USE OF MONOCLONAL-ANTIBODIES TO DETECT CONFORMATIONAL ALTERATIONS IN LACTATE-DEHYDROGENASE ISOENZYME-5 ON HEAT DENATURATION AND ON ADSORPTION TO POLYSTYRENE PLATES
USE OF MONOCLONAL-ANTIBODIES TO DETECT CONFORMATIONAL ALTERATIONS IN LACTATE-DEHYDROGENASE ISOENZYME-5 ON HEAT DENATURATION AND ON ADSORPTION TO POLYSTYRENE PLATES
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DOI：
10.1016/0161-5890(86)90122-7
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发表时间：
1986-09-01
影响因子：
3.6
通讯作者：
KATCHALSKIKATZIR, E
中科院分区：
文献类型：
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作者：
HOLLANDER, Z;KATCHALSKIKATZIR, E
制备抗人乳酸脱氢酶同工酶5（HLDH5）的单克隆抗体（mAb）MF30（IgG）。当 MF30 酶被吸附到聚苯乙烯板上时，发现 MF30 与 HLDH5 高度特异性结合，但在溶液中时不识别同工酶。 mAb MF30 不识别吸附在聚苯乙烯上的同工酶 HLDH1、HLDH2 和 HLDH3。热处理的HLDH5(在70°C、pH 7.5下加热45秒)对MF30的表现与未处理的同工酶相同，即它们之间的相互作用仅在变性的同工酶吸附到ELISA板上之后才发生。第二种单克隆抗体（编号为 2/66）是针对猪乳酸脱氢酶同工酶 5 (PLDH5) 制备的，发现在溶液中以及吸附到聚苯乙烯上时，会与猪同工酶相互作用。然而，在同工酶经过上述热处理后，没有发生这种相互作用。人们发现 mAb 2/66 在溶液中以及吸附到聚苯乙烯上时都能与人同工酶 HLDH5 完全发生交叉反应；然而，与猪同工酶的情况一样，所有这些识别在热变性时都消失了。上述发现表明HLDH5在聚苯乙烯表面的吸附伴随着构象变化。酶的热变性似乎会导致其抗原模式与吸附酶的抗原模式不同的构象的出现。数据表明 mAb MF30 和 2/66 识别 HLDH5 的两个不同抗原位点。被 2/66 识别并存在于天然酶中的抗原位点在溶液中和吸附到聚苯乙烯上时在加热时消失。当 mAb MF30 在热处理之前或之后将酶吸附到聚苯乙烯表面上时，另一个抗原决定簇就会被 mAb MF30 识别。这项研究说明了适当的单克隆抗体可能用作探针来检测各种条件下蛋白质中发生的构象变化。
A monoclonal antibody (mAb) MF30 (IgG) against human lactate dehydrogenase isoenzyme 5 (HLDH5) was prepared. MF30 was found to bind with high specificity to HLDH5 when the enzyme was adsorbed onto a polystyrene plate but did not recognize the isoenzyme when in solution. The isoenzymes HLDH1, HLDH2 and HLDH3 adsorbed onto polystyrene were not recognized by mAb MF30. Heat-treated HLDH5 (heated at 70.degree. C, pH 7.5 for 45 sec) behaved towards MF30 in the same way as the untreated isoenzyme i.e. interaction between them took place only after the denatured isoenzyme had been adsorbed onto an ELISA plate. A second mAb, designated 2/66, prepared against porcine lactate dehydrogenase isoenzyme 5 (PLDH5), was found to interact with the porcine isoenzyme when in solution as well as when adsorbed onto polystyrene. However, no such interaction occurred after the isoenzyme had been subjected to heat treatment as above. The mAb 2/66 was found to cross-react fully with the human isoenzyme HLDH5 both in solution and when adsorbed onto polystyrene; however, as in the case of the porcine isoenzyme, all such recognition was lost upon heat denaturation. The above findings suggest that the adsorption of HLDH5 onto a polystyrene surface is accompanied by a conformational change. Denaturation of the enzyme by heat seems to lead to the appearance of a conformation differing in its antigenic pattern from that of the adsorbed enzyme. The data suggest that the mAbs MF30 and 2/66 recognize two different antigenic sites of HLDH5. The antigenic site which is recognized by 2/66 and is present in the native enzyme both when in solution and when adsorbed onto polystyrene disappears on heating. The other antigenic determinant is recognized by mAb MF30 when the enzyme is adsorbed onto a polystyrene surface either before or after heat treatment. This study illustrates the way in which appropriate mAbs might possibly be used as probes for the detection of conformational alterations occurring in proteins under various conditions.