Judul: Development and Optimization of Immunoassay for Detection of Antibodies to SARS-CoV-2 with In-House Recombinant Receptor Binding Domain (RBD) and Nucleocapsid (N) Protein
Abstrak: Pandemi COVID-19 yang disebabkan oleh SARS-CoV-2 telah menjadi ancaman serius bagi masyarakat global, terutama di Indonesia. Upaya pencegahan penyebaran penyakit ini juga telah dilakukan, antara lain dengan pelaksanaan program vaksinasi. Efektivitas pembentukan antibodi pasca vaksinasi ini dapat dideteksi oleh teknik immunoassay seperti indirect Enzyme- linked Immunosorbent Assay (ELISA). Penelitian ini bertujuan untuk mengembangkan, mengoptimasi, dan mengevaluasi kit deteksi antibodi SARS-CoV-2 berbasis teknik indirect ELISA dengan kit komersial dalam mendeteksi antibodi yang terbentuk setelah vaksinasi. Protein antigen yang digunakan untuk coating plate merupakan protein rekombinan in-house RBD dan N dari SARS-CoV-2. Optimasi kit deteksi melingkupi konsentrasi antigen (5 and 10 ng/mL), konsentrasi blocking agent (2.5% dan 5%), dan pengenceran konjugat (1:1000 dan 1:5000). Kit ELISA komersial yang digunakan sebagai pembanding merupakan kit ELISA MyBioSource Cat No. MBS2614310 (RBD) dan MBS3809906 (N) juga kit ELISA Immunodiagnostics Cat No. 41A235 (RBD) dan 41A225 (N). Kondisi optimal dari kit ELISA in-house untuk kedua protein rekombinan RBD danN adalah konsentrasi antigen 10 ng/mL, kosentrasi blocking agent 5%, dan pengenceran konjugat 1:1000. Kedua kit ELISA in-house protein rekombinan dalam menunjukkan kemampuan yang setara dengan kit komersial Immunodiagnostics dalam mendeteksi antibodi terhadap SARS-CoV-2 sehingga dapat digunakan untuk evaluasi keberhasilan pasca vaksinasi., COVID-19 caused by SARS-CoV-2 poses a major threat to the global community, particularly in Indonesia. Countermeasures to prevent the spread of this disease have also been implemented, including the implementation of a vaccination program. An immunoassay technique that can be used to analyze antibodies that might develop following vaccination is the indirect enzyme-linked immunosorbentassay (ELISA). The goal of this study is to develop, optimize, and evaluate a SARS-CoV-2 antibodydetection kit based on the indirect ELISA technique to a commercial kit for detecting antibodies formed after vaccination. The antigen protein used for plate coating is an in-house recombinant protein RBD (Letko et. al. 2020) and N (Liu et. al. 2020) from SARS-CoV-2. The optimization comprised adjusted concentrations of spike recombinant protein (5 and 10 ng/mL), blocking agent (2.5% and 5%), andconjugate (1:1000 and 1:5000).The commercials ELISA kits used as a comparison were the MyBioSource ELISAkit Cat No. MBS2614310 (RBD) and MBS3809906 (N) and also Immunodiagnostics ELISA kits Cat No. 41A235 (RBD) and 41A225 (N). The best conditions for the in-house ELISA kit for RBD dan N recombinant proteinswere antigen concentration of 10 ng/mL, blocking agent concentration of 5%, and conjugate dilution of 1:1000. The two recombinant protein in-house ELISA kits detected antibodies to SARS-CoV-2 at levels comparable to the Immunodiagnostics commercial kit, indicating that they can be used to evaluate post-vaccination efficacy.
Keyword: SARS-CoV-2, ELISA, Protein, Receptor Binding Domain, Nukleokapsid, Immunoassay

Judul: Ekspresi Gen Sintetik Receptor Binding Domain Spike SARS-COV-2 Pada Sistem Ekspresi Escherichia coli
Abstrak: Receptor Binding Domain (RBD) merupakan bagian dari spike SARS-Cov-2 yang secara spesifik dapat berikatan dengan reseptor ACE-2 (Angiotensin converting enzyme-2). Penelitian ini bertujuan untuk menghasilkan protein rekombinan dari RBD SARS-Cov-2 menggunakan sistem ekspresi Escherichia coli BL21 (DE3). Ketersediaan protein rekombinan ini diharapkan dapat diaplikasikan sebagai antigen untuk mengembangkan uji diagnostik berbasis immunoassay. Perancangan gen sintetik RBD menggunakan cetakan urutan nukleotida pada Genbank MZ306692.1 yang dioptimasi kodonnya dan dilengkapi dengan sisi pemotongan enzim restriksi XhoI dan BamHI untuk selanjutnya disisipkan ke vector pET-14b. Gen sintetik RBD kemudian diekspresikan menggunakan E. coli BL21 dan diinduksi menggunakan 0,1mM IPTG (Isopropyl B-D-1-thiogalactopyranoside). Pemurnian protein rekombinan dilakukan dengan teknik kolom kromatografi afinitas Ni2+ - NTA dan dianalisis hasil ekspresinya menggunakan teknik SDS-PAGE. Sistem pemurnian Ni2+-NTA berhasil menghilangkan beberapa protein non-spesifik dan menghasilkan 0,8 mg/mL protein rekombinan RBD SARS Cov-2 murni dan menunjukkan pita protein spesifik berukuran 35,75 kDa. Aplikasi protein rekombinan RBD SARS Cov-2 murni pada teknik ELISA dengan direaksikan terhadap sampel yang divaksinasi menunjukkan nilai rata-rata konsentrasi antibodi sebesar 0,176 g/mL dan pada sampel yang tidak divaksinasi sebesar 0,062 g/mL. Hasil penelitian ini menunjukkan bahwa protein rekombinan RBD SARS CoV-2 yang dikembangkan dalam sistem ekspresi E. coli dapat digunakan sebagai bahan biologis untuk kit deteksi immunoassay in-house terhadap SARS COV-2 (COVID-19)., Receptor binding domain (RBD) is a part of the S protein in SARS CoV-2 that specifically binds to the ACE-2 (Angiotensin-converting enzyme-2) receptor. This study aims to express a recombinant protein of RBD spike SARS-CoV-2 using Escherichia coli BL21 (DE3). The availability of this recombinant protein is expected to apply an antigen to develop immunoassay-based diagnostic tests. The RBD SARS CoV-2 nucleotide sequences were obtained from the GenBank database of MZ306692.1 which was optimized for codon and equipped with the cleavage site of the XhoI and BamHI restriction enzymes for further insertion into the pET-14b vector, expressed in E. coli BL21 (DE3), and then induced with 1.0 mM IPTG. The recombinant protein purification was performed using the Ni2+-NTA affinity chromatography column technique resulting in 0.80 mg/mL of purified RBD SARS CoV-2 recombinant protein. The expression and refining analyses were conducted using SDS-PAGE and have discovered the successfully recombinant protein with a specific band measuring approx ~36 kDa. This purified recombinant protein has been applied to the ELISA technique using vaccinated and non-vaccinated samples and indicated significant results, such as a vaccinated concentration mean of 0.176 µg/mL and a non-vaccinated concentration mean of 0.062 µg/mL. The constructed RBD SARS CoV-2 gene was successfully expressed and purified. Therefore, the E. coli expression system could be used as an alternative to scale up the production of SARS CoV-2 RBD recombinant protein. This RBD SARS CoV-2 recombinant protein was developed to contribute in-house biological materials to produce an immunoassay detection kit for the SARS COV-2 (COVID-19).
Keyword: Escherichia coli, Receptor Binding Domain, SARS-COV-2

Judul: Tree berdiameter lima adalah graceful
Abstrak: This manuscript presents some proofs that every tree of diameter five is graceful. A graceful tree is a graph which has a graceful labelling. This labelling assigns absolute difference of connected vertices, such that the resulting edge labels are mutually distinct. There are three theorems to be considered. First theorem proves that every tree of diameter four is graceful. Second theorem proves that every tree of diameter five is graceful or nearly graceful. The third theorem improves the second theorem and shows that every tree of diameter five is nearly graceful, which can be transformed into graceful using some certain patterns.
Keyword: Graf, Pelabelan graceful, Bogor Agricultural University, Institut Pertanian Bogor, IPB