CN103361313A - Kit and use of SPA (staphylococcal protein A)-antibody trimer - Google Patents
Kit and use of SPA (staphylococcal protein A)-antibody trimer Download PDFInfo
- Publication number
- CN103361313A CN103361313A CN2013102232795A CN201310223279A CN103361313A CN 103361313 A CN103361313 A CN 103361313A CN 2013102232795 A CN2013102232795 A CN 2013102232795A CN 201310223279 A CN201310223279 A CN 201310223279A CN 103361313 A CN103361313 A CN 103361313A
- Authority
- CN
- China
- Prior art keywords
- antibody
- spa
- tripolymer
- cell
- staphylococcal protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a kit and use of SPA (staphylococcal protein A)-antibody trimer. The antibody trimer is formed by an anti erythrocyte antibody, SPA and antibodies of some anti-leukocyte antigen or other antigens. According to the antibody trimer, the self anti erythrocyte antibody is combined with erythrocyte, the other antibody is combined with corresponding leukocyte antigen (or other antigen), then leukocyte (or other cell, factor) and erythrocyte are deposited by a blood sedimentation process or methods, such as density gradient centrifugation to achieve the purpose of eliminating components of corresponding cell and the like, and the erythrocyte can be also connected with corresponding antigen by virtue of the SPA to be used as means for detecting certain antigens. The kit is convenient and simple and can be directly used for clinical separation and extraction of stem cells/progenitor cells, and the collected and extracted stem cells/progenitor cells do not have any foreign markers. The kit based on the trimer can be industrially produced to realize popularization of human hematopoietic stem cell separation and purification technology.
Description
The present invention is patent of invention that name is called " SPA-antibody tripolymer, contain this trimerical cell processing kit and its production and use " (application number 201010022753.4, applying date on 01 13rd, the 2010) patent of dividing an application.
Technical field
The present invention relates to field of biomedicine technology, particularly relate to cell processing kit of staphylococcal protein A,SPA (staphylococalprotein A is hereinafter to be referred as SPA)-antibody tripolymer and uses thereof.
Technical background
SPA can the Fc fragment in people and multiple mammalian blood serum IgG molecule be combined.In conjunction with the affinity order be pig, dog, rabbit, people, monkey, mouse, mouse and ox successively.SPA can also be combined by a small amount of IgM and IgA in serum except with IgG is combined, and the SPA bacterium is that 90%～98%, IgM is that 2%～30%, IgA is 1.5%～20% to the adsorption rate of each immunoglobulin like protein: IgG.
SPA and IgG that coprecipitated reaction occurs must have two combining sites, and they on Fc and Fab section, can be combined with SPA though lack one respectively, coprecipitated reaction do not occur, and the participation of this Fab is not the SPA-antibody response.Studies show that now the combining site of IgG and SPA is CH
2And CH
3Intersection.
It is very wide that SPA uses, and has binding ability with the Fc section of IgG but the principle of all application all be unable to do without SPA, and mainly application facet is as follows:
1.SPA the coagglutination effect of bacterium
With known standard serum, be adsorbed onto the surface of SPA bacterium, make it to become the carrier of absorption antibody, go to diagnose corresponding unknown antigen and produce macroscopic agglutinating particle with this again, the antigen of diagnosis can be graininess, also can be solubility.This kind aggegation is equivalent to reverse indirect hemagglutination, but saves the complicated formalities such as the antibody purification in the indirect hemagglutination, tanning of red blood corpuscle, and only needs SPA thalline and rough immune serum to get final product.So this method is simple, quick, specificity susceptibility is all more satisfied.
2. as the wide spectrum second antibody
Second antibody with SPA replacement IgG antibody has the following advantages: 1. the SPA preparation is easy, stable in properties, and easily purifying can both be used people and multiple mammiferous IgG, not limited by kind.2. the bonding force of SPA is strong, is equivalent to the bonding force of free antibodies and antigen, and the affinity costant of people and rabbit is 10
3The L/ mol, in conjunction with rear activity on IgG without impact.No matter 0 ℃, 37 ℃, 44 ℃ and 56 ℃ almost all can be immediately in conjunction with and need not to hatch for a long time, therefore can shorten test period; 3. SPA puts on easily
125Therefore I, fluorescein, horseradish peroxidase, ferritin etc. can combine with multiple technologies; 4. SPA numberator height homogeneous, it is not immunoglobulin (Ig), not affected by the Fc acceptor, so higher anti-IgG specificity is arranged.
3. be used for extracting IgG purification and utilize the binding characteristic of SPA and IgG to extract IgG, adopt ammonium sulfate, Sephadex post than routine, it is much easy that the methods such as DEAE post or immune gradient centrifugation are wanted, and purity is good.
4. detection and purification IgM IgM are significant in the early diagnosis of virus, for detection specificity IgM antibody, must at first remove IgG in serum.Adopting SPA bacterium absorption IgG is present quick and desirable way, utilizes this method also to open up road for the purification of IgM.
5. other side is used for the detection etc. of detection, Activation In Vitro complement function test, cell surface marker and the TSA of immunocomplex.
Stem cell (stem cell) refers to have unlimited or longer self-renewal capacity, and can differentiate the cell of at least a height differentiation daughter cell.According to cell derived stem cell is divided into Embryo stem cell and adult stem cell, forms the soma cell and comprise autologous stem cells and allosome stem cell.
Present latest developments both domestic and external impel " bone marrow stem cell " grafting vessel new life's the mechanism of action, organismic internal environment that the impact of 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.
The main mononuclearcell that gathers marrow or derived from peripheral blood is transplanted in the therapeutic process clinically, and think that wherein CD34+ or CD133+/VEGF-R2+ cell are the ancestral cells of angiogenesis, but the proliferation and 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 all are the contents of not yet fully understanding.
Surface antigen (Lineage surface antigen) is when differentiation of stem cells during for the progenitor cell that respectively is and mature cell, the specificity sign that the meeting appearance respectively is, 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 these antigens that occur on the hemocyte surface of particular type are commonly referred to as, the Lin antigen positive just can be defined as progenitor cell 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 be according to the different relevant mature cell systems of removal that require of each section office.
Stem cell can effectively be treated various cell injury 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.The technology of separating and extracting stem cells has much from marrow, Cord blood at present, as: immunomagnetic beads method and flow cytometer method etc.The existing common issue with of these methods is: first can not effectively keep all types of stem cells, and the second cost is expensive, and technical qualification are had relatively high expectations.At clinical application certain difficulty is arranged.
Summary of the invention:
The present invention relates to a kind of
Test kit of SPA-antibody tripolymer and uses thereof.
The invention provides a kind of intermediary with red corpuscle in the blood and other cells/factor combination: the spa-antibody tripolymer, can with red corpuscle and needed white corpuscle or other cells/factor combination, the specific cells in the blood/factor can be removed by methods such as erythrocyte sedimentation rate or gradient centrifugations 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 mono-clonal or polyclonal antibody; B antibody: can select different mono-clonals or polyclonal antibody with the experiment needs according to clinical, 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, and A antibody (anti-erythrocyte monoclonal antibody or polyclonal antibody) and B antibody are diluted with antibody diluent, and various weaker concns all can (recommending spa be 0.1mg/ml, antibody concentration: 0.1mg/ml dilution).
Raw material mass mixture ratio is: SPA:A is anti-: B is anti-to be 1.5:1:1; As: SPA is 1.5ml, and A is anti-to be 1ml, and B is anti-to be 1ml.(concentration is 0.1mg/ml)
Temperature of reaction 0C～40 ℃ grade all can, recommend 37 ℃ of incubations (water-bath or incubator all can).
Reaction times: 20 minutes-2 hours, recommended 37 ℃ of incubations 30 minutes.
Production concentration: 0.1mg/ml.
Storage requirement: 4 ℃ of storages.
This tripolymer can be used for separating with Cord blood from human marrow the in-vitro separation test kit etc. of karyocyte, and it is simple that this technology has method, cheap, can suitability for industrialized production, and the characteristics that are easy to promote.This technology has effectively been removed blood plasma, red corpuscle, thrombocyte, plasma proteins, ion, and monocyte, lymphocyte, the syncyte of other required removal moral maturations, and has kept all stem cells, progenitor cell, mescenchymal stem cell etc.This is the application method of a kind of marrow/umbilical cord that can be directly used in clinical separation, extract stem cell/peripheral hematopoietic stem cells in-vitro separation test kit.
2, cell processing kit: based on above-mentioned tripolymer, the bind lymphocytes parting liquid, the chromatoplate of antibodies, the method that adopts density gradient centrifugation, immunochromatographic method and co-immunoprecipitation method to combine is made cell processing kit.It is characterized in that including in this test kit:
The SPA(SP) and the tripolymer of 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 the anti-erythrocyte monoclonal antibody is 1ml, and antilymphocyte antibody is 1ml.Incubation is 30～60 minutes in 37 ℃ of water baths.Adjusting Spa-antibody tripolymer concentration is 1 μ g/ml, and volume is 100ml, PH7.4.4 ℃ of preservations.
Lymphocyte separation medium (commercially available) 50ml.
The chromatoplate of antibodies: the chromatoplate of polystyrene material, add anti-specificity lymphocyte antibody 0.1～1mg/ml behind the sterilization, be paved with the surface and get final product, 37 ℃ were reacted 4 ℃ of preservations 30～60 minutes.
Above-mentioned each reagent solution is separately deposited.
The method that the present invention adopts density gradient centrifugation, immunochromatographic method and immuno-precipitation to combine, the stem/progenitor cells of collected extraction is without any external marker.Can remove different mature lymphocyte system according to different requirements, convenient and simple, can be directly used in clinical, separate in the laboratory, extract stem/progenitor cells.
3, the range of application of Spa-antibody:
Be used for clinical diagnosis as indicator, special antigen or exotic antigen appear in certain Disease blood, the specific antibody of this antigen is connected on the Spa-antibody tripolymer, make red corpuscle as indicator, cohesion forms " red corpuscle-anti erythrocyte antibody-Spa-specific antibody-specific antigens " mixture, 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 corpuscle-anti erythrocyte antibody-Spa-specific antibody " mixture, remove after making specific antibody and erythrocyte binding by the method such as centrifugal.Can remove patient or experiment specific antibody in the blood.
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 with specific mature cell is connected on the Spa-antibody, form " red corpuscle-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 method is: at first peripheral blood, marrow or Cord blood adding are placed with in the reagent bottle of chromatoplate, again SPA-antibody tripolymer liquid is poured into wherein, leave standstill after 20 minutes and collect supernatant liquor, with careful the joining above the lymphocyte separation medium of supernatant liquor, rear centrifugal 20～30 minutes, collect cellular layer, can use with the dilution of 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 the experiment, simple to operate, expense is low, and can by removing corresponding blood ingredient, be used for studying the compositions such as different mature cell systems and other cell/cytokines to the growth of stem/progenitor cells and the impact of differentiation.
Embodiment:
Following embodiment will help further to understand the present invention, but can not limit content of the present invention.
Embodiment 1: cell processing kit
SPA(SP) and the tripolymer of erythrocyte antibody (EA), specificity lymphocyte antibody 1.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 the anti-erythrocyte monoclonal antibody is 1ml, and anti-cd3 antibody, cd14 antibody, cd19 antibody, cd56 antibody respectively are 0.25ml.Incubation is 30 minutes in 37 ℃ of water baths.Add PBS liquid, adjusting Spa-antibody tripolymer concentration is 1 μ g/ml, and volume is 100ml, PH7.4.4 ℃ of preservations.
2, human lymphocyte parting liquid (commercially available) 100ml, centrifugal density is 1.077g/ml.
3, cell chromatoplate: the chromatoplate of polystyrene material, adding concentration behind the sterilization is the anti-cd3 antibody of 0.1mg/ml, cd14 antibody, cd19 antibody, cd56 antibody, be paved with the surface and get final product, 37 ℃ of reactions were spent the night under the room temperature after 30 minutes, put in the 500ml reagent bottle.4 ℃ of preservations.
Material therefor is by filtration sterilization, and material is through 120 degrees centigrade, 30 minutes autoclave sterilizations, and reagent is degerming after filtration, after the aseptic technique preparation, leaves in the test kit respectively.
Cell processing kit application method: get peripheral blood, marrow or Cord blood 200ml, join in the reagent bottle that is placed with chromatoplate, 37 degree left standstill 30 minutes after adding 50mlSPA-antibody tripolymer mixing again, this tripolymer respectively with white corpuscle, erythrocyte binding, form cell mass, be deposited to the test tube bottom.The clone of the unconjugated maturation of part and the antibodies on the chromatoplate form immune complex.Collect supernatant liquor.Lymphocyte separation medium is added in the centrifuge tube, carefully be added to the supernatant liquor of collecting above above the lymphocyte separation medium again, centrifugal 20 minutes of 700g, utilize density gradient centrifugation to remove remaining red corpuscle, thrombocyte, blood plasma and unprecipitated white corpuscle red corpuscle polymer, collect cellular layer, clean 2-3 time with phosphoric acid buffer (PBS), then use injection physiological saline/glucose saline diluted for use.
Prepared cell can be used for clinical self marrow blood stem-cell therapy, will get involved the liver internal therapy liver cirrhosis that be expelled to patient with liver cirrhosis by blood vessel through the stem/progenitor cells dilution that said process extracts.Self marrow blood stem cell after extracting or cord blood stem cell can be expelled to around the lower limb vascular of patient with diabetic feet and the far-end that is expelled to narrow or occluded artery, the treatment diabetic foot.Also can with the disconnected injury of spinal cord of extracting self marrow blood stem cell behind the purifying or cord blood stem cell and being expelled to patients of acute spinal cord injury, treat acute spinal cord transection.Also can be used for other cell injury disease.
Embodiment 2: cell processing kit
SPA(SP) and the tripolymer of erythrocyte antibody (EA), specificity lymphocyte antibody 1.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 the anti-erythrocyte monoclonal antibody is 1ml, and anti-cd3 antibody, cd14 antibody, cd19 antibody, cd56 antibody are 0.25ml.Incubation is 30 minutes in 37 ℃ of water baths.Add PBS liquid, making Spa-antibody tripolymer concentration is 1 μ g/ml, and volume is 100ml, PH7.4.4 ℃ of preservations.
2, the aqueous solution that forms of the Sodium Diatrizoate of lymphocyte separation medium 100ml:0.3% and 8% ficoll 400#, density is 1.077g/ml.
3, antibody conjugates: will resist the nano particle of cd3 antibody, cd14 antibody, cd19 antibody, cd56 antibody and polystyrene material to mix.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 ℃ of water baths.4 ℃ of preservations.
Material therefor is by filtration sterilization, and material is through 120 degrees centigrade, 30 minutes autoclave sterilizations, and reagent is degerming after filtration, after the aseptic technique preparation, leaves in the test kit respectively.
Cell processing kit application method: get marrow or Cord blood 200ml adding and contain in the reagent bottle of antibody conjugates, adding behind the 50mlSPA-antibody tripolymer mixing 37 ℃ left standstill 30 minutes again, this tripolymer respectively with white corpuscle, erythrocyte binding, form cell mass, be deposited to the test tube bottom.Antibodies on the unconjugated eukocyte of part and the antibody conjugates forms small-particle and suspends.Collect supernatant liquor.Lymphocyte separation medium is added in the centrifuge tube, carefully be added to the cell suspending liquid of collecting above above the lymphocyte separation medium again, 2000 rev/mins centrifugal 20 minutes, utilize density gradient centrifugation to remove remaining red corpuscle, thrombocyte, blood plasma and antibody conjugates, collect cellular layer, clean 2-3 time with phosphoric acid buffer (PBS), then with for subsequent use after injection physiological saline/glucose saline dilution.
Use the cell of this test kit preparation to can be used for clinical self marrow blood stem-cell therapy, will be expelled to through the stem/progenitor cells dilution that said process extracts the liver internal therapy liver cirrhosis of patient with liver cirrhosis by the blood vessel intervention.Self marrow blood stem cell after extracting or cord blood stem cell can be expelled to around the lower limb vascular of patient with diabetic feet and the far-end that is expelled to narrow or occluded artery, the treatment diabetic foot.Also can with the disconnected injury of spinal cord of extracting self marrow blood stem cell behind the purifying or cord blood stem cell and being expelled to patients of acute spinal cord injury, treat acute spinal cord transection.Also can be used for other cell injury disease.
Embodiment 3:Spa-antibody
Chicken Marek's disease (MD) is a kind of height lymphoproliferative diseases contagious that is caused by Mareks disease virus.Its principal character is: lymphocytic infiltration occurs and forms swollen sick in peripheral nerve, various internal organs, iris and skin etc.Marek mainly betides chicken, and the report of birds such as betiding turkey, pheasant, duck, quail, swan is also arranged.This disease transmission is strong, and velocity of propagation is fast, and latent period is long.The acute onset chicken is eliminated and mortality ratio reaches 10%～80%.Up to now, in the diagnosis of Marek, except AGPT, still there is not a kind of practical technique that can be convenient to use in basic unit.
The SPA(SP) and the tripolymer of 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 the anti-erythrocyte monoclonal antibody is 1ml, anti-Marek's virus antibody 1ml.Incubation is 60 minutes in 37 ℃ of water baths.Spa-antibody tripolymer concentration is 0.1mg/ml, PH7.4.4 ℃ of preservations.
During use, this tripolymer is diluted to the 1:1000 working concentration, i.e. 0.1 μ g/ml, blood sampling added behind the tripolymer in 37 ℃ of water baths incubation 1 hour, and sampling is originally examined under a microscope, with physiological saline in contrast, the person is positive the clustering phenomena, has namely infected Mareks disease virus.
Embodiment 4:Spa-antibody
Swine fever (SwnieFever, SF) is a kind of height contagious disease of pig.Being a kind of acute, subacute, chronic and typical, the atypical lysis of the pig that caused by Pestivirus suis (ClassiealWSnieFeverviurs, CSFV), is one of Infectious Diseases of harm pig industry.
The SPA(SP) and the tripolymer of 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 the anti-erythrocyte monoclonal antibody is 1ml, swine fever virus resistant antibody 1ml.Incubation is 60 minutes in 40 ℃ of water baths.Spa-antibody tripolymer concentration is 0.1mg/ml, PH7.4.4 ℃ of preservations.
During use, this tripolymer is diluted to 0.1 μ g/ml, and blood sampling added behind the tripolymer in 37 ℃ of water baths incubation 1 hour, and sampling is originally examined under a microscope, and with physiological saline in contrast, the person is positive the clustering phenomena, and namely this blood sample has infected Pestivirus suis.
Embodiment 5:Spa-antibody
Vibrio parahemolyticus (Vibrio parahaemolyticus, VP) is a kind of halophilism pathogenic bacteria widely distributed in ocean and the salt lake.Extensively be distributed in inshore seawater, marine bottom sediment, fish, the shellfish, be adapted to grow in the environment of high density (6%) salt, in the substratum that does not contain salt, can not grow.This bacterium is a kind of common pathogen enterobacteria that is second to vibrio cholerae, its pollution meeting brings heavy losses to mariculture industry, also can cause intestinal tract disease and the food poisonings such as human diarrhoea, occur diarrhoea be water sample or blood sample just, the symptoms such as abdominal cramps, nauseating, 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 and the number of Vibrio parahemolyticus property food poisoning are in first always.
The SPA(SP) and the tripolymer of 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 the anti-erythrocyte monoclonal antibody is 1ml, anti-Vibrio parahemolyticus polyclonal antibody 1ml.Incubation is 6 hours in 12 ℃ of water baths.Spa-antibody tripolymer concentration is 0.1mg/ml, PH7.4.4 ℃ of preservations.
During use, this tripolymer dilutes 0.1 μ g/ml, gets blood sample to be measured, added behind the tripolymer in 37 ℃ of water baths incubation 1 hour, sampling is originally examined under a microscope, with physiological saline in contrast, the person is positive the clustering phenomena, and namely this blood sample has infected Vibrio parahemolyticus.
Embodiment 6:Spa-antibody
Typhoid fever is the acute infectious disease through transmission that is caused by Corynebacterium diphtheriae.Clinical symptoms is long-range heating, whole body toxicity symptom, relative infrequent pulse, hepatosplenomegaly, roserash and oligoleukocythemia etc.Major complications is enterorrhagia, intestinal perforation.
The SPA(SP) and the tripolymer of 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 the anti-erythrocyte monoclonal antibody is 1ml, anti-typhoid antibody 1ml.Incubation is 90 minutes in 30 ℃ of water baths.Spa-antibody tripolymer concentration is 0.1mg/ml, PH7.4.4 ℃ of preservations.
During use, this tripolymer is diluted to 0.1 μ g/ml, and blood sampling added behind the tripolymer in 37 ℃ of water baths incubation 1 hour, and sampling is originally examined under a microscope, and with physiological saline in contrast, the person is positive the clustering phenomena, has namely infected typhoid fever.
Embodiment 7:Spa-antibody
With spa and anti-erythrocyte mono-clonal/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.
The SPA(SP) and erythrocyte antibody (EA), the antibody of lymphocyte subgroup to be measured is (such 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 the anti-erythrocyte monoclonal antibody is 1ml, lymphocyte antibody 1ml to be measured.Incubation is 60 minutes in 37 ℃ of water baths.Spa-antibody tripolymer concentration is 0.1mg/ml, PH7.4.4 ℃ of preservations.
During use, this tripolymer is diluted to the 1:1000 working concentration, i.e. 0.1 μ g/ml, blood sampling added behind the tripolymer in 37 ℃ of water baths incubation 45 minutes, naked eyes can be seen agglutination phenomenon, sampling is originally examined under a microscope, and the observation aggregated particles is the garland spline structure, and it is positive that three red corpuscle center on, the counting positive cell number, and standard blood sample result (positive control) is relatively.
Embodiment 8:Spa-antibody
With spa and anti-erythrocyte mono-clonal/polyclonal antibody and special cells antibodies to be measured, whether can be used to detect the existence of cell to be measured in the blood.
The SPA(SP) and the tripolymer of the antibody of 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 the anti-erythrocyte monoclonal antibody is 1ml, special cells antibody 1ml to be measured.4 ℃ of refrigerators left standstill 12 hours.Spa-antibody tripolymer concentration is 0.1mg/ml, PH7.4.4 ℃ of preservations.
During use, this tripolymer is diluted to the 1:1000 working concentration, i.e. 0.1 μ g/ml, blood sampling added behind the tripolymer in 37 ℃ of water baths incubation 1 hour, naked eyes can be seen agglutination phenomenon, sampling is originally examined under a microscope, and the observation aggregated particles is the garland spline structure, and it is positive that three red corpuscle center on, the counting positive cell number, and standard blood sample result (positive control) is relatively.
Spa-antibody embodiment 9:
With the antibodies of spa and anti-erythrocyte mono-clonal/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 the blood.
The SPA(SP) and the tripolymer of the antibody of 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 the anti-erythrocyte monoclonal antibody is 1ml, the antibody 1ml of tumor associated antigen to be measured.20 ℃ of refrigerators left standstill 3 hours.Spa-antibody tripolymer concentration is 0.1mg/ml, PH7.4.4 ℃ of preservations.
During use, this tripolymer is diluted to the 1:1000 working concentration, i.e. 0.1 μ g/ml, blood sampling added behind the tripolymer in 37 ℃ of water baths incubation 1 hour, naked eyes can be seen agglutination phenomenon, sampling is originally examined under a microscope, and the observation aggregated particles is the garland spline structure, and it is positive that three red corpuscle center on, the counting positive cell number, and standard blood sample result (positive control) is relatively.
Spa-antibody embodiment 10:
With spa and anti-erythrocyte mono-clonal/polyclonal antibody and cytokine antibodies combination to be measured, whether can be used to detect the existence of the special cells factor in the blood.
The SPA(SP) and the tripolymer of the antibody of erythrocyte antibody (EA), cytokine 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 the anti-erythrocyte monoclonal antibody is 1ml, treats cytokine antibodies 1ml.25 ℃ of refrigerators left standstill 2 hours.Spa-antibody tripolymer concentration is 0.1mg/ml, PH7.4.4 ℃ of preservations.
During use, this tripolymer is diluted to the 1:1000 working concentration, i.e. 0.1 μ g/ml, blood sampling added behind the tripolymer in 37 ℃ of water baths incubation 1 hour, naked eyes can be seen agglutination phenomenon, sampling is originally examined under a microscope, and the observation aggregated particles is the garland spline structure, and it is positive that three red corpuscle center on, the counting positive cell number, and standard blood sample result (positive control) is relatively.
Claims (6)
1. cell reagent box that contains staphylococcal protein A,SPA-antibody tripolymer, it is characterized in that: this test kit comprises that the surface is paved with the chromatoplate of B antibody, staphylococcal protein A,SPA-antibody tripolymer solution and lymphocyte separation medium; Wherein, described staphylococcal protein A,SPA-antibody tripolymer is the combination of staphylococcal protein A,SPA 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; The aqueous solution that described lymphocyte separation medium is comprised of the ficoll 400# of 0.3%～1.5% Sodium Diatrizoate and 8%, its density is 1.077g/ml, and pH value is 7～7.8, and described tripolymer strength of solution is 0.1～1mg/ml.
2. the cell reagent box that contains staphylococcal protein A,SPA-antibody tripolymer as claimed in claim 1, it 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, the anti-typhoid antibody.
3. the cell reagent box that contains staphylococcal protein A,SPA-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 the anti-cd90 antibody.
4. the purposes of the cell reagent box shown in claim 1 is characterized in that: be used for marrow blood, Cord blood, the stem cell of peripheral blood or extraction and the purifying of progenitor cell.
5. the purposes of the cell reagent box shown in claim 4, it is characterized in that: when being used for the extraction of the stem cell of marrow blood, Cord blood, peripheral blood or progenitor cell and purifying, realize by following steps: at first peripheral blood, marrow or Cord blood adding are placed with in the reagent bottle of chromatoplate, again staphylococcal protein A,SPA-antibody tripolymer liquid is poured into wherein, leave standstill after 20 minutes and collect supernatant liquor, with careful the joining above the lymphocyte separation medium of supernatant liquor, rear centrifugal 20～30 minutes, collect cellular layer, can use with the dilution of glucose for injection salt solution.
6. purposes as claimed in claim 1, it is characterized in that: described staphylococcal protein A,SPA-antibody tripolymer is obtained by following preparation method: staphylococcal protein A,SPA is diluted with aseptic phosphoric acid buffer, A antibody and B antibody are diluted with antibody diluent, under 0 ℃ or 4～40 ℃ of temperature, incubation 20 minutes-12 hours, obtain described staphylococcal protein A,SPA-antibody tripolymer, wherein, the mass ratio of described staphylococcal protein A,SPA, A antibody and B antibody is 1.5:1:1; Described tripolymer is the combination of staphylococcal protein A,SPA 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013102232795A CN103361313A (en) | 2010-01-13 | 2010-01-13 | Kit and use of SPA (staphylococcal protein A)-antibody trimer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013102232795A CN103361313A (en) | 2010-01-13 | 2010-01-13 | Kit and use of SPA (staphylococcal protein A)-antibody trimer |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010022753.4A Division CN101799473B (en) | 2010-01-13 | 2010-01-13 | SPA-antibody tripolymer, cell treating kit containing tripolymer, preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103361313A true CN103361313A (en) | 2013-10-23 |
Family
ID=49363575
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013102232795A Pending CN103361313A (en) | 2010-01-13 | 2010-01-13 | Kit and use of SPA (staphylococcal protein A)-antibody trimer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103361313A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104237506A (en) * | 2014-10-16 | 2014-12-24 | 青岛中仁生物科技有限公司 | Method for detecting antigen or antibody |
| CN111308066A (en) * | 2020-03-10 | 2020-06-19 | 北京国科融智生物技术有限公司 | Novel method for enriching and detecting biomedical and environmental samples by magnetic particles |
| CN113252914A (en) * | 2021-06-23 | 2021-08-13 | 天津德祥生物技术有限公司 | Antibody diluent for Rh system parting detection card and detection card |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008039141A1 (en) * | 2006-09-29 | 2008-04-03 | Ge Healthcare Bio-Sciences Ab | Chromatography ligand comprising domain c from staphyloccocus aureus protein a for antibody isolation |
| CN101221181A (en) * | 2007-12-06 | 2008-07-16 | 甘肃省医学科学研究院 | Indirect method for detecting lymphocyte subgroup with mono-clone antibody SPA hematid rosette method |
-
2010
- 2010-01-13 CN CN2013102232795A patent/CN103361313A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008039141A1 (en) * | 2006-09-29 | 2008-04-03 | Ge Healthcare Bio-Sciences Ab | Chromatography ligand comprising domain c from staphyloccocus aureus protein a for antibody isolation |
| CN101221181A (en) * | 2007-12-06 | 2008-07-16 | 甘肃省医学科学研究院 | Indirect method for detecting lymphocyte subgroup with mono-clone antibody SPA hematid rosette method |
Non-Patent Citations (3)
| Title |
|---|
| 全国卫生专业技术资格考试专家委员会: "《卫生专业技术资格考试指导-病理学和病理学技术（上）》", 29 February 2004, 山东大学出版社 * |
| 张明富: "禽传染性支气管炎病毒S1基因与猪IgGFc基因的克隆及在Hela细胞中的融合表达", 《中国优秀硕士学位论文全文数据库 2007年 农业科技辑》 * |
| 陶义训: "《免疫学和免疫学检测（第二版）》", 31 October 1997, 人民卫生出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104237506A (en) * | 2014-10-16 | 2014-12-24 | 青岛中仁生物科技有限公司 | Method for detecting antigen or antibody |
| CN111308066A (en) * | 2020-03-10 | 2020-06-19 | 北京国科融智生物技术有限公司 | Novel method for enriching and detecting biomedical and environmental samples by magnetic particles |
| CN113252914A (en) * | 2021-06-23 | 2021-08-13 | 天津德祥生物技术有限公司 | Antibody diluent for Rh system parting detection card and detection card |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101799473B (en) | SPA-antibody tripolymer, cell treating kit containing tripolymer, preparation method and application thereof | |
| Garratty | Blood groups and disease: a historical perspective | |
| Van Setten et al. | Effects of TNFα on verocytotoxin cytotoxicity in purified human glomerular microvascular endothelial cells | |
| Bücheler et al. | Alloantibodies against A and B blood types in cats | |
| Israels et al. | Platelet Fc receptor as a mechanism for Ag-Ab complex-induced platelet injury | |
| Harris et al. | Transfusion studies in rheumatoid arthritis | |
| JPH06500780A (en) | homozygous immunoglobulin | |
| JPH03501560A (en) | Monoclonal antibody specific to the attachment functional region of cell surface proteins of phagocytes | |
| CN101144070A (en) | Marrow umbilical cord blood stem cell in vitro separating kit and application method thereof | |
| Dwyer | Identifying and Enumerating Human T and B Lymphocytes1 | |
| JPH07507387A (en) | Broadly reactive opsonic antibodies that react with common staphylococcal antigens | |
| Adelson et al. | Studies on platelets: VI. Demonstration and characterization of a heterologous (Forssman) platelet agglutinin | |
| CN103361313A (en) | Kit and use of SPA (staphylococcal protein A)-antibody trimer | |
| Chou et al. | CELLS AS ANTIGEN CARRIERS AND AS IMMUNOGLOBULIN PRODUCERS: Synthesis of Antibody and Allogeneic Immunoglobulin after Transfer of Antigen-Treated Cells to Newborn Rabbits | |
| CN103604927A (en) | SPA-antibody trimer for eliminating specific antibodies in blood | |
| CN103616516A (en) | Applications of SPA-antibody trimer as indicator in detection of blood antigens | |
| Kaufman et al. | Ataxia telangiectasia: An autoimmune disease associated with a cytotoxic antibody to brain and thymus | |
| Witebsky et al. | A simple method for the concentration of Rh agglutinins | |
| CN102174470A (en) | Cell processing kit | |
| Amemiya et al. | The nature of antiplatelet activity in antilymphoblast ALG: with special reference to crossreacting antibody, immunochemical characterization, and Coombs' positive thrombocytopenia in ALG-treated renal recipients | |
| Hook et al. | Appearance of natural antibodies in young rabbits | |
| Soltys et al. | Altered lymphocyte reactivity to E. coli in chronic pyelonephritis | |
| Svec et al. | A variant LE cell factor reactive only with “altered” nuclear material | |
| Baumgarten et al. | Endemic Autoimmunity: Cold Auto‐agglutinins in Melanesia | |
| Williams et al. | Lymphocytes binding C-reactive protein and streptococcal membranes in acute rheumatic fever |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20131023 |