Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-09-30
2002-05-07
Ceperley, Mary E. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007500, C435S188000, C436S532000, C436S545000, C436S546000, C436S817000, C530S388240, C530S402000, C530S404000, C530S405000, C530S406000
Reexamination Certificate
active
06383766
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to haptens and conjugate labels having improved specificity for anti-cortisol antibodies, methods for their preparation and use in immunoassays for the detection of cortisol. More particularly, the present invention relates to conjugates comprising horseradish peroxidase and reduced cortisol.
BACKGROUND OF THE INVENTION
Cortisol is the major glucocorticoid in humans. It is synthesized and secreted by the zona fasciculata and the zona reticularis of the adrenal cortex. It is involved in the regulation of carbohydrate, protein, and lipid metabolism. Cortisol levels can rise ten fold following surgery or other major trauma, as the steroid acts to prevent vascular collapse, reduce inflammation, and suppress the immune system.
There are three primary medical disorders associated with hyperadrenalism: Cushing's syndrome, hyperaldosteronism, and congenital adrenal hyperplasia. Cushing's syndrome is the term used to describe any condition resulting from an increased concentration of circulating glucocorticoid, usually cortisol (
Clinical Chemistry: Theory, Analysis, and Correlation;
Lawrence A. Kaplan, Amadeo J. Pesce, C V Mosby Company, 1989, pp 673-4).
The detection and quantification of cortisol in human serum, plasma or urine is required for proper diagnosis, treatment and follow-up of cortisol related conditions.
Competitive binding immunoassays for cortisol comprise anti-cortisol antibody, usually bound to an immobilized or immobilizable substrate and labeled cortisol, or labeled analogs (derivatives) of cortisol. It shall be understood that whenever reference is made to labeled cortisol, unless otherwise indicated, the term is intended to encompass labeled analogs (derivatives) of cortisol. Labeled cortisol competes with cortisol for a limited number of anti-cortisol antibody binding sites. Signal derived from free or bound labeled cortisol is determined as a measure of the amount of cortisol.
The sensitivity and specificity of an immunoassay for cortisol are dependent on the labeled cortisol. It is important that labeled cortisol effectively compete for the limited number of anti-cortisol binding sites with steroids structurally similar to cortisol that may be present in a sample. Otherwise, a clinically acceptable determination of the amount of cortisol in the sample will not be obtained.
Individuals having a deficiency of the enzyme 11&bgr;-hydroxylase, or who receive metyrapone will have greatly increased levels of 11-deoxycortisol, which is structurally similar to cortisol (
Fundamentals of Clinical Chemistry,
Tietz,N. W., W. B. Saunders Co., 1987,p 569) and potentially can compete with labeled cortisol for binding to anti-cortisol antibody. Other cortisol-like steroids that may be present in a sample, which potentially can compete with labeled cortisol for anti-cortisol antibody, include prednisolone, cortisone, and corticosterone. Such competition with labeled cortisol for anti-cortisol antibody is termed cross-reactivity.
Commercial cortisol assays can exhibit cross-reactivity with all the above-identified steroids. For example, a cross-reactivity with 11-deoxycortisol of greater than 10 percent has been observed; seriously compromising the accuracy of the assay for cortisol.
SUMMARY OF THE INVENTION
The problems associated with prior art assays for cortisol have been overcome using the conjugate labels described hereinbelow.
The present invention relates to compositions comprising novel reduced cortisol conjugates, methods for their preparation and use in immunoassays for cortisol.
In another aspect, it relates to conjugates of reduced cortisol as immunogens or haptens for eliciting anti-cortisol or anti-reduced cortisol antibodies.
It was found unexpectedly that labeled reduced cortisol conjugates of the present invention effectively compete with cortisol-like steroids for binding to anti-cortisol antibodies, thereby exhibiting significantly less cross-reactivity compared with prior art labeled cortisol conjugates. Immunoassays for cortisol comprising labeled reduced cortisol conjugates of the present invention exhibit both improved specificity and sensitivity for the determination of cortisol.
Accordingly, the present invention provides a reduced cortisol conjugate of formula:
wherein X is O, S, sufonyl, or phosphono; X
1
is a labeled or unlabeled natural or synthetic polymer or a label; Y
1
is a linking group or a bond; X
2
is a labeled or unlabeled natural or synthetic polymer or a label; Y
2
is a linking group or a bond; A
1
and A
2
are each hydrogen or A
1
and A
2
together form a single bond, B
1
and B
2
are each hydrogen or B
1
and B
2
together form a single bond, E
1
and E
2
are each hydrogen or E
1
and E
2
together form a single bond provided at least one of A
1
and A
2
, or B
1
and B
2
, or E
1
and E
2
are each hydrogen.
In another aspect, the present invention relates to methods for the preparation of reduced cortisol conjugates. Accordingly, we provide a method for preparing a reduced cortisol conjugate of formula
wherein X, X
1
, Y
1
, X
2
, Y
2
, A
1
, A
2
, B
1
, B
2
, E
1
and E
2
are as defined above, comprising:
reacting a compound of formula
with a reducing agent.
An alternative method is provided for preparing a reduced cortisol conjugate of formula
wherein X, X
1
, Y
1
, X
2
, Y
2
, A
1
, A
2
, B
1
, B
2
, E
1
and E
2
are as defined above, comprising the steps of:
(i) reacting a compound of formula
with a reducing agent, thereby forming compound IA or IC; and
ii) with a first coupling agent, thereby forming a compound of formula
wherein G
1
is a coupling group;
(iii) optionally, reacting X
2
with a second coupling agent, thereby forming X
2
—G
2
, wherein G
2
is a coupling group capable of forming a covalent bond with the coupling group G
1
, and wherein G
1
and G
2
may be the same;
(iv) optionally reacting compound IA or IC with X
2
—G
2
wherein G
2
is capable of forming a covalent bond with a functional group of X
1
, thereby forming reduced cortisol conjugate IB or ID;
(v) optionally reacting compound IIIA or IIIC with X
2
wherein G
1
is capable of forming a covalent bond with a functional group of X
2
, thereby forming reduced cortisol conjugate IB or ID;
(vi) optionally reacting compound IIIA or IIIC with X
2
—G
2
, thereby forming reduced cortisol conjugate IB or ID.
In yet another aspect, the present invention relates to methods for the qualitative or quantitative determination of cortisol that utilize the novel labeled reduced cortisol conjugates.
Accordingly, we provide a method for performing a competitive assay for cortisol comprising the steps of:
A) contacting a sample suspected of containing cortisol with
(i) an immobilized or immobilizable receptor that binds cortisol, thereby forming cortisol that is bound and cortisol that is not bound to the immobilized or immobilizable receptor,
(ii) a labeled reduced cortisol conjugate of formula IA, IB, IC or ID as defined above, thereby forming labeled reduced cortisol conjugate that is bound and labeled reduced cortisol conjugate that is not bound to the immobilized or immobilizable receptor;
B) detecting either the labeled reduced cortisol conjugate that is bound or the labeled reduced cortisol conjugate that is not bound to the immobilized or immobilizable receptor as a measure of the amount of cortisol in the sample.
In an alternative ebodiment, the assay method described above may be combined with a step wherein the labeled reduced cortisol conjugate that is bound is separated from the labeled reduced cortisol conjugate that is not bound to the immobilized or immobilizable receptor;
An alternative method for performing a competitive assay for cortisol using a dry analytical element is also provided, the dry analytical element comprising
a) a spreading zone,
b) one or more reagent zones,
c) a support, and
together or separately in one or more of the zones, an immobilized receptor that binds cortisol and optionally, a labeled reduced cortisol conjugate of formula IA, IB,
Contestable Paul B.
Groth Holly L.
Lynn Shirley Y.
Snyder Brian A.
Sprague Lisa D.
Antar Stacey B.
Ceperley Mary E.
Ortho-Clinical Diagnostics, Inc.
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