Note: Descriptions are shown in the official language in which they were submitted.
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MICROVOLUME DETECTING METHOD AND DEVICE
FIELD OF THE INVENTION
This invention relates to a method and device for carrying out
microvolume assays. In particular, the invention provides an assay for
s affinity reaction, such as an immunoassay.
BACKGROUND OF THE INVENTION
Affinity reaction analysis technique indicators, carried out by
reacting receptors and target substance(s), have been developed for years to
determine the existence of target substances. Radioactive labels,
io fluorescent or other chromogenic indicators are used as labels to determine
the presence of target substances, e.g. immunoassay of antigen specifically
binding to antibody.
Recently, the use of enzyme, colored latex particles, and metallic
colloids have been considered a simple and rapid technique for detection,
i s and the results can be evaluated with naked eyes by non-technical people.
However, convenient and rapid methods have several disadvantages such
as low sensitivity and low accuracy. In particular, it is difficult to obtain
a
desirable result when determining extremely low concentrations of the
target substance in a sample.
2o A dot-immunobinding assay for monoclonal and other antibodies has
been disclosed. The principle of such type of assay is as follows: A diluted
solution or suspension of antigen is dotted on a nitrocellulose piece of
filter
paper, and the dot is incubated with test antibody and with peroxide-
conjugated second antibody directed against the first antibody. A positive
2s reaction is detected as a colored dot. The advantages of this assay are
that
( 1 ) the amounts of the antigen and antibody needed for the assay are low,
and (2) the nitrocellulose paper provides an almost white background color,
a.
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so that the positive reaction color is easily observed. See Analytical
Biochemistry, 119, 142-147 ( 1982). However, the disadvantages of the
immunoassay are ( 1 ) complicated techniques involving more than 20 steps
of dilution, incubation, and washing to carry out the assay, and (2) it is
very
time-consuming. A standard assay takes over 20 hours to perform.
USP 4,774,177 disclosed another immunoassay for detecting the
presence of antibody or antigen in a sample. This method utilizes a
nitrocellulose carrier having a primary ligand ( haptin or antibody ) stably
bound thereto a microsized spot, about three mm in diameter. The
to nitrocellulose carrier is treated with a liquid containing non-specific
globulin for the primary ligand in order to block binding sites other than the
antibody binding sites of the primary ligand. The liquid sample (about SOuI)
suspected of containing the antibody is applied to the spot on the
nitrocellulose carrier. Then the nitrocellulose carrier is incubated at room
t s temperature for a time sufficient to bind any antibody that may be present
in the sample, to the primary ligand to form a Iigand-antibody complex.
This takes about six minutes. A liquid containing labeled antiglobulin is
then added to the complex and incubated at room temperature for about
three minutes to bind the labeled antiglobulin to the antibody-ligand
2o complex. The nitrocellulose carrier is washed and ready to be applied to a
suitable color developer to complete the assay. However, the steps of the
method are complicated. Tf no container is provided, and the liquid is
directly added to the nitrocellulose carrier to form the antiglobulin-
antibody-ligand complex, the liquid usually dries out during incubation.
2s This invention overcomes many of the shortcomings of more
conventional methods and provides a more convenient, more rapid, and
more sensitive method for handling microvolume detection.
DESCRIPTION OF THE INVENTION
The invention provides a microvolume assay method of an affinity
r,
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reaction using a membrane of a small area in a microvolume reaction
container for performing said affinity reaction between a target substance in
a sample and a receptor capable of binding to the target substance, which
method comprises the steps of:
s ( 1 ) taking a microvolume of the sample and loading it to the
membrane to allow the target substance in the sample to attach to the
membrane;
(2) placing the membrane into the microvolume reaction container,
adding a solution containing an antibody capable of binding to the target
io substance, wherein the volume of the solution is sufficient to soak the
entire area of the membrane carrying the sample;
(3) incubating the membrane in the reaction container for a time
sufficient to allow the target substance attached on the membrane to bind to
the receptor by an affinity reaction to form a target substance-receptor
1 s complex; and
(4) detecting the target substance-receptor complex.
The other objective of the invention is to provide a device for
performing the microvolume assay method of the invention.
BRIEF DESCRIPTION OF THE DRAWING
?o Figure 1 is a graphical representation of fluid from healthy pumpkin
tissue (A) and the test samples containing 6.4 (B), 3.2(C), 1.6(D), 0.8(E),
0.4(F), 0.2(G), 0.1 (H) ng/~,1 of zucchini yellow mosaic virusillustrated in
Example 3. The amount of test sample applied to the method is 0.2 ~,1.
Figure 2 is a graphical representation of fluid from healthy pumpkin
2s tissue (A) and the test samples containing 6.4 (B), 3.2(C), 1.6(D), 0.8(E),
0.4(F), 0.2(G) ng/~.l of zucchini yellow mosaic virus illustrated in Example
4. The amount of test sample applied to the method is 0.5 ~,1.
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DETAILED DESCRTPTION OF THE INVENTION
The term "affinity reaction" is defined as a pair of molecules, where
one of the molecules has an area on the surface or in a cavity which
specifically binds to a particular spatial and polar organization of the other
molecule. The members of the specific binding pair include, but are not
limited to, antigen and antibody, complementary nucleic acid fragments, or
a pair of proteins capable of recognizing a particular spatial and polar
organization of each other.
io The term "affinity reaction assay" is defined as an assay for carrying
out the objective of affinityanalysis as immunoassay
reaction such or
hybridization detection method.embodimentof the invention
The is
immunoassay which includes, not limitedto,direct detection
but is
immunoassay or sandwich immunoassay. Desirably, the assay can employ
t 5 a signal producing system for measuring the spectrophotometric property of
affinity binding, e.g. enzyme immunoassay.
The term "target substance" refers to any substance capable of
interacting with another substance which includes, but is not limited to,
ligand, DNA, RNA antigen, pathogen, receptor protein, biotin or other
2o substances combined with avidin or any other substance that has the
specific affinity interaction with another substance.
The term "receptor" usually refers to any substance capable of
recognizing and binding with the target substance. Illustrative receptors
include, but are not limited to, DNAs, RI~TAs antibodies, antigens, haptins,
z5 nucleic acid fragments, proteins or protein fragments, receptor poteins,
biotin or other substances combined with avidin or any other substance that
has the specific affinity interaction with another substance.
The invention relates to a microvolume detection method. The
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characteristics of the method reside in that only a microvolume of test
sample is needed for detection, and permit convenient, rapid and sensitive
detection.
The theory of the invention is based on Michaelis-Menten Kinetics.
s Antigen-antibody complex is used as an example to explain the specific
affinity interaction (test sample being as antigen).
Let: Ag = Antigen; Ab = Antibody; [O]= Concentration of the object O;
Complex = Complex formed by antigen-antibody specific recognition
(binding);
to Ag+Ab -~ complex
Then, the dissociation constant, Kd, can be represented as
Kd =[Ag] X [Ab]/[Complex]
For most antigen-antibody reactions, Kd is around 1 x 10'9M, meaning
that the two reactions, Ag and Ab, will tend to form complexes when the
i s total concentration of reactants is equal to or greater than 1 x 10'9M.
Therefore, if the concentration of antigen is very low, one can increase the
concentration of Ab to still drive the reaction in favor of the formation of
A~ Ab complexes. With the special minimum-volume design, we can
easily increase the effective concentration of the Ab without extra expense.
2o In addition, with the increased concentration of the reactants, the speed
and
sensitivity of the test strips are also improved. This can be shown by the
Michaelis-Menten equation as follows:
'V=Va,aX[S]~~[S]+Km)
Where V and Vm~ stand for the velocity and maximum velocity of
35 the reaction, respectively; [S] stands for the concentration of the
substrate,
and K," stands for the dissociation constant of the reaction at the steady-
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states.
The theory of invention to improve the reaction rate can be
illustrated by means of Michaelis-Menten equation:
V-umax[Ag]~~[Ag°]'~'Kd)
s Where V and Vme~ stand for the velocity and maximum velocity of
the affinity reaction, respectively; [Ag] stands for the concentration of the
substrate, and (Kd] stands for the dissociation constant of the affinity
reaction.
By inverting the above equation, the Lineweaver-Burk equation can
to be obtained:
1 /V=Kd/Vmax[Ag~'~' 1 Nmax
Therefore, it can be derived from the equation that when the
concentration of the substrate [Ag] is increased, V~[Ag] will approach the
maximum; so Kd/Vm~[Ag] approaches zero, 1/V approaches 1/Vm~, that is,
is the velocity of the reaction V approaches V,~.. Hence, with our special
design using a minimum volume of reactants and a higher concentration of
labeled antibodies in the kits, both the speed and sensitivity of the
detection
approach the maximum.
The subject method differs from prior art methods by providing a
2o method for determining extremely low concentrations of target substances
through a simple and time-saving assay. Prior art immunoassay takes even
8 hours to perform the assay, while the method according to this invention
allows for assays to be carried out in a few minutes, up to one hour. This
invention is also superior to the prior art because of its simple protocol and
2s convenient handling.
The invention provides a microvolume assay method of an affinity
reaction using a membrane of a small area in a microvolume reaction
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container for performing said affinity reaction between a target substance in
a sample and a receptor capable of binding to the target substance, which
method comprising the steps of
( 1 ) taking a microvolume of the sample and loading it to the
s membrane to allow the target substance in the sample to attach to the
membrane;
(2) placing the membrane into the microvolume reaction container,
adding a solution containing an antibody capable of binding to the target
substance, wherein the volume of the solution is sufficient to soak the
to entire area of the membrane carrying the sample;
(3) incubating the membrane in the reaction container for a time
sufficient to allow the target substance attached to the membrane to bind to
the receptor by an affinity reaction to form a target substance-receptor
complex; and
is (4) detecting the target substance-receptor complex.
The method and device according to the invention is suitable for any
type of affinity interaction including, but not limited to, direct or sandwich
immunoassay, nuclear acid hybridization, or other known affinity
interaction analyses.
2o Preferably, this invention is useful for detection of any type of body
fluids or tissues from animal, humans, and plants. According to the
invention, fluid samples include, but are not limited to, human or animal
body fluids or fluids in plant tissues. The test samples employed in this
invention include, but are not limited to, antigen, antibody, pathogens,
virus,
2s nucleic acid fragments and protein fragments. It is desirable to remove the
color of the background (e.g., green fluid from plant tissue or red fluid
from blood sample). Discoloring reagent is any reagent that can remove
the color of the background such as bleach, alcohol, chloroform, acetone,
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carbon tetrachloride, or a mixture thereof.
According to the invention, the membrane used to absorb the target
substance of test sample may be any porous material capable of absorbing
the target substance. Such materials include, but are not limited to,
s nitrocellulose, nylon, glass fiber, PVDF, rayon, filter paper, polyvinyl
chloride, polyethylene, polystryene, diazotized paper, activated beads,
protein A (Staphylococcus aureus) beads, polylysine, polyarginine,
polyhistidine, glass and plastic. In a preferred embodiment of the invention,
nitrocellulose is employed. The area of membrane is as small as possible
to so that the membrane can still bind the sample in an amount effective to
form an observable spot when the tagged antiglobulin is assayed. The area
is preferably between 1 mm2 and 200mm2, e.g. 1 OOmm2.
In accordance with the subject invention, a solid phase can
advantageously be employed to substantially support the membrane, e.g.
is plastic strip. A wide variety of materials and shapes may be employed. In
one embodiment of the invention, an integral part of strip or stick, such as a
test strip, is employed to retain the said membrane on the end of the strip.
The size of the solid phase should not be limited. For example, the width is
less than lOmm, preferably less than Smm. Said strip supporting the
2o membrane can be made sharp to penetrate plant tissue, human body, or
animal body surface so that the test sample can touch said membrane or the
target substance of the test sample can be adsorbed onto the membrane.
In one embodiment of the invention, a strip whose width is or is less
than 3mm is employed. In addition, a piece of membrane covers one end of
zs the strip which is sharp to penetrate the plant tissue.
The amount of liquid test sample applied to the method of the
invention is extremely small, preferably O.SuI or less. A variety of methods
of applying the test sample onto said membrane are known in the art and
can be employed to practice the invention. For instance, one can drop or
CA 02392681 2002-07-04
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dot the test sample onto said membrane, and/or rinse the membrane into the
test sample solution. In one embodiment of the invention, a sharp stick or
needle is used to dot the liquid test sample onto the membrane. The
observable spot is preferably between 0.2 and l.Smm in diameter, more
preferably less than O.Smm in diameter.
The microvolume reaction container is used to conduct the affinity
reaction according to this invention. There is no limitation on the shape of
said container, as long as the volume is sufficient to rinse the membrane,
the solid phase, or the strip with the reaction solution.
to In one embodiment of the invention, the microvolume reaction
container has a circle or half circle opening or flask bottom or is divided
into upper and lower portions. The diameter of lower portion is less than
that of the upper portion.
According to the invention, the target substance and receptor
is undergoing affinity reaction is recommended in a solution to be in an
amount sufficient to completely rinse the said membrane, e.g. 3-200.1,
preferably with 20-501.
According to the invention, the membrane absorbed with test sample
should be incubated for a period of time sufficient to ensure complete
affinity reaction and target substance-receptor complex formation. The
reaction volume is so small that it enables the high speed reaction. The
reaction time can even be shortened to a few minutes, which is far less than
that of the conventional methods. '
In order to practice the invention, a receptor capable of binding the
2s target substances can be obtained commercially or prepared by
conventional techniques. For instance, antibodies can be prepared using
conventional immunoiogical techniques.
Detection of the target substance-receptor complex may be
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accomplished using known standard affinity reaction analysis. For instance,
the detection can be carried out with labeled substances capable of
producing signals. According to the invention, these labeled substances
include, but are not limited to
s ( 1 ) enzyme, for example:
- horseradish peroxidase, examples of substrates are 1,1'
trimethylene-bis (4-formylpyridinium bromide) dioxime (TMB), 2,2'0
azinobis (3-ethylbenzthiazoline-sulfonic acid) (ABTS), o
phenylenediamine dihydrochloride (OPD), DAB/Cobalt, Chloronaphthol,
to or any fluorometric or cold light labels;
- Soybean Peroxidase, the substrates are mentioned as above;
- Alkaline Phosphatase, the substrates are p-Nitrophenyl phosphate
(PNPP), nitro-blue tetrazolium/S-bromo-4-chloro-3'-indolylphosphate p-
toluidine salt (NBTBCIP), Fast Red TR/AS and any fluorometric or cold
is light labels;
/3-Galactosidase, substrates are, for example, o-nitrophenyl-~3-D-
galactopyranoside (OI~TPG), isopropyl ~i-D-thiogalactopyranoside/5-bromo-
4-chloro-3'-indolyl-~3-D-galactopyranoside (IPTG/X-Gal);
- Glucose Oxidase, substrates are NBT, phenazine methosulfate
~o (PMS);
(2) Biotine-Avidin;
(3) colored latex particles;
(4) metallic colloid;
(5) flouroometric substances; or
2~ (6) radioisotopes, for example, 125 -iodine and 3 ~ -
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sulfur.
The subject invention achieves a simple technique for detecting a
variety of target substance so that assays can be carried out without highly
trained personnel. This purpose is of great importance when the detection
s measurement is by visual inspection. Particularly, the signals can be
observed by naked eyes, preferably with color development, for example,
peroxidase, colored latex particles and metallic colloid.
Alkalinephosphatase is used for a preferred embodiment of the invention.
The conjugation of the label substance and the receptor may be
performed prior to combining the label-bound-receptor with the target
substance. The enzymes can be covalently bound to a specific anti-
immunoglobulin, which in turn is bound to the antibody whose presence is
to be determined.
In a preferred aspect, the invention relates to the "Sandwich
x s Immunoassay," in which the target substances are bound with receptor and
a second labeled receptor is added to produce signals.
In accordance with the subject invention, the target substance-
receptor complexes should be carefully washed to remove any non-
specifically bound target, receptor, and/or label substances. The washing
zo buffer can be water, phosphate-buffered saline (PBS), Tris-buffered saline
(TBS), or saline solution. A surfactant such as Tween-20, Triton-X 100
can be added if necessary.
According to the invention, after incubation for a sufficient time,
signal generating compounds produce signals. To avoid the misreading of
:.>5 the result, stopping solution can be added to stop the reaction. For
instance,
after the enzyme and substrate interaction is completed, add stopping
solution mentioned above or other stopping reagents to stop the reaction.
The dilution buffer, in accordance with the invention, can be the
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washing buffer with a small amount of protein, e.g. 0.5-15% of non-fat
milk, 0.1-~% bovine serum albumin, egg albumine or the mixture thereof.
A surfactant such as Tween-?0, Triton-X 100 etc, can be added if necessary.
In a preferred embodiment of the invention, a microvolume assay
s method of an affinity reaction uses a membrane of a small area in a
microvolume reaction container for performing said affinity reaction
between a target substance in a sample and a receptor capable of binding to
the target substance, which method comprises the steps of
(1) taking a microvolume of the sample and loading it to the
membrane to allow the target substance in the sample to attach to the
membrane;
(2) optionally rinsing the membrane in discoloring solution for a
sufficient time;
{3) placing said membrane into the microvolume reaction
~ s container, adding a solution containing a receptor capable of binding the
target substance and labeled with a signal generating compound, wherein
the volume of the solution is sufficient to soak the entire area of the
membrane attaching the sample;
(4) incubating the membrane in the reaction container for a time
zo sufficient to allow the target substance attached on the membrane to bind
to
the receptor through an affinity reaction to form a target substance-receptor
complex;
{5) washing said membrane, which is treated in step (4), for a few
times and removing the unbound receptor solution; and
~s (6) observing the signal and determining the existence of the
target substance.
The detection assay can detect one or several target substances
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simultaneously by undergoing the affinity reaction with labeled receptor
and target substance(s).
Tire subject invention also provides a method for detecting the
presence of one or more target substances simultaneously in a single liquid
test sample. One or several spots have been bound to the said membrane
capable of binding to the first receptor. The dilution buffer is added to
block the membrane and avoid interference. Said membrane is incubated
with the second receptor. The method far detecting the presence of one or
more than one target substances comprises the steps of:
(1) stably binding one or more spots of first receptor capable of
binding with the target substances onto the membrane;
(2) blocking the membrane by adding the solution containing a
small amount of protein;
(3) taking a microvolume of the sample and loading it to the
is spots) of the membrane;
(4) optionally rinsing the membrane adsorbed with the sample in
discoloring solution for a time sufficient to discolor it;
(5) placing the membrane into the microvolume reaction container;
(6) adding 2-200u1 second receptor solution capable of binding
2o with target substance to the microvolume reaction container, and incubating
the membrane in the reaction container for a time sufficient to allow the
target substance attached on the membrane of the strip to bind to said
receptor by an affinity reaction to form a target substance-receptor complex;
(7) if necessary, washing the membrane treated with step (6) for
s several times to remove the additional second receptor solution; and
(8) detecting the target substance-receptor complex.
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The invention also provides a device for performing the
microvolume assay method of an affinity reaction between a target
substance in a sample and a receptor capable of binding to the target
substance, which device comprises the components of:
( 1 ) a membrane onto which the sample is to be loaded, so that the
target substance can be adsorbed onto the membrane;
(2) a microvolume reaction container in which the target
substance and the receptor can undergo the affinity reaction and form the
target substance-receptor complex; and
to (3) a component for use in detection of the target substance-
receptor complex.
Further components may be employed to carry out the microvolume
assay of affinity reaction, including
(4) micropipet;
is (5) bottles containing other reagents; and
(6) control strip i.e., a membrane which has adsorbed a sufficient
amount of target substance as compared to sample and to determine the
presence of the target substance.
A further aspect of the invention provides a device for carrying out
zo the affinity reaction assays of the invention, which comprises:
( 1 ) test strip which is a strip or stick supporting a membrane
capable of adsorbing the target substance;
(2) microvolurne reaction container;
(3) a bottle containing reagent capable of binding to the receptor
?5 which is labeled or can be bound to a labeled substance;
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(4) a bottle containing signal generating reagents; and, if
necessary, further comprises
(5) micropipet;
(6) bottles containing other reagents; and
s (7) control strip i.e., a membrane which has adsorbed a sufficient
amount of target substance as compared to sample and to determine the
presence of the target substance.
The following embodiment is offered by way of illustration and not
by way of limitation.
to Example 1 Preparation of labeled antibody
Vigorously mix the purified zucchini yellow mosaic virus (lmg/mL)
with complete or incomplete adjuvants injected into the New Zealand
rabbit and rechallenge every seven days. The optimal antibody response to
virus is obtained after one month. By means of ammonium sulphate
is precipitation and DEAF column chromatography separation, the rabbit
anti-virus IgG is obtained and then conjugated with horseradish peroxidase.
1.5% bovine serum albumin and 0.1 % Tween-20 phoso-buffered saline
(PBS) solution are also added to the reaction solution.
Example 2 Preparation of test strip
Cut the plastic strip into 3 mm. by 75mm., and attach a 3 mm. by
Smm. nitrocellulose to the plastic strip.
Example 3 Microvolume reaction detection Assay
1 Take 0.2p1 fluid from healthy pumpkin tissue and standard test
samples containing 6.4, 3.2. 1.6, 0.8, 0.4, 0.2 and 0.1 ng/p,l zucchini yellow
?s mosaic virus.
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2 The above fluid from healthy pumpkin tissue and the standard
test samples are then dotted on the membrane of test strip based on
Example 2. Preferably, the diameter of the spot is less than O.Smm.
3 Pipet 25p,1 of reaction solution prepared by Example 1 into a
s microvolume reaction container.
4 Rinse the strip which absorbs fluid from pumpkin tissue
prepared from step 2 in the container and incubate it for 40 minutes.
Wash the strip with washing buffer.
6 Place the strip into the bottle containing color developer and
incubate it for 7 minutes.
7 The green background of the strip can be removed by soaking
the strip in discoloring solution. The strip is blotted with clean, absorbent
paper to remove excess discoloring solution.
The result as shown in Figure 1 indicates that the fluid from healthy
us pumpkin tissue has no color change but the test samples containing
6.4ng/p,l (B), ~.2ng/p,l (C), l.6ng/p.l (D), 0.8ng/p,l (E), 0.4ng/p,l (F),
0.2ng/p,l
(G) and 0.1 ng/p.l (H) of zucchini yellow mosaic virus have clear color
development. The method permits a high sensitivity of detection which can
reach 0.02ng.
'?o
Example 4 Microvolume reaction detection Assay
1 Take 0.5 p,1 fluid from healthy pumpkin tissue and standard test
samples containing 6.4, 3.2, 1.6, 0.8, 0.4, 0.2ng/p.l zucchini yellow mosaic
virus.
:?s 2 The above fluid from healthy pumpkin tissue and the standard
samples are then dotted on the membrane of test strip based on Example 2.
Preferably, the diameter of the spot is less than O.Smm.
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3 The green background of the strip can be removed by soaking
the strip in discoloring solution. The strip is blotted with clean, absorbent
paper to remove excess discoloring solution.
4 Pipet 251 of reaction solution prepared by Example 1 into a
s microvolume reaction container.
Rinse the strip which absorbs fluid from pumpkin tissue in the
container and incubate it for 35 minutes to complete the reaction.
6 Wash the strip with washing buffer.
7 Place the strip into the bottle containing color developer and
to incubate it for 5 minutes.
The result as shown in Figure 2 indicates that the fluid from healthy
pumpkin tissue has no color change but the test samples containing
6.4ng/p,l (B), 3.2ng/p.l (C), l.6ng/pl (D), 0.8ng/pl (E), 0.4ng/p,l (F) and
0.2ng/p.l (G) of pumpkin yellow mosaic virus have clear color development.
is The method permits a high sensitivity of detection which can reach O.lng.
