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All Journal Jurnal Farmasi Klinik Indonesia ANNALES BOGORIENSES
TUTUS GUSDINAR
School of Pharmacy, Institut Teknologi Bandung-Jln.Ganesha no.10 Bandung
Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemical Engineering, Chemistry & Bioengineering Chemistry Immunology & microbiology Medicine & Pharmacology

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Analisis Kuantitatif 15-F2t-isoprostan dari Plasma Pasien Obstructive Sleep Apnea (OSA) dengan Metode Enzyme Linked Immunosorbent Assay (ELISA) menggunakan Teknik Ekstraksi Imunoafinitas Rusdi, Bertha; Gusdinar, Tutus; Fattah, Miswar
Indonesian Journal of Clinical Pharmacy Vol 1, No 1 (2012)
Publisher : Indonesian Journal of Clinical Pharmacy

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (792.299 KB)

Abstract

15-F2t-isoprostan merupakan penanda stres oksidatif yang kadarnya dalam cairan biologis relatif rendah serta memiliki banyak isomer sehingga diperlukan ekstraksi sampel sebelum dilakukan pengukuran kadar. Metode ekstraksi yang digunakan diantaranya ekstraksi fase solid/Solid Phase Extraction (SPE) serta ekstraksi imunoafinitas. Perbaikan teknik ekstraksi SPE dan teknik ekstraksi imunoafinitas dilakukan untuk membandingkan hasil perolehan kembalinya. Pengukuran dilakukan dengan metode Enzyme Linked Immunosorbent Assay (ELISA). Penilaian efektivitas proses ekstraksi diamati melalui hasil perolehan kembali dari kedua teknik ekstraksi. Teknik ekstraksi dengan perolehan kembali tertinggi digunakan untuk mengukur kadar 15-F2t-isoprostan dari penderita Obstructive Sleep Apnea (OSA). Teknik ekstraksi imunoafinitas menghasilkan perolehan kembali 15-F2t-isoprostan yang cukup baik. Pada penderita OSA kadar 15-F2t-isoprostan dalam plasma cenderung tinggi sehingga memiliki risiko untuk menderita penyakit yang berhubungan dengan aktivitas biologis 15-F2t-isoprostan seperti arteriosklerosis.Kata kunci: 15-F2t-isoprostan, Solid Phase Extraction (SPE), ekstraksi imunoafinitas, ObstructiveSleep Apnea (OSA) Quantitative Analysis of Free 15-F2t-Isoprostane from Plasma of Obstructive Sleep Apnea Patients Using Enzyme Linked Immunosorbent Assay Method with Immunoaffinity Extraction TechniqueAbstract15-F2t-isoprostane is a biomarker in assessment of oxidative stress status that due to its relatively low concentration in biological fluid and also has many isomers, the 15-F2t-isoprostane sample need to be extracted prior to the quantifying processes. Extraction techniques commonly used to extract 15-F2t-isoprostane are solid phase extraction (SPE) and immunoaffinity extraction. Improvements to the SPE and immunoaffinity extraction techniques had been conducted, and the recovery results was then compared. The quantification of 15-F2t-isoprostane then was conducted using Enzyme Linked Immunosorbent Assay (ELISA) method. Then followed by the examination of the plasma recovery results. Extraction technique which had the highest recovery then was used to quantify 15-F2t-isoprostane from plasma of Obstructive Sleep Apnea (OSA) patients. Immunoaffinity extraction technique has a good recovery result. OSA patients have the tendency to have high 15-F2t-isoprostane concentrations in the plasma, therefore have a potential risk to get diseases related to the biological activities of 15-F2t-isoprostane, such as arteriosclerosis.Key words: 15-F2t-isoprostane, Solid Phase Extraction (SPE), immunoaffinity extraction, ObstructiveSleep Apnea
Construction of an EPO (Human-Erythropoietin) Synthetic Gene Through a Recurvise-PCR Method Fuad, Asrul Muhamad; Gusdinar, Tutus; Retnoningrum, Debbie Sofie; Natalia, Dessy
ANNALES BOGORIENSES Vol 12, No 1 (2008): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (7152.292 KB) | DOI: 10.1234/60

Abstract

Human  erythropoietin (hEPO)  is an  important glycoprotein  in human  that is coded by a single gene named EPO  (eryhtopoietin). EPO  is  a  glycoprotein  hormone  that  promotes  erythropoiesis,  which  is  the  formation process of mature  red  blood  cell  (erythrocytes)  in  human  bodies.  It  is  widely used  for  treatment  of anemia  in patient  With  chronic  renal failure.  Therefore  EPO has  been classified  as hematopoietic cytokine. Recombinant hEPO  (rhEPO)  has  been  commercially  available,  such  a  Epogen.  It  is  produced  in  mammalian  cell, such  as CHO  (Chine  e hamster ovary) cells  for  the  reason of  its complex structure as a glyco-protein. In  an effort  to  use and optimize heterolgous EPO gene expression  in  an  alternative eukaryotic  host  cells  such  as  yeast, an  EPO­synthetic  gene  (EPOsyn)  was  constructed.  The  synthetic  gene  had  been designed to contain  optimaI Pichiapastoris codon usage . It had  been constructed by a  recursive-PCR method  in  two-step PCR reactions. The gene was assembled  from  8 single strands synthetic  oligonuclotides having an average  length of 90 nt with 20  to 30 overlap  region  between  two  adjacent  oligos. The  synthetic  gene  has  less  GC  content  (4-.3 1%)  compared  its native (human) gene (59.08%). The synthetic gene has  been cloned  in  pCR2.1  cloning plasmid and sequenced. From  8  independent clones,  it was revealed  that  the error  rate  was  1.59%,  in which  1.42% was due  to deletions and 0.17% due  to substitutions. Design of  the gene sequences, construction method and DNA sequence analysis of  the gene will  be discussed  in  this  paper.   Keywords: Human erythropoietin (hEPO), erythropoiesis  EPO­synthetic  gene, recursive-PCR, Pichiapastoris, hematopoietic cytokine.
Co-Authors Asrul Muhamad Fuad, Asrul Muhamad Bertha Rusdi DEBBIE SOFIE RETNONINGRUM DESSY NATALIA Fattah, Miswar