Continuous cultures of fused cells secreting antibody of predefined specificity

傲慢与偏见

Continuous cultures of fused cells secreting antibody of predefined specificity
分泌具有预定特异性抗体的融合细胞的持续培养


在这篇著名的论文里，两位作者大胆地把以前用于不同骨髓瘤细胞之间的融合技术延伸为将骨髓瘤细胞与经绵羊红细胞免疫的小鼠脾细胞进行融合，建立了杂交瘤制备单抗技术。1984年，获诺贝尔奖。

傲慢与偏见

这篇文章还有一些地方本楼主还没有看懂，另外因为一些技术现在已经被迭代更新，老的方法技术不很熟悉。
需要一点点解读，并且会不断修订。

傲慢与偏见

THE manufacture of predefined specific antibodies by means of permanent tissue culture cell lines is of general interest. There are at present a considerable number of permanent cultures of myeloma cellsL and screening procedures have been used to reveal antibody activity in some of them. This, however, is not a satisfactory source of monoclonal antibodies of predefined specificity. We describe here the derivation of a number of tissue culture cell lines which secrete anti-sheep red blood cell (SRBC) antibodies. The cell lines are made by fusion of a mouse myeloma and mouse spleen cells from an immunised donor. To understand the expression and interactions of the lg chains from the parental lines, fusion experiments between two known mouse myeloma lines were carried out.
用持续培养组织细胞系产生预定的特异性抗体是当时广泛关注的研究课题，之前已有大量的骨髓瘤持续培养的成功经验，以及建立了一系列关于抗体的筛检方法，但是并没有一个满意的能产生预定的特异性抗体的成功报道。
作者大胆地将两种不同骨髓瘤细胞和绵羊红细胞免疫的小鼠脾细胞进行融合。选两种骨髓瘤细胞是为了更清晰地分析和了解融合后细胞产生的抗体链来源于哪些亲本细胞，选用绵阳红细胞作为抗原是因为已经有相应抗体的检测技术，便于单抗的鉴定和筛选。首先对两个已知的骨髓瘤细胞进行融合。

傲慢与偏见

Each immunoglobulin chain result from the integrated expression of one of several V and C genes coding respectively for its variable and constant sections. Each cell expresses only one of the two possible alleles (allelic exclusion; reviewed in ref. 3).
三年以后，利根川进(Susumu Tonegawa)发表了关于人类抗体基因片段(V(D)J)重排机制的文章，解释了对于抗体的轻重链如何产生。(A complete immunoglobulin gene is created by somatic recombination.CELL，1978 Sep;15(1):1-14）

When two antibody-producing cells are fused, the products of both parental lines are expressed , and although the light and heavy chains of both parental lines are randomly joined, no evidence of scrambling of V and C sections is observed. These results, obtained in an heterologous system involving cells of rat and mouse origin, have now been confirmed by fusing two myeloma cells of the same mouse strain,and provide the background for the derivation and understanding of antibody-secreting hybrid lines in which one of the parental cells is an antibody-producing spleen cell.

有研究表明，当两个产生抗体的细胞（一个来源于大鼠，一个来源于小鼠）进行融合后，两个亲本细胞的抗体能在融合细胞都进行表达，但是没有数据能表明两者的轻重链V区和C区是否互相干扰。在本研究中，用两种同样来源小鼠的不同骨髓瘤细胞进行融合，为能更好理解后面的杂交瘤细胞提供理论依据。

傲慢与偏见

Two myeloma cell lines of BALB/c origin were used. PI Bul is resistant to 5-bromo-2'-deoxyuridine\ does not grow in selective medium (HAT, ref. 6) and secretes a myeloma protein, Adj PC5, which is an lgG2A (K), (ref. 1). Synthesis is not balanced and free light chains are also secreted. The second cell line, P3-X63Ag8, prepared from P3 cclls 2 , is resistant to 20 11g ml-1 8-azaguaninc and does not grow in HAT medium.
本研究中用到的进行融合的两种骨髓瘤细胞均来源于BALB/c小鼠，一种是PI Bul细胞，Brdu抗性，不能在HAT培养基中生长，分泌一种骨髓瘤蛋白Adj PC5，属于lgG2A (K)型。另一种是P3-X63Ag8，来源于P3细胞，抗8-氮鸟嘌呤, 在HAT培养基中也不能生长。分泌MOPC 21蛋白，属于lgG I (K)型，已经进行测序。（应该不是用得第一代测序吧？第一代DNA测序技术用的是1975年由Sanger和Coulson开创的链终止法，并在1977年，桑格用此方法测定了第一个基因组序列，全长5375个碱基的噬菌体X174序列，又十年后才有PCR技术）

Equal numbers of cells from each parental line were fused using inactivated Scndai virus• and samples contining 2 x 10^5 cells were grown in selective medium in separate dishes. Four out of ten dishes showed growth in selective medium and these were taken as independent hybrid lines, probably derived from single fusion events.
将两个细胞等量混合，用灭活的仙台病毒进行促融合，在选择性培养基中进行细胞的培养，10个平皿中有4个平皿细胞得以生长存活，挑选到杂交瘤细胞。

The karyotype of the hybrid cells after 5 months in culture was just under the sum of the two parental lines (Table 1).
经过5个月的培养，分析细胞核型，显示融合后细胞染色体数目比两个亲本细胞染色体数目之和略低一些HY-B(106)<3(65)+P1(55)。

傲慢与偏见

Figure I shows the isoelectric focusing (IEF) pattern of the secreted products of different lines. The hybrid cells (samples c-h in Fig. 1) give a much more complex pattern than either parent (a and b) or a mixture of the parental lines (m). The important feature of the new pattern is the presence of extra bands (Fig. I, arrows).
Figure 1 是不同细胞系分泌产物的等点聚焦电泳图，融合细胞（c-h) 的带型比亲本细胞（a和b) 以及亲本细胞直接混合(m)要复杂得多。箭头所指处为新融合细胞多出的条带。（A为非变性电泳结果，B为变性电泳结果）


These new bands, however, do not seem to be the result of differences in primary structure; this is indicated by the IEF pattern of the products after reduction to separate the heavy and light chains (Fig. I B). The IEF pattern of chains of the hybrid clones (Fig. I B, g) is equivalent to the sum of the IEF pattern (a and b) of chains of the parental clones with no evidence of extra products. We conclude that, as previously shown with interspccics hybridsM, new lg molecules are produced as a result of mixed association between heavy and light chains from the two parents. This process is intracellular as a mixed cell population does not give rise to such hybrid molecules (compare m and g, Fig.1A).
在B变性胶中可以看到，A非变性胶中多出的条带，分离出轻重链，和两种亲本细胞的轻重链蛋白一致，说明这些蛋白的一级结构没有明显区别。
在B图中可见，融合细胞（h) 的条带是亲本细胞（a和b) 的条带的累加。说明来源于两种亲本细胞。融合细胞和两种细胞直接混合的蛋白带型不一致，可能的原因为两种亲本细胞的抗体轻重链之间发生了交叉反应，产生新的Ig蛋白分子。

傲慢与偏见

等电聚焦电泳（Isoelectric focusing，简称IEF）
等电聚焦是六十年代中期出现的新技术。近年来等电聚焦技术有了新的进展，已迅速发展成为一门成熟的近代生化实验技术。目前等电聚焦技术已可以分辨等电点（pI）只差0.001pH单位的生物分子。由于其分辨力高，重复性好，样品容量大，操作简便迅速，在生物化学、分子生物学及临床医学研究中得到广泛的应用。
等电聚焦电泳是利用一种特殊的缓冲液（两性电解质）在凝胶（常用聚丙烯酰胺凝胶电泳）内制造一个pH梯度，电泳时每种蛋白质就将迁移到等于其等电点（pI）的pH处（此时此蛋白质不再带有净的正或负电荷），形成一个很窄的区带。

  蛋白质分子是典型的两性电解质分子。它在大于其等电点的pH环境中解离成带负电荷的阴离子，向电场的正极泳动，在小于其等电点的pH环境中解离成带正由荷的阳离子，向电场的负极泳动。这种泳动只有在等于其等电点的pH环境中，即蛋白质所带的净电荷为零时才能停止。

如果在一个有pH梯度的环境中，对各种不同等电点的蛋白质混合样品进行电泳，则在电场作用下，不管这些蛋白质分子的原始分布如何，各种蛋白质分子将按照它们各自的等电点大小在pH梯度中相对应的位置处进行聚焦，经过一定时间的电泳以后，不同等电点的蛋白质分子便分别聚焦于不同的位置。这种按等电点的大小，生物分子在pH梯度的某一相应位置上进行聚焦的行为就称为“等电聚焦”。等电聚焦的特点就在于它利用了一种称为两性电解质载体的物质在电场中构成连续的pH梯度，使蛋白质或其他具有两性电解质性质的样品进行聚焦，从而达到分离、测定和鉴定的目的。

傲慢与偏见

The individual cells must therefore be able to express both isotypes. This result shows that in hybrid cells the expression of one isotype and idiotype
docs not exclude the expression of another: both heavy chain isotypes ( y I and y2a) and both V11 and both V L regions (idiotypes) are expressed. There are no allotypic markers for the C K region to provide direct proof for the expression of both parental C K regions. But this is indicated by the phenotypic link between the V and C regions.
Figure 1A shows that clones derived from different hybridisation experiments and from subclones of one line are indistinguishable.This has also been observed in other experiments (data not shown). Variants were, however, found in a survey of I00 subclones. The difference is often associated with changes in the ratios of the different chains and occasionally with the total' disappearance of one or other of the chains. Such events are best visualised on IEF analysis of the separated chains (for example, Fig. lh, in which the heavy chain of P3 is no longer observed). The important point that no new chains are detected by IEF complements a previous study of a rat-mouse hybrid line in which scrambling of V and C regions from the light chains of rat and mouse was not observed.

图1中c/d/e/f/g IEF结果相似，来自不同的融合实验或者同一种系的不同亚克隆之间并没有区别。对100个单克隆进行分析，变异株主要的不同在于轻重链的比例上，有的链甚至完全消失，图1B中h就没有检测到P3重链。IEF的结果显示融合后细胞没有产生新的抗体链，佐证了之前大鼠-小鼠融合细胞中没有观察到V区和C区之间的重排，产生新链的观察结果。

In this study, both light chains have identical CK regions and therefore scrambled VL-CL molecules would be undetected. On the other hand, the heavy chains are of different subclasses and we expect scrambled V H-C H to be detectable by IEF. They were not observed in the clones studied and if they occur must do so at a lower frequency. We conclude that in syngeneic cell hybrids (as well as in interspecies cell hybrids) V-C integration is
not the result of cytoplasmic events. Integration as a result of DNA translocation or rearrangement during transcription is also suggested by the presence of integrated mRNA molecules11 and by the existence of defective heavy chains in which a deletion of V and C sections seems to take place in already committed cells .

傲慢与偏见

The cell line P3-X63Ag8 described above dies when exposed to HAT medium. Spleen cells from an immunised mouse also die in growth medium. When both cells are fused by Sendai virus and the resulting mixture is grown in HAT medium, surviving clones can be observed to grow and become established after a few weeks. We have used SRBC as immunogen, which enabled us, after culturing the fused lines, to determine the presence of specific antibody-producing cells by a plaque assay technique13 (Fig. 2a).
细胞系 P3-X63Ag8在HAT培养基中不能存活，来自免疫小鼠的脾细胞在HAT中也不能存活，当两种细胞在灭活仙台病毒的促融合下形成杂交瘤细胞，能在HA中生长。本研究中选用SRBC绵羊红细胞作为免疫原，在培养出融合细胞后，能通过噬斑鉴定鉴定是否有特异性抗体产生的细胞系。
The hybrid cells were cloned in soft agar and clones producing antibody were easily detected by an overlay of SRBC and complement (Fig. 2b).  Individual clones were isolated and shown to retain their phenotype as almost all the clones of the derived purified line are capable of lysing SRBC (Fig. 2c). The clones were visible to the naked eye (for example, Fig. 2d).
杂交瘤细胞在软琼脂上培养，形成的能产生SRBC抗体的细胞克隆能比较容易地被一层SRBC和补体检测到(Fig. 2b). 挑选单克隆，纯化后的细胞克隆依然保留裂解红细胞的功能。(Fig. 2c). 肉眼可见形成的单克隆细胞团(Fig. 2d)，以及形成的溶血环。

a, 6,000 hybrid cells Sp-1; 6000个杂交瘤细胞SP-1
b, clones grown in soft agar from an inoculum of 2,000 Sp-1 cells; 软琼脂上接种的2000个SP-1单细胞
c, 挑选的 一株Sp-1/7单克隆 ;
d, 一株单克隆的放大倍数图；
小鼠通过腹腔注射200ul十倍稀释的SRBC细胞，一月后加强免疫，4天后收集脾细胞。10^8来源于BALB/c小鼠的这种免疫脾细胞和10^7 P3-X67Ag8骨髓瘤细胞进行融合。
融合后，SP-1在HAT培养基中生长8天，1-3天换液一次。
细胞在HT培养基中再培养2周，融合后40天，进行抗SRBC的检测。
噬斑形成/总杂交细胞比例约为1/30。
杂交细胞接种培养在软琼脂上，进行改进的SRBC检测实验。当杂交瘤细胞长到合适的大小，铺上2ml0.6%含有25ulSRBC和0.2ml新鲜豚鼠血清作为补体。 经过37℃过夜培养，阳性/总克隆数比例为1/33。挑阳性单克隆细胞继续培养，命名为sp-1-7，90%的细胞鉴定为阳性。
A second experiment in which I 06 P3- X67 Ag8 cells were fused with 108 spleen cells was the source of a clone giving rise to indirect plaques (clone
Sp-2/3-3). Indirect plaques were produced by the addition of I :20 sheep anti-MOPC 21 antibody to the agarose overlay.

傲慢与偏见

The derived lines (Sp hybrids) are hybrid cell lines for the following reasons. They grow in selective medium. Their karyotype after 4 months in culture (Table 1) is a little smaller than the sum of the two parental lines but more than twice the chromosome number of normal BALB/c cells, indicating that
the lines are not the result of fusion between spleen cells. In addition the lines contain a metacentric chromosome also present in the parental P3-X67Ag8. Finally, the secreted immunoglobulins contain MOPC 21 protein in addition to new, unknown components. The latter presumably represent the
chains derived from the specific anti-SRBC antibody. 认为成功获得两细胞融合的杂交瘤细胞原因如下：能在选择性培养基中生长，4个月后核型进行比较，杂交瘤细胞的染色体数目比两个亲代细胞的染色体数目之和要少，又比正常的小鼠细胞染色体的二倍要多。除此之外，在亲本细胞P3-X67Ag8中存在的等臂染色体在杂交瘤细胞中也存在。最后，分泌的抗体中除了未知成分外还有MOPC21蛋白（来源于骨髓瘤细胞）。

Figure 3A shows the IEF pattern of the material secreted by two such Sp hybrid clones. The IEF bands derived from the parental P3 line are visible in the pattern of the hybrid cells, although obscured by the presence of a number of new bands. The pattern is very complex, but the complexity of hybrids of this type is likely to result from the random recombination of chains (see a bove, Fig. 1). Indeed, IEF patterns of the reduced material secreted by the spleen- P3 hybrid clones gave a simpler pattern of lg chains. The heavy and light chains of the P3 parental line became prominent, and new bands were apparent.