Avian leukosis antigen detection kit and preparation method thereof

Abstract

The present invention discloses an avian leukosis antigen detection kit and a preparation method thereof. The kit comprises an anti-avian leukosis virus P27 protein monoclonal antibody and a horseradish peroxidase-labeled sheep anti-P27 polyclonal antibody as a sandwich secondary antibody. The kit is based on a double-antibody sandwich method and has good specificity for A / B, J, and K subtype avian leukosis viruses prevalent in breeder chickens. The minimum detection limit is 1 ng / mL, the kit has strong anti-interference ability, a low false positive rate, and is easy to operate and highly stable. It is suitable for scenarios such as breeder chicken flock purification, SPF embryo quality monitoring, and epidemiological surveys. It effectively solves the problem of expensive imported products and limited domestic promotion, and has significant economic and social benefits.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology detection, and in particular to an avian leukosis antigen detection kit and a preparation method thereof. Background Art

[0002] Due to the lack of effective vaccines and treatments for avian leukosis, pathogen detection, culling of positive chickens, and stock purification have become the primary means of controlling avian leukosis. However, to date, there is no vaccine or effective treatment for avian leukosis. The primary means of controlling avian leukosis is through pathogen detection, culling of positive chickens, and stock purification.

[0003] Currently, avian leukosis (ALL) detection, both domestically and internationally, primarily relies on ELISA technology. Companies such as IDEXX in the United States have commercialized ALL antigen detection kits, but these are expensive and limited to a limited number of settings, such as large-scale enterprises and SPF breeder farms. This makes them difficult to scale up to the vast majority of small and medium-sized chicken farms in my country, severely hindering the decontamination of domestic breeder flocks. Research on related detection technologies has been underway in China since 1991, and while an ELISA method has been established, the kits have yet to be commercialized. Existing technologies suffer from significant drawbacks: Lack of specificity: The antibodies used in traditional methods have limited recognition of subtypes of ALL viruses, such as A / B, J, and K, prevalent in breeder chickens, leading to missed or false positives; Low sensitivity: When using rabbit antibodies as sandwich secondary antibodies, antibody yields are low and affinity is insufficient, making it difficult to detect low antigen concentrations; and High cost: The reliance on imported key raw materials results in high production costs for the kits, limiting their widespread adoption.

[0004] In addition, the existing detection kits have weak anti-interference ability for clinical samples, and the false positive rate is high in the detection of complex samples (such as meconium and cloacal swabs). The operation process is cumbersome and the detection time is long, which cannot meet the batch testing needs of breeding farms.

[0005] To address the above-mentioned issues, the present invention is dedicated to developing an avian leukosis antigen detection kit with strong specificity, high sensitivity, and low cost. By using bioengineering technology to screen a high-affinity monoclonal antibody as the primary coating antibody and a goat anti-P27 polyclonal antibody as the secondary sandwich antibody, detection performance is significantly improved. Simultaneously, the kit's composition and detection process are optimized, making it more suitable for the large-scale purification needs of the domestic breeder chicken breeding industry. This effectively addresses the monopoly of imported products and the shortcomings of existing technologies, and has significant economic value and social significance. The present invention has broad application prospects in the domestic breeder chicken breeding industry. It can be used as a conventional technical means to purify avian leukosis in high-generation breeder chicken farms, for exogenous virus detection and product sampling in veterinary biological product factories and those of health departments, for monitoring the quality of raw materials (such as SPF embryos), for quality control in SPF chicken production enterprises, for clinical diagnosis and epidemiological surveys by provincial and municipal veterinary epidemic prevention departments, and for scientific research in colleges, universities, and research institutions. Through technology promotion, personnel training, and widespread application, the avian leukosis antigen detection kit of the present invention will bring significant economic and social benefits, contributing to the development of my country's poultry industry. Summary of the Invention

[0006] To address the shortcomings of the aforementioned prior art, the present invention provides an avian leukosis antigen detection kit and its preparation method. The monoclonal antibodies used in the kit are screened and produced using bioengineering methods, resulting in high sensitivity and specificity. A horseradish peroxidase-labeled sheep anti-P27 polyclonal antibody is used as a sandwich secondary antibody, achieving high sensitivity and yield. The avian leukosis antigen detection kit of the present invention exhibits excellent specificity and sensitivity for the different subtypes of avian leukosis virus (A / B, J, and K) currently prevalent in breeder chickens, meeting the requirements for clinical purification.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In one embodiment, the present invention provides an avian leukosis antigen detection kit, comprising an anti-avian leukosis virus P27 protein monoclonal antibody and a horseradish peroxidase-labeled sheep anti-P27 protein polyclonal antibody; the amino acid sequence of the P27 protein is shown in SEQ ID NO: 1.

[0009] In another embodiment, the anti-avian leukosis virus P27 protein monoclonal antibody of the present invention is screened and produced by bioengineering methods.

[0010] In another embodiment, the anti-avian leukosis virus P27 protein monoclonal antibody is coated on the solid phase carrier at a concentration of 2-10 μg / mL, and the working concentration of the horseradish peroxidase-labeled goat anti-P27 protein polyclonal antibody is 5-20 μg / mL.

[0011] In another embodiment, the solid phase carrier is an ELISA plate, and the kit is based on a double antibody sandwich method, with anti-avian leukosis virus P27 protein monoclonal antibody as the primary antibody and horseradish peroxidase-labeled sheep anti-P27 protein polyclonal antibody as the sandwich secondary antibody.

[0012] In another embodiment, the kit comprises a positive control and a negative control; the positive control is a P27 protein solution, and the negative control is SPF chicken serum.

[0013] In another embodiment, the kit further comprises a sample diluent, a TMB color developing solution, a stop solution, and a washing solution, wherein the sample diluent is a PBST buffer, and the washing solution is a phosphate buffer containing Tween-20.

[0014] In one embodiment, the present invention provides a bioengineering method for screening monoclonal antibodies against the P27 protein of avian leukosis virus, characterized in that it comprises the following steps: (1) immunizing rabbits with the P27 protein shown in SEQ ID NO:1 as an antigen to enrich IgG B cells; (2) screening high-affinity IgG B cells using fluorescently labeled P27 protein-specific epitope short peptides through flow cytometry; and (3) sequencing the BCR light chain and heavy chain genes of the screened B cells to obtain specific BCR sequences.

[0015] In one embodiment, the present invention provides a monoclonal antibody against the avian leukosis virus P27 protein screened and produced by the bioengineering method. In a preferred embodiment, the amino acid sequence of CDR-H1 of the heavy chain is shown in SEQ ID NO: 22; the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 23; and the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 24.

[0016] The amino acid sequence of CDR-L1 of the light chain is shown in SEQ ID NO: 25; the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 26; and the amino acid sequence of CDR-L3 is shown in SEQ ID NO: 27.

[0017] In one embodiment, the present invention provides a use of the monoclonal antibody in preparing a kit for detecting avian leukosis virus infection.

[0018] In one embodiment, the present invention provides a horseradish peroxidase-labeled sheep anti-P27 polyclonal antibody, which is prepared by immunizing sheep with the P27 protein shown in SEQ ID NO: 1 as an immunogen.

[0019] The beneficial effects achieved by the present invention are:

[0020] (1) The avian leukosis antigen detection kit and related technologies provided by the present invention significantly improve the accuracy, efficiency and economy of avian leukosis detection through bioengineering screening of monoclonal antibodies, optimization of sandwich secondary antibody system and system process design.

[0021] (2) The present invention has high specificity and broad-spectrum detection capabilities. The cross-subtype virus recognition capability is achieved by using a monoclonal antibody screened using bioengineering methods. It is designed against a conserved epitope on the P27 protein of avian leukosis virus and can simultaneously identify A / B, J, and K subtypes of viruses prevalent in breeder chickens, resolving the problem of insufficient coverage of multi-subtype virus detection in existing technologies. Experimental data show that this monoclonal antibody has no cross-reaction with various avian viruses and bacteria, such as NDV and IBDV, and its specificity is significantly superior to that of traditional mouse antibodies.

[0022] (3) The affinity constant of the monoclonal antibody for the precise antigen-antibody binding of the present invention is 7.6×10 9 L / mol, a high-affinity antibody with a low dissociation rate when binding to the P27 protein, ensures a stable signal during detection and reduces false-negative results. Western blotting results show that the monoclonal antibody produces a clear band at 30 kDa when binding to the P27 protein, confirming its high specificity.

[0023] (4) The present invention has excellent sensitivity and detection efficiency. The low-concentration antigen detection kit can detect a minimum of 1 ng / mL of P27 antigen, which is 5-10 times more sensitive than ordinary kits. In clinical sample testing, the positive detection dilution factor for complex samples such as meconium and virus isolation supernatant is significantly higher than that of international imported products. In particular, the detection ability for K subtype virus is better than that of imported reagents.

[0024] (5) The present invention uses a double antibody sandwich system to enhance the sensitivity of the secondary antibody, using a horseradish peroxidase-labeled goat anti-P27 polyclonal antibody as the sandwich secondary antibody. Its titer is >1:2,000,000, which is more than 10 times higher than the sensitivity of the traditional mouse antibody system (titer of approximately 1:1,000,000). The high yield of goat antibodies (50 mL of serum can be isolated from each sheep in a single blood draw) solves the bottleneck of low rabbit antibody yield, reducing production costs while ensuring detection signal strength.

[0025] (6) The present invention has stable performance and convenient operation. The full-process stability test kit can be stably stored for more than one year at 4°C and can maintain a detection efficiency of more than 70% after accelerated aging at 37°C for 9 days. The batch-to-batch consistency rate reaches 100%, and the vacuum sealing process of the coated plate avoids the attenuation of antibody activity, making it suitable for large-scale production and long-term storage.

[0026] (7) The rapid standardized operation and detection process of the present invention only takes 3 hours, including two incubation and color development times, and supports 96-well plate batch detection. It is suitable for high-throughput scenarios such as chicken breeder farms and biological product factories. The operation steps are highly standardized and do not require complex pre-treatment (samples such as serum and egg white can be directly tested), reducing human error.

[0027] (8) The present invention has significant cost advantages and industrial adaptability. The cost of preparing sheep anti-polyclonal antibodies is more than 60% lower than that of rabbit antibodies due to the localization of raw materials and low cost, and there is no need to rely on imported enzyme-labeled secondary antibodies. The monoclonal antibodies screened by bioengineering are mass-produced through the HEK293F cell expression system, avoiding the risk of ascites preparation contamination in traditional hybridoma technology and further reducing production costs. In line with domestic breeding needs, the detection threshold is optimized according to the characteristics of Chinese chicken breeds, and the clinical consistency rate with international imported products is more than 98%, but the detection cost is only 1 / 3 of that of imported products. It is suitable for scenarios such as purification of high-generation breeder farms and SPF embryo quality monitoring, promoting the independent purification of domestic breeder flocks.

[0028] (9) The bioengineering screening technology of the present invention has made a breakthrough by using flow cytometry combined with BCR sequencing technology to accurately screen high-affinity antibodies from tens of thousands of sequences. The screening efficiency is 50 times higher than that of traditional hybridoma technology, and the light chain / heavy chain gene sequences (SEQ ID NO: 7, 8) can be directly obtained, providing a basis for antibody engineering modification.

[0029] (10) Demonstration of the localization of the detection system of the present invention The present invention breaks the monopoly of imported test kits and establishes a full-chain localization technology from antigen preparation (recombinant engineering bacteria BL21 (DE3)), antibody screening to test kit assembly, promoting the independent control of avian leukosis detection reagents, with significant economic and social benefits.

[0030] In summary, the present invention achieves "high specificity, high sensitivity, low cost, and easy operation" for avian leukosis detection through innovation in bioengineering technology and optimization of the detection system, providing key technical support for disease prevention and control in my country's poultry industry, and has significant industrial application value and market competitiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the gel electrophoresis diagram of the PCR product of the P27 gene, where band 1 represents the DNA marker (5000 bp, 3000 bp, 2000 bp, 1500 bp, 800 bp, 500 bp, 300 bp) and band 2 represents the PCR amplification result of the P27 gene.

[0032] Figure 2The PCR identification results of the recombinant plasmid pGEM-T-P27 are shown. Band 1 represents DNA Marker (5000 bp, 3000 bp, 2000 bp, 1500 bp, 800 bp, 500 bp, 300 bp), band 2 represents the negative control using the empty vector pET-28a as the template, and band 3 represents the PCR amplification result of the recombinant expression plasmid pGEM-T-P27.

[0033] Figure 3 Figure 3 is the SDS-PAGE analysis of the purified cleavage product. Figure 3 is the SDS-PAGE analysis of the purified cleavage product, where band 1 represents low molecular weight standard protein markers (95 kDa, 72 kDa, 55 kDa, 43 kDa, 34 kDa, 26 kDa, 17 kDa, 10 kDa), band 2 represents the protein to be purified, band 2 represents the flowthrough, band 4 represents the impurity removal solution, and band 5 represents the NTA-1000 eluate.

[0034] Figure 4 This is the curve for determining the affinity constant of monoclonal antibodies. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0036] Example 1. Preparation of P27 antigen

[0037] The genetically engineered bacteria BL21 (DE3) used in the present invention are derived from the Escherichia coli K-12 strain; the recombinant genetically engineered bacteria E. coli BL21 (DE3) (P27) is a recombinant E. coli BL21 (DE3) containing the prokaryotic expression plasmid pET28a-P27, which was constructed, identified, and preserved in the inventor's laboratory; E. coli BL21 (DE3) was purchased from Takara Biotechnology (Dalian) Co., Ltd.

[0038] The P27 gene of avian leukosis virus (ALV) was cloned through genetic engineering methods and expressed in Escherichia coli BL21(DE3). This recombinant engineered bacterium, E. coli BL21(DE3), synthesizes the P27 protein. This engineered bacterium was further studied and controlled with an internationally imported avian leukosis diagnostic kit. The amino acid sequence of the P27 protein is shown in SEQ ID NO:1.

[0039] 1. Cloning of the Avian Leukosis Virus (ALV) gag Gene

[0040] 1. Experimental methods

[0041] (1) Virus proliferation: Chicken embryo fibroblasts (CEF) were routinely prepared using 9-11 day old SPF chicken embryos. After the cells grew into a monolayer, the culture medium was removed and 0.5 mL of avian leukosis virus (ALV) was inoculated. At the same time, uninfected cells were set up as negative controls. The cells were placed in a 37°C incubator for adsorption for 3 h. During this period, the cell bottle was gently shaken every 15-20 min to ensure that the inoculum fully contacted the cells. The virus solution was then discarded and an appropriate amount of DMEM containing 1% calf serum was added to maintain the culture for 5-7 days. The cells were repeatedly frozen and thawed twice and centrifuged at 5000 rpm / min for 10 min. The supernatant after centrifugation was detected using an internationally imported avian leukosis antigen detection kit.

[0042] (2) Viral RNA extraction and reverse transcription: The supernatant of cell lysis that tested positive was taken, and the total viral RNA was extracted by TRIzol method. The extracted RNA was reverse transcribed into cDNA using Promega reverse transcription kit.

[0043] (3) PCR reaction:

[0044] PCR reaction was performed using the cDNA obtained by reverse transcription as a template, 5'-CCATGCCTGTAGTGATTA-3' (SEQ ID NO: 2) as the upstream primer and 5'-CCCGACCCAGTTTGTCCAT-3' (SEQ ID NO: 3) as the downstream primer.

[0045] The reaction system was as follows: double-distilled water 13 μL; 10× PCR Buffer 2 μL; Taq DNA polymerase 0.5 μL; dNTPs (10 mmol / L each) 0.5 μL; upstream primer 1 μL, downstream primer 1 μL; and cDNA 2 μL.

[0046] The reaction conditions were: 95°C for 5 min, 94°C for 1 min, 57°C for 1 min, and 72°C for 1 min, for a total of 30 cycles; and 72°C for 10 min. Four μL of the PCR product was analyzed by 1% agarose gel electrophoresis.

[0047] (4) Ligation of RT-PCR products: Ligate the PCR product of the P27 gene to the pGEM-T Easy Vector (refer to the pGEM-T vector instructions for specific methods), and then transform JM109 competent cells by chemical method.

[0048] (5) Screening and identification of positive clones: Pick one blue colony of the negative control and several positive white colonies, inoculate them into 5 mL of LBA medium containing Amp, and culture them at 37°C overnight; perform plasmid mini-extraction on the culture, digest the white spot plasmid with EcoRI, and take the digestion products for electrophoresis analysis.

[0049] (6) gag gene sequencing: The recombinant plasmids identified as positive were sent to Beijing Saibaisheng Company for bidirectional sequencing, and the sequencing results were compared with the ALV standard strain (BAI-A strain).

[0050] 2. Experimental results

[0051] Viral RNA was extracted from ALV-infected CEF cells, and a band of approximately 2.1 kb was specifically amplified by RT-PCR. No band was amplified after RNA was extracted from uninfected CEF cells, indicating that the required ALV gag gene was successfully obtained.

[0052] Electrophoresis analysis of the digested product showed a 3 kb T vector band and a 2.1 kb target band, proving that the recombinant plasmid pGEM-T-gag with the exogenous target gene fragment inserted was obtained.

[0053] The sequencing results of the recombinant plasmid were compared with the sequence of ALV BAI-A strain, and the homology was 97%, indicating that the gene cloned in this experiment was ALV gag gene.

[0054] 2. Subcloning of the Avian Leukosis Virus (ALV) P27 Gene

[0055] 1. Experimental methods

[0056] (1) PCR amplification of the P27 gene: The recombinant plasmid pGEM-T-gag was used as a template, and 5'-CGGGATCCATGCCTGTAGTGATTAAGACAG-3' (SEQ ID NO: 4) was used as the upstream primer and 5'-ACGTCGACCTAGGGCTGGATAGCAGAC-3' (SEQ ID NO: 5) was used as the downstream primer for PCR reaction. While keeping the reading frame unchanged, a BamHI restriction site was introduced at the 5' end of the upstream primer and a SalI restriction site was introduced at the downstream primer. The PCR reaction was carried out using Ex-Taq high-fidelity enzyme. The reaction was pre-denatured at 95°C for 5 min, followed by 30 cycles of 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min. Finally, the reaction was carried out at 72°C for 10 min. The pGEM-T easy empty vector was used as a negative control. 4 μL of PCR product was electrophoresed on a 1% agarose gel (containing 0.5 μg / mL EB).

[0057] (2) Ligation of PCR products: Ligate the PCR product of the P27 gene to the pGEM-T Easy Vector (refer to the pGEM-T vector instructions for specific methods), and then transform JM109 competent cells by chemical method.

[0058] (3) Screening and identification of positive clones: Pick one blue colony of the negative control and several positive white colonies, inoculate them into 5 mL of LBA medium containing Amp, and culture them at 37°C overnight; perform plasmid mini-extraction on the culture, digest the white spot plasmid with EcoRI, and take the digestion products for electrophoresis analysis.

[0059] (4) P27 gene sequencing: The recombinant plasmids identified as positive were sent to Dalian Bao Biotechnology Co., Ltd. for sequencing, and the sequencing results were compared with the ALV standard strain (BAI-A strain).

[0060] 2. Experimental results

[0061] After electrophoresis of the PCR product, a specific band of approximately 800 bp appeared, which was consistent with the expected size ( Figure 1 ).

[0062] After electrophoresis analysis of the enzyme-digested products, a 3kb T-vector band and a specific target band of approximately 800bp appeared, while no target band appeared in the empty vector control, indicating that the recombinant plasmid pGEM-T-P27 was successfully constructed.

[0063] The sequencing results of the pGEM-P27 recombinant plasmid were compared with the sequence of ALV BAI-A strain, and the homology was 97%, indicating that the P27 gene was subcloned in this experiment.

[0064] 3. Construction of recombinant engineered bacteria

[0065] 1. Experimental methods

[0066] The recombinant plasmid pGEM-T-P27 and the empty vector pET-28a plasmid were double-digested with BamHⅠ and SalⅠ. The two enzyme-digested products were electrophoresed on 1.2% agarose gel respectively. The double-digested products of the recombinant plasmid pGEM-T-P27 were used to recover the approximately 800 bp P27 gene according to the operating procedures of the DNA rapid recovery kit. The double-digested products of the empty vector pET-28a plasmid were used to recover the linearized vector by ethanol precipitation and stored at -20℃ for future use.

[0067] The P27 gene with BamHI and SalI cohesive ends and the linearized expression vector pET-28a were mixed at a molar ratio of 3:1. T4 DNA ligase was added and the mixture was ligated overnight at 4°C. JM109 competent cells were then transformed using standard chemical methods. Positive clones were identified using restriction enzyme digestion and PCR. Positive clone plasmids extracted and screened were then transformed into E. coli competent BL21(DE3) cells using a chemical method and cultured for later use.

[0068] 2. Experimental results

[0069] The double enzyme digestion product of the recombinant expression vector pET-28a-P27 showed a 5.4 kb T vector band and a specific target band of about 800 bp ( Figure 2 ), proving that the recombinant expression vector pET-28a-P27 was successfully constructed.

[0070] IV. Inducible expression of P27 protein

[0071] 1. Experimental methods

[0072] Select a single, well-grown positive colony from the previous step and inoculate it into LBK liquid medium (containing 30 μg / mL Kan) for overnight culture at 37°C. Amplify the overnight culture 100-fold in LBK liquid medium (containing 30 μg / mL Kan) and incubate at 37°C for 2–3 hours (OD600 to 0.4–0.6). Add IPTG to a final concentration of 1 mmol / L and induce expression at 30°C for 5 hours. Centrifuge at 5000 rpm at 4°C for 15 minutes, discard the culture medium, collect the bacterial pellet, and analyze by 15% SDS-PAGE.

[0073] 2. Experimental results

[0074] The molecular weight of the protein induced by the recombinant bacteria was 30 kDa, which was consistent with the expected size.

[0075] 5. Ultrasonic lysis of induced expression bacteria

[0076] 1. Experimental methods

[0077] Centrifuge 200 mL of bacterial culture at 5000 rpm / min for 15 minutes, discard the supernatant, and weigh the wet weight of the cells. Resuspend the pellet in 10 mL of pre-chilled NTA-0 buffer. Add the appropriate amount of PMSF to a final concentration of 1 mmol / L and the appropriate amount of lysozyme to a final concentration of 0.2 mg / mL. Mix thoroughly, incubate on ice for 30 minutes, and sonicate the cells. Add 10% Triton X-100 (final concentration 0.05%), mix thoroughly, incubate on ice for 15 minutes, and sonicate in an ice bath to disrupt the cells. Add 1 mol / L MgCl2 to a final concentration of 1 mmol / L, mix thoroughly, and incubate at room temperature for 10 minutes. Centrifuge at 12000 rpm at 4°C for 20 minutes. Take 100 μL of the supernatant and a small amount of the pellet, add 100 μL of 2× SDS loading buffer to each, mix thoroughly, and boil for 5 minutes. A 20 μL sample is analyzed by 15% SDS-PAGE electrophoresis.

[0078] 2. Experimental results

[0079] The results showed that after 15% SDS-PAGE electrophoresis analysis of the expression product of the induced recombinant bacteria, protein bands of the expected size were visible in the supernatant and precipitate after ultrasonic lysis. Thin layer scanning analysis showed that the content of recombinant protein in the supernatant was 22.1%, and the content in the precipitate was 20.7%.

[0080] VI. Purification and Identification of P27 Protein

[0081] 1. Purification of recombinant P27 protein:

[0082] Using the lysate of the recombinant engineered strain BL21 (DE3) expressing the P27 protein as the material, the expressed fusion protein was affinity purified according to the "SNBC 3S NTA Resin Usage Instructions" of Shanghai Shenneng Biotechnology Co., Ltd. The main steps are as follows:

[0083] (1) Load 1 mL of NTA resin into the chromatography column and let it stand.

[0084] (2) Equilibrate the column with 20 mL of NTA-0 buffer.

[0085] (3) Take 10 mL of bacterial lysis supernatant and add it to the NTA chromatography column. Control the flow rate at about 15 mL / h and collect the breakthrough portion, which is marked as "breakthrough liquid".

[0086] (4) Elute with 10 mL of NTA-0 buffer at a flow rate of approximately 30 mL / h.

[0087] (5) Elute with 10 mL of NTA-20, NTA-40, NTA-60, NTA-80, NTA-100, NTA-200, and NTA-1000 buffer in sequence, with the flow rate controlled at around 15 mL / h; collect 1 mL of the eluate in each tube.

[0088] (6) The collected samples were subjected to SDS-PAGE analysis to determine the distribution of the target protein in the eluate.

[0089] (7) Determination of OD of purified P27 protein 260 and OD 280 , and calculate the protein concentration. The calculation formula for protein concentration determined by UV absorption method is: protein concentration (mg / mL) = 1.45×OD 280 -0.74×OD 260 .

[0090] The results of SDS-PAGE analysis showed that the eluate with an imidazole concentration of 100 mmol / L contained a high-purity target protein. The results of thin-layer scanning showed that its purity was greater than 95%. The concentration of the protein was measured by UV absorption method and was approximately 0.53 mg / mL. Figure 3 shown.

[0091] 2. Western Blotting Detection of Induced Expression Products

[0092] Take 10 μL of expression product, add an equal amount of 2× loading buffer, boil for 5 minutes, and then perform SDS-PAGE, and then transfer to NC membrane, block overnight at 4°C, incubate with rabbit anti-P27 polyclonal antibody and enzyme-labeled goat anti-rabbit secondary antibody in sequence, and finally add luminescent liquid to expose in the dark room, and wash the film. As a result, a clear band appeared. Compared with the standard molecular weight, its size is about 30 kDa, indicating that the E. coli expresses a specific P27 protein.

[0093] VII. Characterization of Genetically Engineered E. coli BL21(DE3)

[0094] 1. Morphology: This bacterium is a Gram-negative, non-spore-forming rod, measuring 0.4-0.7 μm x 2-3 μm, with blunt ends, either scattered or in pairs. Most strains motility is by peritrichous flagella, but amotile variants with or without flagella also exist. A capsule is usually absent, but microcapsules are often present. Basic dyes stain this bacterium well, with the ends occasionally showing a slightly darker coloration.

[0095] 2. Culture characteristics: These bacteria are facultative anaerobes. When cultured aerobically at 37°C for 12 to 16 hours on kanamycin-resistant LB agar plates, they form round, raised, smooth, moist, translucent, gray colonies.

[0096] 3. Biochemical Characteristics: This bacterium can ferment most carbohydrates to produce acid and gas, and can rapidly ferment lactose. It produces almost no hydrogen sulfide and does not decompose urea. It tests positive for indole and methyl red, but negative for VP and citrate utilization.

[0097] 4. Purity: Purity should be in accordance with Appendix 169 of Part I of the Veterinary Pharmacopoeia of the People's Republic of China (2000 Edition).

[0098] 5. Identification characteristics: After the bacteria are suspended in PBS and boiled, the specific P27 gene can be amplified using P27 gene specific primers. After induction of expression, specific bands of P27 protein can be seen by SDS-PAGE electrophoresis analysis.

[0099] 6. Safety: This genetically engineered bacterium is used to produce P27 antigen, has no infectious characteristics, and does not involve virulence safety.

[0100] 7. Storage: The freeze-dried bacteria can be stored for 5 years at -80℃ and for 1 year at 4℃.

[0101] 8. Data related to the establishment of databases for original seed batches, basic seed batches, and working seed batches

[0102] 1. Establishment and identification of original seed batch: Take the original recombinant engineered bacteria and subculture them three times in a row, expand the culture of each generation, and identify its characteristics.

[0103] (1) Subculture method: Take the original positive strain and inoculate it into 5 mL of LBK (containing 30 μg / mL Kan) liquid medium, and culture it at 37°C overnight. Take out 4 mL and inoculate it into 400 mL of LBK (containing 30 μg / mL Kan) liquid medium, and culture it at 30°C for 2-3 hours (OD600 reaches 0.4-0.6).

[0104] (2) Generation range: No more than 3 generations.

[0105] (3) Characteristics and identification of bacterial strains: This was carried out based on the characteristics of the genetically engineered bacteria E. coli BL21 (DE3).

[0106] (4) Subculture method: Same as step (1).

[0107] (5) Generation range: No more than 3 generations.

[0108] (6) Basic seed batch identification

[0109] a. Specificity Test: Suspend the cells in PBS, boil, and then amplify with P27 gene-specific primers to amplify the specific P27 gene. After induction of expression, analyze the cells by SDS-PAGE electrophoresis, and a specific band of the P27 protein should be visible.

[0110] b. Purity identification: It should be pure according to Appendix 169 of Part I of the Veterinary Pharmacopoeia of the People's Republic of China (2000 Edition).

[0111] c. Screening and identification of working seed batches: Take a certain number of original seeds, multiply them to a certain number after subculture, mix all cultures of the same generation evenly, package them, and store them at -80℃ or in liquid nitrogen for later use.

[0112] d. Subculture method: Same as step (1).

[0113] e. The generation range does not exceed 2 generations.

[0114] f. Identification and testing of production seed batches: Specificity and purity tests are the same as those for basic seed batch identification; content determination: induce expression in each generation of production seeds, ultrasonically lyse the bacteria, and then purify the P27 protein according to the method in step 6, and determine its content by ultraviolet absorption. The results show that the P27 protein content produced by each generation of bacteria should be basically the same, about 20%, and the protein concentration is close to 0.58 mg / mL.

[0115] Example 2: Preparation and identification of anti-P27 monoclonal antibodies

[0116] 1. Animal Immunization and IgG B Cell Enrichment: The P27 protein that passed the inspection and was purified in Example 1 was emulsified with an equal amount of Freund's complete adjuvant and injected subcutaneously into rabbits at multiple sites, 0.8 mL per rabbit (approximately 400 μg per rabbit). Fourteen days later, rabbits were immunized subcutaneously for a second time with P27 antigen containing an equal amount of Freund's incomplete adjuvant, 0.4 mL per rabbit. 14 days later, rabbits were immunized subcutaneously with only P27 antigen, 0.4 mL per rabbit. Three days later, the spleens were harvested and ground, and IgG B cells were enriched by density centrifugation and magnetic bead sorting.

[0117] 2. Flow cytometry sorting of specific IgG B cells: Use software to predict specific epitopes shared by ALV-A / B, ALV-J, and ALV-K but distinct from ALV-E. These epitopes are then split into 14 fragments, artificially synthesized and tagged with GFP fluorescent tags. The enriched IgG B cells are stained with the fluorescently labeled short peptides. Specific IgG B cells with the highest fluorescence intensity are isolated from 1,000 B cells using flow cytometry and expanded in vitro. The amino acid sequences of the 14 fragments are (SEQ ID NO: 6 to SEQ ID NO: 19): MPVVIKTEGPAWTP, LEPKLITRLADTVRTKG, LRSPITMAEVEALMSSP, LLPHDVTNLMRVIL, GPAPYALWMDAWGVQLQTVIAAATRDP, RHPANGQGRGERTNLDRLKGL, ADGMAGNPEGQAAL, LRPGELVAITASALQAFREVARLAE, PTDPWAEITQGPSE, SFVDFANRLIKAVEGSD, LPPSARAPVIIDCFRQKS, QPDIQQLIRAAPSTLTT, PGEIIKYVLDRQKTAP, LTDQGIAAAMSSAI, which are further used to synthesize short peptides, and the synthesized short peptides are used for flow cytometry screening of specific B cells against ALV-A / B, ALV-J, and ALV-K.

[0118] 3. Preparation of anti-P27 monoclonal antibody

[0119] (1) Construction of light chain and heavy chain plasmids of P27 monoclonal antibody: BCR sequencing was performed on the screened IgG B cells to obtain 3000 sequences. The light chain and heavy chain sequences of each BCR were connected to the eukaryotic expression vector pTT5 to make P27 antibody light chain and heavy chain plasmids. After transfection into cells, they were cultured for three days. The cell supernatant was taken and transferred to a 96-well enzyme-labeled plate (coated with 100 ng / well of P27 antigen) for ELISA detection. The OD values ​​were screened and obtained. 620 The BCR sequence corresponding to the cell well with the highest value (3.2980) is shown in SEQ ID NO: 20 (122DK) and SEQ ID NO: 21 (122DH) for the light chain and heavy chain base sequences of the P27 monoclonal antibody (named 122).

[0120] The P27 monoclonal antibody CDR sequence is as follows:

[0121] CDR-H1 (SEQ ID NO:22): GYSFTGYYMH; CDR-H2 (SEQ ID NO:23): WINPNSGGTNYAQKFQG; CDR-H3 (SEQ ID NO:24): ARDYYGSSGWYFDV;

[0122] CDR-L1 (SEQ ID NO:25): RASQSVSSSYLA; CDR-L2 (SEQ ID NO:26): DASNRAT; CDR-L3 (SEQ ID NO:27): QQYNSYPLT.

[0123] The light chain sequence of the antibody is 122DK and the heavy chain sequence is 122DH. After the light and heavy chains are transfected into cells, the translation products can combine on their own to generate antibodies.

[0124] (5) Expression and purification of P27 monoclonal antibody: HEK293F cells were resuspended in Expi293™ expression medium and transferred into a 1 L shake flask containing 200 mL of Expi293™ expression medium. The cells were cultured in a 37°C 5% CO2 shaker at 130 rpm until the cell density reached 1×10 6 Once the cell number and condition are suitable for transfection, prepare the transfection reagents: Mixture A (mix 2.5 mL of Opti-MEM I Reduced Serum Medium with 100 μg of heavy chain plasmid and 100 μg of light chain plasmid for 10 minutes, freshly prepared and used immediately) and Mixture B (mix 2.5 mL of Opti-MEM I Reduced Serum Medium with 600 μL of PEI for 10 minutes, freshly prepared and used immediately). After preparation, let it stand at room temperature for 20 minutes. Mixture A and B are then combined to create Mixture C, which is then allowed to stand at room temperature for 20 minutes. Mixture C is then added to HEK293F cells, and the cell flask is transferred to a 37°C, 5% CO2 cell shaker at 130 rpm and cultured for 7 days. The cells are harvested, centrifuged at 4500 rpm for 15 minutes at 4°C, and the supernatant is collected and filtered through a 0.45 μm filter. The prepacked column is then washed and equilibrated with 10 column volumes of PBS (pH 7.4). The sample was added from the upper end of the prepacked column, and the flow rate was controlled at 1 mL / min. After the sample was bound, the prepacked column was washed with 20 column volumes of PBS (pH = 7.4) to remove non-specifically adsorbed impurities. The flow rate was controlled at 1 mL / min. 2 mL of 1 mol / L Tris-HCl (pH = 8.8) was pre-added to the collection tube at a ratio of 100 μL of 1 mol / L Tris-HCl (pH = 8.8) per mL of eluent.

[0125] After washing, add 20 mL of 100 mmol / L glycine-HCl buffer (pH 2.0) to elute the bound antibody and collect the eluate. The eluted antibody is then concentrated and buffer exchanged. Expression is assessed by ELISA, and stably expressed antibodies are selected for quantification by SDS-PAGE and BCA analysis.

[0126] 3. Characterization of P27 monoclonal antibody

[0127] (1) Concentration determination: The monoclonal antibody concentration was determined to be 1.86 mg / mL.

[0128] (2) Specificity identification: The purified P27 monoclonal antibody was subjected to conventional Western Blotting test and confirmed, which showed that the P27 monoclonal antibody had good reactivity to the P27 protein.

[0129] (3) ELISA assay: ELISA plates were coated with 200-fold diluted P27 monoclonal antibody, 100 μL / well, incubated at 4°C for 12 h, and blocked with 5% skim milk at 37°C for 2 h. Then, avian viruses and bacteria such as NDV, IBDV, IBV, AEV, and EDSV were added, and their OD values ​​were determined using 1200-fold diluted enzyme-labeled antibody and substrate colorimetric solution. 650 The results are shown in Tables 1 and 2:

[0130] Table 1 Results of monoclonal antibody specificity determination by ELISA

[0131]

[0132] Table 2 Results of monoclonal antibody specificity determination by ELISA

[0133]

[0134] From the results in the table, it can be seen that when the monoclonal antibody is used to detect a variety of avian disease viruses and bacteria, its OD value is around 0.1, which is close to the OD value of the negative control, indicating that the monoclonal antibody has no cross-reaction to the detected viruses and bacteria and has strong specificity.

[0135] (4) Determination of affinity constant: The determination was performed by indirect ELISA method. The specific steps are as follows: The qualified P27 protein of Example 1 was diluted at a ratio of 1:25, 1:100, and 1:200, and added to the enzyme-labeled wells, 100 mL / well, and incubated at 4°C overnight. The plate was washed 3 times with PBST, 3 minutes each time, and 300 mL of 5% skim milk was added to each well, and the plate was blocked at 37°C for 4 hours. The purified P27 monoclonal antibody was diluted from 1:30 to 1:15360 and added to the enzyme-labeled plate wells, 100 mL / well, and incubated at 37°C for 1 hour. The plate was washed and patted dry. HRP-goat anti-rabbit IgG diluted 1:500 was added, 100 mL / well, and incubated at 37°C for 1 hour. The plate was washed and patted dry. Substrate solution was added, 100 mL / well, and the reaction was carried out at room temperature in the dark for 15 minutes. 100 mL of stop solution was added, and the reaction was carried out at room temperature for 5 minutes. The reaction was terminated and the OD was measured. 650 value and draw a curve.

[0136] The affinity constant determination curve of P27 monoclonal antibody is plotted with the logarithm of different dilutions of monoclonal antibody as the horizontal axis and the corresponding OD value as the vertical axis. Figure 4 As shown, take the OD value of the flat section of the upper part of each curve as 100%, find out the antibody concentration corresponding to half of its maximum OD value (i.e. 50% OD value), and substitute it into the following formula to calculate the affinity constant.

[0137]

[0138] Where Ab and Ab' represent the antibody concentration (mol / L) that produces half the absorbance when the antigen concentration is Ag and Ag', respectively, and n = Ag / Ag'. Then, by comparing them pairwise, we can obtain the K values ​​when n = 2, n = 4, and n = 8, and calculate the average of the three K values ​​as the final result.

[0139] By calculation, the affinity constant K values ​​when n=2, n=4, and n=8 are:

[0140] K1=1.05×10 10 L / mol, K2=5.15×10 9 L / mol, K3=7.26×10 9 L / mol

[0141] K = (K1 + K2 + K3) / 3 = 7.6 × 10 9 L / mol

[0142] It is generally believed that the K value is 10 7 ~10 12 The antibody has a high affinity; the K value is 10 5 ~10 7The antibody affinity of P27 is low; based on the K value measured in this experiment, it can be considered that the affinity of P27 monoclonal antibody is higher.

[0143] Example 3: Preparation and testing of anti-P27 monoclonal antibody coated plates

[0144] 1. Preparation of coated plates

[0145] (1) Coating: Under sterile conditions, the purified P27 monoclonal antibody from Example 2 was diluted to 3.68 μg / mL with carbonate buffer, and 0.1 mL was added to each well of a 96-well ELISA plate. The plate was incubated at 2-8°C for 15 h, and the coating solution was discarded. The plate was washed three times with 0.01 mol / L PBST.

[0146] (2) Blocking: Add blocking solution to each well of the coated ELISA plate, 250 μL per well, block at 37°C for 4 h, and discard the blocking solution.

[0147] (3) Drying: Place the coated plate in a dryer and dry at room temperature for 30 minutes. Place it in an aluminum foil bag and vacuum seal.

[0148] 2. Inspection of coated board

[0149] (1) Property inspection: transparent, dry and vacuum packed, without damage or breakage.

[0150] (2) Efficacy test: The sensitivity of each batch of the kit was tested using a sample of the standard quality control solution of the kit. The solution sample consisted of 6 different dilutions of P27 protein solution. The specifications and acceptance criteria are shown in Table 3.

[0151] Qualified coated boards should meet the following standards:

[0152] (1) OD of all quality control solution samples 650 The measured values ​​are all within the qualified range.

[0153] (2) OD of the positive control 650 The value is between 1.00 and 1.60; the negative control OD 650 The value is not greater than 0.20.

[0154] Table 3 Concentration and OD of standard quality control solution of the kit 650 Qualified range of measured values

[0155]

[0156] Example 4. Preparation and testing of HRP-labeled sheep anti-P27 polyclonal antibodies

[0157] 1. Polyclonal Antibody Preparation

[0158] 1. Animal immunization: Adult sheep were immunized with the qualified P27 protein obtained in Example 1 by subcutaneous and intradermal injection combined with intramuscular injection. The specific immunization procedure is as follows:

[0159] (1) First immunization: Use intradermal and intramuscular injection. Take the antigen solution that has passed the test in Example 1, add an equal amount of Freund's complete adjuvant, and completely emulsify it. Inject 0.5 mL of the antigen solution into each sheep's skin and muscle.

[0160] (2) Second immunization: After 3 weeks, the second immunization was performed by subcutaneous and intramuscular injection. The antigen solution that passed the test in Example 1 was added with an equal amount of Freund's incomplete adjuvant and completely emulsified. 0.5 mL of the antigen solution was injected subcutaneously and intramuscularly into each sheep.

[0161] (3) Three and four vaccinations: Three and four vaccinations are carried out every three weeks, and the immunization method is the same as the two vaccinations.

[0162] (4) 10-15 days after immunization, blood is collected from the neck vein to check for specific antibodies. Agar amplification can be used for detection. When the titer is >16, blood can be collected to separate serum and purify Ab. Every two weeks thereafter, the sheep will be given a booster immunization in the same way as the second immunization. Blood is collected from the neck vein of the sheep and serum is collected.

[0163] 2. Serological identification

[0164] (1) Agar-Gal-Gal test (AGP) was performed according to the conventional method. A clear precipitation line appeared between the antigen well and the antibody well. The titer of the goat anti-P27 polyclonal antibody reached 1:32.

[0165] (2) The Western Blotting test steps are as follows: take 10 μL of purified P27 protein, treat it with an equal amount of 2× loading buffer, perform SDS-PAGE, and then transfer it to the NC membrane, block it overnight at 4°C, add 500-fold diluted sheep serum and enzyme-labeled horse anti-sheep IgG in sequence, incubate it, add luminescent liquid for exposure, and rinse the film. As a result, a clear band appears. Compared with the standard molecular weight, the size of this band is about 30 kDa, which is consistent with the expected size, indicating that the sheep serum can specifically bind to the expressed P27 protein.

[0166] 3. Serum Collection and Antibody Purification: Blood was collected from the jugular vein to prepare serum. 100 mL of blood was collected from each sheep and approximately 50 mL of serum was separated.

[0167] (1) Preparation of P27 antigen column:

[0168] Swell the activated Sepharose 4B gel powder for 10 minutes, transfer it to a Buchner funnel, and quickly wash the microspheres three times with HCl. Then, wash the microspheres with binding buffer. Transfer the microspheres to a centrifuge tube and add 500 μL of P27 protein concentrated in binding buffer (at a concentration of 5-10 mg / mL). Mix and incubate at 4°C overnight. Centrifuge the overnight mixture at 500 rpm for 5 minutes, recover the supernatant, measure the protein concentration, and calculate the 000 coupling efficiency. Add 10 mL of binding buffer, mix, and centrifuge to remove unbound P27 protein. Block with ethanolamine at 4°C for 5 hours. Transfer the conjugate to a chromatography column, and equilibrate the antigen column with PBS.

[0169] (2) Purification of P27 polyclonal antibody using P27 antigen column

[0170] Dilute the anti-P27 polyclonal antibody goat serum 3-fold with PBS and add it to the antigen column. Wash the antigen column with PBS until no protein is washed out. Elute the polyclonal antibody with elution buffer and collect the antibody protein in a collection tube containing 0.25 mL of neutralization solution, collecting 0.75 mL per tube. Calculate the collected antibody concentration: protein concentration = 1.45 × OD 280 -0.74×OD 260 .

[0171] 4. Preparation of enzyme-labeled polyclonal antibodies: Horseradish peroxidase (HRP) is used to label the prepared polyclonal antibodies.

[0172] 5. Inspection

[0173] (1) Property inspection: It should be a clear, light yellow solution, odorless, tasteless, and free of sediment.

[0174] (2) Potency test: Concentration determination: The concentration of the enzyme-labeled antibody was determined to be 5.65 mg / mL.

[0175] Identification of polyclonal antibody specificity: ELISA plates were coated with 600-fold diluted purified polyclonal antibodies at 100 μL / well, incubated at 4°C for 12 h, blocked with 5% skim milk at 37°C for 2 h, and then NDV, IBDV, IBV, AEV, EDSV, E. coli, E. Salmon and other viruses and bacteria were added, along with 1000-fold diluted enzyme-labeled antibodies. Finally, substrate colorimetric solution was added, and the OD was measured after termination. 650 The results should be close to 0.1, similar to the OD value of the negative control, and the polyclonal antibody has no cross-reaction to other avian viruses and bacteria.

[0176] Determination of enzyme-labeled antibody titer: Purified P27 protein (initial concentration 0.57 mg / mL) was serially diluted (500-256,000-fold dilutions) and coated on an ELISA plate. Array experiments were then performed with serially diluted enzyme-labeled goat anti-P27 polyclonal antibody (100-12,800-fold dilutions). The highest dilution of the enzyme-labeled antibody that reacted with the highest dilution of P27 protein and produced an OD value of approximately 1.0 was used as the working concentration of the enzyme-labeled antibody. The titer of the enzyme-labeled antibody should be no less than 1:1000. The results are shown in Table 4.

[0177] Table 4 Determination of enzyme-labeled goat antibody titer

[0178]

[0179] Example 5. Preparation and testing of enzyme-labeled antibody working solution

[0180] 1. Preparation of enzyme-labeled antibody working solution

[0181] The enzyme-labeled antibody prepared in Example 4 was diluted with PBST solution at 1:600, and a preservative was added and packaged.

[0182] 2. Inspection:

[0183] Sterility test: Test according to Appendix 169 of Part I of the Veterinary Pharmacopoeia of the People's Republic of China (2000 Edition). It should be sterile.

[0184] Efficacy test: The sensitivity of each batch of enzyme-labeled antibody was tested using a sample of the standard quality control solution of this kit. This sample solution consists of six different dilutions of P27 protein solution. The specifications and acceptance criteria are shown in Table 5.

[0185] Qualified enzyme-labeled antibody working solution should meet the following standards:

[0186] (1) OD of all quality control solution samples 650 The measured values ​​are all within the qualified range.

[0187] (2) OD of the positive control 650 The value is between 1.00 and 1.60; the negative control OD 650 The value is not greater than 0.20.

[0188] Table 5 Concentration and OD of standard quality control solution of the kit 650 Qualified range of measured values

[0189]

[0190] Example 6. Determination of ELISA working conditions

[0191] 1. Determine the concentration of coated monoclonal antibody and enzyme-labeled polyclonal antibody by checkerboard method

[0192] First, the OD values ​​of four P27 protein samples with different concentration gradients were determined using an internationally imported avian leukosis antigen detection kit. 3.75 mg / mL monoclonal antibody was gradiently diluted from 1:1000 to 1:64000 for coating, and 3.28 mg / mL enzyme-labeled polyclonal antibody was gradiently diluted from 1:1000 to 1:10000. ELISA was performed on these four samples and the OD values ​​were compared with the OD values ​​of the internationally imported avian leukosis antigen detection kit. 650 The coating concentration with similar readings was determined as the optimal coating concentration for the monoclonal antibody, and the dilution concentration for the enzyme-labeled polyclonal antibody was determined as the optimal working concentration. The results are shown in Tables 6 and 7.

[0193] Table 6 Determination of standard samples

[0194]

[0195] Table 7 Determination of monoclonal antibody and enzyme-labeled polyclonal antibody concentrations by checkerboard method

[0196]

[0197] The results show that when the monoclonal antibody is coated at a dilution of 1:8000 and the enzyme-labeled polyclonal antibody is diluted at 1:2000, the sample readings are closest to the readings corresponding to the internationally imported kits. Therefore, the optimal coating dilution of the monoclonal antibody is set at 1:8000, and the enzyme-labeled secondary antibody dilution is set at 1:2000.

[0198] 2. Determination of negative control

[0199] According to the general negative control determination method, the aseptically collected SPF chicken serum was gradient diluted with PBS and tested using the optimized homemade kit conditions. At the same time, the internationally imported avian leukosis antigen detection kit was used to detect the gradient diluted SPF serum. When the SPF serum was gradient diluted above 1:100, its OD 650 The value was less than 0.1 and was similar to the test result of the internationally imported avian leukosis antigen detection kit, so the SPF serum with a dilution of 1:100 was used as the standard negative control.

[0200] 3. Determination of positive control

[0201] According to the general positive control determination method, P27 antigen (0.58 mg / mL) was diluted with PBS in a gradient manner and tested using the optimized homemade kit conditions. At the same time, the internationally imported avian leukosis antigen detection kit was used to detect the P27 antigen in the gradient dilution. When the P27 protein dilution was 1:100000 (concentration was 5 ng / mL), OD 650The results were similar to those of the positive standard sample of the internationally imported avian leukosis antigen detection kit. For the convenience of calculation, the standard positive control was set as a solution with a P27 antigen concentration of 5 ng / mL.

[0202] 4. Determination of the critical value of the test kit

[0203] Using the 8 antigen concentrations measured in this test and the standard negative and positive samples of the two products and their corresponding OD 620 The average reading can be used to calculate the OD of this product and international imported products. 620 The positive / negative cutoff OD and the minimum detection concentration of the antigen for internationally imported products are measured based on the curve, and the positive / negative cutoff value S / P is determined according to the formula S / P = (SN) / (PN).

[0204] According to the results, the standard positive OD value of the internationally imported avian leukosis antigen detection kit is 0.943, the standard negative sample OD value is 0.047, and the yin and yang critical S / P value is 0.20. Substituting the standard negative and positive OD values ​​and the yin and yang critical S / P values ​​of the internationally imported kit into the formula, the yin and yang critical OD value can be calculated to be 0.226. The corresponding yin and yang critical concentration is 9.687ng found through the OD-concentration curve, and the corresponding OD value of this product is 0.226. The standard positive OD value of this product is 0.85, with a fluctuation range of 0.831 to 0.853, and the standard negative OD value is 0.040, with a fluctuation range of 0.0370 to 0.0420. Substituting our standard yin and yang mean value into the formula to obtain the S / P critical value of 0.202, substituting the standard yin and yang fluctuation range into the formula to obtain the maximum and minimum values ​​of 0.20 and 0.22 respectively, so the judgment standard is set to S / P equal to 0.20. 5. Determination of the working conditions of the ELISA method

[0205] According to the results, the final conditions of the ELISA method were:

[0206] The ELISA plate was Corning ELISA plate, the coating solution was CB buffer, the coating antigen was purified P27 monoclonal antibody (1.86 mg / mL), the coating dilution was 1:2500, the enzyme-labeled polyclonal antibody was HRP-labeled goat anti-P27 polyclonal antibody (5.65 mg / mL), the diluent was PBST, the optimal dilution was 1:600, the reaction conditions were 4°C coating overnight, the test sample was reacted at 25°C for 1 hour, the enzyme-labeled polyclonal antibody was reacted at 25°C for 1 hour, and the color was developed at 25°C for 15 minutes. Among them, the OD value of negative serum was 0.04. 650 The value was controlled below 0.15, and the positive control OD 650 Subtract the negative control OD 650 Should be greater than 0.2. Determine the positive or negative nature of the sample using the following method:

[0207] Calculate the sample S / P value

[0208]

[0209] When the sample S / P ratio is less than (0.2), the sample is negative for ALV-P27 antigen. When the sample S / P ratio is greater than or equal to (0.23), the sample is positive for ALV-P27 antigen. When the sample S / P ratio is less than (0.23), the sample is suspected to be positive for ALV-P27 antigen.

[0210] Example 7: Use of the detection kit

[0211] (1) Sample preparation

[0212] Egg white: Collect the diluted egg white and add it directly to the ELISA reaction plate as the sample to be tested without dilution.

[0213] Cloacal swab: Add the cloacal swab to 1 mL of culture medium or sample diluent and store frozen. Before testing, return the sample to room temperature, allow it to stand, and then aspirate 100 µL of the supernatant as the test sample.

[0214] Serum: Add 100µL of clarified serum sample directly to the reaction plate without dilution.

[0215] (2) Preparation before the experiment

[0216] Before use, bring the kit components to room temperature; prepare sample dilution plates or microtubes; prepare 1X wash buffer: for example, add 50 mL of 20x concentrated wash buffer to 950 mL of deionized water, mix thoroughly, and set aside; do not dilute the negative and positive controls.

[0217] (3) Operation method

[0218] a. Sample Addition: Secure the required number of strips to the plate rack. For each experiment, set up two positive control wells and two negative control wells. Add 100µL of each positive and negative control solution to the appropriate wells. Add 100µL of the sample to be tested to the remaining wells. Samples do not need to be diluted for testing.

[0219] b. Incubation: Cover the plate with sealing film and incubate at room temperature (25°C ± 2°C) for 60 min.

[0220] c. Wash: Discard the liquid in the wells and fill each well with diluted wash solution (approximately 350µL / well) for washing. Discard the wash solution in the wells. Repeat the wash four times and pat dry.

[0221] d. Add enzyme: Add 100µL of enzyme marker to each well.

[0222] e. Incubation: Cover the plate with sealing film and incubate at room temperature (25°C ± 2°C) for 60 min.

[0223] f. Wash: Discard the liquid in the wells and fill each well with diluted wash solution (approximately 350µL / well) for washing. Discard the wash solution in the wells. Repeat the wash four times and pat dry.

[0224] g. Color development: Add 100 µL of TMB color development solution to each well and incubate at room temperature (25°C ± 2°C) for 15 min.

[0225] h. Stop: Add 100µL of stop solution to each well and gently tap to mix.

[0226] i. Reading: Read the absorbance of each well using a microplate reader at a single wavelength of 650 nm (A(650)). Please complete the reading within 10 minutes after termination.

[0227] (4) Result determination

[0228]

[0229] Negative control standard: ≤ 0.15; positive control standard: ≥0.2.

[0230] If the experimental results do not meet the negative and positive control standards, the experiment is considered invalid and must be repeated.

[0231] Judgment criteria: Calculate the sample S / P value

[0232]

[0233] When the sample S / P ≤ 0.23, the sample is ALV-P27 antigen negative, and when the sample S / P > 0.23, the sample is ALV-P27 antigen positive.

[0234] The presence of P27 antigen in the sample being tested is related to its S / P ratio. The positive control represents a P27 antigen level of approximately 15 ng / mL, so the relative content of P27 in the sample being tested can be determined using the S / P ratio.

[0235] (5) The factors that may affect the experimental results are as follows:

[0236] a. Insufficient washing, such as insufficient amount of washing liquid.

[0237] b. The needle hole of the plate washer is clogged or the position is improperly adjusted, and there is too much washing liquid remaining at the bottom of the well.

[0238] c. The sample amount is inaccurate.

[0239] d. Cross contamination and contamination of enzyme conjugates and other reagents should be avoided during the test.

[0240] e. Not using the correct wavelength for readings.

[0241] f. Avoid drying of the reaction wells during the test.

[0242] g. Using highly hemolyzed specimens, incompletely agglutinated blood, serum containing fibers, or serum contaminated with microorganisms will lead to adverse results.

[0243] (6) Notes

[0244] a. The test kit taken out of the refrigerated environment should be equilibrated to room temperature before opening and use.

[0245] b. Unused pre-coated strips should be stored in a sealed bag with desiccant.

[0246] c. If crystals appear in the 20x concentrated wash solution, please place it at 37°C until it dissolves before use.

[0247] d. Reagent components of different batches cannot be mixed.

[0248] e. Avoid exposing the reagents to direct sunlight and hypochlorous acid gas during testing and storage.

[0249] f. This test kit and all wastes should be treated as infectious items.

[0250] Example 8. Clinical evaluation test kit

[0251] The kit developed in this project was clinically evaluated. The methods used were to compare the consistency of the kit with that of the internationally imported avian leukosis antigen detection kit, using both the kit and the internationally imported kit to test the same batch of egg white samples, virus isolation supernatant samples, and meconium samples. The sensitivity of the kit was compared with that of the internationally imported kit using different kits to test gradiently diluted meconium samples, gradiently diluted virus isolation supernatant samples, and cell culture supernatants from different ALV strains, different inoculation concentrations, and different collection times. The detection limit of the P27 antigen of the kit was compared with that of a common avian leukosis antigen detection kit. The specific evaluation results are as follows.

[0252] 1. Compliance test

[0253] This kit and the internationally imported avian leukosis antigen detection kit were used to simultaneously detect egg white, meconium, and virus-isolated cell culture supernatant. The specific results are shown in Table 8.

[0254] Table 8 Comparison results of avian leukosis antigen detection kits

[0255]

[0256] According to the results, in 460 egg white samples, according to the kit's judgment criteria, this kit detected two more samples than the internationally imported kit, with a concordance rate of 98.91%. In 92 meconium samples, according to the kit's judgment criteria, this kit detected one more sample than the internationally imported kit, with a concordance rate of 97.83%. In 492 virus isolation supernatant samples, according to the kit's judgment criteria, this kit detected 52 more samples than the internationally imported kit, with a concordance rate of 84.96%.

[0257] In summary, the detection rate of this test kit is far superior to that of internationally imported test kits.

[0258] 2. Sensitivity test

[0259] 1. Meconium sample

[0260] A total of 114 meconium samples were tested, and each sample was tested after 9 serial dilutions (1:1, 1:6, 1:12, 1:24, 1:48, 1:96, 1:192, 1:384, and 1:768).

[0261] According to the kit standard, with an S / P ratio ≥ 0.2, the highest detection dilution factor of the homemade kit was higher than that of the imported kit for seven meconium samples. See Table 9 for details.

[0262] Table 9 Maximum dilution factor of meconium test kit

[0263]

[0264] 2. ALV virus liquid

[0265] A total of 7 known positive virus fluids were tested, and each sample was tested after 9 serial dilutions (1:1, 1:6, 1:12, 1:24, 1:48, 1:96, 1:192, 1:384, and 1:768).

[0266] According to the test kit standard, with S / P ≥ 0.2, the maximum detection dilution factor of one virus liquid sample of the homemade test kit was lower than that of the internationally imported test kit, and the maximum detection dilution factors of six samples were consistent, as shown in Table 10.

[0267] Table 10 Highest dilution factor for virus separation supernatant detection in the kit

[0268]

[0269] 3. A / B, J, K subgroup virus cell supernatant

[0270] Cell supernatants containing A / B, J, and K subgroup viruses with determined titers were diluted to three different concentrations (1:1, 1:5, and 1:10) and inoculated into DF-1 cells. Subgroup K, due to its lower titer and poor replication capacity, was diluted two-fold and inoculated into DF-1 cells. Cell supernatants were collected from 3 to 9 days after inoculation for testing at three different concentrations of the three ALV viruses.

[0271] According to the kit standards, with S / P ≥ 0.2, the sensitivity of the homemade kit for detecting A / B, J, and K subgroup avian leukosis viruses is equivalent to that of internationally imported kits.

[0272] 3. Minimum detection limit test

[0273] This kit and the internationally imported avian leukosis antigen detection kit were used to simultaneously detect different concentrations of P27 antigen. The specific results are shown in Table 11.

[0274] Table 11 Minimum detection limit test

[0275] According to the results, the minimum detection limit of this kit for P27 antigen is 1ng / mL, which is higher than the internationally imported avian leukosis antigen detection kit.

[0276] Although the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A kit for detecting avian leukosis antigen, characterized in that: The invention comprises an anti-avian leukosis virus P27 protein monoclonal antibody and a horseradish peroxidase-labeled sheep anti-P27 protein polyclonal antibody; the amino acid sequence of the P27 protein is shown in SEQ ID NO: 1; The anti-avian leukosis virus P27 protein monoclonal antibody comprises a heavy chain and a light chain, wherein the amino acid sequence of CDR-H1 of the heavy chain is shown in SEQ ID NO: 22; the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 23; the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 24; the amino acid sequence of CDR-L1 of the light chain is shown in SEQ ID NO: 25; the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 26; and the amino acid sequence of CDR-L3 is shown in SEQ ID NO:

27.

2. The kit according to claim 1, wherein The anti-avian leukosis virus P27 protein monoclonal antibody is coated on the solid phase carrier at a concentration of 2-10 μg / mL, and the working concentration of the horseradish peroxidase-labeled sheep anti-P27 protein polyclonal antibody is 5-20 μg / mL.

3. The kit according to claim 2, wherein The solid phase carrier is an ELISA plate, and the kit is based on a double antibody sandwich method, with anti-avian leukosis virus P27 protein monoclonal antibody as the primary antibody and horseradish peroxidase-labeled sheep anti-P27 protein polyclonal antibody as the sandwich secondary antibody.

4. The kit according to claim 3, wherein The kit further comprises a positive control and a negative control; the positive control is a P27 protein solution, and the negative control is SPF chicken serum.

5. The kit according to claim 4, characterized in that The kit further comprises a sample diluent, a TMB color developing solution, a stop solution and a washing solution. The sample diluent is a PBST buffer solution, and the washing solution is a phosphate buffer solution containing Tween-20.

6. A monoclonal antibody against avian leukosis virus P27 protein, comprising a heavy chain and a light chain, characterized in that: The amino acid sequence of CDR-H1 of the heavy chain is shown in SEQ ID NO: 22; the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 23; and the amino acid sequence of CDR-H3 is shown in SEQ ID NO:

24. The amino acid sequence of CDR-L1 of the light chain is shown in SEQ ID NO: 25; the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 26; and the amino acid sequence of CDR-L3 is shown in SEQ ID NO:

27.

7. Use of the monoclonal antibody according to claim 6 in preparing a kit for detecting avian leukosis virus infection.

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