Method of sample preparation for urine protein analysis with capillary electrophoresis

Processes are provided for pretreating body fluid compositions and subsequently analyzing the pretreated body fluid compositions for analytes of interest. Processes for pretreating the compositions include providing size exclusion gel having a molecular weight fractionation range or a molecular weight exclusion such that the size exclusion gel is capable of excluding or fractionating the analytes of interest, and then causing the composition to contact the size exclusion gel in order to separate the analytes from low molecular weight composition components which interfere with the separation and analysis of the analytes of interest. Processes for analyzing pretreated compositions include electrophoretic methods such as capillary zone electrophoresis which involve the separation and detection of analytes of interest.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates generally to processes for preparing body 
fluid samples for their subsequent analysis using sample component 
separation techniques. More particularly, the present invention involves 
methods for removing low molecular weight components from urine samples, 
thus providing samples which are free of compounds which interfere with 
the electrophoretic separation and detection of higher molecular weight 
species. 
2. Description of Relevant Art 
Proteins present in mammalian body fluids such as whole blood, serum, 
plasma, cerebrospinal fluids, tears, sweat, saliva and urine are useful as 
indicators of the presence or absence or certain disease states. Thus, the 
ability to identify and quantitate a variety of proteins in body fluid 
clinical samples can provide diagnosticians a great deal of information 
leading to the diagnosis of a variety of diseases. 
For example, patients inflicted with kidney disease will excrete urine 
containing albumin and other serum proteins which are typically absent in 
the urine of healthy individuals. Additionally, the urine of myeloma 
patients is known to have free light chain gamma globulins, proteins not 
normally excreted in the urine of patients free of myeloma. Accordingly, 
techniques for identifying and quantitating these and other protein 
components of clinical urine samples can provide indicators of abnormal 
conditions such as kidney disease and the presence of myeloma in patients. 
A number of techniques involving the analysis of proteins found in body 
fluids are known. These range from wet chemistry methods which simply 
indicate the presence or absence of proteins to relatively complex methods 
involving the separation, identification, and quantitation of proteins 
which may be present at very low concentrations. These methods typically 
involve separating the fluid components using electrophoretic or 
chromatographic techniques followed by detecting the separated proteins. 
Typically, the detection methods involve directly analyzing the separated 
proteins by measuring their uv absorption using a detection wavelength at 
which the protein has a relatively high absorbance. Other optical 
detection methods include those which involve labelling the sample 
proteins with fluorescent labels or chemiluminescent labels and then 
detecting the separated proteins using fluorescent or chemiluminescent 
detectors. All of these methods have the advantage of providing 
qualitative as well as quantitative information when utilized in 
connection with standard curves generated from known proteins of known 
concentration. 
Since mammalian proteins are charged molecules, mixtures of proteins can be 
subjected to electrophoretic separation techniques resulting in the 
separation of the protein components. In particular, recently developed 
capillary electrophoresis techniques provide efficient and rapid 
separations of small concentrations of charged species, and have become 
the method of choice for the rapid separation and analysis of charged 
components of clinical samples. 
In general, capillary gel electrophoresis involves introducing a sample 
into a capillary column and applying an electric field across the column. 
The electric field causes the charged sample components to move within the 
gel filled column with the direction and speed of the movement being 
determined by the electrophoretic mobility of each charged component. The 
electrophoretic mobility in turn is dependent upon the mass of each of the 
sample components with those components having greater mobility travelling 
faster than those with slower mobility. This results in the sample 
components being resolved into discrete zones in the capillary column. 
Another form of capillary electrophoresis or "open tube" CE is similar to 
the above-described gel capillary electrophoresis except that the column 
is filled with an electrically conductive buffer solution. Upon applying 
an electric field to the capillary, the negatively charged capillary wall 
will attract a layer of positive ions from the buffer. Under the influence 
of the electrical potential caused by the electric field, the bulk 
solution must flow toward the cathode in order to maintain 
electroneutrality. This electroendosmotic flow provides a fixed velocity 
component which drives both neutral species and ionic species towards the 
cathode. 
Typically capillary gel electrophoresis and open-tube CE utilize an on-line 
detector such as a uv absorbance detector or other optically based 
detector to monitor separations and provide quantitative and qualitative 
data relating to the separated components. Proteins inherently absorb in 
the ultraviolet spectrum at 214 nm and 280 nm, making uv detectors the 
detector of choice because the proteins do not require special labelling. 
One problem associated with using uv detection methods is the presence of 
low molecular weight sample components which may be present at relatively 
high concentrations in clinical samples. Typically, these low molecular 
weight components are not the analyte of interest, but absorb at the 
preferred monitoring wavelength, which for proteins is 214 nm. Frequently, 
these lower molecular weight components will co-migrate with the proteins 
of interest, thus causing separation and detection problems. Even when the 
lower molecular weight components do not co-migrate they can be present at 
such high relative concentrations that the lower concentration components 
of interest are not detected. Thus, the lower molecular weight components 
can substantially interfere with the detection of the higher molecular 
weight analytes of interest which are typically present at much lower 
concentrations. 
Attempts to overcome problems associated with interfering sample components 
generally involve procedures directed toward removing the unwanted 
components from the sample prior to performing the separations. These 
procedures include subjecting the sample to dialysis to separate low 
molecular weight components, solvent extraction techniques to partition 
low molecular components, precipitation, and centrifugation. In some 
cases, the sample is concentrated in order to increase the concentration 
of analytes which are known to be present at very low concentrations and 
not detectable in the presence of smaller molecular weight interfering 
components. These procedures are tedious and labor intensive, add cost and 
time to the analysis process and are generally considered unacceptable by 
clinical practitioners. 
The practice of separating low molecular weight components from clinical 
samples is associated primarily with procedures involving the assay of 
urine for Bence Jones (BJ) protein in possible myeloma patients and the 
analysis of certain serum proteins in proteinuria patients. The amount of 
Bence Jones protein in urine varies from patient to patient and can be 
difficult to detect in the presence of low molecular weight interfering 
sample components. Membrane dialysis has been effective in removing the 
interfering components. However, this method is cumbersome, slow and 
requires large volumes of buffer solutions. 
Accordingly, it would be desirable to provide methods for pretreating body 
fluid clinical samples in order to remove clinical sample components which 
interfere with the analysis of the clinical samples. Furthermore, it would 
be desirable to provide methods for analyzing body fluid samples for 
certain analytes while eliminating the effects of the presence of 
interfering components. More particularly, there is a need to provide 
methods for analyzing patient urine samples for low concentrations of 
Bence Jones protein and other proteins indicative of certain disease 
states. 
SUMMARY OF THE INVENTION 
The present invention satisfies the above needs by providing efficient high 
yield processes for removing low molecular weight components of clinical 
samples prior to analyzing the clinical samples for higher molecular 
weight analytes. Because the processes of the present invention 
effectively remove sample components which interfere with the analysis of 
analyte components which can be present at much lower concentrations, the 
practice of the present invention allows the direct analysis of small 
amounts of analyte components and can preclude the need to perform sample 
concentration steps. 
In one aspect the present invention provides processes for pretreating 
clinical liquid compositions prior to analyzing the clinical liquid 
compositions for at least one analyte having a molecular weight range. 
More particularly, processes of the present invention generally involve 
providing size exclusion gel having a molecular weight fractionation range 
or a molecular weight exclusion suitable for fractionating or excluding 
the analyte or analytes, and then causing the liquid clinical composition 
to contact the size exclusion gel in order to separate the analyte from 
any low molecular weight components of the liquid composition. 
In preferred embodiments, the processes of the present invention are 
associated with the analysis of clinical urine samples for proteins having 
a molecular weight range greater than about 6,000, and involve the use of 
polyacrylamide or polysaccharide size exclusion gels. The size exclusion 
gel preferably has a molecular weight exclusion greater than about 6,000. 
Typically the size exclusion gel is packed in a column and a urine sample 
is passed through the column, providing a column eluent containing higher 
molecular weight proteins which have been excluded by the size exclusion 
gel or fractionated to the extent that they are included in the column 
eluent. For the analysis of urine clinical sample, higher molecular weight 
proteins preferably include albumin and Bence Jones proteins which are 
indicative of certain disease states such as kidney disease and myeloma, 
respectfully. Advantageously, the column eluent can be separated using 
methods known in the art for separating clinical sample components, and in 
particular, clinical urine samples. 
Accordingly, the present invention additionally includes processes for 
pretreating clinical samples, separating the pretreated clinical sample 
components and determining at least one clinical sample analyte having a 
analyte molecular weight range. These processes typically include 
providing size exclusion gel having a molecular weight fractionation range 
or a molecular weight exclusion (or molecular weight cut-off) suitable for 
fractionating or excluding the analyte or analytes, and causing the 
clinical sample to contact the size exclusion gel, thus providing a column 
eluent which includes analytes which have been fractionated and eluted or 
excluded and eluted as determined by the analyte molecular weight. Then by 
subjecting the column eluent to an electrophoretic separation method and 
detecting analytes, the presence of any clinical sample component having a 
molecular weight greater than the molecular weight cut-off can be 
determined. 
Preferred embodiments of the present invention involve the use of capillary 
electrophoresis (CE) separation methods and in particular open tube CE 
methods. 
These and other advantages associated with the present invention and a more 
detailed explanation of preferred embodiments are described below and 
should be taken in combination with the following drawings.

DETAILED DESCRIPTION OF THE INVENTION 
In general, the present invention involves the treatment and analysis of 
clinical fluid samples for certain component analytes which are of 
interest to clinicians and diagnosticians. More particularly, the present 
invention deals with treating clinical fluid samples with size exclusion 
gel in order to separate lower molecular weight sample components from 
higher molecular weight sample components prior to analyzing the fluid 
sample. 
Typically, the methods described herein involve the efficient and quick 
removal of interfering low molecular weight components from clinical 
samples. Because low molecular weight components found in urine can 
interfere in the electrophoretic separation and analysis of higher 
molecular weight urine proteins, the present invention is particularly 
applicable to the analysis of urine for clinically significant higher 
molecular weight components. For example, Bence Jones protein and albumin 
each has a molecular weight greater than 10,000 and when found in urine 
are associated with the disease states of myeloma and kidney malfunction, 
respectively. However, those skilled in the art will appreciate that the 
present invention also finds application in any processes in which it is 
advantageous to separate low molecular weight body fluid constituents from 
higher molecular weight body fluid constituents. These include but are not 
limited to serum, plasma, tears, sweat, saliva and cerebral spinal fluid. 
A general embodiment of the present invention includes processes for 
treating a clinical liquid composition prior to analyzing the clinical 
liquid composition for at least one analyte having a molecular weight 
range. The process includes the steps of first providing size exclusion 
gel having a molecular weight fractionation range or a molecular weight 
exclusion (also termed molecular weight cut-off) suitable for 
fractionating or excluding the analyte or analytes. Then causing the 
liquid clinical composition to contact the size exclusion gel results in 
the separation of the analyte or analytes from any low molecular weight 
components of the liquid composition. In performing these steps, a gel 
eluent is obtained which includes the analyte or analytes of interest. 
As previously mentioned, the practice of the present invention preferably 
involves the analysis of analytes such as Bence Jones proteins found in 
the urine of myeloma patients as well as the analysis of serum proteins 
including but not limited to albumin, transferrin, .alpha..sub.2 
macroglobin, immunoglobulin, haptoglobin, and .alpha..sub.1 antitrypsin. 
Size exclusion gels suitable in the practice of the present invention 
include porous gel particles or beads prepared of any of a wide variety of 
polymeric materials. These gels are available commercially from a number 
of manufacturers and suppliers including BIO-RAD, Pharmacia, Pierce 
Chemical, and Sigma Chemicals. The particle size and particle size 
distribution of the gel beads is not crucial to their use in the present 
invention, however, appropriate sizes range from less than 20 .mu.m to 300 
.mu.m in diameter. As will be discussed below, the diameter of the beads 
effects the rate at which eluent is collected as a result of contacting 
the liquid composition with the gel. Typically, the smaller the bead 
diameter, the larger the total surface area, and the slower the eluent 
flow rate. 
When placed in contact with liquid solutions of constituents having 
different molecular weights, size exclusion gels having appropriate pore 
sizes, will exclude certain solution constituents and trap or allow other 
constituents to flow into the pores of the size exclusion gel beads. 
Typically, the totally excluded solution constituents will flow freely 
around the gel beads. Solution constituents or components which flow into 
the pores of the size exclusion gel will reside in the pores in the 
presence of flowing liquid for varying lengths of time. This residing time 
is dependent upon the molecular weight of the constituents and the pore 
sizes of the beads with the larger molecular weight constituents residing 
in the pores for less time than smaller molecular weight constituents. 
This phenomenon results in a molecular weight fractionation of the sample 
components by the size exclusion gel and the pore size of the porous gel 
beads determines the molecular weight fractionation properties and the 
molecular weight exclusion properties of the gel. 
As explained in more detail below, in the context of their utility in the 
present invention, size exclusion gels are selected so that the gel pore 
size is sufficiently small to exclude body liquid components which are the 
analytes of interest. Similarly, the pore size is large enough to trap, 
without fractionating, low molecular weight sample constituents which 
interfere with the analysis of analytes of interest. For example, an 
exclusion gel having a molecular weight cut-off of about 6,000 will 
generally exclude molecules having a molecular weight greater than 6,000. 
Those skilled in the art, however, will appreciate that these molecular 
weights are approximate, and different types of molecules may behave 
slightly differently depending upon the liquid or solvent in which the 
molecule is dissolved and the chemical class of the molecule. 
Alternatively, size exclusion gels can be selected so that they exclude 
analytes of interest, and/or, under the pretreatment conditions, 
fractionate sample components so that the analytes of interest elute from 
the gel and the interfering constituents remain in the gel. In this case, 
the gel pore size of suitable size exclusion gels is such that an analyte 
of interest may not be totally excluded by the gel bead pores, but flows 
into the pores and then out of the pores and becomes part of the eluent 
from the gel. In a flowing system, these are fractionated components and 
will elute after the totally excluded larger components. When used in 
accordance with the present invention these size exclusion gels trap and 
remove small molecular weight components which are not analytes of 
interest and allow larger molecular weight components which are analytes 
of interest to elute by totally excluding them or partially fractionating 
them by molecular weight. Those components which are small enough to enter 
the size exclusion gel pores tend to remain in the pores unless forced out 
by sufficient force as the result of flowing liquid. 
Accordingly, in the practice of the present invention, when the appropriate 
size exclusion gel is brought into contact with a body liquid sample, 
smaller molecular weight sample components are trapped in the pores and 
the larger molecular weight components of analytical interest ultimately 
are recovered in an eluent. Thus, a size exclusion gel having a molecular 
weight cut-off of 40,000 and a fractionation range of 2,500 to 30,000 
typically will exclude molecules having a molecular weight greater than 
40,000. However, lower molecular weight molecules will be fractionated 
when placed in contact with these gels and elute under the appropriate 
conditions. As described in more detail below, such appropriate conditions 
depend upon the amount of wash utilized to provide the eluent and the 
ionic strength of the wash. 
In preferred embodiments of the present invention, the size exclusion gels 
are prepared from hydrophilic polymers which are crosslinked in order to 
render the gel beads insoluble in aqueous based systems. Particularly 
useful hydrophilic size exclusion gel beads are prepared of crosslinked 
polyacrylamide or a crosslinked polysaccharide such as crosslinked 
dextran. However, size exclusion gels prepared from other polymers such as 
crosslinked polyvinylalcohol, hydrophilic crosslinked acrylates and 
methacrylates, and crosslinked polyvinylpyrrolidinone can also be used. 
Preferred size exclusion gels have a molecular weight cut-off or molecular 
weight exclusion of at least 6,000. That is, when utilized in the practice 
of the present invention, proteins present in urine or other body fluid, 
and having a molecular weight of about 6,000 or less will become trapped 
or find their way into the pores of these gels once they come into contact 
with the pores of the gel material. Other suitable size exclusion gels 
have a molecular weight cut-off of 30,000 to 40,000, and a fraction range 
of 2,500 to 40,000, thus allowing solution constituents having molecular 
weights of 10,000 and greater to fractionate and become part of a gel 
eluent. 
Commercially available gels suitable for the practice of the present 
invention include the Bio-Gel P-6 and P-30 line of crosslinked 
polyacrylamide gels available from BIO-RAD and the Sephadex G-25 line of 
crosslinked polysaccharide (dextran) gels available from Pharmacia. The 
BIO-RAD polyacrylamide gels are available in gel bead diameters of medium, 
fine, and extra fine with the medium being from 90-180 .mu.m, the fine 
being from 45-90 .mu.m, and extra fine being from 20-30 .mu.m. Similarly, 
the Pharmacia dextran gels are available in fine with a diameter of 20-80 
.mu.m, medium with a diameter of 50-150 .mu.m, coarse with a diameter of 
100-300 .mu.m, and superfine with a diameter of 20-50 .mu.m. 
Since the preferred size exclusion gels are fabricated of hydrophilic 
materials, prior to their use, preferably, the gel beads are swollen in 
distilled water. This step stabilizes the pore size, and helps maintain 
dimensional characteristics of the beads. Following the addition of water 
to the beads, it is additionally preferred that at least 3 volumes of a 
buffer such as phosphate buffer is passed over the size exclusion gel 
beads. 
In accordance with the present invention, a preferred method of providing 
size exclusion gel is to load the gel beads in a column configured with an 
injection port at both ends of the column. The size of the column can vary 
with the volume of fluid being analyzed or brought into contact with the 
size exclusion gel. Preferred column dimensions which allow fast and 
efficient recovery are about 2 cm.times.0.8 cm. These columns have a gel 
bead capacity of less than 1 cm.sup.3 and a typical void volume of about 
0.3 mL. When used in accordance with the present invention one gel filled 
column can be used to pretreat about 0.3 mLs of urine or other body fluid. 
That is, a sufficient amount of fluid sample components having a molecular 
weight greater than the gel molecular weight fractionation range will be 
removed from the fluid sample. Those skilled in the art will recognize 
that the column configuration is non-limiting and any suitably sized 
container can be utilized to house the size exclusion gel. 
In accordance with the present invention, after the column is filled with 
size exclusion gel beads, the urine or body fluid sample is applied 
directly to the center of one end of the column. This step causes the 
urine or body fluid sample to contact directly the size exclusion gel 
which is packed into the column. In preferred embodiments in which the 
column is fitted with an injection port or other opening positioned at the 
center of the end of the column, the fluid is directed to contact the 
beads at the center of the column. This precludes fluid sample simply 
running along the column walls without sufficient contact with the size 
exclusion gel. 
Preferably, the urine or fluid sample is applied drop by drop and allowed 
to drain into the size exclusion gel packed column. In order to elute 
substantially all of the components excluded by the size exclusion gel and 
the higher molecular weight fractionated components, the next step 
includes applying sufficient washing buffer to the packed column and 
collecting the resulting eluent, comprising the excluded and suitably 
fractionated components, from the opposite end of the column. 
The flow rate of all liquids passing through the column is proportional to 
the pressure drop through the column. Without the application of pressure 
in excess of atmospheric pressure, and particularly when fine gel beads 
are utilized, the additional washing step enhances the flow of eluent 
through the column. Additionally, the volume of washing buffer is selected 
so that, of the fractionated sample components, those having a molecular 
weight of over about 10,000 are also eluted. When size exclusion gels 
having a molecular weight fractionation range of 2,500 to 40,000 are 
utilized, urine sample volumes of 300 .mu.l are effectively eluted from a 
column with a 110 mmol ionic strength buffer (ICS.TM.) wash of about 400 
.mu.l. Those skilled in the art will appreciate that the ionic strength of 
the washing buffer will effect the fractionation properties of the size 
exclusion gel. Typically, the higher the buffer solution ionic strength, 
the faster the lower molecular weight components elution. 
After the eluent is collected it can be stored for later analysis 
(preferably frozen) or analyzed immediately for analytes of interest, 
including Bence Jones protein and serum proteins associated with kidney 
disease. Accordingly, processes for separating and determining at least 
one analyte having a analyte molecular weight range greater than the 
exclusion molecular weight of the gel are additionally within the scope of 
the present invention. Such processes include pretreating a clinical 
sample as described above and then separating the eluent components and 
detecting the analyte or analytes of interest. 
Any technique or procedure suitable for separating, identifying and/or 
quantitating proteins is suitable. These techniques include 
chromatographic methods, such as high pressure liquid chromatography, and 
electrophoretic separation methods. Because of the very small sample 
volumes required, efficiency, speed, and ease of operation, capillary zone 
electrophoresis (CZE) methods are preferred. 
Capillary zone electrophoresis methods including capillary gel 
electrophoresis and open tube electrophoresis techniques for analyzing 
serum proteins and urine are known and those skilled in the art are 
credited with having the knowledge and ability to perform CE protein 
separations. When used in connection with analyzing urine samples 
pretreated in accordance with the present invention, CZE analysis can 
involve dilution of the eluent prior to injecting it into the capillary. 
It is noted that in preferred embodiments of the present invention, the 
eluent is diluted by approximately one haft with the washing buffer and 
for this reason, further dilution is not necessary. In rare clinical cases 
where the analyte or analytes of interest is present in extremely high 
concentrations, dilutions can result in enhanced analytical results. When 
the column eluent is diluted, suitable diluents are buffer compositions 
and include the phosphate buffer ICS.TM. available from Beckman 
Instruments, Inc. 
A typical urine protein CZE method utilizes capillaries having an internal 
diameter of 25 .mu.m, a separation length of 20 cm, and a total capillary 
length of 27 cm. The electric field strength applied across the capillary 
can vary substantially, however, for the general analysis of urine and 
serum protein analyses 10 kv for 7 minutes at 24.degree. C is sufficient. 
Detecting the proteins which have migrated under the influence of the 
electric field is conveniently accomplished using an ultra-violet detector 
at 214 nm, a wavelength at which proteins universally absorb. 
The following non-limiting examples illustrate the efficacy and advantages 
associated with treating clinical samples and analyzing the samples for 
certain analytes of interest in accordance with the present invention. It 
is understood that these examples are for illustration purposes only and 
that alternative embodiments such as the use of similar size exclusion 
gels and alternative analytical techniques are contemplated as within the 
scope of the present invention. 
EXAMPLE 1 
Urine samples from normal individuals were obtained and treated with size 
exclusion gel in order to demonstrate the efficacy of removing lower 
molecular weight urine components in accordance with the present 
invention. 
A control untreated normal patient urine sample was subjected to CZE 
analysis by loading the sample into a 25 .mu.m.times.27 .mu.m 150 mM 
borate buffer silica capillary column. The column had an exterior coating 
of polyamide and a uv detector window disposed at 7 cm from a column 
outlet. The sample loading conditions were a 10 second pressure injection. 
After applying a separation voltage of 10 kv for 7 minutes, the migrated 
urine components were detected at a wavelength of 214 nm. 
The electropherogram obtained from the CZE analysis of the normal urine 
sample is shown in FIG. 1a. All of the peaks are small molecular weight 
components found in normal patient urine. 
P-30, a crosslinked polyacrylamide gel having a molecular weight 
fractionation range of about 2,500 to 40,000 was purchased from BIO-RAD. 
About 1 ml of the gel was washed with distilled water, packed into a small 
column having a diameter of 2 cm.times.0.8 cm, and then washed with 
ICS.TM. phosphate buffer. Excess buffer was drained from the top of the 
column and 300 .mu.l of the same normal patient urine shown in the 
electropherogram of FIG. 1a was applied drop-wise to the top of the gel 
without disturbing the surface of the gel. After the 300 .mu.l of urine 
had completely entered the gel bed, 400 .mu.l of phosphate buffer was 
added to the gel packed column to elute proteins having a molecular weight 
larger than about 40,000 and to elute proteins having a molecular weight 
greater than about 10,000 which are fractionated by the gel. The eluent 
was collected and analyzed using CZE using the same conditions used to 
obtained the electropherogram of FIG. 1a. 
FIG. 1b is an electropherogram of the urine sample obtained from a normal 
patient and treated with P-30 polyacrylamide size exclusion gel as 
described above. The electropherogram is flat indicating the complete 
removal of small molecules by the polyacrylamide gel. 
EXAMPLE 2 
In order to show that high molecular weight proteins are excluded or 
fractionated and eluted by size exclusion gel, the normal patient urine 
subjected to the CE analysis of FIG. 1 b was spiked with an aliquot of 
normal human serum at a dilution of one part human serum to 9 parts of 
urine. The resulting combination of human serum and urine was divided into 
two parts. A first part was subjected to direct CE analysis using the same 
analytical conditions utilized to obtain the electropherograms of FIG. 1a 
and FIG. 1b. A second part was treated with the P-30 crosslinked 
polyacrylamide gel beads in the same manner as described above for the 
sample of FIG. 1b. This second part was also subjected to CE analysis 
using the same analytical conditions as the first part. 
FIG. 2 shows the electropherograms obtained from each spiked normal urine 
sample which was pretreated prior to spiking. The electropherogram 
indicated as a) was obtained from a spiked sample which was not treated 
subsequent to spiking the urine. The electropherogram indicated as b) was 
obtained from the spiked sample protreated with P-30 subsequent to spiking 
the urine. As illustrated by the electropherograms of FIG. 2, both samples 
have identical components, indicating that the pretreatment with the 
crosslinked polyacrylamide gel does not take-up or remove the higher 
molecular serum proteins which were added to the urine. Thus, size 
exclusion gel bead filled column allowed complete recovery of the 
proteins. 
EXAMPLE 3 
In order to show that even very low concentrations of proteins are 
recovered using the processes of the present invention, the pre-treated 
normal patient urine sample prepared in EXAMPLE 1 was spiked with human 
serum at a dilution of 1 part serum and 79 parts urine. The spiked sample 
was divided into two parts, a) and b), which were treated in the same 
manner as described in EXAMPLE 2, respectively. A capillary 
electropherogram of each of the spiked urine samples is shown in FIG. 3. 
Electropherogram a) is that of the serum spiked normal urine sample which 
was not treated with the size exclusion gel after spiking. 
Electropherogram b) is that of the serum spiked normal urine sample which 
was treated with the P-30 size exclusion gel after spiking. These 
electropherograms indicate that even at very low serum protein 
concentrations, complete recovery of the proteins occurs subsequent to 
passing the samples through a size exclusion gel column. 
EXAMPLE 4 
In order to demonstrate the efficacy and accuracy of the processes of the 
present invention, a pathological urine sample was obtained from a patient 
known to have Bence Jones proteins. A portion of the sample was first 
analyzed for total protein and albumin on a Beckman CX-7 clinical 
diagnostic instrument and found to have a total protein concentration of 
2.21 mg/ml and an albumin concentration of 0.0166 mg/ml. 
Another portion of the pathological urine sample was treated with P-30 
crosslinked polyacrylamide gel in the same manner as described in EXAMPLE 
1 sample b) . The treated sample was then analyzed by CZE and 
electropherogram b) of FIG. 4 was obtained. This electropherogram clearly 
shows the two Bence Jones light chain peaks. It is also evident that 
albumin is present at a concentration of about 1% of the total protein 
concentration as fully supported by the CX-7 analysis. The results of the 
electropherogram of the treated Bence Jones pathological sample were 
corroborated with a gel electrophoresis analysis of the same sample. The 
gel electrophoresis was performed on a Beckman Paragon SPE II instrument 
with the results shown in electropherogram a) of FIG. 4. 
These results clearly illustrate that the processes of the present 
invention exclude low molecular weight components of urine samples and 
allow full recovery of higher molecular weight proteins which can be 
indicative of a pathologically abnormal condition. 
EXAMPLE 5 
In order to demonstrate the efficacy and accuracy of the processes of the 
present invention on samples containing abnormal amounts of albumin, a 
pathological sample was obtained from a proteinuria patient. A portion of 
the proteinuria pathological urine sample was treated with P-30 
crosslinked polyacrylamide gel in the same manner as described in EXAMPLE 
1, sample b). The treated sample was then analyzed by CZE and 
electropherogram b) shown in FIG. 5 was obtained. This electropherogram 
clearly shows the albumin which was excluded by the size exclusion gel. 
Additionally, low molecular weight sample components are not evident in 
the electropherogram, evidencing their removal by the size exclusion gel. 
The results of the electropherogram of the treated abnormal pathological 
sample were corroborated with a gel electrophoresis analysis of the same 
sample. The gel electrophoresis was performed on a Beckman Paragon SPE II 
instrument with the results shown in electropherogram a) of FIG. 5. The 
albumin concentration of these samples was 5.47 mg/ml and the total 
protein was 7.7 mg/ml. 
EXAMPLE 6 
Urine samples from normal individuals were obtained and treated with size 
exclusion gel having a molecular weight cut-off of up to 6,000 in 
accordance with the present invention. These experiments were performed in 
order to demonstrate the efficacy of utilizing size exclusion gels having 
lower molecular weight cut-offs and molecular weight fractionation ranges 
than those used in EXAMPLES 1-5. 
A control untreated normal patient urine sample was subjected to CZE 
analysis by loading the sample into a 25 .mu.m.times.27 cm polyamide 
coated capillary with a detector window positioned 7 cm from a tube 
outlet. The sample was loaded using a 10 second pressure injection. After 
applying a separation voltage of 10 kv for 7 minutes, the migrated urine 
components were detected at a wavelength of 214 nm. 
The electropherogram obtained from the CZE analysis of the normal urine 
sample is shown in FIG. 6a. All of the peaks are small molecular weight 
components found in normal patient urine. 
P-6, a crosslinked polyacrylamide gel having an upper molecular weight 
fractionation range less than about 6,000 was purchased from BIO-RAD. 
About 1 mL of the gel was washed, packed into a small column, and washed 
with buffer as described in EXAMPLE 1. The normal patient sample was 
applied to the gel column, collected, and analyzed by CZE as described 
previously. FIG. 6b is an electropherogram of the urine sample obtained 
from a normal patient and treated with P-6 polyacrylamide size exclusion 
gel as described above. The electropherogram is flat indicating the 
complete removal of small molecules by the polyacrylamide gel. 
EXAMPLE 7 
In order to demonstrate the efficacy and accuracy of the processes of the 
present invention utilizing a size exclusion gel having a molecular weight 
cut-off of about 6,000, a pathological urine sample was obtained from a 
patient known to have Bence Jones proteins. A first portion of the 
pathological sample was not pretreated with size exclusion gel and 
analyzed by CZE as described in EXAMPLE 6. The resulting electropherogram 
is shown in FIG. 7 electropherogram a) where a number of components are 
shown, many of which appear to co-migrate. 
A second portion of the pathological urine sample was treated with P-6 
crosslinked polyacrylamide gel in the same manner as described in EXAMPLE 
1 sample b). The treated sample was then analyzed by CZE as previously 
described and electropherogram b) of FIG. 7 was obtained. This 
electropherogram clearly shows the Bence Jones protein. 
These results clearly illustrate that the processes of the present 
invention exclude low molecular weight components of urine samples and 
allow full recovery of higher molecular weight proteins which can be 
indicative of a pathologically abnormal condition. 
EXAMPLE 8 
In order to demonstrate the efficacy and accuracy of the processes of the 
present invention utilizing a size exclusion gel having a molecular weight 
cut-off of about 6,000, a pathological urine sample was obtained from a 
proteinuria patient known to have an abnormally high amount of urine 
albumin. A first portion of the pathological sample was not pretreated 
with sized exclusion gel and analyzed by CZE as described in EXAMPLE 6. 
The resulting electropherogram is shown in FIG. 8 electropherogram a) 
where a number of components are shown, many of which appear to 
co-migrate. 
A second portion of the pathological urine sample was treated with P-6 
crosslinked polyacrylamide gel having a molecular weight cut-off of 6,000 
in the same manner as described in EXAMPLE 1, sample b). The treated 
sample was then analyzed by CZE and electropherogram a) shown in FIG. 8 
was obtained. This electropherogram clearly shows that albumin was 
excluded by the size exclusion gel and eluted as column eluent. 
Additionally, low molecular weight sample components are not evident in 
the electropherogram, evidencing their removal by the size exclusion gel. 
EXAMPLE 9 
In order to demonstrate the relative effectiveness of size exclusion gels 
having a molecular weight fractionation range of about 1,000 to 5,000 and 
a molecular weight cut-off of about 6,000 and size exclusion gels having a 
molecular weight cut-off of greater than 40,000 (and a molecular weight 
fractionation range of 2,500 to 40,000), a portion of the treated normal 
urine used in EXAMPLE 1 was spiked with trypsin inhibitor at a 
concentration of 1 mg/ml. Trypsin inhibitor has a molecular weight similar 
to that of Bence Jones proteins. 
A CZE analysis was performed on the trypsin inhibitor spiked treated normal 
urine sample using the CZE analysis procedure described above. This 
provided a control electropherogram. 
Next a portion of trypsin inhibitor spiked treated normal urine was treated 
again with P-30 crosslinked polyacrylamide gel as described in EXAMPLE 1. 
An electropherogram was obtained for the size exclusion gel treated sample 
using the CZE analysis procedure described above. Finally a portion of the 
trypsin inhibitor spiked treated normal urine was treated with crosslinked 
polyacrylamide gel as also described in EXAMPLE 1 except that the size 
exclusion gel was P-6 having a molecular weight cut-off of about 6,000. 
FIG. 9 illustrates electropherograms obtained using the trypsin inhibitor 
spiked urine. Electropherogram a) shows an overlay of the control 
electropherogram and the electropherogram obtained from the spiked sample 
treated with P-30 size exclusion gel. Electropherogram b) shows an overlay 
of the control electropherogram and the electropherogram obtained from the 
spiked sample treated with P-6 size exclusion gel. Finally, 
electropherogram c) shows an overlay of the electropherogram obtained from 
the spiked sample treated with P-6 and that obtained from the spiked 
sample treated with P-30. These clearly indicate the effectiveness of 
using both size exclusion gels in accordance with the present invention. 
The low molecular weight cut-off of the P-6 excluded all of the trypsin 
inhibitor and the molecular weight fractionation range of 2,500 to 30,000 
easily fractionated the trypsin inhibitor so that it eluted from the 
column. 
EXAMPLE 10 
In order to compare the performance of a crosslinked polysaccharide 
(dextran) size exclusion gel and a crosslinked polyacrylamide gel, a 
pathological urine sample was obtained from a patient known to have Bence 
Jones proteins and treated as follows. 
A portion of the pathological urine sample was treated with P-6 crosslinked 
polyacrylamide gel in the same manner as described in EXAMPLE 1 sample b). 
The treated sample was then analyzed by CZE using the same capillary and 
analytical conditions described above. A second portion of the 
pathological urine sample was treated with a crosslinked polysaccharide 
(dextran) size exclusion gel. The dextran gel utilized is available from 
Pharmacia under the trade name Sephadex-G-25 and has a molecular weight 
cut-off of as high as about 5,000. The second portion was treated with the 
gel in the same manner as the first portion and subsequently analyzed by 
CZE in the same manner. 
FIG. 10 illustrates the electropherograms obtained from the two treated 
samples. Electropherogram a) was obtained from the sample pretreated with 
the polyacrylamide P-6 gel and electropherogram b) was obtained from the 
sample pretreated with the dextran gel. These electropherograms clearly 
demonstrate the two Bence Jones light chain peaks. It is also evident that 
size exclusion gels prepared from crosslinked dextran and size exclusion 
gels prepared from crosslinked polyacrylamide are effective and suitable 
in the practice of the present invention. 
These results clearly illustrate that the processes of the present 
invention exclude low molecular weight components of urine samples and 
allow recovery of higher molecular weight proteins which can be indicative 
of a pathologically abnormal condition. 
EXAMPLE 11 
In order to demonstrate and compare the effectiveness of utilizing a 
polysaccharide size exclusion gel with a polyacrylamide gel to pretreat 
urine, a pathological urine sample containing an abnormal amount of 
albumin was obtained from a proteinuria patient. A portion of the 
proteinuria pathological urine sample was treated with P-6 crosslinked 
polyacrylamide gel in the same manner as described in EXAMPLE 1, for 
sample b). The treated sample was then analyzed by CZE utilizing analysis 
conditions described above. 
A second portion of the pathological urine sample was treated in the same 
manner with the same crosslinked dextran size exclusion gel utilized in 
EXAMPLE 10, Sephadex G-25. This treated sample was analyzed subsequently 
using the same CZE procedures. FIG. 11 illustrates each of the 
electropherograms obtained from the CZE analysis of these pretreated 
samples. Electropherogram a), the solid line, was obtained from the sample 
pretreated with the P-6 size exclusion gel and electropherogram b), the 
dotted line, was obtained from the sample pretreated with Sephadex G-25 
gel. 
Both of these electropherograms clearly show the presence of serum proteins 
in urine associated with proteinuria patients. These proteins include 
gamma globulins, transferrin, and albumin. Moreover, these 
electropherograms indicate that interfering low molecular weight urine 
components are not present in the sample and the polyacrylamide gel and 
the polysaccharide gel are effective in allowing recovery of the proteins 
of interest. 
Although the present invention has been described with regard to certain 
preferred methods, other embodiments, versions, and modification are 
contemplated as being within the scope of the present invention. For 
example, the use of size exclusion packed gels column described herein 
lend themselves to the automation of the invention described herein. More 
particularly, suitably sized size exclusion gel packed columns can be 
positioned in-line with a capillary electrophoresis system and samples can 
be pretreated, separated, and detected in an automatic fashion. This 
precludes the need for excess sample handling and can decrease the time 
required for each analysis. Accordingly, the spirit and scope of the 
present invention is limited only by the following claims.