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20
18
Required range & particle size
0.1
1
ng/mL ng/mL
No item
10 ng/mL
100 ng/mL
1 ug/mL
1 PSA 2 PGⅡ 3 PGⅠ 4 AFP 5 FER 6 Mb 7 U-β2M 8 Cys-C 9 CRP 10 S-β2M 11 D-dimer 12 RBP
Notes for developing ＆ manufacturing
latex reagents
1
Principle of latex photometric Immunoassay
Serum（Ag）
λ
I0
Cuvette
(Intensity)
I0
A=In(I0／ Iｔ)
Iｔ
T0
Tt
Absorbance R2
cardiac
cancer other
19
10 ug/mL
100 ug/mL
1000 ug/mL
main
particle size
0.2um ～
0.45um
0.2um ～
0.35um
0.06um ～
0.2um
清聴感謝
“Blockmaster”,”IMMUTEX” are registed Trademarks of JSR Corporation in Japan, People's Republic of China.
Polystyrene Latex
OSO3-
OSO3-
PSL
OSO3-
Physical adsorption
OSO3-
OSO3-
PSL
OSO3-
Carboxylated Polystyrene Latex
adsorption
OSO3-
COO-
COO-
PSL－ COOH
OSO3-
OSO3-
COO-
Covalent conjugation
http://www.horiba.com/scientific/products/particle-characterization/technology/zeta-potential/
6
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
Antibody
-----
-
-----COO-
Hale Waihona Puke Baidu
EDC・HCl
--
--
--COO-
--- - -
Zeta potential (mV) about 40%Down
The absolute value of zeta potential go down when using EDC, antibody adsorption
30-60min Supernatant
Stirring（RT～37℃）
Antibody sol （1Vol） （Quantity;10～200μg/mg-LTX,１hr） EDC sol COOHx1～50 equivalent quantity
Centrifugation
-CONH-
-COOH
Blocking(pH7.2-8.5,1-2hr、BSA:1-2%,Blockmaster CE210, CE510) Redisperse by Vortex etc.
⇒ No worry to change other lots.
2year
JSR Life Sciences is No.1 supplier of latex in Japan.
17
Products & Samples List
Wide range Type
middle range Type
High sensitive Type
Mechanism of TFF purification & Merits
Example) Case of small particle 60-80nm size particle by centrifugation process
⇒ must use ultra centrifugation
Using Ultra Centrifugation ⇒ Max < 5 L / day
+EDC
CH3CH2N=C=NCH2CH2CH2NCH3 HCl
NHOC
OSO3-
OSO3-
PSL－ COOH
CONH OSO3-
CONH
4
Covalent conjugation（１step）
Latex(0.5％×1Vol) Buffer（EX：MES, HEPES、pH6-7.5)
-COOH
30-60min
Conclusion (Sensitization process)
Antibody and EDC cause soft aggregation of particles. Because zeta potential of particles is changed by them.
Sonication can break up aggregation of particles. Saturation of absorbance is time to finish of sonication.
30-60min
Stirring（RT～37℃）
Aggregation is likely to occur at this step!
Supernatant
Centrifugation
R2 Buffer
Redisperse by ultrasonic wave Latex Reagent
5
Why is aggregation occurred?
Scale up
14
△
○
Purification process and calibration curve
15
Profits of our latex particles
Ready to use. No need to wash.
Without purification After purification
12
Purification Process of making latex reagents
Centrifugation
Pellet
Remove
Tangential flow filtration(TFF)
Wash out Hollow fiber
Pump
Circulation
13
Ref) http://cn.spectrumlabs.com/filtration/
Stirring（RT～37℃）
Supernatant
Centrifugation
R2 Buffer
Redisperse by ultrasonic wave
Latex Reagent
(4)
Absorbance of each steps of making latex reagents
(3)
(2)
(1)
(4)
OD570 of every step shows ultra sonication can redisperse completely.
(※) Blockmaster™or BSA in R2 buffer help long-term stability.
9
Calibration curve of reagent using sonication
Using TFF purification ⇒ Max 20L> / day
merits and demerits
Small particle processing
Centrifuge TFF
△
◎
Multi preparation
○
△
Small scale preparation
○
△
Ref) http://cn.spectrumlabs.com/filtration/
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!
Background is high
Sonication is very useful to make good latex reagent.
10
Effect of Temp, pH and EDC at sensitization
Antibody denatured!
11
γ: μg/mg-beads
R1＋
(T)
T0
2
Iｔ （T)
Tt
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
Zeta potential is an important factor to understand stability of particle.
The absolute value of zeta potential is higher, the particle is more stable.
Surfactant etc.. obstacle antibodies are coming. Antibodies are accessible to particle easily.
16
Profits of JLS latex particles
Lot-to-lot difference is very small
Ultrasonic wave
Temperature control
Homogenizer Orifice Tube Temperature control 7
Absorbance
Checking dispersion level of particles
①
③ it’s time to finish!!
8
Dispersion after conjugation
Latex(0.5％×1Vol) (1)
Buffer Stirring（RT～37℃）
(2)
(3)
Supernatant
Antibody sol （1Vol） EDC sol
Centrifugation
Blocking Reagent
Redisperse by Vortex etc.
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.
3) Customized grade is available
COOα
β
OSO3-
COO -
Density：1.05
Refractive Index :1.59
Pure water＋0.09%NaN3
3
TEM Photograph
Methods of Antibody conjugation
IMMUTEXTM is PSL designed as “ready to use” and have most suitable surface to immobilize antibodies
18
Required range & particle size
0.1
1
ng/mL ng/mL
No item
10 ng/mL
100 ng/mL
1 ug/mL
1 PSA 2 PGⅡ 3 PGⅠ 4 AFP 5 FER 6 Mb 7 U-β2M 8 Cys-C 9 CRP 10 S-β2M 11 D-dimer 12 RBP
Notes for developing ＆ manufacturing
latex reagents
1
Principle of latex photometric Immunoassay
Serum（Ag）
λ
I0
Cuvette
(Intensity)
I0
A=In(I0／ Iｔ)
Iｔ
T0
Tt
Absorbance R2
cardiac
cancer other
19
10 ug/mL
100 ug/mL
1000 ug/mL
main
particle size
0.2um ～
0.45um
0.2um ～
0.35um
0.06um ～
0.2um
清聴感謝
“Blockmaster”,”IMMUTEX” are registed Trademarks of JSR Corporation in Japan, People's Republic of China.
Polystyrene Latex
OSO3-
OSO3-
PSL
OSO3-
Physical adsorption
OSO3-
OSO3-
PSL
OSO3-
Carboxylated Polystyrene Latex
adsorption
OSO3-
COO-
COO-
PSL－ COOH
OSO3-
OSO3-
COO-
Covalent conjugation
http://www.horiba.com/scientific/products/particle-characterization/technology/zeta-potential/
6
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
Antibody
-----
-
-----COO-
Hale Waihona Puke Baidu
EDC・HCl
--
--
--COO-
--- - -
Zeta potential (mV) about 40%Down
The absolute value of zeta potential go down when using EDC, antibody adsorption
30-60min Supernatant
Stirring（RT～37℃）
Antibody sol （1Vol） （Quantity;10～200μg/mg-LTX,１hr） EDC sol COOHx1～50 equivalent quantity
Centrifugation
-CONH-
-COOH
Blocking(pH7.2-8.5,1-2hr、BSA:1-2%,Blockmaster CE210, CE510) Redisperse by Vortex etc.
⇒ No worry to change other lots.
2year
JSR Life Sciences is No.1 supplier of latex in Japan.
17
Products & Samples List
Wide range Type
middle range Type
High sensitive Type
Mechanism of TFF purification & Merits
Example) Case of small particle 60-80nm size particle by centrifugation process
⇒ must use ultra centrifugation
Using Ultra Centrifugation ⇒ Max < 5 L / day
+EDC
CH3CH2N=C=NCH2CH2CH2NCH3 HCl
NHOC
OSO3-
OSO3-
PSL－ COOH
CONH OSO3-
CONH
4
Covalent conjugation（１step）
Latex(0.5％×1Vol) Buffer（EX：MES, HEPES、pH6-7.5)
-COOH
30-60min
Conclusion (Sensitization process)
Antibody and EDC cause soft aggregation of particles. Because zeta potential of particles is changed by them.
Sonication can break up aggregation of particles. Saturation of absorbance is time to finish of sonication.
30-60min
Stirring（RT～37℃）
Aggregation is likely to occur at this step!
Supernatant
Centrifugation
R2 Buffer
Redisperse by ultrasonic wave Latex Reagent
5
Why is aggregation occurred?
Scale up
14
△
○
Purification process and calibration curve
15
Profits of our latex particles
Ready to use. No need to wash.
Without purification After purification
12
Purification Process of making latex reagents
Centrifugation
Pellet
Remove
Tangential flow filtration(TFF)
Wash out Hollow fiber
Pump
Circulation
13
Ref) http://cn.spectrumlabs.com/filtration/
Stirring（RT～37℃）
Supernatant
Centrifugation
R2 Buffer
Redisperse by ultrasonic wave
Latex Reagent
(4)
Absorbance of each steps of making latex reagents
(3)
(2)
(1)
(4)
OD570 of every step shows ultra sonication can redisperse completely.
(※) Blockmaster™or BSA in R2 buffer help long-term stability.
9
Calibration curve of reagent using sonication
Using TFF purification ⇒ Max 20L> / day
merits and demerits
Small particle processing
Centrifuge TFF
△
◎
Multi preparation
○
△
Small scale preparation
○
△
Ref) http://cn.spectrumlabs.com/filtration/
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!
Background is high
Sonication is very useful to make good latex reagent.
10
Effect of Temp, pH and EDC at sensitization
Antibody denatured!
11
γ: μg/mg-beads
R1＋
(T)
T0
2
Iｔ （T)
Tt
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
Zeta potential is an important factor to understand stability of particle.
The absolute value of zeta potential is higher, the particle is more stable.
Surfactant etc.. obstacle antibodies are coming. Antibodies are accessible to particle easily.
16
Profits of JLS latex particles
Lot-to-lot difference is very small
Ultrasonic wave
Temperature control
Homogenizer Orifice Tube Temperature control 7
Absorbance
Checking dispersion level of particles
①
③ it’s time to finish!!
8
Dispersion after conjugation
Latex(0.5％×1Vol) (1)
Buffer Stirring（RT～37℃）
(2)
(3)
Supernatant
Antibody sol （1Vol） EDC sol
Centrifugation
Blocking Reagent
Redisperse by Vortex etc.
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.
3) Customized grade is available
COOα
β
OSO3-
COO -
Density：1.05
Refractive Index :1.59
Pure water＋0.09%NaN3
3
TEM Photograph
Methods of Antibody conjugation
IMMUTEXTM is PSL designed as “ready to use” and have most suitable surface to immobilize antibodies