Rabbit-derived single-chain antibody for detecting liver cancer marker GPC3 and kit of rabbit-derived single-chain antibody

Abstract

The invention belongs to the technical field of immunodetection, and discloses a preparation and characterization method of a rabbit-derived single-chain antibody for detecting a liver cancer marker GPC3. The rabbit-derived single-chain antibody has the characteristics of small volume, high specificity, high stability and good sensitivity, and can be used in an immunoassay method for detecting a liver cancer marker GPC3; according to the method, a specific rabbit single-chain antibody is obtained through screening, expression and purification by using a phage display technology, a GPC3 antigen is coated on a solid-phase carrier, the rabbit single-chain antibody is used as a detection carrier, the binding force of the single-chain antibody and the antigen is identified, and the method is high in specificity, good in sensitivity and high in specificity. A novel single-chain antibody with high specificity and high sensitivity is provided for detection of GPC3 in serum.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, specifically to a rabbit-derived single-chain antibody and its kit for detecting the liver cancer biomarker GPC3. Background Technology

[0002] Liver cancer is one of the most common malignant tumors. Hepatocellular carcinoma (HCC) is the most common form of liver cancer. Despite improvements in treatment methods in recent years, the prognosis for HCC remains poor.

[0003] Glypican-3 (GPC3) is a glycoprotein highly expressed in hepatocellular carcinoma (HCC). Composed of 580 amino acids and 70 kDa in size, it contains a 40 kDa N-terminal subunit and a 30 kDa C-terminal subunit and plays a role in promoting tumor growth, invasion, and metastasis. GPC3 is highly expressed in HCC tissues but almost not expressed in normal adult organs, and is absent in both normal liver cells and hepatocytes from benign liver diseases such as hepatitis. Because GPC3 can be released from the cell surface, serum GPC3 levels are considered a potential indicator of HCC.

[0004] ELISA is a rapid, simple, and highly specific method for detecting antibody titers in large quantities of disease samples and serum. It has advantages such as high accuracy, high sensitivity, and good reproducibility.

[0005] Chinese patent CN120865419A discloses an avian single-chain antibody, a kit, and a detection method for detecting the liver cancer biomarker GPC3. The method uses a double-antibody sandwich method, using chicken single-chain antibody as the capture antibody and rabbit polyclonal antibody as the detection antibody to detect GPC3 antigen. However, it has the problem of insufficient sensitivity of chicken single-chain antibody, resulting in excessive dosage of chicken single-chain antibody, and it cannot be directly used as an indirect ELISA method to detect GPC3 antigen, which increases the time and complexity of the detection method.

[0006] Based on this, the technical problem that this invention needs to solve is: how to provide a rabbit-derived single-chain antibody that is small in size, highly specific, highly stable, and highly sensitive, so that the ELISA method containing this antibody has the advantages of being fast, accurate, and reliable for the detection of GPC3. Summary of the Invention

[0007] The purpose of this invention is to provide a rabbit-derived single-chain antibody that is small in size, highly specific, highly stable, and highly sensitive, making the ELISA method containing this antibody fast, accurate, and reliable for the detection of GPC3.

[0008] In addition, the present invention also provides a kit containing the antibody.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] A rabbit-derived single-chain antibody for detecting the liver cancer biomarker GPC3, wherein the rabbit-derived single-chain antibody comprises the FR1 region as shown in SEQ ID NO.1, the CDR1 region as shown in SEQ ID NO.2, the FR2 region as shown in SEQ ID NO.3, the CDR2 region as shown in SEQ ID NO.4, the FR3 region as shown in SEQ ID NO.5, and the CDR3 region as shown in SEQ ID NO.6.

[0011] Preferably, the linkage relationship of each region of the rabbit-derived single-chain antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3.

[0012] Preferably, the method for preparing the rabbit-derived single-chain antibody is as follows:

[0013] 1) Expression of GPC3 antigen TF-A: The full-length GPC3 gene was amplified using primers, digested with enzymes to construct an expression vector, and the expression vector was transformed into competent E. coli cells for expression. The GPC3 antigen TF-A target protein was then obtained by purification.

[0014] 2) Construct a phage library;

[0015] 3) Screening of rabbit-derived single-chain antibodies: The GPC3 antigen TF-A from step 1) was used as the panning coating antigen and added to the phage library constructed in step 2) to screen for positive phage particles; the obtained positive phage particles were expressed in competent E. coli cells and then purified by nickel column to obtain the rabbit-derived single-chain antibody.

[0016] More preferably, the specific method for constructing the phage library is as follows:

[0017] 1) New Zealand white rabbits were immunized with TF-A as the immunogen;

[0018] 2) Mononuclear cells were isolated from the spleen cells after immunization and resuspended in culture medium to obtain a cell resuspension.

[0019] 3) Extract total RNA from the cell resuspension according to the RNAiso Plus instructions to obtain total RNA;

[0020] 4) Using the extracted total RNA as a template, cDNA encoding the antibody gene was synthesized using Oligo(dT) primers and PrimeScript II reverse transcriptase to obtain a cDNA library;

[0021] 5) Using cDNA as a template, the heavy chain variable region gene (VH) and the light chain variable region gene (VL) were amplified by PCR. After agarose gel electrophoresis, the gel was cut, and the PCR products were purified and recovered using the Omega kit.

[0022] 6) Using the VH and VL genes obtained by gel extraction as templates, overlap PCR amplification was performed using primers to obtain the scFv antibody gene fragment;

[0023] 7) The vector pComb3X and the single-chain antibody gene fragment were digested with enzymes and then ligated.

[0024] 8) The ligation product of vector pComb3X and single-stranded gene fragment was electroporated into competent cells. The transformed cells were revived, and helper phages were added for infection. The infected cells were cultured, and the supernatant was collected. The phages were precipitated with PEG / NaCl and resuspended in PBS to obtain a phage library.

[0025] In addition, the present invention discloses a kit for detecting the liver cancer biomarker GPC3, the kit containing the rabbit-derived single-chain antibody as described above.

[0026] Preferably, the kit for detecting liver cancer markers further includes HRP enzyme anti-antibody, PBST buffer, and skim milk powder.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] The rabbit-derived single-chain antibody disclosed in this invention has the characteristics of small size, high specificity, high stability, and good sensitivity. It can be used in an immunoassay method for detecting the liver cancer marker GPC3. The method involves using the rabbit-derived single-chain antibody as a detection carrier to bind to the GPC3 antigen coated on a solid-phase carrier. Using this method, an ELISA immunoassay method for GPC3 is established. This method has high specificity and good sensitivity, providing a rapid, accurate, and reliable detection method for GPC3 in serum. Attached Figure Description

[0029] Figure 1 The map shows the expression vector pCOLD-TF-GPC3-A (TF-A).

[0030] Figure 2 SDS-PAGE electrophoresis image of TF-A protein;

[0031] Figure 3 SDS-PAGE electrophoresis image of rabbit-derived single-chain antibody expression;

[0032] Figure 4This is the GPC3 standard curve. Detailed Implementation

[0033] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0034] Raw material commodity information is shown in Table 1:

[0035] Table 1 Product Information Table

[0036] Experimental reagents Manufacturer Restriction endonucleases (BamH I, Xho I) Takara Agarose gel DNA recovery kit Omega High purity plasmid miniprep kit Omega IPTG Aladdin pCold TF plasmid ZOMANBIO / Zhuangmeng Biology Carbenicillin sodium Bidepharm T4 DNA ligase Takara 5× Loading buffer CW Biotech SDS-PAGE kit CW Biotech Bovine serum albumin Amresco BeyoGold His-tag Purification Resin (Reduction-resistant chelating type) Beyotime BL21(DE3)pLysS competent cells Sangon Biotech LB broth Huankai Microbial High fidelity enzyme PrimeSTAR HS DNA Polymerase Takara Anti-HA-Peroxidase, High Affin Roche

[0037] Example 1

[0038] A rabbit-derived single-chain antibody, the amino acid sequence of which is shown below:

[0039] SEQ ID NO.1 (FR1 region): AVTLDESGGGLQTPGGALSLVCKASGFTFS;

[0040] SEQ ID NO.2 (CDR1 region): KYQMN;

[0041] SEQ ID NO.3 (FR2 region): WVRQAPGKGLEFVA;

[0042] SEQ ID NO.4 (CDR2 region): AINKFANRTTYGAAVRG;

[0043] SEQ ID NO.5 (FR3 region): RATISRDNGQGTVRLQLNNLRAEDTGTYFCT;

[0044] SEQ ID NO.6 (CDR3 region): KYAYGHCGGGGVCGASSIDAWGHGTEVIVSSSTSGQAGQHHHHHHGAYPYDVPDYAS.

[0045] The preparation method of this rabbit-derived single-chain antibody is as follows:

[0046] 1) Expression of GPC3 antigen TF-A

[0047] Primers were designed and synthesized. The upstream primer sequence is shown in SEQ ID NO.7, and the downstream primer sequence is shown in SEQ ID NO.8. The full-length GPC3 gene (sequence shown in SEQ ID NO.9) was amplified, digested with BamHI and XhoI, and then ligated to the double-digested plasmid pCOLD-TF to construct the pCOLD-TF-GPC3-A (TF-A) expression vector. Its map is shown below. Figure 1 As shown. The expression vector was transformed into *E. coli* BL21(DE3)pLysS competent cells using the heat shock method. The cells were plated on carbenicillin plates, and single colonies were picked and cultured to select positive clones. Positive clones with correct sequencing results were induced to express the expression vector in large quantities and characterized by SDS-PAGE electrophoresis. The results are shown in the reference [reference needed]. Figure 2 (In the figure: M: Marker; 1: Empty vector expression; 2: Before induction; 3: After induction), after electrophoresis verification, the GPC3 antigen TF-A target protein with high purity was obtained by purification with 50% BeyoGold™ His-tag Purification Resin.

[0048] Table 2

[0049] Name Sequence Sequence number TF-A upstream primer GGCTCGAGCATGGAAGAAAAATACCAACTA SEQ ID NO.7 TF-A downstream primer ATTGGATCCTCAGTGCACCAGGAAGAAGAA SEQ ID NO.8 Full-length GPC3 gene MNHKVHHHHHHMQVSVETTQGLGRRVTITIAADSIETAVKSELVNVAKKVRIDGFRKGKVPMNIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKINPAGAPTYVPGEYKLGEDFTYSVEFEVYPEVELQGLEAIEVEKPIVEVTDADVDGMLDTLRKQQATWKEKDGAVEAEDRVTIDFTGSVDGEEFEGGKASDFVLAMGQGRMIPGFEDGIKGHKAGEEFTIDVTFPEEYHAENLKGKAAKFAINLKKVEERELPELTAEFIKRFGVEDGSVEGLRAEVRKNMERELKSAIRNRVKSQAIEGLVKANDIDVPAALIDSEIDVLRRQAAQRFGGNEKQALELPRELFEEQAKRRVVVGLLLGEVIRTNELKADEERVKGLIEEMASAYEDPKEVIEFYSKNKELMDNMRNVALEEQAVEAVLAKAKVTEKETTFNELMNQQASAGLEVLFQGPSAGLVPRGSGGIEGRHMELGTLEHGRKIPTNSTIEHGTAASVCKYGAQVLNYSECCGFPRGL SEQ ID NO.9

[0050] (ii) Construction of a phage-displaying nanobody library

[0051] 1. Animal immunization

[0052] A two-month-old female New Zealand white rabbit was used as the experimental animal. Four rounds of immunization were administered via intramuscular injection using TF-A as the immunogen. The primary immunization involved emulsifying Freund's complete adjuvant with an equal volume of immunogen and administering an intramuscular injection at a dose of 1.0 mg / rabbit. Booster immunizations were given every two weeks using Freund's incomplete adjuvant emulsified with an equal volume of immunogen and administered via injection at a dose of 0.5 mg / rabbit. The immunization protocol is shown in Table 3.

[0053] Table 3. Immunization Plan for Hens

[0054] Number of immunizations Interval (days) Immunization dose (μg) Adjuvant type Immune sites Initial exemption 0 1000 Freund's complete adjuvant Multiple injections on the back and subcutaneous tissue Two free 14 500 Freund's incomplete adjuvant Multiple injections on the back and subcutaneous tissue Three exemptions 14 500 Freund's incomplete adjuvant Multiple injections on the back and subcutaneous tissue Four Free 14 500 Freund's incomplete adjuvant Multiple injections on the back and subcutaneous tissue

[0055] 2. Amplification of single-chain antibody genes

[0056] 2.1 Isolation of mononuclear cells from the spleen

[0057] (1) Seven days after the last immunization, the white rabbits were euthanized by injecting air, disinfected by soaking in 75% alcohol, dissected on a UV workbench, the spleen was separated, the white fat on the spleen was gently removed with scissors, and placed in a culture dish containing RPMI-1640 culture medium.

[0058] (2) Place the isolated spleen in a sterile grinding steel mesh with a diameter of 200 mesh and grind it clockwise gently until the spleen is ground into connective tissue shape visible to the naked eye.

[0059] (3) Centrifuge the cell suspension obtained after grinding in a centrifuge tube at 1500g for 5 minutes at room temperature;

[0060] (4) Discard the supernatant, add 6 mL of RPMI-1640 culture medium to resuspend, and then transfer to a centrifuge tube containing 3 mL of lymphocyte separation medium. Keep the liquid level balanced and centrifuge at 2500g for 20 min.

[0061] (5) Take the middle white cloud layer, add it to 6 mL of RPMI-1640 culture medium, and then centrifuge at 1500g at room temperature for 5 min;

[0062] (6) Discard the supernatant and resuspend in 1 mL of RPMI-1640 culture medium.

[0063] 3. Total RNA extraction

[0064] Total RNA was extracted according to the RNAiso Plus instructions. The method is briefly described below:

[0065] (1) Pour the cell resuspension into a 1.5 mL centrifuge tube and centrifuge at 8000 g at 4 °C for 2 min;

[0066] (2) Discard the supernatant gently, being careful not to disturb the cell pellet at the bottom of the centrifuge tube;

[0067] (3) Add RNAiso Plus;

[0068] (4) Mix the lysed cells thoroughly until no obvious cell precipitate is visible to the naked eye in the lysate;

[0069] (5) Let stand at room temperature for 5 minutes;

[0070] (6) Add chloroform (1 / 5 RNAiso Plus, v / v), invert the container to mix thoroughly, and let stand at room temperature for 5 min;

[0071] (7) Centrifuge at 12000g at 4℃ for 15min;

[0072] (8) Carefully remove the centrifuge tube from the centrifuge. You can see that the lysate is clearly divided into three layers.

[0073] (9) Transfer the upper lysate to another new centrifuge tube;

[0074] (10) Add an equal volume of isopropanol to RNAiso Plus, mix thoroughly by inverting the container, and let stand at room temperature for 10 minutes.

[0075] (11) Place the centrifuge tube in the centrifuge and centrifuge at 12000g at 4℃ for 10min;

[0076] (12) Carefully discard the supernatant and add an equal volume of 75 vol% ethanol to RNAiso Plus;

[0077] (13) Place the centrifuge tube in the centrifuge and centrifuge at 7500g at 4℃ for 5min;

[0078] (14) Be careful to discard the Shangqing;

[0079] (15) Invert the centrifuge tubes and dry them at room temperature for 5 minutes;

[0080] (16) Add 25 μL of deionized water to dissolve the precipitate and obtain total RNA;

[0081] (17) The concentration and purity of the extracted RNA were measured by ultraviolet spectrophotometer and stored at -80℃ for later use.

[0082] 4. Total RNA reverse transcription to synthesize cDNA

[0083] Using the extracted total RNA as a template, cDNA encoding the antibody gene was synthesized using Oligo(dT) primers and PrimeScript II reverse transcriptase. The procedure is briefly described below:

[0084] (1) Add the following reagents to the PCR tube, as shown in Table 4:

[0085] Table 4

[0086] reagents Usage Oligo dT Primer (50μM) 1μL dNTP Mixture (10mM each) 1μL Total RNA 5μg RNase-free ddH2O Up to 10μL

[0087] (2) Place the PCR tube in the PCR instrument, keep it at 65℃ for 5 minutes, and then cool it rapidly on ice;

[0088] (3) Add the reverse transcription reaction solution described in Table 5, with a total reaction volume of 20 μL;

[0089] Table 5

[0090] reagents Usage The above reaction solution 10μL 5x PrimeScript II buffer 4μL RNase inhibitor (40 U / μL) 0.5 μL (20 U) PrimeScript II RTase (200 U / μL) 1Ml(200U) Deionized water UP to 20μL

[0091] (4) Mix thoroughly;

[0092] (5) Reverse transcription reaction conditions are referenced in Table 6:

[0093] Table 6

[0094] temperature time 30℃ 10min 42℃ 60min 95℃ 5min 4℃ Hold

[0095] (6) Store the cDNA library obtained by reverse transcription at -80℃ for later use.

[0096] 5. Amplification of single-chain antibody genes

[0097] Table 7

[0098]

[0099] (1) The heavy chain variable region gene (VH) and the light chain variable region gene (VL) were amplified by PCR. Using cDNA obtained from reverse transcription as a template, the antibody heavy chain variable region gene was amplified by using upstream and downstream primer pairs ⑦⑪, ⑧⑪, ⑨⑪, and ⑩⑪; the antibody light chain variable region gene (VL) was amplified by using upstream and downstream primer pairs ①④, ①⑤, ①⑥, ②④, ②⑤, ②⑥, ③④, ③⑤, and ③⑥. The following reagents were added to the PCR tubes, and the system was referenced in Table 8.

[0100] Table 8

[0101] reagents Usage Sterilized water To 50μL 10x PCR buffer 1μL 10mM dNTPmix 1μL Upstream primer (10mM) 1μL Downstream primer (10mM) 1μL cDNA 500ng PrimeStar HS High Fidelity Enzyme 1μL Total 50μL

[0102] (2) Place the PCR tube in the PCR amplification instrument and refer to Table 9 for the reaction conditions;

[0103] Table 9

[0104]

[0105] (3) After agarose gel electrophoresis, the gel was cut, and the PCR products were purified and recovered using the Omega kit. The light chain bands and heavy chain bands could be mixed together for recovery.

[0106] (4) Overlap PCR was used to amplify the scFv antibody gene. Using the VH and VL genes obtained by gel excision and recovery as templates, overlap PCR was performed with upstream primer RSC-F and downstream primer RSC-B to obtain the scFv antibody gene.

[0107] ① Add the following reagents to the PCR tube, referring to Table 10 for the system;

[0108] Table 10

[0109] reagents Usage Sterilized water To 50μL 10x PCR buffer 1μL 10mM dNTP mix 1μL Upstream primer (10 μM) 1μL Lower primer (10 μM) 1μL VH fragments are recycled by cutting rubber. 500ng VL segment of rubber cutting and recycling 500ng PrimeStar HS High Fidelity Enzyme 1μL Total 50μL

[0110] ② Place the PCR tube in the PCR amplification instrument, and refer to Table 11 for the reaction conditions;

[0111] Table 11

[0112]

[0113] ③ After PCR amplification, the product was detected by 1% agarose gel electrophoresis, and the target band was recovered to obtain the scFv antibody gene fragment. The concentration and purity of the recovered DNA were measured using NanoDrop and stored at -20℃ for later use.

[0114] 6. Enzymatic digestion reaction of pComb3X and single-chain antibody gene fragments

[0115] The digestion method was performed according to the instructions for the New Englang Biolab SfiI restriction enzyme. A brief description of the method is as follows:

[0116] (1) According to the enzyme digestion system in Table 12, add the various reagents to the PCR tube in sequence and mix well;

[0117] Table 12

[0118] reagents Usage 10x Buffer M 20μL pComb3X plasmid or scFv gene fragment 10μg SfiI endonuclease 18μL (360U) RNase-free ddH2O Up to 200μL

[0119] (2) Place the PCR tube in a PCR instrument and incubate overnight at 50°C;

[0120] (3) The next day, add 22.5 μL of 0.5 M EDTA and mix well;

[0121] (4) The enzyme digestion effect was detected by 1% agarose gel electrophoresis;

[0122] (5) The enzyme digestion products were recovered using an Omega gel recovery kit.

[0123] 7. Ligation of vector pComb3X and single-stranded gene fragments

[0124] The digestion method was performed according to the instructions for the New Englang Biolab SfiI restriction enzyme. A brief description of the method is as follows:

[0125] (1) Add the various reagents to the PCR tube in sequence according to the enzyme digestion system in Table 13 and mix well;

[0126] Table 13

[0127] reagents Usage pComb3X vector digested with SfiI enzyme 600ng SfiⅠ digested scFv fragment 1320 10x Ligase Buffer 20μL T4 DNA ligase 10μL RNase-free dH2O Up to 200μL

[0128] (2) Place the PCR tube in the PCR instrument and react at 16°C overnight;

[0129] (3) Heat inactivate T4 DNA ligase at 65℃ for 10 min;

[0130] (4) After adding 20 μL of 3M sodium acetate (pH 5.2), add 660 μL of anhydrous ethanol;

[0131] (5) Incubate the PCR tubes at -20°C overnight;

[0132] (6) Centrifuge at 4℃, 10000g for 30min;

[0133] (7) Discard the supernatant, resuspend the precipitate in 70% ethanol, and wash the precipitate.

[0134] (8) Centrifuge at 4℃, 10000g for 15min;

[0135] (9) Discard the supernatant, invert the PCR tube, and let it stand at room temperature for a few minutes to evaporate the residual ethanol in the PCR tube;

[0136] (10) Add 2 μL of sterile water to resuspend the precipitate and store at -20℃ for later use.

[0137] 7. Electroporation of the ligation product of vector pComb3X and single-stranded gene fragment

[0138] (1) Take the TG1 electrocompetent cells out of the -80℃ freezer and place them on ice to thaw for 10 min;

[0139] (2) Add 3 μL of the purified ligation product to each tube, mix well, and incubate on ice for 5 min.

[0140] (3) Quickly transfer the mixture of competent cells and ligation products to a pre-cooled electroporation cup;

[0141] (4) Electrical shock conversion under the conditions of 1.8KV, 200Ω, and 25μF;

[0142] (5) Quickly add 1 mL of preheated SOC medium at 37°C and resuspend the transformed cells;

[0143] (6) Repeat the above steps 5 times to introduce a total of 5 ligation products into TG1 electrocompetent cells.

[0144] (7) Place the transformed cells in a shaker and revive them at 37°C and 250 r / min for 1 hour;

[0145] (8) Take 2 μL of culture for titer determination, add the remaining culture medium to 100 mL of SB medium, add 100 μL of carbenicillin (50 μg / mL) and 100 μL of tetracycline (20 μg / mL), continue culturing for 2 hours, and then add 1 mL of 1 x 10¹³ cfu / mL helper phage VCSM13;

[0146] (9) Place the bacterial culture in a constant temperature incubator and let it stand at 37°C for 30 minutes to allow the bacteriophages to complete the infection;

[0147] (10) Incubate the bacterial culture at 37℃ and 250r / min for 2 hours with shaking.

[0148] (11) Two hours later, kanamycin was added to a final concentration of 70 μg / mL;

[0149] (12) Place the bacterial culture in a shaker at 37°C and 250 r / min and continue to shake and culture overnight;

[0150] (13) The next day, the bacterial solution was centrifuged at 10000g for 20 minutes at 4℃, and the supernatant was collected.

[0151] (14) Add 50 mL of 5x PEG / NaCl solution to the supernatant, mix thoroughly, and then incubate on ice for 2 hours;

[0152] (15) Centrifuge at 4℃, 10000g for 20min;

[0153] (16) Discard the supernatant and invert the centrifuge tube onto absorbent paper to dry for 30 minutes;

[0154] (17) Add 1 mL of PBS solution to the centrifuge tube and resuspend all the phage pellets to obtain the avian single-chain antibody phage library.

[0155] The constructed single-chain antibody phage library was stored at -80℃ for later use.

[0156] (iii) Screening of rabbit-derived single-chain antibodies

[0157] (1) The GPC3 antigen TF-A expressed in step one) was used as the selection coating antigen, diluted to 10 μg / mL with coating buffer, and added to the microplate at 100 μg / well. The plate was coated overnight at 4°C.

[0158] (2) Wash the plate 3 times with PBST, pat dry with absorbent paper, add 350 μL / well of 3wt% skim milk powder solution, and block at 37℃ for 1h;

[0159] (3) Wash the plate 3 times with PBST, pat dry with absorbent paper, add 100 μL / well 1×10¹² pfu / mL of the phage display nanobody library constructed in step 2) (labeled as R1 Input), and incubate at 37 ℃ for 1.5 h;

[0160] (4) Add 200 μL of PBST containing 0.05 vol% Tween-20 and vigorously pipette 10 times to remove the washing solution. Then add another 200 μL of PBST containing 0.05 vol% Tween-20 and vigorously pipette 10 times to remove the washing solution. Repeat this process for a total of 10 washes to remove unbound phages. Pat dry with absorbent paper.

[0161] (5) Add 100 μL / well of 0.1 M glycine-HCl (pH= 2.2) for acid elution, let stand for 2 min, and add 50 μL / well of 1 M Tris-HCl (pH= 8.8) to neutralize;

[0162] (6) Collect the liquid in the well plate, take 2 μL of the collected liquid (marked as R1 Output) to determine the titer, and transfer the remaining liquid to 2 mL of freshly cultured ER2738 bacterial solution, and incubate at 37℃ for 30 min.

[0163] (7) Add 6 mL of LB medium preheated to 37°C and 8 μL of 50 mg / mL carbenicillin sodium, and continue culturing for 2 h;

[0164] (8) Add another 1 mL of 1×10 11 pfu / mL helper phage M13K07, 91 mL LB medium and 92 μL 50 mg / mL carbenicillin sodium, and continue culturing for 2 h;

[0165] (9) Add kanamycin sulfate to bring the final concentration to 70 μg / mL and continue culturing overnight;

[0166] (10) The next day, centrifuge at 11,000 rpm for 20 min at 4℃, collect the supernatant, add 50 mL of 2.5 M PEG / NaCl solution and mix thoroughly, then let stand on ice for 2 h; centrifuge at 11,000 rpm for 30 min at 4℃, and discard the supernatant. Add PBS: 2.5 M PEG / NaCl at a volume ratio of 4:1 to resuspend the precipitate, let stand on ice for 2 h; centrifuge at 11,000 rpm for 30 min at 4℃, discard the supernatant, invert the centrifuge tube on absorbent paper and dry for 3 min; add 1 mL of PBS to dissolve the precipitate, take 2 μL for titer determination, transfer the remainder to a 1.5 mL centrifuge tube, label it R2 Input, and store at -80℃. A total of four rounds of biological panning were performed. The panning conditions in the second, third, and fourth rounds, including the concentration of Tween-20, the number of washes, and the concentration of the coating antigen, were more stringent than those in the previous round. The specific selection criteria are shown in Table 14.

[0167] Table 14 Selection Criteria

[0168] Number of selections Coating antigen concentration (μg / mL) Number of washes Tween-20 concentration (vol%) Round 1 10 10 0.05 Second round 1 15 0.1 Third round 0.1 20 0.5 Fourth round 0.01 30 0.5

[0169] Single colonies were randomly selected from the Output plates after the second, third, and fourth rounds of selection and inoculated into 3 mL of CA-resistant LB medium. The plates were then incubated at 37°C and 250 rpm until the OD reached the target value. 600 =0.8; Take 2 mL of the culture and add 20 μL of 1 mL 1×10 11Pfu / mL helper phage M13K07 was incubated at 37°C for 15 min; then cultured at 37°C and 250 rpm for 2 h; kanamycin sulfate was added to a final concentration of 70 μg / mL, and cultured overnight. The next day, the supernatant was collected by centrifugation. Specific positive phage clones were finally screened using an indirect competitive phage-ELISA method. Positive clones were sent to Shanghai Sangon Biotech for sequencing; correctly sequenced positive phage plasmids were extracted using a plasmid extraction kit and stored at -20°C.

[0170] (iii) Expression of rabbit-derived single-chain antibodies

[0171] Transform 1 μL of the positive phage particles prepared in step two) into 100 μL of *E. coli* TOP10F' competent cells, plate them onto carbenicillin-resistant plates, and incubate overnight at 37°C. Pick single colonies and incubate overnight at 37°C with shaking in 5 mL of CA-resistant LB medium. Inoculate the bacterial suspension at a 1:1000 ratio into 5 mL of CA-resistant LB medium and incubate overnight at 37°C with shaking at 220 rpm. The next day, add the bacterial suspension at a 1:100 ratio to 500 mL of CA-resistant LB medium and incubate at 37°C with shaking at 220 rpm until the bacterial growth rate reaches OD. 600 =Approximately 0.8, take 1 mL of the pre-induction bacterial culture and store at 4℃; add IPTG to a final concentration of 0.5 mM, and induce overnight at 30℃ and 220 rpm. On the third day, centrifuge at 8000 rpm for 10 minutes to collect the bacterial cell pellet, lyse the pellet with lysis buffer, centrifuge the lysis product at 4℃ and 16000 rpm for 5 minutes, collect the supernatant of the bacterial lysis buffer, and mix with 50% BeyoGold™ His-tag Purification Resin at 4℃ with shaking overnight. The next day, load the mixture into an empty affinity chromatography column tube, wash with PBS and 2 mM imidazole PBS sequentially to remove contaminating proteins, and finally elute the target antibody protein with 50 mM imidazole PBS. Finally, perform SDS-PAGE electrophoresis for detection. The detection results are as follows: Figure 3 (In the figure, M: Marker; 1: Before induction; 2: After induction; 3: Flow-through; 4: Purified antibody), the results prove that rabbit-derived single-chain nanobody was successfully expressed.

[0172] Example 2

[0173] An ELISA kit for detecting the liver cancer marker GPC3 contains the rabbit single-chain antibody of Example 1, HRP enzyme-resistant antibody, phosphate buffer and Tween 20 PBST, and skim milk powder.

[0174] Example 3

[0175] An ELISA method for detecting the liver cancer marker GPC3 using an ELISA kit prepared in Example 2.

[0176] Determination of the standard curve

[0177] (1) Coating GPC3 antigen TF-A: Coating 5 μg / mL GPC3 antigen TF-A with carbonate buffer (pH=9.6), 100 μL / well, overnight at 4℃, and washing 4 times with PBST (phosphate buffer + 0.001 vol% Tween 20).

[0178] (2) Blocking: Add 3wt% skim milk powder solution (dissolved in PBS), 350μL / well, incubate at 37℃ for 2 hours, and then wash 4 times with PBST (phosphate buffer + 0.001vol% Tween 20).

[0179] (3) Dilute rabbit single-chain antibody at different ratios and add it: Dilute rabbit single-chain antibody (concentration 0.3 mg / mL) 100, 200, 400, 800, 1600, 3200 and 6400 times in sequence, add PBS in the last row, 100 μL / well, incubate at 37°C for 1 hour, and wash 4 times with PBST (phosphate buffer + 0.001 vol% Tween 20).

[0180] (4) Add 100 μL of Anti-HA-Peroxidase, High Affin (1:10000) diluted with PBS to a well. After incubating at 37°C for 1 hour, wash 5 times with PBST (phosphate buffer + 0.001 vol% Tween 20).

[0181] (6) Color development: Add 100 μL of TMB color development solution to each well. After reacting for 15 minutes, add 50 μL of 0.1 M concentrated sulfuric acid to terminate the reaction. Measure the OD at 450 nm using an ELISA reader.

[0182] (7) Plotting the standard curve for ELISA immunoassay: Using Origin 8.5 software, with absorbance as the ordinate and the rabbit single-chain antibody dilution factor as the abscissa, a curve was fitted to obtain the standard detection curve for GPC3 enzyme-linked immunosorbent assay, as shown below. Figure 4 As shown.

[0183] Depend on Figure 4 Analysis shows that the antibody dilution factor and absorbance fit a curve relationship. When using rabbit-derived single-chain antibody to detect GPC3 antigen, the absorbance is still about 0.5 when the single-chain antibody is diluted 1600 times, indicating that the method has high sensitivity and specificity, and provides a highly sensitive rabbit single-chain antibody for GPC3 enzyme-linked immunosorbent assay.

Claims

1. A rabbit-derived single-chain antibody for detecting the liver cancer biomarker GPC3, characterized in that, The rabbit-derived single-chain antibody comprises the FR1 region as shown in SEQ ID NO.1, the CDR1 region as shown in SEQ ID NO.2, the FR2 region as shown in SEQ ID NO.3, the CDR2 region as shown in SEQ ID NO.4, the FR3 region as shown in SEQ ID NO.5, and the CDR3 region as shown in SEQ ID NO.

6.

2. The rabbit-derived single-chain antibody for detecting the liver cancer biomarker GPC3 according to claim 1, characterized in that, The connection relationship of each region of the rabbit-derived single-chain antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3.

3. A reagent kit for detecting liver cancer markers, characterized in that, Includes the GPC3 rabbit-derived single-chain antibody as described in claim 1 or 2.

4. The reagent kit for detecting liver cancer markers according to claim 3, characterized in that, It also includes HRP enzyme anti-antibody, PBST buffer, and skim milk powder.

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