34 results about "Amoebocyte lysate" patented technology

The utility model discloses a rapid endotoxin detection device based on a recombinant C factor method, which comprises a shell with an opening on the front side; the kit is pushed into or pulled out of the shell through the opening by the bracket; the kit is placed on the bracket, a reaction cavity is formed in the kit, and a recombinant C factor reagent is stored in the reaction cavity; and the fluorescence analyzer is fixedly arranged above the kit in the shell. According to the endotoxin rapid detection device based on the recombinant C-factor method, recombinant C-factor reagents are added into the reagent storage area of the kit, the consistency of the recombinant C-factor reagents is high, batch difference does not exist, the accuracy is high when endotoxin detection is carried out, and the endotoxin rapid detection device is not limited by the yield of tachypleus amebocyte lysate. In the detection process, the recombinant C factor reagent is not exposed in the air, so that the influence of endotoxin in the air on the detection result is reduced.

The invention belongs to the technical field of bacterial endotoxin detection, and particularly discloses a sulpiride bacterial endotoxin detection method which comprises the following steps: sequentially preparing a series of standard substance solutions, a test substance solution, a test substance positive control solution and an interference test solution; adding the series of standard substance solutions into tachypleus amebocyte lysate, detecting by adopting a dynamic nephelometry, and drawing a standard curve; respectively adding the test sample solution, the test sample positive control solution and the interference test solution into a limulus reagent, detecting by adopting a dynamic nephelometry, and calculating the bacterial endotoxin content in the sulpiride sample according to a standard curve linear regression equation. Compared with the prior art, the sulpiride sample is dissolved by using thionyl chloride, and then the sulpiride sample is further diluted by using the bacterial endotoxin inspection water to prepare the test solution, so that the problems that sulpiride is difficult to dissolve in the bacterial endotoxin inspection water and cannot effectively release bacterial endotoxin can be solved.

This invention provides a recombinant horseshoe crab three-factor composition and its application. The composition comprises recombinant horseshoe crab factor C, recombinant horseshoe crab factor B, and recombinant horseshoe crab coagulase factor; all three factors are expressed by mammalian HEK293 cells. This allows for the expression of factors C, B, and PCE in mammalian HEK293 cells. Simultaneous expression of all three factors enhances factor C activity and improves sensitivity; furthermore, the expressed factors are secretible and possess high activity.

Resorbable implants comprising poly(butylene succinate) and copolymers thereof have been developed. The implants implants are preferably sterilized, and contain less than 20 endotoxin units per device as determined by the limulus amebocyte lysate (LAL) assay, and are particularly suitable for use in procedures where prolonged strength retention is necessary, and can include one or more bioactive agents. The implants may be made from fibers and meshes of poly(butylene succinate) and copolymers thereof, or by 3d printing, and the fibers may be oriented. Coverings and receptacles made from forms of poly(butylene succinate) and copolymers thereof have also been developed for use with cardiac rhythm management devices and other implantable devices. These coverings and receptacles may be used to hold, or partially / fully cover, devices such as pacemakers and neurostimulators. The coverings and receptacles are made from meshes, webs, lattices, non-wovens, films, fibers, and foams, and contain antibiotics such as rifampin and minocycline.

The invention belongs to the technical field of medicine quality detection, and particularly relates to a method for detecting bacterial endotoxin of a raw material medicine in bromhexine hydrochloride injection. According to the method, a mode of heating and dissolving the hydrochloric acid solution with the pH value of 4.0 is adopted, and the 001 * 7 type cation exchange resin microcolumn purification is combined, so that the inhibition effect of cation impurities in the raw material medicine on agglutination reaction of the tachypleus amebocyte lysate can be effectively eliminated, and a stable and uniform test solution is prepared. According to the process, the minimum non-interference dilution multiple is obviously reduced to 160 times, and the operation error and the detection sensitivity loss caused by high-multiple dilution are reduced. As the dilution ratio is greatly reduced, a tachypleus amebocyte lysate with the sensitivity of 0.25 EU.mL <-1 > can be selected in the method. Compared with a high-sensitivity reagent needed in the prior art, the reagent of the specification is easier to purchase and lower in cost, solves the problems that the high-sensitivity reagent is large in daily purchase difficulty and high in price, and is more suitable for conventional application in industrial quality control.

The invention discloses an endotoxin concentration quantitative detection device and method, and relates to the field of medical detection.The device comprises a light source, an optical fiber Raman sensor, a capillary tube and a signal detection unit; the light source is used for emitting light signals to the optical fiber Raman sensor; the capillary tube is used for packaging the optical fiber Raman sensor and sucking a to-be-detected liquid sample, so that the to-be-detected liquid sample is in contact with the optical fiber Raman sensor; the liquid sample to be detected contains tachypleus amebocyte lysate protein and endotoxin; endotoxin is combined with tachypleus amebocyte lysate protein, so that the adsorption effect of the surface of the optical fiber Raman sensor on the tachypleus amebocyte lysate protein is reduced, an optical signal is scattered, and a Raman fingerprint characteristic spectrum is formed; the signal detection unit is used for determining the endotoxin concentration of the liquid sample to be detected according to the intensity change of the Raman fingerprint characteristic spectrum. According to the invention, the detection reliability is ensured, and meanwhile, the use amount of limulus lysate protein is reduced.

The invention discloses a recombinant tachypleus amebocyte lyophilized preparation for bacterial endotoxin detection as well as a preparation method and application of the recombinant tachypleus amebocyte lyophilized preparation. The recombinant tachypleus amebocyte lyophilized preparation is obtained by lyophilizing a lyophilized solution, wherein the lyophilized solution comprises the following components: mannitol, cane sugar, polyvinylpyrrolidone, recombinant human albumin, ascorbic acid, sodium chloride, inositol, L-arginine hydrochloride, ethylene glycol diethyl ether diamine tetraacetic acid, Tween 80, Proclin 300, Tris-HCl with the pH value of 7.0-8.0, a recombinant tachypleus amebocyte lyophilized preparation, a chromogenic substrate and a reaction buffer solution. The invention further discloses a preparation method and application of the recombinant tachypleus amebocyte lyophilized preparation. The recombinant tachypleus amebocyte lyophilized preparation is prepared by matching a multi-element synergistic protection system with a seven-stage gradient temperature control freeze-drying process, and can be used for high-sensitivity and high-stability detection of bacterial endotoxin in drugs, medical instruments, biological products and clinical samples.

The invention discloses a method for eliminating interference in LNP type sample bacterial endotoxin detection. The method comprises the following steps: S1, determining a detection standard curve of a tachypleus amebocyte lysate dynamic color development method; s2, preparing a test sample solution A with different dispersing agent volume ratios and a test sample solution B added with the endotoxin with the midpoint concentration in the standard curve, and carrying out an interference elimination test by adopting the standard bacterial endotoxin solution C, the solution A and the solution B; s3, redissolving the tachypleus amebocyte lysate with bacterial endotoxin inspection water according to the specification of the tachypleus amebocyte S4, detecting by adopting a tachypleus amebocyte lysate dynamic color development method; and S5, judging the effectiveness of the experiment. According to the method for eliminating interference in bacterial endotoxin detection of the LNP type sample, the endotoxin dispersing agent is introduced, so that the multi-dimensional interference problems of endotoxin wrapped by lipid in the LNP sample, pH value deviation, osmotic pressure abnormity and the like are solved in a targeted manner.

The present invention is related to compositions comprising clarified limulus amebocyte lysate (LAL), wherein the LAL is substantially free of coagulogen and methods of making such compositions. The invention further relates to a method of detecting an endotoxin in a sample using a chromogenic assay, the method comprising: (a) contacting the sample with a reagent comprising clarified LAL and a chromogenic substrate; and (b) measuring a chromogenic effect resulting from a change in the chromogenic substrate in the presence of endotoxin in the sample, wherein the LAL is substantially free of coagulogen. The invention also relates to kits comprising clarified LAL substantially free of coagulogen, and methods of making such.

The invention discloses a detection method of refenasin bacterial endotoxin and application, and relates to the field of medicine quality control. The invention relates to a method for detecting bacterial endotoxin of refenasin, which comprises the following steps of: preparing a suspension from refenasin by using water containing polysorbate 80, diluting the suspension by using N-methyl pyrrolidone, uniformly mixing and dissolving, diluting by using water, and detecting the bacterial endotoxin by using a tachypleus amebocyte lysate. According to the invention, the problem of dissolution of the test sample is solved, and the interference influence on bacterial endotoxin detection is eliminated.

The disclosure relates to biomaterials, specifically non-pyrogenic bacterial cellulose for implantable medical applications, and to a depyrogenation process. It addresses the problem that sterile bacterial cellulose remains pyrogenic due to endotoxin, particularly lipopolysaccharide, which resists standard sterilization and purification. The process involves providing a purified bacterial cellulose membrane in an aqueous medium and subjects it to depyrogenation at about 140° C.-250° C. under pressure sufficient to maintain the aqueous phase liquid. The treated material is a bacterial cellulose hydrogel having a water holding capacity of at least 100 g water / g dry bacterial cellulose and endotoxin below 0.5 EU / mL as determined by a Limulus amebocyte lysate assay on enzymatically degraded samples. The product and process are useful for manufacturing non-pyrogenic implantable bacterial cellulose devices, including internal implants, tissue scaffolds, and other biocompatible medical components.

The invention relates to the technical field of microbiological detection, and discloses a sodium dihydrogen phosphate monohydrate bacterial endotoxin content detection method, which comprises the steps of sample pretreatment, interference test verification, sample solution preparation, tachypleus amebocyte lysate reaction, result determination and quantitative detection. Wherein the sample pretreatment adopts heatless raw water dissolution and ultrasonic oscillation to ensure that the sample is uniformly dispersed; an interference test verifies that a standard bacterial endotoxin solution is mixed with a sample, the interference degree is evaluated, and the dilution ratio is adjusted; the tachypleus amebocyte lysate reaction is subjected to qualitative and quantitative analysis by adopting a gel method, a dynamic nephelometry and a chromogenic matrix method, so that the detection sensitivity is ensured. By optimizing sample treatment conditions, interference elimination strategies and detection parameters, the accuracy and repeatability of detection are improved, the probability of occurrence of false positive and false negative is reduced, the applicability and reliability of the detection method are improved, and strict quality control requirements of pharmaceutical grade sodium dihydrogen phosphate monohydrate are met.

The invention relates to the technical field of platelet lysate monitoring, and discloses a platelet lysate quality real-time monitoring device based on multi-modal sensing, which comprises a monitor main body, and a plurality of test tubes for bearing lysate samples are arranged in the monitor main body. A liquid storage cylinder for bearing pyrogen-free diluent and tachypleus amebocyte lysate is arranged above the test tube, an instillation mechanism for automatically instilling liquid in the liquid storage cylinder into the test tube is arranged in the monitor main body, and a shaking mechanism for shaking and mixing the test tube is arranged between the monitor main body and the liquid storage cylinder; according to the device, individual operation differences and visual fatigue errors which are difficult to avoid and exogenous pollution risks possibly introduced in the pipetting process, which are caused by manual one-by-one sample adding, are eliminated, and strict standardization and whole-course traceability of key parameters in the detection process are realized; therefore, the requirements of high throughput, high reliability and compliance of endotoxin detection in industrial production of the platelet lysate are met.

The invention discloses a recombinant tachypleus tridentatus C factor based on codon optimization and application of the recombinant tachypleus tridentatus C factor in endotoxin detection, and relates to the technical field of bioengineering. The limulus C factor sequence is used as a starting sequence, the limulus C factor is recoded, and the gene sequence of the recombinant limulus C factor is shown as SEQ ID NO: 1; protein corresponding to the sequence is expressed through an insect cell-baculovirus expression system, and the expression quantity is high, that is, the protein expression quantity of the recombinant horseshoe crab factor C is successfully improved through codon optimization. The production cost of the recombinant tachypleus tridentatus C factor endotoxin detection method kit is reduced, and the recombinant tachypleus tridentatus C factor detection method replaces a tachypleus tridentatus reagent detection method to become a mainstream method for endotoxin detection, so that the problem of raw material supply of tachypleus tridentatus reagent due to the fact that tachypleus tridentatus becomes a protective animal is solved; and the existing recombinant horseshoe crab factor C detection method has the problems of high product price and the like.

The invention discloses a tachypleus amebocyte lysate based on a peptide-rhodamine 110 conjugate and application of the tachypleus amebocyte lysate in endotoxin detection. The tachypleus amebocyte lysate comprises a C factor, a B factor, a clotting zymogen and a peptide-R110 conjugate. The peptide-R110 conjugate is adopted as a limulus reagent substrate, when the limulus reagent substrate is applied to endotoxin detection, a fluorescence detection background signal can be remarkably reduced, the detection sensitivity is greatly improved, meanwhile, the dual detection functions of color development and fluorescence are achieved, end-point method reading can be achieved in a fluorescence mode, and the detection sensitivity is greatly improved. High-precision and high-reliability detection of trace endotoxin is realized, and the method has important significance in quality control in the pharmaceutical production process, safety evaluation of medical equipment and other fields needing endotoxin detection.

This invention provides a rapid detection kit for bacterial endotoxins in recombinant hepatitis B vaccine and its usage method. The kit includes the following items: one working standard for bacterial endotoxins, 10 Limulus amebocyte lysate (LAL) reagents, one 5ml vial of 1×PBS buffer solution (Solution A), one 50ml vial of water for bacterial endotoxin testing (BET) solution (Solution B), six pyrogen-free glass test tubes, two test tube racks, one ampoule opener, two sealing films, and one instruction manual. This kit is designed for the bacterial endotoxin test of recombinant hepatitis B vaccine (CHO cells). The solution concentration is pre-set according to the limits specified in the pharmacopoeia, eliminating the need for testing personnel to recalculate the dilution steps and solution concentrations according to the limits. The test can be completed by simply adding samples according to the instructions. The test is easy to repeat and suitable for personnel with or without experience in endotoxin testing.

Resorbable implants, coverings and receptacles comprising poly(butylene succinate) and copolymers thereof have been developed. The implants are preferably sterilized, and contain less than 20 endotoxin units per device as determined by the limulus amebocyte lysate (LAL) assay, and are particularly suitable for use in procedures where prolonged strength retention is necessary, and can include one or more bioactive agents. The implants may be made from fibers and meshes of poly(butylene succinate) and copolymers thereof, or by 3d printing molding, pultrusion or other melt or solvent processing method. The implants, or the fibers preset therein, may be oriented. These coverings and receptacles may be used to hold, or partially / fully cover, devices such as pacemakers and neurostimulators. The coverings, receptacles and implants described herein, may be made from meshes, webs, lattices, non-wovens, films, fibers, foams, molded, pultruded, machined and 3D printed forms.

The disclosure relates to biomaterials, specifically non-pyrogenic bacterial cellulose for implantable medical applications, and to a depyrogenation process. It addresses the problem that sterile bacterial cellulose remains pyrogenic due to endotoxin, particularly lipopolysaccharide, which resists standard sterilization and purification. The process involves providing a purified bacterial cellulose membrane in an aqueous medium and subjects it to depyrogenation at about 140°C–250°C under pressure sufficient to maintain the aqueous phase liquid. The treated material is a bacterial cellulose hydrogel having a water holding capacity of at least 100 g water / g dry bacterial cellulose and endotoxin below 0.5 EU / mL as determined by a Limulus amebocyte lysate assay on enzymatically degraded samples. The product and process are useful for manufacturing non-pyrogenic implantable bacterial cellulose devices, including internal implants, tissue scaffolds, and other biocompatible medical components.

The invention discloses a method for detecting bacterial endotoxin in arginine hydrochloride. The method comprises the following steps: rechecking sensitivity of tachypleus amebocyte lysate; preparing a test solution; gradient dilution; interference test; and detecting by a gel method. According to the arginine hydrochloride bacterial endotoxin detection method, interference is effectively eliminated through pH value adjustment and a gradient dilution method, and an effective and reliable detection method is provided for arginine hydrochloride bacterial endotoxin detection.

The invention discloses a method for detecting bacterial endotoxin of degradable polyester, and relates to the technical field of insoluble substance impurity detection.The method comprises the steps that the degradable polyester is dissolved with dichloromethane, and an organic phase solution is obtained; bET water is added, and water-phase extract liquor is extracted through oscillation; and carrying out bacterial endotoxin detection on the test solution by using the tachypleus amebocyte lysate. According to the method, the degradable polyester is dissolved by adopting dichloromethane, so that the problem that the material is difficult to dissolve is solved, the interference effect on bacterial endotoxin detection is eliminated, the detection result is accurate and reliable, the reproducibility is good, and the method has important significance in guaranteeing the safety of the polymer medical material; more powerful technical support can be provided for quality control of related medical implant materials, and the safety and reliability in medical application are further improved.

A non-pyrogenic bacterial cellulose characterised as being a hydrogel having a water content of at least 95 wt.% and having less than 0.5 EU / ml of endotoxins, measured with a Limulus amoebocyte lysate

Provided is a technology related to a horseshoe crab factor B variant, and also provided is means for performing endotoxin measurement with high sensitivity. A polypeptide having an amino acid sequence in which the amino acid residue at the 193-position in an amino acid sequence of a polypeptide of horseshoe crab factor B is substituted with a cysteine (Cys) residue, is produced. Endotoxin measurement can be carried out with high sensitivity by combining this polypeptide with horseshoe crab factor C, as a Limulus reagent.

The invention discloses a wide-temperature-range compound enzyme endotoxin detection reagent and a preparation method thereof. According to the reagent, an active enzyme compound in lysate of limulus blood cells serves as a core, a temperature control response material, a modifier and a stabilizer are compounded to construct an intelligent compound enzyme system, the narrow temperature range limitation of a traditional reagent is broken through, the activity can be accurately adjusted within the range of 0-90 DEG C, the relative activity at the temperature of 20-70 DEG C is kept at 80-95%, and the relative activity at the temperature of 80-90 DEG C is improved to 120-130% of the initial activity. The detection limit of the reagent is 0.005-0.02 EU / mL, the activity retention rate at the temperature of 50 DEG C is increased by 75%-90% compared with that of a traditional tachypleus amebocyte lysate, and the reagent is excellent in stability in a high-salt and low-pH environment, adapts to temperature fluctuation scenes and is suitable for endotoxin detection of drugs, food and medical instruments.

The application discloses a method for in vitro isolation and culture of limulus blood ameba-like cells, and belongs to the field of marine biological cell engineering. The method realizes high-purity (≥95%) and high-activity isolation of cells by minimally invasive blood sampling, composite anticoagulant protection, double-mode gradient centrifugal purification, full-chemical-defined serum-free culture, non-enzymatic subculture and GMP quality control. The cells can be stably subcultured for more than 12 generations, the survival rate is ≥65% in 120 days, and the endotoxin response function does not attenuate. The method is suitable for all limulus species in the world, the damage of blood sampling to the limulus body is extremely low, the sustainable utilization of resources can be realized, the culture system is free of animal-derived components, meets the GMP requirements, is suitable for large-scale production in a bioreactor, effectively solves the bottleneck problems of low purity, short survival, difficult amplification and damage to the limulus body in the prior art, and can be applied to the fields of limulus reagent preparation and natural immune research.

Provided is a technology related to a horseshoe crab factor B variant, and also provided is means for performing endotoxin measurement with high sensitivity. A polypeptide having an amino acid sequence in which the amino acid residue at the 193-position in an amino acid sequence of a polypeptide of horseshoe crab factor B is substituted with a cysteine (Cys) residue, is produced. Endotoxin measurement can be carried out with high sensitivity by combining this polypeptide with horseshoe crab factor C, as a Limulus reagent.

The invention discloses a body fluid endotoxin rapid detection method and device based on a localized surface plasma resonance effect. According to the method, based on a refractive index sensing principle of a local plasma sensor, a trace body fluid sample and a tachypleus amebocyte lysate are directly mixed in a detection area of a plasma chip, and specific enzymatic gelation reaction of endotoxin is triggered, so that local refractive index change of the surface of the plasma sensor is caused; an optical detection system is used for monitoring the transmission light field image stripe displacement of the plasma sensor or the red shift of the reflection / transmission spectrum characteristic peak caused by the reaction in real time, and quantitative detection of the endotoxin concentration is realized by analyzing the dynamic process of signal change. The method integrates sample mixing, reaction and detection, does not need complex pretreatment, and has the characteristics of simplicity in operation, extremely low sample consumption (less than or equal to 1 mu L), high sensitivity, quick response and the like. Based on the method, the invention further provides a portable detection device integrated with a temperature control function, the endotoxin in various body fluids can be rapidly and accurately quantified on site, and an effective technical means is provided for infection early warning, disease monitoring and medical safety.

The application discloses a method for detecting endotoxin of collagen solution, and relates to the technical field of collagen detection. The method comprises the following steps: after collagen solution is formed into collagen gel, endotoxin checking water is added to soak the collagen gel, the soaking solution is taken out and mixed with limulus reagent, the mixture is kept at a temperature of 35-38 DEG C, and then whether the mixture forms gel is determined to judge the content of endotoxin of the collagen solution. The method can reduce the probability of false positive of the detection result.

This invention discloses a method for detecting bacterial endotoxins in gelatin microcarriers used for cell culture, comprising the following steps: (1) rechecking the sensitivity of the Limulus Amebocyte Lysate (LAL) reagent to ensure that the sensitivity result of the LAL reagent is between 0.5 and 2.0 λ; (2) determining the limit value and minimum dilution concentration of bacterial endotoxins in the gelatin microcarriers; (3) enzymatically digesting the PBS solution of the gelatin microcarriers to be tested with collagenase solution to obtain an enzymatic digest; (4) checking for bacterial endotoxins in the gelatin microcarriers: mixing a magnesium ion regulator and the LAL reagent to obtain a reconstituted LAL reagent, mixing the enzymatic digest obtained in step (3) with the reconstituted LAL reagent to obtain a mixed solution, and using the gelation method to determine whether bacterial endotoxins are present. By combining the pretreatment enzymatic digestion step with the LAL reagent reconstituted with a magnesium ion regulator, the gelatin microcarriers can be effectively enzymatically digested, fully releasing endotoxins, and the interference of phosphate ions can be effectively eliminated by the magnesium ion regulator, thereby improving the specificity and accuracy of the detection.

The application relates to a recombinant three-factor limulus reagent and a kit, and the bacterial endotoxin detection reagent comprises the following components: a recombinant C factor, a recombinant B factor, a recombinant coagulogen, a chromogenic substrate and a balance enhancer selected from one or more of alkyl pyranoside, polyethylene glycol ether, PEG and dioxane derivatives. The recombinant three-factor limulus reagent of the application can enhance the reaction activity of the recombinant limulus reagent and endotoxin, enhance the detection signal, shorten the detection time, and has good consistency in the reactivity to endotoxins from different species and excellent universality.