CN106940377B - A kind of gel suspension buffer solution, the gel obtained by its processing, detection blocks and its application - Google Patents
A kind of gel suspension buffer solution, the gel obtained by its processing, detection blocks and its application Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The present invention provides that a kind of gel suspension buffer solution, the gel obtained by its processing, detection blocks and its application.The gel suspension buffer solution includes following ingredient：0.7 0.99g/L imidazoles, 0.6 0.9g/LNaCl, 0.05 0.2%Tween 20,0.05 0.2%Proclin 300 and 1 3% glucan.The cushioning effect of the gel suspension buffer solution of the present invention is strong, improve suspending power, slow down the sinking speed of gel, overcome the problems, such as that filling difficulty is big, and the gel suspension buffer solution of the present invention has broad spectrum antibiotic activity, and correlated response with enzyme or antibody is not interfered with, ingredient is simple, it is easy to prepare, makes gel be more easy to suspend using the gel suspension buffer solution when preparing blood type test card, extends the sedimentation time, the bubble excluded in gel is filtered by vacuum simultaneously, it is filling to be easy to gel, obtains bubble-free and foreign matter between gel particle, improves accuracy in detection.
Description
Technical field
The invention belongs to medical material tech field, be related to a kind of gel suspension buffer solution, the gel obtained by its processing,
Detection card and its application.
Background technology
In clinical treatment, blood transfusion is to rescue important means and the measure of sick and wounded's life.To ensure the safety of blood transfusion, choosing
With reliable experimental method, it is very important using specificity, the experimental technique of high sensitivity.Blood group is the master of human blood
One of feature is wanted, the inhereditary feature of the various composition antigens of blood is expressed, wherein abo blood group and RhD blood groups becomes blood transfusion and device
Important blood group system in official's transplanting.Early in 1907, Hoktoen just first proposed the typing of blood to the important for the treatment of of blood transfusion
Property, the concept that Ottenberg etc. was further provided in 1908 for cipient blood cross matching more emphasizes the typing of blood
To the far reaching significance of clinical blood transfusion.The incompatible blood transfusion of abo blood group, will almost occur hemolytic blood transfusion reaction symptom without exception：
Disseminated intravascular coagulation, renal failure are so that death.Therefore, the accurately sizing of abo blood group is most important.The Ministry of Public Health issues within 2000
's《Clinical blood transfusion technical specification》(defending doctor's hair [2000] 184) chapter 4 Article 15 clear stipulaties：" Blood Transfusion Dept. (blood bank) is wanted
Verification blood transfusion request slip, receptor and blood donor's blood sample item by item check receptor and blood donor's abo blood group (positive reverse type), and
Rh (D) blood group of routine inspection patient, can carry out cross matching when correct." " detection ABO is especially emphasized in British Pharmacopoeia
Blood group will detect two aspect of antibody in red cell antigens and serum or blood plasma." nineteen ninety, Lapierre delivered micro-column gel and exempted from
Epidemic disease experimental technique, this is the milestone of human erythrocyte's typology development.U.S.'s transfusion science meeting《Blood transfusion technical manual》12nd edition
It rises within (1996) and just the technology has been included in the routine techniques of erythrocyte blood type detection.It nowadays, should in advanced country in the world
Hemagglutination test of the new technology just in Replacing for many years is applied in the routine clinical inspection work of erythrocyte blood type serology.
Antihuman globulin test, also known as Coombs ' are tested, and are the important of diagnosis of autoimmune hemolytic anemia (AIHA)
Foundation.Straightway testing be check be detected red blood cell on whether there is or not incomplete antibody, indirect test be check serum in dissociate it is endless
Whole antibody.Direct Coombs ' experiments more have diagnostic value compared with indirect test to AIHA.It can be by AIHA with specific monovalent antiserum
It is divided into three types：IgG/C3 is positive, accounts for 67%；Independent IgG, accounts for 20%；Independent C3, accounts for 13%；Antihuman globulin detection card,
It is mainly used for the application of blood group cross matching, irregular antibody screening and irregular antibody identification etc., refers specifically to using micro-
Column gel technique carries out direct or indirect antihuman globulin test, i.e. Coombs ' experiments.Microcolumn gel technology is 1984
Yves lapierre inventions, Switzerland's DiaMed introduces patented product to the market within 1988.Its principle is based on free red blood cell
With aggregation red blood cell whether the colloidal medium being made up of gel particle with molecular sieve effect, to make the red of different conditions
Cell is detached, and is a kind of agglutinating reaction carried out in microtrabeculae Guan Zhongyong gel medias.Micro-column gel broad spectrum antihumanglobulin
Reagent detection card is microcolumn gel technology and the novel product that antihuman globulin test method is combined, and is answered extensively in recent years
With.Cumbersome washing procedure is omitted and in feminine gender in antihuman globulin test of the microcolumn gel technology relative to traditional test tube method
The red blood cell of addition IgG type antibodies sensitization in reaction tube, the step whether accurate and reliable for confirming negative reaction, and it is micro-
Column gel technique is small using specimen amount, easy to operate, interference from human factor is small, is easy to normalizing operation and automation mechanized operation, ties
The advantages that fruit is easy to judge, convenient for storage and check, is suitable for the detection of extensive sample.
Have some producers both at home and abroad at present and produce the micro-column gel card of various different function, but comes with some shortcomings：One
Be gel suspension buffer solution suspension it is poor, when filling, gel sinking speed is fast, and difficulty is big when causing gel filling, second is that
Gel before filling the container, bubble operation is not exhausted, causes product qualified rate after filling card low.
Therefore, this field it is expected exploitation it is a kind of can effectively solve gel it is filling when suspension sex chromosome mosaicism and it is filling after
There is the micro-column gel detection card of air bubble problem.
Invention content
In view of the deficiencies of the prior art, it is handled the purpose of the present invention is to provide a kind of gel suspension buffer solution, by it
Gel, detection card and its application arrived.
To reach the invention purpose, the present invention uses following technical scheme：
On the one hand, the present invention provides a kind of gel suspension buffer solution, and the gel suspension buffer solution includes following ingredient：
In the present invention, by using the gel suspension buffer solution containing ingredient as described above, not only cushioning effect is strong, and
And after there are environment, and imidazole solution is very stable, and glucan is dissolved in water, its suspension energy can be improved closer to human erythrocyte
Power, slows down the sinking speed of gel wherein, and sodium salt then ensure that the ionic strength of gel suspension buffer solution.Using
Proclin300 has broad spectrum antibiotic activity, can inhibit bacterium, fungi and yeasts within the long time as preservative
The growth of equal microorganisms, meanwhile, it has the good compatibility with enzyme again, does not interfere with the correlated response with enzyme or antibody,
Dosage can reach antibacterial purpose less, and dosage is relatively low to person poultry harmless.Using polysorbas20 as lubricant, it
It is nonionic surfactant, has the effects that emulsification, diffusion, solubilising, stablizes, it is main so that red blood cell passes through gel gap
When obtain suitable lubricating ability, enable the red blood cell of no agglutination completely by gel particle gap, and the red blood cell being aggregated is then
It is stayed in gel glue surface or more or gel rubber column gel column by gel barrier.
In the present invention, in the gel suspension buffer solution imidazoles content be 0.7-0.99g/L, such as 0.7g/L,
0.73g/L、0.75g/L、0.78g/L、0.8g/L、0.83g/L、0.85g/L、0.88g/L、0.9g/L、0.93g/L、0.95g/L
Or 0.99g/L.
In the present invention, in the gel suspension buffer solution NaCl content be 0.6-0.9g/L, such as 0.6g/L,
0.63g/L、0.65g/L、0.68g/L、0.7g/L、0.74g/L、0.78g/L、0.8g/L、0.83g/L、0.85g/L、0.88g/L
Or 0.9g/L.
In the present invention, the mass percentage of Tween 20 is 0.05-0.2%, example in the gel suspension buffer solution
Such as 0.05%, 0.08%, 0.1%, 0.12%, 0.14%, 0.16%, 0.18% or 0.2%.
In the present invention, the mass percentage of Proclin 300 is 0.05-0.2% in the gel suspension buffer solution,
Such as 0.05%, 0.08%, 0.1%, 0.12%, 0.14%, 0.16%, 0.18% or 0.2%.
In the present invention, in the gel suspension buffer solution glucan mass percentage be 1%, 1.2%, 1.4%,
1.6%, 1.8%, 2%, 2.3%, 2.5%, 2.7%, 2.9% or 3%.
Ingredient as described above is dissolved in purified water in the present invention and obtains the gel suspension buffer solution.
Preferably, the gel suspension buffer solution includes following ingredient：
Preferably, the pH of the gel suspension buffer solution is 7.2~7.4, such as 7.2,7.25,7.3,7.35 or 7.4.
On the other hand, the present invention provides a kind of gel handled by gel suspension buffer solution as described above.
Preferably, the method for the processing is：Polyacrylamide gel is centrifuged, after discarding supernatant, using isometric
Purified water is cleaned 3-5 times, removes gel breakage fragment and salt ion excessive in the gel particle and gel of aggregation, then again
Gel is cleaned using the physiological saline of equivalent 3 times, after reusing gel suspension buffer solution for cleaning at least twice, is discarded supernatant, is obtained
To the gel.
The gel particle that the present invention is handled is uniform in size and complete spherical in shape, clear using the gel suspension buffer solution
The sinking speed of gel can be slowed down after washing, difficulty when reduction gel is filling is easy to filling.
On the other hand, the present invention provides a kind of detection card, and the detection card includes gel suspension buffering as described above
The gel that liquid is handled.
Preferably, detection card is blood type test card, and the blood type test card includes six micro-pipes, in first micro-pipe
The working solution of the gel and anti-A monoclonal antibodies that are handled containing gel suspension buffer solution as described above, second micro-pipe
In the working solution of gel and anti-B monoclonal antibodies that handles containing gel suspension buffer solution as described above, third is micro-
The working solution of the gel and anti-D monoclonal antibodies that are handled containing gel suspension buffer solution as described above in pipe, the 4th to
It is the neutral gel without any antibody in 6th micro-pipe.
Preferably, the working solution of first micro-pipe moderate resistance A monoclonal antibodies handles to obtain with gel suspension buffer solution
Gel volume ratio be 2:(2-5), such as 2:2、2:2.3、2:2.5、2:2.8、2:3、2:3.3、2:3.5、2:3.8、2:4、
2:4.3、2:4.5、2:4.7 or 2:5, preferably 2:3.
Preferably, the working solution of second micro-pipe moderate resistance B monoclonal antibodies handles to obtain with gel suspension buffer solution
Gel volume ratio be 2:(2-5), such as 2:2、2:2.3、2:2.5、2:2.8、2:3、2:3.3、2:3.5、2:3.8、2:4、
2:4.3、2:4.5、2:4.7 or 2:5, preferably 2:3.
Preferably, the working solution of the third micro-pipe moderate resistance D monoclonal antibodies handles to obtain with gel suspension buffer solution
Gel volume ratio be 2:(2-5), such as 2:2、2:2.3、2:2.5、2:2.8、2:3、2:3.3、2:3.5、2:3.8、2:4、
2:4.3、2:4.5、2:4.7 or 2:5, preferably 2:3.
Preferably, the preparation method of the neutral gel is：By gel suspension buffer solution as described above and gel suspension
The gel that buffer solution is handled is according to volume ratio 2:(2-5) (such as 2:2、2:2.3、2:2.5、2:2.8、2:3、2:3.3、2:
3.5、2:3.8、2:4、2:4.3、2:4.5、2:4.7 or 2:5) it is mixed to get the neutral gel.
Preferably, the gel that the gel suspension buffer solution is handled with gel suspension buffer solution is according to volume ratio 2:3
Mixing.
Preferably, it is coagulated using what gel suspension buffer solution was handled to described before filling to first to the 6th micro-pipe
Glue and neutral gel are filtered by vacuum, and bubble is discharged.So that bubble-free and foreign matter between obtained gel particle.
Gel is filtered by vacuum in the present invention, after bubble is discharged, is added respectively according to 26~30 microlitres of amount of every pipe
Enter to the corresponding position in six micro-pipes of Blank gel card, obtains ABO, RhD blood typing detection card.
The present invention is right in order to ensure the quality of ABO, RhD blood typing prepared detection card and antihuman globulin detection card
Antibody starting material for configuration is screened.
Preferably, working solution potency >=128 of the anti-A monoclonal antibodies.
Preferably, the working solution of the anti-A monoclonal antibodies dilutes anti-A monoclonal antibodies by gel suspension buffer solution and obtains
It arrives.
Preferably, working solution potency >=128 of the anti-B monoclonal antibodies.
Preferably, the working solution of the anti-B monoclonal antibodies dilutes anti-B monoclonal antibodies by gel suspension buffer solution and obtains
It arrives.
Preferably, working solution potency >=64 of the anti-D monoclonal antibodies.
Preferably, the working solution of the anti-D monoclonal antibodies dilutes anti-D monoclonal antibodies by gel suspension buffer solution and obtains
It arrives.
Preferably, the detection card is antihuman globulin detection card, and the antihuman globulin detection card includes six micro-pipes,
Contain antihuman globulin gel in six micro-pipes, the antihuman globulin gel is by antihuman globulin reagent and institute as above
The gel that the gel suspension buffer solution stated is handled is according to volume ratio 2:(2-5) is mixed to get.
I.e. in the present invention, the preparation method of the antihuman globulin gel is：By antihuman globulin reagent and institute as above
The gel that the gel suspension buffer solution stated is handled is according to volume ratio 2:(2-5) (such as 2:2、2:2.3、2:2.5、2:2.8、
2:3、2:3.3、2:3.5、2:3.8、2:4、2:4.3、2:4.5、2:4.7 or 2:5) it is mixed to get the antihuman globulin gel.
Preferably, the gel that the antihuman globulin reagent is handled with the gel suspension buffer solution is according to volume
Than 2:3 mixing.
In the present invention, the antihuman globulin reagent is broad spectrum antihumanglobulin reagent (anti-igg+C3d), with IgG
Positive reaction occurs for the anti-D sensitized erythrocytes of type or C3d sensitized erythrocytes, with not only without IgG antibody but also without C3d antibody sensitizeds it is red carefully
Negative reaction occurs for born of the same parents.
Preferably, it is coagulated using what gel suspension buffer solution was handled to described before filling to first to the 6th micro-pipe
Glue and antihuman globulin gel are filtered by vacuum, and bubble is discharged.So that bubble-free and different between obtained gel particle
Object.
Gel is filtered by vacuum in the present invention, after bubble is discharged, is added respectively according to 26~30 microlitres of amount of every pipe
Enter to the corresponding position in six micro-pipes of Blank gel card, obtains antihuman globulin detection card.
On the other hand, the application being stuck in the present invention provides detection as described above in blood group or antihuman globulin detection.
Compared with the existing technology, the invention has the advantages that：
The present invention gel suspension buffer solution cushioning effect it is strong, closer to human erythrocyte there are environment, improve
Suspending power slows down the sinking speed of gel, overcomes the problems, such as that filling difficulty is big, and the gel suspension buffer solution tool of the present invention
There is broad spectrum antibiotic activity, and do not interfere with the correlated response with enzyme or antibody, ingredient is simple, is easy to prepare, and is preparing blood group inspection
The gel suspension buffer solution is utilized when surveying card, gel is made to be more easy to suspend, extends the sedimentation time, while arranging using vacuum filtration method
Except the bubble in gel, then progress gel is filling, obtains bubble-free and foreign matter between gel particle, improves accuracy in detection.
Description of the drawings
Fig. 1 is the structural schematic diagram of the detection card of the present invention, and wherein 1-6 is respectively first micro-pipe, second micro-pipe, the
Three micro-pipes, the 4th micro-pipe, the 5th micro-pipe and the 6th micro-pipe.
Specific implementation mode
The technical solution further illustrated the present invention below by specific implementation mode.Those skilled in the art should be bright
, the embodiment, which is only to aid in, understands the present invention, should not be regarded as a specific limitation of the invention.
Embodiment 1
(1) in the present embodiment, the formula of gel suspension buffer solution is as follows：
The purifying water dissolution of the above reagent, adjustment pH is 7.2~7.4, obtains gel suspension buffer solution.
(2) the gel suspension buffer solution obtained using the present embodiment prepares gel, and preparation method is as follows：
First polyacrylamide gel is centrifuged, after discarding supernatant, is cleaned 3-5 times using isometric purified water, is removed solidifying
Then glue breakage fragment and salt ion excessive in the gel particle and gel of aggregation reuse the physiological saline cleaning of equivalent
Gel 3 times discards supernatant after reusing gel suspension buffer solution for cleaning twice, obtains even particle size and complete spherical in shape
Gel.
(3) selection of antibody
The anti-A antibody of IgM properties is selected, and makes its potency >=128 using the dilution of gel suspension buffer solution, it is molten to obtain anti-A
Liquid.
The anti-B antibody of IgM properties is selected, and makes its potency >=128 using the dilution of gel suspension buffer solution, it is molten to obtain anti-B
Liquid.
The anti-D of IgM properties is selected, and makes its potency >=64 using the dilution of gel suspension buffer solution, it is molten to obtain anti-D
Liquid.
(4) preparation of gel
The preparation of specific gel：The anti-solution A of step 3, anti-B solution, anti-solution D is taken to be prepared with step 2 respectively solidifying
Glue is according to 2:3 ratios are uniformly mixed, and the methylenum careuleum of final concentration of 0.02mg/mL is then added in the gel that anti-solution A is prepared,
Anti- B solution prepare gel in be added the tartrazines of final concentration of 0.1mg/mL to get respectively contain anti-A monoclonal antibodies,
The specific gel of anti-B monoclonal antibodies, anti-D monoclonal antibodies.
The preparation of neutral gel：Take the gel that the gel suspension buffer solution that step 1 is prepared is prepared with step 2 according to 2:3
Ratio is uniformly mixed to get neutral gel.
(5) gel is filling
Fifty-fifty ready gels that step 4 is prepared carry out suction filtration discharge bubble before packing using vacuum filtration pump,
Then it is added separately to the corresponding position in six micro-pipes of Blank gel card according to the amount of 26~30 microlitres of every pipe, first micro-
The specific gel containing anti-A monoclonal antibodies is added in pipe, is added in second micro-pipe special containing anti-B monoclonal antibodies
Property gel, the solidifying specific gel containing anti-D monoclonal antibodies is added in third micro-pipe, is added in the 4th to the 6th micro-pipe
Neutral gel without any antibody forms ABO, RhD blood typing detection card.
(6) it seals
Heating power plastic packaging is carried out to the nozzle of micro-column gel card using aluminium foil, obtains blood type test card.
Embodiment 2：
ABO, RhD blood typing the detection card being prepared using embodiment 1 carry out Blood grouping, and method is as follows：
ABO, RhD blood typing detection card are filled in six microtrabeculae type pipes on card side by side by polypropylene plastics transparent card
Sephadex is filled out, sequence first to filling anti-A, anti-B, anti-D monoclonal antibodies in third Zhi Weiguan gels respectively from left to right
IgM reagents, referred to as specific gel, detect human red blood cells ABO, RhD antigen.There was only gel in 4th to the 6th micro-pipe,
Referred to as neutral gel, the 4th micro-pipe is as negative control pipe；5th and the 6th micro-pipe are as abo blood group reverse type pipe.
By six gel micro-pipe labels of micro-column gel reagent card, as shown in Figure 1.
0.5%~0.8% red cell suspension of person to be checked is separately added into first to fourth micro-pipe, often 50 μ L of pipe.
Person's serum to be checked is separately added into the 5th and the 6th micro-pipe, often 50 μ L of pipe.
Known A types and 0.5%~0.8% concentration suspension of Type B red blood cell are separately added into the 5th and the 6th micro-pipe, often
50 μ L of pipe.
At once it uses special centrifugal machine to centrifuge 5 minutes (900rpm 2min, 2500rpm 3min), takes out naked eyes judgement knot
Fruit.Judgement result is shown in Table 1.
The standard of result judgement is as follows：
Positive findings：Red cell antigens and the specific antigen-antibody complex that corresponding antibodies are formed in micro-column gel are floating
It is positive reaction in gel surface or glue.
Response intensity：Specific erythrocyte antigen antibody complex is located at glue surface, is strong positive reaction；Compound is in glue
In for weakly positive react；More smaller close to glue bottom particles, reaction is weaker.
Negative findings：Tested red cell antigens are combined without corresponding antibody, specific antigen-antibody complex do not occur, red
Cell is sunken to the bottom of micro-column gel.
Table 1
Embodiment 3
(1) in the present embodiment, the formula of gel suspension buffer solution is as follows：
The purifying water dissolution of the above reagent, adjustment pH is 7.2~7.4, obtains gel suspension buffer solution.
(2) the gel suspension buffer solution obtained using the present embodiment prepares gel, and preparation method is as follows：
First polyacrylamide gel is centrifuged, after discarding supernatant, is cleaned 3-5 times using isometric purified water, is removed solidifying
Then glue breakage fragment and salt ion excessive in the gel particle and gel of aggregation reuse the physiological saline cleaning of equivalent
Gel 3 times discards supernatant after reusing gel suspension buffer solution for cleaning twice, obtains even particle size and complete spherical in shape
Gel.
(3) selection of antibody
Broad spectrum antihumanglobulin reagent (anti-igg+C3d) is selected, with the anti-D sensitized erythrocytes of IgG types or C3d sensitized erythrocytes
Occur positive reaction, with not only without IgG antibody again without the red blood cell of C3d antibody sensitizeds generation negative reaction.
(4) preparation of gel
The preparation of antihuman globulin gel：The broad spectrum antihumanglobulin reagent that step 3 selects is taken to be prepared with step 2 solidifying
Glue is according to 2:3 ratios are uniformly mixed to get semi-finished product antihuman globulin gel.
(5) gel is filling
Fifty-fifty ready gels that step 4 is prepared carry out suction filtration discharge bubble before packing using vacuum filtration pump,
Then it is added separately in six micro-pipes of Blank gel card according to the amount of 26~30 microlitres of every pipe, forms antihuman globulin detection
Card.
(6) it seals
Heating power plastic packaging is carried out to the nozzle of micro-column gel card using aluminium foil, obtains blood type test card..
Embodiment 4
The antihuman globulin detection card being prepared using embodiment 3 carries out antihuman globulin detection, and method is as follows：
This product ties up to filling sephadex and anti-human ball egg in six microtrabeculae type pipes of polypropylene plastics see-through card on piece
(how anti-) in vain, is made antihuman globulin detection card, and gel micro-pipe is divided into reaction chamber and gel splitter two parts.
The method of inspection is as follows：
Direct antiglobulin test (DAT)
The micro-pipe of micro-column gel reagent card is marked, as shown in Figure 1.
It will be made into 0.5%~0.8% red blood cell physiological saline suspension after each person's red blood cell brine to be checked.
Negative control red blood cell：The O-shaped red blood cell of healthy male is made into 0.5%~0.8% red blood cell physiological saline suspension.
Each 0.5%~0.8% red blood cell physiological saline suspension of person, 50 μ l to be checked are added in No. 1 to No. 5 pipe, negative control
50 μ l of red blood cell are added in No. 6 pipes.
At once it uses special centrifugal machine to centrifuge 5 minutes (900rpm 2 minutes, 2500rpm 3 minutes), takes out, naked eyes judge
As a result.
Indirect antihuman globulin test (IAT)
The micro-pipe of micro-column gel reagent card is marked, as shown in Figure 1.
The low solion suspension of the O-shaped red blood cell of 0.5%~0.8% standard (being derived from healthy adult male) (is only applicable to
Cross matching and Irregular antibodies screening) it is separately added into 1-6 pipes, every 50 μ L of pipe.
Negative control sera：It is derived from healthy male AB type serum.
Each 50 μ L of serum to be checked are added in No. 1 pipe to No. 5 pipes, 50 μ L of negative control sera are added in No. 6 pipes.
Reagent card after sample-adding is set in 37 DEG C of couveuses 15 minutes.
At once it uses special centrifugal machine to centrifuge 5 minutes (900rpm 2 minutes, 2500rpm 3 minutes), takes out, naked eyes judge
As a result.
Testing result：
If No. 6 pipes should be negative as a result, being positive as a result, centrifugation one time again, is still such as positive findings, should repeat the examination
It tests or considers gel splitter there may be quality problems.
When No. 6 pipe negative findings are set up：
Direct antiglobulin test
Be negative result in No. 1 pipe to No. 5 pipes, and surface red cell specimen is not in vivo by IgG sensitization.
Be positive result in No. 1 pipe to No. 5 pipes, and surface red cell specimen is in vivo by IgG sensitization.
Indirect antihuman globulin test
It is negative in No. 1 pipe to No. 5 pipes as a result, showing (or other to wait for without containing the O-shaped red blood cell of the standard in tested serum
Examine red blood cell sample) the IgG class incomplete antibodies of antigentic specificity.
It is positive in No. 1 pipe to No. 5 pipes as a result, showing (or other to be checked containing the O-shaped red blood cell of the standard in tested serum
Red blood cell sample) antigentic specificity IgG class incomplete antibodies.
Irregular antibodies screening
Reagent card is marked, as shown in Figure 1.Examinee's serum is detached, screening red blood cell or panel erythrocyte are made into
0.5%~0.8% concentration is separately added into the micro-pipe marked, each 1 drop (or 50 μ L).Examinee's serum 1 is added immediately respectively
It drips (or 50 μ L).
Reagent card after sample-adding is set in 37 DEG C and is incubated 15 minutes.Using special centrifugal machine centrifugation, (900rpm 2 divides within 5 minutes
Clock, 2500rpm 3 minutes), take out visual results.
As a result judge
According to the reaction pattern of the screening red blood cell or panel erythrocyte provided come judging result.
Cross matching
Receptor is detached with the red blood cell serum of blood donor.
Receptor and blood donor's red blood cell are made into 0.5%~0.8% physiological saline suspension respectively.
Receptor's serum 1 is dripped (or 50 microlitres) to drip in (or 50 microlitres) addition main side pipe with blood donor's red blood cell 1.
Receptor's red blood cell 1 is dripped (or 50 microlitres) to drip in (or 50 microlitres) addition time side pipe with blood donor sera 1.
Reagent card after sample-adding is set in 37 DEG C and is incubated 15 minutes.
5 minutes (900rpm 2 minutes, 2500rpm 3 minutes) is centrifuged using special centrifugal machine, takes out visual results.
Inspection result：
Agglutination shows as red blood cell and stays in gel separation media upper surface or be distributed in separating medium；It is not aggregated then table
It is now the bottom that red blood cell stays in microtrabeculae by separating medium.
Erythrocyte agglutination is positive findings, regardless of whether being aggregated, the presence of haemolysis is considered as positive findings：Cross matching
The positive findings of experiment show cross matching blood donor and receptor blood sample it is incompatible, Antibody screening experiment positive findings
Show to contain irregular antibody in person's blood plasma to be checked.
It is negative findings that no haemolysis and red blood cell, which are not aggregated,：Negative findings in cross match blood test show cross matching
The blood sample of blood donor and receptor are compatible, and the negative findings in Antibody screening experiment show that person's blood plasma to be checked does not contain and do not advise accordingly
Then antibody.
HDN Maternal immunoglobulin G antibody titers
Serum to be checked is taken, by the serum of same volume and DTT (or 2-Me) mixing, 37 DEG C are incubated 30~60 minutes.
Card is marked, the maternal serum that doubling dilution is handled through DTT (or 2-Me) in test tube.
50 μ L of doubling dilution serum are added in index aperture successively.
50 O-shaped 0.5%~0.8% red cell suspensions of μ L Healthy Peoples are added in all holes and (measure pregnant woman's ABO blood group system
IgG blood group antibodies in addition) or 50 μ l Healthy People A types are added in all holes or 0.5%~0.8% red cell suspension of Type B (measures
Pregnant woman ABO blood group system IgG blood group antibodies).
37 DEG C are incubated 15 minutes.
It is centrifuged 5 minutes with special centrifugal machine, 900rpm 2 minutes, 2500rpm 3 minutes.
It takes out, naked eyes judging result.
Neonatal hemolytic disease tire (baby) youngster's incomplete antibody
1~3 hole of card is used to detect free anti-A in Neonatal or blood plasma, the IgG type antibody outside anti-B and ABO systems；
The anti-A in liquid, the IgG type antibody outside anti-B and ABO systems are diffused for detecting newborn's red blood cell in 4th~6 hole.
Operating procedure：
Thermolysis：Red blood cell to be checked is taken, is washed 3~4 times with isotonic saline solution, 6% N of packed red cells and same volume is taken
Seralbumin mixing is set 56 DEG C of water-baths 10 minutes, is during which constantly shaken, 2000rpm, centrifuges 2~3 minutes, takes supernatant, that is, put
Dispersion liquid.
Reagent card is marked.
50 μ l of A1 types red cell suspension are separately added into 1,4 holes；50 μ l of Type B red cell suspension are separately added into 2,5 holes；It will
50 μ l of O-shaped red cell suspension are separately added into 3,6 holes.
1~3 hole is separately added into 50 μ l Neonatals, and 4~6 holes are separately added into 50 μ l and diffuse liquid.
37 DEG C are incubated 15 minutes.
It is centrifuged 5 minutes with special centrifugal machine, 900rpm 2 minutes, 2500rpm 3 minutes.
It takes out, according to newborn's blood group sentence read result.
Applicant state, the present invention by above-described embodiment come illustrate the present invention gel suspension buffer solution, by its processing
Obtained gel, detection card and its application, but the invention is not limited in above-described embodiments do not mean that the present invention must be according to
Bad above-described embodiment could be implemented.Person of ordinary skill in the field is it will be clearly understood that any improvement in the present invention, to this hair
The equivalence replacement of bright selected raw material and the addition of auxiliary element, the selection etc. of concrete mode, all fall within the protection model of the present invention
Enclose within the open scope.
Claims (24)
1. a kind of gel handled using gel suspension buffer solution, which is characterized in that the gel suspension buffer solution includes
Following ingredient：
The method of the processing is：Polyacrylamide gel is centrifuged, after discarding supernatant, 3- is cleaned using isometric purified water
5 times, gel breakage fragment and salt ion excessive in the gel particle and gel of aggregation are removed, the life of equivalent is then reused
It manages brine cleaning gel 3 times, after reusing gel suspension buffer solution for cleaning at least twice, discards supernatant, obtain the gel.
2. the gel according to claim 1 handled using gel suspension buffer solution, which is characterized in that the gel
Buffer suspension liquid includes following ingredient：
3. the gel according to claim 1 or 2 handled using gel suspension buffer solution, which is characterized in that described
The pH of gel suspension buffer solution is 7.2~7.4.
4. a kind of detection card, which is characterized in that the detection card includes as claimed in any one of claims 1-3 using solidifying
The gel that colloidal suspension buffer solution is handled.
5. detection card according to claim 4, which is characterized in that the detection card is blood type test card, the blood group inspection
It includes six micro-pipes to survey card, and gel and anti-A monoclonals that the gel suspension buffer solution is handled are contained in first micro-pipe
The working solution of antibody, the gel handled containing the gel suspension buffer solution in second micro-pipe and anti-B monoclonal antibodies
Working solution, the work of the gel and anti-D monoclonal antibodies that are handled containing the gel suspension buffer solution in third micro-pipe
Make liquid, is the neutral gel without any antibody in the 4th to the 6th micro-pipe.
6. detection card according to claim 5, which is characterized in that the work of first micro-pipe moderate resistance A monoclonal antibodies
The volume ratio for making the gel that liquid is handled with gel suspension buffer solution is 2:(2-5).
7. detection card according to claim 6, which is characterized in that the work of first micro-pipe moderate resistance A monoclonal antibodies
The volume ratio for making the gel that liquid is handled with gel suspension buffer solution is 2:3.
8. detection card according to claim 5, which is characterized in that the work of second micro-pipe moderate resistance B monoclonal antibodies
The volume ratio for making the gel that liquid is handled with gel suspension buffer solution is 2:(2-5).
9. detection card according to claim 8, which is characterized in that the work of second micro-pipe moderate resistance B monoclonal antibodies
The volume ratio for making the gel that liquid is handled with gel suspension buffer solution is 2:3.
10. detection card according to claim 5, which is characterized in that the work of the third micro-pipe moderate resistance D monoclonal antibodies
The volume ratio for making the gel that liquid is handled with gel suspension buffer solution is 2:(2-5).
11. detection card according to claim 10, which is characterized in that the third micro-pipe moderate resistance D monoclonal antibodies
The volume ratio for the gel that working solution is handled with gel suspension buffer solution is 2:3.
12. detection according to claim 5 card, which is characterized in that the preparation method of the neutral gel is：It will be described solidifying
The gel that colloidal suspension buffer solution is handled with gel suspension buffer solution is according to volume ratio 2:(2-5) is mixed to get the neutral condensate
Glue.
13. detection card according to claim 12, which is characterized in that the gel suspension buffer solution is buffered with gel suspension
The gel that liquid is handled is according to volume ratio 2:3 mixing.
14. detection card according to claim 5, which is characterized in that described before filling to first to the 6th micro-pipe
The gel and neutral gel handled using gel suspension buffer solution is filtered by vacuum, and bubble is discharged.
15. detection card according to claim 5, which is characterized in that the working solution potency of the anti-A monoclonal antibodies >=
128。
16. detection card according to claim 5, which is characterized in that the working solution of the anti-A monoclonal antibodies is hanged by gel
Floating buffer solution dilutes anti-A monoclonal antibodies and obtains.
17. detection card according to claim 5, which is characterized in that the working solution potency of the anti-B monoclonal antibodies >=
128。
18. detection card according to claim 5, which is characterized in that the working solution of the anti-B monoclonal antibodies is hanged by gel
Floating buffer solution dilutes anti-B monoclonal antibodies and obtains.
19. detection card according to claim 5, which is characterized in that the working solution potency of the anti-D monoclonal antibodies >=
64。
20. detection card according to claim 5, which is characterized in that the working solution of the anti-D monoclonal antibodies is hanged by gel
Floating buffer solution dilutes anti-D monoclonal antibodies and obtains.
21. detection card according to claim 4, which is characterized in that the detection card is antihuman globulin detection card, described
Antihuman globulin detection card includes six micro-pipes, contains antihuman globulin gel, the anti-human ball egg in six micro-pipes
The gel that white gel is handled by antihuman globulin reagent and the gel suspension buffer solution is according to volume ratio 2:(2-5) is mixed
It obtains.
22. detection card according to claim 21, which is characterized in that the antihuman globulin reagent is outstanding with the gel
The gel that floating buffer solution is handled is according to volume ratio 2:3 mixing.
23. detection card according to claim 21, which is characterized in that described before filling to first to the 6th micro-pipe
The gel and antihuman globulin gel handled using gel suspension buffer solution is filtered by vacuum, and bubble is discharged.
24. the detection according to any one of claim 4-20 is stuck in the application in Blood grouping.
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| CN105866444A (en) * | 2016-05-03 | 2016-08-17 | 江阴力博医药生物技术有限公司 | MNSs blood-group system detection reagent card and preparation method thereof |
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