Edwardsiella tarda antibody detection kit and application thereof

Abstract

The invention relates to the field of biological detection, in particular to an Edwardsiella tarda antibody detection kit and application thereof, and provides an anti-Edwardsiella tarda monoclonal antibody 3H5 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. The detection kit containing the monoclonal antibody 3H5 can realize qualitative and quantitative detection of edwardsiella tarda, and has high specificity and sensitivity.

Description

Technical Field

[0001] The present invention relates to the field of biological detection. Specifically, the present invention relates to a detection kit for Edwardsiella tarda antibodies and its application. Background Art

[0002] Edwardsiella tarda is an important Gram-negative bacterium that widely exists in aquatic environments and is an opportunistic pathogen of fish, amphibians, and humans. In the field of aquaculture, Edwardsiella tarda is the main pathogenic bacterium causing Edwardsiellosis, which can lead to severe fish infectious diseases, especially in cultured catfish, tilapia, and bass, manifested as skin ulcers, organ hemorrhage, and septicemia symptoms, resulting in high mortality and economic losses. In addition, Edwardsiella tarda can also infect humans, causing diseases such as gastroenteritis, septicemia, and wound infections, posing a threat to public health.

[0003] Monoclonal antibody technology has been widely used in bacterial detection due to its high specificity and sensitivity. Techniques such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and immunochromatographic test strips based on monoclonal antibodies have been developed for the detection of Edwardsiella tarda. These methods achieve rapid detection of the target bacterium by recognizing specific antigens on the surface of Edwardsiella tarda. Compared with the traditional culture method, the monoclonal antibody detection technology significantly shortens the detection time and has the potential for on-site rapid screening. However, related technologies are still being continuously optimized to further improve the detection effect.

[0004] Although the monoclonal antibody detection technology has significant advantages in sensitivity and specificity compared with traditional methods, there are still certain limitations. On the one hand, the recognition of the target antigen by the antibody may be interfered by the complexity of the sample matrix, resulting in a decrease in detection sensitivity; on the other hand, the non-specific binding of the antibody conjugate may cause false positive results. Currently, the detection methods for Edwardsiella tarda mainly include traditional bacterial isolation and culture, molecular biology detection (such as PCR), and serological detection, but these methods have the disadvantages of complex operation, long time consumption, or insufficient specificity. Therefore, developing a high-specificity and high-sensitivity detection kit based on monoclonal antibodies for the rapid qualitative and quantitative detection of Edwardsiella tarda can not only improve the diagnostic efficiency but also provide technical support for the timely early warning and scientific prevention and control of diseases. Summary of the Invention

[0005] The present invention provides a highly specific and sensitive monoclonal antibody 3H5 against Edwardsiella tarda and its application in a detection kit, aiming to achieve rapid and accurate detection of Edwardsiella tarda.

[0006] In a certain embodiment of the present invention, the monoclonal antibody 3H5 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, wherein the heavy chain variable region comprises 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 comprises 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. These specific CDR sequences endow the antibody with a high recognition ability for specific antigens of Edwardsiella tarda.

[0007] Based on the monoclonal antibody 3H5, the present invention designs an antibody detection kit for Edwardsiella tarda, which kit comprises the following key components:

[0008] The anti-Edwardsiella tarda monoclonal antibody 3H5, which is used to recognize specific antigens of Edwardsiella tarda;

[0009] The HRP-labeled goat anti-mouse IgG secondary antibody, which is used for signal amplification;

[0010] A chromogenic agent or a fluorescent reagent, which is used for detecting the signal generation of the binding of the antibody to the antigen.

[0011] The kit of the present invention realizes the qualitative and quantitative detection of the target bacteria through the high-affinity binding of the monoclonal antibody 3H5 to specific antigens of Edwardsiella tarda. Combining enzyme-catalyzed chromogenic or fluorescent signal amplification technology, the kit has significant advantages in detection sensitivity and specificity, effectively avoiding the common false positive and false negative problems in traditional methods. Description of the Drawings

[0012] Figure 1 SDS-PAGE analysis results of the OMP antigen fragment.

[0013] Figure 2 The titer of the isolated serum of the immunized mice was determined by the indirect ELISA method.

[0014] Figure 3 SDS-PAGE analysis results of the light chain and heavy chain of the monoclonal antibody 3H5.

[0015] Figure 4 The titer curve of the monoclonal antibody 3H5 detected by the indirect ELISA.

[0016] Figure 5 Subtype analysis of the monoclonal antibody 3H5.

[0017] Figure 6 Sensitivity analysis of the monoclonal antibody 3H5 to Edwardsiella tarda. Detailed implementation manners

[0018] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0019] Example 1 Preparation of outer membrane protein (OMP) antigen fragment of Edwardsiella tarda

[0020] Design an immunogenic antigen fragment according to the full-length sequence of Edwardsiella tarda (outer membrane protein [Edwardsiella tarda], GenBank: AGH12865.1) recorded in NCBI:

[0021] AAEIYNKDGNKLDLYGKVDGLHYFSQDHNKDGDQSYVRFGFKGETQINDQLTGYGQWEAQANVNQPESNSSNFFTRLGFAGLKYGNYGSIDYGRNYGVLYDIEGWTDVLPEFGGDTSAQSDNFMA (SEQ ID NO: 1).

[0022] According to the OMP sequence (SEQ ID NO:1) provided by the NCBI database, primers with appropriate restriction sites were designed; using the synthesized primers and the OMP template sequence, the target sequence was amplified by PCR technology; the PCR conditions referred to the standard amplification program: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds, for a total of 30 cycles, and finally extension at 72°C for 10 minutes. The PCR product was recovered using a DNA purification kit for subsequent restriction digestion and ligation; the pET-28a(+) plasmid and the purified PCR product were digested with NdeI and XhoI double enzymes respectively; the digested plasmid vector and PCR fragment were purified using a DNA purification kit; using T4 DNA ligase, the OMP gene was inserted into the pET-28a(+) vector and ligated overnight; the ligation product was transformed into competent Escherichia coli DH5α strain, spread on LB plates (containing kanamycin resistance), and cultured at 37°C for 16 hours. Positive clones were selected and plasmids were extracted, and the correctness of the insertion was confirmed by restriction digestion identification and sequencing; the constructed recombinant plasmid was transformed into Escherichia coli BL21(DE3), and positive clones were selected and cultured in LB medium containing kanamycin; when the OD600 reached 0.6 - 0.8, 1 mM IPTG was added at a final concentration to induce protein expression, and induction was carried out at 30°C or 16°C for 4 - 8 hours. After the induction culture was completed, the cells were collected by centrifugation at 4°C and 5000×g for 10 minutes; the cells were resuspended in lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0), and ultrasonic disruption was used (ultrasonic for 5 seconds, interval for 5 seconds, a total of 20 minutes of cycling); the lysate was centrifuged (4°C, 12000×g, 20 minutes), the supernatant was collected and the target protein with His tag was purified by nickel column affinity purification method; the protein peak was collected by gradient elution (20 mM - 500 mM imidazole); the purified protein was dialyzed into PBS buffer to remove imidazole and other impurities, and finally the protein solution was concentrated. Prepare an SDS-PAGE gel (4% stacking gel and 12% separating gel); take an appropriate amount of purified protein sample, add 1×SDS-PAGE loading buffer, and denature at 100°C in a water bath for 5 minutes; perform electrophoresis at a voltage of 100 V, and adjust the voltage to 120 V when the dye front reaches the separating gel to separate the proteins; stain with Coomassie Brilliant Blue R-250 to observe the band position and purity, see Figure 1 。

[0023] Figure 1 The results showed that the molecular weight of the OMP antigen fragment was 14 kDa, which was in line with expectations.

[0024] Example 2 Screening, Preparation and Identification of Monoclonal Antibodies Against Edwardsiella tarda

[0025] The purified OMP protein in Example 1 was mixed with Freund's complete adjuvant at a ratio of 1:1, and BALB / c mice were immunized subcutaneously with 100 μg of antigen per mouse; after two weeks, the OMP protein was mixed with Freund's incomplete adjuvant again and repeated 3 times, with each dose being 100 μg; two weeks after the three immunizations, blood was collected from the orbital venous plexus or tail vein of the mice. The collected blood was placed in a 37 °C water bath for 30 min, left to stand overnight at 4 °C, centrifuged at 6000 rpm for 5 min to separate the serum, and stored frozen at -40 °C in a refrigerator; the indirect ELISA method was used to measure the titer of the separated serum of the immunized mice, and the serum of non-immunized blank mice was used as a negative control, see Figure 2

[0026] Figure 2 The results showed that the serum titer of Mouse No. 1 was the highest and could be used to prepare splenocytes for cell fusion after booster immunization.

[0027] Mouse No. 1 was sacrificed, the spleen was taken, and splenocytes were isolated for fusion. The murine myeloma cell line (SP2 / 0) was used and resuscitated, cultured in the logarithmic growth phase state, and mouse splenocytes and myeloma cells were mixed at a ratio of 10:1 using 50% PEG medium. Immediately after PEG treatment, the fusion mixture was diluted with RPMI-1640 medium. The fused cells were cultured in HAT selective medium to select hybridoma cells. 10% fetal bovine serum was added to the medium to promote cell proliferation. About 8 days after cell fusion, the selected positive clones were subcloned by the limiting dilution method (0.5 cells / well), and positive hybridoma cells were screened by immunofluorescence. Finally, 1 positive hybridoma cell strain that could stably secrete monoclonal antibodies against OMP antigen fragments was identified and named 3H5. The supernatant of monoclonal hybridoma cell 3H5 was collected, and antibody 3H5 was purified using a Protein G affinity column; an appropriate amount of the purified antibody 3H5 sample was mixed with SDS loading buffer, loaded after heating and denaturation, and SDS-PAGE was used to separate the proteins, and the molecular weight and purity of the antibody were verified by Coomassie Brilliant Blue staining, see Figure 3 。

[0028] Figure 3 The results showed that under reducing conditions, antibody 3H5 had 1 band near 50 kDa and 25 kDa respectively, which was consistent with the theoretical molecular weights of the heavy chain and light chain after reduction; no obvious miscellaneous bands were seen, indicating that the prepared antibody 3H5 had high purity.

[0029] Add 100 μL of 5 - 10 μg / mL OMP antigen fragment solution to each well of the ELISA plate; incubate overnight at 4°C or for 2 hours at 37°C. After coating, wash the plate 3 times with PBS washing solution, soaking for 5 minutes each time; after washing the plate, gently pat it dry. Add 300 μL of 1% BSA - PBS solution to each well and incubate at 37°C for 1 hour to block non - specific binding. After incubation, discard the blocking solution and wash again 3 times with PBS washing solution, 5 minutes each time; serially dilute the monoclonal antibody 3H5 to be tested, add 100 μL of primary antibody at different dilutions to each well, and incubate at 37°C for 1 hour; after incubation, wash 5 times with PBS washing solution, 5 minutes each time; add HRP - labeled anti - mouse IgG secondary antibody (diluted 1:5000), and incubate at 37°C for 1 hour. After incubation, wash 5 times with PBS washing solution, 5 minutes each time. Add 100 μL of TMB chromogenic substrate solution to each well and react in the dark for 10 - 15 minutes until it turns blue. Add 50 μL of 2M H 2 SO 4 stop solution, mix well, and the blue color immediately turns yellow; use an ELISA microplate reader to read the absorbance value (OD value) of each well at a wavelength of 450 nm, plot the curve of antibody dilution factor vs. OD value, and analyze the titer of antibody 3H5 according to the curve, as shown in Figure 4 .

[0030] Figure 4 The results showed that the titer of monoclonal antibody 3H5 detected by indirect ELISA was 1:256000.

[0031] Identification of the variable region sequences of monoclonal antibody 3H5: Total RNA was extracted from the hybridoma cell line producing the 3H5 antibody. RNA extraction was performed using a commercial RNA extraction kit, and the RNA concentration and purity were measured. The extracted mRNA was reverse transcribed into cDNA using reverse transcription reaction (RT-PCR). Appropriate primers, either random primers or specific primers (targeting the framework region sequences of the antibody heavy chain VH and light chain VL), were selected. cDNA was synthesized using a high-efficiency reverse transcriptase and an RT-PCR kit. Specific primers were designed and synthesized targeting the conserved regions of the variable regions of the antibody heavy chain (VH) and light chain (VL). The VH and VL region gene fragments were amplified by PCR reaction. Appropriate PCR conditions (denaturation at 95°C, annealing at 55°C, extension at 72°C) were used for 30 - 35 cycles. The specificity and product length of the PCR amplification were confirmed by agarose gel electrophoresis. The amplified VH and VL fragments were purified by gel purification or using a PCR product purification kit. The concentration of the purified product was further determined to ensure it met the requirements for subsequent sequencing. The purified PCR products were sent to a sequencing company or sequenced bidirectionally using a sequencer by Sanger sequencing. Samples were prepared according to the requirements of the sequencing platform, and appropriate primers were selected for complete sequencing of the VH and VL regions. The raw sequencing data were obtained, and base sequence alignment and correction were performed. The VH and VL sequences were aligned using antibody sequence analysis software (Kabat) to identify the specific positions of the CDR and framework regions (FR), and the amino acid sequences of the VH and VL variable regions of the 3H5 antibody were determined.

[0032] Variable heavy chain (VH) of monoclonal antibody 3H5: MDFGLSFIFLALILKGVQCQVRLQESGPGLVQPAQSLPITCTVSGFSFTDYGVYWIRQSPGKGLDWLGVIWSGGNADYNTPFSGRFSINKDNSKTQVFFKMDSLQSNDTAVYYCAIALTYSDYEKPYWGQGTTTAVSS (SEQ ID NO:2);

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

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

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

[0036] Light chain variable region (VL) of monoclonal antibody 3H5: DIVLTQSPASLAVSLGQRVTINCRASESVDTSNLHWFEQKPGQAPRLMIFDASTRRSGVPSRFSGSGSGTEFTLTISALQAKDVTVYYCQRYGSNPWTLGQGTKVEIR (SEQ ID NO:6);

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

[0038] L-CDR2: DASTRR (SEQ ID NO:8);

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

[0040] Using the ELISA method, it was measured with a monoclonal antibody typing kit. The capture antibody was coated overnight at 4°C. The supernatant of 3H5 cells was used as the primary antibody, and the HRP-labeled goat anti-mouse Ig screening antibody was used as the secondary antibody. ABTS and sodium citrate substrate buffer containing 2 H 2 O 450 was used as the substrate. It was placed at room temperature for 10 min to measure the OD Figure 5 value. The results are as

[0041] shown, indicating that the subtype of monoclonal antibody 3H5 is IgG3 / κ chain.

[0042] Glass slide agglutination tests were performed with standards of Edwardsiella ictaluri, Edwardsiella tarda, Escherichia coli, Edwardsiella hoshinae, Vibrio anguillarum, Streptococcus agalactiae, and Pseudomonas fluorescens, respectively, against monoclonal antibody 3H5. The above standards were inoculated onto LB agar medium and cultured at 37°C for 24 hours. Single colonies were picked into 5 mL of physiological saline to prepare a bacterial suspension, and the bacterial suspension concentration was adjusted to the McFarland 1.0 standard (~10 8 CFU / mL).

[0043] On a glass microscope slide, different strains were marked in separate regions. An equal volume (20 μL) of bacterial suspension was added dropwise to each region, followed by 10 μL of the working solution of antibody 3H5. In the control group, PBS was added dropwise instead of the antibody. The mixture was gently stirred with a micro glass rod, and each slide used an independent glass rod. It was left to stand at room temperature for 2 minutes, and the reaction was observed. The agglutination intensity comparison between Edwardsiella tarda and other related strains was recorded.

[0044] Table 1 Specific identification of monoclonal antibody 3H5

[0045]

[0046] Note: The grading of agglutination reaction intensity: +++ indicates obvious agglutination, ++ indicates moderate agglutination, + indicates slight agglutination, and - indicates no agglutination.

[0047] The results in Table 1 showed that antibody 3H5 exhibited an obvious agglutination reaction of +++ in Edwardsiella tarda, a slight agglutination reaction of + in Edwardsiella hoshinae; while no agglutination (-) occurred in other strains. This indicated that monoclonal antibody 3H5 could specifically recognize Edwardsiella tarda and showed a strong agglutination reaction in the slide agglutination test; it had no obvious cross-reaction with related strains, demonstrating its application potential in the detection and diagnosis of Edwardsiella tarda.

[0048] Example 4 Sensitivity analysis of monoclonal antibody 3H5 to Edwardsiella tarda

[0049] The bacterial solution of Edwardsiella tarda was serially diluted to prepare the following concentration series: 1×10 2 、1×10 3 、1×10 4 、1×10 5 、1×10 6CFU / mL. Dilute the monoclonal antibody 3H5 to 1 μg / mL and add 100 μL to each well of the ELISA plate. Incubate overnight at 4 °C. Add 5% bovine serum albumin (BSA) solution to each well and incubate at room temperature for 1 hour to block unbound sites. Add different concentrations of Edwardsiella tarda bacterial solutions, 100 μL per well, and incubate at room temperature for 1 hour. Add 100 μL of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG secondary antibody to each well and incubate at room temperature for 30 minutes. Add 100 μL of TMB solution to each well and react in the dark for 10 minutes. Add 50 μL of stop solution (2 M sulfuric acid) to stop the reaction. Measure the absorbance value (OD 450 ) at a wavelength of 450 nm using an ELISA reader, and record the OD450 values at different bacterial solution concentrations. Use the bacterial solution concentration as the abscissa and the OD450 value as the ordinate to plot a standard curve, perform a correlation analysis, and calculate the correlation coefficient (R 2 ), as shown in Figure 6 .

[0050] Standard curve equation:

[0051] y = 0.87·log(x) + 0.5

[0052] Correlation coefficient:

[0053] R 2 = 0.9975

[0054] Figure 6 The curve trend shows that the OD 450 value has a good linear correlation with the concentration of Edwardsiella tarda bacterial solution. The correlation coefficient R 2 = 0.9975, and the lowest detection limit for the detection of Edwardsiella tarda antigen obtained in the present invention is 1×10 2 CFU / mL.

[0055] The above is the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A kit for detecting Edwardsiella tarda antibodies, characterized in that: The kit comprises the following components: a) anti-Edwardsiella tarda antibody 3H5 and HRP-labeled goat anti-mouse IgG secondary antibody, wherein the anti-Edwardsiella tarda antibody 3H5 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; b) Colorimetric or fluorescent reagents for detecting binding signals.

2. A monoclonal antibody for detecting Edwardsiella tarda, 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.

3. The antibody according to claim 2, characterized in that The heavy chain variable region comprises 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 comprises 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.

4. Use of the detection kit according to claim 1 and the antibody according to any one of claims 2 to 3 in the preparation of a product for detecting Edwardsiella tarda.

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