Fecal antigen double antibody sandwich sa-elisa test kit for fasciola hepatica infection

Abstract

This invention provides a double-antibody sandwich SA-ELISA detection kit for Fasciola hepatica infection in sheep. A double-antibody sandwich SA-ELISA method for detecting Fasciola hepatica CAg (cag) is established using mouse anti-Fasciola hepatica D38 monoclonal antibody and rabbit anti-Fasciola hepatica D38 polyclonal antibody. This detection uses monoclonal antibodies as coating antibodies, resulting in high specificity. Furthermore, the introduction of a biotin-streptavidin signal amplification system enhances sensitivity. It offers advantages such as high accuracy, good stability, simple operation, rapid detection, and easy interpretation, making it suitable for rapid clinical diagnosis and epidemiological surveys at the grassroots level.

Description

Technical Field

[0001] This invention relates to a method for detecting circulating antigen (CAg) D38 protein in sheep liver fluke infection, and more specifically, to providing a double-antibody sandwich biotin-streptavidin signal amplification ELISA (streptavidin-ELISA / SA-ELISA) detection kit for sheep liver fluke infection. This invention also discloses the preparation method of the kit, belonging to the field of ruminant parasite infection detection technology. Background Technology

[0002] Liver fluke ( Fasciola hepatica Fasciola hepatica is a zoonotic worm that parasitizes the bile ducts of its host. It has a worldwide distribution and primarily affects ruminants such as cattle and sheep. Globally, over 700 million livestock are infected with Fasciola hepatica, resulting in annual economic losses exceeding US$3.2 billion to the livestock industry and severely impacting its development. Currently, control of Fasciola hepatica infection mainly relies on chemical drugs such as triclofenac, but drug resistance is easily developed, and there is currently no effective vaccine available. Therefore, timely detection of Fasciola hepatica-infected animals and the implementation of control measures are of paramount importance.

[0003] Compared to fecal examination and molecular biological detection methods, immunological detection methods have certain advantages due to their high sensitivity, ease of operation, and suitability for batch testing. The basis of immunological detection is the detection of antigens or antibodies. Current methods for detecting liver fluke infection mainly rely on antibody detection; however, antibody detection cannot distinguish between current and past infections, while detecting circulating antigen (CAg) can confirm current infection. Therefore, detection based on circulating antigen in liver fluke infection has certain diagnostic advantages. Currently, there is no ideal commercially available method in China for detecting circulating antigen in sheep liver fluke infection. Summary of the Invention

[0004] This invention discloses the nucleic acid sequence of the liver fluke-specific gene D38 (GenBank: THD28786.1) and a double-antibody sandwich SA-ELISA detection kit for sheep liver fluke infection circulating antigen (CAg) based on the D38 gene, which is used to detect whether sheep are infected with liver fluke, and solves the problem that there is currently no ideal method for detecting sheep liver fluke infection circulating antigen.

[0005] The characteristics of the D38 gene are as follows: The D38 gene coding sequence (CDS) is 633 bp long, encoding 210 amino acids, with a relative molecular mass of approximately 42 kDa and a theoretical isoelectric point of 10.03. The encoded protein has no signal peptide, no transmembrane region, and multiple B cell antigenic epitopes, among which 6 dominant antigenic epitopes are located at amino acid positions 23-33, 68-74, 87-113, 129-136, 143-150, and 202-209, respectively.

[0006] This invention also provides a method for preparing a double-antibody sandwich SA-ELISA detection kit for sheep liver fluke, which is suitable for industrial production.

[0007] Polystyrene plastic reaction plates, coating solution, blocking solution, washing solution, capture antibody, positive and negative reference sera for sheep liver fluke infection, biotin-labeled detection antibody, enzyme-labeled avidin solution, substrate solution, stop solution, and sample dilution solution, etc.

[0008] The capture antibody is a mouse monoclonal antibody against Fasciola hepatica D38 (Anti-Fh-D38-mAb) (titer 204800, subtype G2a).

[0009] The detection antibody was a biotin-labeled polyclonal antibody against Fasciola hepatica D38 (Bio-Anti-D38-pAb) (titer 512000).

[0010] The coating solution was a 0.05 M carbonate buffer solution with a pH of 9.6;

[0011] The sample diluent (PBS) and wash buffer (PBST) are phosphate buffers containing 0.5% Tween-20;

[0012] The blocking solution was a PBST solution containing 1% bovine serum albumin;

[0013] The substrate chromogenic solution is a TMB substrate solution;

[0014] The stop solution is a 2 mol / L H2SO4 solution.

[0015] Mouse anti-D38 monoclonal antibody and rabbit anti-D38 polyclonal antibody were prepared, and a double-antibody sandwich SA-ELISA method was established to detect Fasciolopsis buski (CAg) in sheep serum. This method overcomes the shortcomings of current methods for detecting CAg in sheep serum, such as low specificity and sensitivity, and high requirements for personnel and equipment, providing a new approach for detecting early infection of Fasciolopsis buski in sheep.

[0016] The specific steps are as follows: Using anti-fascicola D38 monoclonal antibody (capture antibody) as the coating antibody, coat the ELISA plate and wash away the free portion; block with blocking buffer and wash away the free portion; then add the test serum. If the serum contains Fascicola CAg, it will specifically bind to the coating antibody to form an immune complex, washing away the free test serum; add biotin-labeled anti-fascicola D38 polyclonal antibody (detection antibody), thus forming an anti-fascicola D38 monoclonal antibody-fascicola CAg-biotin-coated plate. Biotin-labeled anti-fascicola D38 polyclonal antibody complex is prepared by washing away the free biotin label; avidin is added to amplify the reaction signal and wash away the free portion; a chromogenic substrate is added, and the enzyme label will cause the colorless chromogenic agent to turn yellow after the stop solution is added; if the serum being tested does not contain Fascicola CAg, the anti-fascicola D38 monoclonal antibody-Fascicola CAg-biotin-labeled anti-fascicola D38 polyclonal antibody complex cannot be formed, and the enzyme label cannot be immobilized on the ELISA plate, remaining colorless after the addition of the chromogenic substrate and stop solution.

[0017] The absorbance value (OD) at a wavelength of 450 nm was measured using an ELISA reader. 450nm (value), the intensity of the color after the reaction is terminated, OD 450nm The value is positively correlated with the amount of sheep liver fluke CAg in the corresponding serum sample to be tested.

[0018] D38 is a novel Fasciola hepatica-specific gene. A double-antibody sandwich SA-ELISA method for detecting Fasciola hepatica CAg in sheep was established using mouse anti-Fasciola hepatica D38 monoclonal antibody and rabbit anti-Fasciola hepatica D38 polyclonal antibody. This assay uses monoclonal antibodies as coating antibodies, resulting in high specificity. Furthermore, the introduction of a biotin-streptavidin signal amplification system enhances sensitivity. It offers advantages such as high accuracy, good stability, simple operation, rapid detection, and easy interpretation, making it suitable for rapid clinical diagnosis and epidemiological surveys at the grassroots level.

[0019] All reagents in the kit of this invention are pre-prepared working solutions that can be directly used without processing, reducing operational steps and enabling rapid and sensitive detection of CAg in sheep serum. This avoids complex operations and has low equipment requirements. Furthermore, the kit requires small sample volumes, has long reagent shelf life, and is non-toxic to operators. Therefore, the double-antibody sandwich SA-ELISA method for detecting CAg in sheep liver fluke, established using anti-liver fluke D38 monoclonal and polyclonal antibodies, has good market prospects. Attached Figure Description

[0020] Figure 1 Determining the optimal sample dilution;

[0021] Figure 2 Determining the optimal sealing agent;

[0022] Figure 3 Sensitivity testing;

[0023] Figure 4 Specificity test. Detailed Implementation

[0024] The present invention will be further described with reference to the following embodiments, but the content of the present invention is not limited to the following embodiments. Example

[0025] Purified recombinant protein D38 was mixed with Freund's adjuvant and thoroughly emulsified. BALB / c mice were immunized four times, and mice with high serum antibody titers were selected for subsequent experiments. Mouse spleen cells were fused with SP2 / 0 cells using polyethylene glycol (PEG). After fusion, the cells were cultured in a semi-solid medium for initial screening, and hybridoma cell lines were screened a second and third time using indirect ELISA. Culture media of positive hybridoma cells producing high antibody titers against rFgD38 were collected, and immunoglobulin subclasses were identified using the SBA Clonotyping™ System / HRP kit. After preliminary identification, monoclonal antibodies were generated in ascites fluid. Monoclonal antibodies were purified using Protein A / G affinity chromatography resin, and the concentration of monoclonal antibodies was determined using a BCA protein assay kit. Monoclonal antibody titers were measured using indirect ELISA. A fitted reaction curve model was constructed, and the affinity constant was calculated. SDS-PAGE was used to determine the purity of the monoclonal antibodies, and Western blotting was used to observe the reactivity of the mouse monoclonal antibodies. Example

[0026] Purified recombinant protein D38 was mixed with Freund's adjuvant and thoroughly emulsified, and then used to immunize 2-month-old New Zealand rabbits three times. Seven days after the third immunization, blood was collected from the marginal ear vein of the rabbits, and serum was collected. The antibody titer in the rabbit serum was determined using an indirect ELISA method. If the serum antibody titer met the target, blood was collected from the heart, and a large amount of serum was collected. The polyclonal antibody was crudely purified using the saturated ammonium sulfate method, and then further purified using Protein A / G mixed column purification resin. SDS-PAGE was used to analyze the antibody purification effect, and Western blot was used to observe the reactivity of the rabbit polyclonal antibody. Example

[0027] Anti-liver fluke D38 polyclonal antibody was labeled according to the biotin-N-succinimide instructions. The steps are as follows:

[0028] (1) Take out the biotin reagent from the refrigerator and equilibrate to room temperature.

[0029] (2) Weigh 2 mg of biotin and dissolve it in 590 μL of dimethyl sulfoxide to form a 10 mM biotin solution.

[0030] (3) Add an appropriate amount of biotin solution to the purified anti-D38 rabbit polyclonal antibody solution and incubate on ice for 2 hours. The molar ratio of polyclonal antibody to BNHS is 1:20, 1:40, 1:80, and 1:160.

[0031] (4) Add the reacted solution into an ultrafiltration tube and centrifuge at 4000 r / min for 10 min to remove unbound biotin.

[0032] (5) Transfer the liquid in the upper layer of the ultrafiltration tube to a brown EP tube and store it at -20°C in the dark.

[0033] Example 4:

[0034] Select the optimal blocking agent. Dilute the capture antibody and sheep liver fluke reference positive serum to the optimal working concentration. Add biotin-labeled rabbit anti-FhD38 protein polyclonal detection antibody with a molar ratio of polyclonal antibody to biotin of 1:20, 1:40, 1:80, and 1:160 to the ELISA plate sequentially. Add enzyme-labeled avidin and substrate reaction solution for color development and measure OD. 450 Based on the nm value, the optimal molar ratio of polyclonal antibodies to biotin was determined to be 1:40. Example

[0035] 1. Determination of working concentrations for capture and detection antibodies

[0036] Using a checkerboard array method, different concentrations of capture antibody (30 μg / mL, 15 μg / mL, 7.5 μg / mL, 3.75 μg / mL, and 1.87 μg / mL) were used to coat ELISA plates. Blocking agent and reference serum samples were added, and the plates were incubated with detection antibody at different dilutions (1:1000, 1:2000, 1:4000, and 1:8000) for double-antibody sandwich SA-ELISA to determine the optimal working concentrations of the capture and detection antibodies. The optimal coating concentration of the capture antibody was 3.75 μg / mL, and the optimal dilution of the detection antibody was 2000-fold (see Table 1).

[0037]

[0038] 2. Determination of the optimal dilution of the sample

[0039] Under optimized conditions, serial dilutions of liver fluke positive and negative reference serum samples were performed (1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128) to determine the optimal sample dilution. A 4-fold dilution of serum was selected as the optimal sample dilution (see Appendix). Figure 1 ).

[0040] 3. Selection of the best sealant

[0041] At the determined optimal dilution concentrations of capture and detection antibodies, four blocking agents (including 1% gelatin, 10% rabbit serum, 1% BSA, and 5% skim milk powder) were used for blocking. It was found that FhD38 showed higher P / N values ​​when blocked with 1% BSA and 10% rabbit serum. Since commercially available 1% BSA reagent is more readily available, 1% BSA was ultimately determined to be the optimal blocking agent (see appendix). Figure 2 ).

[0042] 4. Determination of the cutoff value for double-antibody sandwich SA-ELISA

[0043] Twenty negative serum samples were tested using the established double-antibody sandwich SA-ELISA method. The X value for the 20 negative serum samples was calculated to be 0.121, and the SD value was 0.0078. The cutoff value for positive and negative results was X + 3SD = 0.145. To avoid false positive results, a doubtful interval was defined by adding or subtracting one standard deviation from the cutoff value.

[0044] Based on statistical principles, the criteria for judging the effectiveness of the double-antibody sandwich SA-ELISA method were derived, including OD. 450nm A value ≥0.153 is considered positive. 450nm Values ​​< 0.137 are considered negative, while values ​​in between, i.e., 0.137 < X ≤ 0.153, are considered suspicious.

[0045] 5. Performance Measurement

[0046] (1) Sensitivity test

[0047] The established double-antibody sandwich SA-ELISA method was used to determine the sensitivity of sheep liver fluke reference positive serum by serial dilutions from 1:2 to 1:128. The sensitivity of this method was 1:64 (see Appendix). Figure 3 ).

[0048] (2) Specificity test

[0049] Using sheep negative serum as a reference, the double-antibody sandwich SA-ELISA method showed no cross-reactivity between sheep liver fluke reference positive serum and sheep schistosome japonicus positive serum, sheep haemophora contorta positive serum, sheep dicocephalus positive serum, and sheep neosporidium positive serum, thus confirming the specificity of this method (see Appendix). Figure 4 The method has been shown to be highly specific.

[0050] (3) Repeatability test

[0051] Twelve sheep serum samples were selected and subjected to batch-to-batch and batch-to-batch repeatability tests using the established double-antibody sandwich SA-ELISA method. The maximum coefficient of variation for the batch-to-batch repeatability test was 5.66%, and the maximum coefficient of variation for the batch-to-batch test was 5.83%, indicating that the method has good repeatability (see Table 2).

[0052]

[0053] Example 6:

[0054] The SA-ELISA assay kit contains the following components:

[0055] 1. Capture antibody, i.e. anti-liver fluke D38 monoclonal antibody: monoclonal antibody diluted with coating solution at pH 9.6 to achieve a concentration of 3.75 μg / mL.

[0056] 2. Sample dilution solution, i.e., PBS (phosphate buffer, pH 7.4): Dissolve 8 g of NaCl, 0.2 g of KCl, 3.63 g of Na2HPO4·12H2O and 0.24 g of KH2PO4 in 800 mL of deionized water in sequence. Adjust the pH of the solution to 7.4 with concentrated HCl, add deionized water to make up to 1000 mL, autoclave and store at room temperature for later use.

[0057] 3. Coating solution (carbonate buffer, pH 9.6): Weigh 2.93 g of NaHCO3 and 1.95 g of Na2CO3 into a 250 mL beaker, add deionized water to bring the volume to 100 mL, autoclave and store at room temperature for later use.

[0058] 4. Blocking solution (1% BSA): Dissolve 0.1 g bovine serum albumin in PBST solution and bring the volume to 10 mL.

[0059] 5. Washing solution (0.01 mol / L, pH 7.4 PBST solution): Dissolve 8 g of NaCl, 0.2 g of KCl, 3.63 g of Na2HPO4·12H2O and 0.24 g of KH2PO4 in 800 mL of deionized water in sequence. Adjust the pH of the solution to 7.4 with concentrated HCl, add 0.5 mL of Tween-20, and add deionized water to make up to 1000 mL. After autoclaving, store at room temperature for later use.

[0060] 6. Substrate solution: Commercially available TMB substrate solution.

[0061] 7. Termination solution (2 mol / L H2SO4): 22.2 mL concentrated sulfuric acid, 177.8 mL deionized water. Slowly pour the concentrated sulfuric acid along the wall of the beaker into the water, mix well, and store at room temperature until needed.

[0062] 8. Preparation of antibody (biotin-labeled rabbit anti-liver fluke D38 polyclonal antibody) solution: Dilute the antibody 1:2000 with PBST solution and store at 4°C.

[0063] 9. Enzyme-labeled avidin: Commercially available enzyme-labeled avidin solution.

[0064] 10. Reference positive serum for sheep liver flukes

[0065] 11. Reference negative serum for sheep liver flukes

[0066] 12. Polystyrene plastic reaction plate

[0067] The kit should be stored at 4 ℃. Liver fluke reference positive and negative sera should be stored at -20 ℃. Example

[0068] 182 samples of sheep serum were provided by sheep farmers in a certain area of ​​Changchun City, Jilin Province, for testing. The steps are as follows:

[0069] (1) Take the capture antibody and coat the microplate with 100 μL per well, and coat overnight at 4 °C;

[0070] (2) Wash with detergent 3 times, each time for 1 minute, with a 5-minute interval, and pat dry;

[0071] (3) Add 100 μL of blocking solution to each well and incubate at 37 ℃ for 2 h;

[0072] (4) Wash with detergent 3 times, each time for 1 minute, with a 5-minute interval, and pat dry;

[0073] (5) Dilute the serum to be tested with PBS (1:4). Set up one well each for negative and blank controls. Dilute the negative control serum to 1:4 as well. Add the blank control directly without dilution. Incubate at 37 ℃ for 2 h.

[0074] (6) Wash 3 times with PBST, each time for 1 min, with a 5 min interval, and pat dry;

[0075] (7) Add 100 μL of antibody detection solution to each well and incubate at 37 °C for 1 h;

[0076] (8) Wash with detergent 3 times, each time for 1 minute, with a 5-minute interval, and pat dry;

[0077] (9) Add enzyme-labeled avidin solution, 100 μL per well, and incubate at 37 °C for 50 min;

[0078] (10) Wash with detergent 4 times, each time for 1 minute, with a 5-minute interval, and pat dry;

[0079] (11) Add 100 μL of TMB substrate solution to each well.

[0080] (12) After developing color at 37 ℃ for 10 min, add 100 μL of stop solution to each well and stop at room temperature;

[0081] (13) Measure OD value: Measure OD value using an enzyme-linked immunosorbent assay (ELISA) reader. 450 nm value; each plate in each experiment includes a reference negative serum and a blank control.

[0082] (14) Result Interpretation: After zeroing the blank control wells, the OD values ​​of each well were measured using a microplate reader at 450 nm. 450 When nm ≥ 0.153, the sample is considered positive; when OD 450 When nm < 0.137, the sample is considered negative; when 0.137 ≤ OD 450 When nm < 0.153, the sample is deemed suspicious and needs to be retested.

[0083] The established double-antibody sandwich SA-ELISA method was used to test 182 serum samples from a livestock farmer in Changchun. The positive rate was 9.34% (17 / 182).

Claims

1. A double antibody sandwich SA-ELISA test kit for circulating antigens of Fasciola hepatica, characterized by It mainly consists of the following parts: Polystyrene plastic reaction plate, coating solution, blocking solution, washing solution, capture antibody, sheep liver fluke infection reference positive and negative serum, biotin-labeled detection antibody, enzyme-labeled avidin solution, substrate solution, termination solution and sample diluent; The capture antibody is anti-Fasciola hepatica D38 mouse monoclonal antibody; The detection antibody is biotin-labeled anti-Fasciola hepatica D38 polyclonal antibody; The coating solution is 0.05M carbonate buffer solution with pH value of 9.6; The sample diluent PBS and the washing solution PBST are phosphate buffer solutions containing 0.5% Tween-20; The blocking solution is 1% bovine serum albumin PBST solution; The substrate developing solution is TMB substrate solution; The termination solution is 2 mol / L H2SO4 solution.

2. The preparation method of the double-antibody sandwich SA-ELISA detection kit for the circulating antigen of Fasciola hepatica according to claim 1, comprising the following steps: 1) Capture antibody, i.e. anti-Fasciola hepatica D38 monoclonal antibody: dilute the monoclonal antibody in the coating solution with pH value of 9.6 to a concentration of 3.75 μg / mL; 2) Sample diluent, PBS phosphate buffer solution, pH value 7.4: dissolve 8 g of NaCl, 0.2 g of KCl, 3.63 g of Na2HPO4·12H2O and 0.24 g of KH2PO4 in 800 mL of deionized water in sequence, adjust the pH value of the solution to 7.4 with concentrated HCl, and make up to 1000 mL with deionized water; 3) Coating solution, carbonate buffer solution, pH value 9.6: weigh 2.93 g of NaHCO3 and 1.95 g of Na2CO3 in a 250 mL beaker, and make up to 100 mL with deionized water; 4) Blocking solution 1% BSA: dissolve 0.1 g of bovine serum albumin in PBST solution, and make up to 10 mL; 5) Washing solution 0.01 mol / L, pH 7.4 PBST solution: dissolve 8 g of NaCl, 0.2 g of KCl, 3.63 g of Na2HPO4·12H2O and 0.24 g of KH2PO4 in 800 mL of deionized water in sequence, adjust the pH value of the solution to 7.4 with concentrated HCl, and add 0.5 mL of Tween-20, and make up to 1000 mL with deionized water; 6) Substrate solution: TMB substrate developing solution; 7) Termination solution 2 mol / L H2SO4: 22.2 mL of concentrated sulfuric acid and 177.8 mL of deionized water, slowly pour the concentrated sulfuric acid along the wall of the cup into the water, and mix well; 8) Preparation of biotin-labeled rabbit anti-Fasciola hepatica D38 polyclonal antibody for use: dilute the detection antibody with PBST solution at a ratio of 1:2000, and store at 4°C; 9) Enzyme-labeled avidin: commercially available enzyme-labeled avidin solution; 10) Sheep liver fluke reference positive serum; 11) Sheep liver fluke reference negative serum; 12) Polystyrene plastic reaction plate.

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