Note: Descriptions are shown in the official language in which they were submitted.
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IMMUNOASSAY FOR ANTIGENS
This invention relates to a method and an
article for the competitive assay of antigens and antigen-
like substances. More particularly, the invention relates
to a gravimetric immunoassay utilizing an antigen-
precoated piezoelectric oscillator.
Generally, the term "antigen" is applied to any
foreign substance which, when present in a host animal,
will stimulate the production of specific antibodies to
the substance. Antibodies show a remarkable ability to
bind selectively the antigen which stimulated their
production. This ability of antibodies to discriminate
between antigens which stimulated their production and
the myriad of other compounds of similar structure that
are found in biological fluids, e.g. serum or urine, is
the basis for virtually all immunoassay techniques.
A number of competitive binding assays are known
in the art for the qualitative and quantitative determin-
ation of antigens and antibodies. These assays are based
on the principle that when a binding element X (antigen
or antibody) is mixed with a substance Y ~or which it has
specific binding sites (corresponding antibody or antigen~,
a complex XY will be ~ormed. Similarly, a form of the
substance Y', which has been labeled in some manner to
distinguish it from Y without disturbing its binding
affinity for X, will form a complex XY'. If the con-
centrat~ons of Y and Y' exceed the number of binding sites
available on X, then they will compete with each other for
binding sites in proport;on to their concentrations. I F
the amounts of X and Y' are kept constant, then as
non-labeled Y is added, it will compete more effectively
with Y', and the amount of XY' complex will fall. Thus,
competi-tive binding assays for antigens require an
antibody (having specific binding sites for the antigen
being assayed) and a labeled form oF the antigen.
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Prior art competitive binding assays for
antigens generally use one of several different types of
labeling systems to distinguish between the two competing
~orms of the antigen being measured. These systems
include radioactive isotope labeling, enzyme labeling,
spin labeling, and ~luorescent labeling.
Assays using radioactive isotope labeling are
the most generally used. In these assays the labeled
antigen is distinguished from the unlabeled antigen by
the presence of a radioactive isotope in the ~ormer. The
amoun~ of labeled antigen bound to the antibody or the
amount Free in solution can readily be determined by
radioactivity measurements. Radioimmuno assays are
described in U.S. Patents 3,555,143 and 3,646~346. The
primary disadvantages of assay techniques using radio-
active isotope labeling include radioactivity hazards,
expensive instrumentation, the necessity to modi~y
chemically the antigen to label it, constant restandard-
ization due to short-lived isotopes and tedious separation
techniques.
Competitive binding assays utilizing enzyme
labeling are described in U.S. Patents 3,654,090,
3,791,932 and 3,850,752. For example, in U.S. 3,850,752,
the labeled antigen is distinguished ~rom unlabeled
antigen by being covalently linked to an enzyme molecule.
The enzyme-antigen conjugate must retain at l~east part of
both its imrnunochemical and enzymatic actlvity. The binding
antibody is perferably insolubilized either by crosslinking
or by covalent linking to an insoluble carrier. When a
given amount of insolubilized antibody is added to a
liquid sample contain3ng a known amount of antigen and an
unknown amount of unlabeled antigen, the two forms of the
antigen compete ~or the binding sites on the antibody.
The concentration of unknown antigen can be measured by
determining the enzymatic activity of either the liquid
or solid phase.
346i~
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Disadvantages associated with competitive binding
assays utili~ing enzyme labeling include the necessity for
chemical modi~ication1o~ the antigen, highly specialized
antisera, very precise timing during assay and reagent
instability. Additionally, the assays are subject to
interference from the unknown sample.
U S. Patent 3,690,83~ describes a competitive
binding assay utilizing spin labeling. In this assay,
the labeled substance is distinguished from the unlabeled
substance by the presence o~ a stable free radical
functionality Disadvantages o~ the spin labeling
technique include the necessity of chemical modification
of the antigen, expensive and cumbersome spin label
detection systems and relati~ely low sensitivity.
In the prior art competitive assay discussed
above, the success of the technique generally depends upon
a good separation of unbound antigen ~both labeled and
unlabeled) from the àntigen-ant~body reaction products.
Separation has been facilitated by attaching the antibody
to a solid carrier such as particles of a water insoluble
carrier (U.S. Patent 3,555,l43) or the interior surface
of a plastic test tube tU.S. Patent 3,646,346). Although
these techniques offer advantages in terms of ease of
separation, they suf~er certain disadvantages such as
loss of precision and reproducabilîty.
The present invention overcomes many o~ the
disadvantages associated with prior art competitive
assays for antigens. The method of the present invention
can be carried out without the necessity of labeling the
antigen by modify~ng it chemically or isotopically.
Furthermore, the antibody is added to the reaction mixture
in its natural state. There is no necessity to attach
the antibody to a solid carrier to facilitate separation.
Piezoelectric oscillators have previously been
used for the direct m~asurement o~ antlbodies. In J.
Biomed. Mater. Res., Vol. 6, pp. 565-576 (1972) Shons et al
describe coating a piezoelectric oscillator with speci~ic
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proteins. The protein-coated oscillator is then placed
in a sample containing an unknown quantity of the
corresponding anti~ody to the protein. As the antibody
binds to the oscilla~or, there is a downward shift in the
frequency of the oscillator. The concentration of anti-
body in the sample can be calculated by reference to a
standard curve. This method di~fers From the method of
the present invention in several significant respects.
Firstly, the prior art method measures the concentration
of antibody in an unknown sample. No operative method is
disclosed for measuring the concentration of the same
antigen as is bound to the surface o~ the oscillator.
The method described in the article is not a competitive
binding assay, nor can it be utilized to measure both
large and small molecules~ Furthermore, the unique
combination of a measuring device (or label) and
separation means in a competitive assay system is not
described, Further differences between the present
invention and the prior art will be apparent from the
description of the invention hereinbelow.
According to the present invention there is
provided a method for the immunochemical determination of
antigen in an unknown liquid sample, The method com-
prises the steps of: (1) contac~ing the liqu`id sample
with a predetermined amount of an antibody specific for
the antigen being determined and a compos;te comprising
a piezoelectric oscillator h~ving immobilized thereon, in
biologically active configuration, an antigen or mixture
of proteins containing the antigen to be determined, and
the composite having a premeasured frequency; (2) allo~ing
the immunochemical reaction to proceed; (3) separating
the oscillator ~rom the liquid sample; and 5~) determining
the changè in frequency of the oscillator. This method
may be used to determine the presence of a particular
antigen in a liquid sample, and by reference to a standard
curve, the change in frequency of the oscillator can be
used to determine the amount of the antigen in the sample.
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The method can be carried out simply and
efficiently with relatively unsophisticated, rugged and
inexpensive instrumentation. Since the competing antigen
is immobilized on the oscillator, there is no necessity
to modify it chemically to distinguish it from the antigen
to be determined. Furthermore, the competing antigen is
in the solid phase, thereby facilitating separation of the
antigen-antibody reaction product ~rom the 1iquid specim~n.
Accordingly9 where prior art techniques require both
lQ labeling the antigen and immobilization of the antibody9
the present invention achieves the combined result by
simply immobilizing the antigen on a piezoelectric
oscillator.
The disadvantages associated with labeling
techniques used in conventional competitive binding assays
have been overcome. Unlike the most commonly used label,
i.e.~ radioisotopes, there is no hazardous radioactivity
or decay of the label with time. There are no unstable
and strongly temperature, pH and time dependent reagents
as in the c~se of assay techniques using enzyme labeling.
~or does the method of the present invention require the
exacting chemical syntheses needed to prepare spin labels.
The method of the present invention can be used
in the qualitative and quantitative analysis of a variety
of antigenic materials ranging from low molecular weight
compounds to large macromolecules which meet the require-
ments of competitive assays and which can be immobilized
on the oscillator. Antigens at a concentration as low as
10 11 moles/liter can be measured using the method of the
present invention. For many significant small molecules,
e.g., medicaments, ~he assay has sensitivities over-
lapping the range of radioimmune assays, i.e., nanograms/ml.
Understanding of the invention will be ~acilitated
by reference to the accompanying drawings wherein:
Figure 1 is an enlarged elevational view of a
piezoelectric quartz crystal oscillator partially cut away
to illustrate coatings thereon; and
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Figure 2 is a graph illustrating a standard
curve obtained by measuring the frequency changes of an
antigen-coated oscillator in response to increased levels
of antigen (IgM).
Piezoelectric oscillators as shown in Figure 1
are commonly used in electronic equipment as frequency
standards. The term "oscillator" as used herein refers to
the piezoelectric material itself which is capable of
oscillat;ng when placed in an appropriately designed
electronic circui~. The oscillator 10 consists of a small
quartz wafer 12 (in this case, 14 mm in diameter and 0.2 mm
thick) having deposited thereon two metal electrodes 14.
(The metal electrode on the opposite side of the quartz
wafer 12 is not shown). Examples of metals which may be
used include nickel, gold, chromium, tantalum and pre
ferably silver. The quartz wafer 12 with electrodes 14 is
supported on pins 16 on a base plug 18. ~Ihen placed in an
electronic oscillator circuit, the portion of quartz wafer
12 between electrodes 14 vibrates with its precise natural
frequency. Preferred resonators are 5 to 10 MHz AT-cut
quartz crystals, although 1-4 MHz and 11-50 MHz crystals of
similar cut may be used. A given mass mechanically coupled
to one or both of the electrodes of the oscillator causes
a downward shift in the fundamen~al or resonant frequency.
Quartz crystals having a plurality of electrode pairs
deposited thereon may also be used, with the portion of
the crystal between each pair having a characteristic
frequency. Each electrode, when coated with the same
mixture of proteins, may be contacted with a different
monospecific antibody, and in this way, multiple assays
may be carried out using the same quartz crystal.
When used according to the present invention,
the oscillator, including the electrode portion, is coated
with a specific antigen layer 20. Antigen layer 20 may
be immobilized directly on the electrode or a primer coat
22 may be applied to the electrode to facilitate
immobilization of the antigen.
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To practice the method of the present invention,
an antigen-coated oscillator such as that illustrated in
Figure 1 having a premeasured fre4uency is contacted with
a liquid sample, ~ypically a biological fluid, e.g. serum
or urine, containing a known concentration of antibody
specific for the antigen and an known concentration of
unbound antigen. Contacting the oscillator with the sample
may be done by immersing the oscillator in the sample or
by simply applying a drop of the mixture of antibody and
sample to the surface of the electrode. Following an
incubation period, generally between five minutes and two
hours, and preferably between 30 and 75 minutes, the
oscillator is rinsed, and its frequency is measured and
recorded. By repeating this procedure, each time varying
the concentration o~ free an~igen in ~he sample, a standard
curve can be obtained showing change in frequency as a
function of free antigen concentration. A sample curve
is shown in Figure 2 for the antigen IgM. (The details of
the experiment are set forth in Example 5 below). By
reference to the standard curve prepared using a
particular antibody the concentration of a particular
antigen in an unknown sample can be readily determined.
If only one side of the oscillator is used in an
assay, the re~erse side can be used subsequently to assay
for the same antigen or other antigens present in the antigen
coat on the oscillator.
The antigen may be attached to the osci11ator by
a number of conventional techniques known in the art for
attaching proteins to solid supports. The antigen may
simply be allowed to adsorb from an aqueous solution onto
the surface of the oscillator. This method is least pre-
ferred because it results in a relatively high degree of
nonspecific adsorption during the assay, and sensitivity
is reduced,
Similarly antigens may be deposited on hydro-
phobic polymer-coated oscillators (such as polystyrene or
fluorinated polymers) in which case attachment occurs by
~L63~L60
dispersion force interaction, which often leads -to non-specific adsorption ~Jhen
the surface is exposed to additional proteins, for example, during the assay.
Another technique for depositing the antigen on the oscillator is by
crosslinking the protein antigen with a conventional agent such as ylutar-
aldehyde.
As especially preferred class of immobilizing agent is described in
United States Patent No. 4,242,096, issued December 30, 1980 and assigned to
the assignee of the present application. A monolayer of these polymers applied
to the surface of the oscillator promotes the deposition of a uniEorm layer of
antigen on the oscillator. Non-specific adsorp-tion during the assay is mini-
mized and a high degree of sensitivity is achieved.
The antigen is immobilized or adsorbed on the oscillator (treated or
untreated) by immersing the oscillator in an aqueous solution of the antigen.
A single antigen may be present in the solution, or the antigen may consist of
a complex mixture of molecules. Preferred antigens include tissue-associated
proteins, plasma proteins, drugs, vitamins, antibiotics, polysaccharides and
nucleic acids. Optimum concentration for attachment (or adsorption) varies from
antigen to antigen according to their solubility or molecular weight, however,
concentrations in the range of 0.5 to 100 milligram per milliliter are generally
preferred. Attachment is accomplished preferably at room temperature and at a
pH so as to maintain activity of the antigen. Optimum time for the attachment
oE antigens to the oscillator varies with the molecular weight and polarity of
the antigen. For example, serum albumin (MW = 60,000; isoelectric point 4.5)
requires as little as five minutes, whereas, IgM (MW = 750,000 - l,000,000;
isoelectric point c.a. 7.5) requires approximately 24 hours. Additionally,
mixtures of proteins may be adsorbed, for example the Cohn fraction II gamma
globulins or the ~-globulin fraction of serum, in which case several different
types of proteins are attached
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simultaneously. Since speci~icity in the assay is achieved
by using nonspecific antibody, oscillators so prepared can
be used to independently assay for several different types
of proteins9 according to the composition of the attached
protein layer. Before removing the oscillator from the
antigen solution, a water wash is directed into the
solution and the oscillator is thoroughly washed without
contacting the air. This wash procedure prevents contact
of the oscillator with denatured protein at an air-protein
solution in~erface. The oscilla-~or is removed and allowed
to dry. The frequency o~ the coated oscillator is then
determined. The oscillator is ready to be used in an
immunochemical assay.
The antigen-coated oscillator is stable and may
be stored for extended periods of time (e.g. several months)
without loss of immunological activity.
A convenient means for carrying out the method o~
the invention is to provide a immunological diagnositc
ki-t comprising a piezoelectric oscilla-tor precoa-ted with
at least one or a combination of antigens to be determined
and having a premeasured frequency. In conjunction with
the antigen-coated oscillator there is provided a pre-
determined amoùnt of an antibody specific for the antigen.
To carry out the method, the antibody is added to the
unknown liquid sample, e.g~ blood or urine, and the sample
is then contacted with the oscillator. The change in
frequency of the oscillator is measured. By reference to
a standard curve~ the presence of the antigen in the sample
and the concentration 'hereof can be determined.
The method of the present invention may be used to
assay for virtually all antigenic materia'ls of clinical
interest including natura'lly occurring and synthetic drugs,
drugs of abuse, hormones, vitamins, antibiotics, and the
like. Other materials which may be used as antigen coats
include tissue-associated proteins such as myoglobin, and
-microglobin; plasma proteins such as albumin, gamma-
globulins (e.g., IgG, IgA, IgM, IgD and IgE), haptoglobin,
;391~
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complement factors, fibrinogen, a1-antitrypsin, and high
and low density lipopr.~teins; organ specific enzymes such
as amylase, lactic dehydrogenase and creatinine
phosphokinase Especially preferred antigens include Cohn
5 II gamma globulins, ~ globulins, serum albumin, thyroxin
and amylase.
The invention may be further illustrated by
reference to the following examples:
. EXAMPLE 1
ïh~ Preparation of Piezoelectric Oscillators
and the Competitive Assay Method
for Human Gamma Globulin tIgG)
Preparation of the Oscillators: 10 MHz
(fundamental) AT cut quartz crystal oscillatorsl (0.3 cm
diameter) mounted in MIL type HC-6/U holders were surface-
treated by soaking overnight in a 0.06% aqueous solution of
poly~2-hydroxy-3-diamethylamino-1,4-butane ) with
stirring at room temperature. The oscilla-tors were
washed of excess polymer by flooding with a copious quantity
of deionized water and then dried in a stream of nitrogen.
After equilibration at room temperature (50% relative
humidity), the base frequency of the oscillators was
determined using a Hewlett-Packard model 5300A digital
frequency meter attached to the output of an International
Crystal OT-13 oscillator circuit. The oscillators thus
prepared had a uniform coating of polymer correponding to
a mean change in frequency (~F) of 285 Hz per oscillator.
The antigen employed was human gamma globulin
(IgG from Cohn Fraction II3). A 10 mg/ml solution of the
antigen in 0.02 M phosphate buffered saline (PBS, pH 7.0)
was prepared and the solution allowed to stand overnight
at 4C. The clear supernatant was decanted and
immediately filtered through Whatman No. 1 filter paper.
Northern Engineering Laboratories7 NE-6.
Prepared according to U.S. Patent 3,740,414
Sigma Chemical Company, St. Louis7 Missouri
3~
1,
The polymer-treated osc;llators were incubated in the
Cohn II gamma globulin solution at room temperature, with
stirring for 4 hours. The solution was then flooded with
PBS, followed by sufficient deionized water to ensure that
no adsorbable substances remained in the oscillator-treat-
ment bath. After drying in a nitrogen stream and
equilibration at 50% relative humidity, the rPsultant
frequency of each oscillator was determined. The com-
posite Cohn IT gamma globulin oscillators had a very
uniform amount of protein attached thereto corresponding,
in this case, to a ~F of 580 ~ 63 Hz per oscillator. This
frequence change is in addition to that observed with the
polymer coating polymer.
Assay Procédure: The procedure is illustrated by
the preparation of a standard curve relating frequency shift
to IgG concentration. Since each determination point of
the standard curve is essentially an unknown before the
assay is performed, the determination of a standard curve
can be taken as illustrative of the analysis of unknowns.
An assay mixture was prepared by combining
sequentially an aliquot of a commercially prepared human
serum standard (Q.I.C ~ 4~5 containing the antigen which
had been diluted 1:10 with phosphate buffered saline,
0.250 ml antihuman IgG (5.0 mg antibody/ml) and sufficient
phosphate buffered saline to provide a final volume of
20.0 ml. Three oscillators (prepared as above) were
immersed in this assay mixture and incubated 75 minutes
at room temperature with stirring. After incubation, the
oscillators were recovered by flood rinsing sequentially
with phosphate buffered saline and suFficient deionized
water to remove all adsorbable species. After drying in
a stream of nitrogen and equilibration at 50% R.H. the
final frequency of the oscillators was determined.
-
Quantitative Immunodiffusion Control trademark of Meloy
Laboratories, Inc.
5For unknown serum samples, 20 microliters of sera would
be used.
~6~
The loss in frequency for the Cohn II gamma
globulin oscilla-tor is directly related to the extent of
antibody reaction with the oscillator, and is a measure
of excess'or unreacted antibody in the assaY. Therefore~
the frequency shift is inversel,y related to IgG con-
centration in the samples.
The table below summarizes the results obtained
using a commercially prepared serum standard, (Q.I.C ~)
diluted 1:10 with PBS~ Mean IgG concentration for the
undiluted sample was 8.07 mg/ml.
~ IgG Concentration
QF (Hz, Average) Q.I.C~Aliquot In Assay Mixture Moles/Liter
511 0.050 ml 2 ~g/ml 1.3x10-8
561 0.100 ml 4 ~g/ml 2.6x10-8
495 0,200 ml 8 ~g/ml 5.3xlO 8
370 0,250 ml 10 ~g/ml 6.6xlO 8
165 0.300 ml 12 ~g/ml 8.0x10-8
100 0.400 ml 16 ~g/ml 10.6xlO-8
EXAMPLE 2
Preparation of Oscillators and the
Competitive Assay Technique for Thyroxine
~Small Molecule Assavs)
~ .
Preparation of the Oscillators: 10 MHz AT cut
quartz crystal oscillators were surface treated as
described in Example 1. The antigen employed was L-
thyroxine6 (sodium salt). A 100 ~g/ml dispersion of this
material in 0.02M PBS was prepared and incubated by
stirring overnight at room temperature with polymer-treated
oscillators. Following the procedure of Example 1, a
composite L-thyroxine~oscillator was obtained having a
very uniform amount of L-thyroxine, corresponding to a
~F of 476 ~ 42 Hz per oscillator.
Assay ProcPdure: The procedure is illustrated
by the preparation of a standard curve relating frequency
6Sigma Chemical Company
- 13 _
shift to L-thyroxine concentration.
The assay mixture was prepared by mixing
sequen~ially an aliquot o~ an L-thyroxine standard, 0.100
ml thyroxine standard, 0.100 ml thyroxine antisera
(y-fraction, 10 mg protein/ml) and sufficient phosphate
buffered saline (PBS) to provide a final volume of 20.0 ml.
The mixture was stirred and incubated 60 minutes at room
temperature with two L-thyroxine coated oscillators. After
incubation, the oscillators were recovered by flood rinsing
consecutively with PBS and sufficient deionized water to
remove all adsorbable species. After drying in a stream
of nitrogen and equili'bration at 50% relative humidity the
final frequency of each oscillator was determined.
The table below summar;zes the results obtained
using aliquots o~ a thyroxine standard (1.0 mg/ml in
0.02M PBS, pH 7.0).
Thyroxine Conc.
~F (Average) Thyroxine Aliquot in Assay Mixture
165 Hz 0.010 ml 0.5 ng/ml
137 Hz 0.050 ml 2.5 ng/ml
70 Hz 0.100 ml 10.0 ng/ml
EXAMPLE 3
Preparation of Oscillators and the Competitive
Assay for Human Serum Albumin Us;ng
5MHz Oscillators and a Small Reaction Volume
5M~Z AT cut guartz crystal oscillators were
surface treated as described in Example 1. Human serum
albumin ~HSA) was attached to the oscillators by incubating
the polymer-treated oscillators at room tempera~ure with
HSA solution (7.5 mg/ml, PBS pH 7.0) for 5 minutes. After
washing, composite oscillators were obtained having a
uniform coating nf human serum album;n corresponding to a
mean ~F of 72 + 15Hz per oscillator.
Assay Procedure: One milliliter of a solution
containing 110 ~g/ml antlhuman serum albumin in PBS pH
7.0 was rapidly combined with 1.0 ml of a commercially
prepared human serum standard (Q.I.C ~, 40 mg HSA/ml)
Meloy Laboratories, Inc.
3~
diluted with PBS to provide HSA concentrations in the
range, 0.5-20 ~g/ml. Immediately after mixing~ 0.5 ml
aliquots of this solution were added to individual
oscillators and the mixture (oscillators and solution)
allowed to incubate at room temperature without stirring
for 75 minutes. Each HSA concentration was assayed in
triplicate. After incubation the oscillators were
recovered by flood rinsing consecutively with pH 7.0 PBS
and sufficient deionized water to remove all adsorbable
species. After drying in a stream of nitrogen and
equilibration at 50% relative humidity the final fre-
quency of the oscillators was determined.
The table below summarizes the results obtained
using a dilute Q.I.C.~ prepared as indicated above.
HSA Concentration
~F, AverageIn Assay Mixture
103 Hz 0 ~g/ml
88.3 Hz 0.5 ~g/ml
69.6 Hz 4.0 ~g/ml
48,~ Hz 20.0 ~g/ml
EXAMPLE 4
Preparation of Oscillators and a Competitive
Assav Usina GlutaraldehYde Immobilized Protein
Pre~aration of Oscillators: A 500 ~1 aliquot
of human y-globulins, Cohn fraction II tl.O mg/ml in
O.lM acetate, pH 5.0) was combined with 25 ~1 of 1%
glutaraldehyde (in O.lM acetate, pH 5.0) and the
mixture very thinly painted on the electrode portion of
10 MHz AT-cut quartz crystal oscillators. The coated
oscillators were kept in a humid atmosphere (water bath)
at room temperature for 45 minutes. Excess salt and
ungelled protein were removed by soaking 30 minutes in
four consecutive changes of distilled water, and the
oscillators dried in a stream of nitrogen. After a 15
minute equilibration at ambient temperature and humid~ty,
the resultant frequency of each oscillator was determined.
A coating of glu~araldehyde crosslinked y-globulins
corresponding to mean ~F of 1320 Hz per oscillator was found.
~63~a61;1
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Assay Procedure: A standard curve relating
frequency shift to human IgG concentration was obtained
following the procedure described in Example 1. Sample
volumes utilized were 10 ml for each assay and contained
53.35 ~g antihuman IgG/ml. Each point was run in
duplicate. The results for a series of aliquots of
dilute Q.I.C ~ 10 with PBS) are given below:
~ IgG Concentration
Q.I.C~Aliquot In Assay Mixture ~F (Hz)
0.050 ml 4 ~g/ml 187.5
0.100 ml 8 ~g/ml 137
0.200 ml 16 ~g/ml ~ 80.5
0.300 ml 24 ~g/ml 74.5
EXAMPLE 5
Assay for the Protein IgM Using One
Face of the Oscillator at a Time
_
Piezoelectric oscillators were prepared as
described in Example 1 except that incubation time was
extended to 24 hours. After washing, a composite
oscillator was obtained having a uni~orm coating of human
Cohn fraction II ~-globulins corresponding to a mean ~F of
1367 Hz per oscillator.
Assay Procedure: The antigen-coated oscillators
were mounted horizontally and placed in a petri dish con-
tainins a dampened f'ilter paper in order to maintain a highrelative humidity during the incubation period, An assay
mixture was then prepared by combining, consecutively,
25 ~1 antihuman IgM~ 1.0 ml PBS and a 10 to 50 ~1 a~iquot '
of undiluted Q.I,C.~, A 50-60 ~1 drop'le~t''of ~his mixture
was immediately ap~lied to one face of the oscillator in
such a manner as to expose the entire electrode surface to
the solution. The petri dish was then covered and the
oscillator incubated for 75 minutes at room temperature.
The droplet was then washed away wtth a stream of dis-
tilled water (about 100 ml). After drying in a stream ofnitrogen and equilibrating at 50% relative humidity, the
final frequency of the oscillator was detPrmined, T~e
results for a series of aliquots of undiluted Q~I,C. are
as follows:
~31 6c.~4f~(1
,
16
IgM Concentration
Q.I.C.~AliquotIn Assay Mixture ~F
0.0 ml O ~g/ml 134 Hz
0.010 ml8.8 ~g/ml 113 Hz
0.0~5 ml22.0 ~g/ml 73 Hz
0.050 ml44.0 ~g/ml 45 Hz
A standard curve plotted from this data is shown in
Figure 2,
EXAMPLE 6
Assay for a Protein (~-antitrypsin) in the
-~lobulin Fraction of Human Sera
Oscillators were prepared as described in
Example 1 except ~hat human ~-globulin fraction IV was
used. A mean ~F of 320 Hz per oscillator was found.
As~ Procedure: The antigen-coated oscillators
were mounted horizontally and placed in a petri dish
containing a dampened filter paper to maintain a high
relati~e humidity during the incubation period. An
assay mixture was prepared by combining 100~ antisera
to human ~Tantitrypsin (0.8 mg Abiml), 0.5 ml PBS and a
10-200~ aliquot o~ undiluted Q.I.C. . The mixture for
each aliquot was vortexed and allowed to stand at room
temperature for 30 minutes, A 50~ droplet of each sample
was then applied to one Face o~ a previously prepare~d
~-globulin oscillator in such a manner as to expose the
entire electrode sur~ace to the solution. The petri dish
was then covered and the oscillator incubated for 30
minutes at room tempeiature. The droplet was then washed
away with a stream of distilled water and dried with
nitrogen, Af~er equilibrating at ambient humidity, the
final frequency of the oscillator was determined. The
results for a series of aliquots of undiluted Q.I.C,~ are
given ~n the following table, All aliquo~s were run in
duplicate.
46~11
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17 -
a,Tanti trypsin
Co n c e nt ra t i o n ( m g /m l )
Q . I . C .(~Al i quot In Assay Mi xture ~F ( Hz )
10~ 30 106
25~ 73 g3
50~ 1 46 75
95~ 250 46
200~ 457 25
