CN1260569C - Reagent box for external blood group and blood serum experimental determination - Google Patents
Reagent box for external blood group and blood serum experimental determination Download PDFInfo
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- CN1260569C CN1260569C CN 200410046105 CN200410046105A CN1260569C CN 1260569 C CN1260569 C CN 1260569C CN 200410046105 CN200410046105 CN 200410046105 CN 200410046105 A CN200410046105 A CN 200410046105A CN 1260569 C CN1260569 C CN 1260569C
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Abstract
The present invention discloses a reagent kit for determination experiments of external blood groups and blood serum, which aims to provide a stable chymopapain reagent kit for determination experiments of extracorporeal blood groups and blood serum. The reagent kit comprises a chymopapain preparation, a chymopapain solvent, an enzyme inhibitor-64 preparation and an enzyme inhibitor-64 solvent, wherein the chymopapain preparation comprises chymopapain, L-cysteine, pullulan, etc.; the enzyme inhibitor-64 preparation comprises trans-epoxide succinyl-L-leucyl (4-guanidino) butane, mannitol, trehalose, pullulan, etc. The provided reagent kit simplifies the operating processes of determination experiments of external blood groups and blood serum, and enhances the safety and the sensitivity of the determination experiments. The reagent kit can be widely used for the screening determination, the identification and the valent titration of blood group antibodies and corresponding blood group antigens of red cells, the washing of the red cells coated by the antibodies, and the determination of immune blood group antibodies of biological products for injection.
Description
Technical field
The present invention relates to the external blood group serology calibrating kit in the enzyme preparation field, particularly a kind of chymopapain kit.
Background technology
Use thiol protease to carry out erythrocyte blood type, the existing nearly 60 years applicating history of immunity blood group antibody calibrating.At present, preparation commonly used has papain, bromelain and cradin in external blood group serology calibrating experiment.The mixed enzyme formed by multiple sulfydryl enzyme of papain wherein, different times results and bigger with various sulfydryl enzyme ratio drift values in the papain of different process production, enzyme kinetics is an on-fixed constant, causes easily producing significantly " false negative ", " false positive " result in the external blood group serology calibrating experiment.
Summary of the invention
The purpose of this invention is to provide a kind of stable used chymopapain kit in external blood group serology calibrating experiment.
External blood group serology calibrating kit provided by the present invention is by chymopapain preparation, chymopapain solvent, enzyme inhibitor-64 preparation and enzyme inhibitor-64 solvent composition; Described chymopapain preparation contains chymopapain, L-halfcystine, Propiram, trehalose; Described enzyme inhibitor-64 contains trans-epoxy succinic acyl-L-leucylamino (4-guanidine radicals) butane, sweet mellow wine, trehalose and Propiram.
Described chymopapain solvent is the same with the basic recipe of enzyme inhibitor-64 solvent, be the aqueous solution of 0.02-0.07mol/L potassium dihydrogen phosphate, 0.003-0.04mol/L sodium hydrogen phosphate, but in actual applications, their concentration is adjusted according to the pH value of external blood group serology calibrating experiment different phase needs, the former is pH5-6, and the latter is pH6-7.
In the chymopapain preparation in the chymopapain of every 200000PU unit the content of L-halfcystine and Propiram be respectively 0.025-0.050mol and 0.05-0.1mmol.Every 400000TU unit is trans in the enzyme inhibitor-64-epoxy succinic acyl-L-leucylamino (4-guanidine radicals) butane in the content of sweet mellow wine, trehalose and Propiram be respectively 0.2-0.3mol, 0.01-0.02mol and 0.05-0.1mol.
In order to make the chymopapain preparation more stable, also contain sweet mellow wine, trehalose and vitamin C salt in the described chymopapain preparation.The content of sweet mellow wine, trehalose and vitamin C salt is respectively 0.2-0.3mol, 0.01-0.02mol and 0.01-0.02mol in the chymopapain of every 200000PU unit.
With chymopapain, auxiliary materials such as L-halfcystine, Propiram are separated with water-soluble, after the aseptic filtration, obtain the chymopapain preparation through freeze drying.Wherein, extract the method for chymopapain, comprise the steps:
1) add chitosan solution in raw material, post precipitation is removed sediment, keeps clear liquid;
2) adding ammonium sulfate in clear liquid is 30%-50% to solution ammonium sulfate saturation degree, removes precipitation, keeps supernatant;
3) in supernatant, add excess tartrate, behind the flocculation sediment, remove precipitation and keep supernatant;
4) adding ammonium sulfate in supernatant is 65-70% to solution ammonium sulfate saturation degree, the collecting precipitation thing;
5) with pure water dissolution precipitation thing, be 1-2 ten thousand daltonian ultra filtration membrane concentrated solutions, collect concentrate with the molecular cut off;
6), collect the eluent of chymopapain with cellulose C-92 chromatographic column separation concentrated solution;
7) be the eluent that 1-2 ten thousand daltonian ultra filtration membranes concentrate chymopapain with the molecular cut off, obtain the chymopapain concentrate.
In order to make the better effects if of separation, in described step 2) supernatant in, add before the excess tartrate, adding ammonium sulfate earlier is 50% to solution ammonium sulfate saturation degree, removes precipitation, the reservation supernatant carries out subsequent step again.
The preparation method of enzyme inhibitor-64 preparation is: sweet mellow wine, trehalose and Propiram in trans-epoxy succinic acyl-L-leucylamino (4-guanidine radicals) butane are separated with water-soluble, after the aseptic filtration, obtained enzyme inhibitor-64 preparation through freeze drying.
Quality control standard according to regulation in " Chinese biological goods rules " carries out hygiene, physics, chemistry, biology to chymopapain kit of the present invention, and aspects such as blood immunology detect, and the result shows that the present invention meets the requirements fully.Can be widely used in human ABO-IgG anti--A, IgG be anti--B, Rh-is anti--D, anti--C, anti--E, anti--c, anti--e, Kell-is anti--K, anti--K, kiad-is anti--JK
a, anti--JK
b, Lewis-resists-Le
a, anti--Le
b, anti--Le
A+b, P-resists-P
1Deng blood group antibody and corresponding blood group antigen carry out screening, the titration of examining and determine, tire; By the erythrocytic cleaning of antibody sandwich; The calibrating of injection biological products immunity blood group antibody.
The present invention is owing to adopted technique scheme, have the following advantages: 1. in external blood group serology calibrating experiment, utilize the hydrolysis modification with chymopapain reagent solution of standardization vigor to erythrocyte membrane of the present invention, make it to possess in the short period of time the corresponding blood group antibody of identification---the ability of immuno agglutination reaction takes place, and overcomes in traditional albumen enzyme one-step experimental technique enzyme to the hydrolysis effect same period of red blood cell, antibody.2. after chymopapain reagent is finished hydrolysis modification to erythrocyte membrane; Use enzyme inhibitor-64 to suppress its enzymatic activity fast, overcome that proteinase reacts erythrocytic excessive property hydrolysis effect-generations " false positive " in the proteinase experiment; And, have the experimental implementation of simplifying process to the hydrolysis effect-generation " false negative " of blood group antibody reaction, improve the security and the sensitivity of calibrating experiment.3. Propiram is a linear macromolecule material, uses as a kind of stablizing-spreading agent, can increase the stability of goods; Form the necessary colloid protection of calibrating experiment, prevent OER haemolysis, stop the generation of " reagent type-false positive " experimental result.4. according to the Technology Need of calibrating experiment different phase, the dedicated solvent that design has corresponding pH, ionic strength has enough sensitivity to guarantee the experiment calibrating.
Embodiment
Embodiment 1, the titration of chymopapain vigor
One, obtain solution
1) 0.067mol/L ph5.5 phosphate solution
Prescription
Potassium dihydrogen phosphate 0.064mol
Sodium hydrogen phosphate 0.003mol
Above-mentioned solute is dissolved in the 900ml pure water, is diluted to the 1000ml constant volume with pure water.
2) sample solution
Get the vigor of indicating to tire 〉=the 400PU/ bottle, 〉=the 1000PU/ bottle, 〉=each 1 bottle in the chymopapain preparation of 2000PU/ bottle, add 2 milliliters, 5 milliliters, 10 milliliters dissolvings of 0.067mol/L ph5.5 phosphate solution, mix.
3) substrate solution
Take by weighing sodium hydrogen phosphate 0.93mol, sodium dihydrogen phosphate 0.06mol uses dissolved in distilled water, is diluted to the 1000ml constant volume.
Take by weighing azo albumin 2.0g, be dissolved in the above-mentioned phosphate solution of 100ml.
Two, chymopapain preparation vigor titration
1) with sample solution, substrate solution is preheated to 37 ± 0.5 ℃.
2) with substrate solution 1.0ml, in the sample solution 100 μ l injecting tubes, mix, put 37 ± 0.5 ℃ of water-baths 10 minutes.
3) add 5% trichloroacetic acid solution 1ml, mix.
4) 1000 * g centrifugal reaction solution is 5 minutes, draws supernatant 1ml.
5) add 0.5mol/L sodium hydroxide solution 1ml, mix.
6) working sample OD value under 430nm.
What under these conditions, hydrolysate produced 0.002 light absorption value at 430nm changes into a vigor potency unit (PU).
Three, the vigor potency unit marker method and the specification of product
The sign of the vigor potency unit of product can be selected arbitrarily from following two kinds of methods.
1, the vigor marker method I that tires
The vigor potency unit that every bottle of enzyme preparation of PU=was had in 1/10 minute.
2, the vigor marker method II that tires
The vigor potency unit that every bottle of enzyme preparation of PU=was had in 10 minutes.
Embodiment 2, the active titration of enzyme inhibitor-64
One, obtain solution
1) 0.067mol/L ph5.5 phosphate solution
Prescription
Potassium dihydrogen phosphate 0.064mol
Sodium hydrogen phosphate 0.003mol
Above-mentioned solute is dissolved in the 900ml pure water, is diluted to the 1000ml constant volume with pure water.
2) chymopapain solution
The vigor of getting is tired to 1 bottle in the chymopapain preparation of 〉=400PU/ bottle, with 2 milliliters of 0.067mol/L ph5.5 phosphate solutions dissolvings, mixes.
3) 0.067mol/L pH7.0 phosphate solution
Prescription
Potassium dihydrogen phosphate 0.027mol
Sodium hydrogen phosphate 0.040mol
Dissolve above-mentioned solute with pure water, be diluted to the 1000ml constant volume.
4) sample solution
Get tire 1 bottle in enzyme inhibitor-64 preparation of 800TU/ bottle, 2000TU/ bottle, 4000TU/ bottle of activity, add 0.067mol/L pH7.0 phosphate solution 2ml, 5ml, 10ml dissolving, mix.
5) substrate solution
Take by weighing sodium hydrogen phosphate 13.2g, sodium hydrogen phosphate 6.8g, use dissolved in distilled water, be diluted to the 1000ml constant volume.
Take by weighing azo albumin 2.0g, be dissolved in the 100ml phosphate solution.
Two, the active titration of enzyme inhibitor-64 preparation
1) with sample solution, chymopapain solution, substrate solution is preheated to 37 ± 0.5 ℃.
2) extracting sample solution 50 μ l in the chymopapain solution 150 μ l injecting tubes, mix, and room temperature was placed 5 minutes.(sample)
3) add substrate solution 1.0ml to above-mentioned test tube, put 37 ± 0.5 ℃ of water-baths 10 minutes.
4) add 5% trichloroacetic acid solution 1ml, mix.
5) 1000 * g centrifugal reaction solution is 5 minutes, draws supernatant 1ml.
6) add 0.5mol/L sodium hydroxide solution 1ml, mix.
7) get chymopapain solution 150 μ l in addition, in the substrate solution 1.0ml injecting tube, mix, by 4)---6) shown in method experimentize.(contrast)
8) working sample OD value under 430nm.
Under these conditions, compared with the control, what sample produced 0.002 extinction value at 430nm changes into an active potency unit of enzyme inhibitor-64 (TU).
Three, the sign of product activity potency unit and specification
The product activity potency unit indicates and can select arbitrarily from following two kinds of methods.
The activity marker method I that tires
The active potency unit that TU=every bottle of enzyme inhibitor-64 preparation was had in 1/10 minute.
The activity marker method II that tires
The active potency unit that TU=every bottle of enzyme inhibitor-64 preparation was had in 10 minutes.
The extraction of embodiment 2, chymopapain
Get the freezing thing of papaya pulp of 1000g, melt, add 1000ml 0.02mol/L phosphate buffer (pH5.5), homogenate in homogenizer with microwave heating.
Add 1% chitosan solution 100ml, homogenate is stirred, and room temperature leaves standstill after 2 hours and filters, and removes precipitation, keeps filtrate.
Add ammonium sulfate to 35% saturation degree in filtrate, room temperature left standstill 60 minutes, centrifugal 20 minutes of 2-8 ℃, 10000 * g, removed precipitation; Add ammonium sulfate to 50% saturation degree in centrifuged supernatant, room temperature left standstill 60 minutes, centrifugal 20 minutes of 2-8 ℃, 10000 * g, removed precipitation; Add excess tartrate solution in supernatant, regulate pH to＜2.5, room temperature left standstill 60 minutes.Centrifugal 20 minutes of 10000 * g discards sediment; Add ammonium sulfate to 70% saturation degree at last again in supernatant, room temperature left standstill 60 minutes, centrifugal 20 minutes of 10000 * g, collecting precipitation thing.
Sediment is dissolved with the 200ml pure water, is 10,000 polysulfone hollow fibre ultra filtration membrane ultrafiltration then with molecular cut off, and gradation adds 2-8 ℃ of water in ultra-filtration process, and loop ultrafiltration 1h under the condition of 0.1MPa obtains ultrafiltration and concentration liquid.
In above-mentioned solution, add 100ml 0.2mol/L sodium phosphate buffer (pH5.5), mixing.With this kaliumphosphate buffer pre-balance cellulose CM-92 chromatographic column, then with the speed of 5ml/min, with the solution upper prop, with 0.7mol/L sodium phosphate buffer (pH5.5) is eluent, collecting the chymopapain eluent, is 10,000 polysulfone hollow fibre ultra filtration membrane concentrate eluant with molecular cut off.
Add Propiram 0.1mmol, L-halfcystine 0.025mol to concentrate, freeze drying obtains the dry thing 30g of chymopapain, and measuring enzyme work with the method for embodiment 1 is 750PU/mg/ minute.
The preparation of embodiment 3, chymopapain kit
One, chymopapain preparation
Prescription:
Propiram 0.05mmol
Sweet mellow wine 0.3mol
Trehalose 0.02mol
Sodium ascorbate 0.02mol
L-halfcystine 0.05mol
Chymopapain 200000PU
Quantitatively take by weighing above-mentioned solute, be dissolved in the pure water, be settled to 1000ml,, be filled to clean cillin bottle by every bottle of 2000PU specification branch then with 0.2 μ m filtering with microporous membrane degerming.Be cooled to-40 ℃ rapidly with freeze drier, kept 4 hours; Start dry drying program then,, frozen product intensification was kept 3 hours for 33 ℃, stop decompression, pack, obtain the chymopapain preparation with the 5 ℃ of speed drying under reduced pressure that per hour heat up.
Two, chymopapain solvent
Prescription:
Potassium dihydrogen phosphate 0.064mol
Sodium hydrogen phosphate 0.003mol
Dissolve above-mentioned solute with pure water, reconcile pH to 5.5, be settled to 1000ml,, divide by every bottle of 10ml then to be filled to polyethylene bottle sealing with 0.2 μ m filtering with microporous membrane degerming.
Three, enzyme inhibitor-64 preparation
Prescription:
Trans-epoxy succinic acyl-L-leucylamino (4-guanidine radicals) butane 40000TU
Propiram 0.05mmol
Sweet mellow wine 0.3mol
Trehalose 0.02mol
Quantitatively take by weighing above-mentioned solute, be dissolved in the pure water, be settled to 1000ml, with 0.2 μ m filtering with microporous membrane degerming.Be filled to clean cillin bottle by every bottle of 4000TU specification branch then.Be cooled to-40 ℃ rapidly with freeze drier, kept 4 hours; Start dry drying program then,, frozen product intensification was kept 3 hours for 33 ℃, stop decompression, pack, obtain finished product enzyme inhibitor-64 preparation with the 5 ℃ of speed drying under reduced pressure that per hour heat up.
Four, enzyme inhibitor-64 solvent
Prescription:
Potassium dihydrogen phosphate 0.027mol
Sodium hydrogen phosphate 0.040mol
Dissolve above-mentioned solute with pure water, reconcile pH to 7.0, be settled to 1000ml,, divide by every bottle of 10ml then to be filled in the polyethylene bottle sealing with 0.2 μ m filtering with microporous membrane degerming.
Chymopapain preparation, chymopapain solvent, enzyme inhibitor-64 preparation, enzyme inhibitor-64 solvent of above-mentioned manufacturing are concentrated and be packaged in the kit, indicate: pawpaw curdled milk protein 20 00PU/ bottle, chymopapain solvent 10ml, enzyme inhibitor-644000TU/ bottle, enzyme inhibitor-64 solvent 10ml, and be equipped with the operation instruction postscript and can dispatch from the factory.
With chymopapain dissolution with solvents chymopapain preparation, form the chymopapain reagent solution before using, tire with 2% azo albumin solution detection vigor.According to the evaluation criterion of external blood group serology experiment calibrating sensitivity, the vigor potency unit that per 50 μ l chymopapain reagent solutions should have is calculated as 10-16PU according to the above-mentioned vigor marker method I that tires; Be calculated as 100-160PU according to the above-mentioned vigor marker method II that tires.
With enzyme inhibitor-64 dissolution with solvents enzyme inhibitor-64 preparation, form enzyme inhibitor-64 reagent solution before using, the azo albumin solution with 2%, chymopapain reagent solution detection of active are tired.According to the evaluation criterion of external blood group serology experiment calibrating security, the active potency unit that the enzyme inhibitor of per 50 μ l-64 reagent solution should have is calculated as 20-32TU by the above-mentioned activity marker method I that tires; Be calculated as 200-320TU by the above-mentioned activity marker method II that tires.
The preparation of embodiment 4, chymopapain kit
Production run is substantially the same manner as Example 3, and just product specification has difference.
One, chymopapain preparation
Prescription:
Propiram 0.048mmol
Sweet mellow wine 0.3mol
Trehalose 0.02mol
Sodium ascorbate 0.02mol
L-halfcystine 0.05mol
Chymopapain 200000PU
Every bottle is filled to clean cillin bottle, freeze drying by 1000PU specification branch.
Two, chymopapain solvent
Prescription:
Potassium dihydrogen phosphate 0.064mol
Sodium hydrogen phosphate 0.003mol
By the packing of every bottle of 5ml specification, sealing.
Three, enzyme inhibitor-64 preparation
Prescription:
Trans-epoxy succinic acyl-L-leucylamino (4-guanidine radicals) butane 40000TU
Propiram 0.01mmol
Sweet mellow wine 0.2mol
Trehalose 0.01mol
By the packing of every bottle of 2000TU specification, freeze drying.
Four, enzyme inhibitor-64 solvent
Prescription:
Potassium dihydrogen phosphate 0.027mol
Sodium hydrogen phosphate 0.040mol
By every bottle of 5ml packing, sealing.
Chymopapain preparation, chymopapain solvent, enzyme inhibitor-64 preparation, enzyme inhibitor-64 solvent of above-mentioned manufacturing are concentrated and be packaged in the kit, indicate: pawpaw curdled milk protein 10 00PU/ bottle, chymopapain solvent 5ml, enzyme inhibitor-64 2000TU/ bottle, enzyme inhibitor-64 solvent 5ml, and be equipped with the operation instruction postscript and can dispatch from the factory.
With chymopapain dissolution with solvents chymopapain preparation, form the chymopapain reagent solution before using, tire with 2% azo albumin solution detection vigor.According to the evaluation criterion of external blood group serology experiment calibrating sensitivity, the vigor potency unit that per 50 μ l chymopapain reagent solutions should have is calculated as 10-16PU according to the above-mentioned vigor marker method I that tires; Be calculated as 100-160PU according to the above-mentioned vigor marker method II that tires.
With enzyme inhibitor-64 dissolution with solvents enzyme inhibitor-64 preparation, form enzyme inhibitor-64 reagent solution before using, the azo albumin solution with 2%, chymopapain reagent solution detection of active are tired.According to the evaluation criterion of external blood group serology experiment calibrating security, the active potency unit that the enzyme inhibitor of per 50 μ l-64 reagent solution should have is calculated as 20-32TU by the above-mentioned activity marker method I that tires; Be calculated as 200-320TU by the above-mentioned activity marker method II that tires.
The preparation of embodiment 5, chymopapain kit
Production run is substantially the same manner as Example 3, and just product specification has difference.
One, chymopapain preparation:
Prescription:
Propiram 0.048mmol
Sweet mellow wine 0.3mol
Trehalose 0.02mol
Sodium ascorbate 0.02mol
L-halfcystine 0.05mol
Chymopapain 200000PU
The packing of every bottle of 400PU specification, freeze drying.
Two, chymopapain solvent
Prescription:
Potassium dihydrogen phosphate 0.064mol
Sodium hydrogen phosphate 0.003mol
By the packing of every bottle of 2ml specification, sealing.
Three, enzyme inhibitor-64 preparation
Prescription:
Trans-epoxy succinic acyl-L-leucylamino (4-guanidine radicals) butane 40000TU
Propiram 0.01mmol
Sweet mellow wine 0.2mol
Trehalose 0.01mol
By the packing of every bottle of 800TU specification, freeze drying.
Four, enzyme inhibitor-64 solvent
Prescription:
Potassium dihydrogen phosphate 0.027mol
Sodium hydrogen phosphate 0.040mol
By the packing of every bottle of 2ml specification, sealing.
Chymopapain preparation, chymopapain solvent, enzyme inhibitor-64 preparation, enzyme inhibitor-64 solvent of above-mentioned manufacturing are concentrated and be packaged in the kit, indicate: pawpaw curdled milk albumen 400PU/ bottle, chymopapain solvent 2ml, enzyme inhibitor-64 800TU/ bottle, enzyme inhibitor-64 solvent 2ml, and be equipped with the operation instruction postscript and can dispatch from the factory.
With chymopapain dissolution with solvents chymopapain preparation, form the chymopapain reagent solution before using, tire with 2% azo albumin solution detection vigor.According to the evaluation criterion of external blood group serology experiment calibrating sensitivity, the vigor potency unit that per 50 μ l chymopapain reagent solutions have calculates and should be 10-16PU according to the above-mentioned vigor marker method I that tires; Be calculated as 100-160PU according to the above-mentioned vigor marker method II that tires.
With enzyme inhibitor-64 dissolution with solvents enzyme inhibitor-64 preparation, form enzyme inhibitor-64 reagent solution before using, the azo albumin solution with 2%, chymopapain reagent solution detection of active are tired.According to the evaluation criterion of external blood group serology experiment calibrating security, the active potency unit that the enzyme inhibitor of per 50 μ l-64 reagent solution should have is calculated as 20-32TU according to the above-mentioned activity marker method I that tires; Be calculated as 200-320TU according to the above-mentioned activity marker method II that tires.
Embodiment 6, utilize kit of the present invention to carry out blood group antibody specificity identification experiment
The step of utilizing kit of the present invention to carry out blood group antibody specificity identification experiment is:
1, get test tube, mark I, II, III, IV, V, VI, VII, VIII, IX, X, XI put test tube rack successively and arrange.
2, add 3% blood group spectrum cell suspension, the 50 μ l that are consistent with its mark to above-mentioned test tube, chymopapain reagent solution 50 μ l mix.
3, test tube being moved into 37 ± 0.5 ℃ of water-baths hatched 5 minutes.
4, respectively add enzyme inhibitor-64 reagent solution 50 μ l to whole test tubes; Respectively add serum specimen 50 μ l to whole test tubes, or red blood cell diffuses liquid 100 μ l, mix.
5, test tube being moved into 37 ± 0.5 ℃ of water-baths hatched 7 minutes.
6, test tube is moved into hydro-extractor, centrifugal 1 minute of 120 * g.
7, shake test tube gently, Red Blood Cells Suspension, visual inspection experimental identification result.According to whether being examined aggegation that serum produces at different blood groups spectrum cells, judge the blood group antibody specificity of being examined serum.
Through adopt room temperature saline experiment, 37 saline experiments, blood group antibody specificity identification experiment method such as antihuman globulin experiment and experimental technique of the present invention be as parallel experiment indirectly, to the people source type Rh-that originates from Immucor anti--D, anti--C, anti--E, anti--c, anti--e, Kell-is anti--K, anti--k, kiad-resist-JK
a, anti--JK
b, Lewi s-is anti--Le
a, anti--Le
b, anti--Le
A+b, P-is anti--P
1Deng blood group antibody serum, and clinical sample serum carries out the antibody specificity evaluation.Experimental result shows that this experimental technique is 100% to the experimental identification accuracy rate of above-mentioned blood group antibody.
Claims (9)
1, a kind of external blood group serology identification kit is basically by chymopapain preparation, chymopapain solvent, enzyme inhibitor-64 and enzyme inhibitor-64 solvent composition; Described chymopapain preparation contains chymopapain, L-halfcystine and Propiram; Described enzyme inhibitor-64 contains trans-epoxy succinic acyl-L-leucylamino (4-guanidine radicals) butane, sweet mellow wine, trehalose and Propiram.
2, external blood group serology identification kit according to claim 1 is characterized in that: described chymopapain solvent is the aqueous solution of 0.02-0.07mol/L potassium dihydrogen phosphate, 0.003-0.04mol/L sodium hydrogen phosphate, pH5-6; Described enzyme inhibitor-64 solvent is the aqueous solution of 0.02-0.07mol/L potassium dihydrogen phosphate, 0.003-0.04mol/L sodium hydrogen phosphate, pH6-7.
3, external blood group serology identification kit according to claim 1 is characterized in that: in the described chymopapain preparation in the chymopapain of every 200000PU unit the content of L-halfcystine and Propiram be respectively 0.025-0.050mol and 0.05-0.1mmol.
4, use kit according to claim 1 or the outer blood group serology calibrating of 2 or 3 described donors experiment, it is characterized in that: also contain sweet mellow wine, trehalose and vitamin C salt in the described chymopapain preparation.
5, kit is used in the outer blood group serology calibrating of donor according to claim 4 experiment, it is characterized in that: in the described chymopapain preparation in the chymopapain of every 200000PU unit the content of sweet mellow wine, trehalose and vitamin C salt be respectively 0.2-0.3mol, 0.01-0.02mol and 0.01-0.02mol.
6, the outer blood group serology calibrating of donor according to claim 4 experiment kit, it is characterized in that: the preparation method of described chymopapain preparation is: chymopapain, L-halfcystine, Propiram, sweet mellow wine, trehalose and vitamin C salt are separated with water-soluble, after the aseptic filtration, obtain chymopapain reagent through freeze drying.
7, kit is used in the outer blood group serology calibrating of donor according to claim 1 experiment, it is characterized in that: every 40000TU unit is trans in the described enzyme inhibitor-64-epoxy succinic acyl-L-leucylamino (4-guanidine radicals) butane in the content of sweet mellow wine, trehalose and Propiram be respectively 0.2-0.3mol, 0.01-0.02mol and 0.05-0.1mol.
8, according to claim 1 or the outer blood group serology calibrating of 2 or 7 described donors experiment kit, it is characterized in that: the preparation method of described enzyme inhibitor-64 is: sweet mellow wine, trehalose and Propiram in trans-epoxy succinic acyl-L-leucylamino (4-guanidine radicals) butane are separated with water-soluble, after the aseptic filtration, obtain enzyme inhibitor-64 reagent through freeze drying.
9, kit is used in the outer blood group serology calibrating of donor according to claim 1 experiment, it is characterized in that: the vigor that the chymopapain reagent solution has is tired and is 1/10 minute 10-16PU/50 μ l or 10 minutes 100-160PU/50 μ l; The activity that enzyme inhibitor-64 reagent solution has is tired and is 1/10 minute 20-32TU/50 μ l or 10 minutes 200-320TU/50 μ l.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410046105 CN1260569C (en) | 2004-05-31 | 2004-05-31 | Reagent box for external blood group and blood serum experimental determination |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410046105 CN1260569C (en) | 2004-05-31 | 2004-05-31 | Reagent box for external blood group and blood serum experimental determination |
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| CN1584596A CN1584596A (en) | 2005-02-23 |
| CN1260569C true CN1260569C (en) | 2006-06-21 |
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| CN 200410046105 Expired - Fee Related CN1260569C (en) | 2004-05-31 | 2004-05-31 | Reagent box for external blood group and blood serum experimental determination |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101851267A (en) * | 2010-04-22 | 2010-10-06 | 江南大学 | Antibody protective agent and application thereof |
| ES2556985T3 (en) | 2011-01-11 | 2016-01-21 | Capsugel Belgium Nv | New hard capsules comprising pululane |
| CN103926415B (en) * | 2014-03-24 | 2015-11-25 | 上海市血液中心 | Blood group antibody associating rapid detection system in human blood |
| CA3059527A1 (en) | 2017-04-14 | 2018-10-18 | Capsugel Belgium Nv | Pullulan capsules |
| BR112019021396A2 (en) | 2017-04-14 | 2020-04-28 | Capsugel Belgium Nv | pullulan manufacturing process |
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