ARTICLE FOR DIAGNOSTIC ASSAYS

Abstract: Article (30) useful in diagnostic assays for delivery of a measured amount of a soluble reagent used in the assay. The article (30) is a shaped porous mass having the soluble reagent dispersed within the pores thereof. The article (30) is prepared by adding the soluble reagent to the shaped mass as a solution to distribute the soluble reagent within the pores of the shaped mass. The shaped mass is then dried, for example, by lyophilization. A solvent poured through the mass will redissolve the reagent. In a preferred embodiment, the reagent is a labeled monoclonal antibody useful in an immunoassay.

Description: DESCRIPTION Article For Diagnostic Assays Field of the Invention The present invention relates generally to the field of diagnostic assays and to apparatus and articles useful therefor. In another aspect, the invention relates to the area of reagents for diagnostic assays. In another aspect it relates to means for the accurate delivery of small amounts of a soluble reagent. Background Highly sensitive diagnostic assays, particularly immunoassays and other ligand-receptor assays permit the precise measurement of very small amounts of analytes occurring in samples of body fluids such as serum or plasma, urine and sputum. These analyses are complicated by the fact that they depend upon the accurate measurement and transfer of small amounts of reagent. For example, many immunoassays employ a soluble antigen or antibody. In these assays, the antigen or the antibody is often provided in a lyophilized form which is dissolved and transferred by pipette. Other problems arise from the use of samples which, as collected, contain a large amount of unwanted cellular debris which inhibit or mask the formation of an antigen- antibody complex, in the case of an immunoassay, or other complexes characteristic of a ligand-receptor assay. The debris may also clog or otherwise block access to surfaces upon which occur some part of the assay process. These complications are not insurmountable when currently available diagnostic kits are used by trained personnel. However, in many cases they substantially reduce, if not eliminate, the possibility that an assay can be reliably carried out by persons lacking such training. This is most serious in the case of products intended for sale over the counter to lay persons. However, means for eliminating these problems would make the assays more convenient for use by even trained personnel. Summary of the Invention The present invention is an article or apparatus which can be used to reduce or eliminate the afore¬ mentioned problems. The article comprises a shaped porous mass containing a measured quantity of a soluble reagent dispersed throughout but adhering or coated to the surfaces of the pores or entrapped within the pores. Introduction of a solvent for the reagent to the shaped porous mass effectively reconstitutes a solution of the soluble reagent so that it can be used in the assay. The article can be used as a prefilter in a diagnostic assay by selection of a pore size which will be small enough to filter"out unwanted cellular debris. Further, since the amount of soluble reagent incorporated into the shaped porous mass can be predetermined and accurately introduced, the subsequent user need only manipulate the shaped porous mass to introduce a measured quantity of reagent into a diagnostic assay, eliminating the need for preparing and pipetting a solution. As de¬ scribed in more detail hereafter, the article has particular utility in immunoassays which employ a soluble mono- or polyclonal antibody preparation as a reagent. Finally, the porous mass can act as a reaction chamber in which formation of the product of the reaction between the soluble reagent and an analyte or other component can occur. Accordingly, an object of the invention is to simplify the conduct of diagnostic assays. A further object is to provide a simple means for accurately introducing a quantity of a soluble reagent to a process, particularly a diagnostic assay. Yet another object is to provide means which simultaneously act as a filtering means in a diagnostic assay and also delivers a premeasured quantity of a soluble reagent used in the assay. The manner of accomplishment of these and other objects will be apparent from the following description of preferred embodiments. Brief Description of Drawings Figure 1 is a side view of a cylindrical cartridge for the article. Figure 2 is a sectional side view of a cylindrical container for a diagnostic assay. Figure 3 is a sectional side view of a cylindrical container of Figure 2 in accordance with the invention. Detailed Description As already indicated, the present invention provides an article which has particular application to diagnostic assays which employ a soluble reagent. Among such assays are ligand-receptor assays generally, including but not limited to immunoassays, which use either a soluble antigen or a polyclonal or monoclonal antibody preparation as a soluble reagent, and DNA probe assays, which use a nucleic acid oligomer as a soluble reagent. The invention is particularly preferred for use in immunometric assays which use a soluble labeled antibody as a component of the assay, most preferably a monoclonal antibody. Accordingly, the invention will be described with reference to the use of a monoclonal antibody as the soluble reagent although it will be appreciated by those skilled in the art that the invention can be used in connection with other soluble reagents and that this application of the invention is, therefore, merely exemplary. The article of the invention comprises a shaped porous mass containing a measured quantity of the soluble reagent dispersed throughout the porous structure, coated or otherwise adhered to the surfaces of, or entrapped within, the porous mass. The particular shape of the porous body is not critical and usually will be of a shape convenient for a specific application. Most often it will be shaped to fit within a vessel or other receptacle adapted to receive the porous mass for its intended use. Referring to Fig. 1, the porous mass is shown as a cylindrical cartridge 30 which will have a diameter which permits snug insertion -into a cylindrical receptacle, although other shapes are equally useful, for example, a conical mass or cubical mass may be better suited for some applications. The porous mass will most often be of a polymeric material such as polystyrene, polyethylene, nylon, polyester, cellulose acetate, and the like. A shaped body of glass or mineral fibers may also be used. The material actually selected must, of course, be non- reactive with the soluble reagent and not bind with the reagent in any other way which substantially impairs the ability of the reagent to redissblve. The physical structure of the shaped mass is not critical and, for example, can be obtained by organizing individual fibers or by release of a blowing agent in a molten mass as it cools to form an open cell foam. The techniques of making such bodies are, of course, well known and make no part of this invention. As will become clearer later, the principal criteria for selecting a particular mass will be pore size considerations and compatibility with the soluble agent. Incorporation of the soluble reagent into the shaped porous mass is achieved by its addition as a solution to the mass followed by a drying operation, preferably lyophilization in the case of temperature sensitive biologicals such as antigens, antibodies or nucleic acid oligomers. Usually a quantity of solution less than that which will completely saturate the shaped mass is used. If this is not done, a substantial portion of the reagent will likely become coated on the exterior surface of the shaped mass where it can easily be dislodged by abrasion. For example, if the saturation volume of the shaped mass is 250 μl, it is desirable to introduce the reagent in only about 200 μl of total solution in a manner which avoids local saturation with resultant migration of a substantial portion of the solution to the outer surface under circumstances where a film of the reagent forms on the outer surface. Preferably, before or after introduction of the soluble reagent, the shaped mass is placed in a container which will protect it during storage and handling. In the case of a cylindrical cartridge, a protective sleeve may be used. The protective container may be adapted to be easily removed or joined to another member used in the assay. A preferred apparatus for conducting diagnostic assays, particularly immunoassays, with which the article May 11, 1984, the disclosure of which is incorporated by reference herein. That apparatus is shown in Fig. 2. Thus, in Fig. 2, there is shown a cylindrical container 10, although it may have any other appropriate shape, having an upper opening 12 defined by sidewall 14. The container may be made of glass or a suitable plastic material. As shown in Fig. 2, container 10 also has a lower opening 16, in which is inserted a removable plug 18, to permit insertion of porous member 20, a circular membrane or filter disc, and an optional member 21, whose function is described below, which rest on cylindrical absorbent member 22, which is also inserted through opening 16. A portion of container 10 is constricted as shown in Fig. 2 by reference numeral 24 to provide an integral funnel to direct sample onto the member 20 and to assure that effective washing of sample and other addends onto the member 20 is accomplished. The size of member 22 and, therefore, the volume of - the portion of container 10 below the constriction is preferably selected so that all of the liquid to be added to the apparatus during an assay can be received in and retained in absorbent member 22. Means for venting air (not shown in Fig. 2), for example, small ports, is provided in container 10, near the bottom, to allow displaced air to escape. Optionally, the bottom of container 10 can be eliminated and liquid allowed to pass through members 20 and 22 and exit the container through the bottom. However, since the article is intended to be disposable and to facilitate the disposal of sample in a simple and hygienic fashion, it is preferred to use a structure shown in Fig. 2. Member 20 may be used to either filter cellular or other material from a sample or, for example, as a support for bound antibody against an antigen being assayed to remove the antigen from solution. In any case, the liquid sample being assayed is applied to the member 20 by introduction through opening 12. After the sample permeates the member 20 and " the liquid is drawn therethrough by and into absorbent member 22, a solution of labeled antibody in the case of a "sandwich assay", preferably a monoclonal antibody, is added through opening 12 to member 20. The labeled antibody then binds either to antigen bound to antibody on the member 20 or associated with cellular material trapped on the surface of 20. If member 20 has a monoclonal antibody bound to it, and the labeled antibody is also a monoclonal antibody, the two antibodies are selected to bind to non-interfering antigen binding sites as described in U.S. 4,376,110 and application Serial Number 323,498 filed June 6, 1981, the disclosures of which are incorporated by reference. Preferably the soluble antibody is labeled with an enzyme although other conventional immunoassay labels may be used in appropriate circumstances. For example, a - fluorescent label or a radionuclide can be used. After the labeled antibody solution has passed through the member 20, a washing liquid is applied to member 20 to flush unbound labeled antibody from member 20 and into member 22. The sloping structure of the walls 24 provides an integral funnel to facilitate application of the washing liquid to the walls to remove adhered residue of the labeled antibody solution. The addition of labeled antibody solution and washing liquid to the member 20 may be preceded by brief incubation periods to permit more extensive binding by antibody or antigen in solutions trapped on or in the interstices of member 20 and, thereby, increase the sensitivity of the assay. However, such incubation steps are either unnecessary or may be very brief, i.e., on the order of 60 seconds or less. The flow of solutions containing antigen or labeled antibody through the member 20 results in a substantially faster rate of binding than is observed in the absence of flow. If the antibody label is an enzyme, after washing to remove unbound antibody from member 20, a solution of the enzyme substrate is added to member 20. If the target antigen is bound either to antibody bound to member 20 or to cellular material on member 20, the antigen will have bound to it a portion of labeled antibody. The enzyme will cause the substrate to react and generate, if properly selected, a color change which can be detected visually or by instrumental means. In the case of the use of cellulose acetate material, the absorbent member 22 may bind labeled antibody non-specifically at its upper surface. Accordingly, some visual color change may occur' at this surface just under the member 20. To avoid this color change being visualized through member 20, a separating member (designated 21 in Fig. 2) of porous polyethylene or other material which does not bind antibody non-specifically is preferably disposed between members 20 and 22. Figure 3 shows the use of the present invention with the article of Fig. 2. Thus, the shaped mass 30 of Fig. 1 is shown positioned within the space defined by the sidewall 14 of container 10. To illustrate its function, the use of the composite article of Fig. 3 to perform an assay for human choriogonadotropin, HCG, a hormone which is elevated during pregnancy and which may be detected in urine, will be described. In this case, the soluble reagent used is a monoclonal antibody against HCG which has been lyophilized within member 30, which is itself fabricated of cellular acetate fibers. The monoclonal antibody is labeled with the enzyme alkaline phosphatase, although other enzymatic, fluorescing or even radioactive labels may be used.. A measured urine sample is added to the top of the shaped porous mass 30, preferably in an amount calculated not to completely saturate the mass. The volume of sample, since it does not saturate the mass, is retained within the pores of the mass and functions to dissolve the antibody, or at least a substantial portion thereof. By proper selection of pore size, the mass also acts as a prefilter to separate any epithelial cells found in the urine which might clog member 20. The urine sample is allowed to remain within member 30 long enough to dissolve the antibody and permit its binding to any HCG within the sample. In this way, member 30 functions as a reaction vessel for the immunoreaction of antibody and HCG. After such incubation, a wash solution, which may be additional urine, is applied to wash the antibody-antigen complex from the member 30. As the antibody-antigen complex is washed from member 30, it contacts and passes through membrane 20. Member 30 is then removed and discarded. This affords an opportunity for the antibody bound to the solid phase to bind the HCG:labeled antibody complex and remove it from the solution. Unbound labeled antibody is washed through member 20 and into the absorbent mass 22. After the sample and wash have passed through member 20, a small amount of solution containing a substrate for alkaline phosphatase, for example, indoxyl phosphate, is added to the top of member 20 to react with any enzyme bound to the filter as the result of the reaction of HCG with the antibody bound to member 20 and the enzyme labeled soluble antibody. Formation of a visible blue color is indication of this reaction. While the stepwise reaction is recommended, in certain circumstances, a volume of urine may be used which exceeds the saturation volume of the porous member. As a result, the soluble antibody and unfiltered material in the sample are conducted directly to member 20. The time for reaction between the antibody and HCG in the sample is, of course, reduced. The stepwise procedure, therefore, usually provides a more sensitive assay. In another application, the shaped porous mass may have contained therein an antibody against an antigen on the surface of a cellular particle. In such a case, the pore size of member 30 must be selected to permit passage of the target material which is then trapped on the surface of member 20. The pore size of member 30 may, however, be selected to inhibit the passage of larger cells which might interfere with the assay by clogging member 20. This procedure is particularly well suited to the detection of bacteria, fungi, parasites and other particulate organisms and viruses which can be filtered and have associated antigens. In another application, the shaped mass may be used merely to conveniently deliver a measured amount of soluble antibody. Thus, after sample has been added to member 20, the member 30 is inserted into opening 12 and a solvent for the reagent within, for example, an antibody, is allowed to flow through the member 30 to dissolve and deliver the antibody to the surface of member 20. The foregoing description is but illustrative of the use to which an article of the present invention may be put. Accordingly, the scope of the invention is to be considered limited only by the appended claims.

Claims: Claim 1. An article for use in a diagnostic assay which uses a measured amount of a soluble reagent comprising a shaped porous mass having the soluble reagent dispersed within the pores of the shaped mass, the soluble reagent being eluted from the porous mass by passage of a solvent for the antibody through the mass. 2. An article according to claim 1 wherein the soluble reagent is lyophilized within the pores of the shaped mass. 3. An article according to claims 1 or 2 wherein the diagnostic assay is a ligand-receptor assay and the soluble reagent is one of said ligand receptor pair. 4. An article according to claim 3 wherein the soluble reagent i-s selected from a preparation of polyclonal antibodies, a monoclonal antibody, an antigen or a nucleic acid oligomer. 5. An article according to claim 4 wherein the soluble reagent is a monoclonal antibody. 6. An article according to claims 1 or 2 wherein the shaped porous mass is a body of polymeric material, glass or mineral fibers. 7. An article according to claim 6 wherein the polymeric material is selected from a nylon, a polyester, a polyolefin, polystyrene or cellulose acetate. 8. An article according to claim 7 wherein the soluble reagent is selected from a preparation of polyclonal antibodies, a monoclonal antibody, an antigen or a nucleic acid oligomer. 9. An article according to claim 8 wherein the soluble reagent is a monoclonal antibody. 10. In a diagnostic assay of a sample wherein a measured quantity of a soluble reagent is employed, the improvement comprising adding the soluble reagent to the assay , by passing a solvent for the reagent through a shaped, porous mass having the soluble reagent dispersed within the pores thereof whereby the soluble reagent is dissolved in the solvent and washed from the shaped mass. 11. An assay according to claim 10 wherein the soluble reagent is selected from a polyclonal antibody preparation, a monoclonal antibody, an antigen or a nucleic acid oligomer. 12. An assay according to claim 10 or 11 wherein the soluble reagent is labeled to permit detection. 13. An assay according to claim 12 wherein the label is selected from an enzyme, a fluorescent member or a radionuclide. 14. An assay according to claim 10 or 11 wherein the shaped porous mass is of a polymeric material, glass or a mineral fiber. 15. An assay according to claim 14 wherein the polymeric material is selected from a nylon, a polyolefin, a polyester, polystyrene or cellulose acetate. 16. A diagnostic assay according to claims 10 or 11 wherein the shaped porous mass is used to filter cellular material from the sample. 17. A diagnostic assay according to claim 10 or 11 wherein the sample is selected from urine, serum, plasma or sputum. 18. A diagnostic assay according to claim 16 wherein the sample is selected from urine, serum, plasma or sputum. 19. A diagnostic assay according to claim 10 or 11 wherein the solvent is the sample being assayed. 20. A diagnostic assay according to claim 19 wherein the sample is urine. 21. A diagnostic assay according to claim 19 wherein the soluble reagent reacts with a component of the sample within the pores of the porous mass.

Inventors: LIU LYNNE HLSELICK, LIU, LYNNE, HLSELICK
Applicants: HYBRITECH INC, HYBRITECH INCORPORATED
Application year: 1985
Main classification: G01N33
IPC codes: C12M1, C12Q1, G01N33