乙型流感病毒診斷試劑（美國(guó)OSOM）

廣州健侖生物科技有限公司

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乙型流感病毒診斷試劑（美國(guó)OSOM）

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【公司名稱(chēng)】 廣州健侖生物科技有限公司
【市場(chǎng)部】    楊永漢

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【騰訊  】 2042552662
【公司地址】 廣州清華科技園創(chuàng)新基地番禺石樓鎮(zhèn)創(chuàng)啟路63號(hào)二期2幢101-103室

非特異性染色
① 是否有效地去除了內(nèi)源性酶和生物素。應(yīng)注意的是，并不是每一種組織均需要進(jìn)行此步驟，但對(duì)于內(nèi)源性酶或生物素豐富的組織，如肝臟、腎臟等，需考慮此原因。處理的方法為：
滅活堿性磷酸酶：zui常用的方法是將左旋咪唑（24mg/m1）加入底物液中，并保持pH 值在7.6~8.2，即能除去大部分內(nèi)源性堿性磷酸酶，對(duì)于仍能干擾染色的酸性磷酸酶，可用50mmol/L 的酒石酸抑制。
飽和處理內(nèi)源性生物素：
消除內(nèi)源性生物素的方法是事先滴加親和素，以飽和內(nèi)源性生物素，使之不再有剩余的結(jié)合位點(diǎn)。具體方法是在ABC 法或SP法染色前將切片浸于25ug/ml親和素溶液中處理15 分鐘，PBS 清洗15 分鐘后即可染色。
② 是否選擇使用了正確的封閉血清。電荷吸附所造成的非特異性背景染色消除方法是以二抗動(dòng)物的非免疫血清，用PBS 稀釋為3%-10%溶液孵育切片，以封閉吸附位點(diǎn)。有時(shí)其它無(wú)關(guān)蛋白，如牛血清白蛋白也常應(yīng)用。另外，取材時(shí)避開(kāi)出血、壞死區(qū)亦極重要。zui近有些國(guó)內(nèi)的實(shí)驗(yàn)室應(yīng)用5%脫脂奶粉替代血清進(jìn)行抗原封閉，效果也不錯(cuò)。
③ 所選擇的抗體是否符合試驗(yàn)要求。因抗原不純、標(biāo)本片中含有與靶抗原相似的抗原決定簇等原因造成的非特異性染色只能通過(guò)采用高純度、高效價(jià)的抗體、或針對(duì)更具特異性抗原決定簇的單克隆抗體來(lái)解決。
④ 一抗的使用濃度是否過(guò)高。
⑤ 清洗是否充分。應(yīng)嚴(yán)格操作規(guī)程。因在緩沖液中含有一定量的鹽，這亦有利于減低背景著色，通常0.05mol/l Tris—HCl，0.15mol/l NaCl已適用于多數(shù)染色方法，溶液內(nèi)加入吐溫20，效果更佳。特殊標(biāo)記時(shí)，試劑公司一般都提供緩沖液的配方。
⑥ DBA 的使用是否正確。DAB 的孵育時(shí)間和配制方式可以產(chǎn)生某些背景顏色，使用濃縮型DAB 試劑盒時(shí)，請(qǐng)嚴(yán)格按照說(shuō)明書(shū)標(biāo)明的滴加順序操作，注意校正蒸餾水的pH 值，以確保實(shí)驗(yàn)結(jié)果的正確性；粉劑DAB 溶解時(shí)，常有一些不溶性顆粒，這些顆粒須經(jīng)過(guò)濾除去，否則可能沉積于切片組織上，產(chǎn)生斑點(diǎn)狀著色。另外，DAB 保存不妥產(chǎn)生氧化物亦可沉積于切片上，因此需將DAB 保存于避光干燥處，現(xiàn)用現(xiàn)配，臨用前加H2O2。孵育時(shí)間過(guò)長(zhǎng)也會(huì)造成背景染色。
⑦ 標(biāo)本染色過(guò)程中是否曾經(jīng)干涸，否則會(huì)造成邊緣部的非特異性染色。
⑧ 檢查二抗與標(biāo)本的內(nèi)源性組織蛋白是否有交叉反應(yīng)。

Non-specific staining
① whether the effective removal of endogenous enzymes and biotin. It should be noted that this procedure is not required for every tissue, but for endogenous enzymes or biotin-rich tissues such as the liver, kidneys, etc., this reason needs to be considered. The method of treatment is:
Inactivation of alkaline phosphatase: The most commonly used method is to add levamisole (24 mg / ml) to the substrate and maintain pH between 7.6 and 8.2 to remove most of the endogenous alkaline phosphatase. For still Can interfere with staining of acid phosphatase, available 50mmol / L of tartaric acid inhibition.
Saturated treatment of endogenous biotin:
The method of eliminating endogenous biotin is to drip the avidin in advance to saturate the endogenous biotin so that it no longer has the remaining binding sites. The specific method is ABC method or SP staining prior to the slice immersed in 25ug / ml avidin solution for 15 minutes, PBS washed for 15 minutes after staining.
② choose to use the correct closed serum. Non-specific background staining caused by the charge-sorption method is based on the non-immune serum of the secondary antibody, incubated with 3% -10% diluted PBS solution to block the adsorption site. Sometimes other unrelated proteins, such as bovine serum albumin are often used. In addition, avoid bleeding when drawing, necrosis area is also very important. Recently some domestic laboratories apply 5% skim milk instead of serum for antigen blocking, the effect is not bad.
③ whether the selected antibodies meet the test requirements. Non-specific staining due to impure antigen, antigenic determinants in the specimen containing antigen similar to the target antigen can only be achieved by using high-purity, high-titer antibodies or monoclonal antibodies against more specific epitopes solve.
④ the use of the first antibody concentration is too high.
⑤ cleaning is adequate. Should be strictly operating procedures. It is also advantageous to reduce the background color because a certain amount of salt is contained in the buffer. Usually, 0.05 mol / l Tris-HCl and 0.15 mol / l NaCl are suitable for most staining methods. Tween 20 is added to the solution to give a better result . Reagents typically provide buffer solutions for special labeling.
⑥ DBA use is correct. DAB incubation time and preparation methods can produce some background color, the use of concentrated DAB kit, in strict accordance with the instructions specified in the order of dropwise addition, pay attention to correct the pH value of distilled water to ensure the correctness of the experimental results; DAB DAB When dissolved, there are often some insoluble particles, these particles have to be removed by filtration, or may be deposited on the tissue sections, resulting in spots colored. In addition, DAB improper storage of oxide can also be deposited on the slice, so the DAB should be stored in a dark place, is now available with, before the addition of H2O2. Long incubation time can also cause background staining.
⑦ specimens have been dried in the dyeing process, otherwise it will cause non-specific edge of the stain.
⑧ check the secondary antibodies and specimens of endogenous tissue proteins cross-reaction.