Monoclonal antibody and fluorescent microsphere immunochromatography detection test strip for echinococcus multilocularis

Abstract

The invention discloses an echinococcus multilocularis monoclonal antibody and a fluorescent microsphere immunochromatography detection test strip, and relates to the technical field of monoclonal antibodies. The echinococcus multilocularis monoclonal antibody comprises an echinococcus multilocularis monoclonal antibody 20B7D9 and an echinococcus multilocularis monoclonal antibody 18F11G9, and the echinococcus multilocularis monoclonal antibody 20B7D9 and the echinococcus The echinococcus multilocularis monoclonal antibody 20B7D9 can be combined with the echinococcus multilocularis monoclonal antibody 18F11G9, so that the echinococcus multilocularis can be detected by a double-antibody sandwich method. The fluorescent microsphere immunochromatography test strip prepared from the 20B7D9 and the echinococcosis multilocularis monoclonal antibody 18F11G9 is used for medical detection of echinococcosis multilocularis, has the advantages of high sensitivity, short reaction time, strong anti-interference capability and accurate and reliable detection result, and has wide clinical application value.

Description

Technical Field

[0001] This invention relates to the field of monoclonal antibody technology, specifically to a monoclonal antibody against multilocular echinococcosis and a fluorescent microsphere immunochromatographic test strip containing the antibody. Background Technology

[0002] Echinococcus multilocularis larvae can parasitize the digestive tracts of various animals, including pigs, cattle, sheep, and humans. It primarily infects humans and animals through the ingestion of undercooked meat containing the parasite's eggs or contaminated drinking water.

[0003] Echinococcosis multilocularis larvae can cause serious damage to multiple systems in the human body. In the intestinal tract, larvae damage the intestinal mucosa, leading to symptoms such as nausea, vomiting, diarrhea, and upper abdominal discomfort. Neurologically, patients may experience muscle paralysis, seizures, or symptoms such as blindness and cognitive impairment. Simultaneously, larvae can also infect the liver, lungs, and other internal organs, causing corresponding diseases. For pregnant women, this parasite can cross the placental barrier, leading to miscarriage, stillbirth, and fetal malformations. In some acute infections, patients may even experience life-threatening conditions such as respiratory failure. Therefore, timely diagnosis and treatment are crucial for Echinococcosis multilocularis infection.

[0004] Currently, serum antibody testing is the sole diagnostic method for Echinococcosis multilocularis infection, but it has several limitations. First, the results are prone to misdiagnosis. Second, in the early stages of infection, antibody levels are low, making negative results common. Furthermore, serum antibody testing cannot distinguish between active and late-stage infections. Therefore, multiple testing is needed to ensure accurate diagnosis and obtain more precise results. While more advanced techniques such as PCR molecular detection and imaging-guided MRI are more accurate and convenient, they are also more expensive and time-consuming. Therefore, serum antibody testing needs further improvement to enhance accuracy, shorten diagnostic time, and increase sensitivity.

[0005] Therefore, developing monoclonal antibodies against Echinococcus multilocularis can play an important role in medical diagnosis and treatment. However, the expression levels, purity, anti-interference properties, specificity, and selectivity of existing publicly available Echinococcus multilocularis monoclonal antibodies do not meet the ideal requirements for medical detection. Furthermore, these antibodies can only be used alone, failing to meet the demands of medical testing. Simultaneously, due to factors such as the spatial structure of the Echinococcus multilocularis antigen and the monoclonal antibody, it is difficult to develop monoclonal antibodies that can be used in pairs for ELISA sandwich assays to detect Echinococcus multilocularis, resulting in a severe lack of early medical detection methods for this disease.

[0006] It is evident that developing multilocular echinococcosis monoclonal antibodies that can simultaneously improve diagnostic accuracy, possess high specificity and selectivity, and enable early understanding of a patient's infection status is of great significance for medical diagnosis. Summary of the Invention

[0007] To overcome the shortcomings of existing Echinococcus multilocularis antibodies and early diagnostic methods for Echinococcus multilocularis, this invention provides a monoclonal antibody against Echinococcus multilocularis and a fluorescent microsphere immunochromatographic test strip containing the antibody.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A monoclonal antibody against Echinococcus multilocularis, namely Echinococcus multilocularis monoclonal antibody 20B7D9, has the amino acid sequence of its heavy chain as shown in SEQ ID NO.1 and the amino acid sequence of its light chain as shown in SEQ ID NO.2.

[0010] The present invention also provides a nucleotide sequence expressing the multilocular echinococcosis monoclonal antibody 20B7D9.

[0011] The present invention also provides expression cells expressing the aforementioned Echinococcus multilocularis monoclonal antibody 20B7D9.

[0012] The present invention also provides an antibody for the detection of Echinococcus multilocularis, comprising the aforementioned Echinococcus multilocularis monoclonal antibody 20B7D9.

[0013] The present invention also provides a monoclonal antibody against Echinococcus multilocularis, namely Echinococcus multilocularis monoclonal antibody 18F11G9, the amino acid sequence of its heavy chain is shown in SEQ ID NO.3, and the amino acid sequence of its light chain is shown in SEQ ID NO.4.

[0014] The present invention also provides a nucleotide sequence for expressing the multilocular Echinococcus monoclonal antibody 18F11G9.

[0015] The present invention also provides an expression cell that expresses the aforementioned Echinococcus multilocularis monoclonal antibody 18F11G9.

[0016] The present invention also provides an antibody for the detection of Echinococcus multilocularis, comprising the aforementioned Echinococcus multilocularis monoclonal antibody 18F11G9.

[0017] Preferably, the antibody used for detecting Echinococcus multilocularis is a dual antibody, consisting of Echinococcus multilocularis monoclonal antibody 20B7D9 and Echinococcus multilocularis monoclonal antibody 18F11G9.

[0018] The present invention also provides the application of the antibody used for the detection of Echinococcus multilocularis in the double antibody sandwich method for the detection of Echinococcus multilocularis.

[0019] Preferably, among the antibodies, Echinococcus multilocularis monoclonal antibody 20B7D9 is the labeling antibody, and Echinococcus multilocularis monoclonal antibody 18F11G9 is the coating antibody.

[0020] The present invention also provides a fluorescent microsphere immunochromatographic test strip for detecting multilocular echinococcosis using a double-antibody sandwich method, characterized in that it contains the aforementioned double antibodies for detecting multilocular echinococcosis.

[0021] Preferably, the fluorescent microsphere immunochromatographic test strip further includes a sample pad, a conjugate pad, fluorescent microspheres, and an NC membrane.

[0022] Preferably, the fluorescent microspheres are carboxylated europium chelate fluorescent nanospheres, and the particle size of the fluorescent microspheres is 285nm-315nm.

[0023] Preferably, the sample pad is made of 8892 glass fiber.

[0024] Preferably, the bonding pad is made of 2010 glass fiber.

[0025] The present invention also provides the structure of the fluorescent microsphere immunochromatographic test strip for detecting multilocular Echinococcus larvae, wherein the fluorescent microsphere immunochromatographic test strip is provided with a sample pad, a conjugate pad and an NC membrane arranged in sequence according to the flow direction of the sample to be tested; the conjugate pad is immobilized with labeled antibodies labeled by fluorescent microspheres; and the NC membrane is immobilized with coated antibodies.

[0026] Preferably, the sample pad, conjugate pad, nitrocellulose membrane and absorbent paper are fixed sequentially on the PVC base plate according to the flow direction of the sample to be tested.

[0027] This invention also provides a non-diagnostic detection method for the fluorescent microsphere immunochromatographic test strip for detecting Echinococcus multilocularis. Specifically, the sample to be tested is dropped onto the sample pad and left for 9-12 minutes. The presence or absence of Echinococcus multilocularis in the sample is determined by the presence or absence of fluorescence in the antibody-coated region fixed on the NC membrane. The content of Echinococcus multilocularis in the sample is calculated based on the fluorescence intensity of the antibody-coated region fixed on the NC membrane. The higher the fluorescence intensity, the higher the content of Echinococcus multilocularis, and vice versa.

[0028] Compared with the prior art, the multilocular echinococcosis monoclonal antibody 20B7D9 provided by the present invention has the advantages of high specificity and good sensitivity for the detection of multilocular echinococcosis AgB antigen. At the same time, the structure size of this antibody is also suitable for labeling antibodies or coating antibodies in the detection of double-antibody sandwich antigens.

[0029] The obtained Echinococcus multilocularis monoclonal antibody 20B7D9 can be combined with Echinococcus multilocularis monoclonal antibody 18F11G9 to establish a Echinococcus multilocularis (EMAGB) test strip based on the double antibody sandwich method.

[0030] Testing has shown that the fluorescent microsphere immunochromatographic test strip for Echinococcus multilocularis provided by this invention can be used as a clinical testing tool, and it is fast and convenient, capable of completing the test within 9 minutes, significantly shortening the sample processing time and having a clear time advantage compared to other testing methods.

[0031] The fluorescent microsphere immunochromatographic test strip of Echinococcus multilocularis provided by this invention exhibits excellent performance in detecting EMAGB in serum, with a wide linear range of 5-1200 ng / mL and a sensitivity of up to 5.7 ng / mL, which can completely cover the possible concentration range of EMAGB in the test sample.

[0032] Quantitative assays and correlation analyses were performed on multiple serum samples using both existing ELISA antibody detection methods and the test strip provided in this invention. The results showed a correlation coefficient (k) of 1.0 between the two methods, indicating a significant correlation and demonstrating the high accuracy of the fluorescent microsphere immunochromatographic test strip for *Echinococcus multilocularis* provided in this invention. Furthermore, the coefficient of variation for repeated detections using the fluorescent microsphere immunochromatographic test strip for *Echinococcus multilocularis* provided in this invention was less than 10%, fully meeting the reagent requirements.

[0033] High concentrations of interfering substances MYO, CTNI, CKMB, and proBNP do not show significant cross-reactivity with the fluorescent microsphere immunochromatographic test strip of Echinococcus multilocularis larvae of the present invention, and the test strip is not affected by high concentrations of hemoglobin, triglyceride mixtures, and hemoglobin when detecting samples.

[0034] In summary, the fluorescent microsphere immunochromatographic test strip for Echinococcus multilocularis provided by this invention has the advantages of high sensitivity, fast reaction time, strong anti-interference ability, and accurate and reliable detection results, and has broad clinical application value. Attached Figure Description

[0035] Figure 1 The detection structure diagram of the fluorescent microsphere immunochromatographic quantitative detection test strip;

[0036] Figure 2 The dose-response curve for the fluorescent microsphere immunochromatographic quantitative detection test strip;

[0037] Figure 3 A graph showing the experimental results of the Hook effect analysis for the fluorescent microsphere immunochromatographic quantitative detection test strip;

[0038] Figure 4 Linear dilution curves of the fluorescent microsphere immunochromatographic quantitative detection test strip for serum sample detection;

[0039] Figure 5Distribution of multilocular Echinococcus larvae in healthy serum as detected by a fluorescent microsphere immunochromatographic quantitative detection test strip. Detailed Implementation

[0040] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0041] Example

[0042] 1. Screening of monoclonal antibodies

[0043] 1.1 Recombinant expression of Echinococcus multilocularis AgB antigen protein

[0044] The *Echinococcus multilocularis* AgB antigen was expressed in *E. coli* expression systems using the PET plasmid. The specific method is as follows: The *Echinococcus multilocularis* AgB gene (as shown in SEQ ID NO. 9) was amplified by PCR and inserted into the PET plasmid to construct a transformation system. The constructed PET plasmid was transformed into *E. coli* host cells, and positive strains containing the PET plasmid were screened using a semi-solid culture medium. Equal-volume induction was used to promote the expression of *Echinococcus multilocularis* AgB antigen protein in *E. coli*, resulting in high expression levels and excellent purity of the *Echinococcus multilocularis* AgB antigen protein.

[0045] After protein purification, DNase I was used for further digestion to ensure the purity and sensitivity of the AgB protein.

[0046] The final recombinant AgB antigen protein had a purity of 95.7% and a water solubility of over 1 mg / mL.

[0047] 1.2 Immunization of mice with recombinant AgB protein

[0048] Recombinant AgB protein was injected into mice (C57BL / 6J strain), and the presence of specific antibodies in the mouse serum was analyzed by ELISA technology to screen out positive mice with high levels of specificity and antigen-binding ability.

[0049] During this process, the mouse recombinant AgB antigen protein immunization produced highly specific and high-titer antibodies. The results of mouse immunization identified by ELISA are shown in Table 1.

[0050] Table 1 Results of mouse immune serum detection

[0051] Negative Hole 1 Negative Hole 2 Mouse serum well 1 Mouse serum well 2 Mouse serum well 3 Mouse serum well 4 absorbance 0.215 0.169 3.125 2.965 2.654 2.865

[0052] As shown in Table 1, the immunization of mice was successful.

[0053] 1.3 Isolation of mouse spleen cells and construction and screening of hybridoma cells

[0054] Immunization of fused animals begins two months before cell fusion and is performed three times.

[0055] Before cell fusion, spleen lymphocytes and myeloma cells are prepared, with the myeloma cells being of the same strain as the immunized animals.

[0056] Cell fusion was performed using the PEG method. After approximately 5 days, cells were screened and selected using HAT and HT media, and were observed and transferred promptly. Limiting dilution was used for three cloning processes.

[0057] HAT medium (50×) contains inosine (5mM), aminopterin (20μM), and thymidine (0.8mM). 10ml of this product can prepare 500mL of medium, with a final working concentration of 100μM inosine, 0.4μM aminopterin, and 16μM thymidine. Instructions for use: Dilute 1 volume of HAT medium (50×) into 49 volumes of cell culture medium.

[0058] HT medium contains hypoxanthine and thymine, and is used to supplement cell culture when purine and pyrimidine synthesis pathways are blocked. The specific components of HT medium (50×) are: 5 mol / L hypoxanthine, 3 mol / L thymine deoxyribonucleotide, and double-distilled water. The method of using HT medium is to dilute 1 volume of HT selective medium (50×) into 49 volumes of cell culture medium.

[0059] Successfully obtained hybridoma cells were cryopreserved for future use. Further identification and validation were performed before using monoclonal antibodies to ensure their specificity and stability.

[0060] The cell supernatant of the obtained hybridoma cells produced highly specific and high-titer antibodies. The results of ELISA identification of the antibody content in the cell supernatant are shown in Table 2 below.

[0061] Table 2. Results of antibody content detection in cell supernatant.

[0062] Negative Hole 1 Negative Hole 2 Supernatant 1 Supernatant 2 Supernatant 3 Supernatant 4 absorbance 0.291 0.274 1.567 1.965 2.063 1.739

[0063] 1.4 Sequencing of antibodies expressed in positive hybridoma cells

[0064] Before preparing monoclonal antibodies, hybridoma cells need to be cultured and collected. RNA is extracted from hybridoma cells using the TRIzol method, and then reverse transcribed to synthesize cDNA. The cDNA is amplified by PCR using the SMART RACE method to obtain the variable regions of the heavy and light chains. After ligating the antibody gene into a T vector, positive clones are selected and sequenced. Bioinformatics programs are used to analyze the sequencing results to obtain the antibody sequence. Finally, by analyzing and comparing the antibody sequences, the antibody type and subtype, as well as the variable and frame regions of the antibody, are determined.

[0065] The immunization process screened for the monoclonal antibody 20B7D9 against Echinococcus multilocularis (the amino acid sequence of the heavy chain is as shown in SEQ ID NO.1, the gene sequence expressing the heavy chain is as shown in SEQ ID NO.5, the amino acid sequence of the light chain is as shown in SEQ ID NO.2, and the gene sequence expressing the light chain is as shown in SEQ ID NO.6).

[0066] The same screening method was used to obtain the monoclonal antibody 18F11G9 for Echinococcus multilocularis (the amino acid sequence of the heavy chain is as shown in SEQ ID NO.3, the gene sequence expressing the heavy chain is as shown in SEQ ID NO.7, the amino acid sequence of the light chain is as shown in SEQ ID NO.4, and the gene sequence expressing the light chain is as shown in SEQ ID NO.8).

[0067] 2. Preparation of fluorescent microsphere immunochromatographic test strips for the detection of multilocular Echinococcus larvae using a double-antibody sandwich method

[0068] 2.1 Materials

[0069] 2.1.1 Reagents

[0070] Antibody and biotechnology products:

[0071] AgB antibody (20B7D9), AgB antibody (18F11G9, the amino acid sequence of the heavy chain is as shown in SEQ ID NO.3, the gene sequence expressing the heavy chain is as shown in SEQ ID NO.7, the amino acid sequence of the light chain is as shown in SEQ ID NO.4, the gene sequence expressing the light chain is as shown in SEQ ID NO.8), chicken IgGY antibody (Guangdong Feipeng Biotechnology Co., Ltd.), goat anti-chicken anti-IgGY antibody (Guangdong Feipeng Biotechnology Co., Ltd.).

[0072] Bovine serum albumin (BSA) produced by Xiamen Wantai Kerry Biotechnology Co., Ltd.

[0073] Chemical reagents from Roche Diagnostics, USA: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxythiosuccinimide (sulfo-NHS), sodium morpholine ethanesulfonate (MES), sodium azide (NaN3).

[0074] Trehalose from Sigma-Aldrich, USA.

[0075] Laboratory consumables from Wako Pure Chemical Co., Ltd. (Japan): centrifuge tubes, nitrocellulose membranes (NC membranes), and glass fiber.

[0076] Merck Millipore, USA: Carboxylated europium chelate fluorescent nanospheres (CM-EUs) with a particle size of 300 nm ± 15 nm.

[0077] Thermo Fisher Scientific, USA: Tween-20, Triton-X100, Tetronic 1307 (S9).

[0078] Other basic chemical reagents from Sigma-Aldrich (USA) and Hangzhou Hangan Technology Co., Ltd.

[0079] 2.1.2 Instruments

[0080] The Milli-Q Systhesis ultrapure water preparation system and the HulaMixerIM Sample Mixer magnetic microsphere mixer are from Merck Millipore, USA.

[0081] The Certrifuge 5427R benchtop high-speed refrigerated centrifuge, ultra-micro electronic balance, and SCIENTA-II ultrasonic cell disruptor were supplied by Eppendorf GmbH, Germany.

[0082] Ningbo Xinzhi Biotechnology Co., Ltd. provides time-resolved fluorescence immunochromatographic test strips.

[0083] Medisensor, a South Korean company, provides the Biodot-XYZ3060 3D inkjet platform.

[0084] The ZQ-2000 strip cutter is supplied by Biodot, an American company.

[0085] The DD-330CH electronic dehumidifier, DGG-9000B electric thermostatic blower dryer, and fully automatic multifunctional microplate reader are supplied by Shanghai Jinbiao Biotechnology Co., Ltd.

[0086] The PB20 pH meter and micropipette are from Zhongshan Beidou Wandefu Electronic Technology Co., Ltd. and Bio-Rad Inc. of the United States, respectively.

[0087] The German company Sartoruis provides the relevant weighing test instruments.

[0088] Thermo Fisher Scientific, a US company, supplies other laboratory equipment and consumables.

[0089] 2.1.3 Buffer Solution

[0090] 1) The activation buffer for carboxylated europium chelate fluorescent nanospheres was prepared with 50 mmol / L MES and the pH was adjusted to 6.0.

[0091] 2) Activating reagent a is prepared from the above MES solution containing 1% EDC.

[0092] 3) Activating reagent b is prepared from the above MES solution containing 1% sulfo-NHS.

[0093] 4) The carboxylated europium chelate fluorescent nanospheres and protein coupling buffer were prepared with 25 mmol / L LPB and the pH was adjusted to 7.0.

[0094] 5) The washing solution was prepared by mixing 25 mmol / L Tris-base, 0.2% Tween 20, 0.15 mol / L NaCl and 0.05% ProClin 300, and the pH was adjusted to 7.8.

[0095] 6) The blocking solution is prepared with 50 mmol / L PB containing 5% BSA by mass, and the pH is adjusted to 8.0.

[0096] 7) The preservation solution was prepared with 25 mmol / L Tris-base, 0.05% Tween 20, 0.15 mol / L NaCl, 0.05% ProClin 300, 1% BSA, and 5% trehalose, and the pH was adjusted to 7.2.

[0097] 8) The labeling microsphere dilution solution was prepared by mixing 25 mmol / L Tris-base, 0.05% Tween-20, 1% BSA, 0.05% ProClin300, 5% trehalose and 20% sucrose, and the pH was adjusted to 9.0.

[0098] 9) The coating solution was prepared by mixing 10 mmol / L Na2HPO4·12H2O, 0.15 mol / L NaCl, 0.3% trehalose and 0.1% NaN3, and the pH was adjusted to 7.4.

[0099] 10) The sample pad pretreatment solution was prepared by mixing 10 mmol / L sodium tetraborate, 1% PVP, 0.2% sodium caseinate, 1% Tritium-X100, 1% S9 and 0.02% NaN3.

[0100] 11) The conjugate pad pretreatment solution was prepared with 50 mmol / L Na2HPO4·12H2O, 0.5% PVA, 0.5% BSA, and 1% Tritium-X100, and the pH was adjusted to 7.4.

[0101] 12) The standard diluent was prepared by mixing 50 mmol / L Tris-base, 0.15 mol / L NaCl, 0.1% ProClin 300, 0.01% Tween 20 and 1.5% BSA, and the pH was adjusted to 7.8.

[0102] All percentage values ​​mentioned in the above buffer solution preparations are volume percentages.

[0103] 2.1.4 Clinical Samples

[0104] Three samples of Echinococcus multilocularis protoscolex and six serum samples from patients infected with Echinococcus multilocularis were provided by the Affiliated Hospital of Qinghai University.

[0105] 2.2 Antibody Pretreatment

[0106] 2.2.1 Preliminary concentration of antibodies:

[0107] Remove 50 μg each of the EMAGB antibody raw materials (AgB antibody 20B7D9 and AgB antibody 18F11G9) used for coupling with carboxylated europium chelate fluorescent nanospheres from a -20°C freezer. Add the antibodies to a 50 kDa ultrafiltration centrifuge tube provided by Millipore, USA. Centrifuge at 6000 × g for 8 min at 4°C.

[0108] 2.2.2 Antibody washing and buffer replacement:

[0109] The pre-concentrated antibody raw material was added to 200 μL of carboxylated europium chelate fluorescent nanospheres and protein conjugation buffer. The mixture was centrifuged at 6000 × g for 8 min at 4 °C, and the filtrate was discarded. Another 200 μL of protein conjugation buffer was added, and the centrifugation process was repeated (4 °C, 6000 × g, 8 min).

[0110] 2.2.3 Collection and purification of antibodies:

[0111] After centrifugation, remove the ultrafiltration tube, discard the filtrate, and replace it with a new collection tube. Invert the ultrafiltration tube and centrifuge at 2500×g for 1 minute at 4°C, collecting the filtrate. Then, invert the filter membrane, add 100 μL of ligation buffer (10 mM phosphate buffer, pH 7.2), and let it stand for 5 minutes. Invert the ultrafiltration tube again and centrifuge at 2500×g for 1 minute at 4°C, collecting the filtrate. Finally, 200 μL each of the two purified antibodies, AgB antibody 20B7D9 and AgB antibody 18F11G9, were collected.

[0112] 2.3 Pretreatment of carboxylated europium chelate fluorescent nanospheres

[0113] 2.3.1 Activation preparation of microspheres:

[0114] The carboxylated europium chelate fluorescent nanospheres were thoroughly mixed using a vortex mixer to ensure uniform dispersion. 200 μL of the microsphere suspension (1% solids content) was taken as the sample to be processed.

[0115] Transfer the prepared microsphere suspension to a 1.5 mL centrifuge tube. Centrifuge at 15000 × g for 15 min at 4 °C. This step precipitates the microspheres and separates the supernatant containing surfactants and preservatives. After centrifugation, remove the supernatant.

[0116] Add 1 mL of carboxylated europium chelate fluorescent nanosphere activation buffer to the test tube containing the precipitated microspheres. Mix thoroughly using a vortex mixer to ensure the microspheres are uniformly suspended. Centrifuge at 15000×g for 15 min at 4°C to remove impurities from the supernatant. Aspirate the supernatant using a micropipette and repeat this step once to ensure complete removal of impurities.

[0117] 2.3.2 Activation of microspheres

[0118] Add 14 μL of carboxylated europium chelate fluorescent nanosphere activator a and 132 μL of activator b to the microsphere precipitate. Mix thoroughly using a vortex mixer to ensure complete reaction between the microspheres and activators. Transfer the microsphere suspension to a 2 mL centrifuge tube and add 854 μL of activation buffer. Incubate at room temperature using a magnetic microsphere mixer for 30 min to promote microsphere activation.

[0119] 2.3.3 Cleaning of microspheres

[0120] Transfer the microsphere suspension from the 2 mL centrifuge tube to a 1.5 mL centrifuge tube. Centrifuge at 15000 × g for 30 min at 4 °C to remove unreacted activating reagents. Remove the supernatant to ensure the microsphere precipitate is pure.

[0121] 2.3.4 Microsphere Preparation Before Coupling

[0122] Add 1 mL of coupling buffer to the microsphere precipitate and resuspend the microspheres. Sonicate the microspheres to ensure uniform dispersion (50 W, 1 second operation, 1 second interval, for 3 minutes; subsequent sonication conditions are the same). Centrifuge at 15000 × g for 30 min at 4°C to remove excess coupling buffer. Carefully remove the supernatant and repeat this step once to ensure the microspheres are fully ready for the coupling reaction.

[0123] 2.4 Coupling of carboxylated europium chelate fluorescent nanospheres with antibodies

[0124] 2.4.1 Initiation of the coupling reaction:

[0125] The prepared microspheres were added to 200 μL of coupling buffer and sonicated to ensure uniform dispersion. The sonicated microsphere suspension was transferred to a 2 mL centrifuge tube. The previously purified and concentrated antibody was added to ensure effective contact between the antibody and the microspheres. Then, an additional 800 μL of coupling buffer was added to dilute the total microsphere concentration to 2 mg / mL. The microspheres and antibody were thoroughly mixed using a vortex mixer to ensure a homogeneous reaction system. The coupling reaction was carried out at 37°C for 2 hours to promote effective binding between the antibody and the microspheres. Using this method, fluorescently labeled Echinococcus multilocularis monoclonal antibodies 20B7D9 and 18F11G9 were obtained.

[0126] 2.4.2. Preliminary washing of antibody-conjugated microspheres:

[0127] Transfer the suspension of the coupling product from the 2 mL centrifuge tube to a 1.5 mL centrifuge tube. Centrifuge at 10000 × g for 15 min at 4 °C to precipitate the microspheres. Carefully remove the supernatant, taking care to avoid disturbing the microsphere precipitation.

[0128] 2.4.3 Further washing of antibody-conjugated microspheres

[0129] Add 1 mL of washing buffer to the precipitated microspheres and sonicate to ensure uniform resuspending. Centrifuge at 10000×g for 15 min at 4°C to remove unbound antibodies and other impurities. Carefully remove the supernatant and repeat the washing step once to ensure the microspheres are clean and all unreacted reagents are removed.

[0130] 2.4.4 Blocking and preservation of antibody-conjugated microspheres

[0131] 2.4.4.1 Closing Mark:

[0132] Add 1 mL of blocking solution to the microsphere precipitate. This solution typically contains substances that react with unreacted active groups to block remaining active sites and reduce nonspecific binding. Sonicate the microspheres to ensure they are uniformly dispersed in the blocking solution. Transfer the sonicated microsphere suspension to a 2 mL centrifuge tube. Vortex thoroughly mix the microspheres to ensure uniform contact between the blocking solution and the microspheres. Rotate the label at room temperature for 1 hour to complete the blocking reaction.

[0133] 2.4.4.2 Washing of antibody-conjugated microspheres:

[0134] Transfer the microsphere suspension from the 2 mL centrifuge tube to a 1.5 mL centrifuge tube. Centrifuge at 10000 × g for 15 min at 4 °C to precipitate the microspheres. Carefully remove the supernatant, taking care to avoid disturbing the microsphere precipitate.

[0135] 2.4.4.3 Further washing and storage of antibody-conjugated microspheres:

[0136] Add 1 mL of preservation solution to the precipitated microspheres. This solution typically contains stabilizers and antifreeze to protect the stability of the microspheres during long-term storage. Sonicate the microspheres to ensure uniform resuspension. Centrifuge at 10000×g for 15 min at 4°C to remove excess preservation solution. Carefully remove the supernatant and repeat the washing step once to ensure the microspheres are clean and all unreacted reagents are removed.

[0137] 2.4.4.4 Dilution and storage of antibody-conjugated microspheres:

[0138] Dilute the microspheres to a working concentration of 2 mg / mL using the preservation solution. Store the diluted microspheres at 2–8°C, avoiding light exposure, to maintain their activity and stability. Label the microspheres with their concentration and preparation date for future traceability and to ensure experimental accuracy.

[0139] 2.4.5 Conjugation of europium chelate fluorescent nanospheres with chicken IgY antibody

[0140] The protocol used for europium chelate fluorescent nanospheres conjugated with chicken IgY antibody is the same as that used for europium chelate fluorescent nanospheres conjugated with AgB monoclonal antibodies (20B7D9 and 18F11G9).

[0141] 2.5NC membrane coating

[0142] 2.5.1 Antibody Dilution

[0143] The EMAGB monoclonal antibodies (20B7D9 and 18F11G9) and goat anti-chicken anti-IgG antibodies were diluted using coating buffer.

[0144] 2.5.2 Pretreatment of NC membrane:

[0145] Before antibody coating, the NC membrane needs to be pretreated to ensure its surface is suitable for antibody adsorption and immobilization. The pretreatment process includes routine NC membrane cleaning steps such as washing, activation, and blocking nonspecific binding sites.

[0146] 2.5.3 Antibody coating was performed using the XYZ3060 three-dimensional spraying platform:

[0147] The diluted antibody solution was loaded onto the Biodot XYZ3060 three-dimensional spraying platform. According to the test strip design requirements, monoclonal antibody 20B7D9 or 18F11G9 solution was precisely sprayed onto the T-line region of the NC membrane, and chicken IgY antibody solution was precisely sprayed onto the C-line region. The antibody concentration for both the T-line and C-line was uniformly set to 2 mg / mL, and the spraying volume was 4 μL / cm.

[0148] 2.5.4 Antibody fixation:

[0149] After printing, the NC membrane is dried, allowing the antibodies adsorbed on the membrane surface to form a stable coating. During the fixation process, the antibodies interact with the NC membrane surface through hydrophobic interactions, hydrogen bonding, or electrostatic adsorption to form a stable coating layer.

[0150] Using this method, two NC membranes were obtained, one for the T-line and the other for the 18F11G9 monoclonal antibody. The C-line of both NC membranes was coated with chicken IgY antibody.

[0151] 2.6 Pretreatment of sample pad (8892 glass fiber) and bonding pad (2010 glass fiber)

[0152] Cut the sample pads into 17mm wide and 300mm long pieces and immerse them in the sample pad pretreatment solution for 1.5 hours. After immersion, drain the sample pads and dry them in a 37°C forced-air drying oven for 3 hours to remove excess liquid and ensure the pads are dry. After drying, the sample pads should be stored in a dehumidifying cabinet for later use.

[0153] The treatment method for the conjugate pad is to soak it in a pretreatment solution and then dry it.

[0154] 2.7 Solid-state construction of carboxylated europium chelate fluorescent nanosphere coupling complexes

[0155] The antibody conjugates (20B7D9 and 18F11G9) and IgY conjugates, which had been successfully coupled to carboxylated europium chelate fluorescent nanospheres, were sonicated and resuspended to ensure uniform dispersion of the microspheres and antibodies. The conjugates were diluted separately using labeled microsphere dilution buffer. The EMAGB antibody (20B7D9 and 18F11G9) conjugates were diluted to 0.32 mg / mL, and the IgY conjugates to 0.064 mg / mL. After dilution, the two conjugates were thoroughly mixed in a 1:1 ratio to ensure uniform distribution and effective binding of the two antibodies (spray volume of both antibodies was 6.5 μL / cm) on the conjugate pad. Subsequently, the mixed antibody conjugates were sprayed using a Biodot XYZ3060 three-dimensional spraying platform. The spray rate was set to 10 μL / cm to ensure uniform antibody distribution on the conjugate pad, which is beneficial for improving the detection performance of the test strip. After spraying, place the conjugate pad in a 37°C drying oven for 2 hours. This step helps fix the antibodies on the sprayed area, ensuring their stability in the test strip. Finally, store the dried conjugate pad in a dehumidifier for later use.

[0156] This yields binding pads of 20B7D9 and lgY bound to labeled fluorescent microspheres, and binding pads of 18F11G9 and lgY bound to labeled fluorescent microspheres.

[0157] 2.8 Assembly of test strips

[0158] PVC base plate: The PVC base plate is the supporting structure of the test strip. Its function is to keep the nitrocellulose membrane, sample pad and absorbent paper in the correct position and ensure the structural integrity of the test strip during use.

[0159] Nitrocellulose membrane: The nitrocellulose membrane (NC membrane) is the core part of the test strip, and it has a detection area (T line) and a control area (C line).

[0160] Sample pad: The sample pad is the first layer of the test strip and is used to receive and absorb the sample to be tested.

[0161] The conjugate pad: The function of the conjugate pad is to adsorb and immobilize the labeled fluorescent nanosphere-antibody conjugate. It ensures that the conjugate remains active within its shelf life and is effectively released as the sample flows through, thereby participating in the detection reaction.

[0162] Absorbent paper: Absorbent paper is usually located at the very end of the sample flow direction of the test strip. Its main function is to absorb excess liquid, control the speed and direction of sample flow, and ensure that the sample can pass through the NC membrane evenly.

[0163] The assembly process is as follows: First, arrange the sample pad, conjugate pad, nitrocellulose membrane, and absorbent paper in sequence. Then, use adhesive to fix these components to the PVC base plate. After assembly, use a strip cutter to cut the PVC base plate and the components fixed to it into 3mm wide test strips. Finally, pack the cut test strips into test strip holders for easy storage and transportation. Store the packed test strips in a dehumidifying cabinet for later use. The use of a dehumidifying cabinet can prevent the test strips from getting damp, maintain their performance stability, and prepare them for subsequent testing.

[0164] 2.9 Preparation of reagents and standards

[0165] In immunochromatographic assays, preparing a series of calibrators at different concentrations is crucial for establishing a standard curve and evaluating the performance of test strips. The multilocular Echinococcus larvae AgB antigen prepared by the Northwest Plateau Institute of Biology, Chinese Academy of Sciences, can serve as a representative analyte. By diluting it into calibrators of different concentrations, it can be used to evaluate key parameters of the test strips, such as sensitivity, specificity, and repeatability.

[0166] The following is a detailed description of the calibration sample preparation process:

[0167] Diluting Echinococcus multilocularis AgB antigen: First, dilute the AgB antigen to predetermined concentration values ​​using the standard diluent, corresponding to six levels from A to F of the standard. The specific concentrations are: Level A 0 ng / mL, Level B 10 ng / mL, Level C 100 ng / mL, Level D 500 ng / mL, Level E 1500 ng / mL, and Level F 6000 ng / mL. These concentration values ​​cover a range from low to high, simulating the antigen concentration in actual samples and helping to evaluate the detection performance of the test strip at different concentration levels.

[0168] Aliquoting of calibrators: Aliquot each diluted calibrator into individual 1mL vials. Aseptic technique must be maintained during the aliquoting process to avoid contamination and cross-contamination.

[0169] Labeling and Storage: Clearly label each 1mL vial with the concentration level and preparation date of the calibrator for easy identification and tracking. Store the dispensed calibrator at 4°C to help maintain the stability and activity of the antigen and prevent performance degradation due to temperature changes.

[0170] Preparation: Store the calibration samples properly in a dehumidifier or refrigerator, and ensure they are fully warmed to room temperature before use to guarantee the accuracy of the test results. Before using the calibration samples for experiments, check their expiration date and storage conditions to ensure the reliability of the reagents.

[0171] 3. Method for detecting Echinococcus multilocularis larvae using fluorescent microsphere immunochromatographic test strips.

[0172] Dilute the serum sample or reagent standard with standard diluent at a ratio of 1:20 and vortex thoroughly. Take 60 μL of the diluted suspension and add it dropwise to the sample pad of the test strip. Incubate at room temperature for 9 minutes to complete the antigen-antibody reaction. Place the test strip in the aQcare TRF time-resolved fluorescence immunochromatography test strip instrument for detection. The instrument will scan the test strip and record the fluorescence intensity on the T and C lines, respectively, using the relative peak height H. T And Hc represents. Calculate H T The ratio of Hc to R(H T / H C The concentration of EMAGB antigen in the sample was analyzed based on the standard curve.

[0173] Experimental Techniques

[0174] When the serum sample (containing EMAGB antigen) is added to the sample pad, it moves forward under capillary action. Within the conjugation pad, the EMAGB antigen specifically binds to the EMAGB antibody (CM-EUs-Ab1) conjugated with carboxylated europium chelate fluorescent nanospheres, forming an antigen-antibody complex (CM-EUs-Ab1-Ag). This complex continues to chromatographically advance through capillary action to the detection zone (T line), where it is captured by the coated EMAGB antibody (Ab2), forming a sandwich complex (CM-EUs-Ab1-Ag-Ab2) and immobilized on the T line. Excess fluorescent microsphere labeling continues to move forward to the control zone (C line), where the IgY conjugated with carboxylated europium chelate fluorescent nanospheres (CM-EUs-RIgY) is captured by the coated goat anti-IgY antibody and immobilized on the C line. Excess fluorescent microsphere labeling is ultimately absorbed by absorbent paper.

[0175] 4. Methodological Evaluation

[0176] 4.1 Establishment of dose-response curves

[0177] Self-made calibration standards A through F were repeatedly tested, with five tests performed for each concentration sample to ensure data reliability. The H values ​​from these tests were calculated. T / H C The average of the values ​​yielded a set of data. The logarithm of the concentration of each standard was plotted on the x-axis, H... T / H CThe logarithm of the mean (M) was used as the ordinate, and a linear fit was performed using a double log-log mathematical model and the least squares method. A dose-response curve was established within the dose concentration range between calibrators B and F, and the correlation coefficient (r) of this curve should reach or exceed 0.9900 to ensure the goodness of fit and the reliability of the detection system. This process is crucial for evaluating the sensitivity and linear range of the test strips, ensuring the accuracy and consistency of the test results.

[0178] 4.2 Sensitivity Experiment

[0179] Perform 20 repeated measurements of the zero calibrator (A) to ensure reliable data. Calculate H from these 20 measurements. T / H C The mean (denoted as M) and standard deviation (SD) of the values ​​provide a statistical description of the background value. To determine the analytical sensitivity, H is used... T / H C The mean (M) is added to twice the standard deviation (M+2SD), and then the background value is subtracted from this value. Finally, the result is substituted into the previously established dose-response curve to calculate the corresponding concentration value. This concentration value is the analytical sensitivity of this test strip, which is the lowest EMAGB concentration that can be reliably detected. This method ensures high sensitivity and accuracy of the detection system, providing reliable test results for clinical applications.

[0180] 4.3 Recovery Rate Test

[0181] To verify the accuracy and reliability of the EMAGB time-resolved fluorescent microsphere immunochromatographic quantitative test strip, different concentrations of pure EMAGB antigen were added to three serum samples with known concentrations at a volume ratio of 1:100. Multiple parallel measurements were performed for each sample with added EMAGB antigen to obtain a set of measured values ​​(M) and standard deviations (SD). These data allowed us to calculate the recovery rate for each sample. Ideally, the recovery rate ranged from 85% to 115%, indicating that the test strip has good accuracy and consistency in the detection of EMAGB antigen.

[0182] 4.4 Precision Experiment

[0183] Samples of different concentrations were selected for testing. Each sample was tested repeatedly, and the mean, standard deviation, and CV (CV, calculated by dividing the standard deviation by the mean) of the test values ​​were calculated to assess the consistency and reliability of the test results.

[0184] 4.5 Hook Effect Experiment

[0185] To investigate the hook effect of the EMAGB time-resolved fluorescent microsphere immunochromatographic quantitative assay test strip, EMAGB antigen was diluted into a series of high-concentration gradient samples in calibration buffer. These samples were then used for detection by the test strip to observe and evaluate the strip's detection performance at high concentrations. The hook effect, also known as the hook-Jorgensen effect, refers to the phenomenon in some immunoassays where, when the concentration of the analyte (EMAGB antigen in this case) exceeds a certain threshold, the detection signal no longer correlates with the analyte concentration but instead decreases. This phenomenon is usually due to antibody or binding site saturation, resulting in excess analyte failing to bind effectively, thus affecting the detection results. By preparing high-concentration gradient samples and using the test strip for detection, the concentration threshold at which the hook effect begins can be determined, and the applicability and reliability of the test strip in the high-concentration range can be evaluated. This experiment is significant for understanding the detection limitations of the test strip, optimizing detection conditions, and ensuring accuracy and consistency in clinical applications.

[0186] 4.6 Dilution Linearity Experiment

[0187] To evaluate the parallelism of the EMAGB time-resolved fluorescent microsphere immunochromatographic quantitative assay test strip at different dilutions, three serum samples of known concentrations were selected. These samples were serially diluted using calibration buffer, with multiple parallel tests performed at each dilution level. Parallelism analysis is a method for assessing the consistency of a detection method by comparing the test results of the same sample at different dilution levels to evaluate the consistency and reproducibility of the assay. In this process, the test values ​​for each dilution level were recorded, and the mean, standard deviation, and coefficient of variation (CV) of these values ​​were calculated. By comparing the test results at different dilution levels, the consistency of the test strip at different dilutions can be evaluated, thereby determining its reliability and applicability in serum sample testing. This analysis is crucial for ensuring the efficiency and accuracy of the test strip in practical applications, especially when processing serum samples, where serum concentrations can vary significantly. Therefore, the evaluation of dilution parallelism can provide important reference information for clinical laboratories, helping to optimize sample processing procedures and improve test quality.

[0188] 4.7 Anti-interference test

[0189] To evaluate the interference resistance of the EMAGB fluorescent microsphere immunochromatographic quantitative test strip, the following experiment was designed. Common interfering substances were added to EMAGB calibrators of known concentrations, and then the test strip was used for detection.

[0190] First, bilirubin solution (5 mg / mL) was added to standards C, D, and E, adjusting the final concentrations to 0.08 mg / mL, 0.04 mg / mL, 0.02 mg / mL, 0.01 mg / mL, and 0 mg / mL, respectively. The total bilirubin molar concentration at 0.08 mg / mL was 204.5 μmol / L. Next, a triglyceride mixture solution (10 mg / mL) was added to the same standards, setting the final concentrations to 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0 mg / mL, corresponding to a triglyceride content of approximately 2.26 mmol / L. Finally, hemoglobin solution (8 mg / mL) was added to the standards, resulting in final hemoglobin concentrations of 4 mg / mL, 2 mg / mL, 1 mg / mL, and 0.5 mg / mL. After adding these interfering substances, the samples were tested according to the specific operating steps described above for the test strip. By analyzing the measurement deviations in the test results, the stability and anti-interference ability of the test strips in the face of these common interfering substances can be evaluated.

[0191] 4.8 Stability Test

[0192] In this experiment, a batch of test strips was selected and placed in an environment of 37°C for 7 consecutive days. This temperature is close to normal human body temperature, simulating the conditions that the test strips might encounter in actual use. During the accelerated aging experiment, we regularly observed the physical appearance of the test strips, checking for any visible changes, such as color changes, cracks, or other damage. These observations help us understand the physical stability of the test strips during long-term storage or use. In addition to the appearance inspection, the performance of the test strips at each stage was also evaluated. This included evaluating their linear range, i.e., whether the relationship between the detection results and the EMAGB concentration remained consistent; analytical sensitivity, i.e., the test strip's ability to detect low concentrations of EMAGB; accuracy, i.e., how close the detection results are to the true values; and precision, i.e., the consistency and reliability obtained from repeated tests.

[0193] 4.9 Determination of Normal Reference Range

[0194] The normal reference range of human serum EMAGB was determined by testing plasma samples from healthy individuals using a self-made EMAGB fluorescent microsphere immunochromatographic test strip.

[0195] 4.10 Methodological Comparison

[0196] The concentration of EMAGB in clinical samples was accurately determined by parallel testing with a self-made EMAGB fluorescent microsphere immunochromatographic quantitative test strip and an ELISA diagnostic kit from Bioporto, Denmark. This parallel testing method helps to validate and compare the performance of two different detection technologies, ensuring the accuracy and reliability of the test results. In the statistical analysis of the data, linear regression analysis was used to assess the consistency between the two detection methods. By constructing a regression model, the relationship between the detection results of the self-made test strip and the ELISA kit was observed. Ideally, the detection results of the two methods should exhibit a high linear relationship, meaning that the detection results of the self-made test strip can well predict the results of the ELISA kit, and vice versa. Pearson's correlation analysis was used to assess the strength of the linear relationship between the two variables. In this study, the correlation coefficient between the detection results of the self-made test strip and the ELISA kit was calculated to quantify the correlation between the two methods. The correlation coefficient value ranges between -1 and 1, with values ​​close to 1 or -1 indicating a strong correlation, and values ​​close to 0 indicating no or very weak linear relationship. SPSS 13.0.0 and OriginPro 7.5 software were used for data processing and plotting.

[0197] The above methods were used to evaluate the methodology of the EMAGB fluorescent microsphere immunochromatographic quantitative test strips.

[0198] EMAGB fluorescent microsphere immunochromatographic quantitative test strips include:

[0199] Fluorescent microsphere immunochromatographic quantitative test strip: The binding pad contains 20B7D9 antibody and chicken IgG antibody labeled with fluorescent microspheres; 18F11G9 antibody is immobilized on the T line of the NC membrane; and anti-IgG antibody is immobilized on the C line of the NC membrane. Figure 1 As shown.

[0200] 20B7D95 Results and Discussion

[0201] Methodological evaluation criteria:

[0202] The microsphere coupling solution was prepared using a sample pad pretreatment buffer containing 1% S9 and 50 mmol / L PB (pH 7.0).

[0203] The coupling ratio of EMAGB antibody to europium chelate fluorescent nanospheres was 25 μg / mg;

[0204] The concentration of the T-line coated antibody was set at 2.0 mg / mL, while the concentration of EMAGB microspheres (microspheres conjugated with antibodies) was 0.5 mg / mL, and the reaction time was determined to be 9 min.

[0205] Under the above conditions, the performance of the test strips was evaluated for multiple indicators, including dose-response curve, analytical sensitivity, recovery rate, precision, hook effect, dilution linearity, interference, stability, and methodological comparison.

[0206] 5.1 Establishing a dose-response curve

[0207] To establish the mathematical model, a self-made calibration sample was first tested, with each concentration sample repeated five times to obtain the average value. Then, the H0 value for each concentration sample was calculated. T / H C The ratios were calculated, and the average of these ratios (M) was obtained. Next, the logarithm of the concentration values ​​of the calibrator was used as the x-axis, and H was... T / H C The logarithm of the mean (M) was used as the ordinate. A double log-log model was used, and the data were fitted to a straight line using the least squares method to obtain a fitted straight line. Dose-response curves were established for the fluorescent microsphere immunochromatographic quantitative detection test strips, and the results are shown below. Figure 2 As shown.

[0208] Analysis of the experimental data yielded the equation for the dose-response curve. The equation for the dose-response curve of the EMAGB time-resolved fluorescent microsphere immunochromatographic quantitative test strip is log(y) = -0.0145 + 0.0135log(x), with a correlation coefficient (r) as high as 0.9995. This result reveals that the EMAGB time-resolved fluorescent microsphere immunochromatographic quantitative test strip exhibits excellent dose-response linearity. Furthermore, the coefficient of variation (CV) of all calibrators was controlled below 10%, and no hook effect was observed within the investigated dose-response curve range, indicating that the obtained data quality meets stringent testing standards.

[0209] 5.2 Sensitivity Analysis

[0210] To determine the analytical sensitivity of the EMAGB fluorescent microsphere immunochromatographic quantitative test strip, 20 replicate assays were performed on the zero standard (A) to calculate H. T / H C The mean (M) and standard deviation (SD) of the ratios. Then, H... T / H C The mean value was added to twice the standard deviation (M+2SD), and the background value was subtracted from this value. The result was then substituted into the previously established dose-response curve equation to calculate the analytical sensitivity. Using this method, the analytical sensitivity of the self-made EMAGB time-resolved fluorescent microsphere immunochromatographic quantitative test strip was determined to be 5.7 ng / mL.

[0211] 5.3 Recovery rate experiment

[0212] To verify the accuracy of the self-made EMAGB time-resolved fluorescent microsphere immunochromatographic quantitative test strip, different concentrations of pure EMAGB antigen were added to three serum samples of known concentrations at a volume ratio of 1:100 (antigen:serum). Following the procedure described above, multiple parallel measurements were performed on each sample, and the measured values ​​(M) and standard deviations (SD) were recorded. The recovery rate was calculated using the formula: Recovery rate = Measured value / (Sample concentration + Added concentration) * 100%. The experimental results are summarized in Table 3, showing the recovery rate data of the self-made EMAGB test strip. Each sample was measured five times.

[0213] Table 3. Experimental results of recovery rate of self-made EMAGB test strips (n=5)

[0214]

[0215]

[0216] Experimental results showed that when pure EMAGB antigen was added to serum samples of different concentrations, the recovery rate of the self-made EMAGB test strips fell within the acceptable range of 85% to 115% at the low, medium, and high concentration levels, indicating that the test strips have high accuracy and meet the test kit's testing standards.

[0217] 5.4 Precision test results

[0218] To evaluate the precision of the EMAGB test strips, samples with medium and high concentrations were tested. Each sample was independently tested five times using the same batch of test strip reagents. The mean, standard deviation, and coefficient of variation (CV) of the test values ​​for each sample were then calculated. The CV was calculated by dividing the standard deviation by the mean. These detailed test results are summarized in Table 4.

[0219] Table 4. Precision experiment of EMAGB test strips (n=5)

[0220]

[0221] Based on the data in Table 4, we can observe that the internal coefficients of variation (CV) for the two samples with different concentrations were 2.56% and 4.62%, respectively, both less than 10%. These results indicate that the self-made EMAGB test strips perform well in terms of precision, and the consistency and repeatability of their test results meet the requirements specified in the kit.

[0222] 5.5 Experimental Analysis Results of the Hook Effect

[0223] After diluting the EMAGB antigen and preparing high-concentration samples at different gradients, the samples were tested using EMAGB test strips. According to... Figure 3 The test results showed that when the EMAGB concentration in the sample exceeded 1200 ng / mL, H was observed. T / H C The ratio did not continue to rise linearly, but instead showed a downward trend. This phenomenon confirms the Hook effect occurring in high-concentration samples, i.e., signal saturation or inhibition due to antigen excess, affecting the accuracy of the test results.

[0224] 5.6 Results of Dilution Linearity Experiment

[0225] For linear analysis of serum samples, three serum samples with known antigen concentrations (200 ng / mL, 400 ng / mL, and 600 ng / mL) were selected and serially diluted using calibration buffer at dilution factors of 0.1, 0.2, 0.4, and 0.8. The diluted samples were then tested using the EMAGB test strip of this invention. The test results are as follows: Figure 4 As shown, the EMAGB test strips exhibited good linearity within the tested dilution range, indicating their ability to perform accurate detection at different dilution ratios.

[0226] 5.7 Analysis Results of Anti-interference Experiment

[0227] To evaluate the interference resistance of the self-made EMAGB test strips, experiments were conducted on calibration samples C, D, and E. Multiple final concentration gradients were established by adding different concentrations of bilirubin solution, triglyceride mixture solution, and hemoglobin solution. Specifically, the final concentration gradients for bilirubin were 0.08 mg / mL, 0.04 mg / mL, 0.02 mg / mL, 0.01 mg / mL, and 0 mg / mL; for the triglyceride mixture, they were 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0 mg / mL; and for hemoglobin, they were 4 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, and 0 mg / mL. At each final concentration, samples were measured according to the specific operating procedures of the test strips. The experimental results are recorded in Tables 5-7, which analyze the performance of the test strips in the presence of these common interfering substances. All measurements in Tables 5-7 are antigen concentrations, in ng / mL.

[0228] Table 5. Results of bilirubin interference experiment using EMAGB test strips (n=5)

[0229]

[0230] Table 6. Results of the experiment on interference from triglyceride mixtures in EMAGB test strips (n=5)

[0231]

[0232] Table 7. Results of hemoglobin interference experiment using EMAGB test strips (n=5)

[0233]

[0234] The interference experiments clearly show that even in the presence of high concentrations of bilirubin (0.08 mg / mL), triglyceride mixture (2 mg / mL), and hemoglobin (4 mg / mL), the results of measurements using the self-made EMAGB test strips on each calibrator remained within acceptable measurement deviations. This finding indicates that the EMAGB test strips remain accurate and reliable even in the presence of these high concentrations of potential interfering substances, unaffected by their interference. Furthermore, all data obtained in the anti-interference experiments met the assay requirements specified in the kit, further confirming the quality and reliability of the test strips.

[0235] 5.8 Stability Experiment Analysis Results

[0236] In the stability study of the EMAGB test strips, the strips were stored at 37°C for 7 days, followed by assays on the calibrators. The results were fitted using a LOG-LOG_B mathematical model, and the linear correlation coefficient r of the dose-response curve was 0.9905, indicating a high linear relationship. The analytical sensitivity reached 5.7 ng / mL, which meets the assay requirements of the kit. Furthermore, the recovery rates were all within the range of 90%-110%, meeting the accuracy evaluation standards of the kit. Regarding precision, the internal CV values ​​were all below 10%, which also meets the precision evaluation requirements of the kit. Based on these stability study results, we can conclude that the self-made EMAGB test strips exhibit good stability and reliability in storage and use, meeting the quality standards of the kit.

[0237] 5.9 Determine the normal reference range

[0238] Using a self-made EMAGB test strip, we conducted extensive testing on 72 serum samples from healthy individuals to analyze the distribution characteristics of EMAGB levels in the serum of healthy individuals. Specific results are shown in Table 8 and... Figure 5 These data provide valuable information about the distribution of EMAGB serum levels in healthy individuals.

[0239] Table 8. Statistical results of EMAGB level detection in serum of healthy individuals using EMAGB test strips (n=72)

[0240] Case number Measured value ng / mL Case number Measured value ng / mL Case number Measured value ng / mL 1 0.50 25 1.03 49 0.32 2 0.23 26 1.00 50 0.76 3 0.34 27 0.60 51 0.62 4 1.13 28 1.16 52 0.49 5 0.51 29 0.64 53 0.34 6 0.32 30 0.93 54 1.04 7 0.92 31 0.15 55 0.85 8 0.68 32 0.71 56 0.76 9 0.49 33 0.77 57 0.86 10 1.17 34 0.47 58 0.54 11 1.17 35 0.38 59 0.86 12 0.13 36 0.71 60 0.23 13 0.64 37 0.39 61 0.38 14 1.02 38 0.51 62 0.62 15 0.98 39 0.90 63 0.35 16 0.78 40 0.28 64 1.07 17 0.78 41 0.11 65 0.81 18 0.29 42 0.46 66 0.22 19 1.10 43 0.90 67 0.95 20 0.88 44 0.52 68 0.24 21 0.72 45 0.45 69 1.07 22 0.35 46 0.55 70 0.26 23 0.15 47 1.10 71 0.90 24 0.60 48 1.09 72 0.81

[0241] In a study of EMAGB detection in serum samples from 72 healthy individuals, analysis of the data in the table showed that EMAGB levels ranged from 0 to 1.5 ng / mL. Overall, serum EMAGB levels in healthy individuals exhibited a skewed distribution. Therefore, 1.5 ng / mL should be the noise limit for the test strip, and a positive cutoff value was determined by multiplying the noise level by four times, ultimately set at 6 ng / mL. Thus, when using the self-made EMAGB quantitative test strip, the normal reference value for EMAGB in the serum of healthy individuals should not exceed 6 ng / mL.

[0242] 5.10 Methodological Comparison Analysis Results

[0243] In the comparative study, the self-made EMAGB time-resolved fluorescent microsphere immunochromatographic quantitative test strip was used as the evaluation reagent, and the echinococcosis antibody detection reagent (clinical diagnostic kit) was used as the control reagent. Parallel tests were performed on 100 clinical serum samples, and the results are shown in Table 9. Statistical analysis showed that the clinical sensitivity concordance rate of the self-made test strip was 100% (40 / 40), while the clinical specificity concordance rate reached 100% (60 / 60). Further k-coefficient testing showed k = 1.0, indicating a high statistically significant agreement between the self-made test strip and the antibody method.

[0244] Table 9. Comparison results of self-made EMAGB test strips and control reagents.

[0245]

[0246] Positive compliance rate = 40 / 40 × 100% = 100%; Negative compliance rate = 60 / 60 × 100% = 100%; Overall compliance rate = 100 / 100 × 100% = 100%; Kappa = 1.0.

[0247] In summary, the above detection process employed a combination of time-resolved fluorescent microspheres and immunochromatography to develop a novel EMAGB test strip using a double-antibody sandwich method. This test strip is a point-of-care testing tool that is rapid and convenient, capable of completing the test in just 9 minutes, significantly reducing sample processing time and offering a clear time advantage compared to other detection methods.

[0248] The self-made EMAGB test strip demonstrated excellent performance in detecting serum EMAGB, with a wide linear range of 5-1200 ng / mL and an analytical sensitivity of 5.7 ng / mL, covering the possible concentration range of EMAGB. Compared with ELISA and CMIA methods, the self-made test strip showed significant advantages in several aspects.

[0249] Quantitative assays and correlation analyses were performed on multiple serum samples using existing ELISA antibody detection methods and the test strip provided in this invention. The results showed a correlation coefficient (k) of 1.0 between the two methods, indicating a significant correlation. Furthermore, the coefficient of variation for repeated tests using the EMAGB test strip provided in this invention was less than 10%, meeting the reagent requirements. High concentrations of MYO, CTNI, CKMB, and proBNP showed no significant cross-reactivity with the self-made EMAGB test strip, and the test strip was not affected by high concentrations of hemoglobin, triglyceride mixtures, or hemoglobin when detecting samples.

[0250] Using a self-made test strip, the reference range for EMAGB in healthy serum samples was determined to be 6 ng / mL through analysis of 72 samples. Based on these results, the self-made EMAGB time-resolved fluorescent microsphere immunochromatographic quantitative test strip exhibits high sensitivity, rapid reaction time, and accuracy and reliability, making it suitable for broad clinical applications.

Claims

1. A monoclonal antibody against multilocular echinococcosis, characterized in that: The monoclonal antibody against Echinococcus multilocularis is 20B7D9. The amino acid sequence of its heavy chain is shown in SEQ ID NO.1, and the amino acid sequence of its light chain is shown in SEQ ID NO.

2.

2. A nucleotide sequence expressing the multilocular Echinococcus monoclonal antibody 20B7D9 as described in claim 1.

3. An expression cell expressing the multilocular echinococcosis monoclonal antibody 20B7D9 as described in claim 1.

4. An antibody for detecting Echinococcus multilocularis, characterized in that... It contains the monoclonal antibody 20B7D9 containing the multilocular echinococcosis larvae as described in claim 1.

5. A monoclonal antibody against multilocular echinococcosis, characterized in that: The monoclonal antibody against Echinococcus multilocularis is 18F11G9, the amino acid sequence of its heavy chain is shown in SEQ ID NO.3, and the amino acid sequence of its light chain is shown in SEQ ID NO.

4.

6. A nucleotide sequence expressing the multilocular Echinococcus monoclonal antibody 18F11G9 as described in claim 5.

7. An expression cell expressing the multilocular echinococcosis monoclonal antibody 18F11G9 as described in claim 5.

8. An antibody for detecting Echinococcus multilocularis, characterized in that... It contains the monoclonal antibody 18F11G9 against the multilocular echinococcosis larvae as described in claim 5.

9. The antibody for detecting Echinococcus multilocularis according to claim 4 or 8, characterized in that: It consists of monoclonal antibodies against Echinococcus multilocularis 20B7D9 and 18F11G9.

10. The application of the antibody for detecting Echinococcus multilocularis as described in claim 9 in the double antibody sandwich method for detecting Echinococcus multilocularis.

11. The application of the antibody for detecting multilocular echinococcosis as described in claim 10 in the double-antibody sandwich method for detecting multilocular echinococcosis, characterized in that: Of the antibodies, Echinococcus multilocularis monoclonal antibody 20B7D9 is the labeling antibody, and Echinococcus multilocularis monoclonal antibody 18F11G9 is the coating antibody.

12. A fluorescent microsphere immunochromatographic test strip for detecting multilocular Echinococcus larvae using a double-antibody sandwich method, characterized in that: It includes the antibody for detecting Echinococcus multilocularis as described in claim 9.

13. The fluorescent microsphere immunochromatographic test strip for detecting Echinococcus multilocularis larvae as described in claim 12, characterized in that: It also includes sample pads, conjugate pads, fluorescent microspheres, and NC membranes.

14. The fluorescent microsphere immunochromatographic test strip for detecting Echinococcus multilocularis larvae as described in claim 13, characterized in that: The fluorescent microspheres are carboxylated europium chelate fluorescent nanospheres, and the particle size of the fluorescent microspheres is 285nm-315nm; And / or the sample pad is made of 8892 glass fiber; And / or the bonding pad is made of 2010 glass fiber.

15. The structure of the fluorescent microsphere immunochromatographic test strip for detecting Echinococcus multilocularis larvae according to any one of claims 12-14, characterized in that: According to the flow direction of the sample to be tested, a sample pad, a conjugate pad, and an NC membrane are set in sequence; the conjugate pad is immobilized with labeled antibodies labeled with fluorescent microspheres; and the NC membrane is immobilized with coated antibodies.

16. The structure of the fluorescent microsphere immunochromatographic test strip for detecting Echinococcus multilocularis larvae according to claim 15, characterized in that: Following the flow direction of the sample to be tested, the sample pad, conjugate pad, nitrocellulose membrane, and absorbent paper are fixed sequentially on the PVC base plate.

17. The non-diagnostic detection method of the fluorescent microsphere immunochromatographic test strip for detecting Echinococcus multilocularis larvae according to any one of claims 12-14, characterized in that: The sample to be tested is dropped onto the sample pad and left for 9-12 minutes. The presence or absence of Echinococcus multilocularis in the sample is determined by the presence or absence of fluorescence in the antibody-coated region fixed on the NC membrane. The content of Echinococcus multilocularis in the sample is calculated based on the fluorescence intensity of the antibody-coated region fixed on the NC membrane.

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