Anti-streptavidin antibodies and related uses thereof

Abstract

The invention provides an anti-streptavidin antibody which is high in specificity, high in sensitivity, less in cross reaction and small in batch difference. The antibody not only can 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 invention relates to the field of immune detection, and in particular to an anti-streptavidin monoclonal antibody or an antigen binding fragment thereof and related applications thereof. Background Art

[0002] Streptavidin (SA) is a secretory protein extracted from the culture of Streptomyces Avidinii. It is composed of four identical peptide chains with a molecular weight of about 65kDa. Due to its high specificity binding to biotin, each streptavidin molecule can bind to four biotin molecules and can bind to a variety of markers. Therefore, signal amplification technology based on streptavidin is widely used to detect biotinylated biomolecules, such as biotin-labeled primary antibodies, secondary antibodies, ligands, toxins, magnetic beads or DNA probes 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 antigens and have high affinity, but sometimes they will produce non-specific recognition, the batch-to-batch differences are relatively large, and the system stability is insufficient.

[0004] Therefore, there is a need in the art for a monoclonal antibody with high specificity, high sensitivity and low cross-reactivity against streptavidin. Summary of the invention

[0005] The inventors immunized mice with recombinant streptavidin protein, took spleen cells from mice and fused them with myeloma cells, screened hybridoma cell lines that specifically bind to streptavidin by ELISA method, constructed recombinant antibodies, and obtained recombinant anti-streptavidin antibodies. Thus, the present invention was completed.

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

[0007] 1) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 1-3, H CDR1, V H CDR2 and V HCDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 4-6 respectively L CDR1, V L CDR2 and V L CDR3;

[0008] 2) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 7-9, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 10-12 respectively. L CDR1, V L CDR2 and V L CDR3; or

[0009] 3) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 13-15, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 16-18, respectively. L CDR1, V L CDR2 and V L CDR3.

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

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

[0012] 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.

[0013] In a fifth aspect, the present invention 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 according to the first aspect of the present invention.

[0014] In a sixth aspect, the present invention provides a method for detecting an antigen to be detected using 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 invention as a quality control system.

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

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

[0017] (1) The anti-streptavidin antibody of the present invention can specifically bind to streptavidin, thereby realizing similar applications of the streptavidin-biotin system, 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) related detection.

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

[0019] (3) The anti-streptavidin antibody of the present invention can be used in combination with a streptavidin-biotin system, such as as a further reagent, to amplify weak or low signals, etc. For example, the anti-streptavidin antibody of the present invention can be used in immunochromatography such as colloidal gold chromatography as a quality control system to establish a detection method based on biotin-streptavidin binding. Compared with the detection method that relies on the formation of a sandwich of colloidal gold labeled with the first antibody-antigen to be detected-second antibody at the detection line, the sensitivity of the related detection using the anti-streptavidin antibody of the present invention is further improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the drawings required for use in the specific implementation or the description of the prior art are 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 DESCRIPTION

[0024] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. It should be understood that the following description is only used to illustrate the present invention by way of example, and is not intended to limit the scope of the present invention. The scope of protection of the present invention shall be subject to the attached claims. In addition, those skilled in the art will understand that the technical solution of the present invention may be modified without departing from the spirit and purpose of the present invention. 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 described herein belongs. Before describing the present invention in detail, the following definitions are provided for a better understanding of the present invention.

[0026] Where a numerical range is provided, such as a concentration range, a percentage range, or a ratio range, it is understood that each intervening value to one-tenth of the unit of the lower limit between the upper and lower limits of the range and any other stated or intervening values ​​in the stated range are included in the subject matter unless the context clearly dictates otherwise. 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, subject to any specifically excluded limit in the stated range. Where the stated range includes one or two limits, ranges excluding either or both of those included limits are also included in the subject matter.

[0027] In the context of the present invention, many embodiments use the expressions "comprising", "including" or "consisting essentially / mainly of..." The expressions "comprising", "including" or "consisting essentially / mainly of..." can usually be understood as open-ended expressions, indicating that it includes not only the elements, components, assemblies, method steps, etc. specifically listed after the expression, but also other elements, components, assemblies, method steps. In addition, in this article, the expressions "comprising", "including" or "consisting essentially / mainly of..." can also be understood as closed expressions in some cases, indicating that it only includes the elements, components, assemblies, method steps specifically listed after the expression, but does not include any other elements, components, assemblies, method steps. At this time, the expression is equivalent to the expression "consisting of..."

[0028] Unless otherwise specified, all steps mentioned in this application may be performed sequentially or randomly, unless the context requires them to be performed sequentially. For example, when it is stated that "the method includes steps (1) and (2)", it means that the method may be implemented by first performing step (1) and then step (2), or by first performing step (2) and then step (1), unless the context can determine that step (1) must be performed before step (2). For another example, when it is stated that "the method may also 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 that is typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and the isotype of the antibody can be defined accordingly as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region (hinge region) of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and the heavy chain constant region (C H ). The heavy chain constant region consists of three domains (C H1 , C H2 and C H3 Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ). The light chain constant region consists of a domain C L The constant region of the antibody can mediate the binding of the immunoglobulin 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. H and V L The variable regions can be further subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). For each heavy or light chain, its variable region each contains three CDRs, namely CDR1, CDR2 and CDR3. H and V L It consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V H and VL ) respectively form the antigen binding site.

[0030] The rules for assigning amino acids to various regions or domains are defined in many papers: 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 / 3Dstructure-DB and IMGT / DomainGapAlign: 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 exact boundaries of CDR have been defined differently according to different systems. The Kabat system not only provides a clear residue numbering system that can be applied to any variable region of an antibody, but also provides precise residue boundaries that define three CDRs, which are called Kabat CDRs; Chothia found that certain sub-portions within the Kabat system CDRs, despite having great diversity at the amino acid sequence level, have almost identical peptide main chain conformations, and these sub-portions are called Chothia CDRs, and Chothia CDRs have boundaries that overlap with Kabat CDRs. The above-mentioned overlapping boundaries are described by Padlan and MacCallum, and the CDR boundary definition may not strictly follow the above-mentioned system, such as the AbM definition. In this article, 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" or "monoclonal antibody" refers to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously. The antibody molecule can be an immunoglobulin, whether it is a natural immunoglobulin or an immunoglobulin obtained partially or completely by a synthetic method. The antibody molecule can also include all polypeptides or proteins with an antibody binding domain. Antibody fragments with antibody domains 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 are relative to monoclonal antibodies, and they usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen. Monoclonal antibodies can usually be obtained using the hybridoma technology 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 can also be obtained by recombinant DNA technology (see US Patent 4,816,567). As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and can be used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and can be used interchangeably; the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by Ala or A. Glycine can be represented by Gly or G, valine can be represented by Val or V, leucine can be represented by Leu or L, isoleucine can be represented by Ile or I, proline can be represented by Pro or P, phenylalanine can be represented by Phe or F, tyrosine can be represented by Tyr or Y, tryptophan can be represented by Trp or W, serine can be represented by Ser or S, threonine can be represented by Thr or T, cysteine ​​can be represented by Cys or C, methionine can be represented by Met or M, asparagine can be represented by Asn or N, glutamine can be represented by Gln or Q, aspartic acid can be represented by Asp or D, glutamic acid can be represented by Glu or E, lysine can be represented by Lys or K, arginine can be represented by Arg or R, and histidine can be represented by His or H.

[0033] As used herein, the term "recombinant antibody" means that the antibody gene is cloned into an expression vector by molecular biological techniques and then the expression vector is transfected into a suitable host cell line for expression. The recombinant antibody encoding gene may be identical or non-identical to the antibody encoding gene of natural origin. For example, the complete encoding gene of the 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 the immunizing animal, or the gene encoding the variable region (including heavy chain variable region and light chain variable region) of the antibody obtained by the immunizing animal can be cloned into an expression vector together with the gene encoding the constant region of the antibody from another species (such as human) source for expression, thereby obtaining an antibody including heavy chain and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody with mouse heavy chain and light chain variable regions connected to a human constant region. The antibody is generally referred to as a "chimeric antibody" in the art.

[0034] As used herein, the term "antigen binding fragment" refers to fragments and antibody analogs from antibodies that are capable of binding to antigens, which generally include at least part of the antigen binding region or variable region (e.g., one or more CDRs) of the parental antibody. The antigen binding fragment retains at least some of the binding activity of the parental antibody. Typically, when the activity is expressed on a molar basis, the antigen binding fragment retains at least 10% of the parental binding activity. Specifically, the antigen binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parental antibody 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. Among them, the "Fab fragment" consists of a light chain, a heavy chain CH1, and a variable region. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab' fragment" contains one light chain and part of one heavy chain (including the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains); thus, an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments to form a F(ab')2 molecule. An "Fv region" contains the variable regions from both the heavy and light chains, but lacks the constant region.

[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 its antigen.

[0036] In the present invention, primers are also used to carry out PCR amplification of the nucleotide sequence encoding the antibody. In the primer sequence, some sites only involve a single base, such as any one of adenine (A), guanine (G), cytosine (C) and thymine (T), while some sites involve a combination of two, three or four bases, in which case these bases are referred to as degenerate bases of each other, which are mainly determined based on the degeneracy of the codons. Degenerate bases can be represented by the letters R, Y, M, K, S, W, H, B, V, D, and N, wherein 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 mentioned above, the present invention aims to provide an anti-streptavidin monoclonal antibody.

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

[0039] 1) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 1-3, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 4-6 respectively L CDR1, V L CDR2 and V L CDR3;

[0040] 2) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 7-9, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 10-12 respectively. L CDR1, VL CDR2 and V L CDR3; or

[0041] 3) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 13-15, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 16-18, respectively. L CDR1, V L CDR2 and V L CDR3.

[0042] In a specific embodiment, the antibody is a complete antibody comprising a variable region and a constant region. For the antibodies of the present invention, any framework 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 amino acid sequence of the original FR or constant region as a source, or it can be a different amino acid sequence obtained by performing one or more amino acid substitutions, deletions, additions and / or insertions on the amino acid sequence of 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 a major part thereof, wherein the CDR or CDR group is located in the naturally occurring V sequence encoded by the rearranged immunoglobulin gene. H and V L The CDR or CDR group of the antibody variable domain corresponds to the position of the antibody variable domain.

[0043] In a specific embodiment, the heavy chain variable region further comprises heavy chain framework regions HFR1, HFR2, HFR3 and HFR4, H CDR1, V H CDR2 and V H CDR3 is arranged 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 is arranged.

[0044] In a further specific embodiment, the heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 respectively have the sequences shown in SEQ ID NOs: 19-22, or sequences having more than 80%, more than 85%, more than 90% or more than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity thereto, 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 embodiment, the light chain variable region further comprises light chain framework regions LFR1, LFR2, LFR3 and LFR4, which are closely related to the V L CDR1, V L CDR2 and V L CDR3 is arranged from amino terminus to carboxyl terminus according to LFR1, V L CDR1, LFR2, V L CDR2, LFR3, V L The sequence of CDR3 and LFR4 is arranged.

[0050] In a further specific embodiment, the light chain framework regions LFR1, LFR2, LFR3 and LFR4 respectively have the sequences shown in SEQ ID NOs: 23-26, or sequences having more than 80%, more than 85%, more than 90%, more than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity thereto, 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 a specific embodiment, 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. Those skilled in the art can make a selection based on needs, and this document does not specifically limit this.

[0056] In yet another specific embodiment, the constant region sequence may be from 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 the anti-streptavidin monoclonal antibody or antigen-binding fragment thereof according to 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, it is well within their capabilities to determine its nucleic acid coding sequence. In addition, in order to obtain a monoclonal antibody by recombinant means, the nucleic acid molecule can be cloned into a vector, and the vector can be further introduced into an expression cell to express the antibody protein using the expression cell.

[0059] In a third aspect, the present invention provides a vector 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 a specific embodiment, 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 expression cell is prepared by introducing the above-mentioned nucleic acid molecule or the above-mentioned vector into a host cell by molecular biological methods well known to those skilled in the art.

[0064] As mentioned above, the inventors immunized mice with recombinant streptavidin immunogens, took the spleen cells of the mice and fused them with myeloma cells, and screened out hybridoma cell lines that can specifically bind to streptavidin by ELISA. After screening out the monoclonal cell strains that secrete the target antibody, the heavy chain and light chain variable region cDNAs can be recovered from the cell strains by reverse transcription-PCR, and the appropriate immunoglobulin constant region can be selected, and then the heavy chain and light chain variable region cDNAs and constant region cDNAs are transferred into host cells such as COS or CHO cells, thereby obtaining the expression cells expressing the target antibody of the present invention. Other antibodies or chimeric molecules that retain the specificity of the original antibody can be produced using the above-mentioned monoclonal antibodies and recombinant DNA technology. These technologies may include introducing DNA encoding the variable region or complementary determining region (CDR) of the antibody immunoglobulin into a eukaryotic expression vector including the constant region of different immunoglobulins or the constant region plus the framework region, or introducing both into a suitable eukaryotic expression vector, and then introducing the eukaryotic expression vector into an expression cell such as a CHO host cell, thereby obtaining each recombinant anti-streptavidin antibody.

[0065] In a specific embodiment, the expression cell can be a mammalian cell, such as a Chinese hamster ovary cell, a mini-hamster kidney cell, a monkey kidney cell, a mouse thymoma cell, or a human embryonic kidney cell. In a more specific embodiment, the expressing cells can be, for example, monkey kidney cells transformed by SV40 (COS-7, ATCC CRL1651), human embryonic kidney cells (HEK293 or HEK293 cells subcloned for growth in suspension culture, Graham et al., 1977, J. Gen Virol. 36:59), baby 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 Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251), monkey kidney cells (CV-1, ATCC CCL70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL2), canine kidney cells (MDCK, ATCC CCL34), buffalo rat liver cells (BRL3A, ATCC CRL1442), human lung cells (W138, ATCC CCL75), human liver cells (HepG2, HB8065), mouse breast tumor (MMT060562, ATCC CCL51), TR1 cells (Mather et al., 1982, Annals NY Acad. Sci. 383:44-68), MRC5 cells, FS4 cells, etc., but not limited thereto.

[0066] In a fifth aspect, the present invention 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 according to the first aspect of the present invention.

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

[0068] In a specific embodiment, the method comprises the following steps:

[0069] (1) allowing the anti-streptavidin monoclonal antibody or the antigen-binding fragment thereof to react with a separation medium to form an anti-streptavidin monoclonal antibody or the antigen-binding fragment thereof coupled to the separation medium;

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

[0071] (3) co-incubating the cell population, the anti-streptavidin monoclonal antibody or antigen-binding fragment thereof coupled to the sorting medium, and the sorting antibody coupled to streptavidin to form a first complex;

[0072] (4) separating the first complex from other components in the cell population using a separation medium in the first complex;

[0073] (5) incubating the separated first complex 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, so that the anti-streptavidin monoclonal antibody or its antigen-binding fragment dissociates from streptavidin to form a second complex of biotin-sorting antibody coupled to streptavidin-target cells, thereby obtaining sorted target cells.

[0074] In a specific embodiment, the separation medium can 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 antigen-binding fragment thereof-streptavidin-sorting antibody.

[0076] In a sixth aspect, the present invention provides a method for detecting an antigen to be detected using 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 invention as a quality control system.

[0077] In a specific embodiment, the anti-streptavidin antibody or antigen-binding fragment thereof of the present invention can be used in combination with a streptavidin-biotin system, and the high affinity of biotin and streptavidin can be used to improve the sensitivity of detection.

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

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

[0080] (2) The sample to be tested is dripped onto the sample pad of the colloidal gold test strip and allowed to react for a period of time, and then whether the test line is colored is determined to determine whether the sample to be tested contains the antigen to be tested.

[0081] In a preferred embodiment, the first antibody and the second antibody are different.

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

[0083] It is understandable that when the antigen to be tested is present in the sample to be tested, as the sample to be tested migrates in the test strip, a colloidal gold-streptavidin-biotin-first antibody-antigen complex will first be formed, and a colloidal gold-streptavidin-biotin-first antibody-antigen-second antibody complex will further be formed at the test line, so that the test line is colored, and the free colloidal gold-labeled streptavidin will further migrate to the quality control line, and form a colloidal gold-streptavidin-anti-streptavidin antibody complex here, so that the quality control line is colored; when the antigen to be tested is not present in the sample to be tested, a complex that can be colored cannot be formed at the test line, and the free colloidal gold-labeled streptavidin will continue to migrate to the quality control line, and form a colloidal gold-streptavidin-anti-streptavidin antibody complex here, so that the quality control line is colored. It should be understood that if the quality control line does not develop color, it indicates that the colloidal gold test strip is invalid and cannot be used for detection.

[0084] The traditional colloidal gold sandwich method detects the antigen to be detected by forming a colloidal gold-antigen to be detected-second antibody complex at the T line marked with the 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 used as the quality control system can be adapted to a variety of detection antibodies to adapt to the detection of different antigens.

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

[0086] In a specific embodiment, the anti-streptavidin monoclonal antibody of the present invention can specifically bind to streptavidin, and thus, when combined with 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 not limited thereto. When combined with fluorescent group-labeled streptavidin, it can be used for immunofluorescence (IF), in situ hybridization (ISH), flow cytometry (FCM), but not limited thereto. When combined with immobilized streptavidin, it can also be used for protein purification and nucleic acid extraction, but not limited thereto.

[0087] Example

[0088] In the following examples, the preparation method of the antibody of the present invention and its characterization and related applications are shown. Unless otherwise specified, the test methods used are all conventional methods, and unless otherwise specified, the test materials used in the following examples are all purchased from conventional reagent stores. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of the present invention.

[0089] It should be noted that the terms used in the specification of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The above summary of the invention and the detailed description below are only for the purpose of specifically explaining the present invention, and are not intended to limit the present invention in any way. Without departing from the spirit and purpose of the present invention, the scope of the present invention is determined by the attached claims.

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

[0091] Antigen coupling and immunization: The purified recombinant streptavidin protein (SA, heavy chain biological, catalog number HP155-2) was used as an immunogen to immunize mice. Immunization was performed 4 times, with an interval of 2 weeks between each immunization, and the immunization dose was 100 μg / mouse. 7 days after the fourth immunization, the mice were cut off from the tail and blood was collected, and the serum was separated. The antibody titer level of the immunized mouse antiserum was detected by indirect ELISA method to observe the immune response effect. 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 the immunized mouse 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 then plated into a 96-well culture plate at 200 μL / well 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 taken for antibody detection.

[0093] Screening of positive hybridoma cells: Take SA protein, dilute to 1 μg / mL, add 100 μL / well to 96-well plate, and coat overnight at 4°C; wash 3 times with phosphate buffered saline (PBST), pat dry and wash with PBST containing 2% BSA The cells were blocked at 37°C for 2 h, washed 3 times, and patted dry. The supernatant of fused cells, 1:1000 diluted positive serum of immune mice (as positive control), and 1:1000 diluted negative serum of mice (as negative control) were added to the corresponding wells at 100 μL / well, incubated at 37°C for 1 h, washed 3 times, and patted dry. 1:4000 diluted horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (purchased from Sigma) was added at 100 μL / well, incubated at 37°C for 1 h, washed 3 times, and patted dry. 100 μL / well of tetramethylbenzidine (3,3',5,5'-Tetramethylbenzidine, TMB) substrate was added, and the reaction was terminated after color development at room temperature in the dark for 10 min.

[0094] Detect the OD of all wells in the ELISA plate at a wavelength of 450 nm on the ELISA reader. 450nm When the OD of negative serum 450nm ≤0.1, to measure the absorbance OD of well (NS1) 450nm The value is the OD of the negative well 450nm The positive result was 2.1 times or more, and the positive hybridoma cells were selected for the next cloning.

[0095] Cloning of positive cell lines: After sampling and counting the positive cell wells that secrete antibodies, dilute to 100 cells / 10mL culture medium, plate the diluted cell suspension to a 96-well cell culture plate at 100μL / well, and culture in a 37°C, 5% CO2 cell culture incubator. After 6-7 days, the formation of cloned cells can be observed under a microscope, mark the single clone growth well, remove the cell supernatant, perform ELISA test (the same as the above fusion test), and select positive monoclonal cells. Perform limiting dilution on the cells in the positive wells, measure the ELISA value 5-6 days after each limiting dilution, and pick the OD obtained by ELISA test. 450nmThe monoclonal wells with higher positive values ​​were subjected to limiting dilution until the results of the 96-well plate were positive for ELISA. The monoclonal strains with high positive values ​​were selected for final determination. Three cell lines that stably secreted anti-SA protein antibodies were obtained, total RNA was isolated, and cDNA was prepared by reverse transcription to clone immunoglobulin sequences from hybridoma cell lines, and the antibody variable region sequences of the above hybridoma cell lines were determined. The sequences of the complementary determining regions of the heavy chain and the light chain are shown in Table 1 below (based on the Chothia numbering system).

[0096] Table 1: Complementarity determining region sequences of heavy and light chains

[0097]

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

[0099] The specific steps of constructing recombinant antibodies, preparing cell lines that stably express antibodies through eukaryotic expression, and culturing and purifying them on a large scale are as follows.

[0100] For antibody V L and V H Gene, the sequence obtained by sequencing is as shown in Table 1 above CDR sequence and as shown in SEQ ID NO: 19-SEQ ID NO: 22 heavy chain framework region (HFR) and as shown in SEQ ID NO: 23-SEQ ID NO: 26 light chain framework region (LFR) sequence are sequentially connected to obtain heavy chain and light chain variable regions, respectively, and then the recombinant antibody eukaryotic expression vector is constructed by molecular cloning method. The eukaryotic expression plasmid is electrotransfected into CHO host cells, and after electrotransfection, it is added to the pressure screening medium (50 μM MSX) and cultured for 20 days, and the supernatant is taken for ELISA detection (using horseradish peroxidase (HRP) labeled sheep anti-mouse IgG as the secondary antibody for screening, the method is the same as above) to screen out the recombinant antibody cell line that stably expresses.

[0101] The selected stable cell lines were cultured on a large scale using the cell roller bottle culture technique to prepare recombinant antibodies. The cells were cultured in a medium (Vega CHO) at a rate of (0.2-0.3)×10 6 cells / mL were inoculated in a roller bottle containing 300mL of culture medium and cultured in a cell culture incubator. After 7-9 days of culture, samples were taken for observation under a microscope. When the cell viability was less than 50%, the samples were collected by centrifugation. The samples were affinity purified using a protein A affinity chromatography column to obtain antibodies, namely recombinant monoclonal antibodies S1', S2' and S3'.

[0102] The SA protein was diluted with 0.05 mol / L carbonate buffer at pH 9.6 to a concentration of 1 μg / mL, and added to a 96-well ELISA plate at 100 μL / well and coated overnight at 4°C, washed 3 times, and patted dry. 2% BSA in PBST was blocked at 37°C for 2 hours, washed 3 times, and patted dry. Anti-SA protein monoclonal antibodies S1', S2', and S3' were graded diluted with pH 7.4, 0.02M PBS buffer, with an initial antibody concentration of 5 μg / mL, and graded diluted three times in sequence to obtain a series of monoclonal antibody samples with different concentrations. The diluted monoclonal antibody sample was added to the above ELISA plate at 100 μL / well, incubated at 37°C for 1 hour, washed 3 times, and patted dry. Add 1:4000 diluted horseradish peroxidase (HRP) labeled goat anti-mouse IgG (purchased from Sigma), 100 μL / well, incubated at 37°C for 1 hour, washed 3 times, and patted dry. TMB substrate was added and the reaction was terminated after color development at room temperature in the dark for 10 min. OD was measured by ELISA 450nm The above ELISA results were analyzed by software to obtain the affinity EC values ​​of monoclonal antibodies S1', S2' and S3'. 50 Value (nM), the results are shown in Table 2 below.

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

[0104] SA protein Recombinant Antibody S1' <![CDATA[4.21×10 -10 M]]> Recombinant Antibody S2' <![CDATA[3.98×10 -10 M <!-- 10 -->]]> Recombinant Antibody S3' <![CDATA[4.46×10 -10 M]]>

[0105] Example 3: Application in cell sorting

[0106] This example 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 the magnetic beads to facilitate the next step of culture and application.

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

[0108] The sorting antibody CD3 antibody was coupled to streptavidin SA: streptavidin was activated with N-succinimidyl maleate and reacted at room temperature for 60 minutes, CD3 antibody reduced with DTT was added and reacted with activated SA, the ratio of CD3 antibody to SA was 5:1, and the reaction was carried out at room temperature for 30 minutes. The reaction was terminated and purified using affinity chromatography.

[0109] Magnetic bead cell sorting:

[0110] 5×10 6The cells to be selected were resuspended in 500 μL buffer, 2 μg SA-sorting antibody (CD3 antibody) was added, and 40 μL SA antibody-coated magnetic beads were added after incubation and washing. After incubation for 15 minutes, the tube containing the cells was placed on the magnetic rack for 2 minutes, and the supernatant was carefully collected (CD3 negative cell group). The tube was removed, 2 μg / mL biotin was added and incubated for 5 minutes, and the tube was placed on the magnetic rack again for 2 minutes, and the supernatant was carefully collected (CD3 positive cell group). The two groups of cells were resuspended in buffer and flow cytometry was performed. The results of using S1', S2' and S3' coated magnetic beads were as follows: 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, the exemplary application of the anti-streptavidin antibody of the present invention as a quality control system (C line) in the detection of new coronavirus is illustrated.

[0113] Preparation of colloidal gold: Add 200 mL of ultrapure water to a conical flask, heat to boiling, add 1 mL of 2% chloroauric acid (Sigma-Aldrich, Catalog No.: 16961-25-4) solution, immediately add 1 mL of 2% trisodium citrate (Sigma-Aldrich, Catalog No.: 6132-04-3) aqueous solution after boiling, continue stirring and boiling for 10 minutes, and cool naturally for use.

[0114] Labeling colloidal gold conjugate: take 10mL of the above colloidal gold and put it into a beaker, add 140μL of 0.2M K2CO3 to adjust the pH to 7.4 while stirring, and continue stirring for 10 seconds; add 200μg streptavidin and continue stirring for 5 minutes; add 0.1ml 10% BSA and continue stirring for 5 minutes; centrifuge at 12000g for 10 minutes, discard the supernatant, and dilute the precipitate to 1mL with colloidal gold diluent (10mM PB, 150mM NaCl, 0.2% BSA, 0.1% TritonX-100, 3% Sucrose, 0.01% Proclin300) as streptavidin-colloidal gold complex.

[0115] Preparation of colloidal gold pad: The colloidal gold complex was diluted 10 times with colloidal gold diluent, soaked in glass fiber (Shanghai Jinbiao Company), and freeze-dried to make a gold label pad. The mouse anti-2019-nCOV NP monoclonal antibody (heavy chain bio HA811-41MB) conjugated with biotin was diluted to 100-200 ng / mL to make a biotin pad.

[0116] Coating of nitrocellulose membrane (NC membrane): dilute anti-streptavidin antibody (S1', S2' or S3') to 1 mg / mL to make the quality control line working solution, dilute mouse anti-2019-nCOV NP monoclonal antibody (heavy chain biological HA811-22M) to 1 mg / mL to make the detection line working solution, and use a membrane spotter to draw lines on the corresponding positions of nitrocellulose membrane (Millipore, catalog number: HF135002) as C line (quality control line) and T line (detection line). Dry at 50℃ for 1 hour and set aside.

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

[0118] Sensitivity test: Detect antigen quality control products of different concentrations. Specifically, dilute the recombinant 2019-nCoVNP antigen quality control product by times, take 80μL and drop it on the sample pad, leave it at room temperature for 5-15 minutes, and determine the result. The activity of antigen-antibody binding in the sample can be indicated according to the depth of the displayed band color. The results are shown in Table 3 below.

[0119] Table 3: Colloidal gold immunochromatographic test results of 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 traditional sandwich method (a detection method in which the first antibody is labeled with colloidal gold and forms a sandwich with the second antibody on the detection line), as shown in Table 4 below.

[0122] Table 4: Comparison of 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] It can be seen from the results in Table 4 that if C7 is used as the positive standard, the colloidal gold sandwich method using the anti-streptavidin antibody of the present invention can increase the minimum detection limit of the 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, the antibody or the antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain complementary determining region V H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementarity determining region V L CDR1, V L CDR2 and V L CDR3; of which: 1) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 1-3, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 4-6 respectively. L CDR1, V L CDR2 and V L CDR3; 2) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 7-9, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 10-12 respectively. L CDR1, V L CDR2 and V L CDR3; or 3) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 13-15, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 16-18 respectively. L CDR1, V L CDR2 and V L CDR3.

2. The anti-streptavidin monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region also includes framework regions HFR1, HFR2, HFR3 and HFR4, which are closely related to the V H CDR1, V H CDR2 and V H CDR3 is arranged from the amino terminus to the carboxyl terminus according to HFR1, V H CDR1、HFR2、V H CDR2、HFR3、V H The light chain variable region further comprises framework regions LFR1, LFR2, LFR3 and LFR4, which are arranged in the order of CDR3 and HFR4, wherein the HFR1, HFR2, HFR3 and HFR4 have the sequences shown in SEQ ID NOs: 19-22, respectively, or sequences having 80% or more, 85% or more, 90% or more identity with the sequences, and the light chain variable region further comprises framework regions LFR1, LFR2, LFR3 and LFR4, which are arranged in the order of CDR3 and HFR4, wherein the HFR1, HFR2, HFR3 and HFR4 have the sequences shown in SEQ ID NOs: 19-22, respectively, or sequences having 80% or more, 85% or more, 90% or more identity with the sequences, L CDR1, V L CDR2 and V L CDR3 is arranged from amino terminus to carboxyl terminus according to LFR1, V L CDR1, LFR2, V L CDR2, LFR3, V L The sequence of CDR3 and LFR4 is arranged, wherein the LFR1, LFR2, LFR3 and LFR4 respectively have the sequences shown by SEQ ID NO: 23-26 or sequences with more than 80%, more than 85%, more than 90% or more than 95% identity with the sequences.

3. A nucleic acid molecule encoding the anti-streptavidin monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. A vector comprising the nucleic acid molecule according to claim 3; preferably, the vector is a plasmid vector, such as any one of pEE12, pCAGGS, pTOPO, pcDNA such as pCDNA3.1, pTT, pTT3, pEFBOS, pBV, pJV and pBJ.

5. An expression cell comprising the nucleic acid molecule according to claim 3 or the vector according to claim 4, preferably, the expression cell is a mammalian cell, for example, selected from Chinese hamster ovary cells, mini-hamster kidney cells, monkey kidney cells, mouse thymoma cells, and human embryonic kidney cells.

6. 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 according to claim 1 or 2.

7. The method according to claim 6, wherein: The method comprises the following steps: (1) allowing the anti-streptavidin monoclonal antibody or the antigen-binding fragment thereof to bind to a sorting medium, such as magnetic particles, to form an anti-streptavidin monoclonal antibody or the antigen-binding fragment thereof coupled to the sorting medium; (2) allowing the sorting antibody to react with streptavidin to form a streptavidin-coupled sorting antibody, wherein the sorting antibody specifically binds to a target antigen expressed in a target cell; (3) co-incubating the cell population, the anti-streptavidin monoclonal antibody or antigen-binding fragment thereof coupled to the sorting medium, and the sorting antibody coupled to streptavidin to form a first complex; (4) separating the first complex from other components in the cell population using a separation medium in the first complex; (5) incubating the separated first complex 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, so that the anti-streptavidin monoclonal antibody or its antigen-binding fragment dissociates from streptavidin to form a second complex of biotin-sorting antibody coupled to streptavidin-target cells, thereby obtaining sorted target cells.

8. 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 according to claim 1 or 2 as a quality control system.

9. The method according to claim 8, wherein: The method comprises the following steps: (1) providing a colloidal gold test strip, the colloidal gold test strip comprising: a gold label pad, a biotin pad, a nitrocellulose membrane and a sample pad, wherein the gold label pad is provided with colloidal gold-labeled streptavidin, the biotin pad is provided with a first antibody coupled with biotin and targeting an antigen to be tested, the nitrocellulose membrane is provided with a detection line and a quality control line, wherein the detection line is formed by a second antibody that is the same as or different from the first antibody and targets the antigen to be tested, and the quality control line is formed by the anti-streptavidin monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2; (2) The sample to be tested is dripped onto the sample pad of the colloidal gold test strip and allowed to react for a period of time, and then whether the test line is colored is determined to determine whether the sample to be tested contains the antigen to be tested.

10. Use of the anti-streptavidin monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 in immunoassay; preferably, the immunoassay comprises immunohistochemistry (IHC), immunocytochemistry (ICC), in situ hybridization (ISH), enzyme-linked immunosorbent assay (ELISA), immunochromatography (ICA) and western blotting (WB), immunofluorescence (IF), in situ hybridization (ISH) or flow cytometry (FCM).

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