JPH06160383A - Immunologically measuring method for human anp - Google Patents

Abstract

PURPOSE:To effectively prevent a decrease in a measured value by using antibody for recognizing an antigen-deciding group existing between an N-terminal part and a cyclic part of a human ANP. CONSTITUTION:A sole antibody to a cyclic part or an N-terminal part or a combination of antibodies to the both can be used in an antibody for recognizing an antigen deciding group existed between the N-terminal part and the cyclic part of a human ANP. The antibody may be not only polyclonal antibody but also monoclonal. When the monoclonal antibody is used, several types of monoclonal antibodies may be combined. The polyclonal antibody and the monoclonal antibody may be mixed for use. A plurality of antigen deciding groups exist between the N-terminal part and the cyclic part of the human ANP, and any antibody for recognizing the parts may be widely selected.

Description

Detailed Description of the Invention

[0001]

The present invention relates to ANP in human blood.
The present invention relates to an immunological measurement method for (atrial natriuretic peptide). More specifically, the present invention relates to human A
The present invention relates to a method for preventing a decrease in measured value which is a problem in immunological measurement of NP. ANP is a peptide hormone reported by Matsuo and Samukawa et al. In 1983 (Reference 1), and is produced in the heart and has potent natriuretic activity and smooth muscle relaxing activity. Human ANP has a molecular weight of about 3
000 α-type, β-type which is an inverse equilibrium dimer of α-type, and γ-type which is a precursor of α-type have been confirmed (hereinafter, human ANP unless otherwise specified). Refers to the α type). It has been reported that the blood concentration of human ANP serves as an index for dialysis of various heart diseases and dialysis patients (Reference 2).
~ 4). Reference 1: Biochem.Biophys.Res.Comm., 118; 131,1984 Reference 2: Biomed.Res., 7; 173,1986 Reference 3: J.Clin.Endocrinol.Metab, 63; 819,1986 Reference 4: J .Endocr., 110; 193,1986

[0002]

[Problems of the prior art] For human ANP, various immunological measurement methods have been tried. The following documents can be referred to as the immunological assay methods for human ANP that have been reported so far.・ RIA competition method using monoclonal antibody against human ANP cyclic region and N-terminal (Life Science, Vol. 43, 761-
768,1988: Reference 5, etc.)-Polyclonal antibody against human ANP, monochrome-
ELISA sandwich method using null antibody (J. Immuno
l. Methods, 124, 25-28, 1989) ・ Monoclonal antibody against human ANP cyclic site and C
ELISA sandwich method using polyclonal antibody against powder (included in the 65th Annual Meeting of the Japanese Society of Endocrinology / Abstract 78) In these immunological measurement methods, the measured value of ANP decreases with the lapse of time after blood collection. To be observed. In any of the reports, the immunological measurement operation itself is a general method applied to ANP, and since it is a measurement system using an antibody recognizing the C-terminal portion or a monoclonal antibody, measurement after blood collection It seems that the value is decreasing.

At present, only the following methods are known as techniques for avoiding a decrease in the measured value of human ANP with the passage of time after blood collection. That is, human ANP is extracted as soon as possible after blood collection, and the extracted human ANP is measured. This method has a problem that it requires an extraction operation of human ANP and that the time until extraction and the extraction conditions may affect the measured value. In order to reduce the influence of these fluctuation factors as much as possible, a protease inhibitor is often required to prevent the reduction of human ANP measurement values by extraction. It is required to suppress degradation of human ANP as much as possible by adding a protease inhibitor to blood in advance. Thus, currently known human AN
The immunological measurement method of P is accompanied by some fluctuation factors of measured values. Therefore, it is desired to provide a method that enables highly accurate measurement with a simpler operation.

[0004]

DISCLOSURE OF THE INVENTION The present invention provides a new technique for preventing a decrease in the measured value with time after blood collection in an immunological assay method for ANP (atrial natriuretic peptide) in human blood. It is an issue. The object of the present invention is to reliably prevent a decrease in measured value without a special operation such as an extraction operation.

[0005]

The present invention provides an immunological assay for human ANP, which comprises using an antibody that recognizes an antigenic determinant present between the N-terminal portion and the cyclic portion of human ANP. Method for preventing decrease in measured value, immunoassay method for human ANP using the method for preventing decrease in measured value,
And a reagent for measurement.

As the antibody recognizing the antigenic determinant present between the N-terminal portion and the cyclic portion of human ANP of the present invention, an antibody against the cyclic portion or the N-terminal portion is used alone, or a combination of these antibodies is used. be able to. The antibody may be a polyclonal antibody or a monoclonal antibody. When using a monoclonal antibody,
A plurality of types of monoclonal antibodies may be used in combination. In addition, a mixture of a polyclonal antibody and a monoclonal antibody may be used. Although a plurality of antigenic determinants are present from the N-terminal portion to the cyclic portion of human ANP, a wide range of antibodies can be selected as long as they are antibodies that recognize this portion. Table 1 exemplifies combinations of antibodies that can be used in the present invention. The “monoclonal antibody” in the table indicates that either one kind or a plurality of kinds can be used.

[0007]

[Table 1]

The antibody required for the present invention can be prepared by a known method. That is, a polyclonal antibody required for the present invention can be prepared by obtaining an antiserum using human ANP or a synthetic ANP peptide fragment as an immunogen and performing immunoaffinity purification with an antigenic determinant at a required site. Alternatively, the same immunogen can be used to obtain a monoclonal antibody by screening based on the reactivity to a necessary site. Known monoclonal antibodies can be used as the monoclonal antibody of the present invention. For example, the above-mentioned document 5 describes a monoclonal antibody that recognizes a cyclic portion and a monoclonal antibody that recognizes an N-terminal portion.

The present invention provides an effect of preventing a decrease in measured value for all peptides having ANP activity such as α-type, β-type and γ-type. Since these ANPs have the same structure in the N-terminal portion and the cyclic structure portion, the measured value preventing method of the present invention can be applied to any of the ANPs. The γ-type has a structure in which the N-terminal portion of the α-type is extended, but since it has an antigenic determinant common to the α-type, it is possible to prevent a decrease in the measured value by using the same antibody.

The immunological measurement method to which the method for preventing reduction of the measurement value in the immunological measurement of human ANP of the present invention can be applied is any immunoassay which can be carried out by utilizing the above-mentioned combination of antibodies. Includes immunological measurement methods. Therefore, specifically, a competitive method, a sandwich method, an agglutination method, or the like can be used as the measurement principle, and a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like can be used as the type of label.

A particularly preferable combination among such antibodies and measuring methods is a competitive reaction method using an antibody that recognizes an antigenic determinant present in the N-terminal portion. In this case, an enzyme, radioisotope or the like can be selected as the label. It is preferable to use one or more monoclonal antibodies. The reason is that it is easy to obtain an antibody specific to human ANP for the N-terminal portion, and the measurement operation is relatively simple in the competitive reaction.

According to the present invention, it is possible to sufficiently obtain the effect of preventing decrease in the measured value in the immunological measurement of human ANP, but it is more complete if a known protease inhibitor such as aprotinin is added to blood in advance. It can be expected to prevent the decrease of measured values.

[0013]

The antibody which recognizes the antigenic determinant existing between the N-terminal portion and the cyclic portion of human ANP of the present invention is human AN.
It has the effect of preventing a decrease in the measured value in the immunological measurement of P. The present inventors have advanced the research on the cause of the decrease in the measured value of human ANP after blood collection, which is caused by the disappearance of the antigenicity of human ANP with time, and the disappearance of the antigenicity is caused by the protease inhibitor. It was confirmed that it could not be sufficiently prevented by addition of. On the other hand, since the N-terminal part or the cyclic part of human ANP is relatively stable in antigenicity even after blood collection, it was discovered that a decrease in the measured value can be prevented by using an antibody that recognizes this part. Was completed. Next, the present invention will be described in more detail based on examples.

[0014]

【Example】

Changes in ANP measurement values after blood collection Human ANP in plasma was measured by four immunological measurement methods using antibodies with different recognition sites, and changes in the measurement values with time after blood collection were compared. The four immunological assay methods are outlined in Table 2 and detailed below. As a sample, a human ANP high-value sample collected from EDTA / aprotinin was used.

[0015]

[Table 2]

Assay a: Competitive Method Using One Monoclonal Antibody Recognizing N-Terminal Part (2 Antibody Method) A peptide corresponding to the amino acid sequence of human ANP 1-28 was synthesized and bound to thyroglobulin by the carbodiimide method. Was used as an immunogen. The amino acid sequence of the 1-28 peptide is shown below. Amino acid sequence: SLRRSSCFGGRMDRIGAQ
SGLGCNSFRY This immunogen was made into an emulsion with Freund's complete adjuvant (5 μg peptide: 0.1 ml FC
A.), BALB-C mice were subcutaneously immunized. After three additional immunizations (once every two weeks) by the same procedure, splenocytes of a mouse with an increased antibody titer against 1-28 peptide were isolated and a plurality of monoclonal antibodies against human ANP were obtained by the cell fusion method. It was Furthermore, among these monoclonal antibodies, the binding of the antibody to the 125I1-28 peptide was confirmed by the synthetic partial peptide of human ANP (for example, 1-11, 1).
Those which recognize the N-terminal portion and those which recognize the cyclic portion were selected from the binding inhibition reaction by 3-28). The obtained monoclonal antibody was 0.1% EDTA2N
a, 0.1% NaN ₃ , 0.15M NaCl, 3.3% dextran T40, 0.5% bovine serum albumin 5
It was diluted to 5 μg / ml with 0 mM phosphate buffer (pH 7.2, hereinafter abbreviated as PBS) and used for measurement. As the second antibody, a commercially available anti-mouse antibody diluted 50 times with the same buffer was used. ^{125 of the} same 1-28 peptide by enzymatic method
It was labeled with I and diluted to 5 KBq / ml with PBS to give a labeled antigen. Also, the same peptide was prepared by a gravimetric method as a standard substance. The measurement operation is as follows. To 100 μl of the standard substance or sample, 100 μl of the monoclonal antibody and 100 μl of the labeled antigen were added and reacted at 4 ° C. for 3 hours. After the reaction, the second antibody was added and the mixture was further reacted at 4 ° C. for 1 hour, washed by centrifugation, and the radioactivity of the precipitate was counted by a γ counter.

Assay b: Competitive Method Using One Monoclonal Antibody Recognizing Cyclic Portion (2 Antibody Method) From the monoclonal antibodies against 1-28 peptide, those recognizing the cyclic portion are selected, and assay a and The measurement was performed by the same operation.

Assay c: Competitive Method Using One Polyclonal Antibody Recognizing C-Terminal Part (2 Antibody Method) Commercially available kit for measuring human ANP "A" using an antibody specific to the C-terminal part of human ANP The HANP kit "Eiken""(manufactured by Eiken Chemical Co., Ltd., trade name) was used. The procedure was in accordance with the designated procedure except that the reaction condition between the sample and the antiserum was 25 ° C. for 3 hours and the reaction condition with the labeled antigen was 4 ° C. overnight.

Assay d: Solid phase sandwich method using one monoclonal antibody that recognizes the C-terminal portion and one monoclonal antibody that recognizes the cyclic portion Commercially available human ANP measurement kit "Shionoria ANP"
(Shionogi Pharmaceutical Co., Ltd., trade name) was used according to the designated operation method.

The results are shown in Table 3. It was confirmed that the method using an antibody that recognizes the C-terminal portion of ANP, compared with the method according to the present invention (assays a and b), showed a marked decrease in the measured value even in the presence of protease.

[0021]

[Table 3]

[0022]

According to the present invention, human ANP can be immunologically measured with high accuracy. According to the present invention, a decrease in the ANP measurement value over time, which cannot be avoided by a method other than extraction by the conventional technique, can be easily prevented without any special operation.
That is, by using an antibody that recognizes a specific antigenic determinant, it is possible to effectively avoid a decrease in human ANP measurement value. In this example, a sample plasma to which aprotinin, which is a protease inhibitor, was added in advance was used as a sample because of the necessity of comparison with other methods, but such a protease inhibitor is not always necessary in the present invention. Therefore, it is possible to measure human ANP with high accuracy by using a sample prepared under the same blood collecting condition as other measurement items as a sample.

Claims (5)

[Reference Number] P-000268 [Claims] 1. A human, which uses an antibody that recognizes an antigenic determinant present between the N-terminal portion and the cyclic portion of human ANP, and which prevents a decrease in the measured value of human ANP that accompanies degradation after blood collection. Immunological measurement method of ANP 2. A method for preventing a decrease in the measured value in an immunological assay of human ANP, which comprises using an antibody that recognizes an antigenic determinant present between the N-terminal portion and the cyclic portion of human ANP. 3. An antibody that recognizes an antigenic determinant present between the N-terminal portion and the cyclic portion of human ANP is an antibody against the cyclic portion or the N-terminal portion, or a combination of these antibodies, and The antibody used is human ANP
3. The method for preventing decrease in measured value in immunological measurement of human ANP according to claim 2, wherein the method does not substantially react with an antigenic determinant existing near the C-terminus of 4. An antibody recognizing an antigenic determinant present between the N-terminal portion and the cyclic portion of human ANP is a monoclonal antibody against the cyclic portion or the N-terminal portion, or a combination of these monoclonal antibodies. The method for preventing decrease in measurement value in immunological measurement of human ANP according to claim 2, 5. A human ANP, which uses an antibody that recognizes an antigenic determinant existing between the N-terminal portion and the cyclic portion of human ANP, and which prevents a decrease in the measured value of ANP that accompanies degradation after blood collection. For immunological assay