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.

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