Two-dimensional polyacrylamide gel electrophoresis analysis of cryoglobulins and identification of an IgM-associated peptide.
The clonality of immunoglobulins (Igs) in cryoprecipitates (n = 41) was studied by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Our series included 24 cryoglobulins characterized by immunofixation electrophoresis (IF), 12 'trace amount' cryoglobulins, defined by a protein content in the precipitate of less than 0.05 mg/ml of serum, and five cryoglobulins of undetermined protein composition by IF. 2-D PAGE analysis showed polyclonal IgG associated either with monoclonal Igs (type II cryoglobulins; n = 14) or with polyclonal IgM (type III cryoglobulins; n = 14). In ten cryoprecipitates (two 'trace amount' cryoglobulins as well as seven of 19 type II and as one of five type III cryoglobulins by IF) polyclonal IgG were associated with a mixture of polyclonal and monoclonal IgM. These cryoglobulins were tentatively named type II-III cryoglobulins. A monoclonal IgM was observed in one cryoprecipitate (type I cryoglobulins). Two cryoglobulins presented unexpected 2-D patterns, characterized by the presence of oligoclonal IgM, with trace amounts of Igs of different isotypes (tentatively named type II-III(variant) cryoglobulins). A peptide of 44 kDa with a pI of 5.45 was observed in all cryoglobulins containing IgM (n = 40). This peptide was also present in purified monoclonal or polyclonal IgM fractions. N-terminal microsequencing (12 amino acid residues) revealed that this IgM-associated peptide was an unknown protein. Our results highlight the role of 2-D PAGE as an aid in the analysis of cryoglobulins.
Tissot
J D
JD
Fondation Centre de Transfusion Sanguine de la Croix-Rouge suisse, Lausanne, Switzerland.
Schifferli
J A
JA
Hochstrasser
D F
DF
Pasquali
C
C
Spertini
F
F
Clément
F
F
Frutiger
S
S
Paquet
N
N
Hughes
G J
GJ
Schneider
P
P
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000097:blood; D003449:Cryoglobulinemia / Q000097:blood / Q000276:immunology; D003450:Cryoglobulins / Q000032:analysis* / Q000145:classification; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D007132:Immunoglobulin Isotypes / Q000097:blood; D007075:Immunoglobulin M / Q000097:blood*; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000097:blood* / Q000737:chemistry / Q000235:genetics
D000911:Antibodies, Monoclonal; D003450:Cryoglobulins; D007132:Immunoglobulin Isotypes; D007075:Immunoglobulin M; D010455:Peptides