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ABSTRACT Background Methods Results Conclusions Diagnosis of allergies is mostly based on the patient's clinical history and allergen provocation tests. Determination of specific IgE (sIgE) profiles can be performed to support allergy diagnosis. This is commonly done in vivo by the skin prick test or in vitro with automated systems. Several platforms exist to quantify sIgE levels, but all these methods require access to specific instruments, often delaying the test's results. The IgE luciferase‐linked immunosorbent assay (LuLISA) allows bioluminescent quantification of IgE against peanut in microliter samples, but this method awaits extension to other allergens. This study aimed to validate a new method, named LuLIPLEX, for multiplexed bioluminescent detection of sIgE against 20 major molecular allergens.Quantification of sIgE against 12 recombinant or purified food allergens and eight aeroallergens was performed by LuLIPLEX versus standard IgE detection methods (ImmunoCAP, ISAC, ALEX, or NOVEOS). Multiplexed detection of IgE against these 20 allergens was performed within 45 min using 50 μL of serum, plasma, or whole blood samples.A head‐to‐head comparison between LuLIPLEX and standard IgE detection methods showed a high correlation among all allergens tested. sIgE profiles in polysensitized subjects could be determined within 45 min in serum and plasma samples, as well as using a single drop of capillary blood.LuLIPLEX is a rapid and sensitive method to quantify sIgE levels against multiple allergens. Given that the test is very fast and can be performed on small and inexpensive luminometers, the IgE LuLIPLEX could allow point‐of‐care testing of sIgE profiles in allergic subjects. [ABSTRACT FROM AUTHOR]

Introduction: VHHs (VH of heavy-chain-only antibodies) represent a unique alternative to Q7 conventional antibodies because of their smaller size, comparable binding affinity and biophysical properties. Method: In this study, we systematically analyzed VHH NGS sequences from 22 Alpacas and structure data from public database. Results: VHHs in Alpaca can be grouped into five main types with multiple distinct sequence and structure features. Based on the existence of hallmark residues in FR2 region, VHHs can be classified into two groups: nonclassical VHHs (without hallmark residues) and classical VHHs (with hallmark residues). Based on VHH hallmark residues at 42 position (IMGT numbering, FR2 region) and number of cysteines, we found that Alpaca classical VHHs can be further separated into three main types: F_C2 VHHs with F (phenylalanine) at position 42 and having 2 cysteines within sequences, Y_C2 VHHs with Y (tyrosine) at position 42 and having 2 cysteines, and F_C4 with F at position 42 and having 4 cysteines. Non-classical VHHs can be further separated into 2 types based on germlines mapped: N_V3 for VHHs mapped to V3 germlines and N_V4 for V4 germlines. Based on whether FR2 residues are involved in binding, two kinds of paratopes can be identified. Different types of VHHs showed distinct associations with these two paratopes and displayed significant differences in paratope size, residue usage and other structure features. Discussion: Such results will have significant implications in VHH discovery, engine e ring, and design for innovative therapeutics. [ABSTRACT FROM AUTHOR]

Summary: Heavy‐chain antibodies (HCAbs) are a unique type of antibodies devoid of light chains, and comprised of two heavy chains‐only that recognize their cognate antigen by virtue of a single variable domain also referred to as VHH, single domain antibody (sdAb), or nanobody (Nb). These functional HCAbs, serendipitous discovered about three decades ago, are exclusively found in camelids, comprising dromedaries, camels, llamas, and vicugnas. Nanobodies have become an essential tool in biomedical research and medicine, both in diagnostics and therapeutics due to their beneficial properties: small size, high stability, strong antigen‐binding affinity, low immunogenicity, low production cost, and straightforward engineering into more potent affinity reagents. The occurrence of HCAbs in camelids remains intriguing. It is believed to be an evolutionary adaptation, equipping camelids with a robust adaptive immune defense suitable to respond to the pressure from a pathogenic invasion necessitating a more profound antigen recognition and neutralization. This evolutionary innovation led to a simplified HCAb structure, possibly supported by genetic mutations and drift, allowing adaptive mutation and diversification in the heavy chain variable gene and constant gene regions. Beyond understanding their origins, the application of nanobodies has significantly advanced over the past 30 years. Alongside expanding laboratory research, there has been a rapid increase in patent application for nanobodies. The introduction of commercial nanobody drugs such as Cablivi, Nanozora, Envafolimab, and Carvykti has boosted confidence among in their potential. This review explores the evolutionary history of HCAbs, their ontogeny, and applications in biotechnology and pharmaceuticals, focusing on approved and ongoing medical research pipelines. [ABSTRACT FROM AUTHOR]

Simple Summary: This study presents a novel method for screening nanobodies (VHHs) against porcine epidemic diarrhea virus (PEDV) using yeast two-hybrid technology. The aim was to develop neutralizing nanobodies that can overcome the limitations of current vaccines and address the shortage of therapeutic antibodies for PEDV. By employing the PEDV spike (S) protein as bait, the researchers screened a synthetically constructed nanobody yeast library and identified seven neutralizing antibodies with potent antiviral activity. Moreover, combining these seven antibodies into a "cocktail" further enhanced their antiviral effects, highlighting their potential as a promising candidate for antibody-based therapeutics in preventing and treating PEDV. Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that infect pigs' intestinal epithelial cells, causing high morbidity and mortality. Due to the rapid mutation of PEDV, vaccine efficacy is uncertain, prompting exploration of alternative treatments. Nanobodies, also known as variable heavy chain domains of heavy chain-only antibodies (VHHs), offer significant potential in biomedical applications due to their small size and high specificity. In this study, yeast two-hybrid technology was employed to screen for eight specific VHH sequences targeting the PEDV S protein from a synthetically constructed nanobody yeast library. The VHH genes were then cloned into expression plasmids for recombinant protein production, and the resulting VHHs (termed PEDV S-VHHs) were purified. Indirect immunofluorescence assay (IFA) and Western blotting analysis confirmed that these VHHs specifically bind to both PEDV and its S protein. Neutralization assays demonstrated that seven PEDV S-VHHs exhibited potent neutralizing activity against PEDV. Additionally, a combination of these seven antibodies showed enhanced antiviral effects. Preliminary predictions were also made regarding the binding sites between these VHHs and PEDV. The PEDV S-VHHs described in this study hold potential as candidates for the prevention and treatment of PEDV infection. [ABSTRACT FROM AUTHOR]