Annales Bogorienses

Number 1, 2004

1-8
SENSITIVITY IMPROVEMENT OF A DIRECT COMPETITIVE ELISA FOR ATRAZINE BY EXPLOITING LOW CROSS-REACTIVITY OF AN ATRAZINE-SPECIFIC RECOMBINANT ANTmODY FAB-FRAGMENT

Authors: Wien Kusharyoto, Rolf D. Schmid

Abstract

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The hapten-binding site of the antibody Fab-fragment K411 B specific towards the herbicide atrazine (2-cllloro-4-(ethylamino)-6-(isopropyJamjno)- 1,3,5-triazine) was modified by means of structural modeling and site-directed mutagenesis. A triple mutant (GlnL89GlulValH3711e/GluL3Val) of the Fab-fragment showed an increased affinity towards the hapten HlCVC6 (4-arnino-6-chloro-l ,3,5-triazine-2-(6-aminohexanecarboxylic acid» compared to the affinity ofthe wild-type Fab-fragment lowards the same hapten. However, the mutant exhibited substantially lower affinity towards the hapten H/CVC6 Ihan towards atrazine and the hapten iPr/ CVC6 (4-chloro-6-(isopropylamino)-1,3,5-triazioe-2-(6-aminohexanecarboxylic acid», which is usual ly used in the synthesis of enzyme tracers in ELISA for atrazine. Advantage was taken of the low cross-reactivity and increased affinity of the mutant Fab fragment towards H/CVC6 to improve the sensitivity of a direct-competitive ELISA tor atrazine. HlCIlC6 was covalently conjugated with horseradish peroxidase (HRP), and the conjugate H/CI/C6-HRPwas used as enzyme tracer in the ELISA for atrazine. An eight-fold improvement in sensitivity ofa direct-competitive ELISA for atrazine could be achieved using the tracer H/CVC6-HRP compared to the sensitivity of ELISA using the tracer iPr/CVC6-HRP. The detection limit for atrazine was as low as 0.01 i g/l.

9-19
AN APPLICATION OF REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION IN A RELATIVE QUANTIFICATION OF GENE EXPRESSION

Authors: Adi Santoso

Abstract

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The ability to quantify steady state levels of individual mes enger-RNA (mRNA) transcripts has been the key issue for study on the control of gene expression. Although two available techniques, Northern blot and nuclease protection assays (NI)A) have been widely used tor detecting mRNA, these techniques have critical limitations. The most obvious limitation of these two techn iques is the required number of target mRNAs to be detected. Reverse transcription-polymerase chain reaction (RT-P R), which has been accepted as a highly sensitive and specific method, provides a means for detecting and quantifying gene expression using, theoretically only a single molecule of mRNA. The ensitivity and reliability ofRT-PCR i dependent upon both the RT and PCR steps. The PCR step ha been problematic because of the exponential nature of this reaction where small variation can lead Lo dramatic changes in final result. here fore, the use of RT-P R for quanti fication of gene expres ion requires pre-experimental planntng and de ign. In thiS experiment, the procedure fOT pre-experimental planning, linear range determination and subsequent relative quantification of gene expression are described in detail. study of ornithine decarboxyJase gene, a gene involved in the polyamine biosynthesis and temporally expressed, dunng embryogenesis of Musca domestica (housefly) was used as the model. The re ults show that during early embryogenesis (t-l to t-4) the expression lev I was very low. The increase In expression profile was observed started at t-5, peaked at t-9, and followed by substantial decrease from t-l 0 to t- 12.

20-26
MATERNAL CONTRIBUTION IN REVEALING THE EFFECTS OF METHOXYACETIC ACID (MAA) ADMINISTERED BEFORE IMPLANTATION ON THE EMBRYONIC DEVELOPMENT OF SWISS WEBSTER MICE (Mus musculus)

Authors: Ekayanti Mulyawati Kaiin, Sony H. Sumarsono, Tien W. Surjono, Sri Sudarwati

Abstract

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Maternal contribution to and direct action of methoxyacetic acid (MAA) on the embryonic development bad been examined by conducting embryo tTansfer. To reveal the maternal contribut ion, compacted morulae and early blastocysts, which were collected from untreated Swiss Webster donor mice on day 3 of gestation, were transferred to day 2 pseudopregnant recipients, after having been treated with 2 .0 mmollkg body weight (b.w.) MAA by gavage on day 1 of pseudopregnancy. Direct effect of MAA on the embryonic development were observed by transferring compacted morulae and early bla tocysts, simi larly recovered from day 3 pregnant donor mice, after MAA treatment on day 2 of gestation with the same method and dosing, to untreated day 2 pseudopregnant recipients. Control donor mice and recipient were given distilled water only as the MAA olvent. Observations on fetuses resulting fTom embryo transfer wert: carried out on day 16 of gestatlOn . Administration of MAA to the donors tended to decrease the unplantatlOn rate and the survival rate of the implanted embryos. W11en MAA was given to the recipients the implantation rate and survival rate of embryos transferred decreased significantly (p<0 .05) but the survival rate of implanted embryos were significantly higher (p<0.05) Lf compared to those of MAA treated donors. The intrauterine death tended to inc rease either in the treated donors or recipients. Th re was no et1ect of MAA on the fetal body weight and in producing fetal malformations . It is concluded that at the beginning of implantation, maternal contribution in revealing the effects of MAA on the embryonic development of Swiss Webster mice is predominant, whereas after Implantation took place, the quality of the embryos become ';lore important for their survival.

27-36
THE USE OF 16s RNA AND NODe GENE SEQUENCE IN RESOLVING THE PHYLOGENET IC RELATIONSHIP OF Rhizobia A SOCIATED WITH Paraseriaflthes fa/cataria (L.) Nielsen PLANT

Authors: Titik K. Prana, Teruo Sone, Hiroko Kawasaki Nakagawa, Atsushi Yokota, Tatsuji Seki, Fusao Tomita

Abstract

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Number of views: 320

Studies on genet ic position of several iso lates liv ing symbiot i a lly on ParaseriCllltli esfalcalaria have been carried out using amplification and se uenc ing tec hni ques of 16S rDNA and specific gene fo r nodulation (/lode) . The "p hylogenetic LTee" cons tructed based On 165S rONA showed that rh izobia growing symbiotically on Paraserianthes fa/ca taria consisted f 3 group . TIl . fi r t group, was fasl- growing rhIzobia wh ich have close r lationshlp to Rhizobium tropicii, the second and the third groups were slow-growing r!lI zobia wh ic h have c lose relationsh ip to Bradyrhizobillnl elkanJi and B j aponiclIfll. However, the "phy logenetic tTee" constructed on the ba is of part ln l ll odC gene md icatcd the existence of an JIldependen t group, si nce they did not show any siglU tica nt degree of relationsh ip w ith the ex isting groups. T hi s was also supported by diffi rences in pby iological characteri tiC i.e Indole Ace ti c Acid productIOn and salt tolerance of the iso lates . These differe nces shows uS that "direct _equenc ing" method of certain specdic genes coul d give a more specific re ult tha n ou ld di p lay more clearly the degree of relat ionship at species leve l.