WO1989000694A1

WO1989000694A1 - Improved turbidimetric rate inhibition assay for haptens

Info

Publication number: WO1989000694A1
Application number: PCT/US1988/002303
Authority: WO
Inventors: Frank J. Lucas; Juan Bedevia; Mark L. Shenkin; Brian J. Mcrae; James H. Carter; Harold R. Crews
Original assignee: Coulter Electronics, Inc.
Priority date: 1987-07-21
Filing date: 1988-07-07
Publication date: 1989-01-26
Also published as: AU2128388A; JPH02500133A; EP0324015A4; EP0324015A1

Abstract

An improved turbidimetric rate inhibition assay for haptens is disclosed. This assay utilizes both monoclonal antibodies and polyantigenic conjugates in precise proportions in a hapten modulated competitive binding regime, wherein the relative concentration of hapten is inversely proportional to the amount of absorbance of a reaction mixture attributable to a precipitating immunocomplex. The dynamic analytical range and signal to noise ratio of this assay is unique for turbidimetric analysis of haptens. This assay is further unique in the specification for the reagents and in the stability of the reagent, even when fully diluted.

Description

Improved Turbidimetric Rate Inhibition Assay For Haptens

BACKGROUND OF THE INVENTION

1. Field of the Invention - This invention is directed to a method. More specifically, this invention is directed to a hapten modulated competitive binding immunoassay, wherein the relative concentration of hapten is determined by measurement of the amount of absorbance resulting from formation of an immunocomplex between an antibody and a polyantigenic molecule (hereinafter "conjugate") which mimics the immunochemical response of the hapten relative to the antibody. The dynamic analytical range and the signal to noise ratio of this assay is unique for turbidimetric analysis of haptens. The method of this invention is also unique in the specification of the reagent which is utilized in this method.

2. Background of the Invention - The manifestation of immunochemical interaction between an antigenic material and an antibody may be perceptible or imperceptible depending upon a number of variables, including the environment in which such interaction occurs; the relative size of the resultant complex; the relative solubility of the resultant complex in the reaction medium; the native fluorescence of the complex; and/or the presence of certain other agents, (whether or not endogenous to the reaction environment), which can introduce a label, indicator or tag into the resultant complex, thereby rendering it detectable with or without the aid of instrumentation.

While all of the various approaches to immunoassay are efficacious to a degree, each has inherent limitations which necessarily result in some trade-off either with respect to speed, sensitivity, convenience and/or safety. More specifically, homogenous immunoassay techniques involve the manifestation of a detectable species, which is indicative of an analyte of interest, in the presence of other sample components which may contribute or mask the monitored phenomenon. The detectable species or label can, in certain instances, be monitored directly, in the case of a fluorophore; or, require an additional reagent, such as a substrate, to manifest the presence of an otherwise invisible label (i.e. enzyme). In heterogenous immunoassay techniques, the sample is contacted with a solid phase having adsorbed reagent specific for interaction with one or more sample components. After the reagents associated with the solid phase interact with one or more of the sample components, the fluid phase of the sample (and sample constituents which remain dissolved and suspended in the fluid) are separated from the solid phase (and the sample constituents which have become bound to the reagent on the solid phase). The solid phase is further washed to remove any unbound materials. This wash fluid can be recovered and combined with the fluid fraction of the sample or simply discarded. The two mutually exclusive fractions which are produced in this manner either contain dissolved analyte (fluid phase) or insolubilized analyte (solid phase). Either one or both of these fractions can thereafter be analyzed for the presence of the analyte of interest. Where the detectable species (label), which is indicative of analyte of interest, is a fluorophore, the fluid phase or solid phase is irradiated with excitation energy and its fluorescence emission monitored. Where the label is an enzyme, a substrate must first be contacted with the fluid or solid phase and the reactive environment monitored for the production of a detectable species.

Another type of heterogenous immunoassay involves the use of a radioisotope as a label and the subsequent partitioning of the fluid phase/solid phases into two mutually exclusive fractions. More specifically, the analyte of interest and the radiolabel are initially contacted and interact with an immunoreagent which has been insolubilized on a solid phase; or, a second antibody used to precipitate an immunocomplex containing the radiolabel. The amount of radiolabel present in the solid phase is monitored on a scintillation counter and the number of "counts" correlated with the concentration of analyte in the sample. All of the above approaches to immunoassay represent a compromise and/or have inherent shortcomings. In most instances (with the exception of RIA), these shortcomings translate into a loss in sensitivity in the assay. More specifically, in virtually all of the immunoassay formats described above, with notable exception of RIA, the signal to noise ratio can be, and usually is, adversely affected by the environment in which the detectable species is monitored. For example, in a homogenous immunoassay, the monitoring of the detectable species can encounter interference from the other constituents which are endogenous to the sample. In a heterogenous immunoassay, similar constraints are obviously encountered where the fluid phase is monitored. The monitoring of the detectable species associated with a solid phase does not totally resolve this problem, particularly where the indicator is a fluorescent compound and the solid phase is itself somewhat fluorescent within either the excitation or emission spectra of the detectable species.

As noted above, the immunoassay based upon detection of a radiolabel does not suffer similar signal to noise loss over the usual time course of the monitored assay interval. Such assays do, however, require expensive instrumentation for measuring the detectable species and have generally fallen into disfavor because of strict regulations associated with disposal of the isotopic materials required for their performance.

The immunoassay systems described above have the capability of providing both qualitative and quantitative results. These assays can also be run as end-point or as kinetic (rate) reactions. The kinetic assay, of course, requires the use of instrumentation for rate (slope) determination and does lend itself to subtraction of interferents which may mask lower level of analyte. The use of kinetic measurements is preferred for systems having high background, provided the signal contribution by the background does not exceed the dynamic analytical range of the monitoring instrument. Li those analysis where qualitative results are all that is needed (i.e. identification of cell surface markers for blood typing), simple coagulation assays have proven adequate. Coagulation/agglutination type assays have been developed for soluble analytes, particulate analytes and stable emulsions containing latex beads. The latex bead based reagents which are used in such assays are present as a stable suspension within the fluid environment. In a typical analytical environment utilizing a latex reagent, their subsequent interaction with an analyte of interest and/or the change in their environment results in changes in the dispersion stability (agglutination) which can be attributed to the presence of the analyte of interest. Agglutination assays utilizing latex bead reagents have been developed for a number of serological determinants. For the most part, these assays are qualitative in nature.

Where quantitative analysis of a particulate dispersion is desired, the choices have generally been limited to nephelometric or turbidimetric analysis. Nephelometry and turbidimetry are not simply alternative techniques for monitoring the photooptical properties of the same particulate dispersion. Each analytical method can be used to measure precipitin reactions based upon immunochemical interactions, however, the relative sensitivity of each can and will vary, depending upon the specific immunoreagents which are used, their relative reactivity

(avidity) and their relative concentration in the fluid environment which is subjected to such analysis. Nephelometry generally involves irradiation of a diluted dispersion with a coherent light source (i.e. laser), and the measurement of the intensity of the reflected light at 90° to the incident radiation. This analytical techmque, as applied to immunoassay, is less sensitive to antibody concentration and titer. Turbidimetric analysis, by way of contrast, utilizes conventional spectrometric analysis (measurement of optical density/absorbance of dispersion), however, reagent specifications and performance are more demanding and offers the potential for a broader dynamic analytical range. The potential for adaptation of turbidimetric monitoring technique to immunoassay have been recognized for some time, see for example U.S. Patent 4,604,365. In addition, latex reagent systems have been "engineered" for enhancement in the precision turbidimetrically monitored immunoassay, see for example U.S. Patents 4,581,337; 4,524,025; 4,521,510; 4,477,346; 4,460,695; and, 4,401,765. These patents describe certain monoclonal antibodies (U.S. 4,524,025), and certain enhancements for polymer particles (which are covalently bonded to compounds of biological interest) (U.S. 4,401,765).

The principles, factors and constraints involved in light scattering and absorbance determinations of precipitin reactions, based upon immunochemical interactions, have been known for some time. In a series of papers co-authored by Pauling, Pressman and others, the inhibiting effects of haptens upon the precipitation of antisera/small molecule complex were reported in the early 1940's. This reported research accurately quantified the amount of free hapten needed to effect such inhibition, permitting accurate prediction of the effect of the free hapten in a precipitin reaction. Pauling, L., et al, J. Am. Chem. Soc, 64: 2994-3002 (1942). By the late 1940^*s, the advances made by Pauling, et al clearly indicated that the principles of the antibody/antigen precipitin reactions were well understood. Most of their work during this period focused upon the ability of the free hapten to solubilize the precipitated immunocomplex and/or generate varying quantities of precipitate, depending upon the component order of addition to the reaction environment. Pauling, L., et al, J. Am. Chem. Soc, 64: 3010-3014 (1942) and Pauling, L., et al, J. Am. Chem. Soc, 64: 3015-3020 (1942).

By the early 1950's, light scattering techniques for measurement of precipitin reactions, based upon immunochemical interactions, became more prevalent with the advances made in instrumentation. Light scattering properties of antigen/antibody interactions were reportedly first observed at 90° to the incident radiation, Goldberg, R.J., et al, J. of Immunol., 66: 79-86 (1951); and, thereafter at low angle of light scatter, Doty, P. et al, Advances in Protein Chemistry. 6: 35-121 (1952). The low angle light scatter measurements by Doty, et al were reportedly more sensitive than measurements made at 90° to the incident radiation (at least for the system that was studied). In the early 1970's, advances in instrumentation led to attempts to automated monitoring measurement of immunoprecipitin reactions by nephelometry, Davies, G.E., Immunology 20 (1970) 779. Precipitin reactions, even with instrumentation, are sometimes difficult to detect. In addition, such reactions were often quite slow and could require anywhere from several minutes to several hours to detect, hi order to enhance the speed, and size of the particles produced in such precipitin reactions, it was found that the addition of certain polymers to the reaction medium could promote more particle growth, Harrington, J.C., et al, Immunochemistry, 8 (1971) 413-421. The interest in immunoprecipitin reactions, apparently kindled by advances in instrumentation continued through the balance of the decade. I 1974 Cohen reported the monitoring the early stages of an agglutination reaction by quasi-light scattering spectroscopy and by turbidimentry, Cohen, R. J. ,et. al., Immunochemistry, 12 (1974) 349. The model system used in the Cohen study was a polystyrene latex wherein the individual polymer particle were coated with bovine serum albumin (BSA) and the agglutinating agent, an antiserum to BSA.

In 1979, a competitive nephelometric immunoassay for theophylline was first described in the technical literature, Nishikawa, T., et al, Clin. Chem. Acta, 91 (1979) 59-65. At about the same time, Beckman Instruments, Inc. (Fullerton, California) introduced a nephelometric instrument and reagent system for performance of in vitro diagnostic tests on biological fluid samples. The reagent systems (ICS™ Reagents) from Beckman available for use on this system included test kits for determination of serum levels of therapeutic drugs (i.e. theophylline). The theophylline test kit consisted of an anti-serum to theophylline and a theophylline conjugate consisting of a theophylline derivative covalently bonded to an equine apoferritin carrier protein. The ratio of theophylline to carrier protein was not disclosed, nor the position of derivatization of theophylline. The analytical protocol for performance of the assay was essentially the same as described in the Nishikawa paper which appears in Clin Chem Acta, 91 (1979), 59-65. The details of the assay protocol described in the package insert for the Beckman reagent for theophylline, are fairly typical of accepted practice. This insert specified that the sample, which is to be subjected to analysis, must be initially diluted (1:6) followed thereafter by yet another dilution step (1:6). The objectives of such dilution are three fold: (a) to reduce the interference (signal noise) from materials which are endogenous to the sample; (b) to reduce the interference (signal noise) which can result from cross-reactivity of one or more of the reagents with one or more of the constituents (other than the analyte) which are present in the sample; and (c) to insure the monitored reaction falls within dynamic analytical range of the reaction for a given instrument.

Shortly thereafter, a review article appeared which discussed the application of different automated monitoring techniques for immunoprecipitin reactions. Deverill, I., et al, J. Immunol. Meth., 38 (1980) 191-204. The Deverill article reviewed the various methodologies historically used in detecting/monitoring immunoprecipitin reactions and concluded that "the direct measurement of turbidity using a high quality ultraviolet absorptiometer (spectrophotometer) currently offers the most sensitive optical method for this characterization of fluid phase immunoprecipitation". Deverill, I., at p.195.

Unfortunately, reagent development for immunoprecipitation reactions useful in analytical testing has lagged behind instrument development. In order to "engineer" immunoprecipitin reagent systems for the high through-put instrument environment, it became apparent that the immunoprecipitin reaction would have to occur within the time course of existing clinical analyzers or that instrumentation would have to be specifically designed to accommodate these immunoprecipitin reagent systems. Since the latter alternative was unattractive, efforts were made to define reagent systems to operate within the operational parameters of existing instrumentation. The developer of such reagent systems were, thus, driven by the economic realities of the marketplace and, thus, designed such reagent systems for use in a turbimetric monitoring environment.

One of the more recent entrants into this environment is the reagent system developed for operation on the duPont aca clinical analyzer; such reagent systems being described in U.S. Patents 4,401,765 and 4,524,025. The duPont reagent system includes a monoclonal antibody and a hapten/latex particle conjugate. The performance characteristics of this type of reagent system must, of necessity, conform to the instrument environment contemplated for its use. The hapten/latex particle reagent, once prepared (diluted) as a working solution, however, suffers from an abbreviated shelf -life (generally less than 8 hours). Accordingly, such reagents must be prepared on a daily basis and then only in limited quantities.

Similar constraints are also present where such reagent systems are prepared utilizing a hapten/carrier protein. The reagent system described in U.S.4,604,365 is representative of such prior art systems. While the hapten/carrier protein described in the '365 patent may be inherently more stable and soluble, its use in an instrument environment necessarily contemplates the addition of polymeric precipitin reaction "enhancers". These enhancers, although intended to accelerate formation of a precipitating immunocomplex, also tend to cause spontaneous (auto) agglutination of the hapten/carrier protein and/or degradation of the hapten/carrier protein.

In addition to reagent stability, the '365 patent brings into focus some of the factors which need be considered in the synthesis of reagents for an analytical protocol involving spectrophotometric monitoring of a precipitin reaction. These factors will, of necessity, have a substantial bearing not only upon the stability of the reagent system, but also its sensitivity. The definition of operational/functional reagent parameters are generally known. Four (4) such systems are specifically identified and precisely described in the '365 patent for certain hapten/HS A carrier protein systems. What is apparent from this patent and from other literature is that reagent systems dynamics vary with the specific assay format, type of analyte, and demands/requirements of the monitoring system. In the definition of reagent parameters for an immunoprecipitin reaction (which is to be momtored by turbidimetry), the relative binding affinity of the reagents (antibody and analyte mimic or hapten analog) is generally critical, as is the composition of the analyte mimic Where the analyte is a "small molecule" the ability to evoke an immune response within a sensitized host is generally not possible. Accordingly, the sensitization of the host to the small molecule is achieved by coupling the small molecule to a larger substance (generally referred to as a "carrier protein") and the utilization of this hapten/protein compound as an immunogen. As is readily appreciated, the response to this immunogen will not only evoke antibodies to the small molecule component of the immunogen, but also to portions of the protein molecule as well. Accordingly, the protein used in this immunogen is selected so that antibodies which are formed to the protein component of this complex (if any) do not cross-react with any proteins which may be present in the sample which is ultimately subjected to an immunoassay. The experience of the biotechnology industry appears to suggest that the preferred free drug inhibited antibodies are produced in response to immunogens consisting of haptens conjugated to bovine serum albumin (BSA).

The manner in which the small molecule is coupled to the protein can also affect the nature of the antibodies which are evoked in the host sample. For example, it may be advantageous under certain circumstances to employ a bridging molecule between the small molecule and the protein. The bridging molecule will distance the small molecule from the protein and, thus, induce the formation of antibodies having greater specificity for the small molecule. It is also potentially possible that the antibodies which are evoked will recognize the bridging unit.

Where the immunoassay is based upon competitive binding inhibition principles, a second reagent (generally referred to as a "conjugate"), is prepared which mimics the immunochemical interaction of the analyte with an antibody (which is specific for the analyte). This analyte mimic generally comprises a hapten, or a chemically distinctive group which is immunochemically identical to a portion of the hapten, and a protein. The protein portion of this conjugate can be active (i.e. enzyme) or inert. Where the protein is inert, it may serve as a binding site for second antibody or a nucleation site for formation of a precipitating immunocomplex. Where the interaction of the conjugate and the antibody specific for a hapten of interest forms a precipitating immunocomplex, the relative concentration of this immunocomplex can be determined by standard light scattering and spectrophotometric techniques. The relative concentration of this precipitating immunocomplex will generally be inversely proportional to the relative concentration of the competing analyte present in the sample undergoing analysis.

One of the more serious impediments to the widespread adaptation of precipitin reaction based immunoassay methodology is the limited stability of the working reagent solutions (notably, the conjugate solution). The various attempts at overcoming the stability problems in working reagent solutions have not been successful and, thus, the commercial adaptation of this methodology to immunochemical analysis has been driven either by the unavailability of a sufficiently competitive method and/or the desire to enhance the test menu for a particular instrument. Notwithstanding the advances made to date, the available reagent systems (i.e. the latex based reagent systems, previously discussed) are only somewhat marginally more stable than the more traditional reagents used in immunoassay and each continue to suffer from the problems associated with abbreviated shelf -life and working solution stability.

SUMMARY OF THE INVENTION

The improved hapten modulated immunoprecipitin reaction of this invention provides a unique approach to overcoming many of the deficiencies in systems for determination of hapten concentration by measurement of a precipitin reaction utilizing turbidimetric monitoring techniques. The approach adapted by this invention accommodates many of the constraints placed upon reagent systems used in this type of measurement without sacrifice in accuracy, speed or reagent stability. It is these features which not only differentiate this invention from the past attempts at enhancement of prior turbidimetric analytical techniques, but also provide significant enhancement in dynamic analytical range and signal to noise ratio.

More specifically, the turbidimetrically monitored assay of this invention is based, in part, upon a unique set of reagent system parameters which (a) provides unexpected reagent stability and controlled formation of the precipitating immunocomplex and, (b) translates into an expanded dynamic analytical range thereby providing the clinician with needed information to make informed judgments as to efficacious and toxic levels of therapeutic agents. This assay is particularly well suited for monitoring therapeutic drug levels of patients with a degree of accuracy, speed and over a dynamic analytical range at least comparable to the more traditional clinical chemistry and immunochemical methods; and, with a degree of accuracy, speed and control not previously attainable by turbidimetric rate inhibition techniques. These enhancements are attributable to proper selection of antibody, precision engineering of a companion reagent (conjugate) for the antibody, and adjustment in the salt concentration of the reaction environment to control/tune the rate of precipitin reaction to provide a dynamic analytical range which affords both low end and high end sensitivity. The reagent systems of this invention permit the adaptation of a hapten modulated turbidimetric rate inhibition assay to automated spectrophotometer based instrumentation without system or equipment modification.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a bar graph illustrating the correlation between the dynamic analytical range of an immunoassay based upon a phenobarbital (hapten) modulated immunoprecipitin reaction (which is momtored by turbidimetry), and the concentration of phosphate buffer in the reaction environment.

Fig. 2 is a bar graph illustrating the correlation between the dynamic analytical range of an immunoassay based upon a phenobarbital modulated immunoprecipitin reaction (which is momtored by turbidimetry), and the concentration of an enhancer (polyethylene glycol).

Fig. 3 is a graphical illustration of the effect of bridge length on sensitivity of a phenobarbital apoferritin conjugate, using two different monoclonal antibodies, A4D6 -2512 and A3D3-2I.

Fig. 4 is a graphical illustration of the effect of the phenobarbita apoferritin mole ratio on assay sensitivity, using two different monoclonal antibodies, A4D6 -2512 and A3D3-2I. Fig. 5 is a graphical illustration of the dynamic anlaytical range of an immunoassay based upon phenobarbital modulated immunoprecipitin reaction which is monitored over a time course by turbidimetry.

Fig. 6 is a graphical illustration of the dynamic analytical range of an immunoassay based upon a phenobarbital modulated immunoprecipitin reaction having a linear (correlation coefficient of the regression line = .994) dose response curve.

DESCRIPTION OF THE INVENTION INCLUDING PREFERRED EMBODIMENTS

Preliminary to a detailed discussion of this invention, it would be helpful to initially define the following terms and phrases.

The phrase "turbidimetric rate inhibition assay", "turbidimetric rate inhibition immunoassay", and "hapten modulated immunoprecipitin reaction" and acronym "TRIA" as used herein are intended as descriptive of an analytical protocol which utilizes free analyte (hapten), at an unknown concentration, in a sample to modulate the rate of an immunoprecipitin reaction. The free analyte is competitive with an analyte mimic for binding to an immunoreagent (i.e. antibody), thereby preventing the formation of a precipitating immunocomplex between the antibody and the analyte mimic The complex formed between the analyte and the antibody are soluble and, thus, the higher the concentration of analyte in the sample, the lesser the quantity of precipitating complex formed.

The terms "nephelometric" and "nephelometry" as used herein are intended as descriptive of a technique for monitoring particulate dispersions by initially irradiating the dispersion with a coherent light beam and monitoring the intensity of the reflected beam at a 90° angle from the incident radiation.

The terms "spectrophotometric", "spectrophotometry", "turbidimetric" and "turbidimetry" are all used interchangeably herein as descriptive of a technique for monitoring the optical density of a particulate dispersion utilizing conventional photometric techniques and equipment. More specifically, these terms describe a monitoring system which measures the amount of light transmitted through a reaction cuvette. The value of light transmission which is reported is based upon the value of the incident light from the source, less the amount of light which is attributable to the absorption and reflectance by the contents of the reaction cuvette. The extent or concentration of particulate matter determined to be present in the dispersion by this technique (in the context of the method of this invention), is preferably inversely proportional to the concentration of free analyte originally contained in the sample subjected to analysis.

The phrases "particulate dispersion" and term "dispersion" as used herein are intended as descriptive of die product of a precipitin reaction of a polyvalent antibody and a polyvalent haptenic compound. This reaction product consists of a network or latice formed between a number of immunocomplexes until the size (molecular weight) of the network exceeds the solubility product constant for this species within the fluid medium in which the assay is conducted.

The phrase "dynamic analytical range" as used herein is intended as descriptive of the range of concentration of an analyte (hapten) within a sample that encompasses a spectrum of values ranging from a imnimum efficacious quantity of an analyte to an upper level at which intervention or remedial action may be appropriate, hi the context of therapeutic drug monitoring, the dynamic analytical range will, of course, include the range value over which the drug is normally prescribed and ideally extends to a level at least double the recommended maximum therapeutic level to alert the clinician of potential toxic effects.

The phrases "reaction mixture" and "reaction envoironment" as used herein are intended as descriptive as the final mixture of reagents and sample in a reaction vessel (i.e. cuvette) which is both necessary and appropriate for performance of a hapten modulated turbidimetric rate inhibition immunoassay. The sample is combined with these reagents without prior dilution and the sample/total volume ratio in the reaction mixture is typically in the range from about 1:25 to 1:101.

The term "avidity" as used herein is intended as descriptive of the relative binding affinity of the antibody to the hapten and polyvalent haptenic compound for which the antibody is specific

Turning now to a discussion of the method of this invention, the criteria for performance of an immunoassay are many and varied. Depending upon the assay format and the sample composition, the values to be obtained will generally be related to the analytical techniques used in the collection of data. Li the context of the method of this invention, the immunoreagent characteristics, the relationship to one another and to the analyte, and the relative salt concentration of the reaction environment are critical to the modeling of a hapten (analyte) modulated turbidimetric rate inhibition assay having a dynamic analytical range having both low end and high end sensitivity. It is further understood that these immunoreagents, both individually and collectively, are useful in other analytical protocols which utilize immunochemical interactions as a basis for identification/quantification of an analyte in a sample.

The method of this invention, in part, involves the preparation (and selection) of immunoreagents specific for a particular analyte of interest and the use of such immunoreagents in a precipitin reaction; the rate of such reaction being modulated by the amount of free analyte initially present in a sample. Many of the general objectives/ parameters governing the preparation of such immunoreagents are known, however, until such materials are actually prepared and evaluated in the specific environment and under actual assay conditions, their compatability with one another and the assay environment cannot be assured. At a minimum, optimization is generally required. For example, in the raising of the antibodies, the immunogen used consists of an analyte mimic conjugated to a carrier protein through a bridging moiety. The relative length of the bridge moiety and the animal source of the carrier protein are consciously selected and differ substantially from die composition of the companion reagent (conjugate) for the antibody. The reasons for such differences are to increase specificity of the antibody for epitope which is indicative of the analyte of interest. The position of attachment of the bridging moiety to the haptenic compound is, of course, the same for both the immunogen and the conjugate to insure the presentation of a common epitope to the antibody. What is not, however, apparent is that the extent to which the length of this bridging moiety is constrained (on the low end) by the need to retain epitope identity (apart from the carrier protein) and, (at the high end) by steric considerations relating to the orientation of the epitope in a manner which is readily identifiable by the antibody. In each reagent system, these same general considerations will control, notwithstanding, that the engineering of the specific immunoreagents will be different from one analyte to another and for different position isomers of the same analyte. Table 1 is illustrative of a series of reagent systems which have been specifically engineered in accordance with the demands/criteria of the turbidimetrically monitored immunoassay of this invention. Each of these reagent systems are suitable for therapeutic drug monitoring for a specific, commonly prescribed, drug. The dynamic analytical range of the reagent system for each drug includes the normal therapeutic range, up to the level which is substantially in excess of die upper limit of the therapeutic range. The reagent systems which are exemplified are specific for theophylline (THEO); phenobarbital (PHENO); phenytoin (PHENY); gentamycin (GENTA); and, tobramycin (TOBRA). TABLE 1

PARAMETER THEO PHENO PHENY GENTA TOBRA

ASSAY FEATURES Dynamic Range (mg/L) 0-60 0-80 0-40 0-16 0-16

Max. rate (m Abs)/ minutes (±10%) 105 165 135 105 105

Serum/Tot. Volume 1:101 1:101 1:101 1:26 1:26

REAGENT SYSTEM COMPONENTS

CONJUGATE

Polymerization <10% <10% <10% <10% <10%

Conc (mg/ml) .19-.394 .19-394 .01-394 .002-.190 .002-.01

Protein** Apo Apo Apo Apo Apo

Mole Ratio 17-21 9-10 23-30 20-23 6-8

HAPTEN

Bridge 8 8 7 4 4

Attachment Site N-l N-l N-3 Random Random PARAMETER THEO PHENO PHENY GENTA TOBRA

ANTIBODY Antibody Yes Yes Yes Yes Yes

Titer (mg/mL) >0.02 >0.02 >0.02 >0.02 >0.02

Affinity 10l0 10l0 ιolO ιolO ιolO

Avidity - As defined above by Assay Features

BUFFER Protease Inhibitors Yes Yes Yes Yes Yes

Salt Cone 100-500 100-500 .100-500 100-500 100-500

PEG Type 6,000 6,000 6,000 6,000 6,000 (Mw)*

PEG 2.2-2.6 2.2-2.6 2.2-2. 2.2-2.6 2.2-2.6

Saline Cone 0.9% 0.9% 0.9% 0.9% 0.9%

Final Salt Cone (Mw) 150 150 150 150 150

Final Protein Cone >.2% >.2% >.2% >.2% >.2%

*Polyethylene glycol (PEG) 4000-20,000 or, altematively, Dextran @

100,000-200,000.

**Apoferritin (Apo) Through judicious selection and engineering of the antibody/conjugate pair, it is possible to effectively determine free hapten concentration of a fluid sample by measurement of optical density of a dispersion by conventional spectrophotometric techniques.

REAGENT SPECIFICATIONS

Conjugate Characteristics - Each of the reagent systems included in the above table consists of a monoclonal antibody and a companion reagent comprising a hapten protein conjugate having a precise ratio (ratio range) of haptenic functional groups attached to the protein. The protein component of this conjugate is preferably apoferritin, although other globular proteins having an organic or inorganic prosthetic group are also suitable. These globular proteins typically function in biological systems as transport/carrier proteins, have a compact tertiary structure, and a quaternary structure consisting of a sufficient number of tertiary units to produce a highly ordered macromolecule with a molecular weight of at least 100,000. It is, of course, understood that only those globular proteins which can form water soluble conjugates with a hapten (at the appropriate hapte protein ratio), are suitable as immunoreagents in the method of this invention.

As is further evident from this table, the determination of the precise ratio of hapten to apoferritin protein is also unique for each conjugate and is critical to its performance in the TRIA method of this invention. Where the ratio of hapten to apoferritin protein is below the preferred value, the relative rate of interaction with the antibody is too slow and may not result in the growth of a complex of sufficient size to form a precipitate. Conversely, where the ratio of hapten to apoferritin protein is in excess of the preferred ratio, the rate of immunocomplex formation is directly, rather than inversely, proportional to the amount of free hapten in the sample. Moreover, only when the correct ratio of hapten to apoferritin protein is employed does hapten inhibition of antibody/conjugate occur consistent with the preferred objectives of turbidimetric rate analysis. More specifically, the ratio of hapten to apoferritin protein in the conjugate is, thus, selected with reference to die anticipated concentration of an analyte and the potential range of deviation in concentration of analyte from the anticipated range. For example, the immunoreagents which are engineered for determination of theophylline are sensitive to detection of this drug within the therapeutic range and up to a level which is twice the upper limit of the recommended therapeutic range.

Antibody Characteristics - One of the principle criteria in the selection of monoclonal antibody for this type of assay is a slight bias for binding to the analyte over binding to the conjugate. As noted previously, the antibody is preferably present in the reaction environment at a slight excess, relative to the available epitopic sites in the conjugate. The combination of these two factors, with respect to the antibody performance and availability, are indeed critical to the dynamic analytical range of a turbidimetric rate inhibition assay. The failure to satisfy either one or both of these requirements of antibody performance will materially diminish the dynamic analytical range of this invention, thus, rendering it of limited value and uncompetitive with alternative techniques in regard to the dynamic analytical range.

Salt Concentration - The relatively high salt concentration of the reaction environment has been unexpectedly found to enhance the affinity of the monoclonal antibody for the analyte. The source of salt can be the fully diluted conjugate reagent solution. In the preferred embodiments of this invention, both the enhancer for the precipitin reaction and the conjugate are combined in a fully diluted single reagent. In order to stabilize this reagent and prevent spontaneous agglutination in the presence of the enhancer, at least about 150 mM sodium chloride, and at least about lOOmM buffer (phosphate buffer) are added to this conjugate/enhancer solution. The salt concentration relative to the conjugate in the conjugate/enhancer solution is selected to effectively and substantially neutralize the charge of the conjugate and thereby enhance the stability of this reagent against spontaneous agglutination. The high salt concentration also modulates the precipitin reaction by inhibiting the effect of the enhancer upon the immunocomplex, thereby controlling the rate of formation of the precipitating immunocomplex, and thus extending the dynamic analytical range of the assay.

The stability of the conjugate in the presence of the enhancer is also believed to be attributable, in part, to the nature of the apoferritin protein. This protein component of the conjugate is resistant to degregation by the enhancer, and yet when reacted with antibody, is responsive to relative rapid, yet controlled precipitin formation.

Assay Characteristics - As noted above, the monoclonal antibodies selected for use in the method of this invention are chosen for their high titer and their preferential affinity toward the free hapten (analyte). More specifically, in order for the free hapten from the sample to be effective in the inhibition of coupling of the antibody to the conjugate, the antibody is selected to have a slight bias toward the free hapten. Thus, once the sample, conjugate reagent and monoclonal antibody are combined in a suitable reaction environment, the precipitin reaction will proceed at a controlled rate. The free hapten from the sample competes with the conjugate for the antibody, displacing some of the conjugate from the available sites on the antibody. Such displacement of the conjugate by the free hapten will reduce the amount of precipitate in dispersion, with a corresponding reduction in the optical density. The rate of such displacement can be correlated with the relative concentration of free hapten in the resulting dispersion. The concentration of free hapten, under the preferred assay conditions of this invention, is inversely proportional to the optical density of the dispersion. As is evident from the above discussion, proper reagent selection and engineering of immunoreagents is determinative of the sensitivity and dynamic analytical range of the method of this invention. The reagent system parameters set forth in Table 1 represent the optimization of a combination of parameters for the individual components of each of these reagent systems; and, their relative concentration to one another in an immunoassay which is based upon turbidimetric monitoring of a hapten modulated immunoprecipitin reaction. The interrelated matrix of factors/requirements of TRIA are both complex and unpredictable. Unfortunately, in such complex systems, a change in one of the parameters of a component of the reagent system impacts a number of other variables (both reagent and process). As this refinement of the parameters of the individual components proceeded, it was surprisingly discovered that the salt concentration of the reagent and of the reaction mixture was critical both with respect to reagent stability and for control over the kinetics of the precipitin reaction. More specifically, it was discovered that relatively high total salt concentrations in the drug- apoferritin reagent solution stabilized this conjugate against agglutination/degradation by precipitin reaction enhancer compounds, thus, affording the capability of preparing a single, fully diluted reagent solution containing both of these components of the reagent system. This relatively high total salt concentration also afforded the additional unexpected capability of controlling/modulating the formation, in the reaction mixture, of an immunocomplex between the conjugate and antiserum in the presence of free drug. The result is an essentially linear relationship between the optical density of the reaction medium and the rate of formation of the precipitating immunocomplex.

The figures which accompany this disclosure illustrate the range of concentration of phosphate buffer in the conjugate reagent solution (Fig. 1); and, the range of concentration of polyethylene glycol (6000 MW) enhancer in die conjugate reagent solution (Fig. 2). It is noted that the values depicted in these figures are indicative of the relative concentration in the conjugate reagent and differ from me values in Table 1. The range of values for each of these components set forth in Table 1 is their concentration in the reaction mixture. Optimization of length of the methylene bridge which connects the drug to the apoferritin was then varied to achieve optimum sensitivity (at both the low and high end of the dynamic analytical range). Fig. 3 illustrates the optimization of bridging length for two phenobarbital/monoclonal antibodies having different hapten/apoferritin protein ratios as optimum. Figs. 3 and 4 illustrate the correlation between such ratios and assay sensitivity. Fig. 4 also demonstrates that variations in bridge length will determine the performance of an antibody/conjugate pair in an assay, even where the ratio of drug:apofeπitin is the same. Once all these parameters of this matrix have been properly defined, then, and only then, can the rate of the precipitin reaction be controlled (i.e. essentially linear) and the dynamic analytical range of the resultant turbidimetric immunoassay be expanded to encompass both the normal therapeutic levels of the drug in the biological sample, but also elevated levels which may require clinical intervention. Fig. 5 illustrates the change in absorbance of a dispersion, having a known concentration of phenobarbital, approximately every eight (8) seconds, over a period of 160 seconds. As the concentration of drag in the reaction medium increases, the plot of absorbance becomes increasingly linear. This feature of the assay can be demonstrated in another way by calculating the correlation coefficient of the linear regression equation using calibrator values over the full dynamic range for phenobarbital. When these values are plotted (Fig. 6), the correlation coefficient approaches perfection at (1.0); a straight line; the phenobarbital value at .994 demonstrates a straight line within the allowed experimental precision of the assay.

In order to more fully appreciate some of the unique features of this invention, the following examples are provided. Parts and percentages appearing in such examples are by weight unless otherwise indicated. Apparatus and techniques used in the preparation of reagents and in dieir evaluation in the method of this invention are standard or as hereinbefore described. EXAMPLE 1

A reagent system was prepared for determination of phenobarbital levels in a biological sample by a drug modulated immunoprecipitin reaction which was to be momtored by turbidimetry. The specification for the individual reagents is set forth in Table 1. The individual components of the reagent system are initially prepared as two separate solutions: (a) an antiserum which had been produced by a clone that was sensitized to an immunogen consisting of phenobarbital conjugate at the 1 -position to keyhole limpet hemocyanin (KLH) through a methylene bridge having three carbon atoms; and, (b) a phenobarbital/apoferritin conjugate solution. Each of these individual reagent solutions also contains about 0.1% sodium azide and from about 100 to 150 mmol/L phosphate buffer. An enhancer for the precipitin reaction, 6.2% polyethylene glycol (6000 MW), is included in the phenobarbital/apoferritin conjugate. The phenobarbital/apoferritin conjugate solution is fully diluted and is surprisingly stable for extended periods of time (up to 8 months), even after the reagent container has been opened.

A turbidimetric rate inhibition immunoassay for a serum sample containing a known concentration of phenobarbital, utilizing the above reagent system, is performed on an automated clinical chemistry analyzer (DACOS Chemistry Analyzer - Coulter Electronics, Inc., Hialeah, Florida). The relative concentration of phenobarbital in the sample is momtored kinetically and the slope data correlated with a standard curve stored in the analyzer's microprocessor. The value reported by the analyzer conforms to the level of the phenobarbital known to be present in the sample.

The foregoing description, example and accompanying figures are intended as illustrative of a number of the preferred embodiments of the method. The metes and bounds of the invention are set forth in die following claims.

Claims

1. hi an immunoassay of a fluid sample wherein the analyte is a hapten and the reaction environment is monitored turbidimetrically, said immunoassay being based upon a hapten modulated precipitin reaction involving the competitive immunochemical interaction of a hapten and hapten mimic with an antibody in the presence of a precipitin reaction enhancer, said assay being characterized by:

controlling the rate of the precipitin reaction by adding to said fluid sample an enhancer modulating effective amount of a mixture consisting essentially of physiological and buffering salts, the relative concentration of salts to enhancer effectively optimizing the kinetics of the precipitin reaction so as to produce an essentially linear change in optical density of the fluid sample over the dynamic analytical range of the hapten.

2. The immunoassay of Claim 1, characterized in that the enhancer is selected from the group consisting of polyethylene glycol and Dextran.

3. The immunoassay of Claim 1, characterized in that the mixture of physiological and buffer salts consists essentially of about 150mM sodium chloride and at least about lOOmM buffering salts.

4. The immunoassay of Claim 1, characterized in that the modulation of rate of the precipitin reaction by the mixture of salts approaches zero order kinetics.

5. The immunoassay of Claim 1, characterized in that the hapten is a therapeutic agent and the dynamic analytical range of the assay is inclusive of a minimum therapeutic level for said agent on the low end of the range, and a toxic level for said agent on the upper end of the range.

6. The immunoassay of Claim 1, characterized in that the antibody is a monoclonal antibody and the hapten mimic consists essentially of a polyhaptenic molecule having a plurality of functional epitopic sites on a globular carrier protein, the ratio of functional epitopic sites to carrier protein being within a range which enables free hapten modulation of a precipitin reaction, between the antibody and the companion reagent, over the dynamic analytical range for the hapten.