WO1991002980A1 - Procede et appareil de mesure d'antigenes organiques - Google Patents

Procede et appareil de mesure d'antigenes organiques Download PDF

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WO1991002980A1
WO1991002980A1 PCT/US1990/004826 US9004826W WO9102980A1 WO 1991002980 A1 WO1991002980 A1 WO 1991002980A1 US 9004826 W US9004826 W US 9004826W WO 9102980 A1 WO9102980 A1 WO 9102980A1
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antibody
antigenic
based materials
substance
sample
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William H. Frey Ii
John W. Schmalz
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Ramsey Foundation
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Ramsey Foundation
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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
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/548—Carbohydrates, e.g. dextran
    • 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
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic

Definitions

  • the present invention relates to methods for the detection and measurement of antibodies and antigenic substances in samples using im unoassay techniques. More particularly, this invention relates to the detection of antibodies and antigenic organic substances, such as pharmaceutical compounds, carbohydrates, proteins and lipids, and the like, in materials such as a tissue, cell, body fluid, hybridoma, bacteria, and the like.
  • the invention further relates to a diagnostic test apparatus for an immunosorbent assay.
  • the apparatus includes a surface coated with an adsorbent material, such as silica gel, alumina, charcoal, and the like.
  • antigens are proteins, polysaccharides, or complex molecules that include a protein or carbohydrate moiety. It is known that certain lipids that contain a carbohydrate moiety, such as glycosphingolipids or simply glycolipids, are highly immunogenic. See S. I. Hakomori, A. Rev. Biochem. 1981, .50, 733. Similarly, glycoproteins, lipoproteins, phospholipids, and proteolipids are known to be immunogenic.
  • the determination of the presence and/or concentrations of antigenic substances associated with a wide variety of body disorders and diseases, in serum or other body fluids and materials may be carried out by using various immunoassay techniques. These techniques are based upon formation of a complex between the antigen being assayed and an antibody or antibodies in which one of the members of the complex is labelled.
  • One type of immunoassay technique is a solid- phase assay based on the principle that plastic surfaces, such as polystyrene or polyvinyl chloride, will tightly adsorb nanogra amounts of proteins.
  • an antigen-containing solution is added to a container made of a polyvinyl material and allowed to incubate for a few hours. The unbound material is then washed away and any remaining protein-binding sites are saturated by a large excess of a noninteracting protein.
  • a solution containing a first antibody to the antigen is then added to the container, allowed to react with the antigen, and any unbound material is washed away. This first antibody may be labelled.
  • a labelled second antibody i.e., a "revealing" reagent
  • This is generally a labelled anti-immunoglobulin antibody.
  • the label may be, for example, a radioactive, fluorescent, phosphorescent, or enzyme group.
  • the common supports used for adsorption of the antigenic substance are generally plastic materials made of polystyrene, polypropylene, polyvinyl chloride, and the like. Other generally useful materials are nitrocellulose, sepharose, agarose, crosslinked dextran, and other polysaccharide polymers. These supports tend to work well for protein antigens and often nonprotein antigens. See A. Voller et al., Bull.
  • Enzyme linked immunosorbent assay hereinafter "ELISA” has been used by a number of investigators to detect and measure specific glycolipids and glycolipid antibodies.
  • ELISA Enzyme linked immunosorbent assay
  • N. asai et al., Brain Res. 1983, 277, 155 used the ELISA procedure to study interaction of the monoclonal antibody A2B5 with the tetrasialoganglioside G Qlc .
  • P. Fredman et al., Biochem. J. 1988, 251, 17 used the ELISA procedure in the study of the interaction of the IgGl monoclonal antibody Sulph I with sulphated glycolipids, such as sulphatide, sulpholacto-sylcera ide, and seminolipid.
  • the ELISA procedure has also been used in the study of a polyclonal antibody to phosphatidylserine. See L. Manneta-Teyret et al., J. Immunol. Methods 1988, 108, 123.
  • TLC is a commonly used method of separating and detecting chemical species such as carbohydrates and lipids.
  • a two-dimensional TLC system was used. See S. Sonnino et al., Anal. Biochem. 1983, 128, 104.
  • a modification of the typical TLC method i.e., immuno- staining on silica gel-coated thin layer chromatography plates (immuno-TLC) has also been used by a number of investigators to detect specific glycolipids and glycolipid antibodies. For example, D. A. Cheresh et al., J. Biol. Che .
  • TLC techniques used TLC techniques in the study of the interaction between monoclonal antibodies and a human melanoma-associated ganglioside, which is a glycolipid containing sialic acid groups. Although, TLC techniques are available for the detection of carbohydrate and lipid antigens, these methods do not allow for the accurate measurement of the concentration of the antigens present.
  • One object of the present invention is to provide a method for the detection, including quantitative measurement, of antigenic organic substances. Also, an object of the invention is to provide a method for detecting antigenic organic substances, including epitopes and haptens, particularly when the desired antigenic substance is of low concentration relative to the total content of the sample material or mixture.
  • Yet another object of the invention is to provide a method for detecting antibodies in a sample material or mixture, as for example, a cellular material, particularly when low in concentration.
  • Another object of the present invention is to provide an apparatus for the detection of an antigenic organic substance, including epitopes and haptens.
  • Yet another object is to provide such an apparatus having enhanced capabilities of detection when the antigenic substance is low in concentration, and especially when the substance is contained in a sample material or mixture of other substances and/or interfering compounds.
  • It is a further object of the invention to provide an apparatus for the detection of antibodies, particularly when low in concentration in a sample, and more particularly when the antibody is part of a crude mixture, with or without interfering compounds.
  • This invention provides a method for the detection of antibodies and antigenic substances using immunoassay techniques. More particularly, the present invention relates to the qualitative and quantitative detection of antibodies and antigenic organic substances in a sample, as for example, a body or cellular material. Typically, the antibodies and antigenic organic substances are naturally present within living tissue or are present as a result of prior exogenous introduction.
  • antigenic organic substance includes any organic substance that will elicit an immunogenic response when injected into a mammal, alone or in combination with a hapten, as for example, carbohydrates, proteins, lipids, vitamins, neurotransmitters, cofactors, plant alkaloids, aromatic compounds, hormones, peptides, pharmaceutical compounds, and the like, and derived epitopes, fragments and haptens thereof.
  • the method for detecting an antigenic organic substance in a sample material or mixture includes contacting a solid, finely divided adsorbent material, with the sample to generally coat the adsorbent material with the antigenic organic substance and immobilize the antigenic substance on the absorbent material.
  • the solid adsorbent material may be comprised of any of a variety of high surface area, particulate adsorbent materials having a high affinity for organic substances.
  • adsorbent materials may include inorganic, or mineral, oxides such as silica gel and silica-based materials, clay-based materials, alumina-based materials, magnesia-based materials, and the like.
  • Other adsorbent materials that are useful according to the invention include charcoal and cellulose-based materials.
  • the absorbent material may also be any material generally used for the partition, affinity, ion exchange, molecular weight distribution or solid-liquid phase purification of organic compounds under conditions, as for example, pH, ionic strength, or solute concentration, which provide for absorption of the antigenic substance but not the first or second antibody, as used according to the invention, such as carboxymethyl cellulose or octyl sepharose.
  • the adsorbent material is coated onto a surface of a solid support, as for example, the surface of a plate or the wells of an ELISA test plate.
  • a surface of a solid support as for example, the surface of a plate or the wells of an ELISA test plate.
  • methods employing the use of solid adsorbent materials not coated onto the surface or a solid support are also within the scope of the present invention.
  • the adsorbent material is generally coated onto the surface of the wells of an ELISA test plate.
  • a blocking solution such as a solution of bovine serum albumin and salt in an appropriate buffer, may be used to block non ⁇ specific binding sites on the absorbent material, i.e., sites not bound by the antigenic substance, and thus preclude first and/or second antibody from binding directly to the surface of the absorbent material.
  • the immobilized antigenic substance may be contacted with a first antibody that is capable of reacting with the antigenic substance, whereby an antigen-antibody complex, or immobilized "primary complex," is formed comprising the first antibody and the antigenic organic substance.
  • the first antibody may be a polyclonal or, preferably, a monoclonal antibody, and may be labelled. Detection of the immobilized and labelled first antibody in the primary complex indicates the presence of the antigenic organic substance in the sample material or mixture. Further, concentration of the antigenic organic substance in the sample may be determined by measuring the immobilized primary complex, as for example, by quantitative colorimetric techniques, and the like.
  • the primary complex may be contacted with a labelled second antibody which is capable of reacting with the first antibody, thus forming an immobilized antigen-(first) antibody-(second) antibody complex, or "secondary complex.”
  • the antigenic organic substance in the sample may then be detected both qualitatively and quantitatively through the detection and measurement of the immobilized labelled second antibody.
  • the first and/or second antibody are capable of providing a measurement of the concentration of the antigenic organic substance in the mixture. It is also preferred that prior to detecting the antigenic substance that any labelled antibody that is not immobilized is removed along with any other nonimmobilized substances.
  • the present invention is also advantageous in that it provides for the detection of an antibody in a sample material or mixture, as for example, a body fluid, tissue, hybridoma supernatant fraction, ascites fluid, and the like which is reactive with an antigenic organic substance.
  • a sample material or mixture as for example, a body fluid, tissue, hybridoma supernatant fraction, ascites fluid, and the like which is reactive with an antigenic organic substance.
  • an antigenic organic substance capable of binding with the antibody being assayed is immobilized onto an adsorbent material and contacted with a sample material or mixture purportedly containing the first antibody thereby forming an immobilized primary complex comprising the antigenic substance and first antibody. If the first antibody is labelled, the primary complex may be detected directly. Detection of the first antibody in the immobilized primary complex indicates the presence and/or the concentration of the first antibody in the sample.
  • the concentration of the antibody in the sample may be determined by measuring the immobilized primary complex, for example, by quantitative colorimetric techniques, and the like.
  • the primary complex may be contacted with a labelled second antibody which is capable of reacting with the first antibody, thereby forming a secondary complex comprising an antigen-(first) antibody- (second) antibody.
  • the presence of the first antibody may then be detected both qualitatively and quantitatively by detecting and measuring immobilized labelled second antibody.
  • the antigenic substance and/or the second antibody are capable of providing a measurement of the concentration of the first antibody of the mixture. It is also preferred that any nonimmobilized antibody or other substance is removed prior to detecting the first antibody, preferably by washing.
  • the invention further provides a diagnostic test apparatus and test kit for an immunogenic assay, specifically an immunosorbent assay.
  • the immunogenic test apparatus comprises a surface onto which is coated a solid, finely divided adsorbent material.
  • the apparatus is preferably adapted for quantitative immunogenic analyses.
  • the preferred apparatus according to the invention is an ELISA test plate having a surface onto which is coated an adsorbent material, such as silica gel or any of a variety of finely divided substances suitable according to the method of the invention.
  • the test kit comprises the test apparatus and optionally, reagents which may be capable of detecting an antibody or antigenic organic substance in a sample material or mixture.
  • Figure 1 is a graph of the optical density obtained in an ELISA assay utilizing MAb A2B5 for 80 ng/well of lipid antigen extracted from cod fish brain, mixed with nonantigenic lipids (75% phosphatidylcholine, 25% cholesterol) to obtain the percent fish brain lipid antigen extract shown on the x-axis, and plated onto polystyrene, polyvinyl chloride (P.V.C.), or silica gel coated ELISA test plates.
  • MAb A2B5 for 80 ng/well of lipid antigen extracted from cod fish brain, mixed with nonantigenic lipids (75% phosphatidylcholine, 25% cholesterol) to obtain the percent fish brain lipid antigen extract shown on the x-axis, and plated onto polystyrene, polyvinyl chloride (P.V.C.), or silica gel coated ELISA test plates.
  • Figure 2 is a graph of the corrected optical density obtained in an ELISA assay utilizing MAb A2B5 for 1.5 ⁇ g/well of the lipid antigen bovine sulfatide, mixed with nonantigenic lipids as in Figure 1, using polystyrene or silica gel coated ELISA test plates.
  • Figure 3 is a ' graph of the corrected optical density obtained in an ELISA assay utilizing MAb 126 for 240 ng/well of total lipid extracts (TLE) of human brain, mixed with nonantigenic lipids as in Figure 1, using polystyrene or silica gel coated ELISA test plates.
  • Figures 4a and 4b are graphs of the corrected optical density obtained in ELISA assays utilizing two different human plasma antisera for 240 ng/well of G ⁇ ganglioside, mixed with nonantigenic lipids as in Figure 1, using polystyrene or silica gel coated ELISA test plates.
  • Figure 5 is a graph of the optical density obtained in ELISA assays, using monoclonal antibody A2B5 for varying amounts of phosphorylated casein antigen purified from bovine milk, as shown on the x-axis, and plated onto polystyrene or silica gel coated ELISA test plates.
  • Figure 6 is a graph of the optical density obtained in ELISA assays, using monoclonal antibody TLE- 41 for varying amounts of dephosphorylated casein antigen purified from bovine milk, as shown on the x-axis, and plated onto polystyrene or silica gel coated ELISA test plates.
  • Figure 7 is a graph of the optical density obtained in ELISA assays, using a monoclonal antibody to chondroitin sulfate, for varying amounts of chondroitin sulfate antigen extracted from bovine trachea, as shown on the x-axis, and plated onto polystyrene or silica gel coated ELISA test plates.
  • This invention is based upon the discovery that the typical ELISA procedure may be improved with respect to sensitivity and selectivity if the plastic ELISA test plate is at least partially coated with an adsorbent material such as silica gel, alumina, talc, and the like. Furthermore, it has been discovered that the presence of such an adsorbent material enhances the binding of a substance and/or improves the orientation of a substance on the surface of a test plate such that the detection and measurement of an antibody or an antigenic organic substance in a sample material or mixture is enhanced. In particular, the method and apparatus of the invention enhance detection, including quantitative measurement, of lipids or lipid-like substances in a sample. This is a significant discovery due to the limited ability to detect lipid antigens in such body materials as serum and cerebrospinal fluid, particularly when the antigen is present in very small concentrations, e.g., less than about 10% of the total lipid content.
  • the present invention relates to methods for the detection and quantitative measurement of antibodies and antigenic organic substances in a sample material or mixture such as a body fluid, tissue, and the like. Because certain organic antigens are capable of acting as markers for a variety of disorders, the method of the present invention is useful in the diagnosis of such disorders. For example, diseases which produce protein substances, as for example, Alzheimer's disease, Pick's disease, cerebrovascular diseases and amyloidoses may be detected according to the method and apparatus of the invention.
  • diseases which produce carbohydrate substances may also be detected.
  • the glycogen storage diseases such as generalized glycogenosis, von Gierke's disease and McArdle's disease, the mucopolysaccharidoses such as Hurler's and Hunter's syndromes, and cystic fibrosis, and carbohydratureas
  • any disease such as a type of cancer that produces a glycolipid, or other lipid abnormality, or lipid storage diseases where lipids are stored in cells and not properly broken down, may also be diagnosed according to the invention. Examples of diseases with lipid abnormalities are Tay-Sachs, Alzheimer's, Sandhoff's, Nieman-Pick, and Fabrey's Disease.
  • This method also is useful in the diagnosis of neuroblastoma, i.e., brain tumor.
  • Patients with neuroblastoma usually have high concentrations of the antigenic lipid G D2 in their body serum.
  • This method is also useful as a method of tracking the success of various therapies by monitoring the presence and amount of the ganglioside G D2 in the serum.
  • the methods and apparatus of the present invention also provide for determining the presence and the concentration of antibodies reactive with antigenic organic substances, in a sample material or mixture such as a body fluid, tissue, hybridoma supernatant fraction, ascites fluid, and the like.
  • a sample material or mixture such as a body fluid, tissue, hybridoma supernatant fraction, ascites fluid, and the like.
  • the amount and location of brain damage may be determined by detecting antibodies produced in response to brain cells and fragments thereof released into the cerebrospinal fluid (CSF) and blood as a result of the stroke, by testing a sample of body material according to the method of the invention. Detection of a buildup of an antibody within the body may thus provide evidence of the diseased state.
  • CSF cerebrospinal fluid
  • the apparatus and methods of the present invention are useful in monitoring various organic substances, as for example, glycolipids, used in the treatment of outpatients.
  • the monosialoganglioside G ⁇ a neuronotrophic and neurotogenic factor
  • the quantity of G M1 in the blood of a patient following treatment with this nerve growth factor may be monitored.
  • the invention relates to methods for detecting and measuring antibodies and antigenic organic substances using immunoassay techniques.
  • the antibodies and organic substances are naturally occurring within living tissue, as for example, body tissue or plant tissue, but may also be present as a result of exogenous introduction.
  • Antibodies which immunoreact with organic substances may be detected according to the method of the invention, as for example, antibodies to heparin, blood group antigens, cytokeratin, tau, sulfatide, gangliosides, and the like. Such antibodies may be of any class, as for example, IgG, IgM, IgA, and the like.
  • the method of the invention is also useful for the detection of antigenic organic substances including those which will elicit an immunogenic response when injected into a mammal, either alone or as a hapten coupled to a carrier.
  • Such substances include vitamins, neurotransmitters, cofactors, plant alkaloids, aromatic compounds, hormones, oligopeptides, pharmaceutical compounds, carbohydrates, proteins, lipids, and the like, and derived epitopes and haptens thereof.
  • Lipids are a heterogenous group of substances that may be categorized by their insolubility in water and by their extractability in organic solvents of low polarity, such as ether, tetrahydrofuran, chloroform or chloroform/methanol mixtures.
  • This broad definition of lipids includes a variety of classes of compounds including phospholipids, sulfolipids, glycolipids, proteolipids, lipopolysaccharides, lipoproteins, fatty acids, neutral fats, aliphatic alcohols and waxes » terpenes, and steroids.
  • Glycolipids are membrane components of mammalian cells, and may be detected in a variety of body fluids, such as serum, ascitic fluid, and cerebrospinal fluid.
  • body fluids such as serum, ascitic fluid, and cerebrospinal fluid.
  • the glycolipids found in body tissues and fluids are capable of acting as markers for a variety of disorders, including certain cancers, Alzheimer's Disease, Tay-Sachs Disease, or myelination and demyelinating disorders. See, for example, P. Fredman et al., Biochem. J. 1988, 251, 17; C. R.
  • glycolipids are known to be capable of immunogenic response.
  • Glycolipids include any of a class of lipids that contain carbohydrate moieties.
  • glycolipids are usually derived from sphingosine, and may be referred to as glycosphingolipids. Included within this group are gangliosides, which are complex glycolipids that contain an oligosaccharide chain containing sialic acid groups, i.e., acidic sugar residues. Also included within this group are sulfolipids, i.e., sulfated glycolipids.
  • any antibody which may be capable of immunoreacting with a organic substance is useful in the method of the present invention.
  • Such antibodies may be monoclonal or polyclonal, or from any class, as for example, IgG, IgM, IgA, and the like.
  • the antibody used in the method of the invention depends upon the antigenic substance to be detected.
  • An example of an antibody to lipid antigens is monoclonal antibody A2B5 which will recognize lipid substances, as for example, sulfatide, G Q1C , codfish brain gangliosides, glycolipids found in blood and cerebrospinal fluid, and phospholipids such as phosphatidylglycerol and phosphatidylserine.
  • Monoclonal antibody 126 will also recognize lipid antigens, as for example, G D2 and other glycolipids present in total lipid extracts of human brain.
  • Other monoclonal antibodies are capable of recognizing brain lipid antigenic substances associated with Alzheimer's disease, myelolukodystrophy sulfatide lipid, and other animal and plant cell lipid antigens.
  • Further examples of monoclonal antibodies to lipid antigens include monoclonal antibody (MAb) 14-18 to G D2) MAb MB-3.6 to G D3j MAb 18B8 to G T3 , MAb C3 to Gm, and MAb 312 (Chemicon Int. Inc., El Segundo, CA) to G Qlc . See D.A. Cheresh et al., Cancer Res. 1986, 4j5, 5112; G.B. Grunwald et al., Proc. Natl. Acad. Sci. USA 1985, 82., 4008; S.P.
  • Examples of monoclonal antibodies to protein antigens include antibodies to A-Cell Adhesion Molecule, cytokeratin,neurofilaments, and tau. See for example, Volk, et al., EMBO J, 1984, 3 , 2249; Gigi-Leitner, et al., Differentiation , 1986, 31, 191; Debus, et al., Differentiation, 1983, 2JL, 193; Papasozomenos, et al., Cell Motil ⁇ Cvtoskel. , 1987, 8 , 210.
  • carbohydrate antigen specific monoclonal antibodies include antibodies to chondrotin sulfate, heparin, glycophorin, and various blood group antigens. See for example, Avnur, et al., Cell, 1984, 38, 811; Chen, et al., J. Biol. Chem. 1985, 260., 13208; Bouhours, et al., Anticancer Res. , 1986, .6, 139; imber, et al., J. Reprod. Immunol. 1988, 12. r 297.
  • Monoclonal antibodies that may be used according to the invention may be synthesized by various known techniques employed to produce hybridomas capable of secreting monoclonal antibodies that would be selectively reactive with antigenic organic substances. Such techniques for antibody preparation are set forth in M. M. Rapport and Y. Huang, "Present Status of the Immunology of Gangliosides," pp. 15-25, in Advances in Experimental Medicine and Biology, Vol. 174, R. W. Ledeen et al., eds. (1984), and in G. S. Eisenbarth et al., Proc. Natl. Acad. Sci. USA, 1979, 7j, 4913, the disclosures of which are incorporated by reference herein.
  • the present invention is not to be limited by a specific type of antibody other than in the antibody's ability to selectively react with organic substances. That is, the antibodies which may be employed in accordance with the present invention are antibodies which are capable of specifically binding to epitopes associated with antigenic organic substances.
  • the preferred antibodies according to the invention are monoclonal antibodies (MAbs).
  • Monoclonal antibody A2B5 a murine IgM, is commercially available from American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland (ATCC Accession No. CRL1520) .
  • Monoclonal antibody A2B5 is secreted by a cloned hybridoma formed by the fusion of the murine myeloma P3X63 Ag8 with spleen cells derived from a BALB/c mouse immunized against chick embryo retina cells. This antibody exhibits only minimal reactivity with G ql ganglioside, the tetrasialoganglioside present in normal adult brain gray matter, but reacts strongly with fetal ganglioside G Qlc . Furthermore, i muno-TLC and ELISA studies utilizing A2B5 have demonstrated that A2B5 binds to and detects the sulfated glycolipid sulfatide from bovine or human brain and certain base-labile glycolipids.
  • Monoclonal antibody 126 a murine IgM, is commercially available from American Type Culture Collection (ATCC Accession No. HB8568) .
  • Monoclonal antibody 126 is secreted by a cloned hybridoma formed by the fusion of the murine myeloma P3X63 Ag8 with spleen cells from BALB/c mice immunized against LAN-1 neuroblastoma cells. This antibody exhibits reactivity with G D2 and other glycolipids. See D.A. Cheresh et al., vide supra.
  • IgM antibodies have been deposited and are available from American Type Culture Collection (ATCC Accession No. HB- 10520 and No. HB-10521, respectively.
  • Monoclonal antibody TLE-41 has demonstrated reactivity with total lipid extract of the cerebral cortex of patients afflicted with Alzheimer's disease, with certain phosphoproteins such as casein, and human and bovine brain sulfatide.
  • GLE-17 has demonstrated reactivity with a crude ganglioside fraction of Alzheimer's cerebral cortex. The antigens detected by these antibodies, although not yet confirmed, are suspected as being a sulfolipid, glycolipid, phosphoprotein or phospholipid.
  • a primary complex is formed between the antigenic organic substance, immobilized on a solid, finely divided adsorbent material, and a first antibody which is capable of reacting with the antigenic organic substance. If the first antibody is labelled, the immobilized primary complex may be directly detected. Alternately, the primary complex may be contacted with a labelled second antibody that is capable of binding to the first antibody, thereby forming an immobilized secondary complex. Detection of the primary or secondary .complex thus indicates the presence and/or concentration of the antigenic substance in the sample.
  • the labelling of the first or second antibody may be achieved by incorporating a radioactive atom, isotope or group, or by coupling an enzyme, a dyestuff or a fluorescent group to the antibody.
  • the labelling step may be performed prior to the immunological reaction, as for example, by reacting a first antibody with a second antibody having been earlier labelled. Alternately, the labelling step may be performed subsequent to the immunological reaction, as for example, by reacting a first antibody with an antigenic substance to form a primary complex, and then reacting the primary complex with a labelled second antibody.
  • a variety of labels are known and suitable according to the invention, as for example, fluorescent labels, radiolabels, enzymatic labels, and the like.
  • the antibody is radiolabelled.
  • the labelling of the primary or secondary complexes with a radioactive isotope may be performed according to any known method and with any isotope suitable according to the invention, as for example, 125 I, 131 I, l ⁇ C, and 3 H
  • the labelled antibody is an antibody-enzyme conjugate such that the antibody is covalently linked to one or more enzyme molecules.
  • the production of antibody-enzyme conjugates which employ a covalent bond may be achieved, for example, by the use of reagents such as diisocyanates, carbodiimides, glutaric aldehyde, and bisdiazobenzidine.
  • the choice of enzyme moiety for the antibody-enzyme conjugate depends upon various properties of the enzyme, as for example, the specific activity of the enzyme, a high conversion rate providing increased sensitivity within the method of the invention, and the simplicity and ease of detecting the enzyme and measuring the enzymatic activity.
  • Enzymes which are suitable according to the invention include any enzyme which may be detected or imaged, for example, colorimetrically, spectrophotometrically, or fluorimetrically.
  • oxidoreductases such as catalase, peroxidase, glucose oxidase, urease, alkaline phosphatase, or any enzyme commonly used according to an ELISA procedure may be used according to the methods and apparatus of the invention.
  • a preferred enzyme label is horseradish peroxidase.
  • Also preferred according to the present invention are commercially available anti-mouse antibodies or reactive antibody fragments incorporating enzyme labels, as for example, peroxidase-labelled affinity purified goat antibody to mouse IgM (Kirkegaard and Perry Code No. 04-18-03).
  • the immobilized primary or secondary complex comprising an enzyme label may be isolated and the enzyme detected and the level of enzymatic activity measured by incubating the complex with a substrate comprising nutrients and other substances otherwise necessary for the enzymatic reaction.
  • peroxidase may be detected, and quantitated colorimetrically, by reacting it with ortho-phenylenediamine or 3,3' ,5,5' - tetramethylbenzidine and hydrogen peroxide.
  • the detection of the immobilized primary or secondary complex in sample material taken from a patient afflicted with a disease as compared to that taken from a healthy individual may indicate the presence and/or concentration of a particular antigenic organic substance, and, in turn, may act as a marker for a particular disease or affliction.
  • the adsorbent material useful in the present invention is any finely divided, solid material which is capable of enhancing the retention of the antigenic organic substance.
  • a typical adsorbent material according to the invention is silica.
  • Other suitable adsorbent materials include inorganic adsorbents such as alumina, diatomaceous earth, talc, calcium carbonate, bentonite, mica, vermiculite, and other finely divided solids typically used on TLC plates. Also, glass, water glass or any other dissolved Si 2 0 3 material may be used.
  • the absorbent material may also be any material generally used for the partition, affinity, ion exchange, molecular weight distribution or solid-liquid phase purification of organic compounds as used, for exmaple, in column chromatography separation, under conditions, i.e., pH, ionic strength or solute concentration, which provide for absorption of the antigenic substance but not the first or second antibody, as used according to the method of the invention.
  • Such absorbent materials include, for example, substances having ion exchange properties such as carboxymethyl (CM) cellulose or DEAE cellulose to absorb charged antigens, materials with hydrophobic properties such as octylsepharose or L-phenylalanine sepharose which are capable of binding hydrophobically to substances, materials which are functional group specific reagents such as resins that are capable of binding to sulfhydryl groups, affinity gels such as Blue separose which is capable of binding enzymes which utilize nucleotide co- factors, and the like.
  • the adsorbent material it is preferred that the adsorbent material have a high surface area and a high affinity for organic substances.
  • the adsorbent material is a finely divided material with a major portion, of the material having a particle size less than about 50 microns, preferably less than about 20 microns, and providing a total surface area of at least about 50 m 2 /gm.
  • material having a smaller particle size provides an adsorbent material having a higher amount of surface area and, accordingly, a higher degree of adherence of the antigenic organic to the adsorbent material.
  • adsorbent materials include, but are not limited to, silica gel and other silica-based materials such as diatomaceous earth and talc, i.e., finely powdered hydrous magnesium silicate, alumina and other alumina- based materials, magnesia-based materials, bentonite and other clay-based materials, other mineral oxide-based materials, charcoal, and cellulose-based materials.
  • the adsorbent is an inorganic- or mineral- oxide-based material. More preferably, the adsorbent material is silica gel. A preferred particle size of the silica gel is 60-200 mesh.
  • Silica, silica gel, and silicic acid all refer to materials produced by acidification of silicate solutions followed by washing and drying of the resulting gel. Such materials generally have a surface area of about 500 m 2 /g. The surface comprises Si-OH groups spaced at intervals of about 5 A.
  • the Si-OH groups are generally the active sites of adsorption. The activity of a given batch of silica adsorbent material depends on the number and availability of these adsorption sites. It is believed that the active sites may interact with polar solutes generally by means of hydrogen bonding or by an electron donor action. Alumina materials will generally retain adsorbates with easily polarizable electrons, as for example Lewis bases, and acidic solutes perhaps by proton donation. Also, aromatic species may be retained perhaps by charge-transfer interaction.
  • the antigenic substance may be immobilized on the adsorbent material by covalent bonding, by adsorption, by complexation such as charge transfer, dipole exchange, chelation, and the like, or other suitable means.
  • the enhanced levels of detection and measurement of antigenic lipid substances in mixtures is due to the lipid being more appropriately oriented by the adsorbent such that the antibody readily binds it, whereas on plastic material, the orientation of the lipid may be such that the antigenic site is hidden from the antibody.
  • the enhanced detection and measurement may be due to an increased concentration of adsorbed lipid antigen on the finely divided high surface area of the adsorbent material as compared to polystyrene or polyvinyl chloride surfaces.
  • the apparatus of the invention comprises a surface, such as a solid support, onto which the solid, finely divided adsorbent material may be coated.
  • the solid support may be any water-insoluble, water- insuspensible material, as for example, polymeric beads, filter paper, test tubes or microliter plates.
  • a typical solid support is comprised of polystyrene or polyvinyl chloride.
  • a preferred solid support is an ELISA test plate, although this type of immunogenic test apparatus is not specifically required according to the invention. It is further preferred that a surface of the solid support is adapted for quantitative measurement.
  • the adsorbent material may be adhered to the surface of the solid support.
  • a water insoluble binder is used to adhere the adsorbent material to the solid support, as for example, a polyacrylate such as poly(isobutyl methylaerylate) .
  • An ELISA test plate apparatus may be prepared according to the present invention by placing an amount of organic solution comprising the binder effective to bind the adsorbent material to the surface of the solid support, for example, 0.05% poly(isobutyl methylaerylate) in cyclohexane, into each well of the test plate.
  • an amount of the adsorbent material may be sprinkled into each well to effectively cover the bottom surface of each well.
  • the bottom surface of the well may be partially or entirely covered with the adsorbent material. It is preferred that the adsorbent material is allowed to dry and excess adsorbent material is removed. It is further preferred that the amount of adsorbent material in each well is similar, although the amount need not be a quantitative amount. According to the invention, the amount of adsorbent in a particular well will not significantly affect the assay results unless the amount of adsorbent material is insufficient to bind the antigen present in the sample or the variations in the amount of absorbent material lead to significant variation in the optical properties of the plate.
  • the adsorbent material on the surface of the solid support may interfere with automated optical density readings of the reaction product.
  • a solid support may be used according to the invention on which the adsorbent material is partially coated.
  • absorbent material such as the silica gel
  • absorbent material such as the silica gel
  • the reaction product is analyzed on the plate or within the well according to kinetic measurement techniques to quantify the rate of the enzymatic reaction.
  • the reaction product is removed from the solid support, and transferred to a surface which is generally transparent or otherwise suitable for the analysis of the reaction product.
  • the invention includes any number of embodiments of apparatus which comprise a solid support coated with the solid, finely divided adsorbent material.
  • a test plate may have a well configured so as to be greater in width than the well of a standard ELISA plate, the absorbent material being coated to only partly cover the bottom surface of the well, the other part being uncoated and generally transparent.
  • an optical density reading would be taken of that portion of the reaction product that overlays the uncoated side of the well.
  • a well may be further divided into two sub-wells, for example, by a low divider wall or partition, the bottom surface of one sub-well being coated with adsorbent material and the bottom surface of the second sub-well being uncoated and generally transparent.
  • the immunological reaction according to the invention would take place in the coated sub-well, and upon completion, the reaction product transferred to the uncoated sub-well, as for example, by shaking or tipping the plate, and the optical density measured.
  • the apparatus of the invention may be a two-part assembly comprising a first plate which is uncoated and generally transparent, and capable of providing a lid-like covering for a second plate onto which adsorbent material has been coated.
  • the immunological reaction would be allowed to proceed on the coated second plate, and, upon completion, the plate assembly would be inverted such that the reaction product is transferred to the uncoated first plate for optical density measurements.
  • the test plates have at least one well, and more preferably, the plates of the assembly are ELISA test plates.
  • the test plates may be attached, as for example, by a detachable hinge or joint.
  • the apparatus of the invention may be a two-part assembly comprising a first plate which is uncoated and generally transparent with at least one well, and a second plate having at least one stalk-like structure or other suitable protuberance which generally extends from the inner surface of the second plate.
  • the protuberance preferably has a surface onto which the adsorbent material may be coated.
  • the protuberances are at least partially coated with adsorbent material.
  • the test plates may be attached, as for example, by a detachable hinge or joint.
  • the adsorbent material on the protuberance is coated with the sample containing the antigenic substance to be assayed.
  • a known concentration of a first antibody, capable of reacting with the antigenic substance and optionally labelled, is placed into the well of the uncoated first test plate.
  • the second plate is then positioned over the first plate such that the antigenic substance on the absorbent material of the protuberance contacts the antibodies on the first plate to form a primary complex.
  • Immobilized primary complex on the protuberance may be detected directly if the first antibody is labelled. Alternately, the immobilized primary complex may be contacted with a labelled second antibody to form an immobilized secondary complex which may then be detected.
  • a preferred embodiment of the method of the invention comprises assaying a sample material or mixture for one or more antigenic organic substances.
  • the sample may comprise a body material, fluid, fragment or extract thereof, as for example, blood serum, blood plasma, tears, urine, cerebrospinal fluid, tissues such as brain tissue, cells such as blood cells, cell membranes, subcellular organelles such as mitochondria, or membrane-bound bodies such as Pick's bodies as associated with Pick's disease or Lewy bodies as associated with Parkinson's disease, or the like.
  • the antigenic substance may be a protein or carbohydrate derivable from the surface of a cell membrane, the sample comprising the intact cell, the cell membrane, or a fragment thereof which contains the antigenic membrane protein or carbohydrate.
  • the mixture need not be from the human body, of course, but could contain an antigenic organic substance from any source including plants, animals, bacteria, or chemically synthesized organic substances.
  • Body material generally contains a relatively high concentration of interfering compounds, or substances generally present and which are capable of interfering with or otherwise reducing the effectiveness of the analysis of antibodies or antigenic organic substances.
  • proteins are capable of interfering with the analysis of antigenic lipid substances.
  • lipid substances are preferably extracted from body material with an organic solvent, organic solvent mixture, or a combined aqueous/organic solvent mixture. Choice of such a solvent is made in accordance with the lipid being analyzed.
  • the solvent extraction of lipids from the sample material to be analyzed generally depends upon the concentration of lipids in the sample. For example, with sample material containing a relatively small proportion of the antigenic lipid substance and a relatively high concentration of interfering compounds, extraction of the antigenic lipid substance is preferably and advantageously carried out prior to the analysis.
  • the antigenic organic substance in the sample is initially immobilized on a solid, finely divided adsorbent material, preferably coated onto a surface of a solid support.
  • the immobilization interaction between the organic substance and the adsorbent material may be of a physical or a chemical nature.
  • the lipid substances are solvent extracted from a sample, such as a tissue or CSF, and then plated, for example, onto an ELISA test plate which has been coated with silica gel, and the solvent allowed to evaporate at room temperature. It is noted that other organic substances in the mixture may also be immobilized onto the adsorbent material along with the antigenic organic substance.
  • nonimmobilized solution as for example, plasma or serum, and any nonimmobilized component
  • nonimmobilized solution are then removed from the plate, preferably by washing the plate with a buffered salt solution.
  • the advantage of the immobilization procedure is that no centrifugation step is needed for the separation of the antigenic organic substance being assayed from the sample.
  • the immobilized portion of the sample is then contacted with a first antibody, preferably monoclonal, that is capable of binding to the antigenic organic substance.
  • a blocking solution such as a solution of bovine serum albumin and salt in an appropriate buffer, may be used to block non ⁇ specific binding sites on the absorbent material, i.e., sites not bound by the antigenic substance, and thus preclude first and/or second antibody from binding directly to the surface of the absorbent material.
  • the primary complex comprising immobilized antigen and a first antibody bound thereto is preferably separated from nonimmobilized, first antibody. Because of the advantageous immobilization step, this may be accomplished by aspiration and/or by washing.
  • the primary complex may be detected and measured directly if the antibody carries a detectable label. Alternately, the primary complex may be contacted with a second antibody that is capable of binding to the first antibody, and which incorporates a detectable label bound thereto, thereby forming an immobilized secondary complex comprising antigen-first antibody-second antibody.
  • labels are known and suitable according to the invention, as for example, fluorescent labels, radiolabels, enzymatic labels, and the like.
  • the labelled second antibody is radiolabelled or is an antibody-enzyme conjugate comprising the second antibody covalently linked to one or more enzyme molecules. Detection of the complexed labelled second antibody in the immobilized secondary comlex may be correlated to the concentration of antigenic organic substance present in the sample. Where the second antibody comprises an antibody-enzyme conjugate, the presence of enzyme and level of enzymatic activity of the immobilized secondary complex may be determined as an indication of the presence and relative amount of the antigenic organic substance in the sample.
  • the labelled second antibody may be added in certain situations without removing nonimmobilized first antibody, i.e., any first antibody not bound to the immobilized antigenic organic substance.
  • any nonimmobilized substances may then be removed.
  • Such nonimmobilized substances may include, for example, unbound labelled second antibody, unbound first antibody, labelled second antibody complexed with a first antibody not associated with the immobilized antigenic organic substance, and other nonimmobilized compounds or components.
  • this removal step is accomplished by washing and aspirating the test apparatus several times, preferably 3- 5 times, with a buffered salt solution. This removal step may also generally remove interfering compounds or substances, as might be present within the sample being analyzed.
  • a method of detecting antibodies that are immunoreactive with antigenic organic substances is also provided.
  • a sample may be tested for the presence and the concentration of a particular antibody.
  • the method for detecting antibodies is similar to the method for detecting organic antigenic substances, except the absorbent material is coated with a known antigenic organic substance that is capable of reacting with the antibody being assayed, or first antibody, and the immobilized antigenic substance is then contacted with the sample purportedly containing the antibody.
  • An immobilized primary complex comprising the known antigenic substance and the antibody is thus formed, and may be detected directly if the antibody carries a detectable label. Detection of the immobilized primary complex indicates the presence of the antibody in the sample.
  • the antigenic substance is capable of providing a measurement of the concentration of the antibody in the sample.
  • concentration of the antibody in the sample may be determined by measuring the immobilized primary complex, as for example, by quantitative colorimetric techniques.
  • the immobilized primary complex may be contacted with a labelled second antibody which is capable of reacting with the first antibody to form an immobilized secondary complex comprising the antigen-first antibody-second antibody. Detecting and measuring the immobilized secondary complex may then be correlated to the presence and the concentration of the first antibody in the sample.
  • the method and apparatus of the invention is also useful in indirect methods of ELISA. See for example, Voller, et al., "The Enyme Linked Immunosorbent Assay (ELISA) , A Guide with Abstracts of Microplate Applications,” Dynatech Laboratories, Inc. (1979), pp. 18- 19.
  • ELISA Enyme Linked Immunosorbent Assay
  • a known antigenic substance may be immobilized on the adsorbent material; and a reagent containing the first antibody incubated with the sample material purportedly containing the antigenic substance to form antibody-antigen complex, and then reacted with the immobilized antigenic substance on the adsorbent material to form immobilized primary complex.
  • Decreased complexing of antibody with antigen on the adsorbent material thus is an indicator of the presence of the antigenic substance in the sample.
  • the primary complex may be measured directly if the first antibody is labelled. Alternately, the primary complex may be contacted with a labelled second antibody reactive with the first antibody to form an immobilized labelled secondary complex. Detection of the immobilized labelled primary or secondary complex may be used to indicate the presence or absence, and/or concentration of the antigenic substance in the sample.
  • test kit Also provided within the scope of the invention is a test kit comprising an immunogenic test apparatus having a surface onto which is coated a solid, finely divided adsorbent material that is suitable according to the method of the invention.
  • the surface of the immunogenic test apparatus is adapted for quantitative immunogenic analysis.
  • the test kit may comprise any number of embodiments of apparatus which comprise a solid support coated with the solid, finely divided absorbent material, such as those embodiments that are described herein. It is preferred that the apparatus comprise an ELISA test plate.
  • the test kit comprise at least one reagent for detecting an antigenic organic substance.
  • the reagent comprises a first antibody which is capable of reacting with the antigenic organic substance being assayed.
  • the first antibody may be labelled.
  • the test kit may further contain a second reagent comprising a labelled second antibody which is capable of reacting with the first antibody, particularly if the first antibody is not labelled.
  • the kit contains a reagent which is capable of providing measurement of the concentration of the antigenic substance in a sample material or mixture.
  • the kit may contain a blocking solution which is capable of blocking non-specific binding sites on the absorbent material, i.e., sites not bound by the antigenic organic substances, but which may bind either the first or second antibodies.
  • the test kit may comprise at least one reagent for detection of an antibody in a sample.
  • the reagent comprises an antigenic organic substance that is capable of reacting with the antibody for which the assay is being performed, or first antibody.
  • this first antibody may be labelled.
  • the test kit may further include a second reagent comprising labelled second antibody capable of reacting with the first antibody, particularly if the first antibody is not labelled. It is preferred that the antigenic substance and/or second antibody are capable of providing a measurement of the concentration of the antibody in the sample.
  • the kit may contain a blocking solution which is capable of blocking non-specific binding sites on the absorbent material, i.e., sites not bound by the antigenic organic substances but which may bind either the first or second antibodies.
  • a blocking solution which is capable of blocking non-specific binding sites on the absorbent material, i.e., sites not bound by the antigenic organic substances but which may bind either the first or second antibodies.
  • ELISA polystyrene plate was coated with silica gel by the following method.
  • High molecular weight poly(isobutyl methacrylate) (30 ⁇ l of a 0.05% cyclohexane solution, Aldrich Chemical Co.) was placed into each well of a polystyrene plate.
  • Silica gel 60-200 mesh, Fisher Scientific
  • the poly(isobutyl methacrylate) was allowed to air dry for approximately 2 hours at room temperature. The plate was inverted to remove excess silica gel.
  • Each well was scanned spectrophotometrically with a Kinetics Microplate Reader (Molecular Devices) to insure uniform coating with the silica gel.
  • the range of the values of optical density for each well was 1.271 to 1.800, with an average of 1.557.
  • a known concentration of glycolipid antigen extracted from cod fish brain was added to a series of test tubes.
  • a mixture of phosphatidylcholine and cholesterol (75:25 by weight) was then added to the tubes to contaminate the glycolipid antigen to the extent shown in Table 1 and Figure 1.
  • the lipids were then allowed to air dry.
  • the lipid mixture was solubilized with a volume of chloroform/methanol/water (60:40:10, by volume) which was no more that 5% to 10% of the final reconstitution volume. After 2 to 5 minutes of incubation, the sample was diluted to its final volume with 100% methanol. All subsequent dilutions were made in 100% methanol.
  • the antigen solutions were plated onto the silica gel ELISA plates at a volume of 50 ⁇ l per well. The solvent was removed upon evaporation at room temperature until the wells appeared dry and the silica gel white.
  • the antigen-coated silica gel ELISA plates were treated with 200 ⁇ l of the diluent per well and incubated at room temperature for 60 minutes. Each well was washed with fresh diluent (3 x 300 ⁇ l) and aspirated between each addition.
  • the substrate solution for the following enzyme reaction was made during the previous incubation step.
  • Freshly made substrate solution (75 ⁇ l) was added to each well except the well designated as the blank.
  • the ELISA plate was incubated for 5 minutes in the dark. If desired, kinetic data could be collected at this time by reading the optical density over time on an ELISA spectrophotometer at a wavelength of 650 nm.
  • Lipidmixtures containing different antigens mixed with phosphatidylcholine and cholesterol as described in Example II were plated onto either polystyrene or silica gel coated ELISA plates. A constant amount of the antigen was plated onto the surface of each well of either polystyrene or silica gel ELISA test plates. The antigen was then allowed to react with its specific antibody using a standard ELISA protocol. The extent of the antibody antigen reaction was described by the optical density that is plotted along the y axis in each of Figures 2, 3, and 4. The x axis describes the lipid antigen percentage of the total lipid in the mixtures for each data point. Each data point represents the average optical density, the error bars represent one standard deviation.
  • the antigen concentration is constant for each data point, however, the percentage of the antigen in the total lipid mixture varies.
  • the y axis is the corrected optical density, that is, the average optical density for a specific antigen percentage minus the average optical density of the background.
  • the antigen was bovine sulfatide and the monoclonal antibody was A2B5.
  • the concentration of sulfatide in each reaction well was 1.5 ⁇ g.
  • Bovine sulfatide is particularly sensitive to contaminating lipids and is effectively masked when the antigen concentration drops below 80%.
  • the silica gel plates show only a small reduction in the reaction signal at 50% sulfatide. It is important to note that A2B5 reacts with both gangliosides and sulfatide.
  • Figure 3 is a graph using the total lipid extracts of human brain with the ganglioside antibody MAb 126. Again, the silica gel plates have better reaction signals than polystyrene plates in highly contaminated samples. At the 10% antigen concentration, the reaction signal when using typical polystyrene plates was reduced approximately 90%, while the signal when using a silica gel plate was only reduced approximately 15%.
  • Figures 4a and 4b describe the reactions of antibodies in the plasma samples of Alzheimer's patients to G M! ganglioside.
  • Two patients who were treated with G M1 ganglioside were found to have high antibody activity for G ganglioside. Although the two plasma samples reacted with the same antigen, they can be considered completely separate antibodies.
  • the reaction signal for the polystyrene plates dropped to half maximum when the percent antigen dropped to approximately 30%.
  • the silica gel plates retained their reaction signal until a much lower percent antigen level was reached.
  • a known concentration of phosphorylated casein antigen extracted from bovine milk was added to a first series of test tubes and air-dried.
  • a known concentration of dephosphorylated casein antigen was added to a second series of test tubes and air-dried.
  • the protein antigen mixtures were dissolved in 50 mM sodium bicarbonate buffer (pH 9.5), and diluted to their final volumes using the same buffer. All subsequent dilutions were also made using the sodium bicarbonate buffer.
  • the diluted protein antigen solutions were plated onto silica gel ELISA plates in concentrations of 10, 30, 60, 100, 500, and 1,000 ng/well, such that each well contained 50 ⁇ l of solution.
  • the diluted phosphorylated casein antigen solution was plated onto a first set of plates, and the diluted dephosphorylated casein antigen solution was plated onto a second set of plates. The plates were incubated overnight at 4 C.
  • the ELISA assay was conducted using standard techniques but, except with the monoclonal antibody A2B5, without the use of Tween or other detergent.
  • the antigen-coated silica gel ELISA plates were treated with 200 ⁇ l of the diluent per well and incubated at room temperature for 60 minutes. Each well was washed with fresh diluent (3 x 300 ⁇ l) and aspirated between each addition.
  • the substrate solution for the following enzyme reaction was made during the previous incubation step.
  • the substrate solution was made by preparing a 2 mM 3,3',5,5'-tetramethylbenzidine solution (Kirkegaard & Perry Labs) in containing 0.030% hydrogen peroxide at pH 4.9.
  • Freshly made substrate solution (75 ⁇ l) was added to each well except the well designated as the blank.
  • the ELISA plates were incubated for 5 minutes in the dark. If desired, kinetic data could be collected at this time by reading the optical density over time on an ELISA spectrophotometer at a wavelength of 650 nm.
  • a known concentration of chondroitin sulfate antigen extracted from bovine trachea was added to a first series of test tubes and air-dried.
  • the carbohydrate antigen mixture was dissolved in water, and diluted to a final volume using methanol. All subsequent dilutions were also made using methanol.
  • the diluted carbohydrate antigen solution was plated onto a silica gel ELISA plate in concentrations of 0.1, 1, 10, 100, and 1,000 ng/well, each well containing 50 ⁇ l of solution. The plates were incubated and allowed to dry overnight at 4 C.
  • the ELISA assay was conducted using standard techniques but without the use of Tween or other detergent.
  • the antigen-coated • 5 silica gel ELISA plates were treated with 200 ⁇ l of the diluent per well and incubated at room temperature for 60 minutes. Each well was washed 3 times with fresh diluent (300 ⁇ l) and aspirated between each addition.
  • the substrate solution for the following enzyme reaction was made during the previous incubation step.
  • the substrate solution was made by preparing a 2 mM 5 3,3',5,5'-tetramethylbenzidine solution (Kirkegaard &
  • Freshly made substrate solution (75 ⁇ l) was added to each well except the well designated as the blank.
  • ELISA plates were incubated for 5 minutes in the dark. If 0 desired, kinetic data could be collected at this time by reading the optical density over time on an ELISA spectrophotometer at a wavelength of 650 nm.

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Abstract

Procédé de détection et de mesure de concentrations d'anticorps et de substances organiques antigéniques dans de la matière et dans des mélanges tels que de la matière d'échantillon biologique et cellulaire, par exemple, du sérum, des larmes, de l'urine, et du fluide cérébro-spinal. Le procédé consiste à employer les techniques de dosage par la méthode ELISA, dans laquelle une lame de test ELISA est enduite d'une matière adsorbante. L'invention concerne également un appareil de test immunogénique quantitatif dont une surface est enduite d'une matière adsorbante solide finement divisée, par exemple, une lame de test ELISA enduite d'un gel de silice. Le procédé permet la détection et la mesure de faibles concentrations d'anticorps et de substances organiques antigéniques, telles que des glucides, des protéines, des lipides et des substances pharmaceutiques. Le procédé permet avantageusement la détermination de lipides spécifiques en présence de substances contaminantes telles que d'autres lipides.
PCT/US1990/004826 1989-08-24 1990-08-24 Procede et appareil de mesure d'antigenes organiques Ceased WO1991002980A1 (fr)

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EP0201079A2 (fr) * 1985-05-07 1986-11-12 Richard Zahradnik Essai immunologique retardé en phase solide
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AU758648B2 (en) * 1998-04-10 2003-03-27 Bio Merieux Method for fixing a biological molecule on a support surface
US6660533B2 (en) 1998-04-10 2003-12-09 Bio Merieux Attaching a biological molecule to a support surface
RU2253871C1 (ru) * 2003-12-15 2005-06-10 Ставропольский научно-исследовательский противочумный институт Способ получения иммуносорбента
CN109512467A (zh) * 2018-10-12 2019-03-26 华中科技大学同济医学院附属协和医院 泪液脂质检测疏水纳米二氧化硅试纸及其制备方法
CN109512467B (zh) * 2018-10-12 2024-07-19 华中科技大学同济医学院附属协和医院 泪液脂质检测疏水纳米二氧化硅试纸及其制备方法

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