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United States Patent |
5,206,139
|
Ward
,   et al.
|
April 27, 1993
|
Method for determining the presence or concentration of a bound enzyme
Abstract
There is disclosed a process and a device for detecting and measuring (1)
the amount of enzyme present as a detecting system following a nucleic
acid hybridization reaction or immunoreaction; (2) the level and activity
of free enzyme in a biological sample; (3) the level of enzyme from
contaminating microorganisms present in a sample; and (4) enzymes from
pure culture isolates for microbial identification and antimicrobial
susceptibility testing.
Inventors:
|
Ward; Jr.; N. Robert (Seattle, WA);
Lozier; Philip J. (Seattle, WA)
|
Assignee:
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BioControl Systems, Inc. (Bothell, WA)
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Appl. No.:
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174848 |
Filed:
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March 29, 1988 |
Intern'l Class: |
C12Q 001/68; G01N 033/53; G01N 033/538 |
Field of Search: |
435/174-182,4,7.9,7.91-7.95,803
436/541,807,824,500
422/59,70
|
References Cited
U.S. Patent Documents
4039652 | Aug., 1977 | Adams et al. | 436/541.
|
Other References
Kuchi et al., Chemical Abstract 10(11):86119, "A fluorometric microassay
for monitoring the enzymic activity of GMI-ganglioside,
beta,-galactosidase by use of high performance liquid chromatography",
Anal. Biochem., 140(1), 146-51. 1984.
|
Primary Examiner: Kepplinger; Esther L.
Assistant Examiner: Spiegel; Carol A.
Attorney, Agent or Firm: Seed and Berry
Claims
We claim:
1. A method for determining the presence or concentration of an enzyme
marker bound to an antibody, antigen or strand of nucleic acid generated
in a hybridization assay or immunoassay, comprising the steps of:
adding a substrate in a selected solution to a first column containing the
enzyme marker further bound to a solid phase as a result of the
hybridination assay or immunoassay, said substrate being reactive with
said bound enzyme marker;
incubating the first column to enzymatically convert said substrate to a
product in an amount proportional to the amount of bound enzyme marker
present;
transferring the product and unreacted substrate onto a second column of no
more than 500 theoretical plates, said second column containing sorbent
selectively binding said product in the presence of said selected
solution;
eluting the product from the sorbent; and
detecting the presence or concentration of the product to determine the
presence or concentration of the bound enzyme marker.
2. The method of claim 1 wherein the solution containing the substrate
flows through the first column and out the second column in a continuous
manner.
3. The method of claim 1 wherein the solution containing the substrate
flows through the first column and out the second column via a stopped
flow cycle.
4. The method of claim 1 wherein the product is detected by absorbance,
fluorescense, luminescence or by electrochemical means.
Description
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method and a device for the rapid and
sensitive detection and measurement of an enzyme which (1) is used as part
of the detection or reporting system for an immunoassay or nucleic acid
hybridization assay, (2) is present in a biological sample, (3) is
associated with a microorganism for detection of the microorganism in a
sample, and (4) is used for pure culture tests, such as microbial
identification and antimicrobial susceptibility tests.
BACKGROUND OF THE INVENTION
Nucleic acid hybridization and antibody immunoassay technologies have been
developed that permit rapid, sensitive and specific measurements of
organic compounds and microorganisms. Recent advances have been directed
toward improving the sensitivity and specificity of these assay systems by
enhancing the detection or "reporting" of the antigen-antibody complex or
nucleic acid hybrid duplex which is formed. Many approaches have been
attempted in this regard. One such example is the multiple labeling of an
antigen, antibody or nucleic acid probe with an enzyme to produce a
nonisotopic and highly sensitive diagnostic test. For example, multiple
copies of enzyme can be chemically coupled to a molecule of avidin. The
avidin can then bind strongly to biotin, which has been chemically linked
to an antibody, antigen or a nucleic acid probe. The result is the
presence of multiple copies of enzyme for every antigen-antibody complex
or nucleic acid hybrid formed. Another approach used in nucleic acid
hybridization assays is the use of multiple enzyme-labeled probes that
hybridize to different sequences on the target genome. Whichever approach
is used to amplify the biological signal, the result of the assay is
usually determined by the development of a distinct color or fluorescence
that is read visually or with an instrument.
The detection of specific nucleic acid sequences through the use of
hybridization probes is a well established procedure. One commonly used
method involves the immobilization of the target polynucleotide sequence
on a solid support (e.g., nitrocellulose, diazobenzyloxymethyl cellulose,
nylon, etc.). The immobilized nucleic acid is then denatured, if it is
double stranded, and subsequently hybridized to a complementary probe. The
probe nucleic acid sequence is labeled isotopically, usually with .sup.32
P, or nonisotopically with direct labeling of the polynucleotide sequence
with an enzyme or indirectly with a biotin-avidin system.
In contrast to radioisotopically labeled probes, nonisotopic systems offer
advantages of safety, relatively low cost, and ease of use. However,
enzyme detection often suffers from high background values from the
nonspecific adsorption of labeled probes to the solid support.
Non-specific adsorption may be reduced with multiple washing steps, which
add to the length and difficulty of the procedure.
A different method for detecting a specific polynucleotide sequence
involves the displacement of a labeled nucleic acid, according to the
method of Vary .et al., "Nonisotopic Detection Methods for Strand
Displacement Assays of Nucleic Acids," Clin. Chem. 32:1696-1701 (1986). A
labeled polynucleotide "signal strand" is hybridized with a larger
sequence (the "probe strand"), which is, in turn, complementary to the
target polynucleotide sequence of interest. Interaction of the target
sequence with the signal-probe hybrid results in the displacement of the
signal strand from the hybrid. After separating the displaced signal
strands from the signal-probe hybrid, the signal strand is measured using
an isotopic label such as .sup.32 p or nonisotopic labels such as an
enzyme. Such assays are potentially more sensitive because of the
reduction of background signal due to nonspecific adsorption.
Two types of enzyme immunoassays are commonly used. The sandwich
immunoassay involves the capturing of antigen molecules in a solution by
solid phase-bound antibody molecules. A second antibody molecule, which is
enzyme-labeled and specific to a different antigenic determinant, is
subsequently added to the solid phase-bound antigen-antibody complex.
Similarly, the competition immunoassay involves the competition of
antigens for antibody binding sites. Enzyme-labeled antigen and unlabeled
antigen from the sample (the antigen of interest) compete for binding
sites on the solid phase bound antibody. In these cases, the amount of
enzyme remaining on the solid support is either proportional, in the first
example, or inversely proportional in the second example, to the amount of
antigen in the sample.
Attempts at increasing the sensitivity of enzyme immunoassays (EIA) and
hybridization assays have frequently focused on increasing the amount of
product generated per antigen-antibody complex or hybrid formed by
increasing the number of labeled enzyme molecules. Enzyme amplification
often results in an increase in false positive reactions due to increased
nonspecific adsorption or an increase in false negative reactions due to
inhibition of antigen and antibody binding or hybridization by
complementary polynucleotide sequences.
Little effort has been directed towards increasing assay sensitivity by
enhancing the measurement of the signal or "product" that is generated by
the enzyme reacting with the substrate. Frequently, the assay sensitivity
is reduced because of a high background signal. The measurement of
extremely low levels of colored or fluorescent enzyme-generated product by
an instrument is often compromised by the inherent color or fluorescence
of the substrate. This problem can be further exacerbated by the common
use of high concentrations of substrate to accommodate a low binding
affinity of the enzyme. Background signal can also result from assay and
sample components that are colored, fluorescent, luminescent or
electrochemically active. In most cases, a positive result is reported
only when the enzyme-generated signal is twice the background signal.
In addition to the use of enzymes for detecting immunoreactions and
hybridization reactions, little progress has been made for increasing
assay sensitivity for detecting free enzymes in a sample as well as
enzymes produced by microorganisms. Assays to measure and detect free
enzymes and microbial enzymes in a biological sample generally utilize
substrates that produce enzyme-generated products that are colored,
fluorescent, luminescent or electrochemically active. The sensitivity of
these assays is most hindered by a high background signal from sample
constituents and assay components including substrate.
One attempt to enhance the measurement of an enzyme-generated product was
described by Kiuchi et al. (A Fluorometric Microassay Procedure for
Monitoring the Enzymatic Activity of GMl-Ganglioside-B-Galactosidase by
Use of High-Performance Liquid Chromatography, 1984, Anal. Biochem.
140:146-151). These investigators utilized a high performance liquid
chromatography (HPLC) system to measure the
GMi-ganglioside-.beta.-galactosidase activity in crude tissue samples by
measuring increased NADH concentration. The biological steps of this
procedure, including the incubation of sample with substrate, were
conducted in a vessel separate and apart from the HPLC instrument.
Following incubation of the substrate and enzyme from the sample, the
reaction solution was injected into an HPLC instrument which separated the
various assay components. The disadvantage of this procedure is that a
conventional HPLC column with a high number of theoretical plates is
required to sufficiently separate the components. This means that the
separation procedure of Kiuchi et al. is a lengthy procedure and requires
the use of an expensive HPLC instrument which is capable of moving fluids
through the large column at high pressures, often in excess of 3,000 psi.
The column used by Kiuchi et al. had the dimensions of 4 mm.times.300 mm
and was packed with reverse phase C18 particles. A column of this type
will typically have in excess of 15,000 theoretical plates at optimal
linear efficiency.
Wehmeyer et al. (Liquid Chromatography with Electrochemical Detection of
Phenol and NADH for Enzyme Immunoassay, 1983, J. Liquid Chromatography
6:2141-56) refers to an enzyme immunoassay procedure with a smaller HPLC
column with the dimensions of 50 mm.times.2 mm to separate phenol from
other components in the reaction solution. Phenol was generated by the
enzymatic cleavage of phenylphosphate. Similar to the procedure at Kiuchi
et al., Wehmeyer et al. performed the enzyme immunoreaction in a vessel
separate from the HPLC instrument. After sufficient incubation time for
the enzyme and substrate in this vessel, the reaction solution was
injected into the HPLC instrument. Wehmeyer et al. needed a long HPLC
column to accomplish sufficient separation of phenol. The problem with a
long HPLC column is an increase in analysis time and the required use of
HPLC rated components which can handle high pressure as a result of the
use of a long column. Also, extraneous materials in the reaction solution
can potentially co-elute with phenol resulting in a significant reduction
in overall assay sensitivity and specificity.
Therefore, there is a need in the art for a method and device to increase
the sensitivity of nonisotopic immunoassays and nucleic acid hybridization
assays that use enzymes for reporting assay results. Additionally, there
is a need in the art for methods for measuring and detecting free enzymes
from microorganisms in a sample and from microorganisms in pure culture.
DISCLOSURE OF THE INVENTION
Briefly stated, the present invention discloses methods and associated
devices for enhancing the detection of a bound marker enzyme which has
been generated by an immunoreaction or by a hybridization reaction in an
assay system. The method generally comprises: (a) adding a substrate in a
selected solution to a first column containing a complementary (to the
substrate) marker enzyme bound to a solid phase; (b) incubating the first
column to enzymatically convert the substrate to a product in an amount
proportional to the amount of enzyme present; (c) transferring the product
and unreacted substrate onto a second column of no more than 500
theoretical plates and preferably approximately 100 theoretical plates,
and most preferably about 50 theoretical plates, the second column
containing a sorbent capable of selectively binding the product in the
presence of the selected solution; (d) selectively eluting the product
from the solution; and (e) detecting the presence or concentration of the
product. The product may be detected by measurement of absorbance,
fluoresence, luminescence or by electrochemical activity and may be
adapted to a continuous flow or stop flow cycle configuration. The sorbent
may selectively retain the product through polar or non-polar
interactions, ion exchange, other specific molecular interactions such as
affinity binding, or combinations thereof. The method may be performed
using a manual format or with an instrument with a fluidics system with a
continuous flow or stopped flow configuration.
The result is a greatly reduced or completely eliminated background signal.
Also, the enzyme-generated product is concentrated from a large reaction
volume to a small detection volume. Further, the use of a sorbent bed with
minimal column length and theoretical plates results in a low pressure
system, a rapid separation of product from other assay components, and a
reduction of reagent requirements.
In a related aspect of the present invention, the device generally
comprises: (a) a first column containing an enzyme bound to a solid
support, the enzyme present from an immunoreaction or a nucleic acid
hybridization reaction; and (b) a second column connected in series to the
first column and containing a sorbent bed of no more than 500 theoretical
plates and preferably approximately 100 theoretical plates and most
preferably approximately 50 theoretical plates, wherein the sorbent is
capable of selectively binding an enzymatically generated product which
has been produced through the addition of a substrate specific to the
enzyme in the first column. In a related aspect of the device, a detection
device is connected in series to the second column, the detection device
being capable of measuring the amount of product eluted from the second
column. The use of connected columns eliminates the necessity of
performing an enzyme immunoassay or hybridization assay in a separate
vessel and then injecting the reaction solution into a second detection
instrument.
In a further related aspect of the present invention, the device comprises
a second column containing a sorbent bed of no more than 500 theoretical
plates, and preferably approximately 100 theoretical plates and most
preferably, 50 theoretical plates, wherein the sorbent is capable of
selectively binding an enzymatically generated product, said product is
produced through the addition of a substrate specific to the free enzyme
of interest in a sample, or to enzymes produced by microorganisms of
interest in a sample, or in a pure culture. In a related aspect, a second
column is connected in series to a detection device capable of measuring
the amount of product eluted from the second column containing the
sorbent.
These and other aspects of the present invention will become evident upon
reference to the following detailed description and attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1a-1c depict various arrangements of the two columns. Column 1 (the
first column) is used to retain the solid support materials upon which the
nucleic acid hybridization reaction or immunoreaction is performed. Column
2 (the second column) is used to contain the sorbent material. The two
columns may be separated with a switching valve located between the
columns as indicated in FIG. 1a or the columns may be present in a single
unit but separated by a barrier, such as a frit or membrane, as indicated
in FIG. 1b. A multiple column arrangement may be configured into an
injection molded plastic unit with flow connectors between columns (FIG.
1c). The paths by which fluids flow through the columns and connectors is
determined by switching valves located external from the plastic unit.
FIG. 2 details a system design for a microprocessor-controlled instrument
that utilizes pressurized gas to move reagents and sample. Metering pumps
can be substituted for the pressurized gas. FIG. 2 illustrates the use of
a plastic unit as detailed in FIG. 1c with switching valves located
external to the plastic unit to direct flow through the columns.
FIG. 3 illustrates a manual plastic disposable device with a two-column
arrangement for enzyme detection and measurement. In this case, the two
columns are separated by a frit. Two directional movement of fluids
through the columns is accomplished by manual movement of the plastic part
containing the columns in and out of the receiving vessel. A rubber "0"
ring located on the insertion end of the unit with the columns forms a
seal with receiving vessel.
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses a method and a device for increasing the
sensitivity of immunoassays or nucleic acid hybridization assays. The
device includes two columns connected in series, permitting low pressure
movement of solutions through columns; the first column is used to perform
a solid phase immunoreaction or nucleic acid hybridization reaction and
the second column is packed with sorbent particles. The first column
contains the means for conducting an immunoreaction or nucleic acid
hybridization reaction or the means for capturing solid support materials
such as latex beads upon which the immunoreaction or hybridization
reaction has been performed. Similarly, the product of the immunoreaction
(antigen-antibody complex) or hybridization reaction (probe-target duplex)
which is formed in solution outside of the first column, may be retained
on the first column by physical means such as on the surface of a membrane
or by chemical means such as through covalently binding onto an affinity
membrane (e.g., ULTRABIND.TM., Gelman Sciences, Ann Arbor, Mich.), through
nonspecific binding of antigen-antibody complexes by Protein A coated to a
solid support material, or through an antigen (or hapten)-antibody
reaction on a solid support material. Once the enzyme is present on the
first column as a result of the immunoreaction or hybridization reaction,
a substrate specific to the enzyme is added to the first column under time
and incubation conditions sufficient to allow the enzyme and substrate to
react to form a product. The product of the enzyme reaction is produced in
proportion to the amount of enzyme present as a result of the
immunoreaction or hybridization reaction.
Briefly, the first column contains an enzyme bound to a solid support as
the result of a nucleic acid hybridization reaction or immunoreaction. A
substrate, which is specific for the enzyme and is in a solution which
permits substantial or complete retention of the enzyme-generated product
onto the sorbent in the second column, is added to the first column and
allowed to incubate under appropriate conditions. The solution containing
the product and unreacted substrate is then allowed to flow into the
second column using an instrument-based fluidics system, such as the one
shown in FIG. 2, or using a manual format as shown in FIG. 3. The second
column functions to bind the product that flows through it, through use of
the sorbent. Preferably, the substrate passes substantially through the
column. The ability of the sorbent to bind the product is dependent upon
the chemical nature of the solution carrying the product and substrate.
This allows the product to be concentrated on the sorbent contained within
the second column. Subsequent elution results in the concentration of the
product, substantially free of substrate and other extraneous materials,
into a volume generally smaller than the original volume of solution
applied to the second column. The use of a second column with a low number
of theoretical plates, as a function of column length and inner diameter,
permits the use of a low pressure diagnostic test that can be performed
rapidly and in a manual format, or with a low pressure instrument. The
detector for the instrument may be located downstream from the second
column, as is shown in FIG. 2.
In another embodiment, the assay system is used for (i) the detection of
free enzymes of interest in a sample, (ii) for detection of microbial
contaminants of interest in a sample, and (iii) for identification and
antimicrobial susceptibility testing of pure culture microorganisms. For
the detection of free enzymes, the assay is performed by adding a sample
to an assay solution containing a substrate, and incubating at an
appropriate temperature. The free enzymes specifically chemically modify
the substrate to produce a product that can be retained on the sorbent
bed.
An example of a free enzyme determination in a biological sample is the
detection of alkaline phosphatase in fluid milk. A loss of alkaline
phosphatase activity in milk is indicative of the effectiveness of
pasteurization of fluid milk. A fluorogenic substrate for alkaline
phosphatase, 4-methyl-umbelliferyl phosphate (MUP) is added as a substrate
to a sample of fluid milk. If the alkaline phosphatase enzyme is present,
it cleaves the MUP to produce the product methylumbelliferone (MU). MU can
be concentrated on a sorbent bed as described herein, and then measured by
fluorescence detection.
Similar to the free enzyme assay, the detection of microbial contaminants
in a sample involves incubation of the sample at an appropriate
temperature in an assay solution containing a substrate. The assay
measures the amount of enzyme produced by the microorganisms in the sample
to detect and estimate the level of microorganisms. These enzymes may be
present within the cell or released from the cell. For example, coliform
bacteria produce the enzyme .beta.-galactosidase and E. coli produce the
enzyme glucuronidase. The incubation of a substrate such as
4-methylumbelliferyl-.beta.-D-galactoside MUGAL with a sample containing
coliform bacteria or the incubation of the substrate
4-methylumbelliferyl-.beta.-D-glucuronide (MUG) with a sample containing
E. coli can produce the fluorescent product MU in proportion to the levels
of these microorganisms in the sample.
Identification and antimicrobial susceptibility tests can be performed on a
suspension of a pure culture isolate. Substrates and other reagents for
these pure culture tests may be similar to those used in European Patent
No. EP-A-91,837 and by Snyder and Wang ("Rapid Characterization of
Microorganisms by Induces Substrate Fluorescence: A Review," Biotechnology
Progress 1:226-230, 1985) and by Snyder, et al. ("Pattern Recognition
Analysis on In Vivo Enzyme Substrate Fluorescence Velocities in
Microorganism Detection and Identification," Appl. Environ. Microbiol.
51:969-977). Useful substrates include indoxyl acetate,
indoxyl-.beta.-D-glucoside, 4MU-D-Glucoside, 4MU-phosphate, indoxyl
phosphate, 4-MU-D galactoside, N-methyl indoxyl acetate, N-methyl indoxyl
myristate, .beta.-naphthyl acetate, .alpha.-naphthyl acetate,
4MU-heptanoate, 4MU-acetate, 5-cromoiodoxyl acetate,
5-bromo-4-chloro-3-phosphate, 3-indoxyl phosphate,
6-bromo-2-naphthyl-.beta.-D-glucoside, 4MU glucuronide, 7-ethoxycoumarin,
glycyl-L-phenyl-.beta.-naphthylamide, .beta.-naphthyl sulfate, 3-indoxyl
sulfate, luminol, resazurin, fluorogenic 4-methylumbelliferyl derivatives,
derivatives of 7-amino-4-methylcoumarin, a mixture of 4-methylumbelliferyl
phosphate and 4-methylumbelliferyl fatty acid ester such as the hexonate,
octanoate, nonanoate or other fatty acid ester with a chain length of
C.sub.6 -C.sub.16, and a mixture of 4-methylumbelliferyl ester such as
phosphate and 7-(N)-(aminoacyl-4-peptidyl)-4-methyl-7-amino-coumarin (such
as 7-(N)-alanyl-4-methyl-7-amino coumarin and the corresponding leucine
derivative. Useful fluorogenic substrates include peptides and esters (in
themselves known materials) of umbelliferone, 4-methylumbelliferone,
3-carboxy-7-hydroxycoumarin, 3-acetyl-7-hydroxy coumarin,
3-carboxyethyl-7-hydroxycoumarin, 3-cyano-7-hydroxycoumarin,
7-amino-4-methylcoumarin, 7-amino-4-trifluoromethylcoumarin,
2-naphthylamine, 4-methoxy-8-naphthylamine, naphthol-AS
(3-hydroxy-2-naphthoic acid anilide, indoxyl 1-(alpha)-and
2-(beta)-naphthol derivatives including N-methyl indoxyl acetate and
indoxyl acetate, resorufin, 1-methyl-7-hydroxyquinolinium iodide, and
6-amino-quinoline. For microbial identification, the culture suspension is
added to solutions, each containing a different substrate, and incubated
at an appropriate temperature. Multiple enzyme tests may be formatted into
a panel of tests. Depending upon the type of microorganism, certain
substrates will be cleaved by specific enzymes produced by the
microorganism to produce a characteristic pattern. The product of the
enzyme reaction may be fluorescent, colored, electrochemically active or
luminescent. Fluorogenic substrates such as 7-amino-4-methylcoumarin are
derivatized to amino acids such as alanine, leucine and valine or to
short-chain peptides or proteins or 4-methylumbelliferone derivatized to
compounds such as acetate, propionate and phosphate, fatty acids such as
oleate and heptanoate, and sugars such as galactose, fucose, arabinose and
glucose. When these fluorogenic substrates are hydrolyzed by the microbial
enzymes, 4-methylumbelliferone (MU) and 7-amino-4-methylcoumarin (AMC) are
liberated.
The products are fluorescent and will strongly bind to a C18 sorbent. The
AMC and MU can be eluted from the C18 sorbent with 100% methanol and can
be measured with a fluorometer.
Recognition of the specific enzymes that a particular microorganism
produces permits identification of the pure culture isolate. Antimicrobial
susceptibility patterns can be obtained by measuring a reduction in the
amount of enzyme produced by a pure culture isolate in the presence of
certain antimicrobial compounds. Reduction in the amount of enzyme
produced by the pure culture isolate is directly related to the
susceptibility of that microorganism to the antimicrobial. Fluorogenic
substrates such as 4-methyl-umbelliferyl phosphate (MUP),
4-methylumbelliferyl nonanoate (MUN) and L-alanyl-7-amido-4-methylcoumarin
(AAMC) can be used for the antimicrobial susceptibility testing. A
reduction in the amount of fluorescent product produced, MU and AMC, from
enzymatic cleavage, is indicative of a decrease in the level of enzyme
related to microbial inhibition by the antimicrobial.
In each of these cases, the incubated solution is added to a second column
containing the sorbent. The second column contains a sorbent of, at most,
500 theoretical plates and preferably approximately 100 theoretical plates
and most preferably approximately 50 theoretical plates. The second column
functions to bind the product formed by incubation of the substrate with
the biological sample. Separation of the product from the substrate is
achieved as described herein.
As noted above, the immunoreaction or hybridization reaction can be
performed on a solid support, such as latex beads or sephacryl beads or
latex-coated frits within the first column. Also, the immunoreaction or
hybridization reaction can be performed outside of the first column on a
solid support system, with the first column being used to retain the solid
support after completion of the immunoreaction or hybridization reaction.
Further, the products of the immunoreaction (antigen-antibody complex) or
hybridization reaction (hybrid duplex) can be retained on the first column
by physical or chemical means. A detection system is provided through use
of the enzyme label which is directly and chemically linked to an antigen,
antibody or nucleic acid probe, or indirectly labeled to any part of the
assay reactants using, for example, avidin-biotin binding. The enzyme is
part of the antigen-antibody complex or the hybrid duplex if the
immunoreaction or hybridization reaction is completed outside of the first
column. Immunoreactions or hybridization reactions conducted on a solid
support or in solution outside of the first column can involve multiple
manual steps, including the sequential addition of reagents, incubations
and washings. These steps may be performed automatically by a programmed
instrument. For example, the addition of reagents to the solid phase
support system, the washing and the incubation steps may all be conducted
by a microprocessor-controlled fluidics system.
In the case of an immunoreaction that is performed on the surface of a
solid support, antigen or antibody is covalently attached or passively
adsorbed to the surface of the solid support. If an antibody is attached
to the support, then the antigen of interest in the .reaction sample can
be detected with a competition immunoreaction or a sandwich
immunoreaction. In the case of a competition immunoreaction, an
enzyme-labeled antigen (the antigen is identical to the antigen of
interest) is mixed into the sample to be tested. This mixture is then
passed over the antibody-coated surface where the unlabeled antigen and
the enzyme-labeled antigen compete for binding sites on the surface of the
solid phase. The amount of enzyme remaining on the solid support surface
provides a quantitative measurement of the level of unlabeled analyte or
antigen of interest in the sample.
In the case of a competition immunoreaction performed in solution,
unlabeled antigen and a standardized amount of enzyme-labeled antigen are
mixed with a standardized concentration of antibody in an assay solution.
The concentration of the enzyme-labeled antigen and antibody should be
standardized such that the ratio of antigen determinant to antibody
binding site is approximately 1:1. After the immunoreaction is completed,
the assay solution is introduced into Column 1 containing solid support
material coated with Protein A. The Protein A binds to the Fc portion of
the antibody molecule and retains the immune complex that has been formed
in solution. The amount of enzyme remaining attached to the Protein A
solid support is inversely proportional to the amount of unlabeled antigen
of interest in the sample.
In a sandwich-type immunoreaction, the antibody molecules on the solid
support surface bind to the specific antigen of interest in the sample to
be tested. Unreacted antigen and extraneous materials are then removed
through a wash step. A second antibody molecule, often specific for a
different antigenic determinant on the antigen molecule, is enzyme labeled
and added to the solid phase system. The solid phase-antibody-antigen
complex will bind the antibody-enzyme. Through this mechanism, the enzyme
label is bound to the solid support as part of an
antibody-antigen-antibody sandwich-type complex. The amount of enzyme
remaining attached to the solid phase after washing is proportional to the
amount of antigen in the sample of interest. The solid support can be part
of the first column. Alternatively, the solid support can be a latex bead
which is later collected in the first column after completion of the
immunoreaction.
In the case of a hybridization reaction, oligonucleotide strands that are
specific for and complementary to the target DNA or RNA are covalently
connected to the surface of the solid phase. Preferably, the
oligonucleotide strands are connected to the solid phase at one end of the
oligonucleotide strand. If a sandwich-type hybridization reaction is
performed, DNA or RNA from the microorganism or another source to be
tested is chemically or mechanically released, denatured, and hybridized
to the strands on the solid phase under appropriate solution conditions.
An enzyme labeled nucleic acid probe that reacts specifically to a
different base sequence of the captured target sequences is then permitted
to hybridize to the complementary nucleic acid strand on the target
strand. This enzyme labeled probe is captured by the target nucleic acid
sequence on the solid phase and acts to report the presence of the target
strand on the solid phase. Therefore, with a hybridization reaction, the
presence of an enzyme bound to the solid phase serves to indicate the
presence or absence of specific nucleic acid sequences of interest.
A competition nucleic acid hybridization reaction is performed by using an
enzyme labeled nucleic acid probe that is complementary to the strand of
nucleic acid on the solid phase. The enzyme-labeled probe also contains a
nucleic acid sequence that is identical to or substantially similar to a
target sequence of interest on a polynucleotide strand from a biological
sample. The enzyme labeled nucleic acid probe is mixed with the target
nucleic acid from the sample of interest. Both the labeled and the
unlabeled target sequence compete to hybridize to complementary sequences
bound to the solid phase. The amount of enzyme remaining on the solid
phase is inversely proportional to the amount of target nucleic acid
sequence in the sample of interest.
A hybridization reaction can also be performed in solution and the hybrid
captured in the first column. An example is the capturing of a nucleic
acid hybrid formed in solution with an antigen and antibody reaction in
the first column. In this case, two polynucleotide probes which are
complementary and specific to two unique sequences on a target
polynucleotide strand are used. One of these probes is labeled with enzyme
and the second probe is labeled with hapten. The probes hybridize with the
target strand under appropriate assay conditions. After the hybridization
reaction is completed, the assay solution containing the hybridization
complex is introduced into the first column which contains solid support
material coated with antibodies specific to the hapten. The antibodies
retain the hybridization complex. This results in the presence of enzyme
on the first column.
A second example involves the use of antibodies that specifically react
with a DNA:DNA hybrid or a RNA:DNA hybrid. In this case, one of the
complementary strands is labeled with enzyme. As was described above,
after hybridization in solution is completed, the assay solution
containing the hybrid is introduced into the first column containing solid
support materials coated with antibodies to the DNA:DNA hybrid or RNA:DNA
hybrid. This retention of the DNA:DNA or RNA:DNA hybrids results in the
presence of enzyme in the first column, the amount of enzyme present in
the first column is proportional to the amount of target nucleic acid in
the sample of interest.
As briefly described above, the first column may contain the solid phase
material upon which the immunoreaction or hybridization reaction is
performed to generate a bound marker enzyme. Alternatively, the first
column may be used to retain the solid phase materials from an
immunoreaction or hybridization reaction which had been performed outside
of the first column to capture the marker enzyme. The retention of the
solid phase materials in the first column can be accomplished with a
physical barrier such as a frit or membrane at one opening of the first
column. Further, the first column may act to capture an antibody-antigen
complex, or a probe-target oligonucleotide duplex, both containing an
enzyme, formed in solution outside of the first column. Each alternative
described above results in the presence of a reporting enzyme bound to a
solid phase material in either direct or inverse proportion to the
molecule, antigen, nucleic acid or microorganism of interest. The present
invention discloses a process and a device for amplifying the signal
generated by the enzyme by reducing the background signal and by
concentrating the product into a smaller detection volume.
As noted above, once the enzyme label is bound to the solid support, a
specific substrate, in a solution which permits complete binding of the
enzyme-generated product, is added to the first column. The substrate
reacts with the bound enzyme, thereby generating a product. The
appropriate reaction conditions, such as time, temperature and pH are
adjusted in accordance with the characteristics of the specific enzyme
involved. Preferably, the substrate is present in a solution which allows
(a) optimal enzyme activity; (b) complete binding of the enzyme-generated
product on the sorbent; and (c) little or no binding of the substrate on
the sorbent. Many enzymes show high activity in buffered solutions such as
0.01-0.1M phosphate buffer (pH 6-8), 0.001-0.1M Tris buffer (pH 6-8),
0.001-0.1M borate buffer (pH 6-9) or 0.001-0.1M bicarbonate buffer (pH
7-9). A further description of the retention of the enzyme-generated
product relative to sorbent and reaction solution is provided below.
After incubation, the solution containing the unreacted substrate and the
product generated from the reaction of the substrate with the bound marker
enzyme is transferred to a second column. The fluidic design for the
solution transfer between columns permits direct contact of the two
columns separated by a permeable device, or by a conduit between columns
or by a microprocessor-controlled fluidics device that can move solutions
from a first column to a second column.
The second column functions to substantially separate the product from the
substrate in solution. The second column contains a sorbent that affects
this separation by retaining the enzyme-generated product relative to the
unreacted substrate and other contaminants. This separation enhances the
detection of extremely low amounts of enzyme bound to the solid support in
the first column.
The sorbent may be any material that can separate the product from
substrate. Suitable sorbents generally affect this separation through
polar, non-polar or ion interactions with the product. The complete
retention of the enzyme generated product on a particular sorbent with
little or no retention of substrate is dependent upon the type of sorbent,
the chemical nature of the product and substrate, and the solution used to
conduct the enzyme reaction. Table 1 illustrates the interdependence of
these components.
TABLE 1
______________________________________
Chemical
Nature of Chemical
Classes
Functional Enzyme- Nature of
Preferred
of Groups on Generated Specific
reaction
Sorbents:
Sorbent Product: Substrate:
Solution:
______________________________________
Non- Octadecyl Substan- Substan-
Polar
polar (C18).sup.1
tially tially solution
Octyl (C8) non-polar polar (general-
Ethyl (C2) ly under
Cyclohexyl 0.1 M)
(CH)
Phenyl (PH)
Polar Cyanopropyl
Substan- Substan-
Non-polar
(CN) tially tially solution
Diol (20H) polar non-polar
Silica (SI)
Aminopropyl
(NH.sub.2)
N-propyl-
ethylene
diamine
(PSA)
Ion- Benzene- Negatively
Substan-
pH between
exchange
sulfonyl- or posi- tially pKa's of
propyl (SCX)
tively uncharged
product
Sulfonyl- charged or has a
and sor-
propyl counter bent; low
(PRS) charge ionic
Carboxy- to the strength
methyl product
(CBA)
Diethyl-
amino-
propyl
(DEA)
Trimethyl-
aminopropyl
(SAX)
______________________________________
.sup.1 Types of functional groups that are chemically linked to the
particulate support such as silica are indicated as well as common
commercial designations for sorbents with these functional groups.
The description of a non-polar sorbent for retention of an enzyme-generated
product is provided for illustration. Preferably, a non-polar sorbent is
utilized, such as a bonded C8 to C22 silica. Most preferably, the
non-polar sorbent is a C18 silica sorbent, a C18 styrene divinyl/benzene,
or a C18 alumina. Non-polar sorbent separates product from substrate based
upon differences in the relative degree of hydrophobicity. The C8 to C18
non-polar sorbents are commonly constructed using activated silica or
alumina with carbon chains of various lengths extending from the surface.
The numeric designation of C8 or C18, for example, refers to the number of
carbon atoms in the chain. The carbon chain creates a non-polar region
around the bonded silica. Compounds that are substantially non-polar in
nature, or compounds that contain non-polar regions are added to the
sorbent bed in a solution that is as polar as possible, such as water. In
the situation of a polar solvent, the substantially non-polar compounds
will associate with the non-polar regions of the sorbent. When non-polar
sorbents are used, the enzyme-generated product and substrate should be
moved to the sorbent bed in the second column by a solvent that is
essentially polar.
A compound will elute off of the sorbent bed when the compound is more
attracted to the eluting solvent than to the sorbent. The sorbent and the
solvent for a particular product and substrate are chosen such that the
product is retained on the sorbent bed until the elution solvent is added
to the second column. An increased assay sensitivity is realized from the
elution of the product from the sorbent bed into a very small volume of
solvent.
The chemical nature of the substrate and product dictate the appropriate
type of sorbent and solvent or reaction solution used. For example, Table
2 below lists examples of enzymes and substrates and resulting products
generated. Table 2 also lists appropriate combinations of sorbent and
eluting solvent that can be used to separate the product from the
substrate.
TABLE 2
__________________________________________________________________________
Retained Eluting
Enzyme Substrate
Product Sorbent
Solvent
Detector
__________________________________________________________________________
.beta.-Galac-
4-Methylum-
Methylum-
C-18 Methanol
Fluoro.
tosidase
belliferyl-
belliferone
.beta.-D-Galac-
toside
.beta.-Glucu-
4-Methylum-
Methylum-
C-18 Methanol
Fluoro.
ronidase
belliferyl-
belliferone
.beta.-D-Glucur-
onide
Glucosi-
4-Methylum-
Methylum-
C-18 Methanol
Fluoro.
dase belliferyl-
belliferone
.alpha.-D-Gluco-
side
Alkaline
4-Methylum-
Methylum-
C-18 Methanol
Fluoro.
Phosphate
belliferyl
belliferone
phosphate
Protease
4-Methylum-
Methylum-
C-18 Methanol
Fluoro.
belliferyl
belliferone
casein
Esterase
4-Methylum-
Methylum-
C-18 Methanol
Fluoro.
belliferyl
belliferone
laurate
Glucose-
NAD+ NADH C-18 Methanol
Flouro
6-Phos- meter or
phate De- electro-
hydrogenase chemical
detector
.beta.-Galac-
Nitrophen-
Ortho- C-18 Aceto- spectro-
tosidase
yl-thio-.beta.-
Nitrophenol nitrile
photo-
D-Galacto- meter or
pyranoside electro-
chemical
detector
Alkaline
p-Nitro-
p-Nitro-
C-18 Aceto- spectro-
Phospha-
phenyl phenol nitrile
photo-
tase phosphate meter or
electro-
chemical
detector
Lactate
Lactic Acid
Pyruvic Cation
Salt Fluoro.
Dehydro-
NADH Acid Exchange
genase NAD+
Peptidase
Peptide
Free Amino
C-18/ Methanol/
Fluoro.
Chain Acids w/
Anion Salt
addition of
exchange
OPA (orth-
ophthaldial-
dehyde)
Aminopep-
L-Arginine
Amino- C-18 Methanol
Fluoro.
tidase B
Aminomethyl
methyl-
coumarin
coumarin
Pyruvate
ADP and
ATP' Ion acidic Lumino-
kinase phospho-
pyruvate
ex- ammonium
meter
enol change
phosphate
pyruvate (NH.sub.2)
buffer
__________________________________________________________________________
Pyruvate Kinase's enzyme generated product, ATP, is mixed with the enzyme
luciterase, or the ATP is passed through a column which contains
immobilized luficerinase, to generate light, which is measured by a flow
luminometer.
For a particular type of sorbent, the appropriate selection of the
substrate-enzyme combination and the reaction solution (solvent) is
essential for the practice of this invention. The substrate and enzyme
generate a product which is retained strongly on the sorbent and is
measurable with a detection device (fluorometer, spectro-photometer,
luminometer, electrochemical detector). Further, the enzyme generates a
product which is substantially chemically distinct from the substrate. For
example, the substrate may contain both polar and non-polar groups. The
enzymatic cleavage of this compound produces a measurable product which is
substantially non-polar in nature. An example of this substrate-enzyme
combination is the substrate 4-methylumbelliferyl B-D-galactoside which
has both a non-polar moiety (the methylumbelliferyl group) and a polar one
(D-galactose). This compound is enzymatically cleaved by the enzyme
B-galactosidase to produce galactose and methylumbelliferone. The
methylumbelliferone moiety is non-polar and fluorescent. Similarly, the
substrate might be substantially ionic (positively or negatively charged),
whereas the product produced by enzyme activity has a countercharge to the
substrate or has no charge. This product can be separated from the
substrate by an ion exchange sorbent.
For a non-polar sorbent, it is preferable that a substrate is substantially
polar when the product produced by the enzyme is substantially non-polar.
If the substrate is sufficiently polar, then the substrate present in a
polar environment will not be retained on the non-polar sorbent and will
be removed from the sorbent column by the flow stream. Substrate molecules
that have some non-polar regions may require the use of solutions that are
made more non-polar so that they are not retained on the sorbent bed. A
solution can be made more non-polar, for example, by the addition of
methanol, acetonitrile, or tetrahydrofuran to water. A non-polar product
can be retained on a non-polar sorbent, such as C18, without the unreacted
substrate being retained. The product is then eluted off the sorbent using
a non-polar elution solvent, such as methanol.
More specifically, in one embodiment of the invention, the first column
containing either antibodies or nucleic acid strands bound to a solid
support is placed into an open ended column. The first column is placed
into a fluidics system such that sample and reagents are permitted to flow
into the column to contact the solid support surface. If a sandwich type
immunoreaction or hybridization reaction is performed, for example, the
sample is passed over the support and the target antibody or target
nucleic acid strand is captured. The enzyme reporter molecule is reacted
with specific material on the solid phase to determine if any material has
been captured. This procedure requires the following steps: (a) wash the
solid support to remove unreacted materials; (b) add the enzyme labeled
antibody or polynucleotide probe; (c) wash the solid support to remove all
unbound enzyme-labeled reagent; (d) add substrate specific for the enzyme
label; and (e) incubate the substrate and enzyme under appropriate
conditions. If enzyme remains bound onto the solid phase, then the enzyme
converts some portion of the substrate to product. The amount of product
produced is proportional to the amount of enzyme residing on the solid
phase.
In the fluidics system, the second column is located at a fixed distance
from the first column. If the fluidics system is part of a
microprocessor-controlled instrument, then a switching valve may be
located between the two columns. This is the two column design as shown in
FIG. 2. Alternatively, the columns may be constructed as a single unit but
with the solid support materials and the sorbent bed connected at a fixed
distance with a conduit or separated by a barrier, such as a frit or a
membrane filter. This is a continuous design as is shown in FIG. 1.
When using either the two column design or the continuous design, the
substrate is added to the solid support of the first column and allowed to
incubate as described above. Preferably, the temperature of the substrate
within the first column is regulated by a heating jacket or block around
the first column to obtain optimal enzyme activity. After an incubation
time that can vary from two minutes to about 2 or more hours, the enzyme
generated product is moved from the solid phase of the first column to the
sorbent bed of the second column by a fluidics system.
The manner in which substrate is added to the solid phase in the first
column and allowed to incubate with enzyme can be varied to optimize
enzyme activity. The method by which substrate is added and incubation
occurs, in turn, dictates the manner that product is brought to the
sorbent bed in the second column. Four representative examples include:
1. The dynamic addition and incubation of substrate. In this embodiment,
the substrate is added to the first column with continuous flow. The flow
out of the first column, containing substrate and product, is applied by a
continuous flow to the sorbent bed of the second column. After a defined
period of continuous flow, wherein a defined amount of substrate has been
added to the first column, substrate addition is terminated. The columns
may be washed with a non-eluting solution to remove substantially all of
the substrate from the two column system. The product is removed from the
second column by an elution solvent. The concentrated product is then
measured by an appropriate detector, such as a spectrophotometer,
fluorometer, luminometer, or electrochemical detector. The advantages of
dynamic addition and incubation are that diffusional constraints
associated with enzyme activity on the solid phase are minimized and the
accumulation of product in the first column that may cause feedback
inhibition of the enzyme is eliminated.
2. Single stopped flow cycle. In this embodiment, substrate is added to the
solid phase of the first column and the flow is stopped. Incubation of the
substrate with the enzyme occurs statically. After the appropriate
incubation time, the substrate and product in the solvent are moved to the
sorbent bed of the second column by the fluidic system. The product
remains bound to the sorbent bed while the substrate is removed. The
product can be removed from the sorbent bed by an appropriate elution
solvent.
3. Multiple stopped flow cycles. In this embodiment, multiple additions of
substrate are made to the solid phase in the first column. After static
incubation for a fixed period of time, the solid support column is
replenished by the addition of new substrate in solvent. The "incubated"
substrate is moved into or toward the sorbent bed in the second column by
the fluidics system. Since the fluidics system is a closed system, the
incubated substrate and product are moved through the sorbent bed in the
second column with each sequential addition of the substrate solution to
the first column. The product is retained on the sorbent bed while the
substrate is substantially not retained. When substrate addition is
completed, the remaining substrate and product is moved into and through
the sorbent column. The product can then be eluted from the sorbent bed by
the addition of an elution solvent into the second column. The multiple
stopped flow embodiment minimizes the problems associated with product
feedback inhibition of enzyme activity. Further, the product is
concentrated into a small volume relative to the substrate volume.
4. Recycling of substrate. In this embodiment, substrate is added to the
solid support in the first column and continuously recirculated through
the first column to provide dynamic incubation of the substrate with the
enzyme on the solid phase. As a corollary, the recirculation can also
occur from the effluent of the second column. In this approach, substrate
is added to the first column, connected in series to the second column.
The flow of solvent containing substrate and product proceeds continuously
through the first column and into the second column wherein the product is
captured in the sorbent bed. The substrate passes through the second
column and is recirculated back into the first column for reaction with
enzyme. The recirculation systems may be beneficial when the substrate is
expensive. Further, the continuous circulation minimizes diffusion
constraints associated with enzyme activity on a solid support surface.
After an appropriate period of recirculation, the product is eluted from
the second column with an appropriate elution solvent.
Irrespective of the method of substrate addition and incubation described,
all embodiments result in the separation of enzyme-generated product from
substrate and concentration of the product for more sensitive detection.
The following examples illustrate the inventive process in an inventive
device when used for a nucleic acid hybridization assay in Example 1 and
for an immunoassay in Example 2. Example 3 illustrates the use of the
invention for the detection of microbial contamination and Example 4
illustrates the use of the invention for the detection of free enzyme in a
sample.
The following examples are offered by way of illustration and not by way of
limitation.
EXAMPLE 1
This example describes a sandwich hybridization assay using an
enzyme-labeled probe that is complementary to a sequence totally contained
within the LT gene of entertoxigenic strains of Escherichia coli. An
enzyme labeled DNA probe is made with .beta.-galactosidase as the enzyme
label on a single-stranded 26-mer oligonucleotide probe. The
.beta.-galactosidase is bound to the probe by the procedure of Jablonski
et al., Nucleic Acids Research 14:6115-28, 1986. A 26-mer complement to
the enzyme-labeled probe is linked via the 5' end to carboxy-modified
latex beads (0.97 .mu.M from Interfacial Dynamics) through a 5-mer
polyadenylate linker arm by the method of Ghosh et al., Nucleic Acids
Research 15:5353- 72, 1987. 50 .mu.l of the latex bead suspension is
hybridized with varying concentrations of the enzyme-labeled probe.
Hybridization conditions are 37.degree. C. for 1 hour in 0.5 ml of 5X SSPE
(4.35% NaCl, 0.69% Na.sub.2 H.sub.2 PO.sub.4, 0.185% EDTA, pH 7.4) and
0.1% SDS (sodium dodecyl sulfate). The latex beads are twice washed by
sedimenting the beads with gentle centrifugation for 10 minutes, followed
by washing with 2X SSC (1.75% NaOH, 0.88% sodium citrate, pH 7.0) and 0.1%
SDS.
The latex beads are added to the first column if the hybridization was not
already conducted in the first column. Substrate,
methylumbelliferyl-.beta.-D-galactoside (MUGAL) at a concentration of 0.5
.mu.g/ml in 0.1M phosphate buffer (pH 7) is added to the first column and
incubated statically at 37.degree. C. for 20 minutes. Incubation of MUGAL
with the bound marker enzyme, .beta.-galactosidase, generates
methylumbelliferone (MU) as the product. After incubation the substrate
and product (MUGAL and MU) are moved in 30% methanol in water by the
fluidics system to the second column. The rate of flow of the fluidics
system is 0.5 ml/min.
The second column contains the C18 non-polar sorbent PRP-1 (Hamilton) and
packed into a column 0.074 inches (i.d.).times.0.75 inches (L) with
approximately 50 to 200 theoretical plates. The MU product will be
retained in the sorbent bed, while the more polar MUGAL will not be
retained on the sorbent when water is the solvent. Elution and
concentration of the MU product from the second column is accomplished
with 100% methanol. The concentration of MU is detected by a flow
fluorometer (Kratos, Spectro flow 980). The amount of MU produced is
directly proportional to the amount of target DNA in the sample that has
been captured onto the solid support.
EXAMPLE 2
This example describes a heterogeneous competitive enzyme immunoreaction
with thyroxine (T4) using fluorescent detection. Monoclonal antibodies
(Immunosearch, Toms River, N.J.) to T4 are covalently linked via
carbodiimide coupling to a carboxy-modified latex beads using the method
of Quash et al. J. Immunoloqical Methods 22:165-74 (1978). The solid
support (latex beads) is added to the first column. A standardized amount
of alkaline phosphatase labeled T4 antigen, which is reactive with the
antibody on the solid support, is mixed with a sample containing the
unlabeled T4 antigen in a 0.1M phosphate buffer (pH 7). The sample
containing the labeled and unlabeled T4 antigens is introduced into the
first column containing the antibody coated latex beads. The labeled and
unlabeled T4 antigens compete for antibody binding sites. The amount of
labeled antigen remaining on the solid support surface is inversely
proportional to the amount of unlabeled antigen in the sample. Unreacted
materials are removed from the first column with a wash using a 0.1M
phosphate buffer (pH 7). The substrate, 0.5 .mu.g/ml 4-methyl-umbelliferyl
phosphate (MUP) in 0.1M Tris buffer with 0.1M NaCl and 50 mM magnesium
chloride (pH 8.5) is added to the first column and allowed to incubate for
5-20 min at 37.degree. C. Incubation of the substrate with the enzyme on
the solid support generates methylumbelliferone (MU) as a product. After
incubation, the substrate and product solution is moved by a fluidics
system to a second column. The second column contains the C18 non-polar
sorbent PRP-1, which is packed in a column 0.074 inch (i.d.).times.0.75
inch (length). This produces a column with approximately 50 theoretical
plates. The product is retained on the C18 sorbent, while the substrate is
not retained due to differences in hydrophobicity. MU is removed from the
second column with an elution solvent consisting of 100% methanol and is
measured in a flow fluorometer (Kratos, Spectroflow 980).
EXAMPLE 3
Microbial Detection and Estimation Tests in Samples
Many different types of samples such as food, water, wastewater, dairy,
clinical and pharmaceutical samples may be tested for microbial
contaminants essentially using the enzyme detection procedure described in
the present invention. The microbial contaminants could include specific
microorganisms such as Escherichia coli or groups of microorganisms such
as the coliform bacteria, the fecal coliform bacteria, the total count or
heterotrophic bacteria, yeasts, and molds. Detection and estimation of
these microbial contaminants would be accomplished by assaying for an
enzyme or enzymes produced by these microorganisms. The use of sorbents in
an inventive device provides earlier testing results by (i) separating the
product of the enzyme reaction from the substrate and soluble sample
constituents using a sorbent bed in a column and (ii) concentrating the
product from the assay solution into a small detection volume.
Coliform bacteria produce the enzyme .beta.-galactosidase for utilization
of lactose. The detection and measurement of activity associated with this
enzyme can be used to rapidly detect and estimate the levels of these
bacteria in a sample such as food. This is accomplished by adding a food
sample (25 grams) into a broth culture medium (225 ml) supplemented with
the fluorogenic substrate 4-methylumbelliferyl-.beta.-D-galactoside
(MU-GAL) at a level of approximately 50-100 .mu.g/ml. The preferential
broth culture medium is one which permits the growth of the coliform
bacteria while inhibiting or suppressing the growth of non-coliform
bacteria. Examples are commonly used media are violet red bile broth, Endo
broth, or lauryl sulfate broth or less commonly used formulations such as
CM (without agar) (Firstenberg-Eden, R. and Klein, C.S., J. Food Science
48:1307, 1983). The fluorogenic substrate MU-GAL is cleaved by the
cellular .beta.-galactosidase enzyme to produce methylumbelliferone and
galactoside. After a specified incubation period, preferably 1-6 hours, at
35.degree. C., a small aliquot of the culture medium is transferred to a
column containing reverse phase C18 sorbent. The methylumbelliferone
strongly binds to the reverse phase sorbent, whereas sample constituents
and remaining substrate, under the appropriate solution conditions, do not
substantially bind. The methylumbelliferone remaining on the sorbent is
eluted from the sorbent with the appropriate elution solvent and measured
using a fluorometer. The amount of fluorescence is proportional to the
number of coliform bacteria in the sample after incubation. The level of
coliform bacteria initially in the sample is estimated from a standard
curve that plots (i) relative fluorescence units versus initial
concentration of bacteria for a specified incubation time or (ii) time
(hours) to detect fluorescence versus initial concentration of bacteria.
EXAMPLE 4
Detection of Free Enzyme in a Sample
The detection and measurement of free enzymes in a sample are used for a
variety of purposes ranging from tests for food and dairy safety and
product quality to tests for clinical diagnosis. Examples of assays for
free enzymes in food and dairy products include the determination of (i)
pasteurization completeness by measuring activity of alkaline phosphatase
in fluid milk and other dairy products and (ii) spoilage potential
(product shelf-life) by measuring the activity of enzymes such as protease
enzymes, trimethylamine oxidase, xanthine oxidase or cytochrome enzymes
such as cytochrome b5 reductase. Clinical diagnostic tests include assays
for creatine kinase activity to assess for damage to the myocardium,
alkaline phosphatase activity in serum for hepatobiliary diseases and bone
diseases, and lactate dehydrogenase activity in cerebrospinal fluid and
serum to determine tissue damage.
Adequate pasteurization results in the inactivation of the enzyme alkaline
phosphatase. Therefore an alkaline phosphatase test in fluid milk measures
pasteurization efficacy. A milk sample is added to a carbonate-magnesium
buffered solution supplemented with the fluorogenic substrate
4-methylumbelliferyl phosphate (MUP). The alkaline phosphatase cleaves the
MUP to produce methylumbelliferone and phosphate. This solution is
incubated under appropriate conditions and an aliquot is transferred to a
column of the present invention, containing a C18 reverse phase sorbent.
The methylumbelliferone is retained on the C18 sorbent while the sample
constituents and remaining substrate are not retained under appropriate
solution conditions. The methylumbelliferone is eluted from the sorbent
with the appropriate solvent and the fluorescence measured. The amount of
fluorescence is proportional to the amount of alkaline phosphatase in the
sample.
From the foregoing it will be appreciated that, although specific
embodiments of the invention have been described herein for purposes of
illustration, various modifications may be made without deviating from the
spirit and scope of the invention.
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