CN106802347A - A kind of method for detecting peripheral blood hepatitis B surface antibody secretory cell - Google Patents
A kind of method for detecting peripheral blood hepatitis B surface antibody secretory cell Download PDFInfo
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Abstract
The present invention relates to a kind of method for detecting peripheral blood hepatitis B surface antibody secretory cell.The present invention activates human peripheral blood single nucleus cell (PBMC) by stimulated in vitro, then using Anti-HBsAg antibody secretory cell in the PVDF plates detection PBMC of pretreatment, detects an Anti-HBsAg antibody secretory cell in 1,000,000 PBMC.Detection difficult part peripheral blood Anti-HBsAg antibody secretory cell quantity instant invention overcomes current peripheral blood Anti-HBsAg antibody secretory cell is few, recall rate is low, experimental repeatability is poor, it is cumbersome, time-consuming, difficulty is big, and need the periphery blood volume taken many, the B cell Activated in Vitro method such as such as CPG, CD40L, PWM, has that activation efficiency is not high, reduces the defects such as the recall rate of Anti-HBsAg antibody secretory cell.The present invention R848, HBsAg and IL 2 stimulate activation human peripheral blood B cell, and capture human peripheral Anti-HBsAg antibody secretory cell with HBsAg pre-coated PVDF plates, reduce detection blood using amount, improve the susceptibility of detection.
Description
Technical field
The present invention relates to a kind of distinct antibodies secretory cell detection method of bio-pharmaceutical engineer technology domain, more particularly to
A kind of method for detecting peripheral blood hepatitis B surface antibody secretory cell.
Background technology
Hepatitis B surface antibody (Anti-HBs antibody) is the mark that body is protected to hepatitis type B virus (HBV) adaptive immune, in HBV
Removing and prevention have a very important role in course of infection again.By plasma cell secretion, body exists Anti-HBs antibody in peripheral blood
After being stimulated by hepatitis B surface antigen (HBsAg), B cell maturation is divided into the thick liquid cell and not secretory antibody of secretion Anti-HBs antibody
Memory B cell, thick liquid cell is short-lived, and memory B cell then can in the case of nonreactive primary stimuli long-term surviving, but its quantity
It is few in peripheral blood.After body is subject to HBsAg to stimulate again, the rapid Proliferation, Differentiation of memory B cell is that plasma cell secretion is anti-
HBs。
In healthy population, after regular Vaccination and Immunoprophylaxis HBV vaccines, serum Anti-HBs antibody level can be gradually reduced, when
When serum Anti-HBs antibody is less than detection level, whether it still has immanoprotection action to still suffer from arguement for human body.At present on evidence
Show, even if dropping to the level that cannot be detected, body through the healthy individuals Anti-HBs antibody of regular Vaccination and Immunoprophylaxis HBV vaccines
The immunoprotection of acquisition is still persistently present, and this is due to there are the memory B cells for being difficult to be detected in vivo, thus outward
All blood Anti-HBs antibody secretory cell quantity may can more reflect continuation immune response of the body to HBV than serum Anti-HBs antibody level.
Before the present invention makes, the detection difficult part of current peripheral blood Anti-HBs antibody secretory cell is：
(1) peripheral blood Anti-HBs antibody secretory cell quantity is few, and recall rate is low, and experimental repeatability is poor；
(2) limiting dilution assay detection antibody secretory cell is cumbersome, time-consuming, difficulty big, and needs the periphery taken
Blood volume is more, it is difficult to carry out popularization and application；
(3) in peripheral blood in addition to the direct secretory antibody of thick liquid cell, memory B cell need to could break up through stimulation oversaturation activation
Be antibody secreting cell, thus without fully activation when, the extremely difficult detection of peripheral blood Anti-HBs antibody secretory cell, and it is existing activation and
It is thin to there is the B such as many deficiencies, such as CPG, CD40L, PWM in the technology of detection peripheral blood Anti-HBs antibody secretory cell
The outer activation method of cell space, has that activation efficiency is not high, it is impossible to B cell is fully converted into antibody secreting cell, drops
The low recall rate of Anti-HBs antibody secretory cell.
The content of the invention
It is an object of the invention to overcome drawbacks described above, a kind of detection peripheral blood hepatitis B surface antibody secretory cell is set up
Method.
The technical scheme is that：
A kind of method for detecting peripheral blood hepatitis B surface antibody secretory cell, it is mainly characterized by by external thorn
Activation human peripheral blood single nucleus cell (PBMC), then using Anti-HBs antibody secretory cell in the PVDF plates detection PBMC of pretreatment,
Detect an Anti-HBs antibody secretory cell in 1,000,000 PBMC.
Specifically include following steps：(1) sterile collection human peripheral；(2) lymphocyte separation medium separates human peripheral, obtains
Obtain PBMC；(3) PBMC is activated using specific process culture；(4) cultured cells is collected, is added in the PVDF plates of pretreatment, put
Enter 5%CO2,37 DEG C, the interior culture of incubator of saturated humidity；(5) PVDF plates are taken out, cell is washed away, is developed the color；(6) detect and count
Number spot, analyzes peripheral blood Anti-HBs antibody secretory cell ratio.
Activating PBMC is stimulated using 1 μ g/mlR848,10ng/ml IL-2,2 μ g/ml HBsAg in the step (3), carefully
Born of the same parents' cultivated days are 5 days.
HBsAg is adopted in the step (4) and pre-processes coated PVDF plates incubated cell, the cell culture time is that 16-24 is small
When.
The PVDF plates are used through alcohol pre-treatment, add the μ l of 10 μ g/ml HBsAg 200 per hole, if plus 15 μ g/ml it is anti-
Human IgG Positive control wells and the negative control hole for adding PBS, 4 DEG C of overnight incubations.
Advantages of the present invention and effect are to stimulate activation human peripheral blood B cell with R848, HBsAg and IL-2, are used in combination
HBsAg pre-coated PVDF plates capture human peripheral Anti-HBs antibody secretory cell, reduces detection blood using amount, improves the quick of detection
Sensitivity, the time required to shortening memory B cell activation and the detection of hepatitis B surface antibody secretory cell.
Other specific advantages of the invention and effect will go on to say below.
Brief description of the drawings
Fig. 1 --- human peripheral Anti-HBs antibody secretory cell testing result schematic diagram in the present invention：
Wherein, a is positive control：Human peripheral total IgG secretory cell/every 2500 PMNCs (PBMC)
Schematic diagram；B is the human peripheral Anti-HBs antibody secretory cell/every 200000 PBMC schematic diagrames for detecting；C is outside the people for detecting
All blood Anti-HBs antibody secretory cells/every 400000 PBMC schematic diagrames；D is negative control schematic diagram.
Specific embodiment
Technical thought of the invention is：
Stimulate peripheral blood human peripheral blood single nucleus cell (PBMC) with R848, HBsAg and IL-2, used after activating B cell
PVDF plates capture human peripheral Anti-HBs antibody secretory cell, and analysis of accounts is carried out after color development treatment.
The present invention is specifically described below：
First, B cell stimulates activation
(1) sterile collection human peripheral；
(2) lymphocyte separation medium is separated and obtains PBMC；
(3) by PBMC with containing 10% hyclone, 1 μ g/ml R848, the 1640 of 2 μ g/ml HBsAg, 10ng/ml IL-2
Nutrient solution re-suspended cell, cell concentration 2 × 106/ ml, is put into 5%CO2, 37 DEG C, culture 5 days in the incubator of saturated humidity.
2nd, PVDF plates detection human peripheral Anti-HBs antibody secretory cell
The HBsAg coating PVDF plates of (1) 10 μ g/ml, while setting the anti-human igg Positive control wells for adding 15 μ g/ml and plus PBS
Negative control hole, 4 DEG C are overnight；
(2) cell of culture 5 days is collected, the RPMI-1640 containing 10% hyclone is resuspended, in addition PVDF plates, per hole
Cell number is 1-4 × 105, it is put into 5%CO2,37 DEG C, culture 16-24 hours in the incubator of saturated humidity；
(3) cell is discarded, PBS board-washings add the detection antibody anti-human igg of 1 μ g/ml (with coating anti-human igg different subtype
And clone number), it is incubated at room temperature 2 hours；
(4) Excess antibody is discarded, PBS board-washings add the Streptavidin-HRP of 1: 1000 dilution, and incubation at room temperature 1 is small
When；
(5) PBS board-washings, plus TMB chromogenic reactions, until obvious spot occurs, a large amount of water rinse color development stopping reaction；
(6) lucifuge dries plate, counts anti-human igg control wells, HBsAg coating hole and negative control hole spot number, calculates anti-
HBs secretory cells number and ratio.
Advantages of the present invention and effect are efficiently to stimulate activation human peripheral blood B cell using R848, HBsAg and IL-2,
And human peripheral Anti-HBs antibody secretory cell is captured with HBsAg pre-coated PVDF plates, and the susceptibility of detection is improve, shorten memory
B cell is activated and detection hepatitis B surface antibody secretory cell required time.
Specifically：
First, B cell stimulates activation
1. sterile collection human peripheral 10ml (anticoagulant heparin), is separated with lymphocyte separation medium and obtains PBMC, with 1640
Nutrient solution is resuspended, and counts, and can obtain PBMC 1 × 107；
2. 1500 turns/min is centrifuged 5 minutes at room temperature, supernatant is removed, with washed cell；
3. with containing 10% hyclone, 1 μ g/mlR848, the RPMI-1640 weight of 2 μ g/ml HBsAg, 10ng/ml IL-2
Outstanding cell, adjustment cell concentration is 2 × 106/ ml, is put into 5%CO2,37 DEG C, culture 5 days in the incubator of saturated humidity.
2nd, PVDF plates detection human peripheral Anti-HBs antibody secretory cell
A. it is coated with PVDF plates (aseptic)
1.PVDF plates add the ethanol of 50 μ l 70% to prewet no more than 2 minutes per hole, rapid board-washing；
2. 200 μ l/ holes board-washing of sterilized water 5 times；
3. add 10 μ g/ml HBsAg, Positive control wells to add 15 μ g/ml anti-human igg per hole, negative control hole adds PBS, 4 DEG C
Overnight incubation.
B. cell incubation (aseptic)
1. 200 μ l/ holes board-washings of aseptic PBS 5 times, wash away excessive antigen or antibody；
2. add 1640 culture mediums of the 200 μ l containing 10%FBS+2% albumin to close per hole.Incubation at room temperature 2 hours；
3. the cell of culture 5 days is collected, and 1500 turns/min is centrifuged 5 minutes, abandons supernatant.RPMI-1640 washed cell, carefully
Born of the same parents count；
4. the RPMI-1640 re-suspended cell of 10%FBS is contained.During cell suspension added into PVDF plates, per hole cell number 1-4
×105；
5. by plate be put into 5%CO2,37 DEG C, be incubated 16-24 hours in the incubator of saturated humidity, period avoids movable plate.
C. Anti-HBs antibody secretory cell spot is detected：(being not required to aseptic)
1.PBS 200 washes PVDF plates 5 times in μ l/ holes, washes away cell；
2. the detection antibody anti-human igg (with coating anti-human igg different subtype and clone number) of the μ g/ml of 100 μ l/ holes 1 is added,
Incubation at room temperature 2 hours；
3. board-washing is with 1；
4. the Streptavidin-HRP of the dilution of 100 μ l/ holes 1: 1000 is added, is incubated at room temperature 1 hour；
5. board-washing is with 1；
6. 100 μ l/ holes TMB chromogenic reactions are added, until obvious spot occurs；
7. a large amount of water rinse to stop chromogenic reaction；
8. lucifuge dries plate, counts anti-human igg control wells, HBsAg coating hole and negative control hole spot number, calculates anti-
HBs secretory cells number and ratio.
Stimulate culture human peripheral PBMC in HBsAg, R848 and IL-2, activate peripheral blood B cell, memory B cell point
Thick liquid cell is turned to, the PVDF plates capture human peripheral Anti-HBs antibody secretory cell of pre-coated HBsAg after 5 days, such as Fig. 1 after color development treatment
Shown, a is positive control, and b, c are respectively every 2 × 105Individual PBMC, every 4 × 105The Anti-HBs antibody secretory cell that individual PBMC is detected,
D is negative control.
Claims (5)
1. it is a kind of detect peripheral blood hepatitis B surface antibody secretory cell method, it is characterised in that by stimulated in vitro activate people outside
All blood mononuclear cells (PBMC), then using Anti-HBs antibody secretory cell in the PVDF plates detection PBMC of pretreatment, can detect one
An Anti-HBs antibody secretory cell in million PBMC.
2. it is according to claim 1 it is a kind of detect peripheral blood hepatitis B surface antibody secretory cell method, it is characterised in that
Specifically include following steps：(1) sterile collection human peripheral；(2) lymphocyte separation medium separates human peripheral, obtains PBMC；
(3) PBMC is activated using specific process culture；(4) cultured cells is collected, is added in the PVDF plates of pretreatment, be put into 5%
CO2,37 DEG C, the interior culture of incubator of saturation temperature；(5) PVDF plates are taken out, cell is washed away, is developed the color；(6) detect and counting spot
Point, analyzes peripheral blood Anti-HBs antibody secretory cell ratio.
3. it is according to claim 2 it is a kind of detect peripheral blood hepatitis B surface antibody secretory cell method, it is characterised in that
Activating PBMC, cell culture day are stimulated using 1 μ g/mlR848,10ng/ml IL-2,2 μ g/ml HBsAg in the step (3)
Number is 5 days.
4. it is according to claim 2 it is a kind of detect peripheral blood hepatitis B surface antibody secretory cell method, it is characterised in that
HBsAg is adopted in the step (4) and pre-processes coated PVDF plates incubated cell, the cell culture time is 16-24 hours.
5. it is according to claim 4 it is a kind of detect peripheral blood hepatitis B surface antibody secretory cell method, it is characterised in that
The PVDF plates are used through alcohol pre-treatment, add the μ l of 10 μ g/ml HBsAg 200 per hole, if plus 15 μ g/ml anti-human igg it is positive
Control wells and the negative control hole for adding PBS, 4 DEG C of overnight incubations.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110964101A (en) * | 2019-12-13 | 2020-04-07 | 山东民康生物科技有限公司 | Preparation method of nano antibody |
| CN111856020A (en) * | 2020-06-19 | 2020-10-30 | 南方医科大学南方医院 | Hepatitis B Virus (HBV) specific T cell detection method and application thereof |
| CN114460849A (en) * | 2022-04-12 | 2022-05-10 | 北京晟海汇泽科技有限公司 | Bionic robot fish motion control method and device and bionic robot fish |
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| WO2008110569A1 (en) * | 2007-03-12 | 2008-09-18 | Bioneer A/S | Method for determination of immunomodulatory effect |
| CN104195110A (en) * | 2014-09-10 | 2014-12-10 | 张明顺 | Human antibody prepared based on fusion of human myeloma cell strain and human B lymphocyte |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110964101A (en) * | 2019-12-13 | 2020-04-07 | 山东民康生物科技有限公司 | Preparation method of nano antibody |
| CN111856020A (en) * | 2020-06-19 | 2020-10-30 | 南方医科大学南方医院 | Hepatitis B Virus (HBV) specific T cell detection method and application thereof |
| CN114460849A (en) * | 2022-04-12 | 2022-05-10 | 北京晟海汇泽科技有限公司 | Bionic robot fish motion control method and device and bionic robot fish |
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Application publication date: 20170606 |