Reagent or kit for detecting E2 by antibody sandwich method and application of reagent or kit

Abstract

The invention relates to the technical field of monoclonal antibody preparation and immunological detection, in particular to a reagent or a kit for detecting E2 through an antibody sandwich method and application of the reagent or the kit. The paired monoclonal antibody has strong specificity and high detection sensitivity, and solves the problems of low content of E2 in human serum and high detection difficulty. The monoclonal antibody is applied to a magnetic particle estradiol content detection kit. The kit is used for detecting the E2 content in serum, is combined with other sex hormone index detection and clinical manifestation, is beneficial to diagnosis of sex hormone disorder diseases, and has clinical significance in detection of diseases such as fertility disorders in thalamus-pituitary-gonad axis, male mammary gland development, estrogenic ovarian and testicular tumors, adrenal cortex hyperplasia and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of monoclonal antibody preparation and immunological detection technology, in particular to a reagent or kit for detecting E2 by antibody sandwich method and application thereof. BACKGROUND

[0002] Estradiol (E2) is an 18-carbon steroid hormone with a benzene ring in the A ring, and its molecular weight is 272.4 Da. It is the main component of human estrogen. 70% of E2 in the blood is combined with sex hormone binding globulin (SHBG), 25% of E2 is combined with plasma albumin, and the rest is free. E2 is mainly produced by ovarian follicles, corpus luteum and placenta during pregnancy, and a small amount is synthesized by testes or is a metabolite of testosterone, and becomes the main source of male estrogen. The synthesis of E2 changes periodically, can promote the formation and development of female reproductive organs, the appearance and maintenance of secondary sexual characteristics, and cooperate with progesterone to form the menstrual cycle. In addition, it also has a wide range of metabolic regulation effects.

[0003] During normal pregnancy, E2 is slightly elevated, and sharply decreases after the placenta is delivered. During abnormal pregnancy, E2 is mostly elevated, such as twin or multiple pregnancy and diabetic pregnant women; if E2 is reduced, it may be severe preeclampsia, fetal death in utero or gestational trophoblastic disease, and should be determined in combination with other examinations. In addition, the pathological causes of E2 elevation may also be ovarian cancer (such as ovarian granulosa cell tumor, ovarian embryoma, ovarian lipoid cell tumor, etc.), heart disease (such as myocardial infarction, angina pectoris, coronary artery stenosis) and other diseases (systemic lupus erythematosus, cirrhosis, male obesity, etc.). There are many pathological factors that cause E2 to decrease, such as low ovarian development, primary ovarian failure, pituitary amenorrhea or infertility, Cushing's syndrome, Addison's disease, malignant tumors, large area of infection, renal insufficiency, brain and pituitary focal lesions, etc. As can be seen, E2 has a wide significance for the diagnosis of clinical diseases, and is one of the important evaluation indexes.

[0004] At present, the detection methods of E2 mainly include chemiluminescence competition method, which is an immunological detection method based on the specific reaction of antibody and antigen, and uses luminescent substances (such as acridinium ester, isoluminol, etc.) to amplify and display the detected signal. Similar immunological detection methods include enzyme-linked method, immunoturbidimetry, radioimmunoassay, fluorescent immunochromatography, etc. The above immunological detection methods all need antibodies against E2. However, the prior art still lacks E2 antibodies with high activity. Therefore, there is a strong demand in the art for antibodies that effectively bind to E2 and detect it. SUMMARY

[0005] In view of the above, the application provides a reagent or kit for detecting E2 by antibody sandwich method and application thereof. The paired monoclonal antibody has high specificity and high detection sensitivity, and solves the problems of low content of E2 in human serum and difficulty in detection. The monoclonal antibody is applied to a magnetic particle estradiol detection kit. The E2 content in serum is detected, and combined with other sex hormone index detection and clinical manifestations, which is helpful for diagnosis of sex hormone disorder diseases, such as detection of diseases in hypothalamus-pituitary-gonadal axis, male breast development, estrogen-producing ovarian and testicular tumors, and adrenal cortical hyperplasia.

[0006] In order to achieve the above-mentioned purpose of the application, the application provides the following technical solutions.

[0007] The application provides a coating antibody, which comprises a heavy chain variable region and a light chain variable region, comprising:

[0008] (I) the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region have the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively; and

[0009] the LCDR1, LCDR2 and LCDR3 of the light chain variable region have the amino acid sequences shown in SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8, respectively; and / or

[0010] (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence of (I), and an amino acid sequence having the same function as the amino acid sequence of (I); or

[0011] (III) an amino acid sequence having more than 90% identity with the amino acid sequence of (I) or (II).

[0012] In some specific embodiments of the application, the coating antibody comprises:

[0013] (I) the heavy chain variable region has the amino acid sequence shown in SEQ ID No. 4; and

[0014] the light chain variable region has the amino acid sequence shown in SEQ ID No. 9; and / or

[0015] (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence of (I), and an amino acid sequence having the same function as the amino acid sequence of (I); or

[0016] (III), an amino acid sequence having more than 90% identity with the amino acid sequence as described in (I) or (II).

[0017] The present application also provides a labeled antibody, comprising a heavy chain variable region and a light chain variable region, comprising:

[0018] (I), the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region have the amino acid sequences of SEQ ID No. 11, SEQ ID No. 12 and SEQ ID No. 13, respectively; and

[0019] the LCDR1, LCDR2 and LCDR3 of the light chain variable region have the amino acid sequences of SEQ ID No. 16, SEQ ID No. 17 and SEQ ID No. 18, respectively; and / or

[0020] (II), an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence as described in (I), and an amino acid sequence having the same function as the amino acid sequence as described in (I); or

[0021] (III), an amino acid sequence having more than 90% identity with the amino acid sequence as described in (I) or (II).

[0022] In some embodiments of the present application, the labeled antibody, comprising:

[0023] (I), the heavy chain variable region has the amino acid sequence of SEQ ID No. 14; and

[0024] the light chain variable region has the amino acid sequence of SEQ ID No. 19; and / or

[0025] (II), an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence as described in (I), and an amino acid sequence having the same function as the amino acid sequence as described in (I); or

[0026] (III), an amino acid sequence having more than 90% identity with the amino acid sequence as described in (I) or (II).

[0027] The present application also provides a pair of monoclonal antibodies for detecting E2 by sandwich method, comprising the coating antibody and the labeled antibody.

[0028] The present application also provides a nucleic acid molecule, comprising a DNA fragment encoding any of the following:

[0029] (I), the coating antibody; and / or

[0030] (I), the coating antibody; and / or

[0031] (III), the pairing monoclonal antibody.

[0032] Preferably, the heavy chain variable region of the coating antibody comprises a nucleic acid molecule as shown in SEQ ID No. 5; and / or

[0033] the light chain variable region of the coating antibody comprises a nucleic acid molecule as shown in SEQ ID No. 10.

[0034] Preferably, the heavy chain variable region of the coating antibody comprises a nucleic acid molecule as shown in SEQ ID No. 5; and / or

[0035] the light chain variable region of the coating antibody comprises a nucleic acid molecule as shown in SEQ ID No. 10.

[0036] The present application also provides an expression vector comprising the nucleic acid molecule.

[0037] The present application also provides a host cell comprising the expression vector.

[0038] The present application also provides the use of any of the following in the preparation of an E2 detection product:

[0039] (I), the coating antibody; and / or

[0040] (II), the labeling antibody; and / or

[0041] (III), the pairing monoclonal antibody.

[0042] The present application also provides a reagent or kit for detecting E2 by antibody sandwich method, comprising any of the following:

[0043] (I), the coating antibody; and / or

[0044] (II), the labeling antibody; and / or

[0045] (III), the pairing monoclonal antibody.

[0046] Preferably, the kit further comprises any one or a combination of at least two of a standard, an antibody diluent, a termination solution or a washing solution.

[0047] The present application includes but is not limited to providing the following benefits:

[0048] The application discloses a monoclonal antibody preparation technology, which comprises phage antibody library screening, preparation of E2 small molecule sandwich method paired antibody by a genetic engineering method, and obtaining a cell strain stably expressing the antibody by using a high expression host cell. The application discloses an immunogen used for immunizing animals during preparation of the antibody, nucleotides and vectors for coding the monoclonal antibody, high expression host cells, an antibody preparation and purification method and application thereof. The paired monoclonal antibody has high specificity and high detection sensitivity, and solves the problems of low content of E2 in human serum and great detection difficulty. The monoclonal antibody is applied to a magnetic particle estradiol detection kit. The monoclonal antibody is used for detecting the content of E2 in serum, and combined with detection of other sex hormone indexes and clinical manifestations, and is helpful to diagnosis of sex hormone disorder diseases, such as detection of diseases in a hypothalamus-pituitary-gonadal axis, male breast development, estrogen-producing ovarian and testicular tumors and adrenal cortex hyperplasia, and has clinical significance. BRIEF DESCRIPTION OF DRAWINGS

[0049] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description.

[0050] Figure 1 Electrophoresis diagram of Ab2;

[0051] Figure 2 E2 sandwich method and liquid quality correlation;

[0052] Figure 3 E2 competition method and liquid quality correlation. DETAILED DESCRIPTION

[0053] The application discloses a reagent or kit for detecting E2 by an antibody sandwich method and application thereof, and a person skilled in the art can refer to the content herein, and appropriately improve process parameters to realize. It is particularly pointed out that all similar replacements and changes are obvious to a person skilled in the art, and they are regarded as being included in the protection scope of the present application. The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application.

[0054] As to the definition and terms in the field, the professionals can refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations of amino acid residues are standard 3-letter and / or 1-letter codes used in the field to represent one of 20 commonly used L-amino acids.

[0055] Antigen refers to a substance capable of stimulating the body to produce (specific) immune response, and can be combined with immune response product antibody and sensitized lymphocytes in vivo and in vitro, and immune effect (specific reaction) occurs. Antigen is divided into complete antigen and hapten. Small molecules are haptens, which have no immunogenicity and cannot cause immune response. However, after the combination of haptens and large molecular proteins, the immunogenicity is obtained and the complete antigen is changed, which can stimulate the immune system to produce antibodies and effector cells.

[0056] Antibody refers to the immunoglobulin produced by B lymphocytes transformed into plasma cells after the animal body is stimulated by antigenic substances, which can have specific binding reaction with corresponding antigens. The preparation method of antibody has experienced three stages: the first stage, the antigen with multiple antigenic determinants is selected as the immunogen, the animal is immunized by the conventional method, the lymphocytes in the body are activated, the antibodies against multiple antigenic determinants are produced, and the obtained antiserum is polyclonal antibody; the second stage, the fusion of hybridoma cells and B cells producing specific antibodies forms hybridoma cells, and the first monoclonal antibody is obtained; the third stage, with the rapid development of DNA recombination technology and other molecular biology technologies, the gene engineering antibody is prepared by using molecular biology means, which is called the third generation antibody. Compared with the hybridoma monoclonal antibody, one immunophages antibody library can enrich antigen specific antibody, can produce more and better antibodies, and even the affinity is higher than that of the antibody obtained from the traditional technology.

[0057] Most phage display technologies commonly use filamentous phages as carriers, such as M13, fd and f1. Filamentous phages mainly have five kinds of coat proteins: PⅢ, PⅥ, PⅦ, PⅧ and PⅨ, and the fusion proteins for antibody display mainly have two kinds: PⅢ and PⅧ. PⅢ is the largest molecule in the coat protein, and its spatial structure has flexibility and variability. The C terminal of PⅢ is a hydrophobic region, which is anchored on the coat, and the N terminal is free, which can insert large fragments of foreign proteins at its N terminal without affecting the structure and function of the phage. The C terminal of PⅧ protein mainly contains basic amino acid residues, which is easy to combine with DNA; the middle region is a hydrophobic region forming a phage coat; the N terminal is free and exposed on the phage surface, and the foreign sequence with 5-6 amino acid residues is generally fused at the N terminal, and each virion has about 2700 copies, and the fusion protein containing PⅧ can be multivalent displayed, and then the target gene is obtained by affinity screening. In the PⅧ protein display system, the copy number of the fusion protein is relatively high, generally about 2700, and generally no more than 6 amino acids of antibody polypeptide, otherwise it will affect the assembly of phage and reduce its infectivity. Because a larger polypeptide or even an entire protein gene fragment can be inserted in gPⅢ, a fragment of more than 50 ku has been successfully displayed, and the copy number of PⅢ is low (about 5 copies), which is beneficial to the screening of high affinity ligands.

[0058] The general procedure for preparing a natural antibody phage library is to extract total RNA from immune antibody cells (pre-B, mature B, memory B cells), which are usually obtained from immune organs (bone marrow, spleen, lymph nodes and tonsils) and peripheral blood. The first strand of cDNA is formed by reverse transcription using 6 nucleotide random primers, poly T or designed PCR downstream primers. The upstream primer is designed according to the 5' end sequence of the FR1 region or the conserved region of the guide sequence of the antibody framework region; the downstream primer of the ScFv library is designed from the conserved sequence of the hinge region (J region), and the Fab antibody library is designed from the constant sequence. One of the defects in screening is that some rare antibodies and low-affinity antibodies are often lost with multiple rounds of phage selection, and the affinity of the antibodies is low. The higher the diversity of the phage library, the higher the success rate of screening high-affinity antibodies.

[0059] An antibody exists as one or more Y-shaped monomers, each Y-shaped monomer consisting of four polypeptide chains, including two identical heavy chains and two identical light chains. The top of the Y-shaped structure is the variable region, which is the antigen binding region. The "variable region" of the heavy chain or light chain of an antibody is the N-terminal mature region of the chain. The heavy chains of human antibodies include five types of a, d, e, g and m, and the corresponding antibodies are called IgA, IgD, IgE, IgG and IgM. Among them, human g can be further divided into g1, g2, g3, g4 and other subtypes, corresponding to IgG1, IgG2, IgG3 and IgG4 four subtypes, and the light chain is of two types of l and k. Among them, IgG1 is the most abundant subtype in plasma.

[0060] An "antibody" includes any isotype of antibody or immunoglobulin, or an antibody fragment that maintains specific binding to an antigen, including but not limited to Fab, Fv, scFv and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies and fusion proteins containing antigen-binding portions of antibodies and non-antibody proteins. Antibodies can be labeled and detected, for example, by radioisotopes, enzymes capable of producing detectable substances, fluorescent proteins, biotin, etc. Antibodies can also be bound to solid phase carriers, including but not limited to polystyrene plates or beads, etc.

[0061] The amino acid sequences of the CDRs of the heavy chain variable region of E2-Ab1 (E2 antibody (Ab1)) are as follows:

[0062] CDR1 (SEQ ID No. 1): SGFSLSRY

[0063] CDR2 (SEQ ID No. 2): GIYGSNK

[0064] CDR3 (SEQ ID No. 3): ARGASAGLDL

[0065] The amino acid sequence of the heavy chain variable region of E2-Ab1 is shown in SEQ ID No. 4:

[0066] QSVEESGGRLVTPGTPLILTCTVSGFSLSRYAMTWVRQVPGKGLEWIGGIYGSNKYSATWAKGRITISKTSTTVDLRITSPTTEDTATYFCARGASAGLDLWGPGTLVTVSS

[0067] The nucleotide sequence of the heavy chain variable region of E2-Ab1 is shown in SEQ ID No. 5:

[0068] CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTAATACTCACCTGCACAGTCTCTGGATTCTCCCTGAGTCGGTATGCAATGACCTGGGTCCGCCAGGTTCCAGGGAAGGGGCTGGAATGGATCGGAGGCATCTATGGCAGTAATAAATACTCCGCGACCTGGGCGAAAGGCCGAATCACCATCTCCAAAACCTCGACCACGGTGGATCTAAGAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGGAGCGAGCGCCGGCCTGGACCTCTGGGGTCCAGGGACCCTCGTCACCGTCTCTTCA

[0069] The amino acid sequences of the CDRs of the light chain variable region of E2-Ab1 are as follows:

[0070] CDR1 (SEQ ID No. 6): QSVDNSNL

[0071] CDR2 (SEQ ID No. 7): YAA

[0072] CDR3 (SEQ ID No. 8): QGYYSGAITA

[0073] The amino acid sequence of the light chain variable region of E2-Ab1 is shown in SEQ ID No. 9: AVVLTQTPSPVSAAVGGTVTISCQSSQSVDNSNLLAWYQQKPGQPPKLLIYYAATLSSGVPSRFKGSGSGTQFTLTISELQCDDAATYYCQGYYSGAITAFGGGTKVEIK

[0074] The nucleotide sequence of the light chain variable region of E2-Ab1 is shown as SEQ ID No. 10:

[0075] GCCGTCGTGCTGACCCAGACACCATCCCCTGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGTCCAGTCAGAGTGTTGATAATAGCAACCTCTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATTATGCAGCCACTCTGTCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGAACTACAGTGTGACGATGCTGCCACTTATTATTGTCAAGGCTATTATAGTGGTGCTATTACTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0076] The amino acid sequence of the CDRs of the heavy chain variable region of E2-Ab1 / E2 complex antibody (Ab2) is as follows:

[0077] CDR1 (SEQ ID No. 11): GYTFTSYW

[0078] CDR2 (SEQ ID No. 12): INPSTGYT

[0079] CDR3 (SEQ ID No. 13): ARWDYGSTSYFDY

[0080] The amino acid sequence of the heavy chain variable region of E2-Ab1 / E2 complex antibody (Ab2) is shown as SEQ ID No. 14:

[0081] QVQLQQSGAELAKPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGYINPSTGYTEYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARWDYGSTSYFDYWGQGTTLTVSS

[0082] The nucleotide sequence of the heavy chain variable region of E2-Ab1 / E2 complex antibody (Ab2) is shown as SEQ ID No. 15:

[0083] CAGGTCCAGCTACAGCAGTCTGGGGCTGAACTGGCAAAACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACTAGCTACTGGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGCACTGGTTATACTGAGTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATGGGACTACGGTAGTACCTCTTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0084] The amino acid sequences of the CDRs of the heavy chain variable region of the E2-Ab1 / E2 complex antibody (Ab2) are as follows:

[0085] CDR1 (SEQ ID No. 15): KASENLYT

[0086] CDR2 (SEQ ID No. 16): QGISSN

[0087] CDR3 (SEQ ID No. 17): HGT

[0088] The amino acid sequence of the heavy chain variable region of the E2-Ab1 / E2 complex antibody (Ab2) is shown in SEQ ID No. 14:

[0089] DIKASENLYTQMKISCKASQGISSNIGWLQQKPGKSFKGLIYHGTNLEDGVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPLTFGAGTKLELK

[0090] The nucleotide sequence of the heavy chain variable region of the E2-Ab1 / E2 complex antibody (Ab2) is shown in SEQ ID No. 15:

[0091] GACATCCTGATGACCCAATCTCCATCCTCCATGTCTGTATCTCTGGGAGACACAGTCAGCATCACTTGCCATGCAAGTCAGGGCATTAGCAGTAATATAGGGTGGTTGCAGCAGAAACCAGGGAAATCATTTAAGGGCCTGATCTATCATGGAACCAACTTGGAAGATGGAGTTCCATCAAGGTTCAGTGGCAGTGGATCTGGAGCAGATTATTCTCTCACCATCAGCAGCCTGGAATCTGAAGATTTTGCAGACTATTACTGTGTACAGTATGCTCAGTTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0092] Unless otherwise specified, the reagents or kits for detecting E2 by antibody sandwich method provided by the present application and the raw materials and reagents used in the application thereof can be purchased from the market.

[0093] The present application is further described below in conjunction with examples:

[0094] Example 1 Preparation process of E2 antibody (Ab1)

[0095] Step 1, immunization of New Zealand white rabbits

[0096] E2-6-cmo-BSA (East Coast Bio) was purchased as the immunogen for immunizing New Zealand white rabbits for 3-5 times, with an immunization cycle of 30 days. The rabbits were injected subcutaneously at multiple points on the back. 1 mg of the immunogen was emulsified with an equal volume of Freund's complete adjuvant to immunize the animals for the first time, and then 0.5 mg of the immunogen was emulsified with an equal volume of Freund's incomplete adjuvant to immunize the animals for 2-4 times, with an interval of 30 days between each immunization. Blood was collected from the marginal vein of the ear 10 days after the third and fifth immunizations, and was allowed to stand at 37℃ for 1 h, and then centrifuged at 6000 r / min for 10 min. The supernatant (antiserum) was collected and subjected to ELISA to detect the immunization effect.

[0097] Step 2, detection of antiserum titer

[0098] Prepare the test plate, anti-rabbit antibody (A0545-1ML, Merk) is added to 0.05 mol / L CB (pH9.6) coating buffer at a coating concentration of 4 μg / mL, and is coated overnight at 4°C. Take the collected supernatant as the test serum, dilute it by 1 / 500, and 50 μL / well, and set the standard factory antibody as the positive control, incubate in a 37°C incubator for 30 min; wash with PBST for 5 times, pat dry, add 1 / 2W diluted E2 enzyme binding agent (horseradish peroxidase labeled antigen), 50 μL / well, incubate in a 37°C incubator for 30 min; wash with PBST for 5 times, pat dry, add luminescent substrate A and B, each 50 μL / well, avoid light reaction for 5 min, and measure the signal value. The titer detection result after the third immunization is shown in Table 1, and the titer reaches 10 5 After the last boost, the animals are killed three days later, the spleen cells are extracted, the total RNA of the spleen tissue is extracted by the Trizol method, and the cDNA is synthesized by reverse transcription.

[0099] Table 1 Serum titer detection result after the third immunization

[0100]

[0101] Step 3, scFv gene splicing and phage screening construction

[0102] (1) scFv gene splicing:

[0103] The light chain variable region and the heavy chain variable region of the antibody are amplified by PCR, and the products after the PCR reaction are recovered by 1% agarose gel. The amplified light chain variable region and the heavy chain variable region are spliced into scFv by using overlap-PCR method, and the product is recovered by 1% agarose gel and stored at -20°C.

[0104] (2) Construction and screening of phage single-chain antibody library

[0105] The phagemid vector pcomb3XSS and the purified and recovered ScFv fragment are subjected to enzyme cutting with SfiI, a recombinant plasmid is constructed, the recombinant plasmid is electroporated into TG1 competent cells to construct a rabbit-derived immune single-chain antibody library, the antibody library recombinant rate bacterial liquid is subjected to PCR identification and a primary phage single-chain antibody library is prepared; the primary phage single-chain antibody library is subjected to enrichment screening for 3 rounds, and a specific phage single-chain antibody library with high affinity and strong specificity is obtained;

[0106] The monoclonal phage supernatant with good gradient is picked, positive clones are identified by Phage-ELISA method to obtain positive sequences, and an E2 antibody (Ab1) is obtained.

[0107] The amino acid sequence of the heavy chain variable region of E2-Ab1 is shown in SEQ ID No. 4, wherein the bolded amino acids are the CDR region amino acid sequences thereof:

[0108] QSVEESGGRLVTPGTPLILTCTVSGFSLSRYAMTWVRQVPGKGLEWIGGIYGSNKYSATWAKGRITISKTSTTVDLRITSPTTEDTATYFCARGASAGLDLWGPGTLVTVSS.

[0109] The amino acid sequence of the light chain variable region of E2-Ab1 is shown in SEQ ID No. 9, wherein the bolded amino acids are the CDR region amino acid sequences thereof:

[0110] AVVLTQTPSPVSAAVGGTVTISCQSSQSVDNSNLLAWYQQKPGQPPKLLIYYAATLSSGVPSRFKGSGSGTQFTLTISELQCDDAATYYCQGYYSGAITAFGGGTKVEIK

[0111] Example 2 Preparation process of anti-E2-Ab1 / E2 complex antibody (Ab2)

[0112] 1. Immunize mice

[0113] The E2 natural product (Sigma) was stored in DMSO, the E2 antibody (Ab1) obtained in Example 1 was dialyzed into 0.01 M PBS, and was incubated in an amount of 10:1 molar ratio of E2 natural product to E2 antibody (Ab1), and was incubated at 37°C for 2 h as an immunogen. BalB / C mice were immunized (3 times), and the specific immunization contents were as follows: the second immunization was performed 21 days after the first immunization, and the third immunization was performed 21 days after the second immunization. Among them, the first immunization was performed by subcutaneous injection + intraperitoneal injection, 100 μg of the immunogen was emulsified with an equal volume of Freund's complete adjuvant, and then was immunized, and 50 μg of the immunogen was emulsified with an equal volume of Freund's incomplete adjuvant for the last two times, and then was immunized (intraperitoneal injection was performed). The blood was collected from the tail vein on the 10th day after the third immunization, and was placed at 37°C for 1 h, and then was centrifuged at 6000 r / min for 10 min, and the supernatant (antiserum) was collected and was subjected to ELISA detection of the immunization effect.

[0114] 2. Detection of antiserum titer

[0115] Prepare the detection plate, Ab1 antibody prepared in Example 1 is added to 0.05 mol / L CB (pH 9.6) coating buffer at a coating concentration of 2 μg / mL, coated overnight at 4°C, as Ab1 antibody plate. In the process of titer detection, add E2 natural marker (Sigma) to the Ab1 antibody plate at a molar ratio of 1:10, react at 37°C for 30 min, wash with PBST 5 times, pat dry, as Ab1 / E2 complex plate. Take the collected supernatant as test serum, dilute by 1 / 1K, add 50 μL / well to the Ab1 / E2 complex plate and the Ab1 antibody plate, respectively, incubate at 37°C for 30 min; wash with PBST 5 times, pat dry, add HRP anti-mouse secondary antibody working solution, 50 μL / well, incubate at 37°C for 30 min; wash with PBST 5 times, pat dry, add luminescent substrate A and B, 50 μL / well each, react in the dark for 5 min, measure the signal value, titer detection is shown in Table 2. From the results, the titer of serum 2 reaches 10 5 Three days after the booster immunization, the animals were sacrificed, the spleen cells were extracted, and the total RNA of the spleen tissue was extracted by Trizol method, and the cDNA was synthesized by reverse transcription.

[0116] Table 2 Titer detection of immune complex animal serum

[0117]

[0118] 3. scFv gene splicing and phage screening construction

[0119] (1) scFv gene splicing:

[0120] Reference Example 1, step 3 (1).

[0121] (2) Construction and screening of phage single-chain antibody library

[0122] Reference Example 1, step 3 (2), the experimental steps for enrichment screening by solid phase panning (1~3 rounds of panning for the first time) are as follows:

[0123] Step 1, coat the positive screening tube (Ab1 / E2 complex): coat overnight at 20 μg / mL*1, the next day after blocking, add E2 small molecule antigen 0.5 μg / m1 (diluted with FBS), 37°C 70 rpm 0.5 h, wash. Antibody concentration 2 rounds 10 μg / m1+ antigen 0.5 μg / m1, 3 rounds of antibody 3 μg / m1+ antigen 0.05 μg / m1.

[0124] Step 2, coating the negative screen tube (Ab1): Ab1 antibody 5 μg / m1*1 overnight coating, the next day, casein blocking and direct use; the negative screen after the immune tube is dissociated, the negative screen tube is 2 rounds of antibody 10 μg / m1, 3 rounds of 15 μg / m1.

[0125] Elution step: phage is added to the negative screen tube (step 2), and then phage is added to the positive screen tube (step 1), and the subsequent steps are the same as the conventional experimental operation, to obtain the E2-Ab1 / E2 complex antibody (Ab2).

[0126] The amino acid sequence of the heavy chain variable region of the E2-Ab1 / E2 complex antibody (Ab2) is shown in SEQ ID No. 14, wherein the bold amino acids are the CDR region amino acid sequences thereof:

[0127] QVQLQQSGAELAKPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGYINPSTGYTEYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARWDYGSTSYFDYWGQGTTLTVSS

[0128] The amino acid sequence of the light chain variable region of the E2-Ab1 / E2 complex antibody (Ab2) is shown in SEQ ID No. 19, wherein the bold amino acids are the CDR region amino acid sequences thereof:

[0129] DILMTQSPSSMSVSLGDTVSITCHASQGISSNIGWLQQKPGKSFKGLIYHGTNLEDGVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPLTFGAGTKLELK

[0130] Example 3 Recombinant monoclonal antibody expression and antibody purification

[0131] 1. Construction of stable cell strain

[0132] The heavy chain constant region and variable region and the light chain constant region and variable region are connected by Overlap-PCR method respectively to obtain the heavy chain and light chain recombinant antibody gene sequence. The heavy chain antibody gene sequence and the light chain antibody gene sequence are double enzyme cut by XmaJI / BstZ17I and EcoRV / PacI respectively, and then linked on the expression vector pCHO 1.0. The constructed recombinant plasmid is transformed into competent DH5α cells, and the positive clones are selected by screening plate, sequenced and plasmid extracted. The extracted plasmid is linearized by RruI enzyme and then transfected into FD-CHOS cells.

[0133] After 48 h of transfection, the expression of the antibody was detected by ELISA, and then the positive cell pool was screened using a pressure culture medium containing 200 nM MTX and 20 μg / mL Puromycin. After the cell viability recovered from the first pressure, the screening concentration was continuously increased (1000 MTX and 50 μg / mL Puromycin) to perform the second pressure. After the cell viability recovered from the second pressure, the construction of the stable cell pool was completed. Then, the stable cell pool was used for monoclonal screening, and a monoclonal antibody cell strain capable of stable high expression was screened.

[0134] 2. Recombinant antibody expression

[0135] The selected monoclonal cell strain with stable high expression was recovered, and was subcultured at a density of 5×10^5 cells / mL. When the cell viability recovered to not less than 95%, the evaluation was started. The feeding strategy was as follows: 1, 3, 5, 7, 9, 11 d... feeding, 4, 6, 8, 10, 12 d... adding sugar to 5 g / L, and then culturing until the cell viability decreased to less than 70%, and then the cell supernatant was harvested, so that the supernatant containing Ab1 and Ab2 was obtained.

[0136] 3. Antibody SPA purification and SDS-PAGE identification

[0137] The chromatography workstation was used to purify 200 mL of the cell supernatant, and the SPA column with a capacity of 10 mL was set. The flow rate was set to 8 mL / min, the chromatography column was flushed with the equilibrium buffer 0.02 mol / L PBS (PH=7.2~7.4), the flow rate for sampling was set to 4 mL / min, and the flow-through was collected when the ultraviolet peak graph started to rise. Re-equilibration: the flow rate was set to 8 mL / min, and the chromatography column was flushed again with the equilibrium buffer 0.02 mol / L PBS (PH=7.2~7.4) until the ultraviolet peak graph decreased to the baseline, and the flow-through collection was stopped. Dissociation: the target protein was dissociated using the dissociation buffer 0.2 mol / L Gly+0.15 mol / L NaCl (PH=2.7), the target protein was collected when the ultraviolet peak graph started to rise, the ultraviolet peak graph decreased to the baseline, the collection of the target protein was stopped, and 1 mol / L Tris (PH=8.5) was added to the collection tube to neutralize to pH 7. The collected proteins were combined and subjected to SDS-PAGE detection. Figure 1 )。

[0138] Example 4 Application of the E2 recombinant rabbit monoclonal antibody kit prepared in the application

[0139] In Example 3 of this invention, the Ab1 antibody prepared was used as the magnetic bead coating antibody, and Ab2 was used as the enzyme-labeled antibody in the kit. Together with standards, sample diluent, substrate A, substrate B, stop solution, and 20× concentrated wash buffer, the antigen in the sample was measured. Specific performance evaluation is as follows:

[0140] (1) Correlation analysis

[0141] Ab1 and Ab2 were used in the reagent kit components, and 50 clinical samples within the selected detection range were randomly tested using the sandwich method and competitive method of this invention. The correlation between the E2 sandwich method (Ab2) and liquid chromatography-mass spectrometry was y = 0.9433x + 4.5952, R... 2 =0.9926 ( Figure 1 The correlation between the competitive method and liquid chromatography-mass spectrometry is y = 1.087x + 33.262, R0. 2 =0.8813 ( Figure 2 Outlier data are shown in Table 3. The correlation data shows that the Ab2 sandwich method has better correlation.

[0142] Table 3 Outlier Results

[0143]

[0144] (2) Precision assessment

[0145] The precision evaluation conducted in this study was intra-assay precision. Samples at high, medium, and low concentration levels were tested using the same batch of reagents. Each sample in each batch was measured 10 times, and the mean, SD, and coefficient of variation (CV%) were calculated. The intra-assay CV for all three levels of samples was less than 3%, as shown in the table below, indicating good reproducibility of the kit.

[0146] Table 4 Precision Measurement

[0147]

[0148] (3) Accelerated stability test

[0149] The materials from the competitive method and sandwich method were subjected to accelerated thermal stability tests. The working solutions of magnetic beads and enzyme conjugate antibodies were placed at 37°C for 7 days, and then clinical samples were tested using a microarray. Component analysis was performed simultaneously at 2–8°C. The results are shown in the table below:

[0150] Table 5 Accelerated Stability Tests

[0151]

[0152] The results show that both the competing material and the sandwich material meet the stability requirements, with the sandwich material exhibiting better stability.

[0153] (4) Matrix effect

[0154] Select at least 10 evenly distributed clinical samples on the same day in the linear range, then prepare samples according to the required matrix such as: calibration matrix, mixed serum, quality control matrix, etc., and can contain commercial quality control samples. The concentration range of the prepared samples should be evenly distributed in the linear range of the kit. According to the evaluation results, the calibration matrix, mixed serum, and quality control matrix to be evaluated are within their 95% confidence intervals, and the application of the antibody does not have a matrix effect.

[0155] (5) Cross-substance evaluation

[0156] The cross-substance is configured into an evaluation concentration with hormone-free serum, and the cross rate is detected, and the results are shown in Table 6. The cross rate with the cross-substance is less than 0.5% when the evaluation concentration is less than 0.5%, which meets the requirements of the kit.

[0157] Table 6 Cross rate results of E2

[0158]

[0159] From the above results, it can be seen that the Ab1 antibody prepared in the application is used as a magnetic bead coated antibody, and Ab2 is used as an enzyme-labeled antibody in the kit, and other components are used together to determine the antigen in the sample. It has reached a high consistency level with the detection results of the current market mainstream manufacturers, and has the advantages of excellent stability, etc. According to the evaluation of each detection performance, it meets the requirements of the kit, and has an important role in the later application and clinical diagnosis.

[0160] The above only describes the preferred embodiments of the present application, and it should be pointed out that for ordinary skilled persons in the technical field, some improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A coated antibody, characterized in that, The coated antibody includes a heavy chain variable region and a light chain variable region, comprising: (I) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region have the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively; and The light chain variable regions LCDR1, LCDR2, and LCDR3 have amino acid sequences as shown in SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, respectively; and / or (II) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids as described in (I), and which has the same function as the amino acid sequence described in (I); or (III) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (I) or (II).

2. The coated antibody as described in claim 1, characterized in that, include: (I) The heavy chain variable region has an amino acid sequence as shown in SEQ ID No. 4; and The light chain variable region has an amino acid sequence as shown in SEQ ID No. 9; and / or (II) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids as described in (I), and which has the same function as the amino acid sequence described in (I); or (III) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (I) or (II).

3. A labeled antibody, characterized in that, The labeled antibody includes a heavy chain variable region and a light chain variable region, including: (I) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region have the amino acid sequences shown in SEQ ID No. 11, SEQ ID No. 12, and SEQ ID No. 13, respectively; and The light chain variable regions LCDR1, LCDR2, and LCDR3 have amino acid sequences as shown in SEQ ID No. 16, SEQ ID No. 17, and SEQ ID No. 18, respectively; and / or (II) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids as described in (I), and which has the same function as the amino acid sequence described in (I); or (III) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (I) or (II).

4. The labeled antibody as described in claim 3, characterized in that, include: (I) The heavy chain variable region has an amino acid sequence as shown in SEQ ID No. 14; and The light chain variable region has an amino acid sequence as shown in SEQ ID No. 19; and / or (II) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids as described in (I), and which has the same function as the amino acid sequence described in (I); or (III) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (I) or (II).

5. A paired monoclonal antibody for the sandwich method of E2 detection, characterized in that, It comprises the coated antibody as described in claim 1 or 2 and the labeled antibody as described in claim 3 or 4.

6. A nucleic acid molecule, characterized in that, Includes DNA fragments encoding any of the following: (I) The coated antibody as described in claim 1 or 2; and / or (II) The labeled antibody as described in claim 3 or 4; and / or (III) The paired monoclonal antibody as described in claim 5.

7. An expression vector, characterized in that, Includes the nucleic acid molecules as described in claim 6.

8. A host cell, characterized in that, Including the expression vector as described in claim 7.

9. Any of the following applications in the preparation of E2 detection products: (I) The coated antibody as described in claim 1 or 2; and / or (II) The labeled antibody as described in claim 3 or 4; and / or (III) The paired monoclonal antibody as described in claim 5.

10. A reagent or kit for detecting E2 using an antibody sandwich method, characterized in that, Includes any of the following: (I) The coated antibody as described in claim 1 or 2; and / or (II) The labeled antibody as described in claim 3 or 4; and / or (III) The paired monoclonal antibody as described in claim 5.

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