WO1992020746A1 - Compositions polymeres a anticorps lies - Google Patents

Compositions polymeres a anticorps lies Download PDF

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Publication number
WO1992020746A1
WO1992020746A1 PCT/US1992/004070 US9204070W WO9220746A1 WO 1992020746 A1 WO1992020746 A1 WO 1992020746A1 US 9204070 W US9204070 W US 9204070W WO 9220746 A1 WO9220746 A1 WO 9220746A1
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Prior art keywords
dye
analyte
antibody
pendent
assay
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PCT/US1992/004070
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English (en)
Inventor
Kenneth F. Buechler
Charles P. Lollo
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Hybritech, Incorporated
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Publication of WO1992020746A1 publication Critical patent/WO1992020746A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B69/00—Dyes not provided for by a single group of this subclass
    • C09B69/10—Polymeric dyes; Reaction products of dyes with monomers or with macromolecular compounds
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/583—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with non-fluorescent dye label

Definitions

  • the polymers contain multiple pendent groups to which are attached one or more marker substances, such as fluorescent and non-fluorescent dyes, as well as various radioisotopes .
  • marker substances such as fluorescent and non-fluorescent dyes, as well as various radioisotopes .
  • one or more marker substances such as fluorescent and non-fluorescent dyes, as well as various radioisotopes .
  • antibodies which are immunospecific for the substances to be detected, are covalently bound to the polymer.
  • the particular substance to be detected by these antibodies is covalently bound to the polymer.
  • reagents will be the antigen of the antibody attached to the polymer.
  • the substances sought to be detected by these polymeric compositions can include various
  • polymeric, dye-labeled immunoreactive reagents can be part of immunoassay systems which can be utilized by a patient at home.
  • immunoassay systems which can be utilized by a patient at home.
  • such a system may be used by a patient to detect blood or urine levels of
  • the immunological bond between the antibody and antigen should display a low dissociation constant, so that various physical manipulations of the polymeric reagent/antigen complex do not dissociate the reagent from the substance to be detected. Under ideal
  • the bond between the immunoreactive, dye-labeled reagent and substance to be detected should be strong and highly specific so that accurate detection and measurement of the substance can be achieved. These conditions are especially important for in-home, patient-controlled assay kits, where controlled
  • compositions do not meet these criteria.
  • Known polymeric, dye-labeled reagents employ covalent binding substrate linking to attach one or more antibodies to the dye-labeled polymers. These binding mechanisms may create such problems as multi-conjugation and cross-linking between the antibody and the dye- polymer which may significantly reduce the specificity of antibody binding to the antigenic
  • Patent No. 4,434,150 This patent discloses an
  • immunoassay reagent formed by coupling an immunoglobulin (antibody) to a water soluble polymer by a covalent bond or a biotin-avidin linkage.
  • the polymer of this patent is capable of additional substitution with various marker substances, particularly fluorescent dyes.
  • the immunological detecting reagents of this patent are capable of attaching
  • hydrophobic dye groups When attached to polymeric molecules, such hydrophobic dye groups may inhibit the water solubility of the polymers.
  • hydrophobic dyes exhibit a low absorption coefficient in aqueous media, thereby decreasing the visual intensity of the dyes, and accordingly the ability to chromagenically detect the dye labeled substance sought to be detected.
  • immunoreactive reagents may only be able to incorporate one antibody per dye-labeled polymer. These polymers often exhibit a coiled, tertiary structure. The single immunoreactive antibody may often be buried in this structure, and be unavailable for complexation with the substance to be detected. The attached antibody may also be in such close physical proximity to several of the dye groups attached to the polymer that its binding specificity and strength are sterically hindered. Azad et al. and Gribman et al, respectively, in U.S. Patent Nos. 4,434,150 and 4,373,932 describe reagents having this character.
  • polymeric, dye-labeled immunoreactive reagents which bind strongly and specifically with the particular substance to be detected in a sample of physiological fluid.
  • the polymers of such reagents should include a multiplicity of antibodies attached by other than covalent means, such that multi-conjugation, cross-linking and steric shielding problems are avoided.
  • the polymers should incorporate water soluble dyes, whose increased dye intensity in aqueous solution enhances the ability to detect and measure the intended substance.
  • the present invention is directed to polymeric compositions for immunologically detecting analytes and is directed to intermediates of those compositions.
  • the present invention is further directed to methods for making and using these polymeric
  • compositions to assay a sample for the presence of analyte are compositions to assay a sample for the presence of analyte.
  • an analyte is a substance to be analytically detected by means of an antibody-antigen conjugation.
  • the polymeric compositions of the present invention can carry dye moieties as well as antibodies, and can accommodate high dye loads without significant reduction in the specificity or binding strength of the bound antibodies. Multi-conjugation and cross-linking problems are avoided by use of a
  • bispecific antibody conjugate which is immunospecific for both the attached dye moieties and the particular analyte to be detected.
  • a plurality of antibodies can be bound to. each dye-loaded polymer.
  • preference for aqueous media allows for the use of more visually intense, water-solvated dyes.
  • the present invention provides an assay dye compound composed of a polymer having an all-carbon backbone, a first pendent alkyl amide or sulfonamide group having one or more N- substituted dye moieties, and a second pendent alkyl carboxy or sulfonoxy acid group.
  • the ratio of the first pendent amide group to second pendent acid group is from about 100:1 to 1:1.
  • the polymeric dye compositions may optionally contain a third pendent acidic N-(sulfonoxyalkyl or aryl)alkyl amide or sulfonamide group. Each alkyl substituent on these groups may contain from one to ten carbon atoms .
  • the aromatic group may be benzene or an alkyl , hydroxy, nitro or cyano substituted derivative thereof.
  • the ratio of the first to third pendent substituent is from about 100:1 to 1:1.
  • the dye moiety or moieties employed in the assay dye compound of the present invention can be any spectrometrically-detectable dye molecule or derivative that contains or is modified to contain a free primary or secondary amine group.
  • the dye exhibits minimal hydrophobic binding to proteins and contains functional groups that render it highly water soluble. Examples include a visual dye, phosphorescent dye, fluorescent dye, chemiluminescent dye, a laser dye, an infrared dye, lanthanide chelate or derivatives thereof.
  • the assay dye compound may advantageously contain two or more distinct dye moieties.
  • the invention provides an immunoreactive dye composition which is a combination of the assay dye compound and a bispecific an.tibody.
  • the bispecific antibody chosen is specifically
  • the portion of the bispecific antibody which is immunospecific for the dye moiety will bind to the dye moiety, while the portion that is immunospecific for the analyte to be detected will remain uncomplexed.
  • combination forms an immunoreactive reagent capable of detecting a corresponding analyte in a physiological or other fluid.
  • the bispecific antibody to be combined with the assay dye compound of the present invention can be based upon a polyclonal antibody, monoclonal antibody, a recombinant antibody in general such as a chimeric antibody, a synthetic antibody formed by recombination of individual L and H chain pairs or fragments of any of those antibodies, such as Fab, Fab', F(ab) 2 , L and H chains, L or H chain fragments and epitopal regions.
  • bispecific antibody When the bispecific antibody is formed of two distinct antibody molecules, i.e., two pairs of coupled
  • the bispecific antibody may be two pairs of coupled light/heavy chains with two different binding sites for binding both the analyte and dye (hereinafter the singlet species).
  • bispecific antibody is the singlet species, it will take the form of a monoclonal antibody, a recombinant
  • antibody in general such as a chimeric antibody, a synthetic antibody of two differing pairs of light and heavy chains, or fragments of any of these antibody forms.
  • the invention provides a detectable analyte dye complex of the immunoreactive dye composition, analyte and a second antibody.
  • the second antibody preferably is immobilized on a solid support such as inert particulate material or a membrane but can also function as a precipitating or weight alteration agent for solution detection as discussed below. It is immunospecific for the analyte to be detected, or species specific for the bispecific antibody, or
  • the second antibody can be a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a chimeric antibody or a synthetic antibody formed from recombination of individual pairs of light and heavy antibody chains.
  • Fragments of these antibodies may also compose the second antibody, these fragments including a Fab, Fab', F(ab) 2 , L chains, H chains, L or H chain fragments and epitopal regions.
  • the immunoreactive dye composition and second antibody should each be sufficient to maintain the corresponding. immun logical conjugations during washing, decanting, separating, exchanging or other physical manipulations of the detectable analyte dye complex.
  • these affinity constants will be at least about 10 8 liters per mole and more preferably 10 9 liters per mole.
  • the assay dye compound can be synthesized by several methods which allow for variations in dye solubility in water as a function of pH. In a first method, an amine- containing dye is reacted with an acid polymer having an all-carbon backbone and a plurality of pendent alkyl carboxylic or sulfonic acid groups.
  • the reaction is conducted in aqueous medium, and under conditions for forming amide or sulfonamide bonds between the amine-containing dye and the acid polymer.
  • the pH of the medium is maintained between about 6 to 12.
  • the molar ratio of acid polymer to amine-containing dye incorporated is about 1: 1 million to about 1:1,
  • the molar ratio of incorporated dye moieties to remaining unreacted pendent alkyl carboxylic or sulfonic acid groups is from about 1,000:1 to about 1:1, preferably about 100:1 to about 1:1, and more preferably about 20:1 to about 1:1.
  • an amine-containing dye and an amino sulfonic (alkyl or aryl) acid compound are reacted with the acid polymer having an all-carbon backbone and a plurality of pendent alkyl carboxylic or sulfonic acid groups.
  • the reaction is conducted in aqueous medium, and under conditions for forming amide or sulfonamide bonds between the acid polymer and the amine groups of (1) the dye, and (2) the amino (alkyl or aryl) sulfonic acid compound.
  • the pH of the medium is maintained between about 4 to 8.
  • the molar ratio of incorporated amine-containing dye moieties to the sum of the incorporated amino sulfonic (alkyl or aryl) acid and unreacted pendent alkyl carboxylic or sulfonic acid groups is from about 1,000:1 to about 1:1, and the molar ratio of acid polymer to amine-containing dye to be incorporated is from ' about 1:1 to about 1:1 million. Preferred molar ratios are as given in the preceding paragraph.
  • the detectable analyte dye complex can be utilized in methods of detecting and measuring the desired analyte. Under these methods, the assay dye compound, the assay dye compound, the
  • bispecific antibody, the second antibody, and a sample containing the analyte to be detected can be combined under conditions for immobilization, or under conditions for solution reaction, and in any order, to yield the detectable analyte dye complex. Thereafter, the
  • immunoreactive dye composition from the detectable analyte dye complex and spectrometric measurement of the dye moieties of the detectable analyte dye complex.
  • Discrimination can be handled for example by
  • the second antibody and sample containing the analyte to be detected be combined first. Thereafter, the bispecific antibody and assay dye compound are added successively. Alternatively, this same order of combination may be maintained, except that the bispecific antibody and assay dye compound are first mixed together before they are added to the second antibody/sample mixture.
  • the second antibody utilized to form the detectable analyte dye complex can be bound either directly or indirectly to a solid support such as a porous or semiporous membrane.
  • a solid support such as a porous or semiporous membrane.
  • the detectable analyte dye complex is formed directly on the support surface. After nonbound components of the testing media have been removed from the membrane, the remaining complex bound to the surface of the membrane can be visually or spectrometrically measured.
  • the detectable analyte dye complex can be indirectly bound to the solid support.
  • Such means include biotin-avidin, an enzyme-irreversible substrate, an antibody to the analyte complex itself (See El Shami for a description of this technique, A.S. El Shami et al., U.S. Patent No. 4,778,751 published October 8, 1988, the disclosure of which is incorporated herein by reference) and microparticles.
  • the microparticles carry bound second antibody and are combined with the immunoreactive dye composition to form a bound but mobile detectable analyte dye complex.
  • the microparticle-bound detectable analyte dye complex is immobilized by its entanglement within the semiporous membrane or fibrous mat.
  • the microparticles are
  • water insoluble polymers such as latex polymer, magnetic particles, glass beads, alumina or polystyrene.
  • the polymeric compositions and methods of the present invention can be combined into an in-home, analyte assay kit including predetermined amounts of the assay dye compound, bispecific antibody and second antibody in soluble or an insoluble form.
  • the assay kit may also provide a container for containing and mixing therein a fixed amount of a sample to be determined, the assay dye compound, the bispecific antibody and second antibody.
  • the second antibody may optionally be bound to a microparticle polymer or a membrane support mounted within the container.
  • FIG. 1 shows, a schematic diagram of the immunoreactive dye composition.
  • FIG. 2 shows a schematic diagram of a
  • detectable analyte dye complex using a solid support or membrane.
  • FIG. 3 shows a schematic diagram of a detectable analyte dye complex using microparticles and a porous membrane.
  • the assay dye compound is an all-carbon backbone polymer with a plurality of pendent dye groups.
  • the pendent dye groups are composed of one or more dye moieties bonded through amide or sulfonamide linkages to alkyl
  • the assay dye compound is composed of a polyacrylic acid or polyvinyl sulfonic acid or a derivative thereof which has been bonded to the amine-containing dye moiety by amide or sulfonamide linkages between the alkylcarboxy or
  • the polymer is derived from a branched or unbranched, olefinically unsaturated carboxylic acid or sulfonic acid monomer of 2 to about 10 carbon atoms.
  • dye refers to any organic radical
  • spectrometrically detectable dye molecule or derivative thereof and amine-containing dye refers to any such dye substituted with a primary or secondary amine, while dye moiety refers to the dye radical covalently bonded to the amine of the amine containing dye.
  • Preferred dye moieties are derived from visual dyes, phosphorescent dyes, fluorescent dyes, lanthanide chelates,
  • radioisotopes radioisotopes, electron opaque substances and their derivatives.
  • soluble visual dyes including solvent dyes, pigments, vat dyes, sulphur dyes, mordant dyes, leucovat dyes and species such as fluorescein, sulforhodamine, rhodamine-hydride, rhodamine hydrazide, Texas Red hydrazine, Congo Red, Trypan Blue, Lissamine Blue and the like, as well as oxazine dyes, cyahine dyes, laser dyes and infrared dyes.
  • solvent dyes including solvent dyes, pigments, vat dyes, sulphur dyes, mordant dyes, leucovat dyes and species
  • fluorescein sulforhodamine, rhodamine-hydride, rhodamine hydrazide
  • Texas Red hydrazine Congo Red
  • Trypan Blue Lissamine Blue and the like
  • the dye is composed of a formula that minimizes non-specific binding of protein to the dye. This property minimizes the effect of non-specific binding on the null control comparison made during the assay. It improves the sensitivity of the assay by minimizing the color or absorbance of the background against which the complex is compared. To maximize this property, it is preferred that the dye be highly water soluble and exhibit minimal hydrophilic binding to protein.
  • the first and second N-substituted dye moieties occur in pairs selected from the group consisting of fluorescein with rhodamine and fluorescein with Texas red. The ratio of first to second N-substituted dye moieties may be from about 100:1 to about 1:100.
  • the assay dye compound of the present invention is derived from an acid polymer having an all-carbon backbone and a plurality of pendent acid groups which form the linkages with the dye moieties.
  • Alkyl carboxy or sulfoxy groups can serve as the pendent acid groups.
  • Particularly preferred polymers include polyacrylic acid, polymethacrylic acid and their derivatives with pendent carboxyl groups. Other pendent groups may also be present, with the sulfoxy, amino and alcohol groups being preferred.
  • the acid polymer employed in the present invention can range in molecular weight from about 10 thousand to 10 million daltons. Preferably the polymer should have a molecular weight over 2 million daltons, most preferably between about 2 million and 8 million daltons. It will be appreciated that the molecular weight of the polymer will be varied in large part according to the number of dye moieties needed to achieve a detectable signal at a designated analyte concentration.
  • the method for formation of the assay dye compound involves linking the acid polymer and amine- containing dye together through amide or sulfonamide bonds. To form this linkage, a first reactive site (carboxy or sulfoxy) on the polymer is reacted with a second, correlative reactive site (amine) on the amine containing dye to form the assay dye compounds of the present invention.
  • Methods for preparation of the assay dye compound by formation of amide or sulfonamide bonds include techniques to activate the carboxyl or sulfoxyl moiety followed by attack of the amine group of the dye. Such techniques are well-known in the art and include carbodiimide reactions, acid halide or pseudo halide
  • Rabjohn Editor-in-Chief, John Wiley and Sons, (1963), pages 6, 34, 62, 88, 154, 263, 285, 339, 348, 411, 513, 521, 554, 608, 616, 620, 715, 739, 780, 900.
  • embodiments of the assay dye compound can be made under acidic or basic conditions.
  • the amidation or sulfonamidation reaction between the acid polymer and the amine-containing dye is run in neutral (up to pH 9) or acid aqueous solution, preferably at a pH from about 4.5 to 7.5, most preferably at a pH of about 6-7.
  • Examples of useful amidation reactions include, without limitation, acid halide or pseudo halide (carbonyl diimidazole) reaction at a pH of from about 7-9 and carbodiimide reaction at a pH from about 4-6.
  • this polymer may exist as a partially coiled molecule In acidic aqueous solution.
  • uncoiled segment contains the ionized groups while the coiled segment contains the un-ionized groups.
  • the ionized carboxylic acid groups react to form amide groups while the un-ionized groups are believed to be relatively less reactive because they are buried in the hydrophobic domain of the polymer.
  • Reaction of the exterior pendent groups with dye is believed to cause a shift in the equilibrium to the open form because of the conversion of the ionized pendent groups to amide or sulfonamide groups, particularly because the dyes in use are hydrophilic. Although this uncoiling proceeds slowly, it is believed to result in further reaction of the amine containing dye and the exterior pendent groups until the counterbalance of nonionizable amide groups shuts down further uncoiling. Consequently, factors for time and for solubility of amide or sulfonamide dye pendent groups are
  • the polymer can be reacted with an additional agent that will promote the solubility and reactability of the intermediate leading to the assay dye compound.
  • an additional agent that will promote the solubility and reactability of the intermediate leading to the assay dye compound.
  • the addition of such an agent in conjunction with the aminecontaining dye aids in the unfolding of the polymer and helps to maintain water solubility of the resulting assay dye compound and subsequent compositions
  • the starting reactants for the acidic reaction method preferably include an amino (alkyl or aryl) sulfonic acid
  • the ratio of dye substituted acid groups i.e., pendent groups with amine-containing dye groups
  • remaining unsubstituted acid groups optionally including those with the amine sulfonic acid substitutions i.e., pendent groups with free carboxy or sulfoxy groups
  • Particularly preferred amine sulfonic acids i.e., pendent groups with amine-containing dye groups
  • amino alkyl sulfonic acids NH 2 -(CH 2 ) n -SO 3 H
  • taurine NH 2 -CH 2 -CH 2 -SO 3 H
  • aryl sulfonic acids such as orthanilic, metanilic and sulfanilic acid (NH 2 - C 6 H 4 -SO 3 H) and the like.
  • reaction to form assay dye compound can be conducted in a medium of higher pH such as greater than 5, preferrably greater than 7, the acid polymer will tend to be in completely ionized form and will be maintained in an uncoiled state. Under such basic conditions, the preference to substitute the starting polymer with sulfonyl groups in order to obtain maximum conversion is lessened. Use of sulfonyl groups as described above is, nevertheless, a preferable option because their presence will help maintain the solubility of the assay dye compound at all pH values especially when the degree of dye substitution is at a maximum.
  • Optimum basic reaction conditions are between about 8.5 and about 9.0. Those skilled in the art will appreciate though that amide synthetic reactions performed at these high pH values will be affected by competing reactions, one of which is hydroxide ion reaction with the
  • the assay dye compound may develop a tendency to become insoluble and precipitate from solution.
  • the maximum equivalent percent substitution for each species of assay dye compound will vary around the targeted upper value. The maximum substitution can be appropriately and routinely determined for each species and the differing polymer molecular weights by making representative samples and extrapolating to the maximum.
  • the amine-containing dye and optional amine sulfonic acid are linked to the pendent groups on the polymer through an amide or sulfonamide linkage.
  • a polyacrylic acid is reacted with a carboxyl or sulfoxyl activating reagent such as a carbodiimide, carbonyl diimidazole, pivaloyl chloride, thionyl chloride or N-hydroxysuccinimide in an amount sufficient to form the desired proportion of activated acid groups within the acid polymer.
  • the reaction is conducted under appropriate acidic pH conditions to maintain the activated acid in solution. Substantially simultaneous with its formation, the activated acid polymer is reacted with the amine
  • invention absorbs or emits photons in the visible
  • a visual dye such as fluorescein or rhodamine will cause the assay dye compound to absorb photons of specific wavelengths in the visible region (e.g., blue), resulting in the transmission of the complementary wavelength of color (e.g., red) to an observer.
  • the assay dye compound is combined with a bispecific
  • the bispecific antibody should be immunoreactive with both the dye group of the assay dye compound and with an analyte to be detected. Immunobinding of the bispecific antibody to the dye moiety is superior to other chemical means of linkage, such as covalent bonding or enzyme linkages. Specifically, the immunobinding employed in the present invention prevents multiconjugation and cross-linking problems between the bispecific antibody and other substituents of the polymer chain.
  • the molar equivalent ratio of bispecific antibody to the assay dye compound can range from a saturating amount of antibody (the amount of which varies depending on the assay dye compound molecular weight) to a ratio of about 1:1.
  • the molar equivalent ratio of antibody to dye ' moieties of the assay dye compound affects the behavior of the complex in the assay; that is, a 1:1 ratio will give the greatest sensitivity but the slowest binding kinetics.
  • Higher molar equivalent ratios of antibody to assay dye compound conversely give faster binding kinetics (i.e., binding of composition to antigen) and lower sensitivity capabilities.
  • compositions of several different ratios determined experimentally by preparing compositions of several different ratios and extrapolating a plot of the molar equivalent ratio compared with kinetics and sensitivity.
  • the bispecific antibody may be any one of the following antibodies.
  • the bispecific antibody may be any one of the following antibodies.
  • the bispecific antibody may correspond to a single chimeric antibody, a single recombinant antibody, or, a single synthetic antibody of two differing pairs of light and heavy chains or fragments thereof wherein the single antibody has binding sites for both antigens (the singlet species).
  • the singlet species the singlet species
  • the bispecific antibody may be two or more polyclonal or monoclonal antibodies, recombinant antibodies, chimeric antibodies or two single chain antibodies chemically bound together, or antibody fragments which have been coupled together through the use of known coupling techniques and reagents such as dimaleimide.
  • the bispecific antibody may be two or more polyclonal or monoclonal antibodies, recombinant antibodies, chimeric antibodies or two single chain antibodies chemically bound together, or antibody fragments which have been coupled together through the use of known coupling techniques and reagents such as dimaleimide.
  • antibodies, fragments of those antibodies such as Fab 1 , Fab 2 and F(ab) 2 ', L chains, H chains, L or H chain fragments and epitopal regions can be incorporated to form either version of the bispecific antibody according to this invention.
  • bispecific antibody and fragment components used in accordance with the present invention are generally known. Methods for their preparation as bifunctional polyclonal, monoclonal and/or recombinant antibodies and all fragments and single chains thereof, as well as methods for developing specific
  • the anti-dye/anti-analyte bispecific antibody . or fragment can also be formed from two or more
  • the two antibodies or fragments are derivatized and linked with appropriate coupling agents known in the art.
  • appropriate coupling agents known in the art.
  • the reagents, procedures and techniques for cross-linking described in such texts as E.A. Rabat, "Structural Concepts in Immunology and Immunochemistry", 2nd Ed., Holt Reinhart and Winston, New York, 1976, or the Pierce Chemical Company Handbook of Reagents provide an appropriate description for accomplishing such
  • reagents such as
  • preferred coupling system is a combination of SPDP and SMCC described below.
  • SPDP N-succinimidyl 3-(2-pyridyldithio) propionate
  • SMC N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate
  • the conjugate is then further purified by gel permeation chromatography to yield the product shown below.
  • the resulting monoclonal antibody prepared by either technique can also be used to prepare the preferred F ab ' fragment by pepsin digestion and partial reduction following the procedure of Masuho (see Example 12). Both the monoclonal antibody and the fragment are preferred for preparing the bispecific antibody
  • the hybridoma technique is preferred for preparing this starting material and follows the
  • mice can be challenged and boosted with antigen composed of the dye in adjuvant with carrier or the analyte in adjuvant with carrier to produce specific immunogenieity.
  • antigen composed of the dye in adjuvant with carrier or the analyte in adjuvant with carrier to produce specific immunogenieity.
  • the spleen cells of the mice are
  • Cell fusions between the spleen cells and mouse myeloma cells can be conducted with a fusion agent such as polyethylene glycol. Cultures from single cell wells can be grown and tested for production of the desired antibody. The test can be conducted, for example by removing aliquots of cell culturation fluid and
  • bispecific antibody according to the present invention.
  • the hybridoma secreting this antibody has been deposited at American Type Culture Collection, as Deposit No.
  • the composition comprises a polymer having an all-carbon backbone (3), and first (4) and second (5) pendent substituents.
  • the second pendent substituent (5) includes an alkyl carboxy group (6).
  • the first. pendent substituent (4) includes a dye moiety (8), linked to an alkyl group through an amide linkage (9).
  • a bispecific antibody (10), herein shown as two antibodies coupled together, is immunologically bound to the dye moiety (8) at the anti- dye portion (12) of the bispecific antibody.
  • the anti- analyte portion (14) of the bispecific antibody (10) remains uncomplexed until reacted with the analyte to be detected. In practice, a fraction of the multiple number of first pendent substituents present will not actually be bound to bispecific antibody (10).
  • bifunctional antibodies (10) bound to the first pendent substituent (4) will not actually bind to the analyte. Consequently, the actual composition in uncomplexed form will also contain the unbound first, pendent substituent and in complexed form will contain the unbound conjugate of bispecific antibody-first pendent substituent as well.
  • the immunoreactive dye compositions in accordance with the present invention are uniquely suited to detect a wide variety of
  • analyte refers to any molecule, such as an antigen or hapten, which can evoke an immunological reaction with an immunoprotein such as an antibody or fragment thereof.
  • analytes may include without limitation, hormones, proteins, vitamins, drugs, viruses, bacteria, enzymes, toxins, carbohydrates, chemicals, and peptides.
  • analytes which are indicative of a particular physiological state in an individual, such as human chorionic gonadotrophin (HCG), human prolactin (PRL), human placental lactogen (HPL), testosterone, human immune deficiency virus (HIV), hepatitis surface antigen, prostate specific antigen (PSA), CA 125, CA 549, CA 19-9, CA 150-3 (Cancer markers), Human growth hormone (hGH), tissue plasminogen activator (TPA), alpha fetoprotein (AFP), bone alkaline phosphatase (BAP), chlamydia, hepatitis A, hepatitis B (surface antigen), group A streptococcus, group B streptococcus, hepatitis A virus (HAV), hepatitis B core antigen (HBc), blood enzymes and the other similar exogenous or endogenous proteins or polynucleic acids that are indicative of human disease or malconditions.
  • HCG human chorionic go
  • the sensitivity of antibodies provides appropriate micro- to nano-molar sensitivity for the method of detection according to the invention.
  • the presence of many dye molecules on the polymer acts as an amplification factor also.
  • the bispecific antibody of the immunoreactive dye composition is sufficiently sensitive to detect the analyte in solution at
  • bispecific antibody to analyte should be maintained from about 1:1 to 100:1 preferably from about 1:1 to 40:1, and most preferably at about 1:10.
  • Conjugation of the immunoreactive dye compositions of the present invention with an analyte and a second antibody yields a detectable analyte dye complex.
  • the second antibody can be immunospecific for the analyte to be detected, or species specific for the bispecific antibody, or immunospecific for the complex formed between the analyte and bispecific antibody.
  • the detectable analyte dye complex provides a convenient means of detecting and measuring a desired analyte, either by a solution technique or preferably by an immobilized technique. Such complexes can be detected by the spectrometric properties of the dye labeled portion of the detectable analyte dye complex.
  • the affinity constants between (1) the bispecific antibody and dye, (2) the bispecific antibody and analyte, and (3) the second antibody and analyte should be sufficient to maintain these immunological conjugations during washing, mixing, decanting,
  • the affinity constants should be at least about 10 8 liters per mole, preferably at least about 10 9 liters per mole.
  • the second antibody may be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a synthetic antibody, a recombinant antibody, or an antibody fragment such as Fab, Fab', F(ab) 2 , and single chains such as the light or heavy chains and fragments thereof carrying the epitopal regions. Its preparation will follow the hybridoma or recombinant techniques outlined above for the bispecific antibody starting material.
  • the second antibody preferably functions as the immobilizing agent for the detectable analyte dye complex by acting as the moiety that binds the complex to a support such as an inert, solid particulate
  • the second antibody also can enable
  • noncomplexed immunoreactive dye composition can be obtained by any of several options.
  • a first option involves precipitation of the detectable dye complex followed by removal of the supernatant and redissolution of the precipitate.
  • the second antibody can act as a precipitating agent in this situation.
  • a second option involves significantly changing the molecular weight of the detectable analyte dye complex relative to the weight of the immunoreactive dye composition. In this option, the second antibody acts as the weight enhancer such that the complex will centrifugally sediment at a significantly greater rate relative to the sedimentation rate of the immunoreactive dye composition. Separation of these two substances by centrifugation permits discriminatory determination of the concentration of the complex.
  • a third option involves quenching the
  • a fourth option involves addition of a known excess amount of immunoreactive dye composition to the unknown
  • concentration of immunoreactive dye composition in solution indicates the concentration of analyte present.
  • concentration of immunoreactive dye composition in solution indicates the concentration of analyte present.
  • options based upon precipitation or molecular weight variation of the complex will follow procedures known in the art such as addition of
  • absorption of the dye moiety of the immunoreactive dye composition group is quenched with a second, distinct dye group.
  • the analyte dye complex is formed as described except that the second antibody complexed with the analyte is bound to a second dye moiety substituent rather than to a solid support membrane.
  • the second dye moiety is designed to
  • the second dye moiety is positioned on the second antibody such that it does not significantly interfere with immunoreactivity but is within the proximity radius of about 30 to 60 angstroms for quenching the absorbance of the first dye moiety.
  • the molar equivalent ratio of second antibody to analyte in the sample should be from about 1:1 to about 10 million:1, preferably from about 90,000:1 to about 1 million:1, and most preferably at about 5,000:1 to about 50,000:1. In this regard, it is preferable to maximize the second antibody to analyte ration to obtain the best (i.e., fastest) kinetics of binding. After formation of the detectable analyte dye complex,
  • detection and measurement of the desired analyte may be performed by a variety of methods.
  • the order for combining the assay dye compound, the bispecific antibody, the second antibody and a sample containing the analyte to be detected may follow any sequence as long as it is compatible with the
  • the second antibody should first be combined with the sample containing the analyte. Thereafter, the bispecific antibody and assay dye compound should be added in successive order.
  • the second antibody and the sample containing the analyte are combined in association with an immobilizing support to form an immobilized
  • bispecific antibody are combined to form the
  • composition are combined to form the immobilized
  • washing the immobilized complex will remove noncomplexed immunoreactive dye composition.
  • FIG's 2 and 3 Illustrations of a detectable analyte dye complex formed by immobilization of the second antibody upon a solid support is given in FIG's 2 and 3. These illustrations include a polymer having an all-carbon backbone (3), and first (4) and second (5) pendent substituents.
  • the second pendent substituent (5) includes an alkyl carboxy group (6).
  • the first pendent substituent (4) includes a dye moiety (8), linked to an alkyl group through an amide linkage (9).
  • a bispecific antibody (10) is immunologically bound by an anti-dye variable region (12) to the dye moiety (8), and an anti-analyte variable region (14) is bound to the analyte (16) to be detected.
  • the analyte (16) to be detected is further bound to an anti-analyte portion (18) of a second antibody (20) which is immobilized by direct means (Fig. 2) or indirect means (Fig. 3) on a solid support (21) so as to form the detectable analyte dye complex.
  • the anti-analyte portion (18) of the second antibody (20) could be made specifically immunoreactive with the bispecific antibody (10) or the complex formed between the bispecific antibody (10) and the dye moiety (8).
  • Detection and measurement of the detectable analyte dye complex can be accomplished through various spectrometric or visual means of detection conducted upon the washed immobilized complex or upon a solution of the complex components obtained from the washed immobilized complex by breaking the immunological conjugates with an appropriate ionic reagent such as, for example, 8M ammonium sulfate.
  • an appropriate ionic reagent such as, for example, 8M ammonium sulfate.
  • spectrophotometer or colorimetric determination, or measurement of photon absorption or emission in a photometer, fluorometer, fluorescimeter, densitometer, and reflectometer.
  • the second antibody (10) may be directly or indirectly bound to a substrate such as the semiporous membrane or fibrous mat (24) as schematically shown, respectively, in FIG's 2 and 3.
  • the membrane may be composed of a flexible or rigid matrix made from any of a variety of filtration or chromatographic materials including glass fibers, nylon, nylon 66, polyethylene and polypropylene based materials, cellulosic and nitrocellulosic based materials, polyacetate and polyvinyl based materials, micro-fibers and natural or synthetic materials. Fluids preferably can flow into and pass easily through the membrane.
  • the membrane also preferably will have pore sizes of at least 0.1 ⁇ and preferably no more than 2 ⁇ .
  • the membrane can be used alone or as part of a more elaborate device.
  • Such devices includes the ICON ® and like devices described in the Valkirs et al. '901 and '019 patents, respectively, herein incorporated by reference. ICON ® is a trademark of Hybritech Incorporated (San Diego, California) for the devices described in the Valkirs et al. patents listed above.
  • Valkirs et al. describe an apparatus for the detection of a target antigen (e.g., analyte) in a liquid sample comprises: (a) a first member which is a porous membrane or filter and to which is bound an antibody against the target antigen
  • Still other devices containing porous and semiporous membranes useful in the present invention include the devices of Bauer et al., U.S. Patent No.
  • the second antibody may be directly bound to the membrane.
  • the direct binding may be covalent or non-covalent and may be accomplished by any method known in the art. Covalent methods include for example, the use of carbodiimide or glutaraldehyde in a solution at a pH from about 4.5 to 10, preferably from about 6 to 8, and most preferably at about 7, and aminosilanes as well as other methods described in
  • Covalent binding may also be accomplished by a cross-linking reaction through linking groups such as the
  • the non-covalent binding takes advantage of the natural adhesion of antibodies to the non-synthetic and especially the synthetic fibers.
  • appropriately buffered solutions can be mixed with the membrane then evaporated leaving a coating of the desired antibody on the membrane.
  • Passively coating the polymer membrane with antibody or by absorbing the second antibody onto the surface of the insoluble polymer at a pH near the isoelectric point of the antibody will also accomplish noncovalent binding.
  • the second antibody may also be immunologically bound to the membrane by employing a second bispecific antibody which immunologically binds to the polymer membrane as well as to the analyte, bispecific antibody or complex.
  • Another version of the immobilization technique according to the present invention involves indirectly binding the second antibody of the detectable analyte dye complex to the membrane through the use of
  • microparticles of a water insoluble polymer such as latex.
  • the microparticles are bound to or entrapped by the membrane, such that the microparticles are within the matrix of the membrane, on the surface of the membrane, or bound to other particles which are in turn bound to the membrane.
  • the microparticles may be any shape, preferably spherical.
  • the size of the particles may vary, but in general they may be slightly larger than the minimum pore size of the membrane and smaller than the maximum pore size, and in addition or in the alternative, may be larger than the maximum pore size.
  • the particles may be bound within the matrix of the membrane, on the surface of the membrane, or to other particles which are in turn bound to the membrane.
  • the particles may be made of a variety of naturally occurring or synthetic materials. Exemplary of such particles are those made from polyethylene,
  • polyacrylates polyacrylamide, and preferably
  • the second antibody may be covalently or non-covalently bound to the microparticle. Such binding may be accomplished by the methods discussed above for binding the antibody directly to the membrane.
  • the micro-particles may be applied (or "spotted") to the membrane in a zone within the surface area of the membrane.
  • spotting localizes the antibody-coated microparticles to a discrete area on the membrane to localize the antibody coated microparticles on or within the membrane.
  • Any of the methods known in the art may be employed.
  • One such method employs various mechanical means such as, for example, the Sandy Springs Spotting Machine (Germantown, Maryland) to apply a suspension, frequently aqueous ("latex”), to the membrane.
  • the microparticle bound detectable analyte dye complex can be poured onto a semiporous membrane or fibrous mat. After the unbound components have passed through the membrane or mat, the remaining microparticle-bound complex adhering to the surface of the membrane or mat can be spectrometrically measured.
  • the surface of the membrane or mat may be optionally washed with an acceptable buffer solution to remove nonbound components before spectrometric
  • buffered solutions include buffers ranging in pH from 4-12 and containing citrate, phosphate, borate, or carbonate and a nonionic detergent such as Triton X100 or Tween 20.
  • immobilized assay techniques may be performed by any of the methods known in the art. For membranes and
  • additional washing with buffer may often be sufficient, preferably drawing the liquid through the membrane or filter or contacting the opposite side of the filter or membrane with a liquid absorbing member that draws the liquid through, for example, by a
  • the ICON ® device Hybritech
  • Moderate temperatures are normally employed for carrying out the assay. Constant temperatures during the period of the measurement are generally required only if the assay is preformed without comparison with a control sample. The temperatures for the determination will generally range from about 15° to 45°C.
  • a signal can be detected by irradiating with light and the level of determining absorption and/or fluorescence through the use of visual observation, a fluorometer or a spectrometer. Where the appropriate equipment is not available, it will normally be desirable to have a chromophore produced which results in a visible color. Where sophisticated equipment is involved, any of the techniques is applicable. For a qualitative positive reaction. For a quantitative analysis, the ICON ® reader and accompanying software (Hybritech Incorporated, San
  • compositions and methods of the present invention can be combined into assay test kits for use in various clinical laboratories and at home settings.
  • assay test kits for use in various clinical laboratories and at home settings.
  • a predetermined amount of second anti-HCG antibody (20), which is immunospecific for human chorionic gonadotrophin, bound to a latex polymer (22) is combined with a measured sample of physiologic fluid containing the HCG analyte to be detected (16) to form a first immunological complex of second antibody and analyte.
  • a measured sample of physiologic fluid containing the HCG analyte to be detected (16) is combined with a measured sample of physiologic fluid containing the HCG analyte to be detected (16) to form a first immunological complex of second antibody and analyte.
  • concentration from about 3 ⁇ 10 -6 to 3 ⁇ 10 -5 mg/ml is provided although this amount will depend upon the assay
  • Such a second antibody solution should be sufficiently sensitive to detect HCG at a
  • a predetermined amount of bispecific antibody (10), which is immunospecific for both the HCG analyte (16)' and an aminosulforhodamine dye moiety (8) is added to the first immunological complex to form the second immunological complex.
  • the predetermined amount will be in the range of about 0.100 to 0.200 ml of a solution containing the
  • bispecific antibody (10) at a concentration from 0.1 to 0.5 mg/ml.
  • the second immunological complex formed in the second step is combined with an assay dye compound (28), incorporating an aminosulforhodamine dye . moiety (8), according to the present invention, to yield a latex-bound detectable analyte dye complex.
  • the amount of complex employed will be about 0.100 to 1 ml of a solution containing the assay dye compound (28).
  • the latex-bound detectable analyte dye complex can be visually detected on a membrane (21) by a doctor, medical technician or patient as previously described.
  • the latex-bound complex may be poured onto a semiporous membrane or fibrous mat (21) to aid in the visual detection of the colored complex containing the HCG analyte to be detected.
  • the membrane or mat is a white or off-white coloration to aid in the accurate color-metric determination of the complex.
  • a female patient could utilize the above method in an in-home test kit to determine if she is pregnant.
  • the above test kit components are
  • spectrometric detection may be determined by a device such as an autoanalyzer, photometer or fluorometer.
  • two or more distinct dyes can be attached to the assay dye compounds according to the present invention, such that the combined dye moieties can be attached to the assay dye compounds according to the present invention, such that the color-metric effect of the combined dye moieties can be intensified.
  • utilizing distinct dye moieties can be utilized to detect two or more analytes in a sample of physiologic fluid.
  • 1-amino-2- (aminosulforhodamine B) ethane it can be prepared as follows. Ethylene-diamine (2.94ml) was combined with methylene chloride (20ml) and the resultant mixture was purged with Argon. The mixture was stirred in an ice bath for ten minutes and lissamine (0.5g) was added over a 5 minute period. The solution was stirred for 3 hours in an ice bath then stored at -20°C overnight. The solution was stirred for 7 hours in an ice bath, stored again overnight at -20°C, then stirred at room
  • Rhodamine-hydride Substituted with Rhodamine-hydride, Aminofluorescein, Rhod-amine Hydrazide, l-Amino-2-(aminosulforhodamine)ethane
  • Polyacrylic Acid (8mg, 2 ⁇ 10 6 daltons,
  • Rhod-amine hydrazide (8.5mg); 3. 1-Amino-2-(aminosulforhodamine)ethane (8.5mg); and
  • Procedure A was repeated through the point where the reaction solution was stirred overnight at ambient temperature. Hexane (80ml) was added to the reaction solution, the mixture was stirred for several minutes and the liquid was decanted. The precipitate was washed 3 times with a methylene chloride/hexane mixture (60ml each). The washes were combined, filtered with suction, and the filtrate was discarded. The filter was placed over the product containing flash and the filter was washed with chloroform. The resultant suspension was taken to dryness in vacuo. The resultant residue was resuspended in dry methylene chloride (4ml), combined with the oily residue left from the decantation procedure, then chromatographed as in Procedure A. Procedure C
  • Procedure B was repeated through the hexane wash decantation.
  • the resultant precipitate was washed 3 times by first dissolving the precipitate in methylene chloride (10ml) then adding hexane (40ml) to precipitate the product. With each wash the liquid was decanted and the supernatants thus obtained were combined and
  • PAA Polyacrylic Acid
  • EDC was added as an aqueous solution with a concentration of 18mg/20 microliters
  • EDAS was added as an aqueous solution with a concentration of 10mg/400 microliters
  • reaction solution was centrifuged (10,000 rpm for 5 minutes) and the supernatant was
  • PAA polyacrylic acid
  • ASR aminosulforhodamine
  • PGAS (1-amino-2,5,7-trioxy-9-(aminosulforhodamine B) was added as an aqueous solution with a
  • Polyacrylic acid (2 ⁇ 10 6 daltons molecular weight) was dissolved in water to give a solution with a concentration of 0.5mg/ml. A portion of this solution (1ml) was combined with additional water (9ml). The pH of the solution was taken to 7.5 by the addition of 0.001N aqueous -potassium hydroxide solution then DMF
  • the reaction solution was stored overnight at 4°C.
  • the solution was chromatographed on Sephadex G25 (2.5 cm x 100 cm) (LPLC) with a mixture of 10 millimolar potassium borate, 1% potassium chloride, 1 millimolar EDTA and 10% acetonitrile in water (pH 8.2) to give a solution of 24 ml of the title product.
  • This product was analyzed by UV spectrometry at 572 nm, where its absorbance was 0.272. Assuming an extension coefficient of 50 cm 2 / ⁇ mol, this absorbance indicated a concentration of 0.054 micromols of dye/ml, or 1.3 micromols of dye on the 0.5 mg of unsubstituted PAP used as starting material. This is approximately 7400 dye molecules per molecule of polymer.
  • EDC (15 mg/ml in water) 200 ul is aliquoted and added to the reaction vessel. The reaction is stirred at room temperature for 2 hours at which point the reaction may be considered complete. At this point the net charge of the product may be changed by binding various moieties to the unreacted carboxyls on the polymer backbone.
  • Ethylene diamine, ammonium hydroxide, ethanolamine and taurine have been used in this respect.
  • reaction product was then exhaustively dialyzed in a 12-14,000 MW cutoff dialysis bag against
  • the product was deeply colored clear solution.
  • the enzyme-deficient, nonsecreting myeloma cell line Sp 2.0-Ag 14 is cultured in Dulbecco's modified minimal essential media (DME) containing penicillin, streptomycin (P,S), nonessential amino acids (NEAA), L-glutamine, 10% heat-inactivated horse serum and 5% heat inactivated calf serum (complete media, CM).
  • DME Dulbecco's modified minimal essential media
  • P,S penicillin, streptomycin
  • NEAA nonessential amino acids
  • L-glutamine 10% heat-inactivated horse serum
  • CM heat inactivated calf serum
  • the 3T6 mouse fibroblast line is used as a feeder cell layer in the cloning of cell hybrids and was cultured in DME containing P,S NEAA, L-glutamine and 10% heat-inactivated calf serum.
  • the immunogen, aminosulforhodamine(AS) -keyhole limpet hemocyanin (KLH) (AS-KLH) is prepared by the covalent conjugation of the dye to the KLH by amide bond formation with carbonyl diimidazole or of an appropriate activated derivative of the dye, such as the
  • Aminosulforhodamine is coupled to Sepharose 6B (Pharmacia) through the introduction of the bisoxirane 1,4-butanedial diglycidyl ether according to the
  • Somatic cell hybrids are generated as previously described in Kranz et al., Immun. Commun., 9., 639-651 (1980).
  • the spleen is removed and approximately 10 8 washed splenic lymphocytes are added to 10 7 washed Sp 2.0-Ag 14 myeloma cells and centrifuged.
  • One milliliter of a 50° polyethylene glycol (PEG 1540 Baker Grade) solution (in HEPES-buffered DME at 37°C) is added to the pelleted cells over a period of 1 min.
  • the cells are washed, resuspended in 100 ml of CM and distributed into 48 culture wells (2 ml were COSTAR 24-well cluster plates). The cells are cultured in selection media containing hypoxanthine, aminopterin and thymidine (HAT) for 2-3 weeks. Supernatants from wells are assayed for anti-AS activity using the radioimmunoassay described later.
  • the cells from anti-AS positive wells are cloned in 0.2% agar (SeaKem) over a 3T6 fibroblast feeder layer as described by Coffino et al. (1972).
  • individual clones are transferred to a 24-well cluster plate, cultured and assayed for antifluorescein activity.
  • the selected clones are grown in a culture until a sufficient number of cells were available for freezing and for injection into BALB-c mice.
  • a liquid-phase radioimmunoassay is used to detect anti-AS secreting hybrids. Four hundred
  • Monoclonal murine anti-AS antibodies are purified from the ascites fluid of BALB-c mice, which have been injected with approximately 5 ⁇ 10 6 of the corresponding hybridoma cells.
  • Antibody purification involves sodium dextran sulfate precipitation,
  • BALB/c mice receive multiple injections of the HCG-KLH antigen emulsified in Freunds adjuvant (10 ⁇ g per injection). After the fourth injection, blood from the immunized animal is collected in 0.5 ml of PBS, and each sample is assayed by ELISA for the presence of antigen- specific antibody.
  • the spleen from a mouse testing positive for immunogenicity is removed aseptically and the cells are isolated by dicing the spleen in 5ml of sterile PBS.
  • the cell suspension is added to a centrifuge tube and tissue fragments are allowed to settle for 1-2 minutes.
  • the cells still in suspension are placed in a similar tube and centrifuged at room temperature.
  • the cells are then washed 3 times by centrifugation in serum-free DMEM (Dulbecco's modified Eagle's medium).
  • Spleen cells are copelleted with P3X63-Ag8.653 myeloma cells at a ratio of 4 spleen cells to 1 myeloma cell.
  • the supernatant fluid is removed, and the pellet is suspended in 1 ml of 35% polyethylene glycol for 1 minute.
  • the polyethylene glycol is gradually diluted by addition of increasing amounts of serum-free DMEM over a period of 15 minutes.
  • HAT medium Monoclonal Antibodies, Kennett, McKean, Backitt, eds. Plenum press 1981
  • HAT medium Monoclonal Antibodies, Kennett, McKean, Backitt, eds. Plenum press 1981
  • the column is washed with appropriate amounts of Tris buffer at pH about 7.5 to elute the F( a b') 2 fragments.
  • the isolated F( ab ') 2 fragments can be treated with a minimum amount of 2-mercaptoethanol (2mM) in buffered aqueous medium (Tris HCL) to reduce the inner fragment disulfide bond and produce the F ab ' fragments.
  • the F ab ' fragments can be dialyzed against isotonic saline to remove buffers and amino acids.
  • the anti-AS F ab ' fragments (2.62 mg dissolved in 1 ml of phosphate-EDTA buffer) can be mixed with a 30- fold molar excess of SMCC (504 ⁇ g of SMCC dissolved in 50.4 ⁇ l of N,N'-dimethylformamide) for 60 min. at room temperature. The reaction is stopped by gel filtration chromatography on Sephadex G-25 (1.5x30cm) equilibrated with phosphate-EDTA buffer. The anti-AS F ab ' -maleimido fractions are pooled and concentrated to approximately 1 ml.
  • a mixture of 2 ml of anti-HCG F ab '-SPDP in phosphate-EDTA (3mg protein per ml) can be combined with a stoichiometric amount (relative to protein) of
  • Polyvinyl chloride microtiter assay plates can be coated with about 100 micromol of antihuman chorionic gonadotropin (anti-HCG) rabbit antibody in aqueous phosphate buffer at pH 7.5 at a concentration of 10 microgram per ml in phosphate buffered saline (PBS).
  • anti-HCG antihuman chorionic gonadotropin
  • the plates are incubated overnight to obtain adsorption of the anti-HCG rabbit antibody onto the plate surfaces.
  • the plates are then washed with fresh phosphate buffer to remove nonadsorbed material then blocked with nonfat dry milk to remove all nonspecific protein binding sites on the assay plates.
  • test wells To half of the plate wells (test wells) is added about 10 ⁇ g of HCG, about 10 ⁇ g of the bifunctional conjugate of Example 13 and about 10 mmol of the ASpolyacrylic acid compound of Example 9 all in PBS while to the other half of the wells (control wells) is added the same mixture without HCG.
  • the plate wells are incubated overnight at room temperature, then washed with fresh PBS buffer to remove nonbound material.
  • test well walls to be the distinct color of AS and the control well walls to be colorless. This difference will indicate that immunospecific binding of the test well walls to be the distinct color of AS and the control well walls to be colorless. This difference will indicate that immunospecific binding of the test well walls to be the distinct color of AS and the control well walls to be colorless. This difference will indicate that immunospecific binding of the test well walls to be the distinct color of AS and the control well walls to be colorless. This difference will indicate that immunospecific binding of the test well walls to be the distinct color of AS and the control well walls to be colorless. This difference will indicate that immunospecific binding of the test well walls to be the distinct color of AS and the control well walls to be colorless. This difference will indicate that immunospecific binding of the test well walls to be the distinct color of AS and the control well walls to be colorless. This difference will indicate that immunospecific binding of the test well walls to be the distinct color of AS and the control well walls to be colorless. This difference will indicate that immunospecific binding of the test well walls to be the distinct color of AS and the control well
  • HCU061 anti-HCG monoclonal antibody bound to it, i.e., the HCG ICON ® Serum II latex prepared according to the procedures given in A.S. Rubenstein EPO application No.
  • HCG Human Chorionic Gonadotrophin
  • Example 13 To this pellet was added 200 ⁇ l of 0.28 mg/ml of the bifunctional Di-F ab ' conjugate of Example 13 which has affinities for both HCG and aminosulforhodamine.
  • a control reaction using a urine sample containing no HCG was prepared in the above manner with the same bifunctional Di-Fab' conjugate and dye assay compound. This sample did not cause any significant colored spot after being passed through the membrane.
  • the same assay was repeated except that the analyte same of urine was reformulated to contain about 500 mIU/ml of HCG. (This is a typical HCG concentration found in the urine of a pregnant woman who is in her third or fourth week of pregnancy . ) Upon incubation and spotting, a colored spot was observed indicating a positive qualitative test for HCG.

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Abstract

Procédé de dosage d'analytes et des synthèse des compositions correspondantes. Un composé de colorant pour dosage composé d'un polymère substitué par des fractions de colorant et par des fractions d'acide sulfonique et/ou carboxylique est conjugué à un anticorps ou fragment d'anticorps bispécifique. Ledit anticorps ou fragment est également immunoréactif avec l'analyte. Un second anticorps qui est également immunoréactif avec l'analyte conjugué de l'anticorps ou du fragment bispécifique est utilisé pour immobiliser ou encore distinguer le conjugué. La mesure de l'intensité de couleur du colorant du conjugué indique la présence et éventuellement la quantité d'analyte.
PCT/US1992/004070 1991-05-14 1992-05-14 Compositions polymeres a anticorps lies WO1992020746A1 (fr)

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Cited By (6)

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Publication number Priority date Publication date Assignee Title
WO1997028447A1 (fr) * 1996-02-01 1997-08-07 Abbott Laboratories Fluorophores polymeres ameliores par des fractions creant un microenvironnement hydrophobe et a restriction conformationnelle
US5661040A (en) * 1993-07-13 1997-08-26 Abbott Laboratories Fluorescent polymer labeled conjugates and intermediates
WO1998008875A1 (fr) * 1996-08-28 1998-03-05 Viva Diagnostika Diagnostische Produkte Gmbh Nouvelles preparations combinees et leur utilisation en immunodiagnostic et en immunotherapie
EP0981748A1 (fr) * 1997-02-26 2000-03-01 Ban-An Khaw Amelioration du signal d'une sonde polymere a anticorps bispecifique pour immunodosages
WO2015118993A1 (fr) * 2014-02-05 2015-08-13 国立大学法人筑波大学 Préparation anti-adhérente comprenant une composition à base d'un copolymère polycationique tribloc et d'un polymère polyanionique
EP2935337A4 (fr) * 2012-12-20 2016-10-05 Akrivis Technologies Llc Polymères contenant une charge utile mélangée et leur utilisation

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EP0155224A2 (fr) * 1984-03-15 1985-09-18 CHROMAGENICS, Inc. Complexe en phase solide porteur d'une fonction sensible à un chromogène pour la détection d'un anticorp, d'un antigène, d'un récepteur ou d'un ligand
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WO1997028447A1 (fr) * 1996-02-01 1997-08-07 Abbott Laboratories Fluorophores polymeres ameliores par des fractions creant un microenvironnement hydrophobe et a restriction conformationnelle
US5994143A (en) * 1996-02-01 1999-11-30 Abbott Laboratories Polymeric fluorophores enhanced by moieties providing a hydrophobic and conformationally restrictive microenvironment
WO1998008875A1 (fr) * 1996-08-28 1998-03-05 Viva Diagnostika Diagnostische Produkte Gmbh Nouvelles preparations combinees et leur utilisation en immunodiagnostic et en immunotherapie
EP0981748A1 (fr) * 1997-02-26 2000-03-01 Ban-An Khaw Amelioration du signal d'une sonde polymere a anticorps bispecifique pour immunodosages
EP0981748A4 (fr) * 1997-02-26 2002-09-18 Ban-An Khaw Amelioration du signal d'une sonde polymere a anticorps bispecifique pour immunodosages
EP2935337A4 (fr) * 2012-12-20 2016-10-05 Akrivis Technologies Llc Polymères contenant une charge utile mélangée et leur utilisation
WO2015118993A1 (fr) * 2014-02-05 2015-08-13 国立大学法人筑波大学 Préparation anti-adhérente comprenant une composition à base d'un copolymère polycationique tribloc et d'un polymère polyanionique
US9962469B2 (en) 2014-02-05 2018-05-08 University Of Tsukuba Adhesion-preventing preparation comprising composition comprising polycationic triblock copolymer and polyanionic polymer

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