CN117285625B - 抗泌乳素蛋白的单克隆抗体及应用 - Google Patents
抗泌乳素蛋白的单克隆抗体及应用 Download PDFInfo
- Publication number
- CN117285625B CN117285625B CN202311579376.8A CN202311579376A CN117285625B CN 117285625 B CN117285625 B CN 117285625B CN 202311579376 A CN202311579376 A CN 202311579376A CN 117285625 B CN117285625 B CN 117285625B
- Authority
- CN
- China
- Prior art keywords
- prl
- prolactin
- clone1
- monoclonal antibody
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 230000001263 anti-prolactin effect Effects 0.000 title claims abstract description 11
- 108010057464 Prolactin Proteins 0.000 claims abstract description 31
- 229940097325 prolactin Drugs 0.000 claims abstract description 31
- 238000001514 detection method Methods 0.000 claims abstract description 16
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 8
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- 102000003946 Prolactin Human genes 0.000 claims abstract 5
- 230000002124 endocrine Effects 0.000 claims description 5
- 210000004027 cell Anatomy 0.000 abstract description 25
- 108090000623 proteins and genes Proteins 0.000 abstract description 25
- 102000004169 proteins and genes Human genes 0.000 abstract description 19
- 238000002156 mixing Methods 0.000 abstract description 10
- 238000012216 screening Methods 0.000 abstract description 10
- 239000006228 supernatant Substances 0.000 abstract description 10
- 238000002965 ELISA Methods 0.000 abstract description 8
- 239000000427 antigen Substances 0.000 abstract description 8
- 102000036639 antigens Human genes 0.000 abstract description 8
- 108091007433 antigens Proteins 0.000 abstract description 8
- 239000011248 coating agent Substances 0.000 abstract description 6
- 238000000576 coating method Methods 0.000 abstract description 6
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 210000002966 serum Anatomy 0.000 abstract description 6
- 238000001261 affinity purification Methods 0.000 abstract description 5
- 239000002671 adjuvant Substances 0.000 abstract description 4
- 238000003556 assay Methods 0.000 abstract description 4
- 210000004369 blood Anatomy 0.000 abstract description 4
- 239000008280 blood Substances 0.000 abstract description 4
- 230000036039 immunity Effects 0.000 abstract description 4
- 230000001804 emulsifying effect Effects 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 3
- 230000004927 fusion Effects 0.000 abstract description 3
- 210000004408 hybridoma Anatomy 0.000 abstract description 3
- 238000001179 sorption measurement Methods 0.000 abstract 1
- 102100024819 Prolactin Human genes 0.000 description 31
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 239000011324 bead Substances 0.000 description 11
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000012258 culturing Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 108020004705 Codon Proteins 0.000 description 4
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 230000001737 promoting effect Effects 0.000 description 4
- 102100021809 Chorionic somatomammotropin hormone 1 Human genes 0.000 description 3
- 108010051696 Growth Hormone Proteins 0.000 description 3
- 108010003044 Placental Lactogen Proteins 0.000 description 3
- 239000000381 Placental Lactogen Substances 0.000 description 3
- 102100038803 Somatotropin Human genes 0.000 description 3
- 230000002159 abnormal effect Effects 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 239000013613 expression plasmid Substances 0.000 description 3
- 239000000122 growth hormone Substances 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 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 2
- 101000687438 Homo sapiens Prolactin Proteins 0.000 description 2
- 102000004407 Lactalbumin Human genes 0.000 description 2
- 108090000942 Lactalbumin Proteins 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 239000004697 Polyetherimide Substances 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 229940011871 estrogen Drugs 0.000 description 2
- 239000000262 estrogen Substances 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 230000005484 gravity Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 208000031424 hyperprolactinemia Diseases 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000002175 menstrual effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229920001601 polyetherimide Polymers 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000003761 preservation solution Substances 0.000 description 2
- 239000000186 progesterone Substances 0.000 description 2
- 229960003387 progesterone Drugs 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000011146 sterile filtration Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- AAYSIOJYGRABGS-UHFFFAOYSA-N 3-[9-[4-(2,5-dioxopyrrolidin-1-yl)oxycarbonyl-2,6-dimethylphenoxy]carbonylacridin-10-ium-10-yl]propane-1-sulfonate Chemical compound C=1C(C)=C(OC(=O)C=2C3=CC=CC=C3[N+](CCCS([O-])(=O)=O)=C3C=CC=CC3=2)C(C)=CC=1C(=O)ON1C(=O)CCC1=O AAYSIOJYGRABGS-UHFFFAOYSA-N 0.000 description 1
- 201000000736 Amenorrhea Diseases 0.000 description 1
- 206010001928 Amenorrhoea Diseases 0.000 description 1
- 201000005670 Anovulation Diseases 0.000 description 1
- 206010002659 Anovulatory cycle Diseases 0.000 description 1
- 208000010228 Erectile Dysfunction Diseases 0.000 description 1
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 1
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 1
- 108700012941 GNRH1 Proteins 0.000 description 1
- 208000001287 Galactorrhea Diseases 0.000 description 1
- 206010017600 Galactorrhoea Diseases 0.000 description 1
- 102000023108 LH Receptors Human genes 0.000 description 1
- 108010011942 LH Receptors Proteins 0.000 description 1
- 206010024419 Libido decreased Diseases 0.000 description 1
- 102000011965 Lipoprotein Receptors Human genes 0.000 description 1
- 108010061306 Lipoprotein Receptors Proteins 0.000 description 1
- 102000009151 Luteinizing Hormone Human genes 0.000 description 1
- 108010073521 Luteinizing Hormone Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 208000007913 Pituitary Neoplasms Diseases 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- -1 acridine ester Chemical class 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-O acridine;hydron Chemical compound C1=CC=CC2=CC3=CC=CC=C3[NH+]=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-O 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 231100000540 amenorrhea Toxicity 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 231100000552 anovulation Toxicity 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000011162 core material Substances 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002069 effect on gonads Effects 0.000 description 1
- 210000000750 endocrine system Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229940028334 follicle stimulating hormone Drugs 0.000 description 1
- 210000002149 gonad Anatomy 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000002267 hypothalamic effect Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 201000001881 impotence Diseases 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000006651 lactation Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000002796 luminescence method Methods 0.000 description 1
- 229940040129 luteinizing hormone Drugs 0.000 description 1
- 210000005075 mammary gland Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000008558 metabolic pathway by substance Effects 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000001817 pituitary effect Effects 0.000 description 1
- 239000000583 progesterone congener Substances 0.000 description 1
- 208000030153 prolactin-producing pituitary gland adenoma Diseases 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 210000002993 trophoblast Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
-
- 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
- G01N2333/575—Hormones
-
- 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)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Analytical Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Endocrinology (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Plasma & Fusion (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了抗泌乳素蛋白的单克隆抗体及应用，单克隆抗体为PRL‑Clone1，PRL‑Clone1的重链可变区氨基酸序列如SEQ ID NO.1所示，PRL‑Clone1的轻链可变区氨基酸序列如SEQ ID NO.2所示。制备方法：将亲和纯化后的PRL蛋白与弗氏佐剂进行等体积混合乳化，采集小鼠血液分离血清，筛选免疫较好的小鼠进行杂交瘤融合实验，以泌乳素作为酶标板包被抗原，取上清进行酶联免疫吸附测定，筛选出优质阳性细胞株，并进行两轮的细胞亚克隆。同时也提供了上述抗体的化学发光诊断检测试剂盒，从而提高诊断试剂的特异性和灵敏度。
Description
技术领域
本发明属于抗体的制备及序列测定领域，具体涉及抗泌乳素蛋白的单克隆抗体及应用。
背景技术
泌乳素（Prolactin，简称PRL）是由垂体前叶的泌乳滋养细胞分泌的蛋白质激素。泌乳素是由199个氨基酸残基组成，分子内部含有3个二硫键，分子量约为22Kd。PRL 与生长激素(GH)、胎盘催乳素(PL) 同属于 PRL/ GH/PL家族，其结构相似、序列同源，并且有重叠的生物学性质。PRL的生物学效应主要为：促进乳腺生长发育、参与应激、物质代谢、调节免疫和性腺功能。PRL对内分泌的作用主要表现在对性腺的影响上。小剂量 PRL对卵巢***和孕激素的合成有促进作用，而大剂量则有抑制作用。PRL对卵巢黄体功能的影响主要是刺激***受体的产生，同时还可促进脂蛋白与膜上受体形成脂蛋白受体复合物，为孕酮的生成提供底物，促进孕酮生成。
PRL的异常升高受多种因素影响，而垂体肿瘤，特别是垂体泌乳素瘤是其常见原因。高泌乳素症患者血清中高浓度的PRL可通过负反馈方式抑制下丘脑***释放激素的分泌，从而减少腺垂体***和***的分泌，致使患者出现无***和***水平低下的情况。高泌乳素血症(HPRL)的女性患者的临床症候群包括溢乳、闭经、初潮延迟、月经量或月经规律的改变，男性患者的临床症候群包括***减退、精致质量降低、***功能障碍、骨质疏松等。血液中异常升高的泌乳素严重影响了人类健康及生活质量，对该指标定量检测可以进行评价内分泌***的功能。
开发性能优异的PRL免疫诊断试剂的核心原料是抗PRL单克隆抗体，一般厂商的特异性和灵敏度不高，所以开发性能优异的PRL诊断试剂非常有必要，同时也期望能够进一步推动诊断试剂的国产化。
发明内容
为解决灵敏性、特异性的问题，本发明提供了抗泌乳素蛋白的单克隆抗体。该抗体性能优异、准确性高。同时提供了基于上述PRL抗体的化学发光试剂盒。
本发明提供如下技术方案：
抗泌乳素蛋白的单克隆抗体，单克隆抗体为PRL-Clone1，
PRL-Clone1的重链可变区氨基酸序列：
EVKLEESGGDLVKPGGSLKLSCAASGFGESSYGLSWVRQTPEKRLESVATISDSGSYSYYIDSVKGRFTISRDNAKNNLYLQMSSSRSEDTALYYCVILTGGAYWGQGTLVTVSAIVSS
PRL-Clone1的轻链可变区氨基酸序列：
MFKVTQSPVVLSASLGERSSLTCRTSQEISGYLSWLLLKPDGTRSRLIYDTSTLDSGVPKGYSGSRSGSDYSLTISSLESEDFAFFYCLQYTTYPLTFGTLTKLELKR
抗泌乳素蛋白的单克隆抗体的制备方法：
泌乳素蛋白的表达生产：在NCBI官网上查找人PRL氨基酸序列，在蛋白氨基酸的C端加上组氨酸标签，利用密码子优化软件在哺乳动物细胞表达***中优化密码子，基因合成后利用EcoRI/BamHI限制性内切酶酶切位点，将基因克隆至pTT5表达载体中，构建pTT5-PRL表达质粒，并测序验证基因序列。
将pTT5-PRL表达质粒与转染试剂PEI混匀后，转染HEK293细胞，同时在OPM-293CD03 Medium细胞培养基中加入增强剂和辅料进行培养，当细胞活率<70%后，收集细胞培养物，离心并收集细胞上清。
泌乳素蛋白的亲和纯化：细胞上清用盐和咪唑充分重悬过滤后，用预装重力柱进行亲和纯化，通过梯度咪唑浓度进行洗杂和洗脱，SDS-PAGE跑胶检测蛋白纯度，将蛋白透析至磷酸盐缓冲液中，0.22μm滤器无菌过滤后，BCA测定蛋白浓度。
将亲和纯化后泌乳素蛋白与弗氏佐剂进行等体积混合乳化，免疫计量为100ug/只小鼠，3周后进行加强免疫，免疫计量为50ug/只小鼠，后期间隔2周每次，采集小鼠血液分离血清，酶联免疫吸附测定抗体效价，筛选免疫较好的小鼠进行杂交瘤融合实验，经过HAT筛选培养基筛选培养后，以泌乳素蛋白作为酶标板包被抗原，取上清进行酶联免疫吸附测定，筛选出优质阳性细胞株，并进行两轮的细胞亚克隆，克隆出单克隆细胞株，用无血清培养和发酵细胞株，收集培养上清。
抗泌乳素蛋白的单克隆抗体在制备诊断泌乳素升高致内分泌失调免疫诊断试剂中的应用。
单克隆抗体用于PRL化学发光试剂盒。试剂盒使用PRL-Clone1作为检测抗体。
与现有技术相比，本发明的有益效果是：
本发明提供了抗泌乳素蛋白，用于制备诊断用单克隆抗体，本发明的抗 泌乳素蛋白的单克隆抗体为PRL-Clone1，能够识别免疫原，此抗体组合具有特异性和高亲和力的特点，同时也提供了基于上述抗体的化学发光检测试剂盒，从而提高诊断试剂的特异性和灵敏度，免疫诊断能够快速帮助医生发现机体因泌乳素异常所致的内分泌失调，精准的检测结果对后续临床治疗指导用药提供重要的医学价值。
附图说明
图1为本发明检测试剂盒与同类产品相关性对比图。
实施方式
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，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。
实施例1 泌乳素蛋白表达载体的构建
在NCBI官网上查找人PRL氨基酸序列，在蛋白氨基酸的C端加上组氨酸标签，利用密码子优化软件在哺乳动物细胞表达***中优化密码子，基因合成后利用EcoRI/BamHI限制性内切酶酶切位点，将基因克隆至pTT5表达载体中，构建pTT5-PRL表达质粒，并测序验证基因序列。
实施例2 泌乳素蛋白的表达生产
转染前一天，细胞以0.8×106/ml接种于细胞培养瓶，以OPM-293 CD03 Medium培养液培养，置于37℃，5%CO2培养箱中，至细胞融合度达到80%-90%，活力大于95%时进行转染，pTT5-PRL表达载体与聚醚酰亚胺混匀后，室温孵育15-30min，缓慢加入到HEK293细胞中，加入增强剂和辅料，于 37℃，5%CO2培养箱中继续培养。当细胞活率<70%后，收集细胞培养物，转速8000 g，离心10 min，收集上清液，再通过转速12000 g，4℃离心30 min，收集细胞上清。
实施例3 泌乳素蛋白的亲和纯化
细胞上清中加入NaCl和咪唑，并将工作浓度分别调整至300 mM和5 mM后再用0.22μm滤膜过滤，Ni TED-6FF beads预装重力柱进行亲和纯化，20mM PB，300mM NaCl，10%甘油，50mM咪唑，pH8.0溶液进行洗杂，20mM PB，300mM NaCl，10%甘油，250mM咪唑，pH8.0溶液进行洗脱，SDS-PAGE跑胶检测蛋白纯度，将蛋白透析至20mM PB，300mM NaCl，10%甘油，pH8.0缓冲液中，0.22μm滤器无菌过滤后，BCA测定蛋白浓度。
实施例4 单克隆抗体的制备
将PRL蛋白与弗氏佐剂进行等体积混合乳化，免疫计量为100ug/只小鼠，皮下多点注射，3周后进行加强免疫，免疫计量为50ug/只小鼠，后期间隔2周每次，采集小鼠血液分离血清，酶联免疫吸附法测定抗体效价，筛选免疫较好的小鼠进行杂交瘤融合实验，经过10天左右的HAT筛选培养基筛选培养后，以PRL作为酶标板包被抗原，取上清进行酶联免疫吸附法测定，筛选出优质阳性细胞株，并进行两轮的细胞亚克隆，克隆出单克隆细胞株，用无血清培养和发酵细胞株，收集培养上清，进行抗体纯化，将纯化后的抗体进行诊断性能分析。
表1第一次亚克隆亲和力检测表
实验参数(IN) ： GAM-HRP:1/3W，TMB:22min
实验参数(CAP)： Biotin:1/24W，SA-HRP:1/3W，TMB:17min
实验结果：选取亲和力高且细胞涨势好的细胞株进行第二次亚克隆。
表2第二次亚克隆亲和力检测表
得到6株单克隆细胞，选取1E12F1，1E9A1，2H12E7三株细胞生产：3*T175。
实施例5 单克隆抗体性能分析
基于双抗体夹心法和吖啶酯发光法原理制备试剂盒。
1、羧基磁珠包被抗体
将购买的羧基磁珠充分混匀，取出相应量的磁珠置于离心管，使用磁力架去除上清液，清洗后加入包被活化液，混匀后加入包被活化液溶解的EDC，充分混匀后室温颠倒活化0.5-1h；
超声分散磁珠，使用包被活化液清洗三次，加入本发明的包被抗体，充分混匀后室温颠倒反应1-2h；
超声分散磁珠，加入封闭剂，充分混匀后室温颠倒封闭1-2h；
超声分散磁珠，使用磁力架去除上清液，清洗三次，加入磁珠保存液将磁珠稀释至浓度为0.23mg/mL待用。
上述磁珠保存液配方为50mM Tris、0.2% Tween-20、0.85% NaCl、2% BSA、0.2%Proclin-300。
2、吖啶酯标记抗体
将NHS酯化的吖啶酯（NSP-DMAE-NHS）与本发明的检测抗体按照分子摩尔比10：1的比例在1×PBS中颠倒混匀标记反应1-2h；
反应结束后将标记液转移至14kD透析袋中，1×PBS中旋转透析，换液6次，每次1-2h；
透析过夜后取出标记液，使用分光光度计测试标记液中蛋白含量，计算并添加保护液，充分混匀后可置于20℃-22℃保存，上机测试时使用标记保存液将蛋白浓度稀释至0.5ug/mL待用。
上述标记保存液的配方为0.1M MES、0.2% Tween-20、0.85% NaCl、2% BSA、0.2%Proclin-300。
3、配制校准品和质控品
使用抗原稀释液将PRL抗原分别稀释至0、0.2、1、2、10、20、100、200、1000 ng/mL，分装至校准品管中。
使用抗原稀释液将PRL抗原稀分别释液至20、100 ng/mL，分装至质控品管中。
上述抗原稀释液配方为1×PBS（磷酸缓冲盐溶液）、1% BSA（牛血清白蛋白）。
检测试剂盒与同类产品对比：
实施例中的PRL化学发光检测试剂盒与贝克曼公司的产品在120例临床标本上进行了相关性对比，数据如图1所示。相关系数R2为0.991。说明本发明的试剂盒能准确检测PRL含量，为临床诊断提供依据，能够充分满足临床体外诊断检测需求。
检测试剂盒灵敏度和精密度考察：
按照临床和实验室标准协会灵敏度验证分析指南文件CLSI:EP17-A2对本发明中的试剂盒的空白限和检测限进行了研究，结果表明空白限和检测限分别为0.1 ng/mL和0.2ng/mL。
按照临床和实验室标准协会精密度验证分析指南文件CLSI:EP5-A3对本发明的试剂盒进行精密度研究，使用3个批号的试剂和校准品，对2个水平的质控品进行了检测，每天上下午各检测两次，连续考察5天，结果表明各批次室内总精密度小于5%。
检测试剂盒抗干扰能力分析：
在PRL浓度约为20 ng/mL和100 ng/mL浓度水平的两个血清样本中分别添加不同浓度的干扰物，以未添加组为对照，添加组和对照组两者相对偏差小于5%为干扰可接受标准，研究不同干扰物的最大浓度水平（不超过临床可见最高浓度）。
表3 检测试剂盒抗干扰能力分析数据
结果表明表3中的浓度时对检测结果干扰不明显。
最后应说明的是：以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (5)
1.抗泌乳素蛋白的单克隆抗体，其特征在于：单克隆抗体为PRL-Clone1，
所述PRL-Clone1的重链可变区氨基酸序列如SEQ ID NO.1所示，PRL-Clone1的轻链可变区氨基酸序列如SEQ ID NO.2所示。
2.权利要求1所述的抗泌乳素蛋白的单克隆抗体在制备诊断泌乳素升高致内分泌失调免疫诊断试剂中的应用。
3.根据权利要求2所述的抗泌乳素蛋白的单克隆抗体在制备诊断泌乳素升高致内分泌失调免疫诊断试剂中的应用，其特征在于：单克隆抗体用于PRL化学发光试剂盒。
4. PRL化学发光试剂盒，其特征在于：含有权利要求1中的PRL-Clone1。
5.根据权利要求4所述的PRL化学发光试剂盒，其特征在于：试剂盒使用PRL-Clone1作为检测抗体。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311579376.8A CN117285625B (zh) | 2023-11-24 | 2023-11-24 | 抗泌乳素蛋白的单克隆抗体及应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311579376.8A CN117285625B (zh) | 2023-11-24 | 2023-11-24 | 抗泌乳素蛋白的单克隆抗体及应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117285625A CN117285625A (zh) | 2023-12-26 |
| CN117285625B true CN117285625B (zh) | 2024-01-23 |
Family
ID=89244748
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311579376.8A Active CN117285625B (zh) | 2023-11-24 | 2023-11-24 | 抗泌乳素蛋白的单克隆抗体及应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117285625B (zh) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250244A (zh) * | 2011-07-07 | 2011-11-23 | 南京医科大学 | 人源抗人催乳素受体抗体及其应用 |
| CN102426250A (zh) * | 2011-08-31 | 2012-04-25 | 内蒙古科慧生物科技有限责任公司 | 垂体泌乳素(prl)定量测定试剂盒及其检测方法 |
| AU2013202084A1 (en) * | 2006-08-18 | 2013-04-18 | Novartis Ag | PRLR-specific antibody and uses thereof |
| CN112175925A (zh) * | 2020-10-12 | 2021-01-05 | 南京佰抗生物科技有限公司 | 一种pivka-ii抗原表位肽和抗pivka-ii单克隆抗体及其应用 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2332995A1 (en) * | 2009-12-10 | 2011-06-15 | Bayer Schering Pharma Aktiengesellschaft | Neutralizing prolactin receptor antibodies and their therapeutic use |
-
2023
- 2023-11-24 CN CN202311579376.8A patent/CN117285625B/zh active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2013202084A1 (en) * | 2006-08-18 | 2013-04-18 | Novartis Ag | PRLR-specific antibody and uses thereof |
| CN102250244A (zh) * | 2011-07-07 | 2011-11-23 | 南京医科大学 | 人源抗人催乳素受体抗体及其应用 |
| CN102426250A (zh) * | 2011-08-31 | 2012-04-25 | 内蒙古科慧生物科技有限责任公司 | 垂体泌乳素(prl)定量测定试剂盒及其检测方法 |
| CN112175925A (zh) * | 2020-10-12 | 2021-01-05 | 南京佰抗生物科技有限公司 | 一种pivka-ii抗原表位肽和抗pivka-ii单克隆抗体及其应用 |
Non-Patent Citations (2)
| Title |
|---|
| Development of a new anti-prolactin receptor (PRLR) antibody, F56, which can serve as a PRLR antagonist;Huanzhong cui 等;《International Journal of Biological Macromolecules》;第95卷;第1223-1227页 * |
| 人泌乳素单克隆抗体制备及其临床应用研究;佟志刚等;《标记免疫分析与临床》(第2期);第97页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117285625A (zh) | 2023-12-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109975536B (zh) | 抗缪勒激素胶乳增强比浊法检测试剂盒及其制备使用方法 | |
| CN109580959B (zh) | 一种检测肝素结合性表皮生长因子的elisa试剂盒 | |
| CN107523552B (zh) | 分泌抗amh单克隆抗体的杂交瘤细胞株、抗amh单克隆抗体及应用 | |
| CN111273042A (zh) | 抗缪勒氏管激素检测试剂盒 | |
| CN116874597B (zh) | 鼠抗人血管内皮生长因子的单克隆抗体及应用 | |
| CN110763839A (zh) | 一种timp-i胶乳增强比浊法检测试剂盒及其制备使用方法 | |
| CN117003878B (zh) | 抗sFLT-1蛋白的单克隆抗体及应用 | |
| CN109721651B (zh) | 一种视黄醇结合蛋白检测试剂盒及其临床应用 | |
| CN113801226A (zh) | 抗人PlGF鼠源单克隆抗体及应用 | |
| CN117285625B (zh) | 抗泌乳素蛋白的单克隆抗体及应用 | |
| CN116874596A (zh) | 抗S100β蛋白的单克隆抗体及其制备方法和应用 | |
| CN106093431B (zh) | 人降钙素原胶体金定量检测卡 | |
| CN116284338A (zh) | 一种重组人载脂蛋白ApoE及其多克隆抗体的制备方法 | |
| CN117003869B (zh) | 抗il-10蛋白的单克隆抗体组合物及应用 | |
| CN106967689B (zh) | sH2a单克隆抗体杂交瘤细胞及其单克隆抗体和应用 | |
| CN112358546B (zh) | 杂交瘤细胞株9c1、plgf-1单克隆抗体及其应用 | |
| CN117285635B (zh) | 抗肝素结合蛋白的单克隆抗体组合物及应用 | |
| CN113817686B (zh) | 一种杂交瘤细胞株及其分泌的单克隆抗体和应用 | |
| CN113956360A (zh) | 一种chi3l1单克隆抗体及其制备方法和应用 | |
| CN109725159B (zh) | 人β2-微球蛋白的定量检测试纸卡与临床应用 | |
| CN113150157A (zh) | 一种用于检测血清中vegf含量的抗体对及其用途 | |
| CN114213534B (zh) | 抗人PlGF鼠源单克隆抗体及应用 | |
| CN111273028A (zh) | rhTSG-6直接竞争ELISA法定量检测试剂盒及其使用方法和应用 | |
| CN117143242B (zh) | 抗Galectin-3蛋白的单克隆抗体组合物及应用 | |
| CN113912719B (zh) | 检测小鼠白介素6的单克隆抗体及其制备方法和应用 |
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 |