Detailed Description
In a first aspect, embodiments of the present invention provide an antibody or a functional fragment thereof, the antibody or functional fragment thereof comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the HCDR1, HCDR2, HCDR3 are amino acid sequences identical to HCDR1, HCDR2, HCDR3 of the heavy chain variable region of SEQ ID No. 17, and wherein the LCDR1, LCDR2, LCDR3 are amino acid sequences identical to LCDR1, LCDR2, LCDR3 of the light chain variable region of any one of SEQ ID nos. 18, 19, 20.
The HCDR1, HCDR2 and HCDR3 are amino acid sequences identical to the HCDR1, HCDR2 and HCDR3 of the same heavy chain variable region defined in the antibody or functional fragment thereof according to the first aspect, and the LCDR1, LCDR2 and LCDR3 are amino acid sequences identical to the LCDR1, LCDR2 and LCDR3 of the same light chain variable region defined in the antibody or functional fragment thereof according to the first aspect.
For example, the HCDR1, HCDR2 and HCDR3 are amino acid sequences identical to HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO. 17, and the LCDR1, LCDR2 and LCDR3 are amino acid sequences identical to LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO. 18.
Under the definition of Kabat, the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region SEQ ID NO. 17 are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the antibody or the functional fragment thereof are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.
Under the definition of Kabat, the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region SEQ ID NO. 18 are respectively shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the antibody or the functional fragment thereof are respectively shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.
In the present invention, the term "antibody" is used in the broadest sense and may include full length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies so long as they exhibit the desired biological activity.
In the present invention, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions comprising one or more or even all of the major amino acid residues responsible for the binding of an antibody or antigen-binding fragment to the antigen or epitope recognized by it. In a specific embodiment of the invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.
In the present invention, the heavy chain complementarity determining region is represented by HCDR and includes HCDR1, HCDR2 and HCDR3, and the light chain complementarity determining region is represented by LCDR and includes LCDR1, LCDR2 and LCDR3.
CDR definition methods are well known in the art and include Kabat definition, chothia definition, IMGT definition, contact definition and AbM definition. As used herein, "Kabat definition" refers to the definition system described by Kabat et al, U.S. Dept. Of HEALTH AND Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" see Chothia et al, J Mol Biol 196:901-917 (1987). Still other CDR definition methods may not strictly follow one of the above schemes, but still overlap at least a portion of the Kabat-defined CDR regions, although they may be shortened or lengthened depending on the predicted or experimental outcome of a particular residue or group of residues. Exemplary defined CDRs are listed in table 1 below with the labels in the different documents being slightly different. Given the variable region amino acid sequence of an antibody, one of skill in the art can routinely determine which residues comprise a particular CDR. It should be noted that CDRs defined by other methods not limited to table 1 are also within the scope of the disclosure.
TABLE 1 CDR definition1
1 The numbering of all CDR definitions in Table 1 is according to the Kabat numbering system (see below), with the amino acid numbers on the heavy chain being indicated by "H+ numbers" and the amino acid numbers on the light chain being indicated by "L+ numbers". The Kabat numbering system can be specifically mapped to any variable region sequence by one of ordinary skill in the art without relying on any experimental data outside of the sequence itself. As described herein in the context of the present application,
"Kabat numbering" refers to the numbering system described by Kabat et al, U.S. Dept. Of HEALTH AND HumanServices, "Sequence of Proteins of Immunological Interest" (1983).
2 The "AbM" as used in table 1 has a lower case "b" referring to CDRs defined by the "AbM" antibody modeling software of Oxford Molecular.
3 CDR-H1 ends at position 35 if both H35A and H35B are absent, CDR-H1 ends at position 35A if only H35A is present, and CDR-H1 ends at position 35B if both H35A and H35B are present.
4 CDR-H1 ends at bit 32 if both H35A and H35B are absent, at bit 33 if only H35A is present, and at bit 34 if both H35A and H35B are present.
5 CDR-H1 ends at position 33 if both H35A and H35B are absent, CDR-H1 ends at position 34 if only H35A is present, and CDR-H1 ends at position 35 if both H35A and H35B are present.
According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or combination of systems Kabat, chothia, IMGT, abM or contacts.
In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Kabat system.
In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Chothia system.
In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by an IMGT system.
In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by an AbM system.
In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Contact system.
In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by Kabat, chothia, IMGT, abM or Contact system combinations.
According to an embodiment of the present invention, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 defined by the Kabat, chothia, abM or IMGT system correspond to the following Kabat numbering positions:
according to an embodiment of the invention, HCDRs and LCDRs are defined by the Kabat system.
In an alternative embodiment, the present examples provide an antibody or functional fragment thereof comprising the following complementarity determining regions:
HCDR1, comprising or consisting of the amino acid sequence set forth in SEQ ID NO. 1.
HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO. 2.
HCDR3, comprising or consisting of the amino acid sequence set forth in SEQ ID NO. 3.
LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 4.
LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 5.
LCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 6.
In the present invention, a "framework region" or "FR" region includes a heavy chain framework region and a light chain framework region, which refers to regions of an antibody heavy chain variable region and a light chain variable region other than CDRs, wherein the heavy chain framework region can be further subdivided into contiguous regions separated by CDRs, including HFR1, HFR2, HFR3, and HFR4 framework regions, and the light chain framework region can be further subdivided into contiguous regions separated by CDRs, including LFR1, LFR2, LFR3, and LFR4 framework regions.
In the present invention, the heavy chain variable region is obtained by ligating the CDRs numbered from HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 with the FRs in a combinatorial arrangement, and the light chain variable region is obtained by ligating the CDRs numbered from LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4 with the FRs in a combinatorial arrangement.
In alternative embodiments, the antibody or functional fragment thereof further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR 4;
the HFR1 comprises/has an amino acid sequence as shown in SEQ ID NO 7 or having at least 80% identity thereto;
the HFR2 comprises/has an amino acid sequence as shown in SEQ ID NO 8 or having at least 80% identity thereto;
the HFR3 comprises/has as SEQ ID NO 9 or an amino acid sequence having at least 80% identity thereto;
the HFR4 comprises/has an amino acid sequence as shown in SEQ ID NO 10 or having at least 80% identity thereto;
the LFR1 comprises/is as SEQ ID No. 11 or an amino acid sequence having at least 80% identity thereto;
the LFR2 comprises/is as SEQ ID No. 12 or an amino acid sequence having at least 80% identity thereto;
the LFR3 comprises/is as SEQ ID No. 13 or an amino acid sequence having at least 80% identity thereto;
the LFR4 comprises/is as SEQ ID No. 14 or an amino acid sequence having at least 80% identity thereto;
In other embodiments, each of the framework region amino acid sequences of the antibodies or functional fragments thereof provided herein may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the corresponding framework region (SEQ ID NO:7, 8, 9, 10, 11, 12, 13 or 14) described above.
In alternative embodiments, the LFR1 comprises/is an amino acid sequence as shown in SEQ ID NO. 11 or 25.
In alternative embodiments, the LFR3 comprises/is an amino acid sequence as shown in SEQ ID NO. 13 or 26.
In alternative embodiments, the antibody or functional fragment thereof binds cTnI with an affinity of KD <2.31 x 10-9 M.
In an alternative embodiment, the antibody or functional fragment thereof binds cTnI with an affinity of KD≤10 10-9M、KD≤10-10M、KD≤10-11M、KD≤10-12 M.
In an alternative embodiment, the antibody or functional fragment thereof binds cTnI with an affinity of KD≤7.62X10-10 M.
Antibody affinity (KD) assays are widely varied and can be classified into thermodynamic, kinetic and dynamic equilibrium assays based on the principle of detection. Among them, thermodynamic detection methods are commonly known as Isothermal Titration Calorimetry (ITC), kinetic detection methods are commonly known as Surface Plasmon Resonance (SPR) and biological membrane light interferometry (BLI), and dynamic equilibrium detection methods are commonly known as enzyme-linked immunosorbent assay (ELISA).
In alternative embodiments, the KD is determined using kinetic detection methods, alternatively surface plasmon resonance, for example, by using techniques such asA biosensor system of the system.
In a second aspect, embodiments of the present invention provide an antibody or functional fragment thereof comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO. 17 and/or a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NO. 18, 19 or 20.
In an alternative embodiment, the antibody or functional fragment thereof of the first or second aspect above comprises the heavy and light chain variable regions of any one of the following combinations:
| Combination of two or more kinds of materials | Heavy chain variable region | Light chain variable region |
| 1 | SEQ ID NO:17 | SEQ ID NO:18 |
| 2 | SEQ ID NO:17 | SEQ ID NO:19 |
| 3 | SEQ ID NO:17 | SEQ ID NO:20 |
In alternative embodiments, the antibody or functional fragment thereof further comprises a constant region.
In alternative embodiments, the constant region comprises a heavy chain constant region and/or a light chain constant region.
In alternative embodiments, the heavy chain constant region is selected from the group consisting of an IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD heavy chain constant region, and the light chain constant region is selected from the group consisting of kappa-type or lambda-type light chain constant regions.
In alternative embodiments, the constant region is of a species derived from a cow, horse, cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, chicken, or human.
In an alternative embodiment, the constant region is of murine species origin.
In an alternative embodiment, the heavy chain constant region sequence (CH) is shown in SEQ ID NO. 15 and the light chain constant region (CL) sequence is shown in SEQ ID NO. 16.
In other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the constant region (SEQ ID NO:15 or 16) described above.
In alternative embodiments, the functional fragment is selected from any one of F (ab ') 2, fab', fab, fv, and scFv of the antibody.
The functional fragments of the above antibodies generally have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the disclosure herein that functional fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The above functional fragments are readily available to those skilled in the art based on the disclosure of the structure of the intact antibodies.
Functional fragments of the above antibodies may also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.
In a third aspect, the invention provides an antibody or functional fragment thereof, comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO. 21 and/or a light chain having an amino acid sequence as shown in any one of SEQ ID NO. 22, 23, 24.
In an alternative embodiment, the antibody of the first, second, third aspects above, comprises a heavy chain and a light chain in any one of the following combinations:
| Combination of two or more kinds of materials | Heavy chain | Light chain |
| 1 | SEQ ID NO:21 | SEQ ID NO:22 |
| 2 | SEQ ID NO:21 | SEQ ID NO:23 |
| 3 | SEQ ID NO:21 | SEQ ID NO:24 |
In a fourth aspect, the invention provides an antibody conjugate comprising an antibody or functional fragment thereof as described above.
In an alternative embodiment, the above antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or a functional fragment thereof.
In alternative embodiments, the antibody conjugate further comprises a label conjugated to the antibody or functional fragment thereof.
In an alternative embodiment, the above-mentioned marker refers to a substance having a property such as luminescence, color development, radioactivity, etc., which can be directly observed by naked eyes or detected by an instrument, by which qualitative or quantitative detection of the corresponding target can be achieved.
In alternative embodiments, the labels include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the invention.
In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-light (FAM), tetrachlorolight (TET), and the like, or analogs thereof), rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof), cy-based dyes and derivatives thereof (including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazosin (preCP), and the like).
In alternative embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose 6-phosphate deoxygenase.
In alternative embodiments, the radioisotope includes, but is not limited to 212Bi、131I、111In、90Y、186Re、211At、125I、188Re、153Sm、213Bi、32P、94mTc、99mTc、203Pb、67Ga、68Ga、43Sc、47Sc、110mIn、97Ru、62Cu、64Cu、67Cu、68Cu、86Y、88Y、121Sn、161Tb、166Ho、105Rh、177Lu、172Lu and 18F.
In alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lomustine and its derivatives, and peroxyoxalate and its derivatives.
In alternative embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
In alternative embodiments, the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.
In an alternative embodiment, the colloidal metal is colloidal gold.
In an alternative embodiment, the antibody conjugate described above further comprises a solid support coupled to the antibody or functional fragment thereof.
In alternative embodiments, the solid support is selected from the group consisting of microspheres, plates, and membranes.
In alternative embodiments, the solid support includes, but is not limited to, magnetic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.
In a fifth aspect, the invention provides a reagent or kit comprising an antibody or functional fragment thereof as described above or an antibody conjugate as described above.
As previously described, the antibodies or functional fragments thereof in some embodiments or examples of the invention are capable of efficiently binding cTnI, and therefore, reagents or kits comprising the cTnI antibodies or functional fragments thereof are capable of efficiently performing qualitative or quantitative detection of cTnI. The reagent or the kit provided by the invention can be used for detection of specific binding performance of cTnI and antibodies thereof, such as immunoblotting, immunoprecipitation and the like. As previously mentioned, the antibodies or functional fragments thereof in some embodiments or examples of the invention have higher binding activity or affinity to cTnI, and thus reagents or kits comprising the antibodies or functional fragments thereof have higher detection sensitivity or specificity.
In a sixth aspect, the invention provides a method of detecting cTnI comprising a) contacting an antibody or functional fragment, antibody conjugate, reagent or kit as described above with cTnI in a sample to be detected under conditions sufficient for an antibody/antigen binding reaction to occur to form an immune complex, and b) detecting the presence of said immune complex, the presence of said complex being indicative of the presence of said antigen in said test sample;
in an alternative embodiment, the immune complex further comprises a second antibody, which binds to the antibody or a functional fragment thereof.
In an alternative embodiment, the immune complex further comprises a second antibody that binds cTnI.
In a seventh aspect, the invention provides a nucleic acid molecule encoding an antibody or functional fragment thereof as described above.
In an eighth aspect, the present invention provides a vector comprising the nucleic acid molecule described above.
In a ninth aspect, the present invention provides a cell comprising the vector described above.
In a tenth aspect, the invention provides a method of producing an antibody or functional fragment thereof comprising culturing a cell as described above.
In an eleventh aspect, the invention provides the use of an antibody or functional fragment thereof, antibody conjugate, or reagent or kit as described above for detecting cTnI or for preparing a product for detecting cTnI.
On the basis of the present invention, which discloses the amino acid sequence of an antibody or a functional fragment thereof, it is easy for a person skilled in the art to prepare the antibody or the functional fragment thereof by genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, by separating and purifying the antibody or the functional fragment thereof from a culture product of recombinant cells capable of recombinantly expressing the antibody or the functional fragment thereof according to any one of the above, and on the basis of this, it is within the scope of the present invention to prepare the antibody or the functional fragment thereof by any technique.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of formulations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.
Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. Such techniques are well explained in the literature, e.g., in the molecular cloning laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989), oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984), animal cell Culture (ANIMAL CELL Culture) (R.I. Freshney, 1987), enzymatic methods (Methods in Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.), experimental immunology Manual (Handbook of Experimental Immunology) (D.M.Weir and C.Blackwell, inc.), mammalian cell gene transfer Vectors (GENE TRANSFER Vectors for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos, 1987), contemporary molecular biology methods (F.M.Ausubel et al, 1987), polymerase chain reactions (28) (J.M.Weir. And C.Blackwell, inc.), PCR methods (J.34.J.37, J.J.37, J.F.37) and PCR methods (J.37, J.F.37, J.J.F.37, J.J.J.J.F.37, J.J.J.J.J.J.J.J.F.J.J.J.J.J.F.J.J.J.L).
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1 preparation of Anti-cTnI 10C7 monoclonal antibodies
Restriction enzymes, PRIME STAR DNA polymerase in this example were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO company. BD SMARTTM RACE cDNA Amplification Kit kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen. Primer synthesis and gene sequencing were accomplished by Invitrogen corporation. The hybridoma cell strain secreting the Anti-cTnI 10C7 monoclonal antibody is an existing hybridoma cell strain, and is recovered for later use.
(1) Antibody Gene production
MRNA is extracted from hybridoma cell strain secreting Anti-cTnI 10C7 monoclonal antibody, DNA product is obtained through RT-PCR method, the product is inserted into pMD-18T vector after A adding reaction by rTaq DNA polymerase, and is transformed into DH5 alpha competent cells, HEAVY CHAIN and LIGHT CHAIN gene clones are respectively taken after colony growth, and each 4 clones are sent to gene sequencing company for sequencing.
(2) Sequence analysis of Anti-cTnI 10C7 antibody variable region Gene
The gene sequence obtained by sequencing is placed in a kabat antibody database for analysis, and VNTI 11.5.5 software is used for analysis to determine that the amplified genes of the heavy chain and light chain primer pair are correct, wherein the VL gene sequence in the LIGHT CHAIN amplified gene fragment is 336bp, the front of the VL gene sequence is 57bp leader peptide sequence, and the VH gene sequence in the HEAVY CHAIN primer pair amplified gene fragment is 360bp, belongs to the VH1 gene family, and the front of the VL gene fragment is 57bp leader peptide sequence.
(3) Construction of recombinant antibody expression plasmids
pcDNATM3.4Vector is a constructed eukaryotic expression vector of recombinant antibody, which has been introduced with HindIII, bamHI, ecoRI and other polyclonal enzyme cutting sites and named pcDNA3.4A expression vector, 3.4A expression vector, VL and VH gene specific primers of the antibody are designed according to the result of gene sequencing of the antibody variable region in pMD-18T, and both ends have HindIII, ecoRI enzyme cutting sites and protective bases respectively, and a LIGHT CHAIN gene fragment of 0.74kb and a HEAVY CHAIN gene fragment of 1.42kb are amplified by a PCR amplification method.
The HEAVY CHAIN and LIGHT CHAIN gene fragments are respectively cut by HindIII/EcoRI double enzyme, the 3.4A vector is cut by HindIII/EcoRI double enzyme, the HEAVY CHAIN gene and LIGHT CHAIN gene after the fragments and the vector are purified and recovered are respectively connected with the 3.4A expression vector, and recombinant expression plasmids of HEAVY CHAIN and LIGHT CHAIN are respectively obtained.
2. Recombinant antibody production
Resuscitate HEK293 cells in advance, subculture to a200 ml system to enable the cell density to reach 3-5×106 cells/ml, the cell density to reach the concentration of selected antibodies and cell viability to be more than 95%, centrifugally clean the cells, re-dissolve the cells with a culture medium, simultaneously adjust the cell density to 2.9×106 cells/ml, re-dissolve the cells with the culture medium, and simultaneously serve as a cell dilution. The medium was used to prepare dilutions of plasmid DNA and transfection reagent, respectively. Adding transfection reagent diluent into plasmid DNA diluent, mixing uniformly, standing at room temperature for 15min, slowly adding the mixture into cell diluent within 1min, mixing uniformly, sampling, counting, recording and observing activity of transfected cells, culturing in a 35 ℃ constant temperature incubator at a rotating speed 120rmp and a CO2 content of 8%, and centrifuging and collecting samples after 13 days. The supernatant was affinity purified using a proteona affinity column. 6ug of the purified antibody was subjected to reducing SDS-PAGE, and the electrophoresed pattern was as shown. Two bands were shown after reducing SDS-PAGE, 1 Mr was 50KD (heavy chain) and the other Mr was 28KD (light chain).
The obtained antibody was designated as Anti-cTnI 10C7Rmb1, and the Anti-cTnI 10C7Rmb1 was subjected to mutation to obtain a mutant antibody, the sequences of the heavy chain (H) and the light chain (L) of which are shown in the following table:
TABLE 2 antibody sequences
Example 2 detection of Performance of antibodies
1. Affinity analysis
The antibody is diluted and purified in advance, and simultaneously, the cTnI recombinant antigen (purchased from the Phpeng organism) is subjected to gradient dilution, and the binding dissociation curve of the antigen-antibody is tested on Biacore 8K+ equipment by utilizing a CM5 chip which is coupled with goat anti-mouse IgG in advance, so that the affinity constant, the binding rate and the dissociation rate are obtained by automatic fitting of an instrument. (KD represents equilibrium dissociation constant, i.e., affinity constant; ka represents binding rate; KD represents dissociation rate)
TABLE 3 affinity data
| Sample name | KD(M) | ka | kd |
| Control | 2.31E-09 | 7.89E+05 | 1.82E-03 |
| Anti-cTnI 10C7 Rmb1 | 7.59E-10 | 1.05E+06 | 7.95E-04 |
| Anti-cTnI 10C7Rmb2 | 7.54E-10 | 1.14E+06 | 8.60E-04 |
| Anti-cTnI 10C7Rmb3 | 7.62E-10 | 1.09E+06 | 8.31E-04 |
2. Activity assay
The coating solution (NaHCO 3 as main component) was diluted to 3ug/ml of cTnI recombinant antigen (purchased from Phpeng organism) and washed at 100uL,37℃for 30min per well for 5 times, at 50 uL/well for 10min for a washing solution (Na2HPO4+Nacl as main component) for a stop solution (20% BSA+80% PBS) for 1h, at 120uL,37℃for 1h for a washing solution, at 100 uL/well for 30min for 5 times for a washing solution for a drying, at 100uL,37℃for 30min for a goat anti-mouse IgG-HRP for 5 times for a washing solution for a drying, at 50 uL/well for a developing solution B for 50 uL/well for a drying time, at 50 uL/well for a stop solution for a drying time, and at 450nm (reference nm) for reading OD values on an microplate reader.
Remarks are liquid A (main component citric acid+sodium acetate+acetanilide+carbamide peroxide), liquid B (main component citric acid+EDTA.2Na+TMB+concentrated HCL), and stop solution (EDTA.2Na+concentrated H2SO 4)
TABLE 4 Activity data
| Concentration (ng/ml) | 3.13 | 1.56 | 0.78 | 0.39 | 0.20 | 0.00 |
| Control | 0.871 | 0.533 | 0.319 | 0.194 | 0.012 | 0.011 |
| Anti-cTnI 10C7 Rmb1 | 1.393 | 0.766 | 0.429 | 0.279 | 0.138 | 0.021 |
| Anti-cTnI 10C7Rmb2 | 1.383 | 0.763 | 0.444 | 0.274 | 0.134 | 0.018 |
| Anti-cTnI 10C7Rmb3 | 1.392 | 0.759 | 0.431 | 0.276 | 0.137 | 0.024 |
3. Stability assessment
Placing the antibody at 4 ℃, -80 ℃ (refrigerator) and 37 ℃ (incubator) for 21 days, taking 7 days, 14 days and 21 days for state observation, and detecting the activity of the 21 days, wherein the results show that no obvious protein state change is seen in the antibody placed for 21 days under three examination conditions, and the activity is not in a descending trend along with the increase of the examination temperature, thus indicating that the antibody is stable. Table 5 below shows the results of the detection of OD by the antibody Anti-cTnI 10C7Rmb for 21 days of enzyme-free activity.
Table 5 stability data
| Sample concentration (ng/ml) | 3.13 | 1.56 | 0 |
| 4 ℃ 21 Day sample | 1.357 | 0.761 | 0.042 |
| -80 ℃,21 Day sample | 1.354 | 0.754 | 0.031 |
| 37 ℃ 21 Day sample | 1.382 | 0.763 | 0.041 |
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
The partial amino acid sequence related to the application is as follows: