112 results about "Blot" patented technology

The invention provides a preparation method and application of a high-sensitivity imprinted membrane for selectively detecting microcystic toxins (MC-LR) in water based on surface enhanced Raman scattering. The preparation method comprises the following steps: step 1, preparation of silver nanocubes; step 2, preparation of Ag NCs-coated TiO2 nanoparticles; step 3, preparation of an Ag NCs-coated TiO2 / PDMS composite membrane; according to the invention, a membrane technology, an SERS technology and MIT are combined, so that the prepared product has excellent physical and chemical properties of a PDMS membrane, high sensitivity of an SERS detection technology and high selectivity of MIT; the invention provides a new method for SERS detection and widens the application range.

The invention relates to the field of biological product preparation, in particular to a preparation method of high-activity oyster-derived small molecule peptide. The method comprises the following steps: selecting fresh meat of pacific oysters or ostrea rivularis as a raw material, degreasing, adding water, crushing, filtering, sequentially carrying out compound protease and neutral protease hydrolysis, implanting a sensor and a probe in hydrolysis, monitoring in real time, and dynamically adjusting reaction conditions; adding a targeting microcapsule wrapping an ascorbic acid glutathione composite protective agent, and releasing the protective agent when a target peptide signal is detected; separating and concentrating through a gradient membrane, and purifying the target peptide by using a molecular imprinting chromatographic column; carrying out alkaline hydrolysis on enzymatic hydrolysis residues to prepare chitosan for cyclic utilization, carrying out enzymatic hydrolysis on residual residues to generate collagen peptide, treating and recycling wastewater, and recovering protease. According to the method, the technical effects that the high-activity and high-purity target peptide is prepared, the resource utilization rate is increased, the production cost is reduced, the target peptide has good oxidation resistance and biological activity, the intestinal enzyme degradation rate is reduced after glycosylation modification, and the blood half-life period is prolonged are achieved.

The invention designs a preparation method of a magnetic molecular imprinting-aptamer sandwich fluorescence sensor for detecting kanamycin. And the magnetic molecular imprinting composite material Fe3O4 (at) UiO-66 (at) MIP with a specific adsorption target object KANA is prepared. When a detection target object KANA exists, the Fe3O4-coated UiO-66-coated MIP can rapidly and specifically capture the KANA in a short time. Then, a DNA single-stranded aptamer marked with FAM is added, after the aptamer and KANA captured on the surface of the Fe3O4 at-UiO-66 at-MIP are subjected to specific recognition and magnetic separation again, an aptamer probe can enter sediment along with the magnetic composite material Fe3O4 at-UiO-66 at-MIP, the fluorescence intensity of supernate is reduced, and quantitative analysis is conducted according to the fluorescence difference value delta F. When the KANA does not exist, the aptamer cannot enter the precipitate, so that the fluorescence signal intensity is not changed. Double recognition is formed through specific binding of an MIP imprinting cavity and the aptamer, matrix interference is reduced by means of fluorescence characteristics and magnetic separation capacity, the method has the advantages of being high in adsorption capacity, specific in recognition, high in sensitivity, wide in detection range, good in selectivity and the like, and rapid and accurate detection of KANA in a complex sample can be achieved.

The application belongs to the technical field of biology and particularly relates to an African swine fever virus pNP868R protein monoclonal antibody and a preparation method and application thereof. The application takes the pNP868R protein prepared by eukaryotic expression as an antigen, immunizes BALB / c mice, uses hybridoma technology, and obtains a hybridoma cell line capable of stably secreting the anti-African swine fever virus pNP868R protein monoclonal antibody through cell fusion, indirect ELISA method screening, limited dilution method subcloning and hybridoma continuous passage culture. The application also obtains an African swine fever virus pNP868R protein monoclonal antibody. The monoclonal antibody has high affinity with the pNP868R protein and can be applied in Western-Blot experiments, indirect immunofluorescence experiments, immunoprecipitation experiments and the capture of proteins interacting with pNP868R, and can be used for the diagnosis, prevention or treatment of African swine fever.

The invention relates to a preparation method of a molecularly imprinted composite filler, and relates to the technical field of analytical chemistry and environmental monitoring technologies, the preparation method of the molecularly imprinted composite filler comprises the following steps: firstly dissolving template molecules and a first monomer, then adding a cross-linking agent and an initiator, carrying out deoxidation and water bath, and then centrifuging to obtain a template molecule solution; and eluting the precipitate with a flushing fluid, and drying to obtain the microspheres. And dissolving the ionic liquid, uniformly stirring with the obtained microspheres, and carrying out centrifugal separation, washing and vacuum drying to finally prepare the molecularly imprinted composite filler. Finally, the hydrophobic property of the molecularly imprinted composite filler is improved through modification of the ionic liquid and the second monomer. The method provided by the invention has excellent hydrophobic effect and enrichment capability on a target object.

The invention relates to the technical field of western blot analysis, and discloses a full-automatic micro western blot analyzer and an analysis method thereof. The full-automatic micro protein immunoblotting analyzer comprises a full-automatic control system, a horizontal control system, a protein separation system, a temperature control system, a liquid path control system, an ultraviolet curing module, an antibody incubation module and a chip scanning module, the horizontal control system controls the protein separation system to horizontally move and correct; the protein separation system carries out protein separation; the temperature control system is used for carrying out constant-temperature control and temperature adjustment; the liquid path control system is used for filling a constant-temperature reagent into the protein separation system and extracting waste liquid; the ultraviolet light curing module is used for carrying out in-situ immobilization on the separated protein; the antibody incubation module is used for performing antibody incubation on the fixed protein marker; and the chip scanning module is used for chip result reading and data processing. According to the invention, full-automatic control, accurate temperature control and automatic horizontal correction can be realized.

This invention provides a peanut allergen peptide, its screening method, and its application. Phage display technology is used to screen for conformational epitopes of Ara h5 and Ara h8 proteins. Polyclonal IgE and IgG antibodies against the target proteins are prepared by immunizing BALB / c mice with Ara h5 and Ara h8 proteins as antigens. ELISA and dotted immunoblotting detection revealed that the titers of polyclonal IgG and IgE against Ara h5 and Ara h8 proteins were 1:64000 and 1:320, respectively, and the antibodies exhibited specificity. Using the antibodies as targets, phage display technology screened the conformational epitope sequences of Ara h5 protein as WETIYSR and FHWWYLK. The conformational epitopes of Ara h8 protein were FPYMKFV, FPYMKFR, SMFARID, and SFHWWLF.

Disclosed herein are compositions comprising protein antigens and RNA encoding the same (eg., compositions comprising protein antigens and RNA encoding antigens) that can be used to induce an immune response against SARS-CoV-2. Also disclosed herein are immunogenic compositions and medical preparations comprising the same, and methods of making and using the same. In some embodiments, the technologies provided herein can be used to address and / or overcome immune imprinting in SARS-CoV-2.

This invention discloses an anti-MSLN antibody and its applications. The anti-MSLN antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3. The amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:7, 8, and 9, respectively; and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:4, 5, and 6, respectively. The antibody provided by this invention has good affinity and specificity for MSLN antigens, effectively detecting target proteins in cells overexpressing MSLN. It can also be widely used as a core ingredient in the development of high-sensitivity MSLN detection kits such as ELISA, Western blotting, and immunohistochemistry.

The invention discloses an efficient ribosome imprinting sequencing library construction method, and belongs to the technical field of biology. In the enzyme digestion treatment step, a mixed enzyme system of RNase I and micrococcus nuclease is adopted to treat the cell lysis buffer, and the trinucleotide periodicity of RPFs can be remarkably improved to 80% or above. In the target fragment recovery step, the sucrose gradient ultracentrifugation step is omitted, the RPFs are directly recovered by gel electrophoresis, a plurality of enzyme reaction steps in library construction are optimized and integrated, and streptavidin magnetic beads are used for solid phase purification, so that the whole library construction period is greatly shortened from 7 days to 2.5 days. The method does not depend on special equipment such as ultracentrifugation, and can be completed in a conventional molecular biology laboratory. Besides, a multi-enzyme synergistic RNA terminal one-step modification system is innovatively constructed, single-tube one-step completion is realized, the terminal treatment efficiency and consistency are improved, and a key guarantee is provided for obtaining a high-quality and high-complexity Ribo-seq library.

The invention provides a fish creatine kinase sensitization and immune cross reactivity analysis method which comprises the following steps: preparing a fish muscle water-soluble protein crude extract, separating 40-45kDa protein components in the fish muscle crude extract through SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and screening a target sensitization protein combined with IgE / IgG through immunoblotting; the method comprises the following steps: extracting total RNA of grass carp and synthesizing cDNA, designing a creatine kinase specific primer for amplification to obtain a creatine kinase gene sequence, and carrying out bioinformatics analysis after sequencing verification; the method comprises the following steps: cloning a creatine kinase gene to an expression vector, transforming escherichia coli BL21, performing IPTG induced expression, and performing urea gradient dialysis renaturation on an inclusion body to obtain the recombinant grass carp creatine kinase. Detecting the IgE binding activity of the recombinant grass carp creatine kinase and the serum of the allergic patient through dot hybridization; the IgG cross reactivity of the recombinant grass carp creatine kinase and the blue crab arginine kinase is verified through indirect ELISA; the IgE cross reaction degree of the recombined grass carp creatine kinase and the blue crab arginine kinase is quantified through inhibitory dot hybridization.

The invention belongs to the field of pharmacy, and relates to a protein degradation targeting chimera (PROTAC) taking PD-L1 as a target spot and medical application thereof. The protein degradation targeting chimera is a compound composed of a target protein ligand, an E3 ligase ligand and a linker and a pharmaceutically acceptable salt of the compound, the specific structure of the compound is as shown in a formula I, and substituent groups are described in the specification in detail. A Western blot experiment (Western Blot) shows that the compound disclosed by the invention can be used for degrading the PD-L1 protein in a targeted manner.

The application relates to a Fenton-like composite material, in particular to a self-activated Fenton-like composite repair material and a preparation method and application thereof, the composite repair material is CaO2@Fe3O4@BIP-BC, a protein imprinting biochar is used as a matrix, magnetite serving as an activator and calcium peroxide serving as a slow-release oxidant are sequentially loaded on the surface. Compared with the prior art, the application has the advantages that (1) the biochar surface active groups and calcium peroxide improve the high efficiency and long-acting property of the Fenton-like process, (2) the protein imprinting improves the selectivity of the removal of target harmful bacteria and harmful bacteria genes, (3) the biochar simultaneously loads the calcium peroxide and the magnetite, the simultaneous addition of the activator and the oxidant is realized, and the use cost and the operation complexity are reduced. The method solves the problems that the traditional Fenton-like oxidation treatment has poor specific removal effect on harmful bacteria and harmful bacteria genes in the environment, the treatment time is short, the required dosage is high, secondary pollution is serious, and the activator and the oxidant need to be used separately.

The application relates to a high-throughput identification method of lncRNA coding peptides and application thereof, and belongs to the technical field of biology. The method combines a translationomics technology and a protein mass spectrometry technology, takes ribosome footprint sequencing data as a starting point, determines an open reading frame located on an lncRNA gene according to a three-base periodicity rule of a sequencing fragment, further constructs a proteogenomics index after systematic filtration, uses a computational proteomics method to detect an lncRNA translation event, and systematically identifies lncRNA coding peptides on the basis. The method has the characteristics of modularity and strong expandability, can be widely used for various types of proteome data, and can high-throughput identify and quantitatively detect lncRNA coding peptides. The application provides a prediction of molecular typing and prognosis of cancer patients based on an lncRNA coding peptide network model, and provides an important reference basis for precise medication of clinical patients.

The invention discloses a visual bifunctional subtype influenza virus molecularly imprinted sensor as well as a preparation method and application thereof. The sensor comprises a fluorescent signal core and a molecularly imprinted polymer layer coated outside the fluorescent signal core, the fluorescent signal core is SiO2 microspheres coated with CdTe quantum dots, the molecularly imprinted polymer layer is internally provided with an imprinting cavity capable of specifically recognizing the subtype influenza virus, the three-dimensional space shape of the imprinting cavity is complementary to that of the subtype influenza virus, and the molecularly imprinted polymer layer is coated with CdTe quantum dots. Sialic acid molecules are covalently modified on the inner surface. The sensor can be used for quantitative detection or visual semi-quantitative detection of subtype influenza viruses such as H7N9 and H5N1, and integrates double functions of rapid screening and accurate quantification. The sensor disclosed by the invention is good in preparation reproducibility, high in selectivity, strong in anti-interference capability and suitable for analysis of complex biological samples, and a novel user-friendly and efficient method is provided for on-site monitoring and early warning of influenza viruses.

The application belongs to the technical field of electrochemical analysis and detection, and discloses a molecular imprinting electrochemiluminescence sensor, a preparation method thereof and application of the sensor in selective detection of trilobatin. The operation comprises the following steps: (1) a prepared g-C3N4@NiMOF composite material is modified on the surface of a glassy carbon electrode; (2) the g-C3N4@NiMOF / GCE is placed in a phosphate buffer solution containing o-phenylenediamine and TRI, and o-PD and TRI are electropolymerized on the electrode surface through cyclic voltammetry in a three-electrode system to obtain MIP-p(o-PD)-TRI / g-C3N4@NiMOF / GCE; and (3) TRI in the polymerization film is eluted to form a specific imprinting cavity, and a MIP-p(o-PD) / g-C3N4@NiMOF / GCE sensor is obtained. The molecular imprinting electrochemiluminescence sensor has the advantages of simple operation, fast analysis speed, good selectivity and high sensitivity in the detection of TRI.

The invention discloses a molecularly imprinted photoelectrochemical sensor for detecting tetracycline and a preparation and detection method, and belongs to the technical field of environmental analysis and detection. The preparation method comprises the following steps: firstly, electrically depositing a BiOI nanosheet on FTO (Fluorine-doped Tin Oxide) conductive glass, and sequentially carrying out vanadium source coating, heat treatment and alkali washing to obtain a high-performance BiVO4 photo-anode; and then the surface of BiVO4 is modified with a CoMn-LDH layer, so that separation and transmission of photo-generated charges are effectively promoted. Finally, tetracycline is taken as a molecular imprinting template, o-phenylenediamine is taken as a functional monomer, electropolymerization is carried out on the surface of the composite electrode, and after elution, the molecular imprinting polymer membrane with specific recognition capability on tetracycline is formed. When the molecularly imprinted polymer is used for detecting tetracycline, a target molecule is specifically combined with an imprinted hole and then is oxidized by a photo-generated hole as an electron donor, so that the separation and migration efficiency of a photo-generated carrier is remarkably enhanced. The method has the advantages of high sensitivity, wide linear range and the like, and high-sensitivity detection of tetracycline residues in an environmental water body can be realized.

The application discloses a single-cell immunoblotting method based on quenchable aptamer probes and relates to the technical field of single-cell protein detection. The method comprises the following steps: S1, a single-cell suspension sample is settled in a microwell array on a composite hydrogel chip; S2, the composite hydrogel chip is placed in an electrophoresis tank, then a lysis solution is slowly poured into the tank, and incubation lysis is carried out; S3, after cell lysis, an electric field is applied to both ends of the chip, and proteins enter a gel coating layer on the surface of the chip under the action of the electric field and start electrophoretic separation; S4, after gel electrophoresis is completed, the chip is placed under an ultraviolet lamp for exposure, and the gel is taken out after exposure is completed; S5, the gel is placed in an aptamer probe solution which is targeted to target proteins, and is soaked and dyed, and the fluorescence signal intensity of the target protein molecules is measured under a laser confocal fluorescence microscope. The application can improve the detection sensitivity of target proteins and has fast imaging speed.

This invention discloses an anti-MSLN antibody and its applications. The anti-MSLN antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3. The amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:7, 8, and 9, respectively; and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:4, 5, and 6, respectively. The antibody provided by this invention has good affinity and specificity for MSLN antigens, effectively detecting target proteins in cells overexpressing MSLN. It can also be widely used as a core ingredient in the development of high-sensitivity MSLN detection kits such as ELISA, Western blotting, and immunohistochemistry.

This invention discloses an antibody (YWHG-4) against HLA-G isoform molecules HLA-G1, HLA-G4, and HLA-G5, and its uses. The antibody (YWHG-4) is produced from a hybridoma with accession number CCTCC NO:2021204, using the antigenic peptide (RGYYNQSEASSHTLQWMIGC) of amino acid position 106-126 of the HLA-G molecule heavy chain as the immunogen. This invention provides the nucleotide encoding the YWHG-4 antibody and its encoding amino acid sequence. Furthermore, this invention provides the uses of the YWHG-4 antibody for the detection of HLA-G isoform molecules HLA-G1, HLA-G4, and HLA-G5 using Western blotting, immunohistochemistry, ELISA, and flow cytometry.

The invention discloses an electrochemical sensor for glycoprotein detection and a glycoprotein detection method. The method comprises the following steps: anchoring an aptamer-protein conjugate on the surface of a gold electrode, and then assembling an anti-fouling peptide around the aptamer-protein conjugate to form an imprinted self-assembled monolayer film. And removing the combined protein through an acid solution to form a biocompatible imprinting cavity capable of being used for recombining a target object. The imprinted cavity can eliminate non-specific adsorption and enhance targeted binding efficiency. The sensor can be used for directly detecting carcino-embryonic antigens with the concentration as low as 0.1 ng / mL through an electrochemical impedance spectroscopy method. Besides, homodimer concanavalin A (ConA) is used as a recognition element and a cross-linking agent, homodimer glucose oxidase (GOx) is assembled on the surface of the electrode in situ, a protein network can be formed, and enzyme catalysis signal amplification detection is achieved. Through in-situ formation of a ConA-GOx assembly, the sensitivity is improved by 100 times.

The invention provides a western blot image automatic analysis method, device, equipment and medium, and relates to the technical field of image understanding, the method comprises the following steps: obtaining a western blot image to be analyzed, converting the western blot image to be analyzed into a gray level image, and carrying out background noise reduction processing on the gray level image; generating a gray scale curve based on the gray scale values of the pixel points in the gray scale image; carrying out trough identification on the gray curve to obtain each trough point in the gray curve; and based on the position of the trough point in the gray curve, adding a segmentation line in the gray image to obtain a lane division result of the to-be-analyzed western blot image, and determining gray value data of each lane based on the lane division result. According to the method, lane automatic division and gray value extraction of the western blot image can be realized, and the analysis efficiency of the western blot image is improved.

The invention belongs to the technical field of gene detection, and particularly relates to a brand new application of a DDX42 gene or an expression product thereof as an acute myelogenous leukemia (AML) diagnosis and prognosis biomarker. Specific high expression of DDX42 in bone marrow and peripheral blood samples of AML patients is revealed for the first time, the expression level of DDX42 is remarkably different from that of a healthy control group, and DDX42 can be detected through real-time fluorescent quantitative PCR, western blot and other methods and is used for diagnosis, differential diagnosis, disease state evaluation or risk stratification of AML. Meanwhile, the invention clarifies a molecular mechanism that DDX42 promotes AML cell proliferation and inhibits apoptosis and differentiation through PLK1 and LIMK1-CREB-DNMT1 signal axes, and verifies that a transcription factor ERG is an upstream forward regulatory factor for the first time. Based on the discovery, the invention not only provides a novel high-sensitivity molecular diagnosis marker for AML, but also lays a solid experimental foundation for developing a novel AML treatment strategy and a drug screening system targeting DDX42 and related signal channels thereof. The method has the advantages of mature detection method, high marker relevance, clear application prospect and the like.

This invention relates to a method for constructing a polycystic kidney disease (PCD) model and its applications. The method involves overexpressing the MYCN gene in a target animal to obtain a PCD model that leads to PCD-related phenotypes. The method includes the following steps: obtaining a first strain of mice with Rosa26 knock-in overexpressing the CAG-LSL-HA tag-MYCN-IRES-BFP-Wpre-polyA gene; obtaining a second strain of mice by inserting Cre-WPRE-polyA into the start codon of the Pax8 gene; and crossing the first and second strains of mice to obtain a MYCN-overexpressing PCD model. This invention employs various experimental methods for validation, including histopathological analysis, immunohistochemical staining, and Western blotting. The model provided by this invention overcomes the limitations of existing in vitro cell models, organoids, and existing animal models in terms of limited phenotypes. The established MYCN-overexpressing PCD animal model exhibits stable disease progression and a short disease cycle, which not only helps to elucidate the disease mechanism but also serves as an ideal platform for drug screening and efficacy evaluation.

The application studies a molecular imprinting electrochemical sensor of a CoMOF-IL sensitive film modified electrode and a preparation method and a detection method thereof, and belongs to the field of electrochemical sensing.The application selects a breast cancer marker carcinoembryonic antigen in serum as a research object, successfully constructs a novel high-sensitivity molecular imprinting electrochemical sensor by combining a nano-composite material, a new imprinting method and a new detection method.Firstly, a CoMOF-IL composite material modified electrode is synthesized by a hydrothermal method, then chitosan and glutaraldehyde are sequentially modified to provide an attachment site of the carcinoembryonic antigen, then a polymer film is directly formed on the surface of the modified electrode by o-phenylenediamine electropolymerization, and finally the template is eluted to obtain the molecular imprinting electrochemical sensor.The molecular imprinting electrochemical sensor combines the molecular imprinting technology and the electrochemical sensing technology, constructs a high-sensitivity and high-selectivity carcinoembryonic antigen sensor, and has been successfully applied to the detection of target proteins in actual serum samples.

The utility model relates to the technical field of molecular biology experimental equipment, in particular to a locating and fast-assembling Western Blot film transferring clamp system which comprises a film transferring clamp base body, a buckle connecting piece capable of being fast connected is installed between the opening ends of the film transferring clamp base body, a sponge layer is arranged in the film transferring clamp base body, and a locating piece is arranged on the sponge layer. A first pressure-sensitive adhesive tape is bonded between the corner of the bottom of the sponge layer and the bottom of the transfer film clamp base body, filter paper is placed on the top of the sponge layer, a second pressure-sensitive adhesive tape is bonded between the corner of the top of the sponge layer and the bottom of the filter paper, and a polyvinylidene fluoride film is placed above the filter paper. Through a bonding, locking and scale cooperative positioning mechanism, the membrane transfer success rate and the experiment repeatability are remarkably improved, meanwhile, the operation complexity is reduced, and a reliable tool is provided for standardization of the western blot technology.

The application discloses a kind of H9N2 subtype AIV multi-copy M2e protein monoclonal antibody, heavy chain and light chain variable region respectively have the amino acid sequence of sequence table SEQ.ID.NO.1 and SEQ.ID.NO.2.The related cell line and its preparation method are also established according to this.The preservation number of hybridoma cell strain 4E10-A10 of the application is CCTCC NO:C202328, and test shows that the anti-multi-copy M2e protein monoclonal antibody secreted by it has high specificity, sensitivity, can specifically recognize 2M2e, 3M2e and 4M2e recombinant protein and the cell expressing H9N2 subtype avian influenza virus 3M2e protein, and is suitable for indirect immunofluorescence detection and Western-blot identification.In conclusion, the application provides material and technical support for the mechanism research of AIV M2e monoclonal antibody to AIV and the application effect evaluation of broad-spectrum vaccine taking AIV M2e protein as target.

The invention relates to a method and a device for enhancing immunoblotting and immunoblotting electrophoresis sensitivity. The method comprises the following steps: performing regular stripe standing wave concentration on protein by utilizing regular stripe standing waves generated by superposing surface sound source ultrasonic waves through a gel electrophoresis carrier which is subjected to electrophoretic separation and contains a low-concentration protein molecule band in a direction vertical to electrophoretic separation; and the protein molecules with different densities are pushed to the node or antinode region of the standing wave and are concentrated. The device comprises an area sound source generator operated by the method and an echo carrier plate with an echo plate. An electrophoresis tank for accommodating an echo carrying disc is additionally arranged, so that the electrophoretic separation step and the concentration step are integrated; and a main bearing body is arranged for bearing the electrophoresis tank, a related power supply and a related switch. According to the method and the device, the concentration of the low-abundance protein in the reaction area reaches or is closer to the optimal concentration for antigen-antibody reaction, in the immunoblotting process, the local luminescence or fluorescence peak intensity after membrane transfer meets the condition of reaching a threshold value or above, and the detection rate of the low-abundance target protein is greatly improved.

The methods, compositions and kits described herein provide signal amplification approaches for the detection of target biomolecules that significantly increase the sensitivity of detection using target-binding ligand molecules. The methods include cyclic addition of nucleic acid repeats to, e.g., target-binding molecules in situ, thereby providing multiple landing pads for labeled probes. The methods are well-suited to performance in multiplex, thereby permitting the sensitive detection of multiple targets in a single assay. These methods and compositions can be applied to, among other things, imaging, flow cytometry, CyTOF, and immunoblotting, providing additional tools for research and diagnostic purposes.

This invention discloses a monoclonal antibody against Oit3 protein in mouse hepatic sinusoidal endothelial cells or its antigen-binding fragment and its applications, belonging to the fields of biotechnology and medical immunology. The monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region contains HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO. 3~SEQ ID NO. 5; the light chain variable region contains LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO. 8~SEQ ID NO. 10. This monoclonal antibody and the recombinant fluorescent antibody exhibit high affinity and high specificity, and can be effectively applied to enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunofluorescence detection. They can serve as important antibody tools for basic research on mouse hepatic sinusoidal endothelial cells and for exploring the mechanisms of liver diseases. Based on its variable region sequence, a recombinant fluorescent antibody, Oit3-scFv-GFP, was also constructed. This recombinant fluorescent antibody enables one-step direct immunofluorescence staining, providing a convenient tool for in situ visualization of mouse hepatic sinusoidal endothelial cells.