抗體簡介

抗體：是高等動物在抗原物質的刺激下由漿細胞產生的一類能與相應抗原發(fā)生特異性結合的免疫球蛋白。抗體通常存在于血清合?馨停ㄗ櫓海┲小?

單克隆抗體及多克隆抗體：由單一克隆的細胞（通常是單一克隆的雜交瘤細胞）所產生的識別某一抗原表位的單一抗體；多克隆抗體是由多種細胞分別接觸相應抗原而產生的多種抗體的總和，多克隆抗體抗原識別抗原的多個表位。

抗體結構：抗體是由四條肽鏈構成的“Y”形對稱的分子，包括兩條重鏈和兩條輕鏈。在“Y”的部位的氨基酸順序在不同的抗體分子中差別很大，是負責與抗原進行特異性結合的區(qū)域，稱為可變區(qū)，余下的區(qū)域則稱為恒定區(qū)。

抗體分類

抗體分類：根據重鏈恒定區(qū)的血清學類型，可將抗體分為IgM，IgG，IgA，IgD，IgE五類，它們的重鏈分別為mu, gamma, alpha, delta, epsilon鏈。在上述每一類別中，按重鏈構造上的變異又可分為幾個亞類，例如人的IgG可分為IgG1，IgG2，IgG3，IgG4四個亞類。

輕鏈分為兩種類型，kappa鏈和lambda鏈，但每種抗體中只存在一種類型的輕鏈。

二抗：二抗是在其它宿主體內制備的能與一抗或一抗片段結合的抗體，上面通常連有酶或熒光素等標簽。由于二抗所具備的優(yōu)點使得其在免疫學實驗中得以應用廣泛，如western blot（通過與特異性抗體結合來鑒定蛋白質），ELISA（以耦聯有酶的抗體或抗原為標記來檢測特異性的蛋白質，尤其是相應的抗原或抗體），免疫組織化學（檢測組織中的特異性抗原），免疫細胞化學（通過免疫學方法檢測細胞的抗原組成），流式細胞術（通過檢測激光所激發(fā)熒光來鑒定分離不同類型的細胞）及免疫沉淀（通過抗原與抗體的特異性結合作用來分離相應抗原）。二抗針對某一特定物種（如小鼠）的所有抗體均具有特異性，因而使用標記的二抗可以免去對每一個一抗進行標記，大大節(jié)省了時間和費用；此外，一個一抗分子可以同時結合幾個二抗分子，從而使信號大大增強，提高了實驗靈敏度。

二抗可以耦聯有幾種不同的標記，可以是酶，熒光素，或生物素。

Table 1. 典型的整個IgG分子的二抗

如何選擇二抗

通常情況下，某一特定的實驗中可能同時有幾種二抗可供選擇，如何能選擇到該實驗的二抗，需要綜合以下幾個方面進行考慮：

一抗的物種來源

一抗是在什么物種來源的，相應的二抗也要是抗該物種的抗體。例如，如果一抗是在小鼠體內制備的，那么你就應當選擇抗小鼠的二抗，如果一抗是在兔體內制備的，那么二抗就應當是抗兔的抗體。至于二抗本身是在什么動物中制備的對二抗的質量并無明顯的影響。

一抗是屬于哪個類或亞類

二抗需與一抗的類別或亞類相匹配。這通常是針對單克隆抗體而言。多克隆抗體主要是IgG類免疫球蛋白，因此相應的二抗就是抗IgG抗體.

單克隆抗體的類別及亞類通常會在產品列表中列出，如果你的一抗是小鼠IgM，那么相應的二抗就應當是抗小鼠IgM，或是抗小鼠IgG抗體。

如果單克隆一抗是小鼠IgG的某一亞類（IgG1，IgG2a，IgG2b，IgG3），那么幾乎所有的抗小鼠IgG都可以與之結合，或者你也可以選擇專門針對這一亞類的二抗，例如，如果你的一抗是小鼠IgG1，那么?憧梢匝≡窨笽gG1的二抗，此種抗體在雙標記實驗中尤其適合。

如果你不知道一抗是哪一類別或亞類，那么抗小鼠IgG是一個不錯的選擇，因為此種抗體可以識別大多數類型的IgG免疫球蛋白。

Table 2. 典型的F（ab’）2片段的二抗

下面總結了幾種具有不同特異性的二抗：針對整個抗體分子（H＋L）具有特異性：如抗IgG（H+L），此類抗體既可以與抗體的重鏈結合也可以與輕鏈結合，即與抗體分子的Fc，F（ab’）2/Fab部分（見圖5）均可反應，抗IgG（H+L）也可以與其他免疫球蛋白家族反應（如IgM和IgA），因為所有的免疫球蛋白都具有相同的輕鏈（kappa鏈或lambda鏈）。

針對Fab片段具有特異性：這類抗體與重鏈輕鏈均可以結合，由于它們可以與輕鏈反應，它們同時也已和具有相同輕鏈的其他種類的免疫球蛋白反應。

這對Fc片段或重鏈具有特異性：這類抗體和重鏈的Fc部分反應，一次它們是類別特異性的（即gamma鏈特異性抗體只與IgG反應，mu鏈特異性抗體只識別IgM，依此類推）。

輕鏈（kappa，labmda）特異性：與所有類別的抗體反應，因為所有類別的抗體均具有相同的lambda鏈或kappa鏈。

經過吸附處理的二抗：不同來源的免疫球蛋白含有相似的結構，抗一個物種的抗體可能與其他物種發(fā)生交叉反應，一次有些二抗經過了動物或人類IgG吸附處理，以減少非特異性背景。例如，如果是小鼠的組織或細胞，那么選擇經小鼠血清或IgG吸附處理過的二抗可以減少非特異性結合。但是此種抗體識別表位的數量被大大減少。

選擇哪種形式的二抗，是整個IgG分子，F（ab’）2片段，還是Fab片段？

整個IgG分子：此種抗體適用于多數情況。

F（ab’）2片段：該種抗體是由兩個靠二硫鍵連接的用于結合抗原的Fab片段組成，這些抗體用在特定的情況中，如需要避免抗體與具有Fc受體的細胞結合的情況。

Fab片段：它們只有一個結合位點，一般用來封閉內源性免疫球蛋白。

選用耦聯有哪種探針的二抗？

一般來講，耦聯到二抗上的探針主要有酶（辣根過氧化酶HRP和堿性磷酸酶AP或其衍生物APAAP，PAP），熒光基團（FITC, RRX, TR, PE）和生物素。選用哪種探針的二抗主要取決于具體的實驗。對于western blot和ELISA，zui常用的二抗是酶標二抗，而細胞或組織標記實驗（細胞免疫化學，組織免疫化學，流式細胞術）中通常使用熒光基團標記的二抗，免疫組化中也可以使用辣根過氧化酶或堿性磷酸酶標記的二抗。如果想要更大程度的放大檢測信號，可以使用biotin/avidin檢測系統。

Table 3. 典型的Fab片段的二抗

下面簡單介紹幾種二抗探針的優(yōu)缺點及其適用的實驗：

酶：主要有兩種不同的酶耦聯物，HRP和AP。比較而言，前者更經濟、快速、穩(wěn)定，而后者較前者更為靈敏，通常情況下，HRP廣泛應用于免疫組化，western blot和ELISA中，而堿性磷酸酶（AP）更適合于固相的免疫檢測實驗，如ELISA和western blot.

熒光基團：

1. FITC：FITC是廣泛使用的熒光基團，但其zui大的缺點是淬滅快，使用抗淬滅劑可以減輕。
2. AMCA：常用在多標記實驗中，如免疫熒光、流式細胞術。其缺點是，淬滅快，且由于肉眼不能很好的檢測其所激發(fā)的藍色熒光，因而AMCA耦聯的二抗更適用于檢測大量存在的抗原。
3. 花青染料（Cy2,Cy3,Cy5）：Cy2比FITC更穩(wěn)定，且在表面熒光顯微鏡下，Cy2的熒光FITC更強。Cy3和Cy5比大多數熒光基團更亮，更穩(wěn)定，背景更弱。二者常在共聚焦顯微實驗中作多標記。但Cy5的缺點是不能用表面熒光顯微鏡觀察，因此通常用具有遠紅外檢測器的共聚焦顯微鏡觀察。
4. TRITC,RRX,TR:TRITC經常和FITC一起用于雙標記實驗中，也可以使用RRX或TR，但TR會產生較高的背景。在使用裝有氪氫燈的激光共聚焦掃描顯微鏡作三標記實驗時，RRX尤其有用，可以和Cy2（或FITC）及Cy5一起使用，因為RRX的發(fā)射波長在Cy2和Cy5中間，且和這兩者的重疊很少。
5. PE：常和FITC一起用于流式細胞術雙標記實驗。
6. 生物素：生物素（biotin）可以和抗生素蛋白（avidin）發(fā)生不可逆反應而緊密結合在一起。首先加入生物素標記的二抗，在加入耦聯有酶或熒光集團的avidin、ExtrAvidin或streptavidin，后者能夠結合于二抗表面的多個位點上，從而極大的放大檢測信號。

抗體的純度：

親和層析純化的抗體一般更受人們歡迎，因為這些產品的純度zui高，產生的非特異性背景zui少。但是有些情況下也要考慮到純化過程中損失的IgG部分，因為其中含有親和力很高的抗體，尤其是當目的抗原存在的量很小的時候，高親和性的IgG部分就顯得尤為重要。

常用抗體綜述

二抗應用實例分析

為分析二抗的使用情況，我們隨機抽取了100篇2007年或2008年發(fā)表于Science 或 Journal of Biological Chemistry雜志上的 文章（英文），這些文章涵蓋了來自于16個國家的約80個研究機構。收集的數據顯示二抗的使用非常廣泛，可以應用到幾乎所有類型的免疫檢測中，尤其是Western blot、免疫組織化學和免疫細胞化學。事實上，二抗在ELISA及流式細胞術中應用也很廣泛，不僅用于科學研究，而且廣泛的應用于臨床分析和疾病診斷。比較而言，二抗在免疫沉淀中的應用相對較少。以下內容是基于對所收集的二抗信息進行統計分析后得出的一些結論，對于您在具體的實驗中如何選擇的抗體具有借鑒作用。

選擇二抗時，您必須考慮到以下因素，如一抗的物種、產生二抗的宿主、一抗的類別或亞類、二抗所耦聯的探針類型及二抗的形式（如整個IgG分子，F （ab’） 2片段，或Fab片段）等等。因此選擇哪種類型的二抗很大程度上決定于您所使用的一抗是什么，尤其針對種屬和類別而言。

通常情況下，一抗可以在山羊、小鼠、兔子、大鼠及綿羊體內制備，其中屬小鼠及兔子zui為常見，相應的，二抗的種屬也應當與一抗的相一致。而對于制備二抗的宿主，則應選擇另外一種不同的動物，一般選用驢、山羊、兔子、綿羊或其他較大的動物，可以實現二抗的大規(guī)模制備，其中又屬山羊使用zui多。

對于二抗類別或亞類的選擇，應遵循與一抗類別或亞類相一致的原則，其中抗IgG使用zui多。這可能是因為除了五種類別的單克隆抗體外，很多一抗是多克隆抗體，屬于IgG類免疫球蛋白。此外anti-IgG可以識別幾乎所有類型的IgG亞類，因此，在不清楚一抗是屬于那個類別或亞類的時候，anti-IgG抗體是個不錯的選擇。從我們的數據來看，值得注意的是， IgG亞類特異性的二抗（如抗IgG1, 抗IgG2, 抗IgG2a, 抗IgG2b）通常用于雙標記實驗中，包括免疫細胞化學、免疫組織化學及流式細胞術。

此外，整個IgG分子是zui常用的一種二抗分子形式，而F （ab’） 2片段或Fab片段一般僅用于特殊實驗中，如需要避免抗體與具有Fc受體的細胞結合的情況，或需要封閉內源性免疫球蛋白的時候。

對于二抗所耦聯的探針的選擇，主要取決于具體的實驗。對于Western blot，zui常用的探針是辣根過氧化酶（HRP），堿性磷酸酶（AP）的使用也很多，但不如HRP使用廣泛。此外，IRDye （如 IRDye 680、IRDye 800） and Alexa Fluor dye （如Alexa 680 ） 等系列染料也偶爾應用于 western blot當中。通過統計分析發(fā)現，來自于Amersham Biosciences、Bio-Rad、Cell Signaling、GE Healthcare Bio-Science、Jackson ImmunoResearch Laboratories、LI-COR Biotechnology、Pierce Biotechnology、Santa Cruz Biotechnology、Sigma 及 Zymed Laboratories公司的HRP標記的二抗在Western blot中均有廣泛使用；而對于AP標記的二抗，您可以選擇Jackson ImmunoResearch Laboratories、Sigma、Bio-Rad 及DAKO公司。

對于免疫熒光檢測實驗（包括免疫細胞化學及免疫組織化學），應用zui廣泛的是耦聯有Alexa Fluor系列染料的抗體（如Alexa Fluor 488, 546, 568, 594, 596, 647, 633）、花青染料（Cyanine）系列標記的抗體（如Cy2, Cy3, and Cy5）、FITC及羅丹明（Rhodamine）。其中對于Alexa Fluor標記的二抗，推薦選擇Invitrogen 公司或Molecular Probes公司（現屬于Invitrogen公司），是的生產Alexa Fluor染料的公司。從我們收集的數據來看，Jackson Immunoresearch Laboratories公司的花青染料（Cyanine）系列標記的二抗使用較多，而對于FITC，Jackson Immunoresearch Laboratories、Santa Cruz Biotechnology、DAKO 及Sigma公司均可選擇。Bio-Rad、Chemicon、Invitrogen或Sigma公司的羅丹明標記的二抗使用較好。另外，如果你想放大檢測信號，你可以選擇生物素標記的二抗，Jackson Immunoresearch Laboratories、Vector Laboratories 或R&D Systems均是不錯的選擇。

對于流式細胞術，FITC是選用zui多的標簽，PE標簽也可以使用。

對于ELISA實驗，HRP 和 AP標簽使用zui多。

在免疫沉淀中，二抗的使用并不是很多，但偶爾也有使用，如用于蛋白的分離，一般選擇無耦聯標記的二抗。

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