HIV-1 infected individuals on antiretroviral therapy with an undetectable viral load do not have completely restored immune function and have a greater risk for developing inflammatory diseases such as cancer, cardiovascular disease and neurocognitive diseases. HIV reservoirs, capable of producing viral proteins and transcripts even in the absence of mature virion production, likely contribute to the development of these diseases. HIV-1 viral protein Nef was shown to induce its own release from infected cells in extracellular vesicles and was detected in the plasma of half of HIV-infected aviremic individuals. It is therefore a good candidate biomarker for the evaluation of active HIV-1 reservoir. Currently, only one commercial immunoassay for Nef detection is available, which has many drawbacks. By using nanobody SdAb19, which shows a broad specificity for Nef from different HIV-1 strains and a nanomolar affinity for Nef binding, we can address some issues and improve the performance of the immunoassay. The hypothesis of this work is to produce sufficient amounts of SdAb19 for detection of viral protein Nef in the immunoassay by expressing His-tagged nanobodies in E. coli and purifying them with nickel affinity chromatography and size exclusion chromatography. We have also produced the bacterial biotin ligase BirA, tagged with maltose binding protein, which will be used to biotinylate nanobody SdAb19-AviTag to ensure immobilization of the nanobody in the immunoassay. By performing optimization of expression, we determined the optimal parameters for expression of SdAb19 in bacteria. Expression in the cytoplasm of E. coli strain BL21[DE3] was most successful, while it was crucial that the rate of expression was slow in order to produce nanobodies in soluble form. Slow rate of expression was ensured by growing cultures at low temperature, shaking at low speed and using lactose for induction. We successfully expressed nanobodies SdAb19 and SdAb19-AviTag, which we purified by using nickel affinity chromatography and size exclusion chromatography. Additionaly, we expressed and purified the bacterial biotin ligase BirA-MBP. Moving forward, we will modify the produced nanobodies and use them in the immunoassay for the capture or detection of Nef.