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Abstrak :
Diabetes melitus merupakan kelainan metabolik yang ditandai hiperglikemia dimana landasan terapinya masih menggunakan human insulin. Telah banyak sediaan yang beredar, namun metode analisis yang standar dan valid belum ditetapkan di dalam negeri. Penelitian ini bertujuan untuk mengembangkan dan memvalidasi metode analisis secara indirect ELISA dibandingkan KCKT UV fase terbalik untuk determinasi sediaan. Antibodi poliklonal IgG dihasilkan dari kelinci yang diimunisasi dengan 1 mg/mL antigen human insulin rekombinan, dimurnikan melalui presipitasi dan kromatografi afinitas, dikuantitasi dengan spektrofotometer UV280nm, dikarakterisasi dengan uji Dot blot menggunakan substrat BCIP-NBT, serta dikarakterisasi melalui uji SDS-PAGE dan Western Blot dengan konsentrasi gel 7,5% dan 17,5%. Sedangkan, metode KCKT menggunakan kolom ReliantTM C-18 (4,6 x 150 mm, 5 µm), fase gerak Na2SO4 pH 2,3 : Na2SO4 pH 2,3 dalam asetonitril (55:45,v/v) rasio 38:62 v/v, standar internal etilparaben 10 µg/mL, detektor UV 215 nm, suhu kolom 40°C, laju alir 1 mL/menit, dan volume injek 20 µL. Validasi kedua metode dilakukan terhadap larutan uji yang mengandung m-kresol dan gliserol. Metode ELISA pada rentang 80,11-200,28 µg/mL (r = 0,99) dan KCKT pada 9,735-146,025 µg/ml (r = 0,9997) terbukti linear. Rekoveri pada ELISA dan KCKT adalah 99,11% ± 5,01 dan 100,71% ± 1,11, sedangkan RSD 3,91% dan 0,64%. LOD dan LOQ metode ELISA 22,05 µg/mL dan 73,51 µg/mL, serta KCKT 0,193 µg/ml dan 0,643 µg/ml. Human insulin bersifat stabil pada suhu 2-8°C selama 24 jam (ELISA) dan suhu 23°C selama 48 jam (KCKT). Kesimpulan, hasil validasi kedua metode valid dan mampu mendeterminasi human insulin tanpa berbeda siginifikan (Uji T, a0,05).  ......Cannot connect to Ginger Check your internet connection or reload the browser. Disable in this text field Rephrase Rephrase current sentence Edit in Ginger Enable Ginger Cannot connect to Ginger Check your internet connection or reload the browser Disable in this text field phrase. Rephrase current sentence. Edit in Ginger Enable Ginger. Diabetes mellitus is a metabolic disorder characterized by hyperglycemia that is still treated with human insulin. Many preparations on the market, but a standard and valid analytical method has not been established in our country. This study aims to develop and validate an indirect ELISA method for determining human insulin comparative to reverse phase UV HPLC. IgG polyclonal antibodies were produced by immunizing rabbits with 1 mg/mL recombinant human insulin antigen, purified by precipitation and affinity chromatography, quantified by UV spectrophotometer 280nm, characterized by dot blot test using BCIP-NBT substrate, and characterized by SDS-PAGE and Western Blot at 7.5% and 17.5% gel concentrations. The HPLC method was performed using a Reliant TM C-18 column (4.6 x 150 mm, 5 µm), with mobile phase Na2SO4 pH 2.3: Na2SO4 pH 2.3 in acetonitrile (55:45, v/v) ratio 38: 62 (v/v), 10 µg/mL ethylparaben internal standard, UV detector 215 nm, 40°C column temperature, 1 mL/minute flow rate, and 20 µL injection volume. The validation of both methods using test solutions containing m-cresol and glycerol. ELISA method in the range of 80.11-200.28 µg/mL (r = 0.99) and HPLC at 9.735-146.025 µg/ml (r = 0.9997) was resulted to be linear. The recovery yields on ELISA and HPLC were 99.11%±5.01 and 100.71%±1.11. RSD on ELISA and HPLC were 3.91%, and 0.64%, respectively. The LOD and LOQ of the ELISA were 22.05 µg/mL and 73.51 µg/mL, while HPLC were 0.193 µg/ml and 0.643 µg/ml. Human insulin is stable at 2-8°C for 24 hours (ELISA) and 23°C for 48 hours (HPLC). In conclusion, the validation results of both methods are valid and able to determine human insulin with no significant difference (T test, a0.05). Cannot connect to Ginger Check your internet connection or reload the browser Disable in this text field Rephrase Rephrase current sentence Edit in Ginger Enable Ginger Cannot connect to Ginger Check your internet connection or reload the browser Disable in this text field Rephrase Rephrase current sentence Edit in Ginger.

Abstrak :
Respons antibodi spesifik SARS-CoV-2 dapat diperoleh dari paparan virus ketika infeksi ataupun dari vaksinasi. Studi mengenai rasio CD4+/CD8+ sebagai penanda status imunitas masih belum banyak dilakukan pada dewasa sehat. Vitamin D yang memiliki efek imunomodulatori pada sistem imun adaptif dan alamiah, mampu memodulasi pembentukan antibodi dan regulasi dari sel T. Penelitian ini bertujuan melihat hubungan kadar 25(OH)D serum terhadap titer antibodi SARS-CoV-2 dan rasio CD4+/CD8+ sebagai penanda status imunitas individu. Studi potong lintang ini dilakukan terhadap tenaga kesehatan di tiga rumah sakit rujukan COVID-19 di Jakarta dan Depok pada periode Juli–Desember 2021. Pengambilan data yang dilakukan berupa wawancara kuesioner data sosiodemografik, pemeriksaan tanda-tanda vital, pengukuran antropometri, dietary assessment menggunakan 24-h food recall dan SQ-FFQ. Pengambilan sampel darah dilakukan untuk menilai kadar 25(OH)D serum, rasio CD4+/CD8+, dan titer antibodi SARS-CoV-2. Didapatkan 154 tenaga kesehatan usia 22-53 tahun dalam kondisi sehat dan tanpa riwayat penyakit kronis. Median asupan vitamin D subjek penelitian sebesar 2,42 mcg/hari (1,23–4,00) dengan 94,7% subjek memiliki asupan vitamin D yang kurang. Median kadar serum 25(OH)D pada subjek sebesar 14,4 ng/mL (9,50–18,62) dengan 81,8% subjek mengalami defisiensi dan 14,9% subjek mengalami insufisiensi vitamin D. Median rasio CD4+/CD8+ 1,14 (0,88–1,34), 85,7% subjek memiliki titer antibodi SARS-CoV-2 >250 U/mL dan 14,3% subjek memiliki titer antibodi ≤250. Tidak ditemukan adanya hubungan yang siginifikan antara kadar 25(OH)D dengan titer antibodi SARS-CoV-2 (p 0,209 OR 4,101 95% CI 0,45–37,04) dan Rasio CD4/CD8 (p 0,385 𝛃 -0,005 95% CI -0,0015–0,006). Asupan dan kadar vitamin D pada subjek penelitian masih tergolong rendah. Penelitian ini tidak berhasil membuktikan adanya hubungan antara kadar serum 25(OH)D dengan rasio CD4+/CD8+ dan titer antibodi SARS-CoV-2. ......SARS-CoV-2 specific antibody response can be generated from exposure to the virus during infection or from vaccination. There is limited data on CD4+/CD8+ ratio in healthy individuals as a marker of immunity status. Vitamin D, which has an immunomodulatory effect on both  innate and adaptive immune systems, is able to modulate antibody formation and regulation of T cells. This study aimed to examine the association between serum 25(OH)D levels and SARS-CoV-2 antibody titers along with CD4+/CD8+ ratio as a marker of immunity status. This cross-sectional study was conducted on healthcare workers at three COVID-19 referral hospitals in Jakarta and Depok in the period of July–December 2021. Data collection was carried out using questionnaire, examination of vital signs, anthropometric measurements, dietary assessment using 24-h food recall, and SQ-FFQ. Blood samples were taken to assess serum 25(OH)D levels, CD4+/CD8+ ratio, and SARS-CoV-2 antibody titers. 154 healthcare workers aged 22-53 years who were in good health and had no history of chronic disease were examined in this study. The median intake of vitamin D was 2.42 mcg/day (1.23-4.00), with 94.7% of participants having insufficient intake of vitamin D. The median serum 25(OH)D level was 14.4 ng/mL (9.50-18.62), with 81.8% participants are vitamin D deficiency and 14.9% are insufficient. Median CD4+/CD8+ ratio was 1.14 (0.88 to 1.34). 85.7% participants had SARS-CoV-2 antibody titers >250 U/mL, while 14.3% were below 250 U/mL. There was no significant relationship of serum 25(OH)D levels to SARS-CoV-2 antibody titers (p 0.209 OR 4.101 95% CI 0.45–37.04) and CD4+/CD8+ ratio (p 0.385 o-0.005 95% CI -0.0015–0.006). Vitamin D intake and serum 25(OH)D levels are relatively low. This study disproves relationship between serum 25(OH)D levels with CD4+/CD8+ ratio and SARS-CoV-2 antibody.