In this study, an anti-rhIFN2b nanobody was discovered and used for the development of a rapid lateral flow strip for the identification of rhIFN2b. nanobody. To develop a rapid test, the nanobody I22 was coupled with a colloidal gold to produce lateral-flow test strips. The developed rhIFN2b detection assay had a limit of detection of 1 1 g/mL. The isolation of I22 and successful construction of a lateral-flow immunochromatographic test strip demonstrated the feasibility of performing ligand-binding assays on a lateral-flow test strip using recombinant protein products. The principle of this novel assay is generally applicable for the rapid testing of other commercial products, 20(S)-Hydroxycholesterol with a great potential for routine use in detecting counterfeit recombinant protein products. Keywords:Recombinant human interferon 2b, Nanobody, Phage display, Screening, Rapid test == Graphical abstract == == Highlights == Discovery of an anti-rhIFN2b nanobody I22. Development of an enzyme-linked immunosorbent assay for semiquantitative detection of rhIFN2b using the novel nanobody I22. Construction of a colloidal gold-based test strip using the nanobody I22 for the detection of rhIFN2b. The detection limit reaches 1 g/mL, which meets the requirements for testing the products of rhIFN2b in the Chinese market. This novel assay reduces the cost associated with cold chain transportation and preservation, simplifies the operation procedure, and largely increases the test speed. == 1. Introduction == Interferon (IFN), a protein with a broad-spectrum antiviral activity in allogeneic cells, was discovered by Isaacs et al. [1,2] in 1957. The recombinant human interferon 2b (rhIFN2b), which was approved for the first time by the United States Food and Drug Administration in 1986, is one of the earliest recombinant proteins used as a drug worldwide [3]. RhIFN2b is currently used as an antiviral agent for treatment of hepatitis 20(S)-Hydroxycholesterol B and hepatitis C. In China, rhIFN2b has been used since 1994 to treat hepatitis and control the spread of the disease. The identification test, one of the most important physicochemical tests applied to recombinant proteins, preliminarily determines known proteins. The identification test is required to be highly specific, repeatable, sensitive, simple, and rapid. Part III of the Chinese Pharmacopoeia (2020) states that identification tests are often performed using immunoblotting and dot immunobinding assays [4]. These methods require antibody reagents with high specificity Rabbit polyclonal to PLEKHG6 and the test should be sensitive and repeatable with the antibody. Most commercial antibodies used for recombinant protein detection are polyclonal or monoclonal. The antibody affinity for the test article varies considerably among different manufacturers and batches, which may affect the reproducibility of the assays. The production of these antibodies is complex and expensive. In addition, monoclonal and polyclonal antibodies must be stored and transported at low temperatures, which hinders the development of rapid testing. Thus, an antibody with high specificity for the target recombinant protein is required to achieve the proper sensitivity and repeatability of the test. Such an antibody would also simplify the identity test, reduce the cost, and increase the efficiency. In 1993, Hamers-Casterman et al.[5] identified heavy chain antibodies (HCAbs) in camels. Clones of heavy chain variable regions are known as 20(S)-Hydroxycholesterol nanobodies or variable domain of heavy chain (VHH) antibodies [6,7]. Nanobodies have a molecular weight of 15 kDa (tenth of the weight of normal antibodies), diameter of 2.2 nm, and length of 4.8 nm [8,9]. Nanobodies 20(S)-Hydroxycholesterol are composed of only one heavy chain variable region and are the smallest fragments that bind antigens in the nature [10]. Nanobodies have several advantages over traditional antibodies, including high water solubility, excellent 20(S)-Hydroxycholesterol stability [[11],[12],[13]], accurate targeting [14,15], low immunogenicity [16], strong tissue penetration [17], simple production [17], controlled half-life, and high modifiability [18]. Thus, nanobodies are a promising substitute for monoclonal and polyclonal antibodies for the identification of recombinant proteins. In this study, we used rhIFN2b to immunize an alpaca. After five rounds of immunization, we extracted total ribonucleic acid (RNA) from the alpaca lymphocytes. The VHH region was amplified using nested polymerase chain reaction (PCR) and a phage vector was constructed. Using phage display techniques.