CN101957364A - Preparation method of lyophilization type platelet antigen spectrum cells - Google Patents
Preparation method of lyophilization type platelet antigen spectrum cells Download PDFInfo
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- CN101957364A CN101957364A CN2010102856085A CN201010285608A CN101957364A CN 101957364 A CN101957364 A CN 101957364A CN 2010102856085 A CN2010102856085 A CN 2010102856085A CN 201010285608 A CN201010285608 A CN 201010285608A CN 101957364 A CN101957364 A CN 101957364A
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Abstract
The invention discloses a preparation method of lyophilization type platelet antigen spectrum cells, belonging to the technical field of biology. The invention aims at solving the problems of clinical deficiency of platelet antigen spectrum cells, easy antibody inspection omission and unavailability of confirming antibody specificity and building an HPA-(1-6),15 genotyping platelet antigen spectrum cell system, and can be widely applied to platelet antigen detection and specificity identification. The preparation method comprises the steps of: a, detecting genotyping of the platelet antigen HPA-(1-6) and HPA-15 system; b, building a platelet antigen spectrum cell system; c, preparing a platelet antigen suspension; d, loading; and e, freezing and drying. The key of the invention is the detection of HPA-(1-6),15 of O-type blood donators and the building of a platelet antigen spectrum of a genotyping result. The platelet antigen spectrum cells of the HPA genotyping are prepared into a lyophilization product with stable antigenicity through freezing and drying, can be used for determining the antibody specificity during platelet antibody detection, meanwhile, the problem that the platelet antigen cannot be stably stored is solved.
Description
Technical field
The invention belongs to biological technical field.
Background technology
Platelet antibody is produced by allosome or self platelet antigen effect body, comprise alloantibody, autoantibody, allo-antibody, DDA and non-specific antibody five classes, these antibody act on human body platelet can be caused blood platelet generation destruction and cause the female alloimmunity thrombopenia (FMAIT) of tire, the invalid disease of blood platelet infusion (PTR), post-transfusion purpura multiple blood platelet immunological diseases such as (PTP).
Accurately, quick, easy detection and identify platelet antibody aspects such as the diagnosis of blood platelet immunity disease, treatment are played an important role.Yet, still lacking standardized platelet antigen spectrum cell at present in the world and use for routine clinical detection, main cause is that its antigenicity very easily takes place to assemble and lose under liquid environment blood platelet.The platelet antigen of preparation voluntarily can only be adopted in each laboratory when detecting platelet antibody at present, because do not know hematoblastic antigen, causes this result who detects in each laboratory of same increment to be difficult to consistent.The blood platelet of selecting for use at random in addition carries out very easily causing when platelet antibody detects the omission of platelet antibody, detection that leads to errors and diagnostic result.
Summary of the invention
The objective of the invention is in order to solve clinical shortage platelet antigen spectrum cell, be prone to the antibody omission and can not determine the problem of antibody specificity, set up HPA-1 ~ 6, the platelet antigen of 15 Genotypings is composed clone, can be widely used in the preparation method of the detection of platelet antibody and the freeze-dried type platelet antigen spectrum cell that specificity is identified.
Step of the present invention is:
The detection of a, platelet antigen HPA-1 ~ 6 and HPA-15 system Genotyping
Gather blood donor EDTA anticoagulated whole blood, extract DNA by salting out method, its purity should be more than 1.6; Blood donor's sample is carried out HPA-1 ~ 6,15 systems carry out Genotyping, amplification condition is as follows: behind 95 ℃ of pre-sex change 5min, and 95 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 50s, 30 circulations, 72 ℃ are extended 5min;
B, establishment platelet antigen spectrum clone
Platelet antigen spectrum cell is set up the spectrotype of 10 groups of HPA-1 ~ 6,15 systems according to O type blood donor's platelet antigen somatotype result, and its antigens genotyping sees Table 1;
The preparation of c, platelet suspension: gather the good fresh EDTA anticoagulated whole blood of 10 blood donors of screening, the centrifugal 30min of 900rpm; Get the upper strata platelet rich plasma, the centrifugal 30min of 3000rpm; Abandon blood plasma, with the frozen-dried protective liquid hematocrit blood platelet that fully suspends, and to regulate PC be 100 ~ 150 * 10
9/ L;
D, load: platelet suspension is placed 37 ℃ of constant temperature oven load 4h, during every 30min vibration mixing once;
E, freeze drying: after the platelet suspension packing after the load, place freeze dryer to carry out freeze drying; Its lyophilisation condition sees Table 2.
Frozen-dried protective liquid of the present invention comprises 0.2M pH7.0 PBS, contains the trehalose of 30 ~ 50 mmol/L and 1 ~ 3% bovine serum albumin(BSA) among the 0.2M pH7.0 PBS.
The present invention with freeze-dried type platelet antigen spectrum cell with physiological saline solution after, be used for carrying out platelet antibody and detect and the specificity identification experiment.
Advantage of the present invention: the blood platelet with the O type blood blood donor of Genotyping is prepared into stable platelet antigen spectrum cell dried frozen aquatic products, provide antigen with long preservation period for clinical, prevent clinical omission platelet antibody, also make platelet antibody detection and evaluation be easy to routinize standardization.
Key of the present invention is to detect O type blood donor HPA-1 ~ 6,15 Genotypings, sets up the platelet antigen spectrum according to the result of somatotype.The platelet antigen spectrum cell of HPA Genotyping is prepared into the stable dried frozen aquatic products of antigenicity through freeze drying, can determine the specificity of antibody when platelet antibody detects, and has solved platelet antigen simultaneously and has been difficult to a stable difficult problem of preserving.
Description of drawings
Fig. 1 is solid phase agglutination reaction pattern diagram----------positive of the present invention;
Fig. 2 is solid phase agglutination reaction pattern diagram----------weak positive of the present invention;
Fig. 3 is solid phase agglutination reaction pattern diagram----------feminine gender of the present invention.
Embodiment
Step of the present invention is:
The detection of a, platelet antigen HPA-1 ~ 6 and HPA-15 system Genotyping
Gather blood donor EDTA anticoagulated whole blood, extract DNA by salting out method, its purity should be more than 1.6; Blood donor's sample is carried out HPA-1 ~ 6,15 systems carry out Genotyping, amplification condition is as follows: behind 95 ℃ of pre-sex change 5min, and 95 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 50s, 30 circulations, 72 ℃ are extended 5min; Observe and the record result with gel imaging system behind the electrophoresis.
B, establishment platelet antigen spectrum clone
HPA is a specific antigen on the blood platelet, has polymorphism.When platelet transfusion, gestation, transplanting, easily cause and produce corresponding antibodies and cause relevant blood platelet correlation immunity disease.
Platelet antigen spectrum cell is set up the spectrotype of 10 groups of HPA-1 ~ 6,15 systems according to O type blood donor's platelet antigen somatotype result, and its antigens genotyping sees Table 1;
The antigens genotyping of HPA-1 ~ 6,15 systems of 10 groups of platelet antigen spectrums of table 1. cell
| ? | ?HPA-1 | ?HPA-2 | HPA-3? | HPA-4? | HPA-5? | HPA-6? | HPA-15? |
| C1 | ?1aa | ?2aa | ?3aa | ?4ab | ?5aa | ?6aa | ?15bb |
| C2 | ?1aa | ?2aa | ?3aa | ?4aa | ?5ab | ?6aa | ?15ab |
| C3 | ?1aa | ?2aa | ?3aa | ?4aa | ?5ab | ?6aa | ?15aa |
| C4 | ?1aa | ?2ab | ?3bb | ?4aa | ?5aa | ?6aa | ?15aa |
| C5 | ?1aa | ?2aa | ?3bb | ?4aa | ?5aa | ?6aa | ?15ab |
| C6 | ?1aa | ?2aa | ?3ab | ?4aa | ?5aa | ?6aa | ?15aa |
| C7 | ?1aa | ?2aa | ?3ab | ?4aa | ?5aa | ?6aa | ?15bb |
| C8 | ?1aa | ?2aa | ?3aa | ?4aa | ?5aa | ?6aa | ?15ab |
| C9 | ?1ab | ?2aa | ?3aa | ?4aa | ?5aa | ?6aa | ?15ab |
| C10 | ?1aa | ?2bb | ?3aa | ?4aa | ?5aa | ?6aa | ?15ab |
The preparation of c, platelet suspension: gather the good fresh EDTA anticoagulated whole blood of 10 blood donors of screening, the centrifugal 30min of 900rpm; Get the upper strata platelet rich plasma, the centrifugal 30min of 3000rpm; Abandon blood plasma, with the frozen-dried protective liquid hematocrit blood platelet that fully suspends, and to regulate PC be 100 ~ 150 * 10
9/ L.
D, load: platelet suspension is placed 37 ℃ of constant temperature oven load 4h, during every 30min vibration mixing once.
E, freeze drying: after the platelet suspension packing after the load, place freeze dryer to carry out freeze drying; Its lyophilisation condition sees Table 2.
The lyophilisation condition of table 2. platelet antigen spectrum cell
| Step | The control temperature | Hold time |
| 1 | -40℃ | 4h |
| 2 | Vacuumize | |
| 3 | -20℃ | 3h |
| 4 | -10℃ | 3h |
| 5 | 5℃ | 2h |
| 6 | 10℃ | 2h |
| 7 | 20℃ | 1h |
| 8 | 30℃ | 1h |
Frozen-dried protective liquid of the present invention comprises 0.2M pH7.0 PBS, contains the trehalose of 30 ~ 50 mmol/L and 1 ~ 3% bovine serum albumin(BSA) among the 0.2M pH7.0 PBS.
The present invention with freeze-dried type platelet antigen spectrum cell with physiological saline solution after, be used for carrying out platelet antibody and detect and the specificity identification experiment.
Using method: redissolve with physiological saline before use, room temperature can experimentize after leaving standstill 5min.
The recovery after the freeze-drying of platelet antigen spectrum cell
Redissolve platelet antigen spectrum cell after the freeze-drying to the freeze-drying front volume with physiological saline, and room temperature is carried out the platelet cell counting to it after leaving standstill and treating that it fully dissolves.And with freeze-drying before platelet count calculate recovery rate relatively, computing formula is as follows:
Behind test of many times, counting, calculate that the recovery is 76.21% after the blood platelet freeze-drying.
Antigenicity after the freeze-drying of platelet antigen spectrum cell
Utilize not homospecific monoclonal antibody, whether the antigenicity that adopts the ELISA method to detect platelet antigen spectrum cell freeze-drying front and back glycoprotein, I a/ II a, I b/ IX, IV changes.Each glycoprotein antigen does not all have marked change before and after found that the blood platelet freeze-drying.Concrete test method is as follows:
1. preparation feedback plate: be cushioned liquid (0.05M, pH 9.6 carbonate buffer solutions) dilution rabbit anti human platelet antibody to 15 μ g/ml with bag, be added in (100 μ l/ hole) in the ELISA Plate, seal back 4 ℃ and spend the night.Discarded the supernatant in the ELISA Plate in second day, add confining liquid to expiring the hole.Hatch 2h for 37 ℃.Use washing lotion detersive enzyme target 5 times then, pat dry.
2. bag is by blood platelet: redissolve platelet antigen spectrum cell after the freeze-drying with physiological saline, room temperature is added in (50 μ l/hole) in the ELISA Plate after leaving standstill and treating that it fully dissolves, and washing is 3 times behind the centrifugal 5min of 1300rpm.
3.ELISA detect: in ELISA Plate, add the monoclonal antibody (3 μ g/mL, 100 μ l/ holes) of antiplatelet glycoprotein, I a/ II a, I b/ IX, IV respectively, make negative control with normal mouse serum simultaneously, hatch 1h for 37 ℃.With washing lotion washing 5 times, add the anti-mouse IgG of AP-50 μ l/ holes, hatch 1h for 37 ℃.With washing lotion washing 5 times, add about substrate (50 μ l/ hole) colour developing 30min, add 3M NaOH solution color development stopping (50 μ l/ hole), measure A
405Value.Sentence read result is with negative control A
4052 times of value are the cutoff value, and it is positive to be higher than the cutoff value, and it is negative to be lower than the cutoff value.
Platelet antigen spectrum cell freeze-drying rear stability
Platelet antigen spectrum cell after the freeze-drying is positioned under 2 ~ 8 ℃ of conditions to be preserved, and every month, Antigen Stability research was carried out in sampling, and method is with embodiment two.Found that this platelet antigen spectrum cell preservation after 24 months, the antigenicity of glycoprotein, I a/ II a, I b/ IX, IV does not all have marked change.
Freeze-dried type platelet antigen spectrum cell is mainly used in monoclonal antibody set platelet antigen and analyzes (MAIPA) and solid phase agglutination, to detect platelet antibody and evaluation antibody specificity in the clinical patients body.
1|, monoclonal antibody set platelet antigen are analyzed (MAIPA)
(1) bag is cushioned liquid dilution sheep anti-mouse igg to 3 μ g/ml by sheep anti-mouse igg with bag in the ELISA Plate, is added in (100 μ l/ hole) in the ELISA Plate.4 ℃ of bags are spent the night.With TBS lavation buffer solution detersive enzyme target 5 times, add then and contain 2%BSA(w/v) the TBS lavation buffer solution, 37 ℃ of sealing 2h.Dry behind the detersive enzyme target 5 times, add drying agent and sealing is preserved.
(2) preparation of platelet suspension is redissolved platelet antigen spectrum cell after the freeze-drying with physiological saline, and room temperature leaves standstill treats that it fully dissolves the centrifugal 5min of back 3800r/min.With twice of PBS-EDTA repeated washing.Suspension blood platelet to concentration is about 200 * 10
9/ l.
(3) add sample to be checked and hatch adding above-mentioned platelet suspension (100 μ l/ hole) in U type microwell plate, the centrifugal 3min of 2500r/min.Abandon supernatant and, hatch 30 min for 37 ℃ behind the mixing with 50 μ l serum to be checked or the resuspended hematocrit blood platelet of blood plasma.Wash 2 times.
(4) adding monoclonal antibody hatches and adds 40 μ l and dilute the mouse monoclonal antibody of getting well with the TBS damping fluid.Hatch 30 min for 37 ℃.With PBS-EDTA washing 3 times.
(5) the cracking blood platelet adds lysis buffer (130 μ l/ hole), 4 ℃ of cracking 30min.Centrifugal 15 min of room temperature 3000 r/min.Other gets a microwell plate and adds TBS damping fluid (130 μ l/ hole), adds the centrifugal back of 100 μ l supernatant again, mixing.
(6) solid-phase capture is got the blood platelet lysate of 100 μ l dilution to wrapping by in the ELISA Plate of sheep anti-mouse igg.Hatch 90min or spend the night for 4 ℃.Detersive enzyme target 4 times.
(7) join anti-human IgG with enzyme and hatch the adding anti-human IgGs of 100 μ l HRP marks (Fc section) in all reacting holes.Hatch 90 min for 4 ℃.Detersive enzyme target 6 times.
(8) colour developing adds 100 μ l substrates, room temperature (22-25 ℃) lucifuge colour developing 30min.
(9) color development stopping adds 100 μ l, 0.5 mol/L H
2SO
4Cessation reaction.
(10) measure to measure the 490nm OD of place value (reference wavelength 630nm ~ 650nm).If can not in time detect, ELISA Plate should be kept in Dark Place and in 30min, finish detection.
(11) interpretation of result OD value is positive findings more than or equal to 2 times of negative control OD value.
2, be used for the solid phase agglutination
(1) compose cell to the freeze-drying front volume with the platelet antigen after the physiological saline redissolution freeze-drying, room temperature leaves standstill treats that it fully dissolves.
(2) take out the platelet antibody detection kit, mark patient, positive control and negative control hole on reaction plate, untapped lath should be stored in the valve bag, after the sealing of adding drying agent, and 2-8 ℃ of storage.
(3) in reacting hole, add 1 (50ul) above-mentioned platelet suspension, jog reaction plate about 10 seconds.
(4) with dull and stereotyped hydro-extractor with reaction plate centrifugal 5 minutes with 50g, make blood platelet be fixed on the reacting hole bottom.
(5) pour out liquid in the reacting hole, and drip the washing working fluid with dropper and clean 3 times, jog microwell plate in the washing process, and then get rid of cleansing solution gently.After the last washing reaction plate is inverted in and blots residual liquid (being sure not to pat) on the thieving paper.
(6) in each reacting hole, add 2 (100ul) low ionic-strength solution immediately, and in respective aperture, add 1 (50ul) patient sample, positive control and negative control respectively.Low ionic-strength solution will be become sky blue or dark green by purple, as still adding sample for purple then illustrates to leak.
(7) reacting hole is sealed with sealing compound, the jog mixing is placed in the wet box 37 ℃ of water-baths and hatched 30 minutes.
(8) reaction plate that finishes has been hatched in taking-up, discards sealing compound.5 washing reaction plates are 5 times set by step.
(9) add 1 (50ul) anti-human IgG and 1 (50ul) indication red blood cell immediately, mixing gently vibrates.
(10) with reaction plate centrifugal 5 minutes with 200g.
(11) result that will detect hole and control wells compares, and interpretation is also write down testing result.
(12) positive findings: the indication red blood cell is tiled in the reacting hole lower surface; If the indication red blood cell only is attached at the bottom of the part hole, and the zone of combination is greatly weak more positive than negative control.Show in patients serum or the blood plasma and contain platelet antibody; Or the blood platelet crossmatch does not conform to.
Negative findings: the indication red blood cell forms erythrocyte aggregation in the reacting hole bottom center.Show and do not contain platelet antibody in patients serum or the blood plasma; Or the blood platelet crossmatch is harmonious.
Claims (3)
1. the preparation method of freeze-dried type platelet antigen spectrum cell is characterized in that:
The detection of a, platelet antigen HPA-1 ~ 6 and HPA-15 system Genotyping
Gather blood donor EDTA anticoagulated whole blood, extract DNA by salting out method, its purity should be more than 1.6; Blood donor's sample is carried out HPA-1 ~ 6,15 systems carry out Genotyping, amplification condition is as follows: behind 95 ℃ of pre-sex change 5min, and 95 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 50s, 30 circulations, 72 ℃ are extended 5min;
B, establishment platelet antigen spectrum clone
Platelet antigen spectrum cell is set up the spectrotype of 10 groups of HPA-1 ~ 6,15 systems according to O type blood donor's platelet antigen somatotype result, and its antigens genotyping sees Table 1;
The preparation of c, platelet suspension: gather the good fresh EDTA anticoagulated whole blood of 10 blood donors of screening, the centrifugal 30min of 900rpm; Get the upper strata platelet rich plasma, the centrifugal 30min of 3000rpm; Abandon blood plasma, with the frozen-dried protective liquid hematocrit blood platelet that fully suspends, and to regulate PC be 100 ~ 150 * 10
9/ L;
D, load: platelet suspension is placed 37 ℃ of constant temperature oven load 4h, during every 30min vibration mixing once;
E, freeze drying: after the platelet suspension packing after the load, place freeze dryer to carry out freeze drying; Its lyophilisation condition sees Table 2.
2. the preparation method of the described freeze-dried type platelet antigen of claim 1 spectrum cell, it is characterized in that: frozen-dried protective liquid comprises 0.2M pH7.0 PBS, contains the trehalose of 30 ~ 50 mmol/L and 1 ~ 3% bovine serum albumin(BSA) among the 0.2M pH7.0 PBS.
3. claim 1 obtains the purposes of freeze-dried type platelet antigen spectrum cell, it is characterized in that: with freeze-dried type platelet antigen spectrum cell with physiological saline solution after, be used for carrying out platelet antibody and detect and the specificity identification experiment.
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| CN103063829A (en) * | 2012-12-21 | 2013-04-24 | 杭州茂天赛科技有限公司 | Frozen stock solution |
| CN104771412A (en) * | 2015-03-13 | 2015-07-15 | ***广州总医院 | Novel platelet freeze-drying treating fluid |
| CN111537326A (en) * | 2020-05-07 | 2020-08-14 | 天津德祥生物技术有限公司 | Method for preparing freeze-dried blood platelet and application thereof |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103063829A (en) * | 2012-12-21 | 2013-04-24 | 杭州茂天赛科技有限公司 | Frozen stock solution |
| CN104771412A (en) * | 2015-03-13 | 2015-07-15 | ***广州总医院 | Novel platelet freeze-drying treating fluid |
| CN104771412B (en) * | 2015-03-13 | 2020-03-31 | ***广州总医院 | Novel platelet freeze-drying treatment solution |
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
| US11740019B2 (en) | 2019-03-14 | 2023-08-29 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11609043B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| US11609042B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11747082B2 (en) | 2019-03-14 | 2023-09-05 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11815311B2 (en) | 2019-03-14 | 2023-11-14 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| US11994343B2 (en) | 2019-03-14 | 2024-05-28 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| CN111537326B (en) * | 2020-05-07 | 2023-05-16 | 天津德祥生物技术股份有限公司 | Method for preparing freeze-dried blood platelet and application thereof |
| CN111537326A (en) * | 2020-05-07 | 2020-08-14 | 天津德祥生物技术有限公司 | Method for preparing freeze-dried blood platelet and application thereof |
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