Anti-streptavidin antibodies and related uses thereof

Abstract

The application provides an anti-streptavidin antibody with high specificity, high sensitivity, few cross reactions and small batch difference. The antibody can not only realize similar application of a streptavidin-biotin system, but also can be used in combination with the streptavidin-biotin system for related applications such as cell sorting, immunochromatography and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunoassay, in particular to an anti-streptavidin monoclonal antibody or antigen-binding fragment thereof and related applications thereof. BACKGROUND

[0002] Streptavidin (SA) is a secretory protein extracted from Streptomyces Avidinii culture, which is composed of four identical peptide chains and has a molecular weight of about 65 kDa. Due to its high specific binding with biotin, each streptavidin molecule can bind four biotin molecules, and can bind with various markers, so that the streptavidin-based signal amplification technology is widely used in the detection of biotinylated biomolecules, such as biotin-labeled primary antibody, secondary antibody, ligand, toxin, magnetic bead or DNA probe in in situ hybridization.

[0003] At present, the anti-streptavidin antibodies on the market are mainly polyclonal antibodies, especially rabbit polyclonal antibodies. Polyclonal antibodies can recognize multiple epitopes on the antigen, have high affinity, but sometimes produce non-specific recognition, and the batch difference is also large, and the system stability is insufficient.

[0004] Therefore, there is a need in the art for a monoclonal antibody specific to streptavidin, which has high specificity, high sensitivity and less cross-reactivity. SUMMARY

[0005] The present inventors immunized mice with recombinant streptavidin protein, fused mouse spleen cells with myeloma cells, and screened hybridoma cell strains that specifically bind to streptavidin by ELISA method, constructed recombinant antibodies, and obtained recombinant anti-streptavidin antibodies. Thus, the present application is completed.

[0006] Therefore, in the first aspect of the present application, an anti-streptavidin monoclonal antibody or antigen-binding fragment thereof is provided, which comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity determining regions V H CDR1, V H CDR2 and V H CDR3, and the light chain variable region comprising light chain complementarity determining regions V L CDR1, V L CDR2 and V L CDR3; wherein:

[0007] 1) the heavy chain variable region comprises heavy chain complementarity determining regions V H CDR1, V H CDR2 and V HCDR3, the heavy chain variable region comprising heavy chain complementarity determining regions V L CDR1, V L CDR2, and V L CDR3.

[0008] 2) the heavy chain variable region comprising heavy chain complementarity determining regions V H CDR1, V H CDR2, and V H CDR3, the heavy chain variable region comprising heavy chain complementarity determining regions V L CDR1, V L CDR2, and V L CDR3; or

[0009] 3) the heavy chain variable region comprising heavy chain complementarity determining regions V H CDR1, V H CDR2, and V H CDR3, the heavy chain variable region comprising heavy chain complementarity determining regions V L CDR1, V L CDR2, and V L CDR3.

[0010] In a second aspect, the present application provides a nucleic acid molecule encoding the anti-streptavidin monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present application.

[0011] In a third aspect, the present application provides a vector comprising the nucleic acid molecule of the second aspect of the present application.

[0012] In a fourth aspect, the present application provides an expression cell comprising the nucleic acid molecule of the second aspect of the present application or the vector of the third aspect of the present application.

[0013] In a fifth aspect, the present application provides a method for sorting target cells from a cell population, comprising the step of using the anti-streptavidin monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present application.

[0014] In a sixth aspect, the present application provides a method for detecting an antigen to be detected by colloidal gold chromatography, comprising the step of using the anti-streptavidin monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present application as a quality control system.

[0015] In a seventh aspect, the present invention provides the use of the anti-streptavidin monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention in immunoassay.

[0016] In summary, this invention provides an anti-streptavidin antibody with high specificity, high sensitivity, low cross-reactivity, and small batch-to-batch variation. The beneficial effects of the anti-streptavidin antibody of this invention include:

[0017] (1) The anti-streptavidin antibody of the present invention can specifically bind to streptavidin, thereby enabling similar applications of the streptavidin-biotin system for related detections such as immunohistochemistry (IHC), immunocytochemistry (ICC), in situ hybridization (ISH), enzyme-linked immunosorbent assay (ELISA), immunochromatography (ICA), Western blotting (WB), immunofluorescence (IF), in situ hybridization (ISH), or flow cytometry (FCM).

[0018] (2) The anti-streptavidin antibody of the present invention has been further proven to be usable for cell sorting. This is because the affinity of the anti-streptavidin antibody to streptavidin is lower than that of streptavidin to biotin. Biotin can be used to separate the sorted positive cells from the magnetic beads, which is convenient for subsequent culture and application.

[0019] (3) The anti-streptavidin antibody of the present invention can be used in conjunction with the streptavidin-biotin system, such as as a further reagent to amplify weak or low signals. For example, the anti-streptavidin antibody of the present invention can be used as a quality control system in immunochromatography such as colloidal gold chromatography to establish a detection method based on biotin-streptavidin binding. Compared with the detection method that relies on the formation of a sandwich between colloidal gold, the antigen to be tested, and the second antibody at the detection line, which depends on the first antibody labeling, the sensitivity of the relevant detection using the anti-streptavidin antibody of the present invention is further improved. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0021] Figure 1 The results of cell sorting using the recombinant monoclonal antibody S1' of the present invention as coated magnetic beads are shown.

[0022] Figure 2 The results of cell sorting using the recombinant monoclonal antibody S2' of the present invention as coated magnetic beads are shown.

[0023] Figure 3 The results of cell sorting using the recombinant monoclonal antibody S3' of the present invention as coated magnetic beads are shown. Detailed Implementation

[0024] The present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the following description is merely illustrative and is not intended to limit the scope of the invention; the scope of protection of the invention is defined by the appended claims. Furthermore, those skilled in the art will understand that modifications can be made to the technical solutions of the present invention without departing from its spirit and intent. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter pertains. Before a detailed description of the invention, the following definitions are provided to better understand it.

[0026] In cases where numerical ranges are provided, such as concentration ranges, percentage ranges, or ratio ranges, it should be understood that, unless the context explicitly specifies otherwise, all intermediate values ​​between the upper and lower limits of the range, up to one-tenth of the lower limit unit, and any other values ​​or intermediate values ​​within the range are included in the subject matter. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and such embodiments are also included in the subject matter, limited by any specific excluded limit values ​​within the range. Where the range includes one or two limit values, the range excluding any one or both of those included limit values ​​is also included in the subject matter.

[0027] In the context of this invention, many embodiments use the expressions "comprising," "including," or "basically / mainly composed of." The expressions "comprising," "including," or "basically / mainly composed of" are generally understood as open-ended expressions, indicating that they include not only the elements, components, parts, or method steps specifically listed after the expression, but also other elements, components, parts, or method steps. However, in this document, the expressions "comprising," "including," or "basically / mainly composed of" can also be understood as closed-ended expressions in certain situations, indicating that they only include the elements, components, parts, or method steps specifically listed after the expression, and do not include any other elements, components, parts, or method steps. In this case, the expression is equivalent to the expression "composed of."

[0028] Unless otherwise specified, all steps mentioned in this application may be performed sequentially or randomly, unless the context dictates that they must be performed sequentially. For example, when stating that "the method includes steps (1) and (2)," it means that the method may be implemented by performing step (1) first and then step (2), or by performing step (2) first and then step (1), unless the context dictates that step (1) must be performed before step (2). As another example, when stating that "the method may further include step (3)," it means that step (3) may be added to the method in any order. Specifically, the method may be implemented in the following order: steps (1), (2), and (3); steps (1), (3), and (2); steps (3), (1), and (2); steps (3), (2), and (1).

[0029] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of a "light" (L) chain and a "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes can be defined accordingly as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "J" regions (hinge regions) of approximately 12 or more amino acids, and the heavy chain also contains "D" regions of approximately 3 or more amino acids. Each heavy chain is further divided by a heavy chain variable region (V... H ) and heavy chain constant region (C H The heavy chain constant region consists of three structural domains (C). H1 C H2 and C H3 It consists of ) light chains. Each light chain is composed of a light chain variable region (V L ) and light chain constant region (C L It consists of a light chain constant region composed of a structural domain C. L Composition. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. V H and V L The region can be further subdivided into highly variable regions (called complementarity-determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). For each heavy or light chain, its variable region contains three CDRs: CDR1, CDR2, and CDR3. Therefore, each V H and V L It consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable region (V) of each heavy chain / light chain pair... H and VL Each of these forms an antigen-binding site.

[0030] The rules for allocating amino acids to various regions or domains have been defined in several publications: Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda Md (1987 and 1991)); Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al., Nature 1989; 342: 878-883; Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3D structure-DB and IMGT / Domain GapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF." Nucleic acids research 2009; 38(suppl_1): D301-D307.

[0031] The precise boundaries of CDRs have been defined differently depending on the system. The Kabat system not only provides a definitive residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining three CDRs, which are referred to as Kabat CDRs. Chothia discovered that certain sub-regions within Kabat system CDRs, despite significant diversity at the amino acid sequence level, have nearly identical peptide backbone conformations; these sub-regions are referred to as Chothia CDRs, which have overlapping boundaries with Kabat CDRs. These overlapping boundaries are further described by Padlan and MacCallum. CDR boundary definitions may not strictly adhere to the aforementioned systems, such as the AbM definition. In this document, CDRs can be defined according to any of these systems, although the preferred embodiment uses the antibody numbering system of Chothia et al. to define CDRs.

[0032] As used herein, the term "monoclonal antibody" refers to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, i.e., a group of identical antibody molecules except for the possibility of spontaneous natural mutations. The antibody molecules can be immunoglobulins, whether they are natural immunoglobulins or partially or wholly obtained through synthetic methods. The antibody molecules may also include all polypeptides or proteins having an antibody-binding domain, and antibody fragments having an antibody domain are molecules such as Fab, scFv, Fv, dAb, Fd, and bifunctional antibodies. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using the hybridoma technique first reported by Kohler et al. G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J]. Nature, 1975; 256(5517):495), but it can also be obtained using recombinant DNA technology (see US Patent 4,816,567). As used herein, the terms “monoclonal antibody” and “monoclonal antibody” have the same meaning and are used interchangeably; the terms “polyclonal antibody” and “polyclonal antibody” have the same meaning and are used interchangeably; the terms “peptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented as Ala or A. Glycine can be represented by Gly or G, valine by Val or V, leucine by Leu or L, isoleucine by Ile or I, proline by Pro or P, phenylalanine by Phe or F, tyrosine by Tyr or Y, tryptophan by Trp or W, serine by Ser or S, threonine by Thr or T, cysteine ​​by Cys or C, methionine by Met or M, asparagine by Asn or N, glutamine by Gln or Q, aspartic acid by Asp or D, glutamic acid by Glu or E, lysine by Lys or K, arginine by Arg or R, and histidine by His or H.

[0033] As used herein, the term "recombinant antibody" refers to an antibody obtained by cloning an antibody gene into an expression vector using molecular biology techniques and then transfecting that expression vector into a suitable host cell line for expression. The encoding gene of a recombinant antibody may or may not be identical to the encoding gene of a naturally derived antibody. For example, the complete encoding gene of an antibody obtained by immunizing an animal can be cloned into an expression vector for expression, thereby obtaining an antibody identical to the antibody obtained by immunizing the animal. Alternatively, the gene encoding the variable region (including the heavy chain variable region and the light chain variable region) of an antibody obtained by immunizing an animal can be cloned together with the gene encoding the constant region of an antibody from another species (e.g., human) into an expression vector for expression, thereby obtaining an antibody comprising heavy chain and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody having mouse heavy chain and light chain variable regions linked to human constant regions. This type of antibody is commonly referred to in the art as a "chimeric antibody."

[0034] As used herein, the term "antigen-binding fragment" refers to a fragment of an antibody capable of binding an antigen, and antibody analogues, which typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. The antigen-binding fragment retains at least some of the binding activity of the parent antibody. Typically, when activity is expressed on a molar basis, the antigen-binding fragment retains at least 10% of the parent antibody's binding activity. Specifically, the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity to the target. Examples of antigen-binding fragments include, but are not limited to: Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. A "Fab fragment" consists of a light chain, a CH1 region of a heavy chain, and a variable region. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule. The “Fab’ fragment” contains a portion of one light chain and one heavy chain (including the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains); thus, interchain disulfide bonds can be formed between the two heavy chains of two Fab’ fragments to form the F(ab’)2 molecule. The “Fv region” contains variable regions from both the heavy and light chains, but lacks constant regions.

[0035] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets.

[0036] In this invention, PCR amplification of the nucleotide sequence encoding the antibody was also performed using primer pairs. In the primer sequences, some sites involve only a single base, such as any one of adenine (A), guanine (G), cytosine (C), and thymine (T), while other sites involve combinations of two, three, or four bases. In these cases, these bases are called degenerate bases, primarily determined based on the degeneracy of the codon. Degenerate bases can be represented by the letters R, Y, M, K, S, W, H, B, V, D, and N, where R represents A / G, Y represents C / T, M represents A / C, K represents G / T, S represents C / G, W represents A / T, H represents A / T / C, B represents G / T / C, V represents G / A / C, D represents G / A / T, and N represents A / T / C / G.

[0037] As previously stated, the present invention aims to provide an anti-streptavidin monoclonal antibody.

[0038] Therefore, in a first aspect of the invention, an anti-streptavidin monoclonal antibody or an antigen-binding fragment thereof is provided, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising a heavy chain complementarity-determining region V. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementarity determination region V. L CDR1, V L CDR2 and V L CDR3; where:

[0039] 1) The heavy chain variable region includes an amino acid sequence defined by the heavy chain complementarity-determining region V shown in SEQ ID NO:1-3. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:4-6. L CDR1, V L CDR2 and V L CDR3;

[0040] 2) The heavy chain variable region includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO:7-9. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:10-12. L CDR1, VL CDR2 and V L CDR3; or

[0041] 3) The heavy chain variable region includes an amino acid sequence defined by the heavy chain complementarity-determining region V shown in SEQ ID NO:13-15. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO:16-18. L CDR1, V L CDR2 and V L CDR3.

[0042] In one specific embodiment, the antibody is a complete antibody comprising a variable region and a constant region. For the antibodies of the present invention, any frame region (FR) and any constant region can be used. The amino acid sequence of the FR or constant region used in the antibodies of the present invention can be the original amino acid sequence of the source FR or constant region, or it can be a different amino acid sequence obtained by substituting, deleting, adding, and / or inserting one or more amino acids into the original FR or constant region. The structure used to support the CDR or CDR group of the present invention generally belongs to the antibody heavy chain or light chain sequence or its major portion, wherein the CDR or CDR group is located in relation to the naturally occurring V region encoded by the rearranged immunoglobulin gene. H and V L At the corresponding position of the CDR or CDR group of the antibody variable domain.

[0043] In one specific implementation, the heavy chain variable region further includes heavy chain frame regions HFR1, HFR2, HFR3, and HFR4, which are related to V H CDR1, V H CDR2 and V H CDR3 progresses from the amino terminus to the carboxyl terminus according to HFR1, V H CDR1, HFR2, V H CDR2, HFR3, V H The order of CDR3 and HFR4.

[0044] In a further specific embodiment, the heavy chain framework regions HFR1, HFR2, HFR3, and HFR4 each have a sequence represented by SEQ ID NO:19-22 or a sequence having a similarity of 80% or more, 85% or more, 90% or more, or even 99% or more, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more, wherein the sequences of HFR1, HFR2, HFR3, and HFR4 are as follows:

[0045] SEQ ID NO:19(HFR1):QVQLQQPGSELVKPGASVKLSCKAS

[0046] SEQ ID NO:20(HFR2):WMHWVKQRPGQGLEWIGEI

[0047] SEQ ID NO:21(HFR3):TKYNEKFKTKATLTVDKSSSTAYMQLSSLTSDDSAVYYCAR

[0048] SEQ ID NO:22(HFR4):WGQGTTLTVSS

[0049] In yet another specific implementation, the light chain variable region further includes light chain framework regions LFR1, LFR2, LFR3, and LFR4, which are related to V L CDR1, V L CDR2 and V L CDR3 progresses from the amino terminus to the carboxyl terminus according to LFR1, V L CDR1, LFR2, V L CDR2, LFR3, V L The order of CDR3 and LFR4.

[0050] In a further specific embodiment, the light chain framework regions LFR1, LFR2, LFR3, and LFR4 each have a sequence represented by SEQ ID NO:23-26 or a sequence having a similarity of 80% or more, 85% or more, 90% or more, or even 99% or more, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more, wherein the sequences of LFR1, LFR2, LFR3, and LFR4 are as follows:

[0051] SEQ ID NO:23(LFR1):DIVLTQSPASLAVSLGQRATMSC

[0052] SEQ ID NO:24(LFR2):WYQQKPGQPPKLLIY

[0053] SEQ ID NO:25(LFR3):GVLDRFSGSGSGTDFTLKISRVEAE DLGVYFC

[0054] SEQ ID NO:26(LFR4):FGGGTKLEIK

[0055] In one specific implementation, the antibody further includes a constant region sequence, such as, but not limited to, a constant region sequence selected from any one of IgG, IgA, IgM, IgE and IgD, which can be selected by those skilled in the art as needed, and there is no particular limitation herein.

[0056] In yet another specific implementation, the species source of the constant region sequence can be rat, mouse, rabbit, goat, sheep, horse, dog, cow, pig, chicken, duck, goose or human, but is not limited thereto.

[0057] In a second aspect, the present invention provides a nucleic acid molecule encoding an anti-streptavidin monoclonal antibody or an antigen-binding fragment thereof of the first aspect of the present invention.

[0058] For those skilled in the art, knowing the amino acid sequence of a protein, such as the anti-streptavidin monoclonal antibody of the present invention, determining its nucleic acid coding sequence is entirely within their capabilities. Furthermore, to obtain a monoclonal antibody via recombination, the nucleic acid molecule can be cloned into a vector, and the vector can be further introduced into expression cells to express the antibody protein.

[0059] In a third aspect, the present invention provides a carrier comprising the nucleic acid molecule of the second aspect of the present invention.

[0060] In a preferred embodiment, the vector may be a plasmid vector, such as pEE12, pCAGGS, pTOPO, pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, and pBJ, but is not limited thereto.

[0061] In one specific implementation, the vector is a pTOPO vector.

[0062] In a fourth aspect, the present invention provides an expression cell comprising the nucleic acid molecule of the second aspect of the present invention or the vector of the third aspect of the present invention.

[0063] The expressed cells are prepared by introducing the aforementioned nucleic acid molecules or the aforementioned vectors into host cells using molecular biology methods well known to those skilled in the art.

[0064] As previously described, the inventors immunized mice with a recombinant streptavidin immunogen, fused mouse spleen cells with myeloma cells, and screened hybridoma cell lines capable of specifically binding streptavidin using ELISA. After screening for monoclonal cell lines secreting the target antibody, the heavy and light chain variable region cDNAs were recovered from the cell lines via reverse transcription-PCR, and suitable immunoglobulin constant regions were selected. The heavy and light chain variable region cDNAs and constant region cDNAs were then transferred into host cells such as COS or CHO cells, thereby obtaining expression cells expressing the target antibody of the present invention. Using the above-described monoclonal antibody and recombinant DNA technology, other antibodies or chimeric molecules that retain the specificity of the original antibody can be generated. These techniques may include introducing DNA encoding the variable region or complementarity-determining region (CDR) of the antibody immunoglobulin into a eukaryotic expression vector comprising the constant region or constant region plus a framework region of different immunoglobulins, or both into a suitable eukaryotic expression vector, and then introducing the eukaryotic expression vector into expression cells such as CHO host cells, thereby obtaining various recombinant anti-streptavidin antibodies.

[0065] In one specific implementation, the expressing cells can be mammalian cells, such as Chinese hamster ovary cells, hamster kidney cells, monkey kidney cells, mouse thymoma cells, and human embryonic kidney cells. In a more specific embodiment, the expressing cells may be, for example, monkey kidney cells transformed with SV40 (COS-7, ATCC CRL1651), human embryonic kidney cells (HEK293 or subcloned HEK293 cells for growth in suspension culture, Graham et al., 1977, J. Gen Virol. 36: 59), juvenile hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells / -DHFR1 (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77: 4216; e.g., DG44), mouse thymoma cells (NSO), mouse testicular supporting cells (TM4, Mather, 1980, Biol. Reprod. 23: 243-251), monkey kidney cells (CV-1, ATCC CCL70), and African green monkey kidney cells (VERO-76, ATCC). CRL-1587, human cervical cancer cells (HELA, ATCCCCL2), canine kidney cells (MDCK, ATCCCCCL34), buffalo rat hepatocytes (BRL3A, ATCCCCRL1442), human lung cells (W138, ATCCCCCL75), human hepatocytes (HepG2, HB8065), mouse mammary tumors (MMT060562, ATCCCCCL51), TR1 cells (Mather et al., 1982, Annals NYAcad. Sci. 383:44-68), MRC5 cells, FS4 cells, etc., but not limited to these.

[0066] In a fifth aspect, the present invention provides a method for sorting target cells from a cell population, comprising the step of using an anti-streptavidin monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention.

[0067] As mentioned earlier, since the affinity of anti-streptavidin antibody to streptavidin is lower than that of streptavidin to biotin, biotin can be used to easily separate the sorted target cells from the sorting medium such as magnetic beads, which facilitates the subsequent culture and application of the target cells.

[0068] In one specific implementation, the method includes the following steps:

[0069] (1) The anti-streptavidin monoclonal antibody or its antigen-binding fragment is made to form an anti-streptavidin monoclonal antibody or its antigen-binding fragment coupled to the sorting medium;

[0070] (2) Form a streptavidin-coupled sorting antibody with a sorting antibody, wherein the sorting antibody specifically binds to the target antigen expressed in the target cell;

[0071] (3) The cell population, the anti-streptavidin monoclonal antibody or its antigen-binding fragment conjugated with the sorting medium, and the sorting antibody conjugated with streptavidin are co-incubated to form a first complex;

[0072] (4) Separate the first complex from other components in the cell population using the sorting medium in the first complex;

[0073] (5) The separated first complex is co-incubated with biotin, wherein the affinity between biotin and streptavidin is greater than the affinity between streptavidin and the anti-streptavidin monoclonal antibody or its antigen-binding fragment, thereby causing the anti-streptavidin monoclonal antibody or its antigen-binding fragment to dissociate from streptavidin, forming a second complex of biotin-streptomycin-sorting antibody-target cell, thereby obtaining the sorted target cells.

[0074] In one specific implementation, the sorting medium may be magnetic microparticles or microspheres.

[0075] In yet another specific embodiment, the first complex is a complex of sorting medium-anti-streptavidin monoclonal antibody or its antigen-binding fragment-streptavidin-sorting antibody.

[0076] In a sixth aspect, the present invention provides a method for detecting an antigen to be tested using colloidal gold chromatography, comprising the step of using an anti-streptavidin monoclonal antibody or its antigen-binding fragment from the first aspect of the present invention as a quality control system.

[0077] In one specific embodiment, the anti-streptavidin antibody or its antigen-binding fragment of the present invention can be used in combination with the streptavidin-biotin system, thereby improving the sensitivity of detection by utilizing the high affinity between biotin and streptavidin.

[0078] In a further specific embodiment, the colloidal gold chromatography method includes the following steps:

[0079] (1) A colloidal gold test strip is provided, the colloidal gold test strip comprising: a gold-labeled pad, a biotin pad, a nitrocellulose membrane and a sample pad, wherein the gold-labeled pad is provided with colloidal gold-labeled streptavidin, the biotin pad is provided with a first antibody conjugated with biotin and targeting the antigen to be tested, and the nitrocellulose membrane is provided with a detection line and a control line, wherein the detection line is formed by a second antibody targeting the antigen to be tested that is the same as or different from the first antibody, and the control line is formed by the anti-streptavidin monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2;

[0080] (2) After the sample to be tested is dropped onto the sample pad of the colloidal gold test strip and allowed to react for a period of time, the presence of the antigen to be tested in the sample is determined by whether the detection line shows color.

[0081] In a preferred embodiment, the first antibody is different from the second antibody.

[0082] It is understood that, in order to successfully implement the method of the present invention, the amount of streptavidin on the colloidal gold test strip needs to be much higher than the amount of biotin, so that the free colloidal gold-labeled streptavidin (i.e. streptavidin that is not bound to biotin) can migrate to the control line and react with the anti-streptavidin antibody there.

[0083] Understandably, when the test sample contains the antigen to be tested, as the sample migrates on the test strip, a complex of colloidal gold-streptavidin-biotin-primary antibody-antigen is first formed. This complex further forms at the test line, resulting in color development of the test line. The free colloidal gold-labeled streptavidin then migrates to the control line, where it forms a complex of colloidal gold-streptavidin-anti-streptavidin antibody, also resulting in color development of the control line. Conversely, when the test sample does not contain the antigen to be tested, no colorimetric complex forms at the test line. However, the free colloidal gold-labeled streptavidin continues to migrate to the control line, forming the same complex, resulting in color development of the control line. It should be understood that if the control line does not develop color, the colloidal gold test strip is invalid and cannot be used for testing.

[0084] Traditional colloidal gold sandwich methods detect the antigen by forming a second antibody complex at the T-line of colloidal gold labeled with a first antibody. In contrast, the colloidal gold chromatography method of the present invention has stronger adaptability and improved detection sensitivity because the anti-streptavidin antibody, which serves as the quality control system, can be adapted to a variety of detection antibodies to detect different antigens.

[0085] In a seventh aspect, the present invention provides the use of the anti-streptavidin monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention in immunoassay.

[0086] In one specific embodiment, the anti-streptavidin monoclonal antibody of the present invention can specifically bind to streptavidin. Therefore, when bound to enzyme-labeled streptavidin, it can be used for immunohistochemistry (IHC), immunocytochemistry (ICC), in situ hybridization (ISH), enzyme-linked immunosorbent assay (ELISA), immunochromatography (ICA), and Western blotting (WB), but is not limited thereto. When bound to fluorescently labeled streptavidin, it can be used for immunofluorescence (IF), in situ hybridization (ISH), and flow cytometry (FCM), but is not limited thereto. When bound to immobilized streptavidin, it can also be used for protein purification and nucleic acid extraction, but is not limited thereto.

[0087] Example

[0088] The following examples illustrate the preparation method, characterization, and related applications of the antibodies of the present invention. Unless otherwise specified, all experimental methods used are conventional methods, and all experimental materials used in the following examples were purchased from conventional reagent stores. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0089] It should be noted that the terminology used in this specification is for the purpose of describing specific embodiments only and is not intended to limit the invention. The foregoing summary section and the following detailed description are for illustrative purposes only and are not intended to limit the invention in any way. The scope of the invention is defined by the appended claims without departing from its spirit and intent.

[0090] Example 1: Preparation of anti-streptavidin monoclonal antibody

[0091] Antigen conjugation and immunization: Purified recombinant streptavidin protein (SA, Heavy Chain Biotechnology, catalog number HP155-2) was used as an immunogen to immunize mice. Mice were immunized four times, with each immunization spaced two weeks apart, at a dose of 100 μg per mouse. Seven days after the fourth immunization, blood was collected from the tails of the mice, and serum was separated. The antibody titer of the antiserum from the immunized mice was detected using an indirect ELISA method to observe the immune response. Mice with serum antibody titers higher than 1:10000 were selected for cell fusion experiments.

[0092] Establishment of hybridoma cells: On the day of fusion, the spleen of immunized mice was removed and a single-cell suspension was prepared. Mouse myeloma cells (SP2 / 0) were fused with the above mouse spleen cells at a ratio of 1:5, and 200 μL / well was seeded into a 96-well culture plate and cultured at 37°C. When the fused cells grew to 1 / 10-1 / 5 of the bottom area of ​​the wells of the 96-well plate, the supernatant was collected for antibody detection.

[0093] Screening for positive hybridoma cells: SA protein was diluted to 1 μg / mL and added to 100 μL / well of a 96-well plate. The plate was incubated overnight at 4°C. The cells were washed three times with phosphate-buffered saline (PBST), patted dry, and then washed with PBST containing 2% BSA. Block at 37℃ for 2 h, wash 3 times, and blot dry. Add 100 μL / well of fusion cell supernatant, 1:1000 diluted positive serum from immunized mice (as positive control), and 1:1000 diluted negative serum from mice (as negative control) to the corresponding wells, incubate at 37℃ for 1 h, wash 3 times, and blot dry. Add 100 μL / well of 1:4000 diluted horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (purchased from Sigma), incubate at 37℃ for 1 h, wash 3 times, and blot dry. Add 100 μL / well of tetramethylbenzidine (TMB) substrate, incubate at room temperature in the dark for 10 min, and then terminate the reaction.

[0094] The OD values ​​of all wells in the ELISA plate were measured at a wavelength of 450 nm using an ELISA reader. 450nm Value. When the OD of negative serum 450nm ≤0.1, measured by the absorbance OD value of the well (NS1). 450nm The value is the negative pore OD. 450nm A positive result was defined as a value 2.1 times or higher than the threshold for cloning. Positive hybridoma cells were then selected for further cloning.

[0095] Cloning of positive cell lines: Positive cell lines secreting antibodies were sampled and counted from wells, then diluted to 100 cells / 10 mL of culture medium. The diluted cell suspension was seeded at 100 μL / well into 96-well cell culture plates and incubated at 37°C in a 5% CO2 incubator. After 6-7 days, clonal cell formation was observed under a microscope. Individual clonal growth wells were marked, and the cell supernatant was collected for ELISA detection (same as the fusion detection described above). Positive monoclonal cells were selected. Limiting dilutions were performed on cells from positive wells, with ELISA values ​​measured 5-6 days after each limiting dilution. The OD values ​​obtained from the ELISA results were then collected. 450nmSingle-clone wells with high positive values ​​were limited dilutions until the entire 96-well plate was positive for ELISA. Single-clone strains with high positive values ​​were selected for consolidation. Three stable cell lines secreting anti-SA protein antibodies were obtained. Total RNA was isolated, and cDNA was prepared by reverse transcription to clone immunoglobulin sequences from the hybridoma cell lines. The variable region sequences of the antibodies from these hybridoma cell lines were then determined. The complementarity-determining regions (CDGs) of the heavy and light chains are shown in Table 1 below (based on the Chothia numbering system).

[0096] Table 1: Complementarity Determinant Region Sequences of Heavy and Light Chains

[0097]

[0098] Example 2: Preparation and purification of recombinant antibodies

[0099] The recombinant antibody was constructed, and a cell line that stably expressed the antibody was prepared through eukaryotic expression. The cell line was then cultured and purified on a large scale. The specific steps are as follows.

[0100] For antibody V L and V H Genes were obtained by sequentially ligating the CDR sequences (as shown in Table 1 above) with the heavy chain framework regions (HFR) sequences (SEQ ID NO:19-SEQ ID NO:22) and the light chain framework regions (LFR) sequences (SEQ ID NO:23-SEQ ID NO:26) to obtain the heavy chain and light chain variable regions, respectively. Subsequently, a recombinant antibody eukaryotic expression vector was constructed using molecular cloning methods. The eukaryotic expression plasmid was electroporated into CHO host cells. After electroporation, the cells were cultured in pressure selection medium (50 μM MSX) for 20 days. The supernatant was then used for ELISA detection (using horseradish peroxidase (HRP)-labeled goat anti-mouse IgG as a secondary antibody for screening, using the same method as above) to select cell lines stably expressing the recombinant antibody.

[0101] The selected stable cell lines were cultured on a large scale using roller flask culture technology for recombinant antibody preparation. Cells were cultured in Vega CHO medium at a concentration of (0.2-0.3) × 10⁻⁶ cells / mL. 6 Cells / mL were seeded in roller flasks containing 300 mL of culture medium and cultured in a cell culture incubator. After 7-9 days of culture, samples were taken and observed under a microscope. When the cell viability was less than 50%, the samples were centrifuged and collected. The samples were purified by affinity chromatography using a protein A column to obtain the antibodies, which are the recombinant monoclonal antibodies S1', S2', and S3'.

[0102] SA protein was diluted with 0.05 mol / L carbonate buffer (pH 9.6) to a concentration of 1 μg / mL. 100 μL was added to each well of a 96-well microplate and incubated overnight at 4°C. The plate was washed three times and then blotted dry. 2% BSA was blocked in PBST at 37°C for 2 hours, followed by washing three times and blotting dry. Monoclonal antibodies against SA protein (S1', S2', and S3') were serially diluted with 0.02 M PBS buffer (pH 7.4) at a starting concentration of 5 μg / mL, with each subsequent 3-fold serial dilution yielding a series of monoclonal antibody samples at different concentrations. 100 μL of each diluted monoclonal antibody sample was added to each well of the microplate and incubated at 37°C for 1 hour, followed by washing three times and blotting dry. 100 μL of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (purchased from Sigma) diluted 1:4000 was added to each well and incubated at 37°C for 1 hour, followed by washing three times and blotting dry. Add TMB substrate, incubate at room temperature in the dark for 10 minutes, then terminate the reaction. OD is measured using a microplate reader. 450nm The affinity EC values ​​of monoclonal antibodies S1', S2', and S3' were obtained by analyzing the ELISA results using software. 50 The value (nM) is shown in Table 2 below.

[0103] Table 2: EC2 binding of recombinant monoclonal antibodies to SA protein 50 value

[0104] SA protein Recombinant antibody S1 ' 4.21 x 10 -10 M]] Recombinant antibody S2' 3.98 x 10 -10 M <!-- 10 -->]]> Recombinant antibody S3' 4.46 x 10 -10 M]]

[0105] Example 3: Application of cell sorting

[0106] This embodiment illustrates a method for sorting positive cells from a cell population using the anti-SA antibody of the present invention, and decoupling the positive cells from magnetic beads to facilitate subsequent culture and application.

[0107] Coating magnetic beads with anti-SA antibodies (S1', S2', or S3') (Dynabeads M-450 Tosylactivated): According to the instructions, conjugate 200 μg of antibody with 1 mL of magnetic beads, and resuspend the conjugated magnetic beads in 1 mL of buffer for later use.

[0108] The sorting antibody CD3 was conjugated with streptavidin SA: streptavidin was activated with N-succinimide maleic acid and reacted at room temperature for 60 minutes. Then, CD3 antibody reduced by DTT was added and reacted with activated SA at a ratio of 5:1. The reaction was stopped at room temperature for 30 minutes and then purified by affinity chromatography.

[0109] Magnetic bead sorting of cells:

[0110] 5×10 6Cells were resuspended in 500 μL buffer, and 2 μg of SA-sorting antibody (CD3 antibody) was added. After incubation and washing, 40 μL of SA antibody-coated magnetic beads were added. After incubation for 15 minutes, the tubes containing cells were placed on a magnetic rack for 2 minutes, and the supernatant was carefully collected (CD3-negative cell group). The tubes were then removed, and 2 μg / mL biotin was added and incubated for 5 minutes. The tubes were again placed on a magnetic rack for 2 minutes, and the supernatant was carefully collected (CD3-positive cell group). Both groups of cells were resuspended in buffer and flow cytometry was performed. The results for using S1', S2', and S3' coated magnetic beads are shown below. Figure 1 , Figure 2 and Figure 3 As shown in the figure, after sorting, the proportion of CD3 Jurkat cells in the cell population increased significantly, from about 45% to about 93%.

[0111] Example 4: Application as a quality control system in immunochromatography

[0112] In this embodiment, an exemplary application of the anti-streptavidin antibody of the present invention as a quality control system (C-line) in the detection of the novel coronavirus is illustrated.

[0113] Preparation of colloidal gold: Add 200 mL of ultrapure water to an Erlenmeyer flask, heat to boiling, add 1 mL of 2% chloroauric acid (Sigma-Aldrich, catalog number: 16961-25-4) solution, and immediately after boiling, add 1 mL of 2% trisodium citrate (Sigma-Aldrich, catalog number: 6132-04-3) aqueous solution, continue stirring and boiling for 10 minutes, and let cool naturally for later use.

[0114] Labeling colloidal gold conjugate: Take 10 mL of the above colloidal gold and put it into a beaker. Add 140 μL of 0.2 Mk2CO3 while stirring to adjust the pH to 7.4, and continue stirring for 10 seconds. Add 200 μg of streptavidin and continue stirring for 5 minutes. Add 0.1 mL of 10% BSA and continue stirring for 5 minutes. Centrifuge at 12000g for 10 minutes, discard the supernatant, and dilute the precipitate to 1 mL with colloidal gold dilution buffer (10 mM PB, 150 mM NaCl, 0.2% BSA, 0.1% Triton X-100, 3% Sucrose, 0.01% Proclin 300) to obtain the streptavidin-colloidal gold complex.

[0115] Preparation of colloidal gold pads: The above colloidal gold complex was diluted 10 times with colloidal gold diluent and then soaked in glass fiber (Shanghai Gold Standard Co., Ltd.), followed by freeze-drying to prepare gold-labeled pads. Biotin-conjugated mouse anti-2019-nCOV NP monoclonal antibody (Heavy Chain Biotech HA811-41MB) was diluted to 100-200 ng / mL to prepare biotin pads.

[0116] Nitrocellulose membrane (NC membrane) coating: Dilute anti-streptavidin antibody (S1', S2', or S3') to 1 mg / mL to prepare the control line working solution. Dilute mouse anti-2019-nCOV NP monoclonal antibody (Heavy Chain Biotechnology HA811-22M) to 1 mg / mL to prepare the test line working solution. Draw lines onto the corresponding positions on the nitrocellulose membrane (Millipore, catalog number: HF135002) using a dotting instrument to serve as the C line (control line) and T line (test line), respectively. Dry at 50°C for 1 hour for later use.

[0117] Assembly of colloidal gold immunochromatographic assay strip: Assemble the above-mentioned gold label pad, biotin pad, antibody-coated nitrocellulose membrane, absorbent paper, polyester plate, and sample pad into a colloidal gold immunochromatographic assay strip.

[0118] Sensitivity testing: Different concentrations of antigen control samples were tested. Specifically, the recombinant 2019-nCoV NP antigen control sample was diluted several times, and 80 μL was added to the sample pad and incubated at room temperature for 5-15 minutes before determining the results. The intensity of the displayed band color indicates the activity of antigen-antibody binding in the sample, as shown in Table 3 below.

[0119] Table 3: Immunochromatographic test results of colloidal gold in the recombinant antibody combination of the present invention

[0120]

[0121] Furthermore, the detection results using the anti-streptavidin antibody of the present invention as the quality control system (C line) were compared with the detection results using the conventional sandwich method (a detection method in which colloidal gold labeled with a first antibody forms a sandwich between the detection line and a second antibody), as shown in Table 4 below.

[0122] Table 4: Comparison between the colloidal gold sandwich method of the present invention and the traditional sandwich method

[0123] Dilution gradient (ng / mL) 10 5 1 0.5 0.1 0.05 0.01 0.005 SA-colloidal gold sandwich C1 C1 C3 C4 C5+ C6 C7+ C7 Traditional sandwich C1 C1 C4+ C4 C5 C6+ C7 C8

[0124] As shown in Table 4, if C7 is used as the positive standard, the colloidal gold sandwich method of the anti-streptavidin antibody of the present invention increases the detection limit of wild-type NP against 2019-nCOV from 10 pg / mL to 5 pg / mL.

Claims

1. An anti-streptavidin monoclonal antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising a heavy chain complementarity-determining region V. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementarity determination region V. L CDR1, V L CDR2 and V L CDR3; where: 1) The heavy chain variable region includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO: 1-3. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO: 4-6. L CDR1, V L CDR2 and V L CDR3; 2) The heavy chain variable region includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO: 7-9. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO: 10-12. L CDR1, V L CDR2 and V L CDR3; or 3) The heavy chain variable region includes an amino acid sequence defined by the heavy chain complementarity-determining region V as shown in SEQ ID NO: 13-15. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes an amino acid sequence defined by the light chain complementarity-determining region V as shown in SEQ ID NO: 16-18. L CDR1, V L CDR2 and V L CDR3.

2. The anti-streptavidin monoclonal antibody or its antigen-binding fragment according to claim 1, wherein, The heavy chain variable region further includes frame regions HFR1, HFR2, HFR3, and HFR4, which are related to V H CDR1, V H CDR2 and V H CDR3 progresses from the amino terminus to the carboxyl terminus according to HFR1, V H CDR1, HFR2, V H CDR2, HFR3, V H The sequence of CDR3 and HFR4, wherein HFR1, HFR2, HFR3, and HFR4 each have a sequence that is more than 80% identical to the sequence shown in SEQ ID NO: 19-22, and the light chain variable region further includes frame regions LFR1, LFR2, LFR3, and LFR4, which are related to V L CDR1, V L CDR2 and V L CDR3 progresses from the amino terminus to the carboxyl terminus according to LFR1, V L CDR1, LFR2, V L CDR2, LFR3, V L The sequence of CDR3 and LFR4, wherein LFR1, LFR2, LFR3 and LFR4 each have a sequence that is more than 80% identical to the sequence shown by SEQ ID NO: 23-26.

3. The anti-streptavidin monoclonal antibody or its antigen-binding fragment according to claim 2, wherein, HFR1, HFR2, HFR3 and HFR4 each have a sequence that is more than 85% identical to the sequence shown by SEQ ID NO: 19-22.

4. The anti-streptavidin monoclonal antibody or its antigen-binding fragment according to claim 3, wherein, HFR1, HFR2, HFR3 and HFR4 each have a sequence that is more than 90% identical to the sequence shown by SEQ ID NO: 19-22.

5. The anti-streptavidin monoclonal antibody or its antigen-binding fragment according to claim 4, wherein, HFR1, HFR2, HFR3 and HFR4 each have a sequence that is more than 95% identical to the sequence shown by SEQ ID NO: 19-22.

6. The anti-streptavidin monoclonal antibody or its antigen-binding fragment according to claim 5, wherein, HFR1, HFR2, HFR3 and HFR4 each have the sequence shown in SEQ ID NO: 19-22.

7. The anti-streptavidin monoclonal antibody or its antigen-binding fragment according to any one of claims 2-6, wherein, The LFR1, LFR2, LFR3 and LFR4 each have a sequence that is more than 85% identical to the sequence shown by SEQ ID NO: 23-26.

8. The anti-streptavidin monoclonal antibody or its antigen-binding fragment according to claim 7, wherein, The LFR1, LFR2, LFR3 and LFR4 each have a sequence that is more than 90% identical to the sequence shown by SEQ ID NO: 23-26.

9. The anti-streptavidin monoclonal antibody or its antigen-binding fragment according to claim 8, wherein, The LFR1, LFR2, LFR3 and LFR4 each have a sequence that is more than 95% identical to the sequence shown by SEQ ID NO: 23-26.

10. The anti-streptavidin monoclonal antibody or its antigen-binding fragment according to claim 9, wherein, The LFR1, LFR2, LFR3 and LFR4 each have the sequence shown in SEQ ID NO: 23-26.

11. A nucleic acid molecule encoding an anti-streptavidin monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1-10.

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

13. The carrier according to claim 12, wherein, The vector is a plasmid vector.

14. The carrier according to claim 13, wherein, The plasmid vector is any one of pEE12, pCAGGS, pTOPO, pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, and pBJ.

15. The carrier according to claim 14, wherein, The pcDNA is pcDNA3.

1.

16. An expression cell comprising the nucleic acid molecule of claim 11 or the vector of any one of claims 12-15.

17. The expression cell according to claim 16, wherein, The expressing cells are mammalian cells.

18. The expression cell according to claim 17, wherein, The mammalian cells were selected from Chinese hamster ovary cells, hamster kidney cells, monkey kidney cells, mouse thymoma cells, and human embryonic kidney cells.

19. A method for sorting target cells from a cell population, comprising the step of using an anti-streptavidin monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1-10.

20. The method according to claim 19, wherein, The method includes the following steps: The anti-streptavidin monoclonal antibody or its antigen-binding fragment is made to form an anti-streptavidin monoclonal antibody or its antigen-binding fragment coupled to the sorting medium; The sorting antibody is reacted with streptavidin to form a streptavidin-conjugated sorting antibody, wherein the sorting antibody specifically binds to the target antigen expressed in the target cell; The cell population, the anti-streptavidin monoclonal antibody or its antigen-binding fragment conjugated with the sorting medium, and the sorting antibody conjugated with streptavidin are co-incubated to form a first complex. The first complex is separated from other components in the cell population using a sorting medium in the first complex. The separated first complex is co-incubated with biotin, wherein the affinity between biotin and streptavidin is greater than the affinity between streptavidin and the anti-streptavidin monoclonal antibody or its antigen-binding fragment, thereby causing the anti-streptavidin monoclonal antibody or its antigen-binding fragment to dissociate from streptavidin, forming a second complex of biotin-streptomycin-conjugated sorting antibody-target cell, thereby obtaining sorted target cells.

21. The method according to claim 20, wherein, The sorting medium is magnetic microparticles.

22. A method for detecting an antigen to be tested using colloidal gold chromatography, comprising the step of using an anti-streptavidin monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-10 as a quality control system, wherein the method is not intended for diagnostic purposes.

23. The method according to claim 22, wherein, The method includes the following steps: A colloidal gold test strip is provided, comprising: a gold-labeled pad, a biotin pad, a nitrocellulose membrane, and a sample pad, wherein the gold-labeled pad is coated with colloidal gold-labeled streptavidin, the biotin pad is coated with a first antibody conjugated to biotin and targeting the antigen to be tested, and the nitrocellulose membrane is coated with a detection line and a control line, wherein the detection line is formed by a second antibody targeting the antigen to be tested, which may be the same as or different from the first antibody, and the control line is formed by an anti-streptavidin monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-10; The sample to be tested is dropped onto the sample pad of the colloidal gold test strip and allowed to react for a period of time. The presence of the antigen to be tested in the sample is determined by whether the detection line develops color.

24. Use of the anti-streptavidin monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-10 in the preparation of a kit for immunoassay.

25. The use according to claim 24, wherein, The immunoassays include immunohistochemistry (IHC), immunocytochemistry (ICC), enzyme-linked immunosorbent assay (ELISA), immunochromatography (ICA), Western blotting (WB), immunofluorescence (IF), in situ hybridization (ISH), or flow cytometry (FCM).

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