抄録
Many cases were previously reported about modifications of serum lactic dehydrogenase (LDH) isoenzymes due to complex formation with immunoglobulins. This LDH-binding immunoglobulin is of interest in connection with circulating autoantibodies. However, the exact nature and properties of this unusual immunoglobulin were still not completely elucidated. This report presents immunochemically unique properties of LDH-binding immunoglobulin and describes an application of affinity chromatography to the isolation of immunoglobulin in question.
The antigenic determinants of LDH-binding immunoglobulin were judged by the method of serum immunoelectrophoresis followed by LDH activity staining. In 14 cases studied presently, each of these immunoglobulins had either alpha or gamma of heavy chain (IgA 7, IgG 7). But the type of light chain was proved to be exclusively kappa regardless of differences in heavy chain class. This fact seems to suggest that LDH-binding immunoglobulin in each case is monoclonal and that unique structures responsible for binding to LDH exist in light chain.
Immunoglobulin A, characterized by binding capacity to LDH, was isolated by two steps of affinity chromatographies which were based upon its binding properties to anti-immunoglobulin A and LDH. Methods of insolubilization of legand protein were as follows. CNBr-activated Sepharose 4B (1g) was used as supporting gel and coupled with each legand protein(10mg)under the condition of 0.1M bicarbonate buffer pH 9.0 (0.5M NaCl). After unreacted functioning groups were blocked with 1M ethanolamine, gel was packed in column (gel bed 3.5ml) and washed with 0.1M borate pH 8.0 (1M NaCl) and 0.1M acetate pH 4.0 (1M NaCl) alternately.
At first 1ml of the patient serum was bufferized against 0.2 M glycine-HC1 buffer pH 2.8 (0.5 M NaCl), and filtered on Sephadex G-200 with the same buffer in order to split off LDH. This LDH-free protein fraction was applied on Sepharose 4B coupled with anti-immunoglobulin A and filtered with 0.2 M Tris-HC1 buffer pH 8.0 (0.5M NaCl). IgA fraction obtained by changing of pH of the elution buffer to 2.8 was then labeled with fluorescein isothiocyanate (FITC). Finally FITC-labeled IgA fraction was applied on Sepharose 4B coupled with LDH which was prepared from human hemolysate. By changing of pH a peak of fluorescence intensity corresponding to LDH binding immunoglobulin was observed to elute. The amount of isolated immunoglobulin was just a trace, but the result of present affinity chromatographic experiments indicates that this new specific purification method is effective and applicable to the isolation of various autoantibodies.
In our 14 cases, all of LDH-binding immunoglobulins had exclusively kappa type of light chain.
LDH-binding immunoglobulin A was isolated by the technique of affinity chromatography.
