Monoclonal antibody for detecting solenopsis invicta as well as application and preparation method of monoclonal antibody

Abstract

The invention relates to the technical field of biology, in particular to a monoclonal antibody for detecting solenopsis invicta as well as application and a preparation method of the monoclonal antibody. Wherein the monoclonal antibody for detecting the solenopsis invicta comprises a first monoclonal antibody or a second monoclonal antibody. The first monoclonal antibody or the second monoclonal antibody is applied to preparation of a colloidal gold reagent, and has the advantages of high sensitivity, strong specificity and good precision performance in detection of solenopsis invicta.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a monoclonal antibody for detecting red imported fire ants, its application, and its preparation method. Background Technology

[0002] Red imported fire ants (Solenopsis invicta Buren) are invasive ants belonging to the genus Solenopsis in the family Formicidae of the order Hymenoptera. They originate from South America and are highly aggressive, causing widespread damage to agriculture, forestry, public safety, and biodiversity due to their omnivorous diet, aggressive nature, and high reproductive rate. When threatened, they immediately climb onto the skin of their prey, bite down with their mandibles, and then inject a toxic acidic liquid into the skin. Symptoms include itching, pain, swelling, dizziness, and fever, with severe cases leading to anaphylactic shock and even death. Red imported fire ants are aggressive and have a wide diet, feeding on plant seeds, fruits, buds, tender stems, and roots, as well as large quantities of other insects and invertebrates, especially ants, bees, and other Hymenoptera. Furthermore, their high reproductive rate and adaptability, coupled with the lack of natural predators in invaded areas, allow them to easily expand on a large scale, reducing local crop yields, causing wildlife population decline, severely damaging the ecological environment, and harming agricultural production. In addition, red imported fire ants tend to proliferate in air conditioner outdoor units, power meters, and household appliances, biting through the insulation of wires and cables and carrying soil into the equipment, causing short circuits and damaging household appliances and public facilities. Currently, the control of red imported fire ants mainly relies on later-stage physical and chemical methods (such as insecticides / pesticides / traps). Early detection and identification of red imported fire ants are primarily achieved through two main methods: physical observation and biotechnology detection. Physical observation involves observing ant nests, identifying ant colony colors, probing ant aggression, and observing head or tail characteristics to identify red imported fire ants. Its effectiveness depends heavily on personal experience and is prone to misjudgment. Biotechnology detection mainly includes two categories: one is rapid detection reagents based on immunological principles, currently unavailable domestically, and only one product (AGDI) internationally, which is expensive; the other is red imported fire ant detection products based on PCR technology, but due to high requirements for equipment and personnel, and cumbersome usage, large-scale promotion is not possible.

[0003] Therefore, developing a convenient, affordable, and high-performance red imported fire ant detection reagent to discover and understand red imported fire ant colony information as early as possible, in order to guide the process of trapping or killing red imported fire ants, is of great significance for the efficient control of red imported fire ants, ecological environment protection, and promotion of crop production, and has significant social and economic value. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a monoclonal antibody for the detection of red imported fire ants, its application and preparation method, aiming to improve the convenience of red imported fire ant detection.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides a monoclonal antibody for detecting red imported fire ants, comprising a first monoclonal antibody or a second monoclonal antibody; The first monoclonal antibody comprises: The heavy chain variable region CDR1 has the following amino acid sequence: SGYTXTSYWM; The heavy chain variable region CDR2 has the following amino acid sequence: NIYXGNGFT; The heavy chain variable region CDR3 has the following amino acid sequence: TRGDXYGSSXPFDY; The light chain variable region CDR1 has the following amino acid sequence: SQDINXYIA; The light chain variable region CDR2 has the following amino acid sequence: HXTST; The light chain variable region CDR3 has the following amino acid sequence: CLXYDNLWTF; Where, X is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, O, S, U, T, W, Y or V; The second monoclonal antibody includes: The heavy chain variable region CDR1 has the following amino acid sequence: SGYTXTDYT; The heavy chain variable region CDR2 has the following amino acid sequence: VISTXYGDA; The heavy chain variable region CDR3 has the following amino acid sequence: ARXEGDVXFAYW; The light chain variable region CDR1 has the following amino acid sequence: SXDINRYL; The light chain variable region CDR2 has the following amino acid sequence: LIYXANR; The light chain variable region CDR3 has the following amino acid sequence: CLQYDEXPWTF; Where, X is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, O, S, U, T, W, Y or V.

[0006] A second aspect of the present invention provides a colloidal gold reagent comprising the monoclonal antibody for the detection of red imported fire ants as described above.

[0007] A third aspect of the present invention provides a kit comprising the colloidal gold reagent as described above.

[0008] A fourth aspect of this invention provides a method for preparing a monoclonal antibody for detecting red imported fire ants, comprising the following steps: S01. Using red imported fire ant venom allergen protein 2 as an immunogenic protein, prepare an antigen; S02. Immunize mice with antigens to obtain post-immunization lymphocytes; S03. Fuse mouse myeloma cells and lymphocytes, screen and subclone the fused cells to obtain target hybridoma cells; S04. The hybridoma cells were domesticated and cultured in suspension to obtain the cell culture supernatant; S05. Monoclonal antibodies are purified from cell culture supernatant.

[0009] The method for preparing the monoclonal antibody, wherein S02 includes: performing an immunization experiment on mice using an antigen, selecting mice with qualified serum titers and weak reactions with cross-linked proteins for shock immunization, and on the 3rd day after shock immunization, sacrificing the mice, taking the mouse spleen, and preparing a lymphocyte suspension.

[0010] The method for preparing the monoclonal antibody, wherein S03 includes: mixing mouse myeloma cells and lymphocytes in the logarithmic growth phase, centrifuging to remove the supernatant, adding fusion buffer to resuspend the cells, then performing electrofusion, repairing after electrofusion, and then screening and subcloning the fused cells to obtain target hybridoma cells.

[0011] In the method for preparing the monoclonal antibody, during fusion, the ratio of mouse myeloma cells to lymphocytes is 1:(1-5).

[0012] The method for preparing the monoclonal antibody, wherein S04 includes: monitoring the cell status in the culture medium daily and identifying the antibody content in the culture medium supernatant; and culturing for 3 to 6 days to stabilize the antibody content in the supernatant.

[0013] The method for preparing the monoclonal antibody, wherein S05 includes: centrifuging the cell culture, collecting the supernatant, purifying it with Protein A magnetic beads, and dialysis to obtain the monoclonal antibody.

[0014] Beneficial Effects: This invention provides a monoclonal antibody for detecting red imported fire ants, comprising a first monoclonal antibody or a second monoclonal antibody. A colloidal gold reagent prepared from this monoclonal antibody exhibits advantages such as high sensitivity, strong specificity, and good precision, enabling accurate and effective identification of red imported fire ants. Furthermore, the colloidal gold reagent based on the monoclonal antibody of this invention is inexpensive, easy to operate, and requires minimal equipment and personnel, making it suitable for various scenarios such as scientific research, customs, agriculture, and forestry. It is highly suitable for widespread application and contributes to the control of red imported fire ants. Attached Figure Description

[0015] Figure 1 The results are negative and positive colorimetric results for the colloidal gold reagent card.

[0016] Figure 2 The results show the detection results when the number of red imported fire ants varies.

[0017] Figure 3 These are test results with different sample loading amounts. Detailed Implementation

[0018] This invention provides a monoclonal antibody for detecting red imported fire ants, its application, and a preparation method. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0019] The first aspect of the present invention provides a monoclonal antibody for detecting red imported fire ants, comprising a first monoclonal antibody or a second monoclonal antibody; The first monoclonal antibody comprises: The heavy chain variable region CDR1 has the following amino acid sequence: SGYTXTSYWM, SEQ ID NO: 1; The heavy chain variable region CDR2 has the following amino acid sequence: NIYXGNGFT, SEQ ID NO: 2; The heavy chain variable region CDR3 has the following amino acid sequence: TRGDXYGSSXPFDY, SEQ ID NO: 3; The light chain variable region CDR1 has the following amino acid sequence: SQDINXYIA, SEQ ID NO: 4; The light chain variable region CDR2 has the following amino acid sequence: HXTST, SEQ ID NO: 5; The light chain variable region CDR3 has the following amino acid sequence: CLXYDNLWTF, SEQ ID NO: 6; Where, X is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, O, S, U, T, W, Y or V; The second monoclonal antibody includes: The heavy chain variable region CDR1 has the following amino acid sequence: SGYTXTDYT, SEQ ID NO: 7; The heavy chain variable region CDR2 has the following amino acid sequence: VISTXYGDA, SEQ ID NO: 8; The heavy chain variable region CDR3 has the following amino acid sequence: ARXEGDVXFAYW, SEQ ID NO: 9; The light chain variable region CDR1 has the following amino acid sequence: SXDINRYL, SEQ ID NO: 10; The light chain variable region CDR2 has the following amino acid sequence: LIYXANR, SEQ ID NO: 11; The light chain variable region CDR3 has the following amino acid sequence: CLQYDEXPWTF, SEQ ID NO: 12; Where, X is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, O, S, U, T, W, Y or V.

[0020] A second aspect of the present invention provides a colloidal gold reagent comprising the monoclonal antibody for the detection of red imported fire ants as described above.

[0021] Preferably, the first monoclonal antibody and the second monoclonal antibody are used together in colloidal gold reagents, such as the first monoclonal antibody for coating and the second monoclonal antibody for labeling.

[0022] A third aspect of the present invention provides a kit comprising the colloidal gold reagent as described above.

[0023] A fourth aspect of the present invention provides a method for preparing a monoclonal antibody for detecting red imported fire ants as described above, comprising the following steps: S01. Using red imported fire ant venom allergen protein 2 as an immunogenic protein, prepare an antigen; S02. Immunize mice with antigens to obtain post-immunization lymphocytes; S03. Fuse mouse myeloma cells and lymphocytes, screen and subclone the fused cells to obtain target hybridoma cells; S04. The hybridoma cells were domesticated and cultured in suspension to obtain the cell culture supernatant; S05. Monoclonal antibodies are purified from cell culture supernatant.

[0024] Example 1: Detection of antibody activity and specificity In this embodiment, antibody one includes: The heavy chain variable region CDR1 has the following amino acid sequence: SGYTKTSYWM, SEQ ID NO: 13; The heavy chain variable region CDR2 has the following amino acid sequence: NIYQGNGFT, SEQ ID NO: 14; The heavy chain variable region CDR3 has the following amino acid sequence: TRGDNYGSSQPFDY, SEQ ID NO: 15; The light chain variable region CDR4 has the following amino acid sequence: SQDINRYIA, SEQ ID NO: 16; The light chain variable region CDR5 has the following amino acid sequence: HNTST, SEQ ID NO: 17; The light chain variable region CDR6 has the following amino acid sequence: CLDYDNLWTF, SEQ ID NO: 18; Antibody II includes: The heavy chain variable region CDR7 has the following amino acid sequence: SGYTMTDYT, SEQ ID NO: 19; The heavy chain variable region CDR8 has the following amino acid sequence: VISTYYGDA, SEQ ID NO: 20; The heavy chain variable region CDR9 has the following amino acid sequence: ARQEGDVNFAYW, SEQ ID NO: 21; The light chain variable region CDR10 has the following amino acid sequence: SADINRYL, SEQ ID NO: 22; The light chain variable region CDR11 has the following amino acid sequence: LIYKANR, SEQ ID NO: 23; The light chain variable region CDR12 has the following amino acid sequence: CLQYDERPWTF, SEQ ID NO: 24; In addition to Antibody 1 and Antibody 2, 11 other antibodies (numbered 3-13#) were simultaneously identified for activity and specificity. However, since they were phased out during the research stage and not put into practical application, amino acid sequencing was not performed.

[0025] The reactivity of antibodies 1, 2, and 3-13# to the Venom allergen2 recombinant protein and eight cross-linked proteins was detected using an indirect ELISA method to identify antibody activity and specificity. The eight cross-linked proteins were recombinant proteins. First, sequence homology analysis was performed on the Venom allergen2 protein to identify ant protein sequences that might cross-link with it. Then, plasmids were constructed, expressed in Ecoli prokaryotes, and purified using Beijing Yiqiao Shenzhou Co., Ltd., ultimately yielding the recombinant proteins.

[0026] ELISA indirect method detection: 1. Coating: Coat each of the proteins described above sequentially at a concentration of 1 μ / mL, 100 μL / well, incubate overnight at 4°C, and wash once with PBS-T; 2. Blocking: Add 200 μL of blocking buffer to each well, block at 37°C for 2 h, and wash once with PBS-T; 3. Add primary antibody: Dilute the monoclonal antibody to a concentration gradient, add it to the wells of the plate, incubate at 37°C for 1 hour, and wash 3 times with PBS-T; 4. Add secondary antibody: Commercially available HRP-labeled goat anti-mouse secondary antibody was diluted at a volume ratio of 1:20000, and 100 μL was added to each well. The mixture was incubated at 37°C for 1 h and washed 3 times with PBS-T. 5. Color development: Add 100 μL of TMB color development solution to each well, develop color for 10 min, and then add 50 μL of 2M sulfuric acid solution to terminate the reaction; 6. Read the values: Use an ELISA reader to measure the absorbance of each well at a wavelength of 450 nm.

[0027] Table 1 shows the results of antibody activity and specificity (OD450 values).

[0028] As can be seen from the results in Table 1, most antibodies showed good activity, but lacked specificity, exhibiting cross-reactivity with at least one of the original coatings. Among the antibodies listed above, Antibody 1, corresponding to the first monoclonal antibody, Antibody 2, corresponding to the second monoclonal antibody, and Antibodies 3#, 4#, and 12# showed good specificity and high activity against the Venom allergen2 recombinant protein, but showed no or very weak reaction to other cross-reactive proteins.

[0029] The amino acid sequence of the Venom allergen2 recombinant protein, Seq1#: DNKELKIIRKDVAECLRTLPKCGNQPDDPLARVDVWHCAMAKRGVYDNPDPAVIKERSMKMCTKIITDPANVENCKKVASRCVDRETQGPKSNRQKAVNIIGCALRAGVAETTVLARKK; The amino acid sequences of the eight cross proteins are as follows: Seq2#: DNKELKIIRKDIAECVRTLRKCANQPDDPLARVDVWHCAMAKRGVFDDPAPAVIKEKCLKMCPKIITDPADVKNCMKVASRCVDRETQRRRSNRQIAINIIACALRAGVVETTVLARKK; Seq3#: DIEAQRVLRKDIAECARTLPKCVNQPDDPLARVDVWHCAMSKRGVYDNPAPAVVKEKNSKMCPKIITDPADVENCKKVVSRCVDRETQRPRSNRQKAINITGCILRAGVVEATVLAREK; Seq4#: DIEAQRVLRKDIAECARTLPKCVNQPDDPLARVDVWHCAMSKRGVYDNPDPAVVKEKNSKMCPKIITDPADVENCKKVVSRCVDRETQRPRSNRQKAINITGCILRAGVVEATVLAREK; Seq5#: MSARYDPLARVDVWHCAMSKRGVYDNPAPAVVKEKNSKMCPKIITDPADVENCKKVVSRCVDRETQRPRSNRQKAINITGCILRAGVVEATVLAREK; Seq6#: DTEKLKILRKDIAKCARTLPKCVNQPDDPLARVDVWHCALAKSGVFDDPDPAAIKKKYKKFCAIAVTDPANVENCKKVTSRCVDKETQCSKSNRQKAINIAACILRSGVTETTVLAREK; Seq7#: DTEKLKVLRKDIAECARTLPKCENQPDDPLARVDVLHCVLAKRGVFDDPAPAAIKKKDEKYCAITITDPADVENCKKVVSRCVDKETQRPRSNRQKAINIVACILRSGVAETTVLARKK; Seq8#: DIEKLKILRKDIAECARTLPKCVNQPDDLLARVDVWHCALAKGGVFDDPTPAAIKRKHKKYCAIAITDPANVENCKKVVSRCIDKETQRPRANRQKAINIIACVMRAGVAETTVLARG; Seq9#: HNEELKVIHKDIAKCARTLPKCVNQPDDPLARVDVWHCAMAKRGVYDNPAPAVIKEKNFKVCSKIITDPANVENCKKVISRCVDRETQRPRSNRQKAINITGCILRAGVVETTVLARKK; The amino acid sequences of Seq1# to Seq9# above were retrieved from the NCBI database. <00001​​Antibodies 1 and 2 from Example 1 were coated and labeled to prepare colloidal gold reagents. The preparation method of the colloidal gold reagents is as follows: 1. Preparation of membrane semi-finished product: Dilute the antibody to 0.5 or 1 mg / mL with coating buffer, streak the membrane at 1 μL / cm, and dry it in a 37℃ drying oven overnight; 2. Preparation of colloidal gold: Using chloroauric acid as a substrate and trisodium citrate as a reducing agent, gold particles of about 40 nm were prepared by boiling in water. 3. Antibody labeling: The antibody and colloidal gold were mixed in a weakly alkaline system and reacted for 10-15 min. The mixture was then blocked with BSA at a concentration of 10 g / 100 mL. The mixture was centrifuged at 10,000 rpm for 10 min. The precipitate was dissolved in the preservation solution and sonicated to ensure uniform dissolution of the particles. The mixture was then stored at 4 °C. 4. Prepare the sample pad: Take a piece of glass fiber, add sample buffer solution according to the ratio, wet both sides evenly, and dry in a 37℃ drying oven overnight; 5. Preparation of semi-finished pad: Cut the glass fiber into 1cm wide pieces, spray the marking material onto the pad at 8μL / cm, and then dry it overnight in a 37℃ drying oven; 6. Plate mounting: According to the specifications of the base plate, cut the film, marking pad, sample pad, absorbent paper, etc. to the specific size in sequence, and then attach the absorbent paper, film, marking pad, sample pad, etc. in sequence; 7. Cutting strips: Cut the attached base plate into strips with a width of 3mm to obtain test strips; 8. Card loading: Each test strip corresponds to a set of card holders. After the test strip is placed, press the upper and lower card holders together and seal it for later use.

[0031] Obtain various types of ants, taking 1-5 ants according to their size (e.g., 1 large ant, 2 medium-to-large ants, and 5 small ants). Then, add 100 μL of PBS grinding buffer (PBS grinding buffer contains 10 mM PB and 150 mM NaCl, pH 7.4) and grind them thoroughly. Take 60 μL of the grinding supernatant and add it to the colloidal gold reagent card. The detection results of the colloidal gold reagents prepared by antibody 1 and antibody 2 are shown in Table 2. Figure 1 The test result was negative for the colloidal gold test card. Figure 1 Left side) and positive ( Figure 1 (Right side) Color development results. The test subject for a negative result was pure water, and the test subject for a positive result was 5 red imported fire ants.

[0032] Table 2

[0033] In Table 2, strong positive is indicated by "++", moderate positive by "+", weak positive by "-", and negative by "×". (Note: The strength of the positive and negative values ​​is determined by the appearance of the colloidal gold precipitation line. "++" indicates that the T line is darker, "+" indicates that there is some color but it is lighter, "-" indicates that the color is very weak and difficult to identify, and "×" indicates that it is not visible at all.)

[0034] In addition, the common ants in Guangdong listed in the table are unidentified ants captured from the surrounding area, and the unknown ants do not conform to the morphological characteristics of red imported fire ants.

[0035] As can be seen from the results in Table 2, the colloidal gold reagents prepared by Antibody 1 and Antibody 2 have excellent sensitivity and good specificity. They can detect even a single red imported fire ant in a sample with high efficiency, and there is no obvious cross-contamination with other common ants.

[0036] Comparative Example 1 A colloidal gold reagent differs from Example 2 in that the antibodies 3# and 12#, which have good specificity in Example 1, are coated and labeled to prepare a colloidal gold reagent.

[0037] The detection results of the colloidal gold reagents prepared from antibody 3# and antibody 12# are shown in Table 3.

[0038] Table 3

[0039] As can be seen from the results in Table 3, the colloidal gold reagent prepared by antibody 3# and antibody 12# has good specificity, but its sensitivity is significantly worse than that of the colloidal gold reagent prepared by antibody 1 and antibody 2.

[0040] Example 3 The preparation method of monoclonal antibodies specifically includes the following steps: Step A01. Using a protein with the amino acid sequence Seq1# as an immunogen, immunize 5 Balb / c mice, continuously immunize and monitor their serum titers. The procedure is as follows: (1) Prepare 0.5 mg of immunogen, dilute it with PBS solution to 1 mg / mL, and then mix it with an equal volume of Freund's complete adjuvant. After repeated extraction and squeezing, a stable emulsion is obtained and injected subcutaneously into mice using a syringe (100 μg / mouse). (2) Two weeks later, the immunogen and Freund's incomplete adjuvant were mixed into an emulsion and injected again using the same dose and emulsification method; (3) Repeat the second immunization method; after the third immunization, on the 7th day after the injection, cut off a section of the mouse tail with sterile scissors, collect the tail blood, put it into a 1.5mL microcentrifuge tube, centrifuge and collect the supernatant; (4) The titer of mouse tail blood was detected by ELISA, and then immunized for 1-2 more times until a sufficient titer level was reached; animals with qualified serum titers (at least ≥81K) and weak reaction with cross-linked proteins were selected for fusion. (5) Three days before fusion, prepare a PBS solution containing 50 μg of protein and inject it into the peritoneum for shock immunization; on the third day after shock immunization, sacrifice the mice and take spleen cells for fusion.

[0041] Step A02. Cell Fusion (1) Take mouse spleen and grind it to make a lymphocyte suspension; (2) Myeloma cells were obtained using SP2 / 0 from Balb / c. Mouse myeloma cells in the logarithmic growth phase were mixed with lymphocytes at a ratio of 1: (1-5). After centrifugation at 1500 rpm for 5 min, the supernatant was discarded and the cells were resuspended in fusion buffer. (3) Slowly transfer the mixture into the fusion tank of the electrofusion instrument, start the instrument to complete the cell fusion, and then transfer the cell suspension into 10 mL of cell repair solution preheated at 37°C; place the cell repair solution in a 37°C incubator and let it stand for 10 min for repair. After the repair is completed, centrifuge at 1200 rpm for 5 min, discard the supernatant, and resuspend the cells in HAT medium. (4) Transfer all solutions to 96-well plates, 100 μL / well, and label them; carefully transfer the 96-well plate to a 37°C, 5% CO2 incubator for incubation; Step A03. Cell screening and subcloning (1) After fusion for 7 to 10 days, the nine proteins described in Example 1 were coated (Venom allergen2 recombinant protein and eight cross proteins). All cell supernatants were screened and tested in 1 to 2 rounds. Cell wells with high ELISA positive values ​​and low cross-reactivity were selected as positive wells, totaling more than 20 strains. (2) Subcloning of the aforementioned positive cells was performed using the limiting dilution method. After each plating culture, the ELISA positivity and cross-reactivity were measured 7–10 days later. Cell culture wells with high positive values ​​and good specificity were selected for further subcloning and plating culture. (3) After ≥2 consecutive rounds of subcloning, the ELISA test results of the entire 96-well plate are positive and all cross proteins are negative, the cells in the corresponding wells can be isolated. (4) Finally, 13 monoclonal strains were obtained, which could stably secrete antibodies against red imported fire ant protein and showed no obvious reaction with 8 cross proteins (or only a weak reaction to individual proteins); the results are shown in Example 1. Step A04. Monoclonal antibody preparation Hybridoma cells were acclimatized and cultured in suspension using serum-free medium. The cell status in the medium was monitored daily, and the antibody content in the supernatant was identified. After 3–6 days of culture, when the antibody content in the supernatant was basically stable, the cell culture was centrifuged, the supernatant was collected, and purified by Protein A magnetic beads and dialysis to finally obtain the corresponding antibodies for each cell line.

[0042] The steps for purifying antibodies using the Protein A magnetic bead method are as follows: 1. Regeneration: After washing the magnetic beads with 0.1M NaOH, rinse 2-3 times with equilibration buffer (0.01M PB plus 0.15M NaCl, pH=7.4) until the pH value stabilizes; 2. Sample loading: Place the equilibrated magnetic beads into the culture supernatant tube and shake to bind for 20 minutes; 3. Equilibration: After sample loading, rinse the magnetic beads 2-3 times with equilibration buffer until no impurities remain; 4. Elution: Rinse the magnetic beads with elution buffer (0.1M glycine solution, pH=3.0) and collect the eluent. The eluent should be neutralized immediately with 1.5M Tris-HCl. 5. Regeneration: After elution, the magnetic beads are washed sequentially with 0.1M NaOH, equilibration buffer, ultrapure water, and 20% ethanol, and then stored at 4℃. 6. Antibody preservation: The eluent was replaced with phosphate buffer at pH 7.4 by dialysis, and 0.05% proclin 300 solution (0.05 g / 100 mL) was added. The solution was then stored at 4 °C.

[0043] Example 4 Product Inclusivity Testing Under standard testing parameters (5 red imported fire ant worker ants, 60 μL sample, 5 min reaction), we explored the effects of factors such as ant concentration, sample volume, and reaction time on the test results to determine the tolerance of the reagent product to operational deviations.

[0044] Take 1-9 red imported fire ant worker ants, add 100 μL of PBS grinding buffer and grind them thoroughly (PBS grinding buffer contains 10 mM PB and 150 mM NaCl, pH 7.4), take 60 μL of the grinding supernatant and add it to the colloidal gold reagent card prepared by antibody 1 and antibody 2, and observe the detection results. Take 5 red imported fire ant worker ants and grind them thoroughly with 100 μL of PBS grinding buffer. Take 40-100 μL of the grinding supernatant and add it to the colloidal gold reagent card prepared with antibody 1 and antibody 2, and observe the detection results. Take 5 red imported fire ant worker ants and grind them thoroughly with 100 μL of PBS grinding buffer. Take 60 μL of the grinding supernatant and add it to the colloidal gold reagent card prepared with antibody 1 and antibody 2. Observe the color development results continuously within 0-30 min.

[0045] The results showed that when 1-9 red imported fire ants were taken or 40-100 μL of sample was added for testing, there was no significant difference between the T and C lines, and the reagent cards showed normal color development. Figure 2 The results show the detection results when the number of red imported fire ants varies. Figure 3 These are test results with varying sample volumes. Color development was complete within 2 minutes after sample addition, and the difference in color development was not significant within 30 minutes. This indicates that general operational deviations do not affect the results, and the reagent product has good compatibility.

[0046] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solution and inventive concept of the present invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A monoclonal antibody for detection of Solenopsis invicta, characterized in that, The first monoclonal antibody or the second monoclonal antibody comprises: The first monoclonal antibody comprises: a heavy chain variable region CDR1, the amino acid sequence of which is SGYTXTSYWM; a heavy chain variable region CDR2, the amino acid sequence of which is NIYXGNGFT; a heavy chain variable region CDR3, the amino acid sequence of which is TRGDXYGSSXPFDY; a light chain variable region CDR1, the amino acid sequence of which is SQDINXYIA; a light chain variable region CDR2, the amino acid sequence of which is HXTST; a light chain variable region CDR3, the amino acid sequence of which is CLXYDNLWTF; wherein X is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, O, S, U, T, W, Y or V. The second monoclonal antibody comprises: a heavy chain variable region CDR1, the amino acid sequence of which is SGYTXTDYT; a heavy chain variable region CDR2, the amino acid sequence of which is VISTXYGDA; a heavy chain variable region CDR3, the amino acid sequence of which is ARXEGDVXFAYW; a light chain variable region CDR1, the amino acid sequence of which is SXDINRYL; a light chain variable region CDR2, the amino acid sequence of which is LIYXANR; a light chain variable region CDR3, the amino acid sequence of which is CLQYDEXPWTF; wherein X is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, O, S, U, T, W, Y or V.

2. A colloidal gold reagent, characterized in that, The monoclonal antibody for red imported fire ant detection comprises the first monoclonal antibody or the second monoclonal antibody.

3. A kit characterized in that, The colloidal gold reagent comprises the first monoclonal antibody or the second monoclonal antibody.

4. A method for preparing a monoclonal antibody for detecting Solenopsis invicta, characterized in that, The method comprises the following steps: S01. Preparing an antigen by using venom sensitization protein 2 of the red imported fire ant as an immunogen protein; S02. Immunizing a mouse by using the antigen to obtain lymphocytes after immunization; S03. Fusing mouse myeloma cells and the lymphocytes, screening and subcloning the fused cells to obtain target hybridoma cells; S04. Domesticating and suspending culturing the hybridoma cells to obtain cell culture supernatant; S05. Purifying the monoclonal antibody from the cell culture supernatant.

5. The method for preparing monoclonal antibodies according to claim 4, characterized in that, The S02 comprises the following steps: immunizing a mouse by using the antigen, selecting the mouse with qualified serum titer and weak cross-protein reaction for impact immunization, killing the mouse on the third day after the impact immunization, taking the mouse spleen, and preparing a lymphocyte suspension.

6. The method for preparing monoclonal antibodies according to claim 4, characterized in that, The S03 comprises the following steps: mixing mouse myeloma cells in a logarithmic growth phase with the lymphocytes, centrifuging to remove supernatant, resuspending the cells by adding a fusion buffer, then performing electrofusion, repairing after the electrofusion, and then screening and subcloning the fused cells to obtain target hybridoma cells.

7. The method for preparing monoclonal antibodies according to claim 4, characterized in that, The number ratio of the mouse myeloma cells to the lymphocytes is 1:(1-5) during the fusion.

8. The method of claim 4, wherein the monoclonal antibody is produced by a hybridoma cell line deposited with the American Type Culture Collection under accession number PTA- 12064. The S04 comprises the following steps: monitoring the cell state in the culture medium every day, identifying the antibody content in the culture medium supernatant, and culturing for 3-6 days to stabilize the antibody content in the supernatant.

9. The method of claim 4, wherein the monoclonal antibody is produced by a hybridoma cell line deposited with the American Type Culture Collection under accession number PTA- 12064. The S05 comprises centrifuging the cell culture, collecting the supernatant, purifying through Protein A magnetic beads, dialysis, and obtaining the monoclonal antibody. The S05 comprises centrifuging the cell culture, collecting the supernatant, purifying through Protein A magnetic beads, dialysis, and obtaining the monoclonal antibody.

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