WO2022100263A1 - 抗amh的抗体、检测amh的试剂和试剂盒 - Google Patents

抗amh的抗体、检测amh的试剂和试剂盒 Download PDF

Info

Publication number
WO2022100263A1
WO2022100263A1 PCT/CN2021/117806 CN2021117806W WO2022100263A1 WO 2022100263 A1 WO2022100263 A1 WO 2022100263A1 CN 2021117806 W CN2021117806 W CN 2021117806W WO 2022100263 A1 WO2022100263 A1 WO 2022100263A1
Authority
WO
WIPO (PCT)
Prior art keywords
cdr
antibody
functional fragment
amh
derivatives
Prior art date
Application number
PCT/CN2021/117806
Other languages
English (en)
French (fr)
Inventor
崔鹏
何志强
孟媛
钟冬梅
叶庆妮
陈晓倩
王晨
范凌云
Original Assignee
东莞市朋志生物科技有限公司
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 东莞市朋志生物科技有限公司 filed Critical 东莞市朋志生物科技有限公司
Publication of WO2022100263A1 publication Critical patent/WO2022100263A1/zh

Links

Images

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones

Definitions

  • the present disclosure relates to the technical field of antibodies, and in particular, to an anti-AMH antibody, a reagent and a kit for detecting AMH.
  • Anti-Mullerian Hormone is a member of the transforming growth factor beta superfamily, first discovered by Professor Alfred Jost in 1974. It is a diglycan protein composed of two 70kD protein subunits through disulfide bonds, with a relative molecular mass of 140kD. Its main function is to affect the growth and differentiation of follicles.
  • the human gene encoding AMH is located on chromosome 19p13.3, with a size of 2.4-2.8 kb and containing 5 exons. Studies have shown that the N-terminal region of AMH plays a key role in maintaining the integrity of the protein.
  • AMH a specific site (amino acid 451) of AMH can be cleaved to form a precursor fragment (pro-AMH, 26-451aar) ) and mature fragments (Mature AMH, 452-560aar), and the cut two fragments can be joined together by non-covalent bonds.
  • AMH plays an important role in the development of gonadal organs and is one of the important markers of male and female gonadal function.
  • transcription factor SOX-9 (SOX-9) activates AMH and is associated with anti-Mullerian Hormone Receptor Type II (AMHR2)
  • AMHR2 anti-Mullerian Hormone Receptor Type II
  • AMH is also a product of women's preantral follicles and small antral follicle granulosa cells, and serum AMH levels slowly decrease over time starting at puberty and decrease to levels undetectable by ELISA at menopause.
  • the normal value of AMH is between 2-6.8ng/ml. The higher the value, the more abundant the egg stock is and the longer the golden period suitable for conception. The lower the value, the worse the ovarian function. After the age of 35, the AMH value will begin to drop sharply. When it is lower than 0.7ng/ml, it means that the egg inventory is seriously insufficient and it is almost difficult to conceive. If the value is greater than 6.8, the constitution of polycystic ovary syndrome can be considered.
  • AMH level can be used to determine the egg stock and ovarian function, and it can be used as the indicator of ovarian responsiveness, polycystic ovary syndrome (PCOS), and ovarian hyperstimulation syndrome (OHSS) in in vitro fertilization combined with embryo transfer (IVF). Diagnose based on.
  • PCOS polycystic ovary syndrome
  • OHSS ovarian hyperstimulation syndrome
  • ELISA enzyme-linked immunosorbent assay
  • electrochemiluminescence and chemiluminescence are widely used methods for clinical detection of AMH at home and abroad.
  • the ELISA method is time-consuming, has low sensitivity, high background value, is prone to false positive and false negative results, and requires professional operation.
  • Electrochemiluminescence and chemiluminescence such as Roche's ELECSYS AMH detector are gradually replacing the ELISA method, but they are expensive and not suitable for single-person and small-batch detection. Higher, not suitable for large-scale promotion and use. All of the above methods require the use of anti-AMH monoclonal antibodies. At present, there are few AMH monoclonal antibody products with differences in performance and defects in specificity and sensitivity.
  • the present disclosure provides an anti-AMH antibody or a functional fragment thereof, the antibody or functional fragment thereof comprising the following complementarity determining regions:
  • CDR-VH1 G-F-X1-F-S-X2-F-G-M-S; where: X1 is S or T; X2 is I, V or L;
  • CDR-VH2 T-X1-S-N-G-G-X2-Y-T-Y-Y-P-X3-S-X4-K-G; where: X1 is L or I; X2 is T or S; X3 is E or D; X4 is I, V or L;
  • CDR-VH3 X1-R-H-P-R-X2-N-G-X3-D-G-A-M; wherein: X1 is A or T; X2 is I, V or L; X3 is Y, F or S;
  • CDR-VL1 A-S-X1-S-X2-D-N-Y-D-X3-S-F-M; wherein: X1 is Q or E; X2 is I, V or L; X3 is I or L;
  • CDR-VL2 A-A-S-N-X1-X2-S; where: X1 is K, Q or R; X2 is G or A;
  • CDR-VL3 Q-Q-S-X1-E-X2-P-W; where: X1 is K and R; X2 is L, V or I.
  • X1 is I
  • X1 is T
  • X3 is I
  • X2 is G
  • X1 is K.
  • X1 is S.
  • X1 is T.
  • X2 is 1.
  • X2 is V.
  • X2 is L.
  • X2 is T.
  • X2 is S.
  • X3 is E.
  • X3 is D in the CDR-VH2.
  • X4 is 1.
  • X4 is V.
  • X4 is L.
  • X2 is 1.
  • X2 is V.
  • X2 is L.
  • X3 is Y.
  • X3 is F.
  • X3 is S.
  • X1 is Q.
  • X1 is E.
  • X2 is 1 in CDR-VL1.
  • X2 is V.
  • X2 is L.
  • X1 is K.
  • X1 is Q.
  • X1 is R.
  • X2 is L.
  • X2 is V.
  • X2 is 1.
  • each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 1-55:
  • the antibody or functional fragment thereof binds to the anti-AMH antigen with an affinity of K D ⁇ 2.6 ⁇ 10 ⁇ 8 mol/L. In some alternative embodiments K D ⁇ 9 ⁇ 10 ⁇ 9 mol/L.
  • X1 is L
  • X1 is A
  • X3 is L
  • X2 is A
  • X1 is R.
  • each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 56-62:
  • the antibody comprises the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L of the sequence shown in SEQ ID NO: 1-4, and/or the sequence of Heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H shown in SEQ ID NOs: 5-8.
  • the antibody further comprises a constant region.
  • the constant region is selected from the constant region of any one of IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.
  • the species source of the constant region is bovine, equine, dairy cattle, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken , duck, goose, turkey, cockfight or man.
  • the constant region is derived from a mouse.
  • the light chain constant region sequence of the constant region is shown in SEQ ID NO:9
  • the heavy chain constant region sequence of the constant region is shown in SEQ ID NO:10.
  • the functional fragment is selected from any one of VHH, F(ab')2, Fab', Fab, Fv and scFv of the antibody.
  • the antibody comprises light chain framework regions FR1-L, FR2-L, FR3- having at least 80% homology with sequences SEQ ID NO: 1, 2, 3, 4, respectively, in order L and FR4-L, and/or, heavy chain framework regions FR1-H, FR2-H, FR3-H, and FR4-H.
  • the present disclosure provides a reagent or kit for detecting AMH, which includes the antibody or a functional fragment thereof.
  • the antibody or functional fragment thereof is labeled with a detectable label.
  • the detectable label is selected from the group consisting of fluorescent dyes, enzymes that catalyze the development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
  • the fluorescent dye is selected from fluorescein dyes and derivatives thereof, rhodamine dyes and derivatives thereof, Cy series dyes and derivatives thereof, Alexa series dyes and derivatives thereof, and proteins Dyes and their derivatives.
  • the enzyme that catalyzes the coloration of the substrate is selected from the group consisting of horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-Phosphate glucose deoxygenase.
  • 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.
  • the chemiluminescent reagent is selected from the group consisting of luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine esters and its derivatives , Dioxetane and its derivatives, Lofenine and its derivatives, and peroxyoxalate and its derivatives.
  • the nanoparticle-based label is selected from the group consisting of nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.
  • the colloid is selected from the group consisting of colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
  • the colloidal metal is selected from colloidal gold, colloidal silver and colloidal selenium.
  • the present disclosure provides a nucleic acid molecule encoding the antibody or functional fragment thereof.
  • the present disclosure provides a vector containing a nucleic acid molecule encoding the antibody or functional fragment thereof.
  • the present disclosure provides a recombinant cell containing the vector.
  • the present disclosure provides the use of the antibody or functional fragment thereof, the reagent or the kit in detecting AMH.
  • the present disclosure provides the use of the antibody or functional fragment thereof, the reagent or the kit for detecting AMH.
  • the present disclosure provides a method of detecting AMH, comprising:
  • the present disclosure provides the use of the antibody or functional fragment thereof, the reagent or the kit in determining the egg stock and ovarian function of a subject.
  • the present disclosure provides a method for determining egg stock and ovarian function in a subject, including:
  • the present disclosure provides a method of diagnosing an AHM-related disease in a subject, comprising:
  • the disease associated with AHM is polycystic ovary syndrome, ovarian hyperstimulation syndrome, premature ovarian insufficiency, ovarian hyporesponsiveness, infertility, secondary ovarian failure, menorrhagia oligo, amenorrhea, endometriosis.
  • the present disclosure provides a method for preparing the antibody or a functional fragment thereof, which comprises: culturing the recombinant cell, and separating and purifying the antibody or the functional fragment thereof from the culture product.
  • FIG. 1 shows the results of reducing SDS-PAGE of the anti-AMH antibody of Example 1.
  • the term "complementarity determining regions” generally refers to: an intact or complete antibody comprises two heavy chains and two light chains; each heavy chain comprises a variable region (VH) and a constant region (CH); Each light chain contains a variable region (VL) and a constant region (CL); the antibody has a "Y" shape, and the stem of the Y consists of the second and the third of the two heavy chains held together by disulfide bonds Composed of three constant regions; each arm of Y includes a variable region and a first constant region of a single heavy chain combined with the variable and constant regions of a single light chain; the variable and heavy regions of the light chain The variable regions of the chains are responsible for antigen binding; the variable regions in both chains typically contain three hypervariable regions, called complementarity determining regions.
  • the term "functional fragment” is intended to refer to a portion of an antibody comprising a heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived.
  • These functional fragments may include, for example, Fd, Fv, Fab, F(ab'), F(ab)2, F(ab')2, single chain Fv (scFv), diabodies, triple chain Antibodies (triabodies), tetrabodies (tetrabodies) and minibodies (minibodies).
  • Other functional fragments may include, for example, heavy or light chain polypeptides, variable region polypeptides or CDR polypeptides or portions thereof, so long as these functional fragments retain binding activity.
  • constant region refers to a relatively stable region of the light and heavy chains of an antibody molecule near the C-terminal amino acid sequence.
  • variable region refers to the region of the light and heavy chains of an antibody molecule that varies greatly in amino acid sequence near the N-terminus.
  • naked anti-stable refers to an antibody or functional fragment thereof that is not labeled, eg, an antibody or functional fragment thereof that is not labeled with a detectable label.
  • Some embodiments of the present disclosure provide antibodies against AMH, reagents and kits for detecting AMH.
  • the antibody provided by the present disclosure can specifically bind to AMH, and has a good affinity for AMH, and the use of the antibody to detect AMH has high sensitivity and specificity.
  • One embodiment of the present disclosure provides an anti-AMH antibody or a functional fragment thereof, wherein the antibody or functional fragment thereof has the following complementarity determining regions:
  • CDR-VH1 G-F-X1-F-S-X2-F-G-M-S; where: X1 is S or T; X2 is I, V or L;
  • CDR-VH2 T-X1-S-N-G-G-X2-Y-T-Y-Y-P-X3-S-X4-K-G; where: X1 is L or I; X2 is T or S; X3 is E or D; X4 is I, V or L;
  • CDR-VH3 X1-R-H-P-R-X2-N-G-X3-D-G-A-M; wherein: X1 is A or T; X2 is I, V or L; X3 is Y, F or S;
  • CDR-VL1 A-S-X1-S-X2-D-N-Y-D-X3-S-F-M; wherein: X1 is Q or E; X2 is I, V or L; X3 is I or L;
  • CDR-VL2 A-A-S-N-X1-X2-S; where: X1 is K, Q or R; X2 is G or A;
  • CDR-VL3 Q-Q-S-X1-E-X2-P-W; where: X1 is K and R; X2 is L, V or I.
  • the antibody against AMH or its functional fragment provided by the present disclosure has the above-mentioned complementarity determining region structure; the above-mentioned antibody or its functional fragment can specifically bind to AMH, and has a good affinity for AMH, and the antibody is used to detect AMH, which has a relatively high performance. High sensitivity and specificity.
  • the present disclosure provides AMH with more and better performing antibody options.
  • X1 is I; in CDR-VH3, X1 is T; in CDR-VL1, X3 is I; in CDR-VL2, X2 is G; in CDR-VL3, X1 is K.
  • the antibody when the above-mentioned mutation site in the above-mentioned complementarity determining region is the above-mentioned amino acid residue, the antibody exhibits better affinity for AMH.
  • X1 is S.
  • X1 is T.
  • X2 is 1 in CDR-VH1.
  • X2 is V.
  • X2 is L.
  • X2 is T.
  • X2 is S.
  • X3 is E.
  • X3 is D.
  • X4 is 1.
  • X4 is V.
  • X4 is L.
  • X2 is 1.
  • X2 is V.
  • X2 is L.
  • X3 is Y.
  • X3 is F.
  • X3 is S.
  • X1 is Q.
  • X1 is E.
  • X2 is 1.
  • X2 is V.
  • X2 is L.
  • X1 is K.
  • X1 is Q.
  • X1 is R.
  • X2 is L.
  • X2 is V.
  • X2 is 1.
  • each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 1-55:
  • the antibody or functional fragment thereof binds to AMH with an affinity of K D ⁇ 2.6 ⁇ 10 ⁇ 8 mol/L.
  • K D ⁇ 9 ⁇ 10 ⁇ 9 mol/L.
  • K D The detection of K D is performed with reference to the methods in the embodiments of the present disclosure.
  • X1 is L; in CDR-VH3, X1 is A; in CDR-VL1, X3 is L; in CDR-VL2, X2 is A; in CDR-VL3, X1 is R.
  • each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 56-62:
  • the antibody comprises the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L, and/or the sequences of the light chain framework regions shown in SEQ ID NOs: 1-4 in sequence.
  • variable region (VH) of the heavy chain and the variable region (VL) of the light chain can be obtained by linking the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L, FR2-L, FR3-L and FR4- L, and/or, heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H having at least 80% homology with sequences of SEQ ID NOs: 5, 6, 7, 8, respectively.
  • the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L, and FR4-L that are at least 80% identical to the sequences of SEQ ID NOs: 1, 2, 3, 4, respectively, in order , and/or, heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H having at least 80% identity to the sequences of SEQ ID NOs: 5, 6, 7, 8, respectively.
  • amino acid sequences of the respective framework regions of the antibodies or functional fragments thereof provided by the present disclosure may be the same as those of the corresponding framework regions (SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7 or 8) 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.
  • the antibody further comprises a constant region.
  • the constant region is selected from the constant region of any one of IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.
  • the species source of the constant region is mammalian or poultry.
  • mammals include cows, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, or humans.
  • poultry animals include chickens, ducks, geese, turkeys or fighting cocks.
  • the species source of the constant region is bovine, equine, dairy cattle, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken , duck, goose, turkey, cockfight or man.
  • the constant region is derived from a mouse.
  • the light chain constant region sequence of the constant region is shown in SEQ ID NO:9
  • the heavy chain constant region sequence of the constant region is shown in SEQ ID NO:10.
  • the functional fragment is selected from any one of VHH, F(ab')2, Fab', Fab, Fv and scFv of the antibody.
  • Functional fragments of the above antibodies generally have the same binding specificity as the antibody from which they were derived.
  • the functional fragments of the above-mentioned antibodies can be cleaved by, for example, methods including but not limited to enzymatic digestion (including but not limited to pepsin or papain) and/or by chemical reduction Sulfur bond method.
  • enzymatic digestion including but not limited to pepsin or papain
  • chemical reduction Sulfur bond method On the basis of the structure of the complete antibody provided in the present disclosure, those skilled in the art can easily obtain the above-mentioned functional fragments.
  • Functional fragments of the aforementioned antibodies can also be obtained 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, including but not limited to, synthetically obtained.
  • An embodiment of the present disclosure provides a reagent or kit for detecting AMH, which includes the antibody or functional fragment thereof according to any one of the above.
  • the antibody or functional fragment thereof in the above reagent or kit is labeled with a detectable label.
  • detectable label refers to a class of substances having properties that can be directly observed by the naked eye or detected or detected by instruments, such as luminescence, coloration, radioactivity, etc., by which the corresponding Qualitative or quantitative detection of a target.
  • the detectable labels include, but are not limited to, fluorescent dyes, enzymes that catalyze color development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
  • the fluorescent dyes include, but are not limited to, fluorescein dyes and derivatives thereof (for example, including but not limited to fluorescein isothiocyanate (FITC) hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or its analogs), rhodamine dyes and their derivatives (such as, but not limited to, red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or the like compounds), Cy series dyes and their derivatives (such as but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc.
  • fluorescein dyes and derivatives thereof for example, including but not limited to fluorescein isothiocyanate (FITC) hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or its analogs
  • Alexa series dyes and their derivatives such as including But not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs
  • protein dyes and their derivatives for example, including but Not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydinoxanthin-chlorophyll protein (preCP), etc.
  • the enzymes that catalyze the coloration of the substrate include but are not limited to horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase enzyme and glucose 6-phosphate deoxygenase.
  • the radioisotopes include but are not limited to 212Bi , 131I , 111In , 90Y , 186Re , 211At , 125I , 188Re , 153Sm , 213Bi , 32P , 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.
  • the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine esters and its derivatives Derivatives, Dioxetane and its Derivatives, Lopine and its Derivatives and Peroxyoxalate and its Derivatives.
  • the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
  • the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
  • the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.
  • An embodiment of the present disclosure provides a nucleic acid molecule encoding the above-mentioned antibody or a functional fragment thereof.
  • One embodiment of the present disclosure provides a vector containing the above-mentioned nucleic acid molecule.
  • One embodiment of the present disclosure provides a recombinant cell containing the above-mentioned vector.
  • Some embodiments of the present disclosure provide the use of the above-mentioned antibodies or functional fragments thereof, reagents or kits for detecting AMH.
  • Some embodiments of the present disclosure provide methods of detecting AMH, comprising:
  • Some embodiments of the present disclosure provide the use of the above-mentioned antibodies or functional fragments thereof, reagents or kits in the determination of egg stock and ovarian function in a subject.
  • a method for determining egg stock and ovarian function in a subject comprising:
  • a method of diagnosing an AHM-related disorder in a subject comprising:
  • diseases associated with AHM include, but are not limited to, polycystic ovary syndrome, ovarian hyperstimulation syndrome, premature ovarian insufficiency, ovarian hyporesponsiveness, infertility, secondary ovarian failure , oligomenorrhea, amenorrhea, endometriosis.
  • One embodiment of the present disclosure provides a method for preparing an antibody or a functional fragment thereof, comprising: culturing the above-mentioned recombinant cells, and separating and purifying the antibody or its functional fragment from the cultured product.
  • restriction endonuclease and rTaq DNA polymerase were purchased from Takara Company.
  • MagExtractor-RNA extraction kit was purchased from TOYOBO Company.
  • BD SMART TM RACE cDNA Amplification Kit was purchased from Takara Company.
  • the pMD-18T vector was purchased from Takara Company.
  • Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were performed by Invitrogen.
  • the mRNA was extracted from the hybridoma cell line that secreted anti-AMH antibody, and the DNA product was obtained by RT-PCR.
  • the product was added to the pMD-18T vector with rTaq DNA polymerase and then inserted into the pMD-18T vector and transformed into DH5 ⁇ competent cells. After the colonies were grown, the heavy chain (Heavy Chain) and the light chain (Light Chain) gene clones were taken respectively, and 4 clones were sent to a gene sequencing company for sequencing.
  • the gene sequences obtained by the above sequencing were placed in the IMGT antibody database (IMGT antibody database from: http://www.imgt.org) for analysis, and the VNTI11.5 software was used for analysis to determine the amplification of heavy chain and light chain primer pairs.
  • the added genes are all correct.
  • the VL gene sequence is 336bp, belonging to the VkII gene family, and there is a 57bp leader peptide sequence in front of it;
  • the gene fragment amplified by the heavy chain primer pair Among them, the VH gene sequence is 366 bp, belonging to the VH1 gene family, and there is a 57 bp leader peptide sequence in front of it.
  • pcDNATM 3.4 vector is the constructed recombinant antibody eukaryotic expression vector.
  • the expression vector has introduced polyclonal restriction sites such as HindIII, BamHI and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector; according to the above pMD-18T
  • 3.4A expression vector pcDNA3.4A expression vector
  • the VL and VH gene-specific primers of the antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively, and a 0.74kb light chain was amplified by PCR amplification method. Gene fragment and 1.46kb heavy chain gene fragment.
  • the heavy chain and light chain gene fragments were digested with HindIII/EcoRI double enzymes respectively, and the 3.4A vector was digested with HindIII/EcoRI double enzymes. After the fragments and vectors were purified and recovered, the heavy chain genes and light chain genes were connected to the 3.4A expression vector to obtain the heavy chain. Chain and light chain recombinant expression plasmids.
  • the recombinant antibody expression plasmid was transiently transfected into CHO cells to determine the activity of the expression plasmid
  • plasmid obtained in step 1-(3) Dilute the plasmid obtained in step 1-(3) to 400ng/ml with ultrapure water, adjust CHO cells to 1.43 ⁇ 10 7 cells/ml in a centrifuge tube, mix 100 ⁇ L of plasmid with 700 ⁇ L of cells, transfer into an electroporation cup, and electroporate, The samples were counted on the 3rd, 5th, and 7th days, and the samples were collected and tested on the 7th day.
  • the coating solution (the main component NaHCO 3 ) diluted the AMH antigen to 1 ⁇ g/ml, 100 ⁇ L per well, overnight at 4°C; the next day, washed twice with the washing solution and patted dry; added blocking solution (20%BSA+80%PBS), 120 ⁇ L per well, 37°C, 1h, pat dry; add diluted cell supernatant, 100 ⁇ L/well, 37°C, 30min; wash 5 times with washing solution, pat dry; add goat anti-mouse IgG-HRP, 100 ⁇ L per well, 37°C, 30min; wash with washing solution 5 times, pat dry; add color developer solution A (50 ⁇ L/well, containing citric acid + sodium acetate + acetanilide + carbamide peroxide), add color developer solution B (50 ⁇ L/well) , containing citric acid+EDTA ⁇ 2Na+TMB+concentrated HCl), 10min; add stop solution (50 ⁇ L/well, containing EDTA ⁇ 2Na+concentr
  • plasmid obtained in step 2-(2) Dilute the plasmid obtained in step 2-(2) to 400ng/ml with ultrapure water, adjust CHO cells to 1.43 ⁇ 10 7 cells/ml in a centrifuge tube, mix 100 ⁇ L of plasmid with 700 ⁇ L of cells, transfer into an electroporation cup, and electroporate, Count the next day; 25umol/L MSX 96-well pressurized culture for about 25 days.
  • step 2-(3) Stably transfect the recombinant antibody expression plasmid in step 2-(3), pressurize the cells obtained by screening the stable cell line, and then culture them in a 125ml shake flask, the inoculation volume is 30ml, and the medium is 100% Dynamis medium. , placed in a shaker with a rotational speed of 120 r/min, a temperature of 37 °C, and 8% carbon dioxide. After culturing for 72 hours, inoculate and expand the culture at an inoculation density of 500,000 cells/ml, and the expansion volume is calculated according to the production demand, and the medium is 100% Dynamis medium. After that, the culture was expanded every 72h. When the cell volume meets the production requirements, the seeding density is strictly controlled to be about 500,000 cells/ml for production.
  • Shaking flask parameters rotating speed 120r/min, temperature 37°C, carbon dioxide 8%.
  • Feed feeding start feeding every day after culturing in the shake flask for 72 hours.
  • HyClone Cell Boost Feed 7a is fed 3% of the initial culture volume every day, and Feed 7b is fed daily at 1/1000 of the initial culture volume. Supplement until the 12th day (feeding on the 12th day).
  • Glucose was supplemented with 3 g/L on the sixth day. Samples were collected on the 13th day.
  • Affinity purification was performed using a protein A affinity chromatography column. Take 4 ⁇ g of purified antibody for reducing SDS-PAGE, and 4 ⁇ g foreign control antibody as control.
  • the electropherogram is shown in Figure 1 below. After reducing SDS-PAGE, two bands are displayed, and one Mr is 50KD (heavy chain, SEQ ID NO: 14), the other Mr is 28KD (light chain, SEQ ID NO: 13).
  • the antibody (WT) sequence of Example 1 was analyzed, and its heavy chain variable region was shown in SEQ ID NO: 12, wherein the amino acid sequence of each complementarity determining region on the heavy chain variable region was as follows:
  • CDR-VH1 G-F-T(X1)-F-S-V(X2)-F-G-M-S;
  • CDR-VH2 T-L(X1)-S-N-G-G-S(X2)-Y-T-Y-Y-P-D(X3)-S-L(X4)-K-G;
  • CDR-VH3 A(X1)-R-H-P-R-V(X2)-N-G-Y(X3)-D-G-A-M;
  • CDR1-VL A-S-E(X1)-S-I(X2)-D-N-Y-D-L(X3)-S-F-M;
  • CDR-VL2 A-A-S-N-K(X1)-A(X2)-S;
  • CDR-VL3 Q-Q-S-R(X1)-E-I(X2)-P-W.
  • the coating solution (the main component NaHCO 3 ) diluted AMH antigen to 1 ⁇ g/ml for microplate coating, 100 ⁇ l per well, overnight at 4°C; the next day, the washing solution (composition: ) was washed twice and patted dry; the blocking solution was added (20%BSA+80%PBS), 120 ⁇ l per well, 37°C, 1h, pat dry; add diluted (WT and mutation 1-6 in Table 1) AMH monoclonal antibody, 100 ⁇ l/well, 37°C, 30min-60min; wash 5 times with washing solution, pat dry; add goat anti-mouse IgG-HRP, 100 ⁇ l per well, 37 °C, 30 min; wash 5 times with washing solution (PBS), pat dry; add chromogenic solution A (50 ⁇ l /well, containing 2.1g/L citric acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L carbamide peroxide), add color developer solution B (50 ⁇ l
  • the purified antibody was diluted to 10ug/mL with PBST, and the AMH antigen was serially diluted with PBST: 45ug/mL, 22.5ug/mL, 11.3ug/mL, 5.60ug/mL, 2.80ug/mL, 1.40 ug/mL, 0.70ug/mL.
  • Running process equilibrate in buffer 1 (PBST) for 60s, immobilize antibody in antibody solution for 300s, incubate in buffer 2 (PBST) for 180s, bind in antigen solution for 420s, dissociate in buffer 2 for 1200s, use 10mM pH 1.69 GLY solution and buffer 3 to regenerate the sensor and output data.
  • K D is the equilibrium dissociation constant or affinity; kon is the association rate; kdis is the dissociation rate.
  • the above antibodies were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days, and samples were taken for 7 days, 14 days, and 21 days for state observation, and the 21-day sample was tested for activity. , the results showed that under the above three test conditions, the antibody was placed for 21 days without obvious changes in protein status, and the activity did not show a downward trend with the increase of the test temperature, indicating that the above antibodies were stable.
  • the following table 7 mutation 1 antibody is the OD results of the enzyme immunoassay activity detection for 21 days.
  • the antibody in Table 3 was paired with another AMH antibody, and the double-antibody sandwich method was used to detect the specificity of 100% and the sensitivity of 99%-99.8% on the chemiluminescence platform.
  • the present disclosure provides an anti-AMH antibody, a nucleic acid molecule and amino acid encoding the antibody, and the reagent and kit for detecting AMH.
  • the antibody provided by the present disclosure can specifically bind to AMH and has a good affinity for AMH, and the antibody is used to detect AMH with high sensitivity and specificity.
  • the prepared reagent and kit for detecting AMH also have the same characteristics as the antibody. The technical effect has broad application prospects and high market value.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Endocrinology (AREA)
  • Hematology (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Peptides Or Proteins (AREA)

Abstract

一种抗AMH的抗体、检测AMH的试剂和试剂盒，涉及抗体技术领域。抗AMH的抗体包括重链互补决定区和轻链互补决定区。该抗体对抗AMH的亲和力较好，使用该抗体检测AMH具有更好的灵敏度和特异性。为AMH的检测提供了更多以及性能更佳的抗体选择。

Description

抗AMH的抗体、检测AMH的试剂和试剂盒
相关申请的交叉引用
本公开要求于2020年11月12日提交中国专利局的申请号为“202011259343.1”名称为“抗AMH的抗体、检测AMH的试剂和试剂盒”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及抗体技术领域，具体而言，涉及一种抗AMH的抗体、检测AMH的试剂和试剂盒。
背景技术
抗穆勒氏管激素(Anti-Mullerian Hormone，AMH)是转化生长因子β超家族中的一员，由Professor Alfred Jost于1974年首先发现。它是由两个70kD的蛋白亚基通过二硫键组成的二聚糖蛋白，相对分子质量为140kD，其主要作用是影响卵泡的成长和分化。人类编码AMH的基因位于19p13.3号染色体，大小2.4～2.8kb，含有5个外显子。研究表明，AMH的N端区域对保持蛋白完整活性起关键作用，在代谢运输过程中，AMH的特定位点(451位氨基酸)可以被切开，形成前体片段(pro-AMH，26-451aar)以及成熟片段(Mature AMH，452-560aar)，而切开的两个片段能够再通过非共价键的形式连接在一起。
AMH在性腺器官发育过程中起着重要作用，是男女性腺功能的重要标记物之一。在男性胎儿的塞尔托利氏细胞中，转录因子SOX-9(Transcription factor SOX-9，SOX9)可激活AMH与抗穆勒激素受体II型(Anti-Mullerian Hormone Receptor Type II，AMHR2)相结合并导致雄性胚胎中的穆勒氏管退化，从而阻止输卵管、子宫和阴道上部的形成，使男性生殖管道正常发育。而在女性胚胎中穆勒氏管将分化为子宫管和输卵管。AMH也是女性的窦前卵泡和小窦卵泡颗粒细胞的产物，从青春期开始，血清中AMH水平随时间慢慢降低，并在更年期降低到ELISA法检测不到的水平。AMH的正常值界于2-6.8ng/ml之间，数值越高，代表卵子存量越丰沛，适合受孕的黄金期较长，数值越低则卵巢功能越差。35岁过后AMH值会开始急剧下降，当低于0.7ng/ml时，表示卵子库存量已严重不足，几乎难以受孕。若数值大于6.8时，可考虑有多囊性卵巢症候群的体质。使用排卵针剂、药物时，卵巢也容易反应过度而排出过多的卵子，造成卵巢过度刺激症候群。因此可通过检测AMH水平来判断卵子存量及卵巢功能，并可以作为体外受精联合胚胎移植术(IVF)的卵巢反应性、多囊性卵巢综合征(PCOS)、卵巢过度刺激综合症(OHSS)的诊断依据。
目前国内外临床上检测AMH的常用方法有酶联免疫吸附法(ELISA)、电化学发光法和化学发光法。其中ELISA方法耗时长、灵敏度低、背景值高、易造成假阳性和假阴性结果，并且需要专业人员进行操作。电化学发光法和化学发光法如罗氏的ELECSYS AMH检测仪目前逐渐替换ELISA法，但价格昂贵，不适合单人份和小批量检测用，加上需要专门的仪器使用人员，维修和检测成本均较高，不适合大范围推广使用。上述方法都需要借助抗AMH的单克隆抗体，而目前AMH单克隆抗体产品较少，性能存在差异，特异性和灵敏度也存在缺陷。
发明内容
本公开提供了一种抗AMH的抗体或其功能性片段，所述抗体或其功能性片段包括如下互补决定区：
CDR-VH1：G-F-X1-F-S-X2-F-G-M-S；其中：X1是S或T；X2是I、V或L；
CDR-VH2：T-X1-S-N-G-G-X2-Y-T-Y-Y-P-X3-S-X4-K-G；其中：X1是L或I；X2是T或S；X3是E或D；X4是I、V或L；
CDR-VH3：X1-R-H-P-R-X2-N-G-X3-D-G-A-M；其中：X1是A或T；X2是I、V或L；X3是Y、F或S；
CDR-VL1：A-S-X1-S-X2-D-N-Y-D-X3-S-F-M；其中：X1是Q或E；X2是I、V或L；X3是I或L；
CDR-VL2：A-A-S-N-X1-X2-S；其中：X1是K、Q或R；X2是G或A；
CDR-VL3：Q-Q-S-X1-E-X2-P-W；其中：X1是K和R；X2是L、V或I。
在一些实施方式中，
CDR-VH2中，X1是I；
CDR-VH3中，X1是T；
CDR-VL1中，X3是I；
CDR-VL2中，X2是G；
CDR-VL3中，X1是K。
在一些实施方式中，CDR-VH1中，X1是S。
在一些实施方式中，CDR-VH1中，X1是T。
在一些实施方式中，CDR-VH1中，X2是I。
在一些实施方式中，CDR-VH1中，X2是V。
在一些实施方式中，CDR-VH1中，X2是L。
在一些实施方式中，CDR-VH2中，X2是T。
在一些实施方式中，CDR-VH2中，X2是S。
在一些实施方式中，CDR-VH2中，X3是E。
在一些实施方式中，CDR-VH2中，X3是D。
在一些实施方式中，CDR-VH2中，X4是I。
在一些实施方式中，CDR-VH2中，X4是V。
在一些实施方式中，CDR-VH2中，X4是L。
在一些实施方式中，CDR-VH3中，X2是I。
在一些实施方式中，CDR-VH3中，X2是V。
在一些实施方式中，CDR-VH3中，X2是L。
在一些实施方式中，CDR-VH3中，X3是Y。
在一些实施方式中，CDR-VH3中，X3是F。
在一些实施方式中，CDR-VH3中，X3是S。
在一些实施方式中，CDR-VL1中，X1是Q。
在一些实施方式中，CDR-VL1中，X1是E。
在一些实施方式中，CDR-VL1中，X2是I。
在一些实施方式中，CDR-VL1中，X2是V。
在一些实施方式中，CDR-VL1中，X2是L。
在一些实施方式中，CDR-VL2中，X1是K。
在一些实施方式中，CDR-VL2中，X1是Q。
在一些实施方式中，CDR-VL2中，X1是R。
在一些实施方式中，CDR-VL3中，X2是L。
在一些实施方式中，CDR-VL3中，X2是V。
在一些实施方式中，CDR-VL3中，X2是I。
在一些实施方式中，所述抗体或其功能性片段的各互补决定区选自如下突变组合1-55中的任意一种：
Figure PCTCN2021117806-appb-000001
Figure PCTCN2021117806-appb-000002
在一些实施方式中，所述抗体或其功能性片段与抗AMH抗原以K D≤2.6×10 -8mol/L的亲和力结合。在一些可选的实施方式中K D≤9×10 -9mol/L。
在一些实施方式中，
CDR-VH2中，X1是L；
CDR-VH3中，X1是A；
CDR-VL1中，X3是L；
CDR-VL2中，X2是A；
CDR-VL3中，X1是R。
在一些实施方式中，所述抗体或其功能性片段的各互补决定区选自如下突变组合56-62中的任意一种：
Figure PCTCN2021117806-appb-000003
Figure PCTCN2021117806-appb-000004
在一些实施方式中，所述抗体包括序列依次如SEQ ID NO:1-4所示的轻链骨架区FR1-L、FR2-L、FR3-L及FR4-L，和/或，序列依次如SEQ ID NO:5-8所示的重链骨架区FR1-H、FR2-H、FR3-H及FR4-H。
在可选的实施方式中，所述抗体还包含恒定区。
在可选的实施方式中，所述恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE和IgD中的任意一者的恒定区。
在可选的实施方式中，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人。
在可选的实施方式中，所述恒定区来源于小鼠。
在可选的实施方式中，所述恒定区的轻链恒定区序列如SEQ ID NO:9所示，所述恒定区的重链恒定区序列如SEQ ID NO:10所示。
在可选的实施方式中，所述功能性片段选自所述抗体的VHH、F(ab’)2、Fab’、Fab、Fv和scFv中的任意一种。
在可选的实施方式中，所述抗体包括分别依次与序列SEQ ID NO:1、2、3、4具有至少80％的同源性的轻链骨架区FR1-L、FR2-L、FR3-L及FR4-L，和/或，分别依次与序列SEQ ID NO:5、6、7、8具有至少80％的同源性的重链骨架区FR1-H、FR2-H、FR3-H及FR4-H。
本公开提供一种检测AMH的试剂或试剂盒，其包括所述抗体或其功能性片段。
在一些实施方式中，所述抗体或其功能性片段标记有可被检测的标记物。
在可选的实施方式中，所述可被检测的标记物选自荧光染料、催化底物显色的酶、放射性同位素、化学发光试剂和纳米颗粒类标记物。
在可选的实施方式中，所述荧光染料选自荧光素类染料及其衍生物、罗丹明类染料及其衍生物、Cy系列染料及其衍生物、Alexa系列染料及其衍生物和蛋白类染料及其衍生物。
在可选的实施方式中，所述催化底物显色的酶选自辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡萄糖氧化酶、碳酸酐酶、乙酰胆碱酯酶以及6-磷酸葡萄糖脱氧酶。
在可选的实施方式中，所述放射性同位素选自 212Bi、 131I、 111In、 90Y、 186Re、 211At、 125I、 188Re、 153Sm、 213Bi、 32P、 94mTc、 99mTc、 203Pb、 67Ga、 68Ga、 43Sc、 47Sc、 110mIn、 97Ru、 62Cu、 64Cu、 67Cu、 68Cu、 86Y、 88Y、 121Sn、 161Tb、 166Ho、 105Rh、 177Lu、 172Lu和 18F。
在可选的实施方式中，所述化学发光试剂选自鲁米诺及其衍生物、光泽精、甲壳动物荧光素及其衍生物、联吡啶钌及其衍生物、吖啶酯及其衍生物、二氧环乙烷及其衍生物、洛粉碱及其衍生物和过氧草酸盐及其衍生物。
在可选的实施方式中，所述纳米颗粒类标记物选自纳米颗粒、胶体、有机纳米颗粒、磁性纳米颗粒、量子点纳米颗粒和稀土络合物纳米颗粒。
在可选的实施方式中，所述胶体选自胶体金属、分散型染料、染料标记的微球和乳胶。
在可选的实施方式中，所述胶体金属选自胶体金、胶体银和胶体硒。
本公开提供一种核酸分子，所述核酸分子编码所述抗体或其功能性片段。
本公开提供一种载体，其含有编码所述抗体或其功能性片段的核酸分子。
本公开提供一种重组细胞，其含有所述的载体。
本公开提供所述抗体或其功能性片段、所述试剂或试剂盒在检测AMH中的用途。
本公开提供所述抗体或其功能性片段、所述试剂或试剂盒，用于检测AMH的用途。
本公开提供一种检测AMH中的方法，包括：
A)在足以发生结合反应的条件下，使上述任一项所述的抗体或其功能性片段与样品接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物。
本公开提供所述抗体或其功能性片段、所述试剂或试剂盒在测定受试者卵子存量及卵巢功能的用途。
本公开提供一种测定受试者卵子存量及卵巢功能的方法，包括：
A)在足以发生结合反应的条件下，使上述任一项所述的抗体或其功能性片段与来自所述受试者的样品接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物。
本公开提供一种诊断受试者中与AHM相关的疾病的方法，包括：
A)在足以发生结合反应的条件下，使上述任一项所述的抗体或其功能性片段与来自所述受试者的样品接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物。
在一些实施方式中，所述与AHM相关的疾病是多囊性卵巢综合症、卵巢过度刺激综合症、早发性卵巢功能不全、卵巢低反应、不孕症、继发性卵巢衰竭、月经过少、闭经、子宫内膜异位。
本公开提供一种制备所述抗体或其功能性片段的方法，其包括：培养所述重组细胞，从培养产物中分离纯化得到所述抗体或其功能性片段。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为实施例1的抗AMH的抗体的还原性SDS-PAGE的结果。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
除非另有定义，否则本文使用的所有技术和科学术语具有与本公开内容所属领域的普通技术人员通常理解的含义相同的含义。尽管与本文描述的那些方法和材料类似或等同的任何方法和材料都可用于本文的制剂或单位剂量的实践或测试，但现在描述一些方法和材料。除非另有说明，否则本文采用或考虑的技术是标准方法。材料、方法和实例仅是说明性而非限制性的。
除非另外指明，否则实践本公开将采用细胞生物学、分子生物学(包含重组技术)、微生物学、生物化学和免疫学的常规技术，所述常规技术在本领域技术人员的能力范围内。文献中充分解释了这种技术，如《分子克隆：实验室手册(Molecular Cloning:A Laboratory Manual)》，第二版(Sambrook等人，1989)；《寡核苷酸合成(Oligonucleotide Synthesis)》(M.J.Gait编，1984)；《动物细胞培养(Animal Cell Culture)》(R.I.Freshney编，1987)；《酶学方法(Methods in Enzymology)》(学术出版社有限公司(Academic Press，Inc.)；《实验免疫学手册(Handbook of Experimental Immunology)》(D.M.Weir和C.C.Blackwell编)；《哺乳动物细胞用基因转移载体(Gene Transfer Vectors for Mammalian Cells)》(J.M.Miller和M.P.Calos编，1987)；《当代分子生物学方法(Current Protocols in Molecular Biology)》(F.M.Ausubel等人编，1987)；《PCR:聚合酶链反应(PCR:The Polymerase Chain Reaction)》(Mullis等人编，1994)；以及《当代免疫学方法(Current Protocols in Immunology)》(J.E.Coligan等人编，1991)，所述文献中的每个文献均通过引用明确并入本文中。
术语定义
如本文所使用，术语“互补决定区”通常是指：一个完整或完全的抗体包含两条重链及两条轻链；每条重链含有可变区(VH)及恒定区(CH)；每条轻链含有一可变区(VL)及一恒定区(CL)；抗体具有“Y”形状，Y的茎部由透过双硫键结合在一起的两条重链的第二及第三恒定区所组成；Y的每个臂部包括与一单个轻链的可变区及恒定区结合的一单个重链的可变区及第一恒定区；该轻链的可变区及重链的可变区负责抗原结合；两条链中的可变区通常包含三个高度可变区，称为互补决定区。
如本文所使用，当用于表示抗体时，术语“功能性片段”是指在表示包含重链或轻链多肽的抗体的一部分，所述多肽保留了片段来源的抗体的一些或所有结合活性。这些功能性片段可以包括(例如)Fd、Fv、Fab、F(ab′)、F(ab)2、F(ab′)2、单链Fv(scFv)、双链抗体(diabody)、三链抗体(triabody)、四链抗体(tetrabody)和微抗体(minibody)。其他功能性片段可以包括(例如)重链或轻链多肽、可变区多肽或CDR多肽或其部分，只要这些功能性片段保留了结合活性即可。
如本文所使用，术语“恒定区”是指抗体分子的轻链和重链中靠近C端氨基酸序列相对稳定的区域。
如本文所使用，术语“可变区”是指抗体分子的轻链和重链中靠近N端氨基酸序列变化较大的区域。
如本文所用，术语“裸抗稳定性”是指未进行标记的抗体或其功能性片段，例如未标记有可被检测的标记物的抗体或其功能性片段。
本公开的一些实施方式提供了抗AMH的抗体、检测AMH的试剂和试剂盒。本公开提供的抗体能够特异性结合AMH，对AMH具有较好的亲和力，采用该抗体检测AMH，具有较高的灵敏度和特异性。
本公开一实施方式提供一种抗AMH的抗体或其功能性片段，所述抗体或其功能性片段具有如下互补决定区：
CDR-VH1：G-F-X1-F-S-X2-F-G-M-S；其中：X1是S或T；X2是I、V或L；
CDR-VH2：T-X1-S-N-G-G-X2-Y-T-Y-Y-P-X3-S-X4-K-G；其中：X1是L或I；X2是T或S；X3是E或D；X4是I、V或L；
CDR-VH3：X1-R-H-P-R-X2-N-G-X3-D-G-A-M；其中：X1是A或T；X2是I、V或L；X3是Y、F或S；
CDR-VL1：A-S-X1-S-X2-D-N-Y-D-X3-S-F-M；其中：X1是Q或E；X2是I、V或L；X3是I或L；
CDR-VL2：A-A-S-N-X1-X2-S；其中：X1是K、Q或R；X2是G或A；
CDR-VL3：Q-Q-S-X1-E-X2-P-W；其中：X1是K和R；X2是L、V或I。
本公开提供的针对AMH的抗体或其功能性片段，具有上述互补决定区结构；上述抗体或其功能性片段能够特异性结合AMH，对AMH具有较好的亲和力，采用该抗体检测AMH，具有较高的灵敏度和特异性。本公开为AMH提供了更多以及性能更佳的抗体选择。
在可选的实施方式中，CDR-VH2中，X1是I；CDR-VH3中，X1是T；CDR-VL1中，X3是I；CDR-VL2中，X2是G；CDR-VL3中，X1是K。
本公开的实施例发现，当上述互补决定区中的上述突变位点为上述氨基酸残基时，该抗体对AMH表现出更好的亲和力。
在可选的实施方式中，CDR-VH1中，X1是S。
在可选的实施方式中，CDR-VH1中，X1是T。
在可选的实施方式中，CDR-VH1中，X2是I。
在可选的实施方式中，CDR-VH1中，X2是V。
在可选的实施方式中，CDR-VH1中，X2是L。
在可选的实施方式中，CDR-VH2中，X2是T。
在可选的实施方式中，CDR-VH2中，X2是S。
在可选的实施方式中，CDR-VH2中，X3是E。
在可选的实施方式中，CDR-VH2中，X3是D。
在可选的实施方式中，CDR-VH2中，X4是I。
在可选的实施方式中，CDR-VH2中，X4是V。
在可选的实施方式中，CDR-VH2中，X4是L。
在可选的实施方式中，CDR-VH3中，X2是I。
在可选的实施方式中，CDR-VH3中，X2是V。
在可选的实施方式中，CDR-VH3中，X2是L。
在可选的实施方式中，CDR-VH3中，X3是Y。
在可选的实施方式中，CDR-VH3中，X3是F。
在可选的实施方式中，CDR-VH3中，X3是S。
在可选的实施方式中，CDR-VL1中，X1是Q。
在可选的实施方式中，CDR-VL1中，X1是E。
在可选的实施方式中，CDR-VL1中，X2是I。
在可选的实施方式中，CDR-VL1中，X2是V。
在可选的实施方式中，CDR-VL1中，X2是L。
在可选的实施方式中，CDR-VL2中，X1是K。
在可选的实施方式中，CDR-VL2中，X1是Q。
在可选的实施方式中，CDR-VL2中，X1是R。
在可选的实施方式中，CDR-VL3中，X2是L。
在可选的实施方式中，CDR-VL3中，X2是V。
在可选的实施方式中，CDR-VL3中，X2是I。
在可选的实施方式中，所述抗体或其功能性片段的各互补决定区选自如下突变组合1-55中的任意一种：
Figure PCTCN2021117806-appb-000005
Figure PCTCN2021117806-appb-000006
Figure PCTCN2021117806-appb-000007
在可选的实施方式中，所述抗体或其功能性片段与AMH以K D≤2.6×10 -8mol/L的亲和力结合。
在可选的实施方式中，K D≤2×10 -8mol/L、K D≤1×10 -8mol/L、K D≤9×10 -9mol/L、K D≤8×10 -9mol/L、K D≤7×10 -9mol/L、K D≤6×10 -9mol/L、K D≤5×10 -9mol/L、K D≤4×10 -9mol/L、K D≤3×10 -9mol/L、K D≤2×10 -9mol/L或K D≤1×10 -9mol/L。
在可选的实施方式中，K D≤9×10 -9mol/L。
在可选的实施方式中，1.52×10 -9mol/L≤K D≤8.94×10 -9mol/L。
K D的检测参考本公开实施例中的方法进行。
在可选的实施方式中，CDR-VH2中，X1是L；CDR-VH3中，X1是A；CDR-VL1中，X3是L；CDR-VL2中，X2是A；CDR-VL3中，X1是R。
在可选的实施方式中，所述抗体或其功能性片段的各互补决定区选自如下突变组合56-62中的任意一种：
Figure PCTCN2021117806-appb-000008
在可选的实施方式中，所述抗体包括序列依次如SEQ ID NO:1-4所示的轻链骨架区FR1-L、FR2-L、FR3-L及FR4-L，和/或，序列依次如SEQ ID NO:5-8所示的重链骨架区FR1-H、FR2-H、FR3-H及FR4-H。
通常情况下，重链可变区(VH)和轻链的可变区(VL)可由以下编号的CDR与FR按如下组合排列连接获得：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
在一些实施方式中，抗体包括分别依次与序列SEQ ID NO:1、2、3、4具有至少80％的同源性的轻链骨架区FR1-L、FR2-L、FR3-L及FR4-L，和/或，分别依次与序列SEQ ID NO:5、6、7、8具有至少80％的同源性的重链骨架区FR1-H、FR2-H、FR3-H及FR4-H。
在一些实施方式中，抗体包括分别依次与序列SEQ ID NO:1、2、3、4具有至少80％的同一性的轻链骨架区FR1-L、FR2-L、FR3-L及FR4-L，和/或，分别依次与序列SEQ ID NO:5、6、7、8具有至少80％的同一性的重链骨架区FR1-H、FR2-H、FR3-H及FR4-H。
需要说明的是，在其他的实施方式和实施例中，本公开提供的抗体或其功能性片段的各自骨架区氨基酸序列可以与上述对应骨架区(SEQ ID NO:1、2、3、4、5、6、7或8)可以具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同源性。
在可选的实施方式中，所述抗体还包含恒定区。
在可选的实施方式中，所述恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE和IgD中的任意一者的恒定区。
在可选的实施方式中，所述恒定区的种属来源为哺乳动物或家禽类动物。在可选的实施方式中，哺乳动物包括牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂或人。在可选的实施方式中，家禽类动物包括鸡、鸭、鹅、火鸡或斗鸡。
在可选的实施方式中，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人。
在可选的实施方式中，所述恒定区来源于小鼠。
在可选的实施方式中，所述恒定区的轻链恒定区序列如SEQ ID NO:9所示，所述恒定区的重链恒定区序列如SEQ ID NO:10所示。
在可选的实施方式中，所述功能性片段选自所述抗体的VHH、F(ab’)2、Fab’、Fab、Fv和scFv中的任意一种。
上述抗体的功能性片段通常具有与其来源抗体相同的结合特异性。本领域技术人员根据本公开记载的内容容易理解到，上述抗体的功能性片段可以通过例如包括但不限于酶消化的方法(包括但不限于胃蛋白酶或木瓜蛋白酶)和/或通过化学还原分裂二硫键的方法获得。在本公开提供了完整抗体的结构基础上，本领域技术人员容易获得上述的功能性片段。
上述抗体的功能性片段还可以通过也是本领域技术人员所知的重组遗传学技术或通过例如自动肽合成仪，例如包括但不限于Applied BioSystems等销售的自动肽合成仪合成获得。本公开一实施方式提供一种检测AMH的试剂或试剂盒，其包括如上任一项所述的抗体或其功能性片段。
在可选的实施方式中，上述试剂或试剂盒中所述抗体或其功能性片段标记有可被检测的标记物。
如本文所用，“可被检测的标记物”是指具有能够被肉眼直接观察或被仪器检测或探测到的特性例如发光、显色、放射性等特性的一类物质，通过该特性可以实现对相应目标物的定性或定量检测。
在可选的实施方式中，所述可被检测的标记物包括但不限于荧光染料、催化底物显色的酶、放射性同位素、化学发光试剂和纳米颗粒类标记物。
在实际的使用过程中，本领域技术人员可以根据检测条件或实际需要选择合适的标记物，无论使用何种标记物，其均属于本公开的保护范围。
在可选的实施方式中，所述荧光染料包括但不限于荧光素类染料及其衍生物(例如包括但不限于异硫氰酸荧光素(FITC)羟基光素(FAM)、四氯光素(TET)等或其类似物)、罗丹明类染料及其衍生物(例如包括但不限于红色罗丹明(RBITC)、四甲基罗丹明(TAMRA)、罗丹明B(TRITC)等或其类似物)、Cy系列染料及其衍生物(例如包括但不限于Cy2、Cy3、Cy3B、Cy3.5、Cy5、Cy5.5、Cy3等或其类似物)、Alexa系列染料及其衍生物(例如包括但不限于AlexaFluor350、405、430、488、532、546、555、568、594、610、33、647、680、700、750等或其类似物)和蛋白类染料及其衍生物(例如包括但不限于藻红蛋白(PE)、藻蓝蛋白(PC)、别藻蓝蛋白(APC)、多甲藻黄素-叶绿素蛋白(preCP)等)。
在可选的实施方式中，所述催化底物显色的酶包括但不限于辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡萄糖氧化酶、碳酸酐酶、乙酰胆碱酯酶以及6-磷酸葡萄糖脱氧酶。
在可选的实施方式中，所述放射性同位素包括但不限于 212Bi、 131I、 111In、 90Y、 186Re、 211At、 125I、 188Re、 153Sm、 213Bi、 32P、 94mTc、 99mTc、 203Pb、 67Ga、 68Ga、 43Sc、 47Sc、 110mIn、 97Ru、 62Cu、 64Cu、 67Cu、 68Cu、 86Y、 88Y、 121Sn、 161Tb、 166Ho、 105Rh、 177Lu、 172Lu和 18F。
在可选的实施方式中，所述化学发光试剂包括但不限于鲁米诺及其衍生物、光泽精、甲壳动物荧光素及其衍生物、联吡啶钌及其衍生物、吖啶酯及其衍生物、二氧环乙烷及其衍生物、洛粉碱及其衍生物和过氧草酸盐及其衍生物。
在可选的实施方式中，所述纳米颗粒类标记物包括但不限于纳米颗粒、胶体、有机纳米颗粒、磁性纳米颗粒、量子点纳米颗粒和稀土络合物纳米颗粒。
在可选的实施方式中，所述胶体包括但不限于胶体金属、分散型染料、染料标记的微球和乳胶。
在可选的实施方式中，所述胶体金属包括但不限于胶体金、胶体银和胶体硒。本公开一实施方式提供一种编码上述抗体或其功能性片段的核酸分子。本公开一实施方式提供含有上述核酸分子的载体。本公开一实施方式提供含有上述载体的重组细胞。本公开一些实施方式提供上述抗体或其功能性片段、试剂或试剂盒在检测AMH中的用途。
本公开一些实施方式提供上述抗体或其功能性片段、试剂或试剂盒，用于检测AMH的用途。
本公开一些实施方式提供检测AMH中的方法，包括：
A)在足以发生结合反应的条件下，使上述抗体或其功能性片段与样品接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物。
本公开一些实施方式提供上述抗体或其功能性片段、试剂或试剂盒在测定受试者卵子存量及卵巢功能的用途。
一种测定受试者卵子存量及卵巢功能的方法，包括：
A)在足以发生结合反应的条件下，使上述抗体或其功能性片段与来自受试者的样品接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物。
一种诊断受试者中与AHM相关的疾病的方法，包括：
A)在足以发生结合反应的条件下，使上述抗体或其功能性片段与来自受试者的样品接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物。
在可选的实施方式中，与AHM相关的疾病包括但不限于多囊性卵巢综合症、卵巢过度刺激综合症、早发性卵巢功能不全、卵巢低反应、不孕症、继发性卵巢衰竭、月经过少、闭经、子宫内膜异位。
本公开一实施方式提供一种制备抗体或其功能性片段的方法，其包括：培养如上所述的重组细胞，从培养产物中分离纯化得到所述抗体或其功能性片段。
在本公开提供了抗体或其功能性片段的氨基酸序列的基础上，本领域技术人员容易想到采用基因工程技术或其他技术(化学合成、杂交瘤细胞)制备得到该抗体或其功能性片段，例如从能够重组表达如上任一项所述的抗体或其功能性片段的重组细胞的培养产物中分离纯化得到该抗体或其功能性片段，这对本领域技术人员来说是容易实现的，基于此，无论采用何种技术制备本公开的抗体或其功能性片段，其均属于本公开的保护范围。
实施例
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实施例1
本实施例中限制性内切酶、rTaq DNA聚合酶购自Takara公司。MagExtractor-RNA提取试剂盒购自TOYOBO公司。BD SMART TM RACE cDNA Amplification Kit试剂盒购自Takara公司。pMD-18T载体购自Takara公司。质粒提取试剂盒购自天根公司。引物合成和基因测序由Invitrogen公司完成。
1重组质粒的构建
(1)抗体基因制备
从分泌抗AMH抗体的杂交瘤细胞株中提取mRNA，通过RT-PCR方法获得DNA产物，该产物用rTaq DNA聚合酶进行加A反应后插入到pMD-18T载体中，转化到DH5α感受态细胞中，长出菌落后分别取重链(Heavy Chain) 及轻链(Light Chain)基因克隆，各4个克隆送基因测序公司进行测序。
(2)抗体可变区基因的序列分析
将上述测序得到的基因序列放在IMGT抗体数据库(IMGT抗体数据库来源于：http://www.imgt.org)中进行分析，并利用VNTI11.5软件进行分析确定重链和轻链引物对扩增出的基因都是正确的，其中轻链扩增出的基因片段中，VL基因序列为336bp，属于VkII基因家族，其前方有57bp的前导肽序列；重链引物对扩增出的基因片段中，VH基因序列为366bp，属于VH1基因家族，其前方有57bp的前导肽序列。
(3)重组抗体表达质粒的构建
pcDNA TM 3.4
Figure PCTCN2021117806-appb-000009
vector为构建的重组抗体真核表达载体，该表达载体已经引入HindIII、BamHI、EcoRI等多克隆酶切位点，并命名为pcDNA3.4A表达载体，后续简称3.4A表达载体；根据上述pMD-18T中抗体可变区基因测序结果，设计该抗体的VL和VH基因特异性引物，两端分别带有HindIII、EcoRI酶切位点和保护碱基，通过PCR扩增方法扩出0.74kb的轻链基因片段和1.46kb的重链基因片段。
重链和轻链基因片段分别采用HindIII/EcoRI双酶切，3.4A载体采用HindIII/EcoRI双酶切，将片段和载体纯化回收后重链基因和轻链基因连接3.4A表达载体中，得到重链和轻链的重组表达质粒。
2稳定细胞株筛选
(1)重组抗体表达质粒瞬时转染CHO细胞，确定表达质粒活性
将步骤1-(3)得到的质粒用超纯水稀释至400ng/ml，调节CHO细胞1.43×10 7个细胞/ml于离心管中，100μL质粒与700μL细胞混合，转入电转杯，电转，第3、5、7天取样计数，第7天收样检测。
包被液(主要成分NaHCO 3)稀释AMH抗原到1μg/ml，每孔100μL，4℃过夜；次日，洗涤液清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔120μL，37℃，1h，拍干；加入稀释后的细胞上清，100μL/孔，37℃，30min；洗涤液清洗5次，拍干；加入羊抗鼠IgG-HRP，每孔100μL，37℃，30min；洗涤液清洗5次，拍干；加入显色液A液(50μL/孔，含柠檬酸+醋酸钠+乙酰苯胺+过氧化脲)，加入显色液B液(50μL/孔，含柠檬酸+EDTA·2Na+TMB+浓HCl)，10min；加入终止液(50μL/孔，含EDTA·2Na+浓H 2SO 4)；酶标仪上450nm(参考630nm)处读OD值。结果显示细胞上清稀释1000倍后反应OD仍大于1.0，未加细胞上清孔反应OD小于0.1，表明质粒瞬转后产生的抗体对AMH抗原有活性。
(2)重组抗体表达质粒线性化
准备下述试剂：Buffer 50μL、DNA 100μg/管、PuvⅠ酶10μL、无菌水补至500μL，37℃水浴酶切过夜；先用等体积酚/氯仿/异戊醇(下层)25:24:1，再用氯仿(水相)依次进行抽提；0.1倍体积(水相)3M醋酸钠和2倍体积乙醇冰上沉淀，70％乙醇漂洗沉淀，去除有机溶剂，待乙醇挥发完全用适量的灭菌水进行复融，最后进行浓度的测定。
(3)重组抗体表达质粒稳定转染，加压筛选稳定细胞株
将步骤2-(2)得到的质粒用超纯水稀释至400ng/ml，调节CHO细胞1.43×10 7个细胞/ml于离心管中，100μL质粒与700μL细胞混合，转入电转杯，电转，次日计数；25umol/L MSX 96孔加压培养约25天。
显微镜下观察标记长有细胞的克隆孔,并记录汇合度；取培养上清，送样检测；挑选抗体浓度、相对浓度高的细胞株转24孔，3天左右转6孔；3天后保种批培，调整细胞密度0.5×10 6个细胞/ml，2.2ml进行批培养，细胞密度0.3×10 6个细胞/ml，2ml进行保种；7天6孔批培上清送样检测，挑选抗体浓度及细胞直径较小的细胞株转TPP保种传代。
3重组抗体生产
(1)细胞扩培
将步骤2-(3)重组抗体表达质粒稳定转染，加压筛选稳定细胞株获得的细胞经复苏之后先在125ml规格的摇瓶中培养，接种体积为30ml，培养基为100％Dynamis培养基，放置于转速120r/min，温度为37℃，二氧化碳为8％的摇床中。培养72h，以50万个细胞/ml接种密度接种扩培，扩培体积根据生产需求进行计算，培养基为100％Dynamis培养基。之后每72h扩培一次。当细胞量满足生产需求时，严格控制接种密度为50万个细胞/ml左右进行生产。
(2)摇瓶生产及纯化
摇瓶参数：转速120r/min，温度为37℃，二氧化碳为8％。流加补料：在摇瓶中培养至72h时开始每天补料，HyClone Cell Boost Feed 7a每天流加初始培养体积的3％，Feed 7b每天流加量为初始培养体积的千分之一，一直补到第12天(第12天补料)。葡萄糖在第六天补加3g/L。第13天收样。用蛋白A(proteinA)亲和层析柱进行亲和纯化。取4μg纯化的抗体进行还原性SDS-PAGE，4μg外来对照抗体作为对照，电泳图如下图1所示，在还原性SDS-PAGE后显示两条带，1条Mr为50KD(重链，SEQ ID NO:14)，另一条Mr为28KD(轻链，SEQ ID NO:13)。
实施例2
抗体的性能检测
(1)实施例1抗体及其突变体的活性检测
分析实施例1的抗体(WT)序列，其重链可变区如SEQ ID NO:12所示，其中，重链可变区上的各互补决定区的氨基酸序列如下：
CDR-VH1：G-F-T(X1)-F-S-V(X2)-F-G-M-S；
CDR-VH2：T-L(X1)-S-N-G-G-S(X2)-Y-T-Y-Y-P-D(X3)-S-L(X4)-K-G；
CDR-VH3：A(X1)-R-H-P-R-V(X2)-N-G-Y(X3)-D-G-A-M；
其轻链可变区如SEQ ID NO:11所示，其中，轻链可变区上的各互补决定区的氨基酸序列如下：
CDR1-VL：A-S-E(X1)-S-I(X2)-D-N-Y-D-L(X3)-S-F-M；
CDR-VL2：A-A-S-N-K(X1)-A(X2)-S；
CDR-VL3：Q-Q-S-R(X1)-E-I(X2)-P-W。
在实施例1的抗AMH抗体(WT)基础上，在互补决定区中对于抗体活性有关的位点进行突变，其中，X1、X2、X3、X4均为突变位点。见下表1。
表1与抗体活性有关的突变位点
Figure PCTCN2021117806-appb-000010
对表1中的抗体结合活性检测：
包被液(主要成分NaHCO 3)稀释AMH抗原到1μg/ml进行微孔板包被，每孔100μl，4℃过夜；次日，洗涤液(组成：)清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔120μl，37℃，1h，拍干；加入稀释后的(表1中的WT和突变1-6)AMH单克隆抗体，100μl/孔，37℃，30min-60min；洗涤液清洗5次，拍干；加入羊抗鼠IgG-HRP，每孔100μl，37℃，30min；洗涤液(PBS)清洗5次，拍干；加入显色液A液(50μl/孔，含2.1g/L柠檬酸、12.25g/L柠檬酸、0.07g/L乙酰苯胺和0.5g/L过氧化脲)，加入显色液B液(50μl/孔，含1.05g/L柠檬酸、0.186g/LEDTA·2Na、0.45g/L TMB和0.2ml/L浓HCl)，10min；加入终止液(50μl/孔，含0.75g/EDTA·2Na和10.2ml/L浓H2SO4)；酶标仪上450nm(参考630nm)处读OD值。结果见下表2。
表2 WT抗体及其突变体的活性数据
抗体浓度(ng/ml) 250 125 62.5 31.25 15.625 0
WT 1.952 1.621 1.182 0.650 0.360 0.046
突变1 2.411 2.382 2.221 1.811 1.016 0.082
突变2 2.45 2.383 2.167 1.503 0.856 0.05
突变3 2.312 2.323 2.107 1.616 0.926 0.063
突变4 2.396 2.046 1.882 0.955 0.860 0.146
突变5 0.653 0.105 - - - -
突变6 0.426 0.063 - - - -
表2数据显示，相较于突变5和突变6来说，WT、突变1至突变4的活性更好，其中，突变1的活性最佳。
(2)抗体及其突变体的亲和力检测
(a)在突变1的基础上，对其他位点进行突变，各突变的序列见下表3。
表3与抗体亲和力有关的突变位点
Figure PCTCN2021117806-appb-000011
Figure PCTCN2021117806-appb-000012
Figure PCTCN2021117806-appb-000013
亲和力分析
利用AMC传感器，将纯化出来的抗体用PBST稀释到10ug/mL，AMH抗原用PBST进行梯度稀释：45ug/mL、22.5ug/mL、11.3ug/mL、5.60ug/mL、2.80ug/mL、1.40ug/mL、0.70ug/mL。
运行流程：缓冲液1(PBST)中平衡60s，抗体溶液中固化抗体300s，缓冲液2(PBST)中孵育180s，抗原溶液中结合420s，缓冲液2中解离1200s，用10mM pH 1.69GLY溶液及缓冲液3进行传感器再生，输出数据。K D表示平衡解离常数即亲和力；kon表示结合速率；kdis表示解离速率。结果见下表4。
表4亲和力检测数据
  K D(M) kon(1/Ms) kdis(1/s)
突变1 8.94E-09 8.86E+04 7.92E-04
突变1-1 4.65E-09 1.73E+05 8.04E-04
突变1-2 2.92E-09 2.58E+05 7.54E-04
突变1-3 2.79E-09 3.12E+05 8.72E-04
突变1-4 4.26E-09 1.96E+05 8.34E-04
突变1-5 2.73E-09 2.34E+05 6.39E-04
突变1-6 3.21E-09 2.86E+05 9.18E-04
突变1-7 2.22E-09 3.34E+05 7.41E-04
突变1-8 2.07E-09 3.93E+05 8.12E-04
突变1-9 4.96E-09 1.99E+05 9.87E-04
突变1-10 2.48E-09 3.68E+05 9.13E-04
突变1-11 3.06E-09 2.88E+05 8.81E-04
突变1-12 4.05E-09 1.31E+05 5.31E-04
突变1-13 6.53E-09 1.34E+05 8.75E-04
突变1-14 3.09E-09 2.98E+05 9.20E-04
突变1-15 2.04E-09 3.24E+05 6.62E-04
突变1-16 3.27E-09 2.42E+05 7.92E-04
突变1-17 4.50E-09 1.44E+05 6.48E-04
突变1-18 1.86E-09 3.38E+05 6.28E-04
突变1-19 2.70E-09 3.56E+05 9.61E-04
突变1-20 1.07E-08 8.90E+04 9.52E-04
突变1-21 4.10E-09 1.64E+05 6.72E-04
突变1-22 4.87E-09 1.42E+05 6.92E-04
突变1-23 1.52E-09 3.35E+05 5.10E-04
突变1-24 6.47E-09 1.18E+05 7.64E-04
突变1-25 5.42E-09 1.63E+05 8.83E-04
突变1-26 5.06E-09 1.81E+05 9.15E-04
突变1-27 6.68E-09 1.19E+05 7.95E-04
突变1-28 1.57E-09 3.13E+05 4.92E-04
突变1-29 3.96E-09 2.47E+05 9.79E-04
突变1-30 1.67E-09 3.42E+05 5.72E-04
突变1-31 4.25E-09 1.50E+05 6.38E-04
突变1-32 2.35E-09 3.47E+05 8.16E-04
突变1-33 3.91E-09 2.09E+05 8.18E-04
突变1-34 1.96E-09 3.06E+05 6.01E-04
突变1-35 2.00E-09 3.27E+05 6.54E-04
突变1-36 3.96E-09 1.82E+05 7.20E-04
突变1-37 2.85E-09 2.83E+05 8.07E-04
突变1-38 2.74E-09 3.50E+05 9.60E-04
突变1-39 4.24E-09 1.54E+05 6.52E-04
突变1-40 3.61E-09 1.74E+05 6.29E-04
突变1-41 7.55E-09 1.26E+05 9.51E-04
突变1-42 2.23E-09 2.63E+05 5.86E-04
突变1-43 3.33E-09 2.79E+05 9.29E-04
突变1-44 1.60E-09 3.25E+05 5.19E-04
突变1-45 4.62E-09 1.74E+05 8.04E-04
突变1-46 4.50E-09 1.83E+05 8.23E-04
突变1-47 4.23E-09 1.22E+05 5.16E-04
突变1-48 2.88E-09 2.54E+05 7.31E-04
突变1-49 3.57E-09 1.57E+05 5.60E-04
突变1-50 2.05E-09 3.66E+05 7.51E-04
突变1-51 3.98E-09 1.41E+05 5.61E-04
突变1-52 6.59E-09 1.41E+05 9.29E-04
突变1-53 3.75E-09 1.98E+05 7.43E-04
突变1-54 4.05E-09 1.65E+05 6.68E-04
表4数据可以看出，突变1及其系列突变体都具有较好的亲和力，说明在突变1的基础上，按表3的突变方式进行突变得到的抗体均具有较好的亲和力。
(b)在WT的基础上，对其他位点进行突变，并检测各突变体的亲和力，各突变的序列见下表5，对应的亲和力数据见表6。
表5以WT为骨架进行的突变
Figure PCTCN2021117806-appb-000014
表6 WT抗体及其突变体的亲和力检测结果
  K D(M) kon(1/Ms) kdis(1/s)
WT 2.57E-08 1.01E+04 2.60E-04
WT 1 1.56E-08 2.43E+04 3.78E-04
WT 2 2.08E-08 1.82E+04 3.79E-04
WT 3 1.70E-08 2.63E+04 4.48E-04
WT 4 1.51E-08 2.65E+04 4.01E-04
WT5 1.30E-08 2.69E+04 3.50E-04
WT6 1.78E-08 2.19E+04 3.89E-04
表6数据显示，WT及其系列突变体都具有不错的亲和力，说明在WT的基础上，按表5的突变方式突变得到的抗体均具有不错的亲和力。
(3)裸抗稳定性考核
将上述抗体置于4℃(冰箱)、-80℃(冰箱)、37℃(恒温箱)放置21天，取7天、14天、21天样品进行状态观察，并对21天样品进行活性检测，结果显示以上三种考核条件下抗体放置21天均未见明显蛋白状态变化，活性也未随考核温度的升高呈下降趋势，说明上述抗体稳定。下表7突变1抗体为考核21天的酶免活性检测OD结果。
表7
样品浓度(ng/ml) 100 10 0
4℃，21天样品 1.985 0.528 0.05
-80℃，21天样品 1.93 0.538 0.045
37℃，21天样品 1.931 0.593 0.051
(4)应用性能检测
用表3的抗体与另一株AMH抗体配对使用，采用双抗体夹心法，在化学发光平台检测特异性达100％，灵敏度达99％-99.8％之间。
以上所述仅为本公开的可选的实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
工业实用性
本公开提供了一种抗AMH的抗体、编码抗体的核酸分子和氨基酸以及该检测AMH的试剂和试剂盒。本公开提供的抗体能够特异性结合AMH，对AMH具有较好的亲和力，采用该抗体检测AMH，具有较高的灵敏度和特异性，制备的检测AMH的试剂和试剂盒同样具有与该抗体相同的技术效果，具有广泛的应用前景和较高的市场价值。

Claims (19)

  1. 一种抗AMH的抗体或其功能性片段，其特征在于，所述抗体或其功能性片段包括如下互补决定区：
    CDR-VH1：G-F-X1-F-S-X2-F-G-M-S；其中：X1是S或T；X2是I、V或L；
    CDR-VH2：T-X1-S-N-G-G-X2-Y-T-Y-Y-P-X3-S-X4-K-G；其中：X1是L或I；X2是T或S；X3是E或D；X4是I、V或L；
    CDR-VH3：X1-R-H-P-R-X2-N-G-X3-D-G-A-M；其中：X1是A或T；X2是I、V或L；X3是Y、F或S；
    CDR-VL1：A-S-X1-S-X2-D-N-Y-D-X3-S-F-M；其中：X1是Q或E；X2是I、V或L；X3是I或L；
    CDR-VL2：A-A-S-N-X1-X2-S；其中：X1是K、Q或R；X2是G或A；
    CDR-VL3：Q-Q-S-X1-E-X2-P-W；其中：X1是K和R；X2是L、V或I。
  2. 根据权利要求1所述的抗AMH的抗体或其功能性片段，其特征在于，
    CDR-VH2中，X1是I；
    CDR-VH3中，X1是T；
    CDR-VL1中，X3是I；
    CDR-VL2中，X2是G；
    CDR-VL3中，X1是K；
    优选的，CDR-VH1中，X1是S；
    优选的，CDR-VH1中，X1是T；
    优选的，CDR-VH1中，X2是I；
    优选的，CDR-VH1中，X2是V；
    优选的，CDR-VH1中，X2是L；
    优选的，CDR-VH2中，X2是T；
    优选的，CDR-VH2中，X2是S；
    优选的，CDR-VH2中，X3是E；
    优选的，CDR-VH2中，X3是D；
    优选的，CDR-VH2中，X4是I；
    优选的，CDR-VH2中，X4是V；
    优选的，CDR-VH2中，X4是L；
    优选的，CDR-VH3中，X2是I；
    优选的，CDR-VH3中，X2是V；
    优选的，CDR-VH3中，X2是L；
    优选的，CDR-VH3中，X3是Y；
    优选的，CDR-VH3中，X3是F；
    优选的，CDR-VH3中，X3是S；
    优选的，CDR-VL1中，X1是Q；
    优选的，CDR-VL1中，X1是E；
    优选的，CDR-VL1中，X2是I；
    优选的，CDR-VL1中，X2是V；
    优选的，CDR-VL1中，X2是L；
    优选的，CDR-VL2中，X1是K；
    优选的，CDR-VL2中，X1是Q；
    优选的，CDR-VL2中，X1是R；
    优选的，CDR-VL3中，X2是L；
    优选的，CDR-VL3中，X2是V；
    优选的，CDR-VL3中，X2是I；
    优选的，所述抗体或其功能性片段的各互补决定区选自如下突变组合1-55中的任意一种：
    Figure PCTCN2021117806-appb-100001
    Figure PCTCN2021117806-appb-100002
    Figure PCTCN2021117806-appb-100003
  3. 根据权利要求2所述的抗AMH的抗体或其功能性片段，其特征在于，所述抗体或其功能性片段与抗AMH抗原以K D≤2.6×10 -8mol/L的亲和力结合；优选的，K D≤9×10 -9mol/L。
  4. 根据权利要求1所述的抗AMH的抗体或其功能性片段，其特征在于，
    CDR-VH2中，X1是L；
    CDR-VH3中，X1是A；
    CDR-VL1中，X3是L；
    CDR-VL2中，X2是A；
    CDR-VL3中，X1是R；
    优选的，所述抗体或其功能性片段的各互补决定区选自如下突变组合56-62中的任意一种：
    Figure PCTCN2021117806-appb-100004
  5. 根据权利要求1-4任一项所述的抗AMH的抗体或其功能性片段，其特征在于，所述抗体包括序列依次如SEQ ID NO:1-4所示的轻链骨架区FR1-L、FR2-L、FR3-L及FR4-L，和/或，序列依次如SEQ ID NO:5-8所示的重链骨架区FR1-H、FR2-H、FR3-H及FR4-H；
    优选的，所述抗体还包含恒定区；
    优选的，所述恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE和IgD中的任意一者的恒定区；
    优选的，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人；
    优选的，所述恒定区来源于小鼠；
    优选的，所述恒定区的轻链恒定区序列如SEQ ID NO:9所示，所述恒定区的重链恒定区序列如SEQ ID NO:10所示；
    优选的，所述功能性片段选自所述抗体的VHH、F(ab’)2、Fab’、Fab、Fv和scFv中的任意一种。
  6. 根据权利要求1-4中任一项所述的抗AMH的抗体或其功能性片段，其特征在于，所述抗体包括分别依次与序列SEQ ID NO:1、2、3、4具有至少80％的同源性的轻链骨架区FR1-L、FR2-L、FR3-L及FR4-L，和/或，分别依次与序列SEQ ID NO:5、6、7、8具有至少80％的同源性的重链骨架区FR1-H、FR2-H、FR3-H及FR4-H。
  7. 一种检测AMH的试剂或试剂盒，其特征在于，其包括如权利要求1-6任一项所述的抗体或其功能性片段。
  8. 根据权利要求7所述的试剂或试剂盒，其特征在于，所述抗体或其功能性片段标记有可被检测的标记物；
    优选的，所述可被检测的标记物选自荧光染料、催化底物显色的酶、放射性同位素、化学发光试剂和纳米颗粒类标记物；
    优选的，所述荧光染料选自荧光素类染料及其衍生物、罗丹明类染料及其衍生物、Cy系列染料及其衍生物、Alexa系列染料及其衍生物和蛋白类染料及其衍生物；
    优选的，所述催化底物显色的酶选自辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡萄糖氧化酶、碳酸酐酶、乙酰胆碱酯酶以及6-磷酸葡萄糖脱氧酶；
    优选的，所述放射性同位素选自 212Bi、 131I、 111In、 90Y、 186Re、 211At、 125I、 188Re、 153Sm、 213Bi、 32P、 94mTc、 99mTc、 203Pb、 67Ga、 68Ga、 43Sc、 47Sc、 110mIn、 97Ru、 62Cu、 64Cu、 67Cu、 68Cu、 86Y、 88Y、 121Sn、 161Tb、 166Ho、 105Rh、 177Lu、 172Lu和 18F；
    优选的，所述化学发光试剂选自鲁米诺及其衍生物、光泽精、甲壳动物荧光素及其衍生物、联吡啶钌及其衍生物、吖啶酯及其衍生物、二氧环乙烷及其衍生物、洛粉碱及其衍生物和过氧草酸盐及其衍生物；
    优选的，所述纳米颗粒类标记物选自纳米颗粒、胶体、有机纳米颗粒、磁性纳米颗粒、量子点纳米颗粒和稀土络合物纳米颗粒；
    优选的，所述胶体选自胶体金属、分散型染料、染料标记的微球和乳胶；
    优选的，所述胶体金属选自胶体金、胶体银和胶体硒。
  9. 一种核酸分子，所述核酸分子编码如权利要求1-6任一项所述的抗体或其功能性片段。
  10. 一种载体，其特征在于，其含有编码如权利要求1-6任一项所述的抗体或其功能性片段的核酸分子。
  11. 一种重组细胞，其特征在于，其含有权利要求10所述的载体。
  12. 如权利要求1-6任一项所述的抗体或其功能性片段或者如权利要求7或8所述的试剂或试剂盒在检测AMH中的用途。
  13. 如权利要求1-6任一项所述的抗体或其功能性片段或者如权利要求7或8所述的试剂或试剂盒，用于检测AMH的用途。
  14. 一种检测AMH的方法，包括：
    A)在足以发生结合反应的条件下，使权利要求1-6任一项所述的抗体或其功能性片段与样品接触以进行结合反应；以及
    B)检测结合反应产生的免疫复合物。
  15. 如权利要求1-6任一项所述的抗体或其功能性片段或者如权利要求7或8所述的试剂或试剂盒在测定受试者卵子存量及卵巢功能的用途。
  16. 一种测定受试者卵子存量及卵巢功能的方法，包括：
    A)在足以发生结合反应的条件下，使权利要求1-6任一项所述的抗体或其功能性片段与来自所述受试者的样品接触以进行结合反应；以及
    B)检测结合反应产生的免疫复合物。
  17. 一种诊断受试者中与AHM相关的疾病的方法，包括：
    A)在足以发生结合反应的条件下，使权利要求1-6任一项所述的抗体或其功能性片段与来自所述受试者的样品接触以进行结合反应；以及
    B)检测结合反应产生的免疫复合物。
  18. 根据权利要求17所述的方法，其中，所述与AHM相关的疾病是多囊性卵巢综合症、卵巢过度刺激综合症、早发性卵巢功能不全、卵巢低反应、不孕症、继发性卵巢衰竭、月经过少、闭经、子宫内膜异位。
  19. 一种制备如权利要求1-6任一项所述的抗体或其功能性片段的方法，其特征在于，其包括：培养权利要求11所述的重组细胞，从培养产物中分离纯化得到所述抗体或其功能性片段。
PCT/CN2021/117806 2020-11-12 2021-09-10 抗amh的抗体、检测amh的试剂和试剂盒 WO2022100263A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011259343.1 2020-11-12
CN202011259343.1A CN114478764A (zh) 2020-11-12 2020-11-12 抗amh的抗体、检测amh的试剂和试剂盒

Publications (1)

Publication Number Publication Date
WO2022100263A1 true WO2022100263A1 (zh) 2022-05-19

Family

ID=81491498

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/117806 WO2022100263A1 (zh) 2020-11-12 2021-09-10 抗amh的抗体、检测amh的试剂和试剂盒

Country Status (2)

Country Link
CN (1) CN114478764A (zh)
WO (1) WO2022100263A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117229409B (zh) * 2022-06-08 2024-04-23 东莞市朋志生物科技有限公司 一种抗苯环己哌啶抗体、检测苯环己哌啶的试剂和试剂盒

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017207694A1 (en) * 2016-06-02 2017-12-07 Kohlmann Angelica Antibodies that bind to human anti-müllerian hormone (amh) and their uses
CN107523552A (zh) * 2017-07-18 2017-12-29 杭州博茵生物技术有限公司 分泌抗amh单克隆抗体的杂交瘤细胞株、抗amh单克隆抗体及应用
CN110218252A (zh) * 2019-05-27 2019-09-10 郑州伊美诺生物技术有限公司 一种抗amh特异性抗体的制备方法以及该抗体在检测amh试剂盒中的应用
CN110407937A (zh) * 2018-04-26 2019-11-05 江苏正大天创生物工程有限公司 一种抗amh单克隆抗体的制备及其应用
CN111004326A (zh) * 2019-12-27 2020-04-14 苏州博方生物技术有限公司 一种抗amh单克隆抗体及其制备方法和应用
CN111196851A (zh) * 2018-11-20 2020-05-26 厦门万泰凯瑞生物技术有限公司 针对人抗缪勒管激素的特异性抗体及其应用
CN111217907A (zh) * 2020-01-21 2020-06-02 四川大学 抗amh单克隆抗体及其应用
US10774142B2 (en) * 2015-09-25 2020-09-15 Inserm (Institut National De La Sante Et De La Recherche Medicale) Anti-mullerian hormone (AMH) neutralizing antibodies and uses thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE602006016267D1 (de) * 2005-05-24 2010-09-30 Beckman Coulter Inc Immunologische assays und antikörper für das anti-müller-hormon
CN111273042B (zh) * 2020-01-21 2023-09-01 迈克生物股份有限公司 抗缪勒氏管激素检测试剂盒

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10774142B2 (en) * 2015-09-25 2020-09-15 Inserm (Institut National De La Sante Et De La Recherche Medicale) Anti-mullerian hormone (AMH) neutralizing antibodies and uses thereof
WO2017207694A1 (en) * 2016-06-02 2017-12-07 Kohlmann Angelica Antibodies that bind to human anti-müllerian hormone (amh) and their uses
CN107523552A (zh) * 2017-07-18 2017-12-29 杭州博茵生物技术有限公司 分泌抗amh单克隆抗体的杂交瘤细胞株、抗amh单克隆抗体及应用
CN110407937A (zh) * 2018-04-26 2019-11-05 江苏正大天创生物工程有限公司 一种抗amh单克隆抗体的制备及其应用
CN111196851A (zh) * 2018-11-20 2020-05-26 厦门万泰凯瑞生物技术有限公司 针对人抗缪勒管激素的特异性抗体及其应用
CN110218252A (zh) * 2019-05-27 2019-09-10 郑州伊美诺生物技术有限公司 一种抗amh特异性抗体的制备方法以及该抗体在检测amh试剂盒中的应用
CN111004326A (zh) * 2019-12-27 2020-04-14 苏州博方生物技术有限公司 一种抗amh单克隆抗体及其制备方法和应用
CN111217907A (zh) * 2020-01-21 2020-06-02 四川大学 抗amh单克隆抗体及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
OH SO RA, CHOE SUN YI, CHO YEON JEAN: "Clinical application of serum anti-Müllerian hormone in women", CLINICAL AND EXPERIMENTAL REPRODUCTIVE MEDICINE, vol. 46, no. 2, 1 June 2019 (2019-06-01), pages 50 - 59, XP055929066, ISSN: 2233-8233, DOI: 10.5653/cerm.2019.46.2.50 *
SUN HUI, MAO HUAJIE, CAI JIANHUI, ZHAO YA: "Research progress on anti-mullerian hormone clinical applications and immunoassay development", FRONTIERS IN LABORATORY MEDICINE, vol. 2, no. 1, 23 March 2018 (2018-03-23), pages 14 - 18, XP055929068, ISSN: 2542-3649, DOI: 10.1016/j.flm.2018.02.002 *
ZHANG XIAO-GANG;LIU XIA;LIU FEI;ZHU XI-QIANG;LING PEI-XUE: "Advances in Assay Technology of AMH", FOOD AND DRUG, vol. 19, no. 5, 20 September 2017 (2017-09-20), pages 372 - 377, XP055929064, ISSN: 1672-979X *

Also Published As

Publication number Publication date
CN114478764A (zh) 2022-05-13

Similar Documents

Publication Publication Date Title
WO2022089045A1 (zh) 抗新型冠状病毒的抗体和检测新型冠状病毒的试剂盒
CA3200148A1 (en) Antibody against novel coronavirus, and reagent and kit for detecting novel coronavirus
WO2022121414A1 (zh) 抗ca24-2的抗体以及检测ca24-2的试剂和试剂盒
WO2022062789A1 (zh) 针对乙型流感病毒的抗体和检测试剂盒
US8604173B2 (en) Dopaminergic neuron progenitor cell marker 187A5
WO2023078447A1 (zh) 抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒
WO2022052781A1 (zh) 抗甲型流感病毒的抗体和试剂盒
WO2022100263A1 (zh) 抗amh的抗体、检测amh的试剂和试剂盒
WO2022057573A1 (zh) 抗甲型流感病毒的抗体、检测试剂盒以及制备方法
CN116693676A (zh) 抗肺炎支原体的抗体、检测肺炎支原体的试剂和试剂盒
CN114516914B (zh) 抗n末端脑钠肽前体的抗体和检测n末端脑钠肽前体的试剂和试剂盒
WO2022121368A1 (zh) 抗胃泌素释放肽的抗体、检测试剂与试剂盒
CN116836277B (zh) 抗雌二醇抗体、检测雌二醇的试剂和试剂盒
WO2022121415A1 (zh) 抗ca19-9的抗体、其应用和检测ca19-9的试剂盒
WO2022100190A1 (zh) 针对抗穆勒氏管激素的抗体以及检测抗穆勒氏管激素的试剂盒
CN114516915B (zh) 抗n末端脑钠肽前体的抗体以及制备抗体的方法
CN116693683B (zh) 抗睾酮抗体、检测睾酮的试剂和试剂盒
CN114516913B (zh) 抗n末端脑钠肽前体的抗体和检测试剂盒
CN116836278B (zh) 抗孕酮抗体、检测孕酮的试剂和试剂盒
CN116836279B (zh) 抗四碘甲状腺素的抗体、检测四碘甲状腺素的试剂和试剂盒
WO2022022532A1 (zh) 可特异性结合25-羟基维生素d的抗体、其应用以及诊断试剂盒
CN116693678B (zh) 抗肺炎支原体的抗体、检测肺炎支原体的试剂和试剂盒
CN116496394B (zh) 抗s100蛋白的抗体、检测s100蛋白的试剂和试剂盒
CN114075280B (zh) 抗ngal的单克隆抗体及其应用和检测试剂盒
CN116836271B (zh) 抗b型链球菌的抗体、检测b型链球菌的试剂和试剂盒

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21890791

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21890791

Country of ref document: EP

Kind code of ref document: A1