Membranes, membrane overlays for exclusion of erythrocytes, and method for immunoassay of whole blood analytes

A diagnostic test strip for chemically determining whole blood analytes comprises a support, a porous detection zone membrane affixed to the support, and an overlay membrane affixed to the support and in overlying and continuous contact with the detection zone membrane. The overlay membrane has a crenating agent for the exclusion of whole red blood cells from the pores of the detection zone membrane.

FIELD OF THE INVENTION 
The present invention relates to a testing system for the chemical or 
immunological assay of whole blood analytes. More specifically, the 
present invention relates to a diagnostic test strip system comprising an 
overlay membrane containing a crenating agent and a detection membrane 
with pores which prevent penetration of erythrocytes into the analyte 
determining detection zone of the test strip system. 
BACKGROUND OF THE INVENTION 
The determination of whole blood analytes is an established diagnostic tool 
in the medical and health care industries. One problem encountered in the 
determination of whole blood analytes is the penetration or permeation of 
red blood cells (RBC) into the reactive, analyte determining sites in the 
testing system. A major cause of immuno assay or chemical assay 
interference in the detection of a signal from an analyte detection zone 
arises from RBC in the test fluid. As the test fluid contacts an absorbent 
detection zone on a membrane carrier, the RBC along with the other 
components of the test fluid are absorbed into and penetrate through the 
membrane and become intermingled in the detection zone. The presence of 
whole RBC in the detection zone results in a discoloration which 
physically and chemically interferes with colorimetric assay procedures. 
A red blood cell is known to comprise an outer membrane enclosing a 
solution that is high in concentration of hemoglobin. The red blood cell 
and the free hemoglobin from hemolysis of the cell can impart a color to 
the detection zone ranging from light pink to dark maroon. As a 
consequence, the production of a visual chemical signal can be partially 
or wholly obscured by the presence of the hemoglobin color in the 
detection zone. Furthermore, the hemoglobin can block the production of 
electromagnetic radiation in a fluorescent-type signal generating 
indicator system. The rapidity of use, accuracy and precision of the dry 
test strip in the qualitative or quantitative analysis of analytes can be 
seriously inhibited by the presence of RBC, hemoglobin and other contents 
of the red blood cell in the detection zone or layer. 
Various techniques have been developed to eliminate the physical and 
chemical interferences created by the presence of whole RBC in the 
detection zones. One alternative, has been the physical elimination of the 
RBC from the sample. Specifically, centrifuge techniques have been used to 
spin down samples thereby expediting the removal of RBC from the samples. 
Agglutinating agents have also been used to clump RBC and facilitate 
physical collection and removal of whole RBC from a sample. Alternatively, 
autonomous or spray applied size exclusion membranes having a definite 
pore size have been used to create and allow analyte penetration of the 
detection zone but exclude whole RBC from the detection zone membrane. 
However, the use of size exclusion processes does not completely eliminate 
interferences created by the presence of RBC at the detection zones. A red 
blood cell can squeeze through a pore having a smaller relative diameter 
than that of the red blood cell due to the malleable or flexible character 
of the cell. Furthermore, the use of smaller pores reduces the real volume 
of analyte which is allowed to pass through any size exclusive membrane 
and actually contact the detection zone. Consequently, the reduced flow of 
analyte to the detection zone may result in variable assay results which 
prove to be undependable in any given instance. 
Accordingly, a need exists for a test system for determining select 
analytes in whole blood samples which is unaffected by the chemical or 
physical interferences normally created by RBC. 
SUMMARY OF THE INVENTION 
The present invention comprises a diagnostic test strip for the chemical or 
immunological assay of whole blood analytes comprising a support, a porous 
detection zone membrane affixed onto the support, and an overlay membrane 
affixed to the support and in overlying and continuous contact with the 
detection zone membrane. The overlay membrane contains an effective amount 
of a crenating agent for the exclusion of whole red blood cells or 
erythrocytes from the pores of the detection zone membrane. 
The detection zone membrane contains a chemical or immuno assay that can 
generate a characteristic signal in the presence of a target analyte. The 
overlay membrane lying in continuous contact with the detection zone 
membrane prevents the passage of RBC through the pores of the detection 
zone membrane through the presence of the crenating agent. The red blood 
cells in passing through the overlay membrane are crenated and made rigid 
and are thereby excluded by the pores of the detection zone membrane. 
Another aspect of the invention is directed to a method of using the 
diagnostic test strip for determining the presence of a target analyte in 
whole blood.

DETAILED DESCRIPTION OF THE INVENTION 
The diagnostic test strip of the present invention for the chemical or 
immunological assay of whole blood analytes comprises a base or substrate, 
a porous detection zone membrane affixed to the substrate and containing a 
reagent system that can generate a detectable signal in the presence of an 
analyte target, and an overlay membrane affixed to the substrate and in 
overlying and continuous contact with the detection zone membrane. The 
detection zone membrane is protected from red blood cell interference in 
the visualization of a signal by the combination of the overlay membrane 
and the pores of the detection membrane. 
SUPPORT 
The base or support of the present invention functions to hold the test 
strip system and provides a handle. The support also provides a basis for 
further mounting of the test strip to make any of a variety of testing 
products. 
Generally, any type of natural or synthetic material which provides the 
necessary rigidity and inertness prior to the use of the test system and 
upon exposure to the fluid analyte may be used as a support. The support 
can be of a variety of shapes and forms, having varied dimensions 
depending on the applicability of the material in production. The support 
typically will have a thickness of at least 0.1 micron, typically greater 
than 1 micron, generally in the range of 10 to 100 microns. The support 
can be opaque, translucent or transparent. However, the signal generated 
by the detection zone should not be masked by the nature of the support. 
Various materials can be employed in the support which avoid interfering 
with signal generation, allow passage of the test fluid, and allow 
reaction of the test components. A wide variety of organic and inorganic 
polymers, both natural and synthetic, may be employed in the support 
including polyethylene, polyvinyl chloride, polypropylene, 
poly-4-methylbutene, polystyrene, polymethacrylate, polyethylene 
terephthalate, rayon, nylon, polyvinyl butyrate, silicone films, 
polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, etc. A 
preferred polymer for the support of the invention is polystyrene. Other 
materials which may be used include paper, glass, fiberglass, ceramics, 
metals, metal foils, metalloids, semi-conductive materials, and others. 
Additionally, natural substances that can form gels or films including 
proteins or protein derivatives, cellulosics, drying oils, and others can 
be used to form the support. The support material is preferably 
nonswellable, and mildly hydrophilic. 
DETECTION ZONE MEMBRANE 
The detection zone membrane of the present invention provides a reactive 
detection system for the identification of any chosen analyte. These 
reactive membranes can be made of a variety of synthetic and natural 
porous polymeric materials that are permeable to the analyte. A primary 
function of the materials making up the detection zone membrane is to act 
as a site or locus for an effective concentration of the detection zone 
components and to provide an effective flow of the test fluid through the 
detection zone to permit reaction between the analyte and the immunoassay 
or chemical assay reagents contained within the test device. The detection 
zone membrane can be of a variety of shapes and forms having varied 
dimensions. The typical membrane material will have a thickness of at 
least 0.1 mil (1 mil equals 0.001 inch), typically greater than 1 mil, 
generally in the range of 10-30 mil. These materials can be semi-opaque, 
translucent or transparent. However, the signal generated by the immuno or 
chemical assay should not be masked by the nature of the membrane 
material. 
A preferred material for forming the reactive detection zone membrane 
comprises a porous nylon membrane formed by casting a porous nylon sheet 
on a nonwoven layer (e.g., polyester). Biodyne and Immunodyne from Pall 
are examples of this preferred material. Such a membrane provides uniform 
pore size (minimum of 0.04 micron, preferably 0.2 to 3 microns), chemical 
inertness to typical solvents and reagents used in forming dry test 
strips, and provides significant mechanical strength and integrity that 
promotes rapid and accurate production. The Immunodyne membrane has 
reactive groups or sites which are used to bind an amine, such as 
polyallylamine, which can be reacted with the membrane to give a charge to 
the membrane. The charge aids in preventing the blood cells from passing 
into the pores of the detection zone membrane. 
The detection zone membrane also comprises an activated colorimetric 
indicator for determining a select analyte constituent in the whole blood. 
The signal which is generated by the indicator in the detection zone 
membrane indicates both presence and concentration of a target analyte. 
Generally any readily available indicator which is adequate for 
determining a chosen analyte and that is compatible with a dry test strip 
format may be used in the detection zone membrane. Preferred indicators 
include glucose and cholesterol indicators. 
The detection zone membrane preferably additionally comprises analyte 
reactive precursors that modify whole blood analytes to provide a 
constituent determinable by the detection zone membrane. The analyte 
reactive precursors preferably comprise cholesterol ester hydrolase and 
cholesterol oxidase when cholesterol is the desired target analyte. The 
analyte reactive precursors preferably comprise glucose oxidase and 
peroxidase when glucose is the target analyte. 
The detection zone membrane may also contain additional constituents and 
materials such as buffers including, for example, 
morpholino-ethanesulfonic acid (MES), citrate, citrate/acetate 
combinations, bishydroxyethylglycine, or other buffers all of which may be 
used to buffer the detection zone membrane to a range appropriate for 
determination of a chosen analyte. The detection zone membrane may also 
contain wetting agents, such as nonionic or anionic surfactants or blood 
thinners such as heparin which may be used to increase the flow of analyte 
to the detection zone. 
The detection zone membrane needs to be made in a buffer solution having a 
pH of about 5 to 7 with a pH of 6 being preferred. When the pH is too 
high, the membrane is not effective in blocking blood cells and when the 
pH is too low the amine functionality of polyallylamine will not attach to 
the reactive sites to generate a charge in the membrane. 
A reactive cholesterol detection zone membrane can be prepared by the 
following method. First a membrane with reactive sites (such as Pall's 
Immunodyne R) is reacted with polyallylamine and then with a dried milk 
preparation to give a charged membrane. The washed and dried membrane is 
then impregnated with an aqueous mixture containing the enzymes 
cholesterol oxidase, cholesterol esterase (or lipase) and peroxidase, a 
buffer, surfactant, heparin, etc., and then dried. The membrane is then 
impregnated with an organic solvent solution of the indicator (such as 
orthotolidine or tetramethylbenzidine), a surfactant, and potentiators. A 
considerable quantity of red blood cells is screened out by this membrane 
treatment, but enough cells get through to still give a red background, to 
inhibit the reaction and to obscure the pure blue color (if 
tetramethylbenzidine is the indicator). A glucose detection zone membrane 
may also be prepared by the above method except for the inclusion of the 
enzyme glucose oxidase instead of the cholesterol enzymes. 
OVERLAY MEMBRANE 
The overlay membrane generally comprises a porous membrane of varying 
thickness containing a crenating agent. The crenating agent functions to 
deplete the volume of fluid within the red blood cell. Once the cell 
becomes crenated or has been shrunk, it is much less malleable and 
flexible and becomes rigid. The overlay membrane should allow for passage 
of the analyte once it has been released from the solution of whole red 
blood cells. Preferably the overlay membrane does not have defined pores, 
but has intermatted fibers. 
Optionally, the overlay membrane may be hydrophilic on one side and 
hydrophobic on the other to prevent the reverse flow of analyte away from 
the direction of the detection zone membrane. The overlay membrane must 
remain in continuous contact over the entire surface of the detection zone 
membrane to provide a uniform size exclusion of RBC as well as to provide 
a uniform and continuous channel for the analyte to flow from the upper 
surface of the overlay membrane to the detection zone membrane. 
The overlay membrane can be made from a synthetic polymeric material and 
has a thickness of about 0.1 to 1,000 microns. While any number of overlay 
membranes may generally be used in the present invention, high density 
polypropylene membranes manufactured under the brand name of HDC 
polypropylene by Pall, Inc., polyvinyl chloride membranes manufactured by 
Labconco, polyester membranes manufactured by DuPont and polypropylene 
non-woven membranes treated with various wetting agents or surfactants 
have all been found useful in the present invention. 
The crenating agent in the overlay membrane functions to shrink RBC by 
extracting fluid from the cells. The shrinking or volume depleting action 
of the crenating agent rigidifies the cells making them less flexible and 
malleable and in turn, less able to penetrate into the pores of the 
detection zone membrane. As a result, the stiffer, less flexible cells 
cannot move easily into the pores and are trapped at the surface of the 
detection membrane. In the meantime, the liquid analyte compositions flow 
through the overlay membrane and penetrate the detection zone membrane to 
provide for a viable signal. 
Generally, the crenating agent may be any constituent or composition which 
effectively reduces the volume of water within the RBC flowing through the 
overlay membrane. Particularly useful are inorganic and organic salts 
which, when present in greater than hypertonic concentration, draw water 
from the interior of the RBC. Inorganic salts are preferred, such as the 
alkali or alkali earth metal salts of a halogen, or any salt of a strong 
acid or base. These salts can include sulfates, nitrates, and chlorides, 
such as sodium chloride, lithium chloride, and potassium chloride. The 
above salts are all useful as they remain outside the cell wall, do not 
interfere with the indicator or analyte and are readily available as 
staple chemicals. A particularly preferred inorganic salt for use in the 
present invention is sodium chloride. 
Generally, the concentration of the salt solution used to treat the overlay 
membrane will range at a level exceeding that which would be hypertonic 
and soluble. For sodium chloride the concentration should be from about 
0.85 to about 35%, preferably from about 1 to about 10% and most 
preferably from about 2 to about 6%. As the most preferred concentration 
of crenating agent is that concentration which is by definition 
hypertonic, the concentration will vary depending upon the crenating agent 
of choice. Accordingly, these concentration ranges should be used as 
guidelines and not be strictly interpreted as limitations on the 
usefulness of the present invention. 
ANALYTES 
Virtually any analyte detectable using an immunological or chemical assay 
system can be detected using the test strip system of the present 
invention. A high molecular weight analyte detected by the device of this 
invention is characterized as typically large molecule polypeptides, 
polysaccharides, polynucleic acids and combinations thereof. Other 
analytes can include somatic cells, germ cells, bacteria, viruses and 
cellular units. 
Subcellular units which can be analytes include viral protein, cell wall 
polysaccharides, DNA, DNA segments, RNA, transfer RNA, messenger RNA, 
mitochondrial DNA, mitochondrial cell nuclei, cell membranes, ribosomes, 
and other varied cell organelles, subunits and constituent parts. Such 
large analytes are typically detected using immunological dry test strips 
of the invention and can have molecular weights in excess of about 50,000. 
Many such analytes can have molecular weights ranging from 50,000 to 
5,000,000 or more. 
The analytical test strips of the present invention can also be used to 
detect and quantitate the presence of analytes having modest molecular 
weights, i.e., molecules with a molecular weight less than about 50,000, 
typically between 5,000 and 50,000. A wide variety of such analytes that 
comprise natural proteins and protein subunits can be detected using the 
device of the invention. Such proteins include histones, globulins, 
nucleoproteins, lipoproteins, glycoproteins, somatotropin, prolactin, 
insulin, pepsin, human plasma protein constituents including human 
albumin, thyroxine, binding globulins, haptoglobulin, cerulo plasmin, 
cholinesterase, myoglobin, fibrinogen, plasminogen, poly and monoclonol 
immunoglobulins of the A, D, E, G, or M classes, free, light or heavy 
chains of amino globulens, Fab fragment or F(ab').sub.2 fragment, immuno 
globulin regions, compliment, blood clotting factors, peptide and protein 
hormones such luteinizing hormone, human chorionic gonadotropin, 
vasopressin, and others. Such proteins are typically detected using a 
immunological detection scheme. Antigenic polysaccharides derived from 
pathogen cell walls also act as an immunological antigen. 
Further, small molecules of natural and synthetic origin can also be 
detected using the dry test strips of the invention. Such small molecules 
having a molecular weight of about 50 to 5,000, typically 100 to 2,000 can 
be detected using both chemical and immunological detection schemes. Such 
analytes include small molecule natural biochemicals, ethical drugs 
(restricted to sale only on a doctor's prescription) and over the counter 
and illicit drugs, hormones, peptides, mono and disaccharides, 
metabolites, pesticides, pollutants and other organic synthetic chemicals. 
Drugs of interest include ethanol, alkaloids, such as morphine, codeine, 
heroin, dextramethorphan, and their derivatives and metabolites. Also 
included are ergotalkaloids such as LSD, steroid alkaloids, quinoline 
alkaloids, and others. Ethical drugs of interest include steroids, bile 
acids, digitoxin, diethylstilbesterol, ethynylestradiol and others. 
Other drugs include barbiturates, such as phenobarbital, secobarbital, and 
others. Additionally, drugs such as amphetamines, catecholamines, 
serotonin, L-dopa, epinephrine, chlorpromazine, benzodiazepine, 
phenolthiazine, theophylline, caffeine, cannabis drugs such as cannabinol 
tetrahydrocannabinol, vitamins, prostaglandins, antibiotics such as 
penicillin and penicillin variants, cephalosporin and variants, 
chloromycetin, actinomycetin, and tetracycline, among others can be 
detected. 
Nucleosides and nucleotides, fragments and derivatives thereof including 
ATP, AND, TMN, AZP, and others can be detected. Additionally, drugs 
including methadone, meprobamate, lidocaine, propanolol, antihistamines, 
anticholinergic drugs and others can be detected. Further, analytes 
specifically detected using the system of the present invention in 
clinical chemical analysis include glucose, cholesterol, triglycerides, 
uric acid, urea and other typical small molecule chemical analytes. 
Antibodies useful in the detection zone of the test strip system of the 
present invention can be prepared by well known polyclonal and monoclonal 
antibody preparing techniques. Polyclonal antibodies can be raised 
conventionally in a variety of test animals including mice, rats, rabbits, 
horses, among others. Monoclonal antibodies can be prepared using well 
known techniques such as that disclosed by Kohler and Milstein, 
"Continuous Cultures of Fused Cells Secreting Antibody of Predetermined 
Specificity", Nature, Vol. 256, pp. 495-497, Aug. 7, 1975. 
The present invention particularly lends itself to the clinical or at home 
detection of analytes and test fluids using oxidant enzymes requiring the 
presence of atmospheric oxygen to generate a unique signal in the presence 
of the test analyte. Particularly useful analysis include glucose 
detection using glucose oxidase, alcohol detection using alcohol oxidase, 
and cholesterol detection using cholesterol oxidase. 
The test strip of the invention functions as follows. The overlay membrane 
treats the red blood cells with a crenating agent such as hypertonic 
sodium chloride so that the cells become rigid and crenated. This 
treatment of the cells could be carried out in solution, but this would 
not be convenient for a solid state system, such as the present invention. 
Normally erythrocytes have dimensions of 2.times.7 microns, but can wiggle 
through pores of 1 micron or less because of their flexibility. The 
detection membrane with pores of 1 micron or less holds back the crenated 
red blood cells while allowing molecules such as glucose, alcohols, etc. 
and aggregations such as cholesterol and its esters to pass through. These 
analytes undergo reaction within and on the back side of the detection 
membrane and an appropriate signal is generated. 
The test strip of the invention can be employed in a variety of testing 
device formats. A detection zone for the detection of analytes can be 
formed on a carrier strip to which a volume of blood can be applied for 
the purpose of determining the presence of the target analyte in the blood 
serum. Alternatively, a "pH paper type strip device" can be used that can 
be unreeled from a strip dispenser. Another alternative is embodied in a 
mechanical device which combines a lance that can penetrate the skin to 
provide a blood sample, and a wicking cloth that contacts the blood sample 
and draws the sample to the dry strip device wherein the unique signal is 
produced with little or no RBC interference. Such devices can be visually 
read or can be read by instrumental methods and are disclosed in Garcia et 
al, U.S. Pat. Nos. 4,637,403 and 4,627,445. 
Preferably the support carrier strip used in preparing the test strip of 
the invention which uses a detection system requiring the presence of 
atmospheric oxygen has a construction that promotes the transfer of oxygen 
from the atmosphere to the reaction site of the detection zone membrane. 
The dry test strip can be formed in such a way to promote atmospheric 
contact. One means comprises forming an aperture in the carrier support 
strip at the contact point between the strip and the detection zone 
membrane. Such an aperture can take the form of an oval, circular, or 
polygonal shaped cut-out in the substrate strip. Alternatively, the 
aperture can comprise a highly oxygen permeable polymeric layer introduced 
into the carrier substrate opposite the color forming detection zone, 
through which oxygen can readily be transported for reaction. Preferably 
the dimensions of the aperture are smaller than the detection zone 
membrane but expose a significant portion of the area of the detection 
zone for oxygen transferred visual detection of the color change when 
analyte is present. 
In another embodiment, the detection zone membrane can attached to the 
support strip using a construction design permitting the flow of 
atmospheric oxygen into the interface between the detection zone and the 
support strip. Such oxygen flow to the interface can be promoted by 
providing attachment means between the detection zone membrane and the 
underlying support such that a significant area volume between the 
detection zone and the support remains unoccupied providing access to 
atmospheric oxygen. Such a construction can be obtained by adhering the 
detection zone membrane to raised adhesive areas or to small areas of 
double sided adhesive tape leaving the majority of the reverse side of the 
detection zone to the contact of atmospheric oxygen. 
The dry test strips of the invention can be manufactured by applying an 
overlaying adhesive on the support carrier having at least one aperture 
and then applying the detection zone membrane onto the support over the 
adhesive. An overlay membrane is then affixed to the support and is in 
overlying and continuous contact with the detection zone membrane. 
Preferably the aperture in the support strip is formed before the 
detection zone membrane is applied to the underlying support. Specific 
methods for manufacturing test strips of the invention are discussed below 
in the Examples. 
A general method for using the diagnostic test strip of the invention 
comprising a support, a detection zone membrane, and an overlay membrane 
for the chemical or immunological assay of whole blood analytes comprises 
the steps of applying a sample of whole blood to the overlay membrane and 
analyzing a signal generated from the detection zone membrane to determine 
the presence of any given analyte. 
A preferred immuno assay for the detection of analytes that can use the 
test system of the present invention is that disclosed in Liotta, U.S. 
Pat. No. 4,446,232. The test strip of the present invention comprising a 
Liotta type device has a matrix of three zones, a first labeled reagent 
zone, a second trapping zone, and a third detection zone for label 
detection. In a Liotta system the first labeled reagent zone contains a 
labeled antigen specific antibody or fragment thereof capable of bonding 
to a target analyte. The second trapping zone contains a boundary of 
immobilized antigen. The third detection zone contains a means for 
detecting the presence of the label on the antigen specific antibody or 
fragment thereof. 
In the operation of the Liotta type device, a test fluid containing target 
analyte is applied to the matrix. The analyte in the fluid binds the 
antigen specific labeled antibody. The presence of the analyte on the 
binding sites of the antibody causes the analyte-antibody labeled complex 
to penetrate the matrix and pass through the trapping zone since the 
presence of analyte prevents the antibody and its label from becoming 
trapped by bound antigen. The protected antibody and label penetrate the 
third zone wherein the presence of the label is detected. In this way, the 
presence of analyte in the test fluid can produce a unique quantitative 
signal in the detection layer. 
In the absence of analyte in the test fluid, no analyte can bond to the 
antigen specific labeled antibody. As the application of the test fluid 
causes the unbound labeled antibody to penetrate the second layer, bound 
antigen reacts with and traps the labeled antibody in the second layer 
preventing any of the label from penetrating and causing a detection 
signal in the third layer. 
DETAILED DISCUSSION OF THE DRAWINGS 
FIG. 1 is a planar view of a support 10 having reading site apertures 12 
and an adhesive strip 18 overlying the reading sites 12 of support 10. 
FIG. 2 is a planar view of the support 10 having a detection zone membrane 
14 overlaying the reading sites 12. The detection zone membrane 14 is 
attached to the support 10 by means of adhesive strip 18. 
FIG. 3 is a planar view of a preferred embodiment of the invention with 
support 10 having an overlay membrane 16 affixed to support 10. The 
overlay membrane 16 is in overlying and continuous contact with the 
detection zone membrane 14. 
FIG. 4 is an enlarged cross-sectional view of the preferred embodiment of 
FIG. 3 showing the support 10 having aperture 12 over which the detection 
zone membrane 14 is placed. Overlay membrane 16 is affixed to support 10 
by means of adhesive strip 18 and is in overlying and continuous contact 
with detection zone membrane 14. Side 20 of support 10 is the blood sample 
contact side and side 22 is the indicator reading side of the test strip. 
WORKING EXAMPLES 
The following working Examples disclose various test strips of the 
invention which were made and tested. The process for preparing the 
detection zone membrane of the test strips follows and was used in making 
the following test strip examples 
The process for preparing the detection zone membrane for the detection of 
cholesterol levels was carried out in three separate stages: (1) reaction 
of the membrane with polyallylamine; (2) impregnation with an aqueous mix; 
and (3) impregnation with an organic solvent mix. An Immunodyne membrane 
(Pall, Inc.) was incubated at ambient temperature in a mix of 10 ml of a 
buffer (0.1 M MES, 0.01 M Bicine, pH 6) and 3 mg of polyallylamine for 30 
minutes. The membrane was then scraped to remove excess solution. The 
polyallylamine treated membrane was then incubated at ambient temperature 
in a milk mix of 20 ml of a buffer (same as above) and 1 g of instant, 
non-fat dry milk (Carnation) for 30 minutes, scraped to remove excess 
solution and then washed 3 times for 5 minutes with deionized water. The 
membrane was then dried for 10-20 minutes with forced air at 50.degree. C. 
The dried treated membrane was then impregnated with an aqueous mix 
containing enzymes. The ingredients and respective amounts in the aqueous 
mix are listed in Table I. 
TABLE I 
______________________________________ 
Ingredient Amount 
______________________________________ 
Aqueous Mix 
Buffer (0.1M MES, 0.01 Bicine, pH 6) 
2 ml 
Heparin (1%) 10 .mu.l 
Dextran (17000 mw) 0.4 gms 
Cholesterol Oxidase (Kodak) 
150 mg 
Cholesterol Esterase (Kodak) 
40 mg 
Peroxidase 20 mg 
Aerosol OT (American Cyanamide) 
240 .mu.l 
Methyl Vinyl Ether/Maleic Anhydride 
160 .mu.l 
(10%, hydrolyzed, adjusted to pH 6) 
Polyvinylpyrrolidone (10%) 
80 .mu.l 
Manganese Chloride 5 mg 
Organic Mix 
Toluene 10 ml 
Tetramethylbenzidine 200 mg 
Quinine 0.5 mg 
Aerosol OT 100 mg 
______________________________________ 
The polyallylamine treated membrane was impregnated with the aqueous mix by 
laying the membrane on the surface of the mix until all the pores were 
filled. The membrane was then dried at 75.degree. C. for 4 minutes. The 
membrane was then coated with an organic mix (see Table I) by drawing the 
membrane through the organic mix. The membrane was then dried at 
75.degree. C. for 4 minutes. 
The overlay membrane was made from a hydrophilic HDC polypropylene membrane 
(Pall, Inc.) which was immersed in a 1 M sodium chloride solution 
containing 5 mg/L heparin until the membrane was saturated. The membrane 
was blotted and then dried at 75.degree. C. for 10 minutes. 
Double side adhesive was placed on a polystyrene support and holes were 
punched through the polystyrene and adhesive. The detection and overlay 
membranes were sequentially placed over the adhesive and rolled to insure 
intimate contact. The combination was then cut into sticks. This assembly 
of the test strip is shown in the drawings (FIGS. 1-3). 
EXAMPLE 1 
The test strip of Example 1 was formed using the above method. 
EXAMPLE 2 
The test strip of Example 2 was formed the same as Example 1, but without 
the crenating overlay membrane. 
EXAMPLE 3 
The test strip of Example 3 was made the same as Example 2, but omitted the 
polyallylamine treatment of the detection membrane. 
TEST RESULTS 
Using a sample of blood to test the exclusion of RBC, the strip of Example 
1 provided the best results in that it had a total absence of red color 
and developed a clear green to blue color proportional to the cholesterol 
content of the blood. The strips of Example 2 allowed some hemoglobin from 
the cells through, giving a muddy colored reaction with cholesterol of the 
blood. The strips of Example 3 were completely hemoglobin stained and gave 
almost no reaction with cholesterol. The overlay membrane in combination 
with the polyallylamine treated detection membrane of Example 1 crenated 
and sequestered the red blood cells thereby allowing the cholesterol 
analyte to penetrate into the detection zone. However, it should be noted 
that adding sodium chloride to the aqueous mix used to make the detection 
zone membrane was not effective in crenating the cells. 
EXAMPLES 4-12 
The preceding analysis was run again to determine appropriate overlay 
membrane substitute materials. The detection zone membranes used on test 
strips 4-12 were made by the same method as used in making the test strips 
of Examples 1-3 above. The various overlay membranes were impregnated with 
a one molar NaCl solution and then dried at 75.degree. C. for a period of 
10 minutes. Various overlay membranes were then mounted on the detection 
zone membranes which had been mounted on a polystyrene support. The 
support was then cut into separate test strips. The various overlay 
membranes that were used are listed in Table II. 
TABLE II 
______________________________________ 
Test 
Strip Ex. 
Overlay Membrane Pore Size 
______________________________________ 
4 Polypropylene (HDC 1025b, Pall, Inc.) 
-- 
5 Polypropylene (HDC 1026b, Pall, Inc.) 
-- 
6 Polypropylene (HDC 1025a, Pall, Inc.) 
-- 
7 Polypropylene (HDC 1026a, Pall Inc.) 
-- 
8 Nylon (Biodyne A, Pall, Inc.) 
0.45 microns 
9 Filter paper #54 hardened (Whatman) 
10 NO OVERLAY MEMBRANE (NaCl 
in organic mixture for coating detection 
membrane) 
11 Nylon (Biodyne A) 1.2 microns 
12 NO OVERLAY MEMBRANE 
(Control) 
______________________________________ 
TEST RESULTS 
Test strips 4, 5, 6, 7 and 10 all provided good analyte results from a 
serum sample. Test strip 9 would be able to provide adequate assay results 
if the two membranes retained better adhesion once contacted with the 
sample solution. Test strips 8 and 11 did not react at all. 
Regarding the exclusion of RBC using a blood sample, all the strips were 
better than the control (strip 12), providing interference free results 
except for test strips 8, 10 and 11. Specifically, test strip 4 provided 
better results than test strips 5, 6, 7, 8 and 9. 
In conclusion, the above working examples establish that sodium chloride 
incorporated in the overlay membrane is effective in shrinking and 
rigidifying RBC. Most of the above membranes worked as overlay layers when 
impregnated with NaCl to crenate RBC. However, Biodyne A having a pore 
size of 0.45 micron did not work at all, neither did Biodyne A having a 
1.2 micron pore size. 
Examples 13 and 14 are specific membrane overlays and reactive detection 
zone membranes prepared for solid state assays of glucose and cholesterol. 
EXAMPLE 13 
Glucose Detection 
The membranes that were formulated for use on the glucose detection test 
strip of Example 13 were produced by a similar method as used in the 
previous Examples. The overlay membrane was formulated from an HDC 
polypropylene membrane (Pall, Inc.) which was treated with a 1 M NaCl 
containing 10 mg heparin per liter and then dried. A reactive membrane of 
Biodyne A (0.2 micron pore diameter--Pall, Inc.) was impregnated with an 
aqueous mix as shown in Table III. The membrane was then dried in a tunnel 
drier and passed through an organic mix (see Table III). 
TABLE III 
______________________________________ 
Ingredient Amount 
______________________________________ 
Aqueous Mix 
Buffer (10 mm Citrate, pH 6) 
100 ml 
Dextran (17000 MW) 20 gms 
Glucose Oxidase 60000 units 
Peroxidase 90000 units 
Methyl Vinyl Ether/Maleic 
0.8 gms 
Anhydride (hydrolyzed) 
Polyvinylpyrrolidone 40 
0.4 gms 
Organic Mix 
Methanol 100 ml 
Gantrez S95 0.3 gms 
Tartrazine 0.03 gms 
o-Tolidine 0.5 gms 
Tetramethylbenzidine 0.2 gms 
______________________________________ 
The membrane was dried again in the tunnel dryer. The reactive membrane was 
then cut into 1/4 inch strips and attached to a polystyrene backing with a 
double sided adhesive (holes were punched through the backing and adhesive 
first.) The adhesive extended slightly beyond the width of the membrane 
strips. The treated overlay membrane was then cut into strips slightly 
wider than the reactive membrane strips. The overlay membrane strip was 
placed over the reactive membrane strip so that the overlay strip was 
attached to the backing with the above adhesive. This combination was then 
cut into single sticks. 
TEST RESULTS 
When blood was placed on the overlay membrane of the sticks, the reverse 
side of the reactive membrane turned from green to blue to black as the 
glucose level of the blood increased. No blood or hemoglobin color 
appeared on the reverse side (reading side) of the reactive detection zone 
membrane. 
EXAMPLE 14 
Cholesterol Detection 
The test strip of Example 14 was made by the following method. The overlay 
membrane was formulated from an HDC polypropylene membrane (Pall, Inc.) 
which was treated with 1 M NaCl containing 10 mg heparin per liter and 
then dried. The reactive membrane Immunodyne (Pall, Inc., 1.2 micron 
diameter pore) was treated with polyallylamine hydrochloride (30 mg/100 
ml) in a pH 6 buffer of 0.1 M morpholinoethanesulfonic acid (MES) and 0.01 
M bis-dihydroxyethylglycine (Bicine). The membrane was washed with 
deionized water and dried. The membrane was then treated with Carnation 
fat-free dried milk (0.5% in the pH 6 buffer), dried, washed with 
deionized water and dried again. 
The treated membrane was then impregnated with an aqueous mix as shown in 
Table IV. 
TABLE IV 
______________________________________ 
Aqueous Mix 
Ingredient Amount 
______________________________________ 
Buffer (pH 6, 0.1M MES, 0.01 Bicine) 
4 ml 
Cholesterol Oxidase (Kodak) 
500 units 
Cholesterol Esterase (Kodak) 
1000 units 
Heparin (1%) 20 .mu.l 
Peroxidase 1000 units 
Dextran (17000 MW) 800 mg 
Aerosol OT (American Cyanamid) (7%) 
480 .mu.l 
Methyl Vinyl Ether/Maleic 
320 .mu.l 
Anhydride (hydrolyzed, 10%) 
Polyvinylpyrrolidone (10%) 
160 .mu.l 
______________________________________ 
In preparing the above mixture, phosphate was removed from the cholesterol 
oxidase and esterase by dissolving the two enzymes in 2 ml of the 
MES-Bicine buffer, placing the solution on a Sephadex G-25 column 
(Pharmacia PD 10) and eluting with buffer until a volume of 4 ml was 
collected. The other ingredients were then added and mixed. The 
impregnated membrane was dried and then treated with a saturated indicator 
solution of tetramethylbenzidine in toluene and dried. 
The reactive detection zone membrane and the overlay membrane were placed 
on a polystyrene backing with a double sided adhesive as described in 
Example 13 and then cut into sticks. 
TEST RESULTS 
When the sticks were contacted with blood, they turned from green to blue 
depending on the level of cholesterol and its esters. The reactivity of 
the sticks may be varied by including inhibitors or other indicators in 
the aqueous toluene mixture used in the detection zone membrane. By 
omitting esterase from the composition, the same system can be used to 
determine free cholesterol. 
The same system for screening out erythrocytes while allowing cholesterol 
and its esters through can be combined with the teaching of Liotta 
described above to determine low density lipoproteins and high density 
lipoproteins using the antisera to the apolipoproteins. 
The purpose of the heparin in the overlay treatments of Examples 13 and 14 
is to prevent the hemolysis of erythrocytes caused by stretching of the 
cells in contact with the membrane. 
While the invention has been described and fully explained in the detailed 
description of the specification and preferred embodiments, many 
embodiments of the invention can be made without departing from the spirit 
and scope of the invention.