Note: Descriptions are shown in the official language in which they were submitted.
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TITL~ OF T~oe INYENTIONo
MULTIPh~_~NTIGEN IMMUNOASSAY
Field of the In~ention
The invention relates to a method for the assay of an
analyte which may be present in a sample by r~acting a sample
suspected o~ containing said analyte with antibodies immobi-
lized to a solid support to form an antibody-antigen complex,
followed by titrating the unoccupied antibody with a second
labeled antibody which is specific to the first immobilized
antibody, ~ollowed by detecting the label.
BAC~GROUND OF THE INVENTION
The detection and quantitation o~ antigenic substances
in biological samples ~requently utilize immunoassay kechni-
ques. These technigues are:based upon the formation of a
¢omplex between the antigenic substance being assayed and an
antibody or antibodies in~which one or the other member of
the ~omplex may be detectably labeled. ~ith competitiYe
immunoassay ~echniques, ~he antigenic substance in a sample
fluid being tested competes with a known quantity o~ labeled
antigen ~or a limited quantity of antibody binding si~es.
The amount of labeled antigen bound to the antibody is
inversely proportional to the amount of antigen in a
sample.
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By contrast, most immunometric assays employ a labeled
antibody. In such an assay, the amount of labeled antibody
associated with the complex is directly proportional to the
amount of antigenic substance in a fluid sample.
In sandwich immunome~ric assays, a ~uantity o~ unlabeled
antibody is bound to a solid support which is insoluble in
the fluid being tested. This immobilized antibody is first
contacted with the sample being tested so that a binary
antigen-antibody complex is ~ormed. After a suitable
incubation period, the solid support is washed to remove
unbound antigens, then contacted with a solution containing a
known quantity of a second antibody. After a second incuba-
tion period, the solid support is then washed a second time
to remove the unreacted antibody. A labeled anti-antibody to
the second antibody is then added, allowed ~o incubate for a
sufficient amount of time, and the complex then ~ashed. The
washed solid support is then tested to detect and quantify
the presence of labeled antibody, for example by measuring
the emitted radiation o~ a radioactive label. The amount of
labeled antibody detected is compared to that for a negative
control sample. This type of assay is frequently referred to
as a two-site or sandwich assay, since the antigen has two
antibodies bonded to its surface at different locations.
Despite their great utility, sandwich immunoassay has been
recognized to be a slow procedure, in part because washing
steps are required and lengthy incubation periods are
required to reach equilibrium. David, et al., U.S. Patent
No. 4,376,110~
To eliminate at least one of the washing steps associa-
ted with this procedure, so-called simultaneous and reverse
assays have been developed. A simultaneous assay inv41ves a
single incubation step as the antibody bound to the solid
support and the labeled antibody are both added to the sample
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being tested at the same time. Af~er incubation, the solid
support is washed to remove unbound analyte and unbound
antibody, and the bound antibody-analyte-labeled antibody
"sandwich" is detected as with a conventional "forward~'
sandwich assay. A reverse assay involves the stepwise
addition ~irst of a solu~ion o labeled antibody to the fluid
sample followed by the addition of unlabeled antibody bound
to a solid support after a suitable incubation. After a
second incubation, the solid phase is washed in conv ntional
~ashion and the amount of labeled complex is detected as
before. United States Patent No. 4,098,876 to Piasio, et al.
However, all of these methods suffer from the requirement for
two antigen-specific antibodies which are able to recognize
separate and distinct epitopes on an antigen. This is a
critical limitation which makes the application of such
immunoassays impractical for small antigens.
Immunoassays which require only one antigen-specific
antibody are pre~erred. Such an immunoassay was described by
Hirano, K. et al., Anal. Biochem. 154:624-631 (1986) who
di~close an assay for tumor-specific alkaline phosphatase
using a nitxocellulose filter coated with monoclonal anti-
bodies specific for alkaline phosphatase. The presence of
the bound analyte was determined by taking advantage of the
enzymatic activity of the bound analyte. Thus, this type of
immunoassay can only be used for detecting analytes with
enzymatic activity which is stable to the conditions of the
assay protocol. In addition, such assays are not applicable
to detection of all enzymes. For example, enæymes such as
thiol protease require the addition of reducing agents to
stabilize them. Such reducing agents destroy antibodies by
cleavinq disulfide bonds.
A dot-blot assay for the detection and quantitation of
~he Leishmania glycoconjugate was developed which involves
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do~-blotting the solubilized protein from parasites onto
nitrocellulose, blocking the ~ree protein-binding si~es with
BLOTT0 (5% w/v skim-powdered milk) and detecting the presence
o~ the glycoconjugate with an iodinated monoclonal anti~ody.
Handmant E. ~ . Imm~nol. ~eth. 83:113-123 (1985). A
second two-site i~munoradiometric assay disclosed by Handman
for glycoconjugate involved i~mobiliza~ion of monoclonal
antibody on nitrocellulose, blocking remaining protein
binding sites with BLOTT0, binding with antigen, followed by
a second incubation wi~h ~he same monoclonal antibody, which
was radioiodinated. This assay was ba~ed on the fact that
Lli~ glycoconjugate possesses a large number of
epitopes recognized by the monoclorlal antibody. This
suggests ~hat IrL~h=~ai~ glycoconjugate contains a repetitive
polymeric structure. Handman, su~ra. Thus, this immunoassay
is limited to antigens capable of binding two or more
identical antibodies. In addition, assays which rely on
radiodinated monoclonal antibody are relatively expensive
because of the high costs of monoclonal antibodies and the
limited shelf lives of most labeled antibodies.
Anothe~ immunoassay which involves a single antihody
comprises immobilization o~ a monoclonal antibody on nitro-
cellulose, blocking the additional binding sites, and using a
labeled antigen to detect khe desired antibodies. Suresh,
.R. et al., Anal. Biochem. 151~19~-195 (1985~. This method
is used to screen hybridoma supernatants and to detect
monoclonal antibodies. ~owever, in many instances, the
antigen i~ not available in 6ufficient quantities to allow
labeling for use in such an assay. Fur~her, labeling the
antigen can re~ult in deleterious al~eration of the immuno-
~pecifici~y of the antigen.
~ nited States Patent No. ~,279,885 to Reese et al.,
describes a solid pha~e competitive pro~ein binding assay
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where an antigen or hapten can be assayed. The method
involves competition between the analyte and a labelPd form
thereof for a limited number of receptor or binding sites
which are i~mobilized to a solid support. The assay may be
conducted by mixing the componen~s simultaneously or
sequentially. The sequential assay involves contacting a
solu~ion of an analyte with a support containing immobilized
receptors or antibodies, ~ollowed by contacting the mixturP
with a tracer. The tracer may be the analyte, or analog
thereo~, which contains a label or tag. Competitive assays
are generally recognized to be less preferable to non-
competitive assays.
It is also pos~ible to have assays which do not utilize
antibodies at all. For example, a sample containing protein
to be assayed is mixed with a marker protein in contact with
a polystyrene latex. A competition is created between the
marker enzyme and the analyte protein for the limited surface
binding sites. The inactivation of the enzyme upon binding
to the hydrophobic latex surface allows m~asurement of the
bound/free enzyme ratio, and thus, the competing protein
concentration~ However, this method is not able to distin-
guish betwean different proteins, and only gives a measure of
the total protein content. Sandwick, et al., Aaal~ Biochem.
147:210-216 (1985).
Further improvements to immunoassay techniques involve
the use of amplification strategies to increase the detection
limit. These strategies include substrate cycling and enzyme
channeling. Mosbach, K., Ann. N. Y. Acad._Sçi. 434:239-248
(1984). However, neither system has been widely adopted.
Thus, it would be desirable to have an immunoassay whioh
is fast and reliable, and requires the preparation of only
one specific antibody. Further, it is de~irable to have an
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immunoassay for a multiplicity of analytes utilizing one
specific antibody for each a~alyte to effect detection.
Summary of the Invention
The invention relates to a method for detecting and
quantitating an analyte in a sample, which include
~ a) contac~ing a sample suspec~ed o~ containing the
analyte with a solid p~ase suppor~ onto which an analyte-
specific first antibody ha been immobilized;
(b) incubating said sample wi~h said support ~or a
sufficient amount of time ~o allow the analyte present in the
sample to bind to said first antibody;
(c) separating said solid suppor~ from the incubation
mixture obtained in step (b);
(d) contacting said solid phase support with a detect-
ably labeled titrating antibody which is specific for said
first antibody;
(e) incubating the mixture formed in step (d) for a
time sufficient to allow ~aid titrating antibody to bind to
said first antibody;
(f) separating said solid phase support from the
incubation mixture obtained in step (e); and
~ g) detecting the analyte in the sample by measuring
the amount of bound labeled antibody.
The invention relates as well to an assay for a multi-
plicity of analytes comprising contacting a ~ample suspected
of containing a multiplicity of analytes with a solid support
containing different analyte-specific antibodies separately
immobilized onto defined areas o~ the solid phase support,
followed by titration and detection with a common titrating
antibody.
The invention also relates to a kit for the detection of
an analyte in a sample comprising a carrier being compart
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mentalized to receiYe in close confinement therein one or
more containers wherein
(a~ a first oontainer contains a solid support
containing a first antibody immobilized to said solid support
wherein said ~irst antibody is speci~ic ~o an analyte;
(b) a second container contains washing buf~ers: and
(c) a third container contains a titrating antibody
specific for the ~irst immobilized antibody.
The invention also relates to a kit for the detection of
a multiplicity o~ analytes in a sample which includes a
carrier means being compar~mentalized to receive in close
confinement therein one or more containers wherein
(a) a first container contains a solid support contain-
ing a multiplicity of analyte-specific first antibodies
separately immobilized to separate de~ined areas of said
solid support:
(h) a second container contains washing buffers; and
(c) a third container contains a second titrating
antibody specific for each analyte-specific first antibody.
The invention offers a convenient, flexible and rapid
method to detect and quantify one or more analytes in
solution. In addition, the invention provides for r~cycling
the solid phase support by elution with a chaotropic salt.
Thus, th~ solid phase support may be reused and the antigen
recovered from the assay system.
DESCRIPTION O~l THE FIGURES
Fiqure 1. This ~igure show~ the general scheme of the
assay, the titration of immobilized IgG not bound to antigen
by iodinated IgG-specific antibody, and subsequent recycling
of ~he nitrocellulose disk.
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Figure 2. This figure is a plot oP the number of disks
per tube versus the amount of bound radioactivity.
Figure 3. This figure shows a comparison of the binding
of iodinated sheep anti-rabbit (SAR) antibodies onto differ-
ent types of immobilization matrices, with and without 20%
methanol.
Fi~ure 4. This figure ranks the relative ability of
different papers to absorb iodinated SAR with or without 20%
methanol.
Fi~ure 5. This figure shows the ef~ect of recycling
antibody coated disks on the amoun~ of residual radioactivity
associated with the absorbed antibody.
Figure 6. This figure shows a standard curve for the
titration of nitrocellulose-immobilized affinity purified SAR
IgG immobilized onto nitrocellulose.
Figure 7. This figure shows a determination of the
amount of IgG bound to nitrocellulose disks for a range of
different SAR dilutions.
Fiqure 8. ~his figure depicts a comparison of a 10-
minute incubation with an overniqht incubation for a series
of SAR dilutions.
Fiqure 9. This figure shows a standard curve for
various concentrations of NIRS using SAR-coated nitrocel-
lulose disks, obtained by ti~ration of unbound free antibody
sites with iodinated titrating antibody.
Fi~ure 10. This ~iqure compares the amount of bound
titrating antibody for two different incubation times and
varying concentrations of NIRS.
FiGurç 11. This figure shows the effect of amplifica-
tion of titratinq antibody f~r dif~erent concentrations of
SAR.
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DESCRIPTION~ OF_THE PREFERRED EMBODIMENTS
This invention is directed towards methods of assay by
immobilizing a first antibody specific to an analyte on a
solid phase support, contacting the sample suspected of
containing the analyte with the immobilized ankibody, and
titrating the unbound antibody with a second labeled antibody
which is specific for the first immobilized antibody.
By '~solid phase support" is intended any support capable
of binding antibodies. Such supports include but are not
limited to nitrocellulose, diazocellulose, microtiter plates,
glass, polystyrene, polyvinylchloride, polypropylene,
polyethylene, dextran, affini~y support gels such as
Sepharose or agar, starch, and nylon. Preferred supports are
nitrocellulose and diazocellulose. Those skilled in the art
will note that many other suitable carriers for binding
monoclonal antibody exist, or will be able to ascertain th~
same by use of routine experimentation.
The term "antibody" refers both to monoclonal antibodies
which have a substantially homogeneous population and to
polyclonal antibodies which have heterogeneous populations.
Both the first and second antibodies may be monoclonal or
polyclonal. Polyclonal antibodies are derived from the
antisera of animals immunized with the analyte. Monoclonal
antibodies to specific antigens may be obtained by methods
known to those skilled in the art~ See, for example Kohler
and Milstein, Nature 256:495-497 (1975). Such antibodies may
be of any immunoglobulin class including IgG, IgM, IgE, IgA,
IgD and any subclass thereof.
The term "antibody'~ i6 meant as wall to include both
intact molecules as well as fragments thereof, such as, for
example, Fab and F(ab')2, which are capable of binding
antigen.
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By the term "analyte" is intended any molecule with an
antigenic site capable of binding to an antibody. Such
analytes may include but are not limited to proteins, drugs,
viruses, cells, haptens, ~ubcellular particles, carbo-
hydrates, hormones, vitamins, metabolites and their binding
materials.
In one e~bodiment, the first analyte-specific antibody
is a polyclonal antibody derived from an animal immunized
with the analyte. The second titrating antibody may be
heterologous polyclonal antibodies which are specific against
the immunoglobulins comprising the firs~ an~ibody. The
second antibody may be obtained by isolating the antibodies
from a second animal species which has been immunized with
antisera from the same species of animal uséd to prepare the
first antibody.
In another embodiment, a first IgG monoclonal antibody
which is specific to the analyte to be assayed is used. The
second titrating antibody comprises a detectably labeled
anti-IgG polyclonal antibody specific to the first antibody.
In still another embodiment, a first IgG polyclonal
antibody, which is specific to the analyte to be assayed, is
used. The second titrating antibody comprises a detectably
labeled anti-IgG monoclonal antibody specific to the first
antibody.
In a preferred embodiment, a number of different
analyte-specific antibodies deriYed from the same animal
species may be separately immobilized on defined areas of a
solid support which is attached to a dip stick. Thus, the
dip stick may be incubated with a sample to assay for many
different analytes simultaneously. The unoccupied antibody
sites on each defined area can then be titrated with co~mon
titrating antibodies which are specific for each antigen-
specific antibody. For instance, each analyte-specific
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antibody may be of the IgG class. The common titrating
antibodies will then be anti-IgG antibodies. As used herein,
the term "common titrating antibodiesl' is used in the plu.al,
although it will be understood that only one class of
antibodies is intended. This asp~ct of the invention
provides for the simultaneous detection and quantitation of a
multiplicity of antigens by a universal labeling method.
The amount of bound analyte is determined indirectly by
measuring the amount of label associated with the second
antibody which binds to the unoccupied Pirst antibody. The
amount of analyte present in a sample is inversely propor-
tional to the amount o~ label present. There are many
different labels and methods of labeling known to those of
ordinary skill in the art. Examples of the types of labels
which can be used in the present invention include, but are
not limited to, enzvmes, radioisotopes, dyes, fluorescent
compounds, chemiluminescent compounds, bioluminescent
compounds and metal chelates. Those of ordinary skill in the
art will know of other suitable labels for binding to the
antibody, or will be able to ascertain the same by the use of
routine experimentation. ~urthermore, the binding of these
labels to the antibodies can be accomplished using standard
techniques commonly known to those of ordinary skill in the
art.
One of the ways in which the titrating antibody of the
present invention can be detectably labeled is by linking the
same to an enzyme. This enzymeO in turn, when later exposed
to its substrate, will react with the substrate in such a
manner as to produce a chemical moiety which can be detected
as, for example, by spectrophotometric, fluorometric or
visual means. Examples of enzymes which can be used to
detectably label the antibody of the present invention
include malate dehydrogenase, staphylococcal nuclease, delta-
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V-steroid isomerase, yeast alcohol dehydrogenase, alpha-
glycerophosphat2 dehydrogenase, triose phosphate isomerase,
horseradish peroxidase, alkaline phosphatase, asparaginase,
glucose oxidase, beta-galactosidase, ribonuclease, urease,
catalase, glucose-VI-phosphate dehydrogenase, glucoamylase
and acetylcholine esterase~ ~vidin-biotin binding may be
used to facilitate the enzyme labeling.
The itrating antibody of the present invention can also
be labeled with a radioactive i~otope which can then be
determined by such means as ~he l~se of a gamma counter or a
scintillation counter. Isotopes which are particularly
use~ul for the purpose of the present invention are: 3H,
125I 131I 32p, 35S, 14c, 51Cr, 36cl, 57Co, 5~Co, 59Fe and
75Se.
It is also po~sible to label the titrating antibody with
a fluorescent compound. When the fluorescently labeled
antibody is exposed to light of the proper wave length, its
presence can then be detected due to the fluorescence of the
dye. Among the most commonly used fluorescent labelling
compounds are fluorescein isothiocyanate, rhodamine,
phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde
and fluorescamine.
The titrating antibody of the invention can also be
detectably labeled using fluorescent emitting metals such a~
52Eu, or others of the lanthanide series. These metals can
be attached to the antibody molecule using such metal
chelating groups as diethylenetriaminepentaacetic acid (DTPA)
or ethylenediaminetetraacetic acid (EDTA).
The titrating antibody o~ the present inven~ion also can
be detectably labeled by coupling it to a chemiluminescent
compound. The presence of the chemiluminescent-tagged
titrating antibody is then determined by detecting the
pre~ence of luminescence that arises during the course o~ a
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chemical reaction. Examples o~ particularly useful chemi-
luminescent labeling compounds are luminol, isoluminol,
theromatic acridinium ester, imidazole, acridinium salt and
oxalate ester.
Likewise, a bioluminescent compound may be used to label
the titratiny antibody of the present in~ention. Biolumi-
nescence is a type of chemiluminescence ~ound in biological
systems in which a catalytic protein increases the Pfficiency
of the chemiluminescent reaction. The presence of a biolumi-
nescent antibody is determined by detecting the presence of
luminescence. Important bioluminescent compounds for
purposes of labeling are luciferin, luciferase and aequorin.
Another techni~ue which may also result in greater
sensitivity when used in conjunction with the present
invention consists of coupling the titrating antibody of the
present invention to low molecular weight haptens. The
haptens can then be specifically detected by means of a
second reaction. For example, it is common to use such
haptens as biotin (reacting with avidin) or dinitrophenyl,
pyridoxal and fluorescamine (reacting with specific anti-
hapten antibodies) in this manner.
In addition, the sensitivity of the assay may be
increased by use of amplification strategies including
substrate cycling and enzyme channeling as taught by Mosbach,
supra, incorporated by reference herein.
For the purposes o~ the present invention, the analyte
which is detected by this assay may be present in a sample
solution. Normally, the sample is a biological sample such
as, for example, saliva, cerebrospinal fluid, blood, serum,
urine, water, food and the like. However, the invention is
not limited to assays using only these samples, it being
possible for one of ordinary skill in the art to determine
suitable conditions which allow the use of other samples.
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For instance, various chemicals and drugs are also capable of
being antigens, and thus may ~e suitable assayed in water,
food, or othPr samples by the methods of this invention.
In carrying out the titrating immunoassay of the presPnt
invention on a sample containing a multiplicity of analytes,
the process comprises:
a~ contacting a sample suspected of containing a
~ultiplicity of analytes with a solid support on which
dif~erent analyte-specific first antibodie have been
separately immobilized to separately defined areas of said
solid support:
b) incubating said sample with said support for a
sufficient amount of time to allow the analytes present in
the sample to bind to said first antibodies;
c) separating the solid phase support from the
incubation mixture obtained in step b~:
d) contacting said solid support with a detectably
labeled titrating antibodies which are specific for said
first antibodies;
e) incubating the mixture formed in step d) for a
time sufficient to allow said titrating antibodies to bind to
said first antibodies:
f) separating said solid phase support from the
incubation mixture obtained in step e); and
g) detecting the analyte in the sample by measuring
the amount of bound labeled titrating antibodies to each
separately defined area.
Of course, the specific concentration~ of label and
analyte, the temperature and timP of incubation, as well as
other assay conditions may be varied, depending on various
factors including the concentration of antigen in the sample,
the n~ture of the sample, and the like. Those skilled in the
art will be able to determine operative and optlmal assay
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conditions for each determina~ion hy employing routine
experimentation. In addition, the eluted titrating antibody
may be separated from the eluted analyte accoding to means
~nown in the art and also recycled for reuse in the assay.
The eluted analyte may further be recovered.
Detection of the labeled antibody may be accompli~hed by
a scintillation count~r, Por example, if the label is a
radioactive gamma emitter, or by a fluorometer, for example,
if the label is a fluorescent ma~erial. In ~he case of an
enzyme label, the detection can be accomplished by colori-
metric methods which employ a substra~e for the enzyme.
Detection may also be accomplished by visual comparison of
the extent of enzymatic reaction of a substrate in comparison
with standards.
Other such stsps as washing, stirring, shaking, filter-
ing and the like may of course be added to the assays as is
customary or necessary for the particular situation.
The solid phase i~munosorbent may be recycled by elution
of the antigen and the titrating antibody with a chaotropic
salt such as MgC12. However, the invention is not limited to
the use of MgC12, it being possible for one of ordinary skill
in the art to determine other chaotropic salts which may be
used to recycle the solid phase immunosorbent, without undue
experimentation. In addition, the eluted titrating antibody
may be separated from the eluted analyte according to means
known in the art and may also then be recovered for re-use.
The eluted analyte may further be recovered.
The assay of the present invention is ideally suited for
the preparation of a kit. Such a kit may comprise a carrier
means being compartmentalized tc receive in close confinement
therewith one or more container mean~ such as vials, tubes
and the like, each of ~aid container means comprising the
separate elements of the immunoassay. ~or example, there may
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be a container means containing the first antibody immobil-
ized on a solid phase support, and further container means
containing detectably labeled titrating antibodiPs in
solution. Further container means may contain standard
solutions comprising serial dilutions o~ analytes to be
detected. The standard solutions of these an~lytes may be
used to prepare a standard curve with the concentration of
the analyte plotted on the abscissa and the detection signal
on the ordinate. The results obtained from a sample ¢ontain-
ing an analyte may be interpolated from such a plot to give
the concentration of the analyte.
In another e~bodiment of the ki~, there may be a
container means containing a dipstick which comprises a
multiplicity of different analyte-specific antibodies
separately immobilized to separate defined areas of the solid
phase support. Further container means may contain a common
titrating antibody which is specific for each analyte-
specific antibody. Further container means may contain
standard solutions of analytes to be detected. The standard
solutions of these analytes may be used to provide a standard
reference dipstick for comparison with the sample dipstick.
The various aspects of the invention are further
described in the following examples. These examples are not
intended to limit the invention in any manner.
EXAMPLES
EXAMPL~ 1
General Procedure For the ImmunoassaY
In the examples that follow, a simple antibody-antigen
system illustrates the protocol used for the assay for one
antigen by isotopic detection.
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Sheep anti-rabbit serum (SAR) was induced by sheep by
standard procedures and heat inactivated at 56C fsr 30
~inutes. Rabbit IgG was purchased from Sigma (St. Louis,
M0). Iodination was achieved using iodo-beads (Pierce
Chemical Company, Rockford, IL) using carrier-free ~odium
iodid~ 25 (Dupont N~w England Nuclear, 8Os~on, MA). All
other chemicals wer~ reagent grade and were purchased from
either 5igma Chemical Corporation (S~. Louis, M0~ or Fisher
(Medford, ~A).
Nitrocellulo~e paper (~illipore filter type HAHY 000-lO,
O.45 uM pore size, Millipore Corporation, 9edford, MA) was
used unless stated otherwise. The other papers te~ted
include: Zeta Probe blotting membrane (cationized nylon,
BioRad, Ric~mond, C~), mixed cellulose acetate and nitrate
paper (GSWP 013-00, O.22 uM pore size3 and Millipore MF
filters (HAWG04750, 0.45 uM pore size, ~illipore Corporation,
Bedford, MA), high binding capacity S&S Nytran (positively
charged hydrophilic nylon-66, pore siæes 0.2 uM and 0.45 uM),
S&S pure 100% hydrophilic nitrocellulose in different pore
sizes (no cellulose acetate added, PH70, 0.025 uM pore size
and BA83, pore size 0.2 uM, and ~A85, pore size 0.45 uM,
Schleicher and Schuell, Xeene, NH), Hydrophilic Hybond-C 87
mm nitrocellulose and Hybond-N 132 mm nylon membranes
(Amersham Corporation, Arlington Heights, ~L).
Sev~n millimeter diameter disks of nitrocellulose
(Millipore ~A 0.45 uM pore, unless stated otherwise) were
made using a standard o~fice hole puncher and transferred by
needle to a 15 x 75 millimeter polystyrene tube. Volumes
between 50-300 ul o~ diluted antisera (SAR) were incubated
with the disXs at room temperature for 10 minu~es. After
washing in Tris-EDTA-azide, the samples were inubated in 1-2
~l of blocker. All ~itration and wash bufPers utilized a
base buffer o~ 50 mM Tris, 5 mM EDTA, O. 01% sodium azide at
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pH 7.4. Blocking buffers included 10% (v/v) bovine serum, 3
BSA, or 5% Carnation non~at milk. The antigen (xabbit IgG)
was t~en added (50~300 ul) and ~ncuba~ed for 30-60 minutes,
as indicated in the following Examples. After washing three
ti~est iodinated non-immune rabbit serum (NIRS-I125) was
~dded (50-300 ul) for the ~ime ~tated (30 minutes minimum).
After a second wash, the ~a~ples were counted for gamma
radiation on a 40% efficient Packard Auto-Gamma Scintillation
Spectrophotometer Model 5220.
EXAMPL~_2
The Effic,iency of ~qG Immobilization
The efficiency of IqG immobilization on nitrocellulose
disks was determined as follows~ Serial dilutions of SAR
were incubated with a ni~rocellulose disk. The nitrocellu-
lose ~ilter were then washed and titrated with iodinated
antisera. A plot o~ the amount of bound IgG, as represented
by the amount o~ radioactivity, appears sc~ematically in the
inset to Figure 2. The competition between IgG and the
larger proportion o~ other serum proteins for protein binding
sites on nitrocellulo6e results in the affinity displacement
(A) of IgG at high serum concentrations. As the antiserum
concentration diminishes, the amount of immobilized IgG
increases until the inflection point (X) is reached, where
all the sample protein i8 absorb~d equally. Diminishing
concentrations of IgG beyond the in~lection point are ~hen
reflected by diminishing counts of ti~rating antibody (B).
This ln~lection point can be displaced to higher serum
concentration~ i~ desired ~imply by providing more nitrocel-
lulose binding 8ite5. Figure 2 shows the relationship
between the number of nitrocellulose disks and the amount o~
bound radioactivityO 500 uL of ~heep anti-rabbit serum was
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incubated ~or 10 minutes with 1-10 disks as shown in Figure
2, washed with Tris buffer, blocked with 1 ml of 10% bovine
serum blocker for 20 minutes, wàshed, ~itrated with 1 ml of
NIRS-I125 for 60 minutes, washed, and the radioactivity bound
to the disk determined. A~ a tenfold increase in nitrocel-
lulose surface area, the number oP binding sites was no
longer limitiny for that concentrat~on of sample. This
illustrates that it is advantageous to use dilute sera for
coating the matrix. Alternatively, puri~ied IgG may be used
to achieve high coating densities.
EXAMPLE 3
Stability of the Immobilized IaG
The stability of immobilized IgG on different immobili-
zation matrices, with and without 20% methanol, was deter-
mined as follows. Disks of cellulose or nylon papers were
incubated with iodinated SAR in Tris-EDTA for 20 minutes,
with or without 20% methanol. The disks were then washed
with Tris-EDTA and the radioactivity bound to the disks
measured. The following papers were tested: 1) BioRad Zeta
Probe, 2) Millipore nitrocellulose HA 0.~5 uM pore, 3)
Millipore mixed esters 0.22 uM pore, 4) Millipore MF 0.45 uM
pore, 5) S&S Nytran 0.2 uM pore, 6) S&S 100% nitrocellulose
0.025 uM pore, 7) S~S Nytran 0.45 uM pore, 8) S&S 100%
nitrocellulose, 0.2 uM pore, 9) S~S 100% nitrocellulose, 0.45
uM pore, 10) Amersham Hybond Nitrocellose, 11) Amersham
Hybond nylon. The amount of bound radioactivity is depicted
in Figure 3. The relative abilities of different papers to
absorb iodinated SA~ under the best conditions (with (+) or
without (-) 20% methanol) is depicted in Figure 4. Millipore
0.45 uM pore nitrocellulose gave the best results and was
chosen for all subsequent experiments. Mixed acetate paper
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also performed well despite reports in the literature that
such papers poorly absorbed proteins (Gershoni, J.M., et al.,
Anal. Biochem. 131:1-15 (1983)~. For S&S pure nitrocellulose
paper, smaller pore sizes performed better (0.02 v~rsus 0.45
13M ) . ~
The e~fsct on stability o~ the immobilized IgG was then
examined during recycling. Nitrocellulose disks in this
experiment, were recycled four times. Nitrocellulose disks
containing immobilized sheep anti-rabbit IgG and titrating
anti~ody NIRS-I125 were e~uted with 2.5 M MgCl2 and the loss
of antibody during the elution cycle determined. Four
elutions with MgCl2 resulted in a 20% loss of absorbed
antibody (as determined by iodinated sheep anti-rabbit serum3
as shown in Figure 5. To test the ~unctional integrity of
the SAR remaining after each cycle of the elution, the disks
were inc~bated with 150 uL o~ iodinated NIRS-antigen for 60
minutes, followed by washing in milk bu~fer, and counting the
radioactivity remaining on the disk. Background counts were
determined using the counts adhering after elution of the
iodinated antigen. All data as shown in Figure 5 is normali-
zed as a percentage of the starting value. During regenera-
tion cycles, nonspecific binding increased gradually, which
indicates the absorption of label onto sites vacated by the
blocker protein or the sheep anti-rabbit serum.
EXAMPLE 4
In thi~ example, the amount of IgG necessary to give a
readable ~ignal was determined. Five ul aliquots of serial
dilutions of SAR were spotted onto disks o~ nitrocellulose
and dried. They were blocked for 30 minutes in 2 ml of 5%
non~at milk, washed in Tris-EDTA, and incubated with 150 ul
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of iodinated NIRS in 3~ BSA for 60 minutes. Following
thorough washing, the radioactivity bound to the disks was
determined. As can be seen in Figure 6, the lowest detect-
able amount of bound IgG under these conditions was approxi
mately 25 ng. A parallel series of experiment~ were con-
ducted by amplifying the detection signal with iodinated S~R.
Again, the lowest detection limit was about 25 ny.
The amount of Ig~ bound to the dis~s after brief
incubation with a range of serum dilutions is depicted in
Figure 7. 100% efficient absorption was achieved at a titre
of approximately 1:500. At lower dilutions, decreasing
amounts of sample IgG were absorbed onto the matrix. The
capacity of the nitrocellulose is approximately 80 ug/cm2.
The area of the disk used was about 1 cm2. Thus, the amount
of specific IgG is far short of saturating the disk. About
50% absorption of total sample IgG was achieved by using
1:128 dilution of serum.
EXAMPLE 5
O~timizatiQn_of Incubation Time for Disk Coatinq
To achieve maximal immobilization of antisera, serial
dilutions of SAR were incubated with nitrocellulose disks for
minutes or overnight. A comparison of a 10 minute
incubation with an overnight incubation for a wide range of
SAR dilutions is shown in Figure 8. A lO-minute incubation
bound more IgG than an overnight incubation. In addition, an
overnight incubation showed an inflection point. The
development of the inflection point only in the longer
incubation time is due to the displacement of IgG by other
serum proteins, due to either mass action or af~inity
differences. At all dilutions o~ SAR, the binding of Ig~ was
diminished with the longer incubation time~ After incuba
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tion, the samples were washed, blocked with 1 ml of 5% nonfat
milk for 20 minutes, washed again and incubated with 450 ml
of iodinated NI~S for 60 minutes in non~at milk buffer.
After thorough washing, the radioactivity bound to the disks
was measured and is presented in Figure 8. ~he relationship
of the time of incubation ~or shorter time periods and the
radioactivity bound to the disks appears in Table 1.
TABLE 1
Radioactivity bound to disk
SAR concentration
Time of
incubation
(minutes) Undiluted 1:4 0 (blank)
-
1 171 + ~0 158 + 12523 ~ 2
155 + 9 199 + 16984 + 14
151 + 33 171 + 26738 + 13
162 ~ 7 318 + 159 1036 + 136
126 ~ 4 172 + 0718 + 147
130 146 + 33 215 + 571190 + 8
.
Undiluted, 1:4, and samples with no SAR were spiked with~
iodinated SA~, and 100 ul incubated with each disk for th~
time shown. After washing with 2 ml volumes of 5% nonfat
milk buf~er, the counts remaining on the disks were deter-
mined. The total counts added initially were 3610 + 596.
Thus, the percentage of the total that became bound ranged
~rom 14% at one minute to 33% at 130 minutes. The counts are
expressed as mPan and SD o~ duplicates. Maximal binding of
IgG in a 100 ul sample occurred at 20 minutes at a 1:4
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dilution~ However, this data was no~ significantly dif~erent
from the 10 minute incubation. Table 2 depicts the percen-
tage of total counts bound to the blank disk for each length
of time. The binding plateaus between 10-20 minutes.
Adequate coating oP disks was achieved using 100 ul of SAR at
a dilution of 1:200 for 10 minutes.
TABLE Z
Time
(minutes) ~ of Total Counts Bound to Blank Disk
_
1 14.5 + 0.3
27.2 + 2
20.4 + 2
28.7 ~ 13
19.9 + 2
134 32O9 + 0.6
LRgend: Timecourse of SAR adsorption onto nitrocellulose
disks. The data is derived from Table 1.
a~
Optimization of Incubation Time With Titratin~ Antibody
~ he relationship between the time of incubation of the
titrating labeled antibody (60 or 140 minutes) and the amount
of bound label for various serial dilutions of NIRS appears
Figure 10. The disks were coated with SAR at a 1:~ dilution
(100 ul for 20 minutes), blocked for 20 minutes in 1 ml of 5%
nonat milk buffer, and incubated with 200 ul of serial
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dilutions of NIRS serum overnight at 4C. After thorough
washing, the free sites were titrated with 100 ul of
iodinated NIRS for ~he ~ime specified. After washing, ~he
radioactivity bound to the disk was determined.
The 140 minute incubation did increase the amount of
bound label and the amplitude of ~he signal, although the
error bars were grea~er. This indicated that there was an
increased amount of nonspecific bin~ing with longer incuba-
tion times. The ampli~ication was grea~est at low concentra-
tions of sample NIRS, where the n~mber of ~ree IgG binding
sites was greatest. The titrating antibody may have a
greater affinity for the immo~ilized IgG than the sample
antigen.
~AMPLE 7
~mplificatlon bv Two Iodinated Titratinq I~G's
The effect of amplification of the signal was then
examined. Antigen-free IgG molecules were incubated with
iodinated NIRS followed by binding the remaining unbound IgG
molecules with an excess of unlabeled anti-IgG molecules to
provide a carpet for the subsequent binding of a second
labeled anti-IgG molecule. Amplification occurs as a result
of the ability of each immobilized primary antibody to bind
to more than one labeled IgG molecule. This second layer of
molecules allows a higher binding of a second labeled
titrating antibody by virtue of mass action. Further, there
is a higher probability of collision between the titrating
IgG molecule and the "carpet" target IgG molecules than
collision with primary immobiliz~d antibody dua to their
number. Amplification resulted in an increase in the signal,
especially where there was less antigen-bound IgG.
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Having now fully described this invention, it will be
appreciated by those skilled in the art that the same can be
performed within a wide range of equivalent parameters and
concentrations and conditions withou~ departing from the
spirit and scope of the invention or any embodiment thereof.
The descriptions disclosed herein refer only to a model
system. Any antibody-antigen combination may be determined
by one o~ ordinary skill in ~he art without undue
experimentation.
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