Note: Descriptions are shown in the official language in which they were submitted.
Case H-542-CIP
This invention broadly relates to an immunological
reagent capable of enhancing the extent of an immunoprecipita-
tion reaction. More specifically, the invention relates to
an immunological reagent solution which can be utilized in a
wide variety of immunological assaying methods.
The invention also relates to immunological assay-
ing methods, in particular those involving a reaction of an
antibody with an antigen to form an antibody-antigen complex,
- the insolubilization of which is substantially increased by
the utilization in the assaying method of the reagent of this
invention.
There are numerous immunological assaying methods
of widely varying methodology which necessitate at one stage
or another in the assay, and for varying purposes, the insolu-
bilization of as much of the antigen-antibody complex as possible.
For example, complex insolubilization plays an important role in
such illustrative important conventional immunological assaying
techniques as electrophoretic analysis, enzymatic assays, radio-
immunoassays (RIA) and nephelometric assays, and much work has
been directed toward developing means for increasing complex
insolubilization in order to improve these assays. Normally,
complex insolubilization is necessary in order to isolate the
- immunological reaction product from the unreacted immunological
reactants involved in a particular assay so that either the
' 25 isolated reaction product or the unreacted reactants can be
separately analyzed to provide a meaningful diagnostic assay.
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For an immunological assaying method involving, for
example, a nephelometric analysis, it is known to dilute test
samples with a solution of polyethylene glycol polymer prior
to both incubation of the sample and making a reading on the
nephelometric instrument. While the polyethylene glycol does
improve the nephelometric analysis by increasing the concentra-
tion of suspended particles, thereby improving the analysis,
there nevertheless remains the problem that satisfactory analysis
of many biological constituents cannot be made because of inade-
quate test range or sensitivity due to insufficient concentra-
tion of suspended particles in the test sample. The broad ob-
jective of the present invention is to solve the foregoing
problem by providing an improved immunological reagent which
is effective to greatly increase the insolubilization of anti-
body-antigen complexes beyond that obtainable from polyethylene
glycol alone, and hence increase the concentration of suspended
particles in the test sample, to permit analysis of biological
constituents not possible with polyethylene glycol alone,
whether nephelometric or other assaying methods are used.
More specifically, we provide in accordance with
the invention a reagent for clinical nephelometric analysis
comprising an aqueous solution of an antiserum to the component
to be assayed and a mixture of about 20% to about 40% by weight
; polyethylene glycol having a molecular weight of about 200 to
about 10,000 and about 80% to about 60% by weight of a block
copolymer of ethylene oxide and polyoxypropylene containing at
least 50% ethylene oxide.
We also provide in accordance with the invention
an immunological assaying method which involves a reaction
between an antigen and an antibody to form an antigen-antibody
complex, the method being characterized by carrying out said
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reaction in the presence of a reagent comprising an aqueous
solution containing about 3 to 6% by weight of a mixture of
polyethylene glycol having a molecular weight of about 200
to about 10,000 and a nonionic surfactant other than poly-
ethylene glycol wherein said mixture contains about 10% to90% by weight polyethylene glycol and about 10% to 90% by
weight nonionic surfactant and said solution has a calculated
HLB value of about 0.7 to 1.7. Significantly, the aqueous
solution when not "in use", as for example in an immunolog-
ical assaying method may have a-mixture concentration which
is less than 3% and more than 6% and is nevertheless
useful to the purpose of this invention providing the concen-
tration of the mixture prior to or "in use" is adjusted to
have a concentration in the range between about 3% to 6%.
Therefore, by the term "in use", it is our intention to cover
a reagent the mixture concentration of which need not initially
; necessarily be in the range between about 3% to 6% so long ~ -
as the concentration range, if outside the specified range of -
3% to 6%~is adjusted to these limits during or prior to an
assaying procedure.
Advantageously, the reagent may be introduced into
the test medium for performing a nephelometric, enzymatic,
electrophoretic or radioimmuno assay.
When a nephelometric assaying method, for example
is conducted for analyzing biological constituents in the form of
- antibody-antigen complexes, the reagent and suspended particles
of the biological constituents are first incubated whereafter the
nephelometric analysis is performed. According to this method, a
light source is made to pass through a liquid test sample whereby
-- 4
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the light rays are directed through the suspended particles.
As these light rays strike the particles, they are scattered
or diffused at any predeter~ined angle, for example, at a
right angle, from the source and are received by photocells.
This scattered light is converted to an electrical signal
which is directly proportional to the amount of particulate
concentration which, in turn, is thereby accurately measured
on a meter face of an lnstrument.
Examples of instruments suitable for nephelometric
analyses are the Laser Nephelometer manufactured by Hyland
Laboratories ; the Aminco-Fluor ~ lorimeter (American Instru~
ment ~ompany); the ~inco-Bowman Spectrophotofluorometer (SPF):
and thè Autoanalyzer II with attached Fluoronephelometer
(Technicon Instruments Corporation).
` By means of ~hese nephelometric principles and equip-
-ment, the clinical technologist can make an accurate determina-
tion of small concentrations of a wide variety of specific pro-
teins, for example the immunoglobulins IgG, IgA, IgM, trans-
ferrin, complement C3, haptoglobin, alphal-antitrypsin, ~-lipo-
protein, albumin, alpha2-macroglobulin, alphal-acid glycoprotein,
and various other biological constituents such as triglyc~rides,
lipoproteins, and human chorionic gonadotropins.
The most important advantages which result from use
of the reagent in accordance with the invention are (a) the
incubation times are significantly reduced and (b) greater con-
centration of insolubilized complex particles are obtained at
sh~rter incubation time with resulting improved test range and
.
~ ~5-
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sensitivity These advan~ages apply to any immunological assaying
method which is benefited by enhanced insolubilization of the
antigen-antibody complex at some point during ~he assay. The
advantages also apply to immunological assay methods which rely
upon an antigen-antibody complex insolubilization step at some
point during the assay procedure.
As above noted, the reagent aqueous solution, in addi-
tion to containing, in use, about 3% to 6% by weight of the mix-
ture of polyethylene glycol and the nonionic surfactant, should
also have a calculated HLB value of between about 0.7 to 1.7.
The HLB value is a well-established measure of the
hydrophilic-lipophilic balance (hence "HLB"~ of a given sur-
factant. The HLB system of surfactant identification was de-
veloped by Atlas Chemical Industries, Inc. and is described
in detail on pp. 28-36 of the Atlas publication entitled "Guide
to the Use of Atlas Surfactants and Sorbitol in Cosmetic and
Pharmaceutical Products" (1965). Each surfactant is
- assigned an HLB number. The-lower the HLB num~er, the more
lipophilic (or oil-loving) the surfactant while the higher the
2~ number, the more hydrophilic (or water-loving? the surfactant.
The method for establishing the HLB value of a~y given sur-
factant is well known and is described, for example, in a publicly
released Atlas printed publication entitled "The Atlas HLB
System" (Code LD~97). The HLB values of numerous surfactants
are also published widely in the literature, particularly liter-
ature put out by the manufacturer of the surfactant in question
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The HLB value of a blend of surfactants, such as
exist in the reagent of this invention, is a function of the
concentration of each surfactant in the blend, and hence the
concentration of the individual surfactants must be taken
into account in computing an HLB value for the blend. The
HLB value of the blend is calculated, in accordance with ac-
- cepted published procedures, by multiplying the assigned HLB
value of each surfactant present in the blend by its concen-
tration in the composition in question, and adding the individual
calculated results. For example, if the reagent of this inven-
tion contained 1% by weight polyethylene glycol (HLB = 20)
and 3% by weight of a nonionic surfactant having an HLB of
30.5, the HLB value of the reagent would be computed as follows:
0.0~ X 20 = 0.2
0.03 X 30.5 = 0~915
1.115 -- HLB value of reagent
To determine whether a given reagent has an HLB value which
falls within the 0.7 to 1.7 range of the reagent of this in-
vention a calculation as above, based on the amount of each
component in the blend, can be readily made, using either pub-
lished HLB values for the components in question or HLB values
determined according to the Atlas method.
The present invention contemplates the use of a
wide variety of nonionic surfactants in conjunction with the
polyethylene glycol provided that (1) the mixture of nonionic
surfactant and polyethylene glycol falls within a range of 3
to 6% by weight at the time the reagent is used in the assay,
1~8~
and (2) that the calculated HLB value of the reagent falls
within a range of 0.7 to 1.7 at the same time. If at the
time of use, the polyethylene glycol-nonionic surfactant mix-
ture accounts for less than 3% of the reagent, or if the re-
agent has an HLB less than about 0.7, it becomes difficultto insolubilize the desired amount of antigen-antibody com-
plex to the extent required for a successful completion of
the immunological assay. On the other hand, if the mixture
accounts for more than 6% of the reagent, or if the reagent
has an HLB greater than about 1.7, other proteins besides the
desired antigen-antibody complex are insolubilized, thereby
destroying the selectivity of the assay and rendering the re-
sults meaningless.
Of course, the reagent solution could, for a variety
of reasons, be prepared and marketed in such manner that it did
not contain the requisite 3 to 6% mixture of polyethylene glycol
and nonionic surfactant, or have the requisite HLB value of 0.7
to 1.7. Poor shelf stability could be one such reason. However,
as above noted, such solutions would require adjustment, prior
to or at some point during their usage in an immunological assay
technique, to the 3 to 6% range and to an HLB value of about 0.7
to 1.7. For this reason, reagents which do not contain the
requisite 3 to 6% of said mixture or an HLB value of about 0.7
to 1.7, but which can be readily adjusted to these values prior
to or during the immunological test procedure, are within the
scope of the invention, even though they may initially be out-
side the specific parameters of concentration and HLB range
--8--
.: ............ .
required during the assay procedure itself.
For example, the marketed reagent solution may re-
quire, if the specific parameters of concentration and HLB
values fall outside the ranges of about 3% to 6% and about
0.7 to 1.7, respectively, dilution, concentration, Qr other
adjustment steps prior to or at some convenient point during
performance of the immunological assay method, for the purpose of
bringing the solution to a mixture level of 3 to 6~/o and a cal-
culated HLB value of 0.7 to 1.7. In such circumstances, the
marketed solution would still fall within the scope of the present
invention Thus the reagent could be marketed at a mixture con-
centration higher than 6%, e.g., 8%, which would be diluted
either prior to or at some suitable point during an immunological
assay procedure to the 3 to 6% range. Similarly, the reagent
could be marketed with a calculated HLB value outside the 0.7
to 1.7 range, but with the requirement that the reagent solution
be in someway altered prior to or at some suitable point during
an immunological assay procedure to bring it to a calculated HLB
range of 0.7 to 1.7.
The reagent could in certain cases be diluted ~o
the proper levels with the antiserum to be used in the test, while
in other cases it could be diluted with the test sample or in
some cases with both the test sample and antiserum. The im-
portant point is that at some point during the immunological
assay procedure, usually either prior to or at the time of the
antigen-antibody reaction, the requisite level of 3 to 6% of
combined polyethylene glycol and nonionic surfactant, and the
requisite HLB value of 0.7 to 1.7, must be provided in order
for the reagent of ~he invention to function properly in the
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assay procedure. The point in the assay procedure at which
these requisite parameters must be p~ovided can vary depend-
ing on the particular procedure involved. For a nephelometric
analysis, for example, it is convenient to prepare and market
the reagent of the invention with a concentration of poly-
ethylene glycol and nonionic surfactant in excess of 6% and
at an HLB value outside the 0.7 to 1.7 range. Then, ~ust
prior to usage in the nephelometric analysis, the reagent is
diluted with the antiserum to be used in the analysis to pro-
vide a concentration of polyethylene glycol and nonionic sur-
factant of between 3 to 6%,preferably about 4%, and a calcu-
lated HLB value within the 0.7 to 1.7 range.
The relative proportions of polyethylene glycol and
nonionic surfactant in the mixture can vary within wide limits,
depending largely on the surfactant being used. Illustratively,
the mixture contains from about 10 to 90% by weight polyethylene
glycol and 10 to 90% nonionic surfactant. Preferably, the mix-
ture contains about 15 to 85% polyethylene glycol and 15 to 85Z
nonionic surfactant.
One or more different forms or types o polyethylene
glycol can be used as the polyethylene glycol component of the
mixture. The polyethylene glycol polymer used generally has a
molecular weight of from about 200 to about 10,000, and preferably-
from about 4,000 to about 6,000- A ~olecular weight range of
a~ut 4,000 is especially preferred.
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~ A -lo-
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The nonionic surfactant component of the mixture
can be any nonionic surfactant which will produce in the re-
agent solution a calculated HLB value of 0.7 to 1.7, and pre-
ferably about 0.7 to 1.3, at a mixture concentration of 3 to
6%. Illustratively, the HLB values of the nonionic surfac-
tant itself can range from about 10 to 30 more.
A particularly preferred nonionic surfactant is
a block copolymer of ethylene oxide and polyoxypropylene.
This particular polymer and its preparation is described in
U.S. Patent 2,674,619. These block copolymers are generally
prepared by condensing ethylene oxide with polyoxypropylene
polymer and can be represented by the following structural formula:
HO(CH2CH2O)a (CH3CHCH2O~b (CH2CH2O)c H-
For purposes of this invention these block copolymers desirablycontain at least 50% ethylene oxide in the molecule and a poly-
- 15 oxypropylene hydrophobic base molecular weight of at least 950,
similarly as described in U~S. Patents 3,450,502, 3,577,522 and
3,590,125.
Illustrative examples of such suitable block copolymers
are the F-38 and F-68 PLURONIC~ polyols sold by Wyandotte Chemicals
Corporation. F-38 contains 80% of polyoxyethylene hydrophilic
; units in the molecule and the polyoxypropylene hydrophobic base
has a molecular weight of 950. F-38 is a particularly preferred
nonionic surfactant. F-68 also contains 80% of polyoxyethylene
hydrophilic units in the molecule but the hydrophobic base has a
molecular weight of 1,750. The total molecular weight of these
two PLURONIC ~ polyols is 4,750 and 8,750, respectively. PLURONIC
L-125 has also been found useful. ~ further description of these
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polyols is found in the bulletin of Wyandotte Chemicals Corpor-
~` ation, "The Pluronic Grid" Sixth Edition.
In the case of the PLURONIC nonionic surfactants, a
mixture consisting of about 20% to 40% by weight polyethylene
glycol and about 80% to 6Q% block copolymer of ethylene oxide
and polyoxypropylene polymer is generally preferred.
A highly preferred reagent solution contains about
one part by weight of polyethylene glycol having a molecular
weight ~ bout 4,000 and about three parts by weight of the
lC Pluronic F-3~ material. This solution can be prepared in a
saline solvent (e.g., 0.9% NaCl) either at, above or below the
desired level of 3 to 6% and then adjusted, if necessary, to -
the desired level of 3 to 6%. Preferably, the solution is pre-
-- pared as an 8% solution o~ the polymer mixture in saline which
is then diluted with antiserum (see Examples 1 and 5 below) or
another suitable diluent prior to its usage in an imm~nological
assay procedure. The diluted reagent thus con~ains about 1%
polyethylene glycol and 3% F-38. Its HLB value of l.lt5 can
be calculated as follows:
1% PEG: 0.01 X 20* = 0.2
3% F-38: 0.03 X 30.5~* = ~.15
1.115
A wide variety of other nonionic surfactants can
i also be used to complement the polyethylene glycol in the re-
agent solution of the invention, provided they provide the
desired HLB value of 0.7 to 1.7 at a mixture concentration of
* the HLB of PEG from the literature i5 20
**the HLB of F-38 from the literature is 30~5
1 2--
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3 to 6%. For example, the TETRONIC ~ series of nonionic sur-
factants available from BASF Wyandotte Corporation and described
in detail in the BASF Wyandotte Brc~chure entitled "Technical
Data on Tetronic ~ Series Nonionic Surfactants" and in U.S. Patent
2,979.52~ haYe been found quite suitable, particularly those
identified as TETROrlIC ~ 707,908, 1107, 1307 and 1508. The
TETRONIC Q products are based on ethylene diamine and have the
general formula:
H(C2H40)y(C3H60)x\ /~C3}~'6)x(C2H4)yH
N-CH2-CH2-N
H(c2H4o)y(c3H6o)x / (C3H6o)x(c2H4o)yH
The molecular weight range of the TETRONIC ~ line can vary from
about 1,650 to over 26,00Q. The more preferred TETRONIC ~ sur-
factants have molecular weights in the range of about 15,000
to 27,000 and HLB values of about 20 to 30.5. The proportion
of polyethylene glycol to TETRONIC ~ surfactant in the mixture
can vary widely, as discussed above.
Another family of useful nonionics is the PLURAFAC
line of products also put out by BASF Wyandotte, particularly- 20 those identified as PLURAFAC ~ 17R8, 25R8~ D25, A38 and A39.
The PLURAFAC ~ products are straight chain, primary aliphatic
oxyalkylated alcohols described in greater detail in the BASF
Wyandotte brochure entitled "Technical Data on Typical Physical
`. Properties of PLURAFAC ~ Nonionic Surfactants", The preferred
PLURAFAC ~ products have HLB values of about 10 to 20 and can
be used in widely varying ratios with the polyethylene glycol.
--13-- -
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Other suitable nonionic surfactants include glycerol
monostearate and the family of alkyl aryl sulonates. A preferred
glycerol monostearate is available from Atlas Industries, Inc.
under the tradename ARLACEL ~ 165 (HLB = 11.0) while a typical
useful alkyl aryl sulfonate is also available from the same
source under the tradename G-3300 (HLB = 11.7).
The above listing on nonionic surfactants is illus-
trative only since other such surfactants are contemplated to
fall within the scope of the invention provided they provide a
reagent which, when used in an immunological assay procedure,
contains about 3 to 6% of a mixture of polyethylene glycol and
surfactant, has a calculated HLB value based on the values of
the individual components present in the solution of about 0.7 to
1.7, and produces the effect of enhancing the insolubilization
of the desired antigen-antibody complex to the extent desired
to improve the assay, and without insolubilizing unwanted com-
ponents or in any other way detrimentally interfering with the
assay procedure.
Of course, more than one nonionic surfactant can be
present with the polyethylene glycol provided the requisite con-
centration of the mixture and HLB value of the solution are
achieved.
In selecting a nonionic surfactant for use in the
invention, one should be used which also has the capability of
providing a clear reagent solution at least at the time the re-
agent solution is first used in the immunological assay procedure.
This is to insure that the reagent does not interfere with the
test results, or does not produce non-specific precipitation of
,~
i~utiis~
the components of the biological sample or the biological
reagents employed in the assay system.
The reagent system of the invention finds broad
utility as an improvement in a host of conventional immunological
assay methods which at one time or another, for whatever the
reason, require a step to insolubilize the antigen-antibody
complex formed during the assay procedure by the antigen~antibody
reaction. Such assay methods are well known to those skilled
in the art and need not be described in detail herein. The
applicability and usefulness of the present invention in these
various assay methods, and the details of how to use the reagent
of the invention in such procedures would be apparent to those
skilled in the art from a reading of this specification and thus
these specifics bear no exhaustive repeating herein~ For example,
the reagent of the invention could be used to enhance any sys-
tem that depends upon the precipitation of antigen-antibody com-
plexes to produce a clear supernatant fluid for fluorescence or
colormetric detection. The reagent could also be used to remove
interfering substances found in biological fluids or reagents
(e.g., lipids, salts, and extraneous proteins) for nephelometry,
enzymatic or other assay systems~ This is acco~plished by the
removal of the reactants from the reaction solution for the
purpose of washing and resuspending these reactants.
Specifically, the reagent of the invention may be
utilized to increase the relative concentration of insoluble
antigen-antibody complexes and increase a given assay system's
test rsnge and sensitLvity. These principles msy be spplied
;
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to quantitative light scattering from immune complexes by
nephelometry, this being at present a preferred embodiment
of the invention. Nephelometric analyses using the reagent
of the invention are particularly useful in the analysis of
various immunoglobulins. However, other test systems that
are based on precipitating antibody may utilize these reagents,
such as radioimmunodiffusion (RID), enzymatic analyses, and
various types of electrophoretic techniques such as immuno-
electrophoresis (IEP), counterelectrophoresis (CEP), and
electroimmunodiffusion (EID).
The reagent of the invention may, for example, be
used in enhancing immunological assays that depend upon the
primary interaction of antigen-antibody coprecipitation tech-
nique commonly used in radioimmunoassays (RIA). This enhanced
; 15 insolubilization characteristic of the present invention can
also be utilized in the various methodologies of attachm~nt
of antibodies or antigen to inert particles used as a carrier
in RIA, nephelometric, and fluorometric assays in a manner well
understood by those skilled in the art.
Numerous radioimmunoassay techniques have been
adopted to quantitate relatively small concentrations of biological
; constituents found in body fluids~ Isotopically labeled antigen
or antibody is reacted with the homologous antigen or antiserum
to produce labeled immune antigen-antibody complexes. These com-
plexes must then normally be rendered insoluble by an insoluble
carrier, precipitating reagent or other known techniques. The
free or non-reacted labeled antigen or antibody can be re ved
by wa~hing techniques. The radioactive concentra~ion of the
precipitated complexes is then determined by gamma or liquid
-16-
io~a~is
scintillation counting. An example of an instrument used for
assa~s of this type is the Autogam~la~ Counter (available from
Nuclear Chicago, Inc.). The reagent in accordance with this
invention is useful to enhance the requisite complex insolubiliza-
tion step, thus improving the analysis. Example 6 below details
the use of the reagent of the invention in a radioimmunoassay
procedure.
Enzymatic techniques have also been adopted to quan-
titate small concentrations of biological constituents found
in body fluids. Enzyme labeled antigen or antibody is reacted
with the specific antibody or antigen to produce immune labeled
antigen-antibody complexes. These complexes are rendered in-
soluble by an insoluble carrier, precipitating reagent or other
technique. The free or non-reacted enzyme labeled antibody
or antigen may be removed by washing techniques. The bound or
reacted enzyme can then produce a reaction with an appropriate
- substrate to produce a colored or fluorescent supernant solu-
tion which can be measured with a spectrophotometer or-fluoro-
meter. C~xa~ les of instruments useful for this purpose are the
Beckman DB ~pectrophotometer (available from Beckman ~nstruments,
Inc.) and the Aminco-Bowma ~ Spectrophotofluorometer (available
from the American Instrument Company). The reagent in accordance
with the invention is also useful to enhance the requisite com-
plex insolu~ilization step, thus improving the analysis.
The usefulness of the reagent in a nephelometric
analysis has been discussed in detail above~ and is further exem~
plified in Example 5 below.
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It is now clear to those skilled in the art that
the improved immunological reagent of the invention has wide
ranging utility in the field of immunological assay procedures.
Using the improved reagent, the clinical technologist can make
accurate determination of a broad range of concentrations of
many specific proteins, for example, the immonoglobulins IgG,
IgA, IgM, transferrin, complement C3, haptoglobin, alphal-antitrypsin,
B-lipoprotein, albumin, alpha2-macroglobulin, alphal-acid glyco-
protein, and various other biological constituents such as tri-
iodothyonine (T3), thyroxine (T4), triglycerides, human chorionicgonadotropins, lipoproteins, and many others whose determination
would benefit from the enhanced insolubilization effect produced
by the present invention.
In st of the applications in which the invention
finds utility, the desired test biological constituent or con-
stituents are admixed with the aqueous reagent solution of the
nvention, incubated for a predetermined period of time such as,
for example, at room temperature (2Q-25~C) for approximately
one hour, and then read on appropriate instrumentation, e..g,
a nephelometer in the case of a nephelometric analysis. The
results of the test samples are compared with reference samples
to determine the unknown concentration.
The following examples are intended to further illus-
trate the invention, although it will be understood that the in-
vention is not limited to these specific examples.',
EXAMPLE 1
. .
A reagent solution is prepared by admixing 25 parts
by weight of polyethylene glycol having a molecular weight of
.
c~
4,000 with 75 parts by weight of PLURONIC F-38, and the mixture
dissolved in normal saline (0.9% NaCl) to a concent~ation of 8%
by weight of said mixture. This solution may either be diluted,such
as with antiserum, to a mixture concentration of 4% (HLB ratio -
1.115) as in step 3 of Example 5 below, prior to incubation, and
used directly in an immunological assay procedure, or it can be
kept in the 8% form until ready for use.
EX~PLE 2
Example 1 is repeated, except that polyethylene glycol
having a molecular weight of 6,000 is substituted for an equivalent
amount of 4,000 material.
EXA~LE 3 ~
Example 1 is repeated except that PLURONI~ F-68 is
substituted for an equivalent amount of F-38.
EXAMPLE 4
Example 1 is repeated except that Tetronic 707, 908,
1107, 1307, 1508; Plurafac 17R8, 25R8, D25, A38 and A39; Pluronic
L125, Arlacel 165; and G-3000 were substituted for the F-38 in
varying amounts in a series ~f experiments to prepare a wide --
~variety of reagent solutions.
EXAMPLE 5
- The immunological reagent solutions prepared in
; Examples 1-4 are used in separate nephelometric assays o~
immunoglobulins (IgA, IgG, IgM), complement-C3 and transferrin,
with each assay being conducted as follows:
1. Reference controls ~ 2, #3, #4, ~5, ~6 ~of
known assay) for each said biological constituent
are diluted 1:100 in saline.
2. The unknowns are then similarly diluted 1:100
i 30 in saline.
3. Prediluted antiserums to IgG, IgM, IgA, C3 and
transferrin are each diluted 1:2 with the 8% mix-
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... . . . . .. . . __ _ .. ... . .. , _.. . . _ . . _. ... . . . _ _~ __ . .. .. _ _ .. _
~ ;' ' - ,
ture of the immonological reagent from
Examples 1, 2, 3, or 4 (as the case may be)
and mixed by inversion to produce a 4% concen-
tration of the polyethylene glycol and nonionic
surfactant in the solution and a calculated HLB
of between 0.7 and 1.7 in all cases.
4. The antiserum is filtered through a 0.45
Millipore filter.
5. A series of test tubes (10 mm x 75 mm disposable
culture tubes) appropriately labeled blank, ref-
erence controls #1, #2, #3, #4, #5, #6, and un-
known, are prepared.
6. To each tube, 1 ml of the diluted mixture of
antiserum and reagent prepared in step 3 is
added.
7. To the appropriate tube 25~1 of reference and
unknown dilutions are added for IgG, IgA, and
transferrin (100 ~1 for Ig~ and C3).
8. The appropriate blank tube correspondingly re-
ceived 25 or 100 ~1 of saline.
9. The tubes are mixed by inversion and incubated
for 1 hour at room temperature (20-25C).
10. Sample blanks are prepared by using 1 ml of the
.~ filtered reagents from Examples 1-4, (as the case
may be) prepared as in step 3 except that the 8% so-
' lution was diluted with saline instead of antiserum.
The blanks are placed in identical labeled tubes as
before steps (5 and 6).
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... . . .
11. The same reference and sample volumes are
added to each tube as before (step 7)
12. All tubes are read in the Laser Nephelometer
PDQTM (Hyland Laboratories) for relative
percent light scatter.
13. The blanks are read and subtracted electronically
from the reaction values by the instrument.
14. The reference results are plotted on linear
graph paper as relative percent light scatter
versus concentration of references.
15. The unknown values are determined by reading
from the reference curve.
The immunological reagents of the invention used in this sample
produced substantially greater precipitation of the antigen-
; 15 antibody complexes, more linearity over a wider range, and
greater sensitivity than was obtained from a reagent containing
- polyethylene glycol alone.
EXAMPLE 6
I This example describes the use of the immonological
reagent of the invention in a radioimmunoassay procedure for
the determination of human thyroid stimulating hormone (HTSH).
A sample of the immunological reagent of the inven-
tion was prepared by mixing 8.4 ml of a 5% solution of polyethy-
lene glycol in 0.9 saline solution with 20 ml of 6% Pluronic F-38
nonionic surfactant. The volume of the mixture was then adjusted
to 30 ml with 0.9 saline solution to give a final reagent con-
centration of 1.4% polyethylene glycol and 4.2% F-38.
The radioimmunoassay was performed as follows:
0.050 ml of Rabbit anti-HTSH absorbed with human chorionic
gonadotropin (HCG) was mixed with 0.200 ml of HTSH standards
.
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. .
of varying strength. 0.050 ml of a phosphare buffer, pH 7.4,
was then added to the mixture and the mixture incubated for
2 hours at 37C. At this point, 0.100 ml of HTSH tagged with
I125 was added and the mixture further incubated for 3 hours
at 37C. 1.0 ml of the reagent of the invention prepared above
was then added and the mixtu~e incubated for one hour at room
temperature. Due to dilution of the reagent upon addition to
the mixture, the concentration of polyethylene glycol and F-38
was reduced from 1.4 and 4.2% to 1 and 3% by weight respectively.
The mixture was centrifuged at 1000 x 9 for 10 minutes. If a
wash of the centrifuged solids is required, the wash solution
must contain the same concentration as the reagent of the inven-
tion at the time the reagent was first used in the assay, i.e.,
1% polyethylene glycol and 3% F-38. The supernatant liquid was
then decanted and the precipitate counted. This procedure was
repeated for a variety of differing HTSH standards and a stan-
dard curve was obtained by plotting the counts in the various
precipitates versus the concentration of corresponding HTSH
standard.
~nce the standard curve had been obtained, the assay
was then performed on unknown test samples, using the procedure
described above, except that the HTSH standard was replaced by
the test sample. The HTSH level in the test sample could then
be readily determined from the location of the precipitate co~nt
` 25 on the standard curve.
; The use of the reagent sol-ution of the invention en-
hanced the extent of precipitate which was formed beyond that
obtainable from a reagent using polyethylene glycol alone, and
resulted in an improved radioimmunoassay.
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- - . . . - - . ~
Suitable nephelometric assay results can also be
achieved without the presence of the polyethylene glycol, for
example, with an aqeous solution containing about 4% by weight
of the block copolymer of ethylene oxide and polyoxypropylene
polymer. However, the mixture of block copolymer and polyethylene
glycol described above is preferred for optimum results.
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. ~
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