Title:  Stabilizing diluent for polypeptides and antigens

United States Patent:  6,579,688

Issued:  June 17, 2003

Inventors:  Steaffens; Jeffrey W. (Lafayette, CO); Panzarella; Laura (Longmont, CO)

Assignee:  Biostar, Inc. (Boulder, CO)

Appl. No.:  737635

Filed:  December 14, 2000

Compositions for stabilizing polypeptides or antigens are described. These compositions are useful for stabilizing polypeptides or antigens stored in aqueous formulations. Such formulations can be used for various analytical or other methods.

SUMMARY OF INVENTION

The present invention features a reagent for stabilizing polypeptides and antigens. The reagent is especially useful for the stabilization of control or reference antigens used in analytical procedures. Antigens such as carbohydrates, proteins, polypeptides, polypeptide fragments, lipoproteins, lipopolysaccharides, polysaccharides, nucleic acids, nucleoproteins, and carbohydrates complexed with polypeptides, lipids, and other compounds are illustrative examples of the types of antigens that may be stabilized using the current invention.

The disclosed reagent surpasses previous formulations for polypeptide and antigen stability at both low and elevated temperatures. Polypeptides and antigens can be stored in the reagent in soluble form for extended periods of time at a variety of temperatures from about 0.5^oC. to more than about 50^oC., preferably from about 2-8^oC., about room temperature (typically from about 23^oC. to about 28^oC., with 25^oC. being particularly preferred) and about 42^oC. to about 43^oC., especially about 45^oC. Stability at 45^oC. is predictive of the reagent's ability to provide long-term antigen stability. Additionally, the disclosed reagent has the advantage of being a single aqueous solution for the purpose of stabilizing polypeptides and antigens.

In a first aspect, the invention features an aqueous reagent composition to enhance polypeptide or antigen stability. In certain preferred embodiments, the reagent comprises one or more of the following: buffer(s), blocking agent(s), solvent(s), salt(s), chelator(s), detergent(s), and preservative(s). Preferably, the reagent does not include N-dodecanoyl-N-methylglycine or decanoyl-N-methylgluconamide. The reagent may also comprise components such as tissue culture medium or commercially available diluents.

The term "buffer" as used herein refers to compositions well known to the skilled artisan that act to minimize the change in pH of a solution. Preferred buffers have a pKa that provides effective buffering at a pH of between 7 and 9. Preferred buffers are ACES, ADA, BES, bicine, bis-tris, CAPS, CHES, diethylmalonate, glycylglycine, glycinamide HCl, HEPES, HEPPS, imidazole, MES, MOPS, PIPES, POPSO, TAPSO, TES, tricine, tris, bicarbonate, and borate. Particularly preferred are phosphate buffers. Preferred buffering agent concentrations are less than 2 M; most preferred concentrations are 0.2 M, 0.1 M, 0.05 M, 0.05 M, 0.02 M, 0.01 M, 0.005, 0.001, and 0.0001 M, having a pH of 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, and 9.

As used herein, the term "blocking agent" refers to a protein rich source containing a mixture of proteins and/or polypeptides, and that may include one or more additional components such as lipids, carbohydrates, salts, and cofactors such as heme. The term "protein rich" is defined herein. Such blocking agents can be used to stabilize one or more proteins, polypeptides, and/or antigens in the compositions and methods described herein. Preferred blocking agents have a pH of between about 6.5 and about 8.0, and/or an osmolality of between about 250 and about 350 mOsm/Kg H₂ O. Particularly preferred blocking agents are sera, such as horse serum, newborn calf serum, calf serum, adult bovine serum, human serum, rabbit serum, sheep serum, etc., and serum replacements known to the skilled artisan. Most preferred as a blocking agent is fetal calf serum.

The term "protein rich" as used herein refers to a solution containing a mixture of proteins and/or polypeptides, and having a total protein concentration between about 1 g % and about 50 g %, most preferably between about 3 g % and about 10 g %. For example, fetal calf serum has a total protein content of between about 3 g % and about 4.5 g %, while adult bovine serum has a total protein content of between about 4.5 g % and about 8.5 g %.

The terms "solvent" and "solubilizing agent" as used herein refer to a liquid substance capable of dispersing the other components of the composition. Preferred solvents are water, glycerol, DMSO, alcohols such as ethanol, methanol, etc., acetone, dimethyl sulfoxide, acetonitrile, and dimethyl formamide.

The term "salt" as used herein refers to one or more compounds that result from replacement of part or all of the acidic hydrogen of an acid by a metal, or an element acting like a metal. Preferred salts are KCl, NaCl, MgCl₂, MgSO₄, and CaCl₂. Preferred salt concentrations are between 4M and 0.1 mM, most preferably between 2M and 50 mM.

The term "chelator" as used herein refers to a molecule that binds metal ions, usually by binding to two or more complexing groups within the molecule. Chelators are well known in the art, and include certain proteins and polypeptides, as well as small molecules such as ethylenediaminetetraacetic acid (EDTA) and ethylene glycol-bis(.beta.-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Preferred chelator concentrations are between 100 mM and 0.01 mM, most preferably between 20 mM and 1 mM.

The term "detergent" as used herein refers to compounds well known in the art that are able to emulsify oils and act as wetting agents. Preferred detergents include CHAPS, cholic acid, deoxycholic acid, digitonin, n-dodecyl-.beta.-D-maltoside, glycodeoxycholic acid, n-lauroylsarcosine, lauryl sulfate, saponin, TWEEN.RTM. 20 (Polyoxyethylene Sorbitan Monolaurate), and TWEEN.RTM. 20 (Polyoxyethylene Sorbitan Monolaurate). Preferred detergent concentrations are between of about 0.001% to about 5%. Most preferably, the composition comprises about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1.0%, and 2%.

The term "preservative" as used herein refers to compounds well known to the skilled artisan that prevent the growth of microorganisms. Preferred preservatives include thimerosal, sorbic acid, BHA, BHT, MICROCIDE II.RTM. (trimethyltetradecylammonium bromide) and antibiotics such as gentamycin, penicillin, streptomycin, etc. Preferred preservative concentrations are between 10% and 0.001 mM, most preferably between 1% and 0.1%.

As used herein "polypeptide" refers to a polymer of amino acids and does not refer to a specific length of the product, thus, peptides, oligopeptides, proteins, and fragments thereof are included within the definition. The term "polypeptide" does not exclude, post-translational modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, and the like.

As used herein "antigens" include carbohydrates, proteins, polypeptides, lipoproteins, lipopolysaccharides, polysaccharides, nucleic acids, and carbohydrates complexed with polypeptides, lipids, and other compounds. Antigens may be capable of eliciting an immune response in an animal having a functional immune system. In a preferred embodiment, the antigens stabilized are polypeptide antigens. These polypeptide antigens may be found alone or in association with other molecules. In a particularly preferred embodiment, the polypeptide stabilized are nucleoprotein antigens. Nucleoprotein antigens may be from any number of sources and may be found alone or in association with other molecules.

By "nucleoprotein antigens" is meant any polypeptide found associated with or attached to a nuclear complex.

A particularly preferred nucleoprotein is a nucleoprotein from influenza virus, particularly influenza type B.

By "antigen stability" is meant the ability to maintain a consistent signal generated by one reagent, in an assay or analytical method, especially a diagnostic assay or an immunoassay, after reagent storage for a given period of time at a set temperature. Typical storage temperatures are from about 2^o-30^oC. Antigen stability can also be assessed by measuring antigen deterioration at elevated temperatures for a shorter period of time. Typical temperatures for accelerated stability tests are from about 30^o to 60^oC.

By "microorganisms" is meant a prokaryote, eukaryote such as yeast, virus, prion or other infectious particle.

By "analytical method" is meant any technique that allows specific detection of one or more antigens. Analytical methods include immunoassays of any detection format and nucleic acid hybridization, numerous examples of which are known in the art. A particularly preferred optical immunoassay method is described in U.S. Pat. Nos. 5,550,063; 5,955,377; and 5,541,057.

In a second aspect, the invention features an aqueous reagent composition to enhance antigen or polypeptide stability wherein the antigen is from a microorganism. In a preferred embodiment, the microorganisms comprise viruses and/or bacteria. The invention is particularly preferred for use with viral analytes. In a most particularly preferred embodiment, the invention features a reagent composition for stabilizing antigens and polypeptides from influenza, especially polypeptides and antigens from influenza B.

In a third aspect, the invention features a reagent composition for stabilizing a nucleoprotein of influenza virus, particularly nucleoprotein from influenza B.

In a fourth aspect, the invention features an aqueous reagent composition for stabilizing an antigen preparation to be used as a control or reference reagent associated with an analytical method.

In a particularly preferred embodiment, the reagent comprises a buffer, a blocking agent, a salt, a chelator, a solubilizing agent, a non-ionic detergent, and a preservative. Most preferably, the reagent does not contain N-dodecanoyl-N-methylglycine or decanoyl N-methylgluconamide.

In further preferred embodiments, the reagent may also contain tissue culture media, STABILCOAT.RTM. buffer (an aqueous solution containing purified bovine protein and other non-toxic chemicals in phosphate buffered saline, pH 7.0-7.4; BSI, Inc.), and formalin-inactivated virus-containing cell culture media.

In yet other preferred embodiments, the reagent comprises sodium phosphate buffer, fetal calf serum, glycerol, sodium chloride, EDTA, TWEEN.RTM. 20 detergent (polyoxyethylene sorbitan monolaurate), MICROCIDE II.RTM. (trimethyltetradecylammonium bromide). Gentamycin Sulfate, and may contain sucrose, tissue culture media and STABILCOAT.RTM. buffer (an aqueous solution containing purified bivine protein and other non-toxic chemicals in phosphate buffered saline, pH 7.0-7.4; BSI, Inc.). The pH of the solution is between about 7 and about 9, most preferably between 7.5 and 8.5. One skilled in the art understands that similar reagents can substitute for those listed above. For example, EDTA can be replaced with EGTA, and MICROCIDE II.RTM. (trimethyltetradecylammonium bromide), or gentamycin can be replaced by other anti-bacterial agents. Preferred concentrations of the above listed reagents are as follows: for sodium phosphate, 0.1 mM to 1000 mM, more preferably 1 mM to 200 mM, most preferably 50 mM to 100 mM; for fetal calf serum, 0.1% to 40% v/v, most preferably 2% to 20% v/v; for glycerol, 0.1% to 30% v/v, most preferably 2.5% to 10% v/v; for sodium chloride, 0.1 mM to 4 M, most preferably 50 mM to 2 M; for EDTA, 0.01 mM to 100 mM, more preferably 1 mM to 20 mM, most preferably 10 mM to 15 mM; for TWEEN.RTM. 20 (Polyoxyethylene Sorbitan Monolaurate), 0.001% to 1%, more preferably 0.01% to 0.1%, most preferably 0.5%; for MICROCIDE II.RTM. (trimethyltetradecylammonium bromide), 0.001% to 1% w/v, most preferably 0.002% to 0.1% w/v; for gentamycin sulfate, 0.01% to 10% w/v; more preferably 0.1% to 2.5%, most preferably 0.25% to 1%; and for sucrose, 0.01% to 5% w/v, most preferably 0.1% to 0.5%.

In especially preferred embodiments, the diluent comprises an amount greater than or equal to about: 50 mM sodium phosphate; 2% v/v fetal calf serum; 10% v/v glycerol; 50 mM sodium chloride; 10 mM EDTA; 0.05% v/v TWEEN.RTM. 20 (Polyoxyethylene Sorbitan Monolaurate) detergent; 0.01% w/v MICROCIDE II.RTM. (trimethyltetradecylammonium bromide) preservative; and 0.5% w/v gentamycin sulfate. The reagent may also comprise up to 0.5% sucrose, 15% STABILCOAT.RTM. buffer (an aqueous solution containing purified bovine protein and other non-toxic chemicals in phosphate buffered saline, pH 7.0-7.4; BSI, Inc.) and 20% tissue culture medium from the antigen preparation or by separate addition. The preferred pH of the solution is between about 7.5 to about 8.5. It may also be possible to replace the buffer and/or salt with a concentration of pre-formulated tissue culture medium (for example, Eagle's Minimum Essential Media or Dulbecco's Modified Eagle Media from BioWhittaker).

In a fifth aspect, the invention features methods for stabilizing polypeptides and antigens derived from microorganisms. In a preferred embodiment said method is used for stabilizing an antigen preparation from a microorganism for use as a control or reference reagent associated with an analytical method or for use in a pharmaceutical preparation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A. Introduction

For ease in understanding the current invention, the development of the reagent composition is described for a specific application, namely stabilizing influenza-derived antigens. However, the general utility of the stabilizing reagent composition can be demonstrated for other antigens or polypeptides by conducting similar experiments.

During the development of reagents for diagnostic assay components or other uses, a determination of a reagent's long-term stability is required. Initially, accelerated stability validations are completed for assessment of a reagent's shelf-life prior to the completion of real-time stability determination. Accelerated stability studies can be performed quickly and are often used by those skilled in the art to predict stability. One means of doing this is to incubate the reagent at elevated temperatures and assay at relatively short time intervals. For example, incubations for 3 to 7 days at 37^oC., 45^oC., and other elevated temperatures are analyzed using the Arrhenius equation to predict whether a reagent will remain stable at about 2^o-8^oC. for long periods of time. This method is currently believed to be most accurate at predicting reagent failure. If a reagent is unable to withstand an elevated temperature challenge, it is not likely to be stable for an extended time under normal storage conditions. Elevated temperature stability is less predictive of long-term stability, as it does not accurately predict rate of decay, but it does give an indication of stability by identifying a probable point of reagent failure. As the loss of stability at elevated temperatures decreases, so does the likelihood of real-world failure.

In addition to accelerated validation, real-time, long-term studies are also necessary to assess a reagent's potential to stabilize polypeptides or antigens. Conditions for real-time studies are known to those skilled in the art. For example, real-time assessment conditions may include storing the reagent at normal storage temperatures of about 2^o-8^oC. or at room temperature (18^o-30^oC.) until the point of reagent failure is reached.

In the development of positive control reagents for an immunoassay for influenza viruses both formalin-inactivated influenza A and B type viruses were assessed for their stability in a protein-based diluent. Where the epitope detected in the assay is from the viral nucleoprotein, inactivated influenza A was significantly more stable in simple diluents and commercially available stabilizing diluents than was inactivated influenza B. To insure commercial viability of a new product for influenza A and B detection, a means of stabilizing inactivated influenza B was required. A diluent was formulated which imparts stability on both types of inactivated influenza viral suspensions. This diluent has demonstrated the ability to improve the stability of inactivated influenza B virus, in solution, for longer periods of time than previous diluent formulations.

Influenza A has been shown to be stable in simple protein based diluents. One such diluent contains bovine serum albumin (BSA) and TWEEN.RTM. 20 (Polyoxyethylene Sorbitan Monolaurate) detergent in phosphate buffered saline (PBS) otherwise called PBT. A second formulation is a commercial product, STABILCOAT.RTM. buffer (an aqueous solution containing purified bovine protein and other non-toxic chemicals in phosphate buffered saline, pH 7.0-7.4; BSI, Inc.), and has also been demonstrated to stabilize influenza A. However, neither of these solutions was able to stabilize influenza B. It is likely that the monoclonal antibodies that detect the different strains in the assays performed recognize different epitopes on the two nucleoproteins. These epitopes may be affected differently by inactivation and storage conditions. Additionally, the immunogens used in the initial preparations for the monoclonal antibodies used in an assay may have been prepared differently and thus, may have favored the production of monoclonals that recognize differently conformed epitopes. We believe that the diluent compositions described herein have similar stabilizing effects on inactivated influenza A nucleoprotein as it does on inactivated influenza B nucleoprotein. In addition, other viral and bacterial proteins are thought to be stabilized as well.

It should be noted that inherent in the process of viral inactivation and dilution into each of the diluent formats is the incorporation of the components of the inactivated viral stock suspensions. The inactivated viral stock suspension is composed of 50% formalin-inactivated virus-containing cell culture media (ICC) and 50% STABILCOAT.RTM. buffer (an aqueous solution containing purified bovine protein and other non-toxic chemicals in phosphate buffered saline, pH 7.0-7.4; BSI, Inc.). Each of the positive control compositions tested comprise some volume of this stock suspension.

B. Formation of New Diluent

The formulation of a new diluent began with the simple protein diluent, PBT, comprising BSA, TWEEN.RTM. 20 (Polyoxyethylene Sorbitan Monolaurate), and PBS solution. Each of the components was evaluated for their ability to stabilize inactivated influenza B virus while maintaining or enhancing the stability of inactivated influenza A virus. An increase in stability of the antigen preparation was assessed in the following manner. Inactivated viral materials were diluted to a desired strength of signal intensity for the analytical testing method with which the control reagent will be marketed. Generally, a low to medium signal strength is selected for the control reagent on its corresponding analytical method. The various reagent compositions at the selected antigen dilution are tested immediately in the appropriate analytical method. The various reagent compositions (containing antigen) are placed at various storage conditions and then periodically re-tested in the analytical method. Reagent compositions that retain the original signal or have only minimal degradation of the original signal strength over the range of storage conditions are determined to provide enhanced stability. Signal strength may be assessed qualitatively, quantitatively, visually, or by instrumentation. During the component selection process, several modifications were made to the PBT diluent. First, a different blocking agent, such as fetal calf serum was substituted for BSA. Next, a solubilizing agent such as glycerol and a chelator, such as EDTA or EGTA, were added to enhance stability. Additionally, it was determined that the inclusion of a non-ionic detergent, such as TWEEN.RTM. 20 (Polyoxyethylene Sorbitan Monolaurate), detergent was essential for stability. Preservatives such as a combination of MICROCIDE II.RTM. (trimethyltetradecylammonium bromide) and Gentamycin sulfate were included as antimicrobial agents.

In a particularly preferred embodiment, the inventive diluent comprises an amount greater than or equal to: 50 mM sodium phosphate, 2% v/v fetal calf serum, 1% v/v glycerol, 50 mM sodium chloride, 5 mM EDTA, 0.05% v/v TWEEN.RTM. 20 (Polyoxyethylene Sorbitan Monolaurate) detergent, 0.01% w/v quaternary ammonium compound, and 0.5% w/v gentamycin sulfate. The reagent may also comprise up to 15% STABILCOAT.RTM. buffer (an aqueous solution containing purified bovine protein and other non-toxic chemicals in phosphate buffered saline, pH 7.0-7.4; BSI, Inc.) 20% tissue culture medium from the antigen preparation or by separate addition. The preferred pH of the solution is between about 7.5 to about 8.5. It may also be possible to replace the buffer and/or salt with a concentration of pre-formulated tissue culture medium (for example, Eagle's Minimum Essential Media or Dulbecco's Modified Eagle Media from BioWhittaker).

Not wishing to be bound by any particular theory, the modifications to the original diluent may enhance polypeptide or antigenic stability by the following mechanisms. Fetal calf serum may provide a richer protein source than BSA, as it is a total serum product instead of purified protein. It may comprise other stabilizing materials. Glycerol may increase hydration of the antigen via hydrogen bonding. The chelator may increase stability by removing cations, which may negatively impact the polypeptide or antigen itself, or which may be necessary for the activity of certain degradative enzymes that could damage the antigen or polypeptide or may inhibit catalytic sites on the antigen. The detergent may maintain important secondary structures and supportive associations of the antigen or polypeptide, while disrupting unimportant or degradative structures/interactions.

The current invention was assessed by first benchmarking antigen stability in a simple BSA protein diluent (PBT), described above. Typically, in reagent development, the retention of positivity to about 3 days at 45^oC. is adequate to assess long-term stability at lower temperatures. Those skilled in the art believe that stability of up to about 14 days at elevated temperature indicates a well-stabilized antigen. In addition, the longer time a reagent retains activity at 45^oC.; the longer it is likely to be stable at lower temperatures, such as at about 2^o-8^oC. and at room temperature. First, formalin-inactivated influenza suspension stocks were made in a manner commonly known in the art and described in Example 1 below. Then, both influenza A and influenza B inactivated stocks were spiked into the simple protein diluent at a 1:2 and 1:4 dilution, respectively. At these dilutions, the test diluent contained, in its entirety, 50% diluent, 25% ICC, and 25% Stabilcoat buffer or 75% diluent, 12.5% ICC, and 12.5% STABILCOAT.RTM. buffer (an aqueous solution containing purified bovine protein and other non-toxic chemicals in phosphate buffered saline, pH 7.0-7.4; BSI, Inc.), respectively. These antigen dilutions were selected to supply a moderate positive signal on visual inspection of a reacted test device. The two resulting solutions were tested at Day 0, Day 3 at 4^oC., and Day 3 at 45^oC. with the FLU OIA.RTM. test kit (Biostar, Inc.) for positivity. Tests were performed as per the package insert. Inactivated influenza B lost all positivity on Day 3 at 45^oC. and inactivated influenza A lost a portion of its positivity. Tests were not run on subsequent timepoints due to the failure of the reagent. The PBT protein diluent failed to maintain the antigen's stability to the desired degree.

In addition, STABILCOAT.RTM. buffer (an aqueous solution containing purified bovine protein and other non-toxic chemicals in phosphate buffered saline, pH 7.0-7.4; BSI, Inc.), a commercially available diluent that is formulated to protect and stabilize antibody coated surfaces, was tested using the antigen dilutions described above. The STABILCOAT.RTM. (an aqueous solution containing purified bovine protein and other non-toxic chemicals in phosphate buffered saline, pH 7.0-7.4; BSI, Inc.)-derived control reagent's stability was assessed as above with the exception that the test points were Day 0 and Day 4 (4^o and 45^oC. storage conditions). Under these conditions the diluent's complete composition was 75% STABILCOAT.RTM. buffer (an aqueous solution containing purified bovine protein and other non-toxic chemicals in phosphate buffered saline, pH 7.0-7.4; BSI, Inc.) and 25% ICC. Even in the presence of tissue culture media, STABILCOAT.RTM. (an aqueous solution containing purified bovine protein and other non-toxic chemicals in phosphate buffered saline, pH 7.0-7.4; BSI, Inc.) was unable to maintain the stability of inactivated influenza B antigen beyond Day 4, indicating its lack of long-term storage potential.

Next, the current invention's reagent composition was tested as above to assess its performance. In this case, the diluent's complete composition contained 50% diluent, 25% ICC, and 25% STABILCOAT.RTM. buffer (an aqueous solution containing purified bovine protein and other non-toxic chemicals in phosphate buffered saline, pH 7.0-7.4; BSI, Inc.) for inactivated influenza A and 75% diluent, 12.5% ICC, and 12.5% STABILCOAT.RTM. buffer (an aqueous solution containing purified bovine protein and other non-toxic chemicals in phosphate buffered saline, pH 7.0-7.4; BSI, Inc.) for inactivated influenza B solutions. At Day 3 at 45^oC., both inactivated influenza A and B lost some positivity but were still clearly positive. The inactivated influenza B reagent was evaluated at Day 7, Day 14, and Day 21. Eventually, after 21 days at 45^oC., inactivated influenza B lost all positivity. Previous studies have shown that inactivated influenza A loses a significant portion of its positivity by Day 21 as well. The enhancement of inactivated influenza A under these particular assay conditions is not shown in the data because it was not tested to failure. However, the stability of inactivated influenza A under these conditions could be very high and extend beyond the timepoints tested. If tested to failure under these or other assay conditions, the inventive diluent should enhance the stability of the influenza A antigen as well. Thus, relative to the simple PBT protein diluent and STABILCOAT.RTM. (an aqueous solution containing purified bovine protein and other non-toxic chemicals in phosphate buffered saline, pH 7.0-7.4; BSI, Inc.), the composition of the current invention is much better able to stabilize antigens in solution for storage.

In another assessment of the current invention, the inventive stabilizing diluent was utilized in a more sensitive optical assay method. The positive control for this method uses antigens diluted to a 1:10 dilution of both formalin-inactivated influenza A and formalin-inactivated influenza B to achieve the moderate signal necessary for such a control solution. Antibodies used in this evaluation were selected based on optimal analytical performance for the type of surface used. Reactive areas on the assay surfaces were made in a preferred method by striping monoclonal anti-influenza A antibody or monoclonal anti-influenza B antibody onto the surface such that each assay surface contained one influenza A reactive area and one influenza B reactive area. The surfaces were dried and overcoated with a preservative solution at room temperature. Antibody surfaces were heat-sealed using heat-staking equipment to a polystyrene ring that allows solution to flow through or around the porous surface. The plastic ring supplies support to facilitate handling and a means to mount the porous surface onto a vacuum source to assist in removal of fluids from and drying of the porous surface. For the purpose of this experiment, assays were conducted in a flow-through manner. Flow-through indicates that sample contacted the assay surface and flowed through, over, or around it during the assay procedure.

This experiment tested the ability of the cell culture media (EMEM, BioWhittaker Cat. #12 136Q)to enhance stability of the antigens as a component of the inventive stabilizing diluent. Four test solutions were made: (A) 75% inventive diluent, 12.5% EMEM and 12.5% STABILCOAT.RTM. buffer (an aqueous solution containing purified bovine protein and other non-toxic chemicals in phosphate buffered saline, pH 7.0-7.4; BSI, Inc.), (B) solution A, where the EMEM additionally contains 2% FCS, 1% L-Glutamine, and 0.5% PenStrep/Fungizone (an antimicrobial solution, BioWhittaker Cat. #17-745H), (C) 75% inventive diluent and 25% Stabilcoat diluent, and (D) inventive diluent only. A 1:10 dilution of inactivated influenza B stock suspension was made in each of the four diluents by first making a 1:4 dilution and subsequently a 1:2.5 dilution with the respective test diluent. Each solution was tested at Day 3 at 4^oC. and Day 3 at 45^oC. Solution A showed the best stability at 3 days at 45^oC., followed by solution B, D and C. The results indicate that incorporating cell culture media/STABILCOAT.RTM. (an aqueous solution containing purified bovine protein and other non-toxic chemicals in phosphate buffered saline, pH 7.0-7.4; BSI, Inc.) into the diluent under increasingly dilute antigen conditions enhances the stability of the antigen (compare stability in solution A to that of solution D). Utilizing a higher concentration of STABILCOAT.RTM. buffer (an aqueous solution containing purified bovine protein and other non-toxic chemicals in phosphate buffered saline, pH 7.0-7.4; BSI, Inc.) as the only other constituent (solution C) proved deleterious to the antigen's stability (relative to solution A).

Therefore, these results clearly indicate that the current invention greatly surpasses other diluents of its type in its ability to stabilize protein antigens in an aqueous medium, especially those of influenza.

Claim 1 of 20 Claims

We claim:

1. An aqueous reagent composition comprising a buffer, a serum, glycerol, a salt selected from the group consisting of potassium chloride, sodium chloride, magnesium chloride, magnesium sulfate, and calcium chloride, a chelating agent selected from the group consisting of EGTA and EDTA, a detergent selected from the group consisting of CHAPS, cholic acid, deoxycholic acid, digitonin, n-dodecyl-.beta.-D-maltoside, glycodeoxycholic acid, n-lauroylsarcosine, lauryl sulfate, saponin, polyoxyethylene sorbitan monooleate, and polyethylene glycol P-1,1,3,3-tetramethylbutylphenyl ether, and a preservative at a final pH of 7.5 to 8.5, wherein said composition does not comprise N-dodecanoyl-N-methylglycine or decanoyl N-methylgluconamide.
 

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