Bruce Jiang

Hybridoma screening in semi-solid medium typically employs antigens labeled with visible fluorophores (e.g., FITC, AF488) to enable single-step identification of antibody-secreting clones. However, conventional chemical conjugation via NHS-esters or isothiocyanate groups frequently modifies lysine residues located within epitopes, potentially abrogating antibody recognition of these critical regions. Here, we describe a SpyTag–SpyCatcher-based site-specific labeling strategy that circumvents epitope damage during semi-solid medium screening. A 16-amino-acid SpyTag was genetically fused to the C-terminus of the target antigen, enabling covalent conjugation to an sfGFP–SpyCatcher fluorescent probe. In semi-solid medium supplemented with SpyTag-antigen and sfGFP–SpyCatcher, positive hybridoma clones were readily identified by distinct fluorescent halos, whereas negative clones showed no detectable signal. Notably, the site-specific method yielded a significantly higher frequency of fluorescence-positive clones compared to the conventional AF488-labeled antigen method, suggesting that epitope preservation enhances screening recovery. Furthermore, this approach did not impair hybridoma growth or final clone positivity, offering a simple, rapid, and epitope-compatible method for monoclonal antibody screening.

The design of high-affinity protein binders is critical for biochemical detection, yet traditional methods remain labor-intensive. AI-driven tools like RFdiffusion, a RoseTTAFold-based diffusion model, offer promising alternatives for generating protein structures with tailored binding interfaces. This study evaluates RFdiffusion’s efficacy in designing de novo binders for six targets: Strep-Tag II (a peptide tag) and five eukaryotic proteins (STAT3, FGF4, EGF, PDGF-BB, and CD4). Five binders were designed for each target and experimentally validated. While two Strep-Tag II binders outperformed streptavidin in Western blot assays, none matched the sensitivity of anti-Strep-Tag II antibodies. Binders for the other targets failed due to low expression, nonspecific binding, or undetectable affinity. Despite generating structurally diverse candidates, RFdiffusion’s success rate was limited by low-affinity designs and inconsistent recombinant expression. These results underscore the need for further optimization of AI-driven protein design tools for practical biochemical applications.

Conjugation to a carrier protein is essential to give rise to the antigenicity of hapten. Three carrier proteins e.g. KLH (Keyhole Limpet hemocyanin), BSA (bovine serum albumin), and OVA (Ovalbumin) were used mostly. KLH is advantageous to the others, majorly owing to its strong immunogenicity and limited usage in other biological assays. However, the cost of obtaining Keyhole Limpet is high and the solubility of KLH is not as well as the other carriers, especially after hapten conjugation. Here, we extracted the shrimp hemocyanin (SHC) from Litopenaeus vannamei (L. vannamei), which is a commonly sea product worldwide. The high pure SHC could be acquired by two-step purification, with a production yield of > 1 g proteins (98% pure) per 1 kg shrimp. Compared to KLH, the peptide-SHC conjugates exhibit higher solubility after hapten conjugation. Meanwhile, compared with KLH, SHC induces comparable antibody production efficiency in mammals, with or without conjugation. Furthermore, rabbit polyclonal antibodies or mouse monoclonal antibodies were generated by immunizing SHC-peptide conjugates, and the subsequent antibodies were confirmed to be used in western blot, immunofluorescence and immunohistochemistry. Therefore, we demonstrated that SHC may be used as a substitute for KLH in future antibody and vaccine development.

Conjugation to a carrier protein is essential to give rise to the antigenicity of hapten, which is a small molecule and cannot induce an immune response by itself alone. Three carrier proteins e.g. KLH (Keyhole Limpet Hemocyanin), BSA (Bovine Serum Albumin), and OVA (Ovalbumin) were used mostly. KLH is advantageous to the others, majorly owing to its strong immunogenicity and limited usage in other biological assays. However, the solubility of KLH is not as well as the others, especially after hapten conjugation. Besides, the high market price of KLH results in high costs in vaccine and antibody development. Here, we extracted the shrimp hemocyanin (SHC) from Penaeus vannamei (P. vannamei) with a production yield of > 1 g proteins (98 % pure) per 1 kg shrimp. Compared to KLH, the peptide-SHC conjugates exhibit higher solubility after hapten conjugation. Furthermore, compared with KLH, SHC induces comparable antibody production efficiency in mammals, with or without conjugation. Finally, rabbit polyclonal antibodies or mouse monoclonal antibodies were generated by immunizing SHC-peptide conjugates, and their applications in western blot, immunofluorescence and immunohistochemistry were confirmed. Therefore, we demonstrated that SHC may be used as a substitute for KLH in future antibody and vaccine development.