JPH0980047A - Method for stably preserving antibody bound insoluble solid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an antibody bound insoluble solid in which inactivation of antibody bound to insoluble solid, desorption of antibody from insoluble solid, and decay due to deterioration of insoluble solid are eliminated and a specified antibody titer can be sustained even after a long term preservation. SOLUTION: An antibody for recognizing an antigen substance to be measured is bound to an insoluble solid and preserved in a buffer solution of pH4.0-6.0.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a stable storage method of an antibody-bound insoluble solid used in a heterogeneous immunoassay.

[0002]

2. Description of the Related Art Conventionally, various immunoassay methods are known for measuring substances in biological fluids. One of the most important immunoassays is the heterogeneous immunoassay. As a heterogeneous immunoassay, radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc. are known. These immunoassays
Since it is possible to measure a trace amount of substance in a sample liquid, it has been rapidly spread in recent years. In the heterogeneous immunoassay, for example, an enzyme immunoassay (edited by Eiji Ishikawa et al., Medical Institute, 1978
Issued annually), the sandwich method and competitive method are widely used. In these methods, an insoluble solid obtained by binding an antibody to glass beads or plastic beads is used. The antibody-bound insoluble solid used in the heterogeneous enzyme immunoassay is stored in a phosphate buffer containing 1% bovine serum albumin at a pH of about 7 until immersion, or in a dry state. It has been done.

[0003]

However, when the antibody-bound insoluble solid is immersed and stored in a buffer solution, the antibody bound to the insoluble solid is deactivated with time, the antibody is released from the insoluble solid, and the insoluble solid is removed. Due to the degradation and the like, the antibody titer of the antibody-bound insoluble solid decreases, which causes a problem that the measurement sensitivity deteriorates.

[0004]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that storage stability can be further improved by adjusting the pH of a buffer solution for storing antibody-bound insoluble solids within a specific range. It was found that an antibody-bound insoluble solid having excellent properties can be obtained, and the present invention was accomplished.

That is, according to the present invention, an antibody that recognizes an antigenic substance to be measured is bound to an insoluble solid, and then the pH is adjusted.
A stable storage method for an antibody-bound insoluble solid, which is characterized by storing in a buffer solution of 4.0 to 6.0.

In the present invention, the antigenic substance to be measured includes the antigenic substance to be measured described in JP-A-2-205774. Among these, the method of the present invention is suitable for the measurement of hormones, tumor-associated antigens and viruses, which require particularly sensitive measurement systems. As the antibody that binds to the insoluble solid, either a monoclonal antibody or a polyclonal antibody that recognizes the antigenic substance to be measured can be used.

As an insoluble solid, Japanese Patent Application Laid-Open No. 2-2057
Insoluble solids described in Japanese Patent Publication No. 74 are listed. Among these, preferred ones are capable of easily and stably binding an antibody,
Further, glass (glass beads, glass test tubes, glass petri dishes, etc.) and plastics (plastic beads, plastic tubes, plastic trays, etc.) that are easy to handle. When glass is used, the glass is harder than plastic, and is therefore preferable in that it is less likely to be scratched or deformed.

A method for binding an antibody onto an insoluble solid is as follows:
The method described in JP-A-2-205774 may be the same. That is, a method in which glass and a monoclonal antibody are chemically bonded (for example, US Pat. Nos. 4,280,992 and 365,2761), and a method in which an antibody is physically adsorbed on a plastic (for example, E. Engbar, J.E. Johnson, Pea Perlman;
Bio Kimika Bio Physica Actor (E. Engvall, JJ
onson, P. Parlmann; Biochimica Biopysica Acta), 251
Volume 1971, pp. 427-434).

In the present invention, the pH of the buffer solution is 4.0 to 4.0.
It is 6.0, preferably 4.5 to 5.5. pH is 6.
If it exceeds 0, erosion and disintegration of insoluble solids such as glass becomes severe, and the antibody titer of the antibody-bound insoluble solids decreases, which is a problem. Further, if the pH is less than 4.0, the antibody bound to the insoluble solid is denatured and inactivated, which is a problem.

The buffer solution of the present invention has a pH of 4.0-4.0.
It is essential to adjust the pH to 6.0, but pH in this range
If so, the type of buffer solution is not particularly limited. For example,
Examples thereof include a phosphate buffer solution, an acetate buffer solution, a citrate buffer solution, and a glycine buffer solution. Acetate buffer and phosphate buffer are preferred. In addition, in order to enhance storage stability, these buffers contain inorganic salts (sodium chloride, potassium chloride, etc.), proteins (bovine serum albumin, I
gG etc.), an antiseptic agent, etc. can be added in an appropriate amount, and in this case also, it is essential to adjust the pH to 4.0 to 6.0 later.

As the heterogeneous immunoassay, the sandwich method described in the literature [for example, enzyme immunoassay (edited by Eiji Ishikawa et al., Published by Medical Institute, published in 1978)], the competitive method, and the assay method described in JP-A-6-130063. Can be used. The sandwich method is obtained by binding an antigenic substance to be measured in a sample liquid to an insoluble solid and binding an antibody that recognizes this antigenic substance and a labeled antibody that recognizes this antigenic substance. It is also a method of measuring the amount of the antigenic substance by measuring the amount of the labeling substance in the immune complex. The competitive method is an immune complex obtained by binding an insoluble solid to the antigenic substance to be measured in the analyte liquid and binding the labeled antigenic substance to the antibody that recognizes this antigenic substance. It is a method of measuring the amount of the antigenic substance by measuring the amount of the labeling substance in the body.

As the labeling substance for labeling the antibody, the labeling substances described in JP-A-2-205774 (enzymes, radioisotopes, fluorescent substances, chemiluminescent substances, etc.) can be used. Of these, preferred are peroxidase and ¹²⁵ , which are easy to label with an antibody and have high sensitivity.
I.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be further described below with reference to examples, but the present invention is not limited thereto.

[0014]

【Example】

Example 1 First, the storage stability of glass beads bound with an antibody against insulin was measured. (1) Preparation of pH 5.0 Acetic Acid Buffer Solution An aqueous solution was prepared by adding 0.1M sodium acetate aqueous solution to 0.1M acetic acid aqueous solution to adjust pH to 5.0. To this aqueous solution, 1 W / V% bovine serum albumin, 0.9 W / V% sodium chloride, 0.1 W / V% sodium azide was added, and then 1 M HCl or 1 M NaOH was added in an appropriate amount to readjust the pH to 5.0. A buffer solution for immersing the antibody-bound insoluble solid was prepared. (2) Production of Insulin Enzyme Immunoassay Reagent a) Production of Anti-Insulin Antibody-Binding Glass Beads The surface of the glass beads was coated with anti-insulin antibody (manufactured by Dako Co.) according to the method of US Pat. No. 652761. After coating the glass beads with the above pH.
Immersed in 5.0 acetate buffer. b) Preparation of peroxidase-labeled anti-insulin antibody An anti-insulin antibody (manufactured by Dako) was used as a reference [S Yoshitake, M. Imagawa, E. Ishikawa, et al .; Jay. Biochem (S.YOSHITAKE, M.IMAGAWA, E.ISHIKAWA, et.al .;
J. Biochem.), 92, 1982, 1413-1424], to give a peroxidase-labeled anti-insulin antibody. This reagent was usually diluted 10 to 5000 times with a buffer for immune reaction and used for measurement. c) Preparation of Insulin Standard Solution Human insulin (Novo) was used in a 0.02 M phosphate buffer (pH 7.2) containing 1 W / V% bovine serum albumin at concentrations of 0, 10, 50, 125 and 250 μU / ml. Diluted so that

(3) Preparation of buffer for immune reaction 1 W / V% bovine serum albumin and 0.02 M phosphate buffer (pH 7.2) containing 0.9 W / V% sodium chloride were used as buffer for immune reaction I was there. (4) Storage stability test of anti-insulin antibody-bound glass beads 50 μl of standard solution or test sample, buffer solution 45 for immune reaction
One glass bead to which anti-insulin antibody was bound was placed in a test tube containing 0 μl, incubated (37 ° C., 15 minutes), and then washed with physiological saline. Next, the beads were transferred to 500 μl of a buffer for immune reaction containing a peroxidase-labeled anti-insulin antibody and incubated (37 ° C., 15 minutes). After washing the beads again with physiological saline, the beads were transferred to 500 μl of a substrate solution (ortho-phenylenediamine solution containing hydrogen peroxide) and incubated (37 ° C., 15 minutes), and then a 1.5 N sulfuric acid aqueous solution was added. The reaction was stopped by adding 3 ml. The absorbance of this solution at 492 nm was measured, and the enzyme activity of the enzyme bound to the beads was measured. Anti-insulin antibody conjugated beads at 40 ° C for 0 months, 1 month, 2 months, 6
Insulin standard solution 250 when stored for 1 month for 1 month
Table 1 shows the U / ml absorbance and the ratio to the 0-month absorbance.

[0016]

[Table 1]

Example 2 Next, the storage stability of glass beads bound with an antibody against β2-microglobulin was measured. (1) Preparation of pH 5.0 acetate buffer The same buffer as the pH 5.0 acetate buffer of Example 1 (1) was used. (2) Preparation of β2-microglobulin enzyme immunoassay reagent a) Preparation of anti-β2-microglobulin antibody-bound glass beads According to the method of US Pat. No. 6,527,61, anti-β2-microglobulin polyclonal antibody ( Dako) was coated. PH after coating
It was immersed in 5.0 acetate buffer. b) Preparation of peroxidase-labeled β2-microglobulin Reference to β2-microglobulin [Nakane PK,
Kawaoi A; J. Histochem. Sightchem (NAK
ANE, PK, KAWAOI, A.; J.Histochem.Cytochem.), 22, 197
4 years, page 1084], and peroxidase was bound by the method described in the above to obtain peroxidase-labeled β2-microglobulin. This reagent is usually 10 to 5,000 as an immune reaction buffer.
It was used after diluting it by a factor of 1. c) Preparation of β2-microglobulin standard solution Human β2-microglobulin (Zelco) at 1 W / V
0.02M Phosphate buffer solution containing 10% bovine serum albumin (pH
In 7.2), the concentration was diluted to 0, 1, 2, 4, 8 mg / L.

(3) Preparation of buffer solution for immune reaction 1 W / V% bovine serum albumin, 0.02 M phosphate buffer solution (pH 7.2) containing 0.9 W / V% sodium chloride was used as the buffer solution for immune reaction I was there. (4) Storage stability test of anti-β2-microglobulin antibody-bound glass beads Standard solution or test sample 50 μl, peroxidase-labeled β2-microglobulin-containing immune reaction buffer 450 μl
Put one glass beads with anti-β2-microglobulin antibody in the test tube containing 1 and incubate (37
(° C, 15 minutes). After washing the beads with physiological saline, the beads were transferred to 500 μl of a substrate solution (hydrogen peroxide-containing ortho-phenylenediamine solution) and incubated (37 ° C., 15 minutes), and then 3 ml of a 1.5 N sulfuric acid aqueous solution was added. In addition, the reaction was stopped. 49 of this liquid
The absorbance at 2 nm was measured, and the enzyme activity of the enzyme bound to the beads was measured. When the anti-β2-microglobulin antibody-bound beads were stored at 40 ° C. for 0 month, 1 month, 2 months, 6 months, 1 year, the absorbance of β2-microglobulin standard solution 0 mg / L and the absorbance at 0 month were measured. Table 2
It was shown to.

[0019]

[Table 2]

Example 3 The anti-β2-microglobulin antibody-bound glass beads were replaced by the following p in place of the pH 5.0 acetate buffer of Example 2 (1).
The time-dependent measurement of storage stability when immersed in H5.5 phosphate buffer was carried out in the same manner as in Example 2 (2) to (4). The results are shown in Table 2. (1) Preparation of pH 5.5 Phosphate Buffer Solution A 0.1 M potassium phosphate aqueous solution was mixed with a 0.1 M disodium phosphate aqueous solution to prepare a pH 5.5 solution. 1 W / V% bovine serum albumin in this aqueous solution,
After adding 0.9W / V% sodium chloride and 0.1W / V% sodium azide, the pH was readjusted to 5.5 by adding an appropriate amount of 1M HCl or 1M NaOH, and a buffer solution for immersing the antibody-bound insoluble solid was prepared. It was made.

Comparative Example 1 For comparison with Example 1, glass beads to which anti-insulin antibody was bound were immersed in the following pH 6.5 phosphate buffer solution instead of the pH 5.0 acetate buffer solution of Example 1 (1). The time-dependent storage stability was measured in the same manner as in Examples 1 (2) to (4). The results are shown in Table 1. (1) Preparation of pH 6.5 phosphate buffer solution 0.05M phosphoric acid 2 in 0.05M potassium phosphate aqueous solution
An aqueous solution having a pH adjusted to 6.5 by adding a sodium aqueous solution was prepared. 1 W / V% bovine serum albumin, 0.9 W / V% sodium chloride and 0.1 W / V% sodium azide were added to this aqueous solution, and then 1 M HCl or 1 M NaO was added.
The pH was readjusted to 6.5 by adding an appropriate amount of H to prepare a buffer solution in which the antibody-bound insoluble solid was dipped.

Comparative Example 2 For comparison with Example 2 and Example 3, the anti-β2-microglobulin antibody-bound glass beads were replaced with the pH 5.0 acetate buffer solution of Example 2 (1), and the following pH 6.5 was used. The time-dependent measurement of storage stability when immersed in a phosphate buffer was performed in the same manner as in Examples 2 (2) to (4). Table 2 shows the results
It was shown to. (1) Preparation of pH 6.5 phosphate buffer solution The same buffer solution as the pH 6.5 phosphate buffer solution of Comparative Example 1 (1) was used.

As is clear from Tables 1 and 2, pH 5.0
Also, when the antibody-bound insoluble solid is immersed in a buffer solution of pH 5.5, it shows excellent storage stability with almost no decrease in absorbance with time, but at pH 6.5, the decrease in absorbance with time is severe, Storage stability is poor.

[0024]

INDUSTRIAL APPLICABILITY The antibody-bound insoluble solid stored in the buffer solution of the present invention having a pH of 4.0 to 6.0 has excellent long-term storage stability. That is, the antibody-bound insoluble solids are
When soaked and stored in a buffer solution of 4.0 to 6.0, inactivation of the antibody bound to the insoluble solid, desorption of the antibody from the insoluble solid, and disintegration due to deterioration of the insoluble solid are eliminated.
It is possible to obtain an antibody-bound insoluble solid that maintains a constant antibody titer even after long-term storage. From the above points, the preservation method of the present invention can be applied to antibody-bound insoluble solids used in all heterogeneous immunoassays, and is particularly used for the measurement of hormones, tumor-associated antigens and viruses that require highly sensitive measurement systems. Useful for antibody-bound insoluble solids.

Claims (2)

[Claims] 1. An antibody that recognizes an antigenic substance to be measured is bound to an insoluble solid, and then pH 4.0 to 6.0.
A stable storage method of an antibody-bound insoluble solid, which is characterized in that it is stored in the buffer solution of 1. 2. The stable storage method according to claim 1, wherein the insoluble solid is glass.