JPH07270414A - Highly sensitive enzyme immunoassay - Google Patents

Abstract

PURPOSE:To detect a slight amount of antigen contained in blood by employing a peroxydase-labelled antibody separated and refined by using a hydroxyapatite column and having an aiming bonding ratio for an enzyme immunoassay EIA. CONSTITUTION:Uniform monochronal antibodies to carry out complete separation are preferably used as an antibody. Any substances commonly used as an antigen are used as an antigen to be made immune in order to obtain an antibody. As an enzyme for bonding with the antibody, a peroxydase is preferably used and glutaric dialdehyde, periodic acid method, etc., are employed for the bonding method. A general or high speed column may be employed for the hydroxyapatite column. A sodium phosphate buffer solution may be used as an eluent for sample elution. To obtain an antibody with 1-2 bonding ratio, absorbance (A280, A403) of the eluted solutions obtained by fractionation while monitoring with ultraviolet absorbance at 280nm is measured, the antibody concentration and HRD concentration are calculated, and the solutions whose bonding ratio is 1-2 are gathered.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a highly sensitive enzyme immunoassay method, and more particularly to an enzyme immunoassay method using a specific peroxidase-labeled antibody.

[0002]

2. Description of the Related Art Enzyme immunoassay (hereinafter abbreviated as EIA)
In addition to the advantage of not using radioactive substances, it has become clear that higher sensitivity measurement is possible than radioimmunoassay. Is widely used as. In particular, in recent years, the automation of inspection has advanced and it has been widely used. In this EIA measurement,
Generally, an enzyme-labeled antibody obtained by labeling the antibody with an enzyme is often used, and the quality of the enzyme-labeled antibody greatly affects the performance of the entire measurement system.

As a method of labeling an antibody with an enzyme, many methods such as the glutaraldehyde method, the periodate method, and the maleimide method have been tried. In any case, the sample after the labeling reaction is an enzyme and an unlabeled antibody. , And a mixture of enzyme-labeled antibodies in which several kinds of enzyme-labeled antibodies having different binding rates are mixed. In order to suppress non-specific adsorption and increase the measurement sensitivity, it is necessary to separate and use only the enzyme-labeled antibody having the desired binding rate from this mixture.

However, it is difficult to separate only the enzyme-labeled antibody having the desired binding rate by the generally known gel filtration method, and other effective and convenient separation means are hardly known. Further, in JP-A-2-002937, a sample after the labeling reaction is purified using a hydroxyapatite column, but the enzyme-labeled antibodies obtained are different in binding rate by simply separating the unlabeled antibody and the enzyme. Since it was a mixture of various enzyme-labeled antibodies, the measurement sensitivity was still insufficient when used in EIA, and the storage stability was not sufficiently satisfactory.

[0005]

In view of this situation, the present invention fractionates the enzyme-labeled antibody after the labeling reaction according to the binding rate of the enzyme to the antibody, has a low background due to non-specific adsorption, and has a high measurement sensitivity. And to obtain an enzyme-labeled antibody having a binding rate with good storage stability and using it for EIA.

[0006]

The above object can be achieved by separating and purifying an enzyme-labeled antibody using a hydroxyapatite column to obtain an enzyme-labeled antibody having a desired binding rate and using it in EIA.

Antibodies used in the present invention include polyclonal antibodies.
Although both null and monoclonal antibodies can be targeted, monoclonal antibodies that are homogeneous as a molecule are more preferred to obtain a more complete separation. Further, the present invention is particularly effective when the antibody is IgG.

Antigens to be immunized to obtain antibodies include cells derived from humans and various animals, high molecular compounds such as proteins and nucleic acids, hormones and other low molecular compounds,
All substances commonly used as antigens can be used.

Further, peroxidase is suitable as an enzyme to be bound to the antibody, particularly horseradish peroxidase.
Ze (hereinafter abbreviated as HRP) is preferably used.

As a method for binding the peroxidase to the antibody, all known methods such as the glutaraldehyde method, the periodate method and the maleimide method can be used. Especially when HRP is used as a peroxidase, the maleimide hinge method is suitable.

The hydroxyapatite column used to achieve the present invention can be used for both general column chromatography and high performance liquid column chromatography. "Biogel HT" and "Biogel HTP" (manufactured by Bio-Rad) as packing materials for general column chromatography, and "HCA-Co" as column for high performance liquid chromatography.
Many commercially available products such as lumn "manufactured by Mitsui Toatsu Chemicals, Inc. are available. To shorten the separation time, 10 m
It is preferable to use a high performance liquid chromatography capable of a high flow rate of about 1 / min.

As the eluent for elution of the sample,
Eluents generally used in hydroxyapatite column chromatography such as sodium phosphate buffer and potassium phosphate buffer can be used. Sample elution is a linear gradient,
Both stepwise concentration gradients are used. In either case, a concentration gradient from low ionic strength to high ionic strength is generally used, but elution with a pH gradient is also possible,
It is more preferable to use both in combination. Also, depending on the sample, low concentration of calcium ions,
Effective separation may be obtained with magnesium ions or the like.

To obtain an antibody with a binding rate of 1 to 2, 280
Achieved by collecting the eluates 1 and 2 by measuring the absorbance (A ₂₈₀ , A ₄₀₃ ) of the eluate obtained by fractionation while monitoring the ultraviolet absorption at nm, and calculating the antibody concentration and HRP concentration. To be done.

As the enzyme immunoassay method of the present invention, various measuring principles such as a sandwich method and a competitive method can be used, but a sandwich method using a peroxidase-labeled antibody is preferably used. The sandwich method using a peroxidase-labeled antibody comprises a) a sample containing an antigen, an immobilized antibody in which an antibody capable of specifically reacting with the antigen to be measured is immobilized on a solid support, and an antigen specifically reacting with the antigen to be measured. The step of reacting with the peroxidase-labeled antibody obtained, b) the step of separating the immobilized antibody generated in step a) -the antigen to be measured-the peroxidase-labeled antibody from the unreacted peroxidase-labeled antibody, and c) being immobilized on a solid substance. The enzyme immunoassay method is characterized by including the step of detecting the complex using a peroxidase contained in the complex. The carrier for the immobilized antibody is appropriately selected, but plastic test tubes, microtiter plates, glass beads, plastic beads, membranes, magnetic beads and the like are used.

The enzyme immunoassay method of the present invention can be used for both the so-called one-step sandwich method and the two-step sandwich method.

[0016]

The present invention will be described in more detail with reference to the following examples.

Example 1 38 mg of anti-CA19-9 mouse monoclonal antibody NS19-9 (Scentcore, class IgG ₁ ) obtained by immunizing human colon cancer cells and HRP (Baehringer-Mannheim, Ltd.) 36 mg of the grade I) was bound by the maleimide hinge method to obtain a reaction solution of 7.9 ml. The reaction solution was filtered through a 0.22 μm filter and then kept at 37 ° C. for a hydroxyapatite column (Mitsui Toatsu Co., Ltd.).
HCA column F2025, φ20mm × L250m
m), and using high performance liquid chromatography (Shimadzu Corporation, Shimadzu LC-6A), 10 mM
500 mM from sodium phosphate buffer (pH 5.8)
Separation and elution were performed with a concentration gradient of sodium phosphate buffer (pH 6.8) at a flow rate of 7 ml / min. The eluate was monitored by ultraviolet absorption at 280 nm to fractionate the protein portion. Absorbance of each fraction (A ₂₈₀ ,
A ₄₀₃ ) was measured and the concentration of NS19-9 was (A ₂₈₀ -A
_From the formula of _{403 /} 3.1) /1.5×160000 (M), the concentration of HRP is A _{403 /} 2.275 × 40000
The rate of HRP binding to NS19-9 was calculated from the formula (M). As a result, HR for NS19-9
Fractions with P binding rates of 2.5, 1.5 and 0.8
10 mg, 25 mg and 3 mg were obtained respectively. 280
The elution pattern monitored by UV absorption at nm is shown in FIG.

Using these three fractions of peroxidase-labeled antibody, a two-step sandwich EIA
And CA19-9 solution of known concentration (0 U / ml, 300
U / ml) was measured and the absorbance was determined. Table 1 shows the measurement results and the S / N ratio {(300 U / ml absorbance) / (0 U / ml absorbance)} that serves as an index of measurement sensitivity.

[0019]

[Table 1]

Next, these fractions were stored at 8 ° C., and after 3 and 6 months, CA19 of known concentration was prepared by the same method.
The absorbance of the -9 solution was measured. The results are shown in Table 2.

[Table 2]

As shown in Table 1, the fraction having a binding rate of 1.5 has the highest S / N ratio and the highest measurement sensitivity. On the other hand, the fraction with a binding rate of 2.5 is 0 U / ml.
Has a high absorbance and the fraction with a binding rate of 0.8 is 3
Absorbance is low at 00 U / ml, both fractions,
Small S / N ratio and low measurement sensitivity. Furthermore, as shown in Table 2, upon storage at 8 ° C., the fractions having a binding rate of 0.8 and 1.5 are stable for 6 months without changing the absorbance, and can be stored for a long period of time. On the other hand, the fraction with a binding rate of 2.5 has a higher absorbance at 0 U / ml and a lower S / N ratio, although the absorbance at 300 U / ml has decreased with storage, and the measurement sensitivity has decreased. Obviously it's falling.

Example 2 Anti-CA15-3 mouse monoclonal antibody DF3 obtained by immunizing a fraction rich in membrane components of human breast cancer metastatic cells
(Cent core company class IgG ₁ ) 69mg and HRP
62 mg were bound by the maleimide hinge method, 13.
9 ml of reaction liquid was obtained. The reaction solution was filtered through a 0.22 μm filter, applied to a hydroxyapatite column kept at 37 ° C., and eluted by high performance liquid chromatography under the same conditions as in Example 1 to fractionate a protein portion. . The elution pattern was almost the same as in Example 1. As a result of measuring the absorbance of each fraction and calculating the binding rate, the HRP binding rate to DF3 was 2.5, 1.
The fractions of 5 and 0.8 were 16 mg and 36, respectively.
mg and 6 mg were obtained.

These fractions were subjected to 2-step sandwich EIA in CA15-3 solution (0 U /
(100 ml / 300 U / ml), the fraction having a binding rate of 1.5 had the highest measurement sensitivity and good storage stability. Table 3 shows S / N which is an index of measurement sensitivity.
The results of storage stability are shown in Table 4.

[0024]

[Table 3]

[0025]

[Table 4]

[0026]

INDUSTRIAL APPLICABILITY The present invention is a method of using a high-quality peroxidase-labeled antibody separated and purified by using a hydroxyapatite column for EIA. The peroxidase-labeled antibody obtained by fractionation has a low background due to non-specific adsorption and a high measurement sensitivity, and thus has a high sensitivity EIA.
It is possible to detect a trace amount of antigen contained in blood and the like. Also, since it has good storage stability, stable quality can be obtained for a long period of time when used in an EIA kit.

[Brief description of drawings]

1 is a separation pattern of a solution after a binding reaction between NS19-9 and HRP in Example 1. FIG.

Claims (2)

[Claims] 1. A highly sensitive enzyme immunoassay method, which comprises using a peroxidase-labeled antibody having a binding ratio of peroxidase to the antibody of 1 to 2. 2. The peroxidase-labeled antibody is purified by a hydroxyapatite column.
The highly sensitive enzyme immunoassay described.