CA1161347A - Continuous release of reagent in an analytical element to reduce assay interference - Google Patents

Abstract

CONTINUOUS RELEASE OF REAGENT IN AN ANALYTICAL
ELEMENT TO REDUCE ASSAY INTERFERENCE
Abstract of the Disclosure Analytical elements and methods for the selective determination of an analyte in aqueous fluids containing the analyte. These elements and methods feature means for continuously releasing chromogenic indicator reagent from a reagent zone to a reaction zone. The continuous release means is responsive to the application of a sample of the fluid to continuously release reagent into the reaction zone at a rate producing color response corresponding to interaction of the indicator with the analyte and reduced interaction of the indicator with interferents. In pre-ferred embodiments, albumin is determined in the presence of interfering proteins such as globulins using buffered chromogenic indicator reagent. In such embodiments, when protein interferents are present, their interference can be substantially eliminated for up to three minutes, dur-ing which time color response is substantially only from the interaction of albumin and reagent. The determination of albumin follows from such color response.

Description

1 lG13~7 CONTINUO~S RELEASE OF REAGE~T I~ AN ANALYTICAL
ELEMENT TO REDUCE ASSAY I~TERFERE~CE
BACKGROUND OF THE INVENTION
Field of the Invention The present invention relates to multizone dry chemistry analytical elements for the determination of an analyte, such as albumin, in aqueous fluids, snd to methods for the use of such elements. In particular, the invention relates to the continuous release within such elements of a chromo~enic indicator reagent composition to a reaction zone to thereby eliminate or reduce inter-ference from competing substances, if present.
Discussion Relative to the Prior Art In the analytical determination for analytes in agueous fluids such as urine and serum or other body fluids, chromo~enic indicator rea~ent compositions that interact with such analytes to produce a color response are freguently employed. The color response is then com-psred with appropriate standards or calibrators to determ~ne the gusntity of analyte present in the fluid.
In many instances, interferin~ ~ubstances accompany the analytes and interact with the rea~ent com-position to produce a color response bearin~ the same color absorption characteristics as those produced by the analyte rea~ent interaction, The analyte-rea~ent and interferent-reagent interactions are additive resultin~ in a biased overall color response from which the component attributable to analyte-rea~ent interaction cannot readily be discerned. For example, in the determination of ma~-nesium in aqueous fluids by interaction with chromogenicindicators such as Eriochrome- Black T (EBT~ or Titan yellow dye to produce a characteristic color response, calcium is a potential interferent. If present, calcium also interacts with either dye to produce a biased color response. Similarly, in the determination of albumin in body fluids by intersction of albumin with buffered chro-mo~enic indicator rea~ents to produce a color respon~e, .,.
~

3 ~ 7 accompanyin~ protein interferents such as globulins or transferrin also interact with the rea~ent to produce 8 biased color response.
The concern for reducin~ interference to snalyte j , assay~ usin~ chromo~enic indicator rea~ents applies to many assays. For the purpose of illustration, the prior art and back~round relative to albumin assays is set forth below.
Albumin is the most abundant of plasma proteins, ~enerally constitutin~ sli~htly over half of the total protein in the plasma. Albumin has a molecular wei~ht of !~
about 69,000, is synthesized in the liver, and has a half-life of about four weeks. It has two important roles:
a) re~ulatin~ the water balance between blood and tissues, and b) functioning as a transport molecule for various materials which are only sli~htly soluble in water, such as bilirubin, fatty acids, cortisol, thyroxine, and a number of dru~s includin~ sulfonamides and bar-biturates, It is frequently important to determine whether patients have a deficiency of serum albumin. A deficiency of albumin in a patient's serum indicates a possibility of edema, nephrosis, cirrhosis, multiple sclerosi~, hepatitis and other disease states. Also, albumin deficiency inter-feres with the transport of insoluble materials, Thus, albumin levels in body fluids are a useful tool for dia~-nosin~ illness. Accordingly, there is a need for pro-cedures and materials which detect and quantify albumin, particularly low values of albumin in body fluids.
The determination of albumin in fluids is now widely practiced usin~ buffered bromcresol ~reen (herein-after referred to as BCG~ solutions or test strip6 as dis-closed in U.S. Patent 3,533,749 issued October 13, 1970 to N. Kleinman and U.S. Patent 3,485,587 issued December 23, 1969 to A. S. Keston. Bromcresol ~reen tBCG~ i~ 8 ~ul-phonphthalein species of chromo~enic indicator materials ,, ~ .
,., 1~13d~7 that exhibit a color response, i.e., a chsn~e in color, by dye-bindin~ interaction with proteins such as slbumin.
Such color response is proportionally related to the amownt of albumin present. As noted above, the indicators are buffered which insures a~ainst color response to p~
chan~es to which the indicators are otherwise sensitive.
ChromoRenic indicators such as BCG are not entirely specific to albumin. Globulins, transferrin and other proteins normally present, for example, in human biolo~ical fluids compete for dye bindin~ with the indi-cator causin~ interference to albumin assays. The inter-ference is particularly si~nificant with albumin at low concentration levels. For example, the normal total pro-tein content, includin~ albumin and ~lobulin, of human serum ran~es from about 6-8 ~/dL. Of this, about 2.9 ~/dL
represents ~lobulins, the balance bein~ predominantly albumin. In normal serum (i.e., from healthy indi-viduals), the albumin-to-globulin ratio is about 1.6:1 while in disease states the ratio may drop to about 0.7:1. A useful clinical assay must therefore avoid bind-in~ with ~lobu~ins and result in a selectlve bindin~ with albumin.
Several authors have su~ested solutions to the problem of non-specificity in 8CG-based albumin determina-tions. In 197~, J. C. Gustafsson reported that the reac-tion of serum albumin with BCG is faster than the reaction of BC~. with other proteins. (Gustafsson, J. F.. C., Clin Chem., 22:616, 1976). Accordin~ly, Gustafsson proposed to measure the absorbance of the solution twice after serum is mixed with BCG rea~ent: immediately and at 6~
minutes. The immediate readin~ when extrapolated back to zero minutes is then employed to determine the albumin concentration. (The sixty minute readin~ is employed in the determination of serum protein components ~n addition to albumin.) The success of this method is predicated on the capability of one bein~ able to obtain an "immediate"

~ ~6î~

absorbance readin~, an impractical requirement for auto-mated systems that have inherent time la~s of one or more minutes before meaningful readin~s can be taken. The Gustafsson zero minute procedure, moreover, is applicable to solution assays wherein for example, lO~L of serum is interacted with 2.0 ml (2000 ~L) of BCG rea~ent 801u-tion, a 2~0-fold dilution. However, with the advent of dry chemistry analytical assay elements, dilution of samples and the use of space-consumin~ liquid rea~ents are no lon~er required. In use, such elements are contacted with undiluted body fluids to produce a color response.
The present inventors have determined, in this re~ard, that in undiluted samples, the total protein-BC~ color response, i.e., the response from both albumin and ~lobu-lins, is si~nificantly more intense than in solutionassays. Accordin~ly, zero minute determination of albumin in dry elements usin~ the procedure of Gustafsson is not only impractical, but less specific to albumin compared to solution assays.
D, Webster (Clin, Chem,, ~3:663, 1977~ confirmed the non-specificity of the BCG-based albumin determination for solution assays. That author concluded that a readin~
taken within 30 seconds was specific to albumin when 3.~
~/liter is subtracted from the result obtained. As in the Gustafsson procedure, Webster's method is predicated on early read times and an assay in dilute solution (~nn-fold dilution of sample fluids). The use of an arbitrary con-stant as a correction factor assumes, moreover, that the albumin result is overstated from the outset and so si~-nifies a non-specific assay. More important, it does not account for real variations from the arbitrary 3.0 ~/liter factor.
After Webster and Gustafsson, the problem of non-specificity of the 8CG-based albumin assay was further disclosed by In~wersen, S. and Raabo, E., Clin. Chim, Acta, 88:545, 1978. In~wersen et al determined that in dilute systems such as those studied by GustafRson and Webster, lowerin~ the concentration of BCG in the rea~ent solution suppressed the bindin~ of globulins to such a degree that a readin~ could be taken as late as one minute after addition of the rea~ent. This readin~ was reported to be almost entirely due to the albumin-BCG complex.
The method accordin~ to In~werson et al permits a readin~ to be taken up to one minute after initiation of the assay in solution. However, the present inventors have determined that when BCG indicator is used in conven-tional dry analytical elements in amounts su~ested byIn~wersen, severe interference from competinR proteins such as ~lobulins is encountered. Still further, the interference is observed immediately, i.e., within less than one minute, thus emphasizin~ the distinction between dilute solution systems and dry chemistry systems employ-in~ undiluted serum.
If one were to consider lowerin~ further the amount of BCG indicator in dry analytical elements in order to reduce the aforementioned Rlobulin interference, it would Renerally not be possible without logs in sensi-tivity and linearity of response to anticipated levels of albumin, particularly hi~h (e.~., 5 ~/dL or hi~her~
albumin levels.
The use of analytical elements for the determina-tion of protein is well known. In the aforementionedKeston U.S. Patent 3,485,587, sin~le zone elements that include absorbent paper impre~nated with buffered indi-cator rea~ent such as BCG are proposed. In use, the element is saturated with a sample of body fluid causin~
instantaneous contact of all the rea~ent with fluid to initiate color development. Such elements are useful to detect the presence or estimate the guantity of total protein. They cannot, however, accurately selectively determine albumin in the presence of ~lobulins becaù~e, 8S
previously noted, ~lobulins compete for available indi-cator.

1 1613d~7 Multizone element~ for the determination of analytes (includin~ albumin) are di~clo~ed in U.S. Patent 3,992,158 issued November 16, 1976 to E. P. Przybylowicz and A. G. Millikan and U.S. Pstent 4,042,335 is~ued Augu~t 16, 1977 to P. L. Clément. In the '158 patent, Fi~ure 2 therein depicts a porous spreadin~ layer on a rea~ent ' layer overlyin~ a support. Numerous matrix material6 guch as ~elatin, a~arose and water ~oluble vinyl polymers, are di~closed a~ useful in the reagent layer. In u~e, the 10 ,~preadin~ layer of element~ accordin~ to the '158 patent is contacted with an aqueous ~ample which di~tribute~
uniformly throu~hout the ~preadin~ layer and into the rea~ent layer. In the reagent layer, a material interact~
with an analyte in the sample to produce a detectable change. Thig patent further di~close~ that the element may be readily adapted for use in the analysis of analyte6 guch as albumin by appropriate choice of te~t rea~ent~ or other interactive materials. Neither ~,G or ~imilar rea~ents, nor the problem of interference to an analyte as~ay u~in~ chromo~enic indicator reaRent iB disclosed in thi~ patent.
Multizone elements for the determination of total protein are disclo~ed in U.S. Patent 4,132,528 i~ued January 2, 1979 to J. N. Eikenberry et al. Such element~
are compo~ed of a ~preadin~ zone for di~tributin~ analyte containin~ fluid and a rea~ent zone composed of rea~ent, an alkaline-providin~ compo~ition, and an alkaline-protec-tive polymer. Alkaline-protective polymers include poly-(vinylpyrrolidone~, poly(acrylamide), agaro~e and copoly-30 mer~ of vinylpyrrolidone and acrylamide. The rea~entemployed to guantitate total protein i~ a modified biuret composition which interact~ indi~criminately with albumin and other proteins. Unlike the pre~ent invention which, in one embodiment, ~electively determines albumin in the pre~ence of protein interferent~, the invention di~clo~ed in the '528 patent can only determine total protein by virtue of the reagent employed.

1 1613~7 Summary of the Invention In the present invention, selective determination of an analyte in aqueous fluids containinR the analyte and possible interferents to an assay for the analyte is carried out in a multizone dry analytical element contain-ing a chromogenic indicator rea~ent. Accordin~ to the invention, chromo~enic indicator reagent is continuously released from a reaRent zone in the element to an adjacent reaction zone containinR the fluid sample containin~ the analyte and possible interferent. The rate at which rea~ent is continuously released to the reaction zone characteristically produces a color response attributable to the selective interaction of indicator with analyte and reduced - often eliminated - interaction of indi-cator with interferents.
The invention set forth below pertains to anelement comprisin~, in part, a confi~uration wherein a reaction zone overlies or is adjacent to a rea~ent zone.
A similar confi~uration is disclo6ed in the aforementioned U.S Patents 3,992,158 and 4,042,335. In contra~t to the disclosure of these patents, however, the present rea~ent zone is impermeable to analytes such as albumin. Hence, the analyte is retained in the reaction zone, while rea~ent, under the influence of fluid in the sample, migrates into the reaction zone to produce a detectable chan~e.
In one aspect, this invention comprises a multi-zone dry analytical element havin~ a reaction zone for receivin~ an aqueous fluid to be subjected to an assay for the selective determination of an analyte in the fluid, which fluid contains the analyte, the element comprisin~:
a) mean~ formin~ a dry rea~ent zone impermeable to the analyte and interferents to an assay for the analyte, said rea~ent zone bein~ in fluid contact with the reaction zone when the element is c~ntacted with a sample of the fluid, the rea~ent zone comprisin~ a 4'~

chromo~enic indicator rea~ent composition which inter-acts with analyte and interferents to produce a color response, and b) means responsive to contact of the sample with the element for continuously releasin~ the reagent compo-sition fro~ the rea~ent zone to the reaction zone at a rate sufficient to produce an analyte color response correspondin~ to 1) the interaction of indicator with analyte, and

2) reduced ioteraction of indicator with inter-ferents.
In a preferred embodiment, an analytical element for the selective determination of albumin in agueous fluids con-tainin~ albumin and possible protein interferents to an albumin assay is set forth. In the albumin selective element, the rea&ent comprises a chromo~enic indicator such as BCG, and the element also contains a buffer, for example, in the rea&ent zone. In accordance with the invention, the rate at which the chromo&enic indicator rea~ent is released is sufficient to produce an albumin color response correspondin~ to the interaction of indi-cator with albumin and reduced interaction of indicator with protein interferents. Most preferably, the rate of rea&ent release is sufficient to produce an albumin IY~
response corresponding to interaction of indicator sub-stantially only with albumin in which case essentially no interaction of iodicator with protein interferents occurs. Unlike the prior art, particularly with re~ard to albumin assays of undiluted body fluids, the present element exhibits albumin selectivity for relatively lon~
periods of time, for example, up to three minutes or more.
A presently preferred means for effectin~ contin-uous release of rea~ent as defined comprises a polymer that serves as binder in the rea&ent zone. ~his polymer is responsive to the application of a fluid sample to the element by continuously releasin& indicator rea&ent from the rea&ent zone to the reaction zone accordin~ to the above-defined rate.

1 16~3d~'~
_9_ In another aspect of this invention, a method for the selective determination of an analyte in aqueous fluids containin~ the analyte and possible interferents to the analyte assay comprises contactin~ a sample of the S fluid with the element to produce an analyte color response as defined, Such response is then employed to determine the analyte such as by comparison ~ith suitable calibrators.
In yet another aspect of the invention, a method 10 for the reduction of interference to an analyte assay com-prises contactin~ an element composed of the above-defined reaction and rea~ent zones with a sample of the fluid to produce an analyte color response as defined above and determininR the analyte.
Detailed Descri tion of the Preferred Embodiments p The present invention facilitates selective determination of an analyte such as albumin or ma~nesium in aqueous fluids such as serum, urine and other fluids usin~ a multizone dry analytical element defined below.
Bia~ed determinations arisin~ from interferents, if present, such as ~lobulins or transferrin, in the case of an albumin assay, or calcium in the case of a ma~nesium assay, are effectively minimized or eliminated. To this end, the present inventors have determined that continuous ~5 release of a chromo~enic indicator rea~ent at a defined rate from one zone in the element to another zone contain-in~ the analyte and possible interferents provides improved analyte selectivity within reasonable periods of time.
The concept of continuous release of indicator reagent to a reaction zone is applicable to the selective determination of various analytes. Accordin~ly, ma~nesium can be determined selectively with the indicator dye [l_ (l-hydroxy-2-naphthylazo~-2-hydroxy-5-nitro-4-naphthalene 35 sulphonic acid sodium salt~ in the presence of calcium as an interferent. This dye is available commercially from Ciba-Gei~y CorP. under the tradename Eriochrome- Black T
(EBT). Alternatively, ma~nesium can be determined with ~ 1~i3~7 Titan yellow dye. The present invention is also employed to selectively determine albumin in the possible presence of protein interferents to an albumin assay. In the albumin assay, a rea~ent composition comprisin~ a chromo-~enic indicator is employed. The rea~ent is buffered asdeflned below. Althou~h this invention contemplates any assays for a variety of analytes and potential inter-ferents, the present invention is set forth more partic-ularly below in the context of an albumin assay for pur-poses of illustration. Correspondin~ elements for otheranalytes operate in the same manner.
Elements accordin~ to the present invention are multizoned. The reaction zone and reagent zone, as well as any other optional zones, are separate and distinct 1~ from one another. Such zones, for example, are hori-zontally disposed layers or vertically disposed bands dependin~ on whether sample application is from the side or the top of the element. The zones are optionally dis-cotinuous rea~ent zone~ within a continuous reaction zoe. Preferably, the zones are in the form of layers.
The reaction zone of the defined element serves several functions. Initially, the zone receives and con-tains a sample of fluid containin~, for example, albumin to be analyzed and protein interferents. Since the reagent zone is impermeable to the albumin and protein interferents, they are effectively retained in the reac-tion zone. Portions of the liguid component in the sample, however, pass from the reaction zone into the reagent zone, causing chromogenic indicator reagent to be continuously released into the reaction zone. Interaction of the rea~ent with albumin follows to produce an albumin color response as defined herein.
The reaction zone optionally serves as a ~pread-ing zone to which a fluid sample is applied or contacted, and within which the sample is uniformly spread. Alterna-tively, a separate spreadin~ zone in fluid contact with the reaction zone is employed to receive and uniformly distribute the fluid sample to the reaction zone.

1 1613d,7 Suitable reaction ~ones include isotropically porous layers as disclosed in U.S. Patent 3,992,158 issued November 16~ 1976 to E.P. Przybylowicz and A.~,. Millikan. Such layers are prepared usin~ 8 variety of component~. In one S a~pect, particulate material~ are u~ed to form the layer~
wherein isotropic porosity is created by interconnected spaces between the particles. Alternatively, ~uch layers are prepared usin~ isotropically porous polymers, for example "blush" polymers as disclosed in U.S. Patent

3,992,158. With re~ard to the present invention, however, the use of pi~ment~ such as TiO2 in the reaction zone i8 to be avoided if such pi~ments mask the color response that occurs in the reaction zone, as described below.
A preferred reaction zone comprises an isotrop-lS ically porous layer containin~ colloidal materials derivedfrom natural or ~ynthetic polymers. Microcrystalline cellulose, which is commercislly available from F~C
Corporation under the name Avicel~ one example of ~uch colloidal ~aterial ~hich ~g preferred for use in the present invention, Example6 of other materials include silica and distomaceous earth, Reaction zones compri~in~ bead layer6 as described in U,S . Patent 4, 258,001 issued March 24, 1981 in the names of Z.R. Pierce an~
D.S. Frank are al~o usefully emPloyed ln multi70n~
elements of the present invention. Such he~
layers contain particles held together by a low amount of adhesive uniquely localized hetwePn adjacent particles where the particles are in clo~t proximity.
The rea~ent zone of the present invention i8, before use, essentially dry. That i~, the rea~ent zone iB
fluid-free, facilitatin~ ~tora~e of the element for lon~
periods of time in a relatively ~mall amount of space.
Also, the rea~ent layer is impermeable to analyte and interferents but otherwi~e permeable to the remainin~

16`i3~ 7 l2-liquid in the fluid sample applied to the reaction zone.
Thus, when a fluid sample is applied, fluid contact between the two zones is established and chromo~enic indi-cator rea~ent is continuously released to the reaction zone to bind to the analyte such as albumin.
The pre~ent reaRent zone is rendered impermeable to analyte and interferents by various means. For example, in the case of albumin, rea~ent zones havin~
pores with a diameter smaller than that of albumin and 10 protein interferent molecules prevent the passa~e of those molecules into the rea~ent zone. On the other hand, such as in the determination of ma~nesium in the presence of calcium interferent havin~ molecular diameters which are extremely small, chelatin~ or ballastin~ a~ents with hi~h 15 molecular wei~hts are desirably employed. Such a~ents interact with the maRnesium and calcium to produce mole-cules with diameters lar~er than the pores in the rea~ent zoe, effectively renderin~ the rea~ent zone impermeable to that analyte and interferent. Useful chelatin& a~ents for ma~nesium and calcium include addition polymers havin~
chelatin~ ~roups appended to the polymer backbone.
Reference herein to fluid contact between the reaction and rea~ent zones in the defined analytical element identifies the sbility of a fluid to pass in such 25 element between superposed or adjacent re~ions of the reaction zone and rea~ent zone. Althou~h the two zones in fluid contact are preferably contiguous, they are optionally separated by intervenin~ zones. However, such intervenin~ zones and any other optional zones included in 30 the element are themselves in fluid contact with each other and with the reaction and rea~ent zones so as not to interrupt the passaze of fluid between the reaction and rea~ent zones.
Rea~ent zones accordin~ to the present invention 35 contain a rea~ent composition comprisin~ a chromo~enic indicator which interacts with analyte (~uch as albumin~
and interferents (such as protein~ to produce a color 16i347 response. Such zones include a matrix in which the rea~ent composition is distributed. The choice of matrix material is variable, except when the matrix is selected as the means for continuously releasin~ the rea~ent into the reaction zone as defined below. When not so selected, however, the matrix ~enerally includes non-proteinaceoùs hydrophilic materials includin~ both naturally occurrin~
and synthetic substances. Examples of such materials are polysaccharides such as a~arose, poly- vinyl compounds lO such as poly(vinyl alcohol), and hydro- philic cellulose derivatives.
Chromo~enic indicators are employed in the reagent zone to determine the analyte, such as albumin, in aqueous fluids. Suitable indicators for albumin are those 15 that are known for their use in solution or in bibulous carriers to determine pH colorimetrically. Such indi-cators produce a color response correspondin~ to the p~ of sample solutions. Many indicators exhibit so-called "pro-tein error" which amounts to a color response of indicator 20 by interaction with protein in ~olution thus ~ivin~ rise to erroneou~ p~ determinations. ~owever, by bufferin~ the indicator, color response attributable to p~ chan~es i~
avoided, thereby renderinR the protein error useful in the determination of protein such as albumin. The buffer is 25 either included in the reaction zone in the rea~ent zone, in both zones or in a separate buffered zone. Preférably, the buffer is included as part of the rea~ent zone. By usin~ buffer, the pH of samples under~oin~ analysis is maintained constant, or at least maintained outside of the 30 pH ran~e in which the indicator normally responds to p~
chan~es. In practicin~ the invention in its broader aspects the use of buffer is optional. In practicin~ an albumin assay, the fore~oin~ principle of bufferin~ the indicator is preferably employed.
Chromo~enic indicators that are used successfully in the present rea~ent zone are indicators disclosed by I. M. Kolthoff in Acid-Base Indicators published by The MacMillan Company, New York (1937~, particularly those l 16-i3~ 7 exhibitin~ protein error as shown on pa~es 350-353.
Examples of such indicators are bromcresol ~reen (BCG), methyl red, bromphenol blue, bromcresol purple, bromthymol blue, phenol red, cresol red, thymol blue, cresolphtha-lein, and those indicators disclosed in U.S. Patent3,438,737 issued April 15, 1969 to R.L. Atkinson et al, columns 3 and 4. Indigo dyes such as indigo carmine are also usefully employed in the practice of the present invention. Of these indicators, the sulphonphthalein indicators such as bromcresol ~reen and bromcresol purple are preferred in the assay for albumin. In the assay for analYtes other than albumin, e.~., ma~nesium, indicators such as Eriochrome Black T- or Titan yellow are suitable for use.
The amount of chromo~enic indicator employed in the present rea~ent zone is sufficient to interact with the lar~est anticipated amount of analyte in the sample of agueous fluid which is to be tested by the present element. In the a~say for albumin, it is preferred that the rea~ent zone comprise sufficient indicator to interact with and produce a linear color response with concentra-tions of albumin up to about 10 g/dL Or higher, mo~t preferably up to 5 g/dL. A linear color response, as used herein, si~nifies a color response that increafies linearly with time durin~ a predetermined time frame after spplica-tion of the sample. For example in the albumin assay, ~uch time frame i5 from about one to about three minutes after sample application. Also, in the albumin assay, BCG
indicator is employed in the rea~ent zone at a concentra-tion ran~e from about .2 ~/m2 to about 3.0 ~/m2,preferably about .5 to about 1.5 g/m2 which has been determined to produce reasonably linear response usin~ 10 ~L fluid samples containin~ up to 5 gtdL albumin. A
concentration of about 1.0 g/m2, in this re~ard, is most preferred and corresponds to a BCG concentration of about 15 millimoles per liter of such fluid. Accordin~ly, for different sample volumes,~adjustment up or down from 1.0 ".
., 1 ~i3ll 7 ~/m2 covera~e in the rea~ent zone to maintain the pre-ferred 15 millimoles per liter is desirable.
Various known types of buffers are useful to maintain the assay for albumin isohydric (i.e., substan-tially constant in pH), and thus avoid p~ effects in thecolor response. The amount and type of buffer depends upon the nature of the fluid bein~ tested and the type of chromo~enic indicator employed. It is also preferable to buffer to a non-alkaline p~. Thus, for a ~iven chromo-~enic indicator with a known pH-sensitive ran~e, one preferably selects a buffer composition that will brin~
the assay to a substantially constant pH belo~ the indi-cator p~-sensitive ran~e. Such a buffer composition, in this instance, comprises acids or acids to~ether with their salts. Suitable acids that are used, sin~ly or in combination with their salts or other listed acids inrlude low molecular wei~ht carboxylic acids such ss malic acid, lactic acid, succinic acid, malooic acid, citric acid, and tartaric acid. Other acid buffers are disclosed in U.S. Patent 3,438,737 issued April 15, 1959 to R, L. Atkin~on et al. Malic acid is 8 preferred buffer.
BCG iR a preferred chromo~enic indicator employed in the pre~ent rea~ent composition for an albumin assay.
For the determination of albumin in neat (undiluted) serum usin~ BCG, the amount of buffer is preferably that amount which will maintain a sample of the serum at a p~ within the ran~e from about 2.0 to about 4,0, most preferably at a pH of about 3.2. For a lQ ~L volume of sample serum, approximately 1~.8 ~/m2 of malic acid buffer has been determined to provide a pH of about 3.2.
The rea~ent composition al~o optionally includes components which do not adversely affect the capability of the chromo~enic indicator to interact with the analyte 8S
discu~sed above. One such optional component i~ a non-ionic surfactant. Repre~entative surfactants includealkylphenoxy polyethoxy ethanols available commercially, for example, from Rohm and Haas Co. under the Triton' l 16i3 tradename series such as Triton X-100- and Triton X-405~; polyoxyethylene oleyl ether such as Brij 98-sold by Atlas Chemical Industries; polyoxyethylene sorbi-tan monolaurate such as Tween 20- also sold by Atlas Chemical Industries; and (p-nonylphenoxy)~lycerol sold by Olirl Matheson Corp. under the tradename Surfactant lOG-.
The element in accordance with the present inven-tion further includes means for continuously releasin~ the defined rea~ent composition from the rea~ent zone to the reaction zone (hereinafter referred to as release means).
Such continuous release, moreover, occurs in response to contact of the element with a fluid sample. The rate of rea~ent release is chosen so as to produce a color response in the reaction zone correspondin~ to the inter-action of the chromo~enic indicator in the rea~ent withanalyte and reduced interaction of the indicator with interferents.
Examples of fore~oin~ release means include several alternative embodiments. In a presently preferred 20 mode, the release mean~ comprise~ a polymeric binder for the rea~ent layer containin~ a polymer that has an affinity for the defined rea~ent composition. This affinity iB slowly disrupted in response to application of a fluid sample to produce continuous rea~ent release.
Preferred polymers exhibitin~ such rea~ent release charac-teristics comprise water-soluble addition polymer~, most preferably vinyl polymers such as those composed of acryl-amide and/or vinylpyrrolidone recurrin~ units, particu-larly poly(vinylpyrrolidone-co-acrylamide) containin~ from about 20 to about 8~ wei~ht percent vinylpyrrolidone and from 80 to about 20 wei~ht percent acrylamide. Especially preferred are copolymers of acrylamide and vinylpyrroli-done prepared from a monomer blend compo~ed of equal wei~ht amounts of acrylamide and vinylpyrrolidone monomers, Useful continuous release of rea~ent has also been achieved with cellulosic polymers such as hydroxy-ethylcellulose as the rea~ent layer binder.

~ 16~ '7 A procedure for screenin~ potential polymers for their use as release means entails formin~ a multilayered analytical element comprisinR a reaction layer overlyin~ a rea~ent layer carried by a support. The rea~ent layer includec the potentially useful polymer and the defined rea~ent composition dispersed in the polymer. The reac-tion layer of t~e element is then contacted with an aqueous control fluid containin~ a known amount of albumin. Alterna~ively, the control fluid is a sample of a solution composed of 0.9% (by wei~ht) NaCl, 0.033%
CaCl2, and 0.3% KCl and the balance water (this solu-tion is commonly referred to as Rin~er's solution). The color response, developed in the reaction layer, measured as the reflection density (DR~ is then monitored versus time until a maximum DR is reached. If the D~ value reaches its maximum immediately, that is, within less than thirty seconds, the polymer bein~ studied is not con-sidered useful to continuously release rea~ent as defined. ~owever, if a maximum DR is reached in more than thirty ~econds, preferably within l-ln minutes, the polymer is uceful. In conducti~ evaluations pursuant to the present invention, it i~ demonstrated, for example, that polymers such as a~arose are not capable of such con-tinuous release, all of its dispersed rea~ent bein~ essen-tially released instantaneously (i.e., in less than thirtyseconds) upon application of the control fluid.
The principle of continuous release, as used herein, denotes a ~radual addition of rea~ent to the reac-tion zone of the present analytical element. It is to be distin~uished from delayed release of rea~ent which con-templates a time period after application of a sample fluid before all available rea~ent is immediately released to the sample fluid, Conti~uous release is essentially synonymous with "meterin~" in the sense that portions of a material are supplied in a regulated amount and rate from a lar~er re~ervoir (i.e., the rea~ent zone~ of such material, as opposed to ~upplyin~ the entire recervoir -~6~

instantaneously. Furthermore, in conductin~ evaluations pursuant to this invention, it has been found that certain polymers, when used as the matrix for the rea~ent zone, not only exhibited continuous release as defined, but also exhibited a so-called la&-phase immediately after applica-tion of the sample fluid. That i~, continuous release of rea~ent was delayed, or at least si~nificantly lowered in rate, for the first 3n to 45 seconds after sample applica-tion. The term "continuous release" is intended to include the combination of a lag-phase and subsequent con-tinuous release of the indicator.
Reference has been made above to continuous release means composed of a certain polymeric mattix for the rea~ent zone in a preferred embodiment. Other embodi-ments to achieve the same purpose include, for example,continuous release zones, impermeable to albumin and pro-tein interferents, interposed between the reaction and rea~ent zones so as to maintain fluid contact between the latter two zones. When the element containin~ such con-tinuous release zone is contacted with a sample of fluid,t~e relea~e zone cooperates with the rea~ent zone to pro-vide continuous rea~ent release to the reaction zone.
Continuous release zones comprise, for example, matrices composed of the continuous release polymers defined above for use in the rea~ent zone, Accordin~ly, such polymers include, for example, water-soluble addition polymers, preferably vinyl polymers such as those comprisinR vinyl-pyrrolidone and/or acrylamide recurrin~ units. Screenin~
of polymers for potential use .n the continuous release zone entails substantially the same procedure employed to screen for useful continuous release polymers for the rea~ent zone matrix. In this instaDce, however, the pro-posed element includes a continuous release zone inter-posed between reaction and rea~ent zones.
The release means is also characterized by exhibitin~ a rate of release that produces an analyte ,, ~ 16i~7 color response correspondin~ to the interaction of indi-cator with analyte and reduced interaction with inter-ferents. The demonstration of such analyte color response in a proposed analytical element is facilitated by compar-S ison with an otherwise identical element containin~ essen-tially no continuous release means (hereinafter referred to as control element~. In this comparison, two of the control elements and two of the proposed elements are employed. The first control element is contacted with a fluid sample, such as neat serum, containin~ analyte, to produce a first color response that is recorded a~ainst time. The second control element is contacted with a sample of t~e same fluid to which an arbitrary, usually 5%
by weight interferent (~-~lobulin in an albumin assay), has been added to produce a second color response that is also recorded a~ainst time. The difference between the two responses at any selected point in time is attribut-able to the presence of added interfèrent (~lobulin). As previously noted, the present invention reduces, and in many c~ses eli~inates, this difference. Accordin~ly, the two proposed elements are contacted with the analyte fluid sample and analyte/added-interferent (&lobulin) fluid sample, respectively, to produce two color response curves with time. In accordance with the present invention, it has been found that the difference between the response curves for the proposed element is inherently less for extensive periods of time after application of a fluid sample compared to the control element. By appropriate selection of components and coatinR levels, the difference between color responses using the proposed elements can be substantially eliminated, preferably for periods of time of at least one minute, most preferaby up to 3 minutes.
After those periods of time, the difference i6 advan-tageously reduced but not eliminated. Eventually, a tran-sition point in time is reached where the color responsedifference for the proposed element is essentially the 1 16i3~7 same as the difference for the control element. There-fore, in using the proposed element, one selects a color response that occurs prior to the transition point, pre-ferably when no difference occurs, as above determined.
Such color response is thereafter employed for the determ-ination of analyte.
The rate of rea&ent release is influenced by several factors includin~, for example, the polymer chosen, the rea~ent zone thickness (for constant levels of chromo&enic indicator), chromo~enic indicator levels and buffer levels when buffer is included in the reaRent zone. How these variables are manipulated is a matter of choice within the skill of the art. It has been found, for example, that the rate of release in certain instances for addition homopolymers of acrylamide is si~nificantly effected by varyin& the buffer level in the rea~ent zone containin~ the homopolymer. In particular, lowerin& the buffer level increased the rate of release from such rea&ent zone.
The analytical elements are self-supportin& or carried on a support. Useful support materials include a variety of polvmeric materials, such as cellulose acetate, poly~ethvlene terephthalate~, polycarhonates and polyvinyl compounds, such as polystyrenes. A support of choice for ~, any particular element is compatible with the intended mode of analyte detection. Preferred supports include radiation-transmissive support materials that transmit electroma&netic radiation of a wavelen&th or wavelen&ths within the region between about 200 nm and about 90n nm, as well as radiation due to radioactivity. When an element includes a support, the reagent zone is usually interposed between the support and the reaction zone, which is preferably the outermost layer in the element.
A variety of different elements, dependin~ on the 35 method of analysis, can be prepared in accordance with the present invention. Elements are confi~ured in a variety of forms, includin& elon&àted tapes of any desired width, sheets or slides.

3 ~ 7 - 2 l The prepared elements are placed in use by apply-in~ a sample of liquid under analysis to the element.
When the reaction zone also serves as a spreadin~ zone, sn applied sample contacts the reaction zone prior to con-tactinR the rea~ent zone and first contacts such reactionzone at its surface furthest removed from such rea~ent zone. ~ecause analytical accuracy of the present elements is not substantially diminished by variations in the volume of applied samples, sample application by hand or 10 machine is acceptable. For reasons of convenience in detectin~ an analytical result, however, reasonable con-sistency in sample volume is desirable.
In an analytical procedure usin~ the present elements, the element is taken from a supply roll, slide 5 packet or other source and positioned to receive a free drop, contact spot or other form of liquid sample, such as from an appropriate dispenser. After sample application, and desirably after the li~uid sample has been taken up bY
the reaction zone, the element is optionally exposed to conditionin~, such aR incubetion or heatin~ to facilitate obtainin~ the test result, Measurement of the analyte color response i~
obtained, usually by passin~ the element throu~h an area in which suitable apparatus for reflection or transmission 25 spectrophotometry is provided. Such apparatus serves to direct a beam of ener~y, such as li~ht, throu~h the support and the rea~ent zone and into the reaction zone.
The li~ht is then reflected back from the reaction zone to a detectin~ means or passes throu~h the element to a 30 detector, in the case of transmission detection.
Alternatively, the beam of ener~y can be directed initially into the reaction zone for reflection or transmission detection in an analo~ous maner, Use of reflection spectrophotometry is preferred. Generally 35 electroma~netic radiation in the range of from about ~00 to about 900 nm has been found useful for such measurements, althou~h it is possible to use any radiation to which the element is ..

I l~^i347 permeable and which is capable of quantifyin~ the analyte color response in the reaction zone. Various known cali-bration techniques are useful to provide calibration curves for the analysis.
Ordinarily, samples of fluid employed with the present element contain both the analyte to be determined and interferents. Accordin~ly, if interferents are cer-tain to be absent, the continuous release means defined above can be dispensed with. ~owever, in practice such lO certainty entails a potentially expensive and time-consum-in~ analytical assessment of the fluid samples for inter-ferents. It is an advanta~e, therefore, that the present element comprises the continuous means of release as defined without re~ard to the presence of interferents in 15 the fluid under analysis. In this manner, the continuous release of rea~ent serves not only to reduce the undesir-able effects of any interferents, but also serves as an assurance a~ainst the possible presence of interferents even thou~h they may be ab~ent. The state of the art is thus advanced by providin~ certainty a~ainst interference and dispensin~ with the need to assay for the presence of the interferin~ substances.
The followin~ E~amples are presented.

EXAMPLES
In the ensuin~ preparations and examples, the followin~ materials and procedures were employed:
Human serum was supplied from hospitals. Samples not immediately used were kept frozeD at -80C.
~ uman Pentex albumin, human Pentex alpha and ~amma ~lobulin fractions were obtained from Miles Laboratories, Inc., Elkhart, Indiana.
Poly(acrylamide-co-N-vinyl-2-pyrrolidone), 50:50 10 monomer wei~ht ratio, was prepared as in Example 1 of U.S.
Patent 4,132,52~ issued January 2 t 1479 to J. N.
Eikenberry et al (hereinafter referred to as polYmer 1).
Poly(acrylamide) was purchased from Eastman Or~anic Chemicals, Rochester, New York (hereinafter 15 referred to as polymer 2).
Sea plaque a~arose was purchased from Marine Colloids, Inc.
Microcrystalline cellulose was purchased as Avicel- from FM~ Corp.
(P-nonylphenoxy~ ~lycerol was purcha~7ed as Surfactant 10G- from Olin Matheson Corp.
All other chemicals were obtained from Eastman Kodak Company, and were rea~ent ~rade, unless otherwise indicated.
Spectrophotometric measurements were made usin~ a Car~ 118 or Beckman 25 instrument. Reflectance measure-mets were made usin~ appropriate reflectometers with interference filters at 630 nm when di-protonated BC~. was monitored and at 42n nm when mono-protonated B~7 was 30 monitored. These instruments were used to measure the reflection density, DR, of a beam of li~ht transmitted throu~h the elements described in the examples and reflected back by the layers in the element.
Element Formation Elements referred to as Elements 41-46 in the followin~ formulations were prepared and used in the ~ 3~7 examples. The coatin~ levels are in ~/m2 of the material indicated.

Resction Microcrystalline Cellulose 53.80 ~/m2 5 Layer Poly(viylpyrrolidone~ 1.35 ~/m2 _ 41 42 43 44 45 46 Polymer 1 32.28 --- --- 16.14 --- ---Polymer 2 --- 32.28 16.14 --- --- ---Rea~ent A~arose --- _-- --- --- 16.14 32.28 10Layer BCG l.n8l.n8n . 54 l.n8 1.08 1.08 Malic Acid 1~.8 5.4 2.7 5.4 5.4 ln.
Surfactant 10~.- n.3~n.~ 0.~2 n.~ n.~ o.~

15 Support / / / polyethylene terephthalate film /// /
-Example 1 Screenin~ for Potentially Useful Release Polymers In this example, the rea~ent release capability 20 Of various p~lymers was evaluated when used as a binder for the rea~ent layer. Elements 44 and 45 were each spotted with 10 ~L samples of solution containin~ 5.0 ~/dL human Pentex albumiD. The DR at 630 nm of Element 45 containin~ a~arose reached a maximum almost immediately ~5 and within less than 3~ seconds, indicatin~ that the a~arose evaluated was not a useful release polymer.' In Element 44, however, the DR at 6~0 nm increased Rrad-ually and reached a maximum after about 4 minutes indicat-in~ that polymer 1 is a useful release polymer.
~he polymers employed as rea~ent layer binder in Elements 41, 42 and 43 were also evaluated for their rea~ent release capability except that rea~ent release was stimulated by contact of each element with lO~L ssmples of Rin~er's solution. DR values at 420 nm were recorded 35 versus time. In Element 41, a msximum DR was reached after about 2 minutes indicatin~ useful release.

61~ 7 In Element 42, no increase in DR was observed for approximately the first l/2 minute after which DR
slowly increased to a maximum after about 4 minutes.
Polymèr 2, therefore, as used in Element 42, exhibits a desirable la~-phase in combination with continuous release a~ defined.
In Element 43, a maximum DR was reached after about 45 seconds indicatin~ useful release.

lO Example 2 Effect of Continuous Release on ~.lobulin Interference Samples of pooled human serum were prepared con-tainin~ added &lobulin in the followin~ amounts: n (con-trol~; 2~/o alpha ~lobulin; 4~ alpha ~lobulin and 2% ~amma 15 ~lobulin. l~ ~L aliquots of these samples were applied to the reaction layer of Element 41, one aliquot to an element. DR at 63n nm was monitored versus time for 5 minutes at 37C. The DR values durin~ the first two minutes for elements contacted with serum containin~ added 20 ~lobulin were essentially identicsl to the DR values for the element contacted with the control indicatin~ e~sen-tially no interferin~ color response from the added ~lobu-lins durin& this time period.

25 Example 3 Comparison of the Effect of Continuous Rea~ent Release and Instantaneous Rea~ent Release on Globulin Interference Elements 41 and 46 (Control A) were contacted with various samples described below. The DR values 30 exhibited at 630 nm by the elements at 1 minute read times were compared.
A third, sin~le-zone element (~,ontrol B~ was pre-pared composed of Whatman-3 Filter paper imbibed with 0.22 ~/m2 BCG; 10.76 ~/m2 malic acid and 0.3 ~/m2 35 Surfactant lOG-. The amount of BC~ imbibed in the paper was one-fifth the amount of dye employed in element 41, Such amount corresponds approximately to the amount of B~,G

i 3~ 7 released in Element 41 from the rea~ent layer to the reac-tion layer durinR the first minute after contact with a liquid sample.
The elements were each contacted with ln ~L l samples of huma Pentex albumin calibrator solutions hav-inR concentrations of 1.7, 2.5 and 5.0 ~ldL albumin respectively, and the DR value at 630 nm exhibited by each element at one minute was recorded. The procedure was repeated except that each calibrator solution con-10 tained an additional 5% Ramma Rlobulin. For each elementa bias attributable to the color response from interaction of 8CG with Ramma ~lobulin was determined by computin~ the percentaRe increase of the D~ for samples containin~
RlobUlin over the DR f the sample containin~ no ~lobu-lS lin. Results are shown in Table I.
Table I
Albumin Albumin &
Amount ~.lobulin P,lement (~/dL~ DR DR / ~ias ~ 41 1.7 .71 .84 18.~
2.5 .88 1.~ 13.6 5.0 1.18 1.24 5 46 (Control A~ 1.7 1.0 1.7 70 2S 2.5 1.3 1.9 46 5.0 1.7 2.4 41 Imbibed 1.7 .17 .42 147 paper 2.5 .26 .46 77 (Control B~ 5.0 .42 .5~ 3~
The lar~er bias percenta~es exhibited by Element 46compared to Element 41 clearly show the advanta~e of continu-ous release of reaRent from the rea~ent laqer, with both reaRent layers containinR the same initial amount of rea~ent, 35 The bias percentaRes of Element 41 and the imbibed paper element substantiates the advanta~e of continuous over the instantaneous use of equal amounts of BC~., ~16~3~7 -~7Fxam~le 4 Reduction of ~alcium Interference In an Assay for Ma~nesium.
Analvtical Elements 41 and 4~ are prepared except that BC~. is replaced with the indicator dye Eriochrome Black T-. A polymeric chelatin~ a~ent for ma~nesium and calcium is also included in the reaction layer to render the rea~ent layer impermeable to ma~nesium and calcium.
10~1 aqueous samples containin~ in one instance n,a~nesium and in another instance ma~nesium and calcium as an lO added interferent are applied to the reaction layers to ~enerate appropriate color response curves a~ainst time. The difference between the response curves, i.e., ma~nesium curve and ma~nesium plus added calcium curve, is substantially less for Element 41 than the difference between the color response lS curves ~enerated for Element 4~ (control). Accordin~ly, calcium interference in a ma~nesium assay is effectively reduced in accordance with the present invention.
The invention ha~ been described in detail ~ith par-ticular reference to preferred e~bodiments thereof, but it will be understood that variations and modifications can be effècted within the spirit and scope of the invention.

Claims (29)

WHAT IS CLAIMED IS:

1. A multizone dry analytical element having a reaction zone for receiving an aqueous fluid to be sub-jected to an assay for the selective determination of an analyte in the fluid, which fluid contains said analyte, said element comprising:
a) means forming a dry reagent zone impermeable to said analyte and interferents to an assay for said analyte, said reagent zone being in fluid contact with said reaction zone when said element is contacted with a sample of said fluid, said reagent zone including a reagent composition comprising a chromogenic indicator which interacts with said analyte and interferents to produce a color response; and b) release means responsive to contact of said sample with said element for continuously releasing said reagent composition from said reagent zone to said reaction zone at a rate sufficient to produce an analyte color response corresponding to:
1) the interaction of said indicator with said analyte, and 2) reduced interaction of said indicator with inter-ferents.

2. A multizone dry analytical element as in Claim 1 wherein said release means comprises a poly- meric binder for said reagent composition, which binder is responsive to contact of a sample of said fluid with said element to effect said continuous release of reagent.

3 A multizone dry analytical element as in Claim 2 wherein said polymeric binder comprises a water-soluble addition polymer.

4. A multizone dry analytical element as in Claim 3 wherein said addition polymer is selected from the group consisting of vinylpyrrolidone, acrylamide and vinylpyrrolidone-acrylamide recurring units.

5. A multizone dry analytical element having a reaction zone for receiving, and for the selective deter-mination of magnesium in, an aqueous fluid sample contain-ing magnesium, said element comprising:
a) means forming a dry reagent zone impermeable to mag-nesium and calcium, in fluid contact with said reac-tion zone when said element is contacted with said sample, said reagent zone comprising a reagent compo-sition comprising a chromogenic indicator which inter-acts with magnesium and calcium to produce a color response; and b) release means responsive to contact of said sample with said element for continuously releasing said reagent composition from said reagent zone to said reaction zone at a rate sufficient to produce a mag-nesium color response corresponding to:
1) the interaction of said indicator with magnesium, and 2) reduced interaction of said indicator with calcium.

6. A multizone dry analytical element as in Claim 5 wherein said chromogenic indicator is Titan yellow or [1-(1-hydroxy-2-naphthylazo)-2-hydroxy-5-nitro-4-naphthalene sulphonic acid sodium salt].

7. A multizone dry analytical element as in Claim 5 wherein said release means comprises a polymeric binder for said reagent composition, which binder is responsive to contact of a sample of said fluid with said element to effect said continuous release of reagent.

8. A multizone dry analytical element as in Claim 7 wherein said polymeric binder comprises a water-soluble addition polymer.

9. A multizone dry analytical element as in Claim 8 wherein said addition polymer is selected from the group consisting of vinylpyrrolidone, acrylamide and vinylpyrrolidone-acrylamide recurring units.

10. A multizone dry analytical element having a reaction zone for receiving, and for the selective deter-mination of albumin in, an aqueous fluid sample containing albumin, said element comprising:
a) a buffer b) means forming a dry reagent zone, impermeable to albumin and protein interferents to an assay for albumin, said reagent zone being in fluid contact with said reaction zone when said element is contacted with said sample, said reagent zone comprising a reagent composition containing a chromogenic indicator which interacts with albumin and protein interferents in the presence of said buffer to produce a color response, and c) means responsive to contact of said sample with said element for continuously releasing said reagent compo-sition from said reagent zone to said reaction zone at a rate sufficient to produce an albumin color response corresponding to the interaction of said indicator with albumin and reduced interaction of said indicator with protein interferents.

11. A multizone dry analytical element as in Claim 10 wherein said chromogenic indicator is a sulphon-phthalein dye.

12. A multizone dry analytical element as in Claim 11 wherein said rate of continuous release is sufficient to produce a color response corresponding to the interaction of said indicator substantially only with albumin.

13. A multizone dry analytical element as in Claim 11 or 12 wherein said chromogenic indicator is brom-cresol green and said buffer is provided in an amount sufficient to maintain said fluid at a pH lower than that of the pH range in which said indicator is pH-sensitive.

14. A multizone dry analytical element having a reaction zone for receiving, and for the selective deter-mination of albumin in, an aqueous fluid sample containing albumin, said element comprising means forming a dry, polymeric reagent zone, impermeable to albumin and protein interferents to assay for albumin, said reagent zone being in fluid contact with said reac-tion zone when the reaction zone is contacted with said sample, said reagent zone comprising, in a polymeric binder, a reagent composition comprising a buffer and a chromogenic indicator which interacts with albumin and protein interferents in the presence of said buffer to produce a color response, said polymer being responsive to contact of said sample with said element for continuously releasing said reagent composition from said reagent zone to said reaction zone at a rate sufficient to produce an albumin color response corresponding to the interaction of said indicator with albumin and reduced interaction of said indicator with protein interferents.

15. A multizone dry analytical element as in Claim 14 wherein said polymeric binder comprises a water-soluble addition polymer, and wherein said chromogenic indicator is water soluble.

16. A multizone dry analytical element as in Claim 15 wherein said addition polymer is selected from the group consisting of vinylpyrrolidone, acrylamide and vinylpyrrolidone-acrylamide recurring units.

17. A multizone dry analytical element as in Claim 15 wherein said chromogenic indicator is a sulphonphthalein dye.

18. A multizone dry analytical element as in Claim 17 wherein said rate of continuous release is sufficient to produce an albumin color response corre-sponding to the interaction of said indicator substan-tially only with albumin.

19. A multizone dry analytical element as in Claim 18 wherein the interaction of said indicator sub-stantially only with albumin occurs for at least a minute after contact of said fluid sample with said element.

20. A multizone dry analytical element as in Claim 18 wherein said indicator is bromcresol green and said buffer is present in an amount sufficient to maintain said fluid at a pH lower than that of the pH range in which said indicator is pH-sensitive.

21. A multizone dry analytical element as in Claim 20 wherein the amount of bromcresol green released from said reagent zone is sufficient to produce an albumin color response that varies linearly with albumin concen-trations up to 5 g/dL in said sample.

22. A multizone dry analytical element as in Claim 20 wherein the amount of said bromcresol green in said reagent layer is sufficient to provide a concentra-tion of at least 15 millimoles bromcresol green per liter of said fluid.

23. A multizone analytical element as in claims 1, 5 and 10 wherein said reaction and reagent zones are separate and distinct layers.

24. A multizone analytical element as in claims 1, 5 and 10 wherein said reaction and reagent zones are separate and distinct layers, and said element comprises, in sequence, a spreading layer, said reaction layer and said reagent layer.

25. A multizone analytical element as in claim 14 wherein said reaction and reagent zones are separate and distinct layers.

26. A multizone analytical element as in claim 25 wherein said element comprises, in sequence, a spreading layer, said reaction layer and said reagent layer.

27. A method for the selective determination of an analyte in an aqueous fluid containing said analyte, said method comprising a) contacting a sample of said fluid with a multizone dry analytical element having a reaction zone to cause a dry reagent zone, otherwise impermeable to said analyte and interferents to an assay for said analyte, to come into fluid contact with said reaction zone, said reagent zone comprising a reagent composition comprising a chromogenic indicator which interacts with said analyte and interferents to produce a color response, and to cause release of said reagent compo-sition from said reagent zone to said reaction zone at a rate sufficient to produce an analyte color response corresponding to 1) the interaction of said indicator with said analyte, and 2) reduced interaction of said indicator with interferents; and b) determining said analyte from said analyte color response.

28. A method for the selective determination of albumin in an aqueous fluid containing albumin, said method comprising a) contacting a sample of said fluid with a multilsyer dry analytical element having a reaction layer to cause a dry reagent layer, otherwise impermeable to albumin and protein interferents to an assay for albumin, to come into fluid contact with said reaction layer, said reagent layer comprising, in a polymeric binder, a reagent composition comprising a buffer end a chromogenic indicator which interacts with albumin and protein interferents in the presence of said buffer to produce a color response, and to cause said polymer to release said reagent composition from said reagent layer to said reaction layer at a rate suffi-cient to produce an albumin color response correspond-ing to the interaction of said indicator with albumin and reduced interaction of said indicator with protein interferents; and b) determining albumin from said albumin color response.

29, A method for reducing protein interference in an analytical determination for albumin is a fluid sample containing albumin and protein interferents to an assay for albumin, said method comprising a) contacting a sample of said fluid with a multilayer dry analytical element having a reaction layer to cause a dry reagent layer, otherwise impermeable to albumin and said protein interferents, to come into fluid contact with said reaction layer, said reagent layer comprising, in a polymeric binder, a reagent composition comprising a buffer sufficient to maintain said fluid at a non-alkaline pH and a sulphonphthalein chromogenic indicator which interacts with albumin and protein interferents in the presence of said buffer to produce a color response, and to cause said polymer to release said reagent composition from said reagent layer to said reaction layer at a rate sufficient to produce an albumin color response corresponding to the interaction of said indicator with albumin and reduced interaction of said indicator with protein interferents; and b) determining albumin from said albumin color response.