JPS61122223A - Purification of antibody - Google Patents

Abstract

PURPOSE:To decrease the heterologous interference of heteroantibodies, and to purify the antibody to the one suitable especially for the low-concentration immunoglobulin assay, by immobilizing an antibody of an antibody fluid col lected from two kinds of animals to an insoluble carrier, and contacting the immobilized antibody with the other antibody liquid. CONSTITUTION:In an immunoassay using a sandwich process wherein the antigen separated from the first animal species is sandwiched stepwise with antibodies collected from the second and the third animal species, the antibody is purified by immobilizing either the second or the third antibody to an insoluble carrier (e.g. CNBR-activated sephalose column), and contacting the antibody to the other antibody. The reactive components in the heteroantibodies can be removed, and the mutual interference can be decreased. Accordingly, an immunoassay can be carried out in high sensitivity. It is especially useful for the purification of an antibody suitable for the assay of a low-concentration immunoglobulin.

Description

[Detailed description of the invention] [Field of application of the invention] The present invention relates to a method for purifying antibodies.

[Background of the invention]

The invention related to the method for purifying antibodies is disclosed in Japanese Patent Application Laid-Open No. 146-1958.
No. 855 is known, but this invention relates to a method for purifying antibodies of a second animal species, and 1.
It is not involved in immunoassays using the Sand-Deutsch method using different antibodies.

[Purpose of the invention]

The present invention relates to a method for purifying antibodies used in immunoassays, and is particularly aimed at providing a method for purifying antibodies suitable for assaying low-concentration immunoglobulins.

[Summary of the invention]

It has been found that in immunoassays using the "sandwich" method, in which the test antigen is sandwiched between two types of antibodies in stages, interference occurs between the two types of antibody components used, which increases the background and tends to cause errors. . Therefore, in order to reduce the mutual interference between antibodies from different species of animals, one of the antibody solutions from two types of animals is immobilized on an insoluble support, and the other antibody solution is brought into contact with this. It has been found that it is advantageous to separately purify antibody solutions from two types of animals by removing reactive components.

[Embodiments of the invention]

The present invention will be explained in detail below.

Example 1 Human immunoglobulin G (hereinafter abbreviated as IgG) to rabbits
A solution of anti-human IgG (containing 5 mg) obtained by immunization with CN
Br-activated Sepharose column (diameter 5 m, length 2
5m), left at room temperature for 24 hours, and anti-human IgG (
rabbit) column was prepared.

Next, anti-human Ig obtained from goat: G solution (anti-human Ig
G (containing 2 mg/mQ) was placed on the above column, and the flow rate was low (0
,, l m Q /a+in ) and 0.1 moQ/Q
, pH 8.0 phosphate buffer (NaCQO, 5mO
Q/Q) was allowed to flow down, and the eluate was collected. Components reactive with rabbit anti-human IgG have been removed and are not present in the eluate. A part of this eluate was used as the first antibody to transfer it to an insoluble support (polystyrene tube, glass tube,
immobilized on latex particles, polyacrylamide gel, agarose gel, etc.).

Next, anti-human IgG obtained from rabbits was purified using a goat antibody column and then labeled with a labeling reagent (eg, isothiocyanine Ml conductor of fluorescein).

The method of attaching the labeling substance is the usual method (Shunsuke Migita Hato, Immunochemistry, Medical Chemistry Experimental Method Course 4. Published by Nakayama Shoten.

(see 1972).

Human IgG was added 10-' to the insoluble support containing the first antibody.
Samples containing in the range of mg/d Q to 10 mg/d Q were contacted and bound (Figure 1b). Next, this was washed with a large amount of phosphate buffer and reacted with a solution containing the second labeled antibody (Fig. 1C). After washing with phosphate buffer again, the concentration of the labeled substance was measured by fluorescence photometry (excitation wavelength 4907 m, fluorescence wavelength 520 nm+). As a result, no matter which insoluble support was used, the blind value (value when using a sample containing no human IgG) was reduced by 70 to 79%. The minimum detectable amount also increased by 175 to 174 times.

Example 2 Anti-human IgG obtained by immunizing a goat with human immunoglobulin G (IgG) was immobilized on a column in the same manner as in Example 1 (rabbit anti-human IgG was used in Example 2).
The eluate was collected.

This purified anti-human 1. When a sandwich immunoassay of human IgG was performed using G, G solution (goat), the blinded value was 62% lower than that before purification. human Ig
The calibration curves for G showed almost similar fluorescence intensities. As a result, the minimum detectable amount was reduced by a factor of five.

Example 3 A CNBr-activated Sepharose column (diameter 5 mm, length 25+ m) was filled with 1 mQ of a solution containing 5 mg of anti-human albumin antibody obtained from a rabbit, and left at room temperature for 24 hours. rabbit) column was prepared. Next, anti-human albumin antibody solution obtained from goat (
2,4 mg/m Q containing) was poured into the above column, and 0.1 mo Q was poured at a flow rate of 0.1 mQ/min.
/Q, pH 8,0 phosphate buffer (Na(Qo, 5mo
(including Q/Q) was allowed to flow down, and the eluate was fractionated after discarding the volume corresponding to the void volume. A part of the eluate was prepared in Example 1.
It was immobilized on an insoluble support in the same manner as above, and the remainder was labeled with the labeling enzyme horseradish peroxidase according to a conventional method. Sand-Germany method using two types of antibodies prepared. −
A calibration curve was created using a colorimetric method using phenylenediamine color development (enzyme immunoassay, see Igaku Shoin publication). Second
An example is shown in the figure. Curve a in FIG. 2 is the case using the antibody purified according to the present invention.

The curve in the figure shows the case where the antibody before purification was used.

In the example, the absorbance decreased to 1/3 at Omg/dQ6.
The minimum detection amount was reduced to about 1/3.

〔Effect of the invention〕

According to the present invention, mutual interference between antibodies derived from two or more types of animals can be significantly reduced, making highly sensitive immunoassay possible.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram showing the Sand-Deutsch method of immunoassay, and FIG. 2 is a calibration curve diagram of human albumin based on a colorimetric method using a purified antibody according to an embodiment of the present invention. 1... Insoluble support, 2... First antibody, 3... Antigen, 4... Second antibody (labeled antibody). ,,r--

Claims (1)

[Claims] 1. In immunoassaying the antigen from the first animal species,
In an immunoassay using antibodies against an antigen of a first animal species obtained from a second and third animal species, either the second or the third is immobilized on an insoluble support; By contacting the antibody of
A method for purifying antibodies characterized by reducing interference phenomena.