Microorganism detection kit and detection method

Abstract

The invention belongs to the technical field of in-vitro diagnosis, and discloses a microbiological detection kit and a detection method. The kit comprises an elisa plate coated with a captured antibody and an HRP labeled antibody working solution, the capture antibody is a monoclonal antibody LG3A-1 specifically combined with a human giardia antigen, the heavy chain amino acid sequence of the capture antibody is as shown in SEQ ID NO.1, and the light chain amino acid sequence of the capture antibody is as shown in SEQ ID NO.2; the HRP labeled antibody is a monoclonal antibody FLSP4 specifically combined with human giardia antigen, the heavy chain amino acid sequence of the monoclonal antibody FLSP4 is as shown in SEQ ID NO.3, and the light chain amino acid sequence of the monoclonal antibody FLSP4 is as shown in SEQ ID NO.4. The microbiological detection kit provided by the invention can be used for detecting the content of giardia in human excrement, is high in specificity, high in sensitivity, good in stability and simple to operate, and has a very good application prospect.

Description

Technical Field

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

[0002] Giardia, also known as Giardia, is a single-cell protozoan widely distributed in the small intestine of humans and mammals. It can cause intestinal diseases with diarrhea as the main symptom. The symptoms of giardiasis vary in severity, with an incubation period of about 1 to 2 weeks and up to 45 days. Some people infected with giardiasis may not show any clinical symptoms and become carriers; those with symptoms are mainly manifested by diarrhea and malabsorption. The typical symptoms in the acute phase are explosive watery diarrhea, foul odor, and large stool volume, which may be accompanied by abdominal distension, abdominal pain, belching, vomiting and other symptoms. No pus or blood can be seen in the feces of patients with giardiasis, but pus cells, red blood cells and trophozoites or cysts can be found under a microscope. If giardiasis is not treated in time, it will 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 who are not cured for a long time will suffer from malnutrition, anemia, developmental disorders, etc.

[0003] At present, giardiasis infection is common in countries around the world, and it faces many challenges in diagnosis and prevention and control, which has attracted widespread attention and high attention from the international community. The key to reducing Giardia infection is early diagnosis and early treatment. The current detection technologies for Giardiasis include morphological examination, nucleic acid detection and other methods. The advantages of morphological examination are rapid, simple and direct, but it is difficult to observe when the number of pathogens is small; nucleic acid detection procedures are cumbersome, and there are many specimen pretreatment steps. It is limited to laboratory operations due to primer design, nucleic acid purification, reaction conditions, etc., and is susceptible to laboratory contamination and false positives. Sample processing and testing must be carried out in an independent space; in order to detect Giardia more quickly, simply and accurately, it is necessary to continuously explore new diagnostic methods and technologies to meet clinical needs.

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

[0005] In order to overcome the shortcomings of the prior art, the first aspect of the present invention is to provide a microorganism detection kit, which includes an enzyme-labeled 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 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 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.

[0006] Preferably, the antibody that specifically binds 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.

[0007] Preferably, the microorganism detection kit further comprises an antigen standard, a washing solution, a color developing solution and a stop solution.

[0008] Preferably, 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.

[0009] Preferably, the concentration of the developing solution is 2 mg / L-3 mg / L; the concentration of the stop solution is 2M-5M.

[0010] Preferably, the capture antibody coating concentration in the ELISA plate coated with the capture antibody is 100 μL / well and the capture antibody concentration is 2 μg / mL.

[0011] Preferably, the preparation method of the HRP labeled antibody working solution is: 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.

[0012] The second aspect of the present invention provides a detection method of the above-mentioned microorganism detection kit, wherein the detection method is: (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), coloring 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 values ​​at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm, and calculate the results using the standard curve.

[0013] Preferably, the sample to be detected is feces.

[0014] The present invention has the following effects compared with the prior art: 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 giardiasis in the tested population, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The standard curve of the kit is used for specific detection of Giardia content. DETAILED DESCRIPTION

[0016] The technical scheme of the present invention is further described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0017] Example 1 (1) Preparation of Giardia antigen: Human Giardia was isolated from feces and cultured in DMEM medium. One week later, human Giardia was resuspended in sterile PBS solution, frozen and thawed three times, ultrasonically broken in an ice bath for 30 min, centrifuged at 12000 rpm for 20 min, and the supernatant was collected to prepare human Giardia antigen. After filtering through a 0.22 μM filter membrane, the concentration of Giardia antigen was determined by the BCA method, and the concentration of Giardia antigen was adjusted to 10 μg / mL and stored at -80°C for later use.

[0018] (2) Preparation of Giardia monoclonal antibodies: S1. Immunization of experimental animals: The prepared Giardia antigen was injected subcutaneously at multiple points in the abdomen to immunize female mice, 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.

[0019] S2. Fusion of hybridoma cells: The mouse with the highest antibody titer obtained in step S1 was killed, and the mouse spleen was taken in a clean bench to prepare a spleen cell suspension. The spleen cell suspension was mixed with myeloma cells at a cell number ratio of 10:1, 50% polyethylene glycol was used as a fusion agent, and fused in a 37°C water bath for 2 minutes. The fused cells were cultured in HAT selection medium, and the cell culture supernatant was collected after one week. The positive hybridoma cells were screened by ELISA, and the screened positive hybridoma cells were subcloned three times by limiting dilution method, and the monoclonal cells with the strongest positive reaction were selected for expansion culture.

[0020] S3. Preparation and purification of monoclonal antibodies: Liquid paraffin was inoculated into the peritoneal cavity of mice at a rate of 0.5 mL / mouse. After 7 days, 8×10 6 The subcloned hybridoma cells were injected into the peritoneum of mice, and the production of abdominal ascites in the mice was observed every day. The ascites of the mice was collected after 8 days. The collected mouse ascites was centrifuged at 3000 rpm for 15 min to remove the cellular components, and filtered with a 0.2μm filter. The collected ascites of different mice were purified by immunochromatography to obtain purified monoclonal antibodies.

[0021] S4. Analysis of monoclonal antibody sequence structure: The activity of purified monoclonal antibodies was accurately measured using Biacore T200, and the two most active monoclonal antibodies were named LG3A-1 and FLSP4, and their variable region sequences were determined. Monoclonal antibody LG3A-1 and monoclonal antibody FLSP4 include light chains and heavy chains, respectively; the heavy chain amino acid sequence of monoclonal antibody LG3A-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 FLSP4 is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4.

[0022] Table 1 Sequence Listing Example 2 Preparation of Giardia lamblia detection kit: (1) The monoclonal antibody LG3A-1 prepared in Example 1 was selected as the capture antibody. An enzyme-labeled 96-well plate was used as a solid phase carrier, and the capture antibody was diluted to 2 μg / mL with a coating buffer (carbonate buffer, 0.05 mol / L) to obtain a coating solution. 100 μL of the coating solution was added to the ELISA plate, and the plate was sealed with a sealing film and placed at 4°C for overnight coating. The coating solution was discarded, and the plate was washed three times with ELISA washing solution. 200 μL of 1% BSA blocking solution was added to each well, and the plate was blocked at room temperature for 3 hours. The liquid in the plate wells was discarded, the plate was patted dry, sealed, and stored at 4°C to obtain an ELISA plate coated with the capture antibody.

[0023] (2) The monoclonal antibody FLSP4 prepared in Example 1 was selected as the labeled antibody. The labeled antibody FLSP4 was dialyzed in 60 mM carbonate buffer for 24 h, during which the solution was changed twice to adjust its concentration to 2 mg / mL. 5 mg of HRP dry powder was weighed and dissolved in 1 mL of distilled water. 21 mg of NaIO4 was weighed and dissolved in 2 mL of distilled water to prepare a 0.1 M NaIO4 solution. 0.2 mL of NaIO4 solution was mixed with 1 mL of HRP solution, stirred at room temperature in the dark for 20 min, and dialyzed overnight in 10 mM NaAc buffer at 4°C. An appropriate amount of 0.2 M carbonate buffer was added to the above-mentioned aldehyded HRP solution to raise its pH to 9.2, and then an equal volume of monoclonal antibody FLSP4 was added and gently stirred at room temperature in the dark for 2 hours. Measure 0.1 mL of 5 mg / L NaBH4 solution, add it all to the reaction solution in the previous step and mix well. Place it at 4°C for 3 hours to obtain a stable enzyme-labeled antibody. Put the above sample into a dialysis bag and dialyze it at 4°C overnight. Centrifuge for 30 minutes to remove the precipitate. The supernatant is the enzyme conjugate, and the HRP-labeled antibody working solution is obtained.

[0024] (3) The HRP-labeled antibody working solution, antigen standard human Giardia antigen, washing solution PBST, color development solution TMB, and stop solution sulfuric acid prepared in step (2) are packaged separately and combined with the ELISA plate coated with the capture antibody prepared in step (1) to form the kit of the present invention.

[0025] Example 3 Drawing of standard curve: (1) Remove the ELISA plate coated with the capture antibody, return to room temperature, wash the plate three times with PBST washing solution and spin dry; add 100 μL of different dilutions of Giardia antigen (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, wash three times with PBST washing solution and spin dry.

[0026] (2) Add 100 μL of HRP-labeled antibody working solution to the reaction wells, incubate at 37°C for 1 h, wash three times with PBST washing solution and spin dry.

[0027] (3) Add 100 μL of 2 mg / L TMB colorimetric solution to the reaction wells, develop the color for 10 min at room temperature in the dark, and then add 100 μL of 2 M sulfuric acid to terminate the reaction.

[0028] (4) 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 subtract the OD value at 630 nm from the OD value at 450 nm. Draw a standard curve with the concentration of the standard as the horizontal axis (ng / mL) and OD450 -OD630 as the vertical axis. The results are as follows: Figure 1 As shown, the detection range of the kit for microbial detection of the present invention is 10-100 ng / mL, R 2 =0.9982.

[0029] Test Example 1 Specificity of the kit: Common intestinal microorganisms in feces, such as Giardia, Staphylococcus aureus, Clostridium, Salmonella and lactic acid bacteria, were cultured and collected, and the bacterial precipitate was resuspended in a sterile PBS solution, and then repeatedly frozen and thawed for 3 times, and then ultrasonically broken in an ice bath for 30 min, and the supernatant was collected after centrifugation at 12000 rpm for 20 min to prepare intestinal microbial antigens. The kit prepared in Example 2 was used for testing.

[0030] 20 min before the experiment, remove the ELISA plate coated with the capture antibody, return to room temperature, wash the plate three times with PBST washing solution and spin dry. Add 100 μL of the sample to be tested, incubate at 37°C for 1 h, wash three times with PBST washing solution and spin dry. Add 100 μL of HRP-labeled antibody working solution to the reaction well, incubate at 37°C for 1 h, wash three times with PBST washing solution and spin dry. Add 100 μL of 2 mg / L TMB color development solution to the reaction well, color at room temperature in the dark for 10 min, and add 100 μL of 2M sulfuric acid to terminate the reaction. Use an ELISA reader for dual wavelength detection, measure the OD value 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 at 450 nm. Calculate the result through the standard curve. If the detected concentration is within the detection range of the kit 10-100 ng / mL, it is positive (+), otherwise it is negative (-). The results are shown in Table 2. The antibody does not react with other similar proteins, and the results show no cross-reaction, indicating that the kit provided by the present invention has high specificity.

[0031] Table 2 Sample test results Test Example 2 Human Giardia Detection: Take 1 g of fecal samples from 16 volunteers who have experienced diarrhea within a week, dilute with 20 mL of sterile PBS solution, shake on an oscillator to mix, centrifuge at 2000 g for 10 min at room temperature, discard the supernatant, and repeat the above operation three times. After repeated freezing and thawing 3 times, ultrasonically break the sample in an ice bath for 30 min, centrifuge at 12000 rpm for 20 min, and take the supernatant to obtain the sample. The samples are recorded as fecal samples 1-16. Take physiological saline as the negative control, and 10 ng / mL of Giardia antigen as the positive control. The detection method is the same as that of Experiment 1, and the results are shown in Table 3.

[0032] Table 3 Clinical sample test results As shown in Table 3, among the 16 volunteers, the stool samples of 10 volunteers tested positive for human Giardia antigen, and the detection 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 stool as a sample to complete Giardia infection detection, and its specificity is relatively strong and sensitivity is relatively high.

[0033] Test Example 3 Stability of Giardia Detection Kit: Take Giardia antigen standards (10ng / mL, 25ng / mL, 50ng / mL, 100ng / mL) as samples to be tested. Place the enzyme-labeled plate coated with capture antibody and the HRP-labeled antibody working solution at 37°C to test their stability for 20 days, and perform antigen standard testing every 10 days. Store the enzyme-labeled plate coated with capture antibody and the HRP-labeled antibody working solution in a 4°C refrigerator for 2 years, and take out the antigen standard testing every 12 months. The detection method is the same as that of Test Example 1, and the results are shown in Table 4.

[0034] Table 4 Clinical sample test results The results in Table 4 show that when the kit is exposed to 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 detection concentration of the Giardia antigen standard is relatively stable, indicating that the kit has good stability and can meet the needs of basic research and clinical diagnosis.

[0035] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within 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. The detection method of a microorganism detection kit according to any one of claims 1 to 7, characterized in that: The detection method is: (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), coloring 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 values ​​at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm, and calculate the results using the standard curve.

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

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