Determination of paraprotein light chain type, identification of heavy chain disease

Abstract Previous studies using immunoneutralization techniques have shown that many factor VIII inhibitors are IgG antibodies of a single light chain type. We have investigated this apparent homogeneity by immunoneutralization assay and liquid isoelectric focusing of inhibitor fractions from five hemophiliacs and two nonhemophiliacs. By immunoneutralization assay, inhibitors from four hemophiliacs and one nonhemophiliac were exclusively k light chain type: the fifth hemophilic inhibitor was predominantly k1 and the second nonhemophilic inhibitor was a mixture of k and gamma. However, heavy chain subtyping of the six predominantly or exclusively k inhibitors showed all to be mixtures of IgG4 and IgG1. By isoelectric focusing, two inhibitors showed multiple peaks of activity between pH 5 and 9. The remaining five showed predominant peaks of activity, which were solely IgGk1 between pH 5.8 and 7, with smaller peaks between pH 7 and 9. The most acidic major peak, focusing at pH 6, was IgG4 in the three cases tested. Two of these acidic peaks neutralized factor VIII more efficiently than other peaks in the same focusing profiles, suggesting greater affinity for factor VIII. These studies demonstrate that factor VIII inhibitors are composed of heterogenous subpopulations of immunoglobulins which can be separated by isoelectric focusing.

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Macro creatine kinase: a study on CK-linked immunoglobulin.

Clinical Chemistry ◽

10.1093/clinchem/26.13.1816 ◽

1980 ◽

Vol 26 (13) ◽

pp. 1816-1820 ◽

Cited By ~ 25

Author(s):

H Yuu ◽

S Ishizawa ◽

Y Takagi ◽

K Gomi ◽

O Senju ◽

...

Keyword(s):

Creatine Kinase ◽

Heavy Chain ◽

Light Chain ◽

Immune Complexes ◽

Gel Filtration ◽

Strict Sense ◽

Light Chain Type ◽

Immunofixation Electrophoresis ◽

Chain Type ◽

Kinase A

Abstract We describe three cases in which creatine kinase (CK, EC 2.7.3.2) was linked to immunoglobulin in serum. In this study, its prevalence was 0.8%. Enzyme-immunofixation electrophoresis revealed that the heavy chain of CK-linked immunoglobulins was of class alpha in all cases. The light-chain type was identified as lambda in two cases and as both lambda and kappa in one case. The complexes were dissociated at pH 3.4 and re-formed with CK isoenzymes MM and MB at pH 7.4. The complex fraction obtained by gel filtration was not inhibited by anti-CK-M antibodies. Treatment of the serum with urea after heating shows residual CK-MM activity; in contrast, normal CK activity disappeared entirely after this treatment. The present study suggests that CK-linked immunoglobulins may be one of the circulating immune complexes and must be distinguished from other macro-CK in the strict sense. The results obtained show that the presence of the complexes results in false-positive CK-B activity in the immuno-inhibition test, and they may provide interesting insights into the mode of binding of the CK-linked immunoglobulins.

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Isotypic discordance of paraproteins and lymphocyte surface immunoglobulins in myeloma

Blood ◽

10.1182/blood.v57.1.192.192 ◽

1981 ◽

Vol 57 (1) ◽

pp. 192-195 ◽

Cited By ~ 9

Author(s):

M Nicholls ◽

PC Vincent ◽

E Repka ◽

J Saunders ◽

FW Gunz

Keyword(s):

Multiple Myeloma ◽

Heavy Chain ◽

Light Chain ◽

B Lymphocyte ◽

Plasma Cells ◽

Light Chains ◽

Lymphocyte Surface ◽

Light Chain Type ◽

Surface Immunoglobulins ◽

Chain Type

Abstract B lymphocyte surface immunoglobulins (Smlg) were studied in 24 patients with multiple myeloma by means of anti-isotypic antisera, and their heavy and light chain isotypes were compared in each patient with those of the paraprotein. In 21 patients, lymphocyte Smlg consisted of only one light chain type, and in 16 of only 1 heavy chain type. However, the Smlg and paraprotein heavy and light chain types were identical in only 5 patients while in 6 they differed in heavy and light chain types, in 7 in light chain type, and in 4 in heavy chain type. In 2 patients with light chain myeloma, Smlg light chains were isotypically the same as the paraprotein. Isotypic discordance between paraprotein and Smlg may signify the proliferation of a second malignant clone with failure to differentiate into secreting plasma cells. Alternatively, it is conceivable that the lymphocyte Smlg could have the same idiotypic specificity as the paraprotein despite the isotypic differences, but this will require further studies using anti-idiotypic antisera.

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Heterogeneity of factor VIII antibodies: further immunochemical and biologic studies

Blood ◽

10.1182/blood.v49.5.807.bloodjournal495807 ◽

1977 ◽

Vol 49 (5) ◽

pp. 807-817 ◽

Cited By ~ 1

Author(s):

MB Hultin ◽

FS London ◽

SS Shapiro ◽

WJ Yount

Keyword(s):

Factor Viii ◽

Isoelectric Focusing ◽

Heavy Chain ◽

Light Chain ◽

Major Peak ◽

Igg Antibodies ◽

Multiple Peaks ◽

Light Chain Type ◽

Apparent Homogeneity ◽

Chain Type

Previous studies using immunoneutralization techniques have shown that many factor VIII inhibitors are IgG antibodies of a single light chain type. We have investigated this apparent homogeneity by immunoneutralization assay and liquid isoelectric focusing of inhibitor fractions from five hemophiliacs and two nonhemophiliacs. By immunoneutralization assay, inhibitors from four hemophiliacs and one nonhemophiliac were exclusively k light chain type: the fifth hemophilic inhibitor was predominantly k1 and the second nonhemophilic inhibitor was a mixture of k and gamma. However, heavy chain subtyping of the six predominantly or exclusively k inhibitors showed all to be mixtures of IgG4 and IgG1. By isoelectric focusing, two inhibitors showed multiple peaks of activity between pH 5 and 9. The remaining five showed predominant peaks of activity, which were solely IgGk1 between pH 5.8 and 7, with smaller peaks between pH 7 and 9. The most acidic major peak, focusing at pH 6, was IgG4 in the three cases tested. Two of these acidic peaks neutralized factor VIII more efficiently than other peaks in the same focusing profiles, suggesting greater affinity for factor VIII. These studies demonstrate that factor VIII inhibitors are composed of heterogenous subpopulations of immunoglobulins which can be separated by isoelectric focusing.

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Isotypic discordance of paraproteins and lymphocyte surface immunoglobulins in myeloma

Blood ◽

10.1182/blood.v57.1.192.bloodjournal571192 ◽

1981 ◽

Vol 57 (1) ◽

pp. 192-195

Author(s):

M Nicholls ◽

PC Vincent ◽

E Repka ◽

J Saunders ◽

FW Gunz

Keyword(s):

Multiple Myeloma ◽

Heavy Chain ◽

Light Chain ◽

B Lymphocyte ◽

Plasma Cells ◽

Light Chains ◽

Lymphocyte Surface ◽

Light Chain Type ◽

Surface Immunoglobulins ◽

Chain Type

B lymphocyte surface immunoglobulins (Smlg) were studied in 24 patients with multiple myeloma by means of anti-isotypic antisera, and their heavy and light chain isotypes were compared in each patient with those of the paraprotein. In 21 patients, lymphocyte Smlg consisted of only one light chain type, and in 16 of only 1 heavy chain type. However, the Smlg and paraprotein heavy and light chain types were identical in only 5 patients while in 6 they differed in heavy and light chain types, in 7 in light chain type, and in 4 in heavy chain type. In 2 patients with light chain myeloma, Smlg light chains were isotypically the same as the paraprotein. Isotypic discordance between paraprotein and Smlg may signify the proliferation of a second malignant clone with failure to differentiate into secreting plasma cells. Alternatively, it is conceivable that the lymphocyte Smlg could have the same idiotypic specificity as the paraprotein despite the isotypic differences, but this will require further studies using anti-idiotypic antisera.

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Immunofixation. III. Application to the study of monoclonal proteins.

Clinical Chemistry ◽

10.1093/clinchem/22.12.1982 ◽

1976 ◽

Vol 22 (12) ◽

pp. 1982-1985 ◽

Cited By ~ 73

Author(s):

R F Ritchie ◽

R Smith

Keyword(s):

Immune System ◽

Heavy Chain ◽

Light Chain ◽

Gel Electrophoresis ◽

Monoclonal Immunoglobulin ◽

Light Chain Type ◽

Monoclonal Proteins ◽

Electrophoretic Polymorphism ◽

Chain Type

Abstract We have shown how immunofixation can be successfully used to study proteins exhibiting electrophoretic polymorphism. An analogous situation is seen in the disorders of the immune system that result in the production of variable quantitites of homogeneous immunoglobulins. Immunoelectrophroesis has been heavily used--sometimes unsuccessfully because of poor resolution--to study these materials. The appearance of a monoclonal immunoglobulin band is identical in both immunofixation and standard gel electrophoresis, a feature that allows unambiguous identification of a protein as to its heavy-chain class and light-chain type. Because antigen and antibody are placed in contact almost immediately after electrophoresis, interfering diffusion does not occur and even extremely small bands can be demonstrated and characterized.

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Rapid typing of serum paraproteins by immunoblotting without antigen-excess artifacts.

Clinical Chemistry ◽

10.1093/clinchem/33.8.1433 ◽

1987 ◽

Vol 33 (8) ◽

pp. 1433-1436 ◽

Cited By ~ 4

Author(s):

A G Norden ◽

L M Fulcher ◽

A D Heys

Keyword(s):

Horseradish Peroxidase ◽

Heavy Chain ◽

Light Chain ◽

Repeat Analysis ◽

Light Chain Type ◽

Serum Immunoglobulins ◽

Protein Staining ◽

Chain Type

Abstract In this new immunoblotting procedure for determining the heavy-chain class and light-chain type of monoclonal serum immunoglobulins, proteins are transferred from agarose electrophoretic gels to nitrocellulose by brief capillary blotting. Paraproteins transferred are detected with appropriate horseradish peroxidase-conjugated antisera to light chain and heavy chain. Examination of 121 serum specimens probably containing a paraprotein (as detected by protein staining) by immunoblotting and by immunofixation gave the same results for 116 specimens: paraproteins were typed in 103 specimens and their presence was excluded in 13. Immunoblotting required one repeat analysis (as compared with 22 for immunofixation). In only 100 min we could type as many as 10 paraproteins, and the procedure did not show antigen-excess artifacts. These results suggest that immunoblotting may be preferable to immunofixation for routine typing of paraproteins.

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