WO2001073437A2 - Essai d'immunosorption relie a un enzyme a base de complexe immun de specificite antigenique - Google Patents

Essai d'immunosorption relie a un enzyme a base de complexe immun de specificite antigenique Download PDF

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Publication number
WO2001073437A2
WO2001073437A2 PCT/US2001/009344 US0109344W WO0173437A2 WO 2001073437 A2 WO2001073437 A2 WO 2001073437A2 US 0109344 W US0109344 W US 0109344W WO 0173437 A2 WO0173437 A2 WO 0173437A2
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capture
antigen
marker
immune complex
antibody
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PCT/US2001/009344
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WO2001073437A3 (fr
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Stanley Paul Racis
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Diagen Corporation
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Publication of WO2001073437A3 publication Critical patent/WO2001073437A3/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • 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
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9

Definitions

  • An individual's immune system responds to foreign matter or antigens (Ag) by synthesizing specific antibodies (Ab) that can interact with initiating antigens and non-covalently bind the antigens to form antigen-antibody complexes or immune complexes (IC).
  • Ab specific antibodies
  • IC immune complexes
  • the normal immune response of antibody binding to antigen to form an immune complex usually benefits the host by eliminating and/or neutralizing the antigen.
  • an immune complex is found as a soluble component of bodily fluids, it is known as a circulating immune complex (CIC).
  • immune complexes are relevant to a large number of diseases. More recently, in vivo and in vitro experiments have helped elucidate many of the factors involved in immune complex formation, removal and localization, as well as the mechanisms of immune complex induced inflammatory reactions. The current belief is that immune complexes can be potentially pathogenic, as well as regulate both cellular and humoral immune responses by interacting with antigen receptor bearing lymphocytes, subpopulations ofT and B cells, and other immune and non-immune cells.
  • a ligand usually antigen or antibody
  • some form of solid support such as cell membranes, microscope slides, micro- titer wells, or diffusion or electrophoretically separated bands that are stabilized in gels.
  • the fluid to be tested usually blood, urine, cerebrospinal fluid or cell cultures, is then placed in contact with the solution or solid support that is displaying the ligand. If analyte, the substance to be measured, usually antibody or antigen, is present in the test fluid, it will bind to the solid support via the ligand display. Unbound materials are washed free of the solid support.
  • a secondary antibody with an attached signal-emitting molecule such as fluorochromes, enzymes, radioisotopes, magnetic substances, or chemiluminescent compounds
  • a signal-emitting molecule such as fluorochromes, enzymes, radioisotopes, magnetic substances, or chemiluminescent compounds
  • a secondary antibody with an attached signal-emitting molecule is incubated with the solid support to facilitate measurement of analyte binding to the ligand.
  • unbound labeled secondary antibody is washed free of the solid support.
  • quantitative detection of the signal is performed to determine the concentration of analyte in the fluid sample.
  • One type of immunoassay is a sandwich assay. Although there are many variations of this technique, the basic concept is simple. First, an antibody that is specific to an analyte of interest is immobilized on a solid-phase support.
  • a sample that potentially contains the analyte is incubated with the bound antibody. Then, a second antibody, which reacts with the analyte at a different site than that of the bound antibody, is added to the mixture. Detection of the second antibody is then used to measure the amount of analyte in the sample.
  • ELISA enzyme-linked immunosorbent assay
  • ELISA is an enzymatic variation on the sandwich assay described above.
  • the goal of an ELISA is to detect an antibody of interest so the roles of binder and ligand are reversed. Therefore, in an ELISA, antigen is bound to a solid-phase substrate. A sample containing an antibody of interest is incubated with the bound antigen. Then, a second, enzyme-labeled antibody directed against the antibody of interest is added to the mixture. Next, an enzyme substrate and any necessary buffers are added so that the enzyme may metabolize the substrate to form a reaction product. Finally, measurement of the reaction product is used to detect the quantity of antibody of interest in the sample.
  • the invention is in the field of immunologic serological in vitro diagnostics.
  • the invention is an ELISA-based diagnostic testing system and method that provides the capability to "look within” and measure a captured circulating immune complex's specific antigen and antibody using typical ELISA microplates and procedures.
  • the system and method of the present invention are more clinically relevant than measuring either antigen or antibody alone, and are useful for the host of diseases and conditions for which markers for the disease or condition have been identified.
  • the technology can also be used to elucidate and/or screen for the humoral immune response's targets within selected individuals, groups of individuals with shared diseases or conditions, and microarray data suggesting sets of activated genes or altered proteomic profiles.
  • One aspect of the invention is a method, and some variations on the same, for detecting antigen and antibody in immune complexes.
  • the method of the present invention comprises: capturing a circulating immune complex, resulting in a captured immune complex; dissociating the captured immune complex; re-associating the captured immune complex with an appropriate reference material, such as labeled antigen, antibody, and/or immune complexes (IC), to form a reformed immune complex; and detecting and quantitating the reference material, and hence antigen, antibody, or both antigen and antibody in the reformed immune complex.
  • a second aspect of the invention is a well design that may be used in the method of the invention.
  • the well design has several important characteristics.
  • the first characteristic of the well is that it has one or more surface area increasing members, such as vertical fins rising from the well bottom, to increase the wells' overall functional surface area.
  • Another characteristic is that the well is designed to remain substantially optically transparent in the appropriate substrate buffer.
  • a third aspect of the invention is a kit for detecting antigen, antibody, or both antigen and antibody in immune complexes.
  • Figure 1 is a flowchart of the core steps of the method for detecting antigen, antibody, or both antigen and antibody in immune complexes.
  • Figure 2 is a flowchart of an embodiment of the method for detecting antigen, antibody, or both antigen and antibody in immune complexes.
  • Figure 3 is a top plan view of the well of the present invention.
  • Figure 4 is a side elevation view of the well of the present invention.
  • the present invention provides a method for detecting antigen and antibody in immune complexes, a well design particularly adapted for use in the method of the invention, and a kit for detecting antigen and antibody in immune complexes.
  • the method is an ELISA-based diagnostic testing method. The method provides the capability to "look within” and measure an immune complex's specific antigen and antibody within both intact and dissociated immune complexes using typical ELISA microplates and procedures.
  • the method of the present invention comprises the core steps of: capturing a circulating immune complex, resulting in a captured immune complex; dissociating the captured immune complex; re-associating the captured immune complex with an appropriate reference material, such as labeled antigen, antibody, and/or immune complexes (IC) to form a reformed immune complex; and detecting and quantitating the reference material, and hence antigen, antibody, or both antigen and antibody in the reformed immune complex, as shown in Figure 1.
  • Immune complexes may be captured by any appropriate means for the disease or disorder in question. Immune complexes are preferably dissociated using agents that cause high salt concentration or low pH, however other methods are also possible.
  • Immune complexes may be re-associated using agents that decrease the salt concentration or increase the pH of the mixture, however other methods are also possible. Finally, detection and quantitation may be accomplished by measuring the optical density of the sample, although other methods of detection and quantitation are possible.
  • the method of the present invention can be used to detect antigen, antibody, or both antigen and antibody for use in diagnosing numerous diseases.
  • the method of the invention can be used to detect proteins that the immune system recognizes as important in disease.
  • proteins include, but are not limited to, coagulation proteins and tumor derived proteins, such as growth factors and their receptors, gene regulation factors, proteins that are important for the development of metastasis, and angiogenic proteins.
  • the first core step of the method is to capture one or more circulating immune complexes from a sample in an initial capture, resulting in one or more captured immune complexes. Capture simply means to selectively bind the sought for type or subtype of circulating immune complex to a solid phase component.
  • solid phase components are wells, gels, polyurethane, polystyrene, magnetic beads or a metallic substrate. Binding to the solid phase can occur by any one of or a combination of the following molecular forces: electrostatic interactions, ionic bonding or covalent bonding.
  • Preferred sample sources are blood, tears, saliva, lymph and urine. However, other sample sources may be used.
  • Circulating immune complexes are captured by any appropriate means for the disease or disorder in question, such as in the reference by Lawley, "Methods of Detection of Circulating Immune complexes", Clinical Immunology & Alert, vol. 1, pp. 383-396 (1981), which is incorporated herein by reference.
  • a capture agent is an agent such as a molecule, chemical, receptor, protein or antibody, that is used to bind the sought for type or subtype of circulating immune complex to a solid phase component.
  • Some of the acceptable capture agents include, without limitation, Staphylococcal proteins, complement component Clq, and anti-complement antibodies.
  • complement activation is the pathological marker desired
  • capture of complement containing immune complexes, Clq or C3 products would be appropriate.
  • Staphylococcal proteins such as protein A or protein G, or antibody directed against human antibody could be used.
  • the exact composition and nature of the capture phase is dependent upon the particular application with regard to the type or subtype of circulating immune complex deemed most clinically relevant for the disease or disorder.
  • the capture system is important to the present invention. However, some variables of the capture system are application specific. For example, if immunopathology involving complement-containing circulating immune complexes is important, then circulating immune complexes displaying complement would be important in terms of the capture system. However, if disease or tumor markers within circulating immune complexes are important, then the capture system would be designed to capture all possible circulating immune complexes rather than just subsets of circulating immune complexes.
  • circulating immune complexes are captured by any appropriate means for the disease or disorder in question.
  • the appropriate means for capturing circulating immune complexes can be readily determined in each case by one of ordinary skill in the art by reference to the literature, using particularly known markers for specific diseases or disorders.
  • DNA is the predominant marker of choice, and complexes containing DNA and anti-DNA that are capable of fixing and activating complement, because of their inherent ability to lead to immunopathology, would probably dictate the use of a complement based capture system comprising Clq or the C3 products.
  • the present invention also provides a method for determining the appropriate capture system for a particular patient's circulating immune complexes.
  • the method comprises pre-screening samples from patients along with appropriate controls for different "types" of circulating immune complexes.
  • the pre-screening tries to detect the presence of elevated circulating immune complex levels in different ELISA capture systems, such as systems based on Clq, Staphylococcal proteins, C3, Raji cell membranes, conglutinnin and MC antibodies.
  • the types of circulating immune complexes can be divided into groups: those which can and/or have bound Clq, those which can or have activated C3, and those which may or may not have activated or bound any complement.
  • the circulating immune complexes are captured with various capture systems for blot analysis, as described in Johnstone & Thorpe, "Immunochemistry in Practice", which is incorporated herein by reference.
  • blot analysis one can identify the individual blots or bands in terms of known markers, potential gene products from microarray data and/or other genetic analysis, suspected markers, or biochemical identification. The result is identification of the circulating immune complex components in each case.
  • Statistical analysis is then performed for correlation of any of the circulating immune complexes type's components with the appropriate clinical parameters for that disease.
  • the user can choose the capture method or methods that are significantly able to capture circulating immune complexes that correlate with the disease's clinical parameters. This procedure enables the user to determine the appropriate capture conditions for the capture phase.
  • the capture of circulating immune complexes may involve the use of a solid substrate or carrier, such as micro-titer wells.
  • a carrier is any molecule or molecular conglomerate that can be utilized as an intermediary to bind circulating immune complexes and facilitate removal from the liquid phase, such as by aggregation or precipitation.
  • the invention comprises a novel well design that will be discussed in greater detail below. For now, it should be realized that the choice of an appropriate well should allow for deposition/attachment of the capture agent or material without disrupting the optics of the well.
  • the preferred method for depositing the capture material on the well is lyophilization. Wells made of immulons, polystyrenes or polyethylenes all work well in the method of the present invention.
  • a cross-linker is any chemical or substance used to facilitate the attachment to the solid phase of the molecule that captures the circulating immune complex.
  • the use of a cross-linker is preferred.
  • Some examples of commercially available cross-linkers are poly-L-lysine, gfuteraldehyde and cyanogen bromide.
  • An acceptable method of pre-treating the wells is described in Voller et al., "The Enzyme Linked Immunosorbent Assay (ELISA)", Dynatech Labs, catalogue no. 011-010-6200.
  • immune complexes exist in either a state of antigen excess, antibody excess or equivalence. Therefore, all three possibilities must be taken into consideration.
  • the method of the invention follows standard ELISA procedures where multiple wells are employed when assuming the captured immune complexes are in antigen excess, antibody excess or equivalence. Simple interaction of the captured immune complex with appropriately labeled antigen or antibody will result in a qualitative determination for the specific presence of target antigens.
  • the circulating immune complexes are dissociated and then re-associated with enzyme labeled markers that are added in a carefully controlled fashion.
  • the next core step of the invention involves dissociating the captured immune complex, resulting in a dissociated immune complex.
  • the dissociation stage is application dependent so the particular dissociation conditions will depend upon the particular subtype of circulating immune complex that was captured. However, agents that cause high salt concentrations or low pH have proven to be effective dissociation agents, with alteration of the pH most preferable.
  • the minimum salt concentration should be approximately 300 mM NaCl while the maximum salt concentration should be approximately 1.5 M NaCl. However, 500 mM NaCl is most preferred.
  • the preferred salt solution has a pH of approximately 7.2 and comprises either 500 mM NaOH, 2 mM EDTA and 50 mM Tris buffer, or 500 mM NaOH, 2 mM EDTA and 50 mM sodium phosphate.
  • the problem with altering the salt concentration is that it makes the re-association stage more difficult in that it requires more time and more wells.
  • the captured immune complexes can be dissociated with low pH.
  • the effective pH maximum for the dissociation stage is approximately 2.8.
  • the preferred pH range is 1.5 to 2.5.
  • the most preferred pH range is 2.0 to 2.5. However, it should be realized that the lower the pH, the shorter the dissociation time that is needed. Acids, such as acetic acid, citric acid and hydrochloric acid, are preferable for lowering the pH.
  • the dissociation conditions should occur for a short period of time and include bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • Several types of BSA may be used, with 1% RIA-grade BSA generally being sufficient.
  • the short period of time for dissociation by lowering the pH is application dependent, but 5 to 30 seconds is generally optimal.
  • each application's specific antigen or marker of interest might require some modification to the time if the antigen or marker proves too sensitive to the lower pH.
  • the maximum time for dissociation has been observed to be 4 to 5 minutes for most glycoproteins, including antibody. However, significant loss of efficiency has been observed to occur generally after 1 minute of exposure to dissociating conditions.
  • an optional intermediate step may be performed.
  • the intermediate step involves adding a metered amount of labeled antigen or antibody so that one can perform a competitive ELISA analysis for the amount of unlabeled antigen or antibody within the originally captured materials.
  • enzyme-labeled antibody is used to target internal antigens and/or markers within the circulating immune complex.
  • enzyme labeled antigen is used when antigen specific antibody is itself the specific target being detected. It is generally preferred that the labeled materials be added in conjunction with the re- association phase.
  • the competitive ELISA of the optional intermediate step takes place in the previously prepared capture wells. It is preferred that the dissociating solution is in a volume at least 4 fold smaller than the re-associating solution. However, the dissociating volume is preferably not less than 10 microliters and not greater than 50 microliters. Re-associating volumes are limited only by the volume of the well. It is most preferred to have a dissociating volume of 20 microliters and a re-associating volume of 20 to 130 microliters. It is desireable to minimize the re-association volume, as a larger re-association volume usually requires increased times for re- association.
  • the invention includes the core step of re-associating the dissociated immune complex with a reference material.
  • the re-association step is coincident with the addition of labeled reference materials.
  • a reference material is any material that is labeled with a marker and that competitively binds the desired component of the dissociated immune complex.
  • Some examples of reference materials are enzyme-labeled markers, such as labeled antigen, antibody, or immune complexes, that are added in a carefully controlled fashion to form a reformed immune complex.
  • Such re- association methods include re-associating agents that decrease the salt concentration or increase the pH in the mixture.
  • re-association may involve simply reversing the dissociation conditions in the original well.
  • the salt concentration could be decreased by dilution, or excess salt could be removed by dialysis.
  • the re-association step will involve increasing the pH. Therefore, the buffer should overcome the low pH from the previous dissociation step and readjust the pH to approximately 7.2 with variable protein loads.
  • re-association may require transfer of the dissociated materials to another prepared well as a function of the specific antigens involved.
  • the antigen is not a protein, such as sterols or nucleic acids, different buffers and solvents might be required which would necessitate a non-aqueous phase for re-association and/or secondary capture.
  • a parameter in the method of the present invention is the time of incubation for re-association.
  • the incubation time for re-association is dependent upon the specific antigens in question and can vary from as low as 20 minutes to as high as 12 or more hours, at differing temperatures. Typically, incubation is for 20 minutes to 2 hours, and preferably 1-2 hours, at room temperature. Re-association should occur for at least 20 minutes, and 60 minutes is preferable. However, if known low affinity reactions are involved, it may be preferable to incubate at 4-10° C for 8-12 hours.
  • Another variable for the re-association stage is the amount of enzyme-labeled marker needed for maximum sensitivity of sample antigen/antibody marker interference. This is determined as follows.
  • Standard reference preparations are determined by generating a standard dose response curve for titrated unlabeled target materials for each of a wide range of enzyme-labeled reactants. Typically, the highest concentration of enzyme-labeled reactants which do not result in background readings (when no unlabeled ligand is present in a sample) above 0.100 are employed. By titrating known amounts of unlabeled target antigen or marker with this concentration of enzyme-labeled reactant, a typical control curve is generated which allows for quantitative determinations.
  • the next core step of the invention involves detecting and quantitating the reference material, and hence, the antigen, antibody, or antigen and antibody within the reformed immune complexes. Detection and quantitation occurs after the labeled and reformed circulating immune complexes are bound to the solid phase.
  • the samples are then washed, the appropriately buffered colorless substrate is added and an optical density (OD) reading of the substrate's color intensity is taken using standard commercially available readers. The OD readings are interpreted using a standard curve as a reference.
  • the degree of inhibition of control enzyme labeled binding relative to controls quantitatively reflects the amount of specific antigen in the original sample.
  • the secondary capture refers to a capture other than the initial capture of circulating immune complex and occurs after the initial capture step.
  • the preferred method of secondary capture is to utilize the same capture system in the same well as the initial capture, but with an antigen- specific labeled antibody that overwhelms the unlabeled aspect, i.e. the antigen of interest in the circulating immune complex, during re-association.
  • individual applications may warrant the direct capture of the specific antigen, either in the same well or in other wells, and/or employing secondary capture systems on other surfaces, such as a well surface, dipsticks, beads or membranes.
  • the secondary capture may proceed in either the same well as the initial capture or in a newly prepared well. If the secondary capture proceeds in the same well as the initial capture, then no secondary capture preparations are needed. However, if the secondary capture proceeds in a newly prepared well, then secondary preparations are needed, based upon the specific antigen or antibody sought.
  • Some acceptable wells for the secondary capture phase include: EB plate, manufactured by Labsystems Co., Ltd. (Atlanta, GA); H type plate, manufactured by Sumitomo Bakelite Co., Ltd (Singapore); C type plate, manufactured by Sumitomo Bakelite Co., Ltd (Singapore); and Maxi-Soap plate, manufactured by Nunc Co., Ltd (Naperville, IL).
  • Beta 2-Glycoprotein I is coated to the desired carrier.
  • Beta 2-Glycoprotein I may be bound to the carrier by appropriately combining and conducting conventional processes under suitable conditions known for immobilization of a protein, such as an enzyme, as discussed in: Liang et al., "Biomedical application of immobilized enzymes", J. Pharm. Sci., vol. 89(8), pp. 979-990 (2000); Wan et al., "Behavior of soluble and immobilized acid phosphatase in hydo-organic media", Biochim Biophysca Ada, vol.
  • carriers are EB plate, H type plate, C type plate, and Maxi- Soap plate.
  • the carrier can be of various types and shapes, including plate-like type, such as microtiter plate or disk; granular type, such as beads; tubular type, such as test tubes; fibrous type; membrane-like type; and fine particulate type, such as latex particles.
  • the appropriate carrier will depend upon the assay method.
  • Beta 2-Glycoprotein I derived from any animal is acceptable, although Beta 2- Glycoprotein I of human origin is preferred.
  • Beta 2-Glycoprotein I may be prepared in any conventional manner, such as by the method of McNeil, as described in Proc. Natl. Acad. Sci., vol.
  • Beta 2-Glycoprotein I amino acid and nucleotide sequences of Beta 2-Glycoprotein I have been disclosed in Rasmussen et al, "Structure of the human beta2-gycoprotein I (apolipoprotein H) gene", European Journal of Biochemistry, vol. 259, pp. 435-440 (1999), which is incorporated herein by reference, so preparation by recombinant DNA techniques or peptide synthesis are also acceptable.
  • Furthennore the sugar chain can be partially or wholly removed and still be used in the present invention. The method of the present invention does not require that Beta 2-Glycoprotein I be highly purified, however, purification is preferred.
  • the method of the present invention allows one to "look within” captured immune complexes for specific antigens and antibodies all within a single ELISA test run. Therefore, the four core steps of capturing, dissociating, re- associating, and detecting and quantitating must occur in this specific order. However, other steps of the invention need not occur in a particular order.
  • the steps of the invention include: determining the appropriate capture conditions; capturing one or more circulating immune complexes from a sample; adding one or more antigen specific reference materials with enzyme-labeled antigen or antibody to the mixture; dissociating the captured immune complexes by adding dissociating agent; re- associating the captured immune complex with reference material by adding re- associating agent; putting a cover with capture fingers into the wells and incubating; transferring the cover and fingers into substrate wells and incubating; and detecting and quantitating the antigen, antibody, or both antigen and antibody by reading the optical density.
  • FIG. 2 Another embodiment of the method of the present invention is shown schematically in Figure 2.
  • This figure shows the sequential steps for the testing of intact captured complexes parallel to the testing of dissociated/re-associated complexes. Except in cases where the target to be quantitated is completely masked or saturated, one can detect the target without dissociation, albeit not in a reasonably quantitative manner. In order to determine the relative amount of target, the complexes must be dissociated and re-associated in the presence of the competing labeled marker.
  • a second aspect of the invention is for a well 10 design that is specifically adapted for use in the method of the invention.
  • One characteristic of the wells 10 are that they have one or more surface area increasing members 20 to increase the well's 10 overall functional surface area.
  • Such members include fins 22, either vertical or horizontal, or any other type of configuration that would increase the overall surface area of the wells 10.
  • the wells 10 would have vertical fins 22 arranged in a radial pattern from the circumference of the well 10 that extend from the well bottom 30 to a height approximately equal to the well radius 40 or about % of the height of the well.
  • the fins 22 are in a radial pattern off the circumference of the well 10 in order to increase the liquid-plastic contact area.
  • the number and thickness of the fins 22 are directly related to their ability to increase the effective surface capture area and the ability to facilitate effective washing and rinsing of the well 10.
  • the preferred design would have fins 22 that have a thickness less than the well bottom 30 but greater than 0.25 of the well bottom 30 thickness.
  • the wells 10 may be made out of any material that will permit the methods of the present invention. However, wells 10 made out of immulon, polystyrene or polyethylene are most preferred. Another characteristic of the wells 10 is that they are designed to remain substantially optically transparent in the appropriate substrate buffer.
  • Substantially optically transparent means that, upon the addition of buffer, the level of diffraction, interference or absorption in the well will cause a low background in an OD reading that can be normalized or accounted for.
  • the level of background should not be above 0.2, and is preferably below 0.05.
  • the surface area increasing members 20 of the wells 10 may have any number of physical configurations, these configurations should not scatter light or cause reflection. Therefore, the surface area increasing members 20 should not be located in the well 10 in a position, or have a configuration that would scatter light when taking an OD reading.
  • a third aspect of the invention is for a kit for detecting antigen, antibody, or both antigen and antibody in immune complexes.
  • Each kit will be application dependent, depending upon the particular subset of circulating immune complexes one wishes to capture. Therefore, each kit will contain a potential marker, receptors for the marker, and/or antibodies against any of these.
  • each kit may contain the appropriate buffers, solutions, wells and other materials necessary to enable a user to perform the method of the present invention. Also, the kits will be designed to take into consideration the states of antigen excess, antibody excess or equivalence based upon the initial pre-screening for which capture method works best. Therefore, the primary purpose of the kit dictates the capture system.
  • kits may contain both positive and negative controls.
  • the kits are particularly suited for use in the detection and treatment of many types of diseases, including: autoimmune disease, oncology (cancer), and infectious diseases. Specific applications for the kits would involve a host of potential markers, their receptors and/or antibodies against any of these.
  • the markers include coagulation proteins and tumor derived proteins, such as growth factors and their receptors, gene regulation factors, proteins that are important for the development of metastasis, and angiogenic proteins.
  • Coagulation proteins include, but are not limited to, fibrin degradation proteins (D-dimer, fibrin split products), plasminogen fragments (angiostatin, endostatin), tissue factor, endothelial cell derived proteins and cleaved antithrombin III.
  • Growth factors and their receptors include, but are not limited to, endothelial growth factor (EGF), Her-2/neu (c-erb-2), c-erb-3 and c-erb-4.
  • Gene regulation factors include, but are not limited to, p53 protein, c-myc protein and cyclins. Proteins that are important for the development of metastasis include, but are not limited to, cathepsin D, urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), matrix metalloproteinasees (MMP), NM23 gene and laminin.
  • uPA urokinase plasminogen activator
  • tPA tissue plasminogen activator
  • MMP matrix metalloproteinasees
  • Angiogenic proteins include, but are not limited to, vascular endothelial growth factor (VEGF), basic fibroblastic growth factor (bFGF), platelet derived growth factor (PDGF) and transforming growth factor- ⁇ (TGF- ⁇ ).
  • VEGF vascular endothelial growth factor
  • bFGF basic fibroblastic growth factor
  • PDGF platelet derived growth factor
  • TGF- ⁇ transforming growth factor- ⁇
  • hsp heat shock proteins
  • PS2 protein PS2 protein
  • any present or new marker associated with a particular condition may be put into a kit.
  • a spectrophotometer capable of reading the absorbance at 490 nm of samples having a volume of 100-200 ⁇ l.
  • Sample blood was obtained by venipuncture. After the sample was obtained, it was allowed to clot for 2 hours at room temperature. The sample was then refrigerated for 15 minutes. Following refrigeration, the sample was centrifuged for 5 minutes at 4,000G and the supernatant (sera) was removed.
  • the sera can be stored at 2-4° C if used within 48 hours. However, if long term storage of the sera is needed, it should be stored at -70° C. Care should be taken to avoid the introduction of ethylenediamine tetraacetic acid (EDTA) and/or other chelating agents because they may affect the binding of the various components of the immune complexes.
  • EDTA ethylenediamine tetraacetic acid
  • the exact volume of sera to be tested can be chosen by the end user, but should be normalized for any one test.
  • the sensitivity of the assay may be increased proportionally to the volume of sample utilized, practical limitations do exist, such as the absolute volume of the well. Therefore, the recommended volume of sera per test sample is 100 microliters ( ⁇ l).
  • all samples should be diluted to give a dilution or a range of dilutions that fall within the range of the standard curve. It is recommended that the patient samples be diluted 1 :50. A 1 :50 dilution is produced by adding 1.45 ml of wash/diluent solution to 50 ⁇ l of prepared sera.
  • the positive control has been standardized to allow for development of a standard curve for the evaluation of the test samples. Lyophilized artificially produced immune complexes are stabilized in human serum. Therefore, 250 ⁇ l of sterile de-ionized type II water was added to lyophilized human antigen immunoglobulin or Heat Aggregated Human IgG (HAI) stock. After reconstitution, there was approximately 500 ⁇ g equivalent/ml in phosphate buffered saline (PBS) with 0.05% bovine serum albumin (BSA) having a pH of approximately 7.2. The positive control, an undiluted solution at 500 ⁇ g equivalent/ml concentration, was then diluted to generate the standard curve.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • the negative control should score in the normal patient range when properly employed in the assay.
  • Lyophilized human serum negative control which should score in the normal patient range was reconstituted in 300 ⁇ l of sterile de-ionized type II water to produce an undiluted negative control. After reconstitution, the negative control was diluted 1 :50 with the wash/diluent solution. A 1 :50 dilution is produced by adding 1.45 ml of wash/diluent solution to 50 ⁇ l of the reconstituted negative control. The negative control was then run as a test sample.
  • Example 4 Plotting the standard curve.
  • the concentrations of immune complexes in the patient samples were read from a standard curve. In order to be as precise and accurate as possible, a standard curve should be produced for each round of testing.
  • the positive control of example 2 was diluted using the wash/diluent solution.
  • the positive control was diluted to the following six concentrations: 62.5, 31.25, 15.625, 7.8125, 1.9531 and 0 ⁇ g/ml.
  • the 62.5 ⁇ g equivalent/ml solution was produced by adding 100 ⁇ l of the reconstituted positive control (HAI stock) to 700 ⁇ l of wash/diluent solution.
  • the 31.25 ⁇ g equivalent/ml solution was produced by adding 400 ⁇ l of the 62.5 ⁇ g solution to 400 ⁇ l of wash/diluent solution.
  • the remaining concentrations were produced by continuing the 1 :2 serial dilution using 400 ⁇ l of the previous solution with 400 ⁇ l of the wash/diluent solution.
  • the OD 490 nm readings for the positive control dilutions were plotted against the appropriate ⁇ g equivalent/ml on semi-log paper. Then, a straight line was drawn that best fits the plotted points. This is the standard curve. If a single point caused an anomalous deformity in the shape of the curve, the point was omitted. If more than one point lied outside the normal expected curve, then the assay was repeated. Experiments have indicated that this standard curve is optimized between 2 and 60 ⁇ g equivalents/ml. Using this standard curve, find the point corresponding to the 1/25 dilution of each patient sample read at 490 nm and record the corresponding ⁇ g equivalent/ml as the value for that sample.
  • a 1/75 dilution reading should be used for determining the ⁇ g equivalent/ml result.
  • the corresponding ⁇ g equivalent/ml result for the 1/75 dilution should be multiplied by a factor of 3, equaling the quantitative ⁇ g equivalent/ml result.
  • test values showed that: A. Any sample recording below 2.5 ⁇ g equivalent/ml should be considered normal. A mean value of 1.8 ⁇ g equivalent/ml should be expected for a normal patient sample with a standard deviation of 1.5.
  • Cyanogen bromide pre-treatment of wells with cross-linkers followed standard procedures for cyanogen bromide, as disclosed in Johnstone & Thorpe, "Immunochemistry in Practice", Blackwell Scientific Publications, pp. 205-208 (1982), which is incorporated herein by reference. Essentially, 15.0 gm of cyanogen bromide solution was slowly added to 20.0 ml of 0.5 M carbonate buffer while 4 N NaOH was used dropwise to keep the pH between 10.5 and 11.0. 200 ⁇ l of this solution was immediately applied to micro-titer wells and allowed to incubate for 10 minutes at room temperature.
  • Gluteraldehyde approximately 200 ⁇ l of 0.1% gluteraldehyde was pipetted into micro-titer ELISA wells and incubated for 1 hour at room temperature. The wells were washed at least 6 times with cold 0.18 M PBS, decanted, and allowed to drain inverted for 5 minutes prior to use.
  • Poly-L-lysine approximately 200 ⁇ l of 2-5 mg/ml solution of poly-L-lysine was pipetted into micro-titer ELISA wells and incubated for 1 hour at room temperature. The wells were washed at least 6 times with cold 0.18 M PBS, decanted, and allowed to drain inverted for 5 minutes prior to use.
  • Plate preparations involve coating the interior of the wells with molecules capable of capturing/binding the desired circulating immune complex set or subset.
  • Staphylococcal proteins can be used for general collection.
  • Clq, anti-Clq, C3d, anti-C3d, conglutinnin, or Raji cell membrane extracts may be used to coat the interior of the wells.
  • the wells may be blocked with bovine serum albumin prior to lyophilization. After lyophilization, the wells are ready for use, i.e., there is no need to rehydrate the wells prior to use.
  • Wells were prepared containing lyophilized Clq on the bottom of each well.
  • the wells were prepared by incubating 150 ⁇ l of a 10 ⁇ g/ml solution of Clq in the wells for 15 minutes at room temperature. The wells were emptied prior to lyophilization. A solution of 1% RIA grade BSA was used to block the wells.
  • Example 7 Well preparation using Staph proteins.
  • Staphylococcus Aureus protein preparations Protein A and Protein G, were purchased from Pharmacia Corp., Piscataway, NJ. 150-200 ⁇ l of the appropriate protein preparation, at approximately 20-100 ⁇ g/ml, were applied to the wells for 1 hour at room temperature. The wells were then washed 3 separate times with PBS. The wells were then incubated for 1 hour with 1 % BSA in PBS to block, followed by one more wash.
  • Staphylococcus Aureus may be employed by following the methods described in Johnstone & Thorpe, Immunochemistry in Practice, pp. 209- 210 (1982), or Agnello, Human Pathology, vol. 14, pp. 343-349 (1983), both of which are incorporated herein by reference.
  • Example 8 Well preparation using Raji cell membrane preps.
  • Raji cell membrane preps were purchased from ATC, Cherry Hill, NJ. 150-200 ⁇ l of the Raji cell prep at approximately 20-100 ⁇ g/ml was applied to the wells for 1 hour at room temperature. The wells were then washed 3 separate times with PBS. The wells were then incubated for 1 hour with 1% BSA in PBS to block, followed by one more wash.
  • Raji cell membrane preps can also be used to prepare wells by following the methods described in Coyle et al., "A micro ELISA Raji cell assay to detect immune complexes", Journal of Immunological Methods, vol. 74, pp. 191-197 (1984).
  • the methods of the present invention have some minor additional modifications, such as the use of fragmented cell membranes as a capture molecule instead of prepared or purified molecules, that can be found in Coyle et al., "A micro ELISA Raji cell assay to detect immune complexes", Journal Immunogical Methods, vol. 74, pp. 191-197 (1984), which is incorporated herein by reference.
  • Example 9
  • Example 10 Method of capturing CICs using Staph proteins.
  • Example 12 Method for detecting CICs with polyethylene glycol (PEG).
  • the enzyme-labeled reactant was then introduced such that the enzyme- labeled reactant and the target molecule formed a new circulating immune complex, which was then captured.
  • the plates were then washed to remove the unbound materials. After adding substrate, OD readings were taken. The OD readings related to the amount of target antigen in the sample.
  • dissociation takes place in the well with the captured circulating immune complex.
  • Re-association with the enzyme-labeled reactant takes place in the same well
  • material was either withdrawn into new wells that were set up for capture or an additional capture surface was introduced into the wells, such as ELISA coverplates with "dipsticks”.
  • Example 14 Method of dissociating captured immune complexes using high salt concentration. This was tested in 2 discrete steps.
  • the synthetic immune complex was prepared by combining each immunoglobulin source (human IgG and murine IgM-anti-human IgG) at molar equivalence for 1 hour at room temperature. The particulates were then centrifuged out at low speeds, and the solution was passed through a Sephadex column. The 3 recovered bands were tested for the presence of each component. The heaviest band was the only one which showed the presence of both human and murine immunoglobulin. Concentrations were determined spectrophotometricly.
  • the solution was then diluted 1 :4 into a dissociating buffer containing 500 mM NaCl, 2 mM EDTA, and 50 mM sodium phosphate at pH 7.3 and 30.5 mmho (milli mho, units of conductance) at approximately 23° C.
  • This solution was then passed through a Sephadex column and 2 bands were identified by absorbance at 280 nm.
  • the bands were collected, dialyzed against PBS overnight, and tested for the presence of human IgG or murine IgM by latex bead agglutination assays with latex beads coated with either anti-human IgG or anti-murine IgM. Each band was found to have only one component, thus suggesting separation, as the original preparation in PBS led to only one band when passed through the Sephadex column.
  • a model circulating immune complex solution was made by mixing molar equivalent solutions of human IgG and murine IgM-anti-human IgG, purchased from Sigma Chemical, St. Louis, Missouri, in physiological saline to reach a concentration greater than 2 mg/ml. This solution was prepared by combining each immunoglobulin source at molar equivalence for 1 hour at room temperature. The particulates were centrifuged out by low-speed centrifugation and collected. The solution was then eluted through a Sephadex G-25 column, purchased from
  • the bands from the G-25 column were diluted 1 :2 in immunoglobulin depleted plasma, i.e., normal human plasma that had been passed through a Protein- A/G Sephadex column, purchased from Pharmacia Corp., Piscataway, NJ, and 100 ⁇ l was added to the wells in each of the capture systems. ELISA measurements were recorded for each system and normalized to a value of one hundred percent for that capture system. The wells from each ELISA system were stopped prior to secondary labeled antibody stages, and were treated with 20 ⁇ l of acid treated saline with a pH of approximately 2.5 or with 20 ⁇ l of normal PBS.
  • the wells were treated with 100 ⁇ l of secondary enzyme-labeled anti-Ig, purchased from Sigma Chemical, St. Louis, Missouri, in 1% BSA in PBS having a pH of 7.2.
  • the well's entire contents were transferred to identical capture system wells that had not been subjected to any acid treatments.
  • 0.5 ml of the acid dissociated CIC preparations (pellets) from example 14 were treated with either 0.5 ml of PBS at a pH of 7.2 or with 0.5 ml of tris-HCl at a pH of 8.0, and incubated at room temperature for 60 minutes. Turbidity was present but gentle centrifugation for 10 minutes yielded a visible pellet that was similar in size and nature to the initial precipitate pellet. This result indicates that acid dissociation could be reversed by returning the pH to over 6.0 within 30-120 seconds of the initial acid exposure. However, as the time of acid treatment exceeded 30 seconds, the amount of the recoverable pellet decreased, and the nature of the pellet was visibly altered.
  • bands representing circulating immune complexes human IgG and murine IgM were independently applied to ELISA systems, except for a few differences.
  • the first difference was that the capturing molecules were cross-linked into the ELISA wells.
  • the second difference was that some wells were not exposed to acid treatment; some wells were exposed to acid treatment alone; and other wells were exposed to acid treatment, followed by treatment with a base, 1 drop of 0.5 M NaOH, to restore the pH to approximately 7.2.
  • other wells saw no removal of materials with the ELISA proceeding as before. While the OD readings varied between the different types of cross-linkers, the relative readings within each system were remarkably consistent.
  • a typical cross-linker response is shown instead of all three.
  • the CIC control response was set to equal 100%) for each capture system. The results are as follows:
  • Enzyme-labeled antibody preparations were purchased from Sigma Chemical, St. Louis, MO, with alkaline phosphatase or horseradish peroxidase as the enzyme- substrate system.
  • the antibodies were directed to either human immunoglobulin heavy chains (to reflect the presence of circulating immune complexes), Beta 2 Glycoprotein, various other anti-cardiolipin antibodies, or enzyme-labeled cardiolipins.
  • Lyophilized anti-human immunoglobulin was complexed to the enzyme horseradish peroxidase.
  • lyophilized horseradish peroxidase labeled goat anti-human immunoglobulin was added to 325 ⁇ l of sterile de-ionized type II water. This stock was then diluted by addition of 10 ml of wash/diluent solution. The diluted solution was then ready for use.
  • Example 20 Substrate reaction. After completing the step of adding the enzyme-labeled conjugate that is illustrated in the previous example, the fluid was decanted from the well and the well inverted. The plate was then tapped several times to remove any remaining solution. 100 ⁇ l of the substrate solution (OPD dissolved in phosphate citrate buffer) was then added to each well. The incubation should proceed for 30 minutes at room temperature. The incubation should also occur in an environment that is protected from exposure to light. The well was wrapped in aluminum foil. Alternatively, the wells could be placed in a box. The user should limit exposure of this solution to light because the substrate solution is light sensitive. In addition, the solution should be made immediately prior to use and should not be re-used.
  • OPD phosphate citrate buffer
  • the incubation time is also important. Decreasing the incubation time will decrease the overall readings, with the background being decreased to a greater extent than the positive readings. On the other hand, increasing the incubation time may increase the apparent sensitivity of the assay, but is often complicated by a concurrent increase in the background readings.
  • Stopping the enzyme-substrate reaction After the 30 minute incubation reaction, 20 ⁇ l of stop solution was added to each well.
  • Our stop solution comprised hydrochloric acid at a concentration of IN.
  • the substrate reaction could be stopped by adding 25 ⁇ l of 2%> hydrogen peroxide in PBS.
  • the results should be read on a standard ELISA reader at the appropriate optical density (OD) for the enzyme-substrate system.
  • a plate reader was used to record the results.
  • the plate reader was zeroed on an empty well.
  • the wells were then read and the optical densities recorded at 490 nm for each patient sample on a patient log. This should occur within one hour of stopping the substrate reaction.
  • the method of the present invention is also able to detect hidden or masked components of circulating immune complexes.
  • a series of immune complexes were constructed such that one component of the complex was available in decreasing amounts.
  • polyethylene glycol (PEG) precipitation was used to collect the immune complexes from the free immunoglobulins. This was followed by placing the re-dissolved PEG precipitates (immune complexes) in ELISA wells coated with murine anti-human IgM. After a 2 hour incubation, the wells were washed and an enzyme-labeled secondary antibody against human IgM and human IgG were added to identical samples. OD readings of the samples and controls were taken.
  • RF-IgM Human Rheumatoid Factor
  • pooled human IgG also purchased from Sigma, starting at equimolar concentrations and then titrating the IgG twofold.
  • the experiment was started with 2 ml of 1 mg/ml RF solution.
  • the RF and IgG solutions were both in PBS at a pH of 7.2, and allowed to incubate at room temperature for 1 hour.
  • PEG was then slowly added to a final concentration of 2%.
  • the resulting solution was centrifuged at approximately 2000G for 10 minutes to pellet the precipitate. The supernatant was discarded and the pellet was washed 3 times with 2%> PEG in PBS.
  • the pellet was then resolubilized in 1 ml of PBS having a pH of 7.2 by vortexing vigorously.
  • Samples from each resolubilized CIC precipitate were then added to ELISA wells that were coated with either murine anti-human IgM or murine anti- human IgG. After incubation, the wells were washed. This was followed by the addition of a secondary enzyme-labeled antibody against either human IgG or human IgM, depending on what the wells were coated with. Next, substrate was added and subsequently stopped. Finally, OD readings were taken.
  • samples A5 and A6 were prepared at double the previous pellet concentration by halving the standard volume of PBS that was used to resombilize the pellet.
  • Sample Al was prepared in the same fashion as above.
  • the same capture systems were employed as above except the antibodies were cross- linked into the wells with cyanogen bromide as previously described.
  • the use of gluteraldehyde as the cross-linking agent gave similar results. Acid treatment of non- cross-linked capture molecules seemed to dissociate them from the wells. After the circulating immune complexes were captured in the wells, acid dissociation was initiated using HCl. Then, enzyme-labeled secondary antibodies were introduced into the well along with buffer to restore the pH, as previously described.
  • the enzyme- labeled secondary antibodies were of murine origin and previously had been shown not to react with human RF. These secondary antibodies were added in excess, limited only by their ability to lead to non-specific background. The results were as follows:
  • proteomics is the science that deals with gene products, namely proteins, and concerns itself with the entire collection of proteins (the proteome) produced by a particular cell or organism.
  • the method of the present invention focuses on antigens that the immune system recognizes as important in disease. This is relevant because certain proteins may serve as sensitive markers for the early detection of various diseases or for the early detection of recurrent disease. Current diagnostic tests look for free proteins in the blood to confirm a diagnosis. On the other hand, the method of the present invention looks at and within molecular conglomerates in the blood so it is able to see what the patient's immune system is recognizing and responding to in terms of a disease. Therefore, the method of the present invention detects markers that current tests miss and is able to detect initial and recurring tumors earlier with less false positive and negative results.
  • the method of the present invention will redefine the way science deals with proteins (selective proteomics) and accelerate the development of such products as disease diagnostics, prognostic markers and therapeutics because the method of the present invention is able to detect various proteins earlier and more precisely than currently available diagnostic techniques due to the fact that the method uses the body's ability to be immune based.
  • the method of the present invention allows the physician to see how well or poorly the patient's immune system is dealing with the disease.
  • the currently available diagnostic modalities such as mammography, MRI and tumor markers, do not meet the needs of breast cancer testing in terms of early detection, response to treatments and in monitoring tumor recurrence.
  • the method of the present invention will help address these shortcoming.
  • the method will aid in diagnosis because there are no FDA (Food and Drug Administration) approved tests available to detect breast carcinoma prior to its detection by mammography.
  • the method will aid as a prognostic marker because there is currently no good FDA approved plasma or serum test for predicting a patient's response to chemotherapy, hormonal therapy or radiation therapy.
  • the method aids in monitoring recurrence because there is no test available that reliably detects recurrent breast cancer until the disease is incurable.
  • the method also finds novel tumor antigens because many of the best selling drugs either act by targeting proteins or are proteins.
  • the current screening methods for breast cancer have a high incidence of false positive results. Because the present invention is able to detect various proteins earlier and more precisely than currently available tests and because the method of the present invention can find evidence of disease in ways existing kits can not, the present invention will have a profound effect on the way diseases are diagnosed, recurrences are detected and molecular therapeutic targets will be discovered.
  • the key to many diseases is early detection so the method of the present invention allows individuals to be treated with drug therapy sooner and more accurately, thereby enhancing a patient's chance for recovery.
  • the method of the present invention will be beneficial to all patient subtypes, but especially beneficial for those younger patients for whom current diagnostic modalities, such as mammograms, are not very sensitive.
  • the method of the present invention can support a range of blood tests, not only for breast cancer, but for various cancers, and also for autoimmune disorders and infectious diseases.
  • Example 25 Feasibility of discovering biomarkers within circulating immune complexes.
  • the Clq captured circulating immune complexes were recovered from the solid phase by washing the individual wells with citric acid (0.05ml @ pH 2.8), with the wash being immediately added to a PBS collection vial (1 ml total of citric wash added to 3 ml of PBS).
  • each group of pooled samples had circulating immune complexes that were recovered by both PEG and Clq.
  • the samples were then either tested intact or after at least partial dissociation for suspected relevant antigens.
  • the sample groups and findings are shown below:
  • the d-dimer assay is a fluorescent slide assay that was performed only on the breast cancer samples. The readings were brighter/higher than any single patient sample ever seen. It is apparent that some suspected relevant biomarkers are contained within circulating immune complexes, however, not all CIC bound biomarkers are detectable on intact circulating immune complexes. Therefore, the clinical significance of any particular sequestered biomarker needs to be elucidated. Whether additional biomarkers are represented within circulating immune complexes of groups of patients, and what their clinical significance may be, both need to be more thoroughly determined.

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Abstract

L'invention concerne le domaine des diagnostics immunologiques sérologiques in vitro. Elle concerne un système d'essai diagnostique à base ELISA et un procédé permettant d'effectuer une 'introspection' et une mesure d'antigènes et d'anticorps spécifique à des complexes immuns au moyen de microplaquettes et de processus classiques ELISA. Un aspect de l'invention consiste en un procédé servant à détecter un antigène et un anticorps dans des complexes immuns. Un deuxième aspect de l'invention consiste en une conception de puits qu'on peut utiliser afin de mettre en application le procédé. Un troisième aspect de l'invention consiste en une trousse servant à détecter un antigène, un anticorps ou à la fois un antigène et un anticorps dans des complexes immuns.
PCT/US2001/009344 2000-03-27 2001-03-23 Essai d'immunosorption relie a un enzyme a base de complexe immun de specificite antigenique WO2001073437A2 (fr)

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EP2367005A1 (fr) * 2003-10-30 2011-09-21 Xeptagen SPA Procédé pour mesurer des valeurs indiquant un cancer
CN102636654A (zh) * 2012-04-28 2012-08-15 上海北加生化试剂有限公司 检测人血清中补体Clq浓度的试剂盒及其方法
CN103575908A (zh) * 2013-10-30 2014-02-12 浙江理工大学 一种古代文物材料中胶原蛋白的检测方法
CN104677889A (zh) * 2015-02-05 2015-06-03 临沂大学 一种基于鲁米诺功能化的磁性免疫探针检测甲胎蛋白的方法
CN105001310A (zh) * 2015-07-14 2015-10-28 上海拜豪生物科技有限公司 一种铬螯合型免疫复合物及其制备方法和应用
CN105017430A (zh) * 2015-07-14 2015-11-04 上海拜豪生物科技有限公司 一种镍螯合型免疫复合物及其制备方法和应用
CN105044369A (zh) * 2015-07-14 2015-11-11 上海拜豪生物科技有限公司 一种铅螯合型免疫复合物及其制备方法和应用
CN105044324A (zh) * 2015-07-14 2015-11-11 上海拜豪生物科技有限公司 一种砷螯合型免疫复合物及其制备方法和应用
CN105137059A (zh) * 2015-07-14 2015-12-09 上海拜豪生物科技有限公司 一种汞螯合型免疫复合物及其制备方法和应用
CN113009135A (zh) * 2021-02-19 2021-06-22 山东省大健康精准医疗产业技术研究院 一种检测cd47的管式磁微粒化学发光免疫定量试剂盒及其制备方法与应用
CN114397454A (zh) * 2021-12-03 2022-04-26 新疆医科大学第三附属医院 一种人c-myc基因快速监测试剂盒及其使用方法

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Cited By (13)

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EP2367005A1 (fr) * 2003-10-30 2011-09-21 Xeptagen SPA Procédé pour mesurer des valeurs indiquant un cancer
CN102636654A (zh) * 2012-04-28 2012-08-15 上海北加生化试剂有限公司 检测人血清中补体Clq浓度的试剂盒及其方法
CN103575908B (zh) * 2013-10-30 2016-01-13 浙江理工大学 一种古代文物材料中胶原蛋白的检测方法
CN103575908A (zh) * 2013-10-30 2014-02-12 浙江理工大学 一种古代文物材料中胶原蛋白的检测方法
CN104677889A (zh) * 2015-02-05 2015-06-03 临沂大学 一种基于鲁米诺功能化的磁性免疫探针检测甲胎蛋白的方法
CN105017430A (zh) * 2015-07-14 2015-11-04 上海拜豪生物科技有限公司 一种镍螯合型免疫复合物及其制备方法和应用
CN105044369A (zh) * 2015-07-14 2015-11-11 上海拜豪生物科技有限公司 一种铅螯合型免疫复合物及其制备方法和应用
CN105044324A (zh) * 2015-07-14 2015-11-11 上海拜豪生物科技有限公司 一种砷螯合型免疫复合物及其制备方法和应用
CN105137059A (zh) * 2015-07-14 2015-12-09 上海拜豪生物科技有限公司 一种汞螯合型免疫复合物及其制备方法和应用
CN105001310A (zh) * 2015-07-14 2015-10-28 上海拜豪生物科技有限公司 一种铬螯合型免疫复合物及其制备方法和应用
CN113009135A (zh) * 2021-02-19 2021-06-22 山东省大健康精准医疗产业技术研究院 一种检测cd47的管式磁微粒化学发光免疫定量试剂盒及其制备方法与应用
CN113009135B (zh) * 2021-02-19 2024-04-02 山东省大健康精准医疗产业技术研究院 一种检测cd47的管式磁微粒化学发光免疫定量试剂盒及其制备方法与应用
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