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Immunosignaturing: An accurate, affordable and stable diagnostic

Identifying diseases at an early, presymptomatic stage may offer the best chance for establishing proper treatment and improving patient outcomes. A new technique known as immunosignaturing harnesses the human immune system as an early warning sentryone acutely sensitive to changes in the body that may be harbingers of illness.

Now, Brian Andrew Chase and Barten Legutki, under the guidance of Stephen Albert Johnston, director of the Center for Innovations in Medicine at Arizona State University's Biodesign Institute have shown that these immunosignatures are not only strong indicators of pre-symptomatic illness, but that samples from serum, plasma, saliva and dried blood can yield reliable and highly stable diagnostic results under a variety of conditions.

As Johnston explains, the new data advance the prospects for applying immunosignaturing as a sensitive, low-cost, universal system for assessing health status. "Our ultimate goal is to monitor the health of healthy people, so it is crucial we have a technique that is cheap, simple and, as we demonstrate here, robust."

The group's results recently appeared in the journal Clinical and Vaccine Immunology.

Immunosignaturing uses random sequence arrays of peptides to trawl for antibodies to disease. Previous work has demonstrated that a glass slide containing an array of some 10,000 such random sequences, each composed of 20 amino acids, can be used to screen the body's full complement of antibodies, when a single drop of blood is spread over its surface.

When the antibodies present in a sample of blood are splayed over the peptide array, they selectively bind to these peptides with varying degrees of affinity. Once the blood is washed away, a machine-readable image of immune activity is left behindthe immunosignaturepotentially providing pre-symptomatic diagnosis for a broad range of ailments, from infectious diseases to chronic afflictions to varied forms of cancer.

The immune fingerprint thus produced will show thousands of spots fluorescing at different levels, corresponding to antibody activity. Immunosignatures may be registered repeatedly over time and will display characteristic changes following exposure to a pathogen, a vaccine or any other factor provoking an alteration in antibody activity.

Johnston notes that this approach to diagnosis represents a new paradigm, in that patients will be able to monitor their health with respect to their own particular baseline immune activity, rather than being measured against some standard established for the population as a whole. The method has been tested as a diagnostic for over 20 diseases to date, with each displaying a characteristic portrait.

In addition to accuracy, immunosignaturing provides a particularly versatile platform for disease diagnosis. Unlike many traditional diagnostic examsfor example, the widely used ELISA (for Enzyme-Linked Immunosorbent Assay)immunosignaturing is not pathogen specific. It therefore permits a general diagnostic screening for multiple disease factors from a single sample. In previous studies, the group demonstrated that immunosignaturing delivers useful diagnostic information for influenza, Alzheimer's disease, pancreatic diseases and lupus.

In the current study, the group demonstrates that immunosignatures remain stable over time and largely unaffected by variance in methods of collection. Such versatility could open the door to the use of vast archival material, for example, samples from prior studies and disease epidemics. It would also allow immunosignaturing to be broadly applied as a diagnostic for routine health monitoring, particularly in developing countries. Samples of blood or saliva could be mailed to a central processing center.

Immunosignatures from the same human donor, derived from both serum and plasma were compared in the study, showing good correlation. Two sequential blood draws were collected into either a serum separator tube or a plasma separator tube, to evaluate the influence of clotting factors on the resultant immunosignature. The data shows that plasma and serum produce equivalent immunosignatures. This fact provides flexibility of use for historical samples, which may have been collected using either blood source.

Next, dried blood spots were examined and serum antibodies recovered to produce an immunosignature. Again, a strong correlation of antibody activity was seen in the dried blood vs. fresh serum samples. Rates of protein recovery from dried bloodincluding functional antibodies ranged from 78 to 100 percent, in both mouse and human samples. The resulting immunosignatures showed higher fluorescence intensities in the case of fresh serum samples, but the effect was uniform across the array.

Once it was clear that dried samples could be used to produce faithful immunosignatures, the group evaluated the stability of samples stored in a simulated mailing environment, noting that large-scale epidemiological studies and health monitoring via immunosignaturing could be carried out if samples retained potency over time and when exposed to heat.

Samples of dried blood were stored at 25 degrees C or 37.8 degrees C. Immunosignatures remained stable after 2 weeks at the lower temperature. At 37.8 degrees C, samples retained stability overnight, but declined in stability after 2 weeks. As antibodies or immunoglobins are sensitive to bacteria, which may be inadvertently introduced during sample collection or transport, the application of protease inhibitors was subsequently used, and demonstrated an improvement in sample stability. Using dried blood, the team also showed that a characteristic immunosignature could be detected in mice previously infected with influenza, when compared with non-infected controls.

Finally, human saliva was assessed for its ability to provide an accurate immunosignature. Although quantities of antibodies of the IgG variety were low, saliva samples did contain sufficient antibodies of the IgA type to produce a useable immunosignature, making collection of saliva a plausible, non-invasive alternative for sample collection.

The new results indicate that immunosignaturing is not only an inexpensive and sensitive diagnostic technique, but also offers good sample stability over a range of conditions. As a versatile tool for the assessment of presymptomatic illness, immunosignaturing can potentially help alleviate the unsustainable burden to the healthcare system, caused in part by late disease diagnosis followed by exorbitantly costly, (and often ineffective) treatment.

Arizona State University

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