CN116693683B - Anti-testosterone antibody, reagent for detecting testosterone and kit - Google Patents

Anti-testosterone antibody, reagent for detecting testosterone and kit Download PDF

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Publication number
CN116693683B
CN116693683B CN202210190274.6A CN202210190274A CN116693683B CN 116693683 B CN116693683 B CN 116693683B CN 202210190274 A CN202210190274 A CN 202210190274A CN 116693683 B CN116693683 B CN 116693683B
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antibody
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CN116693683A (en
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孟媛
钟冬梅
游辉
曹慧方
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Dongguan Pengzhi Biotechnology Co Ltd
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Dongguan Pengzhi Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/548—Carbohydrates, e.g. dextran
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/583—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with non-fluorescent dye label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/60—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances involving radioactive labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones
    • 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]
    • 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
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Abstract

The invention discloses an anti-testosterone antibody, a reagent for detecting testosterone and a kit, and relates to the technical field of antibodies. The anti-testosterone antibodies disclosed herein comprise a heavy chain complementarity determining region and a light chain complementarity determining region. The antibody provides an important raw material source for detecting testosterone.

Description

Anti-testosterone antibody, reagent for detecting testosterone and kit
Technical Field
The invention relates to the technical field of antibodies, in particular to an anti-testosterone antibody, a reagent for detecting testosterone and a kit.
Background
Testosterone (T) is a steroid hormone containing 19 carbon atoms, and the androgen in the blood circulation is mainly Testosterone. Because testosterone is less soluble, free testosterone in the blood is about 2%. About 60% of testosterone binds to Sex Hormone Binding Globulin (SHBG) synthesized by the liver, and this bound testosterone is biologically inactive due to its strong binding capacity. The main physiological role of testosterone is to promote the development and maturation of male sexual organs and maintain secondary sexual characteristics, and in terms of metabolism, testosterone can promote the growth of muscles and the synthesis of proteins, while inhibiting the proteolytic degradation caused by glucocorticoids. In men, testosterone is largely synthesized by the testes; while both the adrenal glands of both men and women synthesize part of testosterone; in addition, the ovaries of women can also secrete small amounts of testosterone. Testosterone tests are performed on men, women and children in many cases. Boys develop delayed puberty or delayed development, often with simultaneous measurement of testosterone, FSH and LH. Judging male sexual precocity of teenagers also requires detection of testosterone, and causes of male sexual precocity caused by testosterone elevation often include various tumors and congenital adrenal hypertrophy. Adult males detect testosterone, often in the suspicion of infertility, reduced sexual function, and erectile dysfunction disease. In women, testosterone is often detected when irregular menstruation or no menstruation occurs, infertility or masculinization characteristics such as hirsutism, profuse voice, etc. Testosterone-elevated tumors occur primarily in the ovaries or adrenal glands, and others are polycystic ovary syndrome (POCS). Therefore, the accurate detection of the content of the gonadal peptide in the organism plays an important role in judging the evaluation of the gonadal peptide and the organism function.
Immunological detection methods are a means for detecting antibodies and antigens based on specific reactions, and are often used for detecting trace amounts of bioactive substances such as proteins and hormones because the detected signals can be amplified and displayed by isotopes, enzymes, chemiluminescent substances, and the like. Currently, immunological detection methods of testosterone mainly comprise a chemiluminescent competition method, and similar immunological detection methods comprise a biochemical immunonephelometry method, a radioimmunoassay method, a fluorescent immunochromatography method and the like. The immunological detection methods described above all require antibodies directed against testosterone. Thus, there is a strong need in the art for antibodies that are effective and bind testosterone and detect it.
In view of this, the present invention has been made.
Disclosure of Invention
Aiming at the fact that the sources of anti-testosterone antibodies on the market are few, the application provides an anti-testosterone antibody with improved affinity and activity, and provides an important raw material source for detecting testosterone.
In order to achieve the above object, according to one aspect of the present invention, there is provided an antibody or a functional fragment thereof comprising HCDR1, HCDR2, HCDR3 having amino acid sequences shown in SEQ ID NO. 1 to SEQ ID NO. 3, and LCDR1 having amino acid sequences shown in SEQ ID NO. 4 or 15, LCDR2 having amino acid sequences shown in SEQ ID NO. 5, and LCDR3 having amino acid sequences shown in SEQ ID NO. 6.
In order to achieve the above object, according to a second aspect of the present invention, there is provided an anti-testosterone antibody or a functional fragment thereof, comprising a heavy chain variable region comprising the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 and/or a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 described above.
In order to achieve the above object, according to a third aspect of the present invention, there is provided an anti-testosterone antibody comprising a heavy chain comprising the heavy chain variable region described above and/or a light chain comprising the light chain variable region described above.
In order to achieve the above object, according to a fourth aspect of the present invention, there is provided an antibody conjugate comprising the above antibody or a functional fragment thereof.
In order to achieve the above object, according to a fifth aspect of the present invention, there is provided a reagent or kit for detecting testosterone, comprising the above antibody or a functional fragment thereof or the above antibody conjugate.
In order to achieve the above object, according to a sixth aspect of the present invention there is provided the use of an antibody as defined above or a functional fragment thereof, an antibody conjugate as defined above or a reagent or kit as defined above in the detection of testosterone.
In order to achieve the above object, the present invention also provides a vector, a cell and a method for preparing the above antibody or a functional fragment thereof.
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 results of reducing SDS-PAGE of Anti-Testo-33A9mut2 to Anti-Testo-33A9mut5.
Detailed Description
The invention provides an antibody or a functional fragment thereof, which comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO. 1-3, LCDR1 with amino acid sequences shown in SEQ ID NO. 4 or 15, LCDR2 with amino acid sequences shown in SEQ ID NO. 5 and LCDR3 with amino acid sequences shown in SEQ ID NO. 6. The antibodies have improved affinity and activity.
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 contributing to the binding affinity of an antibody or antigen binding fragment to the antigen or epitope it recognizes. 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, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining regions are denoted by LCDR and 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. The invention adopts Kabat annotation standard to mark CDR regions, but other methods to mark CDR regions also belong to the protection scope of the invention.
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 alternative embodiments, the antibody or functional fragment thereof further comprises HFR1, HFR2, HFR3, HFR4 having amino acid sequences shown in SEQ ID NO. 7 through SEQ ID NO. 10, and LFR1, LFR2, LFR3 and LFR4 having amino acid sequences shown in SEQ ID NO. 11 through SEQ ID NO. 14.
In other embodiments, each framework region amino acid sequence of an antibody or functional fragment 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% homology to the corresponding framework region (SEQ ID NO:7, 8, 9, 10, 11, 12, 13 or 14) described above.
In an alternative embodiment, the antibody or functional fragment thereof has a KD of 10 or less -7 The affinity of M binds testosterone.
In an alternative embodiment, the antibody or functional fragment thereof has a KD of 3.25X10 ∈ -8 The affinity of M binds testosterone.
In an alternative embodiment, the antibody or functional fragment thereof has a KD of 10 or less -8 M、KD≤10 -9 M、KD≤10 -10 M、KD≤10 -11 The affinity of M binds testosterone.
In alternative embodiments, the antibody orThe functional fragment thereof has KD less than or equal to 8.44×10 -9 The affinity of M binds testosterone.
In an alternative embodiment, LFR1 of said antibody or functional fragment thereof is shown as SEQ ID NO. 16.
In an alternative embodiment, LFR2 of said antibody or functional fragment thereof is shown in SEQ ID NO. 17.
In another aspect, embodiments of the present invention provide an anti-testosterone antibody or a functional fragment thereof, the antibody or the functional fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprises a sequence structure of HFR1-HCDR 2-HFR3-HCDR3-HFR4, the light chain variable region comprises a sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, and the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, the amino acid sequence of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR4 is the amino acid sequence of HFR1, HFR2, HFR3, LFR4, LFR2, LFR4.
In an alternative embodiment, the heavy chain variable region amino acid sequence is set forth in any one of SEQ ID NOs 18 to 22.
In an alternative embodiment, the light chain variable region amino acid sequence is set forth in SEQ ID NOS.23 to 27.
In alternative embodiments, the antibody 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 ovine species.
In an alternative embodiment, the constant region is of a species origin from goat.
In an alternative embodiment, the heavy chain constant region sequence (CH) is shown in SEQ ID NO. 28 and the light chain constant region (CL) sequence is shown in SEQ ID NO. 29.
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% homology to the above-described constant region (SEQ ID NO:28 or 29).
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 another aspect, the invention provides an anti-testosterone antibody comprising a heavy chain and/or a light chain, said heavy chain comprising a heavy chain variable region as described above and a heavy chain constant region as described above; the light chain comprises the light chain variable region described above and the light chain constant region described above.
In an alternative embodiment, the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NOs 30 to 34.
In an alternative embodiment, the amino acid sequence of the light chain is as shown in any one of SEQ ID NOs 35 to 39.
In another aspect, the invention provides an antibody conjugate comprising an antibody or functional fragment thereof as described above.
In an alternative embodiment, the antibody or functional fragment thereof in the above antibody conjugate is labeled with a label.
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 (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 212 Bi、 131 I、 111 In、 90 Y、 186 Re、 211 At、 125 I、 188 Re、 153 Sm、 213 Bi、 32 P、 94 mTc、 99 mTc、 203 Pb、 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn、 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb、 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F。
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 antibody or functional fragment thereof in the above antibody conjugate is coated onto a solid phase.
In alternative embodiments, the solid phase 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 phase is a nitrocellulose membrane.
In another aspect, the invention provides a reagent or kit for detecting testosterone, comprising an antibody or functional fragment thereof or an antibody conjugate as described above.
In another aspect, the invention provides the use of an antibody as described above or a functional fragment thereof, an antibody conjugate or a reagent or kit as described above in the detection of testosterone.
In another aspect, the invention provides a nucleic acid molecule encoding an antibody or functional fragment thereof as described above.
In another aspect, the invention provides a vector comprising the nucleic acid molecule described above.
In another aspect, the invention provides a cell comprising the vector described above.
In another aspect, the invention provides a method of making an antibody or functional fragment thereof comprising: the cells as described above were cultured.
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-Testo-33A9 monoclonal antibody
Restriction enzymes, prime Star DNA polymerase in this example were purchased from Takara Corp. MagExtractor-RNA extraction kit was purchased from TOYOBO Co. 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 Invitrogen corporation.
1 construction of recombinant plasmid
(1) Antibody Gene production
mRNA is extracted from hybridoma cell strains secreting monoclonal antibodies against testosterone, DNA products are obtained through an RT-PCR method, the products are inserted into a pMD-18T vector after an A adding reaction by rTaq DNA polymerase, the products are transformed into DH5 alpha competent cells, after colonies grow out, the Heavy Chain gene clone and the Light Chain gene clone are respectively taken, and 4 clones are sent to a gene sequencing company for sequencing.
(2) Sequence analysis of variable region Gene of Anti-Testo-33A9 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 321bp, 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 375bp, belongs to the VH1 gene family, and a 57bp leader peptide sequence is arranged in front of the VH gene sequence.
(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 introduced into a HindIII, bamHI, ecoRI polyclonal enzyme cutting site, named pcDNA3.4A expression vector and is hereinafter abbreviated as 3.4A expression vector; according to the result of the antibody variable region gene sequencing in pMD-18T, VL and VH gene specific primers of the antibody are designed, hindIII, ecoRI restriction sites and protective bases are respectively arranged at two ends, and a 0.72kb Light Chain gene fragment and a 1.45kb Heavy Chain gene fragment are amplified by a PCR amplification method.
The Heavy Chain gene fragment and the Light Chain gene fragment are respectively cut by HindIII/EcoRI double enzyme, the 3.4A vector is cut by HindIII/EcoRI double enzyme, and the Heavy Chain gene fragment and the Light Chain gene fragment after the fragment and the vector are purified and recovered are respectively connected into the 3.4A expression vector to respectively obtain recombinant expression plasmids of the Heavy Chain gene fragment and the Light Chain gene fragment.
2 stable cell line selection
(1) Recombinant antibody expression plasmid transient transfection CHO cells, determination of expression plasmid activity
The plasmid was diluted to40 ug/100ul with ultrapure water and CHO cells were conditioned 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 (main ingredient NaHCO 3) was diluted to Testo-BSA (available from Boyue organism, testo 401) to 3ug/ml, 100 μl per well, overnight at 4deg.C; the next day, washing with washing liquid for 2 times, and drying; 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 mouse anti-sheep IgG-HRP, 100 μl/well, 37deg.C, 30min; washing with washing liquid for 5 times, and drying; adding a color development solution A (50 mu L/hole, containing citric acid, sodium acetate, acetanilide and carbamide peroxide), and adding a color development solution B (50 mu L/hole, containing citric acid, EDTA, 2Na+TMB and concentrated HCL) for 10min; adding stop solution (50. Mu.L/well, EDTA. 2Na+ concentrated H) 2 SO 4 ) The method comprises the steps of carrying out a first treatment on the surface of the OD was read on the microplate reader at 450nm (reference 630 nm). The results showed that the OD of the reaction was still greater than 1.0 after 1000-fold dilution of the cell supernatant, and that the OD of the reaction was less than 0.1 without cell supernatant, indicating that antibodies generated after transient transformation of the plasmid were active against Testo-BSA (purchased from the bezoar organism, testo 401).
(2) Linearization of recombinant antibody expression plasmids
The following reagents were prepared: buffer 50 mu L, DNA mu g/tube, puv I enzyme 10 mu L, sterile water to 500 mu L, water bath at 37 ℃ for enzyme digestion 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.
(3) Stable transfection of recombinant antibody expression plasmid and pressure screening of stable cell strain
The plasmid was diluted to40 ug/100ul with ultrapure water and CHO cells were conditioned 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; 25umol/LMSX 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
(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 inoculating density, and calculating the expanding culture volume according to production requirements, 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.
(2) Shake 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. 6 μg of purified antibody was subjected to reducing SDS-PAGE, and the electrophoretogram shows two bands after reducing SDS-PAGE, 1 Mr at 50KD and the other Mr at 28KD.
Example 2 affinity and Activity optimization
The Anti-Testo-33A9 monoclonal antibody obtained in example 1 has a testosterone-binding ability, but has insufficient affinity and antibody activity, and thus the applicant has performed directed mutation on the light chain CDRs and the heavy chain CDRs 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. Monoclonal antibodies with remarkably improved affinity and antibody activity are obtained through screening and are named as: anti-Testo-33A9mut1 to Anti-Testo-33A9mut5. The amino acid sequences of the respective monoclonal antibodies are shown in the following table:
TABLE 1 antibody sequences
Sample name Heavy chain sequence number Light chain sequence number
Anti-Testo-33A9mut1 SEQ ID NO:30 SEQ ID NO:35
Anti-Testo-33A9mut2 SEQ ID NO:31 SEQ ID NO:36
Anti-Testo-33A9mut3 SEQ ID NO:32 SEQ ID NO:37
Anti-Testo-33A9mut4 SEQ ID NO:33 SEQ ID NO:38
Anti-Testo-33A9mut5 SEQ ID NO:34 SEQ ID NO:39
Example 3 detection of Performance of antibodies
1 affinity assay
Using the AMC sensor, purified antibodies were diluted to 10ug/ml with PBST, and Testo-BSA (available from Boyue organism, testo 401) was gradient diluted with PBST:
the operation flow is as follows: equilibration for 60s in buffer 1 (PBST), antibody 300s in antibody solution, incubation for 180s in buffer 2 (PBST), binding for 420s in antigen solution, dissociation for 1200s in buffer 2, sensor regeneration with 10mM pH 1.69GLY solution and buffer 3 (PBST), output data. (KD represents equilibrium dissociation constant, i.e., affinity; kon represents binding rate; kdis represents dissociation rate. PBST major component Na2 HPO4+NaCl+TW-20).
Table 2 affinity data
Sample name KD(M) kon(1/Ms) kdis(1/s)
Control 3.25E-08 1.57E+04 5.10E-04
Anti-Testo-33A9mut1 8.44E-09 2.61E+04 2.20E-04
Anti-Testo-33A9mut2 2.34E-10 1.11E+06 2.60E-04
Anti-Testo-33A9mut3 1.99E-10 2.01E+06 4.00E-04
Anti-Testo-33A9mut4 1.26E-10 3.02E+06 3.80E-04
Anti-Testo-33A9mut5 2.22E-10 1.58E+06 3.50E-04
2 Activity assay
Coating (main ingredient NaHCO 3) was diluted to Testo-BSA (available from Boyue organism, testo 401) to 3ug/ml, 100 μl per well, overnight at 4deg.C; the next day, washing with washing liquid for 2 times, and drying; blocking solution (20% BSA+80% PBS) was added, 120. Mu.l per well, 37℃for 1h, and the mixture was dried by pipetting; adding the diluted monoclonal antibody into the mixture at the temperature of 37 ℃ for 30-60 min at the concentration of 100 mu l/hole; washing with washing liquid for 5 times, and drying; adding mouse anti-sheep IgG-HRP, 100 μl/well, 37deg.C, 30min; washing with washing solution (PBS) for 5 times, and drying; adding a color development solution A (50. Mu.l/hole, containing 2.1g/L citric acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L carbamide peroxide), and adding a color development solution B (50. Mu.l/hole, containing 1.05g/L citric acid, 0.186 g/LEDTA.2Na, 0.45g/L TMB and 0.2ml/L concentrated HCl) for 10min; adding stop solution (50. Mu.l/well, 0.75 g/EDTA.2Na and 10.2ml/L of concentrated H) 2 SO 4 ) The method comprises the steps of carrying out a first treatment on the surface of the OD was read on the microplate reader at 450nm (reference 630 nm). The results are shown in Table 3 below. By the same method, the coating solution was used to dilute BSA, and it was confirmed that the reaction OD of the monoclonal antibody with BSA was less than 0.05, and that the monoclonal antibody was not cross-reactive with BSA.
TABLE 3 Activity data
3 stability assessment
Placing the antibody at 4 ℃ (refrigerator), 80 ℃ (refrigerator) and 37 ℃ (incubator) for 21 days, taking 7 days, 14 days and 21 days samples for state observation, and detecting the activity of the 21 days samples, wherein the result shows that no obvious protein state change is seen for 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 4 below shows the results of the detection of OD after 21 days of antibody examination.
Table 4 stability data
Sample concentration (ng/m 1) 125 31.25 0
4 ℃,21 days sample 2.340 1.060 0.048
Sample at-80℃for 21 days 2.356 1.095 0.045
37 ℃ and 21 days of sample 2.345 1.083 0.052
4 detection of correlation
The self-produced antibody is used for detecting clinical standard, and is compared with the detection result of the foreign main stream antibody in the domestic market, and the result shows that: the correlation between the self-produced antibody and the foreign mainstream antibody in clinical standard detection reaches more than 0.95.
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:
numbering device Sequence fragments
SEQ ID NO：1 NNDET
SEQ ID NO：2 GISSFGITYLNPALKS
SEQ ID NO：3 VGGNHYRAYAYGYGSYF
SEQ ID NO：4 SGMNIGSVAVG
SEQ ID NO：5 YDATRAS
SEQ ID NO：6 GSVFGLGY
SEQ ID NO：7 SGMNLGSVAVG
SEQUENCE LISTING
<110> Dongguan City, pengzhi biotechnology Co., ltd
<120> anti-testosterone antibodies, reagents and kits for detecting testosterone
<130> P2022024CN01
<160> 39
<170> PatentIn version 3.3
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Ser Arg Leu Ser Ile Thr Arg Asp Ala Ser Lys Asn Gln Val Ser Leu
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Ser Leu Ser Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val
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Gln Val Arg Leu Gln Glu Ser Gly Pro Asn Leu Val Lys Pro Ser Gln
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Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu
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Tyr Asp Ala Thr Arg Ala Ser Gly Val Pro Asp Arg Phe Ser Ala Ser
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Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu
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Asp Thr Ser Ser Ser Ile Val Thr Leu Gly Cys Leu Val Ser Ser Tyr
20 25 30
Met Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Ile Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ala Ser Thr Ser Gly Ala Gln Thr
65 70 75 80
Phe Ile Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Pro Gly Cys Pro Asp Pro Cys Lys His Cys Arg Cys Pro
100 105 110
Pro Pro Glu Leu Pro Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys
115 120 125
Pro Lys Asp Thr Leu Thr Ile Ser Gly Thr Pro Glu Val Thr Cys Val
130 135 140
Val Val Asp Val Gly Gln Asp Asp Pro Glu Val Gln Phe Ser Trp Phe
145 150 155 160
Val Asp Asn Val Glu Val Arg Thr Ala Arg Thr Lys Pro Arg Glu Glu
165 170 175
Gln Phe Asn Ser Thr Phe Arg Val Val Ser Ala Leu Pro Ile Gln His
180 185 190
Gln Asp Trp Thr Gly Gly Lys Glu Phe Lys Cys Lys Val His Asn Glu
195 200 205
Gly Leu Pro Ala Pro Ile Val Arg Thr Ile Ser Arg Thr Lys Gly Gln
210 215 220
Ala Arg Glu Pro Gln Val Tyr Val Leu Ala Pro Pro Gln Glu Glu Leu
225 230 235 240
Ser Lys Ser Thr Leu Ser Val Thr Cys Leu Val Thr Gly Phe Tyr Pro
245 250 255
Asp Tyr Ile Ala Val Glu Trp Gln Lys Asn Gly Gln Pro Glu Ser Glu
260 265 270
Asp Lys Tyr Gly Thr Thr Thr Ser Gln Leu Asp Ala Asp Gly Ser Tyr
275 280 285
Phe Leu Tyr Ser Arg Leu Arg Val Asp Lys Asn Ser Trp Gln Glu Gly
290 295 300
Asp Thr Tyr Ala Cys Val Val Met His Glu Ala Leu His Asn His Tyr
305 310 315 320
Thr Gln Lys Ser Ile Ser Lys Pro Pro Gly Lys
325 330
<210> 29
<211> 105
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 29
Gln Pro Lys Ser Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Thr Glu
1 5 10 15
Glu Leu Ser Thr Asn Lys Ala Thr Val Val Cys Leu Ile Asn Asp Phe
20 25 30
Tyr Pro Gly Ser Val Asn Val Val Trp Lys Ala Asp Gly Ser Thr Ile
35 40 45
Asn Gln Asn Val Lys Thr Thr Gln Ala Ser Lys Gln Ser Asn Ser Lys
50 55 60
Tyr Ala Ala Ser Ser Tyr Leu Thr Leu Thr Gly Ser Glu Trp Lys Ser
65 70 75 80
Lys Ser Ser Tyr Thr Cys Glu Val Thr His Glu Gly Ser Thr Val Thr
85 90 95
Lys Thr Val Lys Pro Ser Glu Cys Ser
100 105
<210> 30
<211> 456
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 30
Gln Val Arg Leu Gln Glu Ser Gly Pro Asn Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Ile Ser Gly Phe Ser Leu Thr Asn Asn
20 25 30
Asp Glu Thr Trp Val Arg Arg Val Pro Gly Lys Val Pro Glu Trp Leu
35 40 45
Gly Gly Ile Ser Ser Phe Gly Ile Thr Tyr Leu Asn Pro Ala Leu Lys
50 55 60
Ser Arg Leu Ser Ile Thr Arg Asp Ala Ser Lys Asn Gln Val Ser Leu
65 70 75 80
Ser Leu Ser Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val
85 90 95
Gly Gly Asn His Tyr Arg Ala Tyr Ala Tyr Gly Tyr Gly Ser Tyr Phe
100 105 110
Glu Tyr Trp Gly Pro Gly Leu Leu Val Thr Val Ser Ser Ala Ser Thr
115 120 125
Thr Pro Pro Lys Val Tyr Pro Leu Thr Ser Cys Cys Gly Asp Thr Ser
130 135 140
Ser Ser Ile Val Thr Leu Gly Cys Leu Val Ser Ser Tyr Met Pro Glu
145 150 155 160
Pro Val Thr Val Thr Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
165 170 175
Thr Phe Pro Ala Ile Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190
Val Val Thr Val Pro Ala Ser Thr Ser Gly Ala Gln Thr Phe Ile Cys
195 200 205
Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Arg Val Glu
210 215 220
Pro Gly Cys Pro Asp Pro Cys Lys His Cys Arg Cys Pro Pro Pro Glu
225 230 235 240
Leu Pro Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp
245 250 255
Thr Leu Thr Ile Ser Gly Thr Pro Glu Val Thr Cys Val Val Val Asp
260 265 270
Val Gly Gln Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asn
275 280 285
Val Glu Val Arg Thr Ala Arg Thr Lys Pro Arg Glu Glu Gln Phe Asn
290 295 300
Ser Thr Phe Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp
305 310 315 320
Thr Gly Gly Lys Glu Phe Lys Cys Lys Val His Asn Glu Gly Leu Pro
325 330 335
Ala Pro Ile Val Arg Thr Ile Ser Arg Thr Lys Gly Gln Ala Arg Glu
340 345 350
Pro Gln Val Tyr Val Leu Ala Pro Pro Gln Glu Glu Leu Ser Lys Ser
355 360 365
Thr Leu Ser Val Thr Cys Leu Val Thr Gly Phe Tyr Pro Asp Tyr Ile
370 375 380
Ala Val Glu Trp Gln Lys Asn Gly Gln Pro Glu Ser Glu Asp Lys Tyr
385 390 395 400
Gly Thr Thr Thr Ser Gln Leu Asp Ala Asp Gly Ser Tyr Phe Leu Tyr
405 410 415
Ser Arg Leu Arg Val Asp Lys Asn Ser Trp Gln Glu Gly Asp Thr Tyr
420 425 430
Ala Cys Val Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
435 440 445
Ser Ile Ser Lys Pro Pro Gly Lys
450 455
<210> 31
<211> 456
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 31
Gln Val Arg Leu Gln Glu Ser Gly Pro Asn Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Ile Ser Gly Phe Ser Leu Thr Asn Asn
20 25 30
Asp Glu Thr Trp Val Arg Arg Val Pro Gly Lys Val Pro Glu Trp Leu
35 40 45
Gly Gly Ile Ser Ser Phe Gly Ile Thr Tyr Leu Asn Pro Ala Leu Lys
50 55 60
Ser Arg Leu Ser Ile Thr Arg Asp Ala Ser Lys Asn Gln Val Ser Leu
65 70 75 80
Ser Leu Ser Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val
85 90 95
Gly Gly Asn His Tyr Arg Ala Tyr Ala Tyr Gly Tyr Gly Ser Tyr Phe
100 105 110
Glu Tyr Trp Gly Pro Gly Leu Leu Val Thr Val Ser Ser Ala Ser Thr
115 120 125
Thr Pro Pro Lys Val Tyr Pro Leu Thr Ser Cys Cys Gly Asp Thr Ser
130 135 140
Ser Ser Ile Val Thr Leu Gly Cys Leu Val Ser Ser Tyr Met Pro Glu
145 150 155 160
Pro Val Thr Val Thr Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
165 170 175
Thr Phe Pro Ala Ile Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190
Val Val Thr Val Pro Ala Ser Thr Ser Gly Ala Gln Thr Phe Ile Cys
195 200 205
Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Arg Val Glu
210 215 220
Pro Gly Cys Pro Asp Pro Cys Lys His Cys Arg Cys Pro Pro Pro Glu
225 230 235 240
Leu Pro Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp
245 250 255
Thr Leu Thr Ile Ser Gly Thr Pro Glu Val Thr Cys Val Val Val Asp
260 265 270
Val Gly Gln Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asn
275 280 285
Val Glu Val Arg Thr Ala Arg Thr Lys Pro Arg Glu Glu Gln Phe Asn
290 295 300
Ser Thr Phe Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp
305 310 315 320
Thr Gly Gly Lys Glu Phe Lys Cys Lys Val His Asn Glu Gly Leu Pro
325 330 335
Ala Pro Ile Val Arg Thr Ile Ser Arg Thr Lys Gly Gln Ala Arg Glu
340 345 350
Pro Gln Val Tyr Val Leu Ala Pro Pro Gln Glu Glu Leu Ser Lys Ser
355 360 365
Thr Leu Ser Val Thr Cys Leu Val Thr Gly Phe Tyr Pro Asp Tyr Ile
370 375 380
Ala Val Glu Trp Gln Lys Asn Gly Gln Pro Glu Ser Glu Asp Lys Tyr
385 390 395 400
Gly Thr Thr Thr Ser Gln Leu Asp Ala Asp Gly Ser Tyr Phe Leu Tyr
405 410 415
Ser Arg Leu Arg Val Asp Lys Asn Ser Trp Gln Glu Gly Asp Thr Tyr
420 425 430
Ala Cys Val Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
435 440 445
Ser Ile Ser Lys Pro Pro Gly Lys
450 455
<210> 32
<211> 456
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 32
Gln Val Arg Leu Gln Glu Ser Gly Pro Asn Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Ile Ser Gly Phe Ser Leu Thr Asn Asn
20 25 30
Asp Glu Thr Trp Val Arg Arg Val Pro Gly Lys Val Pro Glu Trp Leu
35 40 45
Gly Gly Ile Ser Ser Phe Gly Ile Thr Tyr Leu Asn Pro Ala Leu Lys
50 55 60
Ser Arg Leu Ser Ile Thr Arg Asp Ala Ser Lys Asn Gln Val Ser Leu
65 70 75 80
Ser Leu Ser Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val
85 90 95
Gly Gly Asn His Tyr Arg Ala Tyr Ala Tyr Gly Tyr Gly Ser Tyr Phe
100 105 110
Glu Tyr Trp Gly Pro Gly Leu Leu Val Thr Val Ser Ser Ala Ser Thr
115 120 125
Thr Pro Pro Lys Val Tyr Pro Leu Thr Ser Cys Cys Gly Asp Thr Ser
130 135 140
Ser Ser Ile Val Thr Leu Gly Cys Leu Val Ser Ser Tyr Met Pro Glu
145 150 155 160
Pro Val Thr Val Thr Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
165 170 175
Thr Phe Pro Ala Ile Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190
Val Val Thr Val Pro Ala Ser Thr Ser Gly Ala Gln Thr Phe Ile Cys
195 200 205
Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Arg Val Glu
210 215 220
Pro Gly Cys Pro Asp Pro Cys Lys His Cys Arg Cys Pro Pro Pro Glu
225 230 235 240
Leu Pro Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp
245 250 255
Thr Leu Thr Ile Ser Gly Thr Pro Glu Val Thr Cys Val Val Val Asp
260 265 270
Val Gly Gln Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asn
275 280 285
Val Glu Val Arg Thr Ala Arg Thr Lys Pro Arg Glu Glu Gln Phe Asn
290 295 300
Ser Thr Phe Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp
305 310 315 320
Thr Gly Gly Lys Glu Phe Lys Cys Lys Val His Asn Glu Gly Leu Pro
325 330 335
Ala Pro Ile Val Arg Thr Ile Ser Arg Thr Lys Gly Gln Ala Arg Glu
340 345 350
Pro Gln Val Tyr Val Leu Ala Pro Pro Gln Glu Glu Leu Ser Lys Ser
355 360 365
Thr Leu Ser Val Thr Cys Leu Val Thr Gly Phe Tyr Pro Asp Tyr Ile
370 375 380
Ala Val Glu Trp Gln Lys Asn Gly Gln Pro Glu Ser Glu Asp Lys Tyr
385 390 395 400
Gly Thr Thr Thr Ser Gln Leu Asp Ala Asp Gly Ser Tyr Phe Leu Tyr
405 410 415
Ser Arg Leu Arg Val Asp Lys Asn Ser Trp Gln Glu Gly Asp Thr Tyr
420 425 430
Ala Cys Val Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
435 440 445
Ser Ile Ser Lys Pro Pro Gly Lys
450 455
<210> 33
<211> 456
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 33
Gln Val Arg Leu Gln Glu Ser Gly Pro Asn Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Ile Ser Gly Phe Ser Leu Thr Asn Asn
20 25 30
Asp Glu Thr Trp Val Arg Arg Val Pro Gly Lys Val Pro Glu Trp Leu
35 40 45
Gly Gly Ile Ser Ser Phe Gly Ile Thr Tyr Leu Asn Pro Ala Leu Lys
50 55 60
Ser Arg Leu Ser Ile Thr Arg Asp Ala Ser Lys Asn Gln Val Ser Leu
65 70 75 80
Ser Leu Ser Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val
85 90 95
Gly Gly Asn His Tyr Arg Ala Tyr Ala Tyr Gly Tyr Gly Ser Tyr Phe
100 105 110
Glu Tyr Trp Gly Pro Gly Leu Leu Val Thr Val Ser Ser Ala Ser Thr
115 120 125
Thr Pro Pro Lys Val Tyr Pro Leu Thr Ser Cys Cys Gly Asp Thr Ser
130 135 140
Ser Ser Ile Val Thr Leu Gly Cys Leu Val Ser Ser Tyr Met Pro Glu
145 150 155 160
Pro Val Thr Val Thr Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
165 170 175
Thr Phe Pro Ala Ile Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190
Val Val Thr Val Pro Ala Ser Thr Ser Gly Ala Gln Thr Phe Ile Cys
195 200 205
Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Arg Val Glu
210 215 220
Pro Gly Cys Pro Asp Pro Cys Lys His Cys Arg Cys Pro Pro Pro Glu
225 230 235 240
Leu Pro Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp
245 250 255
Thr Leu Thr Ile Ser Gly Thr Pro Glu Val Thr Cys Val Val Val Asp
260 265 270
Val Gly Gln Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asn
275 280 285
Val Glu Val Arg Thr Ala Arg Thr Lys Pro Arg Glu Glu Gln Phe Asn
290 295 300
Ser Thr Phe Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp
305 310 315 320
Thr Gly Gly Lys Glu Phe Lys Cys Lys Val His Asn Glu Gly Leu Pro
325 330 335
Ala Pro Ile Val Arg Thr Ile Ser Arg Thr Lys Gly Gln Ala Arg Glu
340 345 350
Pro Gln Val Tyr Val Leu Ala Pro Pro Gln Glu Glu Leu Ser Lys Ser
355 360 365
Thr Leu Ser Val Thr Cys Leu Val Thr Gly Phe Tyr Pro Asp Tyr Ile
370 375 380
Ala Val Glu Trp Gln Lys Asn Gly Gln Pro Glu Ser Glu Asp Lys Tyr
385 390 395 400
Gly Thr Thr Thr Ser Gln Leu Asp Ala Asp Gly Ser Tyr Phe Leu Tyr
405 410 415
Ser Arg Leu Arg Val Asp Lys Asn Ser Trp Gln Glu Gly Asp Thr Tyr
420 425 430
Ala Cys Val Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
435 440 445
Ser Ile Ser Lys Pro Pro Gly Lys
450 455
<210> 34
<211> 456
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 34
Gln Val Arg Leu Gln Glu Ser Gly Pro Asn Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Ile Ser Gly Phe Ser Leu Thr Asn Asn
20 25 30
Asp Glu Thr Trp Val Arg Arg Val Pro Gly Lys Val Pro Glu Trp Leu
35 40 45
Gly Gly Ile Ser Ser Phe Gly Ile Thr Tyr Leu Asn Pro Ala Leu Lys
50 55 60
Ser Arg Leu Ser Ile Thr Arg Asp Ala Ser Lys Asn Gln Val Ser Leu
65 70 75 80
Ser Leu Ser Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val
85 90 95
Gly Gly Asn His Tyr Arg Ala Tyr Ala Tyr Gly Tyr Gly Ser Tyr Phe
100 105 110
Glu Tyr Trp Gly Pro Gly Leu Leu Val Thr Val Ser Ser Ala Ser Thr
115 120 125
Thr Pro Pro Lys Val Tyr Pro Leu Thr Ser Cys Cys Gly Asp Thr Ser
130 135 140
Ser Ser Ile Val Thr Leu Gly Cys Leu Val Ser Ser Tyr Met Pro Glu
145 150 155 160
Pro Val Thr Val Thr Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
165 170 175
Thr Phe Pro Ala Ile Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190
Val Val Thr Val Pro Ala Ser Thr Ser Gly Ala Gln Thr Phe Ile Cys
195 200 205
Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Arg Val Glu
210 215 220
Pro Gly Cys Pro Asp Pro Cys Lys His Cys Arg Cys Pro Pro Pro Glu
225 230 235 240
Leu Pro Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp
245 250 255
Thr Leu Thr Ile Ser Gly Thr Pro Glu Val Thr Cys Val Val Val Asp
260 265 270
Val Gly Gln Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asn
275 280 285
Val Glu Val Arg Thr Ala Arg Thr Lys Pro Arg Glu Glu Gln Phe Asn
290 295 300
Ser Thr Phe Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp
305 310 315 320
Thr Gly Gly Lys Glu Phe Lys Cys Lys Val His Asn Glu Gly Leu Pro
325 330 335
Ala Pro Ile Val Arg Thr Ile Ser Arg Thr Lys Gly Gln Ala Arg Glu
340 345 350
Pro Gln Val Tyr Val Leu Ala Pro Pro Gln Glu Glu Leu Ser Lys Ser
355 360 365
Thr Leu Ser Val Thr Cys Leu Val Thr Gly Phe Tyr Pro Asp Tyr Ile
370 375 380
Ala Val Glu Trp Gln Lys Asn Gly Gln Pro Glu Ser Glu Asp Lys Tyr
385 390 395 400
Gly Thr Thr Thr Ser Gln Leu Asp Ala Asp Gly Ser Tyr Phe Leu Tyr
405 410 415
Ser Arg Leu Arg Val Asp Lys Asn Ser Trp Gln Glu Gly Asp Thr Tyr
420 425 430
Ala Cys Val Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
435 440 445
Ser Ile Ser Lys Pro Pro Gly Lys
450 455
<210> 35
<211> 212
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 35
Gln Ala Val Leu Thr Gln Pro Pro Ser Val Ser Glu Ser Leu Gly Gln
1 5 10 15
Arg Val Ser Leu Ser Cys Ser Gly Met Asn Ile Gly Ser Val Ala Val
20 25 30
Gly Trp Phe Gln Gln Val Pro Glu Ser Gly Leu Arg Thr Val Ile Thr
35 40 45
Tyr Asp Ala Thr Arg Ala Ser Gly Val Pro Asp Arg Phe Ser Ala Ser
50 55 60
Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu
65 70 75 80
Asp Glu Ala Glu Tyr Phe Cys Gly Ser Val Phe Gly Leu Gly Tyr Val
85 90 95
Phe Gly Ser Gly Thr Arg Leu Thr Val Leu Gly Gln Pro Lys Ser Ala
100 105 110
Pro Ser Val Thr Leu Phe Pro Pro Ser Thr Glu Glu Leu Ser Thr Asn
115 120 125
Lys Ala Thr Val Val Cys Leu Ile Asn Asp Phe Tyr Pro Gly Ser Val
130 135 140
Asn Val Val Trp Lys Ala Asp Gly Ser Thr Ile Asn Gln Asn Val Lys
145 150 155 160
Thr Thr Gln Ala Ser Lys Gln Ser Asn Ser Lys Tyr Ala Ala Ser Ser
165 170 175
Tyr Leu Thr Leu Thr Gly Ser Glu Trp Lys Ser Lys Ser Ser Tyr Thr
180 185 190
Cys Glu Val Thr His Glu Gly Ser Thr Val Thr Lys Thr Val Lys Pro
195 200 205
Ser Glu Cys Ser
210
<210> 36
<211> 212
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 36
Gln Ala Val Leu Thr Gln Pro Pro Ser Val Ser Glu Ser Leu Gly Gln
1 5 10 15
Arg Val Ser Leu Ser Cys Ser Gly Met Asn Ile Gly Ser Val Ala Val
20 25 30
Gly Trp Phe Gln Gln Val Pro Glu Ser Gly Leu Arg Thr Val Leu Thr
35 40 45
Tyr Asp Ala Thr Arg Ala Ser Gly Val Pro Asp Arg Phe Ser Ala Ser
50 55 60
Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu
65 70 75 80
Asp Glu Ala Glu Tyr Phe Cys Gly Ser Val Phe Gly Leu Gly Tyr Val
85 90 95
Phe Gly Ser Gly Thr Arg Leu Thr Val Leu Gly Gln Pro Lys Ser Ala
100 105 110
Pro Ser Val Thr Leu Phe Pro Pro Ser Thr Glu Glu Leu Ser Thr Asn
115 120 125
Lys Ala Thr Val Val Cys Leu Ile Asn Asp Phe Tyr Pro Gly Ser Val
130 135 140
Asn Val Val Trp Lys Ala Asp Gly Ser Thr Ile Asn Gln Asn Val Lys
145 150 155 160
Thr Thr Gln Ala Ser Lys Gln Ser Asn Ser Lys Tyr Ala Ala Ser Ser
165 170 175
Tyr Leu Thr Leu Thr Gly Ser Glu Trp Lys Ser Lys Ser Ser Tyr Thr
180 185 190
Cys Glu Val Thr His Glu Gly Ser Thr Val Thr Lys Thr Val Lys Pro
195 200 205
Ser Glu Cys Ser
210
<210> 37
<211> 212
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 37
Gln Ala Val Leu Thr Gln Pro Pro Ser Val Ser Glu Ser Leu Gly Gln
1 5 10 15
Arg Val Ser Ile Ser Cys Ser Gly Met Asn Ile Gly Ser Val Ala Val
20 25 30
Gly Trp Phe Gln Gln Val Pro Glu Ser Gly Leu Arg Thr Val Leu Thr
35 40 45
Tyr Asp Ala Thr Arg Ala Ser Gly Val Pro Asp Arg Phe Ser Ala Ser
50 55 60
Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu
65 70 75 80
Asp Glu Ala Glu Tyr Phe Cys Gly Ser Val Phe Gly Leu Gly Tyr Val
85 90 95
Phe Gly Ser Gly Thr Arg Leu Thr Val Leu Gly Gln Pro Lys Ser Ala
100 105 110
Pro Ser Val Thr Leu Phe Pro Pro Ser Thr Glu Glu Leu Ser Thr Asn
115 120 125
Lys Ala Thr Val Val Cys Leu Ile Asn Asp Phe Tyr Pro Gly Ser Val
130 135 140
Asn Val Val Trp Lys Ala Asp Gly Ser Thr Ile Asn Gln Asn Val Lys
145 150 155 160
Thr Thr Gln Ala Ser Lys Gln Ser Asn Ser Lys Tyr Ala Ala Ser Ser
165 170 175
Tyr Leu Thr Leu Thr Gly Ser Glu Trp Lys Ser Lys Ser Ser Tyr Thr
180 185 190
Cys Glu Val Thr His Glu Gly Ser Thr Val Thr Lys Thr Val Lys Pro
195 200 205
Ser Glu Cys Ser
210
<210> 38
<211> 212
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 38
Gln Ala Val Leu Thr Gln Pro Pro Ser Val Ser Glu Ser Leu Gly Gln
1 5 10 15
Arg Val Ser Ile Ser Cys Ser Gly Met Asn Leu Gly Ser Val Ala Val
20 25 30
Gly Trp Phe Gln Gln Val Pro Glu Ser Gly Leu Arg Thr Val Ile Thr
35 40 45
Tyr Asp Ala Thr Arg Ala Ser Gly Val Pro Asp Arg Phe Ser Ala Ser
50 55 60
Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu
65 70 75 80
Asp Glu Ala Glu Tyr Phe Cys Gly Ser Val Phe Gly Leu Gly Tyr Val
85 90 95
Phe Gly Ser Gly Thr Arg Leu Thr Val Leu Gly Gln Pro Lys Ser Ala
100 105 110
Pro Ser Val Thr Leu Phe Pro Pro Ser Thr Glu Glu Leu Ser Thr Asn
115 120 125
Lys Ala Thr Val Val Cys Leu Ile Asn Asp Phe Tyr Pro Gly Ser Val
130 135 140
Asn Val Val Trp Lys Ala Asp Gly Ser Thr Ile Asn Gln Asn Val Lys
145 150 155 160
Thr Thr Gln Ala Ser Lys Gln Ser Asn Ser Lys Tyr Ala Ala Ser Ser
165 170 175
Tyr Leu Thr Leu Thr Gly Ser Glu Trp Lys Ser Lys Ser Ser Tyr Thr
180 185 190
Cys Glu Val Thr His Glu Gly Ser Thr Val Thr Lys Thr Val Lys Pro
195 200 205
Ser Glu Cys Ser
210
<210> 39
<211> 212
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 39
Gln Ala Val Leu Thr Gln Pro Pro Ser Val Ser Glu Ser Leu Gly Gln
1 5 10 15
Arg Val Ser Ile Ser Cys Ser Gly Met Asn Leu Gly Ser Val Ala Val
20 25 30
Gly Trp Phe Gln Gln Val Pro Glu Ser Gly Leu Arg Thr Val Leu Thr
35 40 45
Tyr Asp Ala Thr Arg Ala Ser Gly Val Pro Asp Arg Phe Ser Ala Ser
50 55 60
Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu
65 70 75 80
Asp Glu Ala Glu Tyr Phe Cys Gly Ser Val Phe Gly Leu Gly Tyr Val
85 90 95
Phe Gly Ser Gly Thr Arg Leu Thr Val Leu Gly Gln Pro Lys Ser Ala
100 105 110
Pro Ser Val Thr Leu Phe Pro Pro Ser Thr Glu Glu Leu Ser Thr Asn
115 120 125
Lys Ala Thr Val Val Cys Leu Ile Asn Asp Phe Tyr Pro Gly Ser Val
130 135 140
Asn Val Val Trp Lys Ala Asp Gly Ser Thr Ile Asn Gln Asn Val Lys
145 150 155 160
Thr Thr Gln Ala Ser Lys Gln Ser Asn Ser Lys Tyr Ala Ala Ser Ser
165 170 175
Tyr Leu Thr Leu Thr Gly Ser Glu Trp Lys Ser Lys Ser Ser Tyr Thr
180 185 190
Cys Glu Val Thr His Glu Gly Ser Thr Val Thr Lys Thr Val Lys Pro
195 200 205
Ser Glu Cys Ser
210

Claims (36)

1. An anti-testosterone antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO. 1 to SEQ ID NO. 3, and the light chain variable region comprises LCDR1 with amino acid sequences shown in SEQ ID NO. 4 or 15, LCDR2 with amino acid sequences shown in SEQ ID NO. 5 and LCDR3 with amino acid sequences shown in SEQ ID NO. 6.
2. The antibody or functional fragment thereof according to claim 1, wherein the antibody or functional fragment thereof further comprises HFR1, HFR2, HFR3, HFR4 having the amino acid sequences shown in SEQ ID No. 7 to SEQ ID No. 10 and LFR1, LFR2, LFR3 and LFR4 having the amino acid sequences shown in SEQ ID No. 11 to SEQ ID No. 14 or an amino acid sequence having at least 80% homology with the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 sequences.
3. The antibody or functional fragment thereof according to claim 2, wherein the antibody or functional fragment thereof has a KD.ltoreq.10 -7 The affinity of M binds testosterone.
4. The antibody or functional fragment thereof according to claim 2, wherein LFR1 of said antibody or functional fragment thereof has the amino acid sequence shown in SEQ ID No. 16.
5. The antibody or functional fragment thereof according to claim 2, wherein LFR2 of said antibody or functional fragment thereof has the amino acid sequence shown in SEQ ID No. 17.
6. An anti-testosterone antibody or a functional fragment thereof, wherein said antibody or functional fragment thereof comprises a heavy chain variable region comprising the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 and a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HFR1, HFR2, HFR4, HFR1, LFR2, LFR3, LFR4 is the amino acid sequence of HFR1, HFR2, HFR3, LCDR3, LFR4 as defined in claim 2.
7. An anti-testosterone antibody or a functional fragment thereof, wherein said antibody or functional fragment thereof comprises a heavy chain variable region having an amino acid sequence as set forth in any one of SEQ ID NOs 18 to 22 and a light chain variable region having an amino acid sequence as set forth in any one of SEQ ID NOs 23 to 27.
8. The antibody or functional fragment thereof of any one of claims 1 to 7, wherein the antibody or functional fragment thereof further comprises a constant region.
9. The antibody or functional fragment thereof of claim 8, wherein the constant regions comprise a heavy chain constant region and a light chain constant region.
10. The antibody or functional fragment thereof of claim 9, wherein the heavy chain constant region is selected from the group consisting of the heavy chain constant region of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD; the light chain constant region is selected from kappa-type or lambda-type light chain constant regions.
11. The antibody or functional fragment thereof according to claim 8, wherein the constant region is of bovine, equine, porcine, ovine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human origin.
12. The antibody or functional fragment thereof according to claim 11, wherein the constant region is of ovine species origin.
13. The antibody or functional fragment thereof of claim 9, wherein the heavy chain constant region sequence is as shown in SEQ ID No. 28 or has at least 80% homology thereto and the light chain constant region sequence is as shown in SEQ ID No. 29 or has at least 80% homology thereto.
14. The antibody or functional fragment thereof according to any one of claims 1 to 7, wherein the functional fragment is selected from any one of F (ab ') 2, fab', fab, fv and scFv of the antibody.
15. An anti-testosterone antibody comprising a heavy chain and a light chain, wherein said heavy chain comprises a heavy chain variable region according to any one of claims 6 to 7 and a heavy chain constant region according to any one of claims 8 to 13; the light chain comprises the light chain variable region of any one of claims 6 to 7 and the light chain constant region of any one of claims 8 to 13.
16. An anti-testosterone antibody, which comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in any one of SEQ ID NO. 30-34, and the amino acid sequence of the light chain is shown in any one of SEQ ID NO. 35-39.
17. An antibody conjugate comprising the antibody or functional fragment thereof of any one of claims 1 to 14 or the anti-testosterone antibody of any one of claims 15-16.
18. The antibody conjugate of claim 17, wherein the antibody or functional fragment thereof is labeled with a label.
19. The antibody conjugate of claim 18, wherein the label is selected from the group consisting of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label.
20. The antibody conjugate of claim 19, wherein the fluorescent dye is selected from the group consisting of fluorescein-based dyes and derivatives thereof, rhodamine-based dyes and derivatives thereof, cy-based dyes and derivatives thereof, alexa-based dyes and derivatives thereof, and protein-based dyes and derivatives thereof.
21. The antibody conjugate of claim 19, wherein the enzyme is selected from horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.
22. The antibody conjugate of claim 19, wherein the radioisotope is selected from the group consisting of 212 Bi、 131 I、 111 In、 90 Y、 186 Re、 211 At、 125 I、 188 Re、 153 Sm、 213 Bi、 32 P、 94 mTc、 99 mTc、 203 Pb、 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn、 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb、 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F。
23. the antibody conjugate of claim 19, wherein the chemiluminescent reagent is selected from the group consisting of luminol and derivatives thereof, lucigenin, crustacean fluorescein and derivatives thereof, ruthenium bipyridine and derivatives thereof, acridinium esters and derivatives thereof, dioxane and derivatives thereof, lotensine and derivatives thereof, and peroxyoxalate and derivatives thereof.
24. The antibody conjugate of claim 19, wherein the nanoparticle-based label is a nanoparticle or a colloid.
25. The antibody conjugate of claim 24, wherein the nanoparticle is selected from the group consisting of an organic nanoparticle, a magnetic nanoparticle, a quantum dot nanoparticle, and a rare earth complex nanoparticle.
26. The antibody conjugate of claim 24, wherein the colloid is selected from the group consisting of colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
27. The antibody conjugate of claim 26, wherein the colloidal metal is selected from the group consisting of colloidal gold, colloidal silver, and colloidal selenium.
28. The antibody conjugate of claim 17, wherein the antibody or functional fragment thereof is coated onto a solid phase.
29. The antibody conjugate of claim 28, wherein the solid phase is selected from the group consisting of a microsphere, a plate, and a membrane.
30. The antibody conjugate of claim 29, wherein the solid phase is selected from the group consisting of magnetic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon, and nitrocellulose membranes.
31. A kit or kit for the detection of testosterone, comprising the antibody or functional fragment thereof according to any one of claims 1 to 14, the anti-testosterone antibody according to any one of claims 15 to 16 or the antibody conjugate according to any one of claims 17 to 30.
32. Use of the antibody or functional fragment thereof of any one of claims 1 to 14, the anti-testosterone antibody of any one of claims 15-16, the antibody conjugate of any one of claims 17-30 or the reagent or kit of claim 31 for the preparation of a test testosterone product.
33. A nucleic acid encoding the antibody or functional fragment thereof according to any one of claims 1 to 14, the anti-testosterone antibody according to any one of claims 15 to 16.
34. A vector comprising the nucleic acid of claim 33.
35. A cell comprising the nucleic acid of claim 33 or the vector of claim 34.
36. A method of preparing an antibody or functional fragment thereof according to any one of claims 1 to 14, comprising:
culturing the cell of claim 35.
CN202210190274.6A 2022-02-28 2022-02-28 Anti-testosterone antibody, reagent for detecting testosterone and kit Active CN116693683B (en)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
CN103003697A (en) * 2010-05-17 2013-03-27 得克萨斯系统大学董事会 Rapid isolation of monoclonal antibodies from animals
CN104558174A (en) * 2015-01-29 2015-04-29 东南大学 Testosterone-orientated specific nano-antibody and application thereof
CN111269320A (en) * 2020-02-21 2020-06-12 华南农业大学 Nano antibody NT8 for specifically recognizing 19-nortestosterone and application thereof

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
CN103003697A (en) * 2010-05-17 2013-03-27 得克萨斯系统大学董事会 Rapid isolation of monoclonal antibodies from animals
CN104558174A (en) * 2015-01-29 2015-04-29 东南大学 Testosterone-orientated specific nano-antibody and application thereof
CN111269320A (en) * 2020-02-21 2020-06-12 华南农业大学 Nano antibody NT8 for specifically recognizing 19-nortestosterone and application thereof

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