Note: Descriptions are shown in the official language in which they were submitted.
;- WO94/03631 ~ t~ ~ 2 PCT/US93/0~465
Description
Differential Separation Assay Methods
and Test Kits
Technical Field
This invention is in the field of detecting
analytes using agents which specifically bind to the
analyte to form complexes, such as antigen/antibody
complexes.
Background Art
There is an extensive ~ody of prior art related
analytical techniques based on the formation of complexes
between specific binding substances, such as antigens and
antibodies; hormones or cell modulators and receptors:
avidin/biotin and the like.
~ le Joumal of Clir~nl~to~aplly 539: 177-185 (l991) and
references therein describe the separation of
antigen/antibody complexes by capillary zone electropho-
resis and isoelectric focusing. In that article, the
capillary zone electrophoretic migration of hGH, the an-
tibody to hGH, and the hGH/antibody complex are shown.
Techni~es in Protein C3temis~, Academic Press, Inc.,
N.Y., N.Y. (1984), pp. 456-466, describes the purifica-
tion of antibodies using high performanc~ capillary elec-
trophoresis.
U.S. Patent No,. 4,937,200, Kamazawa, describes
the elution of antigens from an antibody packed affinity
chromatography column wherein one member of the binding
pair is bound to a solid support.
U.S. Patent No. 5,006l473 Bouma, shows the
migration of an alkaline phosphatase labeled antibody in
a liposome embeddea electrophoresis media. After elec-
trophoresis, the liposome is lysed and a staining dye orreactant is released.
U.S. Patent Nos. 4,205,058 and 4,301,139
describe a chromatography column which separates antigen
W094/03631 IJ 1 ~Q` 2 PCT/US93/0546
and antigen/antibody labeled complexes and the reaction
is determined by measuring a radio-labeled antiyen which
- migrates on the column. As an example, T4 I1Zs and anti-
T4 are reacted and separated on a cross-linked polyvinyl
alcohol column. The antibody/T4 complex is retained at
the bottom of the column and the T4 I125 migrates. These
patents represent an example of the direct binding of a
labeled hapten T4 I125 and antibody and separation of
these complexes.
In U.S. Patent No. 4,811,218, M. Hunkapiller et
al. teach a DNA sequencing system using a multiple lane
electrophoresis apparatus. Fluorescent dyes are attached
to molecules moving through the lanes. A moving illumina-
tion and detection system scans the multiple lanes. Four
color data points are recorded for each of several lanes
a~ a particular time at a fixed distance down the gel.
Through a complex analytic procedure, the four colors are
related to the concentrations of four dye-labeled DNA
components. The object is to identify concentrations
of A, C, G, or T which are DNA piece endings where
A = adenosine, C = cytos-ne, G = guanine and T = thymine.
Peak concentrations of a particular dye label are matched
with particular bases in DNA sequences.
In U~S. Patent No. 4,890,247, Sarrine et al.
describe an apparatus which robotlcally handles a
plurality of liquid samples in test tubes, applies the
samples to electrophoresis matrices and then carries out
electrophoresis. The electrophoretically separated
molecules are illuminated with fluorescent light. An
analog signal is produced, representing the scanned field
of view. A compùter stores intensity levels of the
analog signal and performs densitometric analysis to read
the electrophoretic data. Densitometry is a conventional
prior art technique for reading such data.
In an article entitled "Affinity Electrophore-
sis" by Vaclav Horejsi, report in En~mePunficationandRelated
Techni~es, W. Jakoby ed., Academic Press, 1984, p. 275, a
novel type of electrophoresis is described. One lane of
W094f03631 ~ ,?~ ~ PCT/US93/0546
the gel medium is impregnated with immobilized ligands
capable of reacting with a migrating macromolecule, while
another lane, a control gel, is untreated. Thus, a
comparison can be made, using electrophoresis, between a
S macromolecule sample retarded by the affinity gel lane
and a similar sample in the control gel lane. In a
variation of this technique, the gel may incorporate an
antibody which interacts with a migrating antigen. The
two lanes may be calibrated so that different degrees of
retardation, for different concentrations of the migrating
macromolecule, are known. Moreover, microscopic beads
treated with ligands can be entrapped in the gel and
similarly serve as a retardant. Beads have the advantage
of tight packing in the gel if they are of appropriate
size. Activation of the gel involves partial cross-
linking so that the gels do not melt on heating. Alter-
native methods of gel preparation are described, all with
the result that a macromolecular retardant is immobi-
lized. Electrophoresis proceeds in the usual way.
Various types of pulsed electrophoresis are
known for use in separating closely related substances
(see Kreger EPA 457,748; Slater EPA 395,319, Agawa EPA
320,937; and Allington EPA 396,053).
In spite of the above-mentioned advances in
analytical separation chemistryl there is still a problem
in rapidly separating heavy molecules of close weight or
mobility, or charge/mass ratio wherein the analyte and
other substances in a test sample exhibit similar
behavior in separation efforts. An object of the inven-
tion was to devise a method and apparatus for such sepa-
rations.
Disclosure of the Invention
This invention relates to methods and test kits
3S for detecting analytes, in a milieu of substances of
closely related weights or mobilities, which specifically
bind to a labeled binding reagent to form a complex. The
binding agent is labeled with a detectable marker so that
WO94/03631 ~ 2 PCT/US93/0546
binding between the labeled binding agent and analyte
provides a reaction mixture which contains a labele~
binding agent and a complex of analyte and labeled
binding agent. These two labeled substances are placed
on a separation medium and the differential rate of
migration of the labeled specific binding agent and
complex are determined by detecting the label in each as
they are being separated on the separation medium. This
difference in migration identifies the target analytes.
In a preferred embodiment, a second labeled marker is
included which migrates different from the labeled
binding agent and complex and most preferably migrates
faster than the complex or the labeled binding agent.
More specifically, the invention encompasses a
method for detecting an analyte in a test sample compris-
ing:
(a) providing a labeled binding agent which
specifically binds to the analyte to form
a complex;
(b) providing a separation medium on which the
labeled binding agent and complex migrate
with different velocities; then
(c) contacting the test sample with the
labeled binding agent to form a complex
with analyte in the test sample, both free
and bound binding agent having expected
migrations from a starting location to one
or,more measuring places;
(d) separating the labeled binding agent and
complex on the separation medium;
(e) measuring and recording the difference in
migration between the labeled binding
agent and the complex on the separation
medium by detecting the labeled binding
3S agent and labeled binding agent in complex
at said measuring places; and
W~9~/03~31 ~ g o ~ PCT/US93/0~46S
(fj searching said recorded migrations for
bound binding agent in relation to f~ee
binding agent using sa~d expected migra-
tions in comparison to said measured mi-
grations wherein finding of said bound
binding agent indicates presence of said
analyte.
For example, the analyte may be an antigen and
the labeled binding agent a fluorescently labeled anti
body or fragment of an antibody such as an Fab fragment.
The labeled binding agent and complex are
preferably separated by electrophoresis and the label is
detected as each migrates past a fixed point in the
electrophoresis medium.
More particularly, the invention also encom-
passes a method for measuring the concentration of an
analyte in a test sample moving in a single lane
comprlsing:
(a) providing a labeled binding agent which
specifically binds to the analyte to form
a complex;
(b) providing a separation medium on which the
labeled binding agent and complex migrate
in a single lane with different veloci-
ties;
(c3 contacting the test sample with an amount
of the labeled binding agent in excess of
what will react with the analyte to form a
complex with analyte in the test sample,
both free and bound binding agent having
expected mig~ations from a starting loca-
tion to one or more measuring places;
~d) separating th~ excess labeled binding
agent and complex on the separation
medium;
(e) measuring and recording the difference in
migration between the labeled binding
agent and the complex on the separation
WO~4/03631 PCT/US~3/05465
-6-
medium by detecting labeled binding agent
and the labeled binding agent in co~plex
at said measuring places;
(f) searching said recorded migrations for
bound binding agent in relation to free
binding agent using said expected migra-
tions in comparison to said measured mi-
grations wherein finding of said bound
binding agent indicates presence of said
1~ analyte;
(g~ measuring the area under each peak of each
characteristic detected label;
(h) normalizing the areas under each peak, and
(i) relating the normalized measured areas
with the areas of samples containing known
amounts of said analyte.
This method can be applied to the measurement
of multiple analytes in the same lane or to multiple
analytes in different lanes.
A preferred assay utilizes a second labeled
marker which migrates independent of the labeled binding
agent and complex. Preferably, this second labeled
marker migrates first in the medium and serves as a
~uality control check on the system. For example, if the
separation media or other reagent in the test kit are not
operative, the failure of the second labeled marker to
migrate as expected can be readily detected and the reli-
ability of any results from tha~ assay can be discarded.
Also, if the monoclonal antibody or immune complex peak
fails to appear in the proper relationship to the second
labeled marker, this test may be discarded as faulty.
Another aspect of the invention is test kits
which contain an analyte standard, a labeled binding
agent which specifically binds to the analyte to form a
complex, and a separation medium which separates the la-
beled binding agent and complex. Such test kits prefera-
bly contain a second labeled marker which migrates inde-
pendent of the labeled binding agent and complex. The
- W O 94t03631 ~ 2 PC~r/US93/05465
separation medium is preferably in the form of a modular
cartridge having a plurality of electrophoresis lanes as
- shown in Figure lla. Test kits for detecting an antibody
comprise a hapten conjugated labeled carrier, separation
medium, and preferably a second la~eled marker. A test
kit for detecting haptens comprises a hapten conjugated
labeled carrier, a standard sample of the hapten, and an
antibody to the hapten.
Thus, the present invention takes advantage of
10 the specificity of reaction of specific binding mole- -
cules, the ability of separation media to separate small
amounts of materials, the sensitivity of detecting labels
such as fluorescent labels and a quality control agent to
provide rapid, sensitive, and reliable quantitative
results. The methods, test kits, and apparatus of ~his
invention are most advantageously applied to closely
related and difficult to separate complexes of closely
related molecules such as complexes of isoforms of
similar proteins. The term "closely related" means not
only heavy molecules of close molecular weight, but also
molecules whose charge/mass ratio or other characteristic
is such that both molecules exhibit similar migration
rates, e.g., using electrophoresis, so that previous
separation efforts have been difficult.
The term "analyte" refers to a large variety of
chemical substances for which there is a specific binding
partner.
It is contemplated that the present assay may
be applied to the detection of any analyte for which
there is a specific binding partner. The analyte usually
is a peptide, protein, carbohydrate, glycoprotein, ster-
oid, or other organic molecule for which a specific bind-
ing partner exists in biological systems or can be syn-
thesized. The analyte, in functional terms, is usually
selected from the group consisting of antigens and
antibodies thereto; haptens and antibodies thereto; and
hormones, vitamins, metabolites and pharmacological
WO94/03631 ~ lL~I~ a 2 PCT/US93/0546
agents, and their receptors and binding substances. Ex-
amples of analytes are immunologically-active polypep-
tides and proteins of molecular weights between l,OoO and
4,000,000, such as antibodies and antigenic polypeptides
and proteins, as well as haptens of molecular weights
between lO0 and l,500. Representative of such antigenic
polypeptides are angiotensin I and II, C-peptide, oxyto-
cin, vasopressin, neurophysin, gastrin, secretin, and
glucagon. Representative of antigenic proteins are insu-
lQ lin, chorionic gonadotropin (e.g., HCG), carcinoemkryonicantigen ~CEA), myoglobin, hemoglobin, follicle stimulat-
ing hormone, human growth hormone, thyroid stimulating
hormone (TSH), human placental lactogen, thyroxine bind-
ing globulin (TBG), intrinsic factor, transcobalamin,
enzymes such as alkaline phosphatase and lactic dehy-
drogenase, and hepatitis, HTLV-III, influenza, herpes,
and other viral associated antigens. Representative of
antibody ligands are those antibodies of the IgG, Ig~,
IgM and IgA classes specific for any of the antigens or
haptens, or a class thereof, herein described. The class
of hapten ligands is exemplified by thyroxine (T4),
triiodothyronine (T3), the estrogens such as estriol,
prostaglandins, vitamins such as biotin, vitamin B12,
folic acid, vitamin E~ vitamin A, and ascorbic acid
(vitamin C), and drugs such as carbamazepine, quinidine,
digoxin, digitoxin, theophylline, phenobarbital, primi-
done, diphenylhydantoin, morphine, nicotine, and so
forth.
DNA, RNA, and their complementary binding
sequences and binding proteins can be determined by
method of this invention. Cytokines such as interleu-
kins, interferons, G-CSF, GM-CSF, M-CSF, tumor necrosis
factors (TNF), erythropoietin and the like are represen-
tative of cytokines that may be determined by methods of
this invention.
This discussion of analytes is intended to
illustrate the large number and variety of chemical
WO94/03631 PCT/US93/0~6~
_9_
substances which have specific binding agents or for
which specific binding agents can be made.
The term "labeled binding agent" refers to
substances which specifically bind to the analyte and
which have a detectable label. The label may be
covalently linked or bound to the binding agent
indirectly through another specific binding reaction, for
example, a labeled goat antihuman antibody could be used
to label a human antibody~ Those skilled in this art
will recognize a wide variety of techniques to label
protPinaceous, as well as non-protein specific binding
substances. For instance, fluorescent dye lab~ling of
proteins in ge~eral and antibodies or antigens in
partlcular is well-known.
For the determination of small analytes, such
as those ha~ing a molecular weight of 100-1500, it is
necessary to prepare a reagent which is a conjugate of
the analyte to a labeled carrier. The free analyte in
the test sample and analyte on the labeled carrier
compete for the specific binding partner prior to
separation.
Cu~ent Prot~ols in Imm~o~ofo~, edited by John E.
Coligan, Ada M. Kruisbeck, David H. Margolies, Ethan M.
Shevach, and Warren Strober, John Wiley & Sons, N.Y.,
1991, extensive~y describes methods for obtaining
polyclonal and ~lonoclonal antibodies and Fab fragments
thereof, means for fluorescently la~eling these
antibodies and the reactions of these antibodies with
antiqens. Also described are electrophoresis and
electrophoresis media as well as other separation
technigues.
A large number of cytokines and monoclonal
antibodies to these cytokines are known, for example,
interleukin ~ , 2, 3, 4, 5, 6, 7, 8, 9, 10);
interferon (~, ~, y); granulocyte/macrophage colony
stimulating factor (CN-CSF), G-CSF, M-CSF; tumor necrosis
factor (TNF ~ and ~); and Transforming Growth Factor and
their monoclonal antibodies are known. Antibodies to
- .. . .. .... .... ... . . . .. ...... . . . .
W094/03631 ~ 2 PCT/US93/05465
--10--
creatine kinase (skeletal, cardiac, and brain) are
described in U.S. Patent No. 4,353,98~ (Gomez et al.).
Lipopolysaccaride ~LPS), an endotoxin, is a
major outer membrane component of cell walls of Gram-
negative bacteria. Monoclonal antibodies to LPS arewell-known, see U.S. Patent No. 5,092,235 (~illiams et
al. and references therein).
A specific binding substance may be labeled
with any of a variety of dyes, such as fluorescein dyes
or rhodamine dyes by conventional chemical techniques.
Representative fluorescent dyes for making th~ labeling
binding agent are fluorescein isothiocyanate (emission at
520 nm), 4-chloro-7-nitrobenzo-~-oxa-1-diazole (emission
550 nm), tetramethylrhodamine isothiocyanate (emission
lS 580 nm) and Texas Red (emission 610 nm). Th~se dyes are
available from Molecular Probes, Inc. (Eugene, Oregon)~
or can be synthesized with a variety of functional groups
to accommodate the binding of these dyes to various chem-
ical functional groups. For example, fluorescein 5 or 6
succinimidylcarboxylate, fluorescein 5 or 6 iodoaceta-
mide, and fluorescein 5 or 6 maleimide are available.
Similar functional groups are available for tetramethyl-
rhodamine dyes.
It is also well-known to indirectly label a
5pecific binding molecule, such as an antibody by
specifically binding a second labeled antibody to
antibody reagent, such that the detected components are
labeled antibody-antibody jcomplex and labeled antibody-
antibody-antigen complex.
- 30 The term "separation media" refers to size
chromatography, affinity and ion exchange chromatography,
electrophoresis, such as slab get electrophoresis or
capillary electrophoresis. Media, such as polyacryla-
mide, cellulose acetate, agar gel, and agarose gel, are
suitable for electrophoresis. The separation of the
labeled binding agent, complex, and labeled marker can
also be achieved by sedimentation techniques where
centrifugation causes the component to migrate in the
- WO94/03631 ~ PCT/US93/05465
media. These separation techniques are well-known to
those skilled in this art.
In its simplest form, the invention involves
the reaction of a labeled binding agent ~LB*) and an
analyte [A] to form a complex A/LB* and separation of
these species on a separation media, measuring the
differential rate of migration of A/LB* and LB* on the
separation media by detecting the label and then using
the difference in migration or migration rate to identify
the analyte by comparison with calibration data or other
data which establishes expected migration data for the
analyte.
In another embodiment of the invention where
the A/L~* complex is too complicated, the reaction can be
determined by measuring the decrease in the size of-the
LB* peak.
In another embodiment, multiple analytes can be
detected by using different labeled binding agents that
specifically bind to each analyte and detecting the LB*
and AjLB* complex for each label.
- In another aspect of this invention, two
different labeled binding agents can be bound to one
analyte to form a sandwich complex and the migration of
the sandwich complex can be compared with either or both
of the labeled binding agents.
In yet another embodiment of the present
invention, two-dimensional electrophoresis can be used to
separate multicomponentlsystems. In each instance, the
differential migration data is compared with expected
migration information.
Small molecules such as haptens can be detected
by binding the hapten to a labeled carrier [C*] such as a
polypeptide to form a conjugate in which the hapten or
the carrier is labeled (HC*). The hapten in a test
sample is allowed to compete with HC* for antihapten and
th~ mixture is separated on the separation media. The
species HC* and HC* antibody complex are detected as they
` u ~ 2
WO94/03631 - PCT/US93/0546
-12-
separate on the media. This reagent is referred to as a
"hapten conjugated labeled carrier."
All of these embodiments are preferably
practiced by the inclusion of a second labeled marker
which migrates on the separation medium independent of
the labeled binding agent and analyte and preferably the
second marker is a fluorescent dye derivati~e which
migrates more rapidly than the labeled binding agent and
complex in the separation medium. The fluorescent dye
can be bound to a protein or other substance which will
affect its mobility so that it will migrate as des:Lred in
a particular medium. For example, human serum albumin is
a negatively charged protein which migrates very rapidly
toward the positive pole in electrophoresis. The labeled
marker provides for normalization of channel to channel
variability in antibody reaction and detection of signal
amplitude. It provides an early warning that the partic-
ular assay is grossly incorrect. The labeled marker also
provides a reference point for discrimination of both the
labeled binding agent peak and complex peak, thus further
assuring the quality of the assay. The second labeled
marker also provides a means for quantitation of the ana-
lyte.
- This invention is most ad~antageously applied
in the diagnosis of molecular variants of proteins. For
example, creatine kinase (CK) MB isoforms (MB2 and MBl)
have been used in the early diagnosis of muscle injury
following acute myocardial infarction. The determination
of the MB2 and MBl isoforms is also useful in determining
the onset of acute cardiac allograft rejection as well as
injury following coronary artery bypass grafting. The
determination of CXMM isoform is important in monitoring
atropic skeletal muscle changes. There is also known to
be an increase in mitochondrial creatine kinase in
patients with cerebrovascular damages and it is critical
that there be a rapid assay to assess such damage so that
drug therapy can begin.
- WO94/03631 P~T/US93/0~46~
-13-
The determination of alkaline phosphatase iso-
forms is important in liver, bone, and kidney disease, as
well as liver transplant rejection. Those skilled in the
medical arts will recognize a large number of clinically
important proteins which have small differences in struc
ture which can be determined by the method, test kits,
and apparatus of this invention.
Brief Description of the Drawings
10Figure 1 is a plan view of an apparatus for
practicing the present invention.
Figure 2 is a top view of a single gel lane
illustrated in Figure 1.
Figure 3 is a plot of detector signals from
unbound and bound fluorescent material.
Figure 4 is a plot of overlapping detector
signals cf different wa~elength from unbound and bound
fluorescent material.
Figure 5 is a top view of a multiple lane gel
arrangement for electrophoresis.
Figure 6 shows the electrophoretic migration of
Cy5 labeled human serum albumin (HSA) and Cy5 labeled
human serum albumin complexed with an antibody.
Figure 7 shows the electrophoretic migration of
Cy5 labeled bovine serum ~lbumin (BSA) as a second
labeled marker and Cy5 labeled anti-HSA.
Figure 8 is a front perspective view of the
differential separati~n assay instrument.
Figure 9 is a front perspective view of the
- 30 operating components of the differential separation assay
instrument of Figure 8.
Figure 10 is a scAematic drawing of the optics
of the differential separation assay instrument of Figure
8.
35Figure lla is a top plan view of an electropho-
resis cartridge used in the instrument of Figure ~.
Figure llb is a sectional view of the cartridge
of Figure lla through an electrophoresis channel.
WO94~03631 ~ 3 o ~ PCT/US93/0546
Best Mode for Carrying Out the Invention
With reference to Figure l, a single lane gel
- electrophoresis apparatus ll having a well 13 at one end
with a negative voltage terminal 16 and a positive high
voltage electrode terminal lS at an opposite end is
shown. The electrophoresis apparatus consists of a con-
ventional single lane 18 having a substrate 17, a gel
layer l9 and a protective glass cover 2l. The substrate
is usually a self-supporting material which may be glass,
Mylar (Trademark) or any well-known gel support. The gel
itself is usually polyacrylamide or agarose, although
other gel materials such as synthetic acrylamide substi-
tutes may also be used. Uniform polymerization and free-
dom from bubbles and irregularities are desirable proper-
ties~ The glass cover is preferably nonreflective glasswhich merely serves as a protective cover for the gel.
The well 13 is normally positioned vertically so that it
will receive a sample without spillage. The well funnels
a prepared sample toward the gel. The well may combine a
stacking and separating gel and creates a slit of sample
material on the gel. High voltage is then applied to the
gel at terminals 15, 16 and charged ions migrate toward
the positively charged voltage electrode. The end of the
gel near well 13 is maintained at negative or ground po-
tential so that there is a substantial potential differ-
ence from one end of the gel to the distant end.
The sample which is placed in well 13 is a
fluid, fr~quently a fractionated blood sample. Blood may
be pre-processed to remove constituents which will inter-
fere with the assay. Removal may be by filtering, ab-
- sorption, centrifuging or precipitating either the
desired or undesired components so that a desired target
analyte may be obtained for electrophoresis. The desired
target analyt must be one for which there is a specific
binding agent. Fluorescent tags are commercially avail-
able, such as those manufactured by Molecular Probes
Inc. of Oregon which specializes in dyes or dyed beads
that can be covalently attached to binding agents to
WO94/03631 PCr/US93/0546~
) 2
-15-
provide a labeled binding agent. Where target analytes
are found in larger structures, such as pathogenic ~
agents, then such a dye-binding agent conjugate would be
appropriate for tracking that pathogenic agent. Mono-
clonal antibodies can now be manufactured so that thebehavior of this binding agent is uniform and predictable
for many assays. Monoclonal antibodies are more expen-
sive than polyclonal antibodies, but the antibodies have
greater specificity, are directed toward single epitopes,
are easy to produce in large quantities, and are generally
more useful and cause precise separation of bound and
free material.
The labeled binding agent is supplied in excess
so that the reaction with the analyte will be driven to
completion, or nearly to completion, in a reasonabl~ or
convenient amount of time. The amount of excess labeled
binding agent should not be more than twenty times the
amount of expected maximum level bound tag, although the
number may range between 2 and S0, approximately. The
labelins binding agent should alter the mass to charge
ratio when combined with the analyte and subjected to an
electrophoretic field.
A strongly emitting light source, such as light
emitting diode or laser 23 is used to generate a beam 25
The ~ED 23 has an output power of about 50 ~W in a wave-
length band which will excite fluorescence in the fluo-
rescent tagging material. Such excitation radiation is
known as actinic radiation. The beam is intercepted by a
focusing lens 27 which directs the beam through a slit
aperture in barrier 29. Light emerging from the slit is
di~ergent and is intercepted by the collimating lens 31.
The beam is then directed onto a reflecting surface 33
which is part of a prism 35. The reflective surface 33
i5 at a 45 degree angle to the beam so that the reflected
beam makes a 90 degree angle with the incident beam. The
reflected beam is directed toward focusing lens 37 where
the beam passes through one half of the focusing lens,
while the other half is reserved for light traveling in
WO 94/03631 .'..~ 3 ,~ PCI/VS93/0546
--16--
the opposite direction, reflected from gel layer 19.
Light passing through the focusing lens carries an image
of the slit 29 which is directed onto the gel layer 19.
Fluorescent light emitted from the complex and
some reflected light from the gel layer travels in a
retro-beam 39 to the left half of focusing lens 37. Note
that one half of the focusing lens is used by light
travelling in each direction. The right half is used by
the incoming beam, while the left half is used by the
retro-beam. From there, the retro-beam is directed to
reflecting surface 41 which is part of prism 35. The
retro-beam is passed through a filter 43 which rejects
any light other than the desired wavelength from the
fluorescent target. Light transmitted through the filter
is directed toward focusing lens 45. From there the beam
is directed to a light detector, such as photomultiplier
tube 47 with a slit located at the image plane of the
gel.
The time of arrival of the fluorescent sub-
stances is measured relative to the starting time, i.e.,
the application of high voltage which initiates electro-
phoretic migration. Since the arrival time is not pre-
cise, but rather is a Gaussian curve, the peak time is
recorded. The integrated peak area is also used for time
discrimination. ~ach analyte and the corresponding la-
beled binding agent are sub~ect to the same procedure in
the calibration run. In calibration runs a mean migra-
tion time to the measurement slit or pinhole is deter-
mined. Then, the standard deviation is determined for
~ 30 the time of arrival of the free binding agent, as well as
for the bound analyte. In the present invention, it is
necessary to know the mean migration time, i.e., the
~xpected arrival times of bound and labeled binding agent
for analytes because the times will be used to search for
target analyte in a sample where the target substance is
possibly present, but not necessarily present. The
difference in arrival times between the complex and
labeled binding agent may be used to establish a time
W094/03631 ~ 2 PCT~US9~0~465
window so that the arrival of one member may be paired
with the other member in a search for the other member.
If the search reveals that the other member is present
within a standard deviation or two, that other material
is identified as a member of the pair. If nothing is
found within the time window, the first member of the
pair is regarded to be an artifact and is discarded. The
search may be based on the second labeled marker or other
labeled component.
The output of the photomultiplier tube is main-
tained in a buffer memory 49 and a ratio may be formed
between the signals representing complex and labeled
binding agent. A data reader 50 is connected to the
buffer memory 49 for receiving recorded signals which
represent the fluorescent peaks. The data reader is a
computer which correlates the various peaks. Each peak
is recorded in order to search for complex and unbound
labeled binding agent in the recorded data~ Normally,
the time of appearance of the labeled binding agent could
b~ estimated from prior calibration times. Once the
position of the free labeled binding agent is known, a
search is conducted for the corresponding complex which
should be located a certain time interval away, within a
time window defined by statistical limits. A peak within
this window is identified as a complex that will bind
fluorescent substances, i.e., the target analyte. Next
the amplitudes of the identified peaks are examined and a
ratio is computed injthe,data reader 50. The method
whereby labeled binding agent is correlated with complex
is explained further below. The computer also stores
calibrations of known concentrations of target substance
so that ratios may be compared in order to obtain an
estimate of the unknown concentration.
In Figure 2, the top view of gel 11 shows that
the image 29' of slit 29 falls between a positive high
voltage terminal 15 and a slit from well 13, coinciding
with negative voltage terminal 16. In operation the high
voltage applied to terminal 15 causes migration of com-
WO94/03631 ,~1-L.;~ a 2 PCT/US93/05465 ~ :
-18-
plex and labeled binding ayents, which are positively or
negatively charged molecules w~hich respo~d to the elec-
tric field from the high voltage supply. The labeled
binding agent will reach the image 29' of slit 29 which
is fixed in position at a time different than the com-
plex. The labeled binding agent serves as one marker for
a time window which has the bound tagged binding agent as
a corresponding marker, the two markers forming a pair of
markers which are separated in time within the statisti-
cal limit which is defined.
With reference to Figure 3, a plot of thedetector signal is shown where the horizontal axis is
time and the vertical axis is amplitude of the detected
signal. As an example, electrophoresis beings at a first
time, t - 0, and the detector is made operative. At a
second time, t2, a relatively large peak 51 is observed,
representing free fluorescent labeled marker of a first
color. Another signal 54, discussed below, is detected
after peak 51. At a later time, t3, a weaker signal 53 of
the same color is observed. The peak 53 exists in the
mid-region of a window, Wl, between Xl and X2. The
existence of window Wl is established by the labeled
binding agent signal 51. Peak 53 is within window Wl and
is recosnized as a signal from the complex. Peak 54 is
not within window Wl and is treated as a false positive
or artifact, after being checked to determine whether the
signal is not mistaken for the labeled binding agent
signal 5l. A search ofjall signals is made to determine
the most logical positions for free and bound fluorescent
substances. If no signal is found in time window Wl, the
absence of target analyte is inferred. Each window W
acts as a time domain filter, allowing discrimination of
spurious fluorescent signals and noise. Note that all
signals are recorded and signal discrimination occurs
after recording by analyzing recorded data. Even though
gel to gel characteristics may vary, the present inven-
tion has immunity to most variations because the complex
and labeled binding agent traverse the same path.
i WO94/03631 ~ 2 PCT/US93/05465
--19-- :
The ratio of the two signals represented by the
area under the peaks 51 and 53 represents an estimate of
the ratio of a complex to labeled binding agent after
normalizing data relative to calibrations, assuming good
binding efficiency. A further time later, another large
peak 55 is observed. This represents another fluorescent
binding agent. This defines another time window W2 at a
subseguent time and a lesser peak ~7 is measured in the
window. This is taken to represent a complex. Again,
the ratio of complexes to free dye is computed and once
again the target analyte associated with the second dye
may be estimated in concentration.
It is possible for the peaks to overlap each
other as shown in Figure 4. Here, the labeled binding
agent substance peak 61, having a relatively large -
amplitude, overlaps the second peak 6S of similar
amplitude in a test where two different fluorescent
substances were used. The second peak 65 is the second
free fluorescent substance signalO However, because
different colors are used, as separated by the filter 43
in Figure 1, the two peaks may be separately observed.
Peak 61 establishes the time window W3 where a peak 63,
representing a bound fluorescently labeled binding agent
of a color which is the same as that associated with the
unbound peak 61, occurs totally within the second peak
65. Nevertheless, because of the filter 43, peak 63 may
be spatially and optically differentiated from peak 65.
The ratio of bound to unbound signal amplitudes appears
to be about 2:1. The corresponding molecular amounts of
~ 30 ~omplex and labeled binding agent are estimated to be in
the same ratio. For the peak 65, a time window W4 is
established, but no fluorescent signal is found within
the window and so the absence of target analyte is
inferred.
With reference to Figure 5, a multiple lane
electrophoresis sheet gel is shown. The sheet 71 is
provided with two lanes 73 and 75. Each of the lanes has
a respective well 83 and 85 and a respective slit image
W~94~0363~ PCT/U5g3/~546~
1 3 0 2
-20-
87 and 89. The two lanes are constructed similarly, with
the spot image locations in the same position. Lane 73
is used to run a calibrated amount of target analyte, a
known amount of free fluorescently labeled binding agent,
and a fixed amount of a second labeled marker. In lane
75 an unknown amount of target analyte is run with free
fluorescent labeled binding agent and the same fixed
amount of the second labeled marker. The two lanes may
be compared after normalization of peak area between
lanes using the peak area of the second labeled marker to
determine the amount of unknown analyte in lane 75. For
greater accuracy, multiple runs may be made in lane 73 of
various amounts of target analytes so that many ratios
may be st.ored in a memory. A ratio from a run of an
unknown amount of target analyte may then be looked up
and compared with known ratios, with the best match
indicating the amount of target analyte.
Preferred apparatus for practicing the invention
is set out in Figures 8-lla and b. With reference to
Figure 8, measurements are made with the instrument 101
which has two main modules, a computer module 103,
serving to log and display data, and a measurement module
105, serving to receive samples and subject the samples
to testing and measurement in accord with the procedures
disclosed herein. Computer module 103 is a standard PC
of the 386 or 486 MS-DOS kind, running familiar software
suitable for manipulating numbers, such as Excel or Lotus
spreadsheets. A display device 104, a keyboard 106 and a
disk drive 108 are normal input/output devices associated
with the computer. Keyboard 106 i5 used for signalling
commands to the measurement module, such as start, stop,
repeat, and so on. The measurement module 15 includes a
U-shaped frame 107 for receiving an electrophoresis
cartridge 109 and internal optics and electronics covered
by the shroud 201. Once a cartridge 109 is placed on
frame 107, the frame is pulled under the shroud 201 for the
measurements disclosed herein using a signal from
keyboard 106.
WO94/03~31 ~ 0 2 PCT/US93/0546
-21-
Principal features of the measurement modules
are shown in Figure 9. Frame 107, shown without a pro-
tective lip apparent in Figure 8, supporting electro-
phoresis cartridge lO9, is moved by a pinion gear 203, in
the direction indicated by arrows A, to a position
wherein the cartridge rests over a Peltier device 205,
including a fan within housing 207. The Peltier device
is a commercially available refrigeration apparatus which
chills a metallic mass 209 of good thermal conductivity,
such as an aluminum block, to a temperature suitable for
electrophoresis measurements of a particular media. The
mass 209 is kept in thermal contact with cartridge lO9 by
means of a movable pressure plate 301 which is supported
from a slotted plate 303 to apply downward pressure on
oppo~ed edges of cartridge lO9. The pressure plate 301
and the slotted plate 303 are moved downwardly by means
of a spring biased actuator, thereby forcing the car-
tridge lO9 into thermal contact with metallic mass 209
with downward force transferred to the cartridge at edges
of the cartridge which are supported by frame 107.
A first slot 305 in slotted plate 303 admits a
scanning beam 307 which is directed onto a locus of spots
on the cartridge, described below. Light scattered,
reflected or fluorescing from the impinging light is
directed back into optical fibers 309, one fiber corre-
sponding to each beam spot in the locus of spots. Each
of the fibers leads to a single optical detector which is
always on. The detector output is synchronized with the
beam position so that a single fiber is identified as the
- 30 one providing the optical signal at the detector at any
particular time.
In Figure lO, incoming light is seen to
originate at laser 401, which i5 a 5 mw helium-neon
laser, or a laser of any appropriate wavelength. The
beam 307 has its path folded by mirrors 403 and 405,
while the beam is collimated by optics not shown and
focussed by a lens 407 to form a small spot on a scanning
mirror 409. The mirror 409 is locat~d sufficiently close
W094/03631 ,~ t ~ 1 3 ~ 2 PCT~S93/0~65
-22-
to cartridge lO9 that beam spots on the cartridge will
not be significantly out of focus. Mirror 409 is rotated
about a scanning axis in steps, defined by pivots 501 and
503 for mirror motion indicated by arrows B. A motor,
500, steps the mirror in discrete angular amounts of a
few degrees per step starting from a home position at an
edge of the support frame where an optical detector is
located. A beam scan always starts from the home
position and the beam is stepped by known angular amounts
to create a limited number of spots at desired locations
on a cartridge. The number of steps is counted by an
electrical counter, which is a beam synchronizer 502, so
that the beam position is known at all times. In this
way, the beam position can be synchronized with the
detector 505, a photomultiplier or PMT tube which
receives light from the optical fibers 309 which are
gathered in a bundle. A lens or lenses and a filter, not
shown, may be u~ed to optimize coupling between the fiber
bundle and the PMT 505. The filter reduces spurious
light. At the opposite end of the fibers, a holder block
507 is used to secure each optical fiber in a desired
location and to space each fiber very close to the locus
of spots where the incoming beam will impinge.
In Figure lla, a cartridge 109 is seen to
include opposed edges 601 and 603 having apertures 701
and 703 partially extending into the edges. These
apertures are known as wells where fluid samples are
placed. Additional apertures 70S and 707 may be used as
detents to index the location of the cartridge in the
- 30 support frame. Between opposed cartridge edges, slightly
above a heat conducting plate 605, are very thin
capillary tubes 607 having open ends which extend into
the wells 701 and 703 for communicating with any fluid in
the wells. The capillary tubes are clear, low reflection
material or treated with a coating for low reflection.
The tubes have a diameter suitable for electrophoretic
migration of a sample in view of the viscosity of the
sample. In the locus of beam spots mentioned previously,
` W O 94~03631 PC~r/US93~05465
~ ~ ~ 0 2
-23-
one beam spot is provided for each capillary tube so that
the number of angular steps of the beam needs to at least
equal the number of capillary tubes and must be such that
the beam lands squarely on a capillary tube for possibly
exciting a response from a fluid in the capillary. In
Figure llb, electrodes 609 and 709 are seen to extend
into apertures 701 and 703 in order to make contact with
fluid in the well and migrating between the opposed elec-
trodes under the influence of an electric field. In op-
eration, the electrodes come into contact with the wellsat the same time a pressure plate clamps the cartridge in
place as described above. Electrical potential differ-
ences between the electrodes 609 and 709 cause electro-
phoretic migration in the capillary tubes. The beam 307
repetitively scans a locus of spots transverse to the
length of the capillaries, with a spot falling on each
capillary and passing into its center where it illumi-
nates fluid sample material under electrophoresis
migration. Fluorescent light emitted from the sample
falls upon an end of a nearby optical fiber and the light
is guided back to the PMT.
One of the advantages of the present invention
is that analysis of peaks representing bound and free dye
can be computed before electrophoresis is complete, i.e.,
~efore ~he migrating substances reach the distant high
voltage electrode. Another advantage is that the present
system uses only a single lane of an electrophoresis
apparatus so that gel to gel non-uniformities are nulled.
It is possible to use a second lane in an electrophoresis
- 30 device as a reference or calibration, but such calibra-
tions may be done beforehand and results stored in a
memory. It is also possible to use a second or third or
fourth lane for additional analytes of interest creating
panels of relevant analytes. In the prior art, analysis
of target analytes usually requires completion of the
electrophoresis and subsequent analysis by a plurality of
stains, colored or fluorescent substances, etc. Using
the present invention, the analysis may be done in real
W094/03631 ~ PCT/~S93/0546S -~
-24-
time as soon as sufficient separation exists between the
bound and free fluorescent material. Such a separation
- can be at a point which is only twenty-five percent or
thirty-three percent of the length of a lane. Once a
point is found where adequate separation exists, the
image of the slit or pinhole is positioned at that loca-
tion and then all measurements are made from there. It
is also to be noted that this is an open-ended electro-
phoresis system, i.e., there is no need to stop the
electrophoresis at a defined point to get all materials
"on scale." Materials that migrate slowly can be
detected just as well as fast moving target analytes.
Amplitude thresholds may be used as further discrimina-
tion against noise and artificial signals.
To discriminate between two or more target ana-
lytes in the same gel lane, different fluorescent wave-
lengths can be used, so long as filter 43 in Figure 1 can
adequately resolve the different wavelengths. Multiple
tests can be run simultaneously, each test associated
with a particular wavelength.
Industrial Applicability
Example l
Detection of ~roteins ~resent in human blood
Creatine kinase is an enzyme present in various
mammalian tissue. It occurs in three different forms
known as isoenzymes: ÇK-MM (skeletal), CK-MB (cardiac~
and CK-BB (brain). After reléase from tissue and on
circulation in blood the MM and MB forms themselves break
down to smaller fragments known a~ isoforms or subforms.
In the event of myocardial infarction, the MB isoenzyme,
present in cardiac muscle, is released into plasma.
Hence, it serves as a specific diagnostic molecular
marker for cardiac ischemia or necrosis. The early and
rapid detection of this isoenzyme and its isoforms are
very crucial for the diagnosis of myocardial infarction
and for initiating thrombotic therapy.
W O 94/03631 ~ 2 ~c~r/US93/05465
-25-
To perform the test, a blood sample is separat-
ed into plasma and red blood cells. The plasma i5~ mixed
- with excess antibody tagged with a fluorescent dye which
is directed against CK-MB. The attachment of fluorescent
antibodies for a CK assay is known and described in U.S.
Patent No. 4,353,9~2 to M. Gomez et al. If CK-MB is
present in plasma, an immune complex consisting of CK-MB
and fluorescently tagged antibody will be formed. On
application of an electric field, the reaction mixture
consisting of the fluorescent immune complex and the
unreacted fluorescent antibody, will migrate on the gel. `'
Because of charge and mass differences, the labeled
intact immune complex will migrate differently than the
labeled antibody. The fluorescence associated with b~und
and free markers will be detected and arrival times
measured and recorded. Free marker is identified by a
large peak. Any substance within the expected time of
the free substance is regarded to be target analyte.
Anything else is an artifact.
,Example 2
Detection of the Presence
of sexuallY transmitted diseases
Many sexually transmitted pathogens such as
chlamydia, herpes, etc., form lesions in the urogenital
area. For detection of these pathogens, samples are
taken with a swab directly from the lesion and a number of
different types of tests,~lare performed on this extract.
These tests include culture and/or immunochemical tests.
After a lesion is sampled with a swab, the swab
is treated with a solubilization reagent to liberate
micro-organism present. This process will also
solubilize target analytes originating from the micro-
organisms. This extracted solution will be filtered and
reacted with fluorescently tagged antibody so that there
is a substantial excess of unreacted tagging substance.
The differential assay proceeds as described above.
WO94~03631 /J ~ 2 PCT/VS93/05465
-26-
If the difference between the charge, mass,
shape, or combination of these characteristics of the
bound and free substances is great, early separation may
be expected. The results of electrophoresis are predict-
ed at an earlier time than a complete electrophoresisrun.
Exam~le 3
Detection of antibodv to human serum albumin (HSA)
In the following example, an antibody against
HSA is the target substance which is detected by tagging
with fluorescent HSA. HSA, Fraction V, was obtained from
Sigma Chemical Company (St. Louis, Missouri). Monoclonal
anti-HSA was obtained from Biospacific Inc., California.
Cy5-labeled HSA was synthesized by the coupling of Cy5
fluorescent dye to HSA (Biologi~al Detection Systems,
Inc., Pittsburgh, Pennsylvania). This fluorescent sub-
stance is the binding agent.
- Differential separation assay (DSA) was done as
folIows: Cy5-labeled HSA (binding agent) was incubated
with monoclonal anti-HSA (target) at a final concentra-
tion of 400 ng/ml Cy5-HSA and 200 ug/ml anti-HSA in 0.09
M Tris, 0.08 M borate, 0.25 mM EDTA, pH 8.3. A control
sample consisted of CyS-HSA alone at 400 ng~ml without
added antibody. Reactions were performed in l.5 ml
Eppendorf tubes in a total reaction volume of 20 ul.
After incubating the samples at room temperature (20~C)
for 30 minutes, lO ul aliquots were loaded onto 6%
nondenaturing (8 cm x lO cm x 0.75 mm) polyacrylamide`
gels (Jule labs) containing 0.9 M Tris, 0.8 M Borate, 2.6
mM EDTA, pH 8.3. Electrophoresis was performed at lO0 V
for 40 minutes using a Hoefer Mighty Small SE200 system.
The real time detection of fluorescent proteins
during electrophoresis was performed using a He-Ne laser
beam focussed at a point l.3 cm below the wells of the
gel. The emitted fluorescence was collected using a
photomultiplier (PMT) tube. Data was collected using a
Lab-PC from National Instruments (Trademark) (Austin,
~- W094/03631 ~ n 2 PCr/US93/054
-27-
Texas) data acquisition board on the IBM-PC and imported
into a Microsoft Windows (Trademark) Excel (Trademark)
file for analysis and graphics.
Samples containing excess Cy5-HSA were reacted
with excess monoclonal anti-HSA and then were loaded onto
6% acrylamide gels. Separation on this gel system is
based on charge/mass characteristics of the proteins and
more rapidly migrating species migrate past the laser
beam earlier than more slowly migrating protein species.
With reference to Figure 6, the Cy5-HSA peak 91
migrates past the laser beam at approximately 8 minutes.
This is a calibration run to establish a time for free
Cy5-XSA. The immune complex consisting of Cy5-HSA-Anti-
HSA, on the other hand, has a peak 93 which migrates past
the laser spot at 25.5 minutes. This example demon-
strates that the relevant tir~e window for this pair of
binding agent (Anti-HSA) and fluorescent tag (Cy5-HSA~ is
17.5 minutes. The 8 minute peak 91 defines the reference
position in the data acquisition window for finding the
peak of the immune complex Cy5-HSA-Anti-HSA. Peak 95 is
the residual uncomplexed labeled Cy5-HSA.
Exam~le 4
Detectina mediators of sePtic shock
Septic shock is the most common cause of death
in a medical-surgical intensive-care unit. Mortality
rates range from 40% for early phase sepsis to more than
70% for refractory septic shock. Septic shock develops
in a cascade fashion.
Bacterial antigens (including endotoxin)
activate local tissue macrophages, blood monocytes, and
serum complement. Local complement activation induces
~directly and indirectly) migration and activation of
blood neutrophils--as do the macrophage and monocyte
acti~ation products (which include interleukin-1 and
tumor necrosis ~ctor). In addition, activated macro-
phages and lymphocytes produce (again directly or indi-
rectly) molecules that stimulate the endothelial cells to
W094/0363~ 2 Pcr/us93/os46~
-28-
produce more neutrophil chemotactic factors. Several of
these cytokines increase endothelial permeability. This
- in turn promotes blood-neutrophil adhesion and migration.
Once activated, the neutrophil releases cyto-
lytic enzymes and reactive oxidants. Chronic exposure
destroys the local endothelial vasculature. Such
persistent local damage impairs vascular integrity to
such an extent that hemodynamic homeostasis cannot be
restored. Death is the result.
10Unstimulated neutrophils constitutively express
lectin adhesion molecules (LECAM-1). The passive inter- -
action of these molecules with the endothelial cell
ligands, intercellular adhesion molecule-l (ICAM-l), and
endothelial-leukocyte adhesion molecule-l (ELAM-1)
induces neutrophil activation and transmigration. Trans-
migration is promoted by the up-regulation and interac-
tion of neutrophil-specific integrins with endothelial
ICAM-1 and ELAM-l. Tumor necrosis factor, interleukin-8,
lipopolysaccaride (LPS), gamma interferon and
interleukin-l can stimulate endothelial cells to express
neutrophil adhesion molecules.
Endogenous mediators of sepsis are listed in
- Table 1.
- 30
:~ WO94/03631 ~ 2 PCT/US93/05465
29-
Table l
Adhesion molecules
(ELAM-l, ICAM-l, VCAM-l)
Beta-endorphin
Bradykinin
Coagulation factors
(including fibrin, thrombin)
Complement fragments (C3a, C5a)
Elsosenoids (leukotrienes B4, C4, D4, E4,
thromoxane A2, prostaglandins E2)
Endothelin-l
ndothelin-derived relaxing fac~or
Interferon 5
Granulocyte-macrophage colony
stimulating factor
Interleukins (l, 2, 4, 6, 8)
Macrophage derived procoagulant and
inflammatory cytokine
Myocardial depressant substance
Plasminogen activator inhibitors
Platelet activating factor
P~N leukocyte products (toxic oxygen
radicals, proteolytic enzymes)
Serotonin
Transforming growth factor beta
Tumor necrosis factor A
Vascular permeability factor
! These mediators to sepsis can be conveniently
- 30 and rapidly detected by methods and test kits of this
invent~on. In particular, it is important to rapidly
detect LPS, tumor necrosis factor (TNF), interleukin-l,
interleukin-8 and gamma interferon to evaluate the
progress of septic shock. Lipopolysaccaride (LPS) and
monoclonal antibodies to LPS are described in U.S. Patent
No. 5,093,235. Monoclonal anti~odies to TNF interleukin
l and 8 and Y interferon are described in Curre~lt Prorocols in
Immunolo~, suDra.
WO94/03631 ~ PCT/US93/0546~ ~-
-30-
Following the procedures in Example 3, Pxcept
the antibody is labeled and the cytokine is the analyte,
- LPS, interleukin-1, interleukin-2, TNF~, and gamma
interferon are determined.
Example 5
Preparation of second labeled marker
Bovine serine albumin is dissolved at about
1 mg/ml in 50 mM phosphate buffered saline PBS. This
~ovine serum albumin solution is reacted with a solution
of carboxymethylindocyanine succinimidyl ester in
accordance with t~e procedure in Cytomet~ 11: 418-430
~1990). A kit for such labeling is sold by Biological
Detection Systems, Inc., 4617 Winthrop Street,
Pittsburgh, Pennsylvania 15213.
Figure 7 shows the differential migration of
Cy5-BSA peak 97 and Cy5-labeled monoclonal anti-human
serum albumin peak 98. Thus, Cy5-BSA is a suitable
second labeled marker.
The above examples are intended to illustrate
the invention and not to limit it in spirit or scope.
