Biotechnology Methods for Detecting Mercury
Preliminary Data
Shaun Cote, Rebekah Pilling, Caryn K. Prudenté
Abstract
Due to the high levels of mercury contamination in the
environment and a need for better testing methods, we are
exploring the synthesis of a mercury-protein bioconjugate to
facilitate the development of a mercury antibody. Mice have
been immunized with this mercury-protein bioconjugate and
the antibodies will be collected for screening through
Enzyme-Linked Immuno-Sorbent Assay (ELISA) to determine
the effectiveness and sensitivity of the antibody. This poster
will discuss the synthetic processes and structural analysis of
our mercury-protein bioconjugate as well as the testing
method by which we utilize the mercury antibody to detect the
presence of mercury contamination
In the first phase of this research project, a mercury-protein
bioconjugate was developed. An organomercury hapten was
connected to a carrier protein, chicken immunoglobulin, via a linker
arm. Mice were then immunized with this immunogen, causing the
immune system to develop antibodies.
Solid Phase Synthesis
To develop a sensitive and selective ELISA to test with a monoclonal antibody
(mAb), a solid phase must be developed to coat the polystyrene plates. To achieve
maximum selectivity, a solid phase protein-mercury bioconjugate structurally different
than the immunogen must be synthesized.
I ran the synthesis through common organic pathways. First I reacted allyl amine
with Di-tert-butyl dicarbonate to protect the amine. I then reacted the protected allyl
amine with a mercury salt to attach mercury and an amine or alcohol to the allylic
region of the amine.
H
N
O
BrHg
IgG
O
Four mice were screened for antibody development, and the
mouse with the highest titer was used to create mAbs. We were
unsuccessful in developing the mAb, so testing will continue with
the remaining three mice. [2].
Third Bleed Titer
In my second attempt at this reaction, I used a more reactive mercury salt, mercury
nitrate (Hg(NO3)2), and 1,2 Pentane diol. The diol did not have a π bond, so H1-NMR
was not compromised.
Optical Density at 492nm
Mercury is naturally found in environmental systems, but can
be released into the environment through burning coal and
other manufacturing processes. In the environment, mercury
can combine with carbon to form methyl mercury, which is
ingested through organisms, and works its way up through
the food chain to humans. Mercury levels in environmental
systems in Maine are among the highest in North America [1].
mouse 994
3.5
2.5
mouse 1006
mouse 1011
2
mouse 1018
3
1.5
1
0.5
0
0
To deprotect my t-Boc allyl amine structure, I used trifluoro acetic acid. This mercury
group will be connected via a linker arm to a carrier protein, thus creating a solid
phase structurally different than the immunogen
2
4
6
8
10
Synthesis Scheme
+
H2N
O
16
18
20
22
24
26
28
30
32
February 10, 2006
Mouse 1006
2.5
Mouse 1011
2
Mouse 1018
1.5
1
0.5
0
O
O
14
First Bleed Titer
3
O
12
Relative Antibody Concentration
[1/x]
Optical Density at 490 nm
Mercury
In my first reaction, I used mercury acetate (Hg(oAc)2) with an excess of allyl amine.
This mercury salt gave a small yield, and the H1-NMR spectral data was
compromised, as the resulting product had the same π bond as the starting material.
November 3, 2005
4
CH2Cl2
O
0
2
4
6
8
10
Relative Antibody Concentration
[1/X]
RT
Sandwich ELISA Assay
1. Hg(NO3)2/dry CH3CN
O
Hg
O
NH
2. Excess
HO
OH
+
Hg
Sample
containing
antibodies
Immobilized
solid phase
(can vary)
+
Non-specific
Mouse anti-mercury
mercury antibody
antibody trapped on
is washed away
solid phase
Enzyme
O
Hg
O
Acknowledgments
USM Faculty Senate
Professors Ah-Kau Ng and Stephen Pelsue
USM Chemistry Department
References
[1] Maine DEP; http://www.state.me.us/dep/mercury
[2] Research done by Rebekah Pilling
NH
HgCl
O
1. TFAA/CH2Cl2
OH
H2N
HgCl
O
OH
+
An antibody
sandwich!
Enzyme
Enzyme labeled
goat anti-mouse
antibody
Hg
Adapted with permission from Caryn Prudente
Polystyrene ELISA plates are coated with a solid phase containing the
antigen of interest coupled to a protein or sugar. The inherent “stickiness” of
the solid phase protein adheres it to the plate. A serum sample from
immunized mice is then added to the ELISA plate. Mouse anti-mercury
antibodies from the sample are retained on the plate as they bind to the
mercury immobilized in the solid phase. Any non-mercury-specific
antibodies are rinsed away. In a second step an enzyme labeled anti-mouse
antibody is added to the plate and binds to the mouse anti-mercury
antibodies. The enzyme labeled antibody can be used for detection and
quantification. Once the ELISA is optimized if will provide a tool to detect
and quantify mercury in various samples