Format

Send to

Choose Destination
See comment in PubMed Commons below
Optom Vis Sci. 2008 Aug;85(8):653-60. doi: 10.1097/OPX.0b013e3181824e20.

Antibody protein array analysis of the tear film cytokines.

Author information

1
Francis I. Proctor Foundation, University of California-San Francisco, 513 Parnassus, San Francisco, CA 94143, USA.

Abstract

PURPOSE:

Many bioactive proteins including cytokines are reported to increase in dry eye disease although the specific profile and concentration of inflammatory mediators varies considerably from study to study. In part, this variability results from inherent difficulties in quantifying low abundance proteins in a limited sample volume using relatively low sensitivity dot ELISA methods. Additional complexity comes with the use of pooled samples collected using a variety of techniques and intrinsic variation in the diurnal pattern of individual tear proteins. The current study describes a recent advance in the area of proteomics that has allowed the identification of dozens of low abundance proteins in human tear samples.

METHODS:

Commercially available stationary phase antibody protein arrays were adapted to improve suitability for use in small volume biological fluid analysis with particular emphasis on tear film proteomics. Arrays were adapted to allow simultaneous screening for a panel of inflammatory cytokines in low volume tear samples collected from individual eyes.

RESULTS:

A preliminary study comparing tear array results in a small population of Sjögren's syndrome patients was conducted. The multiplex microplate array assays of cytokines in tear fluid present an unanticipated challenge due to the unique nature of tear fluid. The presence of factors that exhibit an affinity for plastic, capture antibodies and IgG and create a complex series of matrix effects profoundly impacting the reliability of dot ELISA, including with elevated levels of background reactivity and reduction in capacity to bind targeted protein.

CONCLUSIONS:

Preliminary results using tears collected from patients with Sjögren's syndrome reveal methodological advantages of protein array technology and support the concept that autoimmune-mediated dry eye disease has an inflammatory component. They also emphasize the inherent difficulties one can face when interpreting the results of micro-well arrays that result from blooming effects, matrix effects, image saturation and cross-talk between capture and probe antibodies that can greatly reduce signal-to-noise and limit the ability to obtain meaningful results.

PMID:
18677223
PMCID:
PMC3786218
DOI:
10.1097/OPX.0b013e3181824e20
[Indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments

    Supplemental Content

    Full text links

    Icon for Wolters Kluwer Icon for PubMed Central
    Loading ...
    Support Center