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Polyacrylamide gel particles having hapten moieties bound thereto as immunoassay reagent    
United States Patent4822747   
Link to this pagehttp://www.wikipatents.com/4822747.html
Inventor(s)Johnson; Richard D. (Elkhart, IN); Runzheimer; H. Volker (Bergisch Gladbach, DE); Sommer; Ronald G. (Elkhart, IN); Yip; Kin F. (Elkhart, IN)
AbstractAn immobilized hapten reagent for use in a specific binding assay for the determination of a hapten or binding analog thereof in a liquid test sample and methods for preparing and using the immobilized hapten reagent. The immobilized hapten reagent comprises a hapten moiety covalently linked substantially only to the external surface of a polyacrylamide gel particle. The immobilized hapten reagent is prepared by forming a reaction mixture comprising the hapten moiety and the carrier material in a nonswelling solvent wherein the gel particle is substantially impervious to the hapten moiety and wherein an analytically insignificant amount of the hapten moiety is nonspecifically bound to the gel particle. The immobilized hapten reagent is characterized by being substantially stable in aqueous solutions and exhibits an insignificant amount of leakage of the hapten moiety into a liquid test medium.
   














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Inventor     Johnson; Richard D. (Elkhart, IN); Runzheimer; H. Volker (Bergisch Gladbach, DE); Sommer; Ronald G. (Elkhart, IN); Yip; Kin F. (Elkhart, IN)
Owner/Assignee     Miles Inc. (Elkhart, IN)
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Publication Date     April 18, 1989
Application Number     06/939,902
PAIR File History     Application Data   Transaction History
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Filing Date     December 9, 1986
US Classification     436/532 436/531 436/535
Int'l Classification     G01N 033/547
Examiner     Marantz; Sidney
Assistant Examiner    
Attorney/Law Firm     Collins; Daniel W.
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USPTO Field of Search     436/531 436/532 436/535
Patent Tags     polyacrylamide gel particles hapten moieties bound thereto as immunoassay reagent
   
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4551426
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What is claimed is:

1. A method of preparing a stable immobilized hapten reagent for use in the specific binding assay determination of a hapten or binding analog thereof in a liquid test sample, the method comprising the steps of:

(a) reacting a hapten moiety with a polyacrylamide gel particle comprising a plurality of external and internal chemically active functional groups wherein the reaction is performed in a solvent in which the gel particle is substantially nonswollen and under conditions to form a covalent bond between the hapten moiety and said external active functional groups which is substantially stable in aqueous solutions;

(b) washing the gel particle resulting from step(a) with a nonswelling solvent;

(c) washing the gel particle resulting from step(b) with an aqueous solution; and

(d) isolating the immobilized hapten reagent resulting from step(c) comprising said gel particle and said hapten moieties bound thereto wherein substantially all of said bound hapten moieties are covalently linked to the external surface functional groups by a linking group which is substantially stable in aqueous solutions.

2. The method of claim 1 wherein the nonswelling solvent is an organic solvent.

3. The method of claim 2 wherein said organic solvent is selected from the group consisting of dimethylformamide, dimethylsulfoxide, acetone, chlorinated hydrocarbons, and cyclic and acyclic alkylethers.

4. The method of claim 1 wherein the hapten moiety is selected from the group consisting of digoxin, digitoxigenin, digitoxin, digoxigenin, and 12-0-acetyldigoxigenin.

5. The method of claim 4 wherein the linking group is selected from the group consisting of bifunctional residues of 1,6-hexamethylenediamine, 6-aminohexanol, 1,12-diamino-4,9-dioxadodecane, 1,17-diamino-3,6,9,12,15-pentaoxaheptadecane, 6-aminocaproic acid, and bovine serum albumin.

6. The method of claim 4 wherein the linking group is a bifunctional residue of 6-aminocaproic acid and the hapten moiety is digitoxigenin.

7. The method of claim 1 wherein the hapten moiety is a glycosylated peptide sequence.

8. The method of claim 7 wherein the linking group is selected from the group consisting of bifunctional residues of 1,1'-[methylenedi-4,1-phenylene]bis-maleimide, bismaleimido-hexane and bismaleimido-hexethylene glycol.

9. The method of claim 7 wherein said glycosylated peptide sequence corresponds with the glucosylated N-terminal peptide sequence in the beta-subunit of human hemoglobin and the linking group is bismaleimido-hexaethylene glycol.

10. The method of claim 1 wherein less than from about 1.times.10.sup.-12 moles hapten/g gel particle dissociates from the gel particle of the immobilized hapten reagent upon standing in an aqueous solution for one week.

11. The method of claim 10 wherein less than from about 1.times.10.sup.-13 moles hapten/g gel particle dissociates from the gel particle of the immobilized hapten reagent upon standing in an aqueous solution for one week.

12. The method of claim 1 wherein the polyacrylamide gel particle in its nonswollen state is substantially impervious to the hapten moiety.

13. A substantially stable immobilized hapten reagent for use in the specific binding assay determination of a hapten or binding analog thereof in a liquid test sample, the immobilized hapten reagent comprising a polyacrylamide gel particle which is substantially swellable in water and a plurality of hapten moieties bound thereto, said gel particle comprising a plurality of external and internal functional groups wherein substantially all of said bound hapten moieties are covalently linked to said external functional groups by a linking group which is substantially stable in aqueous solutions.

14. The immobilized hapten reagent of claim 13 wherein the hapten moiety is selected from the group consisting of digoxin, digitoxigenin, digitoxin, digoxigenin, and 12-0-acetyldigoxigenin.

15. The immobilized hapten reagent of claim 14 wherein the linking group is selected from the group consisting of bifunctional residues of 1,6-hexamethylenediamine, 6-aminohexanol, 1,12-diamino-4,9-dioxadodecane, 1,17-diamino-3,6,9,12,15-pentaoxaheptadecane,, 6-aminocaproic acid, and bovine serum albumin.

16. The immobilized hapten reagent of claim 14 wherein the linking group is 6-aminocaproic acid and the hapten moiety is digitoxigenin.

17. The immobilized hapten reagent of claim 13 wherein the hapten moiety is a glycosylated peptide sequence.

18. The immobilized hapten reagent of claim 17 wherein the linking group is selected from the group consisting of bifunctional residues of 1,1'-[methylene-4,1-phenylene]bismaleimide, bismaleimido-hexane and bismaleimido-hexaethylene glycol.

19. The immobilized hapten reagent of claim 17 wherein said glycosylated peptide sequence corresponds with the glucosylated N-terminal peptide sequence in the beta-subunit of human hemoglobin and the linking group is bismaleimido-hexaethylene glycol.

20. The immobilized hapten reagent of claim 13 wherein less than from about 1.times.10.sup.12 moles hapten/g gel particle dissociates from the gel particle of the immobilized hapten reagent upon standing in an aqueous solution for one week.

21. The immobilized hapten reagent of claim 20 wherein less than from about 1.times.10.sup.-13 moles hapten/g gel particles dissociates from the gel particle of the immobilized hapten reagent upon standing in an aqueous solution for one week.

22. In an immunometric assay method for determining a hapten or analog thereof in a liquid test sample, wherein the test sample is contacted with a labeled antibody reagent capable of binding with said hapten or analog thereof and with an immobilized form of the hapten capable of binding with said antibody reagent, and wherein the amount of labeled antibody reagent which becomes bound to said hapten or analog thereof in the sample compared to that which becomes bound to the immobilized reagent is determined and related to the presence of the hapten or analog thereof in the test sample,

the improvement which comprises employing as the immobilized reagent a substantially stable immobilized hapten reagent comprising a polyacrylamide gel particle which is substantially swellable in water and a plurality of hapten moieties bound thereto, said gel particle comprising a plurality of external and internal function groups wherein substantially all of said bound hapten moieties are covalently linked to said external functional groups by a linking group which is substantially stable in aqueous solutions.

23. The assay method of claim 22 wherein the hapten moiety is selected from the group consisting of digoxin, digitoxigenin, digitoxin, digoxigenin, and 12-0-acetyldigoxigenin.

24. The assay method of claim 22 wherein the linking group is selected from the group consisting of bifunctional residues of 1,6-hexamethylenediamine, 6-aminohexanol, 1,12-diamino-4,9-dioxadodecane, 1,17-diamino-3,6,9,12,15-pentaoxaheptadecane, 6-aminocaproic acid, and bovine serum albumin.

25. The assay method of claim 23 wherein the linking group is 6-aminocaproic acid and the hapten moiety is digitoxigenin.

26. The assay method of claim 22 wherein the hapten moiety is a glycosylated peptide sequence.

27. The assay method of claim 26 wherein the linking group is selected from the group consisting of bifunctional residues of 1,1'-[methylene-4,1-phenylene]bismaleimide, bismaleimido-hexane and bismaleimido-hexaethylene glycol.

28. The assay method of claim 26 wherein said glycosylated peptide sequence corresponds with the glucosylated N-terminal peptide sequence in the beta-subunit of human hemoglobin and the linking group is bismaleimido-hexaethylene glycol.

29. The assay method of claim 22 wherein less than from about 1.times.10.sup.-12 moles hapten/g gel particle dissociates from the gel particle of the immobilized hapten reagent upon standing in an aqueous solution for one week.

30. The assay method of claim 29 wherein less than from about 1.times.10.sup.-13 moles hapten/g gel particle dissociates from the gel particle of the immobilized hapten reagent upon standing in an aqueous solution for one week.

31. In a reagent system for determining a hapten or analog thereof in a liquid test sample, which reagent system comprises (1) a labeled reagent capable of binding with said hapten or analog thereof and (2) an immobilized reagent comprising an immobilized form of the hapten capable of binding with said labeled reagent,

the improvement which comprises employing as the immobilized reagent a substantially stable immobilized hapten reagent comprising a polyacrylamide gel particle which is substantially swellable in water and a plurality of hapten moieties bound thereto, said gel particle comprising a plurality of external and internal functional groups wherein substantially all of said bound hapten moieties are covalently linked to said external functional groups by a linking group which is substantially stable in aqueous solutions.

32. The reagent system of claim 31 wherein the hapten moiety is selected from the group consisting of digoxin, digitoxigenin, digitoxin, digoxigenin, and 12-0-acetyldigoxigenin.

33. The reagent system of claim 32 wherein the linking group is selected from the group consisting of bifunctional residues of 1,6-hexamethylenediamine, 6-aminohexanol, 1,12-diamino-4,9-dioxadodecane, 1,17-diamino-3,6,9,12,15-pentaoxaheptadecane, 6-aminocaproic acid, and bovine serum albumin.

34. The reagent system of claim 32 wherein the linking group is 6-aminocaproic acid and the hapten moiety is digitoxigenin.

35. The reagent system of claim 31 wherein the hapten moiety is a glycosylated peptide sequence.

36. The reagent system of claim 35 wherein the linking group is selected from the group consisting of bifunctional residues of 1,1'-[methylene-4,1-phenylene]bismaleimide, bismaleimido-hexane and bismaleimido-hexaethylene glycol.

37. The reagent system of claim 35 wherein said glycosylated peptide sequence corresponds with the glucosylatjed N-terminal peptide sequence in the beta-subunit of human hemoglobin and the linking group is bismaleimido-hexaethylene glycol.

38. The reagent system reagent of claim 31 wherein less than from about 1.times.10.sup.-12 moles hapten/g of particle dissociates from the particle of the immobilized hapten reagent upon standing in an aqueous solution for one week.

39. The reagent system reagent of claim 38 wherein less than from about 1.times.10.sup.-13 moles hapten/g gel particle dissociates from the particle of the immobilized hapten reagent upon standing in an aqueous solution for one week.
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BACKGROUND OF THE INVENTION

The present invention relates to specific binding assays and reagents for use therein for the determination of an analyte in a liquid test medium. In particular, the present invention relates to heterogeneous immunoassays for determining haptens employing immobilized hapten reagents for the separation of bound and free forms of a labeled reagent.

Various heterogeneous specific binding assays have been developed which generally involve specific binding interactions between the analyte to be detected, a specific binding partner for the analyte, and a labeled reagent, which can be the same or different as the binding partner for the analyte. When performing such assays, the labeled reagent becomes bound to its corresponding binding partner to generate a bound species, any of the labeled reagent not so bound being the free species, wherein the extent of binding is a function of the amount of analyte present. Where the detectable response of the labeled reagent is essentially indistinguishable in the bound species and the free species, it is necessary to physically separate such bound and free species from each other in order to effectively determine the amount of analyte present. Accordingly, once the bound and free species of the labeled reagent have been separated from each other, the amount of label present in either fraction thereof is determined by measuring the activity of the particular label of the labeled reagent and correlating such activity with the amount of analyte present.

The physical separation of the bound species from the free species can be accomplished in many ways. In the heterogeneous specific binding assay known as the immunometric assay, the labeled reagent comprises a labeled form of an anti-analyte antibody. According to such format, separation of the free labeled reagent from the bound labeled reagent is accomplished by addition of an immobilized form of the analyte under determination or an analog thereof which will bind with the antibody of the free labeled reagent. For example, U.S. Pat. No. 4,200,436 discloses an immunoassay for the detection of antigen employing an immobilized form of the antigen to be measured to separate the bound and free forms of a labeled monovalent antibody to the antigen. The immobilized form of the antigen is prepared by chemically binding or physically adsorbing the antigen to solid supports or carrier materials, such as polysaccharides or plastics, according to methods known in the art.

Similarly, U.S. Pat. No. 4,551,426 discloses a heterogeneous immunoassay for the hapten digoxin employing an immobilized form of ouabain (a digoxin analog) to separate the bound and free forms of an anti-digoxin labeled antibody. The immobilized form of ouabain is prepared by coupling ouabain, either directly or through a spacer arm such as a protein, polyamino acid, or synthetic linker, to a support material, such as beaded agarose, beaded dextran, polyacrylamide, or glass, according to methods known in the art.

Such support materials, however, together with the processes employed to couple the desired analyte or analog thereof (ligand) to such support materials, result in relatively unstable reagents, reagents exhibiting substantial release or leakage of the ligand into the surrounding liquid when used in an immunoassay as heretofore described. Such instability is believed to be the result of an instability in the linkage between the ligand and the support as well as nonspecific binding of the ligand to the support material. Such instability of the linkage and nonspecific binding of ligand results in a substantial amount of the ligand being slowly released or leaching into the surrounding medium. The leaching of the ligand into the test medium primarily occurs as a result of the ligand being nonspecifically bound to internal and external portions of the support material. Although inconvenient, ligand nonspecifically bound to the external surface can be removed by washing the support material with an aqueous wash solution prior to use in an assay procedure. However, ligand nonspecifically bound to internal portions cannot be effectively removed and such internalized ligand leaches out from the interior of the support material and into the liquid test medium where it can be substantially indistinguishable from analyte from a test sample and free to bind to the labeled reagent, resulting in an inaccurate measurement of the amount of analyte actually present in the test sample.

The synthesis and use of support materials, particularly crosslinked polymer supports, having chemical structures which are physiochemically compatible with the backbone structure of a peptide have also been described for use in solid-phase peptide synthesis. In particular, techniques for coupling peptides to a polymer (Stahl, et al., J. Amer. Chem. Soc., Vol. 101(18) p. 5383(1979)]and the crosslinking of various polymers [Varadarajan, et al., Biopolymers, Vol. 22, p. 839 (1983)]using reverse-phase suspension polymerization in aqueous organic solvent mixtures have been employed to obtain favorable swelling properties of such support materials in order to provide increased external and internal reaction sites.

Accordingly, it is an object of the present invention to provide an immobilized hapten reagent which is stable in aqueous solution and which does not slowly release or leach hapten into a surrounding aqueous solution.

Further, it is an object of the present invention to provide a process of covalently binding a hapten moiety substantially only to the surface of a carrier material with substantially no internalization of nonspecifically bound hapten which would otherwise be slowly released or leached into the surrounding medium.

Another object of the present invention is to provide a stable, immobilized hapten reagent for use in an immunoassay for the effective immobilization and separation of the free species of a labeled reagent from its bound species.

It is still a further object of the present invention to provide a highly sensitive liquid immunoassay having a low, analytically insignificant initial background signal for the accurate determination of a hapten or binding analog thereof in a liquid test sample.

SUMMARY OF THE INVENTION

The present invention provides an immobilized hapten reagent which is substantially stable in an aqueous environment for use in a specific binding assay, particularly an immunoassay, for the determination of a hapten or binding analog thereof from a liquid test sample. The immobilized hapten reagent comprises a carrier material composed of a polyacrylamide gel particle and a plurality of hapten moieties bound thereto. The gel particle comprises a plurality of external and internal functional groups on the internal and external surfaces of the gel particle, respectively, wherein substantially all of the bound hapten moieties are covalently linked to the external surface functional groups by a linking group which is substantially stable in aqueous solutions, particularly immunoassay test mediums, and an analytically insignificant amount of the hapten moiety remains nonspecifically bound to the carrier material. Accordingly, substantially all of the the hapten moiety remains covalently bound to the carrier material during the performance of an immunoassay and only an insignificant amount, if any, of the hapten moiety dissociates or leaches from the carrier material into the test medium.

According to the present invention, there is also provided a method of preparing the stable immobilized hapten reagent which minimizes the nonspecific binding of the hapten moiety to the gel particle, particularly the nonspecific binding of the hapten moiety to internal surface areas of the gel particle. The method comprises the steps of (a) reacting the hapten moiety with a polyacrylamide gel particle comprising a plurality of external and internal chemically active functional groups in a solvent in which the gel particle is substantially nonswollen and under conditions to form a covalent bond between the hapten moiety and the external functional groups which is substantially stable in aqueous solutions, (b) washing the gel particle resulting from step (a) with a nonswelling solvent, (c) washing the gel particle resulting from step (b) with an aqueous buffer solution, and (d) isolating the immobilized hapten reagent resulting from step (c) comprising the gel particle and the hapten moieties bound thereto wherein substantially all of the bound hapten moieties are covalently linked to the external surface functional groups.

The gel particle is substantially nonswellable when reacted with the hapten moiety in the presence of the nonswelling solvent and, accordingly, is substantially impervious to the hapten moiety and the covalently binding thereof is limited substantially only to the external surface functional groups of the gel particle. Any of the hapten moiety which becomes nonspecifically bound to the external surface of the gel particle in step (a) is removed with the nonswelling and aqueous wash solutions of steps (b) and (c), respectively.

The immobilized hapten reagent is particularly useful in a heterogeneous specific binding assay involving binding between a hapten or binding analog thereof and a labeled reagent comprising a labeled binding partner for the hapten or binding analog thereof, wherein it is necessary to physically separate any of the labeled reagent which becomes bound to the hapten or binding analog thereof from the labeled reagent not so bound. Any of the labeled reagent which does not bind to the hapten or binding analog thereof from the test medium is separated from the bound labeled reagent by binding to the hapten of the immobilized hapten reagent wherein the extent of binding is a function of the amount of hapten or binding analog present in a liquid test sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph which illustrates the dose response to digoxin generated in an immunoassay employing digitoxigenin as the hapten moiety in an immobilized hapten reagent of the present invention.

FIG. 2 is a graph which illustrates the reactivity of an immobolized glycopeptide reagent of the present invention in an immunoassay for the determination of the amount of glycosylated hemoglobin HbAlc in a whole blood sample.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The immobilized hapten reagent of the present invention can be employed in conventional heterogeneous specific binding assay methods, particularly heterogeneous enzyme immunoassays, involving binding between a hapten or binding analog thereof and a labeled reagent comprising a labeled form of a specific binding partner for the hapten or binding analog thereof. Furthermore, the hapten component of the immobilized hapten reagent can be varied for use in such specific binding assay for the detection of any hapten or binding analog thereof for which a specific binding partner exists in biological systems or can be synthesized. Where the specific binding partner for the hapten or binding analog thereof is an anti-hapten, such as an antibody to the hapten or fragment thereof, such specific binding assay method is referred to as an immunometric method.

According to such heterogeneous specific binding assay methods, particularly the immunometric method, the hapten or binding analog thereof being detected is generally combined with the labeled reagent to form a reaction mixture wherein the labeled reagent binds to the hapten being detected. The extent of such binding is then determined and related to the hapten to be determined. The ratio of the amount of labeled reagent bound to the hapten being detected (i.e., the bound species) to the amount of labeled reagent not so bound (i.e., the free species) is a function of the amount of hapten present. Since the signals generated by the label of the labeled reagent of both the bound and free species thereof are indistinguishable, it is necessary to physically separate the free species from the bound species to permit the independent determination of the amount of label present in the one or the other, which determination can then be correlated to the amount of hapten present.

The immobilized hapten reagent of the present invention is particularly useful for the separation of the free species of a labeled anti-hapten antibody from the bound species of such anti-hapten antibody in a specific binding assay wherein the free species binds to and is immobilized by the immobilized hapten reagent to achieve the necessary separation step. In particular, the immobilized hapten reagent of the present invention comprises an appropriately functionalized polyacrylamide gel particle carrier material and a plurality of hapten moieties bound thereto. The gel particle comprises a plurality of external and internal functional groups and substantially all of the bound hapten moieties are covalently linked substantially only to the external surface groups of the gel particle by a linking group. The linking group provides a covalent bond which is substantially stable in aqueous solutions, particularly in aqueous solutions comprising the liquid test medium of an immunoassay, wherein the hapten moiety remains covalently bound to the carrier material during the performance of an immunoassay to effectively separate the free species from the bound species. It is to be appreciated that such stability in aqueous environments prevents the dissociation of the hapten moiety from the carrier material and, accordingly, leaching of the hapten moiety into the test medium environment to thereby result in decreased assay sensitivity and accuracy, as will be described in greater detail hereinafter.

According to a preferred embodiment of the present invention, the immobilized hapten reagent is particularly useful in an immunoassay for the detection of digoxin from a liquid test sampel. The labeled reagent comprises an enzyme-labeled monovalent antibody fragment derived from a monoclonal antibody against digoxin, preferably the Fab' fragment of a monoclonal IgG antibody to digoxin, generally obtained according to methods known in the art and labeled with an enzyme, preferably .beta.-D-galactosidase. Preferably, the labeled reagent is electrophoretically purified on an electrophoretic polyacrylamide gel to result in a substantially pure monoconjugate preparation thereof comprising a single monovalent antibody fragment component covalently linked to a single enzyme component according to the method described in the copending U.S. patent application entitled, "Substantially Pure Enzyme-Antibody Monoconjugate Preparation" (U.S. Ser. No. 939,640), filed on even date herewith.

The immunoassay for digoxin is performed by reacting the labeled reagent, preferably the monoconjugate preparation thereof, with a test sample containing digoxin, and then adding the immobilized hapten reagent of the present invention comprising digoxin or an analog thereof, such as digitoxigenin, covalently linked to the external surface amine groups of an amine functionalized polyacrylamide carrier material by a linking group, as will be described in greater detail hereinafter. Where the hapten moiety of such immobilized hapten reagent comprises digoxin or an analog of digoxin, such as digitoxigenin, the digoxin from the liquid test sample binds to the antibody fragment of the labeled reagent to form the bound species thereof, and any of the free species of the labeled reagent which does not bind to digoxin from the test sample binds to the immobilized hapten reagent for the immobilization and subsequent separation thereof from the bound species, wherein the bound species remains in solution and the free species settles out from solution. The amount of digoxin in the test sample is then determined by measuring the enzyme activity of the bound species of the labeled reagent.

According to another preferred embodiment of the present invention, the immobilized hapten reagent comprises a glycosylated peptide sequence, such as corresponding to the glycolsylated N-terminal peptide sequence in the beta-subunit of human hemoglobin (glycopeptide), covalently linked to the external surface sulfhydryl groups of a polyacrylamide-sulfhydryl derivatized carrier material, such as described in the copending U.S. patent application entitled "Crosslinked Polyacrylamide Sulfhydryl Gel and Sulfhydryl-Functionalized Derivatives Thereof" (U.S. Ser. No. 929,904), filed on even date herewith, which is useful in an immunoassay for the determination of HbAlc. The labeled reagent comprises a monovalent antibody fragment derived from a monoclonal antibody specific for the glucosylated N-terminal peptide sequence in the beta-subunit of human hemoglobin (see European Patent Application No. 185,870) labeled with .beta.-D-galactosidase and purified to a monoconjugate preparation thereof as heretofore described, and the amount of HbAlc in the liquid test sample is determined by measuring the enzyme activity of the bound species of the labeled reagent.

The measurement of the enzyme activity in such immunoassays is accomplished by removing an aliquot of supernatant and depositing the aliquot into a reagent pad incorporated with a chromogenic substrate for the enzyme label, such as resorufin-.beta.-D-galactopyranoside, o-nitrophenol-.beta.-D-galactopyranoside, or, more preferably, a chromogenic acridinone enzyme substrate for .beta.-D-galactosidase comprising a 7-hydroxy-9H-acridin-2-one chromogen derivatized with a .beta.-D-galactose residue such as described in the copending U.S. patent application entitled, "Chromogenic Acridinone Enzyme Substrates" (U.S. Ser. No. 939,855), filed on even date herewith. The detectable signal which is generated by the interaction between the enzyme and the chromogenic substrate is then measured with, for example, a reflectance photometer, and correlated to the amount of hapten present in the liquid test sample.

IMMOBILIZED HAPTEN REAGENT

It is to be appreciated that in order to provide a highly sensitive immunoassay to accurately determined the amount of hapten present in a liquid test sample, nonspecific binding of the hapten moiety to the carrier material should be minimized. Any of such nonspecifically bound hapten moiety otherwise results in the dissociaton of the hapten moiety from the carrier material and the slow release or leaching thereof into the liquid immunoassay test medium as a free hapten species which competes with the hapten from the test sample for binding to the labeled reagent. Accordingly, the label of the labeled reagent bound to such dissociated hapten moiety provides a background signal which will interefere with the detection of the signal provided by the label of the labeled reagent bound to the hapten from the test sample to result in an inaccurate determination of the hapten being detected from the liquid test sample.

Accordingly, an important feature of the present invention is to provide an immobilized hapten reagent which is substantially stable in aqueous solutions and which comprises a hapten moiety covalently linked substantially only to the external surface of an appropriately functionalized polyacrylamide gel particle carrier material, with only an analytically insignificant amount, if any, of the hapten moiety nonspecifically bound thereto.

According to the present invention, control of nonspecifically bound hapten to within acceptable limits is achieved by reacting the hapten moiety with a carrier material comprising an appropriately functionalized or derivatized polyacrylamide resin in a nonswelling solvent in the presence of a linking group which forms a substantially stable, covalent body therebetween to provide an immobilized hapten reagent which is substantially stable in aqueous solutions. The nonswelling solvent limits the extent of covalent binding substantially only to the external surface functional groups of the carrier material, as will be described in greater detail hereinafter.

(a) Carrier Material

According to the preferred embodiment of the present invention, the carrier material of the immobilized hapten reagent is an aminoethyl-derivatized polyacrylamide resin, which is generally swellable in aqueous solutions and which can be prepared according to methods known in the art. According to such methods, a polyacrylamide resin is first prepared by the copolymerization of acrylamide and N,N'-methylenebisacrylamide [S. Hjerten and R. Mosbach, Anal. Chem., Vol. 3, p. 109(1962)], to form, under suitable conditions, crosslinked polyacrylamide chains, followed by treatment with anhydrous ethylene-diamine [J.K. Inman and H.M. Dintzis, Biochemistry, Vol. 8, p. 4074(1969)]to obtain an aminoethyl-derivatized polyacrylamide gel comprising a plurality of amine functional groups for use as the carrier material of the immobilized hapten reagent of the present invention. In addition to the derivatization of polyacrylamide with amine functional groups by direct activation of the polyacrylamide resin as heretofore described, acrylamide and methylenebisacrylamide can be copolymerized with, for example, acrylic acid esters of N-hydroxysuccinimide or N-hydroxyphtalimide which can then be readily reacted with haptens containing primary amino functions, such as an aminohexyl group, which displace the active ester in the resin to provide the immobilized hapten reagent of the present invention [see, J.K. Inman, Methods in Enzymology, Vol. 34B, pp. 30-58(1974); G.L. Stahl, et al., J. Org. Chem., Vol. 44, p. 3424(1979); G.L. Stahl, et al., J. Amer. Chem. Soc., Vol. 101, p. 5383(1979)].

The aminoethyl-derivatized polyacrylamide gel is particularly preferred because of its high capacity for the binding or immobilization of hapten thereto, as well as its low nonspecific adsorption properties. Further, the gel is also commercially available (Bio-Rad Laboratories, Richmond, CA, USA) having various aminoethyl capacities, generally from between about 1.0 and 2.0 meq. per dry gram of the resin. The gel also has a preferred pH range from about pH 2.0 to pH 10.0, and is susceptible to the hydrolysis of amide side groups at higher or lower pH values.

According to another embodiment of the present invention, the carrier material of the immobilized hapten reagent is a novel polyacrylamide sulfhydrylk gel having, as the functional groups, a plurality of sulfhydryl groups, as described in greater detail in the copending U.S. patent application entitled "Crosslinked Polyacrylamide Sulfhydryl Gel and Sulfhydryl-Functionalized Derivatives Thereof" (U.S. Ser. No. 939,904), filed on even date herewith. The polyacrylamide sulfhydryl gel is prepared from the free radical polymerization of acrylamide, bisacrylamide, and N,N'-bisacrylylcystamine and is particularly useful for the preparation of an immobilized glycopeptide reagent for use in the immunoassay determination of the glycosylated form of hemoglobin known as HbAlc. It is to be appreciated that the polyacrylamide sulfhydryl gel is similarly swellable in aqueous solutions. The swelling characteristics can be controlled by the ratio of monomers employed, the degree of crosslinking and the particular crosslinking group, and the active functional groups.

Generally, the physical structure of the polyacrylamide gel particles comprises crosslinked polyacrylamide chains which define an external surface area and an internal surface area wherein the accessibility of the hapten moiety and other reagents to the internal surface area is limited when the gel particle is nonswollen as heretofore described. It is to be understood that the swelling characteristic of the gel particle is the result of the structural network of the crosslinked polyacrylamide chains which result in a generally porous natrue of the gel particle. Accordingly, when the gel particle is nonswollen, the polyacrylamide chains are held tightly together by the crosslinking groups to result in an effective pore size which is smaller or impervious to the hapten moiety or other reagents to thereby prevent the permeation and internalization thereof into the internal surface area of the gel particle. The particles further comprise a plurality of their respective chemically active functional groups at the external and internal surface areas thereof which are essential to the formation of a stable, covalent bond between the hapten moiety and the particle by a linking group, as will be described in greater detail hereinafter. It is to be appreciated that where a covalent bond is not formed between the hapten moiety and a functional group of the particle, such hapten moiety is likely to become nonspecifically bound to the particle by nonspecific binding interactions, such as through ionic and hydrophobic binding interactions and the like. Such nonspecific binding of a hapten moiety to a particle results in a high degree of dissociation of such nonspecifically bound hapten moiety from the carrier material and subsequent leaching or slow release thereof when subjected to immunoassay test conditions or other aqueous environments.

Although a substantially stable, covalent bond can be formed between the hapten moiety and the functional groups as heretofore described in either an aqueous environment or an organic environment, the use of a nonswelling solvent is preferred according to the present invention in order to achieve the least amount of nonspecific binding of the hapten moiety to the gel particle, and, more particularly, substantially only to the external surface thereof according to the present invention. In particular, the substantially high hydrophilicity of the particle results in the undesirable swelling of the gel and increase in the permeation or internalization of the hapten moiety and other reagents into the gel particle when in an aqueous solution, whereas there is essentially no swelling or attendant increase in such permeation or internalization when in an organic solution or solvent containing little or no water. Accordingly, the use of a nonswelling solvent according to the present invention prevents the substantial swelling of the gel particle, which limits the covalent binding of the hapten moiety substantially only to the functional groups on the external surface of the gel particle by minimizing the permeation or internalization of the hapten moiety and other reagents into the particle. Such internalization would otherwise result in the undesirable formation of covalent bonds between the hapten moiety and functional groups which are present on the internal surface area of the gel particle as heretofore described. Such internalization of the hapten moiety and other reagents also results in the greater probability of the nonspecific adsorption of such internalized hapten moiety which would be difficult to remove with a wash solution and which could later leach out from the gel particle during the performance of an immunoassay and thereby result in decreased assay sensitivity and accuracy.

Accordingly, the immobilization of the hapten moiety to the gel particle is performed in a nonswelling organic solvent such as dimethylsulfoxide, dimethylformamide, acetone, chlorinated hydrocarbons, cyclic and acyclic alkylethers, and the like, preferably containing little or no water, which results in essentially no swelling of the gel particle and, accordingly, essentially no attendant increase in the permeation or internalization of the hapten moiety or other reagents into the particle. Since the negligible increase in particle size will result in a gel particle which is substantially impervious to and will effectively exclude the permeation of a hapten moiety and other reagents into the gel particle, any nonspecific binding of the hapten moiety is limited substantially only to the external surface of the gel particle. Any of the hapten moiety which becomes nonspecifically bound to the external surface of the gel particle is effectively removed with a nonswelling wash solution followed by an appropriately buffered rinse or wash solution, such as with an acidic salt solution. Upon the removal of such external, nonspecifically bound hapten moiety, the resulting immobilized hapten reagent comprises substantially all of the hapten moiety covalently bound to the external surface of the gel particle. The reagent thus is substantially stable in aqueous solutions as a result of the insignificant amount of the hapten moiety which is nonspecifically bound to the carrier material. In particular, it is to be appreciated that according to the present invention, less than from about 1.times.10.sup.-12 moles of the hapten moiety/g. of the resin, more usually less than from about 1.times.10.sup.-13 moles of the hapten moiety/g. of the resin, preferably less than about 1.times.10.sup.-14 moles of the hapten moiety/g. of the resin, will dissociate from the gel particle upon standing in an aqueous liquid, such as a buffer solution, e.g., the phosphate-chloride assay buffer described in Example 5, to potentially result in an insignificant amount of leakage thereof during the performance of an immunoassay.

(b) Hapten Moiety

The hapten moiety of the immobilized hapten reagent can be the hapten under determination, or an analog thereof which is capable of binding to the specific binding partner thereof of the labeled reagent, and which hapten or binding analog thereof can be covalently linked to the external surface functional groups of a carrier material particle according to the present invention.

In particular, the hapten moiety of the immobilized hapten reagent of the present invention can be selected for the determination of any hapten for which a binding partner exists in a biological system or can be synthesized, and includes, but is not intended to be limited to, digoxin, digitoxigenin, digitoxin, digoxigenin, 12-0-acetyldigoxigenin, and glycosylated peptide sequences such as the glucosylated N-terminal peptide sequence in the beta-subunit of human hemoglobin, as well as other general classes of drugs, metabolites, hormones, vitamins, toxins and the like organic compounds. Haptenic hormones include thyroxine and triiodothyronine. Vitamins include vitamins A, B, e.g., B.sub.12, C, D, E and K, folic acid and thiamine. Drugs include antibiotics such as aminoglycosides, e.g., gentamicin, tobramycin, amikacin, sisomicin, kanamycin, and netilmicin, penicillin, tetracycline, terramycine, chloromycetin, and actinomycetin; nucelosides and nucleotides such as adenosine diphosphate (ADP) adenosine triphosphate (ATP), flavin mononucleotide (FMN), nicotinamide adenine dinucleotide (NAD) and its phosphate derivative (NADP), thymidine, guanosine and adenosine; prostaglandins; steroids such as the estrogens, e.g., estriol and estradiol, sterogens, androgens, and adrenocortical steroids; and others such as phenobarbital, phenytoin, primidone, ethosuximide, carbamazepine, valproate, theophylline, caffeine, propanolol, procainamide, quinidine, amitryptiline, cortisol, desipramine, disopyramide, doxepin, doxorubicin, nortryptiline, methotrexate, imipramine, lidocaine, procainamide, N-acetylprocainamide, amphetamines, catecholamines, and antihistamines. Toxins include acetyl T-2 toxin, alfatoxine, cholera toxin, citrinin, cytochalasins, staphylococcal enterotoxin B, HT-2 toxin, and the like.

(c) Linking Group

The linking group of the immobilized hapten reagent of the present invention employing the aminoethyl-derivatized polyacrylamide gel particle can be selected from a number of linking groups known in