Development of an immunoassay for the detection of cystatin C dimers.
Human cystatin C (CysC) is a reversible cysteine protease inhibitor, which is abundantly secreted to body fluids. It is a potential marker of kidney dysfunction, but has been suggested to be of diagnostic importance in a number of neurodegenerative diseases, as well. The amyloid formation by a L68Q variant CysC accounts for the hereditary CysC amyloid angiopathy (HCCAA). Also, the wild type CysC forms inactive dimers at partly denaturing conditions through a domain swapping mechanism. Here, we have developed an immunoassay for the detection of dimeric CysC consisting of either a full length or an N-terminally truncated form. A codon optimized gene encoding a full length CysC was expressed in Escherichia coli, where the product was directed to the periplasmic space. Two different forms of CysC were isolated, a full length product and a form proteolytically truncated by 8 N-terminal amino acid residues. In vitro dimerization experiments were conducted in order to enable the selection of monoclonal antibodies for the construction of an immunoassay being able to primarily recognize the dimers. The analytical detection limit of the assay was 0.043 microg/l, with assay imprecision below 16%. The assay was linear in the range of 5-100 microg/l (R(2)=0.997). The dimer assay was employed for the measurement of serum and cerebrospinal fluid (CSF) sample panel of 20 multiple sclerosis (MS) and 22 non-MS patients. A dimer signal was observed in both serum and CSF samples. The dimer signals from CSF were approximately 2-22 times higher (average 13) than the corresponding signals from serum samples. However, the measured signal levels between the different patient groups showed no statistically significant difference in serum or in CSF (P=0.07 and P=0.98 respectively). In conclusion, the immunoassay provides direct means for detecting CysC dimers in serum and CSF in respect to the amount of total CysC.
Hyytiä
Heidi
H
University of Turku, Department of Biotechnology, Tykistökatu 6 A, 20520 Turku, Finland. heidi.hyytia@utu.fi
Ristiniemi
Noora
N
Airas
Laura
L
Pettersson
Kim
K
Hellman
Jukka
J
D000328:Adult; D019943:Amino Acid Substitution; D000682:Amyloid / Q000097:blood / Q000134:cerebrospinal fluid / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D028243:Cerebral Amyloid Angiopathy, Familial / Q000097:blood* / Q000134:cerebrospinal fluid* / Q000235:genetics / Q000276:immunology; D055316:Cystatin C / Q000097:blood* / Q000134:cerebrospinal fluid* / Q000235:genetics / Q000276:immunology; D005260:Female; D006801:Humans; D007118:Immunoassay / Q000379:methods; D008297:Male; D020125:Mutation, Missense; D055503:Protein Multimerization; D011994:Recombinant Proteins; D012680:Sensitivity and Specificity
D000682:Amyloid; D000911:Antibodies, Monoclonal; C525030:CST3 protein, human; D055316:Cystatin C; D011994:Recombinant Proteins