Establishment of Dipstick Development Technology for Detection of Cry1Ac in Transgenic Plants

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International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-2, Issue-6, Nov-Dec- 2017
http://dx.doi.org/10.22161/ijeab/2.6.3 ISSN: 2456-1878
www.ijeab.com Page | 2783
Establishment of Dipstick Development
Technology for Detection of Cry1Ac in Transgenic
Plants
Muhammad Irfan1*, Muhammad Asif1, Aftab Bashir1+, Kauser Abdullah Malik1++
1*Gene isolation lab, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad
1Gene isolation lab, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad.
1+, 1++ Department of Biological Sciences, Forman Christian College, Lahore.
Abstract The insecticidal Bt Cry1Ac protein is, currently
used for transgene ex pression in numerous crops or
deliberating resistance against lepidopteron pests. Since the
introduction of Bt cotton in Pakistan. It has been
demonstrated that the technology has achieved the goal of
providing an effective tool for lepidopteron control.
In this study, single step, sensitive and specific dipstick strip
test for the revealing of recombinant Cry1Ac protein in the
transgenic plants was established. Anti-Bt-Cry1Ac
antibodies and goat anti-rabbit IgG antibodies were used in
test and control lines, respectively. The distance
betweenthese lines were opti mized as 0.5 cm. Polyclonal
rabbit anti Bt-Cry1Ac antibody conjugated to nanocolloidal
gold (20 nm of OD 15 and 40 nm; OD 1 in separate
experiments) at pH 9.2 was used to serve as a probe for
detecting Cry1Ac protein in transgenic Bt cotton samples.
Both conjugate solutions were coated on separate polyester
conjugate pads (0.7 cm × 0.5 cm). The total size of strip
was 7.5 cm × 0.5 cm. For 20 nm gold conjugated strip,
purple color test line and for 40 nm gold red color test line
indicated the binding of gold labeled antibodies to antigen.
The assay was corroborated with transgenic cotton sampl es
with protein extraction buffer 1X PBS of pH 7.
This on-site test offers fast screening for any genetically
modified crop devouring Cry1Ac transgenic protein.
Keywords Nitrocellulose membrane, Cry1Ac, Dipstick,
Nano-colloidal gold particles.
I. INTRODUCTION
Transgenic technology has provided a very powerful tool to
develop crop varieties, which are tolerant to various biotic
and abiotic stresses, and improved qualitative or
quantitative traits. Many genetically modified (GM) crops
have been developed with
different important traits by introducing various transgenes
like insecticidal genes (Cry1Ab, Cry1Ac, Cry1F, Cry2Ab,
Cry3A, Vip3) [1], herbicide tolerant genes ( epsps, bar,
pat, als) [2], virus resistant (cp, prsv-cp, rep, hel),
delayed ripen ing gene s ( sam-k, acc, pg) [3] genes for
color modification (dfr, hfl, bp40) in a numeralcrops
species like canola,cotton, corn, p otato, tomato, brinjal,
papaya, rice, tobacco, soybean, wheat, sunflower, alfalfa,
etc. The two most important traits that have been
successfully introduced in different commercially a vailable
GM crops are insect resistance and herbicide tolerance [4].
Transgenic plants expressing insecticidal genes that were
initially derived fro m common soil bacterium, Bacillus
thuringiensis (Bt), have been found to give an
environmentally safe and efficient control of many insect
pests Bt-cotton containing Cry1Ac gene provides protection
against the lepidopteron insect pests commonly known as
cotton bollworms. Bt cotton has been introduced in many
other countries like Australia (1996), China (199 7),
Argentina (1997), South Africa (1998), Me xico (1998),
Colombia (2002), India (2002) and Pakistan (2010).
(http://www.agbioworld.org/biotech-info/articles/biotech-
art/safety-bt-cotton.html).
Since GM crops have been entered the food chain, public
and scientific domain discussions related to their safety and
manipulability have been continued. Before the commercial
release of any GM crop, their biosafety evaluation is
required to assess the environmental influence and e ffect on
health of the consumers. It was demonstrated that
unauthorized and possibly unsafe GM products may
International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-2, Issue-6, Nov-Dec- 2017
http://dx.doi.org/10.22161/ijeab/2.6.3 ISSN: 2456-1878
www.ijeab.com Page | 2784
sometimes be found in the market [3]. Unauthorized GM
crops altogether p resent a significant socioeconomic risk
through their possible undesired effects on human and
animal health, and the environment. T herefore, several
countries have implemented thresholds for unintended
mixing of GM crops; fixed at 5% in Taiwan and Japan, 3%
in Korea, 1% Australia, New Zealand, Brazil, and 0.9% in
the European Union. He nce, for regulatory co mpliance of
GM labeling, there is a dire need for easy a nd steadfast
detection methods of such GM crops. Protein based GM
crops detection is particularly useful for monitoring
transgene expression both at qualitative and q uantitative
levels [5].
Previously methodologies are being used to detect the
manifestation of GM materials in food stuff which emphasis
on target either transgenic DNA or transgenic protein
expression in GM crops[6]. Recently, Dong et al. have
developed a database “GMO Detection method Database
(GMDD)”which placid almost all the earlier developed and
conveyed GMOs detection methods. The frequently used
DNA based methods includ e polymerase chain reaction
(PCR) and real-time PCR, while protein based methods
include immuno PCR, near infrared (NIR) spectroscopy,
micro fabricated devices, chromatography mass
spectrophotometry and DNA chip technology which offer
solutions to current technical issues in GM crop analysis
[7], but these methods are costly, time taking and
advantages along with the disadvantages[8]. Other protein
based methods include enzyme linked immunosorbent assay
(ELISA) and western blot. These are more accurate, cost
effective but required trained and well equipped lab. Almost
all of the above mentioned methods are not suitable for
onsite testing of transgenic crops. Another protein based
method named ‘dipstick test’ has been found quicker,
simpler, less expe nsive, and suitable for onsite testing and
does not req uire specific skills [9] . The current study is
fixated on immunological based detection of transgenic
plants expressing Bt (Cr y1Ac) gene through d eveloping a
dipstick.
The scientific basis of the lateral flow immunoassay was
consequent from the latex agglutination assay, which was
established in 1956 by Plotz and Singer. Duri ng this period,
plate-based immunoassays were being settled[10]. The
basic principles of the lateral flow technology continual to
be advanced through the early 1980s and were further
recognized during the latter years with the filing of several
major patents on this technology format by companies such
as Becton Dickinson & Co. and Unilever and Carter
Wallace. Since then, at least other 500 patents have been
filed on various aspects of the tech nology [11]. The
technology has been successfull y applied for diagnostic
purposes in the areas of agriculture, veterinary,
environmental health,food and safety, industrial testing, as
well as new areas such as molecular diag nostics and
theranostics[12].
The dipstick (Lateral F low Immunoassay) assay uses a
membrane based detection system. Previous studies shown
a number of names found for the strip based
immunoassay tests, such as lateral flow de vices (LFD),
immunochromatographic (IC) tests, one-step tests,lateral
flow technology (LFT) and dipstick tests[1]. The
dipstick technology is a variant of enzyme-linked
immunosorbent assay (ELISA), using nitroce llulose
membrane strips rather than micro-titer wells and offers a
qualitative or semi-quantitative test.
The objective of this study was to develop a quick,
unpretentious, qualitative, and subtlenanocolloidal gold
based sandwich I C strip assay for one step detection of
transgenic Cry1Ac protein expressed in B t cotton. The
developed dipstick strip can successfully be used in
diagnostic labs and by the cotton growers and farmers to
perform the purity test of seed lots[13].
II. MATERIAL AND METHODS
2.1 Chemicals and reagents
The Material Starter Kit (Cat. No. 010) was purchased from
Diagnostic Consulting Network UK, For nanocolloidal gold
“Gold-ina-Box™ kit (Cat. No. NGIB01-B044) was
procured from BioAssay Works, LLc., USA Highly
purified polyclonal antibodies, which were Rabbit IgG (Cat.
No. 41-GR30) and Bt Cry1Ac antibody (Cat. No. 70r-
BR005) were p urchased from Fitzgerald International,
country.
1X PBS (10 mM), 5% suc rose solution, BSA blocking
buffer (3% BSA in 10 mM PBS) and PBS-Tween 20 (3%)
with 5% BSA and 2 ml polyvinyle alcohol (blocking buffer)
were prepared separately.
2.2 Preparation of Dipstick Strip by using Seven
Different Types of NC Membranes
Dilutions of anti Cry1Ac Antibody (2 mg/ml in 1XPBS
buffer) were prepared. The IEF point for Cry1Ac was
estimated to be 8.8 (CLC bio workbench). Conjugated
solution were prepared having pH 8.8 and 9.2 after
optimized conditions.
Seven membranes of different pore sizes were short listed.
Each membrane was cut into strip of 3 × 1.5 cm size. The
each membrane was treated with sa me protocol. Two lines
were assigned in the middle of the membrane; which were 1
cm apart from each other, upper line was named as control
line the goat anti rab bit antibody (1 μl/line; 1 mg/ml) was
International Journal of Environment, Agriculture and Biotechnology (IJEAB) Vol-2, Issue-6, Nov-Dec- 2017
http://dx.doi.org/10.22161/ijeab/2.6.3 ISSN: 2456-1878
www.ijeab.com Page | 2785
dispensed on control line and lower was named as tes t line
Anti-Bt Cry1Ac antibody (1μl/line; 1 mg/ml) mixed with
3% methanol was immobilized on test line. Each strip of
NC membrane was placed in an incubator at 37ºC to dry for
1 hour. After drying of antibodies the unoccupied sites were
blocked with b locking buffer (10 mM PBS containing 3%
tween-20, 5% BSA and 2% polyvin yl alcohol). These
membranes were incubated with blocking buffer for 4 hrs.
at RT on orbital shaker. When blocking process was
completed then these membranes were soaked in 5%
sucrose solution (w/v) and dried for overnight at 37ºC
finally parts of strip were assembled into coordinating parts
as a one unit assay on adhesive plastic backing. After
assembly of dipstick strips, assay was performed using Bt
positive (Cry1Ac) cotton seeds and commercially available
Immuno Strip (Agdia) were also used as a control.
2.3 Blot test for the determination of antibody and
antigen reaction
A blot test was performed for the confirmation of antibody
and antigen reaction according to protein extraction buffers.
For this purpose following four different experiments were
conducted using Fermentas NC membrane.
1. Nanocolloidal gold conjugated Cry1Ac antibody (3μl)
was dropp ed on NC membrane and labeled this spot
number
2. Seeds of Bt cotton were crushed in 1X SEB4 buffer of
pH 10.5 and used its 2 μl supernatant as anti gen
(Cry1Ac protein) and mixed it with 3 μlnanocolloidal
gold conjugated Cry1Ac antibody. T otal volume of 5 μl
was dropped on NC membrane and labeled it as spot
number 2.
3. A secondary antibody IgG (goat anti rabbit antibody)
was dropped 1 μl on NC membrane and dried at 37ºC
for 30 minutes. After 30 minutes Bt positive seeds were
crushed in 1X SEB4 buffer of pH 10.5 and used its 2 μl
supernatant as antigen (Cry1Ac protein)and mixed it
with 3 μlnanocolloidal gold conjugated Cry1Ac
antibody. Total volume of 5 μl was dropped on NC
membrane exactly at the same place where IgG was
dropped and labeled it as spot number 3.
4. 1 μl of Anti-Cry1Ac Antibody was dropped on NC
membrane and dried it at 37ºC for 30 minutes. After 30
minutes Bt positive cotton seeds were crushed in 1X
SEB4 buffer of pH 10.5 and used its 2 μl supernatant a s
antigen (Cry1Ac protein) and mixed it with 3
μlnanocolloidal gold conjugated Cry1Ac antibody. Total
volume of 5 μl was dropped on NC membrane exactly at
the same place where Anti-Cry1Ac Antibody was
dropped and labeled it as spot number 4.
2.3Blot test for the determination of antibody and
antigen reaction by using 1X PBS as protein extraction
buffer
Again a blot test was performed for the d etermination of
antibody and antigen reaction. For this purpose following
four different experiments were conducted by using
Fermentas Nitro Cellulose Membrane as an experimental
base. Secondly, simple 1X PBS (10mM) buffer was used as
protein extraction buffer.
1. Non-Bt cotton seeds were crushed in 1X PBS buffer of
pH 7.4 and used its supernatant 2 μl and mixed it with 3
μl Cry1Ac antibody conjugated nanocolloidal gold.
Total volume of 5 μl was dropped on NC membrane and
labeled it as spot No. 1.
2. Bt cotton seeds were crushed in 1X PBS buffer o f pH
7.4 and took 2 μl of its supernatant as antigen (Cry1Ac
protein) and mixed with 3 μl Cry1Ac antibody
conjugated nanocolloidal gold. Total volume of 5 μl was
dropped on NC membrane and labeled it as spot No. 2.
3. A secondary antibody IgG (goat anti rabbit antibody)
was dropped (1 μl) on NC membrane and dried at 37ºC
for 30 minutes. After 30 minutes Bt cotton seeds were
crushed in 1X PB S buffer of p H 7.4 and used its (2 μl)
supernatant as an antigen (Cry1Ac protein) a nd mixed
with 3 μlnanocolloidal gold conjugated Cry1Ac
antibody. Total volume of 5 μl was dropped exactly at
the same place on NC membrane where IgG was
dropped and labeled it as spot No. 3.
4. Anti-Cry1Ac Antibody was dropped (1 μl) on NC
membrane and dried at 37ºC for 30 minutes. After 30
minutes Bt cotton seeds were crushed in 1X PBS buffer
(pH 7.4) and used its 2 μl supernatant as an antigen
(Cry1Ac protein) and mixed with 3 μl of nanocolloidal
gold conjugated Cry1Ac antibody. T otal volume of 5 μl
was dropped exactly at the same place on NC membrane
where AntiCry1Ac Antibody was dropped and labeled it
as spot No. 4.
NOTE. The gold antibody co njugate used in all these
experiments for determination of antibody and antigen
reactions was of pH 8.8.
2.4 Preparation of dipstick strips by using different NC
membranes without any treatments
In this experiment three NC membranes of different pore
sizes from different manufacturers were selected. These
were Millipore HF135, Millipore HF120 and Fermentas NC
membranes. Each membrane was cut into strip of 2.5 × 0 .5
cm size. Each membrane was treated with same protocol as
mentioned above. Unoccupied sites on membranes were not
blocked by any treatement.