Methods, immunoassays and devices for detection of anti-lipoidal antibodies

An immunoassay and antibody technology, which can be used in measurement devices, biological tests, material inspection products, etc., and can solve problems such as refractory-lipid antibody similar testing

Inactive Publication Date: 2008-08-13
UNITED STATES OF AMERICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it has been more difficult to develop similar tests for anti-lipid antibodies, at least in part because hydrophobic antigens (e.g., VDRL, USR, or RPR antigens, or cardiolipin) of anti-lipid antibodies are resistant to attachment to immunoassay devices used as immunoassays. A solid support for an element of

Method used

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  • Methods, immunoassays and devices for detection of anti-lipoidal antibodies
  • Methods, immunoassays and devices for detection of anti-lipoidal antibodies
  • Methods, immunoassays and devices for detection of anti-lipoidal antibodies

Examples

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Embodiment 1

[0208] Preparation of USR lipid antigen

[0209] This example describes the preparation of an exemplary lipid antigen, USR antigen, which can be attached to a solid support such as nitrocellulose, using the disclosed method. As described in this example, 100 ml of USR antigen can be limited; however, those skilled in the art will appreciate that the described protocol can be scaled up or down to produce more or less USR antigen, respectively. Furthermore, all method steps, reactions, etc. in this and the following examples are performed at room temperature (eg, from about 20° C. to about 25° C.) unless expressly indicated otherwise.

[0210] Add eight (8) ml of VDRL buffered saline (10 g NaCl, 0.5 ml formaldehyde, 0.093 g disodium hydrogen phosphate, and 0.170 g monobasic potassium phosphate in 1 liter of distilled water) into a 250 ml round glass stopper bottle . Over a period of 40 to 60 seconds, 10 ml of VDRL antigen (0.9% cholesterol, 0.03% bovine cardiac cardiolipin, an...

Embodiment 2

[0212] Ammonium sulfate fractionation of human serum infected with Treponema pallidum

[0213] This example describes a method for isolating serum containing immunoglobulins. The method of this Example (as well as other immunoglobulin isolation methods generally known in the art) can be used to partially purify immunoglobulins from serum of any origin, including serum obtained from non-human subjects such as rabbits.

[0214] Sera from human patients infected with Treponema pallidum (referred to as "hyperimmune antiserum" in this and the following Examples) were obtained from Biomedical Resources (Hatboro, PA). The desired amount of hyperimmune antiserum is placed in a container containing more than twice the amount of serum. Under stirring, 70% saturated ammonium sulfate equal to the amount of serum was slowly added dropwise to the serum. This mixture was stirred at room temperature for about 4 hours and then centrifuged at about 3000 rcf for 30 minutes. After centrifugation...

Embodiment 3

[0216] IgG Purification Using Protein A Affinity Columns

[0217] IgG was separated from the immunoglobulin containing fraction using protein A sepharose chromatography. As is known in the art, Protein A binds specifically to the Fc region of IgG from various mammalian species.

[0218] A buffer reservoir containing 1000 ml of 20 mM sodium phosphate buffer, pH 7.4 ("phosphate buffer") was attached to a 10 ml Protein A Sepharose CL-4B column (Pharmacia). The column was washed with 200 ml of phosphate buffer, and the pH of the eluate was measured. After the pH of the eluate was within the range of 7.3±0.2, the sample containing immunoglobulin was loaded onto the column.

[0219] After disconnection from the buffer reservoir, the immunoglobulin-containing dialysis retentate from Example 2 was applied to the column, taking care not to disturb the surface of the column matrix. Then, 20 ml of phosphate buffer was added to the column, the buffer reservoir was reattached to the col...

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Abstract

The present application describes compositions, methods and devices for detecting anti-lipid antibodies and diagnosing diseases such as syphilis. In particular, methods for immobilizing lipid antigens (including cardiolipin, lecithin, and cholesterol) on solid supports (eg, nitrocellulose membranes) are described. The ability to immobilize lipid antigens on membranes fulfills a long known need for a membrane-based assay for the detection of anti-lipid antibodies. An immunoassay device for simultaneous treponema pallidum and non-treponemal testing is also described.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Patent Application No. 60 / 693,120, filed June 21, 2005, which is hereby incorporated by reference in its entirety. [0003] Thanks for government support [0004] This invention was made by National Center for HIV, STD and TB Prevention, Division of AIDS, STD, and TB Laboratory Research, Laboratory Reference and Research Branch Centers for Disease Control and Prevention, an agency of the United States Government. technical field [0005] The present disclosure relates to methods of immobilizing lipid antigens on solid supports and related immunoassays and immunoassay devices (e.g., test strips, flow-through devices, or lateral flow devices) that can be used, for example, to detect anti- - Lipid antibodies and / or diagnostic disease (eg, syphilis). Background of the invention [0006] Syphilis is a sexually transmitted disease (STD) caused by Treponema pallidum. World...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/571G01N33/92
CPCG01N33/571G01N33/92
Inventor 阿诺德·R·卡斯特罗罗伯特·W·格尔格维多利亚·波普
Owner UNITED STATES OF AMERICA
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