Chromatographic strip having non-compressed edges

A device is disclosed for use in a chromatographic system wherein a component of a mixture is partitioned between a liquid phase and an immobile phase. The device comprises at least one strip of a bibulous material. In the chromatographic system the component traverses at least a portion of the strip. The strip generally has a longitudinal edge substantially corresponding to the direction of traverse of the component. The longitudinal edge has the characteristic of substantially the same rate of traversal by the component along this edge when compared to the rate of traversal of the component along the body of the strip. The strips are prepared from a sheet of a bibulous material by non-deformative or non-compressive cutting of the sheet. The preferred cutting means is a laser beam.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to improved chromatographic strips and methods of 
preparing them. The improved chromatographic strips of the invention are 
useful in analytical chromatography, particularly as immunochromatographic 
strips. 
A number of materials are known for use as chromatographic strips. 
Generally, the material is "bibulous" or "porous," comprising a random or 
oriented pile of fibers such as found, for example, in cellulose, 
fiberglass, woven cloth, cotton, polyester, etc., and the like. The 
preferred material is paper, which is a random pile of cellulose fibers. 
The tortuous interstitial and interconnected capillaries of such a random 
pile of fibers create both a drive for and a resistance to a mobile phase, 
which, in a chromatographic system, traverses at least a portion of the 
strip. The mobile phase is generally a liquid medium, most usually a 
solvent. The transfer of the mobile phase to channels within the random 
pile, which channels have ever smaller diameters, provides the free energy 
gradient to drive the mobile phase through the medium. Dead end pores trap 
the mobile phase and limit its traversal of the chromatographic strip. 
The chromatographic strips are normally prepared from larger sheets from 
which they are cut by mechanical means. The most widely used form of 
mechanical cutting involves a blade or wire. 
Mechanical cutting of the sheet into strips results in a deformation of the 
edge of the strip along the cutting line. This deformation takes the form 
of a compression of the edges of the strip. The fibers which form the 
strip, when cut mechanically, are pushed closer together at the cut edges 
when compared to the distance between the fibers in the body of the strip. 
This deformation of the edges of the strip frequently results in a faster 
rate of traversal for the liquid medium at the edges of the strip than 
through the body of the strip. The fronts of components traversing the 
strip become concave rather than flat. 
In many situations employing a chromatographic strip it is important that 
the shape of the front of the traversing component be flat. In analytical 
and preparative chromatography, it is usually preferable to have a flat 
front. An example of such a situation is affinity chromatography. In such 
a test antibodies are attached to a porous insoluble support. During 
migration of an antigen-containing solution on the porous support, the 
migration of the antigen solute is specifically delayed in comparison to 
the migration of the solvent and other solutes. The relative delay 
decreases with increasing antigen concentration. Accurate quantitations of 
the concentration of analyte in a sample to be analyzed requires that the 
position of the analyte front relative to the solvent front be measured 
accurately. The position of a flat front can usually be measured with 
greater precision and accuracy than that of a concave front, and a higher 
degree of accuracy is thereby obtained in a chromatographic assay. 
Moreover, in preparative chromatography a linear front permits more ready 
separation and isolation of the pure component. 
2. Brief Description of the Prior Art 
U.S. Pat. No. 4,168,146 describes an immunoassay employing 
immunochromatography with antigens followed by contacting the 
immunochromatograph with an aqueous solution containing labelled 
antibodies. An enzyme chromatographic immunoassay is described in U.S. 
Ser. No. 398,505, filed July 15, 1982, now U.S. Pat. No. 4,435,504. 
SUMMARY OF THE INVENTION 
A device is disclosed for use in a chromatographic system wherein a 
component of a mixture is partitioned between a liquid phase and an 
immobile phase. The device comprises at least one strip of a bibulous 
material. In the chromatographic system the component in a mobile phase, 
usually in a liquid medium, traverses at least a portion of the strip. The 
strip has a longitudinal edge that comes in contact with the traversing 
component during the chromatographic process and that lies in a direction 
substantially corresponding to the direction of traverse of the component. 
The longitudinal edge has the characteristic of substantially the same 
rate of traversal of the component along this edge when compared to the 
rate of traversal of the component along the body of the strip. The 
longitudinal edge has substantially the same degree of deformation as the 
body of the strip. Thus, the present device has the characteristic that 
the front of the traversing component remains substantially flat over the 
traversed portion of the strip. The device of the invention is prepared by 
a non-compressive or non-deformative cutting of a sheet of bibulous 
material into strips. Such non-compressive cutting may be achieved, for 
example, by cutting the sheet of bibulous material with a laser beam.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
The benefits of the present invention may be achieved by cutting a sheet of 
bibulous material into chromatographic strips employing means that do not 
compress (non-compressive) or deform (non-deformative) the longitudinal 
side edge of said strip. By the term "non-compressive" or 
"non-deformative" is meant that the cutting does not substantially distort 
the cut edges of the strip when compared to the body of the strip or when 
compared to edges cut by compressive cutting means. That is, the 
relationship of the pores within the bibulous material is substantially 
the same near the cut edge as it is in the body of the strip. The pores 
are not compressed near the cut edge as the pores are near the edge cut by 
compressive cutting means. 
The preferred means for achieving non-deformative or non-compressive 
cutting of a sheet of bibulous material to produce chromatographic strips 
in accordance with the present invention is a laser beam. Laser cutting 
devices are well known in the art. 
The parameters for cutting the sheets, such as intensity of the laser beam, 
the speed of cutting, and the like will be interdependent and will also 
depend on the nature of the bibulous material, the thickness of the 
bibulous material, the ultimate use of the chromatographic strips, and so 
forth. In general, the cutting parameters will be sufficient to cut the 
sheet of bibulous material but insufficient to produce significant 
deformation or compression of the cut edges which would result in 
accelerated traversal of the liquid medium along the side edge of the 
strip when compared to the rate of traversal of the liquid medium along 
the body of the strip or when compared to the rate of traversal along an 
edge cut by compressive means. 
The following are parameters for cutting paper having a thickness from 
about 0.05 to 2 mm, which parameters are provided by way of example and 
not limitation. 
The energy of the laser beam will be from about 5 to 350 watts CW 
(continuous wave), preferably from about 50 to 100 watts CW. The cutting 
speed will depend upon the intensity of the laser beam and should be 
adjusted, where appropriate, to minimize discoloration of the bibulous 
material. The cutting speed is adjusted to maintain optimal cut edge 
quality. At an intensity of the laser beam of about 50 watts CW the 
cutting speed generally will be from about 5-50 centimeters per second, 
preferably from about 20-25 centimeters per second. The particular energy 
and cutting speed to be employed in a specific situation may be easily 
determined by those skilled in the art keeping in mind the above teaching. 
Where discoloration of the chromatographic material might occur during the 
laser cutting operations, an air pressure stream may be focused on the 
area of the sheet being cut. In general, the pressure of the air stream 
should be sufficient to reduce any discoloration of the chromatographic 
material and should not interfere with the cutting operation. The pressure 
may vary from about 20 to 100 psig, preferably from about 50 to 70 psig. 
The non-deformative cutting method of the present invention may be applied 
to all types of bibulous materials which find use in chromatographic 
systems. Chromatographic material means a material susceptible to 
traversal by a mobile material, either a solvent or a solute (traversing 
component) in response to capillary force, gravitational force, 
electrostatic force, positive pressure, or the like. Such materials 
include inorganic powders such as silica, magnesium sulfate, and alumina: 
natural polymeric materials, particularly cellulosic materials, such as 
fiber containing papers, e.g., filter paper, chromatographic paper, etc.; 
synthetic or modified naturally occurring polymers, such as 
nitrocellulose, cellulose acetate, poly(vinyl chloride), polyacrylamide, 
cross linked dextran, agarose, polyacrylate, etc.; either used by 
themselves or in conjunction with other materials; ion exchange resins; 
ceramic materials; and the like. 
The chromatographic strips are generally prepared from sheets of uniform 
thickness of the bibulous material. The strips may have a variety of 
thicknesses, usually from 0.05 to 2 mm, preferably 0.1 to 0.5 mm, and may 
vary in shape usually being rectangular, square, oval, or circular, 
preferably rectangular. The particular dimensions and shape will be 
determined by the chromatographic method in which the strips will be 
employed, normally having a maximum width, perpendicular to the flow, of 
less than 30 cm and a maximum length, parallel to the flow, less than 40 
cm; more frequently a maximum width of 2 cm and a maximum length of 15 cm, 
preferably a maximum width of 1 cm and a maximum length of 10 cm. All that 
is required in the present invention is that the strip have at least one 
longitudinal edge generally corresponding to the direction of flow of the 
traversing component. Longitudinal edge intends a border of the 
chromatographic material and is the boundary where the material begins or 
ends. The structure of the bibulous material may be varied widely and 
includes fine, medium fine, medium, medium coarse and coarse. The surface 
may be varied widely with varying combinations of smoothness and roughness 
combined with hardness and softness. The chromatographic strip may be used 
independently or it may be supported by a variety of inert supports. 
Exemplary of such supports are Mylar.RTM., polystyrene, polyethylene, or 
the like. 
Chromatographic strips prepared in accordance with the teaching contained 
herein find particular use in affinity chromatography, for example, 
immunochromatography. Immunochromatographic methods comprise any number of 
different specific embodiments. The general characteristics of an 
immunochromatographic method are that an antigen or antibody is 
immobilized on the chromatographic material and the complementary binding 
partner, antibody or antigen, in a liquid medium traverses a portion of a 
strip of the bibulous material. 
Exemplary of an immunochromatographic method is the immunoassay disclosed 
in U.S. Pat. No. 4,168,146 (herein incorporated by reference in its 
entirety). The disclosed method is based on utilizing strips of a porous 
carrier material having antibodies bound to it. In the method a portion of 
each of the strips is contacted with an aqueous sample containing the 
antigen to be quantified. Capillary migration is allowed to take place. 
The antigen-containing area of the strip is detected by wetting it with 
antibodies in an aqueous vehicle. The antibodies are normally bound to a 
signal producing system such as, for example, a water soluble fluorescent 
color indicating compound or to an enzyme that catalyzes a 
color-developing reaction. 
Another immunochromatographic method is disclosed in U.S. patent 
application Ser. No. 398,505, filed July 15, 1982, now U.S. Pat. No. 
4,435,504 (herein incorporated by reference in its entirety). The 
disclosed method allows detection of an analyte in a sample where a 
quantitative determination may be readily made without special equipment. 
The sample is immunochromatographed on a bibulous carrier to which is 
conjugated a specific binding partner for the analyte. The 
immunochromatography may be conducted in the presence or absence of a 
labeled conjugate. The label is a member of an enzymatic signal producing 
system, which includes one or more enzymes. After chromatographing the 
samples, if the labeled conjugate was not included in the sample, the 
chromatograph is contacted with a labeled specific binding pair member 
which binds to the chromatograph in relation to the distance travelled by 
the analyte in the sample. By providing appropriate reagents to produce a 
detectable signal, e.g., two enzymes where the substrate of one enzyme is 
the product of another enzyme, a final product is produced which provides 
for a detectable signal. In such a case the distance travelled by the 
analyte may be defined, which distance is related to the amount of analyte 
in the sample. 
The present invention, therefore, comprises a diagnostic device for use in 
immunoassays. The diagnostic device comprises a bibulous material 
providing liquid travel through capillarity and at least one 
non-diffusively bound member of a specific binding pair ("mip"). The 
device may also include one or more members of a signal producing system. 
Generally, the analyte to be measured is a mip selected from the group 
consisting of ligand and receptor. The ligand and receptor are related in 
that the receptor specifically binds to a polar and spacial organization 
of the ligand, being able to distinguish the ligand from other compounds 
having similar characteristics. The signal producing system member, for 
example, may be an enzyme or a fluorescent compound. Generally, the 
immunochromatographic strip contains a plurality of mips attached thereto. 
The thickness of the immunochromatographic strip will generally vary from 
about 0.05 mm to about 2 mm, more usually being about 0.1 mm to 0.5 mm, 
preferably from about 0.2 mm to about 0.4 mm. Usually, the strip will have 
a width of from about 2 to 12 mm, preferably from about 3 to 8 mm, and 
will have a length of from about 20 to 250 mm, preferably from about 30 to 
150 mm. 
Methods for binding a wide variety of materials to a bibulous support are 
found in the literature. See for example, U.S. Pat. No. 4,168,146. The 
amount of a mip which is bound to the bibulous material will vary 
depending upon the size of the immunochromatographic strip and the amount 
required to bind the homologous mip. Generally, the amount of mip will 
range from about 10.sup.-5 to 10.sup.-15 moles per square centimeter, more 
usually from about 10.sup.-7 to 10.sup.-12 moles per square centimeter. 
The number of moles per unit area will be varied in order to insure that 
there is sufficient modification of the distance traversed by the 
traversing component along the affinity chromatographic strip within the 
concentration range of interest. 
Also included within the scope of the present invention are diagnostic kits 
which comprise (1) at least one chromatographic strip prepared in 
accordance with the present invention to which is attached a member of a 
specific binding pair, (2) a member of the specific binding pair 
conjugated to a member of a signal producing system, and (3) any other 
members of the signal producing system as well as any buffers or the like 
for conducting an affinity chromatographic assay. 
The following examples are offered by way of illustration and not by way of 
limitation. 
The following abbreviations are used hereafter: HRP--horse radish 
peroxidase; NHS--N-hydroxy succinimide; EDAC--ethyl dimethylaminopropyl 
carbodiimide; DMF--dimethyl formamide; BSA--bovine serum albumin. 
Temperatures not otherwise indicated are Celsius, while parts are by 
weight except for mixtures of liquids which are by volume. 
EXAMPLE 1 
Preparation of Immunochromatographic Sheets 
A sheet of Whatman 31 ET of about 550 cm.sup.2 was immersed in 1.8 l. 
CH.sub.2 Cl.sub.2, 0.2M in carbonyl-diimidazole, and the mixture gently 
stirred for one hour at room temperature. Additional sheets were activated 
in the same activating solution. Each sheet was then washed with 300 ml 
CH.sub.2 Cl.sub.2 and air dried with an air gun over about 20 sec. The 
sheet was then immersed in a solution of 500 .mu.l of a 49 mg/ml solution 
of antitheophylline and 200 ml of buffer 0.1M sodium phosphate, pH 7.0, 
0.2M NaCl; and the mixture was mildly shaken for 4 hours at room 
temperature. After washing with the phosphate buffer, the solution was 
then immersed in 4% aqueous Dextran T10 solution to serve as a 
preservative, followed by blotting the sheet, freezing and lyophilizing. 
EXAMPLE 2 
Preparation of Immunochromatographic Strips 
A Coherent Model 42, CO.sub.2 laser at 50 watts CW (from Coherent, Inc., 
Palo Alto) and Anomatic II CNC X-Y table were employed. A Coherent Model 
303 coaxial gas jet was used at an air pressure of 60 psig. A standard 
cutting box was used. 
The cutting box was placed on the X-Y table and a sheet of plexiglass was 
placed on the cutting box. A narrow slot was cut in the plexiglass using 
the laser beam. An immunochromatographic sheet prepared in Example 2 was 
placed over the plexiglass. The sheet was cut into strips which were 4.5 
mm wide and 90 mm in length. Cutting speeds of 19 and 26 centimeters per 
second were employed. The performance of the immunochromatographic strips 
cut at the two different speeds was substantially identical. 
EXAMPLE 3 
Preparation of HRP-Oxyamine 
To 5 ml of 10 mg/ml horse radish peroxidase in 5 mM sodium acetate, pH 4.5 
buffer, was added 50 .mu.l 0.2M sodium periodate and the mixture stirred 
for 30 min, followed by chromatography on a G-50 Sephadex column, eluting 
with 2 mM sodium acetate buffer, pH 4.5. The protein fractions were pooled 
to 29 ml, the mixture cooled to 4.degree. C. and 2.9 ml of 0.2M 
2,2'-oxy-bis-ethylamine in 0.5M carbonate buffer, pH 9.5, at 4.degree. 
added. The pH of the mixture was adjusted to 9.5 with 1N sodium hydroxide, 
stirred for 2 hrs and 3.52 ml of a 4 mg/ml sodium borohydride-water 
solution added and the mixture allowed to react for 3 hr, followed by 
chromatography through a Sephadex G-50 column. 
The above procedure was repeated using 400 mg of HRP and 3.5 g of 
2,2'-oxy-bis-ethylamine. No significant change in enzyme activity was 
observed between the native amine and the modified amine, which has about 
four additional amino groups. 
EXAMPLE 4 
Conjugation of Theophylline and HRP 
Into a reaction flask was introduced 8.1 mg of 
1-methyl-3-(3'-carboxypropyl)xanthine, 3.8 mg of NHS, 6.7 mg EDAC and 125 
.mu.l DMF and the mixture allowed to stand overnight at room temperature. 
To four 1.3 ml samples of HRP-oxyamine (1 mg) from Example 3 in 0.1M sodium 
carbonate, pH 9.0 was added varying amounts of the ester prepared above to 
provide for preparations having mole ratios of theophylline to HRP of 400; 
200, and two of 100 each. Into the first reaction mixture (400 mole ratio) 
was added 0.217 ml of DMF and 66 .mu.l of the above ester in 8.25 .mu.l 
increments over a period of about 2 hrs. Into the second reaction mixture 
(200 mole ratio), 0.238 ml of DMF was added and 33 .mu.l of the ester 
added incrementally in 8.25 .mu.l increments. Into the third reaction 
mixture (100 mole ratio), 0.24 ml of DMF was added and 16.5 .mu.l of the 
ester added in 8.2 .mu.l increments, while in the final reaction mixture 
(100 mole ratio), no DMF was added, and 8.25 .mu.l of the ester was added 
in 2.1 .mu.l increments. During the addition, the temperature was 
maintained at 4.degree., and the mixture then allowed to stand overnight 
at 4.degree.. 
The reaction mixtures were then worked up by chromatography on G-25 
Sephadex.RTM. with standard buffer. Folin and UV spectroscopic analysis 
indicated theophylline/HRP ratios of 6.9, 4.0, 1.6 and 2.1, respectively. 
EXAMPLE 5 
Immunochromatographic Assay 
In carrying out the assay, the strips prepared in Example 2 were employed. 
Samples containing 0, 2.5, 5.0, 10, 20 and 40 .mu.g/ml (10 .mu.l) were 
mixed with 0.5 ml of a solution containing 0.1M NaH.sub.2 PO.sub.4, 0.2M 
NaCl, pH 7.0, 1 mg/ml BSA, 0.05% Triton QS-15, 100 .mu.g/ml glucose 
oxidase (Sigma, E.C. 1.1.3.4), and 0.2 .mu.g/ml HRP-theophylline 
conjugate. The end of a strip was dipped into this mixture. After the 
solution had reached the top of the strip by capillary migration (6-12 
min), the strip was removed from the enzyme solution and totally immersed 
in a development solution comprising 15 ml of 50 mM glucose and 200 
.mu.g/ml of 4-chloro-1-naphthol and allowed to stand for 20 min. The 
results are depicted in FIG. 2. 
For purposes of comparison assays were also conducted employing 
immunochromatographic strips prepared from the sheet of Example 2 by 
cutting the sheets on a slitter. Referring to FIG. 1 it can be seen that 
the slitter cut strips exhibit extensive concavity of the front resulting 
from accelerated traversal of the liquid sample along the longitudinal 
edges of the strip when compared to the rate of traversal of the liquid 
sample along the body of the strip. 
FIG. 2 demonstrates that the accelerated traversal of the liquid sample 
along the longitudinal edges of the strips cut in accordance with the 
invention has been minimized when compared to the rate of traversal of the 
liquid sample along longitudinal edges of strips cut using a slitter.