CA2198520A1 - Polyamide membrane - Google Patents

Abstract

Disclosed is a method for determining the presence of an analyte in a fluid sample comprising (a) providing a diagnostic device comprising a membrane which contains a sample application zone and at least one indicator zone, in the indicator zone being affixed a member of a binding pair capable of binding with the analyte or a derivative thereof, wherein the membrane has a lateral flow time of less than about 600 seconds at 4 cm and comprises a polyamide layer and a nonporous support layer; (b) placing the sample on the application zone, the sample being in sufficient quantity to cause the sample to flow from the application zone through the indicator zone; (c) passing the sample from the application zone to the indicator zone; and (d) determining the analyte in the indicator zone. Also disclosed are the membrane and the process of producing the membrane.
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Description

21g852~ ' POLYAMIDE ~FMR~N~

TECHNICAL FIELD OF TEE lNv~N-llON
The present invention is related to microporous membranes in general, and microporous polyamide membranes which have high lateral fluid flow rates and are suitable for use in diagnostic devices, in particular.

BACRGRO~ND OF THE lN V~N-l lON
~ateral flow diagnostic devices are known. An example of such a diagnostic device is the pregnancy test device used to determine the presence of human Chorionic Gonodotropin (hCG) hormone in urine. These devices generally contain a sample application zone, an indicator zone, and a capture zone in conjunction with a porous membrane. The urine sample is placed in the absorption zone which typically contains an absorbent material such as a fibrous felt. The sample application zone also contains a colored reagent capable of binding with hCG.
Typically this reagent consists of latex beads which have been derivatized to be able to bind with hCG. The sample application zone is located on one end of the porous membrane strip.
The membrane strip also contains an indicator zone which contains an immobilized reagent capable of binding with the hCG-latex bead complex advancing from the sample application zone. The binding of the reagent with the complex produces a visible indication that the woman is pregnant.
The membrane strip also contains a capture zone which contains an immobilized reagent which is specific for binding with the latex beads, reacted and unreacted. The capture zone produces a second visible indication to confirm that the test is properly working.
The time required for the test fluid to diffuse from the point at which the test specimen is deposited to a defined point on the membrane is known as the lateral flow 2198:~20 _ 2 time, or LFT. The LFT is critical to the functioning of the device. The magnitude of LFT should not be so small so as to fail to displace a preplaced reagent, and consistent with the displacement time or similar requirements, it should be as small as possible in order to allow the user of the test to reach a conclusion as quickly as possible.
Nitrocellulose membranes have been widely used as the porous membrane strip in diagnostic devices. Nitro-cellulose membrane is not inherently hydrophilic, and forthis reason, to render it suitable for use in diagnostic devices, it is impregnated by the manufacturer with a surface active agent, without which it would not be wetted by and thus not penetrated by the specimens to be analyzed. The presence of the surface active agent has been characterized as undesirable by manufacturers of diagnostic devices because it displace the test sample and may change its-behavior.
The nitrocellulose membrane also has certain other undesirable characteristics. These include the inherent brittleness of the membrane which could present difficulties during the manufacture of the diagnostic devices. The brittleness of the nitrocellulose membrane also limits the number of possible membrane configurations suitable for use in diagnostic devices. For example, bent or twisted configurations are difficult to achieve with nitrocellulose membranes. Nitrocellulose also tends to discolor with time. The discoloration makes it aesthetically unattractive and could interfere with the efficient color development in the indicator and capture zones.
It is also known that nitrocellulose membranes have limited shelf life. The flow properties tend to deteriorate with time possibly due to the migration or fugitivity of the various chemical additives added during the membrane manufacture or post-treatment It also has~
been reported that manufacture of nitrocellulose membranes has been plagued by problems related to lack of reproducibility.
Nylon membranes have been proposed for use in diagnostic devices in view of their hydrophilicity and the more attractive mechanical properties such as flexibility and lack of brittleness. However, nylon membranes heretofore known have high LFT values. High LFT values tend to undesirably increase the response time in the diagnostic testing, and in certain instances, render diagnostic testing infeasible.
Thus, there exists a need for a porous membrane that is less brittle than nitrocellulose membranes and have desirable LFT values. There further exists a-need for a porous membrane with desirable LFT values and that is chemically stable. There also exists a need for a porous membrane with desirable LFT values and that does not discolor. There further exists a need for a porous - membrane with desirable LFT values and that can be manufactured readily and without reproducibility problems.
There also exists a need for a diagnostic device comprising a porous membrane having desirable LFT values.
There further exists for a membrane than can be used in a diagnostic device without the need for pretreating the membrane with large amounts of surfactants.
These and other objects of the present invention will be apparent from the detailed description of the preferred embodiments of the invention set forth below.

SUMMARY OF T~E lN V~ llON
The foregoing needs have been fulfilled to a great extent by the present invention which provides a porous membrane having a lateral flow time of less than about 600 seconds at 4 cm and comprising a polyamide layer and a nonporous support layer. In a preferred embodiment, the LFT of the membrane is less than about 350 seconds at 4 cm.

2198~20 _. 4 The present invention also provides a process for the production of the inventive membrane, the process comprising (a) providing a casting solution comprising a polyamide resin, a solvent, and a nonsolvent; (b) spreading the casting solution on a nonporous support layer to form a thin film thereof on the nonporous support layer; (c) contacting and diluting the film of casting solution with a nonsolvent for the polyamide resin, thereby precipitating the polyamide resin from the casting solution in the form of a thin polyamide layer; (d) washing the membrane to remove the solvent; and (e) drying the membrane by providing a heated gas on at least one side of the membrane. The heated gas is preferably provided on both sides of the membrane.
The present invention further provides a diagnostic device for the detection of an analyte in a fluid, the device comprising a membrane having a lateral flow time of less than about 600 seconds at 4 cm and comprising a polyamide layer, a nonporous support layer, a sample application zone, and at least one indicator zone, in the indicator zone being affixed a member of a binding pair capable of binding with the analyte or a derivative thereof.
The present invention further provides a method for determining the presence of an analyte in a fluid comprising (a) providing a diagnostic device comprising a membrane having a lateral flow time of less than about 600 seconds at 4 cm and comprising a polyamide layer, a nonporous support layer, a sample application zone, and at least one indicator zone, in the indicator zone being affixed a member of a binding pair capable of binding with the analyte or a derivative thereof; tb) placing the fluid on the application zone; (c) passing the fluid from the application zone into the indicator zone; and (d) determining the presence of the analyte in the indicator zone.

Z198~20 _ 5 While the invention has been described and disclosed below in connection with certain preferred embodiments and procedures, it is not intended to limit the invention to those specific embodiments. Rather it is intended to cover all such alternative embodiments and modifications as fall within the spirit and scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS
Fig. l depicts a schematic of the test device used to measure the LFT of the membrane of the present invention.

Fig. 2 depicts a schematic of the floatation dryer employed to dry the wet polyamide membrane.

Fig. 3 depicts the sc~nning electron micrograph (SEM) of the top surface of an inventive polyamide membrane.
The SEM shows that the top surface contains micropores.

Fig. 4 depicts the SEM of the cross-section of an inventive polyamide membrane. The focus is on the region extending down from the top surface towards the bottom surface of the membrane. The SEM shows that this region of the membrane contains micropores of substantially uniform pore size.
Fig. 5 depicts the SEM of the cross-section of an inventive polyamide membrane. The focus is on the region extending up from the bottom surface which is in contact with the~Mylar nonporous layer (shown by the dense white region) towards the top surface of the membrane. The SEM
shows that this region of the membrane also contains micropores of substantially uniform pore size and comparable to the pore size in Fig. 4.

Fig. 6 depicts the schematic of a diagnostic device ~ of the present invention. The device has been opened to show the various components of the device.

Fig. 7 depicts the side view of the membrane and the sample application zone of the diagnostic device.

S DET~TTFn DESCRIPTION OF Tn PREFERRED } ODIMENTS
The present invention provides a membrane having an LFT of less than about 600 seconds at 4 cm and comprising a polyamide layer nonporous support layer. The polyamide layer can have any pore size distribution, preferably a pore size distribution that is substantially symmetric throughout its cross-section. The membrane preferably has an LFT of 350 seconds or less at 4 cm, and more preferably an LFT of from about 90 seconds to about 300 seconds at 4 cm.
The LFT value of the membrane can be measured by any suitable method. A method of measuring LFT follows: A
suspension of blue dyed polystyrene spheres in water was obtained from Bangs Laboratories, Carmel, Indiana, specified as "Royal Blue Al 0.303 mm diameter polystyrene dyed microspheres." Prior to use, the concentration of the microspheres was reduced to 0.04~ by adding one part of the suspension to 250 parts of water.
To perform the test, an apparatus generally in conformance with Fig. 1 is provided. Fig. 1 is an elevation view of a transparent plastic test device 11 comprising a platform 12, an upright post 13, and a shallow cavity 14. Two hundred microliters of the test suspension is placed in cavity 14, thereby forming pool 15. A test strip 16 is cut to 0.5 x 7 cm from the porous membrane to be tested, and marked near one edge at 1, 3, and 5 cm from one end. The unmarked end of the test strip is then fastened to the top of post 13, thus cantilevering the marked end in the air above platform 12. Using tweezers, the cantilevered end of the test strip 16 is dipped into the center of pool 15, in which it is retained by capillarity, and the times are measured for the advancing front(s) to travel 2 cm, from the 1 cm to the 3 cm mark, and the time to travel 4 cm, from the 1 cm mark to the 5 cm mark.
Depending on the nature of the membrane, the blue spheres may advance coincidentally with the liquid front, or the blue spheres may be retarded, in which case a separate blue front is observed, and there is a gap between the blue front and the liquid front. If the blue spheres have reached the 4 cm mark at the same time as the liquid (i.e., the advancing fronts coincide) the "lag" is recorded as zero; if the blue spheres lag behind and have not fully advanced, the magnitude of the lag at 4 cm is recorded accordingly. A lag of more than about 1 mm is undesirable, and a zero lag is highly preferred for proper functioning of a diagnostic test. The membranes of the present invention have been found to be substantially free of lag.
The membrane of the present invention can be made from any suitable polyamide resin. For example,-the polyamide can have the methylene (CH2) to the amide (NHCO) ratio of from about 4:1 to about 7:1. Preferably, the membrane is made from a polyamide selected from the group consisting of polyhexamethylene adipamide, polyhexamethylene sebacamide, polytetramethylene adipamide, polytetramethylene sebacamide, and poly-~-caprolactam, and more preferably from polyhexamethyleneadipamide.
The preparation of the resin solution and the casting of the membrane can be carried out by a variety of methods known to those of ordinary skill in the art. One such method is disclosed in U.S. Patent 4,340,479, the disclosure of which is hereby expressly incorporated by reference in its entirety.
Polyamide resins of any suitable molecular weights can be employed. For-example, the molecular weight can be up to about 200,000, preferably in the range of from about 10,000-150,000. In certain embodiments, the molecular weight can be in the range of about 130,000-140,000.

2198~20 The polyamide resin solution from which the membrane is cast can be a solution in any solvent for the polymer.
A preferred solvent is formic acid at any temperature from its freezing point to its boiling point. Other suitable solvents include other liquid aliphatic acids such as acetic acid and propionic acid, and halogenated aliphatic acids such as trichloroacetic, trichloropropionic, chloroacetic acid, dichloroacetic acids, phenols such as phenol, the cresols, and their halogenated derivatives inorganic acids such as hydrochloric, sulfuric, hydrofluoric and phosphoric; saturated aqueous or alcohol solutions of alcohol-soluble salts such as calcium chloride, magnesium chloride and lithium chloride;
hydroxylic solvents including halogenated alcohols (trichloroethanol, trifluroethanol), benzyl alcohol, and polyhydric alcohols such as ethylene glycol, propylene glycol, and glycerol; and polar aprotic solvents such as ethylene carbonate, diethyl succinate, dimethyl sulfoxide and dimethyl formamide.
The polyamide resin solution, hereafter referenced as the starting resin solution, is prepared by dissolution of the polyamide resin to be used in the membrane in the desired solvent. The resin can be dissolved in the solvent at ambient temperature, but a higher temperature may be used to accelerate dissolution.
The casting solution can be prepared from the starting resin solution by diluting it with a nonsolvent or with a mixture of solvent and nonsolvent, or alternatively, the casting solution can be prepared by dissolving the polyamide resin in a mixture of solvent and nonsolvent.
The casting solution so prepared is then formed into a thin membrane by casting it onto an appropriate support, and the membrane is immersed with minimum delay into a bath containing a nonsolvent for the polyamide resin, together with a substantial proportion of solvent for the resin. If the nonsolvent in the bath is water, and if the solvent is formic acid, the presence of at least about 43 by weight of the bath and usually between about 55~ and 58~ of formic acid by weight of the bath is preferred.
The stability of the casting resin solution can vary depending on the amount of solution prepared. For example, casting resin solutions prepared under small scale batch conditions tend to be relatively unstable compared to casting solutions prepared in a larger scale.
The viscosity of the casting resin solution is preferably adjusted to between about 500 centipoises and - 11,000 centipoises, and in some embodiments, it can be between about 7,000 and about 11,000 centipoises at 30~C.
The casting resin solution can be at any suitable temperature during casting, typically below about 85~C, preferably from about 60~C to about 72~C, and more preferably from about 67~C to about 72~C.
It is important that the casting resin solution be clear, and free from suspended material, before being spread upon the support layer to form a membrane. If suspended material is present, such as undissolved resin particles, these are removed by screening or filtration before casting. The control of the precipitation so as to obtain the formation of a hydrophilic polyamide membrane of desired flow characteristics and pore size requires that the casting solution be controlled with respect to a characteristic referred to herein as "nucleation". The variables that must be controlled include the choice of resin and of solvent and nonsolvent, the concentration of the resin, temperatures of all components, the quantity of nonsolvent, and intensity of mixing. Mixing speeds of as high as about 2700 rpm may be needed to obtain a resin solution suitable for membrane production.
Any suitable nonporous support layer can be used to cast the membrane. For example, the nonporous support layer can be plastic or metal, preferably a plastic. The nonporous layer of the mem~rane as described above can have any suitable thickness, preferably a thickness of 219852~3 from about 12 ~m to about 50 ~m, and more preferably a thickness of from about 25 ~m to about 40 ~m. A preferred plastic support layer is polyethylene terephthalate (PET), also known as Mylar. A particular advantage of PET is that it is transparent to visible light, which makes it attractive for diagnostic devices which can be scanned by a spectrophotometer.
The casting solution is spread as a thin film on the support by means known to those skilled in the art, for example, by using a doctor blade, and the cast membrane is immersed in a quenching bath which typically contains one or more nonsolvents for the polyamide resin. The quench bath preferably contains water as the nonsolvent, and in addition, a solvent such as formic acid. The formic acid lS content should be carefully controlled to obtain a membrane of controlled pore size. Typically, the formic acid content is about 40~ by weight of the quench bath or - higher, preferably between 50~ and 60~ by weight, and more preferably between SS~ and 58~ by weight of the quench bath.
Following the quenching of the cast membrane in the quench or gelation bath, the membrane is washed to remove solvent. Water is suitable, but any volatile liquid in which the solvent is soluble and that can be removed during drying can be used as the washing liquid.
One or several washes or baths can be used as required to reduce solvent content to below the desired minimum. In the continuous process, wash liquid flow is countercurrently to the membrane, which can, for example be passed through a series of shallow washing liquid baths in the washing stage.
It has been discovered that the manner of drying the membrane is critical to obtaining LFT values suitable for use in diagnostic devices. It has been found that by drying the membrane in a floatation dryer using a heated gas, such as heated air, nitrogen, argon, helium, and the like, the desired LFT values can be obtained.

219832~
~ 11 Heated gas is provided to at least one surface of the membrane, preferably to the exposed surface of the polyamide layer. It is further preferred that the heated gas is provided to both sides of the membrane.
Thus,-the membrane is preferably dried as follows. A
schematic of the floatation dryer 200 is shown in Fig. 2.
A roll of the wet membrane 210 having a polyamide layer and a nonporous support layer is placed on a mandrel 211 on the unwind stand associated with the floatation dryer.
Starting with the leading edge, the membrane is threaded through a suitable web path, for example, as defined by tension rollers 212 and 213, into the drying chamber 214.
The membrane is pulled through the drying chamber to the rewind stand 221. The leading edge of the membrane is attached to the empty core of the rewind stand so that the membrane is now ready for drying. The processing parameters are entered into a suitable process control panel and a line run is initiated to drive the membrane through the drying chamber. The driving force is provided by a combination of two drive rollers and the rewind mandrel. In the drying chamber, the membrane is dried by passing between a series of upper air bars (e.g., 215, 216) and lower air bars (e.g., 217, 218). The upper and lower air bars are offset from each other in such a manner as to provide a cushion of air to support the membrane.
The membrane is preferably under minimal tension during drying. The membrane is exposed to hot air from the air bars on both sides to effect the drying.
The drying chamber is typically approximately 10 ft.
long. The residence time of the membrane in the drying chamber can be controlled by the line speed. Any suitable line speed can be employed. For example, a line speed of from about 10 feet per minute (fpm) to about 100 fpm can be employed, preferably a line speed of from about 20 fpm to about 60 fpm, and more preferably a line speed of from about 25 fpm to about 40 fpm, can be employed.

The heated air can be at any suitable temperature.
For example, the air can be at a temperature of from about 65~C to about 160~C, preferably from about 90~C to about 110~C
It is generally preferred to maintain a slight negative pressure inside the drying chamber. For example, a negative pressure of about 1 to 10 inches of water, and preferably about 4 to about inches of water can be maintained.
The polyamide layer of the membrane prepared as above can be of any suitable thickness, for example, a thickness of from about 25 ~m to about 125 ~m. In particular, some embodiments of the present invention have a thickness of from about 50 ~m to about 75 ~m and some others have a thickness of from about 90 ~m to about 100 ~m. In some embodiments of the present invention, for example, where the membrane is to be used as a dipstick to measure the presence of analytes, the polyamide layer can have a thickness of less than 2~ ~m, particularly a thickness of from about S ~m to about 15 ~m. In certain embodiments where the membrane is to be used as a dipstick, the support layer can extend beyond the polyamide layer to form a handle.
The membrane-produced as above has substantially uniform pore structure. The pore structure can be studied by any suitable method known to those of ordinary skill in the art. Sc~nn;ng electron microscopy provides a method of-determining the pore size and pore size distribution.
Fig. 3 depicts the surface morphology of a membrane of the present invention. The surface is porous and substantially uniform. Membranes of the present invention can be made to have any substantially uniform size pores.
In accordance with the present invention, membranes having substantially uniform pores in the range of about 3 to - 35 about 12 ~m can be produced.
Fig. 4 depicts the SEM of the cross-section of an inventive membrane. The focus is on the region extending 2198~20 _ 13 down from the top surface towards the bottom surface of the membrane. The SEM shows that this region of the membrane contains micropores of substantially uniform pore size.
Fig. 5 depicts the SEM of the cross-section of an inventive polyamide membrane. The focus is on the region extending up from the bottom surface which is in contact with the Mylar nonporous layer, shown by the dense white region, towards the top surface of the membrane. The SEM
shows that this region of the membrane also contains micropores of substantially uniform pore size and comparable to the pore size in Fig. 4.
The membrane of the present invention in a preferred embodiment has a protein binding affinity of from about 90 to about 200 ~g/cm2as determined by IgG immersion assay.
The protein binding affinity can be determined by radioactive IgG assay, e.g., by measuring the amount of radioactivity on a membrane of known area that was immersed in a solution containing l2sI labelled IgG
(DuPont, Wilmington, DE), as is known in the art. In some embodiments, the membrane has a water absorption capacity of about 4 ~L/cm2 to about 20 ~L/cm2.
The membrane of the present invention has at least one, and more preferably two or more of the following advantages. The membrane has robust handling characteristics. The membrane is bendable. It is not brittle. It does not discolor with time. It does not require the use of many additives to keep it from cracking. It has a stable chemistry. It is resistant to aging with respect to discoloration and flow rate variation. The polyamide layer does not delaminate from the support layer easily.
The present invention also provides a process for the production of the inventive membrane, the process comprising (a) providing a casting resin solution comprising a polyamide resin, a solvent, and a nonsolvent;
(b) spreading the casting solution on a nonporous support 2198~20 ~ayer to form a thin film thereof on the nonporous support layer; (c) contacting and diluting the film of casting solution with a nonsolvent for the polyamide resin, thereby precipitating the polyamide resin from the casting solution in the form of a thin polyamide layer; (d) washing the membrane to remove the solvent; and (e) drying the membrane by providing a heated gas on at least one side of the membrane.
The present invention further provides a process for preparing the inventive membrane, the process comprising (a) providing a resin solution of a polyamide resin in a solvent; (b) inducing nucleation of the resin solution by controlled addition of a nonsolvent for the polyamide resin and thereby providing a casting solution; (c) lS spreading the casting solution on a nonporous support layer to form a thin film thereof on the nonporous support layer; (d) contacting and diluting the film of casting - solution with a nonsolvent for the polyamide resin, thereby precipitating the polyamide resin from the casting solution in the form of a thin polyamide layer; (e) washing the membrane to remove the solvent; and (f) drying the membrane-by providing a heated gas on at least one side of the membrane.
In a preferred embodiment of the process for preparing the membrane of the present invention, the heated gas is provided on both sides of the membrane.
Multilayered membranes can also be prepared in accordance with the present invention. An example of a multilayered membrane is one in which a nonporous layer is sandwiched between two polyamide layers. The device could also have additional layers and/or media as well as alternative configurations of layers and/or media.
The present invention further provides a diagnostic device for the detection of an analyte in a fluid sample, the device comprising the inventive polyamide membrane.
The membrane further comprises a sample application zone and at least one indicator zone, in the indicator zone 219852~
_ 15 being affixed a member of a binding pair capable of binding with the analyte or a derivative thereof.
Fig. 6 depicts the schematic of a diagnostic device 600 that is similar to the one used for the pregnancy 5 test. The device comprises a bottom half 620 and an upper half 622. In actual use, the two halves will be in closed position. The bottom half accommodates the membrane 610. The membrane has a sample application zone 612, an indicator zone 616, and a capture zone 618. The -10 sample application zone also contains an absorbent pad made of suitable absorbent material such as filter paper, cotton, or other fibrous material, to enhance the sample absorption rate. The opening 624 provides access to the sample application zone.
The sample application zone also contains a first reagent 614 that is capable of binding with the analyte in the sample and/or providing a derivative of the analyte.
The reagent is generally immobilized on a support material such as a polystyrene latex bead or colloidal gold. The latex beads can be of any suitable diameter, for example, of from about 0.02 ~m to about 400 ~m, and preferably a diameter of from about 0.02 ~m to about 5 ~m. The colloidal gold has a diameter of from about 20 nm to about 40 nm. Carbon particles having high surface areas, such 25 as graphite particles, can also be used. Buckyballs may also be suitable.
The derivative in the sample application zone travels toward the indicator zone which has an indicator reagent thereon and is selective to the analyte portion of the 30 derivative. The reaction between the derivative and the indicator reagent results in the development of an indication such as line 630 which can be visually appreciated through the view port 626.
The capture zone has a reagent printed thereon which 35 is selective for the first reagent or the first reagent portion of the derivative. The capture zone develops an indication such as line 632 to confirm that the diagnostic ~ 16 device is functioning properly. The indication in the capture zone can be visually appreciated through the view port 628. Thus, two indications will be seen if the sample contains the analyte and only one indication if the sample does not contain the analyte.
Fig. 7 depicts the side view of the membrane 700 and provides certain additional details of the membrane and the various zones of th-e diagnostic device. The polyamide layer 701 is in contact with the nonporous layer 702. The sample application zone contains an absorbent pad 703 which serves to absorb and distribute the fluid containing the analyte. The sample application zone also contains a reagent release pad 704 which contains the reagent that is capable of binding with the analyte. The indicator zone 705 and capture zone 706 are downstream of the sample application zone.
The device of the present invention can detect a plurality of analytes and can include a plurality of reagents and/or types of beads.
Downstream of the capture zone is located another absorbent pad 707 which serves to absorb the excess fluid sample before the user disposes off the test device. The absorbent pad also serves to accelerate the diffusion of fluid to the capture zone and hastens the color development in the capture zone. The absorbent pad is composed of any suitable absorbent material, including polyesters, polyurethanes, or cellulosic materials.
The present invention also provides a method for determining the presence of an analyte in a fluid sample -comprising (a) providing a diagnostic device of thepresent invention; (b) placing the sample on the application zone; (c) passing the fluid from the application zone into the indicator zone; and (d) determining the presence of the analyte in the indicator zone.
Detection may use any of a variety of labels and/or markers, e.g., enzymes, radioisotopes, liposomes, 2198~20 fluorescent tags, polymer dyes, or colored particles, etc., and detection is by means of, for example, direct visual observation, by developing a color, by spectrophotometry, by radioactive isotope counting, by measuring the magnetic behavior, by fluorescence measurement, or a combination thereof, or by any of many other techniques by which the presence or absence of a chemical or biochemical species may be detected directly or indirectly.
To the indicator zone in the device and method of the present invention is affixed one of the members of a binding pair, which is responsible for the capture of its complementary member. Any suitable means of affixing can be used-to affix the member, e.g., by covalent bonding or, more commonly, by adsorption, e.g., by drying.
Depending on the nature of the material comprising the membrane, derivatization to permit covalent binding for example using glutaraldehyde or a carbodiimide.
Most binding parts employed in the invention are "specificn, e.g., antigen-antibody pairs, and other specific coupling pairs such as antibody-hapten, antibody-cell, antibody-cell fragment, RNA and DNA probes, receptor-receptor ligand, enzyme-substrate, enzyme-inhibitor and other pairs in which a specific binding reaction occurs. However, in some instances the specificity of the assay may be conferred in other ways, such as by a labeling reagent. The requirement for the affixed member is that it must bind the analyte or its derivative. ~or example, for a bacterial analyte such as Streptococcus A, a suitable antibody will be anti-Streptococcus A; for a hormonal analyte such as hCG, a suitable antibody will be anti-hCG; for a drug metabolite analyte such as cocaine, a suitable antibody will be anti-cocaine; and for a protein type analyte such as IgG, a suitable antibody will be anti-hIgG. The essential requirement is that the antigen-antibody pair must be 2198~Q
_ 18 biologically active with respect to each other in the test system.
The affixed member of the pair may bind directly or indirectly to the analyte, or may bind to a derivative S thereof. By "derivativeN is meant any substance whose concentration in the sample is proportional to the analyte. For example, the derivative may be a conjugate of the analyte with an additional component which, in turn, binds to the affixed member, or with an additional component which serves to label the analyte, but not interfere with the analyte's ability to bind to the affixed member. In another illustration, the derivative might be a reaction product formed in stoichiometric relationship to the analyte in a reaction, wherein the reaction product binds to the affixed member. Thus, "derivative~' is a substance quantitatively related to analyte concentration.
It is not necessary that the binding pair member be bound directly chemically or biologically to the membrane.
The binding pair member may be attached to another material wherein said material is physically entrapped in the indicator zone or otherwise affixed in the indicator zone by any physical, chemical or biochemical means. For example, specific binding members can be attached covalently or passively to beads or the like and the beads then affixed on the membrane.
The method of the present invention can be practiced -on any suitable fluid, especially a biological fluid.
Examples include plasma, blood, lymph, urine, serum, ascites fluid, cerebrospinal fluid, gastric or nasal secretions, sputum, pharyngeal exudates, and urethral or vaginal secretions.
The analyte may be insoluble or attached to insoluble supports or may be soluble. Typical cell-bound or solid supported analytes include, e.g., tissue-specific cell surface markers, tissue-shared cell surface markers, viral-associated cell surface markers, and tumor-specific 21~8320 cell surface markers, as discussed in European Patent Application 0 296 724.
In addition to surface markers, soluble analytes can also be detected and measured. Typical soluble analytes include: hormones, enzymes, lipids, nucleic acids, viral antigens, immunoglobulins, lymphokines, cytokines, drugs such as morphine, codeine, heroin, cocaine, steroids, and marijuana, glucose, cholesterol, soluble cancer antigens, bacterial antigens and the like. The analytes also include various proteins such as protamines, histones, phosphoproteins, nucleoproteins, and so forth such as, for example, transcortin, erythropoeitin, transferrin, various globulins, thyroxin-binding globulin, the immunoglobulins of various subclasses A, G, D, E, and M, various complement factors, blood clotting factors such as fibrinogen, Factor VIII, tissue thromboplastin, and thrombin.
Also included are hormones such as insulin, glucagon, relaxin, thyrotropin, somatotropin, gonadotropin, follicle-stimulating hormone, gastrin, bradykinin, vasopressin, and various releasing factors. A wide range of antigenic polysaccharides can also be determined such as those from Neisseria qonorrheae, Pasteurella Pestis, Shi~ella dYsentereae, and certain fungi such as Mycosporum and Aspergillus.
The polyamide membrane can be blocked if needed to eliminate or m;n;m;ze the affinity of certain analytes to the polar functions. Any suitable blocking agent known to those of ordinary skill in the art can be used. Examples include sucrose (0.S~), BSA (0.05-0.5%), casein (0.05-0.5~), and fish gelatin (1-5%). Detergents can also used as the blocking agent. Examples of suitable detergents include the TWEEN~ detergents such as TWEEN 20, which are used in the range of 0.01-0.5%, and other detergents such as TRITON X-100 from Rohm & Haas Co. which is used in an amount of 0.05-0.2%. Combinations of these blocking agents also can be employed. The percentages are by weight of 2198~2d .

the blocking solution which can be prepared in water or phosphate buffered saline.
The present invention further provides a method for processing a biological fluid containing an analyte and other substances comprising:
placing the biological fluid on an application zone of a device comprising a membrane of the present invention comprising a sample application zone and at least one indicator zone, in said indicator zone being affixed a member of a binding pair capable of binding with said analyte or a derivative thereof; and separating the analyte from at least a portion of the other substances.

The following example further illustrates the present invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLE
This example illustrates the properties of the membrane of the present invention.
A membrane comprising a polyhexamethylene adipamide layer and a nonporous PET layer was produced as described above. The viscosity of the casting solution was 9,200 centipoises. The formic acid content of the quench bath was 56.6+0.5~ by weight of the bath. The air in the floatation temperature was at 99~ C at run and at 93~ C at idle. The membrane had an LFT value of 150 seconds at 4 cm. The membrane also has water absorption capacity of 8.5 ~L/c~.
Another membrane comprising a polyhexamethylene adipamide layer and a nonporous PET layer was produced as described above. The formic acid content of the quench bath was 55.2+0.5% by weight of the bath. The membrane had an LFT value of 260 seconds at 4 cm.

219832~

All of the references, including patent and patent application, cited herein are hereby incorporated in their entireties by reference.
While this invention has been described with an emphasis upon the preferred embodiment, it will be obvious to those of ordinary skill in the art that variations of the preferred embodiment may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims.

Claims (60)

1. A method for determining the presence of an analyte in a fluid comprising: (a) providing a diagnostic device comprising a membrane having a lateral flow time of less than about 600 seconds at 4 cm and comprising a polyamide layer, a nonporous support layer, a sample application zone, and at least one indicator zone, in the indicator zone being affixed a member of a binding pair capable of binding with the analyte or a derivative thereof; (b) placing said fluid on the application zone;
(c) passing said fluid from the application zone to the indicator zone; and (d) determining the analyte in the indicator zone.

2. The method of claim 1, wherein said polyamide layer has a pore size distribution that is substantially symmetric throughout its cross-section.

3. The method of claim 2, wherein said membrane has a lateral flow time of about 350 seconds or less at 4 cm.

4. The method of claim 3, wherein said membrane has a lateral flow time of from about 90 seconds to about 300 seconds at 4 cm.

5. The method of claim 4, wherein said polyamide is selected from the group consisting of polyhexamethylene adipamide, polyhexamethylene sebacamide, polytetramethylene adipamide, and poly-.epsilon.-caprolactam.

6. The method of claim 5, wherein said polyamide is polyhexamethylene adipamide.

7. The method of claim 6, wherein said polyamide layer has a thickness of from about 25 µm to about 125 µm.

8. The method of claim 7, wherein said polyamide layer has a thickness of from about 50 µm to about 75 µm.

9. The method of claim 7, wherein said nonporous support layer comprises a nonporous polymer layer.

10. The method of claim 9, wherein said nonporous polymer layer comprises a PET layer.

11. The method of claim 10, wherein said PET layer has a thickness of from about 12 µm to about 50 µm.

12. The method of claim 10, wherein said polyamide layer has a pore size of from about 3 µm to about 12 µm.

13. The method of claim 1, wherein said sample application zone comprises an absobent pad.

14. The method of claim 13, wherein said sample application zone further contains a first reagent capable of reacting with said analyte to provide a first reagent-analyte complex.

15. The method of claim 1, wherein said membrane further comprises a capture zone.

16. The method of claim 15, wherein said capture zone contains a second reagent capable of binding with said first reagent or first reagent-analyte complex.

17. The method of claim 1, wherein said analyte or derivative thereof and the affixed binding pair member are complementary members of a binding pair selected from the group consisting of an antigen/(antibody or antibody fragment) and (antibody or antibody fragment)/antigen.

18. The method of claim 1, wherein the amount of the analyte present in the fluid is determined quantitatively.

19. A diagnostic device for the detection of an analyte in a fluid, said device comprising a membrane comprising a sample application zone and at least one indicator zone, in said indicator zone being affixed a member of a binding pair capable of binding with said analyte or a derivative thereof, wherein said membrane has a lateral flow time of less than about 600 seconds at 4 cm and comprises a polyamide layer and a nonporous support layer.

20. The diagnostic device of claim 19, wherein said polyamide layer has a pore size distribution that is substantially symmetric throughout its cross-section.

21. The diagnostic device of claim 20, wherein said membrane has a lateral flow time of about 350 seconds or less at 4 cm.

22. The diagnostic device of claim 21, wherein said membrane has a lateral flow time of from about 90 seconds to about 300 seconds at 4 cm.

23. The diagnostic device of claim 22, wherein said polyamide is selected from the group consisting of polyhexamethylene adipamide, polyhexamethylene sebacamide, polytetramethylene adipamide, and poly-.epsilon.-caprolactam.

24. The diagnostic device of claim 23, wherein said polyamide is polyhexamethylene adipamide.

25. The diagnostic device of claim 24, wherein said polyamide layer has a thickness of from about 25 µm to about 125 µm.

26. The diagnostic device of claim 25, wherein said polyamide layer has a thickness of from about 50 µm to about 75 µm.

27. The diagnostic device of claim 24, wherein said nonporous support layer comprises a nonporous polymer layer.

28. The diagnostic device of claim 27, wherein said nonporous polymer layer comprises a PET layer.

29. The diagnostic device of claim 28, wherein said PET layer has a thickness of from about 12 µm to about 50 µm.

30. The diagnostic device of claim 24, wherein said polyamide layer has a pore size of from about 3 µm to about 12 µm.

31. The diagnostic device of claim 19, wherein said sample application zone comprises an absobent pad.

32. The diagnostic device of claim 31, wherein said sample application zone further contains a first reagent capable of reacting with said analyte to provide an analyte derivative.

33. The diagnostic device of claim 19, wherein said membrane further comprising a capture zone.

34. The diagnostic device of claim 33, wherein said capture zone contains a second reagent capable of binding with said first reagent or said derivative.

35. The diagnostic device of claim 19, wherein said membrane is used as a dipstick.

36. A membrane having a lateral flow time of less than about 600 seconds at 4 cm and comprising a polyamide layer and a nonporous support layer.

37. The membrane of claim 36, wherein said polyamide layer has a pore size distribution that is substantially symmetric throughout its cross-section.

38. The membrane of claim 37, wherein said membrane has a lateral flow time of from about 90 seconds to about 300 seconds at 4 cm.

39. The membrane of claim 38, wherein said polyamide is selected from the group consisting of polyhexamethylene adipamide, polyhexamethylene sebacamide, polytetramethylene adipamide, and poly-.epsilon.-caprolactam.

40. The membrane of claim 39, wherein said polyamide is polyhexamethylene adipamide.

41. The membrane of claim 40, wherein said polyamide layer has a thickness of from about 25 µm to about 125 µm.

42. The membrane of claim 41, wherein said polyamide layer has a thickness of from about 50 µm to about 75 µm.

43. The membrane of claim 36, wherein said nonporous support layer comprises a nonporous polymer layer.

44. The membrane of claim 37, wherein said membrane has a lateral flow time of from about 90 seconds to about 600 seconds at 4 cm.

45. The membrane of claim 43, wherein said nonporous polymer layer comprises a PET layer.

46. The membrane of claim 45, wherein said PET layer has a thickness of from about 12 µm to about 50 µm.

47. The membrane of claim 36, wherein said polyamide layer has a pore size of from about 3 µm to about 12 µm.

48. A process for preparing a membrane having a lateral flow time of less than about 600 seconds at 4 cm and comprising a polyamide layer and a nonporous support layer, the process comprising (a) providing a casting solution comprising a polyamide resin, a solvent, and a nonsolvent; (b) spreading the casting solution on a nonporous support layer to form a thin film thereof on said nonporous support layer; (c) contacting and diluting the film of casting solution with a nonsolvent for said polyamide resin, thereby precipitating said polyamide resin from the casting solution in the form of a thin polyamide layer; (d) washing the membrane to remove the solvent; and (e) drying the membrane by providing a heated gas on at least one side of the membrane.

49. The process of claim 48, wherein said heated gas is heated air.

50. The process of claim 49, wherein said heated air is at a temperature of from about 65°C to about 160°C.

51. The process of claim 50, wherein said heated air is at a temperature of from about 90°C to about 110°C.

52. The process of claim 48, wherein said solvent for the polyamide resin is formic acid and the nonsolvent added for diluting the casting solution is water.

53. The process of claim 48, wherein said casting solution has a viscosity of from about 7,000 centipoises to about 11,000 centipoises at 30°C.

54. The process of claim 53, wherein said casting solution is cast at a temperature of from about 60°C to about 72°C.

55. The membrane produced by the process of claim 48.

56. The membrane produced by the process of claim 51.

57. The membrane produced by the process of claim 53.

58. The membrane produced by the process of claim 54.

59. A process for preparing a membrane having a lateral flow time of less than about 600 seconds at 4 cm and comprising a polyamide layer and a nonporous support layer, the process comprising (a) providing a polyamide resin solution of a polyamide resin in a solvent; (b) inducing nucleation of said resin solution by controlled addition of a nonsolvent for said polyamide resin and thereby providing a casting solution; (c) spreading the casting solution on a nonporous support layer to form a thin film thereof on said nonporous support layer; (d) contacting and diluting the film of casting solution with a nonsolvent for said polyamide resin, thereby precipitating said polyamide resin from the casting solution in the form solution on a nonporous support layer to form a thin film thereof on said nonporous support layer; (e) washing the membrane to remove the solvent; and (f) drying the membrane by providing a heated gas on at least one side of the membrane.

60. A method for processing a biological fluid containing an analyte and other substances comprising:
placing said biological fluid on an application zone of a device comprising a membrane comprising a sample application zone and at least one indicator zone, in said indicator zone being affixed a member of a binding pair capable of binding with said analyte or a derivative thereof, wherein said membrane has a lateral flow time of less than about 600 seconds at 4 cm and comprises a polyamide layer and a nonporous support layer; and separating the analyte from at least a portion of the other substances.