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