Abstract
Immunoassays based on sandwich immuno-complexes of capture and detection antibodies simultaneously binding to the target analytes have been powerful technologies in molecular analyses. Recent developments in single molecule detection technologies enable the detection limit of the sandwich immunoassays approaching femtomolar (10–15 M), driving the needs of developing sensitive and specific antibodies for ever-increasingly broad applications in detecting and quantifying biomarkers. The key components underlying the sandwich immunoassays are antibody-based affinity reagents, for which the conventional sources are mono- or poly-clonal antibodies from immunized animals. The downsides of the animal-based antibodies as affinity reagents arise from the requirement of months of development timespan and limited choices of antibody candidates due to immunodominance of humoral immune responses in animals. Hence, developing animal antibodies capable of distinguishing highly related antigens could be challenging. To overcome the limitation imposed by the animal immune systems, we developed an in vitro methodology based on phage-displayed synthetic antibody libraries for diverse antibodies as affinity reagents against closely related influenza virus nucleoprotein (NP) subtypes, aiming to differentiating avian influenza virus (H5N1) from seasonal influenza viruses (H1N1 and H3N2), for which the NPs are closely related by 90–94% in terms of pairwise amino acid sequence identity. We applied the methodology to attain, within four weeks, a panel of IgGs with distinguishable specificities against a group of representative NPs with pairwise amino acid sequence identities up to more than 90%, and the antibodies derived from the antibody libraries without further affinity refinement had comparable affinity of mouse antibodies to the NPs with the detection limit less than 1 nM of viral NP from lysed virus with sandwich ELISA. The panel of IgGs were capable of rapidly distinguishing infections due to virulent avian influenza virus from infections of seasonal flu, in responding to a probable emergency scenario where avian influenza virus would be transmissible among humans overlapping with the seasonal influenza infections. The results indicate that the in vitro antibody development methodology enables developing diagnostic antibodies that would not otherwise be available from animal-based antibody technologies.
Highlights
Immunoassays based on sandwich immuno-complexes of capture and detection antibodies simultaneously binding to the target analytes have been powerful technologies in molecular analyses
If the avian influenza virus (H5N1 or H7N9) outbreaks overlap with annual seasonal influenza virus infections (H1N1 and H3N2), there would be immediate challenges to rapidly distinguish the subjects infected with the pandemic avian influenza virus from the subjects infected with seasonal influenza virus strains
NP10 and NP11 in IgG1 form bound to NP of avian influenza virus H5N1 (NPA5) and cross-reacted to NPA1 (NP of H3N1) and NPA2 (NP of H1N1) respectively (Fig. 3)
Summary
Immunoassays based on sandwich immuno-complexes of capture and detection antibodies simultaneously binding to the target analytes have been powerful technologies in molecular analyses. Recent developments in single molecule detection technologies enable the detection limit of the sandwich immunoassays approaching femtomolar (10–15 M), driving the needs of developing sensitive and specific antibodies for ever-increasingly broad applications in detecting and quantifying biomarkers. To overcome the limitation imposed by the animal immune systems, we developed an in vitro methodology based on phagedisplayed synthetic antibody libraries for diverse antibodies as affinity reagents against closely related influenza virus nucleoprotein (NP) subtypes, aiming to differentiating avian influenza virus (H5N1) from seasonal influenza viruses (H1N1 and H3N2), for which the NPs are closely related by 90–94% in terms of pairwise amino acid sequence identity. The ever-increasingly broad applications in detecting and quantifying protein biomarkers, as well as the advancements in sensor technologies, are driving the needs of developing sensitive and specific antibodies as affinity reagents for immunoassays based on sandwich immuno-complexes. One way to confront the challenges would be developing antibodies as affinity reagents capable of distinguishing the subtypes of influenza virus
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