title: [Functionalized Metal Chelates Based on Diethylenetriaminetetraacetic Acids for Chemical Modification of Proteins and Small Biomolecules].
abstract: Bifunctional reagents based on diethylenetriaminetetraacetic acid containing a bound metal ion and a reactive functional group for the interaction with proteins and low-molecular-weight substances have been synthesized. An Amino-derivative of a complexonate was obtained by acylation of monosubstituted diamine with diethylenetriaminepentaacetic acid dianhydride followed by deprotection ofthe amino group, purification by anion exchange chromatography and chelation of Eu3+. This metal chelate derivative was used for labeling 17α-hydroxyprogesterone 3-(O-carboxymethyl)oxime and horseradish peroxidase. The enzyme modified with the Eu3+ complexonate at the carbohydrate component and with a cortisol derivative at the polypeptide chain was used in a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) as well as in an enzyme immunoassay of the steroid hormone. DELFIA showed that labeled 17α-hydroxyprogesterone retained the affinity for corresponding antibodies. A Eu(3+)-complexonate carboxy-derivative N-succinimide ester was obtained by acylation of the aminochelate with p-phthalic acid di-N-succinimide ester. It was used for modification of amino groups of lysine residues in polypeptide chains of human serum albumin and some immunoglobulins G. Purification of Eu3+ complexonate-protein conjugates by gel-chromatography on a Superose- 12 column allowed to separate the modified proteins from unreacted low molecular weight Eu(3+)-derivatives and to determine a degree of lanthanide inclusion into a protein. The amount of Eu3+ covalently attached to a protein was determined by measuring the fluorescence of a conjugate in the dissociative-enhancement solution. The obtained values correlated well with the results of ICP-MS determination of Eu3+ concentration in a conjugate solution. It was shown that conjugates of monoclonal antibodies obtained by the proposed method possessed the required characteristics of fluorescence intensity, signal-to-noise ratio, sensitivity and specificity in DELFIA medical diagnostic systems.
authors.forename: O S
authors.initials: OS
authors.lastname: Kuprienko
authors.forename: L V
authors.initials: LV
authors.lastname: Dubovskaya
authors.forename: P S
authors.initials: PS
authors.lastname: Shabunya
authors.forename: S A
authors.initials: SA
authors.lastname: Fatykhava
authors.forename: O V
authors.initials: OV
authors.lastname: Sviridov
mesh_terms: D019326:17-alpha-Hydroxyprogesterone / Q000737:chemistry*; D002614:Chelating Agents / Q000737:chemistry*; D005063:Europium / Q000737:chemistry*; D005453:Fluorescence; D006801:Humans; D004369:Pentetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D012709:Serum Albumin / Q000737:chemistry*; D013194:Staining and Labeling
chemical_list: D002614:Chelating Agents; D012709:Serum Albumin; C060068:diethylenetriaminetetraacetic acid; D005063:Europium; D019326:17-alpha-Hydroxyprogesterone; D004369:Pentetic Acid