U.S. patent number 3,928,137 [Application Number 05/467,431] was granted by the patent office on 1975-12-23 for reagent formulation for uric acid assay.
This patent grant is currently assigned to Mallinckrodt, Inc.. Invention is credited to Ching Chiang, Alexander A. Monte.
United States Patent |
3,928,137 |
Monte , et al. |
December 23, 1975 |
**Please see images for:
( Certificate of Correction ) ** |
Reagent formulation for uric acid assay
Abstract
An enzyme reagent formulation for use in assaying a biological
specimen for uric acid is prepared as a granular, water-soluble,
anhydrous, storage-stable mixture containing uricase, potassium
chloride, mannitol, gum acacia, bovine serum albumin, glycine,
sodium carbonate and a nitrogen containing polyoxyalkylene nonionic
surfactant obtained by the sequential reaction of ethylenediamine
with propylene oxide and ethylene oxide in the presence of a
catalyst. The surfactant contains polyoxypropylene chains having an
average molecular weight of between about 750 and about 6750, and
polyoxyethylene chains constituting between about 10 and about 80
weight percent of the surfactant. The surfactant has an
advantageous effect upon granulation, dissolution and storage
stability of the reagent formulation.
Inventors: |
Monte; Alexander A. (Glendora,
CA), Chiang; Ching (Glendora, CA) |
Assignee: |
Mallinckrodt, Inc. (St. Louis,
MO)
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Family
ID: |
27392813 |
Appl.
No.: |
05/467,431 |
Filed: |
May 6, 1974 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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200552 |
Nov 19, 1971 |
3816262 |
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190883 |
Oct 20, 1971 |
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Current U.S.
Class: |
435/10;
435/27 |
Current CPC
Class: |
G01N
33/721 (20130101); C12Q 1/62 (20130101) |
Current International
Class: |
C12Q
1/62 (20060101); G01N 33/72 (20060101); G01N
031/14 (); G01N 033/16 () |
Field of
Search: |
;195/63,68,13.5R,100,99
;23/23B |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Naff; David M.
Attorney, Agent or Firm: Madsen; Mathew D.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This application is a division of our copending, coassigned U.S.
Pat. application Ser. No. 200,552, filed November 19, 1971, now
U.S. Pat. No. 3,816,262, which is a continuation-in-part of our
U.S. patent application Ser. No. 190,883, filed Oct. 20, 1971, now
abandoned.
Claims
What is claimed is:
1. An enzyme reagent formulation for use in assaying a biological
specimen for uric acid comprising a granular, water-soluble,
anhydrous, storage-stable mixture containing:
a. uricase;
b. potassium chloride;
c. mannitol;
d. gum acacia;
e. bovine serum albumin;
f. glycine;
g. sodium carbonate; and
h. a nitrogen-containing polyoxyalkylene nonionic surfactant
obtained by the sequential reaction of ethylenediamine with
propylene oxide and ethylene oxide in the presence of a catalyst,
said surfactant containing polyoxypropylene chains having an
average molecular weight of between about 750 and 6750 and
polyoxyethylene chains constituting between about 10 and about 80
weight percent of said surfactant.
2. A reagent formulation as set forth in claim 1 wherein said
nitrogen-containing surfactant is solid and the polyoxypropylene
chains thereof have an average molecular weight of less than about
4000.
3. The method of preparing a granular, water-soluble, free-flowing,
substantially anhydrous, storage-stable enzyme reagent formulation
for use in assaying a biological specimen for uric acid, said
method comprising the steps of:
1. preparing a mixture containing:
a. uricase;
b. potassium chloride;
c. mannitol;
d. gum acacia;
e. bovine serum albumin;
f. glycine;
g. sodium carbonate;
h. a nitrogen-containing polyoxyalkylene nonionic surfactant
obtained by the sequential reaction of ethylenediamine with
propylene oxide and ethylene oxide in the presence of a catalyst,
said surfactant containing polyoxypropylene chains having an
average molecular weight of between about 750 and about 6750 and
polyoxyethylene chains constituting between about 10 and about 80
weight percent of said surfactant;
i. catalase; and
j. a solvent for said surfactant; and
2. removing the solvent to form a substantially anhydrous,
free-flowing, water-soluble, granular solid.
4. The method as set forth in claim 3 wherein the
nitrogen-containing surfactant is solid and the polyoxypropylene
chains thereof have an average molecular weight of less than about
4000.
5. The method as set forth in claim 4 wherein the polyoxypropylene
chains of the surfactant have an average molecular weight of about
2750 and the weight percentage of the polyoxyethylene units thereof
is about 70%.
Description
BACKGROUND OF THE INVENTION
This invention relates to the field of clinical diagnostic testing
and more particularly to novel reagents and methods for making
biological assays on body fluids.
A large variety of test reagents and methods are available for use
in determining the character of various body fluids to assist in
the diagnosis of certain pathological conditions. Tests for
determination of certain types of biological activity or the
presence and quantity of certain biologically active components
provide information indicating the presence or absence of disease
or other physiological disorder. In accordance with such tests, the
biological specimen to be analyzed, for example, a sample of a body
fluid, is typically mixed with a liquid reagent formulation which
contains a reagent capable of effecting a reaction which causes a
measurable change in the specimen/reagent system. Very often the
reaction which takes place in the test is an enzymatic reaction.
Certain tests are designed, in fact, to determine the presence of a
particular enzyme and in such cases the reagent formulation may
contain a substrate upon which the enzyme to be determined is known
to act. In other cases, the determination may be for a material
which is known to be a reactive substrate in an enzymatically
catalyzed reaction. In either case, the reagent formulation very
commonly contains an enzyme, a coenzyme or both. Because the
catalytic activity of most enzymes is specific to a particular
reaction, test reagents can be formulated which are effective to
determine specific biological components or activities even in a
complex body fluid containing a large number of other components
which might interfere with efforts to obtain a purely chemical
analysis. Moreover, many of the components which are to be
determined have highly complex chemical structures which would
render direct chemical analysis difficult even in the absence of
any contaminants.
Unfortunately, enzymes and coenzymes are generally rather delicate
materials which may be readily denatured by heating and which also
tend to degenerate upon storage. Many of the substrate materials
used in biological assay reagent formulations are similarly
unstable. Liquid reagents containing such components are therefore
not generally susceptible to storage and must be freshly prepared
shortly prior to use in clinical diagnostic testing. Because of the
relative expense of enzymes and coenzymes and the skill required to
prepare a reagent formulation containing these materials which can
be utilized to obtain accurate clinical diagnostic test results,
the instability of the liquid formulations has motivated a
substantial amount of research to develop reagents in a relatively
storage-stable form. Much of this effort has been directed to the
development of solid, dry, water-soluble formulations which can be
dissolved in water at the time of testing to provide a fresh liquid
reagent useful in the test. Typical prior art dry reagent
formulations are disclosed in Deutsch U.S. Pat. No. 3,413,198 and
Stern et al. u.S. Pat. No. 3,546,131.
A dry reagent formulation satisfactory for use in preparing liquid
reagents for routine clinical diagnostic tests should satisfy a
number of criteria. It must be readily soluble in a solvent
compatible with the biological specimen, usually water. It should
be capable of solubilizing proteinaceous material in the specimen.
Moreover, it should be readily susceptible to packaging in
convenient sized packages and be adapted for rapid dissolution in
the solvent to provide a liquid reagent of proper strength for a
given test or series of tests.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide improved dry,
water-soluble, reagent formulations for use in conducting clinical
diagnostic tests. It is a further object of the present invention
to provide such formulations which can be readily granulated and
shipped or stored in granular form. It is a particular object of
the invention to provide such reagent formulations in free-flowing,
granular form at consistent bulk densities so that they may be
delivered to a volumetric packaging or tableting operation in
consistent weight amounts. Additional objects of the invention
include the provision of dry reagent formulations having a high
capacity for solubilizing protein; the provision of such
formulations having a high degree of storage stability; the
provision of methods for preparing the dry reagent formulations of
the invention; and the provision of methods for conducting clinical
diagnostic tests utilizing such reagent formulations. Other objects
and features will be in part apparent and in part pointed out
hereinafter.
In one of its aspects, therefore, the present invention is directed
to a reagent formulation for use in conducting a clinical
diagnostic test on a biological specimen. The reagent formulation
comprises a solid, water-soluble, substantially anhydrous,
storage-stable mixture containing a reagent capable of
participating in a test reaction to effect a measurable change in a
test system, and a solid nitrogen-containing polyoxyalkylene
nonionic surfactant. The surfactant has a structure corresponding
to that obtained when ethylene diamine is reacted sequentially with
propylene oxide and ethylene oxide in the presence of a catalyst
and the polyoxypropylene chains of the surfactant have an average
molecular weight of between about 750 and about 6750.
The invention is further directed to a method of conducting a
clinical diagnostic test on a biological specimen using the
aforementioned reagent formulation. The method comprises dissolving
the reagent formulation in water to produce a liquid reagent;
mixing the liquid reagent with a specimen to form a
specimen/reagent test system; and measuring a change in the system
resulting from the reaction between the reagent and the
specimen.
The invention is also directed to a method of preparing the novel
reagent formulation. The method comprises the steps of mixing a
reagent capable of participating in a test reaction to effect a
measurable change in a test system, a nitrogen-containing
polyoxyalkylene nonionic surfactant of the above-noted character,
and a solvent for the surfactant; anad removing the solvent to form
a substantially anhydrous, water-soluble, free-flowing, granular
solid.
DESCRIPTION OF THE DRAWING
The drawing is a grid illustrating the molecular structure of
various commercially available nonionic surfactants useful in the
practice of the invention. The coordinates of each point on the
grid correspond to the chain size of the polyoxyethylene hydrophile
and polyoxypropylene hydrophobe moieties of a particular
surfactant. Boundary lines set out on the grid separate the areas
encompassing surfactants which assume different physical
states.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
To facilitate preparation of liquid reagents from solid
formulations in the clinical laboratory, it is highly desirable to
package the solid formulations in proper unitary amounts. Thus, for
example, the solid formulations may be encapsulated or tabletted
with the proper quantity of reagent in each capsule or tablet for
conducting a single test. Alternatively, a multi-test package can
be provided from which the proper amount of liquid reagent is
prepared for conducting a specified number of tests.
Where a solid reagent formulation is packaged in unitary amounts,
accuracy of metering the solid material into each capsule, tablet
or multi-test package is important. The metering equipment which is
used for delivering solid materials in packaging and tableting
operations, however, almost universally operates on a volumetric
basis. Unless the solid material is free-flowing and has a
consistent bulk density, therefore, it cannot be delivered in
consistent weight amounts to each package, capsule or tableting
station using conventional equipment.
To provide a solid formulation in free-flowing form of consistent
bulk density, it is preferably granulated prior to packaging.
Granulation converts a powdered material into a material
constituted by small agglomerates of relatively uniform size.
Properly prepared, the granular material is free-flowing, has a
consistent bulk density and is readily handled by the metering
devices used in packaging operations. To granulate a powdered
material, the powder is typically mixed with a binder dissolved in
a volatile solvent, wet screened, dried by driving off the solvent,
and dry screened following the drying step. In addition to the
binder, a lubricating substance is normally incorporated in the
granulation mass to further enhance the flow characteristics of the
granules, especially under the compressive stress of tableting
operations.
As noted, solid formulations useful as reagents for conducting
clinical diagnostic tests on biological specimens should have
certain additional properties. Because they are dissolved in water
to produce a liquid reagent, all components, including the binder,
should be readily water-soluble. Because many of the tests involve
enzymatic reactions and/or proteinaceous substrates, the
formulation should possess detergent properties for solubilizing
protein.
It has now been discovered that the above objectives can be met and
that effective clinical reagent formulations for the determination
of certain biological properties of body fluids can be produced in
free-flowing, granular form through the use of particular
nitrogen-bearing polyoxyalkylene nonionic surfactants. Test
formulations granulated with the aid of these surfactants are well
adapted to precision packaging and tableting operations. Because of
their free-flowing character and consistent bulk density, they can
be delivered to either a packaging or tableting operation in
consistent weight amounts by volumetric metering. As a consequence,
clinical test reagents formulated at a central location remote from
a clinical laboratory can be utilized to prepare liquid test
reagents for clinical use without the need for weighing, analyzing,
or other procedures by the clinical chemist or technician.
The nitrogen-containing surfactants which are useful in the
formulations of the invention possess the unique multiple
capability of serving as binders, lubricants and solubilizers for
protein. Moreover, they are themselves water-soluble, thus
promoting the dissolution of the reagent formulations in water to
provide clinical liquid reagents. These surfactants are sold under
the trade designation "Tetronic" by Wyandotte Chemical Corporation.
They are normally prepared by sequential reaction of first
propylene oxide and then ethylene oxide with ethylene diamine in
the presence of an alkaline or acid catalyst. Normally these
surfactants are prepared at elevated temperatures using alkaline
catalysts such as sodium hydroxide, potassium hydroxide, sodium
alkoxide, quarternary ammonium bases and the like. Other methods
are available for the preparation of these surfactants. The
preparation of surfactants such as those utilized in the
formulations of the invention is more fully described in U.S. Pat.
No. 2,979,528.
The properties and physical state of nonionic surfactants having
structures corresponding to those derived from ethylene diamine,
propylene oxide and ethylene oxide vary with the lengths of the
polyoxypropylene and polyoxyethylene chains. As the drawing shows,
the physical state of these surfactants is largely dependent upon
the proportionate weight of the surfactant constituted by the
polyoxyethylene chains, but is also influenced by the average
molecular weight of the polyoxypropylene moieties. The
polyoxypropylene chains are hydrophobic while the polyoxyethylene
chains are hydrophilic. Thus, the surfactants having
polyoxypropylene units of low average molecular weight are more
water-soluble than those having polyoxypropylene units of a higher
average molecular weight. The numbers set out on the face of the
grid correspond to particular members of the Tetronic series. Each
number is located at a point on the grid whose coordinates
correspond to the polyoxyethylene and polyoxypropylene chain sizes
of the particular product which is commercially designated by said
number.
Essentially any surfactant whose structure is defined by the
coordinants of a point lying in the grid of the drawing may be
utilized in the formulations of the invention. It is preferred,
however, that the surfactant be solid or at least semi-solid. A
greater proportion of the solid surfactants can be satisfactorily
incorporated in a reagent formulation and thus a greater binding
and lubricating capacity is obtained without adversely affecting
other properties of the formulation. Desirably, on the order of 2.5
to 5% by weight of the preferred solid surfactants are incorporated
in the reagent formulations. When the liquid formulations are used,
it is not always possible to incorporate more than 2 or 3% by
weight of the surfactant without imparting a somewhat waxy
character to the formulation. The use of 2 to 3% by weight of a
liquid Tetronic surfactant produces a useful product, but the
binding and lubricating capabilities of the surfactant are not
always fully exploited at such a level. Granules having the most
desirable properties are obtained using solid or semi-solid
surfactants.
Since the dry reagent formulations of the invention are dissolved
in water for use in conducting clinical diagnostic tests, it is
also desirable that the surfactant component promote the
dissolution of the granular product. Thus, it is preferred that the
surfactant be as hydrophilic as possible, i.e., that the molecular
weight of the polyoxypropylene hydrophobe moiety of the surfactant
be relatively low. Thus, the preferred surfactants for use in the
formulations of the invention are those which are both solid or
semi-solid in physical state and relatively hydrophilic.
Solid-state surfactants with polyoxypropylene chains having an
average molecular weight of less than about 4000 are especially
preferred, with the most suitable surfactants being those whose
polyoxypropylene chains have an average molecular weight of between
about 2750 and about 3750 and whose weight percentage of
polyoxyethylene units is between about 70% and about 80%. Two
particular surfactants whose weight and structure characteristics
fall within the latter limits are those sold under the trade
designations "Tetronic 707" and "Tetronic 908". "Tetronic 707" has
a polyoxypropylene hydrophobe molecular weight on the order of 2750
and a weight percentage of polyoxyethylene units of about 70 %
while Tetronic 908 has a polyoxypropylene molecular weight of about
3750 and a weight percentage of polyoxyethylene units of about 80%.
Good results are also obtained with surfactants whose
polyoxypropylene chains have an average molecular weight of between
750 and 4000 with a weight percentage of between about 35% and
about 65% polyoxyethylene units. Other surfactants within the grid
of the drawing are reasonably satisfactory but less effective than
those represented by the right lower corner of the grid.
In addition to their advantageous effect upon granulation and
dissolution of dry clinical test reagent formulations, surfactants
of the above-noted character have been found to be effective for
solubilizing protein. As indicated above, this is a highly
advantageous characteristic, since enzymes and other proteinaceous
matter derived from either the reagent formulation or the specimen
commonly participate in the test reactions. By solubilizing
protein, the surfactants function to facilitate the progress of the
test reaction and thus enhance the effectiveness of the reagent
formulation. It may, therefore, be seen that incorporation of these
surfactants in clinical test formulations uniquely provides
multiple advantages in the preparation, packaging, dissolution and
functional operation of clinical reagent formulations.
It has further been discovered that the dry clinical reagent
formulations of the invention are quite stable and generally
possess good shelf life characteristics. Although we cannot
precisely account for the particular ingredient or combination of
ingredients which imparts the high degree of storage stability, it
appears that such stability may be a somewhat general
characteristic of dry clinical reagent formulations which include
the particular nitrogen-containing nonionic surfactants used in our
formulations. If so, the ability to impart storage-stability
represents a further aspect of the unique multiple function of this
type of surfactant in such formulations.
To prepare the reagent formulations of the invention, the
surfactant is mixed with a volatile solvent and at least one
reagent capable of participating in a test reaction to effect a
measurable change in a reagent/specimen test system. The surfactant
should be soluble up to the amount present in the solvent which is
utilized. Solvents which may be used include methylene chloride,
chloroform, methanol, benzene, water, methanol/water, and
chloroform/methylene chloride. After thorough mixing and
appropriate size classification, the solvent is removed to yield a
granular product.
In a preferred embodiment of the invention, the ingredients of the
formulation, in dry particulate form, are thoroughly blended in a
mechanical mixer. With the mixer running, a granulating solution
containing the solvent and the surfactant, preferably that sold
under the trade designation Tetronic 707 or Tetronic 908, is added.
Additional solvent is used as needed to produce granular
agglomerates of the desired size and wetness.
The resulting wet granulation is screened through a coarse screen,
for example 10 mesh, then spread in thin layers in trays and dried
at reduced pressure, for example, 25 inches Hg absolute or less.
Depending on the heat sensitivity of the formulation, drying is
normally carried out at room temperature or at modest elevated
temperature (up to about 37.degree.C.). Generally, the depth of the
wet granules in the trays should not exceed about 1/2 inch to 3/4
inch.
After completion of the drying cycle, the dried granulation is
rescreened through a finer screen, for example, 20 to 30 mesh,
blended thoroughly and packaged in containers essentially
impervious to moisture. Since the components of the reagent
formulation are frequently moisture sensitive, the formulation
should not be exposed to a relative humidity of more than about 5%
after removal from the dryer.
The reagent formulations of the invention are adapted to be
packaged in small unitary packages. For example, sufficient reagent
formulation for a single assay may be tabletted or packaged in a
capsule. The reagent formulations are also adapted to packaging in
such containers as foil strip packets, utilizing automatic
packaging machinery. Utilizing this packaging approach, sufficient
reagent formulation to carry out a suitable predetermined number of
tests, such as 10, 25, or 50 tests, may be accurately packaged in a
single foil packet. The user then simply dissolves the contents of
the multiple test packet in a predetermined volume of water and
uses a suitable aliquot of the resulting liquid reagent in the
preformance of each of a series of assays for the desired
biological substance or property.
In some instances, depending on the nature of the components and
their compatibility, all of the reagents necessary in a single
assay or determination may be included in a single formulation. In
other instances, incompatabilities and/or other considerations may
make it desirable to segregate certain reagents in which case two
or more reagent formulations are prepared in accordance with the
invention.
To conduct a clinical diagnostic test using the formulations of the
invention, the liquid reagent produced by dissolving the dry
formulation in a predetermined amount of water is mixed with the
biological specimen in a predetermined volumetric or weight ratio.
With the aid of appropriate instrumentation as required, the
resulting specimen/reagent system is observed for the presence,
absence, nature and extent of a physical, chemical or biological
change. Such change as does occur is measured to provide the
desired information for use in the clinical diagnosis.
Exemplary reagent formulations prepared in accordance with the
invention and useful for the determination of hemoglobin, blood
urea nitrogen, total protein, serum glutamic oxaloacetic
transaminase, alkaline phosphatase, glucose, inorganic phosphorus,
lactate dehydrogenase-L, serum glutamic pyruvic transaminase, uric
acid (colorimetric) and uric acid (u.v.) are set forth in Table 1.
The preferred compositions of these reagent formulations and
methods for preparing them are described in the examples following
Table 1 which more fully illustrate the invention.
Table 1
__________________________________________________________________________
Exemplary Clinical Test Reagent Formulations Granulating Solution
Poly- No. of Dry Ingredients ethylene Theo- Tests Formu- Type of
Formu- (Reagents, Etc.) TETRONIC glycol CH.sub.2 Cl.sub.2 retical
(Thou- lation lation Name/Formula Wt. (g.) 707 (g.) 6000 (g.) (ml.)
(1) Yield sands)
__________________________________________________________________________
A Reagent Formu- NaHCO.sub.3 300) 30 (a) 200 1000 50 lation for
Hemo- K.sub.3 Fe(CN).sub.6 50) (b) 300 globin Assay KCN 30)
Mannitol 590) T Enzyme Formu- Uricase Formu- lation for Uric lation
(equiva- Acid Assay lent to 0.05 units) Glycine 113.25) 4.50 (a) 25
7.5 Sodium carbonate, anhydrous 39.75) (b) 10 U Copper Reagent
Tris-(hydroxy- Formulation for methyl)-amino- Uric Acid Assay
methane 112.50) 7.05 (a) 15 234 7.5 Sodium Bicar- bonate 112.50)
(b) 5 Copper Sulfate, anhydrous 1.95) V Neocuproine Formu-
Neocuproine .sup.. HCl 5.25) 4.75 (a) 30 160 7.5 lation for Uric
Renex 35 150 (b) 10 Acid Assay W Blank Formu- Uricase Placebo
19.50) 4.50 (a) 25 177 7.5 lation for Uric Glycine 113.25 Acid
Assay Sodium carbonate, anhydrous 39.75
__________________________________________________________________________
(1) (a) indicates amount of CH.sub.2 Cl.sub.2 used as carrier for
Tetroni 707 (b) indicates amount of additional CH.sub.2 Cl.sub.2
used to optimize granulation
EXAMPLE 1
Hemoglobin Reagent Formulation and Assay
Composition of the reagent formulation useful for hemoglobin assay
is set forth as formulation A in Table 1.
To prepare this formulation, sodium bicarbonate (300 g.), milled
potassium ferricyanide (50 g.) and potassium cyanide (30 g.) were
initially added to a Hobart bowl and mixed with a stainless steel
spatula. Mannitol (590 g.) was then added and the resulting blend
was agitated for five minutes in the mixer. While agitation was
continued, a solution of Tetronic 707 (30 g.) in methylene chloride
(200 ml.) was added. An additional amount of methylene chloride
(300 ml.) was then added to produce the proper granulation.
The wet granulation was screened through a No. 10 mesh stainless
steel screen and the wet screened material was transferred to 8
inches .times. 12 inches Pyrex drying trays, at a depth of between
about 1/2 inch and about 3/4 inches in each tray. The granulation
was then dried in a vacuum oven for 15 hours at a temperature of
35.degree.C. and a pressure of 25 inches Hg.
The dried granulation was removed from the vacuum oven in an
environment where the relative humidity was not more than 5%. The
dried granulation was then screened through a No. 20 mesh stainless
steel screen using an Erweka oscillator. The screened, dried
granulation was transferred to a P.K. blender and mixed for 5
minutes, then packaged in tightly closed containers. Approximately
1000 g. of a water-soluble, substantially anhydrous reagent
formulation, sufficient for 50,000 tests, was obtained.
Upon being stored at a temperature of 45.degree.C., the
above-prepared formulation was found to be stable for at least 23
weeks which is equivalent to a stability period of 92 weeks at room
temperature.
Dissolved in water, formulation A yields a liquid reagent useful in
assaying blood hemoglobin. By action of the dissolved reagent,
erythocytes in the blood are hemolyzed releasing hemoglobin which
is oxidized to methemoglobin. Methemoglobin is converted to
cyanmethemoglobin whose formation alters the optical density of the
reagent/specimen system. The optical density of the
reagent/specimen system is measured at 540 nm. using a suitable
spectrophotometer and compared against a reagent blank set at 100%
transmission. The hemoglobin level is then determined by reference
to a standard curve.
To prepare a liquid reagent sufficient for 50 tests, formulation A
(1.00 g.) is dissolved in distilled water and the resulting
solution is diluted to 250 ml. and mixed thoroughly. The reagent
solution thus produced is stable for three months at room
temperature if protected from light.
To conduct the hemoglobin assay test, a reagent/specimen test
system is prepared by adding 20 microliters of well mixed blood
(collected with an anticoagulant) to 5 ml. of the above solution of
formulation A in a clean test tube. The contents of the tube are
mixed thoroughly and allowed to stand at room temperature for at
least five minutes. The optical density is then measured as
described above to determine the hemoglobin level.
EXAMPLE 2
Colorimetric Formulations and Assay for Uric Acid
For the colorimetric uric acid test, three separate formulations
are provided. These three formulations are set forth in Table 1 as
formulations T, U and V. Predetermined amounts of these
formulations are dissolved in separate portions of water to provide
liquid reagents for use in making the uric acid assay.
Preparatory to blending the constituents of enzyme reagent
formulation T, the modified uricase component thereof was produced.
To produce the modified uricase, a borate buffer was initially
prepared by dissolving boric acid (50 g.) in distilled water (3.5
l.) and titrating the resulting solution to a pH of 9 with 10%
solution of sodium hydroxide. The titrated solution was then
diluted to a total volume of 4 l. (0.2 M in borate) and chilled in
refrigerator prior to use. Uricase (about 40 mg.) was transferred
to a 250 ml. beaker by streams of the borate buffer delivered from
a wash bottle. The uricase used was uricase solution in 50%
glycerol obtained from Boehringer Mannheim Corporation (Cat. No.
15074 EVAC) and having a specific activity of about 4.5 U/mg. at
25.degree.C. and about 10.5 U/mg. at 37.degree.C. After the uricase
was transferred, additional borate buffer was added to bring the
total volume of uricase solution in the beaker to approximately 100
ml. The diluted uricase solution was then dialyzed against
approximately 2 l. of 0.2 M borate buffer for 4 hours, contaminated
buffer being replaced with fresh buffer at the end of the first 2
hours of dialysis. While dialysis was in progress potassium
chloride (6 g.), mannitol (4 g.) and gum acacia (4 g.) were
dissolved in 0.2 M borate buffer (100 ml.). The resulting solution
was clarified by centrifugation at 10,000 rpm and 10.degree.C. for
16 minutes using a No. 872 angle rotor in an IEC B-20 refrigerated
centrifuge. Bovine serum albumin (0.4 g.) and approximately 67,200
units of catalase were added to the clarified solution to produce a
solution referred to hereinafter as the inert solution.
After dialysis of the uricase solution was complete, the dialyzed
uricase solution was transferred to a 500 ml. beaker and combined
with a 200 ml. portion of the inert solution. Distilled water (200
ml.) was then added and the resulting solution was thoroughly
mixed. This solution was then transferred into two separate
freeze-drying vessels and shell frozen in a dry ice/alcohol bath at
a temperature of -60.degree.C. or below. The frozen solution was
lyophilized at -60.degree.C. to -70.degree.C. and an absolute
pressure of 5 millimicrons mercury for 20-24 hours. The resultant
lyophilized powder was collected under an atmosphere having a
relative humidity of less than 5% and stored in a dessicator at
4.degree.C. Approximately 19.5 g. of modified uricase was
obtained.
To prepare uricase reagent T, modified uricase (equivalent to 0.05
units/test), glycine (113.25 g.) and anhydrous sodium carbonate
(39.75g.) were blended in a Hobart bowl and thoroughly agitated to
promote intimate mixing. With the mixer running, a solution of
Tetronic 707 (4.50 g.) in methylene chloride (25 ml.) was
introduced. Additional methylene chloride (approximately 10 ml.)
was subsequently added to produce the desired degree of granulation
and wetness. The wet granulation was then screened and dried and
the resulting dry granulation rescreened and packaged in the manner
described in Example 1 for hemoglobin reagent formulation A.
In the preparation of copper reagent formulation U,
tris-(hydroxymethyl)-aminomethane (112.50 g.), sodium bicarbonate
(112.5 g.) and anhydrous cupric sulfate (1.95 g.) were blended in a
Hobart bowl and agitated to promote intimate mixing. With the mixer
running, a solution of Tetronic 707 (7.05 g.) in methylene chloride
(15 ml.) was added. Additional methylene chloride (approximately 5
ml.) was subsequently introduced to produce the desired degree of
granulation and wetness. The wet granulation was then screened and
dried and the resulting dry granulation rescreened and packaged in
the manner described in Example 1 for hemoglobin reagent
formulation A.
To prepare neocuproine reagent formulation V, neocuproine
hydrochloride (5.25 g.), "Renex-35" (150 g.) and Tetronic 707 (4.75
g.) were blended in a Hobart bowl and thoroughly agitated to
promote intimate mixing. Methylene chloride (approximately 40 ml.)
was then introduced to produce the desired degree of granulation
and wetness. The wet granulation was then screened and dried and
the resulting dry granulation rescreened and packaged in the manner
described in Example 1 for hemoglobin reagent formulation A.
Dissolved in separate portions of water, formulations T, U and V
provide liquid reagents useful in assaying a biological specimen
for uric acid. Uric acid in the specimen reduces cupric ion of
formulation U to cuprous ion which in turn reacts with neocuproine
of formulation V in buffered solution to form a color complex. The
resulting optical density of the test system is compared with the
optical density of a blank prepared in the same manner as the test
system but further including uricase from formulation T which
destroys uric acid. The differences in absorbances between the test
system and the blank is proportional to the serum uric acid
content.
To prepare the liquid enzymatic reagent, formulation T (1.18 g.) is
dissolved in distilled water (150 ml.). A uricase blank solution
(formulation W) is prepared by dissolving (1.18 g.) in distilled
water (150 ml.). The copper-bearing liquid reagent is prepared by
dissolving formulation U (1.55 g.) in distilled water (25 ml.). A
neocuproine-bearing liquid reagent is prepared by dissolving
formulation V (1.06 g.) in distilled water (25 ml.). The liquid
reagent solutions of formulations U and V are stable indefinitely
at room temperature while the solution of formulation T should be
prepared fresh daily. The resulting solutions are sufficient for
conducting 50 tests.
In the conduct of the test, a 3 ml. portion of the solution of
formulation T is added to one test tube and 3 ml. portion of
formulation W is added to a second test tube. 0.1 ml. of serum is
then added to both tubes to provide a specimen/reagent test system
in the tube containing distilled water and a blank test system in
the tube containing the solution of formulation T. The contents of
both test tubes are then incubated for 15 minutes at 37.degree.C.,
following which 1 ml. of a combined color reagent mixture, prepared
by mixing equal volumes of the solutions of formulations U and V,
is added to both the specimen/reagent test system and the blank
test system. Both of the test systems are allowed to stand at room
temperature for 15 minutes after addition of the combined color
reagent mixture and the light absorbance of each system is then
measured at 455 nm on a spectrophotometer set at 100% transmission
on a water blank. To provide the data required for the calculation
of uric acid in the serum, another optical density measurement is
taken on a standard reagent blank. The standard reagent blank is
prepared by adding uric acid (100 mg.) and lithium carbonate (60
mg.) to distilled water (about 500 ml.) and warming the mixture to
60.degree.C. to dissolve the additives. The resulting solution is
cooled to room temperature and diluted to a total volume of 1000
ml. with additional quantities of distilled water. 3 ml. of this
reagent blank is then added to a test tube and processed in the
same fashion as the blank and the specimen/reagent test system
including addition of serum, incubation, addition of the
above-noted combined color reagent mixture and a 15-minute hold
prior to measurement of optical density. The mg% uric acid in the
serum specimen is then determined in accordance with the following
calculation: ##EQU1##
EXAMPLE 3
U.V. Formulations and Assay for Uric Acid
Two formulations are used in the uric acid (U.V.) test. One of
these formulations is formulation T of Example 10 while the other
is set forth in Table 1 as formulation W. Predetermined amounts of
these formulations are dissolved in separate portions of water to
provide liquid reagents for use in making the uric acid (U.V.)
assay.
In preparing formulation W, a uricase placebo is used. This is
prepared in the same manner as the modified uricase component of
formulation T as described in Example 10 except that the uricase is
omitted.
To prepare formulation W, uricase placebo (19.50 g.), glycine
(113.25 g.) and anhydrous sodium carbonate (39.75 g.) were blended
in a Hobart bowl and thoroughly agitated to promote intimate
mixing. With the mixer running, a solution of Tetronic 707 (4.50
g.) in methylene chloride (25 ml.) was introduced. Additional
methylene chloride (approximately 10 ml.) was subsequently added to
provide the desired degree of granulation and wetness. The wet
granulation was screened and dried and the resultant dried
granulation rescreened and packaged in the manner described in
Example 1 for hemoglobin reagent formulation A.
A liquid reagent solution of formulation W is used in conjunction
with a liquid reagent solution of formulation T in practicing the
uric acid (U.V.) test. In the presence of the uricase of
formulation T, uric acid from the specimen reacts with water and
oxygen to form allantoin, carbon dioxide, and hydrogen peroxide.
Light absorbance at 293 nm, the absorption peak of uric acid, is
measured before and after treatment of the specimen with uricase
from formulation T with the difference in absorbance being
proportional to the uric acid present in the system. Allantoin, the
product of the uricase catalyzed reaction of water, uric acid and
oxygen, does not absorb at 293 nm.
The liquid reagent solution of formulation T is prepared as
described in Example 10 above. To prepare a blank liquid reagent,
formulation W (1.18 g.) is dissolved in distilled water 9(ml.). As
noted above, the solution of formulation T should be prepared fresh
daily. The liquid reagent solution of formulation W is stable for 1
month when refrigerated. The resulting solution is sufficient for
conducting 50 tests.
In conducting the test, a blank system is prepared by mixing the
solution of formulation W (3.0 ml.) with a specimen of serum (100
.mu.l) while a specimen/reagent test system is prepared by mixing
the solution of formulation T (3.0 ml.) with a specimen of the same
serum (100 .mu.l). Both the blank system and the specimen/reagent
test system are incubated at 37.degree.C. for 15 minutes. The
incubated mixtures are then transferred to cuvettes of a
spectrophotometer having a 1 centimeter light path. The instrument
is zeroed at 0.800 O.D. with the blank at 293 nm. The absorbance of
the unknown is then read and the mg% uric acid in the specimen
determined in accordance with the following calculation:
In view of the above, it will be seen that the several objects of
the invention are achieved and other advantageous results
attained.
As various changes could be made in the above methods and products
without departing from the scope of the invention, it is intended
that all matter contained in the above description shall be
interpreted as illustrative and not in a limiting sense.
* * * * *