88 results about "Keyhole-limpet haemocyanin" patented technology

The invention discloses a method of preparing a bombyx mori silk fibroin specific antibody by utilizing a characteristic polypeptide. The method comprises the following steps: synthesizing a "CGAGAGSGAGAGS" polypeptide sequence by utilizing an Fmoc method, coupling the polypeptide with keyhole limpet hemocyanin (KLH) through the cysteine on the N terminus of the polypeptide so as to obtain a complete antigen; diluting the complete antigen with normal saline, mixing the diluted complete antigen with a complete Freund's adjuvant, adding streptomycin and penicillin to carry out an emulsion treatment so as to obtain a primary immunized antigen emulsion, subjecting a rabbit to a primary immunization by using the primary immunized antigen emulsion, then subjecting the rabbit to a strengthened immunization, wherein the strengthened immunization uses a strengthened immunized antigen emulsion, which is prepared by the following steps: mixing the diluted complete antigen with an incomplete Freund's adjuvant, and then adding streptomycin and penicillin to carry out an emulsion treatment so as to obtain the target product; collecting the blood of the immunized rabbit, when the antibody titer in the rabbit blood sample reaches 1/10000; making the blood blocks fully contract to completely separate out the antiserum, then collecting the antiserum, and subjecting the antiserum to a centrifugation treatment so as to obtain a supernate.

IgG and IgM autoantibody levels against phosphorylcholine in subjects with hypertension (diastolic pressure>95 mmHg) were determined at baseline in order to determine the importance of antibodies for the development of atherosclerosis. The results show that increases in intima-media thickness (IMT) at a follow-up four years after baseline were significantly less prevalent in subjects having high IgM autoantibodies to phosphorylcholine. The presence or absence of IgM autoantibodies against phosphorylcholine is thus related to an increased or decreased risk of developing ischemic cardiovascular diseases. A method to determine IgM antibodies toward phosphorylcholine is proposed in this invention to identify subjects at risk of developing ischemic cardiovascular diseases. Animal experiments show that medium to high levels of IgM antibodies can be detected in plasma after active immunization with a keyhole limpet hemocyanin (KLH)-phosphorylcholine conjugate. A pharmaceutical composition comprising a phosphorylcholine conjugate (active immunization) or a monoclonal antibody with specificity to a phosphorylcholine conjugate (passive immunization) is proposed and the use of these compositions as active or passive immunogens in the treatment or prevention of atherosclerosis.

The invention belongs to the technical field of genetic engineering, and in particular relates to screening of a single-chain antibody against fumonisin and application thereof in immunoassay of fumonisin. The screening method comprises the following steps of: directly setting off from coupled antigen FB1-KLH(keyhole limpet hemocyanin) immunized mouse spleen cells, establishing a single-chain antibody gene library by a molecular cloning method and technology, then screening by a phage display technology, expressing ELISA(enzyme-linked immuno sorbent assay) test, sequencing, and finally obtaining the single-chain antibody against fumonisin named as FB-Mu 1H3 with high affinity and a coding gene thereof. The single-chain antibody can be directly applied to assay of fumonisin after being expressed and purified in a large quantity in escherichia coli, and the applications include assay of fumonisin pollution in field crops, feed, grain or food.

The invention relates to a polyclonal antibody for detecting the residue and the degradation of a CP4-EPSPS protein in a processed food and application thereof. An animal is immunized by using a CP4-EPSPS protein peptide segment of a coupled keyhole limpet hemocyanin (KHL) to obtain antiserum, namely the antibody of the invention. The application comprises a Western blotting and a colloidal gold test strip and has the characteristics of simplicity, practicability, high sensitivity and the like, wherein the Western blotting is used for detecting the residue and the degradation of the CP4-EPSPS protein in food; and test strip technology is used for the rapid detection of the CP4-EPSPS protein in the food. The invention provides a high-practicability method for the safety detection of a genetically modified food, and has a very high practical application value.

The invention relates to a monoclonal antibody of hepatitis B virus X protein (HBx), which is characterized by having specificity reaction to N-end epitope or C-end epitope of HBx, but having no reaction to keyhole limpet hemocyanin (KLH) of carrier protein and other expressed proteins of hepatitis B virus (HBV). The preparation method of the monoclonal antibody comprises the steps of: fusing N-end and C-end antigen epitope polypeptides of HBx respectively with mice immunized with KLH in crosslinking way, and myeloma cells and screening to obtain the monoclonal antibody. The monoclonal antibody can be used for various immunodetection reagents and directed therapeutic drugs of HBx protein or HBx antibody and applied to diagnosis and treatment of diseases such as hepatitis b virus (HBV) infection, hepatocellular carcinoma (HCC) and the like.

The invention provides an anti-aflatoxin general type monoclonal antibody hybridoma cell line and application thereof, belonging to the field of food safety immunodetection. The cell line 1G6 provided by the invention is collected in the China General Microbiological Culture Collection Center (CGMCC) with a collection number of CGMCC No.7210. After an aflatoxin complete antigen is uniformly mixed with equivalent Quick Antibody immunologic adjuvant TM, an obtained mixture is injected to immunize BALB/c mice through leg muscle. B1-KLH (fumonisin B1-Keyhole Limpet Hemocyanin) is adopted for the first time of immunization, M1-KLH (fumonisin M1-Keyhole Limpet Hemocyanin) is adopted for the second time of immunization, and an M1 complete antigen (without containing adjuvant) is adopted for the last time of impaction immunization. Splenocytes of immunized mice are fused with myeloma cells through a PEG (polyethylene glycol) method, and the hybridoma cell line 1G6 is obtained through indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) and indirect competence ELISA screening and three times of subcloning. A monoclonal antibody secreted from the cell line has better affinity and detection sensitivity for aflatoxins B1, B2, G1, G2 and M1, and can be used for immunodetection of the total quantity of the aflatoxins in the food safety.

The invention relates to the detection of a water sample and establishes a rapid colloidal gold immunochromatography detection method and test paper of microcystins MC-LR. The method comprises the following steps of: firstly, preparing a colloidal gold with the average diameter of about 30 nm and utilizing the colloidal gold to mark an anti-microcystins MC-LR monoclonal antibody; covering a conjugate of a microcystins MC-LR hapten and keyhole limpet hemocyanin on a nitrocellulose membrane as a detection belt; taking a goat anti-mouse second antibody as a control belt; and establishing an immunochromatography test paper method for rapidly detecting the microcystins MC-LR. According to the method, the sensitivity can reach to 3 ng/mL and a judged result can be observed by naked eyes only after 3-5 min; and the method has the characteristics of rapidness, direct observation, simplicity in operation, convenience for use and the like, so that the method can be used as an effective way for screening whether a lot of field freshwater and freshwater products are infected by the microcystins MC-LR.

The invention belongs to the technical field of food safety immunodetection and particularly discloses hapten, artificial antigen and antibody directly targeted to alternariol and a preparation method and application thereof. The alternariol hapten is of the structure as indicated in formula (I), wherein n=1, 2. The alternariol artificial antigen is of the structure as indicated in formula (III), wherein n=1, 2; carrier protein is keyhole limpet haemocyanin or bovine serum albumin or ovalbumin. The titer of antiserum obtained from an immune animal of the artificial antigen can reach 1: 128000, the minimum detection limit is 1.15 ng/mL, the half inhibiting concentration is 16.5 ng/mL, the antibody has the remarkable advantages of being high in specificity, good in sensitivity, high in accuracy and the like, therefore the antigen and the antibody can be used for establishing an alternariol enzyme linked immunosorbenption and analysis technology, and thereby the hapten, artificial antigen and antibody directly targeted to alternariol and the preparation method and application thereof can be used for rapidly detecting residual alternariol in food and have wide application prospect.

The invention relates to an ELISA (enzyme-linked immunosorbent assay) kit for an EV (enterovirus) 71 inactivated vaccine antigen. The detection kit comprises an EV71 pre-coated polyclonal antibody ELISA plate, a sample diluent, a second antibody, an enzyme-labeled antibody, a concentrated cleaning solution, an enzyme substrate solution and a stop buffer, wherein the ELISA plate is pre-coated with a polyclonal antibody prepared by taking recombinant EV71 structural protein 1 as an immune source; the second antibody adopts a monoclonal antibody prepared by taking keyhole limpet hemocyanin coupled polypeptide sequence FGEHKQEKDL as the immune source; the enzyme-labeled antibody adopts a horse radish peroxidase labelled goat anti-mouse immunoglobulin antibody; and an antibody standard is placed in the kit. The ELISA kit has better sensitivity when measuring the titer of the EV71 inactivated vaccine antigen, and has better linearity in the range of 5.9-750 ng/ml, and the linearly dependent coefficient r2 is larger than 0.99. The kit for measuring the titer of the EV71 inactivated vaccine antigen simply and conveniently has good specificity, accurate quantification, high sensitivity and good repeatability.

The invention provides an immunogen for obtaining an Nrf1D protein antibody, the Nrf1D protein antibody and an Elisa detection kit. The immunogen is obtained as follows: polypeptide with the amino acid sequence shown in SEQ ID NO.1 is synthesized firstly, Sulfo-SMCC is used as a coupling agent, the polypeptide and KLH (keyhole limpet hemocyanin) are coupled; the antibody is obtained as follows: immunogen is taken as an antigen, the antigen is emulsified with FCA (freund's complete adjuvant) to immunize a New Zealand white rabbit, blood of the immunized New Zealand white rabbit is centrifuged, and a supernatant after centrifugation is collected; the Elisa detection kit comprises components including an ELISA plate, an enveloping solution, a blocking solution, an ELISA secondary antibody, a TMB color developing solution, a stop solution, a detection antigen and the Nrf1D protein antibody, the detection antigen is the polypeptide with the amino acid sequence shown in SEQ ID NO.1, glutaradehyde is used as a coupling agent, and the polypeptide and BSA bovine serum albumin are coupled.

The invention discloses a method for treating septicemia with human neutrophil peptide (HNP) blocking agents. The method comprises the following steps of: preparing small interfering RNA (siRNA) and anti-HNP antibody blocking agents and the like, and blocking, neutralizing and reducing the yield, the function and the biological activity of HNP. The siRNA is prepared in vitro through a design and a chemical synthesis method. A HNP-1 polypeptide is produced through a design to be cross-linked with KLH (Keyhole Limpet Hemocyanin) and then to immunize an experimental rabbit, and a rabbit immune globulin is obtained. The same one HNP-1 polypeptide is used for immunizing a mouse, an anti-HNP antibody variable region is prepared, and then a humanized monoclonal anti-HNP-1 antibody is prepared. Both of the siRNA and the anti-HNP-1 antibody can effectively block, neutralize and reduce the yield, the function and the biological activity of HNP, so that the method can be used for treating septicemia and other related diseases caused by bacterial infections.

The present invention relates to methods and materials for the detection and quantitation 8-OH-Ade in biological specimens. Specifically, the present invention is directed to a group of highly specific monoclonal antibodies reactive with the modified nucleoside structure 8-OH-Ade, and to various immunoassays for 8-OH-Ade utilizing these monoclonal antibodies. The monoclonal antibodies of the present invention may be used in assays for diagnosing or monitoring the progression of certain types of cancer, in addition to a variety of other diseases associated with mutagenesis resulting from oxidative damage of DNA. Assays utilizing the monoclonal antibodies of the present invention may also be used to analyze or monitor toxicant exposure, such as from environmental sources. The monoclonal antibodies of the present invention were prepared with the immunogen 8-OH-adenosine coupled to keyhole limpet hemocyanin (KLH), not to 8-OH-Ade directly. It is believed that the monoclonal antibodies bind with the base portion of the structure (8-OH-Ade) and not the carbohydrate (ribose) or protein linkage region of the conjugate, because, as demonstrated, conjugates bound to nucleosides other than 8-OH-adenosine were unreactive with these antibodies. Therefore, the antibodies of the present invention can be used to detect and quantitate (by the use of a standard curve) the presence of 8-OH-Ade in biological specimens of DNA. Procedures for such an assay include immobilizing the DNA, denaturing it to disrupt the base-pairing scheme exposing the free base structures, and quantitating the amount of 8-OH-Ade present per amount of DAN in a quantitative immunoassay.

The invention relates to a chlordecone antigen, a chlordecone antibody and a preparation method thereof. The antigen is an immune antigen obtained by connecting hydroxyl of single hydroxyl chlordecone with amino in keyhole limpet hemocyanin or bovine serum albumin through N,N-carbonyldiimidazole serving as a coupling agent; and hybridoma prepared from the antigen is subjected to specificity screening, in vitro culture and purification to form the antibody. By a characteristic of high-efficiency specificity competitive combination of the antigen and the antibody, the complicated pretreatment process is not needed, the whole detection process can be completed in 5-8 hours, and a detection threshold for detecting the chlordecone is 6.25 to 0.312mu g/ml. Compared with the conventional instrument detection, the invention is quicker and cheaper, and can be used for analytical detection, quantitative determination, quality standard and purification of chlordecone samples and blockage of bioeffect of the chlordecone. The materials are efficiently, quickly and cheaply detected and analyzed.

The invention relates to a synthetic pokeweed antiviral protein (PAP) antigen and antibody, and a preparation method and application thereof, belonging to the technical field of biology. The synthetic pokeweed antiviral protein antigen comprises a carrier protein with immunogenicity, and a synthetic polypeptide coupled with the carrier protein, wherein the amino acid sequence of the polypeptide is SEQ ID NO 1 or SEQ ID NO 2. The coupler of the synthetic polypeptide and keyhole limpet hemocyanin is used for preparing three PAP antisera; the three PAP antisera can be used for PAP preparation, PAP gene protocaryon expression and detection of eukaryon expression products, transgenic regenerated plants and the like; and the invention can be used for detecting the expression conditions of PAP and gene thereof by immunological means, thereby providing physical foundation for detecting PAP.

The invention relates to the technical field of biological chemistry, and particularly discloses a preparation method of fluoroacetamide hapten and application of a monoclonal antibody. The fluoroacetamide hapten is synthesized by taking ethyl fluoroacetate and p-aminophenylacetic acid as main raw materials; the fluoroacetamide hapten and carrier protein bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) are coupled through an active lipid method, and thus a conjugate is prepared. Through experimental verification, it shows that the prepared conjugate enables a living body to generate the antibody against fluoroacetamide, that is to say, the prepared conjugate is fluoroacetamide artificial antigen. The prepared fluoroacetamide artificial antigen is suitable for enzyme-linked immunological analysis of fluoroacetamide.

The present invention provides a method for altering a T cell mediated pathology in a patient. This method comprises administering a composition comprising at least one and/or two chimeric proteins. Each chimeric protein comprises at least a portion of either the Valpha or Vbeta region of a TCR from particular T cells from a patient having a T cell mediated pathology, and an immunoglobulin constant region. The genes encoding Valpha and/or Vbeta regions and the genes encoding immunoglobulin constant regions are isolated and inserted into an expression vector. The chimeric proteins are produced by introducing the expression vectors into insect cell lines. The chimeric proteins are purified using antibody affinity columns, and then chemically conjugated to an immunogenic carrier, keyhole-limpet hemocyanin (KLH). Since the conjugates comprises chimeric proteins made specifically from particular T cells from a patient having T cell mediated pathology, when it is administered to such a patient, with or without a cytokine, such as granulocyte-macrophage-CSF, or a chemokine, it can induce immune responses to alter such a T cell mediated pathology.

The present invention provides immunogens for generating anti-neonicotinoid antibodies as well as antibodies, methods, reagents, and kits for determining the presence of one or more neonicotinoid insecticides in a sample by immunoassay. The present invention has particular application to determination of the concentration of neonicotinoid insecticides applied to a plant propagation materials such as seed, said antibody is selective for a compound of formula (I) wherein A is 2-chloropyrid-5-yl or 2-chlorothiazol-5-yl; R and R<3> independently are hydrogen or C1-C4alkyl; R<1> and R<2> independently are hydrogen or C1-C4alkyl or R<1> and R<2> are taken together with the nitrogen atoms to which they are attached to form an imidazoline or an oxadiazine ring; and X is N-NO2 or N-CN. Also claimed is a compound of formula (IA) and protein conjugate of formula (II) in which PR is a protein residue of a carrier molecule selected from the group consisting of a purified protein derivative (PPD, Tuberculin) from Diptheria virus, bovine serum albumin, cationized bovine serum albumin, human serum albumin, ovalbumin and keyhole limpet hemocyanin; or an enzyme residue selected from the group consisting of alkaline phosphatase, horseradish peroxidase, and beta -galactosidase.

The invention discloses a synthesis method of an abietic acid artificial antigen. The synthesis method comprises the following steps: taking 12-hydroxyabietic acid (12-HA) as a hapten and carrying outtwo-step 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (DEC) catalysis reaction; taking adipic acid (AA) as a coupling arm to be coupled with keyhole limpet hemocyanin (KLH) and bovine serum albumin(BSA) respectively, so as to synthesize the abietic acid artificial antigens 12-HA-AA-KLH and 12-HA-AA-BSA respectively. A polyclonal antibody obtained by immunizing domestic rabbits through the abietic acid artificial antigen prepared by adopting the synthesis method has ideal valence and can be used for establishing an ELISA method for detecting rosin residues in livestock and poultry meat products.

The invention belongs to the field of biochemistry and molecular immunology, and relates to a porcine neuronal interleukin B polypeptide, and a preparation method and application of a polyclonal antibody thereof. The preparation method comprises the following steps: coupling C-terminal modified polypeptide of the porcine neuronal interleukin B polypeptide with a carrier protein keyhole limpet hemocyanin KLH to obtain an NMB modified polypeptide-KLH coupled protein; emulsifying the NMB modified polypeptide-KLH coupling protein, and immunizing New Zealand white rabbits; collecting and separatingto obtain serum containing a rabbit anti-pig NMB modified polypeptide antibody; and purifying the prepared NMB polyclonal antibody by adopting a Protein G purification column method. A polypeptide antigen and the polyclonal antibody have the characteristics of simple preparation method, low cost and high titer, and can specifically bind to an NMB protein in pig tissues.

The invention relates to a preparation method of an anti-TMP (thrombopoietin mimic peptide) rabbit polyclonal antibody. The preparation method comprises the steps of coupling thrombopoietin mimic peptide (TMP) and keyhole limpet hemocyanin (KLH) to prepare KLH-TMP as an immunizing antigen, collecting blood from a purified antigen immunized New Zealand white rabbit to prepare antiserum, and separating and purifying the anti-TMP rabbit polyclonal antibody from the antiserum. The purity of the antibody serum can reach above 85% after purification by a salting-out method. The antibody prepared bythe method has the characteristics of high purity, specificity and affinity.

The invention discloses a preparation method of a beta2-microglobulin monoclonal antibody. The method comprises the following steps: firstly, screening out two polypeptide epitope sequences with relatively strong immunogenicity as a first antigen epitope and a second antigen epitope respectively; then respectively connecting one cysteine to each carbon-terminal amino acid of the first antigen epitope and the second antigen epitope, and coupling sulfydryl of the cysteine with primary amino of keyhole limpet hemocyanin through an SMCC coupling method so as to obtain an in-vitro synthesized beta2-MG double-epitope polypeptide antigen; then carrying out conventional immunization on the mouse by utilizing the double-epitope polypeptide antigen, and separating out splenic lymphocytes and myelomacells to carry out cell fusion; and finally, screening out positive monoclonal cells, and extracting monoclonal antibody through intraperitoneal injection and ascites recovery. By improving traditional single-epitope antigen immunization methods, the method optimizes the immunogenicity of the antigen, shortens the immunization period, and improves the serum titer in the immunization stage, thereby improving the obtaining efficiency of the positive monoclonal antibody against beta2-MG.

The invention provides a novel specific marker Aire (Autoimmune Regulator) of embryonic stem cells and application thereof. Polypeptide coupling carrier protein KLH (Keyhole Limpet Hemocyanin) composed of a part of amino acid sequences at the C tail end in an Aire sequence is used as immunogen; a prepared Aire polyclonal antibody and a rabbit monoclonal antibody Glut3 (Glucose Transporter 3) and/or GM-CSFRalpha (Granulocyte Macrophage Colony-Stimulating Factor Receptor) prepared by directly taking the embryonic stem cells as the immunogen are used as stem cell identification reagents; the specificity and the sensitivity of detection are higher; furthermore, the specific marker Aire can be used for separation and activity detection of the embryonic stem cells. An embryonic stem cell detection kit containing the Aire polyclonal antibody and the rabbit monoclonal antibody Glut3 and/or GM-CSFRalpha can be used for identifying the totipotency of the embryonic stem cells and also can be used for detecting activity of stem cells simultaneously; therefore, functions of the detection kit are expanded.

The invention discloses a vitamin B5 (VB5, (R)-N-(3,3-dimethyl-2,4-dyhydroxy-1-oxobutyl)-3-calcium alanine) conjugate. The vitamin B5 conjugate is formed by coupling vitamin B5 hapten and keyhole limpet hemocyanin (KLH) which generates immunogenicity and is a carrier material, wherein n refers to number of molecules, of VB5, combined with one keyhole limpet hemocyanin molecule and is an integer, KLH refers to keyhole limpet hemocyanin, and molecular weight ranges from 450KDa to 1300KDa. The invention further discloses a preparation method of the vitamin B5 conjugate. The preparation method includes: connecting vitamin B5 with the carrier material generating immunogenicity to form the coupling material capable of inducing an animal immune system to generate antibodies. The preparation method has the advantages of convenience, quickness, specificity and accuracy. A wide application space is provided for using the coupling material to manufacture an enzyme-linked immunosorbent assay reagent box of VB5.