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The heat aggregation of human IgD
CLINICA
CCA
CHIMICA
75
ACTA
5123
THE
HEAT
M.ENGLISAND
AGGREGATION
OF HUMAN
IgD
J.SINDELAR
Department of Clinical Biochemistry, Postgraduate Vaccines (SE VAC), Praha (Czechoslovakia)
Medical
Institute
and Institute for Sera and
(Received March 27, 1972)
SUMMARY
The effect of heat on IgD immunoglobulin was investigated. _4t 56” an exceptionally rapid and nearly total aggregation occurs. The heated IgD differs in reactivity to anti-light chain sera from the native one.
Recent studies of IgD immunoglobulin have shown several marked differences between IgD and other classes of immunoglobulins. An exceptionally rapid heat aggregation of human IgD is to be described in this study. Eight sera with high concentrations of IgD were investigated. Two IgDL myeloma proteins were examined in fresh sera. Sera with polyclonal IgD were examined from z to IZ months after sampling, being repeatedly thawed and re-frozen during this interval. Control sera with IgG, IgA, IgM monoclonal proteins and both IgDL myeloma sera were diluted with saline so as to obtain a concentration of approximately 5 mg/ml of the corresponding monoclonal component ; sera with polyclonal IgD were examined undiluted. Pooled serum from healthy adults was used as reference material. Concentrations of IgD are stated in multiples of the mean value of this reference: 5.57, 6.25, 8.50, 6.62, 9.87, 19.9, 593.0, 195.0. Effect
ofheat.
Sera with monoclonal IgG, IgA, IgM and IgDL proteins were incubated in a water bath at 37’, 46” and 56” for I, 5, 15, 30 and 60 min. After completed incubation all samples were separated by agar gel electrophoresis. Incubation at 37” and 46” did not show any differences as compared with the non-incubated control samples. Marked changes of both IgD myeloma proteins appeared during incubation at 56”: the narrow band of IgD myeloma protein began to weaken after 5 min and disappeared completely after 30 min of incubation. Simultaneously, an insoluble protein precipitate formed on the anodic side of the starting well. No such changes were observed in sera containing IgG, IgA and IgM proteins. Immmoelectro$koresis Immunoelectrophoresis
with horse polyvalent
serum rich in anti-delta
heavy
Cl&. Chim. Acta, 4r (1972) 75-77
ENGLI:,
SINDEI&
Fig. r . Immunoelectrophoresis of IgDL myeloma serum after increasing intervals of incubation at 56”. The IgD line (D) is marked with arrow-head at the bottom. The line of heated IgD is marked with double arrow-heads.
chain and anti-lambda light chain antibodies (HaHu 358 SEVAC) showed marked differences of the IgD line already after I min incubation at 56” (Fig. I). The anodic end of the IgD line began to lengthen and a new line of heated IgD was forming with proceeding incubation. Changes in sera with polyclonal IgD develop much along the same manner. The line of heated IgD is well demonstrable by means of sera against delta heavy chain (SwHu IgD SEVAC), but not by means of sera against lambda light chains (SwHuBJL SEVAC, RaHuBJL SEVAC).
of IgD rnyelovra @roteins Both IgD myeloma sera were applied to a Sephadex G-zoo column. Similarly as in normal sera three main peaks were found in the elution diagram. The IgD myeloma proteins constituted a portion of the second eluted peak. The first two eluted peaks of sera incubated for 60 min at 56” and of non-incubated control sera were concentrated 20 times and proteins with IgD reactivity examined by means of double diffusion. The results indicate an evident shift of IgD-reacting antigens from the second eluted peak before heat exposure to the first eluted peak after heat exposure. Exposure of proteins to heat may result in association of disbalanced molecules into random and heterogenous aggregate populations. Aggregation of IgG, IgA and IgM immunoglobulins occurs after prolonged exposure to heat and even then involves only a part of the protein molecules present 1- 3. Little is hitherto known about the effect of heat on human IgD immunoglobulin. Spiegelberg et nL4 described an easy spontaneous aggregation of IgD at normal temperature. Killander5 called attention to the heat sensitivity of XgD. Penney etuE.~reported an almost total aggregation of IgD preparations on heating at G3O for 20 min. No changes of “principal” antigenic determinants of the heated IgD were observed. The results of our experiments indicate that in human IgD inlmunoglobulin
Gel $lkntion
Clin. Ckim. A&,
41 (1972) 75-77
EFFECTS OF HEAT ON
heat accounts
IgD
for an exceptionally
77 rapid and nearly total aggregation,
as compared
with other human immunoglobulins. Changes in intramolecular arrangement occurring on heating IgD appear to be rather significant, as may be inferred from the different reactivity of anti-lambda light chain sera to the aggregated IgDL. The easy heat aggregation of IgD immunoglobulin suggests, in agreement with other differences between IgD and the remaining classes of immunoglobulins, a difference in the nature and strength of the stabilising forces involved in the intramolecular arrangement of the IgD molecule. REFERENCES I D. S. ROWE, S. G. ANDERSON AND B. GRAB, Bull. World Health Organ., 42 (1970)535. 2 J. H. MORSE, J. Immunol., g5 (1965) 722. 3 C. S. HENNEY AND K. ISHIZAKA, J. Immunol., 100 (1968) 718. 4 H. J. SPIEGELBERG, J. W.PRAHLANDH.M. GREY, Biochemistry, g(1g7o) zrr5. 5 J. KILLANDER, Nobel Symfiosium, 3 (1967) 614. 6 C.S. HENNEY,H.D.WELCHER,W.D.TERRYANDD.S.ROWE, Immunochemistry,6(1g6g)445. Clin. Chim.
Acta,
41 (1972) 75-77
CCA
CHIMICA
75
ACTA
5123
THE
HEAT
M.ENGLISAND
AGGREGATION
OF HUMAN
IgD
J.SINDELAR
Department of Clinical Biochemistry, Postgraduate Vaccines (SE VAC), Praha (Czechoslovakia)
Medical
Institute
and Institute for Sera and
(Received March 27, 1972)
SUMMARY
The effect of heat on IgD immunoglobulin was investigated. _4t 56” an exceptionally rapid and nearly total aggregation occurs. The heated IgD differs in reactivity to anti-light chain sera from the native one.
Recent studies of IgD immunoglobulin have shown several marked differences between IgD and other classes of immunoglobulins. An exceptionally rapid heat aggregation of human IgD is to be described in this study. Eight sera with high concentrations of IgD were investigated. Two IgDL myeloma proteins were examined in fresh sera. Sera with polyclonal IgD were examined from z to IZ months after sampling, being repeatedly thawed and re-frozen during this interval. Control sera with IgG, IgA, IgM monoclonal proteins and both IgDL myeloma sera were diluted with saline so as to obtain a concentration of approximately 5 mg/ml of the corresponding monoclonal component ; sera with polyclonal IgD were examined undiluted. Pooled serum from healthy adults was used as reference material. Concentrations of IgD are stated in multiples of the mean value of this reference: 5.57, 6.25, 8.50, 6.62, 9.87, 19.9, 593.0, 195.0. Effect
ofheat.
Sera with monoclonal IgG, IgA, IgM and IgDL proteins were incubated in a water bath at 37’, 46” and 56” for I, 5, 15, 30 and 60 min. After completed incubation all samples were separated by agar gel electrophoresis. Incubation at 37” and 46” did not show any differences as compared with the non-incubated control samples. Marked changes of both IgD myeloma proteins appeared during incubation at 56”: the narrow band of IgD myeloma protein began to weaken after 5 min and disappeared completely after 30 min of incubation. Simultaneously, an insoluble protein precipitate formed on the anodic side of the starting well. No such changes were observed in sera containing IgG, IgA and IgM proteins. Immmoelectro$koresis Immunoelectrophoresis
with horse polyvalent
serum rich in anti-delta
heavy
Cl&. Chim. Acta, 4r (1972) 75-77
ENGLI:,
SINDEI&
Fig. r . Immunoelectrophoresis of IgDL myeloma serum after increasing intervals of incubation at 56”. The IgD line (D) is marked with arrow-head at the bottom. The line of heated IgD is marked with double arrow-heads.
chain and anti-lambda light chain antibodies (HaHu 358 SEVAC) showed marked differences of the IgD line already after I min incubation at 56” (Fig. I). The anodic end of the IgD line began to lengthen and a new line of heated IgD was forming with proceeding incubation. Changes in sera with polyclonal IgD develop much along the same manner. The line of heated IgD is well demonstrable by means of sera against delta heavy chain (SwHu IgD SEVAC), but not by means of sera against lambda light chains (SwHuBJL SEVAC, RaHuBJL SEVAC).
of IgD rnyelovra @roteins Both IgD myeloma sera were applied to a Sephadex G-zoo column. Similarly as in normal sera three main peaks were found in the elution diagram. The IgD myeloma proteins constituted a portion of the second eluted peak. The first two eluted peaks of sera incubated for 60 min at 56” and of non-incubated control sera were concentrated 20 times and proteins with IgD reactivity examined by means of double diffusion. The results indicate an evident shift of IgD-reacting antigens from the second eluted peak before heat exposure to the first eluted peak after heat exposure. Exposure of proteins to heat may result in association of disbalanced molecules into random and heterogenous aggregate populations. Aggregation of IgG, IgA and IgM immunoglobulins occurs after prolonged exposure to heat and even then involves only a part of the protein molecules present 1- 3. Little is hitherto known about the effect of heat on human IgD immunoglobulin. Spiegelberg et nL4 described an easy spontaneous aggregation of IgD at normal temperature. Killander5 called attention to the heat sensitivity of XgD. Penney etuE.~reported an almost total aggregation of IgD preparations on heating at G3O for 20 min. No changes of “principal” antigenic determinants of the heated IgD were observed. The results of our experiments indicate that in human IgD inlmunoglobulin
Gel $lkntion
Clin. Ckim. A&,
41 (1972) 75-77
EFFECTS OF HEAT ON
heat accounts
IgD
for an exceptionally
77 rapid and nearly total aggregation,
as compared
with other human immunoglobulins. Changes in intramolecular arrangement occurring on heating IgD appear to be rather significant, as may be inferred from the different reactivity of anti-lambda light chain sera to the aggregated IgDL. The easy heat aggregation of IgD immunoglobulin suggests, in agreement with other differences between IgD and the remaining classes of immunoglobulins, a difference in the nature and strength of the stabilising forces involved in the intramolecular arrangement of the IgD molecule. REFERENCES I D. S. ROWE, S. G. ANDERSON AND B. GRAB, Bull. World Health Organ., 42 (1970)535. 2 J. H. MORSE, J. Immunol., g5 (1965) 722. 3 C. S. HENNEY AND K. ISHIZAKA, J. Immunol., 100 (1968) 718. 4 H. J. SPIEGELBERG, J. W.PRAHLANDH.M. GREY, Biochemistry, g(1g7o) zrr5. 5 J. KILLANDER, Nobel Symfiosium, 3 (1967) 614. 6 C.S. HENNEY,H.D.WELCHER,W.D.TERRYANDD.S.ROWE, Immunochemistry,6(1g6g)445. Clin. Chim.
Acta,
41 (1972) 75-77