The document describes the development of a rapid oral test to detect cotinine, a metabolite of nicotine exposure, at concentrations as low as 2 ng/ml. Antibodies were developed against cotinine and tested in ELISA format. A lateral flow test strip was created using these antibodies and a reader was used to quantify test and control lines to determine cotinine concentration. Pre-treatment of saliva samples was required to prevent nanoparticle aggregation. Initial tests on volunteers detected cotinine in all samples. The inexpensive and sensitive test could help identify second-hand smoke exposure.

1. Detection of Passive Smoke Exposure in Children and Adults using Oral Based Rapid Test Technology1. Abstract
Passive exposure to tobacco smoke causes a variety of illnesses
ranging from allergies to some forms of cancer. Assessment of second
hand tobacco smoke (SHS) exposure, particularly among vulnerable
populations, such as children, may allow intervention and/or prevention
of future disease. A minimally invasive on site test designed to detect
SHS exposure using saliva would constitute a valuable tool for
researchers and clinicians; however, a visual test for that purpose
would be unreliable as its results may be interpreted differently
depending on the operator. Consequently, we have implemented the
use of an inexpensive reader that utilizes a CMOS image sensor to
quantify a signal generated by gold nanoparticles. This reader, from
Alverix Inc., allowed development of a rapid and sensitive test for
cotinine, a metabolite of nicotine.
The rapid test consists of a nitrocellulose strip with a control line,
formed by immobilized Goat anti rabbit IgG, that is used as an internal
standard, and a test line consisting of an immobilized BSA-cotinine
derivative. A sample and an absorbent pad are positioned,
respectively, upstream and downstream from the lines. The sample,
which has been treated with a running buffer containing antibodies
against cotinine is mixed with protein A-gold nanoparticles and then
placed on the sample pad. As the sample runs through the
nitrocellulose membrane, the cotinine molecules bind to the antibodies,
thus blocking their active sites. If no cotinine is present in the sample,
the free antibodies will bind to the BSA-cotinine derivative which was
immobilized on the test line. This way, the concentration of cotinine in
the sample can be inferred from the relative intensity of the test line. In
vitro assays were performed with human saliva containing cotinine
using this test. Results indicated that in conjunction with the Alverix
reader, the test can detect cotinine concentrations as low as 2 ng/ml.
This assay is sensitive, simple and fast and could be easily
implemented at point of care facilities to detect second hand smoke
exposure.
3. Experimental section
The Alverix reader used to quantify the
intensity of the lines is shown below. The
test strip is introduced into the reader, a
graph is generated and integrated.
R. Sam Niedbala (san204@lehigh.edu)
Department of Chemistry
Lehigh University
2. Introduction
The goal of this project is to develop a rapid
test for cotinine using oral fluids. The rapid test
uses the lateral flow format shown below.
Sera from these rabbits were rigorously tested
in ELISA format to determine the performance of
the cotinine antibodies. The specificity of the
assay was determined by testing the cross-
reactivity of compounds structurally similar to
cotinine or nearly ubiquitous in human samples.
This included; caffeine, aspirin, nicotinic acid, and
nicotinamide.The cross-reactivity was determined
by comparing the average signal at a
concentration of 10,000ng/mL for structurally
similar compounds to that of 1ng/mL of cotinine.
A compound was considered non cross-reactive
when its signal was higher than the
corresponding to cotinine. None of the tested
compounds were cross-reactive. The sensitivity
(limit of detection) was determined as the lowest
concentration of cotinine for which the following
was true: average cotinine + 3σ < average blank
– 3σ. Eight replicas were measured for each
condition tested. Sera from rabbit 1799 exhibited
a limit of detection of 4 ng/ml in ELISA plates, the
other sera showed a limit of detection of 6 ng/ml
or higher; consequently, the rapid test was then
developed using sera 1799 (data shown below).
NSB results using the following running buffer formulations: (a) PBS + 1% BSA (b)
PBS+ 2% BSA (c) PBS + 1%PVP (d) PBS + 1%Tween 20 (e) HEPES Buffer + 1%
BSA (g) HEPES buffer + 1% Tween 20 + 1% Tryptone
The sample, diluted in a running buffer
containing antibodies, is applied to the
absorbent pad, and due to capillary effects,
moves across a membrane where it
encounters a capture zone. In this zone the
antibodies are immobilized and labeled.
The test relies on free cotinine in the
sample binding to the anti-cotinine antibodies.
In the absence of cotinine a dark red line will
appear. As higher concentrations of cotinine
are present, the line will fade.
In order to standardize the results, the
control line is used as an internal standard
and is striped upstream of the test line. The
ratio of the lines intensities is used as the
measuring parameter. It was found that this
ratio reaches steady state after 15 minutes,
as shown below.
Signal evolution of a typical cotinine test
0 15 30 45 60
Time (min)
Cotininesignal
Control line
Test line
Standarized ratio
Cotinine
(ng/ml) Average 3SD
Minus
3SD Plus 3SD
0 2.469 0.742 1.727 3.211
0.2 2.362 0.731 1.631 3.094
0.5 2.213 0.75 1.463 2.963
1 2.034 0.733 1.302 2.767
2 1.339 0.477 0.862 1.816
4 1.179 0.528 0.651 1.707
Non Specific Binding (NSB)
NSB refers to the binding of the reporter
to the test line in the absence of antibodies.
This is due most likely to the presence of
“naked” areas (not covered with protein A)
on the surface of the gold nanoparticles. In
order to stabilize the gold, different running
buffer solutions were then prepared and
tested in NSB mode (no antibody in the
running buffer).
NSB was observed in buffers a-c, it is less
visible in buffer d, and is not visible in buffers
e-f, although (e) severely reduces the
intensity of the control line.
Sample pre-treatment
Gold aggregation was observed when
whole saliva was used in the assay. This is
probably due to the presence of proteins
or other chemical entities that act as
“bridges” joining the nanoparticles,
increasing their relative density and
causing their precipitation as shown in the
figure in the next column.
As shown above, common techniques
such as mucin elimination by centrifugation
or antibody removal with protein A, do not
prevent the aggregation of the gold
nanoparticles, to do that, a filtration step with
active adsorbents must be implemented. We
tried different adsorbents, finding that a nylon
filter with a 0.45 µm pore size was the
optimal choice, completely eliminating the
aggregates using a simple, easy to use and
inexpensive filtration system. The
displacement data taken is shown below
Preliminary trial with clinical samples
Samples were taken from three
volunteers who had previously ingested
over the counter nicotine. The samples
were analyzed with the device described
here, cotinine was detected in all the
samples. the results are shown below.
4. Results
Antibody selection:
A 4-hydroxycotinine hapten derivative was
conjugated to KLH and injected in four rabbits,
code named 9429, 1799, 1802 and 2596.
Cotinine derivative selection
Four cotinine derivatives were synthesized,
conjugated to BSA and tested in lateral flow
format. One of them (the 1 spacer) showed a
higher test line intensity reduction
(displacement) than the other derivatives.
CCAG and 3-spacer performed similarly to
each other at 2ng/ml. The 5-spacer derivative
showed very little displacement.
Cotinine analysis of saliva samples using new test
0
0.6
1.2
1.8
2.4
3
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Time (hours)
Signalratio(test/control)
Subject 1
Subject 2
Subject 3
5. Conclusions
The use of an inexpensive reader,
combined with appropriate sample pre-
treatment and carefully selected buffers and
reagents, makes it possible to detect
cotinine concentrations of 2 ng/ml or lower
using rapid oral based technology.
0%
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
1-spacer 3-spacer 5-spacer CCAG
cotinine derivative-BSA conjugate
%Displacement
Displ 0-2
Displ 0-20