CN117659201A - Anti-cocaine antibodies or functional fragments thereof, reagents and kits for detection of cocaine - Google Patents

Anti-cocaine antibodies or functional fragments thereof, reagents and kits for detection of cocaine Download PDF

Info

Publication number
CN117659201A
CN117659201A CN202211015035.3A CN202211015035A CN117659201A CN 117659201 A CN117659201 A CN 117659201A CN 202211015035 A CN202211015035 A CN 202211015035A CN 117659201 A CN117659201 A CN 117659201A
Authority
CN
China
Prior art keywords
antibody
functional fragment
seq
constant region
cocaine
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
Application number
CN202211015035.3A
Other languages
Chinese (zh)
Inventor
孟媛
钟冬梅
覃文新
游辉
曹慧方
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dongguan Pengzhi Biotechnology Co Ltd
Original Assignee
Dongguan Pengzhi Biotechnology Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Dongguan Pengzhi Biotechnology Co Ltd filed Critical Dongguan Pengzhi Biotechnology Co Ltd
Priority to CN202211015035.3A priority Critical patent/CN117659201A/en
Publication of CN117659201A publication Critical patent/CN117659201A/en
Pending legal-status Critical Current

Links

Landscapes

  • Peptides Or Proteins (AREA)

Abstract

The invention discloses an anti-cocaine antibody or a functional fragment thereof, a reagent for detecting cocaine and a kit, and relates to the field of antibodies. The anti-cocaine antibodies disclosed herein comprise a heavy chain complementarity determining region and a light chain complementarity determining region, and provide important sources of raw materials for detection of cocaine with improved affinity and activity.

Description

Anti-cocaine antibodies or functional fragments thereof, reagents and kits for detection of cocaine
Technical Field
The invention relates to the field of antibodies, in particular to an anti-cocaine antibody or a functional fragment thereof, a reagent for detecting cocaine and a kit.
Background
Cocaine (COC) is a tropane alkaloid extracted from the leaves of lithocarpus, also called lithocarpus, which is mostly white crystal, odorless, bitter and tingling, is the first natural alkaloid with local anesthetic effect found by human beings, is a long-acting ester local anesthetic, has high fat solubility, strong penetrability, good affinity to nerve tissues and good surface anesthetic effect. Have been used in medicine as local anesthetics or vasoconstrictors.
Cocaine can increase alertness and cause euphoria, and can feel energetic after being used, but abuse of cocaine can cause human body dependence on medicines, long-term small-dose abuse can cause chronic poisoning of human body, and damage to digestive system, immune system, cardiovascular system and genitourinary system, especially as dose-dependent hepatotoxin, and can cause hepatocyte necrosis. The symptoms of personality disorder or delusional psychosis are usually caused by tension, illness, inability to relax, irritability, blurred vision, night sweat, tinnitus, cramp and loss of coordination for a long time, and if suddenly stopped, the symptoms of anxiety, sudden cold and hot, chicken skin pimple, lacrimation, nasal discharge, sweating, nausea, abdominal pain and the like can appear, and serious depression and drowsiness phenomena, namely addiction attack symptoms, can occur. The pain of the withdrawal reaction, in turn, promotes the drug addict to maintain the drug addict in a drug addict state in a thousand ways to avoid the pain, so the drug addict is classified as a controlled drug in the country, and illegal use is forbidden by the state. The establishment of the rapid detection method of cocaine has very important practical significance and application value for toxin inhibition, poison detection, criminal identification and the like.
At present, the cocaine detection method mainly comprises a colloidal gold immunochromatography method, has the advantages of rapidness, simplicity in operation and the like, can be detected within 24 hours after drugs are abused, is an immunological detection method based on the specific reaction of antibodies and antigens, and simultaneously utilizes colloidal gold to amplify and display detected signals, so that the cocaine-resistant antibody is a key for developing the colloidal gold immunochromatography test paper.
Thus, there is a strong need in the art for antibodies that bind and detect cocaine efficiently and specifically.
In view of this, the present invention has been made.
Disclosure of Invention
The present invention aims to provide an anti-cocaine antibody or a functional fragment thereof, a reagent for detecting cocaine and a kit.
The invention is realized in the following way:
in a first aspect, embodiments of the present invention provide an antibody or functional fragment thereof comprising any one of (a) to (c): (a) The amino acid sequences of HCDR 1-3 and LCDR 1-3, HCDR1, HCDR2 and HCDR3 are shown in SEQ ID No. 1, 2 and 3 or as SEQ ID No. 1, 2 and 4; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are sequentially as shown in any of the following: SEQ ID Nos. 5, 7 and 8; SEQ ID Nos. 6, 7 and 8; SEQ ID Nos. 5, 7 and 9; SEQ ID Nos. 6, 7 and 9;
(b) A heavy chain variable region with an amino acid sequence shown in any one of SEQ ID NO 10-14 and a light chain variable region with an amino acid sequence shown in any one of SEQ ID NO 21-24;
(c) The amino acid sequence has a heavy chain variable region and a light chain variable region which are identical to the sequence shown in (b) by more than 80%, and comprises HCDR 1-3 and LCDR 1-3 of the sequence shown in (a).
In a second aspect, embodiments of the invention provide an antibody conjugate comprising an antibody or functional fragment thereof as described in the previous embodiments.
In a third aspect, embodiments of the invention provide a reagent or kit comprising an antibody or functional fragment thereof as described in the previous embodiments or an antibody conjugate as described in the previous embodiments.
In a fourth aspect, embodiments of the present invention provide a method for detecting cocaine, comprising: mixing the antibody or the functional fragment thereof according to the previous embodiment with a sample to be detected, and contacting the antibody or the functional fragment thereof with cocaine in the sample to be detected to form an immune complex.
In a fifth aspect, embodiments of the invention provide an isolated nucleic acid encoding an antibody or functional fragment thereof according to the previous embodiments.
In a sixth aspect, embodiments of the present invention provide a vector comprising the isolated nucleic acid of the previous embodiments.
In a seventh aspect, embodiments of the invention provide a cell comprising an isolated nucleic acid as described in the previous embodiments or a vector as described in the previous embodiments.
In an eighth aspect, embodiments of the present invention provide a method of preparing an antibody or functional fragment thereof according to the previous embodiments, comprising: the cells described in the previous examples were cultured.
The invention has the following beneficial effects:
the anti-cocaine antibody disclosed by the invention comprises the heavy chain complementarity determining region and the light chain complementarity determining region, and provides an important raw material source for the detection of cocaine, and has improved affinity and activity.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the result of reducing SDS-PAGE of Anti-COC 7F 10.
Detailed Description
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.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Embodiments of the present invention provide an antibody or functional fragment thereof comprising:
(a) The amino acid sequences of HCDR 1-3 and LCDR 1-3, HCDR1, HCDR2 and HCDR3 are shown in SEQ ID No. 1, 2 and 3 or as SEQ ID No. 1, 2 and 4; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are sequentially as shown in any of the following: SEQ ID Nos. 5, 7 and 8; SEQ ID Nos. 6, 7 and 8; SEQ ID Nos. 5, 7 and 9; SEQ ID Nos. 6, 7 and 9. 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 denoted by HCDR, and the 3 CDRs contained in the heavy chain variable region include HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is denoted by LCDR, and the 3 CDRs contained in the light chain variable region include LCDR1, LCDR2 and LCDR3. CDR labeling methods commonly used in the art include: the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering schemes, and the 1997 Lefranc et al, all protein sequences of the immunoglobulin superfamily. Kabat et al were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of Kabat numbering schemes, which are generally considered as widely adopted criteria for numbering antibody residues. In the embodiment of the invention, the CDR regions are marked by adopting Kabat annotation standard, but the CDR regions marked by other methods also belong to the protection scope of the invention.
In another aspect, embodiments of the present invention also provide an antibody or functional fragment thereof, comprising:
(b) A heavy chain variable region with an amino acid sequence shown in any one of SEQ ID NO 10-14 and a light chain variable region with an amino acid sequence shown in any one of SEQ ID NO 21-24.
In the present invention, a "framework region" or "FR" region includes a heavy chain framework region and a light chain framework region, and refers to regions other than CDRs in an antibody heavy chain variable region and a light chain variable region; wherein the heavy chain framework regions can be further subdivided into contiguous regions separated by CDRs comprising HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework regions may be further subdivided into contiguous regions separated by CDRs comprising LFR1, LFR2, LFR3 and LFR4 framework regions.
In the present invention, the heavy chain variable region is obtained by connecting the following numbered CDRs with FRs in the following combination arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by ligating the following numbered CDRs with the FR in the following combination arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.
In another aspect, embodiments of the present invention also provide an antibody or functional fragment thereof, comprising:
(c) The amino acid sequence has a heavy chain variable region and a light chain variable region which are identical to the sequence shown in (b) by more than 80%, and comprises HCDR 1-3 and LCDR 1-3 of the sequence shown in (a).
In alternative embodiments, the antibody or functional fragment thereof comprises a heavy chain framework region in a heavy chain variable region as set forth in any one of SEQ ID NOS: 10-14, and a light chain framework region in a light chain variable region as set forth in any one of SEQ ID NOS: 21-24.
In alternative embodiments, the framework region amino acid sequence of the antibody or functional fragment thereof 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 framework region described above.
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 heavy chain constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE and IgD; the light chain constant region is selected from kappa-type or lambda-type light chain constant regions.
In alternative embodiments, the constant region is of any one of bovine, equine, porcine, ovine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, and human origin.
In an alternative embodiment, the constant region is of murine species origin.
In alternative embodiments, the heavy chain constant region sequence is as shown in SEQ ID NO. 15 or has at least 80% identity thereto.
In alternative embodiments, the light chain constant region sequence is as shown in SEQ ID NO. 25 or has at least 80% identity thereto.
In particular, 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 above-described constant region (SEQ ID NO:15 or 25).
In alternative embodiments, the functional fragment is selected from the group consisting of F (ab') 2 Any of Fab', fab, fv and scFv.
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 another aspect, embodiments of the present invention also provide an antibody or functional fragment thereof, comprising: a heavy chain with an amino acid sequence shown in any one of SEQ ID NO 16-20, and a light chain with an amino acid sequence shown in any one of SEQ ID NO 26-29.
In an alternative embodiment, the antibodies or functional fragments thereof described in the preceding examples are used with a KD < 8.75X10 - 08 The affinity of M binds cocaine.
In an alternative embodiment, the antibodies or functional fragments thereof described in the preceding examples have a KD of 10 or less -08 M、KD≤10 -09 M、KD≤10 -10 M、KD≤10 -11 M or KD is less than or equal to 10 -12 The affinity of M binds cocaine.
In another aspect, embodiments of the invention also provide an antibody conjugate comprising an antibody or functional fragment thereof as described in the previous embodiments.
In alternative embodiments, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or 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 an alternative embodiment, the label is selected from at least one of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label.
In the actual use process, a person skilled in the art can select a suitable marker according to the detection condition or the actual requirement, 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 (chlorophyll), 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, 110 msin, 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, colloidal selenium, disperse dyes, dye-labeled microspheres, and latex.
In alternative embodiments, the colloidal metal includes, but is not limited to, colloidal gold or colloidal silver.
In an alternative embodiment, the colloidal metal is colloidal gold.
In alternative embodiments, the antibody conjugate further comprises a solid support coupled to the antibody or functional fragment thereof. In an antibody conjugate, the antibody is conjugated to a solid support.
In alternative embodiments, the solid support is selected from the group consisting of microspheres, plates, and membranes.
In alternative embodiments, the solid phase includes, but is not limited to, magnetic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.
In an alternative embodiment, the solid support is a nitrocellulose membrane.
In another aspect, embodiments of the invention also provide a reagent or kit comprising an antibody or functional fragment thereof as described in any of the preceding embodiments or an antibody conjugate as described in any of the preceding embodiments. The detection reagent or kit containing the antibody or the functional fragment thereof has improved detection sensitivity and specificity.
In another aspect, an embodiment of the present invention further provides a method for detecting cocaine, including:
mixing the antibody or functional fragment thereof according to any of the preceding embodiments or the antibody conjugate according to any of the preceding embodiments or the reagent or kit according to any of the preceding embodiments with a sample to be tested, and contacting the antibody or functional fragment thereof with cocaine in the sample to be tested to form an immune complex.
In a preferred embodiment, the immune complex further comprises a second antibody, which binds to the antibody or a functional fragment thereof.
In a preferred embodiment, the immune complex further comprises a second antibody, which binds to cocaine.
In another aspect, the embodiment of the present invention further provides an antibody or functional fragment according to any of the preceding embodiments, or an antibody conjugate according to any of the preceding embodiments, or a reagent or kit according to any of the preceding embodiments, for use in the detection of cocaine or in the preparation of a product for the detection of cocaine.
In another aspect, embodiments of the present invention also provide the use of an antibody or functional fragment of any of the embodiments described above or an antibody conjugate of any of the embodiments described above or a reagent or kit of any of the embodiments described above in the preparation of a product having at least one of the following uses, including: at least one of poison detection, criminal identification, etc.
In alternative embodiments, the product comprises a reagent or kit.
In another aspect, embodiments of the invention also provide an isolated nucleic acid encoding an antibody or functional fragment thereof according to any of the preceding embodiments.
In another aspect, embodiments of the invention also provide a vector comprising an isolated nucleic acid as described in any of the previous embodiments.
In another aspect, embodiments of the invention also provide a cell comprising an isolated nucleic acid as described in any of the previous embodiments or a vector as described in any of the previous embodiments.
In another aspect, embodiments of the present invention also provide a method of preparing an antibody or functional fragment thereof according to any of the previous embodiments, comprising: culturing the cells of any of the previous examples.
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. This technique is well explained in the literature, as is the case for 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); methods of enzymology (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C.Blackwell, inc.), gene transfer vectors for mammalian cells (Gene Transfer Vectors for Mammalian Cells) (J.M.Miller and M.P.calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
EXAMPLE 1 preparation of Anti-COC 7F10 monoclonal antibodies
Restriction enzymes, prime Star DNA polymerase in this example were purchased from Takara Corp. MagExtractor-RNA extraction kit was purchased from TOYOBO company. BD SMART TM 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 gene sequencing companies.
1. Construction of expression plasmid
1.1 preparation of Anti-COC 7F10 antibody Gene
mRNA is extracted from hybridoma cell strains secreting Anti-COC 7F10 monoclonal antibodies, DNA products are obtained through an RT-PCR method, the DNA products are inserted into a pMD-18T vector and are transformed into DH5 alpha competent cells, after colonies grow out, positive clones of the Heavy Chain and Light Chain genes are respectively taken, and 4 clones are sent to a gene sequencing company for sequencing.
1.2 sequence analysis of the variable region Gene of Anti-COC 7F10 antibody
The gene sequence obtained by sequencing is placed in a Kabat antibody database for analysis, and VNTI11.5 software is utilized for analysis to determine that the genes amplified by the heavy Chain primer pair and the Light Chain primer pair are correct, wherein in the gene fragment amplified by the Light Chain, the VL gene sequence is 336bp, and a leader peptide sequence of 57bp is arranged in front of the VL gene sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 363bp, belongs to the VH1 gene family, and a 57bp leader peptide sequence is arranged in front of the VH gene sequence.
1.3 construction of recombinant antibody expression plasmids
pcDNA TM 3.4vector is a constructed eukaryotic expression vector of the recombinant antibody, and the expression vector is modified to be introduced into a polyclonal enzyme cutting site, and is hereinafter abbreviated as a 3.4A expression vector; VL and V of Anti-COC 7F10 antibody were designed based on the results of gene sequencing of the antibody variable region in pMD-18T described aboveThe H gene specific primer has restriction enzyme cutting sites and protective bases at two ends, and a 0.74kb Light Chain gene fragment and a 1.42kb Heavy Chain gene fragment are amplified by a PCR amplification method.
The Heavy Chain gene and the Light Chain gene are respectively subjected to double enzyme digestion by adopting restriction enzymes, a 3.4A vector is subjected to double enzyme digestion by adopting restriction enzymes, and the Heavy Chain gene and the Light Chain gene after the fragments and the vector are purified and recovered are respectively connected into a 3.4A expression vector to respectively obtain recombinant expression plasmids of the Heavy Chain gene and the Light Chain gene.
2. Stable cell line selection
2.1 transient transfection of recombinant antibody expression plasmids into CHO cells, determination of expression plasmid Activity
The plasmid was diluted to 40. Mu.g/100. Mu.L with ultrapure water, and CHO cells were regulated to 1.43X 10 7 100. Mu.L of plasmid was mixed with 700. Mu.L of cells in a centrifuge tube, transferred to an electrocuvette, electroblotted, sample counted on days 3, 5, 7, and harvested on day 7.
Coating liquid (main component NaHCO) 3 ) COC-BSA was diluted to 2. Mu.g/mL, 100. Mu.L per well, overnight at 4 ℃; the next day, the washing liquid (main component Na 2 HPO 4 +NaCl) for 2 times, and beating to dry; blocking solution (20% BSA+80% PBS) was added, 120. Mu.L per well, 37℃for 1h, and the mixture was dried by shaking; adding diluted cell supernatant at 100. Mu.L/well, 37℃for 30min (1 h for part of supernatant); washing with washing liquid for 5 times, and drying; adding goat anti-mouse IgG-HRP, 100 mu L of each hole, and 30min at 37 ℃; washing with washing liquid for 5 times, and drying; adding a developing solution A (50 mu L/hole) and a developing solution B (50 mu L/hole) for 10min; adding a stop solution, 50 mu L/well; OD was read on the microplate reader at 450nm (reference 630 nm). The results showed that the reaction OD after 1000-fold dilution of the cell supernatant was still greater than 1.0, and that the reaction OD without cell supernatant wells was less than 0.1, indicating that the antibodies produced after transient plasmid transformation were active on COC-BSA. The reactivity of the cell supernatant with BSA was examined by the same method as described above, and the result showed that the cell supernatant was diluted 1000 times and the OD was less than 0.1, and that the cell supernatant was not added and the OD was less than 0.1, indicating that the antibody produced after transient transformation of the plasmid was inactive against BSA, further demonstrating that the cell supernatant was active against COC.
Remarks: liquid A (main component of citric acid, sodium acetate, acetanilide and carbamide peroxide); liquid B (citric acid+EDTA.2Na+TMB+concentrated HCL as main component); stop solution (EDTA.2Na+ concentrated H) 2 SO 4 )。
2.2 linearization of recombinant antibody expression plasmids
The following reagents were prepared: buffer 50 mu L, DNA ug/tube, pvuI enzyme 10 mu l, sterile water to 500 mu l, water bath at 37℃overnight; firstly, extracting with equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1, and then sequentially extracting with chloroform (water phase); precipitating 0.1 times volume (water phase) of 3M sodium acetate and 2 times volume of ethanol on ice, rinsing the precipitate with 70% ethanol, removing organic solvent, completely volatilizing ethanol, re-thawing with appropriate amount of sterilized water, and measuring concentration.
2.3 stable transfection of recombinant antibody expression plasmid, pressure screening of stable cell lines
The plasmid was diluted to 40. Mu.g/100. Mu.L with ultrapure water, and CHO cells were regulated to 1.43X 10 7 Placing cells/ml in a centrifuge tube, mixing 100 μl of plasmid with 700 μl of cells, transferring into an electrorotor, electrorotating, and counting the next day; 25 mu mol/L MSX 96 wells were incubated under pressure for approximately 25 days.
Observing the clone holes with the cells under a microscope, and recording the confluency; taking culture supernatant, and carrying out sample feeding detection; selecting cell strains with high antibody concentration and relative concentration, turning 24 holes, and turning 6 holes about 3 days; seed preservation and batch culture are carried out after 3 days, and cell density is regulated to be 0.5x10 6 Batch culture was performed with cells/mL,2.2mL, and cell density was 0.3X10 6 Performing seed preservation by using cells/mL and 2 mL; and (3) carrying out sample feeding detection on the culture supernatant of the 6-hole batch culture for 7 days, and selecting cell strains with smaller antibody concentration and smaller cell diameter to transfer TPP for seed preservation and passage.
3. Recombinant antibody production
3.1 cell expansion culture
After cell recovery, the cells were first cultured in 125mL shake flasks with an inoculation volume of 30mL and a medium of 100% Dynamis, and placed in a shaker at a speed of 120r/min at 37℃and with 8% carbon dioxide. Culturing for 72h, inoculating and expanding culture at 50 ten thousand cells/mL, and calculating the expanded culture volume according to the production requirement, wherein the culture medium is 100% Dynamis culture medium. After that, the culture was spread every 72 hours. When the cell quantity meets the production requirement, the inoculation density is strictly controlled to be about 50 ten thousand cells/mL for production.
3.2 shaking flask production and purification
Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8%. Feeding: feeding was started every day until 72h of culture in shake flasks, hyCloneTM Cell BoostTM Feed a fed-batch was 3% of the initial culture volume every day, feed 7b fed-batch was one thousandth of the initial culture volume every day, and fed-batch was continued until day 12 (day 12 Feed). Glucose was fed at 3g/L on day six. Samples were collected on day 13. Affinity purification was performed using a proteona affinity column. 3. Mu.g of 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).
Example 2 affinity and Activity optimization
The Anti-COC 7F10 monoclonal antibody obtained in example 1 had an ability to bind cocaine, but was not satisfactory in affinity and antibody activity, and thus, the applicant had performed directed mutation on the light chain CDR and the heavy chain CDR of the antibody. The method comprises the steps of performing structural simulation of an antibody variable region, structural simulation of an antigen-antibody variable region acting complex, analysis of key amino acids of an antibody and mutation design by using a computer, designing and synthesizing a two-way primer covering a mutation site according to a mutation scheme, synthesizing primers at two ends of target DNA, performing high-fidelity PCR reaction, cloning a PCR product to a vector, and preparing the mutant antibody according to the method described in the example 1. The following monoclonal antibodies Anti-COC-7F10RMb1 to Anti-COC-7F10RMb6 were obtained, the heavy and light chain amino acid sequences of which were as follows:
TABLE 1 antibody sequences
Sample name Heavy chain sequence number Light chain sequence number
Anti-COC-7F10RMb1 SEQ ID NO:16 SEQ ID NO:26
Anti-COC-7F10RMb2 SEQ ID NO:17 SEQ ID NO:26
Anti-COC-7F10RMb3 SEQ ID NO:18 SEQ ID NO:26
Anti-COC-7F10RMb4 SEQ ID NO:16 SEQ ID NO:27
Anti-COC-7F10RMb5 SEQ ID NO:16 SEQ ID NO:28
Anti-COC-7F10RMb6 SEQ ID NO:20 SEQ ID NO:26
Example 3 affinity analysis
Using the AMC sensor, the purified antibodies were diluted to 10. Mu.g/ml with PBST and COC-BSA was diluted in gradient with PBST.
The operation flow is as follows: equilibration for 60s in buffer 1 (PBST), immobilization for 300s in antibody solution, incubation for 180s in buffer 2 (PBST), binding for 420s in antigen solution, dissociation for 1200s in buffer 2, incubation with 10mM pH 1.69GLY solution and bufferLiquid 3 (PBST) was subjected to sensor regeneration and data was output. (K) D Representing equilibrium dissociation constant, i.e. affinity; kon represents the binding rate; kdis represents the dissociation rate. Main component Na of PBST 2 HPO 4 +NaCl+TW-20). The results showed that: the affinity of Anti-COC-7F10RMb1 to Anti-COC-7F10RMb5 was better than that of Anti-COC-7F10RMb6 and control antibody.
Table 2 affinity assay data
Sample name K D (M) kon(1/Ms) kdis(1/s)
Control antibodies 8.75E-08 2.89E+04 2.53E-03
Anti-COC-7F10RMb1 6.66E-09 9.69E+04 6.45E-04
Anti-COC-7F10RMb2 6.42E-09 8.79E+04 5.64E-04
Anti-COC-7F10RMb3 7.86E-09 6.79E+04 5.33E-04
Anti-COC-7F10RMb4 6.96E-09 8.19E+04 5.70E-04
Anti-COC-7F10RMb5 6.48E-09 8.69E+04 5.63E-04
Anti-COC-7F10RMb6 6.00E-08 1.33E+04 7.98E-04
EXAMPLE 4 Activity assay
Coating liquid (main component NaHCO) 3 ) COC-BSA was diluted to 2. Mu.g/ml, 100. Mu.L per well, overnight at 4 ℃; the next day, the washing liquid (main ingredient Na 2 HPO 4 +NaCl) for 2 times, and beating to dry; blocking solution (20% BSA+80% PBS) was added, 120. Mu.L per well, 37℃for 1h, and the mixture was dried by shaking; adding diluted purified antibody and control antibody, 100 μl/well, 37deg.C, 30min; washing with washing liquid for 5 times, and drying; adding goat anti-mouse IgG-HRP, 100 mu L of each hole, and 30min at 37 ℃; washing with washing liquid for 5 times, and drying; adding a developing solution A (50 mu L/hole) and a developing solution B (50 mu L/hole) for 10min; adding a stop solution, 50 mu L/well; OD was read on the microplate reader at 450nm (reference 630 nm).
Remarks: liquid A (main component of citric acid, sodium acetate, acetanilide and carbamide peroxide); liquid B (main component citric acid+EDTA.2Na+TMB+concentrated HCL); stop solution (EDTA.2Na+ concentrated H) 2 SO 4 )。
TABLE 3 Activity data
Concentration (ng/ml) 3.906 1.953 0.977 0.488 0.244 0
Control antibodies 1.336 0.791 0.415 0.241 0.131 0.068
Anti-COC-7F10RMb1 1.746 1.443 0.883 0.466 0.219 0.079
Anti-COC-7F10RMb2 1.756 1.465 0.895 0.487 0.239 0.033
Anti-COC-7F10RMb3 1.646 1.315 0.793 0.396 0.179 0.065
Anti-COC-7F10RMb4 1.716 1.398 0.852 0.455 0.208 0.074
Anti-COC-7F10RMb5 1.709 1.354 0.837 0.402 0.184 0.032
Anti-COC-7F10RMb6 1.422 0.821 0.463 0.267 0.138 0.072
Example 5 assessment of antibody stability
The antibody is placed at 4 ℃ (refrigerator), 80 ℃ (refrigerator) and 37 ℃ (incubator) for 21 days, 7 days, 14 days and 21 days are taken for carrying out state observation, and activity detection is carried out on the 21 days, so that the result shows that no obvious protein state change is seen for the antibody placed for 21 days under three examination conditions, the activity is not in a descending trend along with the increase of the examination temperature, and the stability of the expressed antibody is indicated. The following table shows the OD results of the enzyme-free activity assay for 21 days.
Table 4 stability data
Sample concentration (ng/ml) 1.953 0.488 0
4 ℃,21 days sample 1.421 0.432 0.032
Sample at-80℃for 21 days 1.476 0.479 0.023
37 ℃ and 21 days of sample 1.410 0.422 0.053
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 sequences referred to in this application are as follows:
/>

Claims (11)

1. an antibody or functional fragment thereof, wherein the antibody or functional fragment thereof comprises any one of (a) to (c):
(a) The amino acid sequences of HCDR 1-3 and LCDR 1-3, HCDR1, HCDR2 and HCDR3 are shown in SEQ ID No. 1, 2 and 3 or as SEQ ID No. 1, 2 and 4; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are sequentially as shown in any of the following: SEQ ID Nos. 5, 7 and 8; SEQ ID Nos. 6, 7 and 8; SEQ ID Nos. 5, 7 and 9; SEQ ID Nos. 6, 7 and 9;
(b) A heavy chain variable region with an amino acid sequence shown in any one of SEQ ID NO 10-14 and a light chain variable region with an amino acid sequence shown in any one of SEQ ID NO 21-24;
(c) The amino acid sequence has a heavy chain variable region and a light chain variable region which are identical to the sequence shown in (b) by more than 80%, and comprises HCDR 1-3 and LCDR 1-3 of the sequence shown in (a).
2. The antibody or functional fragment thereof according to claim 1, wherein the antibody or functional fragment thereof is in K D ＜8.75×10 -08 The affinity of M binds cocaine.
3. The antibody or functional fragment thereof of claim 1, wherein the antibody or functional fragment thereof further comprises a constant region;
optionally, the constant region comprises a heavy chain constant region and/or a light chain constant region;
alternatively, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE and IgD; the light chain constant region is selected from a kappa-type or lambda-type light chain constant region;
optionally, the constant region is derived from any one of cow, horse, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, and human;
alternatively, the constant region is of mouse species origin;
alternatively, the heavy chain constant region sequence is as shown in SEQ ID NO. 15 or has at least 80% identity thereto;
alternatively, the light chain constant region sequence is as shown in SEQ ID NO. 25 or has at least 80% identity thereto;
alternatively, the antibody or functional fragment thereof comprises a heavy chain having an amino acid sequence as set forth in any one of SEQ ID NOS.16-20, and a light chain having an amino acid sequence as set forth in any one of SEQ ID NOS.26-29.
4. An antibody or functional fragment thereof according to any one of claims 1 to 3, wherein the functional fragment is selected from the group consisting of F (ab') 2 Any of Fab', fab, fv and scFv.
5. An antibody conjugate comprising an antibody or functional fragment thereof according to any one of claims 1 to 4;
optionally, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or functional fragment thereof;
optionally, the antibody conjugate further comprises a solid support coupled to the antibody or functional fragment thereof;
optionally, the antibody conjugate further comprises a label conjugated to the antibody or functional fragment thereof;
optionally, the label is selected from at least one of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label;
optionally, the label is colloidal gold.
6. A reagent or kit comprising an antibody or functional fragment thereof according to any one of claims 1 to 4 or an antibody conjugate according to claim 5.
7. A method of detecting cocaine, comprising:
mixing the antibody or the functional fragment thereof according to any one of claims 1-4 with a sample to be detected, and contacting the antibody or the functional fragment thereof with cocaine in the sample to be detected to form an immune complex;
preferably, the immune complex further comprises a second antibody, which binds to the antibody or a functional fragment thereof;
preferably, the immune complex further comprises a second antibody, which binds to cocaine.
8. An isolated nucleic acid encoding the antibody or functional fragment thereof of any one of claims 1-4.
9. A vector comprising the nucleic acid of claim 8.
10. A cell comprising the nucleic acid of claim 8 or the vector of claim 9.
11. A method of preparing the antibody or functional fragment thereof of any one of claims 1 to 4, comprising: culturing the cell of claim 10.
CN202211015035.3A 2022-08-23 2022-08-23 Anti-cocaine antibodies or functional fragments thereof, reagents and kits for detection of cocaine Pending CN117659201A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202211015035.3A CN117659201A (en) 2022-08-23 2022-08-23 Anti-cocaine antibodies or functional fragments thereof, reagents and kits for detection of cocaine

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202211015035.3A CN117659201A (en) 2022-08-23 2022-08-23 Anti-cocaine antibodies or functional fragments thereof, reagents and kits for detection of cocaine

Publications (1)

Publication Number Publication Date
CN117659201A true CN117659201A (en) 2024-03-08

Family

ID=90075514

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202211015035.3A Pending CN117659201A (en) 2022-08-23 2022-08-23 Anti-cocaine antibodies or functional fragments thereof, reagents and kits for detection of cocaine

Country Status (1)

Country Link
CN (1) CN117659201A (en)

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050038232A1 (en) * 1999-11-03 2005-02-17 Maxygen, Inc. Antibody diversity generation
US20100143364A1 (en) * 2008-12-05 2010-06-10 Vybion, Inc. Chimeric Antibodies, Compositions and Methods for Treating Cocaine-Related Disorders
US20110092372A1 (en) * 2009-10-14 2011-04-21 Juan Carlos Almagro Methods of Affinity Maturing Antibodies
JP2012232948A (en) * 2011-05-06 2012-11-29 Kagoshima Univ Anti-morphine antibody, method for measuring morphine using the same, and morphine measuring kit containing the same
CN103917872A (en) * 2011-11-02 2014-07-09 优志旺电机株式会社 Fluoroimmunoassay method using polypeptide complex containing fluorolabeled antibody-variable region
CN107988168A (en) * 2017-07-02 2018-05-04 杭州隆基生物技术有限公司 Anti-cocaine monoclonal antibody, the cell line and preparation method for secreting the antibody
EA201991022A1 (en) * 2016-10-27 2019-09-30 Янссен Фармацевтика Нв IMMUNOGLOBULINS AND THEIR APPLICATION
CN114195899A (en) * 2021-12-23 2022-03-18 杭州贤至生物科技有限公司 Anti-cocaine specific antibodies, plasmid vectors and methods
CN114516914A (en) * 2020-11-19 2022-05-20 东莞市朋志生物科技有限公司 Antibody against N-terminal pro-brain natriuretic peptide, and reagent and kit for detecting N-terminal pro-brain natriuretic peptide
CN117487010A (en) * 2022-08-02 2024-02-02 东莞市朋志生物科技有限公司 Anti-tetraiodothyroxine antibody or functional fragment thereof, reagent for detecting tetraiodothyroxine and kit

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050038232A1 (en) * 1999-11-03 2005-02-17 Maxygen, Inc. Antibody diversity generation
US20100143364A1 (en) * 2008-12-05 2010-06-10 Vybion, Inc. Chimeric Antibodies, Compositions and Methods for Treating Cocaine-Related Disorders
US20110092372A1 (en) * 2009-10-14 2011-04-21 Juan Carlos Almagro Methods of Affinity Maturing Antibodies
JP2012232948A (en) * 2011-05-06 2012-11-29 Kagoshima Univ Anti-morphine antibody, method for measuring morphine using the same, and morphine measuring kit containing the same
CN103917872A (en) * 2011-11-02 2014-07-09 优志旺电机株式会社 Fluoroimmunoassay method using polypeptide complex containing fluorolabeled antibody-variable region
EA201991022A1 (en) * 2016-10-27 2019-09-30 Янссен Фармацевтика Нв IMMUNOGLOBULINS AND THEIR APPLICATION
CN107988168A (en) * 2017-07-02 2018-05-04 杭州隆基生物技术有限公司 Anti-cocaine monoclonal antibody, the cell line and preparation method for secreting the antibody
CN114516914A (en) * 2020-11-19 2022-05-20 东莞市朋志生物科技有限公司 Antibody against N-terminal pro-brain natriuretic peptide, and reagent and kit for detecting N-terminal pro-brain natriuretic peptide
CN114195899A (en) * 2021-12-23 2022-03-18 杭州贤至生物科技有限公司 Anti-cocaine specific antibodies, plasmid vectors and methods
CN117487010A (en) * 2022-08-02 2024-02-02 东莞市朋志生物科技有限公司 Anti-tetraiodothyroxine antibody or functional fragment thereof, reagent for detecting tetraiodothyroxine and kit

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
EDWIN POZHARSKI等: "Diversity in Hapten Recognition: Structural Study of an Anti-cocaine Antibody M82G2", 《J.MOL.BIOL》, vol. 349, 31 December 2005 (2005-12-31), pages 570 - 582, XP004893060, DOI: 10.1016/j.jmb.2005.03.080 *
LISA M. EUBANKS等: "A human recombinant monoclonal antibody to cocaine: Preparation, characterization and behavioral studies", 《BIOORG MED CHEM LETT》, vol. 24, no. 19, 1 October 2015 (2015-10-01), pages 4664 - 4666, XP029064437, DOI: 10.1016/j.bmcl.2014.08.035 *
熊英等: "可卡因与其降解抗体15A10的分子对接及分子动力学研究", 《华中师范大学学报（自然科学版）》, no. 2, 31 December 2005 (2005-12-31), pages 205 - 207 *

Similar Documents

Publication Publication Date Title
WO2023078447A1 (en) Antibody against novel coronavirus and reagent and kit for testing novel coronavirus
CN116693676A (en) Anti-mycoplasma pneumoniae antibody, reagent for detecting mycoplasma pneumoniae and kit
CN117487010A (en) Anti-tetraiodothyroxine antibody or functional fragment thereof, reagent for detecting tetraiodothyroxine and kit
CN117343167A (en) Anti-influenza B virus antibody, and reagent and kit for detecting influenza B virus
CN117285638B (en) Anti-buprenorphine antibodies, reagents and kits for the detection of buprenorphine
CN117659201A (en) Anti-cocaine antibodies or functional fragments thereof, reagents and kits for detection of cocaine
CN116444657A (en) Antibodies against novel coronaviruses, reagents and kits for detecting novel coronaviruses
CN117088983B (en) Anti-amphetamine antibodies, reagents and kits for detecting amphetamine
CN117327177B (en) Anti-adiponectin antibody, reagent for detecting adiponectin and kit
CN117088982B (en) Anti-methamphetamine antibodies, reagents and kits for detecting methamphetamine
CN117088984B (en) Anti-benzodiazepine antibody, and kit and reagent for detecting benzodiazepine
CN116444656B (en) Antibody, reagent and method for identifying novel crown mutant antigen
CN117088982A (en) Anti-methamphetamine antibodies, reagents and kits for detecting methamphetamine
CN116836273B (en) Anti-serum amyloid A antibody, reagent for detecting serum amyloid A and kit
CN117229409B (en) Anti-phencyclidine antibody, reagent for detecting phencyclidine and kit
CN117229408A (en) Anti-barbital compound antibody, reagent for detecting barbital drugs and kit
CN116496401B (en) Anti-abnormal prothrombin antibody, reagent for detecting abnormal prothrombin and kit
CN117327177A (en) Anti-adiponectin antibody, reagent for detecting adiponectin and kit
CN117088975A (en) Anti-albumin antibody, reagent and kit for detecting albumin
CN116836274A (en) Anti-serum amyloid A antibody, reagent for detecting serum amyloid A and kit
CN116925216A (en) Anti-serum amyloid A antibody, reagent for detecting serum amyloid A and kit
CN116836271A (en) Antibodies against type B streptococcus, reagents and kits for detecting type B streptococcus
CN116836279A (en) Anti-tetraiodothyroxine antibody, reagent and kit for detecting tetraiodothyroxine
CN117285639A (en) Anti-ethyl glucuronide antibody, and reagent and kit for detecting ethyl glucuronide
CN116693683A (en) Anti-testosterone antibody, reagent for detecting testosterone and kit

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