A puccinia antibody detection kit and application thereof

Abstract

The present application relates to the field of biological detection, in particular to a Puccinia antibody detection kit and application thereof, and provides a monoclonal antibody 1A6 against Puccinia, which comprises a heavy chain variable region as shown in SEQ ID NO: 2 and a light chain variable region as shown in SEQ ID NO: 6, and the qualitative and quantitative detection of Puccinia can be realized by using a detection kit comprising the monoclonal antibody 1A6, and the specificity and sensitivity are relatively high.

Description

Technical Field

[0001] This invention relates to the field of biological detection. Specifically, this invention relates to a *Stemonas stolonifera* antibody detection kit and its application. Background Technology

[0002] Puccinia graminis is a pathogenic fungus that widely infects many important crops such as wheat and barley, causing serious diseases such as wheat stem rust and resulting in significant losses to global agricultural production. This pathogen is highly transmissible and adaptable to various environments, capable of spreading through the air to long distances, posing a threat to food security. Traditional disease control methods mainly rely on chemical control and the breeding of resistant varieties. However, due to the ease with which Puccinia graminis develops resistance and mutations, the effectiveness of single control methods is gradually declining. Therefore, it is necessary to develop effective pathogen detection technologies to achieve early diagnosis and precise control.

[0003] Monoclonal antibodies, due to their high specificity and consistency, have been widely used in the detection of pathogens such as bacteria, viruses, and fungi. By designing monoclonal antibodies targeting the antigenic epitopes of specific pathogens, efficient identification of pathogens can be achieved. Especially in the field of agricultural disease detection, monoclonal antibodies provide a rapid, sensitive, and convenient detection method that can be applied to on-site detection, monitoring, and laboratory analysis. However, the application of monoclonal antibodies against *Strigera stenoptera* remains technologically challenging; existing detection methods lack specific antibodies and cannot meet the demands for high-throughput, low-error pathogen detection.

[0004] While monoclonal antibodies exhibit high specificity in pathogen detection, they face several challenges in terms of specificity and sensitivity in the detection of *Stripetracus rust*. First, *Stripetracus rust* shares antigenic similarities with other related rust pathogens (such as stripe rust and leaf rust), potentially leading to cross-reactivity and affecting detection specificity. Furthermore, due to the low quantity and amount of pathogen and antigen, existing antibodies may lack sufficient sensitivity to detect low concentrations of pathogen in early-stage infections. Further improving antibody binding efficiency and signal amplification capabilities to ensure specificity and sensitivity for *Stripetracus rust* antigens remains a critical technical challenge. Summary of the Invention

[0005] This invention provides a detection kit containing the specific monoclonal antibody 1A6, which enables accurate detection of *Stemonas stem* in samples.

[0006] The antibody detection kit provided by this invention includes a labeled anti-Stemonas stemraceae monoclonal antibody 1A6 and a chromogenic agent or fluorescent reagent. The monoclonal antibody 1A6 is designed against specific antigens of Stemonas stemraceae, possesses high binding affinity, and can effectively recognize and bind to Stemonas stemraceae specific antigens.

[0007] In some embodiments, the heavy chain variable region of the 1A6 antibody contains an amino acid sequence as shown in SEQ ID NO:2 and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:6.

[0008] This invention also provides a method for detecting *Stemona stem*, specifically comprising the following steps: First, the sample to be tested is contacted with a detection kit containing an anti-*Stemona stem* monoclonal antibody 1A6. If the sample contains *Stemona stem* antigen, the 1A6 antibody will bind to it. Next, by detecting the signal intensity generated by the binding of the antibody and antigen, it can be determined whether the sample contains *Stemona stem* antigen.

[0009] In the method of this invention, the detection signal can be read via fluorescence or enzyme-linked immunosorbent assay (ELISA) to achieve highly sensitive detection. The use of fluorescence signals can improve the sensitivity and accuracy of detection, and is suitable for detecting low-concentration samples; ELISA signals are suitable for quantitative analysis, and the specific concentration of *Stemonas styracifolium* antigen can be determined by reading the absorbance. This multi-mode signal detection method makes this invention applicable to a wide range of scenarios and can meet the needs of different experimental environments.

[0010] Furthermore, the present invention also provides a monoclonal antibody 1A6 for detecting *Stemonas stolonifera*. The antibody comprises specific heavy and light chain variable regions, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 sequences as shown in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 sequences as shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0011] Compared with existing technologies, the detection kit and monoclonal antibody 1A6 of the present invention can be used to prepare products for detecting *Stemona stem rust*, including equipment for laboratory diagnosis and rapid field detection. At the same time, the monoclonal antibody 1A6 of the present invention has significant specificity and sensitivity, and can provide accurate detection results at different stages of *Stemona stem rust* infection, providing important technical support for early warning and control of crop diseases. Attached Figure Description

[0012] Figure 1 SDS-PAGE analysis results of recombinant protein Pg-Avr.

[0013] Figure 2 SDS-PAGE analysis results of the light and heavy chains of monoclonal antibody 1A6.

[0014] Figure 3 Subtype identification of monoclonal antibody 1A6 against Avr antigen of *Stripe rust*.

[0015] Figure 4 Specificity analysis of monoclonal antibody 1A6 against *Stemonas stem*.

[0016] Figure 5 Sensitivity analysis of monoclonal antibody 1A6 against *Stemonas stem*. Detailed Implementation

[0017] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0018] Example 1: Preparation of Avr antigen from Puccinia graminis

[0019] Based on the full-length sequence of avirulence protein [Puccinia graminisf.sp.tritici] (GenBank: WOJ46332.1) recorded in NCBI, an immunogenic peptide was designed as follows: MHYITPIILMSIGQFL DILLGAGGLVGAMTPHHQINCNSPYLTFPRLTAKCDSCQLHTKVTNLVSCTS CRKSSLVYEECSTKGCPANWHKSTCQEHKFKRGILHSRCENCQQHKKETQT ISCKNCKNSASTFPYCSSPECHSRW (SEQ ID NO:1).

[0020] PCR was performed using a high-fidelity DNA polymerase. The reaction system included *Striga styracifolium* template DNA, primers Avr-F1 and Avr-R1, dNTPs, and enzyme reaction buffer. PCR cycling conditions were: pre-denaturation (95°C, 5 min), denaturation (95°C, 30 s), annealing (55-60°C, 30 s), extension (72°C, 1 min), 35 cycles, and a final extension (72°C, 10 min). Primers and dNTPs were removed using a PCR product purification kit. The pET-28a vector was digested with restriction endonucleases (NdeI and XhoI) according to the enzyme manufacturer's instructions. The purified PCR product was mixed with the digested vector, and ligation was performed using T4 DNA ligase. The temperature and time were determined according to the ligase manufacturer's instructions. Cells were mixed with the ligation product, incubated at 42°C for 30 s, then quickly returned to ice, and finally allowed to recover growth in LB medium for 1 hour before being plated on antibiotic-containing plates. Positive clones were confirmed to have the correct insert by PCR or restriction enzyme digestion analysis. Positive clones were then selected for small-scale culture in LB medium with an appropriate amount of ampicillin (100 μg / mL). When the culture medium reached OD... 600When the concentration reaches 0.6-0.8, IPTG is added to induce protein expression, with a final concentration of 0.5-1.0 mM. Cells are then cultured at 37°C for 4-6 hours. The induced cell pellet is collected by centrifugation (6000 rpm, 10 minutes). Lysis buffer (PBS buffer with 1 mg / mL lysozyme) is used, and cells are treated on ice for 30 minutes, followed by sonication. The sonication time and power are optimized according to the sample volume and cell concentration. The cells are centrifuged at 12,000 rpm for 30 minutes, and the supernatant is collected for further purification. The supernatant is transferred to a nickel column containing binding buffer (buffered with 20 mM sodium chloride and 10 mM phosphate), thoroughly mixed, and placed on a shaker for 1-2 hours. The nickel column is washed with buffers containing 20, 30, and 40 mM imidazole gradients to remove unbound protein. The bound recombinant protein Pg-Avr is eluted with elution buffer containing 20 mM sodium chloride and 250 mM imidazole, and the eluent is collected. The purity of the protein in the eluent was analyzed by SDS-PAGE. Figure 1 .

[0021] Figure 1 The results showed that the molecular weight of the recombinant protein Pg-Avr was 16.19 kDa, which was in line with expectations.

[0022] Example 2: Screening, preparation, and identification of monoclonal antibodies against *Russula stevia*

[0023] Healthy, well-developed female BALB / C mice aged 6-8 weeks were selected for immunization: 30 μg of purified Pg-Avr recombinant protein was emulsified with an equal volume of Freund's complete adjuvant and injected intraperitoneally into the mice. Two weeks later, the same dose of protein was emulsified with an equal volume of Freund's incomplete adjuvant for a second immunization. Two weeks later, the same dose of protein was emulsified with an equal volume of Freund's incomplete adjuvant for a third immunization. After four immunizations, tail vein blood was collected and serum titers were detected by indirect ELISA. A strong immunization was performed 3 days before fusion. The results are shown in Table 1.

[0024] Table 1. Anti-Pg-Avr antibody titers in the serum of immunized mice

[0025] BALB / C mouse model Antibody titer Mouse1 1：3200 Mouse2 1：6400 Mouse3 1：12800 Mouse4 1：6400 Mouse5 1：12800 Mouse6 1：51200 Mouse7 1：6400 Mouse8 1：25600

[0026] Mouse6 mice with a serum titer of 1A6 reaching 1:51200 were immunized, followed by a booster immunization. 3-4 days later, mouse spleen cells and SP2 / 0 cells were used for cell fusion. Around day 8 post-fusion, positive hybridoma cells were screened using immunofluorescence. A single positive hybridoma cell line stably secreting anti-Pg-Avr monoclonal antibody was identified and named 1A6. A 96-well ELISA plate was coated with 2 μg·mL⁻¹ Pg-Avr protein. The monoclonal antibody 1A6 was serially diluted horizontally starting at 1:1000, and the titer was determined by indirect ELISA to be 1:2048000.

[0027] Prepare 1-2 BALB / c mice in advance, inject 1 mL of paraffin into each mouse intraperitoneally, and inject 5 × 10⁶ 1A6 hybridoma cells into each mouse intraperitoneally one week later. 6 Each animal was sampled individually. Ascites fluid was collected after 10 days, diluted approximately 10-fold with PBS, centrifuged at 3500 rpm for 10 minutes, filtered through a 0.45 μm filter, and purified using a Protein G gel column for the monoclonal antibody 1A6. A small amount of purified sample was retained for SDS-PAGE analysis to determine antibody purity and concentration. (See attached image.) Figure 2 .

[0028] Figure 2 The results showed that under reducing conditions, the 1A6 antibody had one band at 50 kDa and one band at 25 kDa, which were consistent with the theoretical molecular weights of the heavy and light chains after reduction; no obvious impurity bands were observed, indicating that the prepared antibody 1A6 had high purity.

[0029] The purified monoclonal antibody 1A6 was used to identify the monoclonal antibody 1A6 isotype using Mouse-Monoclonal Antibody Isotyping Reagents, following the manufacturer's instructions. The isotype of anti-Pg-Avr monoclonal antibody 1A6 was then identified using ELISA. Figure 3 .

[0030] Figure 3 The results showed that the antibody subtype identification of anti-Pg-Avr monoclonal antibody 1A6 was IgG2a type antibody.

[0031] Identification of the variable region sequence of monoclonal antibody 1A6: Total RNA was extracted from positive hybridoma cells that stably secrete anti-Pg-Avr monoclonal antibody 1A6. High-quality total RNA was obtained using an RNA extraction kit. Strict adherence to the operating procedures and the use of RNase-free consumables and reagents were ensured. The extracted RNA was transcribed into cDNA using reverse transcription PCR (RT-PCR). Random or specific primers were used, with SuperScript III or M-MLV reverse transcriptase selected. Reaction conditions included incubation at 42°C for 60 minutes, followed by inactivation at 70°C for 10 minutes. Specific primers against mouse antibody 1A6 were designed to amplify the variable region gene fragment. The PCR products were validated by agarose gel electrophoresis, and the target band was extracted and purified. The nucleotide sequence of the PCR products was determined using Sanger sequencing. The sequencing results were compared with known antibody gene sequences in the NCBI database to determine the variable region sequences of the light and heavy chains. The sequencing results were analyzed according to the IMGT database standards to determine the sequence locations of CDR1, CDR2, and CDR3.

[0032] Heavy chain variable region (VH) of monoclonal antibody 1A6: QVQLRQSGAELVKPATSIRLSC TASGFTFSTYVMSWVQQAPGKGLEWVAVIFYDGPNKYYADRVKGRYTISR DNAKNTFYHQMNSLRPEDTTVYYCGKDYGGMSGYFDYFGQGTLITVAS (SEQ ID NO:2);

[0033] H-CDR1:TASGFTFSTY(SEQ ID NO:3);

[0034] H-CDR2:AVIFYDGPNKYYADRV (SEQ ID NO:4);

[0035] H-CDR3: GKDYGGMS (SEQ ID NO: 5).

[0036] The light chain variable region (VL) of monoclonal antibody 1A6: DIVMTQSPDSLAFSLGERVTIN CKVSQNVGAAVAWYQEKPGQSAKFLIYWVSTRHTGVPNRFSGSRSGTDFT LTIGYLQAEDVAVYHCQQHYTSPYTFGQGTRVEIKN (SEQ ID NO: 6);

[0037] L-CDR1: INCKVSQNVGA (SEQ ID NO:7);

[0038] ML-CDR2: FLIYWVS (SEQ ID NO:8);

[0039] L-CDR3: CQQHYTSPY (SEQ ID NO:9).

[0040] Example 3: Specificity identification of monoclonal antibody 1A6 against Avr antigen of *Stripe rust*

[0041] Total protein was extracted from plant leaves infected with *Puccinia striiformis*, and impurities were removed by ultracentrifugation. Protein concentration of the extracts was determined using the BCA method, and samples were stored at -80°C for subsequent experiments. Several other control pathogens associated with *Puccinia striiformis* were selected: *Puccinia striiformis*, *Puccinia triticina*, *Uromyces appendiculatus*, *Blumeria graminis*, *Fusarium graminearum*, and *Magnaportheoryzae*. Total protein was extracted from each pathogen and treated in the same manner as the *Puccinia striiformis* samples.

[0042] Enzyme-linked immunosorbent assay (ELISA) identification

[0043] Coating antigen: Extracts of *Stemona stolonifer* and control pathogens (200 ng / well each) were coated onto 96-well ELISA plates using carbonate buffer (pH 9.6) and incubated overnight at 4°C.

[0044] Blocking: Block unbound sites with 5% bovine serum albumin (BSA) and incubate at room temperature for 1 hour.

[0045] Add primary antibody: Add 1A6 antibody (1:500 dilution) and incubate at room temperature for 1 hour.

[0046] Add secondary antibody: Add HRP-labeled anti-mouse IgG secondary antibody (1:5000 dilution) and incubate for 30 minutes.

[0047] Color development: Add TMB color development solution, incubate for 15 minutes, and then terminate the reaction with 2M H2SO4.

[0048] Detection: Absorbance was read at 450 nm using an ELISA reader, see [link to ELISA reader]. Figure 4 .

[0049] Figure 4The results showed that at a wavelength of 450 nm, the absorbance value of *Stemona stem* antigen was significantly higher than that of the control pathogen, while the absorbance of other pathogens was close to that of the negative control. This indicates that the 1A6 antibody has high specificity for *Stemona stem* antigen, which means that the 1A6 antibody can specifically recognize *Stemona stem* protein without cross-reacting with other pathogen samples.

[0050] Example 4: Sensitivity analysis of monoclonal antibody 1A6 against *Stripe rust*

[0051] Monoclonal antibody 1A6 was purified and prepared to a concentration of 1 μg / mL. *Stemona stevia* protein was extracted and purified to prepare a series of dilutions for constructing a standard curve. *Stemona stevia* antigen standards were diluted to the following concentrations: 1000 ng / mL, 500 ng / mL, 250 ng / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, 5 ng / mL, and 1 ng / mL, with each concentration diluted three times to ensure data accuracy and reproducibility. Each concentration of antigen standard was reacted with 1A6 antibody for 30 minutes, followed by incubation with FITC-labeled anti-mouse IgG secondary antibody at room temperature for 30 minutes. Flow cytometry was used to detect at least 10,000 events per sample. Fluorescence intensity was recorded using the FlowJo software, and a standard curve was generated between fluorescence signal and antigen concentration. The mean fluorescence intensity (MFI) of each concentration was analyzed using the FlowJo software, and the MFI value for each concentration was calculated. A standard curve was plotted with the concentration of *Strombus rustus* antigen on the x-axis and fluorescence intensity on the y-axis to determine the linear detection range of the 1A6 antibody. (See [link to relevant documentation]). Figure 5 .

[0052] Figure 5 The results showed that the green dashed line marked the start of the linear range (50 ng / mL), and the red dashed line marked the end of the linear range (1000 ng / mL). The graph clearly demonstrates a good linear relationship (linear correlation coefficient R) between antibody 1A6 and the detection signal (MFI) of *Strigera styracifolium* antigen concentration in the range of 50 ng / mL to 1000 ng / mL. 2 >0.98).

[0053] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A kit for detecting antibodies against *Stemonas stems*, characterized in that, The kit comprises the following components: a) A labeled anti-stipe rust antibody 1A6 for binding to stipe rust-specific antigen, said antibody 1A6 comprising a heavy chain variable region as shown in SEQ ID NO:2 and a light chain variable region as shown in SEQ ID NO:6; b) Chromogenic or fluorescent reagents used to detect the binding of antibodies to antigens.

2. A method for detecting stem rust fungus, characterized in that, The method includes the following steps: a) Contact a sample containing *Stemonas styracifolium* antigen with antibody 1A6 as described in claim 1; b) Detect the binding signal between the antibody and the antigen, and determine whether the sample contains *Stemonas stalkae* based on the detection signal.

3. The method according to claim 2, characterized in that, The detection signal is a fluorescence signal or an enzyme-linked immunosorbent assay (ELISA) signal, in order to quantitatively determine the concentration of *Stemonas stolonifera* antigen.

4. A monoclonal antibody for detecting *Stemonas stolonifera*, characterized in that, The antibody comprises a heavy chain variable region as shown in SEQ ID NO:2 and a light chain variable region as shown in SEQ ID NO:

6.

5. The antibody according to claim 4, characterized in that, The heavy chain variable region includes HCDR1 as shown in SEQ ID NO:3, HCDR2 as shown in SEQ ID NO:4, and HCDR3 as shown in SEQ ID NO:5; the light chain variable region includes LCDR1 as shown in SEQ ID NO:7, LCDR2 as shown in SEQ ID NO:8, and LCDR3 as shown in SEQ ID NO:

9.

6. Use of the test kit of claim 1 or the antibody of any one of claims 4-5 in the preparation of a product for detecting *Stemona stem*.

Images