CN116731186A - Anti-human IgG-Fc rabbit monoclonal antibody and preparation method thereof, polynucleotide molecule, expression vector and host cell - Google Patents
Anti-human IgG-Fc rabbit monoclonal antibody and preparation method thereof, polynucleotide molecule, expression vector and host cell Download PDFInfo
- Publication number
- CN116731186A CN116731186A CN202310836660.2A CN202310836660A CN116731186A CN 116731186 A CN116731186 A CN 116731186A CN 202310836660 A CN202310836660 A CN 202310836660A CN 116731186 A CN116731186 A CN 116731186A
- Authority
- CN
- China
- Prior art keywords
- monoclonal antibody
- variable region
- heavy chain
- light chain
- chain variable
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000283973 Oryctolagus cuniculus Species 0.000 title claims abstract description 47
- 239000013604 expression vector Substances 0.000 title claims abstract description 18
- 108091033319 polynucleotide Proteins 0.000 title claims abstract description 10
- 102000040430 polynucleotide Human genes 0.000 title claims abstract description 10
- 239000002157 polynucleotide Substances 0.000 title claims abstract description 10
- 238000002360 preparation method Methods 0.000 title abstract description 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 15
- 108010047041 Complementarity Determining Regions Proteins 0.000 claims abstract description 13
- 210000004027 cell Anatomy 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 11
- 210000003719 b-lymphocyte Anatomy 0.000 claims description 10
- 239000002773 nucleotide Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 8
- 230000002163 immunogen Effects 0.000 claims description 7
- 239000000427 antigen Substances 0.000 claims description 6
- 102000036639 antigens Human genes 0.000 claims description 6
- 108091007433 antigens Proteins 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- 230000004544 DNA amplification Effects 0.000 claims description 4
- 210000004989 spleen cell Anatomy 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 3
- 230000003053 immunization Effects 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 230000001575 pathological effect Effects 0.000 abstract description 7
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 22
- 239000007788 liquid Substances 0.000 description 13
- 238000001514 detection method Methods 0.000 description 11
- 229940027941 immunoglobulin g Drugs 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 9
- 239000000872 buffer Substances 0.000 description 8
- 238000011534 incubation Methods 0.000 description 8
- 239000012188 paraffin wax Substances 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 238000005406 washing Methods 0.000 description 7
- 239000007853 buffer solution Substances 0.000 description 6
- 238000002791 soaking Methods 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 210000002741 palatine tonsil Anatomy 0.000 description 5
- 238000010166 immunofluorescence Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 229920000936 Agarose Polymers 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000006059 cover glass Substances 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 238000003119 immunoblot Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- 206010009944 Colon cancer Diseases 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 239000003995 emulsifying agent Substances 0.000 description 2
- 230000001434 glomerular Effects 0.000 description 2
- 238000012151 immunohistochemical method Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 101150075350 FL gene Proteins 0.000 description 1
- 101150063409 Fh gene Proteins 0.000 description 1
- 206010017533 Fungal infection Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108700020962 Peroxidase Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- XGZVUEUWXADBQD-UHFFFAOYSA-L lithium carbonate Chemical compound [Li+].[Li+].[O-]C([O-])=O XGZVUEUWXADBQD-UHFFFAOYSA-L 0.000 description 1
- 229910052808 lithium carbonate Inorganic materials 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000011587 new zealand white rabbit Methods 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 210000004180 plasmocyte Anatomy 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of genetic engineering, and discloses an anti-human IgG-Fc rabbit monoclonal antibody, a preparation method thereof, a polynucleotide molecule, an expression vector and a host cell, wherein the monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprises a heavy chain variable region, and the light chain comprises a light chain variable region; the heavy chain variable region comprises 3 heavy chain complementarity determining regions, and the amino acid sequences of the heavy chain complementarity determining regions are shown in SEQ ID NO. 8-10; the light chain variable region comprises 3 light chain complementarity determining regions, the amino acid sequences of which are shown in SE Q ID No. 3-5. The monoclonal antibody can simultaneously meet the requirements of flow type and pathological application, and has the advantages of high specificity, good affinity and good stability.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an anti-human IgG-Fc rabbit monoclonal antibody, a preparation method thereof, a polynucleotide molecule, an expression vector and a host cell.
Background
Immunoglobulin G (IgG) is an antibody produced and released by plasma B cells, and IgG is synthesized mainly by plasma cells in the spleen, lymph nodes and bone marrow. Human IgG is divided into four subclasses, igG1, igG2, igG3 and IgG4, with IgG1 being the most abundant subclass of IgG in human serum.
IgG is the major antibody type of blood and extracellular fluid that can control infection of human tissues. Antibodies can prevent bacterial, fungal and viral infections by binding to a variety of pathogens, such as viruses, bacteria and fungi. Maternal IgG is delivered to the fetus via the placenta, a process critical for neonatal immune defence infections. In some cases, the detection of IgG serves as a diagnostic tool. Clinically, the determination of IgG levels is generally considered to be indicative of the immune status of the body to a particular pathogen. The IgG protein expression abnormality has close correlation with glomerular inflammation, and the proposal of the correlation provides a new approach for early warning and diagnosis of glomerular inflammation and some immune diseases.
At present, most commercial IgG-Fc antibodies mainly meet the requirements of WB, ELIS A, IHC, WB and the like in detection application, few antibodies simultaneously meet the requirements of streaming and pathological application, and most of the existing monoclonal antibodies and polyclonal antibodies have low specificity. Therefore, the development of the humanized rabbit monoclonal antibody with high specificity and high affinity, which can simultaneously meet the requirements of streaming and pathological application, has very important clinical significance.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides an anti-human IgG-Fc rabbit monoclonal antibody, a preparation method thereof, a polynucleotide molecule, an expression vector and a host cell, wherein the monoclonal antibody can simultaneously meet the requirements of flow type and pathological application, and has the advantages of high specificity, good affinity and good stability.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the present invention provides an anti-human IgG-Fc rabbit monoclonal antibody comprising a heavy chain variable region and a light chain comprising a light chain variable region;
the heavy chain variable region comprises 3 heavy chain complementarity determining regions, the amino acid sequences of which are shown in SE Q ID No. 8-10;
the light chain variable region comprises 3 light chain complementarity determining regions, the amino acid sequences of which are shown in SE Q ID No. 3-5.
In the above technical scheme, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2.
In the technical scheme, the amino acid sequence of the heavy chain is shown as SEQ ID NO.6, and the amino acid sequence of the light chain is shown as SEQ ID NO. 1.
In the above technical scheme, the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO.13, and the nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 12.
In the above technical scheme, the nucleotide sequence of the heavy chain is shown as SEQ ID NO.14, and the nucleotide sequence of the light chain is shown as SEQ ID NO. 11.
The invention also provides a polynucleotide molecule which codes for the monoclonal antibody.
The invention also provides an expression vector comprising the polynucleotide molecule.
The invention also provides a host cell transformed with the expression vector.
The invention also provides a preparation method of the anti-human IgG-Fc rabbit monoclonal antibody, which comprises the following steps: the method comprises the steps of taking human IgG-Fc as an immunogen, immunizing a rabbit, separating single antigen specific B lymphocyte from spleen cells, culturing, extracting gene amplification products of a heavy chain variable region and a light chain variable region corresponding to the monoclonal antibody through specific primers, constructing an expression vector, transfecting host cells, obtaining a supernatant containing the monoclonal antibody, and purifying to obtain the monoclonal antibody.
The invention has the beneficial effects that: the anti-human IgG-Fc rabbit monoclonal antibody can identify a cell or tissue sample expressing human IgG-Fc, can simultaneously meet the requirements of flow and pathological application, and has the advantages of high specificity, good affinity and good stability.
Drawings
FIG. 1 is a diagram showing the purification of IgG1 protein detected by SDS-PAGE gel;
FIG. 2 is a schematic diagram of construction of an expression vector containing a heavy chain constant region of a rabbit monoclonal antibody;
FIG. 3 is a schematic diagram of construction of an expression vector containing a rabbit monoclonal antibody light chain constant region;
FIG. 4 shows IgG1 results of experimental rabbit monoclonal antibodies in a sample of cell lysate by immunoblotting;
FIG. 5 shows the detection of IgG1 expression of an experimental rabbit monoclonal antibody in a cell sample by immunofluorescence;
FIG. 6 shows the detection of IgG1 expression in human tonsil tissue by immunofluorescence of experimental rabbit monoclonal antibodies;
FIG. 7 shows the detection of IgG1 expression of an experimental rabbit monoclonal antibody by flow cytometry in a positive cell sample;
FIG. 8 shows the detection of IgG1 expression of an experimental rabbit monoclonal antibody by flow cytometry in 293F cells of a negative sample;
FIG. 9 shows IgG1 expression of experimental rabbit monoclonal antibodies by immunohistochemistry in human colon cancer and tonsil tissue samples;
FIG. 10 shows the detection of IgG1 expression in human brain tissue samples by immunohistochemical method using the experimental rabbit monoclonal antibodies.
Detailed Description
The invention will be further described with reference to specific examples for better illustrating the objects, technical solutions and advantages of the invention. This invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art, and the present invention will only be defined by the appended claims.
The invention provides an anti-human IgG-Fc rabbit monoclonal antibody, named rabbit monoclonal antibody 3E3. The amino acid sequence is as follows:
3E 3-full-length light chain (FL) sequence:
MDTRAPTQLLGLLLLWLPGATFADIVMTQTPSSVSAAVGGTVTINCQASESIANNLAWYQHKPGQRPKLLVFYASTLAPGVPSRFKGSGSGTEFTLTISDLECADAATYYCQSYYYSPTQSYGNTFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQ ID NO.1)
3E 3-light chain variable region (VL) sequence:
ADIVMTQTPSSVSAAVGGTVTINCQASESIANNLAWYQHKPGQRPKLLVF YASTLAPGVPSRFKGSGSGTEFTLTISDLECADAATYYCQSYYYSPTQSYGNTF GGGTEVVVK(SEQ ID NO.2)
3E 3-light chain complementarity determining region (LCDR 1) sequence:
ESIANNLAW(SEQ ID NO.3)
3E 3-light chain complementarity determining region (LCDR 2) sequence:
LVFYASTLAPGV(SEQ ID NO.4)
3E 3-light chain complementarity determining region (LCDR 3) sequence:
QSYYYSPTQSYGNTF(SEQ ID NO.5)
3E 3-heavy chain full-length (FH) sequence:
METGLRWLLLVAVLKGVQCQSLEESGGRLVTPAGSLTLTCTVSGIDLRSYAVAWVRQAPGKGLEWIGMIDNVANTYYATWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCARGSGLDYKYYSIWGPGTLVTVSLGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO.6)
3E 3-heavy chain variable region (VH) sequence:
QSLEESGGRLVTPAGSLTLTCTVSGIDLRSYAVAWVRQAPGKGLEWIGMI DNVANTYYATWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCARGSGLDYKY YSIWGPGTLVTVSL(SEQ ID NO.7)
3E 3-heavy chain complementarity determining region (HCDR 1) sequence:
IDLRSYAVA(SEQ ID NO.8)
3E 3-heavy chain complementarity determining region (HCDR 2) sequence:
IGMIDNVANTYYATWAK(SEQ ID NO.9)
3E 3-heavy chain complementarity determining region (HCDR 3) sequence:
YFCARGSGLDYKYYSI(SEQ ID NO.10)
the invention also provides a preparation method of the anti-human IgG-Fc rabbit monoclonal antibody, which comprises the following steps: the specific recombinant human IgG-Fc is used as an immunogen to immunize rabbits, single antigen specific B lymphocyte culture is separated from spleen cells, gene amplification products of a heavy chain variable region and a light chain variable region corresponding to the antibodies are extracted through specific primers, the gene amplification products are further constructed on an expression vector, cells and other hosts are transfected, and supernatant containing rabbit monoclonal antibody 3E3 is obtained, and the antibody is purified.
The preparation and application of the rabbit monoclonal antibody 3E3 are exemplified below.
Test example 1
The concentration of immunogen (human IgG-Fc) used for preparing the anti-human IgG-Fc rabbit monoclonal antibody is 1.52mg/ml, the purity is more than 95 percent (see figure 1), and the New Zealand white rabbits are immunized. Each white rabbit is immunized by 200 mug, the immunogen is mixed with the equal amount of complete Freund's adjuvant for the first time to prepare an emulsifying agent, 100 mug of the immunogen is mixed with the equal amount of incomplete Freund's adjuvant for the subcutaneous multipoint injection on the abdomen and back at intervals of 3 weeks to prepare the emulsifying agent, the subcutaneous multipoint injection on the abdomen and back is enhanced, the serum titer is measured by ELISA method after four times of immunization, the serum is diluted by 1:243K and the titer is measured by ELISA, the rabbit with OD450nm of more than 0.2 is taken, the subcutaneous multipoint injection of 200 mug of the immunogen is used for enhancing the immunity once, and the spleen is taken three to four days later.
B-lymphocyte sorting is carried out by a method disclosed in patent 201910125091.4, namely a method for efficiently separating single antigen-specific B-lymphocytes from spleen cells, so as to obtain the B-cells. For the single B cells obtained, the method of patent CN202010398174.3, an in vitro B lymphocyte culture system and application, was used for culturing.
Cloning of the genes encoding the rabbit monoclonal antibodies: the cultured B cell supernatants were used to identify positive clones by antigen coated ELISA. Cell collection of Positive clones after lysis according to ZYMO Quick-RNA TM MicroPrep kit (cat# R1051) instructions for extracting RNA and reverse transcription into cDNA, the natural paired rabbit monoclonal antibody light and heavy chain variable region genes (VH and VL) were amplified from the cDNA of the corresponding positive clone by PCR method, and the sequence was determined by sequencing.
Wherein the nucleic acid sequence of the 3E3 monoclonal antibody is as follows:
3E 3-light chain FL Gene sequence:
ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTACTTCTTCTTTGGCTCCCCGGTGCTACTTTTGCCGATATTGTTATGACACAGACTCCCAGTTCTGTGTCCGCAGCCGTCGGGGGAACCGTGACTATCAATTGTCAGGCCAGCGAGAGCATAGCCAATAACCTAGCTTGGTATCAACACAAGCCAGGTCAGCGCCCAAAACTACTGGTGTTCTACGCAAGTACACTTGCTCCTGGGGTGCCTAGCCGCTTTAAGGGATCAGGCAGCGGAACTGAGTTCACCCTCACAATTTCAGATCTGGAATGTGCCGATGCGGCCACCTATTACTGTCAGTCATATTACTACTCTCCTACCCAGTCTTATGGAAACACCTTCGGCGGCGGTACAGAGGTTGTTGTGAAGGGCGACCCGGTGGCGCCCACTGTATTGATCTTTCCACCCGCCGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG(SEQ ID NO.11)
3E 3-light chain variable region VL Gene sequence:
GCCGATATTGTTATGACACAGACTCCCAGTTCTGTGTCCGCAGCCGTCGGGGGAACCGTGACTATCAATTGTCAGGCCAGCGAGAGCATAGCCAATAACCTAGCTTGGTATCAACACAAGCCAGGTCAGCGCCCAAAACTACTGGTGTTCTACGCAAGTACACTTGCTCCTGGGGTGCCTAGCCGCTTTAAGGGATCAGGCAGCGGAACTGAGTTCACCCTCACAATTTCAGATCTGGAATGTGCCGATGCGGCCACCTATTACTGTCAGTCATATTACTACTCTCCTACCCAGTCTTATGGAAACACCTTCGGCGGCGGTACAGAGGTTGTTGTGAAG(SEQ ID NO.12)
3E 3-heavy chain variable region VH Gene sequence:
CAGAGCTTGGAGGAGAGCGGCGGTAGACTGGTTACCCCCGCCGGAAGTCTAACCCTCACCTGCACCGTGTCTGGGATCGATCTGAGGTCTTATGCTGTTGCCTGGGTGCGGCAGGCTCCTGGCAAAGGCCTGGAGTGGATCGGCATGATAGACAACGTAGCCAACACCTATTACGCCACTTGGGCAAAAGGGCGGTTTACAATCAGCAAGACATCCAGCACTACTGTGGATCTGAAGATGACAAGTCTCACCACTGAGGACACCGCCACTTACTTTTGTGCACGTGGCTCTGGGCTTGATTACAAATATTACTCTATCTGGGGCCCCGGTACTTTGGTGACCGTCAGCCTC(SEQ ID NO.13)
3E 3-heavy chain FH Gene sequence:
ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTCCTGAAAGGGGTCCAATGCCAGAGCTTGGAGGAGAGCGGCGGTAGACTGGTTACCCCCGCCGGAAGTCTAACCCTCACCTGCACCGTGTCTGGGATCGATCTGAGGTCTTATGCTGTTGCCTGGGTGCGGCAGGCTCCTGGCAAAGGCCTGGAGTGGATCGGCATGATAGACAACGTAGCCAACACCTATTACGCCACTTGGGCAAAAGGGCGGTTTACAATCAGCAAGACATCCAGCACTACTGTGGATCTGAAGATGACAAGTCTCACCACTGAGGACACCGCCACTTACTTTTGTGCACGTGGCTCTGGGCTTGATTACAAATATTACTCTATCTGGGGCCCCGGTACTTTGGTGACCGTCAGCCTCGGTCAGCCAAAAGCACCGTCCGTGTTTCCACTGGCACCGTGCTGCGGGGATACTCCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCATGTGCCCACCCCCTGAACTCCCGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGACCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATAA(SEQ ID NO.14)
test example 2
The test example provides a method for producing and purifying a rabbit monoclonal antibody, comprising the following steps:
constructing an expression vector:
the mammalian expression vectors used are shown in FIGS. 2-3, wherein pBR322Ori and f1Ori are replication promoters in E.Coli, ampcilin is a plasmid resistance gene, CMV promoter is a promoter in eukaryotes, sr40_PA_terminator is a tailing signal, heavy chain constant (see FIG. 2) and Light chain constant (see FIG. 3) are rabbit heavy chain and light chain constant region sequences, respectively.
Mammalian cell expression female plasmids containing the heavy chain constant region (see FIG. 2) and the light chain constant region (see FIG. 3) of the rabbit monoclonal antibodies were routinely linearized with NheI and XbaI restriction enzymes, respectively.
Amplifying the obtained variable region (VH and VL) genes of the light and heavy chains of the rabbit monoclonal antibody by using PCR primers with homologous sequences at the tail parts consistent with the vector; the PCR reaction system is as follows: 3min at 95 ℃, then 25 cyclic reactions are carried out according to the following conditions, wherein the temperature is 95 ℃ 1min,56 ℃ 1min and 68 ℃ 0.5min; finally, the temperature is 95 ℃ for 10min.
Wherein, the amplification primer sequence of the heavy chain gene is as follows:
5’-tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG-3’；
5’-gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG-3’。
the amplification primer sequences of the light chain genes are as follows:
5’-tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC-3’；
5’-cacacacacgatggtgactgTTCCAGTTGCCACCTGATCAG-3’。
after the amplified PCR product is purified, the heavy chain variable region gene and the light chain variable region gene are respectively constructed into corresponding mammal expression vectors by adopting a homologous recombination mode.
After sequencing verification, the expression plasmid containing the light chain gene and the heavy chain gene of the corresponding rabbit monoclonal antibody is transfected into 293 cells together. After 72-96 hours of transfection, cells were removed by centrifugation, and the culture supernatant was obtained.
Recombinant anti-human IgG-Fc rabbit monoclonal antibodies were purified from the culture supernatant following transfection using protein a affinity gel resin. The experimental steps of the affinity chromatography are as follows: the culture supernatant was transferred to a sterile 50ml centrifuge tube, centrifuged at 1000g, at 4℃or at room temperature for 10 minutes, and the supernatant was collected. The pretreated Protein A Agarose (brand: tiandi manhe; product number: SA 023100) suspension was added to the centrifuged cell supernatant and incubated at room temperature for 3-4 hours or overnight at 4 ℃. After incubation, 1000g was centrifuged for 10min, the Protein A Agarose suspension was transferred to an adsorption column, and centrifuged at room temperature for 1min with a palm centrifuge to separate the solid from the liquid and collect the flow-through. Ten times Protein A Agarose volumes of wash buffer (phosphate buffer pH 7.0) was added and the particles resuspended, centrifuged in a centrifuge, and the wash solution was collected and washed twice more. Adding elution buffer (citrate buffer pH 3.0) into adsorption column, centrifuging with centrifuge to obtain antibody supernatant, placing the antibody supernatant into dialysis bag (Soy Bao; YA 1070), dialyzing overnight, and collecting antibody. The purity of the antibody is more than 90%, the qualified antibody is packaged, and the antibody is preserved at a low temperature of-20 ℃ for standby.
Test example 3
Preliminary identification of antibody affinity was performed on the monoclonal antibody obtained in test example 2, and the affinity of the obtained antibody was preliminarily determined using 293, daudi, and human tonsil samples, and the test was performed by immunoblotting and immunofluorescence detection, and the detection data are shown in fig. 4, fig. 5, and fig. 6: FIG. 4 immunoblotting shows that anti-human IgG1 rabbit monoclonal antibody 3E3 (0.2 mg/ml1:4000 dilution) had a clear and clean band of interest at 52KD position on the 293 extract; FIG. 5 FIG. 6 immunofluorescence assay, anti-human IgG1 rabbit monoclonal antibody 3E3 (0.2 mg/ml1:100 dilution) detected the protein of interest on 93F-IgG (FC) over-expressed, daudi, human tonsil positive samples, and not on 293F untransfected cells; the WB has a band of interest and detection of the protein of interest by IF suggests that anti-human IgG1 rabbit monoclonal antibody 3E3 recognizes human IgG protein and no non-specific recognition.
Test example 4
For the rabbit monock obtained in test example 2Detection of the monoclonal antibody by flow cytometry method, measurement of the obtained antibody by flow application using a CytoFLEX S flow analyser of Beckman Coulter, the experiment was performed by a CytoFLEX S apparatus using a fluorescent secondary antibody as Alexa647-conjugated goat anti-rabit pAb, the method steps are:
1) Cultured Daudi, 293F-IgG and 293F cells were collected in 50ml centrifuge tubes and counted; centrifuging at 400g for 5min, and removing supernatant.
2) The collected cells were resuspended in 1ml of 0.5% BSA/PBS, centrifuged for 3min at 400g, repeated 2 times, and the cells were resuspended to 500ul in 0.5% BSA/PBS. Mu.l of cell suspension was added to each well of a 96-well plate.
3) 200 mu l Intracellular Fixation buffer was added to each well, and the reaction was carried out at room temperature under dark conditions for 30 minutes.
4) 400g was centrifuged for 5min, the supernatant was discarded and washed twice with 1. 1x permeablization buffer.
5) Incubation resistance: 100. Mu.L of primary antibody (2 ug/ml) diluted with permeablization buffer was added and mixed by resuspension; shake in dark and react at room temperature for 30min.
6) 400g was centrifuged for 5min, the supernatant was discarded and washed twice with 1. 1x permeablization buffer.
7) Secondary antibody incubation: the diluted fluorescent secondary antibodies were dispensed into 96-well V-plates at 100uL per well, the cells in each well were resuspended, and mixed and incubated at room temperature for 30min in the dark.
8) Wash 2 times with 1X Permeablization buffer.
9) Each well of cells was resuspended with 200uL 1X Permeablization buffer and stored in the dark.
10 Analysis: analysis was performed using a flow cytometer.
11 See fig. 7 and 8 for experimental data: the anti-human IgG1 rabbit monoclonal antibody 3E3 (2 ug/ml) has no transition signal on a negative sample 293F, and has 1.5 orders of magnitude transition signals on positive samples Daudi and 293F-IgG transient cells, so that the anti-human IgG1 rabbit monoclonal antibody 3E3 can be seen to meet the requirements of flow detection, can specifically identify the anti-human IgG1 protein without non-specific identification, and can effectively avoid false positive results.
Test example 5
The rabbit monoclonal antibody obtained in test example 2 was detected by immunohistochemical method, and the obtained antibody was subjected to pathological application measurement by an optical microscope in this experiment. The experimental method comprises the following steps:
1) Sample preparation, baking of the slices: placing paraffin slices on a slice rack in the same direction, and placing the paraffin slices into a constant temperature box at 56 ℃ to bake the slices for 30min; simultaneously placing the dewaxing liquid 1 cylinders into an incubator at 56 ℃; dewaxing to water: placing paraffin slices and a slice frame into a dewaxing liquid 1 jar, taking out the paraffin slices and the slice frame from an incubator and placing the paraffin slices and the slice frame into a normal temperature dewaxing liquid 2 jar, taking out the paraffin slices and immersing the paraffin slices into the dewaxing liquid 2 jar, and sequentially placing paraffin slices into the dewaxing liquid 2, the dewaxing liquid 3, the absolute ethyl alcohol 1, the absolute ethyl alcohol 2 and the absolute ethyl alcohol 3 according to the sequence of the dewaxing liquid 2, the dewaxing liquid reagent jar for 5min each, and the absolute ethyl alcohol reagent jar for 3min each; washing with running water.
2) Antigen retrieval: the 0.01M sodium citrate repair solution (pH 6.0) was autoclaved.
3) Inactivation of endogenous peroxidases: immersing and washing for 3 times by using PBS buffer solution for 1min each time, and removing the buffer solution on the slice; the sections were completely immersed in 3% hydrogen peroxide solution and incubated at room temperature for 10min.
4) Closing: immersing and washing for 3 times by using PBS buffer solution for 3min each time, and removing the buffer solution on the slice; an immunohistochemical water pen is used for delineating a tissue region to be detected on a slide, and a blocking liquid-PBS blocking liquid is dripped into the delineating region; the sections were placed horizontally in an incubation wet box with water at the bottom, incubated at room temperature for 30min, and counted from the addition of the blocking solution.
5) Incubation resistance: removing the sealing liquid, dripping a primary antibody diluted by an antibody diluent-PBS working liquid on the tissue slice, horizontally placing the tissue slice in an incubation wet box, and incubating for 60min at normal temperature; removing antibody working solution, quickly rinsing with PBS buffer solution for 1 time, soaking and washing with the buffer solution PBS for 3 times, and 3 minutes each time; the soaking and washing period needs to be repeatedly lifted up and down for a plurality of times (the primary anti-dilution ratio is 1:5000).
6) Secondary antibody incubation: dripping a ready-to-use secondary antibody working solution (agent bottle A) on a tissue slice, horizontally placing the tissue slice in an incubation wet box, and incubating for 25min at normal temperature; removing reagents on the sections, quickly rinsing the sections for 1 time by using buffer PBS, soaking and washing the sections for 3 times by using the buffer PBS for 3 minutes each time; the soaking and washing period needs to be repeatedly lifted up and down for a plurality of times.
7) Color development: dropwise adding a color development liquid working solution on a tissue slice, closely observing the color change condition under a microscope, and obtaining proper dyeing intensity; immersing the slices in a large amount of distilled water to terminate the color development; after the development was terminated, the sections were washed in running water for 10 minutes.
8) Counterstaining: the slightly drained sections were counterstained in Mayer's hematoxylin for 1min, after counterstaining was completed, washed with running water for 3min.
9) Returning blue: the slightly drained slices were immersed in a saturated aqueous solution of lithium carbonate for bluing for 3s and washed with running water for 3min.
10 Dewatering: soaking the cleaned slice in absolute ethyl alcohol for 1 time, lifting up and down for several times during the soaking period, and taking out after timing for 10 seconds; and the mixture is placed in a constant temperature blast drying box to be completely dried at a high temperature (54-58 ℃).
11 Sealing plate): and (3) dripping a proper amount of neutral gum into the center of the slice, covering a cover glass, wherein the proper amount of gum is needed, and the cover glass is needed to cover tissues completely after the cover glass is added, so that no gum overflows.
12 Slice scanning; the experimental data are shown in fig. 9 and 10: the anti-Human IgG1 rabbit monoclonal antibody 3E3 (diluted with 0.2mg/ml 1:5000) is significantly more colored on positive samples Human colon carcinoma and Human tonsil tissues than negative samples Human brain, so that the anti-Human IgG-Fc rabbit monoclonal antibody 3E3 can be seen to meet pathological application, and has high affinity and good specificity.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (9)
1. An anti-human IgG-Fc rabbit monoclonal antibody, characterized by: the monoclonal antibodies comprise a heavy chain comprising a heavy chain variable region and a light chain comprising a light chain variable region;
the heavy chain variable region comprises 3 heavy chain complementarity determining regions, the amino acid sequences of which are shown in SE Q ID No. 8-10;
the light chain variable region comprises 3 light chain complementarity determining regions, the amino acid sequences of which are shown in SE Q ID No. 3-5.
2. The monoclonal antibody of claim 1, wherein: the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2.
3. The monoclonal antibody of claim 1, wherein: the amino acid sequence of the heavy chain is shown as SEQ ID NO.6, and the amino acid sequence of the light chain is shown as SEQ ID NO. 1.
4. The monoclonal antibody of claim 1, wherein: the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO.13, and the nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 12.
5. The monoclonal antibody of claim 1, wherein: the nucleotide sequence of the heavy chain is shown as SEQ ID NO.14, and the nucleotide sequence of the light chain is shown as SEQ ID NO. 11.
6. A polynucleotide molecule characterized by: the polynucleotide molecule encodes the monoclonal antibody of any one of claims 1-5.
7. An expression vector, characterized in that; the expression vector comprises the polynucleotide molecule of claim 6.
8. A host cell, characterized in that: the host cell is transformed with the expression vector of claim 7.
9. The method for producing a monoclonal antibody according to claim 1, characterized in that: the method comprises the following steps: the method comprises the steps of taking human IgG-Fc as an immunogen, immunizing a rabbit, separating single antigen specific B lymphocyte from spleen cells, culturing, extracting gene amplification products of a heavy chain variable region and a light chain variable region corresponding to the monoclonal antibody through specific primers, constructing an expression vector, transfecting host cells, obtaining a supernatant containing the monoclonal antibody, and purifying to obtain the monoclonal antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310836660.2A CN116731186B (en) | 2023-07-07 | 2023-07-07 | Anti-human IgG-Fc rabbit monoclonal antibody and preparation method thereof, polynucleotide molecule, expression vector and host cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310836660.2A CN116731186B (en) | 2023-07-07 | 2023-07-07 | Anti-human IgG-Fc rabbit monoclonal antibody and preparation method thereof, polynucleotide molecule, expression vector and host cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116731186A true CN116731186A (en) | 2023-09-12 |
| CN116731186B CN116731186B (en) | 2023-12-26 |
Family
ID=87918570
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310836660.2A Active CN116731186B (en) | 2023-07-07 | 2023-07-07 | Anti-human IgG-Fc rabbit monoclonal antibody and preparation method thereof, polynucleotide molecule, expression vector and host cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116731186B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5215913A (en) * | 1987-11-30 | 1993-06-01 | Roger Williams General Hospital | IgG-1 human monoclonal antibody reactive with an HIV-1 antigen and methods of use |
| US20030175273A1 (en) * | 2001-08-31 | 2003-09-18 | Kenya Shitara | Human CDR-grafted antibody and antibody fragment thereof |
| US20080248042A1 (en) * | 2007-04-05 | 2008-10-09 | Irccs Centro Di Riferimento Oncologico Di Aviano, | Monoclonal antibody cross-reactive against infective agent causing a B-cell expansion and IgG-Fc |
-
2023
- 2023-07-07 CN CN202310836660.2A patent/CN116731186B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5215913A (en) * | 1987-11-30 | 1993-06-01 | Roger Williams General Hospital | IgG-1 human monoclonal antibody reactive with an HIV-1 antigen and methods of use |
| US20030175273A1 (en) * | 2001-08-31 | 2003-09-18 | Kenya Shitara | Human CDR-grafted antibody and antibody fragment thereof |
| US20080248042A1 (en) * | 2007-04-05 | 2008-10-09 | Irccs Centro Di Riferimento Oncologico Di Aviano, | Monoclonal antibody cross-reactive against infective agent causing a B-cell expansion and IgG-Fc |
Non-Patent Citations (2)
| Title |
|---|
| HONGCHENG LIU等: "Impact of IgG Fc-Oligosaccharides on Recombinant Monoclonal Antibody Structure, Stability, Safety, and Efficacy", 《BIOTECHNOLOGY PROGRESS》, vol. 33, no. 5, pages 1173 - 1181, XP055617008, DOI: 10.1002/btpr.2498 * |
| 汪伟立: "结核分枝杆菌免疫球蛋白G与M抗体联合检测诊断肺结核的价值", 《医疗装备》, vol. 34, no. 04, pages 38 - 39 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116731186B (en) | 2023-12-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN117074674B (en) | Colloidal gold test paper for detecting N-acetyl-beta-D glucosaminidase and preparation method thereof | |
| CN117069849B (en) | Monoclonal antibody for recognizing NAG and application thereof | |
| CN116143921B (en) | Rabbit monoclonal antibody aiming at human CD31, and preparation method and application thereof | |
| CN116731181B (en) | Anti-human CD10 protein rabbit monoclonal antibody and application thereof | |
| CN109082413B (en) | Anti-human IgG monoclonal antibody, hybridoma cell strain and application thereof | |
| CN109112113B (en) | Anti-human IgG monoclonal antibody, hybridoma cell strain, kit and application thereof | |
| CN109112114B (en) | Anti-human IgG monoclonal antibody, hybridoma cell strain and application thereof | |
| CN108802368B (en) | Kit for enzyme-linked immunosorbent assay of bovine chlamydia abortus | |
| CN115112881A (en) | Immunomagnetic bead and preparation method and application thereof | |
| CN116462758B (en) | Anti-human PTPRZ1 monoclonal antibody and application thereof in cell flow | |
| CN109280644B (en) | Anti-human IgG monoclonal antibody, hybridoma cell strain and application thereof | |
| CN116731186B (en) | Anti-human IgG-Fc rabbit monoclonal antibody and preparation method thereof, polynucleotide molecule, expression vector and host cell | |
| KR20120132227A (en) | Monoclonal antibody for detecting multiple type Foot and Mouth Disease Virus and method for detecting Foot and Mouth Disease Virus using the same | |
| CN109266620B (en) | Anti-human IgG monoclonal antibody, hybridoma cell strain and application thereof | |
| CN113969265A (en) | Hybridoma cell strain secreting IgG1 type anti-human complement C3d monoclonal antibody and application | |
| CN114560940A (en) | anti-SIRP alpha rabbit recombinant monoclonal antibody and preparation method and application thereof | |
| CN117024578B (en) | IgG antibody for capturing HBV core particles and application thereof | |
| CN116769040B (en) | Mouse anti-GDH hybridoma cell strain, monoclonal antibody and application | |
| CN116082512B (en) | Monoclonal antibody for CDKn1A_p21CIP1 protein, and preparation method and application thereof | |
| CN111434686A (en) | Anti-human PBX1 monoclonal antibody, preparation method thereof and application thereof in clinical diagnosis of recurrent abortion | |
| CN117384291B (en) | Anti-human SALL4 rabbit monoclonal antibody, preparation method and application thereof, polynucleotide molecule, expression vector and host cell | |
| CN117487008B (en) | Anti-human Lin28A protein rabbit monoclonal antibody and application thereof | |
| CN114990074B (en) | Hybridoma cell strain, anti-human fumarate hydratase monoclonal antibody and application thereof | |
| CN117050194B (en) | Antibodies against infectious anemia of horses and their use in semen detection | |
| CN114213542B (en) | CPS-I antibodies and uses 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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |