CN109187958A - A kind of rat CD4 antibody coating magnetic bead and its preparation method and application and the kit containing the magnetic bead - Google Patents
A kind of rat CD4 antibody coating magnetic bead and its preparation method and application and the kit containing the magnetic bead Download PDFInfo
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- CN109187958A CN109187958A CN201811064688.4A CN201811064688A CN109187958A CN 109187958 A CN109187958 A CN 109187958A CN 201811064688 A CN201811064688 A CN 201811064688A CN 109187958 A CN109187958 A CN 109187958A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
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Abstract
A kind of rat CD4 antibody coating magnetic bead of the present invention and its preparation method and application and the kit containing the magnetic bead.Magnetic bead is repeatedly washed by different reagents and supernatant is gone to be made by the preparation method of magnetic bead.As above-mentioned preparation method can made from rat CD4 antibody be coated with magnetic bead.Obtained rat CD4 antibody coating magnetic bead can be used in kit for detecting the application in the immunological rejection after rat renal transplantation.For the prior art, the present invention has the advantages that the preparation method of rat CD4 antibody coating magnetic bead, the rat CD4 antibody made of the above method are coated with magnetic bead and combineImmune cell function assay kit can be used in detecting the immunological rejection after rat renal transplantation.
Description
Technical field
The present invention relates to a kind of rat CD4 antibody coating magnetic bead and its preparation method and application and the reagent containing the magnetic bead
Box.
Background technique
It is existing on the market, there are no a kind of kit, can be used to detect the immunological rejection after rat renal transplantation.
Summary of the invention
It is coated with magnetic bead and its preparation method and application the purpose of the present invention is to provide a kind of rat CD4 antibody and contains and be somebody's turn to do
The kit of magnetic bead.
The purpose of the present invention is achieved through the following technical solutions:
A kind of preparation method of rat CD4 antibody coating magnetic bead, step 1: by magnetic bead by C1 reagent wash, being placed on later
Supernatant is removed after adsorbing on magnet；
Step 2: after antibody, C1 reagent, C2 reagent is added in the pretreated magnetic bead of step 1, being centrifuged at room temperature
Rotate 16-24h；
Step 3: the magnetic bead that step 2 is handled is precipitated by magnet, is cleaned afterwards by HB reagent；
Step 4: removing supernatant after the magnetic bead that step 3 is handled is precipitated by magnet, cleaned afterwards by LB reagent；
Step 5: removing supernatant after the magnetic bead that step 4 is handled is precipitated by magnet, cleaned afterwards by SB reagent；The mistake
Journey can be repeated as many times according to antibody leakage scenarios；
Step 6: the magnetic bead that step 5 is handled adds SB reagent, and centrifugal treating removes after being placed in magnet absorption later
Supernatant completes the preparation of rat CD4 antibody coating magnetic bead；
Wherein, C1 reagent is 60-100% methacrylaldehyde buffer, and C2 reagent is PBS buffer solution.
It further includes step 7: SB reagent being added in the magnetic bead that step 6 is handled, stores at 2-8 DEG C.
It further include preservative sodium azide in step 7.
Further, in step 2, magnetic bead and antibody, C1 reagent, C2 reagent w/v be 1mg: 20ul: 30ul:
50ul。
Further, in step 3, the w/v of magnetic bead and HB reagent is 1mg: 40ul.
Further, in step 4, the w/v of magnetic bead and LB reagent is 1mg: 40ul.
Further, in step 5, the w/v of magnetic bead and SB reagent is 1mg: 40ul.
The HB reagent, LB reagent, SB reagent are 0.01%-0.1%Tween 20.
The magnetic bead is epoxy resin magnetic bead.
The antibody is the antibody of rabbit-anti mouse.
A kind of coating of the rat CD4 antibody as made from above-mentioned preparation method magnetic bead.
A kind of kit, the kit include rat CD4 obtained by the preparation method of rat CD4 antibody coating magnetic bead
Antibody is coated with magnetic bead.
Rat CD4 antibody coating magnetic bead is detecting the application in the immunological rejection after rat renal transplantation.
For the prior art, the present invention has the advantages that the preparation method of rat CD4 antibody coating magnetic bead, passes through
Rat CD4 antibody made of the above method is coated with magnetic bead combination ImmuKnowTM-CylexImmune cell function measures reagent
Box can be used in detecting the immunological rejection after rat renal transplantation.
Detailed description of the invention
Fig. 1 normal rat and rat renal allograft transplantation ATP level measure comparison diagram.
Specific embodiment
The content of present invention is described in detail with embodiment with reference to the accompanying drawings of the specification:
Embodiment 1:
A kind of preparation method of rat CD4 antibody coating magnetic bead, the described method comprises the following steps:
Step 1: being that methacrylaldehyde buffering agents wash epoxy resin magnetic bead with appropriate 75%, be vortexed.Magnet is placed on after washing
Upper absorption 1min, then removes supernatant.During being somebody's turn to do, as long as guaranteeing that the amount of methacrylaldehyde buffer can clean epoxy resin magnetic bead i.e.
Can, even if even excess.
Step 2: after methacrylaldehyde buffer, PBS buffer solution is added in the pretreated epoxy resin magnetic bead of step 1,37
DEG C under room temperature centrifugal rotation 16h；Wherein the accounting of each component is as follows: 5mg epoxy resin magnetic bead, 100ul rabbit anti-mouse antibody,
150ul methacrylaldehyde buffer, 250ul PBS buffer solution.
Step 3: the epoxy resin magnetic bead that step 2 is handled precipitates 1min by magnet, is cleaned afterwards by HB reagent；
The epoxy resin magnetic bead of 5mg is cleaned with 20 reagent of 200ul 0.05%Tween.
Step 4: removing supernatant after the epoxy resin magnetic bead that step 3 is handled is precipitated 1min by magnet, pass through LB reagent afterwards
It is cleaned；The epoxy resin magnetic bead of 5mg is cleaned with 200ul 0.05%Tween 20.
Step 5: removing supernatant after the epoxy resin magnetic bead that step 4 is handled is precipitated 1min by magnet, pass through SB reagent afterwards
It is cleaned；The epoxy resin magnetic bead of 5mg is cleaned with 20 reagent of 200ul 0.05%Tween.
Step 6: the epoxy resin magnetic bead that step 5 is handled is added into 20 reagent of 0.05%Tween, centrifugal treating, later
Supernatant is removed after being placed in magnet absorption, completes the preparation of rat CD4 antibody coating magnetic bead.
Step 7: 20 reagent of 0.05%Tween being added in the magnetic bead that step 6 is handled, is stored at 2-8 DEG C.Wherein, it presses
100ul SB reagent/mg magnetic bead proportion relation adds 20 reagent of 0.05%Tween.
Embodiment 2
A kind of kit, the kit include in embodiment 1 rat CD4 antibody coating magnetic bead preparation method obtained by
Rat CD4 antibody be coated with magnetic bead.
Embodiment 3
ATP detection is carried out to rat CD4 antibody coating magnetic bead obtained is invented, specific experiment process is as follows:
1. testing first part: cytositimulation.
Related component in kit is taken out from refrigerator in advance and is balanced to room temperature.
1.1 are carefully mixed by inversion Quality Control control and rat CD4 whole blood sample, repeatedly to ensure that haemocyte distribution is equal
It is even.:
1.2 with Sample dilution by 1:3 dilution Quality Control and whole blood sample (such as: 250ul whole blood+750ul Sample Dilution
Liquid).
After 1.3 dilutions, each sample uses a 8 well culture plate laths, assembles culture plate according to worksheet.
1.4 25ul Sample dilution is added in preceding 4 holes of each lath, as non-stimulated hole.
1.5 25ul Sample dilution is added in remaining 4 hole of each lath, as stimulation hole.
1.6 will be in 100ul each hole of diluted Quality Control sample addition lath 1.
1.7 according to worksheet, and by 100ul, diluted sample to be tested is added in each hole of corresponding lath.
1.8 cover culture plate lid, shake 30 seconds on oscillator plate.
1.9 are incubated for culture plate with cover 16-18 hours in the incubator of 37 DEG C/5%CO2/ humidification process.
2. testing part 2: cd4 cell selection and ATP release.
Related component in kit is taken out from refrigerator in advance and is balanced to room temperature.
2.1 take out culture plate from culture phase, are placed on oscillator plate and shake 3 minutes.
2.2 carefully overturn CD4 magnetic bead solution reagent bottle several times with the magnetic bead that sufficiently suspends；With single channel pipettor or continuously
It is loaded pipettor and is separately added into 50ul magnetic bead to each hole.Magnetic bead should be sufficiently suspended before absorption to ensure that each hole additional amount is consistent.
Note: because magnetic bead is not easy sufficiently to suspend in reservoir, asking not operate this with multi-channel micropipettor
Step.
2.3 cover culture plate lid, shake 30 seconds on oscillator plate, stand (18-28 DEG C) at room temperature and be incubated for 15 points
Clock.Shake culture plate 30 seconds, room temperature (18-28 DEG C) stationary incubation 15 minutes again.Finally, shake culture plate 30 seconds is again with weight
New suspension magnetic bead.
2.4 take out lath from culture plate, are placed on assembled magnetic support, wait magnetic bead is made to be gathered in hole within 1-2 minutes
Side；Cd4 cell is separated from whole blood according to the following steps.
2.5 with the 8 channel suction nozzles for being connected to negative-pressure ward vacuum pump system protrude into micropore absorb other than magnetic bead containing red
Cell culture fluid.Pay attention to gentle manipulation, avoids siphoning away magnetic bead.
2.6 wash laths three times, to remove other interfering substances in remaining unbonded free cell and hole.
Note: when cleaning solution is added, need to vertically mention and hold multi-channel micropipettor, avoids in suction pipette head touching micropore
Magnetic bead on wall.
It washs #1: 200ul cleaning solution is added to each hole, waits one minute, then siphons away cleaning solution.
It washs #2: 200ul cleaning solution is added to each hole, check that micropore inwall is inhaled if there is blood coagulation spot with micropipettor
Head removes it.It waits 1 minute, then siphons away cleaning solution.
It washs #3: 200ul cleaning solution is added to each hole, lath frame is removed from magnetic support, lath is placed on oscillator plate
Lath frame, is then placed on magnetic support by upper oscillation 1 minute.It waits 1 minute, the side in hole is gathered in magnetic bead, then siphons away and washes
Wash liquid.
2.7 are added 200ul lytic reagent to each hole.
2.8 remove lath frame from magnetic support, vibrate 5 minutes on oscillator plate.
2.9 are placed on lath frame on magnetic support, wait 1-2 minutes.
2.10 continue to test third portion.
Note:, should be by lath freezen protective if test third portion cannot be carried out (in 4 hours) immediately: by lath from magnetic
It takes off, is placed on culture plate framework on seat board item frame；Lath is sealed, culture plate lid, -20 DEG C of freezen protectives are covered.Freezing
Lath keeps stablizing at least in 1 month.To continue subsequent detection test, culture plate is taken out from refrigerator, is put down
Weighing apparatus is put into lath on magnetic support lath frame to room temperature (18-28 DEG C), then repeats above-mentioned 2.8 and 2.9 steps.
3. testing third portion: ATP measurement.
3.1 assemble measurement plate referring to experiment work table: measurement plate includes Quality Control measurement item, rat CD4 sample measurement item, school
Quasi- product measure item.
3.2 take out 50ul pyrolysis product from each hole of culture plate according to worksheet, with multichannel micropipettor, and measurement plate is added
In corresponding aperture；Before each lath is added in pyrolysis product, new suction pipette head is needed to change.
3.3, according to worksheet, respectively take out the corresponding aperture that 50ul is separately added into measurement plate from each concentration level ATP calibration object
(usually doing 2 multiple holes)
3.4 luminescence reagents: before use, gently overturning mixes well it.150ul is taken to shine with multichannel micropipettor
Reagent adds in each hole of measurement plate；Measurement plate is placed on oscillator plate and is vibrated 30 seconds, to ensure to be sufficiently mixed.It is being added
Relative luminous intensity (RLU) is read in 3-10 minutes after luminescence reagent.Final result is referring to Fig. 1, normal rat detection discovery ATP
It as a result is 601.08 ± 10.65ng/mL, and the rat ATP result after kidney transplant is 294.96 ± 48.59ng/mL, and it is normal
Control group compares P < 0.05, and difference has statistical significance.Rat CD4 antibody coating magnetic bead prepared by the present invention can be used in examining
Immunological rejection after surveying rat renal transplantation.
Claims (10)
1. a kind of preparation method of rat CD4 antibody coating magnetic bead, it is characterised in that: the described method comprises the following steps:
Step 1: by magnetic bead by C1 reagent wash, be placed on after being adsorbed on magnet later and remove supernatant；
Step 2: after antibody, C1 reagent, C2 reagent is added in the pretreated magnetic bead of step 1, centrifugal rotary under the conditions of 25-37 DEG C
Turn 16-24h；
Step 3: the magnetic bead that step 2 is handled is precipitated by magnet, is cleaned afterwards by HB reagent；
Step 4: removing supernatant after the magnetic bead that step 3 is handled is precipitated by magnet, cleaned afterwards by LB reagent；
Step 5: removing supernatant after the magnetic bead that step 4 is handled is precipitated by magnet, cleaned afterwards by SB reagent；
Step 6: the magnetic bead that step 5 is handled adds SB reagent, and centrifugal treating removes supernatant after being placed in magnet absorption later
Liquid completes the preparation of rat CD4 antibody coating magnetic bead；
Wherein, C1 reagent is 60-100% methacrylaldehyde buffer, and C2 reagent is PBS buffer solution；The HB reagent, LB reagent, SB
Reagent is 0.01%-0.1%Tween20.
2. the preparation method of rat CD4 antibody coating magnetic bead according to claim 1, it is characterised in that: it further includes step
Rapid 7: SB reagent being added in the magnetic bead that step 6 is handled, is stored at 2-8 DEG C.
3. the preparation method of rat CD4 antibody coating magnetic bead according to claim 1, it is characterised in that: in step 2, magnetic
Pearl and antibody, C1 reagent, C2 reagent w/v be 1mg:15-20ul:20-30ul:40-60ul.
4. the preparation method of rat CD4 antibody coating magnetic bead according to claim 1, it is characterised in that: in step 3, magnetic
The w/v of pearl and HB reagent is 1mg:30-40ul.
5. the preparation method of rat CD4 antibody coating magnetic bead according to claim 1, it is characterised in that: in step 4, magnetic
The w/v of pearl and LB reagent is 1mg:30-40ul.
6. the preparation method of rat CD4 antibody coating magnetic bead according to claim 1, it is characterised in that: in step 5, magnetic
The w/v of pearl and SB reagent is 1mg:30-40ul.
7. the preparation method of rat CD4 antibody according to claim 1 coating magnetic bead, it is characterised in that: the antibody is
The antibody of rabbit-anti mouse.
8. rat CD4 antibody made from preparation method described in any one of -7 is coated with magnetic bead according to claim 1.
9. a kind of kit, it is characterised in that: the kit includes rat CD4 antibody coating magnetic bead according to any one of claims 8.
10. rat CD4 antibody is coated with magnetic bead in detecting the immunological rejection after rat renal transplantation according to claim 8
Application.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811064688.4A CN109187958A (en) | 2018-09-12 | 2018-09-12 | A kind of rat CD4 antibody coating magnetic bead and its preparation method and application and the kit containing the magnetic bead |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811064688.4A CN109187958A (en) | 2018-09-12 | 2018-09-12 | A kind of rat CD4 antibody coating magnetic bead and its preparation method and application and the kit containing the magnetic bead |
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| CN109187958A true CN109187958A (en) | 2019-01-11 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111484975A (en) * | 2020-04-29 | 2020-08-04 | 上海轩锋生物科技有限公司 | Preparation method of human T cell CD3/CD28 activated magnetic beads |
Citations (4)
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|---|---|---|---|---|
| WO2001013947A1 (en) * | 1999-08-26 | 2001-03-01 | Absorber Ab | Reduction of the immunogenicity of non-human grafts |
| CN101006100A (en) * | 2004-06-22 | 2007-07-25 | 托勒克斯股份有限公司 | Optimized dosing with anti-CD4 antibodies for tolerance induction in primates |
| CN102690863A (en) * | 2012-04-11 | 2012-09-26 | 上海云泽生物科技有限公司 | ATP (adenosine triphosphate) testing based lymphocyte proliferation activity analysis reagent kit and preparation and application thereof |
| CN106366194A (en) * | 2016-08-26 | 2017-02-01 | 上海交通大学 | Immunomagnetic bead for peripheral blood lymphocyte separation and preparation and application thereof |
-
2018
- 2018-09-12 CN CN201811064688.4A patent/CN109187958A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001013947A1 (en) * | 1999-08-26 | 2001-03-01 | Absorber Ab | Reduction of the immunogenicity of non-human grafts |
| CN101006100A (en) * | 2004-06-22 | 2007-07-25 | 托勒克斯股份有限公司 | Optimized dosing with anti-CD4 antibodies for tolerance induction in primates |
| CN102690863A (en) * | 2012-04-11 | 2012-09-26 | 上海云泽生物科技有限公司 | ATP (adenosine triphosphate) testing based lymphocyte proliferation activity analysis reagent kit and preparation and application thereof |
| CN106366194A (en) * | 2016-08-26 | 2017-02-01 | 上海交通大学 | Immunomagnetic bead for peripheral blood lymphocyte separation and preparation and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| LIFE TECHNOLOGIES: "《Dynabeads Antibody Coupling Kit》", 30 June 2012 * |
| 李春景 等: "CD4 + T 细胞内ATP 含量检测在肾移植受者免疫状态监测中的应用价值", 《上海交通大学学报（医学版）》 * |
| 王凯阳 等: "Ｔ细胞亚群与CD4+ CD25+调节性T细胞及Foxp3 mRNA移植大鼠急性排斥反应中的作用", 《中华实用诊断与治疗杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111484975A (en) * | 2020-04-29 | 2020-08-04 | 上海轩锋生物科技有限公司 | Preparation method of human T cell CD3/CD28 activated magnetic beads |
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Application publication date: 20190111 |