Download - Uji Pirogen (Lal)
![Page 1: Uji Pirogen (Lal)](https://reader033.vdokumen.com/reader033/viewer/2022061302/54e4ade74a7959a2708b4b0b/html5/thumbnails/1.jpg)
Uji Endotoxin ( Uji Pirogen)
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Endotoxin
• Apa itu endotoksin?
• Endotoksin berasal dari mana?
• Mengapa hal itu menjadi penting?
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Endotoxin
Endotoksin adalah :
a lipopolysaccaride
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Endotoxin
Berasal dari membran sel bakteri gram negatif
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Endotoxin
Mengapa endotoksin sangat penting, karena berkaitan dengan
• pirogen
• bertahan dalam proses sterilisasi
![Page 6: Uji Pirogen (Lal)](https://reader033.vdokumen.com/reader033/viewer/2022061302/54e4ade74a7959a2708b4b0b/html5/thumbnails/6.jpg)
Uji Endotoxin
• Produk apa saja yang diuji?
• Bagaimana uji ini dilaksanakan?
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Endotoxin Testing
Produk apa saja yang diuji?
• injeksi and peralatan medis
dimana akan kontak dengan darah atau larutan spinal.
• Termasuk, bahan baku, air dalam proses monitoring
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Uji Endotoxin
Bagaimana pelaksanaan uji Endotoksin?
• Uji pirogen dengan kelinci
• LAL test
![Page 9: Uji Pirogen (Lal)](https://reader033.vdokumen.com/reader033/viewer/2022061302/54e4ade74a7959a2708b4b0b/html5/thumbnails/9.jpg)
Uji pirogen dengan kelinci
• Metode standar pada abad 20
• Bahan diinjeksikan pada telinga
![Page 10: Uji Pirogen (Lal)](https://reader033.vdokumen.com/reader033/viewer/2022061302/54e4ade74a7959a2708b4b0b/html5/thumbnails/10.jpg)
LAL Test
• Dikembangkan pada 1960’s by Drs. Bang and Levin
• Berdasarkan pada reaksi clotting darah horseshoe crab dengan endotoxin
• Lebih cepat, ekonomis, lebih sensitif dibanding uji pirogen menggunakan kelinci.
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LAL
• Limulus - jenis kepiting
• Amebocyte - sel darah kepiting dimana zat aktifnya telah diderivatisasi
• Lysate - adalah komponen yang ditetapkan dengan pemisahan amebocytes dari plasma kemudian dilysis
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Pengambilan darah kepiting Horseshoe
![Page 13: Uji Pirogen (Lal)](https://reader033.vdokumen.com/reader033/viewer/2022061302/54e4ade74a7959a2708b4b0b/html5/thumbnails/13.jpg)
Darah kepiting Horseshoe
![Page 14: Uji Pirogen (Lal)](https://reader033.vdokumen.com/reader033/viewer/2022061302/54e4ade74a7959a2708b4b0b/html5/thumbnails/14.jpg)
Jenis Uji LAL
• Gel Clot
• Turbidimetric
• Colorimetric
![Page 15: Uji Pirogen (Lal)](https://reader033.vdokumen.com/reader033/viewer/2022061302/54e4ade74a7959a2708b4b0b/html5/thumbnails/15.jpg)
Metode Gel Clot
• Metode Original
• The official “referee test”
• Spesimen diinkubasi dgn LAL yang diketahui sesitivitasnya. Formasi gel clott postif untuk endotoxin.
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Metode Turbidimetric • Sebuah metode kinetik
• Spesimen diinkubasi dgn LAL bersamaan dengan kecepatan peningkatan dalam turbidimetri atau waktu pengambilan untuk mencapai turbiditas kemudian diukur dengan spektrofotometri dan dibandingkan dengan standar.
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Metode Colorimetric
• Endotoxin mengkatalisis aktivasi proenzim dalam LAL dimana akan mengikat substrat tidak berwarna menjadi endoproduk yang berwarna sehingga dapat diukur secara spektrofotometri dibanding kurva standar
• Can be kinetic or endpoint
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Colorimetric Reaction
Endotoxin
1. Proenzyme → Enzyme
Enzyme
2. Substrate → Peptide + p-NA
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Comparison of Methods
Gel Clot Chromogenic
Endpoint
Chromogenic
Kinetic
Turbidimetric
Semi-
quantitative
Quantitative Quantitative Quantitative
Simple Least expensive,
Requires 37 degree bath
Requires
spectrophotometer
or plate reader
Requires
incubating plate or tube reader
Requires
incubating plate or tube reader
Manually read and recorded
Can be automated,
allows electronic
data storage
Can be automated,
allows electronic
data storage
Can be automated,
allows electronic
data storage
Sensitive down
to 0.03 EU/ml
Sensitive down
to 0.1 EU/ml
Sensitive down
to .005 EU/ml
Sensitive down
to .001 EU/ml *
* (Sensitivities vary by reagent manufacturer, instrumentation and testing conditions)
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Steps in Chromogenic Endpoint Procedure
• Read SOP (reagents are prepared)
• Label tubes and make dilutions of stock standard
• Pipette blanks, standards & unknowns into prewarmed plate following template
• Start timer as LAL is added to first well.
Add quickly with repeat pipettor to all wells being used & mix.
• At T = 10 mins add prewarmed chromogenic substrate in same manner as above
• At T = 16 mins. Add stop reagent in same manner as above
• Read plate
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Template
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
![Page 22: Uji Pirogen (Lal)](https://reader033.vdokumen.com/reader033/viewer/2022061302/54e4ade74a7959a2708b4b0b/html5/thumbnails/22.jpg)
Notes
• Peralatan gelas, tip dan air hrs bebas endotoksin
• Temperatur dan waktu adalah kritikal point