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1、1 Notes for developing manufacturing latex reagents 2 Principle of latex photometric Immunoassay I0 (T) T0 Tt (Intensity) I I0 A=In(I0 I) Serum（ （Ag） ） T0 Tt Absorbance （T) Cuvette I R1 R2 3 TEM Photograph Characteristics of IMMUTEXTM 1) Uniform size of polystyrene particle for IVD 0.06um-0.45um 2)
2、Plan type 10200g/mg-LTX,hr） Buffer（EX：MES, HEPES、pH6-7.5) Redisperse by Vortex etc. Blocking(pH7.2-8.5,1-2hr、BSA:1-2%,Blockmaster CE210, CE510) Centrifugation Latex Reagent Stirring（RT37） -COOH -COOH -CONH- Supernatant Redisperse by ultrasonic wave Supernatant 30-60min 30-60min 30-60min 6 Why is agg
3、regation occurred? -COO- -COO- EDCHCl Antibody The absolute value of zeta potential go down when using EDC, antibody adsorption Zeta potential is an important factor to understand stability of particle. The absolute value of zeta potential is higher, the particle is more stable. - - - - - - - - - -
4、- - - - - - - - - - - - - Zeta potential (mV) about 40%Down 7 Ultrasonic wave Homogenizer Orifice Tube Temperature control Mechanism of dispersion is different Ultrasonic wave is generally using Temperature control is important to avoid denature of antibody Temperature control How to redisperse aggr
5、egated particles Sonication ! 8 Checking dispersion level of particles Redispersion Time Absorbance its time to finish! Before sonication, measure absorbance of particles. While sonication, measure absorbance as well, and you can make sure reducing absorbance. Saturation of absorbance is time to fin
6、ish! 9 Latex(0.51Vol) Antibody sol （1Vol） Stirring（RT37） EDC sol Buffer Redisperse by Vortex etc. Blocking Reagent Centrifugation Stirring（RT37） Supernatant Dispersion after conjugation (1) (1) (2) (2) (3) (3) (4) (4) OD570 of every step shows ultra sonication can redisperse completely. Absorbance o
7、f each steps of making latex reagents () Blockmasteror BSA in R2 buffer help long-term stability. Centrifugation R2 Buffer Latex Reagent Supernatant Redisperse by ultrasonic wave 10 Calibration curve of reagent using sonication Sonication is very useful to make good latex reagent. Background is high
8、 11 Effect of Temp, pH and EDC at sensitization : g/mg-beads Antibody denatured! 12 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 absorba
9、nce 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. 13 Remove Pellet Circulation Wash out Hollow fiber Pump Centri
10、fugation Tangential flow filtration(TFF) Purification Process of making latex reagents Ref) 14 Mechanism of TFF purification & Merits Ref) Example) Case of small particle 60-80nm size particle by centrifugation process must use ultra centrifugation Using Ultra Centrifugation Max / day merits and dem
11、erits Centrifuge TFF Small particle processing Multi preparation Small scale preparation Scale up 15 Purification process and calibration curve 16 Profits of our latex particles Ready to use. No need to wash. Surfactant etc. obstacle antibodies are coming. Antibodies are accessible to particle easil
12、y. Without purification After purification 17 Lot-to-lot difference is very small No worry to change other lots. Profits of JLS latex particles JSR Life Sciences is No.1 supplier of latex in Japan. 2year 18 Products & Samples List Wide range Type High sensitive Type middle range Type 19 Required ran
13、ge & particle size NoNoitemitem 1PSAPSA 2PGPG 3PGPG 4AFPAFP 5FERFER 6MbMb 7U-2MU-2M 8Cys-CCys-C 9CRPCRP 10S-2MS-2M 11D-dimerD-dimer 12RBPRBP cardiaccardiaccancercancerotherother main particle size 0.10.1 ng/mL 1 1 ng/mL 1010 ng/mL 100100 ng/mL 1 1 ug/mL 1010 ug/mL 100100 ug/mL 10001000 ug/mL 0.06um0.06um 0.2um0.2um 0.2um0.2um 0.45um0.45um 0.2um0.2um 0.35um0.35um 20 清聴感謝 “Blockmaster”,”IMMUTEX” are registed Trademarks of JSR Corporation in Japan, Peoples Republic of China.