【摘要】：目的：探討磁珠與抗體偶聯(lián)的最佳條件,制備抗單增李斯特菌免疫磁珠；將免疫磁珠與顯色培養(yǎng)法結(jié)合,初步建立起利用免疫磁珠分離技術(shù)對(duì)單增李斯特菌進(jìn)行分離鑒定的檢測(cè)方法。從而為食品中單增李斯特菌的檢測(cè)提供一種快速、高效和靈敏的檢測(cè)新方法。 方法：以單增李斯特菌體作為免疫抗原,免疫新西蘭兔,獲得兔抗單增李斯特菌多克隆抗體,鑒定多抗活性以及與單增李斯特菌體的結(jié)合能力；用激光粒度儀和透射電鏡分析不同活性基團(tuán)修飾磁珠的粒徑大小和分散性,選擇性質(zhì)穩(wěn)定的磁珠用以制備免疫磁珠；設(shè)計(jì)正交試驗(yàn),摸索pH、溫度、時(shí)間對(duì)氨基修飾磁珠與多抗偶聯(lián)的影響,選擇最優(yōu)條件制備免疫磁珠并用于捕獲單增李斯特菌；設(shè)計(jì)不同的偶聯(lián)緩沖溶液、偶聯(lián)時(shí)間、偶聯(lián)溫度,通過比較羧基修飾磁珠與抗體偶聯(lián)后上清中剩余的抗體量來確定最佳偶聯(lián)條件,運(yùn)用制備的免疫磁珠對(duì)樣品中的單增李斯特菌進(jìn)行捕獲,并與顯色平板結(jié)合試驗(yàn),鑒定制備的免疫磁珠捕獲單增李斯特菌的能力。 結(jié)果：制備的兔抗單增李斯特菌多克隆抗體的效價(jià)為1.3×105,該多抗與單增李斯特菌菌體有較好的結(jié)合；通過激光粒度儀和透射電鏡分析,平均粒徑為743nm的EM2-100/40氨基修飾磁珠和粒徑為180nm的PM3-020羧基修飾磁珠具有較好的分散性；正交試驗(yàn)結(jié)果顯示,氨基修飾磁珠與抗體偶聯(lián)的最佳條件組合為pH9.6、37℃偶聯(lián)1h,在該條件下,0.5 mg EM2-100/40氨基修飾磁珠可偶聯(lián)抗體量達(dá)68μg,偶聯(lián)率為45.3%,以最優(yōu)條件制備的1 mg/mL免疫磁珠用于捕菌的最佳工作體積為50μL,其捕菌率為35%；抗體與1 mg PM3-020羧基修飾磁珠在pH6.0的0.01 mol/L一水嗎啉乙磺酸緩沖溶液中,37℃偶聯(lián)2h,偶聯(lián)抗體的量為160μg,偶聯(lián)率約為32%；制得的免疫磁珠對(duì)單增李斯特菌的捕獲率可達(dá)77.0%。 結(jié)論：成功制備了對(duì)單增李斯特菌體具有較好親和力的兔多克隆抗體；獲得磁珠與抗體偶聯(lián)的最佳條件,成功制備了免疫磁珠,并應(yīng)用于樣品中單增李斯特菌檢測(cè)的前處理,表現(xiàn)出良好的捕菌能力,較之常規(guī)平皿增菌培養(yǎng)顯色法,檢測(cè)時(shí)間至少縮短20h。為食品中單增李斯特菌的檢測(cè)建立更為靈敏、快速的檢測(cè)新方法奠定了基礎(chǔ)。
[Abstract]:Objective: to study the optimal conditions for coupling magnetic beads with antibodies, to prepare immune beads against Listeria monocytogenes, and to combine immunomagnetic beads with chromogenic culture. A method for isolation and identification of Listeria monocytogenes by immunomagnetic beads was established. It provides a rapid, efficient and sensitive method for the detection of Listeria monocytogenes in food. Methods: rabbit polyclonal antibody against Listeria monocytogenes was obtained by immunizing New Zealand rabbits with single Listeria monocytogenes as immune antigen, and the activity of polyclonal antibody and its binding ability to Listeria monocytogenes were identified. The particle size and dispersity of magnetic beads modified by different active groups were analyzed by laser particle size analyzer and transmission electron microscope. The immunomagnetic beads with stable properties were selected, and the orthogonal test was designed to explore the pH and temperature. The effect of time on the coupling of amino-modified magnetic beads with polyresistance was studied. The immune beads were prepared under optimum conditions and used to capture Listeria monocytogenes. Different coupling buffer solution, coupling time, coupling temperature, The optimum coupling conditions were determined by comparing the amount of antibodies remaining in the supernatant of carboxyl modified magnetic beads coupled with antibodies. The prepared immunomagnetic beads were used to capture Listeria monocytogenes in the sample and to combine with the chromogenic plate. To identify the ability of the prepared immunomagnetic beads to capture Listeria monocytogenes. Results: the titer of rabbit polyclonal antibody against Listeria monocytogenes was 1.3 脳 10 5, and the antibody was well combined with Listeria monocytogenes. The EM2-100/40 amino modified magnetic beads with average diameter of 743nm and the PM3-020 carboxyl modified magnetic beads with average diameter of 180nm have good dispersion. The optimum conditions of coupling of amino modified magnetic beads with antibodies were pH9.6 ~ (37) 鈩，

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