CN108572253A - A kind of indirect antihuman globulin test accelerates reinforcing agent and its preparation method and application - Google Patents
A kind of indirect antihuman globulin test accelerates reinforcing agent and its preparation method and application Download PDFInfo
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- CN108572253A CN108572253A CN201810205057.3A CN201810205057A CN108572253A CN 108572253 A CN108572253 A CN 108572253A CN 201810205057 A CN201810205057 A CN 201810205057A CN 108572253 A CN108572253 A CN 108572253A
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Abstract
The invention discloses a kind of indirect antihuman globulin test accelerate reinforcing agent, including a concentration of 1~60% polyglycol solution and a concentration of 0.1~5% cohesion amine aqueous solution, the polyglycol solution and cohesion amine aqueous solution volume ratio be (10~20):1.The present invention also provides the preparation method and application of the acceleration reinforcing agent.A kind of indirect antihuman globulin test of the present invention accelerates reinforcing agent, accelerate the preceding cross matching of enhancing blood transfusion and/or red blood cell irregular antibody Screening tests, the reaction incubation time of the reagent is short, high sensitivity, accuracy are strong, it can be with the various weaker red blood cell irregular antibodies of Rapid Detection, suitable for the quick cross matching of emergency treatment and red blood cell irregular antibody screening, it is that patient finds matched blood at the first time, various hemolytic blood transfusion adverse reactions caused by incompatible blood transfusion are reduced, the safer blood transfusion quality of patient is given.
Description
Technical field
The present invention relates to biomedicine technical fields, specifically, are related to a kind of indirect antihuman globulin test's acceleration increasing
Strong agent and its preparation method and application, indirect antihuman globulin test accelerate reinforcing agent that can accelerate match or Antibody screening
Speed.
Background technology
ABO blood group system is first blood group system of the mankind that Landsteiner had found in 1900, as the mankind are to blood
The further research of type finds that mankind's blood group system has 36 kinds so far, and blood group antigens have more than 300.Incompatible blood transfusion can
Significant hemolysis can be caused to react, or even shock is so that dead.Currently, the abo blood group and Rh blood groups of routine clinical detection are 2
Kind most important blood group system, if but remaining 34 kinds of blood group system mismatch, it is possible to caused by antigen-antibody occurs
Adverse transfusion reaction.Therefore, cross matching is particularly important before blood transfusion, it is possible to reduce the generation of Adverse transfusion reaction.
Currently, in traditional antiglobulin test tube cross matching method, main side be added patient blood plasma and blood donor it is red
Cell, secondary side are the red blood cells that the blood plasma and patient of blood donor is added, in 37 DEG C of water-baths three times with brine, most
After antiglobulin reagent is added, observe result after centrifugation.The weakness of this method is that incubation time is long, reaction sensitivity is low, is needed
Be incubated 30 to 60 minutes and differ under 37 DEG C of environment, and may the weaker antibody of missing inspection potency, so as to cause serious blood transfusion
Adverse reaction.When patient is badly in need of with blood, operation may will produce larger danger to this method for a long time.
Yves lapierre in 1984 have invented microcolumn gel technology, and Switzerland's DiaMed is based on micro-column gel skill within 1988
Art and antihuman globulin test method, which are combined, is proposed micro-column gel antihuman globulin reagent card, is used widely in recent years.
Antihuman globulin test of the micro-column gel antihuman globulin reagent card (LISS/Coombs) relative to traditional test tube method, step letter
Just, small using specimen amount, can with automation mechanized operation, suitable for the detection of extensive sample.It is micro- that the country also has many producers to be proposed
Column gel antihuman globulin reagent card.Patents documents such as 101718785 B, CN 101718786 B, CN of CN
102692510 A, CN106950385A etc..
No matter traditional test tube method or micro-column gel agglutination assay, still there is a problem of that incubation time is long, reaction sensitivity is low, have
A kind of acceleration reinforcing agent of necessity exploitation is added in the experiment so that and reaction incubation time shortens, and detection sensitivity is promoted, from
And red blood cell incomplete antibody is detected within a short period of time, ensure patients with transfusion safety.
Invention content
It is indirect that the object of the present invention is to provide a kind of reaction incubation times is short, detection sensitivity is high, patients with transfusion is safe
Antihuman globulin test accelerates reinforcing agent.
It is a further object to provide the preparation methods that above-mentioned indirect antihuman globulin test accelerates reinforcing agent.
There is provided the concrete applications that the indirect antihuman globulin test accelerates reinforcing agent for the third aspect of the present invention.
To achieve the goals above, the technical solution adopted by the present invention is as follows：
One aspect of the present invention provides a kind of indirect antihuman globulin test and accelerates reinforcing agent, including a concentration of 1~
60% polyethylene glycol (PEG) solution and a concentration of 0.1~5% cohesion amine (polybrene) solution, the polyethylene glycol
The volume ratio of solution and cohesion amine aqueous solution is (10~20):1.
Preferably, a concentration of the 1~10% of polyethylene glycol (PEG) solution, cohesion amine (polybrene) solution
A concentration of 0.1~3%, it is furthermore preferred that a concentration of the 3~5% of the polyethylene glycol (PEG) solution, the cohesion amine
(polybrene) a concentration of the 1~3% of solution.
Preferably, the volume ratio of the polyglycol solution and cohesion amine aqueous solution is (12~15):1.
The molecular weight of the polyethylene glycol is 2000~8000, preferably 4000~6000.When the molecular weight of polyethylene glycol is small
When 2000, by research shows that indirect antihuman globulin test can not be enhanced；When the molecular weight of polyethylene glycol is more than 8000
When, by research shows that the false of red blood cell can be caused to assemble, causing the false positive of indirect antihuman globulin test.
Polyethylene glycol (PEG) solution is that polyethylene glycol pulvis is dissolved in distilled water to be made into.
Cohesion amine (polybrene) solution is will to agglomerate amine (polybrene) pulvis and be dissolved in distilled water to be made into.
The present invention also selects low ionic strength solution in the prior art (LISS), bovine serum albumin in pre-stage test
In vain, Sephacryl gel, cross-link dextran G50 etc. accelerate to increase with indirect antihuman globulin test of the present invention
Strong agent is compared, with obvious effects poor, and indirect antihuman globulin test can not be remarkably reinforced.
Preferably, indirect antihuman globulin test of the invention accelerates in reinforcing agent, adds LISS solution and/or ox blood
Pure albumen.
The indirect antihuman globulin test accelerates in reinforcing agent, and the volume ratio of the LISS solution and cohesion amine aqueous solution is
(5~10):1；The volume ratio of the bovine serum albumin(BSA) and cohesion amine aqueous solution is (5~10):1.
The indirect antihuman globulin test of the present invention accelerates reinforcing agent, pH value 6.5-7.
Another aspect of the present invention provides a kind of preparation side of indirect antihuman globulin test's acceleration reinforcing agent
Method includes the following steps：
Polyethylene glycol pulvis is dissolved in be made into distilled water a concentration of 1~60% polyethylene glycol (PEG) solution, will agglomerate
Amine (polybrene) pulvis be dissolved in be made into distilled water a concentration of 0.1~5% cohesion amine (polybrene) solution, then
Cohesion amine aqueous solution is added in polyglycol solution, the volume ratio of polyglycol solution and cohesion amine aqueous solution is (10~20):
1, the pH value for adjusting solution is 6.5-7, obtains the indirect antihuman globulin test and accelerates reinforcing agent.
The indirect antihuman globulin test accelerates reinforcing agent to be dissolved in the result in distilled water to be substantially better than phosphate-buffered
Solution (PBS) and physiological saline.
Another aspect of the present invention provides a kind of preparation side of indirect antihuman globulin test's acceleration reinforcing agent
Method includes the following steps：
Polyethylene glycol pulvis is dissolved in be made into distilled water a concentration of 1~60% polyethylene glycol (PEG) solution, will agglomerate
Amine (polybrene) pulvis be dissolved in be made into distilled water a concentration of 0.1~5% cohesion amine (polybrene) solution, then
Cohesion amine aqueous solution is added in polyglycol solution, the volume ratio of polyglycol solution and cohesion amine aqueous solution is (10~20):
1, LISS solution is added, LISS solution is (5~10) with the volume ratio for agglomerating amine aqueous solution:1；Or add bovine serum albumin
In vain, the volume ratio of bovine serum albumin(BSA) and cohesion amine aqueous solution is (5~10):1；Or LISS solution is added simultaneously and ox blood is pure
The volume ratio of albumen, LISS solution and cohesion amine aqueous solution is (5~10):1, the volume of bovine serum albumin(BSA) and cohesion amine aqueous solution
Than for (5~10):1；The pH value for adjusting solution is 6.5-7, obtains the indirect antihuman globulin test and accelerates reinforcing agent.
There is provided the concrete applications that above-mentioned indirect antihuman globulin test accelerates reinforcing agent for the third aspect of the present invention.
One is the application in traditional test tube method indirect antihuman globulin test.
Specifically, the present invention provides the test methods that a kind of indirect antihuman globulin accelerates match, in the test method
Middle that above-mentioned indirect antihuman globulin test's acceleration reinforcing agent is added, which includes the following steps：It will add in red blood cell
It is uniformly mixed after entering physiological saline cleaning, centrifuges removal supernatant, physiological saline is added and is configured to red cell suspension, so
Blood plasma is added afterwards or serum is uniformly mixed, is eventually adding indirect antihuman globulin test and reinforcing agent is accelerated to be uniformly mixed, be incubated,
Physiological saline is added to centrifuge, jog test tube observing is as a result, according to erythrocyte agglutination or the situation judging result of dispersion.
The indirect antihuman globulin test accelerates reinforcing agent, includes a concentration of 1~60% polyethylene glycol (PEG) solution
With a concentration of 0.1~5% cohesion amine (polybrene) solution, the polyglycol solution and cohesion amine aqueous solution volume ratio
For (10~20):1.
The molecular weight of the polyethylene glycol is 2000~8000, preferably 4000~6000.
Polyethylene glycol (PEG) solution is that polyethylene glycol pulvis is dissolved in distilled water to be made into.
Cohesion amine (polybrene) solution is will to agglomerate amine (polybrene) pulvis and be dissolved in distilled water to be made into.
Preferably, a concentration of the 1~10% of polyethylene glycol (PEG) solution, cohesion amine (polybrene) solution
A concentration of 0.1~3%, it is furthermore preferred that a concentration of the 3~5% of the polyethylene glycol (PEG) solution, the cohesion amine
(polybrene) a concentration of the 1~3% of solution.
Preferably, the volume ratio of the polyglycol solution and cohesion amine aqueous solution is (12~15):1.
Preferably, indirect antihuman globulin test of the invention accelerates in reinforcing agent, adds LISS solution and/or ox blood
Pure albumen.
The indirect antihuman globulin test accelerates in reinforcing agent, and the volume ratio of the LISS solution and cohesion amine aqueous solution is
(5~10):1；The volume ratio of the bovine serum albumin(BSA) and cohesion amine aqueous solution is (5~10):1.
The indirect antihuman globulin test of the present invention accelerates reinforcing agent, pH value 6.5-7.
The indirect antihuman globulin test accelerates the preparation method of reinforcing agent, includes the following steps：
Polyethylene glycol pulvis is dissolved in be made into distilled water a concentration of 1~60% polyethylene glycol (PEG) solution, will agglomerate
Amine (polybrene) pulvis be dissolved in be made into distilled water a concentration of 0.1~5% cohesion amine (polybrene) solution, then
Cohesion amine aqueous solution is added in polyglycol solution, the volume ratio of polyglycol solution and cohesion amine aqueous solution is (10~20):
1, the pH value for adjusting solution is 6.5-7, obtains the indirect antihuman globulin test and accelerates reinforcing agent；
Alternatively, polyethylene glycol pulvis is dissolved in be made into distilled water a concentration of 1~60% polyethylene glycol (PEG) solution,
Will agglomerate amine (polybrene) pulvis be dissolved in be made into distilled water a concentration of 0.1~5% cohesion amine (polybrene) it is molten
Then cohesion amine aqueous solution is added in polyglycol solution by liquid, the volume ratio of polyglycol solution and cohesion amine aqueous solution is
(10~20):1, LISS solution is added, LISS solution is (5~10) with the volume ratio for agglomerating amine aqueous solution:1；Or add ox
The volume ratio of seralbumin, bovine serum albumin(BSA) and cohesion amine aqueous solution is (5~10):1；Or be added simultaneously LISS solution and
The volume ratio of bovine serum albumin(BSA), LISS solution and cohesion amine aqueous solution is (5~10):1, bovine serum albumin(BSA) and cohesion amine are molten
The volume ratio of liquid is (5~10):1；The pH value for adjusting solution is 6.5-7, obtains the indirect antihuman globulin test and accelerates to increase
Strong agent.
The incubation is to be incubated 1~10min, preferably 5min in temperature is 37 DEG C of water-baths.
The rotating speed of the centrifugation is 1000~3500rpm.
The second is the application in micro-column gel agglutination assay indirect antihuman globulin test.
Specifically, the present invention provides a kind of micro-column gel antihuman globulin reagent card, have at least 6 on the reagent card
There is in the micro-column gel pipe a micro-column gel pipe gel suspension medium, the gel suspension medium to contain described indirectly anti-
Human immunoglobulin experiment accelerates reinforcing agent.
There are 6,8 micro-column gel pipes on the reagent card.
Preferably, the formula of the gel suspension medium is as follows：
Potassium dihydrogen phosphate 0.15-0.3g/L；
Disodium hydrogen phosphate 0.35-0.65g/L；
Sodium chloride 1.8-2.0g/L；
The indirect antihuman globulin test of the present invention accelerates reinforcing agent 3-5%；
Polyethylene glycol 0.08-0.15%；
Sodium azide 0.3%；
The purifying water dissolution of the above reagent, adjustment pH value are 6.6-7.
Specifically, the present invention provides a kind of micro-column gel anti-human immunoglobulin crossmatching card, on the reagent card at least
With 6 micro-column gel pipes, there is gel suspension medium, the gel suspension medium to contain described in the micro-column gel pipe
Indirect antihuman globulin test accelerates reinforcing agent.
There are 6,8 micro-column gel pipes on the reagent card.
Preferably, the formula of the gel suspension medium is as follows：
Potassium dihydrogen phosphate 0.15-0.3g/L；
Disodium hydrogen phosphate 0.35-0.65g/L；
Sodium chloride 1.8-2.0g/L；
The indirect antihuman globulin test of the present invention accelerates reinforcing agent 3-5%；
Polyethylene glycol 0.08-0.15%；
Sodium azide 0.3%；
The purifying water dissolution of the above reagent, adjustment pH value are 6.6-7.
Specifically, the present invention provides a kind of red blood cell irregular antibody detection kit, the kit includes separation
The reacting hole endoperidium of chamber, disengagement chamber has the indirect antihuman globulin test to accelerate reinforcing agent.
Due to the adoption of the above technical scheme, the present invention has the following advantages and beneficial effect：
A kind of indirect antihuman globulin test provided by the invention accelerates reinforcing agent, intersects cooperation examination before accelerating enhancing blood transfusion
It tests and/or red blood cell irregular antibody Screening tests, the reaction incubation time of the reagent is short, high sensitivity, accuracy are strong, can
With the various weaker red blood cell irregular antibodies of Rapid Detection, irregularly resist suitable for the quick cross matching of emergency treatment and red blood cell
Body screening finds matched blood for patient at the first time, and it is bad anti-to reduce various hemolytic blood transfusions caused by incompatible blood transfusion
It answers, gives the safer blood transfusion quality of patient.
A kind of indirect antihuman globulin test provided by the invention accelerates in reinforcing agent, and polyethylene glycol and cohesion amine joint are made
With space can be filled with the formation speed of collision frequency between intensified response object and immune complex, increase molecular volume, it can
Improve the sensibility of red cell antigens antibody response.
A kind of indirect antihuman globulin test provided by the invention accelerates reinforcing agent, can be used for accelerating various blood types system
Antigen-antibody reaction, so that traditional anti-globulin technique incubation time was shortened to from 30 to 60 minutes 5 minutes；It can be used for
The antigen-antibody reaction for enhancing various blood types system makes the positive findings of traditional anti-globulin technique enhance 1 to 2 rank
Reaction；The antigen-antibody reaction of various blood types system can be used for detecting, it is equal to the antigen-antibody reaction of various blood types system
Unrestraint acts on, can be with complete detection cross matching.
A kind of indirect antihuman globulin test provided by the invention accelerates reinforcing agent, with a variety of red cell suspensions, physiology salt
Water carries out antiglobulin blank test, equal non-false positive reaction；The antigen-antibody that can be used for accelerating to enhance various blood types system
Reaction, compared with low ionic strength solution (LISS), bovine serum albumin(BSA) reinforcing agent, has a clear superiority.
A kind of indirect antihuman globulin test provided by the invention accelerates reinforcing agent, and it is anti-can not only to accelerate antigen-antibody
It answers, also there is apparent enhancing effect, enhance the recall rate of weaker incomplete antibody, such as the weaker anti-Mur antibody of potency resists
Dia antibody finds the blood of best match for patient, reduces Adverse transfusion reaction.
Description of the drawings
Fig. 1 is 1:Experimental result schematic diagram after 16 times of dilution anti-Ds.
Fig. 2 is 1:Experimental result schematic diagram after 128 times of dilution anti-Ds.
Fig. 3 is 1:Experimental result schematic diagram after 512 times of dilution anti-Ds.
In Fig. 1~3,1 indicates experimental group, and 2 indicate control group.
Fig. 4 is the schematic diagram using the indirect antihuman globulin test's result for accelerating reinforcing agent；Wherein：11 indicate that potency are
1:16 test tube, 12 indicate that potency is 1:32 test tube, 13 indicate that potency is 1:64 test tube, 14 indicate that potency is 1:128
Test tube, 15 indicate that potency is 1:256 test tube, 16 indicate that potency is 1:512 test tube.
Fig. 5 is the schematic diagram of traditional indirect antihuman globulin test's result；Wherein：21 indicate that potency is 1:16 test tube,
22 indicate that potency is 1:32 test tube, 23 indicate that potency is 1:64 test tube, 24 indicate that potency is 1:128 test tube, 25 indicate
Potency is 1:256 test tube, 26 indicate that potency is 1:512 test tube.
Fig. 6 is the various schematic diagrames for accelerating indirect antihuman globulin test's gel microtrabeculae block-regulations of Contrast agent to compare；Its
In：31 indicate the micropore of control group, and 32 indicate that indirect antihuman globulin test prepared by the present invention, which is added, accelerates the micro- of reinforcing agent
Hole, 33 indicate the micropore that LISS liquid is added, and 34 indicate the micropore that bovine serum albumin(BSA) is added.
A is the corresponding test result photos of Fig. 4 in Fig. 7, and B is the corresponding test result photos of Fig. 5 in Fig. 7, and C is in Fig. 7
The corresponding test result photos of Fig. 6.
Specific implementation mode
In order to illustrate more clearly of the present invention, with reference to preferred embodiment, the present invention is described further.Ability
Field technique personnel should be appreciated that following specifically described content is illustrative and be not restrictive, this should not be limited with this
The protection domain of invention.
Polyethylene glycol (PEG) used in the embodiment of the present invention is purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd,
Specification is 500 grams/bottle.
Cohesion amine (polybrene) used in the embodiment of the present invention is purchased from Sigma companies, and specification is 5 grams/bottle.
Standard anti-D (IgG) antibody reagent used in the embodiment of the present invention is public purchased from Shanghai blood biological medicine Limited Liability
Department, specification are 10 milliliters/bottle.
LISS liquid used in the embodiment of the present invention is purchased from Beisuo Biological Technology Co., Ltd., Zhuhai, and specification is 250 milliliters/
Bottle.
Bovine serum albumin(BSA) used in the embodiment of the present invention is purchased from CE-Immundiagnostika GmbH,
Eschelbronn, Germany, specification are 10 milliliters/bottle.
Antihuman globulin reagent used in the embodiment of the present invention is purchased from Shanghai blood biomedical limited liability company, specification
For 10 milliliters/bottle.Instrument used in the embodiment of the present invention：5702R types refrigerated centrifuge (being purchased from EPPENDORF companies of Germany),
KA-2200 immunohematologies centrifuge (being purchased from Kubota companies of Japan), CX31 biomicroscopes are (purchased from Japanese Olympic bar
This company), micropipettor (be purchased from Gilson companies of France), HH.S11-2-S types constant water bath box (cure purchased from Shanghai leap
Treat instrument Co., Ltd), antihuman globulin detection card (being purchased from BIO-RAD companies of the U.S.).
The indirect antihuman globulin test of the embodiment of the present invention accelerates reinforcing agent, matches when in use according to preparation method step
It sets, when not in use, is stored respectively according to the ingredient for being included.
Embodiment 1
A kind of indirect antihuman globulin test provided by the invention accelerates reinforcing agent, including a concentration of 5%, molecular weight be
2000 polyethylene glycol (PEG) solution and a concentration of 0.5% cohesion amine (polybrene) solution, the polyglycol solution
Volume ratio with cohesion amine aqueous solution is 10:1.
The indirect antihuman globulin test accelerates the preparation method of reinforcing agent, includes the following steps：
Polyethylene glycol pulvis is dissolved in be made into distilled water a concentration of 5% polyethylene glycol (PEG) solution, amine will be agglomerated
(polybrene) pulvis be dissolved in be made into distilled water a concentration of 0.5% cohesion amine (polybrene) solution, then will cohesion
Amine aqueous solution is added in polyglycol solution, and the volume ratio of polyglycol solution and cohesion amine aqueous solution is 10:1, with hydrochloric acid or hydrogen
Sodium oxide molybdena is 7 come the pH value for adjusting solution, obtains the indirect antihuman globulin test and accelerates reinforcing agent.It is described indirectly anti-human
Globulin test acceleration reinforcing agent is dissolved in the result in distilled water and is substantially better than phosphate buffer solution (PBS) and physiological saline.
Embodiment 2
A kind of indirect antihuman globulin test provided by the invention accelerates reinforcing agent, including a concentration of 20%, molecular weight
For 8000 polyethylene glycol (PEG) solution and a concentration of 2.5% cohesion amine (polybrene) solution, the polyethylene glycol is molten
The volume ratio of liquid and cohesion amine aqueous solution is 20:1.
The indirect antihuman globulin test accelerates the preparation method of reinforcing agent, includes the following steps：
Polyethylene glycol pulvis is dissolved in be made into distilled water a concentration of 20% polyethylene glycol (PEG) solution, amine will be agglomerated
(polybrene) pulvis be dissolved in be made into distilled water a concentration of 2.5% cohesion amine (polybrene) solution, then will cohesion
Amine aqueous solution is added in polyglycol solution, and the volume ratio of polyglycol solution and cohesion amine aqueous solution is 20:1, with hydrochloric acid or hydrogen
Sodium oxide molybdena is 7 come the pH value for adjusting solution, obtains the indirect antihuman globulin test and accelerates reinforcing agent.
Embodiment 3
A kind of indirect antihuman globulin test provided by the invention accelerates reinforcing agent, including a concentration of 1%, molecular weight be
5000 polyethylene glycol (PEG) solution and a concentration of 0.1% cohesion amine (polybrene) solution, the polyglycol solution
Volume ratio with cohesion amine aqueous solution is 15:1.
The indirect antihuman globulin test accelerates the preparation method of reinforcing agent, includes the following steps：
Polyethylene glycol pulvis is dissolved in be made into distilled water a concentration of 1% polyethylene glycol (PEG) solution, amine will be agglomerated
(polybrene) pulvis be dissolved in be made into distilled water a concentration of 0.1% cohesion amine (polybrene) solution, then will cohesion
Amine aqueous solution is added in polyglycol solution, and the volume ratio of polyglycol solution and cohesion amine aqueous solution is 15:1, with hydrochloric acid or hydrogen
Sodium oxide molybdena is 7 come the pH value for adjusting solution, obtains the indirect antihuman globulin test and accelerates reinforcing agent.
Embodiment 4
A kind of indirect antihuman globulin test provided by the invention accelerates reinforcing agent, including a concentration of 60%, molecular weight
For 4000 polyethylene glycol (PEG) solution and a concentration of 5% cohesion amine (polybrene) solution, the polyglycol solution
Volume ratio with cohesion amine aqueous solution is 12:1.
The indirect antihuman globulin test accelerates the preparation method of reinforcing agent, includes the following steps：
Polyethylene glycol pulvis is dissolved in be made into distilled water a concentration of 60% polyethylene glycol (PEG) solution, amine will be agglomerated
(polybrene) pulvis be dissolved in be made into distilled water a concentration of 5% cohesion amine (polybrene) solution, then will agglomerate amine
Solution is added in polyglycol solution, and the volume ratio of polyglycol solution and cohesion amine aqueous solution is 12:1, with hydrochloric acid or hydrogen-oxygen
The pH value for changing sodium to adjust solution is 6.5-6.8, obtains the indirect antihuman globulin test and accelerates reinforcing agent.
Embodiment 5
A kind of indirect antihuman globulin test provided by the invention accelerates reinforcing agent, including a concentration of 30%, molecular weight
For 6000 polyethylene glycol (PEG) solution and a concentration of 4% cohesion amine (polybrene) solution, the polyglycol solution
Volume ratio with cohesion amine aqueous solution is 16:1.
The indirect antihuman globulin test accelerates the preparation method of reinforcing agent, includes the following steps：
Polyethylene glycol pulvis is dissolved in be made into distilled water a concentration of 30% polyethylene glycol (PEG) solution, amine will be agglomerated
(polybrene) pulvis be dissolved in be made into distilled water a concentration of 4% cohesion amine (polybrene) solution, then will agglomerate amine
Solution is added in polyglycol solution, and the volume ratio of polyglycol solution and cohesion amine aqueous solution is 16:1, with hydrochloric acid or hydrogen-oxygen
The pH value for changing sodium to adjust solution is 6.5-6.8, obtains the indirect antihuman globulin test and accelerates reinforcing agent.
Embodiment 6
A kind of indirect antihuman globulin test provided by the invention accelerates reinforcing agent, including a concentration of 10%, molecular weight
For 5500 polyethylene glycol (PEG) solution and a concentration of 1% cohesion amine (polybrene) solution, the polyglycol solution
Volume ratio with cohesion amine aqueous solution is 18:1.
The indirect antihuman globulin test accelerates the preparation method of reinforcing agent, includes the following steps：
Polyethylene glycol pulvis is dissolved in be made into distilled water a concentration of 10% polyethylene glycol (PEG) solution, amine will be agglomerated
(polybrene) pulvis be dissolved in be made into distilled water a concentration of 1% cohesion amine (polybrene) solution, then will agglomerate amine
Solution is added in polyglycol solution, and the volume ratio of polyglycol solution and cohesion amine aqueous solution is 18:1, with hydrochloric acid or hydrogen-oxygen
The pH value for changing sodium to adjust solution is 7, obtains the indirect antihuman globulin test and accelerates reinforcing agent.
Embodiment 7
Indirect antihuman globulin test's implementation steps：
1. by taking standard anti-D (IgG) antibody reagent as an example, with AB types diluted plasma at corresponding potency, antibody intensity by by force to
Weak distribution is respectively 1 in six test tubes of experimental group and control group:16、1:32、1:64、1:128、1:256、1:512；Each examination
100 microlitres of corresponding antibodies are added in pipe.
2. taking blood donor's O-shaped RhD positive red blood cells suspension brine 1-2 times, it is made into about 3% concentration respectively
Red Blood Cells Suspension are taken respectively in 50 microlitres of each test tubes for being added to step 1.
3. the indirect antihuman globulin test for being separately added into the preparation of the embodiment of the present invention 3 in six test tubes of experimental group accelerates
Reinforcing agent 1mL (including a concentration of 1%, molecular weight be 5000 polyethylene glycol (PEG) solution and a concentration of 0.1% cohesion
The volume ratio of amine (polybrene) solution, the polyglycol solution and cohesion amine aqueous solution is 15:1) it, is incubated 5 minutes；
4. control group is traditional antiglobulin test method, it is added without the indirect antihuman globulin of the preparation of the embodiment of the present invention 3
Experiment accelerates reinforcing agent, is incubated 30 minutes；
5. experimental group and control group use brine 3 times respectively, 1 minute (rotating speed 3400rpm) is centrifuged every time；
6. last time discards supernatant liquid after centrifuging, test tube is buckled to dry on blotting paper；
7. being separately added into 50 microlitres of antihuman globulin reagents, mixing in experimental group and control group；
8. centrifuge 15s (rotating speed 3400rpm), with the naked eye or by om observation as a result, and recorded by degree, as a result such as
Shown in table 1 and Fig. 1~3, Fig. 1 is 1:Experimental result schematic diagram after 16 times of dilution anti-Ds, Fig. 2 is 1:128 times of dilutions are anti-
Experimental result schematic diagram after D antibody, Fig. 3 are 1:Experimental result schematic diagrames after 512 times of dilution anti-Ds, in Fig. 1~3,1
Indicate experimental group, 2 indicate control group.
Table 1 carries out indirect antihuman globulin test's method with standard anti-D and compares
| 1:16 | 1:32 | 1:64 | 1:128 | 1:256 | 1:512 | |
| Experimental group | 3+ | 2+s | 2+s | 2+ | 1+ | 1+ |
| Control group | 2+ | 2+ | 2+ | 1+ | +/- | +/- |
From the data of Fig. 1~3 and table 1 it is found that indirect antihuman globulin prepared by the embodiment of the present invention 3 is added in experimental group
After experiment accelerates reinforcing agent, indirect antihuman globulin test's sensitivity is remarkably reinforced, and incubation time is obviously shortened.
Embodiment 8
Experimental group：Use the indirect antihuman globulin test's cross matching test tube method implementation steps for accelerating Contrast agent：
1. patient respectively uses brine with blood donor's red blood cell 1-2 times, it is made into 2%-5% Red Blood Cells Suspension respectively.
2. taking 3, test tube, it is respectively labeled as main side pipe, secondary side pipe and own control pipe, patients blood plasma 2 is added in the pipe of main side
Drop is added blood donor 2%-5% Red Blood Cells Suspension 1 and drips；Donor plasma 2 is added in secondary side pipe to drip, patient 2%-5% is added
Red Blood Cells Suspension 1 drip；The drop of patients blood plasma 2 is added in own control pipe to drip with patient 2%-5% Red Blood Cells Suspension 1.
3. the indirect antihuman globulin test for being separately added into the preparation of the embodiment of the present invention 3 in above-mentioned 3 test tubes accelerates to increase
Strong agent 1mL (including a concentration of 1%, molecular weight be 5000 polyethylene glycol (PEG) solution and a concentration of 0.1% cohesion amine
(polybrene) volume ratio of solution, the polyglycol solution and cohesion amine aqueous solution is 15:1).
4. being incubated 5 minutes under conditions of temperature is 37 DEG C.
5.3 test tubes use brine 3 times respectively, centrifuge 1 minute (rotating speed 3400rpm) every time.
6. last time discards supernatant liquid after centrifuging, 3 test tubes are buckled to dry on blotting paper.
2 drop antihuman globulin reagents, mixing are separately added into 7.3 test tubes.
8. centrifuging 15s (rotating speed 3400rpm), with the naked eye or by om observation it is aggregated, and result is recorded by degree.
In the detection of 1800 cross matching, accelerate reinforcing agent using indirect antihuman globulin test prepared by the present invention
It carries out enhancing and accelerates reaction, have 1780 cross-sample match negative, there is 20 cross matching positives, non-false positive result.
Aspect between when detecting accelerates reinforcing agent to carry out enhancing and adds using the indirect antihuman globulin test of the invention prepared
Speed reaction, is incubated 5 minutes at 37 DEG C, is compared with the time of the incubation 30 to 60 minutes of conventional method, substantially reduces examination
The time is tested, the quality time has been won in the blood transfusion to rescue patient.
Control group：Traditional indirect antihuman globulin test's cross matching test tube method：
1. patient respectively uses brine with blood donor's red blood cell 1-2 times, it is made into 2%-5% Red Blood Cells Suspension respectively.
2. taking 3, test tube, it is respectively labeled as main side pipe, secondary side pipe and own control pipe, patients blood plasma 2 is added in the pipe of main side
Drop is added blood donor 2%-5% Red Blood Cells Suspension 1 and drips；Donor plasma 2 is added in secondary side pipe to drip, patient 2%-5% is added
Red Blood Cells Suspension 1 drip；The drop of patients blood plasma 2 is added in own control pipe to drip with patient 2%-5% Red Blood Cells Suspension 1.
3. being incubated 30 minutes under conditions of temperature is 37 DEG C.
4.3 test tubes use brine 3 times respectively, centrifuge 1 minute (rotating speed 3400rpm) every time.
5. last time discards supernatant liquid after centrifuging, 3 test tubes are buckled to dry on blotting paper.
2 drop antihuman globulin reagents, mixing are separately added into 6.3 test tubes.
7. centrifuging 15s (rotating speed 3400rpm), with the naked eye or by om observation it is aggregated, and result is recorded by degree.
In the detection of 1800 cross matching, there are 1782 cross-sample match negative, there are 18 cross matching positives,
Non-false positive is as a result, there is 2 samples to be identified as false negative result.The results are shown in Table 2 for experimental group and control group：
2 1800 cross matching situation statistical forms of table
| It is negative | It is positive | |
| Experimental group | 1780 | 20 |
| Control group | 1782 | 18 |
Embodiment 9
Accelerate indirect antihuman globulin test's gel of reinforcing agent using indirect antihuman globulin test prepared by the present invention
Microtrabeculae block-regulations implementation steps：
1. by taking standard anti-D (IgG) antibody reagent as an example, with AB types diluted plasma at corresponding potency, from 1:16 to 1:512
Antibody intensity is by by force to weak distribution, 25 microlitres of antibody-solutions are separately added into six test tubes of experimental group and control group.
2. taking blood donor's O-shaped RhD positive red blood cells suspension brine 1-2 times, 1% Red Blood Cells Suspension are made into,
50 microlitres of red cell suspensions are added in each test tube of step 1.
3. indirect antihuman globulin test prepared by the embodiment of the present invention 4 is added in the test tube of experimental group accelerates reinforcing agent
5ul, be incubated 5 minutes (a concentration of 60%, molecular weight be 4000 polyethylene glycol (PEG) solution and a concentration of 5% cohesion
The volume ratio of amine (polybrene) solution, the polyglycol solution and cohesion amine aqueous solution is 12:1.).
4. the indirect antihuman globulin test for being added without the preparation of the embodiment of the present invention 4 in the test tube of control group accelerates enhancing
Agent is traditional indirect antihuman globulin test, is incubated 15 minutes.
5. centrifuging 10 minutes (rotating speed 1050rpm), detect by an unaided eye agglutination, and is recorded by degree, as a result such as table 3 and figure
Shown in 4 and Fig. 5, Fig. 4 is the schematic diagram (experimental group) using the indirect antihuman globulin test's result for accelerating reinforcing agent；Its
In：11 indicate that potency is 1:16 test tube, 12 indicate that potency is 1:32 test tube, 13 indicate that potency is 1:64 test tube, 14 tables
Show that potency is 1:128 test tube, 15 indicate that potency is 1:256 test tube, 16 indicate that potency is 1:512 test tube；Fig. 5 is to pass
The schematic diagram (control group) for indirect antihuman globulin test's result of uniting；Wherein：21 indicate that potency is 1:16 test tube, 22 indicate
Potency is 1:32 test tube, 23 indicate that potency is 1:64 test tube, 24 indicate that potency is 1:128 test tube, 25 expression potency are
1:256 test tube, 26 indicate that potency is 1:512 test tube.
Table 3 carries out indirect antihuman globulin test's method with standard anti-D and compares
From the data of Fig. 4 and Fig. 5 and table 3 it is found that indirect anti-human ball egg prepared by the embodiment of the present invention 4 is added in experimental group
After white experiment accelerates reinforcing agent, indirect antihuman globulin test's sensitivity is remarkably reinforced, and incubation time is obviously shortened.
Embodiment 10
The various indirect antihuman globulin test's gel microtrabeculae block-regulations for accelerating Contrast agent compare：
1. by taking standard anti-D (IgG) antibody reagent as an example, with AB types diluted plasma at corresponding potency 1:256, select anti-human ball
25 microlitres of antibody-solutions are added in 4 micropores on Protein Detection card, each micropore.
2. taking blood donor's O-shaped RhD positive red blood cells suspension brine 1-2 times, 1% Red Blood Cells Suspension are made into,
It is separately added into 50 microlitres of red cell suspensions in four micropores of step 1.
3. indirect antihuman globulin test prepared by the embodiment of the present invention 4 is added in three micropores of step 2 to accelerate to increase
Strong agent 5ul (a concentration of 60%, molecular weight be 4000 polyethylene glycol (PEG) solution and a concentration of 5% cohesion amine
(polybrene) volume ratio of solution, the polyglycol solution and cohesion amine aqueous solution is 12:1), LISS liquid 5ul, cow's serum
Albumin 5ul is incubated 5 minutes.
4. being added without the indirect antihuman globulin examination of the preparation of the embodiment of the present invention 4 in a remaining micropore for step 2
Acceleration reinforcing agent is tested, is traditional antihuman globulin test, is incubated 5 minutes.
5. centrifuging 10 minutes (rotating speed 1050rpm), detect by an unaided eye agglutination, and is recorded by degree, as a result such as table 4 and figure
Shown in 6, Fig. 6 is the various schematic diagrames for accelerating indirect antihuman globulin test's gel microtrabeculae block-regulations of Contrast agent to compare,
In：31 indicate the micropore of control group, and 32 indicate that indirect antihuman globulin test prepared by the present invention, which is added, accelerates the micro- of reinforcing agent
Hole, 33 indicate the micropore that LISS liquid is added, and 34 indicate the micropore that bovine serum albumin(BSA) is added.The result shows that using of the invention real
The indirect antihuman globulin test for applying the preparation of example 4 accelerates reinforcing agent effect best.
The various indirect antihuman globulin test's gel microtrabeculae block-regulations for accelerating Contrast agent of table 4 compare
Embodiment 11
A kind of indirect antihuman globulin test provided by the invention accelerates reinforcing agent, including a concentration of 3%, molecular weight be
4500 polyethylene glycol (PEG) solution and a concentration of 1% cohesion amine (polybrene) solution, the polyglycol solution and
The volume ratio for agglomerating amine aqueous solution is 12:The volume ratio of 1, LISS solution, LISS solution and cohesion amine aqueous solution is 6:1；
The indirect antihuman globulin test accelerates the preparation method of reinforcing agent, includes the following steps：
Polyethylene glycol pulvis is dissolved in be made into distilled water a concentration of 3% polyethylene glycol (PEG) solution, amine will be agglomerated
(polybrene) pulvis be dissolved in be made into distilled water a concentration of 1% cohesion amine (polybrene) solution, then will agglomerate amine
Solution is added in polyglycol solution, and the volume ratio of polyglycol solution and cohesion amine aqueous solution is 12:1, it is molten to add LISS
The volume ratio of liquid, LISS solution and cohesion amine aqueous solution is 6:1；With hydrochloric acid or sodium hydroxide come adjust solution pH value be 7, obtain
It obtains the indirect antihuman globulin test and accelerates reinforcing agent.
Embodiment 12
A kind of indirect antihuman globulin test provided by the invention accelerates reinforcing agent, including a concentration of 4%, molecular weight be
4800 polyethylene glycol (PEG) solution and a concentration of 2% cohesion amine (polybrene) solution, the polyglycol solution and
The volume ratio for agglomerating amine aqueous solution is 13:1, bovine serum albumin(BSA), bovine serum albumin(BSA) is 5 with the volume ratio for agglomerating amine aqueous solution:1；
The volume ratio of LISS solution, LISS solution and cohesion amine aqueous solution is 8:1；
The indirect antihuman globulin test accelerates the preparation method of reinforcing agent, includes the following steps：
Polyethylene glycol pulvis is dissolved in be made into distilled water a concentration of 4% polyethylene glycol (PEG) solution, amine will be agglomerated
(polybrene) pulvis be dissolved in be made into distilled water a concentration of 2% cohesion amine (polybrene) solution, then will agglomerate amine
Solution is added in polyglycol solution, and the volume ratio of polyglycol solution and cohesion amine aqueous solution is 13:1, it is molten to add LISS
The volume ratio of liquid, LISS solution and cohesion amine aqueous solution is 8:1；Add bovine serum albumin(BSA), bovine serum albumin(BSA) and cohesion amine
The volume ratio of solution is 5:1；The pH value for adjusting solution with hydrochloric acid or sodium hydroxide is 7, obtains the indirect antihuman globulin
Experiment accelerates reinforcing agent.
Embodiment 13
A kind of indirect antihuman globulin test provided by the invention accelerates reinforcing agent, including a concentration of 5%, molecular weight be
5800 polyethylene glycol (PEG) solution and a concentration of 3% cohesion amine (polybrene) solution, the polyglycol solution and
The volume ratio for agglomerating amine aqueous solution is 14:1, bovine serum albumin(BSA), bovine serum albumin(BSA) is 10 with the volume ratio for agglomerating amine aqueous solution:
1；
The indirect antihuman globulin test accelerates the preparation method of reinforcing agent, includes the following steps：
Polyethylene glycol pulvis is dissolved in be made into distilled water a concentration of 5% polyethylene glycol (PEG) solution, amine will be agglomerated
(polybrene) pulvis be dissolved in be made into distilled water a concentration of 3% cohesion amine (polybrene) solution, then will agglomerate amine
Solution is added in polyglycol solution, and the volume ratio of polyglycol solution and cohesion amine aqueous solution is 14:1, add cow's serum
The volume ratio of albumin, bovine serum albumin(BSA) and cohesion amine aqueous solution is 10:1；The pH of solution is adjusted with hydrochloric acid or sodium hydroxide
Value is 7, obtains the indirect antihuman globulin test and accelerates reinforcing agent.
Embodiment 14
An embodiment of the present invention provides a kind of micro-column gel antihuman globulin reagent card, have at least 6 on the reagent card
A micro-column gel pipe, the present embodiment select 6, there is gel suspension medium, the gel suspension to be situated between in the micro-column gel pipe
Matter contains any one indirect antihuman globulin test described in Examples 1 to 6 or embodiment 11~13 and accelerates reinforcing agent.
The formula of the gel suspension medium is as follows：Potassium dihydrogen phosphate 0.15-0.3g/L, disodium hydrogen phosphate 0.35-
0.65g/L, sodium chloride 1.8-2.0g/L, the indirect antihuman globulin test that herein prepared by selection example 3 accelerate reinforcing agent 3-
5%, polyethylene glycol 0.08-0.15%, Sodium azide 0.3%；
The purifying water dissolution of the above reagent, adjustment pH value are 6.6-7.
Embodiment 15
An embodiment of the present invention provides a kind of micro-column gel anti-human immunoglobulin crossmatching card, at least have on the reagent card
There are 6 micro-column gel pipes, the present embodiment to select 8, have gel suspension medium, the gel outstanding in the micro-column gel pipe
Floating medium contains any one indirect antihuman globulin test described in Examples 1 to 6 or embodiment 11~13 and accelerates reinforcing agent.
The formula of the gel suspension medium is as follows：Potassium dihydrogen phosphate 0.15-0.3g/L, disodium hydrogen phosphate 0.35-
0.65g/L, sodium chloride 1.8-2.0g/L, the indirect antihuman globulin test that herein prepared by selection example 3 accelerate reinforcing agent 3-
5%, polyethylene glycol 0.08-0.15%, Sodium azide 0.3%；
The purifying water dissolution of the above reagent, adjustment pH value are 6.6-7.
Embodiment 16
An embodiment of the present invention provides a kind of red blood cell irregular antibody detection kit, the kit includes separation
Chamber, the indirect antihuman globulin test that the reacting hole endoperidium of disengagement chamber has embodiment 4 to prepare accelerate reinforcing agent.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, various changes and improvements may be made to the invention without departing from the spirit and scope of the present invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and
Its equivalent defines.
Claims (10)
1. a kind of indirect antihuman globulin test accelerates reinforcing agent, it is characterised in that：Include a concentration of 1~60% polyethylene glycol
Solution and a concentration of 0.1~5% cohesion amine aqueous solution, the polyglycol solution and cohesion amine aqueous solution volume ratio be (10~
20):1。
2. indirect antihuman globulin test according to claim 1 accelerates reinforcing agent, it is characterised in that：The polyethylene glycol
Molecular weight be 2000~8000；
Preferably, the molecular weight of the polyethylene glycol is 4000~6000.
3. indirect antihuman globulin test according to claim 1 or 2 accelerates reinforcing agent, it is characterised in that：It is described indirect
It further includes LISS solution and/or bovine serum albumin(BSA) that antihuman globulin test, which accelerates reinforcing agent,；
Preferably, the indirect antihuman globulin test accelerates in reinforcing agent, the volume of the LISS solution and cohesion amine aqueous solution
Than for (5~10):1；The volume ratio of the bovine serum albumin(BSA) and cohesion amine aqueous solution is (5~10):1.
4. a kind of indirect antihuman globulin test as claimed in claim 1 or 2 accelerates the preparation method of reinforcing agent, feature to exist
In：Include the following steps：
Polyethylene glycol pulvis is dissolved in be made into distilled water a concentration of 1~60% polyglycol solution, by cohesion amine pulvis it is molten
It is made into a concentration of 0.1~5% cohesion amine aqueous solution in distilled water, cohesion amine aqueous solution is then added to polyglycol solution
In, the volume ratio of polyglycol solution and cohesion amine aqueous solution is (10~20):1, the pH value for adjusting solution is 6.5-7, obtains institute
It states indirect antihuman globulin test and accelerates reinforcing agent.
5. a kind of indirect antihuman globulin test as claimed in claim 3 accelerates the preparation method of reinforcing agent, it is characterised in that：
Include the following steps：
Polyethylene glycol pulvis is dissolved in be made into distilled water a concentration of 1~60% polyglycol solution, by cohesion amine pulvis it is molten
It is made into a concentration of 0.1~5% cohesion amine aqueous solution in distilled water, cohesion amine aqueous solution is then added to polyglycol solution
In, the volume ratio of polyglycol solution and cohesion amine aqueous solution is (10~20):1, add LISS solution, LISS solution and cohesion
The volume ratio of amine aqueous solution is (5~10):1；Or bovine serum albumin(BSA) is added, the volume of bovine serum albumin(BSA) and cohesion amine aqueous solution
Than for (5~10):1；Or LISS solution and bovine serum albumin(BSA) are added simultaneously, LISS solution and the volume ratio of cohesion amine aqueous solution are
(5~10):1, bovine serum albumin(BSA) is (5~10) with the volume ratio for agglomerating amine aqueous solution:1；The pH value for adjusting solution is 6.5-7,
It obtains the indirect antihuman globulin test and accelerates reinforcing agent.
6. a kind of indirect antihuman globulin test as described in any one of claims 1 to 3 accelerates reinforcing agent in traditional test tube method
Application in antihuman globulin test.
7. a kind of indirect antihuman globulin test as described in any one of claims 1 to 3 accelerates reinforcing agent in micro-column gel agglutination assay
Application in antihuman globulin test.
8. a kind of micro-column gel antihuman globulin reagent card, it is characterised in that：There is at least six micro-column gel on the reagent card
It manages, there is gel suspension medium, the gel suspension medium to contain in the micro-column gel pipe such as any one of claims 1 to 3
The indirect antihuman globulin test accelerates reinforcing agent.
9. micro-column gel antihuman globulin reagent card according to claim 8, which is characterized in that the gel suspension medium
Formula it is as follows：
Potassium dihydrogen phosphate 0.15-0.3g/L；
Disodium hydrogen phosphate 0.35-0.65g/L；
Sodium chloride 1.8-2.0g/L；
Indirect antihuman globulin test accelerates reinforcing agent 3-5%；
Polyethylene glycol 0.08-0.15%；
Sodium azide 0.3%；
The purifying water dissolution of the above reagent, adjustment pH value are 6.6-7.
10. a kind of red blood cell irregular antibody detection kit, which is characterized in that the kit includes disengagement chamber, disengagement chamber
Reacting hole endoperidium there is indirect antihuman globulin test as described in any one of claims 1 to 3 to accelerate reinforcing agent.
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