CN104293836A - Making method of immune complex adsorption cells - Google Patents
Making method of immune complex adsorption cells Download PDFInfo
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Abstract
The invention discloses a making method of immune complex adsorption cells. The method comprises the steps of vector construction, virus packaging production, immortalized B lymphocyte production and gene transfection. A set of immortalized cells respectively carrying specific genes is made, has immortalization (infinite proliferation) ability, can express a certain immune complex receptor on the surfaces on the cells, and an immune complex analysis group is established through amplifying cultured cells, and allows all immune complexes in blood or body tissues to be identified, classified and quantified. The method has the characteristics of low cost, infinite proliferation, fast detection speed, simple operation and the like. The aim of the invention is to obtain the immortalized B lymphocytes, the B lymphocytes can stably express an immune complex receptor gene, the receptor can exist on cell membranes, and can be combined with one corresponding immune complex not limited to immortalized B cells, outside the cells,.
Description
Technical field
The present invention relates to a kind of method that cell makes, specifically a kind of making method of immunocomplex adherent cell.
Background technology
Immunocomplex: antibody is combined obtained a kind of mixture and is called immunocomplex with antigen engulfs bacterium by various immunocyte, virus, combines and formed, so also known as antigen after sensitizing substance is common dead--antibody complex.It under normal circumstances, small soluble molecules immunocomplex is discharged by glomerular filtration, and the insoluble immunocomplex of macromole is eliminated by macrophage phagocytic, and this is a part for body defenses mechanism.But in some cases, the immunocomplex that antigen and antibody are formed in vivo, is deposited on vessel wall basis pontis, thus activating complement, the complement be activated, plays the effects such as dissolution of bacteria, virus, tumour cell.
Immortalized cells: a kind of cell biological technics.Refer to zooblast dedifferentiation failure, be subject to virus infection, the transfection of external source malicious code, after radiation or pharmacy effect, frequency dividing cell restriction scheme and DNA inspections-self-destruction mechanism malfunctioning, cause the process with the ability that divide without limit grows.Cellular immortalization be canceration must through approach.
Antigen and corresponding antibodies combine the material formed and are called immunocomplex.Because immunocomplex can detect in blood circulation, therefore be also referred to as circulating immune complex.It and complement, other immunologic active materials combine, and are deposited on vessel wall, can cause tissue injury and vasculitis, cause a series of disease, as lupus erythematosus.Immunocomplex has two kinds of modes in vivo, and one is the circulating immune complex (CIC) be present in blood, and one is immunocomplex fixing in tissue.The detection technique of immunocomplex can be divided into antigen specific method and non-antigen specific method.In most of the cases, the antigenic property in immunocomplex is not clear or very complicated, so antigen specific method being of little use.
CIC is divided into two large classes by the known or unknown of Ag-Ab according to forming immunocomplex, the former is specific immune complex (as the HBsAg-Anti-HBs antibody of hepatitis B, the immunocomplex that thyroprotein antigen-TGA is formed); The latter is non-specific immunity mixture (as glomerulonephritis, systemic lupus erythematous etc.).
The immunocomplex that antigen and antibody are formed in vivo, is deposited on vessel wall basis pontis, thus activating complement, the complement be activated, plays the effects such as dissolution of bacteria, virus, tumour cell.
The disease of immunocomplex has:
1) serum sickness: be by size immune complex deposit at capillary wall, complement, phagocytic cell participate in caused by reaction.
2) immune complex type glomerulitis: be by suis soluble antigen and antibodies, be deposited on glomerular basement membrane, activating complement, attracts neutrophil leucocyte, discharges caused by various enzyme damage renal glomerulus.
3) rheumatoid arthritis: be combined by Rheumatoid factors, polyclonal the immunocomplex formed with immunoglobulin IgG, be deposited on the places such as joint periosteum, subcutis and cause rheumatoid arthritis etc.
4) systemic lupus erythematous (SLE): the one being autoimmune disease, its pathomechanism is also not clear and definite, but it is normal containing multiple autoantibody and a large amount of pathogenic circulating immune complex (circ μ Lating immune complexes in patients serum, CIC), the appearance meeting activating complement of CIC, and be deposited in capillary wall, glomerular basement membrane and other extravascular tissues, cause inflammatory reaction and immunopathogenesis damage.
The detection technique of immunocomplex can be divided into antigen specific method and non-antigen specific method.In most of the cases, the antigenic property in immunocomplex is not clear or very complicated, so antigen specific method being of little use.According to the physics of immunocomplex, immunology and biological characteristics, design a lot of method detecting CIC.
(1) physical measurement
1. polyethylene glycol method
Polyoxyethylene glycol (PEG) be ethylene glycol be polymerized without electric charge shape polysaccharide molecule, the change of molecular weight scope is comparatively large, and conventional molecular weight is 6000.Can optionally be precipitated by macromole immunocomplex with the PEG of 3% ~ 4% concentration, its mechanism of action is still very unclear.PEG solution is mixed with serum to be checked, centrifugal after putting 4 DEG C of refrigerator overnight, throw out PEG solution is fully washed, is again dissolved in the NaOH of 0.01mol/L, under wavelength 280nm, measure the absorbancy of solution; Also scattered light urbidmetry can be utilized directly to measure the immunocomplex of PEG precipitation; Using the thermopolymerization IgG of different concns as the content calculating CIC with reference to standard.
Polyethylene glycol method is simple, can promote in clinical position.But this method is subject to the interference of multiple high molecular weight protein and temperature, specificity is slightly poor.PEG method is also specially adapted to precipitation and obtains CIC, then carries out Libration analysis antigen wherein and antibody.
2. under cryoglobulin is determined at some pathologic condition, the immunocomplex in serum has the characteristic of reversibility cryoprecipitate, places and within 1 ~ 3 day, spontaneously can precipitate (see the 26 chapter) in 4 DEG C of refrigerators; This kind of situation is more common in cryoglobulinemia, involved antigen comprises self IgG, IgM, Nucleotide, tumor associated antigen, renal tubular epithelial, thyroglobulin, Rollet's stroma etc., also has exogenous antigen as hepatitis B virus, Epstein-Barr virus, cytomegalovirus, bovine albumin and horse albumen etc.
(2) complement relevant assay
After IgG and IgM antibody-like is combined with antigen, the complement combining site in heavy chain CH2 district exposes, can fixation of C 1q the serial reaction of activating complement, and this is the basis utilizing complement relevant technologies to detect immunocomplex.
Serum to be checked is heated 56 DEG C of 30min by 1.C1q binding tests in advance, and the C1q be combined with CIC with destruction with deactivation complement wherein, vacates complement combining site.CIC and C1q detects in conjunction with available multiple method, and conventional has following 3 kinds.
(1) liquid phase method: first by the serum specimen mixing effect of isotope-labeled C1q and deactivation, the CIC combining C1q precipitates by the PEG adding 0.5% (final concentration) again, is calculated the content of CIC by the radioactivity detected in throw out.
(2) solid phase method: first C1q is adsorbed in surface of solid phase carriers, adds serum to be checked and CIC and C1q is combined, then add isolabeling or the anti-human igg of enzyme labelling or SPA, finally detect its radioactivity or enzymic activity.
(3) C1q departs from test: first mixed with the serum specimen of deactivation by isotope-labeled C1q, then adds the sheep red blood cell (SRBC) of antibody sensitized, centrifugal after incubation, detects the radioactivity on red corpuscle.The amount of erythrocytic radioactivity and immunocomplex is negative correlation.
2. complement tests the similar complement fixation test (CFT) of this ratio juris.By a certain amount of complement (mostly being mixing guinea pig serum) and the serum mixing incubation to be checked of deactivation, after reaction, add agarose gel.As occurred, haemolysis represents in serum do not have CIC; Haemolysis does not illustrate in sample has CIC to exist.Serum specimen is done different dilution, and compare with known thermopolymerization IgG, the content of CIC can be calculated.The sensitivity of present method is higher, and is easy to carry out in common laboratory, and weak point is that specificity is poor.
3. conglutinin binding tests conglutinin (conglutinin) is a kind of normal protein in bovine serum, can with C3d specific binding; The CIC be combined with complement in body is with C3d, and therefore conglutinin can be combined with CIC.With a certain amount of conglutinin bag by plastics tubing, in pipe, add the serum specimen of dilution, after incubation, add the anti-human IgG antibodies of isotopic labeling or enzyme labelling again, finally detect radioactivity or the enzymic activity of each pipe, calculate the content of CIC.
Conglutinin stable in properties, easily preservation, convenient sources, low price, detection method is also uncomplicated, is convenient to promote.The deficiency of this law is the CIC that can only detect conjugated complement, no matter but which kind of activated pathway all equally detect, and can be used as CIC be separated.
(3) antiglobulin assay method
The autoantibody that Rheumatoid factors, polyclonal (RF) is anti-igg, has stronger avidity with sex change IgG, thermopolymerization IgG and IC.Mono-clonal RF (mRF) can extract from the serum of property cryoglobulinemia pending, and polyclone RF (pRF) can extract from the serum of rheumatoid arthritis.Higher than the susceptibility of pRF with mRF.
MRF is adsorbed on solid phase carrier by 1.mRF solid phase inhibition test, adds serum specimen subsequently, then adds isotope-labeled solubility thermopolymerization IgG.If containing IC in sample, solid phase mRF is combined with IC, and the combination of thermopolymerization IgG and mRF makes suppressed, so the content of radioactivity in solid phase and CIC is negative correlation.
Also first can react with serum specimen with isotope-labeled mRF, then add the sepharose 4B of thermopolymerization IgG attachment, incubation also measures sedimentary intensity of radioactivity after centrifuge washing, the content of measured value and CIC is negative correlation.The susceptibility of this method is higher than front method.
PRF mixes with serum specimen by 2.pRF latex agglutination inhibiting test, then adds the latex suspension of IgG sensitization.If there is CIC to exist in sample, then pRF first combines with it, and agglutination reaction is negative.
3. anti-antibody method anti-antibody can be present in other Healthy Human Serum extremely individual, is the IgM antibody-like of a kind of anti-igg F (ab ') 2, and can react with the IgG of conjugated antigen, but not react with free IgG or thermopolymerization IgG, thus specificity is higher.First anti-antibody is mixed with serum to be checked, then add the people O type Rh+ red corpuscle of IgG sensitization; As there being CIC to exist in sample, anti-antibody is neutralized, and sensitized erythrocyte does not occur aggegation.
Various antiglobulin test is the highest with mRF method susceptibility above, but the more difficult searching of mRF.These class methods are subject to the interference of endogenous RF, preferably checks in advance and after removing the endogenous RF in sample again row test.If there is polymerization Ig in chance sample, also easily there is false negative in RF method; Use anti-antibody method instead and can avoid this phenomenon, but the source of anti-antibody is difficult.
(4) cell technology measures
Some cell surface has Fc acceptor and (or) complement receptor, can composition specific binding corresponding to immunocomplex, thus can be used to detect immunocomplex.
1.Raji test cell line
Raji cell is a kind of B cell strain be separated from Burkin Lymphoma, and there is a large amount of C1q, C3b and C3d acceptor on surface, but without surface immumoglobulin; Therefore Raji cell can be combined with the immunocomplex with complement.In plastics tubing, first add a certain amount of Raji in place, then add serum to be checked, fully centrifuge washing after effect; Finally add fluorescein-labeled anti-human igg, after washing, cell surface manifests fluorescence for the test positive; But fluorescent method can only do qualitative detection.Or add isotope-labeled anti-human igg, detect the radioactivity of sedimentation cell after centrifuge washing; With thermopolymerization IgG standard for referencial use, the typical curve of CIC content and radioactivity can be drawn out, thus try to achieve the content of CIC in sample to be measured.
Raji cell method susceptibility is high, high specificity, method simply, by DNA and endotoxicly to affect; But Raji cell surface also has Fc acceptor, the free IgG therefore in tested serum, by Fc section and Raji Cell binding, causes false positive.Have in sample to be checked during antilymphocyte antibody and also can cause false positive.Moreover the cultivation maintaining Raji cell is more difficult, and the change of culture condition can change number and the affinity of Raji cell surface receptor, affects detection sensitivity.
2. HRBC method human erythrocyte surface has C3b receptor, can combine with the CIC with complement component.By serum to be checked and 4% people O type red cell suspension balanced mix, centrifuge washing after 37 DEG C of incubations; Add isotope-labeled anti-human IgG antibodies, then after inculation washing, measure erythrocytic radioactivity; CIC content in sample is gone out with thermopolymerization IgG criterion calculation for referencial use.But this method can only detect the CIC of conjugated complement.
Other cell methods such as platelet aggregation test, rosettes form inhibition test, ADCC inhibition test, macrophage phagocytic inhibition test and neutrophil leucocyte migration inhibition test etc., also all can be used to detect CIC, the susceptibility of method is also all very high, but the influence factor of these methods is many, repeatable poor, so it is not multiplex to work in reality.
(5) composition detection of immunocomplex
In immunocomplex, the character of antigen and antibody and the immunopathogenesis mechanism of all kinds of detections to clinical diagnosis and treatment disease and further investigation disease have certain values.Such as, but because involved antigen type is a lot, pathogenic micro-organism, Self substances, all kinds of isoantigens etc., detection method can respectively see the detection technique of various antigen.Antibody in immunocomplex relates generally to IgG and subclass, IgM and IgA; Method is separated by immunocomplex in serum, then by the classification of the methods analyst antibody such as double-antibody sandwich ELISA.
The desirable detection method of circulating immune complex should possess following characteristics: 1. susceptibility is high, 2. high specificity, favorable repeatability, 4. easy and simple to handle, 5. widely applicable.Though the method for current detection immunocomplex develops into tens kinds, also do not have a kind of method to possess above-mentioned all features, comprehensively by contrast, the Measures compare such as C1q binding tests, conglutinin binding tests, Raji test cell line and RF inhibition test are quite a lot of.If method is proper, reagent is qualified, sample is fresh, operation is careful, it is careful to analyze, CIC measures and just has larger reference value.
Two, the detection of FIC is organized
Determine that the direct evidence of immune complex disease is not detect CIC, but find fixing IC deposition at diseased region.In some autoimmune diseases and immune complex disease, such as systemic lupus erythematous, part glomerulonephritis, rheumatoid arthritis, polyarteritis nodosa and pemphigus vulgaris etc., tissue deposition immunocomplex detect to the diagnosis of disease and pathogenetic research all more meaningful than detecting of CIC.
Detect tissue precipitation immunocomplex and commonly use immunohistochemistry technology, first tissue sample is taked to cook frozen section from suitable pathogenic site, dye with the anti-human igg of fluorescent marker or anti-human C3, under fluorescent microscope, see corresponding site display fluorescence is positive reaction; Also available enzyme mark anti-human igg or anti-human C3 and sample are cut into slices and are reacted, then develop the color with the substrate solution of enzyme, namely can be observed corresponding site be colored with common biomicroscope.
Three, immunocomplex detect meaning and application
Judge that immunocomplex has three as pathogenetic evidence: 1. pathology local has IC to deposit; 2. CIC level significantly raises; 3. the antigenic property in clear and definite IC.Article 3 evidence is difficult to find sometimes, but at least will possess first two, and the mensuration of independent CIC cannot be taken as proof.Also there is a small amount of CIC (about 10 ~ 20 μ g/mL) in human body, the boundary of its physiology and pathology is not easily distinguishable under state of health.In addition, the method that CIC detects is too many, and its principle is different, is determined as the positive by a kind of method, and it may be negative that another kind of method detects; But detect together with Immunohistochemical Method, its meaning is just much bigger.
The diseases such as current clear and definite systemic lupus erythematous, rheumatoid arthritis, part glomerulonephritis and vasculitis are immune complex disease, CIC detects and is still a kind of auxiliary diagnostic index to these diseases, to judging that disease activity and result for the treatment of also have the certain significance.When finding the situations such as purpura, arthrodynia, proteinuria, vasculitis and serositis, the possibility of immune complex disease can be considered, carry out the detection of CIC and tissue deposition IC.In addition, when suffering from malignant tumour, CIC recall rate also increases, but does not occur the allergic injury symptoms of type III, and the IC being referred to as clinical concealment is sick, but this state is normal relevant with prognosis to the state of an illness of tumour.
There is various deficiency in existing multiple technologies, cannot be separated various different immunocomplex fast and effectively, existing cell adsorption isolation technique mainly uses the existing cell carrying multiple or single complement or immunoglobulin receptor, these extended functionalitys are limited, can in conjunction with limited types.
Summary of the invention
The object of the present invention is to provide a kind of making method of immunocomplex adherent cell, the present invention uses B cell strain constructing technology, construct the B cell of immortalization, plasmid is used to proceed to different complement and Ig receptor gene more respectively, carry the B cell of not isoacceptor, can be used for respectively screening various immunocomplex; And reach the object of separation, screening, quantitative different immunocomplex.
Immunocomplex is that existing various autoimmune disease is (as systemic lupus erythematosus, rheumatoid arthritis, polyarteritis nodosa etc.) pathogenic crucial thing, detecting to it key analyzed is sharp separation from blood, distinguish dissimilar and quantitative analysis is carried out to individual dissimilar immunocomplex, all there is this defect in existing basis, cannot effectively overcome the above problems, the present invention utilizes the immortalised B-cell carrying different immunocomplex acceptor gene as adherent cell, use the immunocomplex that cell adsorption is different respectively, and carry out flow cytometer showed, thus more than quick solution 3 problems, simultaneously, along with the research to individual immunocomplex acceptor, different acceptor genes can be inserted in immortalization bone-marrow-derived lymphocyte gene in the past, produce the immortalization bone-marrow-derived lymphocyte adsorbing different immunity receptor, there is very strong function amplification ability.
For achieving the above object, the invention provides following technical scheme:
A making method for immunocomplex adherent cell, step comprises vector construction, virus packaging makes, immortalization bone-marrow-derived lymphocyte makes and gene transfection,
The step of described vector construction is:
(1.1) designing three genes, is CR1, CR2 and CD93 respectively; These three genes be complement system film on signal protein acceptor, and respectively in conjunction with C3b, C3d and C1q complement proteins in blood, play the effect of catching;
Design primer is:
CR1 upstream primer sequence: 5 '-CTCGAGCGGAGCACAATGATTGGTCA-3 '
CR1 downstream primer sequence: 5 '-CCTAGG TGGCTCCTTTCCCACCAAAA-3 '
CR2 upstream primer sequence: 5 '-CTCGAGGGGTCTCGGAACGCATCC-3 '
CR2 downstream primer sequence: 5 '-CCTAGGTCCAAGCAATGAGGCACACA-3 '
CD93 upstream primer sequence: 5 '-CTCGAGGCCACACAGAGACCGGG-3 '
CD93 downstream primer sequence: 5 '-CCTAGGTGTCTCTAGGGCCACCTCAC-3 '.
(1.2) DNA extraction
In order to clone CR1, CR2 and CD93 sequence of total length, using DNA drawer test kit to extract DNA from HeLa cell, then with above-mentioned primer for template, being reacted the gene fragment cloning three genes respectively by PCR;
(1.3) plasmid construction and amplification, acquisition
According to modern biotechnology molecular engineering, cut enzyme by enzyme and connect technology, pLVX-IRES-Neo is cut from XhoI and BamHI restriction enzyme site, re-uses ligase enzyme and the gene fragment obtained before is connected in pLVX-IRES-Neo; PLVX-IRES-Neo is proceeded in competence bacterial strain, amplification cultivation, the plasmid of last extracting competence bacterial strain; Described XhoI restriction enzyme site sequence is: CTCGAG; BamHI restriction enzyme site sequence is: GGATCC.
As the further scheme of the present invention: the system of the PCR reaction described in step (1.2) is: 2 μ l PCR damping fluids, 2 μ l10mM dNTP and 0.25U exTaq polysaccharase; Pcr amplification program: first 94 DEG C, 30s, 58 DEG C, 30s, 74 DEG C, 50s, 35 circulations; Then 72 DEG C, 10min.
As the further scheme of the present invention: described C1q, C3b and C3d acceptor gene, add XhoI restriction enzyme site at 5 ' end of upstream region of gene primer sequence respectively, add BamHI restriction enzyme site at downstream primer sequence 5 ' end.
As the further scheme of the present invention: the restriction enzyme site that two of described XhoI restriction enzyme site and BamHI restriction enzyme site corresponding carrier pLVX-IRES-Neo are respectively identical.
As the further scheme of the present invention: the step that described virus packaging makes is:
(2.1) 293FT cell cultures
The DMEM substratum of 293FT cell containing 10% inactivated fetal bovine serum, 37 DEG C of saturated humidities, containing 5%CO
2incubator in cultivate;
With the 293FT cell of 0.25% tryptic digestion logarithmic phase, with the substratum adjustment cell density containing 10% serum for 1.0
7individual cell, is re-seeded in 10cm Tissue Culture Dish, 37 DEG C, 5%CO
2cultivate in incubator, when cell confluency degree reaches 90%, can be used for transfection;
(2.2) transfection 293FT cell
(2.2.1) first 2 hours of transfection, changes fresh not containing the dual anti-perfect medium containing 10%FBS;
(2.2.2) in a sterile centrifugation tube, add three kinds of prepared DNA solutions, according to 10 μ g, respectively pLVX-IRES-Neo being joined in 1500 μ L OPTI-MEM of 6.5 μ g, 3.5 μ g, 2.5 μ g mixes;
(2.2.3) shaken up gently by Lipo2000 reagent, get 35 μ L Lipo2000 and join mixing in 1500 μ L OPTI-MEM, both mix rapidly;
(2.2.4), after mixing, static 15 minutes of room temperature, to be formed with the transfection composite of DNA and Lipo2000 diluent;
(2.2.5) transfection composite of DNA and Lipo2000 diluent is dropwise joined in culture dish, 37 DEG C of saturated humidities, containing 5%CO
2incubator in cultivate;
(2.2.6) change after cultivating 12 hours not containing the dual anti-virus harvest substratum containing 2%FBS, continue 37 DEG C of saturated humidities, containing 5%CO
2incubator in cultivate 48 hours;
(2.3) collection of virus and concentrated
(2.3.1) the 293FT cell conditioned medium liquid of after transfection 48 hours is collected, precooling on ice; 1500g, 4 degrees Celsius, centrifugal 30 minutes;
(2.3.2) with 0.45 μm of frit vial supernatant;
(2.3.3), after filtering, viral concentration liquid is added according to optimal conditions; 4 degrees Celsius of hold over night;
(2.3.4) after spending the night, 1500g, 4 degrees Celsius, centrifugal 45 minutes; Remove supernatant, add one milliliter of resuspended virus of serum free medium and assemble agglomerate.
As the further scheme of the present invention: the step that described immortalization bone-marrow-derived lymphocyte makes is:
(3.1) reagent
(3.1.1) lymphocyte separation medium: lucifuge 4 DEG C preservation, heats in 37 DEG C of water-baths with front; In whole sepn process, temperature should control at 8-28 DEG C, too high or too low for temperaturely all affects disintegrate-quality;
(3.1.2) full substratum: RPM1640 substratum, includes 20% foetal calf serum, 56 DEG C of deactivations 30 minutes, the HEPES of 1.6-1.8%1M, 1.2%, 0.2mol/L glutamine, 1% penicillin and Streptomycin sulphate;
(3.1.3) cyclocyto enzyme A: the 5mL of 250mg/ bottle, is diluted to 0.2mg/mL with 1640 substratum, and during use, final concentration is 2 μ g/mL
(3.1.4) EBV liquid: purchased from heredity institute of the Chinese Academy of Sciences, be stored in subzero 80 DEG C; Take out from refrigerator during use, 37 DEG C melt rapidly, with the membrane filtration of 0.22 μm, do not exceed 0.5-1 hour;
(3.2) strain method is built
(3.2.1) get peripheral blood 3-4mL, anticoagulant heparin, application liquid concentration 5 μ g/mL, adds 5-10mL peripheral blood;
(3.2.2) after blood and equivalent RPMI-1640 being mixed, gradually join in the centrifuge tube containing 4mL lymphocyte separation medium along tube wall, mixing blood: lymphocyte separation medium=6: 4,3000 leave the heart 10 minutes;
(3.2.3) draw leukocytic cream, move in another centrifuge tube, add not containing the RPMI-1640 6mL of serum, mix gently, carry out first time washing; 1500 leave the heart 15 minutes;
(3.2.4) abandon supernatant liquor, then add the full nutrient solution of 6mL1640 carry out second time washing, centrifugal 1500 turns 15 minutes;
(3.2.5) abandon supernatant liquor, add the full substratum of 2mL1640, before full substratum adds, put 37 DEG C of water-baths 10 minutes, then add Ciclosporin A and 1.2mL EBV liquid/part, be fully moved into the culturing bottle of 25 square centimeters after mixing, put 37 DEG C, CO
2concentration is in the incubator of 5%;
(3.2.6) often within 2-3 days, add the freshly prepared substratum of 3mL, described freshly prepared substratum is 1.6%1M HEPES damping fluid; 15% foetal calf serum; 1% penicillin and Streptomycin sulphate; PRMI 1640 supplies 100%; About 7 days Microscopic observations, visible lymphocyte obviously increases, and occurs clustering phenomena;
If (3.2.7) cell enlargement is slow, or cell density is low, or medium pH value is in acid, and sucking-off partly measures nutrient solution, carries out equivalent oil changing;
(3.2.8) transfer to when total amount reaches 14mL in 75mL culturing bottle, every 2-3 week adds 5-10mL fresh culture;
(3.2.9) in about 6-8 week, when total amount reaches 45mL, put in 50mL centrifuge tube, centrifugal 1500 turns, 10 minutes; Abandon supernatant, add 3mL freezing media, described freezing media is 5% DMSO, and 95%FBS mixes, and becomes cell suspending liquid; Cryopreservation tube packing, 1mL/ manages, and under zero setting 80 degree immediately, moves in liquid nitrogen container after 1-2 hour; The lymphocyte of preparation is EBV immortalization bone-marrow-derived lymphocyte.
As the further scheme of the present invention: the step of described gene transfection is: get and be in logarithmic phase, immortalization bone-marrow-derived lymphocyte 10 that growth conditions is good
5individually be seeded to T25 Tissue Culture Flask; After the adherent 12h of immortalization bone-marrow-derived lymphocyte, by immortalization bone-marrow-derived lymphocyte at 37 DEG C, CO
2concentration is continue in the incubator of 5% to cultivate; 72h collecting cell is after transfection used for WB and detects; Finally collect containing goal gene cell namely for the purpose of immunocomplex adherent cell, immunocomplex adherent cell is different according to the difference of institute's transgene, respectively to being applied to C3b, C3d and C1q tri-genes.
Compared with prior art, the invention has the beneficial effects as follows: the present invention has produced a set of cell carrying the immortalization of specific gene respectively, there is immortalization (infinite multiplication splitting ability) and express a certain immunocomplex acceptor in the ability of cell surface, by the cell of amplification cultivation, set up immunocomplex analysis combination, once can identify blood or systemic various immunocomplex, classify, quantitatively.Also there is low cost infinite multiplication, detection speed is fast, the feature such as simple to operate.In addition, making immortalization bone-marrow-derived lymphocyte virus used is EBV virus, also known as nerpes vinrus hominis (Human herpesvirus4 (HHV-4)), the object of the invention is the bone-marrow-derived lymphocyte in order to obtain immortalization, being not limited to adopt this virus; The bone-marrow-derived lymphocyte of immortalization used derives from the monocyte in the peripheral blood of human body; Wherein said acceptor gene is separately made into different virus respectively, instead of links together; The final cell of the present invention refers to the bone-marrow-derived lymphocyte having immortalization and express a certain immunocomplex acceptor gene, this bone-marrow-derived lymphocyte is a kind of immunocomplex acceptor gene of energy stably express, and this receptor can exist on cytolemma, and in conjunction with a kind of immunocomplex of extracellular correspondence, immortalised B-cell can be not limited to.
Embodiment
Below in conjunction with the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
A making method for immunocomplex adherent cell, comprise vector construction, virus packaging makes, EBV immortalization bone-marrow-derived lymphocyte makes and gene transfection, concrete steps are as follows:
(1) step of vector construction is:
(1.1) designing three genes, is CR1 (NCBI Gene ID:1378), CR2 (NCBI Gene ID:1380) and CD93 (NCBI Gene ID:22918) respectively; These three genes be complement system film on signal protein acceptor, and respectively in conjunction with C3b, C3d and C1q complement proteins in blood, play the effect of catching;
Design primer is:
CR1 upstream primer sequence: 5 '-CTCGAGCGGAGCACAATGATTGGTCA-3 '
CR1 downstream primer sequence: 5 '-CCTAGG TGGCTCCTTTCCCACCAAAA-3 '
CR2 upstream primer sequence: 5 '-CTCGAGGGGTCTCGGAACGCATCC-3 '
CR2 downstream primer sequence: 5 '-CCTAGGTCCAAGCAATGAGGCACACA-3 '
CD93 upstream primer sequence: 5 '-CTCGAGGCCACACAGAGACCGGG-3 '
CD93 downstream primer sequence: 5 '-CCTAGGTGTCTCTAGGGCCACCTCAC-3 '.
(1.2) DNA extraction
In order to clone CR1, CR2 and CD93 sequence of total length, using DNA drawer test kit to extract DNA from HeLa cell, then with above-mentioned primer for template, being reacted the gene fragment cloning three genes respectively by PCR; Wherein, the PCR reaction system of three gene fragments is: 2 μ l PCR damping fluid (100mM Tris-HCl, pH8.3,500mM KCI, 15mM MgCl
2, 0.01% (0.01g/100mL) gelatin; Takara company, Dalian), 2 μ l10mM dNTP (Takara company, Dalian) and 0.25U exTaq polysaccharase (Takara company, Dalian); Pcr amplification program: first 94 DEG C, 30s, 58 DEG C, 30s, 74 DEG C, 50s, 35 circulations; Then 72 DEG C, 10min; By the gene clone that obtains in pLVX-IRES-Neo carrier.
(1.3) plasmid construction and amplification, acquisition
According to modern biotechnology molecular engineering, cut enzyme by enzyme and connect technology, pLVX-IRES-Neo is cut from XhoI (Forward primer introduces) and BamHI (introducing of Reverse primer place) restriction enzyme site, re-uses ligase enzyme and the gene fragment obtained before is connected in pLVX-IRES-Neo; PLVX-IRES-Neo is proceeded in competence bacterial strain, amplification cultivation, the plasmid of last extracting competence bacterial strain; Described XhoI restriction enzyme site sequence is: CTCGAG; BamHI restriction enzyme site sequence is: GGATCC.
(2) step that virus packaging makes is:
(2.1) 293FT cell cultures
The DMEM substratum of 293FT cell containing 10% inactivated fetal bovine serum, 37 DEG C of saturated humidities, containing 5%CO
2incubator in cultivate;
With the 293FT cell of 0.25% tryptic digestion logarithmic phase, with the substratum adjustment cell density containing 10% serum for 1.0
7individual cell, is re-seeded in 10cm Tissue Culture Dish, 37 DEG C, 5%CO
2cultivate in incubator, when cell confluency degree reaches 90%, can be used for transfection.
(2.2) transfection 293FT cell (the DNA separate operation of three kinds of genes)
(2.2.1) first 2 hours of transfection, changes fresh not containing the dual anti-perfect medium containing 10%FBS;
(2.2.2) in a sterile centrifugation tube, add three kinds of prepared DNA solutions, according to 10 μ g, respectively pLVX-IRES-Neo being joined in 1500 μ L OPTI-MEM of 6.5 μ g, 3.5 μ g, 2.5 μ g mixes;
(2.2.3) shaken up gently by Lipo2000 reagent, get 35 μ L Lipo2000 and join mixing in 1500 μ L OPTI-MEM, both mix rapidly;
(2.2.4), after mixing, static 15 minutes of room temperature, to be formed with the transfection composite of DNA and Lipo2000 diluent;
(2.2.5) transfection composite of DNA and Lipo2000 diluent is dropwise joined in culture dish, 37 DEG C of saturated humidities, containing 5%CO
2incubator in cultivate;
(2.2.6) change after cultivating 12 hours not containing the dual anti-virus harvest substratum containing 2%FBS, continue 37 DEG C of saturated humidities, containing 5%CO
2incubator in cultivate 48 hours.
(2.3) collection of virus and concentrated
(2.3.1) the 293FT cell conditioned medium liquid of after transfection 48 hours is collected, precooling on ice; 1500g, 4 degrees Celsius, centrifugal 30 minutes;
(2.3.2) with 0.45 μm of frit vial supernatant;
(2.3.3), after filtering, viral concentration liquid is added according to optimal conditions; 4 degrees Celsius of hold over night;
(2.3.4) after spending the night, 1500g, 4 degrees Celsius, centrifugal 45 minutes; Remove supernatant, add one milliliter of resuspended virus of serum free medium and assemble agglomerate.
(3) step that EBV immortalization bone-marrow-derived lymphocyte makes is:
(3.1) reagent
(3.1.1) lymphocyte separation medium: lucifuge 4 DEG C preservation, heats in 37 DEG C of water-baths with front; In whole sepn process, temperature should control at 8-28 DEG C, too high or too low for temperaturely all affects disintegrate-quality;
(3.1.2) full substratum (using front configuration): RPM1640 substratum, include 20% foetal calf serum (Gibco) { 56 DEG C deactivation 30 minutes }, the HEPES of 1.6-1.8%1M, 1.2%, 0.2mol/L glutamine, 1% penicillin and Streptomycin sulphate;
(3.1.3) cyclocyto enzyme A (CyA): 5mL (250mg)/bottle, is diluted to 0.2mg/mL with 1640 substratum, and during use, final concentration is 2 μ g/mL (0.5%);
(3.1.4) EBV liquid: purchased from heredity institute of the Chinese Academy of Sciences, be stored in subzero 80 DEG C; Take out from refrigerator during use, 37 DEG C melt rapidly, with the membrane filtration of 0.22 μm, do not exceed 0.5-1 hour.
(3.2) strain method is built
(3.2.1) get peripheral blood 3-4mL, anticoagulant heparin (blood room temperature can place 48 hours), application liquid concentration 5 μ g/mL, can add 5-10mL peripheral blood;
(3.2.2) after blood and equivalent RPMI-1640 being mixed, and in the centrifuge tube that tube wall joins containing 4mL lymphocyte separation medium gradually (mixing blood: lymphocyte separation medium=6: 4), 3000 leave the heart 10 minutes;
(3.2.3) draw leukocytic cream, move in another centrifuge tube, add not containing the RPMI-1640 6mL of serum, mix gently, carry out first time washing; 1500 leave the heart 15 minutes;
(3.2.4) abandon supernatant liquor, then add the full nutrient solution of 6mL1640 carry out second time washing, centrifugal 1500 turns 15 minutes;
(3.2.5) abandon supernatant liquor, add the full substratum of 2mL1640 (full substratum add before, put 37 DEG C of water-baths 10 minutes), then Ciclosporin A (4%) and 1.2mL EBV liquid/part is added, be moved into the culturing bottle of 25 square centimeters after abundant mixing, put 37 DEG C, CO
2concentration is in the incubator of 5%;
(3.2.6) often the freshly prepared substratum of 3mL (1.6%1M HEPES damping fluid within 2-3 days, is added; 15% foetal calf serum (FBS); 1% penicillin and Streptomycin sulphate; PRMI1640 supplies 100%); About 7 days Microscopic observations, visible lymphocyte obviously increases, and occurs clustering phenomena;
If (3.2.7) cell enlargement is slow, or cell density is low, or medium pH value is in acid, and sucking-off partly measures nutrient solution, carries out equivalent oil changing;
(3.2.8) transfer to when total amount reaches 14mL in 75mL culturing bottle, every 2-3 week adds 5-10mL fresh culture;
(3.2.9) in about 6-8 week, when total amount reaches 45mL, put in 50mL centrifuge tube, centrifugal 1500 turns, 10 minutes; Abandon supernatant, add 3mL freezing media (5% DMSO (dimethyl sufoxide), 95%FBS) mixing, become cell suspending liquid; Cryopreservation tube packing, 1mL/ manages, and under zero setting 80 degree immediately, moves in liquid nitrogen container after 1-2 hour; The lymphocyte of preparation is EBV immortalization bone-marrow-derived lymphocyte.
(4) gene transfection:
Get and be in logarithmic phase, immortalization bone-marrow-derived lymphocyte 10 that growth conditions is good
5individually be seeded to T25 Tissue Culture Flask; After the adherent 12h of immortalization bone-marrow-derived lymphocyte, by immortalization bone-marrow-derived lymphocyte at 37 DEG C, CO
2concentration is continue in the incubator of 5% to cultivate; 72h collecting cell is after transfection used for WB and detects; Finally collect containing goal gene cell namely for the purpose of immunocomplex adherent cell, immunocomplex adherent cell is different according to the difference of institute's transgene, respectively to being applied to C3b, C3d and C1q tri-genes.
The present invention has produced a set of cell carrying the immortalization of specific gene respectively, there is immortalization (infinite multiplication splitting ability) and express a certain immunocomplex acceptor in the ability of cell surface, by the cell of amplification cultivation, set up immunocomplex analysis combination, once can identify blood or systemic various immunocomplex, classify, quantitatively.Also there is low cost infinite multiplication, detection speed is fast, the feature such as simple to operate.
To those skilled in the art, obviously the invention is not restricted to the details of above-mentioned one exemplary embodiment, and when not deviating from spirit of the present invention or essential characteristic, the present invention can be realized in other specific forms.Therefore, no matter from which point, all should embodiment be regarded as exemplary, and be nonrestrictive, scope of the present invention is limited by claims instead of above-mentioned explanation, and all changes be therefore intended in the implication of the equivalency by dropping on claim and scope are included in the present invention.
In addition, be to be understood that, although this specification sheets is described according to embodiment, but not each embodiment only comprises an independently technical scheme, this narrating mode of specification sheets is only for clarity sake, those skilled in the art should by specification sheets integrally, and the technical scheme in each embodiment also through appropriately combined, can form other embodiments that it will be appreciated by those skilled in the art that.
Claims (7)
1. a making method for immunocomplex adherent cell, is characterized in that, step comprises vector construction, virus packaging makes, immortalization bone-marrow-derived lymphocyte makes and gene transfection,
The step of described vector construction is:
(1.1) designing three genes, is CR1, CR2 and CD93 respectively; These three genes be complement system film on signal protein acceptor, and respectively in conjunction with C3b, C3d and C1q complement proteins in blood, play the effect of catching;
Design primer is:
CR1 upstream primer sequence: 5 '-CTCGAGCGGAGCACAATGATTGGTCA-3 '
CR1 downstream primer sequence: 5 '-CCTAGG TGGCTCCTTTCCCACCAAAA-3 '
CR2 upstream primer sequence: 5 '-CTCGAGGGGTCTCGGAACGCATCC-3 '
CR2 downstream primer sequence: 5 '-CCTAGGTCCAAGCAATGAGGCACACA-3 '
CD93 upstream primer sequence: 5 '-CTCGAGGCCACACAGAGACCGGG-3 '
CD93 downstream primer sequence: 5 '-CCTAGGTGTCTCTAGGGCCACCTCAC-3 '.
(1.2) DNA extraction
In order to clone CR1, CR2 and CD93 sequence of total length, using DNA drawer test kit to extract DNA from HeLa cell, then with above-mentioned primer for template, being reacted the gene fragment cloning three genes respectively by PCR;
(1.3) plasmid construction and amplification, acquisition
According to modern biotechnology molecular engineering, cut enzyme by enzyme and connect technology, pLVX-IRES-Neo is cut from XhoI and BamHI restriction enzyme site, re-uses ligase enzyme and the gene fragment obtained before is connected in pLVX-IRES-Neo; PLVX-IRES-Neo is proceeded in competence bacterial strain, amplification cultivation, the plasmid of last extracting competence bacterial strain; Described XhoI restriction enzyme site sequence is: CTCGAG; BamHI restriction enzyme site sequence is: GGATCC.
2. immunocomplex adherent cell according to claim 1 and preparation method thereof, is characterized in that, the system of the PCR reaction described in step (1.2) is: 2 μ l PCR damping fluids, 2 μ l10mM dNTP and 0.25U exTaq polysaccharase; Pcr amplification program: first 94 DEG C, 30s, 58 DEG C, 30s, 74 DEG C, 50s, 35 circulations; Then 72 DEG C, 10min.
3. immunocomplex adherent cell according to claim 1 and preparation method thereof, it is characterized in that, described C1q, C3b and C3d acceptor gene, adds XhoI restriction enzyme site at 5 ' end of upstream region of gene primer sequence respectively, adds BamHI restriction enzyme site at downstream primer sequence 5 ' end.
4. immunocomplex adherent cell according to claim 1 and preparation method thereof, is characterized in that, the restriction enzyme site that two of described XhoI restriction enzyme site and BamHI restriction enzyme site corresponding carrier pLVX-IRES-Neo are respectively identical.
5. immunocomplex adherent cell according to claim 1 and preparation method thereof, is characterized in that, the step that described virus packaging makes is:
(2.1) 293FT cell cultures
The DMEM substratum of 293FT cell containing 10% inactivated fetal bovine serum, 37 DEG C of saturated humidities, containing 5%CO
2incubator in cultivate;
With the 293FT cell of 0.25% tryptic digestion logarithmic phase, with the substratum adjustment cell density containing 10% serum for 1.0
7individual cell, is re-seeded in 10cm Tissue Culture Dish, 37 DEG C, 5%CO
2cultivate in incubator, when cell confluency degree reaches 90%, can be used for transfection;
(2.2) transfection 293FT cell
(2.2.1) first 2 hours of transfection, changes fresh not containing the dual anti-perfect medium containing 10%FBS;
(2.2.2) in a sterile centrifugation tube, add three kinds of prepared DNA solutions, according to 10 μ g, respectively pLVX-IRES-Neo being joined in 1500 μ L OPTI-MEM of 6.5 μ g, 3.5 μ g, 2.5 μ g mixes;
(2.2.3) shaken up gently by Lipo2000 reagent, get 35 μ L Lipo2000 and join mixing in 1500 μ L OPTI-MEM, both mix rapidly;
(2.2.4), after mixing, static 15 minutes of room temperature, to be formed with the transfection composite of DNA and Lipo2000 diluent;
(2.2.5) transfection composite of DNA and Lipo2000 diluent is dropwise joined in culture dish, 37 DEG C of saturated humidities, containing 5%CO
2incubator in cultivate;
(2.2.6) change after cultivating 12 hours not containing the dual anti-virus harvest substratum containing 2%FBS, continue 37 DEG C of saturated humidities, containing 5%CO
2incubator in cultivate 48 hours;
(2.3) collection of virus and concentrated
(2.3.1) the 293FT cell conditioned medium liquid of after transfection 48 hours is collected, precooling on ice; 1500g, 4 degrees Celsius, centrifugal 30 minutes;
(2.3.2) with 0.45 μm of frit vial supernatant;
(2.3.3), after filtering, viral concentration liquid is added according to optimal conditions; 4 degrees Celsius of hold over night;
(2.3.4) after spending the night, 1500g, 4 degrees Celsius, centrifugal 45 minutes; Remove supernatant, add one milliliter of resuspended virus of serum free medium and assemble agglomerate.
6. immunocomplex adherent cell according to claim 1 and preparation method thereof, is characterized in that, the step that described immortalization bone-marrow-derived lymphocyte makes is:
(3.1) reagent
(3.1.1) lymphocyte separation medium: lucifuge 4 DEG C preservation, heats in 37 DEG C of water-baths with front; In whole sepn process, temperature should control at 8-28 DEG C, too high or too low for temperaturely all affects disintegrate-quality;
(3.1.2) full substratum: RPM1640 substratum, includes 20% foetal calf serum, 56 DEG C of deactivations 30 minutes, the HEPES of 1.6-1.8%1M, 1.2%, 0.2mol/L glutamine, 1% penicillin and Streptomycin sulphate;
(3.1.3) cyclocyto enzyme A: the 5mL of 250mg/ bottle, is diluted to 0.2mg/mL with 1640 substratum, and during use, final concentration is 2 μ g/mL
(3.1.4) EBV liquid: heredity institute of the Gou Bai Chinese Academy of Sciences, is stored in subzero 80 DEG C; Take out from refrigerator during use, 37 DEG C melt rapidly, with the membrane filtration of 0.22 μm, do not exceed 0.5-1 hour;
(3.2) strain method is built
(3.2.1) get peripheral blood 3-4mL, anticoagulant heparin, application liquid concentration 5 μ g/mL, adds 5-10mL peripheral blood;
(3.2.2) after blood and equivalent RPMI-1640 being mixed, gradually join in the centrifuge tube containing 4mL lymphocyte separation medium along tube wall, mixing blood: lymphocyte separation medium=6: 4,3000 leave the heart 10 minutes;
(3.2.3) draw leukocytic cream, move in another centrifuge tube, add not containing the RPMI-1640 6mL of serum, mix gently, carry out first time washing; 1500 leave the heart 15 minutes;
(3.2.4) abandon supernatant liquor, then add the full nutrient solution of 6mL1640 carry out second time washing, centrifugal 1500 turns 15 minutes;
(3.2.5) abandon supernatant liquor, add the full substratum of 2mL1640, before full substratum adds, put 37 DEG C of water-baths 10 minutes, then add Ciclosporin A and 1.2mL EBV liquid/part, be fully moved into the culturing bottle of 25 square centimeters after mixing, put 37 DEG C, CO
2concentration is in the incubator of 5%;
(3.2.6) often within 2-3 days, add the freshly prepared substratum of 3mL, described freshly prepared substratum is 1.6%1M HEPES damping fluid; 15% foetal calf serum; 1% penicillin and Streptomycin sulphate; PRMI1640 supplies 100%; About 7 days Microscopic observations, visible lymphocyte obviously increases, and occurs clustering phenomena;
If (3.2.7) cell enlargement is slow, or cell density is low, or medium pH value is in acid, and sucking-off partly measures nutrient solution, carries out equivalent oil changing;
(3.2.8) transfer to when total amount reaches 14mL in 75mL culturing bottle, every 2-3 week adds 5-10mL fresh culture;
(3.2.9) in about 6-8 week, when total amount reaches 45mL, put in 50mL centrifuge tube, centrifugal 1500 turns, 10 minutes; Abandon supernatant, add 3mL freezing media, described freezing media is 5% DMSO, and 95%FBS mixes, and becomes cell suspending liquid; Cryopreservation tube packing, 1mL/ manages, and under zero setting 80 degree immediately, moves in liquid nitrogen container after 1-2 hour; The lymphocyte of preparation is EBV immortalization bone-marrow-derived lymphocyte.
7. immunocomplex adherent cell according to claim 1 and preparation method thereof, is characterized in that, the step of described gene transfection is: get and be in logarithmic phase, immortalization bone-marrow-derived lymphocyte 10 that growth conditions is good
5individually be seeded to T25 Tissue Culture Flask; After the adherent 12h of immortalization bone-marrow-derived lymphocyte, by immortalization bone-marrow-derived lymphocyte at 37 DEG C, CO
2concentration is continue in the incubator of 5% to cultivate; 72h collecting cell is after transfection used for WB and detects; Finally collect containing goal gene cell namely for the purpose of immunocomplex adherent cell, immunocomplex adherent cell is different according to the difference of institute's transgene, respectively to being applied to C3b, C3d and C1q tri-genes.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1036987A (en) * | 1988-04-01 | 1989-11-08 | 约翰斯霍普金斯大学 | Human body C3b/C4b acceptor (CR1) |
| CN103232971A (en) * | 2013-04-11 | 2013-08-07 | 哈尔滨体育学院 | A method for B lymphocyte transformation by EB virus (Epstein-barr virus) for elite ice and snow athletes |
-
2014
- 2014-06-24 CN CN201410300880.4A patent/CN104293836A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1036987A (en) * | 1988-04-01 | 1989-11-08 | 约翰斯霍普金斯大学 | Human body C3b/C4b acceptor (CR1) |
| CN103232971A (en) * | 2013-04-11 | 2013-08-07 | 哈尔滨体育学院 | A method for B lymphocyte transformation by EB virus (Epstein-barr virus) for elite ice and snow athletes |
Non-Patent Citations (4)
| Title |
|---|
| SUSAN A. BOACKLE, ET AL.: "CD21 augments antigen presentation in immune individuals", 《EUR. J. IMMUNOL.》 * |
| Y CHEN, ET AL.: "The neural guidance receptor Plexin C1 delays melanoma progression", 《ONCOGENE》 * |
| 刘国卿等: "用Raji细胞酶联兔疫吸附试验测定小鼠循环免疫复合物的探讨", 《中国免疫学杂志》 * |
| 魏军等: "宁夏回族永生B淋巴细胞株的建立及保存", 《宁夏医学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108611322A (en) * | 2018-05-09 | 2018-10-02 | 邹畅 | Method for building up and the application of breast cancer circulating tumor cell system CTC-3, culture medium and CTC-3 |
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