55 results about "Freunds complete adjuvant" patented technology

The invention provides a method for preparing triple vaccine for preventing porcine transmissible gastroenteritis, porcine epidemic diarrhea and porcine rotavirus. The method comprises the following steps: inoculating a host-cell line with a 90 percent grown monostratum against a porcine transmissible gastroenteritis virus, a porcine epidemic diarrhea virus and a porcine rotavirus respectively, and adding a cell maintenance media into the host-cell lines respectively to be cultured at 37 DEG C; after cytopathic effect reaches over 75 percent, collecting viruses to be stored at 20 DEG C below zero for standby; mixing the viruses according to 10 TCID50 in 1:1:1, and simultaneously adding Freund's complete adjuvant and immunopotentiator into the mixture to inactivate the mixture by formaldehyde at 37 DEG C for 24 hours; and adding an oil adjuvant into the mixture to prepare a vaccine of water-oil-water preparation. The method can be used for preparing the triple vaccine for preventing the porcine transmissible gastroenteritis, the porcine epidemic diarrhea and the porcine rotavirus so as to solve the problem that the diseases do not have an effective medicine to treat currently.

An immunoglobulin for preventing the infectious gastroenteritis, epidemic diarrhea and rotavirus of hog is prepared from their immunogens through cell culture, concentrating and proportionally mixing it with complete Frennd's adjuvant. It features that the glycoside-peptide injection is used as its immunopotentiator.

The invention discloses a modeling method of an Sjogren syndrome mouse model. The modeling method comprises the following steps: killing mice, taking out bilateral salivary glands and peeling off capsules and connective tissues; washing with PBS (Poly Butylenes Succinate); adding the PBS according to the amount of adding 0.5ml of the PBS into each salivary gland; shearing the salivary glands into pieces, and uniformly homogenizing and centrifuging in an ice bath; then taking supernatant and quantifying salivary gland antigens by adopting a BCA (Bicinchoninic Acid) protein quantifying method; adjusting the concentration of the salivary gland antigens to be 4mg / ml by utilizing the PBS; adding equal quantity of an FCA (Freund Complete Adjuvant) or an FIA (Freund Incomplete Adjuvant) and diluting the concentration to be 2mg / ml; repeatedly blowing and beating until two liquid phases are dissolved mutually to form an ivory color; randomly grouping C57BL / 6 mice and shaving off furs on the backs of the mice; carrying out intradermal multi-site injection of 2mg / ml mouse salivary gland antigens prepared by the FCA on the back and tails of the mice on the current day and the 7th day, wherein the injection amount is 0.1ml per mouse; injecting equal quantity of the salivary gland antigens prepared by the FIA on the 14th day through the same method; after modeling for about 6 weeks, detecting indexes and screening the successfully modeled mice. The modeling method disclosed by the invention is high in modeling efficiency and short in modeling time.

The invention provides preparation methods for duck hepatitis virus immunogen and hyperimmune serum and application of the duck hepatitis virus hyperimmune serum. According to the preparation methods and the application of the duck hepatitis virus hyperimmune serum, the duck hepatitis virus immunogen is obtained through inoculating a serum 1 type duck hepatitis virus CH60 strain DHAV-1 (Duck Hepatitis A Virus type 1) or a serum 3 type duck hepatitis virus CH1 strain DHAV-3 (Duck Hepatitis A Virus type 3) to an allantoic cavity of a chick embryo of 9-10 days old or a duck embryo of 10-12 days old and carrying out proliferation and treatment, and the hyperimmune serum is obtained through mixing the duck hepatitis virus immunogen with a Freund's complete adjuvant or Freund's incomplete adjuvant to prepare solutions of different concentrations, carrying out repeated immunization on immune animals and then sampling and collecting blood and can be applied to the diagnosis and detection on a duck hepatitis virus. The preparation methods provided by the invention have the advantages that the conditional requirements are low, the operation is simple, and the obtained immunogen can meet the requirements on the preparation of specific antiserum.

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 preparation method of a monoclonal antibody to chloramphenicol (CAP). The method comprises the following steps of: (1) preparing an antigen of CAP-BSA by a diazotization method; (2) conducting lymph immunization to mice with the CAP-BSA antigen, leaving the antigen and a Freund's complete adjuvant to complete emulsification during the first time immunization, and during the second time and the third time immunization, leaving the antigen and a Freund's incomplete adjuvant to complete emulsification, and keeping an interval of 7 days between each time, and 3 days before fusion, carrying out direct antigen injection to the abdominal cavity to booster immunization; (3) measuring serum titers of the mice by an enzyme-linked immunosorbent assay (ELISA) method; (4) selecting a mouse with the highest serum titer, and taking a spleen cell and a myeloma cell for in vitro fusion; (5) culturing and screening a fusion cell in a selective medium, conducting positive cell detection and screening for cloning culture, further performing positive cell cloning and screening for positive cloning, expanding culture and cryopreservation; (6) injecting a cell strain undergoing expanded culture into the abdominal cavities of the mouse so as to produce a lot of ascites; (7) using a chromatographic column to purify the monoclonal antibody against chloramphenicol in the ascites. The prepared monoclonal antibody against CAP-BSA has the advantages of high sensitivity, strong specificity and good practicality.

The invention relates to the technical field for preventing and curing sugarcane ratoon stunting disease, providing a method for preparing germs polyclonal antibody of sugarcane ratoon stunting disease, comprising the following steps: (1) culturing pathogenic germs by means of isolation to obtain antigen; (2) mixing the antigen with equivalent Freund complete adjuvant, emulsifying, and immunizing rabbit; (3) mixing the antigen with equivalent Freund incomplete adjuvant, emulsifying, and again immunizing rabbit, repeating the immunizing process several times, after measuring of titer meets requirement, collecting blood from the heart, isolating antiserum; and (4) purifying the antiserum to obtain the polyclonal antibody. In the invention, the germs of the sugarcane perennial root dwarfing is isolated from sugarcane juice, thus the polyclonal antibody which is obtained thereby can specifically identify the germs of the sugarcane ratoon stunting disease in the sugarcane juice. The germs polyclonal antibody of the sugarcane ratoon stunting disease has the advantages of high specificity, high purity and high titer, can be preserved for a long time, and has important practical significance in measuring the pathogenic germs to diagnose the sugarcane ratoon stunting disease (RSD) by means of immunology in the scientific research and the production of the sugarcane.

The present invention is a preparation method of Vibrio parahaemolyticus toxoid vaccine, which is characterized in that: first amplifying the target gene tdh; then cloning the target gene fragment tdh into the vector pET-28 to construct the expression vector pET-28-TDH, The pET-28-TDH plasmid was transferred into the expression strain BL21; after culturing and induced expression, the cloned expression product was obtained; the cloned expression product was detected by dot blot reaction, and the Vibrio parahaemolyticus toxoid was obtained; the Vibrio parahaemolyticus toxoid was mixed with An equal volume of complete Freund's adjuvant was mixed evenly to obtain the Vibrio parahaemolyticus toxoid vaccine. The vibrio parahaemolyticus toxoid vaccine prepared by the method of the invention can be used as an immune drug for marine fishes attacked by the vibrio parahaemolyticus, and the immune protection rate can reach about 50%.

The invention relates to a preparation method of anti swine fever immunoglobulin, which comprises (1), mixing inactivated swine fever and freund complete adjuvant cultured via tissue at the mol ratio of 1:1 as immunogen immune bovine, while each acceptor animal is injected with 40-60 portions of swine fever vaccines at multipoints on neck, (2), after 10-15 days of immunity, processing reinforced immunity via 150-250 portions of swine fever vaccines, (3), using natural compresson coagulation method to separate immunoglobulin, depositing impure proteins via ammonium sulfate, cutting Fc segment of pepsin, keeping antibody F(ab) second segment, depositing Fc protein via ammonium sulfate, removing Fc protein, adding hydrochloride to adjust pH to 2.0, standing for 2 days at 4.0DEG C, disinfecting, adding NaOH to adjust pH 7.0, killing other animal susceptible viruses, (4) adding antibiotics into blood, standing for 5-10h, separating serum, separating and extracting immunoglobulin via caprylic acid precipitation and ammonium sulfate precipitation, (5) filtering and removing bacterial, packing without germ. The invention has simple preparation and simple application.

An immunoglobulin for preventing the cholera, pseudorabies and parvovirus of hog is prepared from their immunogens through proportional mixing them together and proportionally mixing it with complete Frennd's adjuvant. It features that the glycoside-peptide injection is used as its immunopotentiator.

The invention relates to a recombinant Wzt protein rabbit serum polyclonal antibody and a preparation method thereof, which relate to the field of antibody preparation, and fill the gap of applying recombinant Wzt protein for preparing the specific polyclonal antibody. The preparation method of the polyclonal antibody comprises the steps of after mixing and emulsifying the recombinant Wzt protein and an equivoluminal freund's complete adjuvant, carrying out multiple sites subcutaneous injection on the back parts of female rabbits; carrying out secondary immunizing: after one week, after mixing and emulsifying the recombinant Wzt protein and the equivoluminal freund's incomplete adjuvant, carrying out multiple sites subcutaneous injection on the back parts of the female rabbits; carrying out thrice immunizing and quartic immunizing every other week according to a secondary immunizing process; collecting venous blood of female domestic rabbit ears after immunizing each time, and detecting a rabbit serum antibody titer; at one week after quartic immunizing, collecting rabbit whole blood, centrifugally collecting a supernatant to obtain a rabbit serum, and obtaining the recombinant Wzt protein rabbit serum polyclonal antibody after purifying. The recombinant Wzt protein rabbit serum polyclonal antibody has favorable specificity, and can be used for detecting Brucella Wzt protein.

A hybridoma cell line of secreting cyproheptadine monoclonal antibodies with a preservation number of CGMCC No. 14699 belongs to the field of food safety immunological detection. BALB / c mice are immunized through one time immunization with complete freund's adjuvant, three times of booster immunization with incomplete freund's adjuvant and one time of rush immunization with cyproheptadine complete antigen without adjuvant; the spleen cells from BALB / C mice immunized with high potency and low value of IC50 are fused with murine myeloma cells; and then the hybridoma cell line is obtained through indirect competitive ELISA screening and three sub-clones. The monoclonal antibody secreted by this cell line has good specificity and detection sensitivity to cyproheptadine (value of IC50 is 0.37 ng / ml), being suitable for detection of cyproheptadine in food.

An immunoglobulin for preventing the reproduction-respiration syndrome and gyrate virus B of hog is prepared from their immunogens through cell culture, concentrating and proportionally mixing it with complete Frennd's adjuvant. It features that the glycoside-peptide injection is used as its immunopotentiator.

A preparation method of a grass carp ATG12 polyclonal antibody comprises the steps: firstly, taking grass carp liver RNA as a template, carrying out reverse transcription to synthesize cDNA, then taking the cDNA as a template, amplifying by PCR with primers and recovering a target fragment, and then constructing an ATG12 / pGEX-4T-1 prokaryotic expression vector; transferring the ATG12 / pGEX-4T-1 prokaryotic expression vector into competent cells of escherichia coli DH5[alpha], treating, then carrying out ultrasonic crushing and centrifugation to obtain a supernatant and a precipitate, and then purifying the precipitate to obtain purified ATG12 fusion protein; and finally, fully mixing and emulsifying the purified ATG12 fusion protein as an antigen with a Freund's complete adjuvant with the same volume with the ratio of 1:1, immunizing pure New Zealand white rabbits, extracting heart blood, and separating serum, to obtain the ATG12 polyclonal antibody. The antibody has the advantages of high specificity, low cost and short period, and lays the foundation for obtaining the commercially available ATG12 antibody.

The invention discloses a recombined subunit vaccine of haemaphysalis concinna and a preparation method thereof. The recombined subunit vaccine is formed by mixing antigenic gene recombined protein of the haemaphysalis concinna and Freund's complete adjuvant (FCA), wherein the content of the antigenic gene recombined protein is 50 mg / ml, that is 50 mL rHc-23 and 950 ml Freund's complete adjuvant are mixed to prepare the recombined subunit vaccine of the haemaphysalis concinna; the amino acid sequence of the antigenic gene recombined protein is shown in the table SEQID NO:1; and the nucleotide sequence of the antigenic gene is shown in the table SEQID NO:2. Through screening and cloning, prokaryotic expression and separation and purification of protective antigen gene of the haemaphysalis concinna and the application effect test of the recombined subunit vaccine, the method shows that an expression product of recombined vector bacteria can be identified by rabbit anti-haemaphysalis concinna positive serum. In an animal immunity test, after rHc-23-FCA is subjected to three immune rabbits, the blood saturation rates of haemaphysalis larva, haemaphysalis middle and haemaphysalis imago are 40.3 percent, 45.6 percent and 41.3 percent respectively; and compared with the blood saturation rates of the haemaphysalis larva, haemaphysalis middle and haemaphysalis imago in a contrast set: 90.1 percent, 94.3 percent and 97.7 percent, the differences are remarkable. The method promotes the development of anti-haemaphysalis immunity, haemaphysalis control and haemaphysalis disease spread work.

A hybridoma cell strain secreting a nifursolol residue marker monoclonal antibody with CGMCC No. 14698 is prepared in the following way: BALB / c mice are subjected to the first immunization with a complete Freund's adjuvant, subjected to booster immunization with an incomplete Freund's adjuvant for four times, and subjected to rush immunization once with nifursolol residue marker complete antigen without a Freund's adjuvant so that the BALB / c mice are immunized; the spleen cells of the immunized mice with high titer and low IC50 were fused with mouse myeloma cells by a PEG method, and the fused cells are screened through indirect competitive ELISA and subcloned three times. The monoclonal antibody secreted by this cell strain has good specificity and detection sensitivity (IC50 value of 2 μg / L) to the nifursolol residue marker and can be used for residue detection of the nifursolol residual marker in food.

The invention discloses an egg yolk antibody and a preparation method and device thereof. A test object is used for performing polypide reinvigoration and proliferation on tender eimeriida, giant eimeriida and poisoned eimeriida, after the insects are mixed in equal proportions, antigen is prepared by ultrasonic disruption, the obtained antigen solution is mixed with an equal volume of freund's complete adjuvant and freund's incomplete adjuvant separately, laying hens are immunized three times, egg yolk antibodies in collected egg yolk after three times of immunizationare extracted, purified and identified, and the antibody titer is evaluated; the research on different yolk powder preparation processes is carried out, the effect of lyophilized powder preparation is significantly higher than that of powder spraying technology, the matchingrelationship between the yolk and the dilution, the lyophilization temperature curve and other parameter conditions in the process are prepared, the cost is relatively low, the preparation efficiency is high, antibody loss rate is only 10%, high antibody titer, antibody titer after high-temperature powder spraying shows a high downward trend, and the method is obviously superior to the high-temperature powder spraying technology for preparing yolk powder.

A hybridoma cell line of secreting meloxicam monoclonal antibodies with a preservation number of hybridoma cell line of CGMCC No. 14700 belongs to the field of food safety immunological detection. The. BALB / c mice are immunized through one time immunization with complete freund's adjuvant, three times of booster immunization with incomplete freund's adjuvant and one time of rush immunization with meroxicam complete antigen without adjuvant; the spleen cells from BALB / C mice immunized with high potency and low value of IC50 are fused with murine myeloma cells; and then the hybridoma cell line is obtained through indirect competitive ELISA screening and three sub-clones. The monoclonal antibody secreted by this cell line has good specificity and detection sensitivity to meloxicam (value of IC50 is 0.1 ng / ml), being suitable for detection of meroxicam in food.

The invention relates to a preparation method of bovine anti chicken avian influenza immunoglobulin, which comprises (1), mixing inactivated chicken avian influenza and freund complete adjuvant cultured via tissue at the mol ratio of 1:1 as immunogen immune bovine, while each cattle is injected with 40-100 portions of chicken avian influenza vaccines at multipoints on neck, (2), after 10-15 days of immunity, processing reinforced immunity via 150-300 portions of chicken avian influenza vaccines, (3), adding antibiotics into blood, standing for 5-10h, separating serum, using natural compresson coagulation method to separate immunoglobulin, depositing impure proteins via ammonium sulfate, cutting Fc segment of pepsin, keeping antibody F(ab) second segment, depositing Fc protein via ammonium sulfate, removing Fc protein, adding hydrochloride to adjust pH to 2.0, standing for 2 days at 4.0DEG C, inactivating virus, adding NaOH to adjust pH 7.0, killing other animal susceptible viruses, (4), filtering and removing bacterial, packing without germ. The invention has simple preparation and simple application.

The invention discloses a preparation method of immunoglobulin for resisting porcine epidemic diarrhea, which includes the following steps: (1) mixing tissue-cultured deactivated porcine epidemic diarrhea virus and Freund complete adjuvant at a volume ratio of 1:1, and performing fundamental immunity on cattle immunized with porcine epidemic diarrhea vaccine; (2) performing booster immunization after 7-10 days; (3) slaughtering and sampling blood; (4) adding penicillin and streptomycin, naturally solidifying, standing, and separating serum; (5) precipitating with ammonium sulfate to obtain the hybridprotein, and killing susceptive virus; and (6) filtering and degerming. The prepared immunoglobulin has the advantages of good therapeutic effect, good safety and reliability, no side and toxic effects, low cost, etc.

The invention relates to the technical field of biological protein molecules, in particular to a kenaf mitochondrial protein COX3 antigen polypeptide and a method for preparing a polyclonal antibody and an application. An amino acid sequence of the antigen polypeptide is shown in SEQ ID NO: 2. The method includes coupling the antigen polypeptide with carrier protein KLH to obtain polypeptide-KLH coupling composite protein; mixing with a Freund's complete adjuvant with equal volume, emulsifying, and immunizing animals; collecting animal blood serum, collecting animal blood by carotid artery bloodletting when the titer reaches more than 1: 20000, and preparing antibody serum; and purifying the antibody serum by affinity chromatography to obtain the polyclonal antibody of the kenaf mitochondrial protein COX3. By synthesizing the kenaf COX3 polyclonal antibody, the expression difference of the kenaf COX3 protein in different cytoplasm of kenaf is detected, so as to determine the importantinfluence of COX3 on the development of kenaf anthers.

The invention provides a preparation method of micro-encapsulation coated yolk source cholecystokinin (CCK) antibodies. The preparation method comprises the following steps: preparing an antigens complex through adding freund's complete adjuvant in the mixture formed after a sheep derived CCK and BSA (Bovine Serum Albumin) are crosslinked to immunize laying hens, collecting eggs and obtaining the yolk derived CCK antibodies from the eggs, taking sodium alginate aqueous solution to perform the homogeneous emulsification with yolk derived CCK antibody liquid, dripping the emulsified solution into encystation solution consisting of chitosan and CaCl2 to be coated. The coated yolk derived cholecystokinin antibodies protect the effective actives of the yolk derived cholecystokinin antibodies, and have enteric solubility. According to the preparation method, a micro-encapsulation technology is adopted, and chitosan and sodium alginate are used as the coating materials which are natural polysaccharide, are low in cost, are taken orally and have no any toxic and side effect.

An immunoglobulin for preventing the pest of gooselet is prepared from the allantoic fluid of the embryo of goose or duck suffering from the pest through cell culture, concentrating and proportionally mixing it with complete Frennd's adjuvant. It features that the glycoside-peptide injection is used as its immunopotentiator.

The invention discloses a rabbit monoclonal antibody preparation method. The method comprises the following steps of 1, preparing immune serum; 2, affine purifying an antibody; 3, obtaining a mass spectrum; 4, obtaining an antibody variable region gene spectrum; 5, obtaining the monoclonal antibody, wherein, in step 1, heteromorphic rabbits with bas mutations and wild parents b9 are selected, subcutaneous inoculation of freund's complete adjuvant on the rabbits, immunization is performed every three days, immunization is performed three times, injection is performed one time two weeks after immunization, one month after immunization, NG-XMTTM protein is subjected to intravenous injection at the tails of the rabbits, a sterile small test tube is used for collecting immunized rabbit blood, after marking, the rabbits are delivered to a preparation room, unimmunized rat serum is adopted as control, ELISA is adopted for determining antibody valence, the ELISA is used for determining the OD490 light absorption value quantification and qualitatively judging whether or not the immune serum is positive, and the positive immune serum is selected; the method is more efficient and rapider andhas high affinity.

The invention provides a preparation method for a monoclonal antibody of riemerella anatipestifer. The method comprises the following specific steps: (1) expanding the culture of the riemerella anatipestifer, inactivating formaldehyde, and then preparing suspension by using PBS to complete antigen preparation; (2) mixing the newly prepared antigen with a freund's complete adjuvant uniformly and stirring for 4 hours until a mixture is emulsified, then performing subcutaneous injection on mice, mixing the newly prepared antigen with the freund's incomplete adjuvant uniformly and stirring for 4 huntil a mixture is emulsified after two weeks of immunization, performing subcutaneous injection on the mice again to obtain immunized mice after one week of immunization, and detecting antibody titer is 1 to 6400; (3) performing cell fusion on the spleen cells of the immunized mice and SP2 / 0 cells; 4) performing positive screening and cloning on fusion cells in step (3). According to the method,the antibody titer is increased by improving the immune effect, so that the immunization times are reduced, thereby achieving the effects of saving time and reducing materials.