A kit for detecting microorganisms and use thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a kit for microorganism detection and application thereof. The kit comprises an enzyme label plate coated with a capture antibody, and a HRP-labeled antibody working solution. The capture antibody is a monoclonal antibody L3A-1 specifically combined with an antigen of Brugia malayi, the heavy chain amino acid sequence of which is shown as SEQ ID NO. 1, and the light chain amino acid sequence of which is shown as SEQ ID NO. 2. The HRP-labeled antibody is a monoclonal antibody F4B-2 specifically combined with the antigen of Brugia malayi, the heavy chain amino acid sequence of which is shown as SEQ ID NO. 3, and the light chain amino acid sequence of which is shown as SEQ ID NO. 4. The kit for microorganism detection can detect the content of the antigen of Brugia malayi in human blood, has high specificity and sensitivity, overcomes the defects that Brugia malayi needs professional personnel and equipment, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a reagent kit for microbial detection and its application. Background Technology

[0002] Fasciolopsis buski is a large trematode that primarily resides in the human small intestine, causing fasciolopsiasis. Fasciolopsis buski is a zoonotic pathogen, forming a transmission chain with patients, carriers, and pigs as the main sources of infection. Domestic pigs constitute the primary reservoir host in natural foci. After infection with Fasciolopsis buski, patients may experience abdominal pain, diarrhea, bloating, indigestion, anemia, and edema; children may experience growth retardation and intellectual impairment.

[0003] Traditional etiological diagnosis of Fasciolopsis buski infection mainly relies on direct fecal smears, which involve examining fecal eggs under a microscope to confirm infection. However, this method suffers from poor patient compliance and low detection efficiency. Given the limitations of traditional methods, this invention systematically explores the application value of enzyme-linked immunosorbent assay (ELISA) in the serological diagnosis of Fasciolopsis buski infection, aiming to establish a highly sensitive and specific immunological detection method to provide a scientific basis for improving clinical detection rates and constructing an auxiliary diagnostic system. This research has significant practical implications for optimizing parasitic disease control strategies and improving the diagnostic technology system for foodborne parasitic diseases.

[0004] To achieve the above objectives, the present invention provides a kit for microbial detection and its application. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, one of the objectives of the present invention is to provide a kit for microbial detection.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A kit for microbial detection, the kit comprising an enzyme-linked immunosorbent assay (ELISA) plate coated with a capture antibody and an HRP-labeled antibody working solution; wherein both the capture antibody and the HRP-labeled antibody are antibodies that specifically bind to Fasciolopsis buski antigen;

[0008] The capture antibody is a monoclonal antibody L3A-1 that specifically binds to Fasciolopsis buski antigen, and its heavy chain amino acid sequence is shown in SEQ ID NO.1, and its light chain amino acid sequence is shown in SEQ ID NO.2.

[0009] The HRP-labeled antibody is a monoclonal antibody F4B-2 that specifically binds to Fasciolopsis buski antigen, and its heavy chain amino acid sequence is shown in SEQ ID NO.3, and its light chain amino acid sequence is shown in SEQ ID NO.4.

[0010] Furthermore, the specific preparation method of the antibody that specifically binds to Fasciolopsis buski antigen is as follows:

[0011] (1) Isolate Fasciolopsis buski in feces, make it into insect powder, defatted, cold bathed, ultrasonically crushed and centrifuged to prepare Fasciolopsis buski antigen;

[0012] (2) Take the Fasciolopsis buski antigen prepared in step (1) to immunize mice, and fuse the spleen cells of the immunized mice with myeloma cells to form hybridoma cells;

[0013] (3) ELISA was used to screen hybridoma cells that secrete Fasciolopsis buski antibodies. Positive cell lines were subcloned and expanded, then inoculated into mice. Ascites fluid was collected and purified to obtain monoclonal antibodies L3A-1 and F4B-2.

[0014] Furthermore, the kit also includes antigen standards, washing solution, colorimetric solution, and stop solution.

[0015] Furthermore, the antigen standard is Fasciolopsis buski antigen; the washing solution is PBST; the chromogenic solution is TMB chromogenic solution; and the stop solution is sulfuric acid.

[0016] Furthermore, the concentration of the colorimetric solution is 2 mg / L-4 mg / L; the concentration of the stop solution is 1 M-3 M.

[0017] A second objective of this invention is to provide an application of a kit for microbial detection.

[0018] According to the above-described application of a kit for microbial detection, the kit is used to detect the content of Fasciolopsis buski antigen in a blood sample.

[0019] Furthermore, the specific steps of the detection are as follows:

[0020] (1) Remove the ELISA plate coated with capture antibody, bring it to room temperature, wash the plate with washing buffer and spin dry, add the sample to be tested and the serially diluted antigen standard, incubate at room temperature for 1-3 hours, wash the plate with washing buffer and spin dry;

[0021] (2) Add the HRP-labeled antibody working solution to the system in step (1), incubate at room temperature for 1-3 hours, wash the plate with washing solution and spin dry;

[0022] (3) Add colorimetric solution to the system after the reaction in step (2), develop color at room temperature in the dark for 10-20 minutes, and add stop solution to terminate the reaction;

[0023] (4) Use an enzyme-linked immunosorbent assay (ELISA) reader to perform dual-wavelength detection, measure the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm, and calculate the result using a standard curve.

[0024] Compared with the prior art, the main advantages of the present invention are as follows:

[0025] This invention provides a kit for microbial detection, capable of detecting the content of Fasciolopsis buski antigen in human blood. This kit can effectively assess the risk of fasciolopsiasis in the tested population, and has advantages such as high accuracy, good sensitivity, and simple operation. Furthermore, this kit does not cross-react with other parasitic antigens, exhibiting high specificity and promising application prospects. Attached Figure Description

[0026] Figure 1 Standard curve for a kit to specifically detect Fasciolopsis buski antigen content;

[0027] Figure 2 The diagnostic value of the Fasciolopsis buski detection kit was analyzed using ROC curve analysis. Detailed Implementation

[0028] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0029] Example 1

[0030] (1) Preparation of Fasciolopsis buski antigen:

[0031] Fresh Fasciolopsis buski was extracted from the feces of patients infected with Fasciolopsis buski. The worms were repeatedly washed with physiological saline to remove surface impurities, then chopped, dried with a fan, and ground into powder. The powder was stored in a desiccator at 4°C. 100 mg of the dried powder was weighed and defatted three times with 10 times the volume of acetone, 10 minutes each time, to remove fat. The defatted powder was then soaked in 5 mL of 0.01 M PBS (pH 7.2) at 4°C for one day. The soaked powder was then placed in a cold bath and treated six times with a 100 mA ultrasonic grinder, 1 minute each time. After ultrasonic disruption, the supernatant was collected by centrifugation, filtered through a 0.22 μM filter membrane, and the concentration of Fasciolopsis buski antigen was determined using the BCA method. The powder was stored at -80°C for later use.

[0032] (2) Preparation of antibodies against Fasciolopsis buski:

[0033] S1. Immunization of experimental animals: Antibodies against Fasciolopsis buski were prepared using the Fasciolopsis buski antigen obtained in step (1) as an immunogen. Female mice were immunized via subcutaneous injection at multiple points in the abdomen. For the first immunization, an equal volume of Fasciolopsis buski antigen and complete Freund's adjuvant were mixed and thoroughly emulsified, with an initial protein dose of 50 μg / mouse. For the subsequent three immunizations, an equal volume of Fasciolopsis buski antigen and incomplete Freund's adjuvant were mixed and thoroughly emulsified, with an initial protein dose of 80 μg / mouse. After immunization, mouse serum was collected, and the antibody titer was determined by indirect ELISA. Mice with the highest titer were selected for hybridoma cell fusion.

[0034] S2. Hybridoma cell fusion: Mouse spleen cells and SP2 / 0 myeloma cells were mixed at a ratio of 10:1, and 50% polyethylene glycol fusion agent was added. The mixture was fused in a 37°C water bath for 2 minutes. After fusion, the cells were cultured in HAT medium. One week later, the supernatant was collected, and positive hybridoma cells were screened by ELISA. Three subclonings were performed using the limiting dilution method, and the hybridoma cells with the highest sensitivity and specificity were selected for expansion culture.

[0035] S3. Monoclonal antibody preparation and purification: After pretreating the mouse peritoneum with liquid paraffin, the 5×10⁻⁶ antibody obtained in step S2 was purified. 6 Hybridoma cells were injected into the peritoneal cavity of mice. After observing significant abdominal distension, ascites fluid was collected. The collected ascites fluid was centrifuged at 3000 rpm for 15 minutes to remove cellular components, filtered through a 0.2 μm filter, and then purified into monoclonal antibodies by immunochromatography.

[0036] S4. Determination of Monoclonal Antibody Sequences: The activity of the purified monoclonal antibodies was determined using a Biacore T200 analyzer. The two monoclonal antibodies with the highest activity were named L3A-1 and F4B-2, and their variable region sequences were determined. The heavy chain amino acid sequence of monoclonal antibody L3A-1 is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2; the heavy chain amino acid sequence of monoclonal antibody F4B-2 is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4.

[0037] Table 1 Sequence List

[0038]

[0039] Example 2

[0040] Preparation of Fasciolopsis buski kit:

[0041] (1) Preparation of ELISA plates coated with L3A-1 capture antibody: L3A-1 was used as the capture antibody, and a 96-well ELISA plate was used as the solid phase carrier. The capture antibody was diluted to 2 μg / mL with coating buffer (50 mM carbonate buffer) to obtain the coating solution. 200 μL of the coating solution was added to the ELISA plate, and the plate was sealed and coated overnight at 4°C. The coating solution was discarded, the plate was washed 3 times with washing buffer, 200 μL of blocking buffer was added to each well, and the plate was blocked at room temperature for 4 h. The liquid in the plate was discarded, the plate was patted dry, and the plate was stored at 4°C.

[0042] (2) Preparation of HRP-labeled F4B-2 antibody working solution: Using monoclonal antibody F4B-2 as the HRP-labeled antibody, dialyze the monoclonal antibody F4B-2 in 50 mM carbonate buffer for 24 h, changing the buffer twice during this period to remove impurities, and adjusting the concentration to 2 mg / mL. Weigh 5 mg of HRP dry powder and dissolve it in 1 mL of ddH2O, add 150 μL of 0.1 M NaIO4 solution, and stir at room temperature in the dark for 30 min to oxidize the glycosyl group of HRP to an aldehyde group. Place the aldehyde-treated HRP solution into a dialysis bag and dialyze overnight in 10 mM sodium acetate buffer at 4℃ to remove unreacted NaIO4. Add carbonate buffer to the HRP solution that has been dialyzed overnight to adjust the pH to 9.0. Then, immediately add an equal volume of monoclonal antibody F4B-2, and gently stir at room temperature in the dark for 3 hours. Next, add 0.1 mL of 4 mg / mL NaBH4, mix well, and incubate at 4°C for 2 hours to obtain a stable enzyme-labeled antibody. Place the above sample into 0.15 M PBS (pH 7.4) and dialyze overnight at 4°C to remove unbound antibody and other impurities. Centrifuge the dialysate to remove the precipitate; the supernatant is the HRP-labeled F4B-2 antibody working solution.

[0043] (3) Standard curve determination of the kit: Remove the ELISA plate coated with L3A-1 capture antibody, allow it to return to room temperature, wash the plate three times with PBST washing buffer and agitate dry; add 100 μL of Fasciolopsis buski antigen standards prepared in Example 1 at different dilutions (1.25 ng / mL, 2.5 ng / mL, 5 ng / mL, 7.5 ng / mL, 10 ng / mL, 12.5 ng / mL, 15 ng / mL, 17.5 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL), incubate at room temperature for 1 h, wash three times with PBST washing buffer and agitate dry. Add 100 μL of HRP-labeled F4B-2 antibody working solution to the reaction wells, incubate at room temperature for 1 h, wash three times with PBST washing buffer and agitate dry. Add 100 μL of 3 mg / L TMB chromogenic solution to the reaction wells, incubate at room temperature in the dark for 10 min, and add 100 μL of 1M sulfuric acid to terminate the reaction. A dual-wavelength assay was performed using an ELISA reader, measuring the OD values ​​at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm. The OD value at 450 nm was then subtracted from the OD value at 630 nm. A standard curve was plotted with the standard concentration on the x-axis (ng / mL) and OD450 - OD630 on the y-axis. The results are as follows: Figure 1 As shown, the detection range of the kit for detecting Fasciolopsis buski in this invention is 2.5-30 ng / mL, and the standard curve is y=0.1034x-0.0489, R0. 2 =0.9982.

[0044] (4) Composition of the Fasciolopsis buski kit: The enzyme-labeled plate coated with L3A-1 capture antibody prepared in step (1), the working solution of HRP-labeled F4B-2 antibody prepared in step (2), the antigen standard Fasciolopsis buski antigen, the washing buffer PBST (containing 0.2% Tween 20 PBST buffer), the colorimetric solution 3 mg / L TMB, and the stop solution 1 M sulfuric acid constitute the Fasciolopsis buski kit of the present invention.

[0045] Experimental Example 1

[0046] Specificity of the kit:

[0047] To evaluate the specificity of the kit, we selected Paragonimus westermani, Clonorchis sinensis, Ascaris lumbricoides, and Hookworm, which are similar to Fasciolopsis buski in terms of life history, transmission mode, or pathogenic mechanism, and prepared corresponding antigens. The Fasciolopsis buski antigen prepared in Example 1 was used as a control, and its concentration was adjusted to 10 ng / mL after being determined by the BCA method. Subsequently, these antigens were tested using the Fasciolopsis buski kit prepared in Example 2. The specific steps are as follows:

[0048] (1) Remove the ELISA plate coated with L3A-1 capture antibody, bring it to room temperature, wash the plate 3 times with PBST washing solution and spin dry, add the sample to be tested, incubate at room temperature for 1 h, wash 3 times with PBST washing solution and spin dry.

[0049] (2) Add 100 μL of HRP-labeled F4B-2 antibody working solution to the system in step (1), incubate at room temperature for 1 h, wash 3 times with PBST washing solution and spin dry.

[0050] (3) Add 100 μL of 3 mg / L TMB colorimetric solution to the system after the reaction in step (2), develop color at room temperature in the dark for 10 min, and then add 100 μL of 1 M sulfuric acid to terminate the reaction.

[0051] (4) Dual-wavelength detection was performed using an ELISA reader. The OD value at 450 nm was subtracted from the OD value at 630 nm, and the result was calculated using a standard curve. The results are shown in Table 2. The kit did not cross-react with other parasitic antigens, indicating that the kit prepared in this invention for microbial detection has high specificity.

[0052] Table 2 Sample Test Results

[0053]

[0054] Experimental Example 2

[0055] Blood sample testing:

[0056] Ten blood samples were collected from patients with Fasciolopsis buski disease (numbered 1-10) and ten blood samples were collected from healthy individuals (numbered 11-20). The samples were centrifuged at 6000 rpm for 10 min, and the supernatant was collected as the test samples. The blood samples were tested according to the method in Experiment 1. The test results are shown in Table 3.

[0057] Table 3 Clinical Sample Detection Results

[0058]

[0059] The results are shown in Table 3. The concentrations of fasciolopsiasis detected in the blood samples of the 10 patients with fasciolopsiasis were all within the linear range (2.5-30 ng / mL) of the kit of this invention. This indicates that the detection method established by this invention can use blood as a sample, effectively determine the risk of fasciolopsiasis in the tested population, and has the advantages of high accuracy, good sensitivity, and simple operation, making it suitable for clinical diagnosis.

[0060] Experimental Example 3

[0061] ROC curve evaluation of the diagnostic value of the Fasciolopsis buski detection kit

[0062] To further evaluate the diagnostic value of the Fasciolopsis buski detection kit, we collected 100 samples, including blood samples from 50 healthy individuals and 50 blood samples from patients with Fasciolopsis buski disease. These blood samples were centrifuged at 6000 rpm for 10 min, and the supernatant was used as the test sample. The Fasciolopsis buski antigen levels were detected following the procedures in Example 1. ROC curves were plotted based on the results, and the AUC value was calculated to evaluate the diagnostic value of the kit in Fasciolopsis buski disease.

[0063] The results are as follows Figure 2 The figure shows the ROC curve used to evaluate the diagnostic value of the Fasciolopsis buski detection kit. A higher AUC value indicates a higher diagnostic value for the disease; an AUC above 0.7 indicates good classification ability. As shown in the figure, the AUC value of the Fasciolopsis buski detection kit is 0.974, indicating that the kit can specifically distinguish between healthy individuals and patients with Fasciolopsis buski infection. It can screen for Fasciolopsis buski infection by detecting the content of Fasciolopsis buski antigens, thus possessing high diagnostic value.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A kit for microbial detection, characterized in that, The kit includes an ELISA plate coated with capture antibodies and an HRP-labeled antibody working solution; both the capture antibody and the HRP-labeled antibody are antibodies that specifically bind to Fasciolopsis buski antigen; The capture antibody is a monoclonal antibody L3A-1 that specifically binds to Fasciolopsis buski antigen, and its heavy chain amino acid sequence is shown in SEQ ID NO.1, and its light chain amino acid sequence is shown in SEQ ID NO.

2. The HRP-labeled antibody is a monoclonal antibody F4B-2 that specifically binds to Fasciolopsis buski antigen, and its heavy chain amino acid sequence is shown in SEQ ID NO.3, and its light chain amino acid sequence is shown in SEQ ID NO.

4.

2. The kit for microbial detection according to claim 1, characterized in that, The specific preparation method of the antibody that specifically binds to Fasciolopsis buski antigen is as follows: (1) Isolate Fasciolopsis buski in feces, make it into insect powder, defatted, cold bathed, ultrasonically crushed and centrifuged to prepare Fasciolopsis buski antigen; (2) Take the Fasciolopsis buski antigen prepared in step (1) to immunize mice, and fuse the spleen cells of the immunized mice with myeloma cells to form hybridoma cells; (3) ELISA was used to screen hybridoma cells that secrete Fasciolopsis buski antibodies. Positive cell lines were subcloned and expanded, then inoculated into mice. Ascites fluid was collected and purified to obtain monoclonal antibodies L3A-1 and F4B-2.

3. The kit for microbial detection according to claim 1, characterized in that, The kit also includes antigen standards, washing solution, colorimetric solution, and stop solution.

4. A kit for microbial detection according to claim 3, characterized in that, The antigen standard is Fasciolopsis buski antigen; the washing solution is PBST; the chromogenic solution is TMB chromogenic solution; and the stop solution is sulfuric acid.

5. A kit for microbial detection according to claim 4, characterized in that, The concentration of the colorimetric reagent is 2 mg / L-4 mg / L; the concentration of the stop solution is 1 M-3 M.

6. The application of the kit for microbial detection according to any one of claims 1-5 for non-diagnostic purposes, characterized in that, The method of using the kit is as follows: (1) Remove the ELISA plate coated with capture antibody, bring it to room temperature, wash the plate with washing buffer and spin dry, add the sample to be tested and the serially diluted antigen standard, incubate at room temperature for 1-3 hours, wash the plate with washing buffer and spin dry; (2) Add the HRP-labeled antibody working solution to the system in step (1), incubate at room temperature for 1-3 hours, wash the plate with washing solution and spin dry; (3) Add colorimetric solution to the system after the reaction in step (2), develop color at room temperature in the dark for 10-20 minutes, and add stop solution to terminate the reaction; (4) Use an enzyme-linked immunosorbent assay (ELISA) reader to perform dual-wavelength detection, measure the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm, and calculate the result using a standard curve.

7. The application of the kit for microbial detection according to claim 6 for non-diagnostic purposes, characterized in that, The sample to be tested is blood.

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