Methods, immunoassays and devices for detection of anti-lipoidal antibodies
AssigneeThe United States of America as represented by the Secretary of the Department of Health and Human Services, Centers for Disease Control and Prevention
InventorArnold R. Castro
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Abstract
Compositions, methods and devices for the detection of anti-lipoidal antibodies and the diagnosis of disease, for example, syphilis, are described. In particular, a method for immobilizing a lipoidal antigen, comprising cardiolipin, lecithin, and cholesterol, on a solid support (such as a nitrocellulose membrane) is described. The ability to immobilize a lipoidal antigen on a membrane satisfies a long-felt need for a membrane-based assay for the detection of anti-lipoidal antibodies. Also described are immunoassay devices for concurrently performing treponemal and non-treponemal tests for syphilis.
Claims26
A method of making an immunoassay device for detection of anti-lipoidal antibodies comprising:
- preparing a lipoidal antigen in a non-aqueous solution;
- mixing the lipoidal antigen in a non-aqueous solution with an aqueous solution to generate lipoidal micelles;
- centrifuging the lipoidal micelles to generate a supernatant comprising small lipoidal antigen micelles having an average diameter of less than 5 microns;
- applying the small lipoidal antigen micelles to the microporous membrane having a proximal sample application area and a detection zone with a predetermined pore size, wherein the small antigen micelles enter and are retained in the pores of the detection zone, thereby forming an immunoassay device for detection of anti-lipoidal antibodies.
A method of making an immunoassay device for detection of anti-lipoidal antibodies comprising:
- preparing a synthetic VDRL antigen in a non-aqueous solution comprising ethanol;
- mixing the synthetic VDRL antigen in a non-aqueous solution with an aqueous solution comprising saline solution to generate lipoidal micelles;
- centrifuging the lipoidal micelles to generate a pellet;
- resuspending the pellet in a buffer comprising 3.72% EDTA, 40% choline chloride (v/v), phosphate buffered saline pH 6.9 ±0.1, thereby generating a resuspended pellet;
- centrifuging the resuspended pellet to generate a supernatant comprising small lipoidal antigen micelles having an average diameter of less than 5 microns;
- applying the small lipoidal antigen micelles to the microporous membrane having a proximal sample application area and a detection zone with a pore size of 5 microns to about 10 microns, wherein the small lipoidal antigen micelles enter and are retained in the pores of the detection zone, thereby forming an immunoassay device for detection of anti-lipoidal antibodies.
Dependencies are inferred from the claim text.