WO1988006732A1

WO1988006732A1 - Tagged binding reagents and methods for detecting target analytes

Info

Publication number: WO1988006732A1
Application number: PCT/US1987/003342
Authority: WO
Inventors: Francis Jeffrey Regina; Steven Elliot Diamond; William Johnston Boyle, Jr.; Divakaran Masilamani
Original assignee: Allied Corporation
Priority date: 1987-03-02
Filing date: 1987-12-15
Publication date: 1988-09-07

Abstract

The present invention relates to methods of assay of target analyte binding pair members, and to the reagents useful for carrying out such assays. More specifically, the invention relates to ligand binding or nucleic acid assays using a reagent that comprises a signal moiety, and a moiety complementary to or homologous with all or a portion of the target analyte binding members. Under the appropriate conditions, the signal moiety produces a detectable moiety which can be transferred to a gaseous phase for detection. To serve this purpose, the signal moiety is a moiety capable of reacting with an effective second agent to produce a detectable moiety as a product of that reaction or is a moiety capable of being cleaved from the tagged binding pair member itself, volatilized and detected. The methods and reagents herein described are particularly applicable in the analysis of physiological and biological samples derived from human as well as from animal, plant, and microorganismic sources. In addition, the methods and reagents may be employed in connection with environmental analyses, specifically, in assays for suspect chemical or biological constituents or contaminents in food, water, air, soil samples, and the like.

Description

TAGGED BINDING REAGENTS AND METHODS FOR DETECTING TARGET ANALYTES

Field of the Invention

The present invention relates to methods of assay of target analyte binding pair members, and to the reagents useful for carrying out such assays. More specifically, the invention relates to ligand binding or nucleic acid assays using a reagent that comprises a moiety, complementary to or homologous with all or a portion of the target analyte binding members, said reagent also having a signal moiety. Under the appropriate conditions, the signal moiety produces a detectable compound which can be transferred to a gaseous phase for analysis.

BACKGROUND OF THE INVENTION

Biochemical assays have long been known in which binding reactions, such as between a sample and labeled reagent, are conducted. Such labels include bacteriophages, chromophores, enzymes, fluorophores, free radicals, luminophores, metals, particles, stable isotopes, substrates/coenzymes/inhibitors, and the like. The most widely used labels are radioisotopes, due to their ease of incorporation into biochemical systems, ease and sensitivity of detection, and the like. However, inherent with the use of radioisotopes are decay problems which complicate sample handling and purification, and cause problems with contamination and ultimate disposal. Some radioisotopes are also chemically labile, and all of them are relatively costly.

The desire to eliminate the hazards associated with radioisotopes has led to the development of alternative analytical reagents. Roger W. Giese In "Electrophoric

Release Tags: Ultrasensitive Molecular Labels Providing

Multiplicity", Trends in Analytical Chemistry, vol. 2, no. 7, pp. 166-168 (1983) reports the use of an analytical reagent he terms a 'release-tag', which consists of three molecular groups, a 'signal group',

'release group' and 'reactivity' group. The signal group is for detection purposes, the release group provides a site for specific covalent cleavage of the signal group, and the reactivity group attaches the release tag to a substance of interest. The signal group is a compound that can be detected with high sensitivity by gas chromotagraphy with electron capture detection. Polyhalogenated aliphatics and aromatics are given as examples of gas phase electrophores. However, in another paper, Giese and other workers report that this type of labeling is incompatible with ligand-binding assays. It is said that in the ligand assay, the reagents employed in the release tag system tend to interfere with the ligand/binder interaction. See

Joppick-Kuhn et al "Release Tags: A New Class of

Analytical Reagents", Clin. Chem. 28/9, 1844-1847

(1982).

In U.S. Patent No. 4,629,689 issued to Diamond et al., the use of vapor phase detection in binding assays is reported. However, in this case, the vapor phase detectable moiety is not the label itself and is not generated directly. Rather, the specific binding assay is coupled with an enzymatic reaction which amplifies the binding event. A precursor is then contacted with the enzymatic moiety to produce the vapor phase detectable moiety.

Hence, there is a need for a more direct labeling in binding assays, labeling that will be analogous to isotopic labeling and yet serve as a safer, more economical alternative. SUMMARY OF THE INVENTION The present invention provides a tagged-binding reagent suitable for use in the detection of a target analyte binding pair member contained in a sample under analysis, which comprises a signal moiety and a moiety complementary to or homologous with all or a portion of said target analyte binding member; said tagged reagent adapted to bind to target analyte or to a template containing a binding pair member complementary to said tagged reagent and target analyte.

Also provided is a method for the determination of a target binding pair member in a sample under analysis comprising the steps of: a. contacting the sample with a tagged reagent having a signal moiety capable of producing a known amount of detectable moiety and also a moiety complementary to said target analyte binding pair member for a time sufficient to allow all or a portion of target analyte binding pair member present in said sample to bind to said tagged reagent; b. separating said tagged binding reagent bound to target analyte binding pair member that may be present in the sample from tagged reagent not bound to target analyte binding pair member; c. contacting either said tagged binding reagent bound to target analyte binding pair member, tagged binding reagent not bound to target analyte binding pair member, or a combination thereof, with an effective second agent to activate signal moiety and produce a detectable moiety; d. transferring into a gaseous phase said detectable moiety produced by said signal moiety; and e. detecting said detectable moiety as an indication of the presence of target binding pair member in the sample under analysis.

Further provided is a method for the determination of a target analyte binding pair member in a sample under analysis comprising the steps of: a. providing a template binding pair member capable of binding to a target analyte binding pair member and also capable of binding to a tagged binding reagent, said tagged binding reagent having a signal moiety capable of producing a known amount of a detectable moiety and also having a moiety at least a portion of which is complementary to or homologous with said template binding pair member; b. contacting said template with a known amount of tagged reagent in a fluid phase and also contacting said template with sample possibly containing target analyte binding pair member for binding thereto of tagged reagent, target analyte, or a combination of both; c. separating said template containing, if any, bound tagged reagent, bound target analyte, or a combination thereof from unbound tagged reagent and unbound target" analyte; d. contacting either said template, said unbound tagged reagent, or both with a second effective agent to activate the signal moiety of tagged reagent that may be present to produce detectable moiety; e. transferring said detectable moiety into a gaseous phase for detection thereof; and f. detecting said detectable moiety as an indication of the presence of target binding pair member in the sample under analysis.

As used herein, "binding pair member" means either of a pair of molecules, especially biomolecules, which exhibits a binding affinity and/or specificity for the other molecule of the pair. Examples include the highly specific and high-binding of antibodies or antibody fragments with the respective antigens or haptens; binding of fully- or partially complementary single-stranded nucleic acid polymers or oligomers; the binding of lectins to their corresponding carbohydrates and polysaccharides or the binding of various proteins, including avidin, strepavidin, protein A, and complement protein, to various molecules such as biotin, immunoglobulins, or portions thereof.

"Target analyte binding pair member" means a particular binding pair member complementary to or homologous with the tagged binding pair members, said target analyte present in a biological sample or the like, the presence and/or concentration of which is to be determined or assayed by means of the methods and reagents as described herein.

A "template binding pair member" within the context of the invention is a component with a moiety that is either complimentary to or homologous with either the target analyte, the tagged binding reagent or both. This member directly or indirectly facilitates the separation of the bound and free tagged binding reagent. In some embodiments, the template binding member is attached to an inert solid support.

"Tagged binding reagent" means a binding pair member complementary to or homologous with a target analyte binding pair member, and in some embodiments complementary to or homologous with a template binding member, said reagent also containing a signal moiety.

After a specific binding reaction is carried out, the tagged binding reagent, either bound to target analyte or a template having a complementary binding pair member or the unbound portion , may be reacted with an effective second agent to generate a detectable moiety which can be detected in a gaseous state and thus acts as a signal.

A "signal moiety" within the context of the invention is a component bound to the tagged binding reagent and is responsible for directly or indirectly producing "a detectable moiety." In some embodiments the signal moiety is a detectable moiety itself and need only be cleaved from the binding reagent by the effective second agent for volatilization and gas phase detection thereof. In other embodiments, the signal moiety is reacted with the second agent to produce a product capable of gas phase detection.

"A detectable moiety" within the context of the present invention is a chemical moiety capable of being transferred to a gaseous state and quantitatively detected by means appropriate for gas phase detection.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The tagged binding reagent of the present invention comprises a signal moiety and a moiety complementary to or homologous with all or a portion of a target analyte binding pair member that may be present in a sample under analysis, or a template binding member, or both.

The complementary or homologous moiety of the tagged binding reagent may vary widely and corresponds to the particular target analyte under investigation.

Particularly useful in this regard may be mentioned antigens and haptens such as hormones, of which may be mentioned the various androgen and estrogen hormones, thyroid and parathyroid hormones, pituitary hormones and pancreatic andrenal hormones, and gastrointestinal hormones; immunoglobulins such as IgG, IgE, and the like; human chorionic gonadotropin, digoxin, and various enzymes and other proteins, lipoproteins, glycoproteins, and glycolipoproteins; xenobiotic agents such as pharmaceutical products, therapeutic drugs, and various chemical toxins and pathogenic microorganisms; and target analyte nucleic acids sequences including DNA and

RNA sequences.

The signal moiety is that part of the tagged binding reagent responsible for indirectly or directly producing a chemical moiety that is capable of being volatilized for gas-phase sensing thereof. Detectable moieties are produced directly by the signal moiety when the compound serving as a detectable label itself is attached as the signal moiety of the tagged binding reagent, this compound itself capable of being cleaved from the binding reagent by a second agent, volatilized and detected. Alternatively, the signal moiety is not

"cleaved" from the tagged reagent, but "reacted with" the second agent to produce a detectable moiety as a

"product".

The mode of detection it is desired to use in the analysis of the detectable moiety will govern selection of the particular signal portion of the tagged binding reagent of the present invention. It is within the comtemplation of the present invention that the detectable moieties, once transferred to a gaseous phase, be measured directly as in an ion mobility spectrometer. It is also within the contemplation of the present invention that the detectable moieties in a gaseous phase be partitioned or subjected to selective adsorption, absorption, condensation or the like, such as in a gas chromatograph or the like with the intent to separate the particular detectable moieties sought from other materials which migrate at different rates through the system, after which the signal portion of the tagged binding reagent is detected. The ultimate detection is by charge measurement, absorption, emmission, photoionization, thermal conductivity or the like. Many types of apparati including an election capture detector, a flame ionization detector, an alkaline flame ionization detector, an ion mobility spectrometer or the like, may be used. Thus, choice of the method of analysis governs the choice of the signal portion of the tagged binding reagent that is employed. For example, when an alkali flame detector is to be employed, a signal moiety capable of producing a detectable moiety containing nitrogen, phosphorous or the like would be a good choice. When a detectable moiety contains an electrophore, an electron capture detector may be conveniently employed. In other embodiments if an iin mobility spectrometer is to be used, then a signal moiety that could produce an organic compound capable of being ionized would be applicable. Illustrative of signal moieties containing cleavable detectable moieties falling into the classes described above, are those compounds containing electrophores such as halogenated, nitrated, or conjugated carbonyl compounds which are particularly preferred for an electron capture detector. Of these may be mentioned compounds such as the polyhalogenated aliphatics, aromatics, alcohols, ketones, aldehydes, ethers and nitrates. Illustrative of these are the pentafluorobenzamides, trichloroalcohols, perfluorophenols, perfluorobenzophenone, pentafluorobenzylidines, heptafluorobutyramides, nitrobenzamides, perfluoroctanaldehyde Shiff bases and the like.

Also illustrative of the signal moieties containing cleavable detectable moieties falling into the classes described above are those compounds containing nitrogen and phosphorous which are particularly preferred when using an alkali flame ionization detector. Illustrative of these may be mentioned such compounds as azobenzene, triphenylphosphine, triethylamine, tributylphosphate, N-methylaniline, pyridine, trimethylphosphite and nitrobenzene.

When the ultimate detecting method is through ion mobility spectrometry, labels present in the gas phase capable of undergoing ion-molecule reactions at atmospheric pressure are preferred. Included in this group are nitrated and halogenated alkanes, nitrated and halogenated aromatics, biphenyls, alcohols, nitrated and halogenated phenols, ethers, thioethers, mercaptans, carboxylic acids, esters, ketones, amines, pyridenes, nitrosoamines, organophosphorous compounds and the like. Illustrative of these are nitrophenol, trinitrotoluene, nitrobenzene.

The above examples are directed to signal moieties covalently bonded to the ligand or nucleotide sequences of the present invention, said moiety capable of being cleaved from the ligand or nucleotide sequence at the point of attachment thereto by the second effective agent. These moieties are preferably bound directly to the ligand or nucleotide sequence through functional groups such as esters, thioesters, ethers, thioethers, amides, and the like, or are bonded to spacer groups which in turn are bonded to the ligand or nucleotide sequence. Suitable spacer groups are those that will lend themselves to hydrolysis, reductive or oxidative cleavage, ring closure, photolysis, thermal cleavage, and the like. Of these may be mentioned groups such as dialdehydes, dicarboxylic acid, anhydrides, amino acids, diamines, dimaleimides, diisocyantes, and the like.

Illustrative of effective second agents within the context of the invention are those capable of cleaving the signal moiety from the tagged binding reagent by chemical reactions such as hydrolysis, reductive or oxidative cleavage, ring closure, thermal cleavage, photolysis, or any other appropriate means.

Illustrative of agents useful in this regard are light, heat, enzymes, and chemical reagents. Of the chemical reagents may be mentioned the halogens, pseudo halogens, in particular cyanogen bromide and iodoacetamide, and acids such as hydrochloric, sulfuric, sulfonic, acetic, formic, and the like.

Alternative permutations to the above labeling schemes are also within the contemplation of the present invention. Exemplary of this is the embodiment wherein the signal moiety is not cleaved from the ligand or nucleotide sequence, but rather, chemically reacts with a second reagent to produce a detectable moiety.

Suitable moieties in this regard are those that can react with second agents such as alcohols, carboxylic acids, amines, alkenes, ketones, aldehydes, and the like to produce moieties capable of gas phase detection.

Preferred among those labels are those signal moieties that react with alcohols such as methanol, ethanol, propanol, 2-propanol, 2-butanol, and 1,1,1,3,3,3-hexafluoro-2-propanol to produce formaldehyde. acetaldehyde, propionaldehyde, acetone, methylethylketone and hexafluoroacetone, respectively. Of these labels may preferably be mentioned such compounds as dimethylsulfoxide, methionine sulfoxide, and the like.

In all of the above cases, the detectable moiety ultimately produced by the signal moiety may be further reacted to increase the potential for gas phase detection. For example, detectable moieties such as formaldehyde, acetone, aniline, benzylalcohol, ethanethiol, which are only weakly detectable with various gas chromatographic methods, may be further reacted with pentaflurobenzoyl chloride, pentafluorophenoxyacetyl chloride, heptafluorobutyryl chloride, peαtafluorobenzylhydroxyamine, and the like, to produce compounds with a heightened signal.

In certain embodiments of the present invention, binding of the tagged binding reagent will alter the reactivity of said tagged reagent towards the effective second agent. In these cases, binding of the tagged reagent will affect the reactivity of the signal moiety to the effective second agent in two preferred modes. In the first of these two modes, binding will inhibit reaction of the signal moiety with the effective second agent. In other words, when the tagged reagent is bound in whole or part to its complement, the ability of the signal moiety to be cleaved by the second agent and release detectable moiety is lessened in part and in some cases, completely. By the same token, in embodiments wherein the signal moiety is adapted to react with the second agent to produce the detectable moiety as a product, this capability is also lessened in whole or in part by the binding event. Alternatively, this binding event can produce the opposite results by actually facilitating and thus enhancing reactivity of the signal moiety. In this latter case, tagged reagent in a bound state produces a signal that is greater than that produced by the tagged reagent in an unbound state, and thus the production of detectable moiety becomes directly proportional to the amount of binding taking place.

In certain preferred embodiments of the present invention, a template binding pair member is provided which comprises binding pair members that are complementary to all or a portion of both the target analyte binding members and all or a portion of the tagged binding reagent. The template binding pair member is adapted to be contacted with the tagged reagent and the sample under analysis for binding of complementary portions of each thereto. To serve this purpose, the template may be comprised of complementary binding pair members in free form or may be immobilized onto a support by any conventional technique as follows.

The template binding pair members may be immobilized onto an inert support or other material by a variety of techniques. In general, to immobilize the complementary binding pair members onto the support, the surface should be clean, and preferably of maximum feasible surface area; the latter criterion ensures the largest possible number of sites for attachment of the binding members. Attachment of the binding pair members to the support may be then effected by any of the conventional methods generally known to the art.

For example, a wide variety of functional groups on template molecules may be used as the locus of attachment to the inert surface, as long as the mode of attachment does not substantially block access to the binding site and/or does not alter the structure of the binding site in a manner which substantially diminishes the specificity or affinity of template binding to complementary tagged reagent or target analyte binding pair members.

For protein complements, various amino acid functional groups may be activated for use as sites of attachment, including carboxyl, amino, disulfide, sulfhydryl, thioether and imidazoyl moieties. These and other reactions are reviewed in standard texts such as Chemical Modification of Proteins by A. Glazer, R. DeLange & D. Sigman (Elsevier, New York, 1975) and another book with the same title by G. Means & R. Feeney (Holden-Day, San Francisco, 1971). Additional reviews include the article by R. Feeney, R. Yamasaki & K. Geoghegan in Modification of Proteins, ed. R. Feeney. & J. Whitaker (Amer. Chem. Soc., Washington, 1982) and A. Blair & T. Ghose, J. Immunol. Methods, 59, 129 (1983). Among the preferred chemical treatments, which are described in these reviews and which are well-known to those skilled in the art, are:

1. carbodiimide and imidoester reagents for activation of carboxyl groups;

2. dithiopyridyl-, maleimido- and succimimido-reagents for thiol groups; and

3. treatment with derivatives of acetic, succinic, and maleic anhydride for amino groups;

For templates which are not proteins, other activation chemistries should be selected as appropriate on the basis of the molecular structure of the selected binding pair member. For example, periodate oxidation of saccharides may be appropriate for carbohydrate or carbohydrate-containing species. For nucleic acid probes, a brief and illustrative list of available techniques includes:

Formation of phosphodiester bonds to a support by activation of terminal phosphate groups of the nucleic acids;

Periodate oxidation of ribopolynucleotides and condensation of the resulting dialdehyde with supports bearing amines and hydrazides;

Reaction of terminal diol groups in ribopolynucleotides with supports bearing dihydroxyboryl groups.

These and selected other methods for nucleic acid derivatization are reviewed in "Immobilized

Polynucleotides and Nucleic Acids" by P.T. Gilham in

Immobilized Biochemicals and Affinity Chromatography, ed. R. Dunlap (Plenum Press, New York, 1974), and in H.

Bunemann, P. Westhoff, & G. Herrmann, Nucl. Acids Res., 10, 7163 (1982).

Alternate methods for nucleic acid derivatization include:

Introduction and reaction of a terminal thiol group on nucleic acids [B. Connolly & P. Rider, Nucl. Acids

Res., 13, 4485 (1985); R. Goody & F. Eckstein, J. Amer.

Chem. Soc., 93, 6252 (1971)];

Introduction and reaction of a terminal imidazolide group on nucleic acids [B. Chu, G. Wahl & L. Orgel,

Nucl. Acids Res., 11, 6513 (1983)]; and

Introduction and reaction of a terminal amino group on nucleic acids [J. Coull, H. Weith & R. Bischoff, Tet. Lett., 27, 3991 (1986); L. Smith, S. Fung, M. Hunkapiller, T. Hunkapiller & L. Hood, Nucl. Acids Res., 13, 2399 (1985); L. Wachter, J. Jablonski & K. Ramachandran, Nucl. Acids Res., 14, 7985 (1986)].

In the method of the present invention, certain target analyte binding pair members that may be present in a biological sample, physiological sample, environmental sample, or the like, are detected. In the first step of the method, a known amount of tagged reagent comprising a signal moiety and a binding moiety complementary to or homologous with the target analyte is contacted with the sample under analysis, and/or template, for a time sufficient to allow binding of the tagged reagent binding moiety to the target analyte or to a template having complementary binding members. Any conventional method of contact may be employed. For example, the assay may be carried out in solution by merely mixing the components together in a liquid medium which may be that of the sample under analysis itself, or may be a prepared physiological, or other biological medium such as physiological buffers, saline, or the like. When a template is used, a liquid medium containing both the tagged binding reagent and the target analyte may be conveniently contacted with the template, or a liquid medium containing one or the other may be contacted sequentially with the template for binding of complementary members thereto. The template may be in free form, or immobilized onto a support such as a column, a bead, a microtiter plate, or the like. In some embodiments, the sample under analysis is first purified by precipitation, filtration, centrifugation, electrophoresis, liquid chromatography, and the like.

After this contact, an additional step may be employed to separate tagged reagent bound to target analyte or template from the tagged reagent which did not bind. The separation may be accomplished by conventional techniques such as electrophoresis, affinity chromatography, precipitation, gel filtration chromotagraphy, washing and the like. When a template is immobilized onto a column, unbound tagged reagent will run off, achieving separation of bound from that which did not bind automatically. However, the column should be preferably washed at least once to remove extraneous unbound material. In other embodiments wherein the template is immobilized onto a support, separation may be accomplished by simply removing the support after binding of the tagged binding reagent has been achieved.

A second optional step may be employed after the separation step. In certain embodiments it may be preferred to "unbind" bound members by conventional techniques such as denaturation, displacement, altering the pH and/or ionic strength of buffer, and the like.

However, care should be exercised in selecting a technique that will not compromise the structural integrity of the signal moiety of the tagged binding reagent so as not to interfere with the. amount of detectable moiety that can be ultimately produced. In the next step of the method of the invention, bound or unbound tagged binding reagent, or a combination thereof is contacted with a second agent effective to activate the signal moiety. Many permutations of this contacting step are possible. As discussed above, in some embodiments of the present method, a separating step is not necessary after the specific binding event has taken place. This is due to measurable change in reactivity of the tagged reagent with the second agent when the tagged reagent is in a bound state as compared to an unbound state. In these cases, the second agent may be simply contacted by any conventional means with the sample under analysis which now contains tagged binding reagent that may or may not be bound to target analyte or template. The amount of detectable moiety produced may then be compared against the amount that would be produced in the absence of binding of tagged reagent to target analyte or template.

In other embodiments, tagged reagent possibly bound to target or template is separated from tagged reagent that has not bound. The second agent is then contacted by any convenient means with the sample containing the tagged reagent that is bound, sample containing tagged reagent that did not bind, or both, to activate signal moiety and produce detectable moiety. Activation of the signal moiety will ultimately produce the desired detectable moiety. Preferred agents suitable to activate the signal moiety are those as described hereinabove that will cleave the detectable label from the complex such as by a hydrolysis reaction and the like, or are those that can serve as a reactant which, together with the signal moiety can produce a detectable moiety as a product.

Once the detectable moiety is produced, it is transferred into a gaseous phase. It is contemplated that such transferance can take the simple form of evaporating the detectable moiety from the liquid phase in which the conjugate has been cleaved or the reaction has taken place. It is also contemplated that the detectable moiety can be evaporated from a modification of this liquid phase after various physical or chemical treatments or separations following the contacting of the tagged reagent with the second agent. It is further contemplated that the detectable moiety may be transferred from this liguid phase into another liquid phase and then evaporated. For example, the detectable moiety may be transferred from an aqueous phase where the reaction took place, to an organic phase, by simple extraction. In all of the above cases, the detectable moiety ultimately produced may be further reacted to increase its volatility and therefore ease its transfer from one liquid phase to another or from a liquid to a gas phase. Detectable moieties containing one or more polar groups (i.e. hydroxy, amino, carboxyl or the like) may be derivatized by silylation, methylation, acetylation or acylation. Reagents suitable for this are acetic anhydride, methyliodide, trimethylsilylchloride, triraethylsilylacetamide and the like. In such instances, substances preferably used to extract the detectable moieties are those such as alkanes, acetates, alcohols, nitriles, ethers, ketones; and the like.

In the last step of the method of the invention, the detectable moiety transferred into the gaseous phase is analyzed as a direct or indirect indication of the amount of target analyte that may have been present in the sample under analysis. After transferance to the gaseous phase, the detectable moiety may be recaptured on or in a solid or liquid phase, such as a chromatography column. The gaseous phase may be subjected to selective adsorption, absorption, condensation or the like, such as in a gas chromatography apparatus or ion mobility apparatus, so as to separate the particular detectable moieties sought from other materials which migrate at different rates through the system. An exemplary ion mobility apparatus is described in U.S. Patent 4,311,669 of Spangler et al., 4,390,784 of Browning et al., and 4,378,499 of Soangler et al. In such cases, the ultimate detection is by charge measurement, absorption, emission, ohotoionization, and the like. Suitable detection means bevond those mentioned above include those described in chapter 5 "Detectors" at pages 213-280 of R.L. Grob, Modern Practice of Gas Chromatography (New York 1977).

In the more preferred embodiments of the present method, a template binding pair member as described above is utilized. Both tagged reagent and sample possibly containing target analyte binding pair members are contacted with the template for binding of one or the other, or both thereto.

The geometries applicable for this specific binding event can be any of those described in U.S. Patent No.

4,629,689, herein incorporated by reference insofar as it pertains to the present invention. It should be appreciated, of course, that wherever the enzyme is attached to a ligand or polynucleotide within the context of the geometry described in the referenced patent, labels of the present invention would be substituted for the enzyme moiety.

By way of illustration, assay modes conventionally referred to as "reagent limited modes" and "reagent excess modes" will be discussed.

In "reagent limited mode", target analyte is contacted with a constant and limited amount of a template complementary binding pair member. After reacting until there is no further net association or dissociation of these binding pair members, the proportion of analyte distributed between the bound and the free fractions is determined. A tagged binding reagent containing a moiety homologous with or complementary to either the target analyte, its template binding pair member or both is used in this determination. Several different reaction configurations are possible in this mode. Using antibody-antigen binding pair members for illustrative purposes, several configurations are discussed below.

In cases where the antigen is the target analyte, and a corresponding antibody is selected as the template binding member (i.e. template and target analyte are complementary) a competitive inhibition geometry may be used. Tagged antigen, along with sample (possibly containing target antigen) are contacted simultaneously with the template, and the two species then compete for available binding sites. After equilibrium is reached between the tagged and target antigen and the template antibody, the distribution of tagged antigen between the bound the free fractions is measured. In other configurations involving an antibody that serves as the template binding pair member and tagged and target analyte that are homologous, the order of addition of the reagents becomes critical. For a displacement type geometry, a limited amount of template antibody is saturated with tagged antigen. The template antibody-tagged antigen complex is then contacted with sample possibly containing the target antigen. If no target analyte is present in the sample, the template antibody-tagged antigen complex remains intact. However, when target analyte is present, it binds to the template antibody thereby displacing all or a portion of the tagged antigen. The distribution of tagged antigen between the free and bound fractions is now measured as a function of the target analyte present in the sample. For a sequential saturation type geometry, the template antibody is first incubated with the target analyte. After equilibrium is reached between target analyte and template antibody, tagged antigen is added in sufficient quantity to saturate the remaining template antibody binding sites. Again, the distribution of tagged antigen between the free and bound fractions is determined. Preferred reaction conditions for binding of a tagged or target antibody (antigen) with an immobilized template antigen (antibody) vary over a large range. In general, it is preferred that the binding reaction be carried out in a mostly aqueous buffer solution containing dissolved salts at pH values between about 3 and 11. Reaction temperatures preferably range between about 0° and 60°C, and the percent composition of nonaqueous solution components (e.g. DMF) preferably range between about 0 and 50%. Reaction time periods vary widely, and incubation should preferably be allowed to proceed for a period of time between about 10 seconds and 72 hours. Preferred concentration of salts is between about 1 micromolar and 1 molar. Preferred concentrations of antibodies and antigens is each between about 1 picomolar and 10 micromolar. It is also preferred that the immobilized template members be present on the surface of a support in amounts of about

10^-18 to 10^-8 moles per cm².

In particularly preferred embodiments, the pH values are maintained between about 5 and 8, and the incubation time is about 10 seconds to about 2 hours at about 37°C. In addition, the concentration of binding pair members in solution is about 0.1 nanomolar to 10 micromolar, and the coverage of immobilized template members is between about 10^-13 and 10^-8 moles per cm².

For nucleic acid binding pair members, preferred incubation temperatures are between 0° and 100°C and may involve a first incubation at about 40°-75°C followed by cooling at a second temperature of about 0° to 25°C.

Total incubation time is also preferably about 10 seconds to about 2 hours. The concentration of nucleic acid binding pair members in solution should preferably range between about 10 ^-9 and 10^-21 molar, more preferably between 10^-12 and 10^-18 molar. Either agueous buffers or nonaqueous buffers, or mixtures thereof, may be employed for these incubations, although aqueous buffers at about pH 7 ± 1 are preferred. In contrast to the various geometries discussed above, "reagent excess modes" entail the reaction of target- analyte with a constant amount and molar excess of its complementary binding pair member. After reacting until there is no further net association or dissociation of the target analyte and its binding partner, the proportion of the binding partner distributed between its bound and free fractions is ascertained. Several different reaction configurations are possible in this mode. Using antibody-antigen binding pair members for illustrative purposes, certain configurations are discussed below.

In the basic reagent excess procedure, an immunometric assay, a target antigen is reacted with an excess of a tagged antibody serving as the tagged binding reagent. After this binding reaction, the proportion of tagged antibody distributed between the free and bound fractions is determined. A convenient method of doing this is by removing the free tagged antibody from solution by binding it to a solid phase antigen. The tagged antibody bound to the target antigen still in solution may be treated with an effective second reagent to generate a detectable moiety for analysis. In another permutation of a reagent excess geometry called a two-site immunometric method, an excess of a tagged antibody is reacted with a sample possibly containing a target antigen. After the initial binding reaction, the proportion of tagged antibody distributed between the free and bound fractions is determined by the addition of a solid phase antibody directed towards a second antigenic binding site on the target antigen. This removes the previously bound tagged antibody from solution, thereby separating the free and bound fractions to facilitate analysis.

The choice between reagent excess and reagent limited modes and a particular geometry within that mode will be governed, in most instances, by a number of considerations. Certain analytes may possess properties which influence the choice of analytical mode, such as molecular weight (size), concentration, reaction kinetics or binding affinity. As one example, the sequential saturation mode will be a particularly attractive option over the competitive inhibition modes when the template binding affinity toward target members is higher than toward tagged members. In such situations, the stepwise exposure of the template, which is characteristic of the sequential assay, allows one to derive the full benefit of the high binding affinity of the target members, without the countervailing influence of the higher concentration of the tagged members which would be present in competitive assay mode. In sequential mode, the template is first exposed to the relatively small number of target members, which are able to bind to completion with the template members, due to their high binding affinity and the lack of any competing species. Subsequent exposure to tagged members could be expected to cause only slow or no displacement of the target members already bound, because of the lower binding affinity of the tagged members. Consequently, the tagged members should bind, in the main, only to unoccupied template binding sites. In effect, the slow kinetics of displacement result in an enhanced population of the low- concentration target members on the probe sites.

While the distribution of template binding sites between tagged and target members will approach the same long-time equilibrium value for the competition inhibition modes, short-time nonequilibrium distributions may be obtained due to slow displacement kinetics and/or differential binding affinities exhibited by tagged and target binding pair members.

Such effects may be exploited, as illustrated above, to improve assay sensitivity toward low-concentration analytes, or to permit rapid (nonequilibrium) assays to be performed in situations in which the target, template, and tagged binding pair members have approoriate molecular attributes (e.g. mutual binding affinities, etc.). Additional mathematical details on the kinetics of these and other assay configurations may be found in the article by D. Rodbard in Ligand Assay, ed. J. Langan and J. Clapp, Masson Pub., New York, 1981.

The diagnostic assay kit of the present invention includes preferred forms of the tagged binding reagent as well as a second reagent effective for activating the signal generating moiety of the tagged binding reagent. The tagged binding reagent consists of a moiety homologous or complementary to the target analyte binding pair member and a signal generating moiety. As such, the moiety homologous or complementary to the target analyte preferably contains either an antigen, an antibody, a binding protein, a hapten or a polynucleotide. The signal generating moiety is that component bound to the tagged binding reagent responsible for directly or indirectly producing a detectable moiety. In the directly producing mode, a moiety attached to the .tagged binding reagent, with or without the use of a spacer group, is cleaved. That moiety is preferably an electrophore, a nitrogen- or phosphorous-containing compound, an ionizable compound or the like. In the indirectly producing mode, the tagged reagent reacts with the second agent to yield the detectable moiety.

The second agent for activating the signal generating moiety is included in the analytical kit and is preferably a reagent that cleaves the signal generating moiety from the tagged reagent to afford the detectable moiety. Alternatively, in other embodiments, it is a compound that reacts with and is transformed by the signal generating moiety to yield the detectable moiety. In the former case, the second agent is preferably an acid, a halogen, a pseudo halogen or the like, whereas in the latter, it is preferably a polyhalogenated or nitrated alcohol, carboxylic acid, amine, alkene, ketone, aldehyde or the like. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In certain preferred embodiments of the present invention, a binding member is conjugated to an electroohoric signal moiety which can be activated by cleavage with a reagent. The following represents a particularly preferred tagged binding reagent as well as the reaction scheme to yield detectable moiety:

Wherein R is a polyhalogenic aliphatic or aromatic; X is any binding member that is homologous with target analyte and Y is a cleavage reagent. In particularly preferred embodiments R is pentafluorobenzoyl, pentafluorosulfonyl, trifluoroacetyl, and the like; X is biotin, thyroxine, insulin, and digoxin, and Y is cyanogen bromide, iodoacetamide, and methyl iodide. The cleavage reaction can take place in a neutral, basic or acidic medium. Neutral or basic media that may conveniently be employed are water or acetonitrile. It is preferred, however, that the reaction take place in an acidic medium, preferred acids being formic, hydrochloric, sulfuric, p-toluenesulfonic, acetic, and the like.

In these preferred embodiments, a template, comprising binding pair members complementary to the target analyte and also complementary to the tagged reagent, is contacted with both the sample and the tagged reagent in a fluid phase. The tagged reagent is preferably in a liquid phase comprised of a solution compatible with binding pair members, such as phosphate buffered saline, with a pH range of about 6.8 - 7.4. Concentrations of tagged reagent generally range from about 10^-4M to about 10^-10M with 10^-6M to about 10^-8M particularly preferred. Concentration of template binding pair members generally range from about 10 ^-4M to about 10^-10M, with about 10^-6M to about 10^-8M preferred. Reaction temperatures generally range from about 0°C to about 60°C, with about 37°C particularly preferred.

Separation of members bound to the template from those still in solution is preferably accomplished by an immobilized template binding member that can be physically removed from the sample in which the binding reaction has taken place. The presence of tagged reagent is then determined by contacting it with an effective second agent to activate the signal moiety. Preferred for this purpose is contact with reagents such as cyanogen bromide, methyl iodide, iodoacetamide and ethyl bromoacetate in an acidic medium. It is preferred to add the acid to the system prior to contact with the agent to avoid unwanted side reactions.

The detectable moiety is thus produced by cleavage of the signal moiety, and preferably removed from the system by transferance to an organic medium such as toluene, benzene, cyclohexane or the like. The detectable moiety may be injected into a gas chromatograph equipped with an electron capture detector. Separations may thus be effected by conventional methodology using packed or capillary columns.

The following specific examples present more limited embodiments of the present invention and are not to be construed as limitative thereof. EXAMPLE 1

A series of solutions of the following concentrations of N-pentafluorobenzoyl-L-methionyl-glycylbiotin amide were prepared in 70% formic acid:

1.00X10^-6M 6.25X10^-8 3.91X10^-9

5.00X10^-7 3.13X10^-8 1.95X10^-9

2.50X10^-7 1.56X10^-8 9.77X10^-10

1.25X10⁷ 7.81X10^-9 0

Aliquots (0.5mL) of each were placed in screw cap vials - done in quadruplicate - and charged with 50 microliters of 0.04M cyanogen bromide. The vials were closed tightly and incubated at 70°C for 2 hours. Following this, the volatiles were removed from the vials under reduced pressure at room temperature. The remaining residue was redissolved in 0.5mL of toluene containing 10^-7M lindane (internal standard). The solutions were subsequently analyzed by injecting 1 microliter into a gas chromatograph equipped with an electron capture detector. Separations were effected on a 86% dimethyl - 14% cyanoprop.yl methyl polysiloxane coated fused silica capillary column ( 15M x 0.25 mm with a film thickness of 0.25 microns. Peaks for lindane and

N-pentafluorobenzoyl homoserine lactone were exhibited. The average peak areas for N-pentafluorobenzoyl homoserine lactone normalized with respect to the internal standard are listed below.

log area lactone area lactone

[Biotin Amide ] * [Biotin Amide] (area lindane)ave log (area lindane)ave

3.91 x 10^-9 -8.41 0.008 ± 0.003 -2.120

7.31 x 10^-9 -8.11 0.008 ± 0.002 -2.100 5

1.56 x 10^-8 -7.81 0.017 ± 0.004 -1.780

3.13 x 10^-8 -7.50 0.024 ± 0.001 -1.620

6.25 x 10^-8 -7.20 0.043 ± 0.007 -1.370

1.25 x 1010⁷ -6.90 0.093 ± 0.011 -1.030

2.50 x 10^-7 -6.60 0.198 ± 0.010 -0.700

5.QQ x 10^-7 -6.30 0.447 ± 0.035 -0.350

1.00 x 10^-6 -6.00 1.001 ± 0.060 +0.001

* [Biotin pmide] = concentration of N-pentafluorobenzoyl-2-methionyl glyclbiotin amide.

EXAMPLE II A series of solutions of the following concentrations of biotin are prepared in phosphate buffered saline (pH 7.2) containing 0.05% Tween 20:

1.00 x 10^-5M 1.25 x 10 ^-7 1.56 x 10^-8 5.00 x 10^-7 6.25 x 10^-8 7.81 x 10^-9 2.50 x 10^-7 3.13 x 10^-8 0

An equal volume of 1.00 x 10^-7M N-pentafluorobenzoyl-L- methionylglycylbiotinamide in the same buffer is added to each of the above. Aliquots (0.5 mL) of these combined solutions are then incubated with 50mg of avidin-coated controlled pore glass beads for 2 hours at 37°C. The binding capacity of this support is 1 pico mole of biotin per mg. After the binding reaction, the avidin-coated supports are collected by filtration and washed consecutively with phosphate buffered saline, water, methanol and water. The supports are placed in screw cap vials and charged with 0.5 mL of 70% formic acid and 50 microliters of 0.04 M cyanogen bromide. The vials are closed tightly and incubated at 70°C for 2 hours. Following this, the yolatiles are removed under reduced pressure at room temperature. The remaining residue is extracted with 0.5 mL of toluene containing

10-⁷M lindane. The solutions are analyzed by gas chromatography as described in Example I.

EXAMPLE III

The procedure of Example II except that after the washing step, the avidin-coated controlled pore glass is incubated for 18 hours at 37°C with 0.5 mL of 5 x 10^-5M biotin in phosphate buffered saline to displace any bound N-pentafluorobenzoyl-L-methionylglyelbiotin amide. The aqueous phase is then removed and taken to dryness under reduced pressure. The residue is treated with cyanogen bromide in 70% formic acid and analyzed as described in Example II.

EXAMPLE IV

A 10^-6M stock solution of trichloroethyl biotin ester was prepared in phosphate buffered saline (pH 7.2) containing 0.05% Tween 20. Into glass test tubes containing 50mg of avidin coated carboxyl derivatized controlled pore glass was added lmL of the trichloroethyl biotin ester solution. This reaction was done in duplicate. Also prepared were the following control reactions:

1. 1mL of trichloroethyl biotin ester solution (done in duplicate);

2. 1mL of trichloroethyl biotin ester solution containing 50mg of uncoated carboxyl derivatized controlled pore glass;

3. 1mL of phosphate buffered saline containing

50mg of avidin coated carboxyl derivatized controlled pore glass.

The tubes were then incubated at 37°C for 90 minutes. After this incubation period, the aqueous phase was withdrawn from each of the tubes containing the trichloroethyl biotin ester with the avidin coated carboxy derivatized controlled pore glass. One solution was saved for analysis, whereas the other was treated with an equal volume of 2N HCl for 2 hours at 37°C. The coated and uncoated carboxyl derivatized supports were separately collected on glass frits, washed 4X with (1mL) phosphate buffered saline and dried under vacuum. These were then treated with 2mL of IN HCl for 2 hours at 37°C. For the two tubes containing 1mL of trichloroethyl biotin ester with no support and/or avidin present, one was saved for analysis whereas the other was treated with an equal volume of 2N HCl for 2 hours at 37°C. For the workup, all of the neutral solutions above were saturated with sodium chloride and extracted with ethyl acetate containing 10 ^{- 9}M decafluorobenzophenone. All of the acidic solutions were neutralized with solid sodium bicarbonate prior to extraction with ethyl acetate containing decafluorobenzophenone. The ethyl acetate extracts were subsequently analyzed on a gas chromotograph equipped with electron capture detector. Separations were effected on a 50% cyanopropyl methyl-50% methyl phenyl polysiloxane coated fused silica capillary column (15m x

0.25 mm with a film thickness of 0.15 um). Peaks for

2,2,2-trichloroethanol and decafluorobenzophenone were exhibited in the chromotograms. The results are tabulated below.

Reaction [2,2,2-Trichloroethanol]

TCE-biotin + avidin - CPG (acid hydrolyzed support) 1.7 x 10^-7M

TCE-biotin + avidin - CPG (acid hydrolyzed support) 1.8 x 10^-7

TCE-biotin + avidin - CPG (unhydrolyzed aqueous phase) 0

TCE-biotin + avidin - CPG (acid hydrolyzed aqueous phase) 4.2 x 10^-7

TCE-biotin (unhydrolyzed aqueous phase) 0

TCE-biotin (acid hydrolyzed aqueous phase) 6.1 x 10^-7

TCE-biotin + CPG (acid hydrolyzed support) 0

PBS + avidin - CPG (acid hydrolyzed support) 0

TCE=Trichloroethanol CPG=Controlled pore glass

EXAMPLE V

A series of solutions of the following concentrations of biotin are prepared in phosphate buffered saline (pH 7.2) containing 0.05% Tween 20:

1.00 x 10^-6M 1.12 x 10^-7 1.56 x 10^-8 5.00 x 10^-7 6.25 x 10^-7 7.81 x 10^-9 2.50 x 10^-7 3.13 x 10^-8 0

An equal volume of 1.00 x 10^-7M trichloroethylbiotin ester in the same buffer is added to each of the above. Aliquots (0.5 mL) of these combined solutions are then incubated with 50 mg of avidin-coated controlled pore glass for 2 hours at 37°C. The binding capacity of this support is 1 picomole of biotin per mg. After the binding reaction, the supports are collected on glass frits, washed 4X with (1 mL) phosphate buffered saline and dried under vacuum. The bound ester is hydrolyzed by treatment with 2 mL of

IN HCl for 2 hours at 37°C. Subsequently, the acidic solutions are neutralized with solid sodium bicarbonate and extracted with ethyl acetate containing 10^{- 8}M decafluorobenzophenone. The ethyl acetate extracts are analyzed as described in Example IV.

EXAMPLE VI The following depicts a reaction of a signal moiety in the tagged reagent, an electrophore labelled amine terminus alpha amino acid, with phenylisothiocyanate to yield the detectable moiety, an electrophore labelled phenylthiohydanotin.

EX AMPLE VI I

The following depicts indirect production of detectable moiety by reaction of a methionine sulfoxide moiety in the tagged reagent with an alcohol such as methanol to produce a detectable product, formaldehyde.

The formaldehyde may be further contacted with a second reagent, pentafluorobenzylhydroxyamine hydrochoride, to produce a product, pentafluorobenzyloxime that is strongly electrophoric and suitable for gas chromatography analysis.

Claims

What is claimed is:

1. A tagged binding reagent for detection of a target analyte binding pair member comprising a signal moiety and a moiety complementary to or homologous with all or a portion of said target analyte binding pair member, said tagged binding reagent adapted for use in a specific binding event to bind to all or a portion of a complementary binding pair member; and wherein said signal moiety of said tagged reagent is adapted to activation after said binding event by an effective second agent to produce a detectable moiety.

2. The tagged binding reagent of claim 1 wherein the signal moiety comprises a cleavable compound capable of volatilization for gas phase detection.

3. The tagged binding reagent of claim 2 wherein the cleavable compound contains a nitrogen moiety capable of gas phase detection.

4. The tagged binding reagent of claim 2 wherein the cleavable compound contains phosphorous capable of gas phase detection.

5. The tagged binding reagent of claim 2 wherein the cleavable compound is a strong electrophore.

6. The tagged binding reagent of claim 5 wherein the electrophoric compound is selected from the group consisting of the polyhaiogenated aliphatics. and aromatics.

7. The tagged binding reagent of claim 6 wherein the electrophoric compound is selected from the group consisting essentially of pentafluorobenzoyl, hexafluorobutyryl and trichloromethyl moieties.

8. The tagged binding reagent of claim 1 having a moiety complementary to the target analyte binding pair member.

9. The tagged binding reagent of claim 8 wherein the moiety complementary to target analyte is biotin.

10. The tagged binding reagent of claim 9 comprising n-pentafluorobenzoγl-L-methionylglycyl biotin amide.

11. The tagged binding reagent of claim 1 having a moiety homologous with the target analyte binding pair member.

12. The tagged binding reagent of claim 1 wherein the signal moiety is capable of reacting with a second reagent to produce a gas-phase detectable compound.

13. An analytical kit comprising the tagged binding reagent of claim 1 and a second agent for activation of said signal moiety.

14. A method for the determination of a target analyte binding pair member in a sample under analysis comprising the steps of: a. providing a tagged reagent having a moiety complementary to said target binding pair member and also a signal moiety capable of producing a known amount of detectable moiety; for a time sufficient to allow target binding pair member present in said sample to bind to said tagged reagent; b. contacting said sample with said tagged reagent; c. contacting said sample with an effective second agent to activate said signal moiety in said tagged reagent to produce a detectable moiety; d. transferring into a gaseous phase said detectable moiety produced by said signal moiety; and e. detecting said detectable moiety and comparing against said known amount of detectable moiety as an indication of the presence of target binding pair member in the sample under analysis.

15. The method of claim 14 further including the step of separating said bound tagged binding reagent from sample containing tagged reagent not bound to target analyte binding pair members;

16. A method for the determination of a target binding pair member in a fluid sample under analysis comprising the steps of: a. providing a template binding pair member capable of binding to a target analyte binding pair member in a liquid phase and also capable of binding to a tagged binding reagent in a liquid phase, said tagged binding reagent having a signal moiety and a moiety at least a portion of which is complementary to said template binding pair member; b. contacting said template with said tagged reagent and also contacting said template with said sample possibly containing target analyte for binding thereto of tagged reagent, target analyte, or a combination of both; c. separating said template containing, if any, bound tagged reagent, bound target analyte, or a combination thereof from said sample or liquid phase containing unbound tagged reagent and unbound target analyte; d. contacting said separated tagged binding reagent with a second effective reagent to activate said signal moiety of said tagged reagent to produce detectable moiety; e. transferring said detectable moiety into a gaseous phase for detection thereof; and f. detecting said detectable moiety as an indication of the presence of target binding pair member in the sample under analysis.