Preparation method and application of a rapid immunohistochemical double staining kit

Abstract

The application relates to a preparation method of a rapid immunohistochemical double-staining kit and application thereof, and belongs to the medical detection field.The rapid immunohistochemical double-staining kit comprises two reagents, reagent A is a FITC-coupled double-specific nanobody A, reagent B is a TRITC-coupled nanobody B, the double-specific nanobody A recognizes estrogen receptors (ER) and progesterone receptors (PR), and the nanobody B recognizes human epidermal growth factor receptor 2 (HER2). The immunohistochemical double-staining kit prepared based on the reagent A and the reagent B can rapidly and accurately detect and position specific antigens in tissue slices, and effectively meets the detection and scientific research requirements of ER, PR and HER2 in tissue samples.

Description

Technical Field

[0001] The invention belongs to the field of medical detection, and particularly relates to a preparation method and application of a rapid immunohistochemical double staining kit. Background Art

[0002] Immunohistochemistry (IHC) is a technique used to detect and localize specific antigens in tissue sections. It uses specific antibodies to bind to the target antigen and visualize these binding events with the help of enzymes or fluorescent labels. IHC is crucial in biomedical research and clinical diagnosis.

[0003] Conventional IHC (immunohistochemistry) involves using enzyme-labeled antibodies, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), to observe color changes under a light microscope. IHC is typically used on paraffin-embedded tissue sections and is suitable for tissue-level analysis.

[0004] Immunofluorescence (IF) is a form of immunohistochemistry that uses fluorescently labeled antibodies, such as FITC, TRITC, and Cy3, for observation under a fluorescence microscope. The advantages of IF are that it can be used not only on tissue sections but also on living cells, frozen sections, or tissue culture. It also allows for multi-color detection and simultaneous observation of multiple markers, which is difficult with conventional immunohistochemistry.

[0005] To meet the demands of complex biological sample analysis in biomedical research and clinical diagnostics, multiple staining is required to label different marker sites within a single sample. Conventional multiplex immunohistochemistry (mIHC) can be performed in multiple steps or simultaneously. The multistep method involves multiple rounds of antibody incubation and staining. While this method has the advantage of allowing low-abundance proteins to bind first, preventing them from being masked by steric hindrance, it is also complex and time-consuming. One-step multiplex immunohistochemistry involves the simultaneous addition of a cocktail of antibodies for incubation. Subsequently, a cocktail of secondary antibodies recognizes these primary antibodies and is developed using corresponding substrates, with each substrate producing a distinct color to distinguish the two antigens. While this method offers advantages of simplicity and time savings, it also presents drawbacks and challenges. For example, if one of the targets is a low-abundance antigen, the simultaneous addition of a cocktail of secondary antibodies may mask these low-abundance antigens due to steric hindrance, resulting in poor binding and reduced detection sensitivity. Furthermore, the simultaneous use of multiple antibodies and substrates increases the likelihood of background staining, which can compromise the clarity of the results.

[0006] These complex biological markers, such as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), are three important biomarkers in breast cancer, closely related to the development, progression, prognosis, and treatment of breast cancer. Breast cancer cells that are ER- and PR-positive are often hormone-dependent (estrogen and progesterone), and these tumors are referred to as hormone receptor-positive breast cancer. HER2-positive breast cancer is often more aggressive and has a poorer prognosis, as HER2 overexpression or amplification is associated with enhanced tumor cell proliferation and survival. If a patient's breast cancer is negative for ER, PR, and HER2, this type of breast cancer is referred to as triple-negative breast cancer. Triple-negative breast cancer is often more aggressive, has a poorer prognosis, and lacks targeted treatments. Clinically, ER, PR, and HER2 testing is a crucial component of breast cancer diagnosis and treatment planning. The status of these three markers collectively determines the molecular subtype of the breast cancer, which in turn influences treatment options and patient prognosis. In summary, ER, PR, and HER2 play key roles in the research, diagnosis, prognostic assessment, and treatment decision-making of breast cancer, and their status is crucial for guiding clinical treatment and predicting patient prognosis.

[0007] Therefore, in order to meet the needs of in-depth analysis of complex biological samples in the fields of biomedical research and clinical diagnosis, there is an urgent need to develop a rapid multiplex immunohistochemistry technology that integrates multiplex immunohistochemistry technology with multiplex immunofluorescence technology, which can flexibly adapt to the detection of antigens with different abundances, while simplifying the operation process and shortening the experimental time. Summary of the Invention

[0008] The purpose of the present invention is to solve the above problems in multiple immunohistochemistry technology and to provide a fast, convenient and low-cost double immunohistochemistry reagent and application method.

[0009] In the first aspect, the present invention provides a rapid immunohistochemistry double staining kit, which comprises reagent A and reagent B, wherein the reagent A is a bispecific nanoantibody A coupled with FITC, and the bispecific nanoantibody A recognizes estrogen receptor (ER) and progesterone receptor (PR); the reagent B is a nanoantibody B coupled with TRITC, and the nanoantibody B recognizes human epidermal growth factor receptor 2 (HER2).

[0010] The bispecific Nanobody A (whose amino acid sequence is shown in SEQ ID No: 3) is formed by the fusion expression of Nanobody A1 (whose amino acid sequence is shown in SEQ ID No: 1) that recognizes ER and Nanobody A2 (whose amino acid sequence is shown in SEQ ID No: 2) that recognizes PR. The C-terminus of Nanobody A1 is connected to the N-terminus of Nanobody A2 via a flexible lysine-rich linker, the amino acid sequence of which is shown in SEQ ID No: 5. The tail end of the bispecific Nanobody A also has a Lys-tag and a His-tag.

[0011] The reagent A is a bispecific nanobody A coupled to FITC. Since the bispecific nanobody A has a lysine-rich linker, its abundant lysine residues provide amino groups (-NH2), which react with the isothiocyanate group (-N=C=S) of FITC to form a new carbon-nitrogen bond. FITC is bound to the protein side chain of the linker through a stable covalent bond; and FITC can also be bound to the Lys-tag at the tail end of the bispecific nanobody A to minimize the reaction with the lysine residues in the functional region of the nanobody to ensure the integrity of the affinity of the nanobody.

[0012] The nanobody B (SEQ ID No: 4) is a specific antibody that binds to HER2 and has a Lys-tag and a His-tag at its tail end.

[0013] The reagent B is a nanobody B coupled with TRITC. The Lys-tag at the tail end of the nanobody B is composed of three lysines, which provides an amino group (-NH2) and undergoes an electrophilic addition reaction with the isothiocyanate group (-N=C=S) of TRITC (tetramethylrhodamine isothiocyanate), so that TRITC is bound to the tail end of the nanobody B through a stable covalent bond, minimizing the reaction with the lysine residues in the functional region of the nanobody to ensure the integrity of the affinity of the nanobody.

[0014] In a second aspect, the present invention provides a method for preparing a rapid immunohistochemical double staining kit, the method comprising:

[0015] (1) Alpacas were immunized with recombinant proteins ER, PR, and HER2, respectively, to construct a nanoantibody library, and then nanoantibodies that bind to ER, PR, and HER2 were screened from the nanoantibody library;

[0016] (2) Sequencing and synthesizing the nanobody sequences screened in (1), preparing recombinant plasmids and recombinant bacteria, respectively, and obtaining nanobody A1, A2 and nanobody B through recombinant expression and affinity purification;

[0017] (3) SPR measures the affinity of nanobodies to corresponding antigens, and nanobodies A1 and A2 with higher activity are used to construct bispecific antibodies;

[0018] (4) A flexible linker rich in lysine is designed to construct a fusion protein bispecific nanobody A, and a recombinant bacterium is prepared, and the bispecific nanobody A is obtained through recombinant expression and affinity purification;

[0019] (5) Reagent A is prepared by coupling FITC to the bispecific nanobody A, and reagent B is prepared by coupling TRITC to the nanobody B.

[0020] In a third aspect, the application provides a use of the rapid immunohistochemical double-staining kit, wherein the reagent A can bind to ER and / or PR in a sample, the reagent B can bind to HER2 in the sample, the two reagents can be added to the sample to be tested at the same time, and the binding is completed under the same reaction conditions and time, and finally, nuclear staining is performed with DAPI, and the results are observed under a fluorescence microscope, and the corresponding fluorescence can be observed in a single waveband, such as only green fluorescence or only red fluorescence; or the fluorescence can be observed in a full waveband, such as green, red and blue fluorescence.

[0021] Compared with the prior art, the application has the following beneficial effects:

[0022] The application provides an immunohistochemical double-staining reagent, which comprises reagent A that can recognize ER and PR and reagent B that can recognize HER2, and the two reagents are added to a biological sample at the same time, and the recognition of the three antigens is completed under the same incubation conditions and in the same time, one-step binding is not required, and the secondary antibody and color development steps are not required, thereby greatly simplifying the experimental operation and saving time. After the reaction is completed, the results are observed under a fluorescence microscope, and the staining results of one or two antigens can be observed alone, or the staining results of three antigens can be observed simultaneously, the fluorescence signal is strong, and the results are clear. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a binding dissociation kinetics sensing diagram of nanobodies A1, A2 and nanobody B prepared in Example 4 and corresponding antigens;

[0024] Figure 2 is a linker structure diagram in Example 5;

[0025] Figure 3 is a confocal image obtained after reagent A in Example 6 is used for immunofluorescence staining of a cell slice;

[0026] Figure 4 is a confocal image obtained after reagent A-GS in Comparative Example 1 is used for immunofluorescence staining of a cell slice;

[0027] Figure 5 This is a confocal image obtained after immunofluorescence staining of tissue sections using the rapid immunohistochemistry double staining kit of the present invention in Example 7. DETAILED DESCRIPTION

[0028] The following examples are given to illustrate the specific embodiments of the present invention, but the embodiments of the present invention are not limited to the following examples. Any selection and modification can be made within the scope that does not affect the technical effects to be achieved by the present invention. The technical terms and abbreviations used in the present invention have the conventional meanings known to those skilled in the art; unless otherwise specified, all materials in the following examples were purchased from commercial channels.

[0029] The above and other aspects of the present invention will be further described below, in which:

[0030] (1) Unless otherwise specified or defined, all terms used have their common meanings in the art and are well known to those skilled in the art.

[0031] (2) Unless otherwise indicated, the term "sequence" is used herein (e.g., in similar terms such as "antibody sequence", "variable region sequence", "V HH The term "sequence" or "protein sequence" should generally be understood to include the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the amino acid sequence, unless the context requires a narrower interpretation.

[0032] (3) Unless otherwise indicated, all methods, steps, techniques and operations not specifically described are known and familiar to those skilled in the art, for example, by reference to the cited general background art and other references.

[0033] (4) Amino acid residues are shown according to the standard three-letter or one-letter amino acid code.

[0034] (5) The term "specificity" refers to the ability of a particular antigen-binding molecule (such as a Nanobody or polypeptide of the invention) to bind to different types of antigens or antigenic determinants. The specificity of an antigen-binding molecule can be determined based on its affinity and / or activity. Affinity is expressed as the dissociation equilibrium constant (K) between the antigen and the antigen-binding molecule. D ), is a measure of the binding strength between the antigen and the antigen-binding molecule, K D The smaller the value, the stronger the binding strength between the antigen and the antigen-binding molecule. D The larger the value, the weaker the binding strength between the antigen and the antigen-binding molecule. a represents the binding constant, K a The larger the value, the faster the binding. a The smaller the K, the slower the binding; drepresents the dissociation constant, K d The larger the K, the faster the dissociation. d The smaller the K, the slower the dissociation; and D = K d / K a .

[0035] The specific binding between the above-mentioned antigen and the antigen-binding molecule can be determined by any suitable known method, including Sercatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), as well as other novel methods known in the art, such as plasma resonance technology (SPR) and / or biomembrane interferometry (BLI) technology.

[0036] As is well known to those skilled in the art, the affinity of antigens and antibodies detected by different methods may vary greatly, even by two orders of magnitude. The assay method used in the present invention is the SPR method.

[0037] The Nanobodies, polypeptides, and nucleic acids encoding the same of the invention can be prepared in a known manner, as will be apparent to those skilled in the art from the further description herein. A particularly useful method for preparing such Nanobodies, polypeptides, and nucleic acids generally comprises the following steps:

[0038] (1) expressing a nucleic acid encoding said Nanobody or polypeptide of the invention in a suitable host cell or host organism or in another suitable expression system, optionally followed by;

[0039] (2) Isolating and / or purifying the Nanobodies or polypeptides of the invention thus obtained.

[0040] Alternatively, other methods may be used, including the following steps:

[0041] (3) culturing and / or maintaining the host of the present invention under conditions such that the host of the present invention expresses and / or produces a Nanobody and / or polypeptide of the present invention; optionally followed by;

[0042] (4) Isolating and / or purifying the Nanobodies or polypeptides of the invention thus obtained.

[0043] Example 1: Nanobody library construction

[0044] The phage display library used in the present invention is an immune library with T7 phage as the carrier, and the establishment steps are as follows:

[0045] (1) Immunize alpacas and extract total RNA. Antigens were purchased from commercial channels: recombinant protein ER was purchased from MedChemExpress LLC, catalog number HY-P70248; recombinant protein PR was purchased from Beijing Biolab Technology Co., Ltd., catalog number ZN1701; recombinant protein HER2 was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number 10004-H08H-B. 1 mL of 100 μg / mL recombinant protein ER, PR, and HER2 were mixed with an equal volume of Freund's complete adjuvant, and two alpacas (numbers: 230501 and 230502) were immunized once every 1 to 2 weeks, for a total of 7 immunizations. After the fourth immunization, jugular vein blood was collected from the two alpacas, peripheral blood lymphocytes were isolated, and total RNA was extracted (PuerLink TM RNA Mini Kit, Life Technologies: 12183018A).

[0046] (2) Total RNA was reverse transcribed into cDNA, and the VHH gene was amplified using two rounds of nested PCR.

[0047] In the first round of PCR, cDNA was used as a template, with UP primer 1 and DOWN primer 1 as upstream and downstream primers, respectively. After amplification, a band of 650 to 750 bp was recovered, which was used as a template for the second round of PCR. The upstream and downstream primers of the second round of PCR were UP primer 2 and DOWN primer 2, respectively, and a PCR product of 450 to 500 bp was recovered.

[0048] UP primer1: 5'-GATGGTGGTCCTGGCTGCTCT-3' (SEQ ID NO.8);

[0049] DOWN primer1: 5'-GGTACGTGCTGTTGAACTGTTAG-3' (SEQ ID NO.9);

[0050] UP primer2: 5'-TACGGTAGTCGAATTCCGCCCAGGTGCAGCTC-3' (SEQ ID NO. 10);

[0051] DOWN primer2: 5'-AGCGACTAAGCTTTGAGGAGACGGGACAC-3' (SEQ ID NO. 11).

[0052] (3) The PCR product was double-digested with EcoRI and HindIII, and subjected to agarose electrophoresis to recover the 400-500 bp gene band, which is the VHH gene fragment.

[0053] (4) Ligate T7 vector (T7 Serelect® 10-3 Cloning Kit, Merck Millipore Novagen®: 70550-3) and VHH gene fragment with T4 ligase.

[0054] (5) Mix the ligation product with packaging proteins to form complete T7 phage, and amplify the mixture to obtain a phage original library.

[0055] (6) The titer of the original library is 8.82 x 10 9 pfu / mL, and the diversity is 8.9 x 10 6 .

[0056] Example 2: Nanobody screening

[0057] (1) Bacterial culture

[0058] Inoculate E. coli BLT5403 in Carb-LB liquid medium at a volume percentage of 1%, and culture on a shaker at 37°C and 170 rpm until the OD600 is 0.5-1.0. Estimate the cell density according to the empirical formula for E. coli cell density; ;

[0059] (2) Phage infection and titer determination

[0060] Set the infection multiplicity to 0.001-0.01, and mix the phage and host bacteria E. coli BLT5403 according to this standard. The culture conditions are 37°C and 170 rpm, and the cell lysis is observed at any time. Once lysis is complete, the culture is immediately stopped, and the precipitate is discarded by centrifugation (10000 x g, 15 min). The supernatant is recovered to determine the phage titer.

[0061] Prepare a double-layer medium. The lower medium is Carb-LB solid medium, and the upper medium is Carb-LB solid medium mixed with phage and bacterial liquid. The mixing ratio of the upper medium is: 100 μL of phage with different dilution multiples, 300 μL of host bacteria liquid, and 4 mL of Carb-LB solid medium that has not yet solidified. Mix well and quickly pour into a culture dish containing the lower medium to spread evenly. Three parallel samples are prepared for each sample, and a sample without phage is set as a blank control. After the upper medium solidifies, invert and incubate in a 37°C constant temperature incubator, and observe the appearance of phage plaques at any time. Stop the culture when the size is appropriate. Count the plates with 40-400 phage plaques, and calculate the phage titer (number of phage plaque-forming units per milliliter of sample, pfu / mL) according to the dilution multiple.

[0062] Phage enrichment was performed as follows:

[0063] a) Antigen coating: Add 100 μL of 5 μg / mL antigen to each well and incubate at 37°C for 1 hour and then at 4°C for 8 hours.

[0064] b) Blocking. After antigen coating, wash the 96-well plate three times with 1× TBS and then add 300 μL of skim milk powder to each well for blocking. The blocking conditions were incubation at 37°C for 1 hour and then at 4°C for 8 hours.

[0065] c) Add phage. After blocking, wash the 96-well plate five times with 1× TBST solution. Calculate the volume of phage to be added. If the volume is less than 100 μL, add sterile LB medium. Premix the phage with blocking agent for 1 hour, then add the plate to the 96-well plate and shake at room temperature for 1 hour.

[0066] d) Phage elution. After incubation, discard the supernatant and wash the 96-well plate five times with 1× TBST and five times with 1× TBS. Add 100 μL of T7 elution buffer to each well and shake at 100 rpm at 37°C for 30 minutes to elute the phage. Pipette the eluate into sterile LB medium and determine the phage titer as described above. Calculate the recovery rate:

[0067] .

[0068] e) Enrich phage. Amplify the eluate and determine the titer of the amplified phage. The next round of screening uses the amplified product from the previous round of eluate. After completing two rounds of enrichment, calculate the enrichment. Repeat this process until the enrichment reaches approximately 1.0, then stop enrichment and proceed to the next step. Enrichment is calculated as follows:

[0069] .

[0070] (3) Screening of nanoantibodies

[0071] a) Anti-ER antibody screening:

[0072] First, dilute the ER antigen to 10 μg / mL in TBS, add 100 μL to a 96-well plate, and incubate at 4°C for 12 hours. Aspirate the antigen dilution, wash the plate three times with TBS, pat dry, and add 300 μL / well of 1% protein-free blocking buffer (purchased from Sangon Biotech Co., Ltd.) for 2 hours at room temperature. (Alternate between 1% protein-free blocking buffer and 1% BSA during screening.) Aspirate the blocking buffer, wash the plate six times with TBST, pat dry, and add 100 μL / well of amplified phages for 30 minutes at room temperature. Wash the plate 10 times with TBST, elute the phages with T7 elution buffer (1% SDS), incubate at room temperature for 30 minutes, and amplify the eluate for the next round of screening. This is the anti-ER nanobody library.

[0073] b) Anti-PR antibody screening: The coated antigen is PR, and the other steps are the same as a). This is the anti-PR nanobody library.

[0074] c) Anti-HER2 antibody screening: The coated antigen is HER2, and the other steps are the same as a). This is the anti-HER2 nanobody library.

[0075] Example 3: Construction of genetically engineered bacteria

[0076] The same operation was performed on the three nanobody libraries:

[0077] (1) After four rounds of screening, the screening eluate was solid amplified, and plaques were picked. PCR amplification was performed using the plaque amplification solution as a template and UP primer 3 and DOWN primer 3 as upstream and downstream primers.

[0078] UP primer3: 5'-TTGATTAACATATGGCCCAGGTGCAGCTCGT-3' (SEQ ID NO. 12);

[0079] DOWN primer3: 5'-TTAAGGAACTCGAGCACGGTGACCAGCCTC-3' (SEQ ID NO. 13).

[0080] (2) A portion of the PCR product is outsourced for sequencing to obtain the nanoantibody sequence information.

[0081] (3) Another portion of the PCR product was double-digested with NdeI and XhoI, and the digestion product was recovered. Simultaneously, the digestion product and the vector were recovered using the same method, and the digestion product and the vector were ligated with T4 ligase. The ligation product was then transformed into Escherichia coli to obtain a genetically engineered bacterium expressing a specific nanobody.

[0082] Example 4: Nanobody Preparation and Activity Detection

[0083] Preparation of Nanobodies:

[0084] (1) The basic culture medium of the nanobody is TB medium, which is inoculated at a 1% inoculum and cultured at 37°C for 4 h. The inducer IPTG (final concentration 0.25 mM, the same below) is added for overnight induction.

[0085] (2) After induction, centrifuge at 4000 rpm for 20 min to obtain bacteria containing nanoantibodies.

[0086] (3) Add lysis buffer (10 mM imidazole, 500 mM NaCl, pH 7.4, 0.02 M PB) to the obtained bacteria at a ratio of 1:10, and use a 700 bar high-pressure homogenizer to disrupt the cells.

[0087] (4) Centrifuge at 4°C and 10,000 rpm for 20 min and collect the supernatant.

[0088] (5) The supernatant was filtered through a 0.45 μm filter and then passed through an affinity chromatography column (GE Healthcare, US) for separation and purification of the nanoantibodies, where the affinity chromatography column was filled with Ni Sepharose High Performance.

[0089] (6) The nanoantibodies purified by affinity chromatography were subjected to SDS-PAGE electrophoresis to determine the purity, and the protein solution with higher purity was selected to determine the protein concentration using the BCA method.

[0090] Nanobody activity detection:

[0091] The binding ability of nanobodies to antigens was analyzed using SPR technology. Antigens ER, PR, and HER2 were amino-coupled to a CM5 sensor chip at densities ranging from 500 to 800 RU. Nanobodies were injected at seven different concentrations ranging from 1 to 100 nM, with a flow rate of 45 μL / min in all experiments. Chip regeneration was performed using glycine-HCl, pH 1.5. Binding curves obtained at different nanobody concentrations were used to calculate the kinetic parameters Ka, Kd, ​​and KD. The nanobody with the highest affinity for ER was designated Nanobody A1 (SEQ ID No: 1), the nanobody with the highest affinity for PR was designated Nanobody A2 (SEQ ID No: 2), and the nanobody with the highest affinity for HER2 was designated Nanobody B (SEQ ID No: 4). It is recommended to select several nanobodies with high affinity. These nanobodies can be used as comparisons in subsequent breast cancer testing. Figure 1The middle curves are the response curves of the nanobody at concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 1.5625 nM from top to bottom. The kinetic parameters shown in Table 1 were calculated by equation fitting.

[0092] Table 1 Kinetic parameters of nanobodies

[0093] Antibody ka(1 / Ms) kd(1 / s) kD(M) A1 <![CDATA[2.63×10 4 ]]> <![CDATA[2.20×10 -4 ]]> <![CDATA[8.37×10 -9 ]]> A2 <![CDATA[1.86×10 4 ]]> <![CDATA[5.71×10 -4 ]]> <![CDATA[3.06×10 -8 ]]> B <![CDATA[3.76×10 4 ]]> <![CDATA[8.62×10 -4 ]]> <![CDATA[2.29×10 -8 ]]>

[0094] Example 5: Fusion protein expression

[0095] (1) Linker design. The linker is an important part of the fusion protein, which plays the role of connecting and spacing the two functional domains. Direct fusion of the functional domains without a linker may lead to many adverse consequences, including misfolding of the fusion protein, low yield or no expression, impaired activity, etc. Therefore, selecting or rationally designing a suitable linker is an important part of recombinant fusion protein technology. The amino acid sequence of the flexible linker designed in the present invention is shown in SEQ ID No: 5. Its amino acid composition is mostly hydrophilic amino acids, which can help improve the solubility of the protein, and is rich in lysine, which provides reaction sites for subsequent fluorescent modification and can reduce or avoid the decrease in antibody activity due to non-site-specific modification. Its structure is as shown in Figure 2 As shown, the overall structure of the linker is a flexible structure with a high degree of freedom. The yellow is the N-terminus (G) of the linker, which is used to connect to the C-terminus of the nanoantibody A1; the pink is the C-terminus (T) of the linker, which is used to connect to the N-terminus of the nanoantibody; the blue is the lysine residue (K), which is used to couple FITC.

[0096] (2) Bispecific Nanobody A sequence. The structure of bispecific Nanobody A is A1-linker-A2-Lys-tag-His-tag, and the amino acid sequence of bispecific Nanobody A is SEQ ID No. 3. The bispecific Nanobody A sequence is outsourced for full synthesis to obtain a vector containing the sequence, and a recombinant expression bacterium is constructed.

[0097] (3) The preparation and activity detection of bispecific Nanobody A were carried out as described in Example 4. The SPR technology was used to analyze the binding ability of Nanobody A to ER and PR respectively. The response curves of Nanobody A at concentrations of 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, and 1.5625nM were plotted, and the kinetic parameters shown in Table 2 were calculated by equation fitting. This indicates that bispecific Nanobody A has the ability to bind to both ER and PR simultaneously.

[0098] Table 2 Kinetic parameters of bispecific nanobody A

[0099] Antibody ka(1 / Ms) kd(1 / s) kD(M) ER <![CDATA[3.73×10 4 ]]> <![CDATA[8.20×10 -5 ]]> <![CDATA[2.20×10 -9 ]]> PR <![CDATA[2.06×10 4 ]]> <![CDATA[7.50×10 -4 ]]> <![CDATA[3.64×10 -9 ]]>

[0100] Example 6: Preparation of Rapid Immunohistochemistry Double Staining Kit

[0101] The rapid immunohistochemistry double-staining kit of the present invention comprises reagent A and reagent B. Reagent A is formed by coupling bispecific nanoantibody A with FITC. Reagent A can bind to ER and PR and exhibit green fluorescence under a fluorescence microscope. Reagent B is formed by coupling nanoantibody B with TRITC. Reagent B can bind to HER2 and exhibit red fluorescence under a fluorescence microscope.

[0102] FITC and TRITC were purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0103] The specific preparation process of reagent A is as follows:

[0104] (1) Prepare coupling buffer: 0.1 M sodium carbonate buffer (pH = 9).

[0105] (2) Dissolve bispecific nanobody A in coupling buffer at a concentration of 1 mg / mL.

[0106] (3) Dissolve 200 μg of FITC in 200 μL of coupling buffer and immediately mix with 1 mL of the liquid prepared in step (2).

[0107] (4) Incubate at 37°C in the dark for 1 hour.

[0108] (5) Using Zeba TM Unbound FITC was removed by filtration chromatography using a desalting spin column (Product No. 89891) to obtain bispecific nanobody A coupled to FITC, namely, reagent A of the present invention.

[0109] The specific preparation process of reagent B is as follows:

[0110] (1) Prepare coupling buffer: 0.1 M sodium carbonate buffer (pH = 9).

[0111] (2) Dissolve nanobody B in coupling buffer at a concentration of 1.2 mg / mL.

[0112] (3) Dissolve 200 μg of TRITC in 200 μL of DMSO.

[0113] (4) Take 35 μL of the liquid prepared in step (3) and slowly add it to 1 mL of the liquid prepared in step (2) while stirring and mix well.

[0114] (5) Incubate at room temperature in the dark for 2 hours.

[0115] (6) Using Zeba TM Unbound TRITC was removed by filtration chromatography using a desalting spin column (Product No. 89891) to obtain Nanobody B coupled to TRITC, i.e., Reagent B of the present invention.

[0116] Example 7: Rapid immunohistochemistry double staining kit for biological sample identification

[0117] MCF-7 cells (ER, PR, HER2 positive) were inoculated into the tail vein of mice and fed normally until tumor tissue could be observed. The tumor tissue was then excised and paraffin sections were prepared.

[0118] Dilute Reagent A 10-50 times and Reagent B 10-50 times. Take 100 μL of each, mix well, and then drop onto the slice.

[0119] Incubate at room temperature for 30 minutes in the dark. Wash away any unbound antibodies with PBS, stain the nuclei with DAPI, and mount the slides with an anti-fluorescence attenuation agent (such as PBST or Glycerol) to preserve the fluorescent signal and facilitate microscopic observation.

[0120] Images were captured using a fluorescence confocal microscope. Images were collected at different excitation wavelengths, such as 490-495 nm for FITC, 540-550 nm for TRITC, and 340-350 nm for DAPI. Figure 5 As shown, green fluorescence indicates ER and PR recognized by reagent A, red fluorescence indicates HER2 recognized by reagent B, and blue fluorescence indicates the cell nucleus. This demonstrates that the rapid immunohistochemical double staining kit of the present invention can be used to accurately identify ER, PR, and HER2 in samples. After identification, the fluorescence images can be used to observe the staining results of each antigen individually or all antigens simultaneously, providing flexibility in experimental results.

[0121] Comparative Example 1: Preparation of bispecific nanobodies using GS linker as connecting peptide

[0122] A GS linker, typically a short peptide sequence composed of glycine (G) and serine (S), is used to connect two proteins or protein domains in protein engineering. This linker can be simplified to variants such as "GGGGS" or "G4S." The GS linker is a flexible linker that improves protein solubility and stability, minimally interferes with the function and structure of fusion proteins, and exhibits good compatibility with other proteins, making it suitable for the fusion expression of a variety of proteins. It is the most commonly used fusion protein linker.

[0123] The sequence of the GS linker used in the present invention is shown in SEQ ID No: 6.

[0124] The bispecific nanobody GS sequence was constructed. The structure of the bispecific nanobody GS is A1-GS linker-A2-Lys-tag-His-tag, and the amino acid sequence of the bispecific nanobody GS is SEQ ID No. 7. The bispecific nanobody GS sequence was outsourced for full synthesis to obtain a vector containing the sequence, and a recombinant expression strain was constructed.

[0125] (1) Experimental study on the effect of bispecific nanoantibody GS on the binding ability of ER and PR

[0126] The preparation and activity detection of the bispecific Nanobody GS were carried out as described in Example 4. The SPR technique was used to analyze the binding ability of Nanobody GS to ER and PR, respectively. The response curves of the Nanobody were plotted at concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 1.5625 nM, and the kinetic parameters were calculated by equation fitting as shown in Table 3. This indicates that the bispecific Nanobody GS has the ability to bind to both ER and PR simultaneously. Compared with the bispecific Nanobody A prepared in Example 5, the binding ability of the bispecific Nanobody GS to both ER and PR is reduced.

[0127] Table 3 Kinetic parameters of bispecific nanobody GS

[0128] Antibody ka(1 / Ms) kd(1 / s) kD(M) ER <![CDATA[1.83×10 4 ]]> <![CDATA[1.09×10 -5 ]]> <![CDATA[5.96×10 -9 ]]> PR <![CDATA[9.95×10 3 ]]> <![CDATA[9.60×10 -4 ]]> <![CDATA[9.65×10 -8 ]]>

[0129] (2) Bispecific nanobody A and bispecific nanobody GS for biological sample recognition

[0130] Preparation of a reagent for biological sample recognition using the bispecific nanobody GS: According to the method for preparing reagent A in Example 6, the bispecific nanobody GS with a GS linker was coupled to FITC to prepare reagent A-GS.

[0131] MCF-7 cells (ER, PR, HER2 positive) were cultured and adjusted to the optimal cell state, smeared on glass slides, and cell sections were prepared.

[0132] Dilute reagent A 10-50 times, take 100 μL, mix well and then add dropwise to slice 1.

[0133] Dilute the reagent A-GS by the same multiple, take 100 μL, mix well and then add it dropwise onto slice 2.

[0134] Incubate at room temperature for 30 minutes in the dark. After washing away unbound antibodies with PBS, sections 1 and 2 are stained for nuclei with DAPI and mounted with an anti-fluorescence attenuation agent (such as PBST or Glycerol) to preserve the fluorescent signal and facilitate microscopic observation.

[0135] The images were captured using a fluorescence confocal microscope. The images were collected at different excitation wavelengths, such as 490-495 nm for FITC and 340-350 nm for DAPI. Figure 3 and Figure 4 As shown, Figure 3 The green fluorescence in the middle represents the ER and PR recognized by reagent A, and the blue fluorescence represents the cell nucleus. Figure 4 The green fluorescence represents the ER and PR recognized by reagent A-GS, and the blue fluorescence represents the cell nucleus. By comparison, it was found that the fluorescence intensity of reagent A was greater and the effect was clearer. This is because: on the one hand, the linker of the bispecific nanobody A in reagent A is a lysine-rich linker designed by the present invention, and FITC can be effectively bound to the linker, which increases the fluorescence intensity of reagent A while avoiding the loss of nanobody activity caused by nonspecific binding of FITC; on the other hand, the affinity results of Example 5 and Comparative Example 1 show that the bispecific nanobody A has a stronger affinity for ER and PR, and can more effectively identify ER and PR antigens in the sample.

[0136] In summary, the rapid immunohistochemistry double-staining kit prepared by the present invention utilizes directly labeled antibodies to identify and visualize multiple antigens, which simplifies the traditional multiple immunohistochemistry steps, improves experimental efficiency, and reduces operation time. The antigen recognition process only takes 30 minutes, and does not require a color development step and can be directly used for detection, which greatly simplifies the experimental operation steps and saves several times of operation time. After the reaction is completed, the results are directly observed under a fluorescence microscope, which can provide clear fluorescent images, facilitating accurate analysis of the expression and localization of the antigens; and the staining results of each antigen can be observed separately, or the staining results of all antigens can be observed at the same time, providing flexibility in the experimental results. It has a wide range of applications and is suitable for experiments that require rapid multiple labeling, such as flow cytometry, cell culture, and analysis of tissue sections. It is particularly suitable for biomedical research, clinical diagnosis, and drug development for diseases such as breast cancer.

[0137] Obviously, those skilled in the art may make various changes and modifications to this application without departing from the spirit and scope of this application. Thus, if these modifications and variations of this application fall within the scope of the claims of this application and their equivalents, this application is intended to include these modifications and variations.

Claims

1. A rapid immunohistochemistry double staining kit, characterized in that: The kit comprises reagent A and reagent B, wherein the reagent A is a bispecific nanobody A coupled to FITC, and the amino acid sequence of the bispecific nanobody A is shown in SEQ ID No: 3; the reagent B is a nanobody B coupled to TRITC, and the amino acid sequence of the nanobody B is shown in SEQ ID No:

4.

2. The kit according to claim 1, wherein The bispecific nanobody A is composed of a nanobody A1 that recognizes ER and a nanobody A2 that recognizes PR connected by a linker, the amino acid sequence of A1 is shown in SEQ ID No: 1, the amino acid sequence of A2 is shown in SEQ ID No: 2, and the amino acid sequence of the linker is shown in SEQ ID No: 5; the nanobody B recognizes HER2.

3. A nucleic acid, characterized in that It encodes the bispecific nanobody A in the immunohistochemistry double staining kit as described in claim 1.

4. A nucleic acid, characterized in that It encodes the nanobody B in the immunohistochemical double staining kit as claimed in claim 1.

5. A recombinant vector or recombinant cell containing the nucleic acid according to claim 3 or 4.

6. The method for preparing the rapid immunohistochemistry double staining kit according to claim 1, wherein: The method comprises: (1) Immunize alpacas to prepare libraries and screen nanoantibodies that bind to ER, PR, and HER2 from the nanoantibody library; (2) Prepare recombinant plasmids and recombinant bacteria, express and purify nanoantibodies A1, A2 and B; (3) Screening nanobodies that bind to the corresponding antigens, and using nanobodies A1 and A2 to construct bispecific antibodies; (4) Design linker, construct fusion protein bispecific nanobody A, prepare recombinant bacteria, express and purify; (5) Bispecific nanobody A is coupled to FITC to prepare reagent A, and nanobody B is coupled to TRITC to prepare reagent B.

7. Use of an immunohistochemical double staining reagent in preparing a kit for detecting breast cancer, characterized in that: The immunohistochemical double staining reagent comprises reagent A and reagent B, wherein the reagent A is a bispecific nanobody A coupled to FITC, and the amino acid sequence of the bispecific nanobody A is shown in SEQ ID No: 3; the reagent B is a nanobody B coupled to TRITC, and the amino acid sequence of the nanobody B is shown in SEQ ID No: 4.

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