MXPA97008892A - Stable protein solutions for diagnosis and method to make and use - Google Patents

Abstract

The present invention relates to stable bulking agents and blocking agents for solid phase materials containing proteins. The proteins have thiol groups, which are blocked or are chemically inert, preventing the formation of aggregates. Chemical solutions where the thiol groups, if present, are blocked or chemically inert, are particularly useful in analytical applications and in diagnostic reagents that utilize solid phase materials or particles.

Description

STABLE PROTEIN SOLUTIONS FOR DIAGNOSTICS AND METHOD OF MAKING AND USING THEM BACKGROUND OF THE INVENTION The invention relates to solutions containing dissolved proteins, which are used or stored in plastic containment containers and to a method for making and using said solutions. Said protein solutions are commonly used in analytical or diagnostic applications as bulking agents. That is, the proteins impart an atmosphere of viscosity and an electrogenic character to the aqueous solution, which is consistent with a biological environment. The invention also relates to blocking agents coupled to said materials or particles. Diagnostic products are heavily using solid phase materials, including particles having binding patterns attached thereto in order to effect the capture of an analyte. The solid phase materials or particles are dispersed in the aqueous medium to form a reagent suspension having kinetics, which resembles the solution reactions. Typically, the particles are isolated after the analyte is captured. An especially useful particle is a paramagnetic particle. Paramagnetic particles that have a bound binding pattern are used in diagnostic equipment for the detection of several analytes. In these devices, the binding pattern is specific to a reagent or analyte sought to be detected and quantified. In the presence of the analyte in a test sample, a complex of binding / reagent pattern is formed. This complex can be detected with revealing reagents. The signal developing reagents may comprise additional binding patterns to the analyte, which bears a mark for detection. Paramagnetic particles are typically one size in microns and are normally invisible to the naked eye. The small size of the particle allows that particle to be easily dispersed in an aqueous medium and easily isolated with a magnetic field. The paramagnetic particles are made or processed to provide a reagent or alumina coating on their surface. The coating provides a binding site for reactive species or additional binding patterns on the surface of the particle. The particles are first chemically activated to couple the silane reagent or coating, through known chemistry, to the surface. The silane coating offers an active portion for coupling patterns of attachment to the surface of the particle. The chemically activated particle is then placed in contact with a binding pattern, which needs to be bonded to the particle. For example, an antibody that is specific to an analyte of interest. The reaction is typically performed in an excess of the binding pattern to ensure a complete reaction. After coupling the protein to the protein, the excess protein is removed by reacting the particle with bovine serum albumin (BSA) at 50 ° C. The reaction of the particle with BSA at 50 ° C is commonly referred to as a thermal stress reaction or blocking agent reaction. Then, the finished particle is suspended in a pH buffer containing BSA and another protein, i.e., a bulking agent. In commercial use, reagent products or suspensions of this type are usually packaged in plastic bottles and shipped to end users.

The bulking agent solutions of the prior art are not particularly stable. Even with refrigeration, said solutions show evidence of degradation.

In solutions with suspended particles, the particles, with time, form lumps, which are visible to the naked eye, although the products are stored at 4 ° C. This problem is particularly severe when the pH of the solution is above 7.5. The problem also manifests itself when the bulking agents are kept in containment containers comprising normal laboratory plastic, such as polyethylene, polyethylenetetraate, polystyrene, polycarbonate and polypropylene. The movement or agitation of the solution also increases the regimen of aggregation or fibril formation.

Nomenclature The following definitions are provided to facilitate a clear understanding of the present invention. It is noted that the use of terms in a singular sense should not be constructed so as to limit the applicability of the terms to be used in the plural sense, that is, union pattern or union patterns. The term "binding / reagent pattern" refers to any pair of molecules, which exhibits affinity, complex formation or binding capacity, typically specific binding or interaction (complexing) or reinforcing pairs, such as antibody / antigen, antibody / hapten, enzyme / substrate, enzyme / inhibitor, enzyme / cofactor, binding protein / substrate, carrier protein / substrate, lecithin / carbohydrate, receptor / hormone, receptor / effector, complementary nucleic acid strands, repressor / inducer , and similar. The term "binding pattern" refers to that member of a binding / reactive pattern pair, which is coupled to a solid phase. The term "blocking agent" refers to a material, which, when placed as a coating on a solid phase, reduces non-specific binding, while allowing a lesser load than the maximum binding pattern to the solid phase. The term "reactive" refers to that member of a complex binding / reagent pattern, the existence and / or concentration of which will be determined, the recovery of which is desired, and which will be captured by the binding pattern. of a medium where it is dispersed. An analyte in a test medium is an illustrative reagent, since it is an industrial reagent specifically binding that will be recovered. The term "test assay" generally refers to any procedure in which a member of a binding / reagent pattern in a test sample is to be detected and / or quantified in a medium through various test formats. For example, the "test assay" can be used to describe a diagnostic procedure, analytical procedure, microanalytical procedure, forensic analysis, pharmacokinetic study, cell sorting procedure, affinity chromatogram, industrial or laboratory recovery or analysis of a or more species such as toxins, catalysts, or starting materials or products, and the like. The term can be used to describe a process in which a plurality of reagents, each including a different binding pattern, is employed to capture a plurality of corresponding reagents. A typical test assay is an immunoassay. The term "complex" refers to the specific binding or interaction or association of two or more species, for example, a binding and reagent pattern. The term "capture" refers to the analysis, recovery, detection or other qualitative or quantitative determination of a reagent in a medium, for example, via complex formation in a test assay. As an example, in a sandwich immunoassay, a binding pattern coupled to the surface will form a complex with a reagent, before or after the reagent forms a complex with another binding or reagent pattern (generally labeled). In a competitive assay, labeled and unlabeled reagents typically compete for complex formation with a binding pattern coupled to the surface. The term "inert" refers to a chemical or biological state in which a material does not interact with the binding pattern of the test assay in which it is used in a manner in which the binding pattern and the reagent interact. The term also refers to a chemical or biological state wherein the material does not interact chemically with the other reactive portions in a test reaction or reagent suspension. The term "solid phase" refers to any material, which is insoluble in a medium containing a reagent that will be captured in a particular test assay. In a broader sense, the term describes any entity that can be substantially dispersed within a medium and removed or separated from the medium by immobilization, filtration, division, centrifugation or in which the medium can be supplied, or the like. A blocking agent can be bound to the solid phase to reduce non-specific binding. The term "reagent suspension" generally refers to a processed material modified to participate in a complex formation reaction such as a test assay, more specifically to a solid phase material to which a binding standard is coupled and suspended. in a blocking agent solution. The term "non-specific binding" (NSB) refers to an undesired interaction between a reagent and any other species present in a different test assay than the formation of binding / reagent standard complex. The term "label" refers to an atom or a molecular portion capable of generating a signal for the detection of the binding reaction. This includes, without limitation, radioactive isotopes, enzymes, luminescence agents, precipitating agents, and colorants. The term "sample" refers to any medium that contains a reagent or analyte that will be captured and detected and quantified in a test assay. The term "analyte" refers to either hormone, pharmacological agent, vitamin or cofactor, hematological substances, virus antigens, nucleic acids, nucleotides, allergens, or other markers sought to be detected and quantified. The term "substitutions" refers to the substitution of nitrogen for one or more carbons. The term "derivative thereof" refers to the addition of a nitrogen containing a functional group.

COMPENDIUM OF THE INVENTION The present invention presents articles of manufacture and methods for making a reagent suspension of solid phase material or particles and bulking agents. The suspension delays the formation of protein aggregate or fibril formation. One embodiment of the present invention features a suspension of solid phase materials or particles suspended or arranged in an aqueous medium, comprising a thiol blocked chemical agent, preferably a thiol blocked protein. The thiol blocked protein has the formula: P-X where done P is a protein that has one or more thiol groups. As used above, X is a blocking group covalently bonded to S of the thiol group. Blocking groups for reaction with thiol groups are well known in the art. Representative blocking groups include dC? 2 alkyl, C3-C? 2 cycloalkyl, d-Cn haloalkyl, dC? 2 hydroxyalkyl, C2-C? 2 alkenyl, C2-C? 2 haloalkenyl, C2-C? 2 hydroxyalkyl, C2 alkynyl -C? 2, haloalkynyl C2-C? 2, hydroxyalkynyl C2-C? , C6HS, alkoxy C? -C? 2, C 1 -C 12 haloalkoxy, C 1 -C 12 hydroxyalkoxy, C 1 -C 2 alkylthio, C 1 -C 2 alkynylsulfonyl, C 1 -C 12 alkylsulfonyl, and amine, amide, imide, substitutions and derivatives thereof .

The use of a thiol-blocked protein in a reagent suspension delays the formation of fibrils or protein aggregates, when the suspension of particles is stored in plastic bottles and can also be subject to movement. Preferably, the protein, P, is albumin. Albumin can be derived from any source, which is commercially available. Common forms of albumin include human, bovine, porcine, cat, chicken, dog, donkey, egg, goat, guinea pig, hamster, horse, rat, rabbit, pigeon, sheep, rhesus monkey, turkey, as well as other sources. Alternative chemical compositions can also be used as bulking agents and blocking agents, according to the present invention, provided thiol groups are blocked or chemically inert. Preferably, the particle is a paramagnetic particle. Preferred X is an alkyl thiol group represented by the formula set forth below: MR wherein R is C? -C6 alkyl, C3-C6 cycloalkyl, Ci-C6 haloalkyl, C? -C6 hydroxyalkyl, C2-C6 alkenyl, C2-C6 haloalkenyl, C2-C6 hydroxyalkenyl, C2-C6 alkynyl, C3-C6 haloalkynyl , C2-C6 hydroxyalkynyl, C6H5, d-C4 alkoxy, C? -C4 haloalkoxy, hydroxy alkoxy C? -C 4, C 1 -C 4 alkylthio, C 1 -C 4 alkylsulfinyl, C 4 alkylsulfonyl, and amine, amide, imide, derivatives and substitutions thereof. Preferably, blocking group X may comprise a group set forth below: OR I I -CH2-C-Z As used above, Z is hydroxyl, d-C6 alkyl, C3-C6 cycloalkyl, d-C6 haloalkyl, C-C6 hydroxyalkyl, C2-C6 alkenyl, C2-C6 haloalkenyl, C2-C6 hydroxyalkenyl, C2-C6 alkynyl, C2-C6 haloalkynyl, C2-C6 hydroxyalkynyl, C6HS, C?-C4 alkoxy, haloalkoxy dC, C?-C4 hydroxyalkoxy, C?-C 4 alkylthio, C?-C alqu alkylsulfinyl, C?-C 4 alkylsulfonyl, and amine, amide, imide, derivatives and substitutions thereof, or Z is represented by the formula: U I -N-W wherein U and W are independently hydrogen, C-alkyl. -C6, C3-C6 cycloalkyl, d-C6 haloalkyl, C6-6 hydroxyalkyl, C2-C6 alkenyl, C2-C6 haloalkenyl, C2-C6 hydroxyalkenyl, C2-C6 alkynyl, C2-C6 haloalkynyl, C2-C6 hydroxyalkynyl, C6HS , C 1 -C alkoxy, C 1 -C 4 haloalkoxy, C 1 -C hydroxyalkoxy, C 1 -C alkylthio, C 1 -C 4 alkylsulfinyl, C 1 -C 4 alkylsulfonyl, and amine, amide, imide, derivatives and substitutions thereof. Preferably, Z in NH2.

Preferred X is represented by the formula presented below: NH2 O I I I - (CH2) n-CH-C-OH where n is an integer of 1 -3. Preferably, n is 1. The embodiments of the present invention also provide a method for making a reagent suspension. The method comprises the steps of combining water, a thiol blocked chemical compound, preferably a protein, and one or more particles having a binding pattern attached thereto, to form an aqueous medium. The thiol blocked protein is placed in the solution. One or more of the suspended magnetic particles are suspended in the aqueous medium. The thiol blocked protein has the formula: P-X wherein P is a protein having a thiol group and X is a blocking group covalently linked to the thiol group. Blocking groups are known in the art and include d-C6 alkyl, C3-C6 cycloalkyl, C? -C6 haloalkyl, C? -C6 hydroxyalkyl, C2-C6 alkenyl, C2-C6 haloalkenyl, C2-C6 hydroxyalkenyl, C2- alkynyl C6, C2-C6 haloalkynyl, C2-C6 hydroxyalkynyl, C6Hs, C?-C4 alkoxy, C 1 -C haloalkoxy, C.-C4 hydroxyalkoxy, C?-C 4 alkylthio, d-C 4 alkylsulfinyl, C?-C 4 alkylsulfonyl, and amine , amide, imide, derivatives and substitutions thereof. Preferably, the method further comprises the addition of appropriate pH regulators, inorganic or organic salts, and surfactants, ie, triton, to the reagent suspension. One embodiment of this invention is a method for detecting the presence or absence of an analyte in a test sample. The analyte can be a chemical in a physiological fluid or another fluid or product for which the analysis is made. The method comprises the steps of combining the sample to be tested, which may or may not contain the chemical or analyte or reagent, with the reagent suspension of this invention. The suspension comprises particles coated with a binding pattern capable of reacting with the analyte, a thiol-blocked bulking agent, and water. Ordinary reaction conditions are imposed on the suspension to form a reaction product or complex in the presence of the analyte, if present, and the suspension is verified or detected through a label to determine the presence of the reaction product as a indication of the presence of the analyte. In some cases, the diagnosis or other analytical test for the analyte can be quantitative as well as qualitative according to known practice. Therefore, it is an object of the present invention to provide a diagnostic reagent of a solid phase material or particle suspended in a chemical solution, preferably a protein solution, wherein the thiol groups, if present, are blocked or are chemically inert so as to delay the formation of aggregates or fibrils. Another object of the invention is to provide a diagnostic reagent of a solid phase material having coupled thereto a binding pattern and a blocking agent, which is free of chemically reactive thiol groups. Another object of the invention is to provide a bulking agent for a diagnostic reagent, the bulking agent being a chemical solution, wherein the thiol groups, if present in the solution, are blocked or are typically inert. A further object of the invention is to provide a solid phase material having coupled thereto a binding pattern and a blocking agent including a chemical compound, wherein the thiol groups, if present in the compound, are blocked or are typically inert. . In this manner, the present invention illustrates ways to solubilize solutions containing stabilization protein used in in vitro applications, such as diagnostics. The embodiments of the present invention have particular application with respect to reagent suspensions. Reagent suspensions made in accordance with the present invention are stable and delay the formation of visible aggregates or fibrils. The aspects and advantages of the present invention will be apparent from the following detailed description which, by way of illustration, shows preferred embodiments of the present invention and the principles thereof and what is now considered to be the best mode for apply these principles.

BRIEF DESCRIPTION OF THE DRAWINGS The above aspects, objects, and advantages of the present invention will be better understood from the following specification when read in conjunction with the accompanying drawings, in which: Figure 1 is a series of photographs showing various stages of training of fibrils in a suspension of reagent of the prior art; Figure 2 is a graphical representation of the effect of temperature on BSA monomer content and thiol content; Figures 3a, 3b, 3c and 3d represent size exclusion chromatograms of bovine serum albumin at 4 ° C after 16 hours, at 50 ° C at 16 hours, at 55 ° C at 16 hours and at 60 ° C at 16 hours; Figures 4a, 4b, 4c and 4d show size exclusion chromatograms of bovine serum albumin blocked with thiol at 4 ° C after 16 hours, at 50 ° C after 16 hours, at 55 ° C after 16 hours and at 60 ° C after 16 hours.

DETAILED DESCRIPTION OF THE INVENTION The present invention will be described in detail as an article of manufacture, a suspension of particulate reagent or solid phase materials, and a method for making the same, with the understanding that the present description should be considered as an illustration of the principles of the invention and is not intended to limit the invention to the embodiment described. The present invention has application in an analytical laboratory and diagnostic procedures involving any reagent suspensions, which exhibit the tendency to form clumps or aggregates. The reagent suspension of the present invention comprises a suspension of particles and a thiol blocked protein or chemical solution. A blocking agent comprising a thiol blocked chemical, preferably a protein, is also provided. The method and articles of manufacture of the present application have particular application with the use of paramagnetic particles as the solid phase material. Paramagnetic particles are described in the patent of E.U.A. 4,554,088, the reference of which is incorporated herein and commercially available from Advanced Magnetics, Cambridge, MA. Diagnostic reagents employing such magnetic particles are commercially available from Ciba Corning Diagnostics Corp. The paramagnetic particles described have settling times in aqueous solutions of about 1.5 hours.

The particles have a diameter of approximately 0.1 to 10 μ and are responsible for reducing the magnetic fields of 100-1000 oersteds. This particle size is normally not visible to simple visa. Typically, these particles have an amino silane coating to act as binding sites for additional reactive species or binding patterns. Silane coating processes are described in the patents of E.U.A. 3, 652, 761 and 4, 554, 088, which are incorporated herein by reference. The particles are chemically activated with a reagent, which is coupled to the amino silane and offers a reactive portion for coupling or joining patterns of attachment to the surface of the particle. The prepared reactive particles are placed in contact with a binding pattern, which needs to be coupled to the particle. The reaction is typically performed in an excess of the binding pattern to bring the reaction to term. The excess protein is removed by reacting the reagent with bovine serum albumin (BSA) at 50 ° C. Preferably, in accordance with the present invention, a thiol-blocked BSA is used as a blocking agent. This reaction is referred to as a thermal stress reaction or blocking reaction. The coated particle is then placed in an aqueous pH buffer containing BSA and other proteins to form a reagent suspension. The BSA or other proteins, according to the present invention, are preferably blocked with thiol and act as bulking proteins. These volume-enhancing proteins are biologically compatible, but not reactive, with the desired binding pattern, which is coupled to the surface of the particle and with the same particle. The particles are packed in plastic bottles, marked and placed in storage before being sent to customers. Although stored for periods at 4 ° C, the prior art suspensions maintained in the packaged bottles develop, over time, lumps or aggregates or fibrils, which are visible to the naked eye. In reality, a polystyrene bottle filled with conventional protein solutions and subjected to an oscillating movement for more than an hour, forms what appears to be fibrils or aggregates in the solution. It has been observed that a sheet of material forms and then bends (becomes denser) to appear as a series of strips or fibrils. The process of developing fibrils or protein aggregates is accelerated to a pH of 8.5 as compared to a pH of 7.5. Figure 1 shows a series of photographs illustrating fibrils or aggregates in a suspension of reagent of the prior art. Using anti-BSA antibodies labeled with fluorescent labels, along with other antibodies that react with other protein constituents present in the pH regulator, marked with fluorescent labels without interference, it was possible to identify the type of protein associated with the aggregate. These investigations suggest that the aggregates contain BSA. The fluorescent brand aggregates seem to develop from a film, which entangles the particles. The photographs a-c show the different stages of clod formation. The particles, by themselves, do not seem to contribute to the procedure for making the film. The films were formed on a template or surface, such as an adjoining water-air surface or an adjoining water-plastic surface. The formation of the film suggests an interlacing reaction, which caused the stabilization of the monomer. The agglomeration reaction favored a high pH, high temperatures, the presence of plastic surfaces and the movement of fluid. In accordance with this invention, chemical bulking agents, preferably proteins, are treated to block thiol constituents thereof and thus form stable suspensions of this invention. The protein blocked with thiol has the formula: P-X where done P is a protein that has one or more thiol groups. As used above, X is a blocking group covalently bonded to S of the thiol group. Blocking groups for reaction with thiol groups are well known in the art. Representative blocking groups include d-C ?2 alkyl, C3-C12 cycloalkyl, C1-C12 haloalkyl, C1-C12 hydroxyalkyl, C2-C2 alkenyl, C2-C12 haloalkenyl, C2-C12 hydroxyalkyl. C 2 -C 2 alkynyl, C 2 -C 2 haloalkynyl, C 2 -C 12 hydroxyalkynyl, C 1 H 5, C 1 -C 12 alkoxy, C 1 -C 12 haloalkoxy, C 1 -C 12 hydroxyalkoxy, C 1 -C 12 alkylthio, C 1 -C 12 alkynylsulfinyl, C 1 -C 12 alkylsulfonyl C12, and amine, amide, imide, substitutions and derivatives thereof. Preferred X is an alkyl thiol group represented by the formula set forth below: -MR wherein R is C?-C6 alkyl, C3-C6 cycloalkyl, C ha-C6 halsalkyl, d-Cß hydroxyalkyl, C2-C al alkenyl, C2-C6 haloalkenyl, C2-C6 hydroxyalkenyl, C2-C6 alkynyl, C3-C6 haloalkynyl , C2-C6 hydroxyalkynyl, CeHs, C? -C4 alkoxy, C? -C haloalkoxy, d-C4 hydroxy alkoxy, C1-C4 alkylthio, C? -C4 alkylsulfinyl, C1-C4 alkylsulfonyl, and amine, amide, imide, derivatives and substitutions thereof.

Preferably, blocking group X may comprise a group set forth below: OR I I As used above, Z is hydroxyl, d-Cß alkyl, C3-C6 cycloalkyl, C?-C6 haloalkyl, d-Cß hydroxyalkyl, C2-C al alkenyl, C2-C halo haloalkenyl, C2-C6 hydroxyalkenyl, C2-C6 alkynyl, C2-C6 fvalalkynyl, C2-C6 hydroxyalkynyl, C6HS, C1-C4 alkoxy, C? -C4 haloalkoxy, d-C4 hydroxyalkoxy, C1-C4 alkylthio, C1-C4 alkylsulfinyl, C1-C4 alkylsulfonyl, and amine, amide, imide, derivatives and substitutions thereof, or Z is represented by the formula: U I -N-W wherein U and W are independently hydrogen, d-Cβ alkyl, C3-C6 cycloalkyl, d-C6 haloalkyl, d-C6 hydroxyalkyl d-C C alkenyl, d-C halo haloalkenyl, C2-C6 hydroxy alkenyl, d-C alqu alkynyl, haloalkynyl d-Cβ, C2-Cß hydroxyalkynyl, C6H5, C?-C4 alkoxy, haloalkoxy dC, C 1 -C 4 hydroxyalkoxy, C 1 -C 4 alkylthio, C 1 -C 4 alkylsulfinyl C 1 -C 4 alkylsulfonyl, and amine, amide, imide, derivatives and substitutions of the same. Preferably, Z in NH2. Preferred X is represented by the formula presented below: NH2 O I I I - (CH2) "- CH-C-OH where n is an integer of 1 -3. Preferably, n is 1.

Preferably, the suspensions are aqueous and are regulated in their pH to a pH of 6.0 to 9.0, and preferably of 7.5 to 8.5 and, most preferably, to 7.5. It was observed that the pH of the suspension depends, to some degree, on the binding patterns, with some binding patterns requiring a lower pH for the bulking agents. Conventional pH regulators include phosphate, borate, pH Goods regulators, PIPES, H EPES, MOPSO and TRIS. The particles that can be the paramagnetic particles of the patent of E. U.A. 4, 554,088 above, preferably have sizes on the scale of 0.1 to 10μ in diameter. The particles are commercially used in amounts of 50 micrograms to 250 micrograms of solution, and most preferably 60 to 75 micrograms of solution. The carrier for the particles is preferably water, which may be distilled, deionized or regular water, having minimal amounts of salts and the like, which do not react adversely with the components of the aqueous suspensions of this invention. The chemical bulking agent solutions of this invention are blocked by thiol, if thiol groups are present and are chemically reactive. Preferably, albumin blocked by thiol is used since it is readily available and has good bulking properties. The thiol blocked proteins are preferably used in amounts of 0.05 to 4.0% by weight of the solution and most preferably from 0.05 to 2.0%. further, normal and similar diagnostic reagents can be incorporated into the suspensions, including inorganic and organic salts and surfactants found in typical pH regulators and reagents. Useful salts include sodium chloride, sodium citrate, magnesium and chloride as benefactors include sodium dodecyl sulfate, Tween and detergents In accordance with the invention, the suspensions of this invention can be made by conventional mixing at room temperature. For example, water, pH-regulating reagents, and thiol-blocked protein are mixed at room temperature with moderate agitation through bar to form stable suspensions and then the pH was adjusted as necessary. which particles were added to the solution.The tiot group of the protein can be treated to block through known reactions.The albumin blocked with thiol having less than 0.02 moles of sulfhydryl per mole of albumin, is commercially available from Miles Laboratories (now Bayer), Kankakee, IL, USA (Miles Catalog No. 81 -024 ™). The suspensions of the present invention can be used in conventional diagnostic and detection methods. For example, the presence or absence of an analyte in a sample can be determined by combining the sample with particles for reaction with the analyte, a protein blocked with thiol and water to form a suspension. The suspension was then placed under normal reaction conditions for the reaction to occur if the analyte is present. The suspension was verified for the reaction product as an indication of the presence of an analyte. The following examples are intended to illustrate and not limit the invention.

EXAMPLE 1 This Example describes the relationship of a reactive thiol group in albumin with the formation of polymers. Four samples of BSA were prepared from a 1% BSA supply solution (Catalog No. 81-003 ™) (without blocked thiol) obtained from Miles Laboratories (now Bayer), Elkhardt, IN, USA, at 100 mM of phosphate pH regulator, pH 7.4. A first sample comprised a solution of 1% BSA maintained at 4 ° C for 16 hours. This sample was control for monomer and thiol content. A value of 100% was assigned to Sample 1, with respect to those parameters as set forth in Figure 2. A second sample comprised a solution of 1% BSA maintained at a neutral pH and heated at 50 ° C for 16 hours . A third sample was a 1% solution of BSA at a neutral pH heated at 55 ° C for 16 hours. A fourth sample comprised a solution of 1% BSA at a neutral pH heated at 60 ° C for 16 hours. After 16 hours, size exclusion chromatographies were performed on the four samples. These data are presented in Figure 3a with respect to Sample 1, Figure 3b with respect to Sample 2, Figure 3c with respect to Sample 3, and Figure 3d with respect to Sample 4. These data are presented in the bar graph of Figure 2. In Figure 2, Sample 1 is a control value of 100% with respect to the monomer concentration and the context of thiol. The Figure 1 represents the percentage of monomer and thiol content against temperature in degrees Celsius. The bars with angled lines represent the concentration of monomer. The bars without angled lines represent the concentration of thiol. Compared to Control Sample 1, Sample 2 heated to 50 ° C exhibited a 17% reduction in monomer concentration and a 29% reduction in the thiol present. Compared to Control Sample 1, Sample 3 heated to 55 ° C exhibited a 67% reduction in monomer concentration and a 74% reduction in active thiol. Compared to Control Sample 1, Sample 4 heated to 60 ° C exhibited a monomer reduction of 76% and a reduction in active thiol of 78%. The reduction in thiol concentration content of BSA paralleled the reduction in monomer. Thus, the formation of BSA dimers and polymers was shown to be attributed, in part, to the thiol content of the monomer.

EXAMPLE 2 This Example describes the relationship of aluminum blocked by thiol with the formation of fibrils and protein aggregates. Four samples of thiol-blocked BSA were prepared from a 1% solution of BSA blocked with thiol in 100 mM of phosphate buffer. The BSA blocked with thiol used in this Example, is represented by the formula: P - X where P is albumin and X is represented by the formula: N H2 O I I I (CH2) "- CH-C-OH and n is 1. This composition of thiol-blocked BSA is formed by reacting the thioi group of the cysteine associated with albumin with a second cysteine to form a disulfide bond. This composition is available from Miles, Catalog No. 81 -024 ™ as noted above.

The first sample, Sample 1, comprised a solution of 1% BSA blocked with thiol, said solution was maintained at 4 ° C for 16 hours. This first sample was a control for monomer and reactive thiol content. A second sample, Sample 2, comprised a solution of 1% BSA blocked with thiol, said solution was maintained at a neutral pH and heated at 50 ° C for 16 hours. A third sample, Sample 3, comprised a solution of 1% of the BSA blocked with thiol, said solution was maintained at a neutral pH and heated at 55 ° C for 16 hours. A fourth sample, Sample 4, comprised a 1% solution of a blocked third BSA, said solution was maintained at a neutral pH and heated at 60 ° C for 16 hours. After 16 hours, size exclusion chromatographies were performed on each sample. These data were presented in Figure 4a with respect to Sample 1; Figure 4b with respect to Sample 2, Figure 4c with respect to Sample 3, and, Figure 4d with respect to Sample 4. These data, compared to the data of Example 1, provided that the use of BSA blocked with thiol delayed the formation of lumps and fibrils and aggregates. Samples 1-4 of Example 2 exhibited significantly less dimer formation and maintained a higher monomer concentration than Samples 1-4 of Example 1.

EXAMPLE 3 This example describes further comparisons of protein solutions with unblocked thiol groups with protein solutions in which the thiol group has been blocked. In this example, two supply solutions were made. A first supply solution comprised 5 mM of Barbital pH regulator at pH 8.5, 150 mM NaCl, 0.05% BSA and 0.15% bovine gamma globulin (BGG). The BSA and BGG contained unblocked thiol groups. That is, each had reactive thiol groups. A second supply solution comprised 5 mM of Barbital pH regulator at a pH of 8.5, 150 mM NaCl, 0.05% BSA blocked with thiol and 0.15% BGG. BSA blocked with thiol was as described in Example 2. Five aliquots of the first supply solution were placed in five tissue culture flasks. Similarly, five aliquots of the second supply solution were placed in five tissue culture flasks. The resulting 10 samples, five containing BSA from the unblocked thiol group and five containing thiol blocked BSA were subjected to oscillating movement at room temperature. Samples containing BSA from the unblocked thiol group developed a sheet or membrane in 30 minutes. Samples containing thiol-blocked BSA did not exhibit sheet or membrane formation in about 3 hours. After 20 hours, the amount of lump formation and aggregate exhibited in the samples containing unblocked thiol group BSA exceeded aggregation formation of thiol-blocked BSA.

EXAMPLE 4 This Example further describes the stability of BSA blocked with thiol. In this Example, the protocol of Example 3 was repeated; however, the samples were subjected to oscillating movement while remaining at a temperature of 4 ° C. Samples carrying BSA blocked with thiol were free of aggregates for about 20 hours. However, samples containing unblocked thiol BSA exhibited aggregate formation.

EXAMPLE 5 This Example illustrates the preparation of a thiol blocked protein of the formula: X wherein P is albumin and X is an amine derivative of C? -C4 alkoxy. More specifically, X is: O I I -CHa-C-Z One gram of aliquot of albumin was dissolved in water. The pH of the solution was adjusted to a pH of 8.0 with a solution of sodium hydroxide. A molar excess, 3.3 mg of iodoacetamide was added to the solution and stirred until the reaction was complete, about 2 hours. The thiol group of the amino acid of ciatein of albumin reacts with the iodoacetamide to form an albumin blocked with thiol.

EXAMPLE 6 This Example presents a comparison of pH regulators of thiol blocked and unblocked BSA and movement at different temperatures. Two aqueous supply solutions were made.

The composition of this regulator is established below: 50 mM Sodium Barbital, 150 mM sodium chloride, 1 mM EDTA terasodium satin, 0.5% w / v sodium azide, 0.05% w / v albumin bovine serum (BSA) and 0.15 p / v of bovine globulin range (BGG) with a final pH of 8.5. A supply solution comprised BSA (not blocked with thiol) and was prepared from Miles Lot 482. A second supply solution comprised a blocked BSA sample and was prepared from Miles Lot 1017. They were suspended in each supply solution , paramagnetic particles coupled with mouse monoclonal anti-prolactin. The aliquots of each supply solution were placed in tissue culture flasks. Each sample moved oscillating at room temperature. Each sample was inspected at various times and a determination of the degree of formation of fibrils or aggregates was made. In the Tables provided below, the following notes were used. The largest clod formation is characterized by more "+" signs. The "-" indicates no observed lump. The "+" sign indicates few observed lumps. The "++++" sign indicates a large number of formed lumps. Table 1 establishes the results where BSA Miles Lot 482 and BSA Miles Lot 1017, at a pH of 8.5, moved in an oscillating manner at room temperature.

TABLE 1 The data in Table 1 provide that thiol blocked BSA, BSA Miles Lot 1017, at a pH of 8.5 and subjected to oscillating movement at room temperature provided a more stable reagent suspension. Table 2 establishes the results where BSA Miles Lot 482 and BSA Miles Lot 1017 at a pH of 8.5 and were maintained at 4 ° C and subjected to oscillating movement.

TABLE 2 The data in Table 2 provide that thiol blocked BSA, BSA Miles Lot 1017 at a pH of 8.5 and at 4 ° C and underwent oscillating movement providing more stable reagent suspensions. Two aqueous supply solutions of 0.1 M pH phosphate buffer, pH 7.5, were made with 1% BSA. A supply solution was made with BSA not blocked and BSA Miles Lot 482 designated. A supply solution was made with thiol-blocked BSA and designated BSA Miles Lot 1017. These two solutions also received paramagnetic particles bound to the anti-prolactin mouse monoclonal antibody. Table 3 establishes the results of BSA Miles Lot 482 and BSA Miles Lot 1017, at a pH of 7.5 maintained at room temperature and subjected to oscillating movement.

TABLE 3 The data in Table 3 provide that thiol blocked BSA, BSA Miles Lot 1017, at a pH of 7.5 and at room temperature and underwent oscillating movement, and produced more stable reagent suspensions. In this way, since the preferred embodiments of the invention have been described, the present invention is capable of variation and modification and, therefore, the present invention should not be limited to the precise details established, but should include said changes and modifications. alterations as they fall within the vision of the following claims.

Claims (10)

1. - A reagent suspension comprising: an aqueous suspension agent, a plurality of particles coupling a reagent capable of reacting with an analyte, and a protein blocked with thiol carried by said suspension and having the formula: P - X wherein P is a protein having one or more thiol groups, and X is a blocking group covalently bound to said thiol group, such that the thiol group is blocked or is chemically inert.

2. The suspension according to claim 1, wherein said blocking groups comprise: C? -C? 2 alkyl, C3-C? 2 cycloalkyl, C1-C12 haloalkyl, C? -C? 2 hydroxyalkyl, C2- alkenyl C? 2 C2 haloalkenyl-C? 2 C2 hydroxyalkenyl-C? 2 C2-C? 2, haloalkynyl, C2-C? 2 C2-C12 hydroxyalkynyl, CßH5, alkoxy C? C10, C1-C10 haloalkoxy, C 1 -C 10 hydroxyalkoxy, C 1 -C 10 alkylthio, C 1 -C 10 alkynylsulfonyl, C 1 -C 10 alkylsulfonyl, and amine, amide, imide, substitutions and derivatives thereof.

3. The suspension according to claim 1, wherein P is albumin.

4. The suspension according to claim 3, wherein said albumin is derived from one of the following sources consisting of sources of human, bovine, porcine and poultry.

5. The suspension according to claim 1, wherein X consists of: -MR wherein R is C? -C12 alkyl, C3-d2 cycloalkyl, C? -C? 2 haloalkyl, dC? 2 hydroxyalkyl, C2-C? 2 alkenyl, C2-C? 2 haloalkenyl, C2-C? 2 hydroxyalkenyl, alkynyl AD? 2, haloalkynyl, C2-C? 2, hydroxyalkynyl C2-C? 2, C6H5, alkoxy d-Cio haloalkoxy d-C10 hydroxyalkoxy Ci-do, C1-C10, alquinilsulf ynyl C1-C10 alkylsulphinyl, C1-C10 , and amine, amide, imide, substitutions and derivatives thereof.

6. The suspension according to claim 5, wherein R is: N H2 O I I I (CH2) n-CH-C-OH where n is an integer of 1 -3.

7. The suspension according to claim 6, wherein n is 1.

8. The suspension according to claim 1, wherein X is: O I I -CH2-C-Z wherein Z is hydroxyl, C? C6 alkyl, C3-C6 alkyl, Ci-Css, hydroxyalkyl? C6 alkyl, C2-C6 alkenyl, C2-C6 haloalkenyl, C2-C6 hydroxyalkenyl, C2-C6 alkynyl, haloalkynyl C2 -C6, C2-C6 hydroxyalkynyl, C6H5, C? -C4 alkoxy, C? -C4 haloalkoxy, C? -C hydroxy alkoxy, C? -C alkylthio, C, -C4 alkylsulfinyl, Ci-C4 alkylsulfonyl, and amine, amide , imide, derivatives and substitutions thereof, or Z is: U N-W wherein U or W independently are hydrogen,? C6 alkyl, C3-C6 cycloalkyl, haloC? C6 hydroxyalkyl, d-C6 alkyl, C2-C6 haloalkenyl, C2-C6 hydroxyalkenyl dd, ynyl alk C2-C6, C3-C6 haloalkynyl, C3-C6 hydroxyalkynyl, C6HS, C1-C, haloalkoxy C? -C4 hydroxyalkoxy C? -C4, alkylthio C? -C4, C1-C4 alkylsulfinyl, alkylsulfonyl C -C, and amine, amide, imida, derivatives and substitutions thereof.

9 - The suspension according to claim 3, wherein said particles are paramagnetic and have a diameter of 0.1 to 10μ.

10. - The suspension according to claim 1, wherein said particles are paramagnetic and said suspension is carried by a plastic container. 1 - A method for making a reagent suspension comprising the steps of, combining water and a protein blocked with thiol, and one or more solid phase materials. 12. A method for detecting the presence or absence of an analyte in an aqueous sample placed in a plastic container, said method comprising: combining said sample with particles for the reaction with said analyte, a protein blocked with thiol, and water for forming a suspension of said particles and the protein blocked with thiol, said suspension resists the formation of aggregates, imposing reaction conditions on said suspension to form a reaction product in the presence of an analyte; and checking the suspension for said reaction product as an indication of the presence of said analyte. 13. The method according to claim 12, wherein said thiol blocked protein is composed of the formula: P - X wherein P is a protein having a thiol group, and X is a blocking group covalently bound to said thiol group, said blocking group consisting of C 1 -C 2 alkyl, C 3 -Ci 2 cycloalkyl, C 1 -C 12 haloalkyl, C 1 -C 12 hydroxyalkyl , C2-C12 alkenyl, C2-C12 haloalkenyl, C2-C2 hydroxy alkenyl, C2-C12 alkynyl, C2-C2 haloalkynyl, C2-C2 hydroxyalkynyl, C6H5, d-do alkoxy, C1-C10 haloalkoxy, hydroxyalkoxy C1-C10, C-C10 alkylthio, C1-C10 alkynylsulfinyl, C1-C10 alkylsulfonyl, and amine, amide, imide, substitutions and derivatives thereof. 14. The method according to claim 13, wherein X consists of: -MR wherein R is C? -C6 alkyl, C3-C6 cycloalkyl, C? -C6 haloalkyl, C1-C10 hydroxyalkyl, C2-C6 alkenyl, C2-C6 haloalkenyl, dd hydroxyalkenyl, C2-C6 alkynyl, C2-C6 haloalkynyl, hydroxyalkynyl d-C6, C6H5, C6-C4 alkoxy, haloalkoxy C? -C, C1-C4 hydroxyalkoxy, alkylthio C? -C4, C 1 -C 4 alkynylsulfinyl, C 1 -C 4 alkylsulfonyl, and amine, amide, imide, substitutions and derivatives thereof. 15. The method according to claim 14, wherein said protein is albumin blocked with thiol and said particles are paramagnetic particles having a diameter of about 0.1 to 10 microns.