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Agglutination Immunoassay

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Agglutination Immunoassay Europaisches Patentamt European Patent Office © Publication number: 0 503 454 A1 Office europeen des brevets EUROPEAN PATENT APPLICATION © Application number: 92103667.9 int. CIA G01N 33/53, G01N 33/546 @ Date of filing: 04.03.92 ® Priority: 15.03.91 US 669783 Inventor: Courtney, Jodi Blake 407 Valley Road @ Date of publication of application: Upper Montclair, N.J. 07043(US) 16.09.92 Bulletin 92/38 Inventor: Passarelli, Joseph John 34 Everitts Road © Contracting States: Designated Ringoes, N.J. 08551 (US) BE CH DE ES FR GB IT LI NL Inventor: Salamone, Salvatore Joseph 37 E. Pierrepont Avenue © Applicant: F. HOFFMANN-LA ROCHE AG Postfach 3255 Rutherford, N.J. 07070(US) CH-4002 Basel(CH) @ Inventor: Antonian, Edna © Representative: Notegen, Eric-Andre et al 186 Watchung Drive Grenzacherstrasse 124 Postfach 3255 Hawthorne, N.J. 07506(US) CH-4002 Basel(CH) © Agglutination immunoassay. © A method for enhancing the reactivity of micro- FIGURE - 1 particle based immunoassays has been developed using polyvinylpyrrolidones (PVP). Microparticle ag- gregation reactions are accelerated resulting in in- creased standard curve dynamics and improved sensitivity. 8 z < oa ce o </> ta < o CONCENTRATION (ng/mL) Rank Xerox (UK) Business Services 1 EP 0 503 454 A1 2 The present invention relates to the use of PVP present in a sample, no change in the state of the to improve microparticle based agglutination im- particle dispersion would be detected. The ap- munoassays and significantly enhance the resulting proach is not optimal for the detection of low mo- sensitivity and signal of these immunoassays. lecular weight haptens. However, when a bivalent More particularly, the present invention pro- 5 agglutinator is the soluble binding component the vides an improvement in a microparticle based analyte competes with the hapten on the carrier for immunoassay method for the detection of target agglutinator binding sites. Thus, the presence of molecules in a sample of body fluid by ag- hapten in the sample inhibits particle cross-linking. glutinometric determination, the improvement com- The improvement in sensitivity thus obtained, has prising adding to said sample an amount of poly- io increased the utility of microparticle assays in di- vinylpyrrolidone (PVP) effective to increase the agnostic testing for both haptens, such as abused sensitivity and signal of said immunoassay. and therapeutic drugs, and antigens such as hu- The use of microparticles in diagnostic assays man chorionic gonadotropin (U.S. Patent is now generally well known. The assay method 3,857,931). While these assays depend on the de- may be used in both homogeneous and heteroge- 75 tection of flocculation, the sensitivity obtained with neous formats, for both large molecules (e.g., pro- a soluble agglutinator sacrifices signal where the teins, hormones, enzymes, antibodies, im- agglutinator is not in excess over the range of munocomplexes) and small (e.g. therapeutic drugs, clinical significance. abused drugs and their metabolites), in competitive The use of polyethylene glycol (PEG) and var- and direct binding assays. A variety of polymeric 20 ious detergents as agglutination enhancers has microparticle systems have been developed which been suggested. U.S. Patent No. 4,480,042 at col- allow the covalent or non-covalent attachment of umn 10; Galloway, R.J., Development of Micropar- compounds of biological interest (see, for example, ticle Tests and Immunoassays page 29 (Seradyn, those discussed in U.S. Patent No. 4,480,042 at Inc. Indianapolis, 1988). The stabilization of disper- Column 2). Widely used are particles, of a variety 25 sions of microparticles, as well as inhibition of of sizes, made of monomers co-polymerized with "non-specific agglutination", has been ascribed to styrene. surfactants and water soluble polymers including The various embodiments of microparticle polyvinylpyrrolidone (PVP). Bangs, L. B., Uniform technology in the immunoassay have in common a Latex Particles, pp. 25-26 and 55-56 (Seradyn Inc., solid support which is the carrier for one compo- 30 1984). PVP-360 in low concentrations, i.e., to a final nent of an immunological binding pair. The im- concentration below 0.4%, is incorporated in the munochemical reaction may affect a change in the latex microparticle reagents used in the capillary state of dispersion of the particles or a second mediated agglutinographic slides commercially label could be used when the particles serve only sold as ONTRAK™ (Roche Diagnostic Systems, to isolate a determinant on a solid support. Where 35 Inc.) for drugs of abuse testing. (The final con- the particles are specifically cross-linked (or inhib- centrations referred to in the specification and ited therefrom) as a result of the interaction of the claims herein are referenced to fully made up binding pair, there are a variety of analytical meth- reaction mixtures including all reagents plus sam- ods that may be used for signal detection, such as ple; all percentages are weight/volume unless oth- turbidity, nephelometry, angular anisotropy, quasi- 40 erwise noted.) elastic light scattering and so forth. Not only are there a variety of such agglutinometric techniques Brief Description of the Drawings available, but the method is also applicable to several instruments of varying degrees of automa- Figure 1 graphically demonstrates data gen- tion. 45 erated as in Example 1 , a THC calibration curve. Initial application of microparticles in diagnost- Figure 2 represents the optimization of a THC ics was for the detection of serologically deter- assay by use of PVP 360 at various concentrations minant factors in body fluids (Howanitz J. H., in accordance with Example 2. Immunoassay-lnnovations in Label Technology. Figure 3 demonstrates optimization of a cali- Arch Pathol Lab Med, Vol 112, pp. 775-779, (1988); 50 bration curve for Benzodiazepine assay by use of Hechemy K. E., and Michaelson E. E., Latex Par- PVP-360 and PVP-40, in accordance with Example ticle Assay in Laboratory Medicine. Part I, Labora- 3, versus no addition of PVP-40. tory Management (June 1984). An agglutinator Figure 4 demonstrates enhancement of the selective for the antigen of interest could be bound competitive microparticle aggregation reaction for (covalently or absorbed) on the particle surface. 55 Benzodiazepine by use of PVP-40 at various con- Cross-linking and detection are optimized where centrations, in accordance with Example 4. the antigen has several determinants available for In accordance with the present invention, it was agglutinator binding. Where there is no antigen unexpectedly been found that the addition of effec- 2 3 EP 0 503 454 A1 4 tive amounts of PVP to microparticle based im- (see Figure 2). After a concentration of 1.50% the munoassays results in enhanced reaction kinetics amount of increase in acceleration levels off. In the thereby yielding signals of increased accuracy and case of PVP-40, the 3.27% concentration in the an assay of improved sensitivity and overall signal final reaction mixture more than triples the absor- to noise ratio. When used in sufficient concentra- 5 bance signal from that for PVP at 1.31%, (See tion the PVP works as a reaction enhancer, rather Figure 4). The same 3.27% concentration of PVP- than merely as a stabilizer of the dispersion. PVP 40, however, barely matches the enhancement that of any molecular weight can be utilized in the addition of only 0.77% PVP-360 provides present invention. (Compare Figure 3). The microparticle reagents utilized in the io In a preferred embodiment of the present in- present invention incorporate microparticles that vention the PVP used has an average molecular are coated with one component of an immunolog- weight of about 360 kD and is added to a final ical binding pair which binding pair is diagnostically concentration of at least about 0.6%. selective for the substance of interest. The micro- In certain cases it may be advantageous to use particles remain monodispersed. Upon kinetic inter- is high and low molecular weights together. For ex- action in the presence of the complementary bind- ample, in a benzodiazepine assay reagent pre- ing partner the microparticles aggregate resulting pared for use on the Olympus analyzer (Example in changes of optical density. Enhanced reaction 3), the instrument could not accurately pipet the kinetics yield signals of increased accuracy and an PVP containing reagent if the concentration of assay of improved sensitivity. Significant slopes are 20 PVP-360 in the antibody & diluent reagent reservior obtained for the calibration curves so as to make (R1) was above 1.18%. The addition of PVP-40 to quantitation possible. a concentration of 2.0% in R1 (= 1.31 % final The present invention provides enhancement of concentration) and PVP-360 to a concentration of specific analyte-agglutinator binding mediated by a 1.175% in R1 (= 0.77% final concentration) gave particle carrier so as to permit the design of diag- 25 acceptable kinetics and good pipeting precision. nostc assays with sufficient clinical sensitivity for (Figure 3). application to the detection of haptens and anti- The gross amount of enhancement of signal gens. Specific representative analytes in human and sensitivity will, of course, reflect the inherent body fluid include, for example, A9-carbox- characteristics of each assay and intrinsic factors ytetrahydrocannabinol (COOH-THC), diazepam, hu- 30 such as, for example, antibody affinity, as well as man chorionic gonadotropin, and human gamma extrinsic factors such as apparatus limitations. globulin. However, as can be appreciated from optimization In general, increasing concentrations of PVP graphs such as depicted in Figures 2 and 4, the yield increased sensitivity and signal. Moreover, as skilled worker can readily titrate to a selection of noted previously the same effect is achieved by 35 PVP molecular weight grade and concentration, or increasing the molecular weight of the PVP. PVP is combination thereof, which are optimal for any par- typically available commercially in different grades ticular assay system or apparatus.
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