High-affinity rabbit single-chain recombinant antibody, its application and the resulting reagent kit

Abstract

This invention provides a high-affinity rabbit single-chain recombinant antibody, its application, and the resulting reagent kit, belonging to the field of immunology. The high-affinity rabbit single-chain recombinant antibody provided by this invention has the amino acid sequence shown in SEQ ID NO.1, named 111-scFv-Fc; and has the amino acid sequence shown in SEQ ID NO.5, named 86-scFv-Fc. The antibodies provided by this invention can recognize different antigenic determinants on the surface of avian leukosis virus (ALV). When applied to the detection of trace amounts of ALV, they exhibit advantages such as high specificity, high sensitivity, and good stability. The combination of the two single-chain recombinant antibodies shows high efficiency in ALV antigen detection, meeting the requirements for ALV detection in clinical samples, and has significant implications for clinical diagnosis and research applications.

Description

Technical Field

[0001] This invention belongs to the field of immunology, and particularly relates to a high-affinity rabbit single-chain recombinant antibody, its application, and the resulting reagent kit. Background Technology

[0002] Avian leukosis (AL) is an infectious and toxic tumor disease caused by avian leukosis viruses (ALV), characterized by the proliferation of hematopoietic cells in birds. It has a worldwide distribution and endemic nature, and can infect various chicken flocks worldwide. It is one of the tumor diseases that harm poultry.

[0003] ALV is mainly composed of four structural proteins: surface glycoproteins, transmembrane proteins, matrix proteins, and capsid proteins. The genome, from the 5' to the 3' end, is 5'R-US-gag-pol-env-U3-R3', with the most conserved gag gene encoding nucleocapsid proteins (p12, p15, p19, and p27). Studies have shown that the p27 protein is highly conserved, exhibiting up to 90% homology among exogenous ALV subgroups (A, B, C, D, J). The p27 structure contains numerous easily detectable viral antigenic sites, making it a preferred antigen for preparing detection antibodies. However, avian leukosis virus (ALV) has high cultivation requirements and is difficult to purify, thus preparing antibodies against this virus is not easy, and detection methods based on it are few and not yet widely used. Summary of the Invention

[0004] This invention provides a high-affinity rabbit single-chain recombinant antibody, its application, and the resulting reagent kit. The antibody can recognize different antigenic determinants on the surface of avian leukosis virus and has advantages such as high specificity, high sensitivity, and good stability when applied to the detection of trace amounts of avian leukosis virus.

[0005] To address the aforementioned problems, the present invention provides a high-affinity rabbit single-chain recombinant antibody having the amino acid sequence shown in SEQ ID NO.1, named 111-scFv-Fc; and having the amino acid sequence shown in SEQ ID NO.5, named 86-scFv-Fc.

[0006] Preferably, the rabbit single-chain recombinant antibody 111-scFv-Fc includes a heavy chain variable region V consisting of 108 amino acids as shown in SEQ ID NO. 3. H and the light chain variable region V, consisting of 113 amino acids, as shown in SEQ ID NO. 4. L .

[0007] Preferably, the rabbit single-chain recombinant antibody 111-scFv-Fc comprises rabbit single-chain recombinant antibody 111-scFv, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] Preferably, the rabbit single-chain recombinant antibody 86-scFv-Fc includes a heavy chain variable region V consisting of 116 amino acids as shown in SEQ ID NO. 7. H and the light chain variable region V, consisting of 114 amino acids, as shown in SEQ ID NO. 8. L .

[0009] Preferably, the rabbit single-chain recombinant antibody 86-scFv-Fc comprises rabbit single-chain recombinant antibody 86-scFv, whose amino acid sequence is shown in SEQ ID NO.6.

[0010] Preferably, the heavy chain variable region V of the rabbit single-chain recombinant antibody 111-scFv-Fc or rabbit single-chain recombinant antibody 86-scFv-Fc is... H With light chain variable region V L It is linked by a linker peptide, the amino acid sequence of which is shown in SEQ ID NO.9.

[0011] Preferably, the light chain constant region of the rabbit single-chain recombinant antibody 111-scFv-Fc or the rabbit single-chain recombinant antibody 86-scFv-Fc is the κ chain, and the heavy chain constant region is of the IgG1 type.

[0012] This invention provides a reagent or kit for detecting avian leukosis virus, comprising the high-affinity rabbit single-chain recombinant antibody described in any of the above technical solutions.

[0013] Preferably, the avian leukosis virus is selected from any one of the following: recombinantly expressed avian leukosis virus P27 protein, feces of chickens infected with avian leukosis virus, and egg white infected with avian leukosis virus.

[0014] This invention provides the application of the high-affinity rabbit single-chain recombinant antibody according to any of the above technical solutions in establishing a highly sensitive enzyme-linked immunosorbent assay (ELISA) method for detecting avian leukemia virus P27 protein.

[0015] Preferably, the enzyme-linked immunosorbent assay (ELISA) method is a double-antibody sandwich ELISA method.

[0016] Preferably, in the double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) method, the capture antibody is rabbit single-chain recombinant antibody 111-scFv-Fc, and the detection antibody is rabbit single-chain recombinant antibody 86-scFv-Fc labeled with horseradish peroxidase.

[0017] Preferably, the rabbit single-chain recombinant antibody 111-scFv-Fc and the rabbit single-chain recombinant antibody 86-scFv-Fc bind to different antigenic determinants on the surface of avian leukosis virus.

[0018] In summary, the advantages and positive effects of this invention are as follows:

[0019] The immunogen used in this invention to prepare avian leukosis virus single-chain recombinant antibodies is the full-length avian leukosis virus P27 protein, which is recombinantly expressed in vitro by Escherichia coli and has been verified to have biological activity; the preparation method is a single-chain recombinant antibody development technology based on phage single-chain antibody library screening and culture.

[0020] This invention successfully developed high-affinity rabbit single-chain recombinant antibodies 111-scFv-Fc and 86-scFv-Fc against avian leukosis virus using phage display library screening technology. The phage display library screening technology was used to develop ECs that bind to the 111-scFv-Fc antibody and the recombinant expression of avian leukosis virus P27 protein. 50 2.699×10 -1 μg / mL, EC binding of antibody 86-scFv-Fc and recombinant avian leukosis virus P27 protein 50 It is 1.813×10 -1 μg / mL. Experiments have shown that these two antibodies can recognize different antigenic determinants on the surface of avian leukosis virus (ALV) and can be used to develop a double-antibody sandwich ELISA kit. The double-antibody sandwich ELISA kit developed using these antibodies has advantages such as high specificity, high sensitivity, and good stability. The establishment of this methodology provides a kit that can stably detect trace levels of ALV, which is of great significance in clinical diagnosis and scientific research applications. Attached Figure Description

[0021] Figure 1 The construction of the ALV P27-scFv phage library and the diversity analysis of the immune library provided in the embodiments of the present invention include: (A) antibody titer detection of three immunizations in New Zealand rabbits; (B) amplified bands of VL gene library and VH gene library, M: Marker, 1: VH, 2: VL; (C) VL-linker-VH gene library amplified by overlap extension PCR, M: Marker, 1: VL-linker-VH target band.

[0022] Figure 2The screening of high-affinity ALV P27-scFv provided in the embodiments of the present invention includes: (A) PCR identification results after electroporation; (B) the diversity of the antibody library after screening by the first round of phage display; (C) the diversity of the antibody library after screening by the second round of phage display; and (D) the identification of 9 scFv antibodies that bind highly specifically to the antigen ALV P27 by the second round of phage ELISA.

[0023] Figure 3 The ligation of ALV P27-scFv and pTT5 provided in the embodiments of the present invention includes: (A) a structural diagram of the ALV P27-scFv-Fc vector; (B) gel electrophoresis results of ALV P27-scFv-Fc, M: Marker, 1-9: ALV P27-scFv-Fc-pTT5 plasmid, 10-18: ALV P27-scFv-Fc target band and pTT5 vector after restriction endonuclease digestion with sfiI; (C) SDS-PAGE results of the ALV P27-scFv-Fc recombinant antibody, M: Marker, 1-9: ALV P27-scFv-Fc recombinant antibody target bands 10-1, 2-3, 5-6, 6-3, 68, 86, 87, 96, and 111.

[0024] Figure 4 EC20 is a rabbit single-chain recombinant antibody against avian leukosis virus that binds to recombinantly expressed avian leukosis virus P27 protein. 50 Wherein (A) is the EC of 86-scFv-Fc bound to recombinant avian leukosis virus P27 protein. 50 (B) is the EC2 binding of 111-scFv-Fc to recombinant avian leukosis virus P27 protein. 50 . Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Strains, plasmids, experimental animals and reagents

[0027] Adult New Zealand rabbits were purchased from Yongkang Qingyuan Breeding Farm, Dingyuan County, Anhui Province; ALV P27 protein was prepared and preserved in our laboratory; human IgG Fc-pTT5 plasmid was preserved in our laboratory; HRP-labeled recombinant protein G was purchased from Sangon Biotech (Shanghai) Co., Ltd.; HRP-labeled mouse anti-M13K07 phage was purchased from Chengdu Apak Biotechnology Co., Ltd.; recombinant TEV protease, pET32a plasmid, pComb3xss plasmid, E. coli DH5α and shuffle T7 competent cells were purchased from Shandong Hertz Biotechnology Co., Ltd.; M13K07 helper phage and E. coli ER2738 competent cells were purchased from Nanjing Heming Yinggu Biotechnology Co., Ltd.; SDS-PAGE protein gel kit was purchased from Yamei Biotechnology Co., Ltd.; penicillin-streptomycin solution was purchased from GE; HRP labeling kit was purchased from Beijing MecoWander Biotechnology Co., Ltd.; Freund's adjuvant was purchased from Sigma-Aldrich; ClonExpress II One Step Cloning Kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.; Premix taq and Plasmid mini were also purchased. The kit, gel extract, and transfection reagent were all purchased from TaKaRa.

[0028] Example 1: Preparation of rabbit monoclonal antibody against avian leukosis virus

[0029] 1. Animal Immunization: To obtain rabbit monoclonal antibodies recognizing avian leukosis virus (ALV), purified ALV P27 protein was used as an immunogen to immunize New Zealand white rabbits. Each rabbit was immunized with 100 mg of the immunogen. For the first immunization, the immunogen was mixed with an equal volume of Freund's complete adjuvant to prepare an emulsion, which was injected subcutaneously at multiple sites on the back of the neck and back. Seven days later, 0.5 mL of blood was collected intravenously, and the serum was separated and stored. Two weeks later, 100 mg of the immunogen was mixed with an equal volume of Freund's incomplete adjuvant to prepare an emulsion, which was injected subcutaneously at multiple sites on the back of the neck and back. Seven days later, 0.5 mL of blood was collected intravenously, and the serum was separated and stored. Two booster immunizations were performed. After the third immunization, the serum titer was measured using ELISA. Seven days after the third immunization, the rabbits with high serum titers were sacrificed, and 0.5 mL of blood was collected, the serum was separated and stored, and the spleen was dissected and removed for lymphocyte isolation. Figure 1 A).

[0030] 2. Extraction of total RNA from lymphocytes and amplification of antibody light and heavy chain genes: Total RNA was extracted from lymphocytes using Trizol reagent. Using this RNA as a template, cDNA was synthesized via reverse transcription using Oligo(dT) primers. Primer sequences (SEQ ID NO. 10-SEQ ID NO. 21) were designed based on the light and heavy chain framework regions of the rabbit antibody sequence for PCR amplification of antibody V. H and V LThe coding region was determined. PCR reaction conditions were: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 60 s, 35 cycles, followed by a final extension at 72℃ for 5 min. The PCR product was verified by 1% agarose gel electrophoresis to show a target band at approximately 350 bp. The purified product was then recovered for later use. Figure 1 B).

[0031] 3. Construction of ALV P27-scFv antibody library: Using purified VH and VL as templates, a linker (sequence SEQ ID NO.9) was inserted between VH and VL after overlap PCR to synthesize the scFv fragment VL-linker-VH, which contains sfiI restriction enzyme sites at both ends.

[0032] First round of overlapping extension PCR: Without primers, denature at 98℃ for 1 min, anneal at 45℃ for 30 s, extend at 72℃ for 1 min, repeat 10 cycles, and finally extend at 72℃ for 5 min. Then, add primers overlapping PCR-FP and overlapping PCR-RP (sequences SEQ ID NO. 22~SEQ ID NO. 23), denature at 98℃ for 30 s, then anneal at 58℃ for 30 s, extend at 72℃ for 1 min, repeat 30 cycles, and finally extend at 72℃ for 5 min. The PCR product was verified by 1% agarose gel electrophoresis to yield the target band VL-linker-VH (VH) at approximately 750 bp. Figure 1 C), the amplified products were recovered by gel extraction and stored at -20°C.

[0033] The PCR products and pComb3XSS vector recovered from the gel were digested with the restriction endonuclease sfiI at 50℃ for 30 min. The digested products were verified by 1% agarose gel electrophoresis, purified by column recovery, and ligated with T4 DNA ligase to obtain the scFv-pComb3XSS library. The ligation product was transformed into ER2738 competent cells, and positive clones were screened using tetracycline and ampicillin-resistant 2YT (2×Yeast extract and Tryptone) solid medium. Twenty single clones from the library were randomly selected for PCR identification. Figure 2 A). Wash all clones on the solid medium with 2YT medium and store at 4°C.

[0034] 4. Phage amplification: Take 2 mL of the library bacterial culture stored at 4℃ and inoculate it into 50 mL of 2YT liquid medium, and activate it at 37℃ and 200 rpm to OD. 600When the pH reaches 0.8–1.0, add M13K07 helper phage at a final concentration of 1 × 10¹² pfu / mL for infection. After standing for 30 min, add kanamycin (final concentration 50 μg / mL) and incubate overnight at 30°C and 200 rpm. The next day, centrifuge at 10,000 rpm at 4°C for 20 min, retain the supernatant, and add 1 / 2 volume of PEG8000. Incubate on ice for 2 h to precipitate the phage. Centrifuge at 10,000 rpm at 4°C for 20 min and discard the supernatant. Resuspend the precipitate with 5 mL PBS, add 3 mL PEG8000 for reprecipitation, repeat the centrifugation process, resuspend the precipitate with 4 mL PBS, filter for sterilization, and store at 4°C for later use.

[0035] Example 2 Antibody Screening and Identification

[0036] 1. ALV P27-scFv Affinity Screening: ALV P27 protein was biotinylated beforehand. The amplified svFv library phage, Dynabeads M-280 magnetic beads labeled with ALV P27 protein, and 2% skim milk blocking solution were mixed and incubated at room temperature for 1 hour. The magnetic beads were washed 5–10 times with PBST. The phage on the surface of the magnetic beads was eluted with 0.1 M Glycine-HCl (pH = 2.0), and then neutralized to pH 7.0 with 1 M Tris. The neutralized phage was used to infect ER2738 competent cells in logarithmic growth phase, and the cells were cultured and enriched on LB solid medium. Colonies were harvested for the next round of screening.

[0037] Repeat the above experiment twice. Twenty single clones were randomly selected from the two rounds of enriched affinity screening libraries for PCR validation to assess the diversity of the ALV P27-scFv library. Figure 2 B, 2C)

[0038] 2. Phage ELISA: The purified ALV P27 antigen was coated onto the ELISA plate at a concentration of 50 μg / mL per well and incubated overnight at 4°C. The plate was washed five times with PBST solution containing 0.05% Tween-20, the PBST was discarded, and 2% skim milk blocking buffer was added. The plate was blocked at 37°C for 1 h. The plate was washed five times with PBST solution, the residual liquid in the wells was patted dry, and the antibody supernatant to be screened was mixed with 2% skim milk blocking buffer at a ratio of 3:2. After incubation at room temperature for 10 min to remove interference, the mixture was added to the ELISA plate and incubated at 37°C for 1 h. The plate was washed five times with PBST solution, the residual liquid in the wells was patted dry, and HRP-labeled mouse anti-M13K07 antibody (diluted 1:3000 with blocking buffer) was added. The plate was incubated at 37°C for 1 h. Wash five times with PBST solution, pat dry any residual liquid in the wells, add TMB colorimetric solution, let stand in the dark for 15 min, then add 2 mol / L H2SO4 to stop the color development, and read the absorbance at 450 nm. Figure 2 As shown in D, nine highly sensitive scFv antibodies were screened: 10-1, 2-3, 5-6, 6-3, 68, 86, 87, 96, and 111.

[0039] 3. Homologous recombination and expression of the ALV P27 single-chain recombinant antibody ALV P27-scFv-Fc

[0040] The recombinant pComb3XSS-scFv plasmid and the human IgG-Fc-pTT5-linked plasmid were digested with restriction endonuclease sfiI at 50℃. The products were identified by agarose gel electrophoresis and then recovered from the gel. The ALV P27-scFv-Fc-pTT5 plasmid was obtained by ligation using T4 DNA ligase. Figure 3 A). The product was transformed into E. coli DH5α competent cells for amplification, and the target band was verified by sfiI restriction enzyme digestion and sequencing. Figure 3 B). The recombinant plasmid ALV P27-scFv-Fc-pTT5 was extracted and transfected into HEK293f cells via PEI for eukaryotic expression. After 7 days, the culture supernatant was collected by centrifugation at 10,000 rpm and 4℃ for 20 min. The antibody was purified using a Protein A affinity chromatography column, and the fusion protein was finally preserved in PBS solution.

[0041] The purified sample was added to the reduced protein loading buffer, denatured, and then detected by SDS-PAGE electrophoresis. The results are as follows: Figure 3 As shown in Figure C, the target protein was detected at approximately 50 kDa.

[0042] 4. Indirect ELISA method for detecting the binding of single-chain recombinant antibody ALV P27-scFv-Fc to ALV P27 antigen: The affinity of ALV P27-scFv-Fc to ALV P27 antigen was determined using an indirect ELISA method. Antigen was added to the ELISA plate and incubated overnight at 4°C, followed by blocking with 2% skim milk powder blocking buffer at 37°C for 2 h. The antibody was diluted in 12 gradients with 2% skim milk powder and added to the wells, then incubated at 37°C for 1 h. HRP-labeled goat anti-rabbit IgG secondary antibody was added, and a colorimetric reaction was performed using TMB. The absorbance was measured at 450 nm using an ELISA reader.

[0043] The binding efficacy of the above antibodies to ALV P27 was detected as follows: Figure 4 As shown, ALV P27-scFv-Fc can effectively bind to the ALV P27 protein, and the binding of antibody 86-scFv-Fc and recombinant avian leukosis virus P27 protein to EC2 is also shown. 50 It is 1.813×10 -1 μg / mL ( Figure 4 A), the binding of antibody 111-scFv-Fc and recombinant avian leukosis virus P27 protein to EC 50 2.699×10 -1 μg / mL ( Figure 4 B).

[0044] Example 3 Antibody Characterization

[0045] The gene sequences of rabbit single-chain recombinant antibodies 111-scFv-Fc and 86-scFv-Fc were sequenced, and the amino acid sequences of these antibodies were translated using DNAMAN software, resulting in SEQ ID NO.1 and SEQ ID NO.5, respectively. Amino acid sequence analysis showed that the rabbit single-chain recombinant antibodies 111-scFv-Fc and 86-scFv-Fc are composed of a light chain variable region V... L and heavy chain variable region V H It is formed by linking peptides.

[0046] Among them, the rabbit single-chain recombinant antibody 111-scFv-Fc includes a heavy chain variable region V consisting of 108 amino acids, as shown in SEQ ID NO. 3. H and the light chain variable region V, consisting of 113 amino acids, as shown in SEQ ID NO. 4. L The rabbit single-chain recombinant antibody 86-scFv-Fc includes a heavy chain variable region V consisting of 116 amino acids, as shown in SEQ ID NO. 7. H and the light chain variable region V, consisting of 114 amino acids, as shown in SEQ ID NO. 8. LThe heavy chain variable region V of the rabbit single-chain recombinant antibody 111-scFv-Fc or rabbit single-chain recombinant antibody 86-scFv-Fc. H With light chain variable region V L It is linked by a linker peptide, the amino acid sequence of which is shown in SEQ ID NO.9.

[0047] Based on the above description, the presence of complementary regions for both the light and heavy chains in the rabbit single-chain recombinant antibodies 111-scFv-Fc and 86-scFv-Fc indicates that the antibodies possess complete antigen-binding capacity. Comparison of this amino acid sequence with existing antibody databases reveals that this is a novel rabbit single-chain recombinant antibody, which is one of the greatest innovations of this invention.

[0048] Example 4: Establishment of a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) based on anti-avian leukosis rabbit single-chain recombinant antibodies A and B.

[0049] Horseradish peroxidase (HRP) labeling method: Follow the instructions of the HRP labeling kit (6012-1).

[0050] The specific steps of the double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) are as follows: (1) Dilute the rabbit single-chain recombinant antibodies against avian leukemia virus (ALV) 10-1, 2-3, 5-6, 6-3, 68, 86, 87, 96 and 111 to 5 μg / mL with PBS buffer (pH=7.2~7.4) for coating, incubate overnight at 4℃, and wash the plate with washing buffer; (2) Block with 2% skim milk powder blocking buffer at 37℃ for 2 h; (3) Add 50 ng / mL standard sample (ALV P27 protein), 100 μL per well, incubate at 37℃ for 1 h, and wash the plate with washing buffer; (4) Add 10-1-HRP, 2-3-HRP, 5-6-HRP, 6-3-HRP, 68-HRP, 86-HRP, 87-HRP, 96-HRP and 111-HRP detection antibodies diluted 2000 times, 100 μL per well, and incubate at 37℃ for 1 h. h, wash the plate with washing solution; (4) add TMB colorimetric solution, develop color at room temperature in the dark for 10 minutes, add 2 mol / L H2SO4 to stop the color development, read the absorbance at 450 nm, and determine the best paired antibody.

[0051] Table 1 Results of antibody pairing experiment

[0052]

[0053] As shown in Table 1, after pairing the nine antibodies, when the capture antibody was 111-scFv-Fc and the detection antibody was 86-scFv-Fc, the OD... 450The highest value was found, so rabbit single-chain recombinant antibody 111-scFv-Fc was selected as the capture antibody, and horseradish peroxidase-labeled rabbit single-chain recombinant antibody 86-scFv-Fc was selected as the detection antibody. The combination of the two has the advantages of high specificity, high sensitivity and good stability when used for double antibody sandwich enzyme-linked immunosorbent assay, which is of great significance for clinical diagnosis and scientific research applications.

Claims

1. A high-affinity rabbit single-chain recombinant antibody, characterized in that, It can bind to the avian leukosis virus P27 protein. Its heavy chain variable region is shown in SEQ ID NO. 3, and its light chain variable region is shown in SEQ ID NO. 4; Alternatively, the heavy chain variable region is shown in SEQ ID NO. 7, and the light chain variable region is shown in SEQ ID NO.

8.

2. The high-affinity rabbit single-chain recombinant antibody according to claim 1, characterized in that, The heavy chain variable region and the light chain variable region are connected by a linker peptide, the amino acid sequence of which is shown in SEQ ID NO.

9.

3. The high-affinity rabbit single-chain recombinant antibody according to claim 1 or 2, characterized in that, Single-chain recombinant antibodies include amino acid sequences as shown in SEQ ID NO.2 or 6.

4. The high-affinity rabbit single-chain recombinant antibody according to claim 3, characterized in that, Its light chain constant region is the κ chain, and its heavy chain constant region is the IgG1 type.

5. The high-affinity rabbit single-chain recombinant antibody according to claim 4, characterized in that, Its full-length amino acid sequence is shown in SEQ ID NO.1 or 5.

6. A reagent or kit for detecting avian leukosis virus, characterized in that, Including the high-affinity rabbit single-chain recombinant antibody according to any one of claims 1-5.

7. The reagent or kit according to claim 6, characterized in that, The test sample for the reagent or kit is chicken feces or egg white.

8. The use of the high-affinity rabbit single-chain recombinant antibody according to any one of claims 1-5 in the preparation of an enzyme-linked immunosorbent assay (ELISA) reagent for the high-sensitivity avian leukemia virus P27 protein.

9. The application according to claim 8, characterized in that, The enzyme-linked immunosorbent assay (ELISA) reagent is a detection reagent based on the double-antibody sandwich ELISA method.

10. The application according to claim 9, characterized in that, In the double-antibody sandwich enzyme-linked immunosorbent assay reagent, the capture antibody is rabbit single-chain recombinant antibody 111-scFv-Fc, and the detection antibody is rabbit single-chain recombinant antibody 86-scFv-Fc labeled with horseradish peroxidase. The heavy chain variable region of the rabbit single-chain recombinant antibody 111-scFv-Fc is shown in SEQ ID NO. 3, and the light chain variable region is shown in SEQ ID NO. 4; The rabbit single-chain recombinant antibody 86-scFv-Fc heavy chain variable region is shown in SEQ ID NO. 7, and the light chain variable region is shown in SEQ ID NO.

8.

11. The application according to claim 10, characterized in that, The rabbit single-chain recombinant antibody 111-scFv-Fc and the rabbit single-chain recombinant antibody 86-scFv-Fc bind to different antigenic determinants on the surface of avian leukosis virus.

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