A diagnostic probe for emcrt-specific alveolar echinococcosis and preparation and application thereof

Abstract

The application discloses a diagnosis probe for alveolar echinococcosis of EmCRT specificity and preparation and application thereof, and belongs to the technical field of biomedical imaging. The application first proposes taking the calreticulin specifically secreted by multilocular hydatid tapeworm as a target point, and obtaining a specific polyclonal antibody by immunization and purification, and then coupling the antibody with a detectable marker to construct the target probe, aiming at the defects of the existing alveolar echinococcosis diagnosis method, such as insufficient specificity. The probe can specifically recognize and combine the calreticulin at the lesion site of alveolar echinococcosis, so that the precise detection of the lesion in vivo, non-invasively and visually is realized. The application further provides a diagnosis reagent containing the probe and application of the diagnosis reagent in preparation of an in-vivo imaging diagnosis preparation. The probe has the advantages of high specificity and high sensitivity, and provides a new technical means for early differential diagnosis and curative effect evaluation of alveolar echinococcosis.

Description

Technical Field

[0001] This invention relates to the fields of biomedical technology and molecular imaging, and more specifically, to a diagnostic probe for alveolar echinococcosis (AE) for specific in vivo diagnosis, its preparation method, and its application in the preparation of diagnostic agents or reagents. Background Technology

[0002] Alveolar echinococcosis (AE) is a fatal zoonotic parasitic disease caused by infection with the larvae of *Echinococcus multilocularis*. The lesions primarily infiltrate the liver, resembling malignant tumors, and can metastasize to distant organs such as the lungs and brain. It has an extremely poor prognosis and is sometimes referred to as "parasitic cancer." Currently, the clinical diagnosis of AE mainly relies on imaging examinations (such as ultrasound, CT, and MRI) and serological tests. However, imaging methods have difficulties in differentiating AE from other liver space-occupying lesions such as liver cancer, liver cysts, and hepatic hemangiomas, especially in non-endemic areas, leading to a high rate of misdiagnosis. Conventional serological tests suffer from high false-positive rates and insufficient specificity due to cross-reactivity of the crude antigens used with other parasites (especially cystic echinococcosis, CE).

[0003] Therefore, developing an in vivo detection technology capable of early, highly specific differential diagnosis is crucial for precision medicine in acute exacerbations (AEs). The key to achieving this goal lies in identifying a molecular target with high disease specificity.

[0004] Echinococcus multilocularis calreticulin (EmCRT) is the key target of this invention. Existing research indicates that EmCRT is not only present inside the worm but is also specifically expressed on the surface of the larvae and released in large quantities as an excretory secretory antigen in the microenvironment surrounding the lesion. This characteristic makes EmCRT a highly promising AE-specific biomarker, present locally in the lesion and rarely enriched in other liver diseases or CE lesions, offering a possibility to overcome the lack of specificity in existing diagnostic methods. However, currently, there is a lack of detection probes for Echinococcus multilocularis calreticulin (EmCRT) in existing technologies. Summary of the Invention

[0005] To address the shortcomings of the prior art, this invention provides a diagnostic probe for alveolar echinococcosis using an EmCRT-specific antibody, along with its preparation and application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a diagnostic probe for alveolar echinococcosis in vivo, the probe comprising: Polyclonal antibodies that specifically recognize and bind to the calreticulin (EmCRT) of Echinococcus multilocularis; and Detectable markers covalently linked to the polyclonal antibody; The polyclonal antibody is a polyclonal antibody against EmCRT polypeptide prepared and purified by immunizing animals; The amino acid sequence of the EmCRT polypeptide is selected from one or more of SEQ ID NO:1 (CRT-1) or SEQ ID NO:2 (CRT-2).

[0007] In some embodiments, the detectable marker is a near-infrared fluorescent dye or a radionuclide.

[0008] In some embodiments, the near-infrared fluorescent dye is selected from Cy5.5, Cy7, or IRDye 800CW.

[0009] In some embodiments, the radionuclide is selected from technetium-99m (Turbidium-99m). 99m Tc), Indium-111 111 In), Gallium-68 ( 68 Ga), Zirconium-89 ( 89 Zr), Fluorine-18 ( 18 F), Iodine-131 ( 131 I).

[0010] This invention provides a method for preparing a diagnostic probe for echinococcosis as described above, comprising the following steps: (1) Obtain EmCRT polypeptide antigen and immunize animals to induce the production of specific polyclonal antibodies; the amino acid sequence of the EmCRT polypeptide is selected from one or more of SEQ ID NO:1 (CRT-1) or SEQ ID NO:2 (CRT-2); (2) Anti-EmCRT polyclonal antibodies were isolated and purified from the serum of immunized animals; (3) The anti-EmCRT polyclonal antibody and the activated detectable marker were coupled in a weakly alkaline buffer solution; (4) Purify the reaction product, remove the free marker, and obtain the vesicular echinococcosis diagnostic probe.

[0011] In some embodiments, in step (3), the detectable marker is an N-hydroxysuccinimide ester activated fluorescent dye, and the pH value of the coupling reaction is 8.0-9.0.

[0012] This invention provides a reagent for diagnosing alveolar echinococcosis, comprising an alveolar echinococcosis diagnostic probe as described above and a pharmaceutically acceptable carrier.

[0013] This invention provides the use of the above-described diagnostic probe for alveolar echinococcosis or the above-described reagent in the preparation of an in vivo imaging diagnostic agent for diagnosing alveolar echinococcosis.

[0014] In some embodiments, the in vivo imaging diagnostics include fluorescence imaging, SPECT imaging, or PET imaging.

[0015] This invention provides an immunoassay reagent for in vitro detection of calreticulin from Echinococcus multilocularis, comprising the anti-EmCRT polyclonal antibody prepared as described above.

[0016] The present invention has the following beneficial effects: This invention addresses the lack of specificity in existing diagnostic methods for alveolar echinococcosis by proposing, for the first time, a target protein specifically secreted by *Echinococcus multilocularis*. A specific polyclonal antibody is prepared and purified through immunoassay, and then conjugated with a detectable marker to construct the targeted probe. This probe can highly specifically recognize and bind to the calreticulin at the lesion site of alveolar echinococcosis, thereby achieving precise, non-invasive, and visualized in vivo detection of the lesion.

[0017] Another key to the high specificity of the probe described in this invention lies in its designed physical spatial shielding effect. The CRT-1 and CRT-2 fragments of the EmCRT protein have been shown to be located on a highly exposed polar surface after protein folding, and their amino acid residue distribution facilitates the formation of a stable spatial conformation. During polyclonal antibody preparation, the antibody population induced by this specific conformation exhibits a significantly higher binding energy than general antibodies targeting conserved regions. This targeted induction strategy against 'high-exposure-high-difference' epitopes fundamentally avoids non-specific activation of the immune system caused by parasitic infection (such as background interference from polyclonal B cell activation), enabling this probe to achieve molecular-level discriminative accuracy in clinically differentiating alveolar echinococcosis from cystic echinococcosis, and even from primary liver cancer.

[0018] This invention utilizes a polyclonal antibody prepared by immunizing rabbits with peptides to verify the diagnostic feasibility of the target (EmCRT) and the effectiveness of the probe architecture. This polyclonal antibody can recognize multiple antigenic epitopes on the EmCRT protein and has the advantages of rapid preparation, low cost, and good overall affinity, fully demonstrating the feasibility of the molecular imaging strategy targeting EmCRT. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 Immunofluorescence staining of the prepared EmCRT-specific polyclonal antibody in the scolex of Echinococcus vesicularis. Figure a shows pAb-EmCRT-1, and figure b shows pAb-EmCRT-2.

[0021] Figure 2 Immunofluorescence staining analysis of the prepared EmCRT-specific polyclonal antibody in mouse hepatic alveolar echinococcosis tissue. Figure a shows pAb-EmCRT-1, figure b shows pAb-EmCRT-2, figure c shows the negative control group, and figure d shows the semi-quantitative immunofluorescence analysis of groups a, b, and c.

[0022] Figure 3 Western blot analysis of the prepared polyclonal antibody conjugated with the fluorescent dye Cy7 and the recombinant EmCRT protein. The left side shows pAb-EmCRT-1, and the right side shows pAb-EmCRT-2.

[0023] Figure 4 (a) pAb-EmCRT-1-Cy7; (b) pAb-EmCRT-2-Cy7; (c) Fluorescence imaging of free Cy7 dye 12 h after tail vein injection in mice with hepatic alveolar echinococcosis.

[0024] Figure 5 For (a) 131 SPECT images of I-pAb-EmCRT-2 injected into the tail vein of mice with hepatic alveolar echinococcosis 96 h later; (b) Autoradiography analysis of major organs; (c) Gross image of liver lesions; (d) Quantitative analysis of lesion and non-lesion sites in liver tissue autoradiography results. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto.

[0026] Example 1: Screening of the amino acid sequence of recombinant EmCRT peptide EmCRT (UniProtKB: A0A068YG94) shares a homology with Echinococcus multilocularis within the Echinococcus tapeworm species, namely EmuJ_000653200 (UniProtKB: A0A068Y3H4). To ensure the prepared antibody has good targeting against EmCRT (UniProtKB: A0A068YG94), the amino acid sequences of both were first aligned using GenDOC software to exclude consecutive identical portions. Signal peptide sequence analysis of the EmCRT protein was then performed using a signal peptide prediction tool, identifying the first 18 amino acids as the signal peptide sequence. Transmembrane sequence analysis of the EmCRT protein revealed the absence of a transmembrane region. Finally, the three-dimensional structure of the EmCRT protein was analyzed using peptide selection software developed by Wenyuange Biotechnology Co., Ltd., to select structural regions with higher exposure. Based on the physicochemical property analysis of the EmCRT protein in the above four aspects, two leading EmCRT protein signal peptides, EmCRT-1: CEHAKPKGDFDDRE (SEQ ID NO:1) and EmCRT-2: KQPEDWNEETDGE (SEQ ID NO:2), were selected for subsequent antibody preparation.

[0027] In screening SEQ ID NO: 1 (CRT-1) and SEQ ID NO: 2 (CRT-2) as antigenic epitopes, this invention fully considers the structural differences between the calreticulin (EmCRT) of Echinococcus multilocularis and the homologous proteins of the host (human or rodent).

[0028] Calreticulin is highly conserved evolutionarily. Using full-length proteins or random fragments as antigens can easily lead to cross-reactivity, resulting in high background noise in imaging. This invention, through bioinformatics modeling analysis, reveals that CRT-1 and CRT-2 are located on the surface of the flexible loop region of the EmCRT protein's tertiary structure, and this region exhibits significant sequence divergence between Echinococcus multilocularis and E. granulosus, as well as in the host.

[0029] This specific sequence selection ensures that the prepared polyclonal antibodies can recognize 'non-self' epitopes masked by the host's own proteins, thereby achieving an extremely high signal-to-noise ratio in molecular imaging. This differs from conventional blind screening and is a technical prerequisite for achieving 'accurate identification of alveolar echinococcosis'.

[0030] Example 2: Preparation and purification of anti-EmCRT polyclonal polypeptide antigen I. Polypeptide Synthesis The EmCRT-specific peptide screened in Example 1 was synthesized using a solid-phase synthesis method, specifically as follows: (1) Take an appropriate amount of modified Wang resin for peptide synthesis. First, add 20% pip / DIF to the reactor and shake for 20 min. (2) Filter to remove the solvent, add DMF to the system, react in the shaking reactor for 1 min, and filter to remove the liquid. This operation is repeated three times. (3) Take an appropriate amount of test reagents A and B and a small amount of resin and add them to the test tube. Place the test tube at 100 °C for 30-60 s to check if there is a color change in the resin. If the color changes, it indicates that Fmoc removal is successful. (4) Add the prepared amino acid solution to the reactor, add DIC solution, and shake the reactor for 1 h. (5) Perform the operation as in (3). If there is no color change, it indicates that the condensation is successful. (6) Repeat operation (2) to wash the resin. (7) Repeat the above operation and add the corresponding amino acids until the peptide synthesis is complete. (8) Add condensation reagent (HBTU+HOBt+DIPEA, ureonium salt system) for cyclization modification. (9) Cleavage the resin and perform crude product detection and analysis. (10) The target peptide was purified by RP-HPLC, the fraction was collected and freeze-dried, and finally the purity of the target peptide was >95%.

[0031] II. Peptide Conjugation The synthesized and modified peptide was conjugated with the carrier protein keyhole hemocyanin (KLH) to enhance its immunogenicity. The specific procedure was as follows: 40 mg of KLH was weighed into a 4 mL tube, 2 mL of PBS was added, and the mixture was gently stirred until the KLH dissolved. 8 mg of the conjugating agent Sulfo-SMCC was placed into an Eppendof tube, dissolved in 1 mL of purified water, and stirred thoroughly. The entire solution was then added to the KLH. The KLH solution was incubated at 37 °C for 30 minutes, and the tube was inverted and mixed every 5-10 minutes. The KLH solution was then added to a dialysis bag, placed in 500 mL of 5X PBS, and dialyzed overnight with stirring. The next day, a fresh 500 mL of 5X PBS solution was added, and the tube was dialyzed for 3-6 hours with stirring. The dialysate was collected and placed into a 4 mL tube. Take 4 mg of the modified peptide and dissolve it in 200 μL of peptide solution (0.01 M PBS buffer, pH 7.2-7.4, containing 5 mM EDTA). Add 50 μL of peptide conjugation enhancer TCEP. Take 300 μL of KLH dialysate and add it to 250 μL of the peptide solution, mixing by inverting. Incubate at 37 ℃ for 1-2 hours, mixing by inverting every 20 minutes. Obtain the peptide-KLH conjugate, add PBS to this conjugate to a final volume of 4 mL, and store at -20 ℃ for later use.

[0032] III. Immunity The above-mentioned polypeptide-KLH conjugate was used as an immunogen to immunize New Zealand rabbits and antiserum was obtained. The immunization time and dosage are shown in Table 1 below.

[0033] Table 1

[0034] Immune process: For the first immunization, mix 1 mL of antigen and 1 mL of Freund's complete adjuvant and administer subcutaneously on the back. Select two New Zealand rabbits approximately 7 weeks old for immunization. Each rabbit should have at least 4 immunization points, prioritizing the area near the neck. For the second, third, and fourth immunizations, mix 1 mL of antigen and 1 mL of Freund's incomplete adjuvant and administer subcutaneously on the back. After the third immunization, collect a small blood sample for verification. If the ELISA titer of the antiserum against acetylated peptides is higher than 40kJ, collect blood after the fourth immunization. If it is lower, administer 1-2 more immunizations.

[0035] IV. Thiol Coupling Column Experimental preparation: 1. Prepare the coupling buffer: Add 3.3 mL of 1.5 Tris (pH 8.8) and 1 mL of 0.5 M EDTA to 100 mL of pure water to prepare a final concentration of 50 mM Tris-5 mM EDTA buffer.

[0036] 2. 200 mM cysteine ​​solution: Weigh 0.242 g of cysteine ​​(M = 121.16) and dissolve it in 10 mL of coupling buffer.

[0037] 3. Remove the coupling enhancement solution TCEP from the refrigerator and allow it to dissolve at room temperature.

[0038] Experimental procedure: 1. Remove the chromatography tubes separately and use a homemade pestle to add sand cores to the bottom.

[0039] 2. Remove the peptide-coupled packing material from the 4 ℃ refrigerator and mix thoroughly by inverting the container. Use a 5 mL pipette to add 2 mL of the mixture to a chromatography plastic column (with a white sintered glass core already inserted). Once the liquid has drained completely, you will have 1 mL of packing material.

[0040] 3. Wash the packing material twice with coupling buffer, 4 mL each time.

[0041] 4. After the buffer solution has drained, plug the outlet of the chromatography column with the red cap and add 1 mL of peptide conjugation buffer.

[0042] 5. Add 50 μL of peptide coupling enhancement solution to the chromatography tube, followed by 50 μL of modified peptide and unmodified peptide (already dissolved in peptide solution), and then cover the chromatography column with the white cap.

[0043] 6. Place the chromatography column on the swirl wheel, secure it with the white pipette tip, and mix at low speed for 15 minutes. 7. Remove the chromatography column and let it stand vertically for 15-20 minutes. Open the cap and allow the peptides to dissolve completely. Add white flocculent material to the column. Wash the peptide column twice with coupling buffer, 4 mL each time.

[0044] 8. Add 4 mL of 200 mM cysteine ​​solution to the chromatography column each time, and wash twice, with an interval of 5 minutes between each wash.

[0045] 9. Wash the column twice with 10 mL FBS. The column coupling is now complete and the column can be used immediately for antiserum purification. If storage is required, wash the column with 2 mL of 20% ethanol and cap it with the red cap, then add another 2 mL of 20% ethanol, cap it with the white cap, and store at 4°C.

[0046] V. Antibody Affinity Purification Operating steps: Remove both coupling columns and allow them to drain completely with 20% ethanol. Wash with 10 mL of PBS. Transfer 20 mL of antiserum to a 50 mL centrifuge tube. Centrifuge at 4000 rpm for 5 minutes. Pour the serum into the unmodified peptide-coupled chromatography column and drain completely. Collect the serum. Pour the collected serum into the modified peptide-coupled chromatography column and drain completely. Wash the modified peptide-coupled chromatography column twice with 10 mL of PBS each time. Add 400 μL of pH 8.8 Tris (1.5 M) to a new 15 mL tube as a collection tube. Add 2 mL of glycine (200 mM, pH 2.5) to the column and wash twice with 2 mL of glycine each time, with a 5-minute interval between washes. Mix the solution in the collection tube and check the pH using pH paper to ensure it is close to 8.0. Add 5 mL of glycerol and invert the column thoroughly until there is no interface. Do not mix vigorously; gently mix 20-30 times. Wash the column twice with PBS. The column was washed once with 20% ethanol, drained, and then stored in 20% ethanol. pAb-EmCRT was purified to a concentration of 0.5 mg / mL, and its titer was determined by ELISA. The results are shown in Table 2 below. Table 2

[0047] Example 3: Preparation of fluorescently labeled probe (pAb-EmCRT-Cy7) The purified pAb-EmCRT (1 mg) was dissolved in 1.5 mL PBS, and the pH was adjusted to 8.3 with carbonate buffer to prepare an antibody solution. The NHS-Cy7 dye (1 mg) was dissolved in 400 μL anhydrous DMSO to prepare a dye stock solution. Under light-protected and stirred conditions, 10 μL of the dye stock solution was slowly added to the antibody solution, and the reaction was carried out at room temperature for 2 hours. The reaction solution was transferred to a 10 kDa ultrafiltration centrifuge tube, centrifuged to remove free dye, and washed several times with PBS. The purified antibody-dye conjugate (pAb-EmCRT-Cy7) was collected, aliquoted, and stored in the dark. The labeling rate was determined using a UV-Vis spectrophotometer.

[0048] Example 4: Application of probe in in vivo fluorescence imaging in an orthotopic liver aberration (AE) mouse model Model establishment: Protoscolex of Echinococcus multilocularis (PSCs) were surgically inoculated into the left lobe of the liver of BALB / c mice to establish an orthotopic adenomatous anesthetic (AE) model. Lesion formation was confirmed by magnetic resonance imaging (MRI) 8 weeks later.

[0049] In vivo imaging: AE model mice were randomly divided into experimental and control groups. The experimental group received a tail vein injection of pAb-EmCRT-Cy7 probe (200 μL), while the control group received an equal volume of free Cy7 dye. Imaging was performed using a small animal in vivo fluorescence imaging system at different time points after injection (e.g., 2, 6, 12, 24, 36 h).

[0050] Results: See attached. Figure 4 As shown, the experimental group mice ( Figure 4 b) Twelve hours after injection, the liver lesions showed clear and specific strong fluorescent signals with a high signal-to-noise ratio. In contrast, the control group mice ( Figure 4 c) Only nonspecific systemic distribution and rapid hepatobiliary metabolic signals were observed, with no significant enrichment at the lesion site. Ex vivo organ imaging further confirmed that the fluorescence signal intensity of the probe in the lesion liver tissue was significantly higher than that in normal liver tissue and other major organs (heart, spleen, lung, kidney). pAb-EmCRT-1-Cy7 ( Figure 4 a) Similar specific imaging results were obtained. This indicates that the probe of the present invention has good in vivo targeting and imaging effects.

[0051] Example 5: Radionuclide-labeled probe ( 131 Preparation of I-pAb-EmCRT and its application in SPECT / CT imaging This embodiment aims to demonstrate the labeling of radionuclide iodine-131 for therapeutic diagnosis with the polyclonal antibody described in this invention. 131 I), and used for single-photon emission computed tomography / computed tomography (SPECT / CT) imaging.

[0052] Radioactive marking: a. Labeling was performed using the Iodogen method. 100 µg of purified pAb-EmCRT was dissolved in 100 µL of phosphate buffer (PBS, 0.1 M, pH 7.4).

[0053] b. Add the above antibody solution to a reaction tube pre-coated with 50 µg of Iodogen (1,3,4,6-tetrachloro-3α,6α-diphenylglycourea).

[0054] c. Add approximately 37 MBq (1 mCi) of Na 131 Solution I was reacted at room temperature for 10 minutes, with intermittent light tapping to mix.

[0055] d. Immediately after the reaction is complete, the reaction mixture is centrifuged using a 10 kDa ultrafiltration centrifuge tube, and the radioactive peak in the inner tube is collected to obtain the purified product. 131 I-pAb-EmCRT conjugate.

[0056] e. The radiochemical purity of the labeled products was determined using instantaneous thin-layer chromatography (radio-TLC).

[0057] SPECT / CT live imaging: a. Select mice with orthotopic liver aberration (AE) model (model construction is the same as in Example 3) and healthy control mice.

[0058] b. Inject via tail vein 131 I-pAb-EmCRT probe (approximately 5-10 MBq / mouse, antibody amount approximately 10 µg).

[0059] c. Mice were scanned using a small animal SPECT / CT imaging system at 6, 8, 12, 24, 48, and 72 hours post-injection. CT scans were performed first to obtain anatomical images, followed by SPECT scans to obtain radioactive distribution images, and the two were then fused.

[0060] d. Results: As attached Figure 5 As shown in (a), the SPECT / CT fusion image clearly reveals significant, focal radioactive accumulation in the liver lesion area of ​​the AE model mice, maintaining high uptake even 96 hours after injection (reflecting the targeted retention effect of the antibody). In contrast, the livers of healthy control mice show only a uniform distribution at background levels, or signals are only visible in metabolic organs (such as the thyroid gland, which physiologically uptakes free iodine-131). The CT images provide precise anatomical localization of the radioactive accumulation lesions in the liver.

[0061] The diagnostic probe constructed in this invention exhibits excellent targeting in vivo, and its technical principle is based on the unique dynamic distribution of EmCRT in the microenvironment of alveolar echinococcosis lesions.

[0062] Experiments showed that the probe was highly enriched at the edge of the lesion. The physicochemical mechanism is that EmCRT, as the main excretory secretion product (ESP) of Echinococcus multilocularis, has the highest concentration at the forefront of invasive growth. Moreover, this protein has a hydrophilic C-terminal structure, which makes it easy to form an 'antigen library' at the fibrotic capsule around the lesion.

[0063] When the specific probe described in this invention reaches the lesion through blood circulation, the high affinity of the antibody fragments synergistically binds to the high concentration of free / membrane-bound EmCRT in the lesion at multiple sites. This 'targeted enrichment effect' not only compensates for the lack of uniformity of polyclonal antibodies, but also utilizes the characteristics of multi-site recognition to enhance the intensity of fluorescence / radioactivity signals in complex biological backgrounds, enabling early detection of occult and small lesions.

[0064] Example 6: Autoradiography to verify the tissue distribution and microscopic localization of the probe This embodiment uses autoradiography to visually and at high resolution verify the results at the tissue section level. 131 Specific distribution of I-pAb-EmCRT probe in AE lesions.

[0065] Sample preparation: a. After completing the 96-hour SPECT / CT imaging in Example 5, the AE model mice and healthy control mice were sacrificed.

[0066] b. Quickly remove major organs and tissues such as the liver, heart, spleen, lungs, kidneys, and muscles, and rinse the surface with saline solution to remove any blood.

[0067] c. Place the liver tissue containing the lesion and the control liver tissue in close contact with a high-sensitivity autoradiography phosphorus screen, and then place them together in a light-proof box.

[0068] d. Depending on the radioactivity level of the tissue sample, expose it to room temperature for 1-2 hours.

[0069] e. After exposure, use a phosphor screen imaging scanner to scan the imaging plate and obtain a digitized autoradiographic image.

[0070] Results: See attached. Figure 5 As shown in (c), clear, high-density autoradiographic signal regions can be observed in liver lesion sections of AE model mice, and their shapes highly match the morphology of the lesions. This embodiment not only qualitatively demonstrates the targeting ability of the probe, but its images can also be used for semi-quantitative analysis of the differences in probe distribution in different tissues.

[0071] Conclusion: The targeting probe based on EmCRT-specific polyclonal antibody provided in this invention can achieve highly specific in vivo imaging of AE lesions, effectively distinguishing AE lesions from other liver diseases, and providing a new technical means for the early, non-invasive, and accurate diagnosis of AE.

[0072] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A diagnostic probe for alveolar echinococcosis using an EmCRT-specific antibody, characterized in that, The probe comprises: Polyclonal antibodies that specifically recognize and bind to the calreticulin (EmCRT) of Echinococcus multilocularis; and Detectable markers covalently linked to the polyclonal antibody; The polyclonal antibody is a polyclonal antibody against EmCRT polypeptide prepared and purified by immunizing animals; The amino acid sequence of the EmCRT polypeptide is selected from one or more of SEQ ID NO:1 (CRT-1) or SEQ ID NO:2 (CRT-2).

2. The diagnostic probe for echinococcosis according to claim 1, characterized in that, The detectable marker is a near-infrared fluorescent dye or a radionuclide.

3. The diagnostic probe for echinococcosis according to claim 2, characterized in that, The near-infrared fluorescent dye is selected from Cy5.5, Cy7, or IRDye 800CW.

4. The diagnostic probe for echinococcosis according to claim 2, characterized in that, The radionuclide is selected from technetium-99m ( 99m Tc), Indium-111 111 In), Gallium-68 ( 68 Ga), Zirconium-89 ( 89 Zr), Fluorine-18 ( 18 F), Iodine-131 ( 131 I).

5. A method for preparing a diagnostic probe for alveolar echinococcosis as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) Obtain EmCRT polypeptide antigen and immunize animals to induce the production of specific polyclonal antibodies; the amino acid sequence of the EmCRT polypeptide is selected from one or more of SEQ ID NO:1 (CRT-1) or SEQ ID NO:2 (CRT-2); (2) Anti-EmCRT polyclonal antibodies were isolated and purified from the serum of immunized animals; (3) The anti-EmCRT polyclonal antibody and the activated detectable marker were coupled in a weakly alkaline buffer solution; (4) Purify the reaction product, remove the free marker, and obtain the vesicular echinococcosis diagnostic probe.

6. The method according to claim 5, characterized in that, In step (3), the detectable marker is an N-hydroxysuccinimide ester activated fluorescent dye, and the pH value of the coupling reaction is 8.0-9.

0.

7. A reagent for diagnosing alveolar echinococcosis, characterized in that, It includes the alveolar echinococcosis diagnostic probe as described in any one of claims 1-4 and a pharmaceutically acceptable carrier.

8. The use of the diagnostic probe for alveolar echinococcosis as described in any one of claims 1-4 or the reagent as described in claim 7 in the preparation of an in vivo imaging diagnostic agent for diagnosing alveolar echinococcosis.

9. The application according to claim 8, characterized in that, The in vivo imaging diagnostics include fluorescence imaging, SPECT imaging, or PET imaging.

10. An immunoassay reagent for in vitro detection of calreticulin from Echinococcus multilocularis, characterized in that, It contains a polyclonal antibody against SEQ ID NO:1 or SEQ ID NO:2.

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