CN118063608A - Monoclonal antibody for triiodothyronine, detection reagent based on monoclonal antibody and application of monoclonal antibody - Google Patents
Monoclonal antibody for triiodothyronine, detection reagent based on monoclonal antibody and application of monoclonal antibody Download PDFInfo
- Publication number
- CN118063608A CN118063608A CN202410155456.9A CN202410155456A CN118063608A CN 118063608 A CN118063608 A CN 118063608A CN 202410155456 A CN202410155456 A CN 202410155456A CN 118063608 A CN118063608 A CN 118063608A
- Authority
- CN
- China
- Prior art keywords
- seq
- amino acid
- antibody
- variable region
- acid sequence
- 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
- 238000001514 detection method Methods 0.000 title claims abstract description 63
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 24
- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical compound IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 title claims abstract description 21
- 229940035722 triiodothyronine Drugs 0.000 title claims description 19
- 239000000427 antigen Substances 0.000 claims abstract description 117
- 102000036639 antigens Human genes 0.000 claims abstract description 117
- 108091007433 antigens Proteins 0.000 claims abstract description 117
- 230000027455 binding Effects 0.000 claims abstract description 116
- 239000012634 fragment Substances 0.000 claims abstract description 109
- 238000000034 method Methods 0.000 claims abstract description 31
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 14
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 14
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 14
- 238000002360 preparation method Methods 0.000 claims abstract description 13
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 131
- 238000012360 testing method Methods 0.000 claims description 25
- 108091033319 polynucleotide Proteins 0.000 claims description 17
- 102000040430 polynucleotide Human genes 0.000 claims description 17
- 239000002157 polynucleotide Substances 0.000 claims description 17
- 239000013598 vector Substances 0.000 claims description 11
- 238000003018 immunoassay Methods 0.000 claims description 10
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 108010047041 Complementarity Determining Regions Proteins 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000006467 substitution reaction Methods 0.000 abstract description 3
- 208000024799 Thyroid disease Diseases 0.000 abstract description 2
- 230000002860 competitive effect Effects 0.000 abstract description 2
- 238000013399 early diagnosis Methods 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 30
- 239000000523 sample Substances 0.000 description 24
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 20
- 108090000623 proteins and genes Proteins 0.000 description 17
- 239000007788 liquid Substances 0.000 description 10
- 238000005406 washing Methods 0.000 description 10
- 239000011248 coating agent Substances 0.000 description 9
- 238000000576 coating method Methods 0.000 description 9
- 238000001962 electrophoresis Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 7
- 101100022187 Caenorhabditis elegans mab-10 gene Proteins 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 108090000765 processed proteins & peptides Proteins 0.000 description 6
- 102000004196 processed proteins & peptides Human genes 0.000 description 6
- 238000007789 sealing Methods 0.000 description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 5
- 230000001900 immune effect Effects 0.000 description 5
- 238000003908 quality control method Methods 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 239000000020 Nitrocellulose Substances 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 238000003317 immunochromatography Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 229920001220 nitrocellulos Polymers 0.000 description 4
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 229920002477 rna polymer Polymers 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 101100075829 Caenorhabditis elegans mab-3 gene Proteins 0.000 description 3
- 101100476210 Caenorhabditis elegans rnt-1 gene Proteins 0.000 description 3
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 3
- 229910052782 aluminium Inorganic materials 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 239000002274 desiccant Substances 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 239000011888 foil Substances 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 210000001685 thyroid gland Anatomy 0.000 description 3
- 101100075830 Caenorhabditis elegans mab-5 gene Proteins 0.000 description 2
- 101100075831 Caenorhabditis elegans mab-7 gene Proteins 0.000 description 2
- 101100313161 Caenorhabditis elegans mab-9 gene Proteins 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000007405 data analysis Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 239000004005 microsphere Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000010188 recombinant method Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-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
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000011632 Caseins Human genes 0.000 description 1
- 108010076119 Caseins Proteins 0.000 description 1
- 244000247747 Coptis groenlandica Species 0.000 description 1
- 235000002991 Coptis groenlandica Nutrition 0.000 description 1
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 206010020850 Hyperthyroidism Diseases 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 102100038007 Low affinity immunoglobulin epsilon Fc receptor Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 229920002538 Polyethylene Glycol 20000 Polymers 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 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
- 238000002835 absorbance Methods 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 238000001261 affinity purification Methods 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
- 230000004075 alteration Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000007739 conversion coating Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 230000037029 cross reaction Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000013506 data mapping Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000005831 deiodination reaction Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 208000003532 hypothyroidism Diseases 0.000 description 1
- 230000002989 hypothyroidism Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000011587 new zealand white rabbit Methods 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 239000012562 protein A resin Substances 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 229940080237 sodium caseinate Drugs 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000005495 thyroid hormone Substances 0.000 description 1
- 229940036555 thyroid hormone Drugs 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a monoclonal antibody or antigen binding fragment thereof for triiodothyronine (T3), related nucleic acid products, cell products, detection reagents or kits thereof, and preparation methods and applications thereof. The monoclonal antibody or antigen binding fragment thereof can be combined with T3 with high specificity and high affinity, so that the detection accuracy and sensitivity of T3 are improved; in addition, the invention also develops a high-performance paired antibody suitable for double-antibody sandwich method immunodetection, thereby breaking through the limitations of low sensitivity and poor clinical relevance of the traditional competitive method detection reagent, realizing T3 double-antibody sandwich method immunodetection, and having important significance for early diagnosis and treatment of thyroid diseases and domestic substitution of core antibody raw materials.
Description
Technical Field
The invention relates to the technical field of monoclonal antibody preparation and immunological detection, in particular to a monoclonal antibody aiming at triiodothyronine, a detection reagent based on the monoclonal antibody and application of the detection reagent.
Background
Triiodothyronine (T3 for short) is a hormone secreted by thyroid gland, about 65% is produced directly by thyroid gland in human body, 35% is formed by deiodination of thyroid hormone (T4 for short) in peripheral tissue, and molecular weight is about 650Da.
T3 is an important clinical index for evaluating thyroid function, and has important clinical significance for diagnosing thyroid diseases such as hyperthyroidism, hypothyroidism and the like and detecting curative effect. A part of T3 in the body is synthesized from T4, and only one iodine difference exists between the two structures, so that the specificity requirement on the antibody is high. Currently, most of the T3 antibodies on the market have cross-reaction problems. The existing T3 immunoassay method mainly comprises a chemiluminescence method and an immunochromatography method, and the immunoassay methods mainly adopt the principle of a competition method, so that the detection specificity and sensitivity are deficient, the linear range is narrow, and the graduation of a low-value end region and a high-value end region of a clinical sample is small, so that the detection result is misjudged.
In view of the above problems in the prior art, development of a monoclonal antibody with high specificity and high affinity for T3 is currently urgently needed to meet the requirements of immunological detection, and in particular, development of a high-performance pairing antibody is particularly important to break through the limitations of the conventional competition method and realize the detection of T3 by a double-antibody sandwich method.
Disclosure of Invention
Object of the Invention
Aiming at the problems or needs in the prior art, the invention aims to provide a monoclonal antibody or an antigen binding fragment thereof with high specificity and high affinity aiming at triiodothyronine (T3 for short in English) and develop a high-performance pairing antibody suitable for double-antibody sandwich method immunological detection.
Solution scheme
In order to achieve the above purpose, the invention obtains monoclonal antibodies or antigen binding fragments thereof aiming at triiodothyronine (English abbreviated as T3) with high specificity and high affinity through a large number of screening, related nucleic acids and cell products thereof, a preparation method and application thereof, and an immunodetection reagent based on the same, thereby providing a basis for the high-specificity and high-sensitivity immunodetection of T3.
Specifically, the invention provides the following technical scheme:
In a first aspect, the present invention provides a monoclonal antibody or antigen-binding fragment thereof directed against triiodothyronine, characterized in that said monoclonal antibody or antigen-binding fragment thereof comprises:
(1) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 as HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO.6, QAS and SEQ ID NO.7, LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,
(2) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 15, LAS and SEQ ID NO. 16, LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,
(3) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 24, LAS and SEQ ID NO. 25, LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,
(4) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 28, SEQ ID NO.29 and SEQ ID NO. 30, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NOS: 33, YAS and 34, respectively, LCDR1, LCDR2 and LCDR3; or alternatively, the first and second heat exchangers may be,
(5) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 37, SEQ ID NO. 38 and SEQ ID NO. 39, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 42, SAS, and SEQ ID NO. 43, LCDR1, LCDR2, and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,
(6) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 46, SEQ ID NO. 47 and SEQ ID NO. 48, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 51, SAS and SEQ ID NO. 52 for LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,
(7) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 55, SEQ ID NO. 56 and SEQ ID NO. 57, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 60, DAS and SEQ ID NO. 61, LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,
(8) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 64, SEQ ID NO. 65 and SEQ ID NO. 66, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 69, DAS and SEQ ID NO. 70 for LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,
(9) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 73, SEQ ID NO. 74 and SEQ ID NO. 75 for HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO:78, DAS and SEQ ID NO:79, LCDR1, LCDR2 and LCDR3, respectively;
(10) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 82, SEQ ID NO. 83 and SEQ ID NO. 84 as HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 87, DAS and SEQ ID NO. 88 for LCDR1, LCDR2 and LCDR3, respectively.
Preferably, the monoclonal antibody or antigen binding fragment thereof comprises:
(1) A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO. 4; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 8; or alternatively, the first and second heat exchangers may be,
(2) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 13; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 17; or alternatively, the first and second heat exchangers may be,
(3) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 22; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 26; or alternatively, the first and second heat exchangers may be,
(4) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 31; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 35; or alternatively, the first and second heat exchangers may be,
(5) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 40; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 44; or alternatively, the first and second heat exchangers may be,
(6) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 49; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 53; or alternatively, the first and second heat exchangers may be,
(7) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 58; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 62; or alternatively, the first and second heat exchangers may be,
(8) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 67; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 71; or alternatively, the first and second heat exchangers may be,
(9) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 76; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 80; or alternatively, the first and second heat exchangers may be,
(10) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 85; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 89.
In addition, the monoclonal antibody or antigen binding fragment thereof further comprises a constant region; preferably, the constant region is any one selected from the group consisting of: constant regions of IgG, igA, or IgM antibodies.
Further preferred, the monoclonal antibody or antigen binding fragment thereof comprises:
(1) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 5; and a light chain having an amino acid sequence as shown in SEQ ID NO. 9; or alternatively, the first and second heat exchangers may be,
(2) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 14; and a light chain having an amino acid sequence as shown in SEQ ID NO. 18; or alternatively, the first and second heat exchangers may be,
(3) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 23; and a light chain having an amino acid sequence as shown in SEQ ID NO. 27; or alternatively, the first and second heat exchangers may be,
(4) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 32; and a light chain having an amino acid sequence as shown in SEQ ID NO. 36; or alternatively, the first and second heat exchangers may be,
(5) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 41; and a light chain having an amino acid sequence as shown in SEQ ID NO. 45; or alternatively, the first and second heat exchangers may be,
(6) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 50; and a light chain having an amino acid sequence as shown in SEQ ID NO. 54; or alternatively, the first and second heat exchangers may be,
(7) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 59; and a light chain having an amino acid sequence as shown in SEQ ID NO. 63; or alternatively, the first and second heat exchangers may be,
(8) A heavy chain having an amino acid sequence as set forth in SEQ ID NO. 68; and a light chain having an amino acid sequence as shown in SEQ ID NO. 72; or alternatively, the first and second heat exchangers may be,
(9) A heavy chain having an amino acid sequence as set forth in SEQ ID NO. 77; and a light chain having an amino acid sequence as shown in SEQ ID NO. 81; or alternatively, the first and second heat exchangers may be,
(10) A heavy chain having an amino acid sequence as set forth in SEQ ID NO. 86; and a light chain having an amino acid sequence as shown in SEQ ID NO. 90.
In the first aspect, the monoclonal antibodies defined in items (1) to (10) are hereinafter referred to as antibodies 1 to 10, respectively.
In some possible embodiments, the antigen binding fragment of the monoclonal antibody is selected from the group consisting of Fab, fab ', F (ab') 2, fd, fv, dAb, complementarity determining region fragments, single chain antibodies, human antibodies, chimeric antibodies, or bispecific or multispecific antibodies.
In a second aspect, the present invention provides a polynucleotide encoding a monoclonal antibody or antigen binding fragment thereof as described in the first aspect above. The polynucleotide is not limited to the method of its production and may be obtained using genetic engineering recombinant techniques or chemical synthesis methods.
In a third aspect, the present invention provides a nucleic acid construct comprising a polynucleotide as described in the second aspect above, and, optionally, at least one expression regulatory element operably linked to the polynucleotide.
In a fourth aspect, the present invention provides a recombinant vector comprising a polynucleotide as described in the second aspect above, or a nucleic acid construct as described in the third aspect above.
The vector of the present invention may be a cloning vector or an expression vector, and may be, for example, a plasmid, a cosmid, a phage, or the like.
In some preferred embodiments, the recombinant vector is a recombinant expression vector, preferably a eukaryotic expression vector.
In a fifth aspect, the present invention provides a transformed host cell into which a polynucleotide as described in the second aspect, a nucleic acid construct as described in the third aspect or a recombinant vector as described in the fourth aspect has been transformed;
such host cells include, but are not limited to: prokaryotic cells, such as E.coli cells; eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.). The host cell may also be a cell line, such as a 293T cell line.
Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.
In a sixth aspect, the present invention provides the use of a monoclonal antibody or antigen binding fragment thereof as described in the first aspect, a polynucleotide as described in the second aspect, a nucleic acid construct as described in the third aspect, a recombinant vector as described in the fourth aspect and/or a transformed host cell as described in the fifth aspect, in the manufacture of a detection reagent or kit for detecting triiodothyronine.
In a possible embodiment, the sample is a biological sample of the subject, preferably a serum or plasma sample of the subject.
In a seventh aspect, the present invention provides the use of any two monoclonal antibodies or antigen binding fragments thereof selected from the monoclonal antibodies or antigen binding fragments thereof described in the first aspect above in the preparation of a detection reagent or kit for detecting triiodothyronine.
Preferably, the two monoclonal antibodies or antigen binding fragments thereof are selected from the group consisting of:
(I) Antibody 1 or antigen-binding fragment thereof, and antibody 2 or antigen-binding fragment thereof;
(II) antibody 3 or antigen-binding fragment thereof, and antibody 4 or antigen-binding fragment thereof;
Wherein antibody 1 or an antigen-binding fragment thereof is defined in any of item (1) of the first aspect, antibody 2 or an antigen-binding fragment thereof is defined in any of item (2) of the first aspect, antibody 3 or an antigen-binding fragment thereof is defined in any of item (3) of the first aspect, and antibody 4 or an antigen-binding fragment thereof is defined in any of item (4) of the first aspect.
In an eighth aspect, the present invention provides a detection reagent for detecting triiodothyronine comprising a monoclonal antibody or antigen binding fragment thereof as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, a recombinant vector as described in the fourth aspect above and/or a transformed host cell as described in the fifth aspect above.
Preferably, the detection reagent is a double antibody sandwich immunoassay kit comprising any two monoclonal antibodies or antigen binding fragments thereof selected from the monoclonal antibodies or antigen binding fragments thereof described in the first aspect above, preferably comprising two monoclonal antibodies or antigen binding fragments thereof selected from the group consisting of:
(I) Antibody 1 or antigen-binding fragment thereof, and antibody 2 or antigen-binding fragment thereof;
(II) antibody 3 or antigen-binding fragment thereof, and antibody 4 or antigen-binding fragment thereof;
Wherein antibody 1 or an antigen-binding fragment thereof is defined in any of item (1) of the first aspect, antibody 2 or an antigen-binding fragment thereof is defined in any of item (2) of the first aspect, antibody 3 or an antigen-binding fragment thereof is defined in any of item (3) of the first aspect, and antibody 4 or an antigen-binding fragment thereof is defined in any of item (4) of the first aspect.
In a possible embodiment, the double-antibody sandwich immunoassay kit comprises an immunochromatographic test strip, which is based on the principle of double-antibody sandwich method, and takes any two monoclonal antibodies or antigen binding fragments thereof selected from the monoclonal antibodies or antigen binding fragments thereof described in the first aspect as a capture antibody and a detection antibody respectively;
In some preferred embodiments, the immunochromatographic test strip has (1) any one of antibody 1 or an antigen-binding fragment thereof and (2) antibody 2 or an antigen-binding fragment thereof as a capture antibody, and the other one as a detection antibody; further preferably, the immunochromatographic test strip uses an antibody 1 or an antigen-binding fragment thereof as a detection antibody and an antibody 2 or an antigen-binding fragment thereof as a capture antibody;
In other preferred embodiments, the immunochromatographic test strip has any one of (1) antibody 3 or an antigen-binding fragment thereof and (2) antibody 4 or an antigen-binding fragment thereof as a capture antibody, and the other one as a detection antibody; further preferably, the immunochromatographic test strip uses an antibody 3 or an antigen-binding fragment thereof as a detection antibody and an antibody 4 or an antigen-binding fragment thereof as a capture antibody;
preferably, the monoclonal antibody or antigen-binding fragment thereof as a detection antibody is labeled with a detectable label; such detectable labels are well known to those skilled in the art and include, but are not limited to, radioisotopes, fluorescent materials, luminescent materials, colored materials, enzymes (e.g., horseradish peroxidase), and the like.
In a preferred embodiment, the detectable label is a fluorescent microsphere, a latex microsphere or a colloidal gold particle, preferably a colloidal gold particle.
In a ninth aspect, the present invention provides a method of preparing a monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, the method comprising: allowing the transformed host cell of the fifth aspect described above to express the monoclonal antibody or antigen-binding fragment thereof under conditions suitable for expression of the monoclonal antibody or antigen-binding fragment thereof, and recovering the expressed monoclonal antibody or antigen-binding fragment thereof from a culture of the host cell.
In a tenth aspect, the present invention provides a method of detecting the presence or level of triiodothyronine in a sample, the method comprising using a monoclonal antibody or antigen binding fragment thereof as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above, a transformed host cell as described in the fifth aspect above and/or a detection reagent as described in the eighth aspect above.
In a possible embodiment, the sample includes, but is not limited to, serum, plasma, and the like from a subject.
The method may be used for diagnostic purposes (e.g., the sample is a sample from a patient) or for non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient).
General methods for detecting the presence or level of an antigen of interest in a sample using monoclonal antibodies or antigen binding fragments thereof are well known to those skilled in the art. In certain preferred embodiments, the detection method may use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, competition method, and the like.
Advantageous effects
The monoclonal antibody aiming at the triiodothyronine can be combined with the triiodothyronine with high affinity and high specificity (the combination activity EC50 of the monoclonal antibody reaches ng/ml level, and the KD is 10 -10M~10-11 M), so that the detection of the triiodothyronine with high accuracy and high sensitivity is realized; in addition, the invention also develops a high-performance paired antibody suitable for double-antibody sandwich method immunological detection, thereby breaking through the limitations of low sensitivity and poor clinical relevance of the traditional competitive method detection reagent, realizing the double-antibody sandwich method immunological detection of the triiodothyronine, and the detection method has higher specificity and sensitivity, and the clinical coincidence rate is more than 0.95.
Drawings
One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.
FIG. 1 is a gel diagram of SDS-PAGE detection of 10 anti-T3 monoclonal antibodies expressed and purified in example 3 of the present invention; wherein M represents the electrophoresis band of molecular weight Marker, lane 1 shows the electrophoresis band of each monoclonal antibody under reducing conditions, and lane 2 shows the electrophoresis band of each monoclonal antibody under non-reducing conditions.
FIG. 2 is a graph showing the results of detection of the binding activity of 10 anti-T3 monoclonal antibodies expressed and purified in example 3 and T3-coupled protein according to the present invention, as detected in example 4.
FIG. 3 is a schematic diagram of a colloidal gold immunochromatographic test strip prepared in example 5 of the present invention; wherein 1 is a PVC bottom plate; 2 is a sample pad; 3 is a nitrocellulose membrane; 4 is gold thread; 5 is a detection line; 6 is a quality control line; and 7 is a water absorption pad.
FIG. 4 is a graph showing the correlation between two colloidal gold immunochromatographic test strips and a Roche test reagent based on two paired antibodies of the present invention, as tested in example 6; fig. 4A is a graph showing the result of the colloidal gold immunochromatographic strip 1 based on the T3mAb1+t3mAb2 paired antibody, and fig. 4B is a graph showing the result of the colloidal gold immunochromatographic strip 2 based on the T3mAb3+t3mAb4 paired antibody.
Detailed Description
Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.
In order that the invention may be more readily understood, certain technical and scientific terms are defined as follows. Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The term "about" when used in conjunction with a numerical value is intended to encompass numerical values within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value, including but not limited to ± 5%, ±2%, ±1% and ± 0.1%, as these variations are suitable for carrying out the disclosed methods.
The term "and/or" is understood to mean any one of the selectable items or a combination of any two or more of the selectable items.
As used herein, the term "or" should be understood to have the same meaning as "and/or" as defined above. For example, when items in a list are separated, "or" and/or "should be construed as inclusive, i.e., including at least one of the list of elements or amounts, but also including more than one, and optionally, additional unlisted items. To the extent that only one term is explicitly recited, such as "only one" or "exactly one" or "consisting of" is used in the claims, it will refer to only one number listed or an element of a list.
The term "percent (%) amino acid sequence identity" or simply "identity" is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the reference amino acid sequence after aligning the amino acid sequences (and introducing gaps, if necessary) to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment may be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to obtain the maximum alignment for the full length of sequences compared.
The term "antibody" refers to any form of antibody that has the desired biological activity. Thus, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies.
The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
"Antigen binding fragment" refers to antigen binding fragments of antibodies and antibody analogs, which generally comprise at least a portion of the antigen binding or variable regions of the parent antibody, e.g., one or more CDRs. Fragments of the antibodies retain at least some of the binding specificity of the parent antibody. Antigen binding fragments include peptides selected from the group consisting of Fab, fab '-SH, fv, scFv, F (ab') 2, diabodies, CDRs comprising peptides, and the like.
"Fab fragment" consists of a light chain and a heavy chain CH1 and variable domains.
The "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic effect of the CH3 domain.
"Fab ' fragments" contain a portion of one light chain and one heavy chain comprising a portion of the VH domain, CH1 domain and constant region between the CH1 and CH2 domains, with an inter-chain disulfide bond formed between the two heavy chains of the two Fab ' fragments to form a F (ab ') 2 molecule.
"F (ab') 2 fragments" contain two light chains and portions of two heavy chains comprising portions of the VH domain, CH1 domain, and constant region between CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Thus, a F (ab ') 2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.
The "Fv region" comprises variable regions from both the heavy and light chains, but lacks constant regions.
"Single chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment comprising the VH and VL domains of an antibody, which domains are contained in a single polypeptide chain. In general, scFv polypeptides comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
A "diabody" is a small antigen-binding fragment having two antigen-binding sites. The fragments comprise a heavy chain variable domain (VH) (VH-VL or VL-VH) linked to a light chain variable domain (VL) in the same polypeptide chain. By using a linker that is so short that it is not possible to pair between two domains of the same strand, the domains pair with complementary domains of the other strand and form two antigen binding sites.
"Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the binding rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art.
The term "high affinity" for IgG antibodies refers to KD of 1.0 x 10 -9 M or less for antigen. For other antibody subtypes, "high affinity" binding may vary.
The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form.
Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.
Example 1: preparation of monoclonal antibodies against T3
(1) Animal immunization:
Taking coupled protein T3-BSA (purchased from Nanjing Ming Biotechnology Co., ltd., product No. MY-T3-003) as an antigen, fully emulsifying the antigen with an equal amount of Freund's complete adjuvant, immunizing a rabbit (variety: new Zealand white rabbit, female, 6-8 weeks old) by subcutaneous injection, performing boosting at a time interval of two weeks for 3 times, taking blood from the vein of the rabbit 5-8 days after the last boosting, detecting serum titer by ELISA method, and preparing Peripheral Blood Mononuclear Cell (PBMC) suspension when the serum titer reaches the standard of > 128K;
(2) PBMC preparation:
Adding lymphocyte separation liquid Ficoll into a centrifuge tube, taking anticoagulated peripheral blood (whole blood) and sterile PBS, fully mixing uniformly according to a ratio of 1:1-1:5, slowly superposing on a layered liquid level along a tube wall by using a pipette, horizontally centrifuging for 400g multiplied by 30 minutes, removing part of upper liquid after centrifuging, and inserting the rest about 1mL into a cloud layer by using a pipette, and sucking mononuclear cells. Placing into another centrifuge tube, adding more than 5 times of PBS, centrifuging for 300g×10min, and washing the cells twice. After the last centrifugation, the supernatant was discarded, the red blood cell lysate was added, incubated at room temperature for 2 minutes, the red blood cells were lysed, 10mLPBS was added, and the cells were washed twice by centrifugation for 300g×10 minutes. The supernatant was discarded, RPMI1640 containing 10% FBS was added, the cells were resuspended, and counted;
(3) Obtaining single B cells:
cell screening was performed using a single cell chip screening platform by using prepared PBMCs, through antigen markers, to obtain B cells with antigen specificity.
Through the above steps, 10 monoclonal antibodies against T3 were obtained in total, and identified as IgG antibodies, respectively designated as T3 mAb 1-T3 mAb10. In the following examples, the light and heavy chain gene sequences of these 10 monoclonal antibodies were detected, and expression, purification and binding activity detection were performed.
Example 2: acquisition of the light and heavy chain Gene sequences of monoclonal antibodies against T3
Preparation of cDNA:
Respectively extracting RNA from 10B cells obtained in the embodiment 1, carrying out reverse transcription by taking the RNA as a template to obtain cDNA, and amplifying genes of light chains and heavy chains of the antibody by using a PCR (polymerase chain reaction) mode;
Acquisition of light and heavy chain genes:
Amplifying by utilizing a rabbit-source specific primer, cloning the amplified DNA sequences into vectors, then carrying out conversion coating, picking cloning and extracting plasmids for sequencing to obtain light chain gene sequences and heavy chain gene sequences of 10 antibodies; deducing and obtaining the amino acid sequences of the light chain and the heavy chain of each antibody according to the detected light chain gene sequences and the heavy chain gene sequences of each antibody; specifically, the heavy chain amino acid sequences of the antibodies T3 mAb 1-T3 mAb10 are shown as SEQ ID NO. 5, 14, 23, 32, 41, 50, 59, 68, 77 and 86, and the light chain amino acid sequences are shown as SEQ ID NO. 9, 18, 27, 36, 45, 54, 63, 72, 81 and 90;
analysis of light and heavy chain variable region gene sequences:
And analyzing the VH and VL according to the sequencing result to finally obtain the amino acid sequence of the light chain variable region and the amino acid sequence of the heavy chain variable region.
The amino acid sequences of the heavy chain variable region and the light chain variable region of the 10 monoclonal antibodies are shown below, wherein the CDR regions thereof are underlined.
T3 mAb1:
VH(SEQ ID NO:4)
QSLEESGGDLVKPGASLTLTCTASGFTLSSAWICWVRQAPGKGLEWIACIVGGSSGNTYDATWAKGRFTIPKTSSTTVTLQMTSLTAADTATYFCARDADYGDYGYDLWGPGTLVTVSS;
VL(SEQ ID NO:8)
DVVMTQTPASVSEPVGGTVTIKCQANQNIYSLLAWYQQKPGQPPKLLIYQASKLASGVPSRFKGSGSGTEYTLTISDLECADAATYYCQNDYGISSYGHAFGGGTEVVVK;
T3 mAb2:
VH(SEQ ID NO:13)
QSLEESGGRLVTPGTPLTLTCTASGFSISSYYMSWVRQAPGEGLEWIAMINIGGRAYYANWALGRFTISRTSTTVDLKMTSPTTEDTATYFCVRRVGDSGLYDLWGQGTLVTVSS;
VL(SEQ ID NO:17)
DVVMTQTPASVEAAVGGTVTIKCQASQNIYSWLSWYQQKPGQPPKLLIYLASTLASGVPSRFKGSGSGTEYTLTISDLECADAATYYCQSTYGISSYGAAFGGGTEVVVK;
T3 mAb3:
VH(SEQ ID NO:22)
QSVEESGGRLVTPGTPLTLTCTVSGIDLSNYAMGWVRQAPGKGLEYIGFINTGGSAYYANWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCAKAYVGYVGDGYWVWTLWGQGTLVTVSS;
VL(SEQ ID NO:26)
DIVMTQTPASVEAAVGGTVTIKCQASQSIYSALAWYQQKPGQPPKLLIYLASTLASGVPSRFKGSGSGTQFTLTISDLECADAATYYCQSDYYGSGNTYAVAFGGGTEVVVK;
T3 mAb4:
VH(SEQ ID NO:31)
QSVEESGGRLVTPGTPLTLTCTASGFSLSSYWMIWVRQAPGKGLEWIGCIDAGGATTYYATWAKGRFTISETSTTVDLKITSPATEDTATYFCGRGYDTYDSNTFNIWGPGTLVTVSS;
VL(SEQ ID NO:35)
DVVMTQTPASVEAAVGGTVTIKCQASQSISSWLSWYQQKPGQPPKLLIYYASTLASGVPSRFKGSGSGTEYTLTISDLECADAAIYYCQTTYGISSYGAAFGGGTEVVVK;
T3 mAb5:
VH(SEQ ID NO:40)
QSVEESGGRLVTPGTPLTLTCTVSGFTISSYHMSWVRQAPGKGLEWIGYIWGGGNTDYASWAKGRFTIAKTSSTTVDLKITSPTTEDTATYFCAKAYAGYAGDGYGFDLWGQGTLVTVSS;
VL(SEQ ID NO:44)
DIVMTQTPASVEAAVGGTVTIKCQASQGIYSSLAWYQQKPGQPPKLLIYSASTLASGVPSRFKGSGSGTQFTLTISDLECADAATYYCQCDYYGSGNIYAGSFGGGTEVVVK;T3 mAb6:
VH(SEQ ID NO:49)
EQLKESGGGLVTPGGTLTLTCTVSGFTISTYHMTWVRQAPGKGLEWIGYIWGGDNTDYASWAKGRFTIARTSSSTVDLKITSPTTEDTATYFCAKAYAGYAGDGYGFDLWGQGTLVTVSS;
VL(SEQ ID NO:53)
DIVMTQTPASVEAAVGDTVTIKCQASQSIYSNLAWYQQKPGQPPKLLIYSASTLASGVPSRFKGSGSGTQFTLTISDLECADAATYYCQCDDYGSGNTYNGSFGGGTEVVVK;T3 mAb7:
VH(SEQ ID NO:58)
QSVEESGGRLVTPGTPLTLTCTASGASLSSYAMTWVRQAPGKGLEYIGIIYASSSTWYASWAKGRFTISKTSSTTVDLKITSPTTEDTATYFCARDIHTGSSYYSSTYFNLWGPGTLVTVSS;
VL(SEQ ID NO:62)
AQVLTQTPSPVSAAVGGTVTIKCQASEDIYSLLAWYQQKPGQPPKLLIYDASDLAAGVPSRFSGSGSGTEYTLTISDLECDDAATYYCQGGYYGSGDGAFGGGTEVVVK;
T3 mAb8:
VH(SEQ ID NO:67)
QSVEESGGRLVTPGTPLTLTCTASGFSLSSYAMSWVRQAPGKGLEWIGIIYGSGSTWYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARDIHTGSSYYSSTYFNLWGPGTLVTVSS;
VL(SEQ ID NO:71)
AQVLTQTPSPVSAAVGGTVTIKCQASEDIYSLLAWYQQKPGQPPKLLIYDASDLASGVPSRFSGSGSGTEYTLTISDLECDDAATYYCQGGYYGSGYGAFGGGTEVVVK;
T3 mAb9:
VH(SEQ ID NO:76)
QSVEESGGRLVTPGTPLTLTCTVSGFSLSDYAMSWVRQAPGEGLEWIGTISSGANTYYATWAKGRFTISKTSTTVDLKITRPTTEDTATYFCARELNSYYSSAWGEGYFTLWGQGTLVTVSS;
VL(SEQ ID NO:80)
AAVLTQTPSPVSASVGGTVTISCQASEDIESYLSWYQQKPGQPPKLLIYDASTLASGVPSRFKGSGSGTQFTLTISDLECADAATYYCQSAVYSSTSDAAFGGGTEVVVK;
T3 mAb10:
VH(SEQ ID NO:85)
QSVEESGGRLVTPGTPLTLTCTVSGFSLSDYAMSWVRQAPGKGLEWIGIISTGGTTYYASWAKGRFTISRTSTTVDLKITSPTTEDTATYFCARELNIYYSSSWGEGYFSLWGPGTLVTVSS;
VL(SEQ ID NO:89)
AAVLTQTPSPVSAAVGGTVTIKCQSSDIASYLSWYQQKPGQPPKQLIYDASKLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCQSAVYSSSAVNAFGGGTEVVVK.
Example 3: expression and purification of monoclonal antibodies against T3
(1) Culturing N293 suspension cells, and performing cell transfection when the density reaches (1-3) multiplied by 10 6 cells/ml and the survival rate is more than 80-90%;
(2) Light and heavy chain genes of 10 monoclonal antibodies T3 mAb 1-T3 mAb10 are synthesized according to the light and heavy chain gene sequences of the monoclonal antibodies T3 mAb 1-T3 mAb10 detected in the example 2, and the synthesized light and heavy chain genes are respectively constructed into pcDNA3.4 plasmids; then, the recombinant plasmid containing the antibody light and heavy chain genes is transfected into N293 cells under the following conditions: the mass ratio PEI/plasmid was 3:1, transfected at a ratio of 2. Mu.g plasmid/1 ml N293 cells;
(3) Collecting cell supernatant 5-7 days after transfection, performing affinity purification by using Protein A resin to obtain high-purity monoclonal antibody, replacing elution buffer solution with PBS by dialysis, and measuring antibody concentration by using Nanodrop;
next, the purity of the antibodies was checked by SDS-PAGE and HPLC as follows:
SDS-PAGE detection:
Taking 2 mug of antibody to be detected, adding a proper amount of SDS-PAGE protein loading buffer to make the total volume be 20 mug; simultaneously preparing a reducing SDS-PAGE sample and a non-reducing SDS-PAGE sample, and then loading the samples; firstly, electrophoresis is carried out for 30min at 80V, and then electrophoresis is carried out at 120V until the separation of the strips is clear; and taking down the electrophoresis gel, performing coomassie brilliant blue dyeing, removing the dyeing liquid after 15min, washing with clear water, and then adding a decolorizing liquid for decolorizing until clear bands are seen.
The results are shown in FIG. 1, FIG. 1 showing: under reducing conditions, the 10 monoclonal antibodies all have two electrophoresis bands, and the corresponding molecular weights of the two electrophoresis bands are consistent with the sizes of the respective light chains and heavy chains; under non-reducing conditions, 10 monoclonal antibodies all have a single band, and the corresponding molecular weight of the electrophoretic band is consistent with the size of the respective intact antibody; these results indicate that the 10 monoclonal antibodies are expressed accurately.
In addition, all the electrophoresis bands were clear in edge and free of bands, indicating that the purity of the monoclonal antibodies obtained by the above-described expression and purification steps was high.
Example 4: antigen binding Activity and affinity detection of monoclonal antibodies against T3
In this example, 10 monoclonal antibodies T3mAb1 to T3mAb10 expressed and purified in example 3 were each subjected to antigen activity and affinity detection.
Binding Activity and affinity assays
① Antigen coating: diluting the antigen (i.e., the aforementioned conjugated protein T3-BSA) to a suitable concentration and then adding it to an empty ELISA plate in a volume of 100. Mu.l/well; placing the ELISA plate in a refrigerator at 4 ℃ and incubating overnight;
② Closing: washing the plate for 5 times by using a plate washing machine, buckling, adding sealing liquid into the ELISA plate, placing 150 μl of sealing liquid into a 37 ℃ incubator, and sealing for 1h; washing the board for 5 times by a board washing machine, and buckling;
③ Incubating primary antibodies: diluting the antibody to be tested to a proper concentration gradient by using 1 XPBS; adding diluted antibody to be detected into an ELISA plate, wherein each hole is 100 mu l, and the reaction time is as follows: 37 ℃/1h; washing the board for 5 times by a board washing machine, and buckling;
④ Incubating a secondary antibody: the second enzyme-labeled antibody is diluted according to a proper proportion, then added into an enzyme-labeled plate, and the reaction time is 100 mu l per hole: 37 ℃/30min; washing the board for 5 times by a board washing machine, and buckling;
⑤ Color development: adding the color developing solution into the ELISA plate, wherein each hole is 100 mu l, and the reaction time is as follows: 37 degrees/15 min;
⑥ Termination and reading: adding a stop solution, wherein the volume is 100 mu l/hole; detection wavelength setting: the detection wavelength is 450nm, the reference wavelength is 620nm, and the detection result is derived into an Excel table.
⑦ Data analysis: data analysis and mapping were performed using software, EC50 was fitted, and KD was deduced.
The binding activity and affinity of these 10 monoclonal antibodies to the antigen are shown in FIG. 2 and Table 1 below.
TABLE 1
Antibody numbering | EC50(ng/ml) | KD |
T3 mAb1 | 22.33 | 1.49*10-10M |
T3 mAb2 | 20.27 | 1.35*10-10M |
T3 mAb3 | 11.76 | 7.78*10-11M |
T3 mAb4 | 12.67 | 8.45*10-11M |
T3 mAb5 | 34.45 | 2.3*10-10M |
T3 mAb6 | 20.62 | 1.37*10-10M |
T3 mAb7 | 43.12 | 2.87*10-10M |
T3 mAb8 | 34.08 | 2.27*10-10M |
T3 mAb9 | 22.75 | 1.52*10-10M |
T3 mAb10 | 23.55 | 1.57*10-10M |
Fig. 2 and table 1 show: the EC50 of the 10 monoclonal antibodies combined with antigen reaches ng/ml grade, and KD values are 10 -10M~10-11 M grade.
As mentioned above, for IgG antibodies, it is generally considered in the art that when the KD value of binding to antigen reaches 10 -9 M, it is considered to have high affinity, whereas the KD values of binding to antigen of the 10 monoclonal antibodies of the present invention reach 10 -10M~10-11 M, which is 10 to 100 times the above defined value, and thus can be derived: the monoclonal antibody of the invention has extremely strong antigen binding activity and affinity.
Example 5: preparation of colloidal gold immunochromatography test strip based on anti-T3 monoclonal antibody by double-antibody sandwich method
In this example, two colloidal gold immunochromatographic test strips for detecting 25-OH-VD according to the principle of double-antibody sandwich method are prepared by using the T3 mAb1 and the T3 mAb2, the T3 mAb3 and the T3 mAb4 expressed and purified in example 3 as paired antibodies, and the construction schematic diagram of the test strips is shown in FIG. 3; the preparation method of the test strip comprises the following steps:
(1) Preparation of Au-anti-T3 monoclonal antibody Complex
① Taking 3ml of colloidal gold particles (colloid Jin Lijing: 20nm, maximum absorption peak wavelength is 529nm, absorbance is 2.0A), adding 18 μl of 0.1M K 2CO3, mixing well, adding 10 μg of anti-T3 antibody (i.e. detection antibody), and reacting at normal temperature for 1h;
② Adding 600 μl of 5% BSA blocking solution into the ① system, and blocking at room temperature for 30min;
③ After completion of the blocking, the mixture was centrifuged at 11000rpm at 10℃for 15min, the supernatant was discarded, and the precipitate was redissolved (i.e., concentrated 60-fold, 60X) with 50. Mu.l of a gold-labeled diluent to obtain an Au-anti-T3 monoclonal antibody complex; the gold standard diluent contains 100mM Tris buffer, pH7.5-8.5, 5% sucrose, 1% trehalose and 5% BSA.
(2) Gold drawing
① Closing line is marked: taking 5% BSA solution, setting the water yield of the instrument to be 3 mu L/cm, and placing the scratched bottom plate in a 37 ℃ oven for drying for 2 hours;
② Diluting the final concentration of the Au-anti-T3 monoclonal antibody complex to 50X, setting the water yield to 2 mu L/cm, scribing at the position of a closed line, then placing a bottom plate in a 37 ℃ oven for drying for 2 hours, and then placing a marking pad in an aluminum foil bag with a desiccant added in advance at the humidity of not more than 36%, sealing and preserving for later use;
(3) Coating quality control line C and detection line T
① Preparing a coating liquid of a quality control line C and a detection line T: the goat anti-rabbit antibody is diluted into 1.0mg/mL antibody solution by 10mM PBS buffer solution with pH of 7.4; another anti-T3 monoclonal antibody (i.e., capture antibody) was taken and diluted to 1.0mg/mL in 10mM PBS buffer, ph 7.4;
② Coating: coating the coating liquid of the quality control line C and the detection line T to the positions of the quality control line and the detection line of the nitrocellulose membrane respectively, wherein the intervals between C, T lines are 8mm, and the coating amount is 1 mu L/cm; placing the coated PVC bottom plate in a baking oven at 37 ℃ for 16 hours, then placing the PVC bottom plate in an aluminum foil bag with a desiccant added in advance under the humidity of not more than 36%, and sealing and preserving for later use;
(4) Pretreatment of sample pad
Cleaning a drying net, soaking and wetting a sample pad by using sample pad pretreatment liquid, draining by rotation until no water drops drip, then drying for 4 hours in a 37 ℃ oven, and then placing the sample pad into an aluminum foil bag which is pre-added with a drying agent at the humidity of not more than 36%, sealing and preserving for later use;
The sample pad pretreatment solution contains 10mM Tris buffer with pH of 8.5, 0.1% PVP 10, 0.1% PEG20000, 0.1% Tween20 and 0.5% sodium caseinate;
(5) Assembling and cutting film materials
Taking out the water absorption pad and the treated sample pad under the humidity of not higher than 36%, cutting according to the size of 10mm multiplied by 300mm, taking out the coated PVC base plate, and assembling according to the following procedures: firstly, attaching a sample pad on a PVC bottom plate, enabling a sample pad part to press a nitrocellulose membrane for 2mm, and finally attaching a water absorption pad, wherein the water absorption pad presses the nitrocellulose membrane for 2mm; cutting the assembled PVC bottom plate into 4mm to obtain the colloidal gold immunochromatographic test strip for detecting T3.
The antibody pairing mode of the test strip 1 is as follows: t3mAb 1 (for preparing Au-antibody complex) +t3mAb 2 (for T-wire coating); the antibody pairing mode of the test strip 2 is as follows: t3mAb 3 (used to make Au-antibody complex) +t3mAb4 (used for T-wire coating).
Example 6: detection performance of colloidal gold immunochromatography test strip based on anti-T3 monoclonal antibody by double-antibody sandwich method
In this example, the two colloidal gold immunochromatographic test strips prepared in example 5 were tested for their detection performance for T3 in clinical serum samples and compared with the detection performance of conventional Roche reagents.
Specifically, for the two colloidal gold immunochromatographic test strips prepared in example 5, a clinical serum sample was diluted with a sample dilution (its composition: 10mM PBS+5% sodium dodecyl sulfate+5% disodium EDTA+0.5% guanidine isothiocyanate+0.1% procrin300, pH 7.0), reacted at room temperature for 10min, then 75. Mu.l was added, and after 15 min, detection was performed with a colloidal gold immunoassay meter, the detection values were read, and the data were processed to obtain the T3 concentration in the samples at each dilution; for the Roche detection reagent, the operation is carried out according to the flow of the specification, the serum sample is diluted and then detected, and the T3 concentration data is obtained. Then, the T3 concentration data detected by the two test strips are compared with the T3 concentration data (as a standard) detected by the Rogowski reagent, and a clinical correlation (i.e., a coincidence rate with the Rogowski detection result) curve is made.
The results are shown in FIG. 4; wherein, fig. 4A and 4B are graphs of clinical correlation results of the colloidal gold immunochromatographic test strip 1 and the test strip 2, respectively, which show that the clinical correlation of the two test strips is R 2 > 0.95 and R 2 > 0.97, respectively.
The results show that the T3 colloidal gold immunochromatographic test strip prepared based on the paired antibody provided by the invention has very high coincidence rate (R 2 is more than 0.95) with the detection result of the traditional Roche detection method, so that the T3 colloidal gold immunochromatographic test strip has very good clinical relevance.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (16)
1. A monoclonal antibody directed against triiodothyronine or an antigen binding fragment thereof, characterized in that the monoclonal antibody or antigen binding fragment thereof comprises:
(1) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 as HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO.6, QAS and SEQ ID NO.7, LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,
(2) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 15, LAS and SEQ ID NO. 16, LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,
(3) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 24, LAS and SEQ ID NO. 25, LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,
(4) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 28, SEQ ID NO.29 and SEQ ID NO. 30, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NOS: 33, YAS and 34, respectively, LCDR1, LCDR2 and LCDR3; or alternatively, the first and second heat exchangers may be,
(5) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 37, SEQ ID NO. 38 and SEQ ID NO. 39, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 42, SAS, and SEQ ID NO. 43, LCDR1, LCDR2, and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,
(6) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 46, SEQ ID NO. 47 and SEQ ID NO. 48, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 51, SAS and SEQ ID NO. 52 for LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,
(7) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 55, SEQ ID NO. 56 and SEQ ID NO. 57, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 60, DAS and SEQ ID NO. 61, LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,
(8) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 64, SEQ ID NO. 65 and SEQ ID NO. 66, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 69, DAS and SEQ ID NO. 70 for LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,
(9) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 73, SEQ ID NO. 74 and SEQ ID NO. 75 for HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO:78, DAS and SEQ ID NO:79, LCDR1, LCDR2 and LCDR3, respectively;
(10) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 82, SEQ ID NO. 83 and SEQ ID NO. 84 as HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 87, DAS and SEQ ID NO. 88 for LCDR1, LCDR2 and LCDR3, respectively.
2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof comprises:
(1) A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO. 4; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 8; or alternatively, the first and second heat exchangers may be,
(2) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 13; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 17; or alternatively, the first and second heat exchangers may be,
(3) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 22; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 26; or alternatively, the first and second heat exchangers may be,
(4) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 31; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 35; or alternatively, the first and second heat exchangers may be,
(5) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 40; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 44; or alternatively, the first and second heat exchangers may be,
(6) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 49; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 53; or alternatively, the first and second heat exchangers may be,
(7) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 58; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 62; or alternatively, the first and second heat exchangers may be,
(8) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 67; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 71; or alternatively, the first and second heat exchangers may be,
(9) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 76; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 80; or alternatively, the first and second heat exchangers may be,
(10) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 85; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 89.
3. The monoclonal antibody or antigen-binding fragment thereof according to claim 2, wherein the monoclonal antibody or antigen-binding fragment thereof comprises:
(1) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 5; and a light chain having an amino acid sequence as shown in SEQ ID NO. 9; or alternatively, the first and second heat exchangers may be,
(2) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 14; and a light chain having an amino acid sequence as shown in SEQ ID NO. 18; or alternatively, the first and second heat exchangers may be,
(3) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 23; and a light chain having an amino acid sequence as shown in SEQ ID NO. 27; or alternatively, the first and second heat exchangers may be,
(4) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 32; and a light chain having an amino acid sequence as shown in SEQ ID NO. 36; or alternatively, the first and second heat exchangers may be,
(5) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 41; and a light chain having an amino acid sequence as shown in SEQ ID NO. 45; or alternatively, the first and second heat exchangers may be,
(6) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 50; and a light chain having an amino acid sequence as shown in SEQ ID NO. 54; or alternatively, the first and second heat exchangers may be,
(7) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 59; and a light chain having an amino acid sequence as shown in SEQ ID NO. 63; or alternatively, the first and second heat exchangers may be,
(8) A heavy chain having an amino acid sequence as set forth in SEQ ID NO. 68; and a light chain having an amino acid sequence as shown in SEQ ID NO. 72; or alternatively, the first and second heat exchangers may be,
(9) A heavy chain having an amino acid sequence as set forth in SEQ ID NO. 77; and a light chain having an amino acid sequence as shown in SEQ ID NO. 81; or alternatively, the first and second heat exchangers may be,
(10) A heavy chain having an amino acid sequence as set forth in SEQ ID NO. 86; and a light chain having an amino acid sequence as shown in SEQ ID NO. 90.
4. A monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the antigen-binding fragment is selected from the group consisting of Fab, fab ', F (ab') 2, fd, fv, dAb, a complementarity determining region fragment, a single chain antibody, a human antibody, a chimeric antibody or a bispecific or multispecific antibody.
5. A polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-4.
6. A nucleic acid construct comprising the polynucleotide of claim 5, and, optionally, at least one expression regulatory element operably linked to the polynucleotide.
7. A recombinant vector comprising the polynucleotide of claim 5, or the nucleic acid construct of claim 6.
8. A transformed host cell comprising the polynucleotide of claim 5, the nucleic acid construct of claim 6, or the recombinant vector of claim 7.
9. Use of a monoclonal antibody or antigen binding fragment thereof according to any one of claims 1-4, a polynucleotide according to claim 5, a nucleic acid construct according to claim 6, a recombinant vector according to claim 7 and/or a transformed host cell according to claim 8 for the preparation of a detection reagent or kit for detecting triiodothyronine.
10. Use of any two monoclonal antibodies or antigen binding fragments thereof selected from the group consisting of the monoclonal antibodies or antigen binding fragments thereof of any one of claims 1-4 in the preparation of a detection reagent or kit for detecting triiodothyronine.
11. The use according to claim 10, wherein the two monoclonal antibodies or antigen binding fragments thereof are selected from the group consisting of:
(I) Antibody 1 or antigen-binding fragment thereof, and antibody 2 or antigen-binding fragment thereof;
(II) antibody 3 or antigen-binding fragment thereof, and antibody 4 or antigen-binding fragment thereof;
Wherein antibody 1 or an antigen binding fragment thereof is defined in claim 1 to claim 4 as item (1), antibody 2 or an antigen binding fragment thereof is defined in claim 1 to claim 4 as item (2), antibody 3 or an antigen binding fragment thereof is defined in claim 1 to claim 4 as item (3), and antibody 4 or an antigen binding fragment thereof is defined in claim 1 to claim 4 as item (4).
12. A detection reagent for detecting triiodothyronine comprising the monoclonal antibody or antigen binding fragment thereof of any one of claims 1-4, the polynucleotide of claim 5, the nucleic acid construct of claim 6, the recombinant vector of claim 7 and/or the transformed host cell of claim 8.
13. The detection reagent according to claim 12, wherein the detection reagent is a double antibody sandwich immunoassay kit comprising any two monoclonal antibodies or antigen-binding fragments thereof selected from the monoclonal antibodies or antigen-binding fragments thereof according to any one of claims 1 to 4.
14. The detection reagent of claim 13, wherein the two monoclonal antibodies or antigen-binding fragments thereof are selected from the group consisting of:
(I) Antibody 1 or antigen-binding fragment thereof, and antibody 2 or antigen-binding fragment thereof;
(II) antibody 3 or antigen-binding fragment thereof, and antibody 4 or antigen-binding fragment thereof;
Wherein antibody 1 or an antigen binding fragment thereof is defined in claim 1 to claim 4 as item (1), antibody 2 or an antigen binding fragment thereof is defined in claim 1 to claim 4 as item (2), antibody 3 or an antigen binding fragment thereof is defined in claim 1 to claim 4 as item (3), and antibody 4 or an antigen binding fragment thereof is defined in claim 1 to claim 4 as item (4).
15. The detection reagent according to claim 14, wherein the double-antibody sandwich immunoassay kit comprises a double-antibody sandwich immunochromatographic test strip in which:
Using the antibody 1 or the antigen binding fragment thereof as a detection antibody, and using the antibody 2 or the antigen binding fragment thereof as a capture antibody; or alternatively
Antibody 3 or an antigen-binding fragment thereof is used as a detection antibody, and antibody 4 or an antigen-binding fragment thereof is used as a capture antibody.
16. A method of making the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-4, the method comprising: allowing the transformed host cell of claim 8 to express the monoclonal antibody or antigen-binding fragment thereof under conditions suitable for expression of the monoclonal antibody or antigen-binding fragment thereof, and recovering the expressed monoclonal antibody or antigen-binding fragment thereof from a culture of the host cell.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2023116345449 | 2023-11-30 | ||
CN202311634544 | 2023-11-30 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN118063608A true CN118063608A (en) | 2024-05-24 |
Family
ID=91099917
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202410155456.9A Pending CN118063608A (en) | 2023-11-30 | 2024-02-02 | Monoclonal antibody for triiodothyronine, detection reagent based on monoclonal antibody and application of monoclonal antibody |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN118063608A (en) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH09107972A (en) * | 1995-10-23 | 1997-04-28 | Tosoh Corp | Gene fragment of the like of antibody recognizing thyroid hormone |
CN117024585A (en) * | 2023-08-03 | 2023-11-10 | 珠海重链生物科技有限公司 | Antithyroid globulin monoclonal antibody and application thereof |
-
2024
- 2024-02-02 CN CN202410155456.9A patent/CN118063608A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH09107972A (en) * | 1995-10-23 | 1997-04-28 | Tosoh Corp | Gene fragment of the like of antibody recognizing thyroid hormone |
CN117024585A (en) * | 2023-08-03 | 2023-11-10 | 珠海重链生物科技有限公司 | Antithyroid globulin monoclonal antibody and application thereof |
Non-Patent Citations (2)
Title |
---|
OBERDAN LEO等: "Identification of a monoclonal antibody specific for a murine T3 polypeptide", PNAS, vol. 84, 31 March 1987 (1987-03-31), pages 1374 - 1378, XP001147613, DOI: 10.1073/pnas.84.5.1374 * |
王毅;何咏婷;冯健明;刘银河;: "游离三碘甲状腺原氨酸(FT3)时间分辨荧光免疫分析试剂盒的研制", 分子诊断与治疗杂志, no. 05, 18 September 2013 (2013-09-18), pages 328 - 342 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN102482346B (en) | Antibody specific to methicillin resistant staphylococcus aureus, detection method and kit for methicillin resistant staphylococcus aureus using the same | |
CN114807054B (en) | Mouse anti-human IgG monoclonal antibody hybridoma cell strain, antibody composition and kit | |
CN116396384B (en) | Rabbit monoclonal antibody aiming at mouse adiponectin, application of rabbit monoclonal antibody and double-antibody sandwich method ELISA (enzyme-linked immunosorbent assay) kit | |
CN116120439A (en) | SARS-Cov-2 detection method and reagent kit | |
CN109734803A (en) | Anti-human MYO antibody and its application in detection kit | |
CN114181308A (en) | Procalcitonin antibody and application thereof | |
CN108196061B (en) | Double-sandwich ELISA kit for detecting human PGRN based on monoclonal antibody | |
CN105713091A (en) | Anti-human-CPR (C reactive protein) antibody and application thereof | |
CN106093431B (en) | Human calcitonin original collaurum quantitative test card | |
WO2018227643A1 (en) | Target marker gp73 for detecting steatohepatitis and detection application method | |
CN116284384A (en) | Preparation method and application of progesterone recombinant monoclonal antibody | |
CN113717285B (en) | Anti-human D-dimer antibodies and uses thereof | |
CN113933498B (en) | Double-antibody sandwich ELISA (enzyme-Linked immuno sorbent assay) method for detecting xanthan gum | |
CN118063608A (en) | Monoclonal antibody for triiodothyronine, detection reagent based on monoclonal antibody and application of monoclonal antibody | |
CN109608542A (en) | Anti-human NGAL antibody and its application in Test paper card | |
CN117229411B (en) | Monoclonal antibody specifically binding 25-hydroxy vitamin D, application and detection kit thereof | |
CN105440137B (en) | The antibody of anti-Ractopamine and its application | |
CN117903305A (en) | Monoclonal antibody against estradiol, detection reagent based on monoclonal antibody and application of monoclonal antibody | |
CN105842440A (en) | Fluorogenic quantitative detection test card for human C-reactive protein | |
CN111793136A (en) | NMP22 antibody pair and application thereof | |
CN118344485A (en) | Anti-folic acid and monoclonal antibody of binding protein complex thereof or antigen binding fragment thereof, preparation method and application thereof | |
CN104830805B (en) | Anti-human Clonorchiasis Sinensis monoclonal antibody hybridoma and its monoclonal antibody and application | |
CN109721655A (en) | Anti-human myoglobins antibody and its application in detection kit | |
CN109725151A (en) | Human muscle hemoglobin Test paper card and its clinical application | |
CN117624367B (en) | Anti-human CD141 protein rabbit monoclonal antibody and application thereof |
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 |