CN117074678A - 一种利用流式细胞仪检测tPSA与fPSA的试剂盒 - Google Patents
一种利用流式细胞仪检测tPSA与fPSA的试剂盒 Download PDFInfo
- Publication number
- CN117074678A CN117074678A CN202311032099.9A CN202311032099A CN117074678A CN 117074678 A CN117074678 A CN 117074678A CN 202311032099 A CN202311032099 A CN 202311032099A CN 117074678 A CN117074678 A CN 117074678A
- Authority
- CN
- China
- Prior art keywords
- antibody
- fpsa
- tpsa
- specific antigen
- prostate specific
- 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.)
- Pending
Links
- 238000000684 flow cytometry Methods 0.000 title description 4
- 108010072866 Prostate-Specific Antigen Proteins 0.000 claims abstract description 63
- 238000001514 detection method Methods 0.000 claims abstract description 52
- 239000004005 microsphere Substances 0.000 claims abstract description 36
- 239000000243 solution Substances 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 24
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims abstract description 23
- 238000002156 mixing Methods 0.000 claims abstract description 12
- 239000000427 antigen Substances 0.000 claims abstract description 10
- 102000036639 antigens Human genes 0.000 claims abstract description 10
- 108091007433 antigens Proteins 0.000 claims abstract description 10
- 239000007790 solid phase Substances 0.000 claims abstract description 10
- 239000011259 mixed solution Substances 0.000 claims abstract description 5
- 102100038358 Prostate-specific antigen Human genes 0.000 claims description 60
- 238000006243 chemical reaction Methods 0.000 claims description 7
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 7
- 239000007850 fluorescent dye Substances 0.000 claims description 7
- 239000004793 Polystyrene Substances 0.000 claims description 5
- 102000034287 fluorescent proteins Human genes 0.000 claims description 5
- 108091006047 fluorescent proteins Proteins 0.000 claims description 5
- 229920002223 polystyrene Polymers 0.000 claims description 5
- 239000000975 dye Substances 0.000 claims description 4
- 125000003172 aldehyde group Chemical group 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- 125000003700 epoxy group Chemical group 0.000 claims description 2
- 230000005284 excitation Effects 0.000 claims description 2
- 238000001179 sorption measurement Methods 0.000 claims description 2
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 claims description 2
- 239000004593 Epoxy Chemical group 0.000 claims 1
- 125000003277 amino group Chemical group 0.000 claims 1
- 230000035939 shock Effects 0.000 claims 1
- 239000007787 solid Substances 0.000 claims 1
- 238000004140 cleaning Methods 0.000 abstract description 5
- 210000002307 prostate Anatomy 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 2
- 102000007066 Prostate-Specific Antigen Human genes 0.000 abstract 3
- 108010004469 allophycocyanin Proteins 0.000 description 14
- 108010004729 Phycoerythrin Proteins 0.000 description 10
- 206010060862 Prostate cancer Diseases 0.000 description 8
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 8
- 238000005259 measurement Methods 0.000 description 8
- 239000008055 phosphate buffer solution Substances 0.000 description 7
- 239000011534 wash buffer Substances 0.000 description 7
- 206010028980 Neoplasm Diseases 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 238000004448 titration Methods 0.000 description 6
- 239000011535 reaction buffer Substances 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 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 description 4
- 229920001213 Polysorbate 20 Polymers 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 230000008878 coupling Effects 0.000 description 4
- 238000010168 coupling process Methods 0.000 description 4
- 238000005859 coupling reaction Methods 0.000 description 4
- 231100000673 dose–response relationship Toxicity 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 210000000582 semen Anatomy 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 108090001008 Avidin Proteins 0.000 description 2
- 101000945318 Homo sapiens Calponin-1 Proteins 0.000 description 2
- 101000652736 Homo sapiens Transgelin Proteins 0.000 description 2
- 102100024078 Plasma serine protease inhibitor Human genes 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 102100031013 Transgelin Human genes 0.000 description 2
- 239000008186 active pharmaceutical agent Substances 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 239000003001 serine protease inhibitor Substances 0.000 description 2
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- QGKMIGUHVLGJBR-UHFFFAOYSA-M (4z)-1-(3-methylbutyl)-4-[[1-(3-methylbutyl)quinolin-1-ium-4-yl]methylidene]quinoline;iodide Chemical compound [I-].C12=CC=CC=C2N(CCC(C)C)C=CC1=CC1=CC=[N+](CCC(C)C)C2=CC=CC=C12 QGKMIGUHVLGJBR-UHFFFAOYSA-M 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 206010061218 Inflammation Diseases 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
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102100034569 Pregnancy zone protein Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010001953 Protein C Inhibitor Proteins 0.000 description 1
- 229940122929 Protein C inhibitor Drugs 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 229940122055 Serine protease inhibitor Drugs 0.000 description 1
- 101710102218 Serine protease inhibitor Proteins 0.000 description 1
- 208000000453 Skin Neoplasms Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003541 chymotrypsin inhibitor Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 230000010405 clearance mechanism Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000000295 emission spectrum Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 230000024924 glomerular filtration Effects 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 229910021644 lanthanide ion Inorganic materials 0.000 description 1
- 229910052747 lanthanoid Inorganic materials 0.000 description 1
- 150000002602 lanthanoids Chemical class 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 208000017497 prostate disease Diseases 0.000 description 1
- 210000000064 prostate epithelial cell Anatomy 0.000 description 1
- 201000007094 prostatitis Diseases 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
- 230000019100 sperm motility Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
- G01N15/1436—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement the optical arrangement forming an integrated apparatus with the sample container, e.g. a flow cell
-
- 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
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
-
- 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
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
Abstract
本发明公开了一种同时检测总前列腺特异性抗原及游离前列腺特异性抗原的方法,包括:将样本/校准品加入标记抗总前列腺特异性抗原的单克隆抗体A包被的固相载体溶液中,混匀,震荡孵育,经抗原抗体特异性亲和,微球对样本内tPSA吸附,清洗后加入荧光素一标记tPSA抗体B和荧光素二标记的fPSA抗体C的混合液,混匀孵育,荧光抗体B特异性结合荧光微球上tPSA,荧光单克隆抗体C只与fPSA表位特异性结合;流式细胞仪检测得tPSA和fPSA浓度,计算fPSA/tPSA比值,一次检测获得三个指标。该方法同时检测总前列腺抗原和游离前列腺抗原,实验步骤简单,检测时间短,抗体用量少,降低检测成本,且灵敏度高、准确度高。
Description
技术领域
本发明属于免疫检测分析技术和纳米生物技术领域,具体涉及一种利用流式细胞仪检测tPSA与fPSA的试剂盒。
技术背景
前列腺癌是一种中老年疾病,是男性除皮肤癌之外最常被确诊的癌症类型且是导致男性因癌症死亡的第二大原因。由于前列腺癌早期症状隐匿,不易被察觉,通常确诊时已至进展期;而另一方面,前列腺癌进展缓慢,被发现前常有6-12年的发展时间,因此早期诊断及时治疗至关重要。前列腺特异性抗原(Prostate specific antigen,PSA)作为肿瘤标志物,它的测定对于前列腺癌的诊断具有十分重要的意义。
前列腺特异性抗原PSA是前列腺上皮细胞分泌的一种丝氨酸蛋白酶,是一种糖蛋白分子,包含93%的肽链,7%的糖基,分子量34kDa,等电点6.9,具有液化精液和提高精子活力的作用。一般存在于精液中,主要是精浆中,在血液中的含量极其微少。
大多数的PSA分子都随精液一起排出。90%-95%的PSA与a-1-抗糜蛋白酶形成稳定的共价结构PSA-ACT,而且它们构成了免疫检测PSA复合物的绝大多数(大于等于98%);还有很少量的与丝氨酸蛋白酶抑制剂结合PSA-AT,与蛋白C抑制剂结合的PSA-PCI;血液中fPSA占据PSA的5-15%,且血液中fPSA在大量抗蛋白酶的作用下很不稳定,易失活。
PSA被释放到血液循环后,主要通过两条代谢途径:
1)此途径的PSA分子是一个蛋白水解的过程,最后形成血液tPSA总含量中5-40%这样一个小的比例。由于这个途径缺乏酶活性,从而无法与循环中大量的抗蛋白酶形成复合物,所以它仍然是游离的PSA(或者“非结合”)。fPSA最终通过肾小球滤过作用从血液中被清除,它的半衰期是12-18小时。
2)进入外周血的PSA(60-95%)是具有催化活性的,能够与一些蛋白酶抑制剂形成稳定的共价复合物,如丝氨酸蛋白酶抑制剂(a-1-抗糜蛋白酶ACT,蛋白C抑制剂PCI,a1-抗胰蛋白酶API),以及其他的一系列抗蛋白酶,包括a2-巨球蛋白AMG和妊娠区带蛋白PZP。有活性的PSA优先与AMG结合,大约是PSA-ACT形成的20倍。然而,PSA-AMG很快就被肝脏的清除机制所代谢掉,它的半衰期只有6-7分钟。这就导致血液中PSA浓度接近或低于检测临界线。此外,由于AMG分子的隐藏作用使得PSA所有的抗原表位都被封闭,所以PSA-AMG没有免疫反应。PSA-ACT复合物的清除相对较慢,估计需要一天半的时间,相当于每天减少1ng/mL的PSA-ACT。因此PSA-ACT在血液中累积,并且构成检测PSA抗蛋白酶复合物的重要组成,而PSA-API只占有很小的部分(0.5-5%)。
在血清中,大多数的PSA分子都是以结合态存在,只有少量的游离PSA分子。当前列腺发生炎症时,血清中的PSA含量也会随之发生变化。一般来说,正常的tPSA变化范围是0-4ng/mL,血清中tPSA随着年龄增长,血清中可检测到的tPSA含量是逐渐升高的。当tPSA含量大于10ng/mL时,患有前列腺癌的风险增至67%,并建议50岁以上,应每年做一次PSA检测。当tPSA处于4-10ng/mL这个灰色诊断区域时,由于特异性不强,对于前列腺癌,前列腺增生,前列腺炎的区别很难界定,前列腺癌的患癌风险为25%,所以一般采用fPSA/tPSA的比值作为临床指标来区分。也通过对血清中tPSA衍生物(SA密度,PSA速率,年龄特异性的PSA变化范围等)来试图提高癌症检测的特异性和敏感性。目前PSA被用来作为很多前列腺疾病诊断的主要参数,包括:早期检查及其就诊、疾病监测的临床定位、癌症转移与否、通过病理分期的预测确定临床阶段和组织学分类以及预后的情况。
利用胶体金、酶免法、化学发光法等平台,现在已有多种tPSA或fPSA定量检测试剂盒,中国专利CN 102360011A与CN 218036858 U公开总前列腺特异性抗原tPSA的测定试剂盒及其检测方法;专利CN105044356A与CN103773738A公开检测游离前列腺特异性抗原fPSA的定量检测试剂盒;以上试剂盒检测方法对只对样本内单项指标检测,对tPSA和fPSA不能实现同时检测,单独检测后经计算获得fPSA/tPSA比值,因此不能保证在相同条件下对tPSA与fPSA的检测,检测结果存在误差,影响结果的准确性,且操作繁琐,效率低。专利CN105785017A采用时间分辨荧光免疫分析法对tPSA或fPSA进行同时检测,获得定量结果,为检测提供了极大便利,但该方法仪器和试剂通常更昂贵,且应用必须与特殊镧系染料兼容,易受环境、试剂和容器中的镧系元素离子的污染,使本底增高。
发明内容
本试剂盒用双抗夹心法形成微球+捕获抗体A+tPSA+捕获抗体B+荧光素一及微球+捕获抗体A+fPSA+捕获抗体C+荧光素二模式;通过流式细胞仪一次检测,即得tPSA及fPSA含量,计算fPSA/tPSA比值,一次检测获得三个指标。相较现有的检测方法,该方法在相同条件下对tPSA与fPSA的检测,结果准确、灵敏度高、特异性强且同时检测总前列腺抗原和游离前列腺抗原,实验步骤简单,检测时间短,抗体用量少,降低检测成本。
tPSA/fPSA的检测试剂盒包括清洗液、反应缓冲液、包被PSA单克隆抗体A的微球溶液、PSA校准品、质控品、荧光素一标记tPSA抗体B和荧光素二标记的fPSA抗体C的混合液;本试剂盒使用流式细胞仪同时检测总前列腺特异性抗原及游离前列腺特异性抗原。
第一步:将样本/校准品加入标记前列腺特异性抗原单克隆抗体A(第一抗体)包被固相载体,混匀,震荡恒温孵育,利用抗原抗体特异性亲和完成包被抗体A的微球与tPSA(PSA-ACT与fPSA)亲和吸附;
第二步:加入荧光标记抗体B(第二抗体)和荧光标记单克隆抗体C(第三抗体)的混合液,混匀并孵育,抗体B特异性结合固相载体上的PSA-ACT与fPSA,抗体C与fPSA表位特异性结合;
第三步:流式细胞仪检测第二步反应后结果,获取不同荧光通道读取荧光信号MFI(平均荧光强度),得到tPSA和fPSA浓度。
试剂盒的制备
抗tPSA(总前列腺特异性抗原)的抗体对PSA-ACT,fPSA都有特异性反应,与其它的肿瘤标志物无交叉反应。并且对抗tPSA的抗体对具有等克反应性,排除非等克分子识别。对于抗fPSA的抗体只与fPSA有反应,与PSA-ACT无交叉反应,与其他的肿瘤标志物也无交叉反应,因此选用tPSA抗体为抗体A和抗体B,特异性针对fPSA抗体为抗体C作为试剂盒制备原材料。
包被PSA单克隆抗体A的微球溶液:抗体A包被的固相载体多采用聚苯乙烯微球、磁力微球或磁力荧光微球,固相载体微球表面含有羧基、环氧基、甲苯磺酰基或醛基等活化基团用于包被抗体。固相载体一般采用5-7um的微球,每100万微球可以交联抗体5-10ug。
荧光素一标记tPSA抗体B和荧光素二标记的fPSA抗体C的检测混合液:荧光素采用488或638激发光激发的荧光蛋白或荧光染料如FITC、PE、APC等,荧光染料选择染料之间发射光谱不重叠的染料,避免荧光补偿带来的干扰。荧光素一标记于抗体B特异性结合tPSA,荧光素二标记于抗体C特异性结合fPSA特有表位,采用了两种荧光素作为报告分子,流式细胞仪可同时检测tPSA和fPSA两种信号。荧光染料与抗体B、抗体C可以采用直接的共价标记,也可以采用生物素-亲和素体系进行间接标记。一般荧光蛋白与抗体缀合通常用双功能接头如SMCC进行。SMCC的一端(通过NHS酯)与氨基酸赖氨酸和N-末端中发现的胺(-NH2)反应,另一端(通过马来酰亚胺)与氨基酸中发现的硫醇基团(-SH)反应或采用Buccutite技术。
PSA校准品:用含2mg/mL的BSA和proclin300的1xPBS缓冲液配制成1mg/mL的PSA抗原溶液,将tPSA抗原稀释为250ng/mL、62.5ng/mL、15.625ng/mL、3.9ng/mL、0.97ng/mL、0.244ng/mL、0.056ng/mL、0ng/mL,同理配制fPSA抗原校准品溶液。
反应/清洗缓冲液:用PBS-TBN,1xPBS、0.1%BSA、0.02%Tween20、0.05%叠氮钠,清洗反应试剂,减少非特异性干扰。
附图说明
图1为本发明检测fPSA与tPSA方法的实施例1样本-荧光染料检测结果散点图;
图2为本发明检测fPSA与tPSA方法的实施例2样本-荧光蛋白检测结果散点图;
图3为本发明检测fPSA与tPSA方法的实施例4中总前列腺特异性抗tPSA剂量-反应曲线;
图4为本发明检测fPSA与tPSA方法的实施例4中游离前列腺特异性抗原fPSA剂量-反应曲线。
具体实施方式
下面通过具体实施例对本试剂盒可同时检测总前列腺特异性抗原及游离前列腺特异性抗原的方法进行说明,试剂和仪器均为市售产品:微球采购biolegend或luminex,PSA抗原、tPSA抗体及fPSA抗体采购medix,荧光素FITC、磺化Cy5、PE、SA-PE、APC及偶联试剂盒等采购ATT Bioquest。
实施例1
微球溶液:固相载体采用直径6.5um的聚苯乙烯磁性微球100万可包被10ug的tPSA捕获抗体(第一抗体),检测时微球终浓度为50粒子/μL(滴定验证10ug的tPSA抗体包被1x10^6微球时,效果最佳);
检测溶液:FITC-抗体C(FITC标记的fPSA抗体)和Cy5-抗体B(Cy5标记的tPSA检测抗体)混合物,浓度:FITC-fPSA:50μg/mL,浓度:Cy5-tPSA抗体:20μg/mL,Cy5(花氰染料Cy5)即为前述荧光素一,FITC(异硫氰酸荧光素)即为前述荧光素二,抗体批次及偶联效率存在批间差因此试剂盒最优浓度需要滴定实验加以确认;
清洗/反应溶液:PBS-TBN缓冲液为pH 7.0~7.4的PBS(磷酸盐缓冲液),含有0.02%Tween-20、0.1%BSA、0.05%NaN3。
一种同时检测总前列腺特异性抗原及游离前列腺特异性抗原的方法,包括如下步骤:
第一步:取50μL微球溶液,加入20μL待检测样本/校准品,充分混匀,37℃恒温震荡孵育30min;
第二步:离心或磁分离弃上清,用清洗缓冲液200μL清洗3次微球,加入100μL检测溶液,混匀,在37℃环境下避光震荡孵育30min;
第三步:离心或磁分离去上清,用清洗缓冲液200μL清洗3次微球,加入100μL反应缓冲液,流式细胞仪检测结果;
检测结果:Cy5荧光在流式细胞仪上用APC通道检测,故标注为APC。APC通道MFI数值高即tPSA浓度高,FITC通道MFI数值低即fPSA浓度低。
实施例2
微球溶液:固相载体采用直径5.5um的聚苯乙烯磁性微球100万可包被10ug的tPSA捕获抗体(第一抗体),检测时微球终浓度为50粒子/μL,最优包被量经滴定验证;
检测溶液1:biotin-抗体C(biotin标记的fPSA抗体)浓度:biotin-fPSA抗体50μg/mL
检测溶液2:APC-tPSA抗体(APC标记的tPSA检测抗体)混合物,浓度:APC-tPSA抗体:20μg/mL,APC(藻蓝蛋白APC)即为前述荧光素一,20ug/mL SA-PE(亲和素标记的藻红蛋白streptavidin-PE)PE(藻红蛋白PE)即为前述荧光素二,抗体批次及偶联效率存在批间差因此试剂盒最优浓度需要滴定实验加以确认;
清洗/反应溶液:PBS-TBN缓冲液为pH7.0~7.4的PBS(磷酸盐缓冲液),含有0.02%Tween-20、0.1%BSA、0.05%NaN3。
一种同时检测总前列腺特异性抗原及游离前列腺特异性抗原的方法,包括如下步骤:
第一步:取50μL微球溶液,加入20μL待检测样本/校准品,加入30μL检测溶液1,充分混匀,37℃恒温孵育30min;
第二步:离心或磁分离去上清,用清洗缓冲液200μL清洗3次微球,加入100μL检测溶液2混匀,在37℃环境下避光孵育30min;
第三步:离心或磁分离去上清,用清洗缓冲液200μL清洗3次微球,加入100μL反应缓冲液,流式细胞仪检测结果。
实施例3
微球溶液:捕获微球,采用直径5.5um的聚苯乙烯磁性微球100万可包被10ug的tPSA捕获抗体(第一抗体),检测时微球终浓度为50粒子/μL,最优包被浓度经滴定验证;
检测溶液1:biotin-抗体C(biotin标记的fPSA抗体)和APC-tPSA抗体(APC标记的tPSA检测抗体)混合物,浓度:biotin-fPSA抗体30μg/mL,浓度:APC-tPSA抗体20μg/mL,APC(藻蓝蛋白APC)即为前述荧光素一,抗体批次及偶联效率存在批间差因此试剂盒最优浓度需要滴定实验加以确认;
检测溶液2:SA-PE(亲和素标记的藻红蛋白streptavidin-PE)PE(藻红蛋白PE)即为前述荧光素二;
清洗/反应溶液:PBS-TBN缓冲液为pH7.0~7.4的PBS(磷酸盐缓冲液),含有0.02%Tween-20、0.1%BSA、0.05%NaN3。
一种同时检测总前列腺特异性抗原及游离前列腺特异性抗原的方法,包括如下步骤:
第一步:取50μL微球溶液,加入20μL待检测样本/校准品,充分混匀,37℃恒温孵育30min;
第二步:离心或磁分离去上清,用清洗缓冲液200μL清洗3次微球,加入90μL检测溶液1,加入10μL检测溶液2(10ug/mL)混匀,在37℃环境下避光孵育30min;
第三步:离心或磁分离去上清,用清洗缓冲液200μL清洗3次微球,加入100μL反应缓冲液,实施例2与实施例3采用相同的荧光染料,经流式细胞仪检测表明结果相似;
流式细胞仪读取不同的荧光通道的藻红蛋白(PE)和别藻蓝蛋白(APC)的荧光信号MFI(平均荧光强度),藻红蛋白报告的信号表征fPSA浓度,别藻蓝蛋白的信号表征tPSA浓度。
检测结果:APC通道MFI数值即tPSA浓度,PE通道MFI数值即fPSA浓度。
实施例4
采用实施例3中的试剂组合和检测方法,对产品试剂盒性能进行检测。通道荧光信号(表征tPSA/fPSA浓度)通过国家参考品绘制校准曲线,换算成PSA浓度;
检测限:
利用不同批次的检测试剂盒三批,对校准品稀释液样本或者零背景值的样本同时检测20次,计算检测结果平均值(Mean)及标准差(SD),由方程Mean+2×SD计算出MFI,并计算其浓度值。
实验数据表明tPSA/fPSA最低检测限都在0.056ng/mL即56pg/mL,空白限低于最低检测限56pg/mL。
线性范围:
校准品、质控品是由tPSA/fPSA配制而成,用1xPBS缓冲液配制PSA抗原为1mg/mL,将tPSA抗原稀释为250ng/mL、62.5ng/mL、15.625ng/mL、3.9ng/mL、0.97ng/mL、0.244ng/mL、0.056ng/mL、0ng/mL,同理配制fPSA抗原校准品溶液。缓冲液的组成如下:20mM PBS缓冲液、1%BSA,0.05%proclin300 pH为7.2。
按照实施例2操作最后用流式细胞仪检测,将每一浓度的样本重复检测3次,计算平均值,并计算剂量-反应曲线相关系数(r)值r≥0.990。
tfPSA的测量:
浓度(ng/mL) | 0.056 | 0.244 | 0.97 | 3.9 | 15.625 | 62.5 | 250 |
测定值1 | 542 | 887 | 1203 | 2224 | 5643 | 14645 | 34832 |
测定值2 | 534 | 918 | 1190 | 2287 | 5762 | 14769 | 34584 |
测定值3 | 558 | 878 | 1167 | 2267 | 5653 | 14977 | 33920 |
平均值 | 544.7 | 894.3 | 1186.7 | 2259.3 | 5686 | 14797 | 34445.3 |
SD | 12.2 | 20.9 | 18.2 | 32.2 | 66. | 167.7 | 471.5 |
CV | 2.24% | 2.35% | 1.54% | 1.42% | 1.16 | 1.13% | 1.37% |
fPSA的测量:
浓度(ng/mL) | 0.056 | 0.244 | 0.97 | 3.9 | 15.625 | 62.5 | 250 |
测定值1 | 441 | 1823 | 4583 | 7914 | 18363 | 32802 | 75742 |
测定值2 | 421 | 1835 | 4546 | 8165 | 18416 | 34617 | 75846 |
测定值3 | 436 | 1776 | 4457 | 8112 | 18023 | 33325 | 77606 |
平均值 | 432.7 | 1811.3 | 4528.7 | 8063.7 | 18267.3 | 33581.3 | 76398 |
SD | 10.4 | 31.2 | 64.7 | 132.3 | 213.2 | 934.2 | 1047.5 |
CV | 2.4% | 1.72% | 1.43% | 1.64% | 1.17% | 2.78% | 1.37% |
实验数据表明tPSA/fPSA检测试剂盒的剂量-反应关系存在相关性,通过数据处理可以知道tPSA与fPSA变异系数cv低且r≥0.990。
准确度:
采用罗氏市售前列腺特异性抗原电化学发光法(ECLI)对高值样本和低值样本进行检测,对比实施例3试剂盒检测准确性,重复三次:
实验结果表明,对比市售化学发光试剂盒与实施例三检测误差均小于10%,表明该试剂检测结果准确可靠。
Claims (6)
1.一种同时检测总前列腺特异性抗原及游离前列腺特异性抗原的方法,其特征在于,包括如下步骤:
第一步:将样本/校准品加入标记前列腺特异性抗原单克隆抗体A(第一抗体)包被固相载体,震荡混匀恒温孵育,样本抗原tPSA(PSA-ACT与fPSA)与其抗体特异性亲和吸附;
第二步:加入荧光标记抗体B(第二抗体)和荧光标记单克隆抗体C(第三抗体)的混合液,混匀并孵育,抗体B特异性结合固相载体上的PSA-ACT与fPSA,抗体C与fPSA表位特异性结合;
第三步:流式细胞仪检测第二步反应后结果,获取不同荧光通道读取荧光信号MFI(平均荧光强度),得到tPSA和fPSA浓度。
2.根据权利要求1所述的一种同时检测总前列腺特异性抗原及游离前列腺特异性抗原的方法,其特征在于,所述的固体载体为聚苯乙烯微球、磁力微球或荧光磁性微球,载体表面有可以与抗体氨基结合的活性基团如羧基、环氧基、甲苯磺酰基、醛基,微球尺寸为5-7μm,每100万微球可交联抗体5-10ug。
3.根据权利要求1所述的一种同时检测总前列腺特异性抗原及游离前列腺特异性抗原的方法,其特征在于,荧光素采用不同激发光,采用488或638激发光激发的荧光染料、荧光蛋白或串联染料。
4.根据权利要求3所述的一种同时检测总前列腺特异性抗原及游离前列腺特异性抗原的方法,其特征在于,荧光素为荧光染料FITC、Cy5或荧光蛋白PE、APC。
5.根据权利要求1所述的一种同时检测总前列腺特异性抗原及游离前列腺特异性抗原的方法,其特征在于,第一步与第二步孵育均为37℃恒温避光震荡孵育30min。
6.一种同时检测总前列腺特异性抗原及游离前列腺特异性抗原的试剂盒,其特征在于,包括:包被PSA单克隆抗体A的微球溶液、荧光素一标记tPSA抗体B和荧光素二标记的fPSA抗体C的检测混合液、PSA校准品。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311032099.9A CN117074678A (zh) | 2023-08-16 | 2023-08-16 | 一种利用流式细胞仪检测tPSA与fPSA的试剂盒 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311032099.9A CN117074678A (zh) | 2023-08-16 | 2023-08-16 | 一种利用流式细胞仪检测tPSA与fPSA的试剂盒 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN117074678A true CN117074678A (zh) | 2023-11-17 |
Family
ID=88717827
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311032099.9A Pending CN117074678A (zh) | 2023-08-16 | 2023-08-16 | 一种利用流式细胞仪检测tPSA与fPSA的试剂盒 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117074678A (zh) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20050046265A (ko) * | 2003-11-13 | 2005-05-18 | 주식회사유한양행 | 면역크로마토그래피법을 이용한 전립선암 및 전립선비대증 진단용 키트 |
CN105785017A (zh) * | 2016-05-11 | 2016-07-20 | 江苏省原子医学研究所 | 基于psa磁微粒的双标记时间分辨荧光免疫分析试剂盒 |
JP2016194437A (ja) * | 2015-03-31 | 2016-11-17 | 株式会社Lsiメディエンス | 前立腺特異抗原の測定方法及び測定キット |
CN115494240A (zh) * | 2022-09-15 | 2022-12-20 | 深圳唯公生物科技有限公司 | 一种可同时检测不同亚型免疫球蛋白的方法及试剂组合 |
CN115494037A (zh) * | 2022-09-15 | 2022-12-20 | 深圳唯公生物科技有限公司 | 一种可同时检测甲胎蛋白及其异质体的方法及试剂组合 |
-
2023
- 2023-08-16 CN CN202311032099.9A patent/CN117074678A/zh active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20050046265A (ko) * | 2003-11-13 | 2005-05-18 | 주식회사유한양행 | 면역크로마토그래피법을 이용한 전립선암 및 전립선비대증 진단용 키트 |
JP2016194437A (ja) * | 2015-03-31 | 2016-11-17 | 株式会社Lsiメディエンス | 前立腺特異抗原の測定方法及び測定キット |
CN105785017A (zh) * | 2016-05-11 | 2016-07-20 | 江苏省原子医学研究所 | 基于psa磁微粒的双标记时间分辨荧光免疫分析试剂盒 |
CN115494240A (zh) * | 2022-09-15 | 2022-12-20 | 深圳唯公生物科技有限公司 | 一种可同时检测不同亚型免疫球蛋白的方法及试剂组合 |
CN115494037A (zh) * | 2022-09-15 | 2022-12-20 | 深圳唯公生物科技有限公司 | 一种可同时检测甲胎蛋白及其异质体的方法及试剂组合 |
Non-Patent Citations (1)
Title |
---|
夏术阶: "《前列腺疾病》", 30 April 2009, 中国医药科技出版社, pages: 106 - 108 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Nimse et al. | Biomarker detection technologies and future directions | |
WO2017197914A1 (zh) | 一种基于抗原-适配体的竞争法检测试纸条及其应用以及利用适配体检测黄曲霉毒素b1或m1的试纸条 | |
JP5557450B2 (ja) | 測定対象成分の免疫測定法 | |
US20140193832A1 (en) | Detection of prostate cancer using psa glycosylation patterns | |
WO2016127318A1 (zh) | 心肌肌钙蛋白i超敏检测试剂盒及其超敏检测方法 | |
US20050158877A1 (en) | Novel antibody mediated surface enhanced raman scattering (SERS) immunoassay and multiplexing schemes | |
JP4920415B2 (ja) | プローブ複合体 | |
CN112730839A (zh) | 一种磁微粒化学发光法测定细胞角蛋白19片段含量的试剂盒 | |
WO2021088730A1 (zh) | 游离前列腺特异性抗原检测试剂盒及其制备方法 | |
CN110346560A (zh) | 一种多酶信号颗粒及其制备方法与应用 | |
CN113433318A (zh) | 一种检测甲胎蛋白异质体afp-l3含量的试剂盒及其检测方法和应用 | |
CN110632040B (zh) | 一种血清中前列腺特异性抗原的分析方法 | |
US6379550B1 (en) | Method for detecting PSA and its molecular forms using thiophilic gel | |
CN106645756A (zh) | 一种检测nmp22的试剂盒及其制备方法 | |
CA2017342A1 (en) | Assay method for a substance with a specific sugar chain | |
EP3511712B1 (en) | Method for measuring thyroglobulin | |
JP4515099B2 (ja) | Lasp−1免疫反応性の決定によって炎症性疾患および感染を診断するための方法 | |
CN117074678A (zh) | 一种利用流式细胞仪检测tPSA与fPSA的试剂盒 | |
Cheng et al. | Urinary CD14 as a potential biomarker for benign prostatic hyperplasia–discovery by combining MALDI‐TOF‐based biostatistics and ESI‐MS/MS‐based stable‐isotope labeling | |
JP6578119B2 (ja) | 前立腺特異抗原の測定方法及び測定キット | |
CN115651964A (zh) | 一种磁性探针及制备与检测基质金属蛋白酶的应用 | |
Zhu et al. | Proximity Ligation Measurement of the Complex between Prostate Specific Antigen and α1-Protease Inhibitor | |
CN111735950B (zh) | Fgf18和ca125联合用作早期卵巢癌生物标志物以及试剂盒 | |
CN106556699B (zh) | 用于测定豆荚蛋白的血液水平的方法和组合物 | |
Oh et al. | One-step-immunoassay of procalcitonin enables rapid and accurate diagnosis of bacterial infection |
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 |