A microbial detection kit and detection method

Abstract

The present invention belongs to the field of in vitro diagnostic technology, and discloses a microbial detection kit and a detection method. The kit includes an enzyme-labeled plate coated with a capture antibody and an HRP-labeled antibody working solution; the capture antibody is a monoclonal antibody LG3A-1 that specifically binds to the antigen of Giardia lamblia in humans, and its heavy chain amino acid sequence is as shown in SEQ ID NO.1, and its light chain amino acid sequence is as shown in SEQ ID NO.2; the HRP-labeled antibody is a monoclonal antibody FLSP4 that specifically binds to the antigen of Giardia lamblia in humans, and its heavy chain amino acid sequence is as shown in SEQ ID NO.3, and its light chain amino acid sequence is as shown in SEQ ID NO.4. The microbial detection kit provided by the present invention can detect the content of Giardia lamblia in human feces, has strong specificity, high sensitivity, good stability, and is simple to operate, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnosis, and particularly relates to a microbial detection kit and a detection method. Background Art

[0002] Giardia lamblia, abbreviated as Giardia, is a single-celled protozoan widely distributed in the small intestine of humans and mammals, which can cause intestinal diseases mainly manifested by diarrhea. The symptoms of giardiasis vary in severity, with an incubation period of about 1-2 weeks and a maximum of 45 days. Some infected individuals with giardiasis may not show any clinical symptoms and become carriers; those with symptoms mainly present with diarrhea and malabsorption. The typical symptoms in the acute phase are explosive watery diarrhea, foul smell, and a relatively large amount of stool, and may be accompanied by symptoms such as abdominal distension, abdominal pain, belching, and vomiting. There is no pus and blood visible in the feces of Giardia lamblia patients, but pus cells, red blood cells, trophozoites or cysts can be found by microscopic examination. If giardiasis is not treated promptly, it will mostly develop into a chronic disease, manifested as periodic loose stools, repeated attacks, very smelly stools, and the course of the disease can last for several years; children with long-term illness will lead to malnutrition, anemia, developmental disorders, etc.

[0003] Currently, the infection of giardiasis is widespread in various countries around the world, and faces many challenges in aspects such as diagnosis and prevention and control, which has attracted extensive attention and high emphasis from the international community. The key to reducing Giardia infection is early diagnosis and early treatment. Currently, the detection techniques for giardiasis include morphological examination, nucleic acid detection, etc. The advantage of morphological examination is that it is fast, simple, and direct, but it is not easy to observe when the number of pathogenic protozoa is small; nucleic acid detection has a cumbersome procedure, many specimen pretreatment processes, and is limited by primer design, nucleic acid purification, reaction conditions, etc., and is only limited to laboratory operations. It is easily contaminated by the laboratory and shows false positives, and sample processing and testing must be carried out in an independent space; in order to detect Giardia lamblia more quickly, simply, and with high accuracy, new diagnostic methods and techniques need to be continuously explored to meet the clinical needs.

[0004] Therefore, there is an urgent need to develop an enzyme-linked immunosorbent assay kit for detecting Giardia lamblia infection in feces to benefit more patients. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, a first aspect of the present invention is to provide a detection kit for microorganisms, which includes an enzyme-labeled plate coated with a capture antibody and a working solution of an HRP-labeled antibody; both the capture antibody and the HRP-labeled antibody are antibodies that specifically bind to human Giardia antigen; the capture antibody is a monoclonal antibody LG3A-1 that specifically binds to human Giardia antigen, and its heavy chain amino acid sequence is as shown in SEQ ID NO.1, and its light chain amino acid sequence is as shown in SEQ ID NO.2; the HRP-labeled antibody is a monoclonal antibody FLSP4 that specifically binds to human Giardia antigen, and its heavy chain amino acid sequence is as shown in SEQ ID NO.3, and its light chain amino acid sequence is as shown in SEQ ID NO.4.

[0006] Preferably, the antibody that specifically binds to human Giardia antigen is prepared by the following method:

[0007] (1) Isolate human Giardia from feces, repeatedly freeze-thaw, ultrasonically disrupt and then centrifuge to prepare human Giardia antigen;

[0008] (2) Immunize mice with the human Giardia antigen prepared in step (1), fuse the spleen cells of the immunized mice with myeloma cells to form hybridoma cells;

[0009] (3) Screen for hybridoma cells secreting human Giardia antibody by ELISA, subclone and expand the positive cell line, then inoculate into mice, collect ascites and purify to obtain monoclonal antibody LG3A-1 and monoclonal antibody FLSP4.

[0010] Preferably, the microorganism detection kit further includes an antigen standard, a washing solution, a chromogenic solution and a termination solution.

[0011] Preferably, the antigen standard is human Giardia antigen; the washing solution is PBST; the chromogenic solution is TMB; the termination solution is sulfuric acid.

[0012] Preferably, the concentration of the chromogenic solution is 2 mg / L - 3 mg / L; the concentration of the termination solution is 2 M - 5 M.

[0013] Preferably, the coating concentration of the capture antibody in the enzyme-labeled plate coated with the capture antibody is: 100 μL / well, and the capture antibody concentration is 2 μg / mL.

[0014] Preferably, the preparation method of the HRP-labeled antibody working solution is: add monoclonal antibody FLSP4 to the HRP solution oxidized by NaIO 4 React at room temperature in the dark, add NaBH 4 Continue to react at 4 °C for a period of time, and dialyze overnight with PBS.

[0015] In the second aspect of the present invention, a detection method for the above-mentioned microbial detection kit is provided. The detection method is as follows:

[0016] (1) Centrifuge the sample to be detected and take the supernatant.

[0017] (2) Take out the enzyme-labeled plate coated with the capture antibody, restore it to room temperature, wash the plate with the washing solution and spin dry, add the supernatant of the sample to be detected in step (1), incubate at room temperature, and then wash the plate with the washing solution.

[0018] (3) Add the HRP-labeled antibody working solution to the system in step (2), incubate at room temperature for 1 - 3 h, and then wash the plate with the washing solution.

[0019] (4) Add the chromogenic solution to the system after the reaction in step (3), develop color at room temperature in the dark for 10 - 30 min, and add the stop solution to terminate the reaction.

[0020] (5) Use an enzyme-linked immunosorbent assay (ELISA) reader for dual-wavelength detection, measure the OD values at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm, and calculate the results through the standard curve.

[0021] Preferably, the sample to be detected is feces.

[0022] The present invention has the following effects compared with the prior art:

[0023] The kit for microbial detection provided by the present invention can detect the content of Giardia lamblia in human feces, has strong specificity, high sensitivity, good stability, and simple operation, can effectively judge the risk of Giardia lamblia disease in the population to be tested, and has good application prospects. Description of the Drawings

[0024] Figure 1 Standard curve of the kit for specifically detecting the content of Giardia lamblia. Detailed Embodiments

[0025] The following further describes the technical solutions of the present invention in conjunction with specific embodiments. However, those skilled in the art should understand that the following examples are only used to illustrate the present invention and should not be regarded as a limitation of the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are all conventional products obtained through commercial channels.

[0026] Example 1

[0027] (1) Preparation of Giardia lamblia antigen:

[0028] Giardia duodenalis was isolated from feces and cultured in DMEM medium. After one week, Giardia duodenalis was resuspended in sterile PBS solution, frozen and thawed three times repeatedly, sonicated on ice for 30 min, centrifuged at 12,000 rpm for 20 min, and the supernatant was taken to prepare Giardia duodenalis antigen. After filtration through a 0.22 μM filter membrane, the concentration of Giardia duodenalis antigen was determined by the BCA method, and the concentration of Giardia duodenalis antigen was adjusted to 10 μg / mL and stored at -80 °C for later use.

[0029] (2)Preparation of monoclonal antibody against Giardia duodenalis:

[0030] S1. Immunization of experimental animals: The prepared Giardia duodenalis antigen was used to immunize female mice by multi-point subcutaneous injection in the abdomen, once every 2 weeks for 3 consecutive times. The antibody titer of mouse serum was determined by indirect ELISA method, and the mouse with the highest titer was selected for hybridoma cell fusion.

[0031] S2. Fusion of hybridoma cells: The mouse with the highest antibody titer obtained in step S1 was sacrificed, and the spleen cells were made into a spleen cell suspension in a laminar flow hood. The spleen cell suspension was mixed with myeloma cells at a cell number ratio of 10:1, and 50% polyethylene glycol was used as the fusogen, and fused in a 37 °C water bath for 2 minutes. The fused cells were cultured in HAT selection medium. After one week, the cell culture supernatant was collected, and positive hybridoma cells were screened by ELISA method. The selected positive hybridoma cells were subcloned 3 times by the limiting dilution method, and the monoclonal cells with the strongest positive reaction were selected for expansion culture.

[0032] S3. Preparation and purification of monoclonal antibody: Liquid paraffin was inoculated into the mouse peritoneal cavity at 0.5 mL / mouse. After 7 days, 8×10 6 subcloned hybridoma cells were injected into the mouse peritoneal cavity, and the production of ascites in the mouse abdomen was observed every day. After 8 days, the mouse ascites was collected. The collected mouse ascites was centrifuged at 3,000 rpm for 15 min to remove cell components, filtered through a 0.2 μm filter, and the collected ascites from different mice was purified by immunochromatography to obtain the purified monoclonal antibody.

[0033] S4. Analysis of the sequence structure of monoclonal antibodies: Use Biacore T200 to accurately measure the activity of the purified monoclonal antibodies. Name the two monoclonal antibodies with the highest activity as LG3A-1 and FLSP4, and determine their variable region sequences. Monoclonal antibody LG3A-1 and monoclonal antibody FLSP4 each include a light chain and a heavy chain; among them, the amino acid sequence of the heavy chain of monoclonal antibody LG3A-1 is shown in SEQ ID NO.1, and the amino acid sequence of the light chain is shown in SEQ ID NO.2; the amino acid sequence of the heavy chain of monoclonal antibody FLSP4 is shown in SEQ ID NO.3, and the amino acid sequence of the light chain is shown in SEQ ID NO.4.

[0034] Table 1 Sequence Listing

[0035]

[0036] Example 2

[0037] Preparation of Giardia detection kit:

[0038] (1) Select the monoclonal antibody LG3A-1 prepared in Example 1 as the capture antibody. Use an ELISA 96-well plate as the solid-phase carrier. Dilute the capture antibody with coating buffer (carbonate buffer, 0.05 mol / L) to 2 μg / mL to obtain the coating solution. Add 100 μL of the coating solution to the ELISA plate, seal the plate with a sealing film, and incubate overnight at 4°C. Discard the coating solution, wash 3 times with ELISA washing solution, add 200 μL of 1% BSA blocking solution to each well, block at room temperature for 3 h, discard the liquid in the wells, pat dry, seal, and store at 4°C to obtain the ELISA plate coated with the capture antibody.

[0039] (2) Select the monoclonal antibody FLSP4 prepared in Example 1 as the labeled antibody. Dialyze the labeled antibody FLSP4 in 60 mM carbonate buffer for 24 h, changing the solution twice during this period, and adjust its concentration to 2 mg / mL. Weigh 5 mg of HRP dry powder and dissolve it in 1 mL of distilled water. Weigh 21 mg of NaIO 4 , dissolve it in 2 mL of distilled water to prepare a 0.1 M NaIO 4 solution. Take 0.2 mL of the NaIO 4 solution and mix it with 1 mL of the HRP solution, stir gently in the dark at room temperature for 20 min, and dialyze overnight in 10 mM NaAc buffer at 4°C. Add an appropriate amount of 0.2 M carbonate buffer to the above-mentioned aldehyde-activated HRP solution to raise its pH to 9.2, then add an equal volume of the monoclonal antibody FLSP4, and gently stir in the dark at room temperature for 2 hours. Measure 0.1 mL of 5 mg / L NaBH 4The solution was added to the reaction solution of the previous step and mixed well, and the mixture was reacted at 4°C for 3 hours to obtain a stable enzyme-labeled antibody. The above sample was placed in a dialysis bag and dialyzed overnight at 4°C, and the precipitate was removed by centrifugation for 30 min. The supernatant was the enzyme conjugate, and the HRP-labeled antibody working solution was obtained.

[0040] (3)The HRP-labeled antibody working solution prepared in step (2), the antigen standard Giardia antigen, the washing solution PBST, the chromogenic solution TMB, and the termination solution sulfuric acid were respectively packaged and constituted the kit of the present invention together with the enzyme-labeled plate coated with the capture antibody prepared in step (1).

[0041] Example 3

[0042] Drawing of the standard curve:

[0043] (1)Take out the enzyme-labeled plate coated with the capture antibody, restore it to room temperature, wash the plate 3 times with PBST washing solution and shake dry; add 100 μL of Giardia antigen with different dilution factors (100 ng / mL, 80 ng / mL, 60 ng / mL, 40 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL), incubate at 37°C for 1 h, and wash 3 times with PBST washing solution and shake dry.

[0044] (2)Add 100 μL of the HRP-labeled antibody working solution to the reaction wells, incubate at 37°C for 1 h, and wash 3 times with PBST washing solution and shake dry.

[0045] (3)Add 100 μL of 2 mg / L TMB chromogenic solution to the reaction wells, develop color at room temperature in the dark for 10 min, and add 100 μL of 2 M sulfuric acid to terminate the reaction.

[0046] (4)Use an enzyme-labeled instrument to perform double-wavelength detection, measure the OD values at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm, and subtract the OD value at 630 nm from the OD value measured at 450 nm. The standard curve was drawn with the standard product concentration as the abscissa (ng / mL) and OD450 - OD630 as the ordinate. The results are as Figure 1 shown. The detection range of the kit for microbial detection of the present invention is 10 - 100 ng / mL, and R 2 = 0.9982.

[0047] Test Example 1

[0048] Specificity of the kit:

[0049] Common intestinal microorganisms in feces, such as Giardia lamblia, Staphylococcus aureus, Clostridium, Salmonella, and Lactobacillus, were cultured to collect the bacterial cells. The cell pellet was resuspended in sterile PBS solution, frozen and thawed three times repeatedly, and then sonicated in an ice bath for 30 min. After centrifugation at 12,000 rpm for 20 min, the supernatant was taken to prepare the intestinal microorganism antigen. The test was carried out using the kit prepared in Example 2.

[0050] Twenty minutes before the experiment, the enzyme-linked immunosorbent assay (ELISA) plate coated with the capture antibody was taken out and allowed to return to room temperature. The plate was washed three times with PBST washing solution and then drained. 100 μL of the sample to be tested was added and incubated at 37°C for 1 h. The plate was washed three times with PBST washing solution and then drained. 100 μL of the HRP-labeled antibody working solution was added to the reaction wells and incubated at 37°C for 1 h. The plate was washed three times with PBST washing solution and then drained. 100 μL of 2 mg / L TMB chromogenic solution was added to the reaction wells and the color was developed in the dark at room temperature for 10 min. 100 μL of 2 M sulfuric acid was added to terminate the reaction. A microplate reader was used for double-wavelength detection to measure the OD values at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm. The OD value measured at 450 nm was subtracted from the OD value measured at 630 nm, and the result was calculated through the standard curve. If the detected concentration was within the detection range of the kit, 10 - 100 ng / mL, it was positive (+); otherwise, it was negative (-). The results are shown in Table 2. This antibody did not react with other similar proteins, and the results showed no cross-reaction, indicating that the kit provided by the present invention has high specificity.

[0051] Table 2 Sample test results

[0052]

[0053] Test Example 2

[0054] Detection of Giardia lamblia in humans:

[0055] 1 g of fecal samples were taken from 16 volunteers with diarrhea within one week, diluted with 20 mL of sterile PBS solution, and mixed well with an oscillator. After centrifugation at 2000 g for 10 min at room temperature, the supernatant was discarded, and the above operation was repeated three times. After freezing and thawing three times repeatedly, it was sonicated in an ice bath for 30 min. After centrifugation at 12,000 rpm for 20 min, the supernatant was taken to obtain the sample. The samples were denoted as fecal samples 1 - 16. Normal saline was used as the negative control, and Giardia lamblia antigen at 10 ng / mL was used as the positive control. The detection method was the same as that in Test Example 1, and the results are shown in Table 3.

[0056] Table 3 Clinical sample test results

[0057]

[0058] As shown in Table 3, among the 16 volunteers, the fecal samples of 10 volunteers were detected to be positive for Giardia antigen, and the detected concentrations were all within the linear range of the kit of the present invention, indicating that the detection method established by the present invention can use feces as a sample to complete the detection of Giardia infection, and it has relatively strong specificity and high sensitivity.

[0059] Test Example 3

[0060] Stability of the Giardia detection kit:

[0061] Take the Giardia antigen standard products (10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL) as the samples to be detected. Place the enzyme-labeled plate coated with the capture antibody and the working solution of the HRP-labeled antibody at 37°C to test their stability for 20 days, and perform antigen standard product detection every 10 days. Place the enzyme-labeled plate coated with the capture antibody and the working solution of the HRP-labeled antibody in a 4°C refrigerator for storage for 2 years, and take them out for antigen standard product detection every 12 months. The detection method is the same as that in Test Example 1, and the results are shown in Table 4.

[0062] Table 4 Test results of clinical samples

[0063]

[0064] The results in Table 4 show that: when the kit is exposed at 37°C for 20 days and stored at low temperature for 2 years, the OD value gradient of the standard curve is good, and the detected concentration of the Giardia antigen standard product is also relatively stable, indicating that the kit has good stability and can meet the needs of basic research and clinical diagnosis.

[0065] The above embodiments are only the preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantive changes and substitutions made by those skilled in the art based on the present invention belong to the scope of protection required by the present invention.

Claims

1. A microbial detection kit, characterized in that: The kit comprises an ELISA plate coated with a capture antibody and an HRP-labeled antibody working solution; the capture antibody and the HRP-labeled antibody are both antibodies that specifically bind to human Giardia antigens; The capture antibody is a monoclonal antibody LG3A-1 that specifically binds to human Giardia 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 FLSP4 that specifically binds to human Giardia 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. A microorganism detection kit according to claim 1, characterized in that: The antibody specifically binding to human Giardia antigen is prepared by the following method: (1) Human Giardia lamblia was isolated from feces, and human Giardia lamblia antigen was prepared by repeated freezing and thawing, ultrasonic disruption and centrifugation; (2) immunizing mice with the human Giardia antigen prepared in step (1), and fusing spleen cells of the immunized mice with myeloma cells to form hybridoma cells; (3) ELISA was used to screen hybridoma cells that secreted human Giardia antibodies. The positive cell lines were subcloned and expanded, and then inoculated into mice. The ascites was collected and purified to obtain monoclonal antibodies LG3A-1 and FLSP4.

3. A microorganism detection kit according to claim 1, characterized in that: The microorganism detection kit also includes an antigen standard, a washing solution, a color developing solution and a stop solution.

4. A microorganism detection kit according to claim 3, characterized in that: The antigen standard is human Giardia antigen; the washing solution is PBST; the color developing solution is TMB; and the stop solution is sulfuric acid.

5. A microorganism detection kit according to claim 4, characterized in that: The concentration of the color developing solution is 2mg / L-3mg / L; the concentration of the stop solution is 2M-5M.

6. A microorganism detection kit according to claim 1, characterized in that: The capture antibody coating concentration in the capture antibody-coated ELISA plate is 100 μL / well and the capture antibody concentration is 2 μg / mL.

7. A microorganism detection kit according to claim 1, characterized in that: The preparation method of the HRP labeled antibody working solution is as follows: adding the monoclonal antibody FLSP4 to the HRP solution oxidized by NaIO4, reacting at room temperature in the dark, adding NaBH4 and continuing the reaction at 4°C for a period of time, and dialyzing against PBS overnight.

8. A microorganism detection kit according to claim 4, characterized in that: The method of using the kit is as follows: (1) Centrifuge the sample to be tested and take the supernatant; (2) Take out the ELISA plate coated with the capture antibody, return it to room temperature, wash the plate with a washing solution and spin dry, add the supernatant of the sample to be tested in step (1), incubate at room temperature, and then wash the plate with a washing solution; (3) Add the HRP-labeled antibody working solution to the system in step (2), incubate at room temperature for 1-3 hours, and then wash the plate with washing solution; (4) Adding a color developing solution to the system after the reaction in step (3), developing the color at room temperature in the dark for 10-30 min, and then adding a stop solution to terminate the reaction; (5) Use an 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 results using the standard curve.

9. A microorganism detection kit according to claim 8, characterized in that: The sample to be detected is feces.

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