆抗体的重组表达中国仓鼠卵巢细胞,应理解为,本发明要求保护的技术方案中所涉及的重组载体为将上述抗真菌(1,3)-β-D葡聚糖单克隆抗体的基因插入到本领域技术人员能够购买到的任一表达载体(也可以称为表达质粒)中所获得的重组载体;重组细胞为将上述重组载体导入任一表达细胞后所获得的重组表达细胞。
一种真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒,含有上述抗真菌(1,3)-β-D葡聚糖单克隆抗体。
进一步的,所述真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒中含有采用生物素标记的所述抗真菌(1,3)-β-D葡聚糖单克隆抗体。
进一步的,所述真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒中还含有采用化学发光基团标记的所述抗真菌(1,3)-β-D葡聚糖单克隆抗体。
一种真菌(1,3)-β-D葡聚糖磁微粒化学发光检测方法,利用上述真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒,采用磁微粒化学发光法进行检测。
本发明的抗真菌(1,3)-β-D葡聚糖单克隆抗体、其编码基因及其表达和应用的有益效果为:
本发明提供的来源于抗真菌(1,3)-β-D葡聚糖单克隆抗体与真菌(1,3)-β-D葡聚糖特异性结合,抗体亲和力高,不与内毒素(脂多糖)、半乳甘露聚糖、甘露聚糖、肽聚糖、纤维素、多粘菌素、香菇多糖等物质产生交叉反应。基于该抗体特性研制的真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒灵敏度高、特异性好、定量检测范围宽、检测速度快、可实现机器自动化操作,特别是可以有效避免由内毒素(脂多糖)、(1,3)-β-D葡聚糖结构类似物(半乳甘露聚糖、甘露聚糖、肽聚糖、纤维素)和某些药物(多粘菌素、香菇多糖等)等引起的假阳性检测结果,为临床提供更准确的检测信息,可以满足临床对真菌感染早期诊断的要求,具有很好的临床应用价值。
附图说明
图1是本发明实施例1中克隆细胞筛选情况过程图;
图2是本发明实施例2中Marker及纯化单抗IgG的电泳测定结果图;
图3是本发明实施例3中不同浓度的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体抗体效价测定结果图;
图4是本发明实施例4中纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体与其它干扰检测的物质交叉反应测定结果图。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整地描述。在下面的描述中阐述了很多具体细节以便于充分理解本发明,但是本发明还可以采用其他不同于在此描述的其它方式来实施,本领域技术人员可以在不违背本发明内涵的情况下做类似推广,因此本发明不受下面公开的具体实施例的限制。
(1,3)-β-D葡聚糖购自Sigma-Aldrich公司,CAS号:9051-97-2,货号:89862;
弗氏完全佐剂购自Sigma-Aldrich公司,货号:F5881;
酶标第二抗体为HRP标记的羊抗小鼠IgG,购自美国Jackson公司,货号:115-035-003;
TMB购自全式金公司,货号:HE111-01;
中国仓鼠卵巢细胞购自中国医学科学院基础医学研究所细胞资源中心(细胞编号:1101HAM-PUMC000116)
链霉亲和素磁微粒为DynabeadsTM M-270链霉亲和素,购自Thermo Fisher公司,货号65305;
Sulfo-NHS-LC-Biotin购自上海阿拉丁生化科技股份有限公司,CAS号:191671-46-2,货号:S288926;
NSP-SA-NHS购自苏州美仑生物科技有限公司,CAS号:199293-83-9,货号:MB3332。
实施例1杂交瘤细胞的制备,
一)动物免疫
单克隆抗体可以使用通常的免疫学技术,以(1,3)-β-D葡聚糖为抗原,进行动物免疫,再使用被免疫动物的细胞制备杂交瘤细胞而获得。杂交瘤细胞制备时使用的被免疫动物的种类没有特别限定,如小鼠、大鼠、仓鼠、兔等,本实施例使用BALB/c小鼠作为被免疫动物。抗原在进行动物免疫时,配合使用佐剂效果更佳,佐剂能够提高被免疫动物对抗原的免疫应答。佐剂的种类没有特别限定,例如可使用弗氏完全佐剂、弗氏不完全佐剂、百日咳疫苗、铝佐剂等,其中本实施例初次免疫时使用弗氏完全佐剂,在第二次以后的免疫时使用弗氏不完全佐剂。
取(1,3)-β-D葡聚糖与弗氏完全佐剂等体积混合且充分乳化作为初次免疫试剂;
取6~8周龄BALB/c小鼠(1号鼠、2号鼠和3号鼠),用初次免疫试剂于皮下多点注射,初次免疫试剂使用剂量相当于每只小鼠注射30μg的(1,3)-β-D葡聚糖。以后每隔2周免疫一次,二次免疫开始使用等体积混合且充分乳化的(1,3)-β-D葡聚糖与弗氏不完全佐剂,于腹腔注射,免疫剂量相当于每只小鼠注射30μg的(1,3)-β-D葡聚糖。第四次免疫7天后,取小鼠内眦静脉血用ELISA法测定抗体效价,具体抗体效价测定方法(ELISA法)如下:
11)包被抗原:用包被缓冲液(1.059g的Na2CO3、2.93g的NaHCO3、加超纯水至1000mL,调pH值为9.6)将(1,3)-β-D葡聚糖稀释至1μg/mL加入酶标板,每孔加0.1mL,4℃过夜。次日弃去孔内的液体,同时用PBST洗涤缓冲液(0.2g的KH2PO4、2.9g的Na2HPO4·12H2O、8.0g的NaCl、0.2g的KCl、0.5mL的Tween-20,加超纯水至100mL,调pH值为7.4)洗涤3次,每次洗涤时每孔加PBST洗涤缓冲液300μL(以下未明确说明时,洗涤所用试剂和用量均与本次相同),拍干。
12)封闭:每孔中加入0.1mL的含3%BSA的PBST洗涤缓冲液,置于37℃孵育1h,再经PBST洗涤缓冲液洗涤3次,拍干,得待测用酶标板。
13)加待测的鼠血清样本:取不同小鼠的内眦静脉血,于2000rpm离心5min后取血清,用0.1mol/L的PBS稀释1000倍、1万倍、10万倍、50万倍和100万倍,得不同浓度的免疫小鼠血清;
分别取100μL不同浓度的免疫小鼠血清加至待测用酶标板孔内,同时以未免疫小鼠的血清作为阴性对照,以0.1mol/L的PBS作为空白对照,置于37℃孵育1h,弃去孔中液体,再经PBST洗涤缓冲液洗涤3次,拍干。
14)加酶标第二抗体:先用稀释缓冲液(0.1g的BSA加至PBST洗涤缓冲液中,定容至100mL)将酶标第二抗体稀释至工作浓度,即稀释10000倍,得酶标第二抗体稀释液;
每孔分别加入100μL的酶标第二抗体稀释液,37℃孵育1h,PBST洗涤缓冲液洗涤,拍干。
15)显色:各孔中分别加入100μL的显色底物TMB,37℃孵育15min。
16)终止反应:各孔中分别加入100μL的终止液(浓度为1mol/L的HCL)终止反应。
17)判定结果:在酶标仪上于450nm处以空白对照孔调零后测各孔吸光度值(A450nm),当待测样品A450nm值与阴性对照的A450nm值的比大于2.1时,判定为阳性,此时抗体的最大稀释度即为抗体效价。具体抗体效价测定结果见下表:
表1四次免疫后小鼠抗体效价(A450nm)
由表1可以看出,2号鼠的抗体效价达到1:10万以上,其抗体效价最佳,选取该小鼠进行后续试验。
二)杂交瘤细胞制备
1)骨髓瘤SP2/0细胞悬液的制备
融合前2周常规复苏小鼠骨髓瘤细胞(SP2/0细胞),并接种于培养基中(培养基为含10~20%胎牛血清的RPMIl640培养液,补加青霉素100IU/mL、链霉素100μg/mL),置于5%的CO2温箱中,37℃培养。融合前48~36h改用完全培养液(含20%胎牛血清的RPMIl640培养液),置于37℃、5%CO2温箱培养中进行扩大培养。
融合当天,用吸管将细胞轻轻从瓶壁上吹下,收集于50mL离心管中,于1500rpm离心6min,弃去上清,所得瘤细胞沉淀中加入30mL不完全培养液【不完全培养液是在RPMIl640中补加2%的NaHCO3、3%的4-羟乙基哌嗪乙磺酸(Hepes)、0.011%的丙酮酸钠,并用1mol/L的HCl调节pH为6.8~7.0制得】,再次于1500rpm离心6min,然后用PBST洗涤缓冲液洗涤一次,镜下观察细胞,并选取等大等圆、透亮、活性好的细胞作为SP2/0细胞,收集SP2/0细胞,使SP2/0细胞数达2×107cell,再将SP2/0细胞重悬至10mL不完全培养液,混匀,得骨髓瘤SP2/0细胞悬液。
2)免疫脾细胞悬液的制备:
取2号小鼠在细胞融合前3天,使用(1,3)-β-D等体积混合且充分乳化葡聚糖与弗氏不完全佐剂,通过尾静脉注射对小鼠进行加强免疫,加强免疫的剂量相当于每只小鼠注射10μg的(1,3)-β-D葡聚糖。3天后摘除眼球放血,并分离血清供检测抗体用。将小鼠拉颈处死,浸泡于75%酒精内5min,随即放入超净台中,用无菌手术开腹,取出脾脏,放入已盛有8mL不完全培养液的平皿中冲洗,并细心剥去周围结缔组织,得处理后的脾脏。
将处理后的脾脏移入另一盛有5mL不完全培养液的平皿中,置于200目不锈钢网上,用剪刀将处理后的脾脏剪成小块,然后用注射器内芯挤压研磨,并用平皿内的不完全培养液轻轻冲洗钢网,使脾细胞全部被挤压到液体中去,所得脾细胞溶液转至50mL离心管中,加不完全培养液至30mL混匀,于1500rpm离心6min,弃去上清后,再加不完全培养液至30mL混匀,再次于1500rpm离心6min洗涤一次,所得沉淀细胞加不完全培养液重悬至10mL,混匀,得脾细胞悬液(一般每只鼠可得1~2.5×108个脾细胞。以台盼兰染色用相差显微镜检查,活细胞数高于80%为合格)。
3)细胞融合:
分别吸取含有1×108个脾细胞的脾细胞悬液和含有2×107个骨髓瘤细胞的骨髓瘤SP2/0细胞悬液,加至50mL离心管中,补加不完全培养液至30mL,充分混匀,再于室温下经1200r/min离心6min,弃去上清,轻轻弹击管底,使沉淀细胞松散均匀成糊状,再置于37℃水浴条件下,将1mL的PEG1650沿转动的管壁滴入,静置90s,然后将离心管从水浴中取出,以1000r/min离心6min,弃去上清液,所得沉淀中加入10mL的HAT培养液,轻轻吹吸沉淀的细胞,使其悬浮并混匀,得融合后细胞悬液;
将融合后细胞悬液加入含有HAT培养液的96孔培养板中,每孔2滴,置于37℃温箱培养7d,7d后更换培养液,换液时吸去1/2~2/3培养液(本实施例吸取2/3培养液),加入等量HT培养液,再次培养7d,14d后再次更换培养液,换液时吸去1/2~2/3培养液(本实施例吸取2/3培养液),加入等量的完全培养液(含20%胎牛血清的RPMIl640培养液),得杂交瘤细胞悬液。
4)杂交瘤细胞的筛选与检测
用ELISA法(具体操作步骤参见实施例1)检测杂交瘤细胞悬液的抗体效价,以完全培养液做阴性对照,以0.1mol/L的PBS为空白对照,检测得杂交瘤细胞悬液的A450nm值与阴性对照的A450nm值的比值大于2.1,杂交瘤细胞悬液的抗体效价为阳性。
5)克隆化培养
将抗体效价为阳性的杂交瘤细胞悬液从96孔板扩大至24孔板培养3-5天,再将24孔板中待克隆化的杂交瘤细胞用加样器反复吹打均匀后,取20μL待克隆化的杂交瘤细胞悬液加入到1mL浓度为0.1mol/L的的PBS中,进行50倍稀释,得稀释后的细胞悬液;
用细胞计数板对稀释后的细胞悬液进行细胞计数,计算公式如下:细胞悬液的细胞数/mL=(计数板上4个大格细胞总数/4)×10000×50(稀释倍数),细胞数位3×106/mL。
根据上述细胞计数浓度,取含有230个活细胞的细胞悬液悬浮于4.6mL完全培养液(含20%胎牛血清的RPMIl640培养液)中,所得活细胞液接种于96孔培养板,每孔0.1mL活细胞液,共36孔(记为第一组)。余下的1mL活细胞液中,再加入4mL完全培养液(含20%胎牛血清的RPMIl640培养液),共得5mL细胞液,然后再将细胞液接种至其余36孔,每孔0.1mL细胞液(记为第二组)。剩余的1.4mL细胞液,再补加1.4mL完全培养液(含20%胎牛血清的RPMIl640培养液),接种剩余的24孔,每孔0.1mL(记为第三组)。这样共以三种不同的细胞浓度进行克隆化,第一组36孔,平均每孔5个细胞,第二组36孔,平均每孔1个细胞,第三组24孔,平均每孔0.5个细胞。
将96孔培养板置于37℃、5%CO2温箱培养5d,5d后在倒置显微镜下观察到细胞克隆生长情况,换液并采用ELISA法检测抗体分泌情况,取呈阳性的单克隆孔再次依据上述克隆方法进行克隆,选择两次克隆均为阳性的单克隆孔转入24孔板,再转入培养瓶中扩大培养并冻存,具体克隆细胞筛选过程见图1。
6)杂交瘤细胞的冻存
观察在细胞培养瓶中扩大培养的细胞状态,将处于对数生长期的杂交瘤细胞倒掉上清培养液,用吸管加入1mL不完全培养液,加入0.8mL胎牛血清和0.2mL二甲基亚砜,混合均匀,细胞计数,分装入冻存管,每个冻存管加入细胞数不少于1×106个细胞。将冻存管置于4℃预冷30min,-20℃放置2h,再移至-70℃低温冰箱中过夜,最后投入液氮保存。
实施例2抗真菌(1,3)-β-D葡聚糖单克隆抗体的制备
收集筛选呈阳性的杂交瘤细胞进行克隆,并收集其分泌的抗体,即为抗真菌(1,3)-β-D葡聚糖单克隆抗体,收集抗真菌(1,3)-β-D葡聚糖单克隆抗体,测定抗真菌(1,3)-β-D葡聚糖单克隆抗体的可变区的基因序列,其轻链可变区决定簇互补区的氨基酸序列是由SEQ ID NO:1所示的VL-CDR1,SEQ ID NO:2所示的VL-CDR2和SEQ ID NO:3所示的VL-CDR3组成,其具体氨基酸序列为SEQ ID NO:7,核苷酸序列为SEQ ID NO:9。重链可变区决定簇互补区的氨基酸序列是由SEQ ID NO:4所示的VH-CDR1,SEQ ID NO:5所示的VH-CDR2和SEQ IDNO:6所示的VH-CDR3组成,其具体氨基酸序列为SEQ ID NO:8,核苷酸序列为SEQ ID NO:10。
委托生工生物工程(上海)股份有限公司,构建含有抗真菌(1,3)-β-D葡聚糖单克隆抗体的可变区的基因的真核重组表达质粒pCMVp-NEO-BAN-Anti-glucan。
将真核重组表达质粒pCMVp-NEO-BAN-Anti-glucan导入中国仓鼠卵巢细胞(CHO细胞),得抗真菌(1,3)-β-D葡聚糖单克隆抗体的重组表达中国仓鼠卵巢细胞。
将抗真菌(1,3)-β-D葡聚糖单克隆抗体的重组表达中国仓鼠卵巢细胞接种于含10%胎牛血清的DMEM培养基,添加1mmol/L的异丙基-β-D-硫代半乳糖苷(IPTG)作为诱导剂,于37℃、5%CO2条件下诱导表达48h,收集细胞培养上清(表达产物)。
饱和硫酸铵法制备单克隆抗体IgG的粗提物:取细胞培养上清5mL,分别加5mL生理盐水和10mL饱和硫酸铵水溶液,4℃放置过夜后,于3000rpm离心30min,弃上清液,剩余沉淀中加入5mL生理盐水和5mL饱和硫酸铵水溶液,4℃放置过夜,然后于3000rpm离心30min,弃上清液,得到的沉淀中再次加入5mL生理盐水和2.5mL饱和硫酸铵水溶液,4℃放置过夜,于3000rpm离心30min,弃上清液,所得沉淀溶于2mL生理盐水中,对生理盐水透析18小时,即得单克隆抗体IgG的粗提物。
Protein A凝胶亲和层析法纯化单抗IgG:取1mL的Protein A,装柱后用20mmol/L的磷酸钠缓冲液(取20mmol/L的Na2PO3和20mmol/L的NaH2PO3等体积混合,调PH=7.0)平衡,将单克隆抗体IgG的粗提物5mL在4℃反复上柱20次,以20mmol/L磷酸钠缓冲液充分淋洗柱子,至分光光度测定OD280约等于0,换0.1mol/L的甘氨酸-盐酸缓冲液(pH=2.7)洗脱,每次500μL,洗脱液采用20μL的浓度为1.0mol/L的Tris-HCl缓冲液(pH=9.0)中和,所得抗体洗脱液采用CBG法测定蛋白含量,收集蛋白峰,然后对其采用0.01mol/L的PBS缓冲液(pH=7.4)充分透析,得纯化单抗IgG。
采用SDS-PAGE电泳法测定Marker、单克隆抗体IgG的粗提物和纯化单抗IgG,参见图2,可以看出两条蛋白带根据蛋白分子量回归方程计算出两条染色带分子量约为54kD和25kD,与文献报道的IgG重链及轻链分子量相符。因此,纯化单抗IgG即为纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体。
实施例3纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体的效价测定
31)用包被缓冲液将(1,3)-β-D葡聚糖稀释至1μg/mL加入酶标板,每孔加0.1mL,4℃过夜。次日弃去孔内的液体,同时用PBST洗涤缓冲液洗涤3次,每次洗涤时每孔加PBST洗涤缓冲液300μL,拍干。
32)每孔中加入0.1mL的含3%BSA的PBST洗涤缓冲液,置于37℃孵育1h,再经PBST洗涤缓冲液洗涤3次,拍干,得待测用孔板。
33)将纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体用0.1mol/L的PBS缓冲液分别稀释1000倍、1万倍、10万倍、50万倍、100万倍、200万倍、500万倍和1000万倍,得不同浓度的抗体稀释液;
分别取100μL不同浓度的抗体稀释液加至待测用孔板的孔内,同时以完全培养液做阴性对照,以0.1mol/L的PBS为空白对照,置于37℃孵育1h,弃去孔中液体,再经PBST洗涤缓冲液洗涤3次,拍干。
34)每孔分别加入100μL的酶标第二抗体稀释液,37℃孵育1h,PBST洗涤缓冲液洗涤,拍干。
35)各孔中分别加入100μL的显色底物TMB,37℃孵育15min。
36)各孔中加入100μL的终止液(浓度为1mol/L的HCL)终止反应。
37)在酶标仪上于450nm处以空白对照孔调零后测各孔吸光度值(A450nm),当待测样品A450nm值与阴性对照的A450nm值的比大于2.1时,判定为阳性。参见图3,可以看出纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体稀释500万倍时,A450nm值仍大于对照组2.1倍以上,说明纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体的效价在1:500万以上,其亲和力非常高。
实施例4纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体特异性鉴定
本发明是为了获得仅与(1,3)-β-D葡聚糖强力结合,而与其它干扰检测的物质很少结合甚至不结合的单克隆抗体。杂交瘤细胞经过严格的筛选和单克隆化,对通过基因工程技术重组表达后,纯化所得的抗真菌(1,3)-β-D葡聚糖单克隆抗体,再一次进行了特异性鉴定。该抗体的特异性可以用其与(1,3)-β-D葡聚糖的类似物是否存在交叉反应性及交叉反应的大小来衡量。
具体操鉴定方法如下:
41)用包被缓冲液分别将脂多糖、半乳甘露聚糖、甘露聚糖、肽聚糖、多粘菌素及(1,3)-β-D葡聚糖稀释成10μg/mL,分别加至包被酶标板,每孔加0.1mL,4℃过夜。次日弃孔内液体,用PBST洗涤缓冲液洗涤3次,每次洗涤时每孔加PBST洗涤缓冲液300μL,拍干。
42)每孔中加入0.1mL的含3%BSA的PBST洗涤缓冲液,置于37℃孵育1h,再经PBST洗涤缓冲液洗涤3次,拍干,得含有不同包被多糖的孔板。
43)将纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体0.1mol/L的PBS缓冲液分别稀释至浓度为0.001μg/mL、0.01μg/mL、0.1μg/mL、1μg/mL及10μg/mL的纯化后抗体稀释液。
44)分别取100μL不同浓度的纯化后抗体稀释液加至含有不同包被多糖的孔板内,且含有相同包被多糖的孔内均需分别加入上述5种浓度的纯化抗体稀释液,同时以完全培养液作为阴性对照,PBS作为空白对照,置于37℃孵育60min,弃孔内液体,用PBST洗涤缓冲液洗涤3次,每孔加入100μL酶标第二抗体稀释液,37℃孵育1h,PBST洗涤缓冲液洗涤,拍干。
45)各孔中分别加入100μL的显色底物TMB,37℃孵育15min。
46)各孔中加入100μL的终止液(浓度为1mol/L的HCL)终止反应。
47)用在酶标仪上于450nm处以空白对照孔调零后测各孔吸光度值(A450nm),当待测样品A450nm值/阴性对照的A450nm值大于2.1时为阳性,即表示纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体在该浓度下与包被的多糖存在交叉反应。参见图4,可以看出,纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体在0.001-10μg/mL浓度范围内与脂多糖、半乳甘露聚糖、甘露聚糖、肽聚糖、多粘菌素均无交叉反应,且与(1,3)-β-D葡聚糖交叉反应良好,说明纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体具有非常好的特异性,且受其它物质干扰程度很小。
实施例5真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒的制备和应用
一)真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒的制备
本实施例采用纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体作为捕获抗体和/或检测抗体,制得了一种可用于临床的真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒,该试剂盒主要组分包括:生物素标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体、化学发光基团标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体【本实施例采用吖啶磺酰胺标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体,也可用吖啶磺酰胺、吖啶酯、钌复合物、鲁米诺、(金钢烷)-1,2-二氧乙烷或碱性磷酸酶中的至少一种进行标记】、链霉亲和素磁微粒(DynabeadsTMM-270链霉亲和素)、低值校准品【(1,3)-β-D葡聚糖冻干品,用超纯水复溶后浓度为50pg/mL】、高值校准品【(1,3)-β-D葡聚糖冻干品,用超纯水复溶后浓度为200pg/mL】、质控品【(1,3)-β-D葡聚糖冻干品,用超纯水复溶后浓度为100pg/mL】;化学发光激发液A液(0.1mol/L的HNO3、1.32%的H2O2)、化学发光激发液B液(0.35mol/L的NaOH)和PBST洗涤缓冲液。
采用纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体偶联在固相载体上,所述固相载体为羧基化磁微粒、生物素化磁微粒中的至少一种,本实施例采用羧基化磁微粒作为固相载体。具体真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒如下:
51)生物素标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体的制备
取200μg的(1,3)-β-D葡聚糖抗体,用0.02mol/L的PBS(pH=7.4)稀释至1mg/mL,得抗体溶液。
取1mg的Sulfo-NHS-LC-Biotin(生物素抗体标记用)溶于100μL的二甲基亚砜中,取3μL加至抗体溶液中混匀,避光室温反应1h,反应结束后将反应产物装入透析袋中(排阻MW:8000),以0.02mol/L的PBS(pH=7.4)作为透析液,2~8℃透析24h(本实施例在4℃进行透析),所得透析产物中加入终浓度为0.1%的Proclin300和1%的海藻糖,得生物素标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体,密封避光置于2~8℃备用(本实施例在4℃进行透析)。使用时生物素标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体与链霉亲和素磁微粒1:1混合后即可。
52)吖啶磺酰胺标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体的制备
取200μg的(1,3)-β-D葡聚糖抗体,用0.1mol/L的PBS(pH=7.2)稀释到1mg/mL,待化学标记抗体溶液;
取10μL浓度为2mg/mL的NSP-SA-NHS(吖啶磺酰肼胺内盐)加至待化学标记抗体溶液中混匀,避光室温反应1h,再加4μL浓度为10%的赖氨酸水溶液,混匀,避光置于室温下反应30min,反应结束后将反应产物装入透析袋中(排阻MW:8000),用0.02mol/L的PBS(pH=7.2)缓冲液在2~8℃透析过夜(本实施例在4℃进行透析),产物即为吖啶磺酰胺标记的(1,3)-β-D葡聚糖单克隆抗体。
53)校准品及质控品的制备
低值校准品(50pg/mL)的配制方法:取(1,3)-β-D葡聚糖加入超纯水配成浓度为1ng/mL的水溶液,取其中5mL加入20mL甘露醇溶液、6mL聚乙烯吡咯烷酮(PVP)、20mL新生牛血清,然后加超纯水至100mL,混匀,低温冷冻干燥后即为低值校准品(50pg/mL)。
高值校准品(200pg/mL)的配制方法:取(1,3)-β-D葡聚糖加入超纯水配成浓度为4ng/mL的水溶液,取其中5mL加入20mL甘露醇溶液、6mL聚乙烯吡咯烷酮(PVP)、20mL新生牛血清,然后加超纯水至100mL,混匀,低温冷冻干燥后即为低值校准品(200pg/mL)。
质控品(100pg/mL)的配制方法::取(1,3)-β-D葡聚糖加入超纯水配成浓度为2ng/mL的水溶液,取其中5mL加入20mL甘露醇溶液、6mL聚乙烯吡咯烷酮(PVP)、20mL新生牛血清,然后加超纯水至100mL,混匀,低温冷冻干燥后即为质控品(100pg/mL)。
二)真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒的应用
所制真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒可以采用磁微粒化学发光法,在全自动化学发光分析仪上实现自动检测,具体步骤为:反应杯中加入50μL链霉亲和素磁微粒和50μL生物素标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体,混匀,37℃孵育15min,然后在外加磁场下分离磁微粒,吸去反应杯中液体,向反应杯中加300μL清洗液进行清洗,吸去上清,反复清洗3次,再在反应杯中加入100μL待测样品,混匀,37℃孵育15min;再次在外加磁场下分离磁微粒,吸去反应杯中液体,反应杯中加300μL清洗液进行清洗,吸去上清,反复清洗3次,然后在反应杯中加入100μL吖啶磺酰胺标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体,混匀,37℃孵育15min。第三次在外加磁场下分离磁微粒,吸去反应杯中液体,向反应杯中加300μL清洗液进行清洗,吸去上清,反复清洗3次。在反应杯中加入化学发光激发液A液和化学发光激发液B液各50μL,立即测定化学发光值,即可检测出待测样品中真菌(1,3)-β-D葡聚糖的含量。
本发明的真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒是利用夹心法检测样本中的真菌(1,3)-β-D葡聚糖,羧基化磁珠偶联的捕获抗体(生物素标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体)、(1,3)-β-D葡聚糖和化学发光基团修饰的检测抗体(化学发光基团标记的纯化后的抗真菌(1,3)-β-D葡聚糖单克隆抗体)形成了捕获抗体-抗原-检测抗体免疫复合物,磁分离洗涤去除未与磁珠结合的物质后,加入激发液发光,通过检测化学发光强度来分析样本中真菌(1,3)-β-D葡聚糖的含量,所述试剂盒具有灵敏度高、特异性好、干扰物质少、检测速度快、可实现机器自动化操作等优点,适于临床真菌感染早期诊断。
实施例6真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒的抗干扰效果
本实施例中向含有108pg/mL和413pg/mL的(1,3)-β-D葡聚糖临床样本中分别加入不同浓度的脂多糖、半乳甘露聚糖、甘露聚糖、肽聚糖、多粘菌素(具体添加浓度见表2),添加干扰物的样本再次用实施例5所制的真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒按照实施例5中的应用方法进行测定,并计算干扰物质对(1,3)-β-D葡聚糖检测的干扰程度,计算公式如下:
干扰程度(%)=(添加干扰物后样本(1,3)-β-D葡聚糖实测浓度-未添加干扰物时样本(1,3)-β-D葡聚糖实际浓度)/未添加干扰物时样本(1,3)-β-D葡聚糖实际浓度×100%。
通过计算干扰物质对(1,3)-β-D葡聚糖检测的干扰程度,所得结果见下表:
表2(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒的抗干扰效果
通过上表可以看出,即使添加高浓度的干扰物后,采用本发明所制真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒对(1,3)-β-D葡聚糖进行检测,其受干扰程度依然在5.5%以内,说明本发明所制真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒在检测临床样本的过程中抗干扰能力很强,不会发生因干扰物引起的假阳性结果,能为临床提供更精准的检测数据。
显然,所描述的实施例仅仅是本发明的一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
SEQUENCE LISTING
<110> 河北康立德生物科技有限公司
<120> 抗真菌(1,3)-β-D葡聚糖单克隆抗体、其编码基因及其表达和应用
<130> 202108
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 39
<212> PRT
<213> BALB/c小鼠
<400> 1
Trp Asp Ile Val Glu Thr Gln Ser Glu Thr Pro Ser Tyr Ala Ser Val
1 5 10 15
Ile Pro Val Gly Gly Thr Tyr Val Thr Asp Ile Lys Ser Ile Thr Lys
20 25 30
Cys Ser Asp Lys Asp Ser Glu
35
<210> 2
<211> 31
<212> PRT
<213> BALB/c小鼠
<400> 2
Thr Leu Cys Asp Arg Glu Thr Gln Glu Cys Glu Ser Asp Ile Lys Ala
1 5 10 15
Gly Gln Lys Lys Arg Leu Ser Arg Tyr Val Pro Thr Ile Asn Lys
20 25 30
<210> 3
<211> 39
<212> PRT
<213> BALB/c小鼠
<400> 3
Asp Met Glu Tyr Pro Glu Ser Gln Leu Gly Asp Phe Gly Gly Ser Gly
1 5 10 15
Gly Phe Val Gly Phe Val Glu Val Lys Val Arg Arg Ile Gly Glu Gly
20 25 30
Gly Val Thr Arg Ser Leu Glu
35
<210> 4
<211> 41
<212> PRT
<213> BALB/c小鼠
<400> 4
Val Gln Ser Gln Leu Glu Glu Val Ser Gly Gly Arg Arg Leu Val Gln
1 5 10 15
Pro Thr Pro Gly Ser Thr Pro Lys Leu Thr Ser Ala Phe Ser Asp Arg
20 25 30
Thr Ala Tyr Ser Gly Phe Ser Trp Ser
35 40
<210> 5
<211> 31
<212> PRT
<213> BALB/c小鼠
<400> 5
Gln Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly
1 5 10 15
Asp Arg Ser Thr Tyr Ile Pro Leu Met Ser Leu Leu Lys Glu Val
20 25 30
<210> 6
<211> 40
<212> PRT
<213> BALB/c小鼠
<400> 6
Phe Thr Asp Ser Lys Tyr Thr Pro Thr Lys Val Asp Leu Met Pro Thr
1 5 10 15
Asp Ala Tyr Tyr Phe Cys Ala Asn Tyr Asp Arg Asp Ala Val Met Glu
20 25 30
Trp Gly Pro Glu Ile Thr Val Ser
35 40
<210> 7
<211> 109
<212> PRT
<213> BALB/c小鼠
<400> 7
Trp Asp Ile Val Glu Thr Gln Ser Glu Thr Pro Ser Tyr Ala Ser Val
1 5 10 15
Ile Pro Val Gly Gly Thr Tyr Val Thr Asp Ile Lys Ser Ile Thr Lys
20 25 30
Cys Ser Asp Lys Asp Ser Glu Thr Leu Cys Asp Arg Glu Thr Gln Glu
35 40 45
Cys Glu Ser Asp Ile Lys Ala Gly Gln Lys Lys Arg Leu Ser Arg Tyr
50 55 60
Val Pro Thr Ile Asn Lys Asp Met Glu Tyr Pro Glu Ser Gln Leu Gly
65 70 75 80
Asp Phe Gly Gly Ser Gly Gly Phe Val Gly Phe Val Glu Val Lys Val
85 90 95
Arg Arg Ile Gly Glu Gly Gly Val Thr Arg Ser Leu Glu
100 105
<210> 8
<211> 112
<212> PRT
<213> BALB/c小鼠
<400> 8
Val Gln Ser Gln Leu Glu Glu Val Ser Gly Gly Arg Arg Leu Val Gln
1 5 10 15
Pro Thr Pro Gly Ser Thr Pro Lys Leu Thr Ser Ala Phe Ser Asp Arg
20 25 30
Thr Ala Tyr Ser Gly Phe Ser Trp Ser Gln Lys Trp Val Arg Gln Ala
35 40 45
Pro Gly Lys Gly Leu Glu Trp Ile Gly Asp Arg Ser Thr Tyr Ile Pro
50 55 60
Leu Met Ser Leu Leu Lys Glu Val Phe Thr Asp Ser Lys Tyr Thr Pro
65 70 75 80
Thr Lys Val Asp Leu Met Pro Thr Asp Ala Tyr Tyr Phe Cys Ala Asn
85 90 95
Tyr Asp Arg Asp Ala Val Met Glu Trp Gly Pro Glu Ile Thr Val Ser
100 105 110
<210> 9
<211> 327
<212> DNA
<213> BALB/c小鼠
<400> 9
tgggacatag tagaaacaca aagcgaaaca ccaagctacg caagcgtaat accagtagga 60
ggaacatacg taacagacat aaaaagcata acaaaatgca gcgacaaaga cagcgaaaca 120
ctatgcgaca gagaaacaca agaatgcgaa agcgacataa aagcaggaca aaaaaaaaga 180
ctaagcagat acgtaccaac aataaacaaa gacatggaat acccagaaag ccaactagga 240
gacttcggag gaagcggagg attcgtagga ttcgtagaag taaaagtaag aagaatagga 300
gaaggaggag taacaagaag cctagaa 327
<210> 10
<211> 336
<212> DNA
<213> BALB/c小鼠
<400> 10
gtacaaagcc aactagaaga agtaagcgga ggaagaagac tagtacaacc aacaccagga 60
agcacaccaa aactaacaag cgcattcagc gacagaacag catacagcgg attcagctgg 120
agccaaaaat gggtaagaca agcaccagga aaaggactag aatggatagg agacagaagc 180
acatacatac cactaatgag cctactaaaa gaagtattca cagacagcaa atacacacca 240
acaaaagtag acctaatgcc aacagacgca tactacttct gcgcaaacta cgacagagac 300
gcagtaatgg aatggggacc agaaataaca gtaagc 336
Claims (9)
1.一种抗真菌(1,3)-β-D葡聚糖单克隆抗体,其特征在于,所述抗真菌(1,3)-β-D葡聚糖单克隆抗体包括轻链可变区决定簇互补区和重链可变区决定簇互补区;
所述轻链可变区决定簇互补区的氨基酸序列选自下组的CDR的氨基酸序列:SEQ IDNO:1所示的VL-CDR1、SEQ ID NO:2所示的VL-CDR2及SEQ ID NO:3所示的VL-CDR3;
所述重链可变区决定簇互补区的氨基酸序列选自下组的CDR的氨基酸序列:SEQ IDNO:4所示的VH-CDR1、SEQ ID NO:5所示的VH-CDR2及SEQ ID NO:6所示的VH-CDR3。
2.权利要求1所述的抗真菌(1,3)-β-D葡聚糖单克隆抗体,其特征在于,所述抗真菌(1,3)-β-D葡聚糖单克隆抗体的轻链可变区决定簇互补区的氨基酸序列为SEQ ID NO:7;重链可变区决定簇互补区的氨基酸序列为SEQ ID NO:8。
3.一种编码权利要求1或2所述的抗真菌(1,3)-β-D葡聚糖单克隆抗体的基因,其特征在于,所述轻链可变区决定簇互补区的核苷酸序列为SEQ ID NO:9,所述重链可变区决定簇互补区的核苷酸序列为SEQ ID NO:10。
4.包含权利要求3所述的抗真菌(1,3)-β-D葡聚糖单克隆抗体的基因的重组载体。
5.包含权利要求3所述的抗真菌(1,3)-β-D葡聚糖单克隆抗体的基因的重组表达细胞。
6.一种真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒,其特征在于,它含有权利要求1或2所述的抗真菌(1,3)-β-D葡聚糖单克隆抗体。
7.如权利要求6所述的真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒,其特征在于,所述真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒中含有采用生物素标记的所述抗真菌(1,3)-β-D葡聚糖单克隆抗体。
8.如权利要求6或7所述的真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒,其特征在于,所述真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒中还含有采用化学发光基团标记的所述抗真菌(1,3)-β-D葡聚糖单克隆抗体。
9.一种真菌(1,3)-β-D葡聚糖磁微粒化学发光检测方法,其特征在于,所述真菌(1,3)-β-D葡聚糖磁微粒化学发光检测方法是利用权利要求6-8中任一项所述的真菌(1,3)-β-D葡聚糖磁微粒化学发光检测试剂盒,采用磁微粒化学发光法进行检测。
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111045511.1A CN113929776B (zh) | 2021-09-07 | 2021-09-07 | 抗真菌(1,3)-β-D葡聚糖单克隆抗体、其编码基因及其表达和应用 |
PCT/CN2022/107379 WO2022207014A2 (zh) | 2021-09-07 | 2022-07-22 | 抗真菌(1,3)-β-D葡聚糖单克隆抗体、其编码基因及其表达和应用 |
ZA2022/10155A ZA202210155B (en) | 2021-09-07 | 2022-09-13 | Antifungal (1, 3)-beta-d-glucan monoclonal antibody, encoding gene, expression and application therefor |
US18/070,516 US20230340088A1 (en) | 2021-09-07 | 2022-11-29 | Fungal (1,3)-Beta-D glucan-directed monoclonal antibody, coding genes thereof, expression thereof and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111045511.1A CN113929776B (zh) | 2021-09-07 | 2021-09-07 | 抗真菌(1,3)-β-D葡聚糖单克隆抗体、其编码基因及其表达和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113929776A true CN113929776A (zh) | 2022-01-14 |
CN113929776B CN113929776B (zh) | 2023-06-27 |
Family
ID=79275202
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111045511.1A Active CN113929776B (zh) | 2021-09-07 | 2021-09-07 | 抗真菌(1,3)-β-D葡聚糖单克隆抗体、其编码基因及其表达和应用 |
Country Status (4)
Country | Link |
---|---|
US (1) | US20230340088A1 (zh) |
CN (1) | CN113929776B (zh) |
WO (1) | WO2022207014A2 (zh) |
ZA (1) | ZA202210155B (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2022207014A3 (zh) * | 2021-09-07 | 2022-12-08 | 河北康立德生物科技有限公司 | 抗真菌(1,3)-β-D葡聚糖单克隆抗体、其编码基因及其表达和应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1185161A (zh) * | 1994-09-01 | 1998-06-17 | 生化学工业株式会社 | (1→3)-β-葡聚糖结合性蛋白质、识别该蛋白质的抗体及其利用 |
CN112940114A (zh) * | 2021-02-09 | 2021-06-11 | 丹娜(天津)生物科技股份有限公司 | 一种抗真菌1,3-β-D-葡聚糖单克隆抗体及其应用 |
CN113249336A (zh) * | 2021-07-15 | 2021-08-13 | 天津一瑞生物科技股份有限公司 | 鼠抗(1,3)-β-D葡聚糖杂交瘤细胞株,单克隆抗体及应用 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100423614B1 (ko) * | 2001-05-16 | 2004-03-22 | 주식회사유한양행 | 비형간염바이러스의 에스-표면항원을 인식하는단일클론항체의 가변영역 및 이를 코딩하는 유전자 |
CN110885372A (zh) * | 2019-12-17 | 2020-03-17 | 丹娜(天津)生物科技有限公司 | 一种抗念珠菌甘露聚糖的兔源单克隆抗体及其应用 |
CN113929776B (zh) * | 2021-09-07 | 2023-06-27 | 河北康立德生物科技有限公司 | 抗真菌(1,3)-β-D葡聚糖单克隆抗体、其编码基因及其表达和应用 |
-
2021
- 2021-09-07 CN CN202111045511.1A patent/CN113929776B/zh active Active
-
2022
- 2022-07-22 WO PCT/CN2022/107379 patent/WO2022207014A2/zh unknown
- 2022-09-13 ZA ZA2022/10155A patent/ZA202210155B/en unknown
- 2022-11-29 US US18/070,516 patent/US20230340088A1/en active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1185161A (zh) * | 1994-09-01 | 1998-06-17 | 生化学工业株式会社 | (1→3)-β-葡聚糖结合性蛋白质、识别该蛋白质的抗体及其利用 |
US6156519A (en) * | 1994-09-01 | 2000-12-05 | Seikagaku Corporation | (→)-β- d-glucan binding protein, an antibody recognizing the protein and use thereof |
CN112940114A (zh) * | 2021-02-09 | 2021-06-11 | 丹娜(天津)生物科技股份有限公司 | 一种抗真菌1,3-β-D-葡聚糖单克隆抗体及其应用 |
CN113249336A (zh) * | 2021-07-15 | 2021-08-13 | 天津一瑞生物科技股份有限公司 | 鼠抗(1,3)-β-D葡聚糖杂交瘤细胞株,单克隆抗体及应用 |
Non-Patent Citations (2)
Title |
---|
EI-ICHIRO SUNAMURA ET AL: "A novel enzyme immunoassay for the measurement of plasma (1-3)-β-D-glucan levels", 《J IMMUNOL METHODS》 * |
严俊等: "真菌(1,3)-β-D-葡聚糖多克隆抗体制备及ELISA竞争法检测体系的建立", 《检验医学》, vol. 30, no. 9 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2022207014A3 (zh) * | 2021-09-07 | 2022-12-08 | 河北康立德生物科技有限公司 | 抗真菌(1,3)-β-D葡聚糖单克隆抗体、其编码基因及其表达和应用 |
Also Published As
Publication number | Publication date |
---|---|
CN113929776B (zh) | 2023-06-27 |
WO2022207014A3 (zh) | 2022-12-08 |
US20230340088A1 (en) | 2023-10-26 |
ZA202210155B (en) | 2022-09-28 |
WO2022207014A2 (zh) | 2022-10-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2019000267A1 (zh) | Gm杂交瘤细胞、单克隆抗体、试剂盒及其制备方法与应用 | |
CN114478785A (zh) | 抗人chi3l1鼠源单克隆抗体及应用 | |
CN114181308A (zh) | 一种降钙素原抗体及其应用 | |
CN114133452B (zh) | 一种肝素结合蛋白抗体及其试剂盒、应用 | |
WO2024140618A1 (zh) | 1,3-β-D-葡聚糖结合蛋白、制备方法及应用 | |
CN109142738A (zh) | Ecm1作为血清学检测肝纤维化的标志物及其应用 | |
CN113929776B (zh) | 抗真菌(1,3)-β-D葡聚糖单克隆抗体、其编码基因及其表达和应用 | |
CN113045666B (zh) | 胃蛋白酶原ii单克隆抗体及其应用 | |
CN116925218B (zh) | 小热休克蛋白hspb1的抗体、抗体组合物、杂交瘤细胞株及其应用 | |
CN111505295B (zh) | 一种基于竞争性单克隆抗体的旋毛虫病抗体检测试剂盒及其制备方法 | |
CN110885372A (zh) | 一种抗念珠菌甘露聚糖的兔源单克隆抗体及其应用 | |
CN115073591B (zh) | 一种可识别asfv外膜糖基化修饰蛋白的单克隆抗体及应用 | |
CN113717285B (zh) | 抗人d-二聚体抗体及其应用 | |
CN110894235B (zh) | 一种抗隐球菌夹膜多糖的兔源单克隆抗体及其应用 | |
CN110894236B (zh) | 一种抗曲霉菌半乳甘露聚糖的单克隆抗体及其应用 | |
CN117866082A (zh) | 一种抗新型冠状病毒的全人源单克隆抗体及其用途 | |
CN106771216B (zh) | 提高免疫试剂检测特异性的方法及其应用 | |
CN110702913A (zh) | 一种用于定量检测贝氏柯克斯体i相株的单抗组合物 | |
CN112410306B (zh) | 一种检测l-crmp4的双抗体夹心化学发光免疫检测试剂盒及其应用 | |
CN116925219B (zh) | 小热休克蛋白hspb1的抗体、杂交瘤细胞株及其应用 | |
CN113583123B (zh) | 一种血浆中肝素含量的检测方法 | |
CN113603774B (zh) | 一种单克隆抗体及其在疾病诊断和检测中的应用 | |
CN117447601B (zh) | 猪IgM的抗体、抗体组合物及其应用 | |
CN117447602B (zh) | 猪IgM的抗体及其应用 | |
CN116284415B (zh) | 一种纤溶酶-α2抗纤溶酶复合物单克隆抗体及其制备和应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |