Specific detection method for index species of estuary water ecological evaluation
By preparing specific monoclonal and polyclonal antibodies and combining them with colloidal gold lateral flow chromatography test strips, the problem of on-site detection of medium-sized prawns was solved, enabling rapid and accurate ecological monitoring and red tide early warning.
Patent Information
- Application Number
- CN202610397759.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-03-30
- Publication Date
- 2026-07-03
Smart Images
Figure CN122325577A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of marine environmental ecological monitoring technology, specifically relating to a specific detection method for species as an indicator of estuarine aquatic ecology. Background Technology
[0002] In nearshore marine ecological environment monitoring and aquatic health assessment, the monitoring of indicator species is of great significance. Due to the complex structure and variable environmental factors of marine ecosystems, relying solely on physicochemical indicators often fails to comprehensively reflect the ecosystem's state. In contrast, certain biological groups that are highly sensitive to environmental changes and occupy stable ecological niches within the ecosystem can more directly reflect the marine ecological status through changes in their population size and community structure. Therefore, accurate and rapid detection and monitoring of key indicator organisms are of great value for assessing the primary productivity level of marine areas, determining water trophic status, identifying abnormal ecological changes, and predicting fishery resource potential.
[0003] Lucifer intermedius, a planktonic crustacean belonging to the family Luciferidae, is widely distributed in the nearshore waters and estuaries of my country and is one of the common species in marine zooplankton communities. This species occupies an important intermediate link in the marine food web, converting primary resources such as phytoplankton into energy available to organisms at higher trophic levels. It is an important natural food source for larvae of various economically important fish and filter-feeding organisms. Therefore, changes in its population size and distribution pattern often reflect the structure and energy flow of marine planktonic communities, and have high indicative significance in marine ecological research and fisheries resource assessment. Furthermore, during certain marine ecological events, such as before and after harmful algal blooms like red tides, the composition of zooplankton communities often undergoes significant changes. As a dominant or common species, the abundance fluctuations of Lucifer intermedius are of important reference value for analyzing related ecological processes and environmental changes.
[0004] Currently, the identification and monitoring of medium-sized prawns mainly rely on traditional morphological identification methods. Samples are typically collected using planktonic nets, and then manually identified and counted under a stereomicroscope based on morphological features such as the rostrum morphology, compound eye structure, abdominal appendages, and caudal fork. However, this method has certain limitations in practical applications. On the one hand, sample processing and manual identification are time-consuming, making it difficult to meet the needs of rapid analysis of large-scale samples; on the other hand, the identification results largely depend on the experience level of the identifyers, making them prone to human error. Furthermore, for early larvae or individuals damaged during sample preservation, their morphological features are often incomplete, further increasing the difficulty of identification. Simultaneously, traditional morphological methods are difficult to implement in real-time on-site detection or automated monitoring. Although molecular biological detection techniques have high specificity in species identification, they typically require complex experimental procedures and specialized equipment, resulting in long detection cycles, which is also unfavorable for rapid on-site screening and large-scale application.
[0005] Therefore, developing a detection method that can specifically identify medium-sized prawns, has a simple detection process, and is suitable for rapid on-site monitoring is of great significance for improving the efficiency of nearshore marine ecological environment monitoring, carrying out red tide early warning, and ecological management of aquaculture areas. Summary of the Invention
[0006] The purpose of this invention is to provide a specific detection method for species used as indicators of estuarine aquatic ecosystem evaluation. Specifically, based on the screening and obtaining of the antigenic protein Li-ESP1 and its dominant antigenic peptide of medium-sized prawn, a specific monoclonal antibody mAb-Li1 and a paired antibody (polyclonal antibody pAb-Li2) are prepared. Using these antibodies, a colloidal gold lateral flow chromatography rapid detection test strip is prepared, thereby realizing on-site, rapid, and accurate detection of medium-sized prawn, overcoming the technical bottlenecks of traditional methods that rely entirely on professional morphological identification, are cumbersome to operate, and cannot be applied on-site.
[0007] The present invention first provides an antigenic protein of medium-sized shrimp, wherein the amino acid sequence of the antigenic protein Li-ESP1 is SEQ ID NO: 1.
[0008] Furthermore, the present invention provides a peptide Li-PEP-B obtained from the above-mentioned full-length antigen protein through screening and verification, which has better antigenic effects and higher antibody specificity, and whose amino acid sequence is SEQ ID NO: 2.
[0009] This invention also provides a monoclonal antibody, named mAb-Li1, that specifically recognizes the aforementioned antigen protein Li-ESP1 or the dominant peptide Li-PEP-B. This antibody is prepared by secretion from a hybridoma cell line obtained using hybridoma technology with Li-PEP-B as the immunogen.
[0010] This invention further provides a polyclonal antibody, named pAb-Li2, that can be used in conjunction with the aforementioned monoclonal antibody mAb-Li1. The pairing antibody pAb-Li2 is a polyclonal antibody obtained by immunizing rabbits with full-length Li-ESP1 protein (SEQ ID NO: 1) as an immunogen and purifying the immunoglobulin IgG in their serum. After specific adsorption and purification, the pairing antibody pAb-Li2 can recognize different epitopes on the medium-sized shrimp antigen with the monoclonal antibody mAb-Li1, thereby jointly constituting the core recognition element of a dual-antibody sandwich immunoassay system.
[0011] In another aspect, the present invention provides the use of the monoclonal antibody mAb-Li1 and the paired antibody pAb-Li2 in the preparation of reagents or kits for detecting medium-sized prawns or their antigenic components in water.
[0012] Another aspect of the present invention is to provide a colloidal gold lateral flow chromatography test strip for detecting medium-sized prawns in water, which includes a colloidal gold-labeled mAb-Li1 monoclonal antibody as a detection antibody (tracer), a paired antibody pAb-Li2 immobilized on a nitrocellulose membrane as a detection line (T line), and goat anti-mouse IgG as a control line (C line).
[0013] In another aspect, the present invention provides a rapid on-site screening method for medium-sized prawns in water, which involves using the aforementioned colloidal gold test strip to test a filtered and concentrated water sample.
[0014] This invention utilizes the medium-sized prawn species-specific antigen Li-PEP-B and its corresponding monoclonal antibody mAb-Li1 obtained through screening, combined with the paired antibody pAb-Li2 prepared from the full-length protein, and applies them to the development of a colloidal gold rapid immunoassay product. This achieves, for the first time, high-throughput and rapid on-site screening of this species, significantly improving the efficiency and accuracy of red tide biological composition analysis, marine ecological surveys, and zooplankton monitoring. Attached Figure Description
[0015] Figure 1 : Western blot validation image of mAb-Li1 antibody;
[0016] Figure 2 pAb-Li2 specific reaction diagram;
[0017] Figure 3 : Results of colloidal gold method testing for some samples. Detailed Implementation
[0018] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.
[0019] Example 1: Screening, optimization and preparation of specific antigens for medium-sized shrimp
[0020] 1. Screening and identification of candidate specific antigens
[0021] 1) Sample collection and processing: Zooplankton samples were collected from nearshore waters of my country using a planktonic net (mesh size 330 μm). Medium-sized adult *Lucifer typus* were purified and isolated under a stereomicroscope using micromanipulation techniques and stored at -80℃. A closely related species, *Lucifer typus*, was obtained using the same method as a control.
[0022] 2) Protein extraction: Approximately 800 individuals of medium-sized and normal-sized *Cyprinus praecox* were collected, ground in liquid nitrogen, and total protein was extracted using RIPA lysis buffer containing protease inhibitors. Protein concentration was determined by the BCA method.
[0023] 3) Comparative proteomics analysis: Equal amounts (80 μg) of the two total proteins were subjected to two-dimensional electrophoresis. The first dimension was isoelectric focusing (pH 5-8), and the second dimension was SDS-PAGE (10% separating gel). The gels were stained with Coomassie Brilliant Blue.
[0024] 4) Differential point analysis and identification: Two electrophoresis patterns were compared using a gel imaging analysis system. Protein spots that were stably highly expressed in medium-sized *Cyprinus spp.* gels but completely absent in positive-type *Cyprinus spp.* gels were excised. In-gel enzymatic digestion and liquid chromatography-tandem mass spectrometry analysis were performed on the target gel spots, and the obtained peptide sequences were compared with databases.
[0025] 5) Gene cloning: Based on the mass spectrometry identification results, degenerate primers were designed, and a full-length gene encoding a differentially expressed protein was cloned using cDNA of the medium-sized shrimp as a template via RT-PCR and RACE techniques. The protein encoded by this gene was named Li-ESP1, and its deduced full-length amino acid sequence is shown in SEQ ID NO: 1.
[0026] LAFSSVAHICRDVNYGWLLRTMHANGASFFFICLYMHTGRGMYYGSYMYMHAWSVGVVIFLLTMAAAFLGYVLPWGQMSFWGATVITNLFSAIPYIGNDLVQWIWGGFAVDNATLNRFYTFHFLFPFVVAAAT MIHILFIHQTGSNNPLGITSAMDKIPFHPYFVFKDIVGFVIMLTALTVLTLLDPYLLGDPDNFIPANPLSTPAHIQPEWYFLFAYAILRSIPNKLGGVIALAMSIVILLILPFTHSSSKFRSLTFYPINQI (SEQ ID NO: 1).
[0027] 2. Rational design and optimized screening of key immune-advantage peptides
[0028] 1) Bioinformatics analysis and candidate peptide design: The antigenicity and hydrophilicity of Li-ESP1 protein were analyzed, and its sequence was compared with closely related species such as *Cyprinus punctata*. Three fragments located in the highly hydrophilic region and with significant sequence differences were selected, and three linear peptides were designed and synthesized, named Li-PEP-A, Li-PEP-B (SEQ ID NO:2), and Li-PEP-C, respectively.
[0029] 2) Preparation of positive serum: New Zealand white rabbits were immunized with inactivated medium-sized shrimp homogenate to prepare rabbit anti-medium-sized shrimp polyclonal positive serum.
[0030] 3) Immunoreactivity verification of candidate peptides: Indirect ELISA was used. Three synthetic peptides were coated onto enzyme-labeled plates, and the plates were reacted with serially diluted (1:100 to 1:409600) positive serum. A positive result was defined as an OD450 value greater than the mean of the negative controls by 2.1 times.
[0031] Table 1: Results of Immunoreactivity Gradient Detection of Candidate Antigen Peptides
[0032] serum dilution Li-PEP-A OD450 (P / N) Li-PEP-B OD450 (P / N) Li-PEP-C OD450 (P / N) Negative control OD450 1:100 2.05 (14.6) 2.35 (16.8) 1.88 (13.4) 0.14 1:1600 0.85 (6.1) 1.65 (11.8) 0.72 (5.1) 0.14 1:25600 0.16 (1.1) 0.68 (4.9) 0.15 (1.1) 0.14 1:102400 0.14 (1.0) 0.33 (2.4) 0.14 (1.0) 0.14 1:204800 0.14 (1.0) 0.17 (1.2) 0.14 (1.0) 0.14
[0033] Table 1 shows that Li-PEP-B exhibited the strongest immunoreactivity among all tested peptides, with the highest serum dilution at which it could detect a positive signal reaching 1:102400. Furthermore, the reaction of positive serum adsorbed with positive shrimp protein with Li-PEP-B resulted in an OD value decrease of over 92%, demonstrating its high specificity. Therefore, Li-PEP-B:AVDNATLNRFYTFHF (SEQ ID NO:2) was selected as the optimized immunogen for subsequent preparation of monoclonal antibodies.
[0034] 3. Optimize antigen preparation
[0035] The Li-PEP-B peptide is coupled to keyhole hemocyanin (KLH) via a cysteine residue added to its N-terminus to form the complete antigen Li-PEP-B-KLH, which is used for animal immunization.
[0036] Example 2: Preparation and Identification of Monoclonal Antibodies Against Li-ESP1 Protein
[0037] 1) Animal immunization and cell fusion: BALB / c mice were immunized with Li-PEP-B-KLH (100μg / mouse) for a total of 4 immunizations. Three days after the last immunization, spleen cells were harvested and fused with SP2 / 0 myeloma cells.
[0038] 2) Screening and establishment of positive hybridoma cell lines: Positive wells were screened using an indirect ELISA method (with free Li-PEP-B as the coating antigen). Limiting dilution subcloning was performed on strongly positive wells to obtain a stable monoclonal hybridoma cell line secreting antibodies, named 3D8, and the antibody secreted was named mAb-Li1.
[0039] 3) Antibody production and purification: Ascites fluid was prepared and mAb-Li1 was purified using a Protein G affinity chromatography column.
[0040] 4) Antibody specificity identification: Validated by Western Blot ( Figure 1 mAb-Li1 reacts specifically with total protein from medium-sized *Cyprinus spp.*, but does not cross-react with proteins from closely related or common zooplankton such as *Cyprinus spp.* and *Daphnia spp.*.
[0041] Example 3: Assembly, application and validation of colloidal gold side-flow chromatography rapid detection test strips
[0042] 1) Preparation of paired antibody pAb-Li2 polyclonal antibody
[0043] New Zealand white rabbits were immunized with recombinant, full-length Li-ESP1 protein to obtain antiserum. After initial purification using the caprylic acid-ammonium sulfate method, the antiserum was further purified by affinity chromatography using an affinity column coupled with total positive-type shrimp protein to remove cross-reactivity. The flow-through was collected and further purified by Protein A affinity chromatography to obtain a high-purity, high-specificity rabbit polyclonal antibody, named pAb-Li2. This was subsequently validated. Figure 2 pAb-Li2 can effectively pair with mAb-Li1 to form a sandwich detection signal.
[0044] 2) Assembly and validation of colloidal gold test strips
[0045] mAb-Li1, labeled with 40 nm colloidal gold, was sprayed onto the conjugate pad. pAb-Li2 (T line) and goat anti-mouse IgG (C line) were coated onto a nitrocellulose membrane. The membranes were then stacked and assembled into a test strip.
[0046] Take 100 mL of water sample, filter it through a 50 μm sieve, concentrate and resuspend it, then drop it onto the sample pad of the test strip. Read the result within 15 minutes. Figure 3 ).
[0047] To verify the performance, 35 actual water samples covering different sea areas were subjected to double-blind testing and compared with the microscopic counting method. The results are shown in the table below.
[0048] Table 2: Sample Composition and Microscopic Count Results
[0049] Sample type Number of samples (parts) Microscope counting range (ind. / m³) The result was determined by the reference method. Water samples from the red tide affected area 12 10 - 1500 Positive: 9 samples; Negative: 3 samples Water samples from the estuary mixed zone 13 0 - 600 Positive: 7 samples; Negative: 6 samples Water samples from offshore clean area 10 0 - 80 Positive: 1 sample; Negative: 9 samples total 35 0 - 1500 Positive: 17 samples; Negative: 18 samples
[0050] Table 3: Comparison and Verification Results of Colloidal Gold Test Strips and Microscopic Counting Method
[0051]
[0052] Performance calculations: Sensitivity = TP / (TP+FN) = 94.1%; Specificity = TN / (TN+FP) = 94.4%; Overall concordance rate = (TP+TN) / 35 = 94.3%; Kappa value = 0.88. The visual detection limit of the test strip is 20-40 ind. / m. 3 .
[0053] This invention establishes for the first time a rapid immunoassay technique for medium-sized prawns. Based on the optimized antigen peptide Li-PEP-B and full-length protein selected through screening, highly specific paired antibodies were successfully prepared, and a high-performance colloidal gold test strip was developed. This product enables rapid and accurate on-site screening of medium-sized prawns, providing a new and efficient tool for related ecological monitoring and applications.
Claims
1. An antigenic protein, characterized in that, The amino acid sequence of the antigen protein is SEQ ID NO:
1.
2. A peptide fragment, characterized in that, The amino acid sequence of the peptide is SEQ ID NO:
2.
3. The use of the peptide according to claim 2 as an antigen in the preparation of monoclonal antibodies.
4. A monoclonal antibody, characterized in that, The monoclonal antibody is prepared by using the peptide described in claim 2 as an antigen to prepare a hybridoma cell line, and then secreting it through the hybridoma cell line.
5. The use of the monoclonal antibody according to claim 4 in the preparation of a reagent for detecting medium-sized prawns.
6. The use according to claim 5, wherein the compound is ###0002### The reagent mentioned is a colloidal gold side-flow chromatography test strip.
7. A colloidal gold side-flow chromatography test strip, characterized in that, The colloidal gold lateral flow chromatography test strip includes a colloidal gold-labeled monoclonal antibody as the detection antibody, a polyclonal antibody as the detection line, and goat anti-mouse IgG as the quality control line, all immobilized on a nitrocellulose membrane.
8. The colloidal gold side-flow chromatography test strip as described in claim 7, characterized in that, The polyclonal antibody is obtained by immunizing rabbits with the antigen protein described in claim 1 and purifying the immunoglobulin IgG in their serum.
9. A method for screening medium-sized prawns in water, characterized in that, The method involves using the colloidal gold side-flow chromatography test strip as described in claim 7 for detection.