WO1998019161A1 - Solid phase immunoassay - Google Patents
Solid phase immunoassay Download PDFInfo
- Publication number
- WO1998019161A1 WO1998019161A1 PCT/US1997/018458 US9718458W WO9819161A1 WO 1998019161 A1 WO1998019161 A1 WO 1998019161A1 US 9718458 W US9718458 W US 9718458W WO 9819161 A1 WO9819161 A1 WO 9819161A1
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- WIPO (PCT)
- Prior art keywords
- immunoreagent
- polyethyleneimine
- antibody
- coating
- solid phase
- Prior art date
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Classifications
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/81—Protease inhibitors
- G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/962—Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
Definitions
- the invention relates to a rapid immunochemical method, using a modified latex surface, for determining concentrations of proteins in biological fluids.
- Immunoassays are currently used to determine the concentrations of various substances in biological fluids. Immunoassays using immobilized immunoreagents are in widespread use for the detection and quantification of biological molecules in samples.
- An immunoreagent which may comprise an antibody or antigen, is first immobilized on a solid surface, such as a test tube, microplate, beads or the like.
- the immobilized reagent is then contacted with an analyte solution containing a complementary immunoreagent, whereby an immobilized immunocomplex is formed between the immunoreagent and complementary immunoreagent.
- the immobilized immunocomplex can be separated from the unreacted analyte solution, preferably with repeated washing of the immobilized immunocomplex.
- the separated immunocomplex may be subjected to further processing to quantitate the amount of the immunocomplex.
- Quantitative methods include, for example, radio immunoassay (RIA), wherein the amount of the adsorbed immunocomplex may be determined by counting radioactive disintegrations ; enzyme-linked immunosorbent assay (ELIS A) , wherein the adsorbed immunocomplex, which has an enzyme coupled thereto, is contacted with a substrate for the enzyme to produce a detectable product; immunofluorescent assay, wherein the fluorescent intensity of a fluorescent substance linked to the immunocomplex is measured; and chemiluminescent assay wherein the chemiluminescence of a chemiluminescent agent is measured.
- RIA radio immunoassay
- ELIS A enzyme-linked immunosorbent assay
- PCFIA particle concentration fluorescence immunoassay
- LK is one of two kininogens found in human plasma (Margolis and Bishop,
- LK is present in human plasma at a concentration of 93 ⁇ g/ml and exhibits a molecular weight of 64 kDa.
- LK is composed of a large heavy chain (59 kDa) and a small light chain (5 kDa). The two chains sandwich the nonapeptide, bradykinin, the most potent vasodilatory substance in humans.
- the heavy chain is a potent thiol proteinase inhibitor (Sueyoshi et al., FEBS. Lett. 182: 193-195, 1985). The function of the light chain of LK is not known at this time.
- the release of large quantities of bradykinin into the circulation results in shock which can lead to death.
- the heavy chains of HK and LK are identical. Therefore, antibodies against heavy chain immunodeterminants cross-react with both HK and LK.
- the light chains of HK and LK which grossly differ by size as well as function, share a segment of 12 amino acid residues at their amino terminus which is derived from the common domain, D4, and differ in their carboxy terminal portions represented by domains D5 H and D6 H (HK) or D5 L (LK). Therefore, antibodies to the complete light chains that include the common amino terminal segment, might partially cross-react with the two types of kininogens . In contrast, antibodies to the unique light chain portions of HK and LK allow discrimination between the two kininogens.
- SIRS Systemic Inflammatory Response Syndrome
- Pathol. 71 :587-601, 1990 discloses the lack of rapid, simple, reliable assays for the quantification of its components.
- the lack of rapid, simple, reliable assays for the quantification of its components has prevented in depth analyses of the contribution of the kallikrein-kinin system to the pathogenesis of these diseases.
- Very little is known about the role of LK in health and disease because, until now, there has not been a rapid method to directly quantify LK in biological fluids.
- LK has been measured indirectly in biological fluids by determining the concentration of HK and LK (total kininogens) with antibodies to their common heavy chains (Kitamura et aL, J. Biol. Chem. 260:8610-8617, 1985) and then subtracting the concentration of HK, as determined by an antibody to its unique light chain, from the value for total kininogens (Syvanen et aL, FEBS Letters 129:241- 245, 1981: Kerbiriou-Nabias et aL , Br. J. Haematol. 56: 273-286, 1984; Legris et aL, J. Immunol. Meth. 168: 111-121, 1994).
- One such LK assay method includes incubating acidified plasma with trypsin to release bradykinin, first in plasma plus a kininase inhibitor to prevent the destruction of bradykinin after it is released (total kininogens), then incubating a second portion of plasma with a kininase inhibitor and glass powder to activate the contact system thereby releasing bradykinin from HK, and finally, incubating a third plasma sample with glass powder to deplete HK in the absence of a kininase inhibitor (LK), prior to measuring the bradykinin by a bioassay (Uchida and Katori, Thromb. Res. 15: 127-134, 1979).
- LK kininase inhibitor
- the glass powder is assumed to only activate plasma kallikrein that, in turn, releases bradykinin from HK.
- plasma kallikrein can potentially release bradykinin from LK in plasma. Henderson et aL, Blood 84:474-482, 1994, have observed the rapid disappearance of bradykinin from both LK and HK on neutrophils incubated with purified plasma kallikrein.
- Another LK assay method employs the immunochemical technique, radioimmunoassay, to indirectly measure LK.
- HK is detected by using specific antibodies to HK light chain and another antibody to detect the sum of LK plus HK heavy chains (total kininogens). The difference between the values for total kininogens and HK is assumed to represent the concentration of LK in the sample (Kerbiriou-Nabias et aL , Br. J. Haematol 56: 273-286, 1984).
- Another LK immunochemical assay employs an ELISA using a monoclonal antibody to the common heavy chain of LK and HK that has partial cross-reactivity with HK (Jerabek et aL, Agents Action suppl. 38:257-266 1992). However, it is doubtful that this assay specifically measures LK.
- HK is the contact system cofactor of plasma proteolysis because of its 56 kDa light chain that has the ability to tightly adsorb to negatively-charged surfaces. Because of its ability to form a complex with two of the contact factor zymogens, namely prekallikrein and factor XI, HK can transport these zymogens to the activating surface. This contact system cofactor activity allows HK to be assayed by coagulant activity (Colman et al., J. Clin. Invest.
- HK can also be measured, immunochemically, by several methods, including radial immunodiffusion (Scott and Colman, J. Clin. Invest. 65: 413-421, 1980), radioimmunoassay (Proud et al. , J. Lab. Clin. Med. 95: 563-574, 1980), rocket electrophoresis (Bouma et aL, J. Lab. Clin.
- LK has a small light chain of 5 kDa that does not adsorb to negatively-charged surfaces and its function is, at this time, unknown. Therefore, only antibodies raised against the unique LK light chain or peptides contained therein can specifically capture LK.
- the immobilized immunoreactant which is either an antibody or an antigen
- the solid surface typically comprises, for example, a microparticle or microsphere in the case of a particle assay, or the wells of a polystyrene microplate in the case of an enzyme-linked immunosorbent assay (ELISA).
- ELISA enzyme-linked immunosorbent assay
- HKa the active cofactor form of HK
- fibrinogen IgG
- albumin the form of HK that most readily displaces fibrinogen, IgG, and albumin from surfaces
- fibrinogen and/or HK to displace passively-adsorbed antibodies from the solid matrix can result in high coefficients of variation in these assays, especially for determinations of fibrinogen and HK.
- the primary antibody is covalently attached to the surface, the reproducibility of the assay increases (Scott and Colman, J. Lab. Clin. Med 119: 77-86, 1992).
- LK low molecular weight kininogen
- What is needed is a solid support material for immunoassays which avoids nonspecific adherence of molecules such as HK, which have an affinity for solid surfaces. What is also needed is a solid support for immunoassays which is capable of attaching, per unit of surface area, a high concentration of immunoreagent.
- a solid support for an immunoassay comprises (a) a microplate comprising a negatively charged polymeric material, and (b) a coating of polyethyleneimine on the microplate.
- a solid phase carrier for an immunoassay comprises (a) microparticles comprising a carboxylate-modified latex; (b) a coating of polyethyleneimine on the microparticles.
- the invention is directed to a solid phase carrier for an immunoassay comprising (a) a solid support; (b) a layer of microparticles coated on the solid support, the microparticles being formed of a negatively charged polymeric material; and (c) a coating of polyethyleneimine on the microparticles.
- the invention also provides solid phase immunoreagents for use in immunoassays.
- a solid phase immunoreagent comprises (a) a microplate comprising a negatively charged polymeric material; (b) a coating of polyethyleneimine on the microplate; and (c) an immunoreagent immobilized on the microplate by covalent coupling to the polyethyleneimine coating.
- a solid phase immunoreagent comprises (a) microparticles comprising a carboxy-modified latex; (b) a coating of polyethyleneimine on the microparticles; and (c) an immunoreagent immobilized on the microparticles by covalent coupling to the said polyethyleneimine coating.
- a solid phase immunoreagent for an immunoassay comprises (a) a solid support; (b) a layer of microparticles coated on the solid support, the microparticles being formed of a negatively charged polymeric material ; (c) a coating of polyethyleneimine on the microparticles; and (d) an immunoreagent immobilized on the microparticles by covalent coupling to the polyethyleneimine coating.
- a method of treating a negatively charged polymeric surface is provided to inhibit contact activation of plasma by the surface.
- the surface is coated with polyethyleneimine to inhibit contact activation of plasma brought in contact with the surface.
- the amount of polyethyleneimine comprising the coating should be sufficient to mask the negative charge of the polymeric material comprising the negatively charged polymeric support, e.g. , microplate or microparticles, and should also be sufficient, by masking such negative charges, to reduce or essentially entirely eliminate nonspecific adsorption of biological molecules to the support.
- a first solid phase immunoreagent according to the invention is contacted with a liquid containing or suspected of containing a complementary immunoreagent, whereby an immobilized immunocomplex is formed; and quantitating the immobilized immunocomplex so formed.
- the immobilized immunocomplex may be detected with a detectably labeled second immunoreagent that is immunoreactive with the complementary immunoreagent.
- the second immunoreagent typically comprises an antibody.
- the immobilized immunocomplex may be washed to remove unbound immunoreagent. The amount of label incorporated into the immobilized immunocomplex is then determined.
- the detectable label of the detectably labeled second immunoreagent may comprise, for example, a fluorescent label, a radiolabel or an enzyme label.
- the assay takes the form of a competitive solid phase immunoassay.
- a solid phase immunoreagent according to the invention is contacted with a liquid containing or suspected of containing a complementary immunoreagent and a known amount of labeled complementary immunoreagent, whereby portions of the labeled and unlabeled complementary immunoreagents are bound to the solid phase immunoreagent. The amount of the labeled complementary immunoreagent bound to the solid phase immunoreagent is quantitated.
- the immunoreagent and complimentary immunoreagent together comprise an antigen/antibody pair.
- the complementary immunoreagent is an antibody having binding affinity for the antigen.
- the immunoreagent is an antibody
- the complementary immunoreagent is an antigen for which the antibody has binding affinity.
- the immunoreagent will comprise an antibody.
- a solid support for a high molecular weight kininogen assay comprises a microplate a coating of a negatively charged polymeric material on the microplate.
- the coating on the microplate preferably comprises a layer of microparticles formed of a negatively charged polymeric material, most preferably microparticles comprising a carboxylate-modifled latex.
- the invention provides an assay method for high molecular weight kininogen comprising
- the negatively charged support comprises a microplate, and the microplate contains a coating of a carboxy late-modified latex or has a negative charge imparted via irradiation, for example, as is common in 96 well tissue culture plates.
- the invention provides a kit for kininogen assays comprising: (a) a first solid support comprising a negatively charged surface for capturing high molecular weight kininogen;
- a second solid support comprising a coating of polyethyleneimine thereon, and a capture antibody specific for low molecular weight kininogen immobilized on said second solid support by covalent coupling to said polyethyleneimine coating;
- a third solid support comprising a coating of polyethyleneimine thereon, and a capture antibody specific for kininogen heavy chain immobilized on said third solid support by covalent coupling to said polyethyleneimine coating; and (d) a supply of detector antibody that recognizes both the heavy and light chain of high molecular weight kininogen.
- the solid supports preferably take the form of microplates or microparticles.
- the detector antibody may bear a detectable label.
- a detectable label is carried by a secondary detector antibody which binds to the detector antibody.
- the detectable label comprise, for example, a fluorescent label, a radiolabel or an enzyme label.
- a supply of such secondary detector antibody may be supplied in the kininogen assay kit.
- the kit may further comprise a supply of substrate for the enzyme and a reagent to stop the hydrolysis.
- kininogen assay kit comprises a supply of one or more anticoagulants capable of preventing coagulation of blood samples during collection; a supply of one or more proteinase inhibitors capable of preventing proteolysis of kininogens following sample collection; and a 29 supply of one or more buffers.
- these optional components may be included in a series of containers.
- the kit may comprise:
- a first container comprising a mixture of one or more anticoagulants and one or more proteinase inhibitors
- a third container comprising a wash buffer composition for washing the microplates following kininogen capture.
- the contents of the containers may be in dry powder or liquid form.
- at least the mixture of anticoagulant and proteinase inhibitor contained in the first vial, and the buffer composition contained in the second vial, are in powder form.
- microplate is meant a plate, typically made of an optically transparent plastic material, which contains a plurality of wells suitable of holding samples for analysis.
- nonspecific adsorption binding by means of a mechanism other than a reaction between an antibody and its cognate antigen.
- biological molecule is meant any molecule which may be present in the biological sample under analysis.
- the level of nonspecific adsorption is reduced to a level as low as the limit of detection of the assay.
- the "background” represented by nonspecific adsorption of molecules to the solid support is reduced essentially to zero, or to a value where the signal to noise ratio is greater than 50: 1.
- Figs. 1A, IB and 1C are, respectively, standard curves for HK, LK and TotK plasma assays of normal plasma.
- Normal pooled plasma initially diluted to 1:50 with PCFIA diluent, was serially diluted with PCFIA diluent to generate the standard curves.
- the ordinate values are expressed as RFU (relative fluorescence units). Each point represents the mean +/- the standard deviation of triplicate determinations.
- Figs. 2A, 2B and 2C are, respectively, PCFIA measurements of HK, LK and
- Fig. 3 is a plot of the data from Fig. 1C vs. the data from Fig. IB.
- Kininogen- deficient plasma 0.
- Fig. 3 correlates the levels of HK and LK in the plasma of normal donors.
- Fig. 4 is a plot of the sum of the HK values (light chain of each) vs. values for
- TotK (heavy chain) in the plasma of normal donors.
- Fig. 4 correlates the sum of LK plus HK vs. TotK.
- Figs. 5 A, 5B and 5C and 5D are standard curves for LK plasma assays generated from normal pooled plasma using polyethyleneimine-modified latex microspheres (Fig. 5A) and conventional, positively-charged amino-modified microspheres (Figs. 5B, 5C and 5D).
- Figs. 6A, 6B and 6C and 6D are standard curves for TotK plasma assays generated from normal pooled plasma using polyethyleneimine-modified latex microspheres (Fig. 6A) and conventional, amino-modified latex microspheres (Figs. 6B, 6C and 6D).
- Fig. 7 is a plot of the results of a PCFIA total kininogen assay versus ELISA total kininogen assay results from the total kininogen assay of six plasma samples. An r value of 0.98 was obtained.
- Fig. 8 is a plot of the results of a PCFIA LK assay versus ELISA LK assay results from the LK assay of seven plasma samples. An r value of 0.98 was obtained.
- Fig. 9 is a plot of a total kininogen ELISA of samples of known total kininogen concentration carried out in a 96-well microplate wherein the wells were provided with the following coatings prior to attachment of total kininogen monoclonal antibody: a passive coating of polyethyleneimine (solid circles); a coating of polyethyleneimine covalently attached to the well via EDC. HCl (hollow circles); a coating of carboxy-modified latex particles on to which polyethyleneimine was passively adsorbed (solid triangles); a coating of carboxy-modified latex particles to which polyethyleneimine was covalently coupled via EDC. HCl (hollow triangles).
- Fig. 10 is a plot of a PCFIA determination of goat IgG constructed from serial dilutions of purified goat serum.
- Fig. 11 is a standard curve for an HK plasma ELISA, generated from normal pooled plasma using microplates coated with carboxylate-modified latex as a solid support for capturing HK.
- solid phase immunoassay an assay which utilizes an immunoreagent to capture and detect a complementary immunoreagent, wherein the immunoreagent is bound to a solid support.
- immunoreagent and “complementary immunoreagent” is meant a pair of reagents which bind to one another by an antibody-antigen binding reaction. It may be appreciated that where the immunoreagent is an antibody, the complementary immunoreagent will comprise an antigen which binds the antibody. It may be appreciated that where the immunoreagent is an antigen, the complementary immunoreagent will comprise an antibody which binds that antigen.
- the antibody may comprise a polyclonal or monoclonal antibody which binds the target antigen.
- the antibody may comprise an intact antibody, or fragments thereof capable of binding antigen, including but not necessarily limited to, Fab and F (ab') 2 fragments.
- the term "antibody” includes intact antibody molecules and fragments thereof which retain antigen binding ability.
- the antigen is typically a protein or peptide antigen, but non-protein antigens, e.g. , carbohydrates, lipids, cellular wall components, etc. , may also be detected provided the appropriate antibodies are available.
- An antibody fixed to a solid support, used to capture an antigen from a patient sample is referred to hereinafter as a "capture antibody” .
- the capture antibody binds to a determinant of the antigen.
- the detector antibody binds to another determinant of the captured antigen, which determinant remains exposed on the antigen molecule after the antigen is bound by the capture antibody. It may be appreciated that the detectable label may be affixed directly to the detector antibody (primary detector antibody), or may be fixed to a third antibody which binds to an antigenic determinant on the detector antibody (secondary detector antibody).
- the label may be supplied by donkey anti-sheep IgG.
- the secondary detector antibody may thus comprise a conjugate of donkey anti-sheep
- HRP horseradish peroxidase
- the coating material on the solid support comprises polyethyleneimine.
- polyethyleneimine is meant any polymer which consists of about 85% or more of ethyleneimine (also known as aziridine) monomer.
- the ethyleneimine monomer has the structure (CH 2 CH 2 NH).
- polyethyleneimine is meant to include not only homopolymers of ethyleneimine, but also copolymers thereof comprising at least about 85% ethyleneimine units.
- the polymer is a homopolymer of ethyleneimine.
- a preferred polyethyleneimine has an average molecular weight of about 50,000.
- the antigen which comprises either the capturing immunoreagent or the captured complementary immunoreagent may comprise any substance or antigenic determinant of a substance which may be present in a biological sample of interest, e.g., blood, saliva, urine, feces, etc., the detection or quantification of which is desired.
- the complementary immunoreagent is total kininogen (HK plus LK), or LK alone.
- the solid support which is modified by polyethyleneimine treatment as described herein comprises a negatively charged polymeric material.
- the support may be formed entirely of the negatively charged polymeric material, or may comprise only a coating of negatively charged polymer.
- the surface negative charge of the support permits enhanced coupling of the polyethyleneimine layer which has been found to drastically reduce the level of nonspecific adsorption of biological molecules to the underlying solid support.
- the support may take any convenient physical form, such as a membrane, grid, slide, film, stick, tube or particle.
- carboxylate-modified latex particles are the preferred solid supports.
- the support may take the form of a microplate bearing a coating of carboxylate-modified particles.
- the surface may be coated with a functionalized polymer, most preferably with a carboxylate-modified latex polymer. Where the support is not itself comprised of a negatively charged polymeric material, a coating of such charged material must be applied.
- the underlying support may comprise any of the materials which find use as carriers for immunoreagents in solid phase immunoassays.
- materials include glass, metal, and various plastics such as polystyrene, polycarbonate, polyvinyl chloride , polyethylene , polypropylene , silicone resins , and the like , without regard to the requirement of negative charge, as the necessary negative charge is supplied by the added coating of negatively charged polymeric material.
- the coating carrying the necessary negative charge may be applied, for example, as a suspension of latex particles, e.g., a suspension of carboxylate-modified latex particles such as SERACOAT (Seradyn, Inc., Indianapolis, IN).
- polystyrene microplates which are used in a wide variety of biological assays, are typically formed of virgin polystyrene, which is essentially uncharged. Thus, such standard microplates will not be satisfactory supports according to the practice of the present invention unless coated with a coating of an appropriated negatively charged polymeric material. On the other hand, the added coating of negatively charged polymer is unnecessary in the case of certain modified polystyrenes, which have been modified to impart a negative charge.
- Tissue culture plates are typically formed from irradiated polystyrene, which is negatively charged. The negative charge assists in cell attachment to the plate. It is also known to modify polystyrene by incorporation of carboxyl groups.
- Carboxylate-modified styrene microparticles are commercially available from Seradyn Inc., Indianapolis, IN.
- the negative charge in these carboxylate-modified polystyrene plates arises from sulfate groups which terminate the polymer chains, adsorbed surfactant used in the process of preparing the microparticles and the carboxy late groups of carboxy late-containing monomers.
- the solid support which is either formed of a negatively charged polymeric material or at least bears a surface coating of a negatively charged polymeric material, is then treated to acquire at least a surface coating of polyethyleneimine.
- coatings include not only polyethyleneimine layers formed by passive physical adherence to the underlying support surface, but also coatings of polyethyleneimine which are chemically fixed to the support surface by reaction with an appropriate coupling agent or agents.
- reference herein to a support "coated” or “treated” with polyethyleneimine means not only the application of polyethyleneimine by passive adsorption, but also active chemical coupling of polyethyleneimine to the support surface by means of coupling agents.
- the preferred form of coupling involves formation of a covalent bond between the support surface and the polyethyleneimine coating .
- the application of polyethyleneimine to particles such as microscopic particles, and the deposition of such particles as a coating on the surface of a macroscopic support such as a slide or microplate.
- the polyethyleneimine modification of the solid support when applied to an underlying surface having a negative charge, virtually eliminates nonspecific adsorption of molecules such as HK which otherwise adhere to solid surfaces.
- proteins such as antibodies or protein antigens, may be easily coupled to the polyethyleneimine-modified surface to serve as the immunoreagent.
- polyethyleneimine is used as a coating for negatively charged polymeric materials to inhibit the material's ability to induce contact activation.
- Factor XII in plasma when it contacts with a negatively charged surface, will initiate the intrinsic blood coagulation pathway. For this reason, negatively charged polymeric materials cannot be used as supports or vessels for the conduct of assays where plasma is present.
- the contact activation function of such polymeric materials is substantially reduced, or essentially eliminated, by application of a coating of polyethyleneimine.
- the polymeric material can now be used as a vessel or a support in a biological assay where plasma is present without fear of spurious results due to triggering of the contact activation system.
- the negatively charged polymeric surface comprises a surface of a microplate.
- the negatively charged polymeric surface comprises a surface of a filter or membrane which is used to collect, process, store, or assay plasma or other blood products.
- the negatively charged polymeric surface comprises a surface of a test tube, vacutainer tube, or other vessel tube which is used to collect, store, assay, or process plasma or other blood products.
- the required degree of masking of the negative charge depends upon the time span of the assay or other procedure employing the vessel or solid support. For example, if the support is to be used in a plasma assay which is to be completed within one hour, a modest coating of polyethyleneimine will suffice. If the support is to be used in a plasma assay of several hours duration, then a more extensive polyethyleneimine coating will be required to ensure that substantially all the surface negative charges are adequately masked. The longer the assay incubation time, the more likely contact activation of the plasma sample will occur, unless steps are taken to block the negative surface charges of the support.
- the presence of contact activation in a plasma sample can be conveniently monitored by the appearance of kallikrein, which is released from the zymogen prekallikrein by the action of factor Xlla.
- the generation of kallikrein can be monitored by the substrate S-2302 (Chromagenix, Inc.), for example.
- the solid support comprises microparticles of a polymer having a negative charge.
- Such particles are generally prepared in the form of polymer latices and take the form of microparticles or microspheres.
- latex is intended to have its ordinary meaning, that is, a stable dispersion of a polymeric substance in an essentially aqueous medium.
- Synthetic latices are generally formed by emulsion polymerization techniques from, e.g. , styrene-butadiene copolymer, acrylate resins, polyvinyl acetate, and similar materials.
- the polymeric support material to which the polyethyleneimine treatment is applied comprises carboxylate-modified polymer microparticles, the preparation of which is described, for example, in U.S. Pat. 5,100,805 to Ziege et a , the entire disclosure of which is incorporated herein by reference.
- the primary components of such polymer particles can be, for example, a vinyl aromatic monomer, and a vinyl acrylate ester monomer, the vinyl aromatic monomer can be styrene, vinyl toluene, t-butyl styrene or mixtures thereof. Styrene is preferred.
- the vinyl acrylate ester monomer can be a monomer having pendent alkyl ester groups of from one to six carbon atoms, for example.
- the vinyl acrylate ester monomer can be methyl, ethyl, propyl, n-butyl, s-butyl, or other versions of the acrylate monomer, or versions of a methacrylate vinyl unit, such as methyl methacrylate, ethyl methacrylate, propyl methacrylate, and the like.
- the particles are prepared by conventional emulsion polymerization methods as described in Pat. 5, 100,805.
- a carboxylate containing monomer e.g. acrylic acid
- Acrylic acid is preferred.
- Carboxylate-modified microparticles and latex coating compositions are commercially available, for example, from Seradyn, Inc., Indianapolis, IN under the trade names CML (carboxylate-modified latex microspheres) and SERACOAT ® (carboxy-modified latex coating material).
- CML carboxylate-modified latex microspheres
- SERACOAT ® carboxy-modified latex coating material
- a representative lot of the CML material is composed of microspheres having a diameter of 0.8 micron containing 0.023 COOH acid milliequivalents per gram.
- Polyethyleneimine coating of the immunoassay solid support surface is most advantageously carried out with a suitable coupling agent. The amount of polyethyleneimine applied should be sufficient to completely mask the negative charge on the underlying polymeric support.
- the amount of polyethyleneimine applied should also be an amount effective to reduce, preferably eliminate, nonspecific adsorption of biological molecules to the negatively charged polymeric support.
- the coupling agent may comprise any chemical agent which is effective in enhancing the attachment of the polyethyleneimine to the support, e.g. to the particles of the latex.
- the coupling agent acts to covalently link the polyethyleneimine coating to the support.
- the preferred coupling agent to fix the polyethyleneimine coating is a carbodiimide, e.g., dicyclohexylcarbodiimide (DDC); l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)orhydrochlorideofEDC; or l-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulphonate (CMC).
- EDC hydrochloride is preferred.
- the carbodiimide reacts with the carboxylic acid group of the carboxylate-modified latex and activates the carboxyl group, allowing it to be coupled to amino groups in the polyethyleneimine .
- the polyethyleneimine coating modification may be carried out by combining the latex particles and the polyethyleneimine, followed by addition of the coupling agent, with stirring, at room temperature.
- the amount of polyethyleneimine added to the mixture should be sufficient to achieve a uniform coating on the latex particles .
- the amount of coupling agent is any amount to obtain attachment of the deposited polyethyleneimine onto the latex particle surface.
- the amount of carbodiimide should exceed the particle's carboxylate content on an equivalent basis, preferably by at least about two fold.
- carboxy-modified latex microspheres (“CML", Seradyn, Inc.) (100 ml of 10% solids) are added to 1 % polyethyleneimine with constant stirring.
- EDC hydrochloride dissolved in water is added and the volume is adjusted with water.
- the mixture is stirred and the microspheres are then centrifuged and washed with water.
- the modified microspheres may then be stored at 4°C in 0.02% NaN 3 .
- Protein immunoreagents such as antibodies or protein antigens, are easily covalently attached to the polyethyleneimine-modified supports , such as microspheres .
- the polyethyleneimine treatment does not interfere with protein coupling to the microsphere.
- the desired protein immunoreagent may be coupled to the polyethyleneimine-modified microspheres, for example, using any of the known coupling agents suitable for the modification and coupling of proteins to solid support materials, provided the coupling does not interfere materially with the ability of the protein to bind its complementary immunoreagent.
- Any coupling agent which has a first reactive group which can attach to a reactive amino or imino group of the polyethyleneimine, and a second reactive group which can react with and form a covalent bond with a group on a protein molecule may be used.
- the coupling agent may comprise the same coupling agent used in carrying out the modification of the latex microspheres to attach the polyethyleneimine coating to the particle surface.
- the agent for coupling the polyethyleneimine to carboxylate-modified latex particles, and the agent for coupling the immunoreagent to the polyethyleneimine-modified particle both comprise a carbodiimide, most preferably EDC or EDC hydrochloride.
- the immobilized immunoreagent is contacted with a liquid comprising or containing the biological test sample of interest.
- the test sample may be a raw biological liquid. More typically, the raw sample is diluted or partially purified prior to assay by conventional techniques known to those skilled in the immunoassay art.
- the sample and immobilized immunoreagent are incubated for a time sufficient to permit formation of an immunocomplex of the immunoreagent and its complementary immunoreagent.
- the time for an ELISA should not be less than about 1.5 hours for each incubation step of antibody with antigen. Longer times may be employed. For PCFIA, the time should be not less than 15 minutes. Longer times may be employed. While the temperature and pH of the incubation step may vary, room temperature (23 °C) and a pH between 6.0 and 8.0 are preferred.
- the immobilized immunocomplex is formed, either with or without intervening wash steps, it is contacted with a second immunoreagent which bears a detectable label.
- the second immunoreagent may comprise either an antigen or an antibody, but typically comprises an antibody.
- the second immunoreagent is labeled directly with the detectable label, or is labeled indirectly through coupling to another molecule which bears the detectable label.
- the label may advantageously comprise, for example, a radionuclide in the case of a radioimmunoassay; a fluorescent moiety in the case of an immunofluorescent assay; a chemiluminescent moiety in the case of a chemiluminescent assay; and an enzyme which cleaves a chromogenic or fluorogenic substrate, in the case of an enzyme-linked immunosorbent assay.
- a radionuclide in the case of a radioimmunoassay
- a fluorescent moiety in the case of an immunofluorescent assay
- chemiluminescent moiety in the case of a chemiluminescent assay
- an enzyme which cleaves a chromogenic or fluorogenic substrate in the case of an enzyme-linked immunosorbent assay.
- an indicator substance for example, a chromogenic substrate
- an instrument for quantification, an instrument is required.
- fluorescent labeled moieties can be monitored using excitation at an appropriate wavelength, while chemiluminescent labeled antigens or antibodies can be followed after reaction by chemically activating the chemiluminescent labels to generate light which can be detected by photometric means.
- a calibration curve for the determination of an antigen (or antibody) can be constructed by measuring the amount of antigen- antibody complex formed upon addition of varying and known amounts of antigen (or antibody) to a solution containing a fixed and known amount of antibody (or antigen).
- the immunoassay may also take the form of a competitive immunoassay.
- an immunoreagent e.g. , capture antibody
- a solid phase immunoreagent e.g. , labeled antigen
- Labeled complementary immunoreagent e.g. , labeled antigen
- the labeled and unlabeled antigens will compete for binding to the antibody. The higher the value of the amount of labeled antigen bound to the antibody, the lower the concentration of antigen in the sample.
- HK does not adsorb to the polyethyleneimine-modified solid support.
- such supports are particularly useful in immunoassays for the direct measurement of substances in human biological fluids where HK may also be present (e.g. , plasma).
- the polyethyleneimine-modified support materials are useful in the direct measurement of LK in biological fluids.
- latex microspheres after covalent attachment of purified antibody specific for LK light chain, may be utilized in a particle concentration fluorescence immunoassay (PCFIA) .
- PCFIA particle concentration fluorescence immunoassay
- ELISA enzyme-linked immunosorbent assay
- the ELISA plates are modified in the same way as the microspheres, and the appropriate antibody or antigens are covalently linked to the microplate surface.
- polyethyleneimine-modified microspheres to which a first (capture) antibody specific for LK light chain has been covalently attached, are placed in each well of a 96 well assay plate fitted with a 0.45 ⁇ m membrane (Idexx Corporation, Westbrook, ME).
- the wells of the assay plate contain appropriate dilutions of a reference sample to derive a standard curve.
- Other wells contain diluted unknown samples.
- the samples are incubated with the microspheres, at room temperature, until all antigen (LK) in the sample is captured by the antibody on the microspheres (usually 15-20 minutes).
- a second (detector) antibody to which a fluorophore has been attached, is added and the mixture is incubated until the detector antibody is bound to all antigen in the sample (usually 10-20 minutes).
- FCA fluorescence concentration analyzer
- FCA fluorescence concentration analyzer
- the LK concentration from the unknown samples is determined by comparing the fluorescence value of the sample with the standard curve fluorescence values generated by the reference sample dilutions. Using this method, it is possible to assay 24 samples in triplicate or 40 samples in duplicate together with seven known standards and a buffer control in less than one hour.
- the LK assay using polyethyleneimine-modified microspheres provides for the specific determination of LK that still contains its light chain. In contrast, when conventional microspheres lacking the polyethyleneimine modification are used as the solid matrix, HK strongly adsorbs to the surface.
- the diluent for diluting the samples advantageously comprises, e.g., 10 mM sodium phosphate, pH 7.4, containing 150 mM NaCl, 0.1 % Tween 20, 2% bovine serum albumin, and an assortment of proteinase inhibitors described more particularly below.
- the capture antibody and/or detector antibody may comprise a polyclonal or monoclonal antibody specific for LK light chain, e.g., the monoclonal antibody "LKL-1 " described by Kaufmann et al.. J. Biol. Chem. 268:9079-9091 (1993).
- the capture antibody and/ or the detector antibody may comprise intact antibody, or fragments thereof capable of binding antigen, including, but not necessarily limited to, Fab and F(ab') 2 fragments.
- Monoclonal LK antibody may be purified from the ascites of the appropriate LK antibody-producing hybridoma according to conventional antibody purification techniques.
- antibody may be purified from ascites fluid using commercially available purification kits, such as the IMMUNOPURE ® Protein A/G column (Pierce Chemical Company Rockford, IL) using the buffers and instructions contained within the kit. After purification, the antibody is concentrated and dialyzed, and stored in buffer.
- Hybridomas producing monoclonal antibody specific for LK light chain may be prepared according to the procedure of Kaufmann et al, supra. The entire disclosure of Kaufmann et aL is incorporated herein by reference.
- a monoclonal antibody having the specificity of MAb LK1-1 may be prepared by first generating, as the immunogen, a conjugate consisting of the peptide Cys-Glu-Tyr-Lys-Gly-Arg-Pro-Pro-Lys-Ala-Gly-Ala-Glu-Pro-Ala-Ser-Glu-Arg-Glu- Val-Ser (SEQ ID NO: 1) conjugated to either keyhole limpet hemocyanin or bovine serum albumin as a carrier.
- the peptide (SEQ ID NO: 1) corresponds to amino acid residues 389-409 of human LK.
- Coupling of SEQ ID NO: 1 to the carrier may be carried out via disulfide linkage formation by first treating the carrier protein with maleimidobenzoyl-N-hydroxysuccinimide ester prior to peptide coupling (Liu et al. , Biochemistry 18, 690-697, 1979).
- Murine monoclonal antibody against the peptide conjugate is then generated in BALB/c mice according to standard procedures (K ⁇ hler and Milstein, Nature 256, 495-497 1975) with minor modifications described by Hock etal. , J. Biol. Chem. 265, 12005-12011, 1990.
- X63/Ag 8.653 myeloma cells are used for fusion with the harvested immune spleen cells.
- Colonies producing antibody of the desired specificity are subcloned three times by the limiting dilution method.
- syngeneic BALB/c mice may be injected intraperitoneally with 0.5 ml of 2,6, 10, 14-tetramethylpentadecane (Pristane ⁇ Aldrich Chem. Co.), followed ten days later by application of 5 x 10 6 hybridoma cells in 0.5 ml of RPMI 1640 medium via the same route. Ascites is collected and used for antibody purification.
- the present invention provides an easy method for the determination of LK in plasma and other biological fluids, as well as a method for the simultaneous evaluation of both HK and LK.
- Kininogen assays according to the present invention are linear over, at least, a 30-fold range ( Figure 1). Previous descriptions of LK determinations in plasma were, for the most part, indirect measurements. Alternatively, measures had to be taken to separate HK from LK prior to assay because of the problem of HK and LK sharing a common heavy chain.
- the method of the present invention provides for the first time, a direct, rapid, quantifiable, measurement of LK, using a monoclonal antibody such as LKL- 1.
- the same detector antibody and the same diluted patient sample may be used to assay HK, LK and total kininogen (TotK).
- This feature permits one to perform "kininogen profiling" studies to assess not only the absolute concentration of the kininogens, but also the specific concentration of kininogens, as well as the extent of kininogen cleavage.
- the solid support comprises a conventional negatively -charged microsphere to which no capture antibody need be coupled, since HK is captured by the microsphere by virtue of its ability to adhere to negatively-charged solid surfaces.
- the microsphere comprises a carboxylate-modified latex. The sample is contacted with the microspheres, and HK binds to the negatively charged surface. Washing the microspheres removes the unbound material and leaves immobilized HK on the particle surface.
- the solid support comprises polyethyleneimine-modified microspheres to which an antibody has been attached which recognizes the heavy chains of both HK and LK.
- the antibody may comprise monoclonal antibody 2B5 , obtained from hybridoma ATCC #HB-8963 , the preparation of which is described in U.S. Patent No. 4,908,431. The entire disclosure of Pat. 4,908,431, is incorporated herein by reference.
- the support comprises polyethyleneimine-modified microspheres to which is attached an antibody which is specific for LK light chain, and does not recognize HK, such as the LK-1 MAb.
- a carboxylate-modified latex of smaller particle sizes, 0.1 ⁇ m in diameter (SERACOAT * TC3X, Seradyn, Inc.) is used to coat the wells of a 96-well microplate. The plate is dried.
- SERACOAT * TC3X Seradyn, Inc.
- TC3X TC3X
- the plate is dried.
- EDC. HCl (9.6 mg/ml) was added and the mixture is then immediately added to the latex-coated microplate. The wells are completely filled with the solution and allowed to stand to fix the polyethyleneimine coating on the carboxylate-modified latex particles.
- the immunoreagent coupled to the latex/polyethyleneimine-coated plate comprises an MAb, such as MAb 2B5, which recognizes both HK and LK heavy chains.
- the immunoreagent comprises an MAb such as MAb LKL-1 which is specific for LK light chain.
- the antibody is applied, at a concentration of 5 ⁇ g/well, to each well in a solution containing EDC. HCl as a coupling agent, followed by incubation for at least about two hours.
- the wells of the assay plate are filled with appropriate dilutions of a reference sample to derive a standard curve. Other wells contain diluted unknown samples.
- the wash buffer may comprise, for example, 10 mM sodium phosphate, pH 7.0, containing 0.5 M NaCl, 0.2% NaN 3 and 0.1 % polysorbate 20 (Tween 20).
- sheep anti-human HK diluted in buffer 200 ⁇ l diluted 1: 1500 in buffer
- the plates are incubated for 1.5 hours, then washed.
- donkey anti-sheep IgG to which the enzyme horseradish peroxidase has been conjugated is diluted in buffer and applied to each well.
- Alkaline phosphatase cannot be used because there are phosphatases in human plasma that can hydrolyze the substrate and cause a background. After room temperature incubation for two hours, the plates are washed and then enzyme substrate is added to each well. The optical density of the hydrosylate is determined with respect to standards to provide the TotK or LK concentration in the unknown samples.
- HK in biological fluids can adsorb to negatively charged surfaces without the need for a capture antibody.
- an HK ELISA may be conducted without the need for an immobilized capture antibody, or a polyethyleneimine coating on the ELISA microplate.
- Carboxylate-modified latex of smaller particle sizes e.g., the 0.1 ⁇ m diameter SERACOAT" TC3X particles may again be used to coat the wells of a 96-well microplate.
- the carboxylate-modified latex provides a surface negative charge which binds HK.
- the microplate may comprise an uncharged material, such as virgin polystyrene, or a modified polystyrene, which has been modified to impart a negative charge.
- the surface coating of carboxylate-modified latex may be omitted.
- coating of the plate with the latex is preferred, even in cases where the microplate may already comprise a negatively charged material.
- the wells of the microplate are filled with appropriate dilutions of a reference standard to derive a standard curve. Other wells contain diluted unknown samples. After 1.5 hours of incubation at room temperature, the plates are washed with wash buffer and contacted with sheep anti-human HK as a primary detector antibody, donkey anti-sheep IgG-HRP as the secondary detector antibody, and enzyme substrate, as above.
- the ELISA is completed by determining the optical density of the enzyme hydrosylate, and comparing the value with standard values to provide the HK concentration in the unknown samples.
- the kininogen assay embodiments of the invention are amenable to practice in kit format.
- a kit suitable for HK, LK and TotK assays, contains solid supports, preferably in the form of microplates (for ELISA) of microspheres (for PCFIA).
- a microplate or microsphere for conducting an HK assay has a negatively charged surface for capturing HK.
- a microplate or microsphere for an LK assay carries a coating of polyethyleneimine and a capture antibody specific for LK.
- a microplate or microsphere for TotK assay carries a coating of polyethyleneimine and a capture antibody specific for kininogen heavy chain.
- the capture antibodies are covalently coupled to the polyethyleneimine coating on the microplate or microsphere.
- the kininogen assay kit further contains a supply of detector antibody which recognizes both the heavy and light chain of HK.
- the detector antibody can thus be used to quantitate the kininogen captured in any of the three kininogen assays — HK, LK or TotK.
- a detectable label is carried by the detector antibody.
- the detector antibody may comprise anti- human IgG-FITC, for example.
- the detectable label is carried by a secondary detector antibody which binds the primary detector antibody.
- a supply of the secondary detector antibody e.g. , goat anti-sheep IgG-HRP, is thus optionally included in the kit for ELISA.
- An ELISA kit may further contain a supply of substrate for the enzyme, e.g. , HRP, and a reagent to stop hydrolysis of the substrate by the enzyme, e.g. , citric acid or H 2 S0 4 .
- the kininogen assay kit further optionally includes appropriate auxiliary reagents for collecting, storing and assaying patient samples. These reagents are formulated to stabilize patient samples, to prevent clotting, and to prevent cleavage of kininogens by proteinases in the test sample.
- One such auxiliary reagent is a mixture of proteinase inhibitors and anticoagulant, such as described in Example 2A, below.
- the preferred cocktail contains acid-citrate-dextrose (ACD) as an anticoagulant
- other suitable anticoagulants include heparin or oxalic acid.
- Patient blood samples are advantageously drawn into a plastic syringe containing an amount of proteinase inhibitor cocktail sufficient to prevent coagulation and kininogen cleavage in the sample. A sufficient amount is approximately one tenth of the volume of the blood sample being drawn.
- the proteinase inhibitor cocktail is described herein as a liquid, it may be appreciated that the cocktail can be formulated in dry powder form, which is then diluted with an appropriate amount of sterile water to reconstitute the liquid form prior to use.
- auxiliary reagent for the kininogen assay kit comprises a diluent buffer for diluting patient samples.
- the diluent buffer is advantageously based upon phosphate buffered saline, and includes stabilizers such as polysorbate 20 (Tween 20), bovine serum albumin, and preferably a small amount of proteinase inhibitor cocktail to ensure that the proteinases in the patient sample are not activated upon dilution of the sample with the diluent buffer.
- PBS-TBI a representative diluent buffer
- Example 1C Example 1C, below. While the diluent buffer is described herein as a liquid, it may be appreciated that the buffer can be formulated in dry powder form, which is then diluted with an appropriate amount of sterile water to reconstitute the liquid form prior to use.
- Another auxiliary reagent for the kininogen assay kit comprises a wash buffer for washing unbound material from microplates or microspheres following capture of kininogens.
- a representative wash buffer consists of 10 mM sodium phosphate, pH 7.0, containing 0.5 M NaCl, 0.02% sodium azide and 0.1 % polysorbate 20.
- the wash buffer is advantageously stored in the kit as a 10 X concentrate, which is then diluted 1: 10 with sterile water prior to use.
- the wash buffer may be stored in dry form, and diluted with an appropriate amount of sterile water to reconstitute the liquid form prior to use.
- Patient blood samples (about 5 ml) for the kininogen assays may be collected into plastic syringes containing 0.5 ml of proteinase inhibitor cocktail.
- the blood should be immediately centrifuged to remove cells.
- the cell-free plasma may be stored frozen at -70 °C.
- Microspheres for the direct measurement of LK were prepared as follows with the monoclonal antibody LKL-1, which is directed against the LK light chain, as the capture antibody.
- LKL-1 Monoclonal antibody LKL-1 (Kaufmann et al. , J. Biol. Chem. 268:9079-9091 , 1993) was purified from ascites on an IMMUNOPURE ® Protein A/G column (Pierce Chemical Company, Rockford, IL) using the buffers and instructions contained within the kit.
- LKL-1 is a mouse antibody specific to human LK light chain. After purification, the antibody was concentrated and dialyzed in a CENTRICON 30 concentrator (Amicon Corp., Danvers, MA). The buffer was exchanged with 10 mM sodium phosphate, pH 6.0 + 0.5 mM EDTA. Portions (1.5 ml) of the purified antibody were frozen until needed. It was necessary to completely remove any traces of sodium azide (NaN 3 ) from the buffer in which the protein was stored as it interfered with the subsequent covalent coupling.
- Benzamidine 100 mM
- 400 ⁇ g/ml hexadimethrine bromide 2 mg/ml soybean trypsin inhibitor
- 20 mM EDTA 263 ⁇ M leupeptin (all from Sigma Chemical Co.)
- 1 mM Phe-Phe-Arg-chloromethyl ketone PPACK II
- 20 mM [4-(2-aminoethyl)- benzenesulfonylfluoride.HCl (AEBSF) both from Calbiochem, LaJolla, CA
- ACD anticoagulant acid-citrate-dextrose
- PBS-Tween was composed of 10 mM sodium phosphate, pH 7.0, 150 mM sodium chloride, 0.02% NaN 3 , and 0.1 % Tween 20 (Sigma Chemical Co.).
- PBS-Tween-BSA was prepared by adding 5 ml of 10% bovine serum albumin to 45 ml PBS-Tween.
- a diluent, "PBS-TBI” was prepared consisting of PBS-Tween containing 1 % BSA and 1 % proteinase inhibitor cocktail.
- Covalent linkage of the mouse anti-human LK monoclonal antibody LKL-1 to polyethyleneimine-modified latex microspheres was carried out as follows. In a 50 ml conical screw cap polypropylene centrifuge tube were placed 32.6 ml H 2 0, 2 ml of 100 mM sodium phosphate, pH 6.0 + 5 mM EDTA, and 2 ml of 8% (w/vol.) polyethyleneimine-modified microsphere prepared according to Example 1. The contents were briefly mixed before adding 1.2 ml of monoclonal antibody LKL-1
- the tube was vigorously mixed to thoroughly resuspend the coated particles and then placed on a nutator for 30 minutes.
- the microspheres were then centrifuged and washed twice with phosphate buffered saline containing 0.1 % Tween 20 (Sigma Chemical Co. St. Louis, MO).
- the coated microspheres were then brought to a final volume of 60 ml in PBS- Tween-BSA containing 0.02% NaN 3 and stored at 4°C.
- the coated microspheres were stable for at least 12 months. Sixty ml of the microspheres is sufficient to perform 3,000 LK determinations.
- LKL- 1 -containing microspheres were diluted 1:30 before they were added to the assay plate. Coupling efficiency was similar for each batch of particles prepared. The concentration of LKL- 1 in the final microsphere suspension was 12.12 +/- 1.49 ⁇ g/ml microsphere suspension. F. Binding Capacity of Microsphere for Low Molecular Weight Kininogen
- Microspheres for the simultaneous direct measurement of HK and LK were prepared as follows with a monoclonal antibody, 2B5, directed against the common heavy chains of HK and LK.
- Patent 4,908,431 was purified from ascites according to the procedure of Example 2, part A.
- MAb 2B5 is a mouse monoclonal antibody directed against the common heavy chains of human HK and LK.
- MAb 2B5 was covalently linked to polyethyleneimine-modified microsphere prepared according to Example 1 , in the same manner as Example 2, part D, except that the final volume of microspheres, after coupling, was brought to 40 ml. Coupling efficiency of the antibody to the microsphere was determined as in Example 2, part E.
- the concentration of MAb 2B5 was 7.99 +/- 3.69 ⁇ g/ml. Approximately 2,000 total kininogen determinations could be performed with 40 ml of these microspheres.
- a polyclonal antibody to whole HK (that recognizes both heavy and light chains of HK and the heavy chain of LK), labeled with FITC was purchased from The Binding Site, Ltd., San Diego, CA.
- the same antibody, unlabeled was also used and was combined with the FITC-labeled antibody by adding twenty ⁇ l of the FITC- labeled antibody (10 mg/ml), 100 ⁇ l of unlabeled antibody (14 mg/ml) and 7820 ⁇ l PBS-Tween-BSA to a glass vial.
- the tracer antiserum could be stored at 4°C for up to 2 weeks.
- PCFIA was performed as follows, in triplicate, in 96 well assay plates fitted with a 0.45 ⁇ m membrane (Idexx Corporation). Twenty ⁇ l of LKL- 1 -coated microspheres prepared according to Example 2 were placed in each well followed by 10 ⁇ l of reference plasma diluted in PBS-TBI (ranging from 1 :50 to 1:3200) or 10 ⁇ l of unknown plasma samples from 38 normal donors that had been diluted 1 : 100 in PBS-TBI. Normal pooled plasma containing 93 ⁇ g/ml LK was used so that the value of 186 ⁇ g/ml was chosen for the 1 : 50 diluted reference plasma. PBS-TBI was used as the blank.
- FCA Fluorescence Concentration Analyzer
- the curves were fitted by the Winplate version 1.1 software and the averaged values for the triplicate or duplicate determinations were automatically calculated from the standard curve.
- the standard curve for LK generated with the normal pooled plasma appears as Fig. IB.
- LK could be detected in the unknown samples by PCFIA between 50 and 1900 ng/ml.
- the assay was liner in this range.
- the LK concentration in 38 normal donors ranged from 61 to 156 ⁇ g/ml with a mean of 93 ⁇ g/ml (Fig. 2B).
- HK and LK share a common heavy chain and because MAb 2B5 is directed towards the heavy chain (Schmaier et aL, J. Biol. Chem. 262: 1405-1411 , 1987), using 2B5 as the capture antibody results in the simultaneous measurement of both HK and LK (or total plasma kininogens). For brevity, this determination is called, "TotK” (for total plasma kininogens). TotK-PCFIA was performed as described above for LK-PCFIA except that 20 ⁇ l of 2B5 MAb-coated microspheres (Example 3) were substituted for the LKL-1 MAb-coated microspheres used in the LK-PCFIA.
- a pool of normal plasma containing 173 ⁇ g/ml TotK was used as the standard. The value of 346 ⁇ g/ml was chosen for the 1:50 diluted reference plasma. The standard curve for TotK generated with the normal pooled plasma appears as Fig. 1C. TotK could be detected in the unknown samples by PCFIA between 100 and 3500 ng/ml. The assay was liner in this range. TotK in the 38 normal donors ranged from 129 to 274 ⁇ g/ml with a mean of 176 ⁇ g/ml (Fig. 2C).
- HK adheres to carboxylate-modified latex even in the absence of a capture antibody coating.
- LK does not adhere to such microspheres in the absence of an antibody coating specific for LK.
- uncoated carboxylate-modified latex microspheres CML, Seradyn, Inc, were used as the solid support to capture HK instead of the C11C1 MAb-coated microspheres of Scott and Colman, supra.
- HK-PCFIA was performed using 20 ⁇ l of the uncoated CML microspheres (0.25%) and 20 ⁇ l of diluted reference plasma or donor plasma. Using a normal pooled plasma containing 80 ⁇ g/ml HK, a value of 160 ⁇ g/ml was chosen for the 1 :50 diluted reference plasma. The standard curve for HK generated with the normal pooled plasma appears as Fig. 1A. HK could be detected in the unknown samples by PCFIA between 50 and 1600 ng/ml. The assay was linear in this range. HK in the 38 normal donors ranged from 59 to 114 ⁇ g/ml with a mean of 82 ⁇ g/ml (Fig. 2A).
- the interassay coefficients of variation (c.v.) for HK, LK, and TotK were 3.34, 5.45, and 3.47 %, respectively for a sample containing 1 Unit/ml kininogens (where 1 Unit is defined as the amount in 1 ml of normal, pooled plasma) and 5.23, 6.59, and 7.48 %, respectively, for a sample containing 0.1 Units/ml.
- the intraassay coefficients of variation were 4.79, 5.49, and 4.18 % for HK, LK, and TotK, respectively, for a sample containing 1 Unit/ml kininogens and 3.76, 1.69, and 3.23 %, respectively, for a sample containing 0.7 Units/ml.
- CKI Cleaved Kininogen Index
- HK total protein was measured with Coomassie Plus Protein Reagent (Pierce Chemical Co.) in all donors in order to calculate "specific kininogen levels" (HK, LK, or TotK divided by total protein).
- HK ranged from 1.11-2.19 ⁇ g/mg total protein with a mean value of 1.46 ⁇ g/mg.
- Specific LK ranged from 1.12-3.06 ⁇ g/mg with a mean value of 1.64 ⁇ g/mg.
- Specific TotK ranged from 2.23-5.35 ⁇ g/mg with a mean value of 3.12 ⁇ g/mg (Table 1).
- Example 4 Polyethyleneimine-modified vs. Amino-modified Microspheres
- the LK and TotK concentration determination procedures of Example 4 were followed, except that instead of the inventive polyethyleneimine-modified latex microspheres, commercially available amino-modified latex microspheres from two suppliers were used to prepare the MAb-coated microspheres: SPHEROTEC APS and SPHEROTEC DMS (Spherotech, Inc., Libertyville, IL); and BANGS APS (Bangs Laboratories, Inc., Carmel, IN).
- SPHEROTEC APS and SPHEROTEC DMS Spherotech, Inc., Libertyville, IL
- BANGS APS Boangs Laboratories, Inc., Carmel, IN.
- microspheres were coated with MAb LKL-1 , which is specific for the LK light chain.
- Other microspheres were coated with MAb 2B5, which is directed to the common heavy chain of HK and LK.
- Inventive polyethyleneimine-modified microspheres generated by polyethyleneimine modification of carboxylate-modified latex microspheres according to the present invention were also coated with LKL-1 or 2B5 and used for comparison. Standard curves were generated with normal pooled human plasma, as described in Example 4. Both LK (Fig. 5 A) and TotK (Fig. 6 A) standard curves were linear with the inventive PEI-ML microspheres and had little to no background, indicating no non-specific binding of fluoresceinated antibody tracer. The SPHEROTEC APS microspheres produced a linear assay with TotK (Fig. 6B) but had less than l A the binding capacity of PEI-ML (Fig. ⁇ A).
- SPHEROTEC APS microspheres produced a high background and a signal to noise ratio of ⁇ 2.0, indicating that most of the signal in the LK assay was due to non-specific adsorption of HK and/or detector antibody to the microspheres (Fig. 5B).
- the SPHEROTEC DMS microspheres were totally unsuitable because they produced an extremely high background in both the LK and TotK assays (Fig. 5C, 6C). This result suggests that the tracer is non-specifically adsorbing to the microspheres.
- the BANGS APS microspheres exhibited a high background in both the LK (Fig. 5D) and TotK (Fig. 6D) assays and a signal to noise ratio less than 4.0.
- the BANGS APS curves were similar for both assays, suggesting that both HK and LK were binding in addition to non-specific binding of the tracer.
- HK and/or tracer were incubated with a high concentration of purified HK (527 ⁇ g/ml) and the blank values (zero HK) were subtracted from each.
- PEI-ML bound less than 2% of the HK.
- SPHEROTEC APS and SPHEROTEC DMS bound HK and/or tracer.
- the values for purified HK were higher than the highest value on their respective standard curves even after the high tracer background was subtracted.
- BANGS APS bound 30% of the HK in addition to a sizable amount of tracer.
- Crevicular fluid (10 ⁇ l) was collected from patients, undergoing routine treatment at Temple University School Of Dental Medicine, Philadelphia, P , after obtaining written, informed consent.
- the fluid was collected with a PERIOTRON STRIP (Pro Flow, Amityville, NY) from patients with active periodontal disease and periodontal patients after the disease had been treated where inflammation was no longer evident.
- the PERIOTRON STRIP was immediately placed into a microcentrifuge tube containing 50 ⁇ l 10% protease inhibitor cocktail and frozen at -70°C. These samples were diluted 1 :2 for PCFIA so that the final dilution was 1: 12.
- the antibody was allowed to incubate anywhere from two hours to overnight.
- Normal pooled human plasma was diluted 1 : 1000 in PBS-TBI. Serial dilutions in PBS-TBI were made to construct a standard curve. PBS-TBI was used as the blank. Unknown samples to be measured were diluted 1:2000 in PBS-TBI. All determinations were performed in triplicate. Standards and samples (200 ⁇ l of each) were placed in a well to which MAB 2B5 was linked. After a 1.5 hour incubation at room temperature, the plates were emptied and washed three times with a wash buffer consisting of 10 mM sodium phosphate, pH 7.0, containing 0.5 M NaCl, 0.02% sodium azide and 0.1 % polysorbate 20.
- a wash buffer consisting of 10 mM sodium phosphate, pH 7.0, containing 0.5 M NaCl, 0.02% sodium azide and 0.1 % polysorbate 20.
- the antibody was allowed to incubate anywhere from two hours to overnight. No differences were observed between short and long coupling times.
- Example 7C An ELISA procedure similar to the procedure of Example 7C was followed to determine the LK content of the seven samples used in the TotK analysis of Example 7.
- the above-prepared LKL-1 MAB-coated plates were substituted for the 2B5 MAB-coated plates of Example 7.
- the same seven samples i.e., five normal samples, Williams' plasma sample and Fitzgerald plasma sample) were tested using the LK PCFIA of Example 4C.
- the results of the PCFIA and ELISA for LK are plotted in Fig. 8. An r value of 0.98 was obtained. This value indicates that there is excellent correlation between the PCFIA and ELISA assays in the determination of LK. Williams' plasma was unreactive in this assay.
- the wells of the four quadrants thus contained (1) a passively adsorbed coating of polyethyleneimine, (2) a coating of polyethyleneimine covalently coupled to the plate surface by treatment with EDC.HCl, (3) a coating of carboxy-modified latex particles to which a coating of polyethyleneimine has been passively adsorbed, or (4) a coating of carboxy- modified latex particles to which a coating of polyethyleneimine has been covalently attached by treatment with EDC.HCl.
- the thus-treated plate was allowed to stand for two hours. The excess liquid was poured out and the thus-coated wells were washed with deionized H 2 0 ten times. The plate was then inverted on top of a paper towel and placed in a 37°C air oven to evaporate any residual liquid.
- Example 7B The procedure of Example 7B was followed to attach total kininogen monoclonal antibody 2B5 to all wells of the microplate.
- Example 7C The procedure of Example 7C was followed to generate a total kininogen standard curve for each plate quadrant by adding serial dilutions of normal pooled human plasma diluted in PBS-TBI. PBS-TBI was used as the blank. All determinations were performed in triplicate. The resulting standard curves are shown in Fig. 9: wells passively coated with polyethyleneimine (solid circles); wells to which polyethyleneimine was covalently attached via EDC .
- a 1 wt. % solution of 0.1 ⁇ diameter carboxy-modified latex particles (SERACOAT ® TC3, Seradyn, Inc.) in ethanol was applied to completely fill each well of a MICROTEST III 96-well tissue culture plate (Falcon Plastics). After ten minutes, the nonadherent particles of TC3 were discarded and the plate was rinsed with deionized water. The plate was then inverted on paper towels and dried in a warm air oven at 37 °C for 30 minutes.
- Normal pooled human plasma was diluted 1 : 1000 in PBS-TBI. Serial dilutions in PBS-TBI (1:2) were made to construct a standard curve. PBS-TBI was used as the blank. Unknown samples (Williams plasma and Fitzgerald plasma) to be measured were diluted 1 :2000 in PBS-TBI. Standards and samples (200 ⁇ l of each) were placed in the microplate wells. After a 1.5 hour incubation at room temperature, the plates were emptied and washed three times with a wash buffer consisting of 10 mM sodium phosphate, pH 7.0, containing 0.5 M NaCl, 0.02% sodium azide and 0.1 % polysorbate 20.
- a wash buffer consisting of 10 mM sodium phosphate, pH 7.0, containing 0.5 M NaCl, 0.02% sodium azide and 0.1 % polysorbate 20.
- Fig. 11 The data from the serial dilutions of the normal pooled plasma are plotted in Fig. 11 , which comprises a standard curve for HK concentration in plasma. Williams plasma. The standard curve was then used to determine the HK values of the Williams plasma and Fitzgerald plasma samples. The value for Williams plasma HK was zero. The value for Fitzgerald plasma HK was about 7 ⁇ g/ml. These values are in agreement with Williams and Fitzgerald HK values determined by PCFIA.
- Composition A and Composition B are shown in Table 6. Two plates were left untreated.
- Composition A Composition B polyethyleneimine (10%) 4 ml 4 ml
- EDC.HCl 48 mg/250 ⁇ l 0 2 ml The plates were incubated at room temperature over the weekend. The plates were then washed 10 times with deionized water and dried at 35 °C for 30 minutes.
- Polyethylene-modified microspheres prepared according to Example 1 were coated with anti-goat IgG (Sigma Chem. Co.) at a concentration of 0.1 mg/ml antibody by incubating the mixture of Table 8 overnight at room temperature on a rotating mixer:
- the treated microspheres were then blocked for 30 minutes with 10 mg/ml BS A , centrifuged and washed with water, and resuspended in a final volume of 4 ml PBS- Tween.
- a PCFIA standard curve utilizing serial dilutions of purified goat IgG was generated spanning an IgG concentration of 0 to 1000 ng/ml.
- the diluent for preparation of the samples for the standard curve was PBS-Tween-BSA filtered with a 0.2 ⁇ m syringe filter. Twenty ⁇ l microspheres were added to each well of a 96- well microplate, followed by 20 ⁇ l of diluted sample or reference standard.
- an FITC-labeled donkey anti-goat IgG (anti-goat IgG-FITC) solution prepared by diluting commercially available anti-goat IgG-FITC 1: 100 in PBS-Tween-BSA containing 20% donkey serum was added to the wells as a tracer. The mixture was incubated for 20 minutes. The plate was then read in an FCA as in Example 1 , at an instrument gain of 5. The data appears in Fig. 10.
- the ovalbumin-peptide conjugate was covalently attached to polyethyleneimine- modified CML microspheres according to the attachment procedure of Example 2.
- the peptide-linked polyethyleneimine-modified microspheres were then used to screen the serum of five mice immunized with the ovalbumin-peptide conjugate, using a particle concentration fluorescence immunoassay (PCFIA) according to Example 4.
- PCFIA particle concentration fluorescence immunoassay
- the mouse serum was diluted 1: 1000 with PCFIA diluent, according to Example 4 above, and 20 ⁇ l were placed in the wells containing the microspheres. After 15 minutes, the plate was washed and 20 ⁇ l 1 :50 diluted anti-mouse IgG-FITC (Sigma Chemical Co.) was added and incubated 15 minutes. The plate was washed and read in a Fluorescence Concentration Analyzer, as in Example 4. The data is set forth in Table 9.
- Total mouse IgG was also measured on all samples using commercially available reagents, as in Example 2, so that "specific anti-LK IgG" (RFU divided by total mouse IgG) could be calculated. There were between 6318 and 44622 specific anti- LK IgG in the serum of the mice after immunization. This is compared to a value of 83304 that was determined for ascites from the hybridoma producing MAb LKL- 1.
- One or more of the immune mice were sacrificed.
- the spleens were removed and splenocytes were fused with myeloma cells to prepare hybridomas according to conventional hybridoma procedures.
- the hybridoma tissue culture supernatants were then screened using PCFIA for antibody reactivity to the peptide-linked polyethyleneimine-modified microspheres. There was no measurable fluorescence in the pre-immune samples. Most of the 500 hybridomas screened produced no fluorescence.
- Several samples had low RFUs ( ⁇ 10,000). Only ten samples provided relative fluorescence units (RFUs) between 10,000 and 60,000.
- ADDRESSEE Seidel, Gonda, Lavorgna & Monaco, P.C.
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP52051098A JP2001503861A (en) | 1996-10-30 | 1997-10-14 | Solid-phase immunoassay |
AU52382/98A AU5238298A (en) | 1996-10-30 | 1997-10-14 | Solid phase immunoassay |
EP97947260A EP0963553A1 (en) | 1996-10-30 | 1997-10-14 | Solid phase immunoassay |
CA002270287A CA2270287A1 (en) | 1996-10-30 | 1997-10-14 | Solid phase immunoassay |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US08/741,436 | 1996-10-30 | ||
US08/741,436 US5789261A (en) | 1996-10-30 | 1996-10-30 | Solid phase immunoassay |
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WO1998019161A1 true WO1998019161A1 (en) | 1998-05-07 |
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ID=24980713
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PCT/US1997/018458 WO1998019161A1 (en) | 1996-10-30 | 1997-10-14 | Solid phase immunoassay |
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Country | Link |
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US (1) | US5789261A (en) |
EP (1) | EP0963553A1 (en) |
JP (1) | JP2001503861A (en) |
AU (1) | AU5238298A (en) |
CA (1) | CA2270287A1 (en) |
WO (1) | WO1998019161A1 (en) |
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EP1612557A2 (en) | 1999-10-23 | 2006-01-04 | Plasso Technology Limited | Binding surface |
WO2007023297A1 (en) * | 2005-08-26 | 2007-03-01 | Plasticell Limited | Method |
US8409874B2 (en) * | 2004-04-15 | 2013-04-02 | Corning Incorported | Reversible binding surface |
US9946834B2 (en) | 2009-09-21 | 2018-04-17 | Plasticell Limited | Apparatus and method for processing cell culture data |
US10870115B2 (en) | 2015-08-13 | 2020-12-22 | Dyax Corp. | Evacuated blood collection tubes containing protease inhibitors for the assessment of contact system activation |
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US5132108A (en) * | 1990-11-08 | 1992-07-21 | Cordis Corporation | Radiofrequency plasma treated polymeric surfaces having immobilized anti-thrombogenic agents |
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CA1272127A (en) * | 1985-04-04 | 1990-07-31 | Hybritech Incorporated | Solid phase system for use in ligand-receptor assays |
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US5100805A (en) * | 1989-01-26 | 1992-03-31 | Seradyn, Inc. | Quantitative immunoassay system and method for agglutination assays |
-
1996
- 1996-10-30 US US08/741,436 patent/US5789261A/en not_active Expired - Fee Related
-
1997
- 1997-10-14 EP EP97947260A patent/EP0963553A1/en not_active Withdrawn
- 1997-10-14 AU AU52382/98A patent/AU5238298A/en not_active Abandoned
- 1997-10-14 JP JP52051098A patent/JP2001503861A/en active Pending
- 1997-10-14 CA CA002270287A patent/CA2270287A1/en not_active Abandoned
- 1997-10-14 WO PCT/US1997/018458 patent/WO1998019161A1/en not_active Application Discontinuation
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US4908431A (en) * | 1986-01-22 | 1990-03-13 | Temple University-Of The Commonwealth System Of Higher Education | Monoclonal antibodies to human kininogen and methods of preparing same |
US4921809A (en) * | 1987-09-29 | 1990-05-01 | Findley Adhesives, Inc. | Polymer coated solid matrices and use in immunoassays |
US5132108A (en) * | 1990-11-08 | 1992-07-21 | Cordis Corporation | Radiofrequency plasma treated polymeric surfaces having immobilized anti-thrombogenic agents |
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US8409874B2 (en) * | 2004-04-15 | 2013-04-02 | Corning Incorported | Reversible binding surface |
WO2007023297A1 (en) * | 2005-08-26 | 2007-03-01 | Plasticell Limited | Method |
US8318492B2 (en) | 2005-08-26 | 2012-11-27 | Plasticell Limited | Method for determining the cell culture history of a cell unit labeled with more than one type of tag |
US9946834B2 (en) | 2009-09-21 | 2018-04-17 | Plasticell Limited | Apparatus and method for processing cell culture data |
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Also Published As
Publication number | Publication date |
---|---|
JP2001503861A (en) | 2001-03-21 |
EP0963553A1 (en) | 1999-12-15 |
US5789261A (en) | 1998-08-04 |
CA2270287A1 (en) | 1998-05-07 |
AU5238298A (en) | 1998-05-22 |
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