WO1997006184A1 - Antibody that recognizes serum amyloid a - Google Patents

Abstract

An antibody useful in the immunoassay on the basis of the agglutination of human serum amyloid A (SAA) which serves as a marker of inflammation, etc., and the use of this antibody. The antibody is a novel one which recognizes SAA and also has the activity of binding to SAA contained in the gel filtration fractions of molecular weights of 10 to 40 kD. Also, a reagent and method for the immunoassay of SAA and a method for purifying SAA by using this antibody are provided. This novel antibody having an excellent binding activity enables the assay of SAA based on the immunological agglutination thereof. This antibody is excellent in the titer against SAA and contributes not only to the specific assay of SAA at a high sensitivity but also to the widening of the assay scope.

Description

 Specification

 Antibodies that recognize serum amyloid A

Technical field

 The present invention relates to an antibody that recognizes amyloid A (hereinafter abbreviated as SAA) in human serum, and to the use of this antibody.

 In the field of clinical tests and the like, immunological measurement methods are often used to easily measure substances or to specifically measure substances with high sensitivity. To measure an antigenic substance by an immunological measurement method, an antibody that specifically reacts with the antigen is required. That is, an antibody that recognizes SAA is required for immunological measurement of SAA, which is an antigenic substance.

 S AA is a serum protein with a molecular weight of about 1200, which is a precursor protein of amyloid protein A (hereinafter abbreviated as AA protein) deposited in tissues in certain types of amyloidosis [1] . In recent years, serum levels of SAA have been shown to increase in inflammatory diseases and have been evaluated as sensitive markers of inflammation [2] [3]. Background art

 There have been several reports on immunoassay techniques using antibodies that recognize SAA [4] [5] [8]. In these prior art documents, the measurement is performed using immunological agglutination produced by reacting SAA with an antibody as an index. The measurement technique using immunological agglutination as an index does not require a physical separation operation of unbound components (BZF separation). Therefore, it has the advantages of simple operation and easy automation as compared to heterogeneous analysis methods such as ELISA. On the other hand, the following strict conditions are required for the antibodies used for measurement.

Condition A: Aggregation requires higher avidity.

 Aggregation reactions, while maintaining physically stable aggregates, generally require higher levels of binding activity than required by ELISA.

If the reaction system is composed of antibodies with insufficient binding activity, the number of antibodies that can bind to one antigen molecule will not change, even if the amount of antibody used is increased to cover the low binding activity. No sensitivity can be obtained. Also, when using an antibody bound to a carrier such as latex, the amount of antibody bound is still limited. There is a limit to measures to increase the amount of antibody used.

 Condition B: The positional relationship of the epitope, which is not a problem in ELISA, can be an obstacle in the coagulation method.

 In an agglutination reaction, there must be multiple epitopes recognized by the antibody on the same antigen. This condition is the same as the ELISA sandwich method. However, the agglutination method has the disadvantage of requiring a physically stronger bond, as described above, and therefore, it is disadvantageous to perform the reaction only with a close epitope even if the physical position is different. This is because steric hindrance is likely to occur, and as a result it is difficult to obtain large aggregates.

 In polyclonal antibodies, such positional relationship of the epitopes is unlikely to be a major problem. However, despite the polyclonal antibodies, only a few epitopes are useful for immunoassay of SAA, and the selection of an epitope is one of the most important conditions, though not so much as a monoclonal antibody.

 Antibodies obtained based on the technology disclosed in the prior art documents introduced above did not necessarily satisfy these conditions in + minutes. In particular, regarding the binding activity of the antibody, it was difficult to obtain an antibody satisfying the above conditions, and it was considered that commercial supply of the reagent was difficult.

Specifically, for example, a report using a nephrometric atsushi [5] confirmed linearity between 1 and 13 g / ml. In £ Otsu 1 5 1. 5- 3 0 μ g / ml [6], or _{5 5- 7 5 Ο η 8/} 200 μ 1 [7] reported there were measured.

In addition, in the measurement example by the immunological latex agglutination method [8], the detection limit is 0.5 μg / ml, whereas the calibration curve loses the slope near 30 g / ml. . In other words, in this document, it was only possible to measure the concentration by about 60 times. This value is not necessarily insufficient. However, the blood concentration of SAA varies widely, sometimes reaching several hundreds / zg / ml, and when measuring a large number of samples, it is often observed that the upper limit of the measurement range is exceeded. For samples exceeding the measurement range, dilution and re-measurement will be required, leading to a reduction in processing capacity. There have been reports on SAAs that achieved a measurement range of 1.25 to 160 / ig / ml in a special reaction system combining dot blot and enzyme-labeled antibody [12]. However, in this report In addition, a treatment for enhancing antigenicity by heat treatment is required, which is distinguished from the method of measuring a sample without pretreatment as in the present invention. There is also a report [13] that has achieved a measurement range of 100-fold by solid-phase immunoradiometric assay without denaturing the sample. However, this result was obtained by performing a solid phase washing step using an RI-labeled antibody. Furthermore, since the concentration of SAA used for the standard is unknown, the measurement range cannot be evaluated.

 Alternatively, it is possible to measure a high-value sample by increasing the dilution ratio of the sample or increasing the amount of the antibody used. However, such a measure is not practical because the measurement sensitivity is reduced as a result. In particular, it is very difficult to increase the antibody concentration while maintaining the sensitivity, particularly for an antibody having insufficient binding activity as in the past. In other words, samples with a wide range of concentrations cannot be measured under the same measurement conditions.

 An object of the present invention is to provide a new antibody that enables measurement by SAA agglutination. With such a novel antibody, an immunoassay reagent for SAA having excellent measurement performance and a use such as a measurement method are provided. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a graph showing the reactivity of a reagent with a fraction obtained by fractionating normal human serum by gel filtration. In the figure, the vertical axis shows the measured value of SAA (// g / ml), and the horizontal axis shows the fraction number.

 Figure 2 shows the reactivity of the reagents with fractions obtained by gel filtration of HDL purified from serum containing SAA by ultracentrifugation and diluted with PBS (SAA concentration: 7.δ μg / ml). It is a graph. In the figure, the vertical axis shows the measured value of SAA (μg / ml), and the horizontal axis shows the fraction number.

 Fig. 3 is a graph showing the reactivity of the reagent to fractions obtained by gel filtration of r SAA diluted with PBS (S AA concentration of 7.5 µg / ml). Indicates the measured value of SAA (g / ml), and the horizontal axis indicates the fraction number.

Fig. 4 is a graph showing the reactivity of the reagent to the fraction obtained by gel filtration of normal human serum supplemented with rSAA (SAA concentration 7.5 μg / ml). You. In the figure, the vertical axis shows the measured value of SAA (/ zg / ffll) and the horizontal axis shows the fraction number: Figure 5 shows normal human serum plus SAA purified from human serum (SAA concentration 7. 5 is a graph showing the reactivity of a reagent with a fraction obtained by fractionating (5 μg / ml) by gel filtration. In the figure, the vertical axis shows the SAA measurement value (g / ml), and the horizontal axis shows the fraction number.

 FIG. 6 is a calibration curve obtained by using the antibody of the present invention and a reagent for latex agglutination reaction prepared with a conventional antibody. In the figure, the vertical axis shows the difference in scattered light intensity, and the horizontal axis shows the SAA concentration (g / ml).

 FIG. 7 shows the result of epitope mapping of the antibody of the present invention. In the figure, the vertical axis shows the absorbance at 45 Omn, and the horizontal axis shows the number of the pin on which the synthetic peptide was immobilized. FIG. 8 shows the results of conventional epitope mapping of antibodies. In the figure, the vertical axis indicates the absorbance at 45 Onm, and the horizontal axis indicates the number of the pin on which the synthetic peptide was immobilized. FIG. 9 shows the results of analyzing the binding activity of the antibody by IAsys. In the figure, the vertical axis Kon shows the dissociation rate constant (1 / s), and the horizontal axis shows the antibody concentration (nM). Ab 1 (—Hata) is a conventional antibody, and Ab 2 (—〇 一) is the antibody of the present invention. Disclosure of the invention

 The object of the present invention is solved by an antibody that recognizes SAA and has the following reactivity. That is, the antibody of the present invention is an antibody that recognizes SAA, and has a molecular weight of 10 to 4 OkD, which can be obtained by fractionating serum containing high-density lipoprotein and SAA by gel filtration under non-denaturing conditions. It is an antibody that reacts with SAA contained in the surface. Hereinafter, this condition will be specifically described.

 Most of SAA exists in serum in association with high-density lipoprotein (hereinafter abbreviated as HDL). SAA associated with HDL (hereinafter referred to as HDL-SAA) elutes to a molecular weight of 100-300 kD when fractionated by gel filtration under non-denaturing conditions. Normally, when SAA is purified, it is collected as serum HDL fraction, so SAA recovered by general purification procedures can be called SAA contained in this 100-30 OkD fraction.

However, when the molecular weight is used as the index by gel filtration, the fraction is 10-4 OkD. Also slightly elutes SAA. Conventional antibodies cannot detect the SAA in this fraction with sufficient sensitivity and are difficult to trace, but the antibodies of the present invention clearly show reactivity with the SAA contained in this fraction. HDL-SAA is eluted in a fraction of 100 to 30 OkD when the molecular weight is determined by gel filtration, so that the novel characteristics of the antibody of the present invention can be clearly distinguished.

 A typical SAA eluted in the fraction with a molecular weight of 10-4 OkD by gel filtration is SAA that is not associated with HDL (hereinafter abbreviated as f SAA). In order to obtain purified fSAA, it is advisable to use a method of fractionating fSAA from a material containing SAA such as serum or ascites using the molecular weight as an index. The fSAA, which is slightly present in these body fluids, is recovered.

 Specifically, the following operation is performed. For example, when serum is used as a raw material, first, the specific gravity of serum containing a large amount of SAA (here, SAA also includes HDL-SAA) is adjusted, and fractionation with a specific gravity of 1.23 to 1.063 is performed by ultracentrifugation. Collect. The fraction having a higher specific gravity than the collected HDL is applied to an antibody affinity column chromatography column using an anti-SAA antibody. After washing impurities, fSAA adsorbed on the column is eluted. In this case, the antibody to be used must be an antibody that recognizes fSAA according to the present invention, not a known antibody. This is because conventional antibodies cannot adsorb fSAA. The eluted fSAA is pooled, and the fraction having the target molecular weight of 10-4 OkD is collected by gel filtration. If necessary, this can be concentrated to recover fSAA in pure form [9]. However, fSAA has little power even in high SAA serum, so it is not possible to recover a sufficient amount without preparing a large amount of raw materials. For example, when 10 L of serum containing 388 of 100 // 8/011 is used as a starting material, such a purification method can only recover about 1 mg of purified antigen (the recovery rate is 0% of the total SAA). 1%). When recovering from the HDL fraction, a recovery rate of about 10-20% can generally be expected, indicating that it is difficult to purify SAA.

In order to obtain a large amount of fSAA, a method in which a gene encoding the amino acid sequence of SAA is cloned and inserted into an appropriate host-vector system and expressed can be employed. According to the newly obtained knowledge of the present inventors, the toxicity to the host is high. A gene [16] encoding the amino acid sequence of SAA cloned by a known method [11] into a vector [10] such as PET useful for the expression of a new protein [10], and then introducing into E. coli. For example, it was confirmed that a protein having the amino acid sequence of SAA could be expressed. SAA obtained by purifying a protein having the same amino acid sequence as SAA expressed in this manner (hereinafter abbreviated as rSAA) naturally has the same amino acid sequence as SAA purified from serum. However, the amount of lipid components associated with one molecule is extremely small. For these reasons, it is considered that the antibody of the present invention exhibits almost the same antigenicity as fSAA purified from serum.

 Depending on the expression system, a methionine residue corresponding to the initiation codon may remain at the N-terminus of the product. However, according to the experience of the present inventors, in the case of SAA, addition of a methionine residue to the N-terminus did not particularly affect antigenicity and preservation. Therefore, a peptide having a methionine residue added to the N-terminus can be said to be a protein having substantially the same amino acid sequence as SAA.

 In order to confirm the reactivity between fS AA and the antibody, fS AA may be reacted with the antibody as it is or, if necessary, sensitized to a carrier. The reactivity with the antibody can also be confirmed by the Ottaello-Nii method for observing the immunoprecipitation reaction in agar or the immunoprecipitation reaction for observing the formation of an immunological complex in a solution. In order to quantify the reactivity, a particle agglutination reaction method in which an antibody is sensitized to a particle carrier such as latex and the agglutination of the particles by the antigen is optically measured, an immunoassay for optically quantifying the formation of an immune complex A turbidimetric method (hereinafter abbreviated as TIA), an RIA method in which the antigen is immobilized and the reactivity of the antibody is traced with an enzyme-labeled antibody or the like, or an ELISA method can also be employed.

Western blotting is also effective for confirming reactivity. In other words, the antigen mixture in which HD L-SAA and fSAA are mixed is separated by electrophoresis under non-denaturing conditions of 0rnstein and Davis [14] [15] together with the molecular weight, and then This is blotted on a nitrocellulose membrane. In this way, the antibody whose reactivity is to be checked is brought into contact with the nitrocellulose membrane on which the serum protein is immobilized, washed if necessary, and further reacted with a labeled antibody against the antibody. The binding of the labeled antibody may be determined visually, or quantitatively the reactivity can be determined by measuring the signal intensity of color intensity (enzyme labeling) and photoradiography (RI labeling) with a densitometer. Perform comparison I can. According to this method, the reactivity to both HDL-SAA and fSAA can be simultaneously confirmed, which is convenient.

 For an accurate comparison of antibody reactivity, try to provide as favorable conditions as possible for the immune response. Conditions that affect reactivity can include temperature, pH, and salt concentration. However, it is basically important that the antibody of the present invention has reactivity with fSAA, and it is not always necessary to strictly compare the strength of the reactivity. Known antibodies that recognize SAA do not substantially react with fSAA, and can be clearly distinguished from known antibodies by confirming the presence or absence of reactivity.

 Antibodies having the above-mentioned reactivity have excellent agglutinating activity and are useful as immunoassays for SAA, particularly as antibodies for immunological particle agglutination and immunoturbidimetry.

 In the present invention, the fact that the antibody does not substantially react with SAA of a specific molecular weight fraction means that the antibody does not cause an observable change when the antibody is reacted under the following conditions, for example. Can be defined.

 That is, a fixed amount of SAA is added to normal human serum not containing SAA to prepare a sample. In the examples, r SAA was added to a final SAA concentration of 7.5 μg / ml. The antibody according to the present invention is to confirm the reactivity with SAA which is eluted in the fraction having a molecular weight of 10-4 OkD when SAA of 10 / g / ml, preferably 7.5 // g / ml is added. Can be. Thus, by mixing rSAA with normal human serum containing HDL, HDL-associated SAA and fSAA are generated. This sample is applied to a gel column whose elution pattern has been confirmed in advance by a molecular weight marker. Perform a genre filtration to separate the desired molecular weight fractions (ie, 100-30 OkD and 10-40 kD) and react with the antibody whose reactivity is to be confirmed. The antibody sensitizes the latex particles and immunological agglutination is followed by optical measurements. Under such conditions, if the antibody does not substantially react, the amount of optical change due to the agglutination reaction is less than the detection limit. The details of a series of operations will be described in Examples.

As shown in the Examples, the antibody of the present invention exhibits reactivity with a 10-4 OkD fraction even when a sample obtained by fractionating a PBS solution of rSAA by gel filtration is used as a sample. This division Is nothing but r S AA itself. That is, the antibody of the present invention can be distinguished from a known antibody by showing a clear aggregation activity when bound to latex particles and reacted with rSAA.

The binding activity of the antibody according to the present invention can also be quantified by an analytical method that quantitatively captures the binding state between molecules. For example, a commercially available optical biosensor called IAsys (Affinity SENSORS) is based on the incident angle at which laser light is applied from various angles to a sensor cuvette containing a sample and the evanescent wave resonates. An analytical system that calculates the mass of the molecules bound to the cuvette, enabling real-time observation of the binding and dissociation between molecules in the cuvette. In the cuvette of this analysis system, SAA contained in the domain of the molecular weight of 10-4 OkD by gel filtration or the gene encoding the amino acid sequence shown in SEQ ID NO: 1 is expressed in a microbial cell as a host. After immobilizing the recombinant protein (r SAA) to be used, the anti-SAA antibody is reacted and the dissociation equilibrium constant (M) or the association equilibrium constant is calculated. By defining a dissociation flat ⁵衡定number the binding activity of the antibody of the present invention in a preferred embodiment, 2 X 1 0- ⁷ Μ less when measured r SAA as an antigen, more preferably about 5 values of X 1 0- Show. Note Analysis of the antibodies known in the same conditions, has a value of at most be 5 X 1 0- ⁷ about Micromax, it is clear that excellent binding activity of the antibody according to the invention.

0 The antibody of the present invention can be stably obtained by using highly purified fSAA as an immunogen and employing a special immunization method for further enhancing immunogenicity.

 FSAA used for an immunogen can be obtained by a method as described above. That is, the antibody of the present invention is obtained by using fSAA or rSAA present in serum as an immunogen, and further pooling antibodies satisfying the above conditions using these antigens in antibody screening. Obtainable.

When using purified fSAA or rSAA as an immunogen, various techniques can be applied to enhance its antigenicity. Freund's complete adjuvant (FCA) is one of the components required to enhance immunogenicity. You. In addition, it is effective to add M. tuberculosis as an immunogen or to combine pertussis vaccine intramuscularly at the time of immunization. It has been pointed out that SAA has high homology among many mammals, and thus is unlikely to show antigenicity to immunized animals. One of the reasons that the measurement range of SAA by agglutination reported so far was not necessarily sufficient is presumed to be one of the causes of the low antigenicity of SAA. In the present invention, fSAA is used as an immunogen, and the reactivity is screened with fSAA to obtain an antibody having an unprecedented binding activity. Provided. BEST MODE FOR CARRYING OUT THE INVENTION

 The antibody of the present invention thus obtained has the conditions described above, that is, it has reactivity with SAA eluted with a molecular weight of 10-4 kD by gel filtration, and preferably has the following properties. It has a good coagulation activity. That is, a preferable antibody in the present invention shows a value of 0.05 or more as compared with a blind test when the change in absorbance for 300 seconds immediately after the reaction is measured under the following conditions. . Such large values cannot be obtained with the aggregation activity of conventional antibodies.

 Condition:

Sensitizes antibodies to polymer particles with a particle size of 0.4-0.6 μm

Particle concentration 0.02-0.5%

 Contact with 1.0 / g / ml human purified SAA

The liquid volume ratio is sample: latex emulsion = 1: 3 to 1:15. These reaction conditions are the minimum necessary conditions, and other reaction conditions are the characteristics of the target antibody and the polymer particles to be used. Needless to say, preferable conditions are selected in accordance with other components such as. Other reaction conditions refer to the reaction temperature, measurement wavelength, pH of the reaction solution and buffer components.

The present invention also provides a SAA immunoassay reagent using the antibody. The antibodies of the present invention can be applied to known immunoassay reagents. Preferred reagent forms include the following. The most preferred reagent for immunoassay according to the present invention is an immunoassay reagent obtained by sensitizing an insoluble carrier particle with an antibody. Insoluble carrier particles include polymer particles typified by polystyrene and gelatin, as well as inorganic materials such as silica and various metal sols, and biological materials such as red blood cells and bacterial cells. I have. Antibodies can be physically adsorbed or chemically bound to these insoluble carriers.

 Since the insoluble carrier particles to which the antibody is bound aggregate in the presence of SAA, it is possible to measure SAA by optically following this aggregation or visually determining the aggregation. As optical measurement of particle aggregation, absorbance measurement and scattered light measurement are known. The wavelength of the light source used for optical measurement is selected from an infrared part, a near-infrared part, and a visible part mainly according to the particle size of the insoluble carrier particles. In some cases, a laser is used as the light source.

 As the reagent for immunological measurement according to the present invention, a reaction enhancer that promotes the formation of an insoluble precipitate based on the formation of an immune complex is supplied to the immunological reaction site when the antibody exists in a free state. A form in which the reagent is combined with the reagent may be employed. In this form, the use of the antibodies of the invention with strong aggregating activity is very advantageous, since the immune complexes must be generated in an optically traceable form.

 In the reagent of the present invention, examples of the reaction enhancer that promotes the formation of an insoluble precipitate based on the formation of an immune complex include polyethylene glycol and derivatives thereof, nonionic surfactants, and polyadiones such as dextran sulfate. Are known. These enhancers may be used in appropriate combinations.

 When the antibody of the present invention is used as a reagent for immunological measurement, it may be used as a fragment digested with an appropriate enzyme for the purpose of suppressing nonspecific effects of rheumatoid factor and complement. As antibody fragments, F (ab,) 2 by pepsin, Facb 'by plasmin and the like are known.

Known components can be combined with the SAA immunoassay reagent of the present invention. That is, a buffer that gives a pH required for an immune reaction, a reaction enhancer that promotes an immune reaction, a reaction stabilizer that suppresses a non-specific reaction, Proca, or the like may be used in combination. The following are used as buffering agents.

 G O O D buffer

 2-Monorefolinoethanesulfonic acid (abbreviated as 2- (N-Morpholino) ethanesulfonic acid, MES)

 5 Piperazine monobis (2-ethanesulfonic acid)

 (Abbreviated as Piperazine-N, N'-bis (2-ethanesulfonic acid), PIPES) (2-acetoamide) One 2-aminoethanesulfonic acid

 (Abbreviated as N- (2-Acetamido) -2-aminoethanesulfonic acid, ACES) Bis (2-hydroxetyl) 1- 2-aminoethanesulfonic acid

0 (abbreviated as N, N-Bis (2-hydroxyethyl) -2-aminoehtanesulfonic acid, BES) Bis (2-hydroxyxethyl) imino tris (hydroxymethyl) methane

(Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane _¾ Abbreviated as B is— Tris)

 3-[bis (2-hydroxyethyl) amino] — 2-hydroxypropanesulfonate (3- [N, N-Bis (2-hydroxyethyl) aminoj-2-hydroxypropanesu Ifonic acid,

DIPSO)

 2-Hydroxitytyl piperazine-1-propanesulfonic acid

 (Ν-2-Hydroxyethylpiperazine-N'-3-propanesulfonic acid, abbreviated as EPSPS)

0 Hydroxicetyl piperazine-1-ethanesulfonic acid

 (Ν-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid, abbreviated as HEPS)

 2-Hydroxitytyl piperazine 2-Hydroxypropane-1 3-Snorefonic acid (Ν-2-Hydroxyethylpiperazine-N-2-hydroxypropane-3-sultonic acid, abbreviated as H L Pδ P SO)

 3- (morpholino) prononesulfonic acid (abbreviated as 3- (N-orpholino) propanesulfonic acid, MOPS)

 3-(morpholino) _ 2-hydroxypropanesulfonic acid

(3- (N-Morpholino) -2-hydroxypropanesulfonic acid, abbreviated as MO PSO )

 Piperazine monobis (2-hydroxypropanesulfonic acid)

 (Pioerazine-N, N, -bis (2-hydroxypropanesulfonic acid), abbreviated as POPS〇)

 N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid, abbreviated as TAPS)

 Tris (hydroxymethyl) methyl-2-hydroxy-3-aminopropanesulfonate (abbreviated as -Tris (hydroxymethyl) methyl-2-hydroxy-3-aminooropanesulfonic acid, TAP SO)

 Tris (hydroxymethyl) methyl-2-aminomethanesulfonic acid

 (N-Tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid, abbreviated as TES)

Other buffers

 2-amino-2-hydroxymethyl-1,3-propanediol

 Phosphate buffer, also known as (2-Aminc ^ 2-hydroxymethyl-1,3-propanediol) or Tris (hydroxymethyl) aminomethane

Ammonia buffer

 Among these buffers, GOOD buffers, such as HEPES and PIPES, are mentioned as particularly preferred buffers because they not only give a favorable pH for the immune reaction but also have a small effect on proteins.

 In addition, reaction stabilizers and blockers include BSA (pseudo-albumin), animal serum, IgG, IgG fragments (Fab and Fc), albumin, milk protein, amino acids, polyamino acids, choline, sucrose, etc. It is known that polysaccharides, gelatin, hydrolyzate of gelatin, polyhydric alcohols such as casein, glycerin and the like are effective for stabilizing reactions in immunological reactions and for suppressing nonspecific reactions.

The SAA immunoassay reagent according to the present invention containing these various components can be supplied in a solution state or a dry state. For distribution in a solution state, various surfactants, sugars, inactive proteins, etc. May be added. These stabilizers are also effective as stabilizers or excipients when drying reagents. When the reagent for immunological measurement of SAA according to the present invention is distributed in the form of a solution, it is advantageous to add a preservative that does not affect the immunological reaction with SAA. . Antiseptics such as amphotericin B and microside are used for such preservatives.

 The method for immunological measurement of SAA according to the present invention is realized by the reagent for immunological measurement of SAA described above. If the reagent is for an agglutination reaction, the progress of the agglutination reaction can be performed optically or by visually observing the progress of the agglutination reaction.

 The present invention further provides a novel method for measuring SAA having the following features. That is, a method for immunologically measuring human serum amyloid A using an antibody, wherein the antibody is bound to insoluble carrier particles, and the measured value of the immunological agglutination of the insoluble carrier particles is 10 This is a method for measuring human serum amyloid A corresponding to a human serum amyloid A concentration value of 1: 1 in a 0-fold concentration range. When SAA is measured by the latex agglutination reaction described above, a sample with a SAA concentration of 50 μg / ml, serially diluted up to 100-fold, is measured under the same measurement conditions, and obtained. The results are plotted on a graph. The graph thus drawn is the calibration curve. The same measurement conditions mean that conditions that affect the measurement results, such as sample dilution conditions and the ratio of reagent to sample volume, are unified. Even with a known measurement technique having a narrow measurement range, a concentration range of 100 times can be measured by changing the dilution ratio or the liquid volume ratio between the sample and the reagent according to the concentration. However, changing the measurement conditions according to the concentration does not satisfy the requirements of the present invention of the same measurement conditions, so that the known measurement technology and the measurement technology of the present invention are clearly distinguished.

Here, the same condition means that the condition for measurement in a certain 100-fold concentration range is the same. Therefore, it is not always necessary to use the same measurement conditions. Specifically, the same conditions are used in the concentration range of 0.5-500 / zg / inl (100 times), but the measurement conditions and the concentration of 100-1000 / ig / ml are used. The conditions for measuring the range (100 times) need not be the same. In order to measure 100 to 100 g / ml, the conditions should be set according to this concentration range. In other words, the present invention is capable of measuring a 100-fold concentration range under certain measurement conditions. It is a feature. In the above measuring method, the corresponding measured value in the concentration range of 100 times increases continuously according to the concentration. Alternatively, if the measured value is a measurement system that decreases in accordance with the concentration, the measured value will continue to decrease in this concentration range. In other words, the standard curve drawn by the novel reagent according to the present invention can be defined in a preferred embodiment that the measured value in the concentration range of 100-fold continues to change in a certain direction. In the conventional immunological latex agglutination method, the binding activity of the antibody used as a reagent is insufficient, so that when a wide concentration range of 100 times is measured under the same measurement conditions, a linear calibration curve is obtained. Only part of it changed. In other words, if the concentration shows linearity in the low concentration range, a point appears in which the calibration curve decreases as the concentration increases. This phenomenon is called prozone and makes quantitative measurement in high concentration areas difficult. On the other hand, when the linearity is exhibited in the high concentration range, there is a point where the slope of the calibration curve is lost at a certain concentration and the measurement becomes impossible. In short, the sensitivity is insufficient. In the state where the measured value of the latex agglutination reaction does not reflect the change in the concentration of the component to be measured, such as the prozone phenomenon or insufficient sensitivity, multiple concentrations correspond to one measured value. The defined correspondence does not satisfy the condition of 1: 1.

 In addition, the present invention provides a method for purifying SAA using a novel antibody. Since the antibody of the present invention has a strong binding activity to SAA, SAA can be recovered at a high yield when applied to antibody affinity chromatography. That is, the material containing SAA is applied as it is or after pretreatment by ion exchange chromatography, ultracentrifugation, or the like, to a column on which the antibody of the present invention is immobilized, and the SAA is captured by an immune reaction. After washing the SAA-capture column with an appropriate buffer that does not affect immunological binding, the SAA is removed from the antibody using an eluent that cleaves the immunological binding, and the SAA is recovered. It can be obtained in high yield. For eluents, chaotropic agents such as urea and guanidine at high concentrations of 1 M or more are used. Antibody affinity chromatography can be performed in the presence of a suitable surfactant.

In addition, since the antibody of the present invention is reactive with SAA contained in addition to the HDL surface, fSAA can be recovered when applied to a material that has been previously classified based on molecular weight or specific gravity. SAA purified by the antibody of the present invention can be used for immunogens and immunoassays. Useful as a standard for routine use.

 Further, the antibody of the present invention is useful because it has a high binding activity even in the recovery of rSAA described above. According to the antibody of the present invention having reactivity with SAA contained in the molecular weight of 10-4 OkD by gel filtration, rSAA can be easily recovered.

 The present invention provides a novel antibody having a specific binding activity. The antibody of the present invention has excellent agglutinating activity and expands the measurement range of a reagent based on SAA immunological agglutination. Since the known antibody against SAA uses SAA recovered as an HDL fraction of serum as an immunogen, it is presumed that an antibody having substantial reactivity with fSAA could not be obtained. On the other hand, in the present invention, a new antibody was obtained by using ίS AA, which can be obtained only in a small amount, or r SAA newly obtained as an immunogen.

 The antibody of the present invention not only reacts with fSAA, but also exhibits a higher binding activity to HDL-SAA than conventional antibodies. Since the antibody of the present invention has such binding activity, the performance of the immunoassay reagent can be remarkably improved. Specifically, it provides reliable measurements over a wide range without sacrificing reagent sensitivity. It is not clear why the difference in fSAA binding activity from conventional antibodies leads to improved reagent performance. However, experimentally, a clear effect has been confirmed that cannot be explained simply by reacting with fSAA. Most of SAA exists in the living body in association with HDL, and fSAA is hardly observed in ordinary biological samples. Therefore, the high binding activity of the antibody to fSAA according to the present invention does not lead to an incorrect analysis result. In humans, the major inflammatory symptoms are SAA subtypes such as 1α, 1, 1, 2α, and 2 among the subtypes of human, and the antibody of the present invention has any of these subtypes. It does not respond to subtype SAA-14, which does not show any association with inflammatory symptoms. Example

1. Establishment of SA Α expression strain 1— 1. Synthesis of primer for PCR

 In order to PCR amplify the gene encoding SAA, a primer having the following sequence was synthesized by the solid phase phosphite method. An Ndel site was introduced on the 5 'side of the primer and a BamHI site was introduced on the 3' side of the primer in accordance with the cloning site of the vector, and each recognition sequence was enclosed in [].

 5, Side primer:

 5,-GTAGTTCAGGT [CATATG] CGAAGCTTCnTTCGTTCCnG-3,

 3 'primer:

 5, one GACA [GGATCC] GAGGAAGCTCAGTATTTCTCAG-3,

 1 -2. PC R amplification

 To amplify cDNA encoding SAA, the primers listed in 1-1 and the template of the human liver cDNA library were used for 94 ^ 1 min, 63 ^ 1 min, 72 hr. PCR amplification was carried out for 30 cycles at 1 minute at ° C. The amplified DNA fragment was confirmed to be about 35 Obp by 2% agarose gel electrophoresis. Further, when mapping was performed with each restriction enzyme, a DNA sequence encoding a known human SAA [16] was obtained. A match was confirmed.

 1 -3. Construction of human SAA protein expression vector

 The DNA fragment amplified in step 1-2 was digested with Ndel and BamHI (manufactured by Takara Shuzo) and ligated to the Ndel / BamHI site of a plasmid vector pET21-a (+) having an ampicillin resistance gene.

 1-4. Establishment of expression strain

The obtained plasmid vector was introduced into Escherichia coli BL21 (DE3) p Lys S which had been made competent by the rubidium chloride method, and was transformed. After selection of transformed bacteria by culturing on LB agar medium containing ampicillin (12 δ μg / ml), plasmid was purified and strains having a higher molecular weight than pET21 were selected. Strains expressing a protein with a molecular weight of 12000 that reacts with the anti-SAA antibody were selected. The amino acid sequence of the expressed protein was examined, and it was confirmed that the protein had a sequence in which a methionine derived from the initiation codon was added to the N-terminus of the known SAA1-H1 shown in Sequence 1. In the present specification, the following abbreviations of amino acids are used. A or Ala

Arginine R or Arg

 N or Asn

Aspartic acid D or Asp

Sistine C or Cys

Glutamine Q or Gin

Gunoretamic acid E or Glu

Glycine G or Gly

Histidine H or His

Isoleucine I or lie

Leucine L or Leu

Lysine K or shi ys

Methionin M or Met

Fueninorealanine F or Phe

Proline P or Pro

Serine S or Ser

 Threonine Ding or Thr

 Tryptophan W or Trp

Tyrosine Y or Tyr

Valin V or Val

The pET21 used here is a transformation vector containing the T7 phage promoter, and transcription of the integrated gene is started by T7 RNA polymerase supplied by the host E. coli. The T7 RNA polymerase gene is induced only by adding Isopropyl β-D-thiogalactopyranoside (IPTG) to the culture solution, so that gene transcription can be controlled by IPTG. Expression of a poorly soluble substance such as SAA is generally toxic to the host and cannot be expected to produce a sufficient amount of expression.However, controlling gene transcription in this way will sufficiently increase the bacterial mass. As the expression can be induced at a different stage, the yield of the expression product increases as a result. 1-5. R S AA expression The E. coli PET-SAA1-BL21 strain obtained in 1-4 was inoculated into 300 ml of LB medium containing carbenicillin (500 μg / ml) and cultured at 37 ° C. When the absorbance at 60 Onm is between 0.6 and 0.9, add IPTG to ImM, and then add rifampicin to 200 / g / ml after 30 minutes, then 3 to 6 hours After the culture, the precipitate was collected by centrifugation. With the addition of rifampicin, the expressed SAA can suppress the f¾ bacteria effect on the host, and the amount of recovery is significantly increased compared to the absence of rifampicin.

 1 -6. Purification

 The precipitate was dispersed in guanidine buffer (4 M guanidine hydrochloride, 0.025 M Tris-hydrochloric acid, pH 8.6), followed by sonication to disrupt the cells and solubilize rSAA. The solubilized sample is dialyzed against affinity buffer (0.5M NaCI, 2mM EDTA, 0.01M Tris-HCl · ρΗ8.2, 0.1% Twee η20), and the supernatant is collected by centrifugation. After passing through a Sepharose 4 affinity column to which anti-SAA antibody has been adsorbed, and then adsorbing, elution buffer (0.5 液 NaCl, 2 mM EDTA, 0.01 M Tris-HCl · ρΗ8.2, 0.1% (Twe e η20, 3M KSCN), and eluted fractions are dialyzed against guanidine buffer

The mixture was applied to a Sephacryl S-300 column and separated according to the difference in molecular weight.

When the separated fractions were subjected to disk electrophoresis, it was confirmed that they were separated in a substantially pure state. Finally, approximately 2 _ng of rSAA was recovered from 300 ml of culture.

 2. Antibody

 The rSAA obtained in Step 1 was added to a 0.01 M Tris-HCl buffer (pH 8.6, 0.0

5% Tween20), adjust to 1 mg / ml, mix an equal volume with FCA containing human killed Mycobacterium tuberculosis (4 ing of 1 ml of FCA for 1 ml of FCA), emulsify enough, and mix well. Rabbits were immunized with ml. At the same time, a pertussis vaccine was intramuscularly injected into the base of the hind foot. Immunization was performed every two weeks. The antiserum obtained by partially collecting blood four months later was checked for its reactivity with rSAA by the Octet method. The antiserum of the individual for which a high antibody titer was confirmed was fractionated by 40% ammonium sulfate to collect IgG, which was dialyzed against PBS to obtain an anti-SAA antibody (10 mg / ml) according to the present invention. 3. Preparation of reagents using antibodies

 The anti-SAA antibody (10 mg / ml) obtained in 2 was physically adsorbed to polystyrene latex (average particle size 0.109 μιη) with 37 ^ for 1 hour, washed with 0.1 M HEPES buffer, and finally washed. Suspended in a dispersion medium (0.1 M HEPES buffer containing 1% BSA, pH 7.4) so that the latex concentration becomes 0.4%, and a reagent for the SAA latex agglutination reaction (hereinafter simply referred to as emulsion) I got

 For comparison, a reagent (emulsion B) was prepared by the same procedure for a conventional anti-SAA antibody [17] obtained by immunization with SAA purified from the HDL fraction by a known method.

4. Antibody reactivity (binding activity)

 Using the emulsion obtained in 3, the antibody binding activity was compared by latex agglutination. The operation is as follows.

 300 μl of the reagent was added to sample 501, and reacted at 37 for about 3 minutes. During this period, the change in absorbance at 585 nm based on the immunological agglutination was measured. The SAA concentration was determined based on a calibration curve created based on a standard set by a known method [18]. As samples, those obtained by diluting some SAA-containing samples and fractionating by gel filtration were used. The molecular weight of each fraction was determined in advance by preparing a calibration curve by eluting a molecular weight marker under the same conditions. The following five types of SAA-containing samples were prepared. SAA purified from serum was obtained by a known method [17]. After mixing with purified HDL, the mixture was left at 4 ^-^ after mixing before use in experiments.

 (1) Normal human serum (SAA concentration is below the detection limit, expressed as NHS) only

 (2) PBS solution of purified human HDL (SAA concentration 7.δ μg / ml)

 (3) r SAA in PBS solution (SAA concentration 7.5 μg / ml)

(4) Add rSAA to NHS (final SAA concentration is 7.5 μg / ml)

(5) Add SAA purified from serum to NHS (final SAA concentration is 7.δ μg / ml). The conditions for gel filtration are as follows. Using TSKgel G3000 SWXL (manufactured by Tosoichi) for the column, elute with 20 mM phosphate buffer (pH 7.2, containing 0.15 M NaCl) at a flow rate of lml / min. Fractionated. this Under the conditions, fSAA is eluted at Fr. 13-15 (corresponding to a molecular weight of 10-40 kD), whereas SAA associated with HDL is eluted at Fr.8-10 (corresponding to a molecular weight of 100-300 KD).

 The results are as shown in FIGS. The antibody of the present invention clearly shows reactivity to the fraction eluted in Fr. 13-15 corresponding to fSAA, but the antibody obtained by the conventional method cannot confirm the reactivity in the same area ( Figure 3-5). In addition, the antibodies of the present invention show higher reactivity not only with fSAA but also with HDL-SAA than conventional antibodies (FIGS. 4-15). From such a phenomenon, it was confirmed that rSAA and fSAA expressed in E. coli were immunologically identical. In addition, in the case where rSAA shown in FIG. 3 is dissolved in PBS as a sample, the SAA eluted on the 10-4 OkD surface is nothing but rSAA. Thus, it is clear that the known antibodies show no observable aggregation with rSAA, whereas the antibodies of the present invention react and aggregate with rSAA.

 In addition, FIG. 1 shows that normal serum did not react with any antibody. Regardless of which antibody was used, the reactivity was confirmed only with 100-30 OkD (HDL-SAA) when reacted with SAA recovered as an HDL fraction (Figure 2).

 5. Linearity of reagent

 The linearity of the SAA immunoassay reagent obtained by the present invention was compared with the reagent obtained by the conventional antibody.

A dilution series containing 0-66 / zg / ml of SAA was prepared, and measurement was performed using the emulsion obtained in step 3. The operation is as follows. The dilution series was prepared by diluting the SAA-containing serum whose concentration had been assayed with 5 Om of HEPES buffer (pH 7.4, hereinafter simply referred to as diluent) so that the SAA concentration was 66 g / inl. It was prepared by diluting it twice with a diluent. Diluent 225 1 and 20 μl of each concentration of SAA-containing solution were dispensed into the measurement cell, and after 30 seconds, 75 μl of emulsion was added and scattered light was measured at a wavelength of 66 Onm (scattered light T2). The scattered light was measured after 2 seconds (scattered light T3), and the third scattered light measurement (scattered light T4) was performed after 130 seconds to determine the difference in scattered light intensity between each measurement point. For measurement, a fully automatic immunochemical analyzer LX-3000 (Eiken Chemical 'Analytica) The results were shown in DLSE obtained as the measured value of this instrument (Table 1).

Table 1. Comparison of linearity between reagents of the present invention and conventional antibodies

 Figure 6 shows a standard curve drawn based on the data in Table 1. As is clear from FIG. 6, the measured value of the reagent using the antibody of the present invention continues to increase from 0.52 to 66; zg / ml, and the concentration difference of 100 times or more (specifically, Can be measured under the same conditions. In contrast, conventional antibodies can guarantee linearity only up to about 33 / z g / ffll, and the range over which the measured values continue to increase is slightly more than 60 times the concentration difference. The range of measurement with conventional antibodies may not be inadequate, but the measurable range may be so narrow that some samples require dilution. According to the antibody of the present invention, which has a much wider measurement range than the conventional antibody, it is possible to greatly reduce the chances of encountering sampnolle that must be diluted and remeasured to exceed the measurement range.

6. Antibody reactivity (specificity)

In order to compare the specificity of a known antibody with the antibody of the present invention, using a Multi-Pin Peptide Synthesis Starter Kit Non-Cleable Type (manufactured by Chiron Mimotopes Pty. After peptide synthesis, a conventional anti-SAA antibody and an anti-SAA antibody according to the present invention were respectively reacted, and epitope mapping [19] was attempted based on the difference in reactivity. The amino acid sequence of the synthetic peptide is composed of 10 residues shifted from the N-terminus of the amino acid sequence of SAA shown in Sequence 1 by 2 amino acid residues in order, corresponding to the position of the 96-well microtiter plate. Pin Was fixed to the tip. By immersing this in a well into which the antibody solution has been dispensed, an immune reaction can be performed.

 Antibodies were diluted 2000-fold with 2 OmM phosphate buffer (ρΗ7.2, containing 2% BSA, 0.1% Tween 20, and 0.15 M NaC1), and then added to each well for 175 minutes. The pin with the synthetic peptide immobilized was dipped. After allowing to stand still for 4 hours in step 4, the pins were washed with PBS and reacted with a peroxidase (hereinafter abbreviated as POD) labeled antibody. The POD-labeled antibody is prepared by diluting commercially available POD-labeled anti-rabbit IgG goat serum 2000-fold with 2 OmM phosphate buffer (containing 1% BSA and 0.15 M NaCl). The washed pins were immersed in 175 μl aliquots and reacted at room temperature for 60 minutes. After the reaction, the pins are washed with PBS, then immersed in a well containing dispensed 150 1 substrate solution (containing 0.84 mM tetramethylbenzidine and 2 mM hydrogen peroxide). As a liquid, 3.6 N sulfuric acid was added in 50 1 portions. The absorbance at 45 Onm was measured with a microphone-mouth plate reader.

 The results of the epitope mapping of the present invention are shown in FIG. 7 (antibody of the present invention) and FIG. 8 (conventional antibody). The numbers on the horizontal axis in the figure indicate pin numbers, where 1 is a synthetic peptide consisting of 10 amino acid residues starting from the N-terminal Arg force, and 2 or less are 3, 5, 7, .... Equivalent to 10 amino acid residues shifted. There was almost no difference between the two reaction patterns, and it was speculated that the selection of the epitope could not explain the improvement in binding activity.

 7. Antibody reactivity (binding activity)

 In order to quantify the binding activity of the present invention, rSAA which is an antigen is used as a solid phase ligand of IAsys, and an antibody is used for a liquid phase. Was calculated. IAsys is a system that allows real-time observation of the binding state of binding components (immunoglobulin molecules) in the liquid phase to solid phase ligand (rSAA).

In a cuvette coated with carboxymethyl dextran (CMD), add 1 OmM phosphate buffer (containing 137 mM NaC and 2.7 mM KC1 containing 0.05% TVeen20, hereinafter abbreviated as PBS / T) ) For 200 minutes, allow the temperature to stabilize from 24.5 to 25.5 ° C for about 10 minutes, and then measure the baseline for 7 minutes. Specified. After reacting with 0.2 M of 0.4M EDC in 0.1M NHS for 7 minutes to activate the carboxyl group, unreacted EDC / NHS was washed away with a PBSZT buffer. RSAA (50 μg / ml) previously dissolved in an acetate buffer (10, pH 5.0) was added as ligand solution (200 μl) and immobilized for 10 minutes. Note that EDC refers to e-thy 3- (3-dimethylaminopropyl) carbodiimide, and NHS refers to N-hydroxysuccinimide. After washing off the ligand of unreacted PBS / T, by the this reacting 1M ethanol § Min aqueous _P H8. 5 a 2 00 μ ΐ added 5 minutes, the unreacted carboxyl groups were inactivated. The total amount of rSAA immobilized after washing with PBSZT was quantified as a resonance angle.

Antibody solutions at each concentration were added to the cuvette, and the amount of antibody associated with the ligand, rSAA, was measured in real time. Subsequently, the amount of antibody dissociated by adding PBS was measured in real time. After the measurement, the cuvette was treated with 3 M KSCN and 0.2% EDTA2Na for 2 minutes to completely dissociate the antibody, and then reused by washing with PBS. The antibody concentration added to the cuvette was 31.25 nM, 62.5 nM, 125 nM, 250 nM, and 32.5 nM. Based on the binding state at each concentration, IAsys FASTfit analysis software (registered trademark) , The association rate constant K _ass and the dissociation rate constant K diss (or Kon) were obtained, and the dissociation equilibrium constant KD was calculated using KdissZKass. The reciprocal is the association equilibrium constant KA. The parameters used were a baseline of 120 seconds before the start of each association, the association phase was 5-1800 seconds after the addition of the antibody, and the dissociation phase was PBS. It was up to 511 seconds after the addition.

 As a result of this analysis, the dissociation equilibrium constant KD of the conventional antibody for rSAA was 7.25

2.73 x 1 (は⁷ 、, whereas the antibody of the present invention is 1.01 ± 0.48 x 1

0 is an ⁷ M, are between them were at least three times the opening. That is, it can be said that the antibody of the present invention has a binding activity (avidity) to rSAA that is three times or more as large as that of a conventional antibody. A part of the data for analysis is shown in Fig. 9. Industrial applicability The antibody of the present invention makes it possible to easily obtain a reagent having a wide measurement range that is difficult to achieve with conventional antibodies. In addition, the reagent for immunological measurement of SAA or the immunological measurement method of SAA using the antibody of the present invention enables analysis with a wide measurement range based on immunological agglutination which is easy to automate. The immunoassay reagent for SAA using the novel antibody provided by the present invention is excellent in linearity particularly at a high concentration, and realizes a wide measurement range.

SAA is useful as a sensitive inflammatory marker. If the serum concentration increases with inflammation symptoms of human _{1 α, 1 β, ΐ γ} , 2, and such 2/3 is a sub-class. The use of the antibody according to the present invention makes it possible to provide an immunological drug capable of measuring these subclasses over a wide concentration range. Furthermore, the antibodies of the present invention are useful for purifying rSAA. RSAA obtained using a bacterium which is difficult to purify with a conventional antibody as a host, the antibody of the present invention binds with strong binding activity and can be recovered in high yield.

References

[1] 丄 Clin.lnvest. 53: 1054-1061, 1974

[2] J.CIin.lnvest.55: 746-753,1975

 [3] J.CIin.lnvest.61: 390-394,1978

[4] Marker Protein in Inflamation, Vol. 3 p157-162,1986 [5] Klinische Wochenschrift 67: 447-451, 1989

[6] Clin. Chim. Acta. 179, 169-175, 1989

[7] Scand.J.lmmunol., 18,329-338,1983

[8] Ann. Clin. Biochem., 30, 72-76; 1993

[9] Biophysical Chemistry Vol.36, No.4, 217 (29) -222 (34), 1992

[10] Gene 139 73-75,1994 [11] Biochemistry 24: 2931-2936,1985 [12] J.lmmunol.Methods., 116,131-135; 1989 [13] J.lmmunol.Methods., 54,213-221 , 1982 [14] Ann.NYAcacd.Soc.121 404, 1964 [15] Ann.NYAcacd.Soc.121 321, 1964 [16] J.Biol.Chem.262: 15790-15795,1987

 [17] JP-A-Heisei 4- 25 5 7 6 0

[18] J.lmmunol.Methods.83,217-225,1985 [19] SOUTHEAST ASIAN J TROP MED PUBRIC HEALTH, 24 (4) 523,1990

Sequence listing

SEQ ID NO: 1

Array length: 1 0 4

Sequence type: amino acid

 Topology: linear

Sequence type: peptide

Origin: human

Other information: Human serum amyloid A

Array

 Arg Ser Phe Phe Ser Phe Leu Gly Glu Ala Phe Asp Gly Ala Arg

 1 5 10 15 Asp Met Trp Arg Ala Tyr Ser Asp Met Arg Glu Ala Asn Tyr lie

 20 25 30

Gly Ser Asp Lys Tyr Phe His Ala Arg Gly Asn Tyr Asp Ala Ala

 35 40 45

Lys Arg Gly Pro Gly Gly Val Trp Ala Ala Glu Ala lie Ser Asp

 50 55 60

Ala Arg Glu Asn lie Gin Arg Phe Phe Gly His Gly Ala Glu Asp

 65 70 75

Ser Leu Ala Asp Gin Ala Ala Asn Glu Trp Gly Arg Ser Gly Lys

80 85 90 Asp Pro Asn His Phe Arg Pro Ala Gly Leu Pro Glu Lys Tyr

 95 100

SEQ ID NO: 2

Array length: 39

Sequence type: nucleic acid

Number of chains: 1 strand

 Topology: linear

Sequence type: other nucleic acid synthetic DNA

Other information: 5 'primer sequence for human serum amyloid A cDNA amplification

 GTAGTTCAGG TCATATGCGA AGCTTCTTTT CGTTCCnG 39 SEQ ID NO: 3

Array length: 32

Sequence type: nucleic acid

Number of chains: 1 strand

 Topology: linear

Sequence type: other nucleic acid synthetic DNA

Other information: 3 'primer sequence for human serum amyloid A cDNA width

 GACAGGATCC GAGGAAGCTC AGTATTTCTC AG 32

Claims

The scope of the claims  1. An antibody that recognizes human serum amyloid A and has a molecular weight of 10 that can be obtained by fractionating serum containing high-density lipoprotein and human serum amiloid A by gel filtration under non-denaturing conditions. Reacts with human serum amyloid A contained in the fraction of 44 OkD.饥 body  2. The antibody according to claim 11, wherein the human serum amyloid A contained in the fraction having a molecular weight of 10 to 4 OkD is selected from the following group:  (1) Human serum amyloid A purified from a fraction with a molecular weight of 10 to 40 kD by gel filtration of human serum amyloid A-rich serum  (2) Recombinant protein obtained by expressing a gene encoding the amino acid sequence shown in Sequence 1 in a microbial cell as a host 3. Dissociation equilibrium constant of the antibody, 2 X 1 0- ⁷ M when a gene co one de the Amino acid sequence shown in SEQ 1 and the recombinant protein obtained by expressing the microbial cells as host reacted as antigen The antibody of claim 2, which is 4. The antibody can be used to fractionate serum containing high-density lipoprotein and human serum amyloid A by gel filtration under non-denaturing conditions to obtain a fraction with a molecular weight of 100 to 30 OkD. The antibody according to claim 1, which reacts with the contained human serum amyloid A 5. Immunoassay reagent for human serum amine A containing the antibody according to claim 1 or 2 6. The reagent for measuring human serum amyloid A according to claim 5, wherein the antibody is bound to insoluble carrier particles.  7. The claim 5 wherein the antibody is present in a free state and is combined with a reagent that supplies a reaction enhancer that promotes the formation of an insoluble precipitate based on immune complex formation in the immunological reaction field. For the determination of human serum amyloid A 8. A method for immunologically measuring human serum amyloid A using an antibody, wherein the antibody is bound to insoluble carrier particles, and the measured value of the immunological aggregation reaction of the insoluble carrier particles is: Measurement method of human serum amyloid A corresponding to the 1: 1 serum serum amyloid A concentration value in a 100-fold concentration range 9. A method for immunological measurement of human serum amyloid A using the antibody according to claim 1 or 2.  10. The immunoassay for human serum amide A according to claim 9, wherein the antibody is immobilized on insoluble carrier particles.  5 11. The method for immunologically measuring human serum amyloid A according to claim 10, wherein the optical measurement of agglutination of insoluble carrier particles by immunological reaction is performed.  1 2. Using the antibody in a free state, react with human serum amyloid A in the presence of a reaction enhancer that promotes the formation of an insoluble precipitate based on immune complex formation, and optically measure the resulting insoluble precipitate Claim 9: Immunological measurement of human serum amyloid A 10 ways  13. A method for purifying human serum amyloid A by binding human serum amyloid A to an antibody and separating it from co-existing components, and then recovering human serum amyloid A bound to the antibody, wherein the antibody is any of claims 1 or 2. Using the method described in  15 14. The method according to claim 13, wherein the serum amyloid A to be purified is included in the domain of a molecular weight of 10—4 OkD by gel filtration selected from 1 or 2 shown below.  (1) Human serum amyloid A present in serum and contained in other than high-density lipoprotein domain (2) a recombinant protein comprising at least one antigenic determinant specific to human serum amyloid A and obtained from the gene encoding the amino acid sequence shown in SEQ ID NO: 0 or a partial sequence thereof  15. An antibody recognizing human serum amyloid A for immunological agglutination, which is obtained by fractionating serum containing high-density lipoprotein and human serum amyloid A by Genofiltration under non-denaturing conditions. Antibody for immunological agglutination that reacts with human serum amyloid A contained in the fraction with a molecular weight of 10 to 4 kD that can be obtained  1 6. The antibody according to claim 15, wherein the immunological agglutination reaction is an immunological latex agglutination method.