CA1172960A - Activated apoglucose oxidase and its use in specific binding assays - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A method for increasing the ability of apoglucose oxidase to combine with flavin adenine dinucleotide (FAD) and derivatives thereof to form active glucose oxidase by interacting apoglucose oxidase with an immunologically derived binding substance, e.g., an antibody or a fragment thereof, having a specific bind-ing affinity for glucose oxidase. The apoglucose oxidase/anti-glucose oxidase immune complex is characterized by enhanced ability to combine with FAD
and FAD-derivatives to yield glucose oxidase activity.
The activation is particularly significant at tempera-tures elevated from room temperature, e.g., between 30°-45°C. An improved homogeneous specific binding assay method is provided for determining ligands wherein an FAD label is used and is monitored by its ability to combine with apoglucose oxidase to form active glucose oxidase by including anti-glucose oxidase in the reaction mixture.

Description

~72~60 ACTIVATED APOGLUCOSE OXIDASE
AND ITS USE IN SPECIFIC BINDING ASS~YS

BACKGROUND OF THE INVENTION

1. FIEBD OF TH~ INVENTION

This invention relates to a method for increasing the ability of apoglucose oxidase to combine with flavin adenine dinucleotide (FAD) and FAD derivatives to form active glucose oxidase, i.e., a method for activating apoglucose oxidase. Ihe invention further relates to 10 homogeneous specific binding assay methods, reagent means, and test kits for determining a ligand, such as an antigen, hapten or antibody, in a liquid medium wherein FAD is employed as label and wherein the FAD
label is monitored by its ability to combine with apo-15 glucose oxidase to form active glucose oxidase.

Glucose oxidase is a conjugated enzyme composed ofan enzymatically inactive, high molecular weight protein component (apoenzyme) and FAD (a low molecular weight, nonproteinaceous prosthetic group). Apoglucose oxidase 20 and FAD have a high binding affinity (hinding constant of around 101 molar~l), hut can be effective]y separated by treatment with acidified ammonium sulfate [Swoboda, Biochim. Biophys. Acta 175:365-379(1969)]. Swoboda demonstrated that apoglucose oxidase exists in 25 solution mainly with the molecular configuration of a ~17;~60

- 2 ~

loose flexible coil, and to a lesser extent in a compact globular form, whereas when FAD is added the protein is converted to a compact, nearly spherical form having glucose oxidase activity. FAD is understood to most efficiently bind with the globular form of apoglucose oxidase to yield active glucose oxidase. Stabilization of apoglucose oxidase by intramolecular crosslinking with glutaraldehyde has been reported [Solomon et BiopoZymers 16:1837-1952(1977)].

a 2. BRIEF DESCRIPTIO~ OF THE P~IO~ ART

.
U.S. Patent No. 4,238,565 assigned to the present assignee describes specific binding assay methods wherein FAD is employed as label and is monitored by its ability to combine with apoglucose oxidase to form active glucose oxidase. Both homogeneous and hetero-geneous formats are anticipated. In a homogeneous assay for determining an antigen in a liquid medium, a test sample of the liquid medium is combined with 2~ antibody to the antigen and with a labeled conjugate comprising the antigen or an analog thereof coupled to FAD whereby any antigen from the sample competes with antigen-FAD for bindinx with antibody. Apoglucose oxidase is also present or is added after an appropriatc incubation period and is capable of combining with antigen-FAD which has not been bound by antibody to yield active glucose oxidase. However, antibody-bound antigen-FAD is not capable of such combination with apoglucose oxidase. Consequently, the concentration of antigen in the test sample dictates thc amount of measurable glucose oxidase which results. Glucose oxidase activity is measurable in a wide variety of known manners, including colorimetric methods.

.

:, o - ~ -~ he FAD-labeled homogeneous speci~ic binding assa~
is generall~ applicable to the determination of a ~.ide variety of ligands over a wide range of concentrations.
However, application of the assay method to certain existing analytical instrumcntation requiring incuba-tion of the assay reaction mixture at elevated tempera-tures and for short periods has been found to be limited. It has been found that the ability of FAD
and FAD-derivatives (i.e., ligand-FAD conjugates) to combine with apoglucose oxidase to form active glucose oxidase decreases rapidly with increasing temperature above room temperature, to the point that at tempera-tures above about 30C significantly greater amounts of apoglucose oxidase must be added to the assay reaction mixture to obtain a dose-response curve. At 35-40C, the FAD-apoglucose oxidase recombination reaction is slowed to the point that analytically useful dose-response curves are not possible to obtain with short incubation periods (e.g., 10 minutes or less) for the determination of ligands at concentrations below 10 6M.

The preparation of antibody to ~lucose oxidase is reported by Green et aZ, J. Immuno~ 104:1094-1100(1970).
Apog]ucose oxidase preparations useful in the FAD-25 ]abeled specific binding assay method are described inG3~ladian Patent No. 1,143,309, issued March 22, 1983 and asslgned to the present assignee.

SUMMARY OF THE INVEN~ION

It has now been found that the ability of apo-30 glucose oxidase to combine with FAD to form activeglucose oxidase is increased by interacting apoglucose oxidase with an immunologically derived binding sub-. , .

.~
,~., :1~7;~tiO

stance having a specific binding affinity for glucose oxidase. Such binding substance (hereinafter referred to as "anti-glucose oxidase"~ may be an antibody raised against glucose oxidase or a fragment thereof, e.g., Fab, F(ab'), or F(ab')2, or any other substancc -` having a specific binding affinity for glucose oxidase.
The apoglucose oxidase/anti-glucose oxidase comple~ has been ~ound to be activated to recombination with FAD.
The apoglucose oxidase activation has been found to in-crease with increasing temperatures between 25-37C to the extent that at 37C the formation of active glucose oxidase is enhanced as much as 100 to 1000-fold by the presence of antiglucose oxidase. Thus, FAD-labeied homogeneous specific binding assays for ligands appear-ing in concentrations between 10-5 and 10-8M are made feasible for use with analytical instrumentation requir-ing elevated reaction temperatures (e.g., 37C) and short incubation periods (e.g., 5-7 minutes).

BRIEF DESCRIPTION OF TE~E DRAWINGS

Fig. 1 is a graphical representation of data from the examples set forth below showing the effect of temperature on the FAD/apoglucose oxidase recombination reaction in the presence and absence of anti-glucose oxidase.

~ig. 2 is a graphical representation of data from the examples below showing optimized standard curves for a theophylline assay at 37C with an~ without anti-glucose oxidase.

~:~7~a,~0 ~ESCRI~TION OF THE ~REFERR~D 1.~1B~DIME~TS

- In the context of this disclosure, the following terms shall be defined as follows unless otherwise indicated:
Ligand - the analyte which is the object of an assay, i.e., the substance, or class of related substances, wh~se presence or amount in a liquid medium is under determination.
Specific binding partner of the ligand - any substance, OT class of substances, which has a specific binding affinity for the ligand to the exclusion of other sub-stances.
FAD-labeled specific binding assay - an assay, usually an immunoassay, of any of the homogeneous types described in the aforesaid U.S. Patent No. 4,238,565 wherein the label is FAD.
Reagent means - a composition, test device or test kit comprising the ~eagents used to perform an FAD-labeled specific bind-ing assay.

The anti-glucose oxidase of the present invention may be an antibody, an antibody fragment, or any other substance having a specific binding affinity for glucose oxidase and which is deri~ed from immunological processes. When in the form of whole antibody, anti-glucose oxidase may be of any of the known classes, e.g., IgG, IgM, and so forth, and of any subclasses thereof. Any fragment of any such antibody which ~ . .

1~ 0 retains the specific binding affinity for glucose oxidase may also be employed, for instance, the frag-ments of IgG conventionally known as Fah, ~(ab'), and F(ab')2. Antibodies to glucose oxidase may be obtained by any known means. In particular, such antibodies may be obtained in the form Or antiserum from an animal (e.g , mouse, rabbit, guinea pig, goat, etc.) which has been immunized against glucose oxidase.
Such antibodies can also be obtained by somatic cell hybridization techniques, such antibodies being commonly referred to as monoclonal antibodies. Re-views of such monoclonal antibody techniques are found in ~ymphocyte Hybridom~s, ed. Melchers et ~Z, Springer-Verlag (New York 1978), Nature 26~:495(1977), and Science 208: 692(1980). Activation of apog]ucose oxidase by anti-glucose oxidase is believed to result from the formation of an immune complex which tends to hold the apoenzyme in a molecular configuration favor-ing the formation of active glucose oxidase upon bind-ing with FAD.

The amount of anti-glucose oxidase to be added to a given amount of apoglucose oxidase to give optimum activation will be determined empirically. It has been found in general that as increasing amounts of anti-glucose oxidase are added to a fixed amount of apo~lucoseoxidase, apoenzyme activation increases until a maximum ' activation is attained. The further addition of anti-~- glucose oxidase does not result in further act;vation, but rather in general actually leads to decreased activation. Excess anti-glucose oxidasc accordingly will not be desirable. Titration of anti-glucose oxi-dase against glucose oxidase activity will indicate for a given amount or concentration of apoenzyme the o~ti-mum amount or concentration of anti-glucose oxidase to use.

~7~ iO

The ability of anti-glucose oxidase to activate apoglucose oxidase occurs over a reasonably broad temperature range, running between noC and temperatures at which the proteins denature significantly. The activation is particularly significant over the range of lO~C to 45C, most particularly between about 30C
and ahout 40C.

The ability to activate apoglucose oxidase with anti-glucose oxidase provides particular advantages in application to FAD-labeled homogeneous s~ecific binding assays. Such assays are basically characterized by the monitoring of the FAD label by its ability to combine with apoglucose oxidase to form active glucose oxidase.
In accordance with the present invention, such apoglucose oxidase is activated by interaction with anti-glucose oxidase. The present invention is particularly appli-cable to an FAD-labeled homogeneous immunoassay method for determining a ligand in a liquid medium wherein a reaction mixture is formed by combining the liquid 20 medium with reagent means including (a) a labeled conju-gate comprising the FAD label coupled to said ligand or a binding analog thereof, i.e., a substance which is bound by the ligand binding partner substantially the same as the ligand, (b) an antibody to the ligand, and (c) apoglucose oxidase, and wherein glucose oxidase activity is thereafter measured in the rcaction mixture as a direct function of the amount of ligand in the liquid medium under assay. Such assay method can be used to determine such ligands as antigenic proteins, 5 including antibodies, and polypeptides having molecular weights between l,000 and lO,000,000 and haptens having molecular weights between 100 and l,500.

Alternatively, where the ligand under determination can bind with an FAD-labeled binding partner to ~he 10 ligand to affect the ability of the FAD label to combine with apoglucose oxidase, such as where the ligand is a binding protein and the labeled binding partner is the substance bound thereby, the present invention is appli-cable to an FAD-labeled homogeneous immunoassay method 15 for determining such a ligand in a li4uid medium wherein a reaction mixture is formed by combining the liquid medium with reagent means including (a) a labeled conju-gate comprising the FAD label coupled to said binding partner of the ligand and (b) apoglucose oxidase, and 20 wherein glucose oxidase activity thereafter measured in the reaction mixture is an inverse function of the ~ amount of ligand in the liquid medium under assay. ~Such ~ ligand/binding partner pairs to which such a method is applicable include antibodies/hapten or antigen, 25 thyroxine binding globulin/thyroxinc or triiodothyronine, and the like.

When used in an FAD-labeled specific binding assay, apoglucose oxidase activation may be ohtained by a variety of ways of interacting the apocnzymc with 30 anti-glucose oxidase. In one embodiment, anti-glucose ~L~7~9~iO

oxidase and the principal reagents of thc assay, usually comprising ~a) an FAD-labeled form oi~ the ligand or an analog thereof, (b) a specific binding partner of the ligand, e.g., an antibody, (c) apoglucos( oxidase, and (d) a glucose oxidase indicator system, are coml-inc~l as separate reagents to form the assay reaction mixture.
In another embodiment, some of the reagents- are precombined, e.g., (a) apoglucose oxidase and the hind-ing partner and (b) FAD-labeled conjugate and the lO -glucose oxidase indicator system, and added to the others as combined reagents.

The anti-glucose oxidase and apoglucose oxidase may be added to the assay reaction mixture in the form of their activated complex. In cases of short assay incubation times and low analyte concentrations this may be preferable since in such a case essentially all of the apoglucose oxidase is in its activated form immediately upon formation of the assay reaction mixture. If added as separate reagents, the apoenzyme and anti-glucose oxidase require some finite time for complete activation of apoglucose oxidase by complex-ing with the anti-glucose oxidase. Wherc an apoglucose oxidase/anti-glucose oxidase complex rcagcnt is uscd, it may be preferable to employ monovalent antibody fragments, e.g., Fab or ~(ab'), as the anti-glucose oxidase. In this way one can prevent the possible formation of intermolecular crosslinks between apo-glucose oxidase which otherwise coul~ ]ead to the forma-tion of suspensions or precipitates which could intcrfer with the assay reaction or the mcasured signal.

1172~60 The reagent means of the present invention comprises all of the essential chemical elements required to con-duct a desired assay method of the present invention.
Such reagent means are presented in a commercially 5 packaged form, as a composition or admixture where the compatability of the reagents will allow, in a test device configuration of any present or future type, or as a test kit, i.e., a packaged combination of one or more containers or vessels holding the necessary rea-10 gents. The reagents of the present invention include(a) an FAD-labeled conjugate comprising FAD coupled to the ligand, a binding analog thereof, or a specific binding partner thereof, depending on the particular assay technique intended, (b~ apoglucose oxidase, (c) 15 where the FAD-labeled conjugate comprises the ligand or an analog thereof, a specific binding partner of the ~' ligand (e.g., an antibody or other binding protein), and (d) anti-glucose oxidase as defined herein, and option-ally one or more or all of an indicator composition for 20 detecting glucose oxidase activity (e.g., comprising glucose, peroxidase and a chromogen or chromogen system responsive to hydrogen peroxide produced when glucose oxidase acts on glucose). Of course, the reagent means can include other substances as are known in the art and which may be desirable from a commercial and user standpoint, such as buffers, diluents, standards, and so forth.

The present invention will now be illustrated, but is not intended to be limited by the following examples.

1~7;~

Apoglucose oxidase substantially free from residual glucose oxidase activity is prepared by the method described in Canadian Patent No. 1,143,309, issued March 22, 1983 and assigned to the present ~ssignee.

~ heophylline-FAD conjugates are prepared by the method described in U.S. Patent ~o. 4,238,565 assigned to the present assignee.

Sodium 3,5-dichloro-2-hydroxybenzene sulfonate (sodium DHSA) is prepared by hydrolysis of sodium

3,5-dichloro-2-hydroxybenzene sulfonyl chloride (Aldrich Chemical Co., Milwaukee, WI) with sodium bicarbonate solution.

Antibody to glucose oxidase is obtained by injecting goats on four subcutaneous sites near the shoulders with a total of 3.0 ml adjuvant (1.5 ml Freund's adjuvant and 1.5 ml saline) containing glucose oxidase in the range 0.1-1.0 mg. Complete Freund's adjuvant is used for the initial injection and incomplete Freund's adjuvant is used for the booster injections. Five weeks after the initial injection, the following schedule is followed at one week intervals: boost, bleed, bleed, rest, repcat.

. ~

~7~60 EFFECT OF TEMPERATURE ON T~E ACTIVATION
OF APOGLUCOSE OXIDASE IN THE rRESENCE AND ABSENCE
OF ANTISERUM AGAINST GL[JCOSE OXIDASE
_ A. Reagents Mix A - 2 micromolar (~M) apoglucose oxidase in 0.1 molar ~M) sodium phosphate buffer, pH 7.0, containing 4 millimolar (mM) aminophenazone, 10% glycerol, and 0.1~ (w/v) bovine serum albumin. This mix was incubated at room temperature.

Mix B - 1.0 nanomolar (nM) theophylline-FAD
was added to a glucose oxidase assay reagent solution consisting of 0.1 M
sodium phosphate buffer, pH 7.0, 0.105 M glucose, 2.11 mM sodium DHSA, 1.05% (w/v) bovine serum albumin and 63 micrograms per milliliter (~g/ml) peroxidase. Aliquots of this mix were incubated at the various temperatures indicated below.

B. Assays Mix A [100 microliters (~1)] was mixed with either 10 ~1 of goat antiserum against glucose oxidase (anti-GO serum) or 10 ~1 of nonimmune goat serum (NI
serum) in disposable cuvettes. The solutions were incubated at room temperature for 15 minutes. Mix B
(1.90 ml) equilibrated to the desired assay temperature was then added and the reaction mixture mixed by inversion. After a 15 minute incubation at the desired assay temperature, the absorbance at 520 nano-meters ~nm) was read against Mix B. Blanks were also ~7~t;0 run exactly as above except that theophylline-FAD was absent from Mix B. The results are shown in tabular forr,l below and as a graph in Figure 1 of the drawings.
The absorbance readings are corrected for the blank.

5 Temperature Absorbance (520 nm) (C) NI serum Anti-GO serum 0.041 0.094 0.100 0.212 0.134 0.430 24 0.170 0.682 28 0.073 0.885 32 0.020 0.929 36 NC* 0.778 NC* 0.606 *no measurable color These data demonstrate that the pre.sence of anti-glucose oxidase antiserum enhances the ability of apoglucosc oxidase to combine with the theophylline-FA~ conjug/ltc to form active glucose oxidase. At room temperaturc (25C), the ~resence of anti-glucose oxidase produces a 4-fold increase in apoglucose oxidase activation.
The effect at 37C is estimated at between 100 and 1000-fold.

1~7~6~) IN THE PRESENCE AND ABSENCE OF
ANTISERUM TO GLUCOSE OXIDASE
-I. Assay in the absence of anti-glucose oxidase Reagents: Mix A - Combined apoenzyme/anti-theophylline reagent: 32 ~M apoglucose ; oxidase and 50 ~1 anti-theophylline antiserum per ml were added to 0.1 M
sodium phosphate buffer, pH 7.0, con-taining 0.1% (w/v) bovine serum albumin.
This mix was incubated at 20C.

Mix B - Combined glucose oxidase assay and theophylline - FAD label: 63 nM
theophylline-FAD was added to 0.1 M
sodium phosphate buffer, pH 7.0, con-taining 0.105 M glucose, 2.11 mM sodiùm DHSA, 0.21 mM aminophenazone, 1.05%
(w/v) bovine serum albumin, and 63 ~g/ml horseradish peroxidase. This mix was incubated at 37C.

Theophylline standards: Prepared by gravimetri-cally spiking pooled human serum with different amounts of theophylline. These standards were diluted 10-fold with 0.1 M
sodium phosphate buffer, pH 7Ø

Assay procedure: Mix B ~1.90 ml) was mixed with 50 ~1 of a diluted theophylline standard in an disposable cuvette and equilibrated at 37C. Mix A (0.10 ml) was then added and the reaction mixture mixed by inversion.

:~7;~60 After a 6 minute incubation at 37C, the absorbance at 520 nm was read against Mix B.
The final assay concentrations for the principal reagents were:

apoglucose oxidase 1.60 ~M
theophylline-FAD 60 nM
anti-theophylline 2.5 ~l/ml The results are shown in tabular form below and as a graph in Figure 2 of the drawings.

10Theophylline Absorbance serum concentration (520 nm) ~g/ml) o 0.146 6 0.162 1512 0.174 0.183 0.190 0.198 ':.
The absorbance change over the full range o theo-20 phylline serum concentrations was only 0.052. This data represents the present optimized theophylline assay in the absence of anti-glucose oxidase.
, .
II. Assay in the presence of anti-glucose oxidase :' Reagents: Mix A - 600 nM apoglucose oxidase, 100 ~l/ml rabbit antiserum against glucose oxidase, and 20 ~l/ml anti-theophylline were added to 0.1 M phos-phate buffer, pH 7.0, containing 4 mM
aminophenazone and 10% glycerol. This mix was incubated at room temperature.

~y 7 ~

Mix B - 10.5 nM theophylline-FAD was added to 0.10 M phosphate buffer, pH 7.0, 0.105 ~
glucose, 2.11 mM sodium DHSA, 1.05% (w/v) bovine serum albumin, and 63 ~g/ml horse-radish peroxidase. This mix was incubated at 37C.
Theophylline standard.s: Same as above.
Assay procedure: Mix B (1.90 ml) was mixed with 20 ~1 of a diluted theophylline standard in a disposable cuvette and equilibrated at 37C. Mix A (0.10 ml~
was then added and the reaction mixture mixed by inversion. After a 5 minute incubation at 37C, the absorbance at 520 nm was read against Mix B.
The final assay concentrations for the principal reagents were:
apoglucose oxidase 0.03 ~M
theophylline-FAD 10 nM
anti-theophylline 1.0 ~l/ml The results are shown in tabular form below and as a graph in Figure 2 of the drawings.
Theophylline Absorbance serum concentration ~520 nm) (~g/ml) 0.162 0.298 0.378 0.444 0.495 The absorbance change over the full range of theophylline serum concentrations was 0.333 compared to 0.052 for the optimized assay without anti-glucose oxidase (part I
above). Further, the optimized theophylline assay at 37C with anti-glucose oxidase present re~uired approxi-mately 50-fold less apoglucose oxidase, 6-fold less theophylline-FAD conjugate, and 2.5-fold less anti-theophylline.
~ MS-1171 1~'7~?60 These data demonstrate the greatly improved features of the optimized assay at 37C with anti-glucose oxidase present over that without anti-glucose oxidase.

USE OF ANTIBODY FRAGMENTS
TO ACTIVATE APOGL~COSE OXIDASE

I. Preparation of anti-glucose oxidase Jg~

Goat antiserum against glucose oxidase (35 ml) was dialysed against 2 liters of 15 mM potassium phosphate, pH 8.0, for 16 hours at room ~emperature. The dialysed 10 solution was applied to a column (2.5 x 30 cm) DE-52 cellulose (Whatman Ltd., UK) equilibrated with 15 mM
potassium phosphate, pH 8Ø The column was eluted with the same buffer at a flow rate of 50 ml/hour and 12 ml fractions were collected. The absorbance of the 15 effluent was monitored at 280 nm. An initial absorbance peak was eluted in fractions 14 to 21 and this was pooled and labeled "FI". The eluting buffer was changed to 50 mM potassium phosphate, pH 8.0, and a second major absorbance peak was collected in fractions 39 to 44 20 which was pooled and labeled "FII". The two absorbance peaks correlated well with the abi]ity of the fraction.s to activate apoglucose oxidase.
. .
II. Papain digestion of anti-glucose-oxidase IgG (l;ab fragments) Protein from FI (50 mg) was incorporated into 5 ml of 50 mM sodium phosphate, pH 7.3, containing 0.15 M
sodium chloride, 25 mM cysteine, l mM ethylenediamine tetraacetic acid (EDTA) and l mg papain. The solution was incubated at 37C for 60 minutes.

o Solid iodoacetamide (28 mg) was then addcd to give a 30 mM solution for the purpose of inactivating the papain. This solution was incubated for a further 15 minutes after which it was applied to a column 5 (25 x 90 cm) of Sephadex G150 equilibrated with 50 mM
sodium phosphate, pH 7.0, containing n. 02~ (w/v) sodium azide. The column was developed by elution with the same buffer at a flow rate of 25 ml/hour and 4.6 ml fractions were collected. Absorbance at 280 nm was 10 monitored and a peak eluted between fractions 47 to 72 was shown to contain a mixture of Fab and Fc fragments and correlated well with the ability of the fractions to activate apoglucose oxidase. This material was pooled, concentrated to 7.9 mg/ml protein and labeled 15 "FI(a)". No whole IgG was eluted and it was concluded that complete digestion of IgG had occurred.

Protein from FII (50 mg) was digested as described above. Chromatography on Sephadex G150 was performed as described above. Absorbance peaks at 280 nm were eluted, one between fractions 34 to 50 which was identi-fied as IgG and a second between fractions 56-75 which was shown to contain a mixture of Fab and Fc fragments.
~oth absorbance peaks correlated well with the ability of the fractions to activate apoglucose oxidase. The first peak was pooled, concentrated to 16.8 mg/ml and labeled "FII(a)". The second peak ~as pooled, concen-trated to 6.2 mg/ml and labeled "FII(b)".

III. Activation of apoglucose oxidase by Fab fragments A. Reagents Apoglucose oxidase (3.2 ~M) was prepared in 0.1 M sodium phosphate buffer, pH 7.0, con-taining 8 mM aminophenazone and 10% glycerol.
The glucose oxidase assay reagent contained . "
* Trade Mark - 11'7~$60 0.10 M sodium phosphate buffer, pll 7.0, 0.105 M glucose, 2.11 mM sodium DHSA, 1.05~ ~w/v) bovine serum albumin, 63 ~g/ml peroxidase, and 1.0 nM theophylline~FAD.

5 B. Assays Aliquots (0.20 ml) of 0.10 M sodium phosphate buffer, pH 7.0, containing antibody or anti-body fragments were mixed with 0.03 ml of apoglucose oxidase reagent in a disposable cuvette and incubated at room temperature for 15 minutes. The glucose oxidase reagent (1.90 ml) was equilibrated at 37C and added to the cuvette. The assay solution was incu-bated at 37C for 15 minutes and the absorbance read at 520 nm against the glucose oxidase reagent solution as reference. The recorded absorbances were corrected for a blank (assay solution without theophylline-FAD).

The results were as follows:
~:;
20 Activation by FI(a) - Fab and Fc fragments . . _ Protein Absorbance (~g) (520 nm) 0 0.050 0.300 160 0.555 320 0.700 400 0.900 800 O.g22 1600 0.778 .

1~72~f~0 ~.o Activation b) FII(a) - whole IgG

Protein Absorbance (~g) (520 nm) 0 0.050 84 0.405 168 0.779 252 0.910 420 1.044 840 1.094 1680 1.140 Activation by FII(b) - Fab and Fc fragments Protein Absorbance (~g) (520 nm) 0 0.050 -. 15 62 0.257 124 0.504 186 0.693 .; 310 0.918 620 1.046 1240 1.000 These data demonstrate that fragments (Fab) of anti-glucose oxidase antibody are effective in activat-ing apoglucose oxidase.

Claims (45)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for increasing the ability of apo-glucose oxidase to combine with flavin adenine di-nucleotide and derivatives thereof to form active glucose oxidase comprising the step of interacting apoglucose oxidase with an immunologically derived binding substance having a specific binding affinity for glucose oxidase.

2. The method of Claim 1 wherein said binding substance is an antibody, or a fragment thereof, to glucose oxidase.

3. The method of Claim 2 wherein said antibody is of the IgG class.

4. The method of Claim 3 wherein said binding substance is an Fab, F(ab'), or F(ab')2 fragment of said antibody.

5. A complex of apoglucose oxidase and an immuno-logically derived binding substance having a specific binding affinity for glucose oxidase.

6. The complex of Claim 5 wherein said binding substance is an antibody, or a fragment thereof, to glucose oxidase.

7. The complex of Claim 6 wherein said antibody is of the IgG class.

8. The complex of Claim 7 wherein said binding substance is an Fab, F(ab'), or F(ab')2 fragment of said antibody.

9. In a homogeneous specific binding assay method for determining a ligand in a liquid medium wherein flavin adenine dinucleotide is employed as label and is monitored by its ability to combine with apoglucose oxidase to form active glucose oxidase, the improvement which comprises interacting said apoglucose oxidase with an immunologically derived binding substance having a specific binding affinity for glucose oxidase.

10. The method of Claim 9 wherein said binding substance is an antibody, or a fragment thereof, to glucose oxidase.

11. The method of Claim 10 wherein said antibody is of the IgG class.

12. The method of Claim 11 wherein said binding substance is an Fab, F(ab'), or F(ab')2 fragment of said antibody.

13. In a homogeneous immunoassay method for deter-mining a ligand in a liquid medium, wherein a reaction mixture is formed by combining said liquid medium with reagent means including (a) a labeled conjugate comprising, as label, flavin adenine dinucleotide coupled to said ligand or a binding analog thereof, (b) an antibody to said ligand, and (c) apoglucose oxidase, and wherein glucose oxidase activity is measured in said reaction mixture, the improvement which comprises adding to said reaction mixture an immunologically derived binding substance having a specific binding affinity for glucose oxidase.

14. The method of claim 13 wherein said binding sub-stance is an antibody, or a fragment thereof, to glucose oxi-dase.

15. The method of claim 14 wherein said antibody is of the IgG class.

16. The method of claim 15 wherein said binding sub-stance is an Fab, F(ab'), or F(ab')2 fragment of said antibody.

17. The method of claim 13 wherein said binding sub-stance is added to said reaction mixture in the form of its immune complex with said apoglucose oxidase.

18. The method of claim 14 wherein said binding sub-stance is added to said reaction mixture in the form of its immune complex with said apoglucose oxidase.

19. The method of claim 15 wherein said binding sub-stance is added to said reaction mixture in the form of its immune complex with said apoglucose oxidase.

20. The method of claim 16 wherein said binding sub-stance is added to said reaction mixture in the form of its immune complex with said apoglucose oxidase.

21. The method of claim 17, 18 or 19 wherein said re-action mixture is maintained at a temperature between about 30°C and about 40°C.

22. The method of claim 20 wherein said reaction mix-ture is maintained at a temperature between about 30°C and about 40°C.

23. The method of claim 17, 18 or 19 wherein said re-action mixture is maintained at a temperature of about 37°C.

24. The method of claim 20 wherein said reaction mix-ture is maintained at a temperature of about 37°C.

25. In a reagent means for determining a ligand in a liquid medium by a homogeneous specific binding assay method, which means includes (a) a labeled conjugate wherein the label is flavin adenine dinucleotide and (b) apoglucose oxidase, the improvement which comprises including in said means an immunologically derived binding substance having a specific binding affinity for glucose oxidase.

26. The reagent means of claim 25 wherein said binding substance is an antibody, or a fragment thereof, to glucose oxidase.

27. The reagent means of claim 26 wherein said anti-body is of the IgG class.

28. The reagent means of claim 27 wherein said bind-ing substance is an Fab, F(ab'), or F(ab')2 fragment of said antibody.

29. The reagent means of claim 25, 26 or 27 wherein said binding substance is added to said reaction mixture in the form of its immune complex with said apoglucose oxidase.

30. The reagent means of claim 28 wherein said bind-ing substance is added to said reaction mixture in the form of its immune complex with said apoglucose oxidase.

31. A test kit for determining a ligand in a liquid medium, comprising (1) a labeled conjugate comprising, as label, flavin adenine dinucleotide coupled to said ligand or a binding analog thereof, (2) a specific binding partner of said ligand, (3) apoglucose oxidase, and (4) an immunologically derived binding substance having a specific binding affinity for glucose oxidase.

32. The test kit of claim 31 wherein said binding sub-stance is an antibody, or a fragment thereof, to glucose oxi-dase.

33. The test kit of claim 32 wherein said antibody is of the IgG class.

34. The test kit of claim 33 wherein said binding sub-stance is an Fab, F(ab'), or F(ab')2 fragment of said antibody.

35. The test kit of claim 31, 32 or 33 wherein said binding substance is added to said reaction mixture in the form of its immune complex with said apoglucose oxidase.

36. The test kit of claim 34 wherein said binding sub-stance is added to said reaction mixture in the form of its immune complex with said apoglucose oxidase.

37. The test kit of claim 31 wherein said specific binding partner of said ligand is an antibody to said ligand.

38. The test kit of claim 37 wherein said ligand is a hapten.

39. A test kit for determining a ligand in a liquid medium, comprising (1) a labeled conjugate comprising, as label, flavin adenine dinucleotide coupled to a specific binding partner of said ligand, (2) apoglucose oxidase, and (3) an immunologically derived binding sub-stance having a specific binding affinity for glucose oxidase.

40. The test kit of claim 39 wherein said binding substance is an antibody, or a fragment thereof, to glucose oxidase.

41. The test kit of claim 40 wherein said antibody is of the IgG class.

42. The test kit of claim 41 wherein said binding sub-stance is an Fab, F(ab'), or F(ab')2 fragment of said antibody.

43. The test kit of claim 39, 40 or 41 wherein said binding substance is added to said reaction mixture in the form of its immune complex with said apoglucose oxidase.

44. The test kit of claim 42 wherein said binding sub-stance is added to said reaction mixture in the form of its immune complex with said apoglucose oxidase.

45. The test kit of claim 39 wherein said ligand is an antibody and said binding partner is a hapten or antigen bound thereby.