Patent Application
20100248393
The present invention relates to a method for determination of an antigen and an antibody against the antigen, and a determination reagent for use in the method. More particularly, the present invention relates to a method for determination of both of an antigen and an antibody against the antigen in a sample by an immuno-agglutination determination method by the use of one and the same reagent, for example, a method which permits determination of both of an antigen such as IgA and an antibody against the antigen, such as anti-IgA antibody by the use of one and the same reagent; and a determination reagent for use in such a method.
Determination of IgA in blood is like a routine in the clinical examination field because the level of IgA in blood is high in the case of chronic inflammation, chronic hepatitis, cirrhosis, IgA nephropathy, collagenosis (e.g. rheumatism), IgA type myeloma, etc. As a method for determining IgA, a method using immunoturbidimetry (TIA method) is usually adopted (patent document 1). TIA method is a method for determining an antigen such as IgA in a specimen which comprises subjecting the antigen in the specimen and an antibody as reagent to antigen-antibody reaction, and determining the amount of the antigen on the basis of the degree of the turbidity thus produced. This method is advantageous in that when an antigen (e.g. IgA) present in a high concentration is determined, the determination is possible without diluting a sample. This, method, however, is disadvantageous in that the cost of a reagent for the determination is high because a large amount of the antibody is used.
By the way, it is known that some people have almost no IgA and are deficient in IgA in some cases. It is considered that some of the people deficient in IgA have an antibody against IgA, i.e., anti-IgA antibody. It has been reported that if, by any chance, a person having anti-IgA antibody needs blood transfusion because of a disease or an accident, the transfusion of blood derived from a normal person, i.e., a person having IgA into the patient having anti-IgA antibody causes the antigen-antibody reaction between anti-IgA antibody and IgA in the blood of the patient, so that anaphylactic reaction, i.e., a shock is caused, resulting in a critical condition (non-patent document 2). Even the transfusion of blood derived from a normal person, i.e., a person having IgA into an IgA-deficient person having no anti-IgA antibody causes the production of anti-IgA antibody in the body, so that this person obtains the antibody. Therefore, such a person undergoes a shock reaction when the person receives the transfusion of blood derived from a normal person.
For preventing such a shock reaction, the blood of a person having no IgA is necessary as blood for transfusion to a patient having anti-IgA antibody. Therefore, it is necessary to investigate the presence of anti-IgA antibody in both of the bloods of a patient and a blood donor. However, since no reagent for determining anti-IgA antibody is provided at present in the clinical examination field, no measurement is carried out in order to know who has anti-IgA antibody. Thus, coping with such a problem without delay is considered necessary.
Non-patent document 1: Nobuhiko Kubo et al., Nihon Rinshou, Vol. 57, Extra Number (1999), pp. 10-12
Non-patent document 2: Takako Migita et al., Nihon Yuketsu Gakkai-zasshi, Vol. 50, No. 3, pp. 419-424 (2004)
The present invention is intended to provide a method and a determination reagent which make it possible to determine not only an antigen but also an antibody against the antigen in a sample by adopting a competitive homogeneous immuno-agglutination determination method which has been hardly adopted for determining an antigen or antibody in a sample, and a conventional immuno-agglutination determination method.
Under such a situation, the present inventor investigated a method for determining IgA in a sample. As a result, it was found that the purpose is achieved by the adoption of a competitive homogeneous immuno-agglutination determination method which has not often been adopted in the clinical examination field. It was also found that when the determination of IgA is attempted with a reagent used in this method, the value of IgA level becomes minus in some cases, depending on a specimen. Furthermore, it was found that such a specimen does not contain IgA and contains anti-IgA antibody. As a result, it was found that surprisingly, the reagent permits determination of not only IgA but also anti-IgA antibody in a sample. On the basis of this finding, the present inventor earnestly investigated in order to develop a method for determination of an antigen and an antibody in a sample at the same time by the use of one and the same reagent. The present invention has been accomplished in the course described above.
Therefore, the present invention relates to the items (1) to (11) described below.
(1) A method for determination of both of an antigen and an antibody against the antigen in a sample by the use of one and the same reagent, which comprises
(i) using a determination reagent comprising an antibody capable of causing an antigen-antibody reaction with the antigen contained in the sample and an antigen capable of causing an antigen-antibody reaction with both the antibody contained in the sample and the antibody contained in the reagent, either the antigen or the antibody in the reagent being supported on microparticles,
(ii) mixing the sample with the determination reagent, and
(iii) determining the antigen or the antibody in the sample on the basis of the degree of increase or decrease in agglutination caused by the antigen-antibody reaction.
(2) A method according to the above item (1), wherein as the sample, a sample containing an antigen and not containing an antibody against this antigen, or a sample not containing an antigen and containing an antibody against this antigen, or a sample containing neither an antigen nor an antibody against this antigen is subjected to the determination.
(3) A method for determination of both of an antigen and an antibody against the antigen in a sample by the use of one and the same reagent according to the above item (1) or (2), which comprises
(i) using a determination reagent comprising an antibody capable of causing an antigen-antibody reaction with the antigen contained in the sample and an antigen capable of causing an antigen-antibody reaction with both the antibody contained in the sample and the antibody contained in the reagent, the antigen in the reagent being supported on microparticles,
(ii) mixing the sample with the determination reagent, and
(iii) a step in the case where an antigen is contained in the sample, i.e., a step of allowing the antigen contained in the sample and the antigen supported on the microparticles and constituting the determination reagent, to compete with each other for an antigen-antibody reaction with the antibody constituting the determination reagent, and determining the antigen in the sample on the basis of the degree of decrease in agglutination caused by the antigen-antibody reaction, or
(iv) a step in the case where an antibody is contained in the sample, i.e., a step of subjecting the antigen supported on the microparticles and constituting the determination reagent to an antigen-antibody reaction with the antibody contained in the sample and the antibody constituting the determination reagent, and determining the antibody in the sample on the basis of the degree of increase in agglutination caused by the antigen-antibody reaction.
(4) A method for determination of both of an antigen and an antibody against the antigen in a sample by the use of one and the same reagent according to the above item (1) or (2), which comprises
(i) using a determination reagent comprising an antibody capable of causing an antigen-antibody reaction with the antigen contained in the sample and an antigen capable of causing an antigen-antibody reaction with both the antibody contained in the sample and the antibody contained in the reagent, the antibody in the reagent being supported on microparticles,
(ii) mixing the sample with the determination reagent, and
(iii) a step in the case where an antigen is contained in the sample, i.e., a step of subjecting the antibody supported on the microparticles and constituting the determination reagent to an antigen-antibody reaction with the antigen contained in the sample and the antigen constituting the determination reagent, and determining the antigen in the sample on the basis of the degree of increase in agglutination caused by the antigen-antibody reaction, or
(iv) a step in the case where an antibody is contained in the sample, i.e., a step of allowing the antibody contained in the sample and the antibody supported on the microparticles and constituting the determination reagent, to compete with each other for an antigen-antibody reaction with the antigen constituting the determination reagent, and determining the antibody in the sample on the basis of the degree of decrease in agglutination caused by the antigen-antibody reaction.
(5) A method according to any one of the above items (1) to (4), wherein the antigen in the sample is characterized in that it is usually present in biological samples derived from normal human beings or animals but is absent in biological samples derived from an extremely limited number of human beings or animals and that these human beings or animals have an antibody against the antigen.
(6) A method according to any one of the above items (1) to (5), wherein the antigen in the sample is IgA and the antibody in the sample is anti-IgA antibody.
(7) A method according to any one of the above items (1) to (3) and (5) and (6), which is practiced without dilution of the sample, by using a determination reagent comprising an antibody and an antigen supported on microparticles, to avoid prozone phenomenon.
(8) A reagent for determination of both of an antigen and an antibody against the antigen in a sample, which comprises an antibody capable of causing an antigen-antibody reaction with the antigen contained in the sample and an antigen capable of causing an antigen-antibody reaction with both the antibody contained in the sample and the antibody contained in the reagent, either the antigen or the antibody in the reagent being supported on microparticles.
(9) A determination reagent according to the above item (8), with which as the sample, a sample containing an antigen and not containing an antibody against this antigen, or a sample not containing an antigen and containing an antibody against this antigen, or a sample containing neither an antigen nor an antibody against this antigen is subjected to the determination.
(10) A determination reagent according to the above item (8) or (9), for which the antigen in the sample is characterized in that it is usually present in biological samples derived from normal human beings or animals but is absent in biological samples derived from an extremely limited number of human beings or animals and that these human beings or animals have an antibody against the antigen.
(11) A determination reagent according to any one of the above items (8) to (10), for which the antigen in the sample is IgA and the antibody in the sample is anti-IgA antibody.
The following determination may be carried out by the adoption of a competitive homogeneous immuno-agglutination determination method and a conventional immuno-agglutination determination method by employing the determination method and determination reagent of the present invention. When an antigen is contained in a sample, it may be determined. When an antibody against the antigen is present in the sample, it is also possible to determine the antibody. In addition, the following may be carried out as one run of determination with one and the same reagent by using a small amount of an expensive antibody in this determination reagent. When an antigen is present in a sample, it may be determined. When an antibody against the antigen is present in the sample, it may be determined. Thus, both the antigen and the antibody against the antigen in the sample may be determined. When neither an antigen nor an antibody against the antigen is present in a sample, it is also possible to confirm their absence by employing the determination method and determination reagent of the present invention.
When an antigen present in a high concentration in an ordinary biological sample, such as IgA is determined, the antigen in the sample may be determined without dilution of the sample, by using a determination reagent comprising an antibody against the antigen and an antigen supported on microparticles, to avoid prozone phenomenon.
Therefore, the determination method and determination reagent of the present invention may be employed for investigating the presence of anti-IgA antibody in both of the bloods of a blood donor and a recipient in order to prevent shock reaction at the time of blood transfusion. They may be used also in the field of clinical examinations for diagnosis of chronic inflammation, chronic hepatitis, cirrhosis, IgA nephropathy, collagenosis (e.g. rheumatism), IgA type myeloma or the like on the basis of IgA level.
In the present invention, the sample is, for example, a liquid sample derived from a living body. Specific examples of the sample are plasma, serum, urine, etc. Samples derived from blood, such as plasma and serum are suitable. In the present invention, as the sample, there are imagined three kinds of samples, i.e., a sample containing an antigen and not containing an antibody against this antigen, a sample not containing an antigen and containing an antibody against this antigen, and a sample containing neither an antigen nor an antibody against this antigen. Any of these samples may be subjected to the determination. In the case of a sample containing both of an antigen and an antibody against the antigen, antigen-antibody reaction occurs in a living body from which such a sample is collected, so that shock reaction occurs. Therefore, such a sample cannot exist.
As the antigen in the sample, an antigen suitable for the determination method and determination reagent of the present invention is “an antigen characterized in that it is usually present in biological samples derived from normal human beings or animals but is absent in biological samples derived from an extremely limited number of human beings or animals and that these human beings or animals have an antibody against the antigen”. Such an antigen includes, for example, immunoglobulin A (IgA), haptoglobulin, α2-macroglobulin, complement C9 and complement C4.
As the antibody in the sample, an antibody suitable for the determination method and determination reagent of the present invention is an antibody against the above-exemplified antigen in the sample. Such an antibody includes, for example, anti-IgA antibody, anti-haptoglobulin antibody, anti-α2-macroglobulin antibody, anti-complement C4 antibody and anti-complement C4 antibody against the above-exemplified antigens, respectively.
The antibody in the reagent used in the determination method of the present invention and used as the determination reagent of the present invention is not particularly limited as long as it is capable of causing an antigen-antibody reaction with the antigen contained in the sample. When the antigen to be determined is IgA, haptoglobulin, α2-macroglobulin, complement C9 or complement C4, anti-IgA antibody, anti-haptoglobulin antibody, anti-α2-macroglobulin antibody, anti-complement C9 antibody and anti-complement C4 antibody, respectively, may be exemplified as the antibody in the reagent. As to the source of such an antibody, for example, when an antigen and an antibody against the antigen in a human sample are determined, the source may be any source as long as the antibody in the reagent is capable of causing an antigen-antibody reaction with the antigen contained in the sample. The antibody in the reagent includes, for example, antibodies derived from goat, antibodies derived from rabbit, and antibodies derived from rat. In addition, as the antibody in the reagent used in the present invention, either a monoclonal antibody or a polyclonal antibody may be used. Furthermore, a fragment of any antibody capable of causing an antigen-antibody reaction with the antigen contained in the sample may be used as the antibody contained in the reagent used in the present invention.
The antigen in the reagent used in the determination method of the present invention and used as the determination reagent of the present invention is not particularly limited as long as it is capable of causing an antigen-antibody reaction with both the antibody contained in the sample and the antibody contained in the reagent. When anti-IgA antibody, anti-haptoglobulin antibody, anti-α2-macroglobulin antibody, anti-complement C9 antibody or anti-complement C4 antibody is used as the antibody contained in the reagent, IgA, haptoglobulin, α2-macroglobulin, complement C9 and complement C4, respectively, may be exemplified as the antigen in the determination reagent. The source of the antigen in the reagent and a process for producing the antigen may be any source and any process as long as the antigen is capable of causing an antigen-antibody reaction with both the antibody contained in the sample and the antibody contained in the reagent. For example, when IgA or anti-IgA antibody in a human sample is determined, the antigen in the reagent includes, for example, IgA derived from goat, IgA derived from rabbit, IgA derived from rat, and IgA obtained by recombination. In addition, a fragment of any antigen capable of causing an antigen-antibody reaction with both the antibody contained in the sample and the antibody contained in the reagent may be used as the antigen contained in the reagent.
As the microparticles used in the determination method and determination reagent of the present invention, microparticles usually used in immuno-agglutination reaction may be used as they are. The most conventional microparticles are latex particles. As the microparticles, particles of 0.01 to 0.5 micron are usually used. As a method for supporting the antigen or the antibody on the microparticles in the present invention, there may be adopted conventional supporting methods such as a physical adsorption method utilizing hydrophobic interaction, and a covalent bond method.
The determination method of the present invention is based on the fact that only one of the product and reactants in the immuno-agglutination reaction causes the appearance of a turbidity, while the other components are water-soluble or hardly cause the appearance of a turbidity. The principle of this fact is explained below by taking the case of using a determination reagent comprising an antibody and an antigen supported on microparticles.
At first, the reactants to be mixed are as follows: (i-1) an antigen derived from a sample, in the case where the antigen is present in the sample, (i-2) an antibody derived from the sample, in the case where the antibody is present in the sample, (ii) an antigen supported on microparticles and constituting a determination reagent, and (iii) an antibody constituting the determination reagent. All of these reactants (i) to (iii) are soluble or uniformly dispersible in water.
Then, the reactants are mixed to carry out an antigen-antibody reaction. When an antigen is present in the sample, there are competitively produced two antigen-antibody reaction products, i.e., an antigen-antibody reaction product of (iii) the antibody constituting the determination reagent with (i-1) the antigen derived from the sample and an antigen-antibody reaction product of (iii) the antibody constituting the determination reagent with (ii) the antigen supported on microparticles and constituting the determination reagent. The antigen-antibody reaction product of (iii) the antibody constituting the determination reagent with (ii) the antigen supported on microparticles and constituting the determination reagent is water-insoluble and causes the appearance of a turbidity, while the antigen-antibody reaction product of (iii) the antibody constituting the determination reagent with (i-1) the antigen derived from the sample hardly causes the appearance of a turbidity. Therefore, the turbidity of the reaction solution is increased with an increase in the amount of the antigen-antibody reaction product of (iii) the antibody constituting the determination reagent with (ii) the antigen supported on microparticles and constituting the determination reagent.
In the competitive reaction described above, (i-1) the antigen derived from the sample reacts with a definite amount of (iii) the antibody constituting the determination reagent, in competition with (ii) the antigen supported on microparticles and constituting the determination reagent, to reduce the amount of the insoluble antigen-antibody reaction product produced, i.e., the antigen-antibody reaction product of (iii) the antibody constituting the determination reagent with (ii) the antigen supported on microparticles and constituting the determination reagent and reduce the degree of turbidity produced in the reaction solution. Therefore, the turbidity of the reaction solution is reduced with an increase in the concentration of the antigen contained in the sample. Accordingly, the antigen in the sample may be determined on the basis of the degree of reduction of the turbidity.
When an antibody is present in the sample, antigen-antibody reaction products are produced by the reaction of each of (i-2) the antibody derived from the sample and (iii) the antibody constituting the determination reagent with (ii) the antigen supported on microparticles and constituting the determination reagent. Therefore, the amount of the antigen-antibody reaction product of (i-2) the antibody derived from the sample with (ii) the antigen supported on microparticles and constituting the determination reagent is increased with an increase in the concentration of the antibody contained in the sample, so that the degree of turbidity produced in the reaction solution is increased. Accordingly, the turbidity of the reaction solution is increased with an increase in the concentration of the antibody contained in the sample. Therefore, the antibody in the sample may be determined on the basis of the degree of increase of the turbidity.
Next, the principle is explained below by taking the case of using a determination reagent comprising an antigen and an antibody supported on microparticles.
At first, the reactants to be mixed are as follows: (i-1) an antigen derived from a sample, in the case where the antigen is present in the sample, (i-2) an antibody derived from the sample, in the case where the antibody is present in the sample, (ii) an antigen constituting a determination reagent, and (iii) an antibody supported on microparticles and constituting the determination reagent. All of these reactants (i) to (iii) are soluble or uniformly dispersible in water.
Then, the reactants are mixed to carry out an antigen-antibody reaction. When an antigen is present in the sample, there are produced two antigen-antibody reaction products, i.e., an antigen-antibody reaction product of (iii) the antibody supported on microparticles and constituting the determination reagent with (i-1) the antigen derived from the sample and an antigen-antibody reaction product of (iii) the antibody supported on microparticles and constituting the determination reagent with (ii) the antigen constituting the determination reagent. These antigen-antibody reaction products with the two antigens are water-insoluble and cause the appearance of a turbidity. Therefore, the amount of the antigen-antibody reaction product of (i-1) the antigen derived from the sample with (iii) the antibody supported on microparticles and constituting the determination reagent is increased with an increase in the concentration of the antigen contained in the sample, so that the degree of turbidity produced in the reaction solution is increased. Therefore, the turbidity of the reaction solution is increased with an increase in the concentration of the antigen contained in the sample. Accordingly, the antigen in the sample may be determined on the basis of the degree of increase of the turbidity.
When an antibody is present in the sample, there are competitively produced two antigen-antibody reaction products, i.e., an antigen-antibody reaction product of (ii) the antigen constituting the determination reagent with (i-2) the antibody derived from the sample and an antigen-antibody reaction product of (ii) the antigen constituting the determination reagent with (iii) the antibody supported on microparticles and constituting the determination reagent. In the competitive reaction described above, (i-2) the antibody derived from the sample reacts with (ii) the antigen constituting the determination reagent, in competition with a definite amount of (iii) the antibody supported on microparticles and constituting the determination reagent, to reduce the amount of the insoluble antigen-antibody reaction product produced, i.e., the antigen-antibody reaction product of (iii) the antibody supported on microparticles and constituting the determination reagent with (ii) the antigen constituting the determination reagent and reduce the degree of turbidity produced in the reaction solution. Therefore, the turbidity of the reaction solution is reduced with an increase in the concentration of the antibody contained in the sample. Accordingly, the antibody in the sample may be determined on the basis of the degree of reduction of the turbidity.
In the present invention, as to a method for measuring the degree of turbidity due to agglutination caused by the antigen-antibody reaction, the turbidity produced is usually measured by means of absorbance. The degree of turbidity is measurable also by visually observing a agglutination or by counting microparticles not agglutinated.
Specific examples of a method for practicing the determination method of the present invention are as follows.
When a reagent comprising an antibody and an antigen supported on microparticles is used as the determination reagent, the determination reagent is prepared at first by preparing a homogeneous dispersion of the antibody (e.g. anti-IgA antibody) in a buffer solution (e.g. phosphate buffer) (a first reagent) and a homogeneous dispersion of microparticles (e.g. latex particles) supporting thereon the same antigen (e.g. IgA) as an antigen to be determined (e.g. IgA) in a buffer solution (e.g. phosphate buffer) (a second reagent). Then, using an automatic analyzer (e.g. Autoanalyzer Hitachi Model 7180), an antigen-antibody reaction is carried out by adding the first reagent and then the second reagent to a sample containing the antigen or an antibody, which is to be determined, and the rate of agglutination caused is measured in terms of the degree of absorbance change by a two-point end method at a specific wavelength (for example, 340 nm to 800 nm). On the basis of the measured value thus obtained, the amount of the objective antigen or antibody in the sample may be determined by using a calibration curve previously obtained by the use of standard samples containing known concentrations of the antigen or the antibody.
The calibration curve is suitably obtained as follows: as shown in
The using amounts of the antibody and the microparticles supporting the antigen thereon which constitute the determination reagent used for practicing the determination method are usually varied depending on the amounts of the antigen or the antibody to be determined and the sample used. Basically, the using amounts may be merely determined so that the calibration curve can be obtained by adopting a competitive homogeneous immuno-agglutination method using the determination reagent and that as a result of obtaining the calibration curve, the antigen can be determined. When IgA as antigen and anti-IgA antibody as antibody are determined with an automatic analyzer (e.g. Autoanalyzer Hitachi Model 7180), the using amounts are, for example, as follows.
When 1.5 to 35 μl, preferably 5 to 25 μl of the sample is used, the concentration of anti-IgA antibody contained in the first reagent is preferably adjusted so that its final concentration (the concentration in a mixture of the sample, the first reagent and the second reagent) may be 0.5 to 50 μg/ml (in terms of Becker titer), more preferably 1.5 to 15 μg/ml; the concentration of the IgA-sensitized latex contained in the second reagent is preferably adjusted to 0.005 to 0.5%, more preferably 0.015 to 0.15%; and the concentration of IgA used for the sensitization of latex (in terms of IgA weight) is preferably adjusted to 0.005 to 0.5 mg/ml, more preferably 0.015 to 0.15 mg/ml. In this case, the volume of each of the first reagent and the second reagent is preferably adjusted to 30 to 250 μl, more preferably 50 to 150 μl, and the total volume of the sample, the first reagent and the second reagent is preferably adjusted to 120 to 300 μl, more preferably 150 to 250 μl.
When a reagent comprising an antigen and an antibody supported on microparticles is used as the determination reagent, the determination reagent is prepared at first by preparing a homogeneous dispersion of the antigen in a buffer solution (e.g. Tris buffer) (a first reagent) and a homogeneous dispersion of microparticles (e.g. latex particles) supporting thereon an antibody against an antigen to be determined, in a buffer solution (e.g. Tris buffer) (a second reagent). Then, using an automatic analyzer (e.g. Autoanalyzer Hitachi Model 7180), an antigen-antibody reaction is carried out by adding the first reagent and then second reagent to a sample containing an antigen or an antibody, which is to be determined, and the rate of agglutination caused is measured in terms of the degree of absorbance change by a two-point end method at a specific wavelength (for example, 350 nm to 800 nm). On the basis of the measured value thus obtained, the amount of the objective antigen or the objective antibody in the sample may be determined by using a calibration curve previously obtained by the use of standard samples containing known concentrations of the antigen or the antibody.
The calibration curve is suitably obtained as follows: as shown in
The using amounts of the antigen and the microparticles supporting the antibody which constitute the determination reagent used for practicing the determination method are usually varied depending on the amounts of the antigen or the antibody to be determined and the sample used. Basically, the using amounts may be merely determined so that the calibration curve can be obtained by adopting a competitive homogeneous immuno-agglutination method using the determination reagent and that as a result of obtaining the calibration curve, the antigen can be determined. When the antigen and the antibody are determined with an automatic analyzer (e.g. Autoanalyzer Hitachi Model 7180), the using amounts are, for example, as follows.
When 1.5 to 35 μl, preferably 5 to 25 μl of the sample is used, the concentration of the antibody contained in the first reagent is preferably adjusted so that its final concentration (the concentration in a mixture of the sample, the first reagent and the second reagent) may be 0.5 to 50 μg/ml (in terms of Becker titer), more preferably 1.5 to 15 μg/ml; the concentration of the antibody-sensitized latex contained in the second reagent is preferably adjusted to 0.005 to 0.5%, more preferably 0.015 to 0.15%; and the concentration of the antibody used for the sensitization of latex is preferably adjusted to 0.05 to 10 mg/ml, more preferably 0.5 to 5 mg/ml. In this case, the volume of each of the first reagent and the second reagent is preferably adjusted to 30 to 250 μl, more preferably 50 to 150 μl, and the total volume of the sample, the first reagent and the second reagent is preferably adjusted to 120 to 300 μl, more preferably 150 to 250 μl.
As is clear from the above explanation, the determination reagent for practicing the determination method of the present invention comprises an antibody capable of causing an antigen-antibody reaction with an antigen contained in a sample and an antigen capable of causing an antigen-antibody reaction with both of an antibody contained in the sample and the antibody contained in the reagent, and either the antigen or the antibody in the reagent is supported on microparticles. In addition, if necessary, the determination reagent may contain conventional additives such as a diluting buffer solution, coating buffer solution, blocking reagent, preservative, stabilizer, etc.
The present invention is concretely illustrated with the following examples, which should not be construed as limiting the scope of the invention.
All of Examples 1 to 3 show a method for determining both of an antigen (IgA) and an antibody (anti-IgA antibody) by using a determination reagent comprising an antibody and an antigen supported on microparticles.
IgA was adsorbed on latex particles as follows.
With 4 mL of a 1% solution of polystyrene latex particles with a particle diameter of 98 nm was mixed 4 mL of a solution obtained by dissolving human serum IgA in phosphate buffer to a concentration of 0.5 mg/mL, and the resulting mixture was stirred at room temperature for 1 hour. After the stirred mixture was centrifuged at 20,000 rpm for 45 minutes, the supernatant was disposed and the precipitate was recovered. To the precipitate was added 4 mL of a coating buffer solution to suspend the precipitate, and the resulting suspension was ultrasonicated to disperse the latex particles completely. The thus obtained human IgA-sensitized latex particles suspension having a latex concentration of 1% was kept in cold storage.
A first reagent and a second reagent were prepared as follows by using the latex particles having IgA adsorbed thereon and anti-human IgA antibody.
As the first reagent, a diluting buffer solution containing 0.2% of anti-human IgA goat serum (Becker titier: 8.8 mg/mL) was used.
As the second reagent, there was used a dilution obtained by diluting the human IgA-sensitized latex particles suspension having a latex concentration of 1% described in the above item 1) with diluting buffer solution.
The compositions of the reagents are as follows.
composition of the coating buffer solution
As standard samples, there were used dilutions obtained by diluting serum having a known IgA concentration with the diluting buffer solution. The IgA concentration had been determined by the use of protein standard serum CRM470.
The standard samples were subjected to measurement with Autoanalyzer Hitachi Model 7170S by reacting 100 μL of the first reagent and 100 μL of the second reagent with 15 μL of serum as sample and measuring the degree of absorbance change by a two-point end method between the 19th and 26th photometric points (corresponding to a period between just after the addition of R2 and 2.5 minutes after the addition) at a dominant wavelength of 505 nm and a complementary wavelength of 800 nm.
Table 1 shows the degree of absorbance change caused when the standard samples were subjected to the measurement by the use of the reagents described above, and
As shown in Table 1 and
The same reagents and determination conditions as in Example 1 were employed.
2) Quantitation with an Automatic Analyzer
Quantitation was carried out by using the standard samples described in Example 1 and the function of giving a multipoint calibration curve of Autoanalyzer Hitachi Model 7170S.
As samples, dilutions obtained by properly diluting serum with the diluting buffer solution were used. The correlation was confirmed by using “N-assay TIA IgA-SH” (Nitto Boseki Co., Ltd.), a commercial reagent for TIA method, as a reference reagent. TIA method was practiced according to specified parameters by similarly using Autoanalyzer Hitachi Model 7170S.
The same reagents and determination conditions as in Example 1 were employed.
As samples, there were used dilutions obtained by proper serial dilution of a specimen containing IgA or anti-IgA antibody with the diluting buffer solution.
3) Quantitation with an Automatic Analyzer
Quantitation was carried out by using the standard samples of 0 mg/ml and 1 mg/ml among the standard samples described in Example 1 and the function of giving two-point calibration curve of Autoanalyzer Hitachi Model 7170S.
Table 2 and
As shown in Table 2 and
It was confirmed that both of an antigen and an antibody may be determined by using a determination reagent comprising an antigen and an antibody supported on microparticles.
Specifically, the concentration of human CRP (an antigen) or anti-human CRP antibody (an antibody) in a specimen was measured by the use of a determination reagent comprising a latex sensitized by anti-human CRP antibody (an antibody supported on microparticles) and human CRP (an antigen). When the specimen contained anti-human CRP antibody, a specimen containing anti-human CRP antibody derived from goat was used as a model sample.
Anti-human CRP antibody was adsorbed on latex particles as follows.
With 100 mL of a 1% solution of polystyrene latex particles with a particle diameter of 120 nm was mixed 100 mL of a solution obtained by dissolving anti-human CRP antibody in Tris buffer to a concentration of 3.0 mg/mL, and the resulting mixture was stirred at room temperature for 1 hour. After the stirred mixture was centrifuged at 20,000 rpm for 45 minutes, the supernatant was disposed and the precipitate was recovered. To the precipitate was added 100 mL of a coating buffer solution to suspend the precipitate, and the resulting suspension was ultrasonicated to disperse the latex particles completely, and then was stirred at room temperature for 1 hour. Thereafter, the stirred suspension was centrifuged and to the resulting precipitate was added 100 mL of Tris buffer to suspend the precipitate. The resulting suspension was ultrasonicated to disperse the latex particles completely, whereby a 1% suspension of latex particles sensitized by anti-human CRP antibody was obtained.
A first reagent and a second reagent were prepared as follows by the use of the latex particles sensitized by anti-human CRP antibody and human CRP.
As the first reagent, Tris buffer containing 0.10 mg/dL of human CRP was used.
As the second reagent, there was used a dilution obtained by 5-fold dilution of the 1% suspension of latex particles sensitized by anti-human CRP antibody described in the above item 1) with Tris buffer.
The compositions of the reagents are as follows.
The determination was carried out with Autoanalyzer Hitachi Model 7180 by reacting 120 μL of the first reagent and 120 μL of the second reagent with 2.4 μL of each of samples and measuring the degree of absorbance change by a two-point end method between the 18th and 28th photometric points (corresponding to a period between just after the addition of R2 and 2.9 minutes after the addition) at a dominant wavelength of 570 nm and a complementary wavelength of 800 nm.
As the samples, there were used dilutions obtained by properly diluting a specimen containing human CRP and anti-human CRP antibody with physiological saline.
Table 3 and
As can be seen from the result of determining human CRP and anti-human CRP antibody in the samples which is shown in Table 3 and
By employing the determination method and determination reagent of the present invention, an antigen in a sample may be determined by the use of an immuno-agglutination determination system and when an antibody against the antigen is present in the sample, it is also possible to determine the antibody by the use of the same reagent as for the antigen. In addition, the following may be carried out as one run of determination with one and the same reagent by using a small amount of an expensive antibody in the determination reagent. When an antigen is present in a sample, it may be determined. When an antibody against the antigen is present in the sample, it may be determined. Thus, both the antigen and the antibody against the antigen in the sample may be determined. When neither an antigen nor an antibody against the antigen is present in a sample, it is also possible to confirm their absence by employing the determination method and determination reagent of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2006-244389 | Sep 2006 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2007/067385 | 9/6/2007 | WO | 00 | 2/19/2009 |
