JSR厦门展会资料(胶乳微球)

0.06um ～ 0.2um
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清聴感謝
“Blockmaster”,”IMMUTEX” are registed Trademarks of JSR Corporation in Japan, People's Republic of China. 20
Ultrasonic wave
Temperature control
Homogenizer Orifice Tube
Temperature control
7
Checking dispersion level of particles
①
Absorbance
③ it’s time to finish!!
Stirring（RT～37℃）
30-60min 30-60min Supernatant Antibody sol （1Vol） EDC sol （Quantity;10～200μg/mg-LTX,１hr） -COOH
COOHx1～50 equivalent quantity -CONH-
Centrifugation
Using Ultra Centrifugation Using TFF purification
⇒ Max < 5 L / day ⇒ Max 20L> / day
merits and demerits
Centrifuge Small particle processing Multi preparation Small scale preparation Scale up
Ref) http://cn.spectrumlabs.com/filtration/
TFF ◎ △ △ ○
△ ○ ○ △
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Purification process and calibration curve
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Profits of our latex particles
Ready to use. No need to wash.
Without purification
Surfactant etc.. obstacle antibodies are coming.
After purification
Antibodies are accessible to particle easily.
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Profits of JLS latex particles
Mechanism of TFF purification & Merits
Example) Case of small particle 60-80nm size particle by centrifugation process ⇒ must use ultra centrifugation
OSO3NHOC
+EDC
CH3CH2N=C=NCH2CH2CH2NCH3 HCl
CONH
PSL
OSO3OSO3OSO3-
PSL－ COOH
OSO3CONH
4
Covalent conjugation（１step）
Latex(0.5％×1Vol) Buffer（EX：MES, HEPES、pH6-7.5) -COOH
Sonication can break up aggregation of particles. Saturation of absorbance is time to finish of sonication. Dispersion by sonication is very important. If not using sonication, blank of calibration curve become high. pH, Temp, EDC・HCl amount at sensitization are very important factor to make latex reagents.
Antibody
- - --COO -- --
- -COO -- -
-
-
EDC・HCl
- - - -
Zeta potential (mV) about 40%Down The absolute value of zeta potential go down when using EDC, antibody adsorption
wenku.baidu.com
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Purification Process of making latex reagents
Centrifugation
Tangential flow filtration(TFF)
Wash out
Remove Pellet
Hollow fiber
Pump
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Circulation
Ref) http://cn.spectrumlabs.com/filtration/
Wide range Type
middle range Type
High sensitive Type
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Required range & particle size
0.1 ng/mL 1 ng/mL 10 ng/mL 100 ng/mL 1 ug/mL 10 ug/mL 100 ug/mL 1000 ug/mL
(2)
Redisperse by Vortex etc.
(1)
(4)
Stirring（RT～37℃）
Supernatant Centrifugation R2 Buffer Redisperse by ultrasonic wave Latex Reagent
OD570 of every step shows ultra sonication can redisperse completely.
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Dispersion after conjugation
Latex(0.5％×1Vol) (1) Buffer Stirring（RT～37℃） Absorbance of each steps of making latex reagents
(3)
(2) (3)
Supernatant
Antibody sol （1Vol） EDC sol Centrifugation Blocking Reagent
Polystyrene Latex
OSO3-
Carboxylated Polystyrene Latex
adsorption COOOSO3-
COO-
PSL
OSO3-
PSL－ COOH
OSO3OSO3COOOSO3-
Physical adsorption
OSO3-
Covalent conjugation
http://www.horiba.com/scientific/products/particle-characterization/technology/zeta-potential/
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How to redisperse aggregated particles
⇒ Sonication !
・ Mechanism of dispersion is different ・ Ultrasonic wave is generally using ・ Temperature control is important to avoid denature of antibody
Notes for developing ＆ manufacturing latex reagents
1
Principle of latex photometric Immunoassay
Serum（Ag）
λ
Cuvette
(Intensity)
I0
Iｔ
Absorbance R2 A=In(I0／ Iｔ) R1＋
3) Customized grade is available
COO α
Density：1.05
β
COO -
Refractive Index :1.59
OSO3
Pure water＋0.09%NaN3
TEM Photograph
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Methods of Antibody conjugation
IMMUTEXTM is PSL designed as “ready to use” and have most suitable surface to immobilize antibodies
Lot-to-lot difference is very small
⇒ No worry to change other lots.
2year
JSR Life Sciences is No.1 supplier of latex in Japan.
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Products & Samples List
I0 Iｔ T0
Tt
(T)
2
T0
Tt
（T)
Characteristics of IMMUTEXTM
1) Uniform size of polystyrene particle for IVD 0.06um-0.45um
2) Plan type & Carboxylated PSL
Plan type :Physical adsorption Carboxylated PSL: Covalent conjugation using EDC
No
1 2 3 4 5 6 7 8 9 10 11 12
item PSA PGⅡ PGⅠ AFP FER Mb U-β2M Cys-C CRP S-β2M D-dimer RBP
cardiac cancer other
main particle size
0.2um ～ 0.45um
0.2um ～ 0.35um
Blocking(pH7.2-8.5,1-2hr、BSA:1-2%,Blockmaster CE210, CE510) Redisperse by Vortex etc. 30-60min Supernatant Stirring（RT～37℃） Centrifugation R2 Buffer Redisperse by ultrasonic wave Latex Reagent 5
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Effect of Temp, pH and EDC at sensitization
Antibody denatured!
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γ: μg/mg-beads
Conclusion (Sensitization process)
Antibody and EDC cause soft aggregation of particles. Because zeta potential of particles is changed by them.
②
Redispersion Time
① Before sonication, measure absorbance of particles. ② While sonication, measure absorbance as well, and you can make sure reducing absorbance. ③ Saturation of absorbance is time to finish!
(4)
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(※) Blockmaster™or BSA in R2 buffer help long-term stability.
Calibration curve of reagent using sonication
Background is high
Sonication is very useful to make good latex reagent.
Aggregation is likely to occur at this step!
Why is aggregation occurred?
Zeta potential is an important factor to understand stability of particle.
The absolute value of zeta potential is higher, the particle is more stable.