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"Polyclonal antibodies of aflatoxin B1 were successfully produced from New Zealand White female rabbits after immunization by the hapten of aflatoxin B1-carboxymethyl hydroxylamine hemihydrochloride (AFB1-CMO) conjugated with bovine serum albumin (BSA) as the antigen. The hapten was synthesized using the carbodiimide method with CMO as a linker. Absorption peaks at 362, 264, and 218 nm were observed as a result of characterization with UV-Vis spectroscopy, while IR spectroscopy showed peaks at 3448 cm-1 and 1642 cm-1 attributable to the hydroxyl and nitrile groups, respectively. Furthermore, mass spectrometry showed fragmentation at the m/z of 386, 368.2, and 310, which confirms that the hapten of AFB1-CMO was successfully synthesized. The hapten was then conjugated with BSA to serve as an antigen of AFB1 when it was injected into the rabbits. The specificity of the antigen towards its antibody and the confirmation of hapten-BSA conjugation were characterized using the dot blot immunoassay, which showed a BSA concentration of 1.74 mg/mL. Two weeks after the primary immunization by its antigen, agar gel precipitation testing showed that the rabbit blood serum had positive results for polyclonal antibodiest against AFB1 with the highest concentration of antibodiest of 2.19 mg/mL."

"Sintesa of Poliklonal Antibodi Aflatoxin B1. Antibodi poliklonal aflatoksin B1 telah berhasil diproduksi pada hewan uji kelinci betina New Zealand White setelah diimunisasi dengan hapten aflatoksin B1-carboxymethyl hydroxylamine hemihydrochloride (AFB1-CMO) yang dikonjugasikan dengan bovin serum albumin (BSA) sebagai antigen. Hapten AFB1 disintesis menggunakan metode karbodiimida dengan CMO sebagai linker. Puncak absorbansi pada 362, 264, 218 nm teramati sebagai hasil karakterisasi menggunakan spektrofotometer UV-Visibel, sementara dengan spektrum IR diperoleh puncak pada 3448.126 cm-1 dan 1642.451 cm-1 yang masing-masing mengindikasikan adanya gugus hidroksil dan nitril. Hasil spektrometri massa menunjukkan fragmentasi pada m/z 386, 368.2, dan 310 yang membuktikan hapten AFB1-CMO telah berhasil disintesis. Hapten ini kemudian dikonjugasikan dengan BSA agar dapat berperan sebagai antigen AFB1 ketika diinjeksikan pada kelinci. Kekhususan antigen aflatoksin B1 terhadap antibodinya dan konfirmasi konjugat hapten-BSA menunjukkan hasil positif pada uji dot blot immunoassay dengan konsentrasi BSA sebesar 1.74 mg/mL. Dua pekan setelah imunisasi primer, agar gel precipitation test menunjukkan hasil positif terhadap antibodi poliklonal AFB1 dengan konsentrasi tertinggi 2.19 mg/mL

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Polyclonal antibodies of aflatoxin B1 were successfully produced from New Zealand White female rabbits after immunization by the hapten of aflatoxin B1-carboxymethyl hydroxylamine hemihydrochloride (AFB1-CMO) conjugated with bovine serum albumin (BSA) as the antigen. The hapten was synthesized using the carbodiimide method with CMO as a linker. Absorption peaks at 362, 264, and 218 nm were observed as a result of characterization with UV-Vis spectroscopy, while IR spectroscopy showed peaks at 3448 cm-1 and 1642 cm-1 attributable to the hydroxyl and nitrile groups, respectively. Furthermore, mass spectrometry showed fragmentation at the m/z of 386, 368.2, and 310, which confirms that the hapten of AFB1-CMO was successfully synthesized. The hapten was then conjugated with BSA to serve as an antigen of AFB1 when it was injected into the rabbits. The specificity of the antigen towards its antibody and the confirmation of hapten-BSA conjugation were characterized using the dot blot immunoassay, which showed a BSA concentration of 1.74 mg/mL. Two weeks after the primary immunization by its antigen, agar gel precipitation testing showed that the rabbit blood serum had positive results for polyclonal antibodiest against AFB1 with the highest concentration of antibodiest of 2.19 mg/mL."

"Polyclonal antibodies of aflatoxin B1 were successfully produced from New Zealand White female rabbits afterimmunization by the hapten of aflatoxin B1-carboxymethyl hydroxylamine hemihydrochloride (AFB1-CMO)conjugated with bovine serum albumin (BSA) as the antigen. The hapten was synthesized using the carbodiimidemethod with CMO as a linker. Absorption peaks at 362, 264, and 218 nm were observed as a result of characterizationwith UV-Vis spectroscopy, while IR spectroscopy showed peaks at 3448 cm-1 and 1642 cm-1 attributable to thehydroxyl and nitrile groups, respectively. Furthermore, mass spectrometry showed fragmentation at the m/z of 386,368.2, and 310, which confirms that the hapten of AFB1-CMO was successfully synthesized. The hapten was thenconjugated with BSA to serve as an antigen of AFB1 when it was injected into the rabbits. The specificity of the antigentowards its antibody and the confirmation of hapten-BSA conjugation were characterized using the dot blotimmunoassay, which showed a BSA concentration of 1.74 mg/mL. Two weeks after the primary immunization by itsantigen, agar gel precipitation testing showed that the rabbit blood serum had positive results for polyclonal antibodiestagainst AFB1 with the highest concentration of antibodiest of 2.19 mg/mL.Sintesa of Poliklonal Antibodi Aflatoxin B1. Antibodi poliklonal aflatoksin B1 telah berhasil diproduksi pada hewanuji kelinci betina New Zealand White setelah diimunisasi dengan hapten aflatoksin B1-carboxymethyl hydroxylaminehemihydrochloride (AFB1-CMO) yang dikonjugasikan dengan bovin serum albumin (BSA) sebagai antigen. HaptenAFB1 disintesis menggunakan metode karbodiimida dengan CMO sebagai linker. Puncak absorbansi pada 362, 264,218 nm teramati sebagai hasil karakterisasi menggunakan spektrofotometer UV-Visibel, sementara dengan spektrum IRdiperoleh puncak pada 3448.126 cm-1 dan 1642.451 cm-1 yang masing-masing mengindikasikan adanya gugus hidroksildan nitril. Hasil spektrometri massa menunjukkan fragmentasi pada m/z 386, 368.2, dan 310 yang membuktikan haptenAFB1-CMO telah berhasil disintesis. Hapten ini kemudian dikonjugasikan dengan BSA agar dapat berperan sebagaiantigen AFB1 ketika diinjeksikan pada kelinci. Kekhususan antigen aflatoksin B1 terhadap antibodinya dan konfirmasikonjugat hapten-BSA menunjukkan hasil positif pada uji dot blot immunoassay dengan konsentrasi BSA sebesar 1.74mg/mL. Dua pekan setelah imunisasi primer, agar gel precipitation test menunjukkan hasil positif terhadap antibodipoliklonal AFB1 dengan konsentrasi tertinggi sebesar 2.19 mg/mL."

"[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]"

"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"

"ABSTRAKGelatin merupakan salah satu jenis protein yang diperoleh dari kolagen alami yang terdapat dalam kulit dan tulang hewan. Gelatin yang paling banyak digunakan ialah gelatin babi. Penggunaan gelatin dengan bahan baku hewan babi pada produk makanan dan farmasi seringkali menyebabkan alergi, selain juga tidak diperbolehkan dalam agama islam, sehingga perlu dilakukan analisis kandungan didalam makanan olahan agar aman untuk dikonsumsi. Salah satu analisis yang dapat digunakan ialah metode imunosensor, salah satunya Enzyme-Linked Immunosorbent Assay ELISA yang memiliki tingkat sensitifias yang tinggi, mampu menguji sampel yang tidak murni, dan mampu mengikat secara selektif antigen yang dikehendaki. Metode ini memerlukan antibodi anti-gelatin babi untuk mendeteksi gelatin babi sehingga dilakukan produksi antibodi poliklonal antigen gelatin babi menggunakan hewan uji kelinci. Pada penelitian ini produksi antibodi poliklonal gelatin babi dilakukan dari antigen gelatin babi dengan mengekstraksi gelatin dari kulit babi. Rendemen yang dihasilkan sebesar 20,52 dan dikarakterisasi dengan spektrofotometer UV-Vis dan FTIR serta dianalisis dengan SDS PAGE dihasilkan bahwa gelatin babi memiliki 5 pita pemisahan dan gelatin dapat dijadikan sebagai antigen. Antibodi poliklonal antigen gelatin babi dapat diproduksi dengan hewan uji kelinci dan antibodi yang diperoleh pada hari ke 65 dilakukan pemurnian IgG. Antibodi yang dipurifikasi tersebut dapat dijadikan sebagai konjugat sensor pada imunosensor berbasis ELISA dengan konsentrasi IgG sebesar 0,289 mg/mL.

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ABSTRACTGelatin is one type of protein derived from natural collagen found in the skin and animal bones. The most widely used gelatin is pork gelatin. The use of gelatin with raw materials of pigs on food and pharmaceutical products often causes allergies, as well as not allowed in Islam, so it is necessary to analyze the content in processed foods to be safe for consumption. One of the analysis that can be used is immunosensor method, one of them is Enzyme Linked Immunosorbent Assay ELISA which has high sensitivity level, able to test impure samples, and able to selectively bind antigen desired. This method requires a pig anti gelatin antibody to detect pig gelatin so that the production of polyclonal antibody of pig gelatin antigen using rabbit test animals. In this study the production of polyclonal antigen pig gelatin carried out from pig gelatin antigen by extracting gelatin from pig skin. The yield of gelatin 20.52 and characterized by UV Vis and FTIR spectrophotometers and analyzed by SDS PAGE was found that pig gelatin has 5 separation bands and gelatin can be used as antigen. Polyclonal antibodies of antigen porcine gelatin can be produced with rabbit test animals and antibodies obtained on the 65th day of IgG purification. The purified antibodies can be used as conjugate sensors on immunosensors based ELISA with a concentration of IgG of 0.289 mg mL."

"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."