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"Sejak September 2008, ketika isu kontaminasi melamin diketahui secara luas, telah menjadi perhatian kesehatan internasional untuk memenuhi kebutuhan pemantauan cepat dan akurat deteksi melamin dalam sampel susu. Suatu metode indirect competitive enzyme-linked immunosorbent assay (icELISA) dengan spesifisitas yang tinggi dikembangkan untuk deteksi melamin dalam susu. Sintesis hapten dan pembentukan konjugat hapten-protein dilakukan sebagai immunogen untuk membentuk antibodi yang selektif terhadap melamin. Hapten melamin disintesis dengan mereaksikan asam gamma-amino butirat (GABA) dengan 2- kloro-4,6-diamino-1,3,5-triazin (CAAT). Digunakan disikloheksilkarbodiimida (DCC) dan N-hidroksisuksinimida (NHS) untuk merubah gugus karboksilat hapten menjadi ester aktif sehingga dapat dikonjugasikan ke bouvine serum albumin (BSA). Hapten asam 4-(4,6-diamino-1,3,5-triazin-2-ylamino)-butanoat berhasil disintesis dengan yield 8,16%, dan serapan maksimum UV pada panjang gelombang 233 nm. Karakterisasi hapten menunjukkan adanya serapan inframerah gugus amina sekunder pada bilangan gelombang 1654,92 cm-1 dan spektrum mass spectroscopy pada m/z 212,4 yang menunjukkan massa relatif hapten (C7H12N6O2). Konjugat hapten-BSA menunjukkan serapan UV maksimum pada panjang gelombang 215 nm. Hasil karakterisasi menunjukkan hapten dan konjugat hapten-BSA telah berhasil disintesis. Dua plat coating antigen dipersiapkan menggunakan kopling antara hapten dengan ovalbumin (OVA). Hasil penelitian menunjukkan bahwa antibodi poliklonal dengan titer yang tinggi berhasil diproduksi dan dapat dideteksi menggunakan uji AGPT dan metode icELISA, hasilnya menunjukkan bahwa antibodi yang terbentuk mempunyai spesifisitas yang tinggi untuk deteksi melamin.

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Since September 2008, when the current melamine contamination incident became widely known, have become an international health event to meet the need for rapid and reliable monitoring of melamine in milk samples. An indirect competitive enzyme-linked immunosorbent assay (icELISA) with enhanced specificity for melamine in milk was developed. Hapten and hapten-protein conjugate were prepared as immunogen of antibody production for melamine assay. Hapten was synthesized by reacting gamma amino butyric acid and 2- chloro-4,6-diamino-1,3,5-triazine (CAAT). Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) were used to alter carboxylic group of hapten into active ester goup, which made it possible to be conjugated to bovine serum albumin (BSA). Hapten 4-(4,6-diamino-1,3,5-triazine-2-ylamino) butanoic acid was successfully synthesized with 8.16% yield and 233 nm maximum wavelength of UV absorption.

Characterization of hapten showed the infrared vibrational spectrum of secondary amine at 1654.92 cm-1 and mass spectrum (MS) m/z 212.4 refers to hapten (C7H12N6O2). The hapten-BSA conjugate showed UV absorption at maximum wavelength of 216 nm. These results indicated that hapten and conjugate were successfully synthesized. Two plate coating antigens were prepared by coupling both haptens to egg ovalbumin (OVA). The results showed that polyclonal antibodies with high titers were successfully production and can be detection use AGPT test and icELISA method, the result showed that a high specificity of antibody for detection melamine."

"Terdapat banyak metode untuk mendeteksi kontaminasi melamin dalam makanan atau produk pakan termasuk immunoassay. Dalam penelitian ini, hapten melamin disintesis dengan mereaksikan 2-kloro-4,6-diamino-1,3,5-triazina (CAAT) dengan asam 6-aminokaproat. Karakterisasi hapten dengan spektrometer Fourier Transform Infrared (FTIR) menunjukkan absorpsi ikatan N-H dari gugus amina sekunder alifatik pada frekuensi 3350-3310 cm-1 dan vibrasi ulur ikatan C-N pada daerah sidik jari 1250-1020 cm-1. Karakterisasi dengan 1H-NMR menunjukkan pergeseran kimia pada 4,20 ppm (t, 1H), sedangkan dengan 13C-NMR pada pergeseran kimia 139 ppm. Karakterisasi dengan spektrometer massa menunjukkan M+. m/z 240,4 untuk hapten (C9H16N6O2). Imunogen disintesis dengan cara menkonjugasikan hapten dengan bovine serum albumin (BSA) dengan menggunakan metode ester aktif. Konjugat hapten-BSA menyerap panjang gelombang maksimum UV pada 224 nm. Data menunjukkan bahwa hapten dan imunogen berhasil disintesis. Hapten-BSA kemudian disuntikkan ke kelinci untuk menghasilkan antibodi poliklonal. Setelah tiga minggu imunisasi, serum menunjukkan adanya antibodi melalui Gel Agarose Precipitation Test (AGPT). Dari hasil optimasi awal indirect ELISA diketahui konsentrasi optimum coating antigen hapten-OVA adalah 1 μg/mL dan konjugat HRP-anti rabbit IgG adalah 1:10.000. Dari hasil indirect competitive ELISA diperoleh bahwa pengenceran optimum antibodi yang masih memberikan respon positif berupa absorbansi yang tinggi adalah 1:125 (6 μg/mL). Antiserum yang dihasilkan memiliki sensitivitas yang cukup baik terhadap melamin dengan nilai IC50 sebesar 2,83 ppm dan limit deteksi (IC15) sebesar 0,22 ppm melalui indirect competitive ELISA.

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There are many method to detect the melamine contamination in food or feed product including immunoassay. In this study, hapten of melamine was synthesized by reacting 2-chloro-4,6-diamino-1,3,5-triazine (CAAT) and 6-aminocaproic acid. Characterization of hapten with fourier transform infrared spectroscopy (FTIR) showed secondary N-H stretching vibrational band at 3350-3310 cm-1 and C-N stretching vibrational band at fingerprint region 1250-1020 cm-1. Characterization of hapten with 1H-NMR showed chemical shift at 4,20 ppm (t, 1H), and 139 ppm by 13C-NMR. Hapten also characterized with high-resolution mass spectrometry (HRMS) that showed M+. m/z 240,4 for hapten (C9H16N6O2). The Immunogen was prepared by coupling hapten to bovine serum albumin (BSA) using the active ester method. Hapten-BSA conjugate showed the maximum wavelength of UV absorpstion at 224 nm. The data pointed out that hapten and immunogen was successfully synthesized. Hapten-BSA conjugate then injected into rabbit to produce the polyclonal antibodies. After three weeks immunization, serum showed the presence of antibodies through Agar Gel Precipitation Test (AGPT). The initial optimization of indirect ELISA showed that the optimum concentration of coating antigen hapten-OVA was 1 mg/mL and HRP-conjugated anti-rabbit IgG was 1:10,000. The optimum antibody dilution that still gave a positive response was 1:125 (6 mg/mL) through indirect competitive ELISA. Antiserum produced has a good sensitivity to melamine with IC50 2.83 ppm and a detection limit (IC15) 0.22 ppm using indirect competitive ELISA."

"Sintesis hapten dan pembentukan konjugat hapten-protein dilakukan sebagai immunogen untuk membentuk antibodi yang selektif terhadap melamin. Hapten melamin disintesis dengan mereaksikan asam gamma-amino butirat (GABA) dengan 2-kloro-4,6-diamino-1,3,5-triazin (CAAT). Digunakan disikloheksilkarbodiimida (DCC) dan N-hidroksisuksinimida (NHS) untuk merubah gugus karboksilat hapten menjadi ester aktif sehingga dapat dikonjugasikan ke bouvine serum albumin (BSA). Hapten asam 4-(4,6-diamino-1,3,5-triazin-2-ylamino)-butanoat berhasil disintesis dengan yield 8,16%, dan serapan maksimum UV pada panjang gelombang 233 nm. Karakterisasi hapten menunjukkan adanya serapan inframerah gugus -NH- sekunder pada bilangan gelombang 1654,92 cm-1 dan spektrum mass spectroscopy pada m/z 212,4 yang menunjukkan massa relatif hapten (C7H12N6O2). Konjugat hapten-BSA menunjukkan serapan UV maksimum pada panjang gelombang 216 nm. Hasil karakterisasi menunjukkan hapten dan konjugat hapten-BSA telah berhasil disintesis.

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Hapten and hapten-protein conjugate were prepared as immunogen of antibody production for melamine assay. Hapten mel was synthesized by reacting gamma amino butyric acid and 2-chloro-4,6-diamino-1,3,5-triazine (CAAT). Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) were used to alter carboxylic goup of hapten into active ester goup, which made it able to be conjugated to bovine serum albumin (BSA). Hapten 4-(4,6-diamino-1,3,5-triazine-2-ylamino) butanoic acid was successfully synthesized with 8.16% yield and 233 nm maximum wavelength of UV absorption.

Characterization of hapten showed the infrared vibrational spectrum of secondary –NH- at 1654.92 cm-1 and mass absorbance (MS) m/z 212.4 refers to hapten (C7H12N6O2). Hapten-BSA conjugate showed maximum wavelength of UV absorption at 216 nm. Results pointed out that hapten and conjugate were successfully synthesized."

"Metode immunoassay untuk pendeteksi melamin pada susu formula membutuhkan antibodi melamin. Antibodi melamin dapat dibentuk dengan cara menyuntikkan melamin yang terikat pada hapten agar dikenali sebagai protein pada kelinci. Hapten dibuat dengan cara mereaksikan 2-kloro-4,6-diamino-1,3,5- triazin (CAAT) dengan asam 6-aminokaproat. Agar menjadi immunogen, hapten dikonjugasikan dengan BSA melalui reaksi dengan DCC dan NHS. Sintesis hapten asam 6-(4,6-diamino-1,3,5-triazin-2-ylamino)heksanoat menghasilkan yield 18.64%, serapan maksimum UV pada 241 nm, vibrasi FTIR khas pada vibrasi ulur N-H sekunder 1660 cm-1 , nilai pergeseran kimia khas 1 MR pada 4.2 ppm (q,7H) dengan integrasi 2 untuk H berikatan N sekunder NMR pada 146.92 ppm (1'C) untuk C berikatan N sekunder, dan nilai MS [ ] 240. Konjugat hapten-BSA menghasilkan serapan maksimum UV pada 221 nm.

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Melamine antibody is needed in immunoassay method for the detection of melamine. The antibody could be obtained by the injection of melamine bounded the hapten, to be recognized as the protein into the rabbit. The hapten was formed by the reaction of 2-chloro-4,6-diamino-1,3,5-triazine (CAAT) and 6- aminocaproic acid. In order to behave like immunogen, hapten was conjugated to BSA by reaction with DCC and NHS. Synthesis of hapten 6-(4,6-diamino-1,3,5- triazine-2-ylamino) hexanoic acid produces 18,64% yield with the maximum UV absorption at 241 nm and particular FTIR vibration of secondary N-H stretching vibration at 1660 cm-1, furthermore chemical shift values 1H-NMR showed 4.2 ppm (q, 7H) wi he integration of 2 for H to bind the secondary N, while 13C- NMR at 146.92 'C) for C to bind the secondary N, characterization with MS showed [] 240. Hapten conjugated BSA was characterized by maximum UV absorption at 221 nm."

"[ABSTRAKAeromonas hydrophila merupakan foodborne dan waterborne patogen, yang banyakditemukan pada lingkungan perairan. Bakteri ini dapat menginfeksi manusia, hewanteresterial dan hewan air seperti ikan mas, Infeksi A. hydrophila dapat mengakibatkankomplikasi gastrointestinal dan non-gastrointestinal pada manusia dengan penularanterjadi secara oral maupun luka yang terinfeksi bakteri. Sedangkan infeksi A.hydrophila pada ikan mas dapat menjadi sumber penularan ke manusia danmengakibatkan kematian ikan mas budidaya yang berdampak pada kerugian ekonomi.Hingga saat ini informasi mengenai infeksi A. hydrophila pada manusia masih jarangdilaporkan. Hal tersebut dapat terjadi karena hingga saat ini metode diagnosa antibodianti A. hydrophila belum banyak berkembang, sedangkan uji kekebalan penting bagiskrining, diagnosa banding dan uji konfirmasi terhadap infeksi A. hydrophila. Untukitu penelitian ini bertujuan untuk mengembangkan metode ELISA dan uji deteksicepat berdasarkan modifikasi ELISA menggunakan imunostik untuk mendeteksiantibodi anti A. hydrophila. Sebagai hewan uji digunakan enam ekor kelinci (NewZeland White) dan enam ekor ikan mas (Cyprinid carpio) yang diimunisasi denganantigen sel utuh bakteri A. hydrophila yang diiaktivasi dengan formalin 0,3%.Imunisasi pada ikan mas dilakukan secara intraperitoneal, diulang satu minggu setelahimunisasi pertama, sedangkan imunisasi pada kelinci dilakukan satu kali denganemulsi antigen dan Freund?s complete adjuvant kemudian dilanjutkan dengan dua kalibooster menggunakan emulsi antigen dan Freund?s incomplete adjuvant. Koleksiserum hewan uji dilakukan setiap minggu hingga minggu ke-6 dari koleksi serumpertama. Hasil optimasi terhadap uji ELISA menunjukkan bahwa uji ELISA yangdikembangkan mampu mendeteksi antibodi anti A. hyrophila, demikian pula denganimunostik yang dirakit mampu memdeteksi antibodi anti A. hyrophila pada serum ikanmas dan kelinci.

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ABSTRACTAeromonas hydrophila is foodborne and waterborne pathogens. The bacteria thatoccur ubiquitously and autochthonously in aquatic environments. These bacteria caninfect humans, animals terrestrial and aquatic animals such as goldfish, infection of A.hydrophila can lead to complications of gastrointestinal and non-gastrointestinalinfection in humans and transmitted by oral and infected wounds contamination. Theinfected carp with A. hydrophila is a source of transmission to humans, resulting in thedeath of farmed fish that have an impact on economic disadvantage. Till now there arelacking information of A. hydrophila infection in humans reported. This can be occurbecause to the current diagnostic methods antibodi A. hydrophila undeveloped, whilethe immunity test is important for screening, differential diagnosis and confirmationtest of A. hydrophila infection. Therefore, the aims of this research is to developELISA methods and rapid detection test based on the modified ELISA usingimunostick to detect antibodies against A. hydrophila. For animals models, 6 rabbits(New Zeland White) and 6 carp (Cyprinid carpio) were immunized with whole cellantigen A. hydrophila bacteria which inactivated using 0.3% formalidehide. Carpimmunized intraperitoneally with antigen, and repeated one week after the firstimmunization, whereas immunization in rabbits done once with antigen emulsion andFreund's complete adjuvant followed by two booster using emulsion of antigen andFreund's incomplete adjuvant. Collection of animal serum done every week, till thesixth week from the first serum collection. The results of the optimization of theELISA test showed that the developed ELISA test is able to detect antibodies againstA. hyrophila, as well as the assembled imunostik capable to detect antibodies againstA. hyrophila both in carp and rabbit serum respectively, Aeromonas hydrophila is foodborne and waterborne pathogens. The bacteria thatoccur ubiquitously and autochthonously in aquatic environments. These bacteria caninfect humans, animals terrestrial and aquatic animals such as goldfish, infection of A.hydrophila can lead to complications of gastrointestinal and non-gastrointestinalinfection in humans and transmitted by oral and infected wounds contamination. Theinfected carp with A. hydrophila is a source of transmission to humans, resulting in thedeath of farmed fish that have an impact on economic disadvantage. Till now there arelacking information of A. hydrophila infection in humans reported. This can be occurbecause to the current diagnostic methods antibodi A. hydrophila undeveloped, whilethe immunity test is important for screening, differential diagnosis and confirmationtest of A. hydrophila infection. Therefore, the aims of this research is to developELISA methods and rapid detection test based on the modified ELISA usingimunostick to detect antibodies against A. hydrophila. For animals models, 6 rabbits(New Zeland White) and 6 carp (Cyprinid carpio) were immunized with whole cellantigen A. hydrophila bacteria which inactivated using 0.3% formalidehide. Carpimmunized intraperitoneally with antigen, and repeated one week after the firstimmunization, whereas immunization in rabbits done once with antigen emulsion andFreund's complete adjuvant followed by two booster using emulsion of antigen andFreund's incomplete adjuvant. Collection of animal serum done every week, till thesixth week from the first serum collection. The results of the optimization of theELISA test showed that the developed ELISA test is able to detect antibodies againstA. hyrophila, as well as the assembled imunostik capable to detect antibodies againstA. hyrophila both in carp and rabbit serum respectively]"

"Strip test immunokromatografi telah dikembangkan untuk deteksi kuantitatif kandungan melamin dalam sampel susu dan produk susu. Strip test ini dibuat berdasarkan reaksi kompleks antigen-antibodi (melamin-anti melamin). Nanopartikel emas (AuNP) digunakan sebagai label terhadap antibodi membentuk kompleks nanopartikel emas-antibodi (AuNP-Ab). Studi kuantifikasinya dilakukan dengan mengukur nanopartikel emas secara elektrokimia menggunakan metode anodic stripping voltammetry (ASV). Perangkat elektroda pada strip test difabrikasi dengan menggunakan boron-doped diamond (BDD) sebagai elektroda kerja, Ag/AgCl sebagai elektroda standar, dan platina (Pt) sebagai elektroda penunjang. Limit deteksi (LOD) untuk AuNP sebesar 24.3 μM dapat dicapai menggunakan perangkat ini. Komponen strip test immunokromatografi dilakukan dengan kondisi volume sampel melamin 100μL, nanopartikel emas-antibodi (AuNP-Ab) 60 μL, antibodi penangkap 10 μL, waktu immunoreaksi 7 menit, volume pelarut HClO4 0.02 M sebesar 200 μL, dan dengan kondisi elektrokimia pada rentang potensial dari -0.5 V sampai 1.5 V, scan rate 100 mV/s, serta waktu deposisi 240 sekon. Limit deteksi (LOD) strip test immunokromatografi yang dapat dicapai adalah 0.15 mg melamin/mL PBS (phosphate buffer solution) (150 ppm). Hasil ini menunjukkan bahwa strip test immunokromatografi tersebut dapat digunakan untuk mendeteksi keberadaan melamin secara kuantitatif.

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Immunochromatographic strip test was developed for quantitative detection of melamine in sample milk and milk products. The strip test was developed based on antigen-antibody (melamine-anti melamine) complex reaction. Gold nanoparticle (AuNP) was used as a label to antibody to form antibody-labeled gold nanoparticle (AuNP-Ab). The quantification study was studied by electrochemical measurement of gold nanoparticle using anodic stripping votlammetry (ASV) method. Electrode device for the strip test was fabricated using boron-doped diamond (BDD) as the working electrode, Ag/AgCl as the reference electrode, and platinum (Pt) as the counter electrode. Limit of detection(LOD) of AuNP detectable in the strip test electrode device was 24.3 μM. Immunochromatographic strip test using sample volume of 100μL, AuNP- Ab of 60 μL, capturing antibody of 10 μL, immunoreactions time of 7 min, volume HClO4 0.02M of 200 μL performed by electrochemical condition of scan rate of 100 mV/s and deposition time of 240 s, showing an LOD of 0.15 mg melamine/mL PBS (phosphate buffer solution) (150 ppm). The results indicated that the strip test immunochromatographic could be used to quantitative detection of melamine."

"Pada penelitian ini dilakukan uji optimasi dan aplikasi pada pengembangan strip test immunokromatografi untuk deteksi selektif melamin. Strip test dibuat pada permukaan membran nitroselulosa menggunakan koloid Au-np yang terkonjugasi dengan antibodi melamin (Ab) sebagai label, sehingga Ab dapat secara selektif membentuk kompleks melamin-Ab-Aunp. Pengukuran konsentrasi Aunp diasumsikan berbanding lurus dengan konsentrasi Ab sehingga akan menghasilkan kuantitas melamin. Metode elektrokimia anodic stripping voltammetry digunakan untuk mengukur kuantitas Au-np dengan boron doped diamond (BDD) dan glassy carbon (GC) sebagai elektroda kerja, platina spiral sebagai elektroda pendukung, dan Ag/AgCl sebagai elektroda pembanding. Keadaan optimum diperoleh pada penggunaan elektroda BDD dengan ukuran Au-np optimum adalah sebesar 12,26 nm, optimasi konsentrasi BSA dan Ab berturut-turut 10% dan 5%. Optimasi volume reagen melamin yang digunakan dalam strip test adalah 50 μL, Aunp-Ab 30 μL, Ab 5% 60 μL, dan HClO4 225 μL. Strip test ini memiliki panjang sebesar 27,5 mm dengan ukuran optimum dari strip test ini adalah sample pad 5 mm x 5 mm, conjugate pad 5 mm x 5 mm, nitrocellulose membrane 1 cm x 5 mm, test zone 5 mm x 5 mm, absorbent pad 7,5 mm x 5 mm. Waktu immunoreaksi yang dibutuhkan adalah 10 menit dengan waktu oksidasi Au-np sebesar 3 menit. Limit deteksi (LOD) dari pengukuran melamin standar adalah 5 μg/ mL PBS dengan reprodusibilitas arus yang baik (RSD 6,723% untuk 10 kali pengukuran). Uji interference dengan menggunakan urea dan asam sianurat menghasilkan sinyal negatif dan uji sampel susu mengandung melamin 5 μg/mL memberikan sinyal dengan besar kesalahan relatif sebesar 8%.

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In this research, optimization and applications was done on the development of immunochromatographic strip test for selective detection of melamine. Strip test was made on the surface of nitrocellulose membrane using colloidal gold nanoparticles (GNPs) which was conjugated with melamine antibody (Ab) as a label that can be selectively form complexes melamine-Ab-Aunp. The GNPs concentration are assumed directly proportional to the concentration of Ab therefore it will produce the quantity of melamine. Electrochemical anodic stripping voltammetry method was used to measure the quantity GNPs with boron doped diamond (BDD) and glassy carbon (GC) as the working electrodes, a platinum spiral as the counter electrode, and Ag/AgCl as the reference electrode. The BDD electrode was found to be the optimum working electrode and the optimum size of GNPs was 12.26 nm. The concentration 10 % of BSA and 5 % of Ab were the best concentration. Optimization of the volume of reagents used in the strip test are 50 μL of melamine, 30 μL of GNPs-Ab, 60 μL of Ab with 5% concentration, and 225 μL of HClO4. Strip test which consisted of sample pad, conjugate pad, nitrocellulose membrane, test zone, and absorbent pad created total length about 27.5 mm and 5 mm with the optimum dimensions are 5 mm x 5 mm of a sample pad, 5 mm x 5 mm of a conjugate pad, 1 cm x 5 mm of nitrocellulose membrane, 5 mm x 5 mm of a test zone, 7.5 mm x 5 mm of an absorbent pad. Immunoreaction time required was 10 minutes and oxidation time of GNPs was 3 minutes. Limit of detection (LoD) of melamine measurement standard was 5 μg/mL PBS with current good reproducibility (RSD 6.723% for 10 times measurement). Interference test using urea and cyanuric acid generated negative signal with relative error of 8%."

"[AFB1 merupakan mikotoksin yang dihasilkan jamur Aspergillus terutama A. flavus dan A. parasiticus yang hidup di daerah tropis. AFB1 dapat ditemukan pada berbagai produk pertanian dan makanan pokok mamalia seperti kacang tanah, jagung, kedelai, beras, dan gandum sehingga produk ini beresiko tinggi terkontaminasi toksin terutama AFB1 pada masa panen maupun pada masa penyimpanan. Resiko terbesar yang ditimbulkan toksin ini adalah kanker dan kematian. Telah banyak metode atau upaya yang dikembangkan untuk pendeteksian dini terhadap keberadaan jamur atau aflatoksin. Salah satu metode yang sederhana, terjangkau (murah), dan teruji kehandalannya adalah immunoassay dan immunokromatografi. Immunoassay membutuhkan antibodi sebagai pengenal AFB1. Pada penelitian ini antibodi AFB1 diproduksi. Mula-mula hapten AFB1 yang telah berhasil disintesis dikonjugasikan dengan protein (BSA) untuk dijadikan immunogen melalui metode ester aktif. Immunogen lalu disuntikan ke kelinci untuk menghasilkan antibodi poliklonal. Pembentukan antibodi terjadi pada hari ke-15 terhitung dari hari pertama imunisasi. Keberadaan antibodi dipastikan melalui Gel Agarose Precipitation Test (AGPT). Endapan yang terbentuk menunjukan terjadinya ikatan antara antibodi AFB1 dengan antigennya. Antibodi dimurnikan untuk dikonjugasikan dengan nano CdS hasil sintesis. Hasil konjugasi digunakan untuk mengembangkan biosensor berupa immunostrip pendeteksi aflatoksin B1. Metode immunostrip dijadikan alternatif deteksi karena teknik ini lebih praktis, cepat, mudah dilakukan oleh siapa saja.

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Aflatoxin B1 (AFB1), is mycotoxin produced by the fungus Aspergillus, especially A. flavus and A. parasiticus lived in the tropics. AFB1 composed by coumarin and two rings of furan. AFB1 can be found in a variety of agricultural products and basic food of mammals such as peanuts, corn, soybeans, rice, and wheat therefore they have high-risk products contaminated by toxin mainly AFB1 on the harvest or during storage. The greatest risk of the toxin is cancer or even death. Many methods have been developed for early detection of the presence of aflatoxins. One method that is simple, affordable (cheap), and proven reliability are immunoassay and immunochromatografi. Immunoassay needs antibody of AFB1 for the sensing antigen. The Immunogen synthesized by conjugating hapten of aflatoxin B1 with bovine serum albumin (BSA) by using the active ester method. The hapten – BSA was then injected into rabbits to produce polyclonal antibodies. The antibodies can be detected at the 15th days after the injection had been conducted. The presence of antibodies is confirmed using Agarose Gel Precipitation Test (AGPT). The precipitation showed the bond between AFB1 antibodies with antigens. The purified antibody then conjugated with nano CdS. The conjugate then applied to develop the biosensor for detection of AFB1 using immunostrip based on immunokromatographic method as an alternative method which can provide ease, rapid, simple method and practicable for everyone., Aflatoxin B1 (AFB1), is mycotoxin produced by the fungus Aspergillus,especially A. flavus and A. parasiticus lived in the tropics. AFB1 composed bycoumarin and two rings of furan. AFB1 can be found in a variety of agriculturalproducts and basic food of mammals such as peanuts, corn, soybeans, rice, andwheat therefore they have high-risk products contaminated by toxin mainly AFB1on the harvest or during storage. The greatest risk of the toxin is cancer or evendeath. Many methods have been developed for early detection of the presence ofaflatoxins. One method that is simple, affordable (cheap), and proven reliabilityare immunoassay and immunochromatografi. Immunoassay needs antibody ofAFB1 for the sensing antigen. The Immunogen synthesized by conjugating haptenof aflatoxin B1 with bovine serum albumin (BSA) by using the active estermethod. The hapten – BSA was then injected into rabbits to produce polyclonalantibodies. The antibodies can be detected at the 15th days after the injection hadbeen conducted. The presence of antibodies is confirmed using Agarose GelPrecipitation Test (AGPT). The precipitation showed the bond between AFB1antibodies with antigens. The purified antibody then conjugated with nano CdS.The conjugate then applied to develop the biosensor for detection of AFB1 usingimmunostrip based on immunokromatographic method as an alternative methodwhich can provide ease, rapid, simple method and practicable for everyone]"

"ELISA merupakan metode alternatif untuk mendeteksi residu tetrasiklin pada produk hewan. Síntesis imunogen tetrasiklin dan produksi antibodi anti tetrasiklin merupakan dua tahapan penting yang harus dilakukan jika ingin melakukan analisis residu tetrasiklin dengan metode ELISA. Metode tolidin dan metode NCS dapat digunakan untuk mensintesis imunogen tetrasiklin. Imunogen TC-Tolidin-BSA berwarna ungu dan menyerap pada dua λ maks yaitu 277 nm dan 491 nm, sedangkan imunogen TC-NCS-BSA berwarna kuning kecoklatan dan menyerap pada dua λ maks yaitu 278 nm dan 322 nm. Hasil KLT dan HPLC menunjukan bahwa kedua imunogen yang dihasilkan cukup murni. Dari hasil SDS-PAGE dapat diperkirakan BM dari TC-Tolidin-BSA adalah sebesar 71.219 Da sedangkan BM TC-NCS-BSA sebesar 70.501 Da. Nilai BM dari kedua imunogen tetrasiklin lebih besar dibandingkan BM dari BSA (berbanding terbalik dengan Rf), hal ini menunjukkan bahwa imunogen sudah terbentuk. Produksi antibodi anti tetrasiklin dilakukan dengan cara imunisasi imunogen TC-Tolidin-BSA dan TC-NCS-BSA pada perbandingan 1:75 terhadap kelinci white New Zealand berkelamin jantan. Purifikasi antibodi dilakukan dengan protein A sepharose yang spesifik mengikat IgG. Konsentrasi IgG tertinggi dari kedua imunogen terdapat pada fraksi 1, yaitu sebesar 10.93 mg/mL untuk imunogen TC-Tolidin-BSA dan 10.61 mg/ mLuntuk TC-NCS-BSA. Hasil SDS-PAGE terhadap antibodi menunjukkan bahwa IgG terurai menjadi 2 pita (rantai ringan dan rantai berat).

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ELISA is an alternative method for detecting tetracycline residues in animal products. This method has been known as rapid, sensitive, specific, and cost-effective analysis. Synthesis of tetracycline immunogen and production of anti-tetracycline antibody are two important steps which must be done if we like to analysis of tetracycline residues with ELISA. Tolidine and NCS methods applicable to synthesis of tetracycline immunogens. Tolidine method produce a purple immunogen (TC-Tolidine-BSA) and absorb at two maximum wavelength (277 nm and 491 nm), while NCS method produce a yellowish-brown immunogen (TC-NCS-BSA) and absorb at two maximum wavelength (278 nm and 322 nm). TLC and HPLC result show that both of immunogens have good purity because have not contain free tetracycline and residue of reagent. From result of SDS-PAGE can be estimated molecular weight (MW) of TC-TOLIDINBSA equal to 71.219 Da while MW of TC-NCS-BSA equal to 70.501 Da. The value of MW from both of immunogens is higher than BSA (smaller Rf), this fact indicate that immunogen have been formed. Anti-tetracycline antibody produced by immunizing of immunogens at comparison of the same concentration BSA and Tetrasiklin ( 1:75) to male white New Zealand rabbit. The antibody purified by protein A sepharose that specific coating IgG. The highest concentration of IgG from both immunogen list on fraction 1 (10.93 mg/mL for TC-Tolidin-BSA dan 10.61 mg/ mL for TC-NCS-BSA). SDS-PAGE result show that IgG has been divided into two band (heavy chains and light chains)."

"Pada penelitian ini dilakukan pengembangan strip test immunokromatografi untuk deteksi selektif melamin menggunakan platform magnetic. Strip test dibuat menggunakan membran nitroselulosa dan tersusun dari sample pad, conjugate pad, test zone, dan absorbent pad. Conjugate pad mengandung antibodi melamin berlabel nanopartikel emas (AuNP-Ab), test zone mengandung antibodi penangkap dan digunakan sebagai tempat pengukuran, sedangkan digunakan absorbent pad untuk mengarahkan aliran sampel. Untuk menahan sampel agar tidak bergerak selama pengukuran di test zone, antibodi pada test zone dimodifikasi dengan magnetic beads (MB). MB yang digunakan adalah MB yang mengandung gugus fungsi avidin. Sehingga dengan memodifikasi antibodi dengan biotin membentuk Ab-biotin, Ab-MB dapat terbentuk melalui interaksi avidin-biotin. Bila sampel melamin mencapai conjugate pad, melamin akan berinteraksi dengan AuNP-Ab membentuk kompleks mel-Ab-AuNP yang akan bergerak ke test zone dan membentuk kompleks MB-Ab-mel-Ab-AuNP. Pengukuran dilakukan dengan meletakkan batang magnet di bawah test zone sehingga komples tersebut tidak bergerak selama pengukuran. Dengan asumsi bahwa konsentrasi AuNP berbanding lurus dengan konsentrasi antibodi dan dengan demikian dengan konsentrasi melamin, pengukuran dapat dilakukan dengan metode anodic stripping voltammetry. Boron doped diamond (BDD) 0.1% digunakan sebagai elektroda kerja, kawat platina spiral sebagai elektroda pendukung, dan Ag/AgCl sebagai elektroda pembanding. Strip test dipreparasi menggunakan volume AuNP-Ab 60 μL dan Ab-MB 30 μL. Deteksi melamin pada strip test immunokromatografi dilakukan dengan volume sampel 100 μL, waktu immunoreaksi 7 menit, dan volume pelarut HClO4 0.02 M 200 μL. Kondisi elektrokimia yang digunakan adalah rentang potensial dari -500 mV sampai 1500 mV, scan rate 100 mV/s, potensial deposisi -500 mV, serta waktu deposisi 240 sekon. Limit deteksi melamin yang diperoleh adalah 150 ppm (0.15 mg melamin/mL PBS).

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In this research, immunochromatographic strip test for selective detection of melamine was developed by using platform magnetic. The strip test was made from nitrocellulose membrane and composed of sample pad, conjugate pad, test zone, and absorbent pad. The conjugate pad consisted of gold nanoparticle labeled melamine antibody (AuNP-Ab), while the test zone consisted of the capture antibody and used for the measurements. In addition, there was an absorbent pad used to direct the sample flow. In order to keep the sample in the test zone, the capture antibody was immobilized at the magnetic beads (MB). The MB contained avidin functional groups. Therefore, the antibody was modified by biotin (Ab-biotin) to form antibody modified MB (Ab-MB) through avidin-biotin interaction. When the sample contained melamine reach the conjugate pad, melamine interacts to antibody to form mel-ab-AuNP complexes, which then move to the test zone and form MB-Ab-mel-Ab-AuNP complexes. The measurements were performed by placed a magnetic bar under the test zone to avoid the movements of the complexes. As per assumption that the AuNP concentration is equivalent to the antibody as well as the melamine concentrations, the measurement can be performed by Anodic Stripping Voltammetry method. Boron doped diamond (BDD) 0.1% was applied as the working electrode, while a wire platinum spiral and an Ag/AgCl system were used as the counter and the reference electrodes, respectively. The strip test were prepared by using AuNP-Ab and Ab-MB volumes of 60 μL and 30 μL, respectively. The melamine detection was performed with a sample volume of 100 μL, an immunoreaction time of 7 min, and 200 μL of 0.02 M HClO4, while the electrochemical measurements was performed in a potential range from -500 mV to1500 mV, a scan rate of 100 mV/s, a potential deposition of -500 mV and a deposition time of 240 s. Limit of detection of 150 ppm (0.15 mg melamine/mL PBS) can be achieved."