CN109134653A - A kind of preparation method of Procalcitonin antibody - Google Patents
A kind of preparation method of Procalcitonin antibody Download PDFInfo
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- CN109134653A CN109134653A CN201811053151.8A CN201811053151A CN109134653A CN 109134653 A CN109134653 A CN 109134653A CN 201811053151 A CN201811053151 A CN 201811053151A CN 109134653 A CN109134653 A CN 109134653A
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
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Abstract
The invention discloses a kind of preparation methods of Procalcitonin antibody, this method includes animal immune and splenocyte preparation, the preparation of myeloma cell, immune spleen cell fusion with myeloma cells, the screening of hybridoma, the subclone of positive hybridoma cell, large scale preparation PCT monoclonal antibody, the purifying of PCT antibody, identification monoclonal antibody characteristic and etc., preparation method of the present invention is simple and effective, the PCT monoclonal antibody of preparation has high specificity, the features such as high sensitivity, there is good binding ability with PCT, it can be used for the functional study of PCT, it can be used for developing the diagnostic reagent based on PCT, for bacterium infection early diagnosis, antidiastole and treatment monitoring provide help.
Description
Technical field
The present invention relates to cell engineering fields, and in particular to a kind of preparation method of Procalcitonin antibody.
Background technique
Procalcitonin (procalcitonin, PCT) is a kind of glycoprotein, from being positioned on o.11 chromosome
The single copy gene of (11p15,4).PCT is the preceding peptide material of calcitonin (CT), and PCT precursor is under the effect of endogenous polypeptidase
The end nPro-CT unique sequence is cut, the PCT of 116 amino acid is generated, molecular weight is about 13kD.Its molecular structure is containing 57
The N-terminal (N-ProCT) of amino acid contains 32 amino acid CT (Cakitonin) and contains 21 amino acid katacalcins
(Katacalcin) it forms.Under normal physiological conditions, PCT is to be synthesized by thyroid gland and secreted, in the blood of healthy human body
Content in slurry is very low (being less than 0.1ng/ml), but the stability of PCT albumen in vivo is fine, and half-life period is about 20-
24h.PCT can produce by cytokine influences such as endotoxin, TNF-α in Various Tissues in human infection, when serious bacterial,
Its horizontal in blood plasma increases when pyemia, multiple organ failure, and PCT will not when autoimmunity, allergy and virus infection
It increases, PCT reflects the active degree of systemic inflammatory response.PCT has been considered as a kind of for bacterium infection early diagnosis, mirror
The newest specific index of monitoring Zhen Duan and not be treated.Therefore, the detection of PCT has important value in clinical diagnosis.
Currently, clinically mainly using immunologic method, including colloidal gold immunochromatographimethod, enzyme to exempt from the detection of PCT
Method, immunofluorescence technique, immunochemiluminescence, radio immunoassay etc..And these methods are used it is crucial that obtaining handy
PCT antibody.Since content is very low (< 0.1ng/ml) in normal human serum by PCT, diagnosis dividing value clinically is generally
0.5ng/ml.Therefore also very high to affine force request other than specificity will be got well to the PCT antibody of diagnosis.
Summary of the invention
The purpose of the present invention is to provide a kind of preparation method of Procalcitonin antibody, the antibody specificity of preparation is good, parent
With power height, it can be used in Procalcitonin detection kit.
To achieve the above object, the invention provides the following technical scheme: a kind of preparation method of Procalcitonin antibody, including
Following steps:
(1) animal immune and splenocyte preparation: using BALB/c mouse, and PCT albumen and Freund's complete adjuvant is isometric
It is mixed into the emulsion of Water-In-Oil, subcutaneous multi-point injection mouse, the dosage of inoculation of mouse is 50ug/, is spaced 3 weeks, by PCT albumen
Mixed in equal volume with incomplete Freund's adjuvant, subcutaneous multi-point injection mouse, the dosage of inoculation of mouse be 50ug/ only, interval 3 weeks into
The impact of row antigen, 100ugPCT albumen are not added adjuvant tail vein injection, and after 3d, eyeball of mouse blood sampling separates serum, saves standby
With, mouse is put to death, immune spleen cell is taken under aseptic condition, immune spleen cell is resuspended in incomplete RPMI1640 culture solution,
Adjusting concentration is 1 × 107A/mL；
(2) preparation of myeloma cell: myeloma cell cultivates in the complete RPMI1640 containing 10% (v/v) fetal calf serum
It is cultivated, is merged first 1 day in liquid, carried out cell and change liquid and keep a cell in logarithm growth stage；
(3) cell fusion: immune spleen cell and myeloma cell being mixed with the ratio of 5:1, are put into fusion pipe,
2000r/min is centrifuged 5min, abandons supernatant, flicks tube bottom with finger, makes precipitating loosely at paste；50% polyethylene glycol PEG4000
1mL, left hand uniform rotation fusion pipe, the right hand hold 1mL pipette and draw 50%PEG solution 1mL, and the tube wall along rotation instills, 60s
It inside adds, 25mL DMEM culture medium is added in 5min, terminate reaction, speed is added are as follows: in lmin plus in 1mL, 2min
4mL, remaining 20mL is added to add in 5min, 1000rpm/min is centrifuged 10min, abandons supernatant, 20mL HAT culture medium is added, gently
Featheriness, which is beaten, makes sedimentation cell suspend；Cell suspension addition has been covered in 96 orifice plates of feeder cells, additional amount is the hole 0.1mL/,
Set 37 DEG C, containing 5%CO2Incubator in cultivate, liquid is partly changed with HAT complete culture solution in the 7th day every hole after fusion, the 14th after fusion
It partly changes liquid with HT complete medium, changes liquid with RPMI-1640 complete culture solution within the 21st day after fusion；
(4) when cell length to 1/5 area of bottom hole, it is miscellaneous that ELISA screening positive clone cell screening: is carried out to cell supernatant
Oncocyte is handed over, selects the cell hole that OD value is high, inhibiting rate is big, colony counts are less to be cloned, and expand culture；
(5) it is subcloned: subclone being carried out 2-4 times using positive hybridoma cell of the soft agar assay to screening, until arriving
100% cell positive rate finally obtains high specific PCT hybridoma cell strain, by high specific PCT hybridoma cell strain liquid nitrogen
It saves；
(6) large scale preparation monoclonal antibody: adult BALB/c mouse intraperitoneal inoculation atoleine, every mouse 0.5ml, after 10 days
The diluted PCT monoclonal antibody hybridoma cell of intraperitoneal inoculation serum free medium, cell number is adjusted to 5*105, every
Mouse injects 0.2ml, and mouse ascites production was observed after 5 days in interval daily, and if abdomen obviously expands, when touch, skin has
Tension can acquire ascites, and ascites 2000r/min is centrifuged 5min, collect supernatant to get PCT monoclonal antibody is arrived；
(7) PCT monoclonal antibody purification: is purified using affinity chromatography Protein G Sepharose Fast Flow
Antibody, the purity of PCT monoclonal antibody after purification is measured with SDS-PAGE, and purity reaches 90%；
(8) it identifies monoclonal antibody characteristic: PCT monoclonal antibody is identified using the mouse monoclonal antibody subtype identification kit of Roche company
Hypotype, hypotype be IgG1 type, light chain be k chain.
In step (3), feeder cells the preparation method comprises the following steps: 4 days before cell fusion, pass through neck dislocation and put to death non-immune 8
Week old KM female mice under sterile conditions, is cut with disinfection after 75% alcohol impregnates 5min and starts skin of abdomen, exposure abdomen
Film, 75% alcohol swab wipe peritonaeum, syringe inject 10ml incomplete culture medium to abdominal cavity, syringe needle be retained in it is intraperitoneal, it is another
Only hand-held 75% alcohol swab is gently after abdomen massage 1min, and the culture solution of injection is sucked out in syringe, and 2000r/min is centrifuged 5min,
Supernatant is abandoned, feeder cells will be precipitated with 5ml HAT culture medium and suspended, adjustment feeder cells concentration is 2*105A/mL, will be above-mentioned
96 orifice plates are added in feeder cells suspension, and every hole 0.1ml places 37 DEG C, 5%CO2It is cultivated in incubator spare.
The present invention have the characteristics that the utility model has the advantages that PCT monoclonal antibody of the present invention have high specificity, high sensitivity, can
Using the important tool as diagnosis bacterium infection, the application of this antibody will be the diagnosis of functional study and exploitation based on PCT of PCT
Reagent lays the foundation.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described.
Embodiment 1
A kind of preparation method of Procalcitonin antibody, includes the following steps:
(1) animal immune and splenocyte preparation: using BALB/c mouse, and PCT albumen and Freund's complete adjuvant is isometric
It is mixed into the emulsion of Water-In-Oil, subcutaneous multi-point injection mouse, the dosage of inoculation of mouse is 50ug/, is inoculated with 5 mouse, interval 3
PCT albumen is mixed in equal volume with incomplete Freund's adjuvant, subcutaneous multi-point injection mouse in week, the dosage of inoculation of mouse is 50ug/
Only, it is spaced progresss antigen impact in 3 weeks, 100ugPCT albumen is not added adjuvant tail vein injection, and after 3d, eyeball of mouse is taken a blood sample, and separates
Serum, serum keeping is spare, puts to death mouse, takes immune spleen cell under aseptic condition, immune spleen cell is resuspended in not exclusively
In RPMI1640 culture solution, adjustment concentration is 1 × 107A/mL；
(2) preparation of myeloma cell: myeloma cell cultivates in the complete RPMI1640 containing 10% (v/v) fetal calf serum
It is cultivated, is merged first 1 day in liquid, carried out cell and change liquid and keep a cell in logarithm growth stage；
(3) 4 days before cell fusion, it is small that non-immune 8 week old KM female the preparation of feeder cells: is put to death by neck dislocation
Mouse under sterile conditions, is cut with disinfection after 75% alcohol impregnates 5min and starts skin of abdomen, exposure peritonaeum, 75% alcohol swab
Peritonaeum is wiped, syringe injects 10ml incomplete culture medium to abdominal cavity, and syringe needle is retained in intraperitoneal, and another holds 75% alcohol
For cotton gently after abdomen massage 1min, the culture solution of injection is sucked out in syringe, and 2000r/min is centrifuged 5min, abandons supernatant, uses 5ml
HAT culture medium will precipitate feeder cells and suspend, and adjustment feeder cells concentration is 2*105A/mL adds above-mentioned feeder cells suspension
Enter 96 orifice plates, every hole 0.1ml places 37 DEG C, 5%CO2It is cultivated in incubator spare；
(4) cell fusion: immune spleen cell and myeloma cell being mixed with the ratio of 5:1, are put into fusion pipe,
2000r/min is centrifuged 5min, abandons supernatant, flicks tube bottom with finger, makes precipitating loosely at paste；50% polyethylene glycol PEG4000
1mL, left hand uniform rotation fusion pipe, the right hand hold 1mL pipette and draw 50%PEG solution 1mL, and the tube wall along rotation instills, 60s
It inside adds, 25mL DMEM culture medium is added in 5min, terminate reaction, speed is added are as follows: in lmin plus in 1mL, 2min
4mL, remaining 20mL is added to add in 5min, 1000rpm/min is centrifuged 10min, abandons supernatant, 20mL HAT culture medium is added, gently
Featheriness, which is beaten, makes sedimentation cell suspend；Cell suspension addition has been covered in 96 orifice plates of feeder cells, additional amount is the hole 0.1mL/,
Set 37 DEG C, containing 5%CO2Incubator in cultivate, liquid is partly changed with HAT complete culture solution in the 7th day every hole after fusion, the 14th after fusion
It partly changes liquid with HT complete medium, changes liquid with RPMI-1640 complete culture solution within the 21st day after fusion；
(5) when cell length to 1/5 area of bottom hole, it is miscellaneous that ELISA screening positive clone cell screening: is carried out to cell supernatant
Oncocyte is handed over, selects the cell hole that OD value is high, inhibiting rate is big, colony counts are less to be cloned, and expand culture；
(6) it is subcloned: preparing feeder cells in culture dish, 2.0% agarose normal saline solution and equivalent is double
The HT culture solution of concentration mixes, and is made into 1% agarose culture solution, incubates in 45 DEG C of water-baths, sucks plate culture supernatants, is added
1% agarose culture solution 3ml room temperature 10 minutes, takes hybridoma suspension 1ml, and 1% agarose culture solution 1ml is added and mixes,
It is laid on upper layer, 37 DEG C, 7.5% wet culture 7-14 days；When clonal growth is to 2mm, it is sucked out and is cloned with capillary syring, direct transferred species
In 24 orifice plates containing feeder cells, expand culture, draw supernatant, detect antibody, positive hole can continue cloning, until arriving
100% cell positive rate finally obtains high specific PCT hybridoma cell strain, by high specific PCT hybridoma cell strain liquid nitrogen
It saves；
(7) large scale preparation monoclonal antibody: adult BALB/c mouse intraperitoneal inoculation atoleine, every mouse 0.5ml, after 10 days
The diluted PCT monoclonal antibody hybridoma cell of intraperitoneal inoculation serum free medium, cell number is adjusted to 5*105, every
Mouse injects 0.2ml, and mouse ascites production was observed after 5 days in interval daily, and if abdomen obviously expands, when touch, skin has
Tension can acquire ascites, and ascites 2000r/min is centrifuged 5min, collect supernatant to get PCT monoclonal antibody is arrived；
(8) PCT monoclonal antibody purification: is purified using affinity chromatography Protein G Sepharose Fast Flow
Antibody, the purity of PCT monoclonal antibody after purification is measured with SDS-PAGE, and purity reaches 90%；
(9) it identifies monoclonal antibody characteristic: PCT monoclonal antibody is identified using the mouse monoclonal antibody subtype identification kit of Roche company
Hypotype, hypotype be IgG1 type, light chain be k chain.
Embodiment 2
It is substantially the same manner as Example 1, the difference is that the difference of antibody purification process, purification process are as follows: fresh acquisition
Ascites 0.22um filtering with microporous membrane ascites, to remove biggish grumeleuse and fat drop, 4 DEG C of centrifuge with 10000r/min
Centrifugation 15 minutes removes cell residue and finely ground particle substance, takes the ascites that upper layer is limpid, and the barbital that pH7.2 is added in equivalent is slow
Salt water dilution is rushed, then to add the ratio of 150mg SiO 2 powder that SiO 2 powder is added in every 10ml dilution ascites, is mixed
Even, suspension shook frequently in incubation at room temperature 30 minutes, and centrifuge 2000r/min is centrifuged 20min, and removal precipitating is to get purifying
Antibody, the purity of PCT monoclonal antibody after purification is measured with SDS-PAGE, and purity reaches 75%.
Embodiment 3
It is substantially the same manner as Example 1, the difference is that the difference of antibody purification process, purification process are as follows: fresh acquisition
Ascites 0.22um filtering with microporous membrane ascites, to remove biggish grumeleuse and fat drop, 4 DEG C of centrifuge with 10000r/min
Centrifugation 15 minutes removes cell residue and finely ground particle substance, takes the ascites that upper layer is limpid, and the barbital that pH7.2 is added in equivalent is slow
Salt water dilution is rushed, then to add the ratio of 150mg SiO 2 powder that SiO 2 powder is added in every 10ml dilution ascites, is mixed
Even, suspension shook frequently in incubation at room temperature 30 minutes, and centrifuge 2000r/min is centrifuged 20min, and it is mono- to obtain PCT for removal precipitating
Clonal antibody, then PCT monoclonal antibody is purified using affinity chromatography Protein G Sepharose Fast Flow, it obtains
The PCT monoclonal antibody of high-purity, the purity of PCT monoclonal antibody after purification is measured with SDS-PAGE, and purity reaches 93%.
Experimental animal, biological agent, reagent, instrument used in PCT method for preparing monoclonal antibody provided by the invention
It is bought by market.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (6)
1. a kind of preparation method of Procalcitonin antibody, which comprises the steps of:
(1) animal immune and splenocyte preparation: BALB/c mouse is used, PCT albumen is mixed in equal volume with Freund's complete adjuvant
At the emulsion of Water-In-Oil, subcutaneous multi-point injection mouse, the dosage of inoculation of mouse is 50ug/, is spaced 3 weeks, by PCT albumen and not
Family name's Freund's incomplete adjuvant mixes in equal volume, subcutaneous multi-point injection mouse, and the dosage of inoculation of mouse is 50ug/, and interval is resisted for 3 weeks
Original impact, 100ugPCT albumen are not added adjuvant tail vein injection, and after 3d, eyeball of mouse blood sampling separates serum, saves backup, locates
Dead mouse takes immune spleen cell under aseptic condition, immune spleen cell is resuspended in incomplete RPMI1640 culture solution, adjusts dense
Degree is 1 × 107A/mL；
(2) preparation of myeloma cell: myeloma cell is in the complete RPMI1640 culture solution containing 10% (v/v) fetal calf serum
It is cultivated, is merged first 1 day, carried out cell and change liquid and keep a cell in logarithm growth stage；
(3) cell fusion: immune spleen cell is mixed with myeloma cells, and is put into fusion pipe, and 2000r/min is centrifuged 5min,
Supernatant is abandoned, flicks tube bottom with finger, makes precipitating loosely at paste；50% polyethylene glycol PEG4000 1mL, left hand uniform rotation are melted
It closes and manages, the right hand holds 1mL pipette and draws 50%PEG solution 1mL, and the tube wall along rotation instills, and adds in 60s, adds in 5min
Enter 25mL DMEM culture medium, terminate and react, speed is added are as follows: adds 4mL, remaining 20mL to exist in lmin plus in 1mL, 2min
It is added in 5min, 1000rpm/min is centrifuged 10min, abandons supernatant, and 20mL HAT culture medium is added, and gently piping and druming makes sedimentation cell
It suspends；Cell suspension addition is covered in 96 orifice plates of feeder cells, additional amount is the hole 0.1mL/, sets 37 DEG C, containing 5%CO2
Incubator in cultivate, liquid is partly changed with HAT complete culture solution in the 7th day every hole after fusion, is cultivated completely with HT within the 14th day after fusion
Base partly changes liquid, changes liquid with RPMI-1640 complete culture solution within the 21st day after fusion；
(4) when cell length to 1/5 area of bottom hole, ELISA screening positive clone hybridoma cell screening: is carried out to cell supernatant
Cell, and expand culture；
(5) it is subcloned: subclone being carried out 2-4 times using positive hybridoma cell of the soft agar assay to screening, until thin to 100%
Born of the same parents' positive rate finally obtains high specific PCT hybridoma cell strain, by high specific PCT hybridoma cell strain Liquid nitrogen storage；
(6) large scale preparation monoclonal antibody: adult BALB/c mouse intraperitoneal inoculation atoleine, every mouse 0.5ml, abdominal cavity after 10 days
The inoculation diluted PCT monoclonal antibody hybridoma cell of serum free medium, cell number is adjusted to 5*105, every mouse
0.2ml is injected, mouse ascites production was observed after 5 days in interval daily, and if abdomen obviously expands, when touch, skin has anxiety
Sense, can acquire ascites, and ascites 2000r/min is centrifuged 5min, collect supernatant to get PCT monoclonal antibody is arrived；
(7) PCT monoclonal antibody antibody purification: is purified using affinity chromatography Protein G Sepharose Fast Flow；
(8) it identifies monoclonal antibody characteristic: concentration is measured using the nucleic acid-protein analyzer that BIO-RAD company produces, using Roche company
Mouse monoclonal antibody subtype identification kit identification PCT monoclonal antibody hypotype.
2. a kind of preparation method of Procalcitonin antibody as described in claim 1, which is characterized in that in step (2), the bone
Myeloma cells are SP2/0 cell.
3. a kind of preparation method of Procalcitonin antibody as described in claim 1, which is characterized in that in step (3), spleen is immunized
The ratio of cell and myeloma cell are 5:1.
4. a kind of preparation method of Procalcitonin antibody as described in claim 1, which is characterized in that in step (3), raising is thin
Born of the same parents' the preparation method comprises the following steps: put to death non-immune 8 week old KM female mice, the leaching of 75% alcohol by neck dislocation 4 days before cell fusion
It after steeping 5min, under sterile conditions, is cut with disinfection and starts skin of abdomen, exposure peritonaeum, 75% alcohol swab wipes peritonaeum, injection
Device inject 10ml incomplete culture medium to abdominal cavity, syringe needle be retained in it is intraperitoneal, another hold 75% alcohol swab gently massage abdomen
After portion 1min, the culture solution of injection is sucked out in syringe, and 2000r/min is centrifuged 5min, is abandoned supernatant, will be sunk with 5ml HAT culture medium
Shallow lake feeder cells suspend, and adjustment feeder cells concentration is 2*10596 orifice plates, every hole is added in above-mentioned feeder cells suspension by a/mL
0.1ml places 37 DEG C, 5%CO2It is cultivated in incubator spare.
5. a kind of preparation method of Procalcitonin antibody as described in claim 1, which is characterized in that described pure in step (7)
The purity of PCT monoclonal antibody is 90% after change.
6. a kind of preparation method of Procalcitonin antibody as described in claim 1, which is characterized in that described in step (8)
The hypotype of PCT hybridoma cell strain is IgG1 type, and light chain is k chain.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110105448A (en) * | 2019-05-10 | 2019-08-09 | 上海钹乐诗生物技术有限公司 | A kind of preparation method of the anti-human SAA monoclonal antibody of source of mouse |
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| CN103913579A (en) * | 2014-03-24 | 2014-07-09 | 北京普恩光德生物科技开发有限公司 | Procalcitonin detection kit |
| CN105801697A (en) * | 2015-07-09 | 2016-07-27 | 南京诺尔曼生物技术有限公司 | Monoclonal antibody of human-derived procalcitonin, and preparation method and application thereof |
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2018
- 2018-09-10 CN CN201811053151.8A patent/CN109134653A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103913579A (en) * | 2014-03-24 | 2014-07-09 | 北京普恩光德生物科技开发有限公司 | Procalcitonin detection kit |
| CN105801697A (en) * | 2015-07-09 | 2016-07-27 | 南京诺尔曼生物技术有限公司 | Monoclonal antibody of human-derived procalcitonin, and preparation method and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110105448A (en) * | 2019-05-10 | 2019-08-09 | 上海钹乐诗生物技术有限公司 | A kind of preparation method of the anti-human SAA monoclonal antibody of source of mouse |
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