ALPCO Anti-HAV ELISA Kit 相關(guān)介紹：
HAV是一種單鏈RNA病毒，感染人類肝臟可以引發(fā)病毒性甲型肝炎急性傳染病，通常通過(guò)糞—口途徑傳播。人與人之間的傳播通過(guò)攝取被HAV污染的水和食物 或直接接觸感染者。感染HAV病毒后一般2～6周表現(xiàn)出癥狀，平均潛伏期在28天。甲型肝炎病毒多侵犯兒童及青年，發(fā)病率隨年齡增長(zhǎng)而遞減，40歲上成人 中，80%左右均有抗HAV抗體。在甲型肝炎的顯性感染或隱性感染過(guò)程中，機(jī)體都可產(chǎn)生抗HAV的lgM 和lgG抗體。前者在急性期和恢復(fù)期出現(xiàn)，后者在恢復(fù)后期出現(xiàn)，并可維持多年，對(duì)同型病毒的再感染有免疫力。

ALPCO Anti-HAV ELISA Kit 產(chǎn)品使用：
§  檢測(cè)原理：Mediagnost anti-HAV EIA, E10 是偽競(jìng)爭(zhēng)酶聯(lián)免疫分析。血清或血漿樣本添加到之前已涂有一層HAV滅活抗原的微量滴定板小孔中，在 37℃溫育2小時(shí)。Anti-HAV抗體可以與抗原結(jié)合。然后繼續(xù)添加結(jié)合物（過(guò)氧化物酶標(biāo)記的anti-HAV IgG），37℃溫育1小時(shí)?？乖杂山Y(jié)合位點(diǎn)可以與結(jié)合物，沖洗掉多余的結(jié)合物，添加底物，在室溫下溫育30分鐘，結(jié)合到滅活抗原的結(jié)合物會(huì)使底物變?yōu)?藍(lán)色。zui后用終止液終止反應(yīng)，顏色變?yōu)辄S色。可以在微量滴定板讀數(shù)器上讀出顯色反應(yīng)產(chǎn)物的吸光率。吸光率與anti-HAV滴定量成反比例。定量測(cè)定使用 標(biāo)準(zhǔn)血清。也可以使用標(biāo)準(zhǔn)試劑制作滴定曲線。

§  檢測(cè)過(guò)程：A1/A2孔作空白對(duì)照→每孔加50µl稀釋結(jié)合物（除了A1/A2），不要清空和清洗滴定板 →反應(yīng)室密封，37℃溫育1小時(shí)→清空小孔，用清洗緩沖液清洗三次（300µl/孔）→每孔加100µl底物溶液，A1/A2孔也加，暗處室溫（20 - 25℃）溫育30分鐘→每孔加100µl反應(yīng)終止液，包括A1/A2孔→以TMB空白底物（A1/A2孔）為準(zhǔn)調(diào)節(jié)分光光度計(jì)到450nm，測(cè)定每種樣本 在450nm的分光光度值

§  結(jié)果分析：計(jì)算陰性對(duì)照和樣本的平均分光光度值，陽(yáng)性對(duì)照和陰性對(duì)照平均分光光度值誤差小于0.4，否則必需重新檢測(cè)。定點(diǎn)值計(jì)算：（陽(yáng)性對(duì)照分光光度值+陰性對(duì)照平均分光光度值）/2。如果陽(yáng)性對(duì)照分光光度值小于定點(diǎn)值則認(rèn)為是陽(yáng)性。樣本平均分光光度值大于定點(diǎn)值則被認(rèn)為是陰性。

 英文簡(jiǎn)述：
Positive detection of antibodies directed against the Hepatitis A virus （anti-HAV） is evidence of immunity to Hepatitis-A virus （1）. After natural infection with the Hepatitis A-Virus, neutralising antibodies appear at the same time of Anti-HAV of IgG-Class formation.
Mediagnost anti-HAV EIA, E10 is a pseudo-competitive enzyme immunoassay. Serum or plasma samples are added to the wells of a microtiter plate, which have been previously coated with inactivated HAV antigen, and incubated for 2 hours at 37 °C. Anti-HAV antibodies bind to the antigen. The conjugate （peroxidase labeled anti-HAV IgG） is added and incubated again for 1 h at 37 °C. Free binding sites of the antigen are bound with conjugate. Excess conjugate is washed of the plate and the substrate is added and incubated for 30 min at room temperature. The bound conjugate changes the colour of the subtrate to blue. The reaction is terminated by adding the stopping solution. The colour turns yellow. The absorbance of the coloured reaction product is measured on a microtiter plate reader. The extinction is reciprocal to the anti-HAV titer. For quantitative determination use the included serum standards.
The preparation of titration curve e.g. for calibration of sera by means of standard reagents is also possible.

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