Bilirubin assay using crosslinkable polymers

A colorimetric assay for the determination of conjugated or unconjugated bilirubin in biological fluids can be carried out with an improved analytical element. The element includes a support having thereon a gelatin-free mordant layer with a positively-charged interactive mordant having at least one binding site for bilirubin, a radiation-blocking layer, and a porous spreading layer. The interactive mordant is dispersed in a binder material of crosslinkable copolymers which include a monomer capable of reaction with a crosslinking agent to crosslink the copolymer.

FIELD OF THE INVENTION 
This invention relates to an assay for conjugated or unconjugated bilirubin 
in clinical chemistry. It also relates to a dry analytical element useful 
in this assay and to certain polymers which are particularly useful as 
binder material in the element. 
BACKGROUND OF THE INVENTION 
Bilirubin is a degradation product of hemoglobin. In a healthy individual, 
bilirubin released from aged or damaged red blood cells in the body is 
excreted or degraded into other derivatives. In some cases, however, an 
abnormal amount of bilirubin occurs within the body in the case of 
excessive hemolysis or liver failure. There is evidence that excessive 
amounts of bilirubin in the blood can lead to an undesirable increase in 
bilirubin concentration within the body cells which interferes with 
various cellular processes. The clinical significance of bilirubin 
determination, then, in tests for liver and other related organ functions, 
is apparent. 
In human body fluids such as bile and serum, bilirubin exists in several 
different forms, these forms commonly being referred to in the art as 
conjugated bilirubin (B.sub.c, both mono- and diconjugated forms), 
unconjugated bilirubin (B.sub.u, also known as indirect bilirubin), and 
delta bilirubin (also known as biliprotein). The total bilirubin content 
(B.sub.T), represents the sum of all forms of bilirubin. 
A variety of colorimetric assays for bilirubin are known. For example, U.S. 
Pat. No. 4,069,017 describes an assay for bilirubin carried out on a dry 
multilayer analytical element containing an interactive mordant in a 
reagent layer which binds to bilirubin thereby producing a detectable 
product. The mordant also enhances the molar absorptivity of bilirubin and 
causes a spectral shift in the unconjugated moiety making possible the 
simultaneous analysis of both conjugated and unconjugated bilirubin by 
reading reflectance density at 400 and 460 nm. The element also comprises 
a porous spreading layer and a radiation-blocking layer. Chromophores 
which can cause spectral interference, such as hemoglobin and delta 
bilirubin, are retained in the spreading layer above the radiation 
blocking layer. The bilirubin species, Bu and Bc, migrate through the 
radiation blocking layer to bind with the mordant. The interactive mordant 
is dispersed in a binder material such as gelatin or its derivatives. 
Unfortunately, gelatin and its derivatives have a slight color change over 
time in the 400 to 460 nm region of the spectrum, making for poor 
stability of the system. 
A significant advance in the art is described in U.S. Pat. No. 4,788,153 
(issued Nov. 29, 1988 to Detwiler). The assay for bilirubin described 
therein is carried out on an analytical element substantially free of 
gelatin in the reagent layer. An alternative polymer, 
poly(acrylamide-co-N-vinylpyrrolidone), was used as the reagent layer 
vehicle. 
However, this polymer is not crosslinkable and the structural integrity of 
the element cannot be maintained during the analysis, resulting in 
interferences due to hemoglobin and deltabilirubin. 
It is therefore desirable to obtain for use in the reagent layer binder 
materials that do not absorb light in the 400 to 460 nm range of the 
spectrum, and which maintain structural integrity. 
SUMMARY OF THE INVENTION 
The problems described above have been solved with an analytical element 
for the determination of conjugated or unconjugated bilirubin comprising a 
support having thereon, in order: 
(A) a reagent layer comprising a positively-charged interactive mordant for 
bilirubin, said mordant being dispersed in a binder material which is a 
copolymer derived from: 
(1) one or more monomers selected from the group consisting of acrylamide 
and N-vinylpyrrolidinone; and 
(2) one or more crosslinkable monomers selected from the group consisting 
of (i) primary amino group-containing monomers, (ii) active methylene 
group-containing monomers, and (iii)activated halogen group-containing 
monomers; 
(B) a radiation blocking layer; and 
(C) a porous spreading layer. 
This invention also provides a method for the determination of conjugated 
or unconjugated bilirubin in an aqueous liquid comprising the steps of: 
(A) contacting the aqueous liquid with the above-described analytical 
element; and 
(B) measuring the amount of conjugated or unconjugated bilirubin bound to 
said interactive mordant. 
The element of this invention can be used in an assay for either conjugated 
or unconjugated bilirubin. Because of the crosslinkable polymer vehicle in 
the reagent layer, it is less susceptible to deterioration than prior art 
elements. Certain of these polymers are hydrolytically stable and thus the 
crosslinking will remain intact and maintain coating integrity. The 
element of the invention demonstrates better stability and less 
interference from serum pigments such as hemoglobin. 
It was surprising to find that incorporation of certain monomers capable of 
being crosslinked with common hardeners could produce polymeric binder 
materials showing such significant improvements. These results were 
unexpected because crosslinking affects diffusion in and out of the 
reagent layer and therefore would be expected to have deleterious effects 
on the diffusion of reagents in the element As explained below, it is 
important that all reagents in the element be in fluid contact. 
Further, some monomers of the invention have reactive methylene groups 
which, under high pH conditions, would be expected to form condensation 
products leading to yellow color interferents. Surprisingly, color 
interference did not occur. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to the determination of bilirubin (conjugated 
or unconjugated) in aqueous liquids. In particular, the invention can be 
used to assay biological fluids of either animals or humans, but 
preferably of humans. Such fluids include, but are not limited to, whole 
blood, plasma, serum, lymph, bile, urine, spinal fluid, sputum, 
perspiration and the like as well as stool secretions. It is also possible 
to assay fluid preparations of human or animal tissue such as skeletal 
muscle, heart, kidney, lungs, brains, bone marrow, skin and the like. 
The method of this invention can be practiced with a dry multilayer 
analytical element comprising a support having thereon a multiplicity of 
individual layers. The uppermost layer is a spreading layer to uniformly 
distribute the aqueous liquid over the element. Serum proteins and other 
pigments such as hemoglobin remain in the spreading layer on the basis of 
molecular size. A radiation-blocking layer, directly beneath the spreading 
layer, allows the diffusion of bilirubin to the reagent layer underneath, 
but blocks light from the pigments retained in the spreading layer The 
bottom layer contains a mordant to bind bilirubin. This mordant also 
enhances the molar absorptivity of bilirubin and causes a spectral shift 
in the unconjugated moiety making possible the simultaneous analysis of 
both conjugated and unconjugated bilirubin by reading reflectance density 
at 400 and 460 nm. 
In addition to the spectral enhancement properties of the mordant, this 
system. depends on the successful separation of mordanted bilirubin 
beneath the radiation-blocking layer and other serum pigments above the 
radiation-blocking layer. The mordant/reagent layer, then, must be free of 
pigments that absorb light in the 400 to 460 nm range of the spectrum. 
The support can be any suitable dimensionally stable, and preferably, 
nonporous and transparent (that is, radiation transmissive) material which 
transmits electromagnetic radiation of a wavelength between about 200 and 
about 900 nm. A support of choice for a particular element should be 
compatible with the intended mode of detection (for example, reflectance 
spectroscopy). Useful supports can be prepared from polystyrene, 
polyesters, polycarbonates, cellulose esters and other materials known in 
the art. 
The outermost layer is a porous spreading layer prepared from any suitable 
fibrous or non-fibrous material or mixtures of either or both. The term 
"porous" as used herein means being full of pores such that a fluid can be 
absorbed by capillary action and can pass to other layers in fluid contact 
with the porous layer. The void volume and average pore size of this zone 
can be varied depending upon the fluid to be tested. Useful spreading 
layers can be prepared using fibrous materials, either mixed with a 
suitable binder material or woven into a fabric, as described in U.S. Pat. 
No. 4,292,272 (issued Sep. 29, 1981 to Kitajima et al), polymeric 
compositions or particulate materials, for example, beads bound together 
with or without binding adhesives, as described in U.S. Pat. Nos. 
3,992,158 (issued Nov. 16, 1976 to Przybylowicz et al), 4,258,001 (issued 
Mar. 24, 1981 to Pierce et al) and 4,430,436 (issued Feb. 7, 1984 to 
Koyama et al) and Japanese Patent Publication No. 57(1982)-101760. It is 
desirable that the spreading zone be isotropically porous, meaning that 
the porosity is the same in each direction in the zone as caused by 
interconnected spaces or pores between particles, fibers or polymeric 
strands. 
The elements can have more than one spreading layer, each layer being 
prepared of the same or different materials and having the same or 
different porosity. 
The element also comprises a radiation-blocking layer which contains a 
suitable radiation-blocking pigment, for example titanium dioxide or 
barium sulfate, distributed in a suitable hydrophilic binder material 
which may be the same or different from that used in the reagent layer. 
The interactive mordant needed to bind with bilirubin to provide a 
detectable product is located in a reagent layer located beneath the 
radiation-blocking layer. The interactive mordants useful in the practice 
of this invention correspond to the mordants described in U.S. Pat. No. 
4,069,017 (noted above) and the hydrophobic amines described in U.K. 
Patent Specification No. 2,085,581 (published Apr. 28, 1982) which are 
believed to become positively-charged mordants. In general, these mordants 
have one or more binding sites for bilirubin and comprise at least one 
moiety having a hydrophobic organic matrix and a charge-bearing cationic 
group. Such mordants can be monomeric or polymeric, but preferred mordants 
are homopolymers and copolymers having the properties noted above. They 
bind both conjugated and unconjugated forms of bilirubin. 
The reagent layer also contains a hydrophilic binder material which is 
permeable to bilirubin. As noted above, it is preferred that the binder 
material not contain gelatin or a derivative of gelatin. This binder 
material must also be non-interfering, that is, it must not interfere with 
the mordanting of bilirubin to the mordant described above. In other 
words, it should not be capable of binding or mordanting to bilirubin. 
A list of useful mordants and binder materials is described in U.S. Pat. 
No. 4,788,153 (issued Nov. 29, 1988 to Detwiler) which is incorporated 
herein by reference. The specific hydrophilic binder materials used in the 
present invention are described in more detail below. 
Other layers, for example, subbing or filter layers, can be included in the 
element if desired. All of the layers in the element are generally in 
fluid contact with each other, meaning that fluids and nonmordanted 
reagents and reaction products can pass or be transported between 
superposed regions of adjacent layers. 
The elements of the present invention are free of any interactive 
compositions which give a colorimetric or fluorometric response in the 
presence of bilirubin other than the interactive mordants described below. 
In particular, they are free of the diazonium salts and detectable ligands 
for forming detectable species known in the art for bilirubin 
determination, for example in U.S. Pat. Nos. 4,069,016 (issued Jan. 17, 
1978 to Wu) and 4,548,905 (issued Oct. 22, 1985 to Wu). 
The particularly useful binder materials of the present invention maintain 
the structural integrity of the element without absorbing light at 400 or 
460 nm. The binder materials of the present invention comprise 
crosslinkable copolymers derived from: 
A) one or more monomers comprising about 0 to 99, preferably 40 to 60, and 
most preferably about 45 to 55 weight percent of the total binder polymer, 
said one or more monomers being selected from the group consisting of 
acrylamide monomers and the monomer 1-vinyl-2-pyrrolidone. Examples of 
suitable acrylamide monomers include acrylamide, N-isopropylacrylamide, 
N-(1,1-dimethyl-3-oxobutyl)acrylamide, 
2-acrylamido-2-hydroxymethyl-1,3-propanediol, 
N-(3-dimethylamino-propyl)acrylamide, N,N-dimethylacrylamide, 
N,N-diethylacrylamide, and 3-(2-dimethylaminoethyl)acrylamide. 
Particularly preferred is unsubstituted acrylamide; and 
B) one or more monomers comprising about 1 to 10, preferably about 2 to 5 
weight percent of the total binder polymer, said one or more monomers 
having reactive groups capable of reaction with a crosslinking agent to 
crosslink the copolymer, and being selected from the group consisting of: 
(i) primary amino group-containing monomers and the acid addition salts 
thereof such as N-(3-aminopropyl)methacrylamide hydrochloride, 
2-aminoethyl methacrylate hydrochloride, and p-aminostyrene. 
(ii) active methylene group-containing monomers, i.e., monomers having a 
##STR1## 
group appended thereto wherein R.sub.2 is a cyano, acyl, or alkoxycarbonyl 
group. Suitable examples of acrylic ester monomers containing such groups 
include 2-acetoacetoxyethyl acrylate, 2-acetoacetoxyethyl methacrylate, 
ethyl .alpha.-acetoacetoxymethyl acrylate, and 2-cyanoacetoxyethyl 
methacrylate (described in U.S. Pat. Nos. 3,459,790 and 3,554,987). Vinyl 
monomers containing such groups, for example, ethyl acryloylacetate, 6-(m- 
and p- vinylphenyl)-2,4-hexanedione (60:40); ethyl 5-(m- and p- 
vinylphenyl)-3-oxopentanoate (60:40) and the corresponding methyl ester 
are described in U.S. Pat. Nos. 3,929,482; 3,939,130, and 3,904,418. Amide 
monomers containing such active methylene groups, such as 
N-(2-acetoacetoxyethyl)acrylamide, 
N-(2-acetoacetamidoethyl)-methacrylamide, 
4-acetoacetyl-1-methacryloylpiperazine, acetoacetamidoethyl methacrylate, 
and N-(3-acetoacetamidopropyl)methacrylamide are described in U.S. Pat. 
Nos. 4,247,673 and 4,215,195; 
(iii) activated halogen group-containing monomers which have appended 
halomethylaryl, halomethylcarbonyl, halomethylsulfonyl, haloethylcarbonyl, 
and haloethylsulfonyl groups which will, after polymerization, also 
undergo crosslinking with a suitable crosslinking agent such as a diamine, 
dithiol, diol, etc. Monomers having such halomethylaryl groups, for 
example, vinylbenzyl chloride, and vinylbenzyl bromide are disclosed in 
U.S. Pat. No. 4,017,442. Useful monomers having appended haloethylsulfonyl 
groups such as m- and p-(2-chloroethylsulfonylmethyl)styrene and 
N-(4-chloroethylsulfonylmethylphenyl)acrylamide are described in U.S. Pat. 
Nos. 4,161,407 and 4,548,870. Monomers which provide halomethylcarbonyl 
crosslinkable groups include vinyl chloracetate, 
N-(3-chloroacetamidopropyl) methacrylamide, 2-chloroacetamidoethyl 
methacrylate, 4-chloracetamidostyrene, m- and 
p-chloracetamidomethylstyrene, 
N-(3-chloroacetamidocarbonyliminopropyl)methacrylamide, 
2-chloroacetamidocarbonyliminoethylmethacrylate, 
4-chloracetamidocarbonyliminostyrene, m- and 
p-chloracetamidocarbonyliminomethylstyrene, 
N-vinyl-N'-(3-chloropropionyl)urea, 4-(3-chloropropionamido)styrene, 
4-(3-chloropropionamidocarbonylimino)styrene, 
2-(3-chloropropionamido)ethyl methacrylate, and 
N-[2-(3-chloropropionamido)ethyl]methacrylamide. 
It is well known that the haloethylsulfonyl and haloethylcarbonyl groups of 
polymers derived from monomers containing such groups can be readily 
dehydrohalogenated to vinylsulfonyl and vinylcarbonyl groups which are 
also readily crosslinkable with amine and sulfhydryl groups containing 
crosslinking agents in accordance with this invention, and such derived 
polymers are also within the scope of useful polymers of the present 
invention. 
Polymers having active methylene or primary amine groups are conveniently 
crosslinked with conventional gelatin hardeners such as formaldehyde, 
glyoxal and dialdehydes such as succinaldehyde and glutaraldehyde as 
described in U.S. Pat. No. 3,232,764; active esters such as described in 
U.S. Pat. No. 3,542,558; active halogen compounds such as described in 
U.S. Pat. Nos. 3,106,468 and 3,957,882; s-triazines such as described in 
U.S. Pat. No. 3,325,287; aziridines such as described in U.S. Pat. No. 
3,575,705; active olefins such as described in U.S. Pat. No. 3,490,911 and 
3,640,720; vinylsulfones such as a bis(vinylsulfonylmethyl)ether and 
bis(vinylsulfonyl)methane as described in U.S. Pat. No. 3,841,872 and U.S. 
Pat. No. 3,539,644; halogen-substituted aldehyde acids such as mucochloric 
and mucobromic acids; and polymeric hardeners such as dialdehyde starches; 
poly(acrolein-co-methacrylic acid); 
poly(acrylamide-co-2-chloroethylsulfonylmethylstyrene) and 
poly(acrylamideco-vinylsulfonylmethylstyrene. 
Polymers having activated halogen can be crosslinked with agents having two 
or more amino or mercapto groups such as ethylenediamine, 
1,3-propanediamine, 1,3-propanedithiol, dithiothreitol, dithioerythritol, 
and butylenediamine. 
More specifically, the polymers of this invention are those which conform 
to the structure: 
##STR2## 
wherein: (A) represents recurring units of one or more polymerized 
acrylamide monomers of the type described above; 
(B) represents recurring units of polymerized 1-vinyl-2-pyrrolidone; 
R.sup.1 is hydrogen or methyl; 
L is a linking group which is at least one, and preferably a combination of 
at least two, of the types of groups selected from alkylene of 1 to 30, 
preferably 1 to 10, carbon atoms; arylene groups of 6 to 12 ring carbon 
atoms, such as phenylene, tolylene, xylylene and naphthylene, --Z--, and 
##STR3## 
where alkylene means straight and branched chain alkylene and alkylene 
interrupted or terminated with heteroatoms or heteroatom-containing groups 
such as oxy, thio, imino 
##STR4## 
where R.sup.3 is hydrogen or alkyl of 1 to 6 carbon atoms), ester 
(--COO--), amide (--CONH--), ureylene (--NHCONH--), sulfonyl (--SO.sub.2 
--), and urethane (--NHCOO--) groups; 
Z is 0, imino 
##STR5## 
as defined above) or an N,N'-heterocyclylene group of 5 to 7 carbon and 
hetero ring atoms such as 1,4-piperazinylene; 
R.sup.2 is a reactive group selected from the group consisting of: 
i) primary amino and acid addition salts thereof, i.e., --NH.sub.2 and 
--NH.sub.2.HX where X is an acid anion such as halide, e.g., chloride, 
bromide, fluoride, and iodide; 
ii) an active methylene group, i.e., a group having an acid hydrogen atom 
that is easily displaced by a nucleophile, preferably conforming to the 
structure 
##STR6## 
where R.sup.4 is cyano, acyl of about 1 to 6 carbon atoms such as acetyl, 
propionyl, buryryl, etc., preferably acetyl, or an ester group 
##STR7## 
where R.sup.5 is an alkyl group of about 1 to 5 carbon atoms; and group 
selected 
iii) an activated halogen group selected from halonethylaryl such as 
chloromethylphenyl, halomethylcarbonyl such as chloroacetyl, 
haloethylcarbonyl such as 3-chloropropionyl, and haloethylsulfonyl such as 
2-chloroethylsulfonyl, said activated halogen groups preferably conforming 
to the structure: 
EQU HALO--CH.sub.2 --R.sup.6 -- 
where HALO represents a halogen atom, preferably chloro or bromo and 
--R.sup.6 -- is carbonyl, an ester (--COO--), amide 
##STR8## 
methylenecarbonyl, or methylenesulfonyl group, or a sovalent bond linking 
the HALO--CH.sub.2 group directly to the aromatic ring of an arylene group 
in the linking chain, e.g., to a phenylene, tolylene, xylylene, or 
naphthylene group in the linking chain, and 
x, y, and z represent weight percents, totalling 100, of the recurring 
units such that x is about 0 to 99, preferably 40 to 60, and most 
preferably 45 to 55, y is about 0 to 99, preferably 40 to 60, and most 
preferably 45 to 55, and z is 1 to 10, preferably 2 to 5 weight percent. 
Preparation of the polymers of the invention proceeds via conventional 
addition polymerization techniques such as by using redox initiator 
systems, such as persulfate-bisulfite or hydrogen peroxide, or organic 
soluble free-radical-generating initiating systems such as 
2,2'-azobis(2-methylpropionitrile). We prefer to use a hydrogen peroxide 
initiator in a conventional solution polymerization process, preferably 
using a mixture of water and isopropanol as the solvent. 
The amount of hydrophilic binder material in the mordant layer should be 
sufficient to adequately disperse the mordant therein and to form a 
suitable film. The amount will also depend upon the type of polymeric 
mordant used. Where the mordant is a film-forming polymer, less binder 
material may be needed. Generally, the amount of binder is from about 2 to 
about 20 g/m.sup.2 with amounts of from about 5 to about 20 g/m.sup.2 
being preferred. 
Other optional addenda (including buffers, surfactants and the like) can be 
added to one or more layers of the element, if desired. Also useful in the 
element are one or more bilirubin effectors, or promotors as they are also 
known in the art. Such materials include sodium benzoate, caffeine, gum 
arabic, salicylate, bile salts and mixtures thereof. Preferably, such 
materials are included in the porous spreading layer of the elements. 
A variety of different elements, depending on the method of assay, can be 
prepared in accordance with the present invention. Elements can be 
configured in a variety of forms, including elongated tapes of any desired 
width, sheets, slides or chips. Generally, the elements are individual 
slides which are packaged together in cartridges for use in automated 
analyzers. 
The assay of this invention can be manual or automated. In general, in 
using the dry elements, bilirubin determination is made by taking the 
element from a supply roll, chip packet or other source and physically 
contacting it with a sample (for example up to 200 .mu.l) of the liquid to 
be tested so that the sample and reagents (that is, the interactive 
mordant) within the element become mixed. Such contact can be accomplished 
in any suitable manner, for example, by dipping or immersing the element 
into the liquid or, preferably, by spotting the element by hand or machine 
with a drop of the liquid with a suitable dispensing means. 
After liquid application, the element can be exposed to conditioning, such 
as incubation, heating or the like, that may be desirable to quicken or 
otherwise facilitate obtaining any test result. 
When the mordant binds to bilirubin, a detectable change results which is 
readily measured using suitable apparatus for reflection 
spectrophotometry. Such apparatus is well known in the art. The signal 
from the detectable species so measured is indicative of the amount of 
bilirubin in the fluid tested. 
The method and elements of this invention can be used to measure either 
conjugated or unconjugated forms of bilirubin according to the teaching of 
U.S. Pat. No. 4,338,095, noted above, and which is incorporated herein by 
reference. Generally, this selective measurement of one or both forms of 
bilirubin is accomplished by contact of liquid and element as described 
above, and by measuring the absorption or emission spectra at two or more 
wavelengths and performing the appropriate calculations. 
The crosslinkable copolymers used in the element of the invention are 
prepared as follows. 
Preparation of N-(3-acetoacetamidopropyl)methyacrylamide 
Triethylamine (24 g, 0.24 mole) was added dropwise at 0.degree. C. to a 
solution of N-(3-aminopropyl)methacrylamide hydrochloride (40 g, 0.24 
mole) and diketene (20 g, 0.24 mole) in methanol (800 ml). After addition, 
the temperature was maintained at 0.degree. C. for 2 hours under stirring. 
Stirring was continued at 20.degree. C. for 20 hours. The solvent was then 
removed. The residue was dissolved in chloroform (1 liter), washed with 5% 
hydrochloric acid (200 ml), washed with saturated NaHCO.sub.3 (200 ml), 
dried over anhydrous magnesium sulfate, and filtered. Excess solvent was 
removed. The residue was recrystallized from benzene (500 ml) and ethyl 
ether (500 ml) to give N-(3-acetoacetamidopropyl)methacrylamide (melting 
point =93.degree.-94.degree. C.) at a yield of 50%. 
Preparation of N-(3-Chloroacetamidopropyl)methacrylamide 
In a 3-liter 4-neck flask fitted with condenser, stirrer, and 2-dropping 
funnels were placed N-(3-aminopropyl)methacrylamide hydrochloride (157 g 
0.88 moles) in methanol (1.2 L) and 2,6-di-tert-butyl-p-cresol (1.0 g). In 
one funnel was placed chloroacetyl chloride (100 g, 0.89 mole), and in 
funnel two was placed triethylamine (178 g, 1.76 mole). The solution was 
cooled to 0.degree.-5.degree. C. (ice-methanol), and triethylamine was 
added in a slow stream over 30 minutes, and the chloroacetyl chloride was 
added over 1 hour. After the addition, the temperature was maintained at 
0.degree. C. for 2 hours, the ice bath was removed, and stirring was 
continued at room temperature overnight. The solvent was removed, and to 
the residue was added hot ethyl acetate (500 mL). The mixture was filtered 
to remove triethylamine hydrochloride, then the solid was washed with hot 
ethyl acetate (500 mL), filtered again, the filtrate combined, and the 
solvent was removed on a rotary evaporator. The residue was crystallized 
from ethyl acetate (400 mL) by heating to dissolve, filtering to remove 
any solid present, and cooling to 0.degree. C. to crystallize. The crude 
monomer was purified by chromatography on a silica gel packed column. The 
product was eluted from the column using a 1:1 mixture of ethyl acetate 
and dichloromethane (4 L). The collected solvent was evaporated, and the 
residue crystallized from ethyl acetate (300 mL) with 
2,6-ditert-butyl-p-cresol (500 mg) to give a white crystalline compound, 
mp 85.degree.-90.degree. C., 83 g (43% yield). Analysis Calculated C.sub.9 
H.sub.15 ClN.sub.2 O.sub.2 : C, 49.4; H, 6.9; N, 12.8; Cl, 16.2. Found: C, 
49.0; H, 7.6; N, 13.2; Cl, 17.3. 
Preparation of 
Poly[acrylamide-co-N-vinyl-2-pyrrolidinone-co-N-(3-acetoacetamidopropyl)me 
thacrylamidel (Weight ratio 48.75/48.75/2.5) 
To a solution of acrylamide (105.3 g, 1.4 moles), N-vinyl-2-pyrrolidinone 
(105.3 g, 0.94 mole), N-(3-acetoacetamidopropyl)methacrylamide (5.4 g, 
0.024 mole), and hydrogen peroxide (8.0 g, 30% in water) in H.sub.2 O (1.8 
L) and isopropanol (400 mL) which had been #10 degassed with nitrogen was 
heated at 65.degree.-70.degree.0 C. under a nitrogen atmosphere for 5 
hours and allowed to sit at temperature overnight. The next day the 
solution was concentrated at low heat (40.degree.-50.degree. C.) on a 
rotary evaporator to about 1 L (20.5% solids). This solution was used 
directly for coating. 
Preparation of 
Poly[acrylamide-co-N-vinylpyrrolidinone-co-N-(3-aminopropyl)methacrylamide 
Hydrochloridel (Weight ratio 48.75/48.75/2.5) 
This material was prepared in the same manner as Example 3 except the 
polymer was precipitated in acetone (5 gal), filtered, dried in a vacuum 
oven and redissolved in H20 at 17.4% solids. Also, 
N-(3-aminopropyl)methacrylamide hydrochloride was used instead of 
N-(3-acetoacetamidopropyl)methacrylamide. 
Preparation of 
Poly[acrylamide-co-N-vinylpyrrolidone-co-N-3-chloroacetamidopropyl)methacr 
ylamidel (Weight ratio 48.75/48.75/2.5) 
This material was prepared in the same manner as Example 4 except that 
N-(3-chloroacetamidopropyl)methacrylamide was used instead of 
N-(3-aminopropyl)methacrylamide hydrochloride.

EXAMPLE 1 
Comparison of Elements with Different Binder Materials in Reagent Layer 
This is a comparison between the element of the present invention and two 
control elements. The two control elements were prepared in the same 
manner as the element of the present invention except that one had 
hardened gelatin as the binder material in the reagent layer, and the 
other had poly(acrylamide-co-N-vinyl-2-pyrrolidone) (Weight ratio 50/50). 
The element of the present invention had the format and components 
illustrated below. The term "dry" used herein to describe the weight of 
the components indicates that the coating coverage is determined as dry 
weight after normal coating and drying processes. 
TABLE I 
______________________________________ 
Coating Format 
Useful 
Dry Ranges 
G/m.sup.2 G/m.sup.2 
______________________________________ 
Spreading 
61.388 Anatase TiO.sub.2 10-200 
Layer 10.293 Cellulose Acetate 50-600 
2.027 Triton X-405 0-20 
1.015 Brij 78 0-20 
5.614 Caffeine 0.5-10 
5.293 Sodium Benzoate 0.5-10 
2.321 Polyurethane 0-50 
Sub Layer 
0.390 Poly-N-Isopropylacrylamide 
0.5-5 
Radiation 
21.770 Anatase TiO.sub.2 5-50 
Blocking 0.002 Ottasept 0-.05 
Layer 1.915 Gel 0.2-5 
0.153 Surfactant Olin 10G 
0.01-1 
0.127 Daxad 0.01-1 
Reagent 8.781 Binder (Polymer i, ii, or iii) 
3-15 
Layer 0.003 Ottasept 0-0.05 
1.758 Mordant 0.2-5 
3.619 Bicine 0.5-5 
0.138 Surfactant 10G 0.01-1 
0.110 Crosslinking agent 0.01-0.5 
______________________________________ 
KEY: 
Polymer i is Poly(acrylamideco-N-vinylpyrrolidone 
co-N-(3-aminopropyl)methacrylamide Hydrochloride) 
Polymer ii is 
Poly(acrylamideco-N-vinyl-2-pyrrolidone-co-N-(3-acetoacetamidopropyl)meth 
crylamide) 
Polymer iii is Poly(acrylamideco-N-vinyl pyrrolidone 
co-N-(3-chloroacetamidopropyl)methacrylamide) 
Triton X405 is an octylphenoxy polyethoxy ethanol surfactant sold by Rohm 
and Haas Co. (rights purchased by Union Carbide Co.) 
Brij 78 is a polyoxyethylene stearyl ether surfactant sold by ICI America 
Inc. 
Estane is a polyesterpolyurethane sold by B. F. Goodrich. 
Ottasept is a bactericidal agent. 
Gel is deionized gelatin. 
Surfactant 10G is a nonylphenoxypolyglycidol sold by Olin Chem. Co. 
Daxad is the sodium salt of a carboxylic acid polymeric 
surfactant/dispersing agent sold by W. R. Grace. 
Mordant is a cationic polymer mordant of the type described in U.S. Pat. 
4,338,095. 
Bicine is N,Nbis(2-hydroxyethyl)glycine buffer. 
The effectiveness of the binder materials in the regent layers of five 
different elements were evaluated as follows. A pool of neonate serum was 
divided into five pools and spiked with 0, 50, 100, 200 and 300 mg/dL of 
hemoglobin int he form of a hemolysate. The spiked pools were then run and 
Bu and Bc predictions obtained. Slides to be tested were calibrated on a 
KODAK EKTACHEM analyzer using standard calibrators. The changes in 
predicted Bu and Bc concentrations were tabulated as a function of 
hemoglobin concentration. 
Table II illustrates the results for the five types of elements tested. 
TABLE II 
__________________________________________________________________________ 
Changes in Predicted Concentration Due to Addition of Hemoglobin: All 
Values are in mg/dL 
Bc Bu 
__________________________________________________________________________ 
Hemoglobin 50 100 200 300 50 100 200 300 
Added: 
Standard 0.41 
0.76 
1.54 
2.11 
-0.34 
-0.59 
-1.17 
-1.45 
Formula 
poly[acrylamide-co-N-vinyl-2-pyrrolidone] 
Hardened Gel 0.28 
0.55 
1.08 
1.44 
-0.18 
-0.32 
-0.63 
-0.79 
Polymer i 0.36 
0.55 
1.25 
1.59 
-0.22 
-0.40 
-0.78 
-0.99 
Polymer ii 0.18 
0.32 
0.60 
0.78 
-0.16 
-0.21 
-0.39 
-0.48 
Polymer iii 0.27 
0.50 
1.02 
1.35 
-0.19 
-0.29 
-0.64 
-0.80 
__________________________________________________________________________ 
Polymer iii was crosslinked with dithiothreitol(DTT). The other binders 
were crosslinked with Bis(Vinylsulfonylmethyl ether (BVSME). 
The above results show that all of the crosslinkable binders (gelatin and 
polymers i, ii, and iii) show less change due to hemoglobin. In 
particular, polymer ii shows the least change. Thus, these polymers and 
gelatin are less sensitive to interference by hemoglobin. These polymers 
are hydrolytically stable and thus the crosslinking is expected to remain 
intact and maintain coating integrity. 
The invention has been described in detail with particular reference to 
preferred embodiments thereof, but it will be understood that variations 
and modifications can be effected within the spirit and scope of the 
invention.