CA1289874C - Anti-enzyme antibody immunoassay - Google Patents
Anti-enzyme antibody immunoassayInfo
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- CA1289874C CA1289874C CA000532968A CA532968A CA1289874C CA 1289874 C CA1289874 C CA 1289874C CA 000532968 A CA000532968 A CA 000532968A CA 532968 A CA532968 A CA 532968A CA 1289874 C CA1289874 C CA 1289874C
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- binding component
- immunoassay
- binding
- analyte
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- 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
- G01N33/531—Production of immunochemical test materials
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- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- 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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
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- 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
- G01N33/54306—Solid-phase reaction mechanisms
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Abstract
ABSTRACT
An immunoassay utilizing the inhibition or inactivation of a signal generating molecule to reduce a controlled baseline signal to a lower level of signal corresponding to the amount of analyte present in the sample. one embodiment is a heterogeneous enzyme immunoassay, preferably using monoclonal antibodies wherein an insolublized first antibody binds to an antigen analyte of interest. A second and third antibody, which themselves have been conjugated together, are added so that the second antibody complexes preferentially with the antigen. Then an active enzyme and its specific substrate are added to the complex, simultaneously for example, and enzyme will be bound preferentially to the third antibody which exerts an inhibitory influence on the enzyme. The resultant decrease in enzyme activity is measured and the relative amount of antigen in the sample is calculated.
An immunoassay utilizing the inhibition or inactivation of a signal generating molecule to reduce a controlled baseline signal to a lower level of signal corresponding to the amount of analyte present in the sample. one embodiment is a heterogeneous enzyme immunoassay, preferably using monoclonal antibodies wherein an insolublized first antibody binds to an antigen analyte of interest. A second and third antibody, which themselves have been conjugated together, are added so that the second antibody complexes preferentially with the antigen. Then an active enzyme and its specific substrate are added to the complex, simultaneously for example, and enzyme will be bound preferentially to the third antibody which exerts an inhibitory influence on the enzyme. The resultant decrease in enzyme activity is measured and the relative amount of antigen in the sample is calculated.
Description
ANTI-EN~YME ANTIBODY IMMUNOASSAY
BACR~ROUND OF THE IN~IENTION
FI}:LD OF THE INVENTION
The present invention relates to methods for conducting a solid phase assay. More specifically, the present invention relates to methods for conducting an immunoassay for the detection or quantitation of a biological analyte, using an antibody: antibody conjugate which will inhibit an enzyme indicator bound to the conjugate.
PRIOR ART
The method of the present invention relates to the performance of a solid phase assay for the determination of the presence or quantity of a biological substance suspected of being in a specimen. Commonly, the substance of interest is an antigen but can also be an antibody.
Numerous techniques have been developed to measure the amount antigen present in a sample of biological fluid.
Certain of these techniques involve a solid phase system whereby a substance which will form an immunocomplex with the analyte is bound to an insolublized carrier or material, such as latex, cellulose, and the like. In this manner the immunocomplex when formed can be separated in its solid phase from the liquid phase, which contains excess uncomplexed material. Subsequently, a material which has been labelled or "tagged" with a signal generating molecule is added to the immunocomplex so that the material binds to the analyte. After separating the - ~ : ' . .
bound label from the unbound label, the sample is read and the amount of signal measured is related to the amount of analyte present to which it is bound.
Variations on the basic immunoassay technique have been developed to try to overcome some of the problems associated with immunoassays; sensitivity, reliability and cost effectiveness are the major concerns. Although the immunometric assay techniques have been found to be particularly useful in analyzing for antigens and antibodies, there has been difficulty in the past on establishing an optimum level of sensitivity for the assay to be helpful in the detection or monitoring of disease states or maladies in the body. Some of the original immunoassays developed used radioisotopic labelled substances as a signal generating material. While effective to a large degree, radioimmunoassays suffer from the handling and disposal problems and high costs involved with radioactive materials. To avoid these problems other techniques were developed to increase the sensitivity and reduce the cost and hazards of the immunoassay. Enzyme labels, along with fluorescent and luminescent labels have been introduced to increase sensitivity and reliability of the tests while reducing the cost per test, an expense that at bottom line is born by the patient.
In theory, the more specific the binding material is for the analyte the more sensitive the assay. To that end, multiple binding site assays have been developed so -~i I ' ~
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, 1 2~74 that less analyte is required to produce a reliability measurable signal. For instance, where an antigen is the analyte of interest several assays have been developed wherein two different antibodies are used: one is bound to the solid support and the other is labelled. The two antibodies bind to two different epitopic sites on the antigen. More recently, monoclonal antibodies have been developed for this type of use can be provide much greater selectivity and specificity for a given antigen. By being engineered to bind only to a given particular antigen monoclonals overcome the specificity problem associated with polyclonal antibodies. An example of this use is in EPA No. 0044219, which describes a method of immunoanalysis using monoclonal antibodies specific to two distinct antigenic binding sites.
US Patent No. 4,376,110, issued to David et al., discloses a sandwich immunoassay using two monoclonal antibodies which are site specific, yet may be a product of the same or dif:Ferent cell lines. A "sandwich," or "two-site," immunoassay relies upon the formation of an insolublized antibody:antigen:labelled antibody complex.
The two monoclonal antibodies are designed to minimize cross-reactivity and interference with each other when binding to the antigen. However, there still remains the problem of nonspecific binding and imperfect washing away of unbound materials. Some small amount of labelled antibody can remain in the rsaction area by binding to :
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either the reaction support surface or compounds in the area other than the insolublized antibody. This results in more label remaining in the area and generating a signal even though it is not bound to an antigen. since a falsely high signal level is measured, the overall assay is less sensitive than if the nonspecific binding could be further reduced or eliminated. Attempts have been made at increasing the sensitivity of the assay by increasing the selectivity and specificity of the antibody, e.g., by using monoclonal antibodies, and by improving the wash step to effect a more efficient separation of the bound from unbound label. These methods, however, still have yet to achieve the levels of sensitivity and consistency that are desired by the medical diagnostic industry in detecting minute quantities of analytes.
US Patent No. 4,446,233, issued to Auditore-Hargreaves et al., discloses a homogeneous immunoassay using a covalent hybrid antibody. The antibody contains one binding site specific for an analyte and one binding site specific for an indicator. The unique feature of that invention is the use of a single antibody which has been designed so as to contain two distinct binding sites on the same antibody, joined through inter-heavy-chain disulfide bonding. The antibody is composed of two different heavy-chain light-chain half-molecules. The method has limitations in that there are difficulties in designing a covalently hybrid antibody specific for a given antigen.
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For each different test a new hybrid must be created; as contrasted with the well known procedure for producing monoclonal antibodies. Furthermore, specificity problems relating to the purity and specificity of each half-chain of the hybrid can occur.
US Patent No. 4,134,792, issued to Boguslaski et al., discloses a procedure employing as a label a reversibly binding enzyme modulator in an enzyme inhibition assay.
The modulator is a chemical compound that can inhibit the activity of a particular enzyme. The distinguishing feature is that the enzyme binds to an inhibitor chemical molecule, rather than to an antibody, where the inhibitor molecule has been previously chemically conjugated to an antibody. The function and properties of a modulating antibody substantially different than a chemical molecule.
US Patent No. 4,233,401, issued to Yoshida et al., describes an anti-enzyme homogeneous competitive binding assay using a conjugated enzyme:ligand (an antibody or other molecule) which competes with analyte for binding sites on an antibody. When enzyme:ligand is bound to the antibody the enzyme inhibitor present is unable to inhibit the enzyme, while inhibition is possible when not bound to the antibody. The assay is limited to a homogeneous medium, using no solid phase, and required competition for the antibody binding sites. Furthermore, the enzyme is required to be bound to the li~uid prior to reaction with inhibitor.
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Us Patent No. 4,150,949, issued to Smith, describes a fluorescence quenching competitive assay for gentamicin wherein the ~luorescence of a labelled compound is reduced when the compound binds with the antibody. Reduction of fluorescence is measured to determine the amount of antibiotic in the sample.
US Patent No. 4,220,450, issued to Maggio, discloses a fluorescence immunoassay technique wherein a quencher molecule is conjugated to a member of an immunological pair, such as an antibody:antigen complex, and a chemiluminescent molecule is conjugated to the other member of the pair. When the complex forms the c~uencher is positioned in close proximity to the chemiluminescer and inhibits the emission of light by the chemiluminescent sourçe.
US Patent No. 4,281,061, issued to Zuk et al., describes-a double antibody assay wherein analyte, labelled ligand, antilicJand and polyvalent receptor for the antiligand create a matrix which modulates the approach of a macromolecular member of a signal producing system to the labelled ligand.
The primary concerns with these labelled compound assay systems is the interference of background signal noise. BaGkground noise occurs, in part, because of nonspecific binding of labelled compound to material in the sample other than the analyte. In a fluorescent assay system, naturally fluorescing substances often are present in .' .
the sample fluid which emit a signal that can partially "drown out"
the signal being transmitted by the antigen bound tag. In an enzyme label system enzyme can bind to other proteins in the solution.
To reduce this noise a wash step is necessary to separate the insolubilized bound material from the unbound material in solution.
The result is an improved signal; still, current techniques are still unable to reduce the background noise level to the point where a much more sensitive assay is obtainable.
Present technology is based on the reading of a positive signal against a "blank" background of supposedly zero signal output. In reality, however, there is spurious signal generated from nonspecific binding of label as well as naturally occurring signal emitters in the biological solution. This results in a less sensltive assay.
There is a need then for an immunoassay system that obviates the dependence upon a realistically unachievable zero baseline level, a system that is based upon a user-controlled baseline that would achieve a more sensitive determination of analyte.
SUMMARY OF THE INVENTION
The instant invention overcomes the disadvantages in the prior art and achieves the objectives stated hereinabove. Briefly stated the present invention comprises a heterogeneous immunoassay for the detection or quantitation of an analyte suspected of being in a specimen comprising: (a) providing a specimen suspected of containing an analyte of interest, an insolubilized first binding component capable of immunologically reacting with said analyte, a second binding component capable of immunologically reacting with said analyte and to which has been chemically bound a third binding component incapable of reacting with said analyte but capable of binding to and inhibiting the activity of a signal generating ,~
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74c component: (b) combining said specimen, insolubilized binding component and bound second and third binding components in a manner favoring the formation of an insolubilized complex between insolu-bilized first binding component, any of said analyte present, and bound second and third binding components; (c) separating at least all unbound third binding component from any said insolubilized complex material; ~d) contacting with said insolubilized complex an amount of active signal generating component sufficient and for an incubation period sufficient to bind said complexed third binding component resulting in the inhibition of signal from any signal component and to fit within the linear range of the instrument used to measure said signal; and (e) measuring the amount of signal generated as being inversely related to the presence or amount of analyte in said specimen.
lS DESCRIPTION OF THF INVENTION
A heterog0neous assay employs a two phase system whereby certain components are bound to a solid phase, which must be physically separated from unbound components in a liquid phase. The preferred embodiment is a sandwich assay technique in which two distinct antigenic sites on the antigen are used as binding areas.
An antibody is insolubilized on a solid surface and is used to "capture" the antigen by binding to it at one antigenic site. A
second antibody, which is conjugated to an indicator or label, binds to the bound antigen at another site. The unbound conjugate is separated from the bound conjugate, typically by washing, centrifugation, precipitation, filtration, or other procedure. The indicator is then developed, if necessary, and read to obtain a measurement of signal intensity which is correlative with the presence or amount of antigen present in the specimen.
~t '74 In a competitive heterogeneous assay labelled antigen and sample antigen compete for a limited number of binding sites on an insolublized antibody. The solid phase is washed or separated to remove unbound material. After the label is developed, if necessary, it is read; the activity of the solid phase-bound label is inversely proportional to the amount of unlabelled antigen present on the sample.
An indirect assay can be performed for the detection of antibody whereby antigen is bound to a solid phase, which in turn is reacted with the unknown antibody (such as Rubella virus antibody, HTLV-III virus antibody, and the like) which binds to the insolublized antigen. After separating bound from unbound antibody a labelled anti-antibody is added which binds to the unknown antibody. The solid phase is washed and read as described above and the amount of signal is proportional to the presence or amount of antibody present in the specimen.
The solid phase is typically a solid support surface to which is linked the capture antibody or antigen. The surface can be composed of material such as but not limited to latex, glass, glass beads, cellulose, wood, sticks, plastic or other synthetic polymers, filter paper, and the like.
The analyte is a substance suspected of being in a specimen whose presence or concentration is to be determined. The specimen is any biological fluid including whole blood, serum or plasma, cerebral spinal fluid, ~Zl , , saliva, feces, sputum, mucusr urine, cell or tissue extracts, pus, wound exudate, and the like. The analyte is a material that can include drugs, hormones, vitamins, enzymes, proteins, antibodies, polysaccharides, bacteria, protozoa, parasites, fungi, viruses, cell and tissue antigens, other blood cell or blood fluid substances, and the like.
Binding co~ponent is a material capable of selectively complexing or binding to a recognizable substance: i.e., the analyte or the label indicator material. The binding component commonly will be an antibody, but can also be an antigen such as where Rubella virus is the analyte. Either polyclonal antibodies or monoclonal antibodies are usable in the method of this invention. Monoclonal antibodies are preferable because of their sensitivity, selectivity and specificity, and they are able to be designed for a particular analyte of interest. Monoclonal antibodies are prepared according to the method described by Kholer and Milstein in Nature 256:495-497, 1975. More specifically, the monoclonal antibodies of the present invention are prepared by fusing spleen cells, from a mammal which has been immunized against the antigen with an appropriate myeloma cell line. The resultant product is then cultured in a standard HAT (hypoxanthine, aminopterin, and thymidine) medium. Screening tests for the specific monoclonal antibodies are employed utilizing immunoassay techni~ues which will be described below.
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The immunized spleen cells may be derived from any mammal, such as primates, humans, rodents (i.e., mice, rats, and rabbits), bovine, ovine, canine, or the like, but the present invention will be described in connection with mice. The mouse is first immunized by injection of an antigen chosen generally for a period of eleven weeks.
When the mouse shows sufficient antibody production against the antigen, as determined by conventional assay, it is given a booster injection of the antigen, and then killed so that the immunized spleen may be removed. ~he fusion can then be carried out utilizing immunized spleen cells and an appropriate myeloma cell line.
The fused cells yielding an antibody which give a positive response to the presence of the antigen are removed and cloned utilizing any of the standard methods.
The monoclonal antibodies from the clones are then tested against standard antigens to determine their specificity for the particular antigen. The monoclonal antibody selected, which is specific for the antigen or species, is then bound to an appropriate label.
Amounts of antibody sufficient for labelling and subsequent commercial production are produced by the known techniques, such as by katch or continuous tissue culture or culture ln vivo in mammals, such as mice.
At least one binding component can be a monoclonal antibody, and preferably all three are; moreover, a mixture of monoclonal and polyclonal antibodies are usable.
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As previously discussed, an indicator material is necessary to provide a measurable indication of the extent of reaction between binding component and analyte. The indicator is preferably an enzyme, which, in the presence of an appropriate substrate, catalyzes a reaction the result of which is a visibly colored product. The enzyme should also be inhibitable by an appropriate antibody.
This feature will be described in detail below. Any enzyme with appropriate substrate is usable as an indicator.
Among the common enzyme~substrate combinations include but are not limited to, alkaline phosphate/indoxyl phosphate, horseradish peroxidase/luminol or O-phenylene-diamine, hexakinase/phosphoenolpyruvate, or the like.
A first antibody is selected that will capture or bind to the antigen or analyte of interest, and is in turn bound to the solid phase. A second and third antibody are selected such that the second antibody binds selectively to the antigen. The third antibody is an anti-enzyme antibody capable of exerting an inhibitory effect on the enzyme.
This antibody may be prepared according to the procedure described above for producing monoclonal antibodies to a given antigen; in this case the antigen injected into the mouse would be the enzyme. The inhibitory effect of the anti-enzyme may be by means such as but not limited to ~1) steric hindrance of the enzvme's active site by binding at or near the active site; (2) conformational inhibition by binding to a site on the enzyme distant from the active .~
''1 2~R~74 site which would induce a conformational change of the enzyme structure resulting in the inactivation of the active site; (3) blocking by engulfing a substantial portion of the enzyme molecule effectively preventing access by the substrate; or other inhibitory mechanisms.
While the means of inhibition are noteworthy, it is the end effect of enzyme inhibition that is desired.
Second and third antibodies are conjugated to each other via a chemical or biochemical bond or linkage. One well known method for accomplishing this is by reaction with glutaraldehyde. Another method is indirect binding by attaching avidin to one antibody, biotinylating the other antibody and reacting the two products together, resulting in an avidin-biotin linkage. Indirect binding can also be achieved by attaching a sulfhydryl group to one antibody and an iodoacetyl moiety to the other antibody, then reacting the products together. Other methods are well known in the art and need not to be repeated here.
It is important that the anti-enzyme antibody be nonreactive toward the antigen, and, that the second antibody be nonreactive toward the enzyme. This ensures against both antibodies binding to the antigen or to the enzyme, which would produce a false result. Utilization of this double antibody conjugate provides a novel means for 2~ selectively binding to a signal resulting in its inactivation.
In accordance with the present invention, then, first ~ 2?4~F~74 antibody is bound to the solid phase material. First antibody, specimen, second antibody and third anti-enzyme antibody conjugate are contracted, whether simultaneously or sequentially, depending upon the desired procedure, and permitted to react to form an insolublized complex of first antibody:antigen:second antibody-third anti-enzyme antibody where antigen is present in the specimen. Any of the complex formed is subsequently separated from unbound material. The preferred embodiment employs glass fiber filters as the solid phase and the complex insolublized on or within the filter matrix, which can be washed with an appropriate wash solution. Active enzyme and its substrate are then contracted with the complex in a manner favoring the binding of enzyme by any of the complexed anti-enzyme present. Since initial level of enzyme activity is measurable prior and subsequent to contacting with the anti-enzyme antibod.y, the relative decrease in signal can be determined, which is inversely proportional to the presence or amount of antigen present in the specimen.
An alternative embodiment is a competitive assay wherein analyte suspected of being in a specimen competes with analyte bound to second antibody:anti-enzyme antibody conjugate for a limited number of bonding sites on an insolublized antibody. After allowing for equilibrium to be established, bound material is separated from unbound anti-enzyme conjugated analyte. Enæyme and substrate are added and the reduction of enzyme activity is observed.
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1 2~ 4 Another embodiment of the present invention is an indirect binding assay where the analyte of interest is an antibody. In this method the first binding component is an antigen insolublized on a solid surface and the second binding componenet is an anti-antibody directed against the analyte of interest. The procedure is carried out in the same manner as either the sandwich or competitive assays described hereinabove.
In yet another embodiment of this invention the signal generating component is comprised of an enzyme, its substrate and a cofactor. In such a method thel third binding component will preferentially bind to the coenzyme exerting an inhibitory influence on it, preventing the reaction of enzyme and substrate, and thereby preventing the formation of color. In this type of assay, after the insolublized complex has formed enzyme and substrate can be added to the complex together or separately, and then, at time zero, cofactor is added. Third binding component will inhibit the cofactor, or, alternatively, the enzyme, thereby preventing color from forming. The decreased rate of color formation is inversely related to the presence or amount of analyte present. An example of such a system uses an enzyme such as glyceralclehyde-3-phosphate dehydrogenase, a substrate such as glyceraldehyde-3-phosphate dehydrogenase and a cofactor such as a nicotinamide adenine dinucleotide tNAD). The above example is by way of illustration only and not limitation.
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As in all the above described embodiments the secondary binding components and analyte can be brought into contact with the insolublized first binding component either simultaneously or sequentially. Furthermore, enzyme and substrate may be added to the bound complex simultaneously or sequentially. This flexibility enables the user to configure the method to a variety of applications.
That the signal be inhibited in the presence of the 13 desired antigen is an important feature of this invention.
Where there is no reaction with antigen, the level of signal from the enzyme activity can be preset to lie within the optimal range for detection. Thus the assay can be turned or adjusted for different instrumentation systems that have varying sensitivity ranges or optimum performance ranges of signal detection. In conventional assays a blank background of theoretically zero signal generation is used to compare with a positive reaction signal. While this provides an a~equate basis for measurement, it has an inherent limitation built into the method: it is very difficult to completely eliminate background noise, which is caused by nonspecific binding of label, or the presence of signal generating substances in the sample solution, such as fluorescent materials, which produce a signal not correlative with the amount of antigen present. Such background noise reduces the overall efficiency and sensitivity of the assay. Also, the initial, low level, - . -activity measure of the sample component in question generally begins without the optimal or "linear" portion of the detection system. By eliminating the dependence upon a zero signal level and substituting a baseline level associated with full enzyme activity or signal output a more accurate and sensitive assay results.
An important determination option is the measurement of the differential rate of decrease of color or signal formation between at least two separate readings of the reaction mixture over a period of time between samples and/or standards. An initial reading such as at time zero is taken, enzyme and substrate (and/or cofactor) are added and allowed to react, and an endpoint reading is taken, such as at five minutes, using reagents optimized to give maximum color at that arbitrary cutoff time. The kinetic rate is compared to a reference standard, and the presence or concentration of analyte is determined from this comparison. By controlling the incubation period after adding enzyme and substrate one can extend or contract the endpoint to fit well within the linear range of the particular instrument. The advantage of this is to obviate the dependence upon baseline signal that has any limitation on it which may be based upon background noise. A rate-measuring assay exploits upon the differential rate of reaction, rather that the initial and/or final concentration of reactants.
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Most instrumentation has an optimal range of operation where sensitive results are best obtained. With the present invention the color or signal level is initially relatively high; i.e., at time zero, and consequently at the machine's most accurate and sensitive range. As inhibition occurs, the rate of color or signal production will decrease, but will still be well within the optimal sensitivity range of the instrument, thereby affording more sensitive detection and quantitation of sample component.
To illustrate, consider the absorbance being measured within a typical linear range oP utility of between 0.1 and 0.9 absorbance units. Conventional colorimetric assays, starting from a blank background level of theoretically 0.0, measure absorbance after reaction which might rise to 0.05 or 0.09 at the low end of the scale, which is in the nonlinear range of the instrument and may produce equivocal and less sensitive results. The present invention, however, initially has a reading at the upper end of the range, e.g., 0.9, which decreases as inhibition of signal increases, with the resulting absorbance falling well within the linear~ most sensitive, range of the instrument.
More precise results are thus obtainable with the present invention.
The Examples which follow further describe, define and illustrate a number of different embodiments of this invention. The apparatus and techniques used in the preparation of reagents and/or performance or evaluation of ~P .
the method of this invention are standard or as hereinbefore described.
BXAMPLES
Assay for human chorionic gonadotropin ("hCG").
(A) Procedure for preparing anti-enzyme antibody conjugate:
First, 1.5 mg of a monoclonal antibody directed against hCG ("Ab2") in phosphate buffer and 1.5 mg of a monoclonal antibody directed against al~aline phosphatase ("Ab3") in phosphate buffer are mixed ~Jith lOul glutaraldehyde and incubated for 75 minutes at 25'C. After cooling in an ice bath the mixture is put onto a gel filtration column and eluted with Tris~HCL buffer (50 mmol/l, pH 8.0, containing NaCl, 0.1 mmol/l; MgCl2, mmol/l; Na-azidè, 0.1~ (w~v)) in fractions of ca. lml.
Combine the protein containing fractions, which contain the purified conjugate, free of parts of Ab2 and Ab3 which have not reacted. Up to lOmg/ml bovine serum albumin can be added to the conjugate for stabilization.
tB) Simultaneous procedure for testing a specimen for hCG:
First, 1 drop (50ul) of an insolublized monoclonal antibody directed against hCG ("Abl"), 1 drop of specimen suspected of containing hCG and 1 drop of Ab2-Ab3 conjugate are mixed and incubated. The mixture is added to 12~ 4 a filter and the filtrate washed with 1 ml of Tris-buffered saline to separate bound form unbound material. Then, 1 drop of alkaline phosphatase and 1 drop of indoxyl phosphate substrate are added to the insolublized complex simultaneously. The reaction zone containing the components is read and the decreased rate of color formation is indicative of the presence of hCG.
Assay procedure for Group A St reprococcus ("GAS").
(A) Procedure for preparing anti-enzyme conjugate:
As an alternative procedure for preparing Ab2-Ab3 conju~ate (cf. EXAMPLE l(A)), first 1 ml of a monoclonal antibody directed against GAS ("Ab2") is mixed with 1 ml of a biotin active ester (such as N-hydroxysuccimide) and incubated for 1 hour. The mixture is separated by gel filtration as described above. The same biotinylation procedure is performed substituting for Ab2 a monoclonal antibody directed against an enzyme such as alkaline phosphatase ("Ab3"). Then, 1 ml of the products of the two reactants are mixed with 1 ml of avidin and incubated for 1 hour.
(B) Procedure for testing a specimen for GAS:
Sequential Method:
To 1 drop of an insolublized monoclonal antibody directed against GAS is added 1 drop of specimen suspected of containing GAS and incubated for 10 minutes. Then, 1 drop of Ab2-Ab3 conjugate is added to the mixture and - ::
incubated for 10 minutes. The mixture formed is added to a filter and then washed with 1 ml of Tris-buffered saline to separate bound from unbound material. To any insolublized material is added 1 drop of alkaline phosphatase and 1 drop of indoxyl phosphate substrate. The reaction zone containing the components is read and the decreased rate of color formation is indicative of the presence of GAS.
Assay procedure for other analytes:
The procedures of EXAMPLE l or EXAMPLE 2 are followed, but for the analyte used therein there is separately and in turn substituted one of the following: Rubella virus antibody, herpes simplex virus I or II, Neisseria qonorrhoeae, hepatitis, Chlamydia trachomatis, Candida albicans, Tricho~onas vaainalis, and creatinine phosphokinase. For Abl and Ab2 are substituted monoclonal antibodies directed against the analyte of interest.
In each case a positive result is obtained.
While the invention has been described in connection with certain preferred embodiments, it is not intended to limit the scope of the invention to the particular form set forth, but, on the contrary, it is intended to cover such alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.
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BACR~ROUND OF THE IN~IENTION
FI}:LD OF THE INVENTION
The present invention relates to methods for conducting a solid phase assay. More specifically, the present invention relates to methods for conducting an immunoassay for the detection or quantitation of a biological analyte, using an antibody: antibody conjugate which will inhibit an enzyme indicator bound to the conjugate.
PRIOR ART
The method of the present invention relates to the performance of a solid phase assay for the determination of the presence or quantity of a biological substance suspected of being in a specimen. Commonly, the substance of interest is an antigen but can also be an antibody.
Numerous techniques have been developed to measure the amount antigen present in a sample of biological fluid.
Certain of these techniques involve a solid phase system whereby a substance which will form an immunocomplex with the analyte is bound to an insolublized carrier or material, such as latex, cellulose, and the like. In this manner the immunocomplex when formed can be separated in its solid phase from the liquid phase, which contains excess uncomplexed material. Subsequently, a material which has been labelled or "tagged" with a signal generating molecule is added to the immunocomplex so that the material binds to the analyte. After separating the - ~ : ' . .
bound label from the unbound label, the sample is read and the amount of signal measured is related to the amount of analyte present to which it is bound.
Variations on the basic immunoassay technique have been developed to try to overcome some of the problems associated with immunoassays; sensitivity, reliability and cost effectiveness are the major concerns. Although the immunometric assay techniques have been found to be particularly useful in analyzing for antigens and antibodies, there has been difficulty in the past on establishing an optimum level of sensitivity for the assay to be helpful in the detection or monitoring of disease states or maladies in the body. Some of the original immunoassays developed used radioisotopic labelled substances as a signal generating material. While effective to a large degree, radioimmunoassays suffer from the handling and disposal problems and high costs involved with radioactive materials. To avoid these problems other techniques were developed to increase the sensitivity and reduce the cost and hazards of the immunoassay. Enzyme labels, along with fluorescent and luminescent labels have been introduced to increase sensitivity and reliability of the tests while reducing the cost per test, an expense that at bottom line is born by the patient.
In theory, the more specific the binding material is for the analyte the more sensitive the assay. To that end, multiple binding site assays have been developed so -~i I ' ~
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, 1 2~74 that less analyte is required to produce a reliability measurable signal. For instance, where an antigen is the analyte of interest several assays have been developed wherein two different antibodies are used: one is bound to the solid support and the other is labelled. The two antibodies bind to two different epitopic sites on the antigen. More recently, monoclonal antibodies have been developed for this type of use can be provide much greater selectivity and specificity for a given antigen. By being engineered to bind only to a given particular antigen monoclonals overcome the specificity problem associated with polyclonal antibodies. An example of this use is in EPA No. 0044219, which describes a method of immunoanalysis using monoclonal antibodies specific to two distinct antigenic binding sites.
US Patent No. 4,376,110, issued to David et al., discloses a sandwich immunoassay using two monoclonal antibodies which are site specific, yet may be a product of the same or dif:Ferent cell lines. A "sandwich," or "two-site," immunoassay relies upon the formation of an insolublized antibody:antigen:labelled antibody complex.
The two monoclonal antibodies are designed to minimize cross-reactivity and interference with each other when binding to the antigen. However, there still remains the problem of nonspecific binding and imperfect washing away of unbound materials. Some small amount of labelled antibody can remain in the rsaction area by binding to :
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either the reaction support surface or compounds in the area other than the insolublized antibody. This results in more label remaining in the area and generating a signal even though it is not bound to an antigen. since a falsely high signal level is measured, the overall assay is less sensitive than if the nonspecific binding could be further reduced or eliminated. Attempts have been made at increasing the sensitivity of the assay by increasing the selectivity and specificity of the antibody, e.g., by using monoclonal antibodies, and by improving the wash step to effect a more efficient separation of the bound from unbound label. These methods, however, still have yet to achieve the levels of sensitivity and consistency that are desired by the medical diagnostic industry in detecting minute quantities of analytes.
US Patent No. 4,446,233, issued to Auditore-Hargreaves et al., discloses a homogeneous immunoassay using a covalent hybrid antibody. The antibody contains one binding site specific for an analyte and one binding site specific for an indicator. The unique feature of that invention is the use of a single antibody which has been designed so as to contain two distinct binding sites on the same antibody, joined through inter-heavy-chain disulfide bonding. The antibody is composed of two different heavy-chain light-chain half-molecules. The method has limitations in that there are difficulties in designing a covalently hybrid antibody specific for a given antigen.
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For each different test a new hybrid must be created; as contrasted with the well known procedure for producing monoclonal antibodies. Furthermore, specificity problems relating to the purity and specificity of each half-chain of the hybrid can occur.
US Patent No. 4,134,792, issued to Boguslaski et al., discloses a procedure employing as a label a reversibly binding enzyme modulator in an enzyme inhibition assay.
The modulator is a chemical compound that can inhibit the activity of a particular enzyme. The distinguishing feature is that the enzyme binds to an inhibitor chemical molecule, rather than to an antibody, where the inhibitor molecule has been previously chemically conjugated to an antibody. The function and properties of a modulating antibody substantially different than a chemical molecule.
US Patent No. 4,233,401, issued to Yoshida et al., describes an anti-enzyme homogeneous competitive binding assay using a conjugated enzyme:ligand (an antibody or other molecule) which competes with analyte for binding sites on an antibody. When enzyme:ligand is bound to the antibody the enzyme inhibitor present is unable to inhibit the enzyme, while inhibition is possible when not bound to the antibody. The assay is limited to a homogeneous medium, using no solid phase, and required competition for the antibody binding sites. Furthermore, the enzyme is required to be bound to the li~uid prior to reaction with inhibitor.
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Us Patent No. 4,150,949, issued to Smith, describes a fluorescence quenching competitive assay for gentamicin wherein the ~luorescence of a labelled compound is reduced when the compound binds with the antibody. Reduction of fluorescence is measured to determine the amount of antibiotic in the sample.
US Patent No. 4,220,450, issued to Maggio, discloses a fluorescence immunoassay technique wherein a quencher molecule is conjugated to a member of an immunological pair, such as an antibody:antigen complex, and a chemiluminescent molecule is conjugated to the other member of the pair. When the complex forms the c~uencher is positioned in close proximity to the chemiluminescer and inhibits the emission of light by the chemiluminescent sourçe.
US Patent No. 4,281,061, issued to Zuk et al., describes-a double antibody assay wherein analyte, labelled ligand, antilicJand and polyvalent receptor for the antiligand create a matrix which modulates the approach of a macromolecular member of a signal producing system to the labelled ligand.
The primary concerns with these labelled compound assay systems is the interference of background signal noise. BaGkground noise occurs, in part, because of nonspecific binding of labelled compound to material in the sample other than the analyte. In a fluorescent assay system, naturally fluorescing substances often are present in .' .
the sample fluid which emit a signal that can partially "drown out"
the signal being transmitted by the antigen bound tag. In an enzyme label system enzyme can bind to other proteins in the solution.
To reduce this noise a wash step is necessary to separate the insolubilized bound material from the unbound material in solution.
The result is an improved signal; still, current techniques are still unable to reduce the background noise level to the point where a much more sensitive assay is obtainable.
Present technology is based on the reading of a positive signal against a "blank" background of supposedly zero signal output. In reality, however, there is spurious signal generated from nonspecific binding of label as well as naturally occurring signal emitters in the biological solution. This results in a less sensltive assay.
There is a need then for an immunoassay system that obviates the dependence upon a realistically unachievable zero baseline level, a system that is based upon a user-controlled baseline that would achieve a more sensitive determination of analyte.
SUMMARY OF THE INVENTION
The instant invention overcomes the disadvantages in the prior art and achieves the objectives stated hereinabove. Briefly stated the present invention comprises a heterogeneous immunoassay for the detection or quantitation of an analyte suspected of being in a specimen comprising: (a) providing a specimen suspected of containing an analyte of interest, an insolubilized first binding component capable of immunologically reacting with said analyte, a second binding component capable of immunologically reacting with said analyte and to which has been chemically bound a third binding component incapable of reacting with said analyte but capable of binding to and inhibiting the activity of a signal generating ,~
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74c component: (b) combining said specimen, insolubilized binding component and bound second and third binding components in a manner favoring the formation of an insolubilized complex between insolu-bilized first binding component, any of said analyte present, and bound second and third binding components; (c) separating at least all unbound third binding component from any said insolubilized complex material; ~d) contacting with said insolubilized complex an amount of active signal generating component sufficient and for an incubation period sufficient to bind said complexed third binding component resulting in the inhibition of signal from any signal component and to fit within the linear range of the instrument used to measure said signal; and (e) measuring the amount of signal generated as being inversely related to the presence or amount of analyte in said specimen.
lS DESCRIPTION OF THF INVENTION
A heterog0neous assay employs a two phase system whereby certain components are bound to a solid phase, which must be physically separated from unbound components in a liquid phase. The preferred embodiment is a sandwich assay technique in which two distinct antigenic sites on the antigen are used as binding areas.
An antibody is insolubilized on a solid surface and is used to "capture" the antigen by binding to it at one antigenic site. A
second antibody, which is conjugated to an indicator or label, binds to the bound antigen at another site. The unbound conjugate is separated from the bound conjugate, typically by washing, centrifugation, precipitation, filtration, or other procedure. The indicator is then developed, if necessary, and read to obtain a measurement of signal intensity which is correlative with the presence or amount of antigen present in the specimen.
~t '74 In a competitive heterogeneous assay labelled antigen and sample antigen compete for a limited number of binding sites on an insolublized antibody. The solid phase is washed or separated to remove unbound material. After the label is developed, if necessary, it is read; the activity of the solid phase-bound label is inversely proportional to the amount of unlabelled antigen present on the sample.
An indirect assay can be performed for the detection of antibody whereby antigen is bound to a solid phase, which in turn is reacted with the unknown antibody (such as Rubella virus antibody, HTLV-III virus antibody, and the like) which binds to the insolublized antigen. After separating bound from unbound antibody a labelled anti-antibody is added which binds to the unknown antibody. The solid phase is washed and read as described above and the amount of signal is proportional to the presence or amount of antibody present in the specimen.
The solid phase is typically a solid support surface to which is linked the capture antibody or antigen. The surface can be composed of material such as but not limited to latex, glass, glass beads, cellulose, wood, sticks, plastic or other synthetic polymers, filter paper, and the like.
The analyte is a substance suspected of being in a specimen whose presence or concentration is to be determined. The specimen is any biological fluid including whole blood, serum or plasma, cerebral spinal fluid, ~Zl , , saliva, feces, sputum, mucusr urine, cell or tissue extracts, pus, wound exudate, and the like. The analyte is a material that can include drugs, hormones, vitamins, enzymes, proteins, antibodies, polysaccharides, bacteria, protozoa, parasites, fungi, viruses, cell and tissue antigens, other blood cell or blood fluid substances, and the like.
Binding co~ponent is a material capable of selectively complexing or binding to a recognizable substance: i.e., the analyte or the label indicator material. The binding component commonly will be an antibody, but can also be an antigen such as where Rubella virus is the analyte. Either polyclonal antibodies or monoclonal antibodies are usable in the method of this invention. Monoclonal antibodies are preferable because of their sensitivity, selectivity and specificity, and they are able to be designed for a particular analyte of interest. Monoclonal antibodies are prepared according to the method described by Kholer and Milstein in Nature 256:495-497, 1975. More specifically, the monoclonal antibodies of the present invention are prepared by fusing spleen cells, from a mammal which has been immunized against the antigen with an appropriate myeloma cell line. The resultant product is then cultured in a standard HAT (hypoxanthine, aminopterin, and thymidine) medium. Screening tests for the specific monoclonal antibodies are employed utilizing immunoassay techni~ues which will be described below.
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The immunized spleen cells may be derived from any mammal, such as primates, humans, rodents (i.e., mice, rats, and rabbits), bovine, ovine, canine, or the like, but the present invention will be described in connection with mice. The mouse is first immunized by injection of an antigen chosen generally for a period of eleven weeks.
When the mouse shows sufficient antibody production against the antigen, as determined by conventional assay, it is given a booster injection of the antigen, and then killed so that the immunized spleen may be removed. ~he fusion can then be carried out utilizing immunized spleen cells and an appropriate myeloma cell line.
The fused cells yielding an antibody which give a positive response to the presence of the antigen are removed and cloned utilizing any of the standard methods.
The monoclonal antibodies from the clones are then tested against standard antigens to determine their specificity for the particular antigen. The monoclonal antibody selected, which is specific for the antigen or species, is then bound to an appropriate label.
Amounts of antibody sufficient for labelling and subsequent commercial production are produced by the known techniques, such as by katch or continuous tissue culture or culture ln vivo in mammals, such as mice.
At least one binding component can be a monoclonal antibody, and preferably all three are; moreover, a mixture of monoclonal and polyclonal antibodies are usable.
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As previously discussed, an indicator material is necessary to provide a measurable indication of the extent of reaction between binding component and analyte. The indicator is preferably an enzyme, which, in the presence of an appropriate substrate, catalyzes a reaction the result of which is a visibly colored product. The enzyme should also be inhibitable by an appropriate antibody.
This feature will be described in detail below. Any enzyme with appropriate substrate is usable as an indicator.
Among the common enzyme~substrate combinations include but are not limited to, alkaline phosphate/indoxyl phosphate, horseradish peroxidase/luminol or O-phenylene-diamine, hexakinase/phosphoenolpyruvate, or the like.
A first antibody is selected that will capture or bind to the antigen or analyte of interest, and is in turn bound to the solid phase. A second and third antibody are selected such that the second antibody binds selectively to the antigen. The third antibody is an anti-enzyme antibody capable of exerting an inhibitory effect on the enzyme.
This antibody may be prepared according to the procedure described above for producing monoclonal antibodies to a given antigen; in this case the antigen injected into the mouse would be the enzyme. The inhibitory effect of the anti-enzyme may be by means such as but not limited to ~1) steric hindrance of the enzvme's active site by binding at or near the active site; (2) conformational inhibition by binding to a site on the enzyme distant from the active .~
''1 2~R~74 site which would induce a conformational change of the enzyme structure resulting in the inactivation of the active site; (3) blocking by engulfing a substantial portion of the enzyme molecule effectively preventing access by the substrate; or other inhibitory mechanisms.
While the means of inhibition are noteworthy, it is the end effect of enzyme inhibition that is desired.
Second and third antibodies are conjugated to each other via a chemical or biochemical bond or linkage. One well known method for accomplishing this is by reaction with glutaraldehyde. Another method is indirect binding by attaching avidin to one antibody, biotinylating the other antibody and reacting the two products together, resulting in an avidin-biotin linkage. Indirect binding can also be achieved by attaching a sulfhydryl group to one antibody and an iodoacetyl moiety to the other antibody, then reacting the products together. Other methods are well known in the art and need not to be repeated here.
It is important that the anti-enzyme antibody be nonreactive toward the antigen, and, that the second antibody be nonreactive toward the enzyme. This ensures against both antibodies binding to the antigen or to the enzyme, which would produce a false result. Utilization of this double antibody conjugate provides a novel means for 2~ selectively binding to a signal resulting in its inactivation.
In accordance with the present invention, then, first ~ 2?4~F~74 antibody is bound to the solid phase material. First antibody, specimen, second antibody and third anti-enzyme antibody conjugate are contracted, whether simultaneously or sequentially, depending upon the desired procedure, and permitted to react to form an insolublized complex of first antibody:antigen:second antibody-third anti-enzyme antibody where antigen is present in the specimen. Any of the complex formed is subsequently separated from unbound material. The preferred embodiment employs glass fiber filters as the solid phase and the complex insolublized on or within the filter matrix, which can be washed with an appropriate wash solution. Active enzyme and its substrate are then contracted with the complex in a manner favoring the binding of enzyme by any of the complexed anti-enzyme present. Since initial level of enzyme activity is measurable prior and subsequent to contacting with the anti-enzyme antibod.y, the relative decrease in signal can be determined, which is inversely proportional to the presence or amount of antigen present in the specimen.
An alternative embodiment is a competitive assay wherein analyte suspected of being in a specimen competes with analyte bound to second antibody:anti-enzyme antibody conjugate for a limited number of bonding sites on an insolublized antibody. After allowing for equilibrium to be established, bound material is separated from unbound anti-enzyme conjugated analyte. Enæyme and substrate are added and the reduction of enzyme activity is observed.
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1 2~ 4 Another embodiment of the present invention is an indirect binding assay where the analyte of interest is an antibody. In this method the first binding component is an antigen insolublized on a solid surface and the second binding componenet is an anti-antibody directed against the analyte of interest. The procedure is carried out in the same manner as either the sandwich or competitive assays described hereinabove.
In yet another embodiment of this invention the signal generating component is comprised of an enzyme, its substrate and a cofactor. In such a method thel third binding component will preferentially bind to the coenzyme exerting an inhibitory influence on it, preventing the reaction of enzyme and substrate, and thereby preventing the formation of color. In this type of assay, after the insolublized complex has formed enzyme and substrate can be added to the complex together or separately, and then, at time zero, cofactor is added. Third binding component will inhibit the cofactor, or, alternatively, the enzyme, thereby preventing color from forming. The decreased rate of color formation is inversely related to the presence or amount of analyte present. An example of such a system uses an enzyme such as glyceralclehyde-3-phosphate dehydrogenase, a substrate such as glyceraldehyde-3-phosphate dehydrogenase and a cofactor such as a nicotinamide adenine dinucleotide tNAD). The above example is by way of illustration only and not limitation.
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As in all the above described embodiments the secondary binding components and analyte can be brought into contact with the insolublized first binding component either simultaneously or sequentially. Furthermore, enzyme and substrate may be added to the bound complex simultaneously or sequentially. This flexibility enables the user to configure the method to a variety of applications.
That the signal be inhibited in the presence of the 13 desired antigen is an important feature of this invention.
Where there is no reaction with antigen, the level of signal from the enzyme activity can be preset to lie within the optimal range for detection. Thus the assay can be turned or adjusted for different instrumentation systems that have varying sensitivity ranges or optimum performance ranges of signal detection. In conventional assays a blank background of theoretically zero signal generation is used to compare with a positive reaction signal. While this provides an a~equate basis for measurement, it has an inherent limitation built into the method: it is very difficult to completely eliminate background noise, which is caused by nonspecific binding of label, or the presence of signal generating substances in the sample solution, such as fluorescent materials, which produce a signal not correlative with the amount of antigen present. Such background noise reduces the overall efficiency and sensitivity of the assay. Also, the initial, low level, - . -activity measure of the sample component in question generally begins without the optimal or "linear" portion of the detection system. By eliminating the dependence upon a zero signal level and substituting a baseline level associated with full enzyme activity or signal output a more accurate and sensitive assay results.
An important determination option is the measurement of the differential rate of decrease of color or signal formation between at least two separate readings of the reaction mixture over a period of time between samples and/or standards. An initial reading such as at time zero is taken, enzyme and substrate (and/or cofactor) are added and allowed to react, and an endpoint reading is taken, such as at five minutes, using reagents optimized to give maximum color at that arbitrary cutoff time. The kinetic rate is compared to a reference standard, and the presence or concentration of analyte is determined from this comparison. By controlling the incubation period after adding enzyme and substrate one can extend or contract the endpoint to fit well within the linear range of the particular instrument. The advantage of this is to obviate the dependence upon baseline signal that has any limitation on it which may be based upon background noise. A rate-measuring assay exploits upon the differential rate of reaction, rather that the initial and/or final concentration of reactants.
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Most instrumentation has an optimal range of operation where sensitive results are best obtained. With the present invention the color or signal level is initially relatively high; i.e., at time zero, and consequently at the machine's most accurate and sensitive range. As inhibition occurs, the rate of color or signal production will decrease, but will still be well within the optimal sensitivity range of the instrument, thereby affording more sensitive detection and quantitation of sample component.
To illustrate, consider the absorbance being measured within a typical linear range oP utility of between 0.1 and 0.9 absorbance units. Conventional colorimetric assays, starting from a blank background level of theoretically 0.0, measure absorbance after reaction which might rise to 0.05 or 0.09 at the low end of the scale, which is in the nonlinear range of the instrument and may produce equivocal and less sensitive results. The present invention, however, initially has a reading at the upper end of the range, e.g., 0.9, which decreases as inhibition of signal increases, with the resulting absorbance falling well within the linear~ most sensitive, range of the instrument.
More precise results are thus obtainable with the present invention.
The Examples which follow further describe, define and illustrate a number of different embodiments of this invention. The apparatus and techniques used in the preparation of reagents and/or performance or evaluation of ~P .
the method of this invention are standard or as hereinbefore described.
BXAMPLES
Assay for human chorionic gonadotropin ("hCG").
(A) Procedure for preparing anti-enzyme antibody conjugate:
First, 1.5 mg of a monoclonal antibody directed against hCG ("Ab2") in phosphate buffer and 1.5 mg of a monoclonal antibody directed against al~aline phosphatase ("Ab3") in phosphate buffer are mixed ~Jith lOul glutaraldehyde and incubated for 75 minutes at 25'C. After cooling in an ice bath the mixture is put onto a gel filtration column and eluted with Tris~HCL buffer (50 mmol/l, pH 8.0, containing NaCl, 0.1 mmol/l; MgCl2, mmol/l; Na-azidè, 0.1~ (w~v)) in fractions of ca. lml.
Combine the protein containing fractions, which contain the purified conjugate, free of parts of Ab2 and Ab3 which have not reacted. Up to lOmg/ml bovine serum albumin can be added to the conjugate for stabilization.
tB) Simultaneous procedure for testing a specimen for hCG:
First, 1 drop (50ul) of an insolublized monoclonal antibody directed against hCG ("Abl"), 1 drop of specimen suspected of containing hCG and 1 drop of Ab2-Ab3 conjugate are mixed and incubated. The mixture is added to 12~ 4 a filter and the filtrate washed with 1 ml of Tris-buffered saline to separate bound form unbound material. Then, 1 drop of alkaline phosphatase and 1 drop of indoxyl phosphate substrate are added to the insolublized complex simultaneously. The reaction zone containing the components is read and the decreased rate of color formation is indicative of the presence of hCG.
Assay procedure for Group A St reprococcus ("GAS").
(A) Procedure for preparing anti-enzyme conjugate:
As an alternative procedure for preparing Ab2-Ab3 conju~ate (cf. EXAMPLE l(A)), first 1 ml of a monoclonal antibody directed against GAS ("Ab2") is mixed with 1 ml of a biotin active ester (such as N-hydroxysuccimide) and incubated for 1 hour. The mixture is separated by gel filtration as described above. The same biotinylation procedure is performed substituting for Ab2 a monoclonal antibody directed against an enzyme such as alkaline phosphatase ("Ab3"). Then, 1 ml of the products of the two reactants are mixed with 1 ml of avidin and incubated for 1 hour.
(B) Procedure for testing a specimen for GAS:
Sequential Method:
To 1 drop of an insolublized monoclonal antibody directed against GAS is added 1 drop of specimen suspected of containing GAS and incubated for 10 minutes. Then, 1 drop of Ab2-Ab3 conjugate is added to the mixture and - ::
incubated for 10 minutes. The mixture formed is added to a filter and then washed with 1 ml of Tris-buffered saline to separate bound from unbound material. To any insolublized material is added 1 drop of alkaline phosphatase and 1 drop of indoxyl phosphate substrate. The reaction zone containing the components is read and the decreased rate of color formation is indicative of the presence of GAS.
Assay procedure for other analytes:
The procedures of EXAMPLE l or EXAMPLE 2 are followed, but for the analyte used therein there is separately and in turn substituted one of the following: Rubella virus antibody, herpes simplex virus I or II, Neisseria qonorrhoeae, hepatitis, Chlamydia trachomatis, Candida albicans, Tricho~onas vaainalis, and creatinine phosphokinase. For Abl and Ab2 are substituted monoclonal antibodies directed against the analyte of interest.
In each case a positive result is obtained.
While the invention has been described in connection with certain preferred embodiments, it is not intended to limit the scope of the invention to the particular form set forth, but, on the contrary, it is intended to cover such alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.
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Claims (29)
1. A heterogeneous immunoassay for the detection or quantitation of an analyte suspected of being in a specimen comprising:
(a) providing a specimen suspected of containing an analyte of interest, an insolubilized first binding component capable of immunologically reacting with said analyte, a second binding component capable of immunologically reacting with said analyte and to which has been chemically bound a third binding component incapable of reacting with said analyte but capable of binding to and inhibiting the activity of a signal generating component:
(b) combining said specimen, insolubilized binding component and bound second and third binding components in a manner favoring the formation of an insolubilized complex between insolubilized first binding component, any of said analyte present, and bound second and third binding components;
(c) separating at least all unbound third binding component from any said insolubilized complex material;
(d) contacting with said insolubilized complex an amount of active signal generating component sufficient and for an incubation period sufficient to bind said complexed third binding component resulting in the inhibition of signal from any signal component and to fit within the linear range of the instrument used to measure said signal; and (e) measuring the amount of signal generated as being inversely related to the presence or amount of analyte in said specimen.
(a) providing a specimen suspected of containing an analyte of interest, an insolubilized first binding component capable of immunologically reacting with said analyte, a second binding component capable of immunologically reacting with said analyte and to which has been chemically bound a third binding component incapable of reacting with said analyte but capable of binding to and inhibiting the activity of a signal generating component:
(b) combining said specimen, insolubilized binding component and bound second and third binding components in a manner favoring the formation of an insolubilized complex between insolubilized first binding component, any of said analyte present, and bound second and third binding components;
(c) separating at least all unbound third binding component from any said insolubilized complex material;
(d) contacting with said insolubilized complex an amount of active signal generating component sufficient and for an incubation period sufficient to bind said complexed third binding component resulting in the inhibition of signal from any signal component and to fit within the linear range of the instrument used to measure said signal; and (e) measuring the amount of signal generated as being inversely related to the presence or amount of analyte in said specimen.
2 The immunoassay of claim 1 wherein said first binding component, specimen, bound second and third binding component are added to said reaction zone simultaneously.
3. The immunoassay of claim 1 wherein said first binding component, specimen, conjugated second and third binding component are mixed sequentially in a predetermined order.
4. The immunoassay of claim 1 wherein said signal generating component is composed of a substance or substances capable of being inactivated or inhibited when contacted with an antibody directed against it.
5. The immunoassay of claim 4 wherein said signal generating component is selected from an enzyme, luminescent, fluorescent, bioluminescent, chemiluminescent or radioisotopic material.
6. The immunoassay of claim 5 wherein said signal generating component is an active enzyme and an appropriate substrate.
7. The immunoassay of claim 6 wherein said enzyme is alkaline phosphatase and said substrate is indoxyl phosphate.
8. The immunoassay of claim 5 wherein said enzyme is luciferase and said substrate is luciferol.
9. The immunoassay of claim 6 wherein said enzyme is contacted with said complexed third binding component prior to the addition of said substrate.
10. The immunoassay of claim 1 wherein said analyte is an antigen.
11. The immunoassay of claim 10 wherein said antigen is a material selected from a drug, hormone, vitamin, enzyme, protein, antibody, polysaccharide, bacteria, protozoa, parasite, fungus, virus, cell or tissue antigens or other blood cell or blood fluid substances.
12. The immunoassay of claim 1 wherein said first binding component, second binding component and third binding component are antibodies.
13. The immunoassay of claim 12 wherein said first binding component is an antibody directed against said analyte of interest.
14. The immunoassay of claim 12 wherein said second binding component is an antibody directed against said analyte of interest.
15. The immunoassay of claim 12 wherein said third binding component is an antibody directed against a particular signal generating component exerting an inhibitory influence on said material and is inactive toward said analyte.
16. The immunoassay of claim 12 wherein at least one of said first, second and third binding components are polyclonal antibodies.
17. The immunoassay of claim 12 wherein at least one of said first, second and third binding components are monoclonal antibodies.
18. The immunoassay of claim 12 wherein said first, second and third binding components are a mixture of polyclonal and monoclonal antibodies such that at least one of said binding components is a monoclonal antibody.
19. The immunoassay of claim 12 wherein said second binding component and said third binding component are chemically bound together.
20. The immunoassay of claim 19 wherein said second binding component and said third binding component are bound by a biotin-avidin linkage.
21. The immunoassay of claim 19 wherein said second binding component and said third binding component are bound by reaction with glutaraldehyde.
22. The immunoassay of claim 19 wherein said second and third binding components are added separately to the reaction mixture of step (b) in such a manner that said second and third binding components become conjugated to each other in the process of complex formation.
23. The immunoassay of claim 1 wherein said reaction zone is defined by a solid porous matrix means capable of retaining said reaction components in a delimited area.
24. A method for performing a heterogeneous assay immunoassay for the detection or quantitation of human chorionic gonadotropin suspected of being in solution comprising:
(a) providing a specimen suspected of containing human chorionic gonadotropin, an insolubilized first monoclonal antibody directed against human chorionic gonadotropin, a second monoclonal antibody directed against human chorionic gonadotropin and that has been chemically conjugated to a third monoclonal antibody which is capable of binding to and inactivating or inhibiting luciferase;
(b) combining in a reaction zone said specimen solution, insolubilized first monoclonal antibody and second monoclonal antibody bound to third monoclonal antibody in a manner favoring the formation of an insolubilized complex between insolubilized first monoclonal antibody:human chorionic gonadotropin:second monoclonal antibody - third monoclonal antibody;
(c) separating at least all unbound third monoclonal antibody from any said insolubilized complex material;
(d) contacting with said insolubilized complex an amount of active luceriferase and luciferol sufficient and for an incubation period sufficient such that any of said third monoclonal antibody present in said complex exerts an inhibitory influence on said luciferase and to fit within the linear range of the instrument used to measure any signal generated by said lucerifase and luminol; and (e) measuring the decrease in signal generated by said effective amount of luceriferase and luminol as being inversely related to the presence or amount of human chorionic gonadotropin present in said specimen.
(a) providing a specimen suspected of containing human chorionic gonadotropin, an insolubilized first monoclonal antibody directed against human chorionic gonadotropin, a second monoclonal antibody directed against human chorionic gonadotropin and that has been chemically conjugated to a third monoclonal antibody which is capable of binding to and inactivating or inhibiting luciferase;
(b) combining in a reaction zone said specimen solution, insolubilized first monoclonal antibody and second monoclonal antibody bound to third monoclonal antibody in a manner favoring the formation of an insolubilized complex between insolubilized first monoclonal antibody:human chorionic gonadotropin:second monoclonal antibody - third monoclonal antibody;
(c) separating at least all unbound third monoclonal antibody from any said insolubilized complex material;
(d) contacting with said insolubilized complex an amount of active luceriferase and luciferol sufficient and for an incubation period sufficient such that any of said third monoclonal antibody present in said complex exerts an inhibitory influence on said luciferase and to fit within the linear range of the instrument used to measure any signal generated by said lucerifase and luminol; and (e) measuring the decrease in signal generated by said effective amount of luceriferase and luminol as being inversely related to the presence or amount of human chorionic gonadotropin present in said specimen.
25. A method for performing a competitive heterogeneous assay for the detection or quantitation of an analyte suspected of being in a specimen comprising:
(a) providing a specimen suspected of containing an analyte of interest, an insolubilized first binding component, an effective amount of said analyte bound to a second binding component directed against said analyte, and a third binding component conjugated to said second binding component, said third binding component incapable of reacting with said analyte but capable of binding to and inhibiting the activity of a signal generating component;
(b) combining said specimen, insolubilized first binding component, analyte bound to conjugated second binding component - third binding component in a manner that any analyte present in said specimen and said conjugated analyte compete for available sites on said insolubilized first binding component to form an insolubilized complex;
(c) separating at least all unbound third binding component from said insolubilized complex material;
(d) contacting with said insolubilized complex material an active signal generating component capable of being bound by said third binding component in an amount sufficient and for an incubation period sufficient to bind said third binding component resulting in the inhibition of any signal and to fit within the linear range of the instrument used to measure said signal; and (e) measuring the amount of signal generated as being inversely related to the presence or amount of analyte in said specimen.
(a) providing a specimen suspected of containing an analyte of interest, an insolubilized first binding component, an effective amount of said analyte bound to a second binding component directed against said analyte, and a third binding component conjugated to said second binding component, said third binding component incapable of reacting with said analyte but capable of binding to and inhibiting the activity of a signal generating component;
(b) combining said specimen, insolubilized first binding component, analyte bound to conjugated second binding component - third binding component in a manner that any analyte present in said specimen and said conjugated analyte compete for available sites on said insolubilized first binding component to form an insolubilized complex;
(c) separating at least all unbound third binding component from said insolubilized complex material;
(d) contacting with said insolubilized complex material an active signal generating component capable of being bound by said third binding component in an amount sufficient and for an incubation period sufficient to bind said third binding component resulting in the inhibition of any signal and to fit within the linear range of the instrument used to measure said signal; and (e) measuring the amount of signal generated as being inversely related to the presence or amount of analyte in said specimen.
26. The method of claim 24 wherein said reactants are combined in a reaction zone.
27. The method of claim 25 wherein said reactants are combined in a reaction zone.
28. The method of claim 26 or 27 wherein said reaction zone is a filter matrix means comprising a solid porous inert structure composed of a material capable of containing on or within its pores an immunological reaction and capable of retaining an insolubilized product.
29. The method of claims 1, 24 or 25 further comprising:
(a) contacting with any of said insolubilized third binding component an effective amount of a cofactor material necessary for a particular enzyme-substrate reaction wherein said conjugated third binding component preferentially recognizes and binds said cofactor exerting an inhibitory influence on said cofactor;
(b) permitting an equilibrium of bound and unbound cofactor to form; and then (c) contacting with said complex an effective amount of enzyme and its substrate so that unbound cofactor will react with said enzyme and substrate resulting in the generation of a signal.
(a) contacting with any of said insolubilized third binding component an effective amount of a cofactor material necessary for a particular enzyme-substrate reaction wherein said conjugated third binding component preferentially recognizes and binds said cofactor exerting an inhibitory influence on said cofactor;
(b) permitting an equilibrium of bound and unbound cofactor to form; and then (c) contacting with said complex an effective amount of enzyme and its substrate so that unbound cofactor will react with said enzyme and substrate resulting in the generation of a signal.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US84406786A | 1986-03-26 | 1986-03-26 | |
| US844,067 | 1986-03-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1289874C true CA1289874C (en) | 1991-10-01 |
Family
ID=25291713
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000532968A Expired - Fee Related CA1289874C (en) | 1986-03-26 | 1987-03-25 | Anti-enzyme antibody immunoassay |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU7238087A (en) |
| CA (1) | CA1289874C (en) |
| WO (1) | WO1987006006A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63159759A (en) * | 1986-12-24 | 1988-07-02 | Fujirebio Inc | Enzyme immunoassay |
| US5565326A (en) * | 1994-05-31 | 1996-10-15 | Johnson & Johnson Clinical Diagnostics, Inc. | Separation-free specific binding assays using anti-inhibitor antibodies |
| US5705353A (en) * | 1995-06-07 | 1998-01-06 | Beckman Instruments, Inc. | Method of reducing interferences in assays |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4134792A (en) * | 1976-12-06 | 1979-01-16 | Miles Laboratories, Inc. | Specific binding assay with an enzyme modulator as a labeling substance |
| US4228237A (en) * | 1978-09-21 | 1980-10-14 | Calbiochem-Behring Corp. | Methods for the detection and determination of ligands |
| US4493890A (en) * | 1981-03-23 | 1985-01-15 | Miles Laboratories, Inc. | Activated apoglucose oxidase and its use in specific binding assays |
| US4433059A (en) * | 1981-09-08 | 1984-02-21 | Ortho Diagnostic Systems Inc. | Double antibody conjugate |
| EP0119767B1 (en) * | 1983-03-11 | 1990-11-22 | FUJIREBIO KABUSHIKI KAISHA also trading as FUJIREBIO INC. | Method of measuring ligands |
| JPS59202064A (en) * | 1983-04-30 | 1984-11-15 | Fujirebio Inc | Method for measuring antigen determining group possessing substance |
-
1987
- 1987-03-19 AU AU72380/87A patent/AU7238087A/en not_active Abandoned
- 1987-03-19 WO PCT/US1987/000571 patent/WO1987006006A1/en unknown
- 1987-03-25 CA CA000532968A patent/CA1289874C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| AU7238087A (en) | 1987-10-20 |
| WO1987006006A1 (en) | 1987-10-08 |
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