MAB8256 Sigma-AldrichAnti-Influenza A Antibody, H1N1 Antigen, clone 9B3.2

Biomolecular interactions, such as antibody-antigen binding, are fundamental to many biological processes. At present, most techniques for analyzing these interactions require immobilizing one or both of the interacting molecules on an assay plate or a sensor surface. This is convenient experimentally but can constrain the natural binding affinity and capacity of the molecules, resulting in data that can deviate from the natural free-solution behavior. Here we demonstrate a label-free method for analyzing free-solution interactions between a single influenza virus and specific antibodies at the single particle level using near-field optical trapping and light-scattering techniques. We determine the number of specific antibodies binding to an optically trapped influenza virus by analyzing the change of the Brownian fluctuations of the virus. We develop an analytical model that determines the increased size of the virus resulting from antibodies binding to the virus membrane with uncertainty of ± 1-2 nm. We present stoichiometric results of 26 ± 4 (6.8 ± 1.1 attogram) anti-influenza antibodies binding to an H1N1 influenza virus. Our technique can be applied to a wide range of molecular interactions because the nanophotonic tweezer can handle molecules from tens to thousands of nanometers in diameter.

During the winter-spring season 2006-2007, 38 influenza virus strains were identified in patients admitted to hospital with an acute respiratory tract infection. Infections were diagnosed in parallel by direct fluorescent antibody (DFA) staining using type-specific monoclonal antibodies and real-time reverse transcription (RT)-PCR targeting the gene M (nt 25-124). In addition, virus strains were isolated in MDCK cells. Overall, 37 influenza virus strains were type A, and one type B. Of these, 35 (80.4%) were detected and typed by real-time RT-PCR, 34 (80.1%) by DFA, and 27 (71.0%) by virus isolation. Subtyping of 37 influenza virus A strains by RT-PCR and DFA gave the following results: 4/6 H1 strains were correctly subtyped by both methods, while of the 29 H3 strains subtyped by RT-PCR 7 were missed by DFA. Thus, the overall concordance of the two subtyping methods was 28/37 (75.7%). Viral RNA quantification by real-time PCR showed that when respiratory secretion collection was done within 5 days after the onset of symptoms, viral load was greater than 1 x 10(6) RNA copies/ml. In conclusion, typing and subtyping of influenza virus type A strains may benefit from both MAbs and RT-PCR, while viral RNA quantification may provide an indication of symptom onset.