Use of monoclonal antibodies to detect conformational alterations in lactate dehydrogenase isoenzyme 5 on heat denaturation and on adsorption to polystyrene plates.
A monoclonal antibody (mAb) MF30 (IgGl) 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 degrees 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.
D000327:Adsorption; D000818:Animals; D000911:Antibodies, Monoclonal; D004797:Enzyme-Linked Immunosorbent Assay; D006358:Hot Temperature; D006801:Humans; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase; D011137:Polystyrenes; D011487:Protein Conformation; D011489:Protein Denaturation; D013329:Structure-Activity Relationship; D013552:Swine / Q000276:immunology
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D011137:Polystyrenes; D007770:L-Lactate Dehydrogenase