Skripsi

Optimasi Konsentrasi Antibodi pada Imunofluoresensi Tidak Langsung Protein Fibronectin untuk Analisis Kerangka Biomaterial yang Tersusun dari Matriks Ekstraseluler = Optimization of Antibody Concentration in Indirect Immunofluorescence of Fibronectin Protein for the Analysis of Biomaterial Scaffolds Composed of Extracellular Matrix.

Latar Belakang Ekstraseluler matriks (ECM) merupakan komponen penting dalam pertumbuhan, regenerasi, serta pemeliharaan jaringan. Visualisasi ECM dapat dilakukan melalui teknik imunofluoresensi, namun hasil pewarnaan sangat dipengaruhi oleh konsentrasi antibodi primer dan sekunder. Optimalisasi konsentrasi diperlukan untuk mendapatkan sinyal spesifik dengan background minimal. Metode Penelitian ini merupakan penelitian eksperimental in vitro menggunakan sel fibroblas paru manusia (MRC-5). Pewarnaan dilakukan dengan antibodi primer Rabbit Anti-Human Fibronectin dan antibodi sekunder Goat Anti-Rabbit IgG FITC dengan variasi konsentrasi berbeda. Hasil dianalisis secara kuantitatif menggunakan alat Tali Image-Based Cytometer. Data diuji secara deskriptif dan dianalisis menggunakan uji Mann-Whitney. Hasil Kombinasi antibodi sekunder 1/50 dengan antibodi primer 1/100 menghasilkan jumlah sel terwarnai terbanyak. Namun, hasil uji statistik menunjukkan tidak terdapat perbedaan bermakna (p > 0,05) antar kelompok perlakuan, yang kemungkinan disebabkan oleh jumlah pengulangan sampel yang terbatas. Selain itu, kondisi kultur in vitro sangat memengaruhi viabilitas sel, di mana media tanpa Gentamicin berisiko terkontaminasi jamur. Kesimpulan Konsentrasi antibodi primer dan sekunder memengaruhi kualitas hasil imunofluoresensi tidak langsung, meskipun pada penelitian ini tidak didapatkan perbedaan bermakna. Rasio antibodi yang optimal diperlukan untuk memperoleh sinyal fluoresensi yang kuat dan spesifik dengan background minimal. Kondisi kultur in vitro juga berperan penting dalam menjaga viabilitas sel dan keberhasilan pewarnaan.
Kata Kunci: Imunofluoresensi tidak langsung, antibodi primer, antibodi sekunder, matriks ekstraseluler, MRC-5


Introduction The extracellular matrix (ECM) plays a crucial role in tissue growth, regeneration, and maintenance. Visualization of the ECM can be performed using immunofluorescence techniques; however, the staining quality highly depends on the concentration of primary and secondary antibodies. Optimization of antibody concentration is essential to achieve a specific fluorescence signal with minimal background. Method This study was an in vitro experimental research using human lung fibroblast cells (MRC-5). Indirect immunofluorescence staining was performed using Rabbit AntiHuman Fibronectin as the primary antibody and Goat Anti-Rabbit IgG FITC as the secondary antibody with various dilution ratios. Quantitative analysis was conducted using the Tali Image-Based Cytometer, and the data were analyzed descriptively and statistically using the Mann–Whitney test. Results The combination of secondary antibody 1/50 and primary antibody 1/100 produced the highest number of stained cells. However, statistical analysis showed no significant difference (p > 0.05) between treatment groups, possibly due to the limited number of replicates. Additionally, the in vitro culture conditions significantly affected cell viability, as the absence of Gentamicin in the final culture media led to fungal contamination in some wells. Conclusion The concentrations of primary and secondary antibodies affect the quality of indirect immunofluorescence staining, although this study found no statistically significant difference. An optimal antibody ratio is required to produce a strong and specific fluorescence signal with minimal background. Moreover, maintaining proper in vitro culture conditions is crucial for cell viability and the success of staining procedures.
Keywords: Indirect immunofluorescence, primary antibody, secondary antibody, extracellular matrix, MRC-5