CN1584595A - External blood group and blood serum experimental determining method - Google Patents
External blood group and blood serum experimental determining method Download PDFInfo
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- CN1584595A CN1584595A CN 200410046104 CN200410046104A CN1584595A CN 1584595 A CN1584595 A CN 1584595A CN 200410046104 CN200410046104 CN 200410046104 CN 200410046104 A CN200410046104 A CN 200410046104A CN 1584595 A CN1584595 A CN 1584595A
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Abstract
A method for determining in vitro blood type with serological identification includes mixing chymopapain reagent solution uniformly with red blood cell suspension and hatching the mixture in water both field, microwave field or automatic temperature control oscillator field; adding enzyme inhibitor-64 reagent solution, detected matter and control matter and hatching it in fields as abovementioned, centrifuging 120 xg for one minute; recording detection result.
Description
Technical field
The present invention relates to external blood group serology calibration method, particularly providing a kind of is the external blood group serology experiment calibration method of medium with chymopapain reagent solution, enzyme inhibitor-64 reagent solution.
Background technology
In the external blood group serology of routine-proteinase experiment calibration method, because the thiol protease reagent that uses lacks complete quality arbitration standard, and traditional papain, bromelain single stage method, two step method experimental technique, existence is to cause proteinase calibrating experiment the internal reason of " false positive ", " false negative " experiment verification result to occur to erythrocytic excessive hydrolysis, to the hydrolysis-degradation of blood group antibody.
Summary of the invention
The purpose of this invention is to provide a kind of external accurately blood group serology calibration method.
The method of the outer blood group serology of detection bodies provided by the present invention comprises the steps:
1) chymopapain reagent solution and red cell suspension are mixed, in water-bath field, microwave field or automatic temperature-controlled shaker, hatch; Described chymopapain preparation contains chymopapain, L-halfcystine and Propiram;
2) add enzyme inhibitor-64 reagent solution, add examined object respectively, positive control and negative control thing mix, and hatch in water bath, microwave field or automatic temperature-controlled shaker; Described enzyme inhibitor-64 contains trans-epoxy succinic acyl-L-leucylamino (4-guanidine radicals) butane, sweet mellow wine, trehalose and Propiram;
3) 120 * g is centrifugal 1 minute, the record testing result.
In order to make the better effects if of detection, also contain sweet mellow wine, trehalose and vitamin C salt in the described chymopapain preparation; 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; 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.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 1.
Described water-bath is hatched to hatching 5-10 minute 37-39 ℃ of water-bath; Described microwave is hatched to hatch 200-300 second in the microwave field of setting power 300-360W, output power 30%; Described automatic temperature-controlled shaker is hatched to hatch 5-10 at 37-39 ℃, the automatic temperature-controlled shaker of 300-500r/min, 1-3Level and is divided kind.By thermal effect, finish the hydrolysis modification of chymopapain to erythrocyte membrane, form the absorption-aggegation condition of blood group antigens to blood group antibody.
The red blood cell reagent that the calibrating experiment is used is: blood group spectrum cell suspension beyond the blood group antibody screening red cell suspension beyond the ABO, red blood cell ABO, red blood cells of type A suspension, the Type B red cell suspension, receptor's red cell suspension, blood donor's red cell suspension, patient's red cell suspension.The red blood cell concentration of above-mentioned red blood cell reagent is 2-4%.
The sample thing that calibrating experiment is used is: patient (or receptor) serum sample, and blood donor's serum sample, patient, blood donor IgG be anti--and A, anti--B experiment calibrating use serum sample solution, and red blood cell diffuses liquid; And people source type ABO-IgG-anti--A, anti--B, Rh-is anti--D, anti--C, anti--E, anti--c, anti--e, Kell-is anti--K, anti--k, kiad-be anti--JK
a, anti--JK
b, Lewis-is anti--Le
a, anti--Le
b, anti--Le
A+b, P-is anti--P
1Antibody serum.
In the calibrating experiment, be experiment container as adopting U type porous plate, can adopt scanning microplate reader-blood group interpreting system to read, write down experimental result automatically; With test tube is experiment container, adopts and manually reads, writes down experimental result.
Experiment calibration method provided by the present invention, be used for this ABO-IgG-of human blood sample anti--A, anti--B, Rh-is anti--D, anti--C, anti--E, anti--c, anti--e, Kell-is anti--K, anti--k, kiad-be anti--JK
a, anti--JK
b, Lewis-is anti--Le
a, anti--Le
b, anti--Le
A+b, P-is anti--P
1Deng the calibrating of blood group antibody and corresponding blood group antigen, as the antibody screening originally of clinical blood sample, antibody specificity is identified, antibody titer titration, the anti-A of IgG, anti--B calibrating, blood transfusion compatibility test etc.
The technology that the present invention has overcome traditional proteinase experiment calibrating lacks limit, has improved the security and the sensitivity of experiment calibrating, is in particular in:
1, uses the tailored version vigor to tire and be the chymopapain reagent solution of 10-16PU/50 μ l or 100-160PU/50 μ l, under the experimental temperature-time conditions of regulation, erythrocyte membrane is carried out effective hydrolysis to be modified, exposed blood group antigens acceptor makes it to possess the ability that directly forms visual immune agglutination with corresponding blood group antibody.
2, after chymopapain is finished hydrolysis modification to erythrocyte membrane, use the tailored version activity to tire, suppress the chymopapain vigor fast to enzyme inhibitor-64 reagent solution of 20-32TU/50 μ l or 200-320TU/50 μ l.Prevent organized enzyme to erythrocytic excessive property hydrolysis effect-generation " false positive " experimental result, and organized enzyme is to hydrolysis effect-generation " false negative " experimental result of blood group antibody.Significantly improve the security and the sensitivity of calibrating experiment, fundamentally eliminate the generation of protease reagent " reagent type " experiment calibrating accident in external blood group serology.
3, employed chymopapain reagent solution, enzyme inhibitor-64 reagent solution do not contain protein precipitant, hemagglutinin.Stop the generation of " reagent---false positive " calibrating experimental result.
4, microwave is a kind of ultrashort wave (USW), under the effect of the thermal effect of microwave, chemical effect, biological effect, chymopapain is modified speed to the red blood cell hydrolysis and is accelerated, enzyme inhibitor-64 is accelerated with the speed that biological sulfydryl forms compound, be that significantly the reduction experiment is consuming time, keep the important experimental technique of experiment calibrating security and sensitivity.
5, core technology of the present invention is: utilize chymopapain that red blood cell is carried out hydrolysis effectively and modify, enzyme inhibitor-64 makes that finishing loses vigor fast to the chymopapain after the red blood cell hydrolysis modification tires, can expand in a nearly step and be enzyme pre-service red blood cell reagent, prepare 3% enzyme A type, Type B red blood cell reagent, blood group antibody screening red cell suspension beyond the 3% enzyme ABO is carried out the screening of non-brine media blood group antibody, identification experiment to adapt to the Blood Center mass.
Embodiment
Experiment material
1, cell material
3% is examined red cell suspension, 3% receptor's red cell suspension, 3% blood donor's red cell suspension, the positive red cell suspension of 3%Rho (D), the negative red cell suspension of 3%Rho (D), 3%A type red cell suspension, 3%B type red cell suspension, 3% screening red cell suspension, 3% blood group spectrum cell suspension.
3% is examined red cell suspension in the above-mentioned cell material, and 3% receptor's red cell suspension, 3% blood donor's red cell suspension are taken from the hospital blood bank blood sample originally.Other cell reagent originates from Immucor.
2, serum material
Patient, blood donor's serum specimen, red blood cell diffuses liquid, and patient, blood donor IgG resist-A, anti--B experiment calibrating serum sample solution; People source type ABO-IgG-is anti--A, anti--B, Rh-is anti--D, anti--C, anti--E, anti--c, anti--e, Kell-is anti--K, anti--k, kiad-be anti--JK
a, anti--JK
b, Lewis-is anti--Le
a, anti--Le
b, anti--Le
A+b, P-is anti--P
1Deng the antibody serum sample, and the AB serum specimen.
Patients serum's sample in the above-mentioned serum material, blood donor's serum sample, patient, receptor IgG be anti--and A, anti--B experiment calibrating use serum sample solution, and red blood cell diffuses liquid etc., takes from the hospital blood bank blood sample originally.Other serum material producing is from Immucor.
The extraction of embodiment 1, chymopapain and preparation
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 0.1 MPa 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-C92 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 20g of chymopapain.
The preparation of embodiment 2, chymopapain kit
1, chymopapain preparation
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 diluted to the 1000ml constant volume,, 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 drying program then,, frozen product intensification was kept 3 hours for 33 ℃, stop decompression, finished product is packed, obtain finished product chymopapain preparation with the 5 ℃ of speed drying under reduced pressure that per hour heat up.
2, chymopapain solvent
Potassium dihydrogen phosphate 0.064mol
Sodium hydrogen phosphate 0.003mol
Dissolve above-mentioned solute with pure water, reconcile pH to 5.5, be diluted to the 1000ml constant volume,, be filled to polyethylene bottle by every bottle of 10ml specification branch then, sealing with 0.2 μ m filtering with microporous membrane degerming.
With chymopapain dissolution with solvents chymopapain preparation, form the chymopapain reagent solution before using, detect the vigor of chymopapain reagent solution with 2% even egg albumin-phosphate solution and tire.
According to the evaluation criterion of external blood group serology experiment calibrating sensitivity, it is 10-16PU that the vigor that the chymopapain reagent solution of per 50 μ l should have is tired according to following identification method I; According to following identification method II is 100-160PU.
The vigor vigor that the per 50 μ l reagent solutions of identification method I:PU=were had in 1/10 minute of tiring is tired.
The vigor vigor that the per 50 μ l reagent solutions of identification method II:PU=were had in 10 minutes of tiring is tired.
3, enzyme inhibitor-64 preparation
Trans-epoxy succinic acyl-L-leucylamino (4-guanidine radicals) butane 400000TU
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 diluted to the 1000ml constant volume, 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, finished product is packed, obtain enzyme inhibitor-64 preparation with the 5 ℃ of speed drying under reduced pressure that per hour heat up.
4, enzyme inhibitor-64 solvent
Potassium dihydrogen phosphate 0.027mol
Sodium hydrogen phosphate 0.040mol
Dissolve above-mentioned solute with pure water, reconcile pH to 7.0, be diluted to the 1000ml constant volume,, be filled in the polyethylene bottle by every bottle of 10ml specification branch then, sealing with 0.2 μ m filtering with microporous membrane degerming.
With enzyme inhibitor-64 dissolution with solvents enzyme inhibitor-64 preparation, form enzyme inhibitor-64 reagent solution before using.
With the chymopapain reagent that 2% even egg albumin-phosphate solution, 30PU/150 μ l/min or 300PU/150 μ l vigor are tired, detect enzyme inhibitor-64 reagent solution activity and tire.
According to the evaluation criterion of external blood group serology experiment calibrating security, it is 20-32TU that the activity that the enzyme inhibitor of per 50 μ l-64 reagent solution should have is tired according to identification method I; According to identification method II is 200-320TU.
The activity activity that the per 50 μ l reagent solutions of identification method I:TU=were had in 1/10 minute of tiring is tired.
The activity activity that the per 50 μ l reagent solutions of identification method II:TU=were had in 10 minutes of tiring is tired.
Embodiment 3 preparation enzyme reagent red blood cells
1) 3% red cell suspension, chymopapain reagent solution are put water-bath and be preheated to 37 ± 0.5 ℃.
2) with isopyknic 3% reagent red blood cell suspension, the chymopapain reagent solution mixes, and forms experimental system, moves into 37 ± 0.5 ℃ of water-baths and hatches 5 minutes.
3) add enzyme inhibitor-64 reagent solution of 0.5 volume to experimental system, mix, room temperature was placed 3 minutes.
4) experimental system is moved into hydro-extractor, centrifugal 1 minute of 120 * g; 1000 * g centrifugal 30 seconds are with physiological saline washing, preparation packed red cells.
5) the long-pending red blood cell of pressure prepares 3% red cell suspension with physiological saline.
6) the mentioned reagent red blood cell can be A type, Type B, O type red blood cell, screening red blood cell, erythrocyte blood type spectrum cell.
With the enzyme red blood cell of method for preparing, can routine be used for non-brine media blood group antibody calibrating experiment, significantly improve work efficiency.
4 couples of embodiment are by the erythrocytic cleaning of antibody sandwich
Under special pathological conditions, patient's autoantibody and its red blood cell have more normal affinity, the antibody that can't dissociate and be adsorbed with the conventional method of diffusing, thus influence the evaluation of erythrocyte blood type.That adopts that following method can make the patient redly reaches desirable cleaning performance, carries out the evaluation of erythrocyte blood type smoothly.
1) 1 processed volume red blood cell is prepared 3% red cell suspension with physiological saline, be preheated to 37 ± 0.5 ℃ respectively with the chymopapain reagent solution of 1 volume.
2) above-mentioned red cell suspension, chymopapain reagent solution are mixed, move into 37 ± 0.5 ℃ of water-baths and hatched 5 minutes.
3) enzyme inhibitor-64 reagent solution of adding 1 volume mixes, and room temperature was placed 3 minutes.
4) experimental system is moved into hydro-extractor, centrifugal 1 minute of 120 * g; 1000 * g centrifugal 30 seconds.With physiological saline washing 3 times, prepare 3% red cell suspension, carry out erythrocyte blood type and identify.
Use said method, diffuse method for the contrast experiment, 100 routine autoimmune hemolytic anemia patients' red blood cell is carried out washing test with routine.Success ratio is 100%, " false positive " result do not occur.
Embodiment 5 blood group antibody screenings
One, test tube-water-bath method
1, gets 5 test tubes, mark positive control, negative control, examined 1, examined 2, examined 3.
2, the heliotropism contrast adds the positive red cell suspension 50 μ l of 3%Rho (D); Add the positive red cell suspension 50 μ l of 3%Rho (D) to negative control; Added 3%I screening red cell suspension 50 μ l to examining 1, added 3%II screening red cell suspension 50 μ l, added 3%III screening red cell suspension 50 μ l to examining 3 to examining 2; Respectively add chymopapain reagent solution 50 μ l to whole test tubes, mix.
3, test tube being moved into 37 ± 0.5 ℃ of water-baths hatched 5 minutes.Respectively add enzyme inhibitor-64 reagent solution 50 μ l to whole test tubes; The heliotropism control tube adds IgG and resists-D serum 50 μ l; Add AB serum 50 μ l to the negative control pipe; Xiang Shoujian pipe 1-is subjected to inspection pipe 3 respectively to add serum specimen 50 μ l, or red blood cell diffuses liquid 100 μ l, mixes.
4, test tube being moved into 37 ± 0.5 ℃ of water-bath fields hatched 7 minutes.
5, test tube is moved into hydro-extractor, centrifugal 1 minute of 120 * g.
6, shake test tube gently, Red Blood Cells Suspension, visual inspection experiment verification result.
7, positive control produces red cell agglutination, and negative control does not produce red cell agglutination, and experimental result is set up.Examined pipe and red cell agglutination occurs and be experiment calibrating positive findings, be subjected to the inspection pipe red cell agglutination not occur and be real calibrating negative findings.
Adopting indirect antihuman globulin blood group antibody screening experimental technique and experimental technique of the present invention is parallel experiment, and consistent screening goes out 4 routine positive blood samples in the serum sample to 2000 routine blood donors, does not have " false negative ", " false positive " experimental result.
To 4 routine positive blood samples, adopt room temperature salt solution, 37 ℃ of salt solution, blood group antibody specificity identification experiment method such as antihuman globulin, and embodiment 6 described blood group antibody specificity identification experiment methods indirectly, carry out antibody specificity and identify.Qualification result is consistent to be confirmed, 3 examples for IgG anti--D, 1 example be for resisting-Le
a
Two, test tube-microwave method
1, goes on foot with the 1st in the water-bath method.
2, go on foot with the 2nd in the water-bath method.
3, test tube is moved into microwave field, at power level 360W, under the condition of power output rating 30%, radiation hatched for 200 seconds.
4, go on foot with the 4th in the water-bath method.
5, test tube is moved into microwave field, at power level 360W, under the condition of power output rating 30%, radiation hatched for 300 seconds.
6, go on foot with the 6th in the water-bath method.
7, go on foot with the 7th in the water-bath method.
Adopting above-mentioned test tube-water-bath antibody screening experimental technique, indirect antihuman globulin blood group antibody screening experimental technique and experimental technique of the present invention is parallel experiment, consistent screening goes out out 4 routine positive blood samples in the serum sample to 2000 routine blood donors, does not have " false negative ", " false positive " experimental result.
To 4 routine positive blood samples, adopt room temperature salt solution, 37 ℃ of salt solution, blood group antibody specificity identification experiment method such as antihuman globulin, and embodiment 6 described blood group antibody specificity identification experiment methods indirectly, carry out antibody specificity and identify.Qualification result is consistent to be confirmed, 3 examples for IgG anti--D, 1 example be for resisting-Le
a
Three, U type porous plate-automatic temperature-controlled concussion instrumental method
1, gets U type porous plate, mark positive control hole, negative control hole, be subjected to verify 1, be subjected to verify 2, be subjected to verify 3.
2, the heliotropism control wells adds the positive red cell suspension 50 μ l of 3%Rho (D); Add the positive red cell suspension 50 μ l of 3%Rho (D) to negative control hole; To being subjected to verify 1 to add 3%I screening red cell suspension 50 μ l,, added 3%III screening red cell suspension 50 μ l to examining 3 holes to being subjected to verify 2 to add 3%II screening red cell suspension 50 μ l; Add chymopapain reagent solution 50 μ l to each experimental port, mix.
3, will test porous plate and move in the automatic temperature-controlled shaker, under 37 ℃, 300r/min, 1Level condition, hatch 7 minutes.
4, add enzyme inhibitor-64 reagent solution 50 μ l to each experimental port, the heliotropism control wells adds IgG and resists-D serum 50 μ l; Add AB serum 50 μ l to negative control hole; Each adds and is examined serum 50 μ l to being subjected to verify 1-to be subjected to verify 3, or red blood cell diffuses liquid 100 μ l, mixes.
5, will test porous plate and move in the automatic temperature-controlled shaker, under 37 ℃, 300r/min, 1Level condition, hatch 7 minutes.
6, experimental system is moved into hydro-extractor, centrifugal 1 minute of 120 * g.
7, experimental system is moved in the automatic temperature-controlled shaker, at 1000r/min, 2Level, 1 minute condition of work low suspension red blood cell.
8, with the scanning microplate reader-the blood group interpreting system reads, writes down experimental result automatically.
9, the positive control hole produces red cell agglutination, and negative control hole does not produce red cell agglutination, and experimental result is set up.Be subjected to verify red cell agglutination to occur and be experiment calibrating positive findings, be subjected to verify red cell agglutination not occur and be real calibrating negative findings.
Adopting indirect antihuman globulin blood group antibody screening experimental technique and U type porous plate of the present invention-automatic temperature-controlled concussion instrument blood group antibody screening experimental technique is parallel experiment, consistent screening goes out 4 routine positive blood samples in the serum sample to 2000 routine blood donors, does not have " false negative ", " false positive " experimental result.
To 4 routine positive blood samples, adopt room temperature salt solution, 37 ℃ of salt solution, blood group antibody specificity identification experiment method such as antihuman globulin, and embodiment 6 described blood group antibody specificity identification experiment methods indirectly, carry out antibody specificity and identify.Qualification result is consistent to be confirmed, 3 examples for IgG anti--D, 1 example be for resisting-Le
a
Four, U type porous plate-microwave-automatic temperature-controlled concussion instrumental method
1, goes on foot with the 1st in U type porous plate-automatic temperature-controlled concussion instrumental method.
2, go on foot with the 2nd in U type porous plate-automatic temperature-controlled concussion instrumental method.
3, will test U type porous plate and move into microwave field, at power level 360W, under the condition of power output rating 30%, radiation hatched for 200 seconds.
4, go on foot with the 4th in U type porous plate-automatic temperature-controlled concussion instrumental method.
5, will test U type porous plate and move into microwave field, at power level 360W, under the condition of power output rating 30%, radiation hatched for 200 seconds.
6, go on foot with the 6th in U type porous plate-automatic temperature-controlled concussion instrumental method.。
7, go on foot with the 7th in U type porous plate-automatic temperature-controlled concussion instrumental method.。
8, go on foot with the 8th in U type porous plate-automatic temperature-controlled concussion instrumental method.
9, go on foot with the 9th in U type porous plate-automatic temperature-controlled concussion instrumental method.
Adopting above-mentioned U type porous plate-automatic temperature-controlled concussion instrument blood group antibody screening experimental technique, indirect antihuman globulin blood group antibody screening experimental technique and experimental technique of the present invention is parallel experiment, consistent screening goes out out 4 routine positive blood samples in the serum sample to 2000 routine blood donors, does not have " false negative ", " false positive " experimental result.
To 4 routine positive blood samples, adopt room temperature salt solution, 37 ℃ of salt solution, blood group antibody specificity identification experiment method such as antihuman globulin, and embodiment 6 described blood group antibody specificity identification experiment methods indirectly, carry out antibody specificity and identify.Qualification result is consistent to be confirmed, 3 examples for IgG anti--D, 1 example be for resisting-Le
a
Embodiment 6 blood group antibody specificity identification experiments (test tube-water-bath method)
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, Lewis-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.
The embodiment 7 blood group antibodies titration experiments (test tube-water-bath method) of tiring
1, gets 10 test tubes, mark 1/1,1/2,1/4,1/8,1/16,1/32,1/64,1/128,1/256,1/512; Respectively add physiological saline 100 μ l to 1/2-1/512; Add titrated blood group antibody serum 200 μ l to 1/1.
2, draw serum 100 μ l with sample injector from 1/1, inject 1/2,, mix with sample injector suction repeatedly.Method prepares blood group antibody serum doubling dilution liquid so.
3, get 11 test tubes, mark 1/1,1/2,1/4,1/8,1/16,1/32,1/64,1/128,1/256,1/512, negative control.Each adds positive 3% red cell suspension, the 50 μ l of the homozygote blood group antigens that are complementary with titrated blood group antibody to above-mentioned test tube, and chymopapain reagent solution 50 μ l mix.
4, test tube being moved into 37 ± 0.5 ℃ of water-baths hatched 5 minutes.
5, respectively add enzyme inhibitor-64 reagent solution 50 μ l to whole test tubes; Add the blood group antibody serum doubling dilution liquid 100 μ l be consistent with its mark to the blood group antibody titration test tube of tiring; Add AB serum 50 μ l to negative control, mix.
6, test tube being moved into 37 ± 0.5 ℃ of water-baths hatched 7 minutes.
7, test tube is moved into hydro-extractor, centrifugal 1 minute of 120 * g.
8, shake test tube gently, Red Blood Cells Suspension, visual inspection titration experiments result.Negative control does not have red cell agglutination, and experimental result is set up.The inverse of the minimum aggegation intensity (1+) that red blood cell is produced with the blood group antibody serum of highly diluted multiple is tiring for titrated antibody.
Through adopting indirect antihuman globulin blood group antibody to tire titration experiments method and experimental technique of the present invention as parallel experiment, 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-is anti--JK
a, anti--JK
b, Lewis-resists-Le
a, anti--Le
b, anti--Le
A+b, P-resists-P
1Carry out the antibody titer titration Deng blood group antibody serum and clinical serum sample.Experimental result shows that experimental technique of the present invention is compared with indirect antihuman globulin antibody titer titration experiments method, and average sensitivity exceeds 2-3 the order of magnitude.
Embodiment 8 IgG resist-A, anti--blood group antibody B calibrating experiment
One, test tube-water-bath method
1, get serum, add equivalent PBS-2me solution, 37 ± 0.5 ℃ of insulations 1 hour, preparation IgG is anti--A, anti--B experiment calibrating serum sample solution.
Get IgG anti--A, anti--B experiment calibrating diffuse liquid with serum sample solution or red blood cell, with doubling dilution method preparation 1/1,1/2,1/4,1/8,1/16,1/32,1/64,1/128,1/256,1/512 doubling dilution liquid, as being examined sample doubling dilution liquid.
2, get 24 test tubes, mark A1/1, A1/2, A1/4, A1/8, A1/16, A1/32, A1/64, A1/128, A1/256, A1/512 are as being examined A, A positive control, A negative control; B1/1, B1/2, B1/4, B1/8, B1/16, B1/32, B1/64, B1/128, B256, B1/512 are as being examined B, and B positive control, B negative control are put test tube rack successively and arranged.
3, add 3%A type red cell suspension 50 μ l to the A1/1-A negative control; Add 3%B type red cell suspension 50 μ l to the B1/1-B negative control; Add chymopapain reagent solution 50 μ l to every test tube, mix.
4, test tube being moved into 37 ± 0.5 ℃ of water-baths hatched 5 minutes.
5, respectively add enzyme inhibitor-64 reagent solution 50 μ l to whole test tubes; To A1/1-A1/512 add 1/1-1/512 add be consistent with its mark number examined sample doubling dilution liquid 100 μ l, to the A positive control add IgG anti--A serum 50 μ l, to A negative control adding AB serum 50 μ l; Add the 1/1-1/512 that is consistent with its mark number to B1/1-B1/512 and examined sample doubling dilution liquid 100 μ l, add IgG to the B positive control and resist-B serum 50 μ l, add AB serum 50 μ l, mix to the B negative control.
6, test tube being moved into 37 ± 0.5 ℃ of water-bath fields hatched 7 minutes.
7, test tube is moved into hydro-extractor, centrifugal 1 minute of 120 * g.
8, shake test tube gently, Red Blood Cells Suspension, visual inspection experiment verification result.
9, positive control produces red cell agglutination, and negative control does not produce red cell agglutination, and experimental result is set up.Examined A and red cell agglutination occurs and be experiment calibrating positive findings, examined A and red cell agglutination do not occur and be real calibrating negative findings; Examined B and red cell agglutination occurs and be experiment calibrating positive findings, examined A and red cell agglutination do not occur and be real calibrating negative findings.With the inverse of being examined the minimum aggegation intensity (1+) that sample produces red blood cell of highly diluted multiple is tiring of titrated antibody.
Adopt indirect antihuman globulin IgG anti--A, anti--blood group antibody B calibrating experimental technique and calibrating experimental technique of the present invention are parallel experiment, in the serum sample to 2000 routine O type blood blood donors consistent examine and determine out 612 routine IgG anti--A, 1106 routine IgG are anti--B, do not have " false negative ", " false positive " experimental result.
IgG of the present invention is anti--and 612 examples that A, anti--blood group antibody B calibrating result of experiment shows are anti--A, 1106 routine IgG are anti--the antihuman globulin IgG that tires indirectly of B is anti--and A, anti--blood group antibody B calibrating result of experiment exceed 2-3 the order of magnitude.
Two, test tube-microwave method
1, the step 1 of same test tube-water-bath method.
2, the step 2 of same test tube-water-bath method.
3, the step 3 of same test tube-water-bath method.
4, test tube is moved into microwave field, at power level 360W, under the condition of power output rating 30%, radiation hatched for 200 seconds.
5, the step 5 of same test tube-water-bath method.
6, test tube is moved into microwave field, at power level 360W, under the condition of power output rating 30%, radiation hatched for 300 seconds.
7, the step 7 of same test tube-water-bath method.
8, the step 8 of same test tube-water-bath method.
9, the step 9 of same test tube-water-bath method.
Adopt above-mentioned test tube-water-bath IgG anti--A, anti--blood group antibody B calibrating experimental technique, indirectly antihuman globulin IgG anti--A, anti--blood group antibody B calibrating experimental technique, with IgG of the present invention anti--A, anti--blood group antibody B calibrating experimental technique are parallel experiment, in the serum sample to 2000 routine O type blood blood donors consistent examine and determine out 612 routine IgG anti--A, 1106 routine IgG are anti--B, do not have " false negative ", " false positive " experimental result.
IgG of the present invention is anti--and 612 examples that A, anti--blood group antibody B calibrating result of experiment shows are anti--A, 1106 routine IgG are anti--the antihuman globulin IgG that tires indirectly of B is anti--and A, anti--blood group antibody B calibrating result of experiment exceed 2-3 the order of magnitude.Resist with above-mentioned test tube-water-bath IgG-A, resist-blood group antibody B calibrating result of experiment no significant difference.
Three, U type porous plate-automatic temperature-controlled concussion instrument method
1, the step 1 of same test tube-water-bath method.
2, get porous plate, mark A1/1, A1/2, A1/4, A1/8, A1/16, A1/32, A1/64, A1/128, A1/256, A1/512 are as being examined A, A positive control, A negative control; B1/1, B1/2, B1/4, B1/8, B1/16, B1/32, B1/64, B1/128, B256, B1/512 are as being examined B, B positive control, B negative control.
3, respectively add 3%A type red cell suspension 50 μ l to the A1/1-A negative control; Add 3%B type red cell suspension 50 μ l to the B1/1-B negative control; Add chymopapain reagent solution 50 μ l to per 1 experimental port, mix.
4, will test porous plate and move in the automatic temperature-controlled shaker, under 37 ℃, 300r/min, 1Level condition, hatch 7 minutes.
5, add enzyme inhibitor-64 reagent solvent 50 μ l to per 1 experimental port, to A1/1-A1/512 add 1/1-1/512 add be consistent with its mark number examined sample doubling dilution liquid 100 μ l, or red blood cell diffuses liquid 100 μ l, add IgG to the A positive control and resist-A serum 50 μ l, add AB serum 50 μ l to the A negative control; To B1/1-B1/512 add be consistent with its mark number examined sample doubling dilution liquid 100 μ l, or red blood cell diffuses liquid 100 μ l, to the B positive control add IgG anti--B serum 50 μ l, to B negative control adding AB serum 50 μ l, mix.
6, will test porous plate and move in the automatic temperature-controlled shaker, under 37 ℃, 300r/min, 1Level condition, hatch 7 minutes.
7, porous plate be will test and hydro-extractor, centrifugal 1 minute of 120 * g moved into.
8, with the scanning microplate reader-the blood group interpreting system reads, writes down experimental result automatically.
9, positive control produces red cell agglutination, and negative control does not produce red cell agglutination, and experimental result is set up.Examined A and red cell agglutination occurred, examined A and the negative experiment verification result of red cell agglutination do not occur for experiment calibrating positive findings; Examined B and the positive experiment verification result of red cell agglutination occurred, examined B and the negative experiment verification result of red cell agglutination do not occur.Positive experiment verification result is tiring of antibody with the inverse of being examined the minimum aggegation intensity (1+) that sample doubling dilution liquid produces red blood cell of highly diluted multiple.
Adopt indirect antihuman globulin IgG anti--A, anti--blood group antibody B calibrating experimental technique and IgG of the present invention be anti--to examine and determine experimental technique be parallel experiment for A, anti--blood group antibody B, in serum sample to 2000 routine O type blood blood donors consistent examine and determine out 612 routine IgG anti--A, 1106 routine IgG are anti--B, do not have " false negative ", " false positive " experimental result.
IgG of the present invention is anti--and 612 examples that A, anti--blood group antibody B calibrating result of experiment shows are anti--A, 1106 routine IgG are anti--the antihuman globulin IgG that tires indirectly of B is anti--and A, anti--blood group antibody B calibrating result of experiment exceed 2-3 the order of magnitude.
Four, U type porous plate-microwave-automatic temperature-controlled concussion instrument method
1, with the step 1 in U type porous plate-automatic temperature-controlled concussion instrument method.
2, with the step 2 in U type porous plate-automatic temperature-controlled concussion instrument method.
3, with the step 3 in U type porous plate-automatic temperature-controlled concussion instrument method.
4, will test porous plate and move into microwave field, at power level 360W, under the condition of power output rating 30%, radiation hatched for 200 seconds.
5, with the step 5 in U type porous plate-automatic temperature-controlled concussion instrument method.
6, will test porous plate and move into microwave field, at power level 360W, under the condition of power output rating 30%, radiation hatched for 300 seconds.
7, with the step 7 in U type porous plate-automatic temperature-controlled concussion instrument method.
8, with the step 8 in U type porous plate-automatic temperature-controlled concussion instrument method.
9, with the step 9 in U type porous plate-automatic temperature-controlled concussion instrument method.
Adopt above-mentioned U type porous plate-automatic temperature-controlled concussion instrument IgG anti--A, anti--blood group antibody B calibrating experimental technique, indirectly antihuman globulin IgG anti--A, anti--blood group antibody B calibrating experimental technique, with IgG of the present invention anti--A, anti--blood group antibody B calibrating experimental technique are parallel experiment, in serum sample to 2000 routine O type blood blood donors consistent examine and determine out 612 routine IgG anti--A, 1106 routine IgG are anti--B, do not have " false negative ", " false positive " experimental result.
IgG of the present invention is anti--and 612 examples that A, anti--blood group antibody B calibrating result of experiment shows are anti--A, 1106 routine IgG are anti--the antihuman globulin IgG that tires indirectly of B is anti--and A, anti--blood group antibody B calibrating result of experiment exceed 2-3 the order of magnitude.Resist with U type porous plate-microwave-automatic temperature-controlled concussion instrument method IgG-A, resist-blood group antibody B calibrating experiment gained experimental result no significant difference.
Embodiment 9 blood transfusion compatibility experiments
One, test tube-water-bath method
1, gets 4 test tubes, mark master, inferior side, positive control, negative control.Add 3% blood donor's red cell suspension, 50 μ l to master, add 3% receptor's red cell suspension, 50 μ l to inferior side; Positive control, negative control respectively add the positive red cell suspension 50 μ l of 3%Rho (D), respectively add chymopapain reagent solution 50 μ l to whole test tubes, mix.
2, test tube being moved into 37 ± 0.5 ℃ of water-baths hatched 5 minutes.
3, respectively add enzyme inhibitor-64 reagent solution 50 μ l to whole test tubes; Add receptor's serum 50 μ l to master; Add blood donor's serum 50 μ l to inferior side, the heliotropism contrast adds IgG and resists-D serum 50 μ l; Adding AB serum 50 μ l to negative control mixes.
4, test tube being moved into 37 ± 0.5 ℃ of water-baths hatched 7 minutes.
5, test tube is moved into hydro-extractor, centrifugal 1 minute of 120 * g.
6, shake test tube gently, Red Blood Cells Suspension, visual inspection experimental result.Positive control produces red cell agglutination, and negative control does not produce red cell agglutination, and experimental result is set up.Master, inferior side do not have red cell agglutination and the negative experimental result of haemolysis, and red cell agglutination and the positive experimental result of haemolysis appear in master or inferior side.
Adopt indirect antihuman globulin blood transfusion compatibility experimental technique, with experimental technique of the present invention be parallel experiment, the consistent 2 routine positive samples of examining and determine out in to 1150 example blood transfusion compatibilities experiments do not have " false negative ", " false positive " experimental result.
Above-mentioned positive sample is adopted room temperature salt solution, 37 ℃ of salt solution, blood group antibody specificity identification experiment method such as antihuman globulin, and embodiment 6 described antibody specificity identification experiment methods indirectly, carry out antibody specificity and identify.Similar experiment verification result is consistent to be confirmed, wherein 1 example resists-E for IgG, and 1 example is anti--K.
Two, test tube-microwave method
1, the step of the 1st in same test tube-water-bath method.
2, test tube is moved into microwave field, at power level 360W, under the condition of power output rating 30%, radiation hatched for 200 seconds.
3, the step of the 3rd in same test tube-water-bath method.
4, test tube is moved into microwave field, at power level 360W, under the condition of power output rating 30%, radiation hatched for 300 seconds.
5, the step of the 5th in same test tube-water-bath method.
6, the step of the 6th in same test tube-water-bath method.。
Adopt indirect antihuman globulin blood transfusion compatibility experimental technique, above-mentioned test tube-water-bath blood transfusion compatibility experimental technique, with experimental technique of the present invention be parallel experiment, the consistent 2 routine positive samples of examining and determine out in to 1150 example blood transfusion compatibility experiments do not have " false negative ", " false positive " experimental result.
Above-mentioned positive sample is adopted room temperature salt solution, 37 ℃ of salt solution, blood group antibody specificity identification experiment method such as antihuman globulin, and embodiment 6 described antibody specificity identification experiment methods indirectly, carry out antibody specificity and identify.Similar experiment verification result is consistent to be confirmed, wherein 1 example resists-E for IgG, and 1 example is anti--K.
Three, U type porous plate-automatic temperature-controlled concussion instrument method
1, gets the U porous plate, mark master hole, inferior side opening, positive control hole, negative control hole.Add 3% blood donor's red cell suspension, 50 μ l to the master hole, add 3% receptor's red cell suspension, 50 μ l to inferior side opening; Heliotropism control wells, negative control hole respectively add the positive red cell suspension 50 μ l of 3%Rho (D), respectively add chymopapain reagent solution 50 μ l to whole experimental ports, mix.
2, U is tested porous plate and move in the automatic temperature-controlled shaker, under 37 ℃, 300r/min, 1Level condition, hatched 7 minutes.
3, respectively add enzyme inhibitor-64 reagent solution 50 μ l to whole experimental ports; Add receptor's serum 50 μ l to the master hole; Add blood donor's serum 50 μ l to inferior side opening, the heliotropism control wells adds IgG and resists-D serum 50 μ l; Adding the hole to negative control goes into AB serum 50 μ l and mixes.
4, U is tested porous plate and move in the automatic temperature-controlled shaker, under 38 ± 0.5 ℃, 300r/min, 1Level condition, hatched 7 minutes.
5, porous plate be will test and hydro-extractor, centrifugal 1 minute of 120 * g moved into.
6, will test porous plate and go in the automatic temperature-controlled shaker, at 1000r/min, 2Level, 1 minute condition of work low suspension red blood cell.
7, with the scanning microplate reader-the blood group interpreting system reads, writes down experimental result automatically.
8, the positive control hole produces red cell agglutination, and negative control hole does not produce red cell agglutination, and experimental result is set up.The positive experimental result of red cell agglutination appears in master hole, inferior side opening, and the negative experimental result of red cell agglutination does not appear in master hole, inferior side opening.
Adopt indirect antihuman globulin blood transfusion compatibility experimental technique, with blood transfusion compatibility experimental technique of the present invention be parallel experiment, the consistent 2 routine positive samples of examining and determine out in to 1150 example blood transfusion compatibilities experiments do not have " false negative ", " false positive " experimental result.
Above-mentioned positive sample is adopted room temperature salt solution, 37 ℃ of salt solution, blood group antibody specificity identification experiment method such as antihuman globulin, and embodiment 6 described antibody specificity identification experiment methods indirectly, carry out antibody specificity and identify.Verification result is consistent confirms in experiment, wherein 1 example for IgG anti--E, 1 routine be to resist-K.
Four, U type porous plate-microwave-automatic temperature-controlled concussion instrument method
1, the step of the 1st in U type porous plate-automatic temperature-controlled concussion instrument method.
2, U type experiment porous plate is moved into microwave field, at power level 360W, under the condition of power output rating 30%, radiation hatched for 200 seconds.
3, the 3rd of U type porous plate-automatic temperature-controlled concussion instrument method the step.
4, U type experiment porous plate is moved into microwave field, at power level 360W, under the condition of power output rating 30%, radiation hatched for 300 seconds.
5, the step of the 5th in U type porous plate-automatic temperature-controlled concussion instrument method.
6.U the step of the 6th in type porous plate-automatic temperature-controlled concussion instrument method.
7, the step of the 7th in U type porous plate-automatic temperature-controlled concussion instrument method.
8, the step of the 8th in U type porous plate-automatic temperature-controlled concussion instrument method.
Adopt indirect antihuman globulin blood transfusion compatibility experimental technique, U type porous plate-automatic temperature-controlled concussion instrument blood transfusion compatibility experimental technique, with experimental technique of the present invention be parallel experiment, the consistent 2 routine positive samples of examining and determine out in to 1150 example blood transfusion compatibility experiments do not have " false negative ", " false positive " experimental result.
Above-mentioned positive sample is adopted room temperature salt solution, 37 ℃ of salt solution, blood group antibody specificity identification experiment method such as antihuman globulin, and embodiment 6 described antibody specificity identification experiment methods indirectly, carry out antibody specificity and identify.Verification result is consistent confirms in experiment, wherein 1 example for IgG anti--E, 1 routine be to resist-K.
The IgG-of the low pH Imnunoglobulin injections of embodiment 10 is anti--and A, anti--B examine and determine experiment
This experiment to the low pH Imnunoglobulin injection that provides by medication suppliers carry out immunity anti--A, anti--B examine and determine, and a kind of quick, safe drug inspection method is provided.
1, examined object formulations prepared from solutions: draw 10ml stoste, regulate pH to 7.0, be diluted to 20ml, make and examined sample solution with normal saline solution with 5% disodium phosphate soln.Get and examined sample solution, examined sample solution doubling dilution liquid with doubling dilution method preparation 1/1,1/2,1/4,1/8,1/16,1/32,1/64,1/128,1/256,1/512.
2, get 24 test tubes, mark A1/1, A1/2, A1/4, A1/8, A1/16, A1/32, A1/64, A1/128, A1/256, A1/512 are as being examined A, A positive control, A negative control; B1/1, B1/2, B1/4, B1/8, B1/16, B1/32, B1/64, B1/128, B256, B1/512 are as being examined B, and B positive control, B negative control are put test tube rack successively and arranged.
3, add 3%A type red cell suspension 50 μ l to the A1/1-A negative control; Add 3%B type red cell suspension 50 μ l to the B1/1-B negative control; Add chymopapain reagent solution 50 μ l to every test tube, mix.
4, test tube being moved into 37 ± 0.5 ℃ of water-baths hatched 5 minutes.
5, respectively add enzyme inhibitor-64 reagent solution 50 μ l to whole test tubes, to A1/1-A1/512 add be consistent with its mark number examined sample doubling dilution liquid 200 μ l, add IgG-to the A positive control and resist-A serum 50 μ l, add AB serum 50 μ l to the A negative control; Add the 1/1-1/512 that is consistent with its mark number to B1/1-B1/512 and examined sample doubling dilution liquid 100 μ l, add IgG-to the B positive control and resist-B serum 50 μ l, add AB serum 50 μ l, mix to the B negative control.
6, test tube being moved into 37 ± 0.5 ℃ of water-bath fields hatched 7 minutes.
7, test tube is moved into hydro-extractor, centrifugal 1 minute of 120 * g.
8, shake test tube gently, Red Blood Cells Suspension, visual inspection experiment verification result.
9, positive control produces red cell agglutination, and negative control does not produce red cell agglutination, and experimental result is set up.Examined A and red cell agglutination occurs and be experiment calibrating positive findings, examined A and red cell agglutination do not occur and be real calibrating negative findings; Examined B and red cell agglutination occurs and be experiment calibrating positive findings, examined B and red cell agglutination do not occur and be real calibrating negative findings.With the inverse of being examined the minimum aggegation intensity (1+) that sample produces red blood cell of highly diluted multiple is tiring of titrated antibody.
Through comparing with indirect antihuman globulin calibrating experimental result, it is 100% that the present invention tests the calibration method accuracy rate, and the experiment calibrating is highly sensitive in 1-2 order of magnitude of indirect antihuman globulin calibrating experiment.
Claims (10)
1, the method for the outer blood group serology of a kind of detection bodies comprises the steps:
1) chymopapain reagent solution and red cell suspension are mixed, in water-bath field, microwave field or automatic temperature-controlled shaker, hatch; Described chymopapain preparation contains chymopapain, L-halfcystine and Propiram;
2) add enzyme inhibitor-64 reagent solution, add examined object then respectively, positive control and negative control thing mix, and hatch in water bath, microwave field or automatic temperature-controlled shaker; Described enzyme inhibitor-64 contains trans-epoxy succinic acyl-L-leucylamino (4-guanidine radicals) butane, sweet mellow wine, trehalose and Propiram;
3) 120 * g is centrifugal 1 minute, the record testing result.
2, the outer blood group serology experimental technique of detection bodies according to claim 1 is characterized in that: also contain sweet mellow wine, trehalose and vitamin C salt in the described chymopapain preparation; 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.
3, the outer blood group serology experimental technique of detection bodies according to claim 1 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.
4, according to claim 1 or the outer blood group serology experimental technique of 2 or 3 described detection bodies, it is characterized in that: described water-bath field is hatched and is insulation in 37-39 ℃ of water bath 5-10 minute; Described microwave field is hatched to be incubated 200-300 second in setting power 300-360W, output power 30% microwave field; Described automatic temperature-controlled shaker is hatched to insulation 5-10 in 37-39 ℃, 300-500r/min, the automatic temperature-controlled shaker of 2-3 level and is divided kind.
5, according to claim 1 or the outer blood group serology experimental technique of 2 or 3 described detection bodies, it is characterized in that: described erythrocytic concentration is 2-4%.
6, according to claim 1 or the outer blood group serology experimental technique of 2 or 3 described detection bodies, it is characterized in that: described examined object is patients serum's sample, blood donor's serum sample, patient, blood donor IgG-be anti--and A, anti--B experiment calibrating use serum sample solution, and red blood cell diffuses liquid; And people source type ABO-IgG-anti--A, anti--B, Rh-is anti--D, anti--C, anti--E, anti--c, anti--e, Kell-is anti--K, anti--k, kiad-be anti--JK
a, anti--JK
b, Lewis-is anti--Le
a, anti--Le
b, anti--Le
A+b, P-is anti--P
1Antibody serum.
7, according to claim 1 or the outer blood group serology experimental technique of 2 or 3 described detection bodies, it is characterized in that: the red blood cell reagent that described calibrating experiment is used is blood group antibody screening red cell suspension beyond the ABO, blood group spectrum cell suspension beyond the red blood cell ABO; Red blood cells of type A suspension or Type B red cell suspension.
8, the outer blood group serology experimental technique of detection bodies according to claim 1, it is characterized in that: described detection record experimental result, when being experiment container, adopt scanning microplate reader-blood group interpreting system to read, write down experimental result automatically with U type porous plate; When being experiment container, adopting and manually read, write down experimental result with test tube.
9, the outer blood group serology experimental technique of detection bodies according to claim 1 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.
10, the outer blood group serology experimental technique of detection bodies according to claim 1 is characterized in that: 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.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101167745B (en) * | 2007-11-02 | 2010-06-02 | 中国人民解放军军事医学科学院野战输血研究所 | Small molecular sugar stable buffer used for blood platelet frozen-dried preservation |
| CN103487590A (en) * | 2013-09-03 | 2014-01-01 | 华南农业大学 | Indirect Dot-ELISA (enzyme-linked immunosorbent assay) method and application of blood group antibody |
| CN103675298A (en) * | 2013-12-13 | 2014-03-26 | 江苏中济万泰生物医药有限公司 | Immune hemolytic anemia detection kit and detection method thereof |
| CN103675297A (en) * | 2013-12-13 | 2014-03-26 | 江苏中济万泰生物医药有限公司 | Medicament-induced hemolytic anemia detection kit and detection method thereof |
| CN107014992A (en) * | 2016-11-10 | 2017-08-04 | 王毅 | A kind of foundation for quantitatively detecting human blood type antibody concentration method |
| CN107202898A (en) * | 2017-06-15 | 2017-09-26 | 迈克生物股份有限公司 | Kit for Shanghai irregular antibody |
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2004
- 2004-05-31 CN CN 200410046104 patent/CN1266477C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101167745B (en) * | 2007-11-02 | 2010-06-02 | 中国人民解放军军事医学科学院野战输血研究所 | Small molecular sugar stable buffer used for blood platelet frozen-dried preservation |
| CN103487590A (en) * | 2013-09-03 | 2014-01-01 | 华南农业大学 | Indirect Dot-ELISA (enzyme-linked immunosorbent assay) method and application of blood group antibody |
| CN103487590B (en) * | 2013-09-03 | 2015-04-08 | 华南农业大学 | Indirect Dot-ELISA (enzyme-linked immunosorbent assay) method and application of blood group antibody |
| CN103675298A (en) * | 2013-12-13 | 2014-03-26 | 江苏中济万泰生物医药有限公司 | Immune hemolytic anemia detection kit and detection method thereof |
| CN103675297A (en) * | 2013-12-13 | 2014-03-26 | 江苏中济万泰生物医药有限公司 | Medicament-induced hemolytic anemia detection kit and detection method thereof |
| CN107014992A (en) * | 2016-11-10 | 2017-08-04 | 王毅 | A kind of foundation for quantitatively detecting human blood type antibody concentration method |
| CN107014992B (en) * | 2016-11-10 | 2019-08-02 | 王毅 | A kind of foundation of quantitative detection human blood type antibody concentration method |
| CN107202898A (en) * | 2017-06-15 | 2017-09-26 | 迈克生物股份有限公司 | Kit for Shanghai irregular antibody |
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