WO1989009409A1

WO1989009409A1 - Immunoassays employing novel markers

Info

Publication number: WO1989009409A1
Application number: PCT/US1989/001333
Authority: WO
Inventors: Ram Nunna
Original assignee: Ram Nunna
Priority date: 1988-03-31
Filing date: 1989-03-30
Publication date: 1989-10-05

Abstract

This invention pertains to homogeneous and heterogeneous enzyme immunoassays employing proteins of blood coagulation pathways. The invention describes use of novel conjugates that generate a visible, insoluble aggregate as a signal. Depending upon the assay format, the time needed to form the insoluble aggregate is a function of the amount of analyte originally present in the sample. These methods eliminate the need for colorimetric or radiometric methods of analysis.

Description

IMMUNOASSAYS EMPLOYING NOVEL MARKERS

Description

Background

Immunoassay is a widely used technique for determining the presence of an analyte in a sample. It can be carried out in a variety of different assay formats (e.g., competitive and non-competi¬ tive, homogeneous and heterogeneous), and can make use of numerous separation methods and labelling techniques (e.g., enzymes, co-enzymes, fluorogenic substrates and dyes).

The specificity of such immunoassays depends on the use of a molecule that binds to the analyte or target of interest. Examples of such molecules include an antibody directed against a protein analyte or a lectin binding to a particular glyco- protein target. Detection of this molecule is typically achieved by complexing with, or including in the molecule, a marker group which can produce a detectable signal. The sensitivity of an assay therefore depends on the specificity of the marker system in providing a signal only from the binding molecule, and the intensity of signal generated by the marker system.

Presently available immunoassays utilizing naturally-occurring enzymes as markers (hereinafter enzyme immunoassays) are advantageous because of their lack of toxicity and longer reagent shelf life. However, their general utility is limited because the enzymes may not generate a visible signal. Moreover, many of them require sophis- ticated instrumentation to measure the signal that is generated by those enzyme markers that do produce a visible and/or colorimetric signal.

Summary of the Invention

This invention pertains to a method of quanti- fying an analyte of interest through use of enzyme immunoassays having enzymes and modulators that generate a visible signal.. This method is par¬ ticularly useful because of its enhanced sensi¬ tivity. In particular, this invention is based on use of enzymes and modulators of plasma coagulation pathways or proteins capable of enzymatic participa¬ tion in plasma coagulation pathways to form a visible clot. The time required for clot formation indicates the extent of enzymatic activity which, in turn, is directly proportional to the concentra- tion(s) of the analyte(s) of interest.

Enzymes and modulators useful in the method include those of the coagulation pathways of bac- teria, reptiles, arthropods and mammals. These enzymes catalyse proteolytic reactions and cause polymerization of fibrinogen or fibrinogen-like substrates. The resultant liquid to solid phase change causes formation of an insoluble aggregate, the detection of which does not require expensive or complicated detection methods.

The method of the present invention uses enzyme conjugates in which a proteolytic enzyme of a coagulation pathway or any protein capable of participating in a coagulation pathway (i.e., a modulator) is linked with either the analyte to be determined (i.e., the analyte of interest) or an antibody or antibody fragment) directed against the analyte of interest. Such an antibody or antibody fragment is capable of binding to the analyte of interest. Such conjugates can be used in both homogeneous and heterogeneous enzyme immunoassays and in either a competitive or a non-competitive format. The increased sensitivity of the method of the present invention is a particular advantage. It is possible to join an enzyme or a modulator of a coagulation pathway to a "binding" molecule and then provide for an enzymatic substrate catalysed by the enzyme. In this way, a large number of enzyme- substrate reactions can enhance the initial signal.

Brief Description of the Drawings

Figure 1 is a schematic representation of the enzymatic reactions involved in blood clot for a- tion. Figure 2 is a schematic representation of the steps involved in a competitive homogeneous assay of the present invention.

Figure 3 is a schematic representation of the various homogenous assay formats of this invention. Asterisks (*) denote i munocomple es formed.

Figure 4 is a schematic represenation of the steps involved in a "sandwich" type hetergeneous assay of the present invention.

Detailed Description of the Invention

This invention pertains to a method of immuno- assay which makes use of proteolytic enzymes and other proteins that are capable of entering into reactions leading to a physical phase change of the immunoassay system. This phase change can be detected and measured and is used to detect and/or measure (quantitate) an analyte or analytes of interest in a sample. In particular, the present invention is a method of enzyme immunoassay which makes use of proteolytic enzymes and modulators of coagulation pathways to produce a detectable and measurable phase change by which the presence and/or quantity of an analyte of interest in a sample can be determined. Enzymes and other proteins (e.g.., modulators) used in this invention are proteins, glyσoproteins, and/or lipoproteins from the coagulation pathways of humans, arthropods, reptiles and certain pro- karyotes. These are referred to herein as "proteins" . Formation and Use of Conjugates

The term "conjugate", as used herein refers to a combination of a protein and either an analyte or antibody, or antibody fragment, depending on the assay format. These conjugates can be prepared by standard coupling techniques. For example, proteolytic enzymes of blood coagulation pathways can be conjugated to proteins via amino, carboxyl, sulfhydryl, disulfide, hydroxyl or indole functional groups. Useful techniques for effecting such conjugation include the carbodiimide, N-hydroxysuc- cini ide ester, periodate, m-maleimidobenzoic acid, isothiocyanate, and dimaleimide methods. Kennedy, J.H. et al. , Clin. Chim. Acta, 70, 1 (1976); Schuurs, A.H. et al. , "Enzyme Immunoassay", Clin. Chim. Acta, 8_1, 1 (1977).

The protein-analyte, protein-antibody, or protein-antibody fragment complex so formed is termed a "conjugate". Such a conjugate participates in enzyme immunoassays of the present invention to yield a phase change. The phase change or signal is an insoluble aggregate formed by reactions involved in the enzyme immunoassay.

In one embodiment of this invention, mammalian coagulation proteins and their respective activated forms can be used in the formation of conjugates. The term "activated forms", in this context, refers to the coagulation proteins whose reactivity is promoted or stimulated. See generally, Schuman and Greenberg (Id. ) . Formation of insoluble aggregates in blood is the result of interaction of numerous specific blood plasma proteins in such a way that serial activation of these enzymes amplifies the initial activation of the phase change. Briefly, two mechanisms are involved in the activation of enzymatic reactions leading to signal formation (Figure 1) . The first mechanism is term the ex¬ trinsic pathway. In this pathway, blood is exposed to a phospholipoprotein called tissue factor. Blood coagulation Factor 7 binds to tissue factor and, once bound to tissue factor, will hydrolyze Factor 10, generating the activated Factor 10. Activated Factor 10 converts prothrombin to thrombin.

The second mechanism is termed the intrinsic pathway and involves several activated coagulation factors (Figure 1) . As a result of this serial activation, activation of the final Factor 10 enzyme is amplified several thousand-fold compared to the extrinsic pathway. The reactions that follow activation of Factor 10 by either pathway are identical (Figure 1). See Austen, D.E.G. and C.R. Rizza, "The Biochemistry of Blood Clotting Factors" in Structure and Function of Plasma Proteins, pp 169-193 (ed A.C. Allison), Plenum Press (1974); Shuman, M.A. and C.S. Greenberg, "Platelet Regula- tion of Thrombus Formation" in Biochemistry of Platelets, pp 319-346 (ed. D.R. Phillips and M.A. Shuman), Academic Press (1986).

Proteins from mammalian coagulation pathways, such as Factors 2, 3, 5, 7, 8, 9, 10, 11, and 12 and their respective activated forms, can be linked with analytes or anti-analyte antibodies to form conju¬ gates.

Use of these mammalian blood factors in immuno¬ assays of this invention is particularly useful as a signal modulation and amplification mechanism. For example, Factor 3 ("tissue factor") initiates blood coagulation by augmenting the proteolytic attack of Factor 7 on Factors 9 and 10 (Figure 1). The tissue factor accelerates reactions in both extrinsic and intrinsic pathways and may function _in vivo as an initiation of the entire system. It has therefore been possible to design an enzyme immunoassay comprising components present in excess whose reaction sequences can be represented as follows:

Tissue factor + phospholipid + Ca +2 + Factor 7

Factor 10 . r-Factor 10a

Factor_5__+_Ca +2 +_phosphol.ipids_±_F.actor_1.0a

Prothrombin = r-thro bin

fibrinogen clot

These events lead to formation of fibrin polymers. The amount of functional tissue factor present in the assay system will determine the sensitivity of the assay and the time needed to generate a clot. In an embodiment of this invention the protein moiety of the conjugates are thrombin-like enzymes isolated from the venom of various snakes. Examples of such proteins are acutase (from Agkistrodon acutus) , thrombin-like enzyme (from Agkistrodon contortrix) , ancrod (from Agkistrodon rhodostoma) , atroxin (from Bothrops atrox) , σrotolase (from the Eastern Diamondback rattlesnake, Crotalus adamanteus) , Factor 5 activating enzymes and Factor 10 activating enzyme (from Russell's viper) and ecarin (from Echis σarinatus) . These snake venom proteolytic enzymes can be considered "thrombin- like" in that they cleave one or more of the fibrin¬ opeptides. Fibrinogen is composed of two sets of three, nonidentical polypeptide chains interconnected by disulfide bonds. The triggering event for the transformation of soluble fibrinogen into the insoluble thread-like polymer fibrin is the removal of several short protein chains, fibrinopeptides A and B. See F.R. Doolittle, "Fibrinogen and Fibrin", Sci. Am. 235.126-135 ^"(1981) .

In one embodiment of the method of this inven¬ tion, the clot is generated by the protein, lipopro- tein, or glycoprotein-catalysed removal of fibrino- peptide A, fibrinopeptide B or both fibrinopeptides from fibrinogen. For example, catalysis is initiated by a conjugate whose protein moiety contains the mammalian enzyme thrombin. Thrombin is highly specific proteolytic enzyme involved in the final step in the coagulation of human blood. Coagulant activity of snake venom enzymes can occur at many steps in the coagulation process shown in Figure 1. Such activity simulates the activity of thrombin, although, in some cases, it allows only 05 incomplete fibrin polymerization. Coagulant activity of snake venoms is summarized in Table 1.

Horseshoe crab (Limulus polyphemus) enzymes, which act upon coaglogen to form a visible clot, can also be used in the method of the present invention.

TABLE 1

Mode of action Main Species

Thrombin-like fibrinogen __ toxin Crotalidae Agkistrodon rhodostoma Bothrops atrox profibrin polypeptide

A

Indirect toxin + Ca +2 Viperidae thromboplastin factor V Vipera russellii x Xa

II II

Direct Toxin ± Ca +2 Echis sp. thromboplastin Notechis scutatus Oxyuranus scutellatus

II ••- 11a Assay Mode and Format

The enzyme immunoassays of this invention can be performed in either the homogenous and heterogeneous mode, and in either competitive or non-competitive formats. Homogeneous Assays

A schematic representation of a competitive homogeneous assay method of the present invention is illustrated in Figure 2 using, for example, a conjugate compris"ed of thrombin linked to the analyte of interest. A standard curve is con¬ structed using the identical steps as outlined but employing increasing amounts of analyte.

The homogeneous assay method is based on antibody-mediated changes in enzyme activity.

Generally, a sufficient amount of the analyte to be measured is linked to the above-described protein/ snake venom enzyme to form the protein conjugate. The bound form of analyte and the unbound analyte whose presence is to be assayed are distinguished by addition of both an antibody against the analyte of interest and a substrate for the protein/enzyme. The amount of unbound or free analyte in the solution is directly proportional to the ability of the conjugate and substrate to react and form the insoluble aggregate or signal. (See Figure 2.)

In particular embodiments of this invention employing homogeneous assays, a proteolytic snake venom enzyme such as crotolase is conjugated with the analyte of interest. The activity of this protein-analyte complex or conjugate can be measured using blood plasma as a substrate. An unknown amount of free analyte to be measured is in the sample and antibody directed against the analyte is added to the sample. The concentration of free analyte can be directly correlated with its ability to modulate activity of the crotolase blood plasma reaction. As the concentration of unbound analyte in the sample increases, it will bind to its available antibody, thereby allowing the crotolase-analyte conjugate to react with the fibrinogen in blood plasma. A visible insoluble aggregate forms, the time of appearance being proportional to the analyte concentration. This competitive assay is based upon the ability of free analyte to compete with conjugated-analyte for antibody. Increased levels of analyte inhibit the binding of conjugated-analyte to the limited amount of antibody. As a result of this competitive inhibition, the proportion of the antibody-conjugate complex decreases as the levels of free analyte increase, thus allowing more conjugate to react with the substrate.

In one particular embodiment of the homogeneous assay format, a novel conjugate is formed by linking the analyte of interest to the substrate, (e.g., fibrinogen) rather than to the proteolytic enzyme (thrombin) . The use of this assay is similar to those described above, except that free proteolytic enzyme is added to initiate aggregate formation. The analyte-fibrinogen conjugate formed in this embodiment of the invention is such that proteolytic enzymes can still act on the fibrinogen to cause polymerization. Upon binding of antibodies directed against the analyte, however, the cleavage release of fibrinopeptides and polymer formation is stopped. This is due to the steric hindrance of the antibody molecule with respect to the proteolytic enzyme- substrate interaction. Free analyte will compete with conjugated analyte for the antibodies.

Tremendous signal amplification can be achieved in homogeneous assays of this invention through the use of tissue factor initiation of clotting, as described above. In embodiments of this invention, a conjugate is made comprising an analyte of inter¬ est (e.g., theophylline) and tissue factor. To a liquid sample containing the analyte to be measured, antibodies against the analyte, the analyte-tissue factor conjugate, and substrate(s) for tissue factor are added. If there is no analyte in the sample, the antibodies will bind to the tissue factor conjugate and prevent aggregate formation. If analyte is present, the clotting system will continue to completion.

The method of the present invention can also be used in a non-competitive immunoassay format. In this format, analyte is allowed to react in solution with an excess amount of antibody ("capture" anti¬ body) . The complexed analyte is measured. The amount of complexed analyte is directly proportional to the amount of free analyte in the sample. The immunocomplex between analyte and antibody is assayed with a second antibody (i.e., marker enzyme- antibody conjugate) directed against either the analyte or capture antibody.

In the presence of suitable substrate, the amount of enzyme-substrate complex is directly 5 proportional to the amount of analyte in the sample. In embodiments using a non-competitive format, the marker enzyme can be any of the proteolytic enzymes of the coagulation pathway. The subsequent in¬ soluble aggregate formed upon reaction of the marker 0 enzyme-antibody conjugate with substrate (e.g., blood plasma, fibrinogen) , is a direct function of the free analyte originally present.

Conjugates of this invention are especially useful for detecting high-molecular weight analytes 5 in homogenous assay methods. Protein conjugates used to detect high-molecular weight analytes in homogenous enzyme assays participate by virtue of their ability to react with fibrinogen or other substrates. o There are at least three means by which proteolytic enzyme-substrate interactions are modified in assays of high molecular weight analytes. First, the conformational change in coagulation protein caused by the binding of the 5 high molecular weight analyte to the protein con¬ jugate may result in either increased or decreased clotting, depending upon the protein and polymeriza¬ tion substrate chosen. Second, steric hindrance may be imposed upon the availability of the active site _Q of the protein when high molecular weight analyte is bound to the protein conjugate. This will decrease clotting. Third, conformational changes or steric hindrance imposed upon the active sites of both the coagulation protein and the fibrinogen or coagulogen can result from formation of an immunocomplex. This immunocomplex can be formed by the binding of a high molecular weight analyte (i.e., an antibody) to the protein conjugate (i.e., a snake venom-antigen conjugate) . In this case, the macromolecular substrate (i.e., fibrinogen) cannot penetrate the immunocomplex formed by binding of the analyte to the conjugate. The effect is to decrease clotting activity.

The various components of the homogeneous assays used in this invention can be combined simultaneously or sequentially and need not be combined in any defined or set sequence. The sequence of steps in embodiments of this invention will vary and will be selected to optimize the signal so formed. Moreover, no step to separate the components is needed.

The following is a detailed description and schematic representation of the different homogeneous assay formats which have been described and are representative of the invention. Refer to Figure 3 for a schematic description of these configurations.

Type 1. This assay format is non-competitive. It is useful for detection of high molecular analytes (generally greater than about 30,000) such as certain isozymes, cancer markers, biological active peptides, infectious disease markers and other clinically important molecules. The format employs a conjugate composed of a coagulation protein (P) linked to a Fab fragment of an antibody directed against the high molecular weight analyte of interest. The reagents also include a well-defined substrate such as fibrinogen or coagulogen. Upon addition of sample containing analyte, the analyte of interest will bind to the conjugate, thus inducing a condition of steric hindrance between the active site of the protein conjugate and the fibrinogen substrate. Therefore, substrate is polymerized more slowly and the time for a predetermined amount of clot formation is prolonged. The time for the predetermined amount of clot formation is a direct indication of the concen¬ tration of the substrate.

Type 2. This assay format is also non-competi¬ tive. It is also useful for a wide variety of biologically significant molecules. The format employs a conjugate composed of a coagulation protein (i.e., P = crotolase) linked to an antibody directed against the high molecular weight analyte of interest (i.e., an antigen) and fibrinogen. Upon addition of sample, the analyte of interest and the bivalent antibody-protein conjugate will form an immunocomplex typical of antigen-antibody reactions. This particular immunocomplex, and others formed in homogeneous assay configurations of this invention are identifed by an asterisk (*) in Figure 3. The binding of analyte imposes steric hindrance by its added molecular size and by its ability to prevent the substrate from penetrating the immunocomplex in order to gain access to the active site of the coagulation protein.

Type 3. This assay format is competitive. It is useful for a wide variety of biologically significant molecules. This format employs reagents consisting of a conjugate composed of a coagulation protein linked to -the analyte of interest, an antibody directed against the analyte of interest and fibrinogen. Upon addition of the sample containing the analyte, the analyte will bind to the antibody, thus effectively allowing unrestricted polymerization of fibrinogen substrate. In the absence of the analyte of interest, the antibody will interact with the protein conjugate and impose steric hindrance by its added molecular size and the formation of immunocomplex. Thus, the time for the formation of a predetermined amount of clot is a direct indication of the concentration of the analyte of interest.

Type 4. This assay format is competitive. It is useful for detection of immunoglobulin molecules. They may be immunoglobulins specific for infectious agents or other clinically significant molecules. This format employs a conjugate composed of a coagulation protein linked to an antibody of interest, an antigen specific for the antibody of interest and a substrate such as fibrinogen. Upon addition of the sample containing an unknown amount of antibody, the antibody will interact with the antigen leaving the protein conjugate free to react with the substrate. In the absence of antibody in the sample, the antigen interacts directly with the protein conjugate and imposes steric hindrance by its added molecular size and formation of immuno- complex. Therefore the time for a predetermined amount of clot formation in the absence of antibody is longer than when antibody is present and is a direct reflection of the concentration of antibody present. Type 5. This assay format is non-competitive. This format is also used for detection of immuno- globulins. It employs a conjugate composed of a coagulation protein linked to an antigen, and a substrate such as fibrinogen. Upon addition of sample, absence of antibody in the sample will allow the protein conjugate to react with the substrate at an optimal rate. The presence of antibody will impose steric hindrance by formation of an immuno¬ complex, as described above. In this manner the enzyme activity is again modulated according to the concentration of the antibody. Heterogeneous Assays

A schematic illustration of a "sandwich"-type heterogeneous assay method of the present invention is shown in Figure 4. This is discussed in detail below. A standard curve is constructed using the identical steps as outlined but employing increasing amounts of analyte.

In other heterogeneous assays carried out according to this invention, the bound analyte is separated from the unbound (free) analyte. Heterogeneous assays can be competitive, wherein the free analyte and enzyme-analyte conjugate compete for a limited number of antibody binding sites linked to a solid phase, or non-competitive, in which the analyte is allowed to react alone with an excess of enzyme-antibody conjugate. In either case, the solid phase is washed free of excess or unbound enzyme-conjugates. Appropriate substrate(s) are added after washing. In particular heterogeneous assays of this invention, a non-competitive, heterogeneous "sandwich" enzyme immunoassay can be initiated by linking a proteolytic blood coagulation enzyme (e.g., crotolase, thrombin, etc.) to antibodies, which are directed against (specific for) the analyte to be measured. An immunoadsorbent is also provided which comprises a solid phase to which is affixed identical "capture" antibodies specific for the analyte to be measured. A sample containing free analyte is incubated with immunoadsorbent under conditions sufficient for any analytes in the sample to complex with the capture antibodies. Enzyme- conjugate is then added in excess to the above mixture and allowed to react with any analyte present in the solid or liquid phases in a similar manner. The solid phase and the liquid sample are separated to remove all unbound substances.

The solid phase complexes containing analyte bound to both conjugate and "capture" antibody are then incubated in the presence of suitable substrate (e.g. , blood plasma, fibrinogen) for a time sufficient to allow this substrate to react with bound enzyme conjugates. The aggregation times are related to the amount of analyte originally bound to their "capture" antibodies. This relationship exists because aggregation time is directly propor¬ tional to the amount of enzyme-antibody conjugate bound to the "captured" analyte.

For each incubation step in the various assay formats, the time and conditions of incubation are selected to ensure maximal binding of analyte to the immobilized antibody (the immunoadsorbent) and to the conjugate. Optimal conditions for each incuba¬ tion can be determined empirically using standard techniques. in the heterogeneous solid phase assay of this invention, the immunoadsorbent is separated from incubation mixtures containing the liquid test sample and the conjugate marker. Separation can be accomplished by a conventional separation technique, such as sedimentation or centrifugation. Prefer¬ ably, though not necessarily, the immunoadsorbent is washed prior to measuring the amount of aggregate associated with the immunoadsorbent. The washing removes nonspecific interfering substances or excess labelled antibody which may affect the accuracy and sensitivity of the assay.

The antibodies used in the assays of this invention can be monoclonal antibodies, polyclonal antibodies, or both. In heterogeneous enzyme immunoassays of this invention, the following types of antibody are particularly useful: the capture (solid phase) antibody is a monoclonal antibody and the conjugate contains a polyclonal or monoclonal antibody against the analyte. The use of polyclonal antibody as the antibody component of the conjugates can lead to an amplification of signal because of the multi-epitopic binding of polyclonal antibodies.

The aggregate formed may be quantified visu¬ ally, turbidometrically, photometrically or electro- potentiometrically. A particularly useful measure for determining quantity of analyte present is measurement of the length of time needed to form the insoluble aggregate.

The immunoassay method of this invention is used to detect and quantify analytes in a liquid sample. Liquid samples include essentially all biological fluids that contain proteins of co¬ agulation pathways, such as blood, or components of blood such as plasma, serum and lymph. The sample may also be a liquid medium to which known quanti- ties of components of coagulation pathways have been added (e.g., urine, saliva).

Many types of solid phases can be employed in the preferred heterogeneous assays of this inven¬ tion. These include beads formed from glass, polystyrene, polypropylene, dextran, and other materials, the microwells of a microwell plate or tubes formed from or coated with such materials, etc. The antibody can be either covalently or noncovalently bound to the solid-phase by techniques such as covalent bonding via an amide or ester linkage or adsorption. Those skilled in the art will know many other suitable solid-phases and methods for immobilizing antibodies thereon, or will be able to ascertain such using no more than routine experimentation. To determine the amount of analyte in a liquid sample, either the insoluble aggregate formed that is associated with the immunoadsorbent or the aggregate formed that is associated with an amount of unbound enzyme conjugate (i.e., that which remains in soluble form), is measured. Generally, it is preferable to measure the markers bound to the immunoadsorbent because at very low concentrations of analyte, only small amounts of marker antibody bind the immunoadsorbent. The reagents for performing the assays of this invention may be assembled in assay kits. For instance, a kit for performing a solid phase immuno- metric assay can comprise, (i) a solid phase immunoadsorbent containing capture antibody specific for the analytes to be measured, and (ii) markers comprising conjugates of antibody specific for each analyte linked to a proteolytic enzyme of a blood coagulation pathway.

Virtually all types of analytes can be deter- mined by the method of the invention. These include hormones, vitamins, therapeutic drugs, drugs of abuse, tumor markers, neonatal markers and anti¬ bodies. Choice of analyte is limited only by the availability of sites for the proteolytic enzyme linkage in the conjugate. Examples of specific protein hormones include thyroid stimulating hormone (TSH) , free thyroxine T₄) , luteinizing hormone (LH) , human alpha-fetoprotein, follicle stimulating hormone (FSH) , growth hormone, human chorionic gonadotropin (HCG) and adrenocorticotropic hormone (ACTH) . Steroid hormones include androgens, progestins, estrogens, corticosteroids and aldosterone- Therapeutic drugs include theophylline.

The invention is illustrated further by the following exemplifications which are not intended to be limiting in any way.

Example 1 Development of a Homogeneous Enzyme Immunoassay for Determination of Theophylline This Example demonstrates the employment of cxotolase in constructing a homogeneous enzyme immunoassay for determination of theophylline. Preparation of enzyme-conjugate: rude lyophilized Crotalus adamanteus venom is available from several suppliers and crotolase can be purified by a variety of well-known chroma- tographic filtration ^"and concentration methods, herein incorporated by reference. See Markland, F.S. and P.S. Damus, J. Biol. Che , 246, 6460 (1971).

Crotolase can be stored for long periods of time in buffer at low temperatures at neutral to basic pH.

To approximately 2 ml of a 10 unit/ml crotolase solution in 0.1 M NaHCO (pH 8.5) is added about 5 ul of a 50 mg/ml solution of theophylline-8-butyric acid lacta in DMSO. The mixture is allowed to react at about 4'C for approximately 4 hours. The resulting conjugate is dialysed at ambient tempera¬ ture against a buffer consisting of 0.1 bovine serum 5 albumin. 0.05 M Tris, and 0.9% NaCl at pH 7.4.

Modulation of conjugate enzyme activity by antibodies against theophylline:

Bovine plasma is used as a substrate and mixed with differing concentrations of anti-theophylline o antibody. Enzyme activity is measured by determin¬ ing the clotting time (Table I) .

TABLE I

Substrate Conjugate Buffer Antibody Clot Time

(ul) (ul) (ul) (ul) (sec) 5 200 50 50 0 20.9

20_.0 50 40 10 22.9

200 50 30 20 46.0

200 50 0 50 81.9

The relationship between clotting time and 0 antibody concentration is linear (log clotting time = 0.803 (log[antibody] ) + 0.571; r = 0.99) and, as expected, increasing amounts of anti-theophylline antibodies compete with the enzyme-theophylline conjugate to prolong the clotting time. The regres- sion coefficient (r = 0.99) indicates the data approximates a straight line with almost all of the variability determined by the antibody reactions.

Modulation of enzyme activity by free theophylline:

The effect of varying amounts of analyte was determined on a homogeneous assay system in which the amounts of substrate, conjugate and antibody are 05 held constant. The effect of free analyte on clotting time in a system with 25 ul of anti- theophylline antibody is given in Table II and the effect of 16 ul antibody is given in Table III.

TABLE II

Substrate Conjugate Antibody Theophylline Buffer Clot Time

(ul) (ul) (ul) (ug/ml) (ul) (sec)

200 50 25 0 25 57.4

200 50 25 4 66

200 50 25 10 73.6

200 50 25 25 81

200 50 25 100 96.4 log clotting time = 0.120 (log[theophylline] ) + 1.73, r = 0.999

^* TABLE III

200 50 16 0 34 41

200 50 16 4 15 38

200 50 ^" 16 7 15 35

200 50 16 18 15 30

200 50 16 56 15 25.4

200 50 16 100 15 23.2 log clotting time = 0.156 (log[theophylline] ) + 1.67; r = 0.999

The data of Table II clearly suggests that at this level of antibody, free theophylline is displacing the theophylline covalently linked to crotolase at a position within the polypeptide which inhibited enzyme-substrate polymerization. The data of Table III suggests that, addition of free theo- phylline at this level of antibody promoted enzyme activities. The unbound theophylline can effi¬ ciently displace the anti-theophylline antibody associated with the crotolase -theophylline con¬ jugate.

Example Use of Ancrod in Heterogeneous, Non- Competitive Enzyme Immunoassay for Human Alpha-fetoprotein The snake venom Ancrod was covalently linked to antibodies directed against alpha-fetoprotein in a molar ratio of 3:1 by utilizing the glutaraldehyde periodate or dimaleimide coupling procedures. Antibodies directed against alpha-fetoprotein can be obtained and purified using methods well-known in the art. Galfre, G. and Milstein, C, Methods in Enzymology (Langone, J.J. and Van Vunakis, H., eds.), Vol. 73, pp. 3-47 (Academic Press, FL 1983)

Samples containing the analyte in unknown concentrations and standards were mixed with solid phase antibodies. These anti-alpha-fetoprotein antibodies can be attached, for example, to a polystyrene plastic tube by natural adsorption.

The mixture was allowed to react for about 10 minutes at about 37 'C. Enzyme-conjugate was then added in excess to the above mixture and allowed to react for about 30 minutes at 37 'C. The solid phase complexes were washed several times with neutral buffer to eliminate all unbound enzyme conjugates. The solid immunoadsorbent was then mixed with a predetermined volume of bovine plasma and clotting times either visualized or determined with a fibrometer. A standard curve was constructed and the concentrations of alpha-fetoprotein in the sample is read off the calibration curve (Table IV)

TABLE IV

Concentration of AFP Clott:ing Time

(X) (ng/ml) (sec) Y (Log X) (Log Yl)

5 0.698 38 1.579

10 1.00 30 1.47

15 1.17 22 1.34

25 1.39 17 1.23

log clotting time = -0.515 (log [AFP]) + 1.95 r = 0.989

Equivalents Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.

These and all other equivalents are intended to be encompassed by the following claims.

Claims

1. An enzyme immunoassay for determining an analyte in a liquid comprising combining: a) the liquid; b) a conjugate, the conjugate comprising the analyte of interest linked to a first protein; c) a second protein capable of interacting specifically with the analyte; and d) a third protein capable of reacting with the conjugate to produce a phase change which results in production of an insoluble aggregate and measuring the insoluble aggregate so produced.

2. An immunoassay of Claim 1, wherein the first protein and the third protein are selected from .the group consisting of proteins of the blood coagulation pathway and proteins capable of enzymatic participation in the blood coagula- tion pathway.

3. An immunoassay of Claim 2, wherein the first protein is selected from the group consisting of mammalian blood Factors 2, 5, 7, 8, 9, 10, 11, 12, and Tissue Factor, and their respective activated forms; thrombin; Staphylocoagulase; snake venom protein; and horseshoe crab enzyme and the third protein is selected from the group consisting of fibrinogen and coagulogen.

4. An immunoassay of Claim 3, wherein the snake venom proteins are selected from the group consisting of acutase, thrombin-like enzyme from Agkistrodon contortrix, ancrod, atroxin, 5 crotolase, ecarin, Russell's viper enzyme and Taipan snake venom.

5. An immunoassay of Claim 1, wherein the second protein is an antibody or fragment.

6. An immunoassay of Claim 5, wherein the fragment o s an F(ab) or F(ab')_ fragment.

7. An immunoassay of Claim 1, wherein the insoluble aggregate so formed is an aggregate of the third protein and the conjugate.

8. An immunoassay of Claim 1, wherein the quantity 5 of analyte is determined by measuring the time needed to form a predetermined quantity of the insoluble aggregate and relating the time needed to a quantitative relationship between the quantity of analyte present and time needed 0 to form such an insoluble aggregate.

9. An enzyme immunoassay for determining an analyte in a liquid comprising combining: a) the liquid; b) a conjugate, the conjugate comprising a 5 first protein capable of interacting specifi¬ cally with the analyte linked to a second protein; and c) a third protein capable of reacting with the conjugate to produce a phase change which results in production of an insoluble aggregate 5 and measuring the insoluble aggregate so produced.

10. An immunoassay of Claim 9, wherein the second protein and the third protein are selected from the group consisting of proteins of the blood o coagulation pathway and proteins capable of enzymatic participation in the blood coagula¬ tion pathway.

11. An immunoassay of Claim 10, wherein the second protein is selected from the group consisting 5 of mammalian blood Factors 2, 5, 7, 8, 9, 10, 11, 12, and Tissue Factor and their respective activated forms; thrombin; Staphylocoagulase; snake venom protein; and horseshoe crab enzyme and the third protein is selected from the 0 group consisting of fibrinogen and coagulogen.

12. An immunoassay of Claim 11, wherein the snake venom proteins are selected from the group consisting of acutase, thrombin-like enzyme from Agkistrodon contortrix, ancrod, atroxin, crotolase, ecarin, Russell's viper enzyme and Taipan snake venom.

13. An immunoassay of Claim 9, wherein the first protein is an antibody or antibody fragment.

14. An immunoassay of Claim 13 _r wherein the fragment is an F(ab) or F(ab') fragment.

15. An immunoassay of Claim 9 , wherein the insoluble aggregate so formed is an aggregate of the third protein and the conjugate.

16. An immunoassay of Claim 9, wherein the quantity of analyte is determined by measuring the time needed to form a predetermined quantity of the insoluble aggregate and relating the time needed to a quantitative relationship between the quantity of analyte present and time needed to form such an insoluble aggregate.

17. An enzyme immunoassay for determining an analyte in a liquid sample, comprising the steps of: a. combining: i) the liquid; ii) a conjugate, the conjugate comprising analyte linked to a first protein selected from the group of fibrinogen, and coagulogen; iii) a second protein capable of binding specifically with the analyte; and iv) a third protein, selected, from the group consisting of thrombin, snake venom enzymes, horseshoe crab enzyme, Staphylocogulase, mammalian blood Factors 2, 5, 7, 8, 9, 10, 11, 12, and Tissue Factor, and their respective activated forms, in order to polymerize the first protein, thereby forming an insoluble aggregate; and b. measuring the insoluble aggregate so formed.

18. An immunoassay of Claim 17, wherein the snake venom enzymes are selected from the group consisting of acutase, thrombin-like enzyme from Agkistrodon contortrix, ancrod, atroxin, crotolase, ecarin, Russell's Viper enzyme and Taipan snake venom and the second protein is an antibody selected from the group consisting of monoclonal antibody, polyclonal antibody or fragment thereof.

19. An immunoassay of Claim 17 wherein the measure¬ ment of the insoluble aggregate consists of recording the time needed to form any pre¬ determined amount of aggregate.

20. An enzyme immunoassay for determining an analyte in a liquid sample, comprising the steps of: a. combining: i) the liquid; ii) a conjugate, the conjugate comprising analyte linked to a first protein selected from the group consisting of thrombin, snake venom enzymes, horseshoe crab enzyme, Staphylocogulase, mammalian blood Factors 2, 5, 7, 8, 9, 10, 11, 12, and Tissue Factor, and their respective activated forms; iii) a second protein capable of binding specifically with the analyte; and iv) a third protein, selected from the group consisting of fibrinogen and co- agulogen, in order to polymerize the third protein, thereby forming an insoluble aggregate; and b. measuring the insoluble aggregate so forme .

21. An immunoassay of Claim 20, wherein the snake venom enzymes are selected from the group consisting of acutase, thrombin-like enzyme from Agkistrodon contortrix, ancrod, atroxin, crotolase, ecarin, Russell's Viper enzyme and Taipan snake venom and the second protein is an antibody or fragment thereof.

22. An immunoassay of Claim 21, wherein the anti¬ body fragment is an Fab or F(ab') fragment.

23. An immunoassay of Claim 20 wherein the measure- ment of the insoluble aggregate consists of recording the time needed to form any pre¬ determined amount of aggregate.

24. An enzyme immunoassay for determining an antibody in a liquid sample, comprising the steps of: a. combining: i) the liquid; ii) a conjugate, the conjugate comprising the antibody of interest or a fragment thereof linked to a first protein, the first protein selected from the group consisting of thrombin, snake venom enzymes, horseshoe crab enzyme, Staphylocogulase, mammalian blood Factors 2, 5, 7, 8, 9, 10, 11, 12, and Tissue Factor, and their respective activated forms; iii) a second protein capable of binding specifically with the antibody or antibody fragment; and iv) a third protein, the third protein selected from the group consisting of fibrinogen and coagulogen, in order to polymerize the third protein, thereby forming an insoluble aggregate; and b. measuring the insoluble aggregate so formed.

25. An immunoassay of Claim 24, wherein the snake venom enzymes are selected from the group consisting of acutase, thrombin-like enzyme from Agkistrodon contortrix, ancrod, atroxin, crotolase, ecarin, Russell's Viper enzyme and Taipan snake venom.

26. An immunoassay of Claim 24, wherein the second protein is an antigen.

27. An immunoassay of Claim 24, wherein the anti¬ body fragment is an Fab or F(ab')~ fragment.

28. An immunoassay of Claim 24 wherein the measure¬ ment of the insoluble aggregate consists of recording the time needed to form any pre¬ determined amount of aggregate.

29. An enzyme immunoassay for determining an antibody in a liquid sample, comprising the steps of: a. combining:i) the liquid; ii) a conjugate, the conjugate comprising an antigen linked to a first protein, the antigen capable of binding specifically with the antibody, the first protein selected from the group consisting of thrombin, snake venom enzymes, horseshoe crab enzyme, Staphylocoagulase, mammalian blood Factors 2, 5, 7, 8, 9 , 10, 11, 12, and Tissue Factor, and their respective activated forms; and iii) a second protein, the second protein selected from the group consisting of fibrinogen and coagulogen, in o order to polymerize the second protein, thereby forming an insoluble aggregate; and b. measuring the insoluble aggregate so formed.

30. An immunoassay of Claim 29, wherein the snake 5 venom enzymes are selected from the group consisting of acutase, thrombin-like enzyme from Agkistrodon contortrix, ancrod, atroxin, crotolase, ecarin, Russell's Viper enzyme and Taipan snake venom.

31. An immunoassay of Claim 29 wherein the measure¬ ment of the insoluble aggregate consists of recording the time needed to form any pre¬ determined amount of aggregate.

32. An enzyme immunoassay for determining an analyte in a liσuid sample comprising the steps of: a. incubating: i) an immunoadsorbent comprising a solid phase having affixed thereto capture antibodies specific for the analyte to be determined; and ii) the sample, for sufficient time and under conditions appropriate for analyte in the sample to complex with the capture antibodies; b. adding a conjugate comprising a protein of a coagulation pathway linked to an antibody specific for the analyte; c. incubating the product of (b) for sufficient time and under conditions appropriate for conjugate to complex with analyte bound to the immunoadsorbent to produce immunoadsorbent-conjugate complex; d. separating the immunoadsorbent and the solution; e. incubating the immunoadsorbent-conjugate complex with a second protein of a co¬ agulation pathway capable of reacting with the first protein bound to the immuno¬ adsorbent, to form an insoluble aggregate; and f. measuring the time needed to form any predetermined quantity of aggregate.

33. An immunoassay of Claim 32, wherein the quantity of analyte is determined by relating the time needed to form any predetermined quantity of aggregate and a quantitative rela¬ tionship between the quantity of analyte present and time needed to form such an in¬ soluble aggregate.

34. An immunoassay of Claim 32, wherein the first protein is selected from the group consisting of mammalian blood Factors 2, 5, 7, 8, 9, 10, 11, 12, and Tissue Factor, and their respective activated forms, horseshoe crab enzyme, Staphylocoagulase and snake venom enzymes, and the second protein is selected from the group consisting of fibrinogen and coagulogen.

35. An immunoassay of Claim 32, wherein the capture aannttiibbooddyy consists of a F(ab) or F(ab'). fragment.

36. In an enzyme immunoassay procedure used to measure the presence or absence of an analyte in a liquid, the improvement comprising employ¬ ing a conjugate capable of participating in heterogeneous assays in such a way that an insoluble aggregate is formed, the conjugate consisting essentially of least one component of a blood coagulation pathway.

37. An enzyme immunoassay of Claim 36 wherein the component of a blood coagulation pathway is selected from the group consisting of thrombin, horseshoe crab enzyme, mammalian Factors 2 , 5 , 1 , 8, 9, 10, 11, 12, and Tissue Factor, and their respective activated forms, ancrod, fibrinogen, coagulogen and Staphylocoagulase.

38. An enzyme immunoassay of Claim 36, wherein the conjugate is capable of participating in a homogeneous immunoassay.

39. A conjugate, for use in an enzyme immunoassay for determining an analyte in a liquid sample, selected from the group consisting of: a. a proteolytic enzyme of a blood coagulation pathway or modulator of a blood coagulation pathway linked to an analyte; and b. a proteolytic enzyme of a blood coagulation pathway or modulator of a blood coagulation pathway linked to an antibody or antibody fragment, the antibody or antibody fragment specific for the analyte.

40. A conjugate of Claim 39, wherein the analyte is selected from the group consisting of: hormones, vitamins, nucleic acids, therapeutic drugs, drugs of abuse, antibodies and proteins.

41. A conjugate of Claim 39 wherein the proteolytic enzyme or modulator is selected from the group consisting of thrombin, horseshoe crab enzyme, mammalian Factors 2,5,7,9,10,11,12, and Tissue Factor, and their respective activated forms, ancrod, fibrinogen, coagulogen, and Staphylocoagulase.

42. A conjugate, consisting essentially of the snake venom ancrod, covalently linked to alpha-feto protein.

43. A conjugate, ^• consisting essentially of ancrod, covalently linked to an antibody directed against alp_ha-fetoprotein.

44. A conjugate, consisting essentially of crotolase covalently linked to theophylline.

45. An enzyme immunoassay kit for determining an analyte in a liquid sample comprising: a. an immunoadsorbent having affixed thereto a capture antibody specific for the analyte to be determined; b. a container having therein a conjugate, the conjugate comprising a first protein of a coagulation pathway linked to an antibody specific for the analyte, the first protein selected from the group consisting of thrombin, snake venom enzymes, horshoecrab enzyme, Staphylocoagulase, mammalian blood Factors 2, 5, 7, 8, 9, 10, 11, 12, and Tissue Factor and their respective activated forms; and c. a container having therein a second protein of a coagulation pathway, the second protein capable of reacting with the first protein to form an insoluble aggregate wherein the second protein is selected from the group consisting of fibrinogen and coagulogen.

46. A kit of Claim 45, wherein the snake venom enzymes are selected from the group consisting of acutase, ancrod, thrombin-like enzyme from Agkistrodon contortrix, atroxin, crotolase, ecarin, Russell's Viper enzyme and Taipan snake venom.

47. An enzyme immunoassay kit for determining an analyte in a liquid sample, comprising: a. a container having therein a conjugate, the conjugate comprising the analyte linked to a first protein, the first protein selected from the group consisting of Thrombin, snake venom enzymes, horshoecrab enzyme, Staphylocoagulase, mammalian blood Factors 2, 5, 7, 8, 9, 10, 11, 12, and Tissue Factor, and their respective activated forms; and b.a container having therein an antibody specific for the analyte of interest; and 5 c. a container having therein a second protein, the second protein selected from the group consisting of fibrinogen and coagulogen, the second protein capable of reacting with the o first protein to form an insoluble aggregate.

48. A kit of Claim 47, wherein the snake venom enzymes are selected from the group consisting of acutase, ancrod, thrombin-like enzyme from 5 Agkistrodon contortrix, atroxin, crotolase, ecarin, Russell's Viper enzyme and Taipan snake venom.