Assays, antibodies, and standards for detection of oxidized and MDA-modified low density lipoproteins
AssigneeLeuven Research & Development VZW
InventorsPaul Noel Holvoet, Desire Jose Collen
Life of this patent
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Abstract
Immunoassays for malondialdehyde-modified low density lipoprotein (MDA-modified LDL) and oxidized low density lipoprotein (OxLDL), monoclonal antibodies (and the cell lines for them) for use in the assays, and a storage-stable standard (which may be used as a calibrator and/or control) are disclosed. MDA-modified LDL and OxLDL are implicated in atherosclerosis and its etiology.
Claims55
An immunological assay for the detection and/or quantification of MDA-modified LDL and OxLDL in a sample, said assay comprising:
- (a) contacting the sample with a first antibody that has high affinity for MDA-modified LDL and OxLDL;
- and
- (b) thereafter visualizing and/or quantifying a binding reaction between the first antibody and the MDA-modified LDL and OxLDL present in the sample;
- wherein the MDA-modified LDL and OxLDL for which the first antibody has high affinity contain at least 60 substituted lysine moieties per apo B-100 moiety.
The assay of any of the preceding claims in which the affinity constant of the first antibody for MDA-modified LDL and for OxLDL is at least about 1×1010 M−1.
The assay of any of the preceding claims in which the first antibody has low affinity for native LDL.
The assay of any of the preceding claims in which the first antibody is the monoclonal antibody mAb-4E6 produced by hybridoma Hyb4E6 deposited at the BCCM under deposit accession number LMBP 1660 CB on or about Apr. 24, 1997.
The assay of any of the preceding claims in which the sample is derived from the fluid or tissue of a human being.
An immunological sandwich assay for the detection and/or quantification of MDA-modified LDL in a sample in which assay a first antibody that has a high affinity for MDA-modified LDL is bound to a substrate, said assay comprising:
- (a) contacting the sample with the substrate having bound to it the first antibody under binding conditions so that at least some of any MDA-modified LDL in the sample will bind to the first antibody;
- (b) thereafter removing unbound sample from the substrate;
- (c) thereafter contacting the substrate with a second antibody that has a high affinity for MDA-modified LDL;
- and
- (d) thereafter visualizing and/or quantifying the MDA-modified LDL that was present in the sample;
- wherein the MDA-modified LDL for which the first antibody and the second antibody have high affinity contains at least 60 substituted lysine moieties per apo B-100 moiety.
Monoclonal antibody mAb-4E6 produced by hybridoma Hyb4E6 deposited at the BCCM under deposit accession number LMBP 1660 CB on or about Apr. 24, 1997.
Hybridoma Hyb4E6 deposited at the BCCM under deposit accession number LMBP 1660 CB on or about Apr. 24, 1997.
Monoclonal antibody mAb-8A2 produced by hybridoma Hyb8A2 deposited at the BCCM under deposit accession number LMBP 1661 CB on or about Apr. 24, 1997.
Hybridoma Hyb8A2 deposited at the BCCM under deposit accession number LMBP 1661 CB on or about Apr. 24, 1997.
A stable standard containing MDA-modified LDL whose extent of substitution of its lysine moieties will remain essentially constant over normal periods of time during normal, storage for biological materials, the MDA-modified LDL of said standard being made by contacting malondialdehyde with LDL at a predetermined molar ratio of malondialdehyde to the apo B-100 moiety of the LDL, the standard containing an agent that reduces the ability of any metal ions present to catalyze oxidation of the LDL and/or an anti-oxidant.
A kit for conducting a sandwich assay for the determination of OxLDL or MDA-modified LDL or both in a sample, said kit comprising:
- (a) a substrate on which is bound a first antibody that has high affinity for OxLDL or MDA-modified LDL or both, the OxLDL and MDA-modified LDL each having at least 60 substituted lysine moieties per apo B-100 moiety, and
- (b) a labeled antibody having a high affinity for OxLDL that becomes bound to the first antibody during the assay or for MDA-modified LDL that becomes bound to the first antibody during the assay or for both that become bound to the first antibody during the assay.
Dependencies are inferred from the claim text.