Anti-idiotype scFv-enzyme fusion proteins: a clonable analyte-mimicking probe for standardized immunoassays targeting small biomarkers.
Anal Chem
; 85(23): 11553-9, 2013 Dec 03.
Article
en En
| MEDLINE
| ID: mdl-24256209
ABSTRACT
Most immunoassays use probes that convert concentrations of analytes into signal intensity. To prepare the probes, analytes are usually linked to a reporter protein (e.g., enzymes) with the aid of chemical reagents. However, these conventional methods yield a mixture of heterogeneous products and consequently reduce assay performance. "Clonable" homogeneous probes, i.e., recombinant molecules in which a target protein is genetically fused to a reporter with a defined coupling ratio, are now available for analyzing protein biomarker concentrations. Here, we have expanded this strategy to measure small biomarkers (haptens) by genetically fusing proteinaceous molecules that mimic target haptens with enzymes. 11-Deoxycortisol (11-DC) was chosen as the model hapten, and the ß-type anti-idiotype antibodies (ßId-Abs) that recognize the paratope of anti-hapten antibodies were used as the target hapten mimic. The V(H) and V(L) genes of a ßId-Ab, targeting a mouse anti-11-DC antibody (CET-M8), were assembled to encode a single-chain Fv fragment (ßId-scFv), which was then fused with a gene encoding a variant of alkaline phosphatase. The product, ßId-scFv-ALP' protein, had satisfactory enzyme activity and bound to CET-M8 in a competitive manner with 11-DC. A colorimetric enzyme-linked immunosorbent assay (ELISA) for 11-DC, based on the competitive reaction between the analyte and ßId-scFv-ALP' against immobilized CET-M8, was found to be sensitive (limit of detection = 22 pg/assay) and specific (cross-reactivity with cortisol, 0.24%) for clinical use and could be used to determine serum 11-DC levels after a simple solvent extraction. The anti-idiotype scFv-enzyme fusion proteins proposed here can be prepared reproducibly as homogeneous products with a 11 coupling ratio and would facilitate standardization of immunoassays for small biomarkers.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Anticuerpos Antiidiotipos
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Clonación Molecular
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Anticuerpos de Cadena Única
Tipo de estudio:
Prognostic_studies
Límite:
Adolescent
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Adult
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Animals
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Female
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Humans
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Male
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Middle aged
Idioma:
En
Revista:
Anal Chem
Año:
2013
Tipo del documento:
Article
País de afiliación:
Japón
Pais de publicación:
EEUU
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ESTADOS UNIDOS
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ESTADOS UNIDOS DA AMERICA
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EUA
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UNITED STATES
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UNITED STATES OF AMERICA
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US
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USA