WO2001085979A2

WO2001085979A2 - Stabilized composition

Info

Publication number: WO2001085979A2
Application number: PCT/US2001/014395
Authority: WO
Inventors: Robert Lynn Long; S. Hoy Eric
Original assignee: Immuno Concepts, N.A., Ltd.
Priority date: 2000-05-05
Filing date: 2001-05-04
Publication date: 2001-11-15
Also published as: WO2001085979A3; AU2001261182A1

Abstract

An indicator composition responsive to peroxidase activity is rendered extremely stable by the addition of a non-liquid organic peroxide as the H-acceptor. The composition is extremely stable both in its dry state as well as in aqueous solution. Activation is achieved simply by the addition of deionized water.

Description

STABILIZED COMPOSITION

BACKGROUND OF THE INVENTION

The present invention relates generally to the field of biochemical assays for detecting the presence of peroxidase or other peroxidatively active substances. More specifically, it relates to stabilized indicator compositions employed for use in wet chemical techniques employed for such analyses.

Peroxidase is an enzymatic hemoprotein which catalyzes the oxidation by hydroperoxides of a number of substrates. Of particular interest among the substrates are dyes possessing leuco forms which are colorless prior to oxidation. Under properly controlled conditions, the amount of oxidative coloration will be proportional to the amount of peroxidase activity . Alternatively, the hydrogen donor may be chosen from the class of indicators which emit light or exhibit otlier detectable characteristics in the oxidized state. Hydroperoxides typically used include hydrogen peroxide, methyl peroxide and ethyl peroxide.

The detection of peroxidase and peroxidase-like activity has utility in a variety of medically related fields. Assays based on peroxidase activity have provided a powerful tool in the immunobiological field, especially in the detection of certain proteins. In the area of cytochemistry, the detection of such activity can be used to identify and monitor certain cell types. Peroxidatively active substances are present in a restricted set of cell types including myeloid leukocytes and granulocyte, erythrocytes, and certain neurons and secretory cells. Mitotic cells also exhibit

peroxidase-like activity. Assays sensitive to peroxidase activity can be used for certain medical

determinations based on cytological condition.

In certain instances, the detection of peroxidase activity readily lends itself to the use of "dip-

and-read" reagent bearing strips. Such strips comprise a porous insoluble matrix strip first

impregnated with a suitable hydrogen donor and acceptor and then dried. When immersed in a

solution containing peroxidase, the indicator is oxidized, typically changing color. The "dip-and- read" teclinique has the advantage of being particularly easy to use but obviously, cannot be used

under all circumstances. Many situations require the use of a wet chemical approach.

The use of a wet chemical approach is necessary when a test involves the cell covering of a

microscope slide as for example in testing for the presence of certain types of protein. An antibody specific to a particular protein may be conjugated with the extracted peroxidase which in turn is

readily extractable and available in quantity from horseradish root. After allowing the antibody to

react with the protein antigen, the protein-antibody-peroxidase complex may be detected by

providing appropriate hydrogen donors and acceptors and monitoring the redox reaction

characterized by the conversion of leuco dye forms to colored products. Such methodology has been used in assays for a wide variety of ligands including proteins, peptides, carbohydrates and any other

immunologically active substance. In one such approach, antinuclear antibodies maybe detected in human serum through the use of the peroxidase redox reaction. HEp-2 cells, transfected HEp-2 cells as described in U.S. Patent No. 5,518,881, or other suitable cells, are fixed onto a slide, and then the cells are contacted with a serum which may contain antinuclear antibodies directed against cellular components, particularly, but not limited to, nuclear antigens. If present in the serum, the antibodies will react with antigens within the HEp-2 cells forming an immune complex. After washing off the excess serum, the immune complexes within the cells are then further reacted with a solution containing antihuman immunoglobulins conjugated with peroxidase enzyme. The antihuman immunoglobulin conjugate is specific for the immune complexes formed in the HEp-2 cells so that the peroxidase enzyme will be present on the slide after washing excess solution away only if antinuclear antibodies were present in the serum. Finally, the reacted cells fixed to the slide are contacted with a chromogenic indicator in a buffered aqueous solution which also contains hydroperoxide, so that the peroxidase-catalyzed reaction may occur. By this procedure, the HEp-2 cells will be colored only to the extent that antinuclear antibodies were present in the serum reacted with the HEp-2 cells. With relatively minor modifications to the preparation of the slide and attached cells, other antigen reactions, such as for nucleic acids, cellular cytoplasmic components, or proteins associated with infectious agents may be detected.

A continuing problem with the peroxidase methodology for the wet chemical approach is the gradual but spontaneous oxidation of the indicator. Such deterioration proceeds while the indicator is in its dry form and even more readily while in aqueous solution. Such deterioration also proceeds more rapidly at room temperature, necessitating the storage of the reagent under refrigeration. When one of the previously used hydroperoxides is added to the solution, the deterioration is so rapid that reagent solutions must be prepared fresh within a few hours prior to use, resulting in an excessive waste of valuable preparation time and costly reagents if all of each batch of reagent is not utilized.

Additionally, certain frequently used and desirable indicators, notably 4-chloro-l-naphthol and 3-amino-9-ethylcarbazole, are insoluble in water. Accordingly, theses types of compounds must first be dissolved in an organic solvent such as alcohol or dimethylsulfoxide to effect solubilization of the indicator before further mixing with the aqueous medium. These reagent solutions must also be prepared immediately prior to use as the indicator may precipitate out of solution upon standing.

The limitations are particularly apparent when tests such as the antinuclear antibody test described above are to be performed in a doctor's office, a field clinic, or by relatively unskilled personnel. Where the testing is to be performed at a location where the demand for testing is so low that only one or a few tests are performed each day, a major portion of the costly indicator reagent solution may be discarded because the spontaneous oxidation renders the solution unreliable after short storage periods. When relatively unskilled persons are involved in the testing, the need to dissolve the indicator in an organic liquid prior to mixing with an aqueous solution may be o erly complex, and may result in mixing errors which can invalidate the test procedure. Further, if the peroxide must be added at the time of use because the indicator solution is unstable in the presence of peroxide, further mixing errors may result.

The stability problem has previously been addressed in U. S. Patent No. 4,615,972 which is hereby incorporated by reference in its entirety. In such patent, the indicator is stabilized with water soluble polymers. More specifically, a chromogenic indicator such as 4-chloro-l-napthol and a polymer such as polyethylene glycol, both in their dry state, are intermixed and ground with a mortar and pestle, or more typically, with a ball mill. The resulting powder is stable for a period of at least several months in the dry state. To prepare an indicator solution for a peroxidase assay, the stabilized powder is added to an aqueous medium and stirred to dissolve. In order to activate the solution, dilute hydrogen peroxide (3% v/v) is added. In its aqueous state, with or without the peroxide, the solution is stable for at least several weeks.

The advance in stability achieved with the use of water soluble polymers was significant. Nonetheless, additional stability in both the dry, pre-activated state, as well as in the aqueous, activated form is desired. Moreover, eliminating the need by the end user to add the hydroperoxide component to the indicator system prior to use would be most beneficial, and would reduce the possibility of accidental omission of the hydroperoxide during reagent preparation. Ambient temperature storage of the reagent in both the dry, pre-activated state, as well as in the aqueous, activated form would provide further benefit. SUMMARY OF THE INVENTION

The present invention provides an improved indicator composition for use in wet method

biochemical assays for the detection of peroxidase activity. The indicator composition, either while

in its dry powdered form or in its activated aqueous state, is stable for longer periods of time than

previously known compositions. The waste of costly reagents or personnel time is thereby avoided since the frequent disposal of excess solution and the preparation of new batches is obviated. A

further aspect of the invention is that the dry powdered indicator composition is readily activated by

dissolution in deionized water without further reagent addition. The storage, handling and appropriate dilution of solutions that were employed to activate previously known indicator systems

is thereby obviated. The reagent in both the dry, pre-activated state, as well as in the aqueous, activated form remains stable and active over a wide temperature range, thereby obviating the need

for refrigerated storage. Finally, the reactivity of the indicator composition is substantially greater

than previously known indicator compositions that rely on hydrogen peroxide for activation. This

increased sensitivity is indicated by substrate inhibition if the rather narrow optimal concentration range is exceeded.

In accordance with the invention, a stabilized indicator composition such as is described in

U. S. Patent No. 4,615,972 is combined with an organic hydroperoxide that exists in a dry state and physiologic buffering while all components are in their dry state. The resulting stability is most unexpected in view of the well-known instability of many hydroperoxides that only exist in liquid forms and which often exist only as short-lived intermediate products in organic chemical reactions. The resulting stability of the combination is further unexpected in view of the dry organic peroxide ' s instability while in its uncombined form. For example, urea peroxide is air, light, and moisture sensitive and unstable at room temperature. Such compound must normally be stored under argon at 2-8 °C. However, once the stabilized indicator compound and organic peroxide are intermixed, the dry powder may be maintained in the laboratory at ambient temperature over long periods of time.

Preparation of the indicator solution is achieved by simply the addition of deionized water which again achieves a most unexpected result in view of the organic hydroperoxide ' s instability and moisture sensitivity. In solution, the indicator composition is stable for an indefinite period of time.

Moreover, the gains in stability and reactivity may be achieved with the use of an extremely environmentally friendly substance. Solutions of urea peroxide that are three orders of magnitude more concentrated are approved for use as an oral solution for the brightening of teeth.

These and other features and advantages of the present invention will become apparent from the following detailed description of a preferred embodiments which, illustrate by way of example the principles of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The indicator composition of the present invention is used to detect the presence of peroxidase or peroxidase-like substances. The composition comprises a stable dry powder and is readily converted to its activated aqueous state by simply the addition of deionized water. The composition is highly stable both in its dry state as well as in solution. Additionally, the composition offers much greater sensitivity than was realized using heretofore hydrogen peroxide based indicator systems.

A variety of indicators well known in the art are oxidized to colored forms in the presence of peroxide, and peroxidase or a peroxidase-like substance. Among the compounds that are frequently used are benzidine, dimethylbenzedine, o-phenylenediamine, p-phenylenediamine, 4- chloro-1-napthol and3-amino-ethylcarbazole. All are susceptible to spontaneous oxidation and must therefore be stabilized in order to extend shelflife both in their dry form as well as in their aqueous state.

To prepare the stabilized indicator composition, the selected indicator is combined with a water soluble polymer such as polyethylene glycol, polyethyleneoxide or polyvinylpyrrolidone and derivatives thereof, by mixing and grinding. To prepare the stabilized indicator powder, the two compounds are first mixed with a paddle and then ground together, either with a mortar and pestle or a ball mill for thirty minutes while cold (4° -8° C). The amount of the indicator combined with the stabilizer is not critical, although a ratio of about 1 part of indicator to about 250 parts of stabilizer by weight, has been found effective. Acceptable results have been obtained with stabilizers having a range of molecular weight between 8,000 and 100,000 Daltons, but it is believed that higher and lower molecular weights will also produce acceptable results. Polyethylene glycol has been

• found to be particularly effective as a stabilizer.

In the event the solid indicator and the stabilizer do not mix uniformly, particularly when large quantities are mixed together, an even distribution can be obtained by first mixing the indicator with a salt such as sodium chloride, and then combining the mixture with the stabilizer as described previously. Sodium chloride is preferred, in a weight ratio of about 12 parts salt to 1 part indicator, since saline solutions are widely utilized in assay procedures. The procedure according to this approach is to mix the indicator and salt and then to grind the mixture in a mortar and pestle for about 10 minutes or a ball mill for about 30 minutes. The solid product is then mixed with the stabilizer and ground together as described previously. The final stabilized indicator powder may be used in the same manner as a powder wherein no salt is used.

After the dry stabilizer and indicator compound have been combined, the organic hydroperoxide, also while in its dry state, is added. Only hydroperoxides that exist in a dry state have been found to exhibit the unexpected stability. Again, using a mortar and pestle, or preferably a ball mill, the dry hydroperoxide is intermixed at low temperature (4-8°C). Optimal stability performance has been realized by combining the two reagents in a ratio of about 1 :600 by weight of urea hydroperoxide (carbamide hydroxyperoxide) to powder. A working concentration from 0.05 to 0.25 mM final concentration of urea hydroperoxide was found to be most effective.

The use of other non-liquid organic hydroperoxides besides urea hydroperoxide offer similar advantages in terms of stability and sensitivity. For example, benzoyl hydroperoxide (activated) or sodium percarbonate can readily be substituted for the urea hydroperoxide and suggest that any non- liquid organperoxide would work equally well.

The following examples, while not to be taken as limiting the invention, will illustrate aspects of the invention:

EXAMPLE 1

Two hundred milligrams of 4-chloro-l-naphthol were added to 50 grams of polyethylene glycol (8000 Daltons) and the two compounds mixed by spatula, then transferred to a ball mill jar and ground for at least 30 minutes. Longer grinding times have not been found to be detrimental. To the ball mill jar 100 milligrams of urea hydroperoxide were added and the milling process was continued for greater than 30 minutes to produce a ready-to-use stabilized indicator powder.

EXAMPLE 2 Sixty-six milligrams of 3-amino-9-ethylcarbazole were added to 10 grams of polyethylene glycol (8000 Daltons). The two compounds were mixed by spatula, then transferred to a ball mill jar and ground for at least 30 minutes. Longer grinding times have not been found to be detrimental.

To the ball mill jar 20 milligrams of urea hydroperoxide were added and the milling process was continued for greater than 30 minutes to produce a ready-to-use stabilized indicator powder.

EXAMPLE 3

Fifty milligrams of 4-chloro-l-naphthol were added to 10 grams of polyethylene glycol

(20,000 Daltons). The two compounds were mixed by spatula, then transferred to a ball mill jar and ground for at least 30 minutes. Longer grinding times have not been found to be detrimental. To the ball mill jar 20 milligrams of urea hydroperoxide were added and the milling process was continued for greater than 30 minutes to produce a ready-to-use stabilized indicator powder.

EXAMPLE 4

Fifty milligrams of 4-clιloro-l-naphthol was added to 10 grams of polyvinylpyrrolidone

(40,000 Daltons). These two compounds were mixed by spatula, then transferred to a ball mill and ground for at least 30 minutes. Longer grinding times have not been found to be detrimental. To the ball mill jar 20 milligrams of urea hydroperoxide were added and the milling process was continued for greater than 30 minutes to produce a ready-to-use stabilized indicator powder. EXAMPLE 5

Fifty milligrams of 4-chloro-l-naphthol were added to 10 grams of polyethyleneoxide

(100,000 Daltons). The two compounds were mixed by spatula, then transferred to a ball mill jar and ground for at least 30 minutes. Longer grinding times have not been found to be detrimental. To the ball mill jar 20 milligrams of urea hydroperoxide were added and the milling process was continued for greater than 30 minutes to produce a ready-to-use stabilized indicator powder.

EXAMPLE 6

Sixty-six milligrams of 3,3',5,5'-tetramethyl-benzidine were added to 10 grams of polyethylene glycol (8000 Daltons). The two compounds were mixed by spatula, transferred to a ball mill jar and ground for at least 30 minutes. Longer grinding times have not been found to be detrimental. To the ball mill jar 20 milligrams of urea hydroperoxide were added and the milling process was continued for greater than 30 minutes to produce a ready-to-use stabilized indicator powder.

EXAMPLE 7

The stabilized indicator powders from EXAMPLES 1-6 were placed in clear plastic sealed ners and subjected to accelerated aging for fifteen days at 43 °C, equivalent to three years at refrigerated temperature. No evidence of oxidation or deterioration of the stabilized indicator powders was found.

EXAMPLE 8

The aged powders form EXAMPLE 7 were each dissolved in a ratio of 3 grams per 100 ml of phosphate buffered saline and stored for an additional week in a clear plastic container at 43 °C,

equivalent to one year at refrigerated temperature. No spontaneous oxidation as evidenced by color

change was observed before, during or after 43 °C storage. After storage, the effectiveness of the

solutions was confirmed using the technique described in EXAMPLE 12. The solutions were found to give appropriate results in the peroxidase assay procedure.

EXAMPLE 9

The stabilized indicator powder formulae in EXAMPLES 1-6 were modified to contain

monobasic and dibasic phosphate salts to maintain a pH of 7.4. Sodium chloride was added to produce a physiologic final solution. The powders were ground as described previously.

EXAMPLE 10

The buffered stabilized indicator powders from EXAMPLE 9 were placed in clear plastic sealed containers and subjected to accelerated aging for fifteen days at 43 °C, equivalent to three years at refrigerated temperature. There was no evidence of oxidation or deterioration of the stabilized indicator powders.

EXAMPLE 11

The aged powders from EXAMPLE 10 were each dissolved in a ratio of 3 grams per 100 ml of deionized water and stored for an additional week in a clear plastic container at 43 ° C, equivalent to one year at refrigerated temperature. No spontaneous oxidation as evidenced by color change was observed before, during or after 43 °C storage. After storage, the effectiveness of the solutions was confirmed using the technique described in EXAMPLE 12. The solutions were found to give appropriate results in the peroxidase assay procedure.

EXAMPLE 12

Detection of peroxidase activity for the various solutions was accomplished utilizing a commercially available HEp-2 coated slide (Immuno Concepts, Sacramento California). To these slides a serum containing antinuclear autoantibodies was incubated with the HEp-2 cells. Using a uniform timing for all steps, the serum was washed from the slide using phosphate buffered saline. A drop of IgG-peroxidase conjugate was added to each HEp-2 cell region and incubated for 30 minutes. The unreacted excess conjugate was thoroughly washed form the slide using phosphate buffered saline. The slide was immersed in the appropriate solution under evaluation for 30 minutes and the excess staining solution rinsed away using phosphate buffered saline and placed immediately in a deionized water solution. Each slide was removed from the water and excess water eliminated by tapping the slide on an absorbent paper. The slides were mounted with a glass coverslip using mounting solution. The slides were evaluated using a light microscope (400X) for the presence and intensity of color staining of the HEp-2 cells. Slides were evaluated by multiple experienced technologists.

While a particular form of the invention has been illustrated and described, it will also be apparent to those skilled in the art that various modification s can be made without departing from the spirit and scope of the invention. Accordingly, it is not intended that the invention be limited except by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A stabilized indicator composition for detecting the presence of peroxidase, comprising: stabilized indicator; and a non-liquid organic hydroperoxide.

2. The stabilized indicator composition of claim 1, wherein said stabilized indicator comprises a mixture of an indicator and water soluble polymer.

3. The stabilized indicator of claim 2, wherein the said indicator comprises 4-chloro-l- napthol.

4. The stabilized indicator composition of claim 2, wherein said soluble polymer comprises polyethylene glycol.

5. The stabilized indicator composition of claim 1, wherein said non-liquid organic hydroperoxide composition comprises urea hydroperoxide.

6. The stabilized indicator composition of claim 1, wherein said non-liquid organic hydroperoxide composition comprises benzoyl hydroperoxide (activated).

7. The stabilized indicator composition of claim 1, wherein said non-liquid organic hydroperoxide comprises sodium percarbonate.

8. The stabilized indicator composition of claim 1, wherein said stabilized indicator comprises 4-chloro-l-napthol combined with polyethylene glycol and said organic peroxide comprises urea hydroperoxide wherein said urea hydroperoxide and said stabilized indicator are combined in a ratio range of from 1 :400 to 1 :800.

9. The stabilized indicator composition of claim 8 , wherein said urea hydroperoxide and said stabilized indicator are combined in a ratio of about 1:600.

10. A method for preparing a stabilized indicator composition for use in wet chemical assay procedure to detect peroxidase, comprising the steps of: providing an indicator in its dry state; providing a stabilizer in its dry state; intermixing said indicator and said stabilizer to provide a stabilized indicator; providing a dry organic hydroperoxide while in its dry state; and intermixing said organic hydroperoxide with said stabilized indicator.

11. The method of claim 10, further comprising the step of adding deionized water.

12. The method of claim 10, wherein said indicator comprises 4-chloro- 1 -napthol.

13. The method of claim 10, wherein said stabilizer comprises polyethylene glycol.

14. The method of claim 10, wherein said organic hydroperoxide comprises urea hydroperoxide.

15. The method of claim 10, wherein said organic hydroperoxide comprises benzoyl hydroperoxide (activated).

16. The method of claim 10, wherein said organic hydroperoxide comprises sodium

percarbonate.

17. A test kit for detecting the presence of a component, comprising: a test slide having attached thereto a first binding partner specific to the component; a liquid solution of a second binding partner specific to the component, said second binding partner being conjugated with a substance exhibiting peroxidase activity; and a quantity of a stabilized liquid indicator which is capable of being oxidized in the presence of peroxidase, said indicator being prepared by a process including combining a peroxidase indicator, a stabilizer substance, and an organic peroxide that exists in a dry state.

18. The test kit of claim 17, wherein the first specific binding partner is HEp-2 cells.

19. The test kit of claim 17, wherein the first specific binding partner is transfected HEp-2 cells.

20. The test kit of claim 17, wherein the second binding partner is antihuman IgG, and the substance exhibiting peroxidase activity is peroxidase.