CN101799473B - SPA-antibody tripolymer, cell treating kit containing tripolymer, preparation method and application thereof - Google Patents
SPA-antibody tripolymer, cell treating kit containing tripolymer, preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an SPA-antibody tripolymer, a cell treating kit containing the tripolymer, a preparation method and application thereof. The tripolymer comprises an anti erythrocyte antibody, an SPA and an antibody of a certain anti leukocyte antigen or other antigens. The tripolymer is combined with an erythrocyte through the anti erythrocyte antibody per se, the other antibody is combined with a corresponding leukocyte antigen (or other antigens), and then leukocyte (other cells, factors) and the erythrocyte are deposited through an erythrocyte sedimentation process or other methods of density gradient centrifugation and the like, thereby achieving the purpose of eliminating ingredients of corresponding cells and the like, connecting the erythrocyte with a corresponding antigen by the SPA, and being used as a mean for detecting a certain antigen. The invention has convenience and simpleness, and can be directly used for clinically separating and extracting stem cells/progenitor cells; and the collected and extracted stem cells/progenitor cells have no external markers. Meanwhile, the kit based on the tripolymer can be industrially produced so as to realize the popularization of the separation and purification technology of hematopoietic stem cells.
Description
Technical field
The present invention relates to field of biomedicine technology, particularly relate to staphylococcal protein A (staphylococalprotein A, hereinafter to be referred as SPA)-antibody tripolymer and the cell processing kit based on this aggressiveness and its production and use.
Technical background
SPA can the Fc fragment in people and multiple mammalian blood serum IgG molecule be combined.In conjunction with compatibility order be pig, dog, rabbit, people, monkey, mouse, mouse and ox successively.SPA is except being combined with IgG, and IgM and IgA that can also be a small amount of in serum be combined, and the adsorption rate of SPA bacterium to each immunoglobulin like protein: IgG is that 90%～98%, IgM is that 2%～30%, IgA is 1.5%～20%.
The SPA and the IgG that occur coprecipitated reaction must have two binding sites, and they in Fc and Fab section, can be combined with SPA though lack one respectively, do not occur coprecipitated reaction, and the participation of this Fab is not SPA-antibody response.Now study and show, the binding site of IgG and SPA is CH
2and CH
3intersection.
SPA application is very wide, has and the binding ability of the Fc section of IgG but the principle of all application all be unable to do without SPA, and mainly application aspect is as follows:
The coagglutination effect of 1.SPA bacterium
With known standard serum, be adsorbed onto the surface of SPA bacterium, make it to become the carrier of absorption antibody, then go to diagnose corresponding unknown antigen and produce macroscopic agglutinating particle with this, the antigen of diagnosis can be graininess, can be also solubility.This kind of aggegation is equivalent to reverse indirect hemagglutination, but saves the complicated formalities such as the antibody purification in indirect hemagglutination, tanning of red blood cell, and only needs SPA thalline and rough immune serum.So this method is simple, quick, specificity susceptibility is all more satisfied.
2. as wide spectrum second antibody
The second antibody that replaces IgG antibody with SPA, has the following advantages: 1. SPA preparation is easy, stable in properties, and easily purifying, can apply people and multiple mammiferous IgG, not limited by kind.2. the adhesion of SPA is strong, is equivalent to the adhesion of free antibodies and antigen, and the affinity costant of people and rabbit is to 10
3l/ grammol, in conjunction with rear on the activity of IgG without impact.No matter 0 DEG C, 37 DEG C, 44 DEG C and 56 DEG C almost all can be immediately in conjunction with and without hatching for a long time, therefore can shorten test period; 3. SPA easily puts on
125i, fluorescein, horseradish peroxidase, ferritin etc., therefore can combine with multiple technologies; 4. SPA numberator height homogeneous, it is not immunoglobulin (Ig), not affected by Fc acceptor, so there is higher anti-IgG specificity.
3. utilize the binding characteristic of SPA and IgG to extract IgG for extracting IgG purification, adopt ammonium sulfate, Sephadex post than routine, the methods such as DEAE post or immune gradient centrifugation are wanted easy too much, and purity is good.
4. detection and purification IgM IgM are significant in viral early diagnosis, for detection specificity IgM antibody, must first in serum, remove IgG.Adopting SPA bacterium absorption IgG is current quick and desirable way, utilizes this method also for road has been opened up in the purification of IgM.
5. other side is used for the detection of detection, Activation In Vitro complement function test, cell surface marker and the TSA of immune complex etc.
Stem cell (stem cell) refers to have unlimited or longer self-renewal capacity, and can differentiate the cell that at least one highly breaks up daughter cell.According to cell derived, stem cell is divided into Embryo stem cell and adult stem cell, and adult stem cell comprises autologous stem cells and allosome stem cell.
The impact of the mechanism of action, organismic internal environment that current latest developments both domestic and external impel " stem cell " grafting vessel new life on stem cell curative effect and the effect of transgenosis marrow derived stem cell are further studied, to improve the result for the treatment of of stem cell.
In therapeutic process clinically, the main mononuclearcell that gathers marrow or derived from peripheral blood is transplanted, and think the ancestral cells that CD34+ wherein or CD133+/VEGF-R2+ cell are angiogenesis, but the Proliferation, Differentiation function of this class cell, their ability of the vascular injury site of going back to the nest and the effective quantity etc. that reaches result for the treatment of are all the contents of not yet fully understanding.
Surface antigen (Lineage surface antigen) is in the time of the Stem cell differentiation CFU-GM that is each system and mature cell, there will be the selectivity mark of each system, as Lymphatic System CD19 CD7 granulocyte series CD13, CD11, CD15, CD16, the CD47 of erythron, CD51, CD71, the CD31 of megakaryocytic series CD41 CD42 CD61 CD63 CD107, T NK system CD2 CD25 CD7, it is surface antigen that the antigen that these surfaces of haemocyte in particular type occur is commonly referred to as, Lin antigen positive just can be defined as CFU-GM and mature cell, also have the cell of many Lin antigen negatives to be referred to as primordial stem cell, as CD34 CD133 SCA-1 CD38 etc.We can require the mature cell that removal is relevant to be according to each section office are different.
Stem cell can effectively be treated various cellular damage diseases, and this has been whole world mainstream health care circle admitted facts.But how to obtain stem cell, how to separate, stem cell that purifying is obtained survival, very desirable is the problem of whole world scientist's joint research.From marrow, Cord blood, the technology of separating and extracting stem cells has much at present, as: immunomagnetic beads method and flow cytometer method etc.The existing common issue of these methods is: first can not effectively retain all types of stem cells, and the second cost is expensive, and technical conditions are had relatively high expectations.On clinical application, there is certain difficulty.
Summary of the invention:
The invention provides a kind of by the intermediary of red blood cell in blood and other cell/factor combinations: spa-antibody tripolymer, can, by red blood cell and needed leucocyte or other cell/factor combinations, the specific cells in blood/factor can be removed by the method such as erythrocyte sedimentation rate or gradient centrifugation by this tripolymer.
Technical scheme of the present invention is:
1.Spa-antibody tripolymer and preparation method thereof:
Material: Recombinant staphylococcus protein A, A antibody: anti-erythrocyte monoclonal or polyclonal antibody, B antibody: can need to select different monoclonals or polyclonal antibody according to clinical and experiment, as: specificity lymphocyte antibody (anti-cd2 antibody, anti-cd3 antibody, anti-cd4 antibody, anti-cd11 antibody, anti-cd15 antibody, anti-cd16 antibody, anti-cd8 antibody, anti-cd19 antibody, anti-cd5 antibody, anti-cd7 antibody, anti-cd13 antibody, anti-cd33 antibody, anti-cd10 antibody, anti-cd21 antibody, anti-cd22 antibody, anti-cd23 antibody, anti-cd32 antibody, anti-cd35 antibody, anti-40cd antibody, anti-cd45 antibody, anti-cd54 antibody, anti-cd56 antibody, anti-cd64 antibody, anti-cd133 antibody, anti-cd117 antibody, anti-cd90 antibody), anti-Marek's virus antibody, anti-HBS antibody, swine fever virus resistant antibody, anti-vibrio parahemolyticus antibody, anti-typhoid antibody etc.
Spa dilutes with aseptic PBS liquid, A antibody (anti-erythrocyte monoclonal antibody or polyclonal antibody) and B antibody are diluted with antibody diluent, various dilute concentrations all can (recommending spa be 0.1mg/ml, antibody concentration: 0.1mg/ml dilution).
Raw material mass mixture ratio is: SPA: A is anti-: it is 1.5: 1: 1 that B resists; As: SPA is 1.5ml, and A resists for 1ml, and B resists for 1ml.(concentration is 0.1mg/ml)
4 DEG C～40 DEG C grades of temperature of reaction all can, better temperature of reaction is room temperature～40 DEG C, recommends 37 DEG C of incubations (water-bath or incubator all can).
Reaction time: 20 minutes～12 hours, the better reaction time was 30 minutes～3 hours, recommended 37 DEG C of incubations 30 minutes.
Production concentration: 0.1mg/ml.
Storage requirement: 4 DEG C of storages.
This tripolymer can be for from human marrow and the in-vitro separation kit etc. that separates karyocyte Cord blood, and it is simple that this technology has method, cheap, can suitability for industrialized production, be easy to the feature of promoting.This technology has effectively been removed blood plasma, red blood cell, blood platelet, plasma proteins, ion, and monocyte, lymphocyte, the apocyte of other required removal moral maturations, and has retained all stem cells, CFU-GM, mescenchymal stem cell etc.This is a kind of application process that can be directly used in clinical separation, extract marrow/umbilical cord/peripheral hematopoietic stem cells in-vitro separation kit of stem cell.
2, cell processing kit: based on above-mentioned tripolymer, bind lymphocytes parting liquid, the chromatoplate of antibody combination, the method that adopts density－gradient centrifuga－tion method, immunochromatographic method and co-immunoprecipitation method to combine, makes cell processing kit.It is characterized in that including in this kit:
The tripolymer of SPA (staphylococcal protein A) and erythrocyte antibody (EA), specificity lymphocyte antibody.The reaction density of SPA and anti-erythrocyte monoclonal antibody and various antilymphocyte antibodys is 0.1～1mg/ml.SPA is 1.5ml, and anti-erythrocyte monoclonal antibody is 1ml, and antilymphocyte antibody is 1ml.Incubation 30～60 minutes in 37 DEG C of water baths.Adjusting Spa-antibody tripolymer concentration is 1 μ g/ml, and volume is 100ml, PH7.4.4 DEG C of preservations.
Lymphocyte separation medium (commercially available) 50ml.
The chromatoplate of antibody combination: the chromatoplate of polystyrene material, after sterilization, add anti-specificity lymphocyte antibody 0.1～1mg/ml, be paved with surface, 37 DEG C are reacted 30～60 minutes, 4 DEG C of preservations.
Above-mentioned each reagent solution is separately deposited.
The method that the present invention adopts density－gradient centrifuga－tion method, immunochromatographic method and immuno-precipitation to combine, the stem/progenitor cells of collected extraction is without any external label.Can remove different mature lymphocyte system according to different requirements, convenient and simple, can be directly used in clinical, laboratory separates, extract stem/progenitor cells.
3, the range of application of Spa-antibody:
Be used for clinical diagnosis as indicator, when occurring special antigen or exotic antigen in certain Disease blood, the specific antibody of this antigen is connected on Spa-antibody tripolymer, make red blood cell as indicator, cohesion forms " red blood cell-anti erythrocyte antibody-Spa-specific antibody--specific antigen " compound, similar garland spline structure, can be observed visually condensation product or microscopic examination to Kranz structure, uses as clinical or laboratory diagnosis.
Can pass through Spa-antibody and specific antibody combination, form " red blood cell-anti erythrocyte antibody-Spa-specific antibody " compound, make to remove after specific antibody and erythrocyte binding by the method such as centrifugal.Can remove patient or specific antibody in blood for experiment.
Can be used in the extraction and purification kit of clinical stem/progenitor cells, in order to the stem/progenitor cells of purifying, increase stem/progenitor cells concentration, improve curative effect.Concrete steps: the antibody that is by specific mature cell is connected on Spa-antibody, form " red blood cell-anti erythrocyte antibody-Spa-lymphocyte antibody-mature lymphocyte ", remove mature lymphocyte system etc. by the method such as centrifugal, make stem/progenitor cells obtain purifying, improve concentration, can be used in the clinical treatment and experimental study of corresponding stem/progenitor cells.
4, the range of application of cell processing kit:
Cell processing kit can be used for extraction and the purifying of the stem/progenitor cells of marrow blood/Cord blood/peripheral blood, concrete application process is: first peripheral blood, marrow or Cord blood are added in the reagent bottle that is placed with chromatoplate, again SPA-antibody tripolymer liquid is poured into wherein, leave standstill and collect supernatant after 20 minutes, by careful supernatant joining above lymphocyte separation medium, latter centrifugal 20～30 minutes, collect cellular layer, dilute and can use with glucose for injection salt solution.
This cell processing kit also can be used for clinical stem-cell therapy, the treatments such as autogenous bone marrow blood refluxing; Also can be used in experiment, simple to operate, expense is low, and can be by removing corresponding blood constituent, for studying the growth of composition on stem/progenitor cells and the impacts of differentiation such as different mature cell systems and other cell/cell factors.
Embodiment:
Following embodiment will contribute to further to understand the present invention, but can not limit content of the present invention.
Embodiment 1: cell processing kit
1, the tripolymer of SPA (staphylococcal protein A) and erythrocyte antibody (EA), specificity lymphocyte antibody.The reaction density of this tripolymer preparation process: SPA and anti-erythrocyte monoclonal antibody and anti-cd3 antibody, cd14 antibody, cd19 antibody, cd56 antibody is 0.1mg/ml.SPA is 1.5ml, and anti-erythrocyte monoclonal antibody is 1ml, and anti-cd3 antibody, cd14 antibody, cd19 antibody, cd56 antibody are respectively 0.25ml.Incubation 30 minutes in 37 DEG C of water baths.Add PBS liquid, adjusting Spa-antibody tripolymer concentration is 1 μ g/ml, and volume is 100ml, PH7.4.4 DEG C of preservations.
2, human lymphocyte parting liquid (commercially available) 100ml, centrifugal density is 1.077g/ml.
3, cell chromatoplate: the chromatoplate of polystyrene material, after sterilization, adding concentration is the anti-cd3 antibody of 0.1mg/ml, cd14 antibody, cd19 antibody, cd56 antibody, be paved with surface, 37 DEG C of reactions were spent the night under room temperature after 30 minutes, put in 500ml reagent bottle.4 DEG C of preservations.
Material therefor is by filtration sterilization, and material is through 120 degrees Celsius, 30 minutes autoclave sterilizations, and reagent is degerming after filtration, after sterile working preparation, leaves in respectively in kit.
Cell processing kit application process: get peripheral blood, marrow or Cord blood 200ml, join in the reagent bottle that is placed with chromatoplate, add again 50mlSPA-antibody tripolymer and mix standing 30 minutes of rear 37 degree, this tripolymer respectively with leucocyte, erythrocyte binding, form cell mass, be deposited to test tube bottom.Antibody on clone and the chromatoplate of the unconjugated maturation of part is in conjunction with forming antigen antibody complex.Collect supernatant.Lymphocyte separation medium is added in centrifuge tube, again the supernatant of collecting above is carefully added to above lymphocyte separation medium, centrifugal 20 minutes of 700g, utilize density－gradient centrifuga－tion method to remove remaining red blood cell, blood platelet, blood plasma and unprecipitated leucocyte red blood cell condensate, collect cellular layer, with phosphate buffer (PBS) cleaning 2-3 time, then use injection physiological saline/glucose saline diluted for use.
Prepared cell can be used for clinical self marrow blood stem-cell therapy, after the stem/progenitor cells dilution that will extract through said process, gets involved through blood vessel the liver internal therapy cirrhosis that is expelled to patient with liver cirrhosis.The lower limb vascular that self the marrow blood stem cell after extracting or cord blood stem cell can be expelled to patient with diabetic feet around and be expelled to the far-end of narrow or occluded artery, treatment diabetes.Also can, by extracting self marrow blood stem cell after purifying or cord blood stem cell and be expelled to the disconnected injury of spinal cord of patients of acute spinal cord injury, treat acute spinal cord transection.Also can be used for other cellular damage disease.
Embodiment 2: cell processing kit
1, the tripolymer of SPA (staphylococcal protein A) and erythrocyte antibody (EA), specificity lymphocyte antibody.The reaction density of this tripolymer preparation process: SPA and anti-erythrocyte monoclonal antibody and anti-cd3 antibody, cd14 antibody, cd19 antibody, cd56 antibody is 0.1mg/ml.SPA is 1.5ml, and anti-erythrocyte monoclonal antibody is 1ml, and anti-cd3 antibody, cd14 antibody, cd19 antibody, cd56 antibody are 0.25ml.Incubation 30 minutes in 37 DEG C of water baths.Add PBS liquid, making Spa-antibody tripolymer concentration is 1 μ g/ml, and volume is 100ml, PH7.4.4 DEG C of preservations.
2, the aqueous solution of the Sodium Amidotrizoate of lymphocyte separation medium 100ml:0.3% and 8% ficoll 400# composition, density is 1.077g/ml.
3, antibody conjugates: the nano particle of anti-cd3 antibody, cd14 antibody, cd19 antibody, cd56 antibody and polystyrene material is mixed.Antibody concentration is 0.1mg/ml, and volume is 1ml, and granules of polystyrene is 10mg.Incubation ambient temperature overnight after 1 hour in 37 DEG C of water baths.4 DEG C of preservations.
Material therefor is by filtration sterilization, and material is through 120 degrees Celsius, 30 minutes autoclave sterilizations, and reagent is degerming after filtration, after sterile working preparation, leaves in respectively in kit.
Cell processing kit application process: get marrow or Cord blood 200ml and add in the reagent bottle containing antibody conjugates, add again 50mlSPA-antibody tripolymer mix rear 37 DEG C leave standstill 30 minutes, this tripolymer respectively with leucocyte, erythrocyte binding, form cell mass, be deposited to test tube bottom.Antibody on the unconjugated eukocyte of part and antibody conjugates suspends in conjunction with forming granule.Collect supernatant.Lymphocyte separation medium is added in centrifuge tube, again the cell suspending liquid of collecting above is carefully added to above lymphocyte separation medium, 2000 revs/min centrifugal 20 minutes, utilize density－gradient centrifuga－tion method to remove remaining red blood cell, blood platelet, blood plasma and antibody conjugates, collect cellular layer, with phosphate buffer (PBS) cleaning 2-3 time, then with for subsequent use after the dilution of injection physiological saline/glucose saline.
Use cell prepared by this kit to can be used for clinical self marrow blood stem-cell therapy, after the dilution of the stem/progenitor cells that will extract through said process, get involved through blood vessel the liver internal therapy cirrhosis that is expelled to patient with liver cirrhosis.The lower limb vascular that self the marrow blood stem cell after extracting or cord blood stem cell can be expelled to patient with diabetic feet around and be expelled to the far-end of narrow or occluded artery, treatment diabetes.Also can, by extracting self marrow blood stem cell after purifying or cord blood stem cell and be expelled to the disconnected injury of spinal cord of patients of acute spinal cord injury, treat acute spinal cord transection.Also can be used for other cellular damage disease.
Embodiment 3:Spa-antibody
Chicken Marek's disease (MD) is the one lymphoproliferative diseases highly contagious being caused by MDV.Its principal character is: peripheral nerve, various internal organs, iris and skin etc. lymphocytic infiltration occur and form swollen sick.Marek's disease mainly betides chicken, also has the report of birds such as betiding turkey, pheasant, duck, quail, swan.This disease transmission is strong, and velocity of propagation is fast, and latent period is long.Acute onset chicken is eliminated and mortality ratio reaches 10% ~ 80%.Up to now, in the diagnosis of Marek's disease, except AGPT, still there is no a kind of practical technique that can be convenient in basic unit application.
The tripolymer of SPA (staphylococcal protein A) and erythrocyte antibody (EA), anti-Marek's virus antibody.The reaction density of this tripolymer preparation process: SPA and anti-erythrocyte monoclonal antibody and anti-Marek's virus antibody is 0.1mg/ml.SPA is 1.5ml, and anti-erythrocyte monoclonal antibody is 1ml, anti-Marek's virus antibody 1ml.Incubation 60 minutes in 37 DEG C of water baths.Spa-antibody tripolymer concentration is 0.1mg/ml, PH7.4.4 DEG C of preservations.
When use, this tripolymer is diluted to 1: 1000 working concentration, i.e. 0.1 μ g/ml, blood sampling, adds after tripolymer in 37 DEG C of water baths incubation 1 hour, and sampling is originally examined under a microscope, with physiological saline in contrast, person is positive clustering phenomena, has infected MDV.
Embodiment 4:Spa-antibody
Swine fever (SwnieFever, SF) is a kind of height contagious disease of pig.Being acute, subacute, chronic and typical, the atypical lysis of one of the pig that caused by CSFV (ClassiealWSnieFeverviurs, CSFV), is one of Infectious Diseases of harm pig industry.
The tripolymer of SPA (staphylococcal protein A) and erythrocyte antibody (EA), swine fever virus resistant antibody.The reaction density of this tripolymer preparation process: SPA and anti-erythrocyte monoclonal antibody and swine fever virus resistant antibody is 0.1mg/ml.SPA is 1.5ml, and anti-erythrocyte monoclonal antibody is 1ml, swine fever virus resistant antibody 1ml.Incubation 60 minutes in 40 DEG C of water baths.Spa-antibody tripolymer concentration is 0.1mg/ml, PH7.4.4 DEG C of preservations.
When use, this tripolymer is diluted to 0.1 μ g/ml, and blood sampling adds after tripolymer in 37 DEG C of water baths incubation 1 hour, and sampling is originally examined under a microscope, and with physiological saline in contrast, person is positive clustering phenomena, and this blood sample has infected CSFV.
Embodiment 5:Spa-antibody
Vibrio parahemolyticus (Vibrio parahaemolyticus, VP) is a kind of halophagia pathogen widely distributed in ocean and salt lake.Extensively be distributed in inshore seawater, marine bottom sediment, fish, shellfish, be adapted to grow in the environment of high concentration (6%) salt, can not growing containing in the nutrient culture media of salt.This bacterium is the common pathogenic entero becteria of one that is second to comma bacillus, its pollution meeting brings heavy losses to mariculture industry, intestines problem and the food poisoning such as also can cause that the mankind suffer from diarrhoea, occur diarrhoea be water sample or blood sample just, abdominal cramps, feel sick, the symptom such as vomiting and headache.Since two thousand one, the outburst of vibrio parahemolyticus property food poisoning has become Chinese modal disease caused by food-borne bacteria, and a number of vibrio parahemolyticus property food poisoning and number are always in first.
The tripolymer of SPA (staphylococcal protein A) and erythrocyte antibody (EA), anti-vibrio parahemolyticus polyclonal antibody.The reaction density of this tripolymer preparation process: SPA and anti-erythrocyte monoclonal antibody and anti-vibrio parahemolyticus polyclonal antibody is 0.1mg/ml.SPA is 1.5ml, and anti-erythrocyte monoclonal antibody is 1ml, anti-vibrio parahemolyticus polyclonal antibody 1ml.Incubation 6 hours in 12 DEG C of water baths.Spa-antibody tripolymer concentration is 0.1mg/ml, PH7.4.4 DEG C of preservations.
When use, this tripolymer dilutes 0.1 μ g/ml, gets blood sample to be measured, add after tripolymer in 37 DEG C of water baths incubation 1 hour, sampling is originally examined under a microscope, with physiological saline in contrast, person is positive clustering phenomena, and this blood sample has infected vibrio parahemolyticus.
Embodiment 6:Spa-antibody
Typhoid fever is the acute infectious disease through transmission being caused by typhoid bacillus.Clinical symptoms is long-range heating, whole body toxicity symptom, relative infrequent pulse, hepatosplenomegaly, roserash and leucocyte minimizing etc.Major complications is enterorrhagia, enterobrosis.
The tripolymer of SPA (staphylococcal protein A) and erythrocyte antibody (EA), anti-typhoid antibody.The reaction density of this tripolymer preparation process: SPA and anti-erythrocyte monoclonal antibody and anti-typhoid antibody is 0.1mg/ml.SPA is 1.5ml, and anti-erythrocyte monoclonal antibody is 1ml, anti-typhoid antibody 1ml.Incubation 90 minutes in 30 DEG C of water baths.Spa-antibody tripolymer concentration is 0.1mg/ml, PH7.4.4 DEG C of preservations.
When use, this tripolymer is diluted to 0.1 μ g/ml, and blood sampling adds after tripolymer in 37 DEG C of water baths incubation 1 hour, and sampling is originally examined under a microscope, and with physiological saline in contrast, person is positive clustering phenomena, has infected typhoid fever.
Embodiment 7:Spa-antibody
By spa and anti-erythrocyte monoclonal/polyclonal antibody and different lymphocyte antibody combinations, can be used to detect the ratio of blood lymphocyte subsets, help to judge patient's immunologic function.
SPA (staphylococcal protein A) and erythrocyte antibody (EA), the antibody of lymphocyte subgroup to be measured is (as anti-cd2 antibody, anti-cd3 antibody, anti-cd4 antibody, anti-cd11 antibody, anti-cd15 antibody, anti-cd16 antibody, anti-cd8 antibody, anti-cd19 antibody, anti-cd5 antibody, anti-cd7 antibody, anti-cd13 antibody, anti-cd33 antibody, anti-cd10 antibody, anti-cd21 antibody, anti-cd22 antibody, anti-cd23 antibody, anti-cd32 antibody, anti-cd35 antibody, anti-40cd antibody, anti-cd45 antibody, anti-cd54 antibody, anti-cd56 antibody, anti-cd64 antibody, anti-cd133 antibody, anti-cd117 antibody, anti-cd90 antibody etc.) tripolymer.The reaction density of this tripolymer preparation process: SPA and anti-erythrocyte monoclonal antibody and lymphocyte antibody to be measured is 0.1mg/ml.SPA is 1.5ml, and anti-erythrocyte monoclonal antibody is 1ml, lymphocyte antibody 1ml to be measured.Incubation 60 minutes in 37 DEG C of water baths.Spa-antibody tripolymer concentration is 0.1mg/ml, PH7.4.4 DEG C of preservations.
When use, this tripolymer is diluted to 1: 1000 working concentration, i.e. 0.1 μ g/ml, blood sampling, adds after tripolymer in 37 DEG C of water baths incubation 45 minutes, naked eyes can be seen agglutination phenomenon, sampling is originally examined under a microscope, and observation aggregated particle is garland spline structure, three red blood cells around positive, counting positive cell number, and standard blood sample result (positive control) is relatively.
Embodiment 8:Spa-antibody
By spa and anti-erythrocyte monoclonal/polyclonal antibody and special cells antibody combination to be measured, whether can be used to detect the existence of cell to be measured in blood.
The tripolymer of the antibody of SPA (staphylococcal protein A) and erythrocyte antibody (EA), special cells to be measured.The reaction density of this tripolymer preparation process: SPA and anti-erythrocyte monoclonal antibody and special cells antibody to be measured is 0.1mg/ml.SPA is 1.5ml, and anti-erythrocyte monoclonal antibody is 1ml, special cells antibody 1ml to be measured.4 DEG C of refrigerators leave standstill 12 hours.Spa-antibody tripolymer concentration is 0.1mg/ml, PH7.4.4 DEG C of preservations.
When use, this tripolymer is diluted to 1: 1000 working concentration, i.e. 0.1 μ g/ml, blood sampling, adds after tripolymer in 37 DEG C of water baths incubation 1 hour, naked eyes can be seen agglutination phenomenon, sampling is originally examined under a microscope, and observation aggregated particle is garland spline structure, three red blood cells around positive, counting positive cell number, and standard blood sample result (positive control) is relatively.
Spa-antibody embodiment 9:
By the antibody combination of spa and anti-erythrocyte monoclonal/polyclonal antibody and tumor associated antigen to be measured, whether can be used to detect the existence of tumor associated antigen to be measured in blood.
The tripolymer of the antibody of SPA (staphylococcal protein A) and erythrocyte antibody (EA), tumor associated antigen to be measured.The reaction density of the antibody of this tripolymer preparation process: SPA and anti-erythrocyte monoclonal antibody and tumor associated antigen to be measured is 0.1mg/ml.SPA is 1.5ml, and anti-erythrocyte monoclonal antibody is 1ml, the antibody 1ml of tumor associated antigen to be measured.20 DEG C of refrigerators leave standstill 3 hours.Spa-antibody tripolymer concentration is 0.1mg/ml, PH7.4.4 DEG C of preservations.
When use, this tripolymer is diluted to 1: 1000 working concentration, i.e. 0.1 μ g/ml, blood sampling, adds after tripolymer in 37 DEG C of water baths incubation 1 hour, naked eyes can be seen agglutination phenomenon, sampling is originally examined under a microscope, and observation aggregated particle is garland spline structure, three red blood cells around positive, counting positive cell number, and standard blood sample result (positive control) is relatively.
Spa-antibody embodiment 10:
By spa and anti-erythrocyte monoclonal/polyclonal antibody and cytokine antibodies combination to be measured, whether can be used to detect the existence of the special cells factor in blood.
The tripolymer of the antibody of SPA (staphylococcal protein A) and erythrocyte antibody (EA), cell factor to be measured.The reaction density of this tripolymer preparation process: SPA and anti-erythrocyte monoclonal antibody and cytokine antibodies to be measured is 0.1mg/ml.SPA is 1.5ml, and anti-erythrocyte monoclonal antibody is 1ml, treats cytokine antibodies 1ml.25 DEG C of refrigerators leave standstill 2 hours.Spa-antibody tripolymer concentration is 0.1mg/ml, PH7.4.4 DEG C of preservations.
When use, this tripolymer is diluted to 1: 1000 working concentration, i.e. 0.1 μ g/ml, blood sampling, adds after tripolymer in 37 DEG C of water baths incubation 1 hour, naked eyes can be seen agglutination phenomenon, sampling is originally examined under a microscope, and observation aggregated particle is garland spline structure, three red blood cells around positive, counting positive cell number, and standard blood sample result (positive control) is relatively.
Claims (5)
1. the preparation method of a staphylococcal protein A-antibody tripolymer, it is characterized in that: staphylococcal protein A is diluted with aseptic phosphate buffer, A antibody and B antibody are diluted with antibody diluent, at 0 DEG C or 4～40 DEG C of temperature, incubation 20 minutes-12 hours, obtain described staphylococcal protein A-antibody tripolymer, wherein, the mass ratio of described staphylococcal protein A, A antibody and B antibody is 1.5: 1: 1; Described tripolymer is the combination of staphylococcal protein A synantibody A, antibody B, and described antibody A is anti-erythrocyte monoclonal antibody or polyclonal antibody, and described antibody B is monoclonal antibody to be measured or polyclonal antibody.
2. preparation method as claimed in claim 1, is characterized in that: described heated culture temperature is 37 DEG C.
3. preparation method as claimed in claim 1, is characterized in that: described incubative time is 30 minutes.
4. the preparation method of staphylococcal protein A-antibody tripolymer as claimed in claim 1, is characterized in that: described antibody B is arbitrary antibody in specificity lymphocyte antibody, anti-Marek's virus antibody, anti-HBS antibody, swine fever virus resistant antibody, anti-vibrio parahemolyticus antibody, anti-typhoid antibody.
5. the preparation method of staphylococcal protein A-antibody tripolymer as claimed in claim 4, it is characterized in that: described specificity lymphocyte antibody is anti-cd2 antibody, anti-cd3 antibody, anti-cd4 antibody, anti-cd11 antibody, anti-cd15 antibody, anti-cd16 antibody, anti-cd8 antibody, anti-cd19 antibody, anti-cd5 antibody, anti-cd7 antibody, anti-cd13 antibody, anti-cd33 antibody, anti-cd10 antibody, anti-cd21 antibody, anti-cd22 antibody, anti-cd23 antibody, anti-cd32 antibody, anti-cd35 antibody, anti-40cd antibody, anti-cd45 antibody, anti-cd54 antibody, anti-cd56 antibody, anti-cd64 antibody, anti-cd133 antibody, anti-cd117 antibody, the combination of one or more in anti-cd90 antibody.
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| CN102167746B (en) * | 2010-11-11 | 2013-09-18 | 姜东成 | SPG-(Streptococcal Protein G) antibody polymer and preparation method as well as application thereof |
| CN106350489A (en) * | 2016-08-31 | 2017-01-25 | 上海交通大学 | Human marrow, umbilical cord blood and peripheral blood stem cell isolation kit and isolation method thereof |
| CN109182364A (en) * | 2018-08-30 | 2019-01-11 | 武汉百意欣生物技术有限公司 | A kind of polyclonal antibody and its preparation method and application of specific recognition albumin A |
| CN112326954B (en) * | 2020-10-28 | 2022-07-08 | 厦门宝太生物科技有限公司 | Homogeneous fluoroimmunoassay reagent for rapidly and quantitatively detecting D-dimer |
| CN113252914B (en) * | 2021-06-23 | 2022-08-30 | 天津德祥生物技术股份有限公司 | Antibody diluent for Rh system parting detection card and detection card |
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| CN1140258A (en) * | 1995-07-05 | 1997-01-15 | 中国人民解放军第四军医大学 | Immune colloid gold reagent and method for testing hepatitis B virus antibody |
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