Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
  • G01N33/549—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic with antigen or antibody entrapped within the carrier

Definitions

• the present invention relates generally to the field of diagnostic assays and to apparatus and articles useful therefor.
• the invention relates to the area of reagents for diagnostic assays.
• it relates to means for the accurate delivery of small amounts of a soluble reagent.
• immunoassays 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.
• 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.
• 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.
• 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.
• the article has particular utility in immunoassays which employ a soluble mono- or polyclonal antibody preparation as a reagent.
• 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.
• 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.
• 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.
• the present invention provides an article which has particular application to diagnostic assays which employ a soluble reagent.
• assays include 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.
• 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.
• 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.
• 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.
• 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.
• the saturation volume of the shaped mass is 250 ⁇ l
• the shaped mass is placed in a container which will protect it during storage and handling.
• 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.
• FIG. 2 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.
• 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.
• 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.
• 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.
• 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.
• the liquid sample being assayed is applied to the member 20 by introduction through opening 12.
• 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.
• 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.
• labeled antibody solution and washing liquid 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.
• the antibody label is an enzyme
• a solution of the enzyme substrate is added to member 20.
• 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.
• 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.
• 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
• Figure 3 shows the use of the present invention with the article of Fig. 2.
• the shaped mass 30 of Fig. 1 is shown positioned within the space defined by the sidewall 14 of container 10.
• 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.
• the mass 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.
• member 30 functions as a reaction vessel for the immunoreaction of antibody and HCG.
• a wash solution which may be additional urine, is applied to wash the antibody-antigen complex from the member 30.
• a substrate for alkaline phosphatase for example, indoxyl phosphate
• stepwise reaction is recommended, in certain circumstances, a volume of urine may be used which exceeds the saturation volume of the porous member.
• a volume of urine may be used which exceeds the saturation volume of the porous member.
• 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.
• the shaped porous mass may have contained therein an antibody against an antigen on the surface of a cellular particle.
• 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.
• the shaped mass may be used merely to conveniently deliver a measured amount of soluble antibody.
• 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.

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• Health & Medical Sciences (AREA)
• Immunology (AREA)
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• Engineering & Computer Science (AREA)
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• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Investigating Or Analysing Biological Materials (AREA)

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Publications (1)

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Citations (8)

• 1985
  • 1985-08-26 IL IL76184A patent/IL76184A0/xx unknown
  • 1985-08-26 AU AU47771/85A patent/AU4777185A/en not_active Abandoned
  • 1985-08-26 EP EP85904370A patent/EP0190335A1/en not_active Withdrawn
  • 1985-08-26 BR BR8506892A patent/BR8506892A/pt unknown
  • 1985-08-26 WO PCT/US1985/001614 patent/WO1986001603A1/en not_active Application Discontinuation
  • 1985-08-26 JP JP60503942A patent/JPS62500121A/ja active Pending
• 1986
  • 1986-03-12 NO NO860937A patent/NO860937L/no unknown
  • 1986-03-19 FI FI861149A patent/FI861149A/fi not_active Application Discontinuation
  • 1986-04-23 DK DK186586A patent/DK186586D0/da not_active Application Discontinuation

Patent Citations (8)

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