An antibody that specifically binds to an e2-e2 antibody complex and uses thereof

Abstract

The application provides an antibody specifically binding to an E2-E2 antibody complex and application thereof. The antibody provided by the application can specifically bind to the E2-E2 antibody, and has good binding activity and affinity. The sandwich method is used to detect E2 by using the antibody in the application, so that the sensitivity and anti-interference ability of E2 detection are greatly improved, and the detection rate of low-value samples is improved.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and medical technology, and in particular to an antibody against an anti-estradiol (E2)-estradiol antibody complex and its application. Background Technology

[0002] Estradiol (E2) is a steroid hormone with a basic steroidal structure, namely cyclopentanoperhydrophenanthrene, composed of three cyclohexanes and one cyclopentane fused together. Estradiol is the most abundant and biologically active estrogen, playing a crucial role in the reproductive system, immune responses, and gene expression in mammals. In the blood, E2 is an important diagnostic marker in various clinical conditions in both men and women.

[0003] Clinically, E2 levels are used to assess menstrual disorders, precocious puberty or delayed puberty, and ovarian function in women, as well as hirsutism and polycystic ovary syndrome. In pregnancy-related disease monitoring, E2 levels are monitored throughout pregnancy, in conjunction with progesterone and inhibin B levels, to predict the risk of hydatidiform mole in pregnant women. In men, E2 testing can be used to assess gynecomastia, delayed puberty, or infertility.

[0004] Currently, the mainstream clinical method for detecting E2 is immunoassay. Since E2 is a small molecule hormone compound with a single antigenic determinant, it cannot support sandwich detection with two different antibodies. Therefore, clinical immunoassays generally use a competitive method.

[0005] However, competitive methods for detecting small molecule antigens suffer from poor sensitivity and interference resistance, especially in detecting low-value samples. Therefore, it is urgent to address the low sensitivity and poor interference resistance of existing competitive methods for detecting E2 and to improve the detection rate of low-value samples.

[0006] The currently feasible complex antibody sandwich method works by having one anti-E2 antibody (primary antibody) form an immune complex with the target antigen, and another anti-complex antibody (secondary antibody) binds to this immune complex to form an immune sandwich complex. The recognition site is a new epitope formed after the first antibody binds to the small molecule hapten, and the complex antibody must not bind to either the free first antibody or the small molecule hapten. The use of the complex antibody sandwich method can greatly improve the sensitivity and accuracy of small molecule antigen detection. However, screening complex antibodies is challenging, and high-affinity complex antibodies suitable for the sandwich method have not yet been reported. Summary of the Invention

[0007] The following is an overview of the subject matter described in detail herein. This overview is not intended to limit the scope of the claims.

[0008] This application provides an antibody against an anti-estradiol (E2)-estradiol antibody complex and its application. The anti-estradiol (E2)-estradiol antibody complex provided in this application can specifically bind to the E2 antigen, exhibiting good binding activity and affinity. Using the antibody in this application for sandwich method detection of E2 significantly improves the detection sensitivity and anti-interference ability of the sandwich method, and also increases the detection rate of low-value samples.

[0009] In one aspect, this application provides an antibody or a functional fragment thereof that specifically binds to an E2-E2 antibody complex, said antibody or functional fragment thereof comprising the following complementarity-determining regions:

[0010] The complementarity-determining region CDR1-VH has the amino acid sequence SYGVH (SEQ ID NO:1);

[0011] The complementarity-determining region CDR2-VH has the amino acid sequence VIWX1GGSTNYNSALMS (SEQ ID NO:19), where X1 is A or G;

[0012] The complementarity-determining region CDR3-VH has the amino acid sequence X2EGX3AX4AMDY (SEQ ID NO:20), where X2 is D or G, X3 is Y, S or T, and X4 is G or Y.

[0013] The complementarity-determining region CDR1-VL has the amino acid sequence RASESVDNFGISFMN (SEQ ID NO:8);

[0014] The complementarity-determining region CDR2-VL has the amino acid sequence X5ASNQGS (SEQ ID NO:21), where X5 is A or T;

[0015] The complementarity-determining region CDR3-VL has the amino acid sequence QQSKEVPX6T (SEQ ID NO:22), where X6 is Y, W, or R.

[0016] In embodiments of this application, the mutation sites of each complementarity-determining region of the antibody or its functional fragment are selected from any of the following combinations of mutations:

[0017]

[0018]

[0019] In embodiments of this application, the mutation sites of each complementarity-determining region of the antibody or its functional fragment are selected from any of the following wild-type or mutation combinations:

[0020]

[0021]

[0022]

[0023]

[0024] In embodiments of this application, the antibody or its functional fragment is combined with the E2-E2 antibody complex at K D Affinity binding on the order of ≤E-8.

[0025] In embodiments of this application, the antibody includes light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L as shown in sequence as SEQ ID NO:11-14; and / or heavy chain backbone regions FR1-H, FR2-H, FR3-H and FR4-H as shown in sequence as SEQ ID NO:4-7.

[0026] In embodiments of this application, the antibody further includes a constant region.

[0027] In embodiments of this application, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

[0028] In the embodiments of this application, the species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, or humans.

[0029] In embodiments of this application, the constant region is derived from mice.

[0030] In the embodiments of this application, the light chain constant region sequence is shown as SEQ ID NO:18, and the heavy chain constant region sequence is shown as SEQ ID NO:17.

[0031] In the embodiments of this application, the functional fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv and scFv.

[0032] The functional fragments of the aforementioned antibodies typically possess the same binding specificity as the antibodies from which they originate. Those skilled in the art will readily understand, based on the description herein, that the functional fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Given the complete antibody structure disclosed in this application, those skilled in the art can readily obtain the aforementioned functional fragments.

[0033] The functional fragments of the aforementioned antibodies can also be synthesized using recombinant genetic techniques known to those skilled in the art or using automated peptide synthesizers, such as those sold by Applied BioSystems.

[0034] In another aspect, this application provides a nucleic acid molecule that encodes the aforementioned antibody or a functional fragment thereof.

[0035] In another aspect, this application provides a carrier comprising the aforementioned nucleic acid molecules.

[0036] In another aspect, this application provides a host cell that contains the aforementioned nucleic acid molecules or the aforementioned vector.

[0037] In another aspect, this application provides the use of the antibody or functional fragment thereof of the anti-estradiol (E2)-estradiol antibody complex as described above, the nucleic acid molecule described above, the vector described above, or the host cell described above in the preparation of a product for detecting E2.

[0038] In another aspect, this application provides an E2 detection kit comprising the aforementioned antibody.

[0039] In another aspect, this application provides a method for detecting the amount of E2 in a test sample, comprising:

[0040] a) Under conditions sufficient to induce antibody-antigen binding, the first anti-E2 antibody is contacted with the E2 antigen in the test sample to form an immune complex; and

[0041] b) Bind the antibody or its functional fragment thereto to the immune complex from step a) to form an immune sandwich complex; and

[0042] c) The amount of the E2 antigen is determined by detecting the amount of the immune sandwich complex.

[0043] In the embodiments of this application, the method is selected from any one or more of the following: fluorescence immunoassay, chemiluminescence immunoassay, colloidal gold immunoassay, radioimmunoassay, or enzyme-linked immunosorbent assay (ELISA).

[0044] Preferably, the chemiluminescent immunoassay is a chemiluminescent immunoassay sandwich method;

[0045] Preferably, the method uses a semi-automated or fully automated immunoassay analyzer for analysis.

[0046] In the embodiments of this application, the sample is selected from at least one of whole blood, serum, or plasma;

[0047] Preferably, the whole blood, serum, or plasma is derived from peripheral blood.

[0048] The antibody specifically binding to the E2-E2 antibody complex provided in this application can specifically bind to the E2-E2 antibody complex, exhibiting good binding activity and affinity. Using the anti-E2-E2 antibody complex antibody of this application for sandwich method detection of E2 significantly improves the sensitivity and anti-interference ability of E2 detection, and also increases the detection rate of low-value samples.

[0049] Other features and advantages of this application will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the application. Other advantages of this application can be realized and obtained by means of the solutions described in the description and the accompanying drawings. Attached Figure Description

[0050] The accompanying drawings are used to provide an understanding of the technical solutions of this application and constitute a part of the specification. They are used together with the embodiments of this application to explain the technical solutions of this application and do not constitute a limitation on the technical solutions of this application.

[0051] Figure 1 The graph shows the correlation between the E2 sandwich method and mass spectrometry.

[0052] Figure 2 The graph shows the correlation between the competitive method and mass spectrometry in the low-value region.

[0053] Figure 3 This is a graph showing the correlation between the sandwich method and mass spectrometry in the low-value region. Detailed Implementation

[0054] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be described in detail below. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined arbitrarily. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0055] The present invention will be further described below with reference to specific embodiments.

[0056] Example 1. Screening of hybridoma cell lines with anti-E2-E2 Ab complex antibody

[0057] The antibody screening in the following examples uses hybridoma fusion technology. The principle of hybridoma fusion technology is to fuse B lymphocytes from the spleen of immunized mice with myeloma cells, thereby giving the B lymphocytes the ability to proliferate indefinitely. By detecting secreted antibodies in the cell supernatant using ELISA, cell wells expressing specific antibodies are screened, and further subcloning yields monoclonal antibodies. Through a complete in vivo immunization process, the animal body can produce high-affinity antibodies. Combined with electrofusion, a high fusion rate and a large number of fusions are ensured, enabling the screening of high-performance target antibodies. The specific steps of this example are as follows:

[0058] 1. E2 antibody preparation process (Ab1)

[0059] A) Immunizing mice

[0060] Purchase E2-6-cmo-BSA (EastCoast Bio) as the immunogen, dissolve it, and emulsify it evenly with an equal volume of Freund's complete adjuvant (Sigma). Take 6-8 week old SPF-grade Balb / c mice and inject 200 μg / mouse subcutaneously at multiple sites. After 3 weeks, emulsify the antigen with Freund's incomplete adjuvant and inject 150 μg / mouse subcutaneously at multiple sites. Administer two booster immunizations. Three days before fusion, administer a shock immunization via intraperitoneal injection.

[0061] B) Cell fusion and subcloning screening

[0062] Spleens were harvested from immunized mice, and dispersed single spleen cells were obtained by grinding and separation. Spleen cells and myeloma cells were mixed using PEG. After the culture medium was terminated, the cells were centrifuged, reconstituted, and seeded into 96-well plates. The medium was changed after one week, and the supernatant was collected for indirect enzyme immunoassay. The cells were coated with goat anti-mouse IgG secondary antibody (Jackson) and E2-17β-6-CMO-HRP (Pantex) was used as the enzyme-labeled antigen. The cell supernatant was evaluated using a competitive method. Wells with good competitive performance were selected for further investigation of cross-linking to E3 and E1. Positive wells with low cross-linking were selected for subcloning using the limiting dilution method. After one week of culture, enzyme immunoassay was repeated 3-4 times until all wells were positive and the cells in the wells were single colonies. The specific hybridoma cell line 3F4E6 was obtained by expansion culture.

[0063] C) Preparation of ascites fluid from positive cell lines

[0064] Single-colony cells were expanded and cultured, then inoculated into IFA-prestimulated mice to prepare ascites. The ascites was collected and purified by SPA affinity to obtain antibodies that specifically bind to E2.

[0065] 2. Preparation process of anti-E2-Ab1 / E2 complex antibody (Ab2)

[0066] A) Immunizing mice

[0067] Both E2 natural standard (Sigma) and E2 mouse antibody (3F4E6) were dialyzed into 0.01M PBS and incubated at a molar ratio ≥10:1 at 37°C for 2 hours. After incubation, the antigen was dialyzed into 0.01M PBS to remove excess antigen, which was then used as an immunogen.

[0068] Emulsify the antigen with an equal volume of Freund's complete adjuvant (Sigma) until homogeneous. Take 6-8 week old SPF-grade Balb / c mice and inject 200 μg / mouse subcutaneously at multiple sites. After 3 weeks, emulsify the antigen with Freund's incomplete adjuvant and inject 150 μg / mouse subcutaneously at multiple sites. Boost the immunization twice. Administer a shock immunization via intraperitoneal injection 3 days before fusion.

[0069] B) Cell fusion and subcloning screening

[0070] Spleens were taken from immunized mice, and dispersed individual spleen cells were obtained by grinding and separation. Spleen cells and myeloma cells were mixed using PEG. After the culture medium was terminated, the cells were centrifuged and reconstituted, and then plated into 96-well plates. The medium was changed after one week, and the supernatant was used for indirect enzyme immunoassay.

[0071] The detection method is as follows: Goat anti-mouse IgG Fcγ secondary antibody (Jackson, 1 μg / ml) was used as the coating antigen. After blocking, cell supernatant was added, followed by the addition of biotin-labeled E2 mouse anti-Fab (Bio-Fab, referring to 3F4E6 digested with enzymes and then biotinylated) or a Bio-Fab+E2 complex. The chromogenic agent was HRP-SA. Finally, positive wells that showed no or low reaction with Bio-Fab and strong reaction with Bio-Fab+E2 were selected. Subcloning was then performed using limiting dilution. After one week of culture, enzyme immunoassay was performed again. Positive wells that showed no or low reaction with Bio-Fab and strong reaction with Bio-Fab+E2 were selected and subjected to limiting dilution. This process was repeated 3-4 times until all wells were positive and the cells in the wells were monocolony. Then, the cell wells that showed no reaction with Bio-Fab and strongest reaction with Bio-Fab+E2 were selected for expansion culture to obtain the specific hybridoma cell line 4A8D6.

[0072] C) Preparation of ascites fluid from positive cell lines

[0073] Single-colony cells were expanded and cultured, then injected into mice pre-inoculated with IFA to prepare ascites fluid. The ascites fluid was collected to obtain complex antibodies, which were then purified by affinity SPA.

[0074] Example 2. Construction of expression plasmids

[0075] i) Gene retrieval

[0076] The 4A8D6 hybridoma cell line was expanded, mRNA was extracted, and cDNA product was obtained by reverse transcription. The product was inserted into the pMD-18T vector after being subjected to an A-addition reaction with rTaq DNA polymerase. The cells were then transformed into DH5α competent cells, and 10 plaques of each heavy chain and light chain gene clones were sent to a gene sequencing company for sequencing.

[0077] ii) Sequence analysis of antibody genes

[0078] The gene sequences obtained from the sequencing were analyzed in the IMGT antibody database, and the VNTI11.5 software was used to confirm that the genes amplified by both heavy and light chain primer pairs were correct.

[0079] iii) Construction of recombinant antibody expression plasmid

[0080] pFastBac TM A recombinant antibody expression vector, abbreviated as pFD vector, was constructed using dual as the vector. Based on the sequencing results of the antibody variable region gene in pMD-18T, light and heavy chain specific primer pairs were designed (see the first-stage primers in Appendix 1). Approximately 0.7 kb of light chain and approximately 1.4 kb of heavy chain gene fragments were obtained by PCR amplification. Corresponding primers were designed (see the second-stage primers in Appendix 1), and overlap PCR was used to introduce the signal peptide gp64 and restriction enzyme sites into the gene fragments. The restriction enzyme sites corresponding to the light and heavy chains are as follows: XhoI-gp64-L-KpnI, BamHI-gp64-H-HindIII.

[0081] The pFD vector and the BamHI-gp64-H-HindIII gene fragment were digested with BamHI / HindIII, and the target fragment was recovered by electrophoresis. The gene fragment and vector fragment were ligated and transformed into DH5α competent cells. After positive colony PCR verification, the cells were expanded and cultured. The vector plasmid with the heavy chain ligated was extracted and abbreviated as pFD-H. Then, this plasmid and XhoI-gp64-L-KpnI were digested with XhoI / KpnI, and the target fragment was recovered by electrophoresis. The cells were ligated and transformed. After successful verification, the vector with the light and heavy chains ligated was extracted and abbreviated as pFD-HL.

[0082] The recombinant plasmid was transformed into E. coli DH10Bac competent cells (containing AcNPV bacmid and helper plasmid) and plated on LB agar plates containing kanamycin (50 μg / mL), tetracycline (10 μg / mL), gentamicin (7 μg / mL), IPTG (40 μg / mL), and X-gal (100 μg / mL). White colonies were selected by blue-white screening, and the recombinant bacmid was extracted using the Omega BAC / PAC extraction kit.

[0083] iv) Expression purification

[0084] Following the Gibco liposome transfection instructions, recombinant bacmid was extracted and transfected into Sf9 cells at a passage density of 1*10^6 / ml. Cells were cultured at 28°C for 6 days. The supernatant collected after filtration through a 0.22μm filter under dark conditions was the first (P1) generation virus solution. The P1 virus was then used to re-infect Sf9 cells to obtain high-titer recombinant virus solutions. This process was repeated to obtain P2 / P3 virus solutions. Finally, the P3 generation virus solution was transfected into HF cells and cultured at 28°C for 3 days, after which the supernatant was collected.

[0085] Following the instructions of the GE AKTA Pure protein separation and purification system, the above expression supernatant was purified using a SPA column to obtain purified E2 complex antibody.

[0086]

[0087] Example 3. Recombinant antibody expression

[0088] Following the Gibco liposome transfection instructions, the recombinant bacmid extracted in Example 2 was transfected into Sf9 cells at a passage density of 1*10^6 / ml. Cells were cultured at 28°C for 6 days. The supernatant collected after filtration through a 0.22 μm filter under dark conditions was the first (P1) generation virus solution. The P1 virus was then used to re-infect Sf9 cells to obtain high-titer recombinant virus solutions. This process was repeated to obtain P2 / P3 virus solutions. Finally, the P3 generation virus solution was transfected into HF cells and cultured at 28°C for 3 days, after which the supernatant was collected.

[0089] Following the instructions for the GE AKTA Pure protein separation and purification system, the above expression supernatant was purified using a SPA column to obtain the purified recombinant antibody, the sequence of which is as follows:

[0090] Heavy chain CDR region amino acid sequence

[0091] CDR1-VH:

[0092] SEQ ID NO:1:SYGVH

[0093] CDR2-VH:

[0094] SEQ ID NO:2:VIWGGGGSTNYNSALMS

[0095] CDR3-VH:

[0096] SEQ ID NO:3: GEGSAYAMDY

[0097] Heavy chain framework region amino acid sequence

[0098] FR1-H:

[0099] SEQ ID NO:4:QVQLKESGPGLVAPSQSLSITCTVSGFSLI FR2-H:

[0100] SEQ ID NO:5:WVRQPPGKGLEWLG

[0101] FR3-H:

[0102] SEQ ID NO:6:RLSISKDNSKSQVFLKMNSLQTDDTAMYYCVR FR4-H:

[0103] SEQ ID NO:7:WGQGTSVTVSS

[0104] amino acid sequence of light chain CDR region

[0105] CDR1-VL:

[0106] SEQ ID NO:8: RASESVDNFGISFMN

[0107] CDR2-VL:

[0108] SEQ ID NO:9: TASNQGS

[0109] CDR3-VL:

[0110] SEQ ID NO:10:QQSKEVPYT

[0111] amino acid sequence of the light chain framework region

[0112] FR1-L:

[0113] SEQ ID NO:11:DIVLTQSPASLAVSLGQRATISC FR2-L:

[0114] SEQ ID NO:12:WFQQKPGQPPKLLIY

[0115] FR3-L:

[0116] SEQ ID NO:13:GVPARFSGSGSGTDFSLNIHPMEEDDTAMYFC FR4-L:

[0117] SEQ ID NO:14:FGGGTKLEIK

[0118] Heavy chain variable region amino acid sequence:

[0119] SEQ ID NO:15:

[0120] QVQLKESGPGLVAPSQSLSITCTVSGFSLISYGVHWVRQPPGKGLEWLGVIWGGGSTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCVRGEGSAYAMDYWGQGTSVTVSS

[0121] Light chain variable region amino acid sequence:

[0122] SEQ ID NO:16:

[0123] DIVLTQSPASLAVSLGQRATISCRASESVDNFGISFMNWFQQKPGQPPKLLIYTASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPYTFGGGTKLEIK

[0124] Heavy chain constant region amino acid sequence:

[0125] SEQ ID NO:17:

[0126] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVH TAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0127] amino acid sequence of the light chain constant region:

[0128] SEQ ID NO:18:

[0129] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0130] Example 4. Antibody performance testing

[0131] 1. Activity detection of anti-E2-E2 Ab complex antibodies and their mutants

[0132] Sequence analysis revealed the following amino acid sequence of the complementarity-determining region (CD) of the variable region of the aforementioned monoclonal antibody:

[0133] CDR1-VH: SYGVH (SEQ ID NO: 1);

[0134] CDR2-VH: VIW-(X1)-GGTNYNSALMS (SEQ ID NO:19);

[0135] CDR3-VH: (X2)-EG-(X3)-A-(X4)-AMDY (SEQ ID NO: 20);

[0136] CDR1-VL: RASESVDNFGISFMN (SEQ ID NO:8);

[0137] CDR2-VL: (X5)-ASNQGS(SEQ ID NO:21);

[0138] CDR3-VL: QQSKEVP-(X6)-T (SEQ ID NO:22).

[0139] Based on the aforementioned monoclonal antibodies, mutations were performed on sites in the complementarity-determining region that are related to antibody activity and affinity. Specifically, X1, X2, X3, X4, X5, and X6 are all mutation sites, as shown in Table 1 below:

[0140] Table 1: Mutation schemes for mutation sites related to antibody affinity

[0141]

[0142]

[0143]

[0144]

[0145]

[0146] Verification revealed that the above mutants had comparable or fluctuating affinity to wild-type (WT), but overall, all were ≤E-8, meeting the basic requirements for E2 detection using the immunosandwich assay. Among them, 33 mutant groups showed a significantly higher affinity than WT (see Table 2). The equilibrium dissociation constant K... D (M) represents the degree of antigen-antibody dissociation in equilibrium, K D The smaller the value of (M), the stronger the affinity between the antigen and antibody.

[0147] Table 2: Results of Mutant Antibody Affinity Detection

[0148]

[0149]

[0150]

[0151] 2. Specificity of anti-E2-E2 Ab complex antibodies and their mutants

[0152] Table 3: Binding assays for primary antibodies and antigens

[0153]

[0154]

[0155] The results showed that the complex antibody did not bind to the free primary antibody or antigen in the reaction system.

[0156] Example 5. Mutant 1 applied to the detection of E2

[0157] 1. Kit components and detection principle:

[0158] A self-produced anti-E2 antibody was used to coat magnetic microspheres, and an anti-E2 complex antibody (detection antibody) was labeled with ABEI luminescent reagent. In the first step, the sample, buffer, and magnetic microspheres were mixed and incubated together. The analyte antigen in the sample bound to the antibody coated on the magnetic microspheres to form an immune complex. After incubation, unbound material was removed by magnetic separation washing. In the second step, the luminescent label was added and incubated again. The ABEI-labeled antibody reacted with the immune complex formed on the magnetic microspheres. After incubation, unbound material was removed by magnetic separation washing. Finally, substrate solution for the fully automated immunoassay system was added to initiate the chemiluminescent reaction, generating a light signal. The relative light intensity (RLU) measured by a photomultiplier tube was proportional to the E2 concentration in the sample.

[0159] All materials and supplies used in the above experiments were produced by New Industries Biotechnology Co., Ltd., and the instrument used was the New Industries MAGLUMI 4000P chemiluminescence immunoassay analyzer.

[0160] 2. Sensitivity Test

[0161] High-value E2 samples (mass spectrometry value 4000 pg / ml) were added to human serum to prepare samples with different concentration gradients. The same Maglumi 4000p spectrometer was used to evaluate the samples using both the competitive and sandwich methods. The light intensity (Rlu) data are shown below.

[0162] Table 4. Detection results of competitive method and sandwich method for gradient concentration samples

[0163]

[0164] The concentration gradient between adjacent samples was controlled to be 2. The light intensity gradient between adjacent samples in the competition method and the sandwich method was calculated, which correspond to N / N-1 and N+1 / N in Table 1, respectively. It can be seen that when the concentration gradient is ≤160 pg / ml, the gradient change between adjacent samples in the competition method is <2, while that in the sandwich method is >2. This shows that the sandwich method has higher sensitivity within the detected gradient range.

[0165] 3. Anti-interference verification

[0166] The analytes in the table below are added to a certain value of E2 sample for detection. The deviation between the detection results and the true values ​​is shown in the table below. A deviation within 10% is considered to have no effect.

[0167] Table 5. Detection results of interfering substances using the competitive method and the sandwich method.

[0168]

[0169] The results showed that when using the sandwich method and complex antibody of this scheme to detect E2, the test deviation was less than 1% in the presence of interfering substances. In particular, for estrone, which has a high structural similarity to E2, the detection deviation was much lower than that of the scheme using the competitive method at all three concentrations.

[0170] 4. Relevance to mass spectrometry methodology

[0171] 286 samples were randomly collected, including samples from different age groups and different gestational periods. Each sample was divided into two parts: one part was sent to a third-party testing center for mass spectrometry analysis, and the other part was used for luminescence analysis on the Maglumi 4000p equipment of New Industries.

[0172] like Figure 1 As shown, the correlation is good throughout the entire detection range, R 2 It can reach 0.99.

[0173] 5. Low-value sample detection

[0174] Thirty-five low-value samples (less than 40 pg / ml) were selected from the above cases. The correlation between the competitive method, sandwich method, and mass spectrometry was compared, showing that R... 2 The increase from 0.85 to 0.94 indicates that the sandwich method has improved detection performance in the low-value region. Figure 2 and Figure 3 As shown.

Claims

1. An antibody or its antigen-binding fragment that specifically binds to an E2-E2 antibody complex, characterized in that, The antibody or its antigen-binding fragment includes the following complementarity-determining regions: The complementarity-determining region CDR1-VH has the amino acid sequence SYGVH (SEQ ID NO: 1); The complementarity-determining region CDR2-VH has the amino acid sequence VIWX1GGSTNYNSALMS (SEQ ID NO: 19), where X1 is A; The complementarity-determining region CDR3-VH has the amino acid sequence X2EGX3AX4AMDY (SEQ ID NO: 20), where X2 is D, X3 is Y, and X4 is G; The complementarity-determining region CDR1-VL has the amino acid sequence RASESVDNFGISFMN (SEQ ID NO: 8); The complementarity-determining region CDR2-VL has the amino acid sequence X5ASNQGS (SEQ ID NO: 21), where X5 is A; The complementarity-determining region CDR3-VL has the amino acid sequence QQSKEVPX6T (SEQ ID NO: 22), where X6 is R.

2. The antibody or its antigen-binding fragment according to claim 1, wherein, The antibody or its antigen-binding fragment binds to the E2-E2 antibody complex at K D Affinity binding on the order of ≤E-8.

3. The antibody or its antigen-binding fragment according to claim 1, wherein, The antibody comprises light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L as shown in sequence as SEQ ID NO:11-14; and / or heavy chain backbone regions FR1-H, FR2-H, FR3-H and FR4-H as shown in sequence as SEQ ID NO:4-7.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein, The antibody also contains a constant region.

5. The antibody or its antigen-binding fragment according to claim 4, wherein, The constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

6. The antibody or its antigen-binding fragment according to claim 5, wherein, The species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, or humans.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein, The constant region was derived from mice.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein, The light chain constant region sequence is shown in SEQ ID NO:18, and the heavy chain constant region sequence is shown in SEQ ID NO:

17.

9. The antibody or antigen-binding fragment thereof according to claim 5, wherein, The antigen-binding fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, and scFv.

10. A nucleic acid molecule, said nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1-9.

11. A vector comprising the nucleic acid molecule of claim 10.

12. A host cell comprising the nucleic acid molecule of claim 10, or comprising the vector of claim 11.

13. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-9, the nucleic acid molecule according to claim 10, the vector according to claim 11, or the host cell according to claim 12 in the preparation of a product for detecting E2.

14. An E2 detection kit, the kit comprising the antibody according to any one of claims 1 to 9.

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