CN102998445B - Reagent and preparation method for determining glycocholic acid - Google Patents
Reagent and preparation method for determining glycocholic acid Download PDFInfo
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- CN102998445B CN102998445B CN201210536455.6A CN201210536455A CN102998445B CN 102998445 B CN102998445 B CN 102998445B CN 201210536455 A CN201210536455 A CN 201210536455A CN 102998445 B CN102998445 B CN 102998445B
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Abstract
The invention relates to a reagent and a preparation method for determining glycocholic acid, and aims to provide the reagent having the characteristic of high accuracy and provide the preparation method having the characteristic of convenient production. The technical solution is as follows: the reagent for determining glycocholic acid comprises the following components of a. a glycocholic acid reagent 1, b. a glycocholic acid reagent 2 and c. a liquid type glycocholic acid reference calibrator. The preparation method for determining glycocholic acid comprises the following steps of 1) preparation of the glycocholic acid reagent 1, comprising a step of mixing components according to the formula amount; 2) preparation of the glycocholic acid reagent 2, comprising the steps of (1) taking a fluid suspension; (2) reacting the mixture; (3) obtaining a carboxyl latex microsphere suspension; (4) adjusting concentration; (5) taking the suspension of (3) and adding into (4); (6) reacting; (7) adding ethanolamine; and (8) performing centrifugation treatment; and 3) preparation of the liquid type glycocholic acid reference calibrator, comprising the steps of mixing according to the formula amount to obtain a mixed solution; and arranging according to CG content or adding a CG pure product in the mixed solution to obtain the CG reference calibrator.
Description
Technical field
The present invention relates to a kind of preparation method of reagent and the reagent of measuring glycocholic acid, particularly adopt Immunoturbidimetry to measure reagent and the corresponding preparation method of the glycocholic acid in serum or blood plasma, can be widely used in medical science and technological field of biochemistry.
Background technology
Serum CG (Cholyglycine, CG) is that cholic acid is combined one of mating type cholic acid of secondary with glycocoll, in liver cell, cholesterol through and complicated enzymatic reaction, be transformed into elementary bile acid.Wherein there are cholic acid (CA) and chenodeoxycholic acid (CD-CA).On the steroids core of cholic acid, have three hydroxyls (C3, C7, C12), the hydroxyl of side chain terminal is combined with glycocoll with peptide bond, and CG is synthesized by liver cell, enters gall-bladder through bile capillaries, bile duct, enters duodenum in company with bile, helps food digestion.95% cholic acid heavily absorbs at terminal ileum, returns liver through portal vein again, by liver cell picked-up recycling.In serum, mainly exist with protein combination form.Under normal circumstances, in peripheral blood, cholic acid content is very micro-, and normal adult no matter on an empty stomach or after the meal, its change of serum C G concentration stabilize is low-level.In the time that liver cell is impaired, liver cell picked-up CG ability declines, and causes CG increased content in blood; When cholestegnosis, liver excretion cholic acid generation obstacle, and the sanguimotor CG increased content that backflows also makes blood CG increased content.Measuring Serum CG (CG) is that evaluation hepatocyte function and liver and gall thereof are one of sensitive indicator of material recycle function.Pregnant woman is in the conceived middle and later periods in addition, due to baby's increase, can oppress liver, make liver excretion cholic acid generation obstacle and cause glycocholic acid higher, in addition due to the gestational period placenta synthetic and secrete a large amount of estrogen and progestational hormone and metabolic burden and increase, can bring out the variation of liver and gall, make pregnant woman easily suffer from intrahepatic cholestasis of pregnancy (ICP), fetus is produced serious influence, increase with ICP patients serum CG the amniotic fluid-pollution rate that makes, early productive rate, fetal distress in uterus rate and cut open palace productive rate and increase, severe patient can cause baby's death, therefore measure pregnancy serum glycocholic acid, the order of severity of finding early ICP and understanding ICP is had to very important meaning, the mensuration of glycocholic acid can be used as examination and follows up a case by regular visits to the index of ICP.Thereby reduction the perinatal fetal mortality.
Detect at present the conventional laboratory method of CG and have radioimmunology analytic approach (RIA), chemiluminescence immunoassay (CLIA), euzymelinked immunosorbent assay (ELISA) (ELISA), radioimmunology analytic approach (RIA) detects length consuming time (19～22h), and there is a pollution of radioelement, label poor stability, discarded object is difficult to process and it is restricted.Chemiluminescence immunoassay (CLIA) is to utilize chemiluminescent substance through catalyst and oxygenant oxidation, form the intermediate of an excited state, the intermediate of this excited state is got back to while stablizing ground state, send photon, utilize luminous signal measuring instrument measurement of photon number, thus the concentration of indirect determination CG.Have in the market tubular type and board-like two kinds of kits, but this method exists the luminescence efficiency of acridinium ester, luminol, the direct labelled antibody of different luminol low, the unsettled shortcoming of label; And this direct labelling method belongs to moment light emitting-type, very difficult stability and the repeatability that ensures test result, and detecting instrument that need to be special.Being not easy to Routine Test Lab carries out.And utilize biotin-avidin system to detect the electrochemiluminescence immune reagent kit of CG, and needing the fully automatic electric chemiluminescence detector of supporting costliness, Routine Test Lab cannot be carried out, and brings unnecessary expense to patient, increases patient burden.Euzymelinked immunosorbent assay (ELISA) (ELISA) automaticity is not high, and be affected by human factors larger, poor repeatability, the whole reaction assay time is very long (at least needing 40 minutes) also.
Summary of the invention
Technical matters to be solved by this invention is the deficiency that overcomes above-mentioned background technology, provides a kind of glycocholic acid to detect reagent; Feature simple to operate, that minute is short, accuracy is high, reproducible that the reagent providing should have, and measure applicable to the glycocholic acid of various types of automatic clinical chemistry analyzers; The preparation method of reagent thereof providing should have feature easy to make and that cost is not high.
Technical scheme provided by the invention is:
Measure a reagent for glycocholic acid, comprise following composition and content:
A, glycocholic acid reagent 1:
All the other are purified water,
The pH of reagent 1 is 7.2-8.0;
B, glycocholic acid reagent 2:
All the other are purified water,
The described latex particle with anti-human CG antibody, carboxylated latex microspherulite diameter is wherein 20-500nm;
C, liquid-type glycocholic acid reference calibrations product:
All the other are purified water;
Described liquid-type glycocholic acid reference calibrations product comprise 5 parts of reference calibrations product that form series concentration; CG content in 5 parts of reference calibrations product is respectively 1.25mg/ml, 2.5mg/ml, 5.0mg/ml, 10.0mg/ml, 20.0mg/ml from low to high; Or,
Described liquid-type glycocholic acid reference calibrations product are CG reference calibrations product of single-point high concentration.
Described damping fluid is the mixing of one or more arbitrary proportions in phosphate buffer, trishydroxymethylaminomethane (Tris) damping fluid, Isosorbide-5-Nitrae-piperazine two ethyl sulfonic acids (PIPES) damping fluid, 4-(2-hydroxyethyl) piperazine-1-1 ethane sulfonic acid (HEPES) damping fluid, Pehanorm base propane sulfonic acid (TAPS) damping fluid, glycine buffer, borate buffer, 3-morpholine propane sulfonic acid (MOPS) damping fluid, 3-(N-morpholinyl)-2-hydroxy-propanesulfonic acid (MOPSO) damping fluid, MES (MES) damping fluid.
In described reagent 1, the concentration of damping fluid is 20-200mmol/L, and the pH value of damping fluid is 7.0-8.0.
Described reaction promoter is polyglycol or dextran sulfate (DS-50).
Described reaction promoter concentration is 0.01-50g/L.
The preferred 0.05-30g/L of described reaction promoter concentration.
Described electrolyte is anion electrolyte or cationic polyelectrolyte, and concentration is 50-200mmol/L.
Described stabilizing agent is the mixing of one or more arbitrary proportions in disodium ethylene diamine tetraacetate, bovine serum albumin(BSA), sodium chloride.
Sodium chloride in described electrolyte preferred cationic.
Described glass or plastic containers, cationic surfactant, anionic surfactant or zwitterionic surfactant; Described surfactant concentration is 0.5-1g/L.
Described non-ionic surfactant is the mixing of one or more arbitrary proportions in Theist, Tween, polyoxyethylene laurel ether, polyoxyethylene phenyl ether, polyoxyethylene octyl group phenylate, polyoxyethylene alkyl phenyl ether, polyoxyethylene nonylplenyl ether.
Content with the latex particle of anti-human CG antibody in described glycocholic acid reagent 2 is 0.002-0.01ml/ml.
Measure a preparation method for glycocholic acid reagent, carry out according to following steps:
1) glycocholic acid reagent 1:
Press formula ratio each composition is mixed, then regulate pH value;
2) glycocholic acid reagent 2:
(1) get carboxylated latex microballoon damping fluid and be diluted to the suspending liquid of 0.01mg/ml;
(2) add 20mg EDAC(ethyl dimethyl amine propyl carbodiimide diimine in the carboxylated latex microsphere suspension liquid of 1ml) and the NHS(N-N-Hydroxysuccinimide sulfonate sodium of 40mg) ratio, after mixing immediately, in room temperature, this potpourri is reacted to 15-30 minute, constantly stir;
(3) with damping fluid or purified water washing carboxylated latex microballoon, remove unreacted NHS and EDAC, and latex microsphere is suspended in purified water, obtaining latex microsphere content is the carboxylated latex microsphere suspension liquid of 0.01mg/ml;
(4) anti-human glycocholic acid antibody is dissolved in buffer solution, the protein concentration that makes anti-human glycocholic acid antibody is 0.25mg/ml;
(5) the carboxylated latex microsphere suspension liquid 1ml getting in step (3) adds anti-human glycocholic acid antibody 1ml in step (4) immediately; Make the content of carboxylated latex microballoon in mixed liquor, anti-human glycocholic acid antibody and buffer solution be respectively 0.005mg/ml, 0.125mg/ml, 25mmol/L;
(6) potpourri is reacted 3 hours at 37 DEG C, constantly stir;
(7) add monoethanolamine in the ratio of 1ml reaction mixture 2.5 μ l monoethanolamines, then react 10-30 minute, constantly stir;
(8) centrifugal treating is removed unconjugated protein and monoethanolamine, then with damping fluid dilution, and making in mixed liquor with the concentration of the latex particle of anti-human CG antibody is 0.002-0.01ml/ml, after then adding stabilizing agent, antiseptic to dissolve to mix;
3) liquid-type glycocholic acid reference calibrations product:
First by formula ratio, 5 portions of mixed liquors are mixed and be divided into antiseptic, stabilizing agent, damping fluid and purified water, then CG reference calibrations product concentration as required, the CG sterling of the 1000mg/ml of respective amount is added respectively in above-mentioned mixed liquor, obtain 5 parts of reference calibrations product that form series concentration; CG content in 5 parts of reference calibrations product is respectively 1.25mg/ml, 2.5mg/ml, 5.0mg/ml, 10.0mg/ml, 20.0mg/ml from low to high; Or,
Press after formula ratio mixes antiseptic, stabilizing agent and damping fluid and obtain mixed liquor, then in mixed liquor, add the CG sterling of a certain amount of 1000mg/ml, obtain the CG reference calibrations product of single-point high concentration.
Described anti-human glycocholic acid antibody is goat-anti people glycocholic acid antibody or the anti-human glycocholic acid antibody of rabbit or mouse-anti people glycocholic acid antibody or the anti-human glycocholic acid antibody of chicken.
Described centrifugal treating adopts hydro-extractor, and centrifuge speed is 15000 revs/min.
The principle of work that CG provided by the invention measures reagent is: the latex particle generation antigen-antibody reaction with anti-human CG antibody in the CG in sample and reagent, reactant liquor turbidity is increased, in reactant liquor turbidity and sample, the amount of antigen is linear within the specific limits, can use Biochemical Analyzer or other optical detecting instrument at 540-600nm wavelength place assaying reaction liquid absorbance, reactant liquor absorbance is directly proportional to surveyed CG concentration.
The invention has the beneficial effects as follows: the glycocholic acid of the Immunoturbidimetry of employing detects reagent, by CG antibody is combined with carboxylated latex microballoon, amplified the surface area of CG antigen and antibody response, not only accuracy is high (with the correlativity R of chemiluminescence immunoassay
2for 0.9965-0.9998), reproducible, and detect fast and (start at most only to need 10 minutes to obtaining a result from measuring, even shorter), can on routine biochemistry instrument, carry out sample mensuration in enormous quantities, greatly improved testing efficiency.The preparation method of this reagent is easy and simple to handle, and raw material is easy to get (all selecting outsourcing raw material), and cost is not high, is applicable to all kinds of medical research units and Routine Test Lab application.
Brief description of the drawings
Fig. 1 shows in the embodiment of the present invention 1 by the mutual relationship of the measured value of the method for the invention gained CG reagent and the CG measured value of chemiluminescence immunoassay gained.Through regretional analysis: y=1.0064x+0.0508, R
2=0.9994, demonstrate the embodiment of the present invention 1 and there is good correlativity with chemiluminescence immunoassay.
Fig. 2 shows in the embodiment of the present invention 2 by the mutual relationship of the measured value of the method for the invention gained CG reagent and the CG measured value of chemiluminescence immunoassay gained.Through regretional analysis: y=1.0064x+0.0508, R
2=0.9994 demonstrates the embodiment of the present invention 2 has good correlativity with chemiluminescence immunoassay.
Fig. 3 shows in the embodiment of the present invention 3 by the mutual relationship of the measured value of the method for the invention gained CG reagent and the CG measured value of chemiluminescence immunoassay gained.Through regretional analysis: y=1.0533x-0.0633, R
2=0.9965 demonstrates the embodiment of the present invention 3 has good correlativity with chemiluminescence immunoassay.
Fig. 4 shows in the embodiment of the present invention 4 by the mutual relationship of the measured value of the method for the invention gained CG reagent and the CG measured value of chemiluminescence immunoassay gained.Through regretional analysis: y=1.0001x+0.0485, R
2=0.9998, demonstrate the embodiment of the present invention 4 and there is good correlativity with chemiluminescence immunoassay.
Embodiment
The present invention detects reagent and the required primary raw material of calibration object
1, anti-human glycocholic acid antibody, has much commercial anti-human glycocholic acid antibody available, as DaKo company of Finland, Japanese UNF company; This antibody only reacts with people's glycocholic acid, without immunological cross-reaction, can be polyclonal antibody or monoclonal antibody with other antigen, be not particularly limited here, as long as can with latex microsphere coupling.Its anti-human glycocholic acid antibody can be that goat-anti (is goat-anti people glycocholic acid antibody; As follows), also can be that rabbit is anti-, chicken anti-or mouse-anti.As long as adopt known immunoelectrophoresis to measure, this antibody only and between people CG presents single heavy alignment, and the titre of anti-human glycocholic acid antibody is more than or equal to 1.0mg/ml.
2, latex microsphere, has many commercial Nano microspheres available, as German Merck company, Japanese UNF company, Japanese JSR company and Thermo company; Its basic material is polystyrene or co polystyrene, and surface is modified with carboxylic group (COOH), also can be not used as any modification, will select different balls according to the associated methods of selecting antibody and microballoon is different here.As select chemical crosslink technique, with the surperficial microballoon (crosslinking chemical is preferred: ethyl dimethyl amine propyl carbodiimide diimine) of modifying through carboxyl, amino, aldehyde radical etc.
The latex microsphere that the preferred carboxylic group of this law is modified, the preferred 20nm-500nm of particle diameter of carboxylated latex microballoon, further, preferably 80nm-300nm.
In reagent 2, anti-human CG antibody latex particle can adopt physisorphtion or chemical crosslink technique preparation, the preferred chemical crosslink technique of the present invention.
3, glycocholic acid: purchased from DaKo company of Finland, needing reference calibrations product for the preparation of this reagent place, can be natural glycocholic acid albumen, can be also genetic recombination glycocholic acid albumen.
CG reference calibrations product concentration, can set 5 reference calibrations product that form series concentration, and in 5 reference calibrations product, CG content is respectively 1.25mg/ml, 2.5mg/ml, 5.0mg/ml, 10.0mg/ml, 20.0mg/ml from low to high;
Also can be prepared into the CG reference calibrations product of single-point high concentration (as 100.0mg/ml, or 500.0mg/ml), when use, become again the reference calibrations product of multiple variable concentrations with normal saline dilution; Here have no particular limits, if the CG reference calibrations product that make can with sample comparison, can working sample in the content of CG.
Further illustrate the present invention below in conjunction with embodiment, but these embodiment should not think to limit definition of the present invention.
Embodiment mono-
One) glycocholic acid reagent 1
After above-mentioned raw materials being dissolved with purified water 800ml, then add purified water and be settled to 1000ml, then adjust PH to 7.2 with hydrochloric acid or NaOH.
Two) glycocholic acid reagent 2
1. the carboxylated latex of getting 80nm for microballoon the MES damping fluid of 50mmol/L, PH6.0 be diluted to the suspending liquid of 0.01mg/ml;
2. add 20mg EDAC(ethyl dimethyl amine propyl carbodiimide diimine by the carboxylated latex microsphere suspension liquid of 1ml) and the NHS(N-N-Hydroxysuccinimide sulfonate sodium of 40mg) mix immediately after in room temperature by this potpourri reaction 15-30 minute, constantly stir;
3. with MES damping fluid or the purified water washing carboxylated latex microballoon of 50mmol/L, PH6.0, remove unreacted NHS and EDAC, and the carboxylated latex microsphere suspension liquid that latex microsphere suspended in purified water to obtain, wherein the content of carboxylated latex microballoon is 0.01mg/ml.
4. goat-anti people glycocholic acid antibody is dissolved in the HEPES buffer solution of 50mmol/L, PH8.5, making the protein concentration of goat-anti people glycocholic acid antibody is 0.25mg/ml;
5. get the carboxylated latex microsphere suspension liquid 1ml in step (3), add the goat-anti people glycocholic acid antibody 1ml in step (4); Now in this mixed liquor, the content of carboxylated latex microballoon, goat-anti people glycocholic acid antibody and buffer solution is respectively 0.005mg/ml, 0.125mg/ml, 25mmol/L;
6. potpourri is reacted 3 hours at 37 DEG C, constantly stir;
7. the ratio that adds 2.5 μ l in 1ml reaction mixture adds monoethanolamine, reacts 20 minutes, constantly stirs;
8. with 15000 revs/min of centrifugal unconjugated protein and monoethanolamines removed, with the MOPS damping fluid dilution of 50mmol/L, PH7.2, making in mixed liquor with the concentration of the latex particle of goat-anti people CG antibody is 0.01ml/ml; Then add bovine serum albumin(BSA) (stabilizing agent) 3mmol/L, 3mmol/L Sodium azide (antiseptic) dissolving to obtain glycocholic acid reagent 2 after mixing.
Three) liquid-type CG reference calibrations product
All the other are purified water;
CG reference calibrations product concentration as required adds corresponding CG sterling 1000mg/ml in above-mentioned damping fluid, be prepared into the reference calibrations product of 5 series concentration, in the reference calibrations product of 5 series concentration, CG content is respectively 1.25mg/ml, 2.5mg/ml, 5.0mg/ml, 10.0mg/ml, 20.0mg/ml from low to high.
Pattern detection (contrast respectively and detect 30 samples with chemiluminescence immunoassay method): according to experimental implementation step, to the glycocholic acid reagent 1 that adds 240 μ L in 10 μ L samples, hatch 3 minutes at 37 DEG C, add 2,37 DEG C of 60 μ L glycocholic acid reagent to hatch after 10 seconds and read absorbance A at wavelength 600nm
1, while reacting to 5 minutes, read absorbance A
2, calculate absorbance changing value △ A=A twice
2-A
1.By with the calibration solution comparison of same processing, calculate the CG concentration in sample.From table 1 and Fig. 1 (Fig. 1 draws according to table 1 data), result can be found out, although the inventive method is simple to operate, required time only, in 10 minutes, detects contrast with chemiluminescence immunoassay method, shows very good correlativity (correlativity R
2be 0.9994).
Table 1
Embodiment bis-
One) glycocholic acid reagent 1
After above-mentioned raw materials being dissolved with purified water 800ml, then add purified water to 1000ml(1L), then adjust PH to 8.0 with NaOH.
Two) glycocholic acid reagent 2
1. the carboxylated latex of getting 150nm for microballoon the MES damping fluid of 50mmol/L, PH6.0 be diluted to the suspending liquid of 0.01mg/ml;
2. add the NHS of 20mg EDAC and 40mg by the carboxylated latex microsphere suspension liquid of 1ml, after mixing immediately, in room temperature, potpourri is reacted to 15-30 minute, constantly stir;
3. with MES damping fluid or the purified water washing carboxylated latex microballoon of 50mmol/L, PH6.0, remove unreacted NHS and EDAC, and the carboxylated latex microsphere suspension liquid that latex microsphere suspended in purified water to obtain, wherein the content of carboxylated latex microballoon is 0.01mg/ml;
4. anti-human rabbit glycocholic acid antibody is dissolved in the borate buffer solution of 50mmol/L, PH8.5, the protein concentration that makes the anti-human glycocholic acid antibody of rabbit is 0.25mg/ml;
5. the carboxylated latex microsphere suspension liquid 1ml getting in step (3) adds the anti-human glycocholic acid antibody of the rabbit 1ml in step (4) immediately; Now in this mixed liquor, the concentration of carboxylated latex microballoon, the anti-human glycocholic acid antibody of rabbit and buffer solution is respectively 0.005mg/ml, 0.125mg/ml, 25mmol/L;
6. potpourri is reacted 3 hours at 37 DEG C, constantly stir;
7. add the monoethanolamine of 2.5 μ l by 1ml reaction mixture, react 30 minutes, constantly stir;
8. with 15000 revs/min of centrifugal unconjugated protein and monoethanolamines removed, with 50mmol/L, PH7.4, phosphate buffer dilution, making in mixed liquor with the concentration of the latex particle of the anti-human CG antibody of rabbit is 0.006ml/ml; Then add bovine serum albumin(BSA) (stabilizing agent) 4mmol/L, 2mmol/L Sodium azide (antiseptic) dissolving to obtain glycocholic acid reagent 2 after mixing.Three, liquid-type CG reference calibrations product
All the other are purified water;
CG reference calibrations product concentration as required adds corresponding CG sterling 1000mg/ml in above-mentioned damping fluid, obtains 5 reference calibrations product that form series concentration; In 5 reference calibrations product, CG content is respectively 1.0mg/ml, 2.0mg/ml, 4.0mg/ml, 8.0mg/ml, 16.0mg/ml from low to high.
Pattern detection (contrast respectively and detect 30 samples with chemiluminescence immunoassay method): according to experimental implementation step, to the glycocholic acid reagent 1 that adds 240 μ L in 10 μ L samples, hatch 3 minutes at 37 DEG C, add 2,37 DEG C of 60 μ L glycocholic acid reagent to hatch after 10 seconds and read absorbance A at wavelength 600nm
1, while reacting to 5 minutes, read absorbance A
2, calculate absorbance changing value △ A=A twice
2-A
1.By with the calibration solution comparison of same processing, calculate the CG concentration in sample.From table 2 and Fig. 2 (Fig. 2 draws according to table 2 data), result can be found out, although the inventive method is simple to operate, required time only in 10 minutes, also correlativity (the correlativity R very good with chemiluminescence immunoassay method
2be 0.9997).
Table 2
Embodiment tri-
One) glycocholic acid reagent 1
After above-mentioned raw materials material being dissolved with purified water 800ml, then add purified water to 1000ml(1L), then adjust PH to 8.0 with NaOH.
Two) glycocholic acid reagent 2
1. the carboxylated latex of getting 300nm for microballoon the MES damping fluid of 50mmol/L, PH6.0 be diluted to the suspending liquid of 0.01mg/ml.
2. add the NHS of 20mg EDAC and 40mg by the carboxylated latex microsphere suspension liquid of 1ml, after mixing immediately, in room temperature, this potpourri is reacted to 15-30 minute, constantly stir;
3. with MES damping fluid or the purified water washing carboxylated latex microballoon of 50mmol/L, PH6.0, remove unreacted NHS and EDAC, and the carboxylated latex microsphere suspension liquid that latex microsphere suspended in purified water to obtain, wherein the content of carboxylated latex microballoon is 0.01mg/ml;
4. mouse-anti people glycocholic acid antibody is dissolved in the TAPS salt buffer solution of 50mmol/L, PH8.5, making the protein concentration of mouse-anti people glycocholic acid antibody is 0.25mg/ml;
5. the carboxylated latex microsphere suspension liquid 1ml getting in step (3) adds the mouse-anti people glycocholic acid antibody 1ml in step (4) immediately; Now in this mixed liquor, the content of carboxylated latex microballoon, mouse-anti people glycocholic acid antibody and buffer solution is respectively 0.005mg/ml, 0.125mg/ml, 25mmol/L;
6. potpourri is reacted 3 hours at 37 DEG C, constantly stir;
7. add the monoethanolamine of 2.5 μ l by 1ml reaction mixture, react 15 minutes, constantly stir;
8. with 15000 revs/min of centrifugal unconjugated protein and monoethanolamines removed, with the PIPES damping fluid dilution of 50mmol/L, PH8.0, making in mixed liquor with the concentration of the latex particle of mouse-anti people CG antibody is 0.002ml/ml; Then add bovine serum albumin(BSA) (stabilizing agent) 2mmol/L, 4mmol/L Sodium azide (antiseptic) dissolving to obtain glycocholic acid reagent 2 after mixing.
Three) liquid-type CG reference calibrations product
All the other are purified water;
CG reference calibrations product concentration as required adds corresponding CG sterling 1000mg/ml in above-mentioned damping fluid, be prepared into the reference calibrations product of 5 series concentration, in the reference calibrations product of 5 series concentration, CG content is respectively 2.0mg/ml, 4.0mg/ml, 8.0mg/ml, 16.0mg/ml, 32.0mg/ml from low to high.
Pattern detection (contrast respectively and detect 30 samples with chemiluminescence immunoassay method):
According to experimental implementation step, to the glycocholic acid reagent 1 that adds 240 μ L in 10 μ L samples, hatch 3 minutes at 37 DEG C, add 2,37 DEG C of 60 μ L glycocholic acid reagent to hatch after 10 seconds and read absorbance A at wavelength 600nm
1, while reacting to 5 minutes, read absorbance A
2, calculate absorbance changing value △ A=A twice
2-A
1.By with the calibration solution comparison of same processing, calculate the CG concentration in sample.From table 3 and Fig. 3 (Fig. 3 draws according to table 3 data), result can be found out, although the inventive method is simple to operate, required time only in 10 minutes, also correlativity (the correlativity R very good with chemiluminescence immunoassay method
2be 0.9965).
Table 3
Embodiment tetra-
One) glycocholic acid reagent 1
After above-mentioned raw materials being dissolved with purified water 800ml, then add purified water to 1000ml, then adjust PH to 7.6 with hydrochloric acid or NaOH.
Two) glycocholic acid reagent 2
1. the carboxylated latex of getting 220nm for microballoon the MES damping fluid of 50mmol/L, PH6.0 be diluted to the suspending liquid of 0.01mg/ml;
2 add the NHS of 20mg EDAC and 40mg by the carboxylated latex microsphere suspension liquid of 1ml, after mixing immediately in room temperature by this potpourri reaction 15-30 minute, constantly stir;
3. with MES damping fluid or the purified water washing carboxylated latex microballoon of 50mmol/L, PH6.0, remove unreacted NHS and EDAC, and the carboxylated latex microsphere suspension liquid that latex microsphere suspended in purified water to obtain, wherein the content of carboxylated latex microballoon is 0.01mg/ml;
4. anti-human chicken glycocholic acid antibody is dissolved in the borate buffer solution of 50mmol/L, PH8.5, the protein concentration that makes the anti-human glycocholic acid antibody of chicken is 0.25mg/ml;
5. the carboxylated latex microsphere suspension liquid 1ml getting in step (3) adds the anti-human glycocholic acid antibody of the chicken 1ml in step (4) immediately; Now in this mixed liquor, the concentration of carboxylated latex microballoon, the anti-human glycocholic acid antibody of chicken and buffer solution is respectively 0.005mg/ml, 0.125mg/ml, 25mmol/L;
6. potpourri is reacted 3 hours at 37 DEG C, constantly stir;
7. add the monoethanolamine of 2.5 μ l by 1ml reaction mixture, react 10 minutes, constantly stir;
8. with 15000 revs/min of centrifugal unconjugated protein and monoethanolamines removed, with 50mmol/L, PH7.6, the dilution of HEPES damping fluid, making in mixed liquor with the concentration of the latex particle of the anti-human CG antibody of chicken is 0.004ml/ml; Then adding bovine serum albumin(BSA) (stabilizing agent) 3mmol/L, 3mmol/L Sodium azide (antiseptic) dissolving is glycocholic acid reagent 2 after mixing.
3) liquid-type CG reference calibrations product
All the other are purified water;
CG reference calibrations product concentration as required adds corresponding CG sterling 1000mg/ml in above-mentioned damping fluid, obtains 5 reference calibrations product that form series concentration; In 5 reference calibrations product, CG content is respectively 0.5mg/ml, 1.0mg/ml, 2.0mg/ml, 4.0mg/ml, 8.0mg/ml from low to high.
Pattern detection (contrast respectively and detect 30 samples with chemiluminescence immunoassay method):
According to experimental implementation step, to the glycocholic acid reagent 1 that adds 240 μ L in 10 μ L samples, hatch 3 minutes at 37 DEG C, add 2,37 DEG C of 60 μ L glycocholic acid reagent to hatch after 10 seconds and read absorbance A at wavelength 600nm
1, while reacting to 5 minutes, read absorbance A
2, calculate absorbance changing value △ A=A twice
2-A
1.By with the calibration solution comparison of same processing, calculate the CG concentration in sample.From table 4 and Fig. 4 (Fig. 4 draws according to table 4 data), result can be found out, although the inventive method is simple to operate, required time only, in 10 minutes, also shows very good correlativity (correlativity R with chemiluminescence immunoassay method
2be 0.9998).
Table 4
Be below glycocholic acid reagent measurement operation step of the present invention and on automatic clinical chemistry analyzer, make a concrete analysis of parameter:
Determination step
Analytical parameters (taking the parameter of Hitachi's 7080 biochemical instruments as example)
Method: Two point end assay, temperature of reaction: 37 DEG C
Predominant wavelength: 520-600nm commplementary wave length: 800nm(can not select)
Sample size: 10 μ L, reagent 1 amount/reagent 2 amounts: 240 μ L/60 μ L
The Direction of Reaction: forward (rising), calibration mode: multiple spot is non-linear
The read point time: be respectively and 21 points (suitable 420 seconds of total reaction time) at 5
Taking the parameter of Hitachi's 7080 biochemical instruments as example
Instrument completes the mensuration that can carry out CG after calibration automatically, and instrument is by automatically calculating corresponding CG concentration.
It is to be noted; above-described is only the preferred embodiments of the invention, for the those of ordinary skill in this technology neck city, under the premise without departing from the principles of the invention; can also serve as some improvement and adjustment, these improved adjustment also should be considered as protection scope of the present invention.
Claims (6)
1. measure a reagent for glycocholic acid, comprise following composition and content:
A, glycocholic acid reagent 1:
The pH of reagent 1 is 7.2-8.0;
B, glycocholic acid reagent 2:
The described latex particle with anti-human CG antibody, carboxylated latex microspherulite diameter is wherein 20-500nm;
C, liquid-type glycocholic acid reference calibrations product:
Described liquid-type glycocholic acid reference calibrations product comprise 5 parts of reference calibrations product that form series concentration; CG content in 5 parts of reference calibrations product is respectively 1.25mg/ml, 2.5mg/ml, 5.0mg/ml, 10.0mg/ml, 20.0mg/ml from low to high; Or,
Described liquid-type glycocholic acid reference calibrations product are CG reference calibrations product of single-point high concentration;
Described reaction promoter is PEG-6000;
Described electrolyte is NaCL;
Described surfactant is the one in Tween-20, Theist, polyoxyethylene laurel ether-35;
Described stabilizing agent is the mixing of one or more arbitrary proportions in disodium ethylene diamine tetraacetate, bovine serum albumin(BSA), sodium chloride;
Described antiseptic is the one in Sodium azide, wide-spectrum bactericide.
2. a kind of reagent of measuring glycocholic acid according to claim 1, it is characterized in that described damping fluid is phosphate buffer, trishydroxymethylaminomethane (Tris) damping fluid, 1, 4-piperazine two ethyl sulfonic acids (PIPES) damping fluid, 4-(2-hydroxyethyl) piperazine-1-1 ethane sulfonic acid (HEPES) damping fluid, Pehanorm base propane sulfonic acid (TAPS) damping fluid, glycine buffer, borate buffer, 3-morpholine propane sulfonic acid (MOPS) damping fluid, 3-(N-morpholinyl)-2-hydroxy-propanesulfonic acid (MOPSO) damping fluid, the mixing of one or more arbitrary proportions in MES (MES) damping fluid.
3. a kind of reagent of measuring glycocholic acid according to claim 2, is characterized in that in glycocholic acid reagent 2 that with the content of the latex particle of anti-human CG antibody be 0.002-0.01mg/ml.
4. a kind of preparation method who measures glycocholic acid reagent claimed in claim 1, carries out according to following steps:
1) glycocholic acid reagent 1:
Press formula ratio each composition is mixed, then regulate pH value;
2) glycocholic acid reagent 2:
(1) get carboxylated latex microballoon damping fluid and be diluted to the suspending liquid of 0.01mg/ml;
(2) add the ratio of the NHS (N-hydroxy-succinamide sulfonate sodium) of 20mg EDAC (ethyl dimethyl amine propyl carbodiimide diimine) and 40mg in the carboxylated latex microsphere suspension liquid of 1ml, after mixing immediately, in room temperature, this potpourri is reacted to 15-30 minute, constantly stir;
(3) with damping fluid or purified water washing carboxylated latex microballoon, remove unreacted NHS and EDAC, and latex microsphere is suspended in purified water, obtaining latex microsphere content is the carboxylated latex microsphere suspension liquid of 0.01mg/ml;
(4) anti-human glycocholic acid antibody is dissolved in buffer solution, the protein concentration that makes anti-human glycocholic acid antibody is 0.25mg/ml;
(5) the carboxylated latex microsphere suspension liquid 1ml getting in step (3) adds anti-human glycocholic acid antibody 1ml in step (4) immediately; Make the content of carboxylated latex microballoon in mixed liquor, anti-human glycocholic acid antibody and buffer solution be respectively 0.005mg/ml, 0.125mg/ml, 25mmol/L;
(6) potpourri is reacted 3 hours at 37 DEG C, constantly stir;
(7) add monoethanolamine in the ratio of 1ml reaction mixture 2.5 μ l monoethanolamines, then react 10-30 minute, constantly stir;
(8) centrifugal treating is removed unconjugated protein and monoethanolamine, then with damping fluid dilution, and making in mixed liquor with the concentration of the latex particle of anti-human CG antibody is 0.002-0.01mg/ml, after then adding stabilizing agent, antiseptic to dissolve to mix;
3) liquid-type glycocholic acid reference calibrations product:
First by formula ratio, 5 portions of mixed liquors are mixed and be divided into antiseptic, stabilizing agent, damping fluid and purified water, then CG reference calibrations product concentration as required, the CG sterling of the 1000mg/ml of respective amount is added respectively in above-mentioned mixed liquor, obtain 5 parts of reference calibrations product that form series concentration; CG content in 5 parts of reference calibrations product is respectively 1.25mg/ml, 2.5mg/ml, 5.0mg/ml, 10.0mg/ml, 20.0mg/ml from low to high; Or,
Press after formula ratio mixes antiseptic, stabilizing agent and damping fluid and obtain mixed liquor, then in mixed liquor, add the CG sterling of a certain amount of 1000mg/ml, obtain the CG reference calibrations product of single-point high concentration.
5. a kind of preparation method who measures glycocholic acid reagent according to claim 4, is characterized in that described anti-human glycocholic acid antibody is goat-anti people glycocholic acid antibody or the anti-human glycocholic acid antibody of rabbit or mouse-anti people glycocholic acid antibody or the anti-human glycocholic acid antibody of chicken.
6. a kind of preparation method who measures glycocholic acid reagent according to claim 5, is characterized in that described centrifugal treating adopts hydro-extractor, and centrifuge speed is 15000 revs/min.
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| CN102944673B (en) * | 2012-11-30 | 2014-11-12 | 北京华宇亿康生物工程技术有限公司 | Kit (Latex-enhanced immunoturbidimetry) for detecting content of glycocholic acid in blood serum |
| CN104535758B (en) * | 2013-06-17 | 2016-07-27 | 北京北检·新创源生物技术有限公司 | A kind of coupling method that polypeptide or protein are covalently coupled on microsphere |
| CN103308698B (en) * | 2013-06-17 | 2015-04-29 | 北京北检·新创源生物技术有限公司 | Method for covalently coupling amino-containing molecules to microspheres |
| CN103344763A (en) * | 2013-06-18 | 2013-10-09 | 华东师范大学 | Latex-reinforced AFP detection kit and its preparation method and use |
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| CN103940816B (en) * | 2014-04-18 | 2016-04-06 | 安徽大千生物工程有限公司 | A kind of kit and preparation method measuring human body content of glycocholic acid |
| CN104459113B (en) * | 2014-12-05 | 2016-03-16 | 重庆中元生物技术有限公司 | A kind of glycocholic acid detection kit |
| CN104569434B (en) * | 2015-01-14 | 2016-08-17 | 复旦大学附属中山医院 | Golgi apparatus protein gp73 latex strengthens immunoturbidimetry detection kit and preparation method thereof |
| CN106124439A (en) * | 2016-08-31 | 2016-11-16 | 潍坊市康华生物技术有限公司 | A kind of detection kit of the glycocholic acid eliminating chyle interference |
| CN107703314B (en) * | 2017-11-15 | 2018-11-20 | 迈克生物股份有限公司 | A kind of corticotropin solution and its application |
| CN109444399A (en) * | 2018-10-26 | 2019-03-08 | 重庆中元汇吉生物技术有限公司 | A kind of glycocholic acid detection kit |
| CN109917143A (en) * | 2019-04-09 | 2019-06-21 | 贵州盛世康生物科技有限公司 | A kind of glycocholic acid measurement reagent and preparation method thereof |
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| CN102628864B (en) * | 2011-12-30 | 2014-08-13 | 北京九强生物技术股份有限公司 | Kit for determining heart-type fatty acid binding protein in serum or urine by latex enhanced turbidimetric immunoassay |
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