Materials Research
Microarray
for Contaminated Water Analysis
Continuation
EPA
Grant Number: X83254101-1
Center: CEER at Alfred University
Investigators: DeRosa, Rebecca and Cardinale, Jean
Institution: Alfred University, School of
Engineering and Division of Biology
Research
Category: Congressionally Mandated Center
Description:
Microbial
and fecal contamination of water due to sewage and wastewater release is a
persistent problem in costal and inland waters. Recently much attention has focused on identifying an
appropriate indicator organism or method of detection. Several bacterial species (E. coli,
Enterococci, fecal coliforms, the Bacteroides-Prevotella group, Salmonella,
Clostridium) and viral species (coliphage, Enterovirus, Coxsackie, Hepatitis,
Norwalk, Rotavirus) have been studied to determine their suitability as
indicator organisms of ambient water quality. Although the EPA continues to support the use of E. coli and
Enterococcal species as indicator organisms, there is a growing body of
evidence that suggests that use of these species alone does not always provide
a complete picture of water quality. Our plan is to improve the binding efficiency of indicator-specific
antibody to specially treated glass slides to aid in the development of a
miniaturized enzyme linked immuno-sorbant assay (ELISA) system. As a result of this research, our
systems will have the potential to allow simultaneous testing for numerous
pathogens, both viral and bacteriological, in a shortened time frame (a few
hours), hence increasing the throughput of testing in current ELISA systems. Additionally, due to a change in the
materials and process our work will reduce the waste stream generated from
traditional ELISA analysis.
Objectives/Hypotheses:
Our
objective is to use a pretreatment (stabilization) of antibody prior to attachment
to derivitized glass surfaces, or inclusion of a stabilizing agent during
arraying to reduce or eliminate antibody denaturation. By doing this we will obtain an
increase in the amount of antibody attached to substrate and therefore decrease
the detection limits.
Approach:
We will
prepare antibody microarray patterns on specially treated glass slides and test
the antibody binding efficiency and conformational changes. Previous work suggests that the
antibody undergoes a structural change upon deposition to the glass
surface. We will employ several
types of additive agents in order to eliminate the structural modification upon
deposition to the surface. Further,
we will test the integrity of the printed slides by analyzing antigen binding
efficiency to the deposited antibody. Surface analysis techniques coupled with binding efficiency determined
from fluorescent imaging will be used as our analytical techniques.
Expected
Results:
We expect
to see a change in the antibody to antigen binding as a result of the glass
surface modification and the addition of a stabilization agent that will help
retain the antibody conformation upon deposition.
Supplemental
Keywords:
antibody
binding, protein binding, glass surface, XPS, protein conformation, ELISA
CEER is funded
in large part by the United States Environmental Protection Agency.
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