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WO1997006184A1 - Anticorps reconnaissant l'amyloide a serique - Google Patents

Anticorps reconnaissant l'amyloide a serique Download PDF

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Publication number
WO1997006184A1
WO1997006184A1 PCT/JP1996/002219 JP9602219W WO9706184A1 WO 1997006184 A1 WO1997006184 A1 WO 1997006184A1 JP 9602219 W JP9602219 W JP 9602219W WO 9706184 A1 WO9706184 A1 WO 9706184A1
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WIPO (PCT)
Prior art keywords
antibody
human serum
saa
serum amyloid
immunological
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PCT/JP1996/002219
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English (en)
Japanese (ja)
Inventor
Hiromi Eitoku
Hiroaki Maekawa
Jiro Nemoto
Atsufumi Wada
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Eiken Kagaku Kabushiki Kaisha
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Publication of WO1997006184A1 publication Critical patent/WO1997006184A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • 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.
  • SAA amyloid A
  • immunological measurement methods are often used to easily measure substances or to specifically measure substances with high sensitivity.
  • 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] .
  • AA protein amyloid protein A
  • Aggregation reactions while maintaining physically stable aggregates, generally require higher levels of binding activity than required by ELISA.
  • 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.
  • the agglutination 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.
  • 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.
  • Antibodies obtained based on the technology disclosed in the prior art documents introduced above did not necessarily satisfy these conditions in + minutes.
  • 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.
  • the detection limit is 0.5 ⁇ g / ml
  • the calibration curve loses the slope near 30 g / ml. .
  • 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.
  • An object of the present invention is to provide a new antibody that enables measurement by SAA agglutination.
  • an immunoassay reagent for SAA having excellent measurement performance and a use such as a measurement method are provided.
  • FIG. 1 is a graph showing the reactivity of a reagent with a fraction obtained by fractionating normal human serum by gel filtration.
  • 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. ⁇ ⁇ g / ml). It is a graph. In the figure, the vertical axis shows the measured value of SAA ( ⁇ g / 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 ⁇ g / 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 ⁇ g / ml).
  • 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 ⁇ g / ml) by gel filtration.
  • 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.
  • the vertical axis shows the difference in scattered light intensity
  • the horizontal axis shows the SAA concentration (g / ml).
  • FIG. 7 shows the result of epitope mapping of the antibody of the present invention.
  • the vertical axis shows the absorbance at 45 Omn
  • 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.
  • the vertical axis indicates the absorbance at 45 Onm
  • 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.
  • the vertical axis Kon shows the dissociation rate constant (1 / s)
  • the horizontal axis shows the antibody concentration (nM).
  • Ab 1 (—Hata) is a conventional antibody
  • Ab 2 (— ⁇ ⁇ ) 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.
  • HDL-SAA high-density lipoprotein
  • the fraction 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).
  • f SAA A typical SAA eluted in the fraction with a molecular weight of 10-4 OkD by gel filtration
  • f SAA A typical SAA eluted in the fraction with a molecular weight of 10-4 OkD by gel filtration
  • f SAA A typical SAA eluted in the fraction with a molecular weight of 10-4 OkD by gel filtration
  • f SAA 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
  • the following operation is performed.
  • serum 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.
  • 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.
  • 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.
  • 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%).
  • a recovery rate of about 10-20% can generally be expected, indicating that it is difficult to purify SAA.
  • 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.
  • 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.
  • rSAA a protein having the same amino acid sequence as SAA expressed in this manner
  • 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.
  • a methionine residue corresponding to the initiation codon may remain at the N-terminus of the product.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a fixed amount of SAA is added to normal human serum not containing SAA to prepare a sample.
  • r SAA was added to a final SAA concentration of 7.5 ⁇ g / 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.
  • 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.
  • 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.
  • an analytical method that quantitatively captures the binding state between molecules.
  • IAsys Affinity SENSORS
  • 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.
  • 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.
  • the anti-SAA antibody is reacted and the dissociation equilibrium constant (M) or the association equilibrium constant is calculated.
  • M dissociation equilibrium constant
  • 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.
  • FCA Freund's complete adjuvant
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the antibody of the present invention When 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.
  • an appropriate enzyme for the purpose of suppressing nonspecific effects of rheumatoid factor and complement.
  • antibody fragments F (ab,) 2 by pepsin, Facb 'by plasmin and the like are known.
  • 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.
  • 2-Monorefolinoethanesulfonic acid (abbreviated as 2- (N-Morpholino) ethanesulfonic acid, MES)
  • N N-Bis (2-hydroxyethyl) -2-aminoehtanesulfonic acid, BES)
  • BES Bis (2-hydroxyxethyl) imino tris (hydroxymethyl) methane
  • HEPS ⁇ -2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid
  • TAPS N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid
  • Tris (hydroxymethyl) methyl-2-hydroxy-3-aminopropanesulfonate (abbreviated as -Tris (hydroxymethyl) methyl-2-hydroxy-3-aminooropanesulfonic acid, TAP SO)
  • TES N-Tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid
  • Phosphate buffer also known as (2-Aminc ⁇ 2-hydroxymethyl-1,3-propanediol) or Tris (hydroxymethyl) aminomethane
  • GOOD buffers such as HEPES and PIPES
  • PIPES PIPES
  • 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.
  • various surfactants, sugars, inactive proteins, etc. May be added.
  • These stabilizers are also effective as stabilizers or excipients when drying reagents.
  • 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.
  • SAA is measured by the latex agglutination reaction described above, a sample with a SAA concentration of 50 ⁇ g / 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.
  • 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.
  • 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.
  • the conditions should be set according to this concentration range.
  • the present invention is capable of measuring a 100-fold concentration range under certain measurement conditions. It is a feature.
  • the corresponding measured value in the concentration range of 100 times increases continuously according to the concentration.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ S 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.
  • the high binding activity of the antibody to fSAA according to the present invention does not lead to an incorrect analysis result.
  • the major inflammatory symptoms are SAA subtypes such as 1 ⁇ , 1, 1, 2 ⁇ , 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.
  • 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 [].
  • cDNA encoding SAA 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.
  • 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.
  • 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 ⁇ ⁇ g / 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.
  • 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.
  • amino acids are used. A or Ala
  • 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.
  • T7 RNA polymerase gene is induced only by adding Isopropyl ⁇ -D-thiogalactopyranoside (IPTG) to the culture solution, so that gene transcription can be controlled by IPTG.
  • IPTG Isopropyl ⁇ -D-thiogalactopyranoside
  • SAA Isopropyl ⁇ -D-thiogalactopyranoside
  • 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.
  • 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 ⁇ ⁇ 8.2, 0.1% Twee ⁇ 20), and the supernatant is collected by centrifugation.
  • elution buffer 0.5 ⁇ NaCl, 2 mM EDTA, 0.01 M Tris-HCl ⁇ ⁇ 8.2, 0.1% (Twe e ⁇ 20, 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.
  • 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 ⁇ ) 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
  • 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.
  • the antibody binding activity was compared by latex agglutination.
  • the operation is as follows.
  • 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).
  • 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.
  • the SAA eluted on the 10-4 OkD surface is nothing but rSAA.
  • the known antibodies show no observable aggregation with rSAA, whereas the antibodies of the present invention react and aggregate with rSAA.
  • 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).
  • 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 ⁇ l of each concentration of SAA-containing solution were dispensed into the measurement cell, and after 30 seconds, 75 ⁇ l 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.
  • 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).
  • Figure 6 shows a standard curve drawn based on the data in Table 1.
  • 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.
  • 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.
  • 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.
  • Antibodies were diluted 2000-fold with 2 OmM phosphate buffer ( ⁇ 7.2, 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.
  • 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 ⁇ l 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.
  • FIG. 7 antibody of the present invention
  • 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.
  • rSAA which is an antigen is used as a solid phase ligand of IAsys, and an antibody is used for a liquid phase.
  • 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).
  • RSAA (50 ⁇ g / ml) previously dissolved in an acetate buffer (10, pH 5.0) was added as ligand solution (200 ⁇ l) and immobilized for 10 minutes.
  • EDC refers to e-thy 3- (3-dimethylaminopropyl) carbodiimide
  • NHS refers to N-hydroxysuccinimide.
  • 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.
  • the association rate constant K ass and the dissociation rate constant K diss 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.
  • 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.
  • 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.
  • 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 ⁇ , 1 ⁇ , ⁇ ⁇ , 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.
  • 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.
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acid synthetic DNA

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Abstract

La présente invention se rapporte à un anticorps destiné au dosage immunologique fondé sur l'agglutination de l'amyloïde A sérique humaine (SAA), qui sert de marqueur d'inflammations, etc., et à l'utilisation de cet anticorps. Ce nouvel anticorps reconnaît la SAA et se lie également à la SAA contenue dans les fractions de filtration de gel de poids moléculaires compris entre 10 et 40kD. L'invention concerne également un réactif et un procédé de dosage immunologique de la SAA, ainsi qu'un procédé de purification de la SAA grâce à cet anticorps. Ce nouvel anticorps ayant une excellente activité de liaison, il permet d'obtenir le dosage de la SAA en se basant sur l'agglutination immunologique de cette dernière. Cet anticorps est excellent pour le titrage contre la SAA et ne contribue pas seulement au dosage spécifique de la SAA avec une sensibilité élevée, mais également à l'élargissement du champ d'application du dosage.
PCT/JP1996/002219 1995-08-08 1996-08-07 Anticorps reconnaissant l'amyloide a serique WO1997006184A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1224319A4 (fr) * 1999-08-25 2004-11-24 Accuplex L L C Dosages diagnostiques de fluides biologiques secretes pour la detection d'etats infectieux et inflammatoires
US7214512B2 (en) 1999-10-22 2007-05-08 The Board Of Regents Of The University Of Nebraska Genomic mammary Amyloid a sequence
US7368546B2 (en) 2003-01-21 2008-05-06 The Board Of Regents Of The University Of Nebraska Human SAA3 nucleic acid molecule, protein, and methods of use for same
CN109503713A (zh) * 2017-09-15 2019-03-22 德赛诊断系统(上海)有限公司 抗人saa单克隆抗体及其制备方法和应用
GB2568298A (en) * 2017-11-13 2019-05-15 Univ Stellenbosch Methods, systems and devices for detecting inflammation
CN112816684A (zh) * 2021-01-07 2021-05-18 武汉华美生物工程有限公司 血清淀粉样蛋白a的校准品稀释液、其制备方法及其应用

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JPS61191697A (ja) * 1985-02-15 1986-08-26 ガムブロ　ルンデイア　アクチーボラグ ペプチド化合物およびこの化合物を使用して製造された抗血清

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JPS61191697A (ja) * 1985-02-15 1986-08-26 ガムブロ　ルンデイア　アクチーボラグ ペプチド化合物およびこの化合物を使用して製造された抗血清

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Title
BIOCHEMICAL JOURNAL, Vol. 263, (1989), ALISTAIR F. STRACHAN et al., "Human Serum Amyloid A Protein", p. 365-370. *
BIOPHYSICAL CHEMISTRY, Vol. 36, No. 4, 1992, HIROMI NAGATOKU et al., "Study (1st Report) on Serum Amyloid A(SAA)", p. 29-34. *
BIOPHYSICAL CHEMISTRY, Vol. 37, No. 1, 1993, HIROMI NAGATOKU et al., "Study (2nd Report) on Serum Amyloid A(SAA)", p. 19-23. *
PROC. NATL. ACAD. SCI. U.S.A., Vol. 89, (1992), L. MEEK et al., "Murine Serum Amyloid A3 is a High Density Apolipoprotein and is Secreted by Macrophages", p. 7949-7952. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1224319A4 (fr) * 1999-08-25 2004-11-24 Accuplex L L C Dosages diagnostiques de fluides biologiques secretes pour la detection d'etats infectieux et inflammatoires
US7569338B1 (en) 1999-08-25 2009-08-04 Accuplex, Llc. Diagnostic assays of secreted biological fluids for detection of infection and inflammatory conditions
US7214512B2 (en) 1999-10-22 2007-05-08 The Board Of Regents Of The University Of Nebraska Genomic mammary Amyloid a sequence
US7368546B2 (en) 2003-01-21 2008-05-06 The Board Of Regents Of The University Of Nebraska Human SAA3 nucleic acid molecule, protein, and methods of use for same
CN109503713A (zh) * 2017-09-15 2019-03-22 德赛诊断系统(上海)有限公司 抗人saa单克隆抗体及其制备方法和应用
GB2568298A (en) * 2017-11-13 2019-05-15 Univ Stellenbosch Methods, systems and devices for detecting inflammation
WO2019092678A1 (fr) * 2017-11-13 2019-05-16 Stellenbosch University Procédés, systèmes et dispositifs de détection d'une inflammation
CN112816684A (zh) * 2021-01-07 2021-05-18 武汉华美生物工程有限公司 血清淀粉样蛋白a的校准品稀释液、其制备方法及其应用

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