U.S. patent number 3,876,502 [Application Number 05/467,551] was granted by the patent office on 1975-04-08 for reagent formulations for assaying urea nitrogen in biological specimens and methods of preparing and using same.
This patent grant is currently assigned to Mallinckodt, Inc.. Invention is credited to Ching Chiang, Alexander A. Monte.
United States Patent |
3,876,502 |
Monte , et al. |
April 8, 1975 |
Reagent formulations for assaying urea nitrogen in biological
specimens and methods of preparing and using same
Abstract
A solid, water-soluble substantially anhydrous, storage-stable
reagent formulation for use in conducting a clinical diagnostic
test on a biological specimen is provided. The reagent formulation
comprises a mixture containing a reagent capable of participating
in a test reaction to effect a measurable change in a test system;
and a nitrogen bearing 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. The polyoxypropylene
chains of said surfactant have an average molecular weight of
between about 750 and about 6750. Methods of preparing the reagent
formulations and methods of using them to conduct clinical
diagnostic tests are also provided.
Inventors: |
Monte; Alexander A. (Glendora,
CA), Chiang; Ching (Glendora, CA) |
Assignee: |
Mallinckodt, Inc. (St. Louis,
MO)
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Family
ID: |
27392815 |
Appl.
No.: |
05/467,551 |
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: |
435/12; 435/10;
435/16; 435/25; 435/28; 435/14; 435/21; 435/26 |
Current CPC
Class: |
G01N
33/52 (20130101); G01N 33/721 (20130101); C12Q
1/58 (20130101); C12Q 1/26 (20130101) |
Current International
Class: |
C12Q
1/26 (20060101); C12Q 1/58 (20060101); G01N
33/72 (20060101); G01N 33/52 (20060101); C01n
031/14 () |
Field of
Search: |
;195/13.5R,13.5C,99 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Tanenholtz; Alvin E.
Assistant Examiner: Fan; C. A.
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.
patent application, Ser. No. 200,552, filed Nov. 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 specimen for
blood urea nitrogen comprising a solid, water-soluble,
substantially anhydrous, storage-stable mixture containing (a)
urease; (b) a salt of ethylenediaminetetraacetic acid; (c) a
carbohydrate polymer; (d) mannitol and (e) a nitrogen-containing
polyoxyalkylene nonionic surfactant having a structure
corresponding to that obtained when ethylene diamine is reacted
sequentially with propylene oxide and ethylene oxide in the
presence of a catalyst, the polyoxypropylene chains of said
surfactant having an average molecular weight of between about 750
and about 6750.
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 assaying a specimen for blood urea nitrogen using
a plurality of solid, water-soluble, substantially anhydrous,
storage-stable reagent formulations respectively comprising:
1. an alkaline chlorine reagent formulation comprising a mixture
containing:
a. sodium dichloroisocyanurate;
b. lithium hydroxide;
c. mannitol; and
d. a nitrogen-containing polyoxyalkylene nonionic surfactant having
a structure corresponding to that obtained when ethylenediamine is
reacted sequentially with propylene oxide and ethylene oxide in the
presence of a catalyst, the polyoxypropylene chains of said
surfactant having an average molecular weight of between about 750
and about 6750;
2. a chromogenic reagent formulation comprising a mixture
containing:
a. sodium salicylate;
b. sodium nitroferricyanide; and
c. said nitrogen-containing polyoxyalkylene nonionic surfactant;
and
3. an enzyme reagent formulation comprising a mixture
containing:
a. urease;
b. a salt of ethylenediaminetetraacetic acid;
c. a carbohydrate polymer;
d. mannitol; and
e. said nitrogen-containing polyoxyalkylene nonionic
surfactant;
the method comprising:
i. dissolving each of said reagent formulations in water to produce
a plurality of liquid reagents;
ii. mixing the liquid reagent containing said granular enzyme
reagent formulation with a specimen of blood serum to form a
specimen/reagent test system;
iii. adding the liquid reagent containing said chromogenic reagent
formulation to said system;
iv. adding the liquid reagent containing said alkaline chlorine
reagent formulation to said system; and
v. measuring a change in said system resulting from the interaction
between said reagents and said specimen.
4. The method as set forth in claim 3 wherein said change is
measured by determining the optical density of said system.
5. The method as set forth in claim 3 wherein said
nitrogen-containing surfactant is solid and the polyoxypropylene
chains thereof have an average molecular weight of less than about
4000.
6. The method of preparing a solid, water-soluble, free-flowing,
substantially anhydrous, storage-stable granular enzyme reagent
formulation for use in assaying blood for urea nitrogen, said
method comprising the steps of:
1. preparing a mixture containing:
a. urease;
b. a salt of ethylene diaminetetraacetic acid;
c. a carbohydrate polymer;
d. mannitol;
e. a nitrogen-containing polyoxyalkylene nonionic surfactant having
a structure corresponding to that obtained when ethylene diamine is
reacted sequentially with propylene oxide and ethylene oxide in the
presence of a catalyst, the polyoxypropylene chains of said
surfactant having an average molecular weight of between about 750
and about 6750; and
f. a solvent for said surfactant; and
2. removing the solvent to form a substantially anhydrous,
free-flowing, water-soluble, granular solid.
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 or 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; and 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 operated on a volumetric
basis. Unless the solid material is freeflowing 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
nitrogenbearing 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 test 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
semisolid 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
polyoxypropoylene 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 one-half inch to
three-fourth 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
performance 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/reagents 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 Reagents Formulations Granulating Solution
poly- ethy- lene gly- No. of Dry Ingredients col Theo- Tests Formu-
Type of Formu- (Reagents, Etc.) TETRONIC 6000 CH.sub.2 Cl.sub.2
retical (Thou- lation lation Name/Formula Wt. (g.) 707 (g.) (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 B Alkaline Chlo- Sodium Di- rine Reagent chloroiso- Formulation
cyanurate 120 15 (a) 200 555 30 for BUN Assay LiOH 240 (b) 100
Mannitol 180 C Chromogenic Sodium Reagent Salicylate 1200 44 2 (a)
250 1250 10 Formulation Sodium Nitro- (b) 100 for BUN Assay
ferricyanide 4 D Granular Urease/EDTA Enzyme Formu- Formulation
lation for (36,000 I.U.) BUN Assay Mannitol 579 15 6 (a) 100 600 30
(b) 200 E Reagent Formu- Cupric Tartrate lation for Total
(Anhydrous) 500 100 (a) 1500 3850 50 Protein Assay Sodium Tartrate
(2H.sub.2 O) 750 (b) 250 LiOH 2000 RENEX-35 500 F Coenzyme Formu-
L-Aspartic Acid 1250 80 (a) 800 25 lation for MDH Formulation (b)
525 GOT Assay (containing 6.0 mg. MDH; 1.4 I.U.) NADH Formulation
(containing 0.5 mg. NADH) Tris-(hydroxymethyl)- aminomethane 1875
Succinic Acid 187.5 G Substrate Formu- Sodium Alpha- 200 25 (a) 500
990 50 lation for Ketoglutarate (b) 200 GOT Assay Mannitol 765 H
Reducer Formu- Ascorbic Acid 3250 150 (a) 500 5000 50 lation for
Alkaline Sulfamic Acid 1600 (b) 200 Phosphatase Assay I Molybdate
Formu- Sodium Molyb- 475 37.5 (a) 300 1250 50 lation for Alkaline
date (<2%H.sub.2 O) Phosphatase Assay Duponol ME Dry 737.5 (b)
150 J Substrate Formu- Disodium .beta.-Glycero- lation for Alka-
phosphate 500 87.5 (a) 400 2600 50 line Phosphatase
Tris-(hydroxymethyl)- Assay aminomethane 1500 (b) 100 Succinic Acid
12.5 Duponol ME Dry 500 K Chromogen Formu- o-Dianisidine.2HCl 7.5
14.4 4.5 (a) 150 30 lation for Glu- Mannitol 423.6 (b) 125 cose
Assay L Buffer Formu- NaH.sub.2 PO.sub.4.H.sub.2 O 311.4 15.0 (a)
40.sup.(2) 600 30 lation for Glucose Assay Na.sub.2 HPO.sub.4 190.8
(b) 5-10.sup.(2) Mannitol 52.8 Spray Dried Gum Arabic 30.0 M Enzyme
Formu- Glucose Oxidase lation for Stabilized 420 15.0 3.0 (a)
40.sup.(2) 450 30 Glucose Assay Mannitol (b) 10.sup.(2) Horseradish
Peroxidase 30 Mannitol N Reducer Formu- Ascorbic Acid 3250 125 (a)
1000 4950 50 lation for In- Sulfamic Acid 800 (b) 500 organic Phos-
Duponol ME Dry 775 phorus Assay O Molybdate Formu-
Tris-(hydroxymethyl)- lation for In- aminomethane 1500 80 (a) 475
3000 100 organic Phosphorus Duponol ME Dry 470 (b) 175 Assay Sodium
Molybdate (<2% H.sub.2 O) 950 P Coenzyme Formu- NAD Modified
lation for Lactate (=225 g NAD) 1190 60 (a) 500 1250 50
Dehydrogenase Assay Mannitol (b) 250 Q Substrate Formu- Lithium
Lactate 2250 75 25 (a) 500 3200 50 lation for Tris-hydroxy- Lactate
Dehydro- methyl)-amino- genase Assay methane 225 (b) 1175
NaHCO.sub.3 450 Renex 35 175 R Coenzyme Formu- NADH Formulation
lation for Serum (equivalent to Glutamic Pyruvic 0.4 mg. NADH)
Transaminase Assay LDH Formulation (equivalent to 2 units LDH)
DL-Alanine 1200 50.4 (a) 200 15 Sodium phosphate, dibasic 585 (b)
70 Sodium phosphate, monobasic 75 S Substrate Formu- lation for
Serum (See Formulation G Above) Glutamic Pyruvic Transaminase Assay
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.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 (b) 10 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 (2) Solvent is water instead of
CH.sub.2 Cl.sub.2?
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
inch .times. 12 inch Pyrex drying trays, at a depth of between
about one-half inch and about three-fourths 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 5 minutes. The optical density is then measured as described
above to determine the hemoglobin level.
EXAMPLE 2
Blood Urea Nitrogen Formulation and Assay
Because certain components of the total number of reagents required
for a blood urea nitrogen (BUN) test tend to interact with one
another in the dry state, three separate formulations are provided
to segregate the interacting components. The three formulations
prepared are set forth in Table 1 as formulations B, C and D.
Predetermined amounts of each of these formulations are dissolved
in separate portions of water to provide liquid reagents for use in
making the BUN assay.
To prepare alkaline chlorine reagent formulation B, sodium
dichloroisocyanurate, sold under the trade designation "ACL-60" by
Monsanto Co. (120 g.), anhydrous lithium hydroxide (240 g.), and
mannitol powder (180 g.) were blended in a Hobart bowl and agitated
to promote intimate mixing. With the mixer running, a solution of
"Tetronic 707" (15 g.) in methylene chloride (200 ml.) was
introduced. Additional methylene chloride (100 ml.) was
subsequently added to produce the desired degree of granulation and
wetness. The wet granulation was screened and dried, and the
resultant dry granulation rescreened and packaged, in the manner
described in Example 1 for hemoglobin reagent formulation A.
In the preparation of the chromogenic reagent formulation C, sodium
salicylate (1200 g.) and sodium nitroferricyanide (4 g.) were
blended in a Hobart bowl and agitated to promote intimate mixing.
With the mixer running, a solution of "Tetronic 707" (44 g.) and
polyethylene glycol having a molecular weight of about 6000 (2 g.)
in methylene chloride (250 ml.) was added. Additional methylene
chloride (approximately 100 ml.) was subsequently introduced to
produce the desired degree of granulation. The wet granulation was
screened and dried, and the resultant dry granulation rescreened
and packaged, as described in Example 1 for hemoglobin reagent
formulation A.
Before blending the constituents of granular enzyme formulation D,
the lyophilized urease/ethylenediaminetetraacetic acid (EDTA)
component was prepared. To prepare this component disodium EDTA (24
g.) and tetrasodium EDTA (15.12 g.) were dissolved in distilled
water (about 900 ml.) with heating as required. A carbohydrate
polymer having a number average molecular weight of about 1850, a
dextrose equivalent of 10-13, a pH of 4.5-5.5, and a decomposition
point of 440.degree.F, sold under the trade designation "Amisol" by
Corn Products Company (11 g.) was added and the solution was cooled
to about 4.degree.C. Lyophilized urease powder having a specific
activity of approximately 200 IU/mg., such as that sold by
Worthington Biochemical Corporation, Code No. URPC, (100,000
International Units) was dissolved in this solution. The solution
was filtered through glass wool or cotton to remove any turbidity.
The clarified solution was divided and each portion placed in one
of several freeze drying vessels and the portions frozen in thin
layers by rotating the vessels in a dry ice/alcohol bath at -
60.degree.C. or below. The frozen thin layers were lyophilized at
-60.degree.C. to -70.degree.C. at a total pressure of 5 m.mu. Hg.
for 16 to 24 hours. The resultant lyophilized powder was collected
under an atmosphere having a relative humidity of less than 5% and
stored in a desiccator at 4.degree.C. Assay of the specific urease
activity of the dried powder was in the range of 2 to 3 I.U./mg. at
37.degree.C. It was found that the modified urease product thus
produced is stable at room temperature and does not require
refrigeration.
The following exemplary procedure provides approximately 600 grams
of granular enzyme formulation containing 12 I.U. urease/20 mg.
utilizing a lyophilized urease/EDTA formulation having a specific
urease activity of 3.0 I.U./mg.
To prepare granular enzyme formulation D, lump-free mannitol
(459mg.) and the lyophilized urease/EDTA formulation prepared as
above and having an activity of 3.0 I.U./mg. (120 g.) were blended
in a Hobart bowl and agitated to promote intimate mixing. With the
mixer running, a solution of "Tetronic 707" (15 g.) and
polyethylene glycol having a molecular weight of about 6,000 (6 g.)
in methylene chloride (100 ml.) was added to the blend. Additional
methylene chloride (approximately 200 ml.) was subsequently
introduced to produce the desired degree of granulation. The wet
granulation was then screened and dried at room temperature, and
the resulting dry granulation rescreened and packaged, in the
manner described for hemoglobin reagent formulation A in Example
1.
Upon being stored at a temperature of 45.degree.C. the above
prepared formulations were found to be stable for at least 21 weeks
which is equivalent to a stability period of 84 weeks at room
temperature.
Dissolved in separate portions of water, formulations B, C and D
provide liquid reagents useful in the determination of blood urea
nitrogen. The urease of formulation D specifically hydrolyzes urea
to ammonium carbonate, which is subsequently decomposed by the
addition of the alkali in formulation B to produce carbon dioxide
and ammonia. In the presence of the catalyst, sodium
nitroferricyanide, of chromogenic reagent formulation C, ammonia
reacts with the phenol derivative of formulation C and active
chlorine from formulation B to form an emerald green color complex.
The intensity of the color is measured colorimetrically at 650 nm.
and is proportional to the urea present.
Preparatory to conducting the test, formulation B (0.925 g.),
formulation C (6.25 g.) and formulation D (1.0 g.) are each
dissolved in water in separate containers. The solution of
formulation D is diluted to 50 ml. while the solutions of
formulations B and C are each diluted to 25 ml. All of the diluted
solutions are thoroughly mixed. The solution of formulation D is
stable for one month under refrigeration. The solutions of
formulations B and D are stable for 3 months if kept in amber
bottles under refrigeration. The resulting solutions are sufficient
for conducting 50 tests.
In the conduct of the test, 1.00 ml. of the dilute solution of
formulation D and 0.005 ml. (5 lambda) of serum are placed in a
test tube, mixed well and incubated at 37.degree.C. for 5 minutes
to effect enzymatic hydrolysis. Formulation C solution (0.5 ml.) is
then added with careful mixing, immediately following which the
solution of formulation B (0.50 ml.) is added and thoroughly mixed
with the other components of the solution. Incubation is continued
at 37.degree.C. for an additional 10 minutes for color development.
At the end of this period, distilled water (2.00 ml.) is added and
the optical density at 650 nm. is read using a spectrophotometer
and compared with the optical density of a reagent blank set at
100% transmission. The urea level in the specimen is then
determined by reference to a standard curve.
EXAMPLE 3
Total Protein Formulation and Assay
The reagent formulation utilized in determination of total protein
in a biological specimen is set forth as formulation E in Table
1.
In the preparation of formulation E, anhydrous cupric tartrate (500
g.), dihydrated sodium tartrate (750 g.), lithium hydroxide (2000
g.) and a surfactant comprising a polyoxyethylene ether alcohol and
urea sold under the trade designation "Renex 35" by Atlas Chemical
Industries (500 g.) were blended and thoroughly agitated in a
Hobart bowl mixer. With the mixer running, a solution of "Tetronic
707" (100 g.) in methylene chloride (1500 ml.) was added.
Additional methylene chloride (250 ml.) was then introduced to
produce 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.
Upon being stored at a temperature of 45.degree.C., the
above-prepared formulation was found to be stable for at least four
weeks which is equivalent to a stability period of 16 weeks at room
temperature.
Dissolved in water, formulation E yields a liquid reagent useful in
assaying a body fluid for total protein. The biuret reaction of
proteins with the alkaline cupric tartrate constituent of
formulation E results in the formation of a violet color.
To prepare the liquid reagent, formulation E (1.925 g.) is
dissolved in distilled water and the solution is diluted to 100 ml.
and thoroughly mixed. This reagent solution is stable for at least
three months at room temperature, and is sufficient to conduct 25
tests.
In the conduct of the total protein test, a reagent/specimen assay
system is prepared by adding 50 microliters of serum to 4.0 ml. of
the solution of formulation E in a test tube. The contents of the
tube are mixed thoroughly and incubated at 37.degree.C. for 15
minutes. The optical density at 540 nm. is then read using a
spectrophotometer and compared with a reagent blank set at 100%
transmission. The total protein level is then determined by
reference to a standard curve.
EXAMPLE 4
Serum Glutamic Oxaloacetic Transaminase Formulations and Assay
For the serum glutamic oxaloacetic transminase (SGOT) test, two
separate formulations are provided. The two formulations which are
utilized are set forth in Table 1 as formulations F and G.
Predetermined amounts of these formulations are dissolved in
separate portions of water to provide liquid reagents for use in
making the SGOT assay.
Preparatory to blending the constituents of coenzyme formulation F,
the modified malate dehydrogenase and reduced nicotinamide adenine
dinucleotide components are prepared.
Modified malate dehydrogenase was derived from yellow split peas.
The peas were pulverized to a fine powder using a mill or a
micromill. A saturated solution of potassium chloride was prepared
at room temperature by stirring potassium chloride (500 g.) in
approximately 1 l. of distilled water for 5 minutes and allowing
the solution to stand at room temperature overnight. Pulverized pea
powder (10 g.) was stirred into a 50 ml. portion of the saturated
potassium chloride solution, which extracted the malate
dehydrogenase therefrom. Extraction was carried out at room
temperature for approximately 3 hours with occasional stirring. At
the end of the 3 hour extraction period, the resulting suspension
was centrifuged at 10,000 rpm using a No. 872 angle rotor in an IEC
B-20 refrigerated centrifuge at 10.degree.C. for 10 minutes. The
slightly colloidal supernatant extract fluid was collected and
transferred to a plurality of freeze drying vessels. The extract
was frozen in thin layers by rotating each vessel in a dry
ice/alcohol bath at -60.degree.C. or below. The frozen thin layers
were lyophilized at -60.degree.C. to -70.degree.C. and an absolute
pressure of 5m.mu. Hg for 18 to 20 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 14 g. of dry powder was obtained which was assayed
for the enxymic activities of both malate dehydrogenase (MDH) and
glutamic oxaloacetic transaminase (GOT)-GOT specific activity was
less than about 0.05% in relation to MDH specific activity, and the
extract enzyme was, therefore, suitable for use. Activity of the
MDH obtained was about 1.4 I.U./6 mg.
To prepare modified reduced nitotinamide adenine dinucleotide
(NADH), gum arabic (15 g.), tris-(hydroxymethyl)-aminomethane (12
g.), "Amisol" (10 g.), and bovine serum albumin (0.5 g.) were
dissolved in distilled water (450 ml.) and the resulting solution
was titrated to pH 9.0 with 12N sulfuric acid. After titration, the
solution was diluted to 500 milliliters with distilled water and
clarified by centrifugation at 10,000 rpm with a No. 872 angle
rotor in an IEC B-20 refrigerated centrifuge at 10.degree.C. for 16
minutes. To the supernatant obtained from this centrifugation, 10
g. of NADH power (reduced nicotinamide adenine dinucleotide
disodium salt) was added and the resulting mixture stirred for at
least 5 minutes to insure good mixing. This mixture was transferred
into freeze drying vessels and frozen in thin layers by rotating
the vessels in a dry ice/alcohol bath at -60.degree.C. or below.
The frozen thin layers were lyophilized for 20 to 24 hours at
-60.degree.C. to -70.degree.C. and an absolute pressure of 5 m.mu.
Hg. 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 250 g. of dry lyophilized
powder was obtained.
To prepare coenzyme formulation F L-aspartic acid (1250 g.), the
above modified MDH (equivalent to 1.4 I.U. or 6.0 milligrams MDH),
the above modified NADH (equivalent to 0.5 milligrams NADH),
tris-(hydroxymethyl)-aminoethane (1875 g.), and milled succinic
acid (187.5 g.) were blended in a Hobart bowl mixer and agitated to
promote intimate mixing. With the mixer running, a solution of
"Tetronic 707" (25 g.) in methylene chloride (800 ml.) was added to
the blend. Additional methylene chloride (approximately 525 ml.)
was subsequently introduced to produce the desired degree of
granulation and wetness. The wet granulation was then screened and
dried at room temperature, and the resulting dry granulation
rescreened and packaged, in the manner described for hemoglobin
reagent formulation A in Example 1.
To prepare substrate formulation G, sodium .alpha.-ketoglutarate
(200 g.) and lump-free mannitol (765 g.) were blended in a Hobart
bowl and agitated to promote intimate mixing. With the mixer
running, a solution of "Tetronic 707" (25 g.) in methylene chloride
(500 ml.) was added to the blend. Additional methylene chloride
(approximately 200 ml.) was subsequently introduced to produce the
desired degree of granulation and wetness. The Wet granulation was
then screened and dried at room temperature and 25 inches pressure
or less and the resulting dry granulation rescreened and packaged,
in the manner described for hemoglobin reagent formulation A in
Example 1.
Upon being stored at a temperature of 45.degree.C., the
above-prepared formulations were found to be stable for at least 3
weeks which is equivalent to a stability period of 12 weeks at room
temperature.
Dissolved in separate portions of water, formulations F and G
provide liquid reagents useful in assaying for serum glutamic
oxaloacetic transaminase. SGOT present in the specimen catalyzes
the transamination of L-aspartic acid and .alpha.-ketoglutaric acid
producing oxaloacetate and glutamate. The oxaloacetate and NADH in
the presence of MDH are converted to malate and NAD. The extent of
reaction is indicative of the SGOT present and is measured by
observing a decrease in optical density at 340 nm. at
37.degree.C.
The liquid reagent solution of formulation F is prepared by
dissolving 7.2 g. of the formulation in distilled water, diluting
to 125 ml. and mixing well. The solution of formulation G is
prepared in similar fashion utilizing a 1.0 g. of the formulation
and diluting to 25 ml. The formulation F solution should be
prepared fresh daily while the formulation G solution is stable for
a week under refrigeration.
In the conduct of the test, 2.5 ml. of the formulation F solution
are placed in a test tube, 0.1 ml. of serum are added thereto, and
the resulting solution stirred and incubated at 37.degree.C. in a
heating unit for 7 to 10 minutes. 0.5 ml. of the formulation G
solution is then added and mixed quickly in less than 10 seconds at
room temperature. Exactly 2 minutes after the liquid reagent
solution of formulation G is added, the test tube is removed from
the heating unit and the absorbance of the reagent/specimen system
is measured. The tube is then returned to the heating unit and held
there until exactly 5 minutes after the solution of formulation G
was first added. At this point, the tube is again removed from the
heating unit and the final absorbance of the reagent/specimen
system is measured. The SGOT content is determined from the two
absorbance readings by calculation utilizing the following
equation: ##EQU1## where A.sub.1 equals absorbance read at 2
minutes, A.sub.2 equals absorbance read at 5 minutes, L equals
light path of the absorption cell or the I.D. of the test tube in
centimeters. An SGOT unit is defined as that amount of enzyme which
catalyzes the oxidation of 0.001 micromole of NADH to NAD per
minute, at 37.degree.C. Other conditions for determining SGOT are
known. The Karmen procedure known to the art carries out the above
reactions at 25.degree.C. insteaad of 37.degree.C.
EXAMPLE 5
Alkaline Phosphatase Formulations and Assay
Three separate formulations are provided for the alkaline
phosphatase test. These formulations are set forth in Table 1 as
formulations H, I and J. Predetermined amounts of these
formulations dissolved in separate portions of water provide liquid
reagents for use in making the alkaline phosphatase assay.
Prior to the preparation of chromogenic reagent formulation I, the
sodium molybdate component of the reagent blend was dried. Sodium
molybdate was transferred into tared 8 inch .times. 12 inch Pyrex
trays at a depth of between about one-half inch and three-fourths
inch. The trays were then placed in a vacuum oven and the sodium
molybdate dried at 55.degree.C. and a total pressure of 25 inches
Hg until a weight loss of not less than 13% was observed.
To prepare chromogenic reagent formulation I, the dried sodium
molybdate (475 g.) and a dry sodium lauryl sulfate product sold
under the trade designation "Duponol ME" by E. I. DuPont de Nemours
and Company (737.5 g.) were blended in a Hobart bowl and agitated
to promote intimate mixing. With the mixer running, a solution of
"Tetronic 707" (37.5 g.) in methylene chloride (300 ml.) was
introduced. Additional methylene chloride (150 ml.) was
subsequently added to produce the desired degree of granulation and
wetness. The wet granulation was screened and dried and the
resultant dry granulation rescreened and packaged in the manner
described in Example 1 for hemoglobin reagent formulation A.
In the preparation of reducer quench formulation H, L-ascorbic acid
(3.25 kg.) and sulfamic acid (1.60 kg.) were blended in a Hobart
bowl and agitated to promote intimate mixing. With the mixer
running, a solution of "Tetronic 707" (150 g.) in methylene
chloride (500 ml.) was introduced. Additional methylene chloride
(200 ml.) was subsequently added to produce the desired degree of
granulation and wetness. The wet granulation was screened and dried
and the resultant dry granulation rescreened and packaged in the
manner described in Example 1 for Hemoglobin reagent formulation
A.
Preparation of buffer substrate reagent formulation J was initiated
by blending .beta.-glycerophosphoric acid, disodiuum salt (500 g.)
tris-(hydroxymethyl)-aminomethane (1.5 kg.), milled succinic acid
(12.5 g.) and "Duponol ME" (500 g.) in a Hobart bowl and agitating
the blend to promote intimate mixing. With the mixer running, a
solution of "Tetronic 707" (87.5 g.) in methylene chloride (400
ml.) was introduced. Additional methylene chloride (100 ml.) was
subsequently added to produce the desired degree of granulation and
wetness.. The wet granulation was screened and dried and the
resultant dry granulation rescreened and packed in the manner
described in Example 1 for hemoglobin reagent formulation A.
Dissolved in separate portions of water, formulations H, I and J
provide liquid reagents useful in the determination of the alkaline
phosphatase content of biological specimens such as blood serum.
Alkaline phosphatase in the specimen promotes the release of
phosphate from the .beta.-glycerophosphate constituent of substrate
reagent formulation J. The phosphate thus released reacts with
sodium molybdate of chromogenic reagent formulation I in the
presence of sulfamic acid and ascorbic acid of reducer quench
formulation H. The ascorbic acid reduces the phosphomolybdic acid
thus formed to phosphomolybedenum blue. The color of the latter is
indicative of the concentration of alkaline phosphatase present in
the reagent specimen system.
In preparation for a test, the dry reagent formulations are each
dissolved in water to provide liquid reagents. Formulation I (1.25
g.) is dissolved in 25 ml. of distilled water to provide a
chromogenic liquid reagent; formulation H (5.00 g.) is dissolved in
25 ml. of water to provide a reducer quench liquid reagent; and
formulation J (2.60 g.) is dissolved in 150 ml. of water to provide
a substrate buffer liquid reagent. The solution of formulation I is
stable for a week at room temperature. The solution of formulation
J is stable for two weeks when refrigerated. The liquid reagent
constituted by the solution of formulation H should be prepared
fresh daily and protected from light. The resulting solutions are
sufficient for conducting 50 tests.
Conduct of an alkaline phosphatase determination is initiated by
adding 3 ml. portions of the solution of substrate formulation J to
each of two test tubes and incubating each tube for 2 minutes at
37.degree.C. One of the two tubes is then used for the test
reaction while the other tube is used for a blank.
0.1 ml. (100 lambda) of serum is added to the tube used for the
test reaction and mixed thoroughly with the liquid reagent therein
to provide a specimen/reagent test system. The test system is then
incubated for exactly 15 minutes at 37.degree.C., following which
0.5 ml. portions of the liquid reagent solution of formulation I
and 0.5 ml. portions of the liquid reagent solution of formulation
H are added to both the test system and the tube carrying the test
blank. Both the test system and the blank are then incubated at
37.degree.C. for another 20 minutes, and the optical density of
each determined at 700 nm against a water blank set at 100%
transmission. Using a standard curve, the inorganic phosphorus
content of each test solution is determined from its optical
density compared to the water blank. Since both the test system and
test blank originally contain the same amount or proportion of
.beta.-glycerophosphate, the inorganic phosphorus released by
action of alkaline phosphatase is determined by subtracting the
total inorganic phosphorus in the test system from the total amount
of inorganic phosphorus in the blank.
To calculate the alkaline phosphatase units present in the test
system, the mg% inorganic phosphorus released is multiplied by 4.
In the procedure of Bodansky, known to the art, there is a
one-to-one correspondence between the mg% phosphorus released and
the alkaline phosphatase units. Since the test of the invention
utilizes only a 15 minute incubation time instead of the 60 minute
incubation time of Bodansky, the factor of 4 is applied to obtain
corresponding results.
The standard curve used to determine absolute inorganic phosphorus
is obtained by spectrophotometric measurements of the optical
densities of potassium dihydrogen phosphate solutions containing 0,
2.5, 5.0, 7.5 and 10 mg% phosphorus at 700 nm, against a reagent
blank set at 100% transmission. A standard potassium dihydrogen
phosphate solution for use in establishing the standard curve is
prepared by weighing out KH.sub.2 PO.sub.4 (438.1 mg.) in a 100 ml.
volumetric flask, diluting to volume with high quality distilled
water, and storing the diluted solution at 4.degree.C. This stock
solution contains 100 mg. phosphorus per 100 ml. (100 mg%) and
appropriately diluted aliquots of this stock solution are used in
establishing the standard curve.
As those skilled in the art will appreciate, phosphorus standard
curves obtained from spectrophotometric measurements of varying
concentrations of potassium dihydrogen phosphate are linear only up
to about 15 mg%. Where high phosphorus sera (above 7.5 mg%) are
analyzed for alkaline phosphatase content, therefore, a 50 lambda
serum sample is used and the final result multiplied by 2.
An alkaline phosphatase unit is defined as the amount of enzyme in
100 ml. of serum which releases 1 mg. phosphorus per hour at
37.degree.C.
EXAMPLE 6
Glucose Reagent Formulations and Assay
For the glucose test, three separate formulations are provided.
These are set forth in Table 1 as formulations K, L and M.
Predetermined amounts of these formulations are dissolved in
separate portions of water to provide liquid reagents for use in
making the glucose assay.
To prepare chromogenic reagent formulation K, mannitol (423.6 g.)
and o-dianisidine dihydrochloride (3,3-dimethoxybenzidine
dihydrochloride) (7.5 g.) were blended in a Hobart bowl and
agitated to promote intimate mixing. With the mixer running, a
solution of "Tetronic 707" (14.4 g.) and polyethylene glycol-6000
(4.5 g.) in methylene chloride (150 ml.) was introduced. Additional
methylene chloride (125 ml.) was subsequently added to produce the
desired degree of granulation and wetness. The wet granulation was
screened and dried at room temperature, and the resultant dry
granulation rescreened and packaged in the manner described in
Example 1 for hemoglobin reagent formulation A.
Buffer reagent formulation L was prepared by blending monobasic
sodium phosphate (311.4 g.), dibasic sodium phosphate (190.8 g.),
mannitol (52.8 g.) and spray dried gum arabic (30.0 g.) in a Hobart
bowl and agitating the resulting blend to promote intimate mixing.
With the mixer running, a solution of "Tetronic 707" (15.0 g.) in
distilled water (40 ml.) was introduced. Additional distilled water
(10 ml.) was subsequently added to produce the desired degree of
granulation and wetness. The wet granulation was screened and dried
and the resultant dry granulation rescreened and packaged in the
manner described in Example 1 for hemoglobin reagent formulation
A.
To prepare modified glucose oxidase, gum arabic (60 g.) and
mannitol (40 g.) were dissolved in water (about 1600 ml.) and the
resulting colloidal solution was titrated to pH 7.0 with 2% sodium
hydroxide. After titration, the solution was diluted to 2 liters
with distilled water and clarified by centrifugation at 10,000 rpm
with a No. 872 angle rotor in an IEC B-20 refrigerated centrifuge.
The clear supernatant inert solution was collected and stored at
4.degree.C. Bovine serum albumin (2 g.) was dissolved in 200 ml. of
the above-prepared inert solution with the aid of a magnetic
stirrer. Liquid glucose oxidase (200 ml.) having a specific
activity greater than 1000 titrimetric units per ml. was then
slowly added and the resulting mixture stirred for an additional 5
minutes to insure good mixing. This solution was transferred into
freeze drying vessels, each vessel being filled with 150-200 ml. of
the solution. The solution was frozen in thin layers by rotating
the vessels in a dry ice/alcohol bath at -60.degree.C. or below.
The frozen layers were lyophilized for 16 to 20 hours at
-60.degree.C. to -70.degree.C. and an absolute pressure of 5m
.mu.Hg. 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 14 g. of dry lyophilized
glucose oxidase powder with a specific activity range of 14-16
IU/mg. at 37.degree.C. was obtained.
In the preparation of enzyme reagent formulation M, a glucose
oxidase (GOD) subformulation was initially produced. The above
modified glucose oxidase (sufficient to provide 45 units of
unmodified material/test) and mannitol (sufficient to provide 14
mg. -- total of modified GOD plus mannitol -- per test) were
blended in a Hobart bowl and thoroughly agitated to promote
intimate mixing. With the mixer running, a solution of "Tetronic
707" (15.0 g.) and polyethylene glycol-6000 (3.0 g.) in distilled
water (40 ml.) was introduced. Additional distilled water (10 ml.)
was subsequently added to produce the desired degree of granulation
of wetness. The wet granulation was screened and dried at room
temperature, and the resultant dry granulation rescreened in the
manner described in Example 1 for hemoglobin reagent formulation A.
The dry, rescreened granulated glucose oxidase subformulation was
transferred to a tared 1500 cc amber bottle and stored in a dry
room pending subsequent intermixture with the other constituents of
formulation M, as described below. Each gram of this granulated
glucose oxidase subformulation contains sufficient GOD for the
conduct of 71 glucose tests. The yield of this formulation in terms
of potential test: units may therefore be calculated as
follows:
No. of tests = wt. of GOD subformulation (g.) .times. 71
tests/g.
A peroxidase trituration subformulation for formulation M was
prepared by blending, with mortar and pestle, an amount of
horseradish peroxidase (POD) and an amount of mannitol sufficient
for the number of tests calculated above and determined by the
following respective calculations: ##EQU2## To the trituration thus
provided, 25 g. of the glucose oxidase subformulation was added and
the resultant mixture agitated with a stainless steel spatula to
disperse the trituration.
After preparation of the GOD/POD dispersion 50 g. of the GOD
subformulation, the dispersion, and an additional 50 g. of the GOD
subformulation, were sequentially screened through a 40 mesh
stainless steel screen onto a clean receiving surface. All of the
screened material was then transferred to a PK blender, along with
the remainder of the GOD subformulation, and mixed for more than 5
minutes. The resultant blended granulation was transferred into 8
inch .times. 12 inch Pyrex trays of a depth of one-half to
three-fourth and dried for about 15 hours at room temperature and a
total pressure of 25 inches Hg.
Dissolved in two separate portions of water, formulation K, and
formulation L together with M, provide liquid reagent solutions
useful in the determination of true glucose in a biological
specimen such as blood serum. In the presence of water and the
glucose oxidase component of enzyme formulation M, glucose in the
specimen is oxidized to gluconic acid with hydrogen peroxide formed
as a by product. The by product, hydrogen peroxide, oxidizes the
o-dianisidine constituent of chromogenic reagent formulation K, in
the presence of horseradish peroxidase, producing a colored product
which causes an increase in optical density at 445 nm. The extent
of the increase indicates the concentration of glucose in the test
system.
A chromogenic liquid reagent is prepared by dissolving formulation
K (0.75 g.) in 50 ml. water. A liquid reagent having both enzyme
activity and buffering capacity is prepared by dissolving
formulation M (0.75 g.) and formulation L (1.0 g.) in another 50
ml. portion of distilled water. The chromogenic liquid reagent
should be prepared fresh daily. The liquid reagent containing
formulations L and M is stable for at least 1 week if refrigerated.
The resulting solutions are sufficient for conducting 50 tests.
In the conduct of the test, 1 ml. each of the solution of
formulation K and the solution containing formulations L and M are
added to a clean, dry test tube. 10 microliters of serum are added
to the tube and the resultant mixture is blended thoroughly and
incubated at 37.degree.C. for exactly 15 minutes. The optical
density of the test system is then read at 445 nm against a reagent
blank set at 100% transmission and the proportion of true glucose
in the specimen determined from a standard curve.
Glucose standard curves are linear up to 300 mg% using the
formulations and assay procedure of this example. Sera with high
glucose levels (>300 Mg%) should be diluted with saline (0.9%
NaCl) before analysis and the calculation corrected with the
dilution factor.
EXAMPLE 7
Inorganic Phosphorus Formulations and Assay
Two separate formulations are provided for the inorganic phosphorus
test. The two formulations which are utilized are set forth in
Table 1 as formulations N and O. Predetermined amounts of these
formulations are dissolved in separate portions of water to provide
liquid reagents for use in making the inorganic phosphorus
assay.
In the preparation of reducer quench formulation N, L-ascorbic acid
(3.25 kg.), milled sulfamic acid (800 g.) and dry "Duponol ME" (775
g.) were blended in a Hobart bowl and thoroughly agitated to
promote intimate mixing. With the mixer running, a solution of
"Tetronic 707" (125 g.) in methylene chloride (1000 ml.) was added.
Additional methylene chloride (500 ml.) was subsequently intruduced
to produce the desired degree of granulation and wetness. The wet
granulation was screened and dried and the resultant dry
granulation rescreened and packaged in the manner described in
Example 1 for hemoglobin reagent formulation A.
To prepare chromogenic reagent formulation O, the sodium molybdate
constituent of the formulation was dried in the manner described in
Example 5. Tris(hydroxymethyl)-aminomethane (1.50 kg.), "Duponol
ME" (470 g.) and dried sodium molybdate (950 g.) were then blended
in a Hobart bowl and thoroughly agitated to promote intimate
mixing. With the mixer running, a solution of "Tetronic 707" (80.0
g.) in methylene chloride (475 ml.) was added. Additional methylene
chloride (175 ml.) was then introduced to produce the desired
degree of granulation and wetness. The wet granulation was screened
and dried and the resultant dry granulation rescreened and packaged
in the manner described in Example 1 for hemoglobin reagent
formulation A.
Dissolved in separate portions of water, reagent formulations N and
O yield liquid reagents useful in assaying a body fluid for
inorganic phosphorus. Sodium molybdate from the solution of
chromogenic formulation O reacts with inorganic phosphorus from the
specimen to produce phosphomolybdic acid. Phosphomolybdic acid is
in turn reduced by the ascorbic acid component of the solution of
reducer quench formulation N to produce a phosphomolybdenum blue,
the color of which is indicative of the inorganic phosphorus
content of the specimen.
The liquid reagent solution of formulation N is prepared by
dissolving 1.5 g. of the formulation in 150 ml. of water. The
solution of formulation O is prepared by dissolving 4.9 g. of that
formulation in 50 ml. of water. The chromogenic liquid reagent
solution of formulation N is stable for 1 week at room temperature.
The liquid reagent solution of formulation O should be prepared
fresh daily and protected from light. The resulting solutions are
sufficient for conducting 50 tests.
In the conduct of the test, 3 ml. of the solution of formulation O
and 1 ml. of the solution of formulation N are added to a clean,
dry test tube and intermixed. 0.05 ml. (50 lambda) of serum is
added to the reagent mixture and thoroughly mixed therewith. The
resultant reagent/specimen test system is incubated at 37.degree.C.
for 20 minutes, after which the optical density of the system is
measured at 700 nm and compared with a reagent blank set at 100%
transmission. The inorganic phosphorus content of the specimen is
then determined from a standard curve.
The phosphorus standard curves are linear up to 15 mg%.
EXAMPLE 8
Lactate Dehydrogenase Reagent Formulations and Assay
For the lactate dehydrogenase-(lactate as substrate) (LDH-L), two
separate formulations are provided. The two formulations which are
utilized are set forth in Table 1 as formulations P and Q.
Predetermined amounts of these formulations are dissolved together
in a single portion of water to provide the liquid reagent for use
in making the LDH-L assay.
To prepare coenzyme formulation P, nicotinamide adenine
dinucleotide, modified as described below (equivalent to 225 mg.
nicotinamide adenine dinucleotide) and mannitol (sufficient to
provide 25.0 mg./test) were blended in a Hobart bowl and agitated
to promote intimate mixing. With the mixer running, a solution of
"Tetronic 707" (60.0 g.) in methylene chloride (500 ml.) was added
to the blend. Additional methylene chloride (approximately 250 ml.)
was subsequently introduced to produce the desired degree of
granulation and wetness. The wet granulation was then screened and
dried at room temperature, and the resultant dry granulation
rescreened and packaged in the manner described in Example 1 for
hemoglobin reagent formulation A.
To prepare modified nicotinamide adenine dinucleotide (NAD), NAD
free acid (10 g.) was dissolved in distilled water (approximately
150 ml.) and the resulting solution was titrated slowly with 10%
sodium hydroxide solution to pH 6.4 to 6.5 (approximately 6.5 ml.
of sodium hydroxide solution was required). Lactose (20.5 g.) was
then added together with a sufficient amount of water to make a
total volume of approximately 400 ml. The solution was stirred
until all solids were completely dissolved. The resulting solution
was transferred into freeze drying vessels and shell frozen by
rotating the vessels in a dry ice/alcohol bath at -60.degree.C. or
below. The frozen product was lyophilized for 20 to 24 hours at
-60.degree.C. to -70.degree.C. and an absolute pressure of 5 m.mu.
Hg. 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 30.8 g. of dry modified
NAD powder was obtained.
To prepare substrate formulation Q, lithium lactate (2.25 kg.),
tris-(hydroxymethyl)-aminomethane (225 g.), sodium bicarbonate (450
g.) and "Renex 35" (175 g.) were blended in a Hobart bowl and
agitated to promote intimate mixing. With the mixer running, a
solution of "Tetronic 707" (75 g.) and polyethylene glycol-6000 (25
g.) in methylene chloride (500 ml.) was added to the blend.
Additional methylene chloride (approximately 1175 ml.) was
subsequently introduced to produce the desired degree of
granulation and wetness. The wet granulation was then screened and
dried at room temperature, and the resultant dry granulation
rescreened and packaged in the manner described in Example 1 for
hemoglobin reagent formulation A.
Dissolved together in a single portion of water, formulations P and
Q provide a liquid reagent useful in assaying a biological specimen
for lactate dehydrogenase-L. Catalyzed by LDH-L from the specimen,
lactate from substrate formulation Q and NAD from coenzyme
formulation P are converted to pyruvate and NADH. The extent of
this reaction, which corresponds to the LDH enzymatic activity of
the specimen, is measured by observing an increase in optical
density at 340 nm.
To prepare a quantity of liquid reagent sufficient for 50 tests,
formulation P (1.25 g.) and formulation Q (3.20 g.) are added to
the distilled water (150 ml.) in a suitable container and the
container shaken gently until the granules are completely
dissolved. The liquid reagent solution thus prepared should be used
within 8 hours.
In the conduct of the test, 3 ml. of the liquid reagent solution is
dispensed into a clean absorption cell. Both the reagent in the
cell and the serum sample are separately preincubated in a heating
unit at 37.degree.C. for 7-10 minutes. After preincubation, 0.050
ml. (50 lambda) of a serum sample is transferred to the cell and
thoroughly mixed with the liquid reagent at 37.degree.C. to produce
a specimen/reagent test system. Exactly 1 minute after the serum
specimen is initially mixed with the reagent, the cell is removed
from the heating unit and the absorbance of the specimen/reagent
test system is read with a spectrophotometer which had previously
been set at zero absorbance with distilled water. Immediately after
this measurement is taken, the cell is returned to the heating unit
and held there until exactly 3 minutes after the serum was first
mixed with the reagent, i.e., 2 minutes after the first reading.
Then the cell is again removed from the heating unit and a final
absorbance reading taken. The LDH units present in the specimen are
determined by the following calculation: ##EQU3## where A.sub.1
equals the absorbance read at 1 minute, A.sub.2 equals the
absorbance read at 3 minutes and L equals the light path in
centimeters.
If the absorbancy difference (A.sub.2 - A.sub.1) exceeds 0.14, a
very high LDH activity in the serum is indicated. At this level of
activity, the relationship between the absorbancy difference and
the LDH units per ml. serum is not linear and the coefficient in
the above-noted equation may be inaccurate, yielding inaccurate
results. To provide greater accuracy in the measurement of high LDH
activities, the above-described test is repeated with a 20 .mu.1
serum sample and the activity calculated in accordance with the
following equation: ##EQU4## As those skilled in the art will
appreciate, the accuracy of this LDH test is also highly dependent
on the temperature control. Preferably, therefore, the
spectrophotometer which is used includes a temperature control
means for the cuvette compartment in which the sample cell is held
during measurement, and the temperature of the sample is controlled
at 37.degree.C. Where the instrument is not so-equipped with
temperature control, the absorption cell should not be removed from
the heating unit for more than 10 seconds when reading the
sample.
The LDH unit is defined as that amount of enzyme which catalyzes
the conversion of 0.001 micromoles per minute NAD to NADH under the
above-described test conditions. The Wacker (or Amador) procedure,
also known to the art, employs the same primary reaction but is
carried out at 25.degree.C. instead of 37.degree.C. By definition,
one Wacker (or Amador) unit is equivalent to 1.15 LDH units. Thus,
to convert LDH units to Wacker units, the LDH units are multiplied
by 0.87.
EXAMPLE 9
Serum Glutamic Pyruvic Transaminase Formulations and Assay
For the serum glutamic pyruvic transaminase test, two separate
formulations are provided. The two formulations which are utilized
are set forth in Table 1 as formulations R and S.
Preparatory to blending the constituents of coenzyme reagent
formulation R, the modified lactate dehydrogenase and reduced
nicotinamide adenine dinucleotide components are prepared. The
modified NADH component was prepared in accordance with the method
described in Example 4. Modified lactate dehydrogenase was prepared
as described below.
Gum arabic (15 g.), ammonium sulfate (10 g.),
tris-(hydroxymethyl)-aminomethane (12 g.), and bovine serum albumin
(0.1 g.) were dissolved in distilled water (450 ml.). The resulting
solution was titrated to a pH of 7.4 with 12N sulfuric acid and
then diluted to a total volume of 500 ml. This solution was
clarified by centrifugation at 10,000 rpm and a temperature of
10.degree.C. for 16 minutes, using a No. 872 angle rotor in an IEC
B-20 refrigerated centrifuge. To 300 ml. of the clarified solution
was added an LDH crystalline suspension in ammonium sulfate
solution (10 ml. containing 100 mg. LDH). The resulting enzyme
mixture was stirred for 5 minutes to insure good mixing and then
divided and each portion placed in one of several freeze-drying
vessels. Each portion was then frozen in thin layers by rotating
the vessel containing it in a dry ice/alcohol bath at -60.degree.C.
or below. The frozen thin layers were lyophilized at -60.degree. C.
to -70.degree.C. at a total pressure of 5 m.mu. Hg for a period of
18-20 hours. The lyophilized powder obtained was collected under an
atmosphere having a relative humidity of less than 5% and stored in
a dessicator at 4.degree.C. Approximately 26 g. of dry powder was
obtained. This powder was assayed for the enzymatic activites of
both glutamic pyruvic transaminase and LDH. The GPT specific
activity was found to be less than 0.04% in relation to the LDH
specific activity and the modified LDH lyophilized powder was
therefore suitable for use in preparing formulation R.
In the preparation of enzyme reagent formulation R, modified NADH
(having an equivalent NADH content of 0.4 mg.), modified LDH
(having an LDH equivalent of 2 units), DL-alanine (1.20 kg.),
dibasic sodium phosphate (585 g.), and milled monobasic sodium
phosphate (750 g.) were blended in a Hobart bowl and agitated to
promote intimate mixing. With the mixer running, a solution of
"Tetronic 707" (50.4 g.) in methylene dichloride (200 ml.) was
added to the blend. Additional methylene chloride (approximately 70
ml.) was subsequently introduced to produce the desired degree of
granulation. The wet granulation was then screened and dried at
room temperature and the resultant dry granulation rescreened and
packaged in the manner described in Example 1 for hemoglobin
reagent formulation A.
Substrate reagent formulation S has essentially the same
composition and was prepared in essentially the same manner as was
formulation G of Example 4.
Dissolved in separate portions of water, formulations R and S
provide liquid reagents useful in assaying for serum glutamic
pyruvic transaminase (SGPT). SGPT present in the spectrum catalyzes
the transamination of L-alanine and .alpha.-ketoglutaric acid
producing a pyruvate and glutamate. Pyruvate and NADH, in the
presence of LDH, are converted to lactate and NAD. The extent of
reaction is indicative of the SGPT present and is measured by
observing a change in optical density at 340 nm at 37.degree.C.
To prepare a coenzyme liquid reagent solution, formulation R (6.55
g.) is dissolved in distilled water (125 ml.). A substrate liquid
reagent is prepared by dissolving formulation S (1.00 g.) in
distilled water (25 ml.). The resulting solutions are sufficient
for 50 tests. The substrate liquid reagent is stable for 1 week
when refrigerated. The coenzyme reagent formulation, on the other
hand, should be prepared fresh daily.
In the conduct of the test, an aliquot of the solution of
formulation R (2.5 ml.) is mixed with a serum specimen (100.mu.1)
to form a reagent/specimen test system which is then preincubated
at 37.degree.C. for 7-10 minutes in a heating unit. After
preincubation, an aliquot of the solution of reagent formulation S
(0.5 ml.) is added to the test system and mixed in less than 10
seconds at room temperature. The container holding the system is
returned to the heating unit where it is held at 37.degree.C.
Exactly 2 minutes after the solution of formulation S is added, the
container is removed from the heating unit and the absorbance of
the test system measured using a spectrophotometer which has
previously been set at zero absorbance using distilled water as a
blank. Immediately after the measurement is taken, the container is
returned to the heating unit and held at 37.degree.C. Exactly 5
minutes after the solution of formulation S is added (i.e., three
minutes after the first reading), the container holding the test
system is again removed from the heating unit and the absorbance
again measured on the spectrophotometer. The SGPT content of the
system is then determined in accordance with the following
calculations: ##EQU5## where A.sub.1 equals absorbance read at 2
minutes, A.sub.2 equals absorbance read at 5 minutes and L equals
the light path of the absorption cell or the I.D. of the container
in centimeters.
An SGPT unit is defined as that amount of enzyme which catalyzes
the oxidation of 0.001 micromoles per minute of NADH to NAD at
37.degree.C. under the above test conditions. The Karmen procedure
known to the art carries out the above reactions at 25.degree.C.
instead of 37.degree.C. By definition, one Karmen unit is equal to
1.09 SGPT units, and SGPT units may therefore be converted to
Karmen units by multiplying the SGPT units by 0.917.
If the absorbancy difference (A.sub.1 - A.sub.2) observed in the
above determination exceeds 0.225 a very high SGPT activity in the
serum is indicated. At this level of activity, the relationship
between the absorbancy difference and the LDH units per milliliter
of serum is not linear and the coefficient in the above-noted
equation may be inaccurate, yielding inaccurate results. To provide
greater accuracy in the measurement of high SGPT activities, the
above-described test is repeated with a 20.mu.1 serum sample and
the activity calculated in accordance with the following equation:
##EQU6## It will be appreciated that the accuracy of the SGPT test
is also highly dependent on close temperature control. Preferably,
therefore, the cuvette compartment of the spectrophotometer used
includes means for temperature control and the temperature of the
specimen is controlled at 37.degree.C. Where the instrument is not
so-equipped with temperature control means, the absorption cell
should not be removed from the heating unit for more than 10
seconds when making an optical density determination on the test
system.
EXAMPLE 10
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
a 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 209 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
sytem 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: ##EQU7##
EXAMPLE 11
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 is 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 (150 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:
(0.8 - O.D. of unknown) .times.41.36
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.
* * * * *