117 results about "Indirect elisa" patented technology

The invention discloses a monoclonal antibody and application thereof in quantitative detection of porcine epidemic diarrhea virus, and belongs to the technical field of biological detection. The preparation method comprises the following steps: firstly, obtaining two monoclonal antibodies GC44F3-1 and GC32C11-5 which specifically react with S protein of the porcine epidemic diarrhea virus; secondly, establishing a double-antibody sandwich ELISA (Enzyme-Linked Immunosorbent Assay) method by taking the two monoclonal antibodies as a coating antibody and a detection antibody respectively, and applying the double-antibody sandwich ELISA method to PEDV (Porcine Epidemic Diarrhea Virus) vaccine S protein quantification; the method can be used for detecting semi-finished products and finished products, can be used for process establishment, optimization, vaccine quality evaluation and stability monitoring of porcine epidemic diarrhea virus vaccine antigens, has the advantages of high sensitivity, high accuracy, high repeatability and high flux, is high in detection flux and high in speed, solves the problem that TCID50 cannot meet vaccine quality evaluation at present, and has a good application prospect. The defect that the existing S protein ELISA method can only be used for qualitative judgment is overcome. The monoclonal antibody can also be used for blocking ELISA, capturing ELISA, indirect ELISA, competitive ELISA and a reagent for detecting the porcine epidemic diarrhea virus through a colloidal gold test strip.

The invention discloses an anti-soluble transferrin receptor monoclonal antibody as well as a preparation method and application thereof, and belongs to the technical field of antibody detection. According to the antibody, an L101-F760 amino acid sequence of human TfR protein is used as an immunogen, specific screening of B cells, reverse RNA (Ribonucleic Acid) into cDNA (Complementary Deoxyribose Nucleic Acid) through RT-PCR (Reverse Transcription-Polymerase Chain Reaction), then homologous recombination is performed to construct heavy and light chain vectors, and finally multiple rounds of screening are performed to obtain the purified sTfR monoclonal antibody, so that the sTfR protein can be specifically recognized, and the antibody has the advantages of high specificity and high sensitivity and can be widely applied to the field of immunotherapy. The natural sTfR protein in human serum can be recognized. The kit is applied to the detection and screening fields of immunohistochemistry, immunofluorescence chemistry, western blot, co-immunoprecipitation, indirect ELISA, flow cytometry and the like, especially has low background and no interference of non-specific binding impure protein during immunohistochemistry detection, can obtain more accurate detection and evaluation results, avoids interference of false positive and false negative, and has high detection accuracy. The method has great application value in the aspects of basic scientific research and clinical pathology detection.

The invention is applicable to the technical field of biology, and provides an L7 / L12-PADRE sequence-multi-linked B cell epitope recombinant protein and a kit for detecting an anti-Brucella antibody. The recombinant protein is a multi-epitope tandem recombinant protein composed of Brucella ribosome L7 / L12 protein, a PADRE polypeptide sequence and multiple B cell epitopes, the amino acid sequence of the recombinant protein is as shown in SEQ ID No.1, and the recombinant protein is good in antigenicity. The anti-brucella antibody indirect ELISA (iELISA) detection method and kit established by taking the recombinant protein as the coating antigen have the characteristics of high sensitivity, strong specificity and good repeatability, are suitable for detecting the condition of generating the anti-brucella antibody by an organism, can clarify the immune background of brucella infection or brucellosis vaccine, and can be used for detecting the brucella infection or brucellosis vaccine. Basic data is provided for prevention and control of the Brucella disease of humans and animals, and meanwhile, the kit is conveniently applied to large-scale sample detection and epidemiological monitoring.

The invention discloses an indirect ELISA (enzyme-linked immuno sorbent assay) detection method of a porcine epidemic diarrhea virus antibody SIgA, and belongs to the technical field of animal antibody detection. In order to solve the problems of limited detection range and single immune evaluation dimension of a porcine epidemic diarrhea virus detection method in the prior art, the invention provides an indirect ELISA detection method of a porcine epidemic diarrhea virus antibody SIgA, and the detection method adopts a pichia pastoris eukaryotic secretion platform to prepare a high-activity PEDV S1 antigen; meanwhile, a secreting type IgA antibody is used as a detection target; the method has the advantages of high detection sensitivity, low background noise, better variant compatibility and practical popularization value, and provides a clinical and industrial-level solution for maternal immunity monitoring, piglet passive protection evaluation and vaccine immunity strategy optimization.

The invention discloses an EmAGO2 truncated recombinant protein, an echinococcosis multilocularis indirect ELISA (enzyme-linked immuno sorbent assay) diagnostic kit based on the truncated recombinant protein and a use method of the kit. The method comprises the following steps: designing a specific primer by selecting a sequence of 1-738bp at the N end of an Em-AGO2 gene, carrying out PCR (Polymerase Chain Reaction) amplification by taking reverse transcription echinococcosis multilocularis DNA (Deoxyribonucleic Acid) as a template, connecting an amplification product with a pCE2-TA / Blunt vector, and then transforming into a competent cell for culturing, so as to obtain a pCE2-T-EmAGO2 plasmid; respectively carrying out double enzyme digestion on the plasmid and an expression vector pET-28a (+), and connecting enzyme digestion fragments to obtain a recombinant protein expression plasmid pET-28a-EmAGO2; the preparation method comprises the following steps: taking EmAGO2 as a template, transforming the EmAGO2 into competent cells, selecting positive clones for induced expression, and carrying out ultrasonication treatment, separation and purification, concentration and desalination treatment on expressed thalli to obtain the EmAGO2 truncated recombinant protein. Furthermore, a mouse anti-EmAGO2 truncated recombinant protein and goat anti-rabbit IgG are respectively used as an antigen and an antibody to establish an indirect ELISA diagnostic kit for the echinococcosis multilocularis, and the kit has high sensitivity, specificity and good repeatability, and can be used for early diagnosis of the echinococcosis multilocularis.

The invention discloses a sheep paratuberculosis specific detection kit and application, and relates to the field of biological detection.The kit comprises an elisa plate coated with a recombinant Mycobacterium paratuberculosis membrane protein antigen, the nucleotide sequence of the recombinant Mycobacterium paratuberculosis membrane protein antigen is shown as SEQ ID NO: 1, and the amino acid sequence of the recombinant Mycobacterium paratuberculosis membrane protein antigen is shown as SEQ ID NO: 2; a confining liquid; a serum diluent; carrying out enzyme labeling on a second antibody; a TMB color developing solution and a stop solution; according to the sheep paratuberculosis specific detection kit disclosed by the invention, the recombinant Mycobacterium paratuberculosis membrane protein antigen is adopted as a core detection component, and the antigen has high specificity and can accurately identify paratuberculosis positive serum and effectively distinguish positive and negative samples; meanwhile, prokaryotic expression and purification are carried out on the specific membrane protein of the mycobacterium paratuberculosis for the first time, and a novel sheep paratuberculosis indirect ELISA diagnosis method with high sensitivity and specificity based on the protein as an antigen is established.

The invention discloses a porcine epidemic diarrhea virus S protein monoclonal antibody and application thereof. Specifically, two monoclonal antibodies GC42G2-1 and GC33D8-1 which have broad-spectrum binding characteristics with the porcine epidemic diarrhea virus S protein are obtained firstly, the GC42G2-1 antibody has good universality and can have strong affinity with different strains, and the GC33D8-1 antibody can be used for binding the porcine epidemic diarrhea virus S protein in a broad-spectrum manner. The GC42G2-1 can be independently used, and also can be matched with a polyclonal antibody or GC33D8-1 to be used for sandwich ELISA (enzyme-linked immunosorbent assay) so as to detect and quantify PEDV (porcine epidemic diarrhea virus). Secondly, the two monoclonal antibodies are respectively used as a coating antibody and a detection antibody, a double-antibody sandwich ELISA method is established, and the double-antibody sandwich ELISA method is applied to PEDV vaccine S protein quantification, has better parallelism compared with other antibodies, and can be widely applied to detection of different strains. The method disclosed by the invention has the advantages of high sensitivity, high accuracy, high repeatability and high flux, and can be used for the development of blocking ELISA (Enzyme-Linked Immunosorbent Assay), capturing ELISA, indirect ELISA, competitive ELISA and detection of porcine epidemic diarrhea viruses by colloidal gold test strips.

The invention discloses a recombinant CapA protein of cat astrovirus. The recombinant CapA protein has an amino acid sequence as shown in SEQ ID NO.1 in a sequence table or is coded by a base sequence as shown in SEQ ID NO.2 in the sequence table. Therefore, a cat astrovirus indirect ELISA antibody detection kit is developed and comprises a coated ELISA plate, and the coated ELISA plate takes the recombinant CapA protein as a coating antigen. Meanwhile, a corresponding indirect ELISA antibody detection method is established. Research results show that the critical value of the indirect ELISA antibody detection method and kit is 0.523; the sensitivity is 1: 32000; the in-batch variation coefficient of detected samples is 2.679%-7.910%, the inter-batch variation coefficient is 3.627%-8.982%, both the in-batch variation coefficient and the inter-batch variation coefficient are smaller than 10%, and the clinical application effect is good. Therefore, the kit has the characteristics of high sensitivity, strong specificity, good repeatability and the like, can quickly and effectively detect the FAstV antibody, and provides technical support for accurate diagnosis, timely prevention and control and vaccine effect evaluation of FAstV.

The invention discloses a method for establishing indirect ELISA (enzyme-linked immunosorbent assay) for detecting mycobacterium bovis Lprl, which comprises the following steps: preparing mycobacterium bovis Lprl protein, taking the purified protein as an antigen, and determining conditions such as optimal antigen coating concentration, serum dilution, serum incubation time, secondary antibody dilution concentration, incubation time and the like. The established indirect ELISA detection method does not have cross reaction with other bovine pathogens, the specificity is good, the detection accuracy is high, and the total coincidence rate of test results of intradermal allergy with bovine tubercle bacillus is 95.8%; the intra-batch variation coefficient of the detected samples is 2.43%-3.61%, the inter-batch variation coefficient is 2.83%-3.96%, and the intra-batch variation coefficient and the inter-batch variation coefficient are both smaller than 10%. A new diagnosis target is developed, the established indirect ELISA method has the characteristics of high sensitivity, strong specificity, good repeatability and the like, the operation is easy, and a new method is provided for the detection work of the mycobacterium bovis.

The invention belongs to the technical field of biology, and particularly relates to a hybridoma cell strain capable of secreting a clostridium perfringens ETX monoclonal antibody with neutralizing activity and application of the hybridoma cell strain. On the basis of natural toxin, an indirect ELISA (enzyme-linked immunosorbent assay) detection method is established by utilizing a non-toxic ETX single amino acid site protein mutant, and a series of hybridoma cell strains secreting an anti-clostridium perfringens ETX monoclonal antibody are screened; the hybridoma cell strain can stably and efficiently secrete a monoclonal antibody with neutralizing activity and a monoclonal antibody without neutralizing resistance respectively, and large-scale batch production can be realized. According to the present invention, the clostridium perfringens ETX monoclonal antibody is firstly utilized to establish the ETX antigen ELISA detection method, and the method has characteristics of simple sample operation, low cost, rapid reaction, strong specificity and the like, can provide the reference for the diagnosis of clostridium perfringens, and provides the basis for the research of the related vaccine potency test substitution method.

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 discloses a detection method and a kit for detecting listeria monocytogenes, and relates to the technical field of biology. The kit is an indirect ELISA kit and comprises an elisa plate coated with an antigen, the amino acid sequence of the antigen is as shown in SEQ ID NO. 2. The indirect ELISA detection kit constructed by the invention only reacts with listeria monocytogenes positive serum, has no cross reaction with other serum, and has strong specificity; when the serum dilution is 1: 800, the detection result is still positive, and the sensitivity is high; the intra-batch variation coefficient ranges from 2.79% to 6.64%, the inter-batch variation coefficient ranges from 2.61% to 7.75%, and the intra-batch repeatability and the inter-batch repeatability are good; 24 serum samples are detected, and the total coincidence rate is 95.83%. The kit provided by the invention can effectively detect sample infection of Listeria monocytogenes, and has the advantages of strong specificity, high sensitivity and good repeatability.

The application relates to a detection method of anti-preS1 antibody, an anti-preS1 antibody detection kit and application, the application carries out multi-sequence comparison and conservation analysis on preS1 sequences of different genotypes (A, B, C, D and CD mixed type) of HBV, takes type A (AB697487.1), type C (FJ032351.1) and type D (LC519808.1) as reference sequences, based on the sequence consistency standard of 85% of full length and 90% of the key region of functional antibody binding, a highly conservative preS1 full-length antigen sequence is systematically screened out in natural variation, the sequence can cover antigen variation of A-D type HBV, and the sequence is taken as a coating antigen to establish an indirect ELISA detection system, a good linear relationship is presented in the range of 4.88-625 ng / mL, and the Cut-off value is 33.6 ng / mL; clinical verification shows that the method can effectively distinguish chronic hepatitis B patients from healthy control groups, compared with detection methods based on partial fragments or overlapping short fragments, the false negative rate can be significantly reduced, and the method is suitable for HBV infection immune state evaluation, anti-virus curative effect monitoring and prognosis analysis.

The present invention relates to the field of diagnostic detection of animal virus antibodies, and specifically provides a method for detecting African swine fever virus antibodies and its application. First, the present invention screens the dominant epitope regions of the structural proteins p30, p54, and p72 of African swine fever, tandemly expresses and purifies the three dominant epitope regions using an Escherichia coli expression system, and uses the purified protein as a coating antigen to establish an indirect ELISA method for detecting clinical swine sera. The present invention makes up for the deficiencies of existing domestic and foreign detection methods, and will be more conducive to the monitoring of antibody levels and prevalence generated by ASFV vaccine immunization and wild virus infection.

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.

This invention discloses an indirect ELISA detection kit and method for bovine pasteurellosis, relating to the field of biotechnology. The indirect ELISA kit includes an enzyme-labeled plate coated with an antigen; the amino acid sequence of the antigen is shown in SEQ ID NO.2. The indirect ELISA detection kit constructed in this invention exhibits high specificity and sensitivity; the intra-assay coefficient of variation is between 1.79% and 7.54%, and the inter-assay coefficient of variation is between 2.23% and 6.91%, both below 10%, demonstrating good inter-assay and intra-assay repeatability. In testing 30 clinical samples, the overall concordance rate with the standard "Diagnostic Techniques for Bovine Hemorrhagic Sepsis (GB / T27530-2025)" was 96.67%. The indirect ELISA detection kit demonstrates good overall performance and can be applied to the detection of bovine pasteurellosis in clinical serum samples.

This invention relates to the application of the avian adenovirus serotype 4 multi-epitope recombinant chimeric protein rcF2 in the preparation of a kit for detecting avian adenovirus serotype 4 antibodies. The amino acid sequence of the avian adenovirus serotype 4 multi-epitope recombinant chimeric protein rcF2 is shown in SEQ ID NO.1. This invention utilizes the multi-epitope recombinant chimeric protein rcF2 to establish an indirect ELISA detection method and kit for detecting avian adenovirus serotype 4. This recombinant chimeric protein can be recognized by avian adenovirus serotype 4 positive sera, and the detection sensitivity of positive sera samples can reach 1:16000. It shows no cross-reactivity with antibodies against other pathogens, indicating that the ELISA detection method based on this multi-epitope recombinant chimeric protein rcF2 is simple to operate, enhances detection sensitivity, and has good specificity and repeatability.

This invention discloses anti-leptin monoclonal antibodies and their applications, relating to the field of biotechnology. This invention develops anti-leptin monoclonal antibodies 2A122H7 and 6B62E3, which can effectively recognize leptin. The ELISA kits prepared using these monoclonal antibodies 2A122H7 and 6B62E3 include indirect ELISA and double-antibody sandwich ELISA kits, exhibiting high specificity and sensitivity for the tested samples, and are convenient, efficient, and cost-effective. This invention provides effective technical support for advancing research on the regulatory mechanisms of economic traits such as feed intake, fat deposition, and reproduction in poultry.

The invention belongs to the technical field of biological medicine, and discloses an antibody for treating advanced rectal cancer and application thereof.The antibody is a monoclonal antibody 3C1 of targeted carcino-embryonic antigen (CEA), the sequence of a heavy chain variable region (VH) of the antibody is as shown in SEQ ID NO: 1, the sequence of a light chain variable region (VL) of the antibody is as shown in SEQ ID NO: 5, and the indirect ELISA detection titer reaches 1: 512000; also provided is an antibody conjugate (ADC) in which the antibody and raltitrexed are coupled by a linker, the drug-antibody ratio (DAR) being 3.0-4.0, the monomer purity being greater than or equal to 95%; the antibody conjugate (ADC) can be used for treating CEA positive advanced rectal cancer, animal experiments show that the tumor inhibition rate of ADC is 86% (significantly higher than 35% of free raltitrexed), the weight loss rate of mice is low, the toxicity of small intestines is light, the problems that existing drugs are poor in targeting and high in toxicity are solved, and the antibody conjugate has the advantages of high efficiency and safety.

The invention belongs to the technical field of bioengineering, and particularly relates to a preparation method and application of an N protein polyclonal antibody of a horse coronavirus. The polyclonal antibody is prepared by truncation expression of the horse coronavirus N protein, and a material is provided for research on inhibition of host innate immune mechanism by the horse coronavirus N protein. Prokaryotic expression is carried out on the N protein of the equine coronavirus, the polyclonal antibody is prepared, and a method and a material are provided for molecular research and mechanism research of the equine coronavirus. According to the invention, in combination with bioinformatics analysis of an N protein of the horse coronavirus, a pGEX-6p-N3 recombinant plasmid is constructed, and prokaryotic expression is carried out, so that a purified recombinant protein is obtained. After a mouse is immunized by the recombinant protein, a polyclonal antibody is obtained, an indirect ELISA method detects that the polyclonal antibody has good titer, Western Blot detection has good reactivity, and the polyclonal antibody can be applied to an indirect immunofluorescence test.

The invention discloses a diagnostic method based on mouse lyme disease serology indirect ELISA laboratory, and relates to the technical field of immunoserology detection.The diagnostic method comprises the steps that experimental conditions are optimized through a system, and the optimal reaction system is determined as follows: the antigen coating concentration is 0.2 microgram / microliter, the serum dilution ratio is 1: 200, and the enzyme-labeled secondary antibody concentration is 1: 2000. 0.8), and meanwhile, the antigen dosage is obviously reduced. Dynamic analysis of the antibody shows that the level of the antibody infecting mouse serum reaches a peak 12-25 days after inoculation, and the threshold value of the specific antibody ratio (ArB%) is 71.43%. Researches prove that the method has the characteristics of simplicity and convenience in operation, economy and high efficiency, and is suitable for primary laboratories and large-scale screening. The method provides reliable standardized technical support for lyme disease laboratory diagnosis and epidemiological monitoring.

The invention discloses a polypeptide-based avian reticuloendotheliosis virus indirect ELISA antibody detection method, and belongs to the technical field of biology. According to the kit, potential epitopes of REV gp90 protein are systematically screened to obtain three sections of specific polypeptide antigens for detecting the avian reticuloendotheliosis virus antibody, and a mixed product of the three sections of specific polypeptide antigens is used as a coating antigen to develop the indirect ELISA antibody detection kit for the avian reticuloendotheliosis virus. On the basis of the indirect ELISA antibody detection kit, the avian reticuloendotheliosis virus antibody detection method established by optimizing the coating concentration, the sealing condition, the serum incubation time and the like has the advantages of strong specificity, high sensitivity, good repeatability and convenience in operation; the kit can effectively and rapidly diagnose epidemic diseases caused by the avian reticuloendotheliosis virus, and is beneficial to popularization and application.

The present invention provides monoclonal antibodies against the cat allergen FeI d 1 protein and their applications. First, the present invention obtained two hybridoma cell lines, 2A7 and 6B1, that stably secrete FeI d 1 monoclonal antibodies. Furthermore, a double-antibody sandwich ELISA method was established using 2A7 as the capture antibody and biotin-labeled 6B1 as the detection antibody. This method can be used to monitor FeI d 1 protein antigen levels, replacing existing indirect ELISA detection methods. It exhibits high specificity and sensitivity, is simple to operate, and produces clear results.

This invention constructs pET-32a-CP1 and pET-32a-CP2 expression vectors based on the CP1 and CP2 protein genes of Giardia duodenalis, transforms them into Rosetta (DE3) competent cells, and induces expression using IPTG. After SDS-PAGE analysis of the recombinant protein expression, Ni-NTA affinity chromatography is used to purify the proteins. Two indirect ELISA methods are then established using these as coating antigens. The indirect ELISA methods based on the recombinant proteins CP1 and CP2 of this invention exhibit good specificity, sensitivity, and repeatability, with concordance rates of 98% and 94% with microscopic detection results, respectively.

The invention discloses a swine acute diarrhea syndrome coronavirus subunit vaccine as well as a preparation method and application thereof. The method comprises the following steps: firstly, predicting the antigen epitope of SADS-CoV S1 protein through bioinformatics, synthesizing and screening the obtained antigen epitope peptide in vitro, and further screening the antigen epitope with high immunogenicity through a Dot-blot experiment. And then, constructing a recombinant fusion protein expression vector by using the SADS-CoV S1 gene and the screened antigen epitope peptide coding gene, and preparing the recombinant fusion protein after the recombinant plasmid is transformed into escherichia coli. A vaccine prepared by emulsifying the recombinant fusion protein by using an adjuvant is used for immunizing a mouse to evaluate the immunogenicity of the mouse, detection results of methods such as indirect ELISA and splenic lymphocyte proliferation experiments prove that the vaccine can effectively induce specific humoral immunity and cellular immunity effects, and the antigen epitope has the effect of enhancing the immunogenicity of the SADS-CoV S1 protein. The vaccine has a wide application prospect and can be used for preventing the acute diarrhea syndrome of pigs.

The present invention discloses a monoclonal antibody 2D1B1 against canine distemper virus H protein and its application, belonging to the technical field of monoclonal antibodies. The present invention screened out 9 monoclonal antibodies by indirect ELISA and indirect immunofluorescence identification. After indirect immunofluorescence identification, the 9 monoclonal antibodies obtained were all able to react with the vaccine strain (CDV-Onderstepoort). The results of Western Blot tests showed that the 9 monoclonal antibodies screened out were all able to react specifically with the eukaryotic expressed CDV H protein, among which 2D1B1 was also able to react specifically with the prokaryotic expressed CDV H protein, that is, 2D1B1 was not only able to react with the attenuated vaccine strain and the virulent isolated strain, but also with both eukaryotic and prokaryotic expressed CDV H proteins, and at the same time had a high neutralizing potency (2 6 ), which can be used to prepare broad-spectrum detection reagents and therapeutic preparations for identifying canine distemper virus H protein.

The invention belongs to the technical field of biology, and particularly relates to a recombinant monoclonal antibody of sheep anti-bluetongue virus VP7 protein. A sheep anti-bluetongue virus antibody library is obtained through a single cell V (D) J sequencing technology, a sheep anti-bluetongue virus VP7 protein recombinant monoclonal antibody is screened and successfully expressed, and it is identified through indirect ELISA and cellular immunofluorescence experiments that the recombinant monoclonal antibody has high affinity with bluetongue virus and can be specifically combined with VP7 protein. The recombinant monoclonal antibody is used as a competitive antibody to establish a competitive ELISA detection method aiming at the bluetongue virus VP7 protein, high specificity of bluetongue antibody detection can be realized, and a basis is provided for mastering the epidemiology of bluetongue virus and evaluating the immune effect of vaccines.

The invention provides an indirect ELISA (Enzyme-Linked Immunosorbent Assay) antibody detection method and an indirect ELISA antibody detection kit based on pyruvate Kinase (PK), and relates to the technical field of parasite detection. The kit comprises a coating antigen Italiella mannici PK protein, a primary antibody, a labeled secondary antibody and an initiator solution, wherein the primary antibody is a mouse or cat anti-Italiella mannici PK protein antibody to be detected; the labeled secondary antibody can be combined with the primary antibody combined with the PK protein of the andrometrium mansoni; and the labeled second antibody is provided with a detection marker. The method comprises the following steps: cloning a PK protein gene into a prokaryotic expression vector PQE80L (+), constructing a recombinant plasmid PQE80L-PK, inducing PK recombinant protein expression by utilizing IPTG (isopropyl-beta-d-thiogalactoside), purifying the recombinant protein to coat an elisa plate, and establishing an indirect ELISA (enzyme-linked immuno sorbent assay) detection method of the Mangnocorynth PK protein antibody by optimizing reaction conditions. The detection method has the advantages of high sensitivity and strong specificity, and provides support for the development of the detection technology of the sitonia mansoni and the prevention and control of the schizocyst.

The invention discloses a CD147 nano antibody D2 as well as a preparation method and application thereof, and belongs to the technical field of molecular biology and immunology. The preparation method comprises the following steps: screening from an alpaca nano antibody phage library by utilizing a phage display technology, and sequencing to obtain 9 CD147 nano antibodies; 3 nano antibody genes are selected and cloned into pCold II, and prokaryotic expression recombinant plasmids are successfully constructed; the method comprises the following steps: carrying out inducible expression for 20 hours under the conditions that the final concentration of IPTG (isopropyl-beta-d-thiogalactoside) is 0.2 mmoL / L, the temperature is 16 DEG C and the speed is 100 rpm / min, carrying out nickel column purification, and then carrying out Coomassie brilliant blue dyeing and Western blot identification to determine and obtain the CD147 nano antibody which is named as C36, D2 and H4; the binding activity of the three nano antibodies and the CD147 protein is detected through indirect ELISA (enzyme-linked immuno sorbent assay). The result shows that the CD147 antigen and the three nano antibodies have binding activity and are dose-dependent. Under the same conditions, when the incubation concentration of the nano-antibody is as low as 1.25 micrograms / mL, the D2 binding effect is the best, the amino acid sequence of the nano-antibody is shown as SEQ ID NO.8, and the nucleotide sequence of the nano-antibody is shown as SEQ ID NO.10. The invention provides a more accurate method for detecting the CD147.

The invention discloses a CD147 nano antibody G45 as well as a preparation method and application thereof, and belongs to the technical field of molecular biology and immunology. A phage display technology is used for screening from a natural alpaca nano antibody phage library, finally three nano antibody genes are selected and cloned into pCold II, and prokaryotic expression recombinant plasmids are successfully constructed; the method comprises the following steps: carrying out inducible expression for 20 hours under the conditions that the final concentration of IPTG (isopropyl-beta-d-thiogalactoside) is 0.2 mmoL / L, the temperature is 16 DEG C and the speed is 100 rpm / min, carrying out nickel column purification, and then carrying out Coomassie brilliant blue dyeing and Western blot identification, so as to determine and obtain the CD147 nano antibody which is named as A8, B8 and G45; the binding activity of the three nano antibodies and the CD147 protein is detected through indirect ELISA (enzyme-linked immuno sorbent assay). The result shows that the CD147 antigen and the three nano antibodies have binding activity and are dose-dependent. Under the same conditions, when the incubation concentration of the nano-antibody is as low as about 0.625 g / mL, the G45 binding effect is good, the amino acid sequence of the nano-antibody is shown as SEQ ID NO.7, and the nucleotide sequence of the nano-antibody is shown as SEQ ID NO.10. The invention provides a more accurate method for high-sensitivity detection of CD147.