Stable reagent for ferric ion complex indicator systems

The invention is a stable reagent used in assay systems that form a colored ferric ion complex, such as Prussian Blue. Such reagents are useful for the detection or measurement of an analyte from a fluid sample. Surprisingly, it has been found that the inclusion of certain ferric ion chelating agents, such as 3-sulfobenzoic acid, will inhibit formation of the blank reaction in the reagent.

FIELD OF THE INVENTION 
The invention relates to a stable reagent useful for forming a ferric ion 
chelation complex, as an indicator, in the detection or measurement of an 
analyte from a fluid sample. The invention also relates to a method of 
making a stable reagent, incorporating the reagent into an analytical 
element, and a method of determining the presence of an analyte in a 
liquid sample. 
BACKGROUND OF THE INVENTION 
Many clinical chemistry assays are performed using reagents that form 
colored indicators. In such assays, the intensity of the color of the 
indicator is correlated to the concentration of analyte in the fluid 
sample being measured. 
Exemplary of such assays is the analysis of glucose from a blood sample by 
utilizing a reagent that forms Prussian Blue (or Turnbull's Blue) as a 
colored indicator. The reagent may include the enzyme glucose oxidase 
(GOD), and potassium ferricyanide and ferric sulfate for the formation of 
Prussian Blue. The reaction that generates Prussian Blue may be depicted 
as follows (Scheme I): 
##STR1## 
Accordingly, the more Prussian Blue that is formed by this reaction, then 
the more glucose is present in the blood sample being analyzed. 
However, a particular problem with reagents that are used to form the 
Prussian Blue indicator, and other reagents that are used to form 
indicators that involve ferric ion complexes, is stability. Such reagents, 
when in liquid form, are unstable to light and heat, and, when in 
lyophilized form or included in a film, are additionally unstable to 
humidity. In the case of a reagent useful for forming the Prussian Blue 
indicator in a glucose assay, the instability is manifested by premature 
formation of Prussian Blue ("the blank reaction") in the reagent prior to 
addition of a blood sample containing glucose. 
SUMMARY OF THE INVENTION 
The invention is a stable reagent used in assay systems that form a colored 
ferric ion complex, such as Prussian Blue. Such reagents are useful for 
the detection or measurement of an analyte from a fluid sample. However, 
in liquid form these reagents are unstable to heat and light. In 
lyophilized form or in a film, these reagents are further unstable to 
humidity. This instability is often manifested in the form of a blank 
reaction. For example, in a reagent used for forming the colored complex 
Prussian Blue, Prussian Blue is prematurely formed in the reagent. 
Surprisingly, it has been found that the inclusion of certain ferric ion 
chelating agents, such as 3-sulfobenzoic acid, will inhibit formation of 
the blank reaction in the reagent.

DESCRIPTION OF THE INVENTION 
The present invention is a stable reagent, capable of forming a ferric ion 
colored complex in the presence of an analyte from a fluid sample, and 
useful for the detection or measurement of the analyte from the fluid 
sample. At a minimum, the reagent includes 
a first compound that will react in a reaction involving the analyte to 
form a second compound that complexes with ferric ion to form a colored 
complex; 
a source of ferric ions; 
and a ferric ion chelator of sufficient type and in sufficient amount to 
substantially inhibit formation of the colored complex prior to addition 
of the analyte to the reagent and to substantially not inhibit formation 
of the colored complex after addition of the analyte to the reagent. 
Ferric ion chelators, such as citric acid, aspartic acid, ethylenediamine 
tetraacetic acid (EDTA), and succinic acid, which have too high an 
affinity for ferric ion are not of the type of ferric ion chelators 
included in this invention. 
When the colored complex is Prussian Blue, the first compound may be a 
ferricyanide, such as potassium ferricyanide, the second compound may be a 
ferrocyanide, the source of ferric ions may be ferric sulfate, and the 
ferric ion chelator may be 3-sulfobenzoic acid, 3-hydroxybutyric acid, 
4-hydroxybutyric acid, 5-aminovaleric acid, butyric acid, or propionic 
acid, or salts thereof. (see Scheme I in Background of the Invention 
section.) The ferric ion chelator may also be 2-sulfobenzoic acid, or 
tartaric acid, or a salt thereof. 
Importantly, inclusion of the ferric ion chelator, of the type specified 
herein, stabilizes a liquid reagent against heat and light and stabilizes 
a dry reagent (lyophilized or in a film) against heat, light and humidity. 
A molar ratio of about 3:1 ferric ion chelator:ferric ion in the reagent 
is sufficient to stabilize the reagent. The reagent will be increasingly 
stabilized with molar ratios of ferric ion chelator to ferric ion above 
about 3:1. The increased stabilization will be beneficial until the ratio 
of ferric ion chelator to ferric ion becomes high enough to deleteriously 
affect the reaction kinetics of the assay, that is, the colored complex 
(e.g., Prussian Blue) forms too slowly in the assay, and, as a result, 
assay time becomes too long and precision and accuracy of measurement are 
adversely affected. 
A catalyst is preferably included in the reagent. The catalyst should be of 
sufficient type and in sufficient amount to catalyze the reaction 
involving the analyte and the first compound. When the analyte is glucose, 
the catalyst may be the enzyme glucose oxidase. 
A buffer is also preferably included, and when 2-sulfobenzoic acid is used 
a buffer should be included, in the reagent. The buffer should be of 
sufficient type and in sufficient amount to provide a desired pH for the 
reagent and the reaction involving the analyte and the first compound. 
Further, the buffer should not deleteriously bind to ferric ion. For 
example, phosphate buffer precipitates ferric ion and should not be used 
in the reagent. In diagnostic assays, biological buffers, such as "Good" 
buffers (available from, e.g., Sigma Chemical Company) are often used 
buffers. Pyruvic acid, 2-amino butyric acid, gluconic acid, and 
2-hydroxyisobutyric acid are useful buffers. The particular buffer used 
will depend upon the particular assay system being employed. For a reagent 
that is capable of forming the Prussian Blue indicator in a diagnostic 
assay, 4-amino butyric acid is a useful buffer. 
The buffer may even be a weak chelator of ferric ion as long as the buffer 
does not substantially inhibit formation of the colored complex (the 
indicator) after the addition of the analyte to the reagent. If the buffer 
does weakly chelate ferric ion, then a lesser amount of ferric ion 
chelator (that is, less than a 3:1 molar ratio of ferric ion chelator to 
ferric ion) may be needed to stabilize the reagent. 
The reagent may be formulated in liquid (aqueous) form, in lyophilized 
form, or incorporated into a film or a diagnostic kit. When incorporated 
into a film, a film former, for example, an aqueous vinyl propionate/vinyl 
acetate copolymer dispersion sold under the mark PROPIOFAN.RTM. 70 D 
(obtained from BASF) or a polyvinylacetate ethylene copolymer, such as 
film formers sold under the mark ELVACE (available from Reichhold 
Chemicals) or AIRFLEX 300 (available from Air Products), is needed. Other 
additives may be helpful in producing a film that incorporates the reagent 
of the present invention. For example, a viscosity controlling agent, such 
as hydroxy-methyl cellulose, a surfactant, such as 
polyoxyethylene-sorbitan monolaurate, an anti-foaming agent, such as 
acetone, and a film opener, such as diatomaceous earth or the film openers 
disclosed in Vogel et al., U.S. Pat. No. 4,312,834, issued Jan. 26, 1982, 
the disclosure of which is hereby incorporated by reference, may be 
helpful additives in formulating a film. Addition of a pigment, such as 
titanium dioxide, may be helpful in reflectance films; and addition of an 
oxidizing agent, such as potassium dichromate, may further increase the 
stability of a film that includes the reagent of the present invention by 
providing an oxidizing environment. 
The present invention is generally applicable to any reagent that includes 
a source of ferric ions and that forms, as an indicator, a colored ferric 
ion chelation complex in the detection or measurement of an analyte from a 
fluid sample. Including a ferric ion chelator that substantially inhibits 
formation of the colored ferric ion chelation complex prior to addition of 
the analyte to the reagent and that does not substantially inhibit 
formation of the colored ferric ion chelation complex after addition of 
the analyte to the reagent will protect the reagent from the destabilizing 
effects of heat and light, if the reagent is in liquid form, and 
additionally from humidity, if the reagent is in dry form (lyophilized or 
incorporated in a film). 
The present invention is specifically applicable to the compositions, 
methods, and analytical elements described in Freitag, U.S. Pat. No. 
4,929,545, issued May 29, 1990, the disclosure of which is hereby 
incorporated by reference. In any of the examples found in the U.S. Pat. 
No. 4,929,545, a reagent that is stable to heat and light, if the reagent 
is in liquid form, and additionally stable to humidity, if the reagent is 
incorporated into a film on a test strip, may be prepared by adding to the 
reagent an amount of ferric ion chelator sufficient to make the molar 
ratio of ferric ion chelator to ferric ion about 3:1. Increasing this 
molar ratio will further stabilize the reagent until the ratio becomes 
high enough to deleteriously affect the reaction kinetics of an analyte 
assay that utilizes the reagent. The molar ratio of ferric ion chelator to 
ferric ion may become so high that assay time is too long and assay 
accuracy and precision become poor. 
Another example of a reagent, or coating mass as it is referred to in 
Example 3 of U.S. Pat. No. 4,929,545, is as follows: 
Step No. 1 
Thoroughly mix 1376 grams(g) water, 58 g sodium hydroxide (pellets), 52 g 
4-amino butyric acid, 331 g 3-sulfobenzoic acid, 52 g ferric sulfate, and 
234 g potassium ferricyanide, thereby forming a penultimate aqueous ionic 
mixture. The penultimate aqueous ionic mixture is filtered to remove large 
particles and impurities. 0.17 g potassium dichromate, 288 g diatomaceous 
earth (available from Eagle-Picher Minerals, Inc. under the mark 
CELABRITE), and a sonicated suspension of 414 g titanium dioxide in 907 g 
water are mixed into the resulting filtrate, thereby forming the ultimate 
aqueous ionic mixture. 
Step No. 2 
Thoroughly mix 1067 g water, 1739 g PROPIOFAN.RTM. 70 D film former that 
has been demonomerized, 485 g of 4% aqueous hydroxymethyl cellulose (sold 
under the mark NATROSOL), 75 g acetone, and 4.7 g polyoxyethylenesorbitan 
monolaurate surfactant (sold under the mark TWEEN 20), thereby forming a 
polymer mixture. This mixture is filtered to remove large particles and 
impurities. 
Step No. 3 
Thoroughly mix 368 g water and 64 g glucose oxidase (G02A grade having 185 
kilounits glucose oxidase/g, available from Biozyme Laboratories 
International, Ltd.), thereby forming an enzyme mixture. This enzyme 
mixture is filtered. 
Step No. 4 
With mixing, the ultimate aqueous ionic mixture is slowly added to the 
filtrate of the polymer mixture. Once addition is complete, the filtrate 
of the enzyme mixture is added to form the penultimate coating mass, which 
is filtered to form the ultimate coating mass. 
The ultimate coating mass may be coated onto a clear, polyester foil, as 
described in U.S. Pat. No. 4,929,545, Example 3 (col. 6), and dried to 
form a film coated test strip. This test strip may be used in detecting or 
measuring glucose in a fluid sample as described in Example 3 (col. 6) of 
U.S. Pat. No. 4,929,545. 
The present invention has been disclosed in the above teachings and 
drawings with sufficient clarity and conciseness to enable one skilled in 
the art to make and use the invention, to know the best mode for carrying 
out the invention, and to distinguish it from other inventions and from 
what is old. Many variations and obvious adaptations of the invention will 
readily come to mind, and these are intended to be contained within the 
scope of the invention as claimed below.