CN1423130A - HLA cross matching method for stream measuring fixed complement antibody and kit thereof - Google Patents

Abstract

Based on the reaction principle of the complement-dependent cytotoxicity (CDC), the invention adds the antibody of the anticomplement of the fluorescence labeling or the complement of the fluorescence labeling and one or more specificity antibodies of the anti target cell with different fluorescence labeling in two colours or more. Then, the flow cytometry classifies and differentiates the specific target cell group as well as makes the semiquantitative determination of the quantity of the complement so as to evaluate and measure the CDC effect. The evaluation provides the handy, fast and accurate scientific reference for the crossing pairing between the donor and the recipient in the transplantation. The invented method can replace the DCD HLA method in the traditional serology or PRA method. The method can also be utilized to measure the antibody level of CDC and the complement fixing antibodies levels.

Description

The HLA crossmatch method and the kit of stream measuring fixed complement antibody

One, technical field immunology organ transplant somatotype is joined type.

Two, background of invention

Rejection was the immune response that the living organism immune system produces external graft (non-) during histoorgan was transplanted.When the protein ingredient (non-antigen) of graft cell surface when contacting with receptor's immune system, discern these non-antigenic components and excite receptor's immunity system, cell immune system that graft is started immune attack by immunity, destroy, repel non-graft, cause the histoorgan graft failure.

In the organ transplant, receptor's immune system is carried out dual immunity identification by MHC (tissue intersolubility's compound) recognition mechanism to graft MHC phenotype and small peptide compound.Be called humam leucocyte antigen (HLA) in human MHC, if transplantation donor and receptor HLA type are inconsistent, then receptor's immune system is identified as graft non-and immune response is taken place for it, causes " immunological rejection ".

In immunological rejection, hyperacute rejection is that the antibody molecule at donor HLA that has existed before transplant the recipient's body inherence is mediated by receptor's body fluid immunizing composition mainly.These antibody make its immune system non-molecule of HLA of contact by receptor's pregnancy, acceptance blood transfusion, organ transplant etc. and produce.The feature of this antibody-like is can combine with donor HLA rapidly also to pass through complement-fixing, and activating complement system generation cascade reaction brings out the CDC effect.This hyperacute rejection takes place rapid unusually, be generally a few minutes to a few hours, and acute, chronic immunological rejection is got involved simultaneously by receptor's humoral immunity and cell immune system.Therefore, generation for immunological rejection in the prevention of organ transplant, except that the donor of as far as possible selecting HLA type and receptor HLA type to be complementary, before transplanting, utilize donor lymphocyte and recipient's serum to carry out crossmatch, to differentiate that the antibody that whether exists in the recipient's body at donor HLA is the key that the prevention hyperacute rejection takes place.Simultaneously, after organ transplant,, comprise that PRA (population response hla antibody) measures the monitoring acute, that chronic rejection takes place and transplants the survive prediction in time limit of back receptor and has extremely important value to the detection of hla antibody in the recipient's body.

According to antibody activating complement whether fixedly, whether bring out the CDC effect, human HLA antibody mainly can be divided into two classes: but a class for bringing out the CDC effect with complement-fixing after corresponding HLA combines and activating complement system, such hla antibody claims complement fixation hla antibody (CFAbs:Complement Fixing Antibodies); Its effect in bringing out immunological rejection is very clear and definite, is the humoral immunity composition that directly causes immunological rejection; And another kind of hla antibody combines though can specificity take place with HLA, and complement-fixing does not excite the CDC effect, is called non-complement fixing antibody (Non-CFAbs; Non-Complement Fixing Antibodies).This antibody-like even do not contact at body immune system under the situation of any non-HLA is present in the human body with native form; And can combine (also can combine) with the HLA molecule of self but activating complement not brings out the immune attack effect with the non-molecule of HLA.So far, people are very not clear to the effect of this antibody-like.But most scholars think that these antibody belong to a kind of natural antibody (Natural Antibody) composition, and biosome is had protective effect.The U.S., France etc. once did comparatively deep research to this.External a large amount of experiment and clinical trial all obtain conclusion in full accord: promptly have natural antibody in the normal human; because it has immunoloregulation function, to keeping living organism certain useful protective effect of healthy performance (Protection Effects) and can being effectively applied to suppress immunological rejection in the organ transplant.

At present, method for measuring is divided into two classes to hla antibody in the world: a class is a Terasaki ' s microlymphocytotoxicity test, abbreviates the CDC serological method as, is a kind of classical standard CD C assay method that generally believes.The lymphocyte mortality ratio that this method is brought out with CDC is as judgment criteria, and its principal character is directly to measure the CDC final effect that is brought out by CFAbs, is a kind of CDC biological effect assay method.Crossmatch and PRA that this method has been widely used in clinical organ transplant measure; But there are a lot of shortcomings such as time-consuming length, technical sophistication, experiment condition is wayward in it.Another kind of method can be measured CFAbs and Non-CFAbs simultaneously; Comprise that cells were tested by flow cytometry is incorporated into the assay for antibodies of cell surface, or wrap by the anti-hla antibody determination method of solid phase particles with the HLA of purifying; And use that purifying HLA bag is measured in conjunction with the antibody ELISA method of HLA by 96 hole microwell plates etc.The shortcoming of these method maximums is existence of having ignored natural hla antibody, promptly is generally Non-CFAbs, and they do not cause the CDC effect; On the contrary, these antibody can seal this HLA antigen site with after corresponding HLA combine, and prevention HLA toxicity antibody-CFAbs combines with corresponding HLA's, thereby blocks consequential CDC effect, make the receptor produce immune tolerance to graft.Here it is, and why many laboratories often draw makes us being difficult to explain or the result conflicting with the CDC serology; even lapse to antipodal result with the receptor is clinical: as accept the receptor that IVIG suppresses the immunological rejection treatment; said method is lasting masculin as a result, and the clinical patient state of an illness is normal or take a turn for the better.In addition, in clinical organ transplant, such methods and results often causes the misleading that donor is selected, and causes some anxious patient who treats organ transplant to miss tree-remover meeting and sb.'s illness took a turn for the worse even death.

All another obvious deficiencies of method of measuring hla antibody are that required time is longer at present, and this is the first cause that causes graft failure in cadaver donor's organ transplant.

In sum, organizing crossmatch before organ transplant, the CFAbs that has existed in the recipient's body is measured, is that the decision organ is migrated to one of key factor that loses; After organ transplant, be the important indicator that the monitoring receptor has or not immunological rejection; Simultaneously, the state of an illness of transplanting the receptor is lapsed to have predicting function.

At present, in the world the said determination hla antibody that generally uses organize the crossmatch method, all can not fully satisfy above requirement.Therefore, set up the standardized method that a kind of easy, quick, accurate, sensitive, stable CFAbs measures and become pressing in the organ transplant.

The present invention is according to the occurring principle of immunology CDC, be that CDC takes place and must be incorporated into corresponding target cell by hla antibody, then the classical pathway of fixing first kind of complement component activating complement is or/and alternative pathway, in the presence of a series of enzymes and the multiple factor, the cascade reaction of complement activation taking place, attack thing (MAC) until forming film, makes biological cell membrane impaired, form aperture and cause osmotic pressure reduction in the cell, cytolysis, death.Because the relative populations of CFAbs complement-fixing becomes positive correlation with sample CFAbs concentration and CDC effect degree, therefore, measure the level of HLA-CFAbs complement-fixing or measure arbitrary factor or complement itself and compound thereof in the complement activation pathway, having or not or relative concentration of HLA-CFAbs can be directly reflected on the one hand, the relative extent and the quantity of CDC effect or cellular damage, death can be reflected on the other hand indirectly.

Because the object that the present invention measures is CDC the HLA-CFAbs-complement compound composition in initial stage takes place, so existing relatively method of reaction time of needing of this method is for the shortest.

Because the present invention adopts fluorescence labeling as tracer, therefore has high sensitivity.

Because the present invention utilizes the complement that exists in receptor's autoserum as the response measurement object, and the reactive system chief component is recipient's serum and donor's cells; Therefore, can simulate environment in the interior physiology of recipient's body truly.

Because CFAbs and complement component be under low-temperature condition in the serum, activity can long preservation (several years), so this method has stability and good repeatability on the methodology.

Because adopting flow cytometer is the analysis to measure instrument, therefore in the same reactive system, can be simultaneously homocellular CDC effect is carried out list or/and the multiple labeling relative quantification is measured to occurring in, and can measure respectively the CDC effect that occurs in multiple different cell colonys simultaneously.Also can carry out the statistical analysis processing at last, provide science, accurate, reliable result collecting data; The permanent storage testing data.

In sum, the CFAbs crossmatch method synthesis that the present invention set up the major advantage on the existing multiple crossmatch methodology, remedy its deficiency, had theoretic rationality, and in the experiment of actual crossmatch, obtained the consistance result that fits like a glove with theoretical foundation.Example one, direct crossmatch method: the karyocyte that 1, in reaction tube, adds a certain amount of organ transplant donor (hereinafter to be referred as donor); 2, the serum that in above-mentioned test tube, adds a certain amount of organ transplant recipients (hereinafter to be referred as the receptor); 3, behind the incubation certain hour, wash with the cleansing solution pair cell; 4, cell is suspended in again in the damping fluid that contains at least a fluorescence labeling immuno conglutinin (as anti-C1q of FITC-and/or C3 antibody etc.); 5, the damping fluid that adds the antibody (as PE-anti-cd 3 antibodies, the anti-CD19 of PerCP or CD20 antibody) that contains at least a another fluorescently-labeled anti-cell surface protein composition; 6, behind the incubation certain hour, pair cell washs; 7, with cell suspension in certain damping fluid; 8, collect, analyze with flow cytometer pair cell sample, the fluorescence intensity result to each specific in sample cell mass surface immuno conglutinin carries out corresponding analysis calculating at last, compares with the normal control sample, provides the semi-quantitative results of crossmatch; 9, the crossmatch result can be expressed as: T lymphocyte chiasma type is negative or positive; The bone-marrow-derived lymphocyte crossmatch is negative or positive; The macrophage crossmatch is negative or positive; The mononuclearcell crossmatch is negative or positive; The neutrophil leucocyte crossmatch feminine gender or the positive etc.Example two, indirectly crossmatch method () 1, acceptor serum handled through 56 ℃ handled the deactivation complement in 30 minutes; 2, step is with in the example one 1,2, but with example one step 2 in different be to add a certain amount of recipient's serum who handles through inactivation of complement; 3, add a certain amount of complement component (be human complement or animal complement, be generally C1q or C3) again; 4, following steps are with step 3,4,5,6,7,8,9 in the example one.Indirectly crossmatch method (two) 1, with in the crossmatch method () indirectly 1,2; 2, in above-mentioned reaction, add a certain amount of fluorescently-labeled complement component; 3, step is with step 6,7,8,9 in the example one.Example three, solid phase HLA antigen particles measure the flow cytometer method () 1 of CFAbs, add a certain amount of solid phase particles that is coated with known specific HLA antigen such as HLA-I class, HLA-II class in reaction tube; 2, add a certain amount of complement component again; 3,, but be the particle of HLA bag quilt this moment " cell " with step 3,4 in the example one; 4, above-mentioned particle is suspended in the damping fluid that contains a certain amount of fluorescently-labeled immuno conglutinin; 5, with step 6,7,8 in the example one, wherein " cell " is the antigen coated solid phase particles of HLA; 6, according to the HLA antigenic component difference of solid phase particles bag quilt, the result can be expressed as: HLA-I class CFAbs is positive or/and HLA-II class CFAbs is negative.Annotate: solid phase particles can be one or more in the above-mentioned same reactive system of *; different sizes; be coated with known specific purifying HLA antigen particles or same size respectively but contain isolabeling (as containing the fluorescence difference) not one or more particle and wrap respectively by known specific purifying HLA antigen, on flow cytometer, obtain classification and determination whereby.Solid phase HLA antigen particles measures the flow cytometer method (two) 1 of CFAbs, with example three steps 1; 2, add fluorescently-labeled complement again, as FITC-C1q or/and FITC-C3 etc.; 3, with step 5,6 in the example three.The CFAbs cells were tested by flow cytometry method method (one) of example four, anti-vascular endothelial cell: 1, in test tube, add some human vascular endothelials (as cultured cells system); 2, with step 2,3,4,5,6,7,8 in the example one; 3, the crossmatch results expression is: the endothelial cell crossmatch is negative or positive.Method (two): 1, the recipient's serum is handled 30 minutes deactivation complements through 56 ℃; 2, with the step 1 in the routine cubic method (); 3, the serum in a certain amount of step 1 of adding through the inactivation of complement processing; 4, add a certain amount of complement component (as C1q, C3 etc.); 5, with step 3,4,5,6,7,8 in the example one; 6, with step 3 in the routine cubic method ().Method (three): 1, with step 1,2,3 in the routine cubic method; 2, add a certain amount of fluorescently-labeled complement component (as C1q, C3 etc.); 3, with step 6,7,8 in the example one; 4, with step 3 in the routine cubic method ().

Three, summary of the invention

The method that the present invention set up is that the flow cytometer that a kind of HLA-CFAbs measures is organized the crossmatch method, it is by measuring existence and the level thereof that is fixed on the surface of cell membrane complement by CFAbs, reach to HLA-CFAbs and bring out the measurement of CDC effect degree.This method donor, receptor's in can be used for organ transplant the crossmatch; According to same principle and methodology thereof, can be used for the similar test of other any needs assessment CDC effect or other effect of complement, have methodological versatility.The CDC-FXM method kit that the present invention set up comprises following chief component: (one), sample:

1, liquid phase part: comprise and to contain CFAbs or contain any albumen of causing complement fixation or the liquid of other composition, as the similar liquids of all biological fluids, tissue, cell extract or artificial preparation.

2, solid constituent: for causing CFAbs or complement directly or indirectly in conjunction with any material composition on it with certain geometric shape.The solid constituent that comprises one or more identical or different geometric shapes is present in same reactive system, and can classify on flow cytometer and differentiate any synthetic or naturally occurring abiotic solid constituent and the biosolids composition of measuring; As the spherical particles of the different sizes of synthetic or identical size but have on flow cytometer measurable not isolabeling spherical particle etc.; Various solid constituents in the biosome comprise various cells, blood platelet, bacterium etc.(2), mark tracer:

In the reactive system, comprise a kind of mark that can be measured by flow cytometer at least, be generally various fluorescently-labeled all complement components, and participate in or with the various immediate constituents of complement activation cascade reaction or composition indirectly; Or at the various fluorescent-labeled antibody of mentioned component.As adopting the fluorescence complement component, can directly directly measure in the reactive system the mark complement that is combined in solid phase particles or cell surface as tracer; Anticomplement or anti-other as usage flag in reactive system participate in the composition antibody of complementary reaction, can realize the indirect measurement to the CFAbs complement-fixing.As in reactive system, use one or more different just can being implemented on the flow cytometer heterogeneity in the same reactive system to be carried out category measurement, (as T lymphocyte, bone-marrow-derived lymphocyte, macrophage, tumour cell, hybridoma, various stem cell, genetic recombination cell etc.) with above fluorescently-labeled antibody at surface of cell membrane characteristic chemical constituent (as CD series of markings, acceptor etc.).

Four, the typical method of embodiment the present invention foundation is learned and roughly is divided into following key step (is example with the HLA crossmatch): one, reactive system is set:

(1), incubation reaction: incubation is more than 5 minutes jointly with donor karyocyte (comprising lymphocyte, macrophage, M7, monocyte etc.) for recipient's serum's (containing various antibody components and C1Q, C3 etc.), and heated culture temperature is controlled between 1 ℃ to 50 ℃ above freezing.Be generally 4 ℃ or room temperature (25 ℃ ±) or 37 ℃.By incubation, the hla antibody among the recipient's serum is incorporated into the HLA molecule on donor's cells surface, and the complement component among the recipient's serum optionally is fixed on the specific region that is combined in surface of cell membrane CFAbs.

(2), washing: cleansing solution can be selected for use and contain the physiological buffering liquid that does not influence cellular morphology, activity and do not have dissociated cell-CFAbs-complement compound.By washing, centrifugal, inhale and to abandon supernatant and can remove various floating preteins composition, especially antibody, the soluble antigen-antibody complex that is present in the liquid phase, free complement etc., through washing, reactive system is other solid constituent such as remaining cell only.

(3), mark: carefully cell precipitation partly is suspended in and contains the anti-C1q of FITC-or/and anti-C3 antibody in the damping fluid of (or F (ab), F (ab ') 2 fragments), PE-anti-cd 3 antibodies and/or the anti-CD19 of PerCP-(or CD20) antibody; Continue incubation.At this moment, the anti-C1q of FITC-in the reactive system forms the tetrad compound or/and anti-C3 antibody can combine with the complement in cell in the step (two)-CFAbs-complement three combinations; Be combined in the sxemiquantitative index of cell surface CFAbs as reflection; And the anti-CD19 of PE-anti-cd 3 antibodies and PerCP-(or CD20) antibody can combine with T, bone-marrow-derived lymphocyte respectively, so as to differentiating the lymphocyte of distinguishing these two groups of different characteristics.

(4), washing: same step (two), this moment, so far, the reaction of crossmatch was finished substantially by all not combined free fluorescent-labeled antibody compositions in the washing removal liquid phase.

(5), measure: cell carefully is suspended in the phosphorus enzyme buffer liquid that contains 2%Formadyhyde, uses flow cytometer to carry out cell harvesting, simultaneously the different fluorescence labeling strength signals of each cell surface are measured also respective graphical, the raw data of store collected.

(6), analysis, result judge: the mean value with negative control (can be the Channel Value or the ChannelShift of FITC fluorescence intensity; Before formal experiment, determined already) add the negative upper limit of two standard deviations (X+2SD), exceed this upper limit person, decidable is that T is or/and the bone-marrow-derived lymphocyte crossmatch positive; Be lower than this upper limit person, be judged to be T or/and bone-marrow-derived lymphocyte crossmatch feminine gender.Two, the negative control and the negative upper limit are set:

Serum is the AB-serum that picks up from never the normal health male sex who accepts blood transfusion, transplants, and serum can be from the pooled serum of same individuality or a plurality of individualities.This serum can be through 56C, and 30 minutes inactivation of complement is handled, also can be not treated.Cell can be from the same colony of serum or different normal person colonies, but number of samples must surpass more than 10, calculate the mean value (X) and the standard deviation (SD) of the anticomplement fluorescence intensity level that crossmatch obtains, and judge the upper limit with X+2SD as negative findings.Three, positive control is set:

Can select for use the blood serum sample of a plurality of high PRA to mix, purpose is for comprising the CFAbs at all HLA types as far as possible to greatest extent.By trial test, establish the positive quality control contrast of three kinds of different CFAbs respectively; Be high, medium and low CFAbs concentration serum, wherein low CFAbs should be located at about about 50 Channel Shifts.Four, own control is set:

Add the reactive system that self mononuclearcell is formed by receptor's autoserum; By with above-mentioned normal negative control relatively, to judge in the recipient's body whether existing of self CFAbs.This index can be used as the reference that the prediction rejection takes place.Illustrate 1, the type of CFAbs differentiates:

As the antibody type in the positive reaction of need discriminating crossmatch, can handle (degraded IgM) to serum by DTT; Carry out crossmatch again, if the result transfers feminine gender to, then CFAbs is the IgM type; Otherwise be the IgG type.Illustrate 2, in the above-mentioned crossmatch, step (one) to step (three) can be merged, promptly in reactive system, add serum, cell and above-mentioned various fluorescent-labeled antibody simultaneously or behind serum, cell, fluorescence immuno conglutinin incubation, without washing, directly add fluorescently-labeled anti-CD3 or/and anti-CD19 or CD20 antibody.Illustrate 3, as required, utilize different fluorescence antibodies combinations, can realize independent T or bone-marrow-derived lymphocyte crossmatch.Illustrate 4, the above basic step that just exemplifies crossmatch, in the actual application can according to described in this patent and ultimate principle and concrete detected object and research, observation purpose difference, on methodology, adjust accordingly.What therefore, concrete experiment condition and step were not limited to reach described in this patent gives an example.Brief summary:

The HLA crossmatch flow cytometer method synthesis of the mensuration fixed complement antibody that the present invention set up anti-IgG flow cytometer crossmatch method highly sensitive, stablize the characteristics that good reproducibility, multiple labeling category measurement and traditional C DC serology crossmatch method can directly be measured CDC immunology biological effect; Based on the inducement mechanism of CDC effect, very early time (initial response facilitation effect) formed target cell HLA-CFAbs-of the stage complement compound by measuring the CDC effect is as the index of CFAbs level in the assess sample and CDC effect degree.Because in the CDC generating process, from complement toxicity antibody (CFAbs) combination, complement fixation arrives can be by the whole last effect of the CDC of in-vitro measurements---cell death occurs, and needs the longer time limit; Therefore, the distinguishing feature of the inventive method is the complement component that can utilize sample self to have, and simulates biological body physiological microenvironment, learns the end last effect that reflects CDC immunology biology in conjunction with effect by the early immune of measuring HLA CFAbs complement-fixing; And can in same reactive system, carry out multiple labeling classification analysis mensuration by the CFAbs to various kinds of cell colony.Therefore, said method (kit) has the distinct advantages that existing all similar approach did not have in the world.Can be widely used in crossmatch, PRA in the HLA field and measure etc., and complement and CDC Study on Effect in the immunology.

Increase: description of drawings

Fig. 1 shows the blind type result that joins of list of two kinds of methods of totally 117 routine HLA crossmatchs among the figure.Totally 109 examples that CDC-FXM is consistent with traditional C DC serological method result, two kinds of all positive person's 26 examples of methods and results; Two kinds of methods and resultses are negative patient 83 examples.At the traditional C DC serological method sample of feminine gender as a result, it is positive that CEC-FXM detects 8 examples; The false negative result that confirms traditional C DC serological method is up to 7%.

Fig. 2 will contain the serum dilution in 1: 4 of CFAbs, and through 56 ℃, inactivation of complement in 30 minutes is handled.The people C1q that adds different amounts then respectively, the concentration of required C1q when the positive appears in observation CDC-FXM result.The result shows: the C1q concentration that makes the CDC-FXM positive is 3 μ g/ml (normal C1q serum mean concentration is 75 μ g/ml).

Fig. 3, the blood-serum P rotein-G affinity chromatography that will contain CFAbs extracts the whole IgG compositions of purifying, returns the IgG that adds purifying by various dose respectively then in the CDC-FXM reaction, observes to cause required IgG concentration when CDC-FXM is positive.

The result shows:

1, the CDC-FXM positive serum is after removing IgG, and complement can not be incorporated into cell surface separately; Be that the complement combination rate that CDC-FXM measures has directly reflected the combination rate of CFAbs and target cell, and have the positive relation.

IgG concentration when 2, making CDC-FXM result transfer the positive to is that (normal human serum IgG concentration mean value is 0.4mg/ml: 12.5mg/ml).

Fig. 4 utilizes solid-phase coating that the plastic beads of purifying HLA antigen is arranged, and with a certain amount of IVIG reaction, utilizes fluorescently-labeled anti-human IgG antibody to detect on flow cytometer and HLA antigen I gG (IVIG) content respectively.The result shows: IgG be starkly lower than normal physiological concentration mean value (＜10mg/ml) time, still can cause combining of extremely significant IgG and purifying HLA antigen.

Fig. 5 utilizes CDC-FXM, IgG-FXM, and CDC serology crossmatch method is measured the organ transplant patient of the same IVIG of acceptance treatment respectively: the result shows: before the IVIG treatment, the crossmatch result of three kinds of methods is all positive; And after the IVIG treatment, patient's rejection is subjected to IVIG and suppresses, and clinical sb.'s illness took a favorable turn, but the IgG-FXM utmost point is positive; And CDC-FXM and CDC serology result are negative, and be consistent with clinical observation.

Fig. 6 adds the different IVIG that measure in containing the serum of CFAbs; With the respective target cell effect; Simultaneously with fixing complement and the IgG content in flow cytometer replicate determination target cell surface.The result shows: IVIG has extremely strong inhibiting effect to the ability of CFAbs complement-fixing, but the combination of IgG is not made significant difference.The above results and clinical use IVIG suppress effectively that the result of immunological rejection matches in the organ transplant; Be that IVIG can reach the CDC lethal effect that suppresses in the immunological rejection thereby seal the CFAbs binding site by combining with target cell.

Fig. 7 extracts two normal persons' of purifying IgG respectively with the Protein-G affinity chromatography, with the solid phase particles reaction that is coated with purifying HLA antigen, with fluorescently-labeled anti-human IgG antibody, detect the combination rate of IgG and HLA antigen on flow cytometer then.

The result shows: the repeatedly conceived IgG purification of producing the normal women (53 years old) of (three times) and never accepting the normal young male sex (20 years old) of any blood transfusion, organ transplant all can combine effect with HLA antigen generation conspicuousness.This result further confirms: there has been natural hla antibody (NaturalHLAAntibody) composition in the normal person in its body before not accepting any external HLA stimulation.Criticize the heterogeneous control of interpolation table 3 as a result between specificity qualification result (videing infra) the table 2:CDC-FXM of table 1:CDC-FXM method criticizes: three kinds of crossmatch methods relatively

Crowd heterogeneous control result of interpolation between table 2:CDC-FXM criticizes

CDC-FXM?QC?RESULT

?QC	????N	????????????????CV％
					????QC-L	????QC-M	????QC-H
		?Inter-Assay	????10	????1.88				????3.61	????4.2
?Intra-Assay	????17				????4.6	????6.2	????3

Sheet1

The specificity qualification result of table 1:CDC-FXM method

SPECIFICITY?OF?C1q?FIXING?ANTIBODY?FLOW?CROSSMATCH

(CHANNEL?SHIFT)

CELL?ID	???CELL ???TYPING	????HOLDEN	????WALLAU	??AFRAM	???ADUBIFA	??NOORI	????RIVAS	???ELIZABETH	????MARY
										????B51+	????B51+	??A11+	??? *A11+	??A2+	????A2+	??? *A2+	????A2+
		06FP82119	???A2+；A11+	????19	????13	??287	???137	??593	????269									???232	????672
LF28291	???A11+									????8	????16	??217	???187	??7	????7	???8	????7
		K16297	???A2+	????3	????19	??24	???1	??578	????306									???124	????666
K16355	???A2+；B51+									????662	????302	??20	???9	??660	????492	???374	????549
		M17563	???A2+；B52+	????692	????301	??7	???4	??544	????290									???68	????520

Table 3: three kinds of crossmatch methods relatively

Claims (18)

1, a kind of reaching by mensuration complement fixation amount measured the sxemiquantitative of CFAbs level in the biological sample, and assessment occurs in flow cytometer HLA crossmatch method and the kit that specific cells surface C DC effect takes place whereby.This method and kit comprise following content:

2, in the reactive system that is subjected to sample product and respective target cell or target antigen composition, add one or more tagged ligand, dyestuff or antibody relevant at least with CDC effect process.

3, CFAbs is interpreted as the antibody component of the fixing or conjugated complement ability of having of any source in the claim 1.

4, tagged ligand in the claim 2 or labelled antibody are interpreted as following two classes:

The first kind: the various compositions that directly or indirectly participate in CDC reaction are or/and its corresponding antibody, and it can reflect the arbitrary stage or the state of CDC effect process.

Second class: can reflect or identify the target cell feature, or have various parts or the antibody component of target cell being differentiated classification.

5, two class parts of being addressed in the claim 4 or antibody can use simultaneously or separately; But in the response measurement system, must comprise at least a tagged ligand or antibody or dyestuff, so as to the generating process of reflection CDC.

6, the CDC effect process in the claim 2 is interpreted as being incorporated into target cell or target antigen by the antibody with complement-fixing effect and first complement component and begins to forming final immune complex, or complement membrane attack complex (MAC; C5b-9) to the overall process of cell death.

7, in the claim 2, be subjected to the sample product to comprise any biological sample that may contain CFAbs; The body fluid that comprises people, animal, routine serum, blood plasma, spinal fluid, amniotic fluid, cerebrospinal fluid, saliva, urine etc.

8, in the claim 2, target cell be interpreted as any can combine with CFAbs through artificial treatment or naturally occurring eucaryon or prokaryote; Example human cell (red blood cell, leucocyte, each section's histocyte, various stem cells etc.), zooblast, insect cell, microorganism, bacterium, blood platelet etc.

9, " artificial treatment " in the claim 8 further is interpreted as: change the cell that cytogenetics feature or cell biology method transform (as the clone of genetic recombination, transgenosis, Fusion of Cells, in vitro culture etc.) through molecular biology method.

10, " artificial treatment " in the claim 4 further be interpreted as comprising dead target cell through chemistry and physical method processing (as chemical reagent fix, physics freeze drying etc.).

11, in the claim 2, the target antigen composition is interpreted as: any biological antigens composition that can combine with CFAbs; Refer in particular to the antigen that exists with solid phase form.Example connects by chemical method or directly is coated on the multiple HLA composition on solid phase particles surface or other antigenic component etc. by physisorption.

12, the labelled antibody in the claim 2 is interpreted as the antibody of all participation compositions (as complement and compound thereof, the various factors, acceptor etc.) in the anti-CDC effect process; And the antibody of anti-target cell antibody of specificity and anti-solid phase particles surface HLA target antigen.

13, in the claim 2 mark of labelled antibody be interpreted as can be detected on flow cytometer all fluoresceins or fluorescin; Example: FITC, PE, Cy5, Per-CP, Cy5-PE, Cy7 etc.

14, the dyestuff in the claim 2 is interpreted as entering by damaged cell and makes damaged cell in the cell or/and dead cell is painted and the various dyestuffs that can predict such as ethidium bromide, eosin and other have glimmering pigment of same function etc. on flow cytometer.

15, the tagged ligand in the claim 2 is interpreted as having fluorescence labeling and participates in all the components (comprising that classical complement activation pathway and C3 substitute complement activation pathway and MBL complement activation pathway) in the CDC course of reaction, especially various fluorescently-labeled C1q, C3 etc.

16, the tagged ligand in the claim 2 further is interpreted as: C1(C1q, C1r, C1s), C2, C3, C4, C5, C6, C7, C8, C9; C1qrs , C1qRs , C2a,C2 B, C3a, C3b,C4a, C4b, C4b2, iC3b, C4b2b, C4b2b3b, C3bBb, C3bnBb, C5a, C5b, C5b67, C5b ∽ 8, C5b ∽ 9; C1-inhibiting factor, C4-are in conjunction with albumen, the D factor, the B factor, the P factor (properdin), the I-factor, the H-factor, S-albumen; Ba, Bb, MBP, MCP, DAF (CD55), CR1, CR2, CR3, CR4, CR5, C3aR, C2aR, C4aR, C1qR, CD59 etc.

17, the tagged ligand in the claim 2 source is interpreted as the various compositions of humanized, animal derived and naturally occurring multiple direct or indirect participation CDC reaction purified or not purified or that obtained by molecular biology method.

18, the labelled antibody in the claim 2 is interpreted as the complete molecule and the fragment thereof of the monoclonal and the polyclonal antibody in any source, comprises humanized, complete antibody molecule, chimeric antibody molecule (ChimericAbs), single-chain antibody molecule and fragment thereof animal derived, plant-derived or that obtain by gene engineering method such as F (ab ') 2, Fab, Fv, Facb, F (abc ') 2, ScFv etc.

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