Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is organic
Reexamination Certificate
2001-04-05
2003-09-30
Ceperley, Mary E. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is organic
C436S527000, C436S530000, C436S544000, C436S823000, C530S391100, C530S391500, C530S408000
Reexamination Certificate
active
06627460
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel methods for the preparation of water-soluble cross-linked conjugates and conjugate complexes as well as to novel water-soluble cross-linked conjugates and conjugate complexes per se. The conjugates and conjugate complexes confer an improved sensitivity in immunochemical assays, in particular when used in lateral flow devices and in methods for determining the presence or absence of small amounts of active components present in liquid samples.
BACKGROUND OF THE INVENTION
A large research effort has been devoted to devising ways to improving immunochemical assay reliability and sensitivity in e.g. home pregnancy and fertility tests and, consequently, there is a continuous need for new and improved methods for preparing conjugates which exhibit a high degree of sensitivity and specificity when employed in such immunochemical assays. Clearly, the number of “active” components, such as the number of antibodies or antigens, and the number of “detectable units”, such as dye molecules, present in the conjugate are of utmost importance when developing new conjugates for use in high-sensitive immunochemical assays.
Various strategies for improving the sensitivity and reliability of immunoassays have been reviewed by L. J. Kricka (1994)
Clin. Chem.
40, 347-357.
EP 0 594 772 B1 relates to water-soluble, polymer-based conjugates comprising moieties derived from divinyl sulfone. EP 0 594 772 B1 describes the possibility of enhancing the attachment of molecular species, such as antibodies and antigens, to a water-soluble carrier molecule by taking advantage of the so-called “salting out” effect. It turned out, however, that by increasing the salt concentration to about 1 M an irreversible precipitate was formed.
It has now surprisingly been found that by further increasing the concentration of salt in the reaction mixture, a reversible (i.e. a re-dissolvable) precipitate is formed which contains “large” water-soluble conjugates which are useful in various immunochemical assays, such as in lateral flow devices.
DESCRIPTION OF THE INVENTION
In a first aspect the present invention relates to a method for the preparation of a water-soluble cross-linked conjugate comprising moieties of at least one carrier component, moieties of more than one linking component, moieties of at least one spacer component, moieties of at least one signal component and moieties of at least one primary targeting component, the signal component being covalently attached to the spacer component and the spacer component being covalently attached, via the linking component, to the carrier component, said method comprising:
a) reacting a water-soluble intermediate conjugate comprising moieties of at least one carrier component, moieties of more than one linking component, moieties of at least one spacer component, moieties of at least one signal component, the signal component being covalently attached to the spacer component and the spacer component being covalently attached, via the linking component, to the carrier component,
via reaction of unreacted reactive moieties derived from the linking component, with at least one primary targeting component in an aqueous solution, the conditions being such that a reversible precipitate is formed;
b) re-dissolving the reversible precipitate comprising the water-soluble cross-linked conjugate in an aqueous medium; and
c) optionally subjecting the water-soluble cross-linked conjugate to a purification step.
In the present context the term “water soluble” when used in connection with the cross-linked conjugates means that the conjugates obtained according to the methods disclosed herein should be soluble in an aqueous medium, such as water, at room temperature, i.e. the cross-linked conjugates obtained by the methods disclosed herein should give rise to a solution which is substantially clear and homogenous as judged by visual inspection of the sample.
In a preferred embodiment of the invention the cross-linked conjugates obtained by the methods disclosed herein have a water solubility of at least 0.1, preferably at least 1, such as at least 10, more preferably at least 50, such as at least 100, in particular at least 200 mg dry conjugate per ml water at 25° C.
Before going into a detailed discussion with respect to the above-mentioned precipitation step, it should be noted that the water-soluble intermediate conjugate may be prepared by a method comprising:
I) reacting at least one water-soluble carrier component with more than one linking component in an aqueous solution at a pH above 7, so as to form an aqueous solution containing a water-soluble intermediate precursor comprising water-soluble moieties of the carrier component having covalently attached thereto reactive moieties derived from the linking component;
II) optionally subjecting the water-soluble intermediate precursor to a purification step;
III) reacting the optionally purified water-soluble intermediate precursor, via reaction of said reactive moieties, with
i) at least one spacer component in an aqueous solution at a pH above 7, so as to form a second water-soluble intermediate precursor, the conditions being such that only a fraction of the reactive moieties reacts with the spacer component and that a significant amount of unreacted reactive moieties remain,
ii) optionally subjecting the second water-soluble intermediate precursor to a purification step, and
iii) reacting the optionally purified second water-soluble intermediate precursor, via reaction of the spacer component, with at least one signal component in an aqueous solution at a pH above 7, so as to form a water-soluble intermediate conjugate, the conditions being such that most of the signal components react with the spacer moiety, rather than with the linker component, and that a significant amount of unreacted reactive moieties of the linker component remain unreacted ( i.e. only a small fraction of the signal components react with the reactive moieties of the linker components); and
IV) optionally subjecting the water-soluble intermediate conjugate obtained in step III) to a purification step.
The method outlined in general form above in steps I-IV, and the components alluded to therein, is schematically represented in FIG.
4
. The Figure is merely to be used for purposes of clarity as it represents anecdotal examples of one embodiment. Therein, the various stages of intermediates and precursors that are part of the method are schematically represented to assist the reader to follow the procedure.
The Water-Soluble Carrier Component
The term “carrier component” in the context of the present invention is used to denote the “backbone” of the conjugate, i.e. the carrier component functions as a backbone on which various molecules may be attached.
The water-soluble polymers which function as the carrier component in the method for the preparation of conjugates may be chosen from a wide variety of types of polymers, including:
natural and synthetic polysaccharides, as well as derivatives thereof, for example dextrans and dextran derivatives, starches and starch derivatives, cellulose derivatives, amylose and pectin, as well as certain natural gums and derivatives thereof, such as gum arabic and salts of alginic acid;
homopoly(amino acid)s having suitable reactive functionalities, such as polylysines, polyhistidines or polyornithines;
natural and synthetic polypeptides and proteins, such as bovine serum albumin and other mammalian albumins; and
synthetic polymers having nucleophilic functional groups, such as polyvinyl alcohols, polyallyl alcohol, polyethylene glycols and substituted polyacrylates.
Very suitable polymers for the purposes of the invention are polysaccharides and derivatives thereof, for example: dextrans, carboxymethyl-dextrans, hydroxyethyl- and hydroxypropyl-starches, glycogen, agarose derivatives, and hydroxyethyl- and hydroxypropyl-celluloses. As will be apparent from the working examples herein (vide infra), notably dextrans have proved to be particularly suitable polym
Lihme Allan Otto Fog
Stanley Christopher John
Amdex A/S
Ceperley Mary E.
Foley & Lardner
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