Method of detecting and identifying circulating antigens in human biological samples

Inventors

AuCoin, David P. • Kozel, Thomas R. • Smalley, Claire

Abstract

Disclosed herein is a method of detecting and identifying antigens that are shed into human bodily fluids during infection. The disclosed method allows circulating antigens associated with a particular infection to be detected within minutes or hours from testing as compared to days required with the current methods. Methods of identifying diagnostic indicators/targets for a given condition or disease are disclosed which include immunizing a veterinary subject with biological fluids obtained from a human infected with particular antigens to identify diagnostic targets for immunoassay. Also disclosed are methods of diagnosing and monitoring a B. pseudomallei-associated condition, such as melioidosis. Point-of-care immunoassays are also provided that can be used to diagnose or monitor the efficacy of a B. pseudomallei-associated condition treatment. These immunoassays can also be used for rapid diagnosis of infection produced by B. pseudomallei, such as meliodosis.

Core Innovation

The invention provides In vivo Microbial Antigen Discovery (InMAD) for detecting circulating/secreted microbial antigens in human body fluids and using the detected antigens to identify diagnostic indicators and diagnostic assay targets. The approach uses immunizing naïve veterinary subjects with filtered serum/urine from infected humans and then detecting antigens in biological samples obtained from the immunized veterinary subjects.

Antigen detection in the veterinary subject samples is performed using one-dimensional immunoblots and two-dimensional immunoblots, optionally followed by mass spectroscopy to identify diagnostic indicators or targets. The method includes comparing detected antigens from the immunized veterinary subject sample with a control, wherein alteration in at least one antigen detected relative to the control indicates that the antigen is a diagnostic indicator for the selected condition or disease.

The disclosure applies InMAD to Burkholderia pseudomallei and melioidosis, including diagnosing infection, monitoring therapy efficacy over time, and self-monitoring. It describes proteomic profiles obtained from two-dimensional Western blots and example identified antigen spots, including GroES, GroEL, a GST domain containing protein, ETFA, FtsA/FtsZ, and NusA (Transcription Elongation Factor NusA), as candidate targets for immunoassays.

Claims Coverage

Independent claim clm-00001 recites a method that identifies diagnostic indicators by immunizing an uninfected veterinary subject with a human biological sample from a diseased human, detecting antigens in the veterinary subject sample, comparing with a control, and identifying altered antigens as diagnostic indicators for the condition. The claim includes five core inventive elements: selecting the condition, immunizing, detecting antigens, comparing with a control, and identifying diagnostic indicators based on antigen alterations.

The independent claim centers on a comparative antigen discovery workflow in which antigens detected after immunizing an un-afflicted veterinary subject with a diseased-human sample are compared to a control, and altered antigens are designated as diagnostic indicators for the selected condition.

Stated Advantages

Documented Applications