Specific embodiment
Fig. 1 is the application schematic diagram of the female tire blood group incompatibility haemolysis disease therapeutic apparatus proposed according to the present invention.
Fig. 2 is the internal structure chart of the plasma separator proposed according to the present invention.
Fig. 3 is the internal structure chart of the absorber 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) it is connected with plasma separator (4), plasma separator (4) absorption in parallel with two through blood plasma pump (6) and circulation line (7)Device (8), absorber (9) be connected, be then successively connected with circulation line (10), venous line (5), venous line (5) it is anotherEnd is connected with vein blood vessel.
In Fig. 2,1 is plasma separator, and 2 be plasma separator inner cavity, and 3 be the micropore on the tube wall of plasma separator inner cavity, 4Being cannot be by the haemocyte of micropore (3), and 5 be the small molecule blood plasma components that can pass through micropore (3), and 6 be plasma separator exocoel,7 be blood plasma outflux, and 8 be switchable valve.
In Fig. 3,1 is the Rh antibody moved down into after absorber, and 2 are fixed in the anti-Rh antibody in agar gel, and 3 areThe anti-Rh antibody being fixed in agar gel combines the secure bond object formed after the Rh antibody moved down, and 4 are fixed inRh positive ghost in agar gel, what 5 Rh positive blood shadow cell combinations being fixed in agar gel moved downThe secure bond object formed after Rh antibody, 6 be the agar gel containing micropore, and 7 be by the Rh antibody of agar gel micropore detention.
Below with reference to Fig. 1, Fig. 2 and Fig. 3, preparation to female tire blood group incompatibility haemolysis disease therapeutic apparatus proposed by the present invention and answerEmbodiment is explained in detail.
One, the preparation of the positive ghost of Rh (D)
1, Rh (D) positive red blood cell source: (1) from Zhejiang Province blood station, positive " O " type of the fresh concentration Rh (D) of purchase is red thinBorn of the same parents；(2) positive " O " the type erythrocyte of the discarded umbilical cord Rh (D) for taking newborn in vitro.
2, main agents: 30mmol/L PB buffer: solution A is 0.04mol/L Na2HPO4(Na2HPO4.12H2O14.328g is dissolved in 1000ml deionized water, and room temperature storage is spare)；Second liquid is 0.04mol/L NaH2PO4(NaH2PO4.2H2O 6.24g is dissolved in 1000ml deionized water, and room temperature storage is spare).Solution A 81.0ml and second liquid are taken respectively19.0ml mixing 40mmol/L PB, then on the basis of 40mmol/L PB buffer plus deionized water dilution 0.7530mmol/L PB buffer is become again, is made into 25 and 35mmol/L in proportion with method.Anti- D standard items and Rh (D) standard are redCell is purchased from Guangzhou Lian Tai Bioisystech Co., Ltd.
3, preparation method
(1) the positive O-shaped red blood cell of the fresh Rh of sufficient amount is taken, respectively under conditions of 4 DEG C and 37 DEG C, with 1500r/minThe centrifugal speed of × 5min is cleaned red blood cell 4~5 times with isometric physiological saline, may be adhered to removing erythrocyte surfaceAbo blood group and Rh blood group and other immune antibody or natural antibody.
(2) with 0.04mol/L Na2HPO4With 0.04mol/L NaH2PO4The osmotic concentration of preparation is 25 and 35mmol/LPH7.4 PB lysate, under 4 DEG C and 2500r/min × 10min of centrifugal condition, alternately and repeatedly Washed Red Blood Cells, pass throughThe alternating of osmotic concentration changes, and hemoglobin is promoted to give outside red blood cell and be removed.
(3) finally with physiological saline cleaning until supernatant is colourless, with the blood being harmful to the human body in red blood cell of dialysing completelyLactoferrin, precipitating are the ghost of the antigen containing D.
(4) detection of antigenicity: 1. by ghost and 37 DEG C of incubation 5min of anti-D standard items, the detection of centrifuging and taking supernatantAnti- D standard items potency reduces numerical value to determine the antigenicity of ghost.Specific method is: taking 10, test tube, respectively number 1~10, the 1st pipe is added ghost and precipitates 1.0ml, then other each pipes plus physiological saline 0.5ml inhale ghost from the 1st pipePrecipitating 0.5ml is added to the 2nd pipe, and 0.5ml to the 3rd is inhaled after mixing and is managed, and is diluted to the 10th pipe with same operation, finally pipe is sucked out0.5ml is discarded, and every pipe adds anti-D standard items 0.5ml, 37 DEG C of incubation 5min to mix gently after centrifugation, is aggregated completely with appearance, is severalMaximum dilution multiple without free cell represents the antigenicity of ghost, and extension rate is bigger, and antigenicity is stronger, adsorbs RhThe ability of antibody is stronger.Supernatant antibody titer can also be measured with Rh (D) normal erythrocytes to determine the antigen of ghostProperty；2. measuring supernatant antibody titer with Rh (D) normal erythrocytes, anti-D standard items potency (known) subtracts supernatant humoral antibody effectValence is the antigenicity (potency) of ghost.Supernatant antibody titer detection method are as follows: take 10 test tubes, respectively number 1~10, physiological saline 1ml is added in each pipe, takes supernatant 1ml to be added to removal 1ml after the 1st pipe mixes and goes to the 2nd pipe, equally to graspIt is diluted to the 10th pipe, finally pipe is sucked out 1ml liquid and discards, by the 40 μ l of serum diluted and 10 μ l of Rh (D) normal erythrocytesMixing is incubated for 10 minutes, is centrifuged 5 minutes interpretation results, the agglutination pipe of maximum dilution multiple is antigenic (potency), and potency is answered1: 1500 or more.
(5) content of hemoglobin detects: with hypotonic destruction ghost, with conventional chemical luminescence analysis or fluorescence analysisMethod, the qualitative content of hemoglobin in quantitative detection ghost should be lower than the reference value lower bound of human normal plasma.
(6) ghost form: observing the form and integrality of ghost under the microscope, should be in circle shadow, no-reflection.
(7) for cellular morphology in circle shadow, no-reflection, highly sensitive routine hemoglobin qualitative detection is feminine gender, quantitativeDetection is lower than the reference value lower bound of human normal plasma, adsorbs the ghost of 1: 1500 or more Rh antibody titer, leaves and takes spare.
Two, the preparation of anti-Rh antibody
1, Rh feminine gender lymphocyte is prepared
(1) primary cell obtains: the purchase fresh concentration Rh feminine gender leucocyte 300ml in Zhejiang Province blood station；Separately buy fresh RhPositive red blood cell suspension 300ml.(2) Rh feminine gender separation of lymphocytes prepares that (separating liquid is protected from light 4 degree of preservations, with preceding in 37 degree of waterHeated in bath, entire separation process temperature should be controlled in 8-28 degree, influence disintegrate-quality too high or too low for temperature): sterile pumpingThe fresh concentration Rh feminine gender leucocyte of 20mL is taken, PBS liquid dilutes 3~5 times, 6mL is taken slowly to be folded with dropper along tube wall after mixing wellHorizontal centrifugal in horizontal centrifuge (400r/min, 20 DEG C) is added in the 10mL centrifuge tube for be added 4mL lymphocyte separation medium35min；It is divided into 3 layers after centrifugation in pipe, upper layer is blood plasma and PBS liquid, and lower layer is mainly red blood cell and granulocyte, and middle layer is lymphCell separating liquid, having a white cloud and mist layer narrow band based on mononuclearcell in upper, middle layer interface is that single core is thinBorn of the same parents (PBMC) are inserted into cloud and mist layer with capillary syring, draw PBMC and are placed in another 50mL centrifuge tube, are added 5 times with upper volume PBSIt is centrifuged (300r/min, 20 DEG C) 10min, abandons supernatant, cell is resuspended in 50mLPBS, (350r/min, 20 DEG C) 15min is centrifuged, in abandoningClearly, Buffer (PBS+0.5% newborn bovine serum+2mmol/LEDTA pH7.2) 2mL is added, cell is resuspended, predominantly Rh is negativeLymphocyte (T cell and B cell).
2, Rh feminine gender sensitized lymphocyte is prepared
Rh feminine gender lymphocyte and Rh positive red blood cell suspension are centrifuged 5min with 1000r/min, remove supernatant, regular growthCulture solution washs 1~2 time, by two kinds of sedimentation cell mixed in equal amounts, adds the regular growth culture solutions (RPMI-1640) of 5 times of amounts, and 37DEG C, 5%CO2 is cultivated for 24 hours, is then washed centrifugation (1000r/min, 3min) with RPMI-1640 liquid and is added with removing cell debrisEnter Tris-NH4Cl effect 5min, splitting erythrocyte, Quick spin (1000r/min, 3min), remove supernatant in cracking it is redCell fragment, PBS washing centrifugation 3 times, RPMI-1640 washing centrifugation 1 time, to remove Tris-NH4Cl remaining in suspension,It is avoided to influence the survival of cell, at this point, mainly containing Rh feminine gender sensitized lymphocyte in suspension.
3, prepare the strain of Rh feminine gender sensitization B cell (Epstein-Barr virus converts Rh feminine gender sensitized lymphocyte)
Routinely Epstein-Barr virus Transformed Human Lymphocytes technology, takes Rh feminine gender sensitized lymphocyte, and adjusting concentration is 2x 106AfterwardsSuitable Epstein-Barr virus (EBV) stoste is added, is placed in 37 DEG C, 5%CO2 overnight incubation prepares the cause of Epstein-Barr virus conversion to be hybridizedQuick lymphocyte.(1) 20% fetal calf serum (Gibco) { 56 DEG C inactivate 30 minutes }, 1.6 RPM1640 culture medium: reagent: are included~1.8%HEPES, 1.2% glutamine, 1% penicillin and streptomysin；Cyclocyto enzyme A (CyA): 5ml (250mg)/bottle is used1640 culture mediums are diluted to 0.2mg/ml, when use final concentration of 2ug/ml (0.5%)；Epstein-Barr virus (EBV) liquid: purchased from Chinese sectionHeredity institute of institute, is stored in subzero 80 degree, and when use takes out from refrigerator, and 37 degree melt rapidly, with the membrane filtration of 0.22um,It does not exceed 0.5~1 hour.(2) method: taking Rh feminine gender sensitized lymphocyte, and 1640 full nutrient solution of 6ml is added to carry out secondWashing is centrifuged 1500 turns 15 minutes；Supernatant is abandoned, the full culture medium of 2ml 1640 is added and (before full culture medium is added, sets 37 degree of water-baths10 minutes), Ciclosporin A (4%) and 1.2mlEBV liquid/part is then added, 25 square centimeters of training is moved into after mixing wellBottle is supported, sets 37 degree, in 5%CO2 incubator；If cell increases, slow or cell density is low or medium pH value is in acidity, inhalesHalf amount culture solution out, carries out equivalent oil changing；It is transferred to when total amount reaches 14ml in 75ml culture bottle, every 2-3 weeks addition 5-10ml fresh culture；Culture medium (the 1.6%1M HEPES buffer solution of 3ml Fresh is added within every 2-3 days；15% tire ox blood(FBS) clearly；1% penicillin and streptomysin；PRMI 1640 is supplied to 100%).7 days or so under the microscope, it is seen that lymphocyteIt significantly increases, clustering phenomena occurs；It about 6~8 weeks, when total amount reaches 45ml, sets in 50ml centrifuge tube, 1500 turns of centrifugation, 10Minute, supernatant is abandoned, is made into that cell suspension is spare, and predominantly Rh feminine gender sensitization B cell strain [if to freeze, is added 3ml and freezesCulture medium (5% dimethyl sub-maple (dimethyl sufoxide), 95%FBS) mixes, and at cell suspending liquid, (cell concentration is aboutIt is 105/ml).Cryopreservation tube packing, 1ml/ pipe, sets -20 DEG C of 2h, then set -70 DEG C of 2h, then freeze in -196 DEG C of liquid nitrogen (or it is verticalMoved into liquid nitrogen container after 80 degree, 1-2 hours i.e. under zero setting)].
4, screen the strain of Rh feminine gender effect B cell (Epstein-Barr virus that screening produces Rh antibody converts Rh feminine gender sensitized lymphocyte)
Effect B cell strain refers to the B cell strain that can generate Rh antibody, with antihuman globulin test or conventional ELISA method sieveWhether choosing produces Rh antibody lymphocyte: (1) Direct antiglobulin test: expressing on detection bone-marrow-derived lymphocyte strain (B cell strain) surfaceRh antibody.Anti-humanglobulin serum and anti-D serum are bought, with brine 1 time and is made into 5% lymphocyte strain suspension,It takes 1 drop cell suspension and 2 to drip anti-humanglobulin serum mixing, sets low-speed centrifugal after room temperature 5min, gently mix, under naked eyes or mirrorObservation, discovery lymphocyte strain agglutination person are that cell surface expression has Rh antibody (while it is outstanding to prepare unsensitized 5% lymphocyteLiquid adds anti-humanglobulin serum to do negative control；Anti- D serum is added to do positive control with Rh (D) positive red blood cell).(2) indirectly anti-Human immunoglobulin test: whether detection lymphocyte strain culture supernatant contains Rh antibody.With brine 2 times and it is made intoThe positive O-shaped red cell suspension of 5%Rh (D), respectively takes red cell suspension, lymphocyte strain culture supernatant and anti-humanglobulin serum1 drop mixes, and sets low-speed centrifugal after room temperature 5min, gently mixes, and naked eyes or microscopic observation, discovery erythrocyte agglutination person are that lymph is thinBorn of the same parents' strain culture supernatant contain Rh antibody (while using red cell suspension and anti-humanglobulin serum mixing tube as negative control, withRed cell suspension and anti-D serum mixing tube are positive control).Being filtered out with this direct or indirect antihuman globulin test can divideMore efficient valence (compared with the positive is still tested after high magnification numbe dilution) B cell strain (hole) of Rh antibody is secreted, after resuming for 2~3 generations.
5, Rh feminine gender effect hybridoma (preparation Rh feminine gender effect B cell strain of hybridoma) is prepared
(1) culture medium and main agents: DMEM culture medium, HAT, HT Selective agar medium are purchased from Sigma company, top grade tire oxSerum (FBS) purchases Jinshi City on daytime ocean Hao biological products science and technology responsibility Co., Ltd；DMSO (-- methyl sulfoxide) it is that domestic analysis is pureReagent.(2) myeloma cell prepares: fusion the last week takes out the myeloma cell (SP2/0) that a pipe freezes out of liquid nitrogen container, standsBe put into hot water thaw (using it is most be Sp2/0 cell strain, the cell strain growth and fusion efficiencies are good, itself is not secretedThe highest growth scale of any heavy chain immunoglobulin or light chain, cell is 9 × 105/ ml, doubling time are usually 10~15h；Selection homologous cell strain is considered in practical application relevant to human body, if Shanghai Fu Xiang Biotechnology Co., Ltd is to ATCC cellThe NCI-H929 human myeloma cell strain that library is introduced).Appropriate complete culture solution is added after thawing, 1000r/m is centrifuged 3min；It repeats1 time.Sediment is moved into Tissue Culture Flask, adds DMEM culture solution, sets CO2 incubator culture, once passed within 3~4 daysOr expanding culture, fusion adjusts cell state in first 24 hours, guarantee that cellular morphology is good before merging, growth is vigorous.It is added suitableBasal medium is measured into centrifuge tube, 1000r/min centrifugation 5-10min after mixing is gently beaten, washes repeatedly cell 2 times.(3)Rh feminine gender effect B cell strain to be hybridized prepares: adjusting total cell number to 1 × 10 with basal medium8~2 × 108Melt for cellIt closes, blue dyeing phase-contrast microscopy is expected with platform, viable count should be higher than that 80% is qualified.(4) cell fusion: by Rh feminine genderThe strain of effect B cell and myeloma cell are added in centrifuge tube with 5~10: 1 ratio, are mixed evenly, 1000r/min centrifugation5min is discarded supernatant, and is gently beaten tube bottom to cell grainless and is precipitated, is repeated 2 times.The gently rotation preheating in 37 DEG C of water-bathsThe 50%PEG3000 of 1000 μ L of preheating is added drop-wise in fusion pipe in 60s along tube wall under aseptic condition by centrifuge tube after taking-upGently rotating centrifugal pipe simultaneously, is also added drop-wise to centrifugation along tube wall in 3~5min for the basal medium of the 25mL of preheating laterGuan Zhong, lightly rotating centrifugal pipe during addition are then allowed to stand in 37 DEG C of water-bath 10min, 1000r/min centrifugations5min is discarded supernatant, and 50mL HAT culture medium is added.It is appropriate mix after be inoculated into 96 well culture plates, be placed in 37 DEG C, 5%It is cultivated in CO2 incubator.(5) hybridoma is cultivated: cell growth status in 96 well culture plates of observation, after 7~10 days onlyHybridoma can grow division, discard HAT culture medium at this time, replace complete medium.Cell clone growth area reachesWhen 1/10 cell hole, culture supernatant is gone, the culture hole of the good hybridoma of growth conditions is selected, is marked under microscope thinCell clone is drawn to the complete medium that has newly in the position of mark using sterile pipette tips in the position of born of the same parents' strain growth, sizeIn culture hole, then successively doubling dilution to hole is counted below, and 37 DEG C, 5%CO2 incubator is interior to be cultivated one week or so, under microscopeCell growth status is observed, when cell clone is covered with to 1/10 or more hole floor space, cell or culture supernatant is taken to detect hybridizationOncocyte function.(6) screen Rh feminine gender effect hybridoma: Rh feminine gender effect hybridoma, which refers to, can generate Rh antibodyHybridoma, method produce the effect B cell strain of Rh antibody with screening, filter out energy with direct or indirect antihuman globulin testThe Rh feminine gender effect hybridoma (clone) for secreting more efficient valence Rh antibody repeats next round dilution culture, repeats 2-3Wheel, detection function are taken out after stablizing, are transferred to culture bottle mass propgation and [are such as intended to freeze and recover, then 12 hour adjustment before preservationCell growth state takes one bottle of growth vigorous, the good cell of form, is made cell suspension after appropriate digestion, 1000r/min fromHeart 5min, removes supernatant, and flicking tube bottom keeps cell loose, and the 9 parts of complete culture solutions and 1 part of DMSO of 4 DEG C of preservations are added, and packing is thinCryopreservation tube is put into liquid nitrogen container after taking-up and saves backup by born of the same parents' cryopreservation tube, 1mL/ pipe, -70 DEG C of refrigerator overnights.Prepare before recovery40 DEG C or so of hot water carefully takes out cryopreservation tube from liquid nitrogen, and being immediately placed in hot water uniformly to shake makes cell thaw, and solvesIt is centrifuged 5min in 1000r/min after jelly, opens cryopreservation tube under aseptic condition in superclean bench, by the cell after defrosting with completelyCulture solution washed once, and is then centrifuged 5min in 1000r/min, discards supernatant, in case making to expand culture].(7) amplification Rh is negativeEffect hybridoma: it moves into culture bottle, sets after Rh feminine gender effect hybridoma is gently resuspended using complete culture solutionIt 37 DEG C, cultivates in 5%CO2 incubator.Pass on amplification cultivation repeatedly according to a conventional method.
6, Rh antibody is prepared
(1) it separated in conventional manner from Rh feminine gender effect Hybridoma Cell Culture supernatant, purify Rh antibody.(2)RhThe preparation of antibody MAb mouse ascites: low-speed centrifugal collects the Rh feminine gender effect hybridoma after culture, cultivates by basisBase diluting cells number is 1 × 107/ mL, mouse peritoneal inject 0.2mL/ only, mouse ascites production are observed after injection, to abdomenThe obvious distension in portion rises, and punctures abdominal cavity with syringe needle, and centrifuge tube collects ascites, and acquisition finishes, and injects appropriate basis culture to mouse peritonealBase is spaced 2~3 days, and same method takes ascites again, the ascites being collected into, and 10000r/min is centrifuged 5min, and Aspirate supernatant dividesDress, -20 DEG C of preservations.Contain a large amount of Rh antibody in supernatant, separates according to a conventional method, purifies Rh antibody, with antihuman globulinThe potency of test or ELISA method measurement antibody.
7, anti-Rh antibody is prepared
(1) experimental animal: immune is selected with experimental animal is the white goat of animal institute of Zhejiang University hybridization, 30 jin of weightThe between twenty and fifty ewe two of health of left and right, is smeared at the back of animal with dyestuff before immune, makes specific label, using doing as everybody else doesThe feeding manner of stable breeding guarantees that sheep has to suitable movement daily, and it is left to move general half an hour at dusk every time for run durationThe right side, is conducive to healthy and strong in this way, avoids sheep overfertilization, increases the immunity of body, and drinking-water is sufficient, and give suitable fine fodder,Green grass, corn, wheat bran, wheat, vitamin etc. keep its nutrition balanced.Often check experiment sheep health status.(2) it is immunizedSource: mixing 0.1mL antigen before Rh antibody (Rh antibody, that is, anti-D serum, commercially available product) prepared by the present invention inoculation, 1.9mL withoutIt is spare that immunogen emulsion is made in bacterium PBS, 2mL Freund adjuvant completely (or not exclusively).(3) goat is immune: two goats are chosen,Labeled as goat A, goat B, antigen inoculation.Immune position is front and back groin, and 2 points of every place's groin point are injected, a total of 8A injection point, injection system are subcutaneous injection, and every goat per injection 4mL immunogene contains 250 μ g antigens；Each injection pointInjecting immune original 0.5mL, containing about 32 μ g antigens.Preceding two goats are immunized to draw blood respectively 10mL, are labeled as 0dP1, -20 DEG C of preservations；Draw blood 10mL after 7 days immune, separates serum, is labeled as 7dP1, detects serum titer with ELISA, and -20 DEG C of remaining serum save, and 3Start within~4 weeks to be immunized for second, immunogene is the sterile PBS+2mL incomplete Freund's adjuvant (IFA) of 0.1mL antigen+1.9mL, is exempted fromEpidemic disease is drawn blood 10mL after 7 days, separates serum, is labeled as 7dP2, and ELISA detects serum titer, -20 DEG C of remaining serum preservations.ThirdSecondary immunization time is after 6~8 weeks, and immunogene is the sterile PBS+2mL incomplete Freund's adjuvant (IFA) of 0.1mL antigen+1.9mL, is exempted fromEpidemic disease is drawn blood 10mL after 7 days, separates serum, is labeled as 7dP3, and ELISA detects serum titer, -20 DEG C of remaining serum preservations, ifSerum titer does not reach 1: 10 at this time6More than, then it needs to be immunized again primary；If serum titer is up to 106Do not have to then exempt from again aboveEpidemic disease, draw blood 50mL every other week later, separates serum, -20 DEG C save backup.(4) anti-Rh antibody serum preparation: generally exempt from every timeIt can be detected in sheep jugular vein blood collection within 7~10 days after epidemic disease, by assistant Baoding animal, keep standing position it, neck is cutAfter hair, sterile cotton balls cleaning disinfection, the blood sampling of jugular vein hand syringes is searched out, the fixation of syringe position is taken into 5~10mL of blood.Bioactivity is carried out after isolating serum.7~10 days after the third immunization, it once can use blood 30- after bioactivity is qualified50mL aseptically separates serum, after the blood clotting in plate or triangular flask to be collected in, with sterile dropper in nothingAfter clot and bottle wall removing in collarium border (such as superclean bench), 37 DEG C are put into, 1~2h is put into 4 DEG C overnight after taking out, makeSerum is sufficiently precipitated and (cannot freeze, otherwise generate haemolysis), separates serum through centrifugation, puts low temperature refrigerator preservation into, usesBefore must dispense again and save backup after signing is qualified.(5) measurement of antiserum titre: antiserum titre is measured using ELISAMethod, when coating are after sample is diluted to 1: 1000 concentration with coating buffer, and every hole adds 100 μ L on ELISA Plate, then putsIt is put into aluminium box in 4 DEG C of refrigerators overnight, the next morning, which takes out, pats dry coating buffer, is washed three times with PBST, every minor tickFive minutes, ELISA Plate is patted dry with gauze for the last time, the confining liquid of 10% serum is added, every hole adds I00UL, is put into 37 DEG C, water1~2h is bathed, then takes out again and pats dry confining liquid, wash 3 every minor tick of ELISA Plate five minutes with PBST, use gauze for the last timeIt pats dry, 100 hole μ L/ primary antibodies (1: 2000 dilution is diluted with the PBST of 4% cow's serum, is put into 37 DEG C, 1~2h of water-bath) is added, soIt takes out again afterwards and pats dry primary antibody liquid, PBST washs ELISA Plate three times, and every minor tick 5 minutes pats dry enzyme mark with gauze for the last timePlate is added secondary antibody liquid (rabbit-anti sheep 1: 1000), and every hole adds 100 μ L, is put into 37 DEG C, 1~2h of water-bath, then take out and pat dry secondary antibody liquid3 every minor tick of ELISA Plate is washed five minutes with PBST, is patted dry for the last time with gauze, every hole adds 50 μ L substrate developing solutions, black outColour developing 10~after twenty minutes be added 2M 50 μ L of H2SO4 solution terminate reaction, after with microplate reader survey OD value (in half an hour).Or withPotency of the antihuman globulin test result positive of maximum dilution multiple as antibody.
The Rh antibody prepared by the present invention, that is, anti-D of Ig, anti-Rh antibody, that is, anti-Ig anti-D are same by technical solution of the present inventionIg anti-A, Ig anti-B, Ig anti-E, Ig anti-C and the anti-A of anti-Ig, the anti-B of anti-Ig, the anti-E of anti-Ig, the anti-C of anti-Ig can be prepared, goes forward side by side oneStep preparation immunoadsorption therapy device.The present invention is after preparing Rh antibody, when carrying out the preparation of anti-Rh antibody, except through immuneGoat preparation is outer, can also then take the immune spleen cell of mouse and myeloma cell fused, then by normal by the way that mouse is immunizedRule hybridoma technology prepares anti-Rh antibody.
Three, the preparation of adsorbent
1, the preparation of the adsorbent containing anti-Rh antibody
The prepared anti-Rh antibody for playing absorption Rh antibody is taken, antibody titer is detected, respectively after 100 DEG C dissolve0.9%, 1.0%, 1.1%, 1.2%, the 1.3% agarose C1-4B physiological saline kept the temperature at 37~42 DEG C is made into 1: 700,1:500, the application antibody of 1: 300,1: 200,1: 100 potency gradient, keeps the temperature spare at 37~42 DEG C.
2, the preparation of the adsorbent simultaneously containing anti-Rh antibody and Rh positive ghost
By the prepared ghost for playing absorption Rh antibody, be respectively 1: 700 with anti-Rh antibody titer, 1: 500,1: 300,1: 200,1: 100 adsorbent is configured to contain anti-Rh antibody and Rh sun while 95%Rh positive blood shadow cell concentrationProperty ghost adsorbent, keep the temperature it is spare at 37~42 DEG C.
Four, the preparation of absorber
1, material
For the acrylate close with Human vascular endothelial etc high molecular material, it is desirable that good biocompatibility, no complementActivation and inflammatory reaction, can the change without leucocyte, blood platelet, blood oxygen pressure, complement C 3 C5a, pass through covalently, grafting, polymerizationThe methods of improve uniformity, the hydrophily of material surface, reduce influence to blood coagulation and oxidative stress.Add in absorber inner surfaceHydrophilic gel generates CA/PMB30, CA/PMB80 and CA/PMB30-80 by controlling wet-spinning procedure, biofacies can be improvedCapacitive.Certain anticoagulant substances are solidificated in carrier or absorber inner surface, blood clotting is can inhibit, reduces heparin dosage even realityHeparin, is such as aggregated on polyacrylonitrile-polyethyleneimine film, can reduce the allergic reaction of allergic constitution by existing no-rod tractor；It willHeparin covalent is integrated to polyether sulfone surface, can keep the mechanical property of polyether sulfone and improve the anticoagulant property of absorber inner surfaceEnergy.The covalent immobilisation linoleic acid film on cellulose acetate film, or the linoleic acid for being covalently bound to polyacrylic acid is grafted onto PS membraneSurface can have better histocompatbility and anticoagulant effect.
2, specification
The hydrostatic column that the bottom diameter for being made 50mm × 60mm is small, top diameter is big, or rectangular, infundibulate is made, volume about 200~300ml, top and the bottom are equipped with cell screen clothes, and top diameter sieve mesh number is 500 mesh, and bottom diameter sieve mesh number is 50 mesh, liquid outletPlace's setting mesh number is 200 aim cell strainers, constitutes the second defence line for preventing cell fragment from entering circulation, liquid entranceIt is equipped with buffer area between mesh screen, is conducive to the stability of system circulation.
3, method
55~65ml is successively taken to contain the suction of anti-Rh antibody and Rh positive ghost simultaneously from low to high by antibody titerIn attached dose of addition hydrostatic column, it is cooled to the adsorbent being first added after semi-solid gel and just then adds next time, being made makesGel in container forms antibody titer from high to low and agarose concentration and blood from low to high from sample introduction end to sample outlet endThe equally distributed absorber of shadow cell, when the blood plasma separated in extracorporal circulatory system filters out absorber, Rh antibody is fixed on absorptionGhost and anti-Rh antibody in agent are combined into fixed compound, the red cell debris that is destroyed and it is in combination made ofMacromolecular immune complex by nearly absorber outlet end concentration is gradually high and molecular sieve gradually small special agar gel detention, removal causeThe blood plasma of disease substance is fed back in vivo after filtering out from absorber, is avoided pregnant woman's Rh antibody to enter fetal blood to reach and is mitigated diseaseThe therapeutic purposes of feelings.
Five, the preparation of blood separator
1, preparation principle: according to the size of visible component in blood of human body (cell): normocyte is about 7 microns of (μIt m), is the discoid cell of concave-concave；Leucocyte is divided into 5 kinds, and about 12 μm of neutrophil leucocyte, eosinophil is more bigger, basophilicProperty granulocyte and neutrophil leucocyte it is close, 6-8 μm of small lymphocyte, approximate with red blood cell, monocyte maximum, about 15-20 μm.Blood platelet is disc, and 1~4 micron to 7~8 microns of diameter is differed, and the platelet mean diameter of people is 2-4 microns, thickness 0.5~1.5 micron.
2, it the material of blood separator and requirement: with absorber of the invention, selects poly-vinegar non-woven fabrics, acetate fiber, take offRouge cotton etc., it is desirable that good biocompatibility, permeability height, hardly activating complement do not cause inflammatory reaction and leucocyte, blood smallThe change of plate, blood oxygen pressure, C3a, C5a.
3, the type and spec of blood separator: plasma separator according to the present invention can be bought from businessman, or committeeHold in the palm businessman's preparation.Hollow fibre type filter made of plasma separator high molecular polymer, the hole can permit blood plasma filtration, butIt can stop all cell components.Hollow-fiber film diameter is 270~370 μm, and film thickness is 50 μm, and aperture is 0.2~0.6 μM, fibre length are 13.5~26 μm.
Six, the component of female tire blood group incompatibility haemolysis disease therapeutic apparatus
1, washed corpuscles and blood plasma blood separator: key member: (1) are used for；(2) plasorber: anti-Rh is includedAntibody, Rh positive ghost and agar gel medium are formed for removing Rh antibody, RBC fragment, Rh antibody and RBC fragmentCompound etc..
2, additional member: including blood pump, heparin pump, sound pulse pressure and air monitering, temperature control system, off gas system,The parts such as monitored conductivity system, ultrafiltration monitoring and leakage blood monitoring form.(1) blood pump (Blood Pump): for pushing bloodCirculation is to maintain going on smoothly for Blood index treatment, and usual blood pump part often has rotary test speed function, to monitor patientBlood circumstance, therefore blood pump runner and the setting of groove spacing are accurate, and need often adjustment, according to the feelings of bloody path pump lineSpacing is generally set as 3.2~3.3mm by condition, can not be too loose, and it is inaccurate otherwise to will cause blood flow detection；Also can not be too tight, otherwiseIt will cause pipe breakage.(2) heparin pump (Heparin Pump): heparin pump is equivalent to the micro-injection pump clinically applied, and usesIt to continue the injecting heparin in sieving pipeline (patient blood), contacts, is easy with air since the blood of patient recycles in vitroBlood coagulation phenomenon occurs, anticoagulative can be occurred using heparin pump.(3) sound pulse pressure monitor: arterial blood pressure monitoring mainly toThe stopping state of dynamic monitoring blood separator micropore, in addition to monitor extracorporal circulatory system thrombus, solidification and the variation of pressure.WhenWhen blood flow deficiency, angiosthenia will be reduced；When having blood coagulation, thrombosis, especially separator blockage of the micro orifice, angiosthenia willIt increases；Vein pressure monitoring is used to monitor the pressure of pipeline blood reflux, when separator blockage of the micro orifice, blood coagulation, thrombosis, blood flowWhen insufficient and venous return syringe needle falls off, vein pressure will decline, if bloody path return pipe distortion blocking or reflux syringe needleWhen blocking, vein pressure will be increased.(4) air monitering (Air Detector): the air gas for monitoring blood pathwayBubble, the general principle for using ultrasonic listening, in order to avoid air embolism occurs for patient and is arranged.When having monitored air bubbleWhen, detection system can drive artery and vein bloody path folder to carry out blocking blood flow, prevent dangerous generation.
In short, Import computer regulates and controls and is made the people of operation on the basis of key member of the present invention and additional memberProperty, the personalization for the treatment of, the safety of design and modularization, automatic monitoring and regulation, liquid crystal display, voluntarily judgement is warnedReport the therapeutic equipment of the micro computers such as reason and ring off signal processing.
Seven, the operating method of female tire blood group incompatibility haemolysis disease therapeutic apparatus
1, it installs: with each portions such as sterile working connecting components, including plasma separator, absorber and each circulation line.
2, it is vented: with sterile saline filling liquid separator, absorber and each circulation line, excluding separator, absorberAnd its gas, bubble in circulating line, it goes through, confirmation after gas, bubble without using.
3, lead to liquid: arterial blood line pipe 1 being connected into arteries, goes through again be vented whether complete, liquid in operationWhether stream is unobstructed, and flow liquid in pipe is avoided to pollute.
4, anticoagulant: to be injected from heparin pump into liquid stream anti-coagulants (heparin), be for the first time 2500 ∪ or 20~30 ∪/kg.
5, start: arterial blood line pipe (1) is connected to the arteries of curer, venous line (5) are connected curer'sVein blood vessel, then opens blood pump, and blood flow is 100~150ml/min, in Fig. 1, when arterial blood is through arterial blood line pipe(1) enter plasma separator (4), the blood plasma separated reaches absorber through circulation line (7) under the action of blood plasma pump (6)(8), wait be full of blood plasma, about 10 minutes, blood plasma is begun releasing, is flowed out through circulation line (10), it is synchronous that blood is perfused to absorber (9)Slurry, when the blood plasma in absorber (8) has nearly flowed, starts again at perfusion blood plasma, and absorber (9) begins releasing blood plasma at this time,Two absorbers (8) in parallel, absorbers (9) are alternately.In Fig. 2, when blood to be separated enters plasma separator (1)When inner cavity (2), the effect through valve (8) can enter the outer of separator by the small molecule blood plasma and its composition (5) of micropore (3)Chamber (6) is then flowed out through plasma outlet port (7), and cannot be flowed out by the haemocyte (4) of micropore (3) through valve (8).In Fig. 3,When Rh antibody (1) enters absorber, the anti-Rh antibody (2) being fixed in agar gel respectively is combined into fixed compound(3), the Rh positive ghost (4) being fixed in agar gel is combined into fixed compound (5), and the Rh not being combined is anti-Body (1) is delayed at the smaller agar gel of micropore (7) during downlink.
Eight, the verifying of therapeutic equipment effect
1, the verifying of ghost absorption Rh antibody efficacy
The effect of to understand therapeutic equipment, the present invention devise easy test method test ghost absorption Rh antibodyEffect.Prepared ghost is taken, is added to and keeps the temperature after 100 DEG C dissolve in 37~42 DEG C of 1.0% spare agarose C1-In 4B, it is configured to the adsorbent of 95% ghost concentration；2.5 × 300mm Westergren's blood sedimentation tube 9 of sterilizing are taken, draw 95%The adsorbent of ghost concentration becomes semisolid after adsorbent is cooling to 200mm scale；Shopping center's blood station Fresh Frozen blood200mL is starched, it is another to buy Rh antibody (the anti-D type serum dry powder standard items of people, Guangzhou Lian Tai Bioisystech Co., Ltd), with fresh bloodSlurry is made into 1: 200,1: 300,1: 600 antibody titer, routinely Rh (anti-D) titre detection method (reference book), furtherDetection confirms whether above-mentioned prepared antibody titer is consistent, (Rh) antibody (titre) before referred to as filtering, before then respectively taking 10ml to filterAntibody is injected separately into the upper end blank pipe of 3 blood sedimentation tubes, after flowing through the adsorbent containing ghost of blood sedimentation tube lower layer, collectsEfflux, (Rh) antibody after referred to as filtering, routinely Rh (anti-D) titre detection method confirms titre, then respectively passes through antibody after filterMake to filter out for the 2nd time in adsorbent containing ghost, is so repeated 3 times filtration and antibody titer detection, as a result (table 1) is saidBright, after Rh antibody filters simple absorber, most of Rh antibody is adsorbed by the adsorbent accordingly containing ghost, through theAfter 1 time, the 2nd time, the 3rd time filters, the average titer of Rh antibody is reduced to 1: 183,1: 58,1 after filtering from 1: 367 before filter respectively: 29, illustrate the increase with filtration number, Rh antibody can be adsorbed constantly by ghost and be removed, so that reaching reduces pregnant woman(newborn) blood plasma Rh antibody titer and the purpose for treating female tire blood group incompatibility Hemolysis.
Titre testing result (1/x) before and after adsorbent of 1 Rh antibody of the table filtration containing ghost
2, the verifying of anti-Rh antibody absorption Rh antibody efficacy
The effect of to understand therapeutic equipment, the present invention devise easy test method and test anti-Rh antibody absorption Rh antibodyEffect.Prepared anti-Rh antibody is taken, is added to and keeps the temperature after 100 DEG C dissolve in 37~42 DEG C of 1.0% spare agarose C1-In 4B, titre is 1: 300~500 after mixing；2.5 × 300mm Westergren's blood sedimentation tube 9 of sterilizing are taken, draw 1.0% agar respectivelySugared C1-4B solution is to 200mm scale, and agarose becomes semisolid after cooling；Shopping center blood station fresh frozen plasma 200mL,Another purchase Rh antibody (the anti-D type serum dry powder standard items of people, Guangzhou Lian Tai Bioisystech Co., Ltd), is matched with fresh frozen plasmaAt 1: 128,1: 256,1: 512 antibody titer, routinely RH (anti-D) titre detection method (reference book), detection confirmation are anti-Body titre, (Rh) antibody before referred to as filtering, antibody is injected separately into the upper end blank pipe of 3 blood sedimentation tubes before then respectively taking 10ml to filter, wait flowAfter outflow, efflux is collected through the 1.0% agarose C1-4B containing anti-Rh antibody of blood sedimentation tube lower layer and out of blood sedimentation tube, is claimedFor (Rh) antibody after filter, routinely RH (anti-D) titre detection method confirms titre, then passes through antibody after filter resist containing anti-Rh respectively1.0% agarose blood sedimentation tube of body filters out, and is so repeated 3 times filtration and antibody titer detection, as a result (table 1) illustrates, Rh antibodyAfter filtering simple adsorbent, part Rh antibody is adsorbed by corresponding anti-Rh antibody, after the 1st time, the 2nd time, the 3rd filtration,The average titer of Rh antibody be reduced to filter from 1: 298 before filter respectively after 1: 149,1: 48,1: 17, illustrate with filtering numberIncrease, Rh antibody can be removed constantly, to reach the mesh for reducing Rh antibody titer and treating female tire blood group incompatibility Hemolysis's.
Titre testing result (1/ before and after 1.0% agarose simple adsorbent of 1 Rh antibody of the table filtration containing anti-Rh antibodyx)
In short, above-mentioned easy confirmatory experiment shows the Rh antibody to dissociate in blood plasma, it can be by adsorbent (blood of the inventionShadow cell and anti-Rh antibody) absorption removes, it was demonstrated that and the female tire blood group constituted using blood separator and absorber as critical component is notClosing haemolysis disease therapeutic apparatus has significant therapeutic efficiency.