Isolation and biochemical characterization of plasma monoclonal free light chains in amyloidosis and multiple myeloma: a pilot study of intact and truncated forms of light chains and their charge properties.
BACKGROUND
The presence of monoclonal immunoglobulin free light chains (FLC) in the serum is commonly associated with the gammopathies, including multiple myeloma, systemic light chain amyloidosis and non-amyloid light chain deposition disease. Although a sensitive nephelometry-based assay is used for quantification of serum FLC and patient follow-up, this method does not provide information regarding the biochemical properties of these proteins. The present study focused on the development of the procedure for isolation and biochemical characterization of monoclonal FLC in small plasma specimens from patients with these disorders.


METHODS
Methods used in this study were ultrafiltration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein elution from gel and support membranes, dialysis, lyophilization, isoelectric focusing (IEF) and Western blotting.


RESULTS
The isolation, concentration and partial purification of FLC was based on micro-preparative SDS-PAGE employing analytical scale gels. For the determination of the isoelectric point of FLC, the developed protocol included consecutive IEF, electrotransfer of IEF-separated proteins onto and elution from support membranes, and their analysis by SDS-PAGE-based Western blotting. The procedures, which require only 20-50 microL of starting plasma, allow biochemical characterization of the monomeric, dimeric and truncated forms of FLC, including their charge properties.


CONCLUSIONS
The developed procedure may be applied to reveal distinguishing chemical features of FLC in serum, which could be important in predicting the pathologic form of disease, and in yielding information to better understand the mechanism(s) involved in the deposition of light chains in tissues.
Kaplan
Batia
B
Heller Institute of Medical Research, Sheba Medical Center, Tel-Hashomer, Israel. kaplanb@sheba.health.gov.il
Ramirez-Alvarado
Marina
M
Dispenzieri
Angela
A
Zeldenrust
Steve R
SR
Leung
Nelson
N
Livneh
Avi
A
Gallo
Gloria
G
D000686:Amyloidosis / Q000097:blood / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000097:blood / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007128:Immunoglobulin Fragments / Q000097:blood / Q000737:chemistry / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000097:blood / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D009101:Multiple Myeloma / Q000097:blood / Q000276:immunology*; D010865:Pilot Projects; D055672:Static Electricity
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007147:Immunoglobulin Light Chains