JPH02500133A - Improved turbidity measurement suppression assay method for hapten - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Improved turbidity measurement suppression assay method date for hapten Separate from 1 from G. The present invention is directed to a method. In particular, the present invention provides a hapten-modulated antagonistic binding immunoassay (h apten modulated compatible blinding immunoassay), in which case the relative concentration of hubtan is A multi-antigen molecule (hereinafter referred to as “ by the formation of an immune complex between the conjugate (referred to as J). It is determined by measuring the intensity of the absorbance produced. Dynamic analysis of this test The range and signal-to-noise ratio are unique for hapten turbidity analysis. Also, The present invention is unique in the specification of reagents used in this method.

2. Minomai Omega Sodama The manifestation of immunochemical interactions between antigenic materials and antibodies depends on many variables. Can be recognized or cannot be recognized. This variable is compatible with the above The environment in which the interaction occurs; the relative size of the product complexes; the product complex in the reaction medium relative solubility of the complex; fluorescence of the complex; and/or label, indicator or tag ( tag) can be introduced into the production complex, which can be used with or without the aid of instrumentation. It includes the presence of some other agents that allow it to be detected independently.

Although all of the various proposals for immunoassays are more or less effective, However, there are some trade-offs for speed, sensitivity, convenience, and/or safety. It has inherent limitations that inevitably arise. In particular, homogeneous immunoassay techniques are Tests that are interested in the presence of other sample components that contribute to or mask the observed phenomenon. Contains a description of the detectable species that indicates the body. In some cases, detectable species or the label can be monitored directly in the case of fluorophores; Additional reagents such as those needed to demonstrate the presence of other visible labels (i.e., enzymes) It is. In heterogeneous immunoassay techniques, a sample is combined with one or more sample components. A solid phase is contacted with a specific adsorption reagent that interacts with it. The reagent bound to the solid phase is 1 or the liquid phase of the sample (and dissolved in the liquid) after interacting with two or more sample components. and sample components remaining in suspension) to the solid phase (sample components that bind to reagents in the solid phase). minutes). Additionally, the solid phase is washed to remove any unbound material. This wash The purified liquid can be collected, combined with the liquid fraction of the sample, or simply discarded. be able to. In this procedure, dissolved analytes (liquid phase) or undissolved analytes (solid phase) This results in two mutually exclusive fractions containing . After that, one or both These fractions can be analyzed for the presence of analytes of interest. . If the detectable species (label) that indicates the analyte of interest is a fluorophore, the liquid The phase or solid phase is irradiated with a monitored excitation energy and its fluorescence emission.

When the label is an enzyme, the substrate is first placed in a liquid or solid phase and the species to be detected. Contact with the reaction atmosphere is required, which is monitored for production.

Other types of heterogeneous immunoassays include the use of radioactive isotopes as labels and liquid phase/solid phase It includes the following dividing means for dividing the phase into two mutually exclusive fractions.

In particular, the analyte of interest and the radioactive isotope used for identification are first brought into contact; interact with insolubilized immunoreagents in solid phase; or used for identification. mutually with a second antibody used to precipitate an immune complex containing a radioactive isotope. Let it work. The amount of radioactive isotope present in the solid phase used for identification is determined by scintillation tion counter and correlate the counts with the concentration of analyte in the sample.

All the above-mentioned suggestions for immunoassays are compromised and/or have unique It has its drawbacks. In many cases (with the exception of RIAs), these shortcomings are Replaced by loss of anger. In particular, virtually all immunoassays are They are configured as described above, with the exception of For example, in a homogeneous immunoassay, the Monitoring of species that can be extracted Other components that may be present in the sample interfered with. In heterogeneous immunoassays, similar constraints that monitor the liquid phase are used. receive. Monitoring of detectable species bound to a solid phase completely solves this problem. , especially if the indicator is a fluorescent compound and the solid phase is itself fluoresce within the excitation or emission spectrum of the species that can be detected.

As mentioned above, immunoassays based on the detection of radioactive isotopes used for identification does not suffer similar signal-to-noise ratio losses in the normal time course of the Niter test interval. However However, this assay requires expensive equipment to measure detectable species and is rigorous. Regulations are generally desired in connection with the processing of isotopic materials required to conduct assays. Not good.

The immunoassay systems described above have the ability to obtain qualitative and quantitative results.

Additionally, these assays are performed as end-point reactions or as dynamic reactions (reaction rates). be able to. Of course, dynamic tests require the use of velocity (slope) measurement instruments. and to subtract interferences that mask low levels of analyte. dynamic instrument The use of background signal contribution monitors the dynamic analysis range of the instrument. Preferred for systems with high backgrounds, provided that they are not exceeded. Yes.

For these analyses, if qualitative results are required (cell table for blood type) A simple coagulation assay is used to identify surface markers. ) is appropriate. Coagulation/agglutination type assays are suitable for soluble, particulate, and latte samples. has been developed for stable emulsions containing x beads. for this test The reagents, including the latex beads used, are present as stable suspensions in a liquid environment. There is. In a typical analytical environment using latex reagents, interactions and/or changes in the environment resulting from the presence of the analyte of interest. It can change the dispersion stability (agglomeration). Latex bead reagent Agglutination assays have been developed for a number of serological determinants. In many cases , these tests are qualitative.

Nephelometric or turbidity measurements are commonly used when quantitative analysis of particle dispersions is required. limited. Nephelometry and turbidity measurements are performed using photooptical (ph) measurements of the same particle dispersion. It cannot be easily replaced by technology with optical characteristics. Each analysis method is immunochemical can be used to measure precipitation reactions based on physical interactions, but These relative sensitivities depend on the specific immunoreagent used, its relative reactivity (binding activity), and It is highly sensitive to its relative concentration in the liquid environment in which the analysis is performed.

In general, turbidimetry involves illuminating a dilute dispersion with a coherent light source (i.e., a laser). radiation and the measurement of the intensity of the reflected light at 90° to the incident radiation. immunity If applicable, this analytical technique is insensitive to antibody concentration and titer. Yes. In contrast, turbidity measurement is based on ordinary spectral analysis (optical density/absorption of dispersion). However, the requirements for reagent specifications and performance are extremely high. and the potential for a wide range of dynamic analysis ranges is proposed. become. Regarding the potential application of turbidity monitoring technology to immunoassays, see This has been attempted previously (e.g., U.S. Pat. No. 4,604,365). ). In addition, accurate turbidity measurement monitor immunoassay performance is achieved with latex reagent systems. (e.g. U.S. Pat. No. 4,581,337; 4.5 24.025; 4.521,510; 4,47? , 346 i4.460,6 95 and No. 4,401,765). These U.S. patents include certain single-click patents. Lone antibodies (U.S. Pat. No. 4,524,025), and polymer particles (U.S. Pat. No. 4,401,765). ing.

The factors involved in light scattering and absorbance measurements of precipitation reactions based on immunochemical interactions. The principles, factors and constraints have been known for some time. Paraling (Paulin g), a series of papers co-authored by Mr. Pressman and others. 1 on the inhibitory effect of haptens on the precipitation of antiserum/small molecule complexes. It states that it was reported in the early 940s. In this research paper, it takes Precisely determine the amount of free hapten required for inhibition to control free hapten in precipitation reactions. Accurately predicts the effect of marten (Paraling L0 et al. rJ, Am, Ch. eIIl, Soc, J64: 2994-3002 (1942), In the late 1940s, research by Bararing et al. progressed, and antibody/antigen precipitation reactions The principle behind this has been elucidated. Their research during this period allowed precipitated immune complexes to Activities that alter the amount of precipitate by solubilizing and/or adding components to the reaction environment. Concentrating on the ability of detached hapten (Bubbling Kami0 et al. rJ.

8m, Chem, Soc, J64: 3010-3014 (194 2) and Pauling L0 et al. rJ, Am, Chew, Soc, J 64: 3015-3020 (1942).

In the early 1950s, optical scattering for the measurement of sedimentation reactions based on immunochemical interactions Random technology began to be directed towards the production of equipment. Light on antiserum/antibody interactions The scattering properties should first be observed at 90° to the incident radiation (Goldherg R, J, et al. rJ, of Tmmunol, J 66: 79-86 (1 951) was reported, followed by observation of light scattering at a low angle (Tottei et al. Mr. P. and others rAdvances in Protein Chemistry” 6:35-121 (1952). Low angle by Tottey et al. Light scattering measurements are performed at 90° to the incident radiation (at least in the systems studied). ) are reported to be even more sensitive. In the early 1970s, turbidimetry A device is being developed to attempt automated monitoring and measurement of immunoprecipitation reactions (data Wiss G, E. R. Immunology 20 (1970) 779).

Previously, instrumental precipitation reactions have been difficult to detect. Furthermore, This reaction is extremely slow and requires minutes to hours for detection. This sedimentation Certain polymers are used to increase the speed and size of particles formed in the reaction. It has been confirmed that the growth of particles is promoted by adding it to the reaction medium ( Harrington J, C, et al.’Iw+IIIunoche@1stry J 8 (1971) 413-421) *By promoting the use of equipment, Of interest in immunoprecipitation reactions, which are clearly preferred, is the balance of a set of 10 consecutive It is to pass the target. In 1974, Cohen introduced single light scattering spectroscopy and turbidity. have reported that the initial stage of the agglutination reaction can be monitored by measurement (Cohen R , J, et al. I+++ munoche-mistry J12 (1974 ) 349), the model system used in Cohen's research The combined particles were treated with bovine serum albumin (BSA) and a flocculant, an antiserum against BSA. It was made of polystyrene latex coated with

In 1979, a competitive turbidity immunoassay for theophylline was developed for the first time. In the literature, Mr. Shikawa T8 says, “Cl1n.

Published in Che IIl, Acta, J 91 (1979) 59-65). It is. Around the same time, Beckman Instruments Inc. (B Eckman Instruments, Inc.), California .

Fullerton offers nephelometry equipment and testing equipment for diagnostic testing on biological fluid samples. Introducing the drug system. Beckman Inn is useful for use with this system. Reagent system from Stolment Twin Corporation (product name “IC3Tl”) IC5 “Reagents” are therapeutic drugs (i.e. Contains a test kit to determine serum levels of (ofilin). Theophylline The test kit contains antiserum against theophylline and theophylline conjugated The body contains a theophylline derivative covalently bound to the equine apoferritin carrier protein. I'm here. The ratio of theophylline to carrier protein has not been determined, and theophylline Analytical experiments to test the derivatization of phosphorus, which has not yet been clarified The plan (analytical protocol) is from the above document rc1in, Chem, Acta,” 91 (1979) 59-65. The report was substantially the same as the Nijikawa report. Beckman test for theophylline The details of the proposed assay design in the package insert for the drug are not actually approved. It is acceptable. This insert contains instructions for first diluting ('1:6) the sample to be analyzed. )L, then diluted (1:6) in another dilution step. This rare Interference has three purposes: (a) interference of materials endogenous to the sample (signal-to-noise ratio); (b) one or more reagents and one or more components (test reducing interference (signal to noise ratio) resulting from cross-reactivity with (other than the body); c) bring the monitored response within the dynamic analysis range of the response for a given instrument; There are times when I try to do so.

Also, regarding the application of different automated monitoring techniques for immunoprecipitation reactions. There are documents that describe it (Deverill 0'J, Immunol, Met h, J 38 (1980) 191-204) - This document describes detection/monitoring. Various methodologies historically used for ring immunoprecipitation reactions have been described. , and direct measurement of turbidity using a high-grade UV spectrophotometer is useful for liquid-phase immunoprecipitation. The most sensitive optical method for the characterization of Providing a law is being discussed.

Unfortunately, the development of reagents for immunoprecipitation reactions used in analytical tests is limited to the development of equipment. It's later than that. Immunoprecipitation Reagent System for High Throughput Instrument Environments To establish immunoprecipitation reactions, immunoprecipitation reactions can be performed using existing clinical analyzers. lyzer), and the equipment was used for these immunoprecipitations. Be sure to specifically design the reagents to accommodate them. Or again, the latter is attractive The reagent system should be able to work within the operating parameters of the existing instrument. It can also be done. Therefore, the developer of this reagent system is This reagent system can be used in a turbidity monitoring environment. It is designed to.

One system that has recently appeared in this field is the DuPont ACA clinical analyzer operation. This system is a reagent system developed for U.S. Pat. 401.765 and 4.524.025. Deepon's reagent System includes monoclonal antibodies and hapten/latex particle conjugates . The performance characteristics of this type of reagent system are essential to the instrument environment in which it is intended to be used. It is necessary to adapt it naturally. However, once made as a working solution ( (diluted) hapten/latex particle reagents have a short shelf life (generally less than 8 hours). (below), therefore this reagent should be prepared on a daily dosing basis and only a limited amount should be prepared for that purpose. There is a need to.

In addition, the above reagent system is similar to that prepared using hapten/carrier proteins. There are restrictions. The reagent system described in U.S. Patent No. 4.604.365 system is representative of the conventional system described above. described in this U.S. patent specification. The habten/carrier protein contained in the protein can be made inherently more stable and soluble. However, its use in an instrumental environment necessarily results in a polymerization precipitation reaction "enhancer". is expected to be added. These enhancers also inhibit the formation of precipitated immune complexes. Although attempts to promote spontaneous (automatic) aggregation of hapten/carrier proteins and and/or degradation of the hapten/carrier protein.

In addition to reagent stability, U.S. Pat. In the synthesis of reagents for analytical experimental design including spectrophotometric monitoring of We've focused on a few factors that need to be considered. These factors are , which necessarily has a substantial sensitivity to it, along with the stability of the reagent system. . The definition of operational/functional reagent parameters is generally known. four pieces 4,604.3. No. 65 accurately describes some Habten/H5A carrier protein systems. ing. Reagent system dynamics are specific to a specific assay format. ssay format), specimen type and monitoring system. How it varies depending on your needs/requirements can be determined from the above US patent specifications and other documents. it is obvious. Reagent parameters for immunoprecipitation reactions (monitored by turbidity measurements) relative binding affinities of reagents (antibody and analyte simulants or Putene analogues) are generally critical, as are the compositions of the analyte simulators. The specimen The ability to elicit an immune response within a sensitized host is generally limited when it is a "small molecule". cannot occur; therefore, sensitization of the host to small molecules (commonly referred to as a "carrier protein"), and This is achieved by utilizing this hapten/protein compound as an immunogen. Up As discussed, the response to this immunogen is similar to that of the small molecular components of the immunogen. or trigger antibodies against parts of the protein molecule. Therefore, the protein used in this immunogen Antibodies that select white matter and form on the protein components of this complex (if any) , an organism that does not cross-react with any proteins present in the sample that will ultimately be subjected to the immunoassay. Experience in the engineering industry has shown that the preferred free drug inhibiting antibodies (free drng 1 inhibited antibodies) to bovine serum albumin (BSA) It is implied that this occurs in response to an immunogen containing a conjugated hapten.

Additionally, the means by which small molecules bind to proteins will depend on the nature of the antibodies raised in the host sample. For example, in a certain environment, a cross-linking molecule between a small molecule and a protein It can be used advantageously. Cross-linking molecules separate small molecules from proteins, allowing them to reach the drinker. induces the formation of antibodies with high specificity for small molecules. It also happens It is potentially possible to confirm that the antibody is a bridging unit.

If the immunoassay is based on the competitive binding/inhibition principle, the analyte and antibody (relative to the analyte) Create a second reagent (commonly referred to as a conjugate) that mimics the specific (unique) immunochemical interaction . Generally, this analyte simulant is immunochemically identical to the hapten or portion of the hapten. contains chemically unique groups, and proteins. The protein part of this conjugate can be active (eg enzymes) or inactive. If the protein is inactive , the protein acts as a binding site for a second antibody or forms a precipitating immune complex Acts as a nucleation site. conjugates, and specific antibodies against the hapten of interest. If the interaction of the body forms a precipitated immune complex, the relative concentration of this immune complex The intensity can be measured by standard light scattering and spectrophotometric techniques. in general, The relative concentration of this precipitated immune complex is determined relative to the competing analyte present in the sample being analyzed. Inversely proportional to concentration.

One important point in the widespread use of immunoassay methodology based on precipitation reactions A major hindrance is the limited stability of the working reagent solution (particularly the conjugate solution). work trial Many attempts to overcome stability problems in drug solutions have not been successful, hence this The general adaptation of the methodology to immunochemical analysis is limited by the unavailability of sufficient competitive methods. performance and/or the need to enhance testing programs for specific equipment. It is being promoted more. Although progress has been made to this day, it is still difficult to obtain (i.e., the latex-based reagent system described above) It is only slightly more stable than many common reagents used in immunoassays; Problems related to short shelf life and working solution stability are constantly encountered.

The next day The improved hapten-denatured immunoprecipitation reaction of the present invention uses turbidity measurement monitoring technology. In a system for measuring hapten concentration by measuring precipitation reactions using It is the only solution that overcomes many drawbacks. Solution adapted by the invention can perform this type of measurement without sacrificing accuracy, speed or reagent stability. The aim is to accommodate the many constraints placed on the reagent systems used in the field. these The characteristics of this technology are not only distinguishable in improving conventional turbidity measurement and analysis techniques, The goal is to achieve significant improvements in analysis range and signal-to-noise ratio.

In particular, the turbidity monitor assay of the present invention provides a method for (a) unexpected reagent stability and sedimentation. Controlled formation of immune complexes and (b) translation into an expanded dynamic analytical range for clinicians Information judgment regarding the effectiveness and toxicity level of therapeutic drugs (inforu+ed) A unique sample system that provides the necessary information to create This is due to the meter set. In particular, this test is accurate, fast and Dynamic analysis range at least comparable to many traditional clinical chemistry and immunochemistry methods and turbidity measurement speed control wave i-nete provides accuracy not previously achieved. Monitor patient drug levels with ease, speed, and control. to confederate to. These improvements include proper selection of antibodies, partner reagents for antibodies (com Accurate processing of panion reagents (conjugates) and lower and upper limits Reaction rings that control/harmonize the rate of precipitation reactions to obtain a dynamic analysis range that provides maximum sensitivity. It is characterized by the adjustment of the salt concentration at the boundary. The reagent system of the present invention is capable of measuring hapten-modulated turbidity. Velocity suppression assays can be applied to instruments including automatic spectrophotometers without system or equipment changes do.

Embryo l4j11 arrogant skin Figure 1 shows phenobarbicur (hapten) modulated immunoprecipitation reaction (monitored by turbidity measurement). Dynamic analysis range of immunoassay based on monitoring) and phosphate buffer in reaction environment It is a bar graph showing the relationship between the concentration of

Figure 2 shows phenobarbicool-modulated immunoprecipitation reaction (monitored by turbidity measurement) Dynamic analysis range and enhancer (polyethylene glycol) for immunoassay based on It is a bar graph showing the relationship between the concentration of

Figure 3 shows two different monoclonal antibodies A, D, -2512 and a, o, -21. Crosslink length in susceptibility of phenobarbir/apoferritin conjugate using This is a graph showing the effect of

Figure 4 shows two different monoclonal antibodies A, D, -2512 and AJs-21. The effect of the phenobarbital:apoferrin molar ratio on the assay sensitivity used is shown. This is a graph.

Figure 5 shows phenobarbicool changes monitored over time by turbidity measurements. 1 is a graph showing the dynamic analysis range of an immunoassay based on a controlled immunoprecipitation reaction.

Figure 6 shows a phenolic acid with a linear (correlation coefficient of regression line = 994'') dose-response curve. FIG. 2 is a graph showing the dynamic analysis range based on barbital-modulated immunoprecipitation reactions.

゛ shellfish A-H's i' day In order to provide a detailed explanation of the present invention, the following terms and phrases will be defined in advance.

The term "turbidity measurement rate suppression test" Hibitton assay) J, “Turbidity measurement speed suppression immunoassay (turb idimetric rate 1nhibition immunoassa y) J, and “hapten modulated immunoprecipitation reaction (hapten modula ted　1mluno-preceptive reaction)'' and and acronym rTRIAJ are active in samples at unknown concentrations. Describe an analytical experimental design that modulates the rate of an immunoprecipitation reaction using isolated specimens. It is intended to be Free analyte is an analyte model that binds to immunoreagents (i.e., antibodies). This inhibits the formation of precipitated immune complexes between the antibody and the analyte mock. To prevent. The complex formed between the analyte and the antibody is soluble and therefore The higher the concentration of analyte therein, the lower the amount of precipitated complexes that form.

The terms "nephelometric" and "nephelometric" (elometry) J first irradiates the particle dispersion with a coherent light beam and then By monitoring the intensity of the incident beam at an angle of 90° from the incident radiation. It attempts to describe techniques for monitoring particle dispersions.

The term "spectrophotometric" J, "spectrophotometric" (spectrophotometry) J, “turbidity” ric) J and “turbidimetry” J are all standard Optical analysis of particle dispersions using conventional photometric techniques and equipment It attempts to describe techniques for monitoring density, and It can be used as In particular, these terms permeate through the reaction cuvette. This describes a monitoring system that measures the amount of light. light to transmit The transmittance value of is based on the value of the incident light from the light source, and may be less than the amount of light. depends on absorption and reflection by the contents of the reaction cuvette. This technology (this invention the content of particulate matter present in the dispersion and determined by The concentration is inversely proportional to the concentration of free analyte originally contained in the sample to be analyzed. preferable.

The term "particulate dispersion" and the term “dispersion” refers to the product of the precipitation reaction of multivalent antibodies and multivalent hapten compounds. This is an attempt to explain. This reaction product has a large network size and molecular weight). Many times the product solubility coefficient for this species in the liquid medium being assayed is exceeded. It contains a mesh or lattice formed between several immune complexes.

The term “dynamic analytical range” J ranges from the minimum effective amount of analyte to the upper level that can adequately engage or treat the effect. attempts to explain the concentration range of an analyte (hapten) in a sample that encompasses the range of values for It is something to do.

In the context of therapeutic drug monitoring, the dynamic analytical range is of course range of treatment, and preferred maximum treatment that alerts clinicians to possible toxic effects. It encompasses a range of values that ideally can be increased to at least twice the level.

The terms “reaction mixture” and “reaction environment” refer to hapten-modulated turbidity measurement rate suppression immunity. into the appropriate reaction vessels (i.e., cuvettes) needed to perform the biochemical assay. It is intended to describe the final mixture of reagents and samples in the test.

The sample is mixed with these reagents without pre-diluting and the sample/samples are mixed in the reaction mixture. The total capacity ratio will generally range from about 1:25 to 1:101.

The term “avidity” refers to the hapten and It attempts to explain the relative binding affinity of antibodies to multivalent hapten compounds. Ru.

In the method of the invention, the criteria for performing the immunoassay can vary widely. I can do it. Depending on the assay format and sample composition, the values obtained are generally Relate to the analytical techniques used to collect the data. In connection with the method of the invention, The characteristics of the reagents, their relationship to each other and to the analyte, and the relative salt concentration of the reaction environment are Hapten (analyte) modulated turbidity measurement with dynamic analytical range including upper and lower sensitivities Making it critical for modeling velocity inhibition tests. Furthermore, these immunoreagents Other analytical experiments that utilize immunochemical interactions as a basis for the identification/quantification of analytes Can be used separately and together in planning projects.

The method of the invention allows for the preparation (and selection) of specific immunoreagents for particulate analytes of interest. ) and the use of this immunoreagent in a precipitation reaction; the reaction rate is , modulated by the amount of free analyte initially present in the sample. However, the above Many common objectives/parameters governing the preparation of immunoreagents are specific to such materials. No information is known until it is actually prepared and evaluated in a standard environment and under actual assay conditions. However, the compatibility with the other and the assay environment are unknown. to the lowest In general, optimization is required.

For example, in antibody raising, the immunogen used is cross-linked. It consists of an analyte mimetic conjugated to a carrier protein via a moiety. The relative length of the bridge and The animal sources of the carrier proteins and carrier proteins were consciously selected, and the combination of partner reagents (conjugates) for the antibody to be substantially different from the composition. The reason for this difference lies in the epitope representing the analyte of interest. This is because the specificity of the antibody is enhanced. The crosslinking part to the hapten compound The attachment moiety is, of course, the immunogen and conjugate that the common epitope presents to the antibody. The same is true for both. However, the extent of the length of this cross-linked part (lower limit) Due to the need to preserve epitope identity (apart from the carrier protein) and (at the upper limit) the orientation of the epitope to make it easier to identify with antibodies. It is not clear that this is constrained by three-dimensional considerations; despite this, , for each reagent system, these same general considerations may vary depending on the specific immunoreagent technique. techniques to differentiate one specimen from another, and to differentiate different parts of the same specimen. Table 1 shows the turbidity measurement monitor immunity of the present invention. A series of sample systems specifically treated according to assay requirements/criteria are shown.

The dynamic analytical range of the reagent system for each drug substantially exceeds the upper limit of the therapeutic range. It encompasses the usual therapeutic range up to the non-therapeutic range. As a reagent system, Theophylline (THEO); Phenobarbital (PHENO); Phenyl Phenytoin (PHENY); Gentamicin (GENT A); and tobramycin (TOBRA) are exemplified.

Table-1 Parameter T) IEOPHENOPHENY GENTA TOBRA Fu-U −Dare Trial] Shito Irikuma no 4 assistants So-”L-raw Superposition <10χ <10χ <10χ <10χ <10χ Concentration (mg/+I ljri, 19-. 394. 19-. 394. 01-. 394. 002-. 190.002-. 01 protein” Apo Apo Apo Apo Ap.

Molar ratio 17-21 9-10 23-30 20-23 6-8 Attachment site N -I N-I N-3 Random Random 5-Raw Antibody Yes Yes Yes Yes Yes Titer (mg/+IIL)>0 ．． 02 >0.02 >0.02 >0.02 >0.02 Affinity 10I01 0” 101010” 10” Binding Activity - Specified interaction as described above with assay characteristics 1 plate Salt concentration 100-500 100-500 100-500 100-500 100-500 polyethylene glycol (PEG) 4000-20.000 , or dextran@100.000-200.000゜honhon apoferritin (Apo) Judicious selection and processing of antibody/conjugate pairs allows suspension by conventional spectrophotometric techniques. The free hapten concentration of the final sample can be effectively determined by measuring the optical density of the sample. I can do it.

A normal day about fLfi green beans Each reagent system shown in the table above is suitable for monoclonal antibodies and and a partner reagent containing a hapten-protein conjugate, such a hapten-protein conjugate. The protein has a precise proportion (range of proportions) of hapten functional groups attached.

The protein component of this conjugate may include other globular particles with organic or inorganic prosthetic groups. Proteins are suitable, but apoferrin is preferred, as a transporter/carrier protein. These globular proteins, which commonly act on physical systems, have a dense three-dimensional structure and and a sufficient number of higher order macromolecules with a molecular weight of at least 100,000. It has a quaternary structure consisting of tertiary units. Of course, water-soluble conjugates can be formed by haptens. These globular proteins that can be formed (at appropriate hapten:protein ratios) can be formed using the method of the present invention. It is suitable as an immunoreagent.

Furthermore, as is evident from the above table, the exact relationship between hapten and apoferrin protein is also The measurement of the ratio is unique for each conjugate and in the TRIA method of the present invention. is critical to performance. The hapten to apoferrin protein ratio is lower than the preferred value. the relative rate of interaction with the antibody is too low to form a precipitate. Size complexes stop growing. Conversely, the ratio of hapten to apoferrin protein is greater than the desired ratio, the rate of immune complex formation is lower than that of free haptics in the sample. directly, rather than inversely, proportional to the amount of water. Additionally, hapten vs. apoferri If a precise ratio of protein is used, the antibody/conjugate's hapten inhibition can be measured by turbidity. consistent with the preferred objectives of velocity analysis. In particular, the hapten pair in the conjugate The ratio of poferritin protein to the expected concentration of the analyte and the concentration of the analyte within the expected range. Make choices regarding the potential scope for deviation. For example, processed for the determination of theophylline Immune samples containing this drug within the therapeutic range and twice the upper limit of the desired therapeutic range be sensitive to detection up to the level of

Toyoyu Chosei: One factor in the selection of monoclonal antibodies for this type of assay The rule of thumb is that there is a slight bias for binding to the analyte relative to binding to the conjugate. It is. As mentioned above, the antibody is capable of forming an effective shrimp in the conjugate in a reaction environment. It is preferable that it be present in a slightly larger amount relative to the site.

The combination of these two factors regarding antibody potency and efficacy may reduce the rate of turbidity. Critically necessary for the dynamic analysis range of the assay. one or both of the antibody abilities. If unsatisfied, the dynamic analysis range of the present invention will be substantially reduced and therefore limited. value and cannot compete with alternative technologies for dynamic analysis range.

U degree　A relatively high salt concentration in the reaction environment increases the affinity of the monoclonal antibody for the specimen. It is unexpected that it was found to increase Sources of salt are rare enough A preferred example of the present invention is a diluted conjugate reagent solution. The sensor and conjugate are mixed in a sufficiently diluted reagent. This reagent to stabilize and prevent spontaneous aggregation in the presence of enhancers. at least about 150+aM sodium chloride and at least about 100mM A buffer (phosphate buffer) is added to the conjugate/en/% cer solution. Conjugate/ The salt concentration relative to the conjugate in the enhancer solution effectively and The stability of this reagent was selected to be substantially neutralizing, thereby limiting the stability of this reagent to spontaneous aggregation. Increase against. Also, high salt concentrations inhibit the action of enhancers in immune complexes. Modulate the precipitation reaction by blocking, thereby increasing the rate of formation of precipitated immune complexes. control and expand the dynamic analysis range of the assay.

Furthermore, the stability of the conjugate in the presence of an enhancer is a property of the apoferrin protein. It seems to belong to some extent.

This protein component of the conjugate resists degradation by enhancers and has a place for reaction with antibodies. responds relatively quickly to control sediment formation.

ffi Monoclonal antibodies selected for use in the methods of the invention, as described above due to its high titer and its selective affinity for free haptens (analytes). Make a selection accordingly. In particular, antibodies couple free haptens from samples to conjugates. Antibodies are slightly directed against free haptens in order to effectively block them. Select to bias. Therefore, once the sample, conjugate reagent and monoclonal When the antibody is combined with the appropriate reaction environment, the precipitation reaction proceeds at a controlled rate. Is it a sample? These free haptens compete with the conjugates for the antibody and are not effective sites on the antibody. Some conjugates of these are substituted. This displacement of the conjugate by the free hapten causes the dispersion to This reduces the amount of sediment in the substrate and correspondingly reduces the optical density. this The rate of displacement can be correlated with the relative concentration of free hapten in the resulting dispersion. I can do it. The concentration of free hapten under the preferred analytical conditions of the present invention is Inversely proportional to the optical density of the dispersion.

As is clear from the above, the selection and processing of appropriate immunoreagents is Determining the sensitivity and dynamic analysis range of the method of the invention. Reagent system shown in Table 1 The parameters are for each individual component of these reagent systems. and turbidity monitoring of hapten-modulated immunoprecipitation reactions. Relative concentrations relative to each other in a ring-based immunoassay are shown. TR The IA factor/requirement correlation matrix is complex, unpredictable and unfortunately In this complex system, one parameter of the components of the reagent system Changes in the resulting individual that affect many other variables (reagents and processes) This improvement in the parameters of each component reduces the salt concentration of the reagents and reaction mixtures to surprises that become critical for stability and for control over the kinetics of precipitation reactions. I confirmed what I should do. In particular, in drug-apoferritin reagent solutions, relatively High overall salt concentration aggregates this conjugate by precipitation enhancer compounds. / Ensure that these components of the reagent system are stabilized against degradation; provides the ability to produce one well-diluted reagent solution containing both Ma Additionally, this relatively high overall salt concentration may be due to the presence of free drug in the reaction mixture. to control/modulate the formation of immune complexes between conjugates and antisera in Gives unexpected additional abilities. This result is based on the optical density of the reaction medium and the precipitated immune complexes. It is shown in a linear relationship between the rate of body formation.

The attached diagram shows the concentration range of 4J phosphate buffer in the conjugate reagent solution (Figure 1); polyethylene glycol (6000 MW) enhancer in conjugate and conjugate reagent solutions. - concentration range (Fig. 2). The values shown in these figures are for conjugate samples. It shows the relative concentration in the drug and shows the 0 values shown in Table 1 that differ from the values shown in Table 1. The range of values for each of these components is its concentration in the reaction mixture. . Optimization of the length of the methylene bridge linking the drug to apoferritin provides optimal sensitivity ( (at the lower and upper limits of the dynamic analysis range). Figure 3 is the optimal condition for two fertilizers with different hapten/apoferritin protein ratios. Optimization of crosslink length for nobarbital/monoclonal antibodies is shown. Ru. Figures 3 and 4 show the relationship between this ratio and the test devil. Also, Figure 4 , drug; apoferritin ratio was kept the same, and the change in crosslink length was compared to the antibody in the assay. / conjugate pairs. Obtained turbidity measurement immunoassay behavior The scope of analysis includes both defined therapeutic levels of the drug in the biological sample as well as High level of clinical involvement expand to encompass. Figure 5 shows a known concentration of phenol over a period of 8-160 seconds. It shows the change in absorbance of a dispersion containing barbicool. Drug in the reaction medium As the concentration of is increased, the absorbance plot becomes increasingly linear. of the test This feature explains the calibrator value (cal) over the entire dynamic range for phenobarbicur. Calculate the relationship coefficients of a linear regression equation using ibrator values) This can be proven by other means. Plot these values. (Figure 6), the relationship coefficient is completed at (1,0), that is, it reaches a straight line. The phenobarbicur value at 1.994 is within the accepted experimental precision of the assay. It shows that it is a straight line.

In order to evaluate certain unique characteristics of the present invention, the present invention will be explained based on examples. . Parts and percentages given in this example are by weight, unless otherwise indicated. Main departure The equipment and techniques used for the preparation of reagents and their evaluation in this method are described above. As standard.

Fruit 111L Reagent System to Determine Fuenobarbicur Levels in Biological Samples were prepared by drug-modulated immunoprecipitation reactions monitored by turbidity measurements.

Specifications for the individual reagents are shown in Table 1. The individual components of the reagent system are Initially, two separate solutions: (a) methylene with 3 carbon atoms; Keyhole linbet hemocyanin (Keyhole phenobarbi binding to limpethe+nocyanin) (KLH) antisera produced by clones sensitized to immunogens consisting of tar conjugates; and and (b) prepared as a phenobarbyl/apoferritin conjugate solution. Also , each of these reagent solutions contains about 0.1% sodium azide and about 10% sodium azide, respectively. It contained 0-150 mmol/L of phosphate buffer. Ene for precipitation reaction Hansa-1, 6.2% polyethylene glycol (6000 hw) It was contained in the phenolic rubital/apoferritin conjugate. Phenobar beer/ Even after the apoferritin conjugate solution has been sufficiently diluted and the reagent container has been opened, the We confirmed the surprising fact that the liquid is stable for a long period of time (up to 8 months).

Using the reagent system described above, serum containing a known concentration of phenobarbicur was prepared. Automatic clinical chemistry analyzer (DACO5 version) can perform turbidity measurement speed suppression immunoassay on samples. Analyzer - Coulter Electrox Inc., Florida I went to the state of Bahérea). Dynamically adjust the relative concentration of phenobarbicur in the sample. standard monitoring and slope data stored in the analyzer's microprocessor. related to the curve. The value is determined by the analyzer for the known phenobarbicure present in the sample. recorded according to the level of

The foregoing description, examples and accompanying drawings illustrate many preferred examples of the method. It is. The boundaries and scope of the invention are indicated in the following claims.

O e spear rF5F4+=A++(34Dnm(×10 one mABs+=F;h6t i Hi

Claims (6)

[Claims] 1. The sample is hapten, the reaction environment is monitored by turbidity measurement, and hapten and Hapten mimics induce antagonistic immunochemical interactions with antibodies due to the presence of precipitation enhancers. In immunoassay methods for liquid samples performed by hapten-modulated precipitation reactions containing Add an effective amount of primarily physiological buffering salts to the liquid sample to modulate the rate of the enhancer. Addition of mixtures containing haptens to control linear changes in the optical density of liquid samples The relative concentration of salt to the enhancer is precipitated to occur over the dynamic analysis range of An immunoassay method for liquid samples characterized by effectively optimizing the kinetics of the reaction. 2. Enhancers from the group consisting of polyethylene glycol and dextran The immunoassay method according to claim 1, which is selected. 3. The mixture of physiological buffer salts consists primarily of about 150 mM sodium chloride and a small amount of sodium chloride. 2. The immunoassay of claim 1, comprising at least about 100 mM buffer salt. 4. Modulation of the rate of sedimentation reaction using zero-order kinetics 2. The immunoassay method according to claim 1, which approaches the immunoassay method (inetics). 5. The hapten is the therapeutic agent, and the dynamic analytical range of the assay is for said therapeutic agent at the minimum therapeutic level and at the upper end of the range for said therapeutic agent; 2. The immunoassay of claim 1, comprising all toxicity levels. 6. The antibody is a monoclonal antibody, and the hapten mimic is mainly a particulate carrier protein. It consists of a polyhapten molecule with a large number of functional epitope sites, and The ratio of pitope sites to carrier protein was determined over the dynamic analytical range for the hapten. Claims to bring the precipitation reaction between the body and the partner reagent within the range where free hapten can be modulated The immunoassay method described in Box 1.