Antibody or antigen-binding fragment thereof for detecting thyroxine, preparation method and application thereof

Abstract

The present invention relates to antibodies or antigen-binding fragments thereof for detecting thyroxine, and related protein products, nucleic acid products, cell products, detection reagents, and preparation methods and applications thereof. The antibody for detecting thyroxine of the present invention is an antibody for an immune complex containing thyroxine, which can bind to a thyroxine antigen-antibody immune complex as an immunogen with high specificity and high affinity, without binding to a single thyroxine and an anti-thyroxine antibody in the immune complex, and therefore, it can be used in combination with the anti-thyroxine antibody in the immune complex for immunological detection of thyroxine, and the immunological detection based on the two antibodies has high detection accuracy and sensitivity, thereby breaking through the limitations of low sensitivity and poor clinical relevance of traditional competitive detection reagents, and is of great significance for the early diagnosis and treatment of thyroid diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies and immunological detection technology, and in particular to an antibody or an antigen-binding fragment thereof for detecting thyroxine, and a preparation method and application thereof. Background Art

[0002] Thyroid hormones are hormones secreted by the thyroid gland, including thyroxine (T4) and triiodothyronine (T3). Thyroxine (T4), also known as 3,5,3',5'-tetraiodothyronine, is the main hormone secreted by the thyroid gland, with a molecular weight of about 776Da. Under normal physiological conditions, about 99.95% of T4 in the blood is bound to thyroxine binding protein (TBG), and only 0.05% of free T4 (FT4) has biological activity. T4 is an indispensable component of the hypothalamus-pituitary-thyroid regulatory system, and can be used for the diagnosis of hyperthyroidism, the determination of primary and secondary hypothyroidism, and the monitoring of thyroid stimulating hormone (TSH) suppression therapy. T3 and T4 differ in structure by only one iodine, so the specificity of the antibody is required to be high. At present, most of the detections based on traditional competitive methods have cross-reaction problems.

[0003] At present, the methods for detecting T4 concentration in human serum include radioimmunoassay, chromatography, chemiluminescence, and immunochromatography. The reagents used in radioimmunoassay contain radioactive isotopes, which poses a safety problem. Chromatography is time-consuming and expensive, so it cannot be widely used. Therefore, immunochromatography and chemiluminescence have become the mainstream. At present, the immunochromatography and chemiluminescence methods for detecting T4 concentration in human serum still use the traditional competitive method, which has low detection sensitivity, narrow linear range, and unclear distinction between low-concentration samples and high-concentration samples, which can easily lead to misjudgment of the results.

[0004] In view of the above-mentioned problems existing in the prior art, there is an urgent need to develop monoclonal antibodies for high-specificity and high-sensitivity detection of T4 to meet the needs of immunological detection, thereby breaking through the limitations of low sensitivity and narrow linearity of traditional competitive methods. Summary of the invention

[0005] Technical issues to be solved

[0006] In view of the problems or needs existing in the prior art, the object of the present invention is to provide an antibody or an antigen-binding fragment thereof against an immune complex containing thyroxine (i.e., an immune complex formed between thyroxine and an anti-thyroxine primary antibody), which can bind to the immune complex containing thyroxine with high specificity and high affinity; the antibody is combined with the anti-thyroxine antibody contained in the immune complex and can be used for double antibody sandwich immunological detection of thyroxine, which has higher sensitivity and linearity, and this detection method has a smaller cross-reactivity with antigen T3 (about 0.1%) and has better specificity.

[0007] Technical Solution

[0008] In order to achieve the above-mentioned purpose, the present invention adopts a thyroxine antigen-antibody immune complex (i.e., an immune complex formed between thyroxine and an anti-thyroxine primary antibody) as an immunogen to immunize mice and obtains an antibody or its antigen-binding fragment that can bind to the thyroxine antigen-antibody immune complex with high specificity and high affinity through a large number of screenings, its related proteins, nucleic acids and cell products, its preparation method and application, and immunoassay reagents or kits based thereon, thereby providing a basis for high-specificity and high-sensitivity immunoassay of thyroxine.

[0009] Specifically, the present invention provides the following technical solutions:

[0010] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof against an immune complex containing thyroxine, wherein the antibody or the antigen-binding fragment thereof specifically binds to an immune complex formed between thyroxine and an anti-thyroxine antibody, and comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has three HCDRs, and the light chain variable region has three LCDRs, and the three HCDRs of the heavy chain variable region and the three LCDRs of the light chain variable region are respectively the three HCDRs of the heavy chain variable region and the three LCDRs of the light chain variable region in any one of the following combinations of heavy chain variable regions and light chain variable regions:

[0011] (1) a heavy chain variable region as shown in SEQ ID NO:6, and a light chain variable region as shown in SEQ ID NO:7;

[0012] (2) a heavy chain variable region as shown in SEQ ID NO: 15, and a light chain variable region as shown in SEQ ID NO: 16;

[0013] (3) a heavy chain variable region as shown in SEQ ID NO:24, and a light chain variable region as shown in SEQ ID NO:25.

[0014] In a feasible embodiment, the heavy chain variable region as shown in SEQ ID NO:6 comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the light chain variable region as shown in SEQ ID NO:7 comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:4, GAS and SEQ ID NO:5, respectively.

[0015] In a feasible embodiment, the heavy chain variable region as shown in SEQ ID NO:15 comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 as shown in amino acid sequences of SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively, and the light chain variable region as shown in SEQ ID NO:16 comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3 as shown in amino acid sequences of SEQ ID NO:13, KAS and SEQ ID NO:14, respectively.

[0016] In a feasible embodiment, the heavy chain variable region as shown in SEQ ID NO:24 comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 as shown in amino acid sequences of SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, respectively, and the light chain variable region as shown in SEQ ID NO:25 comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3 as shown in amino acid sequences of SEQ ID NO:22, RAS and SEQ ID NO:23, respectively.

[0017] In a preferred embodiment, the antibody comprises a heavy chain variable region and a light chain variable region selected from any one of the following:

[0018] (1) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:7;

[0019] (2) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16;

[0020] (3) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 25.

[0021] In a preferred embodiment, the antibody comprises a heavy chain and a light chain selected from any one of the following:

[0022] (1) the amino acid sequence of the heavy chain is the amino acid sequence shown in SEQ ID NO:8, and the amino acid sequence of the light chain is the amino acid sequence shown in SEQ ID NO:9;

[0023] (2) the amino acid sequence of the heavy chain is the amino acid sequence shown in SEQ ID NO: 17, and the amino acid sequence of the light chain is the amino acid sequence shown in SEQ ID NO: 18;

[0024] (3) The amino acid sequence of the heavy chain is the amino acid sequence shown in SEQ ID NO: 26, and the amino acid sequence of the light chain is the amino acid sequence shown in SEQ ID NO: 27.

[0025] For the above embodiments, the antibody is preferably a murine antibody, a single domain antibody, a chimeric antibody, a human antibody or a humanized antibody; and the antigen-binding fragment is preferably Fab, Fab', F(ab')2, Fv or a complementarity determining region fragment.

[0026] In a second aspect, the present invention provides a fusion protein comprising the antibody or antigen-binding fragment thereof as described in the first aspect above.

[0027] In a third aspect, the present invention provides a polynucleotide comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof as described in the first aspect. The polynucleotide is not limited to the method of its production and can be obtained by genetic engineering recombination technology or chemical synthesis method.

[0028] In a fourth aspect, the present invention provides a nucleic acid construct comprising the polynucleotide as described in the third aspect above, and, optionally, at least one expression control element operably linked to the polynucleotide.

[0029] In a fifth aspect, the present invention provides a recombinant vector comprising the polynucleotide as described in the third aspect above, or the nucleic acid construct as described in the fourth aspect above.

[0030] The recombinant vector of the present invention may be a cloning vector or an expression vector, for example, it may be a plasmid, a cosmid, a phage, etc.

[0031] In some preferred embodiments, the recombinant vector is an expression vector, preferably a eukaryotic expression vector.

[0032] In a sixth aspect, the present invention provides a transformed host cell, wherein the polynucleotide as described in the third aspect, the nucleic acid construct as described in the fourth aspect, or the recombinant vector as described in the fifth aspect is transformed;

[0033] The host cell includes, but is not limited to, prokaryotic cells, such as Escherichia coli cells, eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The host cell may also be a cell line, such as a 293T cell line.

[0034] In some feasible embodiments, the host cell is a bacterial, yeast or mammalian cell;

[0035] Optionally, the bacteria is Escherichia coli;

[0036] Optionally, the yeast is Pichia pastoris;

[0037] Optionally, the mammalian cells are Chinese hamster ovary cells or human embryonic kidney 293 cells.

[0038] In the seventh aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof as described in the first aspect, the fusion protein as described in the second aspect, the polynucleotide as described in the third aspect, the nucleic acid construct as described in the fourth aspect, the recombinant vector as described in the fifth aspect and / or the transformed host cell as described in the sixth aspect in the preparation of a detection reagent for detecting thyroxine.

[0039] In a feasible embodiment, the sample used for the detection is a biological sample of a subject, preferably a serum or plasma sample of the subject.

[0040] In an eighth aspect, the present invention provides a detection reagent for detecting thyroxine, which comprises the antibody or antigen-binding fragment thereof as described in the first aspect above, the fusion protein as described in the second aspect above, the polynucleotide as described in the third aspect above, the nucleic acid construct as described in the fourth aspect above, the recombinant vector as described in the fifth aspect above and / or the transformed host cell as described in the sixth aspect above.

[0041] Preferably, the antibody or antigen-binding fragment thereof is labeled with a detectable marker; such detectable markers are well known to those skilled in the art, and include, but are not limited to, radioisotopes, fluorescent substances, luminescent substances, colored substances and enzymes (e.g., horseradish peroxidase), etc.

[0042] In a preferred embodiment, the detectable marker is fluorescent microspheres, latex microspheres or colloidal gold particles, preferably colloidal gold particles.

[0043] In a ninth aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof as described in the first aspect above, the method comprising: under conditions suitable for the expression of the antibody or antigen-binding fragment thereof, causing the transformed host cell as described in the sixth aspect above to express the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the culture of the host cell.

[0044] In the tenth aspect, the present invention provides a method for detecting the presence or expression level of thyroxine in a sample, the method comprising using the antibody or antigen-binding fragment thereof as described in the first aspect, the fusion protein as described in the second aspect, the polynucleotide as described in the third aspect, the nucleic acid construct as described in the fourth aspect, the recombinant vector as described in the fifth aspect, the transformed host cell as described in the sixth aspect and / or the detection reagent as described in the eighth aspect.

[0045] In a feasible embodiment, the sample includes but is not limited to serum, plasma, etc. from a subject.

[0046] The method can be used for diagnostic purposes (eg, the sample is a sample from a patient), or for non-diagnostic purposes (eg, the sample is a cell sample rather than a sample from a patient).

[0047] Beneficial Effects

[0048] The antibody or antigen-binding fragment thereof provided by the present invention against the thyroxine antigen-antibody immune complex has a high binding activity with the thyroxine antigen-antibody immune complex (the EC50 of the binding activity reaches the ng / ml level, and the KD reaches 10 -10 M~10 -11 M), on the other hand, it does not bind to the single thyroxine and the anti-thyroxine antibody in the immune complex. Therefore, it can be used in combination with the anti-thyroxine antibody in the immune complex to detect thyroxine by the double antibody sandwich method, which has higher sensitivity and linearity than the traditional competitive method, breaking through the limitations of low detection sensitivity and poor clinical relevance of the traditional competitive method detection reagents. The clinical compliance rates of the three colloidal gold test strips developed based on the antibody of the present invention and the anti-thyroxine antibody in the immune complex for detecting thyroxine are all above 0.96. In addition, the colloidal gold test strips based on the antibody of the present invention and the anti-thyroxine antibody in the immune complex have good specificity for detecting thyroxine. Specifically, it has a small cross-reactivity with triiodothyronine (the cross-reactivity with D-T3 and L-T3 antigens is about 0.1%), which is far better than the current competitive method detection reagents on the market; in view of the above, the combination of the antibody of the present invention and the anti-thyroxine antibody in the immune complex successfully realizes the high accuracy and high sensitivity detection of thyroxine. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0050] Figure 1 This is a gel image of SDS-PAGE detection of the three monoclonal antibodies expressed and purified in Example 4 of the present invention; wherein M represents the electrophoresis band of the molecular weight marker, lane 1 shows the electrophoresis band of each monoclonal antibody under reducing conditions, and lane 2 shows the electrophoresis band of each monoclonal antibody under non-reducing conditions.

[0051] Figure 2 This is a graph showing the results of the binding activity test between the three monoclonal antibodies expressed and purified in Example 4 of the present invention and the T4 antigen-antibody immune complex, as tested in Example 5.

[0052] Figure 3 This is a structural diagram of the colloidal gold immunochromatographic test strip prepared in Example 6 of the present invention; wherein 1 is a PVC base plate; 2 is a sample pad; 3 is a nitrocellulose membrane; 4 is a gold line; 5 is a detection line; 6 is a quality control line; and 7 is a water absorbent pad.

[0053] Figure 4 The results of the colloidal gold immunochromatographic test strips prepared based on the three monoclonal antibodies of the present invention and the clinical sample correlation of the Roche detection reagent are shown in Figure 7; wherein, Figure A is the result of the colloidal gold immunochromatographic test strip 1 and the clinical sample correlation of the Roche detection reagent, Figure B is the result of the colloidal gold immunochromatographic test strip 2 and the clinical sample correlation of the Roche detection reagent, and Figure C is the result of the colloidal gold immunochromatographic test strip 3 and the clinical sample correlation of the Roche detection reagent. DETAILED DESCRIPTION

[0054] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0055] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0056] In order to make the present invention more easily understood, some technical terms are specifically defined as follows. Unless otherwise clearly defined in other parts of this document, the technical terms used herein have the meanings commonly understood by ordinary technicians in the field to which the present invention belongs.

[0057] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as such variations are appropriate for performing the disclosed methods.

[0058] The term "and / or" should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0059] As used herein, the term "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, that is, including at least one of the numbers or elements in the list, but also including more than one, and optionally, additional unlisted items. Only when the contrary terminology is clearly indicated, such as "only one" or "exactly one" or when "consisting of..." is used in the claims, it will refer to only one number listed or one element of the list.

[0060] The term "antibody" refers to any form of antibody having the desired biological activity. Therefore, it is used in the broadest sense, specifically including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies and camelized single domain antibodies.

[0061] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0062] "Antigen binding fragment" refers to an antigen binding fragment of an antibody and an antibody analog, which generally includes at least a portion of the antigen binding region or variable region of the parent antibody, such as one or more CDRs. The antibody fragment retains at least some of the binding specificity of the parent antibody. Antigen binding fragments include those selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, diabodies, peptides containing CDRs, etc.

[0063] The "Fab fragment" consists of a light chain and the CH1 and variable regions of a heavy chain.

[0064] The "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domains.

[0065] A "Fab' fragment" contains a light chain and a portion of a heavy chain including a VH domain, a CH1 domain and a portion of the constant region between the CH1 and CH2 domains, with an interchain disulfide bond formed between the two heavy chains of the two Fab' fragments to form a F(ab')2 molecule.

[0066] A "F(ab')2 fragment" contains two light chains and a portion of two heavy chains including a VH domain, a CH1 domain, and a portion of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, a F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0067] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0068] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art.

[0069] The term "high affinity" for IgG antibodies refers to a KD for the antigen of 1.0 × 10 -9 M or lower. For other antibody subtypes, "high affinity" binding may vary.

[0070] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form.

[0071] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0072] Example 1: Single B cell screening of antibodies against T4 antigen-antibody immune complex

[0073] (1) Preparation of T4 antigen-antibody immune complex

[0074] Take 2 mg of anti-T4 antibody (manufacturer: bioventix, product number: T4.1C4), add the corresponding amount of T4 small molecule antigen (manufacturer: McLean, product number: L819046, and the molar ratio of antibody to T4 small molecule antigen is 1:2), react at 37 degrees Celsius for 6 hours, dialyze with 1xPBS, and use Nano300 to detect the antibody concentration to obtain T4 antigen-antibody immune complex.

[0075] (2) Animal immunization

[0076] The T4 antigen-antibody immune complex prepared in the above step (1) was used as an immunogen. 200 μg of the above immunogen was fully emulsified with an equal volume of Freund's complete adjuvant and then injected subcutaneously to immunize rabbits (breed: New Zealand white rabbit, female, 6-8 weeks old). The booster immunization was performed at intervals of two weeks for a total of 3 immunizations. 5-8 days after the last booster immunization, blood was collected from the rabbit vein and the serum titer was detected by ELISA. The specific experimental information is shown in Table 1 below.

[0077] Table 1. Experimental information

[0078] .

[0079] The serum titer data detected by ELISA are shown in Table 2 below.

[0080] Table 2. Serum titer data detected by ELISA

[0081] ;

[0082] The immune serum with a serum titer of 512K was selected to prepare the peripheral blood mononuclear cell (PBMC) suspension.

[0083] (3) PBMC preparation

[0084] Add Ficoll, a lymphocyte separation solution, to the centrifuge tube, take anticoagulated peripheral blood (whole blood) and sterile PBS and mix thoroughly at a ratio of 1:1-1:5, use a pipette to slowly superimpose on the stratified liquid surface along the tube wall, centrifuge horizontally at 400g×30 minutes, remove part of the upper liquid after centrifugation, and leave about 1mL. Use a pipette to insert into the cloud layer to absorb the mononuclear cells. Place in another centrifuge tube, add more than 5 times the volume of PBS, centrifuge at 300g×10 minutes, and wash the cells twice. After the last centrifugation, discard the supernatant, add red blood cell lysis solution, incubate at room temperature for 2 minutes, lyse the red blood cells, add 10mL PBS, centrifuge at 300g×10 minutes, and wash the cells twice. Discard the supernatant, add RPMI1640 containing 10% FBS, resuspend the cells, and count;

[0085] (4) Obtaining single B cells

[0086] The prepared PBMCs are used to label antigens and perform cell screening using a single-cell chip screening platform to obtain antigen-specific B cells.

[0087] Through the above steps, a total of 400 B cells were obtained.

[0088] Example 2: Plasmid construction of light and heavy chain gene sequences of monoclonal antibodies against T4 antigen-antibody immune complex

[0089] cDNA preparation:

[0090] RNA was extracted from the 400 B cells obtained by sorting in Example 1, and reverse transcription was performed using RNA as a template to obtain cDNA, and the genes of the light and heavy chains of the antibody were amplified by PCR;

[0091] Acquisition of light and heavy chain genes:

[0092] Specific primers are used for amplification, and the amplified DNA sequences are cloned into vectors respectively, and then transformed and plated, clones are picked, and plasmids are extracted.

[0093] Example 3: Expression and specificity detection of monoclonal antibodies against T4 antigen-antibody immune complex

[0094] (1) Cultivate N293 suspension cells until the density reaches (1-3)×10 6 When the cell count is 10 cells / ml and the survival rate is >80-90%, cell transfection is performed;

[0095] (2) The recombinant plasmids of the light and heavy chain gene sequences of the 400 monoclonal antibodies obtained in Example 2 were transfected into N293 cells. The transfection conditions were: the mass ratio of PEI / plasmid was 3:1, and the transfection ratio was 2 μg plasmid / 1 ml N293 cells;

[0096] (3) 5-7 days after transfection, collect cell supernatant;

[0097] (4) Antigen-specific detection of the supernatant using ELISA:

[0098] ① Antigen coating: dilute the antigen to 2µg / ml, then add 100μl / well to an empty ELISA plate, place the ELISA plate in a 4℃ refrigerator, and incubate overnight;

[0099] ② Blocking: Wash the plate 5 times with a plate washer, tap dry, add blocking solution to the ELISA plate, 150 μl per well, place in a 37°C incubator, block for 1 hour; wash the plate 5 times with a plate washer, tap dry;

[0100] ③ Incubate with primary antibody: add the culture supernatant of 96-well plate to the wells of ELISA plate, 100 μl per well, incubate at 37°C for 1 hour; wash the plate 5 times with a plate washer and dry;

[0101] ④ Incubate with secondary antibody: dilute HRP-labeled goat anti-rabbit secondary antibody at 1:10000, then add to the ELISA plate, 100 μl per well, incubate at 37°C for 30 min; wash the plate 5 times with a plate washer and dry;

[0102] ⑤ Color development: add the color development solution to the ELISA plate, 100 μl per well, reaction time: 37 degrees / 15 minutes;

[0103] ⑥ Stop and read: add stop solution, volume 100μl / well; detection wavelength setting: detection wavelength 450nm, reference wavelength 620nm, export the detection results into Excel table.

[0104] ⑦Data analysis: By comparing the signal strengths, three clones with stronger reaction signals and specific binding to the T4-antibody complex (clone numbers: 2M2C1, 1M2D2 and 1M1E3) were finally selected for subsequent amplification, expression and purification.

[0105] Then, the corresponding plasmids of the three clones constructed in Example 2 (clone numbers: 2M2C1, 1M2D2 and 1M1E3) were sent for sequencing to obtain the gene sequences of the three monoclonal antibodies, and the heavy and light chain amino acid sequences of the three monoclonal antibodies were deduced. The results were: the heavy chain amino acid sequences of T4 mAb1 to T4 mAb3 were shown in SEQ ID NOs: 8, 17, and 26, respectively, and the light chain amino acid sequences were shown in SEQ ID NOs: 9, 18, and 27, respectively; then, based on the sequencing results, the heavy chain variable regions and light chain variable regions of the three monoclonal antibodies were analyzed, and finally the amino acid sequences of their heavy chain variable regions and light chain variable regions were obtained, which are shown below, respectively, wherein the underlined CDR regions are underlined.

[0106] T4 mAb1 (corresponding clone number is 2M2C1)

[0107] VH (SEQ ID NO:6):

[0108] QSVEESGGRLVTPGTPLTLTCTVS GFSFSSGFD MSWVRQAPGKGLEYIGI IYTGSGST YYASWAKGRFTISKTSTTVDLKVTSPTTEDTATYFC ARDSTFAVGDYPYFNL WGPGTLVTVSS;

[0109] VL (SEQ ID NO:7):

[0110] DIVMTQTPSSVSAAVGGTVTIKCQAS QSIGSN LAWYQQKPGQPPKLLIY GAS NLESGVPSRFSGSGSGTEFTLTISDLECADAATYYC QCTGYGSTSLYVD FGRGTEVVVK;

[0111] T4 mAb2 (corresponding clone number is 1M2D2)

[0112] VH (SEQ ID NO:15):

[0113] QSLEESGGGLVKPGASLTLTCKAS GFSLSSYW ICWVRQAPGKGLEWIACI ISPTDNI YASWAKGRFTISVTSSTTVTLQMTRLTAADTATYFC AREAWFEGLGL WGPGTLVTVSS;

[0114] VL (SEQ ID NO:16):

[0115] ALVMTQTPASVSATVGGTVTISCQAS ENIYIG WYQQKPGQRPKLLIY KAS TLASGVSSRFKGSGSGTEYTLTISGVQCDDAATYSC QSYYDINKYGYV FGGGTEVVVK;

[0116] T4 mAb3 (corresponding clone number is 1M1E3)

[0117] VH (SEQ ID NO:24):

[0118] QSLEESGGRLVTPGTPLTLTCTVS GIDLYSHA MGWVRQAPGKGLEWIGT IWSDGFS DYGTWAKGRFTISKTSTTVDLKMTSLTTEDTATYFC VRHYGNDYF WGQGTLVTVSS;

[0119] VL (SEQ ID NO:25):

[0120] AYDLTQTPASVEVAVGGTVTVKCQAS ESINSW LAWYQQKPGQPPKLLIY RAS KLASGVPSRFKGSGSGTEFTLTISGVECADAATYYC QQTYTYNHVDNTFGGGTEMVVE.

[0121] Example 4: Amplification and purification of monoclonal antibodies against T4 antigen-antibody immune complex

[0122] (1) Amplification and expression of cloned antibodies:

[0123] ① The recombinant plasmids of the light and heavy chain gene sequences of the three monoclonal antibodies selected in Example 3 were transfected into N293 cells. The transfection conditions were: the mass ratio of PEI / plasmid was 3:1, and the transfection ratio was 2 μg plasmid / 1 ml N293 cells;

[0124] ②5-7 days after transfection, collect cell supernatant;

[0125] ③ Use Protein A resin for affinity purification to obtain high-purity monoclonal antibodies, replace the elution buffer with PBS through dialysis, and use Nanodrop to measure the antibody concentration;

[0126] (2) Detect the purity of the antibody by SDS-PAGE. The specific method is as follows:

[0127] Take 2 μg of the antibody to be tested, add an appropriate amount of SDS-PAGE protein loading buffer to make the total volume 20 μl; prepare reducing SDS-PAGE samples and non-reducing SDS-PAGE samples at the same time, and then load them; first, electrophoresis at 80V for 30 minutes, and then electrophoresis at 120V until the bands are clearly separated; remove the PAGE gel after electrophoresis, stain with Coomassie Brilliant Blue, remove the staining solution after 15 minutes, rinse with clean water, and then add decolorizing solution for decolorization until clear bands are seen.

[0128] The results are as follows Figure 1 As shown; Figure 1 It was shown that under reducing conditions, each monoclonal antibody had two electrophoretic bands, and the corresponding molecular weights of the two electrophoretic bands were consistent with the sizes of their respective light and heavy chains; under non-reducing conditions, each monoclonal antibody had a single band, and the corresponding molecular weights of the electrophoretic bands were consistent with the sizes of their respective complete antibodies; these results indicated that the three monoclonal antibodies were accurately expressed.

[0129] In addition, all electrophoresis bands had clear edges and no foreign bands, indicating that the monoclonal antibodies obtained by the above expression and purification steps had high purity.

[0130] Example 5: Antigen Binding Activity Detection of Monoclonal Antibodies Against T4 Antigen-Antibody Immune Complex

[0131] In this example, the three monoclonal antibodies expressed and purified in Example 4 were tested for antigen activity and affinity.

[0132] ① Antigen coating: dilute the T4 antigen-antibody immune complex, the anti-T4 antibody naked antibody in the immune complex, and T4-BSA to 2µg / ml respectively, and then add 100μl / well to the empty ELISA plate, place the ELISA plate in a 4℃ refrigerator, and incubate overnight;

[0133] ② Blocking: Wash the plate 5 times with a plate washer, tap dry, add blocking solution to the ELISA plate, 150 μl per well, place in a 37°C incubator, block for 1 hour; wash the plate 5 times with a plate washer, tap dry;

[0134] ③ Incubate the primary antibody: First, dilute the antibody to be tested to 4µg / ml with 1×PBS, and then perform a series of 3-fold dilutions to obtain a series of antibody solutions with different concentration gradients; add the diluted antibody solutions to the wells of the ELISA plate, 100μl per well, and incubate at 37℃ for 1h; wash the plate 5 times with a plate washer and dry;

[0135] ④ Incubate with secondary antibody: dilute HRP-labeled goat anti-rabbit secondary antibody at 1:10000, then add to the ELISA plate, 100 μl per well, incubate at 37°C for 30 min; wash the plate 5 times with a plate washer and dry;

[0136] ⑤ Color development: add the color development solution to the ELISA plate, 100 μl per well, reaction time: 37 degrees / 15 minutes;

[0137] ⑥ Stop and read: add stop solution, volume 100μl / well; detection wavelength setting: detection wavelength 450nm, reference wavelength 620nm, export the detection results into Excel table.

[0138] ⑦Data analysis: Use software to analyze and plot data, fit EC50, and derive its KD.

[0139] The results are as follows:

[0140] (1) The reaction test data of the three antibodies and the T4 antigen-antibody immune complex are shown in the following Table 3.

[0141] Table 3. OD450 values ​​of the tested antibodies at various concentrations

[0142] ;

[0143] According to Table 3, the binding activity curve of antibody and antigen was prepared, and the results were as follows Figure 2 As shown, the data in Table 3 were fitted with a four-parameter curve using the "ELISAcalc" software to obtain the EC50 values ​​of the three antibodies; the KD value data derived from the EC50 data fitted in Table 3 are shown in the following Table 4.

[0144] Table 4

[0145] ;

[0146] The above results show that among these three monoclonal antibodies, the EC50 of T4 mAb1 to T4 mAb3 binding to antigen (i.e., T4 antigen-antibody immune complex) are 13.4 ng / ml, 13.9 ng / ml, and 25.2 ng / ml, respectively, all reaching the ng / ml level, and their KDs all reach 10 -10 M~10 -11 M.

[0147] As mentioned above, for IgG antibodies, it is generally believed in the art that when the KD value of binding to the antigen reaches 10 -9 M is considered to have high affinity, and the KD values ​​of the above three monoclonal antibodies for antigen binding all reached 10 -10 M~10 -11 M is as much as 10-100 times the above-defined value, from which it can be concluded that these three monoclonal antibodies have extremely strong antigen-binding activity and affinity.

[0148] (2) The reaction test data of the three antibodies with the naked anti-T4 antibody in the immune complex are shown in the following Table 5.

[0149] Table 5

[0150] ;

[0151] The above results showed that the three antibodies did not react with naked anti-T4 antibodies.

[0152] (3) The reaction test data of the three antibodies with anti-T4-BSA are shown in Table 6 below.

[0153] Table 6

[0154] ;

[0155] The above results showed that the three antibodies did not react with T4-BSA.

[0156] Example 6: Preparation of a double antibody sandwich colloidal gold immunochromatographic test strip based on a monoclonal antibody against T4 antigen-antibody immune complex and the anti-T4 antibody contained in the immune complex

[0157] In this example, T4 mAb1, T4 mAb2, and T4 mAb3 expressed and purified in Example 4 were paired with anti-T4 antibodies to prepare a colloidal gold immunochromatographic test strip for detecting T4 based on the principle of double antibody sandwich method. The schematic diagram of the structure of the test strip is shown in FIG. Figure 3 As shown; the preparation method of the test strip is as follows:

[0158] (1) Preparation of Au-anti-T4 monoclonal antibody complex

[0159] ① Take 3 ml of colloidal gold particles (colloidal gold particle size: 20 nm, maximum absorption peak wavelength of 529 nm, absorbance of 2.0 A), add 18 µl of 0.1 M K2CO3, mix well, add 10 µg of detection antibody (anti-T4 antibody in immune complex), and react at room temperature for 1 h;

[0160] ② Add 600 μl 5% BSA blocking solution to the system in step ① and block at room temperature for 30 minutes;

[0161] ③ After blocking, centrifuge at 11000 rpm at 10°C for 15 min, discard the supernatant, and re-dissolve the precipitate with 50 μl of gold-labeled diluent (i.e., concentrated 60 times, 60×) to obtain the Au-anti-T4 monoclonal antibody complex; the gold-labeled diluent contains 100 mM Tris buffer at pH 7.5-8.5, 5% sucrose, 1% trehalose, and 5% BSA.

[0162] (2) Deposit

[0163] ① Draw a closed line: Take 5% BSA solution, set the instrument water output to 3μL / cm, and place the drawn bottom plate in a 37℃ oven to dry for 2 hours;

[0164] ② Dilute the Au-anti-T4 monoclonal antibody complex to a final concentration of 50×, set the water output to 2 μL / cm, draw a line at the closed line, and then place the bottom plate in a 37°C oven to dry for 2 hours. Then, place the marking pad in an aluminum foil bag pre-added with desiccant at a humidity not higher than 36% and seal it for storage;

[0165] (3) Coating quality control line C, detection line T

[0166] ① Preparation of coating solution for quality control line C and test line T: dilute goat anti-rabbit antibody with 10mM PBS buffer at pH 7.4 to 1.0mg / mL antibody solution; dilute capture antibody with 10mM PBS buffer at pH 7.4 to 1.0mg / mL antibody solution;

[0167] ② Coating: Coat the control line C and test line T coating solutions on the control line and test line of the nitrocellulose membrane, respectively. The interval between the C and T lines is 8 mm, and the coating volume is 1 μL / cm. Place the coated PVC substrate in a 37°C oven for 16 hours, and then place the PVC substrate in an aluminum foil bag pre-added with desiccant at a humidity not higher than 36% and seal it for future use.

[0168] (4) Pretreatment of sample pad

[0169] Clean the drying net, soak the sample pad with the sample pad pretreatment solution, and then drain it by rotating until no water drops. Then, dry it in a 37°C oven for 4 hours, and then seal it in an aluminum foil bag with desiccant added in advance at a humidity not higher than 36% for storage.

[0170] The sample pad pretreatment solution contains 10 mM Tris buffer at pH 8.5, 0.1% PVP 10, 0.1% PEG20000, 0.1% Tween20 and 0.5% casein sodium;

[0171] (5) Assembly and cutting of membrane materials

[0172] At a humidity not higher than 36%, take out the absorbent pad and the treated sample pad, cut them into a size of 10 mm × 300 mm, take out the coated PVC base plate, and assemble them according to the following process: first stick the sample pad on the PVC base plate so that the sample pad part presses the nitrocellulose membrane for 2 mm, and finally stick the absorbent pad so that the absorbent pad presses the nitrocellulose membrane for 2 mm; cut the assembled PVC base plate into 4 mm, which is the colloidal gold immunochromatographic test strip for detecting T4.

[0173] The antibody pairing method of the test strip is:

[0174] ① Colloidal gold immunochromatographic test strip 1: anti-T4 antibody (i.e., anti-T4 antibody in the immune complex, used to prepare Au-antibody complex) + T4 mAb1 (used for T line coating);

[0175] ② Colloidal gold immunochromatographic test strip 2: anti-T4 antibody (i.e., anti-T4 antibody in the immune complex, used to prepare Au-antibody complex) + T4 mAb2 (used for T line coating);

[0176] ③ Colloidal gold immunochromatographic test strip 3: anti-T4 antibody (i.e., anti-T4 antibody in the immune complex, used to prepare the Au-antibody complex) + T4 mAb3 (for T line coating).

[0177] Example 7: Detection performance of a double antibody sandwich colloidal gold immunochromatographic test strip based on a monoclonal antibody against T4 antigen-antibody immune complex and an anti-T4 antibody contained in the immune complex

[0178] In this example, the three colloidal gold immunochromatographic test strips prepared in Example 6 were used to detect T4 and T3 interference in clinical serum samples, and compared with Roche detection reagents.

[0179] (1) Testing clinical serum samples

[0180] Specifically, for the colloidal gold immunochromatographic test strip prepared in Example 6, the clinical serum sample was diluted with a sample diluent (whose composition is: 10mM PBS + 5% sodium dodecyl sulfate + 5% disodium EDTA + 0.5% 8-aniline-1-naphthalenesulfonic acid + 0.1% proclin300, pH7.0), the sample dilution ratio is 1:3 (serum: diluent, v / v), reacted at room temperature for 10 minutes, and then 75µl of the sample was added. After 15 minutes, the colloidal gold immunoassay was used for detection, the detection signal value was read, and the data was processed; for the Roche detection kit, the operation was carried out according to the instructions, the serum sample was diluted, and then detected to obtain the T4 concentration data. Then, the signal value detected by the test strip and the T4 data detected by the Roche reagent were compared to make a clinical correlation curve.

[0181] The specific results are as follows:

[0182] Tables 7, 8 and 9 are the test results of colloidal gold immunochromatographic test strips 1, 2 and 3, respectively. The first column in the table represents the concentration of clinical samples detected by Roche reagent (in nmol / L), and the second column represents the signal value of the colloidal gold immunochromatographic test strip of the present invention for the corresponding clinical samples;

[0183] Table 7

[0184] .

[0185] Table 8

[0186] .

[0187] Table 9

[0188] .

[0189] According to the results in Table 7, Table 8, and Table 9, the clinical relevance curves (i.e., the coincidence rate between the test results of colloidal gold immunochromatographic test strips 1, 2, and 3 and the Roche test results) of T4 in serum samples were prepared respectively. The results are shown in the figure. Figure 4 As shown in Figures A, B and C in the figure; These results show that the colloidal gold immunochromatographic test strip of the present invention has a clinical relevance R for detecting T4 in serum samples 2 All>0.96.

[0190] The above results show that the T4 colloidal gold immunochromatographic test strip prepared based on the antibody of the present invention and the anti-T4 antibody in the immune complex has a high coincidence rate (R 2 above 0.96), thus having good clinical relevance.

[0191] (2) Testing for T3 Interferors

[0192] Specifically, the T4 antigen was diluted with PBS buffer to 5 concentration gradients of 4, 0.8, 0.16, 0.08, and 0 ng / mL, and 75 μl was added to colloidal gold immunochromatographic test strips 1, 2, and 3. After 15 minutes, the colloidal gold immunoassay was used to detect, and the detection value was read and the data was processed to obtain the standard curve of T4 detection; then the L-T3 and D-T3 antigens were diluted with PBS buffer to 2000 and 400 ng / mL respectively, and 75 μl was added to colloidal gold immunochromatographic test strips 1, 2, and 3. After 15 minutes, the colloidal gold immunoassay was used to detect, and the detection value was read. The detected T / C value was brought into the curve to calculate the concentration value of T4, and then the corresponding cross-reaction rate was obtained. The cross-reaction rates detected by the three test strips were summarized to obtain the results of Table 10. The results showed that the cross-reactivity of the three sandwich colloidal gold immunochromatographic test strips to L-T3 and D-T3 was about 0.1%, which was significantly better than the competitive method.

[0193] Table 10

[0194] .

[0195] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof against an immune complex containing thyroxine, characterized in that: The antibody or antigen-binding fragment thereof specifically binds to an immune complex formed between thyroxine and an anti-thyroxine antibody, and the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has three HCDRs, and the light chain variable region has three LCDRs, and the three HCDRs of the heavy chain variable region and the three LCDRs of the light chain variable region are respectively the three HCDRs of the heavy chain variable region and the three LCDRs of the light chain variable region in any one of the following combinations of heavy chain variable region and light chain variable region: (1) a heavy chain variable region as shown in SEQ ID NO:6, and a light chain variable region as shown in SEQ ID NO:7; (2) a heavy chain variable region as shown in SEQ ID NO: 15, and a light chain variable region as shown in SEQ ID NO: 16; (3) the heavy chain variable region shown in SEQ ID NO:24, and the light chain variable region shown in SEQ ID NO:

25.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: (1) the heavy chain variable region as shown in SEQ ID NO:6 comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the light chain variable region as shown in SEQ ID NO:7 comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:4, GAS and SEQ ID NO:5, respectively; or, (2) the heavy chain variable region as shown in SEQ ID NO:15 comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively, and the light chain variable region as shown in SEQ ID NO:16 comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:13, KAS and SEQ ID NO:14, respectively; or, (3) The heavy chain variable region as shown in SEQ ID NO:24 comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, respectively, and the light chain variable region as shown in SEQ ID NO:25 comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:22, RAS and SEQ ID NO:23, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The antibody comprises a heavy chain variable region and a light chain variable region selected from any one of the following: (1) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7; (2) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16; (3) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

25.

4. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The antibody comprises a heavy chain and a light chain selected from any one of the following: (1) the amino acid sequence of the heavy chain is the amino acid sequence shown in SEQ ID NO: 8, and the amino acid sequence of the light chain is the amino acid sequence shown in SEQ ID NO: 9; (2) the amino acid sequence of the heavy chain is the amino acid sequence shown in SEQ ID NO: 17, and the amino acid sequence of the light chain is the amino acid sequence shown in SEQ ID NO: 18; (3) The amino acid sequence of the heavy chain is the amino acid sequence shown in SEQ ID NO: 26, and the amino acid sequence of the light chain is the amino acid sequence shown in SEQ ID NO:

27.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: The antibody is a chimeric antibody or a humanized antibody; And / or, the antigen binding fragment is Fab, Fab', F(ab')2 or Fv.

6. A fusion protein, characterized in that The fusion protein comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. A polynucleotide, characterized in that The polynucleotide comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

8. A nucleic acid construct, characterized in that The nucleic acid construct comprises the polynucleotide according to claim 7.

9. The nucleic acid construct according to claim 8, characterized in that The nucleic acid construct also comprises at least one expression control element operably linked to the polynucleotide.

10. A recombinant vector, characterized in that: The recombinant vector comprises the polynucleotide according to claim 7, or the nucleic acid construct according to claim 8 or 9.

11. A transformed host cell, characterized in that The polynucleotide according to claim 7, the nucleic acid construct according to claim 8 or 9, or the recombinant vector according to claim 10 is transformed therein.

12. The transformed host cell according to claim 11, characterized in that The host cell is a bacterial, yeast or mammalian cell.

13. The transformed host cell according to claim 12, characterized in that The bacteria is Escherichia coli; And / or, the yeast is Pichia pastoris; And / or, the mammalian cell is a Chinese hamster ovary cell or a human embryonic kidney 293 cell.

14. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the fusion protein according to claim 6, the polynucleotide according to claim 7, the nucleic acid construct according to claim 8 or 9, the recombinant vector according to claim 10 and / or the transformed host cell according to any one of claims 11 to 13 in the preparation of a detection reagent for detecting thyroxine.

15. A detection reagent for detecting thyroxine, characterized in that: The detection reagent comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the fusion protein according to claim 6, the polynucleotide according to claim 7, the nucleic acid construct according to claim 8 or 9, the recombinant vector according to claim 10 and / or the transformed host cell according to any one of claims 11 to 13.

16. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that: The method comprises: allowing the transformed host cell according to any one of claims 11 to 13 to express the antibody or antigen-binding fragment thereof under conditions suitable for the expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the culture of the host cell.

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