CN1751130A - Preparation of red blood cells with a modified level of blood group antigen expression and their use in the quality control of blood typing reagents - Google Patents
Preparation of red blood cells with a modified level of blood group antigen expression and their use in the quality control of blood typing reagents Download PDFInfo
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Abstract
The invention is a method of preparing the red corpuscle of low-expression blood group antigen adopting at least one immunodominant glucoamylase, such as N-acetylgalactosaminase or Alpha-galactosidase. The antigen expressed by the red corpuscle which is prepared by the method only reaches the threshold value of the antigen level that can be detected at clinic substantively. The red corpuscle prepared by this process can be applied to the quality control of the blood group reagent and the adjustment of the testing system, therefore can judge the blood group accurately and standardly.
Description
Technical field
This invention relates to the cell that the blood group antigen expression level changes.Particularly, this invention relates to the preparation of these cells and in the quality control of blood grouping reagent and the correction and the affirmation of hematology, immunohematology and immune analysis.
Background technology
The function of Blood Center is to detect the blood group of blood with the accurate individual of judgement.For blood group accurately and judge for multinomial treatment it is essential accurately, comprise blood transfusion, organ transplantation with treat neonatal heredity haemolysis.
For example, a patient must know its blood group before accepting blood transfusion.The mispairing of blood donor and blood recipient's blood group will produce serious consequence, even can cause blood recipient's death.
In blood transfusion serology, the abo blood group classification is most important a kind of sorting technique in the erythrocyte blood type classification.People's blood group mainly is divided into four kinds of blood group: A, B, AB and O type.The red corpuscle of every kind of blood group carries A antigen respectively, B antigen, and A antigen and B antigen had not both had A antigen and had not had B antigen yet.
In everyone blood, there is the antigenic antibody that lacks in anti-abo blood group antigen or the red corpuscle.Therefore, contain the antigenic antibody of anti-B in the human blood of A type blood, contain the antigenic antibody of anti-A in the human blood of Type B blood, contain anti-A antigen and the antigenic antibody of B in the human blood of O type blood, the both also antigenic antibody of nonreactive B of the antigenic antibody of nonreactive A in the human blood of AB type blood.
From the blood donor before blood recipient blood transfusion, must carry out cross matching.Cross matching is by the rejection of direct detection blood donor blood to blood recipient's serum, or the record of the blood donor of root army and blood recipient's blood group matches.Cross matching need guarantee that a kind of red corpuscle of blood group can not supply with the individual that can produce antibody to the antigen of this blood group.
Historically, directly testing blood donor's red corpuscle can detect the error measurement of faint hypotype to the cross matching of blood recipient's serum rejection property and attempt to the uncompatibility between the inconsistent acceptor blood transfusion.Yet directly the cross matching method of test is seldom used at most of Blood Center now, the substitute is the correct record that depends on blood group.
In blood group serology, erythrocytic mensuration is to adopt the reagent (forward grouping) that contains at the antibody of specific antigen, measures serum to expressing the erythrocytic rejection (reverse packet) of known antigens.
Since the eighties in 20th century, monoclonal antibody (Mabs) has been used in the blood group determination reagent.Compare with traditional polyclonal antiserum, grouping by monoclonal reagents provides higher specificity, Yi Zhi reactivity more, and can improve sensitivity under most of situation.
The quality control of blood grouping reagent is necessary for the accuracy and the credibility of blood group determination.The reduction of specificity and/or susceptibility can appear in blood grouping reagent in transportation and storage process, or the pollution that causes in preparation and use.
In the mankind, low-level A antigen of various expression and/or the antigenic ABO hypotype of B are arranged.Antigenic expression level is widely different in each hypotype, is not difficult for finding out if analyze widely usually.The antigen levels of most of A type blood and rare A hypotype is can received (each erythrocytic antigen molecule number) usually in following scope:
A1,8 * 10
5To 1.2 * 10
6
A2,1.5 * 10
5To 4 * 10
5
A3,4 * 10
4To 1.2 * 10
5
Ax, 7 * 10
3To 10
4
Aend, 2 * 10
3To 3 * 10
3
Am, 10
2To 2 * 10
3
Ael, 10
2To 1.5 * 10
3.
The B hypotype also has the corresponding antigen expression level.Blood grouping reagent should be able to detect all significant clinically ABO hypotypes.
From the target of quality control, blood grouping reagent is used for measuring red corpuscle.Therefore antigen levels is expressed low red corpuscle and is more suitable for being used as " quality control cell " (perhaps " standard reagent ").
The red corpuscle that antigenic expression is low is suitable for the blood group determination analysis, because this red corpuscle can provide the more the more information of clonal antibody reagent probable behavior.
These red corpuscle can be used as the quality control cell, be used for measuring the variation of reagent, this variation can cause detecting the red corpuscle of expressing low-level antigenic blood subtypes, for example: the level of the red corpuscle antigen expressed of A2 type is in the low side that trends towards tolerance interval, thereby the mistake that causes blood group to detect takes place.
These red corpuscle can also be used to proofreading and correct and confirming detection system, can be detected with the abo blood group and the hypotype that guarantee all clinical meanings.
In fact, be very difficult with the red corpuscle of expressing the antigenic natural ABO hypotype of low-level A antigen and/or B as the quality control cell.The ratio that has such phenotype among the crowd is very low.For example: according to estimates, the individual of Ax blood group is 0.003% of an A type blood individual quantity.The ratio in the crowd of other hypotypes is also low.
Do not express in the low-level antigenic cell at those, blood group determination reagent is to estimate like this:
Mensuration is to Normocellular rejection.(this comprises the high-level relatively antigenic red corpuscle of use expression, but this method lacks sensitivity); Or
Dilution blood group determination reagent.(this comprises dilution blood group determination reagent and measures it to Normocellular rejection).
A lot of laboratories only depend on measuring the quality control of reagent suppliers.
The evaluation of blood group determination reagent generally is the method that adopts the dilution of blood group determination reagent.Yet the laboratory can only be with weekly or every month be that all groups after group at different time detects blood group determination reagent.
Dilution blood group determination reagent and detection are also unreliable to the method for Normocellular rejection.Normal cell is expressed high-caliber antigen, and for example, each erythrocytic antigen molecule number is greater than 1.5 * 10
5When detecting blood group determination reagent, these reagent are diluted to lower extent of dilution, still can with the red corpuscle effect, thereby produce seropositive result.Handle these results to judge the antigenic expression under the normal extent of dilution with extrapotation.
This method is except this shortcoming that expends time in, and this method has also been done following supposition: the sensitivity of blood group determination reagent prediction can be extended to measure expresses low-level antigenic red corpuscle.Measure the required threshold value of some ABO hypotypes unless the real sensitivity of blood group determination reagent just drops to, otherwise the rotten of reagent can't be detected.The rotten of reagent of measuring this degree only may appear at finishing of dilution experiment further consuming time.
Should be noted that polyclonal antibody reagent often is two clones and has the characteristics of specific performance.As everyone knows, aspect mensuration ABO hypotype, some clone is better than other.Therefore, reagent often occurs with the form of mixture.When blood group determination reagent was diluted, its intrinsic behavioral characteristic was with inoperative.
When lacking believable blood group determination reagent, the laboratory often depends on the performance in the past of the specification sheets and the reagent of manufacturers.
When lacking the quality control of reliable blood group determination reagent, blood group determination is with rotten reagent, and significantly the hypotype of difference may be by the mensuration of mistake clinically.When this reagent goes bad, can't detect the red corpuscle that antigenic expression is positioned at the hypotype of tolerance interval low side.
If be used for blood transfusion, this blood will cause light or heavy transfusion reaction, comprise that death may appear in the blood recipient.
Obviously, need the low-level antigenic red corpuscle of a kind of believable expression as the quality control cell.The correction of the reliable quality control of blood group determination reagent and the system of mensuration is to guarantee accurate and standardized judgement blood group, and it is necessary being reduced to minimum degree for the danger that makes the blood recipient.
The technician who lacks the empirical many technical ability of a large amount of blood transfusions needs this method more.It is more important that the increase of mensuration confidence level reliably is compared to the understanding of measuring the passing performance of reagent.
The purpose of this invention provides and is used for blood group determination reagent quality control and the correction of the system of mensuration and the red corpuscle of affirmation, or is at least the public a useful selection is provided.
Reference:
Davis，M.O.et al.Cloning，sequence，and expression of a blood group Bactive recombinant alpha-D-galactosidase from pinto ean(Phaseolus vulgaris).Biochem.Mol.Biol.Int.42.3(1997)：453-67.
Goldstein，J.Conversion of ABO blood groups.Transfus.Med.Rev.3(1989)：206-12
Goldstein.J.et al.Group B erythrocytes enzymatically converted togroup O survive normally in A，B，and O individuals.Science 215(1982)：168-70.
Hobbs，L.et al.The activity of a blood type B specific exoglycosidasefrom Glycine max.Clin.Chim.Acta 247.1-2(1996)：7-21.
Hoskins，L.C.et al.Blood group A immunodeterminants on human redcells differ in biologic activity and sensitivity toalpha-N-acetylgalactosaminidase.Transfusion 35(1995))：813-821.
Lenny，L.L.et al.The production of group O cells.Biotechnology 19(1991a)：75-100.
Lenny，L.L.et al.Single unit transfusions of RBC enzymaticallyconverted from group B to group O to A and O normal volunteers.Blood 77(199lb)：1383-8.
Lenny，L.L.et al.Transfusions to group O subjects of 2 units of red cellsenzymatically converted from group B to group O.Transfusion 34(1994)：209-14.
Zhu.A.et al.Characterization of recombinant alpha-galactosidase foruse in seroconversion from blood group B to O of human erythrcytes.Arch.Biochem.Biophys.327.2(1996)：324-29.
Summary of the invention
This invention comprises following aspect:
At first, this invention provides expresses low-level antigenic red corpuscle, and this red cell antigens expression level is low, is equal to the red corpuscle of natural generation abo blood group and hypotype in fact.
The low-level antigenic red corpuscle of this expression is in external preparation.
The reduction of this erythrocytic antigen levels is equal to the antigen presentation that obtains on the serology in fact more and is less than 5 * 10
5The red corpuscle of copy number, the antigen presentation that can reach acquisition is less than 1 * 10
5The red corpuscle of copy number, the antigen presentation that preferably can reach acquisition is less than 2 * 10
4The red corpuscle of copy number.
Express low-level antigenic red corpuscle and be equal to the threshold value of antigen significance clinically in fact more.
Express low-level antigenic red corpuscle, in the serology result, the antigen number in its each red corpuscle is greater than 1 * 10
2Copy number, preferably the antigen number in each red corpuscle is greater than 1 * 10
3Copy number.
The erythrocytic sugar chain mode of connection of immunodominance antigen is that the N-acetylgalactosamine of α connection or the semi-lactosi of α connection are connected to H antigen.
Blood group antigen are A antigen or B antigen preferably.
When adopting aggegation experimental analysis red corpuscle, the reduction of antigenic expression reduces by 2～3 units corresponding to the aggegation score value.
The reduction of antigen levels realizes by using the sugar-modified enzyme of at least a advantage immunity on the zymetology.The reduction of better antigen levels is by making the enzyme of bond rupture in junction, 1-3 position on the zymetology.The reduction of better antigen levels is by using alpha-N-Acetylgalactosaminidase or α-galactase, perhaps using this two kinds of enzymes simultaneously on the zymetology.
Low-level antigen presentation is equal to the reduction of aggegation score value more, is equal in fact to measure natural erythrocytic weak expression abo blood group or the aggegation score value of hypotype in the aggegation experiment.
The red corpuscle that red corpuscle is preferably human.
Red corpuscle preferably is in suspended state.
Preferably comprise the cell sanitas in the suspension, such as Celpresol
TM
Preferably comprise the controlling index that can provide extra in the suspension, as the remarkable antibody of meaning clinically.
The advantage of this invention is embodied in, and this invention provides the erythrocytic suspension of low-level antigen presentation on a kind of zymetology, and this low-level antigen presentation is equal to the erythrocytic phenotype that nature exists in fact.And the level of antigen presentation is equal to the threshold value of antigen significance clinically in fact more.
Red corpuscle is used as the quality control cell.
Suspension is as the quality of quality control reagent control blood group determination reagent and/or to the correction and the conclusive evidence of test macro.
Second aspect, this invention provide a kind of preparation to express low-level antigenic erythrocytic method, and step comprises:
The sugar-modified enzyme of at least a advantage immunity is put into the capable resulting mixture of solution with the red corpuscle of initial antigenic expression;
Mixture is placed under the temperature that is enough to reduce antigenic expression to reduce antigenic expression;
Handle this suspension, to stop the further reduction of antigenic expression.
Judge the reduction of antigenic expression by periodic sample and mensuration mixture.
Detect the antigenic expression of mixture by the aggegation experiment.
When measuring red corpuscle in the aggegation experiment, when the reduction of antigenic expression reduced by 2～3 units corresponding to the aggegation score value, treating suspension was to stop the reduction of antigenic expression.
Erythrocytic initial antigenic expression equals: in the Serological testing, each red cell antigens is expressed quantity greater than 5 * 10
5Copy number.
Contain in the red corpuscle place solution of antigen expressed at least a advantage immunity sugar-modified enzyme be red blood cells of type A.
Contain in the red corpuscle place solution of antigen expressed at least a advantage immunity sugar-modified enzyme be the Type B red corpuscle.
Contain in the red corpuscle place solution of antigen expressed at least a advantage immunity sugar-modified enzyme be AB type red corpuscle.
Remove the sugar-modified enzyme of immunodominance to stop the excessive reduction of antigen levels by cleaning red corpuscle.
The reduction of antigenic expression is equal to the threshold value of antigen significance clinically in fact.
Express low-level antigenic red corpuscle, in Serological testing, the antigen number in its each red corpuscle is less than 5 * 10
5Copy number, preferably the antigen number in each red corpuscle is less than 1 * 10
5Copy number, preferably the antigen number in each red corpuscle is less than 2 * 10
4Copy number.
Express low-level antigenic red corpuscle, in the serology result, the antigen number in its each red corpuscle is greater than 1 * 10
2Copy number, preferably the antigen number in each red corpuscle is greater than 1 * 10
3Copy number.
The erythrocytic sugar chain mode of connection of immunodominance antigen is that the N-acetylgalactosamine of α connection or the semi-lactosi of α connection are connected to H antigen.
Blood group antigen are A antigen or B antigen preferably.
Used enzyme is at least a to be the sugar-modified enzyme of advantage immunity.This endonuclease capable makes bond rupture in junction, α 1-3 position.Preferably use alpha-N-Acetylgalactosaminidase or α-galactase, perhaps use this two kinds of enzymes simultaneously.
The reduction of antigenic expression is equal to the reduction of aggegation score value more, is equal in fact to measure the aggegation score value of natural erythrocytic weak expression ABO hypotype in the aggegation experiment in same aggegation experiment.
The red corpuscle that red corpuscle is preferably human.
The third aspect, this invention provide by expressing low-level antigenic erythrocytic preparation method on the zymetology that second aspect provided of invention.
Fourth aspect, this normal plane provide the method for the quality control of blood group determination reagent, comprising:
Blood group determination reagent and red blood cell suspension according to the preparation of first aspect and the third aspect are reacted;
Measure the aggegation value.
The mensuration of aggegation value is by observing agglutinative quantity.
This method all is recursive for a series of extent of dilution of blood group determination reagent.
What can select is that this process can comprise the step of step judgement red cell antigens expression level, just by the known erythrocytic antigenic expression of reference.The red corpuscle of expressing the known antigens level can be prepared by the method that international monopoly is used described in the PCT/NZ02/00219.
The 5th aspect, this invention provide a test kit by two or more red blood cell suspensions, and this erythrocytic preparation is referring to the first aspect and the third aspect of invention.
This test kit comprises expressing the control of A type and the sensitivity of Type B red corpuscle, and erythrocytic preparation is referring to the first aspect and the third aspect of invention.Comprise the cell sanitas in the suspension, such as Celpresol
TMComprise the controlling index that can provide extra in the suspension, as the remarkable antibody of meaning clinically.
Alternatively be, comprise the reagent of sensitivity control in the test kit, comprise and express Rh DCce (Rlr) and the antigenic red corpuscle of Rh ce (rr).
To introduce this invention in detail below.
Detailed Description Of The Invention:
The sugar of the antigenic advantage immunity of A is the N-acetylgalactosamine that a α connects.The sugar of the antigenic advantage immunity of B is the semi-lactosi that a α connects.The sugar of advantage immunity is connected to H antigen.
The removal of passing through enzyme and the modification of the sugar of advantage immunity can cause losing of original antigen.Therefore, A, the erythrocytic A antigen and the antigenic expression level of B of B or AB blood group can be reduced by enzyme.
Antigenic enzymolysis, digestion is based on following standard, enzyme (Glycosylase) can be on erythrocyte membrane the degrade specifically sugar antigen, and can not destroy non-object construction, for example: protein and carbohydrate that non-target position connects.
The sex change of enzyme was used to remove the abo blood group antigen in the red corpuscle in the past, thereby type A cell and type B cell are changed into O type cell.The investigator can remove major part even whole red cell antigenses with the enzyme of high density.
When blood transfusion, these remove antigenic cell can be used as the general (Davis1997 of blood unit; Goldstein 1989; Hobbs 1996; Hoskins 1995; Lenny 1991a; Zhu 1996)." remove antigenic blood " and in clinical blood transfusion, test successfully, although (Goldstein 1982 in not conventional now application; Lenny 1991b; Lenny 1994).
The method that these authors describe does not provide expression decreased but the red corpuscle of limited antigen levels, and this antigenic expression level is in or is higher than the threshold value of significance clinically.In fact, these methods attempt to provide the not red corpuscle of antigen expressed, or are no more than the antigenic red corpuscle of clinical significance number of thresholds at least.
According to this invention, express low-level A antigen or/and the antigenic red corpuscle of B in external preparation.At A antigen or/and aspect the B antigenic expression, the red corpuscle of preparation equals the red corpuscle of naturally occurring ABO hypotype in Serological testing.
The condition of digestion, for example the ratio of the time of Chu Liing, temperature, enzymic activity and red corpuscle and enzyme can regulated aspect the minimizing of control antigenic expression.The antigenic enzymolysis, digestion time also depends on the initial expression level of antigen on the relative concentration of enzyme solution and the cell on the cytolemma.
The change of enzyme concn can be as the antigenic expression level of control.If expect the antigenic cell of weak expression A antigen or B, can use the enzyme of high density.Strong if desired agglutinative cell can use the enzyme of lower concentration.
Used typical quality control cell is by A type or type B cell preparation in the medical blood transfusion, and enzyme will be removed A antigen and/or B antigen under this condition, thereby obtain specific fraction reacted in the antigen measuring analysis.This analysis will comprise plate, test tube, the use agglutinative platform of gel cards and minitype plate method and any craft or automatization, or the antigenic method of any other detection (for example: enzyme linked immunological, flow cytometer etc.).
Traditional method obtains red corpuscle in serology detects, and the result that aggegation is analyzed approaches 2+.Actual serology result need depend on the sensitivity of used analytical system, and for example plate is different with the sensitivity of automatic mode.
The process of digestion can be monitored by the performance of regular sampling and analysing aggegation system.In case the condition of digestion depends on the low-level antigenic red corpuscle of a large amount of expression of experiment ratio, just can prepare the cell that can be provided as quality control.
The aggegation experiment is to measure antigenic a kind of method.The red corpuscle that the is cross-linked to form caking of iuntercellular antibody is called aggegation.Aggegation can be by observing (using eyes) or detecting with the blood type analytical instrument automatic technology.Enhancing is observed agglutinative can be by using specific enzyme or radioactivity or fluorescent mark technology.
According to following scheme manual agglutination reaction is scored:
| Aggegation mark (unit) | Observe |
| (+) +(i.e.1+) ++(i.e.2+) +++(i.e.3+) ++++(i.e.4+) | Big blood clot of the several little blood clots of very little blood clot that no blood clot is fuzzy is the single big blood clot of little blood clot on every side |
Evaluation to level of agglutination can or be induced the agglutinative method to produce non-direct aggegation by use and estimate by the direct viewing aggegation, for example reinforcing effect or use antiglobulin molecule.
Express low-level antigenic red corpuscle, on serology, be equal to the red corpuscle of natural generation abo blood group and hypotype in fact, have special advantage and advantage as the quality control cell.
If desired, the actual antigenic expression of quality control cell (the antigen molecule number on each red corpuscle) can be by the red corpuscle with reference to the known antigens expression level.These can be the antigenic ABO hypotype of weak expression that exists of nature or the red corpuscle of expressing the known antigens level by the red corpuscle of reference, can be prepared by the method that international monopoly is used described in the PCT/NZ02/00219.
Should be noted that the actual antigenic expression that does not need to know the quality control cell.The quality control cell is used to estimate and detect the qualitative variation of the generation of blood group determination reagent.Quality control cell antigen expression level is low and limitedly be only important part.
Can prepare quality control cell with different antigenic expressions.The antigenic expression of one cover quality control cell can will cause significantly transfusion reaction clinically in the antigenic failure of this threshold value determination by selecting significance threshold value clinically.The antigenic expression of another set of quality control cell can be by selecting have enough assurance to measure the red corpuscle of weak hypotype.
The above-mentioned quality control cell of mentioning can pass through control sensitivity within the specific limits, thus the accuracy that the conclusive evidence abo blood group is measured.These sensitivity control cells also can be used in calibrated altitude sensitive machine or use when the quantitative curve of cells were tested by flow cytometry antigen.This can guarantee the security that abo blood group is supplied with.
Stdn and global consistence that this invention makes quality control.This invention makes and can the result of different experiments chamber and different methods be compared.The quality control cell is included in the blood transfusion serology quality assurance planning, the quality control standard that the abo blood group that can set up is measured.
In this invention, be included in the test kit the quality control cell can be used for proving conclusively the phenotype of A type blood (weak) and Type B blood (weak) in the blood group determination.This test kit can comprise the quality control cell, is used for proving conclusively the blood group determination result to Rh DCce (Rlr) and Rh ce (rr) phenotype.This test kit can guarantee that ABO and RhD blood group determination reagent can carry out quality control.Other test kits that comprise the quality control cell of expressing the certain limit antigen levels are very helpful for some specialized laboratories.
When the suspension of preparation quality control cell, resuspended liquid may contain the antibody of significance clinically.Therefore, should introduce extra quality control feature, for example Gong Cun antibody control.
Following definition is provided and explains auxiliary description and the claimed range of understanding the claim of this invention.
" clinical remarkable threshold " is when the antigenic expression of red corpuscle is lower than this standard, if transfuse blood, the failure of Detection of antigen does not have significance clinically.
" the sugar-modified enzyme of advantage immunity " is meant these endonuclease capables modification A antigen or antigenic antigenic determinants of B, thereby causes the reduction of antigenic expression.For example: the sugar-modified enzyme of advantage immunity comprises alpha-N-Acetylgalactosaminidase or α-galactase.
" reduction of antigenic expression on the zymetology " is meant the antigen by the enzymic digestion cell surface, the external antigenic expression that obtains.
" antigen presentation " is meant the antigen that cell surface occurs, and " antigenic expression " is meant the antigen quantity that cell surface occurs.
" quality control cell " is meant the cell of antigen expressed, is typically red corpuscle, is used for estimating the quality of blood group determination reagent and the mensuration system is proofreaied and correct and proves conclusively.The example of the quality control cell of this invention is the cell of the enzyme modification of description in example 1,2 and 3.
The antigen levels that occurs in above-mentioned narration " is equal to " level that is meant this antigen presentation in fact will provide same in fact result in serological analysis.
Antigenic expression is by each erythrocytic antigen copy number definition, antigenic expression is in the antigenic expression that acceptable level is meant known ABO hypotype, and perhaps this antigenic expression can provide the identical result of known ABO hypotype on the serology.
" height " of antigenic expression and " low " are to be used for distinguishing the ABO hypotype that common ABO hypotype such as A1 and uncommon poor antigen are expressed.
Embodiment
To illustrate this invention by specific embodiment below.
Embodiment 1
The reduction of the B antigenic expression of alpha-galactosidase effect
The alpha-galactosidase of 10 units that extract in the green coffee berry is bought from Glyko (Cat.No.X-5001).
The red corpuscle of 100 microlitres of AB blood group cleans three times with phosphoric acid buffer (PBS).
Control reaction (ctrl):
The red corpuscle of 50 microlitres parcels joins in the citrate buffer of the pH6.5 among the 500mM of the citrate buffer of pH6.5 of 40 microlitre 100mM and 22.5 microlitres.
Enzymatic reaction (enz):
The cell of 50 microlitres parcels joins in the citrate buffer of the pH6.5 among the 500mM of the citrate buffer of 100mM pH6.5 of alpha-galactosidase of 4 units that contain 40 microlitres and 22.5 microlitres.
Mixture reacts in 37 ℃ of water-baths, and mixes frequently.This reaction concluding time is respectively 6 hours and 9 hours, and the method for termination is to remove the red corpuscle that is equivalent to 15 microlitres parcel, cleans cell three times with the phosphoric acid buffer that contains 1% bovine serum albumin.
The red corpuscle of 15 microlitres parcel is resuspended in 1 milliliter Celstab (cell listerine), is used for estimating the extent of dilution of blood group determination reagent (antiserum(antisera)).
The cell that cleans in control reaction or the enzyme reaction is resuspended in 0.8% Celstab (Diamed).The suspension of 50 microlitres 0.8% is transferred to the Diamed card, adds with 1: 4 the blood group determination reagent (antiserum(antisera)) of the anti-B of 50 microlitres of 1: 32 and 1: 512 dilution respectively.
With mixture reaction 5 minutes,, Diamed was stuck in the Diamed immunity whizzer centrifugal 10 minutes according to standard operating procedure.
Specification sheets according to manufacturers is given a mark to agglutination reaction.
Table 1: alpha galactosides enzyme modification (enzymatic) measured by the anti-B blood group determination reagent (Alba clone, Scotland) of dilution and unmodified (contrast) is organized the erythrocytic aggegation result of AB type.
| The time of the short reaction of alpha-galactosidase enzyme | ||||
| Anti-B | 6 hours | 9 hours | ||
| Antibody dilution | Control group | The enzymatic reaction group | Control group | The enzymatic reaction group |
| 1∶4 | 4+ | 3+ | 3+ | 2+ |
| 1∶32 | 3+ | 2+ | 3+ | 2+ |
| 1∶512 | 2+ | 1+ | 2+ | 1+ |
Embodiment 2
The reduction of the A antigenic expression of alpha-N-Acetylgalactosaminidase effect
Alpha-N-Acetylgalactosaminidase (Cat.No.X-5001) is bought from Glyco company.
The red corpuscle of 100 microlitres of AB blood group cleans three times with phosphoric acid buffer (PBS).
Control reaction (ctrl):
The red corpuscle of 6 microlitres parcels joins in the citrate buffer of the pH6.5 among the 500mM of the citrate buffer of pH4 of 100 microlitre 100mM and 26.5 microlitres.
Enzymatic reaction (enz):
The cell of 6 microlitres parcels joins in the citrate buffer of the pH6.5 among the 500mM of the citrate buffer of 100mM pH4 of alpha-N-Acetylgalactosaminidase of 100 milliunits (mU) that contain 100 microlitres and 26.5 microlitres.
Mixture reacts in 37 ℃ of water-baths, and mixes frequently.This reaction concluding time is respectively 6 hours, 12 hours and 24 hours, and the method for termination is to take out the red corpuscle that is equivalent to 2 microlitres parcel, joins in the 100 microlitre CelStab reagent, becomes 0.8% suspension.(, need not clean cell with phosphoric acid buffer for avoiding loss cell.)
Suspension is used for estimating the extent of dilution that is used for estimating blood group determination reagent (antiserum(antisera)).
50 microlitre suspension are transferred to the Diamed card, add the blood group determination reagent (Alba clone, Scotland) with the anti-A of 50 microlitres of 1: 32 and 1: 256 dilution respectively.
With mixture reaction 5 minutes,, Diamed was stuck in the Diamed blood bank centrifuge centrifugal 10 minutes according to standard operating procedure.
Specification sheets according to manufacturers is given a mark to agglutination reaction.
Table 1: alpha-N-Acetylgalactosaminidase modification (enzymatic) measured by the anti-A blood group determination reagent (Alba clone, Scotland) of dilution and unmodified (contrast) is organized the erythrocytic aggegation result of AB type.
| The time of alpha-N-Acetylgalactosaminidase enzymatic reaction | ||||||
| Anti-A | 6 hours | 12 hours | 24 hours | |||
| Antibody dilution | Control group | The enzymatic reaction group | Control group | The enzymatic reaction group | Control group | The enzymatic reaction group |
| 1∶32 | 4+ | 3+ | 4+ | 2+ | 4+ | 1+ |
| 1∶256 | 4+ | 2+ | nc | 0 | 3+ | 0 |
Nc represents can not see cell in the test
In example 1 and example 2, the aggegation value a little less than cell provides on the serology.In these two examples, the detection that the cell of enzyme modification reduces for antibody titers is responsive more, has for example obtained lower aggegation score value when the blood group determination reagent that dilutes with the cell detection of enzyme modification.
These cells are suitable for the evaluation quality control cell with oppose serological blood group analysis and blood group determination reagent.Use the cell of enzyme modification to have resolving ability more for the minimizing that detects antibody titers.
Although the real standard of the antigen presentation of enzyme modification cell is unknown, can analyze by the antibodies experiment if desired.Yet the technician does not need to know the real standard of antigen presentation on the enzyme modification cell to the ability of the Sensitive Detection more of the qualitative variation of blood group determination reagent.
Because initial antigen source different (being provided by Different Individual), so experiment condition has tiny difference, although the difference of reaction times, enzyme concn and/or temperature also can obtain to provide the enzyme modification cell as the quality control cell of similar results.Can need obtain the cell of fraction reacted by the monitoring reaction process, just obtain prepared quality control cell after reaction stops.
Embodiment 3
The method that from coffee berry, prepares alpha-galactosidase.
Alpha-galactosidase is to extract from green coffee berry (fruit coffee) according to the method for Courtois and Petek.(Courtois，J.E.and Petek，J.，α-Galactosidase from CoffeeBeans，(1996)Methods of Enzymology，8：565-571)。
The enzyme that extracts is concentrated to centrifugal ultrafiltration device (Millipore) in Citrate trianion phosphoric acid buffer (100mM, pH6.0) middle dialysis.The activity of enzyme crude extract need not to measure.Total protein concentration is 96mg/ml.
Reduce the Enzymology method of B antigen presentation
Control reaction (ctrl):
AB blood group through cleaning, the red corpuscle (300 microlitre) of parcel join be equipped with the Citrate trianion phosphate buffered saline buffer (500 microlitres, 100mM, pH6.0 and 200 microlitres, 500mM is in Eppendorf centrifuge tube pH6.0).
Enzymatic reaction (enz):
The red corpuscle (300 microlitre) through cleaning, parcel of AB blood group joins Citrate trianion phosphate buffered saline buffer (500 microlitres that alpha-galactosidase is housed, 100mM, pH6.0) and the Citrate trianion phosphate buffered saline buffer (200 microlitres, 500mM is in Eppendorf centrifuge tube pH6.0).
Mixture reacted 24 hours in 37 ℃ of water-baths, and mixed by jolting frequently.
The red corpuscle that enzyme is handled cleans three times with the phosphoric acid buffer that contains 1% bovine serum albumin.Then will through cleaning, the red corpuscle of parcel is suspended in the solution of Celstab cell sanitas with 0.9% ratio, is used for joining the Diamed gel cards and manual test tube serology detects.
Cell suspending liquid (50 microlitres, red blood cell concentration is 0.9% among the Cellstab) and the diluent (50 microlitre) of blood group determination reagent (antibody) joined on the Diamed card with suction pipe, act on 5 minutes, in the Diamed whizzer centrifugal 10 minutes then, the record result.
Cell suspending liquid (25-40 microlitre, red blood cell concentration is 0.9% among the Cellstab) and antibody reagent diluent (25 microlitre) react in glass serum test tube, and in immune whizzer centrifugal 15 seconds then, the record result.
The quality control of blood group determination reagent
The antibody reagent that commercial anti-B (mono-clonal and a polyclone people) reagent was stored by the past provides.The reagent of these storages is measured by single batch enzyme modification cell, and the antigenic expression of these cell reality is unknown.
Selecting blood group determination reagent is because these reagent are out of date, therefore may take place to go bad, and it is impaired to measure effect.The diluent of these reagent is also selected to be assessed.The title that has replaced manufacturers with coding.Performance as the blood group determination reagent of a function of reaction conditions is opposite with the supplier of assessment.
The rotten of blood group determination reagent can be detected by the enzyme modification cell.Rotten is significant, because although the diluent of blood group determination reagent can obtain significant aggegation score value with the cell of expressing high-level antigen (control reaction) time, when with expressing low-level antigenic quality control cell (enzymatic reaction) when measuring, these same reagent can not provide the aggegation score value of significance.
What can infer is to produce the strong positive result when detect untreated control cells (active good) with reagent, and produce weak or negative reaction result with enzyme modification cell (inferring that these quality control cells are at the reagent that detects the poor quality).Whether significantly lose activity although can't detect antiserum(antisera) at the cell with unmodified, the form of back is given prominence to and is listed following example.
As previously mentioned, need not to judge the real standard of enzyme modification cell antigen expression.In fact, can select the level of antigen presentation according to user's requirement.Those different expression levels obtain by the control enzymatic reaction.
| The anti-B of LBA (in April, 2004 is expired) | ||||||||
| Platform | Cell | Antiserum(antisera) concentration | ||||||
| 1∶1 | 1∶64 | 1∶128 | 1∶256 | 1∶512 | 1∶1024 | 1∶2048 | ||
| Diame d | Control group | 4+ | 3+ | 3+ | 3+ | 3+ | 3+ | 2+ |
| Treatment group | 4+ | 2+ | 2+ | 1+ | 1+ | 0 | 0 | |
| Tube | Control group | n.d. | 4+ | 4+ | 3+ | 3+ | 2+ | 0 |
| Treatment group | n.d. | 2+ | 1+ | 0 | 0 | 0 | 0 | |
| The anti-B of BC (in November, 1999 is expired) | ||||||||
| Platform | Cell | Antiserum(antisera) concentration | ||||||
| 1∶1 | 1∶16 | 1∶32 | 1∶64 | 1∶128 | 1∶256 | 1∶512 | ||
| Diamed | Control group | 4+ | 4+ | 4+ | 4+ | 4+ | 3+ | 2+ |
| Treatment group | 4+ | 4+ | 3+/2+ | 2+ | 2+ | vw | 0 | |
| Tube | Control group | n.d. | 4+ | 4+ | 3+ | 2+ | 1+ | 0 |
| Treatment group | n.d. | 4+ | 3+ | 1+ | 0 | 0 | 0 | |
| The anti-B of people BLB (in July, 1987 is expired) | |||||||
| Platform | Cell | Antiserum(antisera) concentration | |||||
| 1∶4 | 1∶8 | 1∶16 | 1∶32 | 1∶64 | 1∶128 | ||
| Diamed | Control group | 4+ | 3+ | 3+ | 3+ | 3+ | 2+ |
| Treatment group | 3+ | 2+ | 2+ | vw | 0 | 0 | |
| Tube | Control group | 4+ | 4+ | 4+ | 3+ | 0 | 0 |
| Treatment group | 1+ | 0 | 0 | 0 | 0 | 0 |
| The anti-B of EPI (in May, 2000 is expired) | |||||||
| Platform | Cell | Antiserum(antisera) concentration | |||||
| 1∶1 | 1∶2 | 1∶4 | 1∶8 | 1∶16 | 1∶32 | ||
| Diamed | Control group | 4+ | 4+ | 4+ | 4+ | 3+ | 3+ |
| Treatment group | 3+ | 2+ | 2+/1+ | 0 | 0 | 0 | |
| Tube | Control group | 4+ | 4+ | 3+ | 3+ | 1+ | 0 |
| Treatment group | 1+ | 0 | 0 | 0 | 0 | 0 | |
| The anti-B of LOR (August calendar year 2001 is expired) | ||||
| Platform | Cell | Antiserum(antisera) concentration | ||
| 1∶1 | 1∶2 | 1∶4 | ||
| Diamed | Control group | 4+ | 3+ | 2+ |
| Treatment group | 1+ | vw | 0 | |
| Tube | Control group | 3+ | 3+ | 2+ |
| Treatment group | 0 | 0 | 0 | |
| The anti-B of ORG (in December, 1993 is expired) | |||||
| Platform | Cell | Antiserum(antisera) concentration | |||
| 1∶1 | 1∶4 | 1∶128 | 1∶512 | ||
| Diamed | Control group | 4+ | 4+ | 4+ | 3+ |
| Treatment group | 4+ | 4+ | 3+ | 0 | |
| Tube | Control group | n.d. | n.d. | n.d. | n.d. |
| Treatment group | n.d. | n.d. | n.d. | n.d. | |
N.d. expression is not done.
In the description in front, there has been reference to mention the whole thing or the compound of those known equivalents, then, here these Equivalents mixed, just as respectively it being illustrated.
What particularly point out is, the inventor is intended that biological catalyst beyond dezymotizing and can develops into equally with the sugar-modified enzyme of previously described advantage immunity and have same activity
Although this invention by the embodiment possible concrete operations relevant with it description, it should be noted that not breaking away under the spirit and scope of the present invention prerequisite and can improve and/or revise the present invention.
Claims (62)
1. as the low-level antigenic red corpuscle of the expression of quality control cell.
2. the low-level antigenic red corpuscle of the expression in the claim 1 is equal to the red corpuscle of natural generation abo blood group and hypotype in fact.
3. express low-level antigenic red corpuscle in claim 1 or the claim 2, the antigen number in its each red corpuscle is less than 5 * 10
5Copy number.
4. the low-level antigenic red corpuscle of the expression in the claim 3, the antigen number in its each red corpuscle is less than 1 * 10
5Copy number.
5. the low-level antigenic red corpuscle of the expression in the claim 4, the antigen number in its each red corpuscle is less than 2 * 10
4Copy number.
6. the low-level antigenic red corpuscle of the expression in the claim 1 is equal to the threshold value of antigen significance clinically in fact.
7. claim 1 is to the low-level antigenic red corpuscle of the expression in the claim 6, and the antigen number in its each red corpuscle is greater than 1 * 10
2Copy number.
8. the low-level antigenic red corpuscle of the expression in the claim 7, the antigen number in its each red corpuscle is greater than 1 * 10
3Copy number.
9. any immunodominance antigen erythrocytic sugar chain mode of connection of claim 1 in the claim 8 is that the N-acetylgalactosamine that α connects is connected to H antigen.
10. any immunodominance antigen erythrocytic sugar chain mode of connection of claim 1 in the claim 8 is that the semi-lactosi that α connects is connected to H antigen.
11. erythrocytic antigen is blood group antigen in claim 9 or the claim 10.
12. erythrocytic antigen is A antigen in the claim 9.
13. erythrocytic antigen is B antigen in the claim 10.
14. when adopting aggegation experimental analysis red corpuscle, the reduction of the erythrocytic antigenic expression of claim 1 in the claim 13 reduces by 2～3 units corresponding to the aggegation score value.
15. claim 1 to the reduction of the red corpuscle enzymatic antigenic expression that claim 14 is mentioned in any one is by using at least a epistatic immunized glucose modified enzyme.
16. the enzymatic reaction that the red cell antigens level of being mentioned in the claim 15 reduces is finished by enzymatic action fracture α 1-3 chain.
17. the enzymatic reaction that the red cell antigens level of being mentioned in the claim 15 reduces is by using α-acetylgalactosamine enzyme or α-galactase, perhaps using these two kinds of enzymes to finish simultaneously.
18. the minimizing that meets the aggegation score value of the red cell antigens level that claim 1 to claim 17 is mentioned in any one is equal in fact in same aggegation and analyzes the low-level antigenic abo blood group of naturally occurring expression measured in the experiment or the red corpuscle of hypotype.
19. the red corpuscle that claim 1 is mentioned in any one to claim 18 is human red corpuscle.
20. the red corpuscle that claim 1 is mentioned in any one to claim 19 is the form of suspension.
21. the suspension of mentioning in the claim 20 includes cell sanitas, for example Celpressol.
22. the composition of being mentioned in claim 20 or the claim 21 that suspension contained can provide extra quality control characteristic.
23. the composition in the suspension of being mentioned in the claim 22 is the remarkable antibody of meaning clinically.
24. the suspension purposes that claim 20 to claim 23 is mentioned in any one is the correction and the conclusive evidence of the control of blood group determination reagent quality and/or the system of mensuration.
25. expressing the step of low-level antigenic erythrocytic method, preparation comprises:
The sugar-modified enzyme of at least a advantage immunity is put into the capable resulting mixture of solution with the red corpuscle of initial antigenic expression;
Mixture is placed under the temperature that is enough to reduce antigenic expression to reduce antigenic expression;
Handle this suspension, to stop the further reduction of antigenic expression.
26. the reduction of antigenic expression is judged by periodic sampling and mensuration mixture in the claim 25.
27. the measuring method in the claim 26 is the aggegation experiment.
28. when the suspension in the claim 27 reduces by 2～3 units in the reduction of antigenic expression corresponding to the aggegation score value, handle to stop the further reduction of antigenic expression.
29. claim 25 is to any one method of being mentioned of claim 28, erythrocytic initial antigenic expression is: in the Serological testing, each red cell antigens is expressed quantity greater than 5 * 10
5Copy number.
30. claim 25 to any one method of being mentioned of claim 29, contain in the red corpuscle place solution of antigen expressed at least a advantage immunity sugar-modified enzyme be red blood cells of type A.
31. claim 25 to any one method of being mentioned of claim 29, contain in the red corpuscle place solution of antigen expressed at least a advantage immunity sugar-modified enzyme be the Type B red corpuscle.
32. claim 25 to any one method of being mentioned of claim 29, contain in the red corpuscle place solution of antigen expressed at least a advantage immunity sugar-modified enzyme be AB type red corpuscle.
33. claim 25 to any one method of being mentioned of claim 32, is removed the sugar-modifiedization enzyme of immunodominance by cleaning red corpuscle.
34. claim 25 is to any one method of being mentioned of claim 32, by adding the epistatic immunized glucose modified enzyme stopped reaction.
35. claim 25 is to any one method of being mentioned of claim 32, by adding the emulative substrate stopped reaction of epistatic immunized glucose modified enzyme.
36. claim 25 is to any one method of being mentioned of claim 35, the reduction of antigenic expression is equal to the threshold value of antigen significance clinically in fact.
37. claim 25 is to any one method of being mentioned of claim 35, the minimizing of antigenic expression should be less than 5 * 10
5Copy number.
38. in the method for being mentioned in the claim 37, the minimizing of each red cell antigens expression level should be less than 10
5Copy number.
39. in the method for being mentioned in the claim 38, the minimizing of each red cell antigens expression level is best less than 2 * 10
4Copy number.
40. claim 25 is to any one method of being mentioned of claim 39, the minimizing of each red cell antigens expression level should be greater than 10
2Copy number.
41. in the method for being mentioned in the claim 40, the minimizing of each red cell antigens expression level should be greater than 10
3Copy number.
42. claim 25 to claim 30 and claim 32 to any one method of being mentioned of claim 41 in, the erythrocytic sugar chain mode of connection of immunodominance antigen is that the N-acetylgalactosamine that α connects is connected to H antigen.
43. claim 25 to claim 29 and claim 31 to any one method of being mentioned of claim 41, the erythrocytic sugar chain mode of connection of immunodominance antigen is that the semi-lactosi that α connects is connected to H antigen.
44. in the method that claim 42 or claim 43 are mentioned, antigen is blood group antigen.
45. in the method that claim 42 is mentioned, antigen is A antigen.
46. in the method that claim 43 is mentioned, antigen is B antigen.
47. claim 25 is to any one method of being mentioned of claim 46, enzyme is in α 1-3 position chain rupture.
48. claim 25 is to any one method of being mentioned of claim 46, the enzyme of use is alpha-N-Acetylgalactosaminidase or α-galactase, perhaps uses this two kinds of enzymes simultaneously.
49. claim 25 is to any one method of being mentioned of claim 48, the reduction of antigenic expression is equal to the reduction of aggegation score value, is equal in fact to express the aggegation score value of low-level antigenic naturally occurring erythrocytic ABO hypotype in the aggegation experiment in same aggegation experiment.
50. claim 25 to any one method of being mentioned of claim 49, usefulness be people's red corpuscle.
51. expressing low-level antigenic red corpuscle prepares according to claim 25 to claim 50.
52. red blood cell suspension prepares according to claim 51.
53. the suspension according to claim 52 preparation includes cell sanitas, for example Celpressol.
54. the suspension according to claim 51 or claim 52 preparation includes the material that extra control characteristic can be provided.
55. the composition in the suspension that claim 54 is mentioned also comprises clinically the significantly antibody of meaning.
56. the quality controlling means of blood group determination reagent comprises:
A. will want according to claim 20 to claim 24 or claim 52 to right
Ask 55 any one prepared suspension and blood group determination reagent to react;
B. estimate the agglutinative degree.
57. in the method that claim 56 is mentioned, the mensuration of aggegation value is by observing agglutinative quantity.
58. the method that claim 56 or claim 57 are mentioned, in the certain limit of blood group determination reagent extent of dilution all be recursive.
59. according to containing two or more suspension in claim 20 test kit prepared to claim 24 or claim 52 to claim 55.
60. the test kit according to claim 59 preparation comprises expression A antigen and/or the erythrocytic suspension of B antigen.
61. in the test kit according to claim 59 or claim 60 preparation, erythrocytic antigenic expression is equal to significant clinically threshold value in fact.
62. to the test kit of claim 61 preparation, comprise and express RhDCce (Rlr) and the antigenic red corpuscle of Rhce (rr) according to claim 59.
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| NZ52421703 | 2003-02-17 | ||
| NZ524217 | 2003-02-17 | ||
| NZ527777 | 2003-08-22 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100569942C (en) * | 2007-11-02 | 2009-12-16 | 中国人民解放军军事医学科学院野战输血研究所 | A kind of alpha-N-Acetylgalactosaminidase and encoding gene thereof and application |
| WO2015032340A1 (en) * | 2013-09-06 | 2015-03-12 | Shanghai Sidansai Biotechnology Co., Ltd | Modified cells for production of blood cells |
| CN109239372A (en) * | 2018-11-02 | 2019-01-18 | 上海市血液中心 | Abo blood group antigen detectability verifies product and its application |
-
2004
- 2004-02-17 CN CN200480004362A patent/CN100588719C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100569942C (en) * | 2007-11-02 | 2009-12-16 | 中国人民解放军军事医学科学院野战输血研究所 | A kind of alpha-N-Acetylgalactosaminidase and encoding gene thereof and application |
| WO2015032340A1 (en) * | 2013-09-06 | 2015-03-12 | Shanghai Sidansai Biotechnology Co., Ltd | Modified cells for production of blood cells |
| CN109239372A (en) * | 2018-11-02 | 2019-01-18 | 上海市血液中心 | Abo blood group antigen detectability verifies product and its application |
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| CN100588719C (en) | 2010-02-10 |
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