An antibody test kit for detecting edwardsiella tarda and application thereof

Abstract

The application relates to the field of biological detection, in particular to an Edwardsiella tarda antibody detection kit and application thereof. The application provides a monoclonal antibody 3H5 against Edwardsiella tarda, 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 higher specificity and sensitivity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological detection. Specifically, the present application relates to an Edwardsiella tarda antibody detection kit and its application. BACKGROUND

[0002] Edwardsiella tarda is an important gram-negative bacillus widely existing in aquatic environments, and is a conditional pathogen of fish, amphibians and humans. In the field of aquaculture, Edwardsiella tarda is the main pathogen causing Edwardsiellosis, which can cause serious infectious diseases in fish, especially in farmed catfish, tilapia and grouper, showing symptoms of skin ulceration, organ hemorrhage and septicemia, resulting in high mortality and economic losses. In addition, Edwardsiella tarda can also infect humans, causing diseases such as gastroenteritis, septicemia and wound infection, posing a threat to public health.

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

[0004] Although monoclonal antibody detection technology has significant advantages in sensitivity and specificity over traditional methods, there are still certain limitations. On the one hand, the recognition of antibodies to target antigens can be interfered by the complexity of sample matrix, leading to reduced detection sensitivity; on the other hand, non-specific binding of antibody conjugates can 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-consuming or insufficient specificity. Therefore, developing a high specificity and high sensitivity detection kit based on monoclonal antibodies for rapid qualitative and quantitative detection of Edwardsiella tarda can not only improve diagnostic efficiency, but also provide technical support for timely warning and scientific prevention and control of diseases. SUMMARY

[0005] The present application provides a high specificity and high sensitivity anti-Edwardsiella tarda monoclonal antibody 3H5 and its application in a detection kit, aiming to achieve rapid and accurate detection of Edwardsiella tarda.

[0006] In one 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; and 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 against specific antigens of Edwardsiella tarda.

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

[0008] The monoclonal antibody 3H5 against Edwardsiella tarda is used to recognize specific antigens of Edwardsiella tarda.

[0009] HRP-labeled goat anti-mouse IgG secondary antibody was used for signal amplification;

[0010] Chromogenic agents or fluorescent reagents are used to detect the signal generation of antibody-antigen binding.

[0011] The kit of this invention enables qualitative and quantitative detection of the target bacterium through the high affinity binding of monoclonal antibody 3H5 to Edwardsiella tarda-specific antigen. Combined with enzyme-catalyzed colorimetric or fluorescence signal amplification techniques, this kit offers significant advantages in detection sensitivity and specificity, effectively avoiding the false positive and false negative problems commonly found in traditional methods. Attached Figure Description

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

[0013] Figure 2 Indirect ELISA was used to determine the potency of isolated serum from immunized mice.

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

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

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

[0017] Figure 6 Sensitivity analysis of monoclonal antibody 3H5 against Edwardsiella tarda. Detailed Implementation

[0018] 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.

[0019] Example 1: Preparation of OMP antigen fragments from outer membrane proteins (OMPs) of Edwardsiella tarda

[0020] Immunogen fragments were designed based on 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] Primers with appropriate restriction enzyme sites were designed based on the OMP sequence (SEQ ID NO:1) provided in the NCBI database. The target sequence was amplified using PCR with the synthesized primers and the OMP template sequence. The PCR conditions followed the standard amplification procedure: 95℃ pre-denaturation for 5 minutes, 95℃ denaturation for 30 seconds, annealing at 55℃ for 30 seconds, extension at 72℃ for 30 seconds, for a total of 30 cycles, followed by a final extension at 72℃ for 10 minutes. The PCR product was recovered using a DNA purification kit for subsequent enzyme digestion and ligation. The pET-28a(+) plasmid and the purified PCR product were double-digested with NdeI and XhoI, respectively. The digested plasmid vector and PCR fragment were purified using a DNA purification kit. The OMP gene was inserted into the pET-28a(+) vector using T4 DNA ligase and ligated overnight. The ligation product was transformed into competent *Escherichia coli* DH5α strain, plated on LB agar plates (containing kanamycin resistance), and incubated at 37℃ for 16 hours. Positive clones were selected and plasmids were extracted. The correctness of the insertion was confirmed by enzyme digestion and sequencing. The constructed recombinant plasmid was transformed into BL21(DE3) Escherichia coli. Positive clones were selected and cultured in LB medium containing kanamycin. When the OD600 reached 0.6-0.8, 1 mM IPTG was added to induce protein expression. The induction was carried out at 30℃ or 16℃ for 4-8 hours. After induction culture, bacterial cells were collected by centrifugation at 4℃ and 5000×g for 10 minutes. The bacterial cells were resuspended in lysis buffer (50mM Tris-HCl, 300mM NaCl, 10mM imidazole, pH 8.0) and sonicated (5 seconds sonication, 5-second intervals, for a total of 20 minutes). The lysis buffer was centrifuged (4℃, 12000×g, 20 minutes), and the supernatant was collected. The His-tagged target protein was purified using nickel column affinity purification. The protein peak was collected by gradient elution (20mM-500mM imidazole). The purified protein was dialyzed into PBS buffer to remove imidazole and other impurities, and finally the protein solution was concentrated. Prepare SDS-PAGE gels (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℃ for 5 minutes; perform electrophoresis at 100V, and adjust the voltage to 120V when the dye front reaches the separating gel to separate the protein; stain with Coomassie Brilliant Blue R-250 to observe the band position and purity, see [reference needed]. 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 from Example 1 was mixed with Freund's complete adjuvant at a 1:1 ratio, and BALB / c mice were subcutaneously immunized with 100 μg of antigen per mouse. Every two weeks, OMP protein was mixed with Freund's incomplete adjuvant, repeated three 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, incubated overnight at 4°C, and centrifuged at 6000 rpm for 5 min to separate the serum, which was then frozen at -40°C. The titer of the separated serum from the immunized mice was determined using an indirect ELISA method. Serum from non-immunized blank mice served as a negative control. Figure 2

[0026] Figure 2 The results showed that the serum of mouse No. 1 had the highest titer and could be used to prepare spleen cells for cell fusion after shock immunization.

[0027] Mice were euthanized, and their spleens were harvested. Spleen cells were isolated for fusion and resuscitated using a mouse myeloma cell line (SP2 / 0). The cells were cultured in the logarithmic growth phase, and mouse spleen cells and myeloma cells were mixed at a 10:1 ratio using 50% PEG medium. Immediately after PEG treatment, the fusion medium was diluted with RPMI-1640 medium. The fused cells were cultured in HAT selective medium, and hybridoma cells were selected. 10% fetal bovine serum was added to the medium to promote cell proliferation. Around day 8 post-fusion, the selected positive clones were subcloned using a limiting dilution method (0.5 cells / well). Immunofluorescence was used to screen for positive hybridoma cells, ultimately identifying one positive hybridoma cell line that stably secreted a monoclonal antibody against the OMP antigen fragment, named 3H5. Collect the supernatant from monoclonal hybridoma cells 3H5 and purify antibody 3H5 using a Protein G affinity column. Take an appropriate amount of the purified antibody 3H5 sample, mix with SDS loading buffer, heat to denature, and then load the sample. Separate the protein using SDS-PAGE. Verify the molecular weight and purity of the antibody by Coomassie brilliant blue staining. (See attached image) Figure 3 .

[0028] Figure 3 The results showed that under reducing conditions, the 3H5 antibody had one band at around 50kDa and 25kDa, 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 3H5 had high purity.

[0029] Add 100 μL of 5-10 μg / mL OMP antigen fragment solution to each well of an ELISA plate; incubate overnight at 4°C or for 2 hours at 37°C. After coating, wash the plate three times with PBS, soaking for 5 minutes each time. After washing, gently pat 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 three more times with PBS, 5 minutes each time. Serially dilute the monoclonal antibody 3H5 to be tested, adding 100 μL of different dilutions of primary antibody to each well, and incubate at 37°C for 1 hour. After incubation, wash five times with PBS, 5 minutes each time. Add HRP-labeled anti-mouse IgG secondary antibody (1:5000 dilution), and incubate at 37°C for 1 hour. After incubation, wash five times with PBS, 5 minutes each time. Add 100 μL of TMB substrate solution to each well and react in the dark for 10-15 minutes until a blue color appears. Add 50 μL of 2M H2SO4 stop solution to each well, mix well, and the blue color will immediately turn yellow. Use an ELISA reader to read the absorbance (OD value) of each well at 450 nm. Plot a curve of antibody dilution versus OD value, and analyze the antibody 3H5 titer based on the curve. Figure 4 .

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

[0031] Identification of the variable region sequence of monoclonal antibody 3H5: Total RNA was extracted from hybridoma cell lines producing 3H5 antibody. RNA extraction was performed using a commercially available RNA extraction kit, and RNA concentration and purity were measured. The extracted mRNA was reverse transcribed into cDNA using reverse transcription-PCR. Appropriate primers, either random or specific (targeting the frame 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 conserved regions of the variable regions of the antibody heavy chain (VH) and light chain (VL). VH and VL region gene fragments were amplified by PCR. Appropriate PCR conditions were used (95°C denaturation, 55°C annealing, 72°C extension) for 30-35 cycles. The specificity of PCR amplification and product length 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 subjected to bidirectional Sanger sequencing using a sequencer. Samples were prepared according to the sequencing platform requirements, and appropriate primers were selected for complete sequencing of the VH and VL regions. 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 locations of the CDR and frame region (FR), and to determine the amino acid sequences of the VH and VL variable regions of the 3H5 antibody.

[0032] Heavy chain variable region (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] The 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] The ELISA method was used, employing a monoclonal antibody typing kit. Capture antibodies were coated overnight at 4°C. 3H5 cell supernatant served as the primary antibody, and HRP-labeled goat anti-mouse Ig selection antibody served as the secondary antibody. A sodium citrate substrate buffer containing ABTS and H2O2 was used as the substrate. OD was measured after incubation at room temperature for 10 minutes. 450 Value, result as Figure 5 As shown, this indicates that the isotype of monoclonal antibody 3H5 is the IgG3 / κ chain.

[0041] Example 3: Specificity identification of monoclonal antibody 3H5 against Edwardsiella tarda

[0042] Standards for Edwardsiella ictaluri, Edwardsiella tarda, Escherichia coli, Edwardsiella hoshinae, Vibrio anguillarum, Streptococcus agalactiae, and Pseudomonas fluorescens were used to perform agar agglutination tests with monoclonal antibody 3H5. The standards were inoculated onto LB agar and incubated at 37°C for 24 hours. Single colonies were picked and transferred to 5 mL of physiological saline to prepare bacterial suspensions, and the bacterial concentration was adjusted to McFarland 1.0 standard (~10). 8 (CFU / mL).

[0043] Different strains were labeled in different regions on a glass microscope slide. An equal volume (20 μL) of bacterial suspension was added to each region, followed by 10 μL of antibody 3H5 working solution. PBS was added to the control group instead of the antibody. The mixture was gently mixed with a micro glass rod. Each slide was placed on a separate glass rod and allowed to stand at room temperature for 2 minutes. The reaction was observed, and the agglutination intensity of Edwardsiella tarda was compared with that of other similar strains.

[0044] Table 1. Specificity identification of monoclonal antibody 3H5

[0045]

[0046] Note: Agglutination intensity grading: +++ indicates significant agglutination, ++ indicates moderate agglutination, + indicates slight agglutination, and - indicates no agglutination.

[0047] Table 1 shows that antibody 3H5 exhibited a significant agglutination reaction (+++) in Edwardsiella tarda and a slight agglutination reaction (+) in Edwardsiella hoshinae; however, no agglutination (-) was observed in other strains. This indicates that monoclonal antibody 3H5 can specifically recognize Edwardsiella tarda and exhibits a strong agglutination reaction in the glass plate agglutination test. Its lack of significant cross-reactivity with similar strains demonstrates its potential application in the detection and diagnosis of Edwardsiella tarda.

[0048] Example 4: Sensitivity analysis of monoclonal antibody 3H5 against Edwardsiella tarda.

[0049] Edwardsiella tarda bacterial suspension was serially diluted to prepare the following concentration series: 1×10⁻⁶ 2 1×10 3 1×10 4 1×10 5 1×10 6CFU / mL, dilute monoclonal antibody 3H5 to 1 μg / mL and add 100 μL to each well of an 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 100 μL of Edwardsiella tarda bacterial culture at different concentrations to each 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 (2M sulfuric acid) to stop the reaction. Measure the absorbance (OD) at 450 nm using a microplate reader. 450 Record the OD450 values ​​at different bacterial concentrations. Plot a standard curve with bacterial concentration on the x-axis and OD450 values ​​on the y-axis, perform correlation analysis, and calculate the correlation coefficient (R²). 2 ),See 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 indicates that OD 450 The value showed a good linear correlation with the concentration of Edwardsiella tarda bacterial culture, with a correlation coefficient R. 2 =0.9975, and the lowest detection limit for the Edwardsiella tarda antigen obtained by this invention is 1×10⁻⁶. 2 CFU / mL.

[0055] 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. An Edwardsiella tarda antibody detection kit, characterized by, The kit comprises the following components: a) Anti-E. lata antibody 3H5, the heavy chain variable region amino acid sequence of which is shown as SEQ ID NO: 2 and the light chain variable region amino acid sequence of which is shown as SEQ ID NO: 6, and HRP labeled goat anti-mouse IgG secondary antibody; b) color developing agent or fluorescent reagent for detecting the binding signal.

2. A monoclonal antibody for detecting Edwardsiella tarda, characterized in that, The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO: 2 and the light chain variable region amino acid sequence is shown as SEQ ID NO:

6.

3. The antibody of claim 2, wherein The heavy chain variable region is composed of HCDR1 shown as SEQ ID NO: 3, HCDR2 shown as SEQ ID NO: 4 and HCDR3 shown as SEQ ID NO: 5; the light chain variable region is composed of LCDR1 shown as SEQ ID NO: 7, LCDR2 shown as SEQ ID NO: 8 and LCDR3 shown as SEQ ID NO:

9.

4. Use of the detection kit of claim 1, or the antibody of any one of claims 2-3 in the preparation of a product for detecting E. lata.

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