EP0324015A1 - Analyse amelioree de l'inhibition du taux turbidimetrique pour des haptenes - Google Patents

Analyse amelioree de l'inhibition du taux turbidimetrique pour des haptenes

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Publication number
EP0324015A1
EP0324015A1 EP88906612A EP88906612A EP0324015A1 EP 0324015 A1 EP0324015 A1 EP 0324015A1 EP 88906612 A EP88906612 A EP 88906612A EP 88906612 A EP88906612 A EP 88906612A EP 0324015 A1 EP0324015 A1 EP 0324015A1
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EP
European Patent Office
Prior art keywords
hapten
immunoassay
antibody
reagent
assay
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP88906612A
Other languages
German (de)
English (en)
Other versions
EP0324015A4 (en
Inventor
Frank J. Lucas
Juan Bedevia
Mark L. Shenkin
Brian J. Mcrae
James H. Carter
Harold R. Crews
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Coulter Electronics Inc
Original Assignee
Coulter Electronics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Coulter Electronics Inc filed Critical Coulter Electronics Inc
Publication of EP0324015A1 publication Critical patent/EP0324015A1/fr
Publication of EP0324015A4 publication Critical patent/EP0324015A4/en
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding

Definitions

  • 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.
  • conjugate a polyantigenic molecule
  • 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.
  • 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).
  • the sample is contacted with a solid phase having adsorbed reagent specific for interaction with one or more sample components.
  • the fluid phase of the sample and sample constituents which remain dissolved and suspended in the fluid
  • 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.
  • the detectable species 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.
  • 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.
  • the signal to noise ratio can be, and usually is, adversely affected by the environment in which the detectable species is monitored.
  • the monitoring of the detectable species can encounter interference from the other constituents which are endogenous to the sample.
  • 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.
  • 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 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.
  • 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.
  • 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
  • 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 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.
  • 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.
  • 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.
  • the relative binding affinity of the reagents 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a phenobarbital (hapten) modulated immunoprecipitin reaction which is momtored by turbidimetry
  • 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.
  • turbidimetric rate inhibition assay 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.
  • nephelometric and nephelometry 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.
  • spectrophotometric spectrophotometry
  • turbidimetric turbidimetry
  • spectrophotometry spectrophotometry
  • turbidimetry turbidimetry
  • pillate 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.
  • 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.
  • 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.
  • a reaction vessel i.e. cuvette
  • 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
  • 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.
  • 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 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.
  • 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.
  • 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).
  • ASSAY FEATURES Dynamic Range (mg/L) 0-60 0-80 0-40 0-16 0-16
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • both the enhancer for the precipitin reaction and the conjugate are combined in a fully diluted single reagent.
  • 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.
  • 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.
  • 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.
  • a change in one of the parameters of a component of the reagent system impacts a number of other variables (both reagent and process).
  • 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.
  • FIG. 1 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.
  • 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:apofe ⁇ itin is the same.
  • 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.
  • 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.
  • 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.
  • KLH keyhole limpet hemocyanin
  • 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.

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Abstract

L'analyse améliorée de l'inhibition du taux turbidimétrique pour des haptènes utilise des anticorps monoclonaux et des conjugués polyantigéniques dans des proportions précises dans un régime de liaison compétitive modulée par des haptènes, la concentration relative d'haptènes étant inversement proportionnelle au degré d'absorption d'un mélange de réaction attribuable à un immunocomplexe de précipitation. La plage analytique dynamique et le rapport signal/bruit de cette analyse sont uniques pour l'analyse turbidimétrique d'haptènes. Cette analyse est également unique par la spécificité des réactifs et la stabilité du réactif même lorsqu'il est entièrement dilué.
EP19880906612 1987-07-21 1988-07-07 Improved turbidimetric rate inhibition assay for haptens Withdrawn EP0324015A4 (en)

Applications Claiming Priority (2)

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US7642587A 1987-07-21 1987-07-21
US76425 1987-07-21

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EP0324015A1 true EP0324015A1 (fr) 1989-07-19
EP0324015A4 EP0324015A4 (en) 1990-11-07

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JP (1) JPH02500133A (fr)
AU (1) AU2128388A (fr)
WO (1) WO1989000694A1 (fr)

Cited By (1)

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CN108709993A (zh) * 2018-04-02 2018-10-26 深圳上泰生物工程有限公司 一种胶乳增强免疫比浊检测试剂盒及其制备和检测方法

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DE4034509A1 (de) * 1990-10-30 1992-05-07 Boehringer Mannheim Gmbh Immunologisches praezipitationsverfahren zur bestimmung eines bindefaehigen analyten sowie reagenz zur durchfuehrung dieses verfahrens
DE4124324A1 (de) * 1991-07-23 1993-01-28 Merck Patent Gmbh Verfahren und mittel zur turbidimetrischen oder nephelometrischen bestimmung von analyten
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WO1989000694A1 (fr) 1989-01-26
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EP0324015A4 (en) 1990-11-07

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