Note: Descriptions are shown in the official language in which they were submitted.
84072721
METHODS TO REDUCE EVAPORATION DURING ELEVATED TEMPERATURE
CROSS-REFERENCE TO RELATED APPLICATION
The application is a non-provisional application claiming the benefit of the
earlier
filing date of co-pending U.S. Patent Application No. 14/701,043, filed April
15, 2015.
FIELD
Assay reagent.
BACKGROUND
Immunohistochemical assays and assay techniques based on in situ hybridization
are widely used in medical diagnostics such as to diagnose abnormal cells such
as those
found in cancerous tumors or to diagnose another disease. Many assays involve
the addition
of heat to the sample, such as to a sample in a reagent on a slide. Assay
steps at elevated
temperatures can cause substantial evaporation of assay reagents. When small
volumes are
used, sample denaturation, hybridization, wash and aging steps and the
resulting assay can be
compromised by evaporation.
DETAILED DESCRIPTION
A reagent and a method of use of a reagent including an evaporation reducing
agent in an aqueous solution operable to reduce evaporation of the reagent in
processing of a
tissue or cellular sample is disclosed. Representative processing for which a
reagent as
described finds use include, but are not limited to, dewaxing, cell
conditioning/antigen
retrieval/cell aging, peroxide/phosphatase block, probe denature, probe
hybridization,
washing, linker hybridization, antibody incubation, probe detection, chromogen
precipitation
and counterstain involving an elevated temperature step (e.g. an elevated
temperature step in
an in situ hybridization procedure). The solution can representatively be used
in
denaturation, wash or hybridization steps of a nucleic acid (single or double
stranded probe)
hybridization assay or in an incubation, antigen retrieval or a wash step
using an antibody in
immunohistochemical or immunocytochemical staining. A suitable reagent may
include
other components such as a detergent/surfactant which helps with solution
spreading in a
hybridization solution reagent. In one embodiment, a suitable amount of an
evaporation
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reducing agent in a reagent operable for tissue or cellular processing is an
amount that will
limit a loss of the reagent due to evaporation to 20 percent or less under
industry acceptable
reagent processing conditions such as subjecting a hybridization solution to
an elevated
temperature between 25 C and 50 C for 10 minutes to 24 hours (e.g., a
hybridization or
incubation process) or to an elevated temperature of between 60 C and 100 C
for two to
ninety minutes (e.g., a denaturing or antigen retrieval process). In one
embodiment, a
suitable amount of an evaporation reducing agent is in the range of 11 percent
to 60 percent
by volume of a regent composition (e.g., solution).
In one embodiment, a reagent (composition) includes an effective evaporation
reducing amount of an evaporation reducing agent comprising the formula of
General
Formula I:
Xi- R - X2,
wherein R is an alkyl, an alkenyl, an alkynyl or an aromatic moiety of one or
more carbon
atoms (e.g., one to six carbon atoms in one embodiment and one to four carbon
atoms in
another embodiment where it is appreciated that if R is a moiety of one
carbon, R is an alkyl)
that may be substituted with oxygen atom, sulfur atom and/or or nitrogen atom
(e.g., -OH, -
SH, or -NH, is substituted for a hydrogen atom of one or more hydrocarbons
defining the
alkyl, alkenyl, alkynyl or aromatic) or that may be interrupted with an oxygen
atom, a sulfur
atom and/or a nitrogen atom (e.g., -C-O-C-, -C-SH-C-, -C-NH-C-). An alkyl
moiety is an
alkane containing open points of attachment for connection for Xi and X2. An
alkenyl
moiety is an alkene containing open points of attachment for connection for Xi
and X2. An
alkynyl moiety is an alkyne containing open points of attachment for
connection to Xi and
X2. Representative alkyls include straight or branched chain alkyls (e.g.,
methyl, ethyl,
propyl, isopropyl) which may or may not be further substituted. Representative
alkenyls
include straight or branched chain alkenyls (e.g., ethenyl, propenyl,
isopropenyl).
Representative alkynyls include straight or branched chain alkynyls which may
or may not be
further substituted (e.g., ethynyl, propynyl, isopropynyl). An aromatic moiety
is an
unsaturated ring of atoms that is stabilized by an interaction of the bonds
forming the ring.
An example of an aromatic moiety is benzyl. Examples of interrupted aromatic
moieties are
pyridine (interrupted with a nitrogen atom); furan (interrupted with an oxygen
atom), and
thiophene (interrupted with a sulfur atom).
Xi and X2 in General Formula I are independently selected to be a moiety where
one or both are susceptible to hydrogen bonding and/or contains an
electronegative atom.
Representative moieties for Xi and X2 will include functional groups that are
capable of
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hydrogen bonding such as but not limited to hydroxyl moieties (-OH); carbonyl
moieties (-
CO); amine moieties (-NH2); aldehyde moieties (-CHO); halogen moieties (-Y,
where Y is
cr, F, Br- or I-); ether moieties (-OR', where Rl is an alkyl (e.g., an alkyl
of one to six
carbon atoms) that may be substituted with a moiety that is susceptible or
capable of
.. hydrogen bonding and/or contains an electronegative atom); carboxyl
moieties (-COOR2,
where R2 is a hydrogen atom or an alkyl (e.g., an alkyl of one to six carbon
atoms) that may
be substituted with a moiety that is susceptible or capable of hydrogen
bonding and/or
contains an electronegative atom); and amide moieties (-CONR3R4, where R3 and
R4 are
independently selected from a hydrogen atom or an alkyl (e.g., an alkyl of one
to six carbon
.. atoms) that may be substituted with a moiety that is susceptible or capable
of hydrogen
bonding and/or contains an electronegative atom). Representative of
substitution moieties
that are susceptible or capable of hydrogen bonding for the noted alkyls
associated with ether,
carboxyl and amide moieties for Xi and X2 (RI, R2, R3 and R4) include
hydroxyl, carbonyl,
amine, aldehyde, halogen, ether, carboxyl and amide moieties.
Examples of suitable evaporation reducing agents having General Formula I
include ethylene glycol, glycine, serine, isoethionic acid, ethanolamine,
polyethylene glycol
and 1,3-propanediol. In one embodiment, R is not substituted or interrupted.
Representative
of an evaporation reducing agent where R is not substituted or interrupted is
ethylene glycol
(HO(CH2)20H) where R is ethyl and each of X1 and X2 is a hydroxyl moiety. An
example of
.. an evaporation reducing agent where X2 is a primary amide that is further
substituted with a
moiety susceptible or capable of hydrogen bonding is H2N-(CH2)3-NH(CH2)2COOH,
where
R is -(CH2)3-; Xi is -NH2 and X2 is -NH(CH2)2COOH. In one embodiment,
a suitable
reagent includes one or more evaporation reducing agents having the formula of
General
Formula I (e.g., a reagent includes a combination of two or three evaporation
reducing agents
.. and having the formula of General Formula I).
An evaporation reducing agent may be in a molecular free folin (a molecule) or
an
acceptable salt thereof (a compound) or ionic liquid. An example of a salt is
a hydrohalide
salt of the molecule such as a hydrochloride salt.
In one embodiment, an evaporation reducing agent as a solute reduces the vapor
.. pressure of a solution upon its mixing with one or more other reagents.
Such solute could be
a molecule, an ionic salt, an ionic liquid, a non-ionic agent that may exhibit
complete or
partial solubility or miscibility with water and is capable of reducing a
partial pressure of an
aqueous solution to which it is mixed or otherwise introduced. Additionally,
an evaporation
reducing agent may or may not form an azeotropic mixture with water. If an
evaporation
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reducing agent does form an azeotropic mixture with water, the solute, in one
embodiment,
forms a negative azeotrope with water.
Generally speaking, when a solvent and a solute are mixed in this context, a
resulting chemical potential of the solvent is lowered so that a partial
pressure of the solvent
molecules is reduced and, relative to a solvent-only solution, has a reduced
tendency to
transition into gas phase. Representatively, a partial pressure of water in an
aqueous solution
where a solute is a nonvolatile evaporation reducing agent will be reduced
which would
result in elevation of a boiling point of the mixture as well as a reduction
in evaporation at the
surface.
Hydrogen bonding can occur when in a molecule, a hydrogen atom is attached to
an electronegative atom such as an oxygen, nitrogen or fluorine atom and the
molecule comes
in proximity with an electropositive atom. The electrostatic attraction of
this nature has a
strength on the order of 5-30kJ/mol which is stronger than Van der Waals
attraction but
weaker than covalent or ionic bonding.
In one embodiment, an evaporation reducing agent is mixed as a solute with
water
to reduce a vapor pressure of the water by creating a solution where a partial
pressure of the
water is reduced such that an assay (e.g., an immunohistologic assay) can be
carried out more
efficiently than if the partial pressure was not lowered. In one embodiment, a
solute is
selected such that there is no or minimum residue left behind which may
interfere with an
assay known to a person skilled in the art,
In one embodiment, a reagent operable for use in processing a cellular or
tissue
sample is prepared by combining a base reagent and an evaporation reducing
agent.
Additional components may also be combined in a suitable composition. The
reagent can be
added to an aqueous buffer solution to reduce evaporation during elevated
temperatures. In
one embodiment, the evaporation reducing agent is combined in an amount to
limit any loss
of the combination due to evaporation to 20 percent or less and, in another
embodiment, to 10
percent or less. Representatively, an evaporation reducing agent or
combination of reducing
agents is/are present in a composition in an amount between 11 percent and 60
percent by
volume or an amount between 1 percent and 60 percent of the reducing agent is
a non-glycol-
containing compound or a combination of reducing agents. Following a
combination of a
base reagent, evaporation reducing agent and any other components, the
composition may be
mixed.
In one embodiment, a method operable in processing of a paraffin-free or
deparaffinized tissue or cellular sample for use in an assay at an elevated
temperature is
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84072721
disclosed. In one embodiment, a method is operable to reduce evaporation of
any aqueous
based processing reagent. The method includes contacting a tissue or cellular
sample with
a reagent including an evaporation reducing agent (e.g., as a solute) having
the formula of
General Formula I:
Xi- R - X2.
Such contact includes applying the reagent to a sample on a slide. Following
contacting
the sample with the reagent, the sample may be processed according to
techniques known
in the art.
In one embodiment, a method operable in processing of a paraffin-free or
deparaffinized tissue or cellular sample for use in an assay at an elevated
temperature is
disclosed. In one embodiment, a method is operable to reduce evaporation of
any aqueous
based processing reagent by adding a solute to a solvent. The resulting
chemical potential
of the solvent is lowered so that the partial pressure of the solvent
molecules is reduced
and they have lesser tendency to turn into gas phase. Partial pressure of
water in an
aqueous solution where solute is a nonvolatile substance is reduced which
would result in
elevation of boiling point as well as reduction in evaporation at the surface.
According to one aspect of the present invention, there is provided a method
of
preparing a tissue or cellular sample for assay comprising: contacting a
tissue or cellular
sample on a slide with an aqueous composition comprising a hybridization
solution or a
primary antibody diluent solution, a detergent and at least one evaporation
reducing agent
selected from a glycine, a serine, an isoethionic acid, an ethanolamine, a
glycerol, an
ethylene glycol, a polyethylene glycol, a 1,3-propanediol and
H2N(CH2)3NH(CH2)2)COOH and wherein the at least one evaporation reducing agent
is
present in an amount of greater than 20 percent by volume when the at least
one
evaporation reducing agent is a non-glycol-containing agent or a combination
of more than
one non-glycol-containing evaporation reducing agent or an amount of greater
than
percent by volume or greater when the at least one evaporation reducing agent
is a
glycol-containing agent; and processing the tissue or cellular sample at a
temperature
between 25 C and 50 C for 10 minutes to 24 hours where the composition is a
30 hybridization solution or between 60 C and 100 C for 2 minutes to 90
minutes where the
composition comprises a primary antibody diluent solution.
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84072721
According to another aspect of the present invention, there is provided use of
a
reagent for processing of a tissue or cellular sample on a slide, the reagent
comprising a
hybridization solution or a primary antibody diluent solution, a detergent and
an
evaporation reducing agent present in an amount of greater than 20 percent by
volume
when the evaporation reducing agent is a non-glycol-containing agent or a
combination of
more than one non-glycol-containing evaporation reducing agent or an amount of
greater
than 30 percent by volume when the evaporation reducing agent is a glycol-
containing
agent, wherein the evaporation reducing agent is selected from a glycine, a
serine, an
isoethionic acid, an ethanolamine, a glycerol, an ethylene glycol, a
polyethylene glycol, a
1,3-propanediol and H2N(CH2)3NH(CH2)2)COOH, wherein the use is for processing
the
tissue or cellular sample at a temperature between 25 C and 50 C for 10
minutes to
24 hours where the composition is a hybridization solution or between 60 C and
100 C for
2 minutes to 90 minutes where the composition comprises a primary antibody
diluent
solution.
EXAMPLES
Example 1: Volume loss due to evaporation during oligonucleotide
hybridization at different hybridization temperatures.
400 I of hybridization solution (reagent) was applied to each of six sample
slides and the slides were individually heated to different temperatures for
30 minutes. It
was observed that evaporation occurred on the slide with hybridization
solution during the
hybridization step.
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84072721
Table 1. Effect of temperature on evaporation
Hybridization Ave volume loss due to
Temperature ( C) evaporation ( 1)
25 7.5
35 37.5
37 50.0
39 47.5
41 50.0
43 55.0
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Example 2: Reducing evaporation during oligonucleotide hybridization by
different reagents.
400 1.11 of hybridization solution (reagent) with different evaporation
reducing
agents was applied to respective sample slides and heated to 43 C for 30
minutes. One
sample slide included a hybridization solution with no evaporation reducing
agent. It was
observed that a significantly greater evaporation of the hybridization
solution occurred with
the sample slide containing a hybridization solution without an evaporation
reducing agent
during the 43 C hybridization step but was reduced with the addition of an
evaporation
reducing agent.
Table 2. Ability of added agent to hybridization
solution (reagent) to reduce evaporation
Avg volume loss
Reagent
(1a)
No addition 55.0
20% Ethylene Glycol 20.0
20% Propylene Glycol 45.0
20% Glycerol 42.5
20% Polyethylene Glycol 15.0
Example 3: Reducing evaporation using different concentrations of ethylene
glycol during hybridization.
400 al of hybridization solution (reagent) having different concentrations of
an
evaporation reducing agent of ethylene glycol was applied to respective sample
slides and
heated to 43 C for 30 minutes. For comparison, 400 al of hybridization
solution without
ethylene glycol was also applied to a sample slide and heated to 43 C for 30
minutes. It was
observed that evaporation was reduced with the addition of ethylene glycol to
the
hybridization solution. The assay performance for the hybridization solution
containing
ethylene glycol was found to be similar to hybridization solution without
ethylene glycol.
Table 3. Reduction in volume loss in % ethylene glycol (EG)
in hybridization solution
Comparison to Starting Volume
Starting vol Avg evaporatiion
% EG A) Evaporation
(pi) (1.11)
0% 400 48 12%
7% 400 28 7%
14% 400 18 4%
20% 400 13 3%
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Comparison to Starting Volume
27% 400 10 2%
33% 400 0 0%
40% 400 0 0%
Example 4: Reproducibility in reducing evaporation volume loss during
hybridization.
400 ill of hybridization solution with specific concentrations of an
evaporation
reducing agent of ethylene glycol was applied to each sample slide and heated
to 43 C for 30
minutes. For comparison, 400 ill of hybridization solution was applied to each
sample slide
and heated to 43 C for 30 minutes. It was observed that evaporation was
reduced with the
addition of ethylene glycol to the hybridization solution.
Table 4. Reduction in evaporation due to addition of ethylene glycol
oligonucleotide hybridization oligonucleotide hybridization
solution with 0% ethylene glycol solution with 20% ethylene glycol
Slide Starting vol Evaporation Starting vol Evaporation
(al) ( 1) ( 1) ( 1)
1 400 45 400 12
2 400 50 400 15
3 400 45 400 15
4 400 55 400 17
5 400 40 400 15
6 400 50 400 10
7 400 50 400 12
8 400 45 400 15
9 400 50 400 12
10 400 45 400 12
Avg 48 Avg 14
Example 5: Reduction in evaporation during denaturation.
About 400 ill of hybridization solution with an evaporation reducing agent of
20
percent ethylene glycol was applied to each sample slide and heated to 92 - 98
C for 2
minutes. For comparison, about 400 n.1 of hybridization solution without
ethylene glycol was
applied to each sample slide and heated to 92 - 98 C for 2 minutes. It was
observed that
evaporation was reduced with the addition of ethylene glycol to the
hybridization solution.
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Table 5. Reduction in evaporation with addition of ethylene glycol to
hybridization solution
during denaturation.
oligonucleotide hybridization solution oligonucleotide
hybridization solution
with 0% ethylene glycol with
20% ethylene glycol
Temp Time Starting Evaporation Avg. loss due
Starting vol .. Evaporation .. Avg. loss due
( C) (min) vol (n.1) (pi) to Evaporation
(111) (111) to Evaporation
92 2 400 60 15% 400 15 4%
92 2 400 65 400 17
92 2 400 50 400 15
95 2 400 60 16% 400 30 7%
95 2 , 400 , 70 , 400 , 30
95 2 400 65 400 25
98 2 400 80 19% 400 33 8%
98 2 400 75 400 30
98 2 400 75 400 30
Example 6: Reduction in evaporation during antibody incubation step.
About 400 IA of primary antibody diluent (reagent) with an evaporation
reducing
agent of 20 percent ethylene glycol was applied to each sample slide and
incubated at a
specific temperature for 30 minutes. For comparison, about 400 I.11 of primary
antibody
diluent without ethylene glycol was applied to each sample slide and incubated
at a specific
temperature for 30 minutes. It was observed that evaporation was reduced with
the addition
of ethylene glycol to the antibody diluent.
Table 6. Reduction in evaporation with addition of ethylene glycol to antibody
diluent
Primary antibody diluent Primary antibody diluent
with 0% ethylene glycol with 20% ethylene glycol
Temp Starting vol Evaporation Avg. loss
due Starting vol Evaporation Avg. loss due
( C) (0) (pi) to Evaporation (0) (1,i1)
to Evaporation
400 0 0% 400 0 0%
20 400 0 400 0
400 3 1% 400 0 0%
25 400 5 400 0
31 400 30 9% 400 5 1%
31 400 40 400 5
37 400 60 17% 400 3 1%
37 400 75 400 5
43 400 95 23% 400 20 6%
43 400 90 400 25
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84072721
Example 7: Reducing evaporation during oligonucleotide hybridization by
different reagents.
400 pl of hybridization solution with different evaporation reducing agents
was
applied to respective sample slides and heated to 43 C for 30 minutes. A
hybridization
solution containing no evaporation reducing agent was also applied to a sample
slide. It was
observed that evaporation occurred in the slide with hybridization solution
during the 43 C
hybridization step but was reduced with the addition of specific reagent.
Table 7. Ability of added evaporation reducing agent to
hybridization solution to reduce evaporation
Evaporation Reducing Agent Avg volume loss (Id)
No addition 57.5
20% ethylene glycol 20
20% glycine 27.5
20% serine 17.5
20% isoethionic acid 25
20% ethanolamine 17.5
20% 1,3-propanediol 22.5
Example 8: Reducing evaporation using ethylene glycol during hybridization at
different temperatures.
400 I of hybridization solution (reagent) with ethylene glycol was applied to
respective sample slides and heated to 35, 37 or 40 C for 120 minutes. For
comparison, 400
I of hybridization solution without ethylene glycol was also applied to a
sample slide and
heated to 403 C for 120 minutes. It was observed that evaporation was reduced
with the
addition of ethylene glycol to the hybridization solution. The assay
performance for the
hybridization solution containing ethylene glycol at different hybridization
temperatures was
found to be similar to hybridization solution without ethylene glycol.
Reagents
Hybridization solution
Chemical Final conc.
SSC 2x
Tris-HCl 10 mM
Dextran sulfate 10 %
Denhardt's solution 2 x
TweenThi 0.05 'Yo
TritonIm 0.10 %
Salmon sperm DNA 50 ag/m1
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Chemical Final conc.
Evaporation reducing agent varied
water varied
Primary antibody diluent
Chemical Final conc.
Sodium Azide 0.05%
Sodium Chloride 300 mM
Sodium Phosphate Dibasic 8 mM
Potassium Phosphate monobasic 2 mM
Green Color Dye 0.025%
BSA Powder I%
Tris Base 10 mM
Tris HCI 40 mM
TweenTm 0.05%
TritonTm 0.1%
Evaporation reducing agent varied
water varied
In the description above, for the purposes of explanation, numerous specific
details have been set forth in order to provide a thorough understanding of
the embodiments.
It will be apparent however, to one skilled in the art, that one or more other
embodiments
may be practiced without some of these specific details. The particular
embodiments
described are not provided to limit the invention but to illustrate it. The
scope of the
invention is not to be determined by the specific examples provided above but
only by the
claims below. In other instances, well-known structures, devices, and
operations have been
shown in block diagram form or without detail in order to avoid obscuring the
understanding
of the description. Where considered appropriate, reference numerals or
terminal portions of
reference numerals have been repeated among the figures to indicate
corresponding or
analogous elements, which may optionally have similar characteristics.
It should also be appreciated that reference throughout this specification to
"one
embodiment", "an embodiment", "one or more embodiments", or "different
embodiments",
for example, means that a particular feature may be included in the practice
of the invention.
Similarly, it should be appreciated that in the description various features
are sometimes
grouped together in a single embodiment, figure, or description thereof for
the purpose of
streamlining the disclosure and aiding in the understanding of various
inventive aspects. This
method of disclosure, however, is not to be interpreted as reflecting an
intention that the
invention requires more features than are expressly recited in each claim.
Rather, as the
following claims reflect, inventive aspects may lie in less than all features
of a single
to
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disclosed embodiment. Thus, the claims following the Detailed Description are
hereby
expressly incorporated into this Detailed Description, with each claim
standing on its own as
a separate embodiment of the invention.
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