1. Antibody Activity Against Sialidase Used as a Potential Therapeutic Treatment for Autoimmune Disorders Amber Williams, Department of Biology, York College Introduction  Sialic Acid is distributed in bacteria and animal tissues especially in gangliosides or glycoproteins on the cell surface. Sialic acid creates a negative charge and can “hide” antigens on the surface of host cells. Most research shows that influenza binds to sialic acid and buds to the surface.  Neuraminidase (NANase)- This is a lysosomal enzyme which cleaves terminal sialic acid residues of infected cells and lets the virus spread to other cells.  Sialidase is a homolog of neuraminidase found in mammals. Four types of sialidase have been identified based on location: Neu 1 (lysosomal), Neu 2 (cytosolic), Neu 3 (membrane), and Neu 4 (unknown).  Sialic acid when found on the surface of cells keeps the complement cycle in the immune system from continuing it’s activation. Sialidase cleaves the sialic acid off the cell which allows the complement to carry on it’s cascade. Since sialidase and neuraminidase are similar, it is thought an antibody against neuraminidase will also reduce the activity of sialidase. Blocking sialidase will keep the complement cycle from starting which may stop inflammation. In the future, the antibody could be used in a possible therapy to treat autoimmune disorders (Scheme 1). Questions Asked 1.Does an antibody created for Neuraminidase definitely block the function of Neuraminidase? 2. Will an antibody elicited against Neuraminidase recognize mammalian Sialidase? 3. How does the titer of the antibody differ when reacted with Neuraminidase and Sialidase? Methods  New Zealand White rabbits were immunized with 50 µg of C. perfringens Neuraminidase.  Antibody purified by affinity chromatography on a protein G column.  Concentrations compared to BSA control.  Dilution of Neuraminidase, addition of antibody and 4-Mu- NANA, reading with fluorescence spectrofluorometer.  Neuraminidase and Sialidase added to gel, ran for 1.5 hours at 67 volts, followed by transfer and addition of Neuraminidase antibody.  Neuraminidase and Sialidase plated, incubation with primary and secondary antibody. Read with Versa Max. Acknowledgements I would like to thank Dr. Alan Cross and Dr. Chiquang Feng from the University of Maryland for their help and support in this research. I would also like to thank Dr. Ronald Kaltreider for his help in putting my research together. Results  Protein Concentration - Comparing BSA to the antibody used in this experiment, the concentration of the protein antibody is approximately 1mg/mL (Tables 1 and 2).  4-Mu-NANA Assay – Using the antibody against Neuraminidase, activity was decreased. Original titers were reduced according to the detected titers. 10mU Neuraminidase reduced to 6mU.  At 2 different dilutions, the antibody recognized both neuraminidase as well as sialidase by leaving protein bands on the gel.  The titer for sialidase differed greatly from the titer for neuraminidase. Overall Conclusions (1)The antibody made against Neuraminidase does decrease the activity of Neuraminidase which demonstrates that the antibody works. (2) The antibody elicited against Neuraminidase does recognize Sialidase. (3) The titer of the antibody differs between Neuraminidase and Sialidase. The activity of the antibody is much stronger for Neuraminidase than Sialidase. Dilution of BSABSA Concentrated1.79241.7486 1:21.39931.4085 1:41.03711.0566 1:80.80020.7772 1:160.62340.6213 1:320.53020.5194 1:640.51980.5005 1:1280.48160.4698 Table 1. BSA concentrations at different dilutions used as a control. Table 2. Concentrations of 3 eluted samples of an antibody after purification. Samples 1-3 Diluted 1:10 Samples 1-3 1.06891.04080.45540.4145 1.72011.76330.71370.7913 1.28091.32540.6460.6416 250kD 150 100 75 50 37 25 20 15 Neuraminidase Sialidase 400µg 40µg 20µg 2µg Figure 4. Western Blot of Neuraminidase and Sialidase using the anti-Neuramindase as the primary antibody. Lanes show proteins which shows the antibody/compound recognition. Literature Cited  Cross, A. S, et al. (2002).Recruitment of murine neutrophils in vivo through endogenous sialidase activity. Journal of Biological Chemistry. 278, 4112-4120.  Kindt, T.J. (2006). Kuby immunology. W. H. Freeman. Scheme 1. Complement pathway used in the immune system. Taken from Kuby Immunology textbook (2006, 6 th edition). Figure 1.Neuraminidase structure taken from notexactlyrocketscience. wordpress.com/.../ notexactlyrocketscience. wordpress.com/.../ Figure 2. Sialidase structure taken from www.st-andrews.ac.uk www.st-andrews.ac.uk Figure 3. Antibody used to decrease Neuraminidase activity using methylumbelliferyl-α-D-N-acetylneuraminic acid as a substrate. Figure 5. Titer of two purified antibodies when used against Neuraminidase and Sialidase from an ELISA assay.