82 results about "Phycoerythrobilin" patented technology

A homogeneous immunoassay method and system for quantitative determination of total immunoglobulin E and specific antibody levels to a plurality of allergens, in which a relatively small sampling of blood is required. The method utilizes relatively small microparticles in aqueous suspension. The immunoassay procedure is an immunometric sandwich procedure preferably utilizing biotin-streptavidin signal amplification techniques and R-phycoerytherin fluorescent labels.

The invention provides a method belonging to the field of cytometric bead array with high sensitivity for testing platelet specific antibody based on platelet basic experimental study. Platelet lysate is used to incubate the microsphere which is coated with platelet membrane glycoprotein monoclonal antibody, then goat-anti-human IgG polyclonal antibody labeled with phycoerythrin is added, which is analyzed in cytometric bead array. If autoantibodies exist on the surface of platelet, 'microsphere-platelet membrane glycoprotein monoclonal antibody-platelet membrane glycoprotein autoantibodies-goat-anti-human IgG polyclonal antibody labeled with phycoerythrin' complex structure is formed, the fluorescence intensity of the test microsphere is enhanced. The invention is easy to operate, and has mature technology, high sensitivity for testing platelet autoantibodies, which is good for basic experimental study of platelet antibody.

The present present invention relates to a method of simultaneously extracting phycoerythrin and agar from seaweeds such as asparagus, etc in seaweeds biological chemical field. The concrete operation process is that cells of the asparagus frond are crushed. A phycobiliprotein crude extract is obtained from water soluble extracts after ammoniym sulfate precipitation. The crude extract is extracted by Phenyl-Sepharose expansion columns and phycoerythrin products are obtained. Q-Sepharose iron exchange columns are used to purify the phycoerythrin products to obtain purified phycoerythrin products. Then, the left asparagus residues after the phycoerythrin extract are used to extract the agar. The present invention can simultaneously obtain the phycoerythrin and the agar from the asparagus. And the purified phycoerythrin purity is larger than 2.954, and the yield is 0.097mg per gram of fresh algae. The yield of the obtained agar is 2.31percent (the algae is wetly weighted), which is equivalent to the yield of an agar direct extraction method. Therefore, the utilization ratio of the asparagus can be greatly improved by adopting the method.

The invention discloses a non-diagnostic method for detecting a suspension chip of PCR products. The chip mainly comprises coded microspheres, a biotinylated primer, a capture probe, and streptavidin-biotin-phycoerythrin, and the method comprises the following steps that: the capture probe is coupled with corresponding microsphere of each size respectively, a red laser excites classified fluorescent lights on aspherical substrate, and the types are determined according to different colors of the spherical substrate, wherein the biotinylated primer shows the a primer needs biotinylation labeling during PCR; the microspheres coupled with the probe can be specifically combined with a PCR product labeled by an amplified biotin; and the streptavidin-biotin-phycoerythrin is combined with a biotin on the PCR product captured on the microspheres, a green laser excites the phycoerythrin, and the number of the reported fluorescent molecules combined on the spherical substrate is measured and is used for indirectly determining the content of the PCR product combined on the spherical substrate.

The related autoantibody parallel detection liquid chip comprises: the mcirosphere, self antigen to form coupling body with corresponding microsphere and use red laser to activate red fluorescence on substrate and determine self antigen type by color, and double antigen treated by biotin to combine and activate the phycoerythrin with green laser and determine fluorescent molecule quantity for indirect obtaining content.

The invention discloses a method for separating and purifying phycoerythrin from bangia fusco-purpurea. According to the method, foreign proteins with small molecular weights are removed at the same time of salt removal by a membrane technology, and the phycoerythrin is effectively separated from the foreign proteins based on the principles that the bonding strength between anion exchange resin and the phycoerythrin is obviously changed in solutions of different salt contents and different pH values, and the like, thereby obtaining high-purity and high-yield phycoerythrin. In different components, the purity of the phycoerythrin is generally higher than 3.0 (commonly accepted standard) and is 5.5 at most, and the yield is about 0.8g / 100g bangia fusco-purpurea. The method has a simple process and good separating effect, and realizes quick and effective separation and purification of the phycoerythrin.

The invention relates to a method for preparing high purity phycoerythrin, phycocyanin and allophycocyanin from the algae, belonging to the bioengineering extraction separation technology. The method is as follows: red algae or cyanobacteria is adopted as a raw material, and phosphate buffer is used as an extractant; after algae cells are broken, the raw material of the red algae or the cyanobacteria is salt dissolved, salted out and dialyzed in grade by ammonium sulfate, and crude extract of phycobiliprotein is obtained; the crude extract is in chromatography by a hydroxyapatite column for one time, is gradient eluted by the phosphate buffer, and is frozen and dried, and high purity phycoerythrin, phycocyanin and allophycocyanin are obtained. The purity of the crude extract of the phycobiliprotein prepared by the effective method in the earlier stage reaches the standard of pharmaceutical grade (A620 / A 280 is more than 2.0), thereby simplifying the follow-up purifying procedures, so the high purity phycoerythrin, phycocyanin and allophycocyanin can be respectively obtained by only one time column chromatography. When the method is used to extract high purity phycobiliprotein from the fresh algae and the dry algae, the process is simple, the production cost is low, and the yield of the products is high. Thus, the method is suitable for mass production.

The invention discloses a method for detecting a system for quickly screening a fever pathogen, and mainly relates to detection of a tuberculosis antibody, a flu antibody, a bird flu antibody, a plague antibody and an SARS antibody in blood serum. The chip mainly comprises coding microspheres, a coating antigen, a detection antibody, a biotinylated antibody and a streptavidin-phycoerythrin. The method comprises the following steps that: the coating antigen and the coding microshperes are coupled; the detection antibody is respectively coupled with the corresponding microspheres in separate specificity; the red laser excites the classified fluorescence on the spherical substrate; the type is determined according to different colors of the spherical substrates, wherein the biotinylated antibody is combined with the detection antibody; and the streptavidin-phycoerythrin is combined with the biotin of the detection antibody captured by the microshperes; the green laser excites the phycoerythrin, the number of report fluorescence molecules combined on the spherical substrate is measured, and the number is used for indirectly determining the content of the detection antibody combined onthe spherical substrate, so that whether the pathogen infection exists is determined, and the aim of quickly screening the fever pathogen is achieved.

This invention relates to an extraction method for high-purity phycobiliprotein from lavers. Currently, phycobiliprotein extraction from spirulina suffers some technological drawbacks, which causes the high prices for high-purity phycobiliprotein as a result of commonly low extraction purities and yields. In contrast, it is main characteristics of this invention that phycobiliprotein is extracted from lavers, which includes following steps: lavers are pounded into pieces at a low temperature and a high speed and the mixture is centrifugalized to obtain the supernatant; original phycobiliprotein extract is prepared by multiple ammonium sulphate salting-out; it is then dialyzed and centrifugalized at a high speed with the obtained solution placed unto a hydroxyapatite column; stepwise low-concentration elution is implemented at first to obtain phycoerythrin and phycocyanin respectively and high-concentration is then implemented to obtain allophycocyanin. Generally speaking, the extraction method for high-purity phycobiliprotein from lavers introduced in this invention has the advantages of simple technique, and practicability.

This invention focuses on a novel process in which Porphyridium cruentum biomass first undergoes a stage of cellular disruption and subsequently stages of recovery and purification in order to achieve the purified B-phycoerythin (BFE) protein dye, using isoelectric precipitation and two-aqueous-phase systems. The steps of recovery and purification include isoelectric precipitation followed by a step of liquid / liquid extraction by means of two-aqueous-phase systems that use polyethylene glycol (PEG) and phosphate salts. The BFE protein dye obtained in the two-aqueous-phase extraction step undergoes an ultrafiltration step in order to remove the polymer (PEG) and to obtain a dye with a purity greater than 4.0 defined as the relationship between the absorbencies at 545 and 280 nm (BFE purity=Abs 545 nm / Abs 280 nm).

The invention relates to phycocyanin beta subunits fluorescent protein combined with phycoerythrobilin PEB, fusion protein formed by phycocyanin beta subunits fluorescent protein and streptavidin and a mutant thereof, the sequences include sequence 1, sequence 2, sequence 3 and sequence 4; furthermore, the invention discloses a method of the fluorescent protein fused with streptavidin for fluorescent immunological detection directly; phycocyanin beta subunit conserved cysteine residue can be not only combined with phycocyanobilin PCB by a thioether bond, but the fact that the phycocyanin beta subunit conserved cysteine residue can be combined with the phycoerythrobilin PEB by the thioether bond through the genetic engineering can be realized, so as to obtain the novel fluorescent phycocyanin, the spectroscopy of the protein is completely different from that of the phycocyanin beta subunits fluorescent protein combined with PCB, and the protein has high fluorescence efficiency; in addition, as the protein carries His-tag label, purification is not only convenient, but also the dissolubility of the protein can be improved; the protein is combined with the streptavidin to form the fusion protein for fluorescent immunological detection directly, thereby being beneficial to the application of the protein in all kinds of the field.

A rapid separation method of purified R-phycoerythrin and R-phycocyanin, which pertains to separation and purification of red algae technical field. The invention uses red algae as raw material, extracts R-phycoerythrin and R-phycocyanin by freeze dissolving and intensified swelling with low ions, carries out primary purification after precipitation with ammonia sulfate, then absorb and enrich R-phycoerythrin and R-phycocyanin by anion exchange chromatography, and further elutes highly purified R-phycoerythrin and R-phycocyanin from anion exchange chromatography step by step. The method eliminates conventional complex separation and purification step which uses multiple chromatography combining molecular sieve chromatography with hydroxyapatite chromatography, solves the problem of extensive and rapid preparation. The method is easy in operation, time and energy saving, with little requirements to apparatus, and high in yield.

A novel suspension hybridization assay was used to determine nucleic acid copy number by flow cytometry. The assay was validated with low copy (lc) products ranging in length from 100 to 2304 bp conjugated to spectrally-distinct polystyrene microspheres. In the example provided herein, these conjugated microspheres were used as multiplex hybridization probes to detect homologous sequences in genomic DNA extracted from cytogenetic cell pellets and labeled with biotin-dUTP. Hybridization was detected with phycoerythrin-labeled streptavidin and analyzed by flow cytometry. Copy number differences were distinguishable by comparing the mean fluorescence intensities of test probes with a diploid reference probe in genomic DNA of patient samples and abnormal cell lines. The assay is capable of distinguishing a single allele and three alleles at a test locus from a biallelic reference sequence, regardless of chromosomal context. The assay is an improvement on previous methods which require prior amplification of locus-specific target DNA because, lc probes provide adequate specificity and sensitivity for accurate copy number determination of homologous targets. Because of its high sensitivity and accuracy, the assay is useful for determination of nucleic acid copy number for a variety of applications, including determination of genomic copy number in humans, animal models of disease and in solution, measurement of transcript levels, forensic DNA analysis, and quality control analysis in agriculture.

The invention provides a method for simultaneously extracting phycoerythrin and sulphated porphyra polysaccharide from inferior nori. The method comprises the following steps: firstly, extracting phycoerythrin from fresh inferior nori; then, extracting sulphated porphyra polysaccharide from residues of phycoerythrin; and simultaneously extracting phycoerythrin and sulphated porphyra polysaccharide in a primary extraction process. The method provided by the invention can be used for obtaining phycoerythrin and sulphated porphyra polysaccharide in primary extraction. The obtained phycoerythrin is high in purity and the sulphated porphyra polysaccharide is good in uniformity. According to the method provided by the invention, the operation is simple and convenient, the process is simple, the separating speed is high, the cost is low, the utilization efficiency of inferior nori is improved, and resources are repeatedly used and utilized for many times.

The developed reagent is three-color immunophenotyping reagent for measurement of CD4 positive lymphocytes in peripheral blood. The reagent contains 7-aminoactinomycin D (7-AAD) which intercalates into double stranded DNA and is easily excited at 488 nm. The fluorescence emission of 7-AAD has peak at 670 nm that can be detected with FL3 detector of flow cytometer. The 7-AAD, therefore, stains white blood cells and discriminates it from red blood cells. The reagent also contains fluorescein isothiocyanate (FITC) labeled CD4 monoclonal antibody and phycoerythrin (PE) labeled CD14 monoclonal antibody which are detected with FL1 and FL2 detectors of flow cytometer, respectively. The developed reagent can be used to measure number of CD4 positive lymphocytes in lymphocyte population and monitor monocyte contamination simultaneously. This reagent therefore provides more accuracy results of CD4 positive lymphocyte enumeration.

The invention provides fluorescent analogues of thyroid hormones T3 and T4. Also provided are assays for thyroid hormones utilizing the compounds of the invention. The assays of the invention are in both one-step and two-step formats and allow the determination of both T3 and T4 concentration in a sample.

The invention relates to a preparation method of a phycoerythrin ratio fluorescence sensor based on a magnetic molecular imprinted nucleus / shell polymer. A Fe3O4 magnetic nano-particleis used as the center, the surface of the Fe3O4 magnetic nano-particleis coupled with blue fluorescence emitting carbon quantum dots B-CDs, a SiO2 shell layer loaded with template molecule phycoerythrin generates onthe surfaces of the Fe3O4 / B-CDs, and template molecule elution is conducted to prepare magnetic molecular imprinted nucleus / shell polymer Fe3O4 / B-CDs / SiO2-MIPs. The polymer dispersion liquid fluorescence emission spectra under different phycoerythrin concentration are measured, the linear relation between the fluorescence emission peak intensity ratio I<phycoerythrin> / I<B-CDs> of the phycoerythrinto the B-CDs and the phycoerythrin molar concentration is fitted, and a phycoerythrin ratio fluorescence sensor is constructed. Compared with the prior art, the method is simple to operate and low incost, raw materials are easy to obtain, ratio signal interference resisting capacity is high, accuracy is high, flexibility and selectivity are high, and a novel ratio fluorescence sensor can be developed and used for efficient phycoerythrin detection.

The invention relates to a preparation method of fluorescence antibody for detecting a Newcastle disease virus and a solid-phase immunofluorescence detection assay kit. The preparation method comprises the following steps of: respectively deriving R-phycoerythrin (RPE) and an antibody resisting the Newcastle disease virus (NDV) by using a cross-linking agent SPDP (N-succinimidyl-3-(2-pyridyldithiol) propionate), cross-linking the derivatives in a proper molar ratio, and purifying through HPLC (High Performance Liquid Chromatography) to prepare an RPE marked NDV fluorescence antibody. The solid-phase immunofluorescence detection assay kit is formed from the fluorescence antibody, an NDV-resisting antibody, an agarose microsphere, diluted hydrochloric acid and a washing liquid. The assay kit comprises the following detection flows of: coating an activated microsphere with the antibody, washing, combining with a sample to be measured, washing, combining with the fluorescence antibody, fully washing, exciting by blue and green light in a fluorescence microscope, observing and judging the result. The prepared fluorescence antibody has the advantages of high yield, high purity, bright orange fluorescence and good stability; and the assay kit has signal enrichment action by using a spherical carrier and can increase detection sensitivity. The invention is applicable to the rapid detection of the Newcastle disease virus.

The present invention belongs to the field of fluorescent proteins in biotechnology, and particularly relates to a preparation method of a high fluorescence intensity recombinant phycobiliprotein concatermer. According to the preparation method, a streptavidin gene is linked to an allophycocyanin alpha subunit gene through a linker sequence; on the basis, one or a plurality of allophycocyanin alpha subunit genes are connected in series through linker sequences to form a fusion gene; and the fusion gene, a phycobiliprotein lyase gent and a phycoerythrobilin biosynthetic enzyme gene co-express in Escherichia coli to obtain the recombinant allophycocyanin concatermer with characteristics of biotin binding ability and high fluorescence intensity. According to the present invention, tn the immunofluorescence assay, the recombinant phycobiliprotein concatermer can achieve the strong fluorescent signal compared to the recombinant phycobiliprotein monomer; and the prepared recombinant phycobiliprotein concatermer can be adopted as the fluorescent marker for immunofluorescence detection in the field of biology and biomedicine.

The invention belongs to a preparation technology of marine natural active substances, and concretely relates to an efficient separation and purification method of Gracilaria verrucosa reagent grade R-phycoerythrin. The method comprises the following steps: soaking a crushed raw material marine red alga economic Gracilaria verrucosa into a PBS buffer solution with the volume 10-30 times the crushed raw material, centrifuging under 9000-15000rpm for 20-30min, collecting the obtained supernatant, and concentrating the supernatant; carrying out grading salting-out on the obtained concentrate by ammonium sulfate, collecting the obtained precipitate, dissolving by using the PBS buffer solution, and dialyzing for 24-48h after the dissolution; and carrying out strong anion Source-15Q chromatography column purification on the obtained dialysate, eluting with a PBS buffer solution containing NaCl as an eluent under a flow rate of 0.5ml / min, and collecting 360-510th min eluate to obtain an R-phycoerythrin solution with the purity of above 5.0. The method has the has the characteristics of high recovery rate of the above obtained product, good purification effect, easy amplification, large sample amount, and efficient separation and purification of phycoerythrins.

A Spirulina maxima extract of the present invention and Allophycocyanin (APC), R-phycoerythrin (R-PE), and C-phycocyanin (C-PC), which are components of the Spirulina maxima extract, show an effect of inhibiting cell death and A2E (pyridinium bis-retinoid), oxidation due to blue light, and therefore can be usefully applied as an active ingredient in a composition for preventing and treating retinal disease.

The invention discloses a multi-fluorescent immunoassay method for rapidly distinguishing 6 types of poultry respiratory pathogens. The multi-fluorescent immunoassay method is simple to operate; a target amplified fragment is obtained through a PCR (Polymerase Chain Reaction); then an amplified product, fluorescence coded microspheres and streptavidin-phycoerythrin are hybridized; an MFI (Mean Fluorescence Intensity) value is read through a detector to distinguish viruses of different types. According to the method disclosed by the invention, avian influenza viruses, chicken infectious bronchitis viruses, chicken Newcastle disease viruses, chicken infectious laryngotracheitis viruses, mycoplasma gallisepticum and mycoplasma synoviae can be accurately detected at the same time; the multi-fluorescent immunoassay method has high specificity, high sensitivity and good repeatability. Compared with a traditional detection method, the method disclosed by the invention realizes simultaneous detection of a plurality of types of different target molecules in the same sample; the use amount of the sample is less; the method is simple and rapid to operate and the detection cost can be greatly reduced.

The invention discloses a multiple fluorescence immunoassay method for detecting mycoplasma gallisepticum and mycoplasma synoviae. The multiple fluorescence immunoassay method has the advantages that the operation is simple; a target amplified fragment is obtained through PCR (polymerase chain reaction), then the amplified fragment, fluorescent encoding microspheres, and streptavidin-phycoerythrin are hybridized, and an MFI (mean fluorescence intensity) value is read through a detection instrument, so as to distinguish different types of pathogens; the mycoplasma gallisepticum and mycoplasma synoviae can be simultaneously detected and identified; the specificity is strong, the sensitivity is high, the repeatability is good, and the like; multiple types of molecules with different purposes in the same sample can be simultaneously detected; the flexibility is good, and the type of to-be-detected pathogens can be increased or decreased on the basis according to requirements.

The invention relates to a preparation method of a phycoerythrin labeled polystyrene microsphere fluorescent probe. The method includes: conducting swelling treatment on seed microspheres by two-stepswelling to obtain surface carboxylated polystyrene microspheres, and coupling the carboxyl on the surfaces of the microspheres with phycoerythrin to obtain the phycoerythrin labeled polystyrene microsphere fluorescent probe. The polystyrene microsphere fluorescent probe prepared by the method provided by the invention has the advantages of strong stability, high sensitivity, difficult quenching,high safety factor, and no toxic or side effect.

The invention discloses plural gel containing phycoerythrin, a preparation method and application. The plural gel is phycoerythrin / collagen / carboxylic carbon nano tube / polyacrylamide plural gel. According to the plural gel disclosed by the invention, the phycoerythrin is prepared into a form of the phycoerythrin / collagen / carboxylic carbon nano tube / polyacrylamide plural gel through an appropriate preparation method, the phycoerythrin can be applied to sensitization batteries, and photoelectric converting efficiency is improved.

The invention discloses a primer group, a kit and a method for detecting six mouse viruses by multiple immunity fluorescence. The primer group is designed by the aid of multiple RT-PCR (reverse transcription-polymerase chain reaction) technologies and Luminex liquid chip technologies and can simultaneously detect six high-infection viruses such as TMEV, MHV, MNV, Reo-3, MVM and MPV of mice. By the aid of the primer group, target amplification products are acquired through specific PCR, the amplification products, fluorescence encoding microspheres and streptavidin-phycoerythrin are hybridized, and different types of viruses are distinguished when a mean fluorescence intensity value is read by a Luminex detector. The method has the advantages of rapidness, high efficiency, high specificity and sensitivity, good repeatability and the like and can be applied to quality monitoring, epidemiological investigation and early warning of test mice. The primer group is good in flexibility, and varieties detecting the viruses can be increased and decreased as required on the basis.

The invention belongs to the technical field of marine organism applications, and relates to a preparation method for extracting phycoerythrobilin, polysaccharides and dietary fibers from low valued lavers and an application of the preparation method. According to the method, after the low valued lavers are dewatered, chopped and soaked in entrainers, by the aid of a supercritical CO2 fluid extraction technology, extraction, separation and purification are performed at proper packing liquid-solid ratio, extraction temperature and extraction pressure to obtain the phycoerythrobilin and the laver polysaccharides. Fishy smell removing agents are added into residual algal residues to remove fishy smell, and the residual algal residues are dried, activated and granulated to obtain the laver dietary fibers. The preparation process is simple, stable, green and efficient, and the obtained three products are fine in powder, high in purity and free from algal fishy smell, and can serve as various food raw materials.

The invention discloses a multiple fluorescence immunoassay primer, kit and method capable of fast distinguishing CAV, MDV, REV and IBDV. The multiple fluorescence immunoassay primer, kit and method has the advantages that target amplification fragments are obtained through PCR, \ amplification products, fluorescence encoded microspheres and streptavidin-phycoerythrin are hybridized, MFI values are read through a detector, and different types of pathogens are distinguished; the method can simultaneously and accurately detect chicken infectious anemia viruses, chicken Marek's disease viruses, chicken reticuloendotheliosis viruses and chicken infectious bursal disease viruses and is high in specificity, high in sensitivity and good in repeatability; compared with a traditional method, the method can simultaneously detect various molecules, with different purposes, in the same sample and is low in sample use amount, simple and fast to operate, capable of greatly lowering detecting cost, good in flexibility and capable of increasing and decreasing to-be-detected pathogen types on the basis.

The invention relates to a preparation method of multiple fluorescently-labeled polystyrene microspheres, and the method comprises the steps of preparing surface-carboxylated polystyrene microspheresby two-step seed swelling polymerization, and combining the polystyrene microspheres with quantum dots to prepare quantum dot fluorescent microspheres, coupling carboxyl groups on the surface of the quantum dot fluorescent microspheres with phycoerythrin to obtain the multiple fluorescently-labeled polystyrene microspheres. The method has the advantages of being simple and low in raw material price, the prepared polystyrene microspheres have a stable structure, by use of double fluorescent labeling of the quantum dots and the phycoerythrin, the multiple fluorescently-labeled polystyrene microspheres have the advantages of remarkable performance characteristics, high safety factors in the preparation process, and no safety hazard to the human body.

The invention provides a method for extracting PE from red algae, which is simple in operation, low in cost and suitable for industrial production. The method selects beta-agarase and cellulase to extract phycoerythrin from marine red algae, saves time, reduces cost, greatly simplifies operation, has high recovery rate and high purity by comparing with the prior art, and provides an easy way to industrialize phycoerythrin production.