Avian leukemia antigen detection kit and preparation method thereof

Abstract

The 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 goat anti-P27 polyclonal antibody labeled by horse radish peroxidase, wherein the monoclonal antibody and the goat anti-P27 polyclonal antibody are used as sandwich secondary antibodies. The kit is based on a double-antibody sandwich method, has good specificity on epidemic A / B, J and K subtype avian leukosis viruses of breeding hens, has the lowest detection limit of 1 ng / mL, has strong anti-interference capability, low false positive rate, simple operation and high stability, is suitable for breeding hen flock purification, SPF embryo source quality monitoring, epidemiological investigation and other scenes, effectively solves the problems of high price of imported products and limited domestic popularization, and has a wide application prospect in the fields of breeding hen flock purification, SPF embryo source quality monitoring, epidemiological investigation and the like. And the method has remarkable economic benefits and social benefits.

Description

Technical Field

[0001] The present invention relates to the field of biotechnological detection, and particularly to a kit for detecting avian leukosis antigen and a preparation method thereof. Background Art

[0002] Since there are no effective vaccines and therapeutic drugs for avian leukosis, pathogen detection, culling of positive chickens, and population purification have become the main means of controlling avian leukosis. However, so far, there are neither vaccines available for the prevention and treatment of avian leukosis nor effective drugs for treatment. The main method of controlling avian leukosis is through pathogen detection, culling of positive chickens, and purification of the population.

[0003] Currently, the detection of avian leukosis at home and abroad mainly relies on ELISA technology. Abroad, companies such as IDEXX in the United States have launched commercial kits for detecting avian leukosis antigen, but their prices are high, and they can only be applied in a few scenarios such as large enterprises and SPF chicken breeding farms, making it difficult to promote in the vast majority of small and medium-sized breeding enterprises in China, severely restricting the purification process of domestic chicken flocks. Since 1991, relevant detection technology research has been carried out in China. Although ELISA detection methods have been established, the commercialization of kits has not been achieved, and the existing technologies have significant defects: insufficient specificity: the antibodies used in traditional methods have limited recognition ability for avian leukosis virus subtypes such as A / B, J, and K prevalent in breeding chickens, and false negatives or misjudgments are likely to occur; low sensitivity: when using rabbit anti as the sandwich secondary antibody, the antibody production is low and the affinity is insufficient, making it difficult to detect low-concentration antigens; high cost: relying on imported key raw materials has led to high production costs of the kit, restricting large-scale application.

[0004] In addition, the existing detection kits have weak anti-interference ability for clinical samples, with a high false positive rate in the detection of complex samples (such as meconium and cloacal swabs), and the operation process is cumbersome and the detection time is long, unable to meet the batch detection requirements of chicken breeding farms.

[0005] In view of the above problems, the present invention is committed to developing an avian leukosis antigen detection kit with high specificity, high sensitivity and low cost. By using bioengineering technology to screen high-affinity monoclonal antibodies as the coated primary antibody and using goat anti-P27 polyclonal antibody as the sandwich secondary antibody, the detection performance is significantly improved; at the same time, the composition and detection process of the kit are optimized to make it more suitable for the large-scale purification needs of the domestic breeding industry of breeding chickens, effectively solving the monopoly of imported products and the defects of existing technologies, and having important economic value and social significance. The present invention has a wide application prospect in the domestic breeding industry of breeding chickens. It can be used as a conventional technical means for the purification of avian leukosis in high-generation breeding chicken farms, for the inspection of exogenous viruses and the sampling inspection of products in veterinary biological product factories and biological product factories of health departments, for the monitoring of the quality of raw materials (such as SPF embryo sources), the quality control of SPF chicken production enterprises, for clinical diagnosis and epidemiological investigation by veterinary epidemic prevention departments in various provinces and cities, and for scientific research by universities and research institutions. By promoting the technology, training personnel and widely applying the avian leukosis antigen detection kit of the present invention, greater economic and social benefits will be brought, contributing to the development of the chicken farming industry in China. Summary of the Invention

[0006] In view of the deficiencies of the above-mentioned existing technologies, the present invention aims to provide an avian leukosis antigen detection kit and a preparation method thereof. The monoclonal antibody used in the kit is screened by bioengineering methods, with high sensitivity and good specificity. A goat anti-P27 polyclonal antibody labeled with horseradish peroxidase is used as the sandwich secondary antibody, which has high sensitivity and large yield. The avian leukosis antigen detection kit of the present invention has good specificity and high sensitivity for different subtypes of avian leukosis viruses (A / B, J, K) prevalent in current breeding chickens, meeting the requirements for clinical purification use.

[0007] To achieve the above object, the present invention adopts the following technical solutions: In one embodiment, the present invention provides an avian leukosis antigen detection kit, including a monoclonal antibody against avian leukosis virus P27 protein and a goat anti-P27 protein polyclonal antibody labeled with horseradish peroxidase; the amino acid sequence of the P27 protein is shown as SEQ ID NO:1.

[0008] In another embodiment, the monoclonal antibody against avian leukosis virus P27 protein of the present invention is screened by bioengineering methods.

[0009] In another embodiment, the monoclonal antibody against avian leukosis virus P27 protein is coated on a solid-phase carrier at a concentration of 2-10 μg / mL, and the working concentration of the goat anti-P27 protein polyclonal antibody labeled with horseradish peroxidase is 5-20 μg / mL.

[0010] In another embodiment, the solid-phase carrier is an enzyme-linked immunosorbent assay (ELISA) plate, and the kit is based on the double-antibody sandwich method, using a monoclonal antibody against avian leukosis virus P27 protein as the primary antibody and a horseradish peroxidase-labeled polyclonal antibody against P27 protein as the sandwich secondary antibody.

[0011] In another embodiment, the kit includes a positive control and a negative control; the positive control is a P27 protein solution, and the negative control is SPF chicken serum.

[0012] In another embodiment, the kit further includes a sample diluent, a TMB chromogenic solution, a termination 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.

[0013] In one embodiment, the present invention provides a bioengineering method for screening monoclonal antibodies against avian leukosis virus P27 protein, which is characterized by including 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) using flow sorting technology to screen out high-affinity IgG B cells with a fluorescently labeled P27 protein-specific epitope peptide; (3) sequencing the BCR light chain and heavy chain genes of the screened B cells to obtain specific BCR sequences.

[0014] In one embodiment, the present invention provides a monoclonal antibody against avian leukosis virus P27 protein screened by the bioengineering method described above. In a preferred example, the amino acid sequence of CDR-H1 of its heavy chain is as shown in SEQ ID NO:22; the amino acid sequence of CDR-H2 is as shown in SEQ ID NO:23; the amino acid sequence of CDR-H3 is as shown in SEQ ID NO:24; The amino acid sequence of CDR-L1 of the light chain is as shown in SEQ ID NO:25; the amino acid sequence of CDR-L2 is as shown in SEQ ID NO:26; the amino acid sequence of CDR-L3 is as shown in SEQ ID NO:27.

[0015] In one embodiment, the present invention provides an application of the monoclonal antibody in the preparation of a kit for detecting avian leukosis virus infection.

[0016] In one embodiment, the present invention provides a horseradish peroxidase-labeled polyclonal antibody against P27, which is prepared by immunizing sheep with the P27 protein shown in SEQ ID NO:1 as an immunogen.

[0017] The beneficial effects achieved by the present invention: (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 the sandwich secondary antibody system, and systematic process design.

[0018] (2) The present invention has high specificity and broad-spectrum detection capabilities. The monoclonal antibodies screened by bioengineering methods for cross-subtype virus recognition are designed against the conserved epitopes of the P27 protein of avian leukosis virus, and can simultaneously recognize A / B, J, and K subtype viruses prevalent in breeding chickens, solving the problem of insufficient detection coverage of multi-subtype viruses in the prior art. Experimental data show that the monoclonal antibody has no cross-reaction with various avian viruses and bacteria such as NDV and IBDV, and its specificity is significantly better than the traditional mouse anti-system.

[0019] (3) The affinity constant of the precise antigen-antibody binding monoclonal antibody of the present invention reaches 7.6×10 9 L / mol, belonging to high-affinity antibodies. The dissociation rate of binding to the P27 protein is low, ensuring stable signals during the detection process and reducing false negative results. Western Blotting results show that the monoclonal antibody produces a clear band with the P27 protein at 30 kDa, verifying its high specificity.

[0020] (4) The present invention has excellent sensitivity and detection efficiency. The detection ability of low-concentration antigens The kit can detect P27 antigen as low as 1 ng / mL, with a sensitivity 5-10 times higher than that of ordinary kits. In the detection of clinical samples, the positive detection dilution multiples of complex samples such as meconium and virus isolation supernatants are significantly higher than those of imported international products, especially the detection ability of K subtype virus is better than that of imported reagents.

[0021] (5) The present invention uses a double-antibody sandwich system for synergistic effect. The goat anti-P27 polyclonal antibody labeled with horseradish peroxidase is used as the sandwich secondary antibody, and its titer > 1:2000000, with a sensitivity more than 10 times higher than that of the traditional mouse anti-system (titer about 1:1000000). The high-yield characteristic of goat anti (50 mL of serum can be separated from each sheep's single blood collection) solves the bottleneck of low rabbit anti production, reduces production costs while ensuring the detection signal intensity.

[0022] (6) The present invention has stable performance and convenient operation. The stability of the whole process The kit can be stably stored at 4°C for more than 1 year, and still maintains more than 70% of the detection efficiency after 9 days of accelerated aging at 37°C. The batch coincidence 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.

[0023] (7) The rapid standardized operation detection process of the present invention only takes 3 hours, including two incubation and color development times, supports batch detection in 96-well plates, and is suitable for the high-throughput requirements of breeding chicken farms, biological product factories and other scenarios. The standardization degree of the operation steps is high, without complex pretreatment (samples such as serum and egg white can be directly detected), reducing human error.

[0024] (8) The present invention has significant cost advantages and industrial adaptability. The cost of preparing domestic raw materials and low-cost sheep anti-polyclonal antibodies is reduced by more than 60% compared with rabbit anti-antibodies, and there is no need to rely on imported enzyme-labeled secondary antibodies. The monoclonal antibodies screened by bioengineering are produced on a large scale through the HEK293F cell expression system, avoiding the risk of ascites preparation pollution in traditional hybridoma technology and further reducing production costs. Optimize the detection threshold according to the characteristics of Chinese chicken breeds to meet the domestic breeding needs. The clinical coincidence rate is more than 98% with international imported products, but the detection cost is only 1 / 3 of imported products. It is applicable to scenarios such as purification of high-generation breeding chicken farms and quality monitoring of SPF embryo sources, promoting the autonomy of epidemic disease purification of domestic breeding chicken flocks.

[0025] (9) The breakthrough of the bioengineering screening technology of the present invention adopts flow sorting 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 transformation.

[0026] (10) Demonstration of the localization of the detection system of the present invention. The present invention breaks the monopoly of imported reagent kits, establishes a full-chain localization technology from antigen preparation (recombinant engineering bacteria BL21 (DE3)), antibody screening to reagent kit assembly, promotes the independent control of avian leukosis detection reagents, and has significant economic and social benefits.

[0027] In summary, through bioengineering technology innovation and detection system optimization, the present invention realizes "high specificity, high sensitivity, low cost, and easy operation" for avian leukosis detection, provides key technical support for the prevention and control of epidemic diseases in China's chicken farming industry, and has significant industrial application value and market competitiveness. Brief Description of the Drawings

[0028] Figure 1 It is a gel electrophoresis diagram of the PCR product of the P27 gene. Among them, band 1 represents DNA Marker (5000 bp, 3000bp, 2000 bp, 1500 bp, 800 bp, 500 bp, 300 bp), and band 2 represents the PCR amplification result of the P27 gene.

[0029] Figure 2PCR identification results of recombinant plasmid pGEM-T-P27. Among them, band 1 represents DNA Marker (5000bp, 3000 bp, 2000 bp, 1500 bp, 800 bp, 500 bp, 300 bp), band 2 represents the negative control with empty vector pET-28a as the template, and band 3 represents the PCR amplification result of recombinant expression plasmid pGEM-T-P27.

[0030] Figure 3 SDS-PAGE analysis diagram of purified lysate. Among them, band 1 represents low molecular weight standard protein Marker (95 kDa, 72kDa, 55kDa, 43kDa, 34kDa, 26 kDa, 17 kDa, 10kDa), band 2 represents the protein to be purified, band 2 represents the permeate, band 4 represents the impurity removal solution, and band 5 represents the NTA-1000 eluent.

[0031] Figure 4 Determination curve of the affinity constant of monoclonal antibody. Specific implementation mode

[0032] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0033] Example 1. Preparation of P27 antigen The genetically engineered bacterium BL21(DE3) used in the present invention is derived from Escherichia coli K-12 strain; the recombinant genetically engineered bacterium E.coli BL21(DE3)(P27) is a recombinant Escherichia coli E.coli BL21(DE3) containing the prokaryotic expression plasmid pET28a-P27, which is constructed, identified and stored by the inventor's laboratory; Escherichia coli E.coli BL21(DE3) is purchased from Takara Bio Inc. (Dalian).

[0034] The avian leukosis virus (ALV) P27 gene was cloned by genetic engineering methods, transferred into Escherichia coli BL21(DE3) for expression, and the recombinant engineering bacterium E.coli BL21(DE3) that synthesizes P27 protein was obtained. This engineering bacterium was deeply studied, and a control experiment was carried out using an imported international avian leukosis diagnostic kit. The amino acid sequence of the P27 protein is shown in SEQ ID NO:1.

[0035] I. Cloning of the gag Gene of Avian Leukosis Virus (ALV) 1. Experimental Methods (1) Virus proliferation: Chicken embryo fibroblasts (CEFs) were routinely prepared from 9- to 11-day-old SPF chicken embryos. After the cells grew into a monolayer, the culture medium was poured off, and 0.5 mL of avian leukemia virus (ALV) was inoculated. At the same time, uninoculated cells were set as negative controls. The cells were statically adsorbed in an incubator at 37°C for 3 h, and during this period, the cell flask was gently shaken every 15-20 min to allow the inoculum to fully contact the cells. Then the virus solution was discarded, and appropriate amount of DMEM containing 1% calf serum was added for maintenance culture for 5-7 d. The cells were frozen and thawed twice repeatedly, centrifuged at 5000 rpm / min for 10 min, and the supernatant after centrifugation was detected using an internationally imported avian leukemia antigen detection kit.

[0036] (2) Virus RNA extraction and reverse transcription: The cell lysate supernatant that was detected to be positive was taken, and total virus RNA was extracted by the TRIzol method. The extracted RNA was reverse transcribed into cDNA using a Promega reverse transcription kit.

[0037] (3) PCR reaction: Using the cDNA obtained by reverse transcription as a template, PCR reaction was carried out with 5’-CCATGCCTGTAGTGATTA-3’ (SEQ ID NO:2) as the upstream primer and 5’-CCCGACCCAGTTTGTCCAT-3’ (SEQ ID NO:3) as the downstream primer.

[0038] The reaction system was: 13 μL of double-distilled water; 2 μL of 10×PCR Buffer; 0.5 μL of Taq DNA polymerase; 0.5 μL of dNTPs (10 mmol / L each); 1 μL of upstream primer, 1 μL of downstream primer; 2 μL of cDNA.

[0039] The reaction conditions were: 95°C for 5 min, 94°C for 1 min, 57°C for 1 min, 72°C for 1 min, for a total of 30 cycles; 72°C for 10 min. 4 μL of the PCR product was detected by 1% agarose gel electrophoresis.

[0040] (4) Ligation of RT-PCR products: The PCR product of the P27 gene was ligated with the pGEM-T Easy Vector (the specific method refers to the pGEM-T vector instruction manual), and then chemically transformed into JM109 competent cells.

[0041] (5) Screening and identification of positive clones: Pick one blue colony of the negative control and several white colonies of the positive ones, and inoculate them separately into 5 mL of LBA medium containing Amp, and culture overnight at 37 °C; perform small-scale plasmid extraction on the cultures, digest the white plasmid with EcoRI, and take the digestion product for electrophoresis analysis.

[0042] (6) Sequencing of the gag gene: Send the recombinant plasmid identified as positive to Beijing Saibaisheng Company for bidirectional sequencing, and compare the sequencing results with the ALV standard strain (BAI-A strain).

[0043] 2. Experimental results Viral RNA was extracted from CEF cells infected with ALV, and a band of about 2.1 kb was specifically amplified by RT-PCR. No band was amplified from the RNA extracted from non-infected CEF cells, indicating that the desired ALV gag gene was successfully obtained.

[0044] After electrophoresis analysis of the digestion product, a 3 kb T-vector band and a band of about 2.1 kb of the target were shown, proving that the recombinant plasmid pGEM-T-gag with an inserted foreign target gene fragment was obtained.

[0045] The sequencing results of the recombinant plasmid were compared with the ALV BAI-A strain sequence, and the homology was 97%, indicating that the ALV gag gene was cloned in this experiment.

[0046] II. Subcloning of the avian leukosis virus (ALV) P27 gene 1. Experimental method (1) PCR amplification of the P27 gene: Using the above recombinant plasmid pGEM-T-gag as a template, with 5’-CGGGATCCATGCCTGTAGTGATTAAGACAG-3’ (SEQ ID NO:4) as the upstream primer and 5’-ACGTCGACCTAGGGCTGGATAGCAGAC-3’ (SEQ ID NO:5) as the downstream primer for PCR reaction. On the premise of 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. Use Ex-Taq high-fidelity enzyme for PCR reaction, pre-denature at 95 °C for 5 min, and then perform 94 °C for 1 min, 56 °C for 1 min, 72 °C for 1 min, for a total of 30 cycles; finally react at 72 °C for 10 min; at the same time, set the pGEM-T easy empty vector as the negative control. Take 4 μL of the PCR product and perform electrophoresis detection with 1% agarose gel (containing 0.5 μg / mL EB).

[0047] (2)Ligation of PCR products: The PCR products of the P27 gene were ligated with pGEM-T Easy Vector (for the specific method, refer to the pGEM-T vector instruction manual), and then transformed into JM109 competent cells by the chemical method.

[0048] (3)Screening and identification of positive clones: One blue colony of the negative control and several white positive colonies were picked and inoculated into 5 mL of LBA medium containing Amp respectively, and cultured overnight at 37 °C; plasmid minipreps were performed on the cultures, the white plaque plasmids were digested with EcoRI, and the digestion products were subjected to electrophoresis analysis.

[0049] (4)Sequencing of the P27 gene: The recombinant plasmid identified as positive was sent to TaKaRa Biotechnology (Dalian) Co., Ltd. for sequencing, and the sequencing results were compared with the ALV standard strain (BAI-A strain).

[0050] 2. Experimental results After electrophoresis detection of the PCR products, a specific band of about 800 bp appeared, which was consistent with the expected size ( Figure 1 ).

[0051] After electrophoresis analysis of the digestion products, a 3 kb T-vector band and a specific target band of about 800 bp appeared, while no target band appeared in the empty vector control, proving that the recombinant plasmid pGEM-T-P27 was successfully constructed.

[0052] The sequencing results of the pGEM-P27 recombinant plasmid were compared with the ALV BAI-A strain sequence, and the homology was 97%, indicating that the P27 gene was subcloned in this experiment.

[0053] III. Construction of recombinant engineering bacteria 1. Experimental method The recombinant plasmid pGEM-T-P27 and the empty vector pET-28a plasmid were double-digested with BamHⅠ and SalⅠ. The two digestion products were electrophoresed in 1.2% agarose gel respectively. The double-digestion product of the recombinant plasmid pGEM-T-P27 was used to recover the P27 gene of about 800 bp according to the operation steps of the DNA rapid recovery kit, and the double-digestion product of the empty vector pET-28a plasmid was used to recover the linearized vector by ethanol precipitation and stored at -20 °C for later use.

[0054] Mix the P27 gene with BamHⅠ and SalⅠ sticky ends and the linearized expression vector pET-28a at a molar ratio of 3:1, add T4 DNA ligase, and ligate overnight at 4°C. Then, transform JM109 competent cells using the conventional chemical method, and identify positive clones by restriction endonuclease digestion and PCR methods. Subsequently, transform the plasmid of the screened positive clone into Escherichia coli BL21(DE3) competent cells by the chemical method and culture for standby.

[0055] 2. Experimental Results The double digestion product of the recombinant expression vector pET-28a-P27 showed a 5.4 kb T-vector band and a specific target band of approximately 800 bp ( Figure 2 ), demonstrating the successful construction of the recombinant expression vector pET-28a-P27.

[0056] IV. Inductive Expression of P27 Protein 1. Experimental Method Select a well-grown positive single colony from the previous step and inoculate it into an LBK (containing 30 μg / mL Kan) liquid medium, and culture overnight at 37°C. Take the overnight culture broth and perform a 100-fold expansion culture in an LBK (containing 30 μg / mL Kan) liquid medium, and culture at 37°C for 2 - 3 h (OD600 to 0.4 - 0.6). Add IPTG to a final concentration of 1 mmol / L and induce expression at 30°C for 5 h. Centrifuge at 5000 rpm for 15 min at 4°C, discard the culture broth, collect the bacterial precipitate, and analyze by 15% SDS-PAGE.

[0057] 2. Experimental Results The molecular weight of the protein induced and expressed by the recombinant bacteria was 30 kDa, which was consistent with the expected size.

[0058] V. Ultrasonic Lysis of Induced Expression Bacteria 1. Experimental Method Take 200 mL of bacterial culture, centrifuge at 5000 rpm / min for 15 min, discard the supernatant, and weigh the wet weight of the bacterial cells. Resuspend the pellet with 10 mL of pre-cooled NTA-0 buffer. Add a certain amount of PMSF to a final concentration of 1 mmol / L and a certain amount of lysozyme to a final concentration of 0.2 mg / mL. Mix well, place on ice for 30 min, and lyse the bacterial cells by sonication. Add 10% Triton X-100 (final concentration 0.05%), mix thoroughly, place on ice for 15 min, and sonicate the cells in an ice bath. Add 1 mol / L MgCl2 to a final concentration of 1 mmol / L, mix well, and let stand at room temperature for 10 min. Centrifuge at 12000 rpm at 4°C for 20 min. Take 100 μL of the supernatant and a small amount of the pellet respectively, add 100 μL of 2×SDS loading buffer to each, mix well, and boil for 5 min. Take 20 μL of the sample for 15% SDS-PAGE electrophoresis analysis.

[0059] 2. Experimental Results The results showed that after 15% SDS-PAGE electrophoresis analysis of the expression products of the induced recombinant bacteria, protein bands of the expected size were visible in both the supernatant and the pellet after sonication lysis. After thin layer scanning analysis, the content of the recombinant protein in the supernatant was 22.1% and that in the pellet was 20.7%.

[0060] VI. Purification and Identification of P27 Protein 1. Purification of Recombinant P27 Protein: Using the lysate supernatant of the recombinant engineering bacterium BL21(DE3) expressing P27 protein as the material, refer to the "SNBC 3S NTA Resin Usage Method" of Shanghai Shenneng Bojin Biotechnology Co., Ltd. to perform affinity purification on the expressed fusion protein. The main operation steps are as follows: (1) Load 1 mL of NTA resin into the chromatography column and let it stand still.

[0061] (2) Equilibrate the chromatography column with 20 mL of NTA-0 buffer.

[0062] (3) Take 10 mL of the bacterial cell lysate supernatant and add it to the NTA chromatography column. Control the flow rate at about 15 mL / h, collect the breakthrough fraction, and label it as "breakthrough solution".

[0063] (4) Elute with 10 mL of NTA-0 buffer, control the flow rate at about 30 mL / h.

[0064] (5) Elute successively with 10 mL of NTA-20, NTA-40, NTA-60, NTA-80, NTA-100, NTA-200, and NTA-1000 buffer, controlling the flow rate at about 15 mL / h; collect the eluate, collecting 1 mL per tube.

[0065] (6) Perform SDS-PAGE analysis on the collected samples to determine the distribution of the target protein in the eluate.

[0066] (7) Measure the OD 260 and OD 280 of the purified P27 protein, and calculate the protein concentration. The calculation formula for measuring the protein concentration by the ultraviolet absorption method is: Protein concentration (mg / mL) = 1.45 × OD 280 - 0.74 × OD 260 .

[0067] The SDS-PAGE analysis results showed that the eluate with an imidazole concentration of 100 mmol / L contained the target protein with high purity. The thin layer scanning results showed that its purity was greater than 95%. The protein concentration measured by the ultraviolet absorption method was approximately 0.53 mg / mL, and the results were as Figure 3 shown.

[0068] 2. Western Blotting Detection of the Induced Expression Product Take 10 μL of the expression product, add an equal volume of 2× loading buffer, boil for 5 min and then perform SDS-PAGE, and then transfer it to the NC membrane. Block it overnight at 4°C. Incubate successively with rabbit anti-P27 polyclonal antibody and enzyme-labeled goat anti-rabbit secondary antibody, and finally add the luminescent solution for darkroom exposure and develop the film. As a result, a clear band appeared. Comparing this band with the standard molecular weight, its size was approximately 30 kDa, indicating that the E. coli expressed specific P27 protein.

[0069] VII. Characterization of the Genetically Engineered Bacterium E. coli BL21(DE3) 1. Bacterial Strain Morphology: It is a Gram-negative bacillus without spores, with a size of 0.4 - 0.7 μm × 2 - 3 μm, round and blunt at both ends, scattered or paired. Most strains move with peritrichous flagella, but there are also non-motile variants without flagella or with lost flagella. Usually, no visible capsule is present, but there is often a microcapsule. Basic dyes have good staining properties for this bacterium, and the two ends of the bacterial cells are occasionally slightly darker.

[0070] 2. Culture Characteristics: All of these bacteria are facultative anaerobes. On the kanamycin-resistant LB agar plate, aerobic culture at 37°C for 12 - 16 h forms round, raised, smooth, moist, semi-transparent, and gray colonies.

[0071] 3. Biochemical characteristics: This bacterium can ferment most carbohydrates to produce acid and gas, and can rapidly ferment lactose. It hardly produces hydrogen sulfide and does not decompose urea. Both the indole and methyl red tests are positive, while both the VP test and citrate utilization test are negative.

[0072] 4. Purity: It shall be pure as per Appendix 169 of Part 1 of the Chinese Veterinary Pharmacopoeia (2000 Edition).

[0073] 5. Identification characteristics: After the bacterial cells are suspended in PBS and boiled, the specific P27 gene can be amplified using P27 gene-specific primers. After induced expression, specific bands of P27 protein can be seen by SDS-PAGE electrophoresis analysis.

[0074] 6. Safety: This genetically engineered bacterium is used for the production of P27 antigen, has no infectious characteristics, and is not involved in virulence safety.

[0075] 7. Storage: The freeze-dried bacterial strain can be stored at -80 °C for 5 years and at 4 °C for 1 year.

[0076] VIII. Relevant information on the establishment of the master seed lot, working seed lot and foundation seed lot 1. Establishment and identification of the master seed lot: The original recombinant engineering bacterium is subcultured continuously for 3 generations. Each generation is expanded and cultured, and its characteristics are identified.

[0077] (1) Subculture method: Take the original positive bacterial strain and inoculate it into 5 mL of LBK (containing 30 μg / mL Kan) liquid medium, and culture it overnight at 37 °C. 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 h (OD600 to 0.4 - 0.6).

[0078] (2) Subculture range: The subculture does not exceed 3 generations.

[0079] (3) Bacterial strain characteristics and identification: It is carried out according to the characteristics identification of the above-mentioned genetically engineered bacterium E. coli BL21(DE3).

[0080] (4) Subculture method: The same as step (1).

[0081] (5) Subculture range: The subculture does not exceed 3 generations.

[0082] (6) Identification of the foundation seed lot a. Specificity test: After the bacterial cells are suspended in PBS, boiled and amplified using P27 gene-specific primers, the specific P27 gene can be amplified. After the bacterial cells are induced to express and analyzed by SDS-PAGE electrophoresis, specific bands of P27 protein should be visible.

[0083] b. Purity identification: It shall be carried out in accordance with Appendix 169 of Part I of the Chinese Veterinary Pharmacopoeia (2000 Edition) and shall be pure.

[0084] c. Screening and identification of working seed lots: Take a certain number of original seeds, proliferate them to a certain number after passage, mix all the cultures of the same passage evenly, aliquot, and store them at -80 °C or in liquid nitrogen for later use.

[0085] d. Subculture method: The same as in step (1).

[0086] e. The passage range does not exceed 2 passages.

[0087] f. Identification and inspection of production seed lots: The specificity test and purity test are the same as those for the basic seed lot; Content determination: Induce the expression of the production seeds of each passage, lyse the bacterial cells by ultrasonic wave, then purify the P27 protein according to the method in step six, and determine its content by ultraviolet absorption method. The results show that the content of the P27 protein produced by the strains of each passage should be basically the same, about 20%, and the protein concentration is close to 0.58 mg / mL.

[0088] Example 2 Preparation and Identification of Anti-P27 Monoclonal Antibody 1. Animal immunization and enrichment of IgG B cells: Emulsify the qualified and purified P27 protein in Example 1 with an equal amount of Freund's complete adjuvant, and subcutaneously inject rabbits at multiple sites, 0.8 mL per rabbit (about 400 μg / rabbit). After 14 days, give a secondary subcutaneous immunization with P27 antigen containing an equal amount of Freund's incomplete adjuvant, 0.4 mL per rabbit. After 14 days, give a subcutaneous immunization only with P27 antigen, 0.4 mL per rabbit. After 3 days, take the spleen and grind it, and enrich IgG B cells by density centrifugation and magnetic bead sorting.

[0089] 2. Flow sorting of specific IgG B cells: Use software to predict the specific epitopes shared by ALV-A / B, ALV-J, and ALV-K but distinct from ALV-E, split them into 14 fragments, artificially synthesize them and add GFP fluorescent tags, stain the enriched IgG B cells with the fluorescently labeled short peptides, and through flow sorting technology, sort out the specific IgG B cells with the highest fluorescence intensity from 1000 B cells for in vitro expansion culture. The amino acid sequences of the 14 fragments are respectively (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 for synthesizing short peptides, and the synthesized short peptides are used for flow screening of specific B cells against ALV-A / B, ALV-J, and ALV-K.

[0090] 3. Preparation of anti-P27 monoclonal antibody (1) Construction of light chain and heavy chain plasmids of P27 monoclonal antibody: Perform BCR sequencing on the screened IgG B cells to obtain 3000 sequences, ligate the light chain and heavy chain sequences of each BCR to the eukaryotic expression vector pTT5 respectively to make P27 antibody light chain and heavy chain plasmids, transfect them into cells respectively and culture for three days, take the cell supernatant into a 96-well ELISA plate (coated with 100 ng / well of P27 antigen) for ELISA detection, and screen out the BCR sequence corresponding to the cell well with the highest OD 620 value (3.2980). The light chain and heavy chain base sequences of the P27 monoclonal antibody (named 122) are shown as SEQ ID NO:20 (122DK) and SEQ ID NO:21 (122DH) respectively.

[0091] The CDR sequences of the P27 monoclonal antibody are as follows: CDR-H1 (SEQ ID NO:22): GYSFTGYYMH; CDR-H2 (SEQ ID NO:23): WINPNSGGTNYAQKFQG; CDR-H3 (SEQ ID NO:24): ARDYYGSSGWYFDV; CDR-L1 (SEQ ID NO:25): RASQSVSSSYLA; CDR-L2 (SEQ ID NO:26): DASNRAT; CDR-L3 (SEQ ID NO:27): QQYNSYPLT.

[0092] 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 self-associate to form the antibody.

[0093] (5) Expression and purification of the anti-P27 monoclonal antibody: Resuspend HEK293F cells with Expi293™ expression medium and transfer them into a 1 L shake flask containing 200 mL of Expi293™ expression medium. Incubate in a cell shaker at 37 °C and 5% CO2 with shaking at 130 r / min until the cell density reaches 1×10 6 cells / mL; when the cell number and status are suitable for transfection, prepare the transfection reagent: Mix solution A (mix 2.5 mL of Opti-MEM I serum-free medium with 100 μg of heavy chain plasmid and 100 μg of light chain plasmid and react for 10 min, prepare fresh) and solution B (mix 2.5 mL of Opti-MEM I serum-free medium with 600 μL of PEI and react for 10 min, prepare fresh). After preparation, let it stand at room temperature for 20 min. Prepare solution C by mixing solution A and solution B and let it stand at room temperature for 20 min. Add solution C to HEK293F cells, transfer the cell flask to a cell shaker at 37 °C and 5% CO2 and shake at 130 r / min for 7 d; harvest the above cells, centrifuge at 4500 r / min for 15 min at 4 °C, collect the supernatant, and filter it using a 0.45 μm filter membrane. Wash and equilibrate the pre-packed column with 10 column volumes of PBS (pH = 7.4). Add the sample to the upper end of the pre-packed column and control the flow rate at 1 mL / min; after the sample binds, wash the pre-packed column with 20 column volumes of PBS (pH = 7.4) to remove non-specifically adsorbed impurity proteins, control the flow rate at 1 mL / min; add 100 μL of 1 mol / L Tris-HCl (pH = 8.8) to each milliliter of eluent, and pre-add 2 mL of 1 mol / L Tris-HCl (pH = 8.8) to the collection tube.

[0094] After washing, add 20 mL of 100 mmol / L glycine hydrochloride buffer (pH = 2.0) to elute the bound antibody, collect the eluate; collect the eluted antibody and perform concentration and buffer replacement. Detect the expression by ELISA, and select the stably expressed antibody for SDS-PAGE and BCA quantification.

[0095] 3. Characterization of the anti-P27 monoclonal antibody (1)Concentration determination: The concentration of the monoclonal antibody was determined to be 1.86 mg / mL.

[0096] (2)Specificity identification: The purified P27 monoclonal antibody was detected by conventional Western Blotting, indicating that the P27 monoclonal antibody had a good reactivity to the P27 protein.

[0097] (3)ELISA assay: The ELISA plate was coated with the P27 monoclonal antibody diluted 200-fold, 100 μL / well, at 4 °C for 12 h. After blocking with 5% skim milk at 37 °C for 2 h, avian viruses and bacteria such as NDV, IBDV, IBV, AEV, and EDSV were added respectively. The OD 650 value was measured using the enzyme-labeled antibody diluted 1200-fold and the substrate chromogenic solution. The results are shown in Tables 1 and 2: Table 1 Results of ELISA assay for the specificity of the monoclonal antibody

[0098] Table 2 Results of ELISA assay for the specificity of the monoclonal antibody

[0099] It can be seen from the results in the table that when using this monoclonal antibody to detect various avian disease viruses and bacteria, the OD values are all around 0.1, close to the OD values of the negative control, indicating that the monoclonal antibody has no cross-reaction with the detected viruses and bacteria and has strong specificity.

[0100] (4)Determination of the affinity constant: It was determined by the indirect ELISA method. The specific steps were as follows: The qualified P27 protein in Example 1 was diluted at ratios of 1:25, 1:100, and 1:200, added to the enzyme-labeled wells, 100 μL / well, and incubated overnight at 4 °C. The plate was washed 3 times with PBST, 3 min each time. 300 μL of 5% skim milk was added to each well and blocked at 37 °C for 4 h. The purified P27 monoclonal antibody was serially diluted from 1:30 to 1:15360 and then added to the enzyme-labeled plate wells, 100 μL / well, and incubated at 37 °C for 1 h. The plate was washed and patted dry. 100 μL of HRP-goat anti-rabbit IgG diluted 1:500 was added, incubated at 37 °C for 1 h, and the plate was washed and patted dry. 100 μL of the substrate solution was added, reacted at room temperature in the dark for 15 min, 100 μL of the stop solution was added, and reacted at room temperature for 5 min to terminate the reaction. The OD 650 value was measured and a curve was plotted.

[0101] Using the logarithm of the different dilution ratios of the monoclonal antibody as the abscissa and the corresponding OD value as the ordinate, the affinity constant determination curve of the P27 monoclonal antibody is as Figure 4As shown, taking the OD value at the flat section at the upper part of each curve as 100%, the antibody concentration corresponding to half of its maximum OD value (i.e., 50% OD value) is found and substituted into the following formula to calculate the affinity constant.

[0102]

[0103] In the formula, Ab and Ab′ respectively represent the antibody concentrations (mol / L) that produce half of the absorbance value when the antigen concentrations are Ag and Ag′, n = Ag / Ag′, and then pairwise comparisons are made to obtain the K values when n = 2, n = 4, and n = 8, and the average of the three K values is taken as the final result.

[0104] Through calculation, the affinity constant K values when n = 2, n = 4, and n = 8 are respectively: K1 = 1.05×10 10 L / mol, K2 = 5.15×10 9 L / mol, K3 = 7.26×10 9 L / mol K = (K1 + K2 + K3) / 3 = 7.6×10 9 L / mol Generally, it is considered that antibodies with K values of 10 7 ~10 12 have high antibody affinity; antibodies with K values of 10 5 ~10 7 have low antibody affinity; based on the K values measured in this experiment, it can be considered that the P27 monoclonal antibody has relatively high affinity.

[0105] Example 3 Preparation and Inspection of Anti-P27 Monoclonal Antibody-Coated Plate 1. Preparation of Coated Plate (1) Coating: Under sterile conditions, the purified P27 monoclonal antibody in Example 2 was diluted with carbonate buffer to 3.68 μg / mL, 0.1 mL was added to each well of a 96-well enzyme-linked immunosorbent assay (ELISA) plate, and incubated at 2 - 8°C for 15 h. Then, the coating solution in the wells was discarded, and the wells were washed three times with 0.01 mol / L PBST.

[0106] (2) Blocking: Add blocking solution to each well of the already coated ELISA plate, 250 μL per well, and block at 37°C for 4 h. Then, discard the blocking solution.

[0107] (3) Drying: Place the coated plate in a dryer and dry at room temperature for 30 min. Then, put it into an aluminum foil bag and seal it under vacuum.

[0108] 2. Inspection of Coated Plate (1) Appearance inspection: Transparent, dry, and vacuum-packed, without damage, breakage, etc.

[0109] (2)Performance test: Use the standard quality control solution sample of this kit to detect the sensitivity of each batch of kits. This solution sample consists of P27 protein solutions at 6 different dilutions, and its specifications and passing criteria are shown in Table 3.

[0110] The qualified coated plates shall meet the following criteria: (1)The OD 650 measurement values of all quality control solution samples are within the qualified range.

[0111] (2)The OD 650 value of the positive control is between 1.00 and 1.60; the OD 650 value of the negative control is not greater than 0.20.

[0112] Table 3 Concentrations of the kit's standard quality control solution and the qualified range of its OD 650 measurement value

[0113] Example 4 Preparation and testing of HRP-labeled goat anti-P27 polyclonal antibody I. Preparation of polyclonal antibody 1. Animal immunization: Use the P27 protein qualified in Example 1 to immunize adult sheep by combining subcutaneous, intradermal injection with intramuscular injection. The specific immunization procedure is as follows: (1)Primary immunization: For intradermal and intramuscular injection, take the antigen solution qualified in Example 1, add an equal volume of Freund's complete adjuvant, and perform complete emulsification. Inject 0.5 mL of the antigen solution intradermally and intramuscularly into each sheep.

[0114] (2)Second immunization: 3 weeks later, perform the second immunization by subcutaneous and intramuscular injection. Take the antigen solution qualified in Example 1, add an equal volume of Freund's incomplete adjuvant, and perform complete emulsification. Inject 0.5 mL of the antigen solution subcutaneously and intramuscularly into each sheep.

[0115] (3)Third and fourth immunizations: Perform the third and fourth immunizations every three weeks later, and the immunization method is the same as the second immunization.

[0116] (4)Collect blood from the jugular vein 10 - 15 days after immunization to check for specific antibodies. AGP detection can be used. When the titer > 16, blood can be collected to separate serum and purify Ab. Every two weeks later, boost the immunization of the sheep, and the immunization method is the same as the second immunization. Periodically collect blood from the jugular vein of the sheep and collect the serum.

[0117] 2. Serum identification (1)The AGP test is carried out according to the conventional method. An obvious precipitation line appears between the antigen well and the antibody well, and the titer of the goat anti-P27 polyclonal antibody reaches 1:32.

[0118] (2)The steps of Western Blotting test are as follows: Take 10 μL of purified P27 protein, treat it with an equal volume of 2× loading buffer, perform SDS-PAGE, then transfer it to an NC membrane, block it overnight at 4°C. After sequentially adding a 500-fold dilution of goat serum and incubating with enzyme-labeled horse anti-goat IgG, add the luminescent solution and expose it, wash the film. As a result, a clear band appears. Comparing the size of this band with the standard molecular weight, it is approximately 30 kDa, which is consistent with the expected size, indicating that the goat serum can specifically bind to the expressed P27 protein.

[0119] 3. Serum collection and antibody purification: Collect blood through the jugular vein to prepare serum. Each goat is collected about 100 mL of blood each time and about 50 mL of serum is separated.

[0120] (1)Preparation of P27 antigen column: Swell the activated Sepharose 4B gel powder for 10 min, transfer it to a Buchner funnel, quickly wash it three times with HCl, and then wash the microspheres with the binding solution. Transfer the microspheres to a centrifuge tube, add 500 μL of P27 protein concentrated in the binding solution with a protein concentration of 5 - 10 mg / mL, mix well and incubate overnight at 4°C. Centrifuge the overnight mixture at 500 rpm for 5 min, recover the supernatant to measure the protein concentration, calculate the coupling efficiency of 000, then add 10 mL of the binding solution and mix well, centrifuge to remove the unbound P27 protein. Block it with ethanolamine at 4°C for 5 h. Transfer the conjugate to a chromatography column and equilibrate the antigen column with PBS.

[0121] (2)Purify the P27 polyclonal antibody with the P27 antigen column Dilute the anti-P27 polyclonal goat serum 3-fold with PBS and add it to the antigen column, wash the antigen column with PBS until no protein is eluted. Elute the polyclonal antibody with the eluent, collect the antibody protein with a collection tube containing 0.25 mL of neutralizing solution, and collect 0.75 mL in each tube. Calculate the concentration of the collected antibody: Protein concentration = 1.45×OD 280 -0.74×OD 260 。

[0122] 4. Preparation of enzyme-labeled polyclonal antibody: Label the prepared polyclonal antibody with horseradish peroxidase (HRP).

[0123] 5. Inspection (1)Character inspection: It should be a clear, light yellow solution, odorless, tasteless, and without precipitates.

[0124] (2)Efficacy inspection: Concentration determination: The concentration of the enzyme-labeled antibody is determined to be 5.65 mg / mL.

[0125] Specific identification of polyclonal antibody: Coat the ELISA plate with purified polyclonal antibody diluted 600-fold, 100 μL / well, at 4°C for 12 h. After blocking with 5% skim milk at 37°C for 2 h, add NDV, IBDV, IBV, AEV, EDSV, E. coli, E. Salmon and other viruses and bacteria, enzyme-labeled antibody diluted 1000-fold, and finally add substrate chromogenic solution. Measure the OD value after termination. 650 The results should all be close to 0.1 and similar to the OD value of the negative control, indicating that the polyclonal antibody has no cross-reaction with other avian viruses and bacteria.

[0126] Determination of the titer of enzyme-labeled antibody: Gradient dilute the purified P27 protein (initial concentration 0.57 mg / mL) (500 - 256000-fold serial dilution), coat the ELISA plate, and perform a checkerboard experiment with gradient-diluted enzyme-labeled goat anti-P27 polyclonal antibody (100 - 12800-fold serial dilution). Take the maximum dilution of the enzyme-labeled antibody that can react with the P27 protein at the maximum dilution and has an OD value of about 1.0 as the working concentration of the enzyme-labeled antibody. The titer of the enzyme-labeled antibody should be not less than 1:1000. The results are shown in Table 4.

[0127] Table 4 Determination of the titer of enzyme-labeled goat anti-

[0128] Example 5 Preparation and inspection of the working solution of enzyme-labeled antibody 1. Preparation of the working solution of enzyme-labeled antibody Dilute the enzyme-labeled antibody prepared in Example 4 with PBST solution at a ratio of 1:600, add preservative and dispense.

[0129] 2. Inspection: Sterility inspection: Conduct the inspection according to Appendix 169 of Part I of the Chinese Veterinary Pharmacopoeia (2000 Edition), and it should be sterile.

[0130] Efficacy inspection: Detect the sensitivity of each batch of enzyme-labeled antibody with the standard quality control solution sample of this kit. This solution sample consists of P27 protein solutions at 6 different dilutions, and its specifications and acceptance criteria are shown in Table 5.

[0131] The qualified working solution of enzyme-labeled antibody should meet the following standards: (1) The measured OD values of all quality control solution samples are within the qualified range. 650

[0132] (2) The OD value of the positive control is between 1.00 and 1.60; the OD value of the negative control is not greater than 0.20. 650 650

[0133] Table 5 Concentrations of the standard quality control solution of the kit and their OD650 Measured value qualified range

[0134] Example 6, Determination of working conditions of ELISA method I. Determination of coated monoclonal antibody concentration and enzyme-labeled polyclonal antibody concentration by checkerboard method First, use an imported international avian leukemia antigen detection kit to measure the OD values of 4 P27 protein samples with different concentration gradients. Coat the 3.75 mg / mL monoclonal antibody by gradient dilution from 1:1000 to 1:64000, and dilute the 3.28 mg / mL enzyme-labeled polyclonal antibody from 1:1000 to 1:10000. Perform ELISA on these 4 samples, and compare the OD values with the OD of the imported international avian leukemia antigen detection kit 650 The coating concentration with an OD reading similar to that of the imported international avian leukemia antigen detection kit is defined as the optimal coating concentration of the monoclonal antibody, and the diluted concentration of the enzyme-labeled polyclonal antibody is defined as the optimal working concentration. The results are shown in Table 6 and Table 7

[0135] Table 6 Determination of standard samples

[0136] Table 7 Determination of monoclonal antibody and enzyme-labeled polyclonal antibody concentrations by checkerboard method

[0137] It can be seen from the results that when the monoclonal antibody is coated by dilution at 1:8000 and the enzyme-labeled polyclonal antibody acts by dilution at 1:2000, the sample reading is closest to the corresponding reading of the imported international kit. Therefore, the optimal coating dilution of the monoclonal antibody is set at 1:8000 times, and the dilution multiple of the enzyme-labeled secondary antibody is set at 1:2000

[0138] II. Determination of negative control According to the general method for determining negative control, collect SPF chicken serum aseptically and perform gradient dilution with PBS. Detect using the optimized self-made kit conditions, and at the same time detect the gradient-diluted SPF serum with an imported international avian leukemia antigen detection kit. When the SPF serum is diluted at a gradient of 1:100 or more, its OD 650 value is less than 0.1 and is similar to the detection result of the imported international avian leukemia antigen detection kit. Therefore, the 1:100 diluted SPF serum is used as the standard negative control

[0139] III. Determination of positive control According to the general method for determining positive controls, the P27 antigen (0.58 mg / mL) was serially diluted with PBS and detected using the optimized self-made kit conditions. At the same time, the serially diluted P27 antigen was detected using an internationally imported avian leukosis antigen detection kit. When the dilution of the P27 protein was 1:100,000 (concentration 5 ng / mL), the OD 650 was similar to the measurement result of the positive standard sample of the internationally imported avian leukosis antigen detection kit. For convenience of calculation, the standard positive control was set as a solution with a P27 antigen concentration of 5 ng / mL.

[0140] IV. Determination of the critical value of the kit Using the 8 antigen concentration values measured in this experiment, and the standard negative and positive samples of the two products and their corresponding average OD 620 readings, OD 620 value-concentration curves of this product and the internationally imported product can be made. According to the curve, the positive and negative critical OD values of the internationally imported product and its minimum antigen detection concentration were measured. The positive critical value S / P was determined according to the formula S / P = (S - N) / (P - N).

[0141] According to the results, the standard positive OD value of the internationally imported avian leukosis antigen detection kit was 0.943, the standard negative sample OD value was 0.047, and the positive and negative critical S / P value was 0.20. Substituting the standard negative and positive OD values and the positive and negative critical S / P value of the internationally imported kit into the formula, the positive and negative critical OD value was calculated to be 0.226. The corresponding positive and negative critical concentrations were found to be 9.687 ng through the OD-concentration curve, and the OD value of this product corresponding to this was 0.226. The standard positive OD value of this product was 0.85, with a fluctuation range of 0.831 - 0.853, and the standard negative OD value was 0.040, with a fluctuation range of 0.0370 - 0.0420. Substituting the average values of our standard negative and positive samples into the formula, the S / P critical value was calculated to be 0.202. Substituting the fluctuation ranges of the standard negative and positive samples into the formula to find the maximum and minimum values, which were 0.20 and 0.22 respectively. Therefore, the determination criterion was set as S / P equal to 0.20. V. Determination of each working condition of the ELISA method According to the results, the final conditions of the ELISA method were: The microtiter plate was a Corning microtiter plate, the coating buffer was CB buffer, the coating antigen was purified P27 monoclonal antibody (1.86 mg / mL), the coating dilution factor 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 working condition was coating overnight at 4°C, the test sample was incubated at 25°C for 1 h, the enzyme-labeled polyclonal antibody was incubated at 25°C for 1 h, and color development was carried out at 25°C for 15 min. Among them, the OD of the negative serum 650The value is controlled below 0.15, and the OD of the positive control 650 minus the OD of the negative control 650 should be greater than 0.2. The positive and negative of the sample are determined according to the following method: Calculate the S / P value of the sample

[0142] When the sample S / P < (0.2), the sample is negative for ALV-P27 antigen; when the sample S / P ≥ (0.23), the sample is positive for ALV-P27 antigen. When (0.20) ≤ sample S / P < (0.23), the sample is suspected positive for ALV-P27 antigen.

[0143] Example 7. Use of the detection kit (1) Preparation of samples Egg white: Collect thin egg white and directly add it as the sample to be tested to the ELISA reaction plate without dilution.

[0144] Cloacal swab: Add the cloacal swab to 1 mL of culture medium or sample diluent and then store it frozen. Before detection, restore the sample to room temperature, let it stand, and then aspirate 100 µL of the supernatant as the sample to be tested.

[0145] Serum: Without dilution, directly take 100 µL of the clear serum sample and add it to the reaction plate.

[0146] (2) Preparation before experiment Restore the kit components to room temperature before use; prepare a sample dilution plate or micro test tube; Preparation of 1X washing solution: For example, for 50 mL of 20-fold concentrated washing solution, add 950 mL of deionized water and mix well for standby; Do not dilute the negative control and positive control.

[0147] (3) Operating method a. Sample addition: Fix the required number of strips on the plate rack. Set 2 wells for positive control and 2 wells for negative control for each experiment. Add 100 µL of positive and negative controls to the appropriate wells respectively, and add 100 µL of the sample to be tested to the remaining wells. The sample is used for detection without dilution.

[0148] b. Incubation: Stick on the sealing film and incubate at room temperature (25°C ± 2°C) for 60 min.

[0149] c. Washing: Discard the liquid in the wells, fill each well with the diluted washing solution (about 350 µL / well) for washing, and discard the washing solution in the wells. Repeat the washing 4 times and then pat dry.

[0150] d. Enzyme addition: Add 100 µL of enzyme-labeled substance to each well.

[0151] e. Incubation: Stick on the sealing film and incubate at room temperature (25°C ± 2°C) for 60 min.

[0152] f. Washing: Discard the liquid in the wells, fill each well with the diluted washing solution (about 350 µL / well) for washing, and discard the washing solution in the wells. Repeat the washing 4 times and then pat dry.

[0153] 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.

[0154] h. Termination: Add 100 µL of termination solution to each well and gently tap to mix evenly.

[0155] i. Reading: Read the values with an enzyme-linked immunosorbent assay (ELISA) reader and read the absorbance value A(650) of each well at a single wavelength of 650 nm. Please complete the reading within 10 min after termination.

[0156] (4) Result determination

[0157] Negative control standard: ≤ 0.15; Positive control standard: ≥ 0.2.

[0158] If the experimental results do not meet the negative and positive control standards, the experiment is considered invalid and must be repeated.

[0159] Judgment criterion: Calculate the S / P value of the sample

[0160] When the S / P of the sample ≤ 0.23, the sample is negative for ALV-P27 antigen; when the S / P of the sample > 0.23, the sample is positive for ALV-P27 antigen.

[0161] Whether the tested sample contains P27 antigen is related to its S / P value. The positive control represents a P27 antigen level of approximately 15 ng / mL. Therefore, the relative content of P27 in the tested sample can be determined by the S / P value.

[0162] (5) Factors that may affect the experimental results are as follows: a. Insufficient washing, such as insufficient washing solution volume.

[0163] b. Blockage of the washing machine needle holes or improper position adjustment, resulting in excessive residual washing solution at the bottom of the wells.

[0164] c. Inaccurate sample addition volume.

[0165] d. Cross-contamination and contamination of enzyme conjugates and other reagents should be avoided during the experiment.

[0166] e. Incorrect wavelength reading is not used.

[0167] f. During the testing process, drying of the reaction wells should be avoided.

[0168] g. Using highly hemolytic specimens, incompletely agglutinated blood, serum containing fibrin, or microbe - contaminated serum will lead to adverse results.

[0169] (6) Precautions a. The kit taken out from the refrigerated environment should be balanced to room temperature before opening for use.

[0170] b. The unused pre - coated strip should be placed in a sealed plastic bag with a desiccant and stored in a sealed manner.

[0171] c. If crystals appear in the 20 - fold concentrated washing solution, place it at 37 °C until dissolved and then use.

[0172] d. Reagent components from different batches cannot be mixed.

[0173] e. Avoid direct exposure of the reagent to sunlight and hypochlorous acid gas during testing and storage.

[0174] f. This kit and various wastes should be treated as infectious substances.

[0175] Example 8. Clinical evaluation test of the kit The kit developed in this project was clinically evaluated. The method was to use an internationally imported avian leukosis antigen detection kit and this kit to detect the same batch of egg white samples, virus isolation supernatant samples, and meconium samples respectively, and compare the coincidence rate between this kit and the internationally imported avian leukosis antigen detection kit; use different kits to detect gradient - diluted meconium samples, gradient - diluted virus isolation supernatant samples, and cell culture supernatants with different ALV strains, different inoculation concentrations, and different sample collection times respectively, and compare and analyze the sensitivity between this kit and the internationally imported avian leukosis antigen detection kit; compare and analyze the detection limit of P27 antigen between this kit and a common avian leukosis antigen detection kit. The specific evaluation results are as follows.

[0176] I. Coincidence rate test This kit and the internationally imported avian leukosis antigen detection kit were used to detect egg white, meconium, and virus - isolated cell culture supernatant simultaneously. The specific results are shown in Table 8.

[0177] Table 8 Comparison results of avian leukosis antigen detection kits

[0178] According to the results, among 460 egg white samples, based on the judgment criteria of the kit, this kit detected 2 more samples than the imported international kit, and the coincidence rate between the two was 98.91%. Among 92 meconium samples, based on the judgment criteria of the kit, this kit detected 1 more sample than the imported international kit, and the coincidence rate between the two was 97.83%. Among 492 virus isolation supernatant samples, based on the judgment criteria of the kit, this kit detected 52 more samples than the imported international kit, and the coincidence rate between the two was 84.96%.

[0179] In summary, the detection rate of this kit is much better than that of the imported international kit.

[0180] II. Sensitivity Test 1. Meconium Samples A total of 114 meconium samples were detected. Each sample was respectively diluted at 9 gradients (1:1, 1:6, 1:12, 1:24, 1:48, 1:96, 1:192, 1:384, 1:768) and then detected.

[0181] According to the kit standard, with S / P≥0.2, among which 7 meconium samples, the highest detection dilution multiple of the self-made kit was higher than that of the imported international kit. Specifically as shown in Table 9.

[0182] Table 9 The Highest Dilution Multiple of Kit for Meconium Detection

[0183] 2. ALV Virus Solution A total of 7 known positive virus solutions were detected. Each sample was respectively diluted at 9 gradients (1:1, 1:6, 1:12, 1:24, 1:48, 1:96, 1:192, 1:384, 1:768) and then detected.

[0184] According to the kit standard, with S / P≥0.2, among which for 1 virus solution sample, the highest detection dilution multiple of the self-made kit was lower than that of the imported international kit, and for 6 of them, the highest detection dilution multiples were the same. Specifically as shown in Table 10.

[0185] Table 10 The Highest Dilution Multiple of Kit for Virus Isolation Supernatant Detection

[0186] 3. A / B, J, K Subgroup Virus Cell Supernatants The A / B, J, K subgroup virus cell supernatants with determined titers were respectively diluted to 3 different concentrations (i.e., 1:1, 1:5, 1:10) and inoculated into DF-1 cells. For the K subgroup, due to its lower titer and poor replication ability, it was inoculated into DF-1 cells after 2-fold serial dilution. The cell supernatants of the 3 ALV viruses at 3 different concentrations were respectively collected on the 3rd - 9th day after inoculation for detection.

[0187] According to the kit standard, with S / P≥0.2, the sensitivity of the self-made kit for detecting avian leukosis virus subgroups A / B, J, and K is comparable to that of the internationally imported kit.

[0188] III. Minimum detection limit test This kit and the internationally imported avian leukosis antigen detection kit were used to detect P27 antigens with different concentrations simultaneously. The specific results are shown in Table 11.

[0189] Table 11 Minimum detection limit test

[0190] Based on the results, the minimum detection limit of this kit for P27 antigen is 1 ng / mL, which is higher than that of the internationally imported avian leukosis antigen detection kit.

[0191] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by 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.

2. The kit according to claim 1, characterized in that The anti-avian leukosis virus P27 protein monoclonal antibody is screened and produced by a bioengineering method.

3. The kit according to claim 1 or 2, characterized in that 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.

4. The kit according to any one of claims 1 to 3, characterized in that 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 a primary antibody and horseradish peroxidase-labeled sheep anti-P27 protein polyclonal antibody as a sandwich secondary antibody.

5. The kit according to any one of claims 1 to 4, characterized in that The kit comprises a positive control and a negative control; the positive control is a P27 protein solution, and the negative control is an SPF chicken serum.

6. The kit according to any one of claims 1 to 5, 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.

7. A bioengineering method for screening monoclonal antibodies against avian leukosis virus P27 protein, characterized in that: The following steps are involved: (1) Immunizing rabbits with the P27 protein shown in SEQ ID NO:1 as an antigen to enrich IgG B cells; (2) Through flow cytometry, high-affinity IgG B cells were screened using fluorescently labeled P27 protein-specific epitope short peptides; (3) The BCR light chain and heavy chain genes of the screened B cells were sequenced to obtain specific BCR sequences.

8. The monoclonal antibody against avian leukosis virus P27 protein screened and produced by the bioengineering method of claim 7.

9. The anti-avian leukosis virus P27 protein monoclonal antibody according to claim 8, 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; 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.

10. Use of the monoclonal antibody according to claim 8 or 9 in preparing a kit for detecting avian leukosis virus infection.

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