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"ABSTRAKLatar Belakang. Protein yang berperan penting dalam fungsi sperma berpotensi sebagai target molekul dalam upaya pengembangan bahan kontrasepsi pria. Salah satu protein yang terdapat pada sperma adalah protein kanal Voltage Dependent Anion Channel3 (VDAC3). VDAC3 berfungsi mengatur aliran ion dan metabolit termasuk ATP. Dari penelitian dengan menggunakan teknik knock-out mouse pada gen VDAC3 dilaporkan bahwa mencit jantan mutan VDAC3 homozigot mengalami penurunan yang signifikan dalam motilitas spermanya. Tujuan penelitian ini adalah memproduksi antibodi poliklonal VDAC3 melalui imunisasi protein rekombinan VDAC3 murni dan uji aktivitasnya terhadap motilitas dan viabilitas sperma manusia.Metode. Verifikasi keberhasilan pemotongan His fussion tag beserta 31 asam amino plasmid dari protein rekombinan dilakukan dengan teknik Western blott. ELISA digunakan untuk mengetahui titer IgG anti VDAC3sedangkan uji efektifitas antibodi VDAC3 terhadap fungsi sperma dilakukan dengan menghitung prosentase sperma yang tidak bergerak, waktu yang ditempuh sperma dalam jarak 0,1 mm. Analisa viabilitas sperma dilakukan dengan metode pewarnaan eosin.Hasil. Pada penelitian ini Western blotting dengan menggunakan antibodi Rabbit Anti VDAC Human menghasilkan pita tunggal dengan ukuran ~ 16 kDa, sedangkan penggunaan antibodi terhadap His (C-term) tidak menunjukan adanya pita. Hasil spektofotometri ELISA titer antibodi poliklonal VDAC3 yang berasal dari kelinci menunjukkan adanya peningkatan titer antibodi poliklonal VDAC3 setelah imunisasi dibandingkan dengan titer antibodi sebelum imunisasi (preimun serum). Hasil uji aktivitas antibodi poliklonal VDAC3 menunjukkan terjadi peningkatan jumlah sperma bergerak yang bermakna pada waktu 30 menit (p<0,05) dan 60 menit (p<0,05), juga terjadi peningkatan waktu tempuh sperma yang bermakna pada waktu 0-30 menit (p<0,05) setelah perlakuan. Penambahan antibodi poliklonal VDAC3 juga berpengaruh secara nyata terhadap persentase viabilitas spermatozoa yang hidup (p<0,05).Kesimpulan. VDAC3 poliklonal antibodi berhasil diproduksi melalui imunisasi dari VDAC3 rekombinan murni. Antibodi poliklonal anti-protein rekombinan Voltage Dependent Anion Channel-3 (VDAC3) dapat menurunkan motilitas dan viabilitas sperma manusia invitro secara bermakna.

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ABSTRACT

Background. Sperm-specific proteins that are important for sperm function can potentially be used as a target for developing a male contraceptive. One of the proteins found in the human sperm is Voltage Dependent Anion Channel3 (VDAC3). VDAC3 regulates the flow of ions and metabolites including ATP in the mitochondrial membrane and cell membrane of the eukaryotes. A previous study showed VDAC3 knockout mice had significant reduction in sperm motility. The purpose of this study was to produce polyclonal antibodies through immunization of pure VDAC3 recombinant protein and analyze its effect towards sperm motility and viability.

Methods. Removal of the His fussion tags plus 31 amino acids from the recombinant plasmid was verified using western immunoblotting. The titter of VDAC3 polyclonal antibody was determined by ELISA. The effect of VDAC3 antibodies against sperm qualities namely motility and viability was assessed using standard sperm analyses approved by the WHO.

Results. Western immunoblotting using Rabbit Anti Human VDAC3, produced a single band with size of ~ 16 kDa. No visible band was detected when anti-His (C-term) antibody was used in the analyses. Spectophotometric ELISA showed that the titer of VDAC3 polyclonal antibodies, derived from rabbits, polyclonal antibody increased better than the pre-immune. Analyses of VDAC3 polyclonal antibody against human sperm showed an increase in the number of sperm to move significant at 30 minutes (p < 0.05) and 60 minutes (p < 0.05), as well as an increase in sperm significant travel time at the time of 0-30 minutes (p < 0.05) after treatment. Polyclonal antibodies VDAC3 also significantly affect the percentage of sperm viability (p < 0.05).

Conclusion. Polyclonal antibody anti-VDAC3 was successfully produced via immunization of the pure recombinant VDAC3. Polyclonal antibody anti-recombinant protein Voltage Dependent Anion Channel-3 (VDAC3) may decrease human sperm motility and viability in vitro significantly."

"Pendahuluan: VDAC merupakan saluran ion pada spermatozoa yang berperan penting dalam pengangkutan ATP, ion, dan metabolit di dalam membran sel. Telah diketahui VDAC3 berperan dalam motilitas sperma. Tujuan dari penelitian ini adalah memproduksi antibodi anti-VDAC3 melawan protein rekombinan hasil ekspresi vektor rekombinan VDAC3 dan menguji efeknya terhadap viabilitas dan motilitas sperma ejakulat manusia.Metode: Protein rekombinan VDAC3 diimunisasikan selama 14 minggu ke 2 ekor kelinci. Kemudian serum kelinci minggu ke -6 diuji dengan metode ELISA untuk mengetahui titer antibodi anti-VDAC3 dalam serum dan serum preimun sebagai kontrolnya. Serum mengandung antibodi anti-VDAC3 dan serum preimun dipaparkan terhadap 22 sampel ejakulat sperma manusia dan dilihat efeknya terhadap parameter motilitas sperma, Velocity Average Path (VAP), Curvilinear Velocity (VCL), dan Velocity Average Path (VAP) dengan menggunakan Computer Assisted Sperm Analysis (CASA), serta uji viabilitas sperma dengan pewarnaan Eosin-Y pada 0, 30, dan 60 menit paparan.Hasil: Terdapat perbedaan bermakna viabilitas sperma ejakulat manusia yang dipapar dengan serum antibodi anti-VDAC3 dibandingkan dengan serum preimun dalam waktu 0, 30, dan 60 (p=0,001). Selain itu, terdapat perbedaan presentase motilitas sperma ejakulat yang dipapar dengan serum antibodi anti-VDAC3 dibandingkan dengan serum preimun dalam waktu 0 dan 30 menit (p=0,007; 0,001), sedangkan pada waktu 60 menit tidak bermakna (p=0,062). Pengujian terhadap parameter motilitas VAP, VSL, dan VCL, tidak menunjukkan perbedaan bermakna (p>0,05).Kesimpulan: Serum antibodi anti-VDAC3 rekombinan dapat menurunkan viabilitas dan motilitas sperma ejakulat manusia

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Introduction: VDAC is an ion channel in spermatozoa that plays an important role in the transport of ATP, ions and metabolites in the cell membrane, play a role in sperm motility. The aim of this study was to produce anti-VDAC3 antibodies against VDAC3 recombinant protein that expressed in E coli and evaluated their effect on viability and motility parameters of human ejaculate sperm.

Methods: The VDAC3 recombinant protein produced then immunized for 14 weeks to 2 rabbits. The VDAC3 antiserum on 6 weeks was tested by ELISA method to determine the VDAC3 antibody titer in serum and preimmune serum as control. Then, this antiserum and preimmune serum were exposed to 22 samples of human sperm ejaculate and evaluated the effect on viability parameters with Eosin-Y staining, percentage of motility, VAP, VCL, and VSL using computer assisted sperm analysis for 0, 30, and 60 minutes of exposure.

Results: There were a significant difference in the viability of human ejaculate sperm exposed to anti-VDAC3 antiserum compared to preimmune serum at 0, 30, and 60 minutes (p = 0.001). In addition, there were a significant difference in the percentage of motility of ejaculated sperm that was exposed by anti-VDAC3 antiserum compared to preimmune serum at 0 and 30 minutes (p = 0.007; 0.001; respectively). Furthermore, percentage of motility of ejaculated sperm that was exposed by anti-VDAC3 antiserum compared to preimmune serum at 60 minutes and the effect of antiserum on the VAP, VSL, and VCL parameters did not show any difference (p>0.05).

Conclusions: Anti-VDAC3 antibody could reduce ejaculated sperm motility parameters and decrease ejaculated sperm living cells"

"Latar Belakang. Voltage Dependent Anion Channel3 (VDAC3) merupakan salah satu protein yang terdapat pada sperma. Pada pengembangan imunokontrasepsi, protein tersebut dapat dijadikan antigen potensial dalam menurunkan fungsi sperma. Beberapa penelitian menunjukan bahwa anti-VDAC dapat mengganggu fungsi normal dan meningkatkan abnormalitas morfologi sperma. Imunisasi protein VDAC3 rekombinan terhadap kelinci diharapkan dapat memicu terbentuknya antibodi poliklonal anti-VDAC3. Antibodi tersebut kemudian dipurifikasi dan dievaluasi kemampuannya dalam mengikat antigen VDAC3 yang terdapat pada sperma manusia melalui pengamatan motilitas, viabilitas, integritas membran, dan integritas akrosom.Metode. Protein rekombinan VDAC3 diproduksi dengan cara mengkultur bakteri E.coli BL21 yang mengandung konstruksi vektor rekombinan. Protein rekombinan tersebut diimunisasikan pada kelinci kemudian diambil antiserumnya. Antibodi VDAC3 yang terkandung dalam antiserum dipurifikasi dengan metode kromatografi afinitas matriks sepharose protein A kemudian konsentrasinya diukur dengan metode ELISA dan dianalisis kemurniannya secara kualitatif dengan SDS-PAGE. Antibodi VDAC3 tersebut kemudian diuji kemampuannya terhadap sperma manusia normal dengan parameter: motilitas, kecepatan pergerakan, mortalitas (menggunakan metode pewarnaan Eosin Y), integritas membran ekor (menggunakan metode HOST), dan integritas akrosom (menggunakan metode FITC-PNA).Hasil. Kultur biakan E. coli dengan penambahan IPTG menunjukkan konsentrasi protein terlarut lebih tinggi yaitu 2,051 mg/ml dibandingkan dengan tanpa IPTG yaitu 1,528 mg/ml. Imunisasi protein VDAC3 rekombinan terhadap kelinci menghasilkan antiserum yang mengandung antibodi antiVDAC3 dengan titer tertinggi yaitu 3,504 pada kelinci B. Hasil SDS-PAGE antibodi yang dimurnikan menunjukan dua pita yang mendekati ukuran ~ 55 kDa dan ~25 kDa. Analisis statistik dari pengaruh antibodi VDAC3 murni terhadap motilitas, kecepatan gerakan, mortalitas, integritas membran ekor dan integritas akrosom sperma menunjukkan perbedaan yang signifikan (p <0,05) dengan kontrol preimun serum.

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ABSTRACTBackground. Voltage Dependent Anion Channel 3 (VDAC 3) is one of the proteins found in sperm. On the development of immunocontraception, this protein can be used as potential antigens to reduce normal function of sperm. Several studies have shown that anti-VDAC can reduce normal function of sperm and also increase the morphological abnormalities of sperm. VDAC3 recombinant protein immunization to rabbit is expected to produce of polyclonal antibodies anti-VDAC3. Then, the antibodies are purified and evaluated its ability to bind antigen VDAC3 contained in human sperma by observation of motility, sperm speed, viability, membrane integrity, and the integrity of the acrosome.Method. VDAC3 recombinant protein produced by culturing of E. coli BL21 that contains the construction of recombinant vector. This recombinant protein immunized to rabbits and then the antiserum taken from blood sample. VDAC3 antibodies that contained in antiserum purified by affinity chromatography matrix protein A Sepharose then the concentration was measured by ELISA and the purity analyzed by SDS-PAGE. The ability of VDAC3 antibodies were then tested to normal human sperma with parameters are: sperm motility, sperm speed, mortality (eosin Y staining method), membrane integrity (using HOST), and the integrity of the acrosome (using FITC-PNA).Results. Culture of E. coli with addition of IPTG showed a higher concentrations (2,051 mg/ml) than without IPTG (1,528 mg/ml). Immunization of protein VDAC3 recombinant to rabbit produce antiserum that contains antibodies with the highest titers 3,504 in rabbit B. SDS-PAGE analysis two protein fragmen with size of ~ 55 kDa and ~ 25 kDa. Statistical analysis of the effect of pure VDAC3 antibodies to motility, sperm speed, mortality, membrane integrity, and the integrity of the acrosome showed a significant difference (p < 0.05) with control preimun serum."

"Penelitian bertujuan untuk mendapatkan antibodi poliklonal kelinci yang distimulasi oleh protein rekombinan globular head neuraminidase (NA) dan mengukur titer antibodi poliklonal. Protein rekombinan globular head NA berhasil diekspresikan secara intraseluler pada sel E.coli BL21 codon plus dengan induksi IPTG 0,1 mM dan dipurifikasi menggunakan resin Ni-NTA. Protein rekombinan globular head NA yang telah dipurifikasi digunakan sebagai antigen untuk menstimulasi antibodi poliklonal kelinci. Hasil penelitian menunjukkan bahwa telah dihasilkan antibodi poliklonal terhadap globular head neuraminidase dan titer antibodi paling tinggi dihasilkan sebesar 1,352.

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The aim of this study was to determine rabbit polyclonal antibody stimulated by neuraminidase (NA) globular head recombinant protein and also to measure the polyclonal antibody titer. NA globular head recombinant protein has been expressed in E.coli BL21 codon plus intracellularly induced by 0,1 mM IPTG and has been purified by Ni-NTA resin. The purified of NA globular head recombinant was used as antigen to stimulate rabbit polyclonal antibody.

The result shows that rabbit polyclonal antibody of neuraminidase globular head was produced and the highest antibody titer was 1,352."

"ABSTRAKPendahuluan. VDAC merupakan protein kanal ion yang bertanggung jawab atas aliran ion Ca2+ dan ATP dalam flagela spermatozoa. Defisiensi gen VDAC3 pada mencit dan mutasi gen VDAC3 pada manusia menyebabkan penurunan motilitas spermatozoa, sehingga VDAC3 dapat dijadikan antigen potensial untuk pengembangan vaksin kontrasepsi laki-laki. Tujuan penelitian ini adalah memproduksi protein rekombinan hVDAC3 dari gen hVDAC3 ekson 5-8 spesifik spermatozoa, dan digunakan sebagai antigen untuk produksi antibodi poliklonal pada kelinci.Metode. Gen hVDAC3 ekson 5-8 spermatozoa diperoleh melalui RT PCR, gen disisipkan ke plasmid pET100/D-TOPO dan diklona dalam E coli TOP 10. Analisis gen sisipan dengan PCR, enzim restriksi dan sekuensing DNA. Protein rekombinan hVDAC3 diekspresikan dalam E coli BL21 StarTM (DE3). Karakterisasi protein dilakukan dengan uji Bradford, SDS PAGE, western blot dan purifikasi protein dengan resin Ni-NTA. Antibodi poliklonal diperoleh dengan cara imunisasi protein rekombinan hVDAC3 ke kelinci dan diukur dengan indirect ELISA. Determinasi lokasi hVDAC3 di spermatozoa dengan metode immunoflurosence.Hasil. Amplifikasi PCR gen hVDAC3 ekson 5-8 berukuran 435 pb dan analisis BLAST menunjukkan 100% identik dengan gen VDAC3 manusia dari bank gen. Vektor rekombinan berukuran 6195 pb mengekspresikan protein rekombinan hVDAC3 berukuran 20 kDa. Antibodi poliklonal telah diproduksi kelinci secara bermakna (p<0.05) dengan titer 2.817, dan antibodi dapat berikatan dengan protein hVDAC3 di kepala dan flagela spermatozoa. Selanjutnya, antibodi poliklonal ini akan digunakan dalam pengembangan vaksin kontrasepsi pada laki-laki.

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ABSTRACT

Introduction. Voltage dependent anion channels (VDAC), also known as mitochondrial porins, are group of proteins in mitochondrial outer membrane that allow the passage of metabolites across the mitochondrial outer membrane, and are involved in ions and ATP transport in sperm flagella. Deficiency and mutation of VDAC3 may cause abnormality in structure and motility of human spermatozoa. VDAC3 could be a potential target to develop non hormonal male contraceptive vaccine. The objective of the study was to produce hVDAC3 recombinant proteins from exon 5 to 8 of human sperm VDAC3 spesific gene.

This recombinant protein was subsequenly used as an antigen to produce polyclonal antibodies in rabbits. Methods. hVDAC3 sperm gene obtained by RT PCR, this gene was inserted into plasmid pET 100/D-TOPO and cloned in E coli TOP 10. The gene was analyzed by PCR method, restriction enzymes and DNA sequencing. The proteins expressed in E coli BL21 StarTM (DE3). Characterization of proteins was evaluated by Bradford method, SDS PAGE and western blot. The recombinant protein was purified with NI-NTA resin. Polyclonal antibodies were obtained by immunization of hVDAC3 recombinant protein into rabbits. Indirect ELISA was done to analyze the antibody. Localization of the VDAC3 recombinant protein in human spermatozoa was evaluated by immunofluorescence method.

Result. By doing PCR amplification and BLAST analysis, the study showed that the hVDAC3 gene had 100% identical to hVDAC3 genes in data bank. E coli BL21 StarTM (DE3) containing recombinant vector (6195 bp) expressed the recombinant protein of hVDAC3 in 20 kDa. This protein produced polyclonal antibodies that bound VDAC3 protein on the head and flagella of human spermatozoa."

"[ABSTRAKAntibodi poliklonal anti aflatoksin B1 telah berhasil diproduksi pada hewan uji kelinci betina New Zealand White setelah diimunisasikan hapten aflatoksin B1-CMO yang dikonjugasikan dengan Bovine Serum Albumin (BSA) sebagai antigen. Hapten aflatoksin B1-CMO disintesis menggunakan metode karbodiimida dengan substrat aflatoksin B1 dan carboxymethyl hydroxylamine hemihydrochloride (CMO) sebagai linkernya. Hasil karakterisasi kromatografi lapis tipis dengan nilai Rf rata-rata sebesar 0.395, spektrum UV-Visibel dengan puncak λ maks pada 362, 264, 218 nm, spektrum IR dengan puncak 3448.126 cm-1 (3000-3600 cm-1) : OH, pada 1632.249 cm-1(1540-1725 cm-1) : C=O, dan 1642.451 cm-1 (1640-1690 cm-1) :C=N (Oksim), dan hasil fragmentasi spektrometri massa (MS/MS) pada m/z 386, 368.2, 310 membuktikan hapten aflatoksin B1-CMO berhasil disintesis. Hapten ini kemudian dikonjugasikan dengan BSA membentuk antigen aflatoksin B1-BSA (AFB1-BSA) sebelum diimunisasikan ke kelinci. Spesifitas antigen AFB1-BSA terhadap antibodinya dan uji konjugasi hapten ke BSA menunjukkan hasil positif menggunakan uji Dot Blot Immunoassay dengan konsentrasi BSA di dalam antigennya sebesar 1.74 mg/mL. Serum darah kelinci berdasarkan uji Agar Gel Precipitation Test (AGPT) positif mengandung antibodi poliklonal anti aflatoksin B1 setelah dua pekan (hari ke-11) sejak imunisasi primer antigen AFB1-BSA dilakukan. Dari serum darah bleeding panen, diperoleh konsentrasi antibodinya sebesar sebesar 2.19 mg/mL. Immunokromatogafi strip tes berhasil dibuat dengan nanopartikel iridium oksida (IrO2 NPs) sebagai kandidat label antibodinya dan dapat digunakan untuk mendeteksi sampel H IgG pada rentang 0.1 μg/mL sampai 10 μg/mL. Studi pendahuluan ini menunjukkan bahwa perangkat strip tes ini dapat digunakan untuk aplikasi konjugat sensor antibodi anti aflatoksin B1-nanopartikel iridium oksida untuk deteksi aflatoksin B1.

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ABSTRACTPolyclonal antibody against aflatoxin B1 have been successfully produced in New Zealand White Rabbit after immunized by hapten of aflatoxin B1-CMO conjugated with Bovine Serum Albumin (BSA) as antigen. Hapten of aflatoxin B1-CMO was synthesized using carbodiimide method with afltoksin B1 as substrate and carboxymethyl hydroxylamine hemihydrochloride (CMO) as its linker. The characterization results of thin layer chromatography with Rf value of 0.395, the spectrum of UV-Visible with λ max peaks at 362, 264, 218 nm, the IR spectrum with peak at 3448.126 cm-1 (3000-3600 cm-1): OH , 1632.249 cm-1(1540-1725 cm-1): C = O, 1642.451 cm-1 (1640-1690 cm-1): C = N (oxime), and the results of mass spectrometry fragmentation (MS / MS) at m/ z of 386, 368.2, 310 proved that hapten of aflatoxin B1 -CMO successfully synthesized. Then, the hapten was conjugated to BSA to form antigen of aflatoxin B1-BSA (AFB1-BSA) before immunized to rabbits. The specificity of antigen of AFB1-BSA to its antibody and the confirmation of hapten-BSA conjugated showed positive results using dot blot immunoassay with BSA concentration in the antigen of 1.74 mg/mL. Based on Agar Gel Precipitation Test (AGPT) shown the rabbit blood serum resulted positive for polyclonal antibody against aflatoxin B1 after two weeks (day 11st) since the primary immunization of its antigen. From blood serum bleeding at harvest obtained the concentration of antibodies was 2.19 mg / mL. An Immunochromatogaphic test strip was successfully fabricated using iridium oxide nanoparticles (IrO2 NPs) as a labeled antibody candidate and can be used to detect the IgG H sample between of 0.1 μg/mL to 10 μg/mL. This preliminary study shown that the device can be used for applications of antibody against aflatoxin B1-nanoparticle iridium oxide conjugate for detection of aflatoxin B1, Polyclonal antibody against aflatoxin B1 have been successfully produced in New Zealand White Rabbit after immunized by hapten of aflatoxin B1-CMO conjugated with Bovine Serum Albumin (BSA) as antigen. Hapten of aflatoxin B1-CMO was synthesized using carbodiimide method with afltoksin B1 as substrate and carboxymethyl hydroxylamine hemihydrochloride (CMO) as its linker. The characterization results of thin layer chromatography with Rf value of 0.395, the spectrum of UV-Visible with λ max peaks at 362, 264, 218 nm, the IR spectrum with peak at 3448.126 cm-1 (3000-3600 cm-1): OH , 1632.249 cm-1(1540-1725 cm-1): C = O, 1642.451 cm-1 (1640-1690 cm-1): C = N (oxime), and the results of mass spectrometry fragmentation (MS / MS) at m/ z of 386, 368.2, 310 proved that hapten of aflatoxin B1 -CMO successfully synthesized. Then, the hapten was conjugated to BSA to form antigen of aflatoxin B1-BSA (AFB1-BSA) before immunized to rabbits. The specificity of antigen of AFB1-BSA to its antibody and the confirmation of hapten-BSA conjugated showed positive results using dot blot immunoassay with BSA concentration in the antigen of 1.74 mg/mL. Based on Agar Gel Precipitation Test (AGPT) shown the rabbit blood serum resulted positive for polyclonal antibody against aflatoxin B1 after two weeks (day 11st) since the primary immunization of its antigen. From blood serum bleeding at harvest obtained the concentration of antibodies was 2.19 mg / mL. An Immunochromatogaphic test strip was successfully fabricated using iridium oxide nanoparticles (IrO2 NPs) as a labeled antibody candidate and can be used to detect the IgG H sample between of 0.1 μg/mL to 10 μg/mL. This preliminary study shown that the device can be used for applications of antibody against aflatoxin B1-nanoparticle iridium oxide conjugate for detection of aflatoxin B1]"

"Spermisida merupakan salah satu kontrasepsi non-hormonal yang bahan aktif utamanya adalah Nonoxynol-9 (N-9). Bahan aktif tersebut diketahui mampu merusak membran spermatozoa untuk mencegah kehamilan. Spermisida memiliki efektivitas yang rendah, sehingga perlu digunakan bersama kontrasepsi lain. Penggunaan berulang N-9 dapat menyebabkan iritasi pada organ reproduksi. Bahan biologis diharapkan menjadi bahan spermisida yang lebih aman dan efektif. VDAC3 merupakan protein yang dapat ditemukan pada spermatozoa dan berperan mengatur motilitas spermatozoa. Oleh karena itu, penelitian ini bertujuan membentuk gel kontrasepsi yang mengandung antiserum VDAC3 yang mampu menghambat motilitas, viabilitas, dan integritas membran spermatozoa. Subjek penelitian adalah 25 semen laki-laki usia 25-40 tahun yang sudah memiliki anak dan normozoosperma. Terbagi menjadi empat kelompok yaitu sperm only, gel only, pre-imun serum, dan antiserum VDAC3. Uji karakteristik gel terdiri dari uji daya sebar, uji pH, dan pengamatan perubahan warna. Uji efektivitas yang meliputi motilitas menggunakan Computer Assisted Sperm Analysis (CASA), viabilitas menggunakan eosin Y, dan integritas membran menggunakan uji Hypoosmotic Swelling (HOS). Hasil menunjukkan daya sebar, pH, dan warna yang stabil selama satu bulan. Hasil efektivitas menunjukkan tidak ada perbedaan bermakna antara motilitas total pre-imun dan antiserum VDAC3, namun viabilitas dan integritas membran memberikan hasil yang berbeda bermakna. Gel dengan kandungan antiserum VDAC3 berpotensi sebagai spermisida.

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Spermicide is non-hormonal contraceptive that contains Nonoxynol-9. Nonoxynol-9 can decrease spermatozoa motility and viability. However, it should be used with other contraception because the effectiveness is low and can irritate genitalia organs. Biological materials are expected to be safer and effective. VDAC3 is a protein channel that can be found in spermatozoa and have the function of sperm motility. Therefore, this research aim is to produce a contraceptive gel containing antiserum VDAC3 and determine its effectiveness on sperm motility, viability, and membrane integrity. We use 25 samples of semen from males aged 25-40, who have a child, and normozoospermic—divided into four groups sperm only, gel only, pre-immune serum, and antiserum VDAC3. The characteristics evaluation involves pH measurement, spreadability, and gel color change. The effectiveness experiment involves sperm motility observation using Computer Assisted Sperm Analysis (CASA), viability examination with eosin Y, and membrane integrity examination using the Hypoosmotic swelling test (HOS). This study's results show that the gel has stable characteristics based on pH, spreadability, and color after one month. There is no significant difference between pre-immune serum and antiserum VDAC3 total motility, but have significant differences in sperm viability and membrane integrity. The gel contains antiserum VDAC and has the potential to be a spermicide."

"Latar belakang: Tujuan penelitian ini adalah untuk membuat konstruksi vektor rekombinan gen Voltage-Dependent Anion Channel isoform 3 (VDAC3) spesifik sperma manusia, untuk produksi antibody VDAC3 yang berpotensi sebagai bahan kontrasepsi laki-laki.Metode: Fragmen target untuk pembuatan vektor rekombinan adalah gen VDAC3 spesifik sperma manusia, yang diperoleh dengan cara mengamplifikasi cDNA dari sperma manusia melalui metode PCR menggunakan primer spesifik gen VDAC3 exon 5 sampai dengan exon 8. Vektor rekombinan gen VDAC3 dikonstruksi dengan cara mengklon produk PCR tersebut (435 pb) ke vektor ekspresi pET101/D-TOPO (5753 pb). Selanjutnya bakteri E. coli TOP10 ditransformasi dengan vektor rekombinan di atas. Hasil klon gen VDAC3 pada vektor dikonfirmasi dengan pemotongan vektor rekombinan dengan enzim restriksi XbaI dan metode PCR colony pada bakteri yang tumbuh dengan menggunakan primer VDAC3 exon 5-8.Hasil: Analisis BLAST dari amplifikasi gen VDAC3 sperma manusia dengan primer spesifik exon 5 sampai exon 8 menunjukkan 94% identik dengan data gene bank. Bakteri E. coli transforman yang berhasil tumbuh ada 12 klon. Hasil elektroforesis vektor rekombinan VDAC3 yang telah dipotong dengan enzim restriksi XbaI, dari 12 klon yang tumbuh menunjukkan pita berukuran 6181 pb pada 8 klon bakteri. Setelah dilakukan metode PCR colony diperoleh pita berukuran 435 pb selanjutnya setelah disekuensing diperoleh sekuen amplicon yang 94% identik dengan gen VDAC3 manusia.Kesimpulan: Penelitian ini berhasil membuat konstruksi vektor rekombinan gen VDAC3 spesifik untuk sperma manusia, untuk pengembangan bahan kontrasepsi laki-laki di masa datang.

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AbstractBackground: The aim of this study was to construct a recombinant vector of human sperm specific VDAC3 gene for production of VDAC3 antibody, which is potential as male contraception vaccine.Methods: Target fragment sequence of VDAC3 gene was obtained through amplification of human sperm VDAC3 cDNA with primers covering exon 5 to exon 8. Its PCR product in size of 435 bp was cloned to the pET101/D-TOPO expression vector (5753 bp). E. coli bacteria were transformed with this vector. Cloning of VDAC3 fragment gene to the vector was confirmed by the using of XbaI restriction enzyme and PCR colony method with primers covering exons 5-8 of the human VDAC3 gene.Results: Alignment analysis of amplified fragment covering exon 5 to exon 8 of VDAC3 gene showed 94% homology to human VDAC3 gene from databank. After cloning to the expression vector and transformation to E. coli competent cells, twelve colonies could grow in culture media. Gel electrophoresis of sliced VDAC3 recombinant vector showed a single band in the size of 6181 bp in 8 colonies. After application of PCR colony and amplicon sequencing, the result showed a single band in the size of 435 bp and fragment sequence with 94% identity to human VDAC3 gene.Conclusion: The construction of human sperm specific VDAC3 gene recombinant vector was established in this study. In the future, this recombinant vector will be used to produce VDAC3 antibody for the development of a male contraception vaccine."

"Infeksi dengue DENV adalah penyakit yang diperantarai nyamuk yang manifestasinya dapat mengarah pada dengue hemorrhagic fever DHF dan/atau dengue shock syndrome DSS yang dapat mengakibatkan kematian. Di Indonesia, DHF sudah endemis dan menjadi penyakit yang terjadi sepanjang tahun. Protein non struktural-1 NS1 dari DENV diketahui merupakan biomarker dalam diagnosis dengue karena protein ini bersirkulasi dalam darah selama fase akut penyakit. Penelitian ini bertujuan untuk mengembangkan antibodi monoklonal mAb untuk mendeteksi antigen NS1 dari DENV serotipe 3 DENV3. Sel hibridoma penghasil mAb diperoleh dengan memfusikan sel B dari mencit yang diimunisasi dengan antigen NS1 yang diekspresikan pada sel CHO-K1 dengan sel PAI myeloma. Seleksi hibridoma dengan ELISA indirect diperoleh 16 klona yang berpotensi menghasilkan antibodi anti-NS1. Tujuh klona terbaik dipilih untuk dikarakterisasi dengan metode IFA terhadap antigen rekombinan NS1 dan hasilnya 6 klona positif menunjukkan reaksi sinyal fluoresens. Klona mAb 4-2D, 4-4F, dan 2-7A diuji terhadap protein NS1 native dari DENV1, DENV2, DENV3, dan DENV4, dan ketiga mAb tersebut mampu mengenali secara spesifik protein NS1 dan tidak bereaksi terhadap protein virus yang lain. Terdapat reaktivitas silang dengan 3 serotipe lainnya yang mengindikasikan bahwa mAb yang diujikan mengenali epitop lestari antigen NS1. Analisis prediksi epitop NS1 juga dilakukan secara in silico terhadap beberapa strain DENV lainnya. Namun, studi lebih lanjut mengenai pemetaan epitop dan afinitas pengikatan antigen-antibodi perlu dilakukan untuk menentukan mAb yang paling potensial sebagai bahan baku kit diagnostik.

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Dengue DENV is a mosquito borne infection disease which its manifestation can be lead to a lethal dengue hemorrhagic fever DHF and or dengue shock syndrome DSS . In Indonesia, DHF has been endemic and the disease occurs throughout the year. Non structural 1 NS1 protein of DENV is known to be a biomarker in dengue diagnosis since the protein is abundantly circulating in the blood during acute phase of the disease. The aim of this study to develop monoclonal antibodies mAbs derived from DENV3 to detect NS1. Hybridoma mAb producing cells were obtained by fusing B cells from an immunized mice with NS1 antigen expressed on CHO K1 cells, with PAI myeloma cells. Hybridoma selection with indirect ELISA showed 16 clones that could be potentially produce anti NS1 antibodies.

Seven up to sixteen clones were selected to be characterized by IFA against recombinant NS1 antigen and the result showed 6 clones produce fluorescence signals. Clones mAb 4 2D, 4 4F, and 2 7A were tested against native NS1 proteins from DENV1, DENV2, DENV3, and DENV4, and these mAbs were able to recognize specifically NS1 protein and did not react against other viral proteins. There is a cross reactivity within 3 other serotypes which initially indicate that mAbs recognizes the conserved epitopes determinant of the NS1 antigen. Epitopes prediction analysis was also performed in silico against several others DENV strains. However, further studies of epitope mapping and antigen antibody binding affinity are necessary to determine the most potential mAbs for diagnostic tools. "

"Spermatozoa merupakan sel inaktif dalam proses transkripsi dan translasi, sehingga pematangan spermatozoa dan kapasitasi dipengaruhi oleh perubahan atau modifikasi protein yang sudah ada sebelumnya. Studi proteomik menemukan reseptor prolaktin pada permukaan spermatozoa. Seminal plasma merupakan sumber protein yang cukup kaya, salah satunya Prolactin Inducible Protein (PIP). PIP diekspresikan secara berbeda dalam spermatozoa atau seminal plasma pada laki-laki infertil dan fertil. Dalam penelitian ini peneliti ingin menganalisa efek penambahan pemberian prolaktin pada normozoospermia untuk melihat apakah prolaktin berpengaruh terhadap aktivasi PIP, di daerah mana PIP terkespresi dengan menggunakan imunositokimia, peningkatan fosforilasi tirosin dengan menguji parameter kualitas spermatozoa berupa kinetik menggunakan CASA, dan kapasitasi melalui western blot. Hasil penelitian menunjukkan PIP pada kelompok normozoospermia diekspresikan pada permukaan nuclear membran, mid piece, dan tail spermatozoa. Penambahan prolaktin pada kultur spermatozoa kelompok normozospermia tidak terdapat perbedaan secara signifikan dalam mengaktivasi PIP. Penambahan prolaktin pada kelompok normozoospermia tidak terdapat perbedaan secara signifikan dalam meningkatkan kinetik spermatozoa. Penambahan prolaktin kelompok normozoospermia tidak terdapat perbedaan secara signifikan dalam meningkatkan fosforilasi tirosin.

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Spermatozoa are inactive cells in the processes of transcription and translation, so spermatozoa maturation and capacitation are influenced by changes or modifications of pre-existing proteins. A proteomic study found prolactin receptors on the surface of spermatozoa. Seminal plasma is a rich source of proteins, one of which is prolactin-inducible protein (PIP). PIP is expressed differently in spermatozoa or seminal plasma in infertile and fertile men. In this study, the researchers wanted to analyze the effect of adding prolactin to normozoospermia to see whether prolactin influenced PIP activation, in which areas PIP was expressed using immunocytochemistry, increased tyrosine phosphorylation by testing spermatozoa quality parameters in the form of kinetics using CASA, and capacitation via western blot. The results showed that PIP in the normozoospermia group was expressed on the surface of the nuclear membrane, midpiece, and tail spermatozoa. The addition of prolactin to the spermatozoa culture of the normozoospermia group did not show a significant difference in activating PIP. The addition of prolactin in the normozoospermia group did not show a significant difference in increasing the kinetics of spermatozoa. The addition of prolactin in the normozoospermia group did not show a significant difference in increasing tyrosine phosphorylation."