Double-antibody sandwich ELISA kit for detecting IgM of cynoglossus semilaevis

Abstract

The invention discloses a double-antibody sandwich ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting IgM of cynoglossus semilaevis. The double-antibody sandwich ELISA kit is constructed by taking anti-cynoglossus semilaevis IgM monoclonal antibodies secreted by a hybridoma cell strain CsIgM-12 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.46591 and a hybridoma cell strain CsIgM-39 with the preservation number of CGMCC No.46592 as a capture antibody and a detection antibody respectively. The titers of the two monoclonal antibodies are 128000 or above, the kit is good in specificity and high in sensitivity, the minimum detection concentration of the kit is 0.01 microgram / mL, the IgM content in the serum of the cynoglossus semilaevis can be accurately detected, and the kit has important significance for evaluating the health condition and immune function of the cynoglossus semilaevis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of immunology and biotechnology, and particularly relates to a double antibody sandwich ELISA kit for detecting Cynoglossus semilaevis IgM (immunoglobulin M). BACKGROUND

[0002] Cynoglossus semilaevis belongs to the order Pleuronectiformes, the family Cynoglossidae and the genus Cynoglossus, and is an important marine aquaculture fish. Cynoglossus semilaevis However, Cynoglossus semilaevis is susceptible to invasion by bacteria, viruses and parasites during the aquaculture process, which causes slow growth, decreased immunity and even mass death. The immune system of Cynoglossus semilaevis plays an important role in resisting pathogen invasion and recovering after disease infection. Immunoglobulin M (IgM) is the most important circulating antibody of Cynoglossus semilaevis, and its level directly reflects the humoral immune state of Cynoglossus semilaevis.

[0003] Detecting the IgM content of Cynoglossus semilaevis is a key means for evaluating the overall immune function and health status of Cynoglossus semilaevis. The basic value of IgM of Cynoglossus semilaevis can be used to establish a health benchmark, and a significant increase in the IgM content usually indicates that the immune system is activated, and a persistent low level may indicate immune suppression, malnutrition or a chronic stress state. Regular monitoring of the IgM level of a cultured Cynoglossus semilaevis population can effectively alert potential health risks and is an important basis for scientific aquaculture management.

[0004] At present, double antibody sandwich ELISA is a commonly used method for detecting immunoglobulin content, and ELISA kits on the market are mostly for detecting human, rat, mouse IgM, and some are for detecting pig, cow, horse, sheep, rabbit, chicken and grass carp. However, so far, there has been no related report on an ELISA kit for detecting Cynoglossus semilaevis IgM. SUMMARY

[0005] The present application is directed to the above technical problems existing in the prior art, and provides a double antibody sandwich ELISA kit for detecting Cynoglossus semilaevis IgM.

[0006] The technical solution of the present application is: a double antibody sandwich ELISA kit for detecting Cynoglossus semilaevis IgM, which has a capture antibody and a detection antibody, the capture antibody is produced by a hybridoma cell strain CsIgM-12 with a preservation number of CGMCC No. 46591, and the detection antibody is produced by a hybridoma cell strain CsIgM-39 with a preservation number of CGMCC No. 46592.

[0007] The classification name of the hybridoma cell strain CsIgM-12 is: hybridoma cell strain No. 12 secreting anti-Cynoglossus semilaevis IgM monoclonal antibody; the preservation unit is: China General Microbiological Culture Collection Center; the address of the preservation unit is: No. 1, Beichen West Road, Haidian District, Beijing; the preservation date is: September 11, 2025; and the preservation number is: CGMCC No. 46591. The classification name of the hybridoma cell strain CsIgM-39 is: hybridoma cell strain No. 39 secreting anti-Cynoglossus semilaevis IgM monoclonal antibody; the preservation unit is: China General Microbiological Culture Collection Center; the address of the preservation unit is: No. 1, Beichen West Road, Haidian District, Beijing; the preservation date is: September 11, 2025; and the preservation number is: CGMCC No. 46592.

[0008] The detection antibody is labeled by HRP.

[0009] The kit also has a Cynoglossus semilaevis IgM standard.

[0010] The application is a double-antibody sandwich ELISA kit for detecting Cynoglossus semilaevis IgM, which is constructed by using the anti-Cynoglossus semilaevis IgM monoclonal antibodies secreted by the hybridoma cell strains CsIgM-12 and CsIgM-39 as the capture antibody and the detection antibody, respectively. The titers of the two monoclonal antibodies are all above 128,000, the kit has good specificity and high sensitivity, the minimum detection concentration is 0.01 μg / mL, the content of Cynoglossus semilaevis serum IgM can be accurately detected, and it has important significance for evaluating the health status and immune function of Cynoglossus semilaevis. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 It is an SDS-PAGE result graph of purified Cynoglossus semilaevis serum IgM in the embodiment of the application.

[0012] Figure 2 It is a WB analysis result graph of the monoclonal antibodies secreted by the hybridoma cell strains CsIgM-12 and CsIgM-39 of Cynoglossus semilaevis in the embodiment of the application.

[0013] Figure 3 It is a standard curve graph of the content of IgM in Cynoglossus semilaevis serum and the detection OD value in the embodiment of the application.

[0014] Figure 4 It is a comparison schematic diagram of the content of IgM in Cynoglossus semilaevis serum of different specifications in the embodiment of the application.

[0015] Figure 5 It is a comparison schematic diagram of the content of IgM in Cynoglossus semilaevis serum fed with and without immune stimulants in the embodiment of the application.

[0016] The half-smooth tongue sole hybridoma cell line CsIgM-12 was deposited on September 11, 2025; depositary institution: China General Microbiological Culture Collection Center (CGMCC); accession number: CGMCC No. 46591.

[0017] The date of deposit of the half-smooth tongue sole hybridoma cell line CsIgM-39: September 11, 2025; depositary institution: China General Microbiological Culture Collection Center (CGMCC); accession number: CGMCC No. 46592. Detailed Implementation

[0018] I. Preparation and HRP labeling 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.

[0019] 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.

[0020] The spleen cells of the immunized mice were fused with SP2 / 0 myeloma cells under sterile conditions using 45% PEG1500, and the fused cells were resuspended with HAT selective medium and dropped into 96-well cell culture plates with feeder cells at 0.2 ml per well, and cultured in a 37°C, 5% CO2 incubator. The growth of the hybridoma cells was observed under an inverted phase contrast microscope. After about two weeks, the supernatant of the hybridoma cells was taken for detection.

[0021] 3. Positive screening by indirect enzyme-linked immunoassay (ELISA) The ELISA detection process was as follows: the Cynoglossus semilaevis IgM was used to coat the enzyme-labeled plate at 5 μg / ml, 100 μl / well, and 4°C overnight; PBST was used for washing 3 times, each time for 5 min; 3% bovine serum albumin (BSA) was used for blocking at 200 μl / well, 37°C for 1 h; PBST was used for washing 3 times, each time for 5 min; the culture supernatant of the hybridoma cells to be detected (the culture supernatant of the myeloma cells was used as a negative control) was added at 100 μl / well, 37°C for 1 h; PBST was used for washing 3 times, each time for 5 min; HRP-labeled goat anti-mouse Ig (1:3000) was added at 100 μl / well, 37°C for 1 h; PBST was used for washing 3 times, each time for 5 min; TMB color developing liquid was added at 100 μl / well, and color development was performed at 37°C for 30 min in the dark; color developing stop solution was added at 50 μl / well, and the OD value at 450 nm was read using an enzyme-labeled instrument; when the positive OD / negative OD (P / N) was >2.1, it was determined to be positive.

[0022] 4. Positive hybridoma cell clones secreting Cynoglossus semilaevis IgM monoclonal antibodies and screening The positive hybridoma cells were cloned by limited dilution. The positive hybridoma cells were resuspended with 1640 culture medium, diluted by 10 times in gradient to 10 2 cells / ml, 1 ml of the cell solution was added to 9 ml of culture medium, mixed uniformly, and dropped into 96-well cell culture plates with feeder cells at 0.1 ml per well. Only one hybridoma cell was selected for culture in each well, and when the cells grew to more than 2 / 3 of the bottom of the well, the supernatant was detected according to the method of step 3 above. The positive wells were cloned again for 2 times according to the above method, and the hybridoma cells secreting Cynoglossus semilaevis IgM monoclonal antibodies were screened.

[0023] 5. Preparation of Cynoglossus semilaevis IgM monoclonal antibodies The hybridoma cell culture supernatant was collected, centrifuged at 5000g for 5 min, and the supernatant was collected; after filtration through a 0.45 μm filter membrane, it was loaded into a pre-equilibrated Protein G affinity chromatography column for antibody purification, according to the product instructions. The purified monoclonal antibody was dialyzed against PBS buffer, concentrated by ultrafiltration, and the antibody concentration was determined by the Bradford method.

[0024] 6. Analysis of the antigenic determinant recognized by the C. semilaevis IgM monoclonal antibody The purified C. semilaevis IgM was subjected to SDS-PAGE and transferred to a PVDF membrane at 200 mA for 1.5 h; the PVDF membrane was blocked with 5% BSA at 4 ℃ overnight; after washing with PBST, the PVDF membrane was placed in the IgM monoclonal antibody to be detected and incubated at 4 ℃ overnight; after washing with PBST, the membrane was placed in HRP-labeled goat anti-mouse Ig and incubated at room temperature for 1 h; the membrane was washed with PBST again; an equal amount of Enhanced Luminol Reagent and Oxidizing Reagent was diluted with ddH2O, mixed well, and added dropwise to the sealing film; the front of the PVDF membrane was contacted with the luminescent reagent, and the color was developed for 1.5-2.0 min; the PVDF was turned over, and the results were observed using a gel imaging system.

[0025] 7. Analysis of the titer of the C. semilaevis IgM monoclonal antibody The antibody titer was determined by indirect ELISA: the C. semilaevis IgM was used to coat the enzyme-labeled plate at 5 μg / ml, 100 μl / well, and incubated at 4 ℃ overnight; washed with PBST 3 times, 5 min each time; blocked with 3% bovine serum albumin (BSA) at 200 μl / well, and incubated at 37 ℃ for 1 h; washed with PBST 3 times, 5 min each time; added the IgM monoclonal antibody to be detected at different dilutions (PBS as negative control), 100 μl / well, and incubated at 37 ℃ for 1 h; washed with PBST 3 times, 5 min each time; added HRP-labeled goat anti-mouse Ig (1:3000), 100 μl / well, and incubated at 37 ℃ for 1 h; washed with PBST 3 times, 5 min each time; added TMB color developing liquid, 100 μl / well, and developed at 37 ℃ in the dark for 30 min; added color developing termination liquid, 50 μl / well, and the OD value at 450 nm was read using an enzyme-labeled instrument; when the positive OD / negative OD (P / N) was >2.1, it was determined to be positive, and the highest dilution of the positive was the antibody titer.

[0026] 8. HRP labeling of the C. semilaevis IgM monoclonal antibody Weigh 1.5 times the antibody mass of HRP and dissolve it in sterile ultrapure water to prepare a 15 mg / ml solution. Add an equal volume of NaIO4 (12.8 mg / ml, freshly prepared) to the HRP solution and incubate at 4°C in the dark for 30 minutes. Add an equal volume of ethylene glycol solution (1%, freshly prepared) to the HRP solution and incubate at 4°C in the dark for 40 minutes. Add the antibody, mix thoroughly by inverting, and place in ≥100 volumes of CBS buffer. Dialyze overnight at 4°C in the dark. Remove the solution and add NaBH4 solution (5 mg / ml, freshly prepared) at a concentration of 20 μl / 1 mg antibody. Incubate at 4°C in the dark for 2 hours. Add an equal volume of saturated ammonium sulfate and incubate at 4°C for 3 hours. Centrifuge (4°C, 12000 rpm, 10 minutes) and discard the supernatant. Dissolve the precipitate in PBS to a concentration of 4 mg / ml and dialyze against 100 volumes of PBS for 2 hours. Add BSA to bring the BSA concentration to 20%. Mix the sample at a concentration of 1 mg / ml; add an equal volume of glycerol and mix well. The final antibody concentration is 1-2 mg / ml. Store at -20°C.

[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 2It is found that the IgM antigenic determinant recognized by monoclonal antibody No. 12 secreted by the Csi gM-12 hybridoma cell strain of C. semilaevis is located on the light chain of IgM, and the IgM antigenic determinant recognized by monoclonal antibody No. 39 secreted by the Csi gM-39 hybridoma cell strain of C. semilaevis is located on the heavy chain of IgM.

[0030] 4. Preparation of C. semilaevis IgM monoclonal antibody and HRP labeling After purification by a Protein G affinity chromatography column, 9 mL of monoclonal antibody was obtained from the culture supernatant of the hybridoma cell Csi gM-12, and the concentration was 1.67 mg / mL; after purification by a Protein G affinity chromatography column and HRP labeling of the antibody, 15 mL of HRP-labeled monoclonal antibody secreted by the hybridoma cell Csi gM-39 was obtained, and the concentration was 0.64 mg / mL.

[0031] II. Construction of a double-antibody sandwich ELISA kit for detecting C. semilaevis IgM (1) The kit composition is shown in Table 1: Table 1

[0032] (2) The technical parameters are shown in Table 2: Table 2

[0033] (3) The use method Preparation before detection: (1) Reagent preparation: please restore all reagents and samples to room temperature (25℃±3℃) before detection. If the concentrated reagent appears crystallization, warm it at 37℃ until the crystallization is completely dissolved. The color developing substrate should be colorless before addition, and please keep the color developing substrate in the dark at all times.

[0034] (2) Detection antibody dilution: take 10 μl of HRP-labeled detection antibody, add 10 mL of antibody diluent (i.e. 1:1000 dilution), and mix well.

[0035] (3) 10x concentrated washing solution dilution: according to the experimental requirements, determine the total volume of 1x washing solution required, and add 9 parts of distilled water to 1 part of 10x concentrated washing solution at a ratio of 1:9, gently stir or use a magnetic stirrer to stir uniformly to ensure complete mixing of the solution.

[0036] Standard dilution: the kit provides one bottle of original standard, and the user can prepare standard samples of different concentrations in small test tubes according to the preparation method in Table 3.

[0037] Table 3

[0038] (5) Sample dilution: In order to ensure the success of the experiment, it is recommended to carry out a pre-experiment before using the kit for the first time to explore the best sample dilution, so as to avoid the color reaction exceeding the highest value of the standard curve or the dilution factor being too large to be lower than the sensitivity of the kit due to too high concentration. For sample dilution, please dilute with sample diluent, and serum samples are recommended to be diluted by 1:5-1:40.

[0039] Detection method (1) Washing: wash the plate with 1x washing solution for 3 times, 200ul per well each time, and 5min each time.

[0040] (2) Sample addition: set blank holes (blank control holes without adding sample and enzyme labeled reagent, and the rest of the operation is the same), standard holes, and sample holes, and set three parallels for each hole. Add 100ul of sample to the bottom of the enzyme labeled plate hole, and try not to touch the hole wall.

[0041] (3) Incubation: after sealing the plate with sealing film, incubate at 37℃ for 1 hour.

[0042] (4) Washing: discard the liquid, spin dry, wash the plate with 1x washing solution for 3 times, 200ul per well each time, and 5min each time.

[0043] (5) Enzyme labeled antibody incubation: add 100ul of diluted HRP labeled detection antibody to the standard hole and the sample hole.

[0044] (6) Incubation: after sealing the plate with sealing film, incubate at 37℃ for 1 hour.

[0045] (7) Washing: discard the liquid, spin dry, wash the plate with 1x washing solution for 3 times, 200ul per well each time, and 5min each time.

[0046] (8) Color development: add 100ul of TMB color developing liquid to the blank hole, standard hole and sample hole, and develop color at 37℃ for 30min.

[0047] (9) Termination: add 50ul of termination liquid to each hole, and use an enzyme labeled instrument to read the OD value at 450nm.

[0048] (10) Calculation: take the concentration of the standard as the abscissa and the OD value as the ordinate to draw a standard curve, calculate the corresponding concentration according to the OD value of the sample from the standard curve, and then multiply the dilution factor to obtain the actual concentration of the sample.

[0049] Three. Kit related experiments constructed by the embodiment of the application (I) Sensitivity analysis The IgM of half-smooth tongue sole was serially diluted to 10, 5, 2.5, 1.25, 0.6, 0.3, 0.15, 0.08, 0.04, 0.02, 0.01, 0.005, 0.002, 0.001, and 0.0005 μg / mL. Sensitivity analysis was performed according to the above-described kit usage method. The results are shown in Table 4. The results indicate that the lowest detectable concentration of the kit of this invention is 0.01 μg / mL.

[0050] Table 4

[0051] (II) Detection Specificity Analysis According to the above usage method, apply the semi-smooth tongue sole ( Cynoglossus semilaevis ), turbot ( Paralichthys olivaceus ),turbot( Scophthalmus maximus Pearl Giant Grouper ( Epinephelus fuscoguttatus ♀× Epinephelus lanceolatus ♂), Red-spotted grouper ( Epinephelus akaara ), Cloud-patterned grouper ( Epinephelus moara Golden Tiger Grouper ( Epinephelus fuscoguttatus ♀× Epinephelus tukula ♂), Eastern Star Spot ( Plectropomus leopardus IgM in serum was detected. The serum was diluted 1:10 with PBST and used as the test sample. The results are shown in Table 5.

[0052] Table 5

[0053] The results show that the half-smooth tongue sole IgM double-antibody sandwich ELISA kit constructed in this invention is only suitable for half-smooth tongue sole, and is not suitable for detecting IgM content in the serum of marine fish such as turbot, flounder, pearl grouper, red-spotted grouper, clouded grouper, golden tiger grouper, and red grouper. It has good specificity.

[0054] (III) Detection of IgM content in the serum of half-smooth tongue sole Serum from approximately 500g of tongue sole was diluted 1:10 with PBST and used as the test sample. The IgM content in the tongue sole serum was detected according to the above kit instructions.

[0055] The OD values ​​of different concentrations of half-smooth tongue sole IgM standards are shown in Table 6. A standard curve was plotted using Graphpad Prism 10.1.2 software based on the IgM concentration and the detected OD values. Figure 3The regression equation is y = 1.975 + (0.113 - 1.975) / (1 + (x / 0.969)^0.898), R2= 0.978. The OD value of the detected C. semilaevis serum sample is 1.289, and the IgM concentration in the detection sample is 1.767 μg / mL according to the standard curve. Since the serum sample was diluted 10 times with PBST during determination, the IgM concentration in the serum should be 17.67 μg / mL.

[0056] Table 6

[0057] (Four) Comparison of IgM content in serum of C. semilaevis of different specifications According to the above-mentioned kit using method, the IgM content in serum of C. semilaevis of different specifications was detected and compared. C. semilaevis was divided into two specifications: large specification fish, about 850 g; small specification fish, about 100 g, 10 fish for each specification, and the serum was diluted 1:10 with PBST as the sample to be detected, and the results are shown in Table 7 and Figure 4 .

[0058] Table 7

[0059] The results show that the average IgM content in serum of large specification fish is 34.616 μg / mL, and the average IgM content in serum of small specification fish is 8.407 μg / mL. The IgM content in serum of large specification fish is significantly higher than that of small specification fish.

[0060] (Five) Detection of IgM content in serum of C. semilaevis after feeding immune stimulants Twenty C. semilaevis (about 100 g) were randomly divided into experimental and control groups, 10 fish for each group. The experimental group was fed with immune stimulant—Ekteen, 2 kg / Tn, purchased from Spain Baiwei Company, and the control group was fed with basic feed. After 90 days of cultivation, the fish serum was collected, and the IgM content in serum was detected by using the above-constructed kit, and the results are shown in Table 8 and Figure 5 .

[0061] Table 8

[0062] The results show that the average IgM content in serum of C. semilaevis in the experimental group is 14.655 μg / mL, and the average IgM content in serum of C. semilaevis in the control group is 8.387 μg / mL. Statistical analysis shows that the IgM content in the experimental group is significantly higher than that in the control group.

Claims

1. A double-antibody sandwich ELISA kit for detecting IgM in half-smooth tongue sole, comprising a capture antibody and a detection antibody, characterized in that: The capture antibody was produced by hybridoma cell line CsIgM-12 with accession number CGMCC No.46591, and the detection antibody was produced by hybridoma cell line CsIgM-39 with accession number CGMCC No.46592.

2. The double-antibody sandwich ELISA kit for detecting IgM in half-smooth tongue sole according to claim 1, characterized in that: The detection antibody is HRP-labeled.

3. The double-antibody sandwich ELISA kit for detecting IgM in half-smooth tongue sole according to claim 1 or 2, characterized in that: The kit contains a semi-smooth tongue sole IgM standard.

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