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WO2004061452A1 - Procede de dosage d'un anticorps - Google Patents

Procede de dosage d'un anticorps Download PDF

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Publication number
WO2004061452A1
WO2004061452A1 PCT/JP2004/000040 JP2004000040W WO2004061452A1 WO 2004061452 A1 WO2004061452 A1 WO 2004061452A1 JP 2004000040 W JP2004000040 W JP 2004000040W WO 2004061452 A1 WO2004061452 A1 WO 2004061452A1
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Prior art keywords
antibody
antigen
protein
binding
binding agent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/JP2004/000040
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English (en)
Japanese (ja)
Inventor
Kazuo Kojima
Sayaka Oyobe
Yasunori Yamada
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Medical and Biological Laboratories Co Ltd
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Medical and Biological Laboratories Co Ltd
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Priority to JP2005507959A priority Critical patent/JPWO2004061452A1/ja
Publication of WO2004061452A1 publication Critical patent/WO2004061452A1/fr
Anticipated expiration legal-status Critical
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

Definitions

  • the present invention relates to a method for measuring an antibody.
  • Antibodies are proteins produced by the immune system in order for the body to eliminate foreign substances. Organism, therefore c having antibodies to substances that living body recognizes as foreign, information indicating whether there vivo has antibodies to what is an important information for understanding the state of the living body .
  • infectious pathogens For example, in organisms infected with infectious pathogens or those that cause an allergic reaction to foreign substances (allergens) outside the body, antibodies that recognize antigenic determinants of the pathogens or allergens are produced. Thus, the presence of antibodies to the pathogen's antigens or allergens indicates that the pathogen has been infected.
  • Infectious pathogens can include bacteria, fungi, mycoplasmas, rickettsias, viruses, and the like.
  • antibody detection methods for diagnosing the infection have been put to practical use. 3 Common infectious agents that are routinely tested include, for example, the following pathogens:
  • Hepatitis B virus OffiV Hepatitis C virus 0ICV
  • HIV AIDS virus
  • an antibody against an allergen in the body Indicate the presence of an allergic immune response to this.
  • Allergens include house dust, plant pollen, and proteins in food. Reagents for detecting antibodies to allergens have also been put to practical use.
  • Autoimmune disease is a disease caused by the immune system recognizing and attacking its own tissue as a foreign body. The immune system normally recognizes and eliminates foreign antigens. However, an autoimmune disease is established when, for some reason, the immune system exerts immunity on its own tissue and causes damage. Generally, a disease in which the presence of autoantibodies is considered to be closely related to the establishment of a disease is called an autoimmune disease (Immunology Dictionary, Tokyo Chemical Dojin, published November 15, 1993).
  • Humoral immunity is a mechanism for eliminating foreign substances that depends on the production of antibodies to the foreign substances.
  • Cell '14 immunization is a mechanism for eliminating foreign substances by cytotoxic immune cells. Therefore, when the own tissue is attacked by humoral immunity, there are antibodies against the normal tissue in the living body.
  • Antibodies to normal tissues are called autoantibodies.
  • the antigen recognized by the autoantibody is a self antigen.
  • diagnosis of an autoimmune disease can be made by proving the existence of an antibody against a self antigen.
  • Various autoimmune disease diagnostic methods have been put to practical use using autoantibodies as an index. The following shows autoimmune diseases and autoantigens recognized by antibodies serving as diagnostic indices.
  • Hashimoto's disease rhinoglobulin, thyroid gland / reoxidase
  • Antibodies can be detected using a binding reaction with an antigen.
  • a measurement method utilizing the detection of an antigen-antibody complex formed by binding an antibody and an antigen is referred to as immunoassay.
  • immunoassay A measurement method utilizing the detection of an antigen-antibody complex formed by binding an antibody and an antigen.
  • Various principles are known in Imnoatssey. First, imnoatusei can be classified into two types depending on whether it requires a separation step for the antigen-antibody complex. That is, in a homogenous immunoassay, it is not necessary to separate the antigen-antibody complex. On the other hand, generally heterogenous imnoassay is a technique that requires the separation of an antigen-antibody complex.
  • the binding of the antigen to the antibody can be confirmed by non-competitive or competitive reactions.
  • non-competitive reactions the antigen to be detected
  • the antigen-antibody complex formed by the binding of the corresponding antibody (or antigen) with the (or antibody) is used as an index of the amount of the antigen (or antibody) to be detected.
  • the amount of the antigen primary antibody complex, Mel the amount and directly proportional antigen (or antibody) 0
  • the degree of inhibition of the binding of the antigen (or antibody) to the corresponding antibody (or antigen) by the antigen (or antibody) to be detected is an indicator.
  • the signal obtained is inversely proportional to the amount of antigen (or antibody) in the sample.
  • the amount of an antigen-antibody complex is measured using a labeled reaction component.
  • a labeled antigen is bound to the antibody to be measured.
  • the amount of the antigen-antibody complex can be clarified by using the amount of the antigen that formed the antigen-antibody complex or the amount of the free antigen that was not used for the formation of the complex as an index.
  • Enzymes, coenzymes, luminescent substances, fluorescent substances, colored substances, or radioactive substances are used for labeling.
  • the sample (A) to be measured is first reacted with a substance (B) that specifically binds to this sample (primary reaction). Subsequently, the complex consisting of A and B is separated from the reaction solution and washed. The resulting complex was allowed to react with another substance (C) that specifically reacts with A labeled with the label, and was composed of three components (C)-(A)-(B) Form a complex (secondary reaction). After removing unreacted (C) by washing, it was common to know the amount of A by measuring the amount of labeled C. However, this method requires more than one washing step between reactions. Separation and washing of the reaction solution is a factor that hinders reduction in operation time.
  • Patent Document 1 a method (one-step method) in which A, B, and C are reacted in the same reaction solution to simultaneously perform a primary reaction and a secondary reaction has been devised (Patent Document 1 / Patent Document 1). 1849128).
  • This method can be used when the substance (A) to be measured is an antigen.
  • this method cannot be applied when measuring antibodies.
  • the following method is generally used. First, the sample is reacted with the insoluble carrier to which the antigen is bound (primary reaction). Next, an anti-immunoglobulin antibody (secondary antibody) labeled with a label is reacted (secondary reaction). The amount of the target antibody in the sample is measured by measuring the amount of labeling of the secondary antibody constituting the complex having the following structure formed in this manner.
  • Solid phase one one (antibody to be measured) one (secondary antibody)
  • the blood sample is a mixture of various antibodies. Therefore, if a secondary antibody such as an anti-human antibody is added without washing, unrelated antibodies in the sample will also react with the secondary antibody. As a result, the reaction with the antibody bound to the insoluble carrier to which the secondary antibody is to be bound is inherently inhibited. Go. For these reasons, in the antibody measurement system, it was essential to wash and remove the unreacted antibody after the reaction between the sample and the antigen-bound insoluble carrier.
  • a labeled anti-immunoglobulin antibody (secondary antibody) was used to detect the antibody to be measured.
  • protein A can be used in place of the anti-immunoglobulin antibody. That is, in the secondary reaction, the labeled protein A can be bound to the antibody to be measured.
  • a washing step was required before the secondary reaction. Disclosure of the invention
  • An object of the present invention is to provide a method for measuring an antibody which can minimize the separation and washing operations.
  • protein A binds specifically to the Fc portion of various immunoglobulins. Binding of protein A to the Fc portion is said to have no effect on the antigen-binding activity of immunoglobulin. Furthermore, because of the specific and stable binding of immunoglobulin and protein A, protein A is widely used as a tool for separating and immobilizing immunoglobulin or detecting IgG. The present inventors thought that the use of a substance having a selective binding activity for an antibody having formed an immune complex would allow the antibody to be measured more easily. We focused on protein A as a protein having such properties. It has been reported that the affinity between IgG and protein A is increased several hundred times by binding of IgG to an antigen to form an immune complex (Langone, JJ; J Immunol Methods, 51 (1982)). 3-22).
  • protein A also has a strong affinity for antibodies that do not bind to the antigen.
  • the binding affinity for the antibody has also been used for the purification of free antibody. Therefore, it was thought that it would be difficult to selectively bind Protein A to an antibody that reacted with an antigen in the presence of a free antibody.
  • the present inventors have found that, in practice, the antibody can be measured in the presence of a free antibody by utilizing the binding activity of protein A to the antibody bound to the antigen. Further, the present invention has been clarified that a high signal can be expected without using a washing operation after the primary reaction by using a protein such as protein A. That is, the present invention relates to the following method for measuring an antibody.
  • a method for measuring an antibody comprising the following steps.
  • the binding agent that recognizes and binds to the antibody that has formed the antigen-antibody complex is selected from the group consisting of protein, protein G, and complement, or a protein functionally equivalent thereto The method according to [1], which is any protein.
  • V of the binding agent that recognizes and binds to the antigen and the antibody forming the antigen-antibody complex is immobilized or has a modification that allows immobilization (1) The method described in.
  • the binding agent that recognizes and binds to the antibody forming the antigen-antibody complex has a label or has a modification capable of binding the label.
  • the solid phase is particles, including a step of counting particles having a label on the solid phase,
  • [8] including a step of detecting the particles and the label bound to the particles by a flow meter
  • a plurality of types of particles to which different antigens are bound have signals that can be distinguished from each other, and the method includes a step of identifying the antigen to which the antibody is bound based on the signal of the particles for which the label is detected (8) The method described in.
  • [11] a step of detecting the binding between the antigen-antibody complex and the binding agent after the reaction with the antibody, antigen, and binding agent to be measured, and further adding a reaction terminator, The described method.
  • the concentration of formaldehyde in the reaction solution is 0.1 to 0.5% v / v.
  • an antigen bound by the antibody to be measured and a binding agent that recognizes and binds to the antibody forming the antigen-antibody complex, either one of which is immobilized or immobilized
  • a kit for measuring an antibody which has a modification that can be converted, and the other has a label or a modification that can be labeled.
  • reaction terminator is formaldehyde and / or dodecyl sulfate.
  • the antibody to be measured can be a population of various antibody molecules having different antigen-binding activities.
  • a blood sample collected from a living body for diagnosis of a disease is a collection of antibodies having various reactivity. Therefore, the antibody contained in the blood sample is suitable as the antibody to be measured in the present invention.
  • Blood samples include whole blood as well as serum and plasma that can be separated from whole blood. Whole blood may be lysed red blood cells.
  • the blood sample is not limited to a human sample. Therefore, the antibody of the immunized animal can be measured according to the present invention. Alternatively, antibodies contained in the blood of transgenic animals that produce human antibodies can be measured.
  • a biological material other than a blood sample can be used as a sample.
  • saliva and mucous membranes are known to secrete antibodies.
  • Milk contains maternal IgG.
  • antibodies generated by culturing B cells taken out of a living body can also be measured in the present invention. Methods for collecting these biological samples and preparing samples for immunological measurement methods are known.
  • the biological sample is preferably taken from a living organism and measured ex vivo by the method of the present invention.
  • the method for measuring an antibody according to the present invention includes the following steps (1) to (3).
  • step (1) is performed by adding an antigen to the sample.
  • Any antigenic substance having an antigenic determinant recognized by the antibody to be detected can be used as the antigen. Therefore, not only a complete antigen molecule but also a partial structure of an antigen containing an antigenic determinant can be used as an antigen. Furthermore, molecules that mimic the antigen structure are also useful as antigens.
  • An anti-idiotype antibody can be shown as a molecule that mimics the structure of an antigen.
  • an antigen constituting the microorganism, cells infected by the microorganism, or a fraction thereof can be used as the antigen.
  • substances that can be allergens and extracts thereof can be used as antigens.
  • an antibody against an own tissue autoantibody
  • an antigen derived from that kind of tissue is used.
  • the antigen not only a naturally-occurring substance, but also a recombinant or a chemically synthesized oligopeptide or oligosaccharide can be used.
  • the antibody bound to the antigen is reacted with a binding agent.
  • a binding agent any substance that recognizes and binds to an antibody that has formed an antigen-antibody complex in the presence of free immunoglobulin can be used.
  • Free Imnog Oral Purine refers to Imnoglobulin that has not formed a complex with the antigen.
  • preferred binding agents include protein A, protein G, and proteins functionally equivalent thereto. Protein A and protein G are particularly preferred binders in the present invention.
  • complement can be used as a binder in the present invention. Methods for obtaining these binders are known. For example, protein A and protein G are commercially available.
  • a functionally equivalent protein includes a protein capable of selectively binding an antibody that has formed a complex with an antigen in the presence of free immunoglobulin.
  • Selective binding to an antibody that has formed an antigen-antibody complex means that it is substantially free of interference with coexisting free immunoglobulin.
  • Substantially no interference of free immunoglobulin can be confirmed as follows. That is, if the amount of binding between the antibody forming the antigen-antibody complex and the protein does not show a significant change when the amount of coexisting free immunoglobulin is changed, the protein is not immunogenic. It can be said that it is selectively bound to the complex.
  • protein A and protein G are proteins having a strong binding affinity for the Fc portion of various immunoglobulins. However, since these proteins also have strong binding affinity to free immunoglobulin, it was thought that they could not selectively bind an antibody that formed an antigen-antibody complex. However, in fact, it has been found by the present inventors that these proteins selectively bind the antigen-antibody complex and enable measurement of the antibody.
  • the antibody to which the binding agent binds includes any immunoglobulin.
  • IgG IgA, IgM, IgD, or IgE can be indicated as imnoglobulin.
  • Protein G It can bind to any of the phosphorus.
  • infectious diseases and autoimmune diseases most of the antibodies produced constantly are IgG. Therefore, it can be said that IgG is one of the important antibodies in the antibody measuring method of the present invention.
  • large amounts of IgM are often produced transiently during the early stages of infection.
  • IgE production is an important indicator in allergic immune responses.
  • steps (1) and (2) can be performed separately or simultaneously. That is, a binder may be added after reacting the antigen and the antibody, or the antigen and the binder may be simultaneously added to the sample.
  • the order of adding the sample, the antigen, and the binding agent is arbitrary. Therefore, if a reagent in which an antigen and a binding agent are mixed is prepared, steps (1) and (2) can be started only by adding a sample. Samples that contain high concentrations of antibody may require dilution of the sample to measure the antibody. For example, blood samples contain high concentrations of antibodies. By preparing a reagent in which a diluting solution is prepared in advance for such a sample, dilution and measurement of antibodies can be performed simply by adding the sample to the reagent.
  • step (2) when the step (2) is performed after the step (1), a step of separating free antibody between the steps is unnecessary.
  • substantially simultaneously means that other components are added to the reaction system without separating components that did not participate in the reaction between the components constituting the reaction. Therefore, even when the reaction is not performed completely simultaneously in terms of time, the case where components necessary for the reaction are sequentially mixed without separation or washing is also included in performing the reaction substantially simultaneously.
  • the method of the present invention includes a step of detecting the binding between the antigen-antibody complex formed in step (2) and a binding agent.
  • the binding between the two can be easily detected by labeling either the antigen or the binding agent.
  • an enzyme, a fluorescent substance, a luminescent substance, a radioactive substance, a coloring substance and the like can be shown.
  • Methods for binding these labeling substances to antigens and binding agents are known.
  • a labeling substance can be chemically bonded.
  • biotin into an antigen or a binding agent and then binding labeled avidin, a labeled substance can be bound indirectly.
  • the labeling substance is a protein
  • a fusion protein of the labeling substance and an antigen protein or a binding agent can be obtained by using gene recombination technology.
  • the solid phase it is possible to use a general solid phase used for a well-known heterogeneous system such as fine particles, beads, or the inner wall of a container. Methods for binding an antigen or a binding agent to these solid phases are also known.
  • commercially available protein A or protein G in a state of being bound to a solid phase can also be used in the present invention. For example, gels such as agarose and sepharose, colored particles such as colloidal gold, and protein A immobilized on ELISA plates are sold.
  • a directly detectable signal is a signal that does not require an additional reaction to detect the signal.
  • signals such as fluorescent signals from fluorescent dyes and colored signals from colored dyes can be detected without additional reaction. Therefore, these signals are included in the directly detectable signals.
  • enzyme labels generally cannot produce a signal without going through an enzymatic reaction.
  • a detection system that does not require a washing step will be specifically described. A system that can analyze fine particles one by one using a single system has been put into practical use.
  • Luminex (trade name; manufactured by Luminex Corp.) can detect a fluorescent label bound to one fine particle.
  • Luminex, Flowmet The fine particles and the fluorescent material bound to the fine particles are detected by separate sensors while flowing the fine particles one by one by Lee.
  • a preferred combination in the present invention is a method in which fine particles are used as a solid phase and a labeling substance that generates a signal directly detectable on a label is used.
  • the fine particles refer to particles having a size that can be flowed one by one by a flow system and that can be distinguished from other fine particles mixed by a sensor.
  • particles having a particle size of about 0.2 to 200 / im can be flowed one by one by a flow system, and can be easily identified by a sensor.
  • fine particles that generate a signal can be used. By giving signals to fine particles that are solid phases in addition to labeling substances, simultaneous measurement of multiple items becomes possible. The multi-item simultaneous measurement is described below.
  • the measurement method of the present invention will be described using an example in which an antigen is immobilized on fine particles and a binder is labeled.
  • different antigens are immobilized on fine particles. Fine particles should generate different signals for each antigen.
  • different signals can be given to fine particles by incorporating different fluorescent dyes into the fine particles.
  • different signals can be given by changing the mixing ratio of a plurality of dyes having different fluorescence wavelengths.
  • the method for binding the antigen to the fine particles is arbitrary.
  • polystyrene beads A protein can be immobilized on a hydrophobic surface by physical adsorption.
  • Luminex beads which are fine particles commercially available for Luminex®, have functional groups on the surface. Using this functional group, the antigen can be immobilized on a solid phase by covalent bond.
  • a modification that allows immobilization can be added to the antigen (or binder) to be immobilized.
  • modification that can be immobilized refers to a modification of an antigen or a binding agent with a substance having binding affinity.
  • a substance with binding affinity can be captured by a binding partner of the substance.
  • biotin-modified antigen is captured by immobilized avidin.
  • the same label may be used regardless of the type of antibody.
  • protein A labeled with a commercially available fluorescent dye can be used.
  • Fluorescein isothiocyanate (FITC) or R-Phycoerythrin (PE) is used as the fluorescent dye.
  • FITC Fluorescein isothiocyanate
  • PE R-Phycoerythrin
  • hepatitis B virus HBV
  • hepatitis C virus hepatitis C virus
  • AIDS virus AIDS virus
  • syphilis antibodies against hepatitis B virus (HBV), hepatitis C virus), AIDS virus), or syphilis.
  • HBV hepatitis B virus
  • hepatitis C virus hepatitis C virus
  • AIDS virus AIDS virus
  • syphilis syphilis
  • these antibodies can be measured simultaneously. That is, the antigen of each pathogenic microorganism is immobilized on fine particles that generate different signals. The sample is allowed to react with the fine particles together with the labeled binder, and the binder is labeled for each fine particle. It is only necessary to detect intellect. If these antibody screenings are performed at the same time, the time and cost required for the screening can be significantly reduced.
  • the method of the present invention is also useful for confirming the presence of antibodies in a sample against various types of allergens.
  • the number of potential allergens is growing. Therefore, the identification of substances responsible for an allergic immune response has become increasingly difficult tests.
  • antibodies to various kinds of antigenic substances can be detected simultaneously and rapidly. That is, various allergens are immobilized on fine particles that generate different signals.
  • the sample may be reacted with the fine particles together with the labeled binder, and the label of the binder may be detected for each fine particle.
  • Luminex offers microphone mouth beads that generate 100 different signals. This means that 100 different antibodies can be simultaneously measured by the present invention.
  • multi-item simultaneous measurement of antigen using antibody-bound fine particles is already known. Therefore, by combining the multi-item simultaneous measurement of the known antigen and the multi-item simultaneous measurement of the antibody according to the present invention, it has become possible to simultaneously perform the measurement operation based on all immunological measurement principles. When a large number of samples are screened, the ability to perform the immunoassays simultaneously in a single system has significant time and cost savings. In the measurement method of the present invention, it is preferable to perform the step (3) after adding the reaction terminator. When measuring a large number of samples at the same time, the reaction time for each sample may differ.
  • a difference in reaction time may cause a decrease in measurement accuracy. Then, after a predetermined reaction time has elapsed, the reaction is stopped once by adding a reaction terminator, and then the step (3) can be performed.
  • the reaction terminator may be formaldehyde.
  • the used concentration of formaldehyde is usually 0.1 to 5.0% v / v, for example, 0.25 to 2.0% v / v, preferably 0.5 to 1% v / v in the reaction solution.
  • dodecyl sulfate can be used as the reaction terminator in the present invention.
  • dodecyl sulfate examples include sodium dodecyl sulfate and lithium dodecyl sulfate.
  • sodium dodecyl sulfate (SDS) is a desirable reaction terminator in the present invention.
  • concentration of dodecyl sulfate used is usually 0.1 to 5.0% w / v in the reaction solution, for example 0.25 to 2.0% w / v, preferably 0.5 to 1% w / v. It is.
  • Formaldehyde or dodecyl sulfate can effectively stop an immune reaction or a reaction involving a binder by a denaturing effect of the protein.
  • Formaldehyde and dodecyl sulfate can be used as a reaction terminator in the present invention either individually or as a mixture of both.
  • the present invention relates to any one selected from the group consisting of protein A, protein G, or a protein functionally equivalent thereto under the condition that an antibody constituting an antigen-antibody complex and free immunoglobulin coexist.
  • a method for separating an antigen-antibody complex comprising a step of binding the binding agent comprising the protein with the antibody constituting the antigen-antibody complex.
  • a binding agent capable of selectively binding an antibody which has formed an antigen-antibody complex in the presence of free immunoglobulin can be used as an antigen-antibody complex under the condition that free immunoglobulin coexists with protein A, protein G, or any protein selected from the group consisting of proteins functionally equivalent thereto. It is a binding agent for selectively binding antibodies that compose the body. Such a binding agent is useful as a separating agent for an antibody that has formed an immune complex.
  • the binding agent in the present invention can be immobilized or labeled.
  • the present invention further includes an antigen bound by the antibody to be measured, and a binding agent that recognizes and binds to the antibody that has formed the antigen-antibody complex, either one of which is immobilized or immobilized.
  • the present invention relates to a kit for measuring an antibody, wherein the kit has a modification that can be converted, and the other has a label or a modification that can be labeled.
  • the antigen bound by the antibody to be measured and the binding agent that recognizes and binds to the antibody forming the antigen-antibody complex may be mixed in advance.
  • the kit of the present invention may further comprise a reaction terminator. Formaldehyde can be used as the reaction terminator.
  • kits according to the present invention in which a plurality of types of antigens are immobilized on fine particles each generating a different signal, is useful as a kit for simultaneously measuring a plurality of types of antibodies. If the microparticles produce an identifiable signal, the label of the binder may be common.
  • FIG. 1 is a diagram showing the measurement results of anti-SS-B antibodies when PE-labeled protein A was reacted without a washing step after the reaction between the beads and the sample.
  • the vertical axis shows the fluorescence intensity measured by Luminex®, and the horizontal axis shows the sample dilution factor.
  • Conj. Dilution shows the dilution of PE-labeled protein A.
  • FIG. 2 is a diagram showing the measurement results of anti-SS-B antibodies when a PE-labeled anti-human IgG antibody was reacted without a washing step after the reaction between the beads and the sample.
  • the vertical axis shows the fluorescence intensity measured by Luminex®, and the horizontal axis shows the sample dilution factor.
  • Conj. Dilution indicates the dilution of the PE-labeled anti-human IgG antibody.
  • FIG. 3 is a diagram showing the results of measuring anti-SS-B antibodies when PE ⁇ protein G (Biomeda) was reacted without a washing step after the reaction between the beads and the sample.
  • the vertical axis shows the fluorescence intensity measured by Luminex®, and the horizontal axis shows the sample dilution factor.
  • Conj. Dilution factor indicates the dilution factor of PE-labeled protein G (manufactured by Biomeda).
  • FIG. 4 is a diagram showing the results of anti-SS-B antibody measurement when PE ⁇ protein G (Biogenesis) was reacted without a washing step after the reaction between the beads and the sample.
  • the vertical axis is Lumi
  • the fluorescence intensity measured by nex® is shown on the horizontal axis, which is the sample dilution factor.
  • Con j. Dilution factor indicates the dilution factor of PE ⁇ protein G (manufactured by Biogenesis).
  • FIG. 5 is a graph showing the effect of a reaction stop solution on the measurement of anti-SS-B antibody using PE-labeled protein A.
  • the vertical axis shows the fluorescence intensity measured by Luminex®, and the horizontal axis shows the elapsed time (minutes) after addition of the reaction stop solution.
  • FIG. 6 is a diagram showing the measurement results of anti-SS-B antibodies when POD-labeled protein A was reacted without a washing step after the reaction between the ELISA plate and the sample.
  • the vertical axis shows the absorbance at 450 nm, and the horizontal axis shows the sample dilution factor.
  • FIG. 7 is a diagram showing the measurement results of anti-SS-B antibodies when a POD-labeled anti-human IgG antibody was reacted without a washing step after the reaction between the ELISA plate and the sample.
  • the vertical axis shows the absorbance at 450 nm, and the horizontal axis shows the sample dilution factor.
  • FIG. 8 is a diagram showing the measurement results of anti-SS-B antibodies when POD-labeled protein A was reacted after washing after the reaction between the ELISA plate and the sample.
  • the vertical axis shows the absorbance at 450 nm, and the horizontal axis shows the sample dilution factor.
  • FIG. 9 is a diagram showing the measurement results of the anti-SS-B antibody when the POD-labeled anti-human IgG antibody was reacted after washing after the reaction between the ELISA plate and the sample.
  • the vertical axis shows the absorbance at 450 nm, and the horizontal axis shows the sample dilution factor.
  • FIG. 10 is a diagram showing the stability after the reaction was stopped when SDS was used as the reaction stopping solution.
  • the vertical axis indicates the fluorescence intensity, and the horizontal axis indicates the final concentration (%) of SDS.
  • FIG. 11 is a diagram showing the stability after termination of the reaction when SDS was used as the reaction termination solution.
  • the vertical axis shows the fluorescence intensity, and the horizontal axis shows the elapsed time (minutes) after the addition of SDS.
  • Anti-SS-B antibody is selected as the antibody to be measured, and the antibody measurement method according to the present invention is constructed. did.
  • Anti-SS-B antibody is an autoantibody found in the serum of patients with Siedalen syndrome and is also called La antibody. By double immunodiffusion, three different autoantibodies were found in the sera of patients with Sjoegren Syndrome, which were named SS-A, SS-B, and SS-C antibodies, respectively (E Williams St. et al .: Qu antitative immunoassay of anti La anibodies using purified recombinant La antigen. Arthritis Rheum., 31: 506, 1988).
  • the SS-B antigen which is an antigen recognized by the SS-B antibody, has been identified and its structure has been determined (Japanese Patent Application Laid-Open No. 9-166965, "Method for determining the presence of autoimmune autoantibodies").
  • the SS-B antigen (La antigen) required for the detection of SS-B antibody is commercially available.
  • DIARECT AG Germany markets a recombinant La antigen (catalog number 12800).
  • Purified La antigen is also commercially available (Immunovision, USA).
  • the activated beads were centrifuged at 10, OOOg for 2 minutes to separate the supernatant, and the mixture was dispersed well by adding a coupling buffer (PH7.3 PBS), and the supernatant was discarded and washed. After the washing operation was repeated twice, 10 ⁇ g / mL of a recombinant SS-B antigen solution (250 / zL) was added, and the mixture was stirred by stirring at room temperature for 2 hours in the dark to bind. Then, the plate was washed twice with 500 ⁇ L of a washing buffer ( ⁇ 7.3 PBS, 0.05% Tween20).
  • a washing buffer ⁇ 7.3 PBS, 0.05% Tween20
  • blocking Z preservation buffer pH 7.3 PBS, lmg / mL BSA, 0.05% Sodium Azide
  • Luminex® Luminex automatic analyzer
  • a 100-fold diluted sample L and bead suspension 50; zL are added to the wells of a 96-well filtration plate (Millipore MultiScreen Assay System). After stirring for 30 seconds, the mixture was allowed to react by standing at 25 ° C for 30 minutes, and the solution was removed by suction.
  • the MESACUP-2 test (manufactured by the Institute of Medical Biology Co., Ltd.) After washing twice, add 100 L of wash buffer and AIOO L of fluorescently-labeled antibody or fluorescently-labeled protein, stir for 30 seconds, and let stand at 25 ° C for 30 minutes to react. And the fluorescence intensity was measured.
  • the sample was diluted regardless of whether a fluorescent-labeled anti-human IgG antibody or fluorescent-labeled protein A was used. The increase of the fluorescence intensity according to was observed.
  • reaction stop solution was examined, and it was found that the reaction could be stopped by adding formaldehyde. Therefore, the concentration of formaldehyde and the stability after stopping were examined.
  • An antibody measurement system based on the present invention was constructed using a microplate generally used as a reaction vessel for ELISA.
  • the microplate used was an antigen-binding microplate for MESACUP2 test “SS-B” manufactured by Medical Biology Laboratories.
  • a peroxidase-labeled anti-human IgGfet compound (manufactured by Medical Biology Laboratory Co., Ltd., product number 208) was similarly diluted 200-fold to 25600-fold, and the same operation was performed to confirm the change in color development. In addition, normal two-step measurement was also performed at the same time, and changes in color development were compared.
  • peroxidase-labeled protein A Fig. 8
  • the concentration was 30 times higher, but the same color was obtained. I got it.
  • the concentration was 30 times higher, but the same color was obtained. I got it.
  • the absorbance increased in a concentration-dependent manner from 12800 to 200 times. It was confirmed that the antibody measurement method according to the present invention was also possible using an ELISA plate.
  • a method for measuring an antibody that can be expected to have high measurement sensitivity without a washing step after the primary reaction has been provided. Further, in a desirable embodiment of the present invention, even a method for measuring an antibody which does not require the production of a free antibody and an antigen-antibody complex can be realized. For example, an imnoatssay using a system for counting microparticles to which a label is bound is known. The antibody captured by the fine particles can be labeled by the method of the present invention. Labeled microparticles are counted by such a system. That is, by applying this type of system to the present invention, BZF separation in antibody measurement becomes unnecessary.

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Abstract

L'invention concerne un procédé de dosage d'un anticorps, consistant à permettre à une protéine, laquelle reconnaît un complexe antigène-anticorps et se lie à celui-ci, de se lier à un tel complexe antigène-anticorps. La protéine utilisée à cet effet peut être la protéine A, etc. Une telle protéine se liant de façon sélective à un complexe antigène-anticorps reconnaît ledit complexe sans être perturbée par des anticorps libres. On obtient ainsi un procédé de dosage d'un anticorps qui ne nécessite pas de séparation éléments liés/éléments libres. Ce procédé permet de doser un anticorps de manière simple et rapide.
PCT/JP2004/000040 2003-01-07 2004-01-07 Procede de dosage d'un anticorps Ceased WO2004061452A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1653233A1 (fr) * 2004-10-30 2006-05-03 Roche Diagnostics GmbH Méthode de détection d'anticorps d'une classe donnée à l'aide d'un anticorps ciblant de manière spécifique un complexe anticorps/antigène
WO2007072922A1 (fr) * 2005-12-22 2007-06-28 Rohm Co., Ltd. Appareil d'immunodosage et procédé
JP2012122868A (ja) * 2010-12-09 2012-06-28 Panasonic Corp 抗原抗体複合体を分離する方法
CN111273007A (zh) * 2020-03-13 2020-06-12 内江师范学院 一种用于快速检测鱼类鮰爱德华氏菌的试剂盒及检测方法

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JPS6022662A (ja) * 1974-05-20 1985-02-05 テクニコン、インストルメンツ、コ−ポレ−シヨン 抗体、抗原または抗体：抗原複合体分析用試薬
JPS6395357A (ja) * 1986-10-13 1988-04-26 Showa Denko Kk 微粒子の螢光強度測定による定量方法
JPH04249769A (ja) * 1991-01-08 1992-09-04 Konica Corp 免疫学的測定方法
JPH05126829A (ja) * 1991-04-03 1993-05-21 Syntex Usa Inc 免疫グロブリンのイムノアツセイ
JPH11295311A (ja) * 1998-04-14 1999-10-29 Otsuka Pharmaceut Co Ltd 抗体測定方法

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Publication number Priority date Publication date Assignee Title
JPS6022662A (ja) * 1974-05-20 1985-02-05 テクニコン、インストルメンツ、コ−ポレ−シヨン 抗体、抗原または抗体：抗原複合体分析用試薬
JPS6395357A (ja) * 1986-10-13 1988-04-26 Showa Denko Kk 微粒子の螢光強度測定による定量方法
JPH04249769A (ja) * 1991-01-08 1992-09-04 Konica Corp 免疫学的測定方法
JPH05126829A (ja) * 1991-04-03 1993-05-21 Syntex Usa Inc 免疫グロブリンのイムノアツセイ
JPH11295311A (ja) * 1998-04-14 1999-10-29 Otsuka Pharmaceut Co Ltd 抗体測定方法

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1653233A1 (fr) * 2004-10-30 2006-05-03 Roche Diagnostics GmbH Méthode de détection d'anticorps d'une classe donnée à l'aide d'un anticorps ciblant de manière spécifique un complexe anticorps/antigène
US8664007B2 (en) 2004-10-30 2014-03-04 Roche Diagnostics Operations, Inc. Immune complex-specific antibodies for increased sensitivity in immunoassay array tests
WO2007072922A1 (fr) * 2005-12-22 2007-06-28 Rohm Co., Ltd. Appareil d'immunodosage et procédé
JPWO2007072922A1 (ja) * 2005-12-22 2009-06-04 ローム株式会社 免疫測定装置及び方法
JP2012122868A (ja) * 2010-12-09 2012-06-28 Panasonic Corp 抗原抗体複合体を分離する方法
CN111273007A (zh) * 2020-03-13 2020-06-12 内江师范学院 一种用于快速检测鱼类鮰爱德华氏菌的试剂盒及检测方法
CN111273007B (zh) * 2020-03-13 2023-08-01 内江师范学院 一种用于快速检测鱼类鮰爱德华氏菌的试剂盒及检测方法

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