Time-resolved fluorescence immunochromatography test strip for detecting IgM (Immunoglobulin M) of cynoglossus semilaevis

Abstract

The invention discloses a time-resolved fluorescence immunochromatography test strip for detecting IgM (immunoglobulin M) of cynoglossus semilaevis, which is characterized in that an anti-IgM monoclonal antibody of cynoglossus semilaevis secreted by a hybridoma cell strain CsIgM-39 marked by time-resolved fluorescent microspheres is used as a detection marking antibody on a combination pad; an anti-cynoglossus semilaevis IgM monoclonal antibody secreted by a hybridoma cell strain CsIgM-12 is used as a detection line (T line) coating antibody, so that the time-resolved fluorescence immunochromatography test strip for detecting the cynoglossus semilaevis IgM is constructed. The titers of the two monoclonal antibodies are 128000 or above, the test strip is good in specificity, high in sensitivity, strong in repeatability and convenient and fast to operate, the detection time is only 10 minutes, the detection sensitivity can reach 0.00625 mu g / mL, the IgM content in serum and plasma of the cynoglossus semilaevis can be accurately detected, and the test strip has important significance for evaluating the health condition and immune function of the cynoglossus semilaevis.

Description

Technical Field

[0001] This invention belongs to the fields of immunology and biotechnology, and specifically relates to a time-resolved fluorescence immunochromatographic test strip for detecting immunoglobulin M (IgM) in half-smooth tongue sole. Background Technology

[0002] Half-smooth tongue sole ( Cynoglossus semilaevis As an important marine economic fish in my country, the aquaculture industry of the tongue sole is expanding rapidly. However, with the development of intensive aquaculture, various bacterial (such as Vibrio and Edwardsiella tarda) and viral diseases frequently break out, causing huge economic losses to the aquaculture industry. The immune system of the tongue sole plays a crucial role in resisting pathogen invasion and recovering from disease infection. Immunoglobulin M (IgM), as the most important circulating antibody in the tongue sole, is a major effector molecule of humoral immunity in fish, and its level directly reflects the humoral immune status of the tongue sole. Detecting the IgM content of tongue sole is a key means of assessing its overall immune function and health status. Baseline values ​​can be used to establish a health benchmark, while a significant increase in content usually indicates that the immune system is activated, while persistently low levels may indicate immunosuppression, malnutrition, or a state of chronic stress. Regularly monitoring the IgM levels of farmed tongue sole populations can effectively predict potential health risks and is an important basis for scientific aquaculture management.

[0003] Conventional methods for detecting IgM in fish mainly include enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunoturbidimetry. Among these, ELISA is considered the gold standard due to its high sensitivity and specificity, enabling quantitative or semi-quantitative analysis. However, these methods heavily rely on large-scale instruments, professional operators, and complex laboratory environments. The detection process is cumbersome and time-consuming (usually requiring several hours), and the sample throughput is limited, making them difficult to apply in field settings such as aquaculture ponds and hatcheries.

[0004] Existing rapid on-site detection technologies include colloidal gold immunochromatographic test strips, which primarily rely on visual observation of band color intensity for approximate judgment. While offering advantages such as ease of operation and rapid, intuitive results (within 15 minutes), these are essentially qualitative or semi-quantitative methods. They are highly susceptible to subjective factors, cannot provide precise concentration values, have poor repeatability, and limited sensitivity, failing to meet the needs of applications requiring precise data, such as monitoring subtle changes in antibody levels and assessing immune status. Time-resolved fluorescence immunoassay (TRFIA) technology utilizes lanthanide chelates (such as europium Eu)... 3+Time-Resolved Fluorescent Immunochromatographic (TRFIA) is an ultra-micro detection technology that uses TRFIA as a marker. Leveraging its long fluorescence lifetime, this technology employs a time-delay measurement strategy to completely eliminate interference from short-lived background fluorescence in the sample matrix, resulting in extremely high signal-to-noise ratio and detection sensitivity. Combining the ultra-high sensitivity, wide dynamic range, and excellent quantitative capabilities of TRFIA with the speed and convenience of immunochromatography, the resulting time-resolved fluorescence immunochromatographic test strip (TRFICA) is one of the best solutions for achieving accurate on-site quantitative detection.

[0005] However, to date, there have been no reports on the use of time-resolved fluorescence immunochromatographic strips for detecting IgM in half-smooth tongue sole. Summary of the Invention

[0006] This invention addresses the aforementioned technical problems in the prior art by providing a time-resolved fluorescence immunochromatographic test strip for detecting IgM in half-smooth tongue sole.

[0007] The technical solution of this invention is: a time-resolved fluorescence immunochromatographic test strip for detecting tongue sole IgM, comprising a base plate, wherein the base plate has a sample pad, a conjugate pad, a reaction plate, and an absorbent pad sequentially overlapped; the conjugate pad is coated with a detection label antibody and a quality control label antigen; the reaction plate has a detection line and a quality control line; the detection label antibody is a time-resolved fluorescent microsphere-labeled hybridoma cell line CsIgM-39 secreted by an anti-tongue sole IgM monoclonal antibody; the detection line (T line) is coated with an anti-tongue sole IgM monoclonal antibody secreted by a hybridoma cell line CsIgM-12; the accession number of the hybridoma cell line CsIgM-39 is CGMCC No. 46592, and the accession number of the hybridoma cell line CsIgM-12 is CGMCC No. 46591.

[0008] The hybridoma cell line CsIgM-12 is classified and named as follows: Hybridoma cell line 12 secreting monoclonal antibody against half-smooth tongue sole IgM; depositary institution: China General Microbiological Culture Collection Center; address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; deposit date: September 11, 2025; accession number: CGMCC No. 46591; The hybridoma cell line CsIgM-39 is classified and named as follows: Hybridoma cell line 39 secreting monoclonal antibody against half-smooth tongue sole IgM; depositary institution: China General Microbiological Culture Collection Center; depositary address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; deposit date: September 11, 2025; deposit number: CGMCC No. 46592.

[0009] Preferably, the quality control labeling antigen is rabbit IgG labeled with time-resolved fluorescent microspheres, and the quality control line is coated with goat anti-rabbit IgG antibody.

[0010] Preferably, the test strip is placed inside a cartridge, which has a sample application hole and a detection and observation area. The sample application hole is located at the position of the sample pad, and the detection and observation area is located at the position of the reaction plate.

[0011] This invention utilizes a time-resolved fluorescent immunochromatographic test strip for detecting tongue sole IgM. The strip uses a time-resolved fluorescent microsphere-labeled hybridoma cell line CsIgM-39 as the binding pad marker antibody, and a hybridoma cell line CsIgM-12 as the detection line (T-line) coating antibody. Both monoclonal antibodies have titers above 128,000. The test strip exhibits high specificity, sensitivity, and repeatability, and is convenient and quick to use, requiring only 10 minutes for detection. The detection sensitivity reaches 0.00625 μg / mL, accurately detecting IgM levels in the serum and plasma of tongue sole, which is of great significance for assessing the health status and immune function of tongue sole. Attached Figure Description

[0012] Figure 1 This is an SDS-PAGE result of purified serum IgM from half-smooth tongue sole according to an embodiment of the present invention.

[0013] Figure 2 This is a WB analysis result of monoclonal antibodies secreted by the half-smooth tongue sole hybridoma cell lines CsIgM-12 and CsIgM-39 in an embodiment of the present invention.

[0014] Figure 3 This is a schematic diagram of the internal structure of the time-resolved fluorescence immunochromatographic strip for detecting IgM in half-smooth tongue sole according to an embodiment of the present invention.

[0015] Figure 4 This is a schematic diagram of the external structure of the time-resolved fluorescence immunochromatographic test strip for detecting IgM in half-smooth tongue sole according to an embodiment of the present invention.

[0016] Figure 5 This is a standard curve showing the relationship between the IgM concentration of tongue sole and the T / C value detected by the test strip in an embodiment of the present invention.

[0017] Hybridoma cell line No. 12, which secretes monoclonal antibodies against half-smooth tongue sole IgM, was deposited on September 11, 2025; depositary institution: China General Microbiological Culture Collection Center (CGMCC); accession number: CGMCC No. 46591.

[0018] Hybridoma cell line No. 39, which secretes monoclonal antibodies against half-smooth tongue sole IgM, was deposited on September 11, 2025; depositary institution: China General Microbiological Culture Collection Center (CGMCC); accession number: CGMCC No. 46592. Detailed Implementation

[0019] I. Preparation of the IgM monoclonal antibody against half-smooth tongue sole according to the present invention (a) Methods 1. Purification of IgM from the serum of half-smooth tongue sole Blood was collected from the tail vein of healthy tongue sole. After being placed at room temperature for 1 hour, the sample was incubated overnight at 4 °C. The next day, the sample was centrifuged at 3000 g for 20 min, and the supernatant serum was collected. The serum was mixed with an equal volume of PBS, and saturated ammonium sulfate was added dropwise while stirring until the final ammonium sulfate mass fraction was 50%. After thorough stirring, the sample was incubated overnight at 4 °C. The next day, the sample was centrifuged at 12000 g for 30 min, and the precipitate was collected. The precipitate was dissolved in an appropriate amount of 0.02 M sodium phosphate buffer and placed in a dialysis bag. The ammonium sulfate was removed by dialyzing with the same buffer. The dialyzed sample was filtered through a 0.22 μm filter and then placed in a pre-equilibrated Protein A affinity chromatography column for further purification. The collected sample was further concentrated by ultrafiltration, and the purity of IgM protein was determined by SDS-PAGE electrophoresis. The IgM protein concentration was determined using the Bradford method.

[0020] 2. Preparation of hybridoma cells Four-week-old BALB / c mice were immunized with tongue sole IgM as the antigen in four doses, each dose being 0.1 mL. The specific immunization procedure was as follows: basal immunization: tongue sole IgM was mixed with Freund's complete adjuvant in an equal ratio and administered via intraperitoneal injection; two weeks later, the first booster immunization: tongue sole IgM was mixed with Freund's incomplete adjuvant in an equal ratio and administered via intraperitoneal injection; three weeks later, the second booster immunization was administered via tail vein injection without adjuvant; four weeks later, the third booster immunization was administered via tail vein injection without adjuvant; three days after the third booster immunization, the mice were sacrificed by cervical dislocation, and the spleen was harvested for cell fusion.

[0021] Under aseptic conditions, spleen cells from immunized mice were fused with SP2 / 0 myeloma cells using 45% PEG1500. The fused cells were resuspended in HAT selective culture medium and added to 0.2 ml of feeder cells in 96-well cell culture plates. The plates were incubated at 37°C with 5% CO2 and the growth of hybridoma cells was observed using an inverted phase-contrast microscope. After approximately two weeks, the supernatant from the hybridoma cells was collected for analysis.

[0022] 3. Positive screening using indirect enzyme-linked immunosorbent assay (ELISA) The ELISA assay procedure is as follows: Coat the microplate with tongue sole IgM (5 μg / ml, 100 μl / well) and incubate overnight at 4 °C; wash three times with PBST for 5 min each time; block with 3% bovine serum albumin (BSA) (200 μl / well) and incubate at 37 °C for 1 h; wash three times with PBST for 5 min each time; add 100 μl / well of hybridoma cell culture supernatant (using myeloma cell culture supernatant as a negative control) and incubate at 37 °C for 1 h; wash three times with PBST for 5 min each time; add 100 μl / well of HRP-labeled goat anti-mouse Ig (1:3000) and incubate at 37 °C for 1 h; wash three times with PBST for 5 min each time; add 100 μl / well of TMB chromogenic solution and incubate at 37 °C in the dark for 30 min; add 50 μl of chromogenic stop solution. μl / well, read the OD value at 450 nm using an ELISA reader; a positive result is determined when the positive OD / negative OD (P / N) ratio is greater than 2.1.

[0023] 4. Cloning and screening of hybridoma cells secreting IgM monoclonal antibodies against half-smooth tongue sole Positive hybridoma cells were cloned using the limiting dilution method. The positive hybridoma cells were resuspended in 1640 medium and serially diluted 10-fold to 10⁻⁶. 2 Cells / ml: Take 1 ml of cell culture and add it to 9 ml of culture medium. Mix well and drop 0.1 ml into each well of a 96-well cell culture plate containing feeder cells. Select a well with only one hybridoma cell. When the cell has grown to more than 2 / 3 of the bottom of the well, take the supernatant and test it according to the method in step 3 above. The positive wells are cloned twice more according to the above method to screen for hybridoma cells that secrete half-smooth tongue sole IgM monoclonal antibody.

[0024] 5. Preparation of IgM monoclonal antibody against half-smooth tongue sole Collect the hybridoma cell culture supernatant, centrifuge at 5000g for 5 min, and collect the supernatant. After filtration through a 0.45 μm filter membrane, pack the supernatant into a pre-equilibrated Protein G affinity chromatography column for antibody purification, following the instructions in the product manual. The purified monoclonal antibody is dialyzed against PBS buffer, then concentrated by ultrafiltration, and the antibody concentration is determined using the Bradford method.

[0025] 6. Analysis of the recognition antigenic determinants of IgM monoclonal antibodies against half-smooth tongue sole Purified tongue sole IgM was transferred to a PVDF membrane after SDS-PAGE and electroporated at 200 mA for 1.5 h. The membrane was then blocked overnight with 5% BSA at 4 °C. After washing with PBST, the PVDF membrane was placed in the IgM monoclonal antibody to be detected and incubated overnight at 4 °C. After washing with PBST, the membrane was placed in HRP-labeled goat anti-mouse Ig and incubated on a shaker at room temperature for 1 h. The membrane was then washed with PBST again. Equal amounts of Enhanced Luminol Reagent and Oxidizing Reagent were diluted with an appropriate amount of ddH2O, mixed, and dropped onto a sealing film. The PVDF membrane was placed face down in contact with the luminescent reagent, and color development was performed for 1.5–2.0 min. The PVDF membrane was then flipped over, and the results were observed using a gel imaging system.

[0026] 7. Titer analysis of IgM monoclonal antibodies against tongue sole Antibody titers were determined using indirect ELISA: Semi-smooth tongue sole IgM was coated onto an ELISA plate (5 μg / ml, 100 μl / well) and incubated overnight at 4 °C; washed three times with PBST for 5 min each time; blocked with 3% bovine serum albumin (BSA), 200 μl / well, and incubated at 37 °C for 1 h; washed three times with PBST for 5 min each time; different dilutions of the target IgM monoclonal antibody (with PBS as a negative control) were added, 100 μl / well, and incubated at 37 °C for 1 h; washed three times with PBST for 5 min each time; HRP-labeled goat anti-mouse Ig (1:3000), 100 μl / well, was added and incubated at 37 °C for 1 h; washed three times with PBST for 5 min each time; TMB chromogenic solution was added, 100 μl / well, and incubated at 37 °C in the dark for 30 min; stop chromogenic solution was added, 50 μL / well. μl / well, and read the OD value at 450 nm using an ELISA reader; when the positive OD / negative OD (P / N) > 2.1, it is judged as positive, and the highest positive dilution is taken as the antibody titer.

[0027] (II) Results 1. Purification of serum IgM from half-smooth tongue sole After ammonium sulfate precipitation and Protein A affinity chromatography, the serum of half-smooth tongue sole was analyzed by SDS-PAGE, which showed two major protein bands with relative molecular masses of 82 and 25 kDa, respectively. Figure 1 These correspond to the heavy and light chains of IgM from the tongue sole, respectively. After ultrafiltration concentration, the IgM protein concentration was determined to be approximately 0.7 mg / mL using the Bradford method.

[0028] 2. Screening of hybridoma cells that secrete IgM monoclonal antibodies against half-smooth tongue sole After cell fusion, more than 100 hybridomas were generated after culture in selective medium containing HAT. Screening using indirect ELISA revealed that more than 20 hybridomas tested positive. Further screening using limiting dilution of the positive hybridoma clones yielded two hybridoma cell lines stably secreting anti-tongue sole IgM monoclonal antibodies: CsIgM-12 (CGMCC No. 46591) and CsIgM-39 (CGMCC No. 46592). Antibody titer analysis showed that both monoclonal antibodies had titers above 128,000.

[0029] 3. Analysis of antigenic determinants recognized by IgM monoclonal antibodies against half-smooth tongue sole According to the results of WB testing ( Figure 2 It was found that the monoclonal antibody secreted by hybridoma cell line CsIgM-12 recognizes the half-smooth tongue sole IgM antigenic determinant located on the light chain of IgM, while the monoclonal antibody secreted by hybridoma cell line CsIgM-39 recognizes the half-smooth tongue sole IgM antigenic determinant located on the heavy chain of IgM.

[0030] 4. Preparation of IgM monoclonal antibody against half-smooth tongue sole Monoclonal antibodies were obtained by co-purification from the culture supernatant of hybridoma cells CsIgM-12 using Protein G affinity chromatography, with a concentration of 9 mL and a concentration of 1.67 mg / mL. Monoclonal antibodies were also obtained by co-purification from the culture supernatant of hybridoma cells CsIgM-39 using Protein G affinity chromatography, with a concentration of 0.64 mg / mL.

[0031] II. Construction of a time-resolved fluorescence immunochromatographic strip for detecting IgM in half-smooth tongue sole The internal structure of the time-resolved fluorescence immunochromatographic test strip for detecting IgM in half-smooth tongue sole according to an embodiment of the present invention is as follows: Figure 3As shown, there is a PVC base plate 5, on which are arranged a sample pad 1, a conjugate pad 2, a reaction plate 3, and an absorbent pad 4, which are stacked in sequence. The contact portion between the sample pad 1 and the conjugate pad 2 is overlapped and pressed together. The contact portion between the conjugate pad 2 and one end of the reaction plate 3 is overlapped and pressed together. The other end of the reaction plate 3 and the contact portion of the absorbent pad 4 are overlapped and pressed together. The conjugate pad 2 is coated with time-resolved fluorescent microsphere-labeled hybridoma cell line CsIgM-39 (accession number CGMCC No. 46592) anti-semi-smooth tongue sole IgM monoclonal antibody and time-resolved fluorescent microsphere-labeled rabbit IgG. The reaction plate 3 is arranged with a detection line 6 (T line) coated with hybridoma cell line CsIgM-12 (accession number CGMCC No. 46591) anti-semi-smooth tongue sole IgM monoclonal antibody and a control line 7 (C line) coated with goat anti-rabbit IgG antibody.

[0032] The external structure of the time-resolved fluorescence immunochromatographic test strip for detecting IgM in half-smooth tongue sole according to embodiments of the present invention is as follows: Figure 4 As shown, the device comprises a housing 8 and a test strip housed within the housing 8. The housing 8 may be elongated and adapted to the shape of the test strip inside. The housing 8 includes a sample application hole 9 and a detection observation area 10. The sample application hole 9 is located at the sample pad 1 of the test strip, and the detection observation area 10 is located at the reaction plate 3 of the test strip, used to observe the changes in the test strip after detection.

[0033] The absorbent pad and base plate in the test strip are the same as in the prior art. The preparation methods for the sample pad, conjugate pad, and reaction plate are as follows: (a) Sample pad 1. Preparation of sample pad treatment solution Add 0.15g of bovine serum albumin and 1ml of Triton-100 to 100ml of 0.02M pH7.4 PBS buffer.

[0034] 2. Preparation of the sample pad Immerse the glass cellulose membrane (24*30cm) in 50ml of sample pad treatment solution for 30 minutes, then lay it flat on a mesh screen and dry it overnight in a drying oven. Cut it into 300mm*18.5mm sizes for later use.

[0035] (ii) bonding pad 1. Preparation of time-resolved fluorescent microsphere-labeled monoclonal antibodies (1) Take 400 μl of 0.01M pH 8.0 borate buffer into a 2ml centrifuge tube, add 100 μl of time-resolved fluorescent microspheres, vortex and mix well.

[0036] (2) Add 80 μl of EDC (10 mg / ml) solution and activate at room temperature with shaking for 15 minutes. Note: EDC should be prepared fresh and dissolved in deionized water.

[0037] (3) Centrifuge at 16000 rpm, 10℃, for 10 min, discard the supernatant, and reconstitute with 0.5 ml of 0.01 M / pH 8.0 borate buffer.

[0038] (4) Labeling: Add 30 μg of conjugated antibody (anti-half-smooth tongue sole IgM monoclonal antibody secreted by hybridoma cell line CsIgM-39 or rabbit IgG), mix well, and place in a shaker at 25°C to react in the dark for 2 hours.

[0039] (5) Blocking: Add 50 μl of 10% BSA solution for blocking, and place in a shaker at 25°C in the dark for 2 hours.

[0040] (6) Purification: Centrifuge at 16000 rpm, 10℃, for 10 min, discard the supernatant, reconstitute with 0.5 ml of 0.01 M / pH 8.0 borate buffer, wash 1-2 times, and after the last centrifugation, reconstitute with 100 μl of time-resolved coupling stock solution, sonicate, and store at 4℃ for later use. The time-resolved coupling stock solution is prepared as follows: 100 ml of 0.02 M pH 7.4 PBS buffer + 1.0 g of BSA + 0.5 ml of Tween-20 + 0.1 ml of ProClin 300.

[0041] 2. Preparation of the binding pad The time-resolved conjugate stock solution, time-resolved fluorescent microsphere-labeled monoclonal antibody, and time-resolved fluorescent microsphere-labeled rabbit IgG were diluted at a volume ratio of 100:2.5:1, mixed well, and then sprayed onto a glass cellulose membrane measuring 300 mm long x 15 mm wide using a gold sprayer at a parameter of 5 μl / cm. The membrane was then dried overnight at 37°C.

[0042] (III) Reaction Plate 1. Coating with nitrocellulose membrane: Cut the nitrocellulose membrane into pieces 300mm long and 25mm wide and paste them onto the marked lines on the PVC board. When pasting the membrane, try not to touch the membrane surface to avoid scratching it.

[0043] 2. Quality control line C: Dilute goat anti-rabbit IgG antibody to 1.0 mg / ml with 0.01 M pH 7.4 PBS, and set the coating parameter of the coating instrument to 1.0 μl / cm.

[0044] 3. Detection line T: Dilute the concentration of anti-semi-smooth tongue sole IgM monoclonal antibody secreted by hybridoma cell line CsIgM-12 to 0.6 mg / ml with 0.01 M pH 7.4 PBS, and set the coating parameter of the membrane scraping instrument to 1.0 μl / cm.

[0045] Dry overnight at 4.37℃.

[0046] III. Experiments related to the test strips constructed in the embodiments of the present invention (I) Detection Sensitivity Analysis Sensitivity is a core indicator for evaluating the performance of immunoassay test strips, directly determining the method's ability to detect low concentrations of target molecules. This invention uses a 700 μg / mL standard of tongue sole IgM as the starting material, and prepares a series of concentration gradients from 3, 2, 1.5, 1, 0.5, 0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125, and 0.00156 μg / mL using a dilution method. 100 μL of sample from each concentration gradient is added to the well of the time-resolved fluorescence immunochromatographic test strip constructed in this invention for detecting tongue sole IgM. After reacting at room temperature for 10 minutes, the T / C value is obtained using a dry fluorescence immunoassay analyzer. At least 15 negative control samples are tested under the same testing conditions. The fluorescence intensity ratio (T / C value) of the test line (T line) to the control line (C line) for each sample was recorded. The limit of detection (LoD) was calculated using the formula SLoD = Mean_blank + 3 × SD_blank, resulting in SLoD = 0.063. The results indicate that at a concentration of 0.00625 μg / mL, the T / C value of the test strip is 0.071, which is higher than the detection limit. Samples with concentrations lower than this have T / C values ​​less than 0.063. Therefore, the detection sensitivity of the test strip can reach 0.00625 μg / mL (Table 1).

[0047] Table 1. Sensitivity analysis of time-resolved fluorescence immunochromatographic test strips used for detecting IgM in tongue sole.

[0048] (ii) Suitability of the test sample and optimal dilution To determine the applicability of the test strip of this invention to different sample types of tongue sole and the optimal detection conditions, eight healthy tongue soles of similar weight (approximately 750g) were selected for testing. Blood was collected from the tail vein of each fish, and serum and plasma samples were prepared separately.

[0049] To determine the optimal dilution for serum sample detection, a series of serial dilutions were performed: 1:100, 1:1000, 1:5000, 1:10000, 1:15000, and 1:20000. 100 μL of sample from each concentration gradient was added to the sample well of the test strip. After reacting at room temperature for 10 minutes, the T / C values ​​were obtained using a dry fluorescence immunoassay analyzer. The results showed that at a serum dilution of 1:10000, the signal value crossed the hook effect interference and showed a reliable decreasing inflection point for the first time. This dilution indicates that the antigen-antibody reaction in the serum sample has entered the optimal ratio range, ensuring the accuracy of the detection results. Therefore, the optimal dilution for serum detection using the test strip is 1:10000 (Table 2).

[0050] Table 2. T / C values ​​of semi-smooth tongue sole serum samples at different dilution gradients

[0051] To determine the optimal detection dilution for plasma samples, a series of serial dilutions were performed: 1:100, 1:1000, 1:5000, 1:10000, 1:15000, and 1:20000. 100 μL of sample from each concentration gradient was added to the sample well of the test strip. After reacting at room temperature for 10 minutes, the T / C values ​​were obtained using a dry fluorescence immunoassay analyzer. The results showed that at a plasma dilution of 1:10000, the signal value also crossed the inflection point where a reliable decrease occurred for the first time after the hook effect interference. This dilution indicates that the antigen-antibody reaction in the plasma sample has entered the optimal ratio range, ensuring the accuracy of the detection results. Therefore, the optimal dilution for plasma detection using the test strip is also 1:10000 (Table 3).

[0052] Table 3. T / C values ​​of semi-smooth tongue sole plasma samples at different dilution gradients.

[0053] (III) Detection Specificity Analysis Specificity is a key parameter for evaluating the accuracy of immunoassay methods, reflecting the detection system's ability to specifically identify the target analyte. To comprehensively evaluate the specificity of this test strip, this invention selected eight common marine fish species as research subjects, including the tongue sole (…). Cynoglossus semilaevis ), turbot ( Paralichthys olivaceus ),turbot( Scophthalmus maximus Pearl Giant Grouper ( Epiμephelus fuscoguttatus ♀× Epiμ ephelus laμceolatus ♂), Red-spotted grouper ( Epiμephelus akaara ), Cloud-patterned grouper ( Epiμephelus moara Golden Tiger Grouper ( Epiμephelus fuscoguttatus ♀× Epiμephelus tukula ♂), Eastern Star Spot ( Plectropomus leopardus All serum samples were processed at the determined optimal dilution. 100 μL of each sample was added to the sample well of the test strip and reacted at room temperature for 10 minutes before being detected by a dry fluorescence immunoassay analyzer to obtain the T / C value.

[0054] The results showed that among all the tested fish serum samples, only the half-smooth tongue sole serum sample exhibited clear test and control lines. The serum samples from the other seven fish species only showed control lines, with no color development observed in the test lines, consistent with the negative control results. This phenomenon demonstrates that the time-resolved fluorescence immunochromatographic test strip constructed in this invention for detecting IgM in half-smooth tongue sole is only suitable for half-smooth tongue sole and is not suitable for detecting IgM content in the serum of other marine fish such as turbot, flounder, pearl grouper, red-spotted grouper, marbled grouper, golden tiger grouper, and red snapper (Table 4).

[0055] Table 4. T / C values ​​of serum from different fish species

[0056] (iv) Repeatability analysis of the test The repeatability of a detection method is a core indicator of its precision, reflecting the consistency and stability of results obtained by the method under identical conditions when independently measuring the same sample. To comprehensively evaluate the repeatability of this test strip, This invention selected eight tongue soles of similar size (approximately 750g), and collected blood from the tail vein of each fish to prepare serum and plasma samples. All samples were diluted 1:10000 and tested using time-resolved immunofluorescence assay strips. Each sample was measured 20 times in parallel to assess repeatability.

[0057] The results showed that the coefficient of variation for all serum and plasma samples was less than 10%, indicating that the detection system had excellent repeatability in both sample types (Table 5).

[0058] Table 5. Repeatability analysis of IgM detection in serum and plasma samples of half-smooth tongue sole.

[0059] (v) Determination of IgM content in serum of half-smooth tongue sole The T / C values ​​of different concentrations of half-smooth tongue sole IgM standard are shown in Table 6.

[0060] Table 6. T / C values ​​of different concentrations of tongue sole IgM standards

[0061] A standard curve was plotted using Graphpad Prism 10.1.2 software based on IgM concentration and the detection T / C value. Figure 5 The regression equation is y = 0.033 + (20.592 - 0.033) / (1 + (x / 2.532)^-0.592), R² = 0.996. The T / C value of the tested half-smooth tongue sole (750g) serum sample was 9.228. According to the standard curve, the IgM concentration in the sample was calculated to be 1.679 ug / mL. Since the serum sample was diluted 10,000 times with PBST during the test, the IgM concentration in the tested half-smooth tongue sole serum should be 16.79 mg / mL.

Claims

1. A time-resolved fluorescence immunochromatographic test strip for detecting IgM in half-smooth tongue sole, comprising a base plate (5), wherein the base plate (5) has a sample pad (1), a conjugate pad (2), a reaction plate (3), and an absorbent pad (4) sequentially overlapped thereon, wherein the conjugate pad (2) is coated with a detection label antibody and a quality control label antigen, and the reaction plate (3) has a detection line (6) and a quality control line (7), characterized in that: The detection marker antibody is a time-resolved fluorescent microsphere-labeled hybridoma cell line CsIgM-39 secreted by an anti-smooth tongue sole IgM monoclonal antibody. The detection line (6) is coated with an anti-smooth tongue sole IgM monoclonal antibody secreted by a hybridoma cell line CsIgM-12. The accession number of the hybridoma cell line CsIgM-39 is CGMCC No. 46592, and the accession number of the hybridoma cell line CsIgM-12 is CGMCC No. 46591.

2. The time-resolved fluorescence immunochromatographic test strip for detecting IgM in half-smooth tongue sole according to claim 1, characterized in that: The quality control marker antigen is time-resolved fluorescent microsphere-labeled rabbit IgG, and the quality control line (7) is coated with goat anti-rabbit IgG antibody.

3. The time-resolved fluorescence immunochromatographic test strip for detecting IgM in half-smooth tongue sole according to claim 2, characterized in that: The test strip is placed inside the housing (8), which has a sample application hole (9) and a detection observation area (10). The sample application hole (9) is located at the position of the sample pad (1), and the detection observation area (10) is located at the position of the reaction plate (3).

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