WO1989011654A1

WO1989011654A1 - Synthetic matrices and derived assay calibrators

Info

Publication number: WO1989011654A1
Application number: PCT/US1989/002108
Authority: WO
Inventors: Anand Akerkar
Original assignee: Anand Akerkar
Priority date: 1988-05-16
Filing date: 1989-05-16
Publication date: 1989-11-30
Also published as: EP0437435A1; JPH03505482A; AU3693489A; EP0437435A4

Abstract

The present invention provides a protein-free liquid matrix useful for preparing control standards and calibrators for assays which comprises water, a buffer and between about 0.1 % and 10 % by weight polyvinylpyrrolidone. The invention also concerns a control solution or calibrator for use in immunoassay and chemical analysis which comprises the matrix of the present invention and an analyte and the use of this control solution or calibrator in immunoassays or chemical analysis of unknown biologically active, naturally occurring materials.

Description

SYNTHETIC MATRICES AND DERIVED ASSAY CALIBRATORS

Background of the Invention

The present invention relates to protein-free polyvinylpyrrolidone based formulations for use as matrices in the preparation of calibrators or standards for immunoassays or clinical chemistry procedures.

Human or mammalian blood products, such as human serum or plasma, have been generally accepted and widely used as matrices for in-vitro calibrators and reference controls. For example, two to eight calibrators containing specific levels of analyte are used in an immunoassay for the purpose of con¬ structing a dose-response curve (standard curve) by means of which the signal responses of reference controls and unknown specimens can be calculated in terms of concentration or mass units of a given analyte. Similar reference controls or calibrators are also utilized for verifying assay performance in other clinical chemistry procedures.

The ideal calibratory solution or matrix would be one which is compositionally fully defined and would exactly match the composition of the unknown specimen except for the unknown analyte concentration. These conditions are rarely attainable in practice due to the wide compositional variability of normal and abnormal physiological blood or urine specimens.

The next best calibratory solution would be a matrix that approximately matches the typical specimen composition, such as homologous human serum-based calibrators made from pooled analyte-free human serum for use in the assay of human serum specimens. However, some techniques utilized for removal of endogenous analytes ("stripping") frequently induce major alterations in the lipid and/or protein composi¬ tions and thereby aggravate the mismatch between the specimen and the calibrator. Affinity chro atography, in contrast, selectively removes the analyts without significant alteration of the matrix. However, it is a costly procedure and is generally used only for removal of endogenous analytes present in extremely low concentrations.

A less optimal alternative would be the use of calibrator matrices containing heterologous animal sera or human or bovine albumin fractions in appropriate buffer for¬ mulations Such protein-based matrices (including gelatin- based matrices as in U.S. Pat. 4,379,847) encompass virtually all calibrator formulations that are currently used in commer¬ cial immunoassay kits for the analysis of human and animal blood or urine specimens. The undefined nature and lac of homogeneity of blood-based or urine-based protein matrices, including matrices derived from costly "purified" protein fractions, frequently require that expensive, time-consuming ^•screening and evaluation of matrices from several sources be done before manufacturing of commercial calibrators can com¬ mence. Other shortcomings of protein-based calibrators are enumerated as follows:

a. high lot-to-lot variability complicates manufacture; b. inherent instability of proteins or protein solutions requires storage at refrigerator or freezer temperatures; c. large and expensive inventories must be maintained to min¬ imize screening expenses; d. potential contamination of protein matrices with unaccep¬ table- high levels of endogenous analytes necessitates in-house processing or purchase of more costly "stripped" protein matrices; e. potential contamination with and the need for testing for pathogenic or non-pathogenic micro-organisms including hepatitis viruses and HIV; f. protein matrices are excellent growth media and require aseptic or sterile manipulation and/or use of effective antimicrobial agents; g. the potential for the presence of viruses and infectious agents in approved lots due to the limitations of current testing procedures; h. the presence of variable and frequently diminishing endogeniσ enzyme levels and their unpredictable effect on the stability or efficacy of the analyte; i. the relatively high cost of human and animal products, particularly of purified blood fractions; and j . the potential for serious shortages in the supply of human blood products due to fewer donors, high donor rejection rates, improved whole blood utilization resulting in fewer outdated blood units and unexpected medical emergencies.

Total elimination of protein from calibrator matrices (i.e., use of buffer solutions ) would solve most of these problems, but is rarely applicable due to the serious specimen -calibrator mismatch. Some attempts have been made in the prior art to overcome some of these shortcomings by replacing proteins with certain synthetic or polymeric materials in the formulation of clinical reagents for in-vitro assays. k

Mast (U.S. Pat. No. 3,920,580) prepared controls con¬ taining synthetic polymers such as polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol, dextran and the like for use in the enzymatic analysis of glucose in blood or serum by means of dry reagent strips. U.S. Pat. No. 4,506,018 pro¬ posed blood diluents containing ethoxylated polypropylene con¬ densates. U.S. Pat. No. 3,876,375 utilized alkylene polyols in biological compositions intended as reference controls in diagnostic analyses. Hydroxyethyl starch was proposed as a plasma substitute in U.S. Pat. No. 3,937,821 but only for in- vivo use. Similarly, polyvinylpyrrolidone has been used extensively in-vivo as a plasma extender since the 1940's (Polyvinylpyrrolidone, chap. 21. L. Blecher, et al., from Handbook of ater-Soluble Gums and Resins, R.L.Davidson, McGraw-Hill, 1980) . There is no know prior art on the use of polyvinylpyrrolidone in liquid protein-free calibrators or standards for use in immunoassays or clinical chemistry protocols.

Accordingly, it is an object of this invention to pro¬ vide optimal calibrator matrices suitable for incorporating known quantities of diverse analytes, singly or in combina¬ tion, that are commonly quantified in clinical and immunoassay laboratories. Another object of this invention is to provide compositionally defined synthetic formulations that are com¬ parable to serum or plasma in terms of their physical or immunological characteristics as well as in their responses under assay conditions. A further object of this invention is to provide calibrator solutions that are stable at room temperatures or refrigerator temperatures for an extended period of time. Summary of the Invention

The present invention provides a protein-free liquid matrix useful for preparing control standards and calibrators for assays which comprises water, a buffer and between about 0.1% and 10% by weight polyvinylpyrrolidone. The invention also concerns a control solution or calibrator for use in immunoassay and chemical analysis which comprises the matrix of the present invention and an analyte and the use of this control solution or calibrator in immunoassays or chemical analysis of unknown biologically active, naturally occurring materials.

Brief Description of the Drawings

FIGURE 1 illustrates the results obtained with three synthetic serum formulations of this invention compared to an albumin-based matrix in an enzyme immunoassay (EIA) for T3 (triiodothyronine) .

FIGURE 2 depicts a comparison of a synthetic and an albumin-based matrix in a phenobarbital enzyme immunoassay. Detailed Description of the Invention

The present invention provides a protein-free liquid matrix useful for preparing control standards and calibrators for assays which comprises water, a buffer and between about 0.1 and 10% by weight polyvinylpyrrolidone.

It has unexpectedly been found that effective calibrator solutions for analyzing diverse analytes may be formed by adding polyvinylpyrrolidone to certain buffered solutions containing one or more analytes at known levels. With the incorporation of polyvinylpyrrolidone in such solu¬ tions to form protein-free calibrators, results are obtained in diverse assays that are identical or comparable to the results observed when calibrator solutions are based on human or animal matrices.

Although the mechanism by which polyvinylpyrrolidone improves calibratory solutions in the formulations of this invention is not fully understood, it is believed that polyvinylpyrrolidone exerts this beneficial effect by virtue of its similarity to proteins in terms of physicochemical parameters, such as its polyamide structure, molecular weight, viscosity and osmotic pressure. Polyvinylpyrrolidone, like albumin and prealbumins, also reversibly binds certain lipophilic and hydrophilic molecules and thereby can act anal¬ ogous to these proteins as carrier or storage agent prior to or during assay incubations. In immunoassays, this binding capability is believed to be a critical factor in the rates of antigen-antibody interactions that occur during incubation in the calibrator solutions as well as in the specimen solution. In immunological reactions, these rates must either be matched or controlled to reach a common end point or plateaus at the conclusion of the incubation period in order to obtain valid assay results. In enzymatic chemistry procedures, an analyte- polyvinylpyrrolodine complex, for example, with cholesterol, could modulate the enzyme kinetics.

It was further unexpectedly found that the use of polyvinylpyrrolidone in the calibrator solutions of this invention permits optimization of their compositional and/or their physicochemical parameters (such as osmolality and pH) so as to achieve optimal matching of the calibrator-response curves with the specimen-response curves when the incubations were done at temperatures ranging from 0° to about 60° C, preferably from 2 ' to about 45" C. In other words, for an enzyme assay or immunoassay conducted at a given temperature and an empirically determined optimum incubation time, it was possible to adjust the calibrator composition and/or the physical parameters of this invention, so as to achieve cor¬ respondence of the calibrator and the specimen responses when both contain analytes at the same concentration. This charac¬ teristic of the calibrators of this invention obviates the need to assign nominal or assay-specific concentration units (rather than true or absolute concentrations) which practice is occasionally utilized in clinical assays when the assay response in specimens deviates significantly from the cor¬ responding assay response in the calibrators.

In the preferred embodiments of this invention, the calibrator solution includes, in addition to one or more analytes, between 0.1 and 10% polyvinylpyrrolidone, preferably between 0.1 and 5%. The polyvinylpyrrolidone included in the calibrators is of a high molecular weight of about 10,000 to about 80,000 daltons, preferably about 40,000 daltons, and preferably also of a pharmaceutical grade. Alternatively or additionally,_, it is frequently beneficial to further prepurify the polyvinylpyrrolidone, particularly if it is of technical grade or is old or yellowish in appearance, by means of a pretreatment with a suitable reducing agent, such as hydrogen gas with noble metal catalysts, sodium borohydride, potassium borohydride or the like, in order to reduce reactive func¬ tional groups, such as peroxides, hydroperoxides, epoxides, aldehydes and ketones, or residual polymerization catalysts which, may have a deleterious effect on the functionality or stability of the analyte in the calibrator solution.

It has been found advantageous to include in the calibrators solution common buffers so as to adjust the pH of the solution to the range of 5.0 to 10.0, preferably 7.0 to 9.5. Preferred buffers include phosphates, borates, tris (hydroxymethyl) aminomethane (TRIS) , Good's buffers, such as N,N-bis-(2-hγdroxymethyl)-glycine, salts of acetic or phthalic acid, or like buffers. In addition to providing pH and osmotic control, these buffers also permit modulation of calibrator response by means of appropriate pH changes within the claimed pH range. To date, the most preferred buffers are hydroxyethylpiperazineethanesulfonic acid (HEPES) , mor- pholinopropanesulfonic acid (MOPS) or MOPSO.

Similarly, it has been found advantageous, although not essential, to include in the calibrator solution common salts, such as alkali metal salts of the halogens, a phosphate or a sulfate. Preferred salts include sodium chloride, potas¬ sium chloride, potassium phophate and the like. It is believed that these salts further permit modulation of the osmotic pressure of the calibrator solutions so that they more closely emulate serum or urine matrix effects. Satisfactory calibrator solutions are obtainable with between about 0 to 3% salt, preferable between about 0.1 to 2.0% salt, and osmolality between about 100 and 800 os/Kg.

It is also beneficial to include in the calibrator solutions of this invention a preservative so as to avoid microbial growth therein. Such preservatives may be selected from the numerous materials which are known to have this prop¬ erty and, furthermore, do not interfere in the assay. Repre¬ sentative compositions for this function include sodium azide, thimerosal, Cialit-tm, octhilinone or 2-octyl-4-isothiazolin-3- one (sold under the commercial tradename Kathon-tm by Rohm & Haas) , sodium benzoate, hexachlorophene, sorbic acid, quaternary ammonium compounds, p-chloro-m-xylenol and gentamicin. The preservative may be included in an amount corresponding to acceptable levels for each preservative, such as between about 0.001 to 1.0%. Further, adjuvants may be added to the calibrator solution to obtain a particular color or physical appearance such as red, orange or yellow dyes, to mimic the color of serum or urine.

The results shown in Figures 1 and 2 and Tables l and 2 for two representative analytes, T3 and phenobarbital, indi¬ cate virtually identical performance of the synthetic matrices of this invention with the conventional protein-based (bovine albumin) matrices of the art. The standard curve parameters are comparable for all the matrices of each example and the reference control values likewise show good agreement.

Other calibrators prepared from the synthetic matrices of this invention using, for example, digoxin, T4, gentamicin, TSH, cholesterol, also performed satisfactorily. There is no apparent restriction to the scope and applicability of these calibrators in the analysis of diverse chemical or biochemical entities comprising normal and abnormal metabolities, elec¬ trolytes, vitamins, steroids, therapeutic drugs, drugs of abuse, peptides, polypeptides, polypeptide hormones, proteins, glycoproteins, enzyme polynucleotides, polysaccharides , indus¬ trial toxicants and the like by means of appropriate immunoas¬ says or clinical chemistry procedure.

The invention also concerns a control solution or calibrator for use in immunoassay and chemical analysis which comprises the above described matrix and an analyte and meth¬ ods of using the control solution or calibrator in immunoas¬ says or chemical analysis on unknown biologically active, naturally occurring materials.

The novel calibrator matrices of this invention and the preparation and use thereof will be further described in the following examples which set forth specific embodiments of this invention. The examples are provided to aid in an under¬ standing of the invention but are not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow thereafter. Experimental Details

Example 1

a. 2.0% Polyvinylpyrrolidone-0.05M HEPES:

To 800ml water were added: 20.0g polyvinylpyrrolidone (40K, pharmaceutical grade), 11.9g HEPES and 6.0g sodium chloride. The pH of the solution was adjusted to 7.3+0.1 with dilute sodium hydroxide. After addition of 0.002g methyl red and 0.005g tartrazine, the solution was diluted to l.OL with water and the pH adjusted to 7.4+0.05. The osmolality was found to be 300 mos/Kg.

b. Preparation of T3 Calibrators:

Four T3 calibrators were formulated by adding appropriate amounts of an accurately prepared T₃ stock solu¬ tion to provide T₃ does levels of 0, 0.5, 2.0 and 4.0 ng/mL to:

1. 1.0% bovine serum matrix

2. matrix of Example la. plus 0.003% gentamicin

3. matrix of Example la. plus 0.02% thimerosal

4. matrix of Example la. plus 0.001% Kathon m

c. T₃ EIA Results:

The four sets of T₃ calibrators were compared in a typical T₃ enzyme immunoassay (EIA) format. The results are tabulated in TABLE I and the standard curves are shown in FIG¬ URE l. TABLE 1

matrix (-) (") 50% calibrators (asorbance) control sera corr. slope B/B< 0 0.5 2.0 4.0 (ng/mL) coeff, 1 2 3

Bovine

+ 0.995 1.54 1.37 1.658 1.318 0.646 0.386 0.5 1.6 3.1 gentamicin

synthetic

+ 0.994 1.30 1.56 1.634 1.276 0.716 0.481 0.5 1.8 4.4 gentamicin

synthetic

+ 0.994 1.40 1.71 1.533 1.256 0.726 0.455 0.4 1.7 4.0 thimerosal

synthetic

+ 0.996 1.44 1.58 1.562 1.265 0.712 0.435 0.4 1.7 3.6 Kathon^R

Example 2

a. 2.0% Polyvinylpyrrolidone - 0.05M MOPSO:

To 200ml water in a suitable vessel were added 20.Og polyvinypyrrolidone (4OK, commercial grade) and sufficient diulte sodium hydroxdide to adjust the pH to 11.0 - 11.5. Sodium borohydride (0.20g) was added and the mixture stirred for 30 minutes. After careful acidification with dilute hydrochloric acid to a pH range of 3.0 - 4.0, the solution was stirred for 30 minutes. MOPSO (11.3g) and 5.5g sodium chloride were added and the pH was adjusted to 7.3+0.1 with dilute sodium hydroxide. After dilution to l.OL with water, Kathontm (l.o mL of a 1.5% solution) was added and the pH was re-adjusted to 7.4+0.05. The solution was filtered through a cartridge filter (0.45pm). The osmolality was 285 mos/Kg.

b. Preparation of Phenobarbital Calibrators:

Four phenobarbital calibrators were prepared by spik¬ ing with appropriate amounts of a phenobarbital stock solution to provide phenobarbital dose levels of 0, 5, 25 and 50 ug/roL in:

1. 1.0% bovine serum matrix

2. matrix of Example 2a plus 0.001% Kathon^tm

c. Phenobarbital EIA results:

The two sets of calibrators were compared in a commer¬ cial phenobarbital EIA. The results are shown in TABLE 2 and the standard curves are presented in FIGURE 2. TABLE 2

matrix (-) (") 50% calibrators (asorbance) control sera corr. slope B/B« 0 5 25 50 (ug/mL) coeff . 1 2 3

Bovine

+ 0.998 0.98 15.6 2.012 1.470 0.848ι 0.643 5.9 20.3 47.4 gentamicin

synthetic Kathon^R 0.998 0.96 16.3 1.772 1.387 0.766 0.593 3.7 15.3 47.4

Example 3

a. 1.0% Polyvinylpyrrolidone - 0.025M Phosphate:

To 800ml water were added 3.55g disodium phosphate (anhydrous), 10.Og polyvinylpyrrolidone (40K, pharmaceutical grade) and 6.5g sodium chloride. The pH of the mixture was adjusted to 7.22+0.1 with dilute hydrochloric acid. After addition of gentamicin sulfate (0.02g) and tartrazine (0.002g), the solution was diluted to l.OL and the pH was re¬ adjusted to 7.2+0.05. The os olality was 310 mos/Kg.

b. The matrix of Example 3a proved useful as a synthetic urine substitute.

Example 4

a. 1.5% Polyvinylpyrrolidone - 0.05M HEPES:

To 800mL water were added 15.Og polyvinylpyrrolidone (40K, pharmaceutical grade), 11.9g HEPES and 6.5g sodium chloride. After adjusting the pH to 7.2+0.05 with dilute sodium hydroxide, the solution was diluted to l.OL with water. Gentamicin sulfate (0.02g) was added as a preservative. The osmolality was 305mos/Kg.

b. Preparation of Cholesterol Calibrators:

A stock solution of water-soluble cholesterol (polyoxyethylene cholesterol sebacate, Sigma, Cat. No. #C~ 1145) was preparede and appropriate aliquots were spike into the matrix of Example 4a. to provide total cholesterol levels of 185 and 245mg/dL. These calibrators proved useful in com¬ mercial cholesterol assays.

Claims

Claims :

1. A protein-free liquid matrix useful for preparing control standards and calibrators for assays which comprises water, a buffer and between about 0.1% and 10% by weight polyvinylpyr¬ rolidone.

2 . A matrix of claim 1, wherein the amount of polyvinylpyr¬ rolidone is between about 0.1 and about 5% by weight.

3. A matrix of claim 2, wherein the amount of polyvinylpyr¬ rolidone is about 2% by weight.

4. A matrix of claim 1, wherein the polyvinylpyrrolidone has a molecular weight in the range of about 10,000 to about 80,000 daltons.

5. A matrix of claim 4, wherein polyvinylpyrrolidone has a molecular weight of about 40,000 Daltons.

6. A matrix of claim 6, wherein the buffer is an alkali metal salt of phosphoric acid or mixtures thereof, a salt of acetic acid, a salt of boric acid or a salt of phthallic acid.

7. A amtrix of claim 6, wherein the buffer is present in an amount so that the pH of the matrix is from about 5.0 to about 10.0.

8. A matrix of claim 7, wherein the buffer is hydroxyethyl- piperazineethanesulfonic acid (HEPES) , morpholinopropanesul- fonic acid (MOPS) or MOPSO and the pH of the matrix is in the range of about 7.0 to about 8.0.

9. A matrix of claim 7, wherein the buffer is a phosphate and the H of the matrix is in the range of about 6.5 to about 8.0.

10. A matrix of claim 7, wherein the buffer is a boraste and the pH of the matrix is in the range of about 8.0 to about 10.0.

11. A matrix of claim 1, further comprising an alkali metal salt of a halogen, a phosphate, or a sulfate.

12. A matrix of claim 11, wherein the alkali metal salt is sodium chloride or potassium chloride.

13. A matrix of claim 1 further comprising a preservative.

14. A matrix of claim 13, wherein the preservative is selected from the group consisting of benzoic acid, sodium benzoate, dichlorophene, hexachlorophene, quaternary ammonium compounds, sorbic acide, phospheric acid, esters of p- hydroxybenzoic acid, sodium azide, thimerosal, gentamicin, octhilinone (Kathon^tιn) and p-chloro-m-xylenol.

15. A matrix of claim 14, wherein the preservative is octhilinone (Kathontm) .

16. A matrix of claim 1 further comprising an adjuvant to obtain a particular color or physical appe3arance.

17. A matrix of claim 16, wherein the adjuvant is a red, orange or yellow dye.