CN106267406A

CN106267406A - Acquired immune deficiency syndrome (AIDS) blood purification

Info

Publication number: CN106267406A
Application number: CN201610539279.XA
Authority: CN
Inventors: 翁炳焕; 李兰娟; 李成林; 钱欣; 陈敏
Original assignee: 翁炳焕
Current assignee: Shu Lan (Hangzhou) Hospital Ltd.; Womens Hospital of Zhejiang University School of Medicine
Priority date: 2016-07-01
Filing date: 2016-07-01
Publication date: 2017-01-04
Anticipated expiration: 2036-07-01
Also published as: CN106267406B

Abstract

nullA kind of acquired immune deficiency syndrome (AIDS) blood purification for medical domain，It is characterized in that preparation can washed corpuscles and the separator of blood plasma，Not only retained former macrophage feature with hybridoma technology preparation but also can the hybridoma macrophage strain of indeterminate growth expand parallel，Prepare HIVgp120 and gp41 antibody and combine HIVgp120 and the gp41 antibody of goat-anti Ig，Thing made above is formulated in agar gel，Depurator is formed with high-biocompatibility material parcel，Gel is made to form antibody titer from high to low from top to bottom and the layer distributed of agarose concentration from low to high，Wherein be combined with goat-anti Ig and unconjugated gp120 and gp41 antibody、Hybridoma macrophage is all fixed in agar gel the effect playing absorption HIV，Made depurator is applied in combination with separator and regulatory process，Blood in extracorporeal circulation is divided into blood plasma and hemocyte by separator，The purified device of blood plasma converges with hemocyte after filtering HIV，Then feed back.

Description

Acquired immune deficiency syndrome (AIDS) blood purification

Technical field

The present invention relates to preparation and the application of acquired immune deficiency syndrome (AIDS) blood purification in medical domain, be mainly used in HIV sufferers blood The removing of slurry HIV (human immunodeficiency virus), thus reach to treat the purpose of acquired immune deficiency syndrome (AIDS).

Background technology

Acquired immune deficiency syndrome (AIDS) is the biography caused by HIV (human immunodeficiency virus) (Human Immunodeficiency Virus, HIV) Catch an illness, be widely current in the whole world, according to World Health Organization (WHO) (WHO) and the relevant report of UNAIDS, from Since within 1981, the U.S. finds Patient With Aids case, the whole world has 208 countries and regions to receive the serious of acquired immune deficiency syndrome (AIDS) so far Threatening, there are about 40,000,000 people and infected acquired immune deficiency syndrome (AIDS), death toll is more than 20,000,000, and there are about 6000 people every day becomes acquired immune deficiency syndrome (AIDS) sense Dye person, there are about people more than 300 simultaneously every day and dies from acquired immune deficiency syndrome (AIDS).It is in rapid growth period at HIV infected individuals in China, super Cross 1,000,000.Acquired immune deficiency syndrome (AIDS) has become as the great of after tumor, cardiovascular and cerebrovascular disease, tuberculosis, diabetes another facing mankind Infectious disease, it has also become the serious public health of global concern and social problem.

HIV (human immunodeficiency virus) is a kind of slow virus (Lentivirus) infecting human immune cells, belongs to reverse transcription sick The one of poison, diameter about 120 nanometer, the most spherical in shape.Outer virionic membrane is lipoid peplos (from host cell), and is embedded with virus Albumen gp120 and gp41.Gp41 is transmembrane protein, and gp120 is positioned at surface, and is combined by noncovalent interaction with gp41.To It is inside the sphere matrix (Matrix) formed by albumen p17, and the half-cone capsid (Capsid) that albumen p24 is formed, capsid In high electron density under Electronic Speculum, include virulent rna gene group, enzyme (reverse transcriptase, intergrase, protease) and other Composition from host cell.

After HIV enters human body, first by macrophage phagocytic, but HIV quickly changes macrophage some position interior Sour environment, creates the condition of its existence applicable, is not the most killed and the most within it breeds.Because CD4 is being subject to of HIV Body, thus in macrophage breeding HIV by its envelope protein gp120 and under the auxiliary of gp41, (gp41 plays bridge Effect, utilizes self hydrophobic interaction mediate retroviral cyst membrane and cell membrane fusion) (cell, monokaryon are huge to be bitten carefully to enter CD4+ cell Born of the same parents, dendritic cell etc.), in intracellular rapid propagation, produce 10 every day⁹～10¹⁰Virion, and it is normal constantly to enter other And regeneration the intracellular duplication of CD4+, manufacture more virus infected cell, make peripheral blood CD4+T cell sustaining breakdown, subtract Few.Gp120 with the CD4 receptor of HIV be combined can directly activate infected apoptosis, even infected by HIV T cell express Envelope antigen also can start normal T-cell, indirectly causes a large amount of broken of CD4+ cell by the crosslinking of cell surface CD4 molecule Bad, result causes the severe immune deficiency centered by CD4+T cell defect, and patient mainly shows: periphery lymphocyte reduces, T4/T8 proportional arrangement, to phytohaemagglutinin and the loss for reaction of some antigen, delayed allergy declines, and NK cell, huge bites Cytoactive weakens, and the synthesis of the cytokine such as IL2, IFN-γ reduces.CD4+T cell is most important immunocyte, infects Person once loses a large amount of CD4+T cell, and whole immune system will suffer deathblow, and the infection to various diseases is all lost Go resistance.HIV can also show as hiding for a long time and not showing clinical symptoms after entering host's CD4+ cell, its genome RNA reverse transcription becomes double-stranded DNA, enters in host cell core with viral integrase enzyme, and under the effect of intergrase, double-stranded DNA is integrated In host cell gene group, integrated viral DNA is referred to as provirus, and the several months of can hiding does not replicates, and causes The AIDS several months is to incubation period for many years.In the incubation period of AIDS, HIV is mainly in the macrophage and dendritic cell of lymph node Breeding, these cells are internal HIV depots, releasably to peripheral blood or transfection peripheral blood in CD4+T cell, have silk to divide Splitting former, antigen, TNF, IL-2 and lymph element (LT) can excite HIV provirus gene multiple at the CD4+T Intracellular transcription infected System.After a large amount of propagation, inhibition of HIV granule constantly discharges from destroyed infection cell and is free on blood, then enters back into New CD4+T cell, continues course of infection.And cell is by after HIV, be expressed in infection cell surface gp120 and Gp41 can fusion between mediated infection and non-infected cells, the gp120 that the CD4+T cell of such as infected by HIV is expressed be uninfected by The CD4 of cell combines, and causes cell to merge and form multinucleated giant cell.

Body can be stimulated after HIV to produce envelope protein (Gp120, Gp41) antibody and core protein (P24) antibody.? Measuring low-level antiviral neutralizing antibody in HIV carriers, AIDS patients serum, wherein AIDS patients level is minimum, HIV carriers are the highest, and the most protected effect of this antibody is described.But antibody can not be with the virus that retains in mononuclear phagocyte Contacting, and HIV envelope protein easily occurs antigenic variation, original antibody is ineffective, makes neutralizing antibody not play due Effect.In the latent infection stage, HIV provirus is integrated in host cell gene group, and therefore HIV will not be known by immune system Not, so only relying on autoimmune function and cannot being removed.The critically important reason of another one should be to kill according to antibody Go out, remove antigen mechanism speculate, after immune antibody is combined with antigen, immunological effect to be produced, or pass through activating complement, Mediation A1) CC effect dissolves cellular antigen, but HIV is not cellular antigen；Phagocyte is attracted by chemotaxis Phagocytosis antigen, but HIV is protected in phagocyte on the contrary, is bred；Antibody has been combined neutralization with antigen, is allowed to lose Go appeal, but HIV antigenic structure is changeable, often make antibody be difficult to.

In terms of the treating AIDS method having been used for clinic at present, effect is less preferable: (1) hiv reverse transcriptase suppresses Agent: be only capable of preventing the permissive cell of not yet infected by HIV from infecting, the cell infected do not had therapeutical effect, and toxic and side effects is relatively Many, including gland plastochondria toxicity, bone marrow depression, globular anemia, granulocyte and thrombocytopenia, pancreatitis, intersect resistance in addition The generation of the property of medicine, this kind of medicine is not used alone, and mainly does drug combination, and is easily generated drug resistance mutant, causes under clinical efficacy Fall or inefficacy.(2) hiv protease inhibitor: be easily generated the toxic and side effects such as drug induced hepatic injury, lipid metabolic disorder and drug resistance Property.(3) hiv integrase inhibitor: integrated with host cell DNA by suppression inhibition of HIV DNA and play a role, reverse with HIV Transcriptase inhibitors and protease inhibitor associating use simultaneously and antiviral are had synergism.(4) suppression inhibition of HIV enters suppression Agent: include block gp120 with CD4 be combined, block HIV be combined with accessory receptor, act on gp41 film subunit and act on T pouring Bar cell surface CC-chemokine receptor 5 (CCR5) blocks HIV and enters host cell, but liver and heart are had side effect.(5) Cytokine therapy: be mainly used in regulatory T-cell dynamic equilibrium.(6) HIV vaccine treatment: due to the particularity of HIV, as intrinsic Immunity is not enough to resist HIV and targeting destroys immune system, and virus mutation is rapid, causes the most not yet developing real safety Effective vaccine.(7) gene therapy: the research of HIV gene therapy is never interrupted, does including antisense technology, RNA bait, RNA Disturb, intrabody, dominant negative mutant, suicide gene etc., but enter the II clinical trial phase stage gene therapy almost No.(8) monoclonal antibody passive immunization therapy: reduce the susceptibility of HIV by lowering CD4+T cell surface CCR5, prolong The progress of slow acquired immune deficiency syndrome (AIDS) and the diffusion of minimizing HIV, neutralizing monoclonal antibody 2G12,2F5 and 4E10 are applied to HIV person to be had Good toleration and safety, can delay but can not stop virus bounce-back (9) adoptive immunity cell therapy: external in a large number Virus a large amount of amplification can be caused when cultivating CD4+T cell autologous for HIV, increase the CD4+T cell quantity that virus infects, and feed back CD4+T cell may increase the place of internal virus replication, causes virus load to rebound, and on the whole, adoptive immunity is thin Born of the same parents treat without obvious toxic and side effects, also do not obtain satisfied therapeutic effect.

In gel, Ag-Ab precipitation was initially applied to study Liesegang's phenomenon, application in 1932 in 1905 In identifying bacterial isolates, nineteen forty-six Oudin carries out immunodiffusion in test tube, is used for analyzing antigen mixture, 1948 years Elek and Ouchterlony establishes the two-way double-diffusion process of agar respectively, for identifying simultaneously, compares two or more antigens or anti- Body.The media gel agar of Ag-Ab precipitation in gel or agarose are a kind of polysaccharide bodies containing sulfate, high temperature Time can be dissolved in water, cold after congeal into gel, be internally formed the network structure of a kind of porous, and aperture be very big, can allow macromole Material (molecular weight is up to more than million) pass freely through.The size in aperture further depends on agar concentration, and agar concentration is big, aperture phase To less, agar concentration is little, and aperture is relatively large.The aperture of 1% agar gel is about 85nm, owing to agar or agarose have Well chemical stability, after gel, water content is big, and transparency is good, and convenient sources is disposable, is therefore a kind of well diffusion Medium.The molecular weight of antigen and antibody is general all below 200,000, spreads to low concentration region from area with high mercury in gel Time suffered resistance the least, be substantially in the form of free diffusing form.Due to the different molecular weight of antigen molecule, structure, shape and electricity Lotus amount is different, and therefore its diffusion coefficient is different, and in gel, diffusion velocity is also the most different.When antigen and corresponding antibodies are after diffusion Gel meets, forms antigen antibody complex, if both are suitable in place's ratio of meeting, then form the complex of maximum.By Molecular weight in complex increases, and granule increases, thus does not continues to diffusion and produce precipitation, presents wire or banding, this Planting precipitation to be the formation of one " specific barrier ", all antigen same in immunology or antibody molecule can not pass through, And different those molecules of character can continue diffusion by this barrier, until forming the complex of themselves. So, each have their own position of precipitation that synantigen is not formed.This kind of reaction is the most agar gel diffusion, or agar diffusion, or Immunodiffusion, " specific barrier " of its wire formed or banding is referred to as immuning lines or immunoprecipitation band, is called for short precipitation Line or sealed Belt.It is the normal experiment checkup item at present with known antibodies detection unknown quantity corresponding antigens, is also " middle traditional Chinese medicines Allusion quotation " in 2010 editions regulation for the standard method of influenza virus vaccine hemagglutinin content detection.Generally by a certain amount of goat-anti people Ig antiserum composition is mixed in agar gel, makes containing the specificity goat-anti sero-fast agar plate of people Ig, to be solidified after beat Hole, and in respective aperture, add human serum to be checked (IgG, IgA, IgM etc.), make serum to be checked spread to surrounding in agar plate, Properly locate to combine at antigen and antibody concentration ratio, form macroscopic white precipitate ring and no longer spread.Thus may be used Seeing, when a kind of solution is by semi-solid gel, macromole solute therein is just being coagulated by the gel pore detention of molecular sieve effect In glue, antibodies that antigen the most therein can be fixed in gel in advance and be attracted in gel.So, can root According to agar gel diffusion principle, the depurator of design treatment acquired immune deficiency syndrome (AIDS), i.e. prepare the different HIV antibody infecting strain, HIV is resisted Body is fixed in gel in advance, and when patient flows through depurator through the isolated blood plasma of extracorporeal circulation, the HIV in blood plasma is by advance The corresponding antibodies that is fixed in gel and in detention gel in depurator, simultaneously because of the aperture of 1% agar gel Be about 85nm, also can by 100～120nm HIV detention in gel, through such antibody absorption and gel molecular sieve effect, can HIV is removed outside prosthesis.

The phagocyte of the mankind has large and small two kinds, and microphage is the neutrophilic granulocyte in peripheral blood, macrophagocyte Being the mononuclear cell in blood and the macrophage in multiple organ, tissue, both constitute mononuclear phagocyte system.Mononuclear cell Being formed by the monocyte precursor Development And Differentiation in bone marrow, account for 3% one the 8% of blood middle leukocytes sum, its volume relatively drenches Bar cell is bigger, and mononuclear cell the most only stops 12-24 hour, subsequently into connective tissue or organ, reach maturity into Macrophage, macrophage is the differentiation of mononuclear phagocyte system camber, ripe cell type, has stronger phagocytosis merit Can, wandering macrophage is more than mononuclear cell several times, lasts a long time, some months of can surviving in the tissue, the macrophage settled down Having different titles, be Kupffer Cell, be microglia in brain, be osteoclast etc. in bone in liver, it expresses Fc Receptor, C3b receptor and CD14, play defense function in inherent immunity, is also that the professional antigen participating in adaptive immunity is offered Cell.The CD4 molecule of Expression of Macrophages, is the receptor of HIV (human immunodeficiency virus) (HIV), after HIV enters human body, first suffers huge biting carefully The phagocytosis of born of the same parents, but HIV quickly changes the sour environment at macrophage some position interior, creates condition of its existence applicable, The most it is not killed amount reproduction the most within it to assemble, then HIV is passed to CD4+T cell.So, hybridoma can be used Technology, prepares hybridoma macrophage strain, for preparing the depurator for the treatment of acquired immune deficiency syndrome (AIDS) after a large amount of amplifications, with macrophage Phagocytic function removes the HIV in blood plasma.

In a word, various medicines and biological product cannot effectively kill internal HIV (human immunodeficiency virus), and price, and side effect is big, So far there is no the effective ways for the treatment of acquired immune deficiency syndrome (AIDS), it has also become attack the global problem being unable to for a long time.

Summary of the invention

In order to solve to attack for a long time the global problem in the treating AIDS field being unable to, present inventors have proposed the present invention.

The invention aims to provide acquired immune deficiency syndrome (AIDS) blood purification；Another object is intended to provide acquired immune deficiency syndrome (AIDS) blood purification Preparation and application process.

The object of the present invention is achieved like this: preparation energy washed corpuscles and the separator of blood plasma, prepares HIVgp120 With gp41 antibody, then prepare goat-anti gp120 and gp41 antibody with gp120 and gp41 antibody for antigen, prepare with hybridoma technology Not only retained former macrophage feature but also can the hybridoma macrophage strain of indeterminate growth expand parallel, with high-biocompatibility material Parcel hybridoma macrophage strain and prepare can prevent cell and fragment thereof from leaching and can for remove HIV offer place purification Device, by gp120 and gp41 antibody and goat-anti gp120 and the mixing of gp41 antibody, makes goat-anti gp120 and gp41 antibody be tied completely Close, but surplus gp120 and the gp41 antibody having high titre, antibody is allocated into reverse gradient titre and is incubated after 100 DEG C dissolve The agarose of the gradient concentration of 39～42 DEG C, adds depurator by antibody titer from the low paramount agarose that takes successively, is cooled to Add again next time after semi-solid gel, make the gel in depurator form antibody titer from high to low from top to bottom and from low to The layer distributed of high agar concentration, beneficially plasma perfusion and molecular sieve and the effect of immune clearance, wherein resist with goat-anti HIV Body combines and unconjugated gp120 and gp41 antibody, hybridoma macrophage strain are all fixed on agarose gel and play absorption The effect of HIV, prepared depurator and then be applied in combination with separator and regulatory process, the blood in extracorporeal circulation is separated Device is divided into blood plasma and hemocyte, and the purified device of blood plasma converges with hemocyte after filtering HIV, then feeds back.

The technological core of the present invention is made up of plasma separator and depurator, and wherein plasma separator is used for washed corpuscles And blood plasma, HIV antibody that the cleanser in depurator is combined with goat-anti Ig by the HIV antibody being fixed on agar gel, hybridoma Macrophage strain is made, the molecular weight ratio unconjugated HIV antibody molecule of its conjugate of HIV antibody being wherein combined with goat-anti Ig Amount is big, not easily passs through gel molecular sieve, and contained goat-anti Ig is easily and the feature of agar gel secure bond, HIV antibody also with Be more easy to be fixed in agar gel, can be in conjunction with when the HIV in blood plasma and the HIV antibody being fixed in agar gel are met React and form antigen antibody complex, thus be fixed in agar gel by HIV antibody, because of the sky of hybridoma macrophage So swallow characteristic, when HIV meets therewith, can be fixed in agar gel by phagocytosis therewith, and the filter that agar gel is formed Hole is reduced along with increasing of agarose concentration, and depurator import department agarose concentration is low, and filter opening is the biggest, beneficially plasma perfusion And the association reaction of high titer antibody or cell and HIV；And exit concentration is high, filter opening is the least, it is easy to detention HIV or macromole Conjugate, depurator is combined with multiple special and non-specific HIV and removes composition, in order to avoid extraordinary strain is because of caused by immunity difference Futile treatment, thus in vitro circulation in, after blood is isolated blood plasma and hemocyte by separator, when blood plasma flows through depurator its In HIV be cleaned agent absorption and remove, the blood plasma after purification and hemocyte feed back after converging, and the present invention is with external mechanical removal The method of HIV substitutes the routine internal anti-reverse transcription drug treatment that cannot effectively kill HIV for a long time, it is achieved that artificial By HIV from the treatment new method of internal mechanical removal.

Detailed description of the invention

Fig. 1 is the application schematic diagram of the acquired immune deficiency syndrome (AIDS) blood purification proposed according to the present invention.

Fig. 2 is the internal structure schematic diagram of the separator proposed according to the present invention.

Fig. 3 is the internal structure schematic diagram of the depurator proposed according to the present invention.

In Fig. 1, one end of arterial blood line pipe (1) is connected with arteries, and the other end is through heparin pump (2) and blood pump (3) being connected with plasma separator (4), plasma separator (4) is through the purification in parallel with two of blood plasma pump (6) and circulation line (7) Device (8), depurator (9) are connected, and are connected with circulation line (10), venous line (5) the most successively, another of venous line (5) End is connected with vein blood vessel.

In Fig. 2,1 is plasma separator, and 2 is plasma separator inner chamber, and 3 is the micropore on plasma separator inner chamber tube wall, 4 Being the hemocyte that can not pass through micropore (3), 5 is blood plasma and the chemical analysis that can pass through micropore (3), and 6 is plasma separator exocoel, 7 is blood plasma flow export, and 8 is the hemocyte outlet with switchable valve.

In Fig. 3,1 is free HIV, 2,4 HIV antibody being respectively fixed in agar gel (6), macrophage, 3,5 Being respectively after HIV is combined with HIV antibody, macrophage and be delayed at the conjugate in agar gel (6), 7 for be coagulated by agar The large volume of HIV of glue (6) molecular sieve detention.

Below in conjunction with Fig. 1, Fig. 2 and Fig. 3, the embodiment of the acquired immune deficiency syndrome (AIDS) blood purification that the present invention proposes is made detailed Describe.

One, the preparation of acquired immune deficiency syndrome (AIDS) blood purification agent

(1) preparation of hybridoma macrophage strain

1, primary cell source

(1) single core blood cell: refer to the lymphocyte separated from blood with density－gradient centrifuga－tion method and monokaryon is huge bites Cell.Concrete grammar is: the White Blood Cells Concentrate buying Blood Center or the Cord blood preserved for scientific research, takes 2mL specimen, PBS 6mL anticoagulation dropper, by hemodilution 2～3 times, is fully slowly superimposed on along tube wall after mixing that to have added 4mL lymph thin by liquid Born of the same parents separate in the 10mL centrifuge tube of liquid horizontal centrifugal (400r/min, 20 DEG C) 35min in horizontal centrifuge；It is divided in pipe after Li Xin 3 layers, upper strata is blood plasma and PBS liquid, and lower floor is mainly erythrocyte and granulocyte, and middle level is lymphocyte separation medium, in upper, middle level Interface has the white cloud and mist layer narrow band based on mononuclearcell to be PBMC, is inserted into cloud and mist layer with capillary pipette, draws PBMC inserts in another 50mL centrifuge tube, adds 5 times and is centrifuged (300r/min, 20 DEG C) 10min with upper volume PBS, abandons supernatant 50mLPBS re-suspended cell, centrifugal (350r/min, 20 DEG C) 15min, abandons supernatant, adds Buffer (PBS+0.5% new life Sanguis Bovis seu Bubali + 2mmol/LEDTA clearly, pH7.2) 2mL re-suspended cell, take 15uL cell suspension and add counted under microscope 4 on blood counting chamber Cell (PBMC) sum in individual block plaid.

(2) single core histiocyte: provided by Zhejiang University's Tissue Engineering Study platform.Substantially belong to from spleen Macrophage, its preparation method is: the 1. acquisition of spleen tissue and transhipment: in the approval of reason committee and patient's informed consent Under, take the spleen specimen tissue of excision, shred into volume about 1mm immediately³Small tissue blocks, move into equipped with pre-cooling 4 DEG C In sterile sealing bottle, it is transported to rapidly cell culture chamber.2. the preparation of spleen tissue cell suspension: spleen tissue block is moved to Aseptic operating platform, PBS washs 3 times, and RPMI-1640 washs 2 times, to remove in-house blood and to ensure the aseptic of tissue.Machine Tool grinds spleen tissue, the most just has substantial amounts of histiocyte to hang and is mixed in RPMI-1640 liquid.By 200 mesh stainless steel filtering net mistakes Filter is outstanding is mixed with histiocytic RPMI-1640 liquid, filtrate be spleen tissue cell suspension (mainly containing erythrocyte, lymphocyte, Macrophage etc.).3. erythrocytic cracking in spleen tissue cell suspension: then centrifugal with the washing of RPMI-1640 liquid (1000r/min, 3min), to remove cell debris, adds Tris-NH4Cl effect 5min, splitting erythrocyte, Quick spin (1000r/min, 3min), removes the splitting erythrocyte fragment in supernatant, centrifugal 3 times of PBS washing, RPMI-1640 washing from The heart 1 time, to remove the Tris-NH4Cl of remaining in suspension, it is to avoid it affects the survival of cell, now, mainly contains in suspension Spleen tissue macrophage and lymphocyte.4. the adhere-wall culture of spleen tissue macrophage: using aforementioned suspension as cultivation Cell stock solution, Trypan Blue judges vigor and counts, and adjusting cell concentration with RPMI-1640 liquid is (3～5) × 10⁶/ L, will Adjusting the cell suspension inoculation of concentration in glass culture bottle, condition of culture is 37 DEG C, 50mL/LCO₂, 100% humidity, point Not Pei Yang 2～3h, under phase contrast microscope observe form.The digestion of adherent spleen tissue macrophage: adherent spleen tissue is huge to be bitten The digestion of cell: suck culture supernatant, macrophage is adherent, and PBS repeatedly blows and beats, digests, and the washing of gained cell suspension is centrifugal (1000r/min, 3min), obtains isolated and purified macrophage.Further, it is also possible to take treatment or the discarded specimen of Post operation carries Take preparation, such as peritoneal cavity liquid, alveolar, liver, spleen, peritoneal tissues, small intestinal mucosa etc..

(3) amniotic fluid, villus cell: attached hospital for obstetrics and gynaecology of Zhejiang University reproduction genetic laboratory is standby.Reason committee member Can ratify with under patient's informed consent, treating excess syndrome tests the residue amniotic fluid after diagnosis report, villus cell, selects exponential phase cell Continue to employ.

2, cell is cultivated and the adherent preliminary sorting of macrophage

Cell is cultivated routinely, but according to the difference of cellularity, suitably adjusts incubation time, condition of culture etc., typically Single core blood cell (PBMC) or single core histiocyte (macrophage) are placed in containing RPMI-1640 culture medium by adherent method Culture dish in, in 37 DEG C, 5%CO₂Cell culture incubator (Themo electro corporation CLASS 100, beautiful State) in hatch 2h, after mononuclearcell is adherent, inhale abandon upper strata suspension cell (cell beyond macrophage be difficult to adherent and Remove with upper liquid), PBs buffer washs 3 times gently, adds a small amount of RPMI 1 culture medium, scrapes patch with cell scraper Parietal cell (predominantly macrophage, but also have other attached cells a small amount of).1 000r/min is centrifuged 5min, abandons supernatant.Amniotic fluid Cell, villus cell are cultivated 1～7 day, occur that cell growth clone, cell growth converge rate and reach the logarithmic growth of 60～80% Phase cell, with trypsinization, PBS, obtains cell suspension, is made into proper cell concentration.

3, cd4 cell sorting

Employing immunomagnetic beads method sorting cd4 cell: 1. main agents and instrument: (Germany U.S. sky Ni is biological for CD4 immunomagnetic beads Technology Co., Ltd.)；0.2% Trypan Blue liquid (Shanghai Sheng Gong biotechnology service company)；New-born calf serum (Hyclone company)；MiniMACS magnetic separation system (Mei Tian Ni Bioisystech Co., Ltd of Germany).2. cd4 cell immunity Magnetic bead sorting method: cell suspension is divided equally to two 1.5mLEppendorf pipes, centrifugal (300r/min, 20 DEG C) 10min, discards Supernatant, the every 80uLBuffer of re-suspended cell contains cell number 10⁷Individual, every 10⁷Individual cell add 20uLCD4MicroBeads or CD8MicroBeads, fully mixes, and hatches 15min at 4～8 DEG C, uses 1mLBuffer washed cell, centrifugal (300r/min, 20 DEG C) 10min, supernatant discarded 500uLBuffer re-suspended cell, MS detached dowel is placed in the magnetic field of MACS separator, with 500uLBuffer rinses, and by 500uL cell suspension by detached dowel, rinses detached dowel repetitive operation 3 times with 500uLBuffer, Collect effluent, containing non-cd4 cell in effluent, in separator, take out detached dowel, divide with 1000uLBuffer pressure flush From post, collecting effluent, this is that (cell viability detects cd4 cell: takes 15uL cell suspension before and after cell purification respectively and waits body Long-pending trypan blue solution mixing, the not colored shinny person of basis of microscopic observation is living cells, and the coloring person of swelling is dead cell, calculates 200 The percentage ratio of living cells in individual cell).The cell now sorted is mainly macrophage.

4, CD14 cell (macrophage) sorting

CD14 is mononuclear cell and the distinctive surface marker of macrophage, if in theory from single core histiocyte, sheep Sort in water cell and villus cell, then gained cell is macrophage；If sorted from single core blood cell, then gained Cell includes mononuclear cell and macrophage；But because the mononuclear cell life-span is short, only survives 1 day in peripheral blood and can not show a candle to huge biting Cell is prone to adherent growth, so the most substantially removing in the cell attachment of the present invention is cultivated, the cell sorted out is essentially Macrophage.

Basic skills is analogous to cd4 cell, uses immunomagnetic beads method.1. reagent: people's CD14 immunomagnetic beads test kit (Miltenyi Biotec, Germany), RPMI-1640 culture medium (Hyclone, the U.S.)；2. immunomagnetic beads method: (A) magnetic bead is with special Opposite sex target cell one mononuclear cell combines: every 1 × 10⁸Individual PBMC adds magnetic bead and the 800uL buffering of 200uL coupling CD14 antibody Liquid (containing the bovine serum albumin 2.5mL and 2mol/L EDTA0.5mL of 10%, 4 DEG C of refrigerator pre-coolings), in 15mL centrifuge tube Fully mixing, hatches 15min for 4 DEG C, and centre can slightly shake 1 time.Taking-up centrifuge tube after 15min, every 1 × 10⁷Individual cell adds 1 ～2mL pre-cooling buffer, 1 000r/min, centrifugal 8min, abandon supernatant, add 0.5mL buffer and blow and beat into single cell suspension. (B) collect the mononuclear cell of marked by magnetic bead: be placed on MACS magnetic frame by cell separation post, add 1mL buffer statocyte Detached dowel, treats that no liquid is dripped, and is added by above-mentioned cell suspension in people's cell separation post immediately, with 0.5mL wash buffer cell Detached dowel 3 times.After to be rinsed, add 1mL buffer, emigrated cells detached dowel from magnetic frame, quickly promote with pin post, Go out the cell combined with CD14 antibody one magnetic bead in detached dowel, be the macrophage of CD14+.

Additionally, following 2 kinds of methods sorting also can be used, including 1. adherent method: be placed in by PBMC and cultivate containing RPMI-1640 In the culture dish of base, in 37 DEG C, containing 5%C0: cell culture incubator (Themo electro corporation CLASS 100, The U.S.) in hatch 2h.After adherent mononuclear cells, inhaling and abandon upper strata suspension cell, PBs buffer washs 3 times gently, adds a small amount of RPMI 1 culture medium, scrapes attached cell with cell scraper.1 000r/min is centrifuged 5min, abandons supernatant.2. fluidic cell Art method: CD14 labelling: take PBMC, with buffer (bovine serum albumin 2.5mL and 2mol/LEDTA 0.5mL containing 10%) Adjusting cell density is 1 × 10⁸/ mL, adds CD14+-FITC antibody 100uL, 4 DEG C of lucifuge labellings in every milliliter of cell suspension 18min, then addition 1mL streaming buffer is to terminate dyeing in centrifuge tube, PBs washs 3 times, with the PBS containing 2% mycillin Adjustment cell density is 2 × 107/mL.Selected by flow cytometry apoptosis: by the cell of preparation at flow cytometer (BD FAcsAria II, U.S.) upper sorting, according to the fluorescence intensity of CD14 antibody, the relative size of cell and the relative particle of cell and interior The complexity of portion's structure, collects the cell of CD14+.

5, prepared by CD14 hybridoma cell strain (hybridoma macrophage strain)

(1) culture medium and main agents: DMEM culture medium, HAT, HT Selective agar medium are purchased from Sigma company, top grade tire cattle Jinshi City Hao on daytime ocean biological product science and technology responsibility company limited purchased by serum (FBS)；DMSO (--methyl sulfoxide) it is domestic analytical pure Reagent.

(2) myeloma cell prepares: merges and takes out the myeloma cell (SP2/ that a pipe is frozen the last week in liquid nitrogen container 0), it is immediately placed in hot water and thaws that (applying most is Sp2/0 cell strain, and this cell strain growth and fusion efficiencies are the best, and itself is not Secreting any heavy chain immunoglobulin or light chain, the Seedling height scale of cell is 9 × 10⁵/ ml, the doubling time be usually 10～ 15h；The actual application relevant to human body considers select homologous cell strain, if Fu Xiang bio tech ltd, Shanghai is to ATCC The NCI-H929 human myeloma cell strain that cell bank is introduced).Adding appropriate complete culture solution after thawing, 1000r/m is centrifuged 3min； It is repeated 1 times.Precipitate is moved in Tissue Culture Flask, add DMEM culture fluid, put CO2 incubator and cultivate, within 3-4 days, once pass Generation or amplification culture, adjust cell state in merging first 24 hours, it is ensured that before merging, cellular morphology is good, it is vigorous to grow.Add Appropriate basal medium is in centrifuge tube, and after beaing mixing gently, 1000r/m is centrifuged 5-10min, repeated washing cell 2 times.

(3) CD14 cell (macrophage) to be hybridized prepares: the mononuclear phagocyte of present invention sorting is with basal medium Adjust total cell and count to 1 × 10⁸～2 × 10⁸Merge for cell.Blue dyeing phase-contrast microscopy, viable count is expected with platform Should be higher than that 80% for qualified.

(4) cell merges: by CD14 cell (mononuclear phagocyte) with myeloma cell with 10: the ratio of 1-5: 1 adds In centrifuge tube, being mixed evenly, 1000r/m is centrifuged 5min, supernatant discarded, beats gently at the bottom of pipe to cell without granular precipitate, weight Multiple 2 times.Preheating centrifuge tube is rotated gently, by the 50% of 1000 μ L of preheating under aseptic condition after taking-up in 37 DEG C of water-baths PEG3000 is added drop-wise in fusion pipe rotating centrifugal pipe the most gently along tube wall in 60s, afterwards by the basis training of the 25mL of preheating Support base to be also added drop-wise in centrifuge tube along tube wall in 3-5min, rotating centrifugal pipe lightly during adding, then stand In 37 DEG C of water-bath 10min, 1000r/m is centrifuged 5min, supernatant discarded, adds 50mL HA T culture medium.Suitably it is inoculated into after mixing In 96 well culture plates, it is placed in 37 DEG C, the CO2 incubator of 5% is cultivated.

(5) there is the mononuclear phagocyte strain screening of phagocytic function: observe cell growth status in 96 well culture plates, 7-10 Only have hybridoma after it and can grow division, now discard HAT culture medium, change complete medium.Cell clone grows When area reaches 1/10 cell hole, culture supernatant, selection is gone to have the culture hole of the hybridoma cell strain that growth conditions is good, aobvious The position of labelling cell strain growth, size under micro mirror, using aseptic rifle head to draw cell clone in the position of mark has to new In the culture hole of complete medium, doubling dilution counts hole to below the most successively, 37 DEG C, cultivates one week left side in 5%CO2 incubator The right side, basis of microscopic observation cell growth status, when cell clone covers with to hole floor space more than 1/10, take in cell or cultivation Clear detection hybridoma macrophage (M φ) strain function.

1. hybridoma macrophage strain phagocytosis antibacterial Function detection: macrophage is hanged with staphylococcus or Candida albicans Liquid mixing incubation, smear, fixing, serge blue liquid dyes, and in oil Microscopic observation phagocytosis situation, counts phagocytosis antibacterial and does not gulps down Bite the macrophage number ratio of antibacterial, to swallow antibacterial function strong macrophage alternately positive clone strain.

2. hybridoma macrophage strain phagocytosis HIV Function detection: take acquired immune deficiency syndrome (AIDS) (AIDS) patient's of Disease Control and Prevention Center's preservation After blood plasma is mixed with hybridoma macrophage strain, separates cell strain, PBS 3 times, measure the phagocyte strain through cracking The function of phagocytosis HIV, with specific reference to HIV-1p24 antigen detection kit, (euzymelinked immunosorbent assay (ELISA), Shanghai inspires biotechnology limited Company) operation, with concentration known 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, The p24 antigen of 80pg/ml is less than 5pg/ml, measurement range 0～400pg/ml, the range of linearity as comparison, lowest detectable limit In 0.5pg/ml～80pg/ml, 15min, 450nm measures absorbance (OD), and blank calibration object absorbance is not higher than 0.050,0pg absorbance is not higher than 0.100, and 1000pg/ml absorbance is not less than 1.000, is recognized as absorbance ＞ 0.12 For being the positive.Concrete test kit description of pressing operates.

3. the strain of hybridoma macrophage produces macrophage cytokines detection: with MIF (MIF) ELISA detection kit (stamen bio tech ltd, Shanghai hundred) by specification operates, and detection range is 0～800pg/ml, Sensitivity is 1.0pg/ml, directly can detect by an unaided eye: in reacting hole, color is the deepest under white background, and the positive is the strongest, negative Reaction for colourless or the most shallow, according to the depth in color, with "+", "-" number represents.Also OD value can be surveyed: at ELISA detector On, in 450nm (if developing the color with ABTS, then 410nm) place, survey each hole OD value after returning to zero with blank control wells, if more than regulation 2.1 times of negative control OD value, are the positive, specifically press the operation of test kit description.MIF be collection cytokine, somatomedin, Hormone and enzyme characteristic multi-effect protein molecular, the regulatory factor as inherent immunity and inflammatory reaction plays central Effect, in various infection and active chronic inflammation disease play panimmunity function.

According to testing result, the cell clone selected in the culture hole with stronger macrophage function repeats next Wheel dilution is cultivated, and repeats 2-3 wheel, takes out, proceed to culture bottle mass propgation after detection function-stable.

(6) preservation of hybridoma macrophage strain and recovery: preserve first 12 hours and adjust cell growth state, take one bottle of life Making cell suspension after long vigorous, that form is good cell, suitably digestion, 1000r/min is centrifuged 5min, removes supernatant, flicks Making cell loose at the bottom of pipe, add 4 DEG C of 9 parts of complete culture solutions preserved and 1 part of DMSO, subpackage cell cryopreservation tube, 1mL/ manages, and-70 DEG C refrigerator overnight, puts into cryopreservation tube in liquid nitrogen container after taking-up and saves backup.The hot water of about 40 DEG C is got out before recovery, will Cryopreservation tube carefully takes out from liquid nitrogen, is immediately placed in hot water uniformly shake and makes cell thawing, is centrifuged at 1000r/min after defrosting 5min, opens cryopreservation tube under aseptic condition in superclean bench, and the cell complete culture solution after thawing washed once, then It is centrifuged 5min, supernatant discarded, in case making amplification culture at 1000r/min.

6, prepared by hybridoma macrophage strain treatment cell

The i.e. amplification cultivation of hybridoma macrophage strain.Move after using complete culture solution the most resuspended above-mentioned cell precipitation Enter in culture bottle, put 37 DEG C, 5%CO2 incubator is cultivated.Repeatedly pass on amplification cultivation, until required hybridoma cell strain Quantity, every Secondary Culture positive hybridoma cell strain 10 generation, the function of detection macrophage hybridoma cell strain, see whether steady Fixed.Continue in several bottles, carry out extensive industrialization to prepare, save backup.

(2) preparation of HIV-1gp120 antibody

Antibody involved in the present invention can entrust specialty businessman to prepare, or directly buys, as Shanghai is auspicious from specialty businessman Unit such as neat bio tech ltd and Shanghai Linc-Bio Science Co., Ltd. etc. all specialize in HIV-1gp120 antibody, The preparation of the various antibody such as gp41 antibody and goat anti-human igg and sale.Method includes that hybridoma technology prepares monoclonal antibody, EB Virus Transformation technology is prepared monoclonal antibody, hybridoma technology and is combined with Epstein-Barr virus transformation technology preparation monoclonal antibody and base Because of engineered antibody, specifically it is listed below.

1, use lymphocyte Epstein-Barr virus to convert with hybridoma technology and combine and prepare HIV-1gp120 monoclonal antibody

Specimen origin have following several by way of: take lymphocyte strain frozen in Infectious Diseases Lab Sample Storehouse (through inactivation HIV immunity and the lymphocyte of EBV transfection)；Buy the fresh White Blood Cells Concentrate in blood station, then carried out inactivation HIV strain and exempt from The lymphocyte of epidemic disease；Take from the cord blood lymphocytes cell (through inactivation HIV immunity) preserved as scientific research；Directly take from HIV-1 The peripheral blood lymphocyte (for self) of the infected self, uses Histopaque lymphocyte separation medium separation single core thin Born of the same parents (PBMC), regulation concentration is 2x 10⁶Epstein-Barr virus (EBV) stock solution that rear addition is appropriate, is placed in 370C, 5%CO2 overnight incubation, Preparing B cell to be hybridized, with the positive hole of ELISA method screening anti-HIV-1 outer membrane protein (gp120), transfer cell is to 24 orifice plates Continue to cultivate 2 weeks, repeat to measure anti-gp120 by ELISA method and confirm positive, continuously clone's secondary a large amount of amplification cultivation.Will After positive cell strain myeloma cell heterogeneous with people Mus (being buied by Zhejiang University's siberian crabapple) mixes (3: 1), add 1ml50% PEG makes the two merge, and then re-suspended cell cultivated liquid in IMDM culture fluid, within second day, adds Peritoneal Cells of Mice (by Zhejiang Jiang great Xue siberian crabapple is buied) as trophocyte, screen anti-gp120 antibody with ELISA after continuing to cultivate 3 weeks, select strong positive Hole hybrid tumor cell amplification is cultivated, and repeatedly clones until obtaining stable cell line, cultivates with this cell line, prepares HIV-1 antibody, uses ELISA detection kit, and by specification operates, and measures the Ig subclass of antibody, and surveys with conventional ELISA method Determine titer and the specificity of antibody, select high specificity, antibody that titer is high.

2, use gene recombinaton HIV-1gp120 to combine hybridoma technology and prepare antibody

(1) reagent and recombinant antigen: relate to reagent: HIV-1gp120 genetic fragment, HIV-1gp120 antigen, HIV antigen Nitrocellulose membrane bar by Beijing Wan Tai Pharma Inc. provide BamH I restriction endonuclease Xho I restriction endonuclease, nucleic acid coprecipitator, T4DNALigase is purchased from precious biological company limited；Liagen glue reclaims test kit purchased from QIAquick company；RPMI 1640 is dry Powder culture medium is purchased from Gibco company；Top grade new-born calf serum is purchased from bright marine growth Engineering Co., Ltd；SupersignalWest Pico Trial Kit, TMB Substrate Kit are purchased from Pierce company；Mice Ig subclass detection kit, freund adjuvant And PEG, Hy-poxanthine, Thymidine and Aminoptem are purchased from Sigma company.HIV-antibody diagnosing reagent kit is purchased from Shanghai biology company limited of China of section, the goat anti-mouse IgG antibody of horseradish peroxidase (HRP) labelling is limited purchased from doctor's moral Company.Construction of recombinant plasmid and qualification: vector plasmid PEGX-4T-2 BamH I, Xho I enzyme action, T4DNA Ligase connects Gp120 genetic fragment, recombinant plasmid transformed enters E.colistrain XL1 blue, checks order.The abduction delivering of recombiant protein and mirror Fixed: recombinant plasmid transformed enters in XL1-Blue escherichia coli, under IPTG derivant effect 25 DEG C, 190r/min shakes, overnight, 4 000r/min are centrifuged 10min, collect antibacterial, SDS-PAGE testing goal protein expression situation.Fusion protein purification and qualification: Expression product centrifugal collecting precipitation hangs through PBS, after 30W Ultrasonic Pulverization instrument cracking antibacterial, is centrifuged collection supernatant and filters, Filtrate, with AKTA PURIFYER100 protein purification instrument, GST column purification, obtains fusion protein GST-HIV, concentrates centrifuge tube and carries out Concentrating, S21 type biology spectrophotometer measurement concentration, purifying protein is identified by SDS-PAGE.

(2) animal immune: BALB/c mouse 6 week old, female, 4, before immunity, take mouse vein blood, separation serum stays and does Negative serum.Mixed with equal-volume Freund's complete adjuvant by the GST-HIV fusion protein of 50-100 μ g, the injection of emulsifying pneumoretroperitoneum is exempted from Epidemic disease mice.After initial immunity, booster immunization mice after using incomplete Freund's adjuvant and fusion protein emulsifying every 2 weeks, immunity Dosage and approach are the same, repeat immunity 2-3 time, and the GST-HIV of last booster immunization direct lumbar injection 50-100 μ g melts Hop protein.

(3) foundation of Detection of Monoclonal Antibody: third time immunity one week after tail vein blood, determines positive serum by square formation method Best effort concentration and GST-HIV fusion protein be most preferably coated concentration.Operate as follows: according to 1: 1000,1: 500,1: 200,1: 100 four dilution factor, uses and is coated buffer dilution antigen, be longitudinally coated 96 hole ELISA Plate, every hole 100 μ L, 4 DEG C of bags By overnight, wash 3 times, every minor tick 3 minutes.Positive serum and negative serum are made doubling dilution respectively by 1: 1000, Laterally it is loaded onto the 10th hole, every hole 100 μ L, is placed in 37 DEG C of incubation 1h in wet box, wash 3 times, every minor tick 3 minutes.Enzyme mark resists Body HRP-sheep anti-mouse igg makees 1: 10000 dilution, every hole 100 μ L, 37 DEG C of incubation 1h to specifications, washs 3 times.Add people now to join OPD substrate solution, every hole 100 μ L, 37 DEG C of lucifuges reaction appropriate times, every hole adds 100 μ L stop buffers and terminates reaction, detects it OD492 value.Positive hybridoma cell screening technique is set up.According to experiment condition and method in square formation method, melt with GST-HIV respectively Hop protein is experimental group, and after abduction delivering, recombinant bacterium (containing plasmid pET-32a) albumen is matched group, screening positive clone.Operation Step is as follows: with the suitableeest concentration envelope antigen that is coated in 96 hole ELISA Plate, and 100 μ l/ holes, 4 DEG C of refrigerators are coated overnight.Take out bag Washed 3 times by adding cleaning mixture after plate, wash 3min every time；Every hole adds cell conditioned medium to be measured (1: 5 dilution) 100 μ L, 37 DEG C of temperature Case hatches 50min, and washing 3 times, wash 3min every time afterwards；Every hole adds-resists 100 μ l, 37 DEG C of incubation 30min, washes Wash 3 times, wash 3min every time；Every hole adds the OPD substrate solution 100 μ L now joined, room temperature lucifuge reaction 10-15min；In every hole Add 2mol/L H2SO4 stop buffer 100 μ l to be used for terminating reaction；Detection plate is placed in microplate reader survey OD492 value.Matched group Set up: positive controls is the positive serum of suitably dilution, and negative control group is anti-with one to have the most dilution unrelated list Anti-cell conditioned medium.Indirect ELISA the selection result judges.Often group detection OD492 value, with P (sample value)/N (negative value) >=2.0 Person is judged to positive value.Screening positive clone standard: cell conditioned medium reacts in sun with just screening group (fusion protein is coated after purification) Property, react the detection hole being negative with negative screening group (tropina containing pET-32a plasmid after induction) be positive sample simultaneously Product.

(4) cell merges: myeloma cell prepares: merge the myeloma taking out a pipe the last week in liquid nitrogen container frozen thin Born of the same parents, are immediately placed in hot water and thaw.Adding appropriate complete culture solution after thawing, 1000r/min is centrifuged 3min；It is repeated 1 times.Will precipitation Thing moves in Tissue Culture Flask, adds DMEM culture fluid, puts CO2 incubator and cultivates, within 3-4 days, once passes on or amplification culture, Cell state is adjusted, it is ensured that before merging, cellular morphology is good, it is vigorous to grow in merging first 24 hours.Appropriate pancreatin is used before merging Using centrifuge tube to collect after digestion, add appropriate basal medium in centrifuge tube, after beaing mixing gently, 1000r/min is centrifuged 5-10min, repeated washing cell 2 times.Splenocyte prepares: before fusion, takes a Balb/c mice, wins eyeball and take blood, blood-letting Post-tensioning neck completely is put to death, and soaks in 75% ethanol.Taking-up layback is put and is fixed on dissection plate, takes spleen under gnotobasis, will Spleen moves in plate.Then 10mL RPMI 1640 basal medium is added at plate, with flat mouth tweezers attrition crushing repeatedly After, use asepsis injector repeatedly to aspirate piping and druming, make single cell suspension.After meter viable count, 1000r/min is centrifuged 10min, Add the basal medium total cell of adjustment and count to 1 × 10⁸～2 × 10⁸Merge for cell.Cell merges: by splenocyte and bone marrow Oncocyte is with 10: the ratio of 1-5: 1 adds in centrifuge tube, is mixed evenly, and 1000r/min is centrifuged 5min, supernatant discarded, strikes gently Beat at the bottom of pipe to cell without granular precipitate, be repeated 2 times.Preheating centrifuge tube, aseptic bar after taking-up is rotated gently in 37 DEG C of water-baths Under part, the 50%PEG3000 of 1000 μ L of preheating is added drop-wise in fusion pipe rotating centrifugal pipe the most gently along tube wall in 60s, Afterwards the basal medium of the 25mL of preheating is also added drop-wise in centrifuge tube along tube wall in 3-5min, light during adding Lightly rotating centrifugal pipe, is then statically placed in 37 DEG C of water-bath 10min, and 1000r/m is centrifuged 5min, supernatant discarded, adds 50mL HA T Culture medium.Suitably it is inoculated in 96 well culture plates after mixing, is placed in 37 DEG C, the CO2 incubator of 5% is cultivated.

(5) screening of positive clone strain: observe cell growth status in 96 well culture plates, only has hybridoma thin after 7-10 days Born of the same parents can grow division, now discards HAT culture medium, changes complete medium.Cell clone growth area reaches 1/10 carefully During hilum, go culture supernatant, cloned by the monoclonal antibody screening technique screening positive hybridoma cell set up before.Use improvement The positive cell hole that indirect ELISA Preliminary screening is gone out by limiting dilution assay gradient limiting dilution assay continuously carries out 3-4 wheel Sub-clone.Selection has the culture hole of the positive hybridoma cell strain that growth conditions is good, labelling cell strain growth under microscope Position, size, use aseptic rifle head to draw cell clone in the position of mark in the new culture hole having complete medium, so After successively doubling dilution count hole to below, 37 DEG C, cultivate about one week in 5%CO2 incubator, basis of microscopic observation cell grows Situation, when cell clone covers with to hole floor space more than 1/10, takes cells and supernatant and carries out antibody inspection side.To testing result Repeat next round dilution for the cell clone in positive culture hole to cultivate, repeat 2-3 wheel, after detection supernatant titer plateaus Take out, proceed to culture bottle mass propgation.

(6) preservation of hybridoma cell strain and Secondary Culture: preserve and recover: preserve first 12 hours and adjust cell growth shape State.Taking one bottle of growth vigorous, make cell suspension after the cell that form is good, suitably digestion, 1000r/m is centrifuged 5min, goes Clear liquid, flicks and makes cell loose at the bottom of pipe, add 4 DEG C preserve 9 parts of complete culture solutions and 1 part of DMSO, subpackage cell cryopreservation tube, 1mL/ manages, and cryopreservation tube is put in liquid nitrogen container after taking-up and saved backup by-70 DEG C of refrigerator overnight.40 DEG C of left sides are got out before recovery Right hot water, carefully takes out cryopreservation tube from liquid nitrogen, is immediately placed in hot water uniformly shake and makes cell thawing, after defrosting 1000r/m is centrifuged 5min, opens cryopreservation tube in superclean bench under aseptic condition, and the cell complete culture solution after thawing is washed Washing once, be then centrifuged 5min, supernatant discarded at 1000r/m, cell precipitation moves into training after using complete culture solution the most resuspended Support in bottle, put 37 DEG C, 5%CO2 incubator is cultivated.Secondary Culture positive hybridoma cell passed for 10 generations continuously, used indirectly The method of ELISA measures culture supernatant antibody titer, observes the change of titer, and whether observe this positive hybridoma cell strain can be steady Determine secretory antibody.

(7) preparation of monoclonal antibody mouse ascites: low-speed centrifugal collects the hybridoma after cultivating, dilute through basal medium Releasing cell number is 1 × 10⁷/mL.Mouse peritoneal injection 0.2mL/ only, observes mouse ascites production, treats that abdominal part is bright after injection Aobvious distension rises, and punctures abdominal cavity with syringe needle, and centrifuge tube collects ascites.Gather complete, mouse peritoneal is injected appropriate basal medium, Every 2-3 days, same method took ascites again.The ascites collected, 10000r/m is centrifuged 5min, Aspirate supernatant, subpackage ,-20 DEG C Preserve.

(8) CHARACTERISTICS IDENTIFICATION of monoclonal antibody: specificity: Weston-Blotting experiment is carried out with reference to " molecular cloning " method, half Dry method transfers, and program is as follows: first with recombinant bacterium albumen after the CD4 fusion protein of purification and induction through 12%SDS-PAGE, and one Group is used as comparison, and one group is used as transfer.Electrophoresis is complete, puts into Cathode buffer with an equal amount of 6 filter paper after being cut by glue In；NC film is first used soak with ethanol 3-5min, is then placed in deionized water, big filter onesize with 6 again after 1-3min Paper is put in anolyte together；With Cathode buffer, the minus plate of electrophoresis tank is smeared moistening, take out the filter in Cathode buffer Paper and gel are successively placed on minus plate, extrude bubble gently.Again NC film in anode buffer liquid and 6 filter paper are taken from anolyte Go out and be layered on successively on gel, extrude bubble gently.Finally cover electrophoresis tank positive plate gently.After switching on power, according to NC film Area, 2mA/cm2 size of current, transfer 2h；After transfer terminates, glue dyes after taking out, after transfer membrane takes out, dilute with PBS 5% defatted milk powder released is closed, and 4 DEG C of refrigerators stand overnight.After closing overnight, discard confining liquid, wash 3 with lavation buffer solution Secondary, each 5min.After washed, add the monoclonal antibody cell conditioned medium of 1: 10 dilution, jog on shaking table, room temperature reaction 60min.Abandon Go one to resist, then wash 3 times with lavation buffer solution, each 5min.The condition groped according to Dot-ELISA, adds 1: 5000 dilution The two of degree resist, jog on shaking table, room temperature reaction 50min.Discard two to resist, then wash 3 times with lavation buffer solution, each 5min. The NC film having protein band is carried out labelling, adds DAB developer, terminate with deionized water rinsing anti-after reaction appropriate time Should.Titer, with the CD4 fusion protein of purification for detection antigen, uses indirect ELISA method to measure hybridoma supernatant and list The titer of anti-ascites.Monoclonal antibody subgroup identification selects mouse monoclonal Ig class/subgroup identification ELIAS secondary antibody i.e. with kit measurement, presses Operating according to test kit operating instruction, step is as follows: the fusion protein of CD4 after purification suitably diluted is coated in ELISA Plate, Every hole 100uL, puts 4 DEG C of refrigerator overnight.The liquid that is coated in ELISA Plate is patted dry, then washes once with lavation buffer solution, 3min.So After Hybridoma Cell Culture supernatant to be measured is added in hand-hole, every hole 100 μ L, put 37 DEG C of incubator incubation 30min.With washing after patting dry Wash buffer and wash five times, 3min/ time.6 kinds of enzyme marker in this test kit are separately added in hole, every hole 100 μ L, are placed in 37 DEG C incubation 30min.Continuation lavation buffer solution washes five times, 3min/ time.Add after OPD substrate solution lucifuge develops the color 15 minutes and add eventually Only liquid, detects OD492 value by microplate reader, and OD492 value substantially exceeds the type in other hole and is HIV-1gp120 monoclonal antibody Ig classification.

(9) HIV-1gp120 monoclonal antibody applies (specialty businessman can be entrusted to complete) after the most refined.

(3) preparation of HIV-1gp41 antibody

Preparation with HIV-1gp120 antibody

(4) preparation of goat-anti people-Ig

Being antigen by made HIV-1gp120 antibody and gp41 antibody, immune sheep prepares antibody.

1, laboratory animal: what immunity laboratory animal was selected is that animal institute of Zhejiang University hybridizes white goat, body weight 30 jin The healthy between twenty and fifty ewe two of left and right, the front dyestuff of immunity smears the back animal, makes clear and definite labelling.Employing is done as everybody else does The feeding manner of stable breeding, ensures that sheep has to appropriate motion every day, and between the lights, general half an hour of every time moving is left for movement time The right side, the most healthy and strong, it is to avoid sheep overfertilization, increase the immunity of body, drinking-water abundance, and give appropriate concentrate, Grass, Semen Maydis, wheat bran, Semen Tritici aestivi, vitamin etc. so that it is balanced in nutrition.Often check experiment sheep health status.

2, HIV-1gp120 antibody and gp41 antibody are antigen: HIV-1gp120 antibody prepared by the present invention and gp41 antibody (IgG) concentration is respectively 2.5mg/mL (can be provided) by businessman, and before inoculation, mixing 0.1mL antigen, 1.9mL aseptic PBS, 2mL are not It is standby that immunogen emulsion made by family name's (or incomplete) adjuvant completely.

3, the immunity of goat: choose two goats, is labeled as goat A, goat B, antigen inoculation (HIV-1gp120 antibody and HIV-1gp41 antibody).Immunity position is front and back's groin, and often place's groin divides 2 some injections, a total of 8 injection points.Note The mode of penetrating is subcutaneous injection, and every goat per injection 4mL immunogen, containing 250 μ g antigens；Each injection point injecting immune is former 0.5mL, containing about 32 μ g antigens.Front two goats of immunity are drawn blood 10mL respectively, are labeled as 0dP1 ,-20 DEG C of preservations；Immunity for the first time I.e. 1, immunogen: 0.1mL antigen+1.9mL aseptic PBS+ Freund's complete adjuvant 2mL (CFA) is exempted from；Draw blood after immune 7 days 10mL, Separate serum, be labeled as 7dP1, ELISA and detect serum titer, remain serum-20 DEG C preservation.Within 3～4 weeks, start second time immunity, Two immunogens: 0.1mL antigen+1.9mL aseptic PBS+2mL incomplete Freund's adjuvant (IFA), draw blood after immune 7 days 10mL, separates Serum, is labeled as 7dP2, ELISA and detects serum titer, remain serum-20 DEG C preservation.After immunization time is 6-8 week for the third time, Three exempt from immunogen: 0.1mL antigen+1.9mL aseptic PBS+2mL incomplete Freund's adjuvant (IFA), and draw blood after immune 7 days 10mL, point From serum, it is labeled as 7dP3, ELISA and detects serum titer, remain serum-20 DEG C preservation.If now serum titer does not reaches 1 ∶10⁶Above, then need to exempt from again once；If serum titer has reached 10⁶Below then need not be immune again, draw blood the most week about 50mL, separates serum ,-20 DEG C of preservations.

4, prepared by serum: within after general each immunity 7～10 days, can detect in sheep jugular vein blood collection.By assistant Baoding Animal so that it is keep stance, after cervical region cropping, aseptic cotton balls cleaning disinfection, searches out the blood sampling of jugular vein hand syringes, Blood 5-10mL is taken by fixing for syringe position.Bioactivity is carried out after isolating serum.7～10 days after the third immunization, warp The most desirable blood 30-50mL after bioactivity is qualified.Aseptically separate serum, in plate to be collected in or triangular flask After blood coagulation, after clot being peeled off with bottle wall in gnotobasis (such as superclean bench) with aseptic dropper, put into 37 DEG C, 1～2h take out after put into 4 DEG C overnight, make serum fully separate out (can not freeze, otherwise produce haemolysis), by centrifugation precipitation point Go out serum, put cryogenic refrigerator into and preserve.Before using must through signing qualified after again subpackage save backup.

5, the mensuration of antiserum titre: antiserum titre is to use ELISA assay method: be with being coated when being coated by sample After liquid is diluted to the concentration of 1: 1000, in ELISA Plate, every hole adds 100 μ L, is then placed in aluminum box and puts in 4 DEG C of refrigerators overnight. Take out the next morning to pat dry and be coated liquid, wash three times with PBST, every minor tick five minutes, pat dry enzyme with gauze for the last time Target, adds the confining liquid of 10% serum, and every hole adds 100UL, puts into 37 DEG C, water-bath 1～2h.Take out the most again and pat dry closing Liquid, with the every minor tick of PBST detersive enzyme target 3 five minutes, pats dry with gauze for the last time, adds 100 μ L/ holes one anti-(1: 2000 Dilution, dilutes with the PBST of 4% Ox blood serum, puts into 37 DEG C, water-bath 1～2h).Taking out and pat dry an anti-liquid, PBST washes Wash ELISA Plate three times, every minor tick 5 minutes, pat dry ELISA Plate with gauze for the last time, add two anti-liquid (the anti-sheep of rabbit 1: 1000), Every hole adds 100 μ L, puts into 37 DEG C, water-bath 1～2h.Take out again and pat dry the two anti-every minor ticks five of liquid PBST detersive enzyme target 3 Minute, patting dry with gauze for the last time, every hole adds 50 μ L substrate nitrite ions, and black out adds the H SO of 2M after developing the color 10-20 minute Solution 50 μ L terminate reaction, after with microplate reader survey OD value (in half an hour).

Two, the preparation of acquired immune deficiency syndrome (AIDS) blood purification

1, the preparation of blood purification cell column

Cleaning the hybridoma macrophage prepared of the present invention with physiological saline solution, 1000r/min is centrifuged, after cleaning again with 1000r/min is centrifuged 5min, takes cell precipitation and loads the 200ml hydrostatic column that acrylate etc macromolecular material is made In, it is fills up to 4/5, cell column is sealed standby.

2, the outfit of blood purification gel antibody

Gp120 and gp41 antibody is mixed with goat-anti gp120 and gp41 antibody respectively, makes the goat-anti gp120 in mixed liquor All it is fully tied with gp41 antibody, but surplus free gp120 and the gp41 antibody having sufficiently high titre, then will be containing combining Type and the mixed antibody of sequestered gp120 antibody and the mixed antibody containing conjunction type and sequestered gp41 antibody again with etc. Titer mixes, and is made into final mixed antibody, prepares 5 kinds of gel mixed antibodies with the agarose C1-4B solution of gradient concentration respectively, The titer making HIVgp120 and gp41 antibody is 1: 700,1: 500,1: 300,1: 200,1: 100 and the agarose concentration of correspondence It is followed successively by 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, irrigates standby blood with equal equal-volume from low to high by antibody titer Liquid daf molecule post, makes gel form antibody titer from high to low from top to bottom and the layering of agarose concentration from low to high Distribution.The filter opening that agar gel is formed reduces along with increasing of agarose concentration, and depurator import department agarose concentration is low, Filter opening is just big, beneficially the association reaction of plasma perfusion and high titer antibody or cell and HIV；And exit concentration is high, filter opening The least, it is easy to detention HIV or Large molecular conjugates.Depurator is combined with multiple special and non-specific HIV and removes composition, in order to avoid Extraordinary strain is because of the futile treatment caused by immunity difference.

Formula one: by gp120 and gp41 antibody with play being incubated at 39～42 DEG C after 100 DEG C dissolve of carrier function 0.7%, the agarose C1-4B normal saline of 0.8%, 0.9%, 1.0%, 1.1% is made into the most corresponding 1: 700,1: 500,1 : 300, the gel antibody of the gradient titre of 1: 200,1: 100, take 40ml gel antibody successively by the paramount titre of low titre and add blood In liquid daf molecule post, it is desirable to the gel antibody being initially charged be cooled to 37 DEG C become semi-solid agar gel after the most then add under Once, make the blood purification cell column form antibody titer from high to low from top to bottom (from import to outlet) and from low to high The layer distributed of agarose concentration, wherein with goat-anti HIV (gp120 and gp41) antibodies and unconjugated gp120 antibody, Gp41 antibody and hybridoma macrophage are all fixed in gel the effect playing absorption HIV.

Formula two: by gp120 and gp41 antibody with rise carrier function after 100 DEG C dissolve, be cooled to 39～42 DEG C After the agarose C1-4B of 1% content is made into the titre gradient of 1: 50～1000 by multiple proportions, Reperfu-sion blood purification cell column, make Blood purification cell column forms antibody titer from high to low and agarose from low to high from top to bottom (from import to outlet) The layer distributed of concentration, wherein resists with goat-anti HIV (gp120 and gp41) antibodies and unconjugated gp120 antibody, gp41 Body and hybridoma macrophage are all fixed in gel the effect playing absorption HIV.

Formula three: indicate the titre being made into by multiple proportions of the different antibody prepared by HIV strain and correspondence thereof respectively The valence value of gradient, such as HIV strain 1 titre 1: 100 pipe, 1: 200 pipe ...；HIV strain 2 titre 1: 100 pipe, 1: 200 pipes ..., the rest may be inferred, then will infect strain 1 titre 1: 100 pipe and 10ml39～the liquid agarose C1-of 42 DEG C of insulations Put into blood purification cell column after 4B mixing, to be placed be cooled to semi-solid gel after, then will infect strain 1 titre 1: 200 pipe with The liquid agarose C1-4B mixing of 10ml39～42 DEG C of insulations, mixing liquid puts into the gel upper strata of blood purification cell column, according to this Analogize.A, B, C totally 3 strain is had in actual applications by combination preparation, the HIV strain in such as somewhere period, preparation Antibody has A strain 1: 100 pipe, 1: 200 pipe ...；B strain 1: 100 pipe, 1: 200 pipe ...；C strain 1: 100 pipe, 1: 200 pipe ...；Warp After combination be exactly ABC strain 1: 100 pipe, ABC strain 1: 200 pipe ... ABC strain 1: 1000 manage, then by ABC strain 1: 1000 pipe with Put into blood purification cell column after the liquid agarose C1-4B mixing of 10ml39～42 DEG C of insulations, to be placed be cooled to semisolid After gel, then the liquid agarose C1-4B mixing by ABC strain 1: 900 pipe with 10ml39～42 DEG C of insulations, mixing liquid puts into blood Being cooled to semisolid gel upper strata in daf molecule post, the rest may be inferred, and centre can be spaced selection, the most then selects ABC Strain 1: 500 pipe mixes with the liquid agarose C1-4B of 10ml39～42 DEG C of insulations, and mixing liquid has put into blood purification cell column Being cooled to semisolid gel upper strata, the consumption of liquid agarose C1-4B (can also be made by adjusting in 1ml～10ml The blood purification cell column become, from import to going out interruption-forming gradient antibody content from low to high, in view of antigen and antibody only Just can play immunoreation when proper ratio, form precipitation line and stop moving ahead of comparator antibody or antigen, so different HIV contains When the blood plasma of amount is by cleanser, HIV just adsorbed detention in different aspects, and also the HIV strain of the present invention is permissible The most almost all of infection strain of letter lid, and be combined with goat-anti Ig in adsorbent and unconjugated gp120 and gp41 antibody Being all the HIV aglucon being fixed in advance in Agar Gel, HIV antibody is tied with HIV especially in conjunction with the HIV antibody having goat-anti HIV Close the immune complex molecular weight that formed bigger, be entirely capable of being delayed in Agar Gel and be eliminated, add aperture about For 85nm Agar Gel inherently can the detention a diameter of 100～HIV of 120nm, so inferring theoretically and having hardly The HIV sufferers failed to respond to any medical treatment).

3, the specification of blood purification and material

Blood purification cell column or blood purification are the cylinder that footpath, the end is little, footpath, top is big, or square, infundibulate, volume Being 200～300ml, import and export and be equipped with cell screen cloth, footpath, import department top sieve number is 800 mesh；Footpath sieve mesh at the bottom of exit Number is 2.0～5.0 mesh (2.5～5.0 mesh are equivalent to 0.1～0.2 micron or 100～200 nanometers), specifically can be made into 2.0 mesh, 7 kinds of different sizes of 2.5 mesh, 3.0 mesh, 3.5 mesh, 4.0 mesh, 4.5 mesh and 5.0 mesh, in order to stop 120 nanometers inhibition of HIV or Bigger antibacterial；Liquid outlet arranges the cell strainer that mesh number is 100 mesh (being equivalent to 4 microns), may leach in order to stop Cell；Relief area, the beneficially stability of system circulation it is provided with between liquid entrance and mesh screen.

Blood purification selects the macromolecular material of acrylate etc, it is desirable to good biocompatibility, activates benefit hardly Body, do not cause inflammatory reaction, do not cause the change of leukocyte, platelet, blood oxygen pressure, complement C 3, C5a.Can pass through covalency, The methods such as grafting, polymerization improve the structure of material, the regulation inhomogeneities on surface, hydrophilic, minimizing to blood coagulation and oxidative stress Impact thus improve biocompatibility, reduce complication generation.Hydrophilic gel is added, by 2 methyl-prop at depurator inner surface Alkene acyloxyethyl phosphocholine-butyl methacrylate is solidificated in cellulose acetate membrane, by controlling wet-spinning procedure, can generate CA/PMB30, CA/PMB80 and CA/PMB30-80, have higher blood and cell compatibility.Some had anticoagulation Material be solidificated on the material of carrier or depurator inner surface, can suppress blood coagulation, improve biocompatibility, also can reduce Heparin consumption, and likely realize no-rod tractor.As being aggregated in by heparin on polyacrylonitrile-polymine film, effect may More preferably, and can reduce the anaphylaxis during purification, the polyacrylonitrile surface of solidifying shell polysaccharide and heparin covalent thing displays that Good blood compatibility, and the activity of pseudomonas aeruginosa can be suppressed, reduce cell-cytotoxic reaction.Heparin covalent is combined To polyether sulfone surface, both maintained the mechanical property of polyether sulfone, the anticoagulation function of depurator inner surface can have been improved again.At acetic acid Covalent immobilisation linoleic acid film on fibrous membrane, maybe will be covalently bound to polyacrylic linoleic acid and be grafted onto polysulfone membrane surface, all may be used To have more preferable histocompatibility and anticoagulant effect.Along with macromolecular material and the development of nanotechnology, with mankind's blood The close material of endothelial tube in the near future it would appear that.

Three, the preparation of plasma separator

1, preparation principle: prepare according to the molecular size of hemocyte and blood plasma components.Such as visible component (blood in blood of human body Cell) size be: normocyte is about 7 microns (μm), is the discoid cell of concave-concave；Leukocyte is divided into 5 kinds, neutral Granulocyte about 12 μm, eosinophilic granulocyte is more bigger, and basophilic granulocyte is close with neutrophilic granulocyte, small lymphocyte 6-8 μm, Approximating with erythrocyte, mononuclear cell is maximum, about 15-20 μm.Platelet is disc, and diameter 1～4 microns are to 7～8 microns not Deng, the platelet mean diameter of people is 2-4 micron, thick 0.5～1.5 micron.

2, material: can be selected for poly-vinegar non-woven fabrics, acetate fiber, absorbent cotton etc., it is desirable to good biocompatibility, activate hardly Complement, do not cause inflammatory reaction, do not cause the change of leukocyte, platelet, blood oxygen pressure, complement C 3, C5a.Can be by altogether Valency, be grafted, the method such as polymerization improves the structure of material, the regulation microinhomogeneities on surface, hydrophilic, minimizing be to blood coagulation and oxygen Change stress impact thus improve sieving adequacy and biocompatibility, the generation of minimizing complication.

3, type and spec: for the profile of separator, can prepare as filter element with the material such as acetate fiber or absorbent cotton Become column construction, be prepared as the shapes such as flat structure with materials such as poly-vinegar non-woven fabrics as filter element；By hemocyte to be separated and blood The molecular size of slurry composition determines aperture.Plasma separator stable in properties involved in the present invention, good biocompatibility, penetrating Hollow fibre type filter made by the high molecular polymer that property is high, hollow-fiber film a diameter of 270～370 μm, and film thickness is 50 μm, Aperture is 0.2～0.6 μm, and fibre length is 13.5～26 μm.This hole is only permitted blood plasma and is filtered, but can stop that all of cell becomes Point.

Four, the application component of acquired immune deficiency syndrome (AIDS) blood purification and purposes

1, key member: (1) plasma separator: be used for separating mononuclear blood cell and blood plasma；(2) blood purification: be used for HIV in adsorbed plasma.

2, additional member: include blood pump, heparin pump, sound pulse pressure and air monitering, temperature control system, off gas system, The part compositions such as monitored conductivity system, ultrafiltration monitoring and leakage blood monitoring.

(1) blood pump (Blood Pump): be used for promoting blood circulation to maintain being smoothed out, generally of blood purification treatment Blood pump part often has rotary test speed function, and to monitor the blood circumstance of patient, therefore blood pump runner sets with flute pitch Want accurately and it needs to often adjust, according to the situation of bloody path pump line, typically spacing is set as 3.2～3.3mm, can not be the most loose, Blood flow detection otherwise can be caused to be forbidden；Also can not be too tight, otherwise can cause pipe breakage.

(2) heparin pump (Heparin Pump): heparin pump is equivalent to the micro-injection pump applied clinically, in order to continue to Injecting heparin in sieving pipeline (patient blood), owing to the blood of patient circulates and air contact in vitro, is susceptible to blood coagulation Phenomenon, uses heparin pump to be possible to prevent the generation of blood coagulation.

(3) sound pulse pressure monitoring: arterial blood pressure monitoring is mainly in order to the stopping state of dynamic monitoring blood separator micropore, separately Outer in order to monitor the change of extracorporeal circulation thrombosis, solidification and pressure.When blood flow deficiency, arterial pressure will reduce；When have blood coagulation, Thrombosis, particularly during separator blockage of the micro orifice, arterial pressure will raise；Venous pressure monitoring is used for monitoring pipeline blood backflow Pressure, when separator blockage of the micro orifice, blood coagulation, thrombosis, blood flow is not enough and venous return syringe needle comes off time, venous pressure Will decline, if the distortion blocking of bloody path return duct or backflow syringe needle occur blocking, venous pressure will raise.

(4) air monitering (Air Detector): be used for monitoring the air bubble of blood pathway, typically use ultrasonic listening Principle, in order to avoid patient occurs air embolism to arrange.When having monitored air bubble, detecting system can drive dynamic, Vein bloody path folder carrys out blocking blood flow, prevents the generation of danger.

In a word, on the basis of key member of the present invention and additional member, Import computer regulates and controls and makes the people of operation Property, the personalization for the treatment of, the safety of design, and modularity, automatically monitoring and regulation and control, liquid crystal display, judge voluntarily to warn The blood purifying therapeutical instrument that the report micro computer such as reason and ring off signal processes.

Five, the connecting path of acquired immune deficiency syndrome (AIDS) blood purification and using method

1, install: such as Fig. 1, with sterile working's connecting components, including separator, depurator and each circulation line.

2, aerofluxus: with physiological saline solution topping up separator, depurator and each circulation line, gets rid of separator, depurator And gas in circulation line, bubble, go through, confirm without use after gas, bubble.

3, logical liquid: arterial blood line pipe (1) is connected the arteries of HIV sufferers, the row of going through the most again Gas is the most complete, and liquid stream is the most unobstructed, and avoids managing the pollution of interior flow liquid.

4, anticoagulant: inject anticoagulant (heparin) from heparin pump (2) to liquid stream, be 2500U or 20～30U/kg for the first time.

5, start: one end of arterial blood line pipe (1) is connected with arteries, venous line (5) is connected venous blood Pipe, then opens blood pump (2), and blood flow is 100～150ml/min, such as Fig. 1, when arterial blood enters through arterial blood line pipe (1) Entering plasma separator (4), the blood plasma separated arrives depurator (8) through circulation line (7) under the effect of blood plasma pump (6), Blood plasma to be full of, about 10 minutes, begin paying out blood plasma, through circulation line (10) flow out, synchronize to depurator (9) irrigate blood plasma, When blood plasma in depurator (8) has nearly flowed, starting again at perfusion blood plasma, now depurator (9) begins paying out blood plasma, two The depurator (8) of parallel connection, depurator 9) alternately, the blood plasma after purification is through circulation line (10) and plasma separator (4) institute The hemocyte separated feeds back through venous line (5) after converging.Such as Fig. 2, when blood to be separated enters the interior of plasma separator (1) During chamber (2), through the effect of valve (8), the little molecule blood plasma components (5) that can pass through micropore (3) enters the exocoel (6) of separator, Then flow out through plasma outlet port (7), and the hemocyte (4) that can not pass through micropore (3) flows out through valve (8).Such as Fig. 3, when containing When the blood plasma of HIV (1) enters depurator, HIV antibody (2) that HIV therein (1) is fixed in agar gel (6) respectively, huge Phagocyte (4) is combined into antigen antibody complex (3), macrophage phagocytic body (5), combined after HIV no longer move down, The most combined 100～HIV of 120nm again by the aperture because concentration is higher less be about 85nm 1% concentration under Layer agar gel molecular sieve detention is at (7) place.So remove HIV, until the plasma circulation amount being previously set (usually 9L), control Treat just end, whole therapeutic process is by computer control, and can detect duty at any time, easy to use, automatization and Safety.

Six, the checking of acquired immune deficiency syndrome (AIDS) blood purification effect

1, HIV-1gp120 antibody, the checking of gp41 cleaning antibody HIV effect

The present inventor, according to the basic skills of the present invention, has done following simple confirmatory experiment: take HIV-1gp120 antibody, Gp41 antibody (Rui Qi bio tech ltd, Shanghai), joins and is incubated after 100 DEG C dissolve at 50 DEG C of 1.0% standby fine jades In lipolysaccharide C1-4B, after mixing, titre is 1: 300～500, takes 2.5 × 300mm Westergren's blood sedimentation tube 5 of sterilizing, draws respectively 1.0% agarose C1-4B solution is to 200mm scale, and after cooling, agarose becomes semi-solid.Ling Qu Disease Control and Prevention Center and infectious disease are real Test the specimen of the 5 example HIV sufferers that room Sample Storehouse preserves, remove each about 10mL of the blood plasma after cell, respectively take blood before 9mLAIDS filter Slurry injects blood sedimentation tube upper end blank pipe in batches, the 1.0% agarose C1-4B containing antibody of blood sedimentation tube lower floor to be flowed through from blood After flowing out in immersed tube, collect effluent, blood plasma after referred to as AIDS filter.Take blood plasma after the front blood plasma of AIDS filter and filter, according to HIV- 1p24 antigen detection kit (euzymelinked immunosorbent assay (ELISA), inspiration bio tech ltd, Shanghai) operate, with concentration known 0pg/ml, The p24 antigen of 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml is as comparison, minimum Detection limit is less than 5pg/ml, measurement range 0～400pg/ml, range of linearity 0.5pg/ml～80pg/ml, and in 15min, 450nm surveys Determining absorbance (OD), blank calibration object absorbance is not higher than 0.050, and 0pg absorbance is not higher than 0.100,1000pg/ Ml absorbance is not less than 1.000, is considered as positive as absorbance ＞ 0.12, and testing result (table 1) illustrates, AIDS blood plasma is filtered After crossing simple purifier, part HIV is adsorbed by corresponding antibodies, and after the 1st time filters, HIV total body clearance is 20.01%, After the 2nd time filters, total body clearance is 27.99%, and after the 3rd time filters, total body clearance is 37.36%.Illustrate along with filtration The increase HIV of number of times can constantly be removed, thus reaches to treat AIDS purpose.Table 1 AIDS blood plasma filters simple purifier P24 testing result (pg/ml) front and back

2, the checking of HIV effect is removed in the strain of hybridoma macrophage

In order to effect of HIV is removed in check cross tumor macrophage strain, the present invention devises easy method of testing: takes and goes out 2.5 × 300mm Westergren's blood sedimentation tube of bacterium 5, draws by centrifugation the macrophage hybridization that (1000r/min, 5min) precipitates respectively Tumor cell strain, to 200mm scale, is then drawn and is incubated after 100 DEG C dissolve at 39～42 DEG C of 0.9% standby agarose C1- 4B, reaches the long scale of about 10mm, and after putting blood sedimentation tube cooling, agarose becomes semi-solid, blood sedimentation tube inner cell can be stoped to flow out but The material of water and the chemical analysis that will not stop little molecule etc passes through.Ling Qu Disease Control and Prevention Center and Infectious Diseases Lab Sample Storehouse are protected 5 example blood plasma, the most about 10mL of acquired immune deficiency syndrome (AIDS) (AIDS) patient deposited, before respectively taking 9mLAIDS filter, blood plasma injects blood sedimentation tube (letter in batches Easily purifier) upper end blank pipe, wait flowing through the hybridoma macrophage strain layer of blood sedimentation tube lower floor and after flowing out in blood sedimentation tube, receive Collection effluent, blood plasma after referred to as AIDS filter.Take blood plasma after the front blood plasma of AIDS filter and filter, with MIF (MIF) ELISA detection kit (stamen bio tech ltd, Shanghai hundred) pairing detection, by specification operates, detection range Being 0～800pg/ml, sensitivity is 1.0pg/ml, directly can detect by an unaided eye under white background: in reacting hole, color is more Deeply, the positive is the strongest, and negative reaction is colourless or the most shallow, according to the depth in color, with "+", "-" number represents.Also OD can be surveyed Value: on ELISA detector, in 450nm (if developing the color with ABTS, then 410nm) place, surveys each hole OD after returning to zero with blank control wells Value, if 2.1 times of the negative control OD value more than regulation, it is the positive.Result such as table 1, before filter, in blood plasma, MIF testing result is equal For negative (or because content preserves cause degraded etc. for a long time less than detection sensitivity, blood plasma), and after filtering, in blood plasma, testing result is Positive.Illustrate that macrophage hybridoma cell strain creates MIF cytokine in this process.MIF be collection cytokine, growth because of Son, hormone and enzyme characteristic multi-effect protein molecular, as in the regulatory factor performance of inherent immunity and inflammatory reaction The effect of pivot, plays panimmunity function in various infection and active chronic inflammation disease.Letter is filtered making AIDS blood plasma Easily before and after depurator while MIF pairing detection, the present invention has also done the pairing detection of HIV-1p24, according to HIV-1p24 antigen Detection kit (euzymelinked immunosorbent assay (ELISA), inspiration bio tech ltd, Shanghai) operates, with concentration known 0pg/ml, 0.5pg/ The p24 antigen of ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml is as comparison, lowest detectable limit Less than 5pg/ml, measurement range 0～400pg/ml, range of linearity 0.5pg/ml～80pg/ml, in 15min, 450nm measures extinction Degree (OD), blank calibration object absorbance is not higher than 0.050, and 0pg absorbance is not higher than 0.100,1000pg/ml extinction Degree is not less than 1.000, is considered as positive as absorbance ＞ 0.12, and result (table 2) illustrates that AIDS blood plasma filters simple purification After device, part HIV is by the phagocytosis absorption of macrophage hybridoma cell strain, and the blood plasma HIV after filtration significantly reduces, through the 1st After secondary filtration, HIV clearance rate is 20.55%, and after the 2nd time filters, HIV clearance rate is 42.83%, p ＜ 0.01, has substantially Effect, illustrate along with filter number of times increase HIV can constantly be removed, thus reach treat AIDS purpose.

Table 1 AIDS blood plasma filter MIF testing result before and after the simple purifier containing hybridoma macrophage (quantitatively: pg/ml)

Table 2 AIDS blood plasma filters p24 testing result (p24:pg/ before and after the simple purifier containing hybridoma macrophage ml)

Above-mentioned simple experiment shows, the HIV in blood plasma can by blood purification agent (HIVgp120 antibody, HIVgp41 antibody, Hybridoma macrophage strain, agar gel micropore) adsorption removal, show that the acquired immune deficiency syndrome (AIDS) blood purification of the present invention has significantly Remove the therapeutic efficiency of blood plasma inhibition of HIV.

Claims

1. the acquired immune deficiency syndrome (AIDS) blood purification for medical domain, it is characterised in that prepare hybridoma macrophage strain, HIV Antibody and combine the HIV antibody of goat-anti Ig, is formulated in agar gel, and the exit that perfusion high-biocompatibility material is made sets Put the depurator of screen cloth, make import department to exit form antibody titer from high to low and agarose concentration from low to high Layer distributed but hybridoma macrophage strain accounts for 4/5, and the separator of separated plasma and hemocyte can constitute purging in vitro device Main part, for removing the HIV in blood plasma.

Acquired immune deficiency syndrome (AIDS) blood purification the most according to claim 1, it is characterised in that described depurator is by the sieve in its exit Net, play the fixing and agar gel of molecular sieve effect, prepare hybridoma macrophage strain therein, HIV antibody, combine goat-anti The HIV antibody of Ig collectively forms molecular sieve mechanical removal and cell and the barrier of antibody mediated immunity removing blood plasma HIV.

3. according to the arbitrary described acquired immune deficiency syndrome (AIDS) blood purification of claim 1,2, it is characterised in that the volume of described depurator is 200～300ml, to import and export and be equipped with cell screen cloth, footpath, import department top sieve number is 800 mesh, footpath sieve number at the bottom of exit Being 2.0～5.0 mesh, liquid outlet arranges the cell strainer that mesh number is 100 mesh, arranges promotion between liquid entrance and screen cloth The relief area of system stability circulation.

Acquired immune deficiency syndrome (AIDS) blood purification the most according to claim 3, it is characterised in that sieve mesh numeral system in footpath at the bottom of described exit Become 7 kinds of different sizes of 2.0 mesh, 2.5 mesh, 3.0 mesh, 3.5 mesh, 4.0 mesh, 4.5 mesh and 5.0 mesh.

Acquired immune deficiency syndrome (AIDS) blood purification the most according to claim 1, it is characterised in that described antibody titer from high to low Layer distributed refers to from the mixed antibody titer of the import department of depurator to exit successively with 1: 700,1: 500,1: 300,1: 200,1: 100 point 5 layers.

Acquired immune deficiency syndrome (AIDS) blood purification the most according to claim 1, it is characterised in that described agarose concentration from low to high Layer distributed refer to from the agarose concentration of the import department of depurator to exit successively with 0.7%, 0.8%, 0.9%, 1.0%, 1.1% point 5 layers.

7. according to the arbitrary described acquired immune deficiency syndrome (AIDS) blood purification of claim 1,2,5, it is characterised in that described HIV antibody includes HIV-1gp120 antibody, HIV-1gp41 antibody.

Acquired immune deficiency syndrome (AIDS) blood purification the most according to claim 1, it is characterised in that described separator hollow-fiber film A diameter of 270～370 μm, film thickness is 50 μm, and aperture is 0.2～0.6 μm, and fibre length is 13.5～26 μm, can stop institute Some cells filter.

9. the preparation for the acquired immune deficiency syndrome (AIDS) blood purification cleanser of medical domain, it is characterised in that with sterile physiological salt Water cleans made hybridoma macrophage strain, and 1000r/min is centrifuged, and is centrifuged 5min with 1000r/min again, takes cell after cleaning Precipitation loads in the 200ml hydrostatic column that high-biocompatibility material is made, and is fills up to 4/5, makes cell column, separately will Gp120 and gp41 antibody mixes with goat-anti gp120 and gp41 antibody respectively, makes the goat-anti gp120 in mixed liquor and gp41 antibody All it is fully tied, but surplus free gp120 and the gp41 antibody having sufficiently high titre, then will be containing conjunction type and sequestered The mixed antibody of gp120 antibody and the mixed antibody containing conjunction type and sequestered gp41 antibody again with etc. titer mixing, It is made into final mixed antibody, prepares 5 kinds of gel mixed antibodies with the agarose C1-4B solution of gradient concentration respectively, make The titer of HIVgp120 and gp41 antibody is 1: 700,1: 500,1: 300,1: 200,1: 100 and the agarose concentration of correspondence depends on Secondary is 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, irrigates standby blood with equal equal-volume from low to high by antibody titer Daf molecule post so that it is in gel form antibody titer from high to low from top to bottom and agarose concentration from low to high Layer distributed.

10. the application in preparing purging in vitro device of the claim 1-8 arbitrary described acquired immune deficiency syndrome (AIDS) blood purification, its feature Being, described purging in vitro device includes that one end of arterial blood line pipe (1) separates with blood plasma with blood pump (3) through heparin pump (2) Device (4) is connected, and plasma separator (4) is through blood plasma pump (6) and circulation line (7) depurator (8) in parallel with two, depurator (9) it is connected, confluxes through circulation line (10), venous line (5) the most successively.