CN116400081A - Compound for detection and its preparation method and application - Google Patents

Abstract

A detection complex comprising a target recognition portion, the target recognition portion being an antibody; a nucleic acid fragment covalently linked to the target recognition portion by a coupling agent; and a nucleic acid dye loaded on the nucleic acid fragment. The invention also provides a preparation method of the compound for detection and a detection method.

Description

Compound for detection and its preparation method and application

technical field

The invention belongs to the field of biotechnology, and in particular relates to an antibody-nucleic acid conjugate loaded with nucleic acid dye, its preparation method and application.

Background technique

For a long time, various antibodies and antibody conjugates have been widely used in the field of biomedical science research. Various labeled antibodies are used in immunocytochemistry, flow cytometry, and enzyme-linked immunoassay (ELISA). And western blotting and other fields, has a wide range of applications. In these applications, antibodies (both primary and secondary) that are directly conjugated to the label are often used.

In recent years, people have developed the combination of nucleic acid molecules and antibodies. Nucleic acid molecules are characterized by high payloads. A variety of fluorescent nucleic acid dye molecules or other compound molecules can be combined with double-stranded nucleic acid molecules. For example, nucleic acid intercalators such as ethidium bromide and propidium iodide can be inserted between the base pairs of nucleic acid molecules; 4',6-diamidino-2-phenylindole (DAPI), Hoechst and other dyes , can bind to the minor groove of double-stranded nucleic acid molecules.

Heterobifunctional crosslinkers can be used, such as succinimidyl 4-hydrazinoornithine acetone hydrazone (SANH) and succinimidyl 4-(N-maleimidomethyl)cyclohexyl Alkane-1-carboxylates (SMCC), which form a bridge between nucleic acid molecules and antibodies. At present, antibody-nucleic acid molecule conjugates are usually obtained by directly covalently combining small molecules with nucleic acid molecules using coupling agents. However, such direct covalent binding requires specific modifications to the nucleic acid molecule. The traditional chemical reagent coupling antibody-dye or nucleic acid molecule-dye technology is too complicated, labor-intensive, and expensive, and it is difficult for the laboratory to prepare it by itself.

Therefore, there is an urgent need for a simple method to obtain sensitive and easy-to-use labels in which dye molecules are non-covalently bound to nucleic acid molecules, which can avoid the huge cost of synthesis and modification of nucleic acid molecules on the one hand, and on the other hand On the one hand, it is convenient for self-preparation in the laboratory.

Contents of the invention

In order to solve one of the above-mentioned technical problems existing in the prior art, the present invention provides a detection compound, its preparation method and application.

According to one aspect of the present disclosure, there is provided a complex for detection, the complex comprising: a target recognition part, the target recognition part is an antibody; a nucleic acid fragment covalently linked to the target recognition part through a coupling agent and, a nucleic acid dye loaded on the nucleic acid fragment.

According to some embodiments of the present disclosure, the nucleic acid fragment is a double-stranded DNA fragment or a single-stranded DNA fragment. According to some embodiments of the present disclosure, the nucleic acid fragment is double-stranded DNA. According to some specific embodiments, the nucleic acid fragment is obtained by PCR amplification.

According to some embodiments of the present disclosure, the nucleic acid fragment has 10 to 250 bases or base pairs. According to some specific embodiments, the nucleic acid fragment has 200 to 250 bases or base pairs.

According to some embodiments of the present disclosure, the target recognition part specifically recognizes a target. According to some embodiments of the present disclosure, the target recognition part is an immunoglobulin, Fab, Fab', (Fab') ₂ or a single chain antibody (scFv). According to some specific embodiments, the target recognition part is an antibody, such as a primary antibody or a secondary antibody. According to some specific embodiments, the antibody is dissolved in a buffer containing ethylenediaminetetraacetic acid (EDTA), such as phosphate buffer containing EDTA. EDTA can chelate divalent ions and has a dual effect, inhibiting the formation of disulfide bonds and inhibiting DNaseI, thereby protecting nucleic acid molecules. According to some specific embodiments, after the antibody is desalted, it is then dissolved in phosphate buffer containing EDTA.

According to some embodiments of the present disclosure, the coupling agent is selected from succinimide 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 4-(N- Maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt (Sulfo-SMCC), (1-ethyl-(3-dimethylaminopropyl)carbonyl diimide) (EDC) or N-hydroxysuccinimide (NHS).

According to some embodiments of the present disclosure, the nucleic acid fragment has a linear, circular or other three-dimensional structure.

According to some embodiments of the present disclosure, the nucleic acid dye is a fluorescent dye, preferably selected from anthocyanine dyes, GelGreen ^™ , GelRed ^™ , ethidium bromide, propidium iodide, 4′,6-diamidino- 2-Phenylindole, Hoechst dye, acridine orange dye, ethidium bromide dimer (EthD), 7-aminoactinomycin D (7-AAD) and analogs thereof.

According to another aspect of the present disclosure, there is provided a method for preparing the complex of the present disclosure, the method comprising: using a coupling agent to activate the target recognition part to obtain the target recognition part with the coupling agent; and The target recognition part with the coupling agent undergoes a coupling reaction with the nucleic acid fragment after being desalted to obtain a target recognition part-nucleic acid fragment complex.

According to some embodiments of the present disclosure, before the coupling reaction with the target recognition moiety with a coupling agent, the nucleic acid fragment is incubated with a nucleic acid dye to be loaded with the nucleic acid dye. Then, the nucleic acid fragment loaded with the nucleic acid dye is coupled with the target recognition part to obtain a target recognition part-nucleic acid fragment-nucleic acid dye complex.

According to some embodiments of the present disclosure, after the complex of the target recognition part-nucleic acid fragment is obtained, the target recognition part-nucleic acid fragment is incubated with a nucleic acid dye, so as to load the nucleic acid dye on the nucleic acid fragment , to obtain target recognition part-nucleic acid fragment-nucleic acid dye complex.

According to some embodiments of the present disclosure, the target recognition part-nucleic acid fragment complex is subjected to desalting treatment, and then incubated with a nucleic acid dye to load the nucleic acid dye. According to some embodiments of the present disclosure, after the nucleic acid fragment is incubated with a nucleic acid dye and subjected to desalting treatment, a coupling reaction occurs with the target recognition part with a coupling agent. The loading amount of the nucleic acid dye on the nucleic acid fragment can be significantly increased by desalting treatment, and the fluorescence intensity of the target recognition part-nucleic acid fragment-nucleic acid dye complex of the present disclosure can be correspondingly increased.

According to some embodiments of the present disclosure, the desalination treatment is performed using a desalination column. The desalting column can be filled with agarose gel made by mixing dextran gel and agarose gel. In a specific embodiment, the desalting column may be a SpinOUT ^TM desalting column, a Zeba ^TM desalting spin column, etc.

According to yet another aspect of the present disclosure, there is provided a method of detecting a sample, the method comprising contacting the sample with a complex of the present disclosure.

According to some embodiments of the present disclosure, the sample includes target-containing substances, such as cells, tissues, organs, body fluids, blood, organisms, and the like.

Commonly used DNA dyes include anthocyanine dyes, GelGreen ^TM , GelRed ^TM , ethidium bromide (EB), propidium iodide (PI), 4',6-diamidino-2-phenylindole (DAPI ), Hoechst dyes, acridine orange dyes, ethidium bromide dimer 3 (EthD-3), 7-aminoactinomycin D (7-AAD) and their analogs. Anthocyanine dyes include, for example, SYBR Green, SYBR Gold, and GeneGreen ^™ . Acridine orange-based dyes include, for example, acridine orange, thiazole orange, and Gold View type I/II. The most common Hoechst dyes include Hoechst 33342 and Hoechst 33258.

Acridine orange dyes, 7-AAD, ethidium bromide and propidium iodide can intercalate between the paired bases of the nucleic acid duplex and release orange-red or green fluorescence under ultraviolet excitation.

Desalting treatment can be performed using a desalting column. Commonly used desalting columns include agarose gel filled with a mixture of dextran gel and agarose gel, such as SpinOUT ^TM desalting column, Zeba ^TM desalting spin column, etc.

The disclosure utilizes the high loading capacity of nucleic acid molecules, nucleic acid dye molecules, prepares a target recognition part-nucleic acid-nucleic acid dye complex, and provides the application of the complex in immunolabelling.

According to yet another aspect of the present disclosure, methods for fluorescent labeling using the complexes of the present disclosure are also provided. The complex of the present disclosure can be used for cell labeling in vitro, immunofluorescent labeling such as flow cytometry (Flow Cytometry). Due to the high load of nucleic acid on the nucleic acid fluorescent dye, the number of fluorescent dye molecules carried on the complex is greatly increased, and the fluorescence intensity is obviously enhanced.

For the first time, the disclosure combines the high affinity between the target and the target recognition part with the high loading capacity of the nucleic acid on the nucleic acid dye molecule to obtain the target recognition part-nucleic acid fragment-nucleic acid dye complex of the present disclosure. The complex disclosed in the present invention can be used for immunolabeling and has excellent application prospects. In addition, in the process of synthesizing the target recognition part-nucleic acid fragment-nucleic acid dye complex, the present disclosure refers to each product in the process of the synthesis method, such as target recognition part-coupling agent, target recognition part-nucleic acid fragment, nucleic acid fragment- The nucleic acid dye and the target recognition part-nucleic acid fragment-nucleic acid dye complex are desalted to remove free coupling agents, nucleic acid fluorescent dyes and other small molecule reagents, because the fluorescence brightness depends on the combination of nucleic acid dye-nucleic acid, The specific nucleic acid dye-nucleic acid has a stable binding feature, so the nucleic acid fragment-nucleic acid dye and the target recognition part-nucleic acid fragment-nucleic acid dye complex of the present invention maintain a strong fluorescence brightness.

The following examples and figures are provided to aid understanding of the present invention. However, it should be understood that these embodiments and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention.

Description of drawings

FIG. 1 shows agarose gel electrophoresis pictures of DNA-1 (M13-M13Rev) and DNA-2 (M13-SK) fragments according to Example 1 of the present disclosure.

Figure 2 shows the fluorescence intensity of the DNA-GelGreen complex before and after desalting.

Fig. 3 shows the SDS-PAGE image of the complex of Anti-ErbB2/HER2 and DNA fragments.

Figure 4 shows the photos of goat anti-rabbit IgG-DNA-GelGreen for cell labeling. (A) Photographs observed under a fluorescence microscope, in which the primary antibody is anti-ErbB2/HER2, and the secondary antibody is goat anti-rabbit IgG-DNA-GelGreen. (B) is a bright field photograph of (A). (C) Photographs observed under a fluorescence microscope, where no primary antibody was added, and the secondary antibody was goat anti-rabbit IgG-DNA-GelGreen. (D) is a bright field photograph of (C).

Fig. 5 shows the fluorescent photos of Anti-ErbB2/HER2 antibody-DNA-GelGreen entering into cells through SKBR3 cell endocytosis. (A) SKBR3 living cells labeled with anti-ErbB2/HER2-DNA-GelGreen at 15 minutes, 200×. (B) SKBR3 living cells labeled with anti-ErbB2/HER2-DNA-GelGreen at 15 minutes, 400×. (C) Anti-ErbB2/HER2-DNA-GelGreen labeled SKBR3 living cells, 5 hours photo, 200×. (D) SKBR3 living cells labeled with anti-ErbB2/HER2-DNA-GelGreen, 5 hours photo, 400×.

FIG. 6 shows the results of flow cytometry detection of cells labeled with Anti-ErbB2/HER2 antibody-DNA-GelGreen. (A) is a blank control. (B) Cells labeled with anti-ErbB2/HER2-FITC. (C) Cells labeled with anti-ErbB2/HER2-DNA1-GelGreen. (D) Cells labeled with anti-ErbB2/HER2-DNA2-GelGreen.

Detailed ways

In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. The specific embodiments described here are only used to explain the present invention, and are not intended to constitute any limitation to the present invention. Also, in the following description, descriptions of well-known structures and techniques are omitted to avoid unnecessarily obscuring the concept of the present disclosure. Such structures and techniques are also described in numerous publications.

definition

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly used in the field to which this invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural and vice versa.

As used herein, the expressions "a" and "an" include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells and equivalents known to those skilled in the art, and the like.

As used herein, the term "about" means a range of ±20% of the numerical value that follows. In some embodiments, the term "about" indicates a range of ±10% of the numerical value that follows. In some embodiments, the term "about" indicates a range of ±5% of the numerical value that follows.

The term "immunoglobulin (Ig)" used herein refers to a globulin having antibody activity, or having a structure similar to an antibody. Immunoglobulins can be divided into IgG, IgM, IgA, IgE and IgD according to their chemical structure and antigen.

The term "Fab fragment" used herein is also called an antigen-binding fragment, which is a region of an antibody structure that can bind to an antigen. Fab can be composed of a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH) and a heavy chain constant region (CH1), the light chain and the heavy chain are connected by a disulfide bond , smaller size, molecular weight of 47 ~ 48kDa. Under the action of papain, IgG can be degraded into two Fab fragments and one Fc fragment. Under the action of pepsin, IgG can be degraded into a (Fab') ₂ fragment and a pFc' fragment. The (Fab') ₂ fragment can be further reduced to form two Fab' fragments.

The term "nucleic acid fragment" as used herein refers to a polynucleotide sequence or a fragment thereof. "Nucleic acid" and "polynucleotide" are used interchangeably and generally refer to a strand of at least two base-sugar-phosphate combinations, especially single- and double-stranded DNA, DNA of a mixture of single- and double-stranded regions, single Stranded and double-stranded RNA, RNA of a mixture of single- and double-stranded regions, and hybrid molecules thereof. Nucleic acid can be exogenous or endogenous to the cell. Nucleic acids can be present in a cell-free environment. A nucleic acid may be a gene or a fragment thereof. A nucleic acid may include one or more analogs (eg, altered backbones, sugars, or nucleobases). Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acids, xeno nucleic acids, morpholinos, locked nucleic acids, diol nucleic acids, threose nucleic acids, dideoxynucleic acids nucleotides, cordycepin, thiol-containing nucleotides, biotin-linked nucleotides, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, Pseudouridine, dihydrouridine, queuosine and wyosine. "Nucleic acid", "polynucleotide", "target polynucleotide" and "target nucleic acid" are used interchangeably.

Nucleic acids may contain one or more modifications (eg, base modifications, backbone modifications) to provide new or enhanced characteristics to the nucleic acid (eg, improved stability). Nucleosides can be base-sugar combinations. The base portion of the nucleoside may be a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. A nucleotide may be a nucleoside that further includes a phosphate group covalently linked to the sugar moiety of the nucleoside. For those nucleosides that include pentofuranose sugars, the phosphate group can be attached to the 2', 3' or 5' hydroxyl moiety of the sugar. In forming nucleic acids, phosphate groups can covalently link adjacent nucleosides to each other to form linear polymeric compounds. The respective ends of this linear polymeric compound can then be further joined to form a cyclic compound. In addition, linear compounds may have internal nucleotide base complementarity, and thus may fold in such a way as to produce fully or partially double-stranded compounds. In nucleic acids, phosphate groups can generally be referred to as forming the internucleoside backbone of the nucleic acid. The linkage or backbone can be a 3' to 5' phosphodiester linkage.

A nucleic acid may comprise a polynucleotide backbone formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatoms or heterocyclic internucleoside linkages. These can include those with: morpholino linkages (formed in part from the sugar moieties of nucleosides); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formyl and thioformyl backbones; Acyl and thioformyl backbones; riboseacetyl backbones; olefin-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; Mixed N, O, S and _CH2 components of other substances.

A nucleic acid can include a nucleic acid mimetic so long as the nucleic acid mimetic is capable of binding a nucleic acid dye. The term "mimetic" may be intended to include polynucleotides in which only the furanose ring or both the furanose ring and the internucleotide linkage are replaced by non-furanose groups, the replacement of only the furanose ring may also be referred to as sugar substitution things. The heterocyclic base moiety or modified heterocyclic base moiety can be maintained to hybridize to an appropriate target nucleic acid. One such nucleic acid may be a peptide nucleic acid (PNA). In PNA, the sugar backbone of a polynucleotide can be replaced by an amide-containing backbone, especially an aminoethylglycine backbone. Nucleotides can be retained and bonded directly or indirectly to the aza nitrogen atom of the amide portion of the backbone. The backbone in PNA compounds may contain two or more linked aminoethylglycine units, which provide the PNA with an amide-containing backbone. The heterocyclic base moiety can be bound directly or indirectly to the aza nitrogen atom of the amide portion of the backbone.

Nucleic acids may also include nucleobase (often simply referred to as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases may include purine bases such as adenine (A) and guanine (G), as well as pyrimidine bases such as thymine (T), Cytosine (C) and Uracil (U)). Modified nucleobases may include other synthetic as well as natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-amino Adenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2- Thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH3)uracil and cytosine, and other alkynyl derivatives of pyrimidine bases , 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen, 8-amino, 8-thio, 8-thioalkyl , 8-hydroxy and other 8-substituted adenine and guanine, 5-halogen especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3- Deazaguanine and 3-deazaadenine. Modified nucleobases may include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine Cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps, such as substituted phenoxazine cytidines (e.g. 9-(2 -aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5, 4-b) (1,4)benzothiazin-2(3H)-ones), such as substituted phenoxazine cytidines (eg 9-(2-aminoethoxy)-H-pyrimido(5, 4-(b)(1,4)benzoxazin-2(3H)-one), carbazolidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole Cytidine (H-pyrido(3',2':4,5)pyrrolo[2,3-d]pyrimidin-2-one).

The term "sample" used herein may refer to a substance containing a target, including, for example, cells, tissues, organs, body fluids, blood, organisms, and the like.

Example 1. Amplified DNA fragments

Use the pBluescript II SK (-) plasmid (with the nucleotide sequence shown in SEQ ID NO.: 1) as a template to carry out PCR reaction, the primer is M13-sh: 5'SH C6 gtaaaacgacggccagt 3' (upstream, SEQ ID NO.: 1), M13 rev: 5'caggaaacagctatgac 3' (downstream, SEQ ID NO.:2), SK: 5'tctagaactagtggatc 3' (downstream, SEQ ID NO.:3). According to the following conditions, PCR amplification was carried out to obtain DNA fragments of about 229bp and 137bp, respectively, which were named DNA-1 (SEQ ID NO.:2) and DNA-2 (SEQ ID NO.:3).

The DNA-1 and DNA-2 fragments were subjected to agarose gel electrophoresis, and the results are shown in Figure 1. DNA-1 (Rev) and DNA-2 (SK) fragments were recovered using a DNA recovery kit.

The determination of embodiment 2.DNA-GelGreen binding fluorescence

Take 30 μL of GelGreen ^TM (100 μg/mL) solution, add 5 μL of DNA-1 and DNA-2 fragments (200 μg/mL) amplified in Example 1, and 5 μL of 1×PBS respectively. Use Sangon's centrifugal desalting column (GT-600, 0.1 mL) to desalt, and desalt DNA-1, DNA-2 and PBS solutions according to the manufacturer's instructions to obtain desalted GelGreen-DNA-1 and GelGreen-DNA-2 samples. Then, the fluorescence intensity of the samples before and after desalting was measured using Promega's Glomax Discover multifunctional detector (Ex: 475nm, Em: 500-520nm). Fig. 2 shows the fluorescence measurement results of GelGreen-DNA-1, GelGreen, DNA-1 and PBS before and after desalting.

As can be seen from Figure 2, only GelGreen, DNA-1 or PBS solution, almost no fluorescence can be observed before and after desalting; GelGreen-DNA-1 shows a significant increase in fluorescence intensity after desalting, which shows that the fluorescence brightness depends on the nucleic acid dye -Nucleic acid binding, and specific nucleic acid dyes have strong binding characteristics between nucleic acids.

Example 3. Coupling of Antibodies and DNA Fragments

Antibody-DNA coupling was performed using Sulfo-SMCC (Thermo).

Briefly, the primary antibody Anti-ErbB2/HER2 (Abcam) or the secondary antibody goat anti-rabbit IgG (Shenggong) was diluted with 1×PBS+5mM EDTA, and the final concentration of the antibody was 0.5mg/mL.

Dissolve Sulfo-SMCC in 1×PBS to obtain a 5 mg/mL Sulfo-SMCC solution. Add 50 μL of Sulfo-SMCC solution per mL of antibody, and couple at 25°C for 0.5 hour to obtain Sulfo-SMCC activated antibody. The obtained Sulfo-SMCC activated antibody was desalted with a Sangon centrifugal desalting column (GT-600, 0.1 mL) for later use.

Then, the antibody activated by Sulfo-SMCC was mixed with DNA-1 and DNA-2 fragments with sulfhydryl groups, and coupled for 0.5 hours at 25°C, and 50 μL of reduced cysteine solution (5 mg/mL) was added to each mL of antibody to Terminate the coupling reaction. The obtained antibody-DNA conjugates were detected by reducing SDS-PAGE, and the results are shown in FIG. 3 .

It can be seen from Figure 3 that compared with the Anti-ErbB2/HER2 antibody without DNA coupling, the mass of the antibody coupled with DNA-1 or DNA-2 becomes larger, indicating that the antibody has been coupled to DNA.

Example 4. Antibody-DNA Conjugates and Dye Conjugation

In this example, the DNA was first mixed with GelGreen ^TM to obtain GelGreen-loaded DNA. Then, the Sulfo-SMCC-coupled antibody was mixed with the GelGreen-loaded DNA, and the coupling reaction was carried out according to the steps of antibody-DNA coupling in Example 3. After the coupling reaction, the antibody-DNA conjugate loaded with GelGreen molecules in 1×PBS+5mM EDTA solution was obtained, and placed at 4°C for use. Alternatively, use Sangon’s centrifugal desalting column GT-600, 0.1mL-6kD cut-off desalting column to desalt, in 1×PBS+5mM EDTA solution, place at 4°C for use.

Example 5. Labeling of Cells Using Secondary Antibody-DNA-Dye Conjugates of the Disclosure

Place the coverslips in a twelve-well culture plate. Breast cancer SKBR3 cells were cultured using DMEM (Equixel) containing 10% fetal bovine serum. After the cells adhered to the coverslip and grew well, the coverslip was taken out, washed with PBS, and fixed with 4% paraformaldehyde at room temperature. Dilute the Anti-ErbB2/HER2 antibody with 1×PBS+1% BSA, add the diluted antibody on the glass slide, incubate at room temperature for 1-2 hours, wash with 1×PBS+1% BSA three times. Then, goat anti-rabbit IgG-DNA-GelGreen diluted in 1×PBS+1%BSA was added, incubated at room temperature for 1-2 hours, and washed three times with 1×PBS+1%BSA. Then microscopic observation was carried out under an Olympus IX73 inverted fluorescence microscope, and the results are shown in Figure 4.

Figure 4A is a fluorescent photo obtained by using Anti-ErbB2/HER2 primary antibody combined with goat anti-rabbit IgG-DNA-GelGreen secondary antibody. It can be seen that there are fluorescent markers on the cell surface; Fluorescent photo of GelGreen secondary antibody as a negative control, there is no fluorescent label on the cell surface. The results showed that goat anti-rabbit IgG-DNA-GelGreen secondary antibody could effectively label cells.

Example 6. Labeling of Live Cells Using Primary Antibody-DNA-Dye Conjugates of the Disclosure

Place the coverslips in a twelve-well culture plate. Breast cancer SKBR3 cells were cultured using DMEM (Equixel) containing 10% fetal bovine serum. After the cells adhered to the coverslip and grew well, the culture medium was removed and washed once with 1×PBS+2% BSA. Then, add the Anti-ErbB2/HER2-DNA-GelGreen complex diluted with 1×PBS+2%BSA+2mM EDTA, incubate at room temperature for 15 minutes, half an hour, and 5 hours respectively, and wash three times with 1×PBS+2%BSA , and then microscopically observed under an Olympus IX73 inverted fluorescence microscope, the results are shown in Figure 5. Figures 5A and 5B show that the fluorescent label was mainly attached to the cell surface at 15 minutes of incubation; Figures 5C and 5D showed that the fluorescent label was endocytosed into the cell interior after 5 hours of incubation.

Example 7. Detection of the effect of antibody-DNA-dye conjugates of the present disclosure on labeling cells by flow cytometry

SKBR3 cells were cultured for 1 to 2 days at 37°C in an incubator with 5% carbon dioxide concentration in DMEM (Ecosel) medium containing 10% fetal bovine serum in a twelve-well plate until the cell density reached about 70%. Discard the supernatant and wash three times with 1x PBS. Cells were digested with trypsin (HyClone) at 37°C for 10 minutes. Centrifuge at 1000×g and remove the supernatant. Suspend the cells in 1×PBS containing 2% FBS, centrifuge again at 800×g, and discard the supernatant. Finally, suspend the cells with 1×PBS containing 2% FBS.

Count the cells and adjust the cell concentration to approximately 2.5 x ¹⁰⁵ cells/ml. Take 2.5mL cells and divide into ten 1.5mL centrifuge tubes, 250μL per tube, 2 centrifuge tubes without antibody, as negative control (NC); 2 centrifuge tubes, add 1μL Anti-ErbB2/HER2-FITC ( Abcam) as a positive control (PC); 5 μL of the Anti-ErbB2/HER2-GelGreen complex prepared in Example 4 was added to the other 8 centrifuge tubes. Incubate at room temperature for 1-2 hours. Then, centrifuge at 1000-1200×g at 4°C, and wash 3 times with 1×PBS+1% BSA.

Then, flow cytometry was performed using an Attune NxT ^TM flow cytometer. Flowjo software was used for data processing and graphing, and the results are shown in Figure 6. It can be seen from Figure 6 that both Anti-ErbB2/HER2-DNA1-GelGreen and Anti-ErbB2/HER2-DNA2-GelGreen complexes can effectively label cells, and can be detected by flow cytometry, and the effect is similar to that of Anti-ErbB2/ HER2-FITC is similar.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

sequence listing

<110> Peking University

Qidong Qichen Biotechnology Co., Ltd.

East China Industrial Research Institute of Life Sciences, Peking University

<120> Compound for detection and its preparation method and application

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2961

<212> DNA/RNA

<213> Artificial Sequence

<400> 1

ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 60

ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 120

ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 180

ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg 240

ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt gagtccacg ttctttaata 300

gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt 360

tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat 420

ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg 480

caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540

gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600

taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg 660

gccccccctc gaggtcgacg gtatcgataa gcttgatatc gaattcctgc agcccggggg 720

atccactagt tctagagcgg ccgccaccgc ggtggagctc cagcttttgt tccctttagt 780

gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt 840

atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg 900

cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg 960

gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc 1020

gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 1080

ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 1140

acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 1200

cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 1260

caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 1320

gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 1380

tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 1440

aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 1500

ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 1560

cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 1620

tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc 1680

tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 1740

ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 1800

aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 1860

aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 1920

aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 1980

gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 2040

gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 2100

caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 2160

ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 2220

attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 2280

ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 2340

gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 2400

ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 2460

tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 2520

gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 2580

cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 2640

gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 2700

tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 2760

ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 2820

gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 2880

tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 2940

catttccccg aaaagtgcca c 2961

<210> 2

<211> 229

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gtaaaacgac ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttgggtaccg 60

ggccccccct cgaggtcgac ggtatcgata agcttgatat cgaattcctg cagcccgggg 120

gatccactag ttctagagcg gccgccaccg cggtggagct ccagcttttg ttccctttag 180

tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctg 229

<210> 3

<211> 137

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gtaaaacgac ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttgggtaccg 60

ggccccccct cgaggtcgac ggtatcgata agcttgatat cgaattcctg cagcccgggg 120

gatccactag ttctaga 137

Claims (10)

1. A complex for detection, the complex comprising:

a target recognition portion that is an antibody;

a nucleic acid fragment covalently linked to the target recognition portion by a coupling agent; and

a nucleic acid dye, loaded on the nucleic acid fragment.

2. The complex of claim 1, wherein the nucleic acid fragment is a double-stranded DNA fragment or a single-stranded DNA fragment, preferably a double-stranded DNA fragment.

3. The complex of claim 1 or 2, wherein the nucleic acid fragment is from a Polymerase Chain Reaction (PCR) or an annealing of an artificially synthesized oligonucleotide, having 20 to 250 bases or base pairs.

4. A complex according to any one of claims 1 to 3, wherein the antibody is selected from the group consisting of immunoglobulins, fab ', (Fab') ₂ Or a single chain antibody scFv.

5. The compound according to any one of claims 1 to 4, wherein the coupling agent is selected from succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic sulfosuccinimidyl ester sodium salt, (1-ethyl- (3-dimethylaminopropyl) carbodiimide) or N-hydroxysuccinimide.

6. The complex of any one of claims 1 to 5, wherein the nucleic acid dye is a fluorescent dye capable of non-covalent binding to the nucleic acid fragment by chimeric means, preferably selected from the group consisting of cyanine dyes, gelGreen ^TM 、GelRed ^TM Ethidium bromide, propidium iodide, 4', 6-diamidino-2-phenylindole, hoechst dye, acridine orange dye, ethidium bromide dimer, 7-amino actinomycin D and/or analogues thereof.

7. A method of preparing the detection complex of any one of claims 1 to 6, the method comprising:

activating the target recognition part by using a coupling agent to obtain a target recognition part with the coupling agent; and

the target recognition part with the coupling agent is subjected to desalination treatment and then is subjected to coupling reaction with the nucleic acid fragment to obtain a target recognition part-nucleic acid fragment complex,

wherein the target recognition portion is an antibody;

wherein the nucleic acid fragment is incubated with a nucleic acid dye to load the nucleic acid dye prior to a coupling reaction with the target recognition moiety with a coupling agent; alternatively, after the coupling reaction of the target recognition moiety with the nucleic acid fragment, the target recognition moiety-nucleic acid fragment complex is incubated with a nucleic acid dye to load the nucleic acid dye on the nucleic acid fragment.

8. The method of claim 7, wherein the method further comprises: desalting the target recognition portion-nucleic acid fragment complex,

preferably, the nucleic acid fragment is incubated with a nucleic acid dye, and subjected to desalting treatment, and then subjected to a coupling reaction with the target recognition moiety with the coupling agent.

9. The method according to claim 7 or 8, wherein the desalting treatment is carried out using a desalting column,

preferably, the desalting column is selected from SpinOUT ^TM Desalting columns or Zeba ^TM Desalting centrifugal column.

10. A method of detecting a sample, the method comprising contacting the sample with a complex of any one of claims 1 to 6.

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