抗體簡介

抗體：是高等動(dòng)物在抗原物質(zhì)的刺激下由漿細(xì)胞產(chǎn)生的一類能與相應(yīng)抗原發(fā)生特異性結(jié)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(xì)胞（通常是單一克隆的雜交瘤細(xì)胞）所產(chǎn)生的識(shí)別某一抗原表位的單一抗體；多克隆抗體是由多種細(xì)胞分別接觸相應(yīng)抗原而產(chǎn)生的多種抗體的總和，多克隆抗體抗原識(shí)別抗原的多個(gè)表位。

抗體結(jié)構(gòu)：抗體是由四條肽鏈構(gòu)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(fù)責(zé)與抗原進(jìn)行特異性結(jié)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(jù)重鏈恒定區(qū)的血清學(xué)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(gòu)造上的變異又可分為幾個(gè)亞類，例如人的IgG可分為IgG1，IgG2，IgG3，IgG4四個(gè)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(nèi)制備的能與一抗或一抗片段結(jié)合的抗體，上面通常連有酶或熒光素等標(biāo)簽。由于二抗所具備的優(yōu)點(diǎn)使得其在免疫學(xué)實(shí)驗(yàn)中得以應(yīng)用廣泛，如western blot（通過與特異性抗體結(jié)合來鑒定蛋白質(zhì)），ELISA（以耦聯(lián)有酶的抗體或抗原為標(biāo)記來檢測特異性的蛋白質(zhì)，尤其是相應(yīng)的抗原或抗體），免疫組織化學(xué)（檢測組織中的特異性抗原），免疫細(xì)胞化學(xué)（通過免疫學(xué)方法檢測細(xì)胞的抗原組成），流式細(xì)胞術(shù)（通過檢測激光所激發(fā)熒光來鑒定分離不同類型的細(xì)胞）及免疫沉淀（通過抗原與抗體的特異性結(jié)合作用來分離相應(yīng)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(biāo)記的二抗可以免去對每一個(gè)一抗進(jìn)行標(biāo)記，大大節(jié)省了時(shí)間和費(fèi)用；此外，一個(gè)一抗分子可以同時(shí)結(jié)合幾個(gè)二抗分子，從而使信號(hào)大大增強(qiáng)，提高了實(shí)驗(yàn)靈敏度。

二抗可以耦聯(lián)有幾種不同的標(biāo)記，可以是酶，熒光素，或生物素。

Table 1. 典型的整個(gè)IgG分子的二抗

如何選擇二抗

通常情況下，某一特定的實(shí)驗(yàn)中可能同時(shí)有幾種二抗可供選擇，如何能選擇到該實(shí)驗(yàn)的二抗，需要綜合以下幾個(gè)方面進(jìn)行考慮：

一抗的物種來源

一抗是在什么物種來源的，相應(yīng)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(nèi)制備的，那么你就應(yīng)當(dāng)選擇抗小鼠的二抗，如果一抗是在兔體內(nèi)制備的，那么二抗就應(yīng)當(dāng)是抗兔的抗體。至于二抗本身是在什么動(dòng)物中制備的對二抗的質(zhì)量并無明顯的影響。

一抗是屬于哪個(gè)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(yīng)的二抗就是抗IgG抗體.

單克隆抗體的類別及亞類通常會(huì)在產(chǎn)品列表中列出，如果你的一抗是小鼠IgM，那么相應(yīng)的二抗就應(yīng)當(dāng)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(jié)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(biāo)記實(shí)驗(yàn)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(gè)不錯(cuò)的選擇，因?yàn)榇朔N抗體可以識(shí)別大多數(shù)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(jié)了幾種具有不同特異性的二抗：針對整個(gè)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(jié)合也可以與輕鏈結(jié)合，即與抗體分子的Fc，F(xiàn)（ab’）2/Fab部分（見圖5）均可反應(yīng)，抗IgG（H+L）也可以與其他免疫球蛋白家族反應(yīng)（如IgM和IgA），因?yàn)樗械拿庖咔虻鞍锥季哂邢嗤妮p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(jié)合，由于它們可以與輕鏈反應(yīng)，它們同時(shí)也已和具有相同輕鏈的其他種類的免疫球蛋白反應(yīng)。

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(yīng)，一次它們是類別特異性的（即gamma鏈特異性抗體只與IgG反應(yīng)，mu鏈特異性抗體只識(shí)別IgM，依此類推）。

輕鏈（kappa，labmda）特異性：與所有類別的抗體反應(yīng)，因?yàn)樗蓄悇e的抗體均具有相同的lambda鏈或kappa鏈。

經(jīng)過吸附處理的二抗：不同來源的免疫球蛋白含有相似的結(jié)構(gòu)，抗一個(gè)物種的抗體可能與其他物種發(fā)生交叉反應(yīng)，一次有些二抗經(jīng)過了動(dòng)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(xì)胞，那么選擇經(jīng)小鼠血清或IgG吸附處理過的二抗可以減少非特異性結(jié)合。但是此種抗體識(shí)別表位的數(shù)量被大大減少。

選擇哪種形式的二抗，是整個(gè)IgG分子，F(xiàn)（ab’）2片段，還是Fab片段？

整個(gè)IgG分子：此種抗體適用于多數(shù)情況。

F（ab’）2片段：該種抗體是由兩個(gè)靠二硫鍵連接的用于結(jié)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(xì)胞結(jié)合的情況。

Fab片段：它們只有一個(gè)結(jié)合位點(diǎn)，一般用來封閉內(nèi)源性免疫球蛋白。

選用耦聯(lián)有哪種探針的二抗？

一般來講，耦聯(lián)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(tuán)（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(shí)驗(yàn)。對于western blot和ELISA，zui常用的二抗是酶標(biāo)二抗，而細(xì)胞或組織標(biāo)記實(shí)驗(yàn)（細(xì)胞免疫化學(xué)，組織免疫化學(xué)，流式細(xì)胞術(shù)）中通常使用熒光基團(tuán)標(biāo)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(biāo)記的二抗。如果想要更大程度的放大檢測信號(hào)，可以使用biotin/avidin檢測系統(tǒng)。

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(diǎn)及其適用的實(shí)驗(yàn)：

酶：主要有兩種不同的酶耦聯(lián)物，HRP和AP。比較而言，前者更經(jīng)濟(jì)、快速、穩(wěn)定，而后者較前者更為靈敏，通常情況下，HRP廣泛應(yīng)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(shí)驗(yàn)，如ELISA和western blot.

熒光基團(tuán)：

1. FITC：FITC是廣泛使用的熒光基團(tuán)，但其zui大的缺點(diǎn)是淬滅快，使用抗淬滅劑可以減輕。
2. AMCA：常用在多標(biāo)記實(shí)驗(yàn)中，如免疫熒光、流式細(xì)胞術(shù)。其缺點(diǎn)是，淬滅快，且由于肉眼不能很好的檢測其所激發(fā)的藍(lán)色熒光，因而AMCA耦聯(lián)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(qiáng)。Cy3和Cy5比大多數(shù)熒光基團(tuán)更亮，更穩(wěn)定，背景更弱。二者常在共聚焦顯微實(shí)驗(yàn)中作多標(biāo)記。但Cy5的缺點(diǎn)是不能用表面熒光顯微鏡觀察，因此通常用具有遠(yuǎn)紅外檢測器的共聚焦顯微鏡觀察。
4. TRITC,RRX,TR:TRITC經(jīng)常和FITC一起用于雙標(biāo)記實(shí)驗(yàn)中，也可以使用RRX或TR，但TR會(huì)產(chǎn)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(biāo)記實(shí)驗(yàn)時(shí)，RRX尤其有用，可以和Cy2（或FITC）及Cy5一起使用，因?yàn)镽RX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(xì)胞術(shù)雙標(biāo)記實(shí)驗(yàn)。
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(yīng)而緊密結(jié)合在一起。首先加入生物素標(biāo)記的二抗，在加入耦聯(lián)有酶或熒光集團(tuán)的avidin、ExtrAvidin或streptavidin，后者能夠結(jié)合于二抗表面的多個(gè)位點(diǎn)上，從而極大的放大檢測信號(hào)。

抗體的純度：

親和層析純化的抗體一般更受人們歡迎，因?yàn)檫@些產(chǎn)品的純度zui高，產(chǎn)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?yàn)槠渲泻杏H和力很高的抗體，尤其是當(dāng)目的抗原存在的量很小的時(shí)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(yīng)用實(shí)例分析

為分析二抗的使用情況，我們隨機(jī)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(gè)國家的約80個(gè)研究機(jī)構(gòu)。收集的數(shù)據(jù)顯示二抗的使用非常廣泛，可以應(yīng)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(xué)和免疫細(xì)胞化學(xué)。事實(shí)上，二抗在ELISA及流式細(xì)胞術(shù)中應(yīng)用也很廣泛，不僅用于科學(xué)研究，而且廣泛的應(yīng)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(yīng)用相對較少。以下內(nèi)容是基于對所收集的二抗信息進(jìn)行統(tǒng)計(jì)分析后得出的一些結(jié)論，對于您在具體的實(shí)驗(yàn)中如何選擇的抗體具有借鑒作用。

選擇二抗時(shí)，您必須考慮到以下因素，如一抗的物種、產(chǎn)生二抗的宿主、一抗的類別或亞類、二抗所耦聯(lián)的探針類型及二抗的形式（如整個(gè)IgG分子，F(xiàn) （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(nèi)制備，其中屬小鼠及兔子zui為常見，相應(yīng)的，二抗的種屬也應(yīng)當(dāng)與一抗的相一致。而對于制備二抗的宿主，則應(yīng)選擇另外一種不同的動(dòng)物，一般選用驢、山羊、兔子、綿羊或其他較大的動(dòng)物，可以實(shí)現(xiàn)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(yīng)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?yàn)槌宋宸N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(shí)別幾乎所有類型的IgG亞類，因此，在不清楚一抗是屬于那個(gè)類別或亞類的時(shí)候，anti-IgG抗體是個(gè)不錯(cuò)的選擇。從我們的數(shù)據(jù)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(biāo)記實(shí)驗(yàn)中，包括免疫細(xì)胞化學(xué)、免疫組織化學(xué)及流式細(xì)胞術(shù)。

此外，整個(gè)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(shí)驗(yàn)中，如需要避免抗體與具有Fc受體的細(xì)胞結(jié)合的情況，或需要封閉內(nèi)源性免疫球蛋白的時(shí)候。

對于二抗所耦聯(lián)的探針的選擇，主要取決于具體的實(shí)驗(yàn)。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(yīng)用于 western blot當(dāng)中。通過統(tǒng)計(jì)分析發(fā)現(xiàn)，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(biāo)記的二抗在Western blot中均有廣泛使用；而對于AP標(biāo)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(shí)驗(yàn)（包括免疫細(xì)胞化學(xué)及免疫組織化學(xué)），應(yīng)用zui廣泛的是耦聯(lián)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(biāo)記的抗體（如Cy2, Cy3, and Cy5）、FITC及羅丹明（Rhodamine）。其中對于Alexa Fluor標(biāo)記的二抗，推薦選擇Invitrogen 公司或Molecular Probes公司（現(xiàn)屬于Invitrogen公司），是的生產(chǎn)Alexa Fluor染料的公司。從我們收集的數(shù)據(jù)來看，Jackson Immunoresearch Laboratories公司的花青染料（Cyanine）系列標(biāo)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(biāo)記的二抗使用較好。另外，如果你想放大檢測信號(hào)，你可以選擇生物素標(biāo)記的二抗，Jackson Immunoresearch Laboratories、Vector Laboratories 或R&D Systems均是不錯(cuò)的選擇。

對于流式細(xì)胞術(shù)，F(xiàn)ITC是選用zui多的標(biāo)簽，PE標(biāo)簽也可以使用。

對于ELISA實(shí)驗(yàn)，HRP 和 AP標(biāo)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(lián)標(biāo)記的二抗。

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