WO2004061452A1

WO2004061452A1 - Method of assaying antibody

Info

Publication number: WO2004061452A1
Application number: PCT/JP2004/000040
Authority: WO
Inventors: Kazuo Kojima; Sayaka Oyobe; Yasunori Yamada
Original assignee: Medical And Biological Laboratories Co., Ltd.
Priority date: 2003-01-07
Filing date: 2004-01-07
Publication date: 2004-07-22
Also published as: JPWO2004061452A1

Abstract

It is intended to provide a method of assaying an antibody involving the step of allowing a protein, which recognizes an antigen-antibody complex and binds thereto, to bind to the antigen-antibody complex. As the protein recognizing an antigen-antibody complex and binding thereto, use can be made of protein A, etc. Such a protein selectively binding to an antigen-antibody complex recognizes the antigen-antibody complex without being interfered by free antibodies. Thus, a method of assaying an antibody without resort to B/F separation can be achieved. According to this method, an antibody can be conveniently and quickly assayed.

Description

Antibody assay method

The present invention relates to a method for measuring an antibody. Background art

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 .

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. For many infectious agents, 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)

AIDS virus (HIV) wind pain virus

Mycobacterium tuberculosis Hemolytic streptococci

Chlamydia mycoplasma

Syphilis Spiro ^

Alternatively, the presence of 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.

Whereas the production of antibodies to pathogens is the result of the body's normal immune response, there are diseases in which the very presence of the antibodies is the etiology. 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).

The mechanism by which the immune system attacks and eliminates foreign substances is roughly divided into humoral immunity and cellular immunity. 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. Thus, the 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

Graves' disease: thyrotropin receptor

Myasthenia gravis: acetylcholine receptor

Insulin-dependent diabetes mellitus: Klangerhans Island

Systemic erythematosus: DNA (cell nucleus) Rheumatoid arthritis: IgG

Autoimmune hemolytic anemia: red blood cell membrane antigen

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. 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.

In Imnoassay, the binding of the antigen to the antibody can be confirmed by non-competitive or competitive reactions. In 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. In the non-competitive reaction, the amount of the antigen primary antibody complex, Mel the amount and directly proportional antigen (or antibody) ₀

In a competitive reaction, on the other hand, 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. Thus, in a competitive reaction, the signal obtained is inversely proportional to the amount of antigen (or antibody) in the sample.

Generally, in a heterogeneous Imnoassay, the amount of an antigen-antibody complex is measured using a labeled reaction component. For example, to measure an antibody, a labeled antigen is bound to the antibody to be measured. Next, 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. In a general heterogeneous Imnoassay, 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.

In order to solve this problem, 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. However, this method cannot be applied when measuring antibodies.

As a method for measuring antibodies in a sample such as an autoantibody or a virus antibody by using a heterogeneous Immonoassay, for example, 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 (antigen) one (antibody to be measured) one (secondary antibody)

In the above reaction, separation and washing of the reaction solution after the reaction (primary reaction) between the sample and the antigen-binding insolubilized carrier is essential. 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. In the antibody measurement system described here, a labeled anti-immunoglobulin antibody (secondary antibody) was used to detect the antibody to be measured. As a substance that recognizes and binds to the antibody, 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. However, it was thought that protein A would inevitably interfere with coexisting unrelated antibodies. Therefore, 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.

It is known that 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).

However, 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. However, 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.

[1] A method for measuring an antibody, comprising the following steps.

(1) Step of binding an antibody contained in a sample to an antigen recognized by the antibody

(2) reacting the antigen-antibody complex formed in (1) with a binding agent that recognizes and binds to the antibody that has formed the antigen-antibody complex in the presence of free immunoglobulin; and

(3) Step of detecting the binding between the antigen-antibody complex and the binding agent

(2) 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.

(3) 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.

[4] The method according to [3], wherein the antigen is immobilized or has an immobilizable modification.

[5] Antigen-antigen-anti-! Any of the binding agents that recognize and bind to the combined antibody have a label that generates a detectable signal or a modification that can bind the label The method according to [3], comprising:

[6] The method according to [5], wherein 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. (7) the solid phase is particles, including a step of counting particles having a label on the solid phase,

The method according to [3].

[8] including a step of detecting the particles and the label bound to the particles by a flow meter

The method according to [7].

(9) 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.

[10] The antibody according to [1], wherein the antibody contained in the sample, the antigen recognized by the antibody, and the binding agent that recognizes and binds to the antibody forming the antigen-antibody complex are reacted substantially simultaneously. Method.

[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.

[12] The method according to [1], wherein the reaction terminator is formaldehyde.

[13] The concentration of formaldehyde in the reaction solution is 0.1 to 0.5% v / v.

2].

[14] The method according to [11], wherein the reaction terminator is dodecyl sulfate.

[15] The method according to [14], wherein the dodecyl sulfate is sodium dodecyl sulfate. [16] The method according to [14], wherein the concentration of dodecyl sulfate is 0.5 to 1% w / v. [17] Any one selected from the group consisting of protein A, protein G, and proteins functionally equivalent thereto under the condition that the antibody constituting the antigen-antibody complex and free immunoglobulin coexist. A method for separating an antigen-antibody complex, comprising a step of binding a binding agent composed of a protein and an antibody constituting the antigen-antibody complex.

[18] The separation method according to [17], wherein the binding agent is immobilized or has a modification that allows immobilization. [19] An antigen-antibody complex containing free immunoglobulin containing protein A, protein G, or any protein selected from the group consisting of proteins functionally equivalent thereto, Binder for selectively binding the composed antibody.

[20] The binder according to [19], wherein the binder is immobilized or has a modification that allows immobilization.

[21] 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.

[22] The kit of [21], wherein 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 are mixed.

[23] The kit according to [22], further comprising a reaction terminator.

[24] The kit according to [23], wherein the reaction terminator is formaldehyde and / or dodecyl sulfate.

In the present invention, the antibody to be measured can be a population of various antibody molecules having different antigen-binding activities. For example, usually, 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.

In the present invention, a biological material other than a blood sample can be used as a sample. For example, saliva and mucous membranes are known to secrete antibodies. Milk contains maternal IgG. Furthermore, 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).

(1) binding an antibody contained in the sample to an antigen recognized by the antibody,

(2) reacting the antigen-antibody complex formed in (1) with a binding agent that recognizes and binds to the antibody that has formed the antigen-antibody complex, in the presence of free immunoglobulin; and

In the present invention, 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.

More specifically, when detecting an antibody against a microbial pathogen, an antigen constituting the microorganism, cells infected by the microorganism, or a fraction thereof can be used as the antigen. In the detection of allergic antibodies, substances that can be allergens and extracts thereof can be used as antigens. Furthermore, in an autoimmune disease, an antibody against an own tissue (autoantibody) is detected. When an autoantibody is to be detected, an antigen derived from that kind of tissue is used. As the antigen, not only a naturally-occurring substance, but also a recombinant or a chemically synthesized oligopeptide or oligosaccharide can be used. In the present invention, the antibody bound to the antigen is reacted with a binding agent. As the binder in the present invention, 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. In the present invention, 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. In addition, 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.

In the present invention, 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.

It is known that 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.

Further, in the present invention, the antibody to which the binding agent binds includes any immunoglobulin. Specifically, in addition to IgG, IgA, IgM, IgD, or IgE can be indicated as imnoglobulin. For example, Protein G It can bind to any of the phosphorus. In 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. Also, large amounts of IgM are often produced transiently during the early stages of infection. Furthermore, IgE production is an important indicator in allergic immune responses.

Of the above 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. In the present invention, when the step (2) is performed after the step (1), a step of separating free antibody between the steps is unnecessary. In the present invention, “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.

By performing steps (1) and (2) simultaneously, the reaction time can be reduced.

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. As the label, 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. For example, for bifunctional reagents Therefore, a labeling substance can be chemically bonded. In addition, by introducing biotin into an antigen or a binding agent and then binding labeled avidin, a labeled substance can be bound indirectly. Alternatively, when 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.

In order to easily detect the label, it is preferable to immobilize either the antigen or the binder. That is, by labeling one of the antigen and the binding agent and immobilizing the other, the binding between the antigen-antibody complex and the binding agent can be easily detected. As 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. Alternatively, 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.

In the case where the fine particles are used as the solid phase and the label generates a directly detectable signal, the binding between the antigen-antibody complex and the binding agent can be detected without a washing step. A directly detectable signal is a signal that does not require an additional reaction to detect the signal. For example, 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. For directly detectable signals, enzyme labels generally cannot produce a signal without going through an enzymatic reaction. Hereinafter, 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. For example, 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. By using such a system, it is possible to specifically detect only the labeling substance bound to the fine particles even under the condition where free labeling substances not bound to the fine particles are mixed.

Therefore, 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. By using such a combination, both the washing after the formation of the antigen-antibody complex and the washing after the reaction between the antigen-antibody complex and the binder can be omitted. Eliminating the washing step is very effective in reducing the reaction time.

In the present invention, 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. In general, 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. As the fine particles used in the present invention, 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.

For example, 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. In order to measure a plurality of types of antibodies according to the present invention, different antigens are immobilized on fine particles. Fine particles should generate different signals for each antigen. For example, different signals can be given to fine particles by incorporating different fluorescent dyes into the fine particles. In addition, 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. For example, polystyrene beads A protein can be immobilized on a hydrophobic surface by physical adsorption. Alternatively, 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. Furthermore, a modification that allows immobilization can be added to the antigen (or binder) to be immobilized. The term “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. For example, biotin-modified antigen is captured by immobilized avidin.

The same label may be used regardless of the type of antibody. For example, 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. The sample is mixed with the antigen-bound fine particles and the binder, and the antigen immobilized on the fine particles reacts with the antibodies in the sample to form an antigen-antibody complex. Further, the antigen-antibody complex reacts with the binding agent.

When fine particles having different antigens immobilized thereon are mixed, it is unclear which antigen is to be detected in the mixed state. Here, a system such as Luminex described above is used. In other words, using a system that can identify fine particles one by one, if it is possible to identify which fine particles the label of the binder is detected, it is possible to know the presence or absence of antibodies for each antigen even if different fine particles are mixed You can do it.

For example, donated blood needs to be tested for the presence of multiple antibodies for screening for infection. Specifically, antibodies against hepatitis B virus (HBV), hepatitis C virus), AIDS virus), or syphilis have been measured. By utilizing the present invention, 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.

Alternatively, 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. By using the present invention, 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, for example, offers microphone mouth beads that generate 100 different signals. This means that 100 different antibodies can be simultaneously measured by the present invention. Alternatively, 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. In the present invention, 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. In the present invention, 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. Alternatively, dodecyl sulfate can be used as the reaction terminator in the present invention. Examples of dodecyl sulfate include sodium dodecyl sulfate and lithium dodecyl sulfate. In particular, sodium dodecyl sulfate (SDS) is a desirable reaction terminator in the present invention. The 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.

In addition, 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. According to the present invention, there is provided a binding agent capable of selectively binding an antibody which has formed an antigen-antibody complex in the presence of free immunoglobulin. The binding agent of the present invention 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. In the kit of the present invention, 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.

As described above, by applying fine particles that generate different signals as the solid phase in the method of the present invention, multiple items can be measured simultaneously. Therefore, a kit 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. BRIEF DESCRIPTION OF THE FIGURES

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. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more specifically based on examples.

[Example 1] Construction of measurement system in Luminex® system

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. For example, DIARECT AG (Germany) markets a recombinant La antigen (catalog number 12800). Purified La antigen is also commercially available (Immunovision, USA).

1. Binding of antigen to insoluble carrier

Not: ^ Carrier (Luminex Corporation ^ Carboxylated Microspheres Regionsl24, manufacturer part number L100-C124-01; hereinafter referred to as "beads") After dispersing 200 L with ultrasonic waves, centrifuge at 10,000 g for 2 minutes to remove the supernatant. Was. The following (i) to (iii) were added to the beads, and the beads were left standing for 20 minutes in the B sound place to activate.

(1) Activation Buffer (0.1 M sodium phosphate, pH 6.1) 400 / L,

Ui) 50mg / mLC> Sulf o-NHS (N-Hydroxysulfosuccinimide sodium salt) 50

(iii) 50 mg / mL of 1-Ethyl-3- (3-dimethylaminopropyl) Carbodiimide hydrochloride 50 ^ L

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 μg / 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 μL of a washing buffer (ρΗ7.3 PBS, 0.05% Tween20). After removing the washing buffer, add 500 L of blocking Z preservation buffer (pH 7.3 PBS, lmg / mL BSA, 0.05% Sodium Azide) and add 500 L of blocking buffer. After carrying out the procedure, except for the blocking buffer, the cells were suspended in a blocking Z storage buffer and stored.

2. Measurement

The following (i)-(iii) were mixed, stirred for 30 seconds, and allowed to react for 1 hour at 25 ° C: After the reaction, how to operate a Luminex automatic analyzer (hereinafter referred to as Luminex®) According to the above, the fluorescence intensity obtained by the anti-SS-B antibody in the sample was measured.

(i) 50 L of sample diluted 100 times,

(i i) 50 μL of a suspension prepared by diluting the beads prepared above by 250 times, and

(iii) Fluorescence-labeled antibody (bucoerythrin-labeled anti-human IgG antibody manufactured by Imnotec

(Goat) Code number IM0550, phycobiliprotein concentration 0.5 mg / mL) or fluorescently labeled protein A (PHYCOPROBE PE PROTEIN A, manufactured by biomeda, lmg / ml) lOO ^ L

On the other hand, in order to perform washing after reacting the sample with the beads, 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.

3. Measurement results

The measurement results are as shown in FIGS. In the case where washing was not performed after the reaction between the sample and the beads and before the reaction with the labeled substance, and in the case of using a fluorescently labeled anti-human Ig (¾t form), the fluorescence intensity decreased as the antigen concentration increased. (Fig. 2) This suggests that the fluorescently labeled anti-IgG antibody was interfered by free immunoglobulin. Was. On the other hand, when the fluorescently labeled protein A was used, the fluorescence intensity increased from a dilution factor of 12800 to 400 times of the antigen when the dilution conditions of the labeled substance were appropriate (Fig. 1). As a result of the selective binding of protein A to the antigen-antibody complex, it was demonstrated that interference-free measurement was possible even in the presence of free antibody.

On the other hand, in a system in which the sample was reacted with the beads and washed before the reaction with the labeled substance, 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. In addition, fluorescently labeled protein

When A was used, a stronger fluorescence intensity was observed in the system without washing.

4. Examination of protein G

In the case of using Protein G, which has an affinity for immunoglobulin as well as Protein A, it was examined whether the force could be measured without performing a washing operation. The operation was carried out using Protein G (manufactured by Biomeda or Biogenesis) in the same manner as for Protein A described above. As shown in FIGS. 3 and 4, when Protein G was used, the washing procedure during the reaction was not required similarly to Protein A, and an increase in the fluorescence intensity was observed in accordance with the dilution factor of the sample.

5. Examination of reaction stop solution

If washing was not performed before the reaction with the label, and separation from the unreacted sample was not performed, the reaction would continue.If a large number of samples were measured, the reaction time would be longer at the beginning and end of the measurement. Differences can cause errors in measurements. Therefore, in order to stop the reaction, a 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.

The results are shown in FIG. When a solution containing formaldehyde at a concentration of 1% or more in PBS was used as the reaction stop solution, the fluorescence intensity was stably maintained. In order to more reliably stop the reaction, PBS containing 2% formaldehyde was used as a reaction stop solution. Considering the volume of the microplate, the volume of the bead suspension, sample, and label should be 50 / L each. After reacting them for 1 hour at room temperature and in the B-sound place, adding 50 ^ L of the above-mentioned reaction stop solution and measuring the fluorescence intensity over time using Lumine X®, the measurement was stable for 1 hour after the reaction was stopped. Was confirmed.

[Example 2] Construction of measurement system in microplate system

1. Binding of antigen to microplate

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.

2. Measurement system

The measurement followed the following procedure. First, the following (i) and (ii) were added to the antigen-binding microplate, and reacted at room temperature (20 to 25 ° C) for 1 hour.

(i) (Ltd.) Medical and Biological Laboratories Co., Ltd. MESACUP2 test "SS- B" in the enzyme label diluted solution up to 100-fold to 1 ²⁸ 00 times for Bye Bye diluted specimen 50 L, and

(ii) MESACUP2 test 50 μL of peroxidase-labeled protein A diluted 200-fold to 25600-fold with enzyme diluent for SS-B

After the reaction, wash four times with the washing solution for MESACUP-2 test “SS-B”, remove the excess washing solution by tapping a microplate on a paper towel, and then develop the color substrate for MESACUP-2 test “SS-B” 100 / zL was added and reacted at room temperature (20-25.C) for 30 minutes to develop color. 100 μL of 1N sulfuric acid was added to each well of the microplate to stop the color reaction, and the mixture was stirred. The absorbance of each well at a wavelength of 450 nm was measured using a microplate reader. As a control, 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.

3. Measurement results

The measurement results are shown in FIGS. Performs measurement in two normal steps (with washing) When using peroxidase-labeled protein A (Fig. 8) as compared with the case where the labeled substance was an anti-human IgG antibody (Fig. 9), the concentration was 30 times higher, but the same color was obtained. I got it. On the other hand, when measurement was performed in one step (without washing), no color was obtained when an anti-human IgG antibody was used as the label (FIG. 7), and the measurement could not be performed. When protein A was used (FIG. 6), 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.

[Example 3] Examination of reaction stop solution

In the present invention, it was confirmed that sodium dodecyl sulfate (SDS) can be used as a reaction stopping solution. As in the case of formaldehyde (Example 1),

50 μL of a dilution series of SDS diluted with PBS was added to the Luninex reagent. The fluorescence intensity over time after the addition of SDS was measured, and the reaction termination effect was examined. Beads to which SS-II was bound were used as the Luninex reagent. Antibodies were measured by the Luninex reagent using the serum of a healthy individual and the serum of an anti-SS-A antibody-positive patient as a sample.

The results are shown in Table 1, FIG. 10 and FIG.

table 1

When a final concentration of 0.625% was added, the fluorescence intensity after the reaction was stable without change over time. At 0.3125% or less, the reaction was not stable and the fluorescence intensity tended to increase with time. At 1.25% or more, the fluorescence intensity was too small to obtain a stable measurement value. From this result, when adding SDS as a reaction stop solution, A concentration of 0.5-1% was considered desirable. Industrial potential

According to the present invention, 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.

To automate the separation operation (B_F separation) between the free immunological components and the components captured on the solid phase, the measurement system had to be equipped with complicated mechanisms. The washing process associated with BZF separation made it difficult to reduce the measurement time. Therefore, providing measurement technology that does not require B / F separation not only simplifies the measurement operation, but also facilitates the automation of the measurement operation.

For example, when antibodies must be measured in a large amount of blood sample, automation of the measurement operation is essential. In addition, the more samples, the greater the effect of reducing the measurement time. For example, blood used for blood transfusions and blood products is screened for infectious diseases such as HIV, HBV, and IICV on all blood samples. Detection of these infections is based on antibody detection. Systems such as Luminex (trade name; manufactured by Luminex Corp.) that use particle counting are systems that enable extremely rapid measurement. By using the method of the present invention and such a system, screening for infectious diseases and allergens can be highly accelerated.

Claims

Claims A method for measuring an antibody, comprising the following steps. (1) a step of binding an antibody contained in a sample to an antigen recognized by the antibody; (2) an antigen-antibody complex formed in (1) in the presence of free immunoglobulin; Reacting a binding agent that recognizes and binds to the antibody that has formed, and (3) detecting the binding between the antigen-antibody complex and the binding agent, recognizing and binding the antibody that has formed the antigen-antibody complex 2. The method according to claim 1, wherein the binding agent is a protein selected from the group consisting of protein A, protein G, complement, and proteins functionally equivalent thereto. The method according to claim 1, wherein one 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. . 4. The method according to claim 3, wherein the antigen is immobilized or has a modification that can be immobilized. Any of the binding agents that recognize and bind the antigen and the antibody that formed the antigen-antibody complex have a label that produces a detectable signal or have a modification that can bind the label Item 3. The method according to item 3. 6. The method according to claim 5, wherein the binding agent that recognizes and binds to the antibody that has formed the antigen-antibody complex has a label or has a modification capable of binding the label. 4. The method according to claim 3, wherein the solid phase is a particle, and comprising the step of counting particles having a label on the solid phase. The method according to claim 7, comprising a step of detecting the particles and the label bound to the particles by a flow meter. The method according to claim 8, further comprising a step of identifying the antigen to which the antibody is bound based on the signal of the particle from which the label is detected, wherein the plurality of types of particles each having a different antigen bound thereto have a discriminable signal. Method. The method according to claim 1, wherein the antibody contained in the sample, the antigen recognized by the antibody, and the binding agent that recognizes and binds to the antibody forming the antigen-antibody complex are reacted substantially simultaneously. The method according to claim 1, comprising a step of detecting the binding between the antigen-antibody complex and the binding agent after the reaction with the antibody to be measured, the antigen, and the binding agent, and further after adding a reaction terminator. The method according to claim 11, wherein the reaction terminator is formaldehyde. The method according to claim 12, wherein the concentration of formaldehyde in the reaction solution is 0.1 to 0.5% v / v. The method according to claim 11, wherein the reaction terminator is dodecyl sulfate. The method according to claim 14, wherein the dodecyl sulfate is sodium dodecyl sulfate. The method according to claim 14, wherein the concentration of dodecyl sulfate is 0.5 to: l% w / v. It is composed of protein A, protein G, or any protein selected from the group consisting of proteins functionally equivalent thereto under the condition that the antibody constituting the antigen-antibody complex and free immunoglobulin coexist. A method for separating an antigen-antibody complex, comprising a step of binding a binding agent and an antibody constituting the antigen-antibody complex. 18. The separation method according to claim 17, wherein the binding agent is immobilized or has a modification that allows immobilization. Select an antibody that forms an antigen-antibody complex under the condition that free immunoglobulin coexists, including any protein selected from the group consisting of protein A, protein G, and proteins functionally equivalent thereto. A binder for the purpose of binding. The binding agent according to claim 19, wherein the binding agent is immobilized or has a modification that allows immobilization.

1. 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 of which is immobilized or immobilized A kit for measuring an antibody, wherein the kit has a modification that can be labeled, and the other has a label or a modification that can be labeled.

2. The kit according to claim 21, wherein 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 are mixed.

3. The kit according to claim 22, further comprising a reaction terminator.

4. The kit according to claim 23, wherein the reaction terminator is formaldehyde and / or dodecyl sulfate.