JPH0151782B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for effectively clarifying the turbidity of a biological sample, such as human serum, and a reagent used in the method. More particularly, the present invention relates to a reagent comprising a surfactant and an enzyme and a method of using the reagent as a haze remover. Turbidity of the produced sample causes serious problems. The turbidity can cause unreadable or inaccurate readings, resulting in highly questionable quantitative results. Turbidity of serum and blood plasma samples is always a significant problem in clinical optical analysis, producing erroneous data and often falsifying optical quantification of serum components. The main cause of turbidity appears to be increased triglycerides in the serum of patients with hyperlipoproteinemia, with or without increased total cholesterol levels.
An abnormal increase in serum cholesterol is a very dangerous condition called artherosclerosis.
are known to be related to each other. Quantification of cholesterol and triglycerides is important because accurate values can help doctors diagnose patients with hyperlipoproteinosis and predict heart disease. When testing cloudy samples in other tests such as aspartate aminotransferase (GOT), alanine aminotransferase (GPT), and lactated dehydrogenase (LDH), The same difficulties exist in quantifying cholesterol or triglycerides. Clinical trials of cloudy serum have been conducted by treating samples with high concentrations of surfactants such as polyoxyethylated lauric acid (US Pat. Nos. 3,853,465 and 4,184,848). Because only surfactants are used there, fairly high concentrations of surfactants are required for effective clarity. High concentrations of surfactants interact with other chemicals or enzyme reactions, complicating the analysis. The surfactants used in this invention are at relatively low concentrations. This is due to the action of enzymes (cholesterol esterase or lipase) added to the clarifying reagent. The exact mechanism of transparency according to the present invention is not yet known. However, if the patient has hyperlipoproteinosis, it can be assumed that the turbidity in the serum sample is mainly due to the increased triglyceride content. Triglycerides are water-insoluble and are usually buried inside the fatty core along with cholesterol esters in lipoprotein complexes. Clarification of lipid-containing samples is achieved by first disrupting the lipoproteins with a surfactant such as lauric acid diethanolamide (DEA) and then hydrolyzing the triglycerides with an enzyme base. The surfactant serves to dissolve the released fatty acids. Without enzymes, it cannot become transparent. An object of the present invention is to provide a reagent that has the effect of clarifying turbidity in a biological sample and a method for making it clear using the reagent.
For example, the target is a sample to be optically assayed or analyzed for cholesterol. The present invention is based on the formula (wherein R is an alkyl group or alkenyl group having 5 to 17 carbon atoms, and x and y are each 1
) and an enzyme selected from the group consisting of cholesterol esterase or lipase or mixtures thereof. This article focuses on reagents that are effective in making turbid biological samples transparent. Said reagents in the form of an aqueous buffer include, based on the total reagent composition, about 0.05 to about 2.5 g/dl of surfactant and preferably an enzyme (cholesterol esterase).
0.1 to about 0.5 g/dl and at least about 0.025 U/ml
Preferably, it consists of: The PH value of the resulting composition ranges from about 5.5 to about 7.0. If the composition contains lipase as the enzyme, about 0.05 to
The composition comprises about 2.5 g/dl, preferably from 0.1 to about 0.5 g/dl and at least about 1.0 U/ml of lipase, and the PH value of the composition ranges from about 5.5 to about 8.0. If the enzyme contained in the composition is any of the above, usable buffers include sodium salt or potassium salt of maleic acid, phosphate, borate, citrate, succinate, and imidazole acetate buffers. , Tris et al. Other suitable buffers can also be used. Such a buffer is one that maintains a desired constant pH without interacting with any of the components of the composition.
It is something that can be kept at a value. When using maleate salts, such as potassium or sodium salt buffers, from about 0.05 to about 0.5M;
It is added in an amount that gives the resulting composition a pH value of about 5.0 to about 7.0. Other buffers are used at similar concentrations. In addition to the above-mentioned components, a solubility promoter can also be added to the enzyme composition. Such solubility promoters are materials that help solubilize the surfactant. For example, bile sulfate salts such as sodium colanate, sodium deoxycholanate, etc. are particularly effective. In another preferred example, the surfactant is of the formula above, where R is an alkyl group, such as lauric acid diethanolamide or oleic acid diethanolamide. Enzymes originate from animals such as the pancreas or from microorganisms. Samples suitable for processing according to the invention include human serum and blood. Combining the above reagents with biological samples effectively clears the turbidity. Reagents are usually in the form of aqueous buffer solutions. formula (wherein, R is an alkyl group or alkenyl group having 5 to 17 carbon atoms, and x and y are an integer in total of 11 or less); and cholesterol. Also within the scope of the invention are reagents consisting of enzymes selected from esterases or lipases or mixtures thereof, which have the effect of clarifying the turbidity of biological samples to be assayed or analyzed optically. A preferred reagent is a surfactant having the above formula in which R is an alkyl group, preferably a lauryl group.
A compound in which the sum of and y is 5, and the enzyme is lipase. The reagent composition preferably includes polyethylene glycol-p-isooctylphenyl ether or other suitable surfactant. Preferred reagents that have the effect of clarifying cloudy samples at pH 2-10 also include a mixture of two surfactants: lauric acid diethanolamide (x=y=1) and epoxidized lauric acid (x+y= 5). The enzyme composition described above is prepared using the surfactant described above. Suitable surfactants included in the above compositions include lauric acid diethanolamide, myristic acid diethanolamide, capric acid diethanolamide, oleic acid diethanolamide and coconut acid diethanolamide. When the reagent of the present invention is combined with a biological sample, the turbidity of the sample can be effectively cleared, allowing accurate optical assay or analysis of the sample. Samples to be optically analyzed for cholesterol are advantageously treated in this way. The present invention is primarily directed to a method of effectively clarifying the turbidity of biological samples using specific surfactants and enzymes, and reagents therefor. Here, the surfactant has the formula (wherein R is an alkyl group or alkenyl group having 5 to 17 carbon atoms, and x and y are each 1). Preferably, R is an alkyl group, such as lauric acid diethanolamide. The enzyme component is cholesterol esterase or lipase or a mixture thereof. The resulting composition surprisingly turned out to be very effective in clarifying the turbidity of biological samples. Therefore, it is very useful in converting cloudy biological samples to clear samples. If no inhibition or interference occurs between the interaction between the surfactant and the enzyme and the action of the assay reagent,
Samples treated with the compositions of the invention described above can be subjected to colorimetric assays or analysis for any particular component. Examples of typical assays that can be performed using the reagents are the quantification of cholesterol, triglycerides, and creatine phosphate kinase. The enzyme components are derived from animal sources, for example from the pancreas, or from microorganisms. The present invention is secondly directed to methods and reagents for effectively clarifying turbid biological samples for optical assays. This reagent has a formula that has a broader definition than the first surfactant. (In the formula, R is an alkyl group or alkenyl group having 5 to 17 carbon atoms, and x and y are integers whose sum is 11 or less.) , cholesterol esterase or lipase or a mixture thereof. In a preferred embodiment, the surfactant is represented by the above formula, where R is an alkyl group and x and y are each 1. Examples are lauric acid diethanolamide, myristic acid diethanolamide and capric acid diethanolamide. Preferred surfactants also include those in which R is an alkenyl group and x and y are each 1, such as oleic acid diethanolamide and coconut acid diethanolamide.
Another preferred example is a surfactant in which R is an alkyl group and the sum of x and y is 5. The enzyme components are derived from animal sources, for example from the pancreas, or from microorganisms. When the reagents described above are used in an assay, they are combined with the biological fluid in the form of an aqueous buffer solution. The cloudy sample becomes clear and can be assayed. Samples that can be effectively cleared and assayed include human serum and blood plasma. The methods and reagents according to the invention are unique in at least two respects. Transparent, without interference from turbidity
This makes it possible to quantify components in biological samples in a clear state, and due to the interaction between the specific surfactant used and the enzyme, a smaller amount of enzyme is needed than conventionally used for cholesterol quantification. That's enough. The most common laboratory test for cholesterol in biological fluids is the determination of total cholesterol, including free cholesterol and cholesterol esters. Both cholesterol and its esters exist together with other lipids and types of proteins in small molecule complexes called lipoproteins in serum; cholesterol esters are usually the only component (60-80%) of total cholesterol. %). Cholesterol is generally water-insoluble;
It is usually buried inside the complex and cannot be accessed by enzymes. If total enzyme determination of total cholesterol is to be carried out either by automated or manual methods, both cholesterol and cholesterol esters must first be liberated from the complex by means of suitable detergents. Cholesterol ester is then hydrolyzed by cholesterol esterase to free cholesterol, which is further oxidized by cholesterol oxidase to yield cholestenone and hydrogen peroxide. The method according to the invention can be used in an automated manner using automatic analyzers, or can also be carried out manually. In preparing compositions for use in assays according to the method of the invention, an aqueous solution is prepared which, in addition to surfactants and enzymes, also contains other auxiliary ingredients known in the art and used for such purposes. . For example, in a cholesterol test, the following components are used and their amounts are as follows: Ingredients Cholesterol assay Paoxidase 0.8-2.0U/Cholesterol oxidase 0.025-0.3U/ml Cholesterol esterase 0.025-0.3U/ml Surfactant 0.05-0.5g/dl Sodium colanate 0.05-0.5g/dl p-hydroxybenzoic acid sodium
2.5-6 g/dl 4-aminoantipyrine 0.5-2.0mM Maleic acid 0.1-0.5M PH 5.5-7.0 Sample/reagent ratio 10.0-400 In the above compositions, cholesterol esterases of animal origin, e.g. pancreatic origin, are used. is preferred, but similar results can be obtained using cholesterol esterase of microbial origin. Example 1 Clarification as a function of cholesterol esterase Potassium maleate (0.1M), sodium colanate (0.25g/dl), lauric acid diethanolamide (0.2g/dl), cholesterol esterase (0.08-0.8U/ml) 3 ml of clearing reagent containing 100 ml of clearing reagent with a final pH of 6.4 are mixed with 0.025 ml of fat-containing serum (triglyceride content approximately 1400 mg/dl). 45
Within 10 minutes at °C, the reaction mixture becomes clear. Cholesterol esterase for clarity may be obtained from the pancreas or from a microorganism. Example 2 Use in Cholesterol Assay As described in this example, cholesterol assay components are included in a clarifying reagent to determine the end point of the assay. Cholesterol esterase (0.125U/ml),
Cholesterol oxidase (0.125U/ml), peroxidase (1.6U/ml), 4-aminoantipyrine (0.6mM), sodium hydroxybenzoate (25mM), sodium colanate (0.25g/ml)
dl), lauric acid diethanolamide (0.2g/
dl) and potassium maleate (0.1M),
3ml of PH6.0 composition. Mix all of this reagent with 0.025 ml of lipid-containing serum sample. Then add this mixture to 45
After incubating for 4-5 minutes at 520nm
Quantify total cholesterol by measuring color density. Without the clarifying action of lauric acid diethanolamide and cholesterol esterase, the results of cholesterol quantification in cloudy samples will always be erroneous. Example 3 Candida lipase (Candida silidrasia)
cylindracea) Lauric acid diethanolamide (to 0.2g/
dl), sodium colanate (0.25 g/dl) and lipase (25 U/ml) and maleate buffer (0.1 M), 3 ml of clearing reagent at PH 6.0 are mixed with 0.025 ml of lipid-containing serum. . After incubating at 45°C for 5 minutes, the cloudy sample becomes clear. A similar clearing effect can be obtained using the same amount of a clearing reagent containing a mixture of lauric acid diethanolamide and epoxidized lauric acid (x+y=5). Example 4 formula (In the formula, the sum of x and y is 5) Surfactant (0.2g/dl) represented by TritonX
-100 (0.4g/dl), potassium maleate (0.2M)
3 ml of pH 6.0 clearing reagent containing lipase (25 U/ml) was mixed with 0.05 ml of lipid-containing sample,
Incubate at °C. After 3 minutes, the cloudy sample becomes clear. Increasing the concentration of buffering agent
Increases the speed of invisibility. Similar results are obtained using higher concentrations of epoxidized lauric acid (x+y=5) without the addition of Triton X-100. Example 5 A Composition A diagnostic reagent composition is prepared as an aqueous solution using the following ingredients. Ingredients Concentration Malic acid 11.6g KOH 10.0g EDTA (K ₂ ) 2.7mM Sodium colanate 5.8mM Sodium p-hydroxybenzoate 25.0mM 4-aminoantipyrine 0.6mM Lauric acid diethanolamide 2.0g Cholesterol esterase 125 units Cholesterol oxidase 125 units Horseradish peroxidase
800 unit PH is adjusted to 6.0. The reagent composition can be stored and used in the form of an aqueous solution, which solution can also be lyophilized in conventional manner and reconstituted with water at the time of use. B Assay - Quantification of Total Cholesterol Mix 3 ml of the above reagent with 0.025 ml of serum or regenerated serum standard sample with cholesterol content up to 500 mg/dl. React for 4-5 minutes at 45°C. Using the reagent as a blank, measure the absorbance of the sample at 525 nm. Example 6 Clarification in the determination of creatine phosphate kinase (CPK) activity in lipid-containing serum Lauric acid diethanolamide (0.4%), cholesterol esterase of pancreatic origin (25 U/dl), sodium colanate (0.25 g%) and Contains thiolglycerol (20mM). PH
6.7 imidazole-acetate buffer solution (0.1M) 2ml
Mix with 0.05 ml of lipid-containing serum. When incubated at 37°C for 15 minutes, the turbidity at 340nm is
It decreases from 2.3OD to 0.02OD. The cleared sample is then mixed with 1 ml of CPK reagent. This reagent contains creatine phosphate (116.7mM),
ADP (6.7mM), AMP (16.7mM), EDTA (6.7mM
M), NADP (6.7mM), hexokinase (125U/dl), glycose-6-phosphate dehydrogenase (G6PDH) (100U/dl),
It was prepared as a 0.1M imidazole-acetate buffer with a pH of 6.7. In the usual way, 340nm, 37
Measure CPK activity at °C. It will be apparent to those skilled in the art that changes and modifications can be made to this invention without departing from the spirit and scope of the invention as defined in the claims and described herein. It is within the scope of the present invention.

[Claims]

1 Erythrocyte sedimentation length measurement consisting of a stationary measurement tube support that vertically supports a large number of blood sedimentation measurement tubes, a light source device and an image sensor that face each other across the blood sedimentation measurement tubes and can illuminate the entire measurement section of the blood sedimentation measurement tubes. system and
A mechanism for moving the measurement system only in a direction across the blood sedimentation measuring tubes so that the blood sedimentation measuring tubes are interposed one after another between the light source and the image sensor, and detecting the presence or absence of the measuring tube at each blood sedimentation measuring tube installation position. and a timing mechanism that measures the measurement time for each measurement tube individually.The measurement tube attachment detection mechanism checks the attachment of the blood sedimentation measurement tube, and at the same time, the timing mechanism starts the measurement. Start measuring time for the tube, and measure the height of the whole blood column, the height of the sedimented red blood cell column, or the height of the sedimented red blood cell column in the measuring tube at predetermined time intervals from the start of time measurement. An automatic erythrocyte sedimentation rate measurement device that measures and stores the erythrocyte sedimentation rate in memory, calculates and records the erythrocyte sedimentation length from these values. 2. Claim 1 in which the measuring tube attachment detection mechanism comprises a light source and a light receiver that are attached to the lower part of the erythrocyte sedimentation length measurement system so as to face each other across the measuring tube.

Claims (1)

The method described in Section 1. 4 A surfactant in which x=y=1 in the above formula
The method according to item 1 above, wherein 0.05 to about 2.5 g/dl and at least about 0.025 U/ml of cholesterol esterase as an enzyme are added to form an aqueous buffer solution having a pH of about 5.5 to about 7.0. 5. The method according to item 4 above, wherein about 0.1 to about 0.5 g/dl of surfactant is added. 6 Maleate as a buffering agent at a concentration of about 0.05 to about
The method according to item 4 above, wherein the method is added at a concentration of 0.5M and the pH is adjusted to about 5.0 to about 7.0. 7. The method described in 4 above, in which a solubility promoter is added. 8. The method according to item 7 above, wherein about 0.25 g/dl of sodium colanate or sodium deoxycholanate is added as a dissolution promoter. 9 A surfactant in which x=y=1 in the above formula
The method according to item 1 above, wherein 0.05 to about 2.5 g/dl and at least about 1.0 U/ml of lipase as an enzyme are added to form an aqueous buffer solution with a pH of about 5.5 to about 8.0. 10 Add about 0.1 to about 0.5 g/dl of surfactant,
The method described in the preceding section 9. 11 Maleate as a buffering agent at a concentration of about 0.05 to about
The method according to the preceding item 9, wherein the method is added at a concentration of 0.5M and the pH is adjusted to about 5.0 to about 7.0. 13. The method according to item 12 above, wherein about 0.25 g/sodium colanate or sodium deoxycholanate is added as a solubility promoter. 14. The method according to item 1, wherein a surfactant in which R is an alkyl group and x=y=1 is added as a surfactant. 15. The method according to item 14 above, wherein lauric acid diethanolamide is added as a surfactant. 16. The method according to item 14 above, wherein myristic acid diethanolamide is added as a surfactant. 17. The method according to item 14 above, wherein capric acid diethanolamide is added as a surfactant. 18. Addition of a surfactant in the above formula in which R is an alkenyl group and x=y=1,
The method described in the preceding section 1. 19. The method according to item 18 above, wherein oleic acid diethanolamide is added as a surfactant. 20. The method according to item 18 above, wherein coconut diethanolamide is added as a surfactant. 21. The method according to item 1 above, wherein an enzyme derived from an animal or microorganism is added. 22. The method according to item 21 above, wherein cholesterol esterase derived from animal pancreas is added as the enzyme. 23 A surfactant in which the sum of x and y in the above formula is 5 or less, and about 0.05 to about 2.5 g/dl, and at least about cholesterol esterase as an enzyme.
0.025U/ml to form an aqueous buffer solution having a pH of about 5.5 to about 8.0. 24 Maleate as a buffering agent at a concentration of about 0.05 to about
24. The method according to item 23 above, wherein the method is added at a concentration of 0.5M and the pH is adjusted to about 5.0 to about 7.0. 25 Add about 0.05 to about 0.5 g/dl of a surfactant in which the sum of x and y in the above formula is 5 or less, and at least about 1.0 U/ml of lipase as an enzyme, and adjust the pH to about
1 in the form of an aqueous buffer solution of 2.0 to about 10.0
The method described in. 26 Maleate, citrate, as a buffering agent
Succinate, Tris or Borate from about 0.05 to about
Add at a concentration of 0.5M and adjust the pH to about 2.0 to about 10.0.
The method described in the preceding section 25. 27. The method according to item 1 above, wherein an enzyme derived from an animal or microorganism is added. 28. The method according to item 27 above, wherein cholesterol esterase derived from animal pancreas is added as the enzyme. 29. The method according to item 1, wherein a surfactant in which R is an alkyl group and the sum of x and y is 5 is added as a surfactant, and lipase is added as an enzyme. 30. The method according to item 29 above, wherein polyethylene glycol-p-isooctylphenyl ether (Triton X-100) is added. 31 Add about 0.05 to about 2.0 g/dl of a mixture of lauric acid diethanolamide and epoxidized lauric acid (x+y=5) as a surfactant, and adjust the pH to about
1 in the form of an aqueous buffer solution of 2.0 to about 10.0
The method described in.