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• • C—CHEMISTRY; METALLURGY
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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase

Definitions

• the present invention relates to the Chemical, Pharmaceutical and Biotechnological branches, and in particular to the detection of microorganisms, and of structures associated with its cell wall such as lipopolysaccharides (LPS) or endotoxins of Gram negative bacteria, peptidoglycans (PG) of Gram bacteria negative and positive, and (1, 3) - ⁇ -D-glucans (BG) of fungi and yeasts.
• LPS lipopolysaccharides
• PG peptidoglycans
• BG ⁇ -D-glucans
• the present invention also has application in the evaluation of microbial contamination in ultrapure waters, such as that used in the semiconductor industry, and as a diagnostic means for the clinical laboratory.
• the innate immune system provides very effective response mechanisms for the detection of microorganisms through the recognition of molecular structures conserved and shared by a large number of microbes. These structures called pathogen-associated molecular patterns (PMAPs), are not found in the host and are essential for the survival or pathogenicity of microorganisms.
• PMAPs pathogen-associated molecular patterns
• endotoxins are the PMAP or exogenous pyrogen most relevant to the pharmaceutical and biotechnology industry because of their high biological potency, ubiquity, resistance to conventional sterilization methods, and the high relative probability of their presence in parenteral solutions [ Williams KL Endotoxin Relevance and Control Overview. in Williams K.L (eds). Endotoxins, Pyrogens, LAL Testing and Depyrogenation. Third edition. Informa Healthcare, New York, London. 2007, 27-46], In addition, they are primarily responsible for the endotoxic shock associated with septicemia caused by Gram-negative bacteria, and constitute the main alarm signal of the presence of these bacteria for
• SRIS systemic inflammatory response syndrome
• FMO multiple organ failure
• shock and death
• LAL Limulus Amebocyte Lysate
• the LAL reagent is prepared from an extract or lysate of the amebocytes present in the hemolymph of horseshoe crabs. It is composed of a cascade of trypsin-like serine peptidases enzymes, which is activated by endotoxins and by (1, 3) -p-D-glucans through Factor C and Factor G, respectively.
• the reagent is based on the coagulation response of horseshoe crabs and is part of their innate immune system. A similar system has not been found to date in another invertebrate species [Iwanaga S and Lee BL, J Biochem Mol Biol 38, 128-150, 2005].
• LAL reagents for endotoxins have been developed by separating the G factor or factor sensitive to (1, 3) - -D-glucans from the rest of the components of the LAL enzyme cascade (US5401647, US5605806, US20030104501), or inhibiting their activation ( US5047353, US5155032, US547984, US5702882, US5998389, US5 79006).
• Horseshoe crabs are marine arthropods known as living fossils since they have evolved very little in the last 300 million years. There are four species of horseshoe crabs from which a reagent of similar properties can be obtained.
• Tachypleus tridentatus The species Tachypleus tridentatus, Tachypleus gigas and Carcinoscorpius rotundicauda are found exclusively in the Asian continent. The small population of the last two has never sustained reagent production.
• the Tachypleus tridentatus commonly known as the Chinese horseshoe crab or Horseshoe crab with three spines, had a high population density along the coast of China, especially in the north of the South China Sea and in the Hainan Islands region.
• recent studies of T. tridentatus in Taiwan, Japan, Hong Kong, Thailand and mainland China have indicated that populations have been drastically reduced almost to extinction. The main causes have been overfishing and pollution of the seas.
• the LAL reagent currently marketed is obtained from Limulus polyphemus, also known as American horseshoe crab, pan crab or bayonet crab.
• the Limulus inhabits the Atlantic coast of the United States, from northern Maine to the Yucatan Peninsula and the Gulf of Mexico. In Delaware Bay is the largest known population in this region, which has seen a dramatic decline in its population and spawning activity [Widener JW and Barlow RB, Biological Bulletin (197): 300-302, 1999] .
• Patents US4229541, US5082782, US6790659 and US20030186432 describe the development of strategies for in vitro culture of amebocytes as a possible alternative to hemolymph of horseshoe crabs as a natural source of amebocytes to prepare LAL. To date there is no commercial reagent obtained by this route, nor a project to obtain it.
• the endotoxin-sensitive Factor C of the Singapore horseshoe crab, Carcinoscorpius rotundicauda (US5712144, US5858706, US5985590) has been successfully cloned and produced recombinantly through a novel methodology, establishing its use for detection and quantification of LPS or endotoxins in a manner similar to the natural LAL reagent (US6645724).
• Formulations of this reagent that combine recombinant Factor C and detergents (US20030054432), or Recombinant Factor C or obtained by purification of the natural source have been patented in formulations that exhibit greater sensitivity and stability (US20040235080).
• these formulations lack the sensitivity provided by the amplifying effect of the enzyme cascade, which has been partially resolved by the use of more sensitive fluorescent substrates.
• Other components of the LAL reagent such as the procoagulatory enzyme (US20090208995) and Factor G (US201001 266) have also been produced recombinantly. Both the LAL and the recombinant Factor C detect LPS, but do not detect peptidoglycans.
• recent studies show the urgency of also detecting and controlling the content of peptidoglycans in formulations for parenteral use, and in other solutions and accessories that should be non-pyrogenic. PG
• Gram positive and Gram negative bacteria are invariable components of the cell wall, and are primarily responsible for the inflammatory response and its harmful health consequences caused by Gram positive organisms, including septic shock [Silhavy TJ et al., Cold Spring Harbor perspectives in biology 2, a000414, 2010].
• PGs are capable of inducing the release of proinflammatory cytokines [Verhoef J and Mattsson E, Trends in Microbiology 3: 136-140, 1995; Teti G, Trends in Microbiology 7: 100-101, 1999], are pyrogenic, released into the environment during the growth and death of bacteria, and are resistant to ordinary means of sterilization [Moesby L, European Journal of Pharmaceutical Science 35, 442-446, 2008]. Due to the similarities between the LPS and the PG, it has been proposed that
• the profenoloxidase activating system is an essential component of the innate humoral immune response in invertebrates [Cerenius et al, Trends Immunol. (29): 263-271, 2008].
• the proFO system is composed of a complex array of proteins that includes proteins that recognize PMAPs, the activating enzyme for profenoloxidase (EAPFO), and profenoloxidase. It has been described in several arthropods that the proFO system is activated in the presence of small amounts of certain PMAPs through a mechanism that has not been fully elucidated. In general, it is known that in the presence of PMAPs EAPFO is activated and that, by means of a proteolytic attack, it converts profenoloxidase (inactive zymogen) into active phenoloxidase. Phenoloxidase oxidizes monophenols and o-diphenols to aminochromes, thus initiating the synthesis of melanin.
• US 497052 describes the development and use of a reagent obtained from the silkworm larvae plasma (SLP) for the detection of BG and PG by the determination of BG and PG the phenoloxidase activity or the peptidase activity of EAPFO. There are described reagent modifications that involve the specific determination of BG or PG. Based on this invention, US 5585248 describes the use of more sensitive synthetic chromogenic substrates for the detection of EAPFO activity. In US 6274565 Bl, a modification of the reagent that allows the specific detection of PG is protected by inhibiting the activation pathway mediated by BG. The SLP assay appears to be particularly useful in the detection of bacterial contamination in platelets (US 7598054 B2).
• patents US 6987002 B2 and US 2002/0197662 Al describe a reagent obtained from complete hemolymph of insect larvae (Tenebrio molitor or Holotrichia diomphalia) for the detection and quantification of (1, 3) -3 -D-glucans.
• the proFO system of the spiny lobster P. argus is found in the hemocytes [Perdomo-Morales and cois, Fish Shellfish Immunol (23): 1 187-1 195, 2007], and is able to activate in the presence of small concentrations of PG , BG and LPS.
• the proFO system is regulated by a 5 kDa peptidase inhibitor and positive net charge. The separation of the inhibitor from the rest of the active components of the system substantially increases the sensitivity of the response to PG, BG and LPS.
• the lobster proFO system also includes a protein Recognizer of LPS and BG (LGBP) for lipopolysaccharide and (1, 3) - -D-glucan binding protein), which is located in the plasma fraction of hemolymph.
• LGBP protein Recognizer of LPS and BG
• LPS or BG BG
• the composition which is presented here for the first time, consists of an aqueous extract of the hemocytes of the locust hemolymph, hereinafter referred to as Lobster Hemocyte Lysate (LHL).
• LHL Lobster Hemocyte Lysate
• the active component of interest in the LHL is the proFO system, and the composition is aimed at the detection and quantification of LPS, PG and BG.
• lobster or lobsters in this document refers to the species included in the Astacidea, Palinura (Achelata) and Thalassinidea, Suborder Reptantia (Macrura), Decapoda Order, Crustacean Class, Phylum Arthropoda.
• lobsters are also subject to overfishing in some regions, they are very abundant species that represent the main fishing resource of many countries (eg Cuba, Brazil, Australia, United States, among others).
• the production of the composition that we present does not affect the availability of these animals for human consumption as food because hemolymph is a byproduct that is discarded.
• Lobster is one of the largest crustaceans, and has a significant volume of easily obtained hemolymph.
• hemolymph is extracted from the animal using a suitable anticoagulant that prevents plasma coagulation, but preferably prevents both plasma coagulation and cell activation and coagulation.
• a suitable anticoagulant that prevents plasma coagulation, but preferably prevents both plasma coagulation and cell activation and coagulation.
• an isotonic solution containing methyl xanthine derivatives such as theophylline, theobromine or caffeine, or their salts, or reagents capable of modifying sulfhydroxy groups (-SH) such as cysteine, iodoacetamide and N-ethylmaleimide can be used.
• solutions containing chelating agents and having a pH between 4.5-8 provided by a suitable buffer for example, the modified Alsever anticoagulant (27 mM sodium citrate, 336 mM NaCI, 1 15 mM glucose, 9 mM EDTA , pH 7) or the citrate-EDTA anticoagulant (0.4 M NaCI, 0.1 M glucose, 30 mM trisodium citrate, 26 mM citric acid and EDTA 10 mM, pH 4.6).
• the modified Alsever anticoagulant is used at a hemolymph: anticoagulant 1: 1 (v / v) ratio.
• the hemocytes are separated from the plasma by centrifugation at 700 g for 10 min at 4 ° C.
• the plasma (supernatant) is discarded, and the sedimented hemocytes are washed to remove remains of plasma components.
• the hemocytes are resuspended with anticoagulant at a volume not greater than that corresponding to the initial hemolymph: anticoagulant mixture, followed by another identical centrifugation cycle. At least two washes are performed.
• the washed hemocyte button is suspended in lysis buffer, preferably with a volume between 1 and 10 ml.
• the lysis buffer may contain NaCl at a concentration between 0.001 and 600 mM, agents capable of stabilizing enzymes (stabilizers) and a pH between 5 and 8.5, provided by a suitable buffer that does not sequence divalent cations.
• stabilizers agents capable of stabilizing enzymes
• a pH between 5 and 8.5 provided by a suitable buffer that does not sequence divalent cations.
• 50 mM Tris-HCI, pH 7.5, 450 mM NaCl is used.
• Hemocytes disintegrate using a suitable breakdown method among those commonly used in biochemistry for the preparation of cell extracts, which may be mechanical, chemical or enzymatic. Considering the characteristics of these cells, the most suitable methods of rupture are osmotic shock, freezing / thawing, homogenization with shaking (Vortex) or manual (Dounce and Potter-Elvehjemem), and ultrasound. The hemocyte homogenate is centrifuged at 13,000 rpm at 4 ° C for 30 min to obtain the clarified supernatant or LHL.
• the present invention includes a method for increasing the sensitivity of the proFO system response in the LHL against LPS, PG and BG.
• This modification is based on the elimination or inactivation of peptidase inhibitors present in the LHL.
• the inhibitor can be separated by techniques based on separation by size and molecular shape, charge or affinity.
• molecular exclusion techniques such as gel filtration chromatography, ultrafiltration and diafiltration will be used.
• chromatographic resins with a maximum limit of exclusion between 10 and 60 kDa, preferably 30 kDa, should be used.
• Chromatographic fractionation is performed at a linear flow between 3 and 60 cm / h, preferably at 9 cm / h, and a sample volume between 1 and 5% of the total column volume, preferably 3%, is applied. Under these conditions, the remaining 35 active proteins of the proFO system elute in the dead volume of the column. When ultrafiltration and diafiltration are used, cutting membranes between 5 and 60 kDa are used, preferably between 10 and 40 kDa.
• LHL-M modified LHL
• Both the LHL and LHL-M reagents are used for the detection of PG, BG and LPS. This detection is based on its activation of the lobster proFO system ( Figure 1).
• the detection of PG, BG and LPS is performed by determining the activity of the active phenoloxidase (FO), or the peptidase activity of the active EAPFOs ( Figure 1).
• Phenoloxidase enzyme activity is determined using monophenolic substrates (eg tyramine, tyrosine, 4-hydrophenylacetic acid, 4- hydrophenylpropionic acid), or diphenolic (eg dihydroxy L-phenylalanine (L-DOPA), dopamine, 3,4 acid dihydroxyphenylacetic acid, catechol, methylcaltecol, and 3,4 dihydroxyphenylpropionic acid).
• dopamine at a concentration of 1.2 mM.
• the formation of colored products allows the phenoloxidase activity to be determined visually or spectrophotometrically
• the sensitivity of the method to determine the phenoloxidase activity can be increased by combining the monophenolic or o-diphenolic substrates with 3- methyl-2-benzothiazolinone hydrazone (3-methyl-2-benzothiazolinone hydrazone (MBTH)) or Besthorn hydrazone.
• MBTH is a potent nucleophilic agent that forms a stable adduct colored with o-quinones generated by phenoloxidases (quinones-MBTH), with a molar extinction coefficient or absorptivity much higher than that of the corresponding aminochrome [Spin JC and cois, Eur J.
• MBTH is used at a final concentration between 0.3-15 mM, preferably 2 to 7 mM.
• the peptidase activity of EAPFOs induced by BG, LPS and PG is determined using chromogenic or flurogenic substrates for trypsin-like serine proteases.
• These substrates are of the general formula R-Arg-Y or R-Lys-Y, where Y is a chromophore such as p-nitroaniline (p-NA) or a fluorophore such as 7-amido-4-met.il coumarin, rhodamine , or 7-amido-4-trifluoro methylcoumarin.
• R represents an L or D amino acid protected by its N-terminal by a protective group, or a peptide composed of L or D amino acids, or its combination, protected at its N-terminal by a protective group.
• the tests to detect PG, LPS and BG using LHL or LHL-M can be performed by kinetic, pseudokinetic or endpoint method. Conventional spectrophotometers or fluorometers are used to detect the reaction. However, due to the greater sample processing capacity and reagent savings, readers capable of reading 96-well microplates, equipped to read wavelengths in the visible, fluorescence, or those capable of performing both functions, are preferred.
• the detection of the signal in time immediately after the addition of all the components of the reaction mixture, including the substrate, is defined as a kinetic test.
• Pseudokinetic assays are defined as those where the signal is determined after the addition of the substrate to a previously incubated reaction mixture.
• Endpoint assays are defined as the single reading of the signal after incubation (s) for a fixed time of the mixture of reactants with the substrate.
• the pH of the reaction mixture should be between 6 and 9, preferably 7.5, and may or may not contain divalent cations. It must contain calcium, magnesium or manganese at a minimum concentration of 5 mM, preferably 50 mM.
• Test temperature 0 should be between room temperature and 45 ° C, preferably 37 ° C.
• the present invention also conceives the use of LGBP to increase the sensitivity of both LHL and LHL-M against BG and LPS.
• LGBP can be included in the reagent formulation, or it can be added later to the reaction mixture.
• the LGBP may be in a concentration range in the assay for the detection of PG and BG between 3 and 200 pg / ml, preferably 50 to 125 pg / ml.
• LGBP is obtained from plasma, which is also an abundant byproduct of the preparation of LHL.
• LGBP can be purified by affinity chromatography using linked hydrophilic matrices a (1, 3) -PD-glucans [Vargas-Dawn F and cois, Comp Biochem Physiol B Biochem Mol Biol, 1 16 (4): 453-458, 1997], immunoaffinity [Duvic B and Sóderháll K, J Biol.
• heparin is used as ligand, with which large amounts of LGBP with high degree of purity and high yields are obtained in a single chromatographic step.
• FIG. 1 Scheme of the locust profenoloxidase activating system (proFO system), and principle of the method for the use of Lobster Hemocyte Lysate (LHL) in the detection of lipopolysaccharides (LPS), peptidoglycans (PG) and ( 1, 3) -pD-glucans (BG).
• LHL lipopolysaccharides
• PG peptidoglycans
• BG 1, -pD-glucans
• FIG. 3 LHL response to lipopolysaccharides (LPS), (1, 3) - -D-glucans (BG) and peptidoglycans (PG).
• A-C panels phenoloxidase activity in arbitrary units ( ⁇ OD 490 nm / min).
• Panel DF presents the graphs that represent the linear relationship between the phenoloxidase response and the concentration of each microbial activator LPS (0.185-1850 ng / ml), BG (1.8-18000 ng / ml) and PG (0.19-19000 ng / ml ).
• Threshold time reaction time to reach a specific optical density or OD threshold.
• Figure 4 Influence of MBTH on the sensitivity of the phenoloxidase response.
• Left panel Optical density at 490 nm after one hour of reaction at 37 ° C (end point).
• Right Panel Speed of the formation of dopachrome (Control) and the colored quinone-MBTH complexes at different concentrations of MBTH.
• Figure 7 Lysate response without inhibitor to LPS using dopamine as substrate. Sensitivity of 0.01 ng / ml of LPS.
• Figure 8 Purification of LGBP by affinity chromatography on heparin-Sepharose CL-6B (Panel A). Electrophoresis in SDS-PAGE (Panel B). Lane 1: Patterns of weight ll molecular; Lane 2: Complete plasma; Lane 3 and 4: LGBP obtained by affinity chromatography analyzed under reducing and non-reducing conditions, respectively. The proteins were stained with Coomassie R-250 bright blue. Examples of realization:
• 10 ml of hemolymph were extracted through the cisternal sinus located on the last pair of lobster legs using sterile, pyrogen-free 20 ml disposable syringes, containing 10 ml of cold anticoagulant.
• the modified Alsever solution composed of 27 mM sodium citrate, 336 mM NaCl, 15 mM Glucose, 9 mM EDTA, pH 7 (1000 mOsmol) was used as anticoagulant.
• the hemolymph: anticoagulant mixture was poured into 50 ml sterile and endotoxin-free polypropylene centrifuge tubes, and centrifuged at 700 x g at 4 ° C for 10 min. The supernatant containing the plasma was separated.
• the hemocyte button was suspended with 20 ml of cold anticoagulant, the original volume of the anticoagulant-hemolymph mixture (50 ml) was taken, and centrifuged again as described above. The washing step was repeated once more.
• the washed hemocyte button was resuspended in 3 ml of lysis buffer composed of 50 mM Tris-HCI, 450 mM NaCI, pH 7.5, and transferred to 13 mm x 10 cm borosilicate tubes.
• the hemocyte suspension was lysed by sonication using three cycles of 20 Watts for 10 sec. In ice bath.
• the hemocyte homogenate was centrifuged at 13,000 rpm for 30 min. at 4 ° C to obtain the clarified LHL.
• the LHL (12 mg / ml) was diluted to a concentration of 0.5 mg / ml with 50 mM Tris-HCI, pH 7.5.
• the test was performed in 96-well sterile flat bottom microplates certified sterile and pyrogen free.
• the reaction mixture consisted of 150 ⁇ of 50 mM Tris-HCI, pH 7.5, 50 mM CaCI 2 , 20 pl of LHL, and 50 ⁇ of activator (LPS, PG or BG).
• the control used endotoxin-free water.
• 50 ⁇ of dopamine was added at 3.75 mM.
• the dopaminechrome formation was read kinetically at 490 nm for 1 h every 15 sec at 37 ° C in a microplate reader.
• the speed of the enzymatic reaction in the linear portion of the OD 490 nm vs. time graph was determined.
• the threshold time was determined, defined as the time
• the 0.8 mg / mL LHL was diluted to 0.1 mg / mL with 50 mM Tris-HCI, pH 7.5.
• the test was performed in 96-well flat bottom microplates.
• the reaction mixture consisted of 5 in 40 pL of 50 mM Tris-HCI, pH 7.5, 160 pL of 1 mg / mL trypsin dissolved in 50 mM Tris-HCI, pH 7.5, 10 pL of LHL and 40 pL of MBTH dissolved in water distilled This mixture was incubated for 20 minutes at 37 ° C and finally 50 pL of 3.75 mM dopamine was added.
• the reaction was read kinetically at 490 nm for 1 h every 15 sec at 37 ° C in a microplate reader.
• LHL response to LPS using chromogenic substrates for serine peptidases.
• 50 pl of chromogenic substrate S-2222 (Bz-lle-Glu (Y-OR) -Gly-Arg-pNA-HCI) 0.6 mM were added.
• the released paranitroaniline 5 was detected kinetically at 405 nm for 1 h at 37 ° C.
• Vt 131.4 ml
• the extract was fractionated in a spin column packed with Sephadex G-50 (1.5 ml).
• the 0 column was equilibrated by washing 4 times with centrifugation at 1000 rpm for 1 min with Tris-HCI pH 7.5, 450 mM NaCI.
• 150 pl of lysate prepared according to example 1 were applied to the spin column, and centrifuged at 1000 rpm for 1 min.
• the eluate was collected and the protein concentration, the inhibitory activity against trypsin, and the phenoloxidase activity in both the untreated fraction (LHL) and the fraction obtained from the spin column (F1) were analyzed.
• the lysate without inhibitor was obtained according to Example 5.
• 50 ⁇ of LPS, 50 ⁇ of Tris-HCL, pH 75, 50 mM CaCI2 and 20 ⁇ of lysate at 0.8 mg / ml were mixed and incubated at 37 ° C by 30 min.
• 50 ⁇ of dopamine at 3.75 mM was added and the reaction was read kinetically at 490 nm for 1 hour at 37 ° C.
• the threshold time was calculated, such as the time required for the reaction to reach an optical density of 0.3 and the log of the threshold time was plotted against the log of the LPS concentrations.
• the plasma obtained during the preparation of the LHL was centrifuged at 5000 rpm for 20 min. at 4 ° C for clarification.
• One hundred milliliters of clarified plasma were dialyzed at 4 ° C using dialysis membranes with a cut of 8000 Da vs 5 L of distilled water at 4 ° C for 48 hours, including 4 dialysate water changes every 12 hours during the period.
• the dialysate was centrifuged at 5000 rpm for 20 min at 4 ° C and the supernatant was discarded.
• the protein pellet was suspended in 50 mM Tris-HCI, 0.2 M NaCI, pH 7.5 (Buffer A), and centrifuged again to remove insoluble denatured proteins.

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