臺灣博碩士論文加值系統

血球凝集抑制試驗 (hemagglutinin- inhibition test，HI test)是一至今仍普遍使用來測定抗流行性感冒病毒抗體的方法，此方法雖然快速、簡便，但測定結果容易因為紅血球的品質及來源而受影響，且血球凝集抑制試驗所測定出的抗體效價亦不能反應真正的保護能力。因此我們的目標是研發新的免疫分析平台如微量中和試驗 (Micro-neutralization test)以及以唾液酸為基底的酵素連結免疫吸附法 (Sialic acid-based enzyme linked immunosorbent assay，Sialic acid-based ELISA)，微量中和試驗是利用試管內病毒感染細胞的情形，來測定能阻止病毒感染的抗體，其中和效價是依能中和百分之五十病毒感染的抗體稀釋倍數之倒數而訂定為50%中和效價 (NT50)。酵素連結免疫吸附法主要是偵測能結合流行性感冒病毒的抗體，以鍵結半乳醣的唾液酸 (Neu5Ac��2-6Gal，��2,6-sialic acid)固定於分析盤上，抓住流行性感冒病毒後，再加入含有流行性感冒病毒抗體的病人血清，最後以帶HRP的山羊抗人類IgG抗體進行結合及呈色。我們的實驗顯示抗流行性感冒病毒抗體可以同時在微量中和試驗以及以唾液酸為基底的酵素連結免疫吸附法中被偵測到，且微量中和試驗比血球凝集抑制試驗能偵測到更高的抗體效價，以及更明顯的抗體轉變 (seroconversion)情形。除此之外，我們也利用系列稀釋的抗流行性感冒病毒核蛋白抗體在酵素連結免疫吸附法中製作一個標準曲線，以便換算出病人血清中抗流行性感冒病毒抗體的濃度。最後我們也比較感染不同流行性感冒病毒的病人，其抗體在血球凝集抑制試驗、微量中和試驗、以唾液酸為基底的酵素連結免疫吸附法所測得的結果，微量中和試驗可以偵測到特異性的中和抗體，也同時可以從幾位病人的結果中發現免疫原罪的現象。另外以唾液酸為基底的免疫連結吸附分析法則是可以在感染不同流行性感冒病毒的病人中偵測到交叉反應的抗體。

Currently, the anti-influenza antibody is measured by the hemagglutinin- inhibition (HI) test. Although it is simple, but the result is easily influenced by the quality and species source of the red blood cells used. Antibody titer determined by HI test cannot reflect the protection titer either. Therefore, we aim to develop new immunological platforms such as micro-neutralization test (micro-NT) and sialic-acid based enzyme-linked immunosorbent assay (ELISA) for the evaluation of the anti-influenza antibody titer. Using the neutralization activity that block the virus infection on cell culture, the titer of neutralization antibody (NT50) was determined as the reciprocal dilution that neutralizes 50% of the influenza virus infection of control. ELISA is to detect antibodies that bind to influenza virus. The Neu5Ac��2-6Gal (��2,6-sialic acid) was coated on plate to hold the influenza virus. Human serum that contains the anti-influenza antibody was added to bind the influenza virus, and the HRP- conjugated anti-human IgG was used to develop the color. Our data showed that anti-influenza antibody can be detected by both flow-based micro-neutralization test and sialic acid-based ELISA. The micro-NT test is more sensitive than HI test in detecting the anti-influenza antibodies. A purified monoclonal mouse anti-influenza antibody was used to construct a standard curve, so that the concentration of the anti-influenza antibody in the human serum can be quantitated. The result of micro-NT, ELISA and HI was compared to evaluate the anti-influenza antibody titers in flu patients. Micro-NT could detect type-specific neutralizing antibody and also reveal the original antigenic sin in some patients. Sialic acid-based ELISA showed cross-binding antibody form different virus-infected patient to heterotypic or heterosubtypic influenza virus.

中文摘要……………………………………………………………. I
英文摘要……………………………………………………………. II
致謝………………………………………………………………… III
總目錄………………………………………………………………. V
表目錄……………………………………………………………… VIII
圖目錄……………………………………………………………… IX

主文
壹、緒論……………………………………………………………… 1
貳、材料與方法……………………………………………………… 8
A. 材料
1. 細胞培養…………………………………………………. 8
2. 病毒株……………………………………………………. 8
3. 流感病人之血清…………………………………………. 8 4. 抗體………………………………………………………. 8
5. 試劑………………………………………………………. 9
6. 塑膠、玻璃製品…………………………………………. 12
7. 儀器………………………………………………………. 12
B. 方法
1. 流行感冒病毒之培養……………………………………… 14
2. 流行感冒病毒之定量 (紅血球凝集試驗)………………… 14
3. 血球凝集抑制試驗………………………………………… 15
4. 由小鼠腹水純化抗體……………………………………… 15
5. 以西方墨點法確認抗體之特異性………………………… 16
6. 以流式細胞儀進行微量中和試驗………………………… 16
7. 以流式細胞儀偵測細胞內病毒蛋白……………………… 17
8. 以唾液酸為基礎的酵素免疫分析(ELISA)法偵測抗流行感冒病毒抗體……………………………………………………17
9. 紫外光照射流行感冒病毒………………………………… 17
參、結果………………………………………………………………19
建立以流式細胞儀測定病毒感染細胞的微量中和試驗……………19
流感疫苗注射者及流感病毒感染者中和抗體的力價………………19
利用微量中和試驗測定流感病人的中和抗體………………………20
建立以唾液酸為基底的酵素連結免疫吸附法………………………22
UV-去活化後的流行感冒病毒亦能應用於以唾液酸為基底的酵素連結免疫吸附法…………………………………………………………24
抗流行感冒病毒抗體濃度的測定……………………………………24
流行感冒病毒感染病人抗流行感冒病毒抗體在HI, NT, 以及ELISA的力價比較……………………………………………………………25
肆、討論………………………………………………………………27
新型免疫分析平台……………………………………………………27
流感病毒的交叉反應及免疫原罪……………………………………30
醣生物學與其應用細胞………………………………………………31
伍、參考文獻…………………………………………………………33
作者簡歷

Barman, S., Adhikary, L., Chakrabarti, A.K., Bernas, C., Kawaoka, Y., and Nayak, D.P. (2004). Role of transmembrane domain and cytoplasmic tail amino acid sequences of influenza a virus neuraminidase in raft association and virus budding. J Virol 78, 5258-5269.

Barrette, R.W., Urbonas, J., and Silbart, L.K. (2006). Quantifying specific antibody concentrations by enzyme-linked immunosorbent assay using slope correction. Clin Vaccine Immunol 13, 802-805.

Bhatia, A., and Kast, R.E. (2007). How influenza's neuraminidase promotes virulence and creates localized lung mucosa immunodeficiency. Cell Mol Biol Lett 12, 111-119.

Burlington, D.B., Wright, P.F., van Wyke, K.L., Phelan, M.A., Mayner, R.E., and Murphy, B.R. (1985). Development of subtype-specific and heterosubtypic antibodies to the influenza A virus hemagglutinin after primary infection in children. J Clin Microbiol 21, 847-849.

Doherty, P.C., Topham, D.J., Tripp, R.A., Cardin, R.D., Brooks, J.W., and Stevenson, P.G. (1997). Effector CD4+ and CD8+ T-cell mechanisms in the control of respiratory virus infections. Immunol Rev 159, 105-117.

Doherty, P.C., Turner, S.J., Webby, R.G., and Thomas, P.G. (2006). Influenza and the challenge for immunology. Nat Immunol 7, 449-455.

Epstein, S.L., Lo, C.Y., Misplon, J.A., and Bennink, J.R. (1998). Mechanism of protective immunity against influenza virus infection in mice without antibodies. J Immunol 160, 322-327.

Fish, S., Zenowich, E., Fleming, M., and Manser, T. (1989). Molecular analysis of original antigenic sin. I. Clonal selection, somatic mutation, and isotype switching during a memory B cell response. J Exp Med 170, 1191-1209.

Francis, T., Jr. (1953). Influenza: the new acquayantance. Ann Intern Med 39, 203-221.

Frank, A.L., Puck, J., Hughes, B.J., and Cate, T.R. (1980). Microneutralization test for influenza A and B and parainfluenza 1 and 2 viruses that uses continuous cell lines and fresh serum enhancement. J Clin Microbiol 12, 426-432.

Gerhard, W., Mozdzanowska, K., Furchner, M., Washko, G., and Maiese, K. (1997). Role of the B-cell response in recovery of mice from primary influenza virus infection. Immunol Rev 159, 95-103.

Graham, M.B., and Braciale, T.J. (1997). Resistance to and recovery from lethal influenza virus infection in B lymphocyte-deficient mice. J Exp Med 186, 2063-2068.

Hackett, C.J., Askonas, B.A., Webster, R.G., and Van Wyke, K. (1980). Monoclonal antibodies to influenza matrix protein: detection of low levels of matrix protein on abortively infected cells. J Gen Virol 47, 497-501.

Hangartner, L., Zinkernagel, R.M., and Hengartner, H. (2006). Antiviral antibody responses: the two extremes of a wide spectrum. Nat Rev Immunol 6, 231-243.

Harmon, M.W., Rota, P.A., Walls, H.H., and Kendal, A.P. (1988). Antibody response in humans to influenza virus type B host-cell-derived variants after vaccination with standard (egg-derived) vaccine or natural infection. J Clin Microbiol 26, 333-337.

Hinshaw, V.S., Webster, R.G., Easterday, B.C., and Bean, W.J., Jr. (1981). Replication of avian influenza A viruses in mammals. Infect Immun 34, 354-361.

Jahiel, R.I., and Kilbourne, E.D. (1966). Reduction in plaque size and reduction in plaque number as differing indices of influenza virus-antibody reactions. J Bacteriol 92, 1521-1534.

Klenerman, P., and Zinkernagel, R.M. (1998). Original antigenic sin impairs cytotoxic T lymphocyte responses to viruses bearing variant epitopes. Nature 394, 482-485.

Knossow, M., Gaudier, M., Douglas, A., Barrere, B., Bizebard, T., Barbey, C., Gigant, B., and Skehel, J.J. (2002). Mechanism of neutralization of influenza virus infectivity by antibodies. Virology 302, 294-298.

Knossow, M., and Skehel, J.J. (2006). Variation and infectivity neutralization in influenza. Immunology 119, 1-7.

Lambert, P.H., Liu, M., and Siegrist, C.A. (2005). Can successful vaccines teach us how to induce efficient protective immune responses? Nat Med 11, S54-62.

Lloyd, D.H., Viac, J., Werling, D., Reme, C.A., and Gatto, H. (2007). Role of sugars in surface microbe-host interactions and immune reaction modulation. Vet Dermatol 18, 197-204.

McMichael, A.J. (1998). The original sin of killer T cells. Nature 394, 421-422.

Moscona, A. (2005). Neuraminidase inhibitors for influenza. N Engl J Med 353, 1363-1373.

Mozdzanowska, K., Feng, J., Eid, M., Kragol, G., Cudic, M., Otvos, L., Jr., and Gerhard, W. (2003). Induction of influenza type A virus-specific resistance by immunization of mice with a synthetic multiple antigenic peptide vaccine that contains ectodomains of matrix protein 2. Vaccine 21, 2616-2626.

Mozdzanowska, K., Feng, J., Eid, M., Zharikova, D., and Gerhard, W. (2006). Enhancement of neutralizing activity of influenza virus-specific antibodies by serum components. Virology 352, 418-426.

Mozdzanowska, K., Furchner, M., Maiese, K., and Gerhard, W. (1997). CD4+ T cells are ineffective in clearing a pulmonary infection with influenza type A virus in the absence of B cells. Virology 239, 217-225.

Mozdzanowska, K., Maiese, K., and Gerhard, W. (2000). Th cell-deficient mice control influenza virus infection more effectively than Th- and B cell-deficient mice: evidence for a Th-independent contribution by B cells to virus clearance. J Immunol 164, 2635-2643.

Nguyen, H.H., van Ginkel, F.W., Vu, H.L., McGhee, J.R., and Mestecky, J. (2001). Heterosubtypic immunity to influenza A virus infection requires B cells but not CD8+ cytotoxic T lymphocytes. J Infect Dis 183, 368-376.

Palese, P. (2004). Influenza: old and new threats. Nat Med 10, S82-87.

Pinto, L.H., and Lamb, R.A. (2007). Controlling influenza virus replication by inhibiting its proton channel. Mol Biosyst 3, 18-23.
Plikaytis, B.D., Turner, S.H., Gheesling, L.L., and Carlone, G.M. (1991). Comparisons of standard curve-fitting methods to quantitate Neisseria meningitidis group A polysaccharide antibody levels by enzyme-linked immunosorbent assay. J Clin Microbiol 29, 1439-1446.

Quinn, C.P., Semenova, V.A., Elie, C.M., Romero-Steiner, S., Greene, C., Li, H., Stamey, K., Steward-Clark, E., Schmidt, D.S., Mothershed, E., et al. (2002). Specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin G antibodies to anthrax toxin protective antigen. Emerg Infect Dis 8, 1103-1110.

Rowe, T., Abernathy, R.A., Hu-Primmer, J., Thompson, W.W., Lu, X., Lim, W., Fukuda, K., Cox, N.J., and Katz, J.M. (1999). Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays. J Clin Microbiol 37, 937-943.

Skehel, J.J., and Wiley, D.C. (2000). Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 69, 531-569.

Steinhauer, D.A. (1999). Role of hemagglutinin cleavage for the pathogenicity of influenza virus. Virology 258, 1-20.

Stephenson, I., Das, R.G., Wood, J.M., and Katz, J.M. (2007). Comparison of neutralising antibody assays for detection of antibody to influenza A/H3N2 viruses: an international collaborative study. Vaccine 25, 4056-4063.

Stitz, L., Schmitz, C., Binder, D., Zinkernagel, R., Paoletti, E., and Becht, H. (1990). Characterization and immunological properties of influenza A virus nucleoprotein (NP): cell-associated NP isolated from infected cells or viral NP expressed by vaccinia recombinant virus do not confer protection. J Gen Virol 71 ( Pt 5), 1169-1179.

Tarendeau, F., Boudet, J., Guilligay, D., Mas, P.J., Bougault, C.M., Boulo, S., Baudin, F., Ruigrok, R.W., Daigle, N., Ellenberg, J., et al. (2007). Structure and nuclear import function of the C-terminal domain of influenza virus polymerase PB2 subunit. Nat Struct Mol Biol 14, 229-233.

Taylor, P.M., and Askonas, B.A. (1986). Influenza nucleoprotein-specific cytotoxic T-cell clones are protective in vivo. Immunology 58, 417-420.

Thomas, P.G., Keating, R., Hulse-Post, D.J., and Doherty, P.C. (2006). Cell-mediated protection in influenza infection. Emerg Infect Dis 12, 48-54.

Tompkins, S.M., Zhao, Z.S., Lo, C.Y., Misplon, J.A., Liu, T., Ye, Z., Hogan, R.J., Wu, Z., Benton, K.A., Tumpey, T.M., et al. (2007). Matrix protein 2 vaccination and protection against influenza viruses, including subtype H5N1. Emerg Infect Dis 13, 426-435.

Treanor, J.J., Tierney, E.L., Zebedee, S.L., Lamb, R.A., and Murphy, B.R. (1990). Passively transferred monoclonal antibody to the M2 protein inhibits influenza A virus replication in mice. J Virol 64, 1375-1377.

Turner, R., Lathey, J.L., Van Voris, L.P., and Belshe, R.B. (1982). Serological diagnosis of influenza B virus infection: comparison of an enzyme-linked immunosorbent assay and the hemagglutination inhibition test. J Clin Microbiol 15, 824-829.

van Kooyk, Y., and Rabinovich, G.A. (2008). Protein-glycan interactions in the control of innate and adaptive immune responses. Nat Immunol 9, 593-601.

Wagner, R., Matrosovich, M., and Klenk, H.D. (2002). Functional balance between haemagglutinin and neuraminidase in influenza virus infections. Rev Med Virol 12, 159-166.

Wu, R., Hu, S., Xiao, Y., Li, Z., Shi, D., and Bi, D. (2007). Development of indirect enzyme-linked immunosorbent assay with nucleoprotein as antigen for detection and quantification of antibodies against avian influenza virus. Vet Res Commun 31, 631-641.

Yamada, S., Suzuki, Y., Suzuki, T., Le, M.Q., Nidom, C.A., Sakai-Tagawa, Y., Muramoto, Y., Ito, M., Kiso, M., Horimoto, T., et al. (2006). Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors. Nature 444, 378-382.

Ye, Z.P., Pal, R., Fox, J.W., and Wagner, R.R. (1987). Functional and antigenic domains of the matrix (M1) protein of influenza A virus. J Virol 61, 239-246.

Zebedee, S.L., and Lamb, R.A. (1988). Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions. J Virol 62, 2762-2772.

Zhirnov, O.P., Ikizler, M.R., and Wright, P.F. (2002). Cleavage of influenza a virus hemagglutinin in human respiratory epithelium is cell associated and sensitive to exogenous antiproteases. J Virol 76, 8682-8689.

Zupancic, M.L., Frieman, M., Smith, D., Alvarez, R.A., Cummings, R.D., and Cormack, B.P. (2008). Glycan microarray analysis of Candida glabrata adhesin ligand specificity. Mol Microbiol 68, 547-559.